mart-1-antigen and Melanoma

"Cutaneous melanocytic tumor with CRTC1-TRIM11 fusion" (CMTCT) is a newly described, potentially novel entity that typically presents as a dermal nodule on the head and neck, extremities, and trunk of adults. Histopathologically, it is reported as a nodular or multinodular tumor composed of epithelioid and spindle cells that are variably immunoreactive for S100-protein, SOX10, and MITF along with more specific melanocytic markers such as MelanA and HMB45. With only 11 cases reported in the English literature so far, the neoplasm appears to behave in a relatively indolent fashion. Nevertheless, in one case, local recurrence and synchronous distant metastasis were evident after 13 years. Additional cases with longer follow-up are essential to determine the neoplasm's biologic behavior with more accuracy. Herein, two cases of CMTCT, one arising on the lower back of a 65-year-old female and the other on the arm of a 33-year-old female in addition to a comprehensive literature review are reported.

Topics: Adult; Aged; Aged, 80 and over; Child; Dermis; Female; gp100 Melanoma Antigen; Humans; Immunohistochemistry; MART-1 Antigen; Melanocytes; Melanoma; Microphthalmia-Associated Transcription Factor; Middle Aged; S100 Proteins; SOXE Transcription Factors; Transcription Factors; Treatment Outcome; Tripartite Motif Proteins; Ubiquitin-Protein Ligases

The presence or absence of metastasis in sentinel lymph nodes often drives melanoma staging, prognosis, and treatment. However, distinguishing between metastatic melanoma cells and clusters of benign melanocytic nevus cells is not always straightforward. When morphologic hematoxylin and eosin interpretation alone is not sufficient, additional hematoxylin and eosin sections and immunohistochemical (IHC) studies may be beneficial. This review and small cases series of 3 diagnostically challenging melanocytic sentinel lymph node cases highlights the IHC approach to evaluate intraparenchymal nodal melanocytic nevi, coexistent metastatic melanoma with adjacent melanocytic nevi cells, and nodal blue nevi. In challenging cases, cytological morphology of the melanocytes, location within the lymph node, and IHC studies may assist in diagnosis. If these tools yield conflicting results, expert opinion is recommended.

Topics: Aged; Diagnosis, Differential; gp100 Melanoma Antigen; Humans; Immunohistochemistry; Ki-67 Antigen; Lymphatic Metastasis; Male; MART-1 Antigen; Melanocytes; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasms, Multiple Primary; Nevus, Pigmented; Sentinel Lymph Node; Skin Neoplasms; SOXE Transcription Factors

To report the clinical and histopathologic features of two cases of eyelids metastases from uveal melanoma diagnosed in a metachronous and synchronous fashion.. Monocentric retrospective case series of histopathologically proven eyelids metastases from uveal melanoma at our institution.. Two patients were presented to our hospital for upper eyelids pigmented and firm lesions. Patient 1 had an history of left uveal melanoma treated conservatively with proton beam therapy 5 years earlier. Examination revealed bilateral upper eyelids lesions. Patient 2 had no malignancy history but was incidentally diagnosed with a cerebral nodule few months earlier. Examination revealed a right upper eyelid nodule and a previously unknown right uveal melanoma. Excisional biopsy was performed for both patients. Pathological assessment allowed the presence of melanoma cells. The lack of BAP1 nuclear expression on immunohistochemistry as well as the absence of cutaneous or mucosal melanoma were consistent with an uveal origin. Diffuse metastatic spread was noted for both patients. Systemic therapies were prescribed. Patient 1 died from metastatic spread (62 months and 4 months after uveal melanoma diagnosis and eyelids metastases removal, respectively) whereas patient 2 was still alive (14 months follow up).. Eyelids metastases from uveal melanoma is an exceptional finding. Excisional biopsy and pathological assessment are of main importance to confirm the diagnosis and to identify genetic mutations for further targeted therapies. Currently, prognosis remains poor.

Topics: Aged; Biomarkers, Tumor; Biopsy; Combined Modality Therapy; Eyelid Neoplasms; Fatal Outcome; Humans; Immunotherapy; Male; MART-1 Antigen; Melanoma; Middle Aged; Prognosis; Proton Therapy; Retrospective Studies; Tumor Suppressor Proteins; Ubiquitin Thiolesterase; Uveal Neoplasms

The incidence of primary mucosal malignant melanoma (PMMM) is 1.3% among all malignant melanomas (MM). Cervical involvement is very rare; the number of cases of cervical PMMM reported so far is around 80. In our patient, a dark color, 2-cm diameter, nonulcerated tumor formation was observed upon examination of the cervix. Tumoral tissue consisted of atypical melanocytic cells containing numerous mitotic figures. In immunochemical studies, S-100, Melan-A, and HMB-45 positivity were observed. The tumor was 20 mm in invasion depth, Breslow IV, and FIGO stage IB1. Radical surgery was followed by adjuvant radiotherapy, and subsequently interferon treatment was applied. Examination and scans 20 months after surgery were free from tumor.

Topics: Adult; Antineoplastic Agents; Biomarkers, Tumor; Chemotherapy, Adjuvant; Female; gp100 Melanoma Antigen; Gynecologic Surgical Procedures; Humans; Immunohistochemistry; Interferons; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Mucous Membrane; Neoplasm Staging; Radiotherapy, Adjuvant; S100 Proteins; Uterine Cervical Neoplasms

Since the identification of S100 protein as an immunohistochemical marker that could be useful in the diagnosis of melanoma in the early 1980s, a large number of other melanocytic-associated markers that could potentially be used to assist in the differential diagnosis of these tumors have also been investigated. A great variation exists, however, among these markers, not only in their expression in some subtypes of melanoma, particularly desmoplastic melanoma, but also in their specificity because some of them can also be expressed in nonmelanocytic neoplasms, including various types of soft tissue tumors and carcinomas. This article reviews the information that is currently available on the practical value of some of the markers that have more often been recommended for assisting in the diagnosis of melanomas, including those that have only recently become available.

Topics: Antibodies, Monoclonal; Biomarkers, Tumor; CD146 Antigen; gp100 Melanoma Antigen; Humans; Immunohistochemistry; Interferon Regulatory Factors; MART-1 Antigen; Melanocytes; Melanoma; Melanoma-Specific Antigens; Melanoma, Cutaneous Malignant; Microphthalmia-Associated Transcription Factor; Monophenol Monooxygenase; Nerve Tissue Proteins; Receptor, Melanocortin, Type 1; Receptors, Nerve Growth Factor; S100 Proteins; Skin Neoplasms; SOXE Transcription Factors; Tetraspanin 30

Malignant melanoma biologically can be divided into non-metastatic and metastatic forms which cannot be predicted precisely using classical clinicopathological parameters, therefore studies on novel genetic or protein markers are abundant in the literature. These studies did not result in clinically useful markers because mostly ignored the results of studies on the genetic basis of metastatic potential of malignant melanoma. Accordingly, the list of promising novel markers is short (BCL2, CDK2, MART-1, OPN). Similar to other solid malignancies, introduction of targeted therapy into clinical practice of melanoma turned the attention toward the genetic basis of resistance to chemo- and targeted therapies. These novel data could lead to the development of molecular diagnostics which can help in designing more effective therapeutic strategies of malignant melanoma.. A malignus melanóma, bár megjelenését tekintve igen sokszínû, de biológiailag áttétképzõ és áttétet nem képzõ formákra osztható. A klasszikus klinikopatológiai faktoroknak a betegség lefolyását felbecsülõ képessége limitált, ezért széleskörû kutatások történnek a melanóma progressziós markerei vonatkozásában. Bár sokféle genetikai vagy fehérjemintázatot azonosítottak, ezek klinikai haszna csekély, mert a vizsgálatok alig vették figyelembe a melanóma progressziója alapjainak megismerését célzó kutatások eredményeit, így az ígéretes markerek csoportja igen szûk (BCL2, CDK2, MART-1, OPN). Hasonlóan más szolid daganatokhoz, a célzott terápiák megjelenése ráirányította a figyelmet a melanóma kemo- és célzott terápiára mutatott rezisztenciájának genetikai alapjaira. Ezek az ismeretek új molekuláris patológiai eljárásokhoz és remélhetõen hatékonyabb terápiás protokollok kialakításához adhatnak segítséget.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Cyclin-Dependent Kinase 2; Diagnosis, Differential; Drug Resistance, Neoplasm; Humans; Interferons; Interleukin-2; MART-1 Antigen; Melanoma; Molecular Targeted Therapy; Osteopontin; Patient Selection; Peptide Fragments; Predictive Value of Tests; Prognosis; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-kit; Skin Neoplasms

To investigate the clinical pathological features, diagnosis and differential diagnosis of pigmented dermatofibrosarcoma protuberance (PDFSP).. The clinical history, histopathological features, immunohistochemical characteristics, treatment and prognosis were analyzed in seven cases of PDFSP. Fluorescence in situ hybridization (FISH) was used to detect the expression of COL1A1/PDGFB fusion gene, and related literature was reviewed.. The median age of the seven patients (4 females, 3 males) was 47 years with the tumors involving mostly the trunk (four cases). Histologically, PDFSP showed a cellular lesion composed of spindle cells arranged in short fascicles that form a distinct storiform pattern, and the pigmented bipolar or multipolar dendritic cells were present with tentacle like processes emanating from a nucleus containing zone. One case showed fibrosarcomatous change. The pigment was tinctorially similar to melanin. The spindle cells were positive for CD34 and vimentin, but negative for HMB45, Melan A, S-100, desmin, CD68 or α-SMA. HMB45, Melan A, S-100 and vimentin were expressed in the melanin containing cells in 4, 4, 5 and 7 cases, respectively. The labeling index of Ki-67 was 1%-8%. Among the 4 cases successfully examined by FISH, 3 showed t(17;22)(q21;q13) which suggested COL1A1/PDGFB fusion gene. Three patients were treated by wide local excision and four were treated by simple surgical excision. Two patients developed recurrences during the follow-up period of 12 to 123 months. Of those treated by wide local excision, none developed recurrence. No patient died in the follow-up period.. PDFSP is a rare pigmented variant of DFSP and an intermediate grade malignant tumor. The orgin of the tumor cells is still controversial. Surgical pathologists and dermatopathologists need to be aware of the prototypical histological appearance of PDFSP as there is a risk of misdiagonsing it as either pigmented tumors associated with neurocutaneous syndromes or a highly malignant melanocytic neoplasm.

Topics: Adult; Aged; Antigens, CD34; Child, Preschool; Dermatofibrosarcoma; Diagnosis, Differential; Female; Follow-Up Studies; gp100 Melanoma Antigen; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasm Recurrence, Local; Neurilemmoma; Neurofibroma; Oncogene Proteins, Fusion; Prognosis; Retrospective Studies; S100 Proteins; Skin Neoplasms; Vimentin

We report the first case, to our knowledge, of a possible primary, signet-ring cell melanoma of the gastroesophageal junction. The mass was initially diagnosed as an invasive, poorly differentiated carcinoma; however, on further review and immunohistochemical workup, the diagnosis of signet-ring cell melanoma was made. The lesion consisted of oval to round epithelioid cells undermining the gastric mucosa and infiltrating the muscularis mucosae. Tumor cells demonstrated abundant cytoplasm and eccentrically located nuclei, many with signet-ring cell morphology. The tumor cells were negative for mucin and pancytokeratin, strongly positive for S100 protein and Melan-A, and focally but strongly positive for human melanoma black-45. Diagnostic imaging failed to prove another site of melanoma, and no history of melanoma or cutaneous lesion was reported by the patient. Therefore, it was determined this was likely a primary lesion. We review the literature and previously reported cases of this rare histologic variant of melanoma.

Topics: Aged; Carcinoma, Signet Ring Cell; Diagnosis, Differential; Esophagogastric Junction; Gastrointestinal Neoplasms; gp100 Melanoma Antigen; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; S100 Proteins

With the advent of incorporating the immunoperoxidase staining technique into the processing of frozen tissue, the use of Mohs micrographic surgery (MMS) has been expanded to include several high-risk tumors such as lentigo maligna, malignant melanoma, and dermatofibrosarcoma protuberans.. To thoroughly review the English medical literature pertaining to the use of immunohistochemical staining techniques on frozen sections during MMS and to summarize the basic relevant outcomes from the different relevant studies.. Medline search was conducted, with the following words used in the search criteria: "Mohs surgery,""staining,""immunostaining," and "immunoperoxidase." RESULTS Generally, all immunostains showed advantage over the traditional hematoxylin and eosin approach. Studies of MART-1 in melanoma chemosurgery indicated that it is typically crisp and has less background staining than MEL-5 and better staining consistency than HMB-45. In cases of desmoplastic melanomas, S100 is the stain of choice.. Immunostaining offers an advantage in MMS. Large, randomized, prospective studies comparing the different immunostains are still lacking in the literature. The authors have indicated no significant interest with commercial supporters.

Topics: Antigens, Neoplasm; Dermatofibrosarcoma; Frozen Sections; Humans; Hutchinson's Melanotic Freckle; Immunoenzyme Techniques; Immunohistochemistry; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Mohs Surgery; Neoplasm Proteins; S100 Proteins; Sensitivity and Specificity; Skin Neoplasms

Gastrointestinal malignant melanomas, either primary or metastatic, are rare and overlooked tumors. There is also controversy regarding the actual existence of primary melanoma in the gastrointestinal tract apart from the esophagus and anorectal regions, where melanocytes normally exist. A case of malignant melanoma in the cecum is presented. The patient was a 30- year-old male who presented to the hospital for abdominal pain and diarrhea. The tumor was located mainly in the submucosa and measured 14x11x4.5 cm. The cut surface was solid, gray-white and fleshy. Histologically, tumor cells were arranged in compact nests or wide cords surrounded by fibrous stroma. The tumor cells had pleomorphic nuclei and quite rich cytoplasm; multinucleated, giant tumor cells were intermingled. Although no tumor cells contained apparent brown pigment, most were found to be positive for S-100 protein, HMB-45, Melan-A, and vimentin. The possibility of a metastatic lesion was considered. While the patient had a history of a pathologically examined dorsal nevus excision two years before, there was no evidence of either cutaneous or ocular primary melanoma at the time of diagnosis. Moreover, a thorough postoperative investigation did not reveal any other lesion in any other site favoring a metastatic spread. There was also no evidence of recurrent disease or metastasis one year after the surgery. This case is presented in view of its rare occurrence in the cecum. The difficulties in the diagnostic course are discussed, together with a literature review on distinguishing a primary mucosal melanoma from a metastatic one from an unknown or regressed cutaneous primary tumor.

Topics: Adult; Antigens, Neoplasm; Cecal Neoplasms; Humans; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; S100 Proteins; Vimentin

The genetic introduction of T cell receptor genes into T cells has been developed over the past decade as a strategy to induce defined antigen-specific T cell immunity. With the potential value of TCR gene therapy well-established in murine models and the feasibility of infusion of TCR-modified autologous T cells shown in a first phase I trial, the next key step will be to transform TCR gene transfer from an experimental technique into a robust clinical strategy. In this review, we discuss the different properties of the TCR transgene and transgene cassette that can strongly affect both the efficacy and the safety of TCR gene transfer.

Topics: Animals; Antigens, Neoplasm; Autoimmune Diseases; Clinical Trials, Phase I as Topic; Codon; Dimerization; Feasibility Studies; Genes, Synthetic; Genes, Transgenic, Suicide; Genetic Therapy; Genetic Vectors; Humans; Immunotherapy, Adoptive; MART-1 Antigen; Melanoma; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Proteins; Neoplasms, Experimental; Receptors, Antigen, T-Cell; Species Specificity; T-Lymphocyte Subsets; Transgenes

Melanoma sentinel lymph nodes (SLN) are carefully evaluated to maximize sensitivity. Examination includes hematoxylin and eosin (H+E) stained sections at multiple levels through the node, with subsequent immunohistochemical (IHC) stains for melanocytic markers if H+E sections are negative for melanoma. However, not all IHC-positive cells in SLN are metastatic melanoma, as evidenced by the presence of MART-1 positive cells in SLN from breast cancer patients with no history of melanoma (so-called 'false-positive' cells). These 'false-positive cells' could be nodal nevus, non-melanocytic cells with cross-reacting antigenic determinants, phagocytic cells containing melanocyte antigens, or possibly melanocytes or melanocyte stem cells liberated at the time of biopsy of the cutaneous melanoma. Examination of SLN requires careful correlation of H+E and IHC findings.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; False Positive Reactions; Humans; Immunohistochemistry; Lymph Nodes; Lymphatic Metastasis; MART-1 Antigen; Melanoma; Neoplasm Proteins; Sentinel Lymph Node Biopsy; Skin Neoplasms

Metastases to the oral cavity are unusual, and most cases affect the jaws. The clinicopathologic features of a metastatic melanoma involving the mandibular gingiva and facial skin are described.. A 42-year-old white woman who had been treated for plantar melanoma 10 years ago showed concomitant nodular lesions in the mandibular gingiva and facial skin. Incisional biopsy and fine needle aspiration cytology were performed in the gingival and facial swelling, respectively.. Radiographs revealed irregular bone erosion and widening of the periodontal ligament space associated with the gingival lesion, whereas cranial computed tomography showed an irregular hyperdense mass associated with the dermal tissue. The histopathologic findings were suggestive of melanoma; immunohistochemical analysis revealed, in both lesions, neoplastic cells strongly positive for S100 and HMB-45 antibodies and weak reactivity for melan-A. The final diagnosis of these lesions was metastatic melanoma, and the patient died before restarting treatment.. To the best of our knowledge, this is the first English-language report of metastatic melanoma affecting the gingiva and facial skin concomitantly. The intraoral involvement of disseminated melanoma indicates a very poor prognosis, as confirmed by the current case.

Topics: Adult; Antigens, Neoplasm; Biopsy; Biopsy, Fine-Needle; Facial Neoplasms; Fatal Outcome; Female; Foot Diseases; Gingival Neoplasms; Humans; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; S100 Proteins; Tomography, X-Ray Computed

The sentinel lymph nodes are the most likely site of nodal metastasis. Their focused analysis results in upstaging cancers, although the extra yield from a more intensive work-up is generally dominated by micrometastases and isolated tumor cells. Nodal staging is generally done to reflect systemic spread of solid tumors and guide treatment accordingly. However, in general, the two processes of haematogenous and lymphogenic spread are not causally interrelated, and the extrapolation from low-volume nodal involvement to systemic involvement and therapeutic consequences of this extrapolation are still under investigation.

Topics: Antigens, Neoplasm; Breast Neoplasms; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Lymph Nodes; Lymphatic Metastasis; MART-1 Antigen; Melanocytes; Melanoma; Neoplasm Proteins; Neoplasm Staging; Reverse Transcriptase Polymerase Chain Reaction; Sentinel Lymph Node Biopsy

Adoptive cell transfer therapy has developed into a potent and effective treatment for patients with metastatic melanoma. Current application of this therapy relies on the ex vivo generation of highly active, highly avid tumor-reactive lymphocyte cultures from endogenous tumor infiltrating lymphocytes or on the genetic engineering of cells using antigen receptor genes to express de novo tumor antigen recognition. When autologous anti-tumor lymphocyte cultures are administered to patients with high-dose interleukin (IL)-2 following a lymphodepleting conditioning regimen, the cells can expand in vivo, traffic to tumor, and mediate tumor regression and durable objective clinical responses. Current investigation seeks to improve the methods for generating and administering the lymphocyte cultures, and future clinical trials aim to improve durable response rates and extend the patient populations that are candidates for treatment.

Topics: Adoptive Transfer; Antigens, Neoplasm; Genetic Therapy; Humans; Lymphocyte Depletion; Lymphocyte Subsets; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Neoplasm Metastasis; Neoplasm Proteins; Skin Neoplasms

A potential diagnostic pitfall in the histologic assessment of melanoma is the inability to recognize unusual melanoma variants. Of these, the more treacherous examples include the desmoplastic melanoma, the nevoid melanoma, the so-called 'minimal-deviation melanoma,' melanoma with prominent pigment synthesis or 'animal-type melanoma,' and the malignant blue nevus. Also problematic are the unusual phenotypic profiles seen in vertical growth phase melanomas; these include those tumors whose morphological peculiarities mimic cancers of nonmelanocytic lineage and those melanomas that express aberrant antigenic profiles not commonly associated with a melanocytic histogenesis. Metaplastic change in melanoma, balloon cell melanoma, signet-ring cell melanoma, myxoid melanoma, small cell melanoma and rhabdoid melanoma all have the potential to mimic metastatic and primary neoplasms of different lineage derivations. Abnormal immunohistochemical expression of CD 34, cytokeratins, epithelial membrane antigen, and smooth muscle markers as well as the deficient expression of S100 protein and melanocyte lineage-specific markers such as GP100 protein (ie HMB-45 antibody) and A103 (ie Melan-A) also present confusing diagnostic challenges. In this review, we will discuss in some detail certain of these novel clinicopathologic types of melanoma, as well as the abnormal phenotypic expressions seen in vertical growth phase melanoma.

Topics: Antigens, Neoplasm; Diagnosis, Differential; Humans; Immunohistochemistry; Ki-67 Antigen; MART-1 Antigen; Melanocytes; Melanoma; Melanoma-Specific Antigens; Mucin-1; Neoplasm Proteins; S100 Proteins; Skin; Skin Neoplasms

The search is on for biomarkers for use in the diagnosis, staging, prognosis, and management of patients with melanoma. As with many types of cancer, the hematogenous spread of melanoma is a bad prognostic sign, and many groups have attempted to detect circulating melanoma cells in patients with different stages of melanoma. Some studies have used direct extraction of intact tumor cells from the peripheral blood and others the detection of surrogate markers of circulating melanoma cells, such as tyrosinase or MART-1. However, a correlation between the detection of intact melanoma cells in the circulation and prognosis is controversial. Many other biomarkers have also been studied, including lactate dehydrogenase, S100, TA90, and C-reactive protein. Much progress has been made, and preliminary studies have shown promise with many of these markers. Finally, the detection of tumor-specific circulating DNA has shown promise as a prognostic and diagnostic marker of disease in melanoma as well. In this review we examine the most promising biomarkers for use in patients with cutaneous melanoma.

Topics: Antigens, Neoplasm; Biomarkers; Biomarkers, Tumor; C-Reactive Protein; Cell Line, Tumor; Humans; L-Lactate Dehydrogenase; Loss of Heterozygosity; MART-1 Antigen; Melanoma; Microsatellite Repeats; Models, Biological; Monophenol Monooxygenase; Neoplasm Proteins; Neoplastic Cells, Circulating; Prognosis; S100 Proteins; Skin Neoplasms

The histological diagnosis of malignant melanoma can be challenging. Immunohistochemical techniques may define a critical role in certain cases, specifically in establishing a primary diagnosis of melanoma. CD34 is a hemopoietic stem cell antigen expressed in bone marrow and endothelial cells, and may also be expressed in vascular and spindle cell tumors; it is generally negative in malignant melanoma.. An 83-year-old white female presented with a 3-4 mm area on her right upper back, which had been present for several years. Histologic sections showed a polypoid distortion by sheets and nodules of transformed amelanotic melanocytes lying in intimate apposition to an attenuated epidermis without a concomitant radial growth phase. Tumor cells were extensively S-100 and CD34 positive and showed focal immunoreactivity with melan-A and HMB-45.. We present a case of malignant melanoma of nodular subtype, which strongly expressed CD34. The spectrum of abnormal phenotypes in malignant melanoma is reviewed, and a possible explanation for the presence of CD34 is discussed. This case demonstrates the potential of malignant melanoma to express CD34, defining an infrequently recognized aberrant phenotype. Whether or not expression of this marker is associated with a more aggressive clinical course remains to be determined.

Topics: Aged, 80 and over; Antigens, CD34; Antigens, Neoplasm; Biomarkers, Tumor; Female; Humans; Immunophenotyping; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; S100 Proteins; Skin Neoplasms

Sentinel lymph node dissection (SLND) has become the standard of care for the staging of clinically-node negative melanomas and breast cancers. A large literature documents the efficacy of SLND in the staging of melanoma and breast cancer. The SLND has lower associated patient morbidity in comparison to elective node dissections that remove the closest regional-draining node group. SLND has improved accuracy over traditional regional node dissection for the staging of melanoma. Currently, several multicenter trials are evaluating the prognostic significance of melanoma micrometastases in SLN detected by immunohistochemical and molecular methods. Pending trial outcome analysis, SLND has no proven effect on mortality. However, given the current oncologic emphasis on detection and removal of nodal tumor metastases, the technique has an important role in minimizing the invasiveness of tumor staging for melanoma and breast cancer. As long as lymph node metastases are used for staging solid malignancies, surgical pathologists are likely to encounter SLN excisional biopsies as a part of their routine practice.

Topics: Antigens, Neoplasm; Forecasting; Humans; Lymph Nodes; Lymphatic Metastasis; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; Neoplasm Staging; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; S100 Proteins; Sensitivity and Specificity; Sentinel Lymph Node Biopsy; Skin Neoplasms

Topics: Aged; Antigens, Neoplasm; Biomarkers, Tumor; Eye Enucleation; Female; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasm Proteins; Tumor Suppressor Protein p53; Uveal Neoplasms

Factors that are predictive of prognosis in patients who are diagnosed with malignant melanoma (MM) are widely awaited. Detection of circulating melanoma cells (CMCs) by reverse transcriptase-polymerase chain reaction (RT-PCR) has recently been postulated as a possible negative prognostic factor. Two main questions were addressed: first, whether the presence of CMCs, defined as the patient being positive for any of the three markers, had a prognostic role; and second, what the predictive value of each individual marker was.. A consecutive series of 200 melanoma patients observed between January 1997 and December 1997, with stage of disease ranging from I to IV, was analyzed by semiquantitative RT-PCR. Tyrosinase, p97, and MelanA/MART1 were used as markers to CMCs on baseline peripheral blood samples. Progression-free survival (PFS) was used as a unique end point and was described by the product limit method. Multivariable analysis was applied to verify whether the auspicated prognostic value of these markers was independent of the stage of disease, and a subgroup analysis was performed that excluded patients with stage IV disease.. Overall, 32% (64 of 200) of patients progressed, and a median PFS of 52 months in the whole series was observed. The presence of CMCs and the markers individually or combined was predictive of prognosis in the univariate analysis but did not provide additional prognostic information to the stage of disease in multivariable models. In the subgroup analysis of stage (ie, I-III subgroup), similar results were observed.. Detection of CMCs in peripheral blood samples at the time of MM diagnosis by semiquantitative RT-PCR does not add any significant predictive value to the stage of disease. Thus, this approach should not be used in clinical practice, and further studies are required to determine its usefulness.

Topics: Adolescent; Adult; Aged; Antigens, Neoplasm; Biomarkers, Tumor; Disease-Free Survival; Female; Humans; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm, Residual; Neoplastic Cells, Circulating; Predictive Value of Tests; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Sensitivity and Specificity; Sentinel Lymph Node Biopsy; Skin Neoplasms

The multiple molecular alterations underlying the neoplastic process and clinical characteristics of cutaneous melanoma are currently under intensive investigation. Recent studies have demonstrated that the levels of melanoma-associated proteins in tumor tissue or in patient serum can serve as new markers to predict disease outcome. Similarly, the expression of thousands of genes in melanoma tumors can be surveyed simultaneously using DNA arrays, allowing molecular profiling of individual tumors, which gives the possibility of classifying melanomas based on their biological diversity. Large clinical studies have also identified multiple prognostic factors, such as tumor ulceration, and led to development of a new, more precise melanoma staging system, which emphasizes the biological characteristics of the primary disease. These new findings may have an important role in earlier measurement of the clinical response and provide a basis for tailored melanoma therapy, the development of which will also be discussed in this review.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Cancer Vaccines; Chemokines; Extracellular Matrix Proteins; Genetic Therapy; Humans; Hyaluronan Receptors; Immunohistochemistry; Integrins; MART-1 Antigen; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Melanoma; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Staging; Nerve Growth Factors; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; S100 Calcium Binding Protein beta Subunit; S100 Proteins; Skin Neoplasms

Some cancer patients mount spontaneous T- and B-cell responses against their tumor cells. Autologous tumor reactive CD8 cytolytic T lymphocyte (CTL) and CD4 T-cell clones as well as antibodies from these patients have been used for the identification of genes encoding the target antigens. This knowledge opened the way for new approaches to the immunotherapy of cancer. In this review, we describe the characterization of the structure-function properties of the melanocyte/melanoma tumor antigen Melan-A/MART-1, the assessment of the T-cell repertoire available against this antigen in healthy individuals, and the analysis of naturally acquired and/or vaccine-induced CTL responses to this antigen in patients with metastatic melanoma.

Topics: Amino Acid Sequence; Antigen Presentation; Antigens, Neoplasm; Cancer Vaccines; Cytotoxicity, Immunologic; Epitopes, T-Lymphocyte; HLA-A2 Antigen; Humans; Immunity, Innate; Immunotherapy, Active; MART-1 Antigen; Melanoma; Molecular Sequence Data; Neoplasm Proteins; Peptide Fragments; Protein Interaction Mapping; Receptors, Antigen, T-Cell; Structure-Activity Relationship; T-Lymphocytes, Cytotoxic; Treatment Outcome

Progress in human tumor immunology has recently been accelerated by new assays for antigen-specific cytotoxic T lymphocytes (CTLs). We have used tetrameric MHC class I complexes (tetramers) to study melanoma-specific CTLs both in vivo and in vitro, and have utilized the results to optimize vaccination strategies for patients. Tetramers have provided some of the best evidence to date that CTL responses against melanoma antigens arise spontaneously in patients. However, CTL responses to common (nonmutated) melanoma epitopes are generally weak or localized, and occur mostly in advanced metastatic disease, hence justifying early immunotherapeutic approaches. These observations led us to design a polyvalent vaccine construct for early administration to melanoma patients at high risk of progression. To compare possible vaccination protocols, we encoded this construct in several different vectors, and developed novel tetramers to track responses to the human melanoma epitopes in transgenic mice. Priming and boosting with the same poly-epitope construct encoded in heterologous vectors led to the expansion of CTLs with a single dominant specificity. Separating the antigens for independent presentation by antigen-presenting cells reversed the effect of immunodominance and induced a powerful polyvalent CTL response. These results provide important pointers for future vaccination trials, and tetramers will form an important tool in the immunomonitoring of these clinical studies.

Topics: Animals; Antigen Presentation; Antigens, Neoplasm; Biopolymers; Cancer Vaccines; Clone Cells; Cytotoxicity, Immunologic; Epitopes, T-Lymphocyte; Genetic Vectors; Histocompatibility Antigens Class I; HLA-A2 Antigen; Humans; Immunodominant Epitopes; Immunologic Memory; Immunotherapy, Active; Lymphokines; MART-1 Antigen; Melanoma; Mice; Mice, Transgenic; Neoplasm Proteins; Peptide Fragments; T-Lymphocytes, Cytotoxic; Vaccines, Subunit; Vaccines, Synthetic

Topics: Antigens, Neoplasm; Autoantigens; Cancer Vaccines; CD8-Positive T-Lymphocytes; Hematopoietic Stem Cells; Humans; MART-1 Antigen; Melanoma; Neoplasm Proteins; Thymus Gland

Diagnostic records from 338 canine oral melanomas in 338 dogs received at the Veterinary Medical Diagnostic Laboratory (1992-1999) were reviewed. Of these tumors, 122 plus an additional 7 metastatic melanomas of unknown origin were selected for clinical follow-up, histologic review, and immunohistochemistry. Chow Chow, Golden Retriever, and Pekingese/Poodle mix breeds were overrepresented, whereas Boxer and German Shepherd breeds were underrepresented. There was no gender predisposition and the average age at presentation was 11.4 years. Forty-nine dogs were euthanized due to recurrence or metastasis. The average postsurgical survival time was 173 days. The gingiva and the labial mucosa were the most common sites. Most tumors were composed of either polygonal cells (27 cases, 20.9%), spindle cells (44 cases, 34.1%), or a mixture of the two (polygonal and spindle) (54 cases, 41.9%). Clear cell (3 cases, 2.3%) and adenoid/papillary (1 case, 0.8%) patterns were uncommon. The metastases of 6/6 oral melanomas had morphologic and immunohistochemical features similar to those of the primary tumors. Immunohistochemically, Melan A was detected in 113/122 oral (92.6%) and 5/7 (71.9%) metastatic melanomas. Only 4/163 nonmelanocytic tumors were focally and weakly positive for Melan A. Antibodies against vimentin, S100 protein, and neuron-specific enolase stained 129 (100%), 98 (76%), and 115 (89.1%) of 129 melanomas, respectively. Antibodies against other melanocytic-associated antigens (tyrosinase, glycoprotein 100) did not yield adequate staining. We conclude that Melan A is a specific and sensitive marker for canine melanomas.

Topics: Animals; Antigens, Neoplasm; Databases, Factual; Dog Diseases; Dogs; Female; Gingiva; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Mouth Neoplasms; Neoplasm Proteins; Retrospective Studies

Melan-A/MART-1 is a recently identified new melanocytic differentiation marker, which is recognized as an antigen on melanoma cells by cytotoxic T-lymphocytes. It is of interest for clinicians as potential immunotherapeutic target and it is relevant for pathologists as a novel diagnostic marker, since two antibodies (A103 and M2-7C10) have become available to study Melan-A/MART-1 expression on archival material. Both antibodies are useful in the differential diagnosis of melanocytic tumors, especially metastatic tumors, since they are more sensitive than HMB-45. Both antibodies are also of diagnostic value in the recognition of perivascular epithelioid cell tumors (angiomyolipoma, lymphangioleiomyomatosis, and clear cell tumor). Since A103 has the unique property of staining many steroid hormone producing cells, this antibody is also of value for the recognition of tumors derived from these cells, such as adrenocortical carcinomas. Both antibodies are likely to be included in the routine diagnostic armamentarium of many immunohistochemical laboratories in the near future.

Topics: Adrenal Cortex Neoplasms; Angiomyolipoma; Antibodies, Monoclonal; Antigens, Differentiation; Antigens, Neoplasm; Diagnosis, Differential; Female; Humans; Immunohistochemistry; Lymphangioleiomyomatosis; Lymphatic Metastasis; Male; MART-1 Antigen; Melanocytes; Melanoma; Neoplasm Proteins; Ovarian Neoplasms; Skin Neoplasms; Testicular Neoplasms

Several T cell-recognized epitopes presented by melanoma cells have been identified recently. Despite the large array of epitopes potentially available for clinical use, it is still unclear which of these antigens could be effective in mediating anti-tumor responses when used as a vaccine. Preliminary studies showed that immunization of melanoma patients with epitopes derived from proteins of the MAGE family may result in significant clinical regressions. However, no sign of systemic immunization could be observed in peripheral blood of treated patients. Conversely, significant immunization (detected as increased antigen-specific CTL activity in peripheral blood) was obtained by vaccinating HLA-A2.1+ melanoma patients with the immunodominant epitope (residues 27-35) of the differentiation antigen MART-1, but this immunization was not accompanied by a significant clinical response. To implement immunotherapeuties capable of significantly impacting disease outcome, it is necessary to identify the potential mechanisms responsible for the failure of some antigens to mediate significant anti-tumor responses in vivo. In the case of the MART-1(27-35) epitope, we hypothesize that one of these mechanisms may be related to the existence of natural analogs of this peptide in other human normal proteins.

Topics: Antigens, Neoplasm; Antigens, Viral; Cancer Vaccines; Down-Regulation; Epitopes; Epitopes, T-Lymphocyte; HLA-A1 Antigen; HLA-A2 Antigen; Humans; Immunization; MART-1 Antigen; Melanoma; Neoplasm Proteins; Peptides; T-Lymphocytes; T-Lymphocytes, Cytotoxic; Vaccination

Clinical observations in the interleukin (IL) 2-based immunotherapies suggest that T cells play a central role in the rejection of melanoma. Using cDNA expression cloning, we have isolated genes encoding melanoma antigens recognized by tumor-infiltrating T lymphocytes. These antigens are categorized as (a) melanocyte-specific melanosomal proteins (MART-1/melan A, gp100, tyrosinase, TRP-1, and TRP-2), (b) tumor-specific mutated proteins (beta-catenin), and (c) others (p15). A variety of mechanisms has been identified for the generation of T cell epitopes on tumor cells. Some of the HLA-A2 binding epitopes from the melanosomal antigens appear to be subdominant self-determinants with relatively low major histocompatibility complex binding affinity. The effectiveness of adoptive transfer into patients of cytotoxic T lymphocytes recognizing the melanosomal antigens, the significant correlation between vitiligo development and clinical response in patients receiving IL-2-based immunotherapies, and the sporadic tumor regressions observed in some patients following immunization with the MART-1 or gp100 peptides in incomplete Freund's adjuvant or recombinant viruses expressing the MART-1 antigen suggest that these epitopes may represent tumor rejection antigens. Phase I immunization trials using peptides or recombinant viruses containing genes encoding the melanosomal antigens MART-1 or gp100, with or without co-administration of cytokines such as IL-2, IL-12, or granulocyte-macrophage colony-stimulating factor, are being conducted in the Surgery Branch of the National Cancer Institute. These studies may demonstrate the feasibility of using melanosomal proteins for the immunotherapy of patients with melanoma.

Topics: Antigens, Neoplasm; Epitopes, T-Lymphocyte; Humans; Immunotherapy; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Neoplasm Proteins

Two genes encoding human melanoma antigens MART-1 and gp100 recognized by HLA-A2 restricted melanoma reactive CTL derived from tumor infiltrating lymphocytes (TIL) were isolated by cDNA expression cloning methods. Multiple unmutated self peptides were identified as T cell epitopes in these melanocyte/melanoma specific proteins (2 from MART-1 and 5 from gp100). Most of these melanoma epitopes contain non-dominant anchor amino acids at the primary anchor positions and have intermediate binding affinity to HLA-A2.1. Melanoma reactive CTL were efficiently induced from PBL and TIL of patients by in vitro stimulation with PBMC pulsed with these epitopes. There is a significant correlation between vitiligo development and clinical response to IL2 based immunotherapy, suggesting that autoreactive T cells are involved in melanoma regression in vivo. These results have implications for understanding the nature of tumor antigens recognized by T cells and for the development of new cancer immunotherapies.

Topics: Antigens, Neoplasm; Humans; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; T-Lymphocytes; Vaccination; Vaccines, Synthetic

The adoptive transfer of tumor-infiltrating lymphocytes (TIL) has demonstrated robust efficacy in metastatic melanoma patients. Tumor antigen-loaded dendritic cells (DCs) are believed to optimally activate antigen-specific T lymphocytes. We hypothesized that the combined transfer of TIL, containing a melanoma antigen recognized by T cells 1 (MART-1) specific population, with MART-1-pulsed DC will result in enhanced proliferation and prolonged survival of transferred MART-1 specific T cells in vivo ultimately leading to improved clinical responses.. We tested the combination of TIL and DC in a phase II clinical trial of patients with advanced stage IV melanoma. HLA-A0201 patients whose early TIL cultures demonstrated reactivity to MART-1 peptide were randomly assigned to receive TIL alone or TIL +DC pulsed with MART-1 peptide. The primary endpoint was to evaluate the persistence of MART-1 TIL in the two arms. Secondary endpoints were to evaluate clinical response and survival.. The combination of TIL +DC showed no difference in the persistence of MART-1 TIL compared with TIL therapy alone. Although more patients showed a clinical response to TIL+DC therapy, this study was not powered to resolve differences between groups.. NCT00338377.

Topics: Adolescent; Adult; Cancer Vaccines; Combined Modality Therapy; Dendritic Cells; Female; Humans; Immunotherapy, Adoptive; Lymphocyte Depletion; Lymphocytes, Tumor-Infiltrating; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Staging; Skin Neoplasms; T-Lymphocytes; Time Factors; Treatment Outcome; Young Adult

Immune and molecular profiling of CD8 T cells of patients receiving DC vaccines expressing three full-length melanoma antigens (MAs) was performed. Antigen expression levels in DCs had no significant impact on T cell or clinical responses. Patients who received checkpoint blockade before DC vaccination had higher baseline MA-specific CD8 T cell responses but no evidence for improved functional responses to the vaccine. Patients who showed the best clinical responses had low PD-1 expression on MA-specific T cells before and after DC vaccination; however, blockade of PD-1 during antigen presentation by DC had minimal functional impact on PD-1high MA-specific T cells. Gene and protein expression analyses in lymphocytes and tumor samples identified critical immunoregulatory pathways, including CTLA-4 and PD-1. High immune checkpoint gene expression networks correlated with inferior clinical outcomes. Soluble serum PD-L2 showed suggestive positive association with improved outcome. These findings show that checkpoint molecular pathways are critical for vaccine outcomes and suggest specific sequencing of vaccine combinations.

Topics: Antigens, Neoplasm; Cancer Vaccines; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; CTLA-4 Antigen; Dendritic Cells; Disease-Free Survival; Gene Expression Regulation, Neoplastic; Humans; Immune Checkpoint Inhibitors; Interferon-gamma; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Programmed Cell Death 1 Receptor; Vaccination

The efficacy of T cells depends on their functional avidity, i. e., the strength of T cell interaction with cells presenting cognate antigen. The overall T cell response is composed of multiple T cell clonotypes, involving different T cell receptors and variable levels of functional avidity. Recently, it has been proposed that the presence of low avidity tumor antigen-specific CD8 T cells hinder their high avidity counterparts to protect from tumor growth. Here we analyzed human cytotoxic CD8 T cells specific for the melanoma antigen Melan-A/MART-1. We found that the presence of low avidity T cells did not result in reduced cytotoxicity of tumor cells, nor reduced cytokine production, by high avidity T cells.

Topics: Animals; Antibody Affinity; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cytotoxicity, Immunologic; Heterografts; Humans; MART-1 Antigen; Melanoma; Mice; Tumor Cells, Cultured

A vaccine that could expand melanoma-specific T cells might reduce the risk of recurrence of resected melanoma and could provide an alternative or adjunct to standard immunotherapy options. We tested the safety and immunogenicity of a vaccine coupling a melanoma-associated peptide with a xenogenic peptide (to promote epitope spreading) and/or resiquimod (to activate antigen-presenting cells). HLA-A2-positive patients with resected stage II, III, and IV melanoma were assigned to treatment on one of three schedules. All patients received three subcutaneous doses of the peptide MART-1a mixed with Montanide. In addition, patients on schedule 1 received the xenoantigen peptide Gag267-274, patients on schedule 2 received topical resiquimod, and patients on schedule 3 received both Gag267-274 and resiquimod. Blood samples were tested for the frequency of antigen-specific T cells by tetramer assay, as well as immune cell subtypes and plasma cytokine levels. Patients enrolled from October 2012 to December 2014, with 10 patients enrolling to each schedule. The most common adverse events were injection site reaction (26 patients) and fatigue (15 patients). Tetramer analysis revealed antigen-specific responses (defined as doubling of MART-1a-specific T cells from pretreatment to post-treatment) in 20, 60, and 40% of patients treated on schedules 1, 2, and 3, respectively. Vaccine treatment consisting of MART-1a peptide, Gag267-274, Montanide, and topical resiquimod was well-tolerated. The addition of the Gag267-274 xenoantigen was not associated with an increase in the response to MART-1a, whereas use of topical resiquimod was associated with a higher frequency of MART-1a-specific T-cell responses that did not meet statistical significance.

Topics: Administration, Topical; Aged; Cancer Vaccines; CD8-Positive T-Lymphocytes; Combined Modality Therapy; Cytokines; Female; Gene Products, gag; HLA-A2 Antigen; Humans; Imidazoles; Male; MART-1 Antigen; Melanoma; Middle Aged; Peptide Fragments; Pilot Projects; Skin Neoplasms

Among Immunotherapeutic approaches for cancer treatment, the adoptive transfer of antigen specific T cells is still a relevant approach, that could have higher efficacy when further combined with immune check-point blockade. A high number of adoptive transfer trials have been performed in metastatic melanoma, due to its high immunogenic potential, either with polyclonal TIL or antigen-specific polyclonal populations. In this setting, the extensive characterization of T cell functions and receptor diversity of infused polyclonal T cells is required, notably for monitoring purposes. We developed a clinical grade procedure for the selection and amplification of polyclonal CD8 T cells, specific for two shared and widely expressed melanoma antigens: Melan-A and MELOE-1. This procedure is currently used in a clinical trial for HLA-A2 metastatic melanoma patients. In this study, we characterized the T-cell diversity (T-cell repertoire) of such T cell populations using a new RNAseq strategy. We first assessed the added-value of TCR receptor sequencing, in terms of sensitivity and specificity, by direct comparison with cytometry analysis of the T cell populations labeled with anti-Vß-specific antibodies. Results from these analyzes also confirmed specific features already reported for Melan-A and MELOE-1 specific T cell repertoires in terms of V-alpha recurrence usage, on a very high number of T cell clonotypes. Furthermore, these analyses also revealed undescribed features, such as the recurrence of a specific motif in the CDR3α region for MELOE-1 specific T cell repertoire. Finally, the analysis of a large number of T cell clonotypes originating from various patients revealed the existence of public CDR3α and ß clonotypes for Melan-A and MELOE-1 specific T cells. In conclusion, this method of high throughput TCR sequencing is a reliable and powerful approach to deeply characterize polyclonal T cell repertoires, and to reveal specific features of a given TCR repertoire, that would be useful for immune follow-up of cancer patients treated by immunotherapeutic approaches.

Topics: Adoptive Transfer; Antigens, Neoplasm; Female; High-Throughput Nucleotide Sequencing; Humans; Male; MART-1 Antigen; Melanoma; Neoplasm Proteins; Receptors, Antigen, T-Cell, alpha-beta; T-Lymphocytes

Cancer vaccines aim to generate and maintain antitumor immune responses. We designed a phase I/IIa clinical trial to test a vaccine formulation composed of Montanide ISA-51 (Incomplete Freund's Adjuvant), LAG-3Ig (IMP321, a non-Toll like Receptor agonist with adjuvant properties), and five synthetic peptides derived from tumor-associated antigens (four short 9/10-mers targeting CD8 T-cells, and one longer 15-mer targeting CD4 T-cells). Primary endpoints were safety and T-cell responses.. Sixteen metastatic melanoma patients received serial vaccinations. Up to nine injections were subcutaneously administered in three cycles, each with three vaccinations every 3 weeks, with 6 to 14 weeks interval between cycles. Blood samples were collected at baseline, 1-week after the third, sixth and ninth vaccination, and 6 months after the last vaccination. Circulating T-cells were monitored by tetramer staining directly ex vivo, and by combinatorial tetramer and cytokine staining on in vitro stimulated cells.. Side effects were mild to moderate, comparable to vaccines with Montanide alone. Specific CD8 T-cell responses to at least one peptide formulated in the vaccine preparation were found in 13 of 16 patients. However, two of the four short peptides of the vaccine formulation did not elicit CD8 T-cell responses. Specific CD4 T-cell responses were found in all 16 patients.. We conclude that vaccination with IMP321 is a promising and safe strategy for inducing sustained immune responses, encouraging further development for cancer vaccines as components of combination therapies.

Topics: Antigens, CD; Antigens, Neoplasm; Biomarkers; Cancer Vaccines; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Combined Modality Therapy; Female; Humans; Lymphocyte Activation Gene 3 Protein; Lymphocyte Count; Male; MART-1 Antigen; Melanoma; Peptides; Treatment Outcome; Vaccination

Molecular staging of sentinel lymph nodes (SLNs) may identify patients who are node-negative by standard microscopic staging but are at increased risk for regional nodal recurrence; such patients may benefit from completion lymph node dissection (CLND).. In a multicenter, randomized clinical trial, patients with tumor-negative SLNs by standard pathology (hematoxylin and eosin [H and E] serial sections and immunohistochemistry [IHC]) underwent reverse transcriptase polymerase chain reaction (PCR) analysis of SLNs for melanoma-specific mRNA. Microscopically negative/PCR+ patients were randomized to observation, CLND, or CLND with high-dose interferon (HDI). For this post-hoc analysis, clinicopathologic features and survival outcomes, including overall survival (OS) and disease-free survival (DFS), were compared between PCR+ patients who underwent CLND vs observation. Microscopic and molecular node-negative (PCR-) patients were included for comparison.. A total of 556 patients were PCR+: 180 underwent observation, and 376 underwent CLND. An additional 908 PCR- patients were observed. Median follow-up was 72 months. Disease-free survival (DFS) was significantly better for PCR+ patients who underwent CLND compared with observation (p = 0.0218). No statistically significant differences in OS or distant disease-free survival (DDFS) were seen. Regional lymph node recurrence-free survival (LNRFS) was improved in PCR+ patients with CLND compared to observation (p = 0.0065). The PCR+ patients in the observation group had the worst DFS; those with CLND had similar DFS to that in the PCR- group (p = 0.9044).. Patients with microscopically negative/PCR+ SLN have an increased risk of nodal recurrence that was mitigated by CLND. Although CLND did not affect OS, these data suggest that molecular detection of melanoma-specific mRNA in the SLN predicts a greater risk of nodal recurrence and deserves further study.

Topics: Adult; Aged; Antigens, Neoplasm; Antineoplastic Agents; Disease-Free Survival; Female; gp100 Melanoma Antigen; Humans; Interferons; Male; MART-1 Antigen; Melanoma; Middle Aged; Molecular Diagnostic Techniques; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Recurrence, Local; Neoplasm Staging; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sensitivity and Specificity; Sentinel Lymph Node Biopsy; Skin Neoplasms; Watchful Waiting

Adoptive transfer of autologous tumor infiltrating lymphocytes (TIL) can mediate durable cancer regression in selected patients with metastatic melanoma. However, the tumor antigens associated with these favorable responses remain unclear. We hypothesized that a clinical strategy involving the iterative adoptive transfer of selected autologous antigen-specific T-cell clones could help systematically define immunologic targets associated with successful cancer therapy, without the interpretative ambiguity of transferring polyclonal populations. Here, we evaluated the clinical efficacy of CD8(+) T-cell clones specific for the melanocyte differentiation antigens (MDA), gp100 and MART-1, respectively.. We conducted two consecutive phase II clinical trials involving the adoptive transfer of highly selected autologous antigen-specific CD8(+) T-cell clones against gp100 and MART-1, respectively. Fifteen patients with HLA-A2(+) treatment-refractory metastatic melanoma received highly avid MDA-specific CD8(+) T-cell clones specific for either gp100 (n = 10) or MART-1 (n = 5) with or without intravenous interleukin-2 (IL2) after a lymphodepleting myeloablative preparative regimen.. Of the 15 treated patients, we observed immune-mediated targeting of skin melanocytes in 11 patients (73%) and clonal engraftment in eight patients (53%) after cell transfer. There were only transient minor tumor regressions observed, but no objective tumor responses based on Response Evaluation Criteria in Solid Tumor (RECIST) criteria.. Despite successful clonal repopulation and evidence of in vivo antigen targeting, the poor therapeutic efficacy after the adoptive transfer of autologous MDA-specific T cells raises significant concerns regarding future immunotherapy efforts targeting this class of tumor antigens.

Topics: Adult; Aged; CD8-Positive T-Lymphocytes; Cytotoxicity, Immunologic; Dermatitis; Female; gp100 Melanoma Antigen; HLA-A2 Antigen; Humans; Immunotherapy, Adoptive; Lymphocytes, Tumor-Infiltrating; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; T-Cell Antigen Receptor Specificity; T-Lymphocytes, Cytotoxic; Treatment Outcome; Tumor Burden

Melanoma patients with a high risk of recurrence may benefit from immunotherapy with mRNA-electroporated autologous monocyte-derived dendritic cells (DCs). Further benefit may be found in combining DC-therapy with interferon alfa-2b.. The long-term clinical outcome of AJCC stage III/IV melanoma patients who had no evidence of disease at the time of treatment with autologous mRNA-electroporated DCs in a single-center pilot clinical trial was analyzed. Antigen loading was accomplished by co-electroporation of mRNA encoding a fusion protein between MAGE-A1, -A3, -C2, Tyrosinase, MelanA/MART-1, or gp100, and an HLA class II-targeting sequence. DCs were administered by 4-6 bi-weekly intradermal injections. IFN-α-2b (5 MIU TIW) was initiated either at recurrence (cohort 1), concomitant with DCs (cohorts 2 and 3), or following the fourth DC administration (cohort 4).. Thirty melanoma patients were recruited between April 2006 and June 2009. DC-related adverse events included grade 2 local injection site reactions in all patients, grade 2 fever and flu-like symptoms in one patient, and skin depigmentation in seven patients. After a median follow-up of over 6 years, the median relapse-free survival is 22 months (95% CI 12-32 months). Twelve patients have died. The median overall survival has not been reached; the 2-year and 4-year survival rates are 93 and 70%, respectively.. Adjuvant therapy following the resection of melanoma metastases with autologous mRNA-electroporated DCs, combined with interferon alfa-2b, is tolerable and results in encouraging long-term overall survival rates justifying further evaluation in a randomized clinical trial.

Topics: Adult; Aged; Cancer Vaccines; Dendritic Cells; Electroporation; Female; Humans; Immunotherapy, Adoptive; Interferon alpha-2; Interferon-alpha; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Melanoma, Cutaneous Malignant; Middle Aged; Monophenol Monooxygenase; Neoplasm Metastasis; Pilot Projects; Recombinant Proteins; RNA, Messenger; Skin Neoplasms; Treatment Outcome

It has been demonstrated that large numbers of tumor-specific T cells for adoptive cell transfer (ACT) can be manufactured by retroviral genetic engineering of autologous peripheral blood lymphocytes and expanding them over several weeks. In mouse models, this therapy is optimized when administered with dendritic cell (DC) vaccination. We developed a short 1-week manufacture protocol to determine the feasibility, safety, and antitumor efficacy of this double cell therapy.. A clinical trial (NCT00910650) adoptively transferring MART-1 T-cell receptor (TCR) transgenic lymphocytes together with MART-1 peptide-pulsed DC vaccination in HLA-A2.1 patients with metastatic melanoma. Autologous TCR transgenic cells were manufactured in 6 to 7 days using retroviral vector gene transfer, and reinfused with (n = 10) or without (n = 3) prior cryopreservation.. A total of 14 patients with metastatic melanoma were enrolled and 9 of 13 treated patients (69%) showed evidence of tumor regression. Peripheral blood reconstitution with MART-1-specific T cells peaked within 2 weeks of ACT, indicating rapid in vivo expansion. Administration of freshly manufactured TCR transgenic T cells resulted in a higher persistence of MART-1-specific T cells in the blood as compared with cryopreserved. Evidence that DC vaccination could cause further in vivo expansion was only observed with ACT using noncryopreserved T cells.. Double cell therapy with ACT of TCR-engineered T cells with a very short ex vivo manipulation and DC vaccines is feasible and results in antitumor activity, but improvements are needed to maintain tumor responses.

Topics: Adult; Cancer Vaccines; Dendritic Cells; Female; Humans; Immunotherapy, Adoptive; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Metastasis; Neoplasm Staging; Peptide Fragments; Positron-Emission Tomography; Receptors, Antigen, T-Cell; T-Lymphocytes; Tomography, X-Ray Computed; Transduction, Genetic; Treatment Outcome; Vaccination

Malignant melanoma (MM) often shows multiple chemo-resistance, leading to poor prognosis of the patients. Therapeutic anti-cancer vaccination may be a feasible way to prolong the survival of patients. We have demonstrated that application of antigenic peptides via the tape-stripped, horny layer-removed skin, known as percutaneous peptide immunization (PPI), induces tumor cell-specific cytotoxic T lymphocytes (CTLs) in rodents and humans.. To evaluate clinical significance of PPI in advanced MM patients.. We performed PPI in 59 patients undergoing advanced MM with Melan-A, tyrosinase, MAGE-2, MAGE-3 and gp-100 peptides based on HLA typing in individuals. The induction of CTLs was assessed by the tetramer or pentamer flow cytometry in 35 patients. Patients showing positive CTL responses to all antigens were defined as complete responder (n=18), and those showing negative responses to at least one applied antigen were classified as incomplete responder (n=17). The primary endpoint of the study was overall survival (OS). For statistical analysis, log-rank test, univariate and multivariate Cox proportional hazard model were used.. OS of the complete responders was longer than that of the incomplete responders (median survival time: 55.8 vs 20.3 months, log rank P=0.089). A hazard ratio for the complete responders relative to the incomplete responders was 0.23 (95% confidence interval: 0.06-0.93, P=0.039) in a multivariate Cox proportional hazard model.. The induction of CTLs was a novel independent survival factor, and the induction of peptide-specific CTLs by PPI contributes to the prolonged survival and represents an impact on therapeutic approaches in MM. Unique trial number: UMIN000005706.

Topics: Administration, Cutaneous; Adult; Aged; Cancer Vaccines; Female; gp100 Melanoma Antigen; HLA Antigens; Humans; Immunization; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Monophenol Monooxygenase; Multivariate Analysis; Peptide Fragments; Proportional Hazards Models; Prospective Studies; Skin Neoplasms; T-Lymphocytes, Cytotoxic; Time Factors; Treatment Outcome

Adoptive cell transfer (ACT) of genetically engineered T cells expressing cancer-specific T-cell receptors (TCR) is a promising cancer treatment. Here, we investigate the in vivo functional activity and dynamics of the transferred cells by analyzing samples from 3 representative patients with melanoma enrolled in a clinical trial of ACT with TCR transgenic T cells targeted against the melanosomal antigen MART-1. The analyses included evaluating 19 secreted proteins from individual cells from phenotypically defined T-cell subpopulations, as well as the enumeration of T cells with TCR antigen specificity for 36 melanoma antigens. These analyses revealed the coordinated functional dynamics of the adoptively transferred, as well as endogenous, T cells, and the importance of highly functional T cells in dominating the antitumor immune response. This study highlights the need to develop approaches to maintaining antitumor T-cell functionality with the aim of increasing the long-term efficacy of TCR-engineered ACT immunotherapy.. A longitudinal functional study of adoptively transferred TCR–engineered lymphocytes yielded revealing snapshots for understanding the changes of antitumor responses over time in ACT immunotherapy of patients with advanced melanoma.

Topics: Adult; Female; Humans; Immunotherapy, Adoptive; Male; MART-1 Antigen; Melanoma; Middle Aged; Receptors, Antigen, T-Cell; Skin Neoplasms; T-Lymphocytes

A significant barrier to effective immune clearance of cancer is loss of antitumor cytotoxic T cell activity. Antibodies to block pro-apoptotic/downmodulatory signals to T cells are currently being tested. Because invariant natural killer T cells (iNKT) can regulate the balance of Th1/Th2 cellular immune responses, we characterized the frequencies of circulating iNKT cell subsets in 21 patients with melanoma who received the anti-CTLA4 monoclonal antibody tremelimumab alone and 8 patients who received the antibody in combination with MART-126-35 peptide-pulsed dendritic cells (MART-1/DC). Blood T cell phenotypes and functionality were characterized by flow cytometry before and after treatment. iNKT cells exhibited the central memory phenotype and showed polyfunctional cytokine production. In the combination treatment group, high frequencies of pro-inflammatory Th1 iNKT CD8(+) cells correlated with positive clinical responses. These results indicate that iNKT cells play a critical role in regulating effective antitumor T cell activity.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; CD8-Positive T-Lymphocytes; Female; Humans; Immunity, Cellular; Male; MART-1 Antigen; Melanoma; Natural Killer T-Cells; Th1 Cells; Th2 Cells

Among synthetic vaccines, virus-like particles (VLPs) are used for their ability to induce strong humoral responses. Very little is reported on VLP-based-vaccine-induced CD4(+) T-cell responses, despite the requirement of helper T cells for antibody isotype switching. Further knowledge on helper T cells is also needed for optimization of CD8(+) T-cell vaccination. Here, we analysed human CD4(+) T-cell responses to vaccination with MelQbG10, which is a Qβ-VLP covalently linked to a long peptide derived from the melanoma self-antigen Melan-A. In all analysed patients, we found strong antibody responses of mainly IgG1 and IgG3 isotypes, and concomitant Th1-biased CD4(+) T-cell responses specific for Qβ. Although less strong, comparable B- and CD4(+) T-cell responses were also found specific for the Melan-A cargo peptide. Further optimization is required to shift the response more towards the cargo peptide. Nevertheless, the data demonstrate the high potential of VLPs for inducing humoral and cellular immune responses by mounting powerful CD4(+) T-cell help.

Topics: Adult; Aged; Antibody Formation; Cancer Vaccines; CD4 Antigens; Cells, Cultured; Female; Humans; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Staging; Peptide Fragments; Skin Neoplasms; T-Lymphocytes, Helper-Inducer; Virion

To verify circulating tumor cell (CTC) prognostic utility in stage IV resected melanoma patients in a prospective international phase III clinical trial.. Our studies of melanoma patients in phase II clinical trials demonstrated prognostic significance for CTCs in patients with AJCC stage IV melanoma. CTCs were assessed to determine prognostic utility in follow-up of disease-free stage IV patients pre- and during treatment.. After complete metastasectomy, patients were prospectively enrolled in a randomized trial of adjuvant therapy with a whole-cell melanoma vaccine, Canvaxin, plus Bacille Calmette-Guerin (BCG) versus placebo plus BCG. Blood specimens obtained pretreatment (n = 244) and during treatment (n = 214) were evaluated by quantitative real-time reverse-transcriptase polymerase chain reaction (qPCR) for expression of MART-1, MAGE-A3, and PAX3 mRNA biomarkers. Univariate and multivariate Cox analyses examined CTC biomarker expression with respect to clinicopathological variables.. CTC biomarker(s) (≥ 1) was detected in 54% of patients pretreatment and in 86% of patients over the first 3 months. With a median follow-up of 21.9 months, 71% of patients recurred and 48% expired. CTC levels were not associated with known prognostic factors or treatment arm. In multivariate analysis, pretreatment CTC (> 0 vs. 0 biomarker) status was significantly associated with disease-free survival (DFS; HR 1.64, P = 0.002) and overall survival (OS; HR 1.53, P = 0.028). Serial CTC (>0 vs. 0 biomarker) status was also significantly associated with DFS (HR 1.91, P = 0.02) and OS (HR 2.57, P = 0.012).. CTC assessment can provide prognostic discrimination before and during adjuvant treatment for resected stage IV melanoma patients.

Topics: Adult; Aged; Antigens, Neoplasm; Biomarkers, Tumor; Cancer Vaccines; Chemotherapy, Adjuvant; Double-Blind Method; Female; Humans; Male; MART-1 Antigen; Melanoma; Middle Aged; Multivariate Analysis; Neoplasm Proteins; Neoplasm Staging; Neoplastic Cells, Circulating; Paired Box Transcription Factors; PAX3 Transcription Factor; Prognosis; Real-Time Polymerase Chain Reaction; Recurrence; Reverse Transcriptase Polymerase Chain Reaction; Survival Analysis

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) is in development for the treatment of metastatic melanoma. In phase II clinical trials, patients with metastatic melanoma that received TIL after preconditioning had a 50-70% clinical response rate. The current approach to generate TIL is to culture melanoma enzyme digests in the presence of IL-2 for a 10- to 20-day period followed by 2 weeks of rapid expansion (REP). Prior to administration, cell therapies are characterized and tested for purity. TIL are characterized by CD3 surface marker expression, and purity is assessed by the amount of tumor remaining in culture. Evaluating TIL purity has traditionally been done by immunohistochemistry, which is often considered semiquantitative. To generate a quantitative assay, we used multiparameter flow cytometry to evaluate the presence of viable tumor cells by staining TIL populations with a viability dye and an antibody cocktail that detects intracellular tumor-antigens gp100, Mart-1, tyrosinase, S100, and surface tumor-antigen melanoma chondroitin sulfate proteoglycan (MCSP), and CD3 on T cells. Tumors were identified by gating on the viable CD3(-) population. Antigens in tumors were initially optimized with individual antibodies using both immunohistochemistry and flow cytometry. When eight different tumor cell lines were spiked into an activated T cell culture, flow cytometry was able to distinguish lymphocytes from tumors in all samples tested. Most importantly, the assay was able to detect melanoma cells in all enzyme digests (9/9) from patient samples. After IL-2-induced TIL expansion, there was a significant decrease in tumor cells; tumor cells were detected in only 2 of 12 samples. In eight IL-2-induced TIL samples that were further expanded in REP, no tumor cells were detected. We have demonstrated that flow cytometry is an alternative to immunohistochemistry for defining the purity of a TIL population.

Topics: Antigens, Neoplasm; CD3 Complex; Cell Line, Tumor; Chondroitin Sulfate Proteoglycans; Flow Cytometry; gp100 Melanoma Antigen; Humans; Immunotherapy, Adoptive; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Monophenol Monooxygenase; S100 Proteins; Staining and Labeling

The effectivenes of cancer vaccines in inducing CD8(+) T-cell responses remains a challenge, resulting in a need for testing more potent adjuvants. Our objective was to determine the safety and immunogenicity of vaccination against melanoma-related antigens employing MART-1, gp100, and tysosinase paptides combined with the TLR9 agonist PF-3512676 and local granulocyte macrophage-colony stimulating factor in oil emulsion. Using continuous monitoring of safety and a 2-stage design for immunologic efficacy, 20 immune response evaluable patients were targetted. Vaccinations were given subcutaneously on days 1 and 15 per cycle (1cycle=28 d) for up to 13 cycles. Interferon-γ enzyme-linked immunosorbent spot was used as the primary assay measuring the frequency of peripheral antigen-specific CD8(+) T cells at days 50 and 90 compared with baseline (target ≥ 9/20 immunologic responses). Clinical responses were measured by Response Evaluation Criteria In Solid Tumors every 8 weeks. Twenty-two (including 20 immune response evaluable) melanoma patients were enrolled. All had American Joint Committe on Cancer stage IV (5M1a, 6M1b, 11M1c) and most had previously received therapy. Eight had previously treated brain metastases. An average of 3.5 cycles of vaccination per patient was administered. Clinical response data were available for 21 patients. There were 2 partial response and 8 stable disease lasting 2-7 months. One patient with ongoing partial response continued on treatment. At a median follow-up of 7.39 months (range, 3.22-20.47 mo), median progression-free survival was 1.9 months (90% confidence interval, 1.84-3.68) and median overall survival was 13.4 months (90% confidence interval,11.3-∞). No regimen-related grade 3/4/5 toxicities were observed. There were 9/20 patients with positive enzyme-linked immunosorbent spot at day 50 and/or day 90. Our adjuvant regimen combining PF-3512676 and granulocyte macrophage-colony stimulating factor was safe and is worthy of further testing with these or alternative peptides, potentially in combination with antibodies that target immunoregulatory checkpoints.

Topics: Adjuvants, Immunologic; Aged; Aged, 80 and over; Cancer Vaccines; Female; gp100 Melanoma Antigen; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Kaplan-Meier Estimate; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplasm Metastasis; Oligodeoxyribonucleotides; Treatment Outcome; Vaccines, Subunit

Optimal vaccine strategies must be identified for improving T-cell vaccination against infectious and malignant diseases. MelQbG10 is a virus-like nano-particle loaded with A-type CpG-oligonucleotides (CpG-ODN) and coupled to peptide(16-35) derived from Melan-A/MART-1. In this phase IIa clinical study, four groups of stage III-IV melanoma patients were vaccinated with MelQbG10, given (i) with IFA (Montanide) s.c.; (ii) with IFA s.c. and topical Imiquimod; (iii) i.d. with topical Imiquimod; or (iv) as intralymph node injection. In total, 16/21 (76%) patients generated ex vivo detectable Melan-A/MART-1-specific T-cell responses. T-cell frequencies were significantly higher when IFA was used as adjuvant, resulting in detectable T-cell responses in all (11/11) patients, with predominant generation of effector-memory-phenotype cells. In turn, Imiquimod induced higher proportions of central-memory-phenotype cells and increased percentages of CD127(+) (IL-7R) T cells. Direct injection of MelQbG10 into lymph nodes resulted in lower T-cell frequencies, associated with lower proportions of memory and effector-phenotype T cells. Swelling of vaccine site draining lymph nodes, and increased glucose uptake at PET/CT was observed in 13/15 (87%) of evaluable patients, reflecting vaccine triggered immune reactions in lymph nodes. We conclude that the simultaneous use of both Imiquimod and CpG-ODN induced combined memory and effector CD8(+) T-cell responses.

Topics: Adjuvants, Immunologic; Aminoquinolines; Cancer Vaccines; CD8-Positive T-Lymphocytes; Flow Cytometry; Freund's Adjuvant; Humans; Imiquimod; Immunologic Memory; Ligands; Lipids; MART-1 Antigen; Melanoma; Nanoparticles; Oligodeoxyribonucleotides; Skin Neoplasms; Statistics, Nonparametric; Toll-Like Receptor 7; Toll-Like Receptor 9

Metastatic and chemoresistant melanoma can be a good target of immunotherapy because it is an intractable cancer with a very poor prognosis. Previously, we tested a dendritic cell (DC)-based phase I vaccine, and confirmed that it was safe. In the present study, we performed a phase II trial of a DC vaccine for metastatic melanoma patients with mainly the HLA-A24 genotype, and investigated the efficacy of the vaccine. Twenty-four patients with metastatic melanoma were enrolled into a phase II study of DC-based immunotherapy. The group included 19 HLA-A24-positive (A*2402) patients and 3 HLA-A2-positive (A*0201) patients. The protocol for DC production was similar to that in the phase I trial. Briefly, a cocktail of 5 melanoma-associated synthetic peptides (gp100, tyrosinase, MAGE-A2, MAGE-A3 and MART-1 or MAGE‑A1) restricted to HLA-A2 or A24 and KLH were used for DC pulsing. Finally, DCs were injected subcutaneously (s.c.) into the inguinal region in the dose range of 1-5x107 per shot. The DC ratio (lin-HLA-DR+) of the vaccine was 38.1±13.3% and the frequency of CD83+ DCs was 25.7±20.8%. Other parameters regarding DC processing were not different from phase I. Immune response-related parameters including the ELISPOT assay, DTH reaction to peptide or KLH, DC injection numbers were shown to be related to a good prognosis. The ELISPOT reaction was positive in 75% of the patients vaccinated. The increase of anti-melanoma antigen antibody titer before vaccination was also shown to be a prognosis factor, but that post-vaccination was not. Based on immunohistochemical analysis, CD8 and IL-17 were not involved in the prognosis. Adverse effects of more than grade III were not seen. Overall survival analysis revealed a significant survival prolongation effect in DC-given melanoma patients. These results suggest that peptide cocktail-treated DC vaccines may be a safe and effective therapy against metastatic melanoma in terms of prolongation of overall survival time.

Topics: Aged; Antigens, Neoplasm; Autoantibodies; Cancer Vaccines; Dendritic Cells; Enzyme-Linked Immunospot Assay; Female; Hemocyanins; HLA-A2 Antigen; Humans; Injections, Subcutaneous; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Peptide Fragments; Survival Analysis; Treatment Outcome; Vaccination

The progressive immune dysfunctions that occur in patients with advanced melanoma make them unlikely to efficiently respond to cancer vaccines. A multicenter randomized phase II trial was conducted to test whether immunization with modified HLA class I tumor peptides in the context of adjuvant therapy results in better immunologic responses and improved clinical outcomes in patients with early melanoma (stages IIB/C-III).. Forty-three patients were enrolled to undergo vaccination (n = 22) or observation (n = 21). The vaccine included four HLA-A*0201-restricted modified peptides (Melan-A/MART-1([27L]), gp100([210M]), NY-ESO-1([165V]), and Survivin([97M])) emulsified in Montanide ISA51 and injected subcutaneously in combination with cyclophosphamide (300 mg/m(2)) and low-dose IL-2 (3 × 10(6) IU). The immune responses were monitored using ex vivo IFN-γ-ELISpot, HLA/multimer staining, and in vitro short-term peptide sensitization assays.. Vaccination induced a rapid and persistent increase in specific effector memory CD8(+) T cells in 75% of the patients. However, this immunization was not associated with any significant increase in disease-free or overall survival as compared with the observation group. An extensive immunologic analysis revealed a significantly reduced cross-recognition of the corresponding native peptides and, most importantly, a limited ability to react to melanoma cells.. Adjuvant setting is an appealing approach for testing cancer vaccines because specific CD8(+) T cells can be efficiently induced in most vaccinated patients. However, the marginal antitumor activity of the T cells induced by modified peptides in this study largely accounts for the observed lack of benefit of vaccination. These findings suggest reconsidering this immunization strategy, particularly in early disease.

Topics: Cancer Vaccines; Case-Control Studies; Cross Reactions; Epitopes, T-Lymphocyte; Histocompatibility Antigens Class I; Humans; Immunologic Memory; MART-1 Antigen; Melanoma; Neoplasm Staging; Peptides; T-Lymphocytes; Treatment Outcome; Vaccines, Subunit

This trial tested a dendritic cell (DC) therapeutic cancer vaccine in which antigen is loaded using a novel non-viral transfection method enabling the uptake of plasmid DNA condensed with a cationic peptide. Proof of principle required the demonstration of diverse T lymphocyte responses following vaccination, including multiple reactivities restricted through both major histocompatibility complex (MHC) class I and II. Patients with advanced melanoma were offered four cycles of vaccination with autologous DC expressing melan A and gp100. Disease response was measured using Response Evaluation Criteria in Solid Tumours. Circulating MHC class I- and II-restricted responses were measured against peptide and whole antigen targets using interferon-γ ELIspot and enzyme-linked immunosorbent assay assays, respectively. Responses were analyzed across the trial population and presented descriptively for some individuals. Twenty-five patients received at least one cycle. Vaccination was well tolerated. Three patients had reduction in disease volume. Across the trial population, vaccination resulted in an expansion of effector responses to both antigens, to the human leukocyte antigen A2-restricted modified epitope, melan A ELAGIGILTV, and to a panel of MHC class I- and II-restricted epitopes. Vaccination with mature DC non-virally transfected with DNA encoding antigen had biological effect causing tumour regression and inducing diverse T lymphocyte responses.

Topics: Adult; Aged; Cancer Vaccines; Dendritic Cells; Female; gp100 Melanoma Antigen; Humans; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Metastasis; T-Lymphocytes; Transfection; Vaccines, DNA

The goal of this study was to test the safety and activity of a therapeutic vaccine, MKC1106-MT, in patients with metastatic melanoma.. MKC1106-MT comprises a plasmid (pMEL-TYR) and two peptides (E-MEL and E-TYR), corresponding to Melan A and tyrosinase, administered by intra-lymph node injection in a prime-boost sequence. All 18 patients were HLA-A*0201 positive and received a fixed priming dose of plasmid and a low or a high peptide dose. Enumeration of antigen-specific T cells was done prior to and throughout the treatment. Patients who did not exhibit disease progression remained on study and could receive up to eight cycles of treatment.. The MKC1106-MT regimen was well tolerated and resulted in an overall immune response rate of 50%. The treatment showed disease control, defined as stable disease that lasted for 8 weeks or more in 6 of 18 (33%) of the patients: 14% and 46% in the low and high peptide dose, respectively. Interestingly, four patients, all with tumor burden largely confined to lymph nodes and Melan A-specific T cells at baseline, showed durable disease control associated with radiologic evidence of tumor regression. There was no noticeable correlation between the expansion of antigen-specific T cells in blood and the clinical outcome; yet, there was evidence of active tumor-infiltrating lymphocytes (TIL) in two regressing lesions.. MKC1106-MT showed immunogenicity and evidence of disease control in a defined patient population. These findings support further development of this investigational agent and the concept of therapeutic vaccination in metastatic melanoma.

Topics: Aged; Aged, 80 and over; Cancer Vaccines; Female; Humans; Immunization, Secondary; Lymph Nodes; Male; MART-1 Antigen; Melanoma; Middle Aged; Models, Biological; Monophenol Monooxygenase; Neoplasm Metastasis; Skin Neoplasms; Vaccination

Although advanced-stage melanoma patients have a median survival of less than a year, adoptive T cell therapy can induce durable clinical responses in some patients. Successful adoptive T cell therapy to treat cancer requires engraftment of antitumor T lymphocytes that not only retain specificity and function in vivo but also display an intrinsic capacity to survive. To date, adoptively transferred antitumor CD8(+) T lymphocytes (CTLs) have had limited life spans unless the host has been manipulated. To generate CTLs that have an intrinsic capacity to persist in vivo, we developed a human artificial antigen-presenting cell system that can educate antitumor CTLs to acquire both a central memory and an effector memory phenotype as well as the capacity to survive in culture for prolonged periods of time. We examined whether antitumor CTLs generated using this system could function and persist in patients. We showed that MART1-specific CTLs, educated and expanded using our artificial antigen-presenting cell system, could survive for prolonged periods in advanced-stage melanoma patients without previous conditioning or cytokine treatment. Moreover, these CTLs trafficked to the tumor, mediated biological and clinical responses, and established antitumor immunologic memory. Therefore, this approach may broaden the availability of adoptive cell therapy to patients both alone and in combination with other therapeutic modalities.

Topics: Adoptive Transfer; Aged; Aged, 80 and over; Antibodies, Monoclonal; Antigen-Presenting Cells; Antigens, CD; CD8-Positive T-Lymphocytes; Cell Movement; CTLA-4 Antigen; Epitopes; Female; Humans; Immune Tolerance; Immunologic Memory; Interleukin-15; Interleukin-2; Male; MART-1 Antigen; Melanoma; Middle Aged; Phenotype; T-Lymphocytes, Cytotoxic; Time Factors

Thus far, peptide vaccines used to stimulate tumor-specific immune responses in patients with melanoma have been largely unsuccessful. Granulocyte-macrophage colony-stimulating factor and interleukin-2 are immune-potentiating cytokines that have improved vaccine responses in preclinical models. We hypothesized that higher doses of granulocyte-macrophage colony-stimulating factor and addition of low-dose interleukin-2 might augment responses to vaccine antigens. Patients with resected stage II, III, or IV melanoma were treated with vaccines containing three melanoma-associated peptides [MART-1a, gp100(207-217), and survivin], along with 300 or 500 mcg granulocyte-macrophage colony-stimulating factor in Montanide ISA. Cohorts of patients received low-dose subcutaneous interleukin-2 on days 7-20 after vaccination. Induction of a response was defined as either doubling of cytotoxic T lymphocyte frequency from baseline or increase in frequency from undetectable (<0.05%) to detectable. Leukocyte subsets and plasma cytokines were analyzed before and after vaccination. Cytotoxic T lymphocyte responses to MART-1a, gp100(207-217), and survivin were induced in 11, 16, and 14 of 19 patients, respectively. Responses were not higher in patients receiving 500 mcg granulocyte-macrophage colony-stimulating factor or low-dose interleukin-2 than in patients receiving 300 mcg granulocyte-macrophage colony-stimulating factor only. Interleukin-2 treatment (in nine patients) led to increases in natural killer cells and T regulatory cells compared with no interleukin-2 treatment (nine patients). Multiple plasma cytokines were transiently induced during vaccination. Neither increasing the dose of granulocyte-macrophage colony-stimulating factor nor addition of low-dose interleukin-2 resulted in an increase in the frequency of vaccine-specific cytotoxic T lymphocytes to a melanoma peptide vaccine. The increase in T regulatory cells associated with interleukin-2 treatment suggests that interleukin-2 may be immunosuppressive in this setting.

Topics: Adjuvants, Immunologic; Adult; Cancer Vaccines; Cohort Studies; Female; gp100 Melanoma Antigen; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Inhibitor of Apoptosis Proteins; Interleukin-2; Male; MART-1 Antigen; Melanoma; Middle Aged; Pilot Projects; Survivin; Vaccines, Subunit; Young Adult

An oncolytic herpes simplex virus engineered to replicate selectively in tumor cells and to express granulocyte-macrophage colony-stimulating factor (GMCSF) was tested as a direct intralesional vaccination in melanoma patients. The work reported herein was performed to better characterize the effect of vaccination on local and distant antitumor immunity.. Metastatic melanoma patients with accessible lesions were enrolled in a multicenter 50-patient phase II clinical trial of an oncolytic herpesvirus encoding GM-CSF (Oncovex(GM-CSF)). An initial priming dose of 10(6) pfu vaccine was given by intratumoral injection, followed by 10(8) pfu every 2 weeks to 24 total doses. Peripheral blood and tumor tissue were collected for analysis of effector T cells, CD4(+)FoxP3(+) regulatory T cells (Treg), CD8(+)FoxP3(+) suppressor T cells (Ts), and myeloid-derived suppressive cells (MDSC).. Phenotypic analysis of T cells derived from tumor samples suggested distinct differences from peripheral blood T cells. There was an increase in melanomaassociated antigen recognized by T cells (MART-1)-specific T cells in tumors undergoing regression after vaccination compared with T cells derived from melanoma patients not treated with vaccine. There was also a significant decrease in Treg and Ts cells in injected lesions compared with noninjected lesions in the same and different melanoma patients. Similarly MDSC were increased in melanoma lesions but underwent a significant decrease only in vaccinated lesions.. Melanoma patients present with elevated levels of Tregs, Ts, and MDSC within established tumors. Direct injection of Oncovex(GM-CSF) induces local and systemic antigen-specific T cell responses and decreases Treg, Ts, and MDSC in patients exhibiting therapeutic responses.

Topics: Antigens, Neoplasm; Cancer Vaccines; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Genetic Therapy; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Injections, Intralesional; MART-1 Antigen; Melanoma; Neoplasm Proteins; Neoplasm Staging; Oncolytic Viruses; Simplexvirus; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory; Transduction, Genetic; Vaccination

Safety and cellular immunogenicity of rising doses and varying regimens of a poly-epitope vaccine were evaluated in advanced metastatic melanoma. The vaccine comprised plasmid DNA and recombinant modified vaccinia virus Ankara (MVA) both expressing a string (Mel3) of seven HLA.A2/A1 epitopes from five melanoma antigens.. Forty-one HLA-A2 positive patients with stage III/IV melanoma were enrolled. Patient groups received one or two doses of DNA.Mel3 followed by escalating doses of MVA.Mel3. Immunisations then continued eight weekly in the absence of disease progression. Epitope-specific CD8+ T cell responses were evaluated using ex-vivo tetramer and IFN-gamma ELISPOT assays. Safety and clinical responses were monitored.. Prime-boost DNA/MVA induced Melan-A-specific CD8+ T cell responses in 22/31 (71%) patients detected by tetramer assay. ELISPOT detected a response to at least one epitope in 10/31 (32%) patients. T cell responder rates were <50% with low-dose DNA/MVA, or MVA alone, rising to 91% with high-dose DNA/MVA. Among eight patients showing evidence of clinical benefit-one PR (24 months+), five SD (5 months+) and two mixed responses-seven had associated immune responses. Melan-A-tetramer+ immunity was associated with a median 8-week increase in time-to-progression (P = 0.037) and 71 week increase in survival (P = 0.0002) compared to non-immunity. High-dose vaccine was well tolerated. The only significant toxicities were flu-like symptoms and injection-site reactions.. DNA.Mel3 and MVA.Mel3 in a prime-boost protocol generated high rates of immune response to melanoma antigen epitopes. The treatment was well tolerated and the correlation of immune responses with patient outcomes encourages further investigation.

Topics: Adult; Aged; Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Disease Progression; Epitopes, T-Lymphocyte; Female; HLA-A2 Antigen; Humans; Immunization, Secondary; Interferon-gamma; Lymphocyte Activation; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Staging; Survival Analysis; Vaccinia virus

Recent immunotherapy trials have shown that lymphodepletion induced by short-term chemotherapy favors subsequent expansion of adoptively transferred T cells, by homeostatic mechanisms. To take advantage of this effect, novel regimens are being developed with the aim to enhance tumor immunity and reduce treatment toxicity. We have designed a clinical phase I trial combining chemotherapy, reinfusion of PBMC containing Melan-A(MART-1)-specific T cells, and vaccination with Melan-A peptide in Incomplete Freund's Adjuvant. Treatment with Busulfan plus Fludarabine depleted lymphocytes only weakly. Cyclophosphamide (CTX) plus Fludarabine depleted lymphocytes more profoundly, with a maximal effect using high doses of CTX. It is interesting to note that, the degree of homeostatic T-cell proliferation correlated tightly with the extent of lymphodepletion. As compared with CD4 T cells, CD8 T cells showed higher susceptibility to chemotherapy, followed by more rapid homeostatic proliferation and recovery, resulting in strong inversions of CD4/CD8 ratios. Despite efficient homeostatic proliferation of total CD4 and CD8 T cells, the frequency of CD8 T cells specific for Melan-A and cancer-testis antigens remained relatively low. In contrast, EBV-specific T cells expanded and reached high numbers. We conclude that short-term chemotherapy promoted homeostatic lymphocyte proliferation depending on the intensity of lymphocyte depletion, however without preferential expansion of tumor antigen-specific T cells.

Topics: Aged; Busulfan; Cancer Vaccines; CD8 Antigens; Cell Proliferation; Cyclophosphamide; Cytokines; Female; Freund's Adjuvant; Humans; Immunotherapy, Adoptive; Lymphocyte Depletion; Male; MART-1 Antigen; Melanoma; Middle Aged; Peptide Fragments; Skin Neoplasms; T-Lymphocytes; Vidarabine

Monitoring of circulating melanoma cells in the peripheral blood is a promising method for identifying a subgroup of patients with minimal residual disease.. To evaluate the prognostic impact of melanoma-associated antigens by multimarker real-time RT-PCR for disease-specific survival time.. Five melanoma markers: Melan-A, gp 100, MAGE-3, MIA and tyrosinase were detected by a quantitative multimarker real-time reverse transcription-PCR (RT-PCR). We included 65 patients with resected melanoma in stage II-III. Peripheral blood samples were examined every 3 months for 2 years. The expression of melanoma markers in 2925 RT-PCR assays was correlated with clinical staging results in total of 5 years.. Twenty-seven patients relapsed during the study period and 26 of them revealed positive markers. MAGE-3 was the most sensitive progression marker in single occurrence or in combination with MIA and gp 100. The time distribution of metastases during the screened period was as follows: progression in the first year was observed in 40.7% patients, second year in 25.9%, third year in 18.6%, fourth and fifth year in 7.4% equally.. Statistically significant tumor marker elevation during the first 2 years after the surgical treatment correlates with a worse prognosis of patients. In contrast, the group showing negative real-time RT-PCR results in 24 months serial blood testing was associated with prolonged 5-year disease-specific survival. Therefore, quantitative detection of melanoma-specific molecular markers in the presented setting represents a useful tool for selecting patients in a higher risk of disease recurrence.

Topics: Adult; Aged; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Disease Progression; Disease-Free Survival; Female; Gene Expression; gp100 Melanoma Antigen; Humans; Leukocytes; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Staging; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; Young Adult

Induction of cytotoxic CD8 T-cell responses is enhanced by the exclusive presentation of antigen through dendritic cells, and by innate stimuli, such as toll-like receptor ligands. On the basis of these 2 principles, we designed a vaccine against melanoma. Specifically, we linked the melanoma-specific Melan-A/Mart-1 peptide to virus-like nanoparticles loaded with A-type CpG, a ligand for toll-like receptor 9. Melan-A/Mart-1 peptide was cross-presented, as shown in vitro with human dendritic cells and in HLA-A2 transgenic mice. A phase I/II study in stage II-IV melanoma patients showed that the vaccine was well tolerated, and that 14/22 patients generated ex vivo detectable T-cell responses, with in part multifunctional T cells capable to degranulate and produce IFN-γ, TNF-α, and IL-2. No significant influence of the route of immunization (subcutaneous versus intradermal) nor dosing regimen (weekly versus daily clusters) could be observed. It is interesting to note that, relatively large fractions of responding specific T cells exhibited a central memory phenotype, more than what is achieved by other nonlive vaccines. We conclude that vaccination with CpG loaded virus-like nanoparticles is associated with a human CD8 T-cell response with properties of a potential long-term immune protection from the disease.

Topics: Adult; Aged; Animals; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Differentiation; Cells, Cultured; Female; HLA-A2 Antigen; Humans; Immunologic Memory; Lymphocyte Activation; Male; MART-1 Antigen; Melanoma; Mice; Mice, Transgenic; Middle Aged; Nanoparticles; Oligodeoxyribonucleotides; Peptide Fragments; Skin Neoplasms; T-Lymphocyte Subsets; Treatment Outcome; Vaccines, Virus-Like Particle

Ipilimumab is a monoclonal antibody that blocks the immune-inhibitory interaction between CTL antigen 4 (CTLA-4) and its ligands on T cells. Clinical trials in cancer patients with ipilimumab have shown promising antitumor activity, particularly in patients with advanced melanoma. Often, tumor regressions in these patients are correlated with immune-related side effects such as dermatitis, enterocolitis, and hypophysitis. Although these reactions are believed to be immune-mediated, the antigenic targets for the cellular or humoral immune response are not known.. We enrolled patients with advanced melanoma in a phase II study with ipilimumab. One of these patients experienced a complete remission of his tumor. The specificity and functional properties of CD8-positive T cells in his peripheral blood, in regressing tumor tissue, and at the site of an immune-mediated skin rash were investigated.. Regressing tumor tissue was infiltrated with CD8-positive T cells, a high proportion of which were specific for Melan-A. The skin rash was similarly infiltrated with Melan-A-specific CD8-positive T cells, and a dramatic (>30-fold) increase in Melan-A-specific CD8-positive T cells was apparent in peripheral blood. These cells had an effector phenotype and lysed Melan-A-expressing tumor cells.. Our results show that Melan-A may be a major target for both the autoimmune and antitumor reactions in patients treated with anti-CTLA-4, and describe for the first time the antigen specificity of CD8-positive T cells that mediate tumor rejection in a patient undergoing treatment with an anti-CTLA-4 antibody. These findings may allow a better integration of ipilimumab into other forms of immunotherapy.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Antineoplastic Agents; Autoimmunity; Cytotoxicity, Immunologic; Double-Blind Method; Exanthema; Humans; Ipilimumab; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Neoplasm Proteins; Skin Neoplasms; T-Lymphocytes, Cytotoxic; Tomography, X-Ray Computed

Immunotherapy by adoptive T-cell transfer aims at maximizing tumor antigen-specific T-cell responses. We treated 14 patients at the metastatic stage in a phase II study with Melan-A-specific T-cell clones generated from patient blood. During the period required for T-cell clone generation, the patients were treated by dacarbazine. Every patient received a T-cell clone suspension followed by subcutaneous injections of interleukin 2 and interferon alpha. Patients were monitored until disease progression occurred. We succeeded in obtaining autologous Melan-A-specific cytotoxic T lymphocyte clones, which were highly reactive against tumor cells for all the patients. Of the 14 patients treated, six (43%) experienced an objective response (CR + PR) with long-term complete remission for two patients (1 CR for 5 years and 1 CR for 28 months). Furthermore, we showed that all the clinical responses were significantly associated with in vivo expansion of the Melan-A-specific T-cell repertoire. This phenomenon appeared to be significantly associated with clinical responses. Thus, over the course of an adoptive cell transfer, monitoring this melanoma-specific T-cell expansion in patient blood appears crucial for predicting the clinical efficiency of such an immunological approach.

Topics: Adoptive Transfer; Aged; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Clone Cells; Female; Humans; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Skin Neoplasms; Skin Pigmentation; T-Lymphocytes, Cytotoxic; Treatment Outcome

Toll-like receptor ligands are potentially useful adjuvants for the development of clinical T cell vaccination. Here we investigated the novel Toll-like receptor2 ligand P40, the outer membrane protein A derived from Klebsiella pneumoniae. Seventeen human leukocyte antigen-A*0201 positive stage III/IV melanoma patients were vaccinated with P40 and Melan-A/Mart-1 peptide subcutaneously in monthly intervals. Adverse reactions were mild-to-moderate. Fourteen patients received at least 8 vaccinations and were thus evaluable for clinical tumor and immune responses. Seven patients experienced progressive disease, whereas 2 patients had stable disease throughout the trial period, 1 of them with regression of multiple skin metastases. The remaining 5 patients had no measurable disease. Melan-A/Mart-1 specific CD8 T cells were analyzed ex vivo, with positive results in 6 of 14 evaluable patients. Increased percentages of T cells were found in three patients, memory/effector T cell differentiation in 4 patients, and a positive interferon-gamma Elispot assay in 1 patient. Antibody responses to P40 were observed in all patients. We conclude that vaccination with peptide and P40 was feasible and induced ex vivo detectable tumor antigen specific T cell responses in 6 of 14 patients with advanced melanoma.

Topics: Adjuvants, Immunologic; Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Bacterial Outer Membrane Proteins; Cancer Vaccines; CD8-Positive T-Lymphocytes; Female; HLA-A Antigens; HLA-A2 Antigen; Humans; Klebsiella pneumoniae; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Neoplasm Staging; Peptide Fragments; Skin Neoplasms

To report the findings on sentinel lymph node biopsy (SLNB) in 30 patients with ocular adnexal (conjunctival or eyelid) melanomas.. Prospective nonrandomized clinical trial.. Thirty patients with diagnosis of eyelid or conjunctival melanoma.. All patients with ocular adnexal melanoma who underwent SLNB at The University of Texas MD Anderson Cancer Center between December 2000 and July 2008 are the subject of this report. Sentinel lymph node biopsy was performed as previously described by our group, and patients were prospectively followed up.. Findings on preoperative lymphoscintigraphy, SLNB, histopathologic examination of the primary tumor and sentinel lymph nodes (SNL), and nodal recurrence after SLNB.. Tumor sites were as follows: bulbar conjunctiva only, 14 patients; palpebral conjunctiva only, 8 patients; both bulbar and palpebral conjunctiva, 4 patients; and eyelid skin only, 4 patients. At least 1 SLN was identified in all patients. The median number of SLNs removed was 2 (range, 1-5). The most common basin sampled was the intraparotid (16 patients), followed by submandibular (level I) (11 patients), preauricular (9 patients), and superior cervical (level II) (6 patients). Five patients had SLN metastasis. Among the 25 patients with negative SLNB findings, there were 2 false-negative events. There were no false-negative events among patients treated during the last 4.5 years of the study. The mean Breslow thickness was 2.57 mm (range, 0.62-12 mm) among patients with negative SLNB and 4.86 mm (range, 2.0-7.2 mm) among patients with positive SLNB findings (P = 0.055). Ulceration was present in 11 patients (39%): 4 (80%) of 5 patients with positive SLNB and 7 (28%) of 25 with negative SLNB, including both patients with false-negative results. The median time from SLNB to last contact was 2 years (range, 10 months to 6 years).. Sentinel lymph node biopsy is effective for identifying nodal micrometastasis in patients with ocular adnexal melanoma and provides important prognostic information. The false-negative event rate in our series improved in the last 4 years, most likely because of a better technique and better patient selection for SLNB. We recommend consideration of SLNB for patients with intermediate-thickness ocular adnexal melanoma and those with ulceration.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Conjunctival Neoplasms; Eyelid Neoplasms; False Positive Reactions; Female; Humans; Lymph Nodes; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Predictive Value of Tests; Prognosis; Prospective Studies; Sentinel Lymph Node Biopsy; Tomography, Emission-Computed, Single-Photon; Tomography, X-Ray Computed

Immunotherapy of cancer is often performed with altered "analog" peptide Ags optimized for HLA class I binding, resulting in enhanced immunogenicity, but the induced T cell responses require further evaluation. Recently, we demonstrated fine specificity differences and enhanced recognition of naturally presented Ag by T cells after vaccination with natural Melan-A/MART-1 peptide, as compared with analog peptide. In this study, we compared the TCR primary structures of 1489 HLA-A*0201/Melan-A(26-35)-specific CD8 T cells derived from both cohorts of patients. Although a strong preference for TRAV12-2 segment usage was present in nearly all patients, usage of particular TRAJ gene segments and CDR3alpha composition differed slightly after vaccination with natural vs analog peptide. Moreover, TCR beta-chain repertoires were broader after natural than analog peptide vaccination. In all patients, we observed a marked conservation of the CDR3beta amino acid composition with recurrent sequences centered on a glycyl-leucyl/valyl/alanyl-glycyl motif. In contrast to viral-specific TCR repertoires, such "public" motifs were primarily expressed by nondominant T cell clonotypes, which contrasted with "private" CDR3beta signatures frequently found in T cell clonotypes that dominated repertoires of individual patients. Interestingly, no differences in functional avidity were observed between public and private T cell clonotypes. Collectively, our data indicate that T cell repertoires generated against natural or analog Melan-A peptide exhibited slightly distinct but otherwise overlapping and structurally conserved TCR features, suggesting that the differences in binding affinity/avidity of TCRs toward pMHC observed in the two cohorts of patients are caused by subtle structural TCR variations.

Topics: Amino Acid Sequence; Antigens, Neoplasm; Autoantigens; Base Sequence; Cancer Vaccines; CD8-Positive T-Lymphocytes; HLA-A Antigens; HLA-A2 Antigen; Humans; Immunotherapy; MART-1 Antigen; Melanoma; Molecular Sequence Data; Neoplasm Proteins; Prospective Studies; Protein Conformation; Receptors, Antigen, T-Cell, alpha-beta; Sequence Alignment; Skin Neoplasms

Tumor antigen-loaded dendritic cells (DC) are believed to activate antitumor immunity by stimulating T cells, and CTL-associated antigen 4 (CTLA4)-blocking antibodies should release a key negative regulatory pathway on T cells. The combination was tested in a phase I clinical trial in patients with advanced melanoma.. Autologous DC were pulsed with MART-1(26-35) peptide and administered with a dose escalation of the CTLA4-blocking antibody tremelimumab. Sixteen patients were accrued to five dose levels. Primary end points were safety and immune effects; clinical efficacy was a secondary end point.. Dose-limiting toxicities of grade 3 diarrhea and grade 2 hypophysitis developed in two of three patients receiving tremelimumab at 10 mg/kg monthly. Four patients had an objective tumor response, two partial responses and two complete responses, all melanoma free between 2 and 4 years after study initiation. There was no difference in immune monitoring results between patients with an objective tumor response and those without a response. Exploratory gene expression analysis suggested that immune-related gene signatures, in particular for B-cell function, may be important in predicting response.. The combination of MART-1 peptide-pulsed DC and tremelimumab results in objective and durable tumor responses at the higher range of the expected response rate with either agent alone.

Topics: Adult; Aged; Antibodies, Blocking; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antigens, CD; Antigens, Neoplasm; Antineoplastic Combined Chemotherapy Protocols; Cancer Vaccines; Combined Modality Therapy; CTLA-4 Antigen; Dendritic Cells; Dose-Response Relationship, Drug; Female; Humans; Immunotherapy, Adoptive; K562 Cells; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Metastasis; Neoplasm Proteins; Peptide Fragments

To evaluate the prognostic and predictive importance of detection of melanoma cells in peripheral blood using reverse transcriptase polymerase chain reaction (RT-PCR) in stage III cutaneous melanoma patients after sentinel or regional lymph node dissection.. Serial testing for tyrosinase and Mart-1/Melan-A transcripts in peripheral blood was performed every 6 months over a maximum period of 60 months in a subset of patients enrolled in EORTC 18991 phase 3 trial, comparing pegylated interferon-alpha-2b with observation. Univariate and multivariate analyses were performed to estimate the role of RT-PCR as prognostic and predictive factor for distant metastasis-free survival (DMFS).. Among 299 patients who underwent RT-PCR analyses, 109 (36.5%) had at least one positive sample, either at time of randomisation (N=17) or subsequently (N=92). The cumulative rate of positive results was similar in the two treatment groups, as the DMFS from first RT-PCR positivity. RT-PCR result, positive versus negative, at a given time point, had no prognostic impact on subsequent DMFS. Cox time-dependent analysis indicated a significantly higher risk of developing distant metastasis in patients with a positive sample as compared to those with a negative one: hazard ratio (HR) of 2.23 (95% confidence interval (CI), 1.40-3.55; p<.001). These results were comparable in the 2 treatment groups, indicating that RT-PCR assessment was not predictive for treatment outcome.. Detection of circulating tumour cells by RT-PCR for tyrosinase and Mart-1/Melan-A was a time-dependent moderate prognostic factor for subsequent development of distant metastasis in stage III melanoma patients.

Topics: Adolescent; Adult; Aged; Antigens, Neoplasm; Antineoplastic Agents; Biomarkers, Tumor; Chemotherapy, Adjuvant; Disease-Free Survival; Female; Humans; Interferon alpha-2; Interferon-alpha; Lymph Node Excision; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Staging; Neoplastic Cells, Circulating; Polyethylene Glycols; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Skin Neoplasms; Statistics as Topic; Young Adult

For the treatment of melanoma DNA vaccines are a promising therapeutic approach. In our institute a plasmid encoding a melanoma-associated epitope (MART-1) and an immunostimulatory sequence (tetanus toxin fragment-c) termed pDERMATT was developed. In a phase I study the plasmid will be administered intradermally using a newly developed tattoo strategy to assess the toxicity and efficacy of inducing tumor-specific T-cell immunity. To facilitate this study a Good Manufacturing Practice (GMP)-compliant plasmid manufacturing process was set up and a pharmaceutical dosage form was developed. Each batch resulted in approximately 200mg plasmid DNA of a high purity >90% supercoiled DNA, an A260/280 ratio 1.80-1.95, undetectable or extremely low residual endotoxins, Escherichia coli host cell protein, RNA, and DNA. In the manufacturing process no animal derived enzymes like RNase or potentially harmful organic solvents are used. After sterile filtration the concentration of the plasmid solution is approximately 1.1mg/mL. For the scheduled phase I study a concentration of 5mg/mL is desired, and further concentration of the solution is achieved by lyophilisation. The formulation solution is composed of 1mg/mL pDERMATT and 20mg/mL sucrose in Water for Injections. Upon reconstitution with a five times smaller volume an isotonic sucrose solution containing 5mg/mL pDERMATT is obtained. Lyophilised pDERMATT is sterile with >90% supercoiled DNA, an A260-280 ratio 1.80-1.95, content 90-110% of labeled, and residual water content <2% (w/w). The product yields the predicted profile upon restriction-enzyme digestion, is highly immunogenic as confirmed in an in vivo mouse model, and stable for at least six months at 5 degrees C. We have not only developed a reproducible process to manufacture pharmaceutical grade plasmid DNA but also a stable dosage form for the use in clinical trials.

Topics: Antigens, Neoplasm; Cancer Vaccines; Humans; MART-1 Antigen; Melanoma; Neoplasm Proteins; Plasmids; Quality Control; T-Lymphocytes, Cytotoxic; Vaccination; Vaccines, DNA

A phase I/II trial was performed to evaluate the safety and immunogenicity of a novel melanoma vaccine comprising six melanoma-associated peptides defined as antigenic targets for melanoma-reactive helper T cells. Source proteins for these peptides include MAGE proteins, MART-1/MelanA, gp100, and tyrosinase.. Thirty-nine patients with stage IIIB to IV melanoma were vaccinated with this six-peptide mixture weekly at three dose levels, with a preceding phase I dose escalation and subsequent random assignment among the dose levels. Helper T-lymphocyte responses were assessed by in vitro proliferation assay and delayed-type hypersensitivity skin testing. Patients with measurable disease were evaluated for objective clinical response by Response Evaluation Criteria in Solid Tumors.. Vaccination with the helper peptide vaccine was well tolerated. Proliferation assays revealed induction of T-cell responses to the melanoma helper peptides in 81% of patients. Among 17 patients with measurable disease, objective clinical responses were observed in two patients (12%), with response durations of 1 and 3.9+ years. Durable stable disease was observed in two additional patients for periods of 1.8 and 4.6+ years.. Results of this study support the safety and immunogenicity of a vaccine comprised of six melanoma helper peptides. There is also early evidence of clinical activity.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Differentiation; Antigens, Neoplasm; Cancer Vaccines; Cell Proliferation; Female; gp100 Melanoma Antigen; Humans; Male; MART-1 Antigen; Melanocytes; Melanoma; Membrane Glycoproteins; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Staging; Skin Neoplasms; Skin Tests; T-Lymphocytes, Helper-Inducer; Vitiligo

Early testing of aerosolized sargramostim therapy demonstrated anecdotal clinical responses in patients with metastatic melanoma associated with emergence of systemic antitumor immunity. To improve the clinical and immunologic efficacy of therapy without compromising patient safety, we performed a further dose escalation trial in patients with metastatic melanoma.. We conducted a dose-escalation clinical trial of HLA-A2 patients with metastatic melanoma to the lung treated with aerosolized granulocyte macrophage colony stimulating factor (GM-CSF) (500-2000 microg/dose, with increments of 250 microg/dose/cohort) twice/d on days 1 to 7 and 15 to 21 every 28 days until progression or severe toxicity to find a dose where a majority of patients develop antitumor immunity. Five patients were treated per each dose level. Clinical, immune, and safety parameters were examined.. The study accrued 40 patients. Toxicity was acceptable. All doses levels were exhausted without identifying a dose of GM-CSF at which a majority of patients (> or =3 of 5) demonstrated significant up-regulation of antitumor immunity. Three of 16 patients who were tetramer positive for at least one melanoma antigen (eg, MART-1) pretreatment developed an immune response (IR) to different tumor antigens. Two of 9 patients who were tetramer negative to all melanoma antigens pretreatment developed an IR against gp100. The greatest changes in antitumor immunity occurred at the highest dose levels.. A dose of aerosolized GM-CSF capable of inducing antitumor immunity in the majority of patients was not reached. All tested doses were well tolerated. The greatest increase in antitumor T cell IRs was achieved at the highest doses of GM-CSF.

Topics: Administration, Inhalation; Adult; Aerosols; Aged; Aged, 80 and over; Antigens, Neoplasm; Dose-Response Relationship, Drug; Female; Granulocyte-Macrophage Colony-Stimulating Factor; HLA-A2 Antigen; Humans; Immunologic Factors; Immunophenotyping; Immunotherapy; Lung Neoplasms; Male; MART-1 Antigen; Maximum Tolerated Dose; Melanoma; Middle Aged; Neoplasm Proteins; Neoplasm Staging; Recombinant Proteins; T-Lymphocytes; T-Lymphocytes, Cytotoxic; Treatment Outcome

Detection of melanoma cells in peripheral blood is a promising method for monitoring haematogenous spread of melanoma cells. It enables us to detect early metastasis and to better stratify candidates for adjuvant immunotherapy. Inconsistent data on the sensitivity and clinical relevance of this method have been reported.. We developed a multimarker real-time reverse transcription-PCR (RT-PCR) for quantification of five melanoma markers: Melan-A, gp100, MAGE-3, MIA and tyrosinase. In this prospective study, 65 patients with resected cutaneous melanoma stage IIB-III were screened. Peripheral blood samples were collected every 3 months for the following 18 months, and circulating melanoma cells were examined and compared with clinical staging results.. Eighteen patients relapsed during the trial and showed different types of melanoma progression. All these patients experienced statistically significant tumour marker elevation in the period from 0 to 9 months before the disease progression. MAGE-3 was the most sensitive progression marker. In patients with progression, we observed three concordant positive markers in 39% of cases, two concordant positive markers in 28%, and finally one marker in 33%.. This report describes a multiple-marker real-time RT-PCR, which is able to provide quantitative data on melanoma markers in the peripheral blood of melanoma patients. Measurement of the studied molecular markers in our hands represents a prognostic factor and a useful method for early detection of metastasis and treatment response of melanoma patients.

Topics: Adult; Aged; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Cell Line, Tumor; Disease Progression; Female; gp100 Melanoma Antigen; Humans; Male; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Staging; Prospective Studies; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sensitivity and Specificity; Skin Neoplasms

We performed a phase 1/2 trial testing the safety, toxicity, and immune response of a vaccine consisting of autologous dendritic cells (DCs) transduced with a replication-defective adenovirus (AdV) encoding the full-length melanoma antigen MART-1/Melan-A (MART-1). This vaccine was designed to activate MART-1-specific CD+8 and CD4+ T cells. Metastatic melanoma patients received 3 injections of 10(6) or 10(7) DCs, delivered intradermally. Cell surface phenotype and cytokine production of the DCs used for the vaccines were tested, and indicated intermediate maturity. CD8+ T-cell responses to MART-1 27-35 were assessed by both major histocompatibility complex class I tetramer and interferon (IFN)-gamma enzyme-linked immunosorbent spot (ELISPOT) before, during, and after each vaccine and CD4+ T-cell responses to MART-1 51-73 were followed by IFN-gamma ELISPOT. We also measured antigen response breadth. Determinant spreading from the immunizing antigen MART-1 to other melanoma antigens [gp100, tyrosinase, human melanoma antigen-A3 (MAGE-A3)] was assessed by IFN-gamma ELISPOT. Twenty-three patients were enrolled and 14 patients received all 3 scheduled DC vaccines. Significant CD8+ and/or CD4+ MART-1-specific T-cell responses were observed in 6/11 and 2/4 patients evaluated, respectively, indicating that the E1-deleted adenovirus encoding the cDNA for MART-1/Melan-A (AdVMART1)/DC vaccine activated both helper and killer T cells in vivo. Responses in CD8+ and CD4+ T cells to additional antigens were noted in 2 patients. The AdVMART1-transduced DC vaccine was safe and immunogenic in patients with metastatic melanoma.

Topics: Adenoviridae; Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Cancer Vaccines; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Dendritic Cells; Female; Humans; Immunodominant Epitopes; Immunotherapy, Active; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Staging; Protein Engineering; Transduction, Genetic; Treatment Outcome

Adoptive therapy of cancer has been mostly tested in advanced cancer patients using tumor-infiltrating lymphocytes (TIL). Following discouraging results likely due to poor tumor-specificity of TIL and/or high tumor burden, recent studies reiterate the enormous potential of this therapy, particularly in melanoma. We had performed a phase II/III randomised trial on 88 stage III melanoma patients, who received autologous TIL plus IL-2 or IL-2 alone, after complete tumour resection. We reported previously clinical and immunological results supporting the ability of tumor reactive TIL infusion to prevent further development of the melanoma disease and to increase overall survival of patients bearing only one tumor invaded lymph node. The absence of correlation between overall and disease-free survival and the amount of infused tumor-specific TIL suggested that therapeutic efficiency might depend on other parameters such as antigen specificity, function or persistence of TIL. Here we studied the recognition of a panel of 38 shared tumor-associated antigens (TAA) by TIL infused to the patients included in this assay, in order to determine if treatment outcome could correlate with particular antigen specificities of infused TIL. Results show that the infusion of Melan-A/MART-1 reactive TIL appears to be associated with a longer relapse-free survival for HLA-A2 patients. These results further support the relevance of Melan-A/MART-1 antigen as a prime target for immunotherapy protocols in melanoma.

Topics: Animals; Antigens, Neoplasm; Chlorocebus aethiops; COS Cells; Disease-Free Survival; HLA-A2 Antigen; Humans; Immunotherapy, Adoptive; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Neoplasm Proteins; Survival Analysis; T-Lymphocytes; Transplantation, Autologous

The aim of this study was to investigate whether depletion of CD4(+)CD25(+) regulatory T cells (Treg) from melanoma patients affects immune responses against tumors. By application of recombinant IL-2-diphteria toxin fusion protein, also known as ONTAK, we were able to significantly reduce the frequency of Treg in peripheral blood, whereas other cell populations remained unaffected. The reduction of Treg started immediately after the first bolus of ONTAK with a dose of 5 microg ONTAK per kg bodyweight and lasted for 13 days with subsequent recovery thereafter. Successive ONTAK treatments further reduced the number of circulating Treg. Using the contact sensitizer DCP we show that all patients developed vast eczema after Treg depletion, whereas no or only mild eczematous reactions were detectable before ONTAK treatment. Corresponding induction of DCP-specific CD4(+) and CD8(+) T cells were detectable. Moreover, after immunization of ONTAK treated patients with tumor antigen peptides, MelanA/MART-1 and gp100, significant induction of peptide specific CD8(+) T cells could be observed in 90% of the patients treated. These cells displayed effector functions, as they were able to lyse peptide-pulsed target cells and secreted IFNgamma upon restimulation. In aggregate, our data indicate that ONTAK depletes Treg in vivo significantly, resulting in enhanced immune functions and substantial development of antigen-specific CD8(+) T cells in vaccinated individuals.

Topics: Adult; Aged; Antigens, Neoplasm; Antineoplastic Agents; CD4 Antigens; CD8 Antigens; Cell Proliferation; Cell Survival; Dimerization; Diphtheria Toxin; Dose-Response Relationship, Drug; Eczema; Female; Flow Cytometry; gp100 Melanoma Antigen; Humans; Interleukin-2; Interleukin-2 Receptor alpha Subunit; Leukapheresis; Male; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Middle Aged; Neoplasm Proteins; Recombinant Fusion Proteins; T-Lymphocytes; Time Factors; Treatment Outcome; Vaccination

To determine the ocular safety of CP-675,206 (Pfizer, New York, New York, USA), a fully human anti-cytotoxic T lymphocyte-associated antigen 4 monoclonal antibody in clinical trials of immunotherapy of metastatic melanoma.. Prospective, nonrandomized study of the eye and vision in phase I/II clinical trials of CP-675,206 in metastatic melanoma conducted at the University of California, Los Angeles.. Patients with regional or distant metastatic melanoma were enrolled in phase I/II clinical trials evaluating the safety and antitumor efficacy of CP-675,206 alone or in combination with melanoma antigen peptide-pulsed dendritic cell vaccines. Ophthalmic evaluation was performed at the onset of CP-675,206 immunotherapy (baseline evaluation), two months or more after the onset of CP-675,206 immunotherapy (end-study evaluation), and at two- to three-month intervals thereafter in patients who continued to receive CP-675,206 immunotherapy (poststudy evaluation). Baseline and end-study evaluations included comprehensive ophthalmic examination, psychophysical and electrophysiologic visual function assessment, fundus photography, fluorescein angiography, and visual function assessment.. Twenty patients with metastatic melanoma arising from the skin, mucosa, eye, or unknown site were evaluated. Systemic toxicity attributed to CP-675,206 included dermatologic manifestations, diarrhea, and autoimmune hepatitis with panhypopituitarism. A subset of patients receiving CP-675,206 demonstrated antitumor efficacy with partial response or complete response of metastatic melanoma. Comparison of ophthalmic baseline with end-study evaluations in all 20 patients and limited-term poststudy evaluations showed no adverse effect of CP-675,206 immunotherapy on the eye or vision.. In this study, CP-675,206 immunotherapy for metastatic melanoma did not adversely affect the eye or vision.

Topics: Abatacept; Adult; Aged; Aged, 80 and over; Antibodies, Blocking; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antigens, Neoplasm; Anus Neoplasms; Choroid Neoplasms; Drug Therapy, Combination; Electrooculography; Electroretinography; Female; Fluorescein Angiography; Humans; Immunoconjugates; Immunotherapy; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Neoplasms; Ocular Physiological Phenomena; Prospective Studies; Skin Neoplasms; Treatment Outcome; Vision, Ocular; Visual Acuity

Between March 1999 and May 2000, 18 HLA-A*0201(+) patients with metastatic melanoma were enrolled in a phase I trial using a dendritic cell (DC) vaccine generated by culturing CD34(+) hematopoietic progenitors. This vaccine includes Langerhans cells. The DC vaccine was loaded with four melanoma peptides (MART-1/MelanA, tyrosinase, MAGE-3, and gp100), Influenza matrix peptide (Flu-MP), and keyhole limpet hemocyanin (KLH). Ten patients received eight vaccinations, one patient received six vaccinations, one patient received five vaccinations, and six patients received four vaccinations. Peptide-specific immunity was measured by IFN-gamma production and tetramer staining in blood mononuclear cells. The estimated median overall survival was 20 months (range: 2-83), and the median event-free survival was 7 months (range: 2-83). As of August 2005, four patients are alive (three patients had M1a disease and one patient had M1c disease). Three of them have had no additional therapy since trial completion; two of them had solitary lymph node metastasis, and one patient had liver metastasis. Patients who survived longer were those who mounted melanoma peptide-specific immunity to at least two melanoma peptides. The present results therefore justify the design of larger follow-up studies to assess the immunological and clinical outcomes in patients with metastatic melanoma vaccinated with peptide-pulsed CD34-derived DCs.

Topics: Adult; Aged; Antigens, CD34; Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Dendritic Cells; Flow Cytometry; gp100 Melanoma Antigen; Humans; Interferon-gamma; Lymphocyte Activation; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Stem Cells; Survival Analysis; Time; Treatment Outcome

A significant percentage of stage II melanoma patients (tumor thickness>1 mm) remain at risk of tumor recurrence after primary tumor excision. In this study, we used tumor antigen-pulsed dendritic cells as an adjuvant for immunization of these "high-risk" melanoma patients after resection of the primary tumor. A total of 13 patients were included and vaccinated 6 times every 14 days with autologous dendritic cells pulsed with a MelanA/MART-1 peptide in combination with a recall antigen. Antigen-specific immune responses were monitored before, during and up to 1 year after the last vaccination. The majority of patients exhibited increased recall antigen-specific CD4+ T cell responses upon vaccination. MelanA/MART-1-specific CD8+ T cells were expanded in 9/13 patients resulting in increased frequencies of memory cells in these patients. CD8+ T cells acquired the capacity to secrete IFN-gamma, to proliferate in culture in response to the tumor antigen used for vaccination and postvaccine samples contained MelanA/MART-1-specific T cells that recognized also the natural MelanA/MART-1-antigen expressed by tumor cells. Moreover, vaccination induced a long-lived tumor antigen-specific DTH-reactivity in the majority of the patients, detectable even 12 months after the last immunization. These data demonstrate for the first time that vaccination with tumor antigen-pulsed dendritic cells in a clinically adjuvant setting induces strong and persistent antigen-specific T-cell responses in tumor-free stage II melanoma patients, suggesting that tumor protective T cell immunity can be achieved.

Topics: Adjuvants, Immunologic; Adult; Antigens, Neoplasm; Cancer Vaccines; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Proliferation; Dendritic Cells; Humans; MART-1 Antigen; Melanoma; Neoplasm Proteins; Skin Neoplasms; Treatment Outcome; Vaccination

We did a phase I dose-escalation trial to evaluate the feasibility and safety of intratumoral injections of C Cure 709, an allogeneic, continuous CTL cell line that, restricted by HLA-A2, recognizes MART-1-positive tumor cells through transduction with a T-cell receptor encoding gene.. Cells were administered intratumorally in four dose levels ranging from 10(8) to 10(9) cells/d on days 1, 4, 7, 10, 14, and 28 of each treatment cycle to patients with metastatic melanoma. Main inclusion criteria were HLA-A2 tissue type, MART-1-positive tumor cells, and metastases suitable for ultrasound-guided injections. Patients were assessed for toxicity and response. Three to six patients were treated per dose level. Patients without progressive disease were offered up to three treatment cycles.. Fifteen patients received a total of 24 treatment cycles with a total of 266 injections of C Cure 709. Toxicity was minor to moderate and most common injection site reactions were fever, fatigue, nausea/vomiting, and arthralgia/myalgia. Side effects disappeared in general within 24 hours. Toxicity was not dose dependent. One patient obtained a partial response, encompassing both metastases used and not used for intratumoral injections. Remaining patients did not achieve an overall response. In addition, we observed local regression of metastases used for injection in two patients and of metastases not used for injection in one patient.. Intratumoral injections of C Cure 709 are feasible, safe, and capable of inducing tumor regression. Further investigation in a phase II setting is warranted.

Topics: Adult; Antigens, Neoplasm; Cell Line; Fatigue; Feasibility Studies; Female; Fever; HLA-A2 Antigen; Humans; Immunotherapy, Adoptive; Male; MART-1 Antigen; Melanoma; Middle Aged; Nausea; Neoplasm Proteins; Receptors, Antigen, T-Cell; T-Lymphocytes, Cytotoxic; Transplantation, Homologous; Treatment Outcome

The use of IFN-alpha in clinical oncology has generally been based on the rationale of exploiting its antiproliferative and antiangiogenic activities. However, IFN-alpha also exhibits enhancing effects on T-cell and dendritic cell functions, which may suggest a novel use as a vaccine adjuvant. We have carried out a pilot phase I-II trial to determine the effects of IFN-alpha, administered as an adjuvant of Melan-A/MART-1:26-35(27L) and gp100:209-217(210M) peptides, on immune responses in stage IV melanoma patients. In five of the seven evaluable patients, a consistent enhancement of CD8(+) T cells recognizing modified and native MART-1 and gp100 peptides and MART-1(+)gp100(+) melanoma cells was observed. Moreover, vaccination induced an increase in CD8(+) T-cell binding to HLA tetramers containing the relevant peptides and an increased frequency of CD45RA(+)CCR7(-) (terminally differentiated effectors) and CD45RA(-)CCR7(-) (effector memory) cells. In all patients, treatment augmented significantly the percentage of CD14(+) monocytes and particularly of the CD14(+)CD16(+) cell fraction. An increased expression of CD40 and CD86 costimulatory molecules in monocytes was also observed. Notably, postvaccination monocytes from two of the three patients showing stable disease or long disease-free survival showed an enhanced antigen-presenting cell function and capability to secrete IP10/CXCL10 when tested in mixed leukocyte reaction assays, associated to a boost of antigen and melanoma-specific CD8(+) T cells. Although further clinical studies are needed to show the adjuvant activity of IFN-alpha, the present data represent an important starting point for considering a new clinical use of IFN-alpha and new immunologic end points, potentially predictive of clinical response.

Topics: Adjuvants, Immunologic; Antigen Presentation; Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Dendritic Cells; gp100 Melanoma Antigen; HLA-A Antigens; HLA-A2 Antigen; Humans; Immunophenotyping; Interferon-alpha; Lymphocyte Activation; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monocytes; Neoplasm Proteins; Neoplasm Staging; Pilot Projects

Therapeutic peptide vaccines for melanoma continue to only demonstrate anecdotal success. We set out to evaluate the impact of low-dose GM-CSF emulsified in Montanide ISA-51 on the immunogenicity of HLA-A2 restricted melanoma differentiation antigen peptide vaccines (MART-1, gp100 and tyrosinase) administered in separate subcutaneous injections.. We conducted a randomized phase II clinical trial of HLA-A2+ patients with metastatic melanoma that were immunized every 3 weeks with one of the following vaccine preparations: (A) peptides + Montanide ISA-51; (B) peptides + Montanide ISA-51 + GM-CSF (10 microg); (C) peptides + Montanide ISA-51 + GM-CSF (50 microg). Immunization efficacy was determined by quantification of vaccine specific tetramer positive cytotoxic T cells in peripheral blood. Global assessment of immune competence was ascertained using DTH testing to common recall antigens as well as peripheral blood immunophenotyping.. Twenty-five eligible patients were equally distributed across all 3 treatment groups. Only 9 patients demonstrated evidence of immunization. Most commonly, immune response was achieved to the gp100 peptide. The addition of low-dose GM-CSF did not impact immunization efficacy. DTH reactivity to Candida appeared predictive of successful immunization. Successful immunization with the peptide vaccines was associated with improved clinical outcomes.. The addition of low dose GM-CSF to peptide vaccines did not enhance immunogenicity. Higher doses of GM-CSF may be needed to achieve this effect and this is a testable hypothesis. Likewise, better patient selection based on immunologic status (DTH reactivity) may be helpful to better understand the clinical impact of therapeutic cancer vaccines.

Topics: Adjuvants, Immunologic; Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Cancer Vaccines; Female; gp100 Melanoma Antigen; Granulocyte-Macrophage Colony-Stimulating Factor; HLA-A2 Antigen; Humans; Hypersensitivity, Delayed; Immunophenotyping; Male; Mannitol; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Oleic Acids; Recombinant Proteins; Skin Neoplasms; Survival Analysis; Vaccines, Subunit

Through the adoptive transfer of lymphocytes after host immunodepletion, it is possible to mediate objective cancer regression in human patients with metastatic melanoma. However, the generation of tumor-specific T cells in this mode of immunotherapy is often limiting. Here we report the ability to specifically confer tumor recognition by autologous lymphocytes from peripheral blood by using a retrovirus that encodes a T cell receptor. Adoptive transfer of these transduced cells in 15 patients resulted in durable engraftment at levels exceeding 10% of peripheral blood lymphocytes for at least 2 months after the infusion. We observed high sustained levels of circulating, engineered cells at 1 year after infusion in two patients who both demonstrated objective regression of metastatic melanoma lesions. This study suggests the therapeutic potential of genetically engineered cells for the biologic therapy of cancer.

Topics: Adoptive Transfer; Adult; Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cells, Cultured; Electroporation; Female; Genetic Engineering; Genetic Therapy; HLA-A Antigens; HLA-A2 Antigen; Humans; Interleukin-2; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Receptors, Antigen, T-Cell, alpha-beta; Transduction, Genetic; Transgenes

The adoptive transfer of in vitro generated tumor antigen-specific cytotoxic T lymphocytes (CTL) provides a promising approach to the immunotherapy of cancer. A phase I study was conducted to test the feasibility, safety, and survival of adoptively transferred Melan-A-specific CTL lines in melanoma patients.. Eleven HLA-A2+ patients with metastatic melanoma received at least three intravenous infusions of Melan-A-specific CTL at 2-week intervals. CTL were generated by four rounds of in vitro stimulation of purified CD8+ peripheral blood lymphocytes with autologous dendritic cells pulsed with an HLA-A2 binding Melan-A peptide. Each T-cell infusion was accompanied by a 6-day course of low-dose interleukin-2.. A total of 52 T-cell infusions were administered, averaging 2.1 x 10(8) Melan-A-specific CTL per infusion. Clinical adverse effects were mild and consisted of chills and low-grade fever in seven of 11 patients. Clinical and immunologic responses revealed an antitumor response in three of 11 patients (one complete regression, one partial regression, one mixed response), an elevated frequency of circulating Melan-A tetramer+ T cells up to 2 weeks in all the patients with a maximal frequency of 2% of total CD8+ T cells, an increase in eosinophils to up to 50% in seven of 11 patients, and a selective loss of Melan-A expression in lymph node metastases in two evaluated patients after T-cell transfer.. Our data indicate that the adoptive transfer of antigen-specific T cells in melanoma patients can induce clinical tumor-specific immune responses without major adverse effects.

Topics: Adult; Aged; Antigens, Neoplasm; Biomarkers, Tumor; CD8-Positive T-Lymphocytes; Feasibility Studies; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Immunotherapy, Adoptive; Lymph Nodes; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Positron-Emission Tomography; Skin Neoplasms; Survival Analysis; T-Lymphocytes, Cytotoxic; Tomography, X-Ray Computed; Treatment Outcome

A phase I/II trial was conducted to evaluate clinical and immunologic responses after intralymphatic and intranodal injections of mature dendritic cells.. Fourteen patients with a metastatic melanoma received matured dendritic cells, loaded with Melan-A/MART-1 and/or NA17-A peptides and keyhole limpet hemocyanin. The cells were matured overnight with Ribomunyl, a toll-like receptor ligand, and IFN-gamma, which ensured the production of high levels of interleukin-12p70. Dendritic cells were injected at monthly intervals, first into an afferent lymphatic and then twice intranodally. Immunologic responses were monitored by tetramer staining of circulating CD8(+) lymphocytes and delayed-type hypersensitivity tests.. Dendritic cell vaccination induced delayed-type hypersensitivity reactivity toward NA17-A-pulsed, keyhole limpet hemocyanin-pulsed, and Melan-A-pulsed dendritic cells in 6 of 10, 4 of 11, and 3 of 9 patients, respectively. Four of the 12 patients analyzed by tetramer staining showed a significantly increased frequency of Melan-A-specific T cells, including one patient vaccinated only with NA17-A-pulsed dendritic cells. Furthermore, 2 of the 12 analyzed patients had a significant increase of NA17-A-specific T cells, including one immunized after an optional additional treatment course. No objective clinical response was observed. Two patients were stabilized at 4 and 10 months and three patients are still alive at 30, 39, and 48 months.. Injections into the lymphatic system of mature peptide-loaded dendritic cells with potential TH1 polarization capacities did not result in marked clinical results, despite immunologic responses in some patients. This highlights the need to improve our understanding of dendritic cell physiology.

Topics: Adult; Aged; Aged, 80 and over; Antibody Formation; Antigens, Neoplasm; Cancer Vaccines; Dendritic Cells; Disease-Free Survival; Female; Humans; Immunity, Cellular; Immunotherapy, Adoptive; Injections, Intralymphatic; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Peptides; Skin Neoplasms; Survival Analysis; Treatment Outcome

Previous studies in small groups of patients suggested that immunization of melanoma patients with peptide epitopes recognized by T cells could induce regression of melanoma. This approach was tested in 36 patients with stage IV melanoma. The (MHC class I-restricted) peptides were from gp100, MART-1, tyrosinase, and MAGE-3. The gp100 and MART-1 peptides had been modified to increase their immunogenicity. In half the patients (groups 3 and 4) the peptides were given in the adjuvant Montanide-ISA-720, and half the patients in both groups were given GM-CSF s.c. for 4 days following each injection. Treatment was well tolerated except for two severe erythematous responses to Montanide-ISA-720 and marked inflammatory responses at sites of GM-CSF administration in three patients. There were no objective clinical responses but stabilization of disease for periods from 3 to 12 months were seen in seven patients. Five of these were patients given the peptides in Montanide-ISA-720. Delayed-type hypersensitivity (DTH) skin test responses were also seen mainly in the patients given the peptides in Montanide-ISA-720. GM-CSF did not increase DTH responses in patients in the latter group but may have increased DTH responses in those not given peptides in Montanide-ISA-720. Inflammatory responses around s.c. metastases or regional lymph nodes were observed in two patients. These results suggest that the peptides are more effective when given in the adjuvant Montanide-ISA-720. Nevertheless, results from this study, together with those from a number of comparable studies, indicate that peptide vaccines are currently of minimal benefit to patients and support the need for ongoing development of new strategies in treatment of this disease.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Cytokines; Enzyme-Linked Immunosorbent Assay; Epitopes; Epitopes, T-Lymphocyte; Female; gp100 Melanoma Antigen; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Hypersensitivity, Delayed; Immunity, Cellular; Immunotherapy; Inflammation; Interferon-gamma; Lymphocytes; Male; Mannitol; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Oleic Acids; Peptides; Skin Neoplasms; Time Factors; Treatment Outcome

The induction of potent CD8+ T cell responses by vaccines to fight microbes or tumors remains a major challenge, as many candidates for human vaccines have proved to be poorly immunogenic. Deoxycytidyl-deoxyguanosin oligodeoxynucleotides (CpG ODNs) trigger Toll-like receptor 9, resulting in dendritic cell maturation that can enhance immunogenicity of peptide-based vaccines in mice. We tested whether a synthetic ODN, CpG 7909, could improve human tumor antigen-specific CD8+ T cell responses. Eight HLA-A2+ melanoma patients received 4 monthly vaccinations of low-dose CpG 7909 mixed with melanoma antigen A (Melan-A; identical to MART-1) analog peptide and incomplete Freund's adjuvant. All patients exhibited rapid and strong antigen-specific T cell responses: the frequency of Melan-A-specific T cells reached over 3% of circulating CD8+ T cells. This was one order of magnitude higher than the frequency seen in 8 control patients treated similarly but without CpG and 1-3 orders of magnitude higher than that seen in previous studies with synthetic vaccines. The enhanced T cell populations consisted primarily of effector memory cells, which in part secreted IFN- and expressed granzyme B and perforin ex vivo. In vitro, T cell clones recognized and killed melanoma cells in an antigen-specific manner. Thus, CpG 7909 is an efficient vaccine adjuvant that promotes strong antigen-specific CD8+ T cell responses in humans.

Topics: Animals; Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Freund's Adjuvant; HLA-A2 Antigen; Humans; Interferon-gamma; Lipids; Lymphocyte Activation; MART-1 Antigen; Melanoma; Neoplasm Proteins; Oligodeoxyribonucleotides; Quality Control; Vaccination

Plasmid DNA encoding human interleukin 12 (IL-12) was produced under GMP conditions and injected into lesions of nine patients with malignant melanoma (stage IV) previously treated with both standard and nonstandard therapies. The treatment was based on efficacy in preclinical studies with melanoma in mice and gray horses. The DNA was applied in cycles, three injections per cycle, for up to seven cycles. Three therapy arms comprised low (2 mg), medium (4 mg), and high (10 to 20 mg) amounts of total DNA. The therapy was well tolerated. Three of nine patients experienced a clinical response: two stable disease and one complete remission. One patient receiving a low dose of DNA experienced a long-lasting stabilization of the disease for more than 3 years, whereas the other two responders received high doses of DNA. All patients but one (patient 9) experienced a transient response at the intratumoral injection site. Immunohistochemical staining of responder sections showed local reduction of angiogenesis and lymphocyte infiltrations. All patients, in particular the clinical and local responders (patients 3, 7, and 8), exhibited an antigen-specific immune response against MAGE-1 and MART-1, which in some cases preexisted. Biopsies of responders showed some increase in IL-12, IP-10, and IFN-(). Serum levels revealed fluctuations. The results show that intratumoral injection of DNA produced some beneficial clinical effect. DNA encoding a cytokine may be useful as a therapeutic or adjuvant against various human cancers.

Topics: Aged; Aged, 80 and over; Antigens, Neoplasm; DNA; Female; Genetic Therapy; Humans; Immunotherapy; Injections, Intralesional; Interferon-gamma; Interleukin-12; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasm Proteins; Plasmids; Skin Neoplasms

Detection of micrometastases in the regional tumor-draining lymph nodes is critical for staging and prognosis in melanoma patients. We applied a quantitative multiple-marker RT-PCR assay to improve the detection of occult melanoma cells in the sentinel lymph node (SLN). From 139 patients with primary cutaneous melanoma who underwent sentinel lymphadenectomy, a total of 235 SLN were assessed for Melan-A and tyrosinase expression by real-time quantitative RT-PCR. Twenty-three patients (17%) were positive by histopathology and expressed messenger RNA of one or two markers. Of the patients with histopathologically negative SLN 39 (28%) were reclassified by positive RT-PCR. Patients were examined for tumor recurrences during a median follow-up period of 29 mo. Tumor recurrences were demonstrated in eight patients (35%) with histopathologically positive SLN, in four patients (10%) with submicroscopic tumor cells detected exclusively by real-time RT-PCR, and in none of the patients negative by both methods. The differences in recurrence rates were statistically significant (p=0.01). These data indicate that real-time quantitative RT-PCR for the detection of minimal residual melanoma in SLN improves the prediction of disease-free survival.

Topics: Antigens, Neoplasm; Disease-Free Survival; Follow-Up Studies; Genetic Testing; Humans; Lymph Node Excision; Lymphatic Metastasis; MART-1 Antigen; Melanoma; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Recurrence, Local; Neoplasm, Residual; Predictive Value of Tests; Reverse Transcriptase Polymerase Chain Reaction; Risk Factors; Sentinel Lymph Node Biopsy; Skin Neoplasms

Immunotherapy for the treatment of metastatic melanoma remains a major clinical challenge. The melanoma microenvironment may lead to local T cell tolerance in part through downregulation of costimulatory molecules, such as B7.1 (CD80). We report the results from the first clinical trial, to our knowledge, using a recombinant vaccinia virus expressing B7.1 (rV-B7.1) for monthly intralesional vaccination of accessible melanoma lesions. A standard 2-dose-escalation phase I clinical trial was conducted with 12 patients. The approach was well tolerated with only low-grade fever, myalgias, and fatigue reported and 2 patients experiencing vitiligo. An objective partial response was observed in 1 patient and disease stabilization in 2 patients, 1 of whom is alive without disease 59 months following vaccination. All patients demonstrated an increase in postvaccination antibody and T cell responses against vaccinia virus. Systemic immunity was tested in HLA-A*0201 patients who demonstrated an increased frequency of gp100 and T cells specific to melanoma antigen recognized by T cells 1 (MART-1), also known as Melan-A, by ELISPOT assay following local rV-B7.1 vaccination. Local immunity was evaluated by quantitative real-time RT-PCR, which suggested that tumor regression was associated with increased expression of CD8 and IFN-gamma. The local delivery of vaccinia virus expressing B7.1 was well tolerated and represents an innovative strategy for altering the local tumor microenvironment in patients with melanoma.

Topics: Adult; Aged; Antigens, Neoplasm; B7-1 Antigen; CD8 Antigens; Female; Gene Expression; Genetic Therapy; HLA-A Antigens; HLA-A2 Antigen; Humans; Immunotherapy; Injections, Intralesional; Interferon-gamma; Interleukin-10; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; T-Lymphocytes; Vaccinia virus

Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) blockade with CP-675,206, a fully human anti-CTLA4 monoclonal antibody, may break peripheral immunologic tolerance leading to effective immune responses to cancer in humans. A phase I trial was conducted to test the safety of CP-675,206.. Thirty-nine patients with solid malignancies (melanoma, n = 34; renal cell, n = 4; colon, n = 1) received an intravenous (IV) infusion of CP-675,206 at seven dose levels. The primary objective was to determine the maximum-tolerated dose and the recommended phase II dose.. Dose-limiting toxicities and autoimmune phenomena included diarrhea, dermatitis, vitiligo, panhypopituitarism and hyperthyroidism. Two patients experienced complete responses (maintained for 34+ and 25+ months), and there were two partial responses (26+ and 25+ months) among 29 patients with measurable melanoma. There have been no relapses thus far after objective response to therapy. Four other patients had stable disease at end of study evaluation (16, 7, 7, and 4 months). Additionally, five patients had extended periods without disease progression (36+, 35+, 26+, 24+, and 23+ months) after local treatment of progressive metastases. Longer systemic exposure to CP-675,206 achieved in higher dose cohorts predicted for a higher probability of response.. CP-675,206 can be administered safely to humans as a single IV dose up to 15 mg/kg, resulting in breaking of peripheral immune tolerance to self-tissues and antitumor activity in melanoma.

Topics: Adult; Aged; Antibodies, Blocking; Antibodies, Monoclonal; Antibodies, Neoplasm; Antigens, CD; Antigens, Differentiation; Antigens, Neoplasm; Autoimmune Diseases; Cancer Vaccines; Colonic Neoplasms; CTLA-4 Antigen; Female; Humans; Immune Tolerance; Immunotherapy; Infusions, Intravenous; Kidney Neoplasms; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Neoplasms; Regression Analysis; T-Lymphocyte Subsets; Treatment Outcome

Previous studies have suggested that immunotherapy with dendritic cell (DC) vaccines may be effective in treatment of patients with AJCC stage IV melanoma. We examined this treatment in phase I/II studies in 33 patients with good performance status and low volume disease. Nineteen patients received DCs plus autologous lysates and 14 patients DCs plus peptides from the melanoma antigens MAGE-3.A2, tyrosinase, gp100, and MART-1. Keyhole limpet hemocyanin (KLH) was used as a helper protein and influenza peptide was given as a positive control. DCs were produced from adherent cells in blood lymphocytes (monocytic DCs), grown in IL-4 and GM-CSF without a maturation step. The DCs were injected into inguinal lymph nodes at weekly intervals (x4), 2 weeks (x1), and 4-weekly intervals (x2). There were 3 responses (3 partial responses) and 1 mixed response in the 19 patients treated with DCs plus autologous lysates. No responses were seen in the group treated with DCs plus peptides. Stable disease (defined as no progression over a period of 3 months) was seen in 4 patients in group 1 and 5 patients in group 2. Treatment was not associated with significant side effects. We examined whether DTH skin tests or assays of IFN-gamma cytokine production may be useful predictors of clinical responses. Twenty-two of 30 patients had DTH responses to KLH and 12 of 13 patients had DTH responses to the influenza peptide. Five of 15 DTH responses were seen against autologous lysates. This was strongly correlated with clinical responses. Approximately half the patients had responses to MART-1 peptide and a third to the other melanoma peptides. Similarly, cytokine production assays showed responses to influenza in 7 of 13 patients, and approximately one third of patients had responses to the other peptides. No IFN-gamma responses were seen in 5 patients against their autologous lysates. There was no correlation between assays of IFN-gamma production and clinical responses. The present studies suggest that autologous lysates may be more effective than the melanoma peptides used in the study as the source of antigen for DC vaccines. DTH responses to autologous lysates appear useful predictors of clinical responses, but further work is needed to identify other measures associated with clinical responses.

Topics: Adult; Aged; Antigens, Neoplasm; Cancer Vaccines; Dendritic Cells; Female; Hemocyanins; HLA-A2 Antigen; Humans; Hypersensitivity, Delayed; Interferon-gamma; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Peptide Fragments; Skin Neoplasms; T-Lymphocytes; T-Lymphocytes, Cytotoxic; Treatment Outcome; Vaccination

The purpose of this study was to test melanoma vaccines consisting of peptides and immunological adjuvants for optimal immunogenicity and to evaluate laboratory immune monitoring for in vivo relevance. Forty-nine HLA-A2 positive patients with Melan-A positive melanoma were repeatedly vaccinated with Melan-A peptide, with or without immune adjuvant AS02B (QS21 and MPL) or IFA. Peptide-specific CD8 T cells in PBLs were analyzed ex vivo using fluorescent HLA-A2/Melan-A multimers and IFN-gamma ELISPOT assays. The vaccines were well tolerated. In vivo expansion of Melan-A-specific CD8 T cells was observed in 13 patients (1/12 after vaccination with peptide in AS02B and 12/17 after vaccination with peptide in IFA). The T cells produced IFN-gamma and downregulated CD45RA and CD28. T-cell responses correlated with inflammatory skin reactions at vaccine injection sites (P < 0.001) and with DTH reaction to Melan-A peptide (P < 0.01). Twenty-six of 32 evaluable patients showed progressive disease, whereas 4 patients had stable disease. The two patients with the strongest Melan-A-specific T-cell responses experienced regression of metastases in skin, lymph nodes, and lung. We conclude that repeated vaccination with Melan-A peptide in IFA frequently leads to sustained responses of specific CD8 T cells that are detectable ex vivo and correlate with inflammatory skin reactions.

Topics: Adolescent; Adult; Aged; Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Dermatitis; Disease Progression; Female; Freund's Adjuvant; Humans; Lipids; Lymphocyte Activation; Male; MART-1 Antigen; Melanoma; Middle Aged; Monitoring, Immunologic; Neoplasm Proteins; Peptides; Viral Matrix Proteins

The importance of CD8(+) cytolytic T cells for protection from viral infection and in the generation of immune responses against tumors has been well established. In contrast, the role of CD4(+) T-helper cells in human infection and in cancer immunity has yet to be clearly defined. In this pilot study, we show that immunization of three resected, high-risk metastatic melanoma patients with a T-helper epitope derived from the melanoma differentiation antigen, melanoma antigen recognized by T cells-1, results in CD4(+) T-cell immune responses. Immune reactivity to that epitope was detected by DR4-peptide tetramer staining, and enzyme-linked immunospot assay of fresh and restimulated CD4(+) T cells from patients over the course of the 12-month vaccine regimen. The postvaccine CD4(+) T cells exhibited a mixed T-helper 1/T-helper 2 phenotype, proliferated in response to the antigen and promiscuously recognized the peptide epitope bound to different human leukocyte antigen-DRbeta alleles. For 1 DRbeta1*0401(+) patient, antigen-specific CD4(+) T cells recognized human leukocyte antigen-matched antigen-expressing tumor cells, secreted granzyme B, and also exhibited cytolysis that was MHC class II-restricted. These data establish the immunogenicity of a class II epitope derived from a melanoma-associated antigen and support the inclusion of class II peptides in future melanoma vaccine therapies.

Topics: Age Factors; Alleles; Antigens, Neoplasm; Cancer Vaccines; CD3 Complex; CD4 Antigens; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cell Proliferation; Dendritic Cells; Enzyme-Linked Immunosorbent Assay; Epitopes; Female; Flow Cytometry; Granzymes; HLA Antigens; HLA-DR Antigens; Humans; Immunotherapy; Leukocytes, Mononuclear; Lipid A; Lymphocytes; Male; MART-1 Antigen; Melanoma; Microscopy, Fluorescence; Neoplasm Proteins; Peptides; Pilot Projects; Saponins; Serine Endopeptidases; T-Lymphocytes; Th1 Cells; Th2 Cells; Time Factors; Treatment Outcome

Dendritic cells (DCs) show promise as adjuvants in anticancer immunotherapeutic strategies. Flt3 ligand (FL) is a hematopoietic growth factor that increases the number of immature DCs in the blood and other tissues. We treated 27 patients with metastatic or high-risk resected melanoma with s.c. FL daily for 14 d in three 28 d cycles. Eighteen of these patients also received vaccination with influenza (Flu), Melan-A (Mel), tyrosinase (Tyr), and NY-ESO-1 peptides. To induce local DC maturation, 8 of the vaccinated patients had imiquimod, a Toll-like receptor-7 ligand (TLR7L), applied topically to their vaccine sites. Patients were monitored for clinical and hematological effects. Immune responses were assessed by cutaneous reactivity to vaccination and by the induction of peptide-specific CD8+ T-cells. Eight patients did not complete the protocol due to adverse events related to their cancer. The treatment was generally safe and well tolerated, although some patients developed clinically significant toxicities related to FL. FL induced increases in immature CD11c+ and CD123+ peripheral blood (PB) DCs. Other hematological effects included monocytosis, granulocytosis, and thrombocytosis, which were marked in some patients. Cutaneous reactions to peptide vaccination and circulating peptide-specific CD8+ T-cells were more frequent in imiquimod-treated patients. FL treatment of melanoma patients has pleiotropic clinical and hematological effects. In vivo maturation of FL-generated DCs using imiquimod may increase immune responses to tumor antigens.

Topics: Adjuvants, Immunologic; Adult; Aged; Aminoquinolines; Antigens, Neoplasm; Antineoplastic Agents; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cytokines; Dendritic Cells; Female; Humans; Imiquimod; Immunotherapy, Active; Male; MART-1 Antigen; Melanoma; Membrane Proteins; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Peptide Fragments

The aim of this study was to evaluate the role of melanoma gene expression as a marker of the presence of melanoma cells in lymphatic drainage routinely collected after lymphadenectomy and to correlate reverse transcriptase-polymerase chain reaction (RT-PCR) assay results with recurrence, survival, and prognostic factors.. We collected 24-hour postoperative lymphatic drainage samples (between days 2 and 4) from 93 patients with stage III melanoma who underwent radical lymphadenectomy between May 2002 and November 2003. We used RT-PCR assays with primers specific for the tyrosinase and MART-1 (Melan-A) genes. The samples were considered positive if at least one marker was expressed. Median follow-up time was 12.8 months.. In 18 (19.4%) of 93 patients, the RT-PCR assay results were positive: in 8 of 18 for tyrosinase only, in 7 of 18 for MART-1 only, and in 3 of 18 for both markers. We observed a significantly higher recurrence rate in patients with positive RT-PCR results (15 of 18; 83%) than negative results (26 of 75; 35%; P = .0001). Positive results of RT-PCR correlated with the number of involved lymph nodes (P = .0001) and extracapsular extension of nodal metastases (P = .03). We observed significant differences in overall and disease-free survival for RT-PCR-positive and -negative patients in univariate and multivariate analyses.. We observed positive RT-PCR assay results for melanoma cells in the lymphatic drainages of approximately 20% of patients after lymphadenectomy. This correlated significantly with early recurrence and shorter survival. These results may suggest that the RT-PCR assay could be useful for routinely analyzing postoperatively collected lymphatic drainage in stage III melanoma patients and for predicting disease progression.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Biomarkers, Tumor; Disease-Free Survival; Female; Gene Expression Profiling; Humans; Lymph Node Excision; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Recurrence, Local; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Skin Neoplasms

Adoptive T cell therapy has been successfully used for treatment of viral and malignant diseases. However, little is known about the fate and trafficking of transferred Ag-specific T cells. Using the tetramer (TM) technology which allows for detection and quantification of Ag-specific CTL, we assessed the frequency of circulating Melan-A-specific CTL in advanced melanoma patients during adoptive T cell therapy. Melan-A-specific CTL were generated from HLA-A2.1(+) patients by in vitro stimulation of CD8(+) T cells with dendritic cells pulsed with a mutated HLA-A2-binding Melan-A (ELAGIGILTV) peptide. Eight patients received three infusions of 0.25-11 x 10(8) Melan-A-specific CTL i.v. at 2-wk intervals along with low-dose IL-2. The transferred T cell product contained a mean of 42.1% Melan-A-TM(+) CTL. Before therapy, the frequencies of Melan-A-specific CTL in patients' circulating CD8(+) T cells ranged from 0.01 to 0.07%. Characterization of the TM frequencies before and at different time points after transfer revealed an increase of circulating Melan-A-specific CTL up to 2%, correlating well with the number of transferred CTL. An elevated frequency of TM(+) T cells was demonstrated up to 14 days after transfer, suggesting long-term survival and/or proliferation of transferred CTL. Combining TM analysis with a flow cytometry-based cytokine secretion assay, unimpaired production of IFN-gamma was demonstrated in vivo for at least 24 h after transfer. Indium-111 labeling of Melan-A-specific CTL demonstrated localization of transferred CTL to metastatic sites as early as 48 h after injection. Overall, the results suggest that in vitro-generated Melan-A-specific CTL survive intact in vivo for several weeks and localize preferentially to tumor.

Topics: Antigens, Neoplasm; Cell Division; Cell Line; Cell Movement; Cell Survival; Epitopes, T-Lymphocyte; Humans; Immunophenotyping; Immunotherapy, Adoptive; Indium Radioisotopes; Infusions, Intravenous; Liver; Lung; Lymphocyte Activation; MART-1 Antigen; Melanoma; Neoplasm Proteins; Pilot Projects; Spleen; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

The melanoma tumor antigen epitope peptides MART-1(26-35 (27L)), gp100(209-217 (210M)),and tyrosinase(368-376 (370D)) were emulsified with incomplete Freund's adjuvant and administered with SD-9427 (progenipoietin), an agonist of granulocyte colony-stimulating factor and the FLT-3 receptor, to evaluate the toxicities of and immune responses to this regimen as primary end points and time to relapse and survival as secondary end points.. Fifteen patients with high-risk resected stage III and IV melanoma were enrolled. Each patient received peptides + incomplete Freund's adjuvant with SD-9427 at doses of either 10, 20, or 40 microg/kg s.c. for 3 days before and 7 days after each vaccination. Immunizations were administered every month for 6 months and then administered once 6 months later. A leukapheresis to obtain peripheral blood mononuclear cells for immune analyses as well as skin testing with peptides and recall antigens was performed before and after vaccination. IFN- gamma release assay, ELISPOT, and MHC-peptide tetramer analysis were performed using peripheral blood mononuclear cells collected before and after vaccination to evaluate peptide-specific cytotoxic T-cell responses.. Local pain and granuloma formation and fatigue of grade I or II were the most common side effects. One patient developed antibody-mediated leukopenia and transient grade III neutropenia that resolved after stopping SD-9427. Six of 12 patients tested developed a positive skin test response to one or more of the peptides. Seven of 10 patients tested demonstrated an immune response to at least one peptide when evaluated by IFN-gamma release assay and ELISPOT assay after vaccination, as did 11 of 12 patients analyzed by MHC-peptide tetramer assay. Four of 15 patients have relapsed with a median follow-up of 20 months, and 1 patient in this high-risk group has died of disease.. SD-9427 with a multipeptide vaccine was generally well tolerated, although one patient developed reversible antibody-mediated neutropenia. These data suggest that the majority of patients with resected melanoma mount an antigen-specific immune response against a multipeptide vaccine administered with SD-9427.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Cancer Vaccines; Colony-Stimulating Factors; Cytokines; Dendritic Cells; Epitopes; Female; Flow Cytometry; gp100 Melanoma Antigen; Humans; Immunohistochemistry; Leukapheresis; Male; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Peptides; Recombinant Proteins; Time Factors; Treatment Outcome; Vaccines; Vaccines, Subunit

Malignant melanoma is one of the most rapidly increasing cancer types, and patients with metastatic disease have a very poor prognosis. Detection of metastatic melanoma cells in circulation may aid the clinician in assessing tumor progression, metastatic potential, and response to therapy. Tyrosinase is a key enzyme in melanin biosynthesis. The gene is actively expressed in melanocytes and melanoma cells. Melan A is a differentiation antigen that is expressed in melanocytes. The presence of these molecules in blood is considered a marker for circulating melanoma cells. In this study, we analyzed the usefulness of this marker combination in evaluating the response to therapy in the blood of 30 patients with malignant melanoma. Circulating cells were detected by a reverse-transcriptase-polymerase-chain reaction. The tyrosinase expression was observed in 9 (30%) patients and Melan A in 19 (63.3%) patients before therapy. Following treatment, the tyrosinase mRNA was detected in only one patient, while Melan A transcripts were still present in 14 patients. We suggest that this molecular assay can identify circulating melanoma cells that express melanoma-associated antigens and may provide an early indication of therapy effectiveness.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Female; Gene Expression Profiling; Humans; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Staging; Neoplastic Cells, Circulating; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Treatment Outcome

Preclinical studies showed that immunization with peripheral blood mononuclear cells (PBMC) loaded with tumor antigen peptides plus interleukin-12 (IL-12) induced CD8+ T-cell responses and tumor rejection. We recently determined that recombinant human (rh) IL-12 at 30 to 100 ng/kg is effective as a vaccine adjuvant in patients. A phase II study of immunization with Melan-A peptide-pulsed PBMC + rhIL-12 was conducted in 20 patients with advanced melanoma.. Patients were HLA-A2-positive and had documented Melan-A expression. Immunization was performed every 3 weeks with clinical re-evaluation every three cycles. Immune responses were measured by ELISpot assay before and after treatment and through the first three cycles, and were correlated with clinical outcome.. Most patients had received prior therapy and had visceral metastases. Nonetheless, two patients achieved a complete response, five patients achieved a minor or mixed response, and four patients had stable disease. The median survival was 12.25 months for all patients and was not yet reached for those with a normal lactate dehydrogenase. There were no grade 3 or 4 toxicities. Measurement of specific CD8+ T-cell responses by direct ex vivo ELISpot revealed a significant increase in interferon gamma-producing T cells against Melan-A (P =.015) after vaccination, but not against an Epstein-Barr virus control peptide (P =.86). There was a correlation between the magnitude of the increase in Melan-A-specific cells and clinical response (P =.046).. This immunization approach may be more straightforward than dendritic cell strategies and seems to have clinical activity that can be correlated to a biologic end point.

Topics: Adult; Aged; Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Female; HLA-A2 Antigen; Humans; Interferon-gamma; Interleukin-12; Leukocytes, Mononuclear; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Metastasis; Neoplasm Proteins; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes; Time Factors; Treatment Outcome

Patients with American Joint Committee on Cancer (AJCC) stage III melanoma are at high risk of recurrence and death. We hypothesized that a multiple-marker reverse transcriptase polymerase chain reaction (MM-RT-PCR) blood assay could predict, early in the course of therapy, those patients destined to experience treatment failure with a melanoma vaccine (MV) previously shown to improve survival in a phase II clinical trial.. After complete surgical resection, prospectively collected cryopreserved peripheral-blood lymphocyte specimens (n = 90) from the serial bleeds of 30 patients with AJCC stage III melanoma were studied by MM-RT-PCR, using the markers tyrosinase, melanoma antigen recognized by T cells-1 (MART-1), and universal melanoma antigen gene-A (uMAG-A). All patients were enrolled in a phase II MV trial during the period of blood draws, and were selected for this study in a blinded fashion. Median duration of clinical follow-up was 74 months for the 13 survivors and 11 months for the 17 nonsurvivors.. The presence of at least one melanoma-specific RT-PCR marker was associated with an increased risk of disease recurrence (risk rate, 3.12; P =.02) and decreased risk of survival (relative rate, 2.62; P =.0496) by multivariate analysis.. MM-RT-PCR of the blood provided early prediction of subsequent disease recurrence and death in clinically disease-free AJCC stage III melanoma patients enrolled in a MV phase II trial. On the basis of the results of this pilot study, the MM-RT-PCR blood assay should be considered as a clinically important monitoring tool for assessing patient response to adjuvant therapy, and in the surveillance of clinically disease-free patients for the earliest signs of recurrence.

Topics: Adult; Aged; Antigens, Neoplasm; Biomarkers, Tumor; Cancer Vaccines; Female; Humans; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Multivariate Analysis; Neoplasm Proteins; Neoplasm Recurrence, Local; Predictive Value of Tests; Reverse Transcriptase Polymerase Chain Reaction; Risk Factors; Skin Neoplasms; Survival; Treatment Outcome

Preclinical studies have shown that low dose IL-12 can potentiate cytotoxic lymphocyte responses. Since previous trials have demonstrated significant toxicity from high dose recombinant human IL-12 (rhIL-12), we sought to determine an optimal biological dose for rhIL-12 at lower doses when combined with peptide antigens. Two studies were undertaken. The rhIL-12 was administered at doses of 0 (placebo), 10, 30 and 100 ng/kg, subcutaneously in one study and intravenously in the other. Apart from IL-12 dosing, the studies were identical. Subjects had evaluable stage III or IV melanoma which expressed Melan-A by RT-PCR or immunohistochemistry. Melan-A (26-35) (EAAGIGILTV) and influenza matrix (58-66) (GILGFVFTL) peptides were administered intradermally on weeks 1, 2, 3, 4 and 9. Twenty-eight subjects were enrolled, of whom 24 were evaluable for clinical and immunological responses. Therapy was well tolerated, the main adverse event being influenza-like symptoms. Immunological monitoring included the evaluation of cutaneous reactions and assays for antigen-specific T-cells. Clinical responses included a complete response in a subject with small volume subcutaneous disease, a partial response in a subject with hepatic metastases, and mixed responses in pulmonary, pleural and nodal disease. Biopsies of accessible tumors showed infiltration with CD4+ and CD8+ lymphocytes capable of lysing Melan-A peptide-pulsed targets in vitro. No clear dose-dependent effect of rhIL-12 could be determined. The rhIL-12 given either s.c. or i.v. was well tolerated at doses of 10-100 ng/kg. Clinical and immunological activity has been observed in this study where peptides were administered either with or without low dose rhIL-12.

Topics: Adjuvants, Immunologic; Adolescent; Adult; Aged; Antigens, Neoplasm; Cancer Vaccines; Drug Administration Schedule; Drug Hypersensitivity; Drug Therapy, Combination; Female; Humans; Influenza A virus; Injections, Intravenous; Injections, Subcutaneous; Interleukin-12; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Peptide Fragments; Recombinant Proteins; Viral Matrix Proteins

We performed a phase I/II clinical trial in metastatic melanoma patients with an ultraviolet (UV)-inactivated nonreplicating recombinant vaccinia virus enabling the expression, from a single construct, of endoplasmic reticulum-targeted HLA-A0201-restricted Melan-A/MART-1(27-35), gp100(280-288), and tyrosinase(1-9) epitopes, together with CD80 and CD86 costimulatory proteins. Corresponding soluble peptides were used to boost responses and granulocyte-macrophage colony-stimulating factor was used as systemic adjuvant. Safety and immunogenicity, as monitored with in vitro-restimulated peripheral blood mononuclear cells by cytotoxic T lymphocyte precursor (CTLp) frequency analysis and tetramer staining, were specifically addressed. Of 20 patients entering the protocol, 2 had to withdraw because of rapidly progressing disease. Immune responses were evaluated in 18 patients (stage III, n = 5; stage IV, n = 13) and increases in specific CTLp frequencies were observed in 15. In 16 patients responsiveness against all 3 antigens could be analyzed: 7 (43%), including all stage III cases, showed evidence of induction of CTLs specific for the three epitopes, and 2 (12%) and 4 (25%), respectively, showed reactivity against two or one tumor-associated antigen. In three stage IV patients no specific CTL reactivity could be induced. Increases in CTLp frequency were detected mostly after viral vaccine injections. However, in a majority of patients final CTLp levels were comparable to initial levels. Tetramer characterization of Melan-A/MART-1(27-35)-specific CTLs during the protocol also suggested preferential expansion after recombinant virus administration. Vector-specific humoral responses, frequently undetectable in stage IV patients, did not appear to prevent tumor-associated antigen-specific CTL induction. Aside from a single occurrence of transient grade 3 leukopenia, no major clinical toxicity was reported. Seventeen of 18 patients completed the 3-month trial (one patient died before the last delayed-type hypersensitivity test). Three displayed regression of individual metastases, seven had stable disease, and progressive disease was observed in seven patients. This is the first report on the administration of a UV-inactivated recombinant vaccinia virus coexpressing five transgenes in cancer patients. The results described here, in terms of safety and immunogenicity, support the use of this reagent in active specific immunotherapy.

Topics: Adult; Aged; Antigens, CD; Antigens, Neoplasm; B7-1 Antigen; B7-2 Antigen; Cancer Vaccines; Defective Viruses; Epitopes; Female; Follow-Up Studies; Genetic Vectors; HLA-A Antigens; Humans; Male; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Middle Aged; Neoplasm Proteins; T-Lymphocytes, Cytotoxic; Vaccines, Synthetic; Vaccinia virus

We report here the adoptive transfer, to patients with metastatic melanoma, of highly selected tumor-reactive T cells directed against overexpressed self-derived differentiation antigens after a nonmyeloablative conditioning regimen. This approach resulted in the persistent clonal repopulation of T cells in those cancer patients, with the transferred cells proliferating in vivo, displaying functional activity, and trafficking to tumor sites. This led to regression of the patients' metastatic melanoma as well as to the onset of autoimmune melanocyte destruction. This approach presents new possibilities for the treatment of patients with cancer as well as patients with human immunodeficiency virus-related acquired immunodeficiency syndrome and other infectious diseases.

Topics: Adolescent; Adult; Antigens, Neoplasm; Autoimmunity; CD8-Positive T-Lymphocytes; Clone Cells; Cytokines; Female; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; HLA-A2 Antigen; Humans; Immunotherapy, Adoptive; Interleukin-2; Lymphocyte Count; Lymphocyte Depletion; Lymphocytes, Tumor-Infiltrating; Male; MART-1 Antigen; Melanocytes; Melanoma; Middle Aged; Neoplasm Proteins; Receptors, Antigen, T-Cell, alpha-beta; T-Lymphocytes; Treatment Outcome

Adoptive T cell therapy, involving the ex vivo selection and expansion of antigen-specific T cell clones, provides a means of augmenting antigen-specific immunity without the in vivo constraints that can accompany vaccine-based strategies. A phase I study was performed to evaluate the safety, in vivo persistence, and efficacy of adoptively transferred CD8+ T cell clones targeting the tumor-associated antigens, MART1MelanA and gp100 for the treatment of patients with metastatic melanoma. Four infusions of autologous T cell clones were administered, the first without IL-2 and subsequent infusions with low-dose IL-2 (at 0.25, 0.50, and 1.0 x 10(6) unitsm(2) twice daily for the second, third, and fourth infusions, respectively). Forty-three infusions of MART1MelanA-specific or gp100-specific CD8+ T cell clones were administered to 10 patients. No serious toxicity was observed. We demonstrate that the adoptively transferred T cell clones persist in vivo in response to low-dose IL-2, preferentially localize to tumor sites and mediate an antigen-specific immune response characterized by the elimination of antigen-positive tumor cells, regression of individual metastases, and minor, mixed or stable responses in 8 of 10 patients with refractory, metastatic disease for up to 21 mo.

Topics: Adult; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Cell Movement; Clone Cells; Female; gp100 Melanoma Antigen; Graft Survival; Humans; Immunotherapy, Adoptive; Interleukin-2; Male; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Recurrence; Safety; T-Lymphocyte Subsets; Treatment Outcome

Circulating malignant cells in peripheral blood are thought to be precursors and surrogate markers of distant metastases and hence markers of a poor clinical outcome. In this study, we used the detection of MART-1 and tyrosinase (TYR) mRNA with a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay to identify circulating melanoma cells. Blood samples were obtained from 35 patients with metastatic melanoma before, during and after treatment with interleukin-2, interferon-alpha and cisplatin. In addition, MART-1 and TYR protein was identified by immunohistochemistry in consecutive biopsies from 15 of the patients. Analysis of three daily blood samples for 3 days demonstrated that four out of 11 patients examined were negative for both markers on all occasions, and two patients were positive for both markers on all occasions but one. The remaining five patients showed sporadic low positive results for one or the other of the two markers. By comparing the immunohistochemistry results from consecutive biopsies with the RT-PCR results, we demonstrated that patients with MART-1 and TYR protein in their tumour cells had circulating MART-1 and TYR mRNA in 77% and 54% of the cases, respectively. During treatment, the majority of patients who were positive for MART-1 and TYR mRNA converted to being negative. However, these conversions did not significantly correlate with objective response. The presence of TYR mRNA in one of the first two samples showed a trend towards being an independent prognostic factor for poor survival.

Topics: Adult; Aged; Antigens, Neoplasm; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Cisplatin; Female; Humans; Immunotherapy; Interferon alpha-2; Interferon-alpha; Interleukin-2; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Multivariate Analysis; Neoplasm Proteins; Neoplastic Cells, Circulating; Predictive Value of Tests; Prognosis; Recombinant Proteins; Reference Values; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skin Neoplasms

Vaccination with dendritic cells (DCs) pulsed with tumor antigen peptides has shown promise in the treatment of melanoma. Interleukin (IL)-12 production by DCs is a key component for their efficacy. Murine studies have shown that IL-12 promotes potent antitumor immunization when coadministered with peptides loaded onto other class I MHC+ cells, thus bypassing the need to use DCs. The easiest cell source to obtain in large quantity from human patients is peripheral blood mononuclear cells (PBMCs). A Phase I clinical trial was thus performed in patients with metastatic melanoma using immunization with autologous PBMCs pulsed with a MAGE-3 or a MelanA peptide, coadministered with various doses of recombinant human (rh)IL-12. Patients receiving low-to-moderate doses of rhIL-12 developed increased specific CD8+ T-cell responses. Of the eight patients showing increased immunity, six had evidence of clinical activity, with one complete, one partial, one minor, and three mixed responses observed. In two patients with mixed responses, growing tumors were found to lack expression of the antigen used to immunize. Thus, vaccination with peptide-pulsed PBMCs plus rhIL-12 induces specific immunity and has clinical activity, without the need to generate DCs. Outgrowth of antigen-negative tumors argues for the future development of polyepitope vaccines.

Topics: Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cells, Cultured; Epitopes; Female; HLA-A2 Antigen; Humans; Interferon-gamma; Interleukin-12; Leukocytes, Mononuclear; Male; MART-1 Antigen; Melanoma; Neoplasm Metastasis; Neoplasm Proteins; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes; Time Factors

We characterized the HLA class I alterations in five metastases obtained from two patients with melanoma immunized with Melan A/MART-1, tyrosinase and gp100 tumor peptides. All three metastases analyzed in the first patient (NW145) showed a similar HLA class I alteration with a dual population of melanoma cells. One population was HLA class I antigen positive and the other had loss of heterozygosity (LOH) in the short arm of chromosome 6 leading to an HLA haplotype loss (A02011, B4007, Cw1). The absence of HLA-A2 antigen may explain why this patient did not develop HLA-A2 restricted, Melan A/MART-1 specificity immunization, since this HLA molecule is the restriction element for the tumor peptides used. However, this HLA-deficient population was not selected after peptide immunotherapy. The primary tumor in this patient presented LOH in region 6q, but only in the vertical growth phase of the lesion, whereas LOH at 6p was observed only in DNA from metastatic material. The second patient (NW16) also presented two metastatic lesions with an identical HLA molecular defect, i.e. HLA B locus downregulation (HLA B51011: serological B51; B1503: serological B70). One lesion expressed the tumor antigen (Melan A/ MART-1), but the other did not. Interestingly, the antigen-positive metastasis regressed after peptide immunotherapy, whereas the other progressed rapidly. These findings provide the first indication that multiple metastases generated in the same host can have identically altered HLA class I phenotypes.

Topics: Aged; Antigens, Neoplasm; Chromosomes, Human, Pair 6; Fatal Outcome; Female; Genes, MHC Class I; gp100 Melanoma Antigen; Haplotypes; Humans; Immunotherapy, Active; Loss of Heterozygosity; Male; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins

Thirty-three metastatic melanoma patients were vaccinated according to a phase I-II study with an allogeneic melanoma cell line that was genetically modified by transfection with a plasmid containing the gene encoding human interleukin 2 (IL-2). The cell line expresses the major melanoma-associated antigens and the HLA class I alleles HLA-A1, -A2, -B8, and Cw7. All patients shared one or more HLA class I alleles with this cell line vaccine. Patients were immunized by three vaccinations, each consisting of 60 x 106 irradiated (100 Gy) melanoma cells (secreting 120 ng of IL-2/10(6) cells/24 hr) administered subcutaneously at weekly intervals for 3 consecutive weeks. Side effects of treatment consisted of swelling of locoregional lymph nodes and induration at the site of injection, i.e., a delayed-type hypersensitivity (DTH) reaction. In three patients, vaccination induced inflammatory responses in distant metastases containing necrosis or apoptosis along with T cell infiltration. Apoptosis occurred only in Bcl-2-negative areas, not in Bcl-2-expressing parts of the metastases. Two other patients experienced complete or partial regression of subcutaneous metastases. Seven patients had protracted stabilization (4 to >46 months) of soft tissue metastases, including one patient who developed vitiligo after vaccination. Immune responses to the vaccine could be detected in 67% of the 27 patients measured. Vaccination was shown to induce a variable change in the number of anti-vaccine cytotoxic T lymphocytes (CTLs) in peripheral blood, which did not correlate with response to treatment. However, in two of five patients the frequency of anti-autologous tumor CTLs measured was significantly higher than before vaccination. This study demonstrates the feasibility, safety, and therapeutic potential of vaccination of humans with allogeneic, gene-modified tumor cells, and that frequencies of vaccine-specific CTLs among patient lymphocytes can be determined by using a modified limited dilution analysis (LDA).

Topics: Adult; Aged; Antigens, Neoplasm; Cancer Vaccines; Female; HLA-A1 Antigen; HLA-A2 Antigen; HLA-B8 Antigen; HLA-C Antigens; Humans; Immunotherapy; Inflammation; Interleukin-2; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Survival Rate; T-Lymphocytes, Cytotoxic; Treatment Outcome; Tumor Cells, Cultured

Systemic effects on T-cell-mediated antitumor immunity, on expression of T-cell adhesion/homing receptors, and on the promotion of T-cell infiltration of neoplastic tissue may represent key steps for the efficacy of immunological therapies of cancer. In this study, we investigated whether these processes can be promoted by s.c. administration of low-dose (0.5 microg/kg) recombinant human interleukin-12 (rHuIL-12) to metastatic melanoma patients. A striking burst of HLA-restricted CTL precursors (CTLp) directed to autologous tumor was documented in peripheral blood by a high-efficiency limiting dilution analysis technique within a few days after rHuIL-12 injection. A similar burst in peripheral CTLp frequency was observed even when looking at response to a single tumor-derived peptide, as documented by an increase in Melan-A/Mart-1(27-35)-specific CTLp in two HLA-A*0201+ patients by limiting dilution analysis and by staining peripheral blood lymphocytes (PBLs) with HLA-A*0201-melanoma antigen-A/melanoma antigen recognized by T cells (Melan-A/Mart)-1 tetrameric complexes. The CTLp burst was associated, in PBLs, with enhanced expression of T-cell adhesion/homing receptors CD11a/CD18, CD49d, CD44, and with increased proportion of cutaneous lymphocyte antigen (CLA)-positive T cells. This was matched by a marked increase, in serum, of soluble forms of the endothelial cell adhesion molecules E-selectin, vascular cell adhesion molecules (VCAM)-1 and intercellular adhesion molecules (ICAM)-1. Infiltration of neoplastic tissue by CDS+ T cells with a memory and cytolytic phenotype was found by immunohistochemistry in eight of eight posttreatment metastatic lesions but not in five of five pretreatment metastatic lesions from three patients. Increased tumor necrosis and/or fibrosis were also found in several posttherapy lesions of two of three patients in comparison with pretherapy metastases. These results provide the first evidence that rHuIL-12 can boost the frequency of circulating antitumor CTLp in tumor patients, enhances expression of ligand receptor pairs contributing to the lymphocyte function-associated antigen-1/ICAM-1, very late antigen-4/VCAM-1, and CLA/E-selectin adhesion pathways, and promotes infiltration of neoplastic lesions by CD8+ memory T cells in a clinical setting.

Topics: Antigens, Neoplasm; CD18 Antigens; CD8-Positive T-Lymphocytes; HLA-A Antigens; Humans; Immunohistochemistry; Immunologic Memory; Interleukin-12; Lymphatic Metastasis; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Neoplasm Metastasis; Neoplasm Proteins; Pilot Projects; Recombinant Proteins; T-Lymphocytes, Cytotoxic

In a previous study, the authors tested the combination of fotemustine (FM) 100 mg/m(2) intravenously (i.v.) on Day 1, dacarbazine (DTIC) 250 mg/m(2) i.v. on Days 2-5, and interferon alpha (IFNalpha) 3 MIU intramuscularly three times per week in 43 patients with advanced melanoma. An overall response rate of 40% and a median survival of 40 weeks were obtained. To evaluate whether the addition of cisplatin (CDDP) to this regimen could improve these results, the authors conducted a preliminary Phase I study and concluded that CDDP 25 mg/m(2) i.v. for 2 days can be combined safely with DTIC, FM, and IFNalpha. Herein, the authors report the results of a Phase II trial with this regimen.. From June 1996 to February 1999, 64 patients with metastatic melanoma who were not amenable to surgery were enrolled in this study. Sixty eligible patients (32 males and 28 females; median age, 53 years) were treated with a combination of FM 100 mg/m(2) i.v. on Day 1, DTIC 300 mg/m(2) i.v. on Days 2-4, and CDDP 25 mg/m(2) i.v. on Days 3 and 4 recycled every 3 weeks. IFN alpha2b was administered at a dose of 3 MIU intramuscularly 3 times per week until disease progression.. A total of 189 courses were administered, with a median number of 3 courses per patient (range, 1-8 courses per patient). Eleven complete responses and 12 partial responses were observed, for an overall response rate of 38.3% (95% exact confidence interval, 26.1-51.8%). The median survival was 36 weeks. Neutropenia and thrombocytopenia affected 85% of patients and 68% patients and was World Health Organization Grade 3-4 in 40% and 50%, respectively. The side effects attributable to IFN alpha2b were mild and manageable. The other side effects were moderate and well controlled by supportive therapy.. The schedule used in this study demonstrated significant activity in patients with advanced, untreated melanoma. The addition of CDDP in the management of the patients in this series seemed to increase significantly both the proportion of patients who achieved a complete response and the probability of long term survival compared with a previous series of patients who were treated by the authors. However, considering the currently available therapies, this regimen does not seem to offer a special advantage in the treatment of patients with this disease. New agents and new protocols are needed.

Topics: Adult; Aged; Anemia; Antigens, CD; Antigens, Neoplasm; Antigens, Surface; Antineoplastic Combined Chemotherapy Protocols; CD146 Antigen; Cisplatin; Dacarbazine; Female; Fever; Follow-Up Studies; Humans; Interferon-alpha; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Membrane Glycoproteins; Middle Aged; Monophenol Monooxygenase; Nausea; Neoplasm Proteins; Neural Cell Adhesion Molecules; Neutropenia; Nitrosourea Compounds; Organophosphorus Compounds; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Analysis; Thrombocytopenia; Treatment Outcome; Vomiting

Twenty-five patients with high-risk resected stages IIB, III, and IV melanoma were immunized with a vaccine consisting of the minimal epitope, immunodominant 9-amino acid peptide derived from the MART-1 tumor antigen (AAGIGILTV) complexed with incomplete Freund's adjuvant. The last three patients received the MART-1(27-35) peptide with incomplete Freund's adjuvant mixed with CRL 1005, a block copolymer adjuvant. Patients were immunized with increasing doses of the MART-1(27-35) peptide in a Phase I trial to evaluate the toxicity, tolerability, and immune responses to the vaccine. Immunizations were administered every 3 weeks for a total of four injections, preceded by leukapheresis to obtain peripheral blood mononuclear cells for immune analyses, followed by a post-vaccine leukapheresis 3 weeks after the fourth vaccination. Skin testing with peptide and standard delayed-type hypersensitivity skin test reagents was also performed before and after vaccinations. Local pain and granuloma formation were observed in the majority of patients, as were fevers or lethargy of grade 1 or 2. No vaccine-related grade III/IV toxicity was observed. The vaccine was felt to be well tolerated. Twelve of 25 patients were anergic to skin testing at the initiation of the trial, and 13 of 25 developed a positive skin test response to the MART-1(27-35) peptide. Immune responses were measured by release of IFN-gamma in an ELISA assay by effector cells after multiple restimulations of peripheral blood mononuclear cells in the presence of MART-1(27-35) peptide-pulsed antigen-presenting cells. An ELISPOT assay was also developed to measure more quantitatively the change in numbers of peptide-specific effector cells after vaccination. Ten of 22 patients demonstrated an immune response to peptide-pulsed targets or tumor cells by ELISA assay after vaccination, as did 12 of 20 patients by ELISPOT. Nine of 25 patients have relapsed with a median of 16 months of follow-up, and 3 patients in this high-risk group have died. Immune response by ELISA correlated with prolonged relapse-free survival. These data suggest a significant proportion of patients with resected melanoma mount an antigen-specific immune response against a peptide vaccine and support further development of peptide vaccines for melanoma.

Topics: Antigens, Neoplasm; Cancer Vaccines; Cytokines; Female; Freund's Adjuvant; Humans; Hypersensitivity, Delayed; Lymphocyte Activation; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Peptide Fragments; T-Lymphocytes, Cytotoxic; Vaccination

Polymerase chain reaction (PCR) with tyrosinase and with MART-1 permits detection of small numbers of circulating melanoma cells (CMCs) in patients who have undergone surgical resection of localized disease. In a previous study, we showed that PCR with MART-1 had sensitivity and specificity similar to those of PCR with tyrosinase in terms of detection of CMCs but that PCR with MART-1 seemed to identify a different but overlapping subgroup of patients. In the current study, we examined the utility and prognostic significance of PCR with tyrosinase and with MART-1.. We analyzed the prognostic significance of the patterns of expression of tyrosinase and MART-1 in 186 patients followed sequentially before and after surgical removal of American Joint Committee on Cancer stage I, II, or III melanoma.. PCR with tyrosinase and with MART-1 in the first 3 months after surgery identified 68.5% of 73 patients who developed recurrence in the 2-year period after surgery. Approximately 35% of patients with positive tests remained disease-free at 2-year follow-up. We found that patients with disseminated recurrence had a significantly lower incidence of MART-1-positive CMCs (16%) than of tyrosinase-positive CMCs (63%). Patients with locoregional metastases had CMCs that expressed tyrosinase and MART-1 at similar rates. These differences in expression of the markers in patients with disseminated recurrence were also associated with a much lower disease-free survival, in those who had CMCs that were positive for tyrosinase but negative for MART-1. The reverse applied in those with locoregional disease.. These findings suggest that PCR with MART-1 and with tyrosinase identifies subgroups of patients who develop disseminated or locally recurrent metastases. We hypothesize that immune responses against MART-1 may reduce the establishment of disseminated metastases.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Disease-Free Survival; Female; Humans; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Recurrence, Local; Neoplastic Cells, Circulating; Postoperative Period; Prognosis; Proportional Hazards Models; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity

A minimally invasive standard has yet to be developed for sentinel lymphadenectomy, and many patients undergo this procedure in the main operating room under general anesthesia. These patients often have microscopic metastases in sentinel nodes that could be missed by histopathologic examination. Techniques of reverse transcriptase polymerase chain reaction (RT-PCR) could detect these metastases if the nodes could be preserved intraoperatively.. Fifty patients with melanoma > or = mm thick underwent sentinel lymphadenectomy under local anesthesia in an outpatient surgical unit. Sentinel nodes were identified using blue dye and technetium-99 sulfur colloid and a hand-held gamma probe. Each node was sectioned, with half sent for routine histopathologic study and half preserved in liquid nitrogen. We used RT-PCR to detect mRNA for tyrosinase and Melanoma Antigen Recognized by T cells-1 (MART-1).. All patients were able to tolerate sentinel lymph node biopsy under local anesthesia. Sentinel lymph nodes were obtained in 100% of our patients, and usable mRNA was harvested from all but five. Ten patients had positive sentinel node(s) by standard histopathologic examination, and all of these nodes were also positive for MART-1 and tyrosinase. Three patients with negative results by histopathology had positive results by RT-PCR analysis. The average cost of these outpatient operations was 38% less than the same operations performed in the main operating room under general anesthesia.. Sentinel lymphadenectomy under local anesthesia in an outpatient setting and intraoperative lymph node preservation in liquid nitrogen are both feasible. Both tyrosinase and MART-1 are promising markers in the detection of occult melanoma in lymph nodes.

Topics: Actins; Ambulatory Surgical Procedures; Anesthesia, Local; Antigens, Neoplasm; Biomarkers, Tumor; Cost-Benefit Analysis; Female; Humans; Lymph Nodes; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Staging; Polymerase Chain Reaction; RNA-Directed DNA Polymerase; Skin Neoplasms

The characterization of the genes encoding melanoma-associated antigens MART-1 or gp100, recognized by T cells, has opened new possibilities for the development of immunization strategies for patients with metastatic melanoma. With the use of recombinant adenoviruses expressing either MART-1 or gp100 to immunize patients with metastatic melanoma, we evaluated the safety, immunologic, and potential therapeutic aspects of these immunizations.. In phase I studies, 54 patients received escalating doses (between 10(7) and 10(11) plaque-forming units) of recombinant adenovirus encoding either MART-1 or gp100 melanoma antigen administered either alone or followed by the administration of interleukin 2 (IL-2). The immunologic impact of these immunizations on the development of cellular and antibody reactivity was assayed.. Recombinant adenoviruses expressing MART-1 or gp100 were safely administered. One of 16 patients with metastatic melanoma receiving the recombinant adenovirus MART-1 alone experienced a complete response. Other patients achieved objective responses, but they had received IL-2 along with an adenovirus, and their responses could be attributed to the cytokine. Immunologic assays showed no consistent immunization to the MART-1 or gp100 transgenes expressed by the recombinant adenoviruses. High levels of neutralizing antibody were found in the pretreatment sera of the patients.. High doses of recombinant adenoviruses could be safely administered to cancer patients. High levels of neutralizing antibody present in patients' sera prior to treatment may have impaired the ability of these viruses to immunize patients against melanoma antigens.

Topics: Adenoviridae; Antibodies, Viral; Antigens, Neoplasm; Cancer Vaccines; Clinical Protocols; gp100 Melanoma Antigen; Humans; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Skin Neoplasms; Treatment Outcome; Tumor Cells, Cultured

A critical requirement for cancer vaccines is that they stimulate CD8+ T cell responses. In this study, we tested the ability of a polyvalent melanoma vaccine to induce CD8+ T cell responses to the melanoma associated antigens MAGE-3 and Melan A/MART-1. Fifteen HLA-A2+ patients with resected malignant melanoma were immunized with the vaccine s.c. every 2-3 weeks. CD8+ T cells in peripheral blood reacting to HLA-A2 restricted epitopes on MAGE-3 (FLWGPRALV) and Melan A/MART-1/(AAGIGILTV) were quantitated using a filter spot assay at baseline and following 4 immunizations. Vaccine immunization induced CD8+ T cells reacting to one or both of these peptides in 9 of the 15 (60%) patients. These cells were CD8+ and HLA-A2 restricted, as reactivity was abrogated by monoclonal antibodies (MAbs) to CD8 and class I HLA, but not by anti-CD4. All responding patients remained recurrence-free for at least 12 months (median 15 months, range 12 to >21 months), whereas melanoma recurred within 3-5 months in non-responders. The differences in outcome were unrelated to differences in disease severity or overall immunological competence between responders and non-responders. Our results demonstrate directly that MAGE-3 and Melan A/MART-1 can stimulate CD8+ T cell responses in humans, and suggest that these responses are protective and surrogate markers of vaccine efficacy.

Topics: Amino Acid Sequence; Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Disease-Free Survival; Epitopes; HLA-A2 Antigen; Humans; MART-1 Antigen; Melanoma; Neoplasm Proteins; Neoplasm Staging; Polymerase Chain Reaction; Tumor Cells, Cultured

Melanocytic nevi existing in lymph nodes create a diagnostic challenge by mimicking metastases. PReferentially expressed Antigen in MElanoma (PRAME) immunohistochemical (IHC) stain can differentiate one from another. FLI-1 IHC expression has been shown in malignant melanoma with variable sensitivity while melanocytic nevi were reported to be negative. We hypothesized that FLI-1/Melan-A dual IHC staining may be used in the distinction of metastatic melanoma from nodal nevi and can be an alternative and/or complementary to PRAME. In this study, we examined 13 lymph nodes with metastatic melanoma and 13 lymph nodes with benign deposits. We stained all of the lymph nodes with FLI-1, FLI-1/Melan-A dual, and PRAME IHC stains. In addition, we stained paired skin samples of the metastatic lymph nodes with FLI-1 and PRAME. In primary cutaneous melanomas, 11 of 13 were positive for FLI-1 and PRAME expression (85%). Malignant cells in 12 and 13 lymph nodes showed positive expression of PRAME and FLI-1, respectively. Only one case with a nevic cell deposit was weakly positive for FLI-1 and the remaining benign cases were negative for both FLI-1 and PRAME. Our results show that FLI-1/Melan-A dual stain is as sensitive and specific as PRAME in distinguishing lymph nodes with metastatic melanoma from nodal nevi. Further studies with larger case numbers are needed to support our significant results.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Coloring Agents; Humans; Lymphatic Metastasis; MART-1 Antigen; Melanoma; Melanoma, Cutaneous Malignant; Nevus; Nevus, Pigmented; Proto-Oncogene Protein c-fli-1; Retrospective Studies; Sentinel Lymph Node Biopsy; Skin Neoplasms

Topics: Humans; MART-1 Antigen; Melanoma; Mohs Surgery; Skin Neoplasms

Melanoblasts originate in the neural crest from where they migrate to peripheral tissues and differentiate into melanocytes. Alteration during melanocyte development and life can cause different diseases, ranging from pigmentary disorders and decreased visual and auditory functions, to tumours such as melanoma. Location and phenotypical features of melanocytes have been characterised in different species, yet data on dogs are lacking.. This study investigates the expression of melanocytic markers Melan A, PNL2, TRP1, TRP2, SOX-10 and MITF in melanocytes of selected cutaneous and mucosal surfaces of dogs.. At necropsy, samples from five dogs were harvested from oral mucosa, mucocutaneous junction, eyelid, nose and haired skin (abdomen, back, pinna, head).. Immunohistochemical and immunofluorescence analyses were performed to assess marker expression.. Results showed variable expression of melanocytic markers in different anatomical sites, particularly within epidermis of haired skin and dermal melanocytes. Melan A and SOX-10 were the most specific and sensitive melanocytic markers. PNL2 was less sensitive, while TRP1 and TRP2 were seldomly expressed by intraepidermal melanocytes in haired skin. MITF had a good sensitivity, yet the expression often was weak.. Our results indicate a variable expression of melanocytic markers in different sites, suggesting the presence of subpopulations of melanocytes. These preliminary results pave the way to understanding the pathogenetic mechanisms involved in degenerative melanocytic disorders and melanoma. Furthermore, the possible different expression of melanocyte markers in different anatomical sites could influence their sensitivity and specificity when used for diagnostic purposes.. Les mélanoblastes proviennent de la crête neurale, d'où ils migrent vers les tissus périphériques et se différencient en mélanocytes. L'altération des ménanocytes durant leur développement et leur vie peut induire différentes maladies : des troubles pigmentaires et des fonctions visuelles et auditives, ainsi que tumeurs telles que le mélanome. La localisation et les caractéristiques phénotypiques des mélanocytes ont été précisées dans différentes espèces, mais ces données manquent chez le chien.. L'expression des marqueurs mélanocytaires Melan A, PNL2, TRP1, TRP2, SOX-10 et MITF est étudiée dans des échantillons de peau et de muqueuses de chiens. MATÉRIELS ET MÉTHODES: Durant l'autopsie de cinq chiens, des échantillons de muqueuse buccale, de jonction cutanéo-muqueuse, de paupière, de nez et de peau velue (abdomen, dos, pavillon, tête) ont été prélevés ; des analyses immunohistochimiques et d'immunofluorescence ont été réalisées pour évaluer l'expression des différents marqueurs. RÉSULTATS: Les marqueurs mélanocytaires sont exprimés de façon variable selon les différents sites anatomiques, en particulier au sein de l'épiderme de la peau velue et des mélanocytes dermiques. Melan A et SOX-10 sont les marqueurs mélanocytaires les plus spécifiques et les plus sensibles. PNL2 apparait moins sensible, tandis que TRP1 et TRP2 sont rarement exprimés par les mélanocytes intraépidermiques de la peau velue. MITF avait une bonne sensibilité, mais l'expression était souvent faible.. Les marqueurs mélanocytaires sont exprimés de façon variable dans différents sites anatomiques, suggérant la présence de sous-populations mélanocytaires. Ces résultats préliminaires ouvrent la voie à la compréhension des mécanismes pathogéniques impliqués dans les maladies mélanocytaires dégénératives et le mélanome. De plus, ces éventuelles différences d’expression des marqueurs mélanocytaires selon le site anatomique pourraient influencer la sensibilité et la spécificité des analyses utilisant ces marqueurs à visée diagnostique.. INTRODUCCIÓN: los melanoblastos se originan en la cresta neural desde donde migran a los tejidos periféricos y se diferencian en melanocitos. La alteración durante el desarrollo y la vida de los melanocitos puede causar diferentes enfermedades, que van desde trastornos pigmentarios y disminución de las funciones visuales y auditivas, hasta tumores como el melanoma. La ubicación y las características fenotípicas de los melanocitos se han caracterizado en diferentes especies, pero faltan datos sobre perros. OBJETIVO: Este estudio investiga la expresión de los marcadores melanocíticos Melan A, PNL2, TRP1, TRP2, SOX-10 y MITF en melanocitos de superficies cutáneas y mucosas seleccionadas de perros. MATERIALES Y MÉTODOS: durante las necropsias, se obtuvieron muestras de cinco perros de mucosa oral, unión mucocutánea, párpado, nariz y piel con pelo (abdomen, espalda, pabellón auricular, cabeza); Se realizaron análisis inmunohistoquímicos y de inmunofluorescencia para evaluar la expresión de los marcadores. RESULTADOS: Los resultados mostraron una expresión variable de marcadores melanocíticos en diferentes sitios anatómicos, particularmente en la epidermis de la piel con pelos y en melanocitos dérmicos. Melan A y SOX-10 fueron los marcadores melanocíticos más específicos y sensibles. PNL2 fue menos sensible, mientras que TRP1 y TRP2 rara vez se expresaron en melanocitos intraepidérmicos en la piel con cabello. MITF tenía una buena sensibilidad, pero la expresión a menudo era débil. CONCLUSIONES Y RELEVANCIA CLÍNICA: Nuestros resultados indican una expresión variable de marcadores melanocíticos en diferentes sitios, lo que sugiere la presencia de subpoblaciones de melanocitos. Estos resultados preliminares inician el camino para comprender los mecanismos patogénicos implicados en los trastornos melanocíticos degenerativos y el melanoma. Además, la posible expresión diferente de los marcadores de melanocitos en diferentes sitios anatómicos podría influir en su sensibilidad y especificidad cuando se utilizan con fines de diagnóstico.. Melanoblasten stammen aus der Neuralleiste, von der sie ins periphere Gewebe auswandern und zu Melanozyten ausdifferenzieren. Veränderungen während der Entwicklung der Melanozyten und ihres Lebenslaufs können verschiedene Krankheiten bedingen, von Pigmentunregelmäßigkeiten und verminderten visuellen und auditorischen Funktionen bis hin zu Tumoren wie Melanome. Die Lokalisation und die phänotypischen Merkmale wurden bei verschiedenen Spezies beschrieben, beim Hund gibt es jedoch bisher keine Daten. ZIEL: Diese Studie untersucht die Exprimierung von Melanozytenmarkern Melan A, PNL2, TRP1, TRP2, SOX-10 und MITF an Melanozyten von selektiven kutanen und mucokutanen Oberflächen von Hunden.. Bei der Nekropsie wurden Proben aus der oralen Mukosa, mucokutanen Übergängen, Augenlid, Nase und behaarter Haut (Bauch, Rücken, Pinna, Kopf) von fünf Hunden genommen; immunhistochemische und Immunfluoreszenz-Analyse wurde durchgeführt, um eine Markerexprimierung zu erfassen.. Die Ergebnisse zeigten eine unterschiedliche Exprimierung der Melanozytenmarker an verschiedenen anatomischen Stellen, vor allem innerhalb der Epidermis der behaarten Haut und der dermalen Melanozyten. Melan A und SOX-10 waren die spezifischsten und sensitivsten Melanozytenmarker. PNL2 waren weniger sensitiv, während TRP1 und TRP2 selten an intraepidermalen Melanozyten der behaarten Haut exprimiert wurden. MITF zeigte eine gute Sensibilität, obwohl die Exprimierung oft nur schwach war. SCHLUSSFOLGERUNGEN UND KLINISCHE RELEVANZ: unsere Ergebnisse zeigten eine variable Exprimierung der Melanozytenmarker an verschiedenen Stellen, was auf das Auftreten unterschiedlicher Subpopulationen der Melanozyten hinweist. Diese vorläufigen Ergebnisse ebnen den Weg für ein Verständnis der pathogenen Mechanismen, die bei degenerativen Melanozyten-Veränderungen und beim Melanom eine Rolle spielen. Weiters könnte die möglicherweise unterschiedliche Exprimierung der Melanozytenmarker an verschiedenen anatomischen Stellen ihre Sensibilität und Spezifität beeinflussen, wenn sie für diagnostische Zwecke eingesetzt werden.. 背景: メラノブラストは神経堤で発生し、そこから末梢組織へ移動してメラノサイトに分化する。メラノサイトの発生・生育過程における変化は、色素障害、視覚・聴覚機能の低下からメラノーマなどの腫瘍に至るまで、様々な疾患の原因となる可能性がある。メラノサイトの位置や表現型は、様々な動物種で明らかにされているが、犬に関するデータは不足している。 目的: 本研究の目的は、犬の皮膚・粘膜のメラノサイトにおけるメラノサイトマーカーMelan A, PNL2, TRP1, TRP2, SOX-10, MITFの発現を検討することであった。 材料と方法: 剖検時に、5頭の犬から口腔粘膜、粘膜皮膚接合部、眼瞼、鼻および有毛皮膚(腹部、背部、耳介、頭部)のサンプルを採取し、免疫組織化学および免疫蛍光解析を行い、マーカーの発現を評価した。 結果 その結果、解剖学的な部位によってメラノサイトマーカーの発現が異なり、特に有毛皮膚表皮内および真皮のメラノサイトの発現にばらつきがあることが判明した。Melan AおよびSOX-10は、最も特異的で感度の高いメラノサイトマーカーであった。PNL2は感度が低く、TRP1およびTRP2は有毛皮膚内の表皮内メラノサイトでほとんど発現していなかった。MITFは感度が高いが、発現が弱い場合が多い。 結論と臨床的意義: 今回の結果は、部位によってメラノサイトマーカーの発現が異なることを示し、メラノサイトの亜集団の存在を示唆するものであった。これらの予備的な結果は、変性メラノサイト障害やメラノーマに関わる発症メカニズムの理解に道を開くものである。さらに、解剖学的部位によってメラノサイトマーカーの発現が異なる可能性があることから、診断目的で使用する場合には、その感度や特異性に影響を与える可能性がある。.. 背景: 黑素细胞起源于神经嵴，从那里迁移到周围组织并分化为黑素细胞。黑素细胞发育和生活过程中的变化会导致不同的疾病，从色素性疾病、视觉和听觉功能下降，到黑色素瘤等肿瘤。黑色素细胞的位置和表型特征已经在不同的物种中得到了表征，但关于犬的数据仍然缺乏。 目的: 本研究研究黑素细胞标志物黑素A、PNL2、TRP1、TRP2、SOX-10和MITF在犬皮肤和粘膜表面黑素细胞中的表达。 材料和方法 尸检时，从五只犬的口腔粘膜、粘膜皮肤连接处、眼睑、鼻子和毛发皮肤(腹部、背部、耳廓、头部)采集样本；进行免疫组织化学和免疫荧光分析以评估标记物的表达。 结果: 结果显示黑素细胞标记物在不同解剖部位的表达不同，尤其是在毛发皮肤的表皮和真皮黑素细胞内。黑素A和SOX-10是最特异和敏感的黑素细胞标志物。PNL2较不敏感，而TRP1和TRP2仅在毛发皮肤表皮内黑素细胞中表达。MITF具有良好的敏感性，但表达往往较弱。 结论和临床相关性: 我们的结果表明，不同部位的黑素细胞标志物表达不同，表明存在黑素细胞亚群。这些初步结果为理解退行性黑素细胞疾病和黑色素瘤的发病机制铺平了道路。此外，当用于诊断目的时，黑素细胞标记物在不同解剖部位的可能不同表达可能会影响其敏感性和特异性。.. Os melanoblastos se originam da crista neural, de onde eles migram para os tecidos periféricos e se diferenciam em melanócitos. Alterações durante o desenvolvimento e ao longo da vida dos melanócitos pode causar diferentes doenças, desde alterações pigmentares e redução das funções auditivas e visuais, a tumores como o melanoma. Localização e características fenotípicas dos melanócitos têm sido detalhadas em várias espécies, mas em cães os dados ainda são escassos.. Este estudo investiga a expressão dos marcadores melanocíticos Melan A, PNL2, TRP1, TRP2, SOX-10 e MITF nos melanócitos de determinadas superfícies cutâneas e mucosas em cães. MATERIAIS E MÉTODOS: À necrópsia, foram coletadas amostras de mucosa oral, junção mucocutânea, pálpebra, nariz e pele pilosa (abdômen, dorso, orelha, cabeça); para se avaliar a expressão dos marcadores, realizou-se imuno-histoquímica e imunofluorescência.. Os resultados demonstraram expressão de marcadores melanocíticos em diferentes sítios anatômicos, particularmente na epiderme da pele pilosa e nos melanócitos dérmicos. Melan A e SOX-10 foram os marcadores melanocíticos mais específicos e sensíveis. PNL2 foi menos sensível, enquanto TRP1 e TRP2 foram raramente expressados por melanócitos intraepidérmicos na pele pilosa. MITF apresentou boa sensibilidade, apesar de a sua expressão ser frequentemente fraca. CONCLUSÕES E RELEVÂNCIA CLÍNICA: Nossos resultados indicam uma expressão variável de marcadores melanocíticos em diferentes regiões, sugerindo a presença de subpopulações de melanócitos. Estes resultados preliminares embasam a compreensão dos mecanismos patogenéticos envolvidos nas doenças melanocíticas degenerativas e no melanoma. Além disso, a possível diferença na expressão de marcadores melanocíticos em diferentes sítios anatômicos pode influenciar a sua sensibilidade e especificidade quando utilizado para diagnóstico.

Topics: Animals; Dog Diseases; Dogs; Epidermis; Immunohistochemistry; MART-1 Antigen; Melanocytes; Melanoma; Skin Neoplasms

To report a previously unrecognized choroidal melanoma clinical feature termed tumor-associated retinal pigmentation (TARP) and determine any correlation with tumor biology.. Imaging and histologic analysis of a retrospective cohort of patients.. Patients with choroidal melanoma identified as having TARP on funduscopy at the Liverpool Ocular Oncology Centre (LOOC), United Kingdom, from January 2020 through January 2023.. Clinical and imaging characteristics of patients diagnosed with choroidal melanoma and exhibiting TARP on fundoscopy were documented. Details of these choroidal melanomas were collated and correlated with histopathology and molecular genetic reports. The chromosome 3 status of each tumor was assessed. In enucleated samples, immunostaining was undertaken to determine the nature of the TARP using specific markers (CD68 and MelanA).. Features of TARP on widefield fundus color imaging, fundus autofluorescence (FAF), and OCT were described. Tumor chromosome 3 status and the immunoprofile of the TARP also were collated.. Tumor-associated retinal pigmentation had a prevalence rate of 7.47 per 100 cases of choroidal melanoma at the LOOC. Twenty-three eyes with TARP were analyzed, with a mean age of 71.4 years (range, 51-88 years). The median largest basal diameter was 16.10 mm (range, 9.17-21.32 mm), and the mean tumor thickness was 8.04 mm (range, 1.40-13.80 mm). Tumor-associated retinal pigmentation was observed on widefield color fundus imaging, with hypofluorescence on FAF images and represented hyperreflective foci located in intraretinal and subretinal spaces on OCT scans. Seventeen patients (73.9%) underwent enucleation, and 6 patients (26.1%) underwent globe-sparing treatment. Molecular genetic analysis of 20 choroidal melanomas (after enucleation or radiotherapy biopsy) revealed monosomy 3 in 18 tumors (90%). Immunostaining of the TARP in enucleated eyes showed CD68+ melanophages in all 17 patients appearing as scattered cells and aggregates; MelanA findings were negative.. Tumor-associated retinal pigmentation represents tumor-associated macrophages, not melanocytes, within intraretinal and subretinal spaces of larger choroidal melanomas. Radiation treatments need not involve this area in the treatment plan, minimizing radiation-related complications. This novel clinical sign seems to be linked to tumors of high metastatic-risk clinical and genetic characteristics, with a preponderance having monosomy 3 anomalies.. The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Topics: Aged; Choroid Neoplasms; Fluorescein Angiography; Humans; MART-1 Antigen; Melanoma; Monosomy; Pigmentation; Retrospective Studies

Topics: Humans; Liver Neoplasms; MART-1 Antigen; Melanins; Melanoma; Retrospective Studies

Heavily pigmented lesions are difficult to evaluate histologically, as melanin obscures cellular details. Several classic laboratory techniques aim to clear melanin and allow evaluation. Most of them are old and appeared before immunohistochemistry (IHC) use. Many laboratories perform IHC with aminoethylcarbazole instead of diaminobenzidine (DAB) in heavily pigmented lesions, as red-stained is easy to interpret despite pigmentation. Nevertheless, many laboratories lack alternatives to DAB. The aim of this study is to compare 6 different tissue bleaching techniques and evaluate which is the best for immunohistochemical staining with DAB. In the present study we have selected a case with gross pigmentation because of the high grade of melanin deposition. We have performed 6 different bleaching techniques and subsequently performed 2 different IHC stains, frequently used in melanoma: SOX10 (nuclear) and Melan-A (cytoplasmic). Five different pathologists, 2 of them with expertise in dermatopathology, have blindly reviewed and scored the staining quality. Our results indicate a high grade of interobserver concordance in the evaluation of IHC results between pathologists. All the bleaching techniques that included a sulfuric acid led to tissue detachment from the slide. The best method for SOX10 was that based in potassium permanganate, with a high quality of staining (4 over 5), while the best method for Melan-A was the 1 based in peroxide hydrogen (4 over 5). We consider this study can be quite useful for those laboratories lacking aminoethylcarbazole for IHC techniques, allowing the use of DAB for IHC of heavily pigmented lesions.

Topics: Humans; MART-1 Antigen; Melanins; Melanoma; Skin Neoplasms; Staining and Labeling

Limited data are available to assist the selection between immune checkpoint inhibitors and BRAF/mitogen-activated protein kinase kinase inhibitors as first-line treatment for patients with BRAF-mutant advanced malignant melanoma.. To investigate the outcomes associated with first-line pembrolizumab or dabrafenib/trametinib treatment for advanced melanoma with activating BRAF V600 mutation.. Data of patients with BRAF V600-mutant melanoma who were treated with first-line pembrolizumab (n = 40) or dabrafenib/trametinib (n = 32) were analyzed. Tumor response, progression-free survival, and overall survival were evaluated. Immune evasion accompanied with emerging resistance to BRAF/mitogen-activated protein kinase kinase inhibitors was assessed.. A longer overall survival was observed after first-line pembrolizumab treatment than after first-line dabrafenib/trametinib treatment (hazard ratio = 2.910, 95% CI: 1.552-5.459), although there were no significant differences in progression-free survival (P = .375) and response rate (P = .123). Emergence of resistance to dabrafenib/trametinib co-occurred with immune evasion, enabling melanoma cells to escape recognition and killing by Melan-A-specific CD8. Analysis was conducted in a retrospective manner.. Pembrolizumab may be recommended over BRAF/mitogen-activated protein kinase kinase inhibitors as the first-line treatment in patients with advanced BRAF V600-mutant melanoma.

Topics: Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; CD8-Positive T-Lymphocytes; Humans; Imidazoles; Immune Checkpoint Inhibitors; MART-1 Antigen; Melanoma; Mitogen-Activated Protein Kinase Kinases; Mutation; Oximes; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Pyridones; Pyrimidinones; Retrospective Studies; Skin Neoplasms

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells recognize and eliminate cancer cells. However, immune evasion, downregulation of immune function by the tumour microenvironment and resistance of cancer cells are major problems. Although CTL and NK cells are both important to eliminate cancer, most studies address them individually. We quantified sequential primary human CTL and NK cell cytotoxicity against the melanoma cell line SK-Mel-5. At high effector-to-target ratios, NK cells or melan-A (MART-1)-specific CTL eliminated all SK-Mel-5 cells within 24 h, indicating that SK-Mel-5 cells are not resistant initially. However, at lower effector-to-target ratios, which resemble numbers of the immune contexture in human cancer, a substantial number of SK-Mel-5 cells survived. Pre-exposure to CTL induced resistance in surviving SK-Mel-5 cells to subsequent CTL or NK cell cytotoxicity, and pre-exposure to NK cells induced resistance in surviving SK-Mel-5 cells to NK cells. Higher human leucocyte antigen class I expression or interleukin-6 levels were correlated with resistance to NK cells, whereas reduction in MART-1 antigen expression was correlated with reduced CTL cytotoxicity. The CTL cytotoxicity was rescued beyond control levels by exogenous MART-1 antigen. In contrast to the other three combinations, CTL cytotoxicity against SK-Mel-5 cells was enhanced following NK cell pre-exposure. Our assay allows quantification of sequential CTL and NK cell cytotoxicity and might guide strategies for efficient CTL-NK cell anti-melanoma therapies. KEY POINTS: Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells eliminate cancer cells. Both CTL and NK cells attack the same targets, but most studies address them individually. In a sequential cytotoxicity model, the interdependence of antigen-specific CTL and NK cell cytotoxicity against melanoma is quantified. High numbers of antigen-specific CTL and NK cells eliminate all melanoma cells. However, lower numbers induce resistance if secondary CTL or NK cell exposure follows initial CTL exposure or if secondary NK cell exposure follows initial NK cell exposure. On the contrary, if secondary CTL exposure follows initial NK cell exposure, cytotoxicity is enhanced. Alterations in human leucocyte antigen class I expression and interleukin-6 levels are correlated with resistance to NK cells, whereas a reduction in antigen expression is correlated with reduced CTL cytotoxicity; CTL cytotoxicity is rescued beyon

Topics: Humans; Interleukin-6; Killer Cells, Natural; MART-1 Antigen; Melanoma; T-Lymphocytes, Cytotoxic; Tumor Microenvironment

Clear cell sarcoma of soft tissue (CCSST) and conventional malignant melanoma (MM) are rare and aggressive tumours with similarities in morphology and the expression of melanocytic markers.. We established two CCSST cell lines (FU-CCSST-1 and FU-CCSST-2) from soft tissues of the patella and supraclavicular. A MM cell line (FU-MM-1) was established from lymph node metastases of subungual malignant melanoma.. FU-CCSST-2 cells were transplantable to immunodeficient mice. Immunohistochemical studies demonstrated tumour cells were negative for cytokeratin AE1/AE3 and positive for S100 protein, HMB45, Melan-A, CD146 and SOX10 in all specimens. FU-CCSST-1 and FU-CCSST-2 harboured t(12;22)(q13;q12) translocations with expression of the EWSR1/ATF1 fusion gene. FU-MM-1 demonstrated loss of the short arm of chromosome 9 and harboured wild-type BRAF (codon 469 and 600) and NRAS (codon 12, 13 and 61).. We report the establishment and characterisation of CCSST and MM cell lines that may have utility in the study of pathogenic mechanisms and development of novel therapeutic reagents.

Topics: Animals; CD146 Antigen; Cell Line; Humans; Keratins; MART-1 Antigen; Melanoma; Melanoma, Cutaneous Malignant; Mice; Proto-Oncogene Proteins B-raf; S100 Proteins; Sarcoma, Clear Cell

Dual immunohistochemical (IHC) staining with D2-40 and S100 improves detection of lymphatic invasion (LI) in primary cutaneous melanoma. However, limited data exist evaluating this technique using other melanocytic markers, and thus, the optimal marker for detection of LI is unestablished. To address this knowledge gap, a case-control study was performed comparing melanoma specimens from 22 patients with known lymphatic spread (LS) with a control group of 11 patients without LS. Specimens underwent dual IHC staining with D2-40 and MART-1, SOX-10, and S100 to evaluate for LI. Receiver operating characteristic analysis was used to estimate each stain's accuracy for detection of LI. The LS group was more likely to be ≥65 years (P = 0.04), have a tumor thickness of ≥1 mm (P < 0.01), and have ulcerated tumors (P = 0.02). Detection of LI with D2-40/MART-1 significantly correlated with LS (P = 0.03), and the D2-40/MART-1 stain was most accurate for LI based on receiver operating characteristic curve analysis (area under the curve [AUC] 0.705) in comparison with D2-40/SOX-10 (AUC 0.575) and D2-40/S100 (AUC 0.633). These findings suggest that MART-1 may be the optimal melanocytic marker to combine with D2-40 for detection of LI in melanoma. Further studies are needed to determine the utility of routinely performing these stains for histopathologic analysis of melanoma.

Topics: Adult; Aged; Biomarkers, Tumor; Case-Control Studies; Female; Humans; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Melanoma, Cutaneous Malignant; Middle Aged; Neoplasm Invasiveness; ROC Curve; S100 Proteins; Sentinel Lymph Node Biopsy; Skin Neoplasms

Adoptive immunotherapy based on the transfer of anti-tumor cytotoxic T lymphocytes (CTLs) is a promising strategy to cure cancers. However, rapid expansion of numerous highly functional CTLs with long-lived features remains a challenge. Here, we constructed NIH/3T3 mouse fibroblast-based artificial antigen presenting cells (AAPCs) and precisely evaluated their ability to circumvent this difficulty. These AAPCs stably express the essential molecules involved in CTL activation in the HLA-A*0201 context and an immunogenic HLA-A*0201 restricted analogue peptide derived from MART-1, an auto-antigen overexpressed in melanoma. Using these AAPCs and pentamer-based magnetic bead-sorting, we defined, in a preclinical setting, the optimal conditions to expand pure MART-1-specific CTLs. Numerous highly purified MART-1-specific CTLs were rapidly obtained from healthy donors and melanoma patients. Both TCR repertoire and CDR3 sequence analyses revealed that MART-1-specific CTL responses were similar to those reported in the literature and obtained with autologous or allogeneic presenting cells. These MART-1-specific CTLs were highly cytotoxic against HLA-A*0201

Topics: Animals; Antigen-Presenting Cells; Cell Culture Techniques; Humans; Immunologic Memory; Immunotherapy, Adoptive; Lymphocyte Activation; MART-1 Antigen; Melanoma; Mice; NIH 3T3 Cells; T-Lymphocytes, Cytotoxic

Topics: Animals; Antigens, Neoplasm; Biomarkers, Tumor; Cat Diseases; Cats; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Retrospective Studies; Skin Neoplasms

Anti-programmed cell death protein 1 (PD-1) antibodies are now routinely administered for metastatic melanoma and for increasing numbers of other cancers, but still only a fraction of patients respond. Better understanding of the modes of action and predictive biomarkers for clinical outcome is urgently required. Cancer rejection is mostly T cell-mediated. We previously showed that the presence of NY-ESO-1-reactive and/or Melan-A-reactive T cells in the blood correlated with prolonged overall survival (OS) of patients with melanoma with a heterogeneous treatment background. Here, we investigated whether such reactive T cells can also be informative for clinical outcomes in metastatic melanoma under PD-1 immune-checkpoint blockade (ICB).. Peripheral blood T cell stimulation by NY-ESO-1 and Melan-A overlapping peptide libraries was assessed before and during ICB in two independent cohorts of a total of 111 patients with stage IV melanoma. In certain cases, tumor-infiltrating lymphocytes could also be assessed for such responses. These were characterized using intracellular cytokine staining for interferon gamma (IFN-γ), tumor negrosis factor (TNF) and CD107a. Digital pathology analysis was performed to quantify NY-ESO-1 and Melan-A expression by tumors. Endpoints were OS and progression-free survival (PFS).. The initial presence in the circulation of NY-ESO-1- or Melan-A-reactive T cells which became no longer detectable during ICB correlated with validated, prolonged PFS (HR:0.1; p>0.0001) and OS (HR:0.2; p=0.021). An evaluation of melanoma tissue from selected cases suggested a correlation between tumor-resident NY-ESO-1- and Melan-A-reactive T cells and disease control, supporting the notion of a therapy-associated sequestration of cells from the periphery to the tumor predominantly in those patients benefitting from ICB.. Our findings suggest a PD-1 blockade-dependent infiltration of melanoma-reactive T cells from the periphery into the tumor and imply that this seminally contributes to effective treatment.

Topics: Aged; Aged, 80 and over; Antigens, Neoplasm; Biomarkers, Tumor; CD8-Positive T-Lymphocytes; Female; Follow-Up Studies; Humans; Immune Checkpoint Inhibitors; Leukocytes, Mononuclear; Lymphocytes, Tumor-Infiltrating; Male; MART-1 Antigen; Melanoma; Membrane Proteins; Middle Aged; Prognosis; Programmed Cell Death 1 Receptor; Survival Rate

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Female; Humans; Immunohistochemistry; Male; Margins of Excision; MART-1 Antigen; Melanoma; Middle Aged; Mohs Surgery; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Neoplasm, Residual; Retrospective Studies; Skin Neoplasms; Young Adult

Topics: Adult; Aged; Aged, 80 and over; Female; Head and Neck Neoplasms; Humans; Immunohistochemistry; Male; Margins of Excision; MART-1 Antigen; Melanoma; Middle Aged; Mohs Surgery; Neoplasm Invasiveness; Retrospective Studies; Skin Neoplasms; Surgical Wound; Time Factors; Travel; Wound Closure Techniques

Topics: Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Mohs Surgery; Neoplasm Invasiveness; Reproducibility of Results; Skin Neoplasms

Blue naevi are uncommon dermal melanocytic neoplasms characterised by GNAQ/GNA11 mutations, which very rarely progress to melanoma. Such melanomas also often have BAP1 mutations, and lack genetic events associated with conventional melanoma. Exceptionally, blue naevi arise in extracutaneous locations; one melanoma arising in this setting has been reported. We report the clinicopathological, immunohistochemical and molecular genetic features of two cases of melanoma arising in extracutaneous blue naevus.. Both arose in males, aged 25 and 63 years, with no history of other melanocytic lesions, and presented as large, painful intra-abdominal masses. The tumours were dark-brown/black, multilobulated, involved small intestinal mesentery and consisted of a predominantly fascicular and spindled, but occasionally nested and epithelioid, proliferation of variably pigmented, relatively monotonous cells with pale cytoplasm and ovoid nuclei with mild to moderate atypia. Mitotic activity was variable but generally low. Both cases showed areas of conventional and cellular blue naevus. Recurrent tumour in one case showed predominantly epithelioid morphology and greater cytological atypia and mitotic activity. One case expressed Melan-A, SOX10 and CD117, with absent expression of S100 protein and DOG1; the other expressed Melan-A, HMB45 and S100 protein. Next-generation sequencing identified GNAQ and BAP1 mutations in one case and GNA11 mutation in the other. Both patients developed widespread metastatic disease.. Exceptionally rare, aggressive melanomas arising in extracutaneous blue naevi should be distinguished from metastatic melanoma, gastrointestinal stromal tumour and malignant melanotic nerve sheath tumour, especially given the significant therapeutic and prognostic differences between these different entities.

Topics: Adult; Biomarkers, Tumor; Diagnosis, Differential; Gastrointestinal Neoplasms; Genetic Markers; High-Throughput Nucleotide Sequencing; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanocytes; Melanoma; Middle Aged; Neoplasm Metastasis; Nevus, Blue; Nevus, Pigmented; Oncogenes; Prognosis; S100 Proteins; Skin Neoplasms; Tumor Suppressor Proteins; Ubiquitin Thiolesterase

Topics: Humans; MART-1 Antigen; Melanoma; Mohs Surgery; Skin Neoplasms

Topics: Adult; Biomarkers, Tumor; Cross-Sectional Studies; Cytoreduction Surgical Procedures; Female; Humans; Immunohistochemistry; Male; Margins of Excision; MART-1 Antigen; Melanoma; Middle Aged; Mohs Surgery; Neoplasm Invasiveness; Neoplasm Staging; Practice Patterns, Physicians'; Skin; Skin Neoplasms; Surgeons; Surveys and Questionnaires

Very limited data are available concerning the clinicopathological and molecular features of early subungual melanoma (SM), especially with regard to the Asian population. The aim of this study was to investigate the clinical, histological, immunohistochemical and chromosomal features of early SM.. Fifty-two in-situ and 13 thin (Breslow thickness ≤1.0 mm) SM cases were retrospectively reviewed. All patients presented with longitudinal melanonychia involving a single digit, and the thumb was the most affected digit (35 of 65, 53.8%). Microscopically, most cases showed small to medium nuclear enlargement (58 of 65) and mild to moderate nuclear atypia (57 of 65). Hyperchromatism and irregular contours of nuclei were persistent features in all cases. The variation of melanocyte count (the number of melanocytes per mm dermal-epithelial junction) ranged from 31 to 255. Intra-epithelial mitoses were identified in 34 cases (52.3%). Statistically, features of in-situ lesions including higher melanocyte count (>70), presence of multinucleated melanocytes, inflammatory infiltrate and cutaneous adnexal extension, were associated with early invasion. Melan-A, human melanoma B (HMB)45, mouse monoclonal melanoma antibody (PNL2) and SOX10 antibodies (>95.0%) showed superior diagnostic sensitivity to S-100 protein (83.1%). Fluorescence in-situ hybridisation (FISH) results were positive in 15 of 23 successfully analysed cases.. To the best of our knowledge, this is the largest single-institution study of early SM in an Asian population, and the largest cohort tested by FISH. Early SM mainly showed small to medium nuclear enlargement and mild to moderate nuclear atypia. High melanocyte count, hyperchromatism and irregular contours of nuclei and intra-epithelial mitoses are crucial diagnostic parameters. Immunohistochemistry, especially SOX10 staining, and FISH analysis are valuable in the diagnosis of SM.

Topics: Adult; Biomarkers, Tumor; Cohort Studies; Female; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Male; MART-1 Antigen; Melanocytes; Melanoma; Middle Aged; Nail Diseases; Retrospective Studies; S100 Proteins; Skin; Skin Neoplasms; SOXE Transcription Factors

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Eccrine Glands; Female; Follow-Up Studies; Humans; Male; MART-1 Antigen; Melanoma; Mohs Surgery; Neoplasm Invasiveness; Retrospective Studies; Skin Neoplasms; Treatment Outcome

Lentigo maligna (LM), the most common type of melanoma in situ, is a diagnostically challenging lesion for pathologists due to abundant background melanocytic hyperplasia in sun-damaged skin. Currently, no laboratory methods reliably distinguish benign from malignant melanocytes. However, preferentially expressed antigen in melanoma (PRAME) has shown promise in this regard, and could potentially be applied to diagnosis and margin assessment in difficult cases of LM.. Ninety-six cases with a diagnosis of LM (n = 77) or no residual LM (n = 19) following initial biopsy were identified and stained with an antibody directed towards PRAME. Immunohistochemistry (IHC) was scored as positive or negative, and measurement of histological margins by PRAME was performed and compared to the measurement of histological margins using conventional methods [haematoxylin and eosin (H&E) and/or sex-determining region Y-box 10 (SOX10) and/or Melan-A]. Of cases with LM, 93.5% (72 of 77) were PRAME. PRAME IHC is a sensitive (93.5%) and specific (94.7%) method for diagnosing LM on biopsy and excision, and measurement of histological margins by PRAME shows a high correlation with conventional methods for margin assessment. Furthermore, the nuclear expression of PRAME makes it a good target for use in dual-colour IHC stains.

Topics: Aged; Biomarkers, Tumor; Humans; Hutchinson's Melanotic Freckle; Immunohistochemistry; Male; MART-1 Antigen; Melanocytes; Melanoma; Melanoma, Cutaneous Malignant; Middle Aged; Skin Neoplasms; Staining and Labeling

Uveal melanoma (UM) and conjunctival melanoma (CM) are ocular malignancies that give rise to life-threatening metastases. Although local disease can often be treated successfully, it is often associated with significant vision impairment and treatments are often not effective against metastatic disease. Novel treatment modalities that preserve vision may enable elimination of small tumors and may prevent subsequent metastatic spread. Very few mouse models of metastatic CM and UM are available for research and for development of novel therapies. One of the challenges is to follow tumor growth in-vivo and to determine the right size for treatment, mainly of the posterior, choroidal melanoma. Hence, the purpose of this study was to establish a simple, noninvasive imaging tool that will simplify visualization and tumor follow-up in mouse models of CM and UM. Tumors were induced by inoculation of murine B16LS9 cells into the sub-conjunctival or the choroidal space of a C57BL/6 mouse eye under a surgical microscope. Five to ten days following injection, tumor size was assessed by Phoenix MicronIV™ image-guided Optical Coherence Tomography (OCT) imaging, which included a real-time camera view and OCT scan of the conjunctiva and the retina. In addition, tumor size was evaluated by ultrasound and histopathological examination of eye sections. Tumor growth was observed 5-9 days following sub-conjunctival or sub-retinal injection of seven-thousand or seventy-thousand cells, respectively. A clear tumor mass was detected at these regions using the MicronIV™ imaging system camera and OCT scans. Histology of eye sections confirmed the presence of tumor tissue. OCT allowed an accurate measurement of tumor size in the UM model and a qualitative assessment of tumor size in the CM model. Moreover, OCT enabled assessing the success rate of the choroidal tumor induction and importantly, predicted final tumor size already on the day of cell inoculation. In conclusion, by using a simple, non-invasive imaging tool, we were able to follow intraocular tumor growth of both CM and UM, and to define, already at the time of cell inoculation, a grading scale to evaluate tumor size. This tool may be utilized for evaluation of new mouse models for CM and UM, as well as for testing new therapies for these diseases.

Topics: Animals; Biomarkers, Tumor; Cell Line, Tumor; Conjunctival Neoplasms; Disease Models, Animal; Immunohistochemistry; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Mice; Mice, Inbred C57BL; Monophenol Monooxygenase; Neoplasm Proteins; Tomography, Optical Coherence; Ultrasonography; Uveal Neoplasms

Topics: Adult; Aged; Aged, 80 and over; Eosine Yellowish-(YS); Female; Fluorescent Dyes; Frozen Sections; Hematoxylin; Humans; Male; Margins of Excision; MART-1 Antigen; Melanoma; Middle Aged; Mohs Surgery; Prospective Studies; Sensitivity and Specificity; Skin Neoplasms; Tissue Fixation

Pure and mixed desmoplastic melanomas (DMs) may have different natural histories and behaviors.. We conducted a retrospective review of patients diagnosed with DM at our institution between January 1997 and April 2019. A total of 33 unique DMs were identified and subsequently analyzed based on the histologic type (pure vs. mixed).. The majority (57.6%) of our cases were classified as pure histology. Patients with pure DMs were more likely to be men (P = 0.035) and be older than 65 years (P = 0.019) compared with patients with mixed DMs. Patients with mixed DM were more likely to have mitoses present (P = 0.001) compared with patients with pure DM. There were no differences in ulceration, perineural invasion, vascular invasion, or survival between patients with pure and mixed histologic subtypes. In addition, no differences in sentinel lymph node biopsy, radiation, or chemotherapy were noted between the 2 histologic subtypes. Immunohistochemistry showed that 27.3% of the pure DMs stained with Melan-A and HMB45 were positive for these immunomarkers.. Pure and mixed variants of DM were found to have similar clinicopathologic characteristics. Patients with the mixed histologic subtype were more likely to have mitoses, but no difference in the therapeutic management or patient survival was seen between the 2 subtypes.

Topics: Adult; Aged; Aged, 80 and over; Female; Head and Neck Neoplasms; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Middle Aged; Mitosis; Retrospective Studies; Skin Neoplasms; Survival Rate

Topics: Biomarkers, Tumor; Biopsy; Cross-Sectional Studies; Dermatologists; Dermatology; Female; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Mohs Surgery; Practice Patterns, Physicians'; Prevalence; Skin; Skin Neoplasms; Surgeons

Topics: B7-H1 Antigen; Cell Line, Tumor; Chemokines; Cytokines; Cytotoxicity Tests, Immunologic; Gene Expression Profiling; HEK293 Cells; Humans; Immunotherapy, Adoptive; Inflammation; MART-1 Antigen; Melanoma; Receptors, Antigen, T-Cell; RNA-Seq; Single-Cell Analysis; Skin Neoplasms; T-Cell Antigen Receptor Specificity; T-Lymphocytes; Tumor Microenvironment

Undifferentiated melanoma should be considered in the differential diagnosis of sarcomatoid cutaneous malignancies to ensure that patients receive the correct treatment. Dermatopathologists should recognize the pitfalls of relying too heavily on immunohistochemistry to establish this diagnosis and consider ancillary tests, including single-nucleotide polymorphism (SNP) copy number arrays and targeted next-generation sequencing (NGS), when a definitive diagnosis cannot be rendered on a primary or metastatic tumor. This technology can also help to exclude a collision of melanoma and sarcoma when both differentiated and undifferentiated components are juxtaposed. We describe an exceedingly rare, illustrative example of undifferentiated sarcomatoid melanoma presenting as a pedunculated nodule. The clinical context and presence of a small differentiated component helped to establish the diagnosis; however, the transition from differentiated to undifferentiated melanoma was accompanied by an abrupt loss of S100, Sox10, MITF, MelanA, and HMB45 with gain of CD10 and p63 staining. SNP copy number array and NGS revealed shared chromosomal copy number changes and overlapping mutations with additional aberrances detected exclusively in the sarcomatoid component, thereby excluding a collision tumor and confirming our putative impression of melanoma with progression to an undifferentiated sarcomatoid phenotype.

Topics: Aftercare; Aged; Antibodies, Monoclonal, Humanized; Biomarkers, Tumor; Diagnosis, Differential; gp100 Melanoma Antigen; Humans; Lymphadenopathy; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Membrane Proteins; Microphthalmia-Associated Transcription Factor; Mutation; Neprilysin; Polymorphism, Single Nucleotide; Sarcoma; Skin Neoplasms; Soft Tissue Neoplasms; SOXE Transcription Factors; Treatment Outcome

The use of immunohistochemical (IHC) stains in dermatopathology is commonplace; however, little is known regarding utilization trends in melanoma diagnosis. Current Medicare local coverage determinations (LCDs) state that most pigmented lesions, including melanoma, can be diagnosed using H&E alone.. Histopathology reports for all biopsy-proven melanomas excised between January 1, 2017 and June 30, 2018, at a single dermatology clinic, were identified with the following parameters abstracted: laboratory/dermatopathologist rendering the diagnosis, whether IHC was performed, type/number of stains utilized, presence/depth of invasion, and melanoma subtype. The association of characteristics with IHC utilization was evaluated using χ. Three hundred and fifty six eligible melanomas were identified. IHC was employed in 228 (64%) of the diagnoses. Invasive melanoma was diagnosed in 199 cases (55.9%) while 157 (44.1%) were identified as melanoma in situ (MIS). Of the 228 that utilized IHC, 117 were performed on invasive melanoma (58.8%) and 111 were performed on MIS (70.7%).. Our findings suggest a higher IHC usage for the diagnosis of melanoma than previously reported. Existing LCDs regarding IHC utilization in melanoma do not reflect the current state of practice. Further investigation regarding IHC utilization and the development of appropriate-use criteria for melanoma IHC is necessary.

Topics: Biopsy; Female; Humans; Immunohistochemistry; Male; MART-1 Antigen; Medicare; Melanoma; Neoplasm Invasiveness; Nevus, Pigmented; Retrospective Studies; Skin Neoplasms; SOXE Transcription Factors; United States

It is well known to pathologists that melanoma is "the great mimicker," looking like almost any other tumor, and able to metastasize anywhere in the body. We report a case of a 48-year-old female with a history of metastatic melanoma 4 years before, who presented with a hepatic mass. Microscopic examination of the liver mass revealed sheets of pleomorphic, epithelioid cells, which expressed a pan-melanocytic cocktail (MART1, HMB45, and tyrosinase). These findings were initially interpreted as metastatic melanoma and the case was transferred for dermatopathology consultation. We compared the morphology of this tumor to the primary melanoma and noticed that the primary melanoma showed nevoid cytology, morphologically distinct from the liver lesion. Consequently, we performed additional immunohistochemical studies, which determined that the liver mass was negative for S100 and SOX10, and established a final diagnosis of epithelioid angiomyolipoma. The key for reaching the correct diagnosis was the morphologic comparison with the original lesion and the evaluation of a wider immunohistochemical profile. For appropriate management in patients with new lesions, even in the context of a patient with known metastatic disease, it is essential to consider other neoplasms in the differential diagnosis.

Topics: Angiomyolipoma; Diagnosis, Differential; Epithelioid Cells; Female; gp100 Melanoma Antigen; Humans; Liver Neoplasms; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Monophenol Monooxygenase; Skin Neoplasms

Perineural invasion, or neurotropism, is defined by the presence of cancer cells either within the neuronal sheath or found along the nerves. In melanoma, it is most commonly associated with invasive desmoplastic melanoma, a melanoma that is most commonly associated with malignant melanoma in situ, lentigo maligna type. Initially, perineural invasion was included in the reported Breslow thickness; however, recent data suggest that it should not be included. In this report, we describe a case of malignant melanoma in situ, lentigo maligna type, with associated neurotropism in the absence of invasive component.

Topics: Aged; Biopsy; Dermis; Follow-Up Studies; Humans; Hutchinson's Melanotic Freckle; Male; Margins of Excision; MART-1 Antigen; Melanoma; Neoplasm Invasiveness; Nerve Fibers; Scalp; SOXE Transcription Factors; Treatment Outcome

The increased use of Mohs micrographic surgery (MMS) to treat melanoma has been accompanied by wide variations in practice patterns and a lack of best practice guidelines.. The present study was a nationwide cross-sectional survey of Mohs surgeons to elucidate commonalities and variations in their use of MMS to treat melanoma.. A cross-sectional analysis was performed using survey responses of Mohs surgeons with membership in the American College of Mohs Surgery.. A total of 210/513 (40.9%) participants used MMS to treat melanoma of any subtype and 123/210 (58.6%) participants within this group treated invasive T1 melanoma (AJCC Eighth Edition) with MMS. A total of 172/210 (81.9%) participants debulked melanoma in situ (MIS). Average margin size of the first Mohs stage for MIS was 4.96 ± 1.74 mm. A total of 149/210 (71.0%) participants used immunohistochemical stains, with 145/149 (97.3%) using melanoma antigen recognized by T-cells 1 (MART-1) in 96.5% of melanoma cases treated with MMS.. Over half of surveyed Mohs surgeons treating melanoma with MMS are treating early invasive melanoma with MMS. Most Mohs surgeons treating melanoma with MMS debulk MIS and virtually all use MART-1 when excising invasive melanoma with MMS.

Topics: Adult; Cross-Sectional Studies; Cytoreduction Surgical Procedures; Female; Humans; Immunohistochemistry; Male; Margins of Excision; MART-1 Antigen; Melanoma; Middle Aged; Mohs Surgery; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Neoplasm Staging; Practice Guidelines as Topic; Practice Patterns, Physicians'; Skin; Skin Neoplasms; Surgeons; Treatment Outcome

A strong association has been reported between chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) and malignant melanoma (MM). In rare cases of MM, lymphoid malignancies may be detected incidentally during sentinel lymph node biopsies. In this case, we found a unique collision of MM and CLL infiltration in the skin. An 88-year-old male patient presented with a mass on the nasal root. Histopathological examination of the skin biopsy specimen revealed a deeply infiltrative, atypical spindle cell proliferation in the background of a collagenous stroma. Accompanying this lesion, there were foci of monotonous lymphoid cell populations involving skin appendages. In the immunohistochemical studies, the spindle cells were diffusely positive for S100, and focally positive for Melan-A and HMB45; the lymphoid cells were positive for CD20, CD5, and Bcl-2 and negative for CD3, Bcl-6, CD10, and Cyclin D1. Histopathological and immunohistochemical findings were consistent with diagnoses of spindle cell melanoma and CLL. Interestingly, these two tumors together in their same morphological appearance were confirmed in a subsequent liver biopsy. Active skin surveillance of patients with CLL may be important to detect MM at an early stage that correlates with a better prognosis.

Topics: Aged, 80 and over; Antigens, CD20; Antineoplastic Agents, Alkylating; Biopsy; CD5 Antigens; Fatal Outcome; Genes, bcl-2; gp100 Melanoma Antigen; Humans; Immunohistochemistry; Leukemia, Lymphocytic, Chronic, B-Cell; Liver; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasms, Multiple Primary; S100 Proteins; Sentinel Lymph Node Biopsy; Skin Neoplasms; Temozolomide

Primary leptomeningeal melanoma is an extremely rare disease of the central nervous system. There are no standard treatment protocols with a poor prognosis in very few reported cases. Immunotherapy in primary brain melanoma has not been successfully applied so far.. We describe a female patient 72-year-old diagnosed in the Neurosurgery Department which presented with generalized seizures.. Histological examination confirmed atypical melanocytes immunohistochemically positive for melan A, HMB45 and S-100 protein in the meninges, BRAF V600E negative. Dermatological, ophthalmological examinations, and 18-FDG PET/CT were negative.. The patient was successfully treated with pembrolizumab 2 mg/kg every 3 weeks for 2 years.. The disease was stable for 2 years and the patient had no significant toxicity.. Our report describes durable intracranial tumor response suggesting the efficacy of PD-1 inhibitor pembrolizumab for central nervous system primary leptomeningeal melanoma.

Topics: Aged; Antibodies, Monoclonal, Humanized; Female; gp100 Melanoma Antigen; Humans; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Meningeal Neoplasms; Programmed Cell Death 1 Receptor; S100 Proteins; Seizures; Treatment Outcome

Metastatic melanoma in sentinel lymph nodes is often elusive to detect with morphology alone. Per American Joint Committee on Cancer staging guidelines, a single atypical melanocyte in lymph node qualifies as metastasis, whether identified by morphology or immunohistochemistry, but single cell staining must be convincing. We propose that the use of a second immunohistochemical run performed on a single slide will allow for more confident diagnosis of micrometastases.. We designed a technical study to determine whether a second antibody application on previously stained slides can successfully detect the same population of cells. Melanocytic neoplasms were stained with SOX-10 using Ventana Benchmark Ultra stainers, coverslipped, and examined, followed by coverslip removal and application of MART-1 (Ventana A103). The order of antibody application and chromagen detection kit (AP-RED vs. DAB) was reversed to establish reliability and robustness of the protocol.. All melanocytes marked with SOX-10 and MART-1, and produced a range of staining quality that varied based on order of stain application and chromagen kit were used. The optimal combination was red MART-1 applied first followed by brown SOX-10 applied second.. Consecutive staining of melanocytes with SOX-10 and MART-1 may improve diagnostic confidence of melanocyte identification, particularly in detection of single cell, micrometastases in sentinel lymph nodes or in situations where dual immunohistochemical stains may be unavailable.

Topics: Biomarkers, Tumor; Diagnosis, Differential; Humans; Immunohistochemistry; Lymphatic Metastasis; MART-1 Antigen; Melanocytes; Melanoma; Neoplasm Staging; Practice Guidelines as Topic; Sentinel Lymph Node; Single-Cell Analysis; Skin Neoplasms; SOXE Transcription Factors; Staining and Labeling

To implement a double-staining technique to identify the most sensitive and specific combinations of melanoma antigen recognized by T cells (Melan-A), microphthalmia-associated transcription factor (MITF), human melanoma black 45 (HMB45), and Ki67 aiming to assist in the diagnosis of atypical melanocytic conjunctival lesions that are more prone to malignant progression.. Eight specimens of conjunctival melanoma and of primary acquired melanosis with moderate to severe atypia were double-immunostained with a combination of a cytoplasmic marker (anti-Melan-A or anti-HMB45), and a nuclear marker (anti-MITF or anti-Ki67). Eight specimens of normal conjunctiva and of conjunctival nevi served as controls. The specimens were processed using 3,3-diaminobenzidine substrate for nuclear stains and the fast-red substrate for cytoplasmic stains. Each slide was analyzed by light microscopy and provided a percent scale and a 0 to 4+ score for each nuclear and cytoplasmic component.. Melan-A and MITF were strongly positive markers for all melanocytic cells, whereas Ki67 and HMB45 provided a variable response for identifying potentially proliferative or aggressive cells. HMB45 and MITF proved to be the best combination for differentiating between atypical and benign lesions on a percent scale and a 0 to 4+ scale (p = 0.0004), with the 3 other combinations providing mainly confirmatory diagnostic information (p < 0.05).. Our study used an immunohistochemical double-staining approach to differentiate between atypical and benign melanocytic lesions of the conjunctiva. Our findings should aid in a more complete immunohistopathological diagnosis of conjunctival melanocytic lesions, particularly in diagnostically difficult cases.

Topics: Biomarkers, Tumor; Conjunctival Neoplasms; Female; gp100 Melanoma Antigen; Humans; Immunohistochemistry; Ki-67 Antigen; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Melanosis; Microphthalmia-Associated Transcription Factor; Middle Aged; Nevus, Pigmented; Retrospective Studies; Staining and Labeling

Vulvar melanosis can occasionally be clinically challenging by mimicking an early melanoma.. To report our experience of initial evaluation and follow-up in this peculiar subset of vulvar melanosis using reflectance confocal microscopy (RCM).. We retrospectively evaluated 18 consecutive cases referred for atypical vulvar pigmentation or for which melanoma was considered and that underwent both RCM examination and histopathological assessment. In 13 cases with available dermoscopic pictures, RCM classification was compared to dermoscopic diagnosis, and in all cases, the density of melanocytes was evaluated on biopsies using MelanA immunostaining.. Among the 18 atypical pigmented lesions, 17 vulvar melanosis and one melanoma were histologically determined. RCM concluded a benign vulvar melanosis in 10 of 17 cases, whereas dermoscopy did so in three of 12 cases. RCM identified the only early malignant lentiginous melanoma. In several cases of vulvar melanosis, RCM could identify foci of melanocytic hyperplasia in an otherwise benign pattern.. In this clinically and dermoscopically challenging subset of vulvar pigmentations, RCM appears relevant for initial extensive evaluation, especially to target initial biopsy sampling, and to perform non-invasive monitoring of foci of melanocytic hyperplasia.

Topics: Adult; Aged; Aged, 80 and over; Biopsy; Dermoscopy; Diagnosis, Differential; Female; Humans; MART-1 Antigen; Melanoma; Melanosis; Microscopy, Confocal; Middle Aged; Retrospective Studies; Skin; Vulvar Neoplasms

Topics: Administration, Intravenous; Aged; Asian People; Docetaxel; Fatal Outcome; Humans; Keratin-7; Male; MART-1 Antigen; Melanoma; Neoplasm Metastasis; Neoplasms, Second Primary; Paget Disease, Extramammary; Scrotum; Skin Neoplasms; Tubulin Modulators

Distinguishing benign nodal nevus from metastatic melanoma can be diagnostically challenging, with important clinical consequences. Recently, the loss of epigenetic marker, 5-hydroxymethylcytosine (5-hmC) expression by immunohistochemistry has been found in melanomas and atypical melanocytic neoplasms.. About 41 metastatic melanomas and 20 nodal nevi were retrieved. Nuclear 5-hmC (brown) and cytoplasmic Melan-A Red (red) double immunohistochemical staining was performed.. Total or partial loss of nuclear expression of 5-hmC was noted in 40/41 metastatic melanomas; these tumor cells were strongly positive for Melan-A Red, except in one case of desmoplastic melanoma. All cases of nodal nevus showed uniformly retained nuclear expression of 5-hmC accompanied by strong Melan-A Red cytoplasmic staining. In two cases containing both nodal nevus and metastatic melanoma, all tumor cells were positive for Melan-A Red, but a nuclear expression of 5-hmC was selectively absent only in the melanoma tumor cells.. Dual 5-hmC/Melan-A Red immunohistochemistry is highly specific in distinguishing nodal nevus from metastatic melanoma. Our protocol for brown and red chromogens used in this study provides excellent color contrast and is easy to interpret. Furthermore, this dual staining method allows the preservation of limited tumor tissue, which could be used for potential molecular studies.

Topics: 5-Methylcytosine; Biomarkers, Tumor; Diagnosis, Differential; Humans; Immunohistochemistry; Lymphatic Metastasis; MART-1 Antigen; Melanoma; Melanoma, Cutaneous Malignant; Nevus, Pigmented; Sentinel Lymph Node; Sentinel Lymph Node Biopsy; Skin Neoplasms; Staining and Labeling

Currently, no universal protocol exists for the assessment of sentinel lymph nodes (SLNs) in cutaneous melanoma. Many institutions use a multistep approach with multiple hematoxylin-eosin (H&E) and immunohistochemical stains. However, this can be a costly and time- and resource-consuming task.. To assess the utility for multistep protocols in the analysis of melanoma SLNs by specifically evaluating the Calgary Laboratory Services (CLS) protocol (which consists of 3 H&E slides and 1 S100 protein, 1 HMB-45, and 1 Melan-A slide per melanoma SLN block) and to develop a more streamlined protocol.. Histologic slides from SLN resections from 194 patients with diagnosed cutaneous melanoma were submitted to the CLS dermatopathology group. Tissue blocks were processed according to the CLS SLN protocol. The slides were re-reviewed to determine whether or not metastatic melanoma was identified microscopically at each step of the protocol. Using SPSS software, a decision tree was then created to determine which step most accurately reflected the true diagnosis.. We found with Melan-A immunostain that 337 of 337 negative SLNs (100%) were correctly diagnosed as negative and 55 of 56 positive nodes (98.2%) were correctly diagnosed as positive. With the addition of an H&E level, 393 of 393 SLNs (100%) were accurately diagnosed.. We recommend routine melanoma SLN evaluation protocols be limited to 2 slides: 1 H&E stain and 1 Melan-A stain. This protocol is both time- and cost-efficient and yields high diagnostic accuracy.

Topics: Adult; Aged; Aged, 80 and over; Coloring Agents; Eosine Yellowish-(YS); Female; gp100 Melanoma Antigen; Hematoxylin; Histological Techniques; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Quality Improvement; S100 Proteins; Sensitivity and Specificity; Sentinel Lymph Node; Sentinel Lymph Node Biopsy; Skin Neoplasms

Topics: Adult; Biomarkers, Tumor; gp100 Melanoma Antigen; Humans; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Nasal Mucosa; Nose Neoplasms; S100 Proteins

The HLA-A*02:01-restricted decapeptide EAAGIGILTV, derived from melanoma antigen recognized by T-cells-1 (MART-1) protein, represents one of the best-studied tumor associated T-cell epitopes, but clinical results targeting this peptide have been disappointing. This limitation may reflect the dominance of the nonapeptide, AAGIGILTV, at the melanoma cell surface. The decapeptide and nonapeptide are presented in distinct conformations by HLA-A*02:01 and TCRs from clinically relevant T-cell clones recognize the nonapeptide poorly. Here, we studied the MEL5 TCR that potently recognizes the nonapeptide. The structure of the MEL5-HLA-A*02:01-AAGIGILTV complex revealed an induced fit mechanism of antigen recognition involving altered peptide-MHC anchoring. This "flexing" at the TCR-peptide-MHC interface to accommodate the peptide antigen explains previously observed incongruences in this well-studied system and has important implications for future therapeutic approaches. Finally, this study expands upon the mechanisms by which molecular plasticity can influence antigen recognition by T cells.

Topics: Amino Acids; Antigen Presentation; Binding Sites; Cells, Cultured; Clone Cells; HLA-A2 Antigen; Humans; Immunodominant Epitopes; Immunotherapy, Adoptive; Lymphocyte Activation; MART-1 Antigen; Melanoma; Peptides; Protein Binding; Protein Conformation; Receptors, Antigen, T-Cell; T-Lymphocytes

Single-institution studies show that frozen section Mohs micrographic surgery (MMS) is an effective treatment modality for cutaneous melanoma, but no multi-institutional studies have been published.. To characterize the use of MMS in the treatment of melanoma at 3 academic and 8 private practices throughout the United States, to recommend excision margins when 100% histologic margin evaluation is not used, and to compare actual costs of tumor removal with MMS vs standard surgical excision.. Prospective, multicenter, cohort study of 562 melanomas treated with MMS with melanoma antigen recognized by T cells 1 immunostaining.. Primary trunk and extremity melanomas (noninvasive and invasive melanoma) achieved histologically negative margins in 97% of tumors with 10-mm margins, whereas 12-mm margins were necessary to achieve histologically negative margins in 97% of head and neck melanomas. Overall average cost per tumor treated was $1328.46.. Nonrandomized and noncontrolled study.. MMS with melanoma antigen recognized by T cells 1 immunostaining safely provides tissue conservation and same-day reconstruction of histologically verified tumor-free margins in a convenient, single-day procedure. When comprehensive margin evaluation is not used, initial surgical margins of at least 10 mm for primary trunk/extremity and 12 mm for head/neck melanomas should be used to achieve histologically negative margins 97% of the time.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Female; Hospital Costs; Humans; Male; Margins of Excision; MART-1 Antigen; Melanoma; Middle Aged; Mohs Surgery; Prospective Studies; Skin; Skin Neoplasms; Treatment Outcome; United States; Young Adult

To illustrate a case of sensorineural hearing loss (SNHL) after immunotherapy based on T cell receptor (TCR) gene therapy using modified T cells recognizing melanoma antigen recognized by T cells 1 for disseminated melanoma.. We present a 59-year-old woman with profound subacute bilateral SNHL including unilateral deafness after immunotherapy based on TCR gene therapy using modified T cells recognizing melanoma antigen recognized by T cells 1 for disseminated melanoma. Ten days after treatment, the patient developed hearing loss of 57 dB hearing loss air conduction at pure-tone average 0.5-1-2-4 kHz in the right ear, and >100 dB hearing loss air conduction at pure-tone average 0.5-1-2-4 in the left ear. The right ear recovered partially, while the left ear remained deaf, despite oral prednisolone (1.0 mg/kg) and salvage treatment with three transtympanic injections of 0.5 ml dexamethasone (4.0 mg/ml).. Based on our presented case and a vast amount of literature there is circumstantial evidence that TCR gene therapy for melanoma targets the perivascular macrophage-like melanocytes in the stria vascularis, resulting in SNHL. We suggest that SNHL after TCR gene therapy may be caused by a disruption of the blood-labyrinth-barrier and the endolymphatic potential and/or a sterile inflammation of the stria vascularis. In severe cases like our subject, we posit that endolymphatic hydrops or hair cell loss may cause irreversible and asymmetrical deafness. Steroid prophylaxis via transtympanic application is debatable.

Topics: Female; Hearing Loss, Sensorineural; Humans; Immunotherapy, Adoptive; MART-1 Antigen; Melanoma; Melanoma, Cutaneous Malignant; Middle Aged; Skin Neoplasms

Topics: Aged; Biomarkers, Tumor; Biopsy; Esophageal Neoplasms; Esophagectomy; Esophagoscopy; Humans; Immunohistochemistry; Lymph Node Excision; Male; MART-1 Antigen; Melanoma; S100 Proteins; Skin Neoplasms

Human blood monocytes are very potent to take up antigens. Like macrophages in tissue, they efficiently degrade exogenous protein and are less efficient than dendritic cells (DCs) at cross-presenting antigens to CD8

Topics: Antigen Presentation; CD8-Positive T-Lymphocytes; Cell Differentiation; Cross-Priming; Dendritic Cells; Humans; MART-1 Antigen; Melanoma; Monocytes

Although immune therapies with checkpoint inhibitors have gained increasing attention in advanced and metastatic melanoma, interferon-α remains a standard therapy for nonmetastatic malignant melanoma with risk factors. Interferons can successfully prevent relapse; however, the response rate is still not as high as would be desired. Prognostic tools to predict the response are required, which could lead to more individualized treatment regimens. In numerous studies over the past decade, circulating epithelial tumor cells (CETCs) have been shown to be a promising biomarker for estimating the risk of metastatic relapse, and we sought to determine whether they can also be used for this purpose in malignant melanoma. To establish a prognostic tool for patients with melanoma, we quantified CETCs over the course of interferon treatment in 49 patients. Patients were categorized into two groups according to the behavior of their circulating tumor cells during the interferon treatment: those with increasing and those with decreasing numbers of circulating tumor cells. Patients with increasing numbers of circulating tumor cells had a significantly higher risk of relapse. Kaplan-Meier survival analysis showed a significant difference between patients with increasing CETC numbers (mean survival time: 2.6 years) and patients with decreasing or stable CETC numbers (mean survival time: 12.6 years) (P=0.001). Quantification of CETCs could prove to be a prognostic marker for patients with melanoma receiving interferon immunotherapy. Further studies should determine whether these results are applicable to other immunotherapies, for example, immune checkpoint inhibition.

Topics: Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Epithelial Cell Adhesion Molecule; Epithelial Cells; Female; Follow-Up Studies; Humans; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Neoplasm Recurrence, Local; Neoplastic Cells, Circulating; Prognosis; Skin Neoplasms; Survival Rate

Increased numbers of CD8

Topics: Adult; CD28 Antigens; CD8-Positive T-Lymphocytes; Complementarity Determining Regions; Cytotoxicity, Immunologic; Ear Neoplasms; Humans; Immunity, Cellular; Immunologic Memory; Male; Margins of Excision; MART-1 Antigen; Melanoma; Receptors, Antigen, T-Cell; Recurrence; Remission Induction; T-Cell Antigen Receptor Specificity

Histologic differentiation of melanoma in situ (MIS) from solar keratosis on chronically sun-damaged skin is challenging. The first-line immunostain is usually MART-1/Melan-A, which can exaggerate the epidermal melanocytes, causing a diagnostic pitfall for MIS. By comparing MART-1 and SOX10 immunostaining, we scored the percentage of epidermal melanocytes per 2-mm diameter fields in pigmented actinic keratosis (n = 16), lichenoid keratosis (n = 7), junctional melanocytic nevus (n = 6), keratosis with atypical melanocytic proliferation (n = 17) and MIS (n = 10). These cases represented an older population (68 years median age) and the head and neck (50%) was the most common anatomic site. MART-1 score was significantly higher than SOX10 (P value <.05) in solar keratoses, but showed no difference in detecting melanocytic proliferations, demonstrating their equal detection rate of melanocytes. The sensitivity of both MART-1 and SOX10 was 100%, while their specificities were 17% and 96%, respectively. These results show that SOX10 is more specific than MART-1 in distinguishing epidermal melanocytes on sun-damaged skin by avoiding overdiagnosis of melanoma.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Female; Humans; Hyperplasia; Immunohistochemistry; Keratosis, Actinic; Male; MART-1 Antigen; Melanocytes; Melanoma; Middle Aged; Sensitivity and Specificity; SOXE Transcription Factors; Sunlight

The histopathologic differentiation between Spitz nevus and melanoma is of particular interest in routine diagnostic procedures of melanocytic tumors. Atypical Spitz nevi are sometimes difficult to distinguish from melanoma. There is still no single criterion that ensures a distinction of melanoma and atypical Spitz nevus. The aim of this study was to reevaluate established and new criteria to differentiate Spitz nevus from melanoma more reliably. We analyzed 25 melanomas with a Breslow index ≥ 1 mm and 18 classical compound Spitz nevi concerning their histopathologic, immunohistochemical and molecular genetic characteristics. Moreover, clinical follow-up data for 5 years were collected. We found statistically significant differences between Spitz nevus and melanoma for the following features: pagetoid spread, atypia, maturation, elastosis, Kamino bodies, p16 expression, and the staining pattern of HMB45. BRAF was positive in 7/21 melanomas and in 1/14 Spitz nevi. Fluorescence in situ hybridization confirmed the histopathologic diagnosis in 36/37 cases. The established clinical, histopathologic, and immunohistochemical criteria to differentiate Spitz nevus and melanoma could be reproduced in our collective. Especially, the expression of p16, BRAF analysis and fluorescence in situ hybridization proved to be helpful tools to improve the differentiation of Spitz nevus and melanoma in our study. Nevertheless, there is-until now-no reliable histopathologic and immunohistochemical parameter which can discriminate Spitz nevus and melanoma with absolute certainty.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Cyclin-Dependent Kinase Inhibitor p16; Diagnosis, Differential; Female; Follow-Up Studies; gp100 Melanoma Antigen; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Nevus, Epithelioid and Spindle Cell; Severity of Illness Index; Skin Neoplasms

Melanocytic immunostains can assist in margin evaluation of melanoma in situ (MIS) excisions; however, their accuracy and reliability relative to hematoxylin & eosin (H&E) is yet to be determined.. The objective of this study was to evaluate the sensitivity, specificity, and concordance of 4 melanocyte-specific immunostains for diagnosing MIS occurring on chronically sun-damaged skin.. Serial permanent sections from representative areas of negative margin and residual tumor were stained using H&E, MITF, MART-1, SOX10, and R21 and examined in a blinded fashion. The study set included 100 digital microscopy images from 10 cases of MIS excisions from the face. Two board-certified dermatopathologists, 4 fellowship-trained Mohs surgeons, 2 Mohs fellows, and 2 dermatology residents independently reviewed the 100 images.. The average melanocyte density was 11 versus 28 melanocytes per 0.5 mm for chronically sun-damaged skin versus residual MIS on H&E, respectively. Statistically significantly higher melanocyte densities were observed using MITF, MART-1, and SOX10 on negative margins. The sensitivity and interobserver concordance was highest using MITF and SOX10. The intraobserver agreement on 4 duplicate images was 85%.. In conclusion, the nuclear immunostains (MITF and SOX10) show the most promise for improving the diagnosis of MIS in chronically sun-damaged skin.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Humans; MART-1 Antigen; Melanocytes; Melanoma; Microphthalmia-Associated Transcription Factor; Neoplasm, Residual; Observer Variation; Sensitivity and Specificity; Skin Aging; Skin Neoplasms; SOXE Transcription Factors

Adoptive cell therapy (ACT) consisting of genetically engineered T cells expressing tumor antigen-specific T-cell receptors displays robust initial antitumor activity, followed by loss of T-cell activity/persistence and frequent disease relapse. We characterized baseline and longitudinal T-cell phenotype variations resulting from different manufacturing and administration protocols in patients who received ACT. Patients with melanoma who enrolled in the F5-MART-1 clinical trial (NCT00910650) received infusions of MART-1 T-cell receptors transgenic T cells with MART-1 peptide-pulsed dendritic cell vaccination. Patients were divided into cohorts based on several manufacturing changes in the generation and administration of the transgenic T cells: decreasing ex vivo stimulation/expansion time, increased cell dose, and receiving fresh instead of cryopreserved cells. T-cell phenotypes were analyzed by flow cytometry at baseline and longitudinally in peripheral blood. Transgenic T cells with shorter ex vivo culture/expansion periods displayed significantly increased expression of markers associated with less differentiated naive/memory populations, as well as significantly decreased expression of the inhibitory receptor programmed death 1 (PD1). Patients receiving fresh infusions of transgenic cells demonstrated expansion of central memory T cells and delayed acquisition of PD1 expression compared with patients who received cryopreserved products. Freshly infused transgenic T cells showed persistence and expansion of naive and memory T-cell populations and delayed acquisition of PD1 expression, which correlated with this cohort's superior persistence of transgenic cells and response to dendritic cell vaccines. These results may be useful in designing future ACT protocols.

Topics: Adult; Aged; Cancer Vaccines; Cells, Cultured; Cryopreservation; Female; Humans; Immunologic Memory; Immunophenotyping; Immunotherapy, Adoptive; Lymphocyte Activation; Male; MART-1 Antigen; Melanoma; Middle Aged; Phenotype; Skin Neoplasms; T-Lymphocytes, Cytotoxic

Topics: Aged; Anterior Capsule of the Lens; Biomarkers, Tumor; Cataract Extraction; Eye Neoplasms; Humans; Immunoenzyme Techniques; Lens Diseases; Male; MART-1 Antigen; Melanoma; Skin Neoplasms

Malignant gastrointestinal melanoma is usually a metastatic lesion. We report the case of a 67-year-old female patient who presented with intermittent abdominal pain, fever, rigor and diarrhoea. CT scan of the abdomen revealed a large mass at the right iliac fossa with features concerning for intra-abdominal abscess. Exploratory laparotomy confirmed the preoperative diagnosis of abscess, and a right hemicolectomy was performed. Histopathological examination of the surgical specimen was indicative of malignant melanoma, and immunohistochemical examination showed positivity to S100 protein, Melan-A, HMB-45 and vimentin. A series of postoperative clinical, laboratory and imaging examinations revealed no suspicious lesions in the skin, eye, leptomeninges or other sites. Therefore, the diagnosis of primary colonic melanoma was confirmed. Only 36 additional cases of primary colonic melanoma have been reported to date. These rare neoplasms are challenging to diagnose and usually require a multidisciplinary treatment approach, including surgery, chemotherapy and possibly immunotherapy or radiotherapy.

Topics: Abdomen; Abdominal Pain; Aftercare; Aged; Biomarkers, Tumor; Chemotherapy, Adjuvant; Colectomy; Colon, Ascending; Colonic Neoplasms; Diarrhea; Female; gp100 Melanoma Antigen; Humans; Laparotomy; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Rare Diseases; S100 Proteins; Tomography, X-Ray Computed; Treatment Outcome; Vimentin

Adult wild-type mice are not supposed to be proper models for ultraviolet radiation (UVR)-induced melanoma since melanocytes are confined to hair follicles and cannot be sufficiently reached by UVR. On the other hand, in mutated mouse models used for melanoma research limitations, including an altered immune system and selection of affected pathways, lead to tumors phenotypically quite different from naturally occurring melanomas. We compared the distribution of epidermal melanocytes in UVR and not-UVR-exposed wild-type C57BL/6 mice. Starting at the age of 8 weeks, mice were exposed to physiologic doses of UVR three times weekly over 16 weeks. Back skin biopsies were taken 4, 8, 12 and 16 weeks after initiation of exposure, and stained for Melan-A, representing a highly selective marker for melanocytes. Surprisingly, after exposure to UVR, Melan-A positive cells were detected also in the interfollicular epidermis of C57BL/6 mice. We conclude that UVR is capable of inducing interfollicular epidermal melanocytes in wild-type mice.

Topics: Animals; Biomarkers; Biopsy; Disease Models, Animal; Epidermal Cells; Epidermis; Female; Hair Follicle; Humans; MART-1 Antigen; Melanocytes; Melanoma; Mice; Mice, Inbred C57BL; Ultraviolet Rays

A promising arsenal of targeted and immunotherapy treatments for metastatic melanoma has emerged over the last decade. With these therapies, we now face new mechanisms of tumor-acquired resistance. We report here a patient whose metastatic melanoma underwent dedifferentiation as a resistance mechanism to adoptive T-cell transfer therapy (ACT) to the MART1 antigen, a phenomenon that had been observed only in mouse studies to date. After an initial period of tumor regression, the patient presented in relapse with tumors lacking melanocytic antigens (MART1, gp100) and expressing an inflammation-induced neural crest marker (NGFR). We demonstrate using human melanoma cell lines that this resistance phenotype can be induced

Topics: Cell Dedifferentiation; Cell Line, Tumor; Coculture Techniques; Drug Resistance, Neoplasm; Humans; Immunotherapy, Adoptive; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Metastasis; Nerve Tissue Proteins; Nevus, Pigmented; Receptors, Chimeric Antigen; Receptors, Nerve Growth Factor; Recurrence

Most tumor-associated proteins are located inside tumor cells and thus are not accessible to current marketed therapeutic monoclonal antibodies or their cytotoxic conjugates. Human leukocyte antigen (HLA) class I can present peptides derived from intracellular tumor-associated proteins and somatically mutated proteins on the cell's surface, forming an HLA/peptide complex as tumor-specific antigens for T cell receptor (TCR) recognition. Therefore, HLA-mediated presentation of intracellular tumor antigen peptides provides a viable way to distinguish tumor cells from normal cells, which is important for broadening antigen selection, especially for antibody-drug conjugates (ADCs) regarding their highly cytotoxic payload. We applied sortase A-mediated conjugation to develop TCR-like ADCs (i.e., EA1 HL-vcMMAE) targeting intracellular MART-1 protein, a melanocyte-differentiating antigen specific for metastatic melanomas, via the cell surface HLA-A2/MART-1

Topics: Aminoacyltransferases; Animals; Antibody Specificity; Antigen Presentation; Antineoplastic Agents; Bacterial Proteins; Cell Line, Tumor; Cysteine Endopeptidases; Endocytosis; Female; Histocompatibility Antigens Class I; Immunoconjugates; Intracellular Space; MART-1 Antigen; Melanoma; Mice, SCID; Peptides; Protein Binding; Pyridones; Pyrimidinones; Receptors, Antigen, T-Cell

Actinic keratosis (AK) and squamous cell carcinoma in-situ (SCCIS) within or near melanoma in situ (MIS) can complicate diagnosis due to overlapping clinical and microscopic features. This study aimed to describe basilar melanocyte density and pagetoid spread in AK and SCCIS for improved diagnostic accuracy.. A total of 22 AK and 22 SCCIS biopsies containing a margin of uninvolved epidermis were immunostained with MART-1 (melanoma antigen recognized by T-cells 1). The basilar melanocyte:keratinocyte ratio and the number and distribution of pagetoid melanocytes were compared in AK, SCCIS, and uninvolved epidermis. An in-vitro human skin model was created to assess the impact of keratinocyte atypia on melanocyte distribution.. The median basilar melanocyte:keratinocyte ratio in SCCIS (1:11.49) was lower than in uninvolved epidermis (1:5.59, P = 0.0011), and the ratio in AK (1:6.94) was similar to uninvolved epidermis (P = 0.987). Pagetoid melanocytes were absent in perilesional skin but common in AK (21/22, P < 0.0001) and SCCIS (22/22, P < 0.0001). Pagetoid melanocytes at or above the mid-spinous layer were more common in SCCIS (21/22) vs AK (7/22, P < 0.0001). Pagetoid melanocytes were present in the in-vitro skin model made with neoplastic but not normal keratinocytes.. Pagetoid melanocytes in AK and SCCIS should be interpreted with caution to avoid overdiagnosis of MIS.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma in Situ; Carcinoma, Squamous Cell; Diagnostic Errors; Female; Humans; Keratinocytes; Keratosis, Actinic; Male; MART-1 Antigen; Melanocytes; Melanoma; Middle Aged; Skin Neoplasms

The efficacy of Mohs micrographic surgery (MMS) for atypical intraepidermal melanocytic proliferation (AIMP) is unknown.. To ascertain the frequency of diagnostic change to melanoma (upstaging) and the frequency of local recurrence after MMS for AIMP. A secondary outcome was the frequency of subclinical spread (defined as the requirement for >1 stage of MMS to achieve tumor-free margins).. Retrospective, cross-sectional study of 223 AIMP (with 92.4% located on the head, neck, hand, foot, or pretibial leg) patients treated with MMS with melanoma antigen recognized by T cells 1 (MART-1) immunostaining.. Upstaging to unequivocal melanoma in situ or invasive melanoma was identified in 18.8% (42/223) of all AIMP patients. The local recurrence rate was 0% (0/223) with a mean follow-up time of 2.7 years (998 days). Subclinical spread was present in 23.8% (53/223) of AIMP patients.. Single site, retrospective design, observational study, lack of objective criteria to diagnose AIMP.. MMS with MART-1 immunostaining achieves excellent local control of specialty site AIMP and permits definitive removal of subclinical spread before reconstruction. The central debulking excision should be evaluated with formalin-fixed paraffin-embedded section staining, since a significant percentage of AIMP are reclassified as melanoma in situ or invasive melanoma.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Biopsy; Cross-Sectional Studies; Diagnostic Errors; Epidermis; Female; Follow-Up Studies; Humans; Hutchinson's Melanotic Freckle; Male; MART-1 Antigen; Melanocytes; Melanoma; Middle Aged; Mohs Surgery; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Neoplasm Staging; Precancerous Conditions; Retrospective Studies; Skin Neoplasms; T-Lymphocytes; Young Adult

The diagnosis of malignant peripheral nerve sheath tumour (MPNST) may be challenging, especially in the sporadic setting. Owing to the lack of specific histological criteria, immunohistochemical and molecular diagnostic markers, several differential diagnoses must be considered, especially melanoma. Indeed, although S100 protein usually stains melanoma, other melanocytic markers are often negative, especially in spindle cell/desmoplastic types. This pattern of immunoreactivity resembles that of some nerve-derived tumours such as MPNST. Owing to their different clinical behaviours and therapeutic implications, accurate identification of these two different tumours is crucial.. S100, SOX10, KBA62, MITF, HMB45, Melan-A, tyrosinase PNL2 and BRAF-V600E immunostaining was performed in a pathologically and genetically well-characterised cohort of primary MPNST (n = 124), including 66 (53%) NF1-associated tumours. Sox10 and KBA62 expression were found, respectively, in 102 (84%) and in 101 (83%) MPNST, whereas S100 was expressed in 64 cases (52%). We observed an increased loss of S100 with increasing histological grade (P = 0.0052). We found Melan-A expression in 14% (n = 17) of all MPNST, occurring in 82% (n = 14) of cases in an NF1 context. Six per cent (n = 8) of MPNST showed tyrosinase positivity, including seven (87%) NF1-associated. MITF expression was found in 10 (8%) MPNST. None expressed PNL2, HMB45 or BRAF-V600E.. MPNST (in NF1 and a sporadic setting) can quite often be positive for Melan-A, tyrosinase and MITF. Pathologists should be cognisant of these exceptions to prevent confusion with melanoma.

Topics: Biomarkers, Tumor; Diagnosis, Differential; Female; Humans; Male; MART-1 Antigen; Melanocytes; Melanoma; Monophenol Monooxygenase; Neurofibrosarcoma; Proto-Oncogene Proteins B-raf; S100 Proteins; SOXE Transcription Factors

Non-small cell lung cancer (NSCLC) and melanoma are frequent entities in routine diagnostics. Whereas the differential diagnosis is usually straight forward based on histomorphology, it can be challenging in poorly differentiated tumors as melanoma may mimic various histological patterns. Distinction of the two entities is of outmost importance as both are treated differently. HMB45 and MelanA are recommended immunohistological markers for melanoma in this scenario. SOX10 has been described as an additional marker for melanoma. However, comprehensive large-scale data about the expression of melanoma markers in NSCLC tumor tissue specimen are lacking so far.. Therefore, we analyzed the expression of these markers in 1085 NSCLC tumor tissue samples. Tissue microarrays of NSCLC cases were immunohistochemically stained for HMB45, MelanA, and SOX10. Positivity of a marker was defined as ≥1% positive tumor cells.. In 1027 NSCLC tumor tissue samples all melanoma as well as conventional immunohistochemical markers for NSCLC could be evaluated. HMB45, MelanA, and SOX10 were positive in 1 (< 1%), 0 (0%) and 5 (< 1%) cases. The HMB45 positive case showed co-expression of SOX10 and was classified as large cell carcinoma. Three out of five SOX10 positive cases were SqCC and one case was an adenosquamous carcinoma.. Expression of HMB45, MelanA and SOX10 is evident but exceedingly rare in NSCLC cases. Together with conventional immunomarkers a respective marker panel allows a clear-cut differential diagnosis even in poorly differentiated tumors.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Diagnosis, Differential; Female; gp100 Melanoma Antigen; Humans; Lung Neoplasms; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; SOXE Transcription Factors

Antigen recognition by T cells bearing αβ T cell receptors (TCRs) is restricted by major histocompatibility complex (MHC). However, how antigens are recognized by T cells bearing γδ TCRs remains unclear. Although γδ T cells can recognize nonclassical MHC, it is generally thought that recognition of antigens is not MHC restricted. Here, we took advantage of an in vitro system to generate antigen-specific human T cells and show that melanoma-associated antigens, MART-1 and gp100, can be recognized by γδ T cells in an MHC-restricted fashion. Cloning and transferring of MART-1-specific γδ TCRs restored the specific recognition of the initial antigen MHC/peptide reactivity and conferred antigen-specific functional responses. A crystal structure of a MART-1-specific γδ TCR, together with MHC/peptide, revealed distinctive but similar docking properties to those previously reported for αβ TCRs, recognizing MART-1 on HLA-A*0201. Our work shows that antigen-specific and MHC-restricted γδ T cells can be generated in vitro and that MART-1-specific γδ T cells can also be found and cloned from the naïve repertoire. These findings reveal that classical MHC-restricted human γδ TCRs exist in the periphery and have the potential to be used in developing of new TCR-based immunotherapeutic approaches.

Topics: Adult; Cells, Cultured; Crystallography, X-Ray; Humans; MART-1 Antigen; Melanoma; Models, Molecular; Receptors, Antigen, T-Cell, gamma-delta; T-Lymphocytes

Melanoma is the second most common non-hematopoietic malignancy after carcinomas to metastasize to the breast and often appears as a well-circumscribed, dense nodule on imaging. Although metastatic lesions presenting as bilateral cysts have been reported, this presentation is not common and may mimic benign breast cysts. We present a challenging case of metastatic melanoma presenting as bilateral breast cysts with spindled cytomorphology in a patient with a history of mammary carcinoma. Discordance between the spindled cytomorphology and the morphology of the core biopsy, which was similar to the patient's primary breast cancer, allowed for entertainment of other tumors and disease processes. Confirmatory immunostaining of the cytology material with HMB-45 was important to establish the diagnosis of metastatic melanoma. Diagn. Cytopathol. 2017;45:446-451. © 2017 Wiley Periodicals, Inc.

Topics: Biomarkers, Tumor; Biopsy, Large-Core Needle; Breast Cyst; Breast Neoplasms; Diagnosis, Differential; Female; gp100 Melanoma Antigen; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; S100 Proteins; Skin Neoplasms

Measurement of melanoma depth of invasion (DoI) in skin tissues is of great significance in grading the severity of skin disease and planning patient's treatment. However, accurate and automatic measurement of melanocytic tumor depth is a challenging problem mainly due to the difficulty of skin granular identification and melanoma detection. In this paper, we propose a technique for measuring melanoma DoI in microscopic images digitized from MART1 (i.e., meleanoma-associated antigen recognized by T cells) stained skin histopathological sections. The technique consists of four modules. First, skin melanoma areas are detected by combining color features with the Mahalanobis distance measure. Next, skin epidermis is segmented by a multi-thresholding method. The skin granular layer is then identified based on Bayesian classification of segmented skin epidermis pixels. Finally, the melanoma DoI is computed using a multi-resolution approach with Hausdorff distance measurement. Experimental results show that the proposed technique provides a superior performance in measuring the melanoma DoI than two closely related techniques.

Topics: Humans; MART-1 Antigen; Melanoma; Melanoma, Cutaneous Malignant; Neoplasm Grading; Neoplasm Invasiveness; Skin; Skin Neoplasms

Topics: Aged; Biomarkers, Tumor; Biopsy; Chemoradiotherapy; Disease Progression; Duodenal Neoplasms; Endoscopy, Gastrointestinal; Fatal Outcome; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Melena; Skin Neoplasms; Stomach Neoplasms; Treatment Outcome

Cytotoxic T lymphocyte (CTL)-based immunotherapies have had remarkable success at generating objective clinical responses in patients with advanced metastatic melanoma. Although the melanocyte differentiation antigens (MDA) MART-1, PMEL, and tyrosinase were among the first melanoma tumor-associated antigens identified and targeted with immunotherapy, expression within normal melanocytes of the eye and inner ear can elicit serious autoimmune side effects, thus limiting their clinical potential as CTL targets. Using a tandem mass spectrometry (MS) approach to analyze the immunopeptidomes of 55 melanoma patient-derived cell lines, we identified a number of shared HLA class I-bound peptides derived from the melanocyte-specific transporter protein SLC45A2. Antigen-specific CTLs generated against HLA-A*0201- and HLA-A*2402-restricted SLC45A2 peptides effectively killed a majority of HLA-matched cutaneous, uveal, and mucosal melanoma cell lines tested (18/25). CTLs specific for SLC45A2 showed significantly reduced recognition of HLA-matched primary melanocytes that were, conversely, robustly killed by MART1- and PMEL-specific T cells. Transcriptome analysis revealed that SLC45A2 mRNA expression in normal melanocytes was less than 2% that of other MDAs, therefore providing a more favorable melanoma-to-melanocyte expression ratio. Expression of SLC45A2 and CTL sensitivity could be further upregulated in BRAF(V600E)-mutant melanoma cells upon treatment with BRAF or MEK inhibitors, similarly to other MDAs. Taken together, our study demonstrates the feasibility of using tandem MS as a means of discovering shared immunogenic tumor-associated epitopes and identifies SLC45A2 as a promising immunotherapeutic target for melanoma with high tumor selectivity and reduced potential for autoimmune toxicity.

Topics: Antigen Presentation; Antigens, Neoplasm; Cytotoxicity, Immunologic; Epitopes; gp100 Melanoma Antigen; HLA-A2 Antigen; HLA-A24 Antigen; Humans; Immunotherapy; MART-1 Antigen; Melanocytes; Melanoma; Membrane Transport Proteins; Peptides; Proto-Oncogene Proteins B-raf; T-Lymphocytes, Cytotoxic; Tandem Mass Spectrometry; Transcriptome

Malignant melanoma is one of the most common malignant diseases of the gastrointestinal tract. It has been reported that the malignant melanoma metastasizes not only to the small intestine due to the abundant blood supply, but also to the stomach, colon, and esophagus. Gastrointestinal metastasis is usually suspected depending on the clinical symptoms, as well as based on radiological or endoscopic findings. Imunohistochemical stains, such as Melan-A/Melanoma antigen recognized by T cell-1 or human melanoma black-45, are useful for confirming the diagnosis of malignant melanoma. A 44-year-old male received an operation due to a malignant melanoma at the left thumb two years ago. On the national health screening endoscopy, a submucosal tumor with hyperemic change on the top was found. The final diagnosis was a metastatic malignant melanoma in the stomach, pancreas, and pelvic bone. We recommend that endoscopists should consider the potential malignancy of subepithelial tumor with mucosa change, despite the tumor size being less than 1 cm.

Topics: Adult; Bone Neoplasms; Endoscopy, Digestive System; Gastrointestinal Neoplasms; gp100 Melanoma Antigen; Humans; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Melanoma, Cutaneous Malignant; Pancreatic Neoplasms; Positron Emission Tomography Computed Tomography; Skin Neoplasms

Topics: Adult; Biomarkers, Tumor; Calmodulin-Binding Proteins; Diagnosis, Differential; Gastrointestinal Neoplasms; Gastrointestinal Stromal Tumors; High-Throughput Nucleotide Sequencing; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Keratins; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Recurrence, Local; Neuroectodermal Tumors; RNA-Binding Proteins; S100 Proteins; Sarcoma, Ewing; SOXE Transcription Factors; Synaptophysin

Therapeutic strategies using anti-PD-1-blocking antibodies reported unparalleled effectiveness for melanoma immunotherapy, but deciphering immune responses modulated by anti-PD-1 treatment remains a crucial issue. Here, we analyzed the composition and functions of the large Melan-A-specific T-cell repertoire in the peripheral blood of 9 melanoma patients before and after 2 months of treatment with anti-PD-1. We observed amplification of Melan-A-specific Vß subfamilies undetectable before therapy (thereafter called emerging Vß subfamilies) in responding patients, with a predominant expansion in patients with a complete response. These emerging Vß subfamilies displayed a higher functional avidity for their cognate antigen than Vß subfamilies not amplified upon anti-PD-1 therapy and could be identified by a sustained coexpression of PD-1 and TIGIT receptors. Thus, in addition to the emergence of neoantigen-specific T cells previously documented upon anti-PD-1 therapy, our work describes the emergence of high-avidity Melan-A-specific clonotypes as a surrogate marker of treatment efficacy.

Topics: Antibodies, Monoclonal; Antibody Affinity; Antibody Formation; Antigens, Neoplasm; Cells, Cultured; Clone Cells; Humans; Immunotherapy; MART-1 Antigen; Melanoma; Programmed Cell Death 1 Receptor; Substrate Specificity; Treatment Outcome

To establish a mouse model with the aim of studying the tumour biology and metastasis formation of uveal melanoma.. Two human primary uveal melanoma cell lines (UMT2 and UMT42) were injected into the choroid of BALB/c nude mouse eyes. Intraocular tumour growth and metastasis formation in the liver and lungs were assessed after 13 to 22 weeks. Formalin-fixed, paraffin-embedded material was processed via haematoxylin and eosin staining for histological examination and periodic acid Schiff staining to search for extravascular matrix patterns. Immunohistochemistry for Melan A, CD34 and Ki67 was performed to assess the expression of a melanocytic lineage marker, angiogenesis and proliferative activity.. All eyes injected with UMT2 cells, but only 25% of eyes treated with UMT42, developed intraocular tumour growth. The morphology of intraocular melanomas resembled that of primary tumours and showed signs of malignancy, including retinal invasion, optic nerve invasion and scleral penetration with extraocular tumour growth. UMT2 tumours formed extravascular matrix patterns exclusively. Most of the tumour cells expressed Melan A. Intratumoural angiogenesis was detected in both tumour entities. Proliferative activity was verified in all but one tumour. However, no metastases appeared in the liver or lungs.. The mouse model presented with the UMT2 cell line allows for investigations of tumour biology of the primary UM because of the high degree of similarity between the tumours generated in the mouse eyes and the corresponding primary human UM. Unfortunately, the model is not suitable for investigations of metastasis formation.

Topics: Animals; Antigens, CD34; Biomarkers, Tumor; Cell Line, Tumor; Disease Progression; Follow-Up Studies; Humans; Immunohistochemistry; Ki-67 Antigen; MART-1 Antigen; Melanoma; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasms, Experimental; Neovascularization, Pathologic; Pilot Projects; Retinal Neoplasms; Uveal Neoplasms

Although malignant melanomas exhibit a wide range of immunophenotypes, concurrent loss of all 3 conventional melanocytic markers (S-100, Melan-A, and HMB-45) is relatively rare. We report a case of primary malignant melanoma with lymph node metastasis, both exhibiting loss of immunoreactivity for conventional melanocytic markers, while aberrantly expressing epithelial antigenicity (pancytokeratin, CAM 5.2).

Topics: Biomarkers, Tumor; gp100 Melanoma Antigen; Humans; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Melanoma, Cutaneous Malignant; Middle Aged; S100 Proteins; Skin Neoplasms

The development of BRAF V600 and MEK inhibitors constitutes a breakthrough in the treatment of patients with BRAF-mutated metastatic melanoma. However, although there is an increase in overall survival, these patients generally confront recurrence, and several resistance mechanisms have already been described. In the present study we describe a different resistance mechanism. After several weeks of long‑term in vitro treatment of two different V600E BRAF‑mutated melanoma cell lines with MARK inhibitors, PLX4032 and/or GDC-0973, the majority of the cells died whereas some remained viable and quiescent (SUR). Markedly, discontinuing treatment of SUR cells with MAPK inhibitors allowed the population to regrow and these cells retained drug sensitivity equal to that of the parental cells. SUR cells had increased expression levels of CD271 and ABCB5 and presented senescence-associated characteristics. Notably, SUR cells were efficiently lysed by cytotoxic T lymphocytes recognizing MART-1 and gp100 melanoma differentiation antigens. We propose quiescent plasticity as a mechanism of resistance to BRAF and MEK inhibitors while retaining sensitivity to immune effectors.

Topics: Animals; Apoptosis; Azetidines; Blotting, Western; CD8-Positive T-Lymphocytes; Cell Cycle; Cell Proliferation; Drug Resistance, Neoplasm; Enzyme Inhibitors; Flow Cytometry; gp100 Melanoma Antigen; Humans; Immunoenzyme Techniques; In Vitro Techniques; Indoles; MAP Kinase Signaling System; MART-1 Antigen; Melanoma; Mice; Mice, Inbred NOD; Mice, SCID; Mitogen-Activated Protein Kinases; Piperidines; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sulfonamides; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured; Vemurafenib; Xenograft Model Antitumor Assays

Indications to treat invasive melanoma with Mohs micrographic surgery (MMS) or analogous techniques with exhaustive microscopic margin assessment have not been defined.. Identify clinical and histologic factors associated with subclinical spread of invasive melanoma.. This retrospective, cross-sectional study evaluated 216 invasive melanomas treated with MMS and melanoma antigen recognized by T cells 1 immunostaining. Logistic regression models were used to correlate clinicopathologic risk factors with subclinical spread and construct a count prediction model.. Risk factors associated with subclinical spread by multivariate analysis included tumor localization on the head and neck (OR 3.28, 95% confidence interval [CI] 1.16-9.32), history of previous treatment (OR 4.18, 95% CI 1.42-12.32), age ≥65 (OR 4.45, 95% CI 1.29-15.39), and ≥1 mitoses/mm. This study was conducted at a single academic institution with a small study population using a retrospective study design that was subject to potential referral bias.. Clinical and histologic factors identify invasive melanomas that are at increased risk for subclinical spread and might benefit from MMS or analogous techniques prior to reconstruction.

Topics: Adult; Age Factors; Aged; Cross-Sectional Studies; Female; Head and Neck Neoplasms; Humans; Hutchinson's Melanotic Freckle; Immunocompetence; Male; MART-1 Antigen; Melanoma; Melanoma, Cutaneous Malignant; Middle Aged; Mitotic Index; Mohs Surgery; Neoplasm Recurrence, Local; Neoplasm, Residual; Plastic Surgery Procedures; Retrospective Studies; Risk Factors; Skin Neoplasms; T-Lymphocytes

The detection of primary anorectal melanoma on anal cytology is a rare and challenging diagnosis. We report a case where anorectal cytology showed isolated malignant cells with oval nuclei, prominent nucleoli, and elongated wispy cytoplasmic projections. There was no evidence of squamous dysplasia or melanin pigment identified. To the best of our knowledge, this is the first reported case of a primary anorectal melanoma detected in anorectal cytology. Detection of malignancies other than squamous cell carcinoma can be seen on anorectal cytology and should be considered when there is no evidence of anal intraepithelial neoplasia. Diagn. Cytopathol. 2017;45:452-455. © 2017 Wiley Periodicals, Inc.

Topics: Aged; Anal Canal; Anus Neoplasms; Biomarkers, Tumor; HIV Infections; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; S100 Proteins

Conjunctival melanocytic lesions encompass a group of clinically diverse, benign to malignant, neoplasms that may contain overlapping histopathological features, making definitive diagnosis challenging in some cases. In this series, we compared multiple immunohistochemical (IHC) markers in 11 conjunctival nevi, 10 primary acquired melanosis (PAM) lesions, and 11 conjunctival melanomas. Immunostains included the melanocytic markers HMB-45 and Melan-A, as well as the proliferative marker Ki-67. Loss of beta-catenin expression has been associated with more aggressive clinical disease in cutaneous melanoma, but its status in conjunctival melanocytic lesions is not known, therefore we incorporated beta-catenin immunohistochemical staining in our study. In this series, conjunctival melanomas had a higher Ki-67 proliferative index and HMB-45 immunoreactivity than did PAM lesions and conjunctival nevi (P<0.001). Melan-A was highly expressed in all 3 groups. Beta-catenin was more strongly expressed in melanomas and nevi than in PAM (P<0.001). There was high inter-grader reliability (Kappa=0.53). Overall, IHC labeling of HMB-45 and Ki-67 is increased in conjunctival melanomas compared to PAM or conjunctival nevi. Beta-catenin, an IHC marker previously unstudied in conjunctival melanocytic lesions, is not preferentially expressed in benign lesions and may play a different role in conjunctival atypia than it does in cutaneous melanoma.

Topics: beta Catenin; Cell Proliferation; Conjunctival Neoplasms; Gene Expression Profiling; Genetic Markers; gp100 Melanoma Antigen; Humans; Immunohistochemistry; Ki-67 Antigen; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Melanoma, Cutaneous Malignant; Nevus, Pigmented; Skin Neoplasms

To establish a mouse model with histologic characteristics of uveal melanoma for investigation of intraocular tumor biology of melanoma.. After injection of 1 × 10(5) of HCmel12 melanoma cells, a cutaneous melanoma cell line, into the vitreous of CX3CR1(+/GFP) or C57Bl/6 mice (n = 12), tumor growth patterns, clinicopathological features, angiogenesis and metastatic behavior were analyzed by histology (hematoxylin and eosin, periodic acid-Schiff without hematoxylin) and immunohistochemistry (HMB45/MART-1-Ab, F4/80-Ab, green fluorescent protein (GFP)-Ab and VE-cadherin-Ab).. HCmel12 cells formed intraocularly growing tumor masses, which showed histologic features of intraocular melanoma such as angiotropism, intratumoral endothelial-lined vasculature, vasculogenic mimicry including prognostic significant extravascular matrix patterns, and invasion by inflammatory cells, in particular macrophages. There was no difference in tumor growth characteristics between CX3CR1(+/GFP) and C57Bl/6 mice. Five of 10 mice proceeded to extrascleral tumor growth and three of these developed metastases.. Intraocularly injected HCmel12 cells developed tumor masses with histologic characteristics of aggressive melanoma similar to human uveal melanoma. Since hematogenous dissemination to the liver was not observed, intravitreally injected HCmel12 cells do not qualify as a model for metastasizing intraocular melanoma. However, since the eye represents a semi-closed compartment with access to constant blood supply, these intraocular tumors represent a model for studies of isolated parameters in general tumor biology of intraocular melanoma.

Topics: Animals; Antigens, CD; Antigens, Differentiation; Biomarkers, Tumor; Cadherins; Cell Line, Tumor; Disease Models, Animal; Female; gp100 Melanoma Antigen; Green Fluorescent Proteins; Humans; Intravitreal Injections; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Mice; Mice, Inbred C57BL; Neovascularization, Pathologic; Skin Neoplasms; Transplantation, Heterologous; Uveal Neoplasms

Topics: Biomarkers, Tumor; Gallbladder; Gallbladder Neoplasms; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Microscopy; Middle Aged; Radiography, Abdominal; Tomography, X-Ray Computed

The detection of circulating tumour cells in the bloodstream before and after surgical manipulation, and the qualitative detection of potential shedding of tumour cells during surgical manipulation of patients with uveal melanoma.. 202 patients treated for a newly diagnosed uveal melanoma were included in the study. Blood samples were acquired 24 h before and 30 min after the basic surgical steps. Detection of potential circulating melanoma cells was extrapolated from the presence of tyrosinase and MelanA/Mart1 transcripts by reverse transcription PCR.. Based on the measurement of tyrosinase transcripts, as a result of the first and second surgical manipulation there were three and zero transitions from negative to positive respectively, while there were two and one transitions from positive to negative, respectively. According to MelanA/Mart1 transcripts, there were 19 and 5 transitions from negative to positive respectively, and 15 and 2 transitions from positive to negative, respectively. No statistically significant differences were documented, concerning the presence of circulating tumour cells in the blood samples acquired before and after the first surgical manipulation or the second one.. The change in the percentage of patients with detected tumour cells in their bloodstream was not statistically significant. The frequent shifts from negative to positive samples as well as from positive to negative samples comparing preoperative to postoperative samples indicates discontinuous shedding or variation due to measurements close to the threshold of detection. As a conclusion, the surgical manipulation does not seem to have a measurable contribution to the spread of melanoma cells in the bloodstream.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Female; Humans; Iatrogenic Disease; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplastic Cells, Circulating; Ophthalmologic Surgical Procedures; Prospective Studies; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Uveal Neoplasms

Topics: Aged; Histocytochemistry; Humans; Immunohistochemistry; Liver; Liver Neoplasms; Magnetic Resonance Imaging; Male; MART-1 Antigen; Melanoma; Microscopy; Radiography; Uveal Neoplasms

Digital melanoma is commonly treated with amputation or wide local excision. Mohs micrographic surgery (MMS) may offer an alternative treatment modality.. To describe outcomes of digital melanomas treated with MMS over a 35-year period.. A retrospective series of digital melanomas treated with MMS was studied. Tumor and treatment characteristics were described and follow-up was assessed.. Sixty-two digital (1.2%) tumors were identified from 4995 melanomas, of which 57 (91.9%) were primary and 5 (8.1%) were recurrent on enrollment. Melanocytic antigen recognized by cytotoxic T lymphocytes from melanoma patients (MART)-1 and HMB-45 immunostains were used in 34 (54.8%) and 14 (22.6%) cases, respectively. Five (8.2%) tumors recurred locally during the course of the study, none of which occurred with MART-1 use. Three (60.0%) local recurrences were salvaged with additional MMS. Local recurrence-free survival rates for primary melanomas at 5 and 10 years were 91.8% and 82.6%, respectively. Overall, 55 (96.5%) patients with primary digital melanomas avoided amputation. Five and 10-year melanoma-specific survival rates for all patients were 95.0% and 81.2%, respectively.. A formal comparison group was not studied.. In the management of digital melanoma, MMS conserves function by avoiding amputation and offers a low local recurrence rate. Outcomes are improved with the use of MART-1.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Female; Fingers; Humans; Male; MART-1 Antigen; Melanoma; Middle Aged; Mohs Surgery; Neoplasm Recurrence, Local; Retrospective Studies; Skin Neoplasms; Survival Rate

Mohs micrographic surgery (MMS) with frozen section immunohistochemistry is a treatment option for malignant melanoma in situ (MMIS) and lentigo maligna melanoma (LMM). Melan-A is a cytoplasmic melanocyte immunostain useful on frozen sections but may lack specificity. Microphthalmia transcription factor (MITF) is a more specific nuclear melanocyte immunostain less frequently used in MMS.. To quantify melanocyte density in chronic sun-damaged skin (CSDS), negative margin, and tumor from patients undergoing MMS for MMIS and LMM using MITF and melan-A.. Sixteen patients with MMIS or LMM had frozen sections from CSDS, negative margin, and 12 tumor samples, stained with MITF and melan-A. Melanocyte counts were performed.. Chronic sun-damaged skin mean melanocyte count (MMC) for MITF and melan-A was 9.8 and 13.7, respectively, (p < .001). Negative margin MMC for MITF and melan-A was 8.84 and 14.06, respectively, (p < .001). Tumor MMC for MITF and melan-A was 63.5 and 62.4, respectively.. Although both MITF and melan-A facilitate the identification of tumor during MMS for MMIS and LMM, the apparent melanocyte density on tumor-free CSDS appears higher with melan-A than MITF. Microphthalmia transcription factor provides a crisp outline of melanocyte nuclei and is a useful alternative stain to melan-A for MMS of melanoma.

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma in Situ; Cell Count; Female; Frozen Sections; Humans; Hutchinson's Melanotic Freckle; Immunohistochemistry; Male; MART-1 Antigen; Melanocytes; Melanoma; Microphthalmia-Associated Transcription Factor; Middle Aged; Mohs Surgery; Skin Neoplasms

In successful cancer immunotherapy, T cell responses appear to be directed toward neoantigens created by somatic mutations; however, direct evidence that neoantigen-specific T cells cause regression of established cancer is lacking. Here, we generated T cells expressing a mutation-specific transgenic T cell receptor (TCR) to target different immunogenic mutations in cyclin-dependent kinase 4 (CDK4) that naturally occur in human melanoma. Two mutant CDK4 isoforms (R24C, R24L) similarly stimulated T cell responses in vitro and were analyzed as therapeutic targets for TCR gene therapy. In a syngeneic HLA-A2-transgenic mouse model of large established tumors, we found that both mutations differed dramatically as targets for TCR-modified T cells in vivo. While T cells expanded efficiently and produced IFN-γ in response to R24L, R24C failed to induce an effective antitumor response. Such differences in neoantigen quality might explain why cancer immunotherapy induces tumor regression in some individuals, while others do not respond, despite similar mutational load. We confirmed the validity of the in vivo model by showing that the melan-A-specific (MART-1-specific) TCR DMF5 induces rejection of tumors expressing analog, but not native, MART-1 epitopes. The described model allows identification of those neoantigens in human cancer that serve as suitable T cell targets and may help to predict clinical efficacy.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Line, Tumor; Cyclin-Dependent Kinase 4; DNA Mutational Analysis; Genetic Therapy; HLA-A2 Antigen; Humans; MART-1 Antigen; Melanoma; Mice, Transgenic; Protein Binding; Receptors, Antigen, T-Cell

A major factor hindering the exploration of adoptive immunotherapy in preclinical settings is the limited availability of tumor-reactive human T cells. Here we developed a humanized mouse model that permits large-scale production of human T cells expressing the engineered melanoma antigen MART-1-specific TCR. Humanized mice, made by transplantation of human fetal thymic tissue and CD34+ cells virally-transduced with HLA class I-restricted melanoma antigen (MART-1)-specific TCR gene, showed efficient development of MART-1-TCR+ human T cells with predominantly CD8+ cells. Importantly, MART-1-TCR+CD8+ T cells developing in these mice were capable of mounting antigen-specific responses in vivo, as evidenced by their proliferation, phenotypic conversion and IFN-γ production following MART-1 peptide immunization. Moreover, these MART-1-TCR+CD8+ T cells mediated efficient killing of melanoma cells in an HLA/antigen-dependent manner. Adoptive transfer of in vitro expanded MART-1-TCR+CD8+ T cells induced potent antitumor responses that were further enhanced by IL-15 treatment in melanoma-bearing recipients. Finally, a short incubation of MART-1-specific T cells with rapamycin acted synergistically with IL-15, leading to significantly improved tumor-free survival in recipients with metastatic melanoma. These data demonstrate the practicality of using humanized mice to produce potentially unlimited source of tumor-specific human T cells for experimental and preclinical exploration of cancer immunotherapy. This study also suggests that pretreatment of tumor-reactive T cells with rapamycin in combination with IL-15 administration may be a novel strategy to improve the efficacy of adoptive T cell therapy.

Topics: Animals; CD8-Positive T-Lymphocytes; Cells, Cultured; Cytotoxicity, Immunologic; HLA-A2 Antigen; Humans; Immunization; Immunotherapy; Lymphocyte Activation; MART-1 Antigen; Melanoma; Mice; Mice, Inbred NOD; Mice, SCID; Peptide Fragments; T-Lymphocytes, Cytotoxic

Published appropriate use criteria (AUC) for Mohs micrographic surgery (MMS) for melanoma are based on consensus opinion.. To evaluate whether published AUC identify melanomas for which MMS may benefit patients by detecting subclinical spread or confirming clear microscopic margins before flap or graft reconstruction.. Retrospective cohort study of 591 melanomas in 556 patients evaluating the correlation between current AUC (anatomic location, recurrent status, and tumor stage) and subclinical spread or reconstruction with a flap or graft.. Anatomic location on the head, neck, genitalia, hands, feet, or pretibial leg was associated with a significantly higher frequency of subclinical spread (odds ratio (OR) 1.89, p = .0280) and flap or graft reconstruction (OR 10.3, p = .0001). Compared with primary lesions, recurrent melanomas had a higher frequency of subclinical spread (OR 1.78, p = .0104) and reconstruction with a flap or graft (OR 1.67, p = .0217). The frequencies of subclinical spread and flap or graft reconstruction did not differ between in situ and invasive melanomas.. Anatomic location and recurrent status are useful criteria to identify melanomas that may benefit from MMS. Tumor stage is not a useful criterion, as MMS has similar benefits for subsets of both invasive and in situ melanomas.

Topics: Aged; Cohort Studies; Female; Humans; Male; MART-1 Antigen; Melanoma; Mohs Surgery; Neoplasm Staging; Plastic Surgery Procedures; Retrospective Studies; Skin Neoplasms; Skin Transplantation; Staining and Labeling; Surgical Flaps

Staging of melanoma includes quantification of a proliferation index, i.e., presumed melanocytic mitoses of H&E stains are counted manually in hot spots. Yet, its reproducibility and prognostic impact increases by immunohistochemical dual staining for phosphohistone H3 (PHH3) and MART1, which also may enable fully automated quantification by image analysis. To ensure manageable workloads and repeatable measurements in modern pathology, the study aimed to present an automated quantification of proliferation with automated hot-spot selection in PHH3/MART1-stained melanomas.. Formalin-fixed, paraffin-embedded tissue from 153 consecutive stage I/II melanoma patients was immunohistochemically dual-stained for PHH3 and MART1. Whole slide images were captured, and the number of PHH3/MART1-positive cells was manually and automatically counted in the global tumor area and in a manually and automatically selected hot spot, i.e., a fixed 1-mm(2) square. Bland-Altman plots and hypothesis tests compared manual and automated procedures, and the Cox proportional hazards model established their prognostic impact.. The mean difference between manual and automated global counts was 2.9 cells/mm(2) (P = 0.0071) and 0.23 cells per hot spot (P = 0.96) for automated counts in manually and automatically selected hot spots. In 77 % of cases, manual and automated hot spots overlapped. Fully manual hot-spot counts yielded the highest prognostic performance with an adjusted hazard ratio of 5.5 (95 % CI, 1.3-24, P = 0.024) as opposed to 1.3 (95 % CI, 0.61-2.9, P = 0.47) for automated counts with automated hot spots.. The automated index and automated hot-spot selection were highly correlated to their manual counterpart, but altogether their prognostic impact was noticeably reduced. Because correct recognition of only one PHH3/MART1-positive cell seems important, extremely high sensitivity and specificity of the algorithm is required for prognostic purposes. Thus, automated analysis may still aid and improve the pathologists' detection of mitoses in melanoma and possibly other malignancies.

Topics: Adult; Aged; Algorithms; Automation, Laboratory; Biomarkers, Tumor; Cell Proliferation; Denmark; Female; Histones; Humans; Image Processing, Computer-Assisted; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Middle Aged; Multivariate Analysis; Neoplasm Staging; Phosphorylation; Predictive Value of Tests; Proportional Hazards Models; Prospective Studies; Reproducibility of Results; Skin Neoplasms; Young Adult

Reliable identification of different melanoma cell lines is important for many aspects of melanoma research. Common markers used to identify melanoma cell lines include: S100; HMB-45; and Melan-A. We explore the expression of these three markers in four different melanoma cell lines: WM35; WM793; SK-MEL-28; and MM127. The expression of these markers is examined at both the mRNA and protein level. Our results show that the metastatic cell line, MM127, cannot be detected using any of the commonly used melanoma-associated markers. This implies that it would be very difficult to identify this particular cell line in a heterogeneous sample, and as a result this cell line should be used with care.

Topics: Biomarkers, Tumor; Cell Line, Tumor; Cells, Cultured; gp100 Melanoma Antigen; Humans; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Metastasis; S100 Proteins; Sensitivity and Specificity

Mohs micrographic surgery (MMS) with melanoma antigen recognized by T-cell (MART-1) immunostaining is an effective treatment of cutaneous melanoma.. To determine the efficacy of MMS with MART-1 immunostain in the management of invasive and in situ melanoma.. A retrospective cohort study evaluated 2,114 melanomas in 1,982 patients excised using MMS and MART-1 immunostain. The margins required for excision were calculated based on Breslow thickness, location, and size. Survival and local recurrence rates were calculated and compared with those of historical controls.. The mean follow-up period was 3.73 years. Local recurrence was identified in 0.49% (7/1,419) of primary melanomas. Approximately 82% of melanomas were excised with ≤6-mm margins. The surgical margin was significantly related to tumor location and size but not to Breslow thickness. The five-year Kaplan-Meier local recurrence and disease-specific survival rates were 0.59 ± 0.30 and 98.53 ± 0.42, respectively. Mohs micrographic surgery with MART-1 immunostain achieved lower local recurrence rates and equivalent or higher Kaplan-Meier survival rates than conventional wide local excision.. Mohs micrographic surgery with MART-1 immunostain is an effective treatment of melanoma as evidenced by low local recurrence rates. It offers the advantage of more tissue-conserving margins than those recommended for conventional excision.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Carcinoma in Situ; Child; Female; Humans; Male; MART-1 Antigen; Melanoma; Middle Aged; Mohs Surgery; Neoplasm Invasiveness; Retrospective Studies; Skin Neoplasms; Survival Rate; Treatment Outcome

Inability to distinguish melanomas from benign nevi is the most frequent reason for malpractice lawsuits in surgical pathology. Reliable diagnostic tools to support hematoxylin and eosin (H&E) stains and induce diagnostic vigilance are thus highly needed. Because high diagnostic performance recently was showed using automated image analysis, the immunohistochemical proliferation marker Ki67 seems a potential candidate. This study aimed to investigate if this previously presented automated algorithm could have prevented 10 false-negative melanoma diagnoses. In addition, diagnostic utility of another, but narrower, immunohistochemical proliferation marker, phosphohistone H3 (PHH3), was explored.. A total of 10 formalin-fixed paraffin-embedded melanocytic tumors, initially classified as benign or dysplastic but revised as melanomas at metastatic debut, were dual-stained for Ki67/MART1 and PHH3/MART1. A Ki67 index was automatically calculated in epidermis, dermis, a combination of such, and a dermal hot spot. Dermal PHH3/MART1 scores were established semi-automatically.. The dermal Ki67 index identified all 10 melanomas, the hot-spot index 8 and the epidermal and combined indices only 2 and 5, respectively. Nine melanomas were PHH3 positive and scores correlated with Ki67.. PHH3 added limited information, but supplemental automated Ki67 assessment could possibly have prevented the misdiagnosis of most melanomas had the algorithm been available at the time of diagnosis.

Topics: Adult; Algorithms; Automation, Laboratory; Biomarkers, Tumor; False Negative Reactions; Female; Histones; Humans; Ki-67 Antigen; Male; MART-1 Antigen; Melanoma; Middle Aged; Phosphorylation; Sensitivity and Specificity; Skin Neoplasms; Young Adult

Histologic and molecular heterogeneity is well recognized in malignant melanoma; however, the diversity of expression of new and classic melanoma markers has not been correlated in serial sections of metastases.. We examined and correlated the expression of microphthalmia transcription factor (MITF) with its transcriptional targets, including melastatin (MLSN1/TRPM1), pigment epithelium-derived factor (SERPINF1/PEDF), SILV/PMEL17/GP100 (human melanoma black 45 [HMB-45]), and melanoma antigen recognized by T cells 1 (MART-1)/MLANA, in 13 melanoma metastases in lymph nodes of 13 patients. The expression levels and patterns of marker expression were recorded by a semiquantitative, 4-point ordinal reactivity method.. Our results showed a consistently robust and diffuse expression of MITF protein in 12 (92%) of 13 metastatic tumors compared with variable expression of MLSN1 (46%) messenger RNA or PEDF (75%), HMB-45 (54%), and MART-1 (46%) proteins.. Overall, in melanoma lymph node metastases, MITF protein expression was not tightly correlated with its gene targets. Moreover, the immunoreactivity for MITF, compared with MART-1 and HMB-45, was retained, supporting immunohistochemical detection of MITF as a more sensitive method of detecting metastatic melanoma.

Topics: Eye Proteins; gp100 Melanoma Antigen; Humans; Lymphatic Metastasis; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Microphthalmia-Associated Transcription Factor; Nerve Growth Factors; Serpins; Skin Neoplasms; TRPM Cation Channels

It is unclear why some patients with in situ melanoma develop metastases. Few reports demonstrate occult invasion with immunohistochemistry staining, which were discordant with reports interpreting such staining as false-positive.. To investigate the occurrence of occult invasive disease within in situ melanoma by using methods to circumvent potential limitations in prior study designs.. Unequivocal in situ melanoma without associated nevi or regression was identified using a consecutive sample of 33 cases plus 1 index case in an academic medical center. After cutting deeper into the most representative tissue block, 3 sequential slides were stained with hematoxylin-eosin (H-E), melanoma antigen (melan-A), and again with H-E. Melan-A-stained slides showing definitive invasion were double-stained with Sry-related HMg-Box gene 10 (SOX10) to confirm the melanocytic nature of the cells of interest. The study evaluated the possibilities of occult invasion detected by immunohistochemistry, sectioning deeper into the tissue block, or both. Slides were independently scored by 3 dermatopathologists with interrater reliability assessed. The study was conducted from January 1, 2012, to July 31, 2014.. Assessment of the occurrence of occult invasion, diagnosis of invasion by immunohistochemistry alone vs cutting deeper into the tissue block, and occurrence of false-positive results using immunohistochemistry alone.. Occult invasive melanoma was detected in 11 of 33 consecutive cases (33%) of previously diagnosed unequivocal in situ melanoma. Six of 11 melanomas (55%) were diagnosable only by immunohistochemistry. The remaining 5 tumors (45%) were diagnosable by both melan-A and H-E staining, likely as a result of simply cutting deeper into the tissue block. Four cases (12%) showed a few melan-A-positive cells in the dermis, which was insufficient for a diagnosis of invasive melanoma and most consistent on a cytomorphologic basis with occult nevi.. Although rare, in situ melanoma may metastasize. Occult microinvasion was demonstrated in up to one-third of the specimens in the present study, which provides a plausible explanation for this adverse event. Thus, history and physical examination including regional lymph nodes, education, and surveillance recommendations should be based on a very low, but not zero, risk of metastasis.

Topics: Adult; Biomarkers, Tumor; Female; Hospitals, University; Humans; Male; MART-1 Antigen; Melanocytes; Melanoma; Neoplasm Invasiveness; Predictive Value of Tests; Retrospective Studies; Sensitivity and Specificity; Skin Neoplasms; SOXE Transcription Factors

Development of liver metastasis remains the most common cause of mortality in uveal melanoma (UM). A few cell lines cultured from primary UM tumors have been used widely to investigate the pathobiology of UM. However, the translation of basic knowledge to the clinic for the treatment of the metastatic disease has remained incremental at best. In this study, we examined whether the properties of UM cell lines at various passages were similar to their corresponding primary tumors.. Gene expression profiling by microarray was performed on UM primary tumors and derived cell lines cultured at varying passages. Expression of UM protein markers was monitored by immunohistochemical analyses and Western blotting. The in vivo tumorigenic properties of UM cultures were evaluated using athymic nude mice.. Cell passaging severely reduced the expression of genes encoding markers typical of UM, including those of the prognostic gene signature. Marked differences between gene expression profiles of primary tumors and cell lines could be linked to the infiltrating immune and stromal cells in situ. In addition, the tumorigenic properties of UM cell lines also increased with cell passaging in culture as evaluated by their subcutaneous injection into athymic mice.. Together, these findings demonstrate that the short-term UM primary cultures exhibit molecular features that resemble the respective surgical material and, thus, represent the best model for in vitro-assessed cancer treatments.

Topics: Animals; Blotting, Western; Cell Count; Cell Line, Tumor; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Mice; Mice, Nude; Microscopy, Phase-Contrast; Neoplasms, Experimental; Polymerase Chain Reaction; RNA, Neoplasm; Uveal Neoplasms

Bullous melanoma represents a rare variant of melanoma characterized by variably large subepidermal, basilar, or suprabasilar blisters. We present 7 cases of bullous melanoma (M:F = 4:3; median age, 57 years; age range, 38-86) located on the heel (n = 2), foot (n = 2), arm (n = 2), and back (n = 1). In 5/7 cases, the bulla was due to dyscohesiveness of basilar or suprabasilar melanocytes with subsequent acantholytic features simulating pemphigus vulgaris or Hailey-Hailey disease, whereas in the last 2 cases a subepidermal bulla without clear-cut relation to the melanocytic complexes was observed. Direct and indirect immunfluorescence studies performed in 4 patients on skin near the original surgical scar (including those with subepidermal bullae) were negative. Measurement of the Breslow index in all 7 cases was affected by the presence of the bulla, and in 5 of them, the TNM classification was different depending on the method of measurement (with or without the bulla). We suggest that the Breslow index in these cases should be measured detracting the thickness of the bulla from the total thickness, but follow-up data on larger numbers of patients are necessary to establish whether the presence of bullous features has any prognostic implication.

Topics: Acantholysis; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Biopsy; Female; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanocytes; Melanoma; Middle Aged; Neoplasm Staging; Predictive Value of Tests; Skin Neoplasms

Metastasis of sentinel lymph node (SLN) is generally evaluated on histopathological examination and controversy still exists over the usefulness of PCR assay of SLN.. To investigate the prognostic value of triple-marker PCR assay of SLN.. A total of 165 patients with primary cutaneous melanoma who underwent SLN biopsy were included. Clinical and histopathological data were retrieved from each patient's file and triple-marker PCR assay (tyrosinase, GP-100 and MART-1) was performed on the SLN as well as routine histopathological evaluation. PCR positivity was defined as the expression of all three PCR markers. To evaluate melanoma-specific survival (MSS) and disease-free survival (DFS), we used the Kaplan-Meier method and the log-rank test. Multivariate analyses using the Cox proportional hazards regression model were also performed.. Sentinel lymph nodes were identified in all 165 patients: 61 patients (37.0%) were male and 104 (63.0%) were female, with a mean age of 60.2 years. Of the 165 melanomas, 81 (49.1%) were acral lentiginous melanomas. Compared with the patients with PCR positivity (1-2 markers) or PCR negativity, patients with PCR positivity (3 markers) had significantly poor MSS (5-year survival rate, 58.7% vs. 84.4%; P < 0.0001) and DFS (5-year survival rate, 25.0% vs. 83.9%; P < 0.0001), with median follow-up of 42 months for MSS and 38 months for DFS. These survival rates of patients with PCR positivity (3 markers) were lower than those of patients with histopathologically positive SLN. In multivariate analysis, PCR positivity (3 markers) was an independent prognostic factor for both MSS (hazard ratio [HR], 2.81; 95% confidence interval [CI], 1.07-7.33; P = 0.035) and DFS (HR, 2.48; 95% CI, 1.08-5.69; P = 0.032).. The expression of three PCR markers was a significant prognostic factor for both MSS and DFS and might be closely correlated to a dismal prognosis.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Child; Child, Preschool; Disease-Free Survival; Female; Follow-Up Studies; gp100 Melanoma Antigen; Humans; Lymph Nodes; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Sentinel Lymph Node Biopsy; Sex Factors; Skin Neoplasms; Survival Rate; Young Adult

SOX10 (Sry-related HMg-Box gene 10) is a key nuclear transcription factor in the differentiation of neural crest progenitor cells to melanocytes. It has been shown to be a sensitive and specific marker of malignant melanoma of multiple histologic types. We evaluated the immunohistochemical profile of SOX10 in 107 sentinel lymph nodes and compared it to S100 protein, HMB-45, and Melan-A. Seventy-seven lymph nodes originally reported as positive for metastatic melanoma, and 30 reported as negative were reviewed. SOX10 identified metastatic melanoma in 58 of 58 (100%) positive cases included in the final analysis. SOX10 staining showed a statistically significant increase in staining intensity when compared with S100 protein, HMB-45, and Melan-A (P = 0.000, 0.000, and 0.003, respectively). In addition, there was a statistically significant increase in percentage of tumor cells stained by SOX10, compared with S100 protein, HMB-45, and Melan-A (P = 0.015, 0.000, and 0.001, respectively). SOX10 stained nodal nevi in a similar manner to S100 protein and Melan-A in 4 cases. Interpretation of nodal nevus staining (negative by HMB-45) was significantly aided by the crisp nuclear staining produced by SOX10 as compared with the obscuring cytoplasmic staining produced by S100 protein and Melan-A in 3 cases. Identification of micrometastases was facilitated by the nuclear staining pattern of SOX10 and lack of background dendritic cell staining seen in S100 protein. We conclude that given the sensitivity and specificity of SOX10 for detecting metastatic melanoma in sentinel lymph nodes, it is a reliable marker for supplementing and potentially replacing traditionally utilized immunohistochemical stains.

Topics: Biomarkers, Tumor; Cell Differentiation; Diagnostic Tests, Routine; gp100 Melanoma Antigen; Humans; Immunohistochemistry; Lymphatic Metastasis; MART-1 Antigen; Melanocytes; Melanoma; Melanoma-Specific Antigens; S100 Proteins; Sensitivity and Specificity; SOXE Transcription Factors; Stem Cells

TGF-β and the inhibitors of differentiation (Id) are linked. Smad7 and other TGF-β inhibitors can potently suppress melanomagenesis; however, little work examining Ids has been reported in melanoma, particularly for Id4. Here, we report that Id4, but not Id2 or Id3 expression, surprisingly, activated robust melanin production in xenografts of previously amelanotic (lacking pigment) 1205Lu/Smad7 (S7) cells. Fontana-Masson stain and de-novo expression of MART-1 and tyrosinase proteins confirmed melanin production. Additionally, pigment-laden CD163+ mouse histiocytes with areas of extensive necrosis were found throughout S7/Id4 tumors, but not in parental 1205Lu, S7/Id2 or S7Id3-derived tumors. Mechanistic investigation revealed increased nuclear M-microphthalmia-associated transcription factor (MITF) and MART-1 promoter activation following Id4 expression in 1205Lu and WM852 melanoma cells, suggesting broader implications for Id4 in melanin synthesis. In human tumors, melanin colocalized with Id4 expression establishing a correlation. Current chemotherapeutics for melanoma are only marginally effective. Immunotherapy provides the most promise, yet the role of innate immunity is poorly understood. Here, TGF-β suppression followed by Id4 expression results in extensive melanin synthesis and robust histiocyte recruitment following tumorigenesis, a novel role for Id4. Our results suggest that TGF-β suppression coupled with pigment overproduction triggers an innate immune response resulting in tumor necrosis.

Topics: Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Cell Proliferation; Genetic Vectors; Histiocytes; Humans; Immunity, Innate; Inhibitor of Differentiation Proteins; Keratinocytes; MART-1 Antigen; Melanins; Melanoma; Mice; Mice, Nude; Neoplasm Transplantation; Pigmentation; Promoter Regions, Genetic; Receptors, Cell Surface; Retroviridae; Skin Neoplasms; Transforming Growth Factor beta

Topics: Colonoscopy; Female; Gastrointestinal Hemorrhage; gp100 Melanoma Antigen; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Microscopy; Middle Aged; Rectal Neoplasms; S100 Proteins

We hypothesized that the co-entrapment of melanoma-associated antigens and the Toll-like receptor (TLR) ligands Poly(I:C) and CpG, known to be Th1-immunopotentiators, in mannose-functionalized aliphatic polyester-based nanoparticles (NPs) could be targeted to mannose receptors on antigen-presenting cells and induce anti-tumor immune responses. High entrapment efficiencies of antigens and immunopotentiators in 150nm NPs were obtained. The co-entrapment of the model antigen ovalbumin and the TLR ligands was crucial to induce high IgG2c/IgG1 ratios and high levels of IFN-γ and IL-2. Mannose-functionalization of NPs potentiated the Th1 immune response. The nanoparticulate vaccines decreased the growth rate of murine B16F10 melanoma tumors in therapeutic and prophylatic settings. The combination of mannose-functionalized NPs containing MHC class I- or class II-restricted melanoma antigens and the TLR ligands induced the highest tumor growth delay. Overall, we demonstrate that the multifunctional properties of NPs in terms of targeting and antigen/adjuvant delivery have high cancer immunotherapeutic potential.

Topics: Animals; Cancer Vaccines; Cell Line, Tumor; Cytokines; Disease Models, Animal; Female; gp100 Melanoma Antigen; Granzymes; Immunoglobulin G; Ligands; Male; Mannose; MART-1 Antigen; Melanoma; Mice, Inbred C57BL; Mice, Transgenic; Nanoparticles; Oligodeoxyribonucleotides; Ovalbumin; Peptides; Poly I-C; Polymers; Toll-Like Receptors; Tumor Burden

MelanA is a known melanocyte marker and is important in melanoma diagnostics. Some tumours, however, show loss of MelanA expression and may therefore be difficult to distinguish from tumours of mesenchymal origin. Pure spindle-cell melanoma is a rare event, and little is known about its biological background and prognosis. However, morphological changes towards a more mesenchymal shape and cellular dedifferentiation may correlate with reactivation of important developmental programmes (epithelial-to-mesenchymal transition) and disseminative tumour cell properties. Inflammation and CD163+ macrophages have been shown to be important inducers of E-cadherin and cell-to-cell adhesion loss, a pivotal and final event of epithelial-to-mesenchymal transition. In a cohort of 385 patients with melanoma, we located nine tumours with a clonal MelanA expression, defined as a tumour section with a distinct MelanA-negative clone next to a MelanA-positive clone. Interestingly, MelanA-negative clones correlated significantly with an augmented inflammatory response of tumour-infiltrating macrophages (CD163+), complete loss of E-cadherin and a spindle-shaped morphology, irrespective of ulcerated status. These cases show the inflammatory heterogeneity of melanoma, which may have important diagnostic, prognostic and therapeutic implications for the patients. We show that melanomas harbour cell clones that bear strong resemblance to tumour-associated macrophages, a pivotal player in a tumour-supporting microenvironment. Interestingly, this distinct inflammatory phenotype is associated with loss of MelanA expression, the presence of spindle-shape morphology and complete loss of E-cadherin, considered as possible markers of poorly differentiated and more invasive tumour cells.

Topics: Antigens, CD; Antigens, Differentiation, Myelomonocytic; Biomarkers, Tumor; Cadherins; Humans; Immunohistochemistry; Inflammation; Macrophages; MART-1 Antigen; Melanoma; Phenotype; Receptors, Cell Surface; Skin Neoplasms; Tumor Microenvironment

Topics: Aged, 80 and over; Biomarkers, Tumor; Cytodiagnosis; gp100 Melanoma Antigen; Histocytochemistry; Humans; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Melanoma, Cutaneous Malignant; S100 Proteins; Skin Neoplasms; Urethra; Urethral Neoplasms; Urinalysis

Sentinel lymph node (SLN) biopsies have widely been used for the detection of occult LN metastasis of malignant melanoma (MM). In addition to conventional biomarkers, we assessed the diagnostic and prognostic significance of melanoma-initiating cell (MIC) markers in SLNs of MM. We examined the expressions of gp100, MART-1 and tyrosinase mRNA for routine diagnosis and those of ABCB5, CD133, nestin, KDM5B, NGFR and RANK mRNA as MIC markers. The presence of micrometastasis was confirmed immunohistochemically using antibodies to S-100, HMB-45, MART-1, and tyrosinase. Discordance between immunohistochemical and molecular data was observed in 14 of 70 (20.0%) patients, among whom five (7.1%) were positive for only molecular markers;two of these five patients tested positive for micrometastasis by repeated immunohistochemical stainings. The quantitative expression levels of gp100, MART-1, and tyrosinase mRNA were significantly higher in the metastatic LNs;the cut-off values remain to be elucidated. ABCB5 mRNA expression was detected more frequently in the metastatic SLNs (p＜0.05) and in the group of patients with recurrence. To make a definite diagnosis of metastasis, we still need a combination of immunohistochemical and molecular probes. ABCB5 might be a suitable molecular marker for the detection of melanoma-initiating cells in SLNs.

Topics: Aged; Aged, 80 and over; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biomarkers; Female; Humans; Immunohistochemistry; Jumonji Domain-Containing Histone Demethylases; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplastic Stem Cells; Nuclear Proteins; Repressor Proteins; RNA, Messenger; Sentinel Lymph Node Biopsy

The presence of Melan-A positive dermal cells in excisions for melanoma in situ represents a frequent conundrum for pathologists. These cells may represent superficially invasive melanoma, benign, incidental, dermal nevi or non-specific staining of dermal melanophages. Occasionally, rare, Melan-A positive dermal cells are present which do not clearly correspond to the above three categories. Our objective was to further characterize these Melan-A positive dermal cells. To do this, immunoperoxidase staining for Melan-A and SOX-10 was performed on 188-cutaneous excisions, including examples of melanoma in situ, atypical junctional melanocytic hyperplasia and non-melanocytic tumors. These were evaluated for the presence of Melan-A and SOX-10 positive dermal cells. Dermal cells, positive for both markers, were identified in 17% of the excisions. The cells were present in 10% of cases from the melanocytic group and 31% of the cases from the non-melanocytic group. These cells did not exhibit cytologic atypia and resembled neither the co-existing neoplasm nor melanophages. We conclude that positivity of these rare Melan-A positive cells for SOX-10 argues that they represent true melanocytes and not non-specific staining. The absence of cytologic atypia in these cells and their presence in excisions of non-melanocytic neoplasms argues that they are benign, reactive, dermal melanocytes.

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Female; Humans; Hyperplasia; Immunoenzyme Techniques; Immunohistochemistry; Male; MART-1 Antigen; Melanocytes; Melanoma; Melanoma, Cutaneous Malignant; Nevus, Pigmented; Skin Neoplasms; SOXE Transcription Factors

Various methods of tissue processing have been used to treat melanoma with Mohs micrographic surgery (MMS).. We describe a method of treating melanoma with MMS that combines breadloaf frozen sectioning of the central debulking excision with complete peripheral and deep microscopic margin evaluation, allowing detection of upstaging and comprehensive pathologic margin assessment before reconstruction.. We conducted a retrospective cohort study evaluating for local recurrence and upstaging in 614 invasive or in situ melanomas in 577 patients treated with this MMS tissue processing methodology using frozen sections with melanoma antigen recognized by T cells 1 (MART-1) immunostaining. Follow-up was available in 597 melanomas in 563 patients.. Local recurrence was identified in 0.34% (2/597) lesions with a mean follow-up time of 1026 days (2.8 years). Upstaging occurred in 34 of 614 lesions (5.5%), of which 97% (33/34) were detected by the Mohs surgeon before reconstruction.. Limitations include retrospective study, intermediate follow-up time, and that the recurrence status of 39.6% of patients was self-reported.. Treating melanoma with MMS that combines breadloaf sectioning of the central debulking excision with complete peripheral and deep microscopic margin evaluation permits identification of upstaging and consideration of sentinel lymph node biopsy before definitive reconstruction and achieves low local recurrence rates compared with conventional excision.

Topics: Adult; Aged; Aged, 80 and over; Cohort Studies; Female; Follow-Up Studies; Humans; Male; MART-1 Antigen; Melanoma; Melanoma, Cutaneous Malignant; Middle Aged; Mohs Surgery; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Neoplasm Staging; Retrospective Studies; Skin Neoplasms; T-Lymphocytes

Here, we describe a fatal serious adverse event observed in a patient infused with autologous T-cell receptor (TCR) transduced T cells. This TCR, originally obtained from a melanoma patient, recognizes the well-described HLA-A*0201 restricted 26-35 epitope of MART-1, and was not affinity enhanced. Patient 1 with metastatic melanoma experienced a cerebral hemorrhage, epileptic seizures, and a witnessed cardiac arrest 6 days after cell infusion. Three days later, the patient died from multiple organ failure and irreversible neurologic damage. After T-cell infusion, levels of IL-6, IFN-γ, C-reactive protein (CRP), and procalcitonin increased to extreme levels, indicative of a cytokine release syndrome or T-cell-mediated inflammatory response. Infused T cells could be recovered from blood, broncho-alveolar lavage, ascites, and after autopsy from tumor sites and heart tissue. High levels of NT-proBNP indicate semi-acute heart failure. No cross reactivity of the modified T cells toward a beating cardiomyocyte culture was observed. Together, these observations suggest that high levels of inflammatory cytokines alone or in combination with semi-acute heart failure and epileptic seizure may have contributed substantially to the occurrence of the acute and lethal event. Protocol modifications to limit the risk of T-cell activation-induced toxicity are discussed.

Topics: Adult; Cell- and Tissue-Based Therapy; Fatal Outcome; Female; Humans; Immunotherapy, Adoptive; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Neoplasm Staging; Receptors, Antigen, T-Cell; T-Cell Antigen Receptor Specificity; T-Lymphocytes; Transduction, Genetic

We report an 89-year-old man who presented with a slowly growing pigmented pedunculated tumor. The nodule was diagnosed as a spindle cell pedunculated malignant melanoma (PMM), a rare variant of spindle-cell malignant melanoma. The clinical presentation of this tumor and its histological and immunohistological features are discussed.

Topics: Aged, 80 and over; Biomarkers, Tumor; Facial Neoplasms; gp100 Melanoma Antigen; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens

Topics: Aged; Biomarkers, Tumor; DNA Mutational Analysis; DNA, Neoplasm; Eye Enucleation; Fatal Outcome; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gq-G11; HSP27 Heat-Shock Proteins; Humans; Male; MART-1 Antigen; Melanoma; Multiplex Polymerase Chain Reaction; Neoplasms, Multiple Primary; Polymorphism, Single Nucleotide; Tumor Suppressor Proteins; Ubiquitin Thiolesterase; Uveal Neoplasms; Visual Acuity

Endogenous tumor-specific T cells are detectable in patients with different tumor types including malignant melanoma (MM). They can control tumor growth, have impact on patient survival and correlate with improved clinical response to immune checkpoint therapy. Thus, they may represent a potent biomarker for respective treatment decisions. So far, major target antigens of endogenous MM-reactive T cells have not been determined systematically. Instead, autoantibodies are discussed as surrogate parameter for MM-specific T cells. Throughout a period of more than 60 days after tumor resection, we therefore determined in 38 non-metastasized primary MM patients and in healthy individuals by IFNγ ELISpot and bead-based fluorescent multiplex assay major target antigens of spontaneous T cell and humoral responses using a broad panel of MM antigens and assessed the presence and suppressive impact of MM-reactive regulatory T cells (Tregs). We show that MM-reactive T cells are frequent in MM patients, transiently increase after tumor removal and are mostly directed against Melan-A/MART-1, Tyrosinase, NA17-A and p53. MM-specific Tregs were only detected in few patients and inhibited MM-reactive T cells particularly early after tumor resection. Tumor-specific autoantibodies occurred in most patients, but did not correlate with T cell responses. Thus, endogenous antibodies may not be reliable surrogate parameters of MM-reactive T cells.

Topics: Amino Acid Sequence; Antigens, Neoplasm; Humans; MART-1 Antigen; Melanoma; Molecular Sequence Data; Monophenol Monooxygenase; Neoplasm Staging; T-Lymphocytes; Tumor Suppressor Protein p53

Benign melanocytic rests are a frequent finding in superficial lymph nodes removed during sentinel lymph node biopsies for melanoma. Whereas the histopathology of these deposits is well understood, very little is known regarding melanocytic lymph node deposits in the setting of giant congenital melanocytic nevi.. We analyzed lymph nodes removed from the drainage basin of giant congenital melanocytic nevi in three patients who had developed melanoma within their giant congenital nevi.. Two of three patients showed widespread, capsular and parenchymal melanocytic deposits in multiple nodes (9 of 11 nodes in one patient and 6 of 8 in the other). Melanocytes were small, non-mitotically active and resembled those in the associated giant congenital melanocytic nevus. Melanocytes were arranged singly and in small nests ∼0.05 mm in diameter, with some larger sheets up to 1 mm. Nodal melanocytes stained for Melan A and S100 on immunohistochemical evaluation, but showed negative or minimal HMB-45 reactivity.. Evaluation of lymph nodes in the setting of giant congenital melanocytic nevi is complicated by the presence of often numerous, parenchymal melanocytic nevic deposits. Bland cytology and minimal or absent HMB-45 staining may be helpful in differentiating these nodal melanocytic nevi from metastatic melanoma. We term this phenomena large congenital nodal nevus.

Topics: Adolescent; Aged; Biomarkers, Tumor; Follow-Up Studies; gp100 Melanoma Antigen; Humans; Lymph Nodes; Male; MART-1 Antigen; Melanocytes; Melanoma; Melanoma-Specific Antigens; Middle Aged; Nevus, Pigmented; S100 Proteins; Sentinel Lymph Node Biopsy; Skin Neoplasms

Metastatic melanoma remains incurable, emphasizing the acute need for improved research models to investigate the underlying biologic mechanisms mediating tumor invasion and metastasis, and to develop more effective targeted therapies to improve clinical outcome. Available animal models of melanoma do not accurately reflect human disease and current in vitro human skin equivalent models incorporating melanoma cells are not fully representative of the human skin microenvironment. We have developed a robust and reproducible, fully humanized three-dimensional (3D) skin equivalent comprising a stratified, terminally differentiated epidermis and a dermal compartment consisting of fibroblast-generated extracellular matrix. Melanoma cells incorporated into the epidermis were able to invade through the basement membrane and into the dermis, mirroring early tumor invasion in vivo. Comparison of our novel 3D melanoma skin equivalent with melanoma in situ and metastatic melanoma indicates that this model accurately recreates features of disease pathology, making it a physiologically representative model of early radial and vertical growth-phase melanoma invasion.

Topics: 3T3 Cells; Animals; Biomarkers, Tumor; Cell Culture Techniques; Cell Line, Tumor; Cells, Cultured; Dermis; Epidermis; Extracellular Matrix; Fibroblasts; Humans; MART-1 Antigen; Melanoma; Mice; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Models, Biological; Neoplasm Invasiveness; Neoplasm Metastasis; Skin; Skin Neoplasms; Skin, Artificial; Tumor Microenvironment

In 1985, John Monaco--the discoverer of LMP-2 and -7, the inducible components of the immunoproteasome--asked his advanced immunology class as to why the MHC region contained not only structural genes, but several others as well, whose functions were then unknown. As we drew a blank, he quipped: perchance because many of the MHC genes are induced by IFN-γ! The ensuing three decades have witnessed the unveiling of the profound fundamental and clinical implications of that classroom tête-à-tête. Amongst its multitudinous effects, IFN-γ induces genes enhancing antigen processing and presentation to T cells; such as those encoding cellular proteases and activators of proteases. In this issue, Keller et al. [Eur. J. Immunol. 2015. 45: 3257-3268] demonstrate that the limited success of MART-1/Melan-A-targeted immunotherapy in melanoma patients could be due to inefficient MART-1(26-35) presentation, owing to the proteolytic activities of IFN-γ-inducible β2i/MECL-1, proteasome activator 28 (PA28), and endoplasmic reticulum-associated aminopeptidase-associated with antigen processing (ERAP). Specifically, whilst β2i and PA28 impede MART-1(26-35) liberation from its precursor protein, ERAP-1 degrades this epitope. Hence, critical to effective cancer immunotherapy is deep knowledge of T-cell-targeted tumor antigens and how cellular proteases generate protective epitope(s) from them, or destroy them.

Topics: Animals; Humans; Interferon-gamma; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Proteasome Endopeptidase Complex; Tumor Escape

Malignant melanoma is a type of skin cancer with accelerated evolution and a high metastatic potential, thus being the most aggressive type of skin tumor. Its origin resides in the epidermal melanocytes, which multiply chaotically, therefore becoming malignant cells. The main objective of this study was represented by the clinical, histological and also immunohistochemical analysis of a peculiar case of malignant cutaneous melanoma arised de novo. Patient M.H., age 54, was admitted due to concerns regarding a cancerous growth on the right scapula. Dermoscopy revealed an asymmetrical, polymorphic and polychromatic lesion; therefore, a surgical intervention was scheduled. The histological exam showed a microscopic structure resembling an epithelioid cell malignant melanoma, with inflammatory reaction and central ulcerations. Immunostaining with melanocytic differentiation markers revealed the presence of pagetoid-disseminated cancerous cells into the epidermis, in addition to deep dermis invasion and the extension of cancerous cells in and around the hair follicles. In most cases, malignant melanoma develops on pre-existent nevi, but it can also appear de novo with accelerated evolution, mainly at phototype I young people. The importance of this particular case consists in the fact that the tumors presented some unusual particularities: appearing on healthy skin tissue, with slow evolution, at an age when this pathology is rarely encountered; the patient was phototype III and the cutaneous territory had been rarely exposed to ultraviolet radiations. Therefore, the case has proven interesting and worthy of being taken into consideration by the appropriate literature.

Topics: Dermoscopy; Epidermis; Humans; Ki-67 Antigen; MART-1 Antigen; Melanoma; Melanoma, Cutaneous Malignant; Monophenol Monooxygenase; Skin Neoplasms

IFN-γ released from cytotoxic T lymphocytes (CTLs) during the effector phase is essential for rejecting bulky melanoma tumors. In contrast, IFN-γ is known to induce certain immunosuppressive factors in tumor cells such as programmed cell death 1 ligand 1 (PD-L1). In this study, we have identified candidates for IFN-γ-inducible CTL-suppressive factors in melanoma cells using complementary DNA microarray analysis, and CD271/p75/neurotrophin receptor (NTR) was one of the candidate genes. Recently, CD271 was identified as a marker of the cancer stem cell-like population in human melanoma tissues. In this study, we showed that overexpression of CD271 on melanoma cells suppressed the in vitro activation of melanoma-specific CTLs. This suppression was mediated by CD271 ligation with activated CTL-derived nerve growth factor and the subsequent downregulation of melanoma antigens. Moreover, we found that the expression levels of PD-L1 on melanoma cells correlated with those of CD271, and they additively suppressed the activation of melanoma-specific CTLs. To the best of our knowledge, the role of overexpression of CD271 in an anti-melanoma T-cell response has been unreported.

Topics: B7-H1 Antigen; Biomarkers, Tumor; Cell Line, Tumor; Down-Regulation; Humans; Immune Tolerance; Interferon-gamma; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Nerve Tissue Proteins; Oligonucleotide Array Sequence Analysis; Receptors, Nerve Growth Factor; Skin Neoplasms; T-Lymphocytes, Cytotoxic; Up-Regulation

To analyze the prognostic relevance and relative impact of circulating myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) compared with functional tumor antigen-specific T cells in patients with melanoma with distant metastasis.. The percentage of CD14(+)CD11b(+)HLA-DR(-/low) MDSCs, CD4(+)CD25(+)FoxP3(+) Tregs, and the presence of NY-ESO-1- or Melan-A-specific T cells was analyzed in 94 patients and validated in an additional cohort of 39 patients by flow cytometry. Univariate survival differences were calculated according to Kaplan-Meier and log-rank tests. Multivariate analyses were performed using Cox regression models.. NY-ESO-1-specific T cells, the M-category, and the frequency of MDSCs were associated with survival. The absence of NY-ESO-1-specific T cells and the M-category M1c independently increased the risk of death. In a second Cox model not considering results on antigen-specific T cells, a frequency of >11% MDSCs showed independent impact. Its association with survival was confirmed in the additional patient cohort. Median survival of patients with a lower frequency of MDSCs was 13 months versus 8 months for others (P < 0.001, combined cohorts). We observed a strong correlation between high levels of MDSCs and the absence of melanoma antigen-specific T cells implying a causal and clinically relevant interaction. No prognostic impact was observed for Tregs.. Circulating CD14(+)CD11b(+)HLA-DR(-/low) MDSCs have a negative impact on survival and inversely correlate with the presence of functional antigen-specific T cells in patients with advanced melanoma. Our findings provide a rationale to investigate MDSC-depleting strategies in the therapeutic setting especially in combination with vaccination or T-cell transfer approaches.

Topics: Aged; Antigens, Neoplasm; Female; Flow Cytometry; Humans; Immune Tolerance; Kaplan-Meier Estimate; Male; MART-1 Antigen; Melanoma; Membrane Proteins; Middle Aged; Myeloid Cells; Prognosis; Proportional Hazards Models; T-Lymphocytes; T-Lymphocytes, Regulatory

Topics: Adult; Anus Neoplasms; Biomarkers, Tumor; Gastrointestinal Hemorrhage; gp100 Melanoma Antigen; Humans; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Microphthalmia-Associated Transcription Factor; Pain; Rectum; S100 Proteins

We recently reported three cases of metastatic melanoma that does not express S100, HMB45, Melan A and Tyrosinase. A concurrent cutaneous scalp primary melanoma was identified later in one of the cases, which showed strong expression of these markers. The difference in immunophenotype between the primary melanoma and its metastasis in the parotid gland in this case raised the question of the biological significance of the expression of these markers and metastatic potential. To address this question, we utilized microarray comparative genomic hybridization (aCGH) to compare the cytogenetic features between the primary and metastatic melanoma. We observed chromosomal gains including 6p, entire chromosome 7, and 8q11.1-q24.3 in both primary and metastatic tumors. However, the metastatic lesion showed unique additional copy of chromosomal 7q, and loss of chromosome 9p24.3-q13 and chromosome 4, which included Melan A encoding gene region in 9p24.1. The above findings suggest the unique cytogenetic changes in the parotid lesion are most likely related to the metastatic behavior, as well as responsible for loss of multiple melanocytic marker expression in the metastatic melanoma for this case.

Topics: Biomarkers, Tumor; Comparative Genomic Hybridization; gp100 Melanoma Antigen; Humans; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Monophenol Monooxygenase; Neoplasm Metastasis; S100 Proteins; Skin Neoplasms

Metastasis heterogeneity presents a significant obstacle to the development of targeted cancer therapeutics. In this study, we sought to establish from a large series of human melanoma metastases whether there exists a determined pattern in tumor cellular heterogeneity that may guide the development of future targeted immunotherapies.. From a cohort of 1,514 patients with metastatic melanoma, biopsies were procured over a 17-year period from 3,086 metastatic tumors involving various anatomic sites. To allow specific tumor cell profiling, we used established immunohistochemical methods to perform semiquantitative assessment for a panel of prototypic melanocyte differentiation antigens (MDA), including gp100, MART-1, and tyrosinase. To gain insight into the endogenous host immune response against these tumors, we further characterized tumor cell expression of MHC I and MHC II and, also, the concomitant CD4(+) and CD8(+) T-cell infiltrate.. Tumor cell profiling for MDA expression demonstrated an anatomic site-specific pattern of antigen expression that was highest in brain, intermediate in soft tissues/lymph nodes, and lowest in visceral metastases. Hierarchical clustering analysis supported that melanoma metastases have a phylogenetically determined, rather than a stochastic, pattern of antigen expression that varies by anatomic site. Furthermore, tyrosinase expression was more frequently lost in metastatic sites outside of the brain and was uniquely correlated with both endogenous CD8(+) and CD4(+) T-cell infiltrates.. Site-specific antigen heterogeneity represents a novel attribute for human melanoma metastases that should be considered in future therapy development and when assessing the responsiveness to antigen-specific immunotherapies.

Topics: CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cluster Analysis; Cohort Studies; gp100 Melanoma Antigen; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; Humans; Immunohistochemistry; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Monophenol Monooxygenase; Neoplasm Metastasis; T-Lymphocytes

The MART1-verified Ki-67 proliferation index is a valuable aid to distinguish melanomas from nevi. Because such indices are quantifiable by image analysis, they may provide a novel automated diagnostic aid. This study aimed to validate the diagnostic performance of automated dermal Ki-67 indices and to explore the diagnostic capability of epidermal Ki-67 in lesions both with and without a dermal component. In addition, we investigated the automated indices' ability to predict sentinel lymph node (SLN) status. Paraffin-embedded tissues from 84 primary cutaneous melanomas (35 with SLN biopsy), 22 melanoma in situ, and 270 nevi were included consecutively. Whole slide images were captured from Ki-67/MART1 double stains, and image analysis computed Ki-67 indices for epidermis and dermis. In lesions with a dermal component, the area under the receiver operating characteristic (ROC) curve was 0.79 (95% confidence interval [CI], 0.72-0.86) for dermal indices. By excluding lesions with few melanocytic cells, this area increased to 0.93 (95% CI, 0.88-0.98). A simultaneous analysis of epidermis and dermis yielded an ROC area of 0.94 (95% CI, 0.91-0.96) for lesions with a dermal component and 0.98 (95% CI, 0.97-1.0) for lesions with a considerable dermal component. For all lesions, the ROC area of the simultaneous analysis was 0.89 (95% CI, 0.85-0.92). SLN-positive patients generally had a higher index than SLN-negative patients (P ≤ .003). Conclusively, an automated diagnostic aid seems feasible in melanocytic pathology. The dermal Ki-67 index was inferior to a combined epidermal and dermal index in diagnosis but valuable for predicting the SLN status of our melanoma patients.

Topics: Area Under Curve; Automation; Biomarkers, Tumor; Carcinoma in Situ; Female; Humans; Image Interpretation, Computer-Assisted; Immunohistochemistry; Ki-67 Antigen; Male; MART-1 Antigen; Melanoma; Melanoma, Cutaneous Malignant; Middle Aged; Mitotic Index; Nevus, Pigmented; ROC Curve; Sentinel Lymph Node Biopsy; Skin Neoplasms

Circulating tumor cells have emerged as prognostic biomarkers in the treatment of metastatic cancers of epithelial origins viz., breast, colorectal and prostate. These tumors express Epithelial Cell Adhesion Molecule (EpCAM) on their cell surface which is used as an antigen for immunoaffinity capture. However, EpCAM capture technologies are of limited utility for non-epithelial cancers such as melanoma. We report a method to enrich Circulating Melanoma Cells (CMCs) that does not presuppose malignant cell characteristics. CMCs were enriched by centrifugation of blood samples from healthy (N = 10) and patient (N = 11) donors, followed by RBC lysis and immunomagnetic depletion of CD45-positive leukocytes in a specialized magnetic separator. CMCs were identified by immunocytochemistry using Melan-A or S100B as melanoma markers and enumerated using automated microscopy image analyses. Separation was optimized for maximum sensitivity and recovery of CMCs. Our results indicate large number of CMCs in Stage IV melanoma patients. Analysis of survival suggested a trend toward decreased survival with increased number of CMCs. Moreover, melanoma-associated miRs were found to be higher in CMC-enriched fractions in two patients when compared with the unseparated samples, validating this method as applicable for molecular analyses. Negative selection is a promising approach for isolation of CMCs and other EpCAM -negative CTCs, and is amenable to molecular analysis of CMCs. Further studies are required to validate its efficacy at capturing specific circulating cells for genomic analysis, and xenograft studies.

Topics: Biomarkers, Tumor; Case-Control Studies; Cell Separation; Humans; Immunoenzyme Techniques; Immunomagnetic Separation; Leukocyte Common Antigens; MART-1 Antigen; Melanoma; MicroRNAs; Neoplastic Cells, Circulating; Prognosis; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; S100 Calcium Binding Protein beta Subunit; Survival Rate; Tumor Cells, Cultured

Toll-like receptor (TLR) ligands are attractive candidate adjuvants for therapeutic cancer vaccines, since TLR signaling stimulates and tunes both humoral and cellular immune responses induced by dendritic cells (DCs). Given that human skin contains a dense network of DCs, which are easily accessible via (intra-)dermal delivery of vaccines, skin is actively explored as an antitumor vaccination site. Here we used a human skin explant model to explore the potential of TLR ligands as adjuvants for DC activation in their complex microenvironment. We show that topical application of Aldara skin cream, 5% of which comprises the TLR7 agonist imiquimod, significantly enhanced DC migration as compared with that resulting from intradermal injection of the TLR7/8 ligand R848 or the soluble form of imiquimod. Moreover, Aldara-treated DCs showed highest levels of the costimulatory molecules CD86, CD83, CD40, and CD70. Topical Aldara induced the highest production of pro-inflammatory cytokines in skin biopsies. When combined with intradermal peptide vaccination, Aldara-stimulated DCs showed enhanced cross-presentation of the melanoma antigen MART-1, which resulted in increased priming and activation of MART-1-specific CD8(+) T cells. These results point to advantageous effects of combining the topical application of Aldara with antitumor peptide vaccination.

Topics: Administration, Topical; Aminoquinolines; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cross-Priming; Cytokines; Dendritic Cells; Humans; Imidazoles; Imiquimod; Injections, Intradermal; Ligands; MART-1 Antigen; Melanoma; Skin; Toll-Like Receptor 7

Topics: Adolescent; Carcinoma, Renal Cell; Desmin; Diagnosis, Differential; gp100 Melanoma Antigen; Humans; Leg; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Perivascular Epithelioid Cell Neoplasms; Sarcoma, Clear Cell; Skin Neoplasms

We initially observed that the presence of circulating NY-ESO-1- and/or Melan-A-specific T cells in patients with stage IV melanoma was significantly associated with prolonged survival. Here, we report the ways in which the phenotypes and functions of these T cells differentially affect survival in patients preselected for NY-ESO-1 and/or Melan-A reactivity.. We assayed functional antigen-reactive T cells recognizing NY-ESO-1 and/or Melan-A after in vitro stimulation using overlapping peptide pools. After restimulation, we assayed six cytokines simultaneously by intracellular cytokine staining. This allowed us to analyze the functional antigen response of both CD4(+) and CD8(+) T cells at the single-cell level.. We observed that NY-ESO-1 stimulated mainly CD4(+) T cells, whereas Melan-A more often stimulated CD8(+) T cells. NY-ESO-1 reactivity was not associated with an additional impact on survival, whether CD4(+) T cells, CD8(+) T cells, or both types of T cells were responding. In contrast, recognition of Melan-A by CD4(+) T cells was associated with reduced survival in our cohort of patients preselected for NY-ESO-1 and/or Melan-A reactivity (that is, in patients with exceptionally long survival). We further observed a negative effect on survival in patients with CD4(+) T cells producing IL4 and IL17 upon Melan-A stimulation. Their prognosis was comparable to patients without any Melan-A reactivity.. The nature and prognostic impact of specific T-cell responses is different according to targeted antigen. Independent from phenotype and functional aspects, NY-ESO-1 reactivity is associated with good prognosis. In terms of Melan-A, antigen-specific CD8(+) but not CD4(+) responses are associated with prolonged survival. Clin Cancer Res; 20(16); 4390-9. ©2014 AACR.

Topics: Aged; Antigens, Neoplasm; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Female; Follow-Up Studies; Humans; Interleukin-17; Interleukin-4; Male; MART-1 Antigen; Melanoma; Membrane Proteins; Middle Aged; Neoplasm Staging; Prognosis; Survival Rate

Accurate fine-needle aspiration (FNA) diagnosis of metastatic melanoma is of therapeutic and prognostic significance and often requires ancillary studies. To the authors' knowledge, the reliability of immunostaining using a pan-melanoma cocktail, Sry-related HMG-BOX gene 10 (SOX10), and microphthalmia transcription factor (MITF) in confirming a diagnosis of melanoma on FNA smears has not been studied to date.. This retrospective study included 50 FNA cases with a definitive diagnosis of melanoma. Twenty-nine cases were epithelioid type (group 1), and 21 cases were predominantly spindle cell type with or without an epithelioid component (group 2). Each case was immunostained using pan-melanoma cocktail, SOX10, and MITF. Staining intensity and the percentage of positive cells were recorded.. The pan-melanoma cocktail was positive in 43 cases (86%), SOX10 was positive in 50 cases (100%), and MITF in 45 cases (90%). SOX10 and MITF demonstrated nuclear staining with stronger and more diffuse staining with less or no background staining compared with pan-melanoma cocktail, which displayed cytoplasmic staining. For pan-melanoma cocktail and SOX10, the detection rates were identical in groups 1 and 2 (86% with pan-melanoma cocktail and 100% with SOX10). For MITF, the detection rate was higher in group 1 compared with Group 2 (93% vs 86%).. In the current study, SOX10 was found to have the highest overall detection rate, followed by MITF and pan-melanoma cocktail. The pan-melanoma cocktail and SOX10 performed equally well for groups 1 and 2, and MITF had a higher detection rate in group 1. Overall, SOX10 and MITF appeared to be superior to the pan-melanoma cocktail and SOX10 seemed better than MITF in confirming a diagnosis of melanoma on FNA smears.

Topics: Biomarkers, Tumor; Biopsy, Fine-Needle; Cohort Studies; Cytodiagnosis; Female; gp100 Melanoma Antigen; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Microphthalmia-Associated Transcription Factor; Reproducibility of Results; Retrospective Studies; Skin Neoplasms; SOXE Transcription Factors

Melanoma is one of the most common skin neoplasms in humans and dogs. The tumor microenvironment in melanoma comprises cancer cells and stromal cells that interact to accelerate tumor progression. Several prognostic markers for melanomas have been studied in many human tumors, including fibroblast-specific protein 1 (S100A4). S100A4 is a member of the S100 family of calcium-binding proteins in stromal cells.. The objective of this study was to describe the immunohistochemical patterns of S100A4 in stroma and neoplastic cells of canine skin melanomas and correlate them with some histological parameters.. Forty-eight samples (38 pigmented and 10 non-pigmented melanomas) were first selected and their nature confirmed using S100, Melan A and vimentin. All cases were examined by immunohistochemistry using S100A4 to correlate expression, histotype, and level of invasion.. All the tumors, including 10 non-pigmented, were positive for S100, Melan A, vimentin and negative for cytokeratin AE1/AE3 (consistent with melanomas). The 48 melanomas were classified as epithelioid (n = 21), spindle (n = 14), and mixed (n = 13). S100A4 was preferentially expressed in epithelioid and spindle cell types compared with mixed melanomas and S100A4 expression was not associated with level of invasion (Clark's levels IV to V).. S100A4 expression in melanoma samples varied among histotypes but not between levels of invasion.

Topics: Animals; Brazil; Dog Diseases; Dogs; Immunohistochemistry; MART-1 Antigen; Melanoma; Neoplasm Staging; Skin Neoplasms; Stromal Cells; Vimentin

Patients with melanoma may develop skin depigmentation spontaneously or following therapy, referred to as melanoma-associated leucoderma (MAL). As clinical presentation of MAL may precede primary/metastatic melanoma detection, recognition of MAL is important to prevent its misdiagnosis as vitiligo and the subsequent application of immunosuppressive treatment. To reveal the immunity involved in MAL development, we investigated the presence of antibody and T-cell immune responses directed against the melanocyte-differentiation-antigens MART-1 (Melan-A), tyrosinase and gp100 in patients with MAL, as compared to patients with vitiligo. Autoantibodies to gp100 and tyrosinase were commonly found in both diseases. Interestingly, MART-1 antibodies were only present in patients with MAL. Melanocyte antigen-specific T cells were found in all patients, with relatively more specific T cells in patients with active vitiligo. Although MAL and vitiligo may appear clinically similar, our results indicate that the humoral immune responses against MART-1 differ between these diseases, which can help to differentiate MAL from vitiligo.

Topics: Adolescent; Adult; Aged; Antibody Formation; Antibody Specificity; CD8-Positive T-Lymphocytes; Demography; Female; Humans; Hypopigmentation; Male; MART-1 Antigen; Melanocytes; Melanoma; Middle Aged; Vitiligo; Young Adult

Advances in genetic engineering have made it possible to generate human T-cell products that carry desired functionalities, such as the ability to recognize cancer cells. The currently used strategies for the generation of gene-modified T-cell products lead to highly differentiated cells within the infusion product, and on the basis of data obtained in preclinical models, this is likely to impact the efficacy of these products. We set out to develop a good manufacturing practice (GMP) protocol that yields T-cell receptor (TCR) gene-modified T-cells with more favorable properties for clinical application. Here, we show the robust clinical-scale production of human peripheral blood T-cells with an early memory phenotype that express a MART-1-specific TCR. By combining selection and stimulation using anti-CD3/CD28 beads for retroviral transduction, followed by expansion in the presence of IL-7 and IL-15, production of a well-defined clinical-scale TCR gene-modified T-cell product could be achieved. A major fraction of the T-cells generated in this fashion were shown to coexpress CD62L and CD45RA, and express CD27 and CD28, indicating a central memory or memory stemlike phenotype. Furthermore, these cells produced IFNγ, TNFα, and IL-2 and displayed cytolytic activity against target cells expressing the relevant antigen. The T-cell products manufactured by this robust and validated GMP production process are now undergoing testing in a phase I/IIa clinical trial in HLA-A*02:01 MART-1-positive advanced stage melanoma patients. To our knowledge, this is the first clinical trial protocol in which the combination of IL-7 and IL-15 has been applied for the generation of gene-modified T-cell products.

Topics: Antigens, CD; Cell Engineering; Cell Proliferation; Clinical Trials as Topic; Cytotoxicity, Immunologic; Gene Expression; Genetic Vectors; Humans; Immunologic Memory; Interferon-gamma; Interleukin-15; Interleukin-2; Interleukin-7; MART-1 Antigen; Melanoma; Phenotype; Receptors, Antigen, T-Cell; Retroviridae; Skin Neoplasms; T-Lymphocytes, Cytotoxic; Transduction, Genetic; Tumor Necrosis Factor-alpha

Topics: Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Prognosis; Skin Neoplasms

To detect the caspase-14 expression in malignant melanoma cells and tumor tissues and its effect on tumor resistance to drug.. The mRNA and protein level of caspase-14 in 4 melanoma cell lines (A375, A875, M14, and SK-Mel-1) and the melanocytes, was detected by reverse transcription PCR (RT-PCR) and Western blotting. Caspase-14 expression in 34 malignant melanoma tumor tissues and 10 dermal nevus tissues was determined by in situ hybridization and immunohistochemistry.. Caspase-14 expression was seen in melanoma cells and melanocytes. It was higher in melanoma-associated antigen 1 recognized by T cells (MART-1) positive cells than in MART-1 negative cells. The cells expressing the lower caspase-14 were more sensitive to the treatment with either chemotherapy drugs camptothecin and cisplatin or radiotherapy than the ones expressing the higher caspase-14 (P<0.01). Caspase-14 expression was observed in 70% dermal nevus, as well as 97% in malignant melanoma tissues, and the difference between them was statistically significant (P<0.05).. Caspase-14 is expressed in tissues and cells of malignant melanoma. Our data indicated that the expression level of caspase-14 affected the drug sensitivity of melanoma.

Topics: Caspase 14; Cell Line, Tumor; Humans; MART-1 Antigen; Melanocytes; Melanoma

Targeting antigen to dendritic cells (DCs) is a powerful and novel strategy for vaccination. Priming or loading DCs with antigen controls whether subsequent immunity will develop and hence whether effective vaccination can be achieved. The goal of our present work was to increase the potency of DC-based antitumor vaccines by overcoming inherent limitations associated with antigen stability and cross-presentation. Nanoparticles prepared from the biodegradable polymer poly(lactic-co-glycolic acid) have been extensively used in clinical settings for drug delivery and are currently the subject of intensive investigation as antigen delivery vehicles for vaccine applications. Here we describe a nanoparticulate delivery system with the ability to simultaneously carry a high density of protein-based antigen while displaying a DC targeting ligand on its surface. Utilizing a targeting motif specific for the DC-associated surface ligand DEC-205, we show that targeted nanoparticles encapsulating a MART-127-35 peptide are both internalized and cross-presented with significantly higher efficiency than isotype control-coated nanoparticles in human cells. In addition, the DEC-205-labeled nanoparticles rapidly escape from the DC endosomal compartment and do not colocalize with markers of early (EEA-1) or late endosome/lysosome (LAMP-1). This indicates that encapsulated antigens delivered by nanoparticles may have direct access to the class I cytoplasmic major histocompatibility complex loading machinery, overcoming the need for "classical" cross-presentation and facilitating heightened DC stimulation of anti-tumor CD8(+) T-cells. These results indicate that this delivery system provides a flexible and versatile methodology to deliver melanoma-associated antigen to DCs, with both high efficiency and heightened potency.

Topics: Antigen Presentation; Antigens, CD; Cancer Vaccines; Dendritic Cells; Humans; Lactic Acid; Lectins, C-Type; MART-1 Antigen; Melanoma; Minor Histocompatibility Antigens; Molecular Targeted Therapy; Nanoparticles; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Receptors, Cell Surface

Melanoma of the skin is characterised by a high metastatic potential, but reports of metastasis to the tongue are rare. We report a case of skin melanoma with metastasis to the lymph nodes, tongue and brain.. This report highlights the clinical and histological features of oral metastatic melanoma.. A 72-year-old man was seen with a nodule on the tongue. The differential diagnosis included salivary gland tumour, lymphoma and metastatic melanoma. His medical history revealed treatment for melanoma in the periumbilical region and micrometastases in the inguinal lymph nodes. An incisional biopsy was obtained and histological analysis showed the presence of a solid, epithelioid malignant tumour of monotonous appearance infiltrating the skeletal musculature. Immunohistochemistry showed reactivity of neoplastic cells to anti-HMB45, anti-melan A and anti-S100 antibodies and negativity for anti-PAN cytokeratin, confirming the diagnosis of metastatic melanoma.. The present findings highlight the importance of a complete medical evaluation of the patient by anamnesis to identify possible oral repercussions of primary diseases in other organs and/or systems.

Topics: Aged; Brain Neoplasms; Diagnosis, Differential; gp100 Melanoma Antigen; Humans; Immunohistochemistry; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; S100 Proteins; Skin Neoplasms; Tongue Neoplasms

The immunoreactivity of PNL2, Melan A, and protein gene product (PGP) 9.5 was compared with that of S100 protein in 50 formalin-fixed, paraffin-embedded equine melanocytic neoplasms. PNL2, PGP 9.5, and S100 protein were detected in all 50 neoplasms; none expressed Melan A. PNL2 was not expressed in 62 nonmelanocytic tumors (equine sarcoids, schwannomas, carcinomas, sarcomas, endocrine tumors, sex-cord stromal tumors, germ cell tumors, and leukocytic tumors) or in normal tissues other than epidermis. In summary, antibody PNL2 is a sensitive marker of equine melanocytic neoplasms and is more specific than S100 protein or PGP 9.5. In contrast, the monoclonal antibody to Melan A did not react with any of the equine melanomas.

Topics: Animals; Antibodies, Monoclonal; Biomarkers, Tumor; Cross Reactions; Horses; Immunohistochemistry; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; S100 Proteins; Sensitivity and Specificity; Skin Neoplasms

We have previously reported a model for melanoma progression in which oscillation between melanoma cell phenotypes characterized by invasion or proliferation is fundamental to tumor heterogeneity and disease progression. In this study we examine the possible role of hypoxia as one of the microenvironmental influences driving metastatic progression by promoting a switch from a proliferative to an invasive phenotype. Immunohistochemistry on primary human cutaneous melanoma biopsies showed intratumoral heterogeneity for cells expressing melanocytic markers, and a loss of these markers correlated with hypoxic regions. Furthermore, we show that the downregulation of melanocytic markers is dependent on hypoxia inducible factor 1α (HIF1α), a known regulator of the hypoxic response. In vitro invasion assays showed that a hypoxic environment increases the invasiveness of proliferative melanoma cell cultures in a HIF1α-dependent manner. In contrast, invasive phenotype melanoma cells showed no increase in invasive potential upon exposure to hypoxia. Thus, exposure of proliferative melanoma cells to hypoxic microenvironments is sufficient, in a HIF1α-dependent manner, to downregulate melanocytic marker expression and increase their invasive potential.

Topics: Biomarkers, Tumor; Cell Proliferation; Disease Progression; Gene Expression Regulation, Neoplastic; Genetic Heterogeneity; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; MART-1 Antigen; Melanoma; Neoplasm Invasiveness; Phenotype; Skin Neoplasms; Tumor Cells, Cultured; Tumor Microenvironment

Anoikis resistance is one of the abilities acquired along tumor progression. This characteristic is associated with metastasis development, since tumorigenic cells must survive independently of cell-matrix interactions in this process. In our laboratory, it was developed a murine melanocyte malignant transformation model associated with a sustained stressful condition. After subjecting melan-a melanocytes to 1, 2, 3 and 4 cycles of anchorage impediment, anoikis resistant cells were established and named 1C, 2C, 3C and 4C, respectively. These cells showed altered morphology and PMA independent cell growth, but were not tumorigenic, corresponding to pre-malignant cells. After limiting dilution of 4C pre-malignant cells, melanoma cell lines with different characteristics were obtained. Previous data from our group showed that increased Timp1 expression correlated with anoikis-resistant phenotype. Timp1 was shown to confer anchorage-independent growth capability to melan-a melanocytes and render melanoma cells more aggressive when injected into mice. However, the mechanisms involved in anoikis regulation by Timp1 in tumorigenic cells are not clear yet.. The β1-integrin and Timp1 expression were evaluated by Western blotting and CD63 protein expression by flow cytometry using specific antibodies. To analyze the interaction among Timp1, CD63 and β1-integrin, immunoprecipitation assays were performed, anoikis resistance capability was evaluated in the presence or not of the PI3-K inhibitors, Wortmannin and LY294002. Relative expression of TIMP1 and CD63 in human metastatic melanoma cells was analyzed by real time PCR.. Differential association among Timp1, CD63 and β1-integrins was observed in melan-a melanocytes, 4C pre-malignant melanocytes and 4C11- and 4C11+ melanoma cells. Timp1 present in conditioned medium of melanoma cells rendered melan-a melanocytes anoikis-resistant through PI3-K signaling pathway independently of Akt activation. In human melanoma cell lines, in which TIMP1 and beta-1 integrin were also found to be interacting, TIMP1 and CD63 levels together was shown to correlate significantly with colony formation capacity.. Our results show that Timp1 is assembled in a supramolecular complex containing CD63 and β1-integrins along melanoma genesis and confers anoikis resistance by activating PI3-K signaling pathway, independently of Akt phosphorylation. In addition, our data point TIMP1, mainly together with CD63, as a potential biomarker of melanoma.

Topics: Anoikis; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Culture Media, Conditioned; Disease Progression; Gene Expression; Humans; Integrin beta1; MART-1 Antigen; Melanocytes; Melanoma; Models, Biological; Multiprotein Complexes; Neoplasm Metastasis; Phenotype; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Binding; Proto-Oncogene Proteins c-akt; Signal Transduction; Tetraspanin 30; Tissue Inhibitor of Metalloproteinase-1

In the American Joint Committee on Cancer (AJCC)-TNM (2009) staging system, the key prognostic factor in cutaneous melanoma is the depth of dermal invasion (Breslow thickness) with further refinement according to the presence of epidermal ulceration or dermal mitoses. Immunodetection of phosphohistone H3 has been shown to facilitate the identification of mitotic figures in various neoplasms. We selected 120 cases of primary cutaneous melanoma with completely annotated histopathologic parameters and clinical outcomes and performed double immunohistochemical staining for MLANA (Mart-1/Melan-A) and phosphohistone H3. One hundred and thirteen cases were amenable to antiphosphohistone H3 staining from 66 men and 47 women, with mean age of 64 years (9-93), including 61 superficial spreading type, 24 nodular, 6 lentigo maligna, 8 acral lentiginous, and 14 unclassified. The mean Breslow thickness was 2.53 mm (0.20-25), ulceration was present in 25/113 (22%) and the mean mitotic count was 3.2/mm(2) (<1-29/mm(2)). In 27/113 (24%) of the cases, antiphosphohistone H3 failed to highlight mitotic figures anywhere in the tissue (normal or tumor cell), whereas in 86/113 (76%) antiphosphohistone H3 detected at least one mitotic figure. Among the latter, antiphosphohistone H3 did not detect mitotic figures in dermal tumor cells in 37/86 cases (43%), whereas anti-PHH3 identified at least one melanocytic mitotic figure in the other 49/86 cases (57%; range: 1-66/mm(2)). The relationship between phosphohistone H3 and manual mitotic count was statistically significant (Pearson correlation=0.59, P<0.0001). Logistic regression analyses demonstrated an association between the development of subsequent metastatic disease and the following variables: mitotic figures (odds ratio (OR)=5.7; P=0.0001); phosphohistone H3-positive mitotic figures (OR=3.0; P=0.008); Breslow thickness (OR=4.0 per mm; P=0.0002); ulceration (OR=3.94; P=0.008). The application of phosphohistone H3 immunohistochemistry to the description of primary cutaneous melanoma is useful in identifying mitotic figures, improves upon the specificity of this designation when used together with MLANA, and correlates with an increased risk for metastasis in univariate analyses.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Biopsy; Child; Female; Histones; Humans; Immunohistochemistry; Logistic Models; Male; MART-1 Antigen; Melanoma; Middle Aged; Mitosis; Mitotic Index; Multivariate Analysis; Neoplasm Invasiveness; Neoplasm Staging; Odds Ratio; Phosphorylation; Predictive Value of Tests; Prognosis; Risk Factors; Skin Neoplasms; Young Adult

Adoptive cell transfer (ACT) of T cells has great clinical potential, but the numerous variables of this therapy make choices difficult. A new study takes advantage of a novel technology for characterizing the T-cell responses of patients. If applied systematically, this approach may identify biomedical correlates of protection, thereby supporting treatment optimization.

Topics: Female; Humans; Immunotherapy, Adoptive; Male; MART-1 Antigen; Melanoma; Skin Neoplasms; T-Lymphocytes

Isolated limb perfusion (ILP) with hyperthermia is an effective treatment for in-transit metastases of malignant melanoma in the extremities. Preclinical studies have shown that hyperthermia may induce an immunogenic death of tumour cells. We therefore decided to study whether ILP may induce tumour-specific immune responses in the clinical setting.. The number of Melan-A/Mart-1 specific CD8+ T cells, as well as other phenotypically different immune cells, was recorded in peripheral blood in 12 HLA-A2+ patients with in-transit metastases undergoing hyperthermic ILP with melphalan.. All patients underwent ILP without any complication and with an overall response rate of 83%. No substantial changes in the number of circulating T-cells, B-cells, NK-cells or monocytes were observed during follow-up. Four out of 12 patients showed an elevation of Melan-A+ CD8+ T-cells 4 weeks after ILP.. We here report our preliminary observations that a small increase in tumour-specific T-cells could be seen in a subpopulation of patients after ILP. However, much more work is necessary to fully delineate the systemic immune response to hyperthermic ILP.

Topics: Aged; Aged, 80 and over; Extremities; Female; Humans; Hyperthermia, Induced; Lymphocyte Count; Lymphocyte Subsets; Male; MART-1 Antigen; Melanoma; Middle Aged; Perfusion; Pilot Projects; Skin Neoplasms; T-Lymphocytes, Cytotoxic

Intraocular melanocytoma is a rare naevus variant that can be located at the optic disc or within the uvea, and belongs to the group of non-epithelial-associated melanocytic lesions. We wanted to gain an understanding of the role of GNAQ, GNA11 and BRAF V600E in the pathogenesis of uveal melanocytoma and in cases of transformation to uveal melanoma and also to perform a differential immunohistochemical study comparing melanocytoma with uveal melanoma.. Two patients were identified with melanocytoma, one of which had transformed to melanoma. In the latter case, the melanocytoma exhibited an immunophenotype that featured nuclear p27 and no HMB45 staining, with very low Cyclin D1 expression compared with the melanoma that featured little nuclear but more cytoplasmic p27 positivity, much higher Cyclin D1 expression and HMB45 positivity. The melanocytomas were negative for CD68 allowing distinction from melanophages. Both melanocytomas and the melanoma harboured mutations in GNAQ, with no mutations of GNA11 or BRAF V600E.. GNAQ mutations are present in uveal melanocytomas and in a case of transformation to melanoma, implicating GNAQ-dependent mitogen activation signals, in the pathogenesis of uveal melanocytoma. This assists in explaining why a proportion of uveal melanocytoma can transform to uveal melanoma, known to harbour high-frequency GNAQ mutations at exon 5, codon 209.

Topics: Adolescent; Adult; Cell Transformation, Neoplastic; Chromosomes, Human, Pair 3; Chromosomes, Human, Pair 8; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; gp100 Melanoma Antigen; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gq-G11; Humans; Immunoenzyme Techniques; In Situ Hybridization, Fluorescence; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Mutation; Nevus, Pigmented; Polymerase Chain Reaction; Proto-Oncogene Proteins B-raf; Retrospective Studies; Uveal Neoplasms

Immunohistochemistry is a routine practice in clinical cancer diagnostics and also an established technology for tissue-based research regarding biomarker discovery efforts. Tedious manual assessment of immunohistochemically stained tissue needs to be fully automated to take full advantage of the potential for high throughput analyses enabled by tissue microarrays and digital pathology. Such automated tools also need to be reproducible for different experimental conditions and biomarker targets. In this study we present a novel supervised melanoma specific pattern recognition approach that is fully automated and quantitative.. Melanoma samples were immunostained for the melanocyte specific target, Melan-A. Images representing immunostained melanoma tissue were then digitally processed to segment regions of interest, highlighting Melan-A positive and negative areas. Color deconvolution was applied to each region of interest to separate the channel containing the immunohistochemistry signal from the hematoxylin counterstaining channel. A support vector machine melanoma classification model was learned from a discovery melanoma patient cohort (n = 264) and subsequently validated on an independent cohort of melanoma patient tissue sample images (n = 157).. Here we propose a novel method that takes advantage of utilizing an immuhistochemical marker highlighting melanocytes to fully automate the learning of a general melanoma cell classification model. The presented method can be applied on any protein of interest and thus provides a tool for quantification of immunohistochemistry-based protein expression in melanoma.

Topics: Humans; Image Processing, Computer-Assisted; Immunohistochemistry; MART-1 Antigen; Melanocytes; Melanoma; Pattern Recognition, Automated; Surface Properties; Tissue Array Analysis

Melanocytic nevus rests in lymph nodes are a known diagnostic challenge, especially in patients with a history of melanoma. Reticulin and NM23 have been studied in this context. The pattern of reticulin staining in melanomas surrounds groups/nests of melanocytes but individual cells in benign nevi. NM23, a metastasis-suppressor gene, has an association with metastatic potential in melanomas and some carcinomas. Twenty-eight cases (14 cases of metastatic melanoma to lymph nodes and 14 cases of lymph node nevus rests, all confirmed with Melan-A staining) were stained with reticulin and NM23. The pattern of reticulin staining was reported as surrounding groups if staining was noted in approximately 5-10 melanocytes in greater than 50% of the lesion but was otherwise reported as surrounding individual melanocytes. Cytoplasmic staining was considered to represent reactivity for NM23. Reticulin staining around groups of melanocytes was identified in all 14 cases of metastatic melanoma. Regarding nodal nevus rest cases, 12 of 14 cases (86%) demonstrated staining around individual melanocytes, whereas in 2 cases, reticulin surrounded melanocytic groups. NM23 staining was equivocal in all cases. Reticulin staining reliably invests groups of melanocytes in cases of metastatic melanoma, whereas in nodal nevus rests, it predominantly surrounds individual melanocytes. NM23 demonstrated no discriminatory value in this analysis. In cases in which a collection of melanocytes is present within a lymph node, reticulin deposition around individual melanocytes supports a diagnosis of lymph nodal nevus rest.

Topics: Aged; Biomarkers, Tumor; Biopsy; Female; Humans; Immunohistochemistry; Lymph Nodes; Lymphatic Metastasis; Male; MART-1 Antigen; Melanocytes; Melanoma; Nevus, Pigmented; NM23 Nucleoside Diphosphate Kinases; Reticulin; Skin Neoplasms; United States

Gap junctions (GJs) enable intercellular communication between adjacent cells through channels of connexins. Using a three-dimensional construct, we previously showed that endothelial and tumor cells formed GJs, allowing melanoma-specific T lymphocytes to recognize and kill melanoma-derived endothelial cells. We demonstrate here on histological sections of melanoma biopsies that GJ formation occurs in vivo between tumor and endothelial cells and between T lymphocytes and target cells. We also show an in vitro increase of GJ formation in melanoma and endothelial cells following dacarbazin and interferon gamma (IFN-γ) treatment or hypoxic stress induction. Our data indicate that although connexin 43 (Cx43), the main GJ protein of the immune system, was localized at the immunological synapse between T lymphocyte and autologous melanoma cells, its over-expression or inhibition of GJs does not interfere with cytotoxic T lymphocyte (CTL) clone lytic function. In contrast, we showed that inhibition of GJs by oleamide during stimulation of resting PBMCs with Melan-A natural and analog peptides resulted in a decrease in antigen (Ag) specific CD8(+) T lymphocyte induction. These Ag-specific CD8(+) cells displayed paradoxically stronger reactivity as revealed by CD107a degranulation and IFN-γ secretion. These findings indicate that Cx43 does not affect lytic function of differentiated CTL, but reveal a major role for GJs in the regulation of antigen CD8(+)-naïve T lymphocyte activation.. GJ formation occurs in vivo between T lymphocytes and tumor cells Cx43 localized at the immunological synapse between T and autologous melanoma cells Inhibition of GJs resulted in a decrease in Ag-specific CD8(+) T lymphocyte induction A role for GJs in the regulation of antigen CD8(+)-naïve T lymphocyte activation.

Topics: Antineoplastic Agents, Alkylating; Apoptosis; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Connexin 43; Cytotoxicity, Immunologic; Dacarbazine; Gap Junctions; Gene Expression; Humans; Hypoxia; Immunological Synapses; Interferon-gamma; MART-1 Antigen; Melanoma; Oxidative Stress; Protein Transport; T-Lymphocytes, Cytotoxic

Malignant melanoma is a highly aggressive tumor with increasing incidence and high mortality. The importance of immunohistochemistry in diagnosis of the primary tumor and in early identification of metastases in lymphatic nodes is enormous; however melanoma phenotype is frequently variable and thus several markers must be employed simultaneously. The purposes of this study are to describe changes of phenotype of malignant melanoma in vitro and in vivo and to investigate whether changes of environmental factors mimicking natural conditions affect the phenotype of melanoma cells and can revert the typical in vitro loss of diagnostic markers. The influence of microenvironment was studied by means of immunocytochemistry on co-cultures of melanoma cells with melanoma-associated fibroblast and/or in conditioned media. The markers typical for melanoma (HMB45, Melan-A, Tyrosinase) were lost in malignant cells isolated from malignant effusion; however, tumor metastases shared identical phenotype with primary tumor (all markers positive). The melanoma cell lines also exerted reduced phenotype in vitro. The only constantly present diagnostic marker observed in our experiment was S100 protein and, in lesser extent, also Nestin. The phenotype loss was reverted under the influence of melanoma-associated fibroblast and/or both types of conditioned media. Loss of some markers of melanoma cell phenotype is not only of diagnostic significance, but it can presumably also contribute to biological behavior of melanoma. The presented study shows how the conditions of cultivation of melanoma cells can influence their phenotype. This observation can have some impact on considerations about the role of microenvironment in tumor biology.

Topics: Biomarkers, Tumor; Cell Culture Techniques; Cell Line, Tumor; Cells, Cultured; Coculture Techniques; Culture Media, Conditioned; Fibroblasts; gp100 Melanoma Antigen; Humans; Immunohistochemistry; Immunophenotyping; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Models, Biological; Monophenol Monooxygenase; Nestin; S100 Proteins; Skin Neoplasms; Tumor Cells, Cultured; Tumor Microenvironment

Uveal melanoma (UM) is the most common primary intraocular malignancy and the leading potentially fatal primary intraocular disease in adults. Melanoma antigen recognized by T-cells (MART-1) has been studied extensively as a clinically important diagnostic marker for melanoma, however, its biological function remains unclear. In the present study, the UM cell line SP6.5, which showed a high level of MART-1 expression, was subjected to small interfering RNA-mediated silencing of MART-1. Silencing of MART-1 expression increased the migration ability of SP6.5 cells and down-regulated the expression of the metastasis suppressor NM23. Our results suggest that MART-1 is a candidate target for the development of therapeutic strategies for UM and in particular for the suppression of metastasis associated with this malignancy.

Topics: Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Humans; MART-1 Antigen; Melanoma; NM23 Nucleoside Diphosphate Kinases; RNA Interference; RNA, Messenger; RNA, Small Interfering; Tumor Suppressor Proteins; Ubiquitin Thiolesterase; Uveal Neoplasms

The distribution of the standard melanoma antibodies S100, HMB-45, and Melan-A has been extensively studied. Yet, the overlap in their expression is less well characterized.. To determine the joint distributions of the classic melanoma markers and to determine if classification according to joint antigen expression has prognostic relevance.. S100, HMB-45, and Melan-A were assayed by immunofluorescence-based immunohistochemistry on a large tissue microarray of 212 cutaneous melanoma primary tumors and 341 metastases. Positive expression for each antigen required display of immunoreactivity for at least 25% of melanoma cells. Marginal and joint distributions were determined across all markers. Bivariate associations with established clinicopathologic covariates and melanoma-specific survival analyses were conducted.. Of 322 assayable melanomas, 295 (91.6%), 203 (63.0%), and 236 (73.3%) stained with S100, HMB-45, and Melan-A, respectively. Twenty-seven melanomas, representing a diverse set of histopathologic profiles, were S100 negative. Coexpression of all 3 antibodies was observed in 160 melanomas (49.7%). Intensity of endogenous melanin pigment did not confound immunolabeling. Among primary tumors, associations with clinicopathologic parameters revealed a significant relationship only between HMB-45 and microsatellitosis (P = .02). No significant differences among clinicopathologic criteria were observed across the HMB-45/Melan-A joint distribution categories. Neither marginal HMB-45 (P = .56) nor Melan-A (P = .81), or their joint distributions (P = .88), was associated with melanoma-specific survival.. Comprehensive characterization of the marginal and joint distributions for S100, HMB-45, and Melan-A across a large series of cutaneous melanomas revealed diversity of expression across this group of antigens. However, these immunohistochemically defined subclasses of melanomas do not significantly differ according to clinicopathologic correlates or outcome.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Female; gp100 Melanoma Antigen; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Male; MART-1 Antigen; Melanins; Melanoma; Melanoma-Specific Antigens; Middle Aged; S100 Proteins; Skin Neoplasms; Young Adult

The genetic modification of peripheral blood lymphocytes using retroviral vectors to redirect T cells against tumor cells has been recently used as a means to generate large numbers of antigen-specific T cells for adoptive cell therapy protocols. However, commonly used retroviral vector-based genetic modification requires T cells to be driven into cell division; this potent mitogenic stimulus is associated with the development of an effector phenotype that may adversely impact upon the long-term engraftment potential and subsequent antitumor effects of T cells. To investigate whether the cytokines used during culture impact upon the engraftment potential of gene-modified T cells, a humanized model employing T cells engrafted with a MART-1-specific T cell receptor adoptively transferred into NOD/Shi-scid IL-2rγ(-/-) (NSG) immune-deficient mice bearing established melanoma tumors was used to compare the effects of the common γ chain cytokines IL-2, IL-7, and IL-15 upon gene-modified T cell activity. MART-1-specific T cells cultured in IL-7 and IL-15 demonstrated greater relative in vitro proliferation and viability of T cells compared with the extensively used IL-2. Moreover, the IL-15 culture prolonged the survival of animals bearing melanoma tumors after adoptive transfer. However, the combination of IL-7 and IL-15 produced T cells with improved engraftment potential compared with IL-15 alone; however, a high rate of xenogeneic graft-versus-host disease prevented the identification of a clear improvement in antitumor effect of these T cells. These results clearly demonstrate modulation of gene-modified T cell engraftment in the NSG mouse, which supports the future testing of the combination of IL-7 and IL-15 in adoptive cell therapy protocols; however, this improved engraftment is also associated with the long-term maintenance of xenoreactive T cells, which limits the ultimate usefulness of the NSG mouse model in this situation.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Humans; Immunotherapy, Adoptive; Interleukins; MART-1 Antigen; Melanoma; Mice; Mice, Inbred NOD; Mice, SCID; Models, Animal; Skin Neoplasms; T-Lymphocytes; Xenograft Model Antitumor Assays

Engaging CD4 T cells in antitumor immunity has been quite challenging, especially in an Ag-specific manner, because most human solid tumors usually do not express MHC class II molecules. We have recently shown that human CD4 T cells engineered to express a human melanoma-associated antigenic epitope, MART-127-35, specific MHC class I-restricted transgenic TCR function as polyfunctional effectors that can exhibit a helper as well as cytolytic effector function, in an epitope-specific and MHC class I-restricted manner (Chhabra et al. 2008. J. Immunol. 181: 1063-1070; Ray et al. 2010. Clin. Immunol. 136: 338-347). TCR-engineered (TCReng) CD4 T cells therefore have translational potential, and clinical trials with MHC class I TCReng CD4 T cells are under way. In this study, we show that although TCReng CD4 T cells could be useful in cancer immunotherapy, they are also susceptible to epitope-specific activation-induced cell death (AICD). We also show that the AICD in TCReng CD4 T cells is a death receptor-independent process and that JNK and p53 play critical roles in this process as pharmacological inhibitors targeting JNK activation and p-53-mediated transcription-independent mitochondria-centric death cascade rescued a significant fraction of TCReng CD4 T cells from undergoing AICD without affecting their effector function. Our data offer novel insights toward AICD in TCReng CD4 T cells and identify several potential targets to interfere with this process.

Topics: Blotting, Western; CD4-Positive T-Lymphocytes; Cell Death; Cell Line, Tumor; Genetic Engineering; Histocompatibility Antigens Class I; Humans; MART-1 Antigen; Melanoma; Receptors, Antigen, T-Cell; Transgenes

A 52-year-old man has unresectable locally recurrent melanoma of the left foot (Fig 1) and pulmonary metastases. Nine months before this presentation, he underwent a wide local excision and sentinel node biopsy for an acral melanoma on his left heel. Pathology disclosed Breslow thickness of 4.8 mm, Clark level IV, and tumor ulceration with a mitotic rate of 37 mitoses/mm(2). Both sentinel nodes in the left groin were positive for melanoma cells, which expressed S100, HMB45, and melan A. At subsequent left inguinal dissection, seven more nodes showed no additional nodal metastases. Within 3 months of his original surgery, the patient developed a local recurrence in the foot, and over the subsequent 6 months, he underwent serial local excisions and topical diphencyprone treatment. A recent staging scan showed at least 20 foci of in-transit disease in the left lower leg and foot, as well as a solitary lung metastasis (12 mm). His Eastern Cooperative Oncology Group performance status is 1, with no significant comorbidities. High-resolution melt followed by sequencing of an in-transit metastasis showed there is no BRAF exon 15 mutation. However, Sanger sequencing of KIT exons 9, 11, 13, and 17, performed as screening for a clinical trial enrolling patients with metastatic acral and mucosal melanomas, showed an exon 13 K642E mutation.

Topics: Biomarkers, Tumor; gp100 Melanoma Antigen; Humans; Leg; Lung Neoplasms; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Mutation; Neoplasm Recurrence, Local; Prognosis; Proto-Oncogene Proteins c-kit; S100 Proteins; Skin Neoplasms

A combined squamomelanocytic tumor is an exceedingly rare occurrence; little is known about its pathogenesis. A definitive diagnosis can only be made via histological examination. We describe herein an 83 year-old man who was discovered to have this combined tumor and recommend the appropriate management for such a lesion.

Topics: Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Squamous Cell; Facial Neoplasms; gp100 Melanoma Antigen; Humans; Keratins; Male; MART-1 Antigen; Melanins; Melanoma; Melanoma-Specific Antigens; Neoplasms, Multiple Primary; S100 Proteins; Skin Neoplasms

A number of trials of adoptive transfer of tumor-specific T lymphocytes have been performed in the last 20 years in metastatic melanoma, with increasingly encouraging results as the relevant melanoma antigens were identified and the purity/specificity of injected T cells improved. We have previously described a sorting method of epitope-specific T lymphocytes that uses magnetic beads coated with HLA/peptide complexes and we suggested that this method could be applied to a clinical setting. In the present work, we provide a detailed description of the whole GMP process of sorting and amplification of clinical grade T cells specific for the melanoma antigens Melan-A and MELOE-1. All the reagents used in this process including the sorting reagent were produced in GMP conditions and we document the optimization of the different steps of the process such as peptide stimulation, sorting, and amplification. The optimized procedure, validated in 3 blank runs in a clinical setting, allowed the production of at least 10⁸ pure (>90%) Melan-A- and MELOE-1-specific T cells within 28 days starting with 100 mL of blood from metastatic melanoma patients. This GMP process is thus ready to be used in an upcoming phase I/II clinical trial on metastatic melanoma patients.

Topics: Cell Line, Tumor; Epitopes, T-Lymphocyte; HLA-A2 Antigen; Humans; Immunotherapy, Adoptive; Lymphocyte Activation; MART-1 Antigen; Melanoma; Neoplasm Metastasis; Peptides; T-Lymphocytes

Metastatic melanoma involving the esophagus is rare; the occurrence of metastatic melanoma in a background of Barrett esophagus is rarer still. We report a case of an 80 year-old male who presented to our institution for workup of Barrett esophagus with high-grade dysplasia and who proved to have metastatic melanoma occurring in the background of Barrett esophagus, the first report of this kind, to our knowledge, in the English literature.. An 80 year-old Caucasian male was diagnosed at an outside institution with Barrett's esophagus with high grade dysplasia and presented to our institution for therapy. The patient underwent endoscopic mucosal resection using a band ligation technique of an area of nodularity within the Barrett esophagus. Microscopic examination demonstrated extensive Barrett esophagus with high-grade dysplasia as well as a second tumor which was morphologically different from the surrounding high-grade dysplasia and which was positive for S-100, HMB 45 and Melan-A on immunohistochemistry, consistent with melanoma. Further workup of the patient demonstrated multiple radiologic lesions consistent with metastases. Molecular studies demonstrated that the melanoma was positive for the 1799T>A (V600E) mutation in the BRAF gene. The overall features of the tumor were most consistent with metastatic melanoma occurring in a background of Barrett esophagus with high-grade dysplasia.. This case demonstrates a unique intersection between a premalignant condition (Barrett esophagus with high grade dysplasia) and a separate malignancy (melanoma). This report also shows the utility of molecular testing to support the hypothesis of primary versus metastatic disease in melanoma.

Topics: Aged, 80 and over; Barrett Esophagus; Biomarkers, Tumor; Esophageal Neoplasms; Esophagus; gp100 Melanoma Antigen; Humans; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Mutation; Neoplasm Metastasis; Proto-Oncogene Proteins B-raf; S100 Proteins

Clear cell sarcoma is a rare cancer primarily of tendons, fascia, and aponeuroses that can be difficult to discern from primary cutaneous malignant melanoma. The two cancers share several histological markers, with most cases of both cancers staining positively for S-100, HMB-45, and melanin. Primary therapy of both cancers involves wide local excision, but while systemic therapy has proven benefit for malignant melanoma, it has not been established for clear cell sarcoma.We report the case of a 58 year old woman with a large, ulcerated, fungating mass on her left lower leg. Frozen section of the mass showed a malignant epithelioid and spindle cell tumor confined to the subcutaneous tissue. A provisional diagnosis of soft-tissue sarcoma was made. Through in-depth study of initial biopsy with immunohistochemistry for S-100, HMB-45, MART-1, and MITF, along with karyotyping and FISH analysis for EWS gene rearrangement, the diagnosis of amelanotic malignant melanoma was confirmed. The patient then underwent systemic treatment with ipilimumab upon recurrence with good response.. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1989338475107348.

Topics: Antibodies, Monoclonal; Biomarkers, Tumor; Diagnosis, Differential; Female; Humans; Ipilimumab; MART-1 Antigen; Melanoma; Microphthalmia-Associated Transcription Factor; Middle Aged; Proto-Oncogene Proteins B-raf; Sarcoma, Clear Cell; Skin Neoplasms; Soft Tissue Neoplasms; Treatment Outcome

Topics: Adolescent; Biomarkers, Tumor; Biopsy; Cell Transformation, Neoplastic; Conjunctival Neoplasms; gp100 Melanoma Antigen; Humans; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Nevus, Pigmented; S100 Proteins

To study the clinicopathologic features and differential diagnosis of cutaneous regressing/regressed melanoma.. Histopathologic evaluation and immunohistochemical study by EnVision method were performed in 8 cases of cutaneous regressing/regressed melanoma. The clinical presentation, treatment and follow-up data were analyzed.. The age of the patients ranged from 40 to 69 years (mean 58 years). The male-to-female ratio was 3: 1. Tumors were located on the back (4 cases), sole of the foot (2 cases), ventral aspect of the toes (1 case), and the forearm (1 case). Clinically, 6 patients presented with progressive black mole of the skin, followed by subsequent focal hypopigmentation, even scarring. Two patients presented with multiple foci of dark-brown pigmentation. Microscopically, 3 cases were completely regressed malignant melanoma. Tumoral melanosis was found in 1 of 3 cases. The other 5 cases were melanoma with severe regression. The extent of regression ranged from 75% to 90%. The Breslow depth of the tumors ranged from 0.5 to 1.0 mm. Immunohistochemically, both metastatic and primary tumor cells were diffusely positive for S-100, HMB45 and Melan A, while melanophages were positive for CD68. Follow-up data were available in 8 patients, ranging from 8 to 27 months. Five patients were alive with no evidence of disease, 1 patient was alive with stable disease and 2 patients died of metastatic melanoma.. Correlation between clinical presentation and pathologic features is important for diagnosis of cutaneous regressing/regressed melanoma. Thin melanoma with extensive regression ( ≥ 75%) should not been regarded as low metastatic risk and wide excision combined with sentinel lymph node biopsy is recommended.

Topics: Adult; Aged; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Back; Female; Follow-Up Studies; Foot; Humans; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Melanoma, Cutaneous Malignant; Melanosis; Middle Aged; S100 Proteins; Sentinel Lymph Node Biopsy; Skin Neoplasms; Toes; Treatment Outcome

There are limited studies on the utility of immunostaining in cytologic specimens suspected of melanoma. In this study, we examined the performance of the most commonly used antibodies including monoclonal antibodies against Melan-A (A103), S-100, and HMB-45 antigens. Immunostains were performed on formalin-fixed, paraffin-embedded cell blocks prepared from 100 cytologic specimens. The specimens consisted of 57 melanomas and 43 nonmelanocytic neoplasms. Of 57 melanomas, 53 showed positive reaction to Melan-A antibody while 51 and 41 revealed positive immunostaining for S-100 and HMB-45, respectively. Of 43 nonmelanocytic neoplasms, 10, 4, and 8 specimens stained positive with an antibody against S-100, HMB-45, and Melan-A, respectively. However, the false-positive immunostaining for Melan-A was eliminated in seven of the eight specimens after applying the pretreatment with avidin/biotin blocking reagents. Overall, the highest sensitivity and negative predictive value (NPV) were achieved in Melan-A antibody (93 and 90%) compared with antibodies to S-100 (89 and 85%), and HMB-45 (72 and 71%). Initially, an intermediate specificity and positive predictive value (PPV) were obtained for Melan-A antibody (81 and 87%) that were greater than S-100 (77 and 84%), and lower than HMB-45 (91 and 91%). However, the aforementioned treatment with avidin/biotin blocking reagents improved both specificity (98%) and PPV (98%) for Melan-A antibody. In conclusion, by blocking endogenous biotin, Melan-A antibody offers the greatest performance. In terms of cost-effectiveness, we suggested that Melan-A antibody should be used as the first-line antibody for detecting melanoma in the cytologic specimens.

Topics: Antibodies; gp100 Melanoma Antigen; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Predictive Value of Tests; S100 Proteins; Sensitivity and Specificity

Atypical intraepidermal melanocytic proliferations (AIMP) have random cytologic atypia and other histologic features that are concerning for malignancy and often require immunohistochemistry to differentiate from melanoma in situ. Immunostaining with S100, Melan-A, and microphthalmia-associated transcription factor (MITF) was performed for 49 morphologically well-characterized AIMP lesions. The percentage of cells in the basal layer of the epidermis that were identified as melanocytes by immunohistochemistry was compared with the percentage observed by morphology on hematoxylin and eosin staining, which is the gold standard stain for identifying cytologic atypia within an AIMP. Melan-A estimated the highest percentage of melanocytes and S100 the fewest in 47 of the 49 lesions examined. The estimated percentage of melanocytes was 23.3% (95% confidence interval: 18.6-28.1; P < 0.001) higher for Melan-A compared with hematoxylin and eosin staining. Melanocyte estimates were similar for hematoxylin and eosin and MITF (P = 0.15) although S100 estimated 21.8% (95% confidence interval: -27.2 to -16.4; P < 0.001) fewer melanocytes than hematoxylin and eosin. Melan-A staining produces higher estimates of epidermal melanocytes than S100 and MITF, which may increase the likelihood of diagnosing melanoma in situ. In contrast, melanoma in situ may be underdiagnosed with the use of S100, which results in lower estimates of melanocytes than the other 2 immunostains. Therefore, the best immunohistochemical marker for epidermal melanocytes is MITF.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers; Cell Proliferation; Diagnosis, Differential; Epidermis; Female; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanocytes; Melanoma; Microphthalmia-Associated Transcription Factor; Middle Aged; Predictive Value of Tests; S100 Proteins; Skin Neoplasms; Staining and Labeling; Young Adult

Tumor thickness (Breslow thickness) represents the main prognostic factor in primary melanoma. Potential differences in melanoma tumor thickness measurements between conventional hematoxylin and eosin (H&E) and Melan-A immunohistochemical staining were evaluated. Ninety-nine excisional biopsies were included in the study. From each sample, 2 consecutive histological sections were stained with H&E and Melan-A, respectively. Tumor thickness was measured from both sections by 2 independent observers. In 59 biopsy specimens (59.6%), higher tumor thickness measurements were recorded in Melan-A-stained than in H&E-stained sections. In 42.4% of such cases (25 biopsies), the observed differences were ≥0.2 mm. After Melan-A evaluation, 33% of in situ melanoma cases were reclassified as invasive melanoma, with thickness measurements ranging from 0.15 to 0.35 mm. In 23 biopsies, identical values were recorded with both techniques, whereas in 17 cases, measurements obtained with H&E staining were slightly higher (from 0.01 to 0.18 mm) than those obtained with Melan-A staining. A high rate of interobserver agreement was noted, and significant intertechnique measurement differences were detected. Significant discrepancies (≥0.2 mm) in thickness measurements between the 2 techniques were mainly attributed to the presence of individual or small clusters of melanocytic cells in the papillary dermis. These melanocytic cells could be easily overlooked in H&E-stained sections, especially in sections showing dense lymphohistiocytic inflammatory infiltrates, numerous melanin-containing histiocytic cells in the upper dermis, or extensive fibrotic changes or regression phenomena. This study confirms the practical interest of immunohistochemical staining with Melan-A in evaluating primary melanoma and, specifically, in situ melanoma cases.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Biopsy; Coloring Agents; Eosine Yellowish-(YS); Female; Hematoxylin; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Staging; Observer Variation; Predictive Value of Tests; Reproducibility of Results; Retrospective Studies; Skin Neoplasms; Staining and Labeling

An unusual case is presented of a young adult patient with two black-stained, radio-nucleotide tracer-active sentinel lymph nodes biopsied following her primary cutaneous melanoma treatment. This was subsequently confirmed to be secondary to cutaneous tattoos, averting the need of an elective regional node dissection. History of tattooing and tattoo removal should therefore be obtained as a routine in all melanoma patients considered for sentinel node biopsy (SLN). SLN biopsy and any subsequent completion node dissection should be strictly staged so that proper histologic diagnosis of the sentinel node is available for correct decision making and treatment.

Topics: Adult; Color; Female; Foreign-Body Migration; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; S100 Proteins; Sentinel Lymph Node Biopsy; Skin Neoplasms; Tattooing

Cellular proliferation is correlated with the progression of melanoma. Accordingly, the proliferation index of H&E-stained thin melanomas was recently included in the staging system of the American Joint Committee on Cancer. Yet, the immunohistochemical markers of proliferation phosphohistone H3 and Ki67 may improve such indices. To accurately quantify these markers, they should be combined with a melanocytic marker, for example, MART1 in an immunohistochemical double stain; also enabling automated quantification by image analysis. The aim of the study was to compare the prognostic impact of phosphohistone H3/MART1, Ki67/MART1, and H&E stains in primary cutaneous melanoma, and to determine the difference between indices established in hot spots and the global tumor areas. The study included 153 consecutive stage I/II melanoma-patients. The follow-up time was 8-14 years for event-free melanoma. Recurrent disease occurred in 43 patients; 37 died of melanoma. Both events occurred in only three thin melanomas. Their paraffin-embedded tissue was stained for phosphohistone H3/MART1, Ki67/MART1, and with H&E. And proliferation indices were established in 1-mm(2) hot spots and in the global tumor areas. In multivariate Cox analyses, only hot spot indices of phosphohistone H3/MART1 and Ki67/MART1 were independent prognostic markers. Phosphohistone H3/MART1 tended to be better than Ki67/MART1 with adjusted hazard ratios of 3.66 (95% CI, 1.40-9.55; P=0.008) for progression-free survival and 3.42 (95% CI, 1.29-9.04; P=0.013) for melanoma-specific death. In all stains, prognostic performance was substantially improved by using hot spots instead of the global tumor areas. In conclusion, phosphohistone H3/MART1 and Ki67/MART1 were superior to H&E stains, and hot spots superior to the global tumor areas. Given the potential for automated analysis, these double stains seem to be robust alternatives to conventional mitotic detection by H&E in stage I/II melanomas in general. This was particularly true for thick melanomas whereas no specific analyses for thin melanomas only could be performed.

Topics: Adult; Aged; Cell Proliferation; Coloring Agents; Denmark; Disease-Free Survival; Eosine Yellowish-(YS); Female; Hematoxylin; Histones; Humans; Image Interpretation, Computer-Assisted; Immunohistochemistry; Kaplan-Meier Estimate; Ki-67 Antigen; Male; MART-1 Antigen; Melanoma; Middle Aged; Mitotic Index; Multivariate Analysis; Neoplasm Recurrence, Local; Neoplasm Staging; Paraffin Embedding; Phosphorylation; Predictive Value of Tests; Prognosis; Proportional Hazards Models; Prospective Studies; Risk Factors; Skin Neoplasms; Staining and Labeling; Time Factors; Young Adult

Melanoma can present with protean combinations and permutations of histologic features mimicking a plethora of nonmelanocytic benign and malignant proliferations. Anecdotal cases of melanoma closely simulating fibrohistiocytic proliferations have been reported. At times, the reliable differentiation between melanoma and histiocytic proliferations could be vexing histopathologically. We report an unusual presentation of melanoma in an 87-year-old man strikingly resembling xanthogranuloma both clinically and histopathologically. Histologic sections revealed a diffuse proliferation of pleomorphic cells some with foamy cytoplasm and occasional Touton-like giant cells in the dermis accompanied by inflammatory cells. Rare single-cell pagetoid scatter was evident within the epidermis. The infiltrate had patchy staining on CD163, interpreted as part of the inflammatory component but the atypical cells stained heavily with Melan A and tyrosinase confirming the diagnosis of malignant melanoma. Our case demonstrates yet another face of malignant melanoma and the critical but judicious use of immunohistochemistry in reliably distinguishing between melanoma and histiocytic tumors.

Topics: Aged, 80 and over; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Biomarkers, Tumor; Biopsy; Diagnosis, Differential; Eyelid Neoplasms; Granuloma; Histiocytosis; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Monophenol Monooxygenase; Predictive Value of Tests; Receptors, Cell Surface; Skin Neoplasms; Xanthomatosis

The mammary gland can be a site of metastasis in patients with malignant melanoma, which is easily recognized microscopically if clinical information is available. Nonetheless, metastatic melanoma presenting as an isolated mammary tumor can be more challenging to diagnose because it can simulate a primary breast carcinoma clinically and morphologically.. To review metastatic melanoma to the breast, presenting as primary breast carcinomas clinically and morphologically.. The authors report 20 cases of metastatic melanoma clinically presenting as breast tumors. Cases with widespread metastatic presentation were excluded.. Epithelioid and spindle cell tumors predominated, suggesting mammary ductal, papillary, or sarcomatoid carcinoma. Most cases (16 of 20) were submitted for consultation or second opinion owing to their unusual presentation in the breast, or to perform predictive/prognostic immunohistochemical assays. Seven cases had a remarkable phenotypic spectrum expanding the differential diagnosis to large cell lymphoma, leiomyosarcoma, medullary carcinoma, malignant schwannoma, and liposarcoma. Tumor cells were negative for cytokeratin stains and positive for S100 protein, HMB-45, and Melan-A. Negative staining was also observed for epithelial membrane antigen, CD45, desmin, estrogen and progesterone receptors, and human epidermal growth factor receptor 2.. Metastatic melanoma may simulate a broad spectrum of primary breast malignancies. Although the application of a simple panel of antibodies assists in rendering the correct interpretation, lesions presenting as isolated breast tumors may introduce a significant diagnostic difficulty, especially when there is inadequate patient history and/or limited biopsy material. Further challenges are introduced by the extraordinary phenotypic plasticity of metastatic melanoma. Awareness of this pattern variance is essential to avoid inappropriate treatment, especially in cases simulating a "triple negative," poorly differentiated carcinoma of the breast.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Breast Neoplasms, Male; Diagnosis, Differential; Female; gp100 Melanoma Antigen; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; S100 Proteins; Skin Neoplasms

Elderly cancer patients are often excluded from immune-based clinical trials and therapies based on the belief that they respond poorly to tumor antigens. Using melanoma as a model and melanoma related Mart-127-35 epitope specific T cell receptor (TCR) engineered T cells as a tool we compared the T cell responses from young and elderly to the Mart-127-35 epitope, ex vivo. We also compared the natural Treg (nTreg) activities and the expression of a number of genes associated with immune response by quantitative real-time reverse Transcription Polymerase Chain Reaction (qRTPCR) in formalin fixed primary melanomas, in situ. We detected a significant difference in CD8(+) T cell response to Flu antigen (influenza matrix peptide Flu MP58-66), but the responses of the two cohorts to melanoma antigen were comparable. nTreg activities in the elderly was significantly compromised. The qPCR analyses of tissues from elderly patients revealed lower levels of Fox-P3 expression but comparable levels of expression of IL-2, IFNγ, TNFα, IL-4, IL-10, IDO, and TGFβ. These findings indicate that elderly patients might be capable of responding to tumor antigens, and need not be excluded from immune-based therapies or clinical trials.

Topics: Adult; Aged; CD8-Positive T-Lymphocytes; Cell Proliferation; Cells, Cultured; Coculture Techniques; Cohort Studies; Cytokines; Dendritic Cells; Epitopes, T-Lymphocyte; Flow Cytometry; Gene Expression; Humans; Interleukin-2 Receptor alpha Subunit; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Receptors, Antigen, T-Cell; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory

Programmed death 1 (PD-1) is known as an important factor for the development of tolerogenicity. This has been proven in chronic viral infections and different tumor models. To address the role of PD-1 and its ligand programmed death ligand 1 (PD-L1) in different stages of malignant melanoma, we investigated peripheral blood and tumor tissues in regard to overall survival (OS) and prognostic relevance. One hundred samples of peripheral blood mononuclear cells from HLA-A2(+) patients with malignant melanoma (Stages I-IV) were analyzed in seven color FACS combined with multimer analyses for the immunodominant epitope of Melan-A (peptide A2/Melan-A(p26-35mod) ). Corresponding formalin-fixed paraffin-embedded tissues of primary tumor and distant organ metastases from 37 cases were analyzed by immunohistochemistry for Melan-A, PD-L1 and PD-1 expression. Compared to the total CD8(+) T cell population, PD-1 expression by A2/Melan-A(+) CD8(+) T cells was over-represented in melanoma stages III and IV (p < 0.001). Although elevation of PD-1(+) Melan-A(+) CD8(+) T cells had no significant influence on OS, a positive correlation was observed between PD-L1 expression on melanoma cells and OS (p = 0.05). Correlation of advanced tumor stage with increased A2/Melan-A-multimer(+) PD-1(+) T cells in the peripheral blood suggest that blocking of PD-1 could have therapeutic potential in advanced stage melanoma.

Topics: Aged; B7-H1 Antigen; CD8-Positive T-Lymphocytes; Disease Progression; Female; Humans; Lymphocytes, Tumor-Infiltrating; Male; MART-1 Antigen; Melanoma; Middle Aged; Programmed Cell Death 1 Receptor; Skin Neoplasms

Four young broiler chickens affected by multiple melanotic tumors are described. Grossly, there were multiple tumors composed of melanocytes within the skin, skeletal muscle, and multiple visceral organs. Tumors ranged from flattened macules to masses that extensively replaced viscera. Microscopically, melanocytes were often well pigmented, and while there was moderate nuclear anisokaryosis, mitotic rates were low. Immunohistochemical staining of some melanomas with antibodies to S100 proteins, Melan-A, vimentin, or neuron-specific enolase after bleaching of tumor cells with potassium permanganate revealed lack of immunostaining of tumor cells with antibodies to S100, strong positive staining of tumor cells for neuron-specific enolase, moderate staining with antibodies to vimentin, and faint staining for Melan-A. Only neuron-specific enolase staining was evident in unbleached tumor cells. Attempts to identify exogenous avian leukosis viruses in these tumors were unsuccessful.

Topics: Animals; Avian Leukosis Virus; Chickens; Immunohistochemistry; MART-1 Antigen; Melanocytes; Melanoma; Melanosomes; Mitotic Index; Phosphopyruvate Hydratase; Pigmentation; Poultry Diseases; S100 Proteins; Skin; Skin Neoplasms; Vimentin

The c-kit receptor is responsible for transmission of promigration signals to melanocytes; its downregulation may be involved in malignant progression of human melanocytic neoplasms. Expression of this receptor has not been examined in normal or neoplastic melanocytes from dogs. In this study, 14 benign dermal and 61 malignant mucosal melanocytic tumors were examined for c-kit (KIT) expression. Sites of the mucosal melanomas were gingiva (not further specified; n = 30), buccal gingiva (n = 6), soft palate (n = 4), hard palate (n = 5), tongue (n = 7), lip (n = 6), and conjunctiva (n = 3). Melan A was expressed in all 14 dermal melanocytomas and in 59 of 61 (96.7%) tumors from oral or conjunctival mucosa, confirming melanocytic origin. C-kit receptor expression was strong and diffuse throughout the cytoplasm in all 14 dermal melanocytomas and was identified in basilar mucosal melanocytes over submucosal neoplasms (27 of 61, 44.3%), junctional (neoplastic) melanocytes (17 of 61, 27.9%), and, less commonly, neoplastic melanocytes of the subepithelial tumors (6 of 61, 9.8%). KIT expression anywhere within the resected melanomas correlated with significantly longer survival. These results suggest that c-kit receptor expression may be altered in canine melanomas and may have potential as a prognostic indicator for mucosal melanomas.

Topics: Animals; Biomarkers, Tumor; Conjunctival Neoplasms; Disease Progression; Dog Diseases; Dogs; Female; Gene Expression Regulation, Neoplastic; Immunohistochemistry; Male; MART-1 Antigen; Melanocytes; Melanoma; Mouth Neoplasms; Prognosis; Proto-Oncogene Proteins c-kit; Receptor Protein-Tyrosine Kinases; S100 Proteins; Skin Neoplasms; Survival Analysis

MART-1 and gp100 are prototypical melanoma antigen (Ag), but their clinical use as vaccines or as targets of cytotoxic lymphocytes achieved modest success. Possible explanations could be that as MART-1 and gp100 are melanocyte differentiation Ag, clonogenic Ag-non-expressing cells would be spared by immune effectors, or that clonogenic cells would be intrinsically resistant to cytotoxic lymphocytes. We therefore analyzed the proliferative status of MART-1/gp100-expressing and -non-expressing cells in biopsies, and the clonogenicity and sensitiveness to cytotoxic lymphocytes of the human cutaneous melanoma cell lines MEL-XY1 and MEL-XY3. Analysis of MART-1/gp100 and Ki-67 expression in 22 melanoma tumors revealed that MART-1/gp100-expressing and -non-expressing cells proliferated competitively. MART-1, gp100, tyrosinase, and CD271 expression were studied in MEL-XY1 and MEL-XY3 colonies. At 7 days, colonies displayed positive, negative, and mixed expression patterns. By 14 days, colonies of different sizes developed, showing cells with different clonogenic potential, and Ag were downregulated, suggesting Ag plasticity. Subcloning of MEL-XY1 colonies showed that Ag expression varied with time without interfering with clonogenicity. Finally, clonogenic, MART-1/gp100-expressing cells were lysed by specific CD8 lymphocytes. Thus, MART-1 and gp100 expression and plasticity would not interfere with proliferation or clonogenicity, and clonogenic cells may be lysed by cytotoxic lymphocytes.

Topics: Cell Proliferation; DNA Methylation; gp100 Melanoma Antigen; Humans; Ki-67 Antigen; MART-1 Antigen; Melanoma; Promoter Regions, Genetic; Skin Neoplasms; T-Lymphocytes, Cytotoxic

In mice, vaccination with high peptide doses generates higher frequencies of specific CD8+ T cells, but with lower avidity compared to vaccination with lower peptide doses. To investigate the impact of peptide dose on CD8+ T cell responses in humans, melanoma patients were vaccinated with 0.1 or 0.5 mg Melan-A/MART-1 peptide, mixed with CpG 7909 and Incomplete Freund's adjuvant. Neither the kinetics nor the amplitude of the Melan-A-specific CD8+ T cell responses differed between the two vaccination groups. Also, CD8+ T cell differentiation and cytokine production ex vivo were similar in the two groups. Interestingly, after low peptide dose vaccination, Melan-A-specific CD8+ T cells showed enhanced degranulation upon peptide stimulation, as assessed by CD107a upregulation and perforin release ex vivo. In accordance, CD8+ T cell clones derived from low peptide dose-vaccinated patients showed significantly increased degranulation and stronger cytotoxicity. In parallel, Melan-A-specific CD8+ T cells and clones from low peptide dose-vaccinated patients expressed lower CD8 levels, despite similar or even stronger binding to tetramers. Furthermore, CD8+ T cell clones from low peptide dose-vaccinated patients bound CD8 binding-deficient tetramers more efficiently, suggesting that they may express higher affinity TCRs. We conclude that low peptide dose vaccination generated CD8+ T cell responses with stronger cytotoxicity and lower CD8 dependence.

Topics: Cancer Vaccines; Chemotherapy, Adjuvant; Cytokines; Dose-Response Relationship, Drug; Female; HLA-A2 Antigen; Humans; Male; MART-1 Antigen; Melanoma; Neoplasm Staging; Oligodeoxyribonucleotides; Peptides; T-Lymphocytes, Cytotoxic; Vaccination

Although a series of melanoma differentiation antigens for immunotherapeutic targeting has been described, heterogeneous expression of antigens such as Melan-A/MART-1 and gp100 results from a loss of antigenic expression in many late stage tumors. Antigen loss can represent a means for tumor escape from immune recognition, and a barrier to immunotherapy. However, since antigen-negative tumor phenotypes frequently result from reversible gene regulatory events, antigen enhancement represents a potential therapeutic opportunity. Accordingly, we have developed a cell-based assay to screen for compounds with the ability to enhance T-cell recognition of melanoma cells. This assay is dependent on augmentation of MelanA/MART-1 antigen presentation by a melanoma cell line (MU89). T-cell recognition is detected as interleukin-2 production by a Jurkat T cell transduced to express a T-cell receptor specific for an HLA-A2 restricted epitope of the Melan-A/MART-1 protein. This cellular assay was used to perform a pilot screen by using 480 compounds of known biological activity. From the initial proof-of-principle primary screen, eight compounds were identified as positive hits. A panel of secondary screens, including orthogonal assays, was used to validate the primary hits and eliminate false positives, and also to measure the comparative efficacy of the identified compounds. This cell-based assay, thus, yields consistent results applicable to the screening of larger libraries of compounds that can potentially reveal novel molecules which allow better recognition of treated tumors by T cells.

Topics: Antibody Specificity; Antigens, Neoplasm; Cell Line, Tumor; Cloning, Molecular; Cytokines; Drug Screening Assays, Antitumor; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Genes, Reporter; Genetic Vectors; Green Fluorescent Proteins; Humans; Immunotherapy; Jurkat Cells; Lentivirus; MART-1 Antigen; Melanoma; Receptors, Antigen, T-Cell; T-Lymphocytes; Tumor Necrosis Factor Receptor Superfamily, Member 7

Herpes simplex virus protein ICP47, encoded by US12 gene, strongly downregulates major histocompatibility complex (MHC) class-I antigen restricted presentation by blocking transporter associated with antigen processing (TAP) protein. To decrease viral vector antigenic immunodominance and MHC class-I driven clearance, we engineered recombinant vaccinia viruses (rVV) expressing ICP47 alone (rVV-US12) or together with endoplasmic reticulum (ER)-targeted Melan-A/MART-1(27-35) model tumor epitope (rVV-MUS12). In this study, we show that antigen presenting cells (APC), infected with rVV-US12, display a decreased ability to present TAP dependent MHC class-I restricted viral antigens to CD8+ T-cells. While HLA class-I cell surface expression is strongly downregulated, other important immune related molecules such as CD80, CD44 and, most importantly, MHC class-II are unaffected. Characterization of rVV-MUS12 infected cells demonstrates that over-expression of a TAP-independent peptide, partially compensates for ICP47 induced surface MHC class-I downregulation (30% vs. 70% respectively). Most importantly, in conditions where clearance of infected APC by virus-specific CTL represents a limiting factor, a significant enhancement of CTL responses to the tumor epitope can be detected in cultures stimulated with rVV-MUS12, as compared to those stimulated by rVV-MART alone. Such reagents could become of high relevance in multiple boost protocols required for cancer immunotherapy, to limit vector-specific responsiveness.

Topics: Antigen Presentation; Antigen-Presenting Cells; Antigens, Viral; ATP Binding Cassette Transporter, Subfamily B, Member 2; ATP-Binding Cassette Transporters; B7-1 Antigen; Cancer Vaccines; Cell Proliferation; Cells, Cultured; Cytokines; Endoplasmic Reticulum; Epitopes; Fibroblasts; Genetic Vectors; HLA-A2 Antigen; HLA-DR Antigens; Humans; Hyaluronan Receptors; Immediate-Early Proteins; MART-1 Antigen; Melanoma; T-Lymphocytes, Cytotoxic; Vaccinia virus

The aim of this study was to analyze the histopathological and immunohistochemical characteristics of 22 cases of primary oral melanomas (OM).. Twenty two cases of primary oral melanoma were analyzed by description of their histopathological features and immunohistochemical study using the antibodies S-100, HMB-45, Melan-A and Ki-67.. The mean age was 58 years and 14 cases were female. The main affected sites were the hard palate, followed by the upper gingiva. Microscopically, 15 cases presented level III of invasion, 2 cases were amelanotic and 13 showed a mixed epithelioid and plasmacytoid or spindle cells composition. Some cases showed necrosis, perivascular and perineural invasion. S-100 and HMB-45 were positive in all cases, but 3 cases were negative for Melan-A. The proliferative index with Ki-67 was high, with labeling index ranging from 15.51% to 63% of positive cells.. S-100 and HMB-45 are more frequently expressed than Melan-A in primary oral melanomas and these markers are helpful to confirm the diagnosis.

Topics: Adult; Aged; Aged, 80 and over; Female; gp100 Melanoma Antigen; Humans; Immunohistochemistry; Ki-67 Antigen; Latin America; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Mouth Neoplasms; S100 Proteins; Young Adult

Congenital melanocytic naevi can give rise to secondary melanocytic tumours, such as proliferative nodules and malignant melanoma. The clinical and histological features of both lesions may be nearly identical, which makes an unequivocal diagnosis impossible. In particular, it is difficult to differentiate clearly between benign and malignant proliferation in infants with secondary melanocytic proliferation. Reports on melanocytic proliferation and malignant melanoma within the paediatric age-group are very rare. There is limited expert knowledge on this subject and little is known about prognosis and outcome. We report here a case of an infant with an unusual transformation of a congenital spindle cell naevus of the umbilical region, and discuss clinical, histological and genomic criteria.

Topics: Diagnosis, Differential; Female; gp100 Melanoma Antigen; Humans; Infant; Infant, Newborn; Ki-67 Antigen; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Nevus, Spindle Cell; S100 Proteins; Skin Neoplasms

The availability of molecular-targeted therapies for the treatment of melanoma has emphasised the need to identify mutations in target genes such as BRAF and KIT. Circulating tumour cells (CTC) are present in the peripheral blood of a significant proportion of cancer patients.. High molecular weight melanoma-associated antigen (HMW-MAA) was used to isolate melanoma cells from peripheral blood as it is selectively expressed at high levels on melanomas. The HMW-MAA-positive cells were isolated using immunomagnetic beads. After removing CD45(+) cells, CTC were identified by staining with MART-1- and gp100-specific antibodies (HMW-MAA(+), CD45(-), MART-1/gp100(+)). Single, isolated CTC were then subjected to BRAF and KIT mutational analysis.. CTC (HMW-MAA(+), CD45(-), MART-1/gp100(+)) were isolated from the blood of 11 patients and BRAF and KIT were sequenced in nine and four patients, respectively. The BRAF sequences identified in the CTC were inconsistent with those identified in autologous melanoma tumours in three patients and the KIT sequences were inconsistent in three patients. In addition, polyclonal BRAF mutations were identified in one patient and concomitant mutations in BRAF and KIT were identified in another patient.. Melanoma cells show clonal heterogeneity. Therefore, CTC genotyping may be crucial for successful molecular-targeted therapy.

Topics: Aged; Aged, 80 and over; Antibodies, Monoclonal; Antigens, Neoplasm; Base Sequence; Cell Line, Tumor; Cell Separation; DNA Mutational Analysis; Female; Genes, ras; Genotype; Humans; Immunomagnetic Separation; Male; MART-1 Antigen; Melanoma; Middle Aged; Molecular Targeted Therapy; Mutation; Neoplasm Proteins; Neoplastic Cells, Circulating; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-kit; Sequence Analysis, DNA; Single-Cell Analysis; Skin Neoplasms

The uptake and long-term cross-presentation of tumor Ag long peptides (LP) by dendritic cells (DC) make them attractive cancer vaccine candidates. However, it remains to be established whether LP can prime long-lived tumor-reactive CTL and whether other cell types are able to cross-present them. Using HLA-A2 healthy donor and melanoma patient-derived PBMC, we studied the in vitro cross-priming potential of Melan-A 16-40 LP bearing the HLA-A2-restricted epitope 26-35 or its analog 26-35(A27L) and compared it to the priming capacity of the short analog. We then addressed LP priming capacity in vivo using HLA-A2 mice. We also studied LP cross-presentation by monocyte-derived DC, plasmacytoid DC, monocytes, and B cells. We showed that the modified LP gave rise to high and sustained cross-presentation by monocyte-derived DC. This led to cross priming in vitro and in vivo and to expansion of long-lived tumor-reactive cytotoxic T cells. In contrast, the LP containing the natural 26-35 epitope primed specific T cells poorly, despite its long-lived cross-presentation, and T cells primed against the short analog were short-lived. We further showed that LP cross-presentation is restricted to monocytes and conventional DC. These results document for the first time, to our knowledge, the strong immunogenicity of a human tumor Ag LP. Of note, they underscore that this property is critically dependent on sufficient HLA binding affinity and/or TCR ligand potency of the cross-presented epitope. We conclude that LP fulfilling this requirement should be used as tumor vaccines, together with DC maturating agents, especially the Melan-A 16-40(A27L) LP, for the treatment of HLA-A2(+) melanoma patients.

Topics: Amino Acid Sequence; Animals; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cells, Cultured; Colorectal Neoplasms; Cross-Priming; Dendritic Cells; Epitopes, T-Lymphocyte; HLA-A2 Antigen; Humans; Immunodominant Epitopes; Lymphocyte Activation; MART-1 Antigen; Melanoma; Mice; Mice, Mutant Strains; Molecular Sequence Data; Monocytes; Peptide Fragments; T-Lymphocytes, Cytotoxic

The mitotic rate was introduced as a major prognostic criterion for the staging of thin (≤1.0 mm) melanoma by the 2009 American Joint Committee on Cancer Staging and Classification (seventh edition). The detection of a single mitotic figure changes the tumor stage in thin melanoma. We sought to address the value of a dual staining to facilitate the determination of the mitotic rate and to assign the mitotic activity to melanocytes.. The mitotic rate of melanoma cells was determined by dual phosphohistone-H3 (PHH3)/Melan-A immunohistochemistry. Results were compared with PHH3 staining alone and conventional hematoxylin and eosin (H&E)-stained slides of 15 melanomas with a tumor thickness <1.0 mm.. PHH3 staining clearly labeled cells in the mitotic cell cycle. The mitotic rate in the PHH3/Melan-A dual stain was equal to that derived by H&E staining. Time required for counting mitotic figures was significantly reduced.. The evaluation of mitotic rate with an immunohistochemical dual stain is faster (mean 63.0%) and more reliable than evaluation by routine H&E staining alone. Dual staining immunohistochemistry may be a useful additional tool to standardize the determination of mitotic rate and may be helpful in evaluation of challenging cases.

Topics: Adult; Aged; Aged, 80 and over; Female; Histones; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Middle Aged; Mitosis; Neoplasm Proteins; Staining and Labeling

We present a unique dermal tumor for which we propose the term plexiform melanocytic schwanomma. The proliferation consisted of lobules of epithelioid and spindled cells with S100, Melan-A and HMB-45 positivity but without obvious melanin pigmentation. The nuclei were moderately pleomorphic in some areas, and in a few areas the mitotic index was elevated. Schwannian differentiation was inferred from the presence of areas with nuclear palisading resembling Verocay bodies, from plexiform architecture and from the presence of a thin rim of EMA positivity around the tumor. Array-based comparative genomic hybridization showed genomic losses that overlap with those seen in sporadic schwanomma. The differential diagnosis included melanoma, melanotic schwannoma and cutaneous melanocytoneuroma, and we compare and contrast our case with these entities.

Topics: Adult; Cell Proliferation; Dermis; Female; gp100 Melanoma Antigen; Humans; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neurilemmoma; S100 Proteins; Skin Neoplasms; Skin Pigmentation

Clear cell sarcoma is a unique soft tissue tumor with distinct microscopic features that include a nested or fascicular pattern of spindle cells accompanied by larger wreath-like giant cells scattered throughout. It harbors a unique EWSR1-ATF1 gene fusion secondary to a t(12;22)(q13;q12) translocation. Recently, it was reported that clear cell sarcoma can occur in the skin and mimic a broad spectrum of entities, including spindle cell melanoma. Here, we describe 3 new cases of clear cell sarcoma of the skin, all of which were confirmed molecularly. The patients, a 12-year-old boy, a 29-year-old woman, and a 60-year-old man, had cutaneous lesions on the thigh, dorsum of foot, and sole, respectively. All 3 lesions were originally considered suspicious of spindle cell melanoma. Microscopically, the lesions featured nodular proliferation centered in the dermis that consisted of discrete fascicles of spindle cell enmeshed by thin fibrous strands. Wreath-like cells were present in all cases. Tumor cells were positive for S100 protein (3 of 3 cases), melan A (2 of 3 cases), HMB 45 (1 of 3 cases) although a junctional melanocytic proliferation was seen in 1 case. Sentinel lymph node biopsy was negative in 2 patients. Follow-up was uneventful in 2 patients, whereas the other patient developed a lymph node metastasis 5 months after primary tumor excision. This study confirms that malignant dermal tumors that mimic but do not exactly replicate spindle cell melanoma should raise suspicion for cutaneous clear cell sarcoma and prompt the investigation for the confirmatory gene fusion t(12;22).

Topics: Adult; Child; Diagnosis, Differential; Female; gp100 Melanoma Antigen; Humans; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; S100 Proteins; Sarcoma; Sarcoma, Clear Cell; Skin Neoplasms

The aim of this study was to assess the importance of immunohistochemical markers in the diagnosis of pigmented conjunctival lesions. Due to the difficulty of making an exact clinical diagnosis, the suspicion of malignancy requires the removal of the lesion and performing a histopathology study in which immunohistochemical markers may help to determine the nature of the lesion.. A case is presented of a 25 year-old woman with a pigmented lesion in the caruncle. It appeared recently and was growing fast with increasing pigmentation. Due to a suspicion of malignancy, the total lesion was removed. The microscopic study revealed cellular alterations which suggested malignancy. However, after carrying out immunohistochemical markers the diagnosis was conjunctival compound nevus.

Topics: Antigens, CD; Antigens, Differentiation, Myelomonocytic; Biomarkers, Tumor; Conjunctival Neoplasms; Diagnosis, Differential; Female; gp100 Melanoma Antigen; Humans; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Nevus, Pigmented; Young Adult

We describe the clinical, histopathological and immunohistochemical features of the malignant melanomas in the perineal regions of Kilis goats from Sanliurfa province in Turkey. We studied 13 female Kilis goats between 3 and 8 years old that were brought to Harran University Veterinary School, Department of Surgery, between 2002 and 2010. By macroscopic examination, the masses were determined to have elastic consistency, dark brown-black color, necrotic surfaces and ulceration. Microscopically, pleomorphic cells were observed under the basal layer and these advanced toward the dermis. These cells were polyhedral, round or spindle-shaped, anaplastic, and their cytoplasm contained varying amounts of dark brown-black pigments. Immunohistochemical staining was obtained with anti-melan A, vimentin and S100 antibodies.

Topics: Animals; Female; Goats; Immunohistochemistry; MART-1 Antigen; Melanoma; Neoplasm Proteins; S100 Proteins; Skin Neoplasms; Staining and Labeling; Vimentin

To analyze the prognostic relevance of circulating T cells responding to NY-ESO-1, Melan-A, MAGE-3, and survivin in patients with melanoma with distant metastasis.. We examined 84 patients with follow-up after analysis (cohort A), 18 long-term survivors with an extraordinarily favorable course of disease before analysis (> 24 months survival after first occurrence of distant metastases; cohort B), and 14 healthy controls. Circulating antigen-reactive T cells were characterized by intracellular cytokine staining after in vitro stimulation.. In cohort A patients, the presence of T cells responding to peptides from NY-ESO-1, Melan-A, or MAGE-3 and the M category according to the American Joint Committee on Cancer classification were significantly associated with survival. T cells responding to NY-ESO-1 and Melan-A (hazard ratios, 0.29 and 0.18, respectively) remained independent prognostic factors in Cox regression analysis and were superior to the M category in predicting outcome. Median survival of patients possessing T cells responding to NY-ESO-1, Melan-A, or both was 21 months, compared with 6 months for all others. NY-ESO-1-responsive T cells were detected in 70% of cohort A patients surviving > 18 months and in 50% of cohort B patients. Melan-A responses were found in 42% and 47% of patients in cohorts A and B, respectively. In contrast, the proportion was only 22% for NY-ESO-1 and 23% for Melan-A in those who died within 6 months.. The presence of circulating T cells responding to Melan-A or NY-ESO-1 had strong independent prognostic impact on survival in advanced melanoma. Our findings support the therapeutic relevance of Melan-A and NY-ESO-1 as targets for immunotherapy.

Topics: Antigens, Neoplasm; Cohort Studies; Europe; Female; Humans; Inhibitor of Apoptosis Proteins; Kaplan-Meier Estimate; Male; MART-1 Antigen; Melanoma; Membrane Proteins; Middle Aged; Neoplasm Proteins; Proportional Hazards Models; Skin Neoplasms; Survivin; Survivors; T-Lymphocytes; Time Factors; Treatment Outcome

To understand the audiologic and vestibular toxicities associated with adoptive cell immunotherapy (ACI) targeting pigment-pathway antigens on melanoma and to investigate the use of intratympanic steroid injections in the treatment of these toxicities.. Prospective nonrandomized study.. Tertiary clinical research center.. Thirty-two patients with progressive metastatic melanoma who failed conventional therapy underwent ACI with T cells genetically modified to target MART-1 (n = 18) or gp100 (n = 14). All patients received serial audiometric testing. Vestibular testing was performed on patients with vestibular complaints. Patients with significant deficits received intratympanic steroid injections.. Of 32 patients, 15 had no hearing change, 9 had mild hearing loss, and 8 had moderate hearing loss following treatment. Ten patients received intratympanic steroid injections for mild (n = 2) or moderate (n = 7) hearing loss or for significant imbalance (n = 1). Of those with mild hearing loss (n = 9), all but 1 recovered to pretreatment hearing levels. Four of 8 patients with moderate hearing loss recovered to baseline hearing levels, and 4 had partial recovery. All 7 patients with posttreatment vestibular complaints had demonstrable vestibular dysfunction. Three of these patients demonstrated recovery to normal vestibular function. The number of modified T cells infused for therapy correlated with the degree of audiovestibular deficit.. Adoptive cell immunotherapy targeting pigment-pathway cell proteins, a novel therapy for melanoma, can induce hearing loss and vestibular dysfunction. The presumed mechanism of autoimmune attack on normal melanocytes in the cochlear stria vascularis and in the vestibular organs demonstrates the importance of melanocytes in normal inner ear function.

Topics: Adaptive Immunity; Adult; Audiometry, Pure-Tone; Chi-Square Distribution; Disease Progression; Female; Glucocorticoids; gp100 Melanoma Antigen; Hearing Loss; Humans; Immunotherapy; Interleukin-2; Male; MART-1 Antigen; Melanoma; Middle Aged; Prospective Studies; Vestibular Function Tests

The proliferation marker Ki67 is an important diagnostic and prognostic aid in surgical pathology. However, manual quantification in a counting frame to accurately establish the proliferation rate (Ki67 index) is cumbersome and time-consuming. Instead, digital image analysis of Ki67/MART1 double stains may provide fast and novel index computations for entire tumor sections.. To design and compare image analysis protocols that compute Ki67 indices of Ki67/MART1 double stains, to compare automated indices with previously published manual indices, and to compare the total number of proliferating cells (mimicking a Ki67 single stain) with the number of MART1-verified proliferating cells.. Whole slide images were captured from 48 melanomas and 77 nevi stained with an immunohistochemical cocktail against Ki67 and MART1. Ki67 indices were determined by digital image analysis and different equations based on number or area.. The differences between mean indices of melanomas and nevi were significant (P < .001) in all index computations. Number-based image analysis of lesions with more than 250 melanocytic cells misclassified 1 of 42 melanomas and 4 of 53 nevi, numbers comparable with manual counting. Automated indices were significantly higher than manual indices, as were indices of mimicked Ki67 single stains compared with MART1-verified Ki67 indices (P < .001).. Ki67 indices established by digital image analysis of Ki67/MART1 double stains demonstrated excellent abilities to discriminate melanomas from nevi with diagnostic performances equal to manually performed indices. Testing different definitions of the automated MART1-verified Ki67 index, no single definition stood out; thus, a variety of definitions may be used.

Topics: Biomarkers; Diagnosis, Differential; Humans; Image Processing, Computer-Assisted; Immunohistochemistry; Ki-67 Antigen; MART-1 Antigen; Melanocytes; Melanoma; Nevus; Skin Neoplasms

Antigen-specific T cells predict survival in patients with distant melanoma metastasis.

Topics: Antigens, Neoplasm; Female; Humans; Male; MART-1 Antigen; Melanoma; Membrane Proteins; Skin Neoplasms; Survivors; T-Lymphocytes

Phenotypic and functional cell properties are usually analyzed at the level of defined cell populations but not single cells. Yet, large differences between individual cells may have important functional consequences. It is likely that T-cell-mediated immunity depends on the polyfunctionality of individual T cells, rather than the sum of functions of responding T-cell subpopulations. We performed highly sensitive single-cell gene expression profiling, allowing the direct ex vivo characterization of individual virus-specific and tumor-specific T cells from healthy donors and melanoma patients. We have previously shown that vaccination with the natural tumor peptide Melan-A-induced T cells with superior effector functions as compared with vaccination with the analog peptide optimized for enhanced HLA-A*0201 binding. Here we found that natural peptide vaccination induced tumor-reactive CD8 T cells with frequent coexpression of both memory/homing-associated genes (CD27, IL7R, EOMES, CXCR3, and CCR5) and effector-related genes (IFNG, KLRD1, PRF1, and GZMB), comparable with protective Epstein-Barr virus-specific and cytomegalovirus-specific T cells. In contrast, memory/homing-associated and effector-associated genes were less frequently coexpressed after vaccination with the analog peptide. Remarkably, these findings reveal a previously unknown level of gene expression diversity among vaccine-specific and virus-specific T cells with the simultaneous coexpression of multiple memory/homing-related and effector-related genes by the same cell. Such broad functional gene expression signatures within antigen-specific T cells may be critical for mounting efficient responses to pathogens or tumors. In summary, direct ex vivo high-resolution molecular characterization of individual T cells provides key insights into the processes shaping the functional properties of tumor-specific and virus-specific T cells.

Topics: Cancer Vaccines; CD8-Positive T-Lymphocytes; Cells, Cultured; Epitopes; Gene Expression Profiling; Granzymes; HLA-A2 Antigen; Humans; Immunologic Memory; Interferon-gamma; Lymphocyte Activation; MART-1 Antigen; Melanoma; NK Cell Lectin-Like Receptor Subfamily D; Perforin; Pore Forming Cytotoxic Proteins; Receptors, CCR5; Receptors, CXCR3; Receptors, Interleukin-7; T-Box Domain Proteins; Tumor Necrosis Factor Receptor Superfamily, Member 7; Vaccines, Subunit

Dendritic cells (DC) can achieve cross-presentation of naturally-occurring tumor-associated antigens after phagocytosis and processing of dying tumor cells. They have been used in different clinical settings to vaccinate cancer patients. We have previously used gamma-irradiated MART-1 expressing melanoma cells as a source of antigens to vaccinate melanoma patients by injecting irradiated cells with BCG and GM-CSF or to load immature DC and use them as a vaccine. Other clinical trials have used IFN-gamma activated macrophage killer cells (MAK) to treat cancer patients. However, the clinical use of MAK has been based on their direct tumoricidal activity rather than on their ability to act as antigen-presenting cells to stimulate an adaptive antitumor response. Thus, in the present work, we compared the fate of MART-1 after phagocytosis of gamma-irradiated cells by clinical grade DC or MAK as well as the ability of these cells to cross present MART-1 to CD8(+) T cells. Using a high affinity antibody against MART-1, 2A9, which specifically stains melanoma tumors, melanoma cell lines and normal melanocytes, the expression level of MART-1 in melanoma cell lines could be related to their ability to stimulate IFN-gamma production by a MART-1 specific HLA-A*0201-restricted CD8(+) T cell clone. Confocal microscopy with Alexa Fluor®(647)-labelled 2A9 also showed that MART-1 could be detected in tumor cells attached and/or fused to phagocytes and even inside these cells as early as 1 h and up to 24 h or 48 h after initiation of co-cultures between gamma-irradiated melanoma cells and MAK or DC, respectively. Interestingly, MART-1 was cross-presented to MART-1 specific T cells by both MAK and DC co-cultured with melanoma gamma-irradiated cells for different time-points. Thus, naturally occurring MART-1 melanoma antigen can be taken-up from dying melanoma cells into DC or MAK and both cell types can induce specific CD8(+) T cell cross-presentation thereafter.

Topics: Antigen Presentation; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Dendritic Cells; Gamma Rays; Gene Expression Regulation, Neoplastic; HLA-A2 Antigen; Humans; Interferon-gamma; Macrophages; MART-1 Antigen; Melanoma; Microscopy, Confocal; Phagocytosis

We recently described two proteasome subtypes that are intermediate between the standard proteasome and the immunoproteasome. They contain only one (β5i) or two (β1i and β5i) of the three inducible catalytic subunits of the immunoproteasome. They are present in tumor cells and abundant in normal human tissues. We described two tumor antigenic peptides that are uniquely produced by these intermediate proteasomes. In this work, we studied the production by intermediate proteasomes of tumor antigenic peptides known to be produced exclusively by the immunoproteasome (MAGE-A3(114-122), MAGE-C2(42-50), MAGE-C2(336-344)) or the standard proteasome (Melan-A(26-35), tyrosinase(369-377), gp100(209-217)). We observed that intermediate proteasomes efficiently produced the former peptides, but not the latter. Two peptides from the first group were equally produced by both intermediate proteasomes, whereas MAGE-C2(336-344) was only produced by intermediate proteasome β1i-β5i. Those results explain the recognition of tumor cells devoid of immunoproteasome by CTL recognizing peptides not produced by the standard proteasome. We also describe a third antigenic peptide that is produced exclusively by an intermediate proteasome: peptide MAGE-C2(191-200) is produced only by intermediate proteasome β1i-β5i. Analyzing in vitro digests, we observed that the lack of production by a given proteasome usually results from destruction of the antigenic peptide by internal cleavage. Interestingly, we observed that the immunoproteasome and the intermediate proteasomes fail to cleave between hydrophobic residues, despite a higher chymotrypsin-like activity measured on fluorogenic substrates. Altogether, our results indicate that the repertoire of peptides produced by intermediate proteasomes largely matches the repertoire produced by the immunoproteasome, but also contains additional peptides.

Topics: Amino Acid Sequence; Antigen Presentation; Antigens, Neoplasm; Cell Line, Tumor; Clone Cells; Epitopes, T-Lymphocyte; gp100 Melanoma Antigen; HEK293 Cells; Humans; Hydrophobic and Hydrophilic Interactions; MART-1 Antigen; Melanoma; Monophenol Monooxygenase; Neoplasm Proteins; Peptide Fragments; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational

To study the clinicopathologic features and differential diagnosis of perivascular epithelioid cell tumors (PEComas) occurring in the urinary system.. The clinicopathologic features of 21 cases of PEComa from September 2002 to September 2010 occurring in the urinary system were retrospectively reviewed. Immunohistochemical study for HMB 45, S-100 protein, smooth muscle actin, desmin, Melan A and Ki-67 was carried out.. Amongst the 21 cases studied, there were 5 males and 16 females. The age of patients ranged from 16 to 76 years (median = 51.3 years). Twenty cases occurred in the kidney and 1 in the bladder. The predominant histopathologic subtype of renal PEComas was classic type (10/20), followed by epithelioid type (5/20), smooth muscle type (3/20), inflammatory type (1/20) and sclerosing type (1/20). Immunohistochemical study showed that HMB 45 and smooth muscle actin were positive in 95.2% (20/21) and 80.9% (17/21) cases, respectively. Melan A, desmin and S-100 protein were expressed in 71.4% (15/21), 61.9% (13/21) and 33.3% (7/21) cases, respectively. The mean proliferative index was 1.29% (range = 0 to 5%). HMB 45 and Melan A were expressed in all of the 5 cases of epithelioid PEComas, whereas smooth muscle actin and desmin were only expressed in one of them. There was no significant difference between epithelioid PEComas and non-epithelioid PEComas in the expression of HMB 45, Melan A, smooth muscle actin and desmin. Positive staining for HMB 45 and smooth muscle actin was demonstrated in the case of bladder PEComa.. PEComas of the urinary system predominantly affect the kidney. Epithelioid renal PEComas and bladder PEComa are relatively rare and have unique pathologic features. It is necessary to distinguish PEComas from other malignant tumors. Immunohistochemical study for HMB 45, Melan A and smooth muscle actin is helpful for confirmation of diagnosis.

Topics: Actins; Adolescent; Adult; Aged; Carcinoma, Renal Cell; Desmin; Diagnosis, Differential; Female; Gastrointestinal Stromal Tumors; gp100 Melanoma Antigen; Humans; Immunohistochemistry; Kidney Neoplasms; Leiomyosarcoma; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Perivascular Epithelioid Cell Neoplasms; S100 Proteins; Urinary Bladder Neoplasms; Young Adult

Altered peptide antigens that enhance T-cell immunogenicity have been used to improve peptide-based vaccination for a range of diseases. Although this strategy can prime T-cell responses of greater magnitude, the efficacy of constituent T-cell clonotypes within the primed population can be poor. To overcome this limitation, we isolated a CD8(+) T-cell clone (MEL5) with an enhanced ability to recognize the HLA A*0201-Melan A(27-35) (HLA A*0201-AAGIGILTV) antigen expressed on the surface of malignant melanoma cells. We used combinatorial peptide library screening to design an optimal peptide sequence that enhanced functional activation of the MEL5 clone, but not other CD8(+) T-cell clones that recognized HLA A*0201-AAGIGILTV poorly. Structural analysis revealed the potential for new contacts between the MEL5 T-cell receptor and the optimized peptide. Furthermore, the optimized peptide was able to prime CD8(+) T-cell populations in peripheral blood mononuclear cell isolates from multiple HLA A*0201(+) individuals that were capable of efficient HLA A*0201(+) melanoma cell destruction. This proof-of-concept study demonstrates that it is possible to design altered peptide antigens for the selection of superior T-cell clonotypes with enhanced antigen recognition properties.

Topics: Amino Acid Sequence; Amino Acid Substitution; Antigen Presentation; Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Circular Dichroism; HLA-A2 Antigen; Humans; Kinetics; MART-1 Antigen; Melanoma; Models, Molecular; Peptide Fragments; Protein Binding; Protein Structure, Secondary; Receptors, Antigen, T-Cell; Surface Plasmon Resonance

Clear cell sarcoma (CCS) and malignant melanoma share overlapping immunohistochemistry with regard to the melanocytic markers HMB45, S100, and Melan-A. However, the translocation t(12; 22)(q13; q12) is specific to CCS. Therefore, although these neoplasms are closely related, they are now considered to be distinct entities. However, the translocation is apparently detectable only in 50%-70% of CCS cases. Therefore, the absence of a detectable EWS/AFT1 rearrangement may occasionally lead to erroneous exclusion of a translocation-negative CCS. Therefore, histological assessment is essential for the correct diagnosis of CCS. Primary CCS of the bone is exceedingly rare. Only a few cases of primary CCS arising in the ulna, metatarsals, ribs, radius, sacrum, and humerus have been reported, and primary CCS arising in the pubic bone has not been reported till date.. We present the case of an 81-year-old man with primary CCS of the pubic bone. Histological examination of the pubic bone revealed monomorphic small-sized cells arranged predominantly as a diffuse sheet with round, hyperchromatic nuclei and inconspicuous nucleoli. The cells had scant cytoplasm, and the biopsy findings indicated small round cell tumor (SRCT). Immunohistochemical staining revealed the tumor cells to be positive for HMB45, S100, and Melan-A but negative for cytokeratin (AE1/AE3) and epithelial membrane antigen. To the best of our knowledge, this is the first case report of primary CCS of the pubic bone resembling SRCT. This ambiguous appearance underscores the difficulties encountered during the histological diagnosis of this rare variant of CCS.. Awareness of primary CCS of the bone is clinically important for accurate diagnosis and management when the tumor is located in unusual locations such as the pubic bone and when the translocation t(12; 22)(q13; q12) is absent.

Topics: Aged, 80 and over; Desmoplastic Small Round Cell Tumor; Diagnosis, Differential; gp100 Melanoma Antigen; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Pubic Bone; S100 Proteins; Sarcoma, Clear Cell; Sarcoma, Small Cell

Soluble adenylyl cyclase (sAC) is an enzyme that generates cyclic adenosine monophosphate, a signaling molecule involved in regulating melanocyte functions. R21, a mouse monoclonal antibody against sAC, shows a striking pan-nuclear staining in lentigo maligna, indicating possible utility for diagnosis and margin assessment.. To evaluate R21 in the diagnosis and evaluation of margins in lentigo maligna.. Thirty one re-excision specimens for lentigo maligna were evaluated for R21 expression using previously published protocol. In addition, 153 cases including 41 lentigo malignas, 30 non-lentigo maligna-type melanomas, 38 lentigos, and 44 nevi were evaluated using a modified stringent protocol to eliminate all nonmelanocyte staining.. The sensitivity of nuclear staining with R21 in lentigo maligna was 87.8%. Nuclear expression of sAC was observed in 40% of other melanomas and 2.3% of benign nevi. R21 did not stain nuclei of resting melanocytes but was observed in 28.9% of melanocytic hyperplasias. These cases were easily distinguished from lentigo maligna in routine sections. R21 staining facilitated extent of the lesion in resection margins. In cases examined under the less stringent conditions, interpretation was facilitated by comparing R21 and Mart1/Melan A staining. Greater than 9 pan-nuclear staining melanocytes within one high-power field along with a pan-nuclear sAC/Melan A ratio greater than 0.5 was consistent with a positive margin whereas 5 or less pan-nuclear staining melanocytes along with a sAC/Melan A ratio of less than 0.3 constituted a negative margin.. R21 is a useful diagnostic adjunct in the diagnosis and evaluation of margins in re-excision specimens in lentigo maligna.

Topics: Adenylyl Cyclases; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antibody Specificity; Biomarkers, Tumor; Cell Nucleus; Diagnosis, Differential; Gene Expression Regulation, Neoplastic; Humans; Hutchinson's Melanotic Freckle; Hyperplasia; Immunohistochemistry; MART-1 Antigen; Melanocytes; Melanoma; Neoplasm Proteins; Nevus; Sensitivity and Specificity; Skin; Skin Neoplasms; Solubility

Topics: Aged; Biomarkers, Tumor; Carcinoma in Situ; Cell Proliferation; Humans; Hutchinson's Melanotic Freckle; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; S100 Proteins; Skin Neoplasms

While there are many obstacles to immune destruction of autologous tumors, there is mounting evidence that tumor antigen recognition does occur. Unfortunately, immune recognition rarely controls clinically significant tumors. Even the most effective immune response will fail if tumors fail to express target antigens. Importantly, reduced tumor antigen expression often results from changes in gene regulation rather than irrevocable loss of genetic information. Such perturbations are often reversible by specific compounds or biological mediators, prompting a search for agents with improved antigen-enhancing properties. Some recent findings have suggested that certain conventional chemotherapeutic agents may have beneficial properties for cancer treatment beyond their direct cytotoxicities against tumor cells. Accordingly, we screened an important subset of these agents, topoisomerase inhibitors, for their effects on antigen levels in tumor cells. Our analyses demonstrate upregulation of antigen expression in a variety of melanoma cell lines and gliomas in response to nanomolar levels of certain specific topoisomerase inhibitors. To demonstrate the ability of CD8+ T cells to recognize tumors, we assayed cytokine secretion in T cells transfected with T cell receptors directed against Melan-A/MART-1 antigen. Three days of daunorubicin treatment resulted in enhanced antigen expression by tumor cells, in turn inducing co-cultured antigen-specific T cells to secrete Interleukin-2 and Interferon-γ. These results demonstrate that specific topoisomerase inhibitors can augment melanoma antigen production, suggesting that a combination of chemotherapy and immunotherapy may be of potential value in the treatment of otherwise insensitive cancers.

Topics: Antigen Presentation; CD8-Positive T-Lymphocytes; Coculture Techniques; Daunorubicin; DNA Topoisomerases; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Glioma; Humans; Immunotherapy; Interferon-gamma; Interleukin-2; Jurkat Cells; Lymphocyte Activation; MART-1 Antigen; Melanoma; Receptors, Antigen, T-Cell; Transgenes

To determine the long-term clinical significance of molecular upstaging in histopathology-negative, paraffin-embedded (PE) sentinel lymph nodes (SLNs) from melanoma patients.. Histopathologic evaluation can miss clinically relevant melanoma micrometastases in SLNs. This longitudinal correlative study is the first 10-year prognostic evaluation of a multimarker quantitative real-time reverse transcriptase-polymerase chain reaction (qRT) assay for PE melanoma-draining SLNs.. The SLN sections (n = 214) were assessed by qRT assay for 4 established messenger RNA biomarkers: MART-1, MAGE-A3, GalNAc-T, and PAX3.. The qRT assay upstaged 48 of 161 histopathology-negative (hematoxylin-eosin and immunohistochemistry) SLN specimens. At a median follow-up of 11.3 years for the entire cohort, estimated rates of 10-year overall survival (OS) and melanoma-specific survival (MSS) were 82% and 94%, respectively, for histopathology-negative/qRT-negative patients; 56% and 61%, respectively, for histopathology-positive patients; and 52% and 60%, respectively, for histopathology-negative/qRT-positive patients (P < 0.001 for OS, P < 0.001 for MSS). In a multivariate analysis of known melanoma prognostic factors, qRT positivity was significant (P < 0.05) for disease-free survival (hazard ratio [HR], 4.3; 95% confidence interval (CI), 2.3-7.8), distant disease-free survival (HR, 6.6; 95% CI, 2.9-14.6), MSS (HR, 6.2; 95% CI, 2.6-14.4), and OS (HR, 2.8; 95% CI, 1.6-4.9).. The multimarker qRT assay has prognostic significance for molecular upstaging of PE melanoma-draining SLNs. Molecular upstaging of histopathology-negative SLNs confers a prognosis similar to that associated with SLN micrometastasis, and the number of positive qRT biomarkers is correlated to disease outcome.

Topics: Aged; Antigens, Neoplasm; Biomarkers, Tumor; Female; Humans; Longitudinal Studies; Male; MART-1 Antigen; Melanoma; Middle Aged; N-Acetylgalactosaminyltransferases; Neoplasm Proteins; Neoplasm Staging; Paired Box Transcription Factors; Paraffin Embedding; PAX3 Transcription Factor; Polypeptide N-acetylgalactosaminyltransferase; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sentinel Lymph Node Biopsy; Skin Neoplasms; Survival Analysis

Durable responses in metastatic melanoma patients remain generally difficult to achieve. Adoptive cell therapy (ACT) with ex vivo engineered lymphocytes expressing high affinity T-cell receptors (TCRα/β) for the melanoma antigen MART-1₂₇₋₃₅/HLA-A*0201 [recognized by F5 cytotoxic T lymphocytes (F5 CTL)] has been found to benefit certain patients. However, many other patients are inherently unresponsive and/or relapse for unknown reasons. To analyze the basis for the acquired resistance and strategies to reverse it, we established F5 CTL-resistant (R) human melanoma clones from relatively sensitive parental lines under selective F5 CTL pressure. Surface MART-1₂₇₋₃₅/HLA-A*0201 in these clones was unaltered and F5 CTLs recognized and interacted with them similar to the parental lines. Nevertheless, the R clones were resistant to F5 CTL killing, exhibited hyperactivation of the NF-κB survival pathway, and overexpression of the antiapoptotic genes B cell lymphoma protein 2 (Bcl-2), Bcl-2 related gene (long alternatively spliced variant of Bcl-x gene; Bcl-(xL)), and myeloid cell differentiation 1 (Mcl-1). Sensitivity to F5 CTL-killing could be increased by pharmacological inhibition of the NF-κB pathway, Bcl-2 family members, or the proteasome, the latter of which reduced NF-κB activity and diminished antiapoptotic gene expression. Specific gene-silencing (by siRNA) confirmed the protective role of antiapoptotic factors by reversing R clone resistance. Together, our findings suggest that long-term immunotherapy may impose a selection for the development of resistant cells that are unresponsive to highly avid and specific melanoma-reactive CTLs, despite maintaining expression of functional peptide:MHC complexes, due to activation of antiapoptotic signaling pathways. Though unresponsive to CTL, our results argue that resistant cells can be resensitized to immunotherapy with coadministration of targeted inhibitors to antiapoptotic survival pathways.

Topics: Antigens, Neoplasm; Apoptosis Regulatory Proteins; CD8-Positive T-Lymphocytes; Cell Communication; Cell Line, Tumor; Cytotoxicity, Immunologic; Gene Expression Regulation, Neoplastic; HLA-A Antigens; HLA-A2 Antigen; Humans; MART-1 Antigen; Melanoma; NF-kappa B; Protein Binding; Signal Transduction; Skin Neoplasms; T-Lymphocytes, Cytotoxic; Tumor Escape

Sentinel lymph nodes (SLNs) of melanoma patients show evidence of tumor-induced immune dysfunction. Our previous works have shown that IL-10 and IFNγ co-regulate indoleamine-2,3-dioxygenase (IDO)-expressing immunosuppressive dendritic cells (DCs) in melanoma SLNs. The goal of this study is to examine the relationship between melanoma SLN tumor burden and the degree of SLN immune dysfunction as a model to study tumor-induced immune dysfunction. We hypothesize that SLN tumor burden correlates with the degree of SLN immune dysfunction.. Patients undergoing SLN biopsy for clinical stages I and II melanomas were enrolled in the study under an IRB-approved protocol. During the SLN biopsy, non-hot and non-blue portion of the SLN was harvested, flash-frozen in liquid nitrogen, and mRNA was extracted. By using quantitative real-time PCR, gene expressions of cytokines (IL-4, IL-10, IFNγ, TGFβ, GM-CSF) and the surrogates of immunosuppressive regulatory and effector cells (IDO-expressing DCs and Foxp3-expressing T-regs, respectively) were measured and correlated against the SLN tumor burden (MART1) and against each other. The data were log transformed for normalization. Statistical test used Student's t-test and stepwise multivariate regression analysis. Statistical significance was determined at P < 0.05.. SLNs of 74 patients were analyzed in this analysis. Ten of seventy-four patients (13.5%) had tumor-positive SLNs. MART1 gene expression showed a significant difference between the SLN (+) and SLN (-) groups (P = 0.04). Among the various cytokines, multivariate analysis showed that only IFNγ gene expression correlated independently with MART1 gene expression (P < 0.0001, r = 0.91). Similar multivariate analyses show that IFNγ (P < 0.0001, r = 0.78), IL-10 (P = 0.0037, r = 0.60), and TGFβ (P < 0.0001, r = 0.95) gene expressions correlated independently with IDO gene expression. IFNγ (P < 0.0001, r = 0.87) and GM-CSF (P = 0.042, r = 0.76) gene expressions correlated independently with Foxp3 gene expression. MART1 gene expression showed independent correlation with IDO (P = 0.0002, r = 0.75) and Foxp3 (P = 0.0002, r = 0.75) gene expressions.. SLN tumor burden correlates with immunosuppressive IDO and Foxp3 expressions within the SLNs of melanoma patients. Our data are consistent with our theory that melanoma induces expressions of specific cytokines, which in turn, stimulate immune suppressors within the SLN. This study also supports our previous finding that IL-10 and IFNγ co-regulate IDO within the SLN. In our data, IFNγ is the sole cytokine that correlates with the SLN tumor burden and seems to play a central role in tumor-induced immunological changes in the SLN immune microenvironment.

Topics: Cytokines; Dendritic Cells; Female; Forkhead Transcription Factors; Gene Expression; Humans; Indoleamine-Pyrrole 2,3,-Dioxygenase; Interferon-gamma; Interleukin-10; Lymph Nodes; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Middle Aged; Multivariate Analysis; Polymerase Chain Reaction; Regression Analysis; Sentinel Lymph Node Biopsy; T-Lymphocytes, Regulatory; Tumor Burden

Evaluation of cutaneous pigmented lesions can be diagnostically challenging and represents an activity often supplemented by immunohistochemistry. Immunohistochemical studies typically employ 3,3'-diaminobenzidine (DAB) resulting in brown staining of both melanocytes and melanin. Difficulty may thus arise in distinguishing different cell types in heavily melanized lesions. Azure blue counterstaining has been used in conjunction with melanoma antigen recognized by T-cells (MART-1) to differentiate melanocytes from melanin by highlighting the latter blue-green. Microphthalmia transcription factor (MiTF) represents an alternative immunomarker that shows nuclear reactivity, which facilitates ease of interpretation.. Twenty examples of solar lentigo and melanoma in situ (MIS) were independently evaluated utilizing MiTF and MART-1/Azure blue for melanocyte quantification. Melanocyte counts were averaged over five high-power fields (×400) to obtain a mean melanocytic count.. There was no significant difference in the mean melanocytic count between MART-1/Azure blue and MiTF as assessed in the solar lentigo group and as assessed independently in the MIS group. MiTF nuclear staining facilitated interpretation and required less laboratory preparation, as an additional counterstain was not necessary.. MiTF is as effective as MART-1/Azure blue in identifying melanocytes in the context of solar lentigo or MIS. On the basis of our results, we favor expanding the use of MiTF as an immunohistochemical marker, as it provides an efficient alternative to MART-1 with Azure blue counterstaining in the evaluation of cutaneous pigmented lesions.

Topics: Azure Stains; Biomarkers, Tumor; Carcinoma in Situ; Diagnosis, Differential; Humans; Immunohistochemistry; Lentigo; MART-1 Antigen; Melanoma; Microphthalmia-Associated Transcription Factor; Skin Neoplasms

Topics: Aged; Biomarkers, Tumor; Chromosome Mapping; Female; gp100 Melanoma Antigen; Humans; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Real-Time Polymerase Chain Reaction; Skin Neoplasms

The greater density and unusual patterning of melanocytes in chronically sun-exposed skin complicates interpretation of intraoperative Melan-A immunohistochemical stained margins during margin-controlled surgery for lentigo maligna (LM) and lentigo maligna melanoma (LMM).. To identify the immunohistochemical similarities and differences in melanocyte distribution between LM and LMM and chronically sun-exposed skin.. Retrospective review of Melan-A-stained original biopsy specimens of LM and LMM and uninvolved sun-damaged skin (negative controls), from 70 LM and LMM cases from the University of Utah in 2008.. Histologic features commonly associated with LM were common in negative controls from chronically sun-exposed skin. Melanocyte confluence (27/70, 39%), stacking (34/70, 49%), theque formation (9/70, 13%), adnexal extension (59/68, 87%), and suprabasilar scatter (23/70, 33%) were observed in the negative controls from sun-damaged skin. Such features were present nearly uniformly in the LM and LMM specimens. Epidermal melanocyte density in LM and LMM differed significantly from that in negative controls (82.7 ± 29.3 and 25.6 ± 9.3 per × 400 field, respectively; p<.001).. Epidermal melanocytic features often ascribed to LM, such as melanocyte confluence, stacking, theque formation, adnexal extension, and suprabasilar scatter, are frequently observed in chronically sun-exposed Caucasian skin and may lead to overestimation of surgical margins.

Topics: Adult; Aged; Aged, 80 and over; Biopsy; Chi-Square Distribution; Female; Humans; Hutchinson's Melanotic Freckle; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Middle Aged; Photomicrography; Retrospective Studies; Skin Aging; Skin Neoplasms; Staining and Labeling; Statistics, Nonparametric

In chronic viral infections, CD8⁺ T cells become functionally deficient and display multiple molecular alterations. In contrast, only little is known of self- and tumor-specific CD8⁺ T cells from mice and humans. Here we determined molecular profiles of tumor-specific CD8⁺ T cells from melanoma patients. In peripheral blood from patients vaccinated with CpG and the melanoma antigen Melan-A/MART-1 peptide, we found functional effector T cell populations, with only small but nevertheless significant differences in T cells specific for persistent herpesviruses (EBV and CMV). In contrast, Melan-A/MART-1-specific T cells isolated from metastases from patients with melanoma expressed a large variety of genes associated with T cell exhaustion. The identified exhaustion profile revealed extended molecular alterations. Our data demonstrate a remarkable coexistence of effector cells in circulation and exhausted cells in the tumor environment. Functional T cell impairment is mediated by inhibitory receptors and further molecular pathways, which represent potential targets for cancer therapy.

Topics: Cancer Vaccines; CD8-Positive T-Lymphocytes; CpG Islands; Cytomegalovirus; Cytomegalovirus Infections; Epstein-Barr Virus Infections; Gene Expression Profiling; Herpesvirus 4, Human; Humans; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Molecular Sequence Data; Receptors, Immunologic; T-Cell Antigen Receptor Specificity; T-Lymphocyte Subsets; Vaccination; Virus Latency

Topics: Adult; Diagnosis, Differential; Humans; Magnetic Resonance Imaging; Male; MART-1 Antigen; Medulloblastoma; Melanocytes; Melanoma; Melanoma-Specific Antigens; Meningeal Neoplasms; Neoplasms, Multiple Primary; Neurilemmoma; Nevus of Ota; S100 Proteins; Skin Neoplasms; Vimentin

Distinction between benign and malignant melanocytic lesions may be difficult by today's methods, even for highly skilled dermatopathologists, emphasizing the need for improved diagnostic tools. We have studied the discriminative abilities of immunohistochemical (IHC) double stains using the IHC markers Ki67 combined with MART1, and HMB45 combined with MITF. Paraffin-embedded tissue sections from 50 melanomas and 78 benign nevi were stained using a simple simultaneous IHC double staining technique. Both simple semiquantitative estimates of the immunopositivity in the deepest third of the lesions and full-scale quantitative measurements of the Ki67 and HMB45 indices were performed, and scores for melanomas and nevi were compared. The differences between melanomas and nevi were significant (P < 0.0001) using either analysis or stain. The misclassification rates for melanomas and nevi were generally lower for Ki67/MART1 stains than for HMB45/MITF stains. In the simple semiquantitative Ki67/MART1 analysis, the misclassification rates were 6% (2%-17%) for melanomas and 12% (6%-21%) for nevi. In full-scale quantitative analysis the corresponding rates were 4% (1%-14%) and 8% (4%-16%), and by combining Ki67 and HMB45 indices, the misclassification rates were 0% (0%-7%) for melanomas and 13% (7%-22%) for nevi. We conclude that both semiscale and fullscale quantitative analyses of Ki67/MART1 stains are valuable diagnostic tools to distinguish melanomas and nevi with a large degree of certainty. The HMB45/MITF stains may serve as adjuncts to predict malignancy and the diagnostic potential of combining the HMB45 and Ki67 indices are promising. The IHC double stains may potentially reduce misinterpretations of melanomas in histopathology.

Topics: Biomarkers, Tumor; Diagnosis, Differential; Female; Humans; Ki-67 Antigen; Male; MART-1 Antigen; Melanoma; Microphthalmia-Associated Transcription Factor; Middle Aged; Neoplasm Proteins; Nevus; Skin Neoplasms; Skin Ulcer; Survival Rate

Uveal melanoma primarily metastasizes hematogenously with metastases often confined to the liver. The aim of this study was to investigate the presence of circulating tumor cells (CTC) in patients with metastatic disease as a marker for systemic disease and to determine their prognostic relevance.. Blood samples from 68 patients were collected at the time of initial treatment of metastases. mRNA expression of tyrosinase and MelanA/MART1 as a surrogate marker for the presence of CTC was analyzed by real-time RT-PCR and compared with patient characteristics.. CTC were detected in 63% of all patients and in 67% of the 48 patients with only liver metastases. Univariate and multivariate analyses revealed PCR results and serum lactate dehydrogenase as independent prognostic factors for progression-free (hazard ratios 2.2/3.5) and overall survival (hazard ratios 4.0/6.5). Combination of PCR and lactate dehydrogenase divided the patient cohort into 3 groups with distinct prognosis.. CTC as evidence for systemic disease can be found in the majority of patients with metastatic uveal melanoma, including patients with visible disease confined to the liver. Detection of CTC-specific mRNA transcripts for tyrosinase and MelanA/MART1 by PCR is a poor prognostic factor for progression-free and overall survival. Characterization of CTC could improve the understanding of their biology.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Disease-Free Survival; Female; Humans; Kaplan-Meier Estimate; Karnofsky Performance Status; L-Lactate Dehydrogenase; Liver Neoplasms; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplastic Cells, Circulating; Proportional Hazards Models; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Rate; Uveal Neoplasms

To date, only 12 cases of angiomatoid Spitz nevus have been characterized in the literature. We present the first case of angiomatoid Spitz nevus in which dermatoscopic findings are described.

Topics: Adult; Angiomatosis; Biomarkers, Tumor; Biopsy; Dermoscopy; Diagnosis, Differential; Humans; Male; MART-1 Antigen; Melanoma; Nevus, Epithelioid and Spindle Cell; Skin Neoplasms; Vascular Neoplasms

To predict the potential antitumor effect of antigen-specific T cells in melanoma patients, we investigated T-cell effector function in relation to tumor-escape mechanisms.. CD8(+) T cells isolated from tumor, adjacent normal skin, and peripheral blood of 17 HLA-A2(+) patients with advanced-stage melanoma were analyzed for their antigen specificity and effector function against melanocyte differentiation antigens MART-1, gp100, and tyrosinase by using HLA-A2/peptide tetramers and functional assays. In addition, the presence of tumor-escape mechanisms PD-L1/PD-1 pathway, FoxP3 and loss of HLA or melanocyte differentiation antigens, both required for tumor cell recognition and killing, were studied.. Higher percentages of melanocyte antigen-specific CD8(+) T cells were found in the melanoma tissues as compared with adjacent normal skin and peripheral blood. Functional analysis revealed 2 important findings: (i) in 5 of 17 patients, we found cytokine production after specific peptide stimulation by tumor-infiltrating lymphocytes (TIL), not by autologous peripheral blood lymphocytes (PBL); (ii) CD8(+) T cells from 7 of 17 patients did not produce cytokines after specific stimulation, which corresponded with significant loss of tumor HLA-A2 expression. The presence of other tumor-escape mechanisms did not correlate to T-cell function.. Our data show that functional T-cell responses could be missed when only PBL and not TIL are evaluated, emphasizing the importance of TIL analysis for immunomonitoring. Furthermore, loss of tumor HLA-A2 may explain the lack of T-cell functionality. These findings have important implications for selecting melanoma patients who may benefit from immunotherapy.

Topics: Aged; Aged, 80 and over; B7-H1 Antigen; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cells, Cultured; Cytokines; Cytotoxicity, Immunologic; Female; Forkhead Transcription Factors; gp100 Melanoma Antigen; HLA-A2 Antigen; Humans; Immunotherapy; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Skin; Tumor Escape

CD4 T lymphocytes play a central role in orchestrating an efficient antitumor immune response. Much effort has been devoted in the identification of major histocompatibility complex class II eptiopes from different tumor-associated antigens. Melan-A/MART-1 is expressed specifically in normal melanocytes and tumor cells of 75% to 100% of melanoma patients. Melan-A/MART-1 is considered as an attractive target for cancer immunotherapy. In the past, several human leukocyte antigen (HLA) class II restricted epitopes have been identified and characterized, including Melan-A/MART-11-20 (HLA-DR11 restricted), Melan-A/MART-125-36 (HLA-DQ6 and HLA-DR3 restricted), Melan-A/MART-127-40 (HLA-DR1 restricted), Melan-A/MART-151-73 (HLA-DR4 restricted), Melan-A/MART-191-110 (HLA-DR52 restricted), and Melan-A/MART-1100-111 (HLA-DR1 restricted). Owing to the infrequent expression of the above HLA class II alleles in Asian populations, immunotherapy using these defined Melan-A/MART-1 peptides could potentially only benefit a very small percentage of Asian melanoma patients. In this study, we established several CD4 T-cell clones by in vitro stimulation of peripheral blood mononuclear cells from a healthy donor by a peptide pool of 28 to 30 amino acid long peptides spanning the entire Melan-A/MART-1 protein. These CD4 T-cell clones recognized a peptide that is embedded within Melan-A/MART-121-50, in a HLA-DPB1*0501 restricted manner. Finally, we demonstrated that this epitope is naturally processed and presented by dendritic cells. HLA-DPB1*0501 is frequently expressed in Asian population (44.9% to 73.1%). Therefore, this epitope could provide a new tool and could significantly increase the percentage of melanoma patients that can benefit from cancer immunotherapy.

Topics: Antigens, Neoplasm; Asian People; CD4-Positive T-Lymphocytes; Cell Line, Tumor; Dendritic Cells; Enzyme-Linked Immunosorbent Assay; Epitopes, T-Lymphocyte; Histocompatibility Testing; HLA-DP beta-Chains; Humans; Immunotherapy; MART-1 Antigen; Melanocytes; Melanoma; T-Lymphocytes, Cytotoxic

Vitiligo is a common toxicity associated with immunotherapy for melanoma. Cytotoxic T lymphocytes (CTLs) against melanoma commonly target melanoma-associated antigens (MAAs) which are also expressed by melanocytes. To uncouple vitiligo from melanoma destruction, it is important to understand if CTLs can respond against melanoma and melanocytes at different levels.. To understand the dichotomous role of MAA-specific CTL, we characterized the functional reactivities of established CTL clones directed to MAAs against melanoma and melanocyte cell lines.. CTL clones generated from melanoma patients were capable of eliciting MHC-restricted, MAA-specific lysis against melanocyte cell lines as well as melanoma cells. Among the tested HLA-A*0201-restricted CTL clones, melanocytes evoked equal to slightly higher degranulation and cytolytic responses as compared to melanoma cells. Moreover, MAA-specific T cells from vaccinated patients responded directly ex vivo to melanoma and melanocytes. Melanoma cells express slightly higher levels of MART-1 and gp100 than melanocytes as measured by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and immunohistochemistry.. Our data suggest that CTLs respond to melanoma and melanocytes equally in vitro and directly ex vivo.

Topics: Cancer Vaccines; Cell Degranulation; Cell Line, Tumor; Clone Cells; Cytotoxicity, Immunologic; Flow Cytometry; HLA-A2 Antigen; Humans; Immunohistochemistry; MART-1 Antigen; Melanocytes; Melanoma; Peptides; T-Lymphocytes, Cytotoxic; Vaccination

It is often challenging to reliably assess the number of lesional melanocytes in intraepidermal melanocytic proliferations involving sun-damaged skin. Therefore, dermatopathologists routinely use immunostains to help differentiate melanocytes from surrounding keratinocytes.. Forty-three cases of solar lentigo or melanoma in situ (of the lentigo maligna type) were retrospectively chosen (20 melanomas in situ and 23 solar lentigo). Microphthalmia transcription factor (MiTF), HMB-45, Melan-A and Mel-5 immunostains were performed with an Azure blue counterstain, and the mean melanocyte counts were calculated within a 1-mm segment of epidermis.. In solar lentigines, the mean melanocyte counts were 27 (MiTF), 23 (HMB-45 and Mel-5) and 41 (Melan-A), as compared to hematoxylin and eosin (H&E) (25). In melanoma in situ, the mean melanocyte counts were 112 (MiTF), 149 (Melan-A), 111 (HMB-45) and 80 (Mel-5), as compared to H&E (109).. These results show that Melan-A significantly overestimates the density of melanocytes within dermatoheliotic skin. Compared to other tested stains, nuclear staining MiTF allowed greater distinction of melanocytes from keratinocytes with melanized cytoplasm. These findings indicate that MiTF is a superior marker for quantification of melanocytes in the evaluation of subtle intraepidermal melanocytic proliferations and in the differential diagnosis of solar lentigo.

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Cell Count; Cell Nucleus; Diagnosis, Differential; Female; Glycoproteins; gp100 Melanoma Antigen; Humans; Keratinocytes; Lentigo; Male; MART-1 Antigen; Melanocytes; Melanoma; Melanoma-Specific Antigens; Microphthalmia-Associated Transcription Factor; Middle Aged; Reproducibility of Results; Retrospective Studies; Skin Neoplasms

Topics: Diagnosis, Differential; Humans; Lupus Erythematosus, Cutaneous; MART-1 Antigen; Melanoma; Microphthalmia-Associated Transcription Factor; S100 Proteins; Skin Neoplasms

A proportion of patients who develop regional and distant recurrences of melanoma after a pathologically negative sentinel lymph node (SN) biopsy are reported to have enhanced signals for melanoma-associated messenger ribonucleic acid (mRNA) when sensitive molecular approaches such as reverse transcriptase polymerase chain reaction (RT-PCR) are used to evaluate their SN tissue. The significance of these findings remains controversial, because the cellular source of the augmented signals cannot be known as the nodal tissue is destroyed during preparation for RT-PCR. Nevertheless, it is claimed that the source of the augmented signal is covert metastatic melanoma cells. To determine whether there are histologically occult metastases in SN and whether there are sources of augmentable melanoma-associated mRNA other than melanoma cells, we applied reverse transcriptase in situ polymerase chain reaction (RT in situ PCR) to formalin-fixed paraffin-embedded nodal tissue. This approach amplifies small amounts of melanoma-associated mRNA and permits identification of cells that express that mRNA. Cells containing MART-1 mRNA were detected in 6 of 21 SNs (29%) and 2 of 16 nonsentinel lymph node (NSNs) (13%) that were tumor negative on hematoxylin and eosin and on immunohistochemical assessment for S-100, MART-1, and HMB-45. In patients with microscopic evidence of melanoma in their SN, MART-1 mRNA-positive cells were identified in 2 of 7 NSNs (29%) that were histologically tumor free. MART-1 mRNA-positive cells were also detected in tumor-negative SN sections from 6 of 7 (86%) nodes that had tumor present in areas of the node not represented in the studied sections. Some cells that expressed MART-1 mRNA that was diffusely distributed in the cytoplasm appeared to be melanoma cells, whereas others resembled macrophages. The latter cells expressed augmented mRNA on granules that were intermixed with melanin granules. In other cases, MART-1 mRNA-positive macrophage-like cells contained nuclei and nucleoli more typical of melanoma cells and may represent the macrophage-melanoma hybrids that have been previously reported. Combination of RT in situ PCR for MART-1 mRNA and immunohistochemistry for CD68 revealed that CD68 was colocalized in some cells that expressed MART-1 mRNA. Some lymph nodes that are tumor negative by histology and immunohistochemistry contain cells that express mRNA for MART-1. Some of these cells may be interpreted as "stealth" melanoma cells in which,

Topics: Antigens, CD; Antigens, Differentiation, Myelomonocytic; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Lymph Nodes; Lymphatic Metastasis; Macrophages; MART-1 Antigen; Melanoma; Neoplastic Cells, Circulating; Predictive Value of Tests; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sentinel Lymph Node Biopsy; Skin Neoplasms

A captive adult crevice kelpfish, Gibbonsia montereyensis, developed a cutaneous mass, approximately 9 × 7 mm on the right side of the head in an area of nonscaled skin. Following surgical debulking, examination of both impression smears and histologic sections of the tumor revealed a predominant population of round to spindloid to polygonal cells with a moderate amount of lightly basophilic cytoplasm. The cytoplasm was filled with round, variably-sized reddish-brown granules that often obscured the nucleus. Nuclei were round to ovoid with coarsely granular chromatin. There was minimal anisocytosis and anisokaryosis. The cytoplasmic granules in histologic sections were weakly positive by the Fontana-Masson method, and staining was eliminated with melanin bleach. Immunohistochemical staining was strongly positive with a murine monoclonal antibody for melan A. As the specificity of melan A for melanophores is not clearly defined in nonmammalian species, the tumor was examined by transmission electron microscopy. Melanophores were not detected. Instead, neoplastic cells were filled with numerous intracytoplasmic organelles with triple-limiting membranes composed of concentric lamellae; these structures were most compatible with pterinosomes, which are the pigment-containing organelles of cells called xanthophores and erythrophores. As both of these organelles are ultrastructurally indistinguishable and as kelpfish skin is known to contain both xanthophores and erythrophores, a diagnosis of a mixed pigment cell tumor or chromatophoroma was made. As the tumor was grossly reddish-brown, the possibility of a neoplastic population of only erythrophores could not be excluded. Pigment cell tumors, arising from cells of the embryonic neural crest, are common in reptiles and bony fish.

Topics: Animals; Antibodies, Monoclonal; Cell Nucleus; Chromatophores; Fish Diseases; Fishes; Immunohistochemistry; MART-1 Antigen; Melanoma; Skin Neoplasms

Regressing nevi are considered an example of an efficient early antitumoral response preventing the development of neoplasia. The underlying mechanism has not been elucidated, although an immune-based destruction of melanocytes is supposed. The aim of this study was to provide evidence of an effective immunosurveillance of pigment lesions in a patient at high risk of melanoma.. A patient with the dysplastic nevus syndrome and a history of melanoma was included in this study. Since 2003, a marked regression of almost all nevi was observed. Immunohistochemistry was performed and the antigen specificity of T-cells was analyzed on T-cells isolated from a regressing nevus by flow cytometry using HLA-A2-peptide tetramers containing Mart-1(26-35), gp100(280-288), gp100(209-217) and tyrosinase(369-377). Immunohistochemistry of the regressing nevi showed a strong infiltrate of CD4 + and CD8 + T-cells. Flow cytometric analyses demonstrated the presence of a CD8 + T-cell response against gp100(280-288) and Mart-1(26-35) both in peripheral blood and in a regressing nevus.. These findings indicate that an immune reaction against melanocyte differentiation antigens can target specifically nevi without signs of vitiligo and suggests that boosting the anti-melanocyte immune response in patients at high risk for melanoma may prevent tumor development at an early stage.

Topics: Blotting, Western; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Dysplastic Nevus Syndrome; Flow Cytometry; gp100 Melanoma Antigen; HLA-A2 Antigen; Humans; Immunoenzyme Techniques; Male; MART-1 Antigen; Melanocytes; Melanoma; Middle Aged; Monophenol Monooxygenase; Skin Neoplasms; T-Lymphocytes, Cytotoxic; Vitiligo

Based on their role for the induction of T-cell responses, dendritic cells (DCs) are popular candidates in cancer vaccine development. We established a novel single-step intracellular delivery of peptide/poly(I:C) complexes for antigen loading and Toll-like receptor-3 (TLR3)-mediated maturation of human DCs using a cell-penetrating peptide (tat(49-57): RKKRRQRRR) as delivery vector. Towards this end, a cationic tat-sequence was fused with an antigenic, major histocompatibility complex (MHC) class I-binding melanoma epitope (Melan-A/Mart-1 sequence: ELAGIGILTV) and then mixed with negatively charged poly(I:C) dsRNA to form peptide/nucleic acid complexes. Flow cytometry and confocal laser scanning microscopy confirmed intracellular localization of TLR3 in monocyte-derived immature DCs (iDCs). Peptide/poly(I:C) complexes were readily internalized by iDCs without negatively affecting cell viability. They induced DC maturation and secretion of bioactive interleukin (IL)-12p70. When peptide/poly(I:C) complex-loaded DCs were used for autologous T cell stimulation, epitope-specific interferon-gamma secretion was quantitatively superior in comparison to peptide-loaded DCs matured by a cytokine cocktail, as detected by enzyme-linked immunospot assays. Thus, complexes of cationic antigenic peptides and poly(I:C) might be of great utility for a TLR3-mediated DC maturation and intracellular peptide targeting in a single step. Resulting DCs induce a strong expansion/activation of antigen-specific T cells in the context of an IL-12p70 secretion.

Topics: Antigens, Neoplasm; Cell Survival; Dendritic Cells; Epitopes; Genes, MHC Class I; Humans; Interferon Inducers; Interleukin-12; MART-1 Antigen; Melanoma; Neoplasm Proteins; Poly I-C; T-Lymphocytes; tat Gene Products, Human Immunodeficiency Virus; Toll-Like Receptor 3

We determined how CD8(+) melanoma tumor-infiltrating lymphocytes (TILs) isolated from two distinct phases of expansion in preparation for adoptive T cell therapy respond to melanoma Ag restimulation. We found that TILs isolated after the rapid expansion protocol (REP) phase, used to generate the final patient TIL infusion product, were hyporesponsive to restimulation with MART-1 peptide-pulsed dendritic cells, with many CD8(+) T cells undergoing apoptosis. Telomere length was shorter post-REP, but of sufficient length to support further cell division. Phenotypic analysis revealed that cell-surface CD28 expression was significantly reduced in post-REP TILs, whereas CD27 levels remained unchanged. Tracking post-REP TIL proliferation by CFSE dilution, as well as sorting for CD8(+)CD28(+) and CD8(+)CD28(-) post-REP subsets, revealed that the few CD28(+) TILs remaining post-REP had superior survival capacity and proliferated after restimulation with MART-1 peptide. An analysis of different supportive cytokine mixtures during the REP found that a combination of IL-15 and IL-21 facilitated comparable expansion of CD8(+) TILs as IL-2, but prevented the loss of CD28 expression with improved responsiveness to antigenic restimulation post-REP. These results suggest that current expansion protocols using IL-2 for melanoma adoptive T cell therapy yields largely CD8(+) T cells unable to persist and divide in vivo following Ag contact. The few CD8(+)CD28(+) T cells that remain may be the only CD8(+) TILs that ultimately survive to repopulate the host and mediate long-term tumor control. A REP protocol using IL-15 and IL-21 may greatly increase the number of CD28(+) TILs capable of long-term persistence.

Topics: Antigens, Neoplasm; CD28 Antigens; CD8-Positive T-Lymphocytes; Cell Proliferation; Cell Separation; Cell Survival; Flow Cytometry; Humans; Immunotherapy, Adoptive; Interleukin-15; Interleukins; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Neoplasm Proteins; T-Lymphocyte Subsets

Topics: Antigens, Neoplasm; Biomarkers; Biopsy; Humans; MART-1 Antigen; Melanoma; Neoplasm Proteins; Predictive Value of Tests; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sensitivity and Specificity; Skin Neoplasms

Multimeric MHC I-peptide complexes containing phycoerythrin-streptavidin are widely used to detect and investigate antigen-specific CD8+ (and CD4+) T cells. Because such reagents are heterogeneous, we compared their binding characteristics with those of monodisperse dimeric, tetrameric and octameric complexes containing linkers of variable length and flexibility on Melan-A-specific CD8+ T cell clones and peripheral blood mononuclear cells (PBMC) from HLA-A*0201(+) melanoma patients. Striking binding differences were observed for different defined A2/Melan-A(26-35) complexes on T cells depending on their differentiation stage. In particular, short dimeric but not octameric A2/Melan-A(26-35) complexes selectively and avidly stained incompletely differentiated effector-memory T cells clones and populations expressing CD27 and CD28 and low levels of cytolytic mediators (granzymes and perforin). This subpopulation was found in PBMC from all six melanoma patients analyzed and proliferated on peptide stimulation with only modest phenotypic changes. By contrast influenza matrix(58-66) -specific CD8+ PBMC from nine HLA-A*0201(+) healthy donors were efficiently stained by A2/Flu matrix(58-61) multimers, but not dimer and upon peptide stimulation proliferated and differentiated from memory into effector T cells. Thus PBMC from melanoma patients contain a differentiation defective sub-population of Melan-A-specific CD8+ T cells that can be selectively and efficiently stained by short dimeric A2/Melan- A(26-35) complexes, which makes them directly accessible for longitudinal monitoring and further investigation.

Topics: Antigens, Neoplasm; Case-Control Studies; CD8-Positive T-Lymphocytes; Cell Differentiation; Dimerization; Flow Cytometry; HLA-A Antigens; HLA-A2 Antigen; Humans; Immunologic Memory; MART-1 Antigen; Melanoma; Neoplasm Proteins; Peptide Fragments; Receptors, Antigen, T-Cell

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Cell Proliferation; Humans; Immunoenzyme Techniques; Ki-67 Antigen; Lymph Nodes; MART-1 Antigen; Melanocytes; Melanoma; Neoplasm Proteins; Nevus, Epithelioid and Spindle Cell; Retrospective Studies; Skin Neoplasms

The function of antigen-specific CD8+ T cells, which may protect against both infectious and malignant diseases, can be impaired by ligation of their inhibitory receptors, which include CTL-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). Recently, B and T lymphocyte attenuator (BTLA) was identified as a novel inhibitory receptor with structural and functional similarities to CTLA-4 and PD-1. BTLA triggering leads to decreased antimicrobial and autoimmune T cell responses in mice, but its functions in humans are largely unknown. Here we have demonstrated that as human viral antigen-specific CD8+ T cells differentiated from naive to effector cells, their surface expression of BTLA was gradually downregulated. In marked contrast, human melanoma tumor antigen-specific effector CD8+ T cells persistently expressed high levels of BTLA in vivo and remained susceptible to functional inhibition by its ligand herpes virus entry mediator (HVEM). Such persistence of BTLA expression was also found in tumor antigen-specific CD8+ T cells from melanoma patients with spontaneous antitumor immune responses and after conventional peptide vaccination. Remarkably, addition of CpG oligodeoxynucleotides to the vaccine formulation led to progressive downregulation of BTLA in vivo and consequent resistance to BTLA-HVEM-mediated inhibition. Thus, BTLA activation inhibits the function of human CD8+ cancer-specific T cells, and appropriate immunotherapy may partially overcome this inhibition.

Topics: Animals; Antigens, CD; Antigens, Neoplasm; Apoptosis Regulatory Proteins; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Humans; Interferon-gamma; Lymphocyte Activation; MART-1 Antigen; Melanoma; Neoplasm Proteins; Oligodeoxyribonucleotides; Programmed Cell Death 1 Receptor; Receptors, Immunologic; Receptors, Tumor Necrosis Factor, Member 14; Vaccination

SOX-9 was originally identified as a master regulator gene that plays a role in the differentiation of mesenchymal cells to chondrocytes. Since then, SOX-9 has been implicated in neural crest development and has emerged as a transcriptional regulator of melanogenesis. Because the role of immunohistochemical detection of SOX-9 in the routine diagnosis of melanoma has not been previously described, we attempted to elucidate the spectrum and labeling characteristics of this antibody in a large cohort of patients with metastatic melanoma. We analyzed the expression of SOX-9 on sections of metastatic melanoma in a tissue microarray and compared the expression of this marker with 3 commonly used melanocytic markers (MART-1, HMB-45 antigen, and S-100 protein). SOX-9 expression was noted in 52 of the 62 cases (83.9%). In comparison, HMB-45 was positive in 53 of the 62 cases (85.5%), MART-1 was positive in 59 of the 62 cases (95.1%), and S-100 protein was positive in 57 of the 62 cases (95%). Interestingly, there were 5 tumors that were negative for 2 or more markers while being positive for SOX-9. Furthermore, 3 of these were negative for all markers except for SOX-9. Our study illustrates that SOX-9 is expressed in a high percentage of melanomas. Furthermore, SOX-9 may be a useful adjunct in the work-up of metastatic melanoma, in such cases where the tumor is negative for the other commonly used melanocytic markers. Last, our data confirm that SOX-9 is not a specific marker for tumors of cartilage lineage and may be expressed in other tumors of neural crest origin.

Topics: Adult; Aged; Antigens, Neoplasm; Biomarkers, Tumor; Brain Neoplasms; Cell Nucleus; Diagnostic Errors; Female; Humans; Immunoenzyme Techniques; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasm Proteins; Retrospective Studies; S100 Proteins; Skin Neoplasms; SOX9 Transcription Factor; Tissue Array Analysis; Young Adult

To evaluate the role of immunohistochemical methods in the diagnosis of benign and malignant conjunctival melanocytic proliferations.. Retrospective immunohistopathologic study.. Paraffin-embedded tissue sections from 20 conjunctival nevi and 15 invasive melanomas were immunoreacted with antibodies against cellular antigens S-100 protein, MART-1, HMB-45, CD-45, and Ki-67 nuclear proliferation protein.. All nevi immunostained moderately to strongly for S-100 protein and MART-1. Results for HMB-45 were negative in the middle and lower subepithelial portions of 18 of 20 lesions; it was usually only weakly positive within the superficial junctional zone. Only 1 melanoma did not stain positively for S-100; MART-1 and HMB-45 were positive in all lesions at some level of intensity. Ki-67 positivity was restricted to the junctional zone of nevi and was diffuse in melanomas. The mean Ki-67 proliferation indices were 1.89% for the nevi and 17.3% for the melanomas. CD-45 can help to highlight lymphocytes that immunostain with Ki-67. Melanomas in situ and atypical primary acquired melanoses had more than twice the Ki-67 proliferation counts of intraepithelial junctional nevocytes (P < .001) and more intense HMB-45 cytoplasmic staining than junctional zone nevocytes.. S-100 and MART-1 were not useful in separating benign from malignant lesions. Results for nevus cells beneath the junctional zone were overwhelmingly negative for HMB-45 and Ki-67. Two nevi and all melanomatous nodules were positive for HMB-45 (P < .001). A higher Ki-67 proliferation index convincingly separated melanomas from nevi (P < .001). Immunostaining for HMB-45 and Ki-67 are valuable adjuncts to careful histopathologic evaluation in assessing benign and malignant conjunctival melanocytic tumors.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Biomarkers, Tumor; Child; Conjunctival Neoplasms; Diagnosis, Differential; Female; Humans; Immunoenzyme Techniques; Ki-67 Antigen; Leukocyte Common Antigens; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasm Proteins; Nevus, Pigmented; Retrospective Studies; S100 Proteins; Young Adult

Adoptive cell transfer of expanded, autologous tumor-infiltrating lymphocytes (TIL) into lymphodepleted melanoma patients can induce the regression of bulky, metastatic disease. To generate the large numbers of T cells needed for infusion, TIL undergo a rapid expansion protocol (REP) in vitro using anti-CD3 antibody, interleukin-2, and irradiated peripheral blood feeder cells that typically results in an approximately 1000-fold expansion over 14 days. However, we have found that the conventional REP (C-REP) often favors the expansion of CD4+ T cells at the expense of tumor antigen-specific CD8+ T cells, which are the most potent cytolytic effector cells. In this study, we demonstrate that addition of transforming growth factor (TGF)-beta1 to the TIL culture at the onset of rapid expansion (T-REP ) maintained the percentage of CD8+ T cells while not inhibiting overall T-cell expansion. Of T cells expanded from different melanoma patient tumors, 13 of 15 TIL demonstrated improved yields and percentages of both CD8+ and MART-1 melanoma antigen-specific T cells after 14 days of expansion in TGF-beta1 compared with the C-REP. This was associated with a marked improvement in the antitumor activity of the resulting bulk TIL culture in terms of interferon-gamma production and melanoma tumor-specific cytotoxic T-lymphocyte activity. In addition, T-REP T cells demonstrated a higher potential for continued expansion in vitro for up to 3 weeks after the expansion compared with C-REP T cells, suggesting that they may also be capable of increased persistence after adoptive cell transfer. Our results suggest that TGF-beta1-expanded TIL have attributes that might predict efficacy superior to that of conventional TIL.

Topics: Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Cell Proliferation; Cell Separation; Cell Survival; Cells, Cultured; Cytotoxicity, Immunologic; Flow Cytometry; Humans; Immunotherapy, Adoptive; Interferon-gamma; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Neoplasm Proteins; Transforming Growth Factor beta1

Dysregulated protein phosphorylation is a hallmark of malignant transformation. Transformation can generate major histocompatibility complex (MHC)-bound phosphopeptides that are differentially displayed on tumor cells for specific recognition by T cells. To understand how phosphorylation alters the antigenic identity of self-peptides and how MHC class II molecules present phosphopeptides for CD4(+) T-cell recognition, we determined the crystal structure of a phosphopeptide derived from melanoma antigen recognized by T cells-1 (pMART-1), selectively expressed by human melanomas, in complex with HLA-DR1. The structure revealed that the phosphate moiety attached to the serine residue at position P5 of pMART-1 is available for direct interactions with T-cell receptor (TCR) and that the peptide N-terminus adopts an unusual conformation orienting it toward TCR. This structure, combined with measurements of peptide affinity for HLA-DR1 and of peptide-MHC recognition by pMART-1-specific T cells, suggests that TCR recognition is focused on the N-terminal portion of pMART-1. This recognition mode appears to be distinct from that of foreign antigen complexes but is remarkably reminiscent of the way autoreactive TCRs engage self- or altered self-peptides, consistent with the tolerogenic nature of tumor-host immune interactions.

Topics: Amino Acid Sequence; Antigen Presentation; Antigens, Neoplasm; Binding Sites; CD4-Positive T-Lymphocytes; Crystallography, X-Ray; HLA-DR1 Antigen; Humans; In Vitro Techniques; MART-1 Antigen; Melanoma; Models, Molecular; Molecular Sequence Data; Multiprotein Complexes; Neoplasm Proteins; Peptide Fragments; Phosphopeptides; Phosphorylation; Protein Conformation; Receptors, Antigen, T-Cell; Serine

PNL2 is a recently generated monoclonal antibody (mAb) that recognizes normal and neoplastic melanocytes. Although the antigen recognized by PNL2 remains unknown, recent studies of human and mouse melanomas have confirmed its usefulness as a diagnostic marker. In the current study, the immunoreactivity of PNL2 in canine melanomas was tested and compared with Melan A (A103). Validation of PNL2 was performed by Western blot analysis. PNL2 and Melan A immunoreactivity were tested on frozen samples of canine melanomas and on 69 formalin-fixed, paraffin-embedded melanocytic neoplasms. Normal canine tissues and nonmelanocytic neoplasms were included as negative controls. Western blot confirmed the presence of a protein recognized by the PNL2 antibody in canine melanomas. Immunohistochemically, PNL2 stained the melanocytic neoplastic cells with an intracytoplasmic, granular pattern. Among the melanocytic neoplasms tested, 62% stained positively with PNL2 and 59% with Melan A; 50.7% stained positively with both mAbs. The overall percentage of neoplasms that stained positively with at least 1 of these 2 antibodies was 68%. The extent of staining (i.e., the percentage of cells stained per specimen) was greater with PNL2 than with Melan A. With both mAbs, staining was most intense and diffuse in the epithelioid cell phenotype. Neither nonspecific staining nor staining in cells other than melanocytes was detected with either mAb. In contrast to human granulocytes, canine granulocytes were negative by both Western blot and immunohistochemical analyses. PNL2 mAb proved to be highly specific for the identification of formalin-fixed canine melanocytic neoplasms and should be a valuable diagnostic reagent.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Blotting, Western; Dog Diseases; Dogs; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; Skin Neoplasms

Melanoma is the major cause of skin cancer death worldwide. Parkinson's disease is a neurodegenerative disorder that is caused by mutation of alpha-synuclein or other genes. Importantly, epidemiological studies have reported co-occurrence of melanoma and Parkinson's disease, suggesting that these two diseases could share common genetic components.. Recently, we found that human melanoma cell lines highly express alpha-synuclein, whereas the protein is undetectable in the non-melanoma cancer cell lines tested. To investigate the expression of alpha-synuclein in human melanoma tissues, we immunostained sections of melanoma, nevus, non-melanocytic cutaneous carcinoma, and normal skin. alpha-Synuclein was positively detected in 86% of the primary and 85% of the metastatic melanoma sections, as well as in 89% of nevus sections. However, alpha-synuclein was undetectable in non-melanocytic cutaneous carcinoma and normal skin.. The Parkinson's disease-related protein, alpha-synuclein, is expressed in both malignant and benign melanocytic lesions, such as melanomas and nevi. Although alpha-synuclein cannot be used to distinguish between malignant and benign melanocytic skin lesions, it might be a useful biomarker for the diagnosis of metastatic melanoma.

Topics: Adult; Aged; alpha-Synuclein; Antigens, Neoplasm; Biomarkers, Tumor; Brain Neoplasms; Cell Line, Tumor; Female; Humans; Male; MART-1 Antigen; Melanins; Melanoma; Middle Aged; Neoplasm Proteins; Nevus; Parkinson Disease; Pigmentation; Retinoblastoma; Skin Neoplasms

Topics: Antigens, Neoplasm; Biopsy; Breast Neoplasms; Dermoscopy; Diagnosis, Differential; Female; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasm Proteins; Nipples; Paget's Disease, Mammary; S100 Proteins; Skin Neoplasms

The elucidation of protein biomarkers that are differentially expressed in human melanocytic tumors during tumor progression may lead to the identification of therapeutic targets and novel diagnostic tests. In a meeting chaired by Dr Mihm, a list of biomarkers of interest in melanoma was compiled. The specialized programs of research excellence (SPORE) in skin cancer developed a melanocytic tumor progression tissue microarray (TMA) to evaluate these candidate biomarkers. In addition to markers reported elsewhere, we evaluated c-Kit, MITF, MART1, HMB-45 and bcl-2.. The TMA contains 480 cores of benign nevi, primary cutaneous melanoma and melanoma metastases. Immunohistochemical detection of melanoma biomarkers, including c-Kit, MITF, MART-1, HMB-45 and bcl-2 was performed.. Intense nuclear staining for MITF protein was observed in 83% of nevi, 56% of primary melanomas and 23% of metastases. Bcl-2 expression was reduced with progression to metastasis (detected in 86, 89 and 52% of nevi, primaries and metastases, respectively), contrary to MART-1, which showed no differential expression (74, 85 and 84%). HMB-45 was observed in 18% of nevi and most (72 and 75%) primary melanomas and metastases. c-Kit protein increased with progression from nevi to primary tumor (10 and 77% of cases, respectively) and was decreased in metastases (26% of cases).. Through a collaboration of the Skin SPOREs sponsored by the Organ Systems Branch of the National Cancer Institute (NCI), we identified a list of melanoma biomarkers of interest, developed a melanocytic tumor progression TMA and completed a coordinated analysis of these biomarkers. This TMA has served as a powerful validation tool for newly identified and known melanoma biomarkers by revealing trends in expression during tumor progression and by confirming the heterogeneity of biomarker expression in cutaneous melanocytic tumors.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Cell Nucleus; Disease Progression; Humans; Immunohistochemistry; Inhibitor of Apoptosis Proteins; MART-1 Antigen; Melanocytes; Melanoma; Melanoma-Specific Antigens; Microphthalmia-Associated Transcription Factor; Microtubule-Associated Proteins; Neoplasm Proteins; Protein Array Analysis; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-kit; Skin Neoplasms; Survivin

Although it has been demonstrated that CTLs can be raised against tumor-associated self-antigens, achieving consistent and effective clinical responses has proven challenging. Superagonist altered peptide ligands (APLs) can often elicit potent antitumor CTL responses where the native tumor-associated epitope fails. Current methods have identified a limited number of superagonist APLs, including the prototypic 27L mutant of MART-1. However, more comprehensive screening strategies would be desirable. In this study, we use a novel genetic screen, involving recombinant technology and class I Ag cross-presentation, to search for supraoptimal superagonists of the 27L MART-1 mutant by surveying the effectiveness of virtually every single amino acid substitution mutant of 27L to activate human Ag-specific CTL clones recognizing the wild-type MART-1(26-35) epitope. We identify three novel mutant epitopes with superagonist properties that are functionally superior to 27L; however, the ability of a given analogue to act as superagonist varies among patients and suggests that a given superagonist APL may be ideally suited to different patients. These findings endorse the use of comprehensive methods to establish panels of potential superagonist APLs to individualize tumor peptide vaccines among patients.

Topics: Antigens, Neoplasm; Cell Line, Tumor; Epitopes, T-Lymphocyte; Genetic Techniques; Humans; MART-1 Antigen; Melanoma; Neoplasm Proteins; T-Lymphocytes, Cytotoxic

Mohs micrographic surgery (MMS) has increasingly become an accepted therapy for melanoma in situ on chronically sun damaged skin (CSDS). However, melanocytes are difficult to locate in frozen material on hematoxylin and eosin. In addition, determining the cut-off between the melanoma and the "atypical melanocytic hyperplasia" in CSDS can be challenging in frozen or formalin-fixed paraffin-embedded sections, with or without immunohistochemistry (IHC). In this article, we report the use of a rapid, 35-minute protocol using microphthalmia-associated transcription factor (MITF) IHC for identifying melanocytes in frozen tissue for its potential use in MMS. In contrast to melanoma antigen recognized by T cells (MART-1), MITF is a nuclear stain, which simplifies identification of melanocytes and quantification of melanocytic parameters. In this study, MITF IHC in frozen sections yielded equivalent melanocyte nuclear diameter and density measurements compared with formalin-fixed paraffin-embedded sections. Nuclear diameter measurements obtained with MITF were similar to that previously reported with MART-1, but the melanocyte density figures were lower. Reliable labeling of melanocytes in frozen sections required the use of diaminobenzidine (DAB) chromogen with Giemsa counterstaining and a buffer devoid of surfactant. Our experience with MITF IHC indicates that it is a dependable immunostain in frozen sections, and may prove to be useful in MMS as an adjunct to hematoxylin and eosin and MART-1 IHC for interpretation of margins for melanoma in situ on CSDS.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Frozen Sections; Humans; Immunohistochemistry; MART-1 Antigen; Melanocytes; Melanoma; Microphthalmia-Associated Transcription Factor; Mohs Surgery; Neoplasm Proteins; Skin Neoplasms

To date there is no effective therapy for metastatic melanoma and at the molecular level the disease progression is poorly understood. A recent study by our group led to the development of a novel phenotype switching model for melanoma progression, wherein cells transition back-and-forth between states of proliferation and invasion to drive disease progression. To explore the model's clinical relevance we interrogated phenotype-specific expression patterns in human melanoma patient material. A matched primary/metastasis pair from a human melanoma patient was obtained and immunohistochemically stained for proliferative and invasive phenotype markers. These were also stained for hypoxia and blood vessel markers. Proliferative phenotype markers Melan-A and Mitf showed consistent anti-correlation with invasive phenotype marker Wnt5A and hypoxia marker Glut-1. These also correlated with observed intra-tumoural vascularization patterns. Similar pattern distributions were present in both primary and metastasis samples. Strikingly, we observed that late phase metastatic melanoma cells adopt morphologies and behaviours identical to very early phase cells. The expression patterns observed closely matched expectations derived from previous in vitro and xenografting experiments. These results highlight the likelihood that disease progression involves melanoma cells retaining the capacity to regulate the expression of metastatic potential critical factors according to changing microenvironmental conditions.

Topics: Antigens, Neoplasm; Disease Progression; Female; Gallbladder Neoplasms; Gene Expression; Gene Expression Profiling; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Microphthalmia-Associated Transcription Factor; Middle Aged; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; Phenotype; Skin Neoplasms

Targeted therapy against the BRAF/mitogen-activated protein kinase (MAPK) pathway is a promising new therapeutic approach for the treatment of melanoma. Treatment with selective BRAF inhibitors results in a high initial response rate but limited duration of response. To counter this, investigators propose combining this therapy with other targeted agents, addressing the issue of redundancy and signaling through different oncogenic pathways. An alternative approach is combining BRAF/MAPK-targeted agents with immunotherapy. Preliminary evidence suggests that oncogenic BRAF (BRAF(V600E)) contributes to immune escape and that blocking its activity via MAPK pathway inhibition leads to increased expression of melanocyte differentiation antigens (MDA). Recognition of MDAs is a critical component of the immunologic response to melanoma, and several forms of immunotherapy capitalize on this recognition. Among the various approaches to inhibiting BRAF/MAPK, broad MAPK pathway inhibition may have deleterious effects on T lymphocyte function. Here, we corroborate the role of oncogenic BRAF in immune evasion by melanoma cells through suppression of MDAs. We show that inhibition of the MAPK pathway with MAPK/extracellular signal-regulated kinase kinase (MEK) inhibitors or a specific inhibitor of BRAF(V600E) in melanoma cell lines and tumor digests results in increased levels of MDAs, which is associated with improved recognition by antigen-specific T lymphocytes. However, treatment with MEK inhibitors impairs T lymphocyte function, whereas T-cell function is preserved after treatment with a specific inhibitor of BRAF(V600E). These findings suggest that immune evasion of melanomas mediated by oncogenic BRAF may be reversed by targeted BRAF inhibition without compromising T-cell function. These findings have important implications for combined kinase-targeted therapy plus immunotherapy for melanoma.

Topics: Antigens, Neoplasm; Benzamides; Butadienes; Cell Line, Tumor; Combined Modality Therapy; Diphenylamine; Epitopes, T-Lymphocyte; Extracellular Signal-Regulated MAP Kinases; gp100 Melanoma Antigen; Humans; Intramolecular Oxidoreductases; MAP Kinase Signaling System; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Nitriles; Oxidoreductases; Proto-Oncogene Proteins B-raf; T-Lymphocytes

We assessed molecular (presence of melanoma cells markers in lymph fluid [LY]) and pathological features (sentinel lymph node [SN] tumor burden according to Rotterdam criteria, metastases microanatomic location) and correlated them with survival and melanoma prognostic factors in a group of patients with positive SN biopsy.. We analyzed 368 consecutive SN-positive patients after completion lymph node dissection (CLND). In 321 patients we obtained data on SLN microanatomic location/tumor burden (only 7 cases had metastases <0.1 mm); in 137 we additionally analyzed 24-hour collected LY after CLND (multimarker reverse transcriptase-polymerase chain reaction [MM-RT-PCR] with primers for tyrosinase, MART1 (MelanA), and uMAGE mRNA (27.7% positive samples)]. Median follow-up time was 41 months.. According to univariate analysis, the following factors had a negative impact on overall survival (OS): higher Breslow thickness (P = .0001), ulceration (P < .0001), higher Clark level (P = .008), male gender (P = .0001), metastatic lymph nodes >1 (P < .0001), nodal metastases extracapsular extension (P < .0001), metastases to additional non-SNs (P = .0004), micrometastases size ≥ 0.1 mm (P = .0006), and positive LY MM-RT-PCR (P = .0007). SN tumor burden showed linear correlation with increasing Breslow thickness (P = .01). The 5-year OS rates for SLN tumor burden <0.1 mm, 1-1.0 mm, and >1.0 mm were 84%/66%/44%, respectively, and for positive and negative LY MM-RT-PCR 47%/0%, respectively. The independent factors for shorter OS (multivariate analysis): male gender, primary tumor ulceration, number of involved nodes ≥ 4, micrometastases size >1.0 mm, and, in additional model including molecular analysis-positive MM-RT-PCR results (hazard ratio [HR] 3.2), micrometastases size >1.0 mm (HR 1.13), and primary tumor ulceration (HR 2.17). Similar results were demonstrated for disease-free survival (DFS) data.. SN tumor burden categories according to Rotterdam criteria and the positive result of LY MM-RT-PCR assay demonstrated additional, independent prognostic value in SN-positive melanoma patients, showing significant correlation with shorter DFS and OS.

Topics: Adult; Antigens, Neoplasm; Biomarkers, Tumor; Female; Follow-Up Studies; Humans; Lymph; Lymph Node Excision; Lymph Nodes; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sentinel Lymph Node Biopsy; Skin Neoplasms; Survival Rate; Tumor Burden

Peripheral blood lymphocytes (PBL) genetically modified to express T cell receptors (TCR) specific to known melanoma antigens, such as melanoma antigen recognized by T cells-1 (MART-1), and gp100 can elicit objective tumor regression when administered to patients with metastatic melanoma. It has also been demonstrated that modifications within the constant regions of a fully human TCR can enhance surface expression and stability without altering antigen specificity. In this study, we evaluated the substitution of murine constant regions for their human counterpart within the DMF5 MART-1-specific TCR. Unlike previous studies, all modified TCRs were inserted into retroviral vectors and analyzed for expression and function following a clinical transduction protocol. PBL were transduced with retroviral supernatant generated from stable packaging lines encoding melanoma-specific TCRs. This protocol resulted in high levels of antigen-specific T cells without the need for additional peptide stimulation and selection. Both the human and murinized TCR efficiently transduced PBL; however, the murinized TCR exhibited significantly higher tetramer binding, mean fluorescence intensity, as well as, increased in vitro effector function following our clinical transduction and expansion protocol. Additional TCR modifications including insertion of a second disulfide bond or the linker modifications evaluated herein did not significantly enhance TCR expression or subsequent in vitro effector function. We conclude that the substitution of a human constant region with a murine constant region was sufficient to increase receptor expression and tetramer binding as well as antitumor activity of the DMF5 TCR and could be a tool to augment other antigen-specific TCR.

Topics: Animals; Antigens, Neoplasm; Cell Line; Cell Line, Tumor; Cell Proliferation; Genetic Vectors; Humans; Leukocytes, Mononuclear; MART-1 Antigen; Melanoma; Mice; Neoplasm Proteins; Plasmids; Receptors, Antigen, T-Cell; Recombinant Proteins; Transduction, Genetic; Up-Regulation

Targeted delivery of tumor antigen genes to dendritic cells (DCs) using adenoviral (Ad) vectors holds great potential for cancer immunotherapy. We previously showed that CD40 targeting of Ad vectors enhanced specific transduction of DC in human skin, while simultaneously ensuring their stable maturation and superior allogeneic T-cell stimulatory capacity. In this study, we evaluated whether CD40-targeted Ad encoding the full-length melanoma antigen recognized by T cells-1 (CD40-Ad-MART-1) could be used to efficiently and selectively transduce conventional and plasmacytoid DC to prime melanoma-specific CD8(+) T-effector cells in human melanoma-draining sentinel lymph nodes (SLNs). CD40 targeting of Ad was achieved using a bispecific fusion protein, binding and neutralizing the Ad fiber knob through soluble coxsackie and adenovirus receptor while retargeting the virus to hCD40 through the tumor necrosis factor-like domain of mCD40L. Selective transduction of conventional and plasmacytoid DC subsets by CD40-Ad was observed in suspensions of human melanoma-draining SLN. Moreover, CD40-Ad-MART-1 enhanced the expansion of functional MART-1-specific CD8(+) T cells from SLN with concomitant decreases in CD4:CD8 T-cell ratios and CD4(+)CD25(hi)FoxP3(+) regulatory T-cell rates. Additional studies revealed that transduction and activation of monocyte-derived DCs with CD40-Ad-MART-1 significantly enhanced their priming efficiency of functional CD8(+) effector T cells with high avidity. These findings provide preclinical evidence of possible efficacy of this approach for cancer immunotherapy.

Topics: Adenoviridae; CD4 Antigens; CD40 Antigens; CD40 Ligand; Cytotoxicity, Immunologic; Dendritic Cells; Forkhead Transcription Factors; Humans; Immunotherapy; Interferon-gamma; Interleukin-2 Receptor alpha Subunit; K562 Cells; Lymph Nodes; MART-1 Antigen; Melanoma; Protein Engineering; Recombinant Fusion Proteins; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory; Transduction, Genetic

Adoptive cell transfer using autologous tumor infiltrating lymphocytes or lymphocytes transduced with antitumor T-cell receptor (TCR) is an effective therapy for patients with metastatic melanoma. A limiting factor in the effectiveness of this treatment is the apoptosis of the transferred cells when Interleukin-2 (IL-2) administration is withdrawn. In an attempt to improve persistence of the transferred lymphocytes, we cotransduced human peripheral blood lymphocytes with retroviruses encoding Bcl-2 or Bcl-xL, antiapoptotic genes of the BCL2 family, and the MART-1 melanoma tumor antigen-specific TCR, DMF5. Lymphocytes were cotransduced with 38% to 64% cotransduction efficiency, and exhibited a marked delay in apoptosis after IL-2 withdrawal. Cotransduction with Bcl-2 or Bcl-xL did not affect cytokine secretion or lytic ability of the DMF5-transduced lymphocytes. After 5 days of IL-2 withdrawal, cotransduced lymphocytes produced similar levels of IFN-γ per cell as DMF5-alone transduced lymphocytes in response to tumor cells. Cotransduction did not alter the phenotype of lymphocytes with respect to a panel of T-cell differentiation markers. In a mouse model of melanoma, adoptively transferred T cells transduced with Bcl-2 persisted better in vivo at the site of tumor, 13 and 21 days after adoptive transfer (P=0.0064 and 0.041, respectively), with evidence of enrichment of the Bcl-2-transduced population over time (P<0.0001). Thus, by coexpressing Bcl-2 or Bcl-xL with a tumor-specific TCR, we have engineered a lymphocyte that resists apoptosis owing to IL-2 withdrawal without altering its tumor-specific function or phenotype, and thus may show improved antitumor effectiveness in vivo after cell transfer.

Topics: Adoptive Transfer; Animals; Antigens, Differentiation; Apoptosis; Cancer Vaccines; Cell Survival; Genetic Engineering; Humans; Immunotherapy, Adoptive; Interferon-gamma; Interleukin-2; MART-1 Antigen; Melanoma; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Proto-Oncogene Proteins c-bcl-2; Receptors, Antigen, T-Cell; Retroviridae; T-Lymphocytes

Nail bed melanoma or subungual melanoma is frequently misdiagnosed compared to other melanoma in other anatomic sites. We report the case of a 48-year-old woman presenting with a dystrophic and pigmented lesion of her fourth finger nail. This initial presentation had been mistaken for onychomycosis, but biopsy of nail bed and nail matrix confirmed nail bed melanoma. This case is presented to help increase the awareness of atypical presentations of acral melanoma.

Topics: Amputation, Surgical; Diagnostic Errors; Female; Humans; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Nail Diseases; Onychomycosis; Skin Neoplasms

To assess the functional significance of intraocular tumor-associated lymphatic vessels in ciliary body melanomas with extraocular extension.. Twelve consecutive patients enucleated for a malignant melanoma of the ciliary body with extraocular extension and immunohistochemical presence of intraocular LYVE-1-positive and podoplanin-positive lymphatic vessels were examined for proliferation status and tumor invasion into tumor-associated lymphatics. Proliferating lymphatic vessels were identified using LYVE-1 and podoplanin as specific lymphatic endothelial markers and Ki-67 as the proliferation marker. Tumor invasion into lymphatic vessels was assessed using Melan-A as the melanoma marker. Kaplan-Meier analyses of survival and metastasis were performed.. Intraocular proliferating lymphatic vessels were detected in all 12 ciliary body melanomas with extraocular extension. The ratio of proliferating lymphatics was significantly higher in the intraocular vs extraocular tumor compartment (P < .001). Extraocular lymphatic invasion by tumor cells was observed in 5 patients (42%), intraocular lymphatic invasion in 4 (33%), and synchronous intraocular and extraocular lymphatic invasion in 3 (25%). Detection of melanoma cells in intraocular and extraocular lymphatic vessels was significantly associated with higher risks of lymphatic spread (P < .001) and lower metastasis-free survival rates (P = .03).. Intraocular tumor-associated lymphatic vessels contain proliferating endothelial cells and can be invaded by cancer cells in ciliary body melanomas with extraocular extension. Lymphatic invasion by tumor cells seems to be associated with an increased risk of lymphatic spread and mortality in these affected patients.

Topics: Aged; Antigens, Neoplasm; Biomarkers, Tumor; Ciliary Body; Eye Enucleation; Female; Humans; Immunoenzyme Techniques; Ki-67 Antigen; Lymphangiogenesis; Lymphatic Metastasis; Lymphatic Vessels; Male; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Middle Aged; Neoplasm Invasiveness; Neoplasm Proteins; Uveal Neoplasms; Vesicular Transport Proteins

Combination of chemotherapy and immunotherapy to increase the effectiveness of an antitumor immune response is currently regarded as an attractive antitumor strategy. In a pilot clinical trial, we have recently documented an increase of melanoma antigen A (Melan-A)-specific, tumor-reactive, long-lasting effector-memory CD8(+) T cells after the administration of dacarbazine (DTIC) 1 day before peptide vaccination in melanoma patients. Global transcriptional analysis revealed a DTIC-induced activation of genes involved in the immune response and leukocyte activation. To identify the possible mechanisms underlying this improved immune response, we have compared the endogenous and the treatment-induced anti-Melan-A response at the clonal level in patients treated with the vaccine alone or with DTIC plus vaccine. We report a progressive widening of T-cell receptor (TCR) repertoire diversity, accompanied by high avidity and tumor reactivity, only in Melan-A-specific T-cell clones of patients treated with chemoimmunotherapy, with a trend toward longer survival. Differently, patients treated with vaccine alone showed a tendency to narrowing the TCR repertoire diversity, accompanied by a decrease of tumor lytic activity in one patient. Collectively, our findings indicate that DTIC plus vaccination shapes the TCR repertoire in terms of diversity and antitumor response, suggesting that this combined therapy could be effective in preventing melanoma relapse.

Topics: Antibody Affinity; Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Combined Modality Therapy; Dacarbazine; Epitopes, T-Lymphocyte; Humans; K562 Cells; MART-1 Antigen; Melanoma; Neoplasm Proteins; Receptors, Antigen, T-Cell; T-Lymphocytes, Cytotoxic; Vaccines, Subunit

Topics: Adrenal Gland Neoplasms; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Breast Neoplasms; Cisplatin; Dacarbazine; Fatal Outcome; Female; Humans; Interferon-beta; Iofetamine; Liver Neoplasms; Lung Neoplasms; MART-1 Antigen; Mastectomy, Modified Radical; Melanoma; Middle Aged; Neoplasm Staging; Nimustine; Nipples; Radiopharmaceuticals; S100 Proteins; Skin Neoplasms; Tamoxifen; Tomography, Emission-Computed, Single-Photon; Vincristine

Adoptive immunotherapy for treatment of cancers and infectious diseases is often hampered by a high degree of variability in the final T cell product and in the limited in vivo function and survival of ex vivo expanded antigen-specific cytotoxic T cells (CTL). This has stimulated interest in development of standardized artificial antigen presenting cells (aAPC) to reliably expand antigen specific CTL. However, for successful immunotherapy the aAPC ex vivo generated CTL must have anti-tumor activity in vivo. Here, we demonstrate that HLA-Ig based aAPC stimulated tumor-specific CTL from human peripheral blood T lymphocytes showed robust expansion and functional activity in a human/SCID mouse melanoma model. HLA-Ig based aAPC expanded CTL were detected in the peripheral blood up to 15 days after transfer. Non-invasive bioluminescence imaging of tumor bearing mice demonstrated antigen dependent localization of transferred CTL to the tumor site. Moreover, adoptive transfer of HLA-Ig based aAPC generated CTL inhibited the tumor growth both in prevention and treatment modes of therapy and was comparable to that achieved by dendritic cell expanded CTL. Thus, our data demonstrate potential therapeutic in vivo activity of HLA-Ig based aAPC expanded CTL to control tumor growth.

Topics: Animals; Antigen-Presenting Cells; Antigens, Neoplasm; Cell Proliferation; Female; Flow Cytometry; HLA-A2 Antigen; Humans; Immunotherapy, Adoptive; MART-1 Antigen; Melanoma; Mice; Mice, Knockout; Mice, SCID; Neoplasm Proteins; Peptides; T-Lymphocytes, Cytotoxic

The aim of this study was to evaluate the prognostic potential of quantitative reverse-transcription, polymerase chain reaction (qRT-PCR) in melanoma patients with pathologically negative sentinel lymph nodes (SLN). Our study included 195 node-negative melanoma patients with a Breslow thickness greater than 0.76 mm (n = 158), or less than 0.76 mm but who had Clark level IV-V, microscopic ulceration, or pathological signs of regression (n = 32), and five patients with melanoma of unknown thickness. SLNs were examined by serial-section histopathology. A portion of each SLN was frozen for qRT-PCR analysis using markers Tyrosinase, MART1, SSX2, MAGEA3, PAX3, and GalNAc-T. In addition, two other markers (PLAB and L1CAM) were evaluated for melanoma specificity but not for SLN analysis. Median follow-up was 64 months, during which time there were 15 (7.7%) recurrences. A total of 370 lymph nodes were analyzed by qRT-PCR. No association was found between quantitative expression level of any marker and disease recurrence. Previously published primer designs were tested for PAX3 and GalNAc-T and revealed that alternative PAX3 transcripts are differentially expressed in melanoma and benign lymph nodes. No associations with recurrence were found regardless of the transcripts amplified by different primer sets. PLAB and L1CAM did not appear to differentiate between malignant melanoma and benign melanocytes or lymph nodes in our analysis. We conclude that, in this large cohort of patients, multimarker qRT-PCR analysis of SLNs did not correlate with disease recurrence. Our data support specific PAX3 splice variants but not GalNAc-T, PLAB or L1CAM as possible markers for melanoma metastasis to SLNs.

Topics: Antigens, Neoplasm; Area Under Curve; Biomarkers, Tumor; Female; Growth Differentiation Factor 15; Humans; Lymph Nodes; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; N-Acetylgalactosaminyltransferases; Neoplasm Proteins; Neoplasm Staging; Neural Cell Adhesion Molecule L1; Paired Box Transcription Factors; PAX3 Transcription Factor; Polypeptide N-acetylgalactosaminyltransferase; Prognosis; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sentinel Lymph Node Biopsy; Skin Neoplasms

It is well established that cytotoxic T lymphocytes (CTL) can kill target cells offering a very small number of specific peptide/MHC complexes (pMHC). It is also known that lethal hit delivery is a very rapid response that occurs within a few minutes after cell-cell contact. Whether cytotoxicity is efficient and rapid in the context of CTL interaction with target cells derived from solid tumours is still elusive. We addressed this question by visualizing the dynamics of human CTL interaction with melanoma cells and their efficiency in eliciting cytotoxicity. Our results show that in spite of CTL activation to lethal hit delivery, killing of melanoma cells is not efficient. Time-lapse microscopy experiments demonstrate that individual CTL rapidly polarize their lytic machinery towards target cells, yet the apoptotic process in melanoma cells is defective or 'delayed' as compared to conventional targets. These results indicate that although CTL activation to lethal hit delivery can be viewed as a 'digital' phenomenon rapidly triggered by a few ligands, melanoma cell annihilation is an 'analogue' response requiring multiple hits and prolonged contact time.

Topics: Apoptosis; Cell Proliferation; Cell Separation; Cytotoxicity, Immunologic; Flow Cytometry; Fluoresceins; Immunotherapy; Ligands; Major Histocompatibility Complex; MART-1 Antigen; Melanoma; Microscopy, Confocal; Peptides; T-Lymphocytes, Cytotoxic; Time Factors

Multimarker reverse transcriptase-polymerase chain reaction (RT-PCR) was originally reported to reveal melanoma-associated mRNAs (MAMs) in melanoma cells but not in the peripheral blood of healthy individuals.. To evaluate the expression of MAMs in the peripheral blood of melanoma patients at different American Joint Committee on Cancer (AJCC) stages, and to correlate their presence with early and/or advanced stages of the disease.. One hundred blood samples of melanoma patients (AJCC I-IV) were analysed using multimarker RT-PCR to assess the co-expression of Tyr-OH, MART-1, MAGE-3, MUC-18/MCAM and p97. Patients were stratified into two disease categories: early and advanced stages. The former includes in situ and melanoma stages AJCC I-II, the latter AJCC III-IV. chi(2) and Fisher's exact tests were used to statistically evaluate the association between each MAM and disease categories. The recognized significant associations were subsequently resubmitted to univariate logistic regression. Furthermore, sensitivity and specificity were established.. At least one MAM could be detected in 24% of our series. Tyr-OH was the most common marker (14%), followed by MUC-18 (12%), MART-1 (5%), MAGE-3 (4%) and p97 (3%). No significant association among Tyr-OH, MART-1, MAGE-3, p97 and disease stages were evidenced. Only MUC-18 was statistically associated (P < 0.009) with advanced stages alone or co-expressed with other MAMs. According to logistic regression univariate analysis, MUC-18 increases the probability (odds ratio: 33) being in advanced stages and the incidence of recurrences (95% CI 2.9-374).. MUC-18 RT-PCR assay could be proposed as an adjunctive molecular method in the management of melanoma patients and is useful in the monitoring of study protocols.

Topics: Adult; Aged; Antigens, Neoplasm; Biomarkers, Tumor; CD146 Antigen; Cell Line, Tumor; Female; Humans; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasm Proteins; Neoplasm Staging; Prospective Studies; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sensitivity and Specificity; Skin Neoplasms

The pDERMATT (plasmid DNA encoding recombinant MART-1 and tetanus toxin fragment-c) plasmid is a novel in-house developed anti-cancer vaccine which encodes a melanoma associated epitope (Mart-1) and an immuno stimulatory sequence (tetanus toxin fragment-c). The pharmaceutical development of pDERMATT necessitated the availability of an assay for the quantification and purity determination of pDERMATT active pharmaceutical ingredient (API), the produced bulk drug and its pharmaceutical dosage form. An anion-exchange liquid chromatographic method (AEX-LC) with ultraviolet (UV) detection was developed, which is based on separation on a non porous anion-exchange (NPR AEX) column with a mobile phase gradient of 0.45-0.53M NaCl in 20mM Tris-HCl 10% isopropanol (IPA) pH 9 and UV detection at 260 and 280nm. The method was found to be precise, accurate and linear over a concentration range of 5-150microg/ml. The supercoiled topoisoform of pDERMATT was well separated from the linear and open-circular form, the main degradation products formed during stress testing, confirming its stability-indicating capability. The use of photo diode array (PDA) detection enabled us to confirm all visible peaks to contain DNA. Additionally capillary gel electrophoresis (CGE) showed the same peak profile as the developed HPLC method. The developed LC-UV method will be used for the pharmaceutical quality control of pDERMATT API, bulk drug and its pharmaceutical dosage form.

Topics: Anions; Antigens, Neoplasm; Calibration; Cancer Vaccines; Chromatography, Ion Exchange; DNA; DNA, Circular; Dosage Forms; Drug Stability; Electrophoresis, Capillary; Epitopes; Escherichia coli; Fermentation; Freeze Drying; Guidelines as Topic; MART-1 Antigen; Melanoma; Neoplasm Proteins; Peptide Fragments; Pharmaceutical Preparations; Plasmids; Quality Control; Recombinant Proteins; Reference Standards; Reproducibility of Results; Restriction Mapping; Sensitivity and Specificity; Sequence Analysis, DNA; Solutions; Spectrophotometry, Ultraviolet; Tetanus Toxin

Animal-type melanoma (ATM) is a rare variant of the tumor showing diffuse, heavily pigmented neoplastic cells in the dermis. Despite the high mean thickness of the lesions, reports seem to indicate a less aggressive behavior and a better survival rate for ATM compared with conventional melanoma, but the underlying pathways related to this favorable outcome are still unknown.. Five women and 2 men aged 20 to 92 years presented with pigmented skin nodules (n = 5) or plaques (n = 2), varying in size from 1.0 to 4.5 cm. Findings from microscopic examination showed monotypic-appearing melanocytes with abundant intracytoplasmic melanin in a nodular or fascicular arrangement (mean Breslow thickness, 4.97 mm). Immunohistochemical analysis of ATM cells demonstrated the typical positive staining for S-100, vimentin, HMB-45, and melan-A. The investigation of the pi isoform of glutathione S-transferase, a family of enzymes involved in tumor progression, revealed that nuclear expression is reduced in ATMs compared with control melanomas, whereas results from cytoplasmic staining did not vary. One patient died of cardiac failure without evidence of disease progression; the remaining patients are disease-free at 3 (n = 4) and 5 years (n = 3).. Our findings confirm that ATM is a variant of melanoma with distinctive clinical and histological features. Low nuclear expression of glutathione S-transferase pi expression is a characteristic of ATM and could add new insight to better understand the unusual biological behavior of this rare neoplasm.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Cell Nucleus; Female; Glutathione S-Transferase pi; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasm Proteins; S100 Proteins; Skin Neoplasms; Vimentin; Young Adult

Regulatory T cells (Treg) may inhibit monocyte-derived melanoma antigen-pulsed dendritic cells (DC) vaccination in treatment of melanoma. However, the Treg level in peripheral blood mononuclear cells (PBMCs) following DC vaccination has not been examined in melanoma patients in Japan.. To evaluate differences in the helper T cell and Treg population and mRNA levels of Treg in pre- and post-DC vaccination PBMCs obtained from melanoma patients.. Levels of intracellular forkhead box protein 3 (Foxp3) mRNA as well as levels of CD4(+)CD25(+)Foxp3(+) and CD4(+)CD25(+) T cells were examined by real-time PCR and flow cytometry using PBMCs from 9 patients who received DC vaccination.. Eight of the 9 cases and 7 of the 9 cases showed increased populations of CD4(+)CD25(+) T cells and CD4(+)CD25(+)Foxp3(+) T cells, respectively after repeated DC vaccination. Five of 8 cases showed an increase of Foxp3 mRNA after treatment. Four of these 5 cases also had increased CD4(+)CD25(+) and CD4(+)CD25(+)Foxp3(+) T cells, but the fifth case showed a decrease in CD4(+)CD25(+)Foxp3(+) T cells. Three cases showed a decrease of Foxp3 mRNA. One of these 3 cases showed decreased population of CD4(+)CD25(+)Foxp3(+) T cells, but two cases showed increased population of CD4(+)CD25(+)Foxp3(+) T cells. In 3 of 8 cases Foxp3 expression at the cellular (protein) and mRNA level were inconsistent.. Repeated DC vaccination may commonly induce Treg and helper T cells at the cellular level. However, there are a few discrepancies of Treg expression at cellular and mRNA level.

Topics: Aged; Antigens, Neoplasm; Cancer Vaccines; Dendritic Cells; Female; Forkhead Transcription Factors; Humans; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Skin Neoplasms; T-Lymphocytes, Helper-Inducer; T-Lymphocytes, Regulatory; Vaccination

The use of melanoma-associated antigen recognized by T cells (MART-1) immunostain has been proposed as a useful adjunct to overcome the inherent difficulties in the use of frozen sections during Mohs surgery for the treatment of melanoma, but no studies have compared MART-1 frozen sections with MART-1 permanent sections. Current MART-1 1-hour protocols add significant time to the procedure.. To determine whether there is a significant difference between frozen and permanent MART-1 immunostained sections using a rapid 19-minute protocol.. Frozen and permanent sections stained with MART-1 were made from dog-ears excised during 25 reconstructions. A rapid 19-minute protocol was used to stain the frozen tissue. The sections were examined blinded, and statistical analysis was performed to analyze the data.. No significant difference was found in number of keratinocytes, nuclear diameter of keratinocytes, number of melanocytes, melanocytic nuclear diameter, confluence, pagetoid spread, melanocytic nesting, or atypical melanocytes.. The 19-minute protocol is a rapid and effective MART-1 immunostain. Frozen sections stained with MART-1 provide information equivalent to that obtained from MART-1 stained permanent sections. Mohs surgeons using MART-1 can feel confident that they have the same information as they would have obtained using permanent sections using the slow Mohs method.

Topics: Adult; Aged; Aged, 80 and over; Animals; Antigens, Neoplasm; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Dogs; Female; Frozen Sections; Humans; Hutchinson's Melanotic Freckle; Immunohistochemistry; Keratinocytes; Male; MART-1 Antigen; Melanocytes; Melanoma; Middle Aged; Mohs Surgery; Neoplasm Proteins; Skin; Skin Neoplasms

Immunotherapy is well-practiced as one of the main adjuvant therapies for melanoma patients. Until now, many immunotherapeutic investigations have focused on improving the effector side of the antitumor response, but only a few studies have been concerned with preventing the loss of tumor-associated antigen (TAA) expression. Loss of TAA should be an important problem for the recognition of tumor cells by cytotoxic T lymphocytes. If any agents that augment the expression of melanoma antigens were found, they could improve the efficacy of immunotherapy by increasing the antigens. To detect effective chemicals, we made a fluorescent cellular reporter system for screening promising candidate chemicals. In this system, the fusion gene of the Melan-A/MART-1 promoter sequence followed by the green fluorescent protein (GFP) coding region was stably transfected into MUX human melanoma cells which are known to express little or no Melan-A/MART-1. Melan-A/MART-1 is a well-known melanoma antigen recognized by autologous cytotoxic T cells, and is a glycoprotein associated with the melanosome, the organelle in which melanin synthesis proceeds. By using this screening system, daunorubicin, doxorubicin and cytochalasin D, which enhanced the green fluorescent, were selected and then were confirmed to actually increase the expression of Melan-A/MART-1 mRNA and protein in human melanoma cells of MU89, MM96L(+) and SK-MEL-28, but also in low-antigen presenting cells such as MM96L(-), MUX, and A375. In conclusion, we have successfully established a well-functioning screening system, which will allow us to find candidate chemicals that up-regulate or maintain the melanoma antigen expression.

Topics: Antigens, Neoplasm; Cell Line, Tumor; Cloning, Molecular; Cytochalasin D; Daunorubicin; DNA Primers; Doxorubicin; Flow Cytometry; Gene Expression Regulation, Neoplastic; Green Fluorescent Proteins; Humans; Immunotherapy; MART-1 Antigen; Melanoma; Neoplasm Proteins; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction

Assessment of T-cell diversity, besides giving insights about the molecular basis of tumor antigen recognition, has clinical implications since it provides criteria for evaluating antigen-specific T cells clinically relevant for spontaneous and vaccine-induced anti-tumor activity. Melan-A is one of the melanoma antigens most frequently recognized by peripheral and tumor-infiltrating lymphocytes in HLA-A2+ melanoma patients. Many clinical trials involving anti-tumor vaccination have been conducted using modified versions of this peptide.. We conducted an in-depth characterization of 210 T-cell receptor beta chain (TRB) clonotypes derived from T cells of HLA-A2+ melanoma patients displaying cytotoxic activity against natural and A27L-modified Melan-A peptides. One hundred and thirteen Melan-A-specific clonotypes from melanoma-free subjects, 199 clonotypes from T-cell clones from melanoma patients specific for melanoma antigens other than Melan-A, and 305 clonotypes derived from T cells of HLA-A2+ individuals showing unrelated specificities, were used as control. After sequence analysis, performed according to the IMGT definitions, TRBV and TRBJ usage, CDR3 length and amino acid composition were compared in the four groups of clonotypes.. TRB sequences of Melan-A-specific clonotypes obtained from melanoma patients were highly heterogeneous, but displayed a preferential usage of few TRBV and TRBJ segments. Furthermore, they included a recurrent "public" amino acid motif (Glycine-Leucine-Glycine at positions 110-112-113 of the CDR3) rearranged with dominant TRBV and TRBJ segments and, in one case, associated with a full conservation of the entire TRB sequence.. Contrary to what observed for public anti-Melan-A T-cell receptor alpha motifs, which had been identified in several clonotypes of both melanoma patients and healthy controls, the unexpectedly high contribution of a public TRB motif in the recognition of a dominant melanoma epitope in melanoma patients may provide important information about the biology of anti-tumor T-cell responses and improve monitoring strategies of anti-tumor vaccines.

Topics: Amino Acid Motifs; Amino Acid Sequence; Antigens, Neoplasm; Base Sequence; Clone Cells; Complementarity Determining Regions; HLA-A2 Antigen; Humans; MART-1 Antigen; Melanoma; Molecular Sequence Data; Neoplasm Proteins; Receptors, Antigen, T-Cell, alpha-beta; T-Lymphocytes

The histopathologic diagnosis of desmoplastic melanoma (DM) is usually based on typical conventional microscopic findings coupled with expression of S100 protein by neoplastic cells. Important differential diagnostic considerations include atypical fibroxanthoma (AFX) and spindle cell squamous carcinoma. Spindle cell squamous cell carcinoma is characterized by positivity for cytokeratin, whereas the diagnosis of AFX has been one of exclusion. Procollagen 1 (PC1) has been identified as a relatively sensitive marker of AFX. In this study, we sought to analyze the expression of PC1, S100 protein, and Melan-A in a series of 37 DMs (27 males and 10 females; ages 36-92 years, mean age 74 years). All lesions displayed a spindle cell morphology with varying degrees of desmoplasia. The neoplastic cells avidly expressed S100 protein in 37 of 37 neoplasms. A complete lack of PC1 expression was noted in 24 of 37 (64.9%). There was a weak PC1 expression by 9 DMs (24.3%) and a moderate expression by 4 DMs (10.8%). Melan-A expression was found in 19 of 37 DMs (51.4%), but in 10 lesions, the expression was only faint and focal. We conclude that PC1 labeling of DM is not uncommon but poses little risk for misdiagnosis, provided the stain is performed as part of panel that includes S100 protein. Melan-A offers little for the diagnosis of DM, as less than a quarter of lesions exhibit a strong reaction with this antibody. It is critical to employ a broad panel of antibodies, including S100 protein, Melan-A, cytokeratin, PC1, and others, in the immunohistochemical evaluation of spindle cell neoplasms of the skin.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Biomarkers, Tumor; Biopsy; Diagnosis, Differential; Female; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Procollagen; S100 Proteins; Sensitivity and Specificity; Skin Neoplasms

Topics: Aged; Antigens, Neoplasm; Antineoplastic Agents; Combined Modality Therapy; Dysphonia; Female; Humans; Interferon alpha-2; Interferon-alpha; Laryngeal Neoplasms; MART-1 Antigen; Melanoma; Mitomycin; Neoplasm Proteins; Neoplasm Recurrence, Local; Neoplasm Staging; Phenotype; Recombinant Proteins; S100 Proteins; Skin Neoplasms; Vocal Cords

We encountered recently 3 cases with a histopathologic diagnosis of melanoma in situ on sun-damaged skin (male = 2, female = 1; median age: 59 years; range: 52-60 years). The diagnosis was based mainly on the finding of actinic elastosis in the dermis and increased number of melanocytes in the epidermis and was confirmed by strong positivity for Melan-A in single cells and in small nests ("pseudomelanocytic nests"), located at the dermoepidermal junction. Indeed, examination of slides stained with hematoxylin and eosin revealed the presence of marked hyperpigmentation and small nests of partially pigmented cells at the dermoepidermal junction, positive for Melan-A. The histologic and especially the immunohistochemical features were indistinguishable from those of melanoma in situ on chronic sun-damaged skin. In addition, a variably dense lichenoid inflammation was present. Clinicopathologic correlation, however, showed, in all patients, the presence of a lichenoid dermatitis (phototoxic reaction, 1 case; lichen planus pigmentosus, 1 case; and pigmented lichenoid keratosis, 1 case). Our cases clearly show the histopathologic pitfalls represented by lichenoid reactions on chronic sun-damaged skin. Immunohistochemical investigations, especially if performed with Melan-A alone, may lead to confusing and potentially disastrous results. The unexpected staining pattern of Melan-A in cases like ours raises concern about the utility of this antibody in the setting of a lichenoid tissue reaction on chronic sun-damaged skin. It should be underlined that pigmented lesions represent a paradigmatic example of how immunohistochemical results should be interpreted carefully and always in conjunction with histologic and clinical features.

Topics: Antigens, Neoplasm; Biopsy; Dermatitis, Phototoxic; Diagnosis, Differential; Diagnostic Errors; Female; Humans; Hyperpigmentation; Immunohistochemistry; Lichenoid Eruptions; Male; MART-1 Antigen; Melanocytes; Melanoma; Middle Aged; Neoplasm Proteins; Predictive Value of Tests; Skin; Skin Neoplasms; Sunlight

Peptide Ags presented by class I MHC molecules on human melanomas and that are recognized by CD8(+) T cells are the subjects of many studies of antitumor immunity and represent attractive candidates for therapeutic approaches. However, no direct quantitative measurements exist to reveal their expression hierarchy on the cell surface. Using novel recombinant Abs which bind these Ags with a peptide-specific, MHC-restricted manner, we demonstrate a defined pattern of expression hierarchy of peptide-HLA-A2 complexes derived from three major differentiation Ags: gp100, Melan-A/Mart-1, and tyrosinase. Studying melanoma cell lines derived from multiple patients, we reveal a surprisingly high level of presentation of tyrosinase-derived complexes and moderate to very low expression of complexes derived from other Ags. No correlation between Ag presentation and mRNA expression was found; however, protein stability may play a major role. These results provide new insights into the characteristics of Ag presentation and are particularly important when such targets are being considered for immunotherapy. These results may shed new light on relationships between Ag presentation and immune response to cancer Ags.

Topics: Antigen Presentation; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Epitopes, T-Lymphocyte; Flow Cytometry; Fluorescent Antibody Technique; Gene Expression; gp100 Melanoma Antigen; HLA-A2 Antigen; Humans; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

A critical factor determining the effectiveness of currently used dendritic cell (DC)-based vaccines is the DC activation or maturation status. We have recently shown that the T-cell stimulatory capacity of DCs pulsed with tumor-antigen-derived peptides can be considerably increased by activating the DCs through electroporation with mRNA encoding CD40 ligand, CD70, and a constitutively active Toll-like receptor 4 (TriMix DCs). Here, we investigate whether TriMix DCs can be coelectroporated with whole tumor-antigen-encoding mRNA.. The T-cell stimulatory capacity of TriMix DCs pulsed with the immunodominant MelanA-A2 peptide and that of TriMix DCs coelectroporated with MelanA mRNA were compared in vitro. TriMix DCs were also coelectroporated with mRNA encoding Mage-A3, Mage-C2, tyrosinase, or gp100. The capacity of these DCs to stimulate tumor-antigen-specific T cells in melanoma patients was investigated both in vitro before vaccination and after DC vaccination.. Like peptide-pulsed TriMix DCs, TriMix DCs coelectroporated with MelanA mRNA are very potent in inducing MelanA-specific CD8(+) T cells in vitro. These T cells have an activated phenotype, show cytolytic capacity, and produce inflammatory cytokines in response to specific stimulation. TriMix DCs coelectroporated with tyrosinase are able to stimulate tyrosinase-specific CD8(+) T cells in vitro from the blood of nonvaccinated melanoma patients. Furthermore, TriMix DCs coelectroporated with Mage-A3, Mage-C2, or tyrosinase are able to induce antigen-specific CD8(+) T cells through therapeutic DC vaccination.. TriMix DCs coelectroporated with whole tumor-antigen mRNA stimulate antigen-specific T cells in vitro and induce antigen-specific T-cell responses in melanoma patients through vaccination. Therefore, they represent a promising new approach for antitumor immunotherapy.

Topics: Antigens, Neoplasm; Cancer Vaccines; CD27 Ligand; Cytokines; Dendritic Cells; Electroporation; Female; Flow Cytometry; gp100 Melanoma Antigen; Humans; Lysosomal-Associated Membrane Protein 1; Male; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Staging; RNA, Messenger; Toll-Like Receptor 4; Tumor Necrosis Factor Receptor Superfamily, Member 9; Vaccination

Tumor antigen-specific T cells are found within melanomas, yet tumors continue to grow. Although the tumor microenvironment is thought to influence the suppression of tumor-reactive T cells, the underlying mechanisms for this T-cell dysfunction are not clear. Here, we report that the majority of tumor infiltrating T lymphocytes (TIL), including MART-1/Melan-A melanoma antigen-specific CD8 T cells, predominantly expressed PD-1, in contrast to T cells in normal tissues and peripheral blood T lymphocytes (PBL). PD-1(+) TIL expressed CTLA-4 and Ki-67, markers that were not expressed by PD-1(-) TIL and T cells in the normal tissues and PBL. Moreover, PD-1(+) TIL were primarily HLA-DR(+) and CD127(-), in contrast to PD-1(-) TIL. Effector cytokine production by PD-1(+) TIL was impaired compared with PD-1(-) TIL and PBL. Collectively, the phenotypic and functional characterizations of TIL revealed a significantly higher frequency and level of PD-1 expression on TIL compared with normal tissue T-cell infiltrates and PBL, and PD-1 expression correlated with an exhausted phenotype and impaired effector function. These findings suggest that the tumor microenvironment can lead to up-regulation of PD-1 on tumor-reactive T cells and contribute to impaired antitumor immune responses.

Topics: Adolescent; Adult; Aged; Antigens, CD; Antigens, Neoplasm; Apoptosis Regulatory Proteins; CD8-Positive T-Lymphocytes; Child; Cytotoxicity, Immunologic; Female; Humans; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Programmed Cell Death 1 Receptor; Tumor Escape; Up-Regulation; Young Adult

We present the case of 10-year-old girl who have had from birth a plane tumor, of tan color, 3-4 mm of diameter, localized on the face on the cutaneous part of the superior lip. This tumor has been stabile until 8-year-old. Then, after repeated sunlight exposures, the lesion has become more stark, hemispheric in shape, has increased in size becoming about 5-6 mm, with irregular borders, and after an accidental traumatism it began to bleed. We have performed the electroexcision of the lesion for diagnostic and therapeutic purpose. The histopathologic exam distinguished typical images of Spitz nevus on some of the histological sections but also of melanocytary tumor with uncertain malignant potential on the others where atypical mitoses localized in the deeper component of the tumor are being noticed. The immunohistochemical assessment of the tumoral cells showed positivity for the melanocytic markers HMB45 and Melan A, within junctional intraepidermic nevic cells and in the nevic cells from superficial dermis, and also for CD44 protein (belonging to the adhesion molecules family). However, cyclin D1 was positive in rare nevic cells, and the proliferation rate of the tumor was small, with a proliferation index for Ki67 lesser than 5%. The correlation between histopathological and immunohistochemical data conducive to final diagnosis of Spitz nevus with uncertain malignant potential. The clinical evolution confirmed the histopathological diagnosis by the fact that the patient did not presented clinical signs of local recurrences or metastasis at three years after the excision of the tumor.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Child; Cyclin D1; Diagnosis, Differential; Female; Humans; Hyaluronan Receptors; Ki-67 Antigen; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; Nevus, Epithelioid and Spindle Cell; Skin Neoplasms

Topics: Antigens, CD34; Antigens, Neoplasm; Biomarkers, Tumor; Biopsy; Cell Proliferation; Dermatofibrosarcoma; Dermis; Diagnosis, Differential; Female; Humans; MART-1 Antigen; Melanoma; Middle Aged; Mohs Surgery; Neoplasm Proteins; S100 Proteins; Skin Neoplasms

The activation and recruitment of CD4(+) T cells are critical for the development of efficient antitumor immunity and may allow for the optimization of current cancer immunotherapy strategies. Searching for more optimal and selective targets for CD4(+) T cells, we have investigated phosphopeptides, a new category of tumor-derived epitopes linked to proteins with vital cellular functions. Although MHC I-restricted phosphopeptides have been identified, it was previously unknown whether human MHC II molecules present phosphopeptides for specific CD4(+) T cell recognition. We first demonstrated the fine specificity of human CD4(+) T cells to discriminate a phosphoresidue by using cells raised against the candidate melanoma antigen mutant B-Raf or its phosphorylated counterpart. Then, we assessed the presence and complexity of human MHC II-associated phosphopeptides by analyzing 2 autologous pairs of melanoma and EBV-transformed B lymphoblastoid lines. By using sequential affinity isolation, biochemical enrichment, mass spectrometric sequencing, and comparative analysis, a total of 175 HLA-DR-associated phosphopeptides were characterized. Many were derived from source proteins that may have roles in cancer development, growth, and metastasis. Most were expressed exclusively by either melanomas or transformed B cells, suggesting the potential to define cell type-specific phosphatome "fingerprints." We then generated HLA-DRbeta1*0101-restricted CD4(+) T cells specific for a phospho-MART-1 peptide identified in both melanoma cell lines. These T cells showed specificity for phosphopeptide-pulsed antigen-presenting cells as well as for intact melanoma cells. This previously undescribed demonstration of MHC II-restricted phosphopeptides recognizable by human CD4(+) T cells provides potential new targets for cancer immunotherapy.

Topics: Amino Acid Sequence; Antigens, Neoplasm; CD4-Positive T-Lymphocytes; Histocompatibility Antigens Class II; HLA-DR Antigens; Humans; Immunotherapy; MART-1 Antigen; Melanoma; Molecular Sequence Data; Mutation; Neoplasm Proteins; Phosphopeptides; Proto-Oncogene Proteins B-raf

CD8(+) T-cells specific for MART-1-(26-35), a dominant melanoma epitope restricted by human leukocyte antigen (HLA)-A*0201, are exceptionally common in the naive T-cell repertoire. Remarkably, the TRAV12-2 gene is used to encode the T-cell receptor alpha (TCRalpha) chain in >87% of these T-cells. Here, the molecular basis for this genetic bias is revealed from the structural and thermodynamic properties of an archetypal TRAV12-2-encoded TCR complexed to the clinically relevant heteroclitic peptide, ELAGIGILTV, bound to HLA-A*0201 (A2-ELA). Unusually, the TRAV12-2 germ line-encoded regions of the TCR dominate the major atomic contacts with the peptide at the TCR/A2-ELA interface. This "innate" pattern of antigen recognition probably explains the unique characteristics and extraordinary frequencies of CD8(+) T-cell responses to this epitope.

Topics: Amino Acid Sequence; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Germ Cells; HLA-A Antigens; HLA-A2 Antigen; Humans; Immunity, Innate; Immunodominant Epitopes; MART-1 Antigen; Melanoma; Models, Molecular; Neoplasm Proteins; Protein Conformation; Receptors, Antigen, T-Cell; Solubility; Substrate Specificity; Thermodynamics

Ability to cross-present exogenous antigens in the human leukocyte antigen class I pathway is key to the antigen presenting function of mature tumor cell-loaded dendritic cells (DC). Conditions of DC maturation have been shown to be important for DCs ability to produce proinflammatory cytokines and induce T cell effector functions. However, it remains unknown if the different pathways of maturation are associated with modulation of the ability of mature DCs to cross-present tumor antigens (TA). Here, we compare DC matured with 3 clinically relevant cytokine combinations including interleukin (IL)-1 beta, tumor necrosis factor-alpha, IL-6 (termed DC-0), DC-0 cells incubated with prostaglandin-2 (termed DC-0+prostaglandin-2), or DC treated with interferon-gamma, interferon-alpha, tumor necrosis factor-alpha, Poly I:C, and IL1-beta (termed DC-1). We found that these DC vary in their ability to cross-present TA to cytotoxic T lymphocytes (CTL), with the DC-1 cytokine combination being significantly more effective than the other 2. TA cross presentation and CTL priming were strongly correlated with level of expression of the antigen processing machinery components, TAP1 and TAP2, indicating that these components could be used as biomarkers to standardize DC preparations for optimal function. However, the up-regulation of TAP1/TAP2 was not sufficient to explain the enhanced cross-presentation ability of DC-1 cells, as the use of IFN-gamma alone to up-regulate TAP1/TAP2 did not generate DC as effective at cross-presentation as the full DC-1 maturation cytokine combination. These data indicate for the first time that the pathways of DC maturation modulate antigen processing machinery component expression to different extents and that differently matured DC vary in the ability to cross-present TA to human leukocyte antigen class I-restricted CTL.

Topics: Antigen Presentation; Antigens, Neoplasm; ATP Binding Cassette Transporter, Subfamily B, Member 2; ATP Binding Cassette Transporter, Subfamily B, Member 3; ATP-Binding Cassette Transporters; Cancer Vaccines; Carcinoma, Small Cell; CD8-Positive T-Lymphocytes; Cell Differentiation; Cell Line, Tumor; Cytokines; Dendritic Cells; Gene Expression Regulation; HLA-A Antigens; HLA-A2 Antigen; Humans; Lymphocyte Activation; MART-1 Antigen; Melanoma; Neoplasm Proteins; Peptide Fragments; Prostaglandins

Topics: Adipose Tissue; Age Factors; Aged; Antigens, Neoplasm; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Diagnosis, Differential; Female; Humans; Lymphatic Metastasis; MART-1 Antigen; Melanoma; Neoplasm Invasiveness; Neoplasm Proteins; Neoplasms, Multiple Primary

Recent studies indicate that WT1 and Bcl2 protein are detected in melanocytic lesions of the skin. We examined, for the first time, WT1 and Bcl2 expression in a variety of conjunctival melanocytic lesions to evaluate their diagnostic utility compared with other melanocytic markers.. Protein expression and localization of WT1 and Bcl2 were studied by means of immunolabeling and semiquantification in 123 conjunctival melanocytic lesions (71 benign nevi, 21 atypical nevi, 11 primary acquired melanosis, and 20 malignant melanomas). Ancillary immunohistochemical studies were performed with Bcl2, S100, HMB45, and Melan A antibodies.. WT1 showed a graded increase in expression in lesions with increasing atypia. Higher mean numbers of WT1-positive cells correlated with increasing atypia in melanocytes. In all cases, Bcl2 expression was positive and more robust than was S100, HMB45, or Melan A expression. WT1 and HMB45 frequently showed diffuse and strong staining in atypical nevi, primary acquired melanosis with atypia, and malignant melanomas compared with benign lesions.. Bcl2 is a highly sensitive immunohistochemical marker for melanocytic tumors of the conjunctiva; HMB45 and WT1 staining distinguishes benign from malignant lesions.. Our results show that HMB45 and WT1 immunolabeling is helpful in the evaluation of conjunctival melanocytic lesions. Accordingly, we recommend the development of an immunohistochemical panel to classify these lesions.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Biomarkers, Tumor; Child; Child, Preschool; Conjunctival Diseases; Conjunctival Neoplasms; Dysplastic Nevus Syndrome; Female; Humans; Immunoenzyme Techniques; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Melanosis; Middle Aged; Neoplasm Proteins; Nevus, Pigmented; Proto-Oncogene Proteins c-bcl-2; S100 Proteins; WT1 Proteins; Young Adult

Topics: Animals; Antigens, Neoplasm; Biomarkers, Tumor; Choroid Neoplasms; Female; Fluorescein Angiography; Macaca fascicularis; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Monkey Diseases; Neoplasm Proteins; Tomography, Optical Coherence; Ultrasonography

We investigated the possibility of introducing exogenous T cell receptor (TCR) genes into T cells by lentiviral transduction, without prior stimulation of endogenous TCR with anti-CD3. TCR transfer is used to impose tumor antigen specificity on recipient T cells, but sustained activation required for retroviral transduction may affect the clinical efficacy of engineered T cells. Cytokine stimulation makes T cells susceptible to lentiviral transduction in the absence of TCR triggering, but this advantage has never been exploited for TCR transfer. Autoimmune diseases are a source of high-affinity TCRs specific for self/tumor antigens. We selected, from a patient with vitiligo, a Mart1-specific TCR based on intrinsic interchain pairing properties and functional avidity. After lentiviral transduction of human peripheral blood mononuclear cells, preferential pairing of exogenous alpha and beta chains was observed, together with effective recognition of Mart1(+) melanoma cells. We tested transduction efficiency on various T cell subsets prestimulated with interleukin (IL)-2, IL-7, IL-15, and IL-21 (alone or in combination). Both naive and unfractionated CD8(+) T cells could be transduced without requiring endogenous TCR triggering. IL-7 plus IL-15 was the most powerful combination, allowing high levels of transgene expression without inducing T cell differentiation (34 +/- 5% Mart1-TCR(+) cells in naive CD8(+) and 16 +/- 6% in unfractionated CD8(+)). Cytokine-prestimulated, Mart1-redirected naive and unfractionated CD8(+) cells expanded better than CD3-CD28-prestimulated counterparts in response to both peptide-pulsed antigen-presenting cells and Mart1(+) melanoma cells. This strategy allows the generation of tumor-specific T cells encompassing truly naive T cells, endowed with an intact proliferative potential and a preserved differentiation stage.

Topics: Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Genetic Therapy; Genetic Vectors; Humans; Immunologic Memory; Lentivirus; MART-1 Antigen; Melanoma; Neoplasm Proteins; Receptors, Antigen, T-Cell; Transduction, Genetic; Vitiligo

Malignant melanoma causes significant health problems. The identification of tumour-associated antigens has led to novel approaches to increase T cell mediated anti-tumour immune response. Melan-A/MART-1 has been use as target antigen for several T cell based immunotherapeutic treatments. More recently, the critical role of CD4+ T cells in inducing and maintaining anti-tumour immunity has been increasingly recognized. In order to optimize tumour immunotherapy, greater efforts have been concentrated on the identification of tumour antigens presented by MHC class II molecules to CD4+ T cells. In a publication, Tiwari et al. (2004) [1] have identified by a computational approach the 15-mer amino-acid sequence 101-115 (PPAYEKLSAEQSPPP) of the Melan-A/MART-1 as a good target for a vigorous and safe immunotherapy. Therefore, we have investigated the in vivo anti-tumour activity of this peptide in a murine melanoma model. For the prophylactic treatment, 20microg or 50microg peptide was subcutaneously injected in mice once a week during 3 weeks before tumour induction. Treatment with 50microg peptide significantly affected tumour development. Thus, our preliminary data demonstrate potential in vivo prophylactic activity of the 101-115 peptide-based vaccine to control melanoma growth.

Topics: Amino Acid Sequence; Animals; Antigens, Neoplasm; Cancer Vaccines; CD4-Positive T-Lymphocytes; Cell Line, Tumor; Female; Immunotherapy; MART-1 Antigen; Melanoma; Mice; Molecular Sequence Data; Neoplasm Proteins; Peptides

Topics: Aged, 80 and over; Antigens, Neoplasm; Biomarkers, Tumor; Carcinosarcoma; Cell Transformation, Neoplastic; Female; Humans; Lymph Nodes; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; S100 Proteins; Skin Neoplasms

We have previously shown that vaccination of HLA-A2 metastatic melanoma patients with the analogue Melan-A(26-35(A27L)) peptide emulsified in a mineral oil induces ex vivo detectable specific CD8 T cells. These are further enhanced when a TLR9 agonist is codelivered in the same vaccine formulation. Interestingly, the same peptide can be efficiently recognized by HLA-DQ6-restricted CD4 T cells. We used HLA-DQ6 multimers to assess the specific CD4 T-cell response in both healthy individuals and melanoma patients. We report that the majority of melanoma patients carry high frequencies of naturally circulating HLA-DQ6-restricted Melan-A-specific CD4 T cells, a high proportion of which express FOXP3 and proliferate poorly in response to the cognate peptide. Upon vaccination, the relative frequency of multimer+ CD4 T cells did not change significantly. In contrast, we found a marked shift to FOXP3-negative CD4 T cells, accompanied by robust CD4 T-cell proliferation upon in vitro stimulation with cognate peptide. A concomitant reduction in TCR diversity was also observed. This is the first report on direct ex vivo identification of antigen-specific FOXP3+ T cells by multimer labeling in cancer patients and on the direct assessment of the impact of peptide vaccination on immunoregulatory T cells.

Topics: Adult; Aged; Antigens, Neoplasm; Cancer Vaccines; Case-Control Studies; CD4-Positive T-Lymphocytes; Female; Forkhead Transcription Factors; HLA-A2 Antigen; HLA-DQ Antigens; Humans; Immunotherapy; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Peptide Fragments; Receptors, Antigen, T-Cell; Th1 Cells; Vaccination

To evaluate the prognostic value of melanocytic differentiation antigens and angiogenesis biomarkers in sentinel lymph nodes (SLNs) with melanoma micrometastases.. Prognostic study of an inception cohort.. Academic research. Patients Between July 1, 1999, and July 31, 2002, all patients who had primary cutaneous or mucosal melanomas that have a Breslow depth of 1.5 mm or greater, ulceration, or Clark level IV or V, or had SLN biopsies.. By the use of quantitative reverse transcription-polymerase chain reaction, the expression of the following was analyzed in SLNs: 2 melanocytic differentiation antigens (tyrosinase [P17646] and melanoma antigen recognized by T cells [MART-1; Q16655]) and genes involved in angiogenesis (VEGF [NM_001025366] and VEGFR2 [AF035121]), lymphangiogenesis (VEGFC [NM_005429], VEGFR3 [X68203], LYVE1 [NM_016164], and PROX1 [002763]), and invasion (uPA [NM_002658], PAI1 [NM_00602], and EMMPRIN [L10240]). Outcome measures were the association of these melanocytic differentiation antigens and angiogenesis biomarkers with clinicopathologic characteristics of patients, and an evaluation of the prognostic value for relapse-free survival and overall survival.. Ninety-one patients were included, with a median follow-up period of 41 months. Micrometastases were present in 15% (14 of 91) of patients. Tyrosinase (P < .001), MART-1 (P < .001), vascular endothelial growth factor 121 (VEGF(121)) (P = .007), and PAI1 (P = .02) expression was significantly associated with micrometastasis. In univariate analysis, histologic findings and tyrosinase and MART-1 expression were significantly associated with relapse-free survival. Tyrosinase and MART-1 expression was associated with overall survival. A multiple Cox proportional hazards regression model identified negative histologic findings and tyrosinase expression that exceeded 27 copies/copy of TATA box-binding protein (third quartile) as significantly associated with an increased risk of relapse or death.. Quantitative assessment of melanocytic differentiation antigens in SLNs, which has prognostic value, is more specific than qualitative assessment. Prognosis may be more effectively predicted by the combination of quantitative assessment of melanocytic differentiation antigens in SLNs with histologic assessment. A significant association was found between the presence of micrometastases and the expression of angiogenesis biomarkers.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Analysis of Variance; Antigens, Differentiation; Antigens, Neoplasm; Biomarkers, Tumor; Biopsy, Needle; Cohort Studies; Disease-Free Survival; Female; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplasm Invasiveness; Neoplasm Proteins; Neoplasm Recurrence, Local; Neoplasm Staging; Neovascularization, Pathologic; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; Risk Assessment; Sensitivity and Specificity; Sentinel Lymph Node Biopsy; Skin Neoplasms; Survival Analysis; Young Adult

The antibody HMB45 is used to diagnose lymphangioleiomyomatosis, a hyperproliferative disorder of lung smooth muscle cells with mutations in both alleles of either TSC1 or TSC2. A subset of these tumor cells expresses the melanoma-associated antigens gp100 and melanoma antigen recognized by T cells (MART-1). To explore the feasibility of targeting tumors in lymphangioleiomyomatosis by melanoma immunotherapy, we therefore assessed melanoma target antigen expression and existing immune infiltration of affected tissue compared with normal lung and melanoma as well as the susceptibility of cultured lymphangioleiomyomatosis cells to melanoma reactive cytotoxic T lymphocytes in vitro. Tumors expressed tyrosinase-related proteins 1 and 2 but not tyrosinase, in addition to gp100 and MART-1, and were densely infiltrated by macrophages, but not dendritic cells or T cell subsets. Although CD8(+) lymphocytes were sparse compared with melanoma, cells cultured from lymphangioleiomyomatosis tissue were susceptible to cytotoxic, gp100 reactive, and major histocompatibility complex class I restricted CD8(+) T cells in functional assays. Responder T cells selectively clustered and secreted interferon-gamma in response to HLA-matched melanocytes and cultured lymphangioleiomyomatosis cells. This reactivity exceeded that based on detectable gp100 expression; thus, tumor cells in lymphangioleiomyomatosis may process melanosomal antigens different from melanocytic cells. Therefore, boosting immune responses to gp100 in lymphangioleiomyomatosis may offer a highly desirable treatment option for this condition.

Topics: Antigens, Neoplasm; Cell Separation; Cytotoxicity, Immunologic; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; gp100 Melanoma Antigen; Humans; Immunohistochemistry; Immunotherapy; Intramolecular Oxidoreductases; Lymphangioleiomyomatosis; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Microscopy, Electron, Transmission; Neoplasm Proteins; Oxidoreductases; Skin Neoplasms; T-Lymphocytes, Cytotoxic

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Humans; Immunohistochemistry; Lymphatic Metastasis; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; S100 Proteins; Sensitivity and Specificity; Sentinel Lymph Node Biopsy; Skin Neoplasms; SOXE Transcription Factors

The diagnosis of malignant melanoma can be challenging given the wide variation in morphologic features and immunohistochemical stains are often used to confirm the diagnosis. We report a case of melanoma with loss of staining for S100 protein, HMB-45, and Melan-A, with retained expression of tyrosinase. Regional lymph node metastases showed positive S100 protein staining. Although the loss of phenotypic markers including S100 protein has been reported in metastatic melanoma, loss of S100 in primary melanoma is rare. Discordant staining between primary and metastatic lesions further emphasizes the protean nature of melanoma.

Topics: Antigens, CD; Antigens, Differentiation, Myelomonocytic; Antigens, Neoplasm; Antineoplastic Agents; Biomarkers, Tumor; Combined Modality Therapy; Facial Neoplasms; Fatal Outcome; Humans; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Palliative Care; S100 Proteins; Shoulder; Skin Neoplasms

Spheroid cells which can grow as nonattached spheroids in vitro culture condition are considered as tumor-initiating cells that have properties similar to those of stem cells. However, the existence of spheroid cells in WM-266-4, a human malignant metastatic melanoma cell line, has not yet been reported.. Accordingly, we investigated whether WM-266-4 can form spheroids, and characterized these spheroids using qRT-PCR, histology, immunohistochemistry and xenograft.. WM-266-4 contains a small subpopulation of cells that grow as spheroids and express genes strongly related to tumor malignancy and stem-like factors. Second, histological analysis of the spheres revealed that they consist of 300-400 round cells per sphere with a high karyoplasmic ratio. They have a basophilic cytoplasm and are highly pleomorphic in size, and sometimes multinucleated and giant. Third, although there were differences between the spheroid and bulk cells, they both have high tumorigenic potential, as both cell types formed a tumor mass upon injection of only 100 cells in nude mice.. We characterized the spheroid cells in an established melanoma cell line. We suggest that enriched spheroid cells might contain more dedifferentiated progenitor cells, but we could not conclude spheroid cells are cancer stem cells.

Topics: AC133 Antigen; Animals; Antigens, CD; Antigens, Neoplasm; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Biomarkers, Tumor; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Glycoproteins; Homeodomain Proteins; Humans; Immunohistochemistry; Intermediate Filament Proteins; MART-1 Antigen; Melanoma; Melanoma, Experimental; Mice; Mice, Nude; Neoplasm Proteins; Nerve Tissue Proteins; Nestin; Nuclear Proteins; Peptides; Polycomb Repressive Complex 1; Proto-Oncogene Proteins; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; Spheroids, Cellular; Time Factors; Transplantation, Heterologous; Tumor Burden; Tumor Suppressor Proteins; Vascular Endothelial Growth Factor Receptor-3; Wnt Proteins; Wnt-5a Protein

T-cell receptor (TCR) gene transfer for cancer immunotherapy is limited by the availability of large numbers of tumor-specific T cells. TCR alpha and beta chains were isolated from a highly lytic HLA-A2-restricted cytotoxic T lymphocyte (CTL) clone recognizing the melanoma-associated Melan-A/MART-1 antigen and inserted into a lentiviral vector carrying a bidirectional promoter capable of robust and coordinated expression of the two transgenes. Lentiviral vector-based gene delivery systems have shown increased transfer efficiency and transgene expression compared with the widely used gamma-retroviral vectors. This vector performed more efficiently than a gamma-retrovirus-based vector containing the same expression cassette, resulting in a T-cell population with 60% to 80% of transgenic TCR expression with mainly CD8(+) intermediate effector phenotype. Transgenic T cells specifically produced cytokine in response to and killed antigen-expressing melanoma cells, retained an overlapping functional avidity in comparison with the TCR donor CTL clone, and exerted significant therapeutic effects in vivo upon adoptive transfer in melanoma-bearing severe combined immunodeficient mice. Optical imaging showed their accumulation in the tumor site. Overall, our results indicate that lentiviral vectors represent a valid tool for stable and high-intensity expression of transgenic TCR and support clinical exploitation of this approach for therapeutic application.

Topics: Animals; Antigens, Neoplasm; Epitopes; Female; Genes, T-Cell Receptor alpha; Genes, T-Cell Receptor beta; Genetic Vectors; HLA-A2 Antigen; Humans; Immunologic Memory; Immunotherapy, Adoptive; Jurkat Cells; Lentivirus; Leukocytes, Mononuclear; MART-1 Antigen; Melanoma; Mice; Mice, SCID; Neoplasm Proteins; Receptors, Antigen, T-Cell, alpha-beta; T-Lymphocytes; Transduction, Genetic

Childhood dermal tumors with melanocytic features is an unusual tumor that can create diagnostic confusion. Among them, paraganglioma-like melanocytic tumors- previous included in melanocytic tumors of uncertain malignant potential- has some particular histopathologic and immunohistochemical features. We describe a case of 13 years old girl with a paraganglioma-like dermal melanocytic tumor of the left leg.

Topics: Adolescent; Antigens, Neoplasm; Biopsy; Cell Lineage; Dermis; Female; Humans; Immunohistochemistry; MART-1 Antigen; Melanocytes; Melanoma; Neoplasm Proteins; Paraganglioma; Proto-Oncogene Proteins c-kit; S100 Proteins; Skin Neoplasms; Vimentin

Detection of micrometastasis in melanoma-draining lymph nodes is important for staging and prognosis. Immunohistochemical staining (IHC) using S-100p-HMB-45-, and MART-1-specific antibodies is used for detecting metastases in sentinel lymph nodes (SLN). However, improvement in IHC is needed for melanoma micrometastasis detection.. Paraffin-embedded archival tissue (PEAT) specimens were obtained from 42 non-SLN macrometastases, 42 SLN metastases, and 16 tumor-negative SLNs of 100 melanoma patients who underwent SLN biopsy. PEAT specimens were assessed by IHC with high molecular weight-melanoma-associated antigen (HMW-MAA)-specific monoclonal antibodies (mAb) and with S-100p-, HMB-45-, and MART-1-specific antibodies. Quantitative real-time reverse-transcriptase PCR assay was used for HMW-MAA and MART-1 mRNA detection.. Expression frequency and immunostaining intensity were higher for HMW-MAA than MART-1 in nodal macrometastases (P < 0.0001 and P < 0.0001, respectively) and micrometastases (P < 0.0001 and P = 0.004, respectively). All 52 (100%) macrometastases were positive with HMW-MAA-specific mAbs, whereas 43 (83%) were positive with MART-1-specific mAbs. In a comparison analysis, 23 of 23 (100%) micrometastases were HMW-MAA-positive, whereas 21 (91%) and 18 (78%) specimens were S-100p- and HMB-45-positive, respectively. Quantitative real-time reverse-transcriptase PCR analysis of 48 nodal metastases showed HMW-MAA mRNA detection in SLNs with metastases.. HMW-MAA is more sensitive and specific than MART-1, S-100p, and HMB-45 for IHC-based detection of SLN micrometastases. SLN PEAT-based detection specificity of melanoma micrometastases can be improved by IHC with HMW-MAA-specific mAbs.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Calcium-Binding Proteins; Humans; Immunohistochemistry; Lymphatic Metastasis; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; Paraffin Embedding; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sensitivity and Specificity; Sentinel Lymph Node Biopsy; Skin Neoplasms

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Disease-Free Survival; Follow-Up Studies; Humans; Immunohistochemistry; Lymph Nodes; Lymphatic Metastasis; MART-1 Antigen; Melanoma; Neoplasm Proteins; Sentinel Lymph Node Biopsy; Skin Neoplasms; Survival Rate; Time Factors

Topics: Antigens, Neoplasm; Humans; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; Neoplasm Staging; Prognosis; Risk Assessment; S100 Proteins; Sentinel Lymph Node Biopsy; Skin Neoplasms

Melan-A specific CD8+ T cells are thought to play an important role against the development of melanoma. Their in vivo expansion is often observed with advanced disease. In recent years, low levels of Melan-A reactive CD8+ T cells have also been found in HLA-A2 healthy donors, but these cells harbor naive characteristics and are thought to be mostly cross-reactive for the Melan-A antigen. Here, we report on a large population of CD8+ T cells reactive for the Melan-A antigen, identified in one donor with no evidence of melanoma. Interestingly, this population is oligoclonal and displays a clear memory phenotype. However, a detailed study of these cells indicated that they are unlikely to be directly specific for melanoma, so that their in vivo expansion may have been driven by an exogenous antigen. Screening of a Melan-A cross-reactive peptide library suggested that these cells may be specific for an epitope derived from a Mycobacterium protein, which would provide a further example of CD8+ T cell cross-reactivity between a pathogen antigen and a tumor antigen. Finally, we discuss potential perspectives regarding the role of such cells in heterologous immunity, by influencing the balance between protective immunity and pathology, e.g. in the case of melanoma development.

Topics: Antigens, Bacterial; Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cells, Cultured; Clone Cells; Cross-Priming; Cytotoxicity, Immunologic; Humans; Immunologic Memory; Immunophenotyping; MART-1 Antigen; Melanoma; Mycobacterium tuberculosis; Neoplasm Proteins; Peptides; T-Cell Antigen Receptor Specificity; Tuberculosis, Pulmonary

There is a need for biomarkers to identify patients at risk for disease progression after resection of melanoma regional lymph node metastasis.. This study assessed the prognostic value of multimarker reverse transcriptase-polymerase chain reaction (RT-PCR) assay in lymphatic drainage (LY) after lymph node dissection (LND) and of preoperative serum lactate dehydrogenase (LDH) levels in American Joint Committee on Cancer (AJCC) stage III melanoma patients.. We collected 24-h LY from 255 stage III melanoma patients after radical LND [114, completion LND after positive sentinel node biopsy (CLND); 141, therapeutic LND for clinically/cytologically detected regional nodal metastases (TLND)]. For detection of melanoma cells, RT-PCR assays with primers specific for tyrosinase, MART1 (MelanA) and uMAGE mRNA were conducted. The LY sample was deemed positive if at least one marker was detected. In 244 patients, the preoperative serum LDH level was available. Median follow-up time was 25 months (range 5-60).. The LY multimarker RT-PCR assay results were positive in 82 of 255 patients (32%). A significantly higher rate of melanoma recurrence was observed in patients with positive LY multimarker RT-PCR results (P = 0.01). Significant relationships were observed between positive LY multimarker RT-PCR results and shorter 3-year overall survival (OS) and disease-free survival (DFS), both in univariate and multivariate analyses (P = 0.007). Preoperative serum LDH level was increased in 79 of 244 patients (32%) [40.5% in TLND group and 23.0% in CLND group (P = 0.003)]. There were significant differences in OS between patients with normal and high preoperative LDH levels (P = 0.007), and these differences were seen mainly in patients in the TLND group.. The multimarker RT-PCR assay detected melanoma cells in approximately 32% of LY after LND, which correlated significantly with early melanoma recurrence and shorter survival. Increased pre-LND serum LDH level had an additional negative impact on OS of melanoma patients with palpable nodal metastases after TLND.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Biomarkers; Biomarkers, Tumor; Disease-Free Survival; Female; Follow-Up Studies; Humans; L-Lactate Dehydrogenase; Lymph; Lymph Node Excision; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Multivariate Analysis; Neoplasm Proteins; Neoplasm Recurrence, Local; Neoplasm Staging; Predictive Value of Tests; Proportional Hazards Models; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skin Neoplasms; Survival Rate

Melan-A is widely used in the diagnostics of human melanoma. The immunogenicity of this glycoprotein makes it a potential target in immunotherapy and several authors have suggested its potential as a prognostic factor. Up to now there has been no clear direct evidence of changes of Melan-A expression during the progression of melanoma. We have performed objective immunohistochemical assessment of the expression of Melan-A in benign naevi and melanomas at different stages of progression. Our results show a complex pattern of changes in the expression of Melan-A in melanomas depending on the location of melanoma cells within individual skin layers. The expression of the antigen during tumour progression significantly decreases for melanoma cells located in the granular/spinous layer (r=-0.94, P=0.02) and increases for the papillary layer (r=0.99, P=0.002) and reticular layer (r=0.89, P=0.04). It should also be emphasized that from the Clark II level of progression the melanomas can be detected with high sensitivity and specificity using a simple cut-off test based on the determination of Melan-A expression in tumour cells located within the papillary layer.

Topics: Antigens, Neoplasm; Biomarkers; Disease Progression; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Neoplasm Proteins; Nevus; Sensitivity and Specificity; Skin; Skin Neoplasms

Most gastric gastrointestinal stromal tumors (GISTs) express CD117/c-kit, as do a subset of metastatic melanomas, leading to a diagnostic dilemma in some cases.. To further differentiate GISTs from melanoma, we investigated expression of melanoma markers in GISTs using a well-characterized set of gastric lesions on tissue microarrays.. Tissue microarrays from paraffin-embedded tissue cores from 38 patients were stained with S100 protein, HMB-45, and Melan-A antibodies. All cases had been previously stained with CD117/c-kit and CD34 antibodies. All were reactive with CD117/c-kit, and 88.2% expressed CD34.. S100 protein was focally expressed in 2 (5.3%) of 38 GISTs; these lesions lacked HMB-45 and Melan-A labeling. No tumor labeled with HMB-45, but 4 (10.6%) of 38 cases labeled with Melan-A antibodies. The Melan-A-reactive cases were all S100 negative and CD34 positive. The S100-reactive cases were spindle cell neoplasms, whereas the Melan-A-reactive cases were epithelioid neoplasms (4/9; 44%). An additional 15 standard sections of separate cases of epithelioid GISTs were then labeled with Melan-A, and 5 (33%) of 15 showed at least focal labeling.. Melan-A staining can be encountered in a subset of epithelioid GISTs, a finding that can suggest a differential diagnosis of melanoma. In this series, the Melan-A-reactive cases lacked S100 protein and expressed CD34, both of which would be unlikely in melanoma. As such, a panel approach is best in differentiating epithelioid GISTs from melanoma.

Topics: Adult; Aged; Aged, 80 and over; Antigens, CD34; Antigens, Neoplasm; Biomarkers, Tumor; Diagnosis, Differential; Female; Gastrointestinal Stromal Tumors; Humans; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Microarray Analysis; Middle Aged; Neoplasm Proteins; S100 Proteins

Melanoma-associated antigens, MART-1, tyrosinase, gp100 and MAGEs, are typical melanoma-specific tumor antigens which can potently induce immune responses in metastatic melanoma patients treated with peptide vaccines. In the present study, we established a dendritic cell (DC)-based HLA-A2 melanoma-associated peptide (MART-1 or gp100)-specific CTL induction method and characterized the CTLs using HLA-A2 tetramer staining in 6 cases of HLA-A2+ melanoma treated with DC vaccines. Peripheral blood mononuclear cells (PBMC) from patients were stimulated twice with MART-1 A2 peptide-pulsed DCs in the presence of a low dose of IL-2. To boost CTL populations, CTL lines were further stimulated twice with MART-1 A2 peptide-pulsed T2 cells. The frequency of MART-1 A2 tetramer-positive CTLs increased from 0.16% (prior to stimulation) to 2.15% (after DC stimulation), and reached 46.5% on average (after additional T2 stimulation) in 4 cases which showed a successful expansion. The absolute numbers of MART-1 A2 tetramer-positive CTLs increased from 187- to 619-fold (average, 415-fold) compared to prior to DC stimulation. CTL assays using MART-1-specific CTL lines demonstrated potent killing activity against MART-1 peptide-pulsed T2 cells or HLA-A2+ melanoma cell lines in accordance with the frequency of tetramer-positive CTLs. Finally, we were successful in identifying melanoma peptide-specific T-cell receptor (TCR) cDNAs in 2 cases for MART-1 and 1 case for gp100 using the anti-TCR MoAb-based sorting as a novel approach instead of a conventional cell cloning, and confirmed peptide-specific IFN-gamma production in TCR cDNA-transduced naïve T cells. The results showed that cloned TCR cDNAs were efficient in reconstituting tumor-specific cytotoxicity and good candidates for novel immunotherapy.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Cancer Vaccines; Cell Line; Cell Separation; Cytotoxicity, Immunologic; Dendritic Cells; Flow Cytometry; gp100 Melanoma Antigen; HLA-A2 Antigen; Humans; Interferon-gamma; Japan; Lymphocyte Activation; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Receptors, Antigen, T-Cell; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes, Cytotoxic; Transduction, Genetic; Tumor Cells, Cultured

HLA-A2 tetramer flow cytometry, IFNgamma real time RT-PCR and IFNgamma ELISPOT assays are commonly used as surrogate immunological endpoints for cancer immunotherapy. While these are often used as research assays to assess patient's immunologic response, assay validation is necessary to ensure reliable and reproducible results and enable more accurate data interpretation. Here we describe a rigorous validation approach for each of these assays prior to their use for clinical sample analysis.. Standard operating procedures for each assay were established. HLA-A2 (A*0201) tetramer assay specific for gp100209(210M) and MART-126-35(27L), IFNgamma real time RT-PCR and ELISPOT methods were validated using tumor infiltrating lymphocyte cell lines (TIL) isolated from HLA-A2 melanoma patients. TIL cells, specific for gp100 (TIL 1520) or MART-1 (TIL 1143 and TIL1235), were used alone or spiked into cryopreserved HLA-A2 PBMC from healthy subjects. TIL/PBMC were stimulated with peptides (gp100209, gp100pool, MART-127-35, or influenza-M1 and negative control peptide HIV) to further assess assay performance characteristics for real time RT-PCR and ELISPOT methods. Validation parameters included specificity, accuracy, precision, linearity of dilution, limit of detection (LOD) and limit of quantification (LOQ). In addition, distribution was established in normal HLA-A2 PBMC samples. Reference ranges for assay controls were established.. The validation process demonstrated that the HLA-A2 tetramer, IFNgamma real time RT-PCR, and IFNgamma ELISPOT were highly specific for each antigen, with minimal cross-reactivity between gp100 and MelanA/MART-1. The assays were sensitive; detection could be achieved at as few as 1/4545-1/6667 cells by tetramer analysis, 1/50,000 cells by real time RT-PCR, and 1/10,000-1/20,000 by ELISPOT. The assays met criteria for precision with %CV < 20% (except ELISPOT using high PBMC numbers with %CV < 25%) although flow cytometric assays and cell based functional assays are known to have high assay variability. Most importantly, assays were demonstrated to be effective for their intended use. A positive IFNgamma response (by RT-PCR and ELISPOT) to gp100 was demonstrated in PBMC from 3 melanoma patients. Another patient showed a positive MART-1 response measured by all 3 validated methods.. Our results demonstrated the tetramer flow cytometry assay, IFNgamma real-time RT-PCR, and INFgamma ELISPOT met validation criteria. Validation approaches provide a guide for others in the field to validate these and other similar assays for assessment of patient T cell response. These methods can be applied not only to cancer vaccines but to other therapeutic proteins as part of immunogenicity and safety analyses.

Topics: Antigens, Neoplasm; Calibration; Cell Line, Tumor; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; gp100 Melanoma Antigen; HLA-A2 Antigen; Humans; Interferon-gamma; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Reference Standards; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity

Topics: Antigens, Neoplasm; Humans; Keratins, Type II; MART-1 Antigen; Melanoma; Neoplasm Proteins; Skin Neoplasms; Staining and Labeling

Melanoma patients may exhibit a T(H)2-skewed cytokine profile within blood and tumor-infiltrating lymphocytes. Therapies that induce beneficial T(H)1-type tumor-specific immune responses, therefore, are highly desirable. Dendritic cells (DC) are widely used as immune adjuvants for cancer. Before their administration, DC are generally induced to mature with a cocktail of recombinant cytokines [interleukin (IL)-1beta, tumor necrosis factor alpha, and IL-6] and prostaglandin E(2) (PGE(2)), which is added to preserve the ability of DC to migrate to draining lymph nodes. However, PGE(2) suppresses the production of IL-12p70, a cytokine essential for differentiation of T(H)1 responses. In this study, human DC were transfected with IL-12p70 mRNA and tested for their ability to alter the T(H)2 type bias manifested by blood T cells of patients with melanoma. Transfected DC secreted high levels of bioactive IL-12p70, as indicated by their capacity to enhance natural killer cell activity, skew T(H)1 responses in allogeneic mixed lymphocyte reactions through reduction of IL-4 and IL-5, and prime CD8(+) T cells to the melanoma-associated antigen Melan A/MART-1. Furthermore, T-cell lines primed in vitro from the blood of melanoma patients showed strong type 2 skewing that was dramatically reversed by IL-12p70 transfection of autologous DC. Thus, IL-12p70 transfection of clinical DC preparations may enhance type 1 antitumor responses and may thereby contribute to effective immune-based therapy.

Topics: Amino Acid Sequence; Antigens, Neoplasm; Cells, Cultured; Dendritic Cells; Humans; Interferon-gamma; Interleukin-12; Interleukin-5; Killer Cells, Natural; MART-1 Antigen; Melanoma; Molecular Sequence Data; Neoplasm Proteins; RNA, Messenger; Th1 Cells; Th2 Cells; Transfection

The incidence of cutaneous melanoma, the most lethal form of skin cancer, is increasing all over the world. Malignant melanoma spreads through hematogenous, lymphagenous path, or by iatrogenic implantations. In this report, we present an unusual case in which a patient experienced the development of melanoma within the split-thickness skin graft donor site on the contralateral lower extremity. Metastases of malignant melanoma occurred in the donor site of the graft, 8 weeks after primary tumor excision, ipsilateral axillary dissection, and reconstruction with a split-thickness skin graft harvested from the contralateral upper thigh. As eight melanoma nodules were seen in donor area, we strongly suspect that spreading of melanoma occurred via a true hematogenous pathway in this case.

Topics: Antigens, Neoplasm; Humans; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasm Proteins; Skin Neoplasms; Skin Transplantation

Vaccine strategies that utilize human DCs to enhance antitumor immunity have yet to realize their full potential. Approaches that optimally target a spectrum of antigens to DCs are urgently needed. Here we report the development of a platform for loading DCs with antigen. It is based on killed but metabolically active (KBMA) recombinant Listeria monocytogenes and facilitates both antigen delivery and maturation of human DCs. Highly attenuated KBMA L. monocytogenes were engineered to express an epitope of the melanoma-associated antigen MelanA/Mart-1 that is recognized by human CD8+ T cells when presented by the MHC class I molecule HLA-A*0201. The engineered KBMA L. monocytogenes induced human DC upregulation of costimulatory molecules and secretion of pro-Th1 cytokines and type I interferons, leading to effective priming of Mart-1-specific human CD8+ T cells and lysis of patient-derived melanoma cells. KBMA L. monocytogenes expressing full-length NY-ESO-1 protein, another melanoma-associated antigen, delivered the antigen for presentation by MHC class I and class II molecules independent of the MHC haplotype of the DC donor. A mouse therapeutic tumor model was used to show that KBMA L. monocytogenes efficiently targeted APCs in vivo to induce protective antitumor responses. Together, our data demonstrate that KBMA L. monocytogenes may be a powerful platform that can both deliver recombinant antigen to DCs for presentation and provide a potent DC-maturation stimulus, making it a potential cancer vaccine candidate.

Topics: Animals; Antigen Presentation; Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cytokines; Dendritic Cells; Histocompatibility Antigens Class II; HLA-A Antigens; HLA-A2 Antigen; Humans; Listeria monocytogenes; MART-1 Antigen; Melanoma; Mice; Mice, Inbred BALB C; Neoplasm Proteins; Recombinant Proteins; Th1 Cells

Human primary melanoma cells (T1) were found to be more susceptible to lysis by a Melan-A/MART-1-specific CTL clone (LT12) than their metastatic derivative (G1). We show that this differential susceptibility does not involve antigen presentation by target cells, synapse formation between the metastatic target and CTL clone, or subsequent granzyme B (GrB) polarization. Although PI-9, an inhibitor of GrB, was found to be overexpressed in metastatic G1 cells, knockdown of the PI-9 gene did not result in the attenuation of G1 resistance to CTL-induced killing. Interestingly, we show that whereas T1 cells express high levels of intercellular adhesion molecule-1 (ICAM-1), a dramatically reduced expression was noted on G1 cells. We also showed that sorted ICAM-1+ G1 cells were highly sensitive to CTL-induced lysis compared with ICAM-1- G1 cells. Furthermore, incubation of metastatic G1 cells with IFN-gamma resulted in the induction of ICAM-1 and the potentiation of their susceptibility to lysis by LT12. More importantly, we found that the level of ICAM-1 expression by melanoma cells correlated with decreased PTEN activity. ICAM-1 knockdown in T1 cells resulted in increased phosphorylation of PTEN and the subsequent activation of AKT. We have additionally shown that inhibition of the phosphatidylinositol (3,4,5)-triphosphate kinase (PI3K)/AKT pathway by the specific inhibitor wortmannin induced a significant potentiation of susceptibility of G1 and ICAM-1 small interfering RNA-treated T1 cells to CTL-induced lysis. The present study shows that a shift in ICAM-1 expression, which was associated with an activation of the PI3K/AKT pathway, can be used by metastatic melanoma cells to escape CTL-mediated killing.

Topics: Antigen Presentation; Antigens, Neoplasm; Cell Line, Tumor; Down-Regulation; Enzyme Activation; G1 Phase; Humans; Intercellular Adhesion Molecule-1; Interferon-gamma; MART-1 Antigen; Melanoma; Neoplasm Proteins; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; RNA, Small Interfering; Serpins; T-Lymphocytes, Cytotoxic

To determine the effect of activation of specific anti-tumor cytotoxic T lymphocytes (CTL) and the ability of cross-presentation in vitro by fusion of HLA-A2+ human dendritic cells (DCs) with HLA-A2- melanoma cells.. The HLA-A2+ human dendritic cells and HLA-A2- melanoma cells were fused by PEG and were cultivated in complete RPMI1640 media containing FCS (10%) and GM-CSF for 24-48 h, and then co-cultured fusion cells with Melan-A specific T cells. HLA-A2- melanoma cells were negative control,While T2 cells and DC+Pts were positive control. The activation of anti-tumor CTL elicited by the fusion cells was detected by intracellular cytokine staining.. The immature DC could express CD80, CD83, CD86, HLA-DR, and HLA-ABC,but the mature DC induced by TNF-alpha, PGE-2, and CD40L further highly expressed above molecules. The rate of specific CTL cells primed by the fusion cells was 16.72%+/-4.26%, negative control was 0.21%+/-1.84%,and positive control was 28.60%+/-5.67%. The CTL from vaccine by fusing DC and LAR6 induced lysis of HLA-A2+ LAR1 cells.. The HLA-A2 restricted specific anti-tumor CTL can be induced in vitro by fusion of HLA-A2+ human dendritic cells with HLA-A2- melanoma cells.

Topics: Antigen Presentation; Antigens, Neoplasm; Cancer Vaccines; Cell Fusion; Cell Line, Tumor; Dendritic Cells; HLA-A2 Antigen; Humans; MART-1 Antigen; Melanoma; T-Lymphocytes, Cytotoxic

At the histological examination of an increasing number of melanocytic tumors there is a need to use various immunohistochemical methods. Currently, we are supplied by several antibodies working well on formalin-fixed, paraffin-embedded samples. We have tested five antibodies (S-100, HMB-45, Melan-A, MITF, PNL-2) on 34 benign and 34 malignant melanocytic tumors. We examined the specificity and sensitivity in the junctional and dermal component separately, with special consideration to features disturbing the evaluation (regression, halo-like inflammation, etc.). We have concluded that the histological diagnosis of melanocytic tumors is based on the detailed examination of traditional HE slides and the immunohistochemical methods only confirm or weaken our opinion.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Biomarkers, Tumor; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Microphthalmia-Associated Transcription Factor; Neoplasm Proteins; Nevus; Nevus, Blue; Nevus, Epithelioid and Spindle Cell; Nevus, Spindle Cell; Paraffin Embedding; Polysaccharide-Lyases; S100 Proteins; Skin Neoplasms

There are currently no effective therapies for metastatic melanoma and targeted immunotherapy results in the remission of only a very small percentage of tumors. In this study, we show that the noncanonical Wnt ligand, Wnt5A, can increase melanoma metastasis in vivo while down-regulating the expression of tumor-associated antigens important in eliciting CTL responses (e.g., MART-1, GP100, and tyrosinase). Melanosomal antigen expression is governed by MITF, PAX3, and SOX10 and is inhibited upon signal transducers and activators of transcription 3 (STAT3) activation, via decreases in PAX3 and subsequently MITF expression. Increasing Wnt5A in Wnt5A-low cells activated STAT3, and STAT3 was decreased upon Wnt5A knockdown. Downstream targets such as PAX3, MITF, and MART-1 were also affected by Wnt5A treatment or knockdown. Staining of a melanoma tissue array also highlighted the inverse relationship between MART-1 and Wnt5A expression. PKC activation by phorbol ester mimicked Wnt5A effects, and Wnt5A treatment in the presence of STAT3 or PKC inhibitors did not lower MART-1 levels. CTL activation studies showed that increases in Wnt5A correspond to decreased CTL activation and vice versa, suggesting that targeting Wnt5A before immunotherapy may lead to the enhancement of current targeted immunotherapy for patients with metastatic melanoma.

Topics: Animals; Antigens, Neoplasm; Humans; Lymphocyte Activation; MART-1 Antigen; Melanoma; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasm Metastasis; Neoplasm Proteins; Phosphorylation; RNA, Small Interfering; STAT3 Transcription Factor; T-Lymphocytes; Transcription, Genetic; Transfection; Wnt Proteins; Wnt-5a Protein

Choosing a reliable source of tumor-specific T lymphocytes and an efficient method to isolate these cells still remains a critical issue in adoptive cellular therapy (ACT). In this study, we assessed the capacity of MHC/peptide based immunomagnetic sorting followed by polyclonal T cell expansion to derive pure polyclonal and tumor-reactive Melan-A specific T cell populations from melanoma patient's PBMC and TIL. We first demonstrated that this approach was extremely efficient and reproducible. We then used this procedure to compare PBMC and TIL-derived cells from three melanoma patients in terms of avidity for Melan-A A27L analog, Melan-A(26-35)and Melan-A(27-35), tumor reactivity (lysis and cytokine production) and repertoire. Regardless of their origin, i.e., fresh PBMC, peptide stimulated PBMC or TIL, all sorted populations (from the three patients) were cytotoxic against HLA-A2+ melanoma cell lines expressing Melan-A. Although some variability in peptide avidity, lytic activity and cytokine production was observed between populations of different origins in a given patient, it differed from one patient to another and thus no correlation could be drawn between T cell source and reactivity. Analysis of Vbeta usage within the sorted populations showed the recurrence of Vbeta3 and Vbeta14 subfamilies in the three patients but differences in the rest of the Melan-A repertoire. In addition, in two patients, we observed major repertoire differences between populations sorted from the three sources. We especially documented that in vitro peptide stimulation of PBMC, used to facilitate the sort by enriching in specific T lymphocytes, could significantly alter their repertoire and reactivity towards tumor cells. We conclude that PBMC which are easily obtained from all melanoma patients, can be as good a source as TIL to derive high amounts of tumor-reactive Melan-A specific T cells, with this selection/amplification procedure. However, the conditions of peptide stimulation should be improved to prevent a possible loss of reactive clonotypes.

Topics: Antigens, Neoplasm; Cell Line, Tumor; Cells, Cultured; Cytotoxicity, Immunologic; Genes, T-Cell Receptor beta; Humans; Immunomagnetic Separation; Immunotherapy, Adoptive; Leukocytes, Mononuclear; Lymphocytes, Tumor-Infiltrating; Major Histocompatibility Complex; MART-1 Antigen; Melanoma; Neoplasm Proteins; T-Lymphocytes

Adoptive therapy with antigen-specific T cells is a promising approach for the treatment of infectious diseases and cancer. However, cloning of antigen-specific T cells by the traditional approach of limiting dilution is a time-consuming, laborious, and inefficient process. Here, we describe a novel flow cytometric strategy for rapid isolation of human tumor antigen-specific T-cell clones by using T-cell receptor (TCR) Vbeta antibodies in combination with carboxyfluorescein succinimidyl ester (CFSE)-based proliferation assay. The CFSE dilution following antigen stimulation identified proliferating antigen-specific T cells, and the TCRVbeta antibodies allowed distinguishing T cells at the clonal level from a heterogeneous T-cell population. This method of TCRVbeta/CFSE dilution was used for the isolation of four different human lymphoma and melanoma-specific CD4(+) and CD8(+) T-cell clones reactive against defined and undefined tumor antigens. Isolated tumor-specific T-cell clones could be expanded to large numbers ex vivo while maintaining phenotype, function, and tumor antigen specificity. The method was simple, efficient, and reproducible, and may have potential application for the development of adoptive immunotherapeutic strategies.

Topics: Antigens, Neoplasm; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line; Cell Separation; Clone Cells; Flow Cytometry; Humans; Immunotherapy, Adoptive; Lymphoma; MART-1 Antigen; Melanoma; Neoplasm Proteins; Receptors, Antigen, T-Cell, alpha-beta

Topics: Adult; Aged; Antigens, Neoplasm; Dendrites; Female; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanocytes; Melanoma; Middle Aged; Neoplasm Proteins; Paget Disease, Extramammary; Skin Neoplasms; Staining and Labeling

Melan-A/MART1 is a melanocytic differentiation antigen recognized on melanoma tumor cells by CD8+ and CD4+ T cells. In this study, we describe a new epitope of this protein recognized in the context of HLA-Cw*0701 molecules by a CD8+ tumor infiltrating lymphocyte (TIL) clone. This CD8+ TIL clone specifically recognized and killed a fraction of melanoma cells lines expressing Melan-A/MART1 and HLA-Cw*0701. We further show that the Melan-A/MART1(51-61) peptide is the optimal peptide recognized by this clone. Together, these data significantly enlarge the fraction of melanoma patients susceptible to benefit from a Melan-A/MART1 vaccine approach.

Topics: Amino Acid Sequence; Antigen Presentation; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Epitopes, T-Lymphocyte; Flow Cytometry; HLA-C Antigens; Humans; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Molecular Sequence Data; Neoplasm Proteins

The generation of melanoma-reactive T cells with the characteristics necessary for in vivo effectiveness remains a considerable obstacle to the application of adoptive cell therapy. Recent clinical success with adoptive cell therapy for melanoma is motivating additional investigation to improve the technology of generating such tumor reactive lymphocytes. Here we describe a novel solid phase T-cell selection system, in which monocytes are immobilized on solid support for antigen-specific T-cell purification. We hypothesized and proved that antigen-specific T cells recognize their cognate antigens and bind to them faster than nonantigen-specific T cells and are concentrated on the surface after removing the nonadherent cells by washing. Moreover, activated antigen-specific T cells proliferated more rapidly than nonspecific T cells, further increasing the frequency and purity of antigen-specific T cells. Optimal selection times for Melan-A-specific T cells are studied. Our data demonstrated that T-cell selection can usually increase the frequency of tumor antigen-specific T cells by >10-fold, whereas T-cell expansion after the selection boost the frequency of tumor antigen-specific T cells by another approximately 10-fold. More importantly, these T cells are generated under more physiologic conditions. This new T-cell selection system is superior to traditional repeated stimulation methods in generating tumor antigen-specific T cells for adoptive cell immunotherapy. This inexpensive and simple T-cell selection system can produce large quantity of highly purified Melan-A-specific T cells within 2 weeks after T-cell activation.

Topics: Antigens, Neoplasm; Cell Communication; Cell Culture Techniques; Cell Line, Tumor; Cell Separation; Cytotoxicity Tests, Immunologic; Dendritic Cells; Epitopes, T-Lymphocyte; HLA-A Antigens; HLA-A2 Antigen; Humans; Immunotherapy, Adoptive; Interferon-gamma; Leukocytes, Mononuclear; MART-1 Antigen; Melanoma; Monocytes; Neoplasm Proteins; T-Lymphocytes, Cytotoxic

A brown cornea is relatively rare. We report a case of progressive brown corneal pigmentation in a patient with a primary acquired melanosis of the conjunctiva. Later the patient developed an iris melanoma.. Case report with clinico-pathological correlation and discussion of possible mechanisms of particle clearance of the cornea.. A 36-year-old female developed a corneal stromal pigmentation adjacent to a pigmented conjunctival lesion of the left eye. The corneal pigmentation had progressed through 8 years. The conjunctival lesion was surgically removed, and proved histopathologically to be a compound nevus with slight atypia and an acquired melanosis. Despite surgery the corneal pigmentation increased, and visual acuity dropped in the diseased eye. A perforating keratoplasty was performed, and two small pigmented iris nodules were now noted. Three years after grafting, growth of the two iris tumours was obvious. In addition, pigmentation of the trabecular meshwork and large, pigmented endothelial precipitates were observed. The corneal pigmentation also increased. The eye was enucleated. Histopathologic evaluation demonstrated a marked accumulation of melanophages on the endothelium of the graft. The host cornea contained pigmented cells in the mid-stroma. The iris contained two melanomas.. The brown pigmentation of the cornea was due to pigment granules from the iris tumours liberated to the anterior chamber. The pigment was transported into the cornea through the endothelium and accumulated in melanophages between corneal lamellas. The pigment subsequently cleared via the corneal limbus in a process resembling clearance of corneal haemochromatosis.

Topics: Adult; Antigens, Neoplasm; Conjunctival Neoplasms; Corneal Diseases; Corneal Stroma; Eye Enucleation; Female; Humans; Iris Neoplasms; Keratoplasty, Penetrating; MART-1 Antigen; Melanocytes; Melanoma; Melanoma-Specific Antigens; Melanosis; Neoplasm Proteins; Nevus, Pigmented; S100 Proteins

Melanoma antigen recognized by T cells 1 (MART-1) and tyrosinase-related protein-2 (TRP-2) are two useful markers for immunohistochemical detection of melanocytic tumors. However, these markers may be passively acquired (phagocytosed) rather than actively synthesized. Reverse transcriptase in situ polymerase chain reaction (RT in situ PCR) can amplify even small amounts of specific mRNA in cells and therefore confirm the cellular source of a marker. We developed a one-step RT in situ PCR procedure in which Thermus thermophilus DNA polymerase synthesizes and amplifies cDNA from mRNA in a single reaction mixture. To examine its practicability and feasibility with formalin-fixed, paraffin-embedded (FFPE) tissue, we compared the results of one-step RT in situ PCR with those of immunohistochemistry (IHC). MART-1 mRNA was identified in the cytoplasm of lesional cells from 23/26 primary melanomas (92%), 9/9 metastatic melanomas (100%) and 5/6 nevi (83%). MART-1 epitope was detected by IHC in 23/24 primary melanomas (96%), 9/9 metastatic melanomas (100%) and 5/6 nevi (83%). TRP-2 mRNA was identified in the cytoplasm of lesional cells from 17/26 primary melanomas (65%), 6/9 metastatic melanomas (67%) and 4/6 nevi (67%). TRP-2 epitope was detected by IHC in 20/24 primary melanomas (83%), 9/9 metastatic melanomas (100%) and 4/6 nevi (67%). Both techniques detected MART-1 and TRP-2 in FFPE melanoma cell lines. Neither marker was detected in squamous cell carcinomas or basal cell carcinomas by RT in situ PCR or IHC. We conclude that the RT in situ PCR technique can be successfully applied to FFPE tissue to determine the cellular sources of gene expression observed by conventional PCR approaches.

Topics: Antigens, Neoplasm; Cell Line, Tumor; Formaldehyde; Humans; Immunohistochemistry; Intramolecular Oxidoreductases; MART-1 Antigen; Melanoma; Neoplasm Proteins; Nevus; Paraffin Embedding; Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Sensitivity and Specificity; Skin Neoplasms; Tissue Fixation

Immunohistochemical staining has been used to help detect malignant melanoma on Mohs surgery frozen sections. Previous investigators have developed protocols for reliable MART-1 immunostaining of frozen sections, but these protocols are time-consuming.. The objective was to report a rapid 20-minute MART-1 immunostaining protocol for frozen sections.. The protocol was utilized on 30 melanomas treated with Mohs micrographic surgery.. The stain clearly highlighted normal background melanocytes, as well as melanocytic hyperplasia and malignant melanoma.. The 20-minute protocol provides a rapid and reliable method for immunostaining of malignant melanoma. The availability of more rapid immunostaining methods improves efficiency of the Mohs laboratory and significantly reduces patient and physician waiting time. The authors have indicated no significant interest with commercial supporters.

Topics: Antigens, Neoplasm; Frozen Sections; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Mohs Surgery; Neoplasm Proteins; Reproducibility of Results; Skin Neoplasms; Staining and Labeling; Time Factors; Tissue Culture Techniques

PEComas arising in somatic soft tissue or skin are rare. To further characterize the clinicopathologic spectrum, we herein report a series of 10 primary cutaneous PEComas. Eight patients were female and 2 patients were male. The age at presentation ranged from 15 to 81 years (median, 52y). None had tuberous sclerosis. Tumor size ranged from 0.7 to 2 cm (median size, 1.5 cm). Eight tumors were located on the limbs and 2 on the back. The tumors involved the dermis and commonly showed infiltration into the subcutaneous fat. Architecturally, a nested, focally trabecular growth pattern with prominent vasculature composed of delicate thin-walled capillaries was observed. Six tumors were composed of epithelioid cells and 4 showed mixed epithelioid and spindle cell morphology. Tumor cells had clear, palely eosinophilic or granular cytoplasm. Multinucleate tumor giant cells were observed in 3 cases. Mitotic activity ranged from 0 to 2 mitoses per 30 high power fields (mean < 1/10 HPF). Pleomorphism or necrosis was absent. All cases showed at least focal positivity for HMB-45. Melan A was positive in 5/7. In cases where HMB-45 was positive in fewer than 5% of tumor cells (5/10), microphthalmia transcription factor was positive in the majority of the tumor cell nuclei. Desmin positivity was observed in 5 and smooth muscle actin in 1 case, respectively. The other muscle markers (caldesmon, calponin) and also pan-keratin and epithelial membrane antigen were negative. Follow-up data were available in 6 cases and ranged between 3 and 108 months (median, 47 mo). None has recurred to date. Primary cutaneous PEComas are rare and thus far apparently benign cutaneous tumors. Accurate recognition of this entity is essential because of potential misdiagnosis as malignant tumors, especially malignant melanoma.

Topics: Actins; Adolescent; Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Biomarkers, Tumor; Calcium-Binding Proteins; Calmodulin-Binding Proteins; Calponins; Dermis; Desmin; Diagnosis, Differential; Female; Giant Cells; Humans; Immunohistochemistry; Keratins; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Microfilament Proteins; Microphthalmia-Associated Transcription Factor; Middle Aged; Mitotic Index; Mucin-1; Neoplasm Invasiveness; Neoplasm Proteins; Skin Neoplasms; Subcutaneous Fat

Extensive pathological workup of sentinel lymph nodes (SLNs) in melanoma detects more patients with metastasis-positive SLNs than do routine protocols, but at the cost of high laboratory workloads. We aimed to design a protocol that reduced this workload without compromising metastasis detection.. We analyzed 920 SLNs from 321 consecutive patients with melanoma by complete step sectioning and immunohistochemistry. We designed different models to theoretically reduce the number of histological sections examined and compared the results from these simulations with results obtained with our extended protocol, with the restricted national Danish protocol, and with the protocol recommended by the European Organization for Research and Treatment of Cancer (EORTC).. The extended protocol increased the metastasis detection rate by 22% (95% confidence interval, 11-34; 30.8% vs. 25.2%, P < .0001) compared with the national Danish protocol, and it had a similar rate to that reported by the EORTC (30.8% vs. 29.4%, P = .6229). The workload associated with complete step sectioning could be reduced by 40% by focusing step sectioning on the SLNs with the highest gamma counts, while examining SLNs with lower gamma counts with one to three central sections. The false-negative rate for detecting metastases with this reduced protocol was 6% compared with complete step sectioning.. Combining complete step sectioning of SLNs with immunohistochemistry ensures high metastasis detection rates. Workloads can be markedly reduced with only a slight increase in the false-negative rate by focusing analysis on the SLNs most likely to contain metastases, i.e., those with the highest gamma counts.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Female; Gamma Rays; Humans; Immunoenzyme Techniques; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Radionuclide Imaging; Radiopharmaceuticals; S100 Proteins; Sentinel Lymph Node Biopsy; Skin Neoplasms; Technetium Tc 99m Aggregated Albumin

Antigen-loaded dendritic cells (DCs) have been intensively investigated as potential cellular antitumor vaccines. Several recent reports have indicated that loading DCs with whole tumor derived mRNA or defined tumor-antigen-encoding mRNA represents an effective nonviral strategy to stimulate T cell responses both for in vitro and in vivo models. Here, we describe the electroporation method as a tool for introducing in vitro transcribed capped mRNA into human DCs for tumor vaccination. We use MART-1/Melan-A as a model tumor-associated antigen for the generation of a DC-based vaccine against melanoma cancer. In addition to efficient antigen loading, it is important to obtain a maximal number of potent antigen-presenting cells. Another prerequisite for the development of a DC-based cancer vaccine is to obtain mature DCs. In this chapter, we describe the basic techniques required for the successful genetic modification of DCs by using the mRNA electroporation method.

Topics: Antigens, Neoplasm; Base Sequence; Cancer Vaccines; Cell Separation; Dendritic Cells; DNA Primers; Electrochemotherapy; Electroporation; Humans; MART-1 Antigen; Melanoma; Mutagenesis, Site-Directed; Neoplasm Proteins; Plasmids; RNA Caps; RNA, Messenger; T-Lymphocytes

Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) for specific melanoma markers is more sensitive than histology for detecting cells of melanocytic origin in sentinel lymph nodes (SLNs) in cutaneous melanoma. The clinical significance of a positive qRT-PCR analysis is unclear. We performed qRT-PCR for the presence of MART-1 and tyrosinase in SLNs from 93 melanoma patients, and then followed these patients clinically (median follow-up time 43.5 months). We found a significant correlation between disease progression and presence of MART-1 mRNA in SLNs (p=0.02), but no correlation with the amount of MART-1 mRNA as measured by qRT-PCR. No correlation between histology and recurrence was detected, recurrence rates being low in both histology-negative (12%) and -positive (15%) patients. We found a significant difference in disease recurrence between patients positive by both histology and RT-PCR and patients negative by both methods (15% vs 0%, p=0.02). However, a significant difference in disease recurrence was also found when comparing patients negative by both methods with patients positive by RT-PCR but negative by histology (0% vs 19%, p=0.009). This suggests that the presence of submicroscopic metastases may influence prognosis, indicating that RT-PCR detection of melanocytic cells in SLNs may be an important diagnostic marker.

Topics: Antigens, Neoplasm; Disease-Free Survival; Female; Humans; Male; MART-1 Antigen; Melanocytes; Melanoma; Middle Aged; Neoplasm Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sentinel Lymph Node Biopsy

Distinguishing lentigo maligna from solar lentigo, and pigmented actinic keratosis can sometimes be problematic. Melan-A is an immunohistochemical marker which that can be helpful in decorating the melanocytes of pigmented lesions. A recent report has suggested that Melan-A may spuriously label nests of junctional keratinocytes, potentially leading to the misdiagnosis of melanoma in situ. We compared Melan-A immunohistochemical staining in pigmented actinic keratosis , non-pigmented actinic keratoses , melanoma in situ of lentigo maligna type and solar lentigines. We found a statistically significant increase of Melan-A staining in melanoma in situ, but no statistical difference in the number of junctional Melan-A positively staining cells, in solar lentigines, pigmented actinic keratoses, and non-pigmented actinic keratoses, respectively. In the non non-melanoma samples, the Melan-A A-positive cells located at the dermal-epidermal junction were interspersed and not observed in clusters. Increased staining with Melan-A, in an actinic keratosis, or solar lentigo should raise the possibility of a contiguous melanoma in situ.

Topics: Aged; Antigens, Neoplasm; Biomarkers, Tumor; Humans; Hutchinson's Melanotic Freckle; Immunohistochemistry; Keratosis, Actinic; Lentigo; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Skin Neoplasms

In human gene therapy applications, lentiviral vectors may have advantages over gamma-retroviral vectors in several areas, including the ability to transduce nondividing cells, resistance to gene silencing and a potentially safer integration site profile. However, unlike gamma-retroviral vectors it has been problematic to drive the expression of multiple genes efficiently and coordinately with approaches such as internal ribosome entry sites or dual promoters. Using different 2A peptides, lentiviral vectors expressing two-gene T-cell receptors directed against the melanoma differentiation antigens gp100 and MART-1 were constructed. We demonstrated that addition of amino-acid spacer sequences (GSG or SGSG) before the 2A sequence is a prerequisite for efficient synthesis of biologically active T-cell receptors and that addition of a furin cleavage site followed by a V5 peptide tag yielded optimal T-cell receptor gene expression. Furthermore, we determined that the furin cleavage site was recognized in lymphocytes and accounted for removal of residual 2A peptides at the post-translational level with an efficiency of 20-30%, which could not be increased by addition of multiple furin cleavage sites. The novel bicistronic lentiviral vector developed herein afforded robust anti-melanoma activities to engineered peripheral blood lymphocytes, including cytokine secretion, cell proliferation and lytic activity. Such optimal vectors may have immediate applications in cancer gene therapy.

Topics: Antigens, Neoplasm; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Furin; Genes, T-Cell Receptor; Genetic Engineering; Genetic Therapy; Genetic Vectors; gp100 Melanoma Antigen; Humans; Immunotherapy, Adoptive; Lentivirus; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Receptors, Antigen, T-Cell; Skin Neoplasms; Transduction, Genetic

Aiming at more precise detection of melanoma cells in sentinel lymph nodes and better understanding of the mechanisms underlying metastatic spread, expression of L1, CEACAM1, and binding of the lectins HPA, ML-I and PNA, was assessed in benign nevi (n=12), primary melanomas (PTs: n=67), their corresponding sentinel lymph nodes (SLNs: n=40), and distant metastases (DMs: n=35). Sensitivity and specificity of CEACAM1 (95-97%; 66%) and L1 (90-93%; 100%) exceeded that of the standard markers MelanA, S100, and HMB45 in single marker use. Lectin binding was found in PTs and DMs (HPA: 69% and 77%; ML-I: 82% and 77%, respectively), but rarely in SLNMs (HPA: 20%, ML-I: 20%, PNA: 5%, respectively). The highly specific and sensitive L1-11A against L1 and 4D1/C2 against CEACAM1 antibodies are a worthy completion to standard antibody panels for diagnosis of melanoma cells. Both CAMs seem to be functionally involved in lymphatic and haematogenous spread, and are thus promising target molecules for immunotoxins.

Topics: Antigens, CD; Antigens, Neoplasm; Biomarkers, Tumor; Cell Adhesion Molecules; Glycoproteins; Humans; Immunohistochemistry; Lectins; Leukocyte L1 Antigen Complex; Lymph Nodes; Lymphatic Metastasis; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; Prognosis; Protein Binding; S100 Proteins; Sentinel Lymph Node Biopsy; Skin Neoplasms

The expansion of cytotoxic CD8+ T lymphocytes (CTLs) which recognize peptide epitopes of tumour or viral origin has been a major aim of immunotherapy research for the past decade. Alongside the established dendritic cell-based methods, more recent approaches using recombinant MHC class I peptide complexes have been developed.. In this study we have explored the potential of a simplified system using soluble streptavidin-linked MHC class I tetramers to expand antigen-specific CTLs in vitro and in vivo.. In vitro tetramer-mediated expansion of CD8+ CTLs recognizing HLA-2/Melan-A and HLA-A2/Gag complexes was demonstrated with PBMCs from healthy donors or HIV+ donors, respectively. With 3 weekly rounds of tetramer stimulation, cell numbers expanded 100-fold from 0.05 to 5.0%. The lytic function of HLA-A2/Melan-A-expanded cells was demonstrated in 51Cr release assays by specific killing of T2 cells pulsed with Melan-A, but not other peptides. Similarly, murine CD8+ T cells specific for the HY epitope H2-Db/Uty could be expanded in vitro over a wide range of tetramer concentrations (0.008-1.0 microg/ml), with a single exposure producing substantial T cell expansion from 0.11 to 36%. Intraperitoneal administration of H2-Db/Uty tetramers to primed C57BL/6 mice produced over 5-fold expansion of Db/Uty-specific CTL in vivo.. The results in this paper demonstrate that simple, multimeric MHC complexes may be of value in expanding CTLs in vitro for adoptive immunotherapy and also potentially in vivo. Further studies will be necessary to clarify the optimum protocols and schedules of administration for T cell expansion using recombinant MHC multimers.

Topics: Adoptive Transfer; Animals; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Epitopes; Female; Flow Cytometry; Gene Products, gag; Genes, MHC Class I; HIV; HLA-A2 Antigen; Humans; Leukocytes, Mononuclear; MART-1 Antigen; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Proteins; Peptide Fragments; T-Lymphocytes, Cytotoxic

Granular cell tumors (GCTs), especially if atypical or malignant, may share cytomorphologic and architectural features with malignant melanoma, when the latter shows granular cell change. In many cases, these neoplasms can be differentiated from each other on histologic grounds, but distinction may sometimes be challenging. By immunohistochemistry, both tumors are strongly positive for S-100 protein and frequently express other nonspecific markers such as CD68, NSE, and NKIC3. In the current study, we reviewed 60 cases of conventional cutaneous, mucosal, and visceral GCT and studied the use of immunoperoxidase staining for the differential diagnosis between malignant melanoma and GCT. Immunohistochemical stains for S-100 protein, A, HMB-45, and microphthalmia transcription factor (MITF) were performed in all cases. All of the tumors were positive for S-100 protein. MITF immunostaining was diffusely positive in 53 (88%) cases, focally positive in three (5%) cases, and negative in four (7%). Fifty-seven (95%) tumors were negative for Melan-A, one case was focally positive, and two cases showed rare positive tumor cells. None of the tumors expressed HMB-45. In conclusion, GCT and malignant melanoma can be reliably differentiated on the basis of immunohistochemical stains in the majority of cases. Although not always positive in malignant melanoma, in this context, HMB-45 expression seems to be 100% specific for the diagnosis of melanoma. Melan-A is slightly less specific, with rare cases of GCT showing focal positivity. MITF is not useful in this differential-93% of the GCTs in our series showed nuclear reactivity for this marker. The latter finding highlights the limited specificity of this antibody in the diagnosis of melanocytic tumors.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Diagnosis, Differential; Granular Cell Tumor; Humans; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Microphthalmia-Associated Transcription Factor; Neoplasm Proteins; Retrospective Studies; S100 Proteins; Sensitivity and Specificity; Skin Neoplasms

The aim of this study was to determine in patients with high-risk primary uveal melanoma whether the detection of circulating tumor cells by quantitative reverse transcription-PCR (RT-PCR) is of prognostic relevance.. Blood samples from 110 patients with high-risk nonmetastatic uveal melanoma were collected on the occasion of primary treatment or follow-up visit. mRNA expression of tyrosinase and MelanA/MART1 were analyzed by real-time RT-PCR and compared with clinical data at presentation and follow-up by univariate and multivariate analyses.. The RT-PCR assay yielded a positive result in 11 of 110 patients, with five positive findings for tyrosinase and five for MelanA/MART1, and one sample positive for both markers. At a median follow-up of 22 months, 25% of patients had developed metastases and 15% had died. Univariate statistical analysis revealed RT-PCR and the largest tumor diameter as important prognostic factors for the development of metastases and for survival. In a Cox proportional hazard model, RT-PCR result and largest tumor diameter predicted metastases (hazard ratios 7.3 and 2.6, respectively), whereas PCR result, largest tumor diameter, and Karnofsky performance status were significant variables for disease-specific survival (hazard ratios 22.6, 4.7, and 6.0, respectively). Analysis of individual RT-PCR results revealed both tyrosinase and MelanA/MART1 transcripts as independent prognostic factors.. The presence of tyrosinase or MelanA/MART1 transcripts is an independent prognostic factor in patients with high-risk primary uveal melanoma for subsequent development of metastases and for survival and can be used to select patients for adjuvant treatment studies.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Cohort Studies; Female; Humans; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplasm Metastasis; Neoplasm Proteins; Neoplastic Cells, Circulating; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Uveal Neoplasms

Melan-A/MART1 is a melanocytic differentiation antigen expressed by tumor cells of the majority of melanoma patients and, as such, is considered as a good target for melanoma immunotherapy. Nonetheless, the number of class I and II restricted Melan-A epitopes identified so far remains limited. Here we describe a new Melan-A/MART-1 epitope recognized in the context of HLA-DQa1*0101 and HLA-DQb1*0501, -DQb1*0502 or -DQb1*0504 molecules by a CD4+ T cell clone. This clone was obtained by in vitro stimulation of PBMC from a healthy donor by the Melan-A51-73 peptide previously reported to contain a HLA-DR4 epitope. The Melan-A51-73 peptide, therefore contains both HLA-DR4 and HLA-DQ5 restricted epitope. We further show that Melan-A51-63 is the minimal peptide optimally recognized by the HLA-DQ5 restricted CD4+ clone. Importantly, this clone specifically recognizes and kills tumor cell lines expressing Melan-A and either HLA-DQb1*0501, -DQb1*0504 or -DQb1*0502 molecules. Moreover, we could detect CD4+ T cells secreting IFN-gamma in response to Melan-A51-63 and Melan-A51-73 peptides among tumor infiltrating and blood lymphocytes from HLA-DQ5+ patients. This suggests that spontaneous CD4+ T cell responses against this HLA-DQ5 epitope occur in vivo. Together these data significantly increase the fraction of melanoma patients susceptible to benefit from a Melan-A class II restricted vaccine approach.

Topics: Amino Acid Sequence; Antigen Presentation; Antigens, Neoplasm; CD4-Positive T-Lymphocytes; Cell Line, Tumor; Epitope Mapping; Epitopes, T-Lymphocyte; HLA-DQ alpha-Chains; HLA-DQ Antigens; HLA-DQ beta-Chains; Humans; MART-1 Antigen; Melanoma; Molecular Sequence Data; Neoplasm Proteins; Peptides; Skin Neoplasms

Uveal melanoma is the most common primary malignant ocular cancer in adults. This tumor has a distinct expression pattern of markers compared with cutaneous melanoma. MC1R is under study as a potential target for antitumor immunity. Because of the potential immunogenicity of MC1R, it is important to evaluate its expression on uveal melanomas.. Two novel monoclonal antibodies (MP1.1C11 and MP1.1B7) were used to examine the expression of MC1R in uveal melanomas. Tissue samples obtained from 17 patients were analyzed for expression of MC1R by immunohistochemistry. Additionally, uveal melanoma cell lines were treated with proinflammatory cytokines, after which MC1R cell surface expression was analyzed by flow cytometry.. Results demonstrated that MC1R is expressed by uveal melanoma to a significantly greater extent than other melanoma markers. With the use of MP1.1C11 or MP1.1B7, MC1R was detected in 95% of the tested melanoma tissues, including one liver metastasis. In contrast, MART-1, S100-specific protein, and gp-100 were only expressed by 66%, 33%, and 67% of the analyzed samples, respectively. Results also demonstrated that even though MC1R is mainly located intracellularly, its cell surface expression can be promoted by cytokines such as IFN-gamma, TNF-alpha, IL-4, and IL-10.. These observations support the inclusion of MC1R in the panel of markers for the diagnosis of uveal melanoma. Therapeutic use of MC1R-specific antibodies targeting cytokine-induced MC1R potentially requires expression of the target molecule on the surfaces of tumor cells. Data presented here support MC1R as a new marker and a putative therapeutic target for uveal melanoma.

Topics: alpha-MSH; Antigens, Neoplasm; Biomarkers, Tumor; Blotting, Western; Cytokines; Flow Cytometry; gp100 Melanoma Antigen; Humans; Immunoenzyme Techniques; Immunotherapy; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Receptor, Melanocortin, Type 1; S100 Proteins; Tumor Cells, Cultured; Uveal Neoplasms

Cytotoxic T lymphocytes (CTL) are crucial in viral clearance and tumor growth control. Thus the induction of CTL activity is an important aim in vaccine development. We investigate an innovative delivery system for peptide transfer to the MHC class I processing pathway of APC with the aim to trigger CTL in the context of an antitumoral response. The strategy relies on a novel antigen delivery system termed "chimeric immunopotentiating reconstituted influenza virosomes" (CIRIV) targeting plasmacytoid dendritic cells (PDC). By using virosomes containing encapsulated Melan-A peptide and a PDC line developed in our laboratory, we evaluated the response of Melan-A specific T cells. Virosomes have the capacity to bind PDC and are endocyted within vesicles in the cytosol. This endocytosis is inhibited by neuraminidase, suggesting that it is mediated by sialic acid present on cell surface. Furthermore, PDC loaded with Melan-A virosomes can induce a Melan-A specific T cell activation. Interestingly, they activate T cells with a better efficiency than PDC loaded with a free peptide and when PDC where previously activated by a TLR ligand. These results indicate that virosomes could be a suitable delivery system for tumor peptide in immunotherapy of cancer.

Topics: Antigen Presentation; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Dendritic Cells; Humans; Immunotherapy, Active; Lymphocyte Activation; MART-1 Antigen; Melanoma; Neoplasm Proteins; Orthomyxoviridae; Virosomes

Cancer cells' growth in three-dimensional (3D) architectures promotes resistance to drugs, cytokines, or irradiation. We investigated effects of 3D culture as compared to monolayers (2D) on melanoma cells' recognition by tumour-associated antigen (TAA)-specific HLA-A(*)0201-restricted cytotoxic T-lymphocytes (CTL). Culture of HBL, D10 (both HLA-A(*)0201+, TAA+) and NA8 (HLA-A(*)0201+, TAA-) melanoma cells on polyHEMA-coated plates, resulted in generation of 3D multicellular tumour spheroids (MCTS). Interferon-gamma (IFN-gamma) production by HLA-A(*)0201-restricted Melan-A/MART-1(27-35) or gp 100(280-288)-specific CTL clones served as immunorecognition marker. Co-culture with melanoma MCTS, resulted in defective TAA recognition by CTL as compared to 2D as witnessed by decreased IFN-gamma production and decreased Fas Ligand, perforin and granzyme B gene expression. A multiplicity of mechanisms were potentially involved. First, MCTS per se limit CTL capacity of recognising HLA class I restricted antigens by reducing exposed cell surfaces. Second, expression of melanoma differentiation antigens is downregulated in MCTS. Third, expression of HLA class I molecules can be downregulated in melanoma MCTS, possibly due to decreased interferon-regulating factor-1 gene expression. Fourth, lactic acid production is increased in MCTS, as compared to 2D. These data suggest that melanoma cells growing in 3D, even in the absence of immune selection, feature characteristics capable of dramatically inhibiting TAA recognition by specific CTL.

Topics: Antigens, Neoplasm; Cell Culture Techniques; Fas Ligand Protein; Granzymes; Histocompatibility Antigens Class I; HLA-A1 Antigen; HLA-A2 Antigen; Humans; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Perforin; Pore Forming Cytotoxic Proteins; Spheroids, Cellular; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

Antitumor lymphocytes can be generated ex vivo unencumbered by immunoregulation found in vivo. Adoptive transfer of these cells is a promising therapeutic modality that could establish long-term antitumor immunity. However, the widespread use of adoptive therapy has been hampered by the difficulty of consistently generating potent antitumor lymphocytes in a timely manner for every patient. To overcome this, we sought to establish a clinical grade culture system that can reproducibly generate antigen-specific cytotoxic T lymphocytes (CTL).. We created an off-the-shelf, standardized, and renewable artificial antigen-presenting cell (aAPC) line that coexpresses HLA class I, CD54, CD58, CD80, and the dendritic cell maturation marker CD83. We tested the ability of aAPC to generate tumor antigen-specific CTL under optimal culture conditions. The number, phenotype, effector function, and in vitro longevity of generated CTL were determined.. Stimulation of CD8(+) T cells with peptide-pulsed aAPC generated large numbers of functional CTL that recognized a variety of tumor antigens. These CTLs, which possess a phenotype consistent with in vivo persistence, survived ex vivo for prolonged periods of time. Clinical grade aAPC(33), produced under current Good Manufacturing Practices guidelines, generated sufficient numbers of CTL within a short period of time. These CTL specifically lysed a variety of melanoma tumor lines naturally expressing a target melanoma antigen. Furthermore, antitumor CTL were easily generated in all melanoma patients examined.. With clinical grade aAPC(33) in hand, we are now poised for clinical translation of ex vivo generated antitumor CTL for adoptive cell transfer.

Topics: Antigen Presentation; Antigen-Presenting Cells; Antigens, Neoplasm; Biomimetics; CD8-Positive T-Lymphocytes; Cell Proliferation; Half-Life; Humans; Immunotherapy, Adoptive; Interferon-gamma; Interleukin-15; K562 Cells; MART-1 Antigen; Melanoma; Neoplasm Proteins; Tumor Cells, Cultured

Topics: Antigens, Neoplasm; Biomarkers, Tumor; gp100 Melanoma Antigen; Humans; Lymph Nodes; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Staging; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Skin Neoplasms; Survival Analysis

The most commonly used melanocytic markers are S100, HMB45, Melan-A, or MART-1 and tyrosinase. Melanoma with complete, concordant loss of these markers has not been reported. We report a case of metastatic melanoma with complete loss of staining for S100, HMB45, Melan-A, and tyrosinase. Interestingly, both the primary melanoma and its metastasis were strongly positive for CD99.

Topics: 12E7 Antigen; Aged; Antigens, CD; Antigens, Neoplasm; Biomarkers, Tumor; CD56 Antigen; Cell Adhesion Molecules; Down-Regulation; Humans; Immunohistochemistry; Immunophenotyping; Lymph Nodes; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Monophenol Monooxygenase; Neoplasm Proteins; S100 Proteins; Skin Neoplasms; Vimentin

We evaluated 35 cases of malignant melanomas with substantial necrosis immunostained with S-100, HMB-45, Melan-A, tyrosinase, PNL2, and microphthalmia transcription factor (MITF). Staining patterns were evaluated in viable and necrotic areas of the tumors. S-100 was the most sensitive marker (97%) in the viable tumors, but necrotic areas demonstrated nonspecific staining. Viable tumors stained variably for HMB-45 (25 [71%]), Melan-A (28 [80%]), tyrosinase (30 [86%]), and PNL2 (23 [66%]). Necrotic areas focally reacted to the same antibodies. The necrotic areas that retained immunoreactivity for these markers corresponded to areas where the outline of the tumor cells could still be recognized as ghost cells on the H&E-stained section. Areas that showed complete coagulative necrosis were negative for melanoma markers. MITF variably stained in the viable tumors but was completely negative in necrotic areas. Our study demonstrated that a combination of antibodies to HMB-45, tyrosinase, and PNL2 detected melanocytic differentiation in necrotic areas in 80% of cases.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Microphthalmia-Associated Transcription Factor; Monophenol Monooxygenase; Necrosis; Neoplasm Proteins; S100 Proteins

In the present study, we demonstrate, in rigorous fashion, that human monocyte-derived immature dendritic cells (DCs) can efficiently cross-present tumor-associated antigens when co-cultured with a mixture of human melanoma cells rendered apoptotic/necrotic by gamma irradiation (Apo-Nec cells).. We evaluated the phagocytosis of Apo-Nec cells by FACS after PKH26 and PKH67 staining of DCs and Apo-Nec cells at different times of coculture. The kinetics of the process was also followed by electron microscopy. DCs maturation was also studied monitoring the expression of specific markers, migration towards specific chemokines and the ability to cross-present in vitro the native melanoma-associated Ags MelanA/MART-1 and gp100.. Apo-Nec cells were efficiently phagocytosed by immature DCs (iDC) (55 +/- 10.5%) at 12 hs of coculture. By 12-24 hs we observed digested Apo-Nec cells inside DCs and large empty vacuoles as part of the cellular processing. Loading with Apo-Nec cells induced DCs maturation to levels achieved using LPS treatment, as measured by: i) the decrease in FITC-Dextran uptake (iDC: 81 +/- 5%; DC/Apo-Nec 33 +/- 12%); ii) the cell surface up-regulation of CD80, CD86, CD83, CCR7, CD40, HLA-I and HLA-II and iii) an increased in vitro migration towards MIP-3beta. DC/Apo-Nec isolated from HLA-A*0201 donors were able to induce >600 pg/ml IFN-gamma secretion of CTL clones specific for MelanA/MART-1 and gp100 Ags after 6 hs and up to 48 hs of coculture, demonstrating efficient cross-presentation of the native Ags. Intracellular IL-12 was detected in DC/Apo-Nec 24 hs post-coculture while IL-10 did not change.. We conclude that the use of a mixture of four apoptotic/necrotic melanoma cell lines is a suitable source of native melanoma Ags that provides maturation signals for DCs, increases migration to MIP-3beta and allows Ag cross-presentation. This strategy could be exploited for vaccination of melanoma patients.

Topics: Antigens, Neoplasm; Apoptosis; CD8-Positive T-Lymphocytes; Cell Differentiation; Cell Line, Tumor; Cell Movement; Chemokine CCL19; Coculture Techniques; Cross-Priming; Dendritic Cells; Epitopes; Gamma Rays; gp100 Melanoma Antigen; Humans; Interleukin-10; Interleukin-12; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monocytes; Necrosis; Neoplasm Proteins; Phagocytosis; Receptors, CCR7; Time Factors

Metastatic malignant melanoma is a devastating disease with a poor prognosis. Recent therapeutic trials have focused on immunotherapy to induce development of endogenous antitumor immune responses. To date, such protocols have shown success in activation of tumor-specific CTL but no overall improvement in survival. To kill tumor, antigen-specific CTL must efficiently target and enter tumor tissue. The purpose of this study was to examine the pathway of leukocyte migration to metastatic melanoma.. Peripheral blood and metastatic melanoma tissues (n = 65) were evaluated for expression of adhesion molecules using immunohistochemistry of tumor sections and flow cytometry of tumor-associated and peripheral blood CTL and compared with healthy controls. CTL expressing T-cell receptors for the melanoma antigen MART-1 were identified in a subset of samples by reactivity with HLA-A2 tetramers loaded with MART-1 peptide.. Results show that the majority of metastatic melanoma samples examined do not express the vascular adhesion receptors E-selectin (CD62E), P-selectin (CD62P), and intercellular adhesion molecule-1 (CD54) on vessels within the tumor boundaries. Strong adhesion receptor expression was noted on vessels within adjacent tissue. Tumor-associated T lymphocytes accumulate preferentially in these adjacent areas and are not enriched for skin- or lymph node-homing receptor phenotype.. Expression of leukocyte homing receptors is dysregulated on the vasculature of metastatic melanoma. This results in a block to recruitment of activated tumor-specific CTL to melanoma metastases and is a likely factor limiting the effectiveness of current immunotherapy protocols.

Topics: Antigens, Neoplasm; CD8 Antigens; Cell Adhesion; E-Selectin; Humans; Intercellular Adhesion Molecule-1; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Neoplasm Proteins; P-Selectin; Receptors, Antigen, T-Cell; Receptors, Lymphocyte Homing; Skin Neoplasms; T-Lymphocytes, Cytotoxic

Previously, we found that human dendritic cells (hDCs) pulsed with a melanoma cell lysate (MCL) and stimulated with TNF-alpha (MCL/TNF) acquire a mature phenotype in vitro and are able to trigger tumor-specific immune responses when they are used in melanoma immunotherapy in patients. In this study, we describe that MCL/TNF induces gap junction (GJ)-mediated intercellular communications and promotes melanoma Ag transfer between ex vivo produced hDCs from melanoma patients. hDCs also exhibit increased expression of the GJ-related protein connexin 43, which contributes to GJ plaque formation after MCL/TNF stimulation. The addition of GJ inhibitors suppresses intercellular tumor Ag transfer between hDCs, thus reducing melanoma-specific T cell activation. In summary, we demonstrate that MCL/TNF-stimulated hDCs can establish functional GJ channels that participate in melanoma Ag transfer, facilitating Ag cross-presentation and an effective dendritic cell-mediated melanoma-specific T cell response. These results suggest that GJs formed between hDCs used in cancer vaccination protocols could be essentials for the establishment of a more efficient antitumor response.

Topics: Antigen Presentation; Antigens, Neoplasm; Cell Communication; Cell Differentiation; Cell Line, Tumor; Cross-Priming; Dendritic Cells; Fluorescent Dyes; Gap Junctions; Humans; Isoquinolines; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; T-Lymphocytes, Cytotoxic; Tumor Necrosis Factor-alpha

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Fluorescent Antibody Technique, Indirect; Humans; Immunoenzyme Techniques; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanocytes; Melanoma; Neoplasm Proteins; Precancerous Conditions; Skin; Skin Neoplasms

Examine the accuracy of sentinel lymph node biopsy (SNB) in scalp melanoma (SM), patterns of nodal metastases, patient outcomes, and the utility of immunohistochemistry (IHC) in SNB evaluation.. Retrospective.. There were 22 patients, 4 females and 18 males. Sentinel lymph nodes (SLN) were localized via preoperative lymphoscintigraphy, intraoperative gamma probe, and Lymphazurin injection. SLNs were stained with hematoxylin-eosin, S-100, HMB-45, Melan-A, micropthalmia transcription factor, and tyrosinase. SLNs were grouped into cervical (levels 1-5) and extracervical (parotid, suboccipital, retroauricular) regions.. There were 13 posterior and 9 anterior SMs. The first SNB were mapped to the extracervical regions in 77% of posterior and 78% of anterior lesions. SLN number ranged from 1 to 5. Ten patients had positive SLNs (PSLN). Forty percent of the PSLN group had SLNs mapped in both cervical and extracervical sites. Six underwent completion lymphadenectomy, with no additional positive nodes identified. No significant difference between PSLN and negative sentinel node (NSLN) patients was seen when compared by SLN number, Breslow's thickness, tumor ulceration, and clinical outcomes. Mean follow-up was 35 months. One patient died of disease. One isolated regional recurrence occurred. Sixty percent of PSLN and 92% of NSLN patients were recurrence free at last follow-up. One distant metastasis occurred in the NSLN group, and one local, one regional, and two patients with distant metastases were in the PSLN group at the time of last follow-up. Additional IHC did not detect other metastases in the NSLN group.. SM is aggressive, as demonstrated by the high rate of SLN metastases, and there were no significant histopathologic factors in the primary tumor that predicted the presence of SLN metastases. SNB was accurate. The majority of first SLNs were localized in extracervical basins.

Topics: Adolescent; Adult; Aged; Antigens, Neoplasm; Biomarkers, Tumor; Child; Female; Follow-Up Studies; Head and Neck Neoplasms; Humans; Immunohistochemistry; Lymph Node Excision; Lymph Nodes; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Microphthalmia-Associated Transcription Factor; Middle Aged; Monophenol Monooxygenase; Neck; Neoplasm Proteins; Prognosis; Retrospective Studies; S100 Proteins; Scalp; Sentinel Lymph Node Biopsy; Skin Neoplasms

The histopathologic distinction between pigmented actinic keratosis (PAK) and atypical junctional melanocytic proliferations (AJMP) is a common problem, and it is one with meaningful clinical significance. Previous publications have suggested that Melanocyte Antigen Related to T-cells-1 (MART-1)--a melanocytic marker related to host immune response--was not useful in making this interpretative separation. To revisit that assertion, the authors selected 68 specimens that concerned the diagnosis of PAK vs. AJMP. The degree of morphologic difficulty attached to each case was rated semiquantitatively using a three-tiered scale, and interpretative problems were caused by cytologic similarity between atypical keratinocytes and aberrant melanocytes, obscuring lichenoid inflammation, subepidermal fibrosis, and an absence of clearly defined cell nests at the dermoepidermal junction. Each biopsy sample was immunostained for MART-1 (using antibody clone A103) with azure-B counterstaining; the principal criterion for a diagnosis of AJMP was that of confluent cellular positivity over at least 1 high-power (x400) microscopic field, in conjunction with nested cell growth. The specimens were then re-examined diagnostically. Immunostaining definitely improved interpretative certitude in 65 examples (96% effectiveness); the final diagnosis was that of PAK for 21 lesions and AJMP for 47. Three specimens--all of which represented AJMP--did not benefit by MART-1 staining. It is concluded that MART-1 immunostaining with azure-B counterstaining is a useful adjunct in the interpretation of problematic intra-epidermal pigmented lesions.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Biopsy; Cell Division; Diagnosis, Differential; Epidermis; Humans; Immunohistochemistry; MART-1 Antigen; Melanocytes; Melanoma; Neoplasm Proteins; Skin Neoplasms

The detection of micrometastases (defined as groups of malignant cells) in the sentinel lymph node (SLN) is an important prognostic tool in melanoma. The use of immunohistochemistry with melanocytic markers such as HMB45 and Melan A increases the detection rate of micrometastases but there are also cases with isolated immunohistochemically positive cells (IPC). To determine the prognostic significance of isolated HMB45 and/or Melan A positive cells in melanoma SLN, we compared the clinical course of 47 patients with IPC to 308 patients with negative SLN and to 122 patients with micrometastases. The mean follow-up was 38.1 months. By Kaplan-Meier analyses, relapse free survival and overall survival of patients with IPC were similar to SLN negative patients, whereas patients with micrometastases had a significantly worse relapse free survival and overall survival. In the 47 patients with IPC, 6 relapses (12.8%) and 3 melanoma-related death (6.4%) occurred, in the SLN negative patients 36 relapses (11.7%) and 17 melanoma-related deaths (5.5%), in the patients with micrometastases 46 relapses (37.7%) and 29 melanoma-related deaths (23.8%). Prognosis of patients with IPC in SLN did not correlate with type of positive staining (HMB45, Melan A, or both), capsular involvement, number of cells, presence of cytologic atypias of IPC, or tumor penetrative depth. In conclusion, with short-term follow-up IPC in melanoma SLN are without prognostic significance.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Biomarkers, Tumor; Child; Disease-Free Survival; Female; Humans; Immunohistochemistry; Lymph Nodes; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasm Proteins; Sentinel Lymph Node Biopsy; Skin Neoplasms; Survival Rate

Melanocytes are specialized pigmented cells that are found in all healthy skin tissue. In certain individuals, diseased melanocytes can form malignant tumours, melanomas, which cause the majority of skin-cancer-related deaths. The melanoma-associated antigenic peptides are presented on cell surfaces via the class I major histocompatibility complex (MHC). Among the melanoma-associated antigens, the melanoma self-antigen A/melanoma antigen recognized by T cells (Melan-A/MART-1) has attracted attention because of its wide expression in primary and metastatic melanomas. Here, a preliminary X-ray crystal structural study of a soluble cognate T-cell receptor (TCR) in complex with a pMHC presenting the Melan-A peptide (ELAGIGILTV) is reported. The TCR and pMHC were refolded, purified and mixed together to form complexes, which were crystallized using the sitting-drop vapour-diffusion method. Single TCR-pMHC complex crystals were cryocooled and used for data collection. Diffraction data showed that these crystals belonged to space group P4(1)/P4(3), with unit-cell parameters a = b = 120.4, c = 81.6 A. A complete data set was collected to 3.1 A and the structure is currently being analysed.

Topics: Antigens, Neoplasm; Crystallization; DNA, Complementary; Humans; Major Histocompatibility Complex; MART-1 Antigen; Melanocytes; Melanoma; Neoplasm Proteins; Plasmids; Receptors, Antigen, T-Cell; Recombinant Proteins; Skin Neoplasms; X-Ray Diffraction

Topics: Aged, 80 and over; Antigens, Neoplasm; Biopsy; Diagnosis, Differential; Female; Giant Cells; Humans; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; S100 Proteins; Skin Neoplasms; Xanthomatosis

Negative co-stimulatory signaling mediated via cell surface programmed death (PD)-1 expression modulates T and B cell activation and is involved in maintaining peripheral tolerance. In this study, we examined the effects of a fully human PD-1-abrogating antibody on the in vitro expansion and function of human vaccine-induced CD8+ T cells (CTLs) specific for the melanoma-associated antigens glycoprotein 100 (gp100) and melanoma antigen recognized by T cells (MART)-1. PD-1 blockade during peptide stimulation augmented the absolute numbers of CD3+, CD4+, CD8+ and gp100/MART-1 MHC:peptide tetramer+ CTLs. This correlated with increased frequencies of IFN-gamma-secreting antigen-specific cells and augmented lysis of gp100+/MART-1+ melanoma targets. PD-1 blockade also increased the fraction of antigen-specific CTLs that recognized melanoma targets by degranulation, suggesting increased recognition efficiency for cognate peptide. The increased frequencies and absolute numbers of antigen-specific CTLs by PD-1 blockade resulted from augmented proliferation, not decreased apoptosis. Kinetic analysis of cytokine secretion demonstrated that PD-1 blockade increased both type-1 and type-2 cytokine accumulation in culture without any apparent skewing of the cytokine repertoire. These findings have implications for developing new cancer immunotherapy strategies.

Topics: Antibodies, Monoclonal; Antigens, CD; Antigens, Neoplasm; Apoptosis Regulatory Proteins; Cytokines; gp100 Melanoma Antigen; Humans; Lymphocyte Activation; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Programmed Cell Death 1 Receptor; Skin Neoplasms; T-Lymphocytes, Cytotoxic

Gastrointestinal involvement by malignant melanoma is predominantly a metastatic phenomenon. Although primary malignant melanoma of the gastrointestinal tract has been documented in the esophagus, stomach, small bowel, and anorectum, the incidence of primary melanoma of the colon is rare and remains controversial in most cases. We present a case of solitary malignant melanoma of the transverse colon occurring in a 64-year-old African American male patient. Complete dermatologic and ophthalmologic examinations revealed no evidence of a cutaneous or an ocular primary lesion. Microscopic examination of the resection specimen revealed malignant melanoma, which was confirmed by immunohistochemical positivity for S100 and melan-A. We believe that this tumor represents a primary colonic malignant melanoma.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Colonic Neoplasms; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; S100 Proteins; Treatment Outcome

HLA-A2-restricted cytolytic T cells specific for the immunodominant human tumor Ag Melan-A(MART-1) can kill most HLA-matched melanoma cells, through recognition of two naturally occurring antigenic variants, i.e., Melan-A nonamer AAGIGILTV and decamer EAAGIGILTV peptides. Several previous studies have suggested a high degree of TCR cross-reactivity to the two peptides. In this study, we describe for the first time that some T cell clones are exclusively nonamer specific, because they are not labeled by A2/decamer-tetramers and do not recognize the decamer when presented endogenously. Functional assays with peptides gave misleading results, possibly because decamers were cleaved by exopeptidases. Interestingly, nonapeptide-specific T cell clones were rarely Valpha2.1 positive (only 1 of 19 clones), in contrast to the known strong bias for Valpha2.1-positive TCRs found in decamer-specific clones (59 of 69 clones). Molecular modeling revealed that nonapeptide-specific TCRs formed unfavorable interactions with the decapeptide, whereas decapeptide-specific TCRs productively created a hydrogen bond between CDR1alpha and glutamic acid (E) of the decapeptide. Ex vivo analysis of T cells from melanoma metastases demonstrated that both nonamer and decamer-specific T cells were enriched to substantial frequencies in vivo, and representative clones showed efficient tumor cell recognition and killing. We conclude that the two peptides should be regarded as distinct epitopes when analyzing tumor immunity and developing immunotherapy against melanoma.

Topics: Antigens, Neoplasm; Binding Sites; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cloning, Molecular; Humans; Immunodominant Epitopes; MART-1 Antigen; Melanoma; Models, Molecular; Neoplasm Proteins; Peptide Fragments; Protein Conformation; Protein Structure, Secondary; Receptors, Antigen, T-Cell

We present two cases of melanoma arising in a dysplastic nevus that contained intradermal sebocyte-like melanocytes characterized by a scalloped dark-staining nucleus surrounded by the pale multivacuolated cytoplasm imitating sebaceous differentiation. Both patients were women, aged 49 and 55 years. Location included the back and temporal area. Microscopically, both cases had the following features in common: the melanoma in situ, which was of the superficially spreading type, was associated with a dysplastic compound nevus, in which the sebocyte-like cells were identified in the intradermal nevus part of the lesion. The sebocyte-like cells comprised a minor component but were immediately recognizable and appeared as cells with multivacuolated neoplasm and scalloped nuclei arranged in nests. Some contained melanin in the cytoplasm. They stained positively for S-100 protein and melan A but were EMA and HMB-45 negative. The conventional part of the nevi demonstrated the same phenotype, with the exception of the melanocytic nests located at the dermoepidermal junction that were HMB-45 positive. The cases are discussed in the context of the pertinent literature.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Dysplastic Nevus Syndrome; Female; Humans; MART-1 Antigen; Melanocytes; Melanoma; Middle Aged; Neoplasm Proteins; Neoplasms, Multiple Primary; S100 Proteins; Sebum; Skin Neoplasms

We observed seborrheic keratoses with many basilar clear cells, creating a microscopic pattern that mimicked a seborrheic keratosis involved by melanoma in situ.. We sought to report a series of these seborrheic keratoses and the immunohistochemical stains used to reach a proper diagnosis.. We reviewed 9 cases of seborrheic keratosis that had a distinctive pattern of basal clear cells with ample cytoplasm. All cases were evaluated by conventional microscopy, and Melan-A, S-100, and high molecular weight keratin 903 immunostains.. The basal clear cells failed to react with Melan-A and S-100 protein antisera. In contrast, these cells labeled with an antikeratin antibody in all cases. In all, 7/9 (78%) showed immunopositivity only at the peripheries of cells, creating a pattern that could be mistaken for a negative stain if not examined at high magnification.. This is a retrospective review of cases limited to a large referral dermatopathology service.. We describe a previously uncharacterized pattern of seborrheic keratosis that can microscopically mimic melanoma in situ. Careful conventional microscopy coupled with a panel of immunostains can allow the proper diagnosis to be reached.

Topics: Aged; Aged, 80 and over; Antibodies; Antigens, Neoplasm; Diagnosis, Differential; Female; Humans; Immune Sera; Immunoenzyme Techniques; Keratins; Keratosis, Seborrheic; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Retrospective Studies; S100 Proteins; Skin Neoplasms; Staining and Labeling

The detection of melanoma cells in peripheral blood has been proposed to select patients with a high risk of relapse. In this study, tyrosinase and melanoma antigen recognized by T cells 1 (MART-1) mRNA expression was evaluated in serial samples obtained before definitive surgery and during follow-up in patients with American Joint Committee on Cancer stage I-II melanoma. Serial samples (n=2,262) were collected from 236 patients from 1997 to 2002. Analyses of the RNA samples were performed with a calibrated reverse transcriptase-PCR assay. Gender, age, primary tumor site, ulceration, thickness, Clark level, and histological subtype were analyzed together with tyrosinase and MART-1 mRNA treated as updated covariates in a Cox proportional-hazard model. After a median follow-up time of 66 months, 42 out of 236 patients (18%) had relapsed. The following variables were significantly associated with relapse-free survival in the univariate analyses: tyrosinase, MART-1, gender, ulceration, thickness, Clark level, and histological subtype. Entering these covariates into a multivariate Cox analysis resulted in thickness as the single independent prognostic factor (P<0.0001), whereas MART-1 (P=0.07) approached significance at the 5% significance level. The serial measurements of tyrosinase and MART-1 mRNA in peripheral blood of stage I-II melanoma patients cannot be demonstrated to have independent prognostic impact on relapse-free survival.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Humans; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; RNA, Messenger; Skin Neoplasms

The success of active cancer immunotherapy entails a robust induction of tumor-reactive effector and memory CD8+ T cells. We compared the in vivo immunogenicity of the melanoma-associated antigen Melan-A(26-35) encoded by third-generation recombinant lentivector (rec. lv) or as peptide admixed with a strong adjuvant. Ex vivo analyses of immunized HLA-A2/H-2K(b) mice showed that rec. lv triggered a stronger anti-Melan-A CD8+ T -cell response than peptide vaccine. Importantly, the majority of anti-Melan-A T cells elicited by rec. lv expressed the memory marker CD127 at the peak of the primary response. In those mice, memory T cells were detectable several months after priming and could be activated by recall peptide vaccination. These results show that immunization with rec. lv induces not only a strong antigen-specific CD8+ T -cell response but also a long-lasting T-cell memory against a bona fide tumor-associated antigen.

Topics: Animals; Antibody Formation; Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Genetic Vectors; HLA-A2 Antigen; Immunologic Memory; Lentivirus; MART-1 Antigen; Melanoma; Mice; Mice, Transgenic; Neoplasm Proteins; Peptide Fragments; Skin Neoplasms

The authors investigated the expression of S100A1, S100A6, S100B, MelanA, and CEA in conjunctival naevi, primary acquired melanosis (PAM), conjunctival melanoma, and uveal melanoma in order to assess their potential usefulness in the pathological differential diagnosis of these entities.. Paraffin embedded sections of 18 conjunctival naevi, 14 PAM, 16 conjunctival melanomas, and 20 uveal melanomas were immunostained for S100A1, S100A6, S100B, MelanA, and CEA, and expression was scored semiquantitatively.. Expression of S100A1 differed significantly between conjunctival naevi and conjunctival melanoma, with percentages of positive cells of 30.6% and 71.4%, respectively. Conjunctival melanomas had high average scores for S100A1 and S100B (71.4%, 62.9%, respectively), while uveal melanomas also had high S100A1 but low S100B scores (88.5%, 18.5%, respectively). MelanA was highly variable; naevi and uveal melanoma had higher average scores than conjunctival melanoma. CEA was hardly detectable in all four groups.. S100A1 seems to be a possible candidate to differentiate conjunctival naevi from conjunctival melanoma. S100B seems to differentiate between uveal melanoma and conjunctival melanoma. However, the study size was small and therefore the data have to be confirmed by others.

Topics: Antigens, Neoplasm; Biomarkers; Carcinoembryonic Antigen; Cell Cycle Proteins; Conjunctival Diseases; Conjunctival Neoplasms; Diagnosis, Differential; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Melanosis; Neoplasm Proteins; Nerve Growth Factors; Nevus; S100 Calcium Binding Protein A6; S100 Calcium Binding Protein beta Subunit; S100 Proteins; Uveal Neoplasms

Clinical trials have shown that strong tumor antigen-specific CD8 T-cell responses are difficult to induce but can be achieved for T-cells specific for melanoma differentiation antigens, upon repetitive vaccination with stable emulsions prepared with synthetic peptides and incomplete Freund's adjuvant. Here, we show in four melanoma patients that ex vivo detectable T-cells and thus strong T-cell responses can also be induced against the more universal cancer-testis antigens NY-ESO-1 and Mage-A10. Interestingly, all patients had ex vivo detectable T-cell responses against multiple antigens after serial vaccinations with three peptides emulsified in incomplete Freund's adjuvant. Antigen-specific T-cells displayed an activated phenotype and secreted IFNgamma. The robust immune responses provide a solid basis for further development of human T-cell vaccination.

Topics: Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Differentiation; Cross Reactions; Epitopes, T-Lymphocyte; HLA-A2 Antigen; Humans; Immunologic Memory; Lymphocyte Activation; MART-1 Antigen; Melanoma; Membrane Proteins; Neoplasm Proteins; Skin Neoplasms

Topics: Antigens, Neoplasm; Colon; Colonic Neoplasms; Colonoscopy; Female; Humans; Ileal Diseases; Intussusception; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasm Proteins

The adjuvant activities of the human lymphocyte activation gene-3 (LAG-3) molecule have been evaluated in a human setting by investigating the ability of a soluble recombinant human LAG-3 protein (hLAG-3Ig) to enhance the in vitro induction of viral- and tumor-specific CTLs. We found that soluble human LAG-3 significantly sustained the generation and expansion of influenza matrix protein Melan-A/MART-1 and survivin-specific CD8+ T lymphocytes in peripheral blood mononuclear cells (PBMC) of both cancer patients and healthy donors, showing its ability to boost CD8+ T-cell memory response or to prime naive T cells in vitro. The peptide-specific T cells generated in the presence of hLAG-3Ig were endowed with cytotoxic activity and enhanced release of type 1 cytotoxic T (Tc1) cytokines and were able to recognize tumor cells expressing their nominal antigen. Phenotype and cytokine/chemokines produced by antigen-presenting cells (APC) of PBMCs exposed in vitro for 2 days to peptide and hLAG-3Ig indicate that the LAG-3-mediated adjuvant effect may depend on a direct activation of circulating APCs. Our data revealed the activity of hLAG-3Ig in inducing tumor-associated, antigen-specific CD8+ T-cell responses in a human setting and strongly support the conclusion that this recombinant protein is a potential candidate adjuvant for cancer vaccines.

Topics: Adjuvants, Immunologic; Animals; Antigen-Presenting Cells; Antigens, CD; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; CHO Cells; Colorectal Neoplasms; Cricetinae; Cytokines; Epitopes, T-Lymphocyte; Humans; Immunoglobulin G; Interferon-gamma; Lymphocyte Activation; Lymphocyte Activation Gene 3 Protein; MART-1 Antigen; Melanoma; Neoplasm Proteins; Peptides; Recombinant Proteins; T-Lymphocytes, Cytotoxic

There is a need for new technologies to study tissue-based biomarkers. The current gold standard, immunohistochemistry, is compromised by variability in tissue processing and observer bias. Reverse transcription-PCR (RT-PCR), immunocytochemistry, and reverse-phase lysate microarrays (RPM) are promising alternative technologies but have not yet been validated, or correlated, on the same patient-derived tissues. Furthermore, RPM is currently limited by time-consuming microdissection and low amounts of evaluable protein lysates.. Metastatic melanoma was surgically excised from 30 patients and macroscopically dissected from surrounding stroma. Each specimen was processed by formalin-fixation (immunohistochemistry), cytospin (immunocytochemistry), or disaggreagation and enrichment (RT-PCR and RPM). The latter protocol uses immunochromatography to remove hematopoetic-derived cells, thus enriching for melanoma cells. Each sample was measured for the expression of gp100 or MART-1 normalized to actin.. Immunochromatography coupled with RPM (I-RPM) is reproducible (r >/= 0.70) and, for gp100, correlates strongly with immunohistochemistry and immunocytochemistry (r = 0.78 and 0.76, respectively) and moderately with transcript levels, measured by RT-PCR (r = 0.61). In contrast, for MART-1, I-RPM correlates strongly with transcript level (r = 0.78) but only moderately strong correlations are noted with immunohistochemistry and immunocytochemistry (r = 0.64 and 0.59, respectively). In general, transcript levels show only moderately strong correlations with immunohistochemistry and immunocytochemistry (r = 0.41-0.64).. I-RPM is a promising technology for quantitative grading of tissue biomarkers; however, antigen-dependent correlations are noted.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Cell-Free System; Gene Expression Regulation, Neoplastic; gp100 Melanoma Antigen; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Neoplasms; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Skin Neoplasms

Pigment synthesizing melanoma (so-called animal type melanoma) (PSM) is a rare histopathological variant of melanoma so termed because of prominent melanin production and its similarity to a variant of melanoma seen in grey horses. The aim of this study was to report the clinicopathological characteristics of 14 cases of animal type melanoma.. Six patients were female and eight were male with ages ranging from 5 to 52 years (mean 31 years, median 39 years). The head and neck represented the most common site. The clinical diagnosis was of melanoma in seven cases, blue naevus in three cases, benign naevus in three cases and a pigmented basal cell carcinoma in one case. The histological diagnosis of PSM was predicated on the basis of an asymmetrical, predominantly intradermal tumour formed of deeply pigmented, round or short, spindle-shaped dendritic melanocytes with some degree of hyperchromatism and a single nucleolus. Cytological atypia was always present but was not pronounced. A prominent population of macrophages was invariably present. Four tumours were compound and 10 tumours were predominantly intradermal. The mitotic count was usually low, ranging from 1 to 5 per 10 high-power fields (mean 2). Perineural and lymphovascular invasion was not seen. The Breslow thickness ranged from 1.1 to 7.5 mm (mean 3.3 mm). Follow-up was available in 13 patients. The median follow-up period was 5 years. Six patients had no recurrence, three had local recurrence in the form of satellite nodules adjacent to the scar, four had spread to the regional lymph nodes and one patient had distant metastases to the liver. There were no deaths.. This study demonstrates that PSM is a distinctive, possible low-grade variant of melanoma usually lacking the histological features predictive of aggressive behaviour seen in ordinary melanoma. It should be managed in the same way as other melanomas with wide local excision.

Topics: Adolescent; Adult; Antigens, Neoplasm; Child; Child, Preschool; Female; Follow-Up Studies; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanocytes; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasm Proteins; Neoplasm Recurrence, Local; Nevus, Blue; Nevus, Pigmented; S100 Proteins; Skin Neoplasms

The aim of T cell vaccines is the expansion of antigen-specific T cells able to confer immune protection against pathogens or tumors. Although increase in absolute cell numbers, effector functions and TCR repertoire of vaccine-induced T cells are often evaluated, their reactivity for the cognate antigen versus their cross-reactive potential is rarely considered. In fact, little information is available regarding the influence of vaccines on T cell fine specificity of antigen recognition despite the impact that this feature may have in protective immunity. To shed light on the cross-reactive potential of vaccine-induced cells, we analyzed the reactivity of CD8(+) T cells following vaccination of HLA-A2(+) melanoma patients with Melan-A peptide, incomplete Freund's adjuvant and CpG-oligodeoxynucleotide adjuvant, which was shown to induce strong expansion of Melan-A-reactive CD8(+) T cells in vivo. A collection of predicted Melan-A cross-reactive peptides, identified from a combinatorial peptide library, was used to probe functional antigen recognition of PBMC ex vivo and Melan-A-reactive CD8(+) T cell clones. While Melan-A-reactive CD8(+) T cells prior to vaccination are usually constituted of widely cross-reactive naive cells, we show that peptide vaccination resulted in expansion of memory T cells displaying a reactivity predominantly restricted to the antigen of interest. Importantly, these cells are tumor-reactive.

Topics: Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cells, Cultured; Cross-Priming; Epitopes, T-Lymphocyte; Humans; MART-1 Antigen; Melanoma; Neoplasm Proteins; Peptide Fragments; Vaccines, Subunit

Although previous studies have separately shown the utility of circulating tumor cells (CTC) or cell-free tumor-related DNA in blood of cancer patients, there has been no investigation of their association and/or the prognostic value of combining these assessments. To date, the true source of tumor-related DNA in serum remains unknown. We hypothesized that CTC is a possible origin of serum tumor-related methylated DNA and their combination can predict disease outcome. To test this hypothesis, we obtained matched pairs of peripheral blood lymphocytes and serum specimens simultaneously from 50 American Joint Committee on Cancer stage IV melanoma patients before administration of biochemotherapy. Peripheral blood leukocytes were analyzed for three mRNA markers of CTC: MART-1, GalNAc-T, and MAGE-A3. Sera were analyzed for two methylated DNA markers: RASSF1A and RAR-beta2. CTC were detected in 13 of 15 (86%) patients with serum tumor-related methylated DNA and only in 13 of 35 (37%) patients without methylated DNA (P = 0.001). The number of CTC markers detected significantly correlated with methylated DNA (P = 0.008). CTC and methylated DNA were significantly correlated with biochemotherapy-treated patients' outcome. Patients with both CTC and methylated DNA showed significantly poorer response to biochemotherapy (P = 0.02) and worse time to progression and overall survival (P = 0.009 and 0.02, respectively). The correlation between CTC and serum tumor-related methylated DNA and the significant effect of this correlation on disease outcome indicate that a composite molecular assessment in blood may be a useful determinant of disease status and efficacy of systemic therapy for melanoma.

Topics: Adolescent; Adult; Aged; Antigens, Neoplasm; DNA Methylation; DNA, Neoplasm; Female; Humans; Male; MART-1 Antigen; Melanoma; Middle Aged; N-Acetylgalactosaminyltransferases; Neoplasm Proteins; Neoplasm Staging; Neoplastic Cells, Circulating; RNA, Messenger

To evaluate the prognostic significance of molecular staging using reverse transcriptase polymerase chain reaction (RT-PCR) in detecting occult melanoma cells in sentinel lymph nodes (SLNs) and circulating bloodstream.. In this multicenter study, eligibility criteria included patient age 18 to 71 years, invasive melanoma > or = 1.0 mm Breslow thickness, and no clinical evidence of metastasis. SLN biopsy and wide excision of the primary tumor were performed. SLNs were examined by serial-section histopathology and S-100 immunohistochemistry. A portion of each SLN was frozen for RT-PCR. In addition, RT-PCR was performed on peripheral-blood mononuclear cells (PBMCs). RT-PCR analysis was performed using four markers: tyrosinase, MART1, MAGE3, and GP-100. Disease-free survival (DFS), distant-DFS (DDFS), and overall survival (OS) were analyzed.. A total of 1,446 patients with histologically negative SLNs underwent RT-PCR analysis. At a median follow-up of 30 months, there was no difference in DFS, DDFS, or OS between the RT-PCR-positive (n = 620) and RT-PCR-negative (n = 826) patients. Analysis of PBMC from 820 patients revealed significant differences in DFS and DDFS, but not OS, for patients with detection of more than one RT-PCR marker in peripheral blood.. In this large, prospective, multi-institutional study, RT-PCR analysis on SLNs and PBMCs provides no additional prognostic information beyond standard histopathologic analysis of SLNs. Detection of more than one marker in PBMC is associated with a worse prognosis. RT-PCR remains investigational and should not be used to direct adjuvant therapy at this time.

Topics: Adult; Aged; Antigens, Neoplasm; Female; gp100 Melanoma Antigen; Humans; Leukocytes, Mononuclear; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Staging; Neoplastic Cells, Circulating; Prognosis; Prospective Studies; Reverse Transcriptase Polymerase Chain Reaction; Sentinel Lymph Node Biopsy; Skin Neoplasms; Survival Analysis

Because of its expression pattern restricted to cells of the melanocytic lineage and to melanoma cells, Melan-A is an important target of immunotherapeutic approaches for the treatment of melanoma. Identification of Melan-A derived sequences recognized by specific T cells is therefore of great interest for the development of these therapeutic strategies. Using circulating CD4(+) T cells from healthy donors, we identified two Melan-A-derived CD4(+) T cell epitopes mapping to the 1-20 and 91-110 regions of the protein and restricted by HLA-DR11 and HLA-DR52 molecules, respectively. CD4(+) T cells specific for the identified epitopes were able to recognize the native antigen when endogenously expressed by antigen presenting cells and tumor cells. In addition, CD4(+) T cells specific for Melan-A 91-110 recognized the epitope after exogenous processing and presentation of Melan-A recombinant protein. Identification of these epitopes will be instrumental for the evaluation of the immune response to Melan-A in cancer patients.

Topics: Alleles; Antigens, Neoplasm; CD4-Positive T-Lymphocytes; Cell Line, Transformed; Cell Line, Tumor; Clone Cells; Epitopes, T-Lymphocyte; Histocompatibility Antigens Class II; HLA-DR Antigens; HLA-DR Serological Subtypes; HLA-DR5 Antigen; Humans; MART-1 Antigen; Melanoma; Neoplasm Proteins; Peptide Mapping

Despite major progress in T lymphocyte analysis in melanoma patients, TCR repertoire selection and kinetics in response to tumor Ags remain largely unexplored. In this study, using a novel ex vivo molecular-based approach at the single-cell level, we identified a single, naturally primed T cell clone that dominated the human CD8(+) T cell response to the Melan-A/MART-1 Ag. The dominant clone expressed a high-avidity TCR to cognate tumor Ag, efficiently killed tumor cells, and prevailed in the differentiated effector-memory T lymphocyte compartment. TCR sequencing also revealed that this particular clone arose at least 1 year before vaccination, displayed long-term persistence, and efficient homing to metastases. Remarkably, during concomitant vaccination over 3.5 years, the frequency of the pre-existing clone progressively increased, reaching up to 2.5% of the circulating CD8 pool while its effector functions were enhanced. In parallel, the disease stabilized, but subsequently progressed with loss of Melan-A expression by melanoma cells. Collectively, combined ex vivo analysis of T cell differentiation and clonality revealed for the first time a strong expansion of a tumor Ag-specific human T cell clone, comparable to protective virus-specific T cells. The observed successful boosting by peptide vaccination support further development of immunotherapy by including strategies to overcome immune escape.

Topics: Antigen Presentation; Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Differentiation; Clone Cells; Cytotoxicity Tests, Immunologic; Disease Progression; Epitopes, T-Lymphocyte; Humans; Immunization, Secondary; Immunodominant Epitopes; Lymphatic Metastasis; Lymphocyte Count; MART-1 Antigen; Melanoma; Neoplasm Proteins; Receptors, Antigen, T-Cell; Time Factors; Vaccines, Subunit

Desmoplastic melanoma is a diagnostic and therapeutic challenge. Immunohistochemical analysis with antibodies to melanoma antigens can complement morphologic evaluation. Although staining for S100 protein is generally positive, staining for other melanoma differentiation antigens, particularly gp100, Melan-A/MART1 and tyrosinase, is often negative despite being commonly positive in other melanoma types. A high clinical index of suspicion and better diagnostic techniques are essential as atypical features and incorrect diagnosis can lead to poor clinical outcomes. Antigens associated with melanoma, such as the melanocyte differentiation and cancer testis antigen, may become important targets for immune therapies. We characterized the patterns of antigen expression of desmoplastic melanoma from 32 patients, including gp100, Melan-A/MART-1, tyrosinase, MAGE-A1, MAGE-A4 and NY-ESO-1. Consistent positive staining with S100 was observed. Differentiation antigens were expressed more frequently than cancer testis antigens regardless of the histological subtype of desmoplastic melanoma. When present, cancer testis antigen expression correlated to positive staining with differentiation antigens. The diagnostic yield of desmoplastic melanoma did not increase with the addition of cancer testis antigen typing. Low levels of expression of cancer testis antigen may indicate that they are suboptimal targets for vaccine development in desmoplastic melanoma.

Topics: Antigens, Differentiation; Antigens, Neoplasm; Biomarkers, Tumor; Cell Differentiation; gp100 Melanoma Antigen; Humans; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Membrane Glycoproteins; Membrane Proteins; Monophenol Monooxygenase; Neoplasm Proteins; Skin Neoplasms

To help distinguish early melanoma from normal sun-damaged skin by quantifying the density, confluence, and depth of follicular penetration of melanocytes in long-standing sun-exposed skin of the face and neck.. Case series.. Referral center.. Random selection of 149 patients undergoing Mohs surgery for basal cell and squamous cell carcinomas of the face and neck.. Frozen-section slides were made from long-standing sun-exposed normal skin and stained with MART-1 (melanoma antigen recognized by T cells 1 staining) immunostain.. The number, confluence, and depth of penetration of melanocytes along the follicular epithelium were quantified per high-power field (original magnification x 400, equivalent to 0.5 mm of skin). Confluence was categorized by the number of adjacent melanocytes (none, 0-1; mild, 2; moderate, 3-6; and severe, >6).. The mean number of melanocytes per high-power field was 15.6 (range, 6-29). Confluence was severe in 1.0% of the specimens, moderate in 34.0%, mild in 54.0%, and absent in 11.0%. Focal areas of increased melanocyte density occurred in 24.2% of the specimens; in these areas, the mean number of melanocytes per high-power field was 20.3 (range, 7-36) and the confluence of melanocytes was severe in 13.0%, moderate in 50.0%, and mild in 37.0%. The mean depth of follicular epithelium penetration by melanocytes was 0.38 mm. Pagetoid spread and nesting of melanocytes were not seen. Nonspecific scattered MART-1-staining dermal cells were in half of the slides.. Melanocytes in long-standing sun-exposed skin have an increased density and a confluence that is often moderate (3-6 adjacent melanocytes), but they do not exhibit pagetoid spread or nesting. Nonspecific MART-1-staining dermal cells should not be interpreted as invasive melanoma.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Female; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanocytes; Melanoma; Middle Aged; Neoplasm Proteins; Predictive Value of Tests; Skin; Skin Neoplasms; Sunlight

Although increasing evidence suggests that CTL are important to fight the development of some cancers, the frequency of detectable tumor-specific T cells is low in cancer patients, and these cells have generally poor functional capacities, compared with virus-specific CD8(+) T cells. The generation with a vaccine of potent CTL responses against tumor Ags therefore remains a major challenge. In the present study, ex vivo analyses of Melan-A-specific CD8(+) T cells following vaccination with Melan-A peptide and CpG oligodeoxynucleotides revealed the successful induction in the circulation of effective melanoma-specific T cells, i.e., with phenotypic and functional characteristics similar to those of CTL specific for immunodominant viral Ags. Nonetheless, the eventual impact on tumor development in vaccinated melanoma donors remained limited. The comprehensive study of vaccinated patient metastasis shows that vaccine-driven tumor-infiltrating lymphocytes, although activated, still differed in functional capacities compared with blood counterparts. This coincided with a significant increase of FoxP3(+) regulatory T cell activity within the tumor. The consistent induction of effective tumor-specific CD8(+) T cells in the circulation with a vaccine represents a major achievement; however, clinical benefit may not be achieved unless the tumor environment can be altered to enable CD8(+) T cell efficacy.

Topics: Adult; Aged; Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Movement; Clone Cells; CpG Islands; Cytomegalovirus; Epitopes, T-Lymphocyte; Herpesvirus 4, Human; Humans; Immunophenotyping; Lymphocyte Activation; Lymphocyte Count; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Neoplasm Proteins; Oligodeoxyribonucleotides; T-Lymphocytes, Regulatory; Tumor Cells, Cultured; Vaccines, Subunit

The sentinel lymph node (SLN) biopsy has become an increasingly important procedure used in the primary staging of malignant melanoma. However, micrometastases in a lymph node can be easily missed on routine H&E-stained sections. Therefore, S-100 and HMB-45 IHC stains are standardly performed on grossly negative SLNs for detection of metastatic melanoma. Each of these IHC markers, however, is not ideal. The authors investigated whether the newer IHC marker Melan-A would improve the detection of metastatic melanoma in SLN biopsies. Forty lymph nodes previously diagnosed with metastatic melanoma were retrospectively evaluated for S-100, HMB-45, and Melan-A expression. In addition, 42 SLN biopsies for metastatic melanoma detection were prospectively collected and evaluated for S-100, HMB-45, and Melan-A expression. All lymph nodes with metastatic melanoma from the retrospective study demonstrated S-100 reactivity. Five of the lymph nodes with metastatic melanoma from the retrospective study failed to express either HMB-45 or Melan-A, all of which displayed a desmoplastic morphology. One of the metastases positive for S-100 and HMB-45 failed to show reactivity with Melan-A (3%). The prospective study found 10 lymph nodes from 42 cases to be positive for metastatic melanoma, which were positive for S-100 (100%). Nine of the involved lymph nodes were positive for HMB-45(90%), and nine were positive for Melan-A (90%). Melan-A, although very specific, cannot replace the use of S-100 and HMB-45 for the detection of metastatic melanoma in SLNs. It can, however, substitute for HMB-45 with equally good results.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Humans; Lymph Nodes; Lymphatic Metastasis; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; Prospective Studies; Retrospective Studies; S100 Proteins; Sentinel Lymph Node Biopsy

The identification of tumor antigens recognized by cytotoxic and T helper lymphocytes has led to the development of specific cancer vaccines. Immunization with tumor antigen-pulsed dendritic cells has proved effective at eliciting elevated levels of tumor antigen-specific T cells in patient blood, but objective clinical responses remain rare, suggesting that vaccine-induced T cells are not trafficking optimally to site(s) of tumor burden. Accumulating evidence from animal models suggests that route of immunization can have a substantial influence on the subsequent migration of primed, activated T cells in vivo.. In a clinical trial designed to elicit more effective cytotoxic T-cell mediated antitumor responses, metastatic melanoma patients were immunized directly via a peripheral intralymphatic route with autologous dendritic cells pulsed with HLA-A*0201-restricted melanoma-associated peptide antigens derived from MART-1 and gp100.. Within 10 days of intralymphatic dendritic cell vaccination, four of six patients developed dramatic and diffuse erythematous rashes in sun-exposed areas of skin that showed extensive T-cell infiltration. CTLs grown from rash biopsies were strongly enriched for tumor antigen-specific T cells that had elevated expression of cutaneous lymphocyte antigen and chemokine receptor-6, consistent with a skin-homing phenotype. Of note, the only patient in the study with cutaneously localized disease showed a significant regression of metastatic lesions following the development of a surrounding rash.. The evidence presented here is consistent with immunization studies in animal models and supports the concept that T cells are "imprinted" in peripheral lymph node sites to express specific ligands and chemokine receptors that allow them to migrate to skin. Furthermore, the preferential migration of the T cells to sun-exposed cutaneous sites suggests that inflammation plays a critical role in this migration. These observations suggest that further study of the effects of immunization route and inflammation on T-cell migration in humans is warranted, and could lead to vaccination approaches that would more reliably direct trafficking of activated T cells to diverse sites of metastatic disease.

Topics: Adult; Antigens, Neoplasm; Cancer Vaccines; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cells, Cultured; Cytotoxicity Tests, Immunologic; Dendritic Cells; Female; gp100 Melanoma Antigen; HLA-A2 Antigen; Humans; Immunotherapy; Male; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Middle Aged; Neoplasm Proteins; Skin Neoplasms; T-Lymphocytes, Cytotoxic; Vaccination

Topics: Adoptive Transfer; Animals; Antigens, Neoplasm; Cancer Vaccines; Epitopes; Genetic Engineering; Genetic Therapy; Humans; Interleukin-2; Lymphocyte Activation; Lymphocyte Count; MART-1 Antigen; Melanoma; Neoplasm Proteins; Receptors, Antigen, T-Cell; T-Lymphocytes, Cytotoxic; Transgenes

Heterogeneous expression of melanocytic antigens occurs frequently in melanomas and represents a potent barrier to immunotherapy. We previously showed that coordinated losses of several melanocytic antigens are generally attributable to down-regulation of antigen gene expression rather than irreversible mutation. Treatment of melanoma cells with mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors blocks ERK activation and increases steady-state levels of mRNAs and corresponding protein expression for the melanocytic antigens Melan-A/MART-1, gp100, and tyrosinase. Although the degree of MEK inhibitor enhancement of antigen expression varied among different cell lines irrespective of their antigen expression status, all showed detectable responses. Notably, the antigen-enhancing effects of the MEK inhibitors could not be attributed to the master melanocytic regulator MITF-M. Because MAPK pathway activation via constitutively active mutant forms of BRAF is common in melanomas, correlation between BRAF function and antigen expression was investigated. No simple correlation of endogenous BRAF mutational status and antigen levels was observed, but transient overexpression of V600E BRAF increased ERK activation and reduced Melan-A/MART-1 levels in antigen-positive cell lines. These data indicate that whereas multiple factors may regulate antigen expression in melanomas, enhancement of MAPK signaling can act as a negative influence. Blocking such signaling with MEK inhibitors accordingly augments antigen levels, thereby enhancing Melan-A/MART-1-specific cytotoxic T-cell responses to antigen-negative cells following MEK inhibition treatment. Consequently, MAPK inhibition may assist targeting of melanomas for immunotherapy.

Topics: Antigens, Neoplasm; Butadienes; Cell Line, Tumor; Enzyme Inhibitors; Humans; MAP Kinase Signaling System; MART-1 Antigen; Melanocytes; Melanoma; Microphthalmia-Associated Transcription Factor; Mitogen-Activated Protein Kinases; Neoplasm Proteins; Nitriles; Organic Chemicals; Polymorphism, Restriction Fragment Length; Proto-Oncogene Proteins B-raf; T-Lymphocytes, Cytotoxic; Transcription, Genetic; Transfection

Over the past decade, many efforts have been made to identify MHC class II-restricted epitopes from different tumor-associated Ags. Melan-A/MART-1(26-35) parental or Melan-A/MART-1(26-35(A27L)) analog epitopes have been widely used in melanoma immunotherapy to induce and boost CTL responses, but only one Th epitope is currently known (Melan-A51-73, DRB1*0401 restricted). In this study, we describe two novel Melan-A/MART-1-derived sequences recognized by CD4 T cells from melanoma patients. These epitopes can be mimicked by peptides Melan-A27-40 presented by HLA-DRB1*0101 and HLA-DRB1*0102 and Melan-A25-36 presented by HLA-DQB1*0602 and HLA-DRB1*0301. CD4 T cell clones specific for these epitopes recognize Melan-A/MART-1+ tumor cells and Melan-A/MART-1-transduced EBV-B cells and recognition is reduced by inhibitors of the MHC class II presentation pathway. This suggests that the epitopes are naturally processed and presented by EBV-B cells and melanoma cells. Moreover, Melan-A-specific Abs could be detected in the serum of patients with measurable CD4 T cell responses specific for Melan-A/MART-1. Interestingly, even the short Melan-A/MART-1(26-35(A27L)) peptide was recognized by CD4 T cells from HLA-DQ6+ and HLA-DR3+ melanoma patients. Using Melan-A/MART-1(25-36)/DQ6 tetramers, we could detect Ag-specific CD4 T cells directly ex vivo in circulating lymphocytes of a melanoma patient. Together, these results provide the basis for monitoring of naturally occurring and vaccine-induced Melan-A/MART-1-specific CD4 T cell responses, allowing precise and ex vivo characterization of responding T cells.

Topics: Amino Acid Sequence; Antigen Presentation; Antigens, Neoplasm; Cancer Vaccines; CD4-Positive T-Lymphocytes; Cell Separation; Clone Cells; Epitopes, T-Lymphocyte; HLA-D Antigens; HLA-DQ Antigens; HLA-DQ beta-Chains; Humans; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Molecular Sequence Data; Neoplasm Proteins; Peptide Fragments; Signal Transduction

Topics: Adolescent; Antigens, Neoplasm; Diagnosis, Differential; Epithelioid Cells; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanocytes; Melanoma; Neoplasm Proteins; Nevus, Blue; Nevus, Pigmented; S100 Proteins; Sentinel Lymph Node Biopsy; Shoulder; Skin Neoplasms

Nineteen patients with high-risk resected stage III and IV melanoma were immunized with three tumor antigen epitope peptides from gp100, MART-1, and tyrosinase emulsified with adjuvant Montanide ISA 51 and received a fully human anti-cytotoxic T-lymphocyte antigen-4 (anti-CTLA-4) monoclonal antibody MDX-010. Each of three cohorts received escalating doses of antibody with vaccine primarily to evaluate the toxicities and maximum-tolerated dose (MTD) of MDX-010 with vaccine. MDX-010 pharmacokinetics and immune responses were secondary end points.. Peptide immunizations with MDX-010 were administered every 4 weeks for 6 months and then every 12 weeks for 6 months. A leukapheresis to obtain peripheral-blood mononuclear cells for immune analyses was performed before treatment and after the sixth vaccination. Patients were observed until relapse.. Grade 3 gastrointestinal (GI) toxicity (diarrhea or abdominal pain) was observed in three patients in the highest dose cohort and one in the middle dose cohort who seemed to be autoimmune. That defined the MTD with vaccine on this schedule at 1 mg/kg. Of eight patients with evidence of autoimmunity, three have experienced disease relapse. Of 11 patients without autoimmune symptoms, nine have experienced disease relapse. Significant immune responses were measured by tetramer and enzyme-linked immunospot assays against gp100 and MART-1.. Dose-related autoimmune adverse events, predominantly skin and GI toxicities, were reversible. Patients mounted an antigen-specific immune response to a peptide vaccine when combined with a human anti-CTLA-4 antibody.

Topics: Antibodies, Monoclonal; Antigens, CD; Antigens, Differentiation; Antigens, Neoplasm; Autoimmunity; Cancer Vaccines; CTLA-4 Antigen; Female; Flow Cytometry; gp100 Melanoma Antigen; Humans; Male; Mannitol; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Staging; Oleic Acids; Peptide Fragments; Receptors, CCR; Receptors, Chemokine; Vaccination

Downregulation of melanoma-associated antigens (MAAs), against which natural cytolytic T lymphocytes (CTLs) exist in humans, is one of the mechanisms that aids in evasion of immune surveillance. In view of putative re-expression strategies for MAAs during immunotherapy, this study was conducted to investigate MAA silencing in malignant melanoma.. The expression of the MAA Melan-A/MART-1 was analyzed in 10 uveal and 10 cutaneous patient-derived melanoma cell lines by Western blot analysis and RT-PCR. Expression characteristics of four other MAAs-Tyr, Tyrp1, Dct, and gp100/Pmel17-were analyzed by RT-PCR. DNA methylation patterns at the Melan-A/MART-1 promoter region were investigated by methylation-sensitive restriction enzyme digestion and subsequent Southern blot analysis. Exogenous promoter activity was assessed in all 20 melanoma cell lines to correlate the DNA methylation patterns with Melan-A/MART-1 expression.. MAA expression was observed in 15 of the 20 melanoma cell lines. Furthermore, there is a direct correlation between DNA methylation patterns at the Melan-A/MART-1 promoter region, exogenous Melan-A/MART-1 promoter activity, and Melan-A/MART-1 protein expression. These data reveal the division of patient-derived melanoma cell lines into two distinct subsets, which are identical for both uveal and cutaneous tumor types.. The authors propose a categorization of melanoma cell lines into two different panels based on shared MAA-expression characteristics: panel I, MAA-expressing cell lines, and panel II, MAA-deficient cell lines. This categorization can be used to obtain knowledge about the regulation of MAA-expression and for further research concerning MAA-based immunotherapy.

Topics: Antigens, Neoplasm; Blotting, Southern; Blotting, Western; DNA Methylation; DNA, Neoplasm; Gene Expression; Genes, Reporter; Humans; MART-1 Antigen; Melanoma; Neoplasm Proteins; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skin Neoplasms; Tumor Cells, Cultured; Uveal Neoplasms

Heat shock has been shown to have pleiotropic effects on tumor physiology besides a direct cytotoxic effect. In the present study, we address the question whether heat shock treatment has an impact on the antigenicity of human melanoma cells and their specific recognition by cytotoxic lymphocytes. The heat shock response was induced by treating the cells with two different thermal isoeffect doses, which resulted in equivalent clonogenic survival, mimicking doses achieved during clinical hyperthermia treatment of tumors. Antigen expression and immune recognition by cytotoxic T cells was studied using the human melanoma cell lines 624.38-MEL, SK-MEL23, WM115 and WM266-4, which naturally express, process and present tyrosinase and Melan-A/melanoma antigen recognized by T cells (MART)-1-derived peptides in the context of HLA-A2 molecules. We demonstrate that during the heat shock response following the two thermal doses, heat shock protein 70 (Mr 72 kDa) (HSP70) was induced with differential kinetics; tyrosinase protein and mRNA levels dissociated with a significant increase in tyrosinase protein and a decrease in transcript levels. A similar dissociation was not observed for Melan-A/MART-1. Furthermore, tyrosinase-specific T-cell recognition did not correlate with changes in HSP70 and antigen protein levels. These results suggest that caution has to be taken when considering protein levels as a marker for the antigenic status of a tumor. Moreover, these results document the maintenance of immunological homeostasis during recovery from heat treatment, thus challenging the view that tumor cells subjected to heat shock become resistant to CTL recognition.

Topics: Antigen Presentation; Antigen-Presenting Cells; Antigens, Neoplasm; Cell Line, Tumor; Heat-Shock Response; Histocompatibility Antigens Class I; HLA-A2 Antigen; HSP70 Heat-Shock Proteins; Humans; MART-1 Antigen; Melanoma; Monophenol Monooxygenase; Neoplasm Proteins

Melanoma-specific T cells can occur spontaneously or in response to vaccination or other therapies, but the frequency is much lower than observed in viral infections. The presence of tumor-specific T cells does not necessarily translate into clinical regressions for a variety of reasons such as an insufficient frequency, activation state or homing capacity of the T cells or escape strategies of the tumor. Having screened melanoma patients prior to inclusion in vaccination trials for spontaneous tumor-specific T cells either by Elispot or tetramer-staining, we have identified 3 patients with sufficient numbers of tumor-reactive T cells to more than 1 TAA and at least 1 virus-antigen to perform phenotypic and functional analysis directly ex vivo. These stage IV melanoma patients showed specific CTL against melan-A.A2, tyrosinase.A2 and influenza matrix peptide (IMP).A2 readily detectable in peripheral blood. T-cell receptor (TCR) staining using the tetramer technology was combined with phenotypic characterization and functional assays. In contrast to IMP-specific CTL, melanoma-specific CTL were predominantly terminally differentiated effector cells. However, analysis of melan-A- and tyrosinase-specific T-cell lines showed that only a part of the melanoma-specific CTL were able to lyse peptide-loaded target cells. Interestingly, the described phenotypic and functional differences of melan-A- and tyrosinase-specific CTL appeared not only between patients but were also evident within patients, suggesting that the immune response against various tumor antigens is regulated independently.

Topics: Adult; Aged; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Female; HLA-A Antigens; HLA-A2 Antigen; Humans; Immunophenotyping; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Peptide Fragments; Phenotype; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

We have comparatively evaluated the proliferative response of CTL induced in metastatic melanoma patients upon immunization against Melan-A/MART-1(27-35) tumor associated antigen (TAA) to IL-2, IL-7 or IL-15 cytokines, sharing a receptor common gamma-chain (c gamma-c cytokines). Twenty-eight CTL clones were generated from CD8+ T cells obtained from 3 patients during the contraction phase of immune response following a successful vaccine mediated expansion of specific effectors. All clones were able to kill tumor cell lines expressing HLA-A0201 and Melan-A/MART-1, and displayed phenotypic characteristics of effector/memory (CD45RA-/CCR7-) or CD45RA+/CCR7- effector cells in intermediate to late developmental stage (CD28-/CD276+/-) CTL. Proliferative responses could be elicited or enhanced by IL-2 and IL-15, but not IL-7, in the absence or in the presence of T-cell receptor (TCR) triggering, respectively. Accordingly, only IL-2 and IL-15 were able to promote the survival of the CTL clones under investigation. While all clones expressed high amounts of receptor c gamma-c (CD132), lower, but detectable, expression of IL-7 receptor alpha chain was also observed. CD8+ cells from one of the patients treated were obtained 6 months after the last vaccine boost and were cultured in the presence of Melan-A/MART-1(27-35) and each of the 3 cytokines under investigation. Consistent with data from CTL clones, expansion of Melan-A/MART-1(27-35) tetramer positive cells was only observed in the presence of IL-2 or IL-15 but not IL-7. Instead, when CD8+ cells from the same patient were sampled shortly (14 days) after an additional vaccination only IL-2 was able to promote the expansion of Melan-A/MART-1(27-35) tetramer positive cells. Taken together these data suggest a selective responsiveness of TAA-specific CTL to different c gamma-c cytokines.

Topics: Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Cell Proliferation; HLA-A Antigens; HLA-A2 Antigen; Humans; Immunologic Memory; Immunotherapy; Interleukin-15; Interleukin-2; Interleukin-7; MART-1 Antigen; Melanoma; Neoplasm Proteins; Receptors, Antigen, T-Cell; Receptors, Interleukin-7; Skin Neoplasms; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

More than 125 genes that regulate pigmentation have been identified to date. Of those, MART-1 has been widely studied as a melanoma-specific antigen and as a melanosome-specific marker. Whereas the functions of other melanosomal proteins, such as tyrosinase, tyrosinase-related protein-1, dopachrome tautomerase, and Pmel17, are known, the function of MART-1 in melanogenesis, is unclear. A role for MART-1 in pigmentation is expected because its expression pattern and subcellular distribution is quite similar to the other melanosomal proteins and usually correlates with melanin content. We investigated the function of MART-1 using a multidisciplinary approach, including the use of siRNA to inhibit MART-1 function and the use of transfection to re-express MART-1 in MART-1-negative cells. We show that MART-1 forms a complex with Pmel17 and affects its expression, stability, trafficking, and the processing which is required for melanosome structure and maturation. We conclude that MART-1 is indispensable for Pmel17 function and thus plays an important role in regulating mammalian pigmentation.

Topics: Animals; Antigens, Neoplasm; Cell Line; Endoplasmic Reticulum; Golgi Apparatus; gp100 Melanoma Antigen; Humans; MART-1 Antigen; Melanoma; Melanosomes; Membrane Glycoproteins; Neoplasm Proteins; Pigmentation; Protein Transport; Proteins; RNA, Small Interfering; Subcellular Fractions

Molecular analysis of melanoma sentinel nodes (SN) is sensitive, but poorly specific because metastases cannot be distinguished from benign nevus inclusions (BNI). We investigated whether quantitative reverse transcription-PCR (RT-PCR) detection of MART-1 and tyrosinase mRNAs could improve this specificity and contribute to SN assessment.. Two hundred twenty SNs from 95 melanoma patients analyzed by extensive immunohistopathology and real-time quantitative RT-PCR.. Using histopathology, SNs and patients were allotted to three diagnostic groups: (a) metastasis positive, (b) BNI positive, and (c) melanocyte-free. Median MART-1 and tyrosinase mRNA levels in SNs were significantly different in patients with metastasis compared with patients with BNIs (P < 0.05) and patients without melanocytic lesions (P < 0.001). However, a "gray-zone" was observed where distinction, based on mRNA levels, could not be made between the three groups. For both genes, the highest mRNA level recorded in each RT-PCR-positive patient was positively correlated with Breslow's tumor thickness. For SNs with metastases, tumor burden was significantly correlated to the mRNA level. Using the presence of a MART-1 RT-PCR signal to detect patients with metastases, a sensitivity of 100% and a negative predictive value of 100% were achieved when extensive immunohistology was used as reference.. Quantitative RT-PCR MART-1 and tyrosinase mRNA analysis cannot be used alone for SN diagnosis because of its poor specificity for melanoma metastasis. However, in approximately one third of cases without RT-PCR evidence of MART-1 expression, extensive histopathologic SN investigation is not necessary, thus substantially reducing the cost of SN analysis. The level of melanocyte-associated mRNA is associated with both tumor thickness and tumor burden as measured histopathologically, suggesting that this may be of prognostic value.

Topics: Adult; Aged; Antigens, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Lymph Nodes; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Predictive Value of Tests; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sentinel Lymph Node Biopsy

This paper describes the first documented case of mucoepidermoid carcinoma (MEC) with melanin pigmentation manifested in the palate. Histopathological sections showed a neoplasm composed of epidermoid, mucous-producing and intermediate cells. Numerous large cells contained dark pigmented materials. Fontana Masson staining revealed dendritic melanocytes and melanin granules. HMB-45, Melan A and S-100 protein were all positive for melanocytes. Histopathological examination was not typical for malignant melanoma; the lesion was diagnosed as a low-grade MEC with melanin pigmentation.

Topics: Adult; Antigens, Neoplasm; Biomarkers, Tumor; Carcinoma, Mucoepidermoid; Dendritic Cells; Diagnosis, Differential; Female; Humans; Immunohistochemistry; MART-1 Antigen; Melanins; Melanocytes; Melanoma; Melanoma-Specific Antigens; Melanosis; Neoplasm Proteins; Palatal Neoplasms; S100 Proteins; Salivary Glands, Minor

Dermatofibromas are common lesions that are often associated with epidermal hyperplasia and basal layer hyperpigmentation. A single case of lentiginous melanocytic hyperplasia overlying a dermatofibroma has been reported, however, nevi and melanoma have to the best of our knowledge, not been previously reported. We present 14 cases of melanocytic lesions associated with dermatofibromas. The clinical data and hematoxylin- and eosin- stained sections were obtained and formalin-fixed, paraffin-embedded tissue was immunostained with antibodies against S-100, Mart-1, Factor XIIIa, and CD117. There were nine females and five males ranging in age from 30 to 64 years and anatomic sites included back (five), arm (six), flank (two), and leg (one). The clinical diagnosis ranged from dermatofibroma to desmoplastic melanoma. Histologically, the melanocytic lesions included junctional, compound, and dermal nevi, and malignant melanoma in situ. In four cases the dermal component appeared to merge with the dermatofibroma. In the case of the melanoma in situ, the dermatofibroma abutted the epidermis. Immunohistochemically, the melanocytic lesions were S-100/ Mart-1+, FXIIIa-, and the dermatofibromas were S-100/Mart-1-, FXIIIa+. Melanocytic neoplasia may appear in association with dermatofibromas. The fibrohistiocytic proliferation may be misinterpreted as a spindle or pleomorphic melanocytic process. Awareness of this association will aid in the correct diagnosis, and immunohistochemical studies will help in the differentiation of these two cell populations.

Topics: Adult; Antigens, Neoplasm; Factor XIIIa; Female; Histiocytoma, Benign Fibrous; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanocytes; Melanoma; Middle Aged; Neoplasm Proteins; Nevus; Proto-Oncogene Proteins c-kit; S100 Proteins; Skin Neoplasms

Detection of melanoma cells in circulation may be important in assessing tumor progression. The objective of this study was to develop a specific, reliable multimarker quantitative real-time reverse transcription-PCR (qRT) assay for detecting melanoma cells in patients' blood.. We developed qRT assays for the mRNA of four melanoma-associated markers: MART-1, GalNAc-T, PAX-3, and MAGE-A3. In optimization studies, we tested 17 melanoma cell lines and 49 peripheral blood leukocyte (PBL) samples from volunteers. We performed RNA and melanoma cell dilution studies to assess the detection limits and imprecision of the assays. We measured the mRNAs in blood specimens from 94 melanoma patients [American Joint Committee on Cancer (AJCC) stage I, n = 20; II, n = 20; III, n = 32; IV, n = 22].. All markers were frequently detected in melanoma cell lines, whereas none of the markers was detected in PBLs from volunteers. The qRT assay could detect 1 melanoma cell in 10(7) PBLs in the melanoma cell-dilution studies. Markers were detected in 15%, 30%, 75%, and 86% of melanoma patients with AJCC stage I, II, III, and IV disease, respectively. The number of positive markers and AJCC stage were significantly correlated (Spearman correlation coefficient = 0.58; P < 0.0001).. Multimarker qRT can detect circulating melanoma cells in blood. Measurement of the studied molecular markers in blood may be useful in detection of metastasis and monitoring treatment response of melanoma patients.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Cell Line, Tumor; DNA-Binding Proteins; Double-Blind Method; Gene Dosage; Humans; Leukocytes; Lymph Nodes; MART-1 Antigen; Melanoma; N-Acetylgalactosaminyltransferases; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Staging; Neoplastic Cells, Circulating; Paired Box Transcription Factors; PAX3 Transcription Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skin Neoplasms; Transcription Factors

The infiltration of tumors by T cells has been shown to correlate with prolonged patients' survival. However, it remains unclear why only some tumors are infiltrated with T cells. This study was designed to investigate possible correlations between intratumoral T-cell infiltrates and the expression of cancer-associated antigens and MHC class I and II molecules in patients with melanoma. Fresh frozen samples from 124 stage IV melanoma patients were analyzed by immunohistochemistry for the expression of Melan-A/MART-1, tyrosinase, gp100, NY-ESO-1, and MHC class I and II. Intratumoral T-cell and B-cell infiltrates were detected by staining with anti-CD4, anti-CD8, anti-CD3, and L26 antibodies. The NY-ESO-1 serum antibody status was assessed by Western blot analysis. Intratumoral CD8+ and CD4+ T cells were detected in 63.9% and 71.3% of patients, respectively. We observed a significant heterogeneity of the expression of the melanocyte differentiation antigens, NY-ESO-1, and MHC class I and II molecules. The only significant correlation was found between the expression of MHC class I and the presence of CD4+ and CD8+ T cells (P < 0.0001). There was a strong association between these two variables with respect to the density and distribution of infiltrating T cells and the pattern of MHC class I expression (focal versus homogenous). Intratumoral T-cell infiltration is closely correlated with the MHC class I expression but not with the expression of differentiation antigens, cancer-associated antigens, or MHC class II molecules. These results may have implications for the definition of prognostic variables and for the identification of patients who may benefit from antigen-specific cancer immunotherapy.

Topics: Antibodies, Neoplasm; Antigens, Neoplasm; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; gp100 Melanoma Antigen; Histocompatibility Antigens Class I; Humans; Immunohistochemistry; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Membrane Proteins; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Staging

Topics: Antigens, Neoplasm; Humans; Immunoenzyme Techniques; MART-1 Antigen; Melanoma; Mohs Surgery; Neoplasm Proteins; Skin Neoplasms

The genes for the alpha and beta chains of a highly reactive anti-MART-1 T-cell receptor were isolated from T-lymphocytes that mediated in vivo regression of tumor in a patient with metastatic melanoma. These genes were cloned and inserted into MSCV-based retroviral vectors. After transduction, greater than 50% gene transfer efficiency was demonstrated in primary T-lymphocytes stimulated by an anti-CD3 antibody. The specificity and biologic activity of TCR gene-transduced T-cells was determined by cytokine production after coculture of T-cells with stimulator cells pulsed with MART-1 peptide. The production of interferon-gamma and granulocyte macrophage-colony stimulating factor (GM-CSF) was comparable to highly active MART-1 specific peripheral blood lymphocytes (PBL) in the amount of cytokine produced and transduced cells recognized peptide pulsed cells at dilutions similar to cytotoxic T lymphocyte (CTL) clones. Human leukocyte antigen (HLA) class I restricted recognition was demonstrated by mobilization of degranulation marker CD107a, by cell lysis, by cytokine production, and by proliferation in the presence of HLA-A2-positive but not HLA-A2-negative melanoma cell lines. Similar data was obtained when tumor-infiltrating lymphocytes (TIL) were transduced with the TCR genes, converting previously nonreactive cells to tumor reactive cells. TCR-transduced T-cells are thus attractive candidates for evaluation in cell transfer therapies of patients with cancer.

Topics: Antigens, CD; Antigens, Neoplasm; Cell Proliferation; Coculture Techniques; Cytokines; Gene Transfer Techniques; Genes, T-Cell Receptor; Genetic Vectors; HLA-A2 Antigen; Humans; Immunotherapy; Leukemia, T-Cell; Lysosomal Membrane Proteins; Lysosomal-Associated Membrane Protein 1; MART-1 Antigen; Melanoma; Neoplasm Proteins; T-Lymphocytes; Transduction, Genetic; Tumor Cells, Cultured

Immune-mediated control of tumors may occur, in part, through lysis of malignant cells by CD8(+) T cells that recognize specific Ag-HLA class I complexes. However, tumor cell populations may escape T cell responses by immune editing, by preventing formation of those Ag-HLA complexes. It remains unclear whether the human immune system can respond to immune editing and recognize newly arising escape variants. We report an example of shifting immune responses to escape variants in a patient with sequential metastases of melanoma and long-term survival after surgery alone. Tumor cells in the first metastasis escaped immune recognition via selective loss of an HLA haplotype (HLA-A11, -B44, and -Cw17), but maintained expression of HLA-A2. In the second metastasis, immune escape from an immunodominant MART-1-specific T cell response was mediated by HLA class I down-regulation, resulting in a failure to present this epitope, but persistent presentation of a tyrosinase-derived epitope. Consequent to this modification in tumor Ag presentation, the dominant CTL response shifted principally toward a tyrosinase-targeted response, even though tyrosinase-specific CTL had been undetectable during the initial metastatic event. Thus, in response to immune editing of tumor cells, a patient's spontaneous T cell response adapted, gaining the ability to recognize and to lyse "edited" tumor targets. The observation of both immune editing and immune adaptation in a patient with long-term survival after surgery alone demonstrates an example of immune system reactivity to counteract the escape mechanism(s) developed by tumor cells, which may contribute to the clinical outcome of malignant disease.

Topics: Antigen Presentation; Antigens, Neoplasm; Apoptosis Regulatory Proteins; Binding, Competitive; Cell Line, Tumor; Cell Membrane; Cytotoxicity Tests, Immunologic; Down-Regulation; Epitopes, T-Lymphocyte; HLA-A2 Antigen; Humans; Immunodominant Epitopes; Lymphatic Metastasis; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Recurrence, Local; Nuclear Proteins; Skin Neoplasms; Survivors; T-Lymphocytes, Cytotoxic; Trans-Activators; Transcription Factors; Tumor Escape

The efficiency of melanoma immunotherapy appears to depend on both melanoma- and immune system-specific factors. Melanoma-specific factors include melanoma-associated antigen (MAA) expression as well as HLA class I molecule expression. We investigated the expression of five MAA - Melan-A/MART-1, tyrosinase, gp100, MAGE-1 and MAGE-3 - by means of FACS analysis in 50 melanoma cell cultures and compared them to the cultures of human foreskin-derived melanocytes and melanoma cell line UKRV-Mel2. Melan-A, tyrosinase and gp100 expression was frequently reduced in melanoma cell cultures, compared to that in foreskin melanocytes, whereas MAGE-1 and MAGE-3 expression showed variable degree of upregulation, compared to that in foreskin melanocytes. The expression of all tested MAA demonstrated high interindividual variability. We further show that cell cultures derived from the same tissue sample are oligoclonal in nature, by demonstrating the presence of up to three cell populations bearing distinct MAA profile. Analysing samples derived from the same patient but each at a different time point, we show that MAA expression profile changes over time either in positive (increase) or in negative (decrease) direction. Finally, we demonstrate that brain metastasis-derived cell cultures significantly overexpress Melan-A and MAGE-3, compared to primary tumours and other metastatic sites (P-value range: 0.05-0.001). Elucidation of the MAA expression patterns and the kinetics within the same patient as well as during the course of the disease may help improve current and develop new immunotherapeutic strategies.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Female; gp100 Melanoma Antigen; Humans; Immunotherapy; Interferon Type I; Kinetics; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Membrane Glycoproteins; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Recombinant Proteins; Tumor Cells, Cultured

Improved detection of early-disseminated melanoma cells may eventually translate into more effective patient care. We present a novel strategy for detection of melanoma cells in sentinel lymph nodes and confirm their malignant descent by genomic characterization.. In sentinel lymph nodes from 358 melanoma patients, we prospectively compared the rates of tumor cell detection between immunocytochemistry using HMB45 and Melan A antibodies on disaggregated lymph node samples and standard histopathology (H&E staining and immunostaining on tissue sections). Immunocytochemical melanoma cell detection was controlled by testing lymph node samples from 59 nonmelanoma patients and by isolation and comparative genomic analysis of 30 antigen-positive cells.. Of the 358 patients, 43 (12%) were positive by standard histopathology, whereas HMB45 immunocytochemistry detected 159 of 358 (44%) positive patients. None of the control samples reacted with the HMB45 antibody. Reexamination of samples that were classified as negative by histopathology revealed that extensive serial sectioning would be necessary to achieve sensitivity similar to HMB45 immunocytochemistry. Interestingly, both the number of immunocytochemically positive samples and the number of positive cells in the sentinel node correlated with the thickness of the primary tumor (r = 0.34; P = 0.001 and P < 0.0001, respectively). Twenty-four of 30 isolated immunocytochemically positive cells (80%) displayed chromosomal aberrations, some of which were isolated from histopathologically negative nodes.. Immunocytochemical detection of melanoma cells in sentinel lymph nodes is superior to standard histopathology. It remains to be determined whether the detection and genomic characterization of isolated melanoma cells in sentinel lymph nodes will provide relevant prognostic information.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Child; Chromosome Aberrations; Female; Genome; Humans; Immunohistochemistry; Lymph Nodes; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasm Proteins; Nucleic Acid Hybridization; Prognosis; Sentinel Lymph Node Biopsy

This study was designed to determine the debated prognostic significance of reverse transcriptase-polymerase chain reaction (RT-PCR) positivity in melanoma patients' sentinel lymph node (SLN) negative by conventional histopathology (PATH).. Patients with primary stage I-II cutaneous melanoma underwent radioguided sentinel lymphadenectomy. Their SLNs were assessed for tyrosinase (Tyr) and melanoma antigens recognized by T-cells (MART-1) mRNA expression using RT-PCR, in parallel with hematoxylin and eosin staining and immunohistochemistry. Tyr and MART-1 expression in the SLNs were correlated with PATH assay results, standard prognostic factors, time to progression and overall survival.. Twenty-three of the 124 patients (18.5%) had positive SLNs by both PATH and RT-PCR (PATH+/PCR+). Sixteen patients (13%) were negative by PATH and positive by RT-PCR (PATH-/PCR+). Eighty-five patients (68.5%) had SLNs that were negative by both PATH and RT-PCR (PATH-/PCR-). At a median follow-up of 30 months, recurrence rates among the three cohorts were statistically different (PATH+/PCR+, 60%; PATH-/PCR+, 31%; PATH-/PCR-, 9.4%). Seven of 23 (30%) and two of 16 (12.5%) patients died in the PATH+/PCR+ and PATH-/PCR+ SLN groups, respectively, whereas no patient died in the PATH-/PCR- SLN group.. RT-PCR is more sensitive than PATH to detect SLN metastases and it is a reliable predictor of disease relapse in stage I-II melanoma patients.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Cohort Studies; Disease Progression; Female; Follow-Up Studies; Head and Neck Neoplasms; Humans; Lymph Node Excision; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sentinel Lymph Node Biopsy; Skin Neoplasms; Survival Rate; Time Factors

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Diagnosis, Differential; Humans; Immunohistochemistry; Inflammation; Lichen Planus; MART-1 Antigen; Melanocytes; Melanoma; Neoplasm Proteins; Skin Neoplasms

Primary mucosal melanomas (MMs) of the head and neck are a rare entity. Melanomas with characteristic melanin-pigmented tumor cells are easy to diagnose, but those without melanin-pigmented tumor cells, amelanotic melanomas, are difficult to identify and need immunohistochemistry (IHC) to confirm the final diagnosis. In this study, we examined the expression of three melanocytic differentiation markers, HMB-45, S-100, and Melan-A in primary oral and nasal MMs. We tried to evaluate whether HMB-45, S-100, and Melan-A were useful for diagnosis of primary oral and nasal MMs and to find out which marker was the best of the three.. This study used IHC to examine the expression of HMB-45, S-100, and Melan-A in 17 formalin-fixed paraffin-embedded specimens of primary oral and nasal MMs. The staining intensities (SIs) and labeling indices (LIs) of HMB-45, S-100, and Melan-A in 17 MMs were calculated and compared between any two markers.. Immunostaining results showed that the positive rate was 94% (16 of 17) for HMB-45, 88% (15 of 17) for S-100, and 71% (12 of 17) for Melan-A in 17 MMs. The SI of HMB-45 was significantly higher than that of S-100 (P = 0.0011) or of Melan-A (P = 0.0034). In addition, the mean LI of Melan-A (59 +/- 43%) was significantly lower than that of HMB-45 (83 +/- 28%, P = 0.0065) or of S-100 (79 +/- 33%, P = 0.0237).. Our results indicate that both HMB-45 and S-100 show a high positive rate and LI in MMs and therefore may be good markers for immunohistochemical diagnosis of primary oral and nasal MMs. In addition, HMB-45 may be a more sensitive marker than S-100 because HMB-45 shows a significantly higher SI than S-100 in this study.

Topics: Adult; Aged; Antigens, Neoplasm; Biomarkers, Tumor; Cell Nucleus; Coloring Agents; Cytoplasm; Female; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanocytes; Melanoma; Melanoma-Specific Antigens; Middle Aged; Mouth Mucosa; Mouth Neoplasms; Nasal Mucosa; Neoplasm Proteins; Nose Neoplasms; S100 Proteins; Sensitivity and Specificity

The new monoclonal antibody SM5-1 has been shown to have significant advantages in immunohistochemistry of melanoma over currently used antibodies such as HMB-45 or anti-S100. In this study we compared the immunohistological staining pattern of SM5-1 with that of the more recently described antibodies A103 (anti-MART-1) and T311 (anti-Tyrosinase) in 344 paraffin-embedded melanoma specimens, consisting of 101 primary melanomas (77 SSM, 16 NM, 6 ALM, 2 LMM) and 243 melanoma metastases. The overall reactivity of SM5-1 for all the specimens was 92% (318/344) compared with 83% (285/344) for MART-1 and 71% (245/344) for Tyrosinase. Staining of melanoma metastases with SM5-1 was found in 91% (222/243), but only in 77% (187/243) with A103 and 63% (154/243) with T311, respectively. Staining with SM5-1 was more homogenous with 196 of 243 (80%) of metastatic lesions showing 50% or more positively stained cells within the lesions, whereas A103 and T311 did so in 141 of 243 (58%) or 117 of 243 (48%) of the lesions. With regard to staining intensity of SM5-1, 157 of 243 (64%) showed a strong or very strong staining intensity, whereas A103 and T311 did so in 85 of 243 (35%) or 70 of 243 (29%) of the lesions. Staining intensity and percentage positivity correlated well for SM5-1, because from the 58 very strong positive metastases 55 showed staining in more than 75% of the cells within a lesion. Importantly, 52 of 56 MART-1-negative metastases and 81 of 89 Tyrosinase-negative metastases were positive for SM5-1. Thirty-eight metastases (15.6%) were negative for both A103 and T311. Of those, 35 (92.1%) were positive for SM5-1, demonstrating the value of SM5-1 in identifying melanoma-associated antigen-negative lesions. We conclude that SM5-1 could be of value in immunohistochemistry of melanoma.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Biomarkers, Tumor; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Monophenol Monooxygenase; Neoplasm Metastasis; Neoplasm Proteins; Skin Neoplasms

In this study, we report the adoptive transfer of highly tumor-reactive Melan-A-specific T cell clones to patients with metastatic melanoma, and the follow-up of these injected cells. These clones were generated from HLA-A*0201 patients by in vitro stimulations of total PBMC with the HLA-A*0201-binding Melan-A peptide analog ELAGIGILTV. Ten stage IV melanoma patients were treated by infusion of these CTL clones with IL-2 and IFN-alpha. The generated T cell clones, of effector/memory phenotype were selected on the basis of their ability to produce IL-2 in response to HLA-A*0201 Melan-A-positive melanoma lines. Infused clones were detected, by quantitative PCR, in the blood of three patients for periods ranging from 7 to 60 days. Six patients showed regression of individual metastases or disease stabilization, and one patient experienced a complete response, but no correlation was found between the detection of the infused clones in PBMC or tumor samples and clinical responses. Nonetheless, frequencies of Melan-A/A2-specific lymphocytes, measured by tetramer labeling, increased after treatment in most patients. In one of these patients, who showed a complete response, this increase corresponded to the expansion of new clonotypes of higher avidity than those detected before treatment. Together, our results suggest that infused CTL clones may have initiated an antitumor response that may have resulted in the expansion of a Melan-A-specific CTL repertoire.

Topics: Adoptive Transfer; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Cell Count; Cell Line, Tumor; Clone Cells; Epitopes, T-Lymphocyte; Humans; Immunophenotyping; Lymphocyte Count; MART-1 Antigen; Melanoma; Neoplasm Proteins; T-Lymphocytes, Cytotoxic

This study was conducted to examine the prognostic impact of four biomarkers [tyrosinase and MART-1 messenger RNA (mRNA), S100beta protein and lactate dehydrogenase (LDH)] in patients with metastatic melanoma, together with established clinical factors. Tyrosinase and MART-1 mRNA were measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). S100beta was measured using a commercially available immunoassay, and LDH was analysed conventionally. All markers were measured in blood samples before interleukin-2-based immunotherapy in 85 patients with metastatic melanoma. LDH, S100beta, tyrosinase, number of metastatic sites, location of metastatic sites and performance status were all significant factors for survival in univariate analyses. In multivariate analysis, tyrosinase [hazard ratio (HR)=1.6; 95% confidence interval (CI), 1.1-2.6; P=0.04] and LDH (HR=2.0; 95% CI, 1.1-3.5; P=0.02) were both independent prognostic factors for survival. A combination variable of tyrosinase and LDH remained independently associated with survival (P=0.04) after adjusting for the American Joint Committee on Cancer (AJCC) stage IV classification in a multivariate analysis involving both models. It can be concluded that tyrosinase mRNA and elevated LDH are independent prognostic factors for poor survival in this group of 85 patients. Additional studies are needed before the prognostic value of tyrosinase mRNA in metastatic melanoma can be firmly established. Further evaluation of the combined measurement of tyrosinase mRNA and LDH is warranted.

Topics: Adult; Aged; Antigens, Neoplasm; Biomarkers, Tumor; Female; Humans; Immunotherapy; Interleukin-2; L-Lactate Dehydrogenase; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Multivariate Analysis; Neoplasm Proteins; Nerve Growth Factors; Prognosis; RNA, Messenger; S100 Calcium Binding Protein beta Subunit; S100 Proteins

Vitiligo is a skin disorder characterized by depigmented macules secondary to melanocyte loss. An unusual facet is its relation to melanoma: cytotoxic T lymphocytes directed to melanocyte antigens are found in both conditions and imply a breakdown of tolerance, yet the resulting immune reaction is the opposite. The mechanisms at the basis of these opposite effects are not known. Here, we performed a direct comparison of whole melanocyte-specific T cell populations in the two diseases. We demonstrate that neither precursor frequencies of Melan-A/MART-1-specific T lymphocytes nor their status of activation differ significantly. However, by using a tetramer-based T cell receptor down-regulation assay, we documented a higher affinity of vitiligo T cells. We calculated that the peptide concentration required for 50% of maximal receptor down-regulation differed by 6.5-fold between the two diseases. Moreover, only vitiligo T cells were capable of efficient receptor down-regulation and IFN-gamma production in response to HLA-matched melanoma cells, suggesting that this difference in receptor affinity is physiologically relevant. The differences in receptor affinity and tumor reactivity were confirmed by analyzing Melan-A/MART-1-specific clones established from the two diseases. Our results suggest that the quality, and not the quantity, of the melanocyte-specific cytotoxic responses differs between the two pathologies.

Topics: Antigens, Neoplasm; Cell Line; Clone Cells; Down-Regulation; Female; Humans; Male; MART-1 Antigen; Melanocytes; Melanoma; Neoplasm Proteins; Receptors, Antigen, T-Cell; T-Lymphocytes, Cytotoxic; Vitiligo

Increasing evidence suggests that adoptive transfer of antigen-specific CD8(+) T cells could represent an effective strategy in the fight against chronic viral infections and malignancies such as melanoma. None the less, a major limitation in the implementation of such therapy resides in the difficulties associated with achieving rapid and efficient expansion of functional T cells in culture necessary to obtain the large numbers required for intravenous infusion. Recently, the critical role of the cytokines interleukin (IL)-2, IL-7 and IL-15 in driving T cell proliferation has been emphasized, thus suggesting their use in the optimization of expansion protocols. We have used major histocompatibility complex (MHC) class I/peptide multimers to monitor the expansion of antigen-specific CD8 T lymphocytes from whole blood, exploring the effect of antigenic peptide dose, IL-2, IL-7 and IL-15 concentrations on the magnitude and functional characteristics of the antigen-specific CD8(+) T cells generated. We show here that significant expansions of antigen-specific T cells, up to 50% of the CD8(+) T cell population, can be obtained after a single round of antigen/cytokine (IL-2 or IL-15) stimulation, and that these cells display good cytolytic and interferon (IFN)-gamma secretion capabilities. Our results provide an important basis for the rapid in vitro expansion of autologous T cells from the circulating lymphocyte pool using a simple procedure, which is necessary for the development of adoptive transfer therapies.

Topics: Adoptive Transfer; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Cell Culture Techniques; Cell Division; Cell Line; Dose-Response Relationship, Immunologic; Epitopes, T-Lymphocyte; Humans; Immunophenotyping; Interleukin-15; Interleukin-2; Interleukin-7; MART-1 Antigen; Melanoma; Neoplasm Proteins

To develop an effective pharmaceutical treatment for a disease, we need to fully understand the biological behavior of that disease, especially when dealing with cancer. The current available treatment for cancer may help in lessening the burden of the disease or, on certain occasions, in increasing the survival of the patient. However, a total eradication of cancer remains the researchers' hope. Some of the discoveries in the field of medicine relied on observations of natural events. Among these events is the spontaneous regression of cancer. It has been argued that such regression could be immunologically-mediated, but no direct evidence has been shown to support such an argument. We, hereby, provide compelling evidence that spontaneous cancer regression in humans is immunologically-mediated, hoping that the results from this study would stimulate the pharmaceutical industry to focus more on cancer vaccine immunotherapy. Our results showed that patients with >3 primary melanomas (very rare group among cancer patients) develop significant histopathological spontaneous regression of further melanomas that they could acquire during their life (P=0.0080) as compared to patients with single primary melanoma where the phenomenon of spontaneous regression is absent or minimal. It seems that such regression resulted from the repeated exposure to the tumor which mimics a self-immunization process. Analysis of the regressing tumors revealed heavy infiltration by T lymphocytes as compared to non-regressing tumors (P<0.0001), the predominant of which were T cytotoxic rather than T helper. Mature dendritic cells were also found in significant number (P<0.0001) in the regressing tumors as compared to the non regressing ones, which demonstrate an active involvement of the different arms of the immune system in the multiple primary melanoma patients in the process of tumor regression. Also, MHC expression was significantly higher in the regressing versus the non-regressing tumors (P <0.0001), which reflects a proper tumor antigen expression. Associated with tumor regression was also loss of the melanoma common tumor antigen Melan A/ MART-1 in the multiple primary melanoma patients as compared to the single primary ones (P=0.0041). Furthermore, loss of Melan A/ MART-1 in the regressing tumors significantly correlated with the presence of Melan A/ MART-1-specific CTLs in the peripheral blood of these patients (P=0.03), which adds to the evidence that the phenomenon of r

Topics: Antigens, Neoplasm; Cancer Vaccines; Cytotoxicity Tests, Immunologic; Drug Industry; Humans; MART-1 Antigen; Melanoma; Motivation; Neoplasm Proteins; Neoplasm Regression, Spontaneous; Neoplasms, Unknown Primary; Observation; T-Lymphocytes, Cytotoxic; Technology, Pharmaceutical

Circulating tumor cells (CTCs) in blood may be important in assessing tumor progression and treatment response. We hypothesized that quantitative real-time reverse transcriptase polymerase chain reaction using multimarker mRNA assays could detect CTCs and be used as a surrogate predictor of outcome in patients receiving neoadjuvant biochemotherapy (BC) for melanoma.. Blood specimens were collected at four sampling points from 63 patients enrolled on a prospective multicenter phase II trial of BC before and after surgical treatment of American Joint Committee on Cancer stage III melanoma. Each specimen was assessed by quantitative real-time reverse transcriptase polymerase chain reaction for expression of four melanoma-associated markers: melanoma antigen recognized by T cells 1; beta1 --> 4-N-acetylgalactosaminyltransferase; paired box homeotic gene transcription factor 3; and melanoma antigen gene-A3 family, and the changes of CTCs during treatment and prognostic effect of CTCs after overall treatment on recurrence and survival were investigated.. At a median postoperative follow-up time of 30.4 months, 44 (70%) patients were clinically disease free. In relapse-free patients, the number of detected markers significantly decreased during preoperative BC (P = .036), during postoperative BC (P = .002), and during overall treatment (P < .0001). Marker detection after overall treatment was associated with significant decreases in relapse-free and overall survival (P < .0001). By multivariate analysis using a Cox proportional-hazards model, the number of markers detected after overall treatment was a significant independent prognostic factor for overall survival (risk ratio, 12.6; 95% CI, 3.16 to 50.5; P = .0003).. Serial monitoring of CTCs in blood may be useful for indicating systemic subclinical disease and predicting outcome of patients receiving neoadjuvant BC for metastatic melanoma.

Topics: Adolescent; Adult; Aged; Antigens, Neoplasm; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Chemotherapy, Adjuvant; Cisplatin; Dacarbazine; Female; Humans; Interferon-alpha; Interleukin-2; Male; MART-1 Antigen; Melanoma; Middle Aged; N-Acetylgalactosaminyltransferases; Neoadjuvant Therapy; Neoplasm Invasiveness; Neoplasm Proteins; Neoplasm Recurrence, Local; Neoplastic Cells, Circulating; Paired Box Transcription Factors; PAX3 Transcription Factor; Prospective Studies; Reverse Transcriptase Polymerase Chain Reaction; Risk Factors; RNA, Messenger; RNA, Neoplasm; Skin Neoplasms; Survival Rate; Treatment Outcome; Vinblastine

To assess the effects of the culture of melanoma cells in 3-dimensional (3D) architectures on their immunorecognition by cytotoxic T lymphocytes (CTLs) specific for tumor-associated antigens.. Growth in 3D architectures has been shown to promote the resistance of cancers to treatment with drugs, cytokines, or irradiation, thereby potentially playing an important role in tumor expansion. We investigated the effects of 3D culture on the recognition of melanoma cells by antigen-specific HLA class I-restricted CTLs.. Culture of HBL melanoma cells expressing Melan-A/Mart-1 tumor-associated antigen and HLA-A0201 on poly-2-hydroxyethyl methacrylate (polyHEMA)-coated plates resulted in the generation of aggregates of 400- to 500-microm diameters containing on average 30,000 cells and characterized by slower proliferation, as compared with monolayer (2-dimensional) cultures. HLA-A0201 restricted Melan-A/Mart-127-35-specific CTL clones were used to evaluate tumor cell immunorecognition measured as specific IFN-gamma production. Comparative gene and protein expression in 2D and 3D cultures was studied by real-time PCR and flow cytometry, respectively. Overall differences in gene expression profiles between 2D and 3D cultures were evaluated by high-density oligonucleotide array hybridization.. HLA-A0201 restricted Melan-A/Mart-127-35 specific CTL clones produced high amounts of IFN-gamma upon short-term (4-24 hours) coincubation with HBL cells cultured in 2D but not in 3D, thus suggesting altered antigen recognition. Indeed, Melan-A/Mart-1 expression, at both gene and protein levels, was significantly decreased in 3D as compared with 2D cultures. Concomitantly, a parallel decrease of HLA class I molecule expression was also observed. Differential gene profiling studies on HBL cells showed an increased expression of genes encoding molecules involved in intercellular adhesion, such as junctional adhesion molecule 2 and cadherin-like 1 (>20- and 8-fold up-regulated, respectively) in 3D as compared with 2D cultures.. Taken together, our data suggest that mere growth of melanoma cells in 3D architectures, in the absence of immunoselective pressure, may result in defective recognition by tumor-associated antigen-specific CTL.

Topics: Analysis of Variance; Antigens, Neoplasm; Cell Culture Techniques; Clone Cells; Coculture Techniques; Flow Cytometry; Gene Expression Profiling; HLA-A Antigens; HLA-A2 Antigen; MART-1 Antigen; Melanoma; Neoplasm Proteins; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

There is increasing awareness of the population about the risk of cutaneous melanoma. In recent years, some improvement was gained in the early recognition of clinical warning signs using dermoscopy. As a result, the number of puzzling cases exhibiting limited classical clues for malignancy are submitted to the dermatopathologist. Thus, the risk of microscopic uncertainty or misdiagnosis may be increasing. The aim of the study was to assess retrospectively the contribution of immunohistochemistry in establishing the histological diagnosis of 520 melanocytic neoplasms. According to the disease evolution and the histological presentation, 6 profiles of phenotypical expressions were distinguished.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Biomarkers, Tumor; Factor XIIIa; Glycoproteins; Humans; Immunohistochemistry; Ki-67 Antigen; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Monophenol Monooxygenase; Neoplasm Proteins; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Plant Lectins; Prognosis; Proteins; Retrospective Studies; S100 Proteins; Skin Neoplasms

Immunization with heat shock proteins (hsp) isolated from cancer cells has been shown to induce a protective antitumor response. The mechanism of hsp-dependent cellular immunity has been attributed to a variety of immunological activities mediated by hsp. Hsp have been shown to bind antigenic peptides, trim the bound peptides by intrinsic enzymatic activity, improve endocytosis of the chaperoned peptides by APCs, and enhance the ability of APCs to stimulate peptide-specific T cells. We have investigated the potential capacity of hsp70 and gp96 to function as a mediator for Ag-specific CTL stimulation in an in vitro model for human melanoma. Repetitive stimulation of PBLs by autologous DCs loaded with melanoma-derived hsp did not increase the frequency of T cells directed against immunodominant peptides of melanoma-associated Ags Melan-A and tyrosinase. In contrast, repeated T cell stimulation with peptide-pulsed DCs enhanced the number of peptide-specific T cells, allowing HLA/peptide multimer-guided T cell cloning. We succeeded in demonstrating that the established HLA-A2-restricted CTL clones recognized HLA-A2(+) APCs exogenously loaded with the respective melanoma peptide as well as melanoma cells processing and presenting these peptides in the context of HLA-A2. We were not able to show that these melanoma-reactive CTL clones were stimulated by autologous dendritic cells pulsed with melanoma-derived hsp. These results are discussed with respect to various models for proving the role of hsp in T cell stimulation and to recent findings that part of the immunological antitumor activities reported for hsp are independent of the chaperoned peptides.

Topics: Antigen Presentation; Antigen-Presenting Cells; Antigens, Neoplasm; Cell Line, Transformed; Cell Line, Tumor; Clone Cells; Coculture Techniques; Dendritic Cells; Epitopes, T-Lymphocyte; Heat-Shock Proteins; HLA-A2 Antigen; HSP70 Heat-Shock Proteins; Humans; Interferon-gamma; K562 Cells; Lymphocyte Activation; Lymphocyte Count; MART-1 Antigen; Melanoma; Monophenol Monooxygenase; Neoplasm Proteins; Peptides; T-Lymphocytes, Cytotoxic; Up-Regulation

Tumour-associated antigens (TAA)-specific vaccination requires highly immunogenic reagents capable of inducing cytotoxic T cells (CTL). Soluble peptides are currently used in clinical applications despite an acknowledged poor immunogenicity. Encapsulation into liposomes has been suggested to improve the immunogenicity of discrete antigen formulations. We comparatively evaluated the capacity of HLA-A2.1 restricted Melan-A/MART-1 epitopes in soluble form (S) or following inclusion into sterically stabilised liposomes (SSL) to be recognised by specific CTL, to stimulate their proliferation and to induce them in healthy donors' peripheral blood mononuclear cells (PBMC), as well as in melanoma-derived tumour-infiltrating lymphocytes (TIL). HLA-A2.1(+), Melan-A/MART-1-NA-8 melanoma cells served as targets of specific CTL in 51Cr release assays upon pulsing by untreated or human plasma-treated soluble or SSL-encapsulated Melan-A/MART-1 27-35 (M27-35) or 26-35 (M26-35) epitopes. These reagents were also used to stimulate CTL proliferation, measured as 3H-thymidine incorporation, in the presence of immature dendritic cells (iDC), as antigen-presenting cells (APC). Induction of specific CTL upon stimulation with soluble or SSL-encapsulated peptides was attempted in healthy donors' PBMC or melanoma-derived TIL, and monitored by 51Cr release assays and tetramer staining. Na-8 cells pulsing with SSL M27-35 resulted in a five-fold more effective killing by specific CTL as compared with equal amounts of S M27-35. Encapsulation into SSL also provided a partial (50%) protection of M27-35 from plasma hydrolysis. No specific advantages regarding M26-35 were detectable in these assays. However, at low epitope concentrations (

Topics: Antigens, Neoplasm; Epitopes; Humans; Immunotherapy; Liposomes; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Neoplasm Proteins; Skin Neoplasms; T-Lymphocytes, Cytotoxic

Several recent reports have described the detection of circulating, cancer-related RNA molecules in serum or plasma from cancer patients, but little is known about the biology of this extracellular RNA. We aimed to determine how RNA is protected against degradation in serum, to optimize RNA isolation from large volumes of serum, and to test our optimized assays for serum-based cancer detection.. We used quantitative reverse transcription-PCR (QRT-PCR) analysis to investigate the isolation and biology of extracellular plasma RNA. We then examined the presence of amplifiable RNA transcripts in plasma and serum from controls and from patients with esophageal cancer and malignant melanoma.. We found that extracellular RNA in plasma is highly degraded and can be isolated most efficiently by guanidinium-phenol extraction followed by precipitation. Extracellular RNA is stable in serum for up to 3 h but is destroyed immediately by addition of detergents. Extracellular RNA can be captured on 0.2 microm filters, allowing concentration of RNA from several milliliters of plasma. When we concentrated RNA from up to 4 mL of serum, detection of cancer-related transcripts in serum from cancer patients and controls was infrequent and inconsistent.. Extracellular RNA is most likely protected within protein or lipid vesicles, possibly apoptotic bodies, which can be disrupted by detergents. Despite optimizing many aspects of plasma RNA detection, we were unable to reproducibly detect cancer-related transcripts. Our data suggest that measurement of circulating RNA may not be a good approach to early cancer diagnosis.

Topics: Animals; Antigens, Neoplasm; Centrifugation; Esophageal Neoplasms; Humans; Intermediate Filament Proteins; Keratin-20; MART-1 Antigen; Melanoma; Mice; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasms; Plasma; Polymerase Chain Reaction; Reproducibility of Results; Ribonucleases; RNA, Messenger; RNA, Neoplasm; Sensitivity and Specificity

Despite the increasing use of dendritic cells (DCs) in clinical trials, questions regarding the optimal means of DC preparation, in particular how to achieve optimal maturation, remain unanswered. We hypothesized that delivering two separate sequential maturation signals to DC in vitro, mimicking the process of DC maturation that occurs in vivo, would enhance the ability of DCs to generate antigen-specific effector T cells in an experimental in vitro antimelanoma model.. Human monocyte-derived DCs were transfected with mRNA encoding melanoma-associated antigen Mart-1 (MART) or influenza M1 matrix protein (M1). After mRNA transfection, DCs were left untreated or exposed to different maturation stimuli either added simultaneously or delivered sequentially 18 h after first stimulation. Phenotypic DC cell-surface marker changes and IL-12 secretion were analyzed. Specific antigen presentation by DCs was measured by IFN-gamma release Elispot assay using a CD8(+) MART peptide-specific T cell clone. RNA-transfected and treated DCs were cultured with autologous naive T cells and the induction of antigen-specific effector T cells were assessed by IFN-gamma release Elispot assay.. DCs transfected and matured had increased cell-surface expression of CD40 and costimulatory molecules CD80, and CD86. DCs matured and further treated by soluble CD40 ligand (sCD40L) had a 10- and 2-fold increase in MART antigen presentation compared to untreated (immature) DCs and DCs treated only with a first maturation signal, respectively (Elispot P = 0.02). Delivery of sequential maturation stimuli resulted in maximal DC IL-12 secretion compared to simultaneous stimuli. Last, generation of antigen-specific effector T cells more than doubled with the sequential addition of sCD40L to mature DC stimulators (Elispot P = 0.009).. Maturation of DCs following mRNA transfection increases expression of cell-surface costimulatory molecules. Delivery of a second sequential maturation stimulus enhances antigen presentation, increases IL-12 secretion, and augments immunogenicity as evidenced by generation of tumor antigen-specific effector T cells. This strategy should be considered in the future development of RNA-based DC vaccine strategies for the treatment of cancer.

Topics: Antigen-Presenting Cells; Antigens, CD; Antigens, Neoplasm; B7-1 Antigen; B7-2 Antigen; CD40 Antigens; CD40 Ligand; Cell Differentiation; Cells, Cultured; Cellular Senescence; Dendritic Cells; Epitopes; Granulocytes; Humans; Interferon-gamma; Interleukin-12; Interleukin-2; Isoantigens; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Phenotype; Signal Transduction; Solubility; T-Lymphocytes; T-Lymphocytes, Regulatory; Viral Matrix Proteins

Melanoma antigen recognized by T cell 1 (MART-1) is regarded as a candidate peptide for vaccination against malignant melanoma, and it is of importance to develop strategies to improve the vaccine-elicited T-cell activation towards MART-1. T-cell activation is, among other determinants, dependent on the density of specific major histocompatibility complex-peptide complexes on the surface of the antigen-presenting cell. In this study, we explored the cell-surface presentation of a substituted MART-1 peptide encoded by transfected minigenes. We investigated the potential of proteasomal targeting compared to non-proteasomal targeting of the epitope to increase its cell-surface presentation. Furthermore, we explored the potential of incorporating multiple minigenes instead of one to increase cell-surface presentation. We show that both proteasomal targeting and repetition of the minigene increase cell-surface presentation of the epitope and propose both these approaches as potential strategies in DNA vaccines to increase MART-1-specific T-cell activation.

Topics: Amino Acid Sequence; Antigen Presentation; Antigens, Neoplasm; Blotting, Western; Cancer Vaccines; Cell Line; Cysteine Endopeptidases; Cytotoxicity Tests, Immunologic; Epitopes; Humans; MART-1 Antigen; Melanoma; Molecular Sequence Data; Multienzyme Complexes; Neoplasm Proteins; Plasmids; Polymerase Chain Reaction; Proteasome Endopeptidase Complex; Repetitive Sequences, Nucleic Acid; T-Lymphocytes; Transfection; Vaccines, DNA

Atypical fibroxanthoma (AFX) is a cutaneous tumor that primarily occurs in the sun-damaged skin of the head and neck of adults. It is often a rapidly growing, solitary lesion that may clinically resemble squamous cell carcinoma, malignant melanoma, or lobular hemangioma. The histologic differential diagnosis primarily includes spindle cell squamous carcinoma and spindle cell melanoma, and immunohistochemical studies are often needed to establish the diagnosis.. We report an unusual case of an AFX with aberrant HMB-45 and MART-1 (melanoma antigen recognized by T cells-1) immunohistochemical expression. Clinical information was obtained. Histologic examination and immunohistochemical studies were performed.. A 54-year-old woman presented with a 1.5 cm posterior scalp lesion, which was excised. Microscopic examination revealed a dermal tumor composed of pleomorphic and spindled cells with numerous giant cells. The tumor cells expressed CD68 but did not express either keratin or S-100. In addition, there was focal gp100 (with HMB-45) and MART-1 expression limited to the large, multinucleated cells with vacuolated cytoplasm. A diagnosis of AFX was subsequently made.. This is the first reported case of an AFX with HMB-45 and MART-1 reactivity.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Carcinoma, Squamous Cell; Diagnosis, Differential; Female; Giant Cells; Histiocytoma, Benign Fibrous; Humans; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasm Proteins; Scalp; Skin Neoplasms

The diagnosis of melanoma metastatic to lymph node remains a difficult problem given its histological diversity. We examined the staining patterns of S-100, NK1/C3, HMB-45, and MART-1 (DC10) in melanoma metastases to lymph nodes. Immunohistochemical stains were performed on tissue sections of 126 formalin-fixed lymph nodes from 126 patients with an established diagnosis of metastatic melanoma. A total of 98% of cases (123 of 126) stained positive for S-100, 93% (117 of 125) stained positive for NK1/C3, 82% (103 of 126) stained positive for MART-1, and 76% (95 of 125) stained positive for HMB-45. The distribution and intensity of staining varied among these markers. A diffuse staining pattern, defined as >50% of tumor cells stained, was observed in 83% of MART-1-positive cases but in only 56% of S-100-positive cases, 48% of NK1/C3-positive cases, and 34% of HMB-45-positive cases. A maximally intense signal was almost always observed for MART-1 (83% of positive cases) but was rarely observed for NK1/C3 (20%). S-100 and HMB-45 showed maximally intense staining in 50% and 54% of cases, respectively. S-100 and NK1/C3 stained both histiocytes and melanocytes, whereas MART-1 and HMB-45 stained only melanocytes. Seventy-eight cases (63%) stained positive for all 4 markers, 17 cases (14%) stained for all markers except HMB-45, 13 cases (10%) stained for all markers except MART-1, 6 cases (5%) stained only with S-100 and NK1/C3, 4 cases (3%) stained only with S-100 and HMB-45, and 2 cases stained for all markers except S-100. One case each stained for the following: only S-100, only S-100 and HMB-45, and all markers except NK1/C3. One case exhibited absence of staining for any of these markers. We demonstrate that lymph node metastases of melanoma are heterogeneous with regard to tumor marker expression. S-100 and NK1/C3 were the most sensitive stains for detecting metastatic melanoma; however, they both also stain other nontumor cells in lymph nodes. MART-1 did not stain histiocytes and exhibited a more frequently intense and diffuse staining pattern than NK1/C3. HMB-45 was less sensitive and demonstrated less diffuse staining than MART-1.

Topics: Antigens; Antigens, Neoplasm; Antigens, Surface; Biomarkers, Tumor; Complement C3; Humans; Immunohistochemistry; Lectins, C-Type; Lymph Nodes; Lymphatic Metastasis; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; NK Cell Lectin-Like Receptor Subfamily B; Proteins; S100 Proteins; Skin Neoplasms

In melanoma patients, CD8+ cytotoxic T cells have been found recognizing self-proteins of which the expression is restricted to the melanocytic lineage. These melanocyte differentiation antigens are expressed in normal melanocytes as well as in 80-100% of primary and metastatic melanoma. In this report, six HLA-A*0201-subtyped metastatic melanoma patients vaccinated with dendritic cells (DCs) pulsed with autologous tumor lysates and keyhole limpet hemocyanin (KLH) were screened for the presence of CD8+ T cells specific for three HLA-A*0201-binding peptides derived from the melanosomal antigens MART-1/Melan-A, gp100, and tyrosinase. For this purpose, nonstimulated as well as in vitro peptide-stimulated peripheral blood mononuclear cells (PBMCs) were tested for peptide-specific IFN-gamma release by enzyme-linked immunosorbent spot (ELISpot) assays. Furthermore, expression of the melanosomal antigens MART-1/Melan-A, gp100, and tyrosinase in tumor lesions was analyzed by immunohistochemistry before and after vaccination. We also used the ELISpot technique to investigate whether KLH-specific T cells were induced and whether these cells released type 1 (IFN-gamma) and/or type 2 (IL-13) cytokines. Our data show induction of CD8+ T cells specific for the melanosomal peptides MART-1/Melan-A(27-35) or tyrosinase(1-9), as well as IFN-gamma-releasing KLH-specific T cells, in two of six vaccinated melanoma patients, but do not support an association between the induction of these T cells and clinical responses.

Topics: Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Cytotoxicity, Immunologic; Dendritic Cells; gp100 Melanoma Antigen; Hemocyanins; HLA-A Antigens; HLA-A2 Antigen; Humans; Hypersensitivity, Delayed; Immunoenzyme Techniques; Interferon-gamma; Lung Neoplasms; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Peptide Fragments; Skin Neoplasms; Vaccination

Memory CD8 T cells mediate rapid and effective immune responses against previously encountered Ags. However, these cells display considerable phenotypic and functional heterogeneity. In an effort to identify parameters that correlate with immune protection, we compared cell surface markers, proliferation, and cytokine production of distinct virus- and tumor-specific human CD8 populations. Phenotypic analysis of epitope-specific CD8 T cells showed that Ag specificity is associated with distinct CCR7/CD45RA expression profiles, suggesting that Ag recognition drives the expression of these molecules on effector/memory T cells. Moreover, the majority of central memory T cells (CD45RAlowCCR7dull) secreting cytokines in response to an EBV epitope produces both IL-2 and IFN-gamma, whereas effector memory CD8 cells (CD45RAdullCCR7-) found in EBV, CMV, or Melan-A memory pools are mostly composed of cells secreting exclusively IFN-gamma. However, these various subsets, including Melan-A-specific effector memory cells differentiated in cancer patients, display similar Ag-driven proliferation in vitro. Our findings show for the first time that human epitope-specific CD8 memory pools differ in IL-2 production after antigenic stimulation, although they display similar intrinsic proliferation capacity. These results provide new insights in the characterization of human virus- and tumor-specific CD8 lymphocytes.

Topics: Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Cell Division; Cell Separation; Cytokines; Epitopes, T-Lymphocyte; Herpesvirus 4, Human; Humans; Interleukin-2; Leukocyte Common Antigens; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; Peptide Fragments; Receptors, CCR7; Receptors, Chemokine

Inconsistent reports on the detection of melanoma cells in peripheral blood by reverse transcriptase-PCR (RT-PCR) have resulted in uncertainty on the prognostic value of circulating melanoma cells.. We developed real-time RT-PCR assays for quantitation of tyrosinase, MelanA/MART1, and gp100 and for porphobilinogen deaminase housekeeping gene. Melanoma tissue (n = 18), peripheral blood samples from healthy donors (n = 21), and patients with cutaneous (n = 122) and uveal (n = 64) melanoma from our institution were analyzed. For quality control, an additional 251 samples from ongoing multicenter studies were compared with in-house samples.. Tyrosinase was not detected in healthy donor blood samples. For the two other markers, cutoff values had to be defined to distinct patient samples from controls. Patients with stage IV uveal and cutaneous melanoma expressed all three markers more frequently and at higher levels in peripheral blood as compared with earlier stages. The variation of expression was 4 logs and correlated with tumor load and serum lactate dehydrogenase. In 2 of 3 uveal melanoma patients, detection of circulating tumor cells preceded the development of liver metastases. The diagnostic sensitivity was optimal in blood samples containing >0.1pg/ microl porphobilinogen deaminase (95.7% of in-house samples and 57.4% of multicenter samples).. Real-time RT-PCR is able to quantitatively define the quality of a sample and provides quantitative data for melanoma markers. Disparities in the results of previous studies may be attributable to undetected differences in sample quality. The prognostic relevance of this assay is currently under evaluation in several prospective randomized trials.

Topics: Antigens, Neoplasm; Base Sequence; DNA Primers; Eye Neoplasms; gp100 Melanoma Antigen; Humans; Hydroxymethylbilane Synthase; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Plasmids; Reference Values; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic

The clinical relevance of dendritic cells (DCs) at the tumor site remains a matter of debate concerning their role in the generation of effective antitumor immunity in human cancers. We performed a comprehensive immunohistochemical analysis using a panel of DC-specific antibodies on regressing tumor lesions and sentinel lymph nodes (SLNs) in melanoma patients. Here we show in a case report involving spontaneous regression of metastatic melanoma that the accumulation of DC-Lamp+ DCs, clustered with tumor cells and lymphocytes, is associated with local expansion of antigen-specific memory effector CTLs. These findings were extended in a series of 19 melanoma-positive SLNs and demonstrated a significant correlation between the density of DC-Lamp+ DC infiltrates in SLNs with the absence of metastasis in downstream lymph nodes. This study, albeit performed in a limited series of patients, points to a pivotal role of mature DCs in the local expansion of efficient antitumor T-cell-mediated immune responses at the initial sites of metastasis and may have important implications regarding the prognosis, staging, and immunotherapy of melanoma patients.

Topics: Adult; Antigens, CD; Antigens, Neoplasm; Dendritic Cells; Humans; Immunophenotyping; Ligands; Lymph Nodes; Lymphatic Metastasis; Lymphocytes, Tumor-Infiltrating; Lysosomal Membrane Proteins; Male; MART-1 Antigen; Melanoma; Neoplasm Proteins; Receptors, CCR6; Receptors, CCR7; Receptors, Chemokine; Sentinel Lymph Node Biopsy; Skin Neoplasms; T-Lymphocytes, Cytotoxic

The status of the sentinel lymph node (SLN) is an important prognostic factor in patients with cutaneous melanoma. Reverse transcription-polymerase chain reaction (RT-PCR) has been used as a sensitive means of detecting tumour cells in SLNs.. To determine whether RT-PCR analysis of the SLN using both the central and the peripheral slices is more sensitive than molecular analysis of the central slice only.. Eighty-three SLNs from 59 patients with primary cutaneous melanoma were identified by SLN mapping. All SLNs were bisected along their longitudinal axis to produce two equal halves. One half was used for histology and immunohistochemistry, and the other was analysed by RT-PCR for tyrosinase and MelanA. Parallel to the longitudinal axis, one central slice (approximately 2 mm in thickness) was cut manually. This central slice was used for our standard RT-PCR protocol. In the current study, up to eight additional peripheral slices (each approximately 2 mm in thickness) were cut parallel to the existing cut surface. These peripheral slices were analysed by additional RT-PCR.. Standard RT-PCR of the central slice yielded positive results in 34 of 59 patients (57%). Additional RT-PCR of peripheral slices demonstrated positive findings in six additional patients (10%) who were initially negative by standard RT-PCR of the central slice. In detail, seven of those 34 patients positive by standard RT-PCR of the central slice had positive histological findings. In each of these seven patients, RT-PCR was positive both in the central slice as well as in the peripheral slices. The remaining 27 patients with positive RT-PCR results of the central slice showed negative histological findings. Only nine (33%) of these 27 patients had a positive RT-PCR also in the peripheral slices. Finally, all 25 patients with negative RT-PCR results in the central slice showed negative histological findings. Six of these patients (24%) revealed positive RT-PCR results on the analysis of peripheral slices. However, three of these patients expressed only MelanA but not tyrosinase. Thirty lymph nodes from 24 nonmelanoma patients served as negative controls for RT-PCR. In three of these 24 patients (13%) expression of MelanA but not tyrosinase was detected by RT-PCR.. Molecular analysis of peripheral slices yielded six additional patients (10%) positive by RT-PCR who were initially negative by standard RT-PCR of the central slice. However, three of these six patients were found to express only MelanA but not tyrosinase. As MelanA expression was also found in 13% of control lymph nodes, positive MelanA expression alone in SLNs of melanoma patients requires cautious interpretation in order to avoid false-positive findings. Thus, additional molecular processing of peripheral slices did not significantly increase the number of patients with RT-PCR-positive SLNs.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Female; Humans; Lymph Nodes; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Reverse Transcriptase Polymerase Chain Reaction; Sentinel Lymph Node Biopsy; Skin Neoplasms

Although tumor-specific CD8 T-cell responses often develop in cancer patients, they rarely result in tumor eradication. We aimed at studying directly the functional efficacy of tumor-specific CD8 T cells at the site of immune attack. Tumor lesions in lymphoid and nonlymphoid tissues (metastatic lymph nodes and soft tissue/visceral metastases, respectively) were collected from stage III/IV melanoma patients and investigated for the presence and function of CD8 T cells specific for the tumor differentiation antigen Melan-A/MART-1. Comparative analysis was conducted with peripheral blood T cells. We provide evidence that in vivo-priming selects, within the available naive Melan-A/MART-1-specific CD8 T-cell repertoire, cells with high T-cell receptor avidity that can efficiently kill melanoma cells in vitro. In vivo, primed Melan-A/MART-1-specific CD8 T cells accumulate at high frequency in both lymphoid and nonlymphoid tumor lesions. Unexpectedly, however, whereas primed Melan-A/MART-1-specific CD8 T cells that circulate in the blood display robust inflammatory and cytotoxic functions, those that reside in tumor lesions (particularly in metastatic lymph nodes) are functionally tolerant. We show that both the lymph node and the tumor environments blunt T-cell effector functions and offer a rationale for the failure of tumor-specific responses to effectively counter tumor progression.

Topics: Adolescent; Adult; Aged; Antigens, Neoplasm; Dose-Response Relationship, Immunologic; Epitopes, T-Lymphocyte; Female; HLA-A2 Antigen; Humans; Immune Tolerance; Interleukin-2; Interleukin-7; Lymph Nodes; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Receptors, Antigen, T-Cell; T-Lymphocytes, Regulatory

Sentinel lymph node (SLN) biopsy in melanoma is an increasingly used procedure. Pathologic evaluation of SLNs using immunohistochemistry improves diagnostic accuracy, yet no universally accepted standard protocol for pathologic processing of SLNs exists.. The primary purpose of this study was to evaluate our experience with the sensitivity of the immunostains S-100, HMB-45, and Melan-A for SLN biopsy.. Ninety-nine positive SLNs from 72 patients were retrospectively reviewed for the presence of microscopic metastatic melanoma on hematoxylin and eosin (H&E), S-100, HMB-45, and Melan-A stained sections and sensitivities of each immunohistochemical stain were determined.. The sensitivities of S-100, HMB-45, and Melan-A were 97%, 75%, and 96% respectively.. Given the lower sensitivity of HMB-45, our practice for evaluation of SLN biopsy specimens was modified using combinations of H&E, S-100, and Melan-A without HMB-45. If the H&E sections are negative or equivocal for metastatic melanoma, immunohistochemistry staining with S-100 protein and Melan-A is performed. New and improved protocols will undoubtedly be forthcoming as the field advances.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Humans; Immunohistochemistry; Lymph Nodes; Lymphatic Metastasis; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; S100 Proteins; Sensitivity and Specificity; Sentinel Lymph Node Biopsy; Skin Neoplasms

Ovarian malignant melanoma (MM), primary or metastatic, is an extremely rare tumor and in the absence of a previous diagnosis can represent a diagnostic challenge. We present the clinicopathologic and immunohistochemical features of 23 cases seen in our institution over a period of 40 years (1962-2001). The patients' age ranged from 14 to 53 years (mean 35.7 years). Ethnicity was known in 19 patients: 14 white, 4 Hispanic, and 1 black. A previous history of MM was definitively obtained in 14 patients; in these cases, the interval between the primary MM and the ovarian metastasis ranged from 15 to 228 months (mean 77.7 months). The tumor was unilateral in 19 and bilateral in 4 cases. The tumor size ranged from 4.5 to 23 cm (average 10 cm); the melanoma arising in a cystic teratoma was 0.2 mm in thickness. The tumor was grossly pigmented in 8 cases (35%). The architectural pattern was nodular (8 cases), diffuse (6 cases), nodular and diffuse (5 cases), nested (3 cases), and lentiginous arising in a teratoma (1 case). Follicle-like spaces were seen in 8 cases, pseudo-glandular areas in 1 case, pseudo-myxoid areas in 1 case, and cords in 1 case. The tumor cell type was epithelioid in 19 cases, spindled in 2 cases, mixed epithelioid and spindled in 1 case, and small cell in 1 case. Nucleoli were prominent in 18 cases, and nuclear inclusions were present but rare in the majority of cases. Nuclear grooves were seen in 3 cases. Necrosis was extensive in 8 cases, focal in 10 cases, and was absent in 5 cases. In 8 cases, initial diagnoses included sex cord stromal tumor, germ cell tumor, sarcoma, or undifferentiated carcinoma. S-100 was positive in 18 of 19 cases, HMB-45 in 17 of 20 cases, MART-1 in 13 of 15 cases, tyrosinase in 10 of 15 cases, and Mitf in 8 of 14 cases. Inhibin was positive in 3 of 14 cases. Calretinin was focally positive in 1 of 12 cases. Treatment performed in 18 of the cases are as follows: oophorectomy with/without chemotherapy (10); total abdominal hysterectomy with bilateral salpingo-oophorectomy with/without chemotherapy (6); vaginal hysterectomy, bilateral salpingo-oophorectomy, and chemotherapy (1); and total abdominal hysterectomy with salpingo-oophorectomy (1). Follow-up ranging from 2 to 96 months was available in 18 patients. All but one had metastases in other organs, most often in the lungs. Thirteen patients died of disease (range 2-76 months), 3 are alive with disease (6-18 months), and 2 have no evidence of disease at 24 and 96

Topics: Adolescent; Adult; Antigens, Neoplasm; Calbindin 2; DNA-Binding Proteins; Ethnicity; Female; Humans; Hysterectomy; Immunohistochemistry; Inhibins; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Microphthalmia-Associated Transcription Factor; Middle Aged; Monophenol Monooxygenase; Neoplasm Metastasis; Neoplasm Proteins; Ovarian Neoplasms; S100 Calcium Binding Protein G; S100 Proteins; Teratoma; Transcription Factors; Treatment Outcome

The success and further evolution of the sentinel lymph node (SLN) concept decisively depend on histological techniques. Fundamental standards were agreed on by a panel of international experts from various disciplines in 1999 and published as "The Augsburg Consensus" in 2000. Conventional histology (hematoxylin and eosin [H&E]) has to be supplemented by immunohistochemistry (eg, S100 and HMB45) using adequate series of paraffin sections. Melanoma cells in SLNs must be carefully differentiated from capsular and trabecular nevocytes, from immigrated Langerhans cells, from interdigitating dendritic leukocytes, and from nerve sheath cells, which all share S100 positivity in the cytoplasm. The micromorphometric S classification is based on the maximum distance of intranodal melanoma cells from the interior margin of the SLN capsule. It has proven its practicability under routine circumstances, as well as its predictive value regarding further nodal and distant metastases as well as overall survival. This has to be considered in prospective randomized trials dealing with the issues of completion lymphadenectomy and adjuvant therapies of melanoma patients. Reverse-transcriptase polymerase chain reaction (RT-PCR) techniques, when performed as a supplement to histology on the basis of additional paraffin sections, can further enhance the diagnostic sensitivity for detecting melanoma cells in SLNs.

Topics: Antigens, Neoplasm; Humans; Immunohistochemistry; Lymphatic Metastasis; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Monophenol Monooxygenase; Neoplasm Proteins; Prognosis; Radionuclide Imaging; Reverse Transcriptase Polymerase Chain Reaction; S100 Proteins; Sentinel Lymph Node Biopsy; Skin Neoplasms

Detection of circulating tumor cells (CTCs) might improve current staging procedures by identifying a subgroup of patients with minimal residual disease and thus a higher risk of disease recurrence. Forty patients with > or =2-mm-thick cutaneous melanoma with or without lymph node metastasis were enrolled. After standard radical surgery and adjuvant therapy in case of lymph node metastasis, patients were followed up with routine physical and radiologic assessments as well as serial PCR-based analysis of CTCs using 2 melanoma markers (tyrosinase and Melan-A/Mart-1). After a median follow-up of 30 months, 18 patients had disease recurrence and 28 were PCR-positive before the disease became clinically evident. The sensitivity of the molecular test was 83%. Median time to PCR positivity and median PCR-to-relapse time were 12 and 8 months, respectively. At multivariate analysis, PCR positivity was an independent predictor of disease recurrence (hazard ratio=2.06, 95% CI 1.07-3.35; p=0.03). Among high-risk melanoma patients, serial PCR-based analysis of CTCs can identify a subgroup at higher risk of disease recurrence, with clinically significant advance. Therefore, CTC detection might be employed for the selection of patients for adjuvant treatment and during follow-up for early indication of therapeutic failure.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Biomarkers, Tumor; Chemotherapy, Adjuvant; Female; Follow-Up Studies; Humans; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Neoplastic Cells, Circulating; Patient Selection; Polymerase Chain Reaction; Prognosis; Risk Factors; Skin Neoplasms; Time Factors

Cytotoxic T-cell immunity directed against melanosomal differentiation antigens is arguably the best-studied and most prevalent form of tumor-specific T-cell immunity in humans. Despite this, the role of T-cell responses directed against melanosomal antigens in disease progression has not been elucidated. To address this issue, we have related the presence of circulating melanoma-specific T cells with disease progression and survival in a large cohort of patients with advanced-stage melanoma who had not received prior treatment. In 42 (68%) of 62 patients, melanoma-specific T cells were detected, sometimes in surprisingly large numbers. Disease progression during treatment was more frequent in patients with circulating melanoma-specific T cells, and mean survival of patients with circulating melanoma-specific T cells was equal to the survival of patients without melanoma-specific T cells. These data suggest that the induction of melanosomal differentiation antigen-specific T-cell reactivity in advanced stage melanoma is a late event most likely due to antigen load and spreading and is not accompanied by a clinically significant antitumor effect. These melanoma-specific T cells may be functionally distinct from T cells raised during spontaneous regression or up vaccination.

Topics: Adult; Aged; Antigens, Neoplasm; CD8 Antigens; CD8-Positive T-Lymphocytes; Disease Progression; gp100 Melanoma Antigen; HLA-A Antigens; HLA-A2 Antigen; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Staging; Neoplastic Cells, Circulating; Survival Analysis

Although reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of melanocyte-associated mRNA can detect sentinel node melanoma metastases, most published assays are semi-quantitative methods of unknown sensitivity and precision, unsuitable for clinical use. We describe a single-step real-time quantitative RT-PCR assay for MART-1 and tyrosinase mRNAs, suitable for sentinel node analysis in a clinical setting. Using serial dilutions of melanoma cell line SK-MEL-28 RNA in water as a calibrator, we obtained linear calibration curves covering the range 0.5 to 10,000 arbitrary units (SK-MEL-28 melanoma cell equivalents). The sensitivity limit was 0.32 (MART-1) and 5 (tyrosinase) arbitrary units. Analytical imprecision was between 11% and 34%. MART-1 PCR efficiency was unaffected when samples were diluted with negative lymph node RNA rather than water, whereas tyrosinase PCR efficiency was halved. To evaluate the clinical suitability of our assay, we quantified melanocyte mRNAs in sentinel nodes with histologically verified micrometastases (n = 10) and benign nevus inclusions (n = 10), and in sentinel nodes without evidence of intranodal melanocytes (n = 10). We found significant differences in median melanocyte-derived mRNA levels comparing the three types of lymph nodes, suggesting that this quantitative molecular protocol may increase assay precision and be useful for the clinical evaluation of sentinel nodes.

Topics: Antigens, Neoplasm; Biomarkers; Female; Humans; Lymph Nodes; Male; MART-1 Antigen; Melanoma; Monophenol Monooxygenase; Neoplasm Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sentinel Lymph Node Biopsy

To report the protocol for surveillance of the eye and vision in a clinical trial of MART1-transduced dendritic cells for metastatic melanoma.. In a phase I/II clinical trial of dendritic cell-based genetic immunotherapy for metastatic cutaneous melanoma, ophthalmic evaluation is performed prior to immunization (Baseline Evaluation), 56+/-7 days after first vaccination (mid-study evaluation), when dendritic cell injections are complete 112+/-7 days after first vaccination (end-study evaluation) and 168+/-7 days after first vaccination (post-study evaluation).. The protocol for baseline, mid-study and end-study evaluations of the eye and vision includes ophthalmic history, comprehensive ophthalmic examination, psychophysical and electrophysiological visual function assessment, fundus photography and fluorescein angiography. Post-study evaluation consists of the 25-item visual functioning questionnaire augmented to elicit autoimmune manifestation with complete ophthalmic evaluation if vision-related symptoms or abnormalities are noted during or after the vaccination.. Limited adverse effects on the eye and vision have been reported in melanoma immunotherapy trials, although this novel mode of therapy has the potential to induce melanoma paraneoplastic syndromes known to severely impair vision. Therefore, surveillance of the eye and vision should be considered in melanoma immunotherapy trials.

Topics: Antigens, Neoplasm; Cancer Vaccines; Cell Transformation, Neoplastic; Clinical Trials as Topic; Contrast Sensitivity; Dendritic Cells; Dose-Response Relationship, Immunologic; Electroretinography; Eye Diseases; Genetic Therapy; Humans; Immunotherapy; Injections, Intradermal; MART-1 Antigen; Melanoma; Neoplasm Proteins; Population Surveillance; Skin Neoplasms; Transduction, Genetic; Vaccination; Vision Disorders; Visual Acuity; Visual Fields

The lack of melanoma-associated antigen (MAA) expression has been associated with the reduced overall survival in melanoma patients. In order to investigate whether the MAA expression detected on cell cultures established from melanoma patients might relate to the overall survival in these patients, we screened primary cell cultures derived from 37 melanoma metastases for the expression of five known MAA: Melan-A, tyrosinase, gp-100, MAGE-1 and MAGE-3 by polymerase chain reaction (PCR) and fluorescence-activated cell sorting (FACS). MAA expression detected by PCR was found at a high percentage in evaluated melanoma cell lines: 25 of 28 (89%) were positive for Melan-A, 22 of 28 (79%) were positive for tyrosinase, 26 of 28 (93%) were positive for gp-100, and 18 of 28 (64%) were positive for MAGE-3 expression. Using the FACS method the percentage of MAA-positive cell lines was much lower: 14 of 31 (45%) cell lines were positive for Melan-A, eight of 31 (26%) were positive for tyrosinase, 13 of 31 (42%) were positive for gp-100, six of 31 (19%) were positive for MAGE-1, and 14 of 31 (45%) were positive for MAGE-3 expression. Kaplan-Meier survival analysis demonstrated that the patients whose cell lines were positive for Melan-A expression by PCR had significantly longer overall survival time as Melan-A PCR-negative cases (P=0.0038). This could not be shown for any of the markers tested by FACS. Our results suggest that the expression of Melan-A/MART-1 in patient-derived cell cultures may help to identify a group of melanoma patients with prolonged survival.

Topics: Adult; Aged; Antigens, Neoplasm; Biomarkers, Tumor; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Staging; Prognosis; RNA, Messenger; Survival Rate; Tumor Cells, Cultured

Tumor-reactive T cells play an important role in cancer immunosurveillance. Applying the multimer technology, we report here an unexpected high frequency of Melan-A-specific CTLs in a melanoma patient with progressive lymph node metastases, consisting of 18 and 12.8% of total peripheral blood and tumor-infiltrating CD8+ T cells, respectively. Melan-A-specific CTLs revealed a high cytolytic activity against allogeneic Melan-A-expressing target cells but failed to kill the autologous tumor cells. Loading of the tumor cells with Melan-A peptide reversed the resistance to killing, suggesting impaired function of the MHC class I antigen processing and presentation pathway. Mutations of the coding region of the HLA-A2 binding Melan-A26-35 peptide or down-regulation of the MHC class I heavy chain, the antigenic peptide TAP, and tapasin could be excluded. However, PCR and immunohistochemical analysis revealed a deficiency of the immunoproteasomes low molecular weight protein 2 and low molecular weight protein 7 in the primary tumor cells, which affects the quantity and quality of generated T-cell epitopes and might explain the resistance to killing. This is supported by our data, demonstrating that the resistance to killing can be partially reversed by pre-exposure of the tumor cells to IFN-gamma, which is known to induce the immunoproteasomes. Overall, this is the first report of an extremely high frequency of tumor-specific CTLs that exhibit competent T-cell-effector functions but fail to lyse the autologous tumor cells. Immunotherapeutic approaches should not only focus on the induction of a robust antitumor immune response, but should also have to target tumor immune escape mechanisms.

Topics: Antigens, Neoplasm; Cysteine Endopeptidases; HLA-A2 Antigen; Humans; Lymph Nodes; Male; MART-1 Antigen; Melanoma; Middle Aged; Multienzyme Complexes; Mutation; Neoplasm Proteins; Proteasome Endopeptidase Complex; T-Lymphocytes, Cytotoxic

Pigmented actinic keratosis is one of the simulators of early melanoma in situ from severely sun-damaged skin. Close scrutiny of the hematoxylin and eosin stained section does not always allow an unequivocal diagnosis, because it is sometimes difficult to distinguish pigmented keratinocytes from melanocytes. Immunohistochemical stains, such as S-100 and HMB-45, are used routinely to address this problem. Melan-A, also known as MART-1, is an additional melanocytic marker and has proved to be useful in identifying metastatic tumors of melanocytic origin. The usefulness of this marker to discriminate pigmented actinic keratosis from early melanoma in situ, however, has not yet been a subject of investigation. In this study we evaluated Melan-A expression in ten unequivocal cases of pigmented actinic keratosis and compared the staining pattern with that of S-100, HMB-45, and tyrosinase. In all ten cases the number of cells highlighted with Melan-A was by far larger than those labeled with S-100, HMB-45, and tyrosinase. Four cases showed clusters of Melan-A positive cells being suggestive of melanocytic nests. Even areas of normal skin adjacent to the actinic keratosis featured prominent staining of Melan-A, but only inconsistent labeling of intraepidermal melanocytes with S-100, HMB-45, and tyrosinase. We therefore believe that Melan-A is a more sensitive marker for intraepidermal melanocytes than S-100, HMB-45, and tyrosinase. In addition there may be expression of Melan-A in keratinocytes and nonmelanocytic cells. To avoid an erroneous diagnosis of malignant melanoma one should therefore interpret results obtained from Melan-A stained slides carefully and in the context with other melanocytic markers.

Topics: Aged; Aged, 80 and over; Antigens, Neoplasm; Biomarkers, Tumor; Carcinoma in Situ; Diagnosis, Differential; Female; Humans; Immunohistochemistry; Keratosis; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; S100 Proteins; Skin Neoplasms; Sunlight

Great attention has been recently given to a flavonoid of the anthocyanin class, cyanidin-3-O-beta-glucopyranoside (C-3-G), which is widely spread throughout the plant kingdom, and is present in both fruits and vegetables of human diets. In this study, we investigated the effect of C-3-G on proliferation and differentiation of human melanoma cells. Both morphological and functional parameters were evaluated, using electron and confocal microscopy, cytofluorometric analysis, HPLC assay, Western blot analysis, and enzymatic assay, as appropriate. A treatment with a single dose of C-3-G decreased cell proliferation without affecting cell viability and without inducing apoptosis or necrosis. The mitotic index and cell percentage in S phase were significantly lower in C-3-G treated cells compared with untreated control. C-3-G treatment induced, in a dose- and time-dependent manner, melanoma cell differentiation characterized by a strong increase in dendrite outgrowth accompanied with a remodeling of the microtubular network, a dramatic increase of focal adhesion and an increased expression of "brain specific" cytoskeletal components such as NF-160 and NF-200 neurofilament proteins. C-3-G treatment also induced increase of cAMP levels and up-regulation of tyrosinase expression and activity resulting in an enhanced melanin synthesis and melanosome maturation. Up-regulation of the melanoma differentiation antigen Melan-A/MART-1 in treated cells respect to the untreated control was also recorded. Data obtained provide evidence that a single treatment with C-3-G is able to revert the human melanoma cells from the proliferating to the differentiated state. We conclude that C-3-G is a very promising molecule to include in the strategies for treatment of melanoma; also because of its nutritional relevance.

Topics: Anthocyanins; Antigens, Neoplasm; Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclic AMP; Cytoskeleton; Humans; MART-1 Antigen; Melanins; Melanoma; Melanosomes; Monophenol Monooxygenase; Neoplasm Proteins

Melanoma lesions that develop in the same patient at different times or simultaneously at different locations may differ antigenically, because malignant melanoma is heterogeneous in terms of its biological, immunological and metastatic properties. The objective of this study was to characterize the molecular profiles of melanoma cells in peripheral blood, lymph nodes and metastatic tissues, employing the messenger RNA (mRNA) expression of tyrosinase, melanoma-inhibiting activity (MIA) and melanoma antigen recognized by T cells-1 (MART-1) as markers. Samples of cells propagated from metastatic sites were obtained from 17 stage III/IV melanoma patients and assayed by reverse transcriptase-polymerase chain reaction (RT-PCR), using specific primers for each marker. In eight patients, marker profiles were analysed in simultaneously obtained specimens of peripheral blood, lymph nodes and metastatic tissues originating from the same patient. Tyrosinase, MIA and MART-1 were expressed in 59%, 76% and 76% of the metastases, respectively. Simultaneously obtained specimens of peripheral blood, lymph nodes and metastatic tissues showed a high degree of homogeneity: 60%, 75% and 20% for tyrosinase, MIA and MART-1, respectively. Our findings suggest that the rather homogeneous expression pattern found in different tumour sites analysed in the same patient is of potential prognostic and therapeutic importance. Furthermore, melanoma lesions may be negative for the expression of antigens such as MART-1, and discrepancies in expression patterns between peripheral blood and metastatic tissues may occur, especially for this marker. Finally, our findings support the notion that molecular screening using an RT-PCR approach is appropriate in this kind of investigation.

Topics: Adult; Aged; Antigens, Neoplasm; Biomarkers, Tumor; Cell Line, Tumor; DNA Primers; Female; Humans; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplasm Metastasis; Neoplasm Proteins; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; Skin Neoplasms

We have mapped the linear antigenic determinant of a commercial MAb raised in the mouse against the melanoma-associated-antigen Melan-A/MART-1. The B cell epitope on the Melan-A/MART-1 oncoprotein is located in the 15-mer amino acid sequence 101-115 PPAYEKLSAEQSPPP, within residues 102-106. The definition of the antigenic sequence on Melan-A/MART-1 oncoprotein was reached following analyses of MHC II binding potential and similarity level to the mouse proteome, that put into evidence the 15-mer amino acid sequence 101-115 PPAYEKLSAEQSPPP as the top scoring peptide in binding H2-A(d) molecules and the epitopic sequence residues 102-106 (i.e., the peptide sequence PAYEK) as having low-similarity level to the mouse proteome. Dot-blot epitope mapping immunoassay identified proline residue 102 as critical, based on its effect on antibody recognition. The present study adds to previous companion reports in validating the hypothesis that low-similarity to the host's proteome and binding potential to MHC II molecules are essential concurring factors in the modulation of the pool of epitopic sequences.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antigens, Neoplasm; B-Lymphocytes; Computational Biology; Epitope Mapping; Epitopes; Humans; Immunoassay; Immunoblotting; Major Histocompatibility Complex; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Mice; Models, Molecular; Molecular Weight; Neoplasm Proteins; Oncogene Proteins; Peptides; Proline; Protein Binding; Proteome

The melanoma-associated protein Melan-A contains the immunodominant CTL epitope Melan-A(26/27-35)/HLA-A*0201 against which a high frequency of T lymphocytes has been detected in many melanoma patients. In this study we show that the in vitro degradation of a polypeptide encompassing Melan-A(26/27-35) by proteasomes produces both the final antigenic peptide and N-terminally extended intermediates. When human melanoma cells expressing the corresponding fragments were exposed to specific CTL, those expressing the minimal antigenic sequence were recognized more efficiently than those expressing the N-terminally extended intermediates. Using a tumor-reactive CTL clone, we confirmed that the recognition of melanoma cells expressing an N-terminally extended intermediate of Melan-A is inefficient. We demonstrated that the inefficient cytosolic trimming of N-terminally extended intermediates could offer a selective advantage for the preferred presentation of Melan-A peptides directly produced by the proteasomes. These results imply that both the proteasomes and postproteasomal peptidases limit the availability of antigenic peptides and that the efficiency of presentation may be affected by conditions that alter the ratio between fully and partially processed proteasomal products.

Topics: Amino Acid Sequence; Animals; Antigen Presentation; Antigens, Neoplasm; Cell Line; Cell Line, Tumor; Cytosol; Epitopes, T-Lymphocyte; HLA-A Antigens; HLA-A2 Antigen; Humans; Hydrolysis; Intracellular Fluid; MART-1 Antigen; Melanoma; Mice; Molecular Sequence Data; Neoplasm Proteins; Peptide Fragments; Peptide Hydrolases; Proteasome Endopeptidase Complex; Protein Precursors; Protein Processing, Post-Translational; T-Lymphocytes, Cytotoxic

Dendritic cells (DC) play critical roles in generating an immune response and in inducing tolerance. Diverse microenvironmental factors can 'polarize' DC toward an immunogenic or non-immunogenic phenotype. Among the various microenvironmental factors, interleukin-10 (IL-10) exhibits a potent immunosuppressive effect on antigen-presenting cells (APC). Here, we show that monocyte-derived DC generated in the presence of IL-10 exhibit a profound down-regulation of many genes that are associated with immune activation and show that the IL-10-grown DC are poor stimulators of CD8(+) T cells in a strictly autologous and major histocompatibility complex (MHC) class I-restricted melanoma antigen recognized by T cells (MART-1) epitope presentation system. However, these IL-10-grown DC can efficiently activate the epitope-specific CD8(+) T cells when they are made to present the epitope following transduction with an adenoviral vector expressing the MART-1 antigen. In addition, we show that the MART-1 protein colocalizes with the MHC class I protein, equally well, in the iDC and in the DC cultured in presence of IL-10 when both DC types are infected with the viral vector. We also show that the vector transduced DC present the MART-1(27-35) epitope for a sustained period compared to the peptide pulsed DC. These data suggest that although DCs generated in the presence of IL-10 tend to be non-immunogenic, they are capable of processing and presenting an antigen when the antigen is synthesized within the DC.

Topics: Adenoviridae; Antigen Presentation; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Cytotoxicity, Immunologic; Dendritic Cells; Genetic Vectors; Humans; Immunophenotyping; Interferon-gamma; Interleukin-10; Lymphocyte Activation; MART-1 Antigen; Melanoma; Neoplasm Proteins; Transduction, Genetic

Osteoid malignant melanoma is a rare type of melanoma described in humans and dogs with some areas of bone differentiation. In this tumour, the origin of the bone matrix remains unclear. We report one case of this variant with, for the first time, a cutaneous origin in a dog. Malignant melanomas are aggressive tumours. Amelanotic tumours are sometimes difficult to recognize as they require immunohistochemical evaluation for an adequate diagnosis and we have used anti-vimentin, S100, and melan A antibodies for identification. Melan A is less sensitive but more specific than S100 in identifying amelanotic melanomas. This tumour was positive for vimentin, S100 and melan A, including the areas of osteoid. These results suggest osteoid differentiation of tumour cells rather than induced stromal metaplasia.

Topics: Animals; Antigens, Neoplasm; Biomarkers, Tumor; Bone Neoplasms; Dog Diseases; Dogs; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Neoplasm Proteins; S100 Proteins; Vimentin

Desmoplastic melanoma (DMM) is an uncommon melanoma variant with a distinct morphology, including a prominent spindle cell component with fibrosis, as well as a distinct immunohistochemical profile. Histologically, the spindle cell component of DMM can be confused with sclerotic/desmoplastic nevi, nonpigmented blue nevi, scar, and neural tumors. The histological distinction between sclerotic/desmoplastic/blue nevi and DMM using standard light microscopic techniques can be exceedingly subtle. Therefore, we investigated whether immunohistochemical staining for Melan-A and Ki-67 expression can be used to discriminate these lesions, distinguishing between epithelioid and spindle cell compartments of the lesions.. Fifty cases of DMM and 13 cases of sclerotic/desmoplastic/blue nevi were identified. Standard immunohistochemical techniques were used with antibodies towards HMB-45, Melan-A (A103), and Ki-67; 43 of 50 DMM cases were available for staining with Melan-A, 42 of 50 for HMB-45, and 31 of 50 cases were stained with Ki-67. All 13 nevi were stained for Melan-A and 8 for Ki-67. Immunoreactivity to Ki-67 antibody was scored as 0 to 5%, 6 to 10%, 11 to 30%, or greater than 30% positive tumor cells.. Only 3 of 43 and 3 of 42 of spindle cell compartments of DMMs were positive for Melan-A and HMB-45, respectively. Focal staining of epithelioid cells in the junctional component or superficial dermis was observed in 33% (14/43). In contrast, 100% of the 13 nevi were strongly positive for Melan-A (P < 0.001). Seventeen melanomas (55%) were 0 to 5% positive for Ki-67, five (16%) fell into the 6 to 10% category, three (10%) were between 11 and 30%, and six (19%) were at least focally greater than 30% positive. All 8 nevi (100%) had less than 5% positive cells for Ki-67 (P = 0.02), with only 2 cases having more than 2% positive cells.. The sclerotic/desmoplastic and hypopigmented blue nevi were uniformly positive for Melan-A, while the vast majority of DMM were negative in their spindle cell compartments. Melan-A is very useful in distinguishing between DMM and sclerotic nevi. Ki-67 appears to be an inconsistent marker for DMM. However, a high labeling index (over 5%) may be used as a clue in diagnosing DMM.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Diagnosis, Differential; Humans; Immunohistochemistry; Ki-67 Antigen; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; Nevus; Skin Neoplasms

We report on 10 patients with resected American Joint Committee on Cancer (AJCC)stage IIA-IIIC melanoma receiving individualized adjuvant peptide vaccinations derived from the melanosomal antigens MelanA/MART1, gp100 and tyrosinase, according to patient tumor associated HLA restricted antigen expression, in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF). Except for 1 patient, all patients had received systemic pretreatment with immunotherapy (n = 8), chemoimmunotherapy (n = 1), chemotherapy (n = 1), or cefalectin therapy (n = 1). Upon prior therapy, 7 of 10 patients had progressed with subcutaneous/cutaneous (n = 2), lymph node (n = 3), or subcutaneous/cutaneous and lymph node (n = 2)metastases, which were subsequently resected prior to vaccination. After a mean of 6.5 vaccination cycles, progression-free survival was 6 months (median, range 2-10). Five patients were relapse-free for 1+ up to 21+ months, 3 patients developed a solitary cutaneous metastasis, and 2 patients developed multiple metastases during vaccination. Overall, vaccine treatment was well tolerated, with no severe side-effects. Eight of 10 patients developed local delayed type hypersensitivity (DTH)reactions to synthetic peptides after the first or second injection. In 2 patients, transient fever, nausea, diarrhea, and muscle pain of National Cancer Institute (NCI)Grade I occurred. In summary, individualized synthetic peptide vaccination, combined with GM-CSF, was feasible and warrants further clinical investigation.

Topics: Adult; Aged; Antigens, Neoplasm; Cancer Vaccines; Disease-Free Survival; Female; gp100 Melanoma Antigen; Granulocyte-Macrophage Colony-Stimulating Factor; HLA Antigens; Humans; Lymph Nodes; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Staging; Peptide Fragments; Skin Neoplasms; Vaccination

Cutaneous melanomas have been found to express several immunogenic differentiation melanoma-associated antigens (MAAs) that have been suggested to play an important role in disease outcome. Adaptive host immunity to MAAs has shown some level of control on melanoma progression. To date, there has been no definitive report correlating the level of differentiated MAAs gene expression in melanomas with overall disease outcome. Metastasis of melanoma to distant visceral organ sites usually indicates a survival of less than 1 year; however, a subset of patients who undergo cytoreductive surgery of distant metastases survive for a longer period. We hypothesized that the gene expression level of differentiation MAAs in metastatic melanoma (AJCC stage IV) lesions would be predictive of survival. We focused on three known differentiation MAAs: tyrosinase (TYR), TYR-related protein 2 (TRP-2), and melanoma antigen recognized by T cells 1 (MART-1); all three of them are known to induce immune responses in melanoma patients and are frequently expressed in melanomas. A quantitative reverse-transcriptase RealTime PCR (qRT) assay was developed for these MAAs to assess mRNA expression in metastatic melanoma tumors obtained from cytoreductive surgery of AJCC stage IV melanoma patients (n = 35). Patients were followed up for over 60 months. There was a variation in mRNA copy levels for individual MAAs in melanoma tumors. Elevated MAA mRNA copy levels of TYR and TRP-2 significantly (P < 0.03 and < 0.009, respectively) correlated with improved overall survival. Patients having at least one MAA expressed in their tumors had a significantly (P = 0.01) better overall survival (median 16 months). These studies demonstrate that levels of differentiated MAA mRNA expression of advanced-stage metastatic melanomas can be used as molecular predictive factors of disease outcome. The studies also imply that an assessment of melanoma tumor MAAs may provide a stratification factor targeted for active-specific immunotherapy.

Topics: Antigens, Neoplasm; Gene Expression; Humans; Intramolecular Oxidoreductases; MART-1 Antigen; Melanoma; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Staging; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured

Improved extraction techniques combined with sensitive real-time reverse transcriptase-polymerase chain reaction may allow detection of mRNA in formalin-fixed, paraffin-embedded (FFPE) materials, but the factors affecting mRNA quantification in clinical material using these methods have not been systematically analyzed. We designed analyses using real-time reverse transcriptase-polymerase chain reaction for quantification of MART-1, beta-actin, and beta(2)-microglobulin mRNAs. The analytical intra- and interassay imprecision (coefficient of variation) was in the range 10 to 20% for all three genes studied. Using these protocols, we studied the influence of tissue autolysis and length of formalin-fixation on mRNA detection in metastatic melanoma. Delay in freezing reduced detectable mRNA, although this was less than predicted and mostly occurred early in autolysis. MART-1, beta-actin, and beta(2)-microglobulin mRNAs were consistently detected in FFPE metastatic melanoma even after fixation for up to 3 weeks, although the total mRNA detected was markedly reduced in fixed compared with fresh tissues (up to 99%). Quantification of MART-1 was, however, possible if this was expressed relative to a housekeeping gene. The polymerase chain reaction product from FFPE tissues could be increased up to 100-fold amplifying short (<136 bp) compared with long amplicons. Variations in time before tissue processing and in fixation length seem to be less important sources of imprecision than previously assumed. Our findings suggest that quantitative analysis of mRNA in archive and routine diagnostic tissues may be possible.

Topics: Actins; Antigens, Neoplasm; Base Sequence; Benzothiazoles; beta 2-Microglobulin; Diamines; DNA Primers; Fluorescent Dyes; Formaldehyde; Humans; Lymphatic Metastasis; MART-1 Antigen; Melanoma; Molecular Diagnostic Techniques; Neoplasm Proteins; Organic Chemicals; Paraffin Embedding; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tissue Fixation

The application of lymphoscintigraphy followed by sentinel lymph node (SN) biopsy to patients with primary melanoma has revolutionised the ability to identify accurately, yet conservatively, those patients who harbour occult nodal metastases. The molecular detection of SN micrometastases facilitates the cost effective analysis of the entire SN using multiple markers. Currently, a lack of marker specificity is the main barrier preventing the molecular evaluation of SN tissue from becoming clinically applicable.. To develop a reproducible multimarker reverse transcription-polymerase chain reaction (RT-PCR) assay, with the emphasis on achieving high specificity for the accurate detection of melanoma metastases in nodal tissue.. Three pigment cell specific (PCS) markers-tyrosinase, Pmel-17, and MART-1-and one cancer testis antigen (CTA)-MAGE-3-were selected for use in a multimarker RT-PCR assay. The conditions for this assay were optimised.. High specificity was achievable for each marker by optimising the PCR cycle number such that unwanted transcripts (that is, illegitimate transcripts and/or specific transcripts from other low abundance nodal cell types) remained undetectable in appropriate controls (normal visceral nodes). Tyrosinase was 100% specific at 40 PCR cycles, MAGE-3 and MART-1 at 35 PCR cycles, and Pmel-17 at 30 PCR cycles. Tyrosinase proved to be the most sensitive marker, detecting 10 melanoma cells in 0.1 g of nodal tissue.. Excellent reproducibility of the entire nodal processing and RT-PCR protocol for the detection of very low numbers of melanoma cells in nodal tissue was shown, although there is a risk of false positives using the PCS markers alone, because of an approximate 4-8.5% incidence rate of nodal nevi in melanoma draining SNs (these nevi being absent in all other normal nodes). MAGE-3 was shown to be the only marker that is not expressed by melanocytes. However, because not all melanomas express MAGE-3, it is recommended that more emphasis should be placed on the development of a panel of CTA markers to ensure a zero false positive rate and to provide optimum detection.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; gp100 Melanoma Antigen; Humans; Lymphatic Metastasis; MART-1 Antigen; Melanocytes; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Nevus; Proteins; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Tumor Cells, Cultured

As compared with natural tumor peptide sequences, carefully selected analog peptides may be more immunogenic and thus better suited for vaccination. However, T cells in vivo activated by such altered analog peptides may not necessarily be tumor specific because sequence and structure of peptide analogs differ from corresponding natural peptides.. Three melanoma patients were immunized with a Melan-A peptide analog that binds more strongly to HLA-A*0201 and is more immunogenic than the natural sequence. This peptide was injected together with a saponin-based adjuvant, followed by surgical removal of lymph node(s) draining the site of vaccination.. Ex vivo analysis of vaccine site draining lymph nodes revealed antigen-specific CD8+ T cells, which had differentiated to memory cells. In vitro, these cells showed accelerated proliferation upon peptide stimulation. Nearly all (16 of 17) of Melan-A-specific CD8+ T-cell clones generated from these lymph nodes efficiently killed melanoma cells.. Patient immunization with the analog peptide leads to in vivo activation of T cells that were specific for the natural tumor antigen, demonstrating the usefulness of the analog peptide for melanoma immunotherapy.

Topics: Amino Acid Sequence; Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Flow Cytometry; Lymph Nodes; Lymphocyte Activation; MART-1 Antigen; Melanoma; Neoplasm Proteins; Peptide Fragments; T-Lymphocytes; T-Lymphocytes, Cytotoxic

We report the clinicopathologic features of three cases of malignant melanoma metastatic to the vagina; only one similar case has been previously reported. The age of the patients ranged from 33 to 70 years; two presented with vaginal bleeding. Two patients had a history of a previous primary malignant melanoma, one a preauricular melanoma treated 7 years earlier and the other a vulvar melanoma treated 2 years earlier. In the third case, a primary malignant melanoma was found on the sole of the right foot after the patient had presented with the vaginal metastases. On gross examination, the vaginal tumors were polypoid; two were on the posterior wall and one was on the anterior wall. All were of epithelioid cell type and were positive with S-100 and microphthalmia-transcription factor. Tyrosinase was positive in two cases, HMB-45 was positive in one case, and MART-1 was focally positive in one case. One patient underwent wide local excision of tumor followed by chemotherapy, one patient had intracavitary radiotherapy, and one had palliative radiotherapy to the brain only. The patients died after a follow-up of 3, 14, and 48 months.

Topics: Adult; Aged; Antigens, Neoplasm; Biomarkers, Tumor; DNA-Binding Proteins; Fatal Outcome; Female; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Microphthalmia-Associated Transcription Factor; Monophenol Monooxygenase; Neoplasm Proteins; Retrospective Studies; S100 Proteins; Skin Neoplasms; Transcription Factors; Vaginal Neoplasms

Topics: Adult; Antigens, Neoplasm; Biomarkers, Tumor; Facial Neoplasms; Humans; Immunoenzyme Techniques; Male; MART-1 Antigen; Melanocytes; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; Sensitivity and Specificity; Skin; Skin Neoplasms

Cutaneous melanoma is the most aggressive form of skin carcinoma in humans, frequently with a rapid progression of disease. To detect early developing metastasis, laboratory tests to determine levels of lactate dehydrogenase (LDH) and alkaline phosphatase (AP) form part of the regular follow-up, but often cannot discover recurrent disease at a sufficiently early stage.. To evaluate the diagnostic accuracy of protein S-100beta (S-100beta), melanoma-inhibitory activity (MIA), LDH, AP, and tyrosinase/MART-1 reverse transcription-polymerase chain reaction (RT-PCR), the authors included 296 consecutive AJCC Stage II or III clinically disease-free melanoma patients. Follow-up examinations were performed every 3 months and blood samples were drawn to determine the levels of these tumor markers.. Metastasis occurred in 41 of the 296 patients during a median follow-up period of 19 months (range, 1-33 months). The sensitivity to detect new metastases was 29% for protein S-100beta, 22% for MIA, 2% for LDH, 17% for AP, and 24% for RT-PCR. The diagnostic accuracy was best for MIA (86%) and S-100beta (84%), whereas AP (79%), LDH (77%), and RT-PCR (72%) demonstrated lower values. Elevated values of S-100beta and MIA during follow-up examinations were associated with decreased survival rates in the further course of the disease, but pathologic findings of the other tumor markers showed no prognostic impact.. To the authors' knowledge, the current study is the first comparison of the diagnostic accuracy of currently available tumor markers in the follow-up of high-risk melanoma patients. Protein S-100beta and MIA demonstrated a higher sensitivity, specificity, and diagnostic accuracy in the diagnosis of newly occurring metastasis compared with to the tumor markers AP, LDH, and RT-PCR diagnostics. Therefore, the tumor markers S-100beta and MIA may be useful in the follow-up of disease-free Stage II and III melanoma patients.

Topics: Adult; Antigens, Neoplasm; Biomarkers, Tumor; Extracellular Matrix Proteins; Female; Follow-Up Studies; Humans; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Recurrence, Local; Nerve Growth Factors; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; Risk; S100 Calcium Binding Protein beta Subunit; S100 Proteins; Skin Neoplasms; Time Factors

Suboptimal activation of T lymphocytes by tumor cells may contribute to the failure of the immune system to control tumor growth. We recently demonstrated that Melan-A/MART-1-reactive CTLs can be anergized by peptide analogues with partial agonist/antagonist functions, which selectively impair interleukin (IL)-2 release. Here we analyze the potential expression of partial agonist/antagonist peptides by tumor cells and their role in suboptimal T-cell activation. HLA-bound peptide fractions were eluted from HLA-A*0201/Melan-A/MART-1(+) melanoma cells and analyzed for reconstitution of the MART-1-specific T-cell epitope. Among the peptide fractions able to induce IFN-gamma release by MART-1-specific T cells, only fraction 43-44 activated IL-2 production by anti-MART-1 T cells, whereas the remaining two fractions acted as peptide antagonists by inhibiting IL-2 release in response to the native epitope. A comparable down-modulation of IL-2 release could also be induced by the MART-1-derived peptide 32-40, previously identified in one of the two anergizing fractions. A substantial deficit in IL-2 release was additionally detected in tumor-specific CD8(+) T cells infiltrating melanoma lesions. To overcome IL-2 impairment by peptide antagonists, anti-MART-1 T cells were generated by in vitro sensitization with the two optimized analogues Melan-A/MART-1(27-35) 1L (with superagonist features) and Melan-A/MART-1(26-35) 2L (with improved HLA-A*0201 binding). T cells raised with the superagonist Melan-A/MART-1(27-35) 1L showed resistance to the inhibition of IL-2 release mediated by melanoma-derived peptide fractions, whereas Melan-A/MART-1(26-35) 2L-specific T cells appeared to be as sensitive as T cells raised with the parental epitope. This resistance was associated with the enhanced ability of Melan-A/MART-1(27-35) 1L-specific T cells to release IL-2. Taken together, these data indicate that melanoma cells can process and present on their surface peptides inhibiting optimal T-cell activation against immunodominant epitopes and that the usage of optimized peptide analogues could represent a promising approach for overcoming tumor-induced immunosuppression and possibly designing more successful vaccines for cancer patients.

Topics: Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Clonal Anergy; Epitopes, T-Lymphocyte; HLA-A Antigens; HLA-A2 Antigen; Humans; Interferon-gamma; Interleukin-2; Ligands; Lymphocyte Activation; MART-1 Antigen; Melanoma; Neoplasm Proteins; Peptide Fragments; Receptors, Antigen, T-Cell; Tumor Cells, Cultured

Plasmacytoid dendritic cells (PDC) are a small population of leukocytes specialized in the production of type I IFN. It has been shown that PDC have a potent T cell stimulatory capacity in allogeneic mixed lymphocyte reaction, However, their role in initiating primary immune responses remains elusive. We report that blood PDC efficiently prime naive CD8(+) lymphocytes specific for the melan-A(26-35) epitope to become IFN-gamma producing cells in vitro. In addition, we found that CD40L-stimulated PDC induce expression on primed melan-A-specific T cells of cutaneous lymphocyte antigen and L-selectin (CD62L), homing receptors that allow the migration of effector cells to the inflamed skin. Finally, we show that PDC can be found in the peri-tumoral area of most primary cutaneous melanomas in vivo and that type I IFN-containing supernatants derived from PDC increase melanoma cell surface expression of CD95 and MHC class I and class II molecules in vitro. Our results suggest a new immunomodulatory role for tissue infiltrating PDC, which may prime tumor-specific T cell responses and affect tumor growth via soluble factors.

Topics: Antigens, Differentiation, T-Lymphocyte; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Dendritic Cells; fas Receptor; Histocompatibility Antigens; Humans; Interferon Type I; Interferon-gamma; Interleukin-12; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Tumor Cells, Cultured

Though acral lentiginous melanoma (ALM) is a major type of malignant melanoma, no immunohistochemical study on this type of melanoma has been reported.. The purpose of this study is to analysis the immunohistochemical findings of ALM using routinely used immune markers.. An immunohistochemical study was performed on paraffin sections of 20 ALMs using S-100 protein, HMB-45, MART-1, vimentin, epithelial membrane antigen (EMA) and CAM 5.2.. S-100 protein (95%) was found to be a more sensitive marker than either HMB-45 (80%) or MART-1 (70%) for recognizing ALM. Melanin bleaching was useful for recognizing heavily pigmented ALM using both S-100 protein and HMB-45. The intensity of HMB-45 correlated well with the melanin content. However, there was no significant correlation between the intensity of S-100 protein and the melanin content. One and two out of 20 cases stained focally with EMA and CAM5.2, respectively, but these cases stained also with HMB-45 and/or S-100 protein.. S-100 protein and HMB-45 were relatively sensitive markers for recognizing ALM. Despite the occasional positivity for the epithelial markers in ALM, all epithelial marker-positive cases stained also with HMB-45 and/or S-100 protein. Therefore, we recommend that the panel of antibodies used for recognizing ALM should contain at least S-100 protein and HMB-45.

Topics: Aged; Antigens, Neoplasm; Biomarkers; Biomarkers, Tumor; Female; Humans; Immunohistochemistry; Keratins; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Mucin-1; Neoplasm Proteins; S100 Proteins; Skin Neoplasms

Topics: Antigens, Neoplasm; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Staging; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Sentinel Lymph Node Biopsy; Skin Neoplasms

Polymerase chain reaction (PCR) is a molecular biology technique that can detect a single metastatic cell in 10(6) to 10(7) normal cells. Its use has been proposed as an additional new method for the detection of malignant melanoma nodal metastases in the sentinel lymph node (SLN) to improve the detection rate guaranteed so far by standard histology (hematoxylin and eosin; H&E) and immunohistochemistry (IHC).. Since October 1995, 137 patients with primary cutaneous melanoma (Breslow thickness,.75-4 mm) have undergone surgery for selective lymphadenectomy. To identify the SLNs, every patient had preoperative lymphoscintigraphy and a vital dye perilesional injection, followed by a gamma probe-guided operation.. In 134 patients at least one SLN was detected, with a detection rate of 98%. Every SLN was examined by H&E and IHC (S-100 antigen and HMB-45 protein). The messenger RNA codifying for tyrosinase and MART-1 (melanoma antigen recognized by T cells) was used as the target sequence for the reverse transcriptase (RT)-PCR. The results showed 11% positive SLNs with IHC and H&E examination and 63% with RT-PCR. No recurrence was noted at follow-up in the group with RT-PCR-negative nodes (absence of false-negative cases).. In our experience, RT-PCR SLN negativity is achieving a very favorable prognostic significance. However, RT-PCR positivity is still to be evaluated. Furthermore, results obtained with this method have been shown so far to be independent of Breslow's tumor thickness.

Topics: Antigens, Neoplasm; Disease-Free Survival; Female; Humans; Immunohistochemistry; Lymphatic Metastasis; MART-1 Antigen; Melanoma; Neoplasm Proteins; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; Skin Neoplasms; Survival Analysis; Tyrosine

The specificity of recognition of pMHC complexes by T lymphocytes is determined by the V regions of the TCR alpha- and beta-chains. Recent experimental evidence has suggested that Ag-specific TCR repertoires may exhibit a more V alpha- than V beta-restricted usage. Whether V alpha usage is narrowed during immune responses to Ag or if, on the contrary, restricted V alpha usage is already defined at the early stages of TCR repertoire selection, however, has remained unexplored. Here, we analyzed V and CDR3 TCR regions of single circulating naive T cells specifically detected ex vivo and isolated with HLA-A2/melan-A peptide multimers. Similarly to what was previously observed for melan-A-specific Ag-experienced T cells, we found a relatively wide V beta usage, but a preferential V alpha 2.1 usage. Restricted V alpha 2.1 usage was also found among single CD8(+) A2/melan-A multimer(+) thymocytes, indicating that V alpha-restricted selection takes place in the thymus. V alpha 2.1 usage, however, was independent from functional avidity of Ag recognition. Thus, interaction of the pMHC complex with selected V alpha-chains contributes to set the broad Ag specificity, as underlined by preferential binding of A2/melan-A multimers to V alpha 2.1-bearing TCRs, whereas functional outcomes result from the sum of these with other interactions between pMHC complex and TCR.

Topics: Amino Acid Sequence; Antigens, Neoplasm; Autoantigens; Cell Differentiation; Clone Cells; Cytotoxicity, Immunologic; Epitopes, T-Lymphocyte; Gene Rearrangement, alpha-Chain T-Cell Antigen Receptor; Gene Rearrangement, beta-Chain T-Cell Antigen Receptor; Hematopoietic Stem Cells; HLA-A2 Antigen; Humans; MART-1 Antigen; Melanoma; Molecular Sequence Data; Neoplasm Proteins; Receptors, Antigen, T-Cell, alpha-beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

MART-1, Melan-A, and Tyrosinase have shown encouraging results for evaluation of melanoma micrometastases in sentinel lymph nodes, as compared to conventionally used S-100 protein and HMB-45. To achieve higher sensitivity, some studies recommend evaluation of three sections, each at intervals of 200 micron. This would mean, routine staining of three adjacent sections in each of the three clusters at intervals of 200 micron, requiring nine slides resulting in added expense. If a cocktail of these antibodies could be used, only one section would be required instead of three generating significant cost savings.. We prepared a combination of monoclonal antibodies to these three immunomarkers in optimized dilutions (MART-1, clone M2-7C10, dilution 1:500; Melan-A, clone A103, dilution 1:100; and Tyrosinase, clone T311, dilution 1:50) and designated it as 'MCW melanoma cocktail'. Formalin-fixed paraffin-embedded tissue sections of sentinel lymph nodes from patients with cutaneous melanoma, without macro-metastases were evaluated with this cocktail.. Melanoma micrometastases were easily detectable with the cocktail in 41 out of 188 slices (8/24 cases). The diagnostic accuracy amongst five pathologists did not show statistically significant difference. Out of 188 slices, 78 had adjacent sections immunostained individually with MART-1 and Melan-A during our previous study. Of these 78 slices, 21 were positive for melanoma micrometastases with MART-1 and Melan-A individually. However, the adjacent section of these slices immunostained with the cocktail detected metastases in four additional slices. Thus, MART-1 and Melan-A could not detect melanoma micrometastases individually in 16% (4/25) of slices positive with the cocktail. Benign capsular nevi were immunoreactive for the cocktail in 4.8% (9/188) slices. All 81 slices of negative test controls (sentinel lymph nodes of mammary carcinoma) were interpreted correctly as negative for melanoma micrometastases.. The melanoma cocktail facilitated easy interpretation of melanoma micrometastases in sentinel lymph nodes with high interobserver agreement. There was improvement in detection rate with the cocktail as compared to MART-1 and Melan-A individually. Furthermore, this approach facilitates cost savings.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Humans; Immunohistochemistry; Lymph Nodes; Lymphatic Metastasis; MART-1 Antigen; Melanoma; Monophenol Monooxygenase; Neoplasm Proteins; Observer Variation; Reproducibility of Results; Sentinel Lymph Node Biopsy; Skin Neoplasms

Activation of CTL-mediated antitumor immunity to self-epitopes expressed by neoplastic cells is thought to be prevented, at any stage of tumor progression, by tolerance mechanisms. In contrast, in 74 American Joint Committee on Cancer stages I-IV melanoma patients, we found that development of lymph node metastases is a key event triggering CD8(+) T-cell-mediated immunity to self-epitopes encoded by melanocyte differentiation antigens. This was shown by the increased peripheral precursor frequency to Melan-A/Mart-1, gp100, and tyrosinase epitopes in stage III and IV compared with stage I and II patients, and by accumulation of functional memory T cells directed to Melan-A/Mart-1(26-35) in tumor-invaded lymph nodes. However, in tumor-invaded lymph nodes of most patients, CD8(+) T cells directed to melanocyte differentiation antigens or to tumor-restricted antigens (MAGE-3 and NY-ESO-1 epitopes), showed a CCR7(+) CD45RA(+) CD27(+) CD28(+) perforin(-) "precursor" phenotype. Only in 7 of 23 cases antigen-specific CD8(+) T cells in invaded lymph nodes showed a predominant CCR7(-) CD45RA(-) CD27(+) CD28(-) perforin(+) "preterminally differentiated" phenotype. In the latter subset of patients, by immunohistochemistry in lymph node lesions, we found that CD8(+) T lymphocytes intermingling with the neoplastic tissue expressed a CCR7(-) CD45RO(+)/RA(-) phenotype, whereas CD4(+) lymphocytes did not infiltrate the tumor. Furthermore, perforin and granzyme B were expressed on a higher fraction of the CD8(+) cells surrounding the invading tumor compared with the lymphocytes infiltrating the neoplastic tissue. In addition, no evidence for tumor regression was found in such metastatic lesions, as documented by absence of neoplastic cell necrosis or apoptosis. These data indicate that neoplastic cells in the lymph nodes and/or increased tumor burden in metastatic disease activate CD8(+) T-cell-mediated antitumor immunity to self-epitopes. However, the paucity of terminally differentiated CD8(+) T cells at tumor site suggests that immunotherapy strategies may require not only the boosting of tumor immunity, but also effective means to promote CD8(+) T-cell differentiation in the neoplastic tissue.

Topics: Antigens, Neoplasm; Autoantigens; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Differentiation; Epitopes, T-Lymphocyte; gp100 Melanoma Antigen; Granzymes; HLA-A Antigens; HLA-A2 Antigen; Humans; Immunologic Memory; Leukocyte Common Antigens; Lymph Nodes; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Membrane Proteins; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Staging; Peptide Fragments; Perforin; Pore Forming Cytotoxic Proteins; Proteins; Receptors, CCR7; Receptors, Chemokine; Serine Endopeptidases

To develop a reverse transcriptase-polymerase chain reaction (RT-PCR) assay to detect canine melanoma-associated antigens (MAAs) and to use this technique to screen aspirates of lymph nodes (LNs) for evidence of metastatic spread of oral malignant melanoma.. 7 dogs with oral malignant melanoma and 4 dogs with multicentric lymphosarcoma.. We prepared cDNA from melanoma tumor biopsies and fine-needle aspirates obtained from submandibular LNs of dogs with oral malignant melanoma or multicentric lymphosarcoma. The RT-PCR assay was performed by use of tyrosinase, Melan-A, gp100, tyrosinase-related protein 2 (TRP-2), or melanoma antigen-encoding gene B (MAGE-B)-specific primers.. We detected MAGE-B mRNA in canine testicular tissue but not in melanoma biopsy specimens. Tyrosinase, Melan-A, gp100, and TRP-2 mRNAs were detected in tumor biopsy specimens and in 2 of 5 LN aspirates from dogs with melanoma, suggesting metastatic spread in those 2 dogs. We did not detect MAAs in LN aspirates obtained from dogs with multicentric lymphosarcoma. Sequencing of canine Melan-A and gp100 PCR products confirmed the specificity of the assay for these genes.. Clinical staging of dogs with oral malignant melanoma is useful to assist in designing appropriate treatments. However, results of histologic examination of LN biopsy specimens can be inconclusive and, in humans, can underestimate the number of patients with metastatic disease. Molecular staging of melanomas in dogs can be achieved by screening LN aspirates for MAA mRNA, and this can be performed in combination with cytologic examination to aid in detection of metastatic disease.

Topics: Animals; Antigens, Neoplasm; Base Sequence; Biomarkers; DNA, Complementary; Dog Diseases; Dogs; Female; Humans; Lymph Nodes; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Molecular Sequence Data; Mouth Neoplasms; Neoplasm Proteins; Polymerase Chain Reaction; RNA, Neoplasm; Testis

The clinically important melanoma diagnostic antibodies HMB-45, melan-A, and MITF (D5) recognize gene products of the melanocyte-lineage genes SILV/PMEL17/GP100, MLANA/MART1, and MITF, respectively. MITF encodes a transcription factor that is essential for normal melanocyte development and appears to regulate expression of several pigmentation genes. In this report, the possibility was examined that MITF might additionally regulate expression of the SILV and MLANA genes. Both genes contain conserved MITF consensus DNA sequences that were bound by MITF in vitro and in vivo, based on electrophoretic mobility shift assay and chromatin-immunoprecipitation. In addition, MITF regulated their promoter/enhancer regions in reporter assays, and up- or down-regulation of MITF produced corresponding modulation of endogenous SILV and MLANA in melanoma cells. Expression patterns were compared with these factors in a series of melanoma cell lines whose mutational status of the proto-oncogene BRAF was also known. SILV and MLANA expression correlated with MITF, while no clear correlation was seen relative to BRAF mutation. Finally, mRNA expression array analysis of primary human melanomas demonstrated a tight correlation in their expression levels in clinical tumor specimens. Collectively, this study links three important melanoma antigens into a common transcriptional pathway regulated by MITF.

Topics: Animals; Antigens, Neoplasm; Biomarkers, Tumor; DNA-Binding Proteins; Enhancer Elements, Genetic; Gene Expression Regulation, Neoplastic; Genes, Reporter; gp100 Melanoma Antigen; Humans; MART-1 Antigen; Melanocytes; Melanoma; Membrane Glycoproteins; Mice; Microphthalmia-Associated Transcription Factor; Mutagenesis, Site-Directed; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Mas; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

Melanoma treatment is based on surgery. In metastatic cases, vaccine therapy has been recently developed to overcome T cell or dendritic cell dysfunction. HLA tetramerbased assays are useful for immunologic monitoring of this trial and to quantitate CD8+ specific lymphocytes. In the present work, we used tetrameric technology to detect expanded populations of tumor specific CD8+ Tcells specific of Melan-A/Mart-1 Antigen have been quantified using flow cytometry. The feasibility of routinely detection of 0,1% +/- 0,03 of CD8 T cells has been demonstrated, without any difference the levels observed before and after (day 30) surgery. The value of experimentation of these cells should be determined in clinic and particularly to analyze surgical practice.

Topics: Adult; Aged; Antigens, Neoplasm; CD8 Antigens; Female; HLA-A2 Antigen; Humans; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Skin Neoplasms

Spontaneous histopathological regression of cancer has been reported. The involvement of the immune system in such regression has been advocated, leading to the theory of immunological surveillance against cancer. A prediction of this theory is that common tumour antigens can be recognized upon repeated exposure by cell-mediated immunity, which leads to tumour regression and the subsequent appearance of tumour antigen-loss variants. However, no direct evidence has been provided in non-viral-induced experimental animal models of primary malignancy or in human primary cancer. This study examined two groups of melanoma patients where histopathological regression of the primary tumour was observed. Many of the 23 patients with multiple (> or =3) primary melanomas showed significant regression of their last melanoma (median 33%, mean 40) compared with matched melanomas from patients with a single primary melanoma (median 0%, mean 12) (p=0.0080), or compared with their first primary melanoma (p=0.0013). Regression was consistent with an 'immunization effect' seen in murine tumour transplantation studies, where inoculation with > or =3 asynchronous tumours induces transplantation rejection on subsequent challenge. A significant decrease in the expression of the melanoma common tumour antigen MART-1 in the last primary tumour from multiple melanoma patients (median 8%, mean 24) versus matched single melanoma patients (median 79%, mean 68) (p=0.0041) and in the last versus first tumour in multiple primary patients was found (p=0.0083). Metastases from 17 patients whose primary skin melanomas had completely regressed (occult primary melanoma) also showed significant MART-1 loss (median 0%, mean 11) compared with matched metastases from patients with non-regressing primary melanoma (median 51%, mean 50) (p=0.0013). MART-1 antigen-loss variants observed in the multiple primary and occult primary patients correlated with the presence of peripheral blood MART-1-specific cytotoxic T lymphocytes (CTLs) (p=0.03). No similar effects were observed with two other melanoma antigens, gp100 and CD63. Thus, in two groups of human melanoma patients, evidence is provided for histopathological tumour regression associated with cancer immune surveillance.

Topics: Antigens, CD; Antigens, Neoplasm; Enzyme-Linked Immunosorbent Assay; Female; gp100 Melanoma Antigen; Humans; Male; MART-1 Antigen; Melanocytes; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Neoplasm Regression, Spontaneous; Platelet Membrane Glycoproteins; Skin Neoplasms; T-Lymphocytes, Cytotoxic; Tetraspanin 30

The goal of adoptive T cell therapy in cancer is to provide effective antitumor immunity by transfer of selected populations of tumor Ag-specific T cells. Transfer of T cells with high TCR avidity is critical for in vivo efficacy. In this study, we demonstrate that fluorescent peptide/MHC class I multimeric complexes incorporating mutations in the alpha3 domain (D227K/T228A) that abrogate binding to the CD8 coreceptor can be used to selectively isolate tumor Ag-specific T cells of high functional avidity from both in vitro expanded and ex vivo T cell populations. Sorting, cloning, and expansion of alpha3 domain mutant multimer-positive CD8 T cells enabled rapid selection of high avidity tumor-reactive T cell clones. Our results are relevant for ex vivo identification and isolation of T cells with potent antitumor activity for adoptive T cell therapy.

Topics: Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Cell Adhesion; Cell Line; Cell Separation; Clone Cells; Cytotoxicity Tests, Immunologic; Epitopes, T-Lymphocyte; HLA-A2 Antigen; Humans; MART-1 Antigen; Melanoma; Mutagenesis, Site-Directed; Neoplasm Proteins; Peptide Fragments; Protein Binding; Protein Structure, Tertiary; Staining and Labeling; Tumor Cells, Cultured

Differentiation of CD8(+) T cells at the tumor site toward effector and memory stages may represent a key step for the efficacy of antitumor response developing naturally or induced through immunotherapy. To address this issue, CD8(+) T lymphocytes from tumor-invaded (n = 142) and tumor-free (n = 42) lymph nodes removed from the same nodal basin of melanoma patients were analyzed for the expression of CCR7, CD45RA, perforin, and granzyme B. By hierarchical cluster analysis, CD8(+) T cells from all tumor-free lymph nodes and from 56% of the tumor-invaded lymph node samples fell in the same cluster, characterized mainly by CCR7(+) CD45RA(+/-) cytotoxic factor(-) cells. The remaining three clusters contained only samples from tumor-invaded lymph nodes and showed a progressive shift of the CD8(+) T cell population toward CCR7(-) CD45RA(-/+) perforin(+) granzyme B(+) differentiation stages. Distinct CD8(+) T cell maturation stages, as defined by CCR7 vs CD45RA and by functional assays, were identified even in melanoma- or viral Ag-specific T cells from invaded lymph nodes by HLA tetramer analysis. Culture for 7 days of CCR7(+) perforin(-) CD8(+) T cells from tumor-invaded lymph nodes with IL-2 or IL-15, but not IL-7, promoted, mainly in CCR7(+)CD45RA(-) cells, proliferation coupled to differentiation to the CCR7(-) perforin(+) stage and acquisition of melanoma Ag-specific effector functions. Taken together, these results indicate that CD8(+) T cells differentiated toward CCR7(-) cytotoxic factor(+) stages are present in tumor-invaded, but not in tumor-free, lymph nodes of a relevant fraction of melanoma patients and suggest that cytokines such as IL-2 and IL-15 may be exploited to promote Ag-independent maturation of anti-tumor CD8(+) T cells.

Topics: Antigens, Neoplasm; Antigens, Viral; CD8-Positive T-Lymphocytes; Cell Differentiation; Cells, Cultured; Epitopes, T-Lymphocyte; Humans; Interleukin Receptor Common gamma Subunit; Interleukin-15; Interleukin-2; Lymph Nodes; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Neoplasm Proteins; Receptors, CCR7; Receptors, Chemokine; Receptors, Interleukin-7; T-Lymphocyte Subsets; Tumor Cells, Cultured

The diagnosis of malignant melanoma remains one of the most difficult to render in surgical pathology, partially because of its extreme histologic variability. Limits in the sensitivity and/or specificity of the currently available melanocytic markers such as anti-S100, HMB45, and anti-MelanA further complicate this problem. Previous work has demonstrated that the B-cell proliferation/differentiation marker MUM1/IRF4 is detected in malignant melanoma and hematolymphoid malignancies, but not in any other neoplasm tested (including colonic, lung, breast, and ovarian carcinomas). In the current study, we have examined MUM1 protein expression in 61 melanocytic lesions and compared the diagnostic usefulness of this marker with that of anti-S100, HMB45, and anti-MelanA. The results indicate that MUM1 is positive in 33/36 (92%) cases of melanoma (21/22 [95%] conventional primary melanomas and 12/14 [86%] metastatic melanomas). In comparison, positivity was seen with anti-S100 in 36/36 cases (100%, 22 primary and 14 metastatic), HMB45 in 28 cases (78%, 17 primary and 11 metastatic), and anti-MelanA in 27 cases (75%, 19 primary and 8 metastatic). Although negative in schwannomas, neurofibromas, and malignant peripheral nerve sheath tumors, MUM1 is detected in only one in eight cases of spindle cell and desmoplastic melanomas. With the exception of desmoplastic and spindle cell melanomas, MUM1 appears to be a sensitive and specific immunohistochemical stain for melanocytic lesions and may prove to be a useful addition to the current panel of melanoma markers.

Topics: Antigens, Neoplasm; B-Lymphocytes; Biomarkers, Tumor; Diagnosis, Differential; DNA-Binding Proteins; gp100 Melanoma Antigen; Humans; Immunohistochemistry; Interferon Regulatory Factors; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Nerve Sheath Neoplasms; Nevus; S100 Proteins; Transcription Factors

Myeloid-origin dendritic cells (DCs) can develop into IL-12-secreting DC1 or non-IL-12-secreting DC2 depending on signals received during maturation. Through rapid culture techniques that prepared either mature, CD83+ DC1 or DC2 from CD14+ monocytes in only 2 days followed by a single 6-7 day DC-T cell coculture, we sensitized normal donor CD8+ T cells to tumor Ags (HER-2/neu, MART-1, and gp100) such that peptide Ag-specific lymphocytes constituted up to 16% of the total CD8+ population. Both DC1 and DC2 could sensitize CD8+ T cells that recognized peptide-pulsed target cells. However, with DC2, a general decoupling was observed between recognition of peptide-pulsed T2 target cells and recognition of Ag-expressing tumor cells, with peptide-sensitized T cells responding to tumor only about 15% of the time. In contrast, direct recognition of tumor by T cells was dramatically increased (to 85%) when DC1 were used for sensitization. Enhanced tumor recognition was accompanied by 10- to 100-fold increases in peptide sensitivity and elevated expression of CD8beta, characteristic of high functional avidity T cells. Both of these properties were IL-12-dependent. These results demonstrate the utility of rapid DC culture methods for high efficiency in vitro T cell sensitization that achieves robust priming and expansion of Ag-specific populations in 6 days. They also demonstrate a novel function of IL-12, which is enhancement of CD8+ T cell functional avidity. A new approach to DC-based vaccines that emphasizes IL-12 secretion to enhance functional avidity and concomitant tumor recognition by CD8+ T cells is indicated.

Topics: Adjuvants, Immunologic; Adult; Antigen Presentation; Antigens, Neoplasm; CD8 Antigens; CD8-Positive T-Lymphocytes; Cell Adhesion; Cell Differentiation; Cell Line, Tumor; Cells, Cultured; Culture Media, Conditioned; Cytotoxicity, Immunologic; Dendritic Cells; Epitopes, T-Lymphocyte; Female; gp100 Melanoma Antigen; Humans; Immunization; Interleukin-12; Lymphocyte Activation; Male; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Peptide Fragments; Receptor, ErbB-2; T-Lymphocyte Subsets

Dendritic cell (DC)-based immunotherapy is a promising form of adjuvant therapy for high-risk tumors. DCs transfected with tumor-associated antigens are capable of stimulating antigen-specific T cells, but cytolytic responses have been disappointing. Activation of DC surface CD40 influences DC cytokine production, particularly that of interleukin (IL)-12, which favors a Th1 (cytotoxic) helper T cell response. This study evaluated the effects of exogenous soluble CD40 ligand (sCD40L) on RNA-transfected DC preparations and their subsequent ability to generate antimelanoma cytolytic T cells.. Human monocyte-derived DCs were cultured and transfected with mRNA encoding full-length melanoma-associated antigen, Mart-1, and matured with and without sCD40L. DC IL-12 secretion and the ability to stimulate naïve T cells were assessed by enzyme-linked immunosorbent assay (ELISA), tetramer analysis, Elispot, and (51)Cr release assay.. Mature DCs stimulated with sCD40L secreted higher levels of IL-12 compared with immature DCs and DCs matured without sCD40L (P <.001). DCs treated with sCD40L generated a greater number of antigen-specific T cells (P <.05) by tetramer and Elispot analyses, and yielded specific T cells with significant cytotoxicity against HLA-matched melanoma cell lines.. CD40L augments DC IL-12 secretion and is essential to potentiate specific antimelanoma cytolytic responses stimulated by the Mart-1 antigen. sCD40L should be considered a crucial adjuvant in DC preparations for RNA-based DC vaccine therapies.

Topics: Antigens, Neoplasm; Cancer Vaccines; CD40 Ligand; Cells, Cultured; Cellular Senescence; Dendritic Cells; Humans; Immunotherapy; Interleukin-12; MART-1 Antigen; Melanoma; Neoplasm Proteins; RNA, Messenger; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory; Transfection

A sentinel lymph node (SLN) that is melanoma negative by pathologic examination implies absence of melanoma metastasis to that regional lymph node field. However, a small proportion of patients develop regional node field recurrence after a negative SLN biopsy. In this study, we reviewed the histopathology of negative SLNs from such patients to determine whether occult melanoma cells were present in the SLNs, to characterize the pathologic features of false-negative SLNs, and to provide recommendations for the histopathologic examination of these specimens. Between March 1992 and June 2001, of 1152 patients who had undergone SLN biopsy for primary melanomas at the Sydney Melanoma Unit, 976 were diagnosed with negative SLNs by initial pathologic examination (using 2 hematoxylin and eosin stained sections, and 2 immunostained sections for S-100 protein and HMB45), and follow-up was available in 957. Of these, 26 (2.7%) developed regional lymph node recurrence during a median follow-up period of 35.7 months. For 22 of them, the original slides and tissue blocks were available for reexamination. The original slides of each block were reviewed. Multiple further sections were cut from each block and stained with hematoxylin and eosin, for S-100, HMB45, and Melan A. Deposits of occult melanoma cells were detected in 7 of the 22 cases (31.8%). In 5 of the 7 cases, deposits of melanoma cells were present only in the recut sections. There were no significant differences in clinical and pathologic variables for those patients in whom occult melanoma cells were found by pathologic reexamination of their SLNs, compared with those in whom no melanoma cells were detected. The detection of melanoma cell deposits in only 7 of 22 false-negative SLNs suggests that mechanisms other than failure of histopathologic examination may contribute to the failure of the SLN biopsy technique in some patients. The failure rate for melanoma detection in SLNs by our routine pathologic examination, using the current protocol at our institution, was <1% (7 of 957 patients). Routinely performing more intensive histopathologic examination of SLNs is difficult to justify from a cost benefit perspective; we therefore recommend examining two hematoxylin and eosin stained sections and two immunostained sections (for S-100 and HMB45) routinely on SLNs from melanoma patients.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; False Negative Reactions; Female; Follow-Up Studies; Humans; Immunohistochemistry; Lymph Nodes; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasm Proteins; Neoplasm Recurrence, Local; S100 Proteins; Sentinel Lymph Node Biopsy; Staining and Labeling

Osteocartilaginous metaplasia is known to occur rarely in melanomas, particularly in subungual melanomas. We present a case of a calcified subungual soft tissue tumour in which biopsy of the lesion showed malignant round and spindle-shaped tumour cells, many of which were associated with the formation of cartilage and osteoid-like material. Subsequent resection showed clear histological evidence of a subungual melanoma. Tumour cells expressed S100, melan-A and neurone-specific enolase but were negative for HMB45. Diagnostic radiological and histological features and the nature of the osteocartilaginous differentiation within this lesion is discussed.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Biopsy; Bone Neoplasms; Cartilage; Diagnosis, Differential; Fingers; Humans; Magnetic Resonance Imaging; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasm Proteins; Osteochondroma; Osteosarcoma; Phosphopyruvate Hydratase; Soft Tissue Neoplasms; Tomography, X-Ray Computed

The recent identification and molecular characterization of tumor-associated antigens recognized by tumor-reactive CD8+ T lymphocytes has led to the development of antigen-specific immunotherapy of cancer. Among other approaches, clinical studies have been initiated to assess the in vivo immunogenicity of tumor antigen-derived peptides in cancer patients. In this study, we have analyzed the CD8+ T cell response of an ocular melanoma patient to a vaccine composed of four different tumor antigen-derived peptides administered simultaneously in incomplete Freund's adjuvant (IFA). Peptide NY-ESO-1(157-165) was remarkably immunogenic and induced a CD8+ T cell response detectable ex vivo at an early time point of the vaccination protocol. A CD8+ T cell response to the peptide analog Melan-A(26-35 A27L) was also detectable ex vivo at a later time point, whereas CD8+ T cells specific for peptide tyrosinase(368-376) were detected only after in vitro peptide stimulation. No detectable CD8+ T cell response to peptide gp100(457-466) was observed. Vaccine-induced CD8+ T cell responses declined rapidly after the initial response but increased again after further peptide injections. In addition, tumor antigen-specific CD8+ T cells were isolated from a vaccine injection site biopsy sample. Importantly, vaccine-induced CD8+ T cells specifically lysed tumor cells expressing the corresponding antigen. Together, these data demonstrate that simultaneous immunization with multiple tumor antigen-derived peptides can result in the elicitation of multiepitope-directed CD8+ T cell responses that are reactive against antigen-expressing tumors and able to infiltrate antigen-containing peripheral sites.

Topics: Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cytotoxicity Tests, Immunologic; Drug Administration Schedule; Epitopes, T-Lymphocyte; Freund's Adjuvant; HLA-A2 Antigen; Humans; Injections, Subcutaneous; Lipids; Longitudinal Studies; Lymphocyte Activation; MART-1 Antigen; Melanoma; Membrane Proteins; Monophenol Monooxygenase; Neoplasm Proteins; Peptide Fragments; Proteins; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured; Uveal Neoplasms; Vaccines, Subunit

Modification of the parental immunodominant Melan-A/MART-1 peptide (MART-1(26-35)) by replacing the alanine with leucine (A27L) enhances its immunogenicity. Because of the reported advantages of RNA over peptides in DC vaccines, we sought to mutate the MART-1 gene to encode a full-length MART-1 antigen with an A27L amino acid substitution. Human DC were transfected with A27L-mutated MART-1 RNA (A27L RNA) or native MART-1 RNA, and then used to stimulate autologous T cells from a series of 8 HLA-A2+ volunteers. After three stimulations, all CTL induced with DC/A27L RNA exhibited more tetramer+ cells, and demonstrated stronger antigen-specific IFNgamma-secreting activity compared to CTL induced with DC/native RNA. A potent MART-1-specific, and predominantly class-I-restricted lysis was detected in most CTL induced with DC/A27L RNA, while native RNA-induced CTL showed minimal and non-specific lysis. HLA-A2+ DC and MART-1 negative/A2+ melanoma cells transfected with the A27L RNA were recognized and killed by MART-1-specific CTL, suggesting that these APC efficiently processed the A27L RNA and presented correct MART-1-specific epitope(s). In summary, introducing an A27L mutation into the MART-1 full-length mRNA sequence enhanced the immunogenicity of the encoded MART-1 Ag. The ease with which such a mutation can be made in RNA presents another potential advantage of using RNA for immunotherapy. Our results support considering this strategy for enhancing the immunogenicity of DC-based RNA vaccines.

Topics: Amino Acid Substitution; Antigens, Neoplasm; Cell Line, Tumor; Dendritic Cells; Humans; Immunophenotyping; Interferon-gamma; MART-1 Antigen; Melanoma; Mutation; Neoplasm Proteins; RNA, Messenger; T-Lymphocytes, Cytotoxic; Transfection

An HLA-A2-positive patient with advanced stage IV melanoma was vaccinated with dendritic cells (DCs) pulsed with melanoma antigens, whereby the rapid progression of disease stalled for a period of 10 months. Monitoring of the cellular immune response against one of the vaccinated HLA-A2-restricted epitopes demonstrated both induction and subsequent decline in the number of interferon-gamma (IFN-gamma)-producing MART-1-reactive cells present in the blood. Enumeration of reactive T cells by MART-126-35/HLA-A2 tetramer staining revealed an induction of such cells after three vaccinations and a subsequent decline that most prominent at times of rapid disease progression. However, a substantial number of reactive cells were present even when no MART-1 reactivity was detectable by functional assays. Isolation of such MART-126-35-reactive T cells by means of peptide/HLA-A2-coated magnetic beads demonstrated the persistence of a TCRVbeta14+ T-cell clone in this population over the whole observation period. Intracellular fluorescence-activated cell sorter staining of such TCRVbeta14+ T cells for IFN-gamma and interleukin-2 after maximal stimulation with phorbol 12-myristate 13-acetate/ionomycin revealed an impairment in their capacity to produce cytokines at the end of the observation period. Thus, functional changes of individual T-cell clones, e.g. clonal exhaustion, seem to be responsible for the known discrepancy between functional and phenotype assays for immune monitoring of tumour patients.

Topics: Antigens, Neoplasm; Disease Progression; HLA-A2 Antigen; Humans; Interferon-gamma; Interleukin-2; Longitudinal Studies; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Receptors, Antigen, T-Cell; Receptors, Antigen, T-Cell, alpha-beta; T-Lymphocytes

We previously described HLA-B35-restricted melanoma tumor-infiltrating lymphocyte responses to frequently expressed melanoma-associated Ags: tyrosinase, Melan-A/MART-1, gp100, MAGE-A3/MAGE-A6, and NY-ESO-1. Using clones derived from these TIL, we identified in this study the corresponding epitopes. We show that five of these epitopes are new and that melanoma cells naturally present all the six epitopes. Interestingly, five of these epitopes correspond to or encompass melanoma-associated Ag epitopes presented in other HLA contexts, such as A2, A1, B51, and Cw3. In particular, the HLA-B35-restricted Melan-A epitope is mimicked by the peptide 26-35, already known as the most immunodominant melanoma epitope in the HLA-A*0201 context. Because this peptide lacked adequate anchor amino acid residues for efficient binding to HLA-B35, modified peptides were designed. Two of these analogues were found to induce higher PBL- and tumor-infiltrating lymphocyte-specific responses than the parental peptide, suggesting that they could be more immunogenic in HLA-B*3501 melanoma patients. These data have important implications for the formulation of polypeptide-based vaccines as well as for the monitoring of melanoma-specific CTL response in HLA-B*3501 melanoma patients.

Topics: Animals; Antigen Presentation; Antigens, Neoplasm; Cell Division; Cell Line, Transformed; Cell Line, Tumor; Clone Cells; COS Cells; Cytotoxicity Tests, Immunologic; Epitopes, T-Lymphocyte; gp100 Melanoma Antigen; HLA-B35 Antigen; Humans; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Membrane Proteins; Mice; Monophenol Monooxygenase; Neoplasm Proteins; Peptide Fragments; Protein Binding; Proteins; T-Lymphocytes, Cytotoxic

Treatment of melanoma cell lines with IFN-gamma induces the switch from proteasome (PS) to immunoproteasome (iPS). This finding has profound implications for the immunobiology of melanoma cells since certain peptides (such as Melan-A(mart1)(27-35)) are cleaved differently by iPS, thus implying a different ability to be presented by HLA class I molecules. IFN-alpha is a cytokine not only produced during infectious diseases, but also used in the treatment of certain cancers. Nevertheless, the effects of IFN-alpha on the switch of PS to iPS are largely unknown. A comparison of the effect of both IFN-alpha and IFN-gamma was thus carried out on melanoma cell lines. RT-PCR showed that mRNA for iPS subunits (i.e. LMP-2, LMP-7 and MECL-1) was detectable both in untreated and IFN-treated melanoma cells. Immunoblotting analysis revealed that while IFN-gamma was able to consistently induce the switch from PS to iPS, IFN-alpha treatment did not, possibly due to post-transcriptional event(s) blocking the expression of iPS-specific subunits. Finally, Melan-A(mart1)(27-35) peptide was found only in the HPLC-MS spectra from both untreated and IFN-alpha-treated cells, but not upon IFN-gamma treatment. Altogether, these data demonstrate that IFN-alpha does not induce the switch from PS to iPS.

Topics: Antibodies; Antibodies, Monoclonal; Antigens, Neoplasm; Blotting, Western; Cell Line, Tumor; Cysteine Endopeptidases; Flow Cytometry; Gas Chromatography-Mass Spectrometry; Gene Expression Regulation, Neoplastic; Histocompatibility Antigens Class I; Humans; Interferon-alpha; Interferon-gamma; MART-1 Antigen; Melanoma; Multienzyme Complexes; Neoplasm Proteins; Proteasome Endopeptidase Complex; Reverse Transcriptase Polymerase Chain Reaction; Up-Regulation

In contrast with the low frequency of most single epitope reactive T cells in the preimmune repertoire, up to 1 of 1,000 naive CD8(+) T cells from A2(+) individuals specifically bind fluorescent A2/peptide multimers incorporating the A27L analogue of the immunodominant 26-35 peptide from the melanocyte differentiation and melanoma associated antigen Melan-A. This represents the only naive antigen-specific T cell repertoire accessible to direct analysis in humans up to date. To get insight into the molecular basis for the selection and maintenance of such an abundant repertoire, we analyzed the functional diversity of T cells composing this repertoire ex vivo at the clonal level. Surprisingly, we found a significant proportion of multimer(+) clonotypes that failed to recognize both Melan-A analogue and parental peptides in a functional assay but efficiently recognized peptides from proteins of self- or pathogen origin selected for their potential functional cross-reactivity with Melan-A. Consistent with these data, multimers incorporating some of the most frequently recognized peptides specifically stained a proportion of naive CD8(+) T cells similar to that observed with Melan-A multimers. Altogether these results indicate that the high frequency of Melan-A multimer(+) T cells can be explained by the existence of largely cross-reactive subsets of naive CD8(+) T cells displaying multiple specificities.

Topics: Amino Acid Sequence; Antigen Presentation; Antigens, Neoplasm; Autoantigens; CD8-Positive T-Lymphocytes; Clone Cells; Cross Reactions; Humans; Immunodominant Epitopes; Macromolecular Substances; MART-1 Antigen; Melanoma; Neoplasm Proteins

A common problem in the routine examination of melanoma re-excision scars occurs when a few or rare mildly atypical cells are present within the scar, raising the question of residual disease. Little is known about the derivation of these cells. Because the normal cutaneous wound-healing process is reparative, we hypothesized that these atypical cells may be reactive proliferating Schwann cell precursors.. The expression of the Schwann cell differentiation markers p75NGFR, CD56/N-CAM and GAP-43 was examined by immunohistochemistry in scars of wide local re-excisions for melanoma and non-melanoma tumors. Expression of S100, gp100 (with HMB45) and MART1 was also analyzed by immunohistochemistry.. All melanoma and non-melanoma re-excision specimens contained mildly atypical, spindled or epithelioid cells within the scar. They varied in number from case to case and expressed S100, p75NGFR, CD56/N-CAM or GAP-43 but not gp100 (with HMB45) or MART1. Rare epithelioid non-melanoma cells within the superficial dermis expressed MART-1.. Atypical cells are present in re-excision scars from melanoma and non-melanoma cases. They demonstrate early Schwann cell differentiation and appear to proliferate during the scarring process. The use of anti-MART-1 alone in the examination of melanoma re-excisions specimens may be inadequate as it may label rare, superficially located, non-melanoma cells within the scar.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Biomarkers; Biomarkers, Tumor; CD56 Antigen; Cell Differentiation; Cicatrix; Female; GAP-43 Protein; gp100 Melanoma Antigen; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Middle Aged; Neoplasm Proteins; Neoplasm Recurrence, Local; Receptor, Nerve Growth Factor; Reoperation; S100 Proteins; Schwann Cells; Skin Neoplasms

The use of reverse transcription polymerase chain reaction (RT-PCR) analysis of melanoma-specific transcripts for the identification of circulating melanoma cells has shown very variable results in different studies on melanoma patients. We have therefore developed quantitative methods to study both analytical and biological variations as possible causes of this phenomenon. Pigment-related and S-100 beta transcripts were quantified in 12 different melanoma cell lines and related to the amounts of 5-S-cysteinyldopa, pigment and S-100B protein. A real-time PCR method was used and the results were expressed as absolute number of transcripts per cell. Tyrosinase, tyrosinase-related protein (TRP)-1, TRP-2 and MART-1/Melan-A mRNA varied from undetectable (< 10(-4) transcripts/cell) to 10(3) transcripts/cell, i.e. by a factor > 10(7) in the different cell lines. S-100 beta mRNA varied from 2.8 to 165 transcripts/cell, i.e. by a factor of 60. Tyrosinase, TRP-1 and TRP-2 mRNA correlated significantly with the amount of 5-S-cysteinyldopa, an intermediate pigment metabolite (P < 0.001, P < 0.001 and P < 0.01, respectively). The amount of S-100 beta mRNA correlated significantly with the amount of S-100B protein (P < 0.001). No cross-correlations were seen between the pigment-related and S-100-related analytes. We conclude that one reason behind the negative results of RT-PCR measurement of pigment-related mRNA may be that these transcripts are not always expressed in the particular cells present in the patient's blood. Furthermore, variation in the expression of the order of 10(7) must have great impact on the diagnostic sensitivity. Measurement of S-100 beta mRNA would be more sensitive, but the use of this transcript is hampered by its presence in the blood cells.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Gene Expression Profiling; Humans; Intramolecular Oxidoreductases; MART-1 Antigen; Melanins; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Neoplastic Cells, Circulating; Nerve Growth Factors; Oxidoreductases; Pigmentation; Polymerase Chain Reaction; Proteins; Pyrroles; RNA, Messenger; RNA, Neoplasm; S100 Calcium Binding Protein beta Subunit; S100 Proteins; Tumor Cells, Cultured; Tyrosine

Aggressive melanoma cells may express endothelial markers that can be used to calculate microvascular density (MVD). High MVD has been associated with adverse outcome in uveal melanoma. If tumor cells label with endothelial cell markers, then MVD may not accurately reflect a tumor's vascularity. This study was designed to study the influence of melanoma cell labeling by endothelial cell markers on MVD.. Tissue sections of 200 ciliary body or choroidal melanomas were stained with CD34 alone, and the MVD was calculated by counting discrete foci of CD34 labeling in hot spots, as described previously. From adjacent sections double labeled by fluorescent immunohistochemical stains for S100 protein and CD34, tumor cells labeling with both markers were identified. The relationship between marker coexpression and MVD was tested. Tissue sections were also double labeled for Melan-A and CD34.. MVD was found to be associated with death from metastatic melanoma as reported previously. However, colocalization of both Melan-A and S100 protein with CD34 was demonstrated. The labeling of tumor cells by CD34 was associated with an elevated calculation of MVD (P < 0.0001) but not with cell type, mitotic figures, tumor-infiltrating lymphocytes, or PAS-positive patterns.. CD34 may label uveal melanoma cells and may contribute to computation of MVD. Although MVD is prognostically significant in uveal melanoma, this feature is not an exclusive measure of tumor vascularity.

Topics: Antigens, CD34; Antigens, Neoplasm; Biomarkers, Tumor; Cell Count; Endothelium, Vascular; Fluorescent Antibody Technique, Indirect; Humans; Immunoenzyme Techniques; MART-1 Antigen; Melanoma; Neoplasm Proteins; Neovascularization, Pathologic; Prognosis; S100 Proteins; Staining and Labeling; Survival Rate; Uveal Neoplasms

This study was performed to detect circulating melanoma cells in peripheral blood using a novel method based on magnetic-activated cell separation (MACS) followed by a nested reverse transcriptase-polymerase chain reaction (RT-PCR) for tyrosinase and MART-1 mRNA. Samples to be tested were enriched for tumour cells either by isolating melanoma cells using two anti-melanoma antibodies (MART-1 and HMB-45) or by CD45 depletion of the non-melanoma cell fraction. The tumour cell-enriched fractions were subjected to mRNA isolation using oligo-deoxythymidylate (oligo-dT) magnetic beads followed by a nested RT-PCR. Sensitivity was assessed by spiking experiments and compared with a commonly used total RNA isolation system previously established in our department. Positive isolation of melanoma cells showed insufficient sensitivity, whereas negative isolation by depletion of leukocytes showed a detection limit of at least one melanoma cell per millilitre of whole blood. In further experiments, the depletion assay was applied to 25 peripheral blood samples of melanoma patients. The preliminary data obtained from the new method indicate a comparable detection rate to the established total RNA extraction method. However, not all the results were concordant. Therefore, future experiments need to be performed with a statistically greater number of patients.

Topics: Adult; Aged; Antigens, Neoplasm; CD4 Antigens; Female; Humans; Immunomagnetic Separation; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Microspheres; Middle Aged; Neoplasm Proteins; Neoplasm Staging; Neoplastic Cells, Circulating; Oligodeoxyribonucleotides; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Sensitivity and Specificity; Tumor Cells, Cultured

The authors investigated a group of 51 patients (29 men and 22 women) with intraocular melanoma: 41 patients with melanoma of the chorioid, 10 patients with melanoma of the ciliary body. They evaluated the clinical and pathological finding according to the TNM classification recommended by UICC (International Union Against Cancer). In all investigated patients they assessed circulating tumour cells (melanocytes) in the peripheral blood stream based on the detection of mRNA tyrosinase and marker MART 1. When evaluating the presence of markers according to the diagnosis irrespective of time, they found in patients in the clinical stage of T2 choroidal melanoma a 19% positivity of different markers and very rare a concurrent positivity of both markers. Patients in the clinical stage T3 had a 51% positivity of one marker and 34% concurrent positivity of both markers. In melanoma of the ciliary body evidence of individual markers was positive in 17% and only in 11% both markers were positive concurrently. On comparison of therapeutic procedures from the aspect of development in time in patients treated by brachytherapy only rare positivity was found at the time of administration the radioactive plaque, following an eight-month interval after brachytherapy the positivity of markers increased to 28%. On evaluation of markers of choroidal melanoma and ciliary body melanoma resolved by enucleation had their positivity at the time of operation was 36%, and during check-ups up to one year or longer it persisted at similar levels. Concurrent presence of both markers before this operation was rare, during postoperative check-up examinations it was within a range of 23 and 33%. The presence of both markers was repeatedly proved in five patients with chooidal melanoma after enucleation of the eye, in four of them in direct correlation with a metastatic process.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Eye Neoplasms; Female; Humans; Male; MART-1 Antigen; Melanoma; Monophenol Monooxygenase; Neoplasm Proteins; Neoplastic Cells, Circulating; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Somatic cell hybrids of HLA-A2(+) EBV-transformed B- or dendritic cells (DC) and allogeneic HLA-A2(-) melanoma cell line Me15 were obtained by in vitro electrofusion using an electroporator. Before fusion, melanoma cells were stably transfected with green fluorescent marker protein (GFP) and neomycin resistance gene (neo(+)). Stably growing hybrid antigen-presenting cells (HAPC) expressing HLA-DR and HLA-A2 (or HLA-A30/31), and melanoma-associated antigens (MART-1, gp100) were selected by a double strategy of immunomagnetic MACS and neomycin selection. Fusion efficiency ranged between 3% and 18% (mean: 8.0+/-4.7%) as defined by simultaneous GFP and HLA-A2 detection. Expression of melanoma-associated antigens (MART-1, gp100) in hybrid cells was determined by reverse transcription-polymerase chain reaction (RT-PCR). HLA-restricted antigen-specific presentation of melanoma antigens was demonstrated by killing of semi-allogenic HAPC by HLA-A2-restricted MART-1 or gp100-specific cytotoxic T lymphocyte (CTL) clones. HLA restriction and antigen specificity were confirmed by inhibition of specific cytotoxicity by anti-HLA antibodies and cold target inhibition. During long-term (42-70 days) neomycin selection of HAPC, a drastic loss of antigen-presenting cell (APC)-derived determinants (e.g. HLA-DR, HLA-A2) was observed which, however, could be "reversed" by repeated MACSorting (days 10, 21 and 49). Our method allows the generation of semi-allogenic HAPC that constitutively proliferate in vitro. This opens the possibility of establishing a number of tumor-APC hybrids expressing defined HLA haplotypes and tumor antigens, of investigating their specific properties (e.g. antigen processing), and testing their diagnostic or therapeutic potential.

Topics: Antigen-Presenting Cells; Antigens, Neoplasm; B-Lymphocytes; Cancer Vaccines; Cell Division; Cell Fusion; Cell Line, Transformed; Cytotoxicity, Immunologic; Dendritic Cells; gp100 Melanoma Antigen; Haplotypes; Herpesvirus 4, Human; HLA-A1 Antigen; HLA-A2 Antigen; HLA-A3 Antigen; Humans; Hybrid Cells; Immunomagnetic Separation; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Reverse Transcriptase Polymerase Chain Reaction; Selection, Genetic; Tumor Cells, Cultured

Mucosal melanomas of the oral cavity are rarely seen in the United States. The hard palate is the most common intraoral site. This unusual case occurred in the oral cavity of a 17-year-old Asian girl, who presented to her dentist with complaints of pain and swelling in the upper jaw. The lesion was distal and palatal to the maxillary left second molar, which was vital. Interestingly, the clinical presentation was a hyperplastic, tender lesion that bled when probed. Histopathologically, the biopsy demonstrated a sheet of spindle-shaped cells arranged in nests and fascicles. The nuclei were vesicular, oval to spindle-shaped, and some contained nucleoli that were distinguishable but not prominent. No melanin pigment was observed in the lesion. Tumor cells strongly expressed S100 protein, gp100 (HMB-45), and microphthalmia transcription factor, and variably expressed MART1, but not cytokeratins, CD34, or muscle-specific actin. The histopathologic features and immunohistochemical findings are consistent with a diagnosis of malignant melanoma.

Topics: Adolescent; Antigens, Neoplasm; Biomarkers, Tumor; DNA-Binding Proteins; Female; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Microphthalmia-Associated Transcription Factor; Mouth Mucosa; Mouth Neoplasms; Neoplasm Proteins; S100 Proteins; Transcription Factors; Treatment Outcome

It is widely accepted that the repertoire of Melan-A-specific T cells naturally selected in melanoma patients is diverse and mostly nonoverlapping among different individuals. To date, however, no studies have addressed the TCR profile in different tumor sites and the peripheral blood from the same patient. We compared the TCR usage of Melan-A-specific T cells from different compartments of a single melanoma patient to evaluate possible clonotype expansion or preferential homing over a 4-mo follow-up period. Using HLA-A2 peptide tetramers, CD8(+) T cells recognizing the modified Melan-A immunodominant ELAGIGILTV peptide were isolated from four metastatic lesions resected from a single melanoma patient, and their TCR repertoire was studied. A panel of T cell clones was generated by cell cloning of tetramer-positive cells. Analysis of the TCR beta-chain V segment and the complementarity-determining region 3 (CDR3) length and sequence revealed a large diversity in the TCR repertoire, with only some of the clones showing a partial conservation in the CDR3. A similar degree of diversity was found by analyzing a number of T cell clones obtained after sorting a Melan-A-specific population derived from PBLs of the same patient after in vitro culture with the immunodominant epitope. Moreover, clonotypes found at one site were not present in another, suggesting the lack of expansion and circulation of one or more clonotypes. Taken together, these results buttress the notion that the CTLs recognizing the immunodominant Ag of Melan-A comprise a high number of different clonotypic TCR, of which only some exhibit common features in the CDR3.

Topics: Amino Acid Sequence; Antigens, Neoplasm; Base Sequence; Clone Cells; Epitopes, T-Lymphocyte; Flow Cytometry; Genes, T-Cell Receptor beta; HLA-A2 Antigen; Humans; Immunoglobulin Variable Region; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Molecular Sequence Data; Neoplasm Proteins; Receptors, Antigen, T-Cell, alpha-beta; T-Lymphocyte Subsets; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

Nodal nevi represent a potential diagnostic pitfall in the analysis of lymph nodes. They may be confused with metastatic melanoma or carcinoma. Although several morphologic guidelines exist for the recognition of nodal nevi, on occasion immunohistochemical studies may be helpful for diagnosis, especially when melanocytes extend into the lymph node parenchyma. To learn more about the immunohistochemical profile of nodal nevi we examined 15 nodal nevi for the expression of S-100 protein, gp100 (HMB-45), Melan-A/MART-1 (A103), and tyrosinase (T311), and we studied the expression of Ki-67 (MIB-1) in nodal nevi and 40 melanoma metastases (35 lymph node and five cutaneous metastases). All nodal nevi were homogeneously immunoreactive for S-100 protein, tyrosinase, and Melan-A/MART-1. Two nodal nevi were focally positive for gp100. Fourteen of 15 nodal nevi were completely negative for Ki-67. One large cellular nodal nevus showed nuclear labeling in <0.2% of melanocytes. All metastases showed MIB-1 labeling. However, the percentage of labeled tumor cells varied widely, ranging from 2% to 80%. These results demonstrate that MIB-1 and HMB-45 are helpful reagents for the distinction of nodal nevi from melanoma. Immunohistochemistry for S-100 protein, Melan-A/MART-1, or tyrosinase facilitates the recognition of melanocytes but does not distinguish between nodal nevus and metastatic melanoma.

Topics: Adult; Aged; Antigens, Neoplasm; Biomarkers, Tumor; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasm Proteins; Nevus, Intradermal; S100 Proteins; Skin Neoplasms

Modifications in tumour antigen-derived epitopes that stabilize the major histocompatibility complex (MHC)-peptide complex result in enhanced stimulatory capacity and improved immunogenicity of the altered peptide. These epitope analogues are attractive candidates for the development of peptide-based vaccine trials. Any modification, however, in tumour antigens may induce T-cell responses that could either fail to react against the naturally occurring peptides or represent only a subset of the total antigen-specific repertoire. In the present study, we performed a critical analysis of the ability of cytotoxic T-lymphocyte (CTL) clones, derived from two melanoma patients through stimulation with the A27L peptide analogue, to cross-react with the naturally processed Melan-A/MART-1 (Melan-A) peptides in terms of T-cell receptor (TCR) affinity, functional avidity and fine antigen specificity. We found that all the A27L-specific clones analysed possessed a very low avidity for the natural Melan-A peptides, and that their binding affinity for human leukocyte antigen (HLA) tetramers complexed with both the modified and the natural Melan-A peptides did not strictly correlate with their functional avidity. We also observed that these clones were able to cross-recognize both natural Melan-A peptides in one patient, but only one peptide in the second patient. We discuss the capability of the A27L peptide analogue to stimulate all the available Melan-A-specific repertoire.

Topics: Antibody Affinity; Antigens, Neoplasm; Cancer Vaccines; Dose-Response Relationship, Drug; Flow Cytometry; HLA Antigens; Humans; MART-1 Antigen; Melanoma; Neoplasm Proteins; Peptides; Protein Binding; Receptors, Antigen, T-Cell; T-Lymphocytes, Cytotoxic

Topics: Antigens, Neoplasm; Female; Humans; MART-1 Antigen; Melanoma; Neoplasm Proteins; Neoplasms, Squamous Cell; Radiotherapy; Uterine Cervical Neoplasms; Vulvar Neoplasms

A 10-year-old male cross-breed dog was referred for investigation of oral malignant melanoma. Fine-needle aspirates were taken from the draining submandibular lymph node. The presence of metastatic melanoma cells was confirmed by cytological examination and reverse transcription polymerase chain reaction (RT-PCR) using primers for the melanoma-associated antigens: tyrosinase and mart-1/melan A. Cytokine expression in the lymph node was evaluated by multiplex RT-PCR, which demonstrated the presence of mRNA for IL-10 and TGF-beta1. However, IL-2, IL-4 and IFNgamma mRNA could not be detected, suggesting a lack of immune activation. Thoracic radiographs showed a lesion within the caudal lung fields suggestive of pulmonary metastasis. The dog developed signs of dyspnoea and collapse and was euthanased four days later. This case illustrates that molecular techniques can be used to aid clinical staging of canine oral malignant melanoma, and suggests that immunosuppressive cytokines could be involved in the pathogenesis of disease.

Topics: Animals; Antigens, Neoplasm; Biopsy, Needle; Cytokines; Diagnosis, Differential; DNA Primers; Dog Diseases; Dogs; Lung Neoplasms; Lymph Nodes; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Monophenol Monooxygenase; Mouth Diseases; Neoplasm Proteins; Radiography; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Synthetic combinatorial peptide libraries in positional scanning format (PS-SCL) have recently emerged as a useful tool for the analysis of T cell recognition. This includes identification of potentially cross-reactive sequences of self or pathogen origin that could be relevant for the understanding of TCR repertoire selection and maintenance, as well as of the cross-reactive potential of Ag-specific immune responses. In this study, we have analyzed the recognition of sequences retrieved by using a biometric analysis of the data generated by screening a PS-SCL with a tumor-reactive CTL clone specific for an immunodominant peptide from the melanocyte differentiation and tumor-associated Ag Melan-A. We found that 39% of the retrieved peptides were recognized by the CTL clone used for PS-SCL screening. The proportion of peptides recognized was higher among those with both high predicted affinity for the HLA-A2 molecule and high predicted stimulatory score. Interestingly, up to 94% of the retrieved peptides were cross-recognized by other Melan-A-specific CTL. Cross-recognition was at least partially focused, as some peptides were cross-recognized by the majority of CTL. Importantly, stimulation of PBMC from melanoma patients with the most frequently recognized peptides elicited the expansion of heterogeneous CD8(+) T cell populations, one fraction of which cross-recognized Melan-A. Together, these results underline the high predictive value of PS-SCL for the identification of sequences cross-recognized by Ag-specific T cells.

Topics: Antigens, Bacterial; Antigens, Neoplasm; Antigens, Viral; Autoantigens; Clone Cells; Combinatorial Chemistry Techniques; Cytotoxicity Tests, Immunologic; Cytotoxicity, Immunologic; Epitopes, T-Lymphocyte; HLA-A2 Antigen; Humans; MART-1 Antigen; Melanoma; Neoplasm Proteins; Peptide Fragments; Peptide Library; Protein Binding; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

TCR-alpha and -beta chains are composed of somatically rearranged V, D, and J germline-encoded gene segments that confer Ag specificity. Recent crystallographic analyses revealed that TCR-alpha has more contacts with peptide than TCR-beta, suggesting the possibility that peptide recognition predominantly relies on TCR-alpha. T cells specific for the self Ag Melan-A/MART-1 possess an exceptionally high precursor frequency in human histocompatibility leukocyte Ag-A2 individuals. This provided a unique situation for assessment of the structural relationship between TCR and peptide/MHC ligand at both the pre- and postimmune levels. Molecular and phenotypic analysis of many different Melan-A-specific T cell populations revealed that a structural constraint is imposed on the TCR for engagement with Melan-A peptides presented by HLA-A2, namely the highly preferential use of a particular TCRAV segment, AV2. Examination of CD8 single-positive thymocytes indicated that this preferential use in forming the Melan-A-specific TCR is mainly imposed by intrathymic positive selection. Our data demonstrate a dominant function of TCRAV2 segment in forming the TCR repertoire specific for the human self Ag Melan-A/MART-1 and support the view that Ag recognition is mediated predominantly by TCR-alpha.

Topics: Amino Acid Sequence; Antigen Presentation; Antigens, Neoplasm; Autoantigens; Base Sequence; Cell Line; Conserved Sequence; DNA, Complementary; Genes, T-Cell Receptor alpha; HLA-A2 Antigen; Humans; In Vitro Techniques; MART-1 Antigen; Melanoma; Neoplasm Proteins; Receptors, Antigen, T-Cell, alpha-beta; T-Lymphocytes

Polyclonal and monoclonal antibodies (MAbs) have been raised against B16F10 cells collected from growing tumors in vivo or grown in culture media supplemented with normal mouse serum to avoid xenogeneic reactivity. Antibody binding to glutaraldehyde-fixed melanoma cells and Melan A melanocytes was assayed using chemiluminescent-enzyme-linked immunosorbent assay (CL-ELISA) for increased sensitivity. Most of the reactivity of antitumor polyclonal IgG (92%) was inhibitable by a carbohydrate pool consisting of melibiose, mannose, lactose, and sialic acid. Two monoclonal IgG(2a) antibodies, A4 and B11, had their reactivity to melanoma cells completely and specifically inhibited by melibiose. MAb A4 did not bind to alpha-galactosyl residues abundantly expressed in a protozoan mucin used as substrate, and its binding to the tumor cells was not affected by alpha-galactosidase treatment or addition of alpha-methyl-galactopyranoside or raffinose. Recognition of a mimotope similar to melibiose is suggested. MAb is cytotoxic in vitro in a complement-mediated reaction and effectively neutralizes melanoma cells protecting syngeneic mice against tumor development in vivo. This MAb is thus an important tool for further studies on antitumor adjuvant therapy combined with other agents associated with immuno- and chemotherapy of invasive melanoma.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neoplasm; Antigens, Neoplasm; Cancer Vaccines; Carbohydrate Metabolism; Carbohydrates; Cell Separation; Complement System Proteins; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Epitopes; Flow Cytometry; Galactose; Glutaral; Humans; Hybridomas; Immunoglobulin G; Immunotherapy; Lactose; Mannose; MART-1 Antigen; Melanoma; Melanoma, Experimental; Melibiose; Mice; Mice, Inbred BALB C; Mice, Nude; Microscopy, Fluorescence; Mucins; N-Acetylneuraminic Acid; Neoplasm Proteins; Protein Conformation; Trypanosoma cruzi

We determined the sensitivity and specificity of 3 novel antibodies (microphthalmia transcription factor [Mitf], Melan-A, and tyrosinase) as markers for melanoma in cytologic preparations and compared the results with those of commonly used markers (S-100 protein [S-100] and HMB-45). We stained 72 cell blocks from 40 patients with melanoma and 32 with nonmelanocytic malignant neoplasms with antibodies against S-100, HMB-45, Mitf, Melan-A, and tyrosinase. Histologic correlation was available in more than 95% of cases. Nuclear stainingfor Mitf and cytoplasmic stainingfor S-100, HMB-45, Melan-A, and tyrosinase in more than 10% of tumor cells was considered positive. All 3 novel markers demonstrated sensitivity superior to S-100 and HMB-45. HMB-45, Melan-A, and Mitf demonstrated specificities of 97%. S-100 protein and tyrosinase were less specific. Sensitivity and specificity for the combination Mitf+/Melan-A+ were 95% and 100%, respectively, whereas they were 80% and 100%, respectively, for S-100+/HMB-45+. Mitf Melan-A, and tyrosinase are sensitive markersfor epithelioid melanoma. Mitf and Melan-A seem more specific than S-100 and tyrosinase. An antibody panel consisting of Mitf and Melan-A is superior to a panel of S-100 and HMB-45 in the diagnosis of melanoma in cytologic specimens.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; DNA-Binding Proteins; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Microphthalmia-Associated Transcription Factor; Monophenol Monooxygenase; Neoplasm Proteins; S100 Proteins; Sensitivity and Specificity; Transcription Factors

Numerous immunohistochemical stains have been employed to detect metastatic melanoma in sentinel lymph node (SLN) biopsies. HMB-45 is considered by some as a specific tool to detect early metastatic melanoma (1). Occasionally, one or two isolated HMB-45-positive cells may cause complications in diagnostic interpretation. The goal of this study was to evaluate the reliability of HMB-45 staining of SLNs with sparse isolated positive cells and to compare its staining with anti-Melan A antibody. HMB-45 and anti-Melan A antibody immunostaining was performed on (Group A) 15 histologically negative SLNs excised from patients with malignant melanoma (MM) and on (Group B) 15 histologically negative SLNs excised from patients with breast carcinoma (BC). None of the patients had clinical evidence of systemic metastasis at the time of SLN biopsy. Five cutaneous biopsies with changes of postinflammatory hyperpigmentation (PIHP) were also stained with both antibodies. HMB-45 staining was repeated in all Group B SLNs after blocking endogenous biotins. Electron-microscopic studies were performed on all cases of PIHP. Isolated HMB-45-stained cells were present in 6 of 15 SLNs removed for MM; 8 of 15 for BC; and 3 of 5 cutaneous biopsies of PIHP. HMB-45 reactivity persisted after blocking endogenous biotins in 6 of 8 positive SLNs from Group B. Anti-Melan A antibody was negative in all SLNs of group A and B and in dermal melanophages of all five cases of PIHP. HMB-45 positivity was demonstrated in histologically negative SLNs and cutaneous biopsies, especially in the milieu of aggregated melanophages. Phagocytosis of premelanosomes by macrophages in the draining lymph nodes may account for isolated cell positivity and can hinder correct diagnostic interpretation. HMB-45 may not be a reliable marker for the detection of micro-metastasis of MM and requires correlation with other immunohistochemical markers, such as anti-Melan A antibody, to enhance specificity.

Topics: Antigens, Neoplasm; Humans; Immunohistochemistry; Lymphatic Metastasis; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; Predictive Value of Tests; Sentinel Lymph Node Biopsy; Skin

We have studied the TCR features and functional responses of three sets of human cytolytic T cell (CTL) clones, recognizing antigenic peptides presented by HLA-A2 and derived from the Epstein-Barr virus proteins BMLF1 and BRLF1 and from the melanoma protein Melan-A/MART-1. Within each set, a majority of clones used a recurrent V alpha region, even though they expressed highly diverse TCR beta chains and V(D)J junctional sequences. Functional assays and peptide/MHC multimer binding studies indicated that this restricted V alpha usage was not associated with the affinity/avidity of the CTL clones. The V alpha dominance, which may be a frequent feature of antigen-specific T cells, likely reflects a restricted geometry of TCR/peptide/MHC complexes, primarily determined by V alpha CDR.

Topics: Amino Acid Sequence; Antigens, Neoplasm; Complementarity Determining Regions; Herpesvirus 4, Human; HLA-A2 Antigen; Humans; Immediate-Early Proteins; MART-1 Antigen; Melanoma; Neoplasm Proteins; Phosphoproteins; Receptors, Antigen, T-Cell, alpha-beta; T-Lymphocytes, Cytotoxic; Trans-Activators; Viral Proteins

Topics: Antigens, Neoplasm; Female; Humans; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Neurilemmoma; Organelles; Tracheal Neoplasms; Treatment Outcome

Recent studies in mouse models have suggested that genetic transfer of tumor antigen-specific high affinity T cell receptors (TCR) into host lymphocytes could be a viable strategy for the rapid induction of tumor-specific immunity. A previously proposed approach for the isolation of such TCRs consists in circumventing tolerance to self-restricting HLA/peptide complexes by deriving them from PMBCs of allogenic donors. Towards this aim, we used fluorescent HLA-A2 class-I/peptide soluble multimers to isolate A2-restricted CD8+ T cells specific for a previously described Melan-A peptide enhanced analog (Melan-A 26-35 A27L) from an HLA-A*0201 (A2) negative donor. We isolated two distinct groups of Melan-A 26-35 A27L-specific clones. Clones from the first group recognized the analog peptide with high avidity but showed very low recognition of Melan-A parental peptides. In contrast, clones from the second group efficiently recognized Melan-A parental peptides. Surprisingly however, most clones recognized not only A2+ Melan-A+ targets, but also A2+ Melan-A- targets suggesting that they can also recognize endogenous peptides other than Melan-A. In addition, one clone showed full cross-recognition of an antigenically unrelated peptide. Together, our data show that HLA-A2/peptide multimers can be successfully used for the isolation of allorestricted CD8+ T cells reactive with tumor antigen-derived peptides. However, as the cross-reactivity of these apparently peptide-specific allorestricted TCRs is presently unpredictable, a careful in vitro analysis of their reactivity to the host's normal cells is recommended.

Topics: Animals; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; HLA-A Antigens; HLA-A2 Antigen; Humans; MART-1 Antigen; Melanoma; Mice; Neoplasm Proteins; Tumor Cells, Cultured

A majority of desmoplastic melanomas and some of the other forms of melanomas are S-100 positive and HMB45 negative; this pattern of immunoreactivity is similar to certain nerve-derived tumors such as malignant peripheral nerve sheath tumor. In this study the immunostaining profile of HMB45-negative malignant melanomas was evaluated by a panel of antibodies against markers associated with melanoma and melanocytic differentiation, including microphthalmia transcription factor, tyrosinase, Melan-A, and MAGE-1. Immunodetection was performed on paraffin sections of 22 cases of HMB45-negative malignant melanomas (including 8 spindle cell melanomas, 8 desmoplastic melanomas, and 6 epithelioid melanomas), 8 HMB45-and S-100-positive malignant melanomas, 15 malignant peripheral nerve sheath tumors, 16 schwannomas, and 11 neurofibromas. Of eight HMB45-positive malignant melanomas, all were positive for Melan-A, tyrosinase, and melanocyte-specific transcription factor, and three were positive for MAGE-1. In the 14 HMB-45 negative, nondesmoplastic melanomas, melanocyte-specific transcription factor was positive in 9, Melan-A in 9, tyrosinase in 6, and MAGE-1 in 11. In eight desmoplastic malignant melanomas, MAGE-1 was positive in three, and all other markers were negative. The five markers tested were negative in all but two schwannomas, one with focal melanocyte-specific transcription factor and the other with tyrosinase and weak MAGE-1 reactivity. MAGE-1, melanocyte-specific transcription factor, tyrosinase, and Melan-A are useful markers in the diagnosis of malignant melanocytic lesions when HMB45 is negative. MAGE-1 may be useful in differentiating melanocytic lesions from nerve-derived lesions, but its sensitivity is relatively low. The immunostaining profile of desmoplastic malignant melanomas more closely resembles that of malignant peripheral nerve sheath tumor than that of other types of malignant melanoma. Melanocyte-specific transcription factor is not a useful marker for desmoplastic melanoma.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; DNA-Binding Proteins; Humans; Immunophenotyping; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Microphthalmia-Associated Transcription Factor; Monophenol Monooxygenase; Neoplasm Proteins; Nerve Sheath Neoplasms; Peripheral Nervous System Neoplasms; S100 Proteins; Transcription Factors

MHC class I tetramers containing peptide epitopes are sensitive tools for detecting antigen-specific CD8(+) T-cell responses. We demonstrate here that binding of HLA-A2 tetramers to CD8(+) T cells specific for the melanoma-associated antigen Melan-A/MART-1 can be fine-tuned by altering either the bound peptide epitope or residues in the alpha 3 domain of HLA-A2, which is important for CD8 binding. Antigen-specific T cells expressing high levels of CD8 could be detected using HLA-A2 tetramers containing the peptide AAGIGILTV, an epitope which is naturally processed and presented from Melan-A/MART-1. In contrast, low CD8-expressing, antigen-specific T cells could be detected efficiently only by using a mutated HLA-A2 tetramer with an altered CD8 binding site or, less efficiently, using the wild-type HLA-A2 tetramer loaded with the peptide analogue ELAGIGILTV, which is superior in stimulating antigen-specific T-cell responses. Our results suggest ways to optimize the identification and expansion of antigen-specific T cells with different requirements for the costimulatory CD8 molecule in facilitating T-cell receptor binding to peptide variants.

Topics: Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Cytokines; Cytotoxicity, Immunologic; Epitopes; Flow Cytometry; HLA-A2 Antigen; Humans; MART-1 Antigen; Melanoma; Neoplasm Proteins; Neoplasm Staging; Protein Binding; Receptors, Antigen, T-Cell; Tumor Cells, Cultured

Sinonasal melanomas are rare neoplasms whose diagnosis may require confirmatory immunohistochemical stains. S-100 protein and HMB-45, the stains most commonly used, have varying sensitivities and specificities. Melan-A, a more recent melanoma-specific marker, may prove helpful when S-100 protein and HMB-45 stains are negative or equivocal.. Seven cases of sinonasal melanoma were assessed for reactivity with Melan-A, S-100 protein and HMB-45.. The study group consisted of two women and five men ages 40 to 83. Six of the neoplasms were strongly positive for S-100 protein. One case was negative for S-100 protein and HMB-45 but positive for Melan-A. HMB-45 staining varied between diffusely positive (three cases), focally positive (two cases), and negative (two cases). All cases were positive for Melan-A either diffusely (four cases) or focally (three cases).. Because Melan-A can be positive in cases that are S-100 protein or HMB-45 negative, it is a useful component in the immunohistochemical panel for the diagnosis of sinonasal melanomas.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Biopsy, Needle; Culture Techniques; Female; Humans; Immunohistochemistry; Magnetic Resonance Imaging; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Paranasal Sinus Neoplasms; Sampling Studies; Sensitivity and Specificity; Staining and Labeling

We have performed a detailed analysis of the recognition of melanoma Ags by the tumor-infiltrating lymphocytes (TIL) 1790, isolated from a patient who experienced a dramatic tumor regression following immunization with peptides from the gp100, MART-1, and tyrosinase Ags. This TIL was found to recognize HLA-A2-restricted CTL epitopes in tyrosinase-related protein (TRP)-2 (clone MR7) and NY-ESO-1 (clone M8). These epitopes were the same as the previously identified nonapeptide TRP-2: 180-188, and the overlapping NY-ESO-1 peptides, obtained by using lymphocytes from in vitro stimulation. We also cloned a previously unknown TRP-2 mRNA isoform (TRP-2-6b) that contained two novel exons alternatively spliced from the sixth intron between exons 6 and 7 of TRP-2 mRNA. The isoform encoded an HLA-A2-restricted antigenic epitope recognized by TIL clone MB4. An immunologic analysis of the patient's PBMC obtained before treatment showed the presence of high reactivity against NY-ESO-1 and both TRP-2 Ags, but not the Ags used for immunization. Because immune response against these Ags was less pronounced, it is possible that NY-ESO-1, TRP-2, and TRP-2-6b may be of importance in the generation of CTL-mediated tumor destruction and may have played a role in the dramatic tumor regression seen in this patient.

Topics: Amino Acid Sequence; Antigens, Neoplasm; Base Sequence; Cell Line; Cell Separation; Clone Cells; Epitopes, T-Lymphocyte; Female; Gene Library; gp100 Melanoma Antigen; Humans; Intramolecular Oxidoreductases; Isoenzymes; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Membrane Proteins; Middle Aged; Molecular Sequence Data; Monophenol Monooxygenase; Neoplasm Proteins; Peptide Fragments; Proteins; Transfection; Tumor Cells, Cultured

In malignant melanoma, tumor-infiltrating lymphocytes are frequently reactive with melanosomal antigens. Achieving complete remissions by peptide therapy is frequently hampered by metastases evading immune recognition. The tumor microenvironment seems to favor reduced expression of target antigens by melanoma cells. Among candidate factors, interferon-gamma (IFN-gamma) (10(2) to 10(3) U/ml) suppressed expression of antigens MART-1, TRP-1, and gp100 by M14 melanoma cells as shown by immunohistology and fluorescence-activated cell sorting analysis, reducing MART-1 expression by >65%. Northern blot analysis revealed that reduced expression was regulated at the transcriptional level, demonstrating a 79% reduction in MART-1 transcript abundance after 32 hours of IFN-gamma treatment. To evaluate consequences of IFN-gamma exposure for immune recognition, MART-1-responsive T cells were reacted with pretreated HLA-matched melanoma cells. Cytotoxicity was reduced up to 78% by IFN-gamma pretreatment, and was restored by addition of MART-1 peptide AAGIGILTV for 2 hours. Examination of melanoma lesions by quantitative reverse transcriptase-polymerase chain reaction revealed up to 188-fold more abundant IFN-gamma transcripts when compared to control skin. Laser capture microdissection and immunohistology localized most IFN-gamma-producing T cells to the tumor stroma. Reduced MART-1 expression was frequently observed in adjacent tumor cells. Consequently, IFN-gamma may enhance inflammatory responses yet hamper effective recognition of melanoma cells.

Topics: Antigens, Neoplasm; Cell Separation; Flow Cytometry; Humans; Immunohistochemistry; Interferon-gamma; MART-1 Antigen; Melanoma; Microscopy; Neoplasm Proteins; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

Many new types of vaccines against infectious or malignant diseases are currently being proposed. Careful characterization of the induced immune response is required in assessing their efficiency. While in most studies human tumor antigen-specific T cells are analyzed after in vitro re-stimulation, we investigated these T cells directly ex vivo using fluorescent tetramers. In peripheral blood lymphocytes from untreated melanoma patients with advanced disease, a fraction of tumor antigen (Melan-A/MART-1)-specific T cells were non-naive, thus revealing tumor-driven immune activation. After immunotherapy with synthetic peptides plus adjuvant, we detected tumor antigen-specific T cells that proliferated and differentiated to memory cells in vivo in some melanoma patients. However, these cells did not present the features of effector cells as found in cytomegalovirus specific T cells analyzed in parallel. Thus, peptide plus adjuvant vaccines can lead to activation and expansion of antigen specific CD8(+) T cells in PBL. Differentiation to protective CD8(+) effector cells may, however, require additional vaccine components that stimulate T cells more efficiently, a major challenge for the development of future immunotherapy.

Topics: Adjuvants, Immunologic; Antigens, Neoplasm; Antigens, Viral; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Differentiation; Cytomegalovirus; HLA-A2 Antigen; HLA-DR Antigens; Humans; Immunologic Memory; Immunotherapy; Influenza A virus; Lymphocyte Activation; Mannitol; MART-1 Antigen; Melanoma; Neoplasm Proteins; Oleic Acids; Peptide Fragments; Vaccines, Subunit; Viral Matrix Proteins

Regional lymph node status is an important predictor of survival in patients with malignant melanoma. Mapping of sentinel lymph nodes using sensitive molecular techniques has recently been introduced. Malignant melanoma is heterogeneous in terms of its biological, immunological and metastatic properties, and melanoma cells exhibit a polymorphous expression of tumour markers. Thus, assays that include multiple markers appear to be more sensitive than single-marker assays.. To characterize the molecular profiles of melanoma cells in sentinel lymph nodes employing the mRNA expression of tyrosinase, MIA and MART-1 as markers.. Samples of sentinel lymph nodes from 17 melanoma patients and 18 control nodes from non-melanoma patients were assayed by reverse transcriptase-polymerase chain reaction, using specific primers for each marker.. We found that both tyrosinase and MIA expression were sensitive indicators of micrometastases in sentinel lymph nodes that were negative on routine histopathological examination, and that the finding of micrometastases expressing MART-1 in sentinel lymph nodes was negatively correlated with overall survival.. Characterization of the molecular profiles of melanoma cells constitutes a valid means of detecting metastatic melanoma cells in sentinel lymph nodes, and of predicting the survival of melanoma patients.

Topics: Adult; Antigens, Neoplasm; Biomarkers, Tumor; Extracellular Matrix Proteins; Female; Gene Expression; Humans; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Sentinel Lymph Node Biopsy; Survival Rate

Selection and activation of T cells is tightly regulated by both antigen-specific receptors and co-receptors to ensure that responses to self antigens are largely avoided. By T cell receptor clonotypic mapping and staining with tetrameric HLA-peptide complexes, we demonstrate the presence of melanocyte differentiation antigen MART-1 specific T cells in the areas of destruction of both neoplastic and normal melanocytic cells in a case of a primary melanoma and its associated hypopigmentation. These self reactive T cells expressed CD94/NKG2 major histocompatibility complex class I specific C-type lectin-like receptors. This family of receptors includes both activating and inhibitory isoforms. Thus, we performed a detailed analysis that revealed the exclusive presence of inhibitory NKG2-A/B receptors in the vitiligo-like leukoderma, whereas both the inhibitory receptors and the activating NKG2-C/E isoforms were present within the tumor. Our data suggest the differential expression of killer inhibitory receptors as a possible mechanism to regulate T cell responses to self antigens.

Topics: Antigens, CD; Antigens, Neoplasm; Autoimmunity; Clone Cells; Gene Expression Regulation, Neoplastic; Humans; Immune Tolerance; Immunohistochemistry; Lectins, C-Type; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; NK Cell Lectin-Like Receptor Subfamily C; NK Cell Lectin-Like Receptor Subfamily D; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Immunologic; Receptors, Natural Killer Cell; Skin Neoplasms; T-Lymphocytes; Vitiligo

Monitoring of CD8+ T-cell responses in cancer patients during peptide vaccination is essential to provide useful surrogate markers and to demonstrate vaccine efficacy. We have longitudinally followed CD8+ T-cell responses in 3 melanoma patients who were immunized with peptides derived from Melan-A/MART-1. Recombinant HLA-A2 tetramers loaded with the naturally presented Melan-A/MART-1 nonamer peptide (AAGIGILTV) and the Melan-A/MART-1 analog (ELAGIGILTV) were used in combination with phenotypical analysis for different T-cell subsets including naive T cells, effector T cells, "true memory" T cells and "memory effector" T cells, based on CD45RA/RO and CCR7-expression. At least in a single patient, T cells binding to the AAGIGILTV epitope were detected in naive, precursor (CD45RA+/CCR7+) CD8+ T cells, and CD8+ T cells binding to the analog ELAGIGILTV peptide were identified in the terminally differentiated (CD45RA+/CCR7-) T-cell subset. Molecular and functional analysis of tetramer-binding T cells revealed that the T-cell receptor (TCR) repertoire was oligo/polyclonal in AAGIGILTV-reactive T cells, but different and restricted to a few TCR clonotypes in ELAGIGILTV-reactive T cells prior to vaccination. The TCR repertoire reactive with Melan-A/MART-1 peptide epitopes was broadened during vaccination and exhibited a different profile of cytokine release after specific stimulation: tetramer-binding T cells from 2/3 patients secreted granulocyte/macrophage colony-stimulating factor (GM-CSF) and interferon-gamma but not interleukin-2 (IL-2) in response to Melan-A/MART-1 peptides. In the third patient, tetramer-binding T cells secreted IL-2 exclusively. Our results show that T-cell responses to peptide vaccination consist of different T-cell subsets associated with different effector functions. Complementary analysis for TCR CDR3 and cytokine profiles may be useful to define the most effective CD8+ T-cell population induced by peptide vaccination.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; ATP-Binding Cassette Transporters; Cancer Vaccines; CD8-Positive T-Lymphocytes; Complementarity Determining Regions; Flow Cytometry; HLA-A2 Antigen; Humans; MART-1 Antigen; Melanoma; Neoplasm Proteins; Oligopeptides; Phenotype; Protein Binding; Receptors, Antigen, T-Cell; Skin Neoplasms; Tumor Cells, Cultured; Vaccination; Vaccines, Subunit

To determine the immunophenotypic differences between uveal and cutaneous melanomas, employing standard melanoma markers as well as p75 neurotrophin receptor (p75NTR) and microphthalmia transcription factor (MITF).. Fifteen uveal melanomas (5 spindle, 5 epithelioid, and 5 mixed uveal subtypes) were immunolabeled with a panel of antibodies that included S100, tyrosinase, melan-A, HMB-45 and HMB-50 combination, MITF, and p75NTR. The results were tabulated on the basis of intensity and pervasiveness of the labeling and compared with a prior study on cutaneous spindle and epithelioid melanomas.. In contrast to its strong labeling of cutaneous melanomas, S100 immunolabeling of uveal melanomas was weak and variable. p75NTR, known to differentiate spindle from epithelioid melanomas of the skin, did not immunolabel uveal melanomas. HMB-45, HMB-50, tyrosinase, melan-A, and MITF immunolabeled all uveal melanomas strongly, irrespective of the histologic subtype, but not cutaneous melanomas. Microphthalmia transcription factor was especially clear in its labeling of uveal melanomas.. Although cutaneous and uveal melanomas share many molecular markers in common, there are differences between the 2 types of melanoma. First, the level of expression of S100 differs between cutaneous and uveal melanomas. Second, while cutaneous melanomas can be further subdivided into spindle and epithelioid types based on their immunophenotype, the uveal melanomas cannot.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Choroid Neoplasms; DNA-Binding Proteins; Humans; Immunophenotyping; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Microphthalmia-Associated Transcription Factor; Monophenol Monooxygenase; Neoplasm Proteins; Receptor, Nerve Growth Factor; Receptors, Nerve Growth Factor; S100 Proteins; Skin Neoplasms; Transcription Factors

Both the underlying molecular mechanisms and the kinetics of TCR repertoire selection following vaccination against tumor Ags in humans have remained largely unexplored. To gain insight into these questions, we performed a functional and structural longitudinal analysis of the TCR of circulating CD8(+) T cells specific for the HLA-A2-restricted immunodominant epitope from the melanocyte differentiation Ag Melan-A in a melanoma patient who developed a vigorous and sustained Ag-specific T cell response following vaccination with the corresponding synthetic peptide. We observed an increase in functional avidity of Ag recognition and in tumor reactivity in the postimmune Melan-A-specific populations as compared with the preimmune blood sample. Improved Ag recognition correlated with an increase in the t(1/2) of peptide/MHC interaction with the TCR as assessed by kinetic analysis of A2/Melan-A peptide multimer staining decay. Ex vivo analysis of the clonal composition of Melan-A-specific CD8(+) T cells at different time points during vaccination revealed that the response was the result of asynchronous expansion of several distinct T cell clones. Some of these T cell clones were also identified at a metastatic tumor site. Collectively, these data show that tumor peptide-driven immune stimulation leads to the selection of high-avidity T cell clones of increased tumor reactivity that independently evolve within oligoclonal populations.

Topics: Amino Acid Sequence; Antigens, Neoplasm; Base Sequence; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Adhesion; Cell Differentiation; Clone Cells; Complementarity Determining Regions; DNA Primers; Epitopes, T-Lymphocyte; Gene Rearrangement, beta-Chain T-Cell Antigen Receptor; Genes, T-Cell Receptor beta; HLA-A2 Antigen; Humans; Immunoglobulin Variable Region; Immunophenotyping; Longitudinal Studies; MART-1 Antigen; Melanoma; Molecular Sequence Data; Neoplasm Proteins; Peptide Fragments; Sequence Analysis, DNA; T-Lymphocyte Subsets

The production of cytotoxic T cells with specificity for cancer cells is a rapidly evolving branch of cancer therapeutics. A variety of approaches aim to amplify anti-tumour cytotoxic T cell responses using purified peptides, tumour cell lysates or recombinant HLA/peptide complexes in differing antigen presenting systems. Using a two-step biotin-streptavidin antibody targeting system, recombinant HLA-class I/peptide complexes were attached to the surface of B cells via the anti-CD20 B9E9-scFvSA antibody-streptavidin fusion protein. Flow cytometry with a conformation dependant monoclonal antibody to HLA class I indicated that targeted HLA-class I/peptide complexes remain on the surface of B cells in culture for periods in excess of 72 h. PBMCs were stimulated in vitro for 8-14 days using the autologous B cells as antigen presenting cells. Following a single cycle of stimulation specific cytotoxic T cell responses to targeted HLA-A2 complexes containing the M1, BMLF1 and Melan A peptides could be demonstrated by tetramer staining and Cr release assays. With the HLA-A2/BMLF1 complex up to 2.99% of CD8+ve cells were tetramer positive producing 20% lysis (E : T 10 : 1) of CIR-A2 target cells in an in vitro cytotoxicity assay compared to baseline levels of 0.09% tetramer +ve and 2% lysis in the unstimulated population. PBMCs from a healthy donor treated with two cycles of stimulations with targeted HLA-A2/Melan A complexes, demonstrated expansion of the melanA tetramer +ve population from 0.03% to 1.4% producing 15% lysis of Melan A pulsed target cells. With further consideration to the key variables of HLA/peptide complex density, the ratio of stimulator to effector cells and optimum cytokine support, this system should offer an easy and effective method for the in vitro amplification of specific cytotoxic T cell responses and warrants development for the in vivo induction of cytotoxic T cell responses in cancer therapy.

Topics: Antibodies; Antibody Specificity; Antigens, Neoplasm; B-Lymphocytes; Flow Cytometry; HLA-A2 Antigen; Humans; MART-1 Antigen; Melanoma; Neoplasm Proteins; Neoplasms; T-Lymphocytes, Cytotoxic; Time Factors; Tumor Cells, Cultured; Viruses

Cytotoxic T cells represent a powerful strategy for antitumor treatment. Depending on the route of injection, an important role for CD4 T cell-mediated help was observed in the induction of this response. For this reason, we investigated whether induction of a CTL response to the HLA-A2-restricted immunodominant peptide melanoma antigen Melan-A was improved by using rVVs expressing the CTL-defined epitope alone or in combination with an SAg. In the latter case, the few infected dendritic cells simultaneously presented an SAg and an antigen, i.e., peptide. Here, we show that the anti-Melan-A response was efficiently induced but not significantly improved by coexpression of the SAg.

Topics: Adjuvants, Immunologic; Animals; Antigens, Neoplasm; Antigens, Viral; Flow Cytometry; Fluorescent Antibody Technique; Gene Expression; Green Fluorescent Proteins; HLA-A Antigens; HLA-A2 Antigen; Luminescent Proteins; Mammary Tumor Virus, Mouse; MART-1 Antigen; Melanoma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Neoplasm Proteins; Recombinant Fusion Proteins; Superantigens; T-Lymphocytes, Cytotoxic; Transfection; Vaccinia virus

Reports of vitiligo associated with metastases and rare cases of spontaneous regression of disease have fueled enthusiasm for immunologic approaches to the treatment of advanced melanoma. More recent strategies have focused on using antigen-presenting dendritic cells as vaccines.. We observed 3 cases of leukoderma associated with a novel adenovirus-mediated gp100/MART-1-transduced dendritic cell (MART indicates melanoma antigen recognized by T cells). All 3 patients had advanced metastatic melanoma. Despite the development of this leukodermic response, all patients experienced disease progression while under treatment.. We provide the initial evidence for effective induction of a leukodermic response with a gp100/MART-1-transduced dendritic cell vaccine.

Topics: Adenoviridae; Adult; Aged; Antigens, Neoplasm; Biopsy, Needle; Cancer Vaccines; Female; Follow-Up Studies; gp100 Melanoma Antigen; Humans; Hypopigmentation; Male; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Neoplasm Staging; Risk Assessment; Skin Neoplasms

Early and correct diagnosis of malignant melanoma is of utmost importance to ensure adequate treatment and the best outcome. Prompted by the death of a patient with an apparent metastasising melanoma in situ, we reassessed 104 people with this malignant disorder, whose diagnosis had been histopathologically verified. We did immunohistochemical analysis of cells with the melanocytic marker melan-A/MART-1, and results of this analysis showed that 30 (29%) of 104 patients had invasive melanomas. One patient died of distant metastases, and the tumour recurred in another. Our finding could be relevant for diagnosis and treatment of melanoma in situ.

Topics: Antigens, Neoplasm; Fatal Outcome; Hematoxylin; Humans; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Reproducibility of Results; Skin Neoplasms

The staining patterns of the monoclonal antibodies S-100 and Melan-A in canine melanoma were assessed based on cytological specimens of six canine melanomas (four benign, two malignant). In addition, eight regional lymph nodes of the two dogs with malignant melanomas were stained using these markers. For reference, all specimens were also evaluated immunohistochemically using S-100 and Melan-A. To assess the immunocytochemical specificity of both antibodies, various canine tumours and normal tissues were stained. The immunocytochemical staining results of the canine melanomas and the regional lymph nodes showed high conformity with the immunohistochemical reactivity patterns for S-100 and Melan-A. The specificity of Melan-A was higher compared with S-100. Melan-A, in particular, may be helpful for the cytological diagnosis of canine melanoma.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Dog Diseases; Dogs; Female; Immunohistochemistry; Lymph Nodes; Male; MART-1 Antigen; Melanoma; Neoplasm Proteins; S100 Proteins

Immunohistochemistry, using a monoclonal antibody to Melan A and a polyclonal antibody to S100 protein, was applied to 48 formalin-fixed, paraffin-embedded specimens of feline melanoma. Forty-two cutaneous, three oral, one mucocutaneous, and two metastatic melanomas comprised the tumors. Thirty-two tumors (67%) were positive for Melan A and 42 (87.5%) were positive for S100. All but one of the tumors that were positive for Melan A were also positive for S100. S100 was detected in 11 of 16 tumors that were negative for Melan A. Seventy-five percent (9 of 12) of amelanotic melanomas were negative for Melan A. Normal adrenal cortex, the cerebellum, and the skin had cells that were positive for Melan A. Sebaceous adenoma was the only nonmelanocytic tumor examined that reacted with antibody to Melan A. Although less sensitive than S100 protein, Melan A is more specific for melanoma and is useful in differentiating feline cutaneous melanoma from the more common pigmented basal cell tumor.

Topics: Animals; Antigens, Neoplasm; Cat Diseases; Cats; Immunohistochemistry; MART-1 Antigen; Melanoma; Neoplasm Proteins; S100 Proteins; Skin; Skin Neoplasms

Commonly used methods for detection of melanoma cells in blood, including RT-PCR and immunocytochemistry, display only a limited sensitivity and specificity. Reliable detection of less than one melanoma cell per ml of blood is hardly possible using these methods. To obtain greater sensitivity so that a single melanoma cell in up to 25 ml of blood can be detected (5 x 10(7) peripheral blood mononuclear cells, or PBMC), we developed a new assay for combined enrichment and immunocytochemical detection of disseminated melanoma cells from PBMC of patients with malignant melanomas. Melanoma cells are directly magnetically labeled using colloidal superparamagnetic microparticles approximately 60 nm in diameter conjugated to the anti-melanoma monoclonal antibody 9.2.27, with no reactivity to normal cells in blood. Magnetically labeled melanoma cells are enriched from PBMC by magnetic cell separation and detected by a new approach for immunocytochemical staining with monoclonal mouse anti-melanoma antibodies (anti-MelanA and HMB-45). The efficiency of this assay was demonstrated in a model system in which 5-500 tumor cells from the melanoma cell line SK-MEL-28 were seeded into PBMC samples from healthy donors containing 5 x 10(7) leukocytes. Mean recovery of the seeded tumor cells was 47.4 +/- 13.99% (n = 15). Applying the assay to 20-50 ml blood samples of patients with stage III-IV malignant melanomas, we were able to detect melanoma cells in two of eight patients (25%).

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Humans; Immunohistochemistry; Immunomagnetic Separation; Leukocytes, Mononuclear; MART-1 Antigen; Melanoma; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Staging; Neoplastic Cells, Circulating; Tumor Cells, Cultured

MART-1 is a good candidate antigen for immunotherapy against HLA-A2 patients with melanoma, since it is a highly immunogenic antigen recognized by HLA-A2 and HLA-B45 restricted CD8+ cytotoxic T cells and expressed in the majority of melanoma lesions. In the present study the expression of MART-1 and HLA-A2 on melanocytic cells and CD8+ T cell infiltration was immunohistochemically analyzed. MART-1 was expressed in most melanocytic lesions, while HLA-A2 was down-regulated with melanoma disease progression. Furthermore, concomitant down-regulation of MART-1 and HLA-A2 in melanoma cells was correlated with poor prognosis. These findings suggest both MART-1 and HLA-A2 expression in melanoma lesions should be analyzed for selection of patients eligible for MART-1 based immunotherapy and monitoring for emergence of melanoma cells resistant to T cell therapy.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Child; Female; Histocompatibility Antigens Class I; HLA-A2 Antigen; Humans; Immunohistochemistry; Lymphocytes, Tumor-Infiltrating; Male; MART-1 Antigen; Melanocytes; Melanoma; Middle Aged; Neoplasm Proteins; Skin Diseases; Skin Neoplasms; Staining and Labeling

The diagnosis of metastatic malignant melanoma (MMM) may be difficult in surgical pathology, often complicated by the unpredictable spread of this tumor and its great variability on histologic evaluation. Traditionally used immunohistochemical markers on melanomas are insufficient because of either a relative lack of specificity (S100 protein) or variably reported sensitivity (HMB45). Information about some newer markers, such as tyrosinase (TYR) and Melan A, is more limited. Recently, based on the study of a small number of tumors, it was suggested that microphthalmia transcription factor (MITF) is 100% sensitive in the identification of metastatic melanoma. In the current study, we compared the diagnostic usefulness of MITF with that of four other markers in 266 cases of conventional metastatic melanomas from different sites, 33 cases of desmoplastic melanomas, and 1 case of melanoma with rhabdoid features. The specificity of MITF was evaluated by using a representative sample of control tumors. Microphthalmia transcription factor with nuclear positivity was seen in 235 of 266 cases of conventional MMM (88%), usually in more than 30% of tumor cells. However, some melanomas had only foci of MITF- and TYR-positive cells, whereas the majority of cells were generally S100 protein-positive. Only 1 of 30 desmoplastic melanomas (3%) had MITF-positive cells, representing epithelioid foci resembling conventional melanoma. Two cases had TYR in a similar pattern; all were HMB45-negative. One metastatic melanoma with rhabdoid features was negative for MITF and other markers except the S100 protein. Half of the S100 protein negative conventional melanomas (6 of 12) were MITF-positive, whereas 4 of 20 (20%) TYR-negative tumors had reactivity for MITF. The percentages of positive cases of MMM (10% or more tumor cells positive) diagnosed with the four other markers in descending order were 90% (S100 protein and TYR), 78% (melan-A), and 66% (HMB45). Microphthalmia transcription factor appeared to be specific, because significant reactivity was not found in 112 carcinomas, 20 lymphomas, 20 angiosarcomas, 20 fibrous histiocytomas, and 20 malignant peripheral nerve sheath tumors. However, positive nuclei were found focally among reactive histiocytes, especially in osteoclasts, epithelioid histiocytes, and sporadic other histiocytes. Microphthalmia transcription factor may be a valuable addition to the marker panel used in diagnosing melanoma, in combination with S100, TYR,

Topics: Angiomyolipoma; Antibodies, Monoclonal; Antigens, Neoplasm; Biomarkers, Tumor; DNA-Binding Proteins; Fluorescent Antibody Technique, Indirect; Histiocytes; Humans; Lymphangiomyoma; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Microphthalmia-Associated Transcription Factor; Monophenol Monooxygenase; Neoplasm Proteins; S100 Proteins; Sensitivity and Specificity; Skin Neoplasms; Transcription Factors

While tumor-associated antigen (TAA)-specific CD8(+) T lymphocytes have been detected in metastatic melanoma patients, immune response in early disease phases has not yet been carefully evaluated. We looked for circulating cytotoxic T lymphocytes (CTL) directed against Melan-A / MART1, tyrosinase, gp100 and MAGE-3 antigens in patients with a diagnosis of primary cutaneous melanoma by using fluorescent HLA-A2 tetramers. In five out of six cases high numbers of CD8(+)/tetramer(+) cells could be detected by flow cytometry, and in four patients lymphocyte populations specific for two different melanoma antigens (Melan-A/MART1 and tyrosinase) were contemporaneously present. The TAA-specific cells could represent as much as 1/220 T lymphocytes in the circulating CD8(+) population. When tetramers were used to monitor the in vitro expansion of TAA-specific CTL precursors upon antigen-specific stimulation, a diverse expansion potential was evidenced in CTL from the different donors and, more strikingly, in CTL specific for the different TAA. Melan-A/MART1-specific CTL clones derived from two patients exhibited a broad range of avidity. Only the highest avidity clones, representing about 50 % of the cases analyzed, were tumor specific. By correlating tetramer staining with clone avidity, we found that tetramer fluorescence intensity could represent a good indicator of TCR affinity, but not of overall clone avidity.

Topics: Adult; Antigens, Neoplasm; Female; Fluorescence; gp100 Melanoma Antigen; HLA-A2 Antigen; Humans; Male; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Receptors, Antigen, T-Cell; T-Lymphocytes, Cytotoxic

The discovery of human melanoma rejection Ags has allowed the rational design of immunotherapeutic strategies. One such Ag, MART-1, is expressed on >90% of human melanomas, and CTL generated against MART-1(27-35) kill most HLA A2.1(+) melanoma cells. However, variant tumor cells, which do not express MART-1, down-regulate MHC, or become resistant to apoptosis, will escape killing. Cytotoxic lymphocytes kill by two main mechanisms, the perforin/granzyme degranulation pathway and the TNF/Fas/TNF-related apoptosis-inducing ligand superfamily of apoptosis-inducing ligands. In this study, we examined whether cis-diaminedichloroplatinum (II) cisplatin (CDDP) sensitizes MART-1/HLA A2.1(+) melanoma and melanoma variant tumor cells to non-MHC-restricted, Fas ligand (FasL)-mediated killing by CTL. MART-1(27-35)-specific bulk CTL cultures were generated by pulsing normal PBL with MART-1(27-35) peptide. These CTL cultures specifically kill M202 melanoma cells (MART-1(+), HLA A2.1(+), FasR(-)), and MART-1(27-35) peptide-pulsed T2 cells (FasR(+)), but not M207 melanoma cells (MART-1(+), HLA A2.1(-), FasR(-)), FLU(58-66) peptide-pulsed T2 cells, or DU145 and PC-3 prostate cells (MART-1(-), HLA A2.1(-), FasR(+)). CDDP (0.1-10 microg/ml) sensitized non-MART-1(27-35) peptide-pulsed T2 to the CD8(+) subset of bulk MART-1-specific CTL, and killing was abolished by neutralizing anti-Fas Ab. Furthermore, CDDP up-regulated FasR expression and FasL-mediated killing of M202, and sensitized PC-3 and DU145 to killing by bulk MART-1-specific CTL cultures. These findings demonstrate that drug-mediated sensitization can potentiate FasL-mediated killing by MHC-restricted CTL cell lines, independent of MHC and MART-1 expression on tumor cells. This represents a novel approach for potentially controlling tumor cell variants found in primary heterogeneous melanoma tumor cell populations that would normally escape killing by MART-1-specific immunotherapy.

Topics: Adjuvants, Immunologic; Animals; Antigens, Neoplasm; Antineoplastic Agents; Apoptosis; Cells, Cultured; Cisplatin; Cytotoxicity Tests, Immunologic; Cytotoxicity, Immunologic; Drug Resistance, Neoplasm; Epitopes; Epitopes, T-Lymphocyte; Fas Ligand Protein; fas Receptor; Humans; Hybridomas; Immunization; Major Histocompatibility Complex; Male; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Neoplasm Proteins; Perforin; Pore Forming Cytotoxic Proteins; Prostatic Neoplasms; RNA, Messenger; T-Lymphocyte Subsets; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured; Up-Regulation

Ex vivo ELISPOT analysis of peripheral blood lymphocytes obtained from stage IV melanoma patients demonstrated reactivity against peptides derived from MART-1 and gp100. However, the number of reactive T cells was < 1% that of total lymphocytes as detected by flow cytometry using tetrameric MHC/peptide complexes. Despite this low frequency, we were able to directly isolate these populations ex vivo by means of magnetic beads coated with MHC/peptide complexes and to subject these cells to T-cell receptor clonotype mapping. This analysis revealed that the MART-1/A*0201- and gp100/A*0201-reactive T-cell populations are composed of oligoclonal T cells that engage several T-cell receptor beta chain families. Longitudinal studies using this approach may result in a better correlation between T-cell reactivity and the course of neoplastic disease.

Topics: Aged; Antigens, Neoplasm; Biomarkers, Tumor; Clone Cells; Electrophoresis, Polyacrylamide Gel; Epitopes, T-Lymphocyte; Flow Cytometry; gp100 Melanoma Antigen; HLA-A Antigens; Humans; Male; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Middle Aged; Neoplasm Proteins; Neoplasm Staging; Receptors, Antigen, T-Cell; Receptors, Antigen, T-Cell, alpha-beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; T-Lymphocytes

An immunoselected melanoma cell line that had lost expression of the dominant melanoma antigens MART-1 and gp100 was generated in an attempt to identify previously unknown tumor antigens. After repeated stimulation with the autologous immunoselected tumor line, a number of HLA-A*0201-restricted T-cell clones were established from the peripheral blood of a single melanoma patient. One T-cell clone (C-22) recognized 14 of 16 HLA-A2+ melanoma cell lines, as well as HLA-A2+ melanocytes but recognized neither HLA-A2+ fibroblasts nor autologous B cells. Screening of an autologous cDNA library resulted in the isolation of a transcript identical to an entry in the expressed sequence tag database. Northern blot analysis revealed that this gene was expressed in most melanoma cell lines and melanocytes but not in normal tissues. The peptide epitope (AMF-GREFCYA) recognized by clone C-22 was identified based on studies of the recognition of truncated cDNAs and the use of the consensus HLAA*0201 binding motif. A second T-cell clone (C-29) was found to recognize a new tyrosinase-related protein 2 epitope (455-463; YAIDLPVSV) in an HLA-A*0201-restricted manner. Together, these results provide additional targets that can be used for the development of immunotherapeutic protocols in HLA-A2+ melanoma patients and demonstrate the utility of immunoselected tumor lines for the identification of new melanoma antigens.

Topics: Amino Acid Sequence; Antigen Presentation; Antigens, Neoplasm; Base Sequence; Cloning, Molecular; Crystallins; DNA, Complementary; DNA, Neoplasm; Epitope Mapping; Epitopes, T-Lymphocyte; gp100 Melanoma Antigen; HLA-A2 Antigen; Humans; Intramolecular Oxidoreductases; Lymphocyte Culture Test, Mixed; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Membrane Proteins; Molecular Sequence Data; Neoplasm Proteins; Protein Biosynthesis; Proteins; T-Lymphocytes; T-Lymphocytes, Cytotoxic; Transfection; Tumor Cells, Cultured

In this study we assessed the expression of the Melan-A/MART-1 antigen by immunohistochemistry using monoclonal antibody A103 in 73 primary cutaneous melanomas and its correlation with tumor staging and patient survival. Melan-A/MART-1 was expressed in 90% of primary tumors, with loss of expression increasing with Breslow thickness. Kaplan-Meier analysis demonstrated a significantly reduced disease-free interval and overall survival rate for patients not expressing this antigen. The poor prognosis of such patients was even worse for those presenting with a primary melanoma and a Breslow thickness of > or = 1 mm. Thus, Melan-A/MART-1 is not only a useful and specific additional marker for the diagnosis of primary cutaneous melanoma, but it may also help refine the prognosis of patients with malignant melanoma.

Topics: Antigens, Neoplasm; Disease Progression; Disease-Free Survival; Female; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Multivariate Analysis; Neoplasm Proteins; Prognosis; Skin Neoplasms; Time Factors

There is a current need for a reliable prognostic marker for melanoma patients, particularly those with stage 2 and stage 3 disease, so that adjuvant therapies can be directed appropriately.. To establish whether or not the use of tyrosinase-specific or melanA/MART-1-specific reverse transcriptase-coupled-polymerase chain reaction (RT--PCR) of peripheral blood cells detects preclinical disease progression in patients with malignant melanoma.. Two hundred and ninety-nine patients with melanoma in clinical stages 1--4 were observed in this study. Samples were obtained sequentially from 153 of these patients at 4-week intervals over a period of up to 2 years and correlated with clinical evidence of disease activity. Tyrosinase and melanA/MART-1 amplicons were analysed by agarose gel electrophoresis and Southern blot hybridization subsequent to a single round of amplification.. We demonstrated a statistically significant increase in tyrosinase RT--PCR positivity with advancing stage of melanoma progression. The percentage tyrosinase positivity in 910 samples tested was: stage 1, 135 samples, 34% positive; stage 2, 196 samples, 51% positive; stage 3, 423 samples, 50% positive; and stage 4, 156 samples, 65% positive. The positivity rate for individual patients tested sequentially was higher if only one positive test was required to label a patient positive, at 42%, 65%, 82% and 81% for patients in stages 1--4, respectively. However, we did not find a clear pattern of conversion from negativity to positivity in patients who progressed during the study from stage 2 to stage 3 or stage 3 to stage 4, and found no clear evidence of increased positivity rates in the 6-week period following melanoma-related surgery in patients with stage 3 and 4 disease. The positivity rate for melanA/MART-1 was lower for both patients and samples, and no melanA/MART-1-positive sample was negative for tyrosinase.. We conclude that the presence of circulating tyrosinase-positive cells as detected by this method appears to be a discontinuous rather than a continuous phenomenon, even in patients with stage 4 disease. For this reason the assay cannot be recommended as a method of sequentially monitoring individual patients in a clinical setting.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Disease Progression; Humans; MART-1 Antigen; Melanoma; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Staging; Neoplastic Cells, Circulating; Postoperative Period; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Tumor Cells, Cultured

To compare the sensitivity and staining pattern of the new immunohistochemical antibody to tyrosinase (T311) with S-100, HMB45, and the recently evaluated antibody to melan-A (A103) in a range of melanocytic lesions.. Archival, formalin fixed, paraffin wax embedded sections from 50 benign and malignant melanocytic lesions were stained immunohistochemically with anti-tyrosinase, A103, S-100, and HMB45. They were scored semiquantitatively for the distribution and intensity of staining.. All melanomas, with the exception of desmoplastic melanoma, showed some staining with all four antibodies. Overall, T311 and A103 showed an intermediate sensitivity compared with that of S-100 and HMB45. T311 stained most benign and malignant lesions strongly and diffusely with minimal background staining. Immunoreactivity was found to be patchy in some naevi, with weak or absent staining of the mature melanocytes. A103 showed strong and diffuse staining of all benign lesions and most melanomas with minimal background staining. S-100 was the most sensitive, with diffuse staining of most lesions, including desmoplastic and metastatic melanoma, but lacked specificity. HMB45 was the least sensitive antibody, frequently demonstrating patchy staining with absent staining in some benign naevi.. S-100 remains the most sensitive marker of melanocytes. However, because of its lack of specificity, it should be used with at least one other more specific antibody. HMB45 is more specific, but lacks sensitivity; T311 is a reliable marker of melanocytes in paraffin wax embedded sections and is worth consideration for use in a staining panel, although it shows no additional benefit over A103.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Biomarkers, Tumor; Clinical Enzyme Tests; Diagnosis, Differential; Humans; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Monophenol Monooxygenase; Neoplasm Proteins; Nevus; S100 Proteins; Sensitivity and Specificity; Skin Neoplasms

The aim of this study was to develop a highly sensitive two-marker assay for the detection of circulating melanoma cells in patients' blood using a reverse transcriptase-polymerase chain reaction (RT-PCR). We analysed the usefulness of two different sets of markers: tyrosinase and MUC-18 (TYR/MUC-18), and tyrosinase and MART 1 (TYR/MART 1). Total cellular RNA was isolated from 337 blood samples from 80 melanoma patients at different stages of the disease. All patients had undergone primary surgery. Assay sensitivity and specificity were confirmed using three different melanoma cell lines and two different fibroblast lines. In addition, blood from 47 healthy subjects and 10 patients with non-melanoma cancer was used as a negative control. We found that two-marker analysis is more accurate than the single tyrosinase assay. The frequency of melanoma cell detection in patients' blood was about 10% higher when the TYR/MART 1 two-marker assay was used. Using this assay we did not find any statistical correlation between the molecular markers and the UICC stage of disease or the Breslow thickness or Clark level of the primary melanoma. The frequency of melanoma cell detection with the TYR/MUC-18 two-marker assay was even higher than the TYR/MART 1 assay, but unfortunately the MUC-18 transcript was also present in about 20% of healthy subjects. Therefore we do not recommend the use of MUC-18 as a standard value marker.

Topics: Adult; Aged; Antigens, Neoplasm; Case-Control Studies; DNA Primers; Female; Humans; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Mucins; Neoplasm Proteins; Neoplastic Cells, Circulating; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Skin Neoplasms; Time Factors; Transcription, Genetic; Tumor Cells, Cultured

HLA-A2+ melanoma patients develop naturally a strong CD8+ T cell response to a self-peptide derived from Melan-A. Here, we have used HLA-A2/peptide tetramers to isolate Melan-A-specific T cells from tumor-infiltrated lymph nodes of two HLA-A2+ melanoma patients and analyzed their TCR beta chain V segment and complementarity determining region 3 length and sequence. We found a broad diversity in Melan-A-specific immune T-cell receptor (TCR) repertoires in terms of both TCR beta chain variable gene segment usage and clonal composition. In addition, immune TCR repertoires selected in the patients were not overlapping. In contrast to previously characterized CD8+ T-cell responses to viral infections, this study provides evidence against usage of highly restricted TCR repertoire in the natural response to a self-differentiation tumor antigen.

Topics: Amino Acid Sequence; Antigens, Neoplasm; Base Sequence; Complementarity Determining Regions; Epitopes, T-Lymphocyte; HLA-A2 Antigen; Humans; Immunodominant Epitopes; Immunoglobulin Variable Region; Lymph Nodes; MART-1 Antigen; Melanoma; Molecular Sequence Data; Neoplasm Proteins; Peptide Fragments; Receptors, Antigen, T-Cell, alpha-beta; T-Lymphocytes, Cytotoxic

Peptide-based vaccines are currently being tested for their ability to induce or augment tumor antigen (Ag)-specific CD8+ T-cell responses in cancer patients. Here we report that the frequency of circulating CD8+ T cells directed against the Melan-A/MART-1 Ag increased >20-fold in an HLA-A2 melanoma patient immunized repeatedly with the corresponding antigenic peptide, as assessed by staining with HLA-A2/peptide tetramers. Multiparameter flow cytometric analysis demonstrated that the increase in total Melan-A-specific cell number was accompanied by a marked increase in the proportion of the cells that expressed an activated/memory surface phenotype. As assessed by ELISPOT assays and intracellular staining, the absolute number of Melan-A-specific cells able to secrete IFN-gamma increased >50-fold upon vaccination. When tested directly after cell sorting on the basis of tetramer staining, Melan-A-specific cells were weakly cytolytic but became highly active after in vitro restimulation. Altogether, these results indicate that large numbers of functionally active tumor Ag-specific CD8+ T cells can be obtained and maintained at high levels after in vivo activation by repeated peptide-based vaccination.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Division; Flow Cytometry; Granzymes; Humans; Interferon-gamma; Lymphocytes; MART-1 Antigen; Melanoma; Neoplasm Proteins; Neoplasms; Peptides; Phenotype; Serine Endopeptidases; Time Factors

A 49-year-old patient with primary, recurrent melanoma on the lower extremity developed metastatic leptomeningeal melanoma that did not respond to treatment with radiation therapy or intrathecal interleukin 2 (IL-2). Disease was characterized by neurological symptoms, including loss of hearing, loss of short-term memory, and gait disturbance. CD8+ CTLs were generated in vitro using autologous dendritic cells pulsed with peptides from the melanoma-associated antigens tyrosinase (145-156), Melan-A/MART-1 (26-35), and gp100/Pmel 17 (209-217). The CTLs exhibited up to 74% specific lysis against peptide-pulsed autologous EBV-transformed B cells, with Melan-A-specific CTLs yielding the greatest lytic activity. CD8+ CTLs possessed a type 1 cytokine profile, expressing tumor necrosis factor alpha and IFNgamma but not IL-4. Infusions of CTLs were supported with systemic low-dose IL-2 administration. 111In labeling and computerized gamma imaging were used to monitor the distribution of CTLs up to 48 h after infusion. Intra-arterial delivery via the right carotid artery was followed by redistribution of the CTLs to the lungs, liver, and spleen within 16 h. In contrast, delivery via an indwelling Ommaya reservoir resulted in prolonged retention of CTLs within the brain for at least 48 h after infusion. Marked but transient elevations in tumor necrosis factor alpha, IFN-gamma, and IL-6 in the cerebrospinal fluid were observed within 4 h of CTL infusion. There was no evidence of tumor progression throughout the treatment period, and clinically the patient showed some resolution of neurological symptoms.

Topics: Antigens, Neoplasm; B-Lymphocytes; CD8-Positive T-Lymphocytes; Cytokines; Dendritic Cells; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; gp100 Melanoma Antigen; Humans; Immunotherapy; Immunotherapy, Adoptive; Indium; Interferon-gamma; Interleukin-2; Interleukin-4; Interleukin-6; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Meningeal Neoplasms; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Proteins; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes, Cytotoxic; Time Factors; Tissue Distribution; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

To elucidate the functional heterogeneity of Ag-specific T lymphocyte populations, we combined labeling of lymphocytes with MHC/peptide tetramers and a cell surface affinity matrix for IFN-gamma. Magnetic cell sorting of IFN-gamma-positive lymphocytes allowed the selective enrichment and identification of live Ag-specific cytokine-secreting cells by flow cytometry. Naive, memory, and effector Ag-specific populations were evaluated in healthy HLA-A2 individuals. Significant fractions of influenza- and CMV-specific cells secreted IFN-gamma upon challenge with cognate peptide, consistent with an effector/memory status. The sensitivity of the approach allowed the detection of significant numbers of CMV-specific IFN-gamma-secreting cells ex vivo (i.e., without Ag stimulation). This was not apparent when using previously described assays, namely, ELISPOT or intracellular IFN-gamma staining (cytospot). CD8+ T cells specific for the melamoma-associated Ag Melan-A/MART-1 did not produce IFN-gamma upon challenge with cognate peptide, reminiscent with their naive functional state in healthy individuals. In contrast, CD45RA(low) Melan-A/MART-1 tumor-specific cells from three of three melanoma patients presented levels of activity similar to those found for influenza- or CMV virus-specific lymphocytes, compatible with a functional differentiation into competent effector/memory T lymphocytes in vivo. Notably, a sizable fraction of Melan-A/MART-1-specific cells from a patient secreted IFN-gamma ex vivo following peptide-based vaccination. Thus, the high sensitivity of the assay provides a valuable tool to monitor effector T cell responses in different clinical situations.

Topics: Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Cell Differentiation; Cytomegalovirus; Enzyme-Linked Immunosorbent Assay; Epitopes, T-Lymphocyte; Flow Cytometry; HLA-A2 Antigen; Humans; Immunomagnetic Separation; Immunophenotyping; Infant, Newborn; Influenza A virus; Interferon-gamma; Lymphocyte Count; MART-1 Antigen; Melanoma; Monitoring, Immunologic; Neoplasm Proteins; Peptide Fragments; Sensitivity and Specificity; Viral Matrix Proteins

Microphthalmia transcription factor (Mitf), a melanocytic nuclear protein critical for the embryonic development and postnatal viability of melanocytes, is a master lineage regulator and modulates extracellular signals. Recently, Mitf expression was shown to be both a sensitive and specific marker of epithelioid melanoma. Because loss of specific melanocytic markers in melanomas with spindle cell morphology is more common compared with those tumors with epithelioid morphology, we investigated the sensitivity of D5, an anti-Mitf antibody, for diagnosis in this diagnostically problematic subset of melanomas. Twenty of 21 (95%) spindle cell and desmoplastic melanomas examined were reactive for S-100 protein. Only 4 of 21 (19%) spindle cell and desmoplastic melanomas were reactive for HMB-45. Six of 21 tumors (29%) were reactive for D5, including one case that was non-reactive for S-100 and HMB-45. Melan-A reactivity was seen in 2 of 13 cases (15%) studied. Eight of 24 (33%) non-melanocytic spindle cell tumors were reactive for D5, including 4 of 6 dermatofibromas, 1 of 6 schwannomas, 1 of 2 leiomyomas, and 2 of 6 leiomyosarcomas. Although D5 was shown in a previous study to be a highly sensitive and specific marker for epithelioid melanomas, the results of this study show it is not a sensitive or specific marker of spindle cell and desmoplastic melanomas. Nevertheless, we believe that diffuse positive staining for D5 when taken in clinical, histologic and immunohistochemical context may be diagnostically useful in selected cases of melanoma.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Biomarkers, Tumor; DNA-Binding Proteins; Female; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Microphthalmia-Associated Transcription Factor; Middle Aged; Neoplasm Proteins; S100 Proteins; Sensitivity and Specificity; Skin Neoplasms; Transcription Factors

Malignant melanomas of the oral and sinonasal mucosa are rare tumors. Amelanotic variants can, on occasion, be difficult to recognize by routine light microscopy. Immunohistochemical studies may be needed for a final diagnosis. A number of new monoclonal antibodies to melanocytic differentiation antigens have been studied recently on primary cutaneous and metastatic melanoma. However, little is known about these antibodies for the diagnosis of mucosal melanomas. In this study the authors analyzed 79 oral and sinonasal mucosal melanomas of 65 patients. A total of 35 tumors originated from the oral mucosa (21 primary tumors, eight local recurrences, and six metastases) and 44 melanomas were from the sinonasal tract (27 primary tumors, nine local recurrences, and eight metastases). Immunohistochemical studies were performed on paraffin-embedded tissues, using the following antibodies: anti-S-100 protein, T311 (anti-tyrosinase), A103 (anti-Mart-1/Melan-A), D5 (antimicrophthalmia-associated transcription factor), and HMB-45 (anti-gp100). Of 35 oral mucosal tumors, 34 (97%) were positive with anti-S-100 protein, 33 (94%) with T311, 30 (85%) with A103, 26 (74%) with D5, and 25 (71%) with HMB-45. All five desmoplastic melanomas of the oral mucosa were positive for S-100 protein, four for tyrosinase, and one each for HMB-45 and A103. No desmoplastic melanoma was positive with D5. All 44 sinonasal melanomas were positive for tyrosinase and Mart-1/Melan-A (100%). Forty-three (98%) were positive with HMB-45, 42 (95%) with anti-S-100 protein, and 40 (91%) with D5. These results reveal that T311 is the most sensitive marker for sinonasal melanomas and closely approaches the sensitivity of anti-S-100 protein for oral mucosal melanomas. For desmoplastic mucosal tumors, anti-S-100 protein remains the most sensitive marker.

Topics: Antigens, Neoplasm; DNA-Binding Proteins; gp100 Melanoma Antigen; Humans; MART-1 Antigen; Melanocytes; Melanoma; Membrane Glycoproteins; Microphthalmia-Associated Transcription Factor; Monophenol Monooxygenase; Mouth Mucosa; Mouth Neoplasms; Nasal Mucosa; Neoplasm Proteins; Nose Neoplasms; S100 Proteins; Transcription Factors

Interferon alpha (IFN-alpha) represents an adjuvant therapy of proven effectiveness in increasing disease-free interval and survival in subgroups of melanoma patients. Since high doses of cytokine are required, the treatment is often accompanied by toxic side effects. Furthermore, naturally occurring insensitivity to IFN-alpha may hamper its therapeutic efficacy. Clinical, molecular or immunological markers enabling the selection of potential responders have not been identified so far. To explore the molecular basis of IFN-alpha responsiveness, we analysed the expression pattern of about 7000 genes in IFN-alpha sensitive and resistant cell lines and we compared the transcription profiles of cells cultured in the presence or absence of the cytokine using high-density oligonucleotide arrays. Melanoma cell lines were screened for their sensitivity to proliferation inhibition and HLA class I induction upon IFN-alpha treatment by standard 3H-thymidine incorporation and flow-cytometry. The study of 4 sensitive and 2 resistant cell lines allowed the identification of 4 genes (RCC1, IFI16, hox2 and h19) preferentially transcribed in sensitive cells and 2 (SHB and PKC-zeta) preferentially expressed in resistant cells. IFN-alpha stimulation resulted in the expression of a panel of 19 known inducible genes in sensitive but not in resistant cells. Moreover a group of 30 novel IFN-alpha inducible genes was identified. These data may provide a useful basis to develop diagnostic tools to select potential IFN-alpha responders eligible for treatment, while avoiding unnecessary toxicity to non-responders. Furthermore, by extending the knowledge of the polymorphic effects of IFN-alpha on gene expression, they offer novel clues to the study of its pleiotropic toxicity.

Topics: Antigens, Neoplasm; Antineoplastic Agents; Cell Division; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Genetic Markers; gp100 Melanoma Antigen; Histocompatibility Antigens Class I; HLA Antigens; Humans; Interferon alpha-2; Interferon-alpha; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Multigene Family; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; Proteins; Recombinant Proteins; Transcriptional Activation; Up-Regulation

We have observed that malignant melanoma cells produce a soluble protein factor(s), which down-regulates melanocyte lineage Melan-A/MART-1 Ag expression by melanoma cells with concomitant loss of recognition by Melan-A/MART-1-specific T cells. This down-modulation of Melan-A/MART-1 expression, which we refer to as "Ag silencing," is mediated via its minimal promoter, whereas the promoter for the restricting Ag-presenting HLA-A2 molecule is not affected. Significantly, this Ag silencing is reversible, as removal of factor-containing supernatants from Melan-A/MART-1-expressing cells results in up-regulation of the promoter for the gene encoding this Ag, and renewed expression of the protein. We have evaluated over 20 known factors, none of which accounts for the Ag-silencing activity of the melanoma cell culture supernatants. The existence of this autocrine pathway provides an additional novel explanation for melanoma tumor progression in vivo in the presence of CTL specific for this melanocyte lineage Ag. These observations may have important implications for Melan-A/MART-1-specific CTL-mediated immunotherapy of melanoma tumors.

Topics: Antigens, Neoplasm; Autocrine Communication; Coculture Techniques; Cytotoxicity Tests, Immunologic; Down-Regulation; Gene Silencing; Humans; Jurkat Cells; MART-1 Antigen; Melanocytes; Melanoma; Neoplasm Proteins; Promoter Regions, Genetic; RNA, Messenger; Solubility; T-Lymphocytes, Cytotoxic; Transcription, Genetic; Tumor Cells, Cultured; Tumor Escape

We have investigated the possible usefulness of recombinant canarypox virus (ALVAC) encoding the melanoma-associated Ag, Melan-A/MART-1 (MART-1), in cancer immunotherapy, using a dendritic cell (DC)-based approach. ALVAC MART-1-infected DC express, and are able to process and present, the Ag coded by the viral vector. One consistent feature of infection by ALVAC is that these viruses induce apoptosis, and we show cross-presentation of Ag when uninfected DC are cocultured with ALVAC MART-1-infected DC. Uptake of apoptotic virally infected DC by uninfected DC and subsequent expression of tumor Ag in the latter were verified by flow cytometry analysis, image cytometry, and confocal microscopy. Functional activity was monitored in vitro by the stimulation of a MART-1-specific cytotoxic T cell clone. Heightened efficiency in Ag presentation is evidenced in the 2- to 3-fold increase in IFN-gamma production by the T cell clone, as compared with the ALVAC-infected DC alone. Cocultures of ALVAC MART-1-infected and uninfected DC are able to induce MART-1-specific T cell immune responses, as assessed by HLA class I/peptide tetramer binding, IFN-gamma ELISPOT assays, and cytotoxicity tests. Overall, our data indicate that DC infected with recombinant canarypox viruses may represent an efficient presentation platform for tumor Ags, which can be exploited in clinical studies.

Topics: Antigen Presentation; Antigens, Neoplasm; Apoptosis; Avipoxvirus; Cancer Vaccines; Cell Differentiation; Cells, Cultured; Clone Cells; Coculture Techniques; Cytotoxicity, Immunologic; Dendritic Cells; Genetic Vectors; Humans; Leukocytes, Mononuclear; Lymphocyte Activation; MART-1 Antigen; Melanoma; Neoplasm Proteins; Phagocytosis; Tumor Cells, Cultured; Vaccines, Synthetic; Viral Vaccines

The melanoma patient's immune response to tumor has been extensively studied. Yet, the frequently observed coexistence of tumor-associated Ag (TAA)-specific T cells with their target cells in vivo remains unexplained. Loss of TAA expression might contribute to this paradox. We studied TAA expression in metastases by obtaining fine-needle aspirations from 52 tumor lesions in 30 patients with melanoma before and soon after immunotherapy. Limitations due to low amounts of starting material were overcome with a high fidelity antisense RNA amplification method. TAA expression was measured by quantitative real-time PCR of anti-sense RNA. Decrease in gp100/Pmel-17 TAA preceded tumor disappearance in several instances and could be best explained by immune selection because most patients had received gp100/Pmel-17-specific vaccination. Conversely, immune selection was absent in nonregressing lesions. These observations suggest that vaccination, when successful, triggers a broad inflammatory reaction that can lead to tumor destruction despite immune selection. Additionally, lack of clinical response might be attributed to lack of this initiating event rather than immune escape. This study provides an insight into the natural history of tumors and defines a strategy for the characterization of gene expression in tumors during therapy.

Topics: Adult; Aged; Antigens, Neoplasm; Cancer Vaccines; Female; Gene Amplification; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Neoplasm Regression, Spontaneous; RNA, Antisense; Testis; Time Factors; Tumor Cells, Cultured

Topics: Adrenal Gland Neoplasms; Antibodies, Neoplasm; Antibody Specificity; Antigens, Neoplasm; Biopsy, Needle; Bone Neoplasms; Humans; Liver Neoplasms; Lung Neoplasms; MART-1 Antigen; Melanoma; Neoplasm Metastasis; Neoplasm Proteins; Pancreatic Neoplasms

We evaluated the expression of vimentin, S100a, and Melan A/MART-1 (melanoma antigen recognized by T cells 1) in seven cell lines established independently from dogs with canine melanoma. We also compared routine immunostaining of 29 clinical specimens from melanoma cases using vimentin, S100a, and neuron-specific enolase (NSE) with staining for Melan A/MART-1 as part of a diagnostic panel. All the cell lines were positive for expression of vimentin and S-100a. MelanA/MART-1 expression was seen consistently in only two of the seven cell lines. Staining for Melan A/MART-1 was most intense near areas of heavy melanin pigmentation. All except one of the clinical specimens were positive for vimentin. S 100a was expressed in the majority of both pigmented (15/20, 75%) and amelanotic (8/9, 88.8%) tumors. Seventeen of 29 (58.6%) tumors were positive for NSE. Melan A/MART-1 was expressed in 18/29 (62%) tumors, including 90% of pigmented tumors, but in no amelanotic tumors. Intensity of Melan A/MART-1 staining correlated positively with biologic behavior, with seven malignant tumors showing negative to weak staining and 10 benign tumors showing moderate to strong staining. Three malignant tumors showed moderate to intense staining for Melan A/ MART-1. Our results suggest that expression of Melan A/MART-1 may be unstable in cultured cell lines. Assessment of both S100a and Melan A/MART-1 expression is useful to confirm a diagnosis of canine melanoma, and Melan A/MART-1 may be especially informative regarding the biologic behavior of these tumors.

Topics: Animals; Antigens, Neoplasm; Biomarkers, Tumor; Cell Differentiation; Cell Lineage; Dog Diseases; Dogs; Female; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Neoplasm Proteins; Phosphopyruvate Hydratase; Retrospective Studies; S100 Proteins; Tumor Cells, Cultured; Vimentin

Some immunotherapeutic approaches based on melanoma-associated antigens rely on in vitro cultivation of melanoma cells. A beneficial effect of interferons has been shown in melanoma. This study aimed to determine whether stimulation of patient-derived melanoma short-term cell cultures using interferon-alpha and -gamma changes the expression pattern of melanoma-associated antigens. Lymph node, skin and brain metastases were cultivated for up to 3 weeks and treated with interferon-alpha, interferon-gamma or mock stimulation. Expression of the melanoma-associated antigens MAGE-3, MelanA/MART-1 and tyrosinase was determined by flow cytometry and compared with the expression pattern of HLA class I molecules. We found consistently enhanced expression of HLA class I molecules, whereas the melanoma-associated antigens showed mixed responses, with moderate induction, suppression or no visible effect. The reaction to interferon stimulation was similar for all the antigens examined within a single melanoma cell culture. In contrast to the HLA class I molecules, which showed induced expression with interferon, the melanoma-associated antigens showed a varied response to interferon stimulation. Differential reaction to interferon stimulation is of importance to immunotherapeutic modalities and might influence progression of the disease. We therefore suggest that evaluation of variation in melanoma-associated antigen expression in the clinical setting may help to identify patients who would profit from adjuvant interferon therapy.

Topics: Animals; Antibodies, Monoclonal; Antigens; Antigens, Neoplasm; Brain Neoplasms; Cell Line; Cell Separation; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Genes, MHC Class I; gp100 Melanoma Antigen; Humans; Interferon-alpha; Interferon-gamma; Interferons; Lymphatic Metastasis; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Mice; Monophenol Monooxygenase; Neoplasm Proteins; Reverse Transcriptase Polymerase Chain Reaction; Skin Neoplasms; Tumor Cells, Cultured; Up-Regulation

Accurate diagnosis of micrometastases in sentinel lymph nodes of cutaneous melanoma is critical for proper clinical management. S-100 protein and HMB-45 are the traditional immunomarkers widely used for this purpose. However, the interpretation of micrometastases by these markers is difficult with significant reduction in the diagnostic accuracy. S-100 protein demonstrates immunoreactivity for other nonmelanoma cells and obscures nuclear details, which are crucial for the interpretation of single cell metastases. We compared the new melanoma markers, Melan-A (clone A103) and MART-1 (clone M2-7C10), with S-100 protein and HMB-45, by examining 77 formalin-fixed paraffin-embedded sections of sentinel lymph nodes from 13 cases of primary cutaneous melanoma. CD68 (PG-M1) and hematoxylin-eosin-stained sections were also studied. Four pathologists interpreted the staining pattern after concealing the identity of each immunomarker. Az values (area under receiver operating characteristic curve) with receiver operating characteristic curve were higher with Melan-A (0.9742) and MART-1 (0.9779) compared with S-100 protein (0.8034) and HMB-45 (0.8651), demonstrating a higher diagnostic accuracy with Melan-A and MART-1 with superior detection of melanoma micrometastases. Melan-A and MART-1 showed sharp cytoplasmic immunoreactivity, almost exclusively restricted to the melanoma cells. Therefore, Melan-A and MART-1 are recommended for the evaluation of micrometastases in sentinel lymph nodes of cutaneous melanoma as a routine alternative to S-100 protein and HMB-45.

Topics: Antigens, CD; Antigens, Differentiation, Myelomonocytic; Antigens, Neoplasm; Humans; Lymph Nodes; Lymphatic Metastasis; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; Observer Variation; Reproducibility of Results; ROC Curve; S100 Proteins; Sentinel Lymph Node Biopsy; Skin Neoplasms

Blood lymphocytes from HLA-A*0201-subtyped melanoma patients and healthy controls were screened for the presence of T cells specific for HLA-A*0201-binding melanoma and viral peptide antigens by the enzyme-linked immunoSPOT (ELISPOT) assay. CD8(+) cells were tested for peptide-specific IFN-gamma release immediately after selection as well as after 2 weeks of in vitro stimulation. After in vitro stimulation, CD8(+) T cells specific for influenza were measured in all patients and controls, whereas these T cells could be detected among nonstimulated CD8(+) cells in only 52% of individuals. Similarly, T cells specific for EBV were more frequently measured among in vitro-stimulated than nonstimulated CD8(+) cells. In nonstimulated CD8(+) cells, T cells specific for MART-1/Melan-A, gp100, tyrosinase and CAMEL were present in 4 (33%), 1 (8%), 1 (8%) and 3 (25%) of 12 patients, respectively. Only MART-1/Melan-A-specific CD8(+) T cells were found in 1 (11%) of 9 healthy controls. CD8(+) T cells specific for MAGE-2 were not observed. After in vitro stimulation, CD8(+) T cells specific for MART-1/Melan-A could be demonstrated in 6 (46%) of 13 patients and 2 (20%) of 10 controls. CD8(+) T cells specific for gp100 were detected in 1 patient after in vitro stimulation. No CD8(+) T cells specific for tyrosinase, MAGE-2 or CAMEL could be measured after in vitro stimulation. These data show that the ELISPOT assay allows direct ex vivo detection of CD8(+) T cells specific for viral and melanoma antigens. Furthermore, the data show that the sensitivity of the ELISPOT assay to measure influenza- and EBV-specific CD8(+) T cells can be enhanced by a short in vitro stimulation step, whereas opposing effects on numbers of CD8(+) T cells specific for melanoma antigens have been observed.

Topics: Antigens, Neoplasm; Antigens, Viral; CD8-Positive T-Lymphocytes; Chromium Radioisotopes; Cytotoxicity, Immunologic; Epitopes, T-Lymphocyte; gp100 Melanoma Antigen; HLA-A2 Antigen; Humans; Immunoenzyme Techniques; Interferon-gamma; Lymphocyte Activation; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Peptide Fragments; Viral Matrix Proteins

Melanoma frequently metastasizes to the central nervous system (CNS). The diagnosis of CNS metastases typically is made following the onset of clinical symptoms. Thus, more sensitive diagnostic approaches are needed to identify subclinical CNS metastases. Currently, standard cytologic analysis of the cerebrospinal fluid (CSF) is limited by its poor sensitivity. A more sensitive assay was therefore developed using multiple reverse transcriptase-polymerase chain reaction (RT-PCR) markers. CSF was collected and assessed by RT-PCR for three known melanoma-associated markers (MAGE-3, MART-1, and tyrosinase) to detect occult metastatic melanoma cells in the CSF of 37 American Joint Committee on Cancer (AJCC) stage IV melanoma patients. Cytologic analysis of CSF was performed on all patients, and immunohistochemistry (IHC) analysis was performed on 33 CSF samples using anti-S100 and anti-HMB-45 antibodies. Only one patient (3%) had tumor-positive CSF cytology and IHC upon entry into the study, whereas 12 patients (32%) were positive for at least one RT-PCR marker. The correlation between CSF RT-PCR positivity of MART-1 and/or MAGE-3 and the development of CNS metastases at 3 mo was significant (p = 0.04). Fifteen of 37 patients (41%) had either positive MRI and/or positive RT-PCR results. Multimarker RT-PCR is more informative and sensitive than cytology/IHC in assessing the CSF of melanoma patients.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Biomarkers, Tumor; Brain Neoplasms; Disease-Free Survival; DNA, Neoplasm; Female; Humans; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Predictive Value of Tests; Reverse Transcriptase Polymerase Chain Reaction; Skin Neoplasms

Tyrosinase reverse transcription-polymerase chain reaction (RT-PCR) has been shown to be highly sensitive in detecting tumour cells in melanoma patients.. To assess whether the detection of minimal residual disease by RT-PCR is improved by concomitant analysis of sentinel lymph nodes (SLNs), bone marrow (BM) and peripheral blood (PB) in patients with primary melanoma.. Thirty-five SLNs, 41 BM samples and 26 PB specimens from 26 patients with primary cutaneous melanoma (tumour thickness > or = 0.75 mm) were examined by nested RT-PCR for tyrosinase and Melan-A. SLNs and BM samples were also analysed by histopathology. RT-PCR findings were related to tumour thickness of the primary melanoma.. Overall, melanoma cells were detected by RT-PCR in 13 of 26 patients (50%). Seven patients had positive RT-PCR results in their SLNs (27%), including all patients (n = 4) with histologically positive SLNs, two patients had positive findings in their BM exclusively detected by RT-PCR (8%) and six patients in PB (23%). The presence of tumour cells detected by RT-PCR in SLNs was not related to the presence of melanoma cells in BM and/or PB. The incidence of RT-PCR-positive SLNs was significantly associated with greater tumour thickness (P = 0.004). Both patients with positive RT-PCR findings in their BM had a large tumour thickness (> or = 2 mm). No association between positive RT-PCR findings in PB and greater tumour thickness was observed.. RT-PCR-positive SLNs were strongly associated with greater tumour thickness, underlining the prognostic significance of SLN positivity. Similar to certain epithelial malignancies, molecular investigation of the BM might provide complementary prognostic information in the early stages of melanoma. In contrast, no association between positive RT-PCR results in PB and increasing tumour thickness was found, implying that RT-PCR findings in PB are of doubtful clinical relevance in primary melanoma.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Biomarkers, Tumor; Bone Marrow Examination; Female; Humans; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Staging; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Sentinel Lymph Node Biopsy; Skin Neoplasms; Statistics, Nonparametric

We have determined high-resolution crystal structures of the complexes of HLA-A2 molecules with two modified immunodominant peptides from the melanoma tumor-associated protein Melan-A/Melanoma Ag recognized by T cells-1. The two peptides, a decamer and nonamer with overlapping sequences (ELAGIGILTV and ALGIGILTV), are modified in the second residue to increase their affinity for HLA-A2. The modified decamer is more immunogenic than the natural peptide and a candidate for peptide-based melanoma immunotherapy. The crystal structures at 1.8 and 2.15 A resolution define the differences in binding modes of the modified peptides, including different clusters of water molecules that appear to stabilize the peptide-HLA interaction. The structures suggest both how the wild-type peptides would bind and how three categories of cytotoxic T lymphocytes with differing fine specificity might recognize the two peptides.

Topics: Amino Acid Sequence; Antigens, Neoplasm; Binding Sites; Crystallography, X-Ray; HLA-A2 Antigen; Humans; Macromolecular Substances; MART-1 Antigen; Melanoma; Models, Molecular; Neoplasm Proteins; Peptide Fragments; Pliability; Protein Binding; Protein Conformation; T-Lymphocytes, Cytotoxic; Water

The effect on immunogenicity of different tumor T cell epitope formulations was evaluated in vitro using nonreplicating recombinant vaccinia vector expressing two forms of the melanoma-associated MART-1/Melan-A antigen. The first recombinant virus expressed a minigene encoding a fusion product between an endoplasmic reticulum (ER)-targeting signal and the HLA-A201 binding 27-35 peptide. The second viral construct encoded the complete MART-1/Melan-A protein. The capacity of HLA-A201 cells infected with either viral construct to generate and to stimulate MART-1/Melan-A 27-35 specific cytotoxic T-lymphocytes (CTL), was comparatively characterized. The results obtained here with a tumor antigen confirmed the capacity of vaccinia virus-encoded ER-minigene to generate a very strong antigenic signal. In cytotoxicity assays, recognition of target cells infected with high amounts of both recombinant viruses with activated specific CTL clones, resulted in similar lytic activity. With regard to calcium mobilization, TCR down-regulation, IFN-gamma release, and T cell proliferation assays, the targeted epitope elicited 10- to 1000-fold stronger responses. Remarkably, the immunogenic difference between the two formulations, in their respective capacity to generate CTL from naive HLA-A2 peripheral blood mononuclear cells in vitro as measured by tetramer detection, was lower (2- to 3-fold). Recombinant vectors expressing complete antigens have demonstrated their capacity to generate specific responses and such vaccines might take advantage of a broader potential of presentation. However, as demonstrated here for the HLA-A201-restricted MART-1/Melan-A immunodominant epitope, nonreplicative vaccinia virus expressing ER-targeted minigenes appear to represent a significantly more immunogenic epitope vaccine formulation.

Topics: Antigen Presentation; Antigens, Neoplasm; Antigens, Viral; Calcium; Cytotoxicity, Immunologic; Down-Regulation; Epitopes; HLA-A2 Antigen; Humans; Immunization; Interferon-gamma; Lymphocyte Activation; MART-1 Antigen; Melanoma; Neoplasm Proteins; Receptors, Antigen, T-Cell; Recombinant Proteins; T-Lymphocytes; T-Lymphocytes, Cytotoxic; Transfection; Tumor Cells, Cultured; Vaccinia virus; Viral Vaccines; Virus Replication

The use of tyrosinase-based polymerase chain reaction (PCR) tests for the detection of circulating tumour cells in the blood of melanoma patients has led to highly controversial results. We here report on the analysis of 120 blood samples from 76 stage I to IV melanoma patients using a new MART-1/Melan-A PCR system in conjunction with the tyrosinase-specific assay reported in the literature. While there were no positive results in localized disease (stages I and II), identification of specific PCR products in stage III melanoma patients was restricted to the MART-1/Melan-A tests, with positive results in 11% (two out of 19) of the blood specimens analysed. Stage IV melanoma patients presented with the highest incidence of detectable mRNA levels, with positive results for tyrosinase in 38% (12 out of 32) and for MART-1/Melan-A in 22% (seven out of 32). By delineating 64 follow-up specimens covering sampling periods of up to 33 weeks, stable mRNA expression profiles were identified in nearly 95%. Four patients, however, showed PCR changes towards positive MART-1/Melan-A expression that were linked to metastatic melanoma progression. Taken together, PCR tests for tyrosinase and MART-1/Melan-A seem to lack sufficient detection frequencies for the routine monitoring of melanoma disease. Regarding the link between MART-1/Melan-A seroconversion and the development of metastatic disease, further studies are needed to clarify the clinical value of this observation.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Disease Progression; Female; Follow-Up Studies; Humans; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Staging; Polymerase Chain Reaction; RNA Stability; RNA, Neoplasm; Sensitivity and Specificity; Skin Neoplasms

The assessment of tumor-associated antigens (TAA) recognized by T lymphocytes is a prerequisite for diagnosis and immunotherapy of melanoma. Different reverse transcription-polymerase chain reaction (RT-PCR) protocols allowing the quantification of the TAA mRNA expression in the solid tumor or the detection of circulating melanoma cells have been described. We have recently shown a positive correlation between the amount of specific product formed by RT-PCR and the staining intensity in immunohistochemical analysis of the corresponding sample. Here we describe a quantification procedure based on the direct digitization of the PCR products after separation on ethidium bromide-stained agarose gels, followed by computer-assisted densitometry. To standardize our method, we examined the linear range of the densitometric quantification procedure as reflected by the correlation of signal intensity to the amount of the corresponding DNA. As an internal measure for the so-termed cDNA in the different samples after RNA isolation and reverse transcription, a beta-actin PCR was introduced. Subsequently, we chose four sets of primers for the melanoma-associated antigens MAGE1, tyrosinase, Melan A/MART-1 and gp100/Pmel17 and performed PCR analysis over a range of cycle numbers. In each case, the amplification rate remained constant up to at least 26 cycles under the respective conditions. Plotting the logarithm of the amount of product against the cycle number yields a slope that equals the logarithm of the amplification rate. The amount of starting material can be determined from the intercept with the ordinate. In summary, the method introduced in the present work allows the quantification of TAA in melanoma which might be important for the monitoring of disease. Technically the method is sound and sensitive, avoids post-PCR manipulations and can be performed with the standard equipment of a molecular biology laboratory. It can be applied also to other solid tumors and leukemias.

Topics: Antigens, Neoplasm; DNA Primers; gp100 Melanoma Antigen; Humans; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sensitivity and Specificity; Skin Neoplasms

Intense efforts of research are made for developing antitumor vaccines that stimulate T cell-mediated immunity. Tumor cells specifically express at their surfaces antigenic peptides presented by MHC class I and recognized by CTL. Tumor antigenic peptides hold promise for the development of novel cancer immunotherapies. However, peptide-based vaccines face two major limitations: the weak immunogenicity of tumor Ags and their low metabolic stability in biological fluids. These two hurdles, for which separate solutions exist, must, however, be solved simultaneously for developing improved vaccines. Unfortunately, attempts made to combine increased immunogenicity and stability of tumor Ags have failed until now. Here we report the successful design of synthetic derivatives of the human tumor Ag Melan-A/MART-1 that combine for the first time both higher immunogenicity and high peptidase resistance. A series of 36 nonnatural peptide derivatives was rationally designed on the basis of knowledge of the mechanism of degradation of Melan-A peptides in human serum and synthesized. Eight of them were efficiently protected against proteolysis and retained the antigenic properties of the parental peptide. Three of the eight analogs were twice as potent as the parental peptide in stimulating in vitro Melan-specific CTL responses in PBMC from normal donors. We isolated these CTL by tetramer-guided cell sorting and expanded them in vitro. The resulting CTL efficiently lysed tumor cells expressing Melan-A Ag. These Melan-A/MART-1 Ag derivatives should be considered as a new generation of potential immunogens in the development of molecular anti-melanoma vaccines.

Topics: Aminopeptidases; Antigens, Neoplasm; Cancer Vaccines; Carboxypeptidases; Cells, Cultured; Cytotoxicity Tests, Immunologic; HLA-A Antigens; HLA-A2 Antigen; Humans; Kinetics; Lymphocyte Activation; MART-1 Antigen; Melanoma; Neoplasm Proteins; Peptides; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

It has been suggested that extracellular mRNA might be released from tumor cells and might be protected from serum RNase by proteins or proteolipid complexes. Our group has recently assessed the potential value of extracellular RNA detection by RT-PCR in the peripheral blood of patients with metastatic melanoma.

Topics: Antigens, Neoplasm; Humans; MART-1 Antigen; Melanoma; Monophenol Monooxygenase; Neoplasm Metastasis; Neoplasm Proteins; Neoplastic Cells, Circulating; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm

The membrane receptor 2B4 is a CD2 family member that is involved in lymphocyte activation. A fraction of human CD8+ alphabeta T cells up-regulate 2B4 in vivo, and here we demonstrate that this correlates with the acquisition of effector cell properties such as granzyme B and perforin expression, rapid IFN-gamma production, and down-regulation of the lymph node homing chemokine receptor CCR7. In PBLs from healthy donors, cytomegalovirus-specific effector T cells were 2B4 positive, whereas naive melanoma Ag (Melan-A/melanoma Ag recognized by T cells-1)-specific T cells were 2B4 negative. In melanoma patients, Melan-A-specific T cells up-regulated 2B4 in parallel with in vivo differentiation. This occurred in PBLs after vaccination with Melan-A peptides and in tumor-infiltrated lymph nodes, likely through disease-associated activation of Melan-A-specific T cells. Thus, 2B4 expression correlates with CD8+ T cell differentiation in vivo.

Topics: Antigens, CD; Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Differentiation; Cell Separation; Epitopes, T-Lymphocyte; Granzymes; Humans; Interferon-gamma; Interphase; Lymphocyte Activation; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Perforin; Pore Forming Cytotoxic Proteins; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Immunologic; Serine Endopeptidases; Signaling Lymphocytic Activation Molecule Family; T-Lymphocyte Subsets; Up-Regulation

A wide variety of differentiation patterns may be found in malignant melanoma. Schwannian features are unusual, and mostly present in the desmoplastic variant. We report the first description of psammoma bodies in malignant melanoma.. A malignant melanoma arose in an intradermal naevus of the scalp in a 51-year-old woman, displaying focal neural-like features in the form of rosette-like pseudo-meissnerian alveolar nests, as well as numerous psammoma bodies grouped in a few areas. Tumour cell immunostaining for S100, HMB45, NKI-C3, and vimentin was detected. In addition, both malignant and benign melanocytic cells showed widespread MART-1 immunoreactivity. Differential diagnosis with psammomatous melanotic schwannoma, a feature of Carney's complex, is particularly emphasized, since dermal variants of this nerve sheath neoplasm have been described. In addition, its potential relationship with cutaneous malignant melanotic neurocristic tumour is discussed.. This is, to our knowledge, the first reported case of cutaneous malignant melanoma with psammoma bodies.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Female; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasm Proteins; Nevus, Intradermal; S100 Proteins; Scalp; Skin; Skin Neoplasms; Vimentin

A prediction of the theory of immunologic surveillance is that tumour antigens can be recognised by cell-mediated immunity during early development of the primary tumour by formation of tumour antigen-specific cytotoxic lymphocytes (CTLs) and that such recognition leads to destruction of those tumour cells (tumour regression) with subsequent appearance of tumour antigen-loss variants. However, this has never been shown in nonviral-induced experimental animal models of primary malignancy or in human primary cancer. We examined 2 groups of human melanoma patients where primary tumour regression was observed. Twenty-three patients with multiple (>/=3) primary melanoma showed significant histologic regression of their last tumour (median tumour regression 33%) compared to matched tumours from patients with a single primary melanoma (median 0%) (p = 0.008) or compared to their first primary tumour (median 0%) (p = 0.001). This increased regression is consistent with an "immunisation effect" seen in murine tumour transplantation studies where innoculation with >/=3 asynchronous tumours induces transplantation rejection on subsequent challenge. A significant decrease in MART-1-positive stained tumour area in the last primary tumour from multiple melanoma subjects (median 8%) vs. matched single melanoma patients (median 79%) (p = 0.004) and in the last vs. first tumour (median 76%) in multiple primary subjects was found (p = 0.008). Metastatic tumours from 17 patients whose primary skin melanomas had completely regressed (occult primary melanoma) also showed significant MART-1 tumour-loss variants (median 0% MART-1-positive tumour) compared to matched metastatic tumours from patients with nonregressing primary tumours (median 51%) (p = 0.001). A correlation with the presence of peripheral blood MART-1-specific CTLs (MHC class I-restricted IFN-gamma producing T lymphocytes) and MART-1 tumour antigen-loss variants was found (p = 0.001). Thus, in 2 groups of human melanoma subjects, we provide evidence of tumour regression associated with Melan A/MART-1 tumour antigen-loss variants correlating with formation of specific Melan A/MART-1 CTLs.

Topics: Antigens; Antigens, Neoplasm; Female; Humans; Interferon-gamma; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Neoplasm Metastasis; Neoplasm Proteins; Skin Neoplasms; T-Lymphocytes; T-Lymphocytes, Cytotoxic; Treatment Outcome

Topics: Antigens, Neoplasm; Humans; MART-1 Antigen; Melanoma; Monophenol Monooxygenase; Neoplasm Proteins; Paraffin Embedding; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Sarcoma, Kaposi

Dendritic cells (DCs) are potent antigen-presenting cells that have been shown to play a critical role in the initiation of host immune responses against tumor antigens. In this study, a recombinant adenovirus vector encoding the melanoma-associated antigen, MART-1, was used to transduce murine DCs, which were then tested for their ability to activate cytotoxic T lymphocytes (CTLs) and induce protective immunity against B16 melanoma tumor cells implanted intracranially.. Genetic modification of murine bone marrow-derived DCs to express MART-1 was achieved through the use of an E1-deficient, recombinant adenovirus vector (AdVMART1). Sixty-two C57BL/6 mice were immunized by subcutaneous injection of AdVMART-1-transduced DCs (23 mice), untransduced DCs (17 mice), or sterile saline (22 mice). Using the B16 murine melanoma, which naturally expresses the MART-1 antigen, all the mice were then challenged intracranially with viable, unmodified syngeneic B16 tumor cells 7 days later. Splenocytes obtained from representative animals in each group were harvested for standard cytotoxicity and enzyme-linked immunospot assays. The remaining mice were followed for survival. Immunization of C57BL/6 mice with DCs transduced with AdVMART1-DC elicited the development of antigenspecific CTL responses. As evidenced by a prolonged survival curve when compared with control-immunized mice harboring intracranial B16 tumors, AdMART1-DC vaccination was able to elicit partial protection against central nervous system (CNS) tumor challenge in vivo. However, this CNS antitumor immunity was weaker than that previously demonstrated against subcutaneous B16 tumors in which the same vaccination strategy was used.. These data suggest that immune responses generated against CNS tumors by DC-based vaccines may be different from those obtained against subcutaneous tumors.

Topics: Adenoviridae; Animals; Antigens, Neoplasm; Brain Neoplasms; Cells, Cultured; Cytokines; Dendritic Cells; Disease Models, Animal; Female; Humans; Immunization; MART-1 Antigen; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Proteins; Neoplasm Transplantation; Spleen; T-Lymphocytes, Cytotoxic; Transduction, Genetic; Xenograft Model Antitumor Assays

Lymphocytes expanded from excised specimens can be used to characterize intratumoral T cell responses. These analyses, however, are limited to one time point in the natural history of the removed tumor. The expansion of autologous tumor cells and tumor-infiltrating lymphocytes (TIL) from fine needle aspirates (FNA) of tumors potentially allows a dynamic evaluation of T cell responses within the same lesion at moments relevant to the disease course or response to therapy. Fourteen TIL cultures and 8 tumor cell lines were generated from 18 FNA (12 patients). Five of six TIL that could be tested against autologous tumor demonstrated specific reactivity. Two additional TIL for which no autologous tumor was available demonstrated recognition of HLA-matched melanoma cell lines. Serial FNA of the same lesions were performed in five HLA-A*0201 patients vaccinated with the emulsified melanoma Ag (MA) epitopes: MART-1:27-35; tyrosinase:368-376(370D); gp100:280-288(288V); and gp100:209-217 (210M). FNA material was separately cultured for a short time in IL-2 (300 IU/ml) after stimulation with irradiated autologous PBMC pulsed with each peptide or FluM1:58-66 (1 micromol/ml). No peptide-specific TIL could be expanded from prevaccination FNA. However, after vaccination, TIL specific for gp100:280(g280), gp100:209 (g209), and MART-1:27-35 (MART-1)-related epitopes were identified in three, three, and two patients, respectively. No Flu reactivity could be elicited in TIL, whereas it was consistently present in parallel PBMC cultures. This excluded PBMC contamination of the FNA material. This analysis suggests the feasibility of TIL expansion from minimal FNA material and localization of vaccine-specific T cells at the tumor site.

Topics: Antigens, Neoplasm; Biopsy, Needle; Cancer Vaccines; Cell Culture Techniques; Cell Division; Epitopes, T-Lymphocyte; gp100 Melanoma Antigen; Humans; Immunodominant Epitopes; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Peptide Fragments; Peptides; T-Lymphocytes; Tumor Cells, Cultured

We have isolated, from an individual patient with metastatic melanoma, a series of eight TIL clones capable of lysing autologous melanoma cell targets. Six of the eight clones expressed TCRAV2S1 and lysed targets expressing HLA-A2 and the Melan-A/MART-1 peptide: AAGIGILTV. Polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) analysis showed that the Melan-A/MART-1-specific clones were predominant in the bulk culture prior to cloning. However, the tumour progressed in vivo even in the presence of these tumour cell-lytic clones. Using the anti-Melan-A/MART-1 MoAb (A-103), we noted that Melan-A/MART-1 expression on three melanoma cell lines varied considerably during in vitro culture, in the absence of T cell immunoselection, relative to cell density. Tumour cells which spontaneously decreased Melan-A/MART-1 expression were less susceptible to specific TIL lysis. Melan-A/MART-1 expression and susceptibility to lysis increased in cells cultured at lower density. These data suggest that modulation of tumour antigen may account for tumour progression in the presence of tumour cell-lytic T lymphocytes. The observations suggest a possible explanation for the common finding of Melan-A/MART-1-specific lytic TIL in clinically progressing melanomas, as well as a possible pathway for therapeutic intervention.

Topics: Amino Acid Sequence; Antigens, Neoplasm; Base Sequence; Clone Cells; Cytotoxicity, Immunologic; DNA; Gene Expression; Humans; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Molecular Sequence Data; Neoplasm Proteins; Phenotype; Polymorphism, Single-Stranded Conformational; Receptors, Antigen, T-Cell, alpha-beta; Tumor Cells, Cultured

The human Melan-A/MART-1 gene encodes an HLA-A2-restricted peptide epitope recognized by melanoma-reactive CD8(+) cytotoxic T lymphocytes. Here we report that this gene also encodes at least one HLA-DR4-presented peptide recognized by CD4(+) T cells. The Melan-A/MART-1(51-73) peptide was able to induce the in vitro expansion of specific CD4(+) T cells derived from normal DR4(+) donors or from DR4(+) patients with melanoma when pulsed onto autologous dendritic cells. CD4(+) responder T cells specifically produced IFN-gamma in response to, and also lysed, T2.DR4 cells pulsed with the Melan-A/MART-1(51-73) peptide and DR4(+) melanoma target cells naturally expressing the Melan-A/MART-1 gene product. Interestingly, CD4(+) T cell immunoreactivity against the Melan-A/MART-1(51-73) peptide typically coexisted with a high frequency of anti-Melan-A/MART-1(27-35) reactive CD8(+) T cells in freshly isolated blood harvested from HLA-A2(+)/DR4(+) patients with melanoma. Taken together, these data support the use of this Melan-A/MART-1 DR4-restricted melanoma epitope in future immunotherapeutic trials designed to generate, augment, and quantitate specific CD4(+) T cell responses against melanoma in vivo.

Topics: Amino Acid Sequence; Antigens, Neoplasm; Cancer Vaccines; CD4-Positive T-Lymphocytes; Epitopes; HLA-DR4 Antigen; Humans; Interferon-gamma; MART-1 Antigen; Melanoma; Molecular Sequence Data; Neoplasm Proteins; Peptide Fragments; Tumor Cells, Cultured

There is considerable interest in the development of vaccination strategies that would elicit strong tumor-specific CTL responses in cancer patients. One strategy consists of using recombinant viruses encoding amino acid sequences corresponding to natural CTL-defined peptide from tumor Ags as immunogens. However, studies with synthetic tumor antigenic peptides have demonstrated that introduction of single amino acid substitutions may dramatically increase their immunogenicity. In this study we have used a well-defined human melanoma tumor Ag system to test the possibility of translating the immunological potency of synthetic tumor antigenic peptide analogues into recombinant vaccinia viruses carrying constructs with the appropriate nucleotide substitutions. Our results indicate that the use of a mutated minigene construct directing the expression of a modified melanoma tumor Ag leads to improved Ag recognition and, more importantly, to enhanced immunogenicity. Thus, recombinant vaccinia viruses containing mutated minigene sequences may lead to new strategies for the induction of strong tumor-specific CTL responses in cancer patients.

Topics: Amino Acid Sequence; Animals; Antigen-Presenting Cells; Antigens, Neoplasm; Cancer Vaccines; Cytotoxicity, Immunologic; Epitopes, T-Lymphocyte; Genes, Synthetic; Genetic Vectors; Humans; Injections, Intraperitoneal; Lymphocyte Activation; MART-1 Antigen; Melanoma; Mice; Mice, Transgenic; Molecular Sequence Data; Mutagenesis, Site-Directed; Neoplasm Proteins; Peptides; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured; Ubiquitins; Vaccines, Synthetic; Vaccinia virus; Viral Vaccines

By stimulating human lymphocytes with an autologous renal carcinoma, we obtained CTL recognizing an antigen derived from a novel, ubiquitous protein. The CTL failed to lyse autologous EBV-transformed B cells, even though the latter express the protein. This is due to the presence in these cells of immunoproteasomes, which, unlike standard proteasomes, cannot produce the antigenic peptide. We show that dendritic cells also carry immunoproteasomes and fail to present this antigen. This may explain why the relevant CTL escape thymic deletion and are not regularly activated in the periphery. Lack of cleavage by the immunoproteasome was also observed for melanoma differentiation antigen Melan-A26-35/HLA-A2, currently used for antitumoral vaccination. For immunization with such antigens, proteins should be less suitable than peptides, which do not require proteasome digestion in dendritic cells.

Topics: Antigen Presentation; Antigens, Neoplasm; B-Lymphocytes; Base Sequence; Cell Line, Transformed; Cysteine Endopeptidases; Dendritic Cells; DNA, Complementary; Herpesvirus 4, Human; Humans; Kidney Neoplasms; MART-1 Antigen; Melanoma; Molecular Sequence Data; Multienzyme Complexes; Neoplasm Proteins; Peptides; Proteasome Endopeptidase Complex; Proteins; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

Fine-needle aspiration (FNA) of the adrenal is a useful modality for the evaluation of primary and metastatic neoplasms. Until now, however, few reliable markers existed for the positive identification of adrenal cortical cells. Originally studied as a melanoma marker, Melan-A, as detected by the murine monoclonal antibody, A103, has gained recent attention as a marker for steroid-producing cells. Formalin-fixed, paraffin-embedded cell blocks from 24 adrenal FNA specimens were stained for cytokeratins (AE1/AE3) and Melan-A (A103). Seven of 8 cases containing normal, hyperplastic, and neoplastic adrenal cortical cells were positive for A103. Among 16 cases of metastatic carcinoma, tumor cells in 14 samples were positive for cytokeratins but negative for A103. The A103 monoclonal antibody is a sensitive marker for the identification of normal, hyperplastic, and neoplastic adrenal cortical cells in cell blocks of adrenal FNA specimens. With the exception of melanoma, A103 reactivity is restricted to adrenal cortical and other steroid-producing cells. A103 should be used routinely for the evaluation of FNA specimens of adrenal mass lesions.

Topics: Adrenal Cortex; Adrenal Cortex Neoplasms; Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Biomarkers, Tumor; Biopsy, Needle; Cell Nucleus; Humans; Hyperplasia; Immunohistochemistry; Keratins; MART-1 Antigen; Melanoma; Mice; Neoplasm Proteins

Class I expression in context with T-cell receptor expression is crucial for peptide presentation and induction of CD8+ cytotoxic T lymphocytes (CTL). Presentation of class I bound peptides is dependent on transporter-associated proteins (TAP) expression and function. Tumor infiltrating lymphocytes from a patient with melanoma were isolated, expanded in vitro in the presence of interleukin-2, and tested for cytotoxicity against HLA-A2 positive, MART-1 positive autologous tumor cells, an HLA-A2-positive, MART-1 positive melanoma cell line (Mel-501), and HLA-A2-negative melanoma cells. Significant killing occurred against both A2-positive cell lines (63% and 65%, respectively), but not against the A2-negative line (18%) or A2-positive autologous tumor (1.5%). These CTL preferentially recognized the MART-1 peptide F119, 27-35, and gp100 peptide F125, 280-288, resulting in a 30% to 60% enhancement of lysis when autologous tumor or major histocompatibility complex class I "empty" T2 cells were pulsed with either peptide. To address whether the deficiency in autologous tumor recognition might be related to a deficiency in Ag presentation, we screened for the presence of TAP1 and TAP2 transcripts by polymerase chain reaction, Southern blotting, and scanning densitometry using sequence-specific primers and probes. Both TAP1 and TAP2 expression levels in the autologous tumor were minimal, yet were upregulated 7- to 18-fold, respectively, by interferon-gamma. Despite this increase, a similar increase in cytotoxicity did not occur. In short, deficiencies in TAP presentation may have functional significance for tumor escape from immunosurveillance and with respect to impending vaccine trials.

Topics: Abdominal Neoplasms; Antigens, Neoplasm; ATP Binding Cassette Transporter, Subfamily B, Member 2; ATP Binding Cassette Transporter, Subfamily B, Member 3; ATP-Binding Cassette Transporters; Cytotoxicity, Immunologic; Female; Gene Expression; HLA-A2 Antigen; Humans; Interferon-gamma; Lymphocytes, Tumor-Infiltrating; Major Histocompatibility Complex; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Metastasis; Neoplasm Proteins; Peptides; Phenotype; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

Immunization with tumor-specific-associated antigen--pulsed dendritic cells has proved to be efficacious in various animal models and is being evaluated for the treatment of cancer in humans. Use of dendritic cells pulsed with specific peptides or transfected with tumor-associated antigen genes has been a focused area of investigation for inducing potent tumor and viral immune responses. In this study, the authors demonstrate transgene expression, including the lacZ and MART-1 genes, in dendritic cells infected with adenoviral constructs. These transiently transduced dendritic cells, derived from melanoma patients' monocytes cultured with granulocyte-macrophage colony-stimulating factor and interleukin-4, express the transgene and can stimulate patients' CD8+ T cells to elicit an antitumor immune response comparable to dendritic cells loaded with a defined peptide. These cytotoxic T lymphocytes were able to recognize both known and unknown tumor-associated antigen epitopes and exhibited cytolytic activity against HLA-matched tumor cells expressing the antigen. The ability to induce tumor-specific cytotoxic T lymphocytes in vitro using gene-modified dendritic cells that transiently express tumor-associated antigens demonstrates the potential use of these antigen-presenting cells for developing in vivo cancer vaccines.

Topics: Adenoviruses, Human; Antigens, Neoplasm; Cells, Cultured; Dendritic Cells; Gene Expression; Genetic Vectors; Humans; Immunotherapy, Adoptive; MART-1 Antigen; Melanoma; Neoplasm Proteins; Peptides; T-Lymphocytes, Cytotoxic; Transgenes; Tumor Cells, Cultured

The histopathologic differential diagnosis of Spitz nevus (SN) from malignant melanoma (MM) may be difficult.. We attempted to elucidate the pattern of expression of a newly recognized melanocyte-specific melanosomal protein MART-1 in routinely processed specimens of SNs, MMs, and ordinary melanocytic nevi (MNs) and to see whether it can help to differentiate between them.. Twenty SN, 22 MM, and 27 ordinary MN were immunostained with anti-MART-1 monoclonal antibody (clone A103).. All SNs, MNs, and MMs demonstrated cytoplasmic staining for MART-1 in some of their tumor cells, of which 17 of 20 (85%) and 24 of 27 (89%) of SN and MN, respectively, demonstrated positive stainings in more than half of their tumor cells, as compared with only 10 of 22 (45%) of the MM (P <.05). The majority of lesions in all 3 types of tumors showed a homogeneous mode of staining, although MM tended to show a more heterogeneous pattern. A consistent pattern of stratification of staining with progressive descent into the dermis was not demonstrated in these tumors.. MART-1 does not differentiate between SN, MM, and ordinary MN in a consistent pattern, but it may be used as a marker for these tumors.

Topics: Antigens, Neoplasm; Diagnosis, Differential; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Neoplasm Proteins; Nevus, Epithelioid and Spindle Cell; Nevus, Pigmented; Skin Neoplasms

Frequencies of vaccine-responsive T-lymphocyte precursors in peripheral blood mononuclear cells (PBMC) prior to and after administration of peptide-based vaccines in patients with cancer can be measured by limiting-dilution assays (LDA) or by ELISPOT assays. We have used a modified version of the ELISPOT assay to monitor changes in the frequency of gamma interferon (IFN-gamma)-producing T cells in a population of lymphocytes responding to a relevant peptide or a nonspecific stimulator, such as phorbol myristate acetate-ionomycin. Prior to its use for monitoring of patient samples, the assay was validated and found to be comparable to the LDA performed in parallel, using tumor-reactive cytolytic T-lymphocyte (CTL) lines. The sensitivity of the ELISPOT assay was found to be 1/100,000 cells, with an interassay coefficient of variation of 15%, indicating that it could be reliably used for monitoring of changes in the frequency of IFN-gamma-secreting responder cells in noncultured or cultured lymphocyte populations. To establish that the assay is able to detect the T-cell precursor cells responsive to the vaccine, we used CD8(+) T-cell populations positively selected from PBMC of HLA-A2(+) patients with metastatic melanoma, who were treated with dendritic cell-based vaccines containing gp100, MELAN-A/MART-1, tyrosinase, and influenza virus matrix peptides. The frequency of peptide-specific responder T cells ranged from 0 to 1/2,600 before vaccination and increased by at least 1 log unit after vaccination in two patients, one of whom had a clinical response to the vaccine. However, no increases in the frequency of peptide-responsive T cells were observed in noncultured PBMC or PBMC cultured in the presence of the relevant peptides after the melanoma patients enrolled in another trial were treated with the intramuscular peptide vaccine plus MF59 adjuvant. Thus, while the ELISPOT assay was found to be readily applicable to assessments of frequencies of CTL precursors of established CTL lines and ex vivo-amplified PBMC, its usefulness for monitoring of fresh PBMC in patients with cancer was limited. In many of these patients antitumor effector T cells are present at frequencies of lower than 1/100,000 in the peripheral circulation. Serial monitoring of such patients may require prior ex vivo amplification of specific precursor cells.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Biological Assay; Cancer Vaccines; Cell Line; Clinical Trials as Topic; Evaluation Studies as Topic; gp100 Melanoma Antigen; Humans; Interferon-gamma; Ionomycin; Leukocytes, Mononuclear; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Reproducibility of Results; Sensitivity and Specificity; T-Lymphocytes, Cytotoxic; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Vaccines, Synthetic

Replication-deficient recombinant adenovirus (Ad) encoding human gp100 or MART-1 melanoma Ag was used to transduce human dendritic cells (DC) ex vivo as a model system for cancer vaccine therapy. A second generation E1/E4 region deleted Ad which harbors the CMV immediate-early promoter/enhancer and a unique E4-ORF6/pIX chimeric gene was employed as the backbone vector. We demonstrate that human monocyte-derived DC are permissive to Ad infection at multiplicity of infection between 100 and 500 and occurs independent of the coxsackie Ad receptor. Fluorescent-labeled Ad was used to assess the kinetics and distribution of viral vector within DC. Ad-transduced DC show peak transgene expression at 24-48 h and expression remains detectable for at least 7 days. DC transduced with replication-deficient Ad do not exhibit any unusual phenotypic characteristics or cytopathic effects. DC transduced with Ad2/gp100v2 can elicit tumor-specific CTL in vitro from patients bearing gp100+ metastatic melanoma. Using a panel of gp100-derived synthetic peptides, we show that Ad2/gp100v2-transduced DC elicit Ag-specific CTL that recognize only the G209 and G280 epitopes, both of which display relatively short half-lives ( approximately 7-8 h) on the surface of HLA-A*0201+ cells. Thus, patients with metastatic melanoma are not tolerant to gp100 Ag based on the detection of CD8+ T cells specific for multiple HLA-A*0201-restricted, gp100-derived epitopes.

Topics: Adenoviruses, Human; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Cells, Cultured; Dendritic Cells; Enterovirus B, Human; Epitopes, T-Lymphocyte; Fluorescent Dyes; Genetic Vectors; gp100 Melanoma Antigen; Half-Life; HLA-A2 Antigen; Humans; Immunodominant Epitopes; Immunophenotyping; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Peptide Fragments; Receptors, Virus; T-Lymphocytes, Cytotoxic; Virion; Virus Replication

Melanoma-reactive HLA-A x 0201-restricted cytotoxic T lymphocyte (CTL) lines generated in vitro lyse autologous and HLA-matched allogeneic melanoma cells and recognize multiple shared peptide antigens from tyrosinase, MART-1, and Pme117/gp100. However, a subset of melanomas fail to be lysed by these T cells. In the present report, four different HLA-A x 0201+ melanoma cell lines not lysed by melanoma-reactive allogeneic CTL have been evaluated in detail. All four are deficient in expression of the melanocytic differentiation proteins (MDP) tyrosinase, Pme117/gp100, gp75/ trp-1, and MART-1/Melan-A. This concordant loss of multiple MDP explains their resistance to lysis by melanoma-reactive allogeneic CTL and confirms that a subset of melanomas may be resistant to tumor vaccines directed against multiple MDP-derived epitopes. All four melanoma lines expressed normal levels of HLAA x 0201, and all were susceptible to lysis by xenoreactive-peptide-dependent HLA-A x 0201-specific CTL clones, indicating that none had identifiable defects in antigen-processing pathways. Despite the lack of shared MDP-derived antigens, one of these MDP-negative melanomas, DM331, stimulated an effective autologous CTL response in vitro, which was restricted to autologous tumor reactivity. MHC-associated peptides isolated by immunoaffinity chromatography from HLA-A1 and HLA-A2 molecules of DM331 tumor cells included at least three peptide epitopes recognized by DM331 CTL and restricted by HLA-A1 or by HLA-A x 0201. Recognition of these CTL epitopes cannot be explained by defined, shared melanoma antigens; instead, unique or undefined antigens must be responsible for the autologous-cell-specific anti-melanoma response. These findings suggest that immunotherapy directed against shared melanoma antigens should be supplemented with immunotherapy directed against unique antigens or other undefined antigens, especially in patients whose tumors do not express MDP.

Topics: Antigen Presentation; Antigens, Differentiation; Antigens, Neoplasm; Cell Differentiation; Chromatography, Affinity; Cytotoxicity, Immunologic; Epitopes; gp100 Melanoma Antigen; HLA-A1 Antigen; Humans; Male; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Oxidoreductases; Pigmentation; Proteins; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

Xanthomas of the skin may mimic balloon cell melanoma because 1) both lesions may exhibit a diffuse dermal proliferation of cytologically similar large vacuolated or clear cells with distinct cytoplasmic membranes, 2) dermal maturation (smaller deep dermal nuclei) is absent in both lesions, 3) melanin pigment is usually absent in balloon cell melanoma, 4) cellular atypia may be minimal in balloon cell melanoma, and 5) mitoses may be absent or rare in balloon cell melanoma. We report a unique xanthoma, which further simulated melanoma by exhibiting epidermotropism and a pseudonesting pattern at the dermal-epidermal junction. The correct diagnosis was made with an immunohistochemical panel revealing tumor cell positivity for CD68 and negativity for S-100 protein and MART-1. Immunohistochemical studies may be required in the critical differential diagnosis of epidermotropic xanthoma and amelanotic balloon cell melanoma.

Topics: Antigens, CD; Antigens, Differentiation, Myelomonocytic; Antigens, Neoplasm; Diagnosis, Differential; Epidermis; Female; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; S100 Proteins; Skin Neoplasms; Xanthomatosis

To design an efficient procedure to expand high avidity melanoma reactive T cells and to perform immunotherapies, we compared conditions of peripheral blood lymphocyte (PBL) stimulation by Melan-A/MART-1 peptides. Avidity of induced CTLs was evaluated by measuring their lysis and cytokine secretion to peptide-pulsed transporter-associated protein-deficient cells and to melanoma cells. In side-by-side experiments, we show that melanoma cells, either allogeneic or autologous, induced the growth of high avidity Melan-A-reactive CTLs from all donors, whereas essentially low avidity T cells were induced by peptide-pulsed PBLs. We also show that at least two cytokines, interleukin-6 and interleukin-2, were required to promote the growth of high avidity CTLs. Once sorted by tetramer labeling or cloning, the specificity and reactivity to tumor cells of peptide-specific T cells induced by allogeneic melanoma cells were confirmed. We then describe a relatively simple and efficient procedure that allowed us to obtain systematically high amounts (in the range of billion) of high avidity Melan-A/ MART-1-specific T cells from the PBLs of HLA-A2 melanoma patients and healthy donors in 3 months. Because this antigen is expressed by most melanoma tumors, this procedure should be useful for checking the efficiency of adoptive immunotherapy of melanoma tumors and using functionally well-defined Melan-A/MART-1-specific CTLs in a large group of patients.

Topics: Amino Acid Sequence; Antigens, Neoplasm; Cell Division; Clone Cells; Cytotoxicity, Immunologic; Dose-Response Relationship, Drug; Flow Cytometry; Humans; Lymphocytes; MART-1 Antigen; Melanoma; Neoplasm Proteins; Oligopeptides; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

Peptides derived from human tumor antigens have been used in a number of clinical trials to induce specific immune responses against autologous tumors in cancer patients. Although favorable clinical results were observed in single patients, immune responses correlating with tumor regression were either not detected or in case of responses, the T-cell specificity was difficult to demonstrate. In this study, we analyzed antigen-specific T-cell responses induced in the skin and in peripheral blood lymphocytes (PBL) in an HLA-A2-positive melanoma patient. The patient showed major regression of metastatic melanoma under continued immunization with peptides derived from the melanocyte differentiation antigens Melan A/MART-1, tyrosinase and gp100/Pmel17. Based on the identification of different T-cell receptor (TCR) families reactive with Melan A/MART-1, we have demonstrated that i.d. immunization with peptides alone leads to oligoclonal expansion of Melan A/MART-1-specific cytotoxic T lymphocytes (CTL), detectable in local delayed-type hypersensitivity (DTH) reactions and PBL. A monoclonal expansion of a Melan A/MART-1-specific TCR VB 16 CTL was reproducibly observed after in vitro stimulation with Melan A/MART-1 peptides. The same TCR VB 16 CTL clone was detected in skin biopsies taken from vitiligo areas. Our findings provide strong evidence for the effective induction of specific T-cell responses to Melan A/MART-1 by i.d. immunization with peptide alone, which accounts for dermal depigmentation, specific cytotoxicity against Melan A/MART-1-expressing melanoma cells and clinical tumor regression.

Topics: Antigens, Neoplasm; Female; Humans; Hypersensitivity, Delayed; Immunization; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasm Proteins; T-Lymphocytes, Cytotoxic; Vitiligo

Melanoma cell detection in peripheral blood by tyrosinase reverse transcription-polymerase chain reaction (RT-PCR) is usually performed on RNA isolated from whole blood using a guanidinium isothiocyanate (GITC)/phenol extraction method or from Ficoll Hypaque isolated mononuclear cells. The first method contains environmentally harmful reagents, and the second is laborious in the preanalytical steps. Cell preparation tubes (CPTs) are ready-to-use Ficoll Hypaque-based tubes that avoid the time-consuming and critical loading on Ficoll Hypaque. We examined whether CPTs can be used to determine melanoma cell dissemination in peripheral blood. We first investigated whether melanoma cells were retained in the mononuclear cell layer. All six morphologically different melanoma cell lines studied in the spiking experiments were retained in the upper layer. In further experiments, we were able to detect low dilutions of added SK-MEL-28 cells more consistently after nested RT-PCR for tyrosinase or MART-1 in the RNA isolated from mononuclear cells from CPTs than from RNA isolated with the GITC method. In addition, RNA was extracted from paired blood samples from 24 analysable stage III and stage IV melanoma patients and analysed for the presence of tyrosinase and MART-1 RNA using both the CPT/RNeasy and the whole blood/GITC method. The quality of the CPT/RNeasy RNA was better than the RNA isolated from whole blood with GITC/phenol. However, the RT-PCR results were less unequivocal: MART-1 mRNA was more often detected with CPTIRNeasy compared with whole blood/GITC (six versus three), whereas tyrosinase mRNA was found less often in CPT/RNeasy RNA (two versus eight). Taken together these results suggest that the CPT isolation method is suitable for the isolation of mononuclear cells, including melanoma cells.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Chromatography, Liquid; Diatrizoate; Ficoll; Guanidines; Humans; MART-1 Antigen; Melanocytes; Melanoma; Monophenol Monooxygenase; Neoplasm Proteins; Neoplastic Cells, Circulating; Neoplastic Stem Cells; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Sensitivity and Specificity; Thiocyanates; Tumor Cells, Cultured

The assessment of the TCR repertoire expressed by tumor-specific CD8+ T lymphocytes has been hampered to date by the difficulty of targeting the analysis to lymphocytes directed against a single epitope. In the present study we have used fluorescent A2/Melan-A tetramers in conjunction with anti-CD8 and anti-TCR beta-chain variable (BV) mAbs to analyze by flow cytometry the BV segment usage by Melan-A-specific CD8+ T cells in tumor-infiltrated lymph nodes (TILN) and tumor-infiltrating lymphocytes (TIL) from A2 melanoma patients. Analysis of TILN populations revealed small proportions of A2/Melan-A tetramer+ cells expressing many different BV together with over-representation of A2/Melan-A tetramer+ cells expressing certain BVs. The BV usage by A2/Melan-A tetramer+ lymphocytes in TIL was more restricted than that in TILN. Moreover, the predominant BV segments were quite distinct in populations derived from different patients. A2/Melan-A tetramer+ cells expressing the dominant BVs found in TILN could also be found in the corresponding peptide-stimulated autologous PBMC, although A2/Melan-A tetramer+ lymphocytes expressing additional BVs were also identified. Together, these results suggest that a large and diverse repertoire of Melan-A-specific T cells using different BV TCR segments is available in A2 melanoma patients.

Topics: Adult; Aged; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Epitopes, T-Lymphocyte; Gene Rearrangement, beta-Chain T-Cell Antigen Receptor; Genes, T-Cell Receptor beta; Humans; Leukocytes, Mononuclear; Lymphocyte Activation; Lymphocyte Count; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Peptide Fragments; Tumor Cells, Cultured

We stained benign melanocytic nevi and malignant melanoma with antibodies to melanoma antigen recognized by T cells (Mart-1) to determine if this was useful in differentiating benign from malignant melanocytic neoplasms. Forty-five primary malignant melanomas and 71 benign melanocytic nevi were stained with antibodies to Mart-1. Two cases of malignant melanoma metastatic to lymph node and three cutaneous metastases of malignant melanoma were also stained. The degree of staining was graded into diffuse positive staining, focal positive staining, and negative staining. Thirty-six of 45 primary malignant melanomas stained diffusely positive with antibodies to Mart-1. This included three of five desmoplastic malignant melanomas that showed positive staining. Four melanomas showed faint or focal positive staining. One of two metastases to lymph node showed strong positive staining and one showed no staining. All three cutaneous metastases showed diffuse positive staining. Sixty-one of 71 melanocytic nevi showed no staining or faint staining with antibodies to Mart-1. Ten of 71 melanocytic nevi showed strong positive staining. The majority of these were congenital nevi. Staining with antibodies to Mart-1 antigen was a useful marker of malignant melanoma. However, staining may also be seen in benign melanocytic neoplasms. The presence or absence of staining for Mart-1 antigen cannot be used to differentiate benign melanocytic nevi from malignant melanocytic tumors.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Diagnosis, Differential; Humans; Immunoenzyme Techniques; Lymphatic Metastasis; MART-1 Antigen; Melanoma; Neoplasm Proteins; Nevus, Pigmented; Skin Neoplasms

For the development of peptide-based immunotherapies, the identification of additional tumor antigens and T-cell epitopes is required. Because HLA-A(*)0201 is the most common allele in Caucasians, who represent the majority of patients with melanomas, 6 peptides carrying an HLA-A(*)0201 motif were synthesized from tyrosinase-related protein-2 (TRP2) melanoma antigen and tested for binding affinity to the HLA allele using processing-defective T2 cells. These peptides were then pulsed onto autologous dendritic cells and used to stimulate in vitro CD8(+)-enriched T cells isolated from peripheral blood of HLA-A(*)02(+) healthy donors or melanoma patients for the induction of specific cytotoxic T lymphocytes (CTLs). One peptide, TRP2(288-296) (SLDDYNHLV), the best HLA-A(*)0201 binder, elicited specific CTLs from 1 of 4 patients and 3 of 4 healthy donors. The induced CTLs from the patient and from 1 donor efficiently recognized HLA-A(*)02(+) TRP2(+) melanomas as well as COS-7 cells expressing HLA-A(*)0201 and TRP2 in an HLA class I-restricted manner, as assessed by cytokine production and direct cytolysis. The remaining 2 CTL lines derived from 2 donors displayed low T-cell receptor avidity, which could lyse melanoma cells in the presence of exogenous peptide. Since TRP2 is an antigen expressed in most melanomas, identification of the TRP2/HLA-A(*)0201 peptide SLDDYNHLV may facilitate the design of present peptide-based immunotherapies for the treatment of a large fraction of melanoma patients.

Topics: Amino Acid Motifs; Animals; Antigen Presentation; Antigens, Neoplasm; COS Cells; Dendritic Cells; Epitopes; HLA-A2 Antigen; Humans; Intramolecular Oxidoreductases; Lymphocyte Activation; MART-1 Antigen; Melanoma; Neoplasm Proteins; Peptide Fragments; Recombinant Fusion Proteins; T-Lymphocytes, Cytotoxic; Transfection; Tumor Cells, Cultured

Cytotoxic T lymphocytes (CTL) recognize minimal peptides of eight to ten residues which are the products of intracellularly processed proteins and are presented at the cell surface by MHC class I molecules. An important step in this process is the translocation of processed proteins from the cytosol across the endoplasmic reticulum membrane mediated by transporter associated with antigen processing (TAP) proteins, or as an alternative, by endoplasmic reticulum insertion signal sequences. We report here that the addition of synthetic signal sequences at the N terminus, but not at the C terminus, of an epitope from the human melanoma antigen MART-1 greatly enhances its presentation in both TAP-deficient and TAP-expressing cells. A newly designed peptide construct, composed of the epitope replacing the hydrophobic part of a natural signal sequence, was also very effective. Interestingly, an artificial signal sequence containing the same epitope was the most efficient construct for enhancing its presentation. These peptide constructs facilitated epitope presentation when loaded into the cytosol of TAP-deficient T2 cells, TAP-expressing melanoma cells and human dendritic cells. These findings may be of practical significance for the development of synthetic anti-cancer vaccines and in vitro immunization of CTL for adoptive immunotherapy.

Topics: Amino Acid Sequence; Antigen Presentation; Antigens, Neoplasm; ATP Binding Cassette Transporter, Subfamily B, Member 2; ATP-Binding Cassette Transporters; Cancer Vaccines; Endoplasmic Reticulum; Histocompatibility Antigens Class I; Humans; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Molecular Sequence Data; Neoplasm Proteins; Peptide Fragments; Protein Sorting Signals; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

There is now considerable evidence that human tumors often express antigens that render them susceptible to lysis by cytotoxic T lymphocytes (CTLs). These findings have raised hope for the development of cancer vaccines to trigger a tumor-specific immune response in cancer patients. To optimize the immunogenicity of cancer vaccines, it is important to improve the monitoring of the immune response. The use of tetrameric soluble major histocompatibility complex (MHC) class I/peptide complexes ("tetramers") to identify tumor-specific CTLs has shown that these novel reagents allow rapid and accurate analysis of human CTL responses in cancer patients. We have used fluorescence-driven cell sorting to clone tumor-specific CTLs after staining with tetrameric MHC class I/peptide complexes. Analysis of melanoma-infiltrated lymph nodes revealed that strong CTL responses often occur in vivo, and that the reactive CTLs have substantial proliferative and tumoricidal potential.

Topics: Antigens, Neoplasm; beta 2-Microglobulin; Epitopes, T-Lymphocyte; Flow Cytometry; Histocompatibility Antigens Class I; HLA Antigens; HLA-A2 Antigen; Humans; Lymph Nodes; MART-1 Antigen; Melanocytes; Melanoma; Monitoring, Immunologic; Neoplasm Proteins; T-Lymphocytes, Cytotoxic; Vitiligo

Dendritic cells (DCs) phagocytose apoptotic influenza-infected monocytes and cross-present influenza antigen to CD8+ T cells, generating a specific CTL response. We investigated whether apoptotic melanoma cells, presented by this mechanism, can lead to CTL responses to tumor-associated antigens and melanoma cells. Apoptotic HLA-A2- MEL-397 melanoma cells were internalized by HLA-A2+ immature monocyte-derived DCs but failed to induce maturation of DCs. When exposed to interleukin 6, interleukin 1beta, tumor necrosis factor alpha, and prostaglandin E2, DCs containing apoptotic MEL-397 cell material matured normally [cross-presenting DCs (cp-DCs)]. Autologous CD8+ CTL lines generated with cp-DCs produced tumor necrosis factor when stimulated with HLA-A2-binding immunodominant peptides from MelanA/MART1 and MAGE-3 (expressed by MEL-397 cells) but not tyrosinase (absent in MEL-397). T2 target cells loaded with the respective peptides were lysed by these cell lines, although to a lesser extent than by CTL lines generated in the presence of mature DCs and peptides from melanoma-associated h antigens. In contrast, lines generated with cp-DCs lysed HLA-A2+ MEL-526 melanoma cells or allogenic HLA-A2+ cp-DCs efficiently, whereas the CTL generated with DCs and peptides had little lytic activity. Mature DCs containing apoptotic tumor cells may thus represent an alternative approach for the therapy of malignant tumors.

Topics: Antigen Presentation; Antigens, Neoplasm; Apoptosis; CD8-Positive T-Lymphocytes; Cytotoxicity, Immunologic; Dendritic Cells; Epitopes, T-Lymphocyte; HLA-A2 Antigen; Humans; Immunodominant Epitopes; Immunotherapy; Lymphocyte Activation; MART-1 Antigen; Melanoma; Monophenol Monooxygenase; Neoplasm Proteins; Peptide Fragments; Phagocytosis; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

A novel antibody A103, which recognizes melan-A/MART-1, has been found to be more sensitive than the antibody HMB-45, which recognizes gp100, in melanocytic lesions of the skin and might therefore also be useful in the diagnosis of uveal and conjunctival melanocytic lesions. In this study we compared the staining characteristics of anti-melan-A, anti-S100 protein and HMB-45 in 13 conjunctival, 11 iris and 37 ciliary and choroidal malignant melanomas. The ciliary and choroidal melanomas comprised 13 spindle cell (10 spindle B and three spindle A), 14 mixed cell and 10 epithelioid cell tumours. In the conjunctival melanomas the diagnostic sensitivity was 100% for anti-S100 and anti-melan-A and 85% for HMB-45. In the iris melanomas the sensitivity was 100% for anti-S100 and anti-melan-A and 55% for HMB-45. A high staining intensity of anti-melan-A was particularly noticed in iris melanomas. In the choroidal malignant melanomas, the spindle cell and mixed cell types showed a sensitivity of only 69-79% with all three antibodies. In the epithelioid cell type the sensitivity was 80% for anti-S100 and 100% for HMB-45 and anti-melan-A. In conclusion, anti-melan-A was found to be a useful addition to antibody panels for ocular melanocytic lesions. Anti-melan-A has a higher sensitivity than HMB-45 in conjunctival and iris melanomas, but the sensitivity is similar to HMB-45 in choroidal melanomas. Anti-melan-A stains in a very similar pattern to anti-S100, but the staining intensity of anti-melan-A is higher than that of anti-S100 in iris melanoma.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Conjunctival Neoplasms; Diagnosis, Differential; Humans; Immunoenzyme Techniques; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; S100 Proteins; Uveal Neoplasms

The presence or absence of melanoma cells in human peripheral blood has recently been shown to be associated with disease prognosis, including overall survival. The detection of tyrosinase mRNA-positive circulating melanoma cells by reverse transcription-polymerase chain reaction (RT-PCR) has been limited to disseminated tumours expressing measurable amounts of this melanocyte-specific enzyme. To biologically classify both melanotic and amelanotic melanomas and to evaluate the clinical and prognostic relevance of tumour cell microcontamination, we examined autologous peripheral blood stem cell (PBSC) harvests from patients with advanced malignant melanoma prior to dose-escalated chemotherapy. To assay heterogeneous melanoma cell antigen expression, we developed a highly sensitive RT-PCR using four melanoma- and one tumour-associated antigen as molecular markers. Expression of the melanocyte-associated transcripts of tyrosinase, MART1/Melan-A, tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2) as well as the tumour-specific transcript of MAGE-3 was analysed by RT-PCR in PBSC harvests from 31 patients. Seven of the 31 PBSC harvests tested positive for one or more molecular markers: two patients for tyrosinase only, and one patient for MAGE-3 only, one patient for tyrosinase and MAGE-3, one for tyrosinase and MART1/Melan-A, and two patients for tyrosinase, MART1/Melan-A, TRP-2 and MAGE-3. mRNA-positive patients exhibited a significantly impaired overall survival (P = 0.0032), with a median survival of 3 months as opposed to 10 months in PBSC mRNA-negative patients. In conclusion, the use of this multiple-marker microcontamination assay allowed for molecular and prognostic classification of advanced malignant melanoma.

Topics: Adult; Antigens, Neoplasm; Biomarkers, Tumor; Female; Hematopoietic Stem Cells; Humans; Immunohistochemistry; Interferon Type I; Intramolecular Oxidoreductases; Male; MART-1 Antigen; Melanoma; Melanoma, Amelanotic; Middle Aged; Neoplasm Proteins; Neoplastic Cells, Circulating; Pregnancy Proteins; Prognosis; RNA, Messenger; RNA, Neoplasm; Sensitivity and Specificity; Tumor Cells, Cultured

To describe the clinical and histopathological features of a rare variant of naevoid melanoma, small cell melanoma, and discuss the histological differential diagnoses.. The clinical and histological features of cases of malignant melanoma with the histological features of small (non-Merkel like) melanoma were reviewed and documented. In addition, five cases had available material for immunohistochemistry and this was performed using antibodies to the S100 protein and melan-A, and the HMB-45 antibody.. There were 15 cases of small cell melanoma from 14 (10 female, four male) patients, aged between 30 and 77 (mean, 48.6) years. The trunk was the most common location. In more than half the cases, the provisional diagnosis was melanoma/borderline lesion. All shared similar histological appearances of an intraepidermal component of in situ melanoma and a dermal component of nests of cells with hyperchromatic nuclei and scanty cytoplasm, usually in tightly packed nests. All components (junctional and intradermal) of the lesions investigated by immunohistochemistry were positive both for S100 protein and melan-A. All junctional components were positive with HMB-45, but with variable staining of the dermal components with this antibody.. Small cell malignant melanoma is postulated to be a distinct histopathological entity and a rare variant of naevoid melanoma. Such lesions can be difficult to interpret and easily missed at scanning magnification because the cells of the dermal component mimic benign naevus cells.

Topics: Adult; Aged; Antigens, Neoplasm; Diagnosis, Differential; Female; Follow-Up Studies; Humans; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasm Proteins; S100 Proteins; Skin Neoplasms

With the discovery of increasing numbers of tumor antigens, there is a need to rapidly determine whether these antigens and the individual peptides they express are able to stimulate immune responses in vivo and thus, can be used to construct cancer vaccines. In this study we used the method of vaccine-induced immune response (VIIR) analysis to identify multiple immunogenic peptide epitopes derived from several melanoma associated antigens and presented by HLA-A*03, A*11 and B*07. Thirty-one patients with melanoma were immunized to a polyvalent vaccine containing multiple antigens, including MAGE-3, Melan A/MART-1, gp100 and tyrosinase. Their peripheral blood was tested for peptide-specific, vaccine-induced CD8+ T cell responses before and after immunization using an enzyme-linked immune spot (ELISPOT) assay with panels of peptides restricted by these three alleles. The peptides were selected for immunogenic potential based on their strong binding affinity in vitro to HLA-A*03, A*11 or B*07. Overall, 60% of the 20 peptides studied were recognized by at least one patient and 50% of the patients showed a vaccine-induced CD8+ T cell response to at least one peptide that matched their HLA specificity. We conclude that VIIR analysis is an effective strategy to directly identify immunogenic peptides that are good candidates for vaccine construction.

Topics: Alleles; Antigen Presentation; Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Epitopes, T-Lymphocyte; gp100 Melanoma Antigen; HLA Antigens; HLA-A Antigens; HLA-A11 Antigen; HLA-A3 Antigen; HLA-B7 Antigen; Humans; Immunotherapy, Adoptive; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Peptide Fragments; Vaccines, Combined

In a significant proportion of melanoma patients, CTL specific for the melan-A(26/7-35) epitope can be detected in peripheral blood using HLA-A2/peptide tetramers. However, the functional capacity of these CTL has been controversial, since although they prove to be effective killers after in vitro expansion, in some patients they have blunted activation responses ex vivo. We used phenotypic markers to characterize melan-A tetramer(+) cells in both normal individuals and melanoma patients, and correlated these markers with ex vivo assays of CTL function. Melanoma patients with detectable melan-A tetramer(+) cells in peripheral blood fell into two groups. Seven of thirteen patients had a CCR7(+) CD45R0(-) CD45RA(+) phenotype, the same as that found in some healthy controls, and this phenotype was associated with a lack of response to melan-A peptide ex vivo. In the remaining six patients, melan-A tetramer(+) cells were shifted toward a CCR7(-) CD45R0(+) CD45RA(-) phenotype, and responses to melan-A peptide could be readily demonstrated ex vivo. When lymph nodes infiltrated by melan-A-expressing melanoma cells were examined, a similar dichotomy emerged. These findings demonstrate that activation of melan-A-specific CTL occurs in only some patients with malignant melanoma, and that only patients with such active immune responses are capable of responding to Ag in ex vivo assays.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Cell Line, Transformed; Cell Movement; Epitopes, T-Lymphocyte; Female; Humans; Immunophenotyping; Lymph Nodes; Lymphocyte Subsets; Lymphocytes, Tumor-Infiltrating; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

A panel of three melanocyte differentiation antibodies has been compared with anti-S100 protein and NKIC3 in an assessment of benign and malignant melanocytic lesions. Anti-polyclonal S100 protein labelled all cases of primary cutaneous malignant melanoma, metastatic melanoma, desmoplastic melanoma and myxoid melanomas. In addition all benign and dysplastic naevi were positive. Conversely, HMB 45 was the least sensitive marker, labelling 24/31 primary cutaneous melanomas, 14/24 metastatic melanomas and only 1/6 desmoplastic melanomas. In the case of naevi, only junctional forms labelled consistently. Results for anti-melan-A and anti-tyrosinase were similar, although anti-tyrosinase proved slightly more sensitive in cases of malignant melanoma. NKIC3 revealed similar results to anti-tyrosinase, but had the disadvantage of reduced selectivity. It is concluded that anti-tyrosinase and anti-melan-A are useful additions to the panel of melanocytic monoclonal antibodies. In addition, both antibodies appear to have greater sensitivity for malignant melanoma than the conventionally used HMB 45 and could be considered as supportive markers to polyclonal anti-S100 protein in the diagnosis of malignant melanoma.

Topics: Antibodies, Monoclonal; Antibody Specificity; Antigens, Neoplasm; Biomarkers, Tumor; Cell Differentiation; Cell Transformation, Neoplastic; Humans; Immunohistochemistry; MART-1 Antigen; Melanocytes; Melanoma; Melanoma-Specific Antigens; Monophenol Monooxygenase; Neoplasm Proteins; S100 Proteins

The goal of tumor immunotherapy is to elicit immune responses against autologous tumors. It would be highly desirable that such responses include multiple T cell clones against multiple tumor antigens. This could be obtained using the antigen presenting capacity of dendritic cells (DCs) and cross-priming. That is, one could load the DC with tumor lines of any human histocompatibility leukocyte antigen (HLA) type to elicit T cell responses against the autologous tumor. In this study, we show that human DCs derived from monocytes and loaded with killed melanoma cells prime naive CD45RA(+)CD27(+)CD8(+) T cells against the four shared melanoma antigens: MAGE-3, gp100, tyrosinase, and MART-1. HLA-A201(+) naive T cells primed by DCs loaded with HLA-A201(-) melanoma cells are able to kill several HLA-A201(+) melanoma targets. Cytotoxic T lymphocyte priming towards melanoma antigens is also obtained with cells from metastatic melanoma patients. This demonstration of cross-priming against shared tumor antigens builds the basis for using allogeneic tumor cell lines to deliver tumor antigens to DCs for vaccination protocols.

Topics: Antigens, Neoplasm; Cancer Vaccines; Cell Differentiation; Cells, Cultured; Dendritic Cells; gp100 Melanoma Antigen; Humans; K562 Cells; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Staging; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

Current strategies for the immunotherapy of melanoma include augmentation of the immune response to tumor antigens represented by melanosomal proteins such as tyrosinase, gp100, and MART-1. The possibility that intentional targeting of tumor antigens representing normal proteins can result in autoimmune toxicity has been postulated but never demonstrated previously in humans. In this study, we describe a patient with metastatic melanoma who developed inflammatory lesions circumscribing pigmented areas of skin after an infusion of MART-1-specific CD8(+) T cell clones. Analysis of the infiltrating lymphocytes in skin and tumor biopsies using T cell-specific peptide-major histocompatibility complex tetramers demonstrated a localized predominance of MART-1-specific CD8(+) T cells (>28% of all CD8 T cells) that was identical to the infused clones (as confirmed by sequencing of the complementarity-determining region 3). In contrast to skin biopsies obtained from the patient before T cell infusion, postinfusion biopsies demonstrated loss of MART-1 expression, evidence of melanocyte damage, and the complete absence of melanocytes in affected regions of the skin. This study provides, for the first time, direct evidence in humans that antigen-specific immunotherapy can target not only antigen-positive tumor cells in vivo but also normal tissues expressing the shared tumor antigen.

Topics: Antigens, Neoplasm; Female; Humans; Immunotherapy, Adoptive; MART-1 Antigen; Melanocytes; Melanoma; Middle Aged; Neoplasm Proteins; Skin; Skin Neoplasms; T-Lymphocytes, Cytotoxic; Vitiligo

Reverse transcription-PCR (RT-PCR) amplification of melanoma cell-specific mRNA can detect melanoma cells in the peripheral blood of patients with malignant melanoma. We present a method to quantify mRNA coding for the melanoma-specific melanoma antigen recognized by T cells #1 (MART-1) in RNA isolated from peripheral blood.. To establish a calibration curve, we measured the concentration of MART-1 mRNA in SK-MEL-28 melanoma cells grown in vitro by competitive RT-PCR. Serial dilutions of these cells were used as calibrators in the assay. The assay was conducted by adding a fixed amount of a RNA internal standard to RNA isolated from either peripheral blood or the calibrators before RT-PCR amplification with MART-1 primers in a nested PCR design. The amount of MART-1 mRNA in blood samples was calculated from the calibration curve.. Addition of melanoma cells grown in vitro to blood from healthy donors demonstrated that the method can detect a single SK-MEL-28 melanoma cell in 1 mL of blood (1.5 x 10(-21) mol MART-1 mRNA/mL). MART-1 mRNA was observed in 4 of 12 blood samples from patients with malignant melanoma, at concentrations of 3-18 x 10(-21) mol MART-1 mRNA/mL of blood. No MART-1 mRNA was detected in blood samples from 25 controls without malignant melanoma. Intra- and interassay CVs were 15% (n = 12; mean = 44 x 10(-21) mol MART-1 mRNA/mL) and 33% (15 samples analyzed in two different analytical runs; mean = 30 x 10(-21) mol MART-1 mRNA/mL), respectively.. Our method is the first competitive RT-PCR assay for quantification of melanoma cells in blood samples that compensates for the variation of both the reverse transcription and PCR reactions. The method allows the inclusion of control samples for continuous quality assessment.

Topics: Adult; Antigens, Neoplasm; Biomarkers, Tumor; Calibration; Female; Humans; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Metastasis; Neoplasm Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured

Melanoma inhibitory activity (MIA) is a small soluble protein secreted by malignant melanoma cells and chondrocytes. Prior studies suggested that MIA expression was relatively tissue-specific, making it a potentially useful marker for melanoma. The current investigations sought to more clearly define the range of tumor/tissue-types where MIA is expressed, compared with expression of 4 other potential melanoma marker genes (tyrosinase melanoma antigen recognized by T cells [MART-1/MelanA], gp100, and melanoma growth-stimulatory activity [MGSA/Gro alpha]). Expression of these genes was assayed by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry in 23 melanoma tumor specimens and in 25 additional nonmelanoma or nonmalignant specimens. MIA, tyrosinase, and MGSA were expressed in most melanoma specimens. Specificity was highest for MART-1, followed by MIA and tyrosinase. Increasing the number of cycles of amplification from 35 to 40 increased sensitivity but decreased specificity of most markers, though MIA was relatively robust. MIA mRNA was also detected in carcinomas of the colon, ovary, kidney, and head/neck, as well as in normal laryngeal epithelium. Although MIA discriminated melanoma from nonmelanoma at least as well as tyrosinase, no single mRNA marker had accuracy greater than 71%, raising potential concern about application of these particular mRNA markers to the minimal disease setting. HUM PATHOL 31:1381-1388.

Topics: Adult; Aged; Antigens, Neoplasm; Biomarkers, Tumor; DNA Primers; DNA, Neoplasm; Extracellular Matrix Proteins; Female; gp100 Melanoma Antigen; Humans; Laryngeal Mucosa; Male; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Middle Aged; Neoplasm Proteins; Proteins; Receptors, Cytokine; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Skin Neoplasms; Tumor Cells, Cultured

Typically, melanocytic nevi "mature" (i.e., exhibit a morphologic shift to smaller or spindle cells with progressive depth in the dermis). In contrast, most malignant melanomas (conventional MMs) lack maturation, and are composed of large pleomorphic cells throughout. The authors describe a series of melanomas with paradoxical maturation mimicking the pattern of nevi. Seventeen primary invasive melanomas with paradoxical maturation (IMPs), two epidermotropic metastatic melanomas with maturation (EMMMs), 13 compound nevi (CN), and 14 conventional MMs without apparent maturation were analyzed by histologic, cytomorphometric, and immunohistochemical techniques. With increasing dermal depth, both CN and IMPs had smaller nuclear and cellular areas, and decreased expression of Ki-67, glycoprotein (gp)100 (with HMB-45), and tyrosinase. IMPs had significant differences from conventional MMs; namely, smaller nuclear and cytoplasmic areas (deep), and decreased expression of Ki-67 (superficial and deep), gp100 (deep), and tyrosinase (deep). IMPs also had notable differences from CN: namely, larger nuclear and cellular areas, more confluence, more mitotic figures, increased Ki-67 and gp100 expression in both the superficial and deep portions, and more melanin (deep). The two EMMMs exhibited histologic and immunohistochemical features similar to the primary IMPs. IMP, because of its mimicry of nevus, can present a diagnostic hazard. The authors propose histologic, morphometric, and immunohistochemical criteria that facilitate recognition and accurate diagnosis of this unusual variant of melanoma.

Topics: Adolescent; Adult; Aged; Antigens, Neoplasm; Biomarkers, Tumor; Cell Differentiation; Child; Diagnosis, Differential; Female; gp100 Melanoma Antigen; Humans; Image Processing, Computer-Assisted; Immunohistochemistry; Ki-67 Antigen; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Membrane Glycoproteins; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Nevus, Intradermal; Nevus, Pigmented; Skin Neoplasms

A 47-year-old woman developed metastatic melanoma to the right ovary 14 years after the enucleation of the right eye for a choroidal spindle cell melanoma. An immunohistochemical study was performed on paraffin sections of both primary and metastatic melanoma specimens to identify markers of both aggressive phenotype and metastatic potential with particular attention to the anomalous expression of cytokeratin intermediate filament proteins. Neoplastic cells of both primary and metastatic tumors immunostained positively for S-100, HMB45, MART-1, and vimentin antibodies, but they were negative for cytokeratins 1-19, 8, 18, and 8,18; <10% of neoplastic cells in both the primary and the metastatic melanomas immunostained for Ki-67 proliferating antigen using MIB-1 antibody. We speculate that the indolent behavior of this ovarian metastasis is reflected by the absence of coexpression of cytokeratins 8 and 18 with vimentin. This case supports the practical value of using this panel of antibodies to evaluate the aggressive potential of uveal melanomas.

Topics: Antigens, Neoplasm; Carcinoma; Choroid Neoplasms; Female; Humans; Immunohistochemistry; Immunophenotyping; Intermediate Filament Proteins; Keratins; Ki-67 Antigen; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Middle Aged; Neoplasm Proteins; Ovarian Neoplasms; S100 Proteins; Time Factors; Vimentin

The functional characteristics of CD8+ T cells specific for melanoma antigens (MAs) have often been defined after in vitro culture using nonprofessional antigen-presenting cells. We have examined CD8+ T-cell immunity to MAs and a viral antigen (influenza) in uncultured T cells of healthy donors and melanoma patients using autologous, mature, monocyte-derived dendritic cells (DCs) pulsed with peptide antigens and viral vectors. Antigen-specific IFN-gamma-producing T cells reactive with HLA-A*0201-restricted peptides from four melanoma antigens (MelanA/MART-1, MAGE-3, tyrosinase, and gp100) were detected only at low frequencies (<30 per 2 x 10(5) peripheral blood mononuclear cells for each of the MAs) from HLA-A2.1-positive healthy donors (n = 12) and patients with stages III/IV melanoma (n = 8). Detection of MA-specific, but not influenza matrix peptide (Flu-MP)-specific, T cells required a high concentration (10 microg/ml) of the peptide in this assay. Furthermore, these T cells did not recognize endogenously processed antigen on tumor cell lines or cells infected with viral vectors capable of expressing MAs. The use of autologous, mature DCs led to a significant increase in the number of Flu-MP, but not MA-specific, T cells in 16-h ELISPOT assays for both melanoma patients and healthy donors. In 1-week cocultures with DCs pulsed with 10 microg/ml peptide, MelanA/MART-1-specific T cells did not readily proliferate or differentiate into lytic effectors, in contrast to strong influenza-specific lytic responses. Therefore, despite distinct memory responses to influenza antigens, melanoma patients and healthy controls have a paucity of MA-reactive memory T cells, failing to rapidly generate IFN-gamma-secreting lytic effectors in short-term assays, even when stimulated by DCs.

Topics: Antigens; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Coculture Techniques; Dendritic Cells; gp100 Melanoma Antigen; HLA-A Antigens; Humans; Immunologic Memory; Interferon-gamma; Leukocytes, Mononuclear; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasms; Peptides; T-Lymphocytes; Time Factors; Tumor Cells, Cultured

Immunogenic peptides of human tumor Ag have been used to generate antigen-specific CTL. However, the vast majority of these peptide-specific CTL clones are of low avidity and are peptide, but not tumor, reactive. Peptide-MHC tetramers have been shown to bind specific TCRs with sufficient affinity to be useful reagents for flow cytometry. In this paper we demonstrate that peptide-MHC tetramers can also be used to selectively identify high avidity tumor-reactive CTL and enrich, from a heterogeneous population, the subpopulation of peptide-reactive T cells that can lyse tumor targets. The melanoma proteins, MART-1 and gp100, were used to induce potentially tumor-reactive T cells, and the intensity of T cell staining by TCR binding of specific peptide-MHC tetramers was assessed. A range of fluorescence intensity was detected, and the magnitude of tetramer binding was correlated with T cell avidity. The population of peptide-reactive T cells was phenotypically similar with regard to expression of TCR and adhesion molecules, suggesting that this differential avidity for tumor cells reflected differential affinity of the TCR for its peptide-MHC ligand. Sorting, cloning, and expansion of tetramerhigh CTL from a heterogeneous population of peptide-stimulated PBMCs enabled rapid selection of high avidity tumor-reactive CTL clones, which retained their functional and tetramerhigh phenotype on re-expansion. These results demonstrate that the avidity of a T cell for its tumor target is due to the specific affinity of the TCR for its peptide-MHC ligand, that this interaction can be described using peptide-MHC tetramers and used to isolate high avidity tumor-reactive CTL.

Topics: Antigen-Presenting Cells; Antigens, Neoplasm; Cell Line; Cell Separation; Clone Cells; Cytotoxicity Tests, Immunologic; Epitopes, T-Lymphocyte; gp100 Melanoma Antigen; HLA-A2 Antigen; Humans; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Oligopeptides; Staining and Labeling; T-Lymphocyte Subsets; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

Previous studies have shown that substitution of single amino acid residues in human Melan-A immunodominant peptides Melan-A27-35 and Melan-A26-35 greatly improved their binding and the stability of peptide/HLA-A*0201 complexes. In particular, one Melan-A peptide analogue was more efficient in the generation of Melan-A peptide-specific and melanoma-reactive CTL than its parental peptide in vitro from human PBL. In this study, we analyzed the in vivo immunogenicity of Melan-A natural peptides and their analogues in HLA-A*0201/Kb transgenic mice. We found that two human Melan-A natural peptides, Melan-A26-35 and Melan-A27-35, were relatively weak immunogens, whereas several Melan-A peptide analogues were potent immunogens for in vivo CTL priming. In addition, induced Melan-A peptide-specific mouse CTL cross-recognized natural Melan-A peptides and their analogues. More interestingly, these mouse CTL were also able to lyse human melanoma cell lines in vitro in a HLA-A*0201-restricted, Melan-A-specific manner. Our results indicate that the HLA-A*0201/Kb transgenic mouse is a useful animal model to perform preclinical testing of potential cancer vaccines, and that Melan-A peptide analogues are attractive candidates for melanoma immunotherapy.

Topics: Alleles; Animals; Antigens, Neoplasm; Arginine; Cell Line; Cytotoxicity, Immunologic; Epitopes, T-Lymphocyte; H-2 Antigens; HLA-A Antigens; Humans; Injections, Subcutaneous; Leucine; Lymphocyte Activation; MART-1 Antigen; Melanoma; Mice; Mice, Transgenic; Neoplasm Proteins; Oligopeptides; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Helper-Inducer; Tumor Cells, Cultured

Topics: Antigens, Neoplasm; Cancer Vaccines; Eye Diseases; Humans; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; Vaccination

HMB-45, an antibody directed against a premelanosome glycoprotein, has thus far been considered the most specific antibody for the immunocytochemical substantiation of the diagnosis of malignant melanoma (MM). A recently described antigen, MART-1, is a transmembrane protein that is present in normal melanocytes and widely expressed in MM. Antibodies to MART-1 have recently become commercially available. Both HMB-45 and MART-1 form the basis of ongoing immunotherapy protocols at the National Institutes of Health/National Cancer Institute.. The authors evaluated 207 lesions from 160 patients with metastatic MM procured via fine-needle aspiration (FNA) for expression of MART-1 (clone M2-7C10) and HMB-45 prior to commencement of immunotherapy. FNAs were performed on subcutaneous soft tissue masses (190 lesions), lung (8 lesions), liver (5 lesions), pancreas (3 lesions), and brain (1 lesion). To test the specificity of the monoclonal antibody directed against MART-1, the authors evaluated its reactivity in normal tissues as well as in various nonpigmented neoplasms that are often included in the differential diagnosis of MM.. Of all lesions tested, 13 (6%) were negative for both MART-1 and HMB-45. Of all patients tested, 20% had 1 or more lesions that were non-immunoreactive with HMB-45, whereas only 10% had 1 or more lesions that were nonimmunoreactive for MART-1. Eight percent of the lesions tested were negative for MART-1 only, whereas 16% of lesions tested were negative for HMB-45 only. In 35% of the lesions, MART-1 stained more cells than HMB-45. In 13%, MART-1 stained fewer cells than HMB-45, and in 52% both antibodies stained an equivalent number of cells. All samples of normal tissue were negative for staining with MART-1, as were the nonpigmented lesions tested. Melanocytes in normal skin samples stained positively for MART-1.. The MART-1 antibody is a superior immunohistochemical marker for the diagnosis of MM. It has the potential to become the preferred antibody over HMB-45 for the diagnosis of metastatic MM in FNA material, as MART-1 stains a higher percentage of lesions in a higher percentage of patients than does HMB-45.

Topics: Antibodies, Monoclonal; Antibodies, Neoplasm; Antigens, Neoplasm; Biopsy, Needle; Diagnosis, Differential; Humans; Immunoenzyme Techniques; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; Paraffin Embedding; Sensitivity and Specificity; Soft Tissue Neoplasms

Previous attempts to treat human malignancies by adoptive transfer of tumor-specific CTLs have been limited by the difficulty of isolating T cells of defined antigen specificity. The recent development of MHC class I/antigenic peptide tetrameric complexes that allow direct identification of antigen-specific T cells has opened new possibilities for the isolation and in vitro expansion of tumor-specific T cells. In the present study, we have derived polyclonal monospecific cell lines from circulating Melan-A-specific CTL precursors of HLA-A*0201+ melanoma patients by combining stimulation with recently identified peptide analogues of the immunodominant epitope from the melanoma-associated antigen Melan-A with staining with fluorescent HLA-A*0201/Melan-A peptide tetramers. In vitro expansion of antigen-specific CD8+ T cells was monitored by flow cytometry with the fluorescent tetramers and anti-CD8 monoclonal antibody. This analysis revealed that Melan-A 26-35 peptide analogues were much more efficient than the parental peptides in stimulating a rapid in vitro expansion of antigen-specific CD8+ T cells. These cells were then isolated by tetramer-guided cell sorting and subsequently expanded in vitro by mitogen stimulation. The resulting polyclonal but monospecific CTLs fully cross-recognized the parental peptides and were able to efficiently lyse Melan-A-expressing tumor cells. Altogether, these results pave the way to a molecularly defined approach to antigen-specific adoptive transfer therapy of cancer.

Topics: Antigens, Neoplasm; Biotinylation; Cell Line; Cell Separation; Cytotoxicity, Immunologic; Flow Cytometry; Fluorescent Antibody Technique, Indirect; HLA-A2 Antigen; Humans; Immunodominant Epitopes; Immunotherapy, Adoptive; Interleukin-2; Lymphocyte Activation; Macromolecular Substances; MART-1 Antigen; Melanoma; Neoplasm Proteins; Peptide Fragments; Recombinant Fusion Proteins; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

Topics: Adrenal Gland Neoplasms; Antigens, Neoplasm; Biomarkers, Tumor; Cancer Vaccines; gp100 Melanoma Antigen; HLA-A2 Antigen; Humans; Immunoenzyme Techniques; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; National Institutes of Health (U.S.); Neoplasm Proteins; United States

Immunization with tumor-associated antigen pulsed dendritic cells (DC) has been shown to elicit both protective and therapeutic antitumor immunity in a variety of animal models and is currently being investigated for the treatment of cancer patients in clinical trials. In this study we show that DC can be generated from peripheral blood mononuclear cells of healthy donors as well as breast and melanoma cancer patients using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-13 (IL-13) and that these DC have many of the same characteristics as DC differentiated using GM-CSF and IL-4. The DC generated in GM-CSF and IL-13 are CD14- and express high levels of the cell surface markers CD86, HLA-DR, and CD58, as do DC generated in GM-CSF and IL-4. The purity and yield of both DC populations are not significantly different. Furthermore, both populations of DC are effective at presentation of alloantigen as determined in a mixed lymphocyte response, and both are able to process and present soluble tetanus toxoid antigen to CD4+ T cells. Because we are interested in the generation of DC for antigen-specific cytotoxic T lymphocyte (CTL) generation, we compared the ability of peptide-pulsed DC differentiated in GM-CSF and IL-4 versus GM-CSF and IL-13 for the generation of influenza and MART-1 specific CTL. Both populations of DC induced CD3+ CD8+ CD4- and CD56- CTL, which could lyse the appropriate targets in an antigen-specific manner. Finally, both GM-CSF and IL-4 DC and GM-CSF and IL-13 DC yielded similar beta galactosidase expression levels after transduction with recombinant adenovirus containing the LacZ gene. These results suggest that DC generated in GM-CSF and IL-13 may be useful for immunotherapy and gene therapy protocols.

Topics: Antigens, Neoplasm; Breast Neoplasms; Cell Differentiation; Dendritic Cells; Genetic Therapy; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunophenotyping; Immunotherapy; Interleukin-13; Interleukin-4; Leukocytes, Mononuclear; Lymphocyte Culture Test, Mixed; MART-1 Antigen; Melanoma; Neoplasm Proteins; Neoplasms; T-Lymphocytes, Cytotoxic

In recent years, large discrepancies were described in the success rate of the tyrosinase reverse transcription polymerase chain reaction (RT-PCR) for detecting melanoma cells in the peripheral blood of melanoma patients. We present a quality control study in which we analysed the reproducibility of detection of tyrosinase and MART-1 transcripts in 106 blood samples from 68 melanoma patients (mainly stages III and IV). With this study, we aimed to improve insight in the reproducibility of a RT-PCR for the detection of (minimal) amounts of circulating melanoma cells. We performed two reverse transcriptions on each mRNA sample and performed tyrosinase and MART-1 nested PCRs in duplicate per cDNA sample. Thus, four tyrosinase and four MART-1 measurements were performed per blood sample. In our study, the majority of blood samples was negative for tyrosinase (80%) or MART-1 (66%). Only four samples were positive in all four determinations for tyrosinase and seven for MART-1. Variable results (1-3 times positive results) were obtained for tyrosinase and MART-1 in 16% and 27% respectively. MART-1 PCR had a better performance than tyrosinase PCR. Sensitivity increased when both markers were used. We reasoned that the low number of melanoma marker PCR-positive blood samples can be explained by differences in mRNA quality. By using real-time quantitative PCR, we found that this was not the case: amplification of porphobilinogen deaminase (PBGD), a low copy household gene, was not different in blood samples in which a melanoma marker was not detected from groups in which this marker was detected more or less consistently (1-4 times). When applying real-time quantitative PCR for tyrosinase and MART-1, we found that a low amount of SK-MEL-28 cell equivalents was present in the blood of melanoma patients, with a higher number of equivalents in the group with a consistently positive result. We conclude that low reproducibility of a repeated assay for the detection of circulating melanoma cells is not caused by differences in mRNA quality between the samples, but due to low numbers of amplifiable target mRNA molecules in the mRNA sample. Use of more than one marker and repetition of the assay will increase the probability of finding positive PCR results.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Female; Gene Amplification; Humans; Hydroxymethylbilane Synthase; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Neoplastic Cells, Circulating; Quality Control; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic

Peptides derived from the melanoma-associated MART-1/Melan-A antigen are currently implemented in immunotherapy for inducing or augmenting T-cell responses directed against peptides expressed by autologous tumor cells in HLA-A2+ patients with melanoma. Here, we describe the specificity of the T-cell clone SK29-FFM1.1, which secretes GM-CSF in response to a panel of synthetic MART-1/Melan-A-derived peptides, including the naturally presented ILTVILGVL(32-40), but exhibits cytotoxicity and IFN-gamma secretion exclusively to the MART-1/Melan-A derived peptide AAGIGILTV(27-35). In addition, cytotoxic T-lymphocyte (CTL) clone SK29-FFM1.1 recognizes 3 different naturally processed and presented peptides on HLA-A2+ MART-1/Melan-A+ melanoma cells, as defined by cytotoxicity and IFN-gamma and GM-CSF secretion. Processing and presentation of MART-1/Melan-A peptides appears to be different in cells of non-melanocytic origin, as shown by the characterization of naturally presented peptides displayed by HLA-A2+ colorectal cancer cells transduced with a MART-1/Melan-A gene-containing retrovirus. Our data suggest that multiple epitopes, including ILTVILGVL and different isoforms of AAGIGILTV derived from MART-1/Melan-A may be naturally presented by melanoma cells to the immune system.

Topics: Amino Acid Sequence; Antigens, Neoplasm; Clone Cells; Cytotoxicity, Immunologic; HLA-A2 Antigen; Humans; Interferon-gamma; MART-1 Antigen; Melanoma; Neoplasm Proteins; Protein Isoforms; Receptors, Antigen, T-Cell, alpha-beta; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes; T-Lymphocytes, Cytotoxic; Transfection; Tumor Cells, Cultured

We identified circulating CD8+ T-cell populations specific for the tumor-associated antigens (TAAs) MART-1 (27-35) or tyrosinase (368-376) in six of eleven patients with metastatic melanoma using peptide/HLA-A*0201 tetramers. These TAA-specific populations were of two phenotypically distinct types: one, typical for memory/effector T cells; the other, a previously undescribed phenotype expressing both naive and effector cell markers. This latter type represented more than 2% of the total CD8+ T cells in one patient, permitting detailed phenotypic and functional analysis. Although these cells have many of the hallmarks of effector T cells, they were functionally unresponsive, unable to directly lyse melanoma target cells or produce cytokines in response to mitogens. In contrast, CD8+ T cells from the same patient were able to lyse EBV-pulsed target cells and showed robust allogeneic responses. Thus, the clonally expanded TAA-specific population seems to have been selectively rendered anergic in vivo. Peptide stimulation of the TAA-specific T-cell populations in other patients failed to induce substantial upregulation of CD69 expression, indicating that these cells may also have functional defects, leading to blunted activation responses. These data demonstrate that systemic TAA-specific T-cell responses can develop de novo in cancer patients, but that antigen-specific unresponsiveness may explain why such cells are unable to control tumor growth.

Topics: Antigens, Neoplasm; Antineoplastic Agents; CD28 Antigens; CD57 Antigens; CD8-Positive T-Lymphocytes; Female; Flow Cytometry; gp100 Melanoma Antigen; HLA-A2 Antigen; Humans; Hyaluronan Receptors; Leukocyte Common Antigens; Lymphatic Metastasis; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Peptide Fragments; Receptors, IgG; Recombinant Proteins; T-Lymphocytes

The purpose of this study was to test different malignant non-melanocytic tumours with the commercially available antibody Melan-A to examine its diagnostic specificity and to compare the S100, Melan-A and HMB-45 reactivity in various melanocytic lesions.. Seventy-three benign and malignant melanocytic lesions and 31 cases of non-melanocytic tumours, sarcomas, carcinomas and carcinoids, were selected. Immunohistochemical staining of paraffin sections, following a high temperature antigen unmasking technique, was performed. Melan-A stains junctional and dermal melanocytes in all benign melanocytic lesions with the exception of neuro-naevoid areas. The epithelioid and the spindle cells in malignant melanomas did not show considerable difference in their Melan-A reactivity. The predominantly spindle cell type mucosal melanomas contained more Melan-A-positive cells than HMB-45-positive cells and similar results were observed in metastatic malignant melanomas. In desmoplastic melanomas the positivity of Melan-A was not consistent. None of the sarcomas, carcinomas and carcinoids expressed Melan-A. Almost all soft tissue tumours, except for two malignant gastrointestinal stromal tumours, were unreactive for HMB-45. These two cases did not react with Melan-A antibody.. Melan-A is a useful additional marker to differentiate non-melanocytic tumours from primary or metastatic melanoma. In melanocytic lesions the Melan-A staining pattern is similar to S100, but seems to be more specific. In desmoplastic melanomas, however, the variable Melan-A staining further necessitated detailed histological examination and the use of the S100 reaction.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Carcinoid Tumor; Carcinoma; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; S100 Proteins; Sarcoma; Skin Neoplasms; Soft Tissue Neoplasms

The tumor-associated-Ag MART-1 is expressed by most human melanomas. The genes encoding an alphabeta TCR from a MART-1-specific, HLA-A2-restricted, human T cell clone have been efficiently transferred and expressed in human PBL. These retrovirally transduced PBL cultures were MART-1 peptide reactive, and most cultures recognized HLA-A2+ melanoma lines. Limiting dilution clones were generated from three bulk transduced PBL cultures to investigate the function of individual clones within the transduced cultures. Twenty-nine of 29 CD8+ clones specifically secreted IFN-gamma in response to T2 cells pulsed with MART-1(27-35) peptide, and 23 of 29 specifically secreted IFN-gamma in response to HLA-A2+ melanoma lines. Additionally, 23 of 29 CD8+ clones lysed T2 cells pulsed with the MART-1(27-35) peptide and 15 of 29 lysed the HLA-A2+ melanoma line 888. CD4+ clones specifically secreted IFN-gamma in response to T2 cells pulsed with the MART-1(27-35) peptide. TCR gene transfer to patient PBL can produce CTL with anti-tumor reactivity in vitro and could potentially offer a treatment for patients with metastatic melanoma. This approach could also be applied to the treatment of other tumors and viral infections. Additionally, TCR gene transfer offers unique opportunities to study the fate of adoptively transferred T cells in vivo.

Topics: Antigens, Neoplasm; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Clone Cells; Cytotoxicity Tests, Immunologic; Gene Transfer Techniques; Genetic Vectors; Humans; Immunophenotyping; MART-1 Antigen; Melanoma; Moloney murine leukemia virus; Neoplasm Proteins; Receptors, Antigen, T-Cell, alpha-beta; T-Lymphocyte Subsets; Tumor Cells, Cultured

Conflicting results have been obtained by various research groups using tyrosinase reverse transcription-polymerase chain reaction (RT-PCR) for detecting micrometastases in the blood of melanoma patients, with positive results ranging from 0 to 100% in disseminated melanoma. Methodological differences in the processing of blood samples may in part account for these discrepancies. The aim of this study was to standardize and optimize the experimental conditions for RT-PCR detection of melanoma cells in peripheral blood. We analysed the effect of different parameters of sample processing on the sensitivity of the tyrosinase RT-PCR using peripheral blood spiked with defined numbers of SKMEL28 melanoma cells. Purification of the mononuclear cell fraction using a Ficoll gradient with a density of 1.077 g/mL prior to RNA isolation gave the highest sensitivity, with the detection of two SKMEL28 cells in 5 mL of blood. In addition, the RNA isolation method and the kind of RT enzyme used had a significant impact on the sensitivity and reproducibility of tyrosinase detection, whereas variations in the PCR conditions had only a minor influence. Furthermore, we showed that amplification of MelanA in addition to tyrosinase resulted in an approximately 10% enhanced sensitivity of melanoma cell detection, whereas gp100/pMel17 and MUC18 gene products were also detected in blood from non-melanoma patients. MelanA could serve as a sensitive marker in addition to tyrosinase for detecting micrometastases.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Cell Separation; Humans; MART-1 Antigen; Melanoma; Monophenol Monooxygenase; Neoplasm Proteins; Neoplastic Cells, Circulating; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sensitivity and Specificity; Skin Neoplasms; Tumor Cells, Cultured

An improved protocol for reverse transcription-polymerase chain reaction (RT-PCR), amplifying tyrosinase and MelanA/MART-1 mRNA from peripheral blood, was used to test 340 blood samples from 225 patients with malignant melanoma for the presence of circulating tumour cells. Positive results for tyrosinase or MelanA were obtained in 19% of patients in stage I (n = 74), 31% in stage II (n = 45), 29% in stage III (n = 48) and 52% in stage IV (n = 58). Amplification of MelanA in addition to tyrosinase resulted in a 30% enhanced sensitivity of melanoma cell detection compared with amplification of tyrosinase alone. The sensitivity was further enhanced by analysis of at least two blood samples per patient and performing at least two PCR analyses per sample. During a median follow-up of 4 months, patients with a positive PCR showed a 2. 4-fold increased risk for relapse compared with PCR-negative patients. These data indicate that the detection of circulating melanoma cells in peripheral blood using our optimized protocol for RT-PCR correlated with the clinical stage of disease and is therefore likely to be a prognostic marker for recurrence. MelanA is a sensitive additional marker to tyrosinase in detecting micrometastases using RT-PCR.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Chi-Square Distribution; Female; Humans; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Neoplastic Cells, Circulating; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sensitivity and Specificity; Skin Neoplasms

Melanoma-reactive human cytotoxic T lymphocytes (CTLs) mediate tumor regression in vivo through specific recognition of MHC-associated peptide epitopes, many of which are encoded by the melanocytic tissue differentiation proteins gp100/Pme117 and MART-1/Melan-A. Vaccines using these peptides may induce protective or therapeutic immunity against melanoma. Rational design of such approaches is aided by a clear understanding of the identity of these antigenic peptides; however, most CTL epitopes described to date were identified indirectly. Especially where these peptides may be used in human clinical trials for the treatment or prevention of cancer, there is substantial need for direct evaluation of HLA-A*0201-associated peptides from MART-1 and gp100 that are naturally processed and presented. To that end, we have isolated peptides directly from HLA-A*0201 molecules of human melanoma cells and have determined that naturally processed epitopes for HLA-A*0201-restricted, melanoma-reactive CTLs include the nonamers MART-1(27-35) (AAGIGILTV), gp100(154-162) (KTWGQYWQV), gp100(209-217) (ITDQVPFSV) and gp100(280-288) (YLEPGPVTA) and the decamer gp100(476-485) (VLYRYGSFSV). Among these, the one that appears to be most abundant at the cell surface is gp100(154-162) (KTWGQYWQV). The others are among the less abundant peptides. HLA-A*0201-restricted CTLs from one melanoma patient who has survived metastatic disease recognized MART-1(27-35) (AAGIGILTV), gp100(280-288) (YLEPGPVTA) and gp100(154-162) (KTWGQYWQV) and were cross-reactive on longer peptides that contained these nonamer sequences. These peptides, identified by both an indirect genetic approach and by a direct peptide approach, can be used for tumor vaccine strategies with confidence that they are identical to the naturally processed peptide epitopes presented at the surface of melanoma cells in association with HLA-A*0201 molecules.

Topics: Antigens, Neoplasm; Epitopes, T-Lymphocyte; Evaluation Studies as Topic; gp100 Melanoma Antigen; HLA-A Antigens; Humans; MART-1 Antigen; Mass Spectrometry; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Peptide Fragments; Peptides; Protein Processing, Post-Translational; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

Melanoma antigen (MA)-specific vaccination strongly enhances antitumor reactivity in vivo and is capable of producing strong cytotoxic T lymphocyte responses in vitro. Furthermore, specific human leukocyte antigen-restricted T cell activation is hypothesized to occur in response to peptide-based immunotherapy, which may lead to the preferential killing of tumor cells bearing the relevant MA. The development of melanoma antigen-loss variants may subsequently occur in vivo.. Analysis of 532 melanoma lesions from 204 patients was performed on fine-needle aspiration biopsy specimens. Lesions were graded for the expression of the MAs gp100 and MART-1 with use of immunocytochemistry. A total of 351 melanoma lesions were divided into cohorts on the basis of the treatment received. The pretreatment group (n = 175) consisted of lesions obtained before any form of gp100 immunotherapy, with the posttreatment group (n = 176) consisting of lesions obtained after vaccination with a modified gp100 epitope, gp209-2M +/- interleukin 2 (IL-2).. The percentage of lesions not expressing the gp100 antigen is greater than the percentage not expressing MART-1 (26% vs 14%). The frequency of lesions with high expression (> 75%) of gp100 significantly decreased with therapy (47% vs 34%) and conversely negative lesions increased (18% vs 29%). Treatment of lesions with peptide alone (no IL-2) revealed a significant decrease in gp100 expression (47% vs 32%), enhanced with the addition of IL-2 to therapy (47% vs 35%). The expression of MART-1 remained essentially unchanged unless IL-2 was added (54% vs 54%, MART-1 peptide alone, 54% vs 43%, MART-1 peptide + IL-2). Of 94 patients (181 lesions) assessed for gp100 expression before treatment, 10 patients responded to therapy. Pretreatment lesions in responding patients expressed some level of gp100 in all cases compared with 27% of nonresponding lesions, which were negative for gp100 expression. CONCLUSIONS. This study indirectly demonstrates that vaccination with an MA-derived peptide can result in immune selection in vivo. Furthermore, it provides strong immunologic evidence for the specificity of MA vaccines and to the relevance of MA expression in predicting the response to vaccination.

Topics: Antigens, Neoplasm; Biopsy, Needle; HLA-A2 Antigen; Humans; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; T-Lymphocytes, Cytotoxic; Vaccination

Reverse transcriptase (RT) polymerase chain reaction (PCR) with multiple markers has been demonstrated to be highly sensitive in detecting circulating cells from patients with malignant melanoma (MM). We evaluated the clinical significance of the presence in peripheral blood of specific PCR-positive mRNA markers as an expression of circulating melanoma cells.. Total cellular RNA was obtained from the peripheral blood of 235 patients with either localized (n = 154) or metastatic (n = 81) melanoma. We performed RT-PCR using tyrosinase, p97, MUC18, and MelanA/MART1 as gene markers. The PCR products were analyzed by gel electrophoresis and Southern blot hybridization. In addition, 20 healthy subjects and 21 patients with nonmelanoma cancer were used as negative controls.. Although detected at various levels among assessable patients, each mRNA marker was significantly correlated with disease stage. A significant correlation with disease stage was demonstrated for patients who were positive to all four markers (P < .0001) or to at least three markers (P < .001). Univariate analysis showed a significant correlation between risk of recurrence (evaluated in stage I, II, and III patients) and increasing number of PCR-positive markers (P = .0002). Logistic regression multivariate analysis indicated that each single marker (except tyrosinase) and, more especially, the presence of four PCR-positive markers remained statistically independent prognostic factors for tumor progression.. Our data establish the existence of a significant correlation among clinical stages, tumor progression, and presence of circulating melanoma-associated antigens in peripheral blood of MM patients. Preliminary assessment of a subset of patients with a higher risk of recurrence needs longer follow-up and further studies to define the role of RT-PCR in monitoring MM patients.

Topics: Adult; Aged; Aged, 80 and over; Antigens, CD; Antigens, Neoplasm; Biomarkers, Tumor; CD146 Antigen; Disease Progression; Female; Humans; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Membrane Glycoproteins; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Neoplastic Cells, Circulating; Neural Cell Adhesion Molecules; Polymerase Chain Reaction; RNA-Directed DNA Polymerase; RNA, Messenger; Sensitivity and Specificity; Skin Neoplasms

Topics: Antigens, Neoplasm; Biopsy; Humans; Immunohistochemistry; Lymph Node Excision; MART-1 Antigen; Melanoma; Monophenol Monooxygenase; Neoplasm Proteins

It is not known if immune response to T cell-defined human histocompatibility leukocyte antigen (HLA) class I-restricted melanoma antigens leads to an expanded peripheral pool of T cells in all patients, affects cytotoxic T lymphocyte (CTL) generation, and correlates with anti-tumor response in metastatic lesions. To this end, a limiting dilution analysis technique was developed that allowed us to evaluate the same frequency of peptide-specific T cells as by staining T cells with HLA-peptide tetrameric complexes. In four out of nine patients, Melan-A/Mart-1(27-35)-specific CTL precursors (CTLp) were >/=1/2,000 peripheral blood lymphocytes and found mostly or only in the CD45RO(+) memory T cell subset. In the remaining five patients, a low (<1/40,000) peptide-specific CTLp frequency was measured, and the precursors were only in the CD45RA(+) naive T cell subset. Evaluation of CTL effector frequency after bulk culture indicated that peptide-specific CTLs could be activated in all patients by using professional antigen-presenting cells as dendritic cells, but CTLp frequency determined the kinetics of generation of specificity and the final number of effectors as evaluated by both limiting dilution analysis and staining with HLA-A*0201-Melan-A/Mart-1 tetrameric complexes. Immunohistochemical analysis of 26 neoplastic lesions from the nine patients indicated absence of tumor regression in most instances, even in patients with an expanded peripheral T cell pool to Melan-A/Mart-1 and whose neoplastic lesions contained a high frequency of tetramer-positive Melan-A/Mart-1-specific T cells. Furthermore, frequent lack of a "brisk" or "nonbrisk" CD3(+)CD8(+) T cell infiltrate or reduced/absent Melan-A/Mart-1 expression in several lesions and lack of HLA class I antigens were found in some instances. Thus, expansion of peripheral immune repertoire to Melan-A/Mart-1 takes place in some metastatic patients and leads to enhanced CTL induction after antigen-presenting cell-mediated selection, but, in most metastatic lesions, it does not overcome tumor escape from immune surveillance.

Topics: Adult; Aged; Amino Acid Sequence; Antigen-Presenting Cells; Antigens, Neoplasm; Female; HLA Antigens; Humans; Immunohistochemistry; Immunologic Memory; Immunologic Surveillance; Lymphocyte Activation; Male; MART-1 Antigen; Melanocytes; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; T-Lymphocytes; T-Lymphocytes, Cytotoxic

Using fluorescent HLA-A*0201 tetramers containing the immunodominant Melan-A/MART-1 (Melan-A) tumor-associated antigen (Ag), we previously observed that metastatic lymph nodes of melanoma patients contain high numbers of Ag-experienced Melan-A-specific cytolytic T lymphocytes (CTLs). In this paper, we enumerated and characterized ex vivo Melan-A-specific cells in peripheral blood samples from both melanoma patients and healthy individuals. High frequencies (>/=1 in 2,500 CD8(+) T cells) of Melan-A-specific cells were found in 10 out of 13 patients, and, surprisingly, in 6 out of 10 healthy individuals. Virtually all Melan-A-specific cells from 6 out of 6 healthy individuals and from 7 out of 10 patients displayed a naive CD45RA(hi)/RO(-) phenotype, whereas variable proportions of Ag-experienced CD45RA(lo)/RO(+) Melan-A-specific cells were observed in the remaining 3 patients. In contrast, ex vivo influenza matrix-specific CTLs from all individuals exhibited a CD45RA(lo)/RO(+) memory phenotype as expected. Ag specificity of tetramer-sorted A2/Melan-A(+) cells from healthy individuals was confirmed after mitogen-driven expansion. Likewise, functional limiting dilution analysis and interferon gamma ELISPOT assays independently confirmed that most of the Melan-A-specific cells were not Ag experienced. Thus, it appears that high frequencies of naive Melan-A-specific CD8(+) T cells can be found in a large proportion of HLA-A*0201(+) individuals. Furthermore, as demonstrated for one patient followed over time, dramatic phenotype changes of circulating Melan-A-specific cells can occur in vivo.

Topics: Adult; Aged; Antigens, Neoplasm; Case-Control Studies; CD8-Positive T-Lymphocytes; HLA-A2 Antigen; Humans; Immunologic Memory; Interferon-gamma; Lymphocyte Count; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Phenotype; Protein Conformation; Time Factors

The induction of an in vitro T cell response against tumour-associated antigens with subsequent expansion of the individual cytotoxic T lymphocyte (CTL) clones still is not routine and the only tumour-associated antigen that has been found to easily induce the establishment of CTL clones is the MART-1/Melan-A antigen. In this paper, we describe a new approach for in vitro immunization based on the use of preselected melanoma cell clones. The human melanoma cell subline FM3.P was cloned and the immunological properties of individual clones were compared. Melanoma cell clone FM3.29, having a high level of expression of melanoma differentiation antigens, as well as high levels of the HLA class I and class II antigens and adhesion molecules, was used for the establishment of a CTL line that was subsequently cloned. For optimization of the conditions of growth of established CTL clones, a particular melanoma subline FM3.D/40 was selected for supporting the proliferation of CTL clones. The majority of the established CTL clones recognized the melanoma-associated differentiation antigens gp100 and MART-1/Melan-A. Epitope analysis indicated that two different epitopes derived from gp100 (154-162 and 280-288) and a single epitope from MART-1/Melan-A (27 35) were recognized by these CTL clones. The gp100-specific CTL clones were found to be significantly more sensitive to the culture conditions than the MART-1/Melan-A-specific CTL clones. In addition, the presence of excess peptide in the culture medium induced autokilling of the gp100-specific, but not the MART-1/Melan-A-specific CTL clones. Taken together, these results demonstrate that, by careful preselection of melanoma cell lines and clones both for the induction of CTL line from patients' peripheral blood lymphocytes and subsequent cloning, it is possible to obtain a large number of stable CTL clones even against such an inherently "difficult" differentiation antigen as gp100.

Topics: Antigens, Neoplasm; Cell Culture Techniques; Clone Cells; Coculture Techniques; Dose-Response Relationship, Drug; Flow Cytometry; gp100 Melanoma Antigen; Humans; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Peptides; Receptors, Antigen, T-Cell; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

Peptide epitopes for tumor-reactive cytotoxic T-lymphocytes (CTL) have been identified on human cancers and are being used in tumor vaccine trials. However, the pharmacokinetics and pharmacodynamics of such peptides have been inadequately studied. It is predicted that immunogenic tumor peptides would have short half-lives in vivo. The goal of the present work was to evaluate the stability of the immunogenic peptide MART-1(27-35) in fresh normal human plasma (NHP) and to identify modifications that convey protection against enzymatic destruction without loss of immunogenicity. We evaluated the stability of the MART-1(27-35) peptide (AAGIGILTV) and modified forms of that peptide for stability and immune recognition in an in vitro model. The peptides were incubated in plasma for varied time intervals and evaluated for their ability to reconstitute the epitope for MART-1(27-35)-reactive CTL. Loss of CTL reactivity signaled loss of immunoreactive peptide. When 1 microM MART-1(27-35) peptide was incubated in plasma prior to pulsing on target cells, CTL reactivity was lost within 3 hr, and the calculated half-life of this peptide was 22 sec. This degradation was mediated by peptidases. The stability of MART-1(27-35) was markedly prolonged by C-terminal amidation and/or N-terminal acetylation (peptide capping), or by polyethylene-glycol modification (PEGylation) of the C-terminus. These modified peptides were recognized by CTL. The MART-1(27-35) peptide is very unstable in plasma. It is probable that it and other immunogenic peptides will be similarly unstable in vivo. Immunogenicity of these peptides might be enhanced by creating modifications that enhance stability.

Topics: Antigens, Neoplasm; Cancer Vaccines; Cell Line; Endopeptidases; Epitopes, T-Lymphocyte; Humans; MART-1 Antigen; Melanoma; Neoplasm Proteins; Peptide Fragments; T-Lymphocytes, Cytotoxic

Detection of micrometastases in the regional tumor-draining lymph nodes is critical for accurate staging and prognosis in melanoma patients. We hypothesized that a multiple-mRNA marker (MM) reverse transcriptase-polymerase chain reaction (RT-PCR) assay would improve the detection of occult metastases in the sentinel node (SN), compared with hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC), and that MM expression is predictive of disease relapse.. Seventy-two consecutive patients with clinical early-stage melanoma underwent sentinel lymphadenectomy (SLND). Their SNs were serially sectioned and assessed for MAGE-3, MART-1, and tyrosinase mRNA expression by RT-PCR, in parallel with H&E staining and IHC, for melanoma metastases. MM expression in the SNs was correlated with H&E and IHC assay results, standard prognostic factors, and disease-free survival.. In 17 patients with H&E- and/or IHC-positive SNs, 16 (94%) expressed two or more mRNA markers. Twenty (36%) of 55 patients with histopathologically negative SNs expressed two or more mRNA markers. By multivariate analysis, patients at increased risk of metastases to the SN had thicker lesions (P =.03), were 60 years of age or younger (P <.05), and/or were MM-positive (P <.001). Patients with histopathologically melanoma-free SNs who were MM-positive, compared with those who were positive for one or fewer mRNA markers, were at increased risk of recurrence (P =.02). Patients who were MM-positive with histopathologically proven metastases in the SN were at greatest risk of disease relapse (P =. 01).. H&E staining and IHC underestimate the true incidence of melanoma metastases. MM expression in the SN more accurately reflects melanoma micrometastases and is also a more powerful predictor of disease relapse than are H&E staining and IHC alone.

Topics: Adult; Age Factors; Aged; Antigens, Neoplasm; DNA, Neoplasm; False Negative Reactions; Female; Humans; Immunohistochemistry; Lymph Node Excision; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Middle Aged; Neoplasm Proteins; Neoplasm Staging; Predictive Value of Tests; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; Risk Assessment; RNA, Messenger; Sensitivity and Specificity; Skin Neoplasms

Many mechanisms have been proposed to explain why immune responses against human tumor antigens are generally ineffective. For example, tumor cells have been shown to develop active immune evasion mechanisms. Another possibility is that tumor antigens are unable to optimally stimulate tumor-specific T cells. In this study we have used HLA-A2/Melan-A peptide tetramers to directly isolate antigen-specific CD8(+) T cells from tumor-infiltrated lymph nodes. This allowed us to quantify the activation requirements of a representative polyclonal yet monospecific tumor-reactive T cell population. The results obtained from quantitative assays of intracellular Ca(2+) mobilization, TCR down-regulation, cytokine production and induction of effector cell differentiation indicate that the naturally produced Melan-A peptides are weak agonists and are clearly suboptimal for T cell activation. In contrast, optimal T cell activation was obtained by stimulation with recently defined peptide analogues. These findings provide a molecular basis for the low immunogenicity of tumor cells and suggest that patient immunization with full agonist peptide analogues may be essential for stimulation and maintenance of anti-tumor T cell responses in vivo.

Topics: Antigens, Neoplasm; Calcium; Cell Line; Cytokines; Cytotoxicity, Immunologic; Humans; Lymphocyte Activation; MART-1 Antigen; Melanoma; Neoplasm Proteins; Receptors, Antigen, T-Cell; T-Lymphocytes

The cloning of genes encoding melanoma antigens has opened new possibilities for the treatment of patients with cancer; however, most tumor rejection antigens recognized by tumor infiltrating lymphocytes are the products of genes that are also expressed by normal melanocytes. Hence, a large set of antigenic determinants of the self have not induced self-tolerance and these peptide determinants furnish target structures for immune responses directed against tumors. The notion that the immunotherapeutic targets involved in cancer regression comprise normal differentiation antigens is stressed by the association between vitiligo-like leukoderma, due to destruction of normal melanocytes, and melanoma regression, due to destruction of cancer cells. Nevertheless, this is the first report to demonstrate by means of a new technique based on reverse transcription polymerase chain reaction and denaturing gradient gel electrophoresis, the presence of clonally expanded T cells with identical BV regions in areas of destruction of both normal and neoplastic cells.

Topics: Amino Acid Sequence; Antigens, Neoplasm; Base Sequence; Humans; Lymphocytes, Tumor-Infiltrating; Male; MART-1 Antigen; Melanoma; Middle Aged; Molecular Sequence Data; Neoplasm Proteins; Receptors, Antigen, T-Cell, alpha-beta; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes; Vitiligo

From the peripheral blood lymphocytes (PBLs) of melanoma patient SK29(AV) we have previously isolated 2 independent cytolytic T lymphocyte (CTL) clones (CTL7/147 and CTL13/211), which lysed autologous tumor cells in association with HLA-B45.1. As demonstrated here, both CTL clones were directed against melanocyte differentiation antigen Melan-A/MART-1, which also was recognized by HLA-A2.1-restricted CTLs from the same patient. By generating and transfecting 3'-deletion mutants of Melan-A/MART-1 cDNA, we localized its peptide-coding regions. The HLA-B45.1-presented peptides were derived from a hydrophobic region of the protein and largely overlapped the peptides recognized by CTLs from the same patient in association with HLA-A2.1. We determined the fine specificity of these CTL clones with synthetic peptides. CTL clone CTL7/147 recognized the 11-mer peptide AEEAAGIGILT (residues 24-34) at the lowest concentrations. The absence of threonine-34 abrogated the recognition by CTL7/147. The truncated peptide AEEAAGIGIL (residues 24-33) proved to be the optimal synthetic peptide for sensitization against lysis by CTL13/211. This indicated that C-terminal threonine-34 was not involved in binding to HLA-B45.1 but, rather, was part of the epitope for CTL7/147. HLA-B45.1-associated peptides of Melan-A/MART-1 were regularly processed and presented by other melanomas and other cell types. Three of 4 independent HLA-A2.1-restricted SK29-CTL clones recognized the 10-mer peptide EAAGIGILTV (residues 26-35) at 10- to 100-fold lower concentrations than the nonamer AAGIGILTV (residues 27-35), previously described as the common immunodominant peptide antigen for all known anti-Melan-A/MART-1 CTLs restricted by HLA-A2.1. Different melanoma peptide antigens currently are applied in therapeutic vaccination studies. Our findings emphasize that restricting to peptides of minimal length might exclude relevant T-cell epitopes.

Topics: Amino Acid Sequence; Animals; Antigens, Neoplasm; Base Sequence; COS Cells; DNA, Neoplasm; Epitopes, T-Lymphocyte; HLA-A2 Antigen; HLA-B Antigens; Humans; MART-1 Antigen; Melanoma; Neoplasm Proteins; Sequence Homology, Amino Acid; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

A variety of human melanoma-associated antigens (MAA) have been identified that can be recognized by T lymphocytes in a major histocompatibility complex-restricted fashion. Among them, tyrosinase, MART-1/Melan- A, and gp100 are derived from nonmutated melanocyte lineage-specific antigens (Ag). These Ag can be recognized by CD8+ and, in the case of tyrosinase, CD4+ T cells. The in situ expression of these MAA may be a significant cofactor in determining the recognition of melanoma targets by Ag-specific T cells. In this study, we examined the patterns of expression of these MAA using immunohistochemical methods on 30 metastatic tumor deposits derived from 25 patients. MAA expression was heterogeneous among the 30 specimens and also within individual lesions. Of note, 23% of the samples examined failed to express the gp100 protein, and 17% of samples had no detectable expression of MART-1. In contrast, all lesions demonstrated some degree of tyrosinase expression even in cases where both gp100 and MART-1 were not detectable. In addition, 60% of samples (18 of 30) showed strong positivity for tyrosinase (> 75% of cells staining) compared with 40% for gp100 and 36% for MART-1. Currently, a number of experimental immunotherapies for melanoma are directed against the MAA tyrosinase, MART-1, and gp100. Although threshold levels of Ag required for T-cell recognition have not yet been defined, tumor-associated Ag expressed in high density, such as tyrosinase, may be better targets for future immunotherapy trials.

Topics: Antigens, Neoplasm; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; gp100 Melanoma Antigen; Humans; Immunotherapy; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Metastasis; Neoplasm Proteins

MART-1/MelanA and Pmel17/gp100 are melanoma-associated antigens (MAAs) that can be recognized by tumor-infiltrating lymphocytes (TILs) capable of mediating successful adoptive therapy in vivo. Analysis of melanoma cell lines in vitro has demonstrated that heterogeneous antigen expression in the context of class I MHC is a significant co-factor in determining the recognition of melanoma targets by cytotoxic lymphocytes (CTLs). In this study, 217 specimens from 103 patients with metastatic melanoma were examined for the expression of MART-1/MelanA (monoclonal antibody [MAb] M27C10) and Pmel17/gp100 (HMB45 MAb) by immuno-histochemistry. Marked heterogeneity in the expression of both MAAs was confirmed by analysis of the percentage of positively staining tumor cells or the average intensity of tumor staining. We also noted heterogeneity of expression among multiple lesions taken from different anatomic sites within a patient. A dissociation was noted in the detection of MART-1 and gp100 in some lesions, with gp100 being undetectable in 24% of the lesions and MART-1 being undetectable in 11%. In several cases, loss of one MAA was not associated with loss of the other MAA, suggesting that MART-1 can represent a useful additional marker for the diagnosis of melanoma in gp100 (HMB45)-negative lesions. Of the 217 specimens, 155 were obtained from HLA-A*0201 patients, of which 6% were negative for HLA-A2, 8% were negative for MART-1/MelanA and 21% were negative for Pmel17/gp100. The potential significance of our findings is illustrated by a case study in which a patient with melanoma experienced rapid tumor progression in association with loss of either MAA or HLA expression in several lesions.

Topics: Antigens, Neoplasm; gp100 Melanoma Antigen; HLA-A2 Antigen; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Metastasis; Neoplasm Proteins; Prospective Studies; Proteins; Tumor Cells, Cultured

The Melan-A/MART-1 gene, which is expressed by normal melanocytes as well as by most fresh melanoma samples and melanoma cell lines, codes for Ags recognized by tumor-reactive CTL. HLA-A*0201-restricted Melan-A-specific CTL recognize primarily the Melan-A(27-35) (AAGIGILTV) and the Melan-A(26-35) (EAAGIGILTV) peptides. The sequences of these two peptides are not necessarily optimal as far as binding to HLA-A*0201 is concerned, since both lack one of the dominant anchor amino acid residues (leucine or methionine) at position 2. In this study we introduced single amino acid substitutions in either one of the two natural peptide sequences with the aim of improving peptide binding to HLA-A*0201 and/or recognition by specific CTL. Surprisingly, analogues of the Melan-A(27-35) peptide, which bound more efficiently than the natural nonapeptide to HLA-A*0201, were poorly recognized by tumor-reactive CTL. In contrast, among the Melan-A(26-35) peptide analogues tested, the peptide ELAGIGILTV was not only able to display stable binding to HLA-A2.1 but was also recognized more efficiently than the natural peptide by two short-term cultured tumor-infiltrated lymph node cell cultures as well as by five of five tumor-reactive CTL clones. Moreover, in vitro generation of tumor-reactive CTL by stimulation of PBMC from HLA-A*0201 melanoma patients with this particular peptide analogue was much more efficient than that observed with either one of the two natural peptides. These results suggest that the Melan-A(26-35) peptide analogue ELAGIGILTV may be more immunogenic than the natural peptides in HLA-A*0201 melanoma patients and should thus be considered as a candidate for future peptide-based vaccine trials.

Topics: Antigens, Neoplasm; Cell Line, Transformed; Cells, Cultured; Clone Cells; Cytotoxicity, Immunologic; Epitopes, T-Lymphocyte; HLA-A Antigens; Humans; Immunodominant Epitopes; Lymph Nodes; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Neoplasm Proteins; Peptide Fragments; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

The staining pattern of the recently described antibody melan-A was compared with those of S100 protein and HMB-45 in a variety of melanocytic lesions to assess the specificity and sensitivity of these antibodies.. Immunohistochemical staining of paraffin sections of a range of melanocytic lesions was carried out following a high temperature antigen retrieval technique. The pattern and intensity of staining was semiquantitatively scored. S100 remains the most sensitive marker of melanocytic differentiation being diffusely positive in all benign and all primary and secondary malignant lesions including naevoid melanomas, and in most desmoplastic/spindle cell melanomas. Of the two more specific melanocytic markers melan-A stains the majority of benign and malignant lesions diffusely but with occasional patchy positivity only in some secondary melanoma deposits and with little staining of desmoplastic/spindle cell melanomas. HMB-45 is the least sensitive of the three showing little positivity of benign mature naevus cells, only variable patchy positivity of primary and secondary melanoma cells and limited positivity in naevoid, desmoplastic and metastatic melanomas.. Melan-A is a useful addition to antibody panels as it is apparently specific for melanocytic lesions and is more sensitive than HMB-45; however, it has less value than S100 in the detection of spindle cell and desmoplastic melanomas.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; Staining and Labeling

Evidence is growing for both humoral and cellular immune recognition of human tumor antigens. Antibodies with specificity for antigens initially recognized by cytotoxic T lymphocytes (CTLs), e.g., MAGE and tyrosinase, have been detected in melanoma patient sera, and CTLs with specificity for NY-ESO-1, a cancer-testis (CT) antigen initially identified by autologous antibody, have recently been identified. To establish a screening system for the humoral response to autoimmunogenic tumor antigens, an enzyme-linked immunosorbent assay (ELISA) was developed using recombinant NY-ESO-1, MAGE-1, MAGE-3, SSX2, Melan-A, and tyrosinase proteins. A survey of sera from 234 cancer patients showed antibodies to NY-ESO-1 in 19 patients, to MAGE-1 in 3, to MAGE-3 in 2, and to SSX2 in 1 patient. No reactivity to these antigens was found in sera from 70 normal individuals. The frequency of NY-ESO-1 antibody was 9.4% in melanoma patients and 12.5% in ovarian cancer patients. Comparison of tumor NY-ESO-1 phenotype and NY-ESO-1 antibody response in 62 stage IV melanoma patients showed that all patients with NY-ESO-1(+) antibody had NY-ESO-1(+) tumors, and no patients with NY-ESO-1(-) tumors had NY-ESO-1 antibody. As the proportion of melanomas expressing NY-ESO-1 is 20-40% and only patients with NY-ESO-1(+) tumors have antibody, this would suggest that a high percentage of patients with NY-ESO-1(+) tumors develop an antibody response to NY-ESO-1.

Topics: Antibodies, Neoplasm; Antigens, Neoplasm; Autoantibodies; Breast Neoplasms; Colonic Neoplasms; Enzyme-Linked Immunosorbent Assay; Female; Humans; Lung Neoplasms; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Membrane Proteins; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasms; Ovarian Neoplasms; Proteins; Recombinant Proteins; Repressor Proteins

Crosslinking of CD28 receptors on resting T lymphocytes by B7 costimulatory molecules expressed by antigen-presenting cells (APCs) plays a critical role in T-cell activation. Human melanomas express major histocompatibility complex (MHC)-restricted tumor-associated antigens that can be recognized by cytotoxic T lymphocytes (CTL), yet they remain poorly immunogenic. One mechanism for the failure of T-cell response is the lack of expression of costimulatory molecules by human melanoma cells. We have transfected the B7-1 gene into three HLA-A2-expressing human melanoma cell lines, and studied their capacity to stimulate primary human T cells. B7-expressing melanoma cells were excellent inducers of T-cell proliferation, cytokine production, and cytolytic activity in allogeneic mixed lymphocyte cultures through a process dependent on the function of the T-cell receptor as well as interactions between B7:CD28, CD2:LFA-3, and LFA-1:ICAM-1. Subset analysis demonstrated that CD4+ T cells or addition of exogenous interleukin-2 was required for the induction of CD8+ CTL. Untransfected parental melanoma cells were inert as APCs in these cultures. Rotating stimulation of T cells with the three B7-expressing cell lines led to the generation of T-cell lines that were cytolytic for HLA-A2+ melanoma cells and other HLA-A2+ targets that were pulsed with HLA-A2-restricted MART-1 peptides. These data demonstrate that expression of B7-1 by human melanoma cells converts them into effective APCs for the in vitro induction of MHC-restricted, melanoma-specific CTL.

Topics: Antigen-Presenting Cells; Antigens, Neoplasm; B7-1 Antigen; Cell Communication; Cell Membrane; Cells, Cultured; Cytokines; HLA-A2 Antigen; HLA-B7 Antigen; Humans; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; MART-1 Antigen; Melanoma; Neoplasm Proteins; Receptors, Antigen, T-Cell; Signal Transduction; T-Lymphocytes; T-Lymphocytes, Cytotoxic; Time Factors; Transfection; Tumor Cells, Cultured

DNA-based immunization strategies designed to elicit cellular antitumor immunity offer an attractive alternative to protein- or peptide-based approaches. In the present study we have evaluated the feasibility of DNA vaccination for the induction of CTL reactivity to five different melanoma Ags in vitro. Cultured, monocyte-derived dendritic cells (DC) were transiently transfected with plasmid DNA encoding human MART-1/Melan-A, pMel-17/gp100, tyrosinase, MAGE-1, or MAGE-3 by particle bombardment and used to stimulate autologous PBMC responder T cells. CTL reactivity to these previously identified melanoma Ags was reproducibly generated after two or three stimulations with genetically modified DC. Co-ordinate transfection of two melanoma Ag cDNAs into DC promoted CTL responders capable of recognizing epitopes from both gene products. Coinsertion of genes encoding the Th1-biasing cytokines IL-12 or IFN-alpha consistently enhanced the magnitude of the resulting Ag-specific CTL reactivity. Importantly, DC transfected with a single melanoma Ag cDNA were capable of stimulating Ag-specific CTL reactivity restricted by multiple host MHC alleles, some of which had not been previously identified. These results support the inherent strengths of gene-based vaccine approaches that do not require prior knowledge of responder MHC haplotypes or of relevant MHC-restricted peptide epitopes. Given previous observations of in situ tumor HLA allele-loss variants, DC gene vaccine strategies may elicit a greater diversity of host therapeutic immunity, thereby enhancing the clinical utility and success of such approaches.

Topics: Antigens, Neoplasm; Cell Line; Cytotoxicity, Immunologic; Dendritic Cells; DNA, Complementary; Epitopes, T-Lymphocyte; gp100 Melanoma Antigen; Humans; Immunophenotyping; Interferon-gamma; Interleukin-12; Luciferases; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monocytes; Mutagenesis, Insertional; Neoplasm Proteins; Peptides; T-Lymphocytes, Cytotoxic; Th1 Cells; Transfection; Tumor Cells, Cultured; Vaccines, DNA

The detection of melanoma cells in the circulation by polymerase chain reaction (PCR) assays has been shown by several investigators to correlate with the stage of the disease and possibly with prognosis.. We performed prospective studies on 276 patients with primary melanoma and regional lymph node (LN) metastases to assess the predictive value of PCR detection of tyrosinase and melanoma antigen recognized by T cells-1 (MART-1) in the blood for recurrence of melanoma.. PCR tests for gp 100, Muc-18, and p97 reacted with RNA in blood from healthy subjects and were considered unsuitable for patient monitoring. The tests were most frequently positive in the first 3 months after surgery. There were 47 recurrences in 123 patients who had been followed up for 18 months. Assays within 3 months of surgery predicted recurrence from melanoma in 66% of the latter (tests for tyrosinase alone detected 51% and MART-1 alone 21% of the patients). Hence, 34% of recurrences were not predicted by tests in the early postoperative period. This did not appear to be because of marker-negative melanoma because summation of tests over the first year identified 89% of those with recurrent disease.. Positive tests were recorded in 35% of patients who remained disease free, but it is too early to assess whether these represent false-positive results. The false-negative results raise the question of whether the assays will provide a reliable basis for selection of patients for adjuvant therapy.

Topics: Antigens, CD; Antigens, Neoplasm; Biomarkers, Tumor; CD146 Antigen; gp100 Melanoma Antigen; Humans; Lymphatic Metastasis; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Neoplastic Cells, Circulating; Neural Cell Adhesion Molecules; Polymerase Chain Reaction; Prognosis; Prospective Studies; Sensitivity and Specificity; Skin Neoplasms

Melan-A is a previously defined, melanocyte differentiation antigen, and an anti-Melan-A murine monoclonal antibody, A103, was recently developed by our group. In this study, we evaluated A103 immunoreactivity on formalin-fixed, paraffin-embedded tissues, exploring the potential of A103 in the diagnosis of metastatic melanoma. Seventy-five metastatic melanomas, 10 primary melanomas, and 10 benign melanocytic nevi were tested. The reactivity of A103 was compared with HMB-4, an anti-gp100 antibody. Results showed that all nevi were A103 positive, and most primary melanomas were A103 and HMB45 positive. Of 75 metastatic melanomas, 61 (81%) were A103 positive, and 56 (75%) were HMB45 positive. Of 19 HMB45-negative lesions, 8 were A103 positive; of 14 A103-negative lesions, 3 were HMB45 positive. Eleven metastatic lesions, as well as 2 of 10 primary melanomas, were dual negative. These negative cases consisted mainly of the spindle cell and desmoplastic variants. Of the positive cases, A103 showed homogeneous staining in a significantly higher proportion of cases than HMB45 (72% versus 52%). In addition, focal staining with less than 5% reactive tumor cells was seen more frequently in HMB45 (12 of 56) than in A103 (5 of 61). These results indicated that A103 can be used as a first-line antibody in the diagnosis of metastatic melanoma. Our results also showed that A103 reacted with angiomyolipoma, which is known to be HMB45 positive. Of normal tissues, unexpected A103 reactivity was observed in the adrenal cortex, granulosa and theca cells of the ovary, and Leydig cells of the testis. This A103 immunoreactivity in benign and neoplastic tissues of nonmelanocytic origin, the basis of which is unclear, could also be of potential diagnostic value.

Topics: Angiomyolipoma; Antibodies, Monoclonal; Antigens, Neoplasm; Female; Humans; Immunohistochemistry; Male; MART-1 Antigen; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; Nevus; Paraffin Embedding; Skin Neoplasms; Tissue Distribution

The exclusiveness of the relationship between peptide and HLA alleles, combined with their extensive polymorphism, emphasizes the need for immunization strategies based on endogenous processing of full length proteins (containing multiple epitopic determinants) for presentation to T cells. This could allow vaccination regardless of the patient's HLA phenotype, assuming that individual molecules can be efficient T cell Ags in association with various HLA alleles. An endogenous system of Ag presentation was developed using dendritic cells infected with recombinant viral vectors expressing the melanoma-associated Ag MART-1/Melan A. CD8+ T cells from melanoma patients were activated in vitro by coincubation with infected dendritic cells and tested for recognition of HLA-A-matched melanoma targets. This allowed the analysis of T cell induction in association with any HLA-A allele of a given patient's phenotype. In this system, MART-1/Melan A could not efficiently immunize in association with HLA-A alleles other than A*0201, including the one residue variant from A*0201: HLA-A*0226. Clonal analysis of MART-1/Melan A-specific CTL confirmed that MART-1/Melan A immunodominance is strongly restricted to the AAGIGILTV/HLA-A*0201 combination. The stringent epitope/allele requirements for MART-1/Melan A/TCR interactions were not associated with limitations in the TCR repertoire. In conclusion, autologous induction of MART-1/Melan A CTL by whole Ag processing and presentation is restricted to a unique allele/ligand combination and is excluded by minimal changes in HLA structure. Thus, whole protein vaccination for small m.w. Ags may provide no further advantage over a peptide-based approach.

Topics: Alleles; Amino Acid Sequence; Antibodies, Neoplasm; Antigen Presentation; Antigens, Neoplasm; Cells, Cultured; Cytotoxicity Tests, Immunologic; Epitopes, T-Lymphocyte; Fowlpox virus; HLA-DQ alpha-Chains; HLA-DQ Antigens; Humans; Immunodominant Epitopes; MART-1 Antigen; Melanoma; Molecular Sequence Data; Neoplasm Proteins; Peptide Fragments; Recombination, Genetic; T-Lymphocytes, Cytotoxic; Vaccinia virus

Topics: Antibodies; Antigens, Neoplasm; Humans; Immunoenzyme Techniques; MART-1 Antigen; Melanoma; Neoplasm Proteins; Skin Neoplasms

The Melan-A (MART1) gene encodes an antigen recognized by cytotoxic T cells. Although its expression in metastatic melanoma has been documented in the literature by several investigators, little is known about its distribution in primary melanomas and benign melanocytic nevi. In this study, we evaluated Melan-A expression immunohistochemically on sections from paraffin-embedded material of 50 benign nevi and 40 primary cutaneous melanomas using the monoclonal antibody A103. To evaluate a potential role of A103 in the differential diagnosis of melanocytic from nonmelanocytic tumors, we also analyzed a number of benign and malignant peripheral nerve sheath tumors, fibrohistiocytic tumors, and leiomyosarcomas. Immunoreactivity with A103 was present in all "nonneurotized" nevi and in all nondesmoplastic primary melanomas, both in the intraepidermal and the dermal component. Only two nevi that underwent prominent neurotization showed no staining with A103. Although all melanomas with epithelioid cells tended to be strongly positive with A103, only 4 of 13 spindle cell and desmoplastic melanomas (all positive with anti-S-100 and negative with HMB-45) were immunoreactive with A103 (two focally, two diffusely). None of the nonmelanocytic lesions expressed Melan-A. Our results confirm that Melan-A protein is broadly expressed in the majority of benign and malignant melanocytic lesions and suggest that A103 can be helpful diagnostically, not only for metastatic tumors, but also for primary skin lesions. Its use in distinguishing between melanocytic and peripheral nerve sheath tumors, however, is limited because of the low or absent expression of Melan-A in nevi that underwent neurotization and spindle cell and desmoplastic melanomas.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Diagnosis, Differential; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Neoplasm Proteins; Nevus, Pigmented; Skin Neoplasms

Several tumor antigens have been described as candidates for immunotherapy. Our study compared HLA-A2-restricted epitopes from 5 antigens commonly expressed by melanomas, i.e., Melan-A/MART-1 peptides (26-35 and 27-35), tyrosinase (368-376), gp-100 (280-288), MAGE-3 (271-279) and NA17-A (1-10), for their relative capacity to promote the development of cytotoxic and cytokine-producing specific CD8+ lymphocytes within melanoma-invaded lymph nodes. We used short-term cultured melanoma-invaded lymph node lymphocytes (MILLs) and tested responses developed by these cells to peptide-pulsed TAP-deficient T2 cells. We measured both the lytic response developed by MILLs and the fraction of these cells that secreted interferon-gamma (IFN-gamma), as deduced from intracellular cytokine labeling. Reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze the expression of the 5 antigens within melanoma-invaded lymph nodes. Melan-A/MART-1, tyrosinase and gp-100 peptides were recognized by MILLs derived, respectively, from 8 of 20, 5 of 19 and 4 of 20 melanoma-invaded lymph nodes expressing these antigens. Most MILLs specific for Melan-A/MART-1 and tyrosinase exhibited both lysis and IFN-gamma responses, whereas most of those specific for gp-100 developed only lysis. Weak lysis without IFN-gamma secretion was developed against NA17-A and MAGE-3 peptides by MILLs from, respectively, 3 of 9 and 2 of 14 lymph nodes expressing these antigens. Our data show a prevalence of both cytotoxic and IFN-gamma-secreting effector T cells specific for differentiation antigens within HLA-A2 melanoma-invaded lymph nodes, which makes these antigens attractive targets for specific immunotherapy.

Topics: Antigens, Neoplasm; Epitopes; HLA-A2 Antigen; Humans; Interferon-gamma; Interleukin-2; Lymph Nodes; MART-1 Antigen; Melanoma; Monophenol Monooxygenase; Neoplasm Invasiveness; Neoplasm Proteins; Sensitivity and Specificity; T-Lymphocytes; T-Lymphocytes, Cytotoxic; Tumor Necrosis Factor-alpha

Topics: Antigens, CD; Antigens, Neoplasm; CD146 Antigen; Enzyme-Linked Immunosorbent Assay; Gene Expression; gp100 Melanoma Antigen; Humans; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Neural Cell Adhesion Molecules; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skin; Skin Neoplasms; Tumor Cells, Cultured

Monoclonal antibody (MAb) A103 specifically detects Melan A/MART-1 protein expression. Melan A/MART-1-derived peptides are recognized by CD8+ T-cells and are used in immunotherapy. We examined formalin-fixed paraffin-embedded tissue from 57 melanomas (34 primary, 23 metastatic) and 39 control cases (junctional, dermal, compound, Spitz, Reed and balloon-cell naevi) using the alkaline phosphatase and anti-alkaline phosphatase immunochemical method after antigen retrieval. Immunoreactivity was rated as low, medium or high, and staining pattern as homogeneous or heterogeneous. Staining with MAb A103 showed a sensitivity of 88% for melanoma, with a very high specificity for melanocytic cells. Immunopositivity decreased along with clinical stage, with stage I showing 100%, stage II 88%, stage III 90% and stage IV 75% immunoreactivity. Staining changed from an exclusively homogeneous pattern in the early clinical stages to a more heterogeneous pattern in the later stages. Melanocytic control tissues consisting of naevi of different subtypes all showed weak to moderate homogeneous immunoreactivity, with polarity towards the epidermis. Analysis of short-term melanoma cell cultures using reverse transcription-polymerase chain reaction (RT-PCR) enzyme-linked immunosorbent assay (ELISA) demonstrated mRNA expression in only one third of the originally immunopositive tumours, suggesting rapid mRNA expression loss in culture. MAb A103 allows the detection of melanoma-associated Melan A/MART-1 protein expression in routine archival tissue and thus enables the profiling of melanomas suited for immunotherapy approaches involving Melan A/MART-1 derived epitopes.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Enzyme-Linked Immunosorbent Assay; Fixatives; Formaldehyde; Humans; Immunoenzyme Techniques; MART-1 Antigen; Melanoma; Neoplasm Proteins; Neoplasm Staging; Nevus, Pigmented; Paraffin Embedding; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Skin Neoplasms; Tissue Fixation; Tumor Cells, Cultured

Characterization of cytolytic T lymphocyte (CTL) responses to tumor antigens has been impeded by a lack of direct assays of CTL activity. We have synthesized reagents ("tetramers") that specifically stain CTLs recognizing melanoma antigens. Tetramer staining of tumor-infiltrated lymph nodes ex vivo revealed high frequencies of tumor-specific CTLs which were antigen-experienced by surface phenotype. In vitro culture of lymph node cells with cytokines resulted in very large expansions of tumor-specific CTLs that were dependent on the presence of tumor cells in the lymph nodes. Tetramer-guided sorting by flow cytometer allowed isolation of melanoma-specific CTLs and confirmation of their specificity and their ability to lyse autologous tumor cells. Our results demonstrate the value of these novel reagents for monitoring tumor-specific CTL responses and for generating CTLs for adoptive immunotherapy. These data also indicate that strong CTL responses to melanoma often occur in vivo, and that the reactive CTLs have substantial proliferative and tumoricidal potential.

Topics: Adult; Aged; Amino Acid Sequence; Antigens, Neoplasm; Female; Histocompatibility Antigens Class I; Humans; Immunotherapy, Adoptive; Indicators and Reagents; Lymphatic Metastasis; Lymphocytes, Tumor-Infiltrating; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Peptide Fragments; Protein Conformation; Staining and Labeling; T-Lymphocytes, Cytotoxic

In the treatment of cutaneous melanoma, provisional therapeutic strategies have been designed to combat tumour load using T cells that are sensitized with peptides derived from melanoma autoantigens, such as glycoprotein 100 (gp100), melanoma antigen recognized by T cells 1 (MART-1 or MelanA), tyrosinase and tyrosinase-related protein 1 (TRP-1). We recently found that gp100, MART-1 and tyrosinase are heterogeneously expressed in human cutaneous melanoma (De Vries et al (1997) Cancer Res 57: 3223-3229). Here, we extended our investigations on expression of these immunotherapy candidate proteins to uveal melanoma lesions. Cryostat sections from 11 spindle-type, 21 mixed and epithelioid tumours and four metastasis lesions were stained with antibodies specifically recognizing gp100, MART-1, tyrosinase and TRP-1. In addition, we used the DOPA reaction to detect tyrosinase enzyme activity as a confirmation of the tyrosinase immunohistochemical results. High expression of gp100, MART-1 and tyrosinase was found in the uveal melanoma lesions: 80% of the lesions displayed 75-100% positive tumour cells. TRP-1 positivity was slightly less: approximately 65% of the lesions stained in the 75-100% positive tumour cell category. All uveal melanoma lesions were positive for the four markers studied, this being in contrast to cutaneous melanoma where 17% of the advanced primary lesions and metastases were negative. The presence of these antigens was a little lower in metastases. We conclude that uveal melanomas and their metastases express melanocyte-lineage immunotherapy candidate proteins very abundantly. Uveal melanomas differ in this respect from cutaneous melanoma, in which the expression of these immunotherapy antigens was much more heterogeneous. This makes uveal melanoma a suitable candidate tumour for immunotherapeutic approaches.

Topics: Antigens, Neoplasm; Autoantigens; gp100 Melanoma Antigen; Humans; Immunohistochemistry; Immunotherapy; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Oxidoreductases; Protein Biosynthesis; Uveal Neoplasms

Dendritic cells (DC) are potent stimulators of primary T cell responses. In this study, we demonstrate that DC, genetically engineered to express the MART-1/Melan-A (MART-1) tumor-associated Ag, express MART-1 mRNA and protein, correctly process and present the HLA-A2.1-restricted immunodominant MART-1 peptide (MART-1(27-35)), and serve as potent stimulators of MART-1-specific CTL in vitro. A replication-defective E1-deleted adenovirus (AdV) was constructed that expresses MART-1 (AdVMART1). Transduced DC produce full length MART-1 mRNA as well as MART-1 protein. AdVMART1 does not significantly down-regulate cell surface class I expression despite having an intact E3 region. Transduction of an HLA-A2-positive/MART-1-negative cell line with AdVMART1 renders these cells sensitive to lysis by CTL specific for the MART-1(27-35) immunodominant peptide. In addition, DC transduced with AdVMART1 stimulated MART-1(27-35)-specific tumor-infiltrating lymphocytes to synthesize IFN-gamma. Finally, AdVMART1-transduced DC were able to generate MART-1(27-35) peptide-specific, class I-restricted CTL in PBL cultures from normal donors. This study supports the use of tumor Ag-engineered DC in genetic immunotherapy.

Topics: Adenoviridae; Antigens, Neoplasm; Clone Cells; Cytotoxicity, Immunologic; Dendritic Cells; Epitopes; Epitopes, T-Lymphocyte; Genetic Engineering; Histocompatibility Antigens Class I; Humans; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Neoplasm Proteins; T-Lymphocyte Subsets; T-Lymphocytes, Cytotoxic

Melan-A/MART-1 is a melanoma differentiation antigen that is recognized by a high proportion of cytolytic T lymphocyte (CTL) clones derived from human leukocyte antigen (HLA)-A2+ melanoma patients. Whereas peptide Melan-A/ MART-1(27-35) was originally defined as the immunodominant CTL epitope, we have previously reported that peptide Melan-A/MART-1(26-35) was recognized more efficiently by the majority of tumor-reactive CTL clones. As demonstrated here, CTL populations generated from blood lymphocytes of either melanoma patients or healthy individuals after in vitro stimulation with peptide Melan-A/MART-1(26-35) killed specifically HLA-A2+ Melan-A+ allogeneic melanoma cells, thus suggesting their potential use in adoptive immunotherapy. We characterized the surface phenotype of the circulating CTL precursors (CTLp), which respond to in vitro stimulation with peptide Melan-A/MART-1(26-35). In melanoma patients, these CTLp predominantly expressed the CD45RO memory marker. In contrast, they were mainly, although not exclusively, found in the CD45RA subpopulation of CD8 T cells in healthy individuals. The demonstration that Melan-A/MART-1-specific CTLp in peripheral blood lymphocytes from HLA-A2+ patients with metastatic melanoma express a memory phenotype provides direct evidence that in vivo priming of this antigen may occur during tumor progression.

Topics: Antigens, Neoplasm; CD8-Positive T-Lymphocytes; HLA-A2 Antigen; Humans; Immunologic Memory; Leukocyte Common Antigens; MART-1 Antigen; Melanoma; Neoplasm Proteins; T-Lymphocytes, Cytotoxic

Both CD8+ and CD4+ T cells have demonstrated roles in antitumor immune response in many animal tumor systems. In many human tumor systems, although abundant literature exists on the evidence of tumor antigen-specific CD8+ CTL response, only limited information is available on tumor antigen-specific CD4+ T-cell response. Using the MART-1/Melan-A (MART-1) antigen system as a prototype human tumor-associated antigen (TAA)- and dendritic cell (DC)-based MART-1 antigen presentation system (i.e., DCs transduced with an adenoviral vector-based construct carrying the MART-1 gene), we explored, in vitro, the feasibility of generating both CD8+ and CD4+ T-cell responses in the same individual. Here, we show that autologous DCs from both HLA-A2-positive melanoma patients and normal healthy individuals that are transduced with an adenoviral vector containing the MART-1 antigen are capable of inducing both MART-1-specific CD8+ and CD4+ T cells in in vitro coculture. After several rounds of stimulation, both the CD4+ and CD8+ T cells synthesized IFN-gamma when they were specifically stimulated. The CD8+ T cells generated in such cocultures also recognized the MART-1(27-35) peptide, AAGIGILTV, in 4-h cytotoxicity assays. These observations, therefore, suggest that Th1-type responses can be generated, in vitro, by stimulation with DCs that are genetically modified to express a TAA. Although the outcome of this type of genetically engineered DC-based stimulation may vary from system to system, this type of in vitro antigen presentation may be very useful in more comprehensive analyses of CD4+ T-cell response to defined TAAs, and such genetically engineered autologous DCs might be better candidates to serve as surrogate cancer vaccines.

Topics: Antigens, Neoplasm; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Communication; Dendritic Cells; Epitopes; Epitopes, T-Lymphocyte; Humans; Lymphocyte Activation; MART-1 Antigen; Melanoma; Neoplasm Proteins; Transduction, Genetic

There is ample evidence for spontaneous antimelanoma immune reactivity mediated by melanocyte-differentiation-antigens (MDAs). Our aim was to determine whether MDA immunoreactivity is associated with increased tumour-infiltrating lymphocytes (TIL) and macrophages (TIM). A retrospective study was conducted in 30 medium and high grade primary cutaneous melanomas (PCM) as identified by CART-analysis. All of the cases had developed clinical evidence for metastasis within 3 years following surgical excision of the PCM. We used immunohistochemistry and computerized image analysis to quantify MDAs positive cells (Melan A/MART-1, gp100/Pmel 17/HMB45, tyrosinase), CD45R0-positive TIL and LI-protein-positive TIM. A stochastic relationship was present between the MDA immuno-reactivities and the densities in TIL and TIM. An inverse relationship was yielded between TIL and TIM. No specific pattern of PCM immunoreactivity for MDAs, TIL and TIM was found to predict metastases.

Topics: Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Cell Differentiation; gp100 Melanoma Antigen; Humans; Immunohistochemistry; Leukocyte Common Antigens; Leukocyte L1 Antigen Complex; Macrophages; MART-1 Antigen; Melanocytes; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Neural Cell Adhesion Molecules; Proteins; T-Lymphocytes

HLA-A*0201 melanoma patients often develop a CTL response to an immunodominant peptide derived from the melanocyte lineage-specific protein Melan-A/MART-1. We have shown previously that the antigenic peptide most often involved is the decapeptide Melan-A(26-35) (EAAGIGILTV). We also observed some clonal diversity in the fine specificity of Melan-A-specific CTL. To substantiate this observation, we have now tested a series of Melan-A(26-35) variant peptides containing single alanine substitutions for binding to HLA-A*0201 and recognition by polyclonal and monoclonal Melan-A-specific CTL. Substitution of several residues with alanine reduced peptide binding activity by > 10-fold. In contrast, substitution of E26 with alanine (AAAGIGILTV) resulted in a 5-fold higher binding activity as well as in stronger stability of the corresponding HLA-A*0201/peptide complexes. Interestingly, the peptide variant AAAGIGILTV was recognized more efficiently than the natural decapeptide by short term cultured, tumor-infiltrated lymph node cell cultures and a number of Melan-A-specific CTL clones derived from different individuals. Moreover, this analysis revealed that the fine specificity of the CTL response to the Melan-A immunodominant epitope is quite diverse at the clonal level. At least three distinct patterns of fine specificity were identified. This diversity appears to reflect the diversity of the TCR repertoire available for this Ag, since similar results were obtained with a panel of Melan-A-specific CTL clones derived from a single melanoma patient. These findings have important implications for the formulation of Melan-A peptide-based vaccines as well as for the monitoring of Melan-A-specific CTL responses in melanoma patients.

Topics: Alleles; Amino Acid Substitution; Antigens, Neoplasm; Cell Line, Transformed; Cells, Cultured; Gene Rearrangement, T-Lymphocyte; HLA-A2 Antigen; Humans; Immunodominant Epitopes; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Neoplasm Proteins; Peptide Fragments; Protein Binding; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

An important element in melanoma vaccine construction is to identify peptides from melanoma-associated Ags that have immunogenic potential in humans and are recognized by CD8+ T cells in vivo. To identify such peptides, we evaluated HLA-A*02+ melanoma patients immunized to a polyvalent vaccine containing multiple Ags, including MAGE-3, Melan-A/MART-1, gp100, tyrosinase, melanocortin receptor (MC1R), and dopachrome tautomerase (TRP-2). Using a filter spot assay, we measured peripheral blood CD8+ T cell responses, before and after immunization, to a panel of 45 HLA-A*0201-restricted peptides derived from these Ags. The peptides were selected for immunogenic potential based on their strong binding affinity in vitro to HLA-A*0201. Vaccine treatment induced peptide-specific CD8+ T cell responses to 22 (47.8%) of the peptides. The most striking finding was the HLA-independent heterogeneity of responses to both peptides and Ags. All responding patients reacted to different combination of peptides and Ags even though the responding patients were all A*0201+ and the peptides were all A*0201-restricted. From 9 to 27% of patients developed a CD8+ T cell response to at least one peptide from each Ag, but no more than 3 (14%) reacted to the same peptide from the same Ag. This heterogeneity of responses to individual peptides and Ags in patients with the same haplotype points to the need to construct vaccines of multiple peptides or Ags to maximize the proportion of responding patients.

Topics: Alleles; Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Disease-Free Survival; gp100 Melanoma Antigen; HLA-A2 Antigen; Humans; Immunization; Intramolecular Oxidoreductases; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Peptide Fragments; Receptors, Corticotropin; Receptors, Melanocortin; Treatment Outcome

The spontaneous cytotoxic T cell responses to melanoma differentiation antigens and influenza matrix peptide were compared in 20 HLA-A2+ melanoma patients and 17 healthy A2+ individuals. Cytotoxic T lymphocyte (CTL) responses were determined by mixed lymphocyte peptide culture (MLPC) involving two stimulations of unfractionated peripheral blood lymphocytes (PBLs) with peptide in vitro. CTL responses to Melan-A 9-mer (amino acids 27-35, AAGIGILTV) peptide were detected in 4 out of 16 normal individuals, but in none of the melanoma patients. CTL specific for influenza matrix peptide were frequently found in both normal individuals and melanoma patients, suggesting that generalized immuno-suppression was not responsible for this difference. No significant responses were observed in either normal individuals or melanoma patients to Melan-A 10-mer (26-35, EAAGIGILTV), two gp1OO epitopes (280-288, YLEPGPVTA; 457466, LLDGTATLRL) and two tyrosinase epitopes (1-9, MLLAVLYCL; 368-376, YMDGMSQV). Melan-A (27-35)-specific CTL cells generated by normal individuals and melanoma patients recognized both synthetic peptide-pulsed T2 cells and two HLA-A2+, Melan-A+ melanoma cell lines (ME272, LAR1) in an antigen-specific, MHC class I restricted manner. T cells generated against Melan-A 9-mer were also able to recognize Melan-A 10-mer-pulsed target cells. Spontaneous CTL responses to Melan-A 9-mer from three known responder normal individuals were further evaluated over a prolonged time course (6-11 months). All 3 subjects demonstrated specific Melan-A 9-mer responses throughout the study period, although lytic activity fluctuated over time for a given individual. We found the MLPC assay to be reliable and easy to perform for monitoring T cell responses, although it may still not be sufficiently sensitive to detect low numbers of precursor T cells.

Topics: Adjuvants, Immunologic; Amino Acid Sequence; Antigens, Differentiation; Antigens, Neoplasm; Humans; Lymphocyte Activation; MART-1 Antigen; Melanoma; Neoplasm Proteins; Oligopeptides; T-Lymphocytes, Cytotoxic

Topics: Alleles; Amino Acid Sequence; Antigens, Neoplasm; Choroid Neoplasms; Ciliary Body; Eye Neoplasms; gp100 Melanoma Antigen; HLA-A Antigens; Humans; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Peptide Fragments; T-Lymphocytes, Cytotoxic

Topics: Antigens, Neoplasm; Epitopes; Fas Ligand Protein; fas Receptor; HLA-A2 Antigen; Humans; Immunotherapy; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Peptide Fragments; T-Lymphocytes, Cytotoxic

Several epitopes in the human melanoma antigens recognized by HLA-A2-restricted CTLs have a relatively low MHC-binding affinity and as a result may be expressed at very low densities on the cell surface, indicating that these epitopes may not be efficient immunogens. To express these epitopes at higher densities on the surface of antigen-presenting cells and therefore improve their immunogenicity, a DNA construct in which a cDNA fragment encoding the melanoma epitope MART-1(27-35) or gp100(280-288) was inserted between sequences encoding the leader and the HLA-A*0201 protein. Cells transfected with these epitope-HLA fusion constructs were recognized by HLA-A2-restricted melanoma-reactive CTLs specific for the MART-1 or gp100 epitope. In addition, tumor-reactive CTLs could be induced from PBMCs of patients with metastatic melanoma by in vitro stimulation with HMY-C1R B-cell lines expressing the MART-1 or gp100 epitope-HLA-A*0201 fusion protein. These epitope-HLA fusion constructs may be useful for the development of immunotherapies for patients with melanoma.

Topics: Animals; Antigens, Neoplasm; COS Cells; DNA; Epitopes; gp100 Melanoma Antigen; HLA-A2 Antigen; Humans; Lymphocyte Activation; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Peptide Fragments; Peptides; Recombinant Proteins; T-Lymphocytes, Cytotoxic; Transfection; Tumor Cells, Cultured

A line of tumor-infiltrating lymphocytes (660TIL) specifically lysed the autologous HLA-A2+ melanoma (660MEL) and also most A2+ melanoma cell lines. We immunoprecipitated A2 from a large number (>10(12)) of 660MEL cells, extracted naturally processed peptides, fractionated them by HPLC, screened the fractions for recognition by 660TIL, and found a single predominant and a minor peak of activity. Although too little was recovered of the major 660MEL peptide to establish its sequence, HPLC fingerprinting showed that it did not correspond to any of the known A2-associated melanoma peptides recognized by T cells, including peptides from tyrosinase, MART-1/Melan-A, gp100 and MAGE-3. The major 660MEL antigenic peptide appears to be derived from MART-1/Melan-A but is neither AAGIGILTV nor ILTVILGVL nor any other MART-1/Melan-A peptide containing the A2 consensus motif. The multiplicity of melanoma peptides recognized by CD8+ T cells, most of which are non-mutated (including most likely the present 660MEL peptide), suggests the existence of unknown mechanisms, perhaps similar to those operating in autoimmune disorders, whereby T cells that recognize normal 'self' sequences become activated.

Topics: Antigen Presentation; Antigens, Neoplasm; Autoantigens; Autoimmunity; Biomarkers, Tumor; Consensus Sequence; Cytotoxicity Tests, Immunologic; Cytotoxicity, Immunologic; Humans; Immunodominant Epitopes; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Neoplasm Proteins; Peptides; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

Recombinant human MART-1 protein was produced by bacterial and baculoviral-insect cell expression systems. By immunization with bacterial MBP-MART-1 fusion protein or MBP cleaved MART-1 protein, a rabbit polyclonal and two murine monoclonal antibodies specific for MART-1 were produced. These antibodies specifically detected MART-1 in immuno-precipitation, Western blotting, flow cytometric assays and in immunohistochemical analysis of tissue sections. They also stained cytoplasmic components in melanocytes and most melanoma cells in frozen or paraffin embedded tissue sections, indicating that these antibodies may be useful for the diagnosis of melanoma. One of the monoclonal antibodies M2-7 C10 recognized only human MART-1, but the other monoclonal antibody M2-9 E3 recognized both human and murine MART-1. The size of the human MART-1 molecule detected by SDS-PAGE with these antibodies was approximately 18 kDa, suggesting possible posttranslational modifications in the MART-1 protein. Subcellular fractionation studies suggested that MART-1 was present in melanosomes and endoplasmic reticulum, although known melanogenic enzymatic activities were not detected in the MART-1 protein. These reagents may be useful for biological studies on melanocytes and melanoma cells as well as for the development and monitoring of immunotherapy for patients with melanoma.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neoplasm; Antibody Specificity; Antigens, Neoplasm; Humans; Immune Sera; Immunohistochemistry; MART-1 Antigen; Melanocytes; Melanoma; Mice; Neoplasm Proteins; Rabbits; Recombinant Proteins; Skin Neoplasms

The recent identification of genes encoding melanoma-associated antigens has opened new possibilities for the development of cancer vaccines designed to cause the rejection of established tumors. To develop a syngeneic animal model for evaluating antigen-specific vaccines in cancer therapy, the murine homologues of the human melanoma antigens MART1 and gp100, which were specifically recognized by tumor-infiltrating lymphocytes from patients with melanoma, were cloned and sequenced from a murine B16 melanoma cDNA library. The open reading frames of murine MART1 and gp100 encode proteins of 113- and 626-amino acids with 68.8 and 77% identity to the respective human proteins. Comparison of the DNA sequences of the murine MART1 genes, derived from normal melanocytes, the immortalized nontumorgenic melanocyte line Melan-a and the B16 melanoma, showed all to be identical. Northern and Western blot analyses confirmed that both genes encoded products that were melanocyte lineage proteins. Mice immunized with murine MART1 or gp100 using recombinant vaccinia virus failed to produce any detectable T-cell responses or protective immunity against B16 melanoma. In contrast, immunization of mice with human gp100 using recombinant adenoviruses elicited T cells specific for hgp100, but these T cells also cross reacted with B16 tumor in vitro and induced significant but weak protection against B16 challenge. Immunization with human and mouse gp100 together [adenovirus type 2 (Ad2)-hgp100 plus recombinant vaccinia virus (rVV)-mgp100], or immunization with human gp100 (Ad2-hgp100) and boosting with heterologous vector (rVV-hgp100 or rVV-mgp100) or homologous vector (Ad2-hgp100), did not significantly enhance the protective response against B16 melanoma. These results may suggest that immunization with heterologous tumor antigen, rather than self, may be more effective as an immunotherapeutic reagent in designing antigen-specific cancer vaccines.

Topics: Amino Acid Sequence; Animals; Antigens, Neoplasm; Base Sequence; Cell Line; Cloning, Molecular; Genetic Code; gp100 Melanoma Antigen; Humans; Immunotherapy; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Neoplasm Proteins; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Tumor Cells, Cultured

MART-1 is a transmembrane protein that appears to be melanocyte specific. This study evaluated the use of a new monoclonal antibody, directed against MART-1, in fresh and paraffin-embedded specimens. Twenty-three paraffin-embedded tumors were evaluated for MART-1 immunoreactivity both before and after microwave-based antigen retrieval. After this comparison, 53 fine-needle aspirates from 43 patients with malignant melanoma were assessed for MART-1 immunoreactivity. Immunocytochemistry was performed on both cytospins and paraffin-embedded tissues that were pretreated with antigen retrieval. Seventeen (74%) of 23 tumors evaluated for immunoreactivity before and after antigen retrieval showed a significant increase in both staining intensity and the number of cells stained. When cytospins and antigen-retrieved cell blocks from 53 fine-needle aspirates were compared. 38 (72%) showed good correlation. In 13 (25%) of 53 tumors, MART-1 immunoreactivity was more intense in the cytospins, although the differences were marked in only two cases: Microwave-based antigen retrieval renders paraffin-embedded tissues nearly as sensitive as fresh material for use in the immunocytochemical detection of MART-1. This technique will allow the evaluation of MART-1 immunoreactivity in archival malignant melanomas.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Neoplasm Proteins; Paraffin Embedding; Specimen Handling

Melanoma is heterogeneous for its biological properties and melanoma-associated antigens (MAAs). This diversity is partially observed in the expression of the MAAs involved with the melanin synthesis pathway. We therefore developed a sensitive multimarker reverse transcription-PCR plus Southern blot assay using five MAAs as molecular markers to detect primary and metastatic melanoma cells. Melanoma cell lines, melanocytes (cultured), primary and metastatic malignant melanoma tissues, and blood from patients with American Joint Committee on Cancer stage I-IV melanoma were assessed for tyrosinase, tyrosinase-related proteins 1 and 2, Pmel 17, and MART-1/Melan-A. All of the MAA mRNA markers were expressed in 100% of melanoma cell lines and cultured melanocytes, 74% of primary and metastatic tumors (excluding tumor-draining lymph nodes), 43% of tumor-involved lymph nodes, and 43% of patients' bloods. Hypomelanotic melanoma tissues expressed a lower frequency of individual mRNA markers. Overall, at least one mRNA marker was expressed in more than 86% of specimens assayed. Normal tissue specimens from patients and blood from normal volunteer donors were negative for MAA mRNA expression. The multimarker MAA reverse transcription-PCR plus Southern blot analysis was more reliable and sensitive than a single-molecular marker assay for the detection of melanoma cells. This molecular assay can also provide information on MAA mRNA expression of metastatic melanoma cells that may assist in monitoring the therapeutic efficacy of active specific immunotherapy toward specific MAA-bearing melanomas.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Blotting, Southern; gp100 Melanoma Antigen; Humans; Interferon-gamma; Intramolecular Oxidoreductases; Isomerases; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Polymerase Chain Reaction; Proteins; RNA Probes; RNA, Messenger; Skin Neoplasms; Tumor Cells, Cultured

Peptides derived from melanocyte differentiation antigens have been identified as targets for MHC class I-restricted cytolytic T lymphocytes (CTLs) in human melanoma Regression of antigen-expressing tumors as well as selection of antigen-loss variants in the presence of antigen-specific CTLs have previously been reported. In the present study, we determined the expression of the melanocyte differentiation antigens Melan A/MART-1 and tyrosinase by mRNA analysis and by immunohistochemical staining with the monoclonal antibodies (MAbs) A103 and T311. Co-expression of Melan A/MART-1 and tyrosinase was detected by both methods in 18/20 melanomas tested. However, immunohistochemistry provided additional information on intensity and microheterogeneity of antigen expression that cannot be detected by mRNA analysis as a molecular basis for the escape from CTL recognition of antigen-negative tumor cells. Comparative analysis of repeated biopsies of metastatic lesions in 5 HLA-A2+ patients showed a gradual loss of Melan A/MART-1 expression in 4/5 and of tyrosinase in 2/5 samples in association with tumor progression. However, 3 of these patients had growing antigen-positive tumors in the presence of antigen-specific CTLs. This led us to assess the expression of MHC class I, the essential restriction element for CTL recognition, and of HLA-A2. We found an unexpectedly high frequency of MHC class I-negative tumors (9/20). Loss of MHC class I expression was detected in 3/5 progressive tumors and isolated loss of HLA-A2 in 1/5 tumors. Our results suggest that strategies enhancing the expression of MHC class I and tumor-associated antigens need to be considered in attempts at making vaccination more effective.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Biopsy; DNA Primers; Female; Genes, MHC Class I; HLA-A2 Antigen; Humans; Immunohistochemistry; Liver Neoplasms; Lymphatic Metastasis; Male; MART-1 Antigen; Melanoma; Middle Aged; Monophenol Monooxygenase; Neoplasm Proteins; Polymerase Chain Reaction; RNA, Messenger; Skin Neoplasms

The MART-1/Melan-A human melanoma tumor antigen can be recognized by T lymphocytes and appears to be involved in tumor regression. To study the transcriptional regulation of this important gene, the 5' untranslated (UT) region of the MART-1/Melan-A gene was cloned and sequenced. Human melanoma cell lines were screened for MART-1/Melan-A mRNA expression. Primer extension and northern analysis were performed to confirm the mRNA size and start site. Several overlapping fragments of 5'UT were isolated from genomic DNA by polymerase chain reaction (PCR) from the previously described sequence for an additional 700 bp upstream. The fragments isolated (ranging from 838 bp to 160 bp in length) were used to drive luciferase reporter gene expression in melanoma and non-melanoma cell lines. Tissue-specific promoter activity was found in a 233-bp fragment of 5' UT with an average index of induction of 35 fold. The 233-bp MART-1/Melan-A promoter does not appear to have cytokine (IL-2, IL-4, IL-7, GM-CSF, TNF-alpha or IFN-gamma) responsive elements when studied in transient transfection assays.

Topics: Antigens, Neoplasm; Base Sequence; Cloning, Molecular; Cytokines; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Luciferases; MART-1 Antigen; Melanoma; Molecular Sequence Data; Neoplasm Proteins; Polymerase Chain Reaction; Promoter Regions, Genetic; RNA, Messenger; Sequence Analysis, DNA; Transfection; Tumor Cells, Cultured

Dendritic cells (DC) are potent professional antigen-presenting cells that can activate naive T lymphocytes and initiate cellular immune responses. As adjuvants, DC may be useful in enhancing the immunogenicity of tumor antigens and mediating tumor regression. Endogenous expression of antigen by DC offers the potential advantage of allowing prolonged constitutive presentation of endogenously processed epitopes and exploitation of multiple restriction elements for the presentation of the same antigen. In this report, we show that human DC are (a) capable of infection by recombinant poxviruses encoding melanoma-associated antigen (MAA) genes and (b) capable of efficiently processing and presenting these MAA to cytotoxic T cells. In 6/6 HLA A*0201-expressing melanoma patients tested, the virally driven expression of MART-1/Melan A MAA by DC was sufficient to generate CD8+ T lymphocytes that could recognize naturally processed epitopes on tumor cells. In most cases, specific anti-MART-1 reactivity could be detected after a single stimulation. Analysis of epitope dominance revealed that the amino acid sequence recognized by these cytotoxic T lymphocytes (CTL) corresponded to the MART-1(27-35) residues previously shown to be most commonly recognized by cytotoxic T lymphocytes expanded from metastatic melanoma lesions. These data show that the virally driven expression of MAA by DC can be exploited for the efficient induction of clinically relevant cytotoxic T-cell responses. This has clinical implications for active immunization therapy, and currently vaccine trials have been proposed for patients with metastatic melanoma.

Topics: Antigen Presentation; Antigens, Neoplasm; Cells, Cultured; Dendritic Cells; Histocompatibility Antigens Class I; Humans; Immunodominant Epitopes; MART-1 Antigen; Melanoma; Neoplasm Proteins; Poxviridae; T-Lymphocytes, Cytotoxic

In recent years, it has become evident that T cells can recognize peptides of melanocytic lineage antigens such as gp100, MART-1, and tyrosinase at the tumor cell surface and can subsequently destroy these cells. It is thus feasible to develop immunotherapeutic approaches based on the melanocytic marker profiles of melanoma cells. One of the predictors of the success rate of such a treatment is the extent of positive (target) tumor cells within the lesions of the patient. First, we investigated the presence of these three proteins in 18 human melanoma cell lines using RT-PCR and immunohistochemistry. In 11 cell lines, mRNA and protein of all three markers could be detected; in one cell line, only two markers were present, and six melanoma cell lines showed no evidence for these markers. Secondly, we stained frozen sections of 105 human melanocytic lesions, 13 common nevocellular nevi, 13 atypical nevi, 13 early primary melanomas (Breslow < 1.5 mm), 25 advanced primary melanomas (aPM; Breslow > or =1.5 mm), and 41 melanoma metastases (MM) with antibodies against glycoprotein 100, melanoma antigen recognized by T cells, and tyrosinase. In addition, we used the 3,4-dihydroxy-L-phenylalanine reaction to detect tyrosinase enzyme activity as a confirmation of the tyrosinase immunohistochemical results in a subset of the lesions. In the benign lesions, glycoprotein 100 was more prominently expressed in epidermal melanocytes, whereas melanoma antigen recognized by T cells was encountered in all or nearly all dermal melanocytes in all nevocellular nevi and atypical nevus lesions. Tyrosinase was found in a lower percentage of melanocytes, both in the epidermis and in the dermis within these lesions. With regard to heterogeneity of staining within the malignant lesions, we found that 54% (early primary melanomas), 48% (aPMs), and 56% (MM) of the lesions stained within the same staining category for all three proteins studied. Approximately 17% of the aPM and MM lesions did not show positive tumor cells for any of the three proteins. We conclude that a subgroup of patients with high expression should be selected for immunotherapeutic treatment approaches based on the presence of these proteins.

Topics: Antigens, Neoplasm; Dihydroxyphenylalanine; gp100 Melanoma Antigen; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Prognosis; RNA, Messenger; Skin Neoplasms; Tumor Cells, Cultured

The Melan-A/MART-1 gene product is frequently recognized by tumor-specific HLA-A2-restricted CTL. An immunodominant nonapeptide has been localized to the region spanning residues 27-35. However, the decapeptide including residues 26-35 (the nonapeptide extended NH2 terminally by one residue) appeared to be recognized as efficiently as the nonapeptide. In this study, we show that the optimal length immunodominant peptide appears to correspond to the decapeptide 26-35, as assessed by quantitative analyses of both 4 polyclonal and 13 monoclonal populations of specific CTL. Functional assays of peptide binding to HLA-A2 indicate that the decapeptide is significantly a more efficient binder than the nonapeptide. Moreover, analogues of the decapeptide including substitutions at a secondary HLA-A2 peptide anchor further improve decapeptide binding. Finally, we show that the functional (9 CTL clones analyzed) and structural TCR repertoire (7 CTL clones) of a group of specific CTL clones is rather diverse. The findings reported here may have important implications for future peptide-based melanoma vaccination trials as well as for the monitoring of specific CTL responses in vivo.

Topics: Amino Acid Sequence; Antigen Presentation; Antigens, Neoplasm; Base Sequence; Clone Cells; Gene Rearrangement, T-Lymphocyte; HLA-A2 Antigen; Humans; Immunodominant Epitopes; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Molecular Sequence Data; Neoplasm Proteins; Point Mutation; Protein Binding; Receptors, Antigen, T-Cell, alpha-beta; Substrate Specificity; T-Lymphocytes, Cytotoxic

This study investigates the generation of primary melanoma cell-specific cytotoxic T lymphocytes (CTLs) in vitro. Induction of peptide-specific CTLs from unfractionated naive peripheral blood mononuclear cells from HLA-A2 healthy donors was assessed using 2 recently described 9-mer epitopes from the melanoma tumor antigen Melan-A/MART-1. The need for help from CD4+ T lymphocytes for the long-lasting induction of CTLs and the capacity of the peptide-induced CTL lines to recognize many melanoma cells were evaluated. CTL lines were obtained reproducibly when CD4+ T-lymphocyte help was provided during the primary stimulation either in an autologous way, in the case of tetanus toxoid antigen (TT) responder donors, or with allogeneic TT-activated T-helper cells, separated by an insert well, in the case of tetanus toxoid non-responder donors. We also investigated helper T-cell-derived factors that are produced by TT-activated lymphocytes. Our results strongly suggest that a complex network of cytokines like interleukin-2 (IL-2), interferon-gamma, IL-6 and IL-1 exerts stimulatory effects for the initiation process of CTLs. In contrast, cytokine-like IL-4 might inhibit generation of cytolytic activity if provided by TT-activated T cells at early stages of induction. Our approach can be used to generate CTLs of a desired specificity for clinical use in adoptive immunotherapy protocols.

Topics: Antigens, Neoplasm; Cells, Cultured; Cytokines; Cytotoxicity, Immunologic; HLA-A2 Antigen; Humans; Interferon-gamma; Leukocytes, Mononuclear; Lymphocyte Activation; MART-1 Antigen; Melanoma; Molecular Sequence Data; Neoplasm Proteins; Oligopeptides; Polymerase Chain Reaction; Receptor-CD3 Complex, Antigen, T-Cell; Receptors, Antigen, T-Cell, alpha-beta; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Helper-Inducer; Tumor Necrosis Factor-alpha

Characterization of tumor-associated antigens (TAAs) recognized by CTLs makes the consideration of therapeutic strategies based on peptide stimulation of peripheral blood lymphocytes (PBLs) feasible. Several such approaches are adoptive transfer of peptide-stimulated PBLs, ex vivo peptide stimulation of dendritic cells, and direct vaccination with TAA-derived peptides. A critical component of any of these peptide-based strategies is the requirement that the patient's PBLs are able to react productively against the presented TAA. The purpose of this study, through the study of T-cell receptor (TCR) usage, was to evaluate the T-cell response in matched MART-1(27-35) peptide-stimulated PBLs and tumor-infiltrating lymphocytes (TILs). MART-1(27-35)-reactive PBL and TIL cultures were generated from three patients by in vitro stimulation with an immunodominant peptide of MART-1 (MART-1(27-35)). All cultures had a human leukocyte antigen A2-restricted, MART-1(27-35)-specific CTL response. The TCR usage of each was assessed by the DNA sequence analysis of 50 TCR beta clones obtained by rapid amplification of cDNA ends per culture. TCR analysis suggests a TCR repertoire that differed from patient to patient (8-16 subfamilies were used) and a predominant usage of a different variable beta chain (BV) by each of these MART-reactive T cells. These predominant BV rearrangements were derived from multiple clonotypes because different variable, diversity, and junctional regions were observed. However, a similar pattern of expansion was present for both PBLs and TILs; the relative usage of each prevailing BV was more marked in TILs (36, 50, and 78% of TILs versus 26, 20, and 24% of PBLs, respectively), a broader TCR repertoire was used by PBLs (P > 0.05), and similar TCR subfamily usage was noted when TIL and PBL cultures from the same patient were compared (8 of 11, 7 of 9, and 7 of 8 for patients 1, 2, and 3, respectively). Furthermore, the exact same clonotypes derived from predominant TCR subfamilies in the PBLs and TILs were present in each patient, suggesting peptide-stimulated expansion in both biological compartments. These studies suggest that there will not be a limited and predictable TCR subfamily response to a specific TAA, although reproducible patterns of PBL and TIL expansion are present from patient to patient. Additionally, identical T-cell clonotypes having the same potential for antigen-driven expansion were present in a patient's PBLs and TILs. As such

Topics: Amino Acid Sequence; Antigens, Neoplasm; Base Sequence; Cells, Cultured; Humans; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Molecular Sequence Data; Neoplasm Proteins; Peptide Fragments; Receptors, Antigen, T-Cell; Sequence Alignment; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; T-Lymphocytes; Tumor Cells, Cultured

Twenty-eight primary and 29 metastatic melanoma lesions and 18 pigmented nevi lesions were analyzed by using the immunoperoxidase reaction with anti-MART-1 and anti-gp100 monoclonal antibodies (mAbs). The MART-1 was expressed in 28, 29, and 18, and gp100 was expressed in 27, 28, and eight of these lesions, respectively. Intensity and percentage of stained cells with anti-MART-1 mAb were stronger and higher than those with anti-gp100 mAb. MART-1 was expressed homogeneously in primary melanoma and pigmented nevi, whereas it was heterogeneously expressed in metastatic melanoma lesions. The level of expression of MART-1 in primary melanoma lesions did not correlate with any clinicopathologic parameters. These results suggest that anti-MART-1 mAb is a useful tool for immunohistochemical analysis of melanocytic lesions and also is useful for patients' selection and monitoring of antigen-loss variants in clinical trials with the MART-1-based immunotherapy.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Antigens, Neoplasm; Female; gp100 Melanoma Antigen; HLA-A2 Antigen; Humans; Immunoenzyme Techniques; Male; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Middle Aged; Neoplasm Metastasis; Neoplasm Proteins; Nevus, Pigmented

The human immune repertoire appears to be capable of recognizing normal antigens expressed by tumor cells. Among these antigens, those of differentiation, characterized by a restricted tissue expression, could be of clinical interest since they may represent a target for immunotherapeutic protocols. In this context we have evaluated, in benign and malignant lesions of the melanocytic lineage, the expression of the Melan-A/MART-1 antigen, which has been shown to be recognized by T cells, of HLA-A2 melanoma patients. The immunohistochemical analysis conducted with a Melan-A/MART-1 monoclonal antibody demonstrated that the antigen expression does not correlate with transformation or tumor progression. At variable levels Melan-A/MART-1, differently from other differentiation antigens, is homogeneously expressed by multiple autologous metastases and by melanoma metastases at different body sites. This tissue distribution adds further biological support to the ongoing use of Melan-A/MART-1-related peptides in active immunotherapy.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Eye Neoplasms; HLA-A2 Antigen; Humans; Immunoenzyme Techniques; MART-1 Antigen; Melanoma; Neoplasm Metastasis; Neoplasm Proteins; Nevus; Skin Neoplasms

The human melanoma tumor Ags, MART1 and gp100, are specifically recognized by HLA-A2-restricted CD8+ CTLs derived from melanoma patients and appear to be involved in tumor regression. In order to develop immunizing vectors for the treatment of patients with metastatic melanoma, replication-defective recombinant adenoviruses, Ad2CMV-MART1 and Ad2CMV-gp100, which encode these tumor Ags, have been generated. Infection of non-Ag expressing HLA-A2+ cell lines A375 and MDA-231 with the vectors resulted in recognition by Ag-specific CTLs as demonstrated by specific target cell lysis and release of cytokines, including IFN-gamma, TNF-alpha, and granulocyte-macrophage-CSF. Sodium butyrate and TNF-alpha can further augment adenovirus-mediated transgene expression and increase recognition by specific CTLs. Although adenovirus-infected cells expressed the E3/19K protein at detectable levels, significant reduction of surface MHC class I expression was observed in only 3 of 10 tumor cell lines infected with either Ad2CMV-MART1 or Ad2CMV-gp100. Because of the suspected homology between the human MART1 and gp100 genes and their murine counterparts, we immunized C57BL/6 mice with these recombinant adenoviruses and demonstrated that immunization with Ad2CMV-gp100 could protect mice from murine melanoma B16 challenge administered intradermally. Depletion of CD8+ but not CD4+ T cells in vivo from Ad2CMV-gp100-vaccinated mice eliminated the protective effect. The anti-gp100 T cells induced by Ad2CMV-gp100 vaccinated appeared to be responsible for the protection. Thus, these recombinant adenoviruses encoding tumor Ags may be useful as vaccines to induce specific T cell immunity for cancer therapy.

Topics: Adenoviridae; Adenovirus E3 Proteins; Animals; Antigens, Neoplasm; Butyrates; Butyric Acid; Cell Line; Defective Viruses; DNA, Recombinant; Gene Expression Regulation, Viral; Genetic Vectors; gp100 Melanoma Antigen; HLA-A2 Antigen; Humans; Immunodominant Epitopes; Immunotherapy, Active; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Melanoma, Experimental; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Neoplasm Proteins; Neoplasm Transplantation; Recombinant Fusion Proteins; T-Lymphocytes, Cytotoxic; Tumor Necrosis Factor-alpha; Vaccination; Vaccines, Synthetic

Cellular immune responses to melanoma-associated Ags are the focus of ongoing studies aimed at developing immunotherapies for treatment of malignant melanoma. Melanoma predominantly affects Caucasians, a population in whom expression of HLA-A2 is prevalent. Among HLA-A2 subtypes, HLA-A*0201 is widely expressed, and HLA-A*0201-restricted, tumor-reactive CTL responses are well studied. We have observed in a group of melanoma patients an unexpectedly high frequency (approximately 20%) of non-HLA-A*0201 subtypes (*0202, *0204, and *0205), and little is known regarding antimelanoma response profiles in patients expressing such subtypes. We analyzed non-HLA-A*0201 peptide response profiles using HLA-A*0201-restricted epitopes from melanoma Ags MART-1/Melan A and glycoprotein 100. Most of these peptides bound to the majority of subtypes tested with 50% inhibitory concentrations less than 500 nM. Recognition of cells pulsed with different peptides (MART-1(27-35), G9(154), and G9(280) Flu M1(58-66)) and expressing different subtype molecules by HLA-A*0201-restricted CTL was limited to only a subset of non-HLA-A*0201 molecules, and the peptide/subtype complexes recognized varied among the effector populations tested. CTL responses elicited from PBL of patients and healthy donors expressing subtypes HLA-A*0202 and HLA-A*0205 suggested significant differences among HLA-A2 subtype function in the context of melanoma Ag presentation. These observations imply the necessity of subtyping patients considered for peptide-based protocols and highlight the need for further study of melanoma-directed cellular responses among patients expressing non-HLA-A*0201 subtypes.

Topics: Alleles; Amino Acid Sequence; Antigen Presentation; Antigens, Neoplasm; gp100 Melanoma Antigen; HLA-A2 Antigen; Humans; Immunotherapy, Adoptive; Lymphocyte Activation; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Molecular Sequence Data; Neoplasm Proteins; Peptides; T-Lymphocytes, Cytotoxic

Antigenic peptides derived from differentiation antigens of the melanocyte lineage were recently identified in human melanomas as targets for MHC-restricted cytotoxic T lymphocytes (CTL). CTL directed against peptides derived from the Melan A/MART-1, tyrosinase and gp100/Pmel17 antigens can be detected in melanoma patients and in healthy controls. The presence of defined antigenic peptides and corresponding precursor CTL in patients with metastatic melanoma opens perspectives for the development of antigen-specific tumor vaccines. In this study, we examined the expression of Melan A/MART-1, tyrosinase and gp100lPmel17 in fresh melanoma tissues of HLA-A2+ patients and the spontaneous CTL reactivity against antigenic peptides derived from these antigens. Our results demonstrate an inverse correlation of antigen expression and CTL response to Melan A/MART-1 and tyrosinase in patients with metastatic melanoma. In 2 patients with advanced disease, CTL responses against Melan A/MART-1 and tyrosinase were induced by intradermal immunization with synthetic nona- or deca-peptides derived from these antigens. Metastases increasing in size over time showed a loss of Melan A/MART-1 expression in the presence of CTL in one patient. The regression of a metastasis with persistent tyrosinase expression was observed in the other patient after the induction of CTL, reactive against tyrosinase. We conclude that CTL responses against melanocyte differentiation antigens may mediate regression of antigen-positive tumors and select for antigen-loss variants in vivo.

Topics: Amino Acid Sequence; Antigens, Neoplasm; Base Sequence; CD8-Positive T-Lymphocytes; Cell Differentiation; Cytotoxicity, Immunologic; DNA Primers; Epitopes; gp100 Melanoma Antigen; Humans; Immunity, Cellular; MART-1 Antigen; Melanocytes; Melanoma; Membrane Glycoproteins; Molecular Sequence Data; Monophenol Monooxygenase; Neoplasm Proteins; Proteins; Tumor Cells, Cultured

Coculture of melanoma cells and T cell clones derived from tumor-infiltrating lymphocytes (TIL) generally results in lysis of the antigen-bearing tumor cells but to inefficient proliferation and IL-2 secretion by responder T cells. This suboptimal activation is classically explained by an inability of tumor cells to provide costimulatory signals. Here we analyzed the responses to synthetic peptides of HLA-A2.1-restricted CTL clones specific for melanoma antigens MART-1 and NA17-A. We showed that peptide concentrations ranging from 1 pM to 10 nM efficiently sensitized the peptide transporter-deficient T2 cells to lysis. T2 cells pulsed with melanoma peptides also induced TIL proliferation and detectable secretion of IL-2, IFN-gamma and GM-CSF, but only for peptide concentrations 10- to 10,000-fold higher than those required for lysis. Hence this suggests that partial triggering of TIL clones by melanoma cells could be due to expression of appropriate MHC-peptide complexes at subthreshold levels. In support of this, we showed that melanoma cells, unable to trigger IL-2 secretion, developed this ability when incubated with the appropriate peptide. These results indicate that the level of antigens expressed on melanoma tumors critically affects TIL activation status and thus, the efficiency of specific immune reactions mediated by these cells.

Topics: Antigens, Neoplasm; Clone Cells; Coculture Techniques; Cytokines; Cytotoxicity, Immunologic; HLA Antigens; HLA-A2 Antigen; Humans; Interleukin-2; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Major Histocompatibility Complex; MART-1 Antigen; Melanoma; Neoplasm Proteins; Peptide Fragments; Receptors, Antigen, T-Cell; T-Lymphocytes; Tumor Cells, Cultured

CTL reactivity to the epitope MART-1(27-35), of the melanoma (self) antigen MART-1/melan A is frequently observed in tumor-infiltrating lymphocytes and may be readily elicited from the peripheral blood of melanoma patients that express HLA-A*0201. Available data suggest that these observations contrast with those made for other HLA-A*0201-presented melanoma self antigens regarding the regularity of observed CTL responses. Based on preliminary findings, we hypothesized that the CTL response to MART-1 might be augmented in part by T cell encounters with peptides derived from sources other than MART-1, which show sequence similarity to MART-1(27-35). To test this idea, a protein database search for potential MART-1 epitope mimics was done using criteria developed from analyses of effector recognition of singly-substituted peptide analogues of MART-1(27-35). Synthetic peptides were made for a portion of the sequences retrieved; 12/40 peptides tested were able to sensitize target cells for lysis by one or more anti-MART-1 effectors. The peptides recognized correspond to sequences occurring in a variety of proteins of viral, bacterial, and human (self) origin. One peptide derives from glycoprotein C of the common pathogen HSV-1; cells infected with recombinant vaccinia virus encoding native glycoprotein C were lysed by anti-MART-1 effectors. Our results overall indicate that sequences conforming to the A2.1 binding motif and possessing features essential to recognition by anti-MART-1 CTL occur frequently in proteins. These findings further suggest that T cells might encounter a variety of such sequences in vivo, and that epitope mimicry may play a role in modulating the CTL response to MART-1(27-35).

Topics: Amino Acid Sequence; Antigens, Neoplasm; Cells, Cultured; Cross Reactions; Cytotoxicity, Immunologic; Epitopes; Humans; Immunity, Cellular; MART-1 Antigen; Melanoma; Molecular Sequence Data; Neoplasm Proteins; Receptors, Antigen, T-Cell, alpha-beta; Sequence Homology, Amino Acid; Structure-Activity Relationship; T-Lymphocytes, Cytotoxic

MART-1 and gp100 melanoma associated antigens (MAA) are expressed by cells of the melanocytic lineage and are recognized by the majority of HLA-A2 restricted tumor-infiltrating lymphocytes. Heterogeneity of expression of MAA in tumor deposits may affect the natural history or response to therapy of patients with melanoma. In this study, we evaluated the expression of these MAA with a new monoclonal antibody (mAb) directed against MART-1 (M2-7C10) and the commercially available HMB45 mAb directed against gp100. Expression was tested in vitro by intracellular fluorescence analysis and in vivo by immunophenotyping of tissue specimens. Nine melanoma cell lines and 25 tissue specimens from metastatic melanoma were analyzed. One cell line did not express MART-1 or gp100. The expression of both antigens was more heterogeneous and significantly reduced (p < 0.01) in melanoma cell lines compared with melanocytes, suggesting progressive loss of expression of MAA by neoplastic cells. None of the nonmelanoma cancer lines tested stained for MART-1 or gp100. Analysis of melanoma lesions by immunohistochemistry showed significant heterogeneity of expression of both MART-1 and gp100 MAA either as a percentage of cells expressing MAA or as intensity of expression. Ten of 25 frozen sections expressed MART-1 in < 50% of the cells. In 6 of 25 lesions, immunoreactivity for MART-1 was totally absent. Fine needle aspiration of metastatic lesions seemed to yield information accurately about amount and heterogeneity of expression of MAA in tumor lesions in vivo. Heterogeneity of expression of MAA may be one of several mechanisms leading to tumor escape from immune recognition, and pretreatment evaluation of tumor lesion for expression of these antigens may help in selecting patients best suited to antigen-specific vaccine therapies.

Topics: Antigens, Neoplasm; gp100 Melanoma Antigen; Histological Techniques; Humans; Immunohistochemistry; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Skin Neoplasms; Tumor Cells, Cultured

In the last few years, mutiple protein target antigens for immunorecognition by T cells have been identified on human melanoma. How melanoma lesions escape from functional antigen-specific immune recognition remains poorly understood. We have identified the concomitant loss of the immunodominant T cell-defined MART-1/Melan-A antigen and downregulation of the TAP-1 gene in a recurrent metastatic melanoma that was resected in 1993. This phenotype was not observed for an earlier autologous melanoma lesion resected in 1987. The "antigen loss" could be restored in the variant tumor cell line by simultaneously providing both the MART-1/Melan-A gene (by retroviral transfer) and the TAP-1 gene (by a bioballistic approach) resulting in tumor cell sensitivity to MART-1/Melan-A-specific cytotoxic T lymphocytes. This suggests that tumor escape from immune surveillance may have occurred in vivo as a sequential result of (a) antigen loss, and (b) downregulation of the peptide-transporter protein TAP-1 expression by this patient's tumor over a 6-yr period from 1987 to 1993. These results suggest that the characterization of the T cell response to melanoma in individual patients and definition of the immunologically relevant genetic defects in tumors may be required to select the most effective therapeutic strategies for a given patient.

Topics: Antigens, Neoplasm; ATP Binding Cassette Transporter, Subfamily B, Member 2; ATP-Binding Cassette Transporters; CD8-Positive T-Lymphocytes; Epitope Mapping; Female; Gene Transfer Techniques; HLA-A2 Antigen; Humans; Immunodominant Epitopes; In Situ Hybridization, Fluorescence; MART-1 Antigen; Melanoma; Neoplasm Proteins; T-Lymphocytes, Cytotoxic; Tumor Escape

MART-1 is expressed ir both normal and neoplastic cells of melanocytic origin. Peripheral blood mononuclear cells (PBMC) from melanoma patients recognize and lyse tumor cells after repetitive in vitro stimulation with the immunodominant peptide MART-1(27-35). In this study, we compared the characteristics of the cytotoxic T lymphocyte (CTL) response to MART-1 in PBMC from 13 HLA-A2 melanoma patients with PBMC from 9 normal healthy donors stimulated in vitro with MART-1(27-35) (AAGIGILTV) or FluM1(58-66) (GILGFVFTL) peptides. The expansion rate among CTLs from different patients was variable and did not correlate with the development of specificity against the MART-1(27-35) or FluM1(58-66) peptides. Specific anti-MART-1(27-35) cytotoxicity could be generated in 13 of 13 melanoma patients but only in 5 of 9 healthy donors (p < 0.001). Anti-FluM1(58-66) activity could be generated in six of seven melanoma patients and six of seven healthy donors. Specific activity against MART-1(27-35), but not FluM1(58-66), was detectable significantly earlier after repetitive in vitro stimulation in melanoma patients (22.7 +/- 2.0 days compared with 32.7 +/- 1.7 days for healthy donors, p < 0.01). This report provides the first evidence of an enhanced level of sensitization of tumor-bearing hosts compared with normal individuals against a differentiation antigen shared by tumor and normal cells of the same lineage. These findings may have important implications for delineating events involved in the biology of tumor rejection naturally or in response to active specific immunotherapy.

Topics: Antigens, Neoplasm; Cytotoxicity, Immunologic; HLA-A2 Antigen; Humans; Immunization; Immunodominant Epitopes; Immunophenotyping; MART-1 Antigen; Melanoma; Neoplasm Proteins; Orthomyxoviridae; Skin Neoplasms; T-Lymphocytes, Cytotoxic; Viral Matrix Proteins

Topics: Antigens, Neoplasm; Eukaryotic Cells; Humans; MART-1 Antigen; Melanoma; Neoplasm Proteins; Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Templates, Genetic; Tumor Cells, Cultured

CD8+ T lymphocytes recognize antigenic peptides presented by major histocompatibility complex (MHC) class I molecules. Individual peptide termini appear to be fixed at the C- and N-terminal ends. In contrast, central peptide side chains residues may point in different directions and exhibit limited flexibility, dependent on the MHC class I structural variation. For instance, position 97 in HLA-A201 has been shown to shift individual peptide species into different coordinations, one oriented towards the peptide N terminus, or more towards the C-terminal end. The conformational shape of such non-anchor peptide residues may affect the affinity of MHC/peptide/TCR interaction, resulting in quantitative, or qualitative different T cell effector functions. To characterize the impact of different amino acid residues occupying position 97 in HLA-A2 on peptide binding and presentation to CTL, we generated a panel of mutated HLA-A2 molecules containing either M, K, T, V, G, Q, W, P or H at position 97. The HLA-A0201 presented melanoma-associated MART-1/Melan-A derived peptide AAGIGILTV was employed to assess the impact of such position-97 mutations on HLA-A2 in peptide binding measured in an HLA-A2 reconstitution assay and presentation to AAGIGILTV-specific polyclonal or clonal T lymphocytes as measured by cytotoxicity, or interferon (IFN)-gamma and granulocyte/ macrophage colony-stimulating factor (GM-CSF) secretion. The high-affinity AAGIGILTV peptide bound to all position-97 mutants, albeit with differential efficiencies, and elicited specific release of IFN-gamma and GM-CSF by CTL. CTL responses were triggered only by the HLA-A2 wild type, by HLA-A2-H97 (histidine position 97 mutant), and HLA-A2-W97. The HLA-A2-M97 presenting molecule elicited enhanced cytokine release and CTL effector functions by polyclonal and by clonal effector T cells. These results indicate that MHC class I-bound peptides can trigger specific cytokine release by effector T cells independently of their ability to induce cytolysis. We conclude that relatively minor changes in the MHC class I peptide binding groove, including substitutions at position 97, can affect recognition by antigen-specific T cells. Mutant MHC class I molecules, such as those described here, may act as partial peptide antagonists and could be useful for inducing T lymphocytes with qualitatively different effector functions.

Topics: Amino Acid Sequence; Antigens, Neoplasm; Cytokines; Cytotoxicity, Immunologic; HLA-A2 Antigen; Humans; MART-1 Antigen; Melanoma; Neoplasm Proteins; Protein Binding; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

Antigenic peptides derived from several differentiation antigens of the melanocyte lineage were recently identified in human melanomas as targets for HLA-A2.1-restricted cytotoxic T lymphocytes (CTLs). To examine their potential role in tumour-directed immune responses in vivo, we determined CTL reactivity against seven antigenic peptides derived from the Melan A/MART-1, tyrosinase and gp100/Pmel17 antigens in the peripheral blood of 10 HLA-A2+ healthy controls and 26 HLA-A2+ melanoma patients. The influenza matrix peptide (GILGFVFTL) presented by HLA-A2.1 was used as a control peptide. CTL reactivity was assessed in a mixed lymphocyte 'peptide' culture assay. Reactivity against Melan A/MART-1-derived peptide antigens was readily detectable in both melanoma patients and controls. Reactivity directed against tyrosinase-derived peptide antigens was also detected in both melanoma patients and healthy individuals, but less frequently. A measurable response against gp100/Pmel17-derived antigens was found in 1/10 controls and in 1/26 of the melanoma patients. Reactivity against the influenza matrix peptide was common in both melanoma patients and controls. Our findings show that precursor CTLs against melanocyte differentiation antigens can be detected in peripheral blood of melanoma patients and healthy individuals. The pattern of CTL reactivity directed against melanoma-associated antigens does not seem to be altered in melanoma patients. Despite antigen-specific CTL reactivity, tumour growth was not prevented in melanoma patients and autoimmune phenomena were not detected in healthy individuals. It remains to be determined whether precursor CTLs recognizing melanocyte differentiation antigens can be activated by immunization and lead to effective tumour rejection in vivo.

Topics: Amino Acid Sequence; Antigens, Differentiation; Antigens, Neoplasm; gp100 Melanoma Antigen; Humans; MART-1 Antigen; Melanoma; Membrane Glycoproteins; Monophenol Monooxygenase; Neoplasm Proteins; Proteins; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

Several antigens, including the products encoded by the genes MAGE-1 and MAGE-3, are recognized on human melanoma cells by HLA-A1, HLA-A2, or HLA-Cw*1601*-restricted T cells on autologous or HLA-matched melanoma cell lines. T-cell recognition of naturally processed MHC class I-presented peptides, or alternatively synthetic peptides derived from MAGE-1 or MAGE-3, leads to cytokine release as well as to a cytotoxic T-cell response in these antimelanoma-directed polyclonal or clonal effector T-cell populations. Recent reports suggest that the activity of T lymphocytes infiltrating melanoma in vivo appears to be impaired. We report here the characterization of the in vitro (in the presence of 6000 IU interleukin 2) expanded tumor-infiltrating lymphocyte (TIL) T-cell line PM2-B2 derived from a patient with rapidly progressing and therapy-resistant head and neck melanoma. The TIL cell line PM2-B2 did not lyse, but instead released granulocyte-macrophage colony-stimulating factor in response to the autologous tumor or HLA-A1-matched allogeneic tumor cell lines. The TIL line PM2-B2 did not kill the MHC class I natural killer/lymphokine-activated killer target cell lines Daudi or K562. The fine specificity of the TIL line PM2-B2 restricted by HLA-A1 was further characterized by evaluating specific granulocyte-macrophage colony-stimulating factor release in response to MHC class I-eluted peptides derived from HLA-A1(+) melanoma cell lines. TIL PM2-B2 failed to recognize the recently described HLA-A1-presented peptides derived from the gene products encoded by MAGE-1 or MAGE-3. PCR-based analysis of the freshly harvested tumor from patient PM2-B2 revealed the presence of message for the melanoma-associated gene products MAGE-1 and MAGE-3, but not for tyrosinase or MART-1/MELAN-A. Acid elution and high performance liquid chromatography fractionation of MHC class I-presented peptides from HLA-A1-matched melanoma cell lines 397 or 888 revealed that TIL PM2-B2 recognized at least three distinct peptide epitopes eluting in high performance liquid chromatographic bioactive fractions 5/6, 36, and 51/52. These bioactive peaks appeared to be shared among HLA-A1(+) melanoma cell lines. We suggest, based on this report, that HLA-A1-presented melanoma-derived peptides (other than those previously reported peptides derived from MAGE-1 or MAGE-3) may represent targets for TIL recognition as defined by cytokine release, but not cytotoxicity. Such an immune response differentially

Topics: Antigen Presentation; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Cytotoxicity, Immunologic; Epitopes, T-Lymphocyte; Granulocyte-Macrophage Colony-Stimulating Factor; HLA-A1 Antigen; Humans; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Neoplasm Proteins; Tumor Cells, Cultured

Specific CD8(+) CTL recognition of melanoma requires expression of MHC class I molecules as well as melanoma-associated peptide epitopes. Human melanoma cells may escape immune recognition by a variety of means, including global or allelic down-regulation of MHC class I molecules. Stable MHC class I cell surface expression requires delivery of cytosolic peptides into the endoplasmic reticulum by the peptide transporter molecules TAP1 and TAP2, with peptides subsequently transported to the cell surface in complexes containing MHC class I heavy chain and beta2-microglobulin. We have evaluated a series of mechanisms resulting in MHC class I down-regulation in a human melanoma cell line, Mz18, typed as HLA-A2(+), A3(+), B7(+), B57(+), Cw1(+), and Cw6(+) by genomic PCR analysis. The melanoma cell line Mz18 exhibits a global down-regulation of MHC class I heavy chain transcripts; beta2-microglobulin; the proteasome subunits LMP2/7, involved in generating cytosolic peptide fragments; and the peptide transporter molecules TAP1 and TAP2, involved in peptide transport from the cytosol into the endoplasmic reticulum. IFN-gamma treatment of Mz18 melanoma cells leads to up-regulation of LMP2/7 and TAP1/2, as well as to up-regulation of HLA-B and HLA-C MHC loci alleles, but not HLA-A2 or HLA-A3. Karyotypic analysis and fluorescence in situ hybridization with chromosome 6 and MHC class I-specific probes showed complex rearrangement of one chromosome 6 involving the MHC class I locus on 6p and translocation of 6q to the long arm of chromosome 19. To evaluate the capability of melanoma Mz18 to present tumor-specific peptides to HLA-A2-restricted, melanoma-specific CTLs, we restored HLA-A2 surface expression by retroviral-mediated transfer of functional HLA-A2 cDNA. Melanoma peptides could only be presented and recognized by CTLs if the HLA-A2-transfected Mz18 cell line was first treated with IFN-gamma, thereby restoring LMP2/7 and TAP1/2 expression and function. Because several melanoma antigens recognized by T cells have been reported to be presented by HLA-A2 (MART-1/Melan-A, tyrosinase, gp100, and MAGE-3), the loss of HLA-A2 molecules may represent an important mechanism by which many melanomas evade immune recognition. These findings suggest that patients entering clinical trials for immunotherapy with melanoma vaccines should be carefully examined for tumor cell allelic MHC class I loss and whether such MHC class I antigen down-regulation can be restored by cytokines

Topics: Antigens, Neoplasm; ATP Binding Cassette Transporter, Subfamily B, Member 2; ATP-Binding Cassette Transporters; Chromosomes, Human, Pair 6; Gene Rearrangement; Gene Transfer Techniques; HLA-A2 Antigen; Humans; Interferon-gamma; MART-1 Antigen; Melanoma; Neoplasm Proteins; RNA, Messenger; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured; Viral Matrix Proteins

Experimental models of vaccination with tumor cells engineered to produce interleukin-4 (IL-4) have shown that the local release of this cytokine is associated with the development of antitumor immunity that may induce regression of established cancer. The aim of this study was to transduce a human melanoma cell line with the gene coding for human IL-4, and to analyze cytokine production, phenotypic characteristics, and antigen expression after transduction. A retroviral vector, constructed by inserting IL-4 cDNA into the LXSN vector, was used to infect the human melanoma cell line Me14932, known to express the MHC class I HLA-A2 and the melanoma-associated antigen Melan-A/MART-1, recognized by HLA-A2-restricted T-cells. The confluence of all G418-resistant cells (Me14932/IL-4) was then analyzed for proviral integration and IL-4 mRNA expression. Substantially stable IL-4 release was detected by ELISA in the supernatant of transduced cells, ranging from 1.6 to 4.6 ng/ml per 10(5) cells per 24 hr; such a cytokine displayed a specific biologic activity, as revealed by the stimulation of blast cell proliferation and the inhibition of lymphokine activated killer cell (LAK) induction by IL-2. After 200 Gy irradiation, IL-4 release remained detectable for 5 weeks, whereas cell proliferation ceased within 7 days. Morphology and immunophenotypic characteristics of the parental cell line (expression of MHC classes I and II, ICAM-1, LFA 3, melanoma-associated antigens, etc.) were retained by the IL-4 gene-transduced melanoma as assayed by microscopy and immunofluorescence; likewise, susceptibility to lysis by LAK cells as well as a T-cell clone recognizing the Melan-A/MART-1 antigen did not change. These results, together with the lack of replication-competent retrovirus, suggest that the Me14932/IL-4 cell line displays suitable characteristics for its use in the treatment of HLA-matched melanoma patients.

Topics: Antigens, Neoplasm; Cell Division; Cells, Cultured; Gene Expression; Gene Transfer Techniques; Genetic Vectors; Humans; Interleukin-4; MART-1 Antigen; Melanoma; Neoplasm Proteins; Phenotype; Retroviridae; Tumor Cells, Cultured

Tumor-infiltrating lymphocytes (TILs) were grown from four distinct anatomic sites from a patient with metastatic melanoma. The metastatic sites included a tumor-involved lymph node, a subcutaneous lesion obtained from the chest wall, a portion of bowel, and adrenal gland. TILs grown from each anatomic site over the course of 20 days in the presence of 6,000 IU/ml recombinant interleukin-2 exhibited comparable growth rates. Between days 30 and 45, the TILs were a mixture of CD3+ CD4+ and CD3+ CD8+ lymphocytes expressing the alpha beta form of the T-cell receptor. TILs derived from each anatomic site specifically lysed autologous tumor obtained from all four anatomic sites. In fine specificity analysis, the TILs exhibited human leukocyte antigen (HLA-A2)-restricted lysis of fresh tumor targets and cultured melanoma cell lines. Each TIL recognized a product of the MART-1 gene, and specifically, the monomer peptide MART-1(27-35). Thus lymphocytes reactive with the MART-1 melanoma antigen appeared to be widely distributed in diverse metastases in this patient. This information, along with previous data on the reactivity of multiple patients to this antigen, attests to its dominance in the immune reactivity of humans to melanoma.

Topics: Adrenal Gland Neoplasms; Antigens, Neoplasm; Axilla; Cell Division; Cell Line; Colonic Neoplasms; Cytotoxicity, Immunologic; Humans; Immunophenotyping; Lymph Nodes; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Neoplasm Proteins; Thorax

By cDNA expression cloning we have isolated a gene encoding a shared human melanoma antigen recognized by HLA-A2 restricted autologous and allogenic tumor-infiltrating lymphocytes (TILs) from patients with metastatic melanoma. By using both transient and stable expression systems, transfection of this gene into non-antigen-expressing HLA-A2+ cell lines resulted in recognition by the antigen-specific TILs. The sequence of this cDNA revealed a previously undescribed putative transmembrane protein whose expression was restricted to melanoma and melanocyte cell lines and human retina but no other fresh or cultured normal tissues tested or other tumor histologies. Thus, we have identified a gene encoding a melanocyte lineage-specific protein (MART-1; melanoma antigen recognized by T cells 1) that is a widely shared melanoma antigen recognized by the T lymphocytes of patients with established malignancy. Identification of this gene opens possibilities for the development of immunotherapies for patients with melanoma.

Topics: Amino Acid Sequence; Antigens, Neoplasm; Base Sequence; Cloning, Molecular; Gene Expression; HLA-A2 Antigen; Humans; Lymphocytes, Tumor-Infiltrating; MART-1 Antigen; Melanoma; Molecular Sequence Data; Neoplasm Proteins; RNA, Messenger; T-Lymphocytes; T-Lymphocytes, Cytotoxic