bromochloroacetic-acid and Neoplasms

Fibroblastic reticulum cell tumor (FRCT) is a rare dendritic neoplasm arising from fibroblastic reticulum cells (FBRCs) and exhibiting peculiar cytokeratin expression. FRCTs usually involve the lymph nodes, although they can also be encountered in the spleen and soft tissues. FRCTs are composed of mildly atypical spindle or ovoid cells, arranged in loose whorls, which express almost invariably low-weight cytokeratins, smooth muscle actin, and CD68. An admixed lymphoplasmacytic infiltrate is also frequently present in solid organ sites. The clinical picture may vary from very indolent to aggressive disease exhibiting features of malignancy, such as cytological pleomorphism, necrosis, or high mitotic rate and metastatic potential. FRCT is a challenging diagnosis, due to its rarity and deceptive cytokeratin expression. Hereafter, we revise the most recent literature regarding such condition and report the case of an extremely indolent splenic FRCT, with no features of malignancy.

Topics: Histiocytic Disorders, Malignant; Humans; Keratins; Neoplasms; Splenic Neoplasms

KRT80 is a human epithelial intermediate filament type II gene; its expression product is a component of intracellular intermediate filaments (IFs) and is involved in the assembly of the cytoskeleton. There is evidence that IFs form a dense network mainly in the perinuclear area, but they can also reach the cortex. They are essential for mechanical cushioning of cells, organelle positioning, cell apoptosis, migration, adhesion, and interactions with other cytoskeletal components. Humans possess 54 functional keratin genes, and KRT80 is one of the more unique genes. It is widely expressed in almost all epithelial cells, although it is structurally more similar to type II hair keratins than to type II epithelial keratins.. In this review, we summarize the basic facts about the keratin family and KRT80, the essential role of KRT80 in neoplasms, and its potential as a therapeutic target. We hope that this review will inspire researchers to at least partially focus on this area.. In many neoplastic diseases, the high expression status of KRT80 and its role in regulating the biological functions of cancer cells have been well established. KRT80 can effectively enhance the proliferation, invasiveness and migration of cancer cells. However, the effects of KRT80 on prognosis and clinically relevant indices in patients with various cancers have not been extensively studied, and even opposite conclusions have been reached in different studies of the same cancer. Based on this, we should add more clinically relevant studies to clarify the prospect of clinical application of KRT80. Many researchers have made great progress in studying the mechanism of action of KRT80. However, their studies should be extended to more cancers to find common regulators and signaling pathways of KRT80 in different cancers. KRT80 may have far-reaching effects on the human body, and this marker may play a crucial role in the function of cancer cells and the prognosis of cancer patients, so it has a promising future in the field of neoplasms.. In neoplastic diseases, KRT80 is overexpressed in many cancers and plays an essential role in promoting proliferation, migration, invasiveness and poor prognosis. The mechanisms of KRT80 functions in cancer have been partially elucidated, suggesting that KRT80 is a potentially useful cancer therapeutic target. However, more systematic, in-depth and comprehensive studies are still needed in this field.

Topics: Cytoskeleton; Epithelial Cells; Humans; Keratins; Neoplasms

Keratins, the epithelial-predominant members of the intermediate filament superfamily, are expressed in a pairwise, tissuespecific and differentiation-dependent manner. There are 28 type I and 26 type II keratins, which share a common structure comprising a central coiled coil α-helical rod domain flanked by two nonhelical head and tail domains. These domains harbor sites for major posttranslational modifications like phosphorylation and glycosylation, which govern keratin function and dynamics. Apart from providing structural support, keratins regulate various signaling machinery involved in cell growth, motility, apoptosis etc. However, tissue-specific functions of keratins in relation to cell proliferation and differentiation are still emerging. Altered keratin expression pattern during and after malignant transformation is reported to modulate different signaling pathways involved in tumor progression in a context-dependent fashion. The current review focuses on the literature related to the role of keratins in the regulation of cell proliferation, differentiation and transformation in different types of epithelia.

Topics: Acetylation; Animals; Apoptosis; Carcinoma, Squamous Cell; Cell Differentiation; Cell Proliferation; Cell Transformation, Neoplastic; Epithelial Cells; Gene Expression Regulation, Neoplastic; Glycosylation; Humans; Keratins; Neoplasms; Phosphorylation; Protein Processing, Post-Translational; Protein Structure, Secondary; Signal Transduction

Most patients with cancers died of distant metastasis. It is always difficult to find cancer metastasis in early time, let alone to prevent or cure it. Currently, oncologists place high hopes on circulating tumor cell (CTC), which, compared to current imaging methods, is found more sensitive for early metastasis. Recently, techniques for CTC enrichment and identification are developing quickly. However, there are great challenges in the clinical interpretation of CTC assessments. Increasing studies have shown the heterogeneity of CTCs, which may play different roles in cancer metastasis. Epithelial-mesenchymal transition is not only the main mechanism of the cancer cells invading the circulation system but also a distinguished characteristic of CTCs. Investigators are trying to differentiate specific subgroups of CTCs that are truly responsible for cancer metastasis. Here, we reviewed the current evidences on epithelial-mesenchymal transition of CTCs from perspectives of enrichment methods, biology, and its subgroups.

Topics: Biomarkers, Tumor; Blood Cell Count; Cell Lineage; Cell Separation; Epithelial Cell Adhesion Molecule; Epithelial Cells; Epithelial-Mesenchymal Transition; Humans; Keratins; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms; Neoplastic Cells, Circulating; Phenotype; Prognosis; Vimentin

Intermediate filament (IF) constitutes an important cytoskeletal component in nearly all the vertebrate cells. IFs are present both in the cytoplasm and in the nucleus. They play an important role in providing mechanical strength of the cell and tissue, growth and regeneration, cell survival and apoptosis, and finally cell migration. IFs are also expressed differentially in different body tissues. Therefore, judicious use of IF may provide the diagnosis and confirmation of different malignancies. This is particularly helpful in the diagnosis of metastatic malignant tumor from an unknown primary. Expression of IFs particularly cytokeratin and vimentin is also related to prognosis of tumors. In this review, we have discussed the basic structure, dynamics, distribution of IF in cells, and its role in diagnosis of cytology. Possible prognostic roles of IF are also discussed.

Topics: Animals; Biomarkers, Tumor; Desmin; Humans; Intermediate Filaments; Keratins; Lamins; Neoplasms; Nestin; Prognosis; Protein Structure, Quaternary; Vimentin

Tissue sections offer the opportunity to understand a patient's condition, to make better prognostic evaluations and to select optimum treatments, as evidenced by the place pathology holds today in clinical practice. Yet, there is a wealth of information locked up in a tissue section that is only partially accessed, due mainly to the limitations of tools and methods. Often tissues are assessed primarily based on visual analysis of one or two proteins, or 2-3 DNA or RNA molecules. Even while analysis is still based on visual perception, image analysis is starting to address the variability of human perception. This is in contrast to measuring characteristics that are substantially out of reach of human perception, such as parameters revealed through co-expression, spatial relationships, heterogeneity, and low abundance molecules. What is not routinely accessed is the information revealed through simultaneous detection of multiple markers, the spatial relationships among cells and tissue in disease, and the heterogeneity now understood to be critical to developing effective therapeutic strategies. Our purpose here is to review and assess methods for multiplexed, quantitative, image analysis based approaches, using new multicolor immunohistochemistry methods, automated multispectral slide imaging, and advanced trainable pattern recognition software. A key aspect of our approach is presenting imagery in a workflow that engages the pathologist to utilize the strengths of human perception and judgment, while significantly expanding the range of metrics collectable from tissue sections and also provide a level of consistency and precision needed to support the complexities of personalized medicine.

Topics: Animals; Automation; Biomarkers, Tumor; Breast Neoplasms; DNA; Female; Fluorescent Dyes; Humans; Image Processing, Computer-Assisted; Immunohistochemistry; Keratins; Neoplasms; Pattern Recognition, Automated; Perception; Precision Medicine; RNA; Software; Tyramine

Intermediate filaments are assembled from a diverse group of evolutionary conserved proteins and are specified in a tissue-dependent, cell type-dependent, and context-dependent fashion in the body. Genetic mutations in intermediate filament proteins account for a large number of diseases, ranging from skin fragility conditions to cardiomyopathies and premature aging. Keratins, the epithelial-specific intermediate filaments, are now recognized as multi-faceted effectors in their native context. In this review, we emphasize the recent progress made in defining the role of keratins towards the regulation of cytoarchitecture, cell growth and proliferation, apoptosis, and cell motility during embryonic development, in normal adult tissues, and in select diseases such as cancer.

Topics: Animals; Apoptosis; Cell Movement; Cell Nucleus; Embryonic Development; Epithelium; Homeostasis; Humans; Intermediate Filaments; Keratins; Neoplasms; Skin Diseases; Stress, Physiological

This issue of Seminars in Nuclear Medicine deals with a watershed event in cancer treatment -- the combined use of functional and anatomical information to guide therapeutic interventions. The use of positron emission tomography/computed tomography (PET/CT) in radiation treatment planning and tumor response evaluation brings a paradigm change in the development of image-guided therapies into routine clinical practice. The implications, as seen in the following articles, are not only promising but also groundbreaking. And, as in every new scientific breakthrough, each step forward generates a myriad of additional important clinical and research questions. Functional imaging takes advantage of the subtle differences between normal and malignant tissues at the cellular level to reveal in vivo unique functional characteristics of neoplasms. The ultimate goal of the partnership between nuclear medicine physicians and radiation oncologists is to use this information with absolute clarity in target definition for radiation treatment planning and therapy, as well as response evaluation. Functional imaging can provide metabolic information and behavioral correlation along with the anatomical imaging for correlative target delineation. Additionally, as a purely diagnostic instrument, PET/CT provides a tool for oncologists to make critical decisions regarding radiation treatment planning modifications secondary to changes in tumor staging (up or down), treatment field modifications, localized control, sites of residual and/or metastatic disease and post therapy response evaluation. The articles in this issue of the seminars provide insights into the current state-of-the-art of functional imaging techniques, mostly centered on the use of (18)F-fluorodeoxyglucose PET/CT in image guided oncologic therapies. Because it is a novel science, the future of image-guided functional treatment planning is bright with technologic and biologic innovations, translational research and new clinical applications.

Topics: Biomarkers, Tumor; DNA Methylation; Humans; Keratins; Magnetic Resonance Imaging; Neoplasms; Oncogenes; Phosphorylation; Positron-Emission Tomography; Radiotherapy Planning, Computer-Assisted; Tomography, X-Ray Computed

The keratins are the typical intermediate filament proteins of epithelia, showing an outstanding degree of molecular diversity. Heteropolymeric filaments are formed by pairing of type I and type II molecules. In humans 54 functional keratin genes exist. They are expressed in highly specific patterns related to the epithelial type and stage of cellular differentiation. About half of all keratins--including numerous keratins characterized only recently--are restricted to the various compartments of hair follicles. As part of the epithelial cytoskeleton, keratins are important for the mechanical stability and integrity of epithelial cells and tissues. Moreover, some keratins also have regulatory functions and are involved in intracellular signaling pathways, e.g. protection from stress, wound healing, and apoptosis. Applying the new consensus nomenclature, this article summarizes, for all human keratins, their cell type and tissue distribution and their functional significance in relation to transgenic mouse models and human hereditary keratin diseases. Furthermore, since keratins also exhibit characteristic expression patterns in human tumors, several of them (notably K5, K7, K8/K18, K19, and K20) have great importance in immunohistochemical tumor diagnosis of carcinomas, in particular of unclear metastases and in precise classification and subtyping. Future research might open further fields of clinical application for this remarkable protein family.

Topics: Biomarkers, Tumor; Cytoskeleton; Epidermis; Epithelium; Humans; Keratins; Neoplasms; Organ Specificity

Plakophilins 1-3 are members of the p120(ctn) family of armadillo-related proteins. The plakophilins have been characterized as desmosomal proteins, whereas p120(ctn) and the closely related delta-catenin, ARVCF and p0071 associate with adherens junctions and play essential roles in stabilizing cadherin mediated adhesion. Recent evidence suggests that plakophilins are essential components of the desmosomal plaque where they interact with desmosomal cadherins as well as the cytoskeletal linker protein desmoplakin. Plakophilins stabilize desmosomal proteins at the plasma membrane and therefore may function in a manner similar to p120(ctn) in the adherens junctions. The three plakophilins reveal distinct expression patterns, and although partially redundant in their function, mediate distinct effects on desmosomal adhesion. Besides a structural role, a function in signaling has been postulated in analogy to other armadillo proteins such as beta-catenin. At least plakophilins 1 and 2 are also localized in the nucleus, and all three proteins occur in a cytoplasmic pool. This review aims to summarize the current knowledge of plakophilin function in the context of cell adhesion, signaling and their putative role in diseases.

Topics: Actins; Animals; Armadillo Domain Proteins; Catenins; Cell Adhesion; Cell Adhesion Molecules; Cell Nucleus; Cytoplasm; Cytoskeleton; Delta Catenin; Desmosomes; Disease Models, Animal; Gene Expression Regulation; Humans; Keratins; Neoplasms; Phosphoproteins; Plakophilins; Signal Transduction

Cytokeratins have been extensively used as serum tumour markers for monitoring of disease progression in cancer patients. The source of cytokeratins in the circulation as well as the mechanisms of release from cells have long been unclear. Recent evidence suggests that cytokeratins present in the circulation of cancer patients are released from apoptotic or necrotic tumour cells. CK18 is cleaved by caspases during apoptosis and a monoclonal antibody (M30) specific to caspase-cleaved forms is available. The molecular form of CK18 released from cells (caspase-cleaved or not) can conveniently be determined by immunoassays (M30-Apoptosense and M65 ELISA assays; Peviva AB, Bromma, Sweden) to determine cell death mode--apoptosis or necrosis. Recent studies where these assays were used to evaluate the response to cytotoxic anticancer drugs using cancer patient serum have been encouraging. CK18 is attracting considerable interest as a response biomarker during clinical trials of anticancer drugs. Properties such as excellent antigen stability and the epithelial specificity of cytokeratins contribute to make this biomarker attractive.

Topics: Antibodies, Monoclonal; Apoptosis; Biomarkers, Tumor; Cell Proliferation; Enzyme-Linked Immunosorbent Assay; Humans; Immunoassay; Keratin-18; Keratins; Models, Biological; Necrosis; Neoplasms

Topics: Apoptosis; Breast Neoplasms; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Keratins; Lung Neoplasms; Lymphatic Metastasis; Male; Neoplasm Metastasis; Neoplasm Seeding; Neoplasms; Neoplastic Cells, Circulating

Ubiquitin regulates cell functions by modifying various proteins, and cytokeratin (CK) is one of the targets of ubiquitilation. Accumulation of modified CK in various cancers has been demonstrated, and the modified CK increases the aggressiveness of the cancer by disrupting the cytoplasmic CK network and allows them to move freely. The phenotype of the cancer cells may be altered in such a way as to facilitate invasion and metastasis. Modified CK also deregulates mechanisms of mitosis and apoptosis, and leads to immortalization. Therapeutic targeting of ubiquitin or ubiquitilated proteins may reduce the malignant potential of cancer cells.

Topics: Humans; Keratins; Neoplasms; Proteasome Endopeptidase Complex; Ubiquitins

Cell death is as important as cell division in both physiological and pathological processes. Three major types of cell death have been described: apoptosis, autophagy and necrosis. Apoptosis is a form of programmed cell death, mediated by caspases. Autophagy is an evolutionarily conserved process involving lysosomes, implicated in both cell survival and death. Necrosis is believed to be an unregulated process, followed by release of intracellular components. The epithelial-specific intermediate filament cytokeratin 18 (Kl8) has different fates depending on the type of cell death. During apoptosis, K18 is cleaved at two sites into three fragments, one of which is specifically recognized by the monoclonal antibody, M30. During autophagy K18 is reported to stay uncleaved. Necrotic cells are considered to release K18. Thus, serum levels of different forms of K18 would reflect the type of cell death occurring in the body. Two enzyme-linked immunosorbent assays have been developed: one for the cleaved fragments of K18 and the other for total K18. Detection of serum levels of cleaved and total K18 showed that the ratios between cleaved and total K18 were highly variable among patients with endometrial cancer. Monitoring serum levels of cleaved and total K18 during chemotherapy showed an association between increases in total K18 levels and clinical responses. Monitoring serum levels of K18 may be a promising approach for early detection of therapeutic effects and the levels of different forms of K18 might indicate the mode of cell death occurring in the body.

Topics: Antineoplastic Agents; Biomarkers; Cell Death; Epithelial Cells; Humans; Keratins; Neoplasms

Cytokeratins, belonging to the intermediate filament (IF) protein family, are particularly useful tools in oncology diagnostics. At present, more than 20 different cytokeratins have been identified, of which cytokeratins 8, 18, and 19 are the most abundant in simple epithelial cells. Upon release from proliferating or apoptotic cells, cytokeratins provide useful markers for epithelial malignancies, distinctly reflecting ongoing cell activity. It appears that motifs in certain cytokeratins make them likely substrates for caspase degradation, and their subsequent release occurs during the intermediate events in apoptosis. The clinical value of determining soluble cytokeratin protein fragments in body fluids lies in the early detection of recurrence and the fast assessment of the efficacy of therapy response in epithelial cell carcinomas. The three most applied cytokeratin markers used in the clinic are tissue polypeptide antigen (TPA), tissue polypeptide specific antigen (TPS), and CYFRA 21-1. TPA is a broad spectrum test that measures cytokeratins 8, 18, and 19. TPS and CYFRA 21-1 assays are more specific and measure cytokeratin 18 and cytokeratin 19, respectively. By following patients with repeated testing during management, the oncologist may obtain critical information regarding the growth activity in symptomatic patients. Although their main use is to monitor treatment and evaluate response to therapy, early prognostic information particularly on tumor progression and metastasis formation is also provided for several types of cancers. Cytokeratin tumor markers can accurately predict disease status before conventional methods and offer a simple, noninvasive, cheap, and reliable tool for more efficient management.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Female; Humans; Keratin-19; Keratins; Male; Neoplasm Metastasis; Neoplasms; Peptides; Predictive Value of Tests; Prognosis; Tissue Polypeptide Antigen

Topics: alpha-Fetoproteins; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Biomarkers; Biomarkers, Tumor; Carcinoembryonic Antigen; Female; Humans; Keratin-19; Keratins; Kidney Failure, Chronic; Male; Neoplasms; Peptide Fragments; Peptides; Peritoneal Dialysis; Phosphopyruvate Hydratase; Prostate-Specific Antigen; Protein Precursors; Prothrombin; Recombinant Proteins; Renal Dialysis; Serpins; Tissue Polypeptide Antigen

Intracellular macromolecules are released from dying tumor cells and may subsequently be detected in patient blood. In this review, we will discuss the use of cytokeratin-18 as a serum biomarker for monitoring therapy-induced cell death. Cytokeratins are abundant intracellular proteins expressed by most types of carcinoma, but not by treatment-sensitive cells from bone marrow and other tissues. Release of cytokeratins into blood is therefore expected to show some specificity for tumor cell death. Cytokeratin-18 (CK18) is cleaved by caspases specifically during apoptosis, and the molecular form of this protein (caspase-cleaved vs. non-cleaved) released from dying tumor cells is therefore diagnostic as to the type of cell death (apoptosis vs. necrosis). Analyses of different CK18 forms in patient sera have suggested that tumor apoptosis may not necessarily be the dominating death mode in many tumors in vivo. Measurements of increased levels of CK18 in serum during therapy of prostate and breast cancer patients have been encouraging with regard to the possible future use of CK18 as a biomarker for monitoring therapy efficiency.

Topics: Antineoplastic Agents; Apoptosis; Biomarkers, Tumor; Endpoint Determination; Humans; Keratins; Necrosis; Neoplasms

Simple epithelial keratins K8 and K18 are components of the intracellular cytoskeleton in the cells of the single-layered sheet tissues inside the body. As members of the intermediate filament family of proteins, their function has been a matter for debate since they were first discovered. Whilst there is an indisputable case for a structural cell-reinforcing function for keratins in the mutilayered squamous epithelia of external barrier tissues, some very different stress-protective features now seem to be emerging for the simple epithelial keratins. Even the emerging evidence of pathological mutations in K8/K18 looks very different from mutations in stratified epithelial keratins. K8/K18-like keratins were probably the first to evolve and, whilst stratified epithelial (keratinocyte) keratins have diversified into a large group of keratins highly specialised for providing mechanical stability, the simple epithelial keratins have retained early features that may protect the internal epithelia from a broader range of stresses, including osmotic stress and chemical toxicity.

Topics: Animals; Apoptosis; Biological Evolution; Epithelium; Gene Expression; Humans; Keratins; Mice; Models, Biological; Mutation; Neoplasms; Skin Diseases, Genetic

Keratin expression in human tissues and neoplasms Keratin filaments constitute type I and type II intermediate filaments (IFs), with at least 20 subtypes named keratin 1-20. Since certain keratin subtypes are only expressed in some normal human tissues but not others, and vice versa, various tissues have been subclassified according to the pattern of keratin staining. Simple epithelia generally express the simple epithelial keratins 7, 18, 19, and 20, while complex epithelia express complex epithelial keratins 5/6, 10, 14, and 15. When an epithelium undergoes malignant transformation, its keratin profile usually remains constant. The constitution and expression patterns of keratin filaments in human epithelial neoplasms are complex and often distinctive. In this article, we first briefly review the molecular and cell biology of keratin filaments. We then focus on the expression patterns of keratin filaments in various human neoplasms.

Topics: Chromosome Mapping; Epithelium; Gene Expression; Genetic Diseases, Inborn; Humans; Keratins; Mutation; Neoplasms

There have been several studies attempting to detect the existence of cancer cells in the peripheral blood of patients with solid malignancies. The use of RT-PCR assays for cytokeratin (CK), carcinoembryonic antigen (CEA), alpha-fetoproteins (AFP), prostate specific antigen (PSA) and prostate specific membrane antigen (PMSA) is likely to be practicable in the detection of circulating tumor cells from epithelial-derived malignancies. The positive rates varied widely among the studies attempting to detect circulating cancer cells in peripheral blood, even when the same targets were used, which may be explained by the sensitivity of the RT-PCR assays and the target genes used. Both false negative results and false positive results can be obtained with the RT-PCR assay system. Furthermore, cancer cells may be released from the primary cancer into the circulation intermittently rather than constantly, and the results may vary among the samples obtained at different time points. In this report, we review our recent studies and related studies by other researchers to show the advances and the problems in detecting circulating cancer cells in patients with solid malignancies. The clinical significance of detecting circulating cancer cells in peripheral blood remains to be determined.

Topics: alpha-Fetoproteins; Biomarkers, Tumor; Carcinoembryonic Antigen; Humans; Keratins; Neoplasms; Neoplastic Cells, Circulating; Polymerase Chain Reaction; Prostate-Specific Antigen

Epithelial cells such as hepatocytes exhibit highly polarized properties as a result of the asymmetric distribution of subsets of receptors at unique portions of the surface membrane. While the proper targeting of these surface receptors and maintenance of the resulting polarity depend on microtubules (MTs), the Golgi sorting compartment, and different actin-filament networks, the contribution of keratin intermediate filaments (IFs) has been unclear. Recent data show that the latter cytoskeletal network plays a predominant role in providing resistance to various forms of stress and to apoptosis targeted to the surface membrane. In this context, we first summarize our knowledge of the domain- or assembly-related features of IF proteins and the dynamic properties of IF networks that may explain how the same keratin pair K8/K18 can exert multiple resistance-related functions in simple epithelial cells. We then examine the contribution of linker protein(s) that integrate interactions of keratin IFs with MTs and the actin-cytoskeleton network, polarity-dependent surface receptors and cytoplasmic organelles. We next address likely molecular mechanisms by which K8/K18 can selectively provide resistance to a mechanical or toxic stress, or to Fas-mediated apoptosis. Finally, these issues on keratin structure-function are examined within a context of pathological anomalies emerging in tissue architecture as a result of natural or targeted mutations, or posttranslational modifications at specific amino acid residues. Clearly. the data accumulated in recent years provide new and significant insights on the role of K8/K18, particularly under conditions where polarized cells resist to stressful or apoptotic insults.

Topics: Animals; Apoptosis; Cell Nucleus; Cytoskeleton; Desmosomes; Epithelial Cells; fas Receptor; Golgi Apparatus; Hepatocytes; Humans; Keratin-8; Keratins; Liver Diseases; Microtubules; Models, Biological; Models, Genetic; Mutation; Neoplasms; Protein Binding; Protein Processing, Post-Translational; Protein Structure, Tertiary; Stress, Mechanical

Despite an increasing molecular-genetic understanding of the development of malignant epithelial neoplasias, the frontline therapy for patients with carcinomas is still surgery. Systemic adjuvant treatments such as chemotherapy or immunotherapy have had limited success perhaps because they are based on analysis of the primary tumour or on cell lines derived from metastasis. However, the characteristics of systemically disseminated tumour cells can be very different from that of the primary tumour or end-stage metastasis. Consequently, there is a need to study the evolution and nature of systemic cancer directly in order to identify new target structures for therapy present on the potential precursors of metastasis--the disseminated tumour cells.

Topics: Animals; Bone Marrow; Bone Marrow Neoplasms; Disease Progression; DNA Mutational Analysis; Epithelial Cells; Humans; Keratins; Neoplasm Metastasis; Neoplasm, Residual; Neoplasms; Tumor Escape

The knowledge about the molecular genetic features of cancer has been vastly increased within the last few years. Tumor markers used so far for diagnosis, monitoring, and follow-up of cancer patients are tumor-associated rather than tumor-specific. Therefore, the development of more sensitive and specific tumor markers is urgently needed. In this review, the possibilities of using molecular genetic characteristics of cancer as specific and sensitive tumor markers are discussed.

Topics: Biomarkers, Tumor; Genes, p53; Genes, ras; Humans; Keratins; Microsatellite Repeats; Molecular Biology; Mutation; Neoplasms

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Diagnosis, Differential; Female; Humans; Keratin-19; Keratins; Lung Neoplasms; Male; Neoplasms; Neoplasms, Hormone-Dependent; Prostate-Specific Antigen; Prostatic Neoplasms; Telomerase

Topics: Biomarkers, Tumor; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Neoplasms

While not all circulating tumor cells survive, one could postulate that patients with circulating tumor cells might be candidates for adjuvant chemotherapy because of the risk of relapse. Recently, reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of circulating tumor cells has been suggested as a potential technique for staging of cancer. In malignant melanoma, the detection of circulating melanoma cells by tyrosinase RT-PCR correlated with the clinical stage of patients and was an independent prognostic factor for recurrence. A strong association was found between the detection of neuroblastoma cells in circulation by tyrosine hydroxylase RT-PCR with bone marrow micrometastasis. This method may be of use to identify early signs of relapse in disease-free patients. In prostatic cancer, the frequency of positivity for detection of circulating tumor cells in peripheral blood by PSA RT-PCR increased with tumor stages but a significant proportion of patients with metastatic disease were negative. The prognostic significance of the detection of tumor cell in blood in other epithelial tumors is not established and will require further long-term follow-up study.

Topics: alpha-Fetoproteins; Biomarkers, Tumor; Genes, ras; Humans; Keratins; Melanoma; Monophenol Monooxygenase; Neoplasms; Neoplastic Cells, Circulating; Point Mutation; Polymerase Chain Reaction; RNA; Tyrosine 3-Monooxygenase

Clinical and experimental experience indicate that differentiation and malignancy are inversely correlated. However, more recent experimental studies using mouse and human keratinocyte systems have demonstrated that complete or even substantial loss in overall epithelial differentiation is not a prerequisite for malignant growth of cancer cells. Major defects in differentiation are also not a prerequisite for premalignant stages, in particular for cell immortalization, which is considered an early and essential step in the transformation process. Moreover, progressive dedifferentiation, often associated with advanced tumor stages, is also found in immortalized cell lines which are, however, nontumorigenic. On the other hand, malignant cell lines may have maintained a high degree of their normal differentiation program and sensitivity to differentiation modulators. However, to date no transformed keratinocyte cell lines with completely normal differentiation have been observed. Since epidermal keratinization is a very complex process involving many different parameters and is fully expressed only under in vivo conditions, an exact and quantitative comparison of such ill-defined phenomena (differentiation and malignancy) is still problematic. Obviously, both phenomena are under separate control and not causally linked. Nevertheless, a better understanding of factors and mechanisms regulating differentiation and of their disturbance in carcinogenesis would offer new possibilities to design novel tumor therapeutic strategies in the field of differentiation therapy.

Topics: Biomarkers; Carcinoma, Squamous Cell; Cell Differentiation; Cell Survival; Cell Transformation, Neoplastic; Disease Progression; Epidermal Cells; Epithelial Cells; Humans; Keratins; Neoplasm Proteins; Neoplasms; Protein Precursors; Skin Neoplasms; Tumor Cells, Cultured

Topics: Adult; Digestive System Neoplasms; Endocrine Gland Neoplasms; Epithelium; Female; Humans; Keratins; Lung Neoplasms; Male; Neoplasms; Precancerous Conditions; Skin Neoplasms; Urogenital Neoplasms

The immunohistochemical approach to tumor typing has dramatically improved our possibilities in the objective diagnosis of neoplasms. Use of optimal material and careful techniques will help to maintain good sensitivity, specificity, and reproducibility of immunohistochemistry. However, the complexity of antigen patterns in tumors, and lack of comprehensive knowledge about them requires caution in the interpretation of results, and may prohibit the simple use of diagnostic algorithms. Especially it is not certain whether the results obtained from typical representatives of various tumor entities will pertain to borderline cases and to undifferentiated variants of the same entities. Use of panels of antibodies rather than the use of single "diagnostic" tests will help to avoid these diagnostic pitfalls. However, all tumor types do not have immunohistochemically distinctive features. This emphasizes the need to use other techniques in such cases, and also suggests that some entities, such as malignant fibrous histiocytoma, are from the point of view of immunohistochemistry diagnoses only made by exclusion rather than being specifically diagnosable entities. All diagnostic immunohistochemistry has to be interpreted in the context of standard histological examination.

Topics: Antigens, Neoplasm; Diagnosis, Differential; Fixatives; Formaldehyde; Frozen Sections; Humans; Immunohistochemistry; Keratins; Neoplasms; Peptide Hydrolases

A Monoclonal Antibody Program was launched at the National Institute of Oncology and Radiobiology (INOR) in Cuba in 1985. Eleven MAbs have been obtained which recognize different leukocyte antigens. Four MAbs a series of reagents raised against different types of cytokeratins, carcino-embryonic antigen and the membrane receptor for Epidermal Growth Factor. These MAbs are especially useful in immunohistochemistry for the study of human tumours, leukemias and lymphomas. Three ultramicro-ELISA systems have being deviced using MAbs. First trials are being made with radioactive MAbs for immunoscintigraphy. The use of anti T-cell MAbs for the prevention of rejection crisis in patients receiving organ transplantation and for the treatment of patient with cutaneous T-cell lymphoma is still mostly a matter for research.

Topics: Academies and Institutes; Antibodies, Monoclonal; Carcinoembryonic Antigen; Cuba; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Humans; Immunoassay; Immunoglobulins; Immunohistochemistry; Keratins; Leukocytes; Medical Oncology; Neoplasms; Radiobiology; Research

The tissue specific expression shown by intermediate filament proteins, their ease of extraction and their antigenicity has led to the use intermediate filament antibodies in diagnostic pathology, particularly antibodies to keratin intermediate filaments because of the predominant involvement of epithelia in cancers. We review the principles of differentiation-specific keratin expression and detail 100 of the best-known monoclonal antibodies to keratins which are currently in use: in many circumstances antibodies can now be reliably used as in situ markers of keratin expression. We also suggest a database scheme for better dissemination of information on available monoclonal antibodies to keratins in the future.

Topics: Antibodies, Monoclonal; Humans; Keratins; Neoplasms

The reported incidence of cytokeratin and vimentin intermediate filament coexpression is rapidly expanding. An up-do-date list of the instances in which this occurs is presented and the implications of this event are discussed.

Topics: Adult; Cells, Cultured; Female; Gene Expression Regulation; Histocytochemistry; Histological Techniques; Humans; Keratins; Male; Neoplasms; Phenotype; Vimentin

The past few years have seen great progress in our knowledge of the biology and the chemistry of intermediate filaments and other filamentous proteins. This has translated into the development of antibodies that, together with new, highly sensitive immunohistochemical methods, have resulted in much improved diagnostic accuracy. New, more specific antibodies to these and other useful markers will no doubt be developed in the future. More efficient techniques for screening of these antibodies will contribute to their rapid evaluation. Libraries of antibodies that identify subclasses of filamentous proteins will very likely become routine tools in the histopathology laboratory in the near future. Moreover, as the chemical structure of the proteins that form the complex family of intermediate filaments becomes better understood, and the DNA sequences coding them are determined, it may become feasible to use molecular genetic approaches such as in situ hybridization to gain further insight into neoplastic processes of unclear histogenesis and to develop more precise diagnostic and taxonomic instruments. Nevertheless, it is worth repeating that application of these molecular tools cannot be successful unless it is backed by a thorough understanding of the principles of histopathologic diagnosis that have been painstakingly established by generations of anatomic pathologists.

Topics: Actins; Antibodies; Antibodies, Monoclonal; Biomarkers, Tumor; Desmin; Diagnosis, Differential; Humans; Immunohistochemistry; Intermediate Filament Proteins; Intermediate Filaments; Keratins; Neoplasms; Vimentin

Topics: Antibodies, Monoclonal; Carcinoma; Cross Reactions; Diagnosis, Differential; Epitopes; Humans; Immunohistochemistry; Keratins; Neoplasms; Pathology, Surgical; Vimentin

It is commonly accepted, that a histogenetic correlation can be established between tumour and mother tissue by means of intermediate filament typing. Recent results concerning the co-expression of different intermediate filaments within one cell, the occurrence of epithelial-specific intermediate filaments in soft tissue tumours, and the modulation of intermediate filament type expression caused by some bioactive substances and viral infections cast doubts on the general acceptance of the histogenetic classification of human tumours by intermediate filament typing. Its characterization determines only the actual morphological differentiation of the neoplastic cells and represents a valuable tool for the diagnostic assessment of the tumour under the viewpoint of the functional differentiation of its cells. A doubtless conclusion concerning the mother tissue of the neoplasia (histogenetic origin) is therefore not possible.

Topics: Animals; Antibodies, Monoclonal; Cytoskeleton; Histocytochemistry; Humans; Intermediate Filaments; Keratins; Neoplasms; Rats; Vimentin

The development and refinement of immunohistochemical methodologies over the past decade has had a major impact in many different areas of diagnostic pathology. The generation of increasing numbers of well-characterized polyclonal antisera and monoclonal antibodies directed to a variety of antigenic determinants has made the phenotyping of neoplasms a reality. The results of such immunohistochemical analyses have provided an important starting point for the further workup and management of patients with undifferentiated or poorly differentiated malignant neoplasms of unknown origin. It should also be apparent, however, that the results of immunohistochemical analyses in such clinical settings are subject to a large number of variables and that these procedures must be controlled rigorously. The results of extensive performance testing, particularly with new reagents, must be available in order to establish their specificities and sensitivities under different conditions of tissue fixation and processing. Such studies not only will establish more precise and reproducible diagnostic criteria but also will be important for the definition of new clinical and pathological entities and for the characterization of novel prognostic parameters.

Topics: Antibodies, Monoclonal; Antibodies, Neoplasm; Antigens, Neoplasm; Antigens, Surface; Carcinoembryonic Antigen; Cell Differentiation; Cytoskeletal Proteins; Desmoplakins; Epithelium; Hematopoietic Stem Cells; Immunologic Techniques; Keratins; Melanoma; Membrane Proteins; Mucin-1; Neoplasms

Inorganic metals and minerals for which there is evidence of carcinogenicity are identified. The risk of cancer from contact with them in the work place, the general environment, and under conditions of clinical (medical) exposure is discussed. The evidence indicates that minerals and metals most often influence cancer development through their action as cocarcinogens. The relationship between the physical form of mineral fibers, smoking and carcinogenic risk is emphasized. Metals are categorized as established (As, Be, Cr, Ni), suspected (Cd, Pb) and possible carcinogens (Table 6), based on the existing in vitro, animal experimental and human epidemiological data. Cancer risk and possible modes of action of elements in each class are discussed. Views on mechanisms that may be responsible for the carcinogenicity of metals are updated and analysed. Some specific examples of cancer risks associated with the clinical use of potentially carcinogenic metals and from radioactive pharmaceuticals used in therapy and diagnosis are presented. Questions are raised as to the effectiveness of conventional dosimetry in accurately measuring risk from radiopharmaceuticals.

Topics: Animals; Biological Availability; Chromosome Aberrations; DNA; DNA Damage; Humans; Keratins; Lithium; Metals; Minerals; Mutation; Neoplasms; Platinum; Radioisotopes; Radiotherapy Dosage; Risk

Topics: Adenocarcinoma; Adenoma, Islet Cell; Animals; Antibodies, Monoclonal; Breast Neoplasms; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Cytoskeletal Proteins; Desmoplakins; Desmosomes; Electrophoresis, Polyacrylamide Gel; Granulosa Cell Tumor; Humans; Immunosorbent Techniques; Intermediate Filament Proteins; Intermediate Filaments; Keratins; Kidney Neoplasms; Lung Neoplasms; Melanoma; Membrane Proteins; Meningioma; Mesothelioma; Microscopy, Electron; Microscopy, Fluorescence; Neoplasms; Neurosecretory Systems; Skin Neoplasms

Keratin protein immunohistochemistry is a powerful diagnostic tool whose role has already been firmly established in many surgical pathology laboratories. Recent studies of the biology of keratin proteins have defined the heterogeneity of keratin protein expression among various epithelial tissues and their tumors and provide the basis for understanding the immunoreactivity of epithelial tumors with various keratin antibodies. Successful execution of the procedure requires attention to technical details such as the fixation of tissue, use of proteolytic enzymes such as trypsin for formalin-fixed tissues, and the choice of the appropriate antibody and controls. Broadly reactive polyclonal and monoclonal antibodies to keratins have been remarkably useful in identifying poorly differentiated and undifferentiated carcinomas. Monoclonal antibodies of restricted specificity and monospecific antibodies to keratin are under development and may prove helpful in defining the organ of origin of metastatic carcinomas. Keratin protein immunohistochemistry supplements existing information used by pathologists in diagnosis, and the immunohistochemical results should be interpreted in the light of the clinical findings, gross and microscopic pathology, and the results of any other special studies.

Topics: Animals; Antibodies; Antibodies, Monoclonal; Bone Neoplasms; Carcinoid Tumor; Carcinoma; Humans; Immunoassay; Keratins; Neoplasms; Sarcoma; Soft Tissue Neoplasms

Most mammalian nucleated cells contain a cytoplasmic fibril system called intermediate filaments. Unlike other cytoskeletal proteins, the subunit proteins of intermediate filaments show a remarkable cell-type-specificity in their expression: mesenchymal, muscle, epithelial, glial and neuronal cells containing each their cell type specific filaments. In this review we discuss the possibilities to use antibodies to these filaments in the diagnosis and histogenetic analysis of human tumors. This approach is based on findings which indicate that these filaments retain their cell-type specific expression also in tumors.

Topics: Antibodies; Carcinoma; Cytoskeleton; Desmin; Glial Fibrillary Acidic Protein; Glioma; Humans; Intermediate Filaments; Keratins; Mesothelioma; Neoplasms; Rhabdomyosarcoma; Sarcoma; Vimentin

Topics: Adult; Antibodies, Monoclonal; Cytoskeleton; Desmin; Diagnosis, Differential; Fluorescent Antibody Technique; Glial Fibrillary Acidic Protein; Humans; Immunoenzyme Techniques; Intermediate Filament Proteins; Intermediate Filaments; Keratins; Neoplasms; Vimentin

A panel of monoclonal antibodies to human intermediate filament proteins was tested on an unselected series of 246 neoplasms. The antibody panel includes two different anti-cytokeratin antibodies, an anti-vimentin antibody, and an anti-neurofilament antibody (Gown and Vogel, Am J Pathol 114:309, 1984). The studies were done on Carnoy's or methacarn-fixed, paraffin-embedded tissue. When used as a panel, they can unequivocally distinguish carcinomas, melanomas, and lymphomas. All carcinomas react with at least one of the anti-cytokeratin antibodies, and carcinomas can be subtyped based upon the pattern of reactivity with the two anti-cytokeratin antibodies. Melanomas react only with the anti-vimentin antibody, and lymphomas react with none of the antibodies. Neural and neuroendocrine tumors can be identified with the anti-neurofilament antibody. A minority of neoplasms, including lymphomas, seminomas, and some sarcomas, do not react with any of the antibodies. These antibodies are reliable diagnostic reagents that are useful in distinguishing different categories of human tumors.

Topics: Antibodies, Monoclonal; Antibody Specificity; Carcinoma; Cytoskeleton; Humans; Intermediate Filament Proteins; Keratins; Melanoma; Neoplasms; Nervous System Neoplasms; Vimentin

The cytoskeleton (CSK) of eukaryotic cells is composed of a complex interconnected network of filaments which is important in a wide variety of cellular functions including changes in cell shape, cell motility, mitosis, anchorage-dependent growth, and the localization of cellular organelles such as mitochondria, polyribosomes, and secretory granules. The various proteins comprising the cytoskeleton include actin in microfilaments, tubulin in microtubules, and the heterogeneous group of intermediate filament proteins that are associated with different cell types (keratin in epithelial cells, vimentin in fibroblasts, desmin in muscle cells, glial filament protein in glial cells, and the neurofilament protein subunits in neural tissue). Many other proteins in glial cells, and the neurofilament protein subunits in neural tissue). Many other proteins are closely associated with the cytoskeleton and influence its organization. In neoplastic cells, the expression of these different CSK proteins, especially the intermediate filament proteins, reflects their morphologic and functional differentiation. The carcinomas contain keratin; identification of individual keratin components may allow further sub-classification of carcinomas which is consistent with their tissue of origin. The sarcomas of muscle origin contain desmin. Vimentin is found primarily with cells of mesenchymal origin, but may coexist with other intermediate filament proteins in other tumors. One example is the coexistence of keratin and vimentin in tumors, such as mesotheliomas, which are derived from epithelial cells of embryonic origin. Glial fibrillary acidic protein is the most specific marker for glial tumors. Tumors of neural origin are characterized by the presence of neurofilament subunits. Therefore, analysis of CSK composition would be useful in diagnosis of clinical specimens and aid in studies of lineage relationships of neoplasms. Although no consistent differences in cytoskeletal structure between neoplastic and normal cells have been identified so far, the presence of more subtle biochemical alterations in the cytoskeletal structure of neoplastic cells that contributes to malignant behavior has not been ruled out. Since the cytoskeletal network plays an important role in cell shape and cell locomotion, which in turn are thought to be involved in growth control, invasion, and metastasis, further work is directed at identifying the various alterations in cytoskeletal architecture that

Topics: Actin Cytoskeleton; Actins; Animals; Astrocytes; Carcinoma; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Cytoskeletal Proteins; Cytoskeleton; Desmin; Epithelium; Glial Fibrillary Acidic Protein; Glioma; Granulocytes; Humans; Intermediate Filament Proteins; Keratins; Leukemia; Lymphoma; Macrophages; Microtubules; Molecular Weight; Muscles; Neoplasm Metastasis; Neoplasms; Neoplasms, Nerve Tissue; Neurons; Sarcoma; Tissue Distribution; Vimentin

Topics: Animals; Antibodies, Monoclonal; Cytoskeleton; Diagnosis, Differential; Glial Fibrillary Acidic Protein; Humans; Intermediate Filaments; Keratins; Neoplasms

Topics: Antibodies, Monoclonal; Carcinoma, Small Cell; Cell Line; Desmosomes; Humans; Intermediate Filament Proteins; Keratins; Lung Neoplasms; Neoplasms; Neuroectodermal Tumors, Primitive, Peripheral; Rhabdomyosarcoma

Topics: Antibodies, Monoclonal; Antibody Specificity; Cross Reactions; Epithelium; Epitopes; Genetic Markers; Humans; Intermediate Filament Proteins; Keratins; Neoplasms; Phenotype; Skin

Immunohistochemistry of intermediate filaments (IF) is a new and important way to evaluate the epithelial, mesenchymal, muscular, glial, or neural differentiation in tumors. This is based on the stable cell-type-specific expression of IF proteins in normal and neoplastic tissues. Immunohistochemical studies with antibodies to intermediate filaments have also given new perspectives in the histogenesis and biologic nature of many tumors. This article reviews both the recent findings and the authors' experience in the use of intermediate filament antibodies in tumor diagnosis and classification.

Topics: Adenocarcinoma; Antibodies, Neoplasm; Carcinoma, Squamous Cell; Desmin; Diagnosis, Differential; Glial Fibrillary Acidic Protein; Histocytochemistry; Humans; Immunochemistry; Keratins; Melanoma; Mesothelioma; Microfilament Proteins; Microscopy, Electron; Neoplasm Proteins; Neoplasms; Nervous System Neoplasms; Sarcoma; Soft Tissue Neoplasms; Thyroid Neoplasms; Urinary Bladder Neoplasms; Vimentin

Immunofluorescence and immunoperoxidase (peroxidase-antiperoxidase, PAP) techniques for the demonstration of neural and non-neural cell markers are contributing greatly to increase the diagnostic accuracy of difficult tumors of the central nervous system. Well characterized nervous system markers include glial fibrillary acidic (GFA) protein, the three protein subunits of neurofilaments, neuron-specific enolase (NSE), myelin basic protein, and S-100 protein. The most important and reliable of these is GFA protein, which is widely in use for the immunohistochemical diagnosis of tumors of the glioma group. Its many practical applications are reviewed and illustrated. Other neural markers, in particular the specificity of NSE and S-100 protein, need to be critically evaluated. Problems related to the immunohistochemical diagnosis of central neuroepithelial tumors of putative neuroblastic origin remain complex and still need to be resolved. Non-neural markers, such as vimentin, desmin, cytokeratins, Factor VIII, alpha-fetoprotein, human chorionic gonadotropin, and immunoglobulins have well defined, although more restricted, applications in surgical neuropathology.

Topics: alpha-Fetoproteins; Antibodies, Monoclonal; Antigens; Carcinoma; Central Nervous System Diseases; Chorionic Gonadotropin; Cytoskeleton; Desmin; Factor VIII; Fluorescent Antibody Technique; Glial Fibrillary Acidic Protein; Histocytochemistry; Humans; Immune Sera; Immunoenzyme Techniques; Immunoglobulins; Intermediate Filament Proteins; Keratins; Lymphoma; Medical Oncology; Meningeal Neoplasms; Myelin Basic Protein; Neoplasm Metastasis; Neoplasms; Neoplasms, Germ Cell and Embryonal; Neurology; Oligodendroglia; Phosphopyruvate Hydratase; S100 Proteins; Sarcoma; Vascular Diseases; Vimentin; von Willebrand Factor

Topics: Animals; Cells, Cultured; Epithelium; Histocytochemistry; Humans; Intermediate Filament Proteins; Keratins; Neoplasms

In most cell types intermediate or 10-mm filaments (IF) are a major cytoskeletal organization and, thus, directly or indirectly influence the structural appearance of the cytoplasm. In line with the cell type-specific expression patterns of different IF proteins in normal animal and human tissue, IF typing distinguishes the major tumor groups, as documented by results with several hundred human tumors classified by conventional histologic methods. Carcinomas are characterized by cytokeratins, sarcomas of muscle cells by desmin, nonmuscle sarcomas by vimentin, and gliomas by glial fibrillary acidic protein. Furthermore, certain tumors originating from the sympathetic nervous system, e.g., ganglioneuroblastoma, pheochromocytoma, and at least some neuroblastomas, are characterized by the presence of neurofilaments. Carcinomas can often be further subdivided with regard to their possible derivation by examining their cytokeratin profiles. The IF type characteristic of the cell of origin seems to be kept not only in the primary tumor but usually also in solid metastases. In general, tumors do not acquire additional IF types. Therefore, IF typing can provide an unambiguous and rapid characterization in certain cases, that are difficult to diagnose by conventional techniques. Some useful examples are the small cell tumors of childhood and the discrimination between undifferentiated carcinoma and lymphoma. IF typing of a few tumors has already led to a revision or reconsideration of the original light microscopic diagnosis. The combined results indicate that at least certain carcinomas, as well as certain other tumor types, seem to arise by the selective multiplication of a particular and identifiable cell type present in the normal tissue. The procedure is not restricted to tumor material. IF typing of Mallory bodies, Alzheimer's disease tangles, certain myopathies, and the cells of the amniotic fluid offers further interesting applications. Thus, IF typing should become a valuable new tool both in histology and surgical pathology.

Topics: Amniotic Fluid; Animals; Carcinoma; Cells, Cultured; Cytoskeleton; Desmin; Embryo, Mammalian; Fluorescent Antibody Technique; Ganglioneuroma; Glial Fibrillary Acidic Protein; Glioma; Humans; Intermediate Filament Proteins; Keratins; Muscular Diseases; Neoplasm Metastasis; Neoplasms; Neuroblastoma; Pheochromocytoma; Sarcoma; Vimentin

Topics: Adenocarcinoma; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Cell Differentiation; Cells, Cultured; Digestive System Neoplasms; Epithelium; Female; Histocytochemistry; Humans; Keratins; Molecular Weight; Neoplasms

Intermediate-sized filaments represent a class of morphologically similar but biochemically and immunologically distinguishable cytoplasmic protein polymer structures. Five major filament types have been identified (cytokeratin, vimentin, desmin, neurofilament protein, glia filament protein) and antibodies to these proteins have been used for distinguishing different cell types and tumors derived therefrom. Epithelial and carcinoma cells are characterized by the presence of cytokeratin filaments and desmosomal elements identified by antibodies to certain high molecular weight proteins of desmosomal plaques. However, the specific pattern of cytokeratin polypeptides is different in different epithelia. The potential value of cell type identification by immunological reactions with antibodies to cytoskeletal proteins in tumor diagnosis is discussed.

Topics: Animals; Antigens, Neoplasm; Cattle; Cell Membrane; Cytoskeleton; Desmosomes; Epithelium; Intermediate Filament Proteins; Keratins; Neoplasms; Protein Precursors; Rats

Topics: Animals; Autoradiography; Biological Assay; Circadian Rhythm; Cyclic AMP; Dialysis; Electrophoresis; Epinephrine; Epithelial Cells; Epithelium; Feedback; Growth Inhibitors; In Vitro Techniques; Keratins; Mice; Mitosis; Neoplasms; RNA; Skin; Time Factors

To examine whether a reported history of cataract, a possible marker of aging, is associated with future mortality.. Participants were 18,669 of the 22,071 U.S. male physicians enrolled in the Physicians' Health Study I who had complete information at study entry, including self-report of presence or absence of baseline cataract. Participants were without a previous history of myocardial infarction, stroke, transient cerebral ischemia, or cancer (except non-melanoma skin cancer). Reported deaths were confirmed by an End Points Committee of physicians.. A total of 581 participants reported a personal history of cataract at baseline. During an average of 12.4 years of follow-up, there were 1,514 deaths including 496 due to cardiovascular (CV) and 1,018 due to non-CV causes. After adjustment for differences in age, men who reported cataract at baseline had a non-significant 9% increased risk of death from any cause compared to men who did not report cataract (RR, 1.09; 95% CI, 0.91-1.30). The RRs were 1.03 (95% CI, 0.75-1.41) for CV death and 1.12 (95% CI, 0.90-1.40) for non-CV death. Adjustment for other risk factors had little effect on these estimates. Similar results were obtained in analyses conducted separately among those with and without self-reported diabetes at baseline.. These results from a population of generally healthy physicians indicate that a report of a history of cataract is not associated with any material increase in mortality after adjustment for differences in age between men with and without cataract. Additional investigation of this cohort is in progress to determine whether incident age-related cataracts as well as their subtypes, confirmed by medical record review, are associated with increased mortality.

Topics: Adult; Aged; Aged, 80 and over; Aging; Aspirin; Cardiovascular Diseases; Cataract; Cause of Death; Humans; Keratins; Male; Middle Aged; Neoplasms; Physicians; Platelet Aggregation Inhibitors; Self Disclosure; Surveys and Questionnaires; Survival Rate; United States

To study the expression of keratin 19, 20 (K19, K20) in different tumor cell lines and tumor tissues and its clinical implication.. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was employed to examine the specific expression of K19 and K20 mRNA in eleven tumor cell lines and 33 corresponding tumor tissue specimens.. The expression of K19 mRNA was detected in 4 kinds of tumor cell lines and all tumor tissues examined, but the magnitude of expression differed, with a difference ranging from 1.7 to 10 folds for the same type of cancer. In some patients, the level of expression was as low as 12% of the positive control. K20 mRNA expression was negative for lung and esophageal tumor cell lines and the corresponding carcinoma specimens. In one of 6 bladder cancer specimens and in 4 of 5 colorectal cancer tissues, K20 expression was positive, at a level of 41%-77% of the positive control. There was no expression of K20 in bladder tumor cell line EJ1 and colorectal tumor cell line SW480.. These results demonstrate that K19 and K20 may be used as a valuable marker for detecting circulating cancer cells, but the low level of expression in some cases of carcinoma would probably result in false negative results.

Topics: Biomarkers, Tumor; Colorectal Neoplasms; Down-Regulation; Esophageal Neoplasms; Humans; Intermediate Filament Proteins; Keratin-20; Keratins; Lung Neoplasms; Neoplasms; Neoplastic Cells, Circulating; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Orally administered retinoids are synthetic derivatives of vitamin A. This new group of drugs (not yet available for general use in the United States) has been effective in experimental trials for treatment of a wide range of skin diseases. The current status of two of these drugs, isotretinoin (13-cis-retinoic acid) and etretinate (Ro 10-9359), is herein reviewed.

Topics: Acne Vulgaris; Administration, Oral; Child; Clinical Trials as Topic; Facial Dermatoses; Female; Humans; Isomerism; Isotretinoin; Keratins; Keratitis; Neoplasms; Psoriasis; Skin Diseases; Tretinoin; Xerostomia

Postoperative spindle cell nodule (PSCN) is a pseudosarcomatous proliferative lesion of unclear molecular genetic origins.. We examined seven patients with PSCN, using routine haematoxylin-eosin (H&E) slide preparations and a series of immunostains. The latter targeted keratin, vimentin, α-smooth muscle actin (SMA), anaplastic lymphoma kinase (ALK [D5F3]), and other proteins. Ubiquitin-specific peptidase 6 (USP6) and anaplastic lymphoma kinase (ALK) gene rearrangements were also analysed by fluorescence in situ hybridization (FISH). There were histories of prior surgical intervention (n = 6) or trauma (n = 1) in all seven patients. All lesions were highly cellular and mitotically active spindle cell proliferations, with no cytologic atypia, nuclear pleomorphism, or aberrant mitoses. Immunohistochemical (IHC) staining disclosed focal, weak keratin positivity in two lesions, whereas vimentin (diffuse, strongly positive) and SMA (tram-track pattern) were present in each instance, and ALK (D5F3) was entirely negative. FISH analysis confirmed USP6 gene rearrangements in all seven cases, showing no ALK gene rearrangements. RNA sequencing results showed an MYH9::USP6 gene fusion in only one lesion (No. 6).. A subset of PSCN is marked by USP6 gene rearrangements, a genetic feature of nodular fasciitis (NF). Given its similarity to NF, a designation as USP6-associated neoplasm (UAN) seems reasonable, signifying a transient clonal neoplastic lesion.

Topics: Anaplastic Lymphoma Kinase; Fasciitis; Gene Rearrangement; Humans; In Situ Hybridization, Fluorescence; Keratins; Neoplasms; Proto-Oncogene Proteins; Ubiquitin Thiolesterase; Vimentin

Two-dimensional transition metal dichalcogenides have attracted widespread attention in cancer theranostics due to their high specific surface area and excellent photothermal conversion properties. However, their dimensions and biodegradability have limited the exploration of the therapeutic properties of transition metal dichalcogenides. Herein, we explore the mechanism of the keratin α-helix-to-random coil transition, as an actuation mechanism for the controllable design and precise synthesis of two-dimension copper sulfide nanoflakes (CuS NFs) with high absorption in the NIR-II window. Upon mixing keratin and Cu

Topics: Biomimetics; Copper; Humans; Keratins; Neoplasms; Precision Medicine; Sulfides

Topics: Aged, 80 and over; Autopsy; Diagnosis, Differential; Female; Fibroblasts; Humans; Immunohistochemistry; Keratins; Lymph Nodes; Neoplasms; Neoplasms, Bone Tissue; Skull

Feather keratin (FK) was used as a multifunctional crosslinker for the fabrication of both pH responsive ionically and reduction responsive covalently crosslinked hyaluronic acid (HA) nanogels, as pH-activated surface charge-reversal double-crosslinked protein/polysaccharide complex nanocarriers for the tumor-targeting DOX delivery. The effect of the preparation condition on the diameter and the drug-loading capacity of the resultant drug delivery systems (DDSs) were investigated in detail. The in vitro controlled release profiles indicated the pH/reduction dual-responsive sustained release of DOX from the proposed double-crosslinked DOX@C-FK/HA nanogels. The in vitro experiments demonstrated that the DOX@C-FK/HA nanogels could be internalized into the HCT 116 cells via a CD44-receptor-mediated endocytosis pathway, exhibiting an enhanced anti-tumor efficacy than the free DOX.

Topics: Animals; Cross-Linking Reagents; Delayed-Action Preparations; Doxorubicin; Feathers; Gels; HCT116 Cells; Hep G2 Cells; Humans; Hydrogen-Ion Concentration; Keratins; Nanoparticles; Neoplasms

Gold nanorods (AuNRs) have been widely applied to photothermal therapy against cancer. However, the chemically synthesized AuNRs such as that via seed-mediate method usually demonstrated a high cytotoxicity due to the existence of cetyltrimethylammonium bromide (CTAB) coating. In this work, keratin, a family of cysteine-rich structural fibrous proteins was used for the first time to encapsulate AuNRs by a simple mixing method. Compared with CTAB-AuNRs, the keratin-encapsulated AuNRs (AuNRs@Kr) showed an improved colloid stability and good biocompatibility including low cytotoxicity and hemolytic effect. Moreover, AuNRs@Kr exhibited great potential as drug carriers with redox-responsive drug release behavior, due to the high concentration of disulfide crosslinking in keratin coating, and the DOX-loaded AuNRs@Kr demonstrated higher chemo-photothermal synergistic therapy efficiency against 4T1 cells compared with either free DOX or AuNRs@Kr alone, suggesting a promising nanoplatform for cancer therapy.

Topics: Doxorubicin; Gold; Humans; Keratins; Nanotubes; Neoplasms; Phototherapy

The combination of chemotherapy and photodynamic therapy (PDT) is considered a valuable strategy for increasing therapeutic response in cancer treatment, and the re-formulation of pharmaceuticals in biocompatible nanoparticles (NPs) is particularly appealing for the possibility of co-loading drugs exerting cytotoxicity by different mechanisms, with the aim to produce synergic effects. We report the in-water synthesis of a novel keratin-based nanoformulation for the co-delivery of the antimitotic Docetaxel (DTX) and the photosensitizer Chlorin e6 (Ce6). The drug-induced aggregation method allowed the formation of monodisperse NPs (DTX/Ce6-KNPs) with an average diameter of 133 nm and loaded with a drug ratio of 1:1.8 of Ce6 vs DTX. The efficacy of DTX/Ce6-KNPs was investigated in vitro in monolayers and spheroids of DTX-sensitive HeLa (HeLa-P) and DTX-resistant HeLa (HeLa-R) cells. In monolayers, the cytotoxic effects of DTX/Ce6-KNPs toward HeLa-P cells were comparable to those induced by free DTX + Ce6, while in HeLa-R cells the drug co-loading in KNPs produced synergic interaction between chemotherapy and PDT. Moreover, as respect to monotherapies, DTX/Ce6-KNPs induced stronger cytotoxicity to both HeLa-P and HeLa-R multicellular spheroids and reduced their volumes up to 50%. Overall, the results suggest that KNPs are very promising systems for the co-delivery of chemotherapeutics and PSs, favoring synergic interactions between PDT and chemotherapy.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Biocompatible Materials; Cell Membrane Permeability; Cell Survival; Chlorophyllides; Docetaxel; Drug Carriers; Drug Compounding; Drug Liberation; Drug Synergism; HeLa Cells; Humans; Keratins; Nanoparticles; Neoplasms; Photochemotherapy; Photosensitizing Agents; Porphyrins; Spheroids, Cellular

Immunocytochemistry (ICC) is utilized as an advanced technique in veterinary cytology. In tumor diagnosis, cytokeratin and vimentin are markers used to distinguish the origin of tumor cells. Standard enzyme-based ICC has limitations in clinical use; and therefore, more convenient and reliable methods are needed.. The purpose of this study was to develop a rapid multiple immunofluorescent (RMIF) detection method for dual cytokeratin and vimentin staining on cytology slides in dogs.. Air-dried smear samples from solid tumors and sediments of pleural effusions were prepared from dogs (n = 14) that were admitted to the Veterinary Teaching Hospital, Kagoshima University, Japan. Mouse monoclonal anti-human cytokeratin (AE1/AE3) and rabbit monoclonal anti-human vimentin (SP20) antibodies were used as primary antibodies, followed by staining with Alexa Fluor-conjugated secondary antibodies. Staining using the RMIF method was compared with enzyme-based ICC staining.. Rapid multiple immunofluorescent immunostaining was clear and specific in the evaluated smears, whereas the enzyme-based ICC showed nonspecific signals. By using the RMIF staining method, epithelial cells, mesenchymal cells, and mesothelial cells could be classified on a single smear of a pleural effusion. In smears of lymph nodes with epithelial tumor metastases, the RMIF method successfully detected metastatic epithelial tumor cells.. The RMIF method might be a useful tool for diagnostic cytology in veterinary medicine.

Topics: Animals; Biomarkers, Tumor; Cytodiagnosis; Dog Diseases; Dogs; Fluorescent Antibody Technique; Keratins; Neoplasms; Time Factors; Vimentin

Genomics has provided a detailed structural description of the cancer genome. Identifying oncogenic drivers that work primarily through dosage changes is a current challenge. Unrestrained proliferation is a critical hallmark of cancer. We constructed modular, barcoded libraries of human open reading frames (ORFs) and performed screens for proliferation regulators in multiple cell types. Approximately 10% of genes regulate proliferation, with most performing in an unexpectedly highly tissue-specific manner. Proliferation drivers in a given cell type showed specific enrichment in somatic copy number changes (SCNAs) from cognate tumors and helped predict aneuploidy patterns in those tumors, implying that tissue-type-specific genetic network architectures underlie SCNA and driver selection in different cancers. In vivo screening confirmed these results. We report a substantial contribution to the catalog of SCNA-associated cancer drivers, identifying 147 amplified and 107 deleted genes as potential drivers, and derive insights about the genetic network architecture of aneuploidy in tumors.

Topics: Aneuploidy; Animals; Cell Line, Tumor; Cell Proliferation; Chromosome Mapping; Chromosomes; E2F1 Transcription Factor; Female; Gene Library; Genomics; Humans; Keratins; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasms; Oncogenes; Open Reading Frames; RNA Interference; RNA, Small Interfering

Frozen sections (FS) of tumor samples represent a cornerstone of pathological intraoperative consultation and have an important role in the microscopic analysis of specimens during surgery. So far, immunohistochemical (IHC) stainings on FS have been demonstrated for a few markers using manual methods. Microfluidic technologies have proven to bring substantial improvement in many fields of diagnostics, though only a few microfluidic devices have been designed to improve the performance of IHC assays. In this work, we show optimization of a complete pan-cytokeratin chromogenic immunostaining protocol on FS using a microfluidic tissue processor into a protocol taking <12 min. Our results showed specificity and low levels of background. The dimensions of the microfluidic prototype device are compatible with the space constraints of an intraoperative pathology laboratory. We therefore anticipate that the adoption of microfluidic technologies in the field of surgical pathology can significantly improve the way FSs influence surgical procedures.

Topics: Breast; Coloring Agents; Equipment Design; Female; Humans; Immunohistochemistry; Keratins; Male; Microfluidic Analytical Techniques; Neoplasms; Prostate; Ureter

Circulating tumor cells (CTCs) are disseminated tumor cells that reflect the tumors of origin and can provide a liquid biopsy that would potentially enable noninvasive tumor profiling, treatment monitoring, and identification of targeted treatments. Accurate and rapid purification of CTCs holds great potential to improve cancer care but the task remains technically challenging. Microfluidic isolation of CTCs within microscale vortices enables high-throughput and size-based purification of rare CTCs from bodily fluids. Collected cells are highly pure, viable, and easily accessible, allowing seamless integration with various downstream applications. Here, we describe how to fabricate the High-Throughput Vortex Chip (Vortex-HT) and to process diluted whole blood for CTC collection. Lastly, immunostaining and imaging protocols for CTC classification and corresponding CTC image galleries are reported.

Topics: Antibodies, Monoclonal; Biomarkers, Tumor; Cell Count; Cell Separation; Cell Size; Dimethylpolysiloxanes; Equipment Design; Fluorescein-5-isothiocyanate; Fluorescent Antibody Technique; Fluorescent Dyes; Humans; Immunoconjugates; Keratins; Lab-On-A-Chip Devices; Leukocyte Common Antigens; Microfluidic Analytical Techniques; Neoplasms; Neoplastic Cells, Circulating; Nylons; Phycoerythrin; Protein Binding; Rheology

Isolation by size using a filter membrane offers an antigen-independent method for capturing rare cells present in blood of cancer patients. Multiple cell types, including circulating tumor cells (CTCs), captured on the filter membrane can be simultaneously identified via immunocytochemistry (ICC) analysis of specific cellular biomarkers. Here, we describe an automated microfluidic filtration method combined with a liquid handling system for sequential ICC assays to detect and enumerate non-hematologic rare cells in blood.

Topics: Antibodies, Monoclonal; Biomarkers, Tumor; Cell Line, Tumor; Cell Separation; Cell Size; Endothelial Cells; Epithelial Cell Adhesion Molecule; Equipment Design; Filtration; Fluorescent Antibody Technique; Fluorescent Dyes; Humans; Immunoconjugates; Keratins; Mesenchymal Stem Cells; Microfluidic Analytical Techniques; Neoplasms; Neoplastic Cells, Circulating; Neoplastic Stem Cells; Protein Binding; Rheology

The RareCyte CyteFinder instrument is an automated scanner that allows rapid identification of circulating tumor cells (CTCs) on microscope slides prepared by the AccuCyte process (see Chapter 13 ) and stained by immunofluorescence. Here, we present the workflow for CyteFinder scanning, analysis, and CyteMapper scan review which includes CTC confirmation and report generation.

Topics: Antibodies, Monoclonal; Automation, Laboratory; Biomarkers, Tumor; Cell Count; Cell Line, Tumor; Cell Separation; Cells, Immobilized; Centrifugation; Epithelial Cell Adhesion Molecule; Equipment Design; Fluorescent Antibody Technique; Fluorescent Dyes; Humans; Image Processing, Computer-Assisted; Immunoconjugates; Keratins; Leukocyte Common Antigens; Neoplasms; Neoplastic Cells, Circulating; Protein Binding; Single-Cell Analysis

The identification of therapeutically targetable mutations in circulating tumor cells (CTCs) from cancer patient blood is increasingly used to personalize patient care. Here, we describe a novel approach for the enumeration, capture, and molecular analysis of CTCs from blood using an FDA-approved CTC enrichment and enumeration platform followed by dielectrophoretic capture and next-generation sequencing.

Topics: Antibodies, Monoclonal; Biomarkers, Tumor; Cell Count; Cell Line, Tumor; Cell Separation; Electrophoresis; Epithelial Cell Adhesion Molecule; Equipment Design; Gene Library; High-Throughput Nucleotide Sequencing; Humans; Keratins; Leukocyte Common Antigens; Neoplasms; Neoplastic Cells, Circulating; Nucleic Acid Amplification Techniques; Precision Medicine; Single-Cell Analysis

Metastasis is the cause of 90% of human cancer deaths. Circulating tumor cells (CTCs) in the peripheral blood and/or lymphatic vessels are cells shed from primary tumors and considered to be precursors of metastasis. Study of CTCs allows the serial monitoring of tumor progression and may provide predictive and prognostic biomarkers in clinic. Current CTC isolation and detection technologies encounter several challenges, including: heterogeneity of CTCs, low cell viability and/or high rate of contamination post-isolation, and the inability to distinguish viable/invasive from nonviable/nonfunctional CTCs, all of which can limit in vitro and in vivo characterization of CTCs. Here, we describe a new method to detect and enumerate of CTCs based on their invasive property.

Topics: Antibodies, Monoclonal; Biomarkers, Tumor; Cell Count; Cell Line, Tumor; Cell Movement; Cell Separation; Cell Survival; Centrifugation, Density Gradient; Collagen; Diffusion Chambers, Culture; Drug Combinations; Ficoll; Fluorescent Dyes; Humans; Keratins; Laminin; Leukocyte Common Antigens; Microscopy, Fluorescence; Neoplasms; Neoplastic Cells, Circulating; Protein Binding; Proteoglycans

Immunocytochemistry (ICC) is an advanced diagnostic technique used in the field of veterinary cytology. We recently developed a rapid ICC method for the detection of cytokeratin and vimentin in dogs, which helps to determine whether tumor cells are of epithelial or nonepithelial origin. However, the diagnostic value of this rapid ICC method in neoplastic diseases of dogs has not been assessed yet.. The aim of the present study was to assess the diagnostic accuracy of rapid ICC compared to standard immunohistochemistry (IHC).. Air-dried smear samples and formalin-fixed paraffin sections were prepared from tumors excised from dogs (n = 30). Immunosignals for cytokeratin and vimentin were detected in smear samples by rapid ICC, and in paraffin sections by standard IHC. Signals in smear samples detected by rapid ICC were compared with positive staining in paraffin sections detected by standard IHC and analyzed for statistical significance (kappa statistic).. Rapid ICC detected specific immunosignals in 25/30 cases (83.3%), and nonspecific signals were detected in 5/30 cases. Statistical analysis revealed fair agreement in epithelial tumors (n = 16) with cytokeratin (κ = 0.236) and vimentin (κ = 0.294). In nonepithelial tumors (n = 14), almost perfect agreement was demonstrated with cytokeratin (κ = 0.857) and vimentin (κ = 0.857).. The rapid ICC method can be a useful tool for the diagnostic cytology of neoplastic tissues in dogs.

Topics: Animals; Biomarkers, Tumor; Dog Diseases; Dogs; Immunohistochemistry; Keratins; Neoplasms; Paraffin Embedding; Vimentin

The variable quality of histochemical and immunohistochemical staining of tissues may be attributed to pre-analytical and analytical variables. Both categories of variables frequently are undefined or inadequately controlled during specimen collection and preparation. Pre-analytical variables may alter the molecular composition of tissues, which results in variable staining; such variations may cause problems when different tissues are used as staining controls. We developed a standard tissue for use as a staining control. Our standard tissue contains five components: 1) nine combined human cell lines mixed with stroma from human spleen; 2) a squamous cancer cell line, A431; 3) fungus; 4) transverse sections of the mosquitofish and 5) normal human spleen. The first three components were embedded in HistoGel(™) and all components were processed to paraffin and used to construct a single standard paraffin block. The muscles of mosquitofish and arteries of the spleen are positive controls for eosin staining, while other tissues are useful for assessing hematoxylin staining. The mosquitofish tissues also are excellent controls for the Masson trichrome stain and all mucin-related histochemical stains that we tested. The goblet cells of the intestine and skin stained strongly with Alcian blue, pH 2.5 (AB-2.5), mucicarmine, colloidal iron, periodic acid Schiff (PAS) or PAS-hematoxylin (PASH) and combination stains such as colloidal iron-PASH. Cell lines were not useful for evaluating histochemical stains except for PASH. The splenic stroma was a useful control for AB-2.5; however, eosin and mucin stains stained cell lines poorly, probably due to their rapid growth and associated loss of some differentiated characteristics such as production of mucins. Nevertheless, the cell lines were a critical control for immunohistochemical stains. Immunostaining of specific cell lines was consistent with the presence of markers, e.g., EGFr in DU145 cells. The cell lines expressed a wide range of markers, so they were useful controls for immunohistochemical staining including EGFr, HER2, E-cadherin, cytokeratins, Ki67, PCNA, estrogen receptor, progesterone receptor, CD3, CD20 and CD45, activated (cleaved) caspase 3 and Bcl-2. The cell lines also were a control for the TUNEL stain.

Topics: Biomarkers; Cadherins; Carmine; Humans; Immunohistochemistry; Keratins; Neoplasms; Staining and Labeling

Topics: AC133 Antigen; Antigens, CD; Glycoproteins; Humans; Keratins; Leukocytes, Mononuclear; Neoplasms; Neoplastic Cells, Circulating; Peptides

Accurate detection and analysis of circulating tumor cells plays an important role in the diagnosis and treatment of metastatic cancer treatment.. A cell microarray chip was used to detect spiked carcinoma cells among leukocytes. The chip, with 20,944 microchambers (105 µm width and 50 µm depth), was made from polystyrene; and the formation of monolayers of leukocytes in the microchambers was observed. Cultured human T lymphoblastoid leukemia (CCRF-CEM) cells were used to examine the potential of the cell microarray chip for the detection of spiked carcinoma cells. A T lymphoblastoid leukemia suspension was dispersed on the chip surface, followed by 15 min standing to allow the leukocytes to settle down into the microchambers. Approximately 29 leukocytes were found in each microchamber when about 600,000 leukocytes in total were dispersed onto a cell microarray chip. Similarly, when leukocytes isolated from human whole blood were used, approximately 89 leukocytes entered each microchamber when about 1,800,000 leukocytes in total were placed onto the cell microarray chip. After washing the chip surface, PE-labeled anti-cytokeratin monoclonal antibody and APC-labeled anti-CD326 (EpCAM) monoclonal antibody solution were dispersed onto the chip surface and allowed to react for 15 min; and then a microarray scanner was employed to detect any fluorescence-positive cells within 20 min. In the experiments using spiked carcinoma cells (NCI-H1650, 0.01 to 0.0001%), accurate detection of carcinoma cells was achieved with PE-labeled anti-cytokeratin monoclonal antibody. Furthermore, verification of carcinoma cells in the microchambers was performed by double staining with the above monoclonal antibodies.. The potential application of the cell microarray chip for the detection of CTCs was shown, thus demonstrating accurate detection by double staining for cytokeratin and EpCAM at the single carcinoma cell level.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Carcinoma; Cell Adhesion Molecules; Cell Line, Tumor; Cell Separation; Epithelial Cell Adhesion Molecule; Humans; Keratins; Leukocytes; Microscopy, Fluorescence; Neoplasm Metastasis; Neoplasms; Neoplastic Cells, Circulating; Oligonucleotide Array Sequence Analysis; Polystyrenes; Reproducibility of Results; T-Lymphocytes

Presence of circulating tumor cells (CTC) in metastatic carcinoma is associated with poor survival. Phenotyping and genotyping of CTC may permit "real-time" treatment decisions, provided CTCs are available for examination. Here, we investigate what is needed to detect CTC in all patients.. CTCs enumerated in 7.5 mL of blood together with survival from 836 patients with metastatic breast, colorectal, and prostate cancer were used to predict the CTC concentration in the 42% of these patients in whom no CTCs were found and to establish the relation of concentration of CTCs with survival. Influence of different CTC definitions were investigated by automated cell recognition and a flow cytometric assay without an enrichment or permeabilization step.. A log-logistic regression of the log of CTC yielded a good fit to the CTC frequency distribution. Extrapolation of the blood volume to 5 L predicted that 99% of patients had at least one CTC before therapy initiation. Survival of patients with EpCAM+, cytokeratin+, CD45- nucleated CTCs is reduced by 6.6 months for each 10-fold CTC increase. Using flow cytometry, the potential three-fold recovery improvement is not sufficient to detect CTC in all patients in 7.5 mL of blood.. EpCAM+, cytokeratin+, CD45- nucleated CTCs are present in all patients with metastatic breast, prostate, and colorectal cancer and their frequency is proportional to survival. To serve as a liquid biopsy for the majority of patients, a substantial improvement of CTC yield is needed, which can only be achieved by a dramatic increase in sample volume.

Topics: Adult; Aged; Antigens, Neoplasm; Biomarkers, Tumor; Cell Adhesion Molecules; Cell Count; Cell Line, Tumor; Epithelial Cell Adhesion Molecule; Female; Flow Cytometry; Humans; Keratins; Leukocyte Common Antigens; Male; Middle Aged; Neoplasm Metastasis; Neoplasm Staging; Neoplasms; Neoplastic Cells, Circulating; Prognosis

This unit contains a detailed protocol for the simultaneous flow cytometric measurement of tumor cells, stromal cells, and DNA content of formalin-fixed, paraffin-embedded (FFPE) tissues. The vimentin-positive stromal cell fraction can be used as an internal reference for DNA content assessments. This allows clear detection of keratin-positive tumor cells with a DNA index lower than 1.0 and of keratin-positive tumor cells with a DNA close to 1.0 in overall DNA aneuploid samples, thus improved DNA ploidy assessment in FFPE carcinomas. Furthermore, the protocol is useful for studying molecular genetic alterations and intratumor heterogeneity in archival FFPE samples. Keratin-positive tumor cell fractions can be flow-sorted for further molecular genetic analysis, while DNA from the sorted vimentin-positive stromal cells can serve as a reference when normal tissue of the patient is not available.

Topics: Cell Separation; DNA; DNA, Neoplasm; Flow Cytometry; Humans; Keratins; Methods; Neoplasms; Paraffin Embedding; Ploidies; Stromal Cells; Vimentin

Pan-cytokeratin (pan-CK) and low molecular weight cytokeratin (LMWCK) tests are the most common immunohistochemistry (IHC) tests used to support evidence of epithelial differentiation. Canadian Immunohistochemistry Quality Control (CIQC), a new provider of proficiency testing for Canadian clinical IHC laboratories, has evaluated the performance of Canadian IHC laboratories in two proficiency testing challenges for both pan-CK and LMWCK.. CIQC has designed a 70-sample tissue microarray (TMA) for challenge 1 and a 30-sample TMA for challenge 2. There were 13 participants in challenge 1, and 62 in challenge 2. All results were evaluated and scored by CIQC assessors and compared with reference laboratory results.. Participating laboratories often produced false-negative results that ranged from 20% to 80%. False-positive results were also detected. About half of participating clinical laboratories have inappropriately calibrated IHC tests for pan-CK and LMWCK, which are the most commonly used markers for demonstration of epithelial differentiation. The great majority of laboratories were not aware of the problem with calibration of pan-CK and LMWCK tests because of inappropriate selection of external positive controls and samples for optimisation of these tests. Benign liver and kidney are the most important tissues to include as positive controls for both pan-CK and LMWCK.. Participation in external quality assurance is important for peer comparison and proper calibration of IHC tests, which is also helpful for appropriate selection of positive control material and material for optimisation of the tests.

Topics: Biomarkers, Tumor; Canada; False Negative Reactions; False Positive Reactions; Female; Humans; Keratins; Laboratories; Male; Molecular Weight; Neoplasm Proteins; Neoplasms; Quality Control; Tissue Array Analysis

Tumour cells employ a variety of mechanisms to invade their environment and to form metastases. An important property is the ability of tumour cells to transition between individual cell invasive mode and collective mode. The switch from collective to individual cell invasion in the breast was shown recently to determine site of subsequent metastasis. Previous studies have suggested a range of invasion modes from single cells to large clusters. Here, we use a novel image analysis method to quantify and categorise invasion. We have developed a process using automated imaging for data collection, unsupervised morphological examination of breast cancer invasion using cognition network technology (CNT) to determine how many patterns of invasion can be reliably discriminated. We used Bayesian network analysis to probabilistically connect morphological variables and therefore determine that two categories of invasion are clearly distinct from one another. The Bayesian network separated individual and collective invading cell groups based on the morphological measurements, with the level of cell-cell contact the most discriminating morphological feature. Smaller invading groups were typified by smoother cellular surfaces than those invading collectively in larger groups. Interestingly, elongation was evident in all invading cell groups and was not a specific feature of single cell invasion as a surrogate of epithelial-mesenchymal transition. In conclusion, the combination of cognition network technology and Bayesian network analysis provides an insight into morphological variables associated with transition of cancer cells between invasion modes. We show that only two morphologically distinct modes of invasion exist.

Topics: Bayes Theorem; Cell Communication; Cell Line, Tumor; Humans; Keratins; Neoplasm Invasiveness; Neoplasms; Pathology; Pseudopodia

A limitation of positive selection strategies to enrich for circulating tumor cells (CTCs) is that there might be CTCs with insufficient expression of the surface target marker which may be missed by the procedure. We optimized a method for enrichment, subsequent detection and characterization of CTCs based on depletion of the leukocyte fraction.. The 2-step protocol was developed for processing 20 mL blood and based on red blood cell lysis followed by leukocyte depletion. The remaining material was stained with the epithelial markers EpCAM and cytokeratin (CK) 7/8 or for the melanoma marker HMW-MAA/MCSP. CTCs were detected by flow cytometry. CTCs enriched from blood of patients with carcinoma were defined as EpCAM+CK+CD45-. CTCs enriched from blood of patients with melanoma were defined as MCSP+CD45-. One-hundred-sixteen consecutive blood samples from 70 patients with metastatic carcinomas (n = 48) or metastatic melanoma (n = 22) were analyzed.. CTCs were detected in 47 of 84 blood samples (56%) drawn from carcinoma patients, and in 17 of 32 samples (53%) from melanoma patients. CD45-EpCAM-CK+ was detected in pleural effusion specimens, as well as in peripheral blood samples of patients with NSCLC. EpCAM-CK+ cells have been successfully cultured and passaged longer than six months suggesting their neoplastic origin. This was confirmed by CGH. By defining CTCs in carcinoma patients as CD45-CK+ and/or EpCAM+, the detection rate increased to 73% (61/84).. Enriching CTCs using CD45 depletion allowed for detection of epithelial cancer cells not displaying the classical phenotype. This potentially leads to a more accurate estimation of the number of CTCs. If detection of CTCs without a classical epithelial phenotype has clinical relevance need to be determined.

Topics: Antigens, Neoplasm; Ascites; Biomarkers, Tumor; Calibration; Cell Adhesion Molecules; Cell Line, Tumor; Epithelial Cell Adhesion Molecule; Humans; Immunomagnetic Separation; Keratins; Leukocyte Common Antigens; Lymphocyte Depletion; Melanoma; Microspheres; Nanoparticles; Neoplasms; Neoplastic Cells, Circulating; Pleural Effusion

Exponential advances in the quantitation of DNA variation and epigenetic states seem poised to convert much of biological research into a statistical exercise. But these developments also invite us to reimagine well-worn biological concepts on a grander scale. Somatic mosaicism refers to postzygotic mutations persisting in the individual, occasionally conspicuous to dermatologists as Blaschkoid, checkerboard, phylloid and patchy morphologies. A thoughtful examination of cutaneous mosaicism suggests, however, that virtually all of us may be somatic mosaics. Such genetic variability within individuals might explain localized presentations of disease and implies that some tissues literally evolve throughout life. We discuss here (i) the likely ubiquity of somatic mosaicism, (ii) the broad range of possible biological consequences and (iii) how experimentalists and clinicians may begin establishing genotype-to-phenotype correlates.

Topics: Cell Lineage; Clone Cells; Disease; Genetic Association Studies; Humans; Keratins; Mosaicism; Mutation; Neoplasms; Nevus; Sequence Analysis, DNA; Skin; Skin Diseases

An early diagnosis of malignancies correlates directly with a better prognosis. Yet for many malignancies there are no readily available, noninvasive, cost-effective diagnostic tests with patients often presenting too late for effective treatment. This article describes for the first time the use of fiber diffraction patterns of skin or fingernails, using X-ray sources, as a biometric diagnostic method for detecting neoplastic disorders including but not limited to melanoma, breast, colon and prostate cancers. With suitable further development, an early low-cost, totally noninvasive yet reliable diagnostic test could be conducted on a regular basis in local radiology facilities, as a confirmatory test for other diagnostic procedures or as a mass screening test using suitable small angle X-ray beam-lines at synchrotrons.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Child; Child, Preschool; Double-Blind Method; Female; Humans; Infant; Infant, Newborn; Keratins; Male; Middle Aged; Nail Diseases; Nails; Neoplasms; Sensitivity and Specificity; Skin; Skin Neoplasms; Synchrotrons; X-Ray Diffraction; Young Adult

To better understand the role of transcription factor NF-E2-related factor (NRF) 2 in the human and its contribution to cancer chemoprevention, we have knocked down its negative regulators, Kelch-like ECH-associated protein 1 (KEAP1) and broad-complex, tramtrack and bric à brac and cap'n'collar homology 1 (BACH1), in HaCaT keratinocytes. Whole-genome microarray revealed that knockdown of KEAP1 resulted in 23 messenger RNAs (mRNAs) being up-regulated > or = 2.0-fold. mRNA for aldo-keto reductase (AKR) 1B10, AKR1C1, AKR1C2 and AKR1C3 were induced to the greatest extent, showing increases of between 12- and 16-fold, whereas mRNA for glutamate-cysteine ligase catalytic and modifier subunits, NAD(P)H:quinone oxidoreductase-1 and haem oxygenase-1 (HMOX1) were induced between 2.0- and 4.8-fold. Knockdown of BACH1 increased HMOX1 135-fold but induced the other genes examined to a maximum of only 2.7-fold. Activation of NRF2, by KEAP1 knockdown, caused a 75% increase in the amount of glutathione in HaCaT cells and a 1.4- to 1.6-fold increase in their resistance to the electrophiles acrolein, chlorambucil and cumene hydroperoxide (CuOOH), as well as the redox-cycling agent menadione. Inhibition of glutathione synthesis during KEAP1 knockdown, by treatment with buthionine sulfoximine, abrogated resistance to acrolein, chlorambucil and CuOOH, but not to menadione. In contrast, knockdown of BACH1 did not increase glutathione levels or resistance to xenobiotics. Knockdown of NRF2 in HaCaT cells decreased glutathione to approximately 80% of normal homeostatic levels and similarly reduced their tolerance of electrophiles. Thus, the KEAP1-NRF2 pathway determines resistance to electrophiles and redox-cycling compounds in human keratinocytes through glutathione-dependent and glutathione-independent mechanisms. This study also shows that AKR1B10, AKR1C1 and AKR1C2 proteins have potential utility as biomarkers for NRF2 activation in the human.

Topics: 20-Hydroxysteroid Dehydrogenases; Basic-Leucine Zipper Transcription Factors; Cells, Cultured; Cytoprotection; Fanconi Anemia Complementation Group Proteins; Gene Expression Profiling; Glutathione; Heme Oxygenase-1; Humans; Hydroxysteroid Dehydrogenases; Intracellular Signaling Peptides and Proteins; Kelch-Like ECH-Associated Protein 1; Keratinocytes; Keratins; Neoplasms; NF-E2-Related Factor 2; Response Elements; Signal Transduction

This unit contains a detailed protocol for the simultaneous flow cytometric measurement of tumor cells, stromal cells, and DNA content of formalin-fixed, paraffin-embedded (FFPE) tissues. The vimentin-positive stromal cell fraction can be used as an internal reference for DNA content assessments. This allows clear detection of keratin-positive tumor cells with a DNA index lower than 1.0 and of keratin-positive tumor cells with a DNA close to 1.0 in overall DNA aneuploid samples, thus improved DNA ploidy assessment in FFPE carcinomas. Furthermore, the protocol is useful for studying molecular genetic alterations and intratumor heterogeneity in archival FFPE samples. Keratin-positive tumor cell fractions can be flow-sorted for further molecular genetic analysis, while DNA from the sorted vimentin-positive stromal cells can serve as a reference when normal tissue of the patient is not available.

Topics: Cell Separation; DNA, Neoplasm; Flow Cytometry; Humans; Keratins; Molecular Biology; Neoplasms; Paraffin Embedding; Ploidies; Tissue Fixation; Vimentin

Squamous cell carcinoma of the mammary gland is rare in both veterinary and human medicine. Whereas human metaplastic and squamous variants are known, the objectives of the current study were to ascertain the presence of such entities in canine mammary tumors and to distinguish them from other (epidermal, sweat gland) squamous tumors that may develop in the same area. A panel of antibodies (anti-cytokeratin [CK] 19, CK 14, CK 5/6, pancytokeratin, and vimentin) was used on 18 mammary gland malignancies with squamous features and 16 malignant skin tumors (11 squamous cell carcinomas of the skin and 5 sweat glands). Fifteen of the 18 mammary carcinomas were classified as metaplastic carcinomas, and the remaining 3 were classified as squamous cell carcinomas. The 2 most useful markers to establish the histogenesis of mammary tumors were pancytokeratin and CK 19. All other antibodies were equally expressed (CK 14 and 5/6) in all histotypes. The antibody panel discriminated primary epidermal squamous tumors (pancytokeratin positive and CK 19 negative) from gland-derived squamous neoplasms (pancytokeratin positive and CK 19 positive) but failed to distinguish primary mammary tumors from other squamous tumors of glandular origin.

Topics: Animals; Carcinoma, Squamous Cell; Dog Diseases; Dogs; Keratins; Mammary Glands, Animal; Neoplasm Proteins; Neoplasms; Skin Neoplasms; Vimentin

Immunocytology substantially improves the diagnostic accuracy of conventional cytology in the diagnosis of carcinomatous effusions. Due to the unequivocal characterization of the various cell populations, a sensitivity of 92% and specificity of 100% was achieved by immunocytology, examining samples of 1234 serous effusions.. Cytology plays a central role in the aetiological clarification of serous effusions. The sensitivity of this method for the diagnosis of carcinomatous effusions varies between 40% and 80%. The aim of the present study was to investigate whether immunocytology substantially improves the diagnostic quality of the cytological examination in the diagnosis of carcinomatous effusions.. Consecutive serous effusions were examined by conventional cytology and by immunocytology. The immunocytological examination was performed on smears, using a standard panel of three antibodies against pancytokeratin, human epithelial antigen 125 and calretinin.. Altogether, 1234 effusion samples were examined. A total of 603 effusions were caused by carcinomas, five by malignant mesotheliomas, 11 by malignant lymphomas and 615 by non-malignant disorders. In conventional cytology, carcinomatous effusions were correctly diagnosed in 314 samples, corresponding to a sensitivity of 52%. In 31 specimens (5%) tumour cells without further specification were described and in 161 samples (27%) the presence of tumour cells was suspected (84% overall sensitivity). A total of 97 carcinomatous effusions (16%) were diagnosed false-negatively and 50 (8%) of the 615 non-malignant effusions false-positively (92% specificity). In immunocytology, 561 carcinomatous samples were correctly diagnosed, representing a sensitivity of 93%. In six cases (1%) the presence of tumour cells was suspected. A total of 36 carcinomatous effusions (6%) were diagnosed false-negatively (94% over-all sensitivity). Out of the 615 non-malignant specimens, there were no false-positive diagnoses (100% specificity).. Immunocytology is a simple, cost-effective, routinely practicable method which substantially improves the diagnostic accuracy of conventional cytology in the diagnosis of carcinomatous effusions. Therefore, we recommend the use of immunocytology in all those cases where cytology on its own is not completely unequivocal.

Topics: CA-125 Antigen; Calbindin 2; Carcinoma; Cytodiagnosis; Exudates and Transudates; Humans; Immunohistochemistry; Keratins; Neoplasms; S100 Calcium Binding Protein G; Sensitivity and Specificity

The diagnosis of prostatic carcinoma (Pca) on routine biopsies may be challenging, and to date the commonly used marker to distinguish prostate carcinoma from benign prostatic lesions has been High Molecular Weight-Cytokeratin (HMW-CK). However, the antigen of HMW-CK is susceptible to the effect of formalin fixation and causes frequent loss or patchy staining in the obviously benign glands. More recently, antibodies to p63 have been reported to be more sensitive than HMW-CK for the detection of prostatic basal cells. p63, a homologue of tumour suppressor gene p53, is essential for prostate development and is selectively expressed in the nuclei of basal cells of normal prostate glands. The objective of this study is to compare the sensitivity and specificity of HMW-CK and p63 in distinguishing prostatic carcinomas from benign prostatic lesions, as well as determining their positive predictive values. Seventy-two cases from HUKM (comprising 29 prostatic carcinomas and 43 benign prostatic hyperplasias) were stained for both HMW-CK and p63. The sensitivity of p63 and HMW-CK in identifying basal cells in benign glands was 88.37% and 90.70% respectively. The specificity of both reagents was 100%, and the positive predictive value for both reagents was also 100%. Thus, p63 is a useful complementary basal cell specific stain to HMW-CK, and would be very helpful to practicing pathologists in dealing with difficult cases.

Topics: Biomarkers; Diagnosis, Differential; Humans; Keratins; Malaysia; Male; Membrane Proteins; Molecular Weight; Neoplasms; Neoplasms, Basal Cell; Prostatic Neoplasms

Determination of multidrug resistance (MDR) activity of tumor cells could provide important information for the personalized therapy of cancer patients. The functional calcein assay (MultiDrug Quant Assay, Solvo Biotechnology, Budaörs, Hungary) has been proven to be clinically valuable in hematological malignancies by determining the transporter activity of MDR protein 1 (MDR1, ATP-binding cassette protein [ABC] B1, P-glycoprotein-170) and MDR-related protein 1 (MRP1, ABCC1). In this study, we evaluated if the same functional test was adaptable for the analysis of MDR activity in solid tumors. For this purpose, tissue specimens of human colorectal cancer samples were subjected to limited enzymatic digestion by collagenase to provide a single-cell suspension; dead cells were excluded by 7-aminoactinomycin D staining, and epithelial cancer cells were detected by Cy5-conjugated anti-BerEP4 monoclonal antibody. The transporter functions of MDR1 and MRP1 in viable epithelial cells were assessed by flow cytometry detecting the intracellular accumulation of calcein dye after exposing cells to various MDR inhibitors. Collagenase disintegration preserved the MDR activity and the antigenicity of tumor cells. Thus using the extended calcein assay provided sufficient viable and functionally active tumor cells from surgical biopsies to determine the functional MDR activity. In conclusion, the newly described modified calcein assay may be applicable for evaluating the MDR phenotype in solid tissue specimens from colorectal forceps biopsy to surgical samples.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Antigens, Surface; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biomarkers; Biopsy; Body Fluids; Carcinoma; Cell Separation; Cell Survival; Fluoresceins; Fluorescent Antibody Technique; Humans; Keratins; Leukemia P388; Neoplasms

We have described previously a cDNA library made from membrane-bound polysomal mRNA prepared from breast and prostate cancer cell lines. The library is highly enriched for cDNAs encoding membrane proteins, secreted proteins, and cytokeratins. To characterize this library, 25,277 cDNA clones were sequenced and aligned with various databases; 1,439 clones did not align with known genes. From this set of clones we identified a previously uncharacterized gene encoding a 334-aa protein. Although protein structural motif prediction programs indicate that the gene encodes a membrane protein comprising a signal sequence, a series of leucine-rich repeats, and a single transmembrane domain with a cytoplasmic tail, confocal microscopy of MCF7 breast cancer cells demonstrates that the protein is not directly associated with the plasma membrane or intracellular membranes but instead colocalizes with intermediate filaments and cytokeratins within the cell. Immunofluorescence studies also show that protein expression is increased greatly in mitotic MCF7 cells, and immunohistochemistry demonstrates its expression in human breast cancer cells. Analysis of mRNA levels in 25 different normal tissues by RT-PCR shows that this gene is expressed highly in normal prostate and salivary gland, very weakly in colon, pancreas, and intestine, and not at all in other tissues. RT-PCR studies on human cancer samples show that the RNA is expressed highly in many cancer cell lines and cancer specimens, including 26 of 33 human breast cancers, 3 of 3 prostate cancers, 3 of 3 colon cancers, and 3 of 3 pancreatic cancers. We name the protein CAPC, cytokeratin-associated protein in cancer.

Topics: Amino Acid Sequence; Breast Neoplasms; Cell Line, Tumor; Cloning, Molecular; Female; Gene Library; Humans; Keratins; Male; Mitosis; Molecular Sequence Data; Neoplasms; Open Reading Frames; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Owing to the explosion of information generated by human genomics, analysis of publicly available databases can help identify potential candidate genes relevant to the cancerous phenotype. The aim of this study was to scan for such genes by whole-genome in silico subtraction using Expressed Sequence Tag (EST) data.. Genes differentially expressed in normal versus tumor tissues were identified using a computer-based differential display strategy. Bcl-xL, an anti-apoptotic member of the Bcl-2 family, was selected for confirmation by western blot analysis.. Our genome-wide expression analysis identified a set of genes whose differential expression may be attributed to the genetic alterations associated with tumor formation and malignant growth. We propose complete lists of genes that may serve as targets for projects seeking novel candidates for cancer diagnosis and therapy. Our validation result showed increased protein levels of Bcl-xL in two different liver cancer specimens compared to normal liver. Notably, our EST-based data mining procedure indicated that most of the changes in gene expression observed in cancer cells corresponded to gene inactivation patterns. Chromosomes and chromosomal regions most frequently associated with aberrant expression changes in cancer libraries were also determined.. Through the description of several candidates (including genes encoding extracellular matrix and ribosomal components, cytoskeletal proteins, apoptotic regulators, and novel tissue-specific biomarkers), our study illustrates the utility of in silico transcriptomics to identify tumor cell signatures, tumor-related genes and chromosomal regions frequently associated with aberrant expression in cancer.

Topics: Algorithms; bcl-X Protein; Blotting, Western; Chromosome Mapping; Chromosomes; Computational Biology; Data Interpretation, Statistical; Databases, Genetic; Down-Regulation; Expressed Sequence Tags; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Library; Genome, Human; Humans; Keratins; Liver Neoplasms; Models, Genetic; Neoplasms; Up-Regulation

We reported the identification of tumour-associated antigens from head and neck carcinomas, including cytokeratin 8 (CK8). These antigens were isolated based on the humoral immune response they elicit in vivo using the antibody-mediated identification of antigens technology. Unlike healthy squamous epithelium, tumour cells displayed CK8 at the plasma membrane. However, the actual presence of CK8 at the plasma membrane is still a matter of debate. Here, we have analyzed the expression of CK8 in detail using confocal laser scanning microscopy and circumstantiated its localization at the plasma membrane of carcinoma cells. Healthy human tissues were devoid of CK8 at the membrane, with the exception of hepatocytes. Moreover, membrane-associated CK8 molecules experienced a re-distribution throughout mitosis, which was associated with phosphorylation at serine 73. Phosphorylated CK8 redistributed into dense speckles and relocated to the plasma membrane upon cytokinesis. Thus, CK8 possesses genuine extracellular epitopes on tumour cells, which may represent valuable targets for therapy.

Topics: Biomarkers, Tumor; Cell Cycle; Cell Line, Tumor; Cell Membrane; Humans; Keratins; Neoplasms; Organ Specificity; Tissue Distribution

To examine the properties of shadow and ghost cells, 3 kinds of antibodies were raised against human hair proteins and their immunoreactivity was examined in tumors expressing those cells: pilomatrixoma, 14 cases; craniopharyngioma, 17 cases; and calcifying odontogenic cyst (COC), 14 cases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses demonstrated that 2 polyclonal antibodies, PA-HP1 and PA-HP 2, reacted strongly with type I acidic and type II neutral/basic hard alpha-keratins. The other monoclonal antibody, MA-HP1, reacted with type II neutral/basic hard alpha-keratins. Immunohistochemical examination revealed that all 3 antibodies reacted only with the hair shaft in sections of normal skin and dermoid cyst. In all pilomatrixoma cases, 3 antibodies reacted with the cytoplasm of transitional and shadow cells but not with that of basophilic cells. Positive reactions were found only in shadow cells of all 13 adamantinomatous craniopharyngiomas. In all COCs, the antibodies reacted only with ghost cells, not with other epithelial components. Immunoreactivity for phosphothreonine, detected in hard alpha-keratins, also was found in transitional, shadow, and ghost cells. The appearance of shadow or ghost cells might represent differentiation into hair in these 3 kinds of tumors.

Topics: Animals; Biomarkers, Tumor; Blotting, Western; Cells, Cultured; Craniopharyngioma; Hair; Hair Diseases; Humans; Hybridomas; Immunoenzyme Techniques; Jaw Neoplasms; Keratins; Mice; Mice, Inbred BALB C; Neoplasm Proteins; Neoplasms; Odontogenic Cyst, Calcifying; Pilomatrixoma; Pituitary Neoplasms; Skin Neoplasms

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Biomarkers, Tumor; Biotechnology; Diagnostic Techniques and Procedures; Growth Substances; Humans; Industry; Keratin-19; Keratins; Neoplasms; Technology Transfer

Activated oncogenes have been detected in a variety of malignant tumors and altered expressions of certain genes are known to play a functional role in the cancer process. The chemical carcinogen, BP, and the insertion of c-Ha-ras, induced characteristics of transformed phenotypes in a suitable human breast epithelial cell line. Carcinogen-treated and Ha-ras-transfected cells showed a progression of changes in the morphology, anchorage independent growth, invasiveness and tumorigenicity in SCID mice. Tumor growth occurs after a series of molecular events that parallel morphological changes. The aim of this work was to determine the neoplastic phenotypes following treatment with benzo(a)pyrene (BP) and transfection with c-Ha-ras oncogene changes and PCNA, Neu, ErbB-3 and Cytokeratin 18 protein expression in MCF-10F cells, a spontaneously immortalized human breast epithelial cell line. Protein expression was determined by immunofluorescent staining coupled with confocal microscopy. An increased oncoprotein expression in comparison to MCF-10F cells was observed in PCNA, Neu, ErbB-3 and Cytokeratin 18 protein expression in breast epithelial cells transformed with a chemical carcinogen and/or oncogene transfected that are not present in the MCF-10F. This in vitro cancer model can be used as a valuable model in the study of breast carcinogenesis.

Topics: Benzo(a)pyrene; Breast Neoplasms; Carcinogens; Cell Differentiation; Cell Line, Transformed; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Genes, ras; Humans; In Vitro Techniques; Keratins; Microscopy, Confocal; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Neoplasms; Oncogene Proteins; Phenotype; Proto-Oncogene Proteins p21(ras)

The correlation between detection of occult neo-plastic cells (ONCs) in lymph node sinuses and the recurrence/metastasis of primary breast, lung, esophageal, and gastric cancer was examined. Among patients with Stage I-III cancer treated by radical resection with dissection of at least 10 lymph nodes, 40 patients who suffered recurrence/metastasis within 5 years post-operatively and 94 patients who did not have recurrence within 5 years were randomly selected. A total of 1,340 lymph nodes were subjected to immunohistochemical staining for cytokeratin to identify ONCs. Then the sensitivity, specificity, positive predictive value, and negative predictive value of ONCs were determined for predicting the recurrence of each cancer. These parameters were respectively 40.0, 75.9, 62.4, and 55.8% for breast cancer, while the respective values were 50.0, 77.4, 68.9, and 60.8% for lung, 57.1, 64.3, 61.5, and 60.0% for esophageal, and 68.8, 65.0, 66.3, and 67.5% for gastric cancer. All of the parameters exceeded 65% for gastric cancer. ONCs also showed a high specificity for breast and lung cancer, but both the sensitivity and specificity were low for esophageal cancer.

Topics: Breast Neoplasms; Esophageal Neoplasms; Humans; Immunohistochemistry; Keratins; Lung Neoplasms; Lymph Nodes; Lymphatic Metastasis; Neoplasm Recurrence, Local; Neoplasms; Predictive Value of Tests; Random Allocation; Sensitivity and Specificity; Single-Blind Method; Stomach Neoplasms

Telomerase is a ribonucleoprotein complex mainly composed of a reverse transcriptase catalytic subunit (telomerase reverse transcriptase gene, hTERT) that copies a template region of its RNA subunit to the end of the telomere. For detecting telomerase activity in a tissue specimen the TRAP assay is a relatively sensitive and specific method, but it can be used only on fresh tissue extracts and offers no information at the single cell level. Immunohistochemistry (IHC) allows to detect hTERT protein expression at an individual cell level in human tissues. We have tested commercially available anti-hTERT antibodies in formalin-fixed and paraffin-embedded human tissues by IHC. Only one monoclonal antibody (NCL-hTERT; Novacastra) was sufficiently specific and this was applied to human tissues in which telomerase activity had been shown by TRAP assay and hTERT mRNA expression by RT-PCR. hTERT protein localized diffusely in the nucleoplasm and more intensely in the nucleoli of cancer cells and proliferating normal cells. Mitotic cells showed diffuse staining of the entire cell. Granular cytoplasmic staining was occasionally found in some tumor cells. In telomerase-positive tumors not all the tumor cells showed hTERT immunoreactivity. A significantly heterogeneous hTERT protein expression was observed in human tumor tissues. The hTERT immunostaining in fixed tissues was concordant with telomerase activity and hTERT mRNA expression in corresponding non-fixed samples. Quantitative RT-PCR of microdissected sections showed that hTERT mRNA expression was higher in cells with nuclear expression than in those with cytoplasmic expression. Double staining with the M30 antibody showed that a subpopulation of hTERT-negative cells is apoptotic. We conclude that: (1) hTERT protein can be detected by IHC in fixed human tissues, but the choice of the antibody, tissue processing, and reaction conditions are critical, (2) hTERT protein localizes in the nucleoplasm, more strongly in the nucleolus, and occasionally in the cytoplasm, (3) telomerase-positive tumors show significant heterogeneity of hTERT protein expression, and (4) a subpopulation of hTERT protein negative tumor cells is identified as apoptotic cells.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Apoptosis; Breast Neoplasms; Cell Nucleolus; Cell Nucleus; Colonic Neoplasms; Cytoplasm; DNA-Binding Proteins; Female; Gene Expression; Humans; Immunohistochemistry; Keratins; Lung Neoplasms; Lymphocytes; Male; Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Sarcoma; Spermatogonia; Telomerase; Urinary Bladder Neoplasms

Metastases in distant organs are the major cause of death for cancer patients, and bone marrow is a prominent homing organ for early disseminated cancer cells. However, it remains still unclear which of these cells evolve into overt metastases. We therefore established a new approach based on the analysis of viable and proliferating cancer cells by single-cell comparative genomic hybridization.. The bone marrow of early-stage breast tumor patients (pN(0)M(0)) was screened for tumor cells by immunostaining. By applying special short-term culturing, we selected for viable and proliferative tumor cells. The short-term culturing allowed us to evaluate the proliferative potential of micrometastatic cells, which we had previously shown to represent an independent prognostic marker. We assessed genomic changes in single disseminated cancer cells by single-cell comparative genomic hybridization.. We found that these viable disseminated cancer cells already had a plethora of copy number changes in their genome. All of these cells showed chromosomal copy number changes with a substantial intercellular heterogeneity and differences to the matching primary tumors.. The established experimental strategy might pave the way for the identification of metastatic stem cells in cancer patients. Our preliminary results support the new concept that early disseminated cancer cells evolve independently from their primary tumor.

Topics: Adult; Aged; Biomarkers, Tumor; Bone Marrow; Bone Marrow Cells; Breast Neoplasms; Cell Proliferation; Cell Survival; Cells, Cultured; Chromosome Mapping; Cluster Analysis; Female; Genome; Humans; Image Processing, Computer-Assisted; Keratins; Lasers; Microsatellite Repeats; Middle Aged; Multigene Family; Neoplasm Metastasis; Neoplasms; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Prognosis; Time Factors

There are many molecular tumor markers for diagnosing and monitoring cancer patients. Especially, quantitative assay for serum levels of tumor markers; such as AFP, CEA, PSA, hCG, CA 19-9 and CA 125, are frequently used in daily practice because of their relative specificities and usefulness to the common cancers. Though not suitable for early diagnosis, but they are used in monitoring patients with advanced caner, especially after treatments. Two of them, AFP and PSA, are also used in the screening and monitoring of high-risk groups, namely patients with chronic viral hepatitis and old male, who are the high risk for hepatoma and proste cancer respectively. Problems in using serum markers are; relatively low specificity and low sensitivity to cancer, confusing naming for similar markers that recognize almost the same molecule of cancer. Users must understand that CA 19-9, CA 50, KM-O 1 and SPAN-1 are in the same sialylated Lewis A group, and CA 125, CA 130 and CA 602; in the mucin antigen group, and STN, CA 54/61 and CA 72-4; in the sialyl Tn antigen group. Combination of two or more markers may inform us the biological characteristics of the cancer. For example, a germ-cell tumors may produce hCG and placental marker. That is of the choriocarcinoma type. Those with hCG and fetal antigens are the ordinal type of germ cell tumors, and those with AFP, CEA and cytokeratin are teratoma, and those with LDH and ALP only but negative for hCG and AFP must be seminoma. For the bronchial and alveolar carcinomas, CEA, SCC, NSE and cytokeratin 19 fragments are useful. Combination may be difficult for beginners but once understood, it will be an art in clinical oncology.

Topics: alpha-Fetoproteins; Antigens, Neoplasm; Biomarkers; Biomarkers, Tumor; Carcinoembryonic Antigen; Diagnosis, Differential; Female; Hepatitis, Viral, Human; Humans; Keratin-19; Keratins; Lewis X Antigen; Male; Neoplasms; Prostate-Specific Antigen; Prostatic Neoplasms; Protein Precursors; Prothrombin

Topics: Animals; Apoptosis; Caspases; Cell Culture Techniques; Cell Differentiation; Cell Proliferation; Enzyme Activation; Flow Cytometry; Hepatocytes; Humans; Intermediate Filament Proteins; Intermediate Filaments; Keratin-18; Keratin-8; Keratins; Mice; Mice, Knockout; Microscopy, Fluorescence; Neoplasms

Topics: Amino Acid Sequence; Apoptosis; Flow Cytometry; Humans; Immunohistochemistry; Immunophenotyping; Intermediate Filaments; Keratins; Molecular Sequence Data; Neoplasm Metastasis; Neoplasms; Optics and Photonics; Phenotype

To extend flow cytometry (FC) to the diagnosis of nonhematopoietic neoplasms, we have developed new flow cytometric assays to identify expression of cytokeratin, epithelial cell adhesion molecule (EpCAM)/epithelial glycoprotein-2, myogenin, and CD99. To validate these assays, we correlated the flow cytometric results with the histologic and immunohistochemical results on paraffin-embedded tissue in a series of 21 cases, including 17 carcinomas, 1 atypical carcinoid, 2 rhabdomyosarcomas, and 1 Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET). Six of 7 assayed carcinomas and the carcinoid were positive for cytoplasmic cytokeratin by the flow cytometric assay. EpCAM was expressed by 11 of 12 carcinomas that were assayed by FC. Both rhabdomyosarcomas expressed myogenin by FC, and the ES/PNET case expressed CD99. Interestingly, the blast-associated antigen CD90 was expressed uniformly on the ES/PNET case and on subsets of cells in the rhabdomyosarcoma and carcinoma cases. Potential applications of the flow cytometric assay to nonhematopoietic neoplasms will include evaluating samples with limited material, monitoring disease persistence and recurrence in patients with previous diagnoses, and making rapid diagnoses in urgent cases.

Topics: 12E7 Antigen; Antigens, CD; Antigens, Neoplasm; Carcinoma; Cell Adhesion Molecules; Cell Lineage; Epithelial Cell Adhesion Molecule; Feasibility Studies; Flow Cytometry; Immunohistochemistry; Immunophenotyping; Keratins; Leukocyte Common Antigens; Myogenin; Neoplasms; Rhabdomyosarcoma; Thy-1 Antigens

We have developed an apoptosis assay based on measurement of a neoepitope of cytokeratin-18 (CK18-Asp396) exposed after caspase-cleavage and detected by the monoclonal antibody M30. The total amount of caspase-cleaved CK18 which has accumulated in cells and tissue culture media during apoptosis is measured by ELISA. The sensitivity is sufficient for use in the 96-well format to allow high-through-put screening of drug libraries. We here describe strategies allowing classification of pro-apoptotic compounds according to their profiles of induction of apoptosis in the presence of pharmacological inhibitors. The time course of induction of CK18 cleavage can furthermore be used to distinguish structurally similar compounds. We propose that compounds that induce rapid CK18 cleavage have mechanisms of actions distinct from conventional genotoxic and microtubuli-targeting agents, and we present one example of an agent that induces almost immediate mitochondrial depolarization and cytochrome c release. Finally, CK18-Asp396 cleavage products are released from cells in tissue culture, and presumably from tumor cells in vivo. These products can be measured in sera from cancer patients. We present evidence suggesting that it will be possible to use the M30-ELISA assay for measuring chemotherapy-induced apoptosis in patient sera, opening possibilities for monitoring therapy.

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Apoptosis; Aspartic Acid; Caspases; Cell Line, Tumor; Cytochromes c; Drug Evaluation, Preclinical; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Epitopes; Humans; Keratins; Membrane Potentials; Mitochondria; Neoplasm Recurrence, Local; Neoplasms; Peptide Fragments; Predictive Value of Tests; Reproducibility of Results; Treatment Outcome

We report here that human esophageal keratinocyte stem cells are characterized by the expression of the low-affinity neurotrophin receptor p75(NTR) and differentially expressed cell adhesion molecules, the beta1 and beta4 integrins. The candidate stem cells could be fractionated from keratinocytes as a minor cell subset by means of immunocytochemical cell sorting based on the different levels of expression of these cell surface molecules. Flow cytometric analysis revealed that this minor cell subset retained a relatively slow-cycling phenotype in vitro. These cells expressed low levels of involucrin and cytokeratin 13, indicating that the p75(NTR)-positive cell subset is immature relative to the other predominant subpopulations coexpressing beta1 integrin at higher levels. The p75(NTR)-positive cell subset was crucial for achieving longevity and the greatest output of keratinocytes comprising all distinguishable subpopulations in vitro. This process was associated with self-renewal and self-amplification of the p75(NTR)-positive cell subset. These findings strongly implicate p75(NTR) as a stem cell marker, which will be valuable for prospectively investigating stem cell regulation in association with different biological processes including neoplastic transformation of regenerative epithelia.

Topics: Blotting, Western; Carcinoma, Squamous Cell; Cell Adhesion; Cell Cycle; Cell Division; Cell Membrane; Cell Transformation, Neoplastic; Epithelial Cells; Epithelium; Esophagus; Flow Cytometry; Humans; Immunohistochemistry; Integrin beta1; Integrin beta4; Keratinocytes; Keratins; Neoplasms; Phenotype; Propidium; Protein Precursors; Receptor, Nerve Growth Factor; Receptors, Nerve Growth Factor; Reverse Transcriptase Polymerase Chain Reaction; Stem Cells; Subcellular Fractions; Time Factors

Topics: Confidence Intervals; Diagnosis, Differential; Humans; Immunohistochemistry; Keratins; Neoplasms; Pathology

p63, cytokeratin (CK) 5/6 and CK 14 have been employed in diagnostic pathology as markers of basal, squamous and myoepithelial differentiation in several types of human neoplasms; however, there is scant data on the concurrent expression of these markers in large series of human neoplasms. We analyzed the distribution of these three immunohistochemical markers in 51 normal human tissue samples, 350 carcinomas, 25 malignant melanomas (MMs), and 25 glioblastomas using three serial sections of tissue array research program (TARP)-4 multi-tumor tissue microarray. Also, we performed double immunostainings to characterize the differential distribution of p63/CK 5/6 and p63/CK 14 in normal breast, salivary gland and skin. p63, CK 5/6 and CK 14 were expressed in basal cells of the prostate and respiratory epithelia and in breast and bronchial myoepithelial cells. p63 was also expressed in cytotrophoblast cells of human placenta and in scattered cells of lymph node germinal center. CK 5/6 and CK 14 also stained the cytoplasm of basal cells of esophageal stratified squamous epithelium and transitional epithelial cells of the bladder. No mesenchymal, neural, endothelial, smooth muscle or adipose cells were stained by any of the markers. p63, CK 5/6, and CK 14 were respectively expressed in 92.6%, 75.0%, and 52.9% of the squamous cell carcinomas of the lung, 10.2%, 20.0%, and 7.4% of the ductal carcinomas of the breast, 12.9%, 34.4%, and 11.8% of the serous and 25.0%, 0%, and 0% of the endometrioid carcinomas of the ovary. Lung, prostate and colonic adenocarcinomas, as well as MMs and glioblastomas were only rarely decorated by one of the markers. Only matched samples of 16 squamous cell carcinomas and two ductal carcinomas of the breast co-expressed these three markers. In double immunostainings, p63-CK 5/6, as well as p63-CK 14 were co-expressed by basal/myoepithelial cells of the salivary glands and basal cells of the epidermis. Our results demonstrate that p63, CK 5/6 and CK 14 may be used together in immunohistochemical panels to characterize squamous differentiation in poorly differentiated carcinomas or carcinomas of unknown origin.

Topics: Biomarkers, Tumor; DNA-Binding Proteins; Female; Genes, Tumor Suppressor; Histocytological Preparation Techniques; Humans; Immunoenzyme Techniques; Keratins; Male; Membrane Proteins; Neoplasms; Phosphoproteins; Trans-Activators; Transcription Factors; Tumor Suppressor Proteins

Numerous studies of circulating epithelial cells (CECs)have been described in cancer patients, and genetic abnormalities have been well documented. However, with one exception in colorectal cancer, there has been no report of matching the genetic abnormalities in the CECs with the primary tumor. The purpose of this investigation was to determine (a) whether CECs in patients including those with early tumors are aneusomic and (b) whether their aneusomic patterns match those from the primary tumor, indicating common clonality.. Thirty-one cancer patients had CECs identified by immunofluorescence staining using a monoclonal anti-cytokeratin antibody. Their CECs were analyzed by enumerator DNA probes for chromosomes 1, 3, 4, 7, 8, 11, or 17 by dual or tricolor fluorescence in situ hybridization. Touch preparations of the primary tumor tissue were available from 17 of 31 patients and hybridized with the same set of probes used to genotype the CECs.. The number of CECs from each patient ranged from 1-92 cells/cytospin. CECs showed abnormal copy numbers for at least one of the probes in 25 of 31 patients. Touch preparations from the primary tumors of 13 patients with aneusomic CECs were available. The pattern of aneusomy matched a clone in the primary tumor in 10 patients.. We conclude that the vast majority of CECs in breast, kidney, prostate, and colon cancer patients are aneusomic and derived from the primary tumor.

Topics: Chromosome Aberrations; Chromosomes, Human; Cytogenetic Analysis; DNA, Neoplasm; Epithelial Cells; Female; Humans; In Situ Hybridization, Fluorescence; Keratins; Male; Neoplasm Staging; Neoplasms; Neoplastic Cells, Circulating; Receptor, ErbB-2

The diagnostic value of tumor markers Cyfra 21-1 and neuron-specific enolase in blood serum and pleural fluid in differential diagnosis of pleural exudation in cancer patients and patients with nontumor pleurisy was evaluated. The most pronounced changes were characteristic of Cyfra 21-1. In patients with pleurisy caused by malignant tumors the degree and incidence of increased Cyfra 21-1 concentrations in the serum and pleural fluid were higher than in patients with pleural exudation of nontumor origin. These data attest to high diagnostic sensitivity and specificity of Cyfra 21-1. Complex measurements of the marker in the serum and pleural fluid will improve diagnosis of pleural exudation of tumorous etiology.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Biomarkers, Tumor; Case-Control Studies; Diagnosis, Differential; Exudates and Transudates; Female; Humans; Keratin-19; Keratins; Male; Middle Aged; Neoplasms; Neurons; Phosphopyruvate Hydratase; Pleural Effusion; Sensitivity and Specificity

There has been considerable variability in the reported results of immunohistochemical staining for some diagnostically relevant antigens. Our objectives in this study were to (1) use a multitumor tissue microarray with tissue from 351 cases received in our department, representing 16 normal tissues and 47 different tumor types, to compare immunohistochemical staining results in our laboratory with published data, using a panel of 22 antibodies; (2) assess interlaboratory variability of immunohistochemical staining for S-100 using this microarray; and (3) test the ability of hierarchical clustering analysis to group tumors by primary site, based on their immunostaining profile. Tissue microarrays consisting of duplicate 0.6-mm cores from blocks identified in the hospital archives were constructed and stained according to our usual protocols. Antibodies directed against the following antigens were used: B72.3, bcl-2, carcinoembryonic antigen, c-kit, pankeratin, CD 68, CD 99, CK 5/6, CK 7, CK 8/18, CK19, CK 20, CK 22, epithelial membrane antigen, estrogen receptor, melan-A, p53, placental alkaline phosphatase, S-100, synaptophysin, thyroid transcription factor-1, and vimentin. Staining results on the array cases were compared with published results, and hierarchical clustering analysis was performed based on the immunohistochemical staining results. Unstained slides of the multitumor tissue microarray were sent to five other diagnostic immunohistochemistry laboratories and stained for S-100 protein. The staining results from the different laboratories were compared. Staining results using our current methods and samples from our laboratory were compatible with those described in the literature for most antigens. Placental alkaline phosphatase staining was not specific with our protocol, showing staining of a broad spectrum of different tumors; this finding initiated a review of our recent requests for placental alkaline phosphatase immunostaining and revealed two instances in which placental alkaline phosphatase positivity was incorrectly interpreted as evidence of a germ cell tumor. S-100 staining was less sensitive but more specific for the diagnosis of melanoma or neural tumor in our laboratory, compared to some published reports. Assessment of interlaboratory variability of S-100 immunostaining showed that there was more frequent staining of carcinomas in some laboratories, resulting in decreased specificity of S-100 staining in distinguishing melanoma

Topics: Biomarkers, Tumor; Cluster Analysis; Diagnosis, Differential; Female; Histocytological Preparation Techniques; Humans; Immunohistochemistry; Keratins; Male; Neoplasms; Quality Control; Reproducibility of Results; S100 Proteins

Cytokeratin 5/6 (CK 5/6) immunoreactivity has been observed in the vast majority of cases of malignant mesothelioma but only rarely in pulmonary adenocarcinomas. Thus, CK 5/6 has been used to distinguish malignant mesothelioma from pulmonary adenocarcinoma. However, the utility of CK 5/6 in distinguishing pleural malignant mesothelioma from pleural metastases from nonpulmonary adenocarcinoma, as well as peritoneal malignant mesothelioma from peritoneal metastatic adenocarcinoma, has not yet been adequately addressed because the tissue expression of CK 5/6 in nonpulmonary neoplasms has not been well defined. We have studied the CK 5/6 expression in 509 cases of various epithelial tumors by immunohistochemistry. We found that the vast majority of cases of squamous cell carcinoma, basal cell carcinoma, thymoma, salivary gland tumor, and biphasic malignant mesothelioma were positive for CK 5/6. In addition, CK 5/6 immunoreactivity was detected in 15 of 24 cases (62%) of transitional cell carcinoma, in 5 of 10 cases (50%) of endometrial adenocarcinoma, in about one third of cases of pancreatic adenocarcinoma (38%) and breast adenocarcinoma (31%), and in one quarter of cases of ovarian adenocarcinomas (25%). Our study confirms the diagnostic utility of CK 5/6 immunohistochemistry in distinguishing biphasic mesothelioma from pulmonary adenocarcinoma but raises caution about its use for the differential diagnosis of pleural or peritoneal malignant mesothelioma versus pleural or peritoneal metastatic nonpulmonary adenocarcinoma, because many types of nonpulmonary adenocarcinomas may be positive for CK 5/6.

Topics: Adenocarcinoma; Biomarkers, Tumor; Carcinoma, Squamous Cell; Diagnosis, Differential; Female; Humans; Immunoenzyme Techniques; Keratin-14; Keratin-5; Keratin-7; Keratins; Male; Mesothelioma; Neoplasms

Topics: Actins; Animals; Annexin A5; Apoptosis; Caspases; Epitopes; Histological Techniques; Humans; In Situ Nick-End Labeling; Keratins; Membrane Proteins; Necrosis; Neoplasms; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Polymerase Chain Reaction; Proteins; Rats

Topics: alpha-Fetoproteins; Antigens, CD; Antigens, Differentiation; Antigens, Neoplasm; Biomarkers, Tumor; CA-19-9 Antigen; Carcinoembryonic Antigen; Cell Adhesion Molecules; Humans; Kallikreins; Keratin-19; Keratins; Neoplasm Proteins; Neoplasms; Serpins

Keratin polypeptides of the nonhair type, numbered 1 through 20 in the Moll catalog, are selectively expressed in normal and neoplastic tissues. Keratin 1 (K1), the highest-molecular-weight keratin (67 kd), was generally considered specific for keratinizing squamous epithelia. However, recent studies have shown that it is an integral component of the multiprotein kininogen receptor of endothelial cells. A library of formalin-fixed, paraffin-embedded tissue samples was evaluated immunohistochemically (avidin-biotin peroxidase complex method) for K1 expression using a specific monoclonal antibody (Novocastra clone 34betaB4). The study group included a wide variety of normal tissues and 541 tumors of epithelial or mesenchymal derivation. The specificity of the antibody to K1 was verified in normal epithelial tissues, where the staining was essentially limited to the epidermis and Hassal corpuscles of the thymus and focally to other squamous epithelia. Among carcinomas, it was essentially limited to keratinizing squamous carcinomas. It was also regularly found in endothelial cells of normal capillaries, veins, and arteries. Capillary, cavernous, and venous hemangiomas often had endothelia with K1 positivity. Among the malignant vascular tumors, epithelioid hemangioendotheliomas were consistently positive (8 of 8). However, angiosarcomas had more variable expression (59 of 81 were positive), with well-differentiated tumors generally having greater reactivity than poorly differentiated examples. Mesenchymal tumors with K1 expression included schwannomas (10 of 16), epithelioid sarcomas (26 of 37), and synovial sarcomas (19 of 68). In the last 2 tumor types, K1 reactivity was detected in both epithelioid and spindled neoplastic populations. In addition to its specificity for keratinizing squamous epithelia, K1 can be immunohistochemically detected in normal vascular endothelial cells and a spectrum of vascular tumors. However, its expression in poorly differentiated vascular tumors is variable, suggesting that this marker is poorly conserved in highly transformed endothelia. The unexpected K1 immunoreactivity in nonvascular soft tissue tumors, such as synovial sarcoma, epithelioid sarcoma, and schwannomas, requires further study.

Topics: Adult; Carcinoma; Endothelium; Female; Fetus; Humans; Immunoenzyme Techniques; Keratins; Male; Mesoderm; Neoplasms; Sarcoma, Synovial; Vascular Neoplasms

The suitability of "real-time" quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of isolated carcinoma cells in bone marrow was investigated by evaluating the expression of cytokeratin (CK)7, CK8, CK18, CK19, and CK20 in 17 gastrointestinal cancer cell lines, 64 control bone marrow specimens from noncancer patients, and 30 bone marrow specimens from patients with gastric or colorectal cancer. RT-PCR products for CK8 and CK18 were detected in all cancer cell lines, but only 16, 5, and 11 cell lines provided evidence for CK19, CK7, and CK20 transcription. Variable numbers of bone marrow specimens from noncancer patients demonstrated background transcription of CK8 (78.1%), CK18 (95.3%), CK19 (35.9%), CK20 (29.6%), and CK7 (16.7%). Maximal background transcription for CK8, CK18, and CK19 ranged from 52.2 to 56.1 copies/10(3) copies glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the corresponding values of 0.06 and 0.76 copies for CK7 and CK20 being distinctly lower. When maximal background values were used as a threshold value to define positivity in tumor cell dilution experiments, sensitivity levels of one tumor cell in 10(4) bone marrow cells were determined for CK7 and CK20 RT-PCR assays. Maximal background expression values of the different CKs as obtained in the control series were exceeded once (CK20), twice (CK18 and CK19), and 18 times (CK7) in bone marrow specimens from cancer patients, with none of these specimens exceeding the maximal background expression value of CK8. We conclude that RT-PCR for CK8, CK18, and CK19 cannot be recommended for the detection of isolated tumor cells in bone marrow of cancer patients. On the other side, the limited number of gastric and colorectal cancer cell lines expressing CK7 and CK20 indicates that assay sensitivity for these CKs might be limited because of their selective expression by carcinoma cells.

Topics: Animals; Bone Marrow Cells; Gene Expression Regulation; Humans; Keratins; Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Tumor Cells, Cultured

Apoptosis has an important role in carcinogenesis and cancer treatment. The end result of this complex pathway is the formation of apoptotic bodies. These can be difficult to quantify accurately, but quantitation is important if we wish to study this process. Several techniques are available which can help. 'TUNEL' is discussed, with its potential drawbacks, and newer antibody techniques, such as M30 and caspase 3, are then reviewed.

Topics: Animals; Apoptosis; Caspase 3; Caspases; Cell Physiological Phenomena; In Situ Nick-End Labeling; Keratins; Neoplasms

The aim of this study was to evaluate the relationship between renal function and the blood level of some tumor markers that are low molecular weight proteins, that is, tumor-associated trypsin inhibitor (TATI), squamous cells carcinoma antigen (SCC), cytokeratin 19 fragments (CYFRA 21-1), tissue polypeptide antigen (TPA), and M3-specific epitope of tissue polypeptide antigen (TPS). In 41 adult patients without evidence of neoplastic disease, glomerular filtration rate (GFR) and the blood levels of creatinine and of the tumor markers were determined. The decrease in GFR was accompanied by an increase in serum levels of TATI, SCC, CYFRA 21-1, and TPA. The serum level of tumor markers increased particularly when GFR fell below 40 ml/min. On the basis of these results, the renal function must be taken into account for the clinical evaluation of the studied tumor markers.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Biomarkers, Tumor; Creatinine; Female; Glomerular Filtration Rate; Humans; Keratin-19; Keratins; Male; Middle Aged; Neoplasms; Peptides; Renal Insufficiency; Serpins; Tissue Polypeptide Antigen; Trypsin Inhibitor, Kazal Pancreatic

The epidermal growth factor receptor (EGFR) is overexpressed in 50-70% of human primary breast, lung, and colon carcinomas, whereas it is not usually expressed in hematopoietic cells. We developed a novel reverse transcription-PCR (RT-PCR)-Southern blot assay for the detection of circulating, EGFR mRNA-expressing tumor cells in carcinoma patients. The assay was set up by increasing the amount of cDNA step by step in the PCR reaction. The highest sensitivity and specificity were found when using 800 ng of cDNA in the PCR reaction. Peripheral blood samples from 91 patients with either colon (38), lung (30), or breast (23) carcinomas and from 38 healthy volunteers were analyzed. EGFR transcripts were found in 44 of 75 (59%) patients with metastatic carcinoma and in 4 of 38 (10.5%) healthy donors (P < 0.001; chi2 test). The expression of EGFR, cytokeratin 19, and carcinoembryonic antigen mRNA in blood samples from patients with metastatic colon carcinoma was compared. EGFR, cytokeratin 19, and carcinoembryonic antigen transcripts were found in 8 of 11 (73%), 3 of 11 (27%), and 5 of 11 (45%) patients, respectively. Furthermore, two of seven (29%) Dukes' B and five of nine (55%) Dukes' C colon carcinoma patients were found to express EGFR mRNA in the peripheral blood. All patients that expressed EGFR transcripts in the peripheral blood were found to express the EGFR protein in the corresponding primary carcinoma, as assessed by immunohistochemistry. These data suggest that the EGFR assay that we developed is a highly specific and sensitive technique to detect circulating tumor cells in patients affected by different carcinoma types.

Topics: Breast Neoplasms; Carcinoembryonic Antigen; Colonic Neoplasms; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Keratins; Lung Neoplasms; Neoplasm Staging; Neoplasms; Neoplastic Cells, Circulating; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Transcription, Genetic; Tumor Cells, Cultured

It was recently reported that certain antigens can be retrieved from paraffin sections of formalin-fixed tissues by ultrasonication using either an expensive and sophisticated ultrasonic cell disrupter probe (cytokeratins 13 and 16) or an inexpensive and generally available ultrasonic cleaning bath (bcl-1). We wished to investigate the routine use of the latter method and therefore tried to retrieve from various tissues 11 antigens that usually require heat-mediated retrieval in citrate buffer. We applied ultrasonic vibration for periods of 30 seconds to 1.5 minutes in a cleaning bath containing citrate buffer or water, with and without the addition of heat, or for 1 minute in hot citrate buffer after microwaving for 10 minutes in the same buffer. Although a slight effect of ultrasound was noted for a few antigens, in no case did the immunostaining reach the level achieved after standard microwave heating in citrate buffer. We conclude that, under the conditions we used, ultrasonic antigen retrieval cannot be used for immunocytochemistry in a routine histopathology laboratory.

Topics: Antigens; Antigens, CD; Antigens, Surface; Biomarkers, Tumor; Cadherins; CD79 Antigens; Citric Acid; Desmin; Hot Temperature; Humans; Hydrogen-Ion Concentration; Immunohistochemistry; Keratins; Ki-67 Antigen; Neoplasms; Proto-Oncogene Proteins c-bcl-2; Receptors, Antigen, B-Cell; Receptors, Estrogen; Receptors, Progesterone; Temperature; Time Factors; Tumor Suppressor Protein p53; Ultrasonics

Cytokeratin 7 (CK 7) and cytokeratin 20 (CK 20) are low molecular weight cytokeratins. Their anatomic distribution is generally restricted to epithelia and their neoplasms. We surveyed 435 epithelial neoplasms from various organ systems by immunohistochemistry using CK 7 and CK 20 monoclonal antibodies. Expression of CK 7 was seen in the majority of cases of carcinoma, with the exception of those carcinomas arising from the colon, prostate, kidney, and thymus; carcinoid tumors of the lung and gastrointestinal tract origin; and Merkel cell tumor of the skin. The majority of cases of squamous cell carcinoma of various origins were negative for CK 7, except cervical squamous cell carcinoma, in which 87% of cases were positive. Approximately two thirds of cases of malignant mesothelioma were CK 7-positive. CK 20 positivity was seen in virtually all cases of colorectal carcinomas and Merkel cell tumors. CK 20-positive staining was also observed in cases of pancreatic carcinomas (62%), gastric carcinoma (50%), cholangiocarcinomas (43%), and transitional cell carcinomas (29%). The expression of CK 20 was virtually absent in carcinomas from other organ systems and in malignant mesothelioma. CK 7- and CK 20-negative epithelial neoplasms included adrenal cortical carcinoma, germ cell tumor, prostate carcinoma, renal cell carcinoma, and hepatocellular carcinoma.

Topics: Biomarkers, Tumor; Carcinoma; Diagnosis, Differential; Female; Humans; Immunoenzyme Techniques; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Male; Neoplasms

It is possible that post-transplant relapse in patients with breast cancer may result, in part, from residual tumor in the autologous PBSC product. It is unclear from the literature what effect residual breast tumor cells have on clinical outcome and whether purging tumor cells would be beneficial. We hypothesized that lack of standardization of assays for detection of residual breast tumor may be responsible for the inconclusive clinical data.. We compared two assays routinely for detection of cytokeratin (CK)-positive cells in stem-cell grafts, immunohistochemistry (IHC) and flow cytometry (FCM). The patient population consisted of individuals with breast cancer, non-epithelial cell-derived tumors and normal donors. A rigorous statistical model was developed for evaluation of the data.. We found that the IHC assay out-performed the FCM assay. Importantly, both assays detected CK-positive cells in PBSC collections of patients with non-epithelial cell-derived tumors and in normal donors. No distinguishing morphological characteristics could be identified to differentiate potentially malignant from non-malignant CK-positive cells. Due to the inability to distinguish true positive from false positive results, we developed a statistical model to establish a quantitative threshold to discriminate positive from negative samples. Among the patients tested, no clinical outcome differences were detected, regardless of where the threshold of CK-positive cells was set.. We conclude the more stringent criteria and more specific markers, rather than the presence or absence of CK-positive cells, need to be established to determine the clinical significance of minimal residual disease in autologous breast-cancer

Topics: Breast Neoplasms; Cell Line, Tumor; False Positive Reactions; Flow Cytometry; Hematopoietic Stem Cells; Humans; Immunohistochemistry; Keratins; Models, Statistical; Neoplasm, Residual; Neoplasms; Prognosis; Sensitivity and Specificity; Stem Cell Transplantation; Time Factors

Topics: Humans; Keratins; Mutation; Neoplasm Metastasis; Neoplasms; Polymerase Chain Reaction

This study was undertaken to assess the utility of combined cytokeratin (CK) 7/20 immunoprofile determination in malignant cytologic cell blocks as an aid to the identification of tumor primary site of origin. Fifty-one cases in which CK 7/20 immunocytochemistry was performed as part of the initial workup were retrieved. Their contribution to the final cytologic diagnosis of tumor primary site of origin was analyzed. CK reactivity patterns were 7+/20- (n = 34), 7-/20+ (n = 9), 7-/20- (n = 7), and 7+/20+ (n = 1). The CK 7+/CK 20- immunophenotype was the most common one obtained, and due to its wide expression in a number of common carcinomas, the least informative. The second most common immunophenotype was CK 7-/20+, which is associated with colorectal origin, and as such was very useful when obtained. The CK immunoprofile was more useful in the setting of a prior carcinoma, being a major diagnostic determinant in 13 cases (55%) from group 1 (those with a prior history of malignancy), compared to 8 cases (29%) from group 2 (those with no prior history of malignancy). In the setting of prior carcinoma, the CK immunoprofile is most useful when carcinomas under consideration have different expected immunoprofiles (e.g., CK 7+/CK20- carcinomas, including lung, breast, ovary, endometrium, and others, vs. CK 7-/CK 20+ carcinomas, primarily colorectal). When similar immunoprofiles are obtained, their usefulness is greater if they are immunoprofiles other than the most common 7+/20- pattern. Similarly, in newly diagnosed carcinomas, the CK immunoprofile either helps to narrow the differential diagnosis or points to a specific diagnosis.

Topics: Biomarkers; Biopsy; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Neoplasms; Phenotype

Flow cytometry of single-cell suspensions prepared by enzymatic digestion from formalin-fixed, paraffin-embedded tissue suffers from several major drawbacks. The most important factors that influence the results are the high and unpredictable coefficients of variation (CVs) of the G0/G1 peak in the DNA histogram and reduction of propidium iodide (PI) intercalation with DNA, resulting from protein cross-linking by formalin.. In this study we introduce a heating step (2 h incubation in citrate solution at 80 degrees C) prior to a brief pepsin digestion of tissue sections in the protocol for DNA content analysis of formalin-fixed and paraffin-embedded tissue. This new method is compared with established methods for the preparation of cell suspensions from frozen and paraffin-embedded tissues with respect to cell yield, DNA histogram resolution, DNA dye saturation kinetics, cell cycle parameters, and antigen retrieval in various epithelial and nonepithelial tissues.. The recovery of single cells from the paraffin sections was doubled by the heat treatment step, while the limited time of proteolysis resulted in decreased cell debris. Furthermore, an increased fraction of cells became cytokeratin-positive, while these immunocytochemically stained cells also exhibited a higher mean fluorescence intensity. The DNA histograms prepared from cell suspensions obtained according to this new protocol showed a significantly improved resolution, leading to a better identification of peridiploid cell populations. Heat pretreatment of paraffin-embedded archival tissue sections showed PI saturation kinetics similar to, or even better than, those of fresh unfixed tissues, independent of duration of fixation.. This new method, making use of routinely available antigen retrieval principles, thus allows high-resolution DNA analysis of routinely fixed and paraffin-embedded tissue samples. Using external reference cells, inter- and intralaboratory standardization of DNA histograms can be achieved.

Topics: Breast Neoplasms; Cell Separation; Colon; Colorectal Neoplasms; Dose-Response Relationship, Drug; Flow Cytometry; Formaldehyde; Hot Temperature; Humans; Immunohistochemistry; Keratins; Kinetics; Meningeal Neoplasms; Neoplasms; Paraffin Embedding; Pepsin A; Propidium; Time Factors

To the authors' knowledge the role of tumor marker determination in the differential diagnosis of pleural effusions has not been established definitively. The current article reports the results of a study of CYFRA 21-1, carcinoembryonic antigen (CEA), cancer antigen 125 (CA 125), squamous cell antigen (SCC), and neuron specific enolase (NSE) in the serum and pleural fluid of patients with pleural effusions of diverse etiologies.. One hundred forty-six patients with pleural effusions (43 malignant, 47 tuberculous, 32 miscellaneous benign, and 24 paramalignant) were studied prospectively. Levels of CYFRA 21-1, CA 125, CEA, NSE, and SCC were measured by radioimmunoassay in the pleural fluid in all patients and in the serum in 118 patients.. There were no significant differences between the serum and pleural fluid levels of tumor markers with the exception of CA 125, which was higher in the pleural fluid. With maximum specificity, the highest sensitivity in the diagnosis of pleural malignancy was obtained with a combination of CYFRA 21-1 (with a cutoff value of 150 U/L), CEA (with a cutoff value of 40 ng/mL), and CA 125 (with a cutoff value of 1000 ng/mL) in pleural fluid. NSE and SCC added no diagnostic value. The simultaneous use of tumor markers and cytology in pleural fluid increased the sensitivity from 55.8% to 81%.. These findings suggest that a combination of CYFRA 21-1, CEA, and CA 125 in the pleural fluid can be a useful addition to pleural cytology in the diagnosis of malignant pleural effusion.

Topics: Adult; Aged; Antigens, Neoplasm; Biomarkers, Tumor; CA-125 Antigen; Carcinoembryonic Antigen; Exudates and Transudates; Female; Humans; Immunoradiometric Assay; Keratin-19; Keratins; Male; Middle Aged; Neoplasms; Phosphopyruvate Hydratase; Pleural Effusion; Pleural Effusion, Malignant; Prospective Studies; Serpins; Tuberculosis

Rapid immunostaining of frozen sections within a tolerable time span would be very helpful for intraoperative diagnosis. A protocol was therefore established using the enhanced polymer one-step staining (EPOS) system (Dako) with antibodies against leucocyte common antigen (LCA), cytokeratin (CK), and anti-melanoma (MEL). Best results with reliable and specific immunostaining and a labelling intensity comparable to standard immunostaining protocols were achieved with fixation of samples in 100% acetone for 20 seconds (CK, LCA) or two minutes (MEL), followed by incubation of the primary antibody and development of the chromogen reaction with 3,3'diaminobenzidine (DAB) for three and five minutes at 37 degrees C, respectively. The total procedure takes only 12 minutes, thus enabling rapid immunostaining on intraoperative frozen sections. Apart from its use in tumour classification, this method is especially useful in detecting tumour cells in sentinel lymph nodes.

Topics: Antigens, Neoplasm; Frozen Sections; Humans; Immunohistochemistry; Intraoperative Period; Keratins; Leukocyte Common Antigens; Lymph Nodes; Melanoma; Neoplasms; Staining and Labeling

CYFRA 21-1 assay, measuring cytokeratin 19 fragments, was compared with carcinoembryonic antigen (CEA) assay, as an addition to cytological analysis for the diagnosis of malignant effusions. Both markers were determined with commercial enzyme immunoassays in pleural fluid from 196 patients. Cytological analysis and/or pleural biopsy confirmed the malignant origin of the effusion in 99 patients (76 carcinomas, nine pleural mesotheliomas and 14 non-epithelial malignancies). Effusions were confirmed as benign in 97 patients (33 cardiac failures, 39 infectious diseases--including 12 tuberculosis-- and 25 miscellaneous effusions). Both markers were significantly higher in malignant than in benign effusions. All the patients with non-epithelial malignancies presented CYFRA and CEA values lower than the 95% diagnostic specificity thresholds (100 and 6 ng ml(-1) respectively). The diagnostic sensitivity in the group of carcinomas and mesotheliomas was similar for CYFRA (58.8%) and CEA (64.7%). However, CEA had a significantly higher sensitivity in carcinomas (72.4% vs 55.3%), while CYFRA had a clearly higher sensitivity in mesotheliomas (89.9% vs 0%). Interestingly, 12 out of the 16 malignant effusions with a negative cytology were CEA and/or CYFRA positive. Regarding their high diagnostic sensitivity and their complementarity, CEA and CYFRA appear to be very useful for the diagnosis of malignant pleural effusions when cytology is negative.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Biomarkers, Tumor; Carcinoembryonic Antigen; Child; Female; Humans; Keratin-19; Keratins; Male; Middle Aged; Neoplasms; Pleural Effusion; Retrospective Studies; Sensitivity and Specificity

Topics: Biopsy; Humans; Keratins; NAD; Neoplasms

Topics: Antigens, Neoplasm; Antigens, Surface; Carcinoma, Squamous Cell; Coloring Agents; DNA, Neoplasm; Female; Flow Cytometry; Humans; Keratins; Neoplasms; Ovarian Neoplasms; Tumor Cells, Cultured

Immunohistochemistry occasionally is used to determine the lineage of entirely necrotic tumors. However, the sensitivity and specificity of antibodies on necrotic tissue are unknown. To determine the usefulness of immunohistochemistry with necrotic lesions, a series of 24 known tumors consisting of 14 carcinomas, 2 lymphomas, 2 melanomas, and 6 sarcomas (all with extensive necrosis) was examined for reactivity with 6 cytokeratin antibodies, S100, and LCA. Carcinomas stained positively with at least 1 cytokeratin antibody in 78% of the cases. The cytokeratin antibodies with the highest sensitivity were AE1, AE1/3, S903, and PANCK. These antibodies also retained specificity for epithelial differentiation; no reactivity was observed in the 10 necrotic nonepithelial tumors. LCA retained its reactivity with necrotic lymphoma, but S100 reacted with only one third of the necrotic lesions. Unexpectedly, reactivity for LCA and S100 occurred in some necrotic carcinomas. Keratin markers can be used on necrotic tissue to determine epithelial differentiation, but the results obtained with S100 and LCA on necrotic tissue should be interpreted with caution.

Topics: Antibodies; Antibody Specificity; Carcinoma; Diagnosis, Differential; Humans; Immunohistochemistry; Keratins; Leukocyte Common Antigens; Lymphoma; Melanoma; Necrosis; Neoplasm Metastasis; Neoplasms; S100 Proteins; Sarcoma

The increasingly popular immunohistochemical techniques for assay of the estrogen receptor (ER) allow localization of receptor positivity to specific cell populations. Heterogeneity of the ER in tumor cell populations may have important implications for analytic cell selection and for prognosis in ER-positive carcinomas. We studied 84 tissue blocks for level-to-level and geographic heterogeneity within level of the ER and cytokeratin by staining alternate serial sections for ER and cytokeratin. Distribution of ER and cytokeratin positivity was manually assessed. Homogenous positive staining was seen in 63 of 84 cases for ER and 71 of 84 cases for cytokeratin. Distinct geographic variability constant from level to level was seen in 7 cases for ER. In each of these cases, the cell populations stained uniformly for cytokeratin. Artifactual heterogeneity seemed to be uncommon for ER. Automated image analysis and manual ER estimation resulted in more positive cases than did the dextran-coated charcoal (DCC) technique. Interobserver correlation for the manual method seemed high, as did correlation between the manual method and image analysis. Because a majority of the immunohistochemical staining heterogeneity for ER seemed to be biologic, we believe it may mark carcinomas that are less responsive to tamoxifen and more likely to progress than would be predicted by more traditional methods of ER analysis.

Topics: Humans; Immunohistochemistry; Keratins; Neoplasms; Receptors, Estrogen; Reproducibility of Results

A series of 33 tumor cases (13 fine-needle aspirates and 20 effusion specimens) were evaluated for the expression of two cytokeratin (CK) subtypes; CK 20, expressed primarily in tumors of the GI tract, mucinous ovarian tumors and transitional cell carcinomas, and CK 7, found chiefly in non-GI tract adenocarcinomas, including breast and lung. CK 20 expression was demonstrated immunocytochemically in seven of seven metastatic colon and two of three metastatic transitional cell carcinomas tested. CK 20 was absent in all metastatic carcinomas of breast, ovary, lung, and uterus examined (23 cases). Anti-CK 7 stains were negative in four of six metastatic colonic carcinomas, with equivocal results in the remaining two cases. Metastatic lung, breast, and ovarian carcinomas were strongly positive for CK 7. This study demonstrates that the combined use of anti-CK 20 and anti-CK 7 antibodies is highly sensitive and specific for metastatic colonic adenocarcinoma in cytologic material and thus could play an important role in distinguishing this entity from other common primary carcinomas.

Topics: Antibodies, Monoclonal; Biomarkers, Tumor; Biopsy, Needle; Carcinoma; Diagnosis, Differential; Female; Humans; Immunoenzyme Techniques; Intermediate Filament Proteins; Keratin-20; Keratins; Neoplasms

Immunohistochemical identification of intermediate filaments and other cell identity markers is of immense value in diagnostic tumor pathology. However, the literature contains many examples of inappropriate expression of markers by various tumors or coexpression of two or more markers supposedly specific for different cell types. The present study investigated this subversion of antigen differentiation in tumors by using developing tissues as a model.. Twenty-five human embryos and fetuses of 4 to 24 weeks' gestation were studied. An indirect immunoperoxidase method was applied to formalin-fixed, paraffin-embedded sections using a panel of 13 commonly used cell identity markers including intermediate filaments.. During early development, vimentin was found to be coexpressed with cytokeratin in surface ectoderm, developing notochord, renal tubule epithelium, and intestinal mucosa. It coexpressed with glial fibrillary acidic protein in developing neuroglial tissue, with S100 in cartilage, and with skeletal muscle markers in myotubules. Hence, vimentin represents immaturity of tissues and is coexpressed with specific cell markers to be eventually replaced by the latter. Transbarrier expression of cytokeratin in smooth muscle was also noted.. The aberrant expression of antigens in neoplastic tissues, as reported in the literature, simulates the varying expression of antigens in immature tissues during development. Hence, it is proposed that the phenomenon of antigenic subversion in neoplasia is related to the process of maturation and differentiation.

Topics: Antibodies, Monoclonal; Antigens; Cell Differentiation; Embryo, Mammalian; Fetus; Gestational Age; Glial Fibrillary Acidic Protein; Humans; Immunoenzyme Techniques; Immunohistochemistry; Intermediate Filaments; Keratins; Neoplasms; Vimentin

High-grade prostatic intraepithelial neoplasia (HGPIN) is the most likely precursor proliferation of peripheral zone, moderately to poorly differentiated prostatic adenocarcinomas. The usual cell type of the epithelial lining of HGPIN is a glandular epithelial cell with characteristic nuclear abnormalities. Here we report nine cases of unusual types of HGPIN, including three cases of signet-ring cell HGPIN, one case of small cell neuroendocrine HGPIN, and five cases of HGPIN with distinctive mucinous features. The three examples of signet-ring cell PIN were all associated with an invasive primary signet-ring cell carcinoma of the prostate. The HGPIN assumed a classical tufted and micropapillary architectural growth pattern, with the constituent cells exhibiting a morphologic appearance identical to that of the invasive signet-ring cells. The intraepithelial and invasive signet-ring cells were mucin negative and were immunoreactive for prostate-specific antigen (PSA). A fourth case displayed a mixed intraepithelial glandular-small cell neoplastic proliferation, where intraepithelial small cells were histologically identical to surrounding invasive small cell carcinoma cells. The small cell HGPIN and invasive small cell carcinoma cells were positive for the neuroendocrine markers chromogranin, synaptophysin, and neuron-specific enolase. In five cases, mucinous distension of HGPIN glands, producing a flat pattern of the epithelial lining layer, comprised the third unusual pattern of HGPIN. These blue mucinous secretions were readily detected by hematoxylin and eosin staining and were composed of both neutral (periodic acid-Schiff-positive) and acidic (alcian blue-positive) mucins. Herein we document the existence of an intraepithelial proliferation of neoplastic cell types-small cell neuroendocrine and signet-ring cell-that are usually considered as stromal-invasive cells in the prostate. The presence of these rare prostatic cell types in both HGPIN and invasive carcinoma provides further support for a close relationship between HGPIN and invasive carcinoma of the prostate. All three unusual types of HGPIN-signet-ring cell, small cell neuroendocrine, and mucinous-are important to diagnostically recognize because of the strength of association of HGPIN with invasive carcinoma.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Chromogranins; Humans; Keratins; Male; Microscopy, Electron; Middle Aged; Neoplasms; Precancerous Conditions; Prostate-Specific Antigen; Prostatic Intraepithelial Neoplasia; Prostatic Neoplasms

There are a variety of routine and immunohistochemical stains used to diagnose mammary and extramammary Paget's disease (MPD and EMPD). Most of the stains commonly used, however, show a positive reaction in the Paget's cells in all cases. We wanted to assess which immunohistochemical stain is the best for the diagnosis of MPD and EMPD, as well as the best stain for identifying small foci of tumors in evaluating tumor margins. We evaluated nine cases of MPD and nine cases of EMPD, which were randomly chosen, with a battery of immunohistochemical stains. These stains included cytokeratin 7, cytokeratin 20, carcinoembryonic antigen, Ber-EP4, and CAM 5.2. Cytokeratin 7 was the only immunohistochemical stain that stained all of the cases diffusely, and, in all of the cases, the staining of the Paget's cell was intense and specific within the epidermis. We concluded that cytokeratin 7 is the immunohistochemical stain of choice in the diagnosis of Paget's disease. Because cytokeratin 7 seems to identify single cells, it might also be valuable in evaluating surgical margins for small foci in a tumor such as EMPD, which might have a multifocal origin.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoembryonic Antigen; Female; Humans; Immunoenzyme Techniques; Keratins; Male; Middle Aged; Neoplasms; Paget Disease, Extramammary; Paget's Disease, Mammary; Staining and Labeling

In this study the recently developed keratin 19 antibody RCK108 is biochemically and immunohistochemically characterized. Its applicability as a keratin marker in routinely processed histological tissue specimens is assessed.. The keratin 19 antibody RCK108 antibody was tested on normal and malignant routinely formalin-fixed, paraffin-embedded tissue specimens. It stains most, although not all, glandular epithelia and showed (focal) reactivity in the basal cell compartment of stratified epithelia. It was found to react with most epithelial tumours, including adenocarcinomas, squamous cell carcinomas and endocrine tumours of various origins.. Its reproducible and highly sensitive staining characteristics make RCK108 a useful antibody to be applied as a broad epithelial marker for carcinoma detection in routinely processed paraffin sections. As such, RCK108 is a specific reagent for practically all epithelial tumours. A few types of epithelial malignancies, known not to contain keratin 19, were negative for RCK108. Therefore the antibody is also useful in some narrow differential diagnostic considerations such as cholangiocellular carcinoma (RCK108 positive) vs. hepatocellular carcinoma (RCK108 negative). Another important feature of this antibody is that it shows very little reactivity in mesenchymal tissues, or mesenchymally derived tumours, as is frequently described for other keratin antibodies. A few leiomyosarcomas showed sporadic reactivity.

Topics: Animals; Antibodies, Monoclonal; Antigen-Antibody Reactions; Biomarkers, Tumor; Cell Line; Epithelial Cells; Humans; Immunohistochemistry; Keratins; Mice; Mice, Inbred BALB C; Neoplasms; Plasmacytoma; Reproducibility of Results; Sensitivity and Specificity

When tumour cells are fused with normal ones, malignancy is suppressed. It has been shown that this suppression is associated with the imposition on the hybrid cell of the terminal differentiation programme of the normal parent cell. We report here the consequences of imposing the synthesis of keratin 1 and keratin 10, markers of terminal differentiation in the epidermal keratinocyte, on malignant cells of keratinocyte and non-keratinocyte lineage. We find that there is extreme selection in vivo against cells making keratin 1: tumours arising from inocula of such cells are invariably produced by the selective overgrowth of cells in which keratin 1 synthesis has been drastically reduced, usually to trace levels. No such selection operates against keratin 10. It is possible that if substantial synthesis of keratin 1 could be induced in malignant cells in a clinical context, some therapeutic benefit might accrue.

Topics: Animals; Biomarkers; Cell Differentiation; Cell Fusion; Drug Resistance; Humans; Hybrid Cells; Keratinocytes; Keratins; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms; Osteosarcoma; Transfection; Transplantation, Heterologous; Tumor Cells, Cultured

A syngeneic, high-affinity, anti-idiotypic monoclonal antibody (MAb; alpha TS1) raised against an anticytokeratin monoclonal antibody (TS1) was evaluated as a second antibody to promote the rapid clearance of radiolabeled TS1 from the blood during experimental radioimmunolocalization. By using a novel biosensor technology (BIAcore), association rate dissociation rate, and affinity constants between the idiotype and the anti-idiotype could be determined. The in vivo results in nude mice carrying HeLa Hep 2 tumors demonstrate the possibility of selectively regulating the amount of the idiotypic 125I-labeled circulating MAb by in vivo injection of this high-affinity, anti-idiotypic antibody. Injection of the anti-idiotype in a molar ratio of 0.75 to the idiotype cleared the blood pool from circulating radiolabeled idiotype within 24 h, with a concomitant rapid excretion of 125I in urine. The total amount of remaining radioactivity in the animals decreased to 15-20% during these 24 h, with the tumors still retaining 60-65% of their initial radioactivity. This approach, using syngeneic primary and secondary MAbs with minimized immunogenicity, significantly improves the tumor:nontumor ratio, thus improving efficiency in experimental radioimmunolocalization and radioimmunotherapy, leaving the endogenous antibody repertoire of the host unaffected.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; HeLa Cells; Humans; Iodine Radioisotopes; Keratins; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms; Radioimmunodetection

A total of 291 enlarged lymph nodes showing a range of reactive-inflammatory processes, primary and metastatic neoplasms were studied to determine the distribution and immunoprofile of their cytokeratin-positive interstitial reticulum cells (CIRC) in comparison with normal nodes. In 258/291 nodes (89%), CIRC numbers were distinctly increased in the subcapsular, paracortical and, occasionally, in the medullary zones; often, these increased CIRC formed networks around follicles, sinuses and vessels. CIRC had comparatively small, irregularly shaped bodies and dendritic processes; occasionally, giant forms were noted. CIRC contained cytokeratins (CK) 8 and 18 but not 19, as shown by immunohistochemistry, and by gel electrophoresis with subsequent immunoblotting. They co-expressed vimentin consistently, alpha-smooth-muscle actin frequently, and desmin less frequently. They did not contain desmoplakins, Factor VIII, S-100, LCA, B and T lymphocyte- and macrophage-associated antigens, chromogranin A, synaptophysin or the A-80 glycoprotein. We found no clear correlation between the increased CIRC and given nodal disease processes. However, CIRC were most abundant in nodes free of but draining malignant tumours; bizarre CIRC assemblies were noted in HIV lymphadenopathy. CIRC appear to represent a subset of the so-called "fibroblastic reticulum cells" of lymph nodes. Their function remains undetermined; their increase in diverse lymphadenopathies suggests that they partake in nodal reactions to injury. It remains unclear whether the increase in CIRC relative number is due to proliferation or to CK gene induction processes but their presence and potential capability to undergo hyperplasia with dysplastic forms should alert pathologists to possible diagnostic pitfalls. In addition, we discuss that CIRC may undergo transformation and represent the "cell of origin" of certain CK-positive tumours restricted to lymph nodes.

Topics: Cytoskeletal Proteins; Dendritic Cells; Humans; Hyperplasia; Immunohistochemistry; Keratins; Lymph Nodes; Lymphangitis; Lymphatic Diseases; Lymphoma; Microscopy, Fluorescence; Neoplasms

Twenty-eight epithelial and 22 nonepithelial feline tumors were studied immunohistochemically. Epithelial tumors were 10 squamous cell carcinomas, two basal cell tumors, two sebaceous gland carcinomas, three apocrine gland carcinomas, three thyroid papillary carcinomas, one thyroid solid carcinoma, one renal clear cell carcinoma, one renal papillary carcinoma, one endometrial carcinoma, and four lung bronchioloalveolar carcinomas. Nonepithelial tumors were 10 fibrosarcomas, one liposarcoma, one leiomyosarcoma, one rhabdomyosarcoma, one hemangiosarcoma, two mast cell tumors, one osteosarcoma, three melanomas, and two lymphomas. Commercially available antibodies directed against high- and low-molecular-weight keratins (keratin, RCK-102, NCL-5D3), vimentin, desmin, glial fibrillary acidic protein (GFAP), and neurofilament intermediate filament (IF) proteins were used in the avidin-biotin-peroxidase complex technique on formalin-fixed, paraffin-embedded tumor tissue samples. All epithelial tumors except the endometrial carcinoma expressed some type of keratin protein. Squamous cell carcinomas expressed high-molecular-weight keratins exclusively. Coexpression of high- and low-molecular-weight keratins was observed in one basal cell tumor, sebaceous and apocrine adenocarcinomas, and thyroid, renal, and lung carcinomas. In addition to keratins, vimentin immunoreactivity was found in all basal cell tumors, all sebaceous gland, thyroid papillary, renal, and lung adenocarcinomas, and one of the apocrine gland adenocarcinomas. Immunoreactivity with GFAP antibody was found in one basal cell tumor and one sebaceous gland adenocarcinoma. The endometrial carcinoma did not react with any of the antibodies applied. Nonepithelial tumors analyzed expressed either vimentin (fibrosarcomas, liposarcoma, haemangiosarcoma, mast cell tumors, osteosarcomas, melanomas) or vimentin and desmin (leiomyosarcoma, rhabdomyosarcoma, one fibrosarcoma) IF proteins exclusively. Lymphomas did not react with any of the antibodies employed. These findings indicate that IF proteins antibodies can be included in diagnostic panels of antibodies for immunocharacterization of feline tumors. In addition, they can be used as a basis for the diagnoses of poorly differentiated or undifferentiated feline neoplasms.

Topics: Animals; Cat Diseases; Cats; Desmin; Glial Fibrillary Acidic Protein; Immunohistochemistry; Intermediate Filament Proteins; Keratins; Kidney; Kidney Neoplasms; Lung Neoplasms; Neoplasms; Neoplasms, Glandular and Epithelial; Neurofilament Proteins; Skin Neoplasms; Thyroid Gland; Thyroid Neoplasms; Vimentin

We evaluated the newly produced tumor marker assay kit, "Ball ELSA CYFRA21-1", which detects cytokeratin 19 fragment in the sera of patients with malignancies, especially lung cancers. The assay procedure is simple based on the one-step radioimmunometric assay method. The measured values depend somewhat on incubation temperature and time. Reproducibility and recovery were good. The minimum measurable level was 1 ng/ml. The dilution test was satisfactory. The CYFRA21-1 levels were gradually decreased by repeated freezing and thawing and after seven such exercises its activity dropped to about 70% of that of first assay. The presence of CYFRA21-1 antigen was strongly correlated with TPA antigen and, although some discrepancies could be observed in clinical samples, CYFRA21-1 activity was completely absorbed by anti-TPA antibody-coated beads in one sample. CYFRA21-1 levels of 44 normal controls were below 1.0 ng/ml. Assuming a cut-off value of 2.0 mg/ml, 32.7% of all cases with benign disease had values greater than 2.0 ng/ml. This fell to 21.4% on exclusion of cases of interstitial pulmonary disease. Those with malignant diseases had high CYFRA21-1 levels whether associated with lung cancer or not. The most high positive ratios were observed in squamous cell lung cancer, small cell lung cancer, and uterine cervical cancer. In conclusion, CYFRA21-1 may be a good tumor marker comparable to TPA not only for lung cancer but also other malignancies as well. High false positives for lung cancer, however, were observed in other pulmonary diseases.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Biomarkers, Tumor; Child; Evaluation Studies as Topic; Female; Humans; Immunoradiometric Assay; Keratin-19; Keratins; Lung Neoplasms; Male; Middle Aged; Neoplasms; Reagent Kits, Diagnostic

The monoclonal antibody OV-TL 12/30, which detects keratin 7, was tested for its usefulness in cytologic diagnosis by reincubating previously Papanicolaou-stained slides. For this purpose malignant effusions of 73 patients with histologically confirmed cancers of the colon, ovary, mesothelium, breast, lung, esophagus, pancreas, urinary bladder, stomach, kidney, and prostate were used. All malignant cells from ovarian adenocarcinomas were positive, whereas malignant cells from colonic adenocarcinomas and malignant mesotheliomas were negative. Adenocarcinomas of gastric, renal, pancreatic, esophageal, and mammary origin demonstrated variable staining. Transitional cell carcinomas were positive, whereas squamous and small cell lung carcinomas were negative. Because OV-TL 12/30 does not react with normal and atypical mesothelial cells in these preparations, this reagent is a valuable tool in distinguishing benign mesothelial cells and adenocarcinoma cells. The authors' results demonstrate that this antibody is an excellent tool in the differential diagnosis of malignant cells in effusions and can be used in routinely stained cytologic specimens to determine primary tumor localization. In addition to its ability to distinguish between ovarian and colonic adenocarcinomas, its negativity in mesotheliomas may prove helpful in several diagnostic considerations.

Topics: Antibodies, Monoclonal; Carcinoma; Diagnosis, Differential; Female; Humans; Immunohistochemistry; Keratins; Male; Neoplasms; Papanicolaou Test; Vaginal Smears

The authors present an improved method for rapid two-color staining with direct conjugated antibodies to cytokeratin and CD45 antigen (leukocyte common antigen) for whole-cell, ethanol-fixed preparations of human carcinomas. This method was quality controlled with the T24 human bladder tumor cell line and compared in parallel analysis of 24 fresh human carcinomas with the original two-color method of multiparametric analysis that had been published in 1989. This rapid method was designed to achieve comparable staining intensities of both green (phenotype directed monoclonal antibody label) and red (propidium iodide labeled DNA) fluorescence, identical DNA indexes, comparable coefficients of variation, and subjective visual quality of DNA histograms. This is accomplished in a single (one-shot), abbreviated incubation with monoclonal antibody diluted in propidium iodide-RNase, thereby eliminating two incubations and three wash steps required with the original method. The single rinse is done in the propidium iodide-RNase staining solution with resuspension in fresh staining solution before analysis. With the rapid method, the preparation time is reduced by 130 minutes, resulting in a 60% time savings in batch staining mode compared with the original method. The time reduction and fewer wash steps, which should avoid excessive cell loss and cytoplasmic stripping, may advance the adoption of this two-color method in clinical practice.

Topics: Color; DNA, Neoplasm; Flow Cytometry; Humans; Keratins; Leukocyte Common Antigens; Neoplasms; Ploidies; Regression Analysis; Staining and Labeling; Tumor Cells, Cultured

The present study was designed to determine whether CYFRA 21-1, measuring cytokeratin 19, could be a specific and sensitive tumour marker for non-small cell lung cancer (NSCLC). Serum measurements were made at diagnosis in 2250 patient samples by an immunoradiometric "sandwich type" assay, using two cytokeratin 19 specific monoclonal antibodies. Among healthy individuals (n = 711) and patients with benign lung disease (n = 546), 95 percentiles were 1.2 and 2.95 ng/ml, respectively. Cumulative distribution analysis curves were established. From these data, 3.3 ng/ml gave 96% specificity. Using this cutoff, the sensitivity for small cell lung cancer was 16% (n = 74) compared to 41% for NSCLC (n = 547). In histological sub-groups, sensitivity was 57% for squamous cell lung cancer, 34% for undifferentiated large cell carcinoma and 27% for adenocarcinoma, the level of CYFRA 21-1 was correlated with tumour size and UICC stage. In squamous cell lung cancer, the sensitivity of the squamous cell carcinoma marker was 30%, 25% for carcinoembryonic antigen and 46% for tissue polypeptide antigen, using the same series of samples and cutoffs defined at 96% specificity. In conclusion, CYFRA 21-1 is a sensitive tumour marker for NSCLC, especially squamous cell lung cancer.

Topics: Adolescent; Adult; Aged; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Female; Humans; Keratins; Lung Diseases; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Neoplasms; Peptide Fragments; Retrospective Studies; Sensitivity and Specificity

A simple and rapid one-step method for demonstrating immunohistochemical markers (leukocyte common antigen, cytokeratin, etc.) is described, which can help define the nature of poorly differentiated neoplasms for diagnosis using frozen section. Microwave irradiation was used to speed immunohistochemical analysis using "Enhanced Polymer One-step Staining" (EPOS) reagents on cryostat sections from a variety of pathologic samples. Reproducible results were obtained using EPOS reagents for leukocyte common antigen and cytokeratin. The overall procedure takes less than 10 min and can be completed during surgery.

Topics: Frozen Sections; Humans; Immunohistochemistry; Keratins; Leukocyte Common Antigens; Lymph Nodes; Microwaves; Neoplasms; Thymus Gland

Recently, a new 'specific tissue polypeptide antigen (TPA)' test was introduced and designated tissue polypeptide-specific antigen (TPS); it is based on the monoclonal antibody (MAb) anti-TPS, M3. We have tested the specificity of this antibody by immunocyto- and immunohistochemistry, gel electrophoresis and immunoblotting. MAb M3 bound to intermediate filaments of epithelial cells and revealed a staining pattern identical to cytokeratin (CK) 18-specific MAb (DE-K18) on tissue sections of various human tissues. On immunoblots of proteins extracted from various epithelial cell lines, M3 reacted with a 45-kD protein corresponding to CK18, and on immunoblots of proteins isolated from MCF-7 culture fluid M3 stained three bands, 45, 33 and 29 kD. The same bands were stained with CK18-specific MAb, indicating that they represent CK18 and its degradation products. TPA, used as a tumor marker in clinical diagnoses and follow-up, was shown to be a degradation product of CK 8, 18 and 19. In contrast to TPA, MAb M3 did not stain CK8 and CK19 present on immunoblots.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Antigens, Neoplasm; Biomarkers, Tumor; Epithelium; Humans; Immunoblotting; Immunohistochemistry; Keratins; Mice; Microscopy, Fluorescence; Neoplasms; Peptides; Reagent Kits, Diagnostic; Tissue Polypeptide Antigen; Tumor Cells, Cultured

Topics: Antibodies; Biomarkers, Tumor; Breast Neoplasms; Cell Separation; DNA, Neoplasm; Female; Flow Cytometry; Humans; Intermediate Filament Proteins; Keratins; Molecular Probes; Neoplasms; Ploidies; Staining and Labeling; Urinary Bladder Neoplasms

Serum levels of cytokeratin subunit 19 (CYFRA 21-1) were measured in 42 healthy volunteers, 104 cases of malignant diseases, 30 patients with chronic renal failure and 13 patients with nonmalignant and infectious diseases. The reliability of the method was demonstrated after dilution of serum samples and intra- and inter-assay reproducibility. Serum CYFRA-21-1 concentrations were less than 2.00 ng/ml in all healthy controls and 86% of the malignant cases had high serum CYFRA 21-1 levels. However slightly elevated values of CYFRA 21-1 were observed in most chronic renal failure patients. High correlation was observed between serum CYFRA 21-1 and Tissue Polypeptide Antigen (TPA) values (r = 0.90, n = 10) but not with serum alpha-feto protein (AFP) concentrations. Furthermore, cross binding tests with the CYFRA 21-1 tracer/CYFRA 21-1 antibody-coated beads and CYFRA 21-1 tracer/TPA antibody-coated beads also gave an almost linear graph. These results indicate that CYFRA 21-1 and TPA share similar type of antigens.

Topics: Biomarkers, Tumor; Communicable Diseases; Cross Reactions; Female; Humans; Immunoradiometric Assay; Keratins; Kidney Failure, Chronic; Macromolecular Substances; Metabolic Diseases; Neoplasms; Reference Values; Reproducibility of Results

An increasing interest in fish species as sentinels of environmental pollution and in carcinogenesis research has led to the identification of diagnostically challenging neoplasms of uncertain cellular origin and the need for additional diagnostic methods. To determine the potential of using commercially available antibodies to intermediate filament proteins on paraffin-embedded fish tissues for immunocytochemistry in tumor diagnosis, the application of three antikeratin antibodies to normal adult tissues from two fish species was assessed. Multiple tissues from 12-14-in. striped bass (Morone saxatilis) and 6-month-old medaka (Oryzias latipes) of both sexes were fixed in Bouin's or formalin fixatives. Formalin-fixed neoplasms from several mammalian species, including cat, dog, hedgehog (Atelerix albiventris, Erinaceus europaeus), rhesus macaque (Macaca mulatta), and sloth bear (Melursus ursinus), were also used as positive controls. Using a strepavidin horseradish peroxidase method on paraffin-embedded tissues, the broad spectrum antibodies AE1/AE3 (Boehringer Mannheim, Indianapolis, IN) and MAK-6 (Triton Biosciences, Alameda, CA), which recognize most of the 19 human cytokeratins, and CAM 5.2 (Becton Dickinson, Mountain View, CA), which recognizes cytokeratins present in human liver, were used as primary antibodies. Epithelia from skin, gills, cornea, bile ducts, renal tubules, gastrointestinal tract, and thymus were strongly positive with AE1/AE3 and MAK-6 in striped bass, but nonepithelial tissues such as bone and muscle were negative. Skin, gills, cornea, and portions of the gastrointestinal tract were strongly positive in medaka with the same antibodies, whereas bile duct, renal, and intestinal epithelia were less so. Tissue digestion improved the intensity of staining, and fixation with Bouin's fixative improved results somewhat compared with formalin fixation.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Bass; Fish Diseases; Immunoenzyme Techniques; Keratins; Neoplasms; Oryzias; Species Specificity

We evaluated "BALL ELSA CYFRA21-1" kit, an immunoradiometric assay kit for cytokeratin 19. Monoclonal antibodies KS19-1 and BM19-21 are used for immunoadsorbent and indicator, respectively. There was no problems in reproducibility, dilution test and recovery test. Minimum detectable concentration was 0.42 ng/ml. The antigen measured by this kit was immunologically cross-reactive with tissue polypeptide antigen (TPA) and CYFRA21-1 concentration was closely correlated with TPA concentration in patient's serum. One of twenty-six healthy subjects showed serum concentration over a cut-off value of 2.0 ng/ml. Serum CYFRA21-1 concentration elevated in 5 of 10 esophageal cancer patients, 5 of 10 gastric cancer patients, 7 of 10 colorectal cancer patients and 6 of 10 pancreatic cancer patients. Positive rate in patients with benign disease including hepatopathy was low. BALL ELSA CYFRA21-1 kit is reliable and CYFRA21-1 could be a useful tumor marker in gastrointestinal cancer.

Topics: Adult; Antibodies, Monoclonal; Antigens, Neoplasm; Biomarkers, Tumor; Evaluation Studies as Topic; Female; Humans; Immunoradiometric Assay; Keratin-19; Keratins; Male; Middle Aged; Neoplasms; Reagent Kits, Diagnostic

We performed an immunohistochemical and ultrastructural study on 67 specimens of malignant fibrous histiocytoma (MFH) from 65 patients. Most of the tumors were musculoskeletal in origin and all presented clinically as a primary malignancy. The tumors were high grade and 57 of 67 were the storiform-pleomorphic subtype. Immunohistochemical studies were performed in 34 cases with both fresh-frozen and formalin-fixed tissue; in 33 cases only formalin-fixed tissue was available. The immunohistochemical panel included vimentin, various molecular weight keratins, epithelial membrane antigen (EMA), desmin, alpha-1-antitrypsin, and alpha-1-antichymotrypsin. Seventeen of 67 (25.4%) cases stained with one or more keratin antibodies. The low molecular weight cytokeratins demonstrated the most widespread and intense staining and, using fresh-frozen tissue, increased sensitivity. Epithelial membrane antigen was detected in 20.6% of cases and six of these cases also stained with keratin. The EMA staining was more focal and less intense than the keratin reactivity. The keratin- or EMA-positive cases were not distinguished by their light microscopic or ultrastructural features. Desmin staining was focally present in 16.9% of cases. The vast majority of tumors stained with vimentin and alpha-1-antitrypsin or alpha-1-antichymotrypsin. There was no staining of tumor cells for S-100. Appropriately fixed tissue was available for electron microscopic evaluation in 15 of 23 MFHs that stained with keratin or EMA. Ultrastructurally, all tumors were composed of an admixture of cells that had the features of fibroblasts, myofibroblasts, and histiocytes; no epithelial structures were identified. This study confirms that MFH may express epithelial markers. It emphasizes the importance of using electron microscopy and clinical findings to distinguish keratin or EMA-positive MFH from carcinoma. This distinction is important because of the significant differences in therapy and prognosis.

Topics: Adult; Aged; Aged, 80 and over; alpha 1-Antichymotrypsin; alpha 1-Antitrypsin; Desmin; Female; Histiocytoma, Benign Fibrous; Humans; Immunohistochemistry; Keratins; Male; Membrane Glycoproteins; Microscopy, Electron; Middle Aged; Mucin-1; Musculoskeletal Diseases; Neoplasms

Eighty-one epithelial lesions were studied immunohistologically for cytokeratin protein expression using three anticytokeratins CAM 5.2, NCL5D3 and RCK102. Consistent differences were noted between squamous and glandular neoplasms. Squamous and cutaneous carcinomas were found to preferentially express higher molecular weight cytokeratins than adenocarcinomas. In addition, tumours of similar morphology from different sites showed differences in expression of these markers. Differences in pattern were also found between benign and malignant lesions in the case of liver and urinary bladder. Thus the value of cytokeratin profile analysis in characterisation of epithelial neoplasms is confirmed and may be useful in distinguishing benign from malignant tumours in some instances.

Topics: Antibodies, Monoclonal; Biomarkers, Tumor; Carcinoma, Squamous Cell; Epithelium; Humans; Immunohistochemistry; Keratins; Neoplasms; Neoplasms, Glandular and Epithelial

Keratin filament are characteristically present in epithelial cells and tumors, but have also been detected in many normal and neoplastic non-epithelial cell types using immunohistochemical techniques. To investigate the validity of this seemingly aberrant protein expression, we applied the highly sensitive polymerase chain reaction (PCR) technique to study keratin gene expression in a variety of non-epithelial tissues. Total RNA was extracted from nine samples of leiomyosarcoma, four non-Hodgkin's lymphoma, seven normal bone marrows, normal lymph node, normal peripheral blood cells, freshly isolated and cultured endothelial cells, cultured skin fibroblasts, and the myeloid leukemia cell line HL-60. Amplification primers and probes for the three most primitive keratin types (8, 18, and 19) were synthesized using published gene sequences. RNA from the breast carcinoma cell line MCF-7, known to be rich in all three keratins, was used as positive control. Concurrently run actin primers were used to confirm RNA integrity. After an initial cycle with reverse transcriptase, PCR amplification was performed for 30 cycles. Southern blots of the PCR products showed variably intense bands corresponding to keratin 8 and 18 gene products in all samples, offering conclusive evidence of keratin gene expression in cells of both stromal and hematopoietic derivation. However, keratin 19 gene transcription was not nearly so ubiquitous, being detected in normal fibroblasts and endothelial cells, two of four non-Hodgkin's lymphoma and four of nine leiomyosarcoma, but not in normal lymph node, peripheral blood cells, HL-60 cells, or any of the seven normal bone marrows examined. Dilutional experiments showed PCR to be highly sensitive in the detection of keratin 19 gene expression, capable of registering one MCF-7 cell in 10(6) HL-60 cells. These studies show that variable levels of keratin 8 and 18 gene expression may be detected by PCR in a wide variety of non-epithelial tissues, supporting previous immunohistochemical and phylogenetic studies. However, keratin 19 gene expression appears to be more restricted and was not evident in any hematopoietic cells devoid of contaminating stromal elements. These findings suggest a role for PCR in the detection of epithelial micrometastasis in certain sites, particularly bone marrow.

Topics: Base Sequence; Gene Expression; Hematopoietic System; Humans; Keratins; Mesoderm; Molecular Probes; Molecular Sequence Data; Neoplasms; Polymerase Chain Reaction; Sensitivity and Specificity

It is known that cytokeratin 19 is particularly abundant in carcinoma of the lung.. A sandwich enzyme-linked immunosorbent assay called CYFRA 21-1 was, therefore, developed to detect soluble cytokeratin 19 fragments in serum using two specific monoclonal antibodies (Ks 19.1 and BM 19.21). The authors investigated the clinical significance of this new marker compared with the established markers carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC), neuron-specific enolase (NSE), carbohydrate antigen (CA) 19-9, CA 125, CA 15-3, CA 72-4, alpha-fetoprotein, and prostate-specific antigen in a pilot study on 1741 serum samples from patients with various benign and malignant diseases.. Postulating a specificity of 95% versus benign diseases of the lung, the diagnostic sensitivity of CYFRA 21-1 in lung cancer (independent of histologic type) at primary diagnosis was superior (47%) to CEA (27%), SCC (15%), and NSE (16%). Especially in squamous cell carcinomas of the lung, the true-positive test results were much higher for CYFRA 21-1 (60%) than for CEA (18%) or SCC (31%).. In small cell lung carcinomas, NSE was confirmed as the marker of first choice. For all of the other solid tumors investigated, CYFRA 21-1 showed no better profile of specificity and sensitivity than the established markers.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Biomarkers, Tumor; Carcinoma, Squamous Cell; Enzyme-Linked Immunosorbent Assay; Female; Humans; Keratins; Lung Neoplasms; Male; Neoplasms; Phosphopyruvate Hydratase; Pilot Projects; Retrospective Studies; Sensitivity and Specificity; Serpins

During the last several decades, immunohistochemical studies of tumors, along with other approaches, have suggested that the clinical and biological progression results, at least in part, from the sequential appearance within the neoplasm of cellular subpopulations whose new characteristics reflect specific somatic genetic changes. However, CNS may provide a different microenvironment for activation and proliferation than other tissues. The tissue-specific distribution of intermediate filament proteins, in particular the keratins, permits their use as marker in histopathology, but several important exceptions are recognized. In this connection, it is of interest that, according to the other reports, glial tumors may be positive for different anti-keratin antibodies. However, the gliomas did not show an immunoreaction in any of the cases when HEA-125 and Ber-EP4 were applied. The great number of multihormonal pituitary adenomas and possible change of the immunohistochemically detectable hormone status in cases of recurrent tumors have particularly re-emphasized the need for new thinking about patterns of classification. The diagnosis of malignant melanoma has been considerably facilitated recently by the introduction of immunohistological labelling with antibodies selective against melanoma antigen (HMB-45). Our results confirmed the necessity of cautious interpretation of HMB-45 immunoreactivity because a HMB-45 expression can be observed in several non-melanotic tumors.

Topics: Adenoma; Antibody Specificity; Biomarkers, Tumor; Central Nervous System Neoplasms; Humans; Immunohistochemistry; Keratins; Neoplasm Metastasis; Neoplasms; Pituitary Neoplasms

We examined the feasibility of performing non-radioactive in situ hybridization (ISH) in flow cytometrically sorted (tumor) cells with a chromosome #1 specific centromere probe. The study was performed in a model system of HL60 cells mixed with different quantities of HeLa cells. These latter cells were sorted directly onto poly-l-lysine coated glass slides on the basis of their keratin content, a cytoskeletal component not present in HL60 cells. Overall morphology of the separated HeLa cells was excellent and, after the ISH procedure, the appropriate number of ISH spots was observed in more than 85% of the sorted cells. This percentage did not differ significantly in cell mixtures with different percentages of HeLa cells (down to 1%). Sorting of HeLa cells in different phases of the cell cycle, and subsequent ISH, revealed the same spot number for chromosome #1 in all cell cycle stages, including mitosis. In the latter phase of the cell cycle we did not find a duplication of the chromosome #1 centromere, not even after sorting of the mitotic cells on the basis of specific labeling with an antibody to mitotin. The early G2 mitotin negative fraction, however, showed a significant percentage of cells with a duplicate spot number, most likely representing a tetraploid cell fraction in this HeLa cell culture. The protocol that evolved from these model studies was applied to cell suspensions of malignant body cavity effusions as well as solid bladder carcinomas. In several of these cases numerical chromosome aberrations could be detected by ISH more evidently after sorting on the basis of keratin labeling.

Topics: Antibodies, Monoclonal; Cell Cycle; Cell Cycle Proteins; Cell Separation; Centromere; Chromosomes, Human; Chromosomes, Human, Pair 1; DNA Probes; Flow Cytometry; HeLa Cells; Humans; Interphase; Keratins; Leukemia, Promyelocytic, Acute; Minichromosome Maintenance Complex Component 2; Neoplasms; Nuclear Proteins; Nucleic Acid Hybridization; Pleural Effusion; Tumor Cells, Cultured; Urinary Bladder Neoplasms

The application of immunohistochemical markers against epithelial antigens has proved useful for studying tumor differentiation and in aiding tumor diagnosis. However, the reactivity of various epithelial markers with poorly differentiated carcinomas (the situation in which they are most often used) has not been well established. As a result, it is unclear how negative results should be interpreted and how often more than one antibody may be needed to document the epithelial nature of poorly differentiated neoplasms. We studied 98 poorly differentiated epithelial tumors with AE1, CAM 5.2, and EMA to assess the use of these markers in their diagnosis. Both CAM 5.2 and EMA provided support for epithelial differentiation in 71% (70/98) of the cases, while AE1 stained 50% (49/98) of the tumors; CAM 5.2 was the single most useful marker in the subset of poorly differentiated neuroendocrine carcinomas, staining 20 (77%) of 26 tumors. Use of these markers in pairs increased the recognition of epithelial differentiation (at least one marker showing positive staining) as follows: AE1/CAM 5.2, 80% (78/98); AE1/EMA, 87% (85/98); and CAM 5.2/EMA, 99% (97/98). Thirty carcinomas stained with all three markers, 34 with two markers, and in 34 cases only one antibody supported epithelial differentiation. Twelve (21%) of 58 tumors showed evidence of S100 reactivity. None of the 71 cases to which PD7 was applied showed staining This study indicates that poorly differentiated carcinomas are heterogeneous in their expression of antigens recognized by AE1, CAM 5.2, and EMA. Moreover, these results quantitate the probability of reactivity with poorly differentiated carcinomas for each marker and support the use of one or more antibodies in a "backup" panel when a negative result is obtained with a single antibody and the diagnosis of carcinoma is still suspected.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Biomarkers, Tumor; Cell Differentiation; Epithelium; Humans; Immunoenzyme Techniques; Keratins; Membrane Glycoproteins; Mucin-1; Neoplasms; S100 Proteins

Three commonly used keratin monoclonal antibodies (MAB)--AE1:AE3, CAM 5.2, and MAK-6--were compared with routinely used cytokeratin antibody. The expression of these antibodies was analyzed in several tissues obtained from clinically normal dogs and in a variety of neoplasms from dogs. Using appropriate enzymatic digestion, paraffin-embedded tissues processed in routine manner retained their typical keratin expression. Differentiated and poorly differentiated epithelial neoplasms, lymphomas, and melanomas were studied by use of the avidin-biotin-peroxidase technique. All 4 of the aforementioned antibodies had similar staining profiles. Of 3 anaplastic carcinomas, 2 had positive reaction to all 4 antibodies. All lymphomas, plasma cell tumors, and amelanotic melanomas had negative reaction to MAK-6, CAM 5.2, AE1:AE3, and cytokeratin MAB. Three basal cell epitheliomas had positive reaction to all 4 antibodies, whereas 1 basal cell tumor with a solid pattern had negative staining reaction. Two carcinoids had negative reaction to all markers and 1 of 2 malignant chemodectomas and 1 transitional cell carcinoma had staining reaction to only AE1:AE3 MAB. Comparing the 4 antibodies, use of AE1:AE3 MAB produced the strongest staining intensity followed by cytokeratin, MAK-6, and CAM 5.2 MAB. All 4 antibodies had low background staining. In conclusion, AE1:AE3 and MAK-6 MAB are as useful as cytokeratin MAB for identification of poorly differentiated epithelial neoplasms in dogs and cats.

Topics: Animals; Antibodies, Monoclonal; Carcinoma; Dog Diseases; Dogs; Immunohistochemistry; Intestine, Small; Keratins; Lung Neoplasms; Neoplasms; Predictive Value of Tests; Urinary Bladder

Five commercially available cytokeratin antibodies (lu-5, AE1/AE3, CAM 5.2, MFN116 and anti-cytokeratin 18) were used to stain a wide range of normal and neoplastic epithelial and non-epithelial tissues to assess their potential value in diagnostic histopathology. All five showed good specificity, with some cross-reactivity in smooth muscle cells. The wider reactivity of AE1/AE3, lu-5, and MFN 116, which includes cytokeratins 8,18 (Moll's catalogue) expressed in simple epithelia and their tumours, as well as cytokeratins expressed in complex stratified squamous epithelia, permits identification of a wider range of epithelial derived tumours. This wider spectrum of reactivity may allow these antibodies to be used in a diagnostic panel for the identification of poorly differentiated tumours.

Topics: Antibodies, Monoclonal; Antibody Specificity; Epithelium; Female; Humans; Immunoenzyme Techniques; Keratins; Male; Neoplasms

A series of 14 new mouse monoclonal antibodies (MAbs) to keratins is described and the data suggesting their potential value in the differential diagnosis of human tumours are reported. The specificities of individual MAbs of the 'C-series' presented here range from monospecificity for keratin No. 7 (MAbs C-18, C-35, C-62, and C-68), keratin No. 8 (MAbs C-15, C-43, and C-15), and keratin No. 18 (MAbs C-04 and C-08) up to the broadly reacting 'pan-keratin' MAb C-11, with the target epitopes of the remaining four MAbs being shared by different pairs of keratin polypeptides. The results of the biochemical characterization of the MAbs, together with their immunohistochemical staining patterns on frozen as well as on paraffin sections of normal human tissues, suggest that they represent a significant contribution to the growing list of anti-keratin MAbs applicable in both research and routine diagnostic pathology. The immunohistochemical examination of a wide range of human neoplasms with the new MAbs not only confirmed their value in making distinctions between carcinomas, on the one hand, and lymphomas, and gliomas, on the other, but also verified the possibility of more subtle subdivisions within the group of adenocarcinomas and their metastases. Furthermore, the identification of small subsets of breast carcinomas with decreased levels or apparent loss of the keratin No. 7 polypeptide and some cases of stomach carcinoma with apparently induced expression of this keratin suggests that such 'exceptions' must be considered when using keratin spectra as one of the criteria in differential diagnosis.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Cell Line; Electrophoresis, Gel, Two-Dimensional; Epithelium; Fluorescent Antibody Technique; Humans; Immunoblotting; Keratins; Mice; Neoplasms

The immunoreactivity of OV-TL 12/30, a monoclonal anti-keratin 7 antibody (Mab), was investigated on frozen as well as paraffin-embedded human tissues. Its reactivity patterns were compared with another well-characterized monoclonal antibody to keratin 7 (RCK 105), and with broadly cross-reacting monoclonal (OV-TL 12/5) as well as polyclonal (pKer) keratin antisera. In frozen sections of normal and malignant human tissues both keratin 7 Mabs gave similar staining patterns. The immunoreactivity for OV-TL 12/30 and the polyclonal antibody (pKer) in tissue sections fixed in 4 per cent formalin or Bouin solution, was completely restored when pretreated with 0.1 per cent pronase, 0.1 per cent trypsin in phosphate-buffered saline (PBS) or with 0.5 per cent pepsin in 0.01 N HCl. Except for loss of immunoreactivity on human normal stomach surface epithelium and glandular mucous cells, Mab OV-TL 12/30 reacted strongly positive with essentially all those formalin- or Bouin-fixed paraffin-embedded tissues that had been shown to stain in non-fixed, frozen sections. In addition to the good correlation in human tissues, a complete correlation between the reactivity on frozen and paraffin-embedded human carcinomas (n = 86) was found as well. While both RCK 105 (anti-keratin 7) and OV-TL 12/5 (anti-keratin 5, 7, 14, 19) did not stain on paraffin-embedded sections, the polyclonal control antiserum (pKer) lost immunoreactivity in some cell types (e.g. mucous cells in compound glands, hepatocytes, pancreatic acinar cells, and proximal and distal convoluted tubules of the kidney). Our study shows that the keratin 7 Mab OV-TL 12/30 is an excellent marker for tumour histopathology since it is reactive in paraffin-embedded formalin-fixed human tissues.

Topics: Antibodies, Monoclonal; Biomarkers, Tumor; Humans; Immunoenzyme Techniques; Keratins; Neoplasms; Tissue Distribution

Reactivity patterns of seven mouse monoclonal antibodies to human keratin 7 were compared using immunoblotting and immunohistochemistry on cultured cells and normal human and animal tissues. Differences in keratin specificities as determined by two-dimensional immunoblots and interspecies cross-reactivity data on 8 mammalian species suggest that at least six nonidentical epitopes of the keratin 7 molecule are recognized by this panel of reagents. Immunohistochemical examination of a panel of various human neoplasms with monoclonal antibodies monospecific for keratins 7, 18 and 19 revealed potential value of keratin subtyping in differential diagnosis of tumors in general and in subclassification of carcinomas in particular.

Topics: Animals; Antibodies, Monoclonal; Breast Neoplasms; Colonic Neoplasms; Cross Reactions; Fluorescent Antibody Technique; Humans; Intermediate Filament Proteins; Keratins; Neoplasms; Staining and Labeling; Tumor Cells, Cultured; Urinary Bladder Neoplasms

The cell differentiation properties of thirty-four sacrococcygeal teratomas (SCT) and their five recurrences were immunohistochemically studied for the expression of different classes of intermediate filament proteins, muscle actin (MA) and S-100 protein. Out of thirty-nine tumors twenty-three were SCTs with only mature tissue elements, seven immature teratomas, five pure endodermal sinus tumors (EST) and four ESTs or embryonal carcinomas (EC) combined with mature components. Cytokeratin positivity was found in all epithelial structures and sometimes also in smooth muscle and primitive mesenchymal cells. An intense cytokeratin immunoreactivity was observed in EC and EST components. Muscle markers, desmin and MA were present in smooth and striated muscle cells. Focal desmin positivity was also found in some epithelial structures in two cases. Glial tissue positive for glial fibrillary acidic protein (GFAP) was found in twenty-eight out of thirty-nine tumors. Some cases with no apparent glial tissue in hematoxylin and eosin staining showed glial differentiation as proved by GFAP positivity. In six out of eleven choroid plexus-like tissues GFAP positive cells were observed. S-100 protein showed an intense distribution of immunoreactivity outside neural tissue, and focal positivity was observed in malignant epithelial structures. Immunohistochemical markers did not reveal any prognostic significance in teratomas. Our findings, however, showed some aberrant features of cell differentiation from normal mature tissue components but closely parallel to those found in normal fetal development.

Topics: Actins; Cell Transformation, Neoplastic; Child; Desmin; Female; Follow-Up Studies; Glial Fibrillary Acidic Protein; Humans; Infant; Infant, Newborn; Keratins; Male; Mesonephroma; Neoplasms; Prognosis; S100 Proteins; Sacrococcygeal Region; Teratoma

A monoclonal enzyme-linked immunosorbent assay (ELISA) was developed for the determination in biological fluids of cytokeratin 8, a potential marker for malignant diseases. Two monoclonal antibodies (MAbs), TS 3 and TS 4, with different epitope specificity, were selected from 4 cytokeratin-8 reactive antibodies. TS 3 was used for coating and TS 4 as HRP-conjugate, respectively. Antibodies were selected with the aim of optimizing the discriminatory capacity between cytokeratin 8 levels in sera from healthy persons and from patients with malignant diseases. In sera from healthy individuals the mean value was determined to be 3.1 +/- 2.3 ng/ml with an upper cut-off level of 7.8 ng/ml (+ 2 SD) using purified cytokeratin 8 as standard. Sera from patients with colon cancer and pancreatic cancer were found to have significantly elevated levels, showing a 4- to more than 10-fold increase compared with the normal level. In patients with ovarian cancer no significant elevation was seen. Cytokeratin 8 monitoring may be of value for patients with colon and pancreatic cancer.

Topics: Antibodies, Monoclonal; Colonic Neoplasms; Enzyme-Linked Immunosorbent Assay; Female; Humans; Keratins; Male; Neoplasms; Ovarian Neoplasms; Pancreatic Neoplasms

One of the great challenges in the cytodiagnosis of effusions is the distinction between reactive mesothelium/histiocytes and cancer cells. This is notably true in patients having undergone radiation and/or chemotherapy. To establish whether monoclonal antibodies (MoAbs) could be used as reliable diagnostic adjuvants, the authors retrospectively and blindly studied 60 cases diagnosed by standard cytologic criteria (malignant, benign, and equivocal), with a panel of seven readily available MoAbs (cytokeratins, vimentin, EMA, B72.3, alpha-CEA, HMFG-2, and Leu-M1) and the lectin Ulex europaeus I. All 18 (100%) malignant cases showed reactivity with EMA and HMFG, whereas 17 (95%) and 11 (61%) reacted with B72.3 and alpha-CEA, respectively. Combinations of (1) EMA + B72.3, (2) EMA + alpha-CEA, and (3) EMA + alpha-CEA + B72.3 displayed positivity in 17 (95%), 11 (61%), and 10 (56%) malignant cases, respectively. Of the 18 benign cases, 7 reacted with HMFG and 2 each with EMA and B72.3. Only one case (5.5%) reacted with both EMA and B72.3. Based on these results, the 24 equivocal cases were regrouped into 14 malignant and 10 benign cases. Follow-up effusions obtained within the ensuing three months in all these patients allowed the authors to unequivocally confirm the diagnosis in all but five. The combination of EMA and B72.3 MoAbs detected malignant cells in 95% of the cases, with a 3.5% incidence of false positive cases in this study. A panel of EMA, B72.3, and alpha-CEA MoAbs should prove the most useful and simple approach to the correct diagnosis in most questionable effusions. Some of the potential pitfalls are discussed.

Topics: Antibodies, Monoclonal; Ascitic Fluid; Carcinoembryonic Antigen; Follow-Up Studies; Humans; Immunohistochemistry; Keratins; Membrane Glycoproteins; Mucin-1; Neoplasms; Pleural Effusion, Malignant; Retrospective Studies; Vimentin

Specimens of neoplastic tissues from 19 dogs and 4 cats were examined immunohistochemically for intermediate filament expression, using commercially available antibodies. Staining was observed in a wide range of tumor tissues and in normal internal controls by use of antibodies to vimentin, desmin, glial fibrillary acidic protein, and low and high molecular weight cytokeratins. Intermediate filament expression was found to be consistent with light and/or electron microscopic findings, and hence believed to be an accurate indicator of tumor histogenesis in cats and dogs. Three fixatives were evaluated for their relative abilities to preserve antigenicity. Absolute alcohol was superior to B5 fixative and both were superior to formalin. Some tissues that clearly displayed intermediate filament antigens with alcohol and B5 fixative failed to stain when fixed in formalin.

Topics: Adenocarcinoma; Animals; Astrocytoma; Cat Diseases; Cats; Cytoskeleton; Desmin; Dog Diseases; Dogs; Immunohistochemistry; Intermediate Filament Proteins; Intermediate Filaments; Keratins; Leiomyosarcoma; Melanoma; Neoplasms; Neurilemmoma

Keratins are the most heterogenic class of intermediary filament molecules and form tonofilaments in all types of epithelial cells. At least 19 different human keratins have been demonstrated. On the basis of biochemical and immunological criteria, these may be subdivided into two subtypes (type I and type II). Recent molecular biological investigations have provided considerable insight into the construction of the keratin molecule and the tonofilament structure and the corresponding gene structure. The basic structure in the tonofilament is a dimer composed of one type I and one type II molecule which is the background for the coordinated paired expression of type I and type II keratins which are observed in all epithelial cells. A given epithelial cell type expresses a constant pattern of keratin pairs which serve as markers for the cell type concerned. In general, the characteristic keratin pattern is retained in neoplastic differentiation of the epithelial cell. With the production of monospecific antikeratin antibodies, it is now possible to identify carcinomata immunologically which improves the possibilities of determine the degree of differentiation and the source. Employment of antikeratin antibodies in tumour diagnosis may be anticipated to be of significance for assessment of the prognosis and monitoring and choice of therapy.

Topics: Humans; Keratins; Molecular Biology; Neoplasms

The use of antibodies specific for the various types of intermediate filament proteins has become an important part of the immunohistochemical approach to surgical pathology. These proteins are expressed in a cell lineage-specific pattern, which is reproduced with great fidelity even in poorly differentiated neoplasms. The historic discovery, embryology, and structure of these proteins are reviewed. A brief discussion of the major applications and precautions in the choice and use of these antibodies is presented.

Topics: Amino Acid Sequence; Antibodies; Cell Line; Cross Reactions; Cytoskeleton; Epithelial Cells; Epithelium; Humans; Immunohistochemistry; Intermediate Filament Proteins; Intermediate Filaments; Keratins; Neoplasms

We describe a new monoclonal antibody (designated Bu20a) against bromodeoxyuridine (BrdU). This antibody was selected by screening against human tissues using the APAAP technique, and shows no crossreactivity with normal nuclei. It stains BrdU incorporated into the nuclei of a wide range of cell types, including human tonsil lymphoid cells, normal mouse tissues, and human tumors growing in nude mice. A double-labeling technique is described using this antibody in which cell smears or tissue sections are first labeled by an immunoperoxidase procedure for a cellular antigen (e.g., mouse or human histocompatibility class II antigen, T-lymphocyte antigen, keratin) and BrdU is then detected by indirect immunofluorescence. This procedure, which was applied to a variety of human and animal cells and tissues, is of wide potential value in analyzing the phenotype of S-phase cells and in co-localizing antigen expression and BrdU incorporation in tissue sections.

Topics: Animals; Antibodies, Monoclonal; Antigens, Differentiation, T-Lymphocyte; Bromodeoxyuridine; Cell Nucleus; Histocompatibility Antigens Class II; Humans; Immunoenzyme Techniques; Immunohistochemistry; Interphase; Keratins; Lymphoid Tissue; Neoplasms; Rats; Tumor Cells, Cultured

Postirradiation sarcomas are an unusual but well-recognized late effect of cancer therapy. In this article, a fine-needle aspiration (FNA) series of four cases is presented. There were three female patients and one male patient, with an age range of 28-55 yr (mean, 41). Two of the patients were irradiated for uterine cervical carcinoma while the other two received irradiation for malignant lymphoma. The time interval to the development of the postirradiation sarcoma ranged from 10 to greater than 20 yr. There were a postirradiation synovial sarcoma of the buttock region, malignant fibrous histiocytoma of the bone (femur), and rhabdomyosarcoma and angiosarcoma of the retroperitoneum. A spectrum of cytologic findings was encountered, reflecting the specific types of sarcomas. Immunocytochemical studies performed on the aspirated material from the angiosarcoma demonstrated the utility of immunoperoxidase stains for ULEX europaeus agglutinin-1 (UEA-1) and, to a lesser degree, factor VIII-related antigen antibody, confirming the vascular nature of this malignancy. The FNA findings from all four cases demonstrated cytologic features that allowed recognition of this unusual complication of irradiation treatment. This article confirms the utility of FNA cytology in following patients with previous malignancies and differentiating a postirradiation sarcoma from recurrent carcinoma.

Topics: Adult; Aged; Biopsy, Needle; Female; Hemangiosarcoma; Humans; Immunoenzyme Techniques; Keratins; Male; Middle Aged; Neoplasms; Neoplasms, Radiation-Induced; Radiotherapy; Vimentin

Twenty normal canine tissue specimens, both fetal and adult; 19 epithelial neoplasms; and 18 nonepithelial neoplasms were examined using 6 commercially available monoclonal antibodies differing in their recognition of various molecular weight cytokeratins in human tissues. Fresh tissue samples were fixed in 100% ethanol and paraffin embedded prior to sectioning. The intermediate filament proteins were identified by an avidin-biotin-immunoperoxidase method. Primary antisera used included AE1/AE3, CAM-5.2, 35BH11, 34BE12, PKK1, MAK-6 cytokeratins, and vimentin. Monoclonal antibodies detected cytokeratins in a wide variety of canine epithelial tissues and neoplasms. Normal mesenchymal tissues and neoplasms, and stromal elements of epithelial tissues, showed no reactivity with anti-cytokeratins, but reacted positively with vimentin. Although PKK1, CAM-5.2, and MAK-6 were the most consistently reactive anti-cytokeratins, the full panel of monoclonals was required to detect cytokeratins in all of the epithelia evaluated.

Topics: Animals; Antibodies, Monoclonal; Dog Diseases; Dogs; Epithelium; Immunohistochemistry; Keratins; Neoplasms

Topics: Antibodies, Monoclonal; Biomarkers, Tumor; Humans; Keratins; Membrane Glycoproteins; Mucin-1; Neoplasms

Seven monoclonal antibodies of rat origin were generated against human epidermal keratins and were assayed by immunoblot and by immunoperoxidase staining of frozen sections from various human epithelial tissues. By immunoblotting, the antibodies recognized two different subsets of high molecular weight cytokeratin polypeptides. Unlike the majority of the reported mouse monoclonal antibodies obtained to similar immunogens, these rat monoclonal antibodies showed an unexpected degree of specificity, i.e., they stained only stratified squamous epithelia and not simple epithelia nor urothelia. The potential of using rats and the appropriate immunogen to generate more tissue-specific anticytokeratin monoclonal antibodies is discussed.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Epithelium; Humans; Hybridomas; Immunoenzyme Techniques; Immunologic Techniques; Keratins; Male; Neoplasms; Rats; Rats, Inbred Strains

Two monoclonal antibodies, UCD/AB 6.11 and UCD/PR 10.11, were evaluated for their patterns of immunohistochemical reactivity in a survey of paraffin-embedded human tissues by the avidin-biotin-immunoperoxidase technique. By two-dimensional immunoblotting, UCD/AB 6.11 reacts with keratin number 18 and UCD/PR 10.11 identifies both keratin numbers 8 and 18, the major keratins found in human simple epithelial cells. Both antibodies have excellent signal-to-noise ratios, and specifically react with simple epithelia, transitional epithelia, mesothelia and tumors derived from such tissues, making them superior immunological reagents. They do not react with neural, muscle, hematopoietic, connective or most epidermal tissues. However, within a given tissue or cell type, differences in the distributions of the antigens recognized by these two antibodies can be observed, raising the possibility of differential expression, modification, or masking of the keratin epitopes revealed by UCD/AB 6.11 and UCD/PR 10.11.

Topics: Antibodies, Monoclonal; Biomarkers, Tumor; Humans; Immunohistochemistry; Keratins; Neoplasms

Topics: Antibodies, Monoclonal; Antibody Specificity; Antigens, Neoplasm; Ascites; Biomarkers, Tumor; Carcinoembryonic Antigen; Diagnosis, Differential; Humans; Immunoenzyme Techniques; Keratins; Membrane Glycoproteins; Mucin-1; Neoplasms; Pericardial Effusion; Pleural Effusion

Ten commercially available antikeratin antisera were tested immunohistochemically on fresh frozen and formalin-fixed paraffin-embedded tissue. Eight of the antisera were in addition tested on protein-immunoblottings. For six of the antisera a good correspondence was found between our immunoblots and data given by the manufacturers. Two monoclonal antisera did not react with keratin proteins. On immunohistochemical testing two of the antibodies showed qualitatively identical staining on both frozen and paraffin sections without background staining. Three of the antibodies reacted weakly or not at all on paraffin sections but gave acceptable staining on frozen sections. Three of the antibodies showed acceptable staining on paraffin sections, but background staining on frozen sections and one antibody gave the reverse staining pattern. For one of the antibodies it was impossible to obtain an acceptable staining due to high non-specific binding of the secondary antibody. None of the antikeratins were true panepithelial tumour markers as all of them failed to detect keratin in at least one of the epithelial tumours. However, a combination of two or three antikeratins (Hybritech AE1 + AE3, Becton Dickinson No 7650, DAKO A622) covered most or all epithelial tumours examined. It is concluded that commercially available antisera show great variability with respect to quality and reactivity indicating that the majority need further purification, characterization and testing on tissues before they are introduced on the commercial market.

Topics: Antibodies; Antibodies, Monoclonal; Colon; Cross Reactions; Epidermis; Epithelium; Esophagus; Frozen Sections; Humans; Immune Sera; Immunoblotting; Immunohistochemistry; Keratins; Neoplasms; Trachea

Topics: Desmin; Humans; Intermediate Filament Proteins; Keratins; Neoplasms; Neurofilament Proteins

In this study we examined 198 sarcomas, 38 carcinomas, 13 'tumours with a spindle cell component' and 22 malignant melanomas with a commercial monoclonal vimentin antibody. All histopathological material was formalin fixed and paraffin embedded. The results show this antibody to be a sensitive and specific marker of mesenchymal derivation or differentiation. It is a useful tool in separating sarcomas from most carcinomas, and in separating malignant melanomas from carcinomas. When used in combination with a cytokeratin antibody it identifies carcinosarcomas and synovial sarcomas.

Topics: Antibodies, Monoclonal; Carcinoma; Carcinosarcoma; Diagnosis, Differential; Humans; Immunoenzyme Techniques; Keratins; Melanoma; Neoplasms; Retrospective Studies; Sarcoma; Vimentin

Topics: Adenocarcinoma; Breast Neoplasms; Carcinoma, Squamous Cell; Carcinoma, Transitional Cell; Cytoskeleton; Diagnosis, Differential; Epithelium; Female; Gastrointestinal Neoplasms; Humans; Intermediate Filament Proteins; Intermediate Filaments; Keratins; Lung Neoplasms; Neoplasms; Skin Neoplasms; Urinary Bladder Neoplasms; Uterine Cervical Neoplasms

The present study describes the distribution of Leu M1 (CD15) immunoreactivity in 13 normal epithelia and 14 epithelial neoplasms. The findings are compared with those obtained using other epithelial markers. Cautionary points and broad recommendations are made with regard to use of anti Leu M1 antibody.

Topics: Antibodies, Monoclonal; Antigens; Antigens, Differentiation, T-Lymphocyte; Antigens, Neoplasm; Antigens, Surface; Antigens, Tumor-Associated, Carbohydrate; Carcinoembryonic Antigen; Epithelium; Humans; Immunoenzyme Techniques; Keratins; Membrane Proteins; Mucin-1; Neoplasms

Metastatic poorly differentiated carcinomas often represent diagnostic difficulties in surgical pathology. Therefore, the expression of cytokeratins of different molecular weights (54, 57, and 66 kd) were compared in paraffin sections of 37 primary carcinomas with their lymph node metastases by an avidin-biotin complex (ABC) method, using monoclonal antibodies. The epithelial tumors consisted of 16 squamous cell carcinomas (SCCs) and 17 adenocarcinomas with different degrees of differentiation (well, moderately, or poorly differentiated), a renal cell carcinoma, a hepatocellular carcinoma, a transitional cell carcinoma of the bladder, and a carcinoid tumor of the stomach. The primary and metastatic tumors showed the same cytokeratin profiles. All SCCs and their metastases were positive for 57-kd cytokeratin and negative for 54-kd cytokeratin. All adenocarcinomas and their metastases were positive for 54-kd cytokeratin and negative for 66-kd cytokeratin. The extent of reactions varied with the differentiation of the carcinomas, with well-differentiated tumors showing more diffuse staining. Cases of lymphoma, sarcoma, and melanoma were negative for the three types of cytokeratins. The results indicate that identification of different molecular weight cytokeratins may be used to distinguish poorly differentiated SCCs from poorly differentiated adenocarcinomas, even in metastatic tumors. In addition, demonstration of these cytokeratins is useful in substantiating presence and identity of small foci of metastases in lymph nodes.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Carcinoma, Squamous Cell; Diagnosis, Differential; Histocytochemistry; Humans; Immunoenzyme Techniques; Keratins; Lymphatic Metastasis; Molecular Weight; Neoplasms; Staining and Labeling

Among the monoclonal antibodies capable of detecting epithelial lineage, some recognize keratin and others identify antigens of epithelial membranes. Of the latter, the most commonly used is directed against an antigen present in cell membranes derived from milk fat globules, epithelial membrane antigen (EMA). To determine their relative sensitivity and specificity and hence their diagnostic value, we compared four commercially available monoclonal antibodies to low-molecular-weight keratins--AE1, CAM 5.2, PKK1, and 35 beta H11--with the monoclonal antibody to EMA (anti-EMA). We studied 383 samples of human neoplasms of diverse histogenetic types and degrees of differentiation. Anti-EMA was found to be less sensitive than the monoclonal antibodies to keratin in several epithelial tumors, including tumors of the prostate (11 of 13 negative), gastrointestinal tract (13 of 34 negative), and thymus (seven of eight negative). Anti-EMA was also less sensitive in mesotheliomas (nine of 24 negative). Of the embryonal carcinomas, all stained with the monoclonal antibodies to keratin, whereas none stained with anti-EMA. False-positive staining with anti-EMA was seen in two of 14 T-cell lymphomas. We conclude that the monoclonal antibodies to low-molecular-weight keratins are more sensitive and specific for the identification of epithelial origin of neoplasms than is monoclonal anti-EMA. Anti-EMA should not be used as the sole marker of epithelial differentiation in tumor diagnosis.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Colonic Neoplasms; False Positive Reactions; Histocytochemistry; Humans; Keratins; Lymphoma; Male; Membrane Proteins; Mucin-1; Neoplasms; Prostatic Neoplasms

The avidin-biotin-peroxidase-antiperoxidase complex method (ABPAP) of immunohistochemistry employs the sequential application of the peroxidase-antiperoxidase complex (PAP) and the avidin-biotin-peroxidase complex (ABC) technics. To assess the efficacy of ABPAP in the detection of cellular antigens and to compare its utility with that of protease digestion technics, the authors studied cytokeratin (CK) reactivity in six examples each of colon, prostate, and breast cancer and Leu-M1 reactivity in five ductal breast carcinomas. ABC, PAP and ABPAP were applied to these cases alone and in combination with prior pepsin digestion of deparaffinized sections. With respect to the number of cells that stained and their intensity in each case, there was significant enhancement of CK and Leu-M1 reactivity with ABPAP, as compared with ABC and PAP when pepsin digestion was not employed. Further, ABPAP provided undigested CK staining results comparable to those seen with the use of ABC or PAP with pepsin digestion. Protease treatment was uniformly detrimental to the visualization of Leu-M1, regardless of the method employed. In summary, ABPAP was clearly superior to ABC and PAP in all settings analyzed. It may prove to be useful adjunct in the immunohistochemical visualization of tissue antigens, when more routine technics yield suboptimal results.

Topics: Antibodies, Monoclonal; Antigens, Surface; Avidin; Biotin; Female; Histocytochemistry; Humans; Immunoenzyme Techniques; Keratins; Male; Neoplasms

Vimentin has been regarded as the intermediate filament characteristic of normal and neoplastic mesenchymal cells and keratins as typical of epithelial cells and the neoplasms derived from them. However, many epithelial cells in tissue culture or in effusion, as well as cells in some solid epithelial neoplasms such as renal, endometrial, ovarian, pulmonary, and thyroid adenocarcinomas, have been shown to coexpress vimentin and keratin. The recent availability of monoclonal antibodies that work reasonably well in paraffin-embedded tissue led us to carry out a comprehensive immunohistochemical study on formalin- and alcohol-fixed specimens of neoplasms in which we used monoclonal antibodies against vimentin. These results were compared with our previous study in which the same tumor tissues were investigated using antibodies against keratin. The antigenicity of vimentin was found to be preserved in all alcohol-fixed specimens and in 63% of formalin-fixed tissues. Vimentin was the sole intermediate filament present in virtually all sarcomas, meningiomas, schwannomas, and melanomas. In addition, variable percentages (10-57%) of carcinomas, neuroendocrine carcinomas, neuroblastomas, thymomas, and mesotheliomas were positive for vimentin, which, except in the neuroblastomas, was coexpressed with keratins. Among the adenocarcinomas, more than 50% of papillary carcinomas of the thyroid, as well as renal, endometrial, ovarian, and lung carcinomas, coexpressed keratins and vimentin. A distinctive paranuclear and basal localization of vimentin was observed in the cells of many of these tumors, in contrast to the predominantly apical distribution of the keratins. The authors conclude that coexpression of vimentin and keratin is more widespread than previously reported and that antibodies to vimentin, by themselves, are of limited value for the differentiation of epithelial from mesenchymal neoplasms.

Topics: Antibodies, Monoclonal; Epithelium; Ethanol; Fixatives; Formaldehyde; Humans; Keratins; Neoplasm Proteins; Neoplasms; Vimentin

Two routinely used antikeratin monoclonal antibodies, AE1:AE3 (Hybritech Inc., La Jolla, CA) and CAM-5.2 (Becton-Dickinson, Mountain View, CA), were compared with a new antikeratin monoclonal antibody mixture, MAK-6 (Triton Biosciences, Inc., Alameda, CA). Various poorly differentiated epithelial neoplasms, lymphomas, melanomas, and sarcomas were studied with the use of the avidin-biotin-peroxidase technic. All three antibodies had similar staining profiles, however, some differences were noted. The MAK-6 and CAM-5.2 antibodies illustrated stronger staining for some grade III transitional cell carcinomas, whereas the AE1:AE3 was variably positive to negative in all cases. Three renal cell carcinomas were positive with all three antibodies, two were negative with all three, and one had scattered positive cells with MAK-6 only. All lymphomas and plasmacytomas were negative with MAK-6 and CAM-5.2, however, AE1:AE3 faintly stained two of three plasmacytomas and two of the seven large cell lymphomas. Two out of three hepatomas evaluated were strongly positive with CAM-5.2 and MAK-6 and variably positive with AE1:AE3. The third case had both positive and negative cells with all three antibodies. In conclusion, MAK-6 antikeratin antibody is as useful as AE1:AE3 and CAM-5.2 for identification of poorly differentiated epithelial neoplasms.

Topics: Antibodies, Monoclonal; Antibody Specificity; Evaluation Studies as Topic; Humans; Immunoenzyme Techniques; Keratins; Neoplasm Proteins; Neoplasms; Predictive Value of Tests

A rapid immunohistologic method is described to analyze the possible cell origin of a given neoplasm during surgery by using selected examples of monoclonal and polyclonal antibodies (anti-cytokeratins, anti-vimentin, anti-leukocyte common antigen, and anti-keratin). This rapid (26-28 min) procedure, consisting of a two-step immunoperoxidase method, did not differ in terms of intensity and specificity of the immunoreaction from the control procedure for which conventional longer times of incubation and washing were used. The results demonstrate the feasibility and effectiveness of the procedure.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Biopsy; Freezing; Histocompatibility Antigens; Humans; Immunohistochemistry; Keratins; Leukocyte Common Antigens; Neoplasms; Vimentin

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) dependent growth and differentiation of 6 tumor cell lines has been determined by the use of the monolayer proliferation assay. Cell lines of 4 gastro-intestinal carcinomas, 1 malignant schwannoma, and 1 malignant histiocytoma have been established and characterized. Cells were incubated for 4, 7, and 11 days in the presence of 0.8 or 8 nM 1,25(OH)2D3 and for control without addition of the hormone. Proliferation rates of 1,25(OH)2D3 treated cells were compared with cell growth in the untreated controls. Five out of 6 cell lines showed a 1,25(OH)2D3 dependent growth pattern. With 8 nM 1,25(OH)2D3 they were all inhibited. With 0.8 nM, 3 of them were inhibited at any time of the test period, whereas 1 was stimulated at day 4 and inhibited at days 7 and 11. One cell line was stimulated at days 4, 7, and 11 when incubated with 0.8 nM 1,25(OH)2D3. No striking morphological changes could be observed in the presence of 1,25(OH)2D3. We conclude that 1,25(OH)2D3 dependent cells in vitro are not necessarily growth-inhibited by this compound. Thus, 1,25(OH)2D3 is not an exclusively proliferation inhibiting agent.

Topics: Antigens, Neoplasm; Calcitriol; Cell Division; Gastrointestinal Neoplasms; Histiocytoma, Benign Fibrous; Humans; Immunoenzyme Techniques; Keratins; Neoplasms; Neurilemmoma; Tumor Cells, Cultured; Vimentin

When malignant human cells are crossed with diploid human keratinocytes, malignancy, as defined by progressive growth in vivo, is suppressed so long as the hybrid cells continue to produce involucrin, a protein that characterizes terminal differentiation in the keratinocyte. When, on continued cultivation in vitro, the cells lose the ability to produce involucrin, they reacquire the ability to grow progressively in the animal.

Topics: Cell Division; Cell Line; Electrophoresis, Polyacrylamide Gel; Epidermal Cells; Epidermis; Humans; Hybrid Cells; Immunoenzyme Techniques; Keratins; Neoplasms; Protein Precursors

Production of a new monoclonal antibody designated NCL-5D3 is described. The antibody recognizes several low molecular weight cytokeratins, in particular cytokeratin Moll number 8 as determined by immunoblotting studies, and is highly effective for immunocytochemistry using routinely processed paraffin-embedded material. Staining is enhanced by prior treatment of the sections with trypsin. Assessment using a wide variety of normal and neoplastic tissue indicates reactivity with all tissues of simple or glandular epithelial origin, and in addition with many squamous carcinomas. Thus the antibody should prove of value in diagnostic histopathology.

Topics: Antibodies, Monoclonal; Antibody Specificity; Histological Techniques; Humans; Immunoenzyme Techniques; Keratins; Molecular Weight; Neoplasms; Paraffin; Trypsin; Tumor Cells, Cultured

Three monoclonal antibodies, 1C7, 2D7 and 6B10, directed against cytokeratins of human esophagus were isolated and characterized by one- and two-dimensional gel electrophoresis and by immunohistochemical staining on sections of human epithelial tissues. In immunoblot experiments, antibodies of clones 1C7 (IgG2a) and 2D7 (IgG2b) react only with cytokeratin no. 13 of the acidic (type I) subfamily of cytokeratin polypeptides (Mr 54000; pI 5.1); antibodies of clone 6B10 (IgG1) detect only cytokeratin no. 4 (Mr 59000; pI 7.3) of the basic (type II) cytokeratin subfamily and allows the detection of this protein and possible degradation products at high sensitivity. In immunohistochemical staining all three antibodies stain non-cornifying squamous epithelium (e.g., tongue, esophagus, anus) and transitional epithelium of the bladder. Antibodies of clone 6B10 also stain cells in certain ciliated pseudostratified epithelia and ductal epithelia of various exocrine glands. These monoclonal antibodies are the first examples of antibodies specific for individual cytokeratin polypeptides characteristic of certain complex epithelia. They allow the identification of distinct minor populations of cells present in certain complex and glandular epithelia and in tumors derived therefrom which hitherto have not been distinguished. The possible reasons for the occurrence of cell type heterogeneity of cytokeratin expression in complex epithelia and in some carcinomas are discussed.

Topics: Antibodies, Monoclonal; Antibody Specificity; Cell Line; Epithelial Cells; Epithelium; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; Isoelectric Point; Keratins; Molecular Weight; Neoplasms

Topics: Animals; Cell Differentiation; Cell Line; Histocytochemistry; Humans; Immunochemistry; Keratins; Neoplasms; Neurosecretory Systems; Vimentin

Immunoperoxidase staining for human keratin proteins was performed cytologically on samples from 90 patients with malignant tumors, and histologically on samples from 164 patients with malignant tumors. At the cytological level, almost all tumor cells not only in squamous cell carcinoma but also in nonsquamous cell carcinoma were positive for keratin proteins, in contrast with the apparent abscence of keratin proteins in sarcoma. At the histological level, almost all neoplastic cells of squamous cell carcinoma were positive for keratin proteins, the same as at the cytological level. In contrast, among cases of nonsquamous cell carcinoma, the frequency of appearance of keratin proteins varied according to the organ; it tended to be low in tumors with relatively good prognosis, such as carcinomas in the digestive system or thyroid cancer, and to be high in tumor with poor prognosis, such as pulmonary cancer, gallbladder cancer and endometrial cancer. However, there was a marked difference between the frequency of appearance of keratin proteins at the cytological level and that at the histological level, particularly in the cases of gastric cancer.

Topics: Antibodies; Breast Neoplasms; Colonic Neoplasms; Female; Gallbladder Neoplasms; Histocytochemistry; Humans; Keratins; Lung Neoplasms; Neoplasms; Sarcoma; Stomach Neoplasms; Thyroid Neoplasms; Uterine Neoplasms

The method described below combines an immunoreaction with Papanicolaou's stain on cytological smears. For the immunoreaction, the avidin-biotin-complex (ABC) method was used. The method was tested on various cytological material with the monoclonal antibody lu-5 and two polyclonal antibodies (anti-keratin and anti-chymotrypsin). Wet fixation of the smears with a modified Delaunay's solution is recommended. Drying of the material impairs immunoreactivity. The main advantages of the technique are the clear-cut permanent immunostaining and the preservation of the nuclear structure, permitting a combined immuncytological characterization of cellular products and conventional cyto-diagnosis.

Topics: Antibodies, Monoclonal; Avidin; Biotin; Chymotrypsin; Cytodiagnosis; Cytological Techniques; Epithelium; Histocytochemistry; Humans; Immunologic Techniques; Keratins; Neoplasms; Pleural Effusion; Staining and Labeling

Antibodies to intermediate filament proteins react in a tissue-specific manner and can be used to characterize tumor cells present in thin-needle aspirates from solid tumors, from palpable lymph nodes and cells present in samples from peritoneal and pleural effusions. From our studies so far the following conclusions can be drawn: Polyclonal antisera to cytokeratins can identify carcinoma metastases in thin-needle aspirates from palpable lymph nodes and distinguish them from malignant lymphomas and nonmalignant lesions such as chronic lymphadenitis, which show only vimentin-positive cells. Monoclonal antibodies to specific cytokeratin polypeptides are able to distinguish between different types of epithelial tumor metastases, i.e. metastases from adenocarcinomas and metastases from squamous cell carcinomas. Cells present in peritoneal and pleural effusions can be partly characterized using intermediate filament antisera. We have found that metastatic adenocarcinoma cells from breast, ovary, endometrium, cervix, colon and stomach, as well as squamous cell carcinomas and malignant mesothelioma stain specifically with antibodies to cytokeratin while mesenchymally derived tumors such as malignant lymphomas, malignant melanomas, and fibrosarcomas, are positive for vimentin only. Metastatic tumor cells of epithelial origin present in aspirates from human serous body cavity fluids may coexpress vimentin next to their original cytokeratin intermediate filaments. Benign mesothelial cells present in body cavity fluids frequently coexpress cytokeratins and vimentin. Tumor cells present in thin-needle aspirates from solid tumors such as pleomorphic adenomas of the parotid gland can be identified as such because of their typical patterns of intermediate filament (co-)expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenoma; Biopsy, Needle; Clinical Laboratory Techniques; Cytoskeletal Proteins; Fluorescent Antibody Technique; Humans; Immune Sera; Keratins; Lymph Nodes; Neoplasms; Vimentin

Tumours of uncertain tissue of origin were investigated by immunohistochemistry on formalin fixed paraffin embedded sections. Two antibodies--PD7/26, an anti common leucocyte antigen, and CAM5.2, an anticytokeratin--recognised most lymphomas and carcinomas, respectively: 88% of these tumours were identified by the two antibodies alone. These antibodies permitted the separation of the cases into groups: positive with CAM5.2, positive with PD7/26, and a third comprising those negative with both. The negative group contained other tumours and a small number of carcinomas and lymphomas; many of the lymphomas were, apparently, of histiocytic origin. Comparison of CAM5.2 with other epithelial markers showed that it was the most effective. Some further classification of the tumours was carried out with a panel of organ and cell specific antibodies: mesotheliomas were recognised by their pattern of reactivity with epithelial markers. Overall, the tumour type was determined in 90% of cases. Immunohistochemistry performed as described can be a potent aid to the diagnostic histopathology of tumours.

Topics: Antibodies, Monoclonal; Breast Neoplasms; Carcinoma; Carcinoma, Squamous Cell; Histocompatibility Antigens; Humans; Keratins; Leukocyte Common Antigens; Lymphoma; Mesothelioma; Neoplasms; Sarcoma; Skin Neoplasms; Thyroid Neoplasms

Epithelial membrane antigen and keratin proteins represent markers of epithelial differentiation that may be detected in routine formalin-fixed, paraffin-embedded tissues. Eighty-seven neoplasms, including 48 adenocarcinomas of various types, squamous and transitional cell carcinomas, small-cell anaplastic carcinomas, carcinoid tumors, mesotheliomas, hepatomas, melanomas (metastatic), adrenal cortical carcinomas, germ cell tumors, and extramammary Paget's disease, were assessed to determine the relative effectiveness of these antigens as tumor markers. Immunoperoxidase studies were performed using monoclonal antibodies to epithelial membrane antigen and monoclonal (combined AE1 and AE3) and polyclonal (bovine muzzle keratins) antibodies to keratin proteins. In more than half the cases (50/87%), both markers yielded comparable results. However, in 29 cases (33%), keratin proteins were clearly superior to epithelial membrane antigen as a tumor cell marker. Particular discrepancies were apparent for some gastrointestinal adenocarcinomas, squamous cell carcinomas, hepatomas (hepatocellular type), spindle cell components of mesotheliomas, and carcinoid tumors. Epithelial membrane antigen represented a better marker in eight cases (9%), mainly for small-cell anaplastic carcinomas and some renal cell and pulmonary adenocarcinomas. Adrenal cortical carcinomas, melanomas, and seminomas were nonimmunoreactive for both antigens. Epithelial membrane antigen and keratin proteins represent useful complementary markers in diagnostic surgical pathology.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Antigens, Surface; Carcinoma; Humans; Immunoenzyme Techniques; Keratins; Lymphoma; Membrane Proteins; Mucin-1; Neoplasms

A neoplastic epithelial cell line, TYS, was isolated from a well-differentiated squamous cell carcinoma expressing carcinoembryonic antigen (CEA) that arose in human oral mucosa. Expressions of CEA and amylase as well as ample tonofilaments were detected in cultured TYS cells. Transplantation of the cells into athymic nude mice resulted in production of adenoid squamous cell carcinoma containing CEA and amylase. Cultivation of TYS cells in the presence of sodium butyrate resulted in suppression of cell growth and production of secretory granules with amylase in the cytoplasm of the cells. When the sodium butyrate-treated cells were transplanted into nude mice, a small mass developed transiently at the inoculation site and then disappeared. This mass was histopathologically interpreted as acinic cell carcinoma with squamoid lesion. These findings suggest that we have established a human adenoid squamous carcinoma cell line presumably derived from a minor salivary gland present in oral mucosa.

Topics: Adenoids; Aged; Aged, 80 and over; Carcinoembryonic Antigen; Carcinoma, Squamous Cell; Cell Line; Female; Humans; Keratins; Lymphatic Diseases; Mouth Neoplasms; Neoplasm Transplantation; Neoplasms

Rabbits were immunized with 10 nm filaments of a mixture of cytokeratins which has been isolated from human heel callus material and reconstituted to filaments in vitro. The antisera to keratins (ASK) have been tested histologically at fixed and unfixed tissue samples by means of the indirect immunofluorescence and PAP technique. The ASK recognized specifically only the epithelial cells of skin, of the mucous membranes of mouth and digestive tract, of salivary glands, sweat gland and mammary gland, but did not react with hepatocytes or kidney cells. The following tumors, tested till now, reacted with the antikeratin antisera: epithelial and lymphoepithelial carcinomas of skin, mouth and digestive tract, carcinomas of salivary glands, mammary gland and thyroid gland, adamantinoma, basalioma of skin, and metastases from carcinomas.

Topics: Animals; Electrophoresis, Polyacrylamide Gel; Epithelial Cells; Epithelium; Fluorescent Antibody Technique; Gastric Mucosa; Humans; Immune Sera; Immunoenzyme Techniques; Intestinal Mucosa; Keratins; Microscopy, Electron; Mouth Mucosa; Neoplasms; Rabbits; Rats; Salivary Glands; Sebaceous Glands; Skin; Sweat Glands

We describe here antigenic cross-reactivity between the human 40-kilodalton cytokeratin polypeptide [Moll et al] and components of bovine desmosomal plaque, namely desmoplakins I and II. This relationship was revealed by an antibody (KM 4.62), raised against cytoskeletal preparation of cultured human breast adenocarcinoma cells (MCF-7) and selected by immunoblotting and immunofluorescent labeling. In cultured human cells that contain the 40-kD cytokeratin, antibody KM 4.62 labeled arrays of filaments throughout the cytoplasm. This antibody labeled the basal layer of nonkeratinizing squamous epithelia as well as various simple (normal and malignant) epithelia and epithelial elements of the thymus. In liver tissue, labeling was obtained only in bile ducts and canaliculi but not in the hepatocytes. In bovine cells and tissues, on the other hand, immunofluorescent labeling with antibody KM 4.62 was strictly confined to desmosomes. This was verified by double immunolabeling with both antibody KM 4.62 and specific cytokeratin or desmosomal antibodies. Immunoblotting analysis indicated that the former antibody reacts specifically with the high molecular weight components of the bovine desmosomal plaque, namely desmoplakins I and II. These immunochemical results suggest that bovine desmoplakins share same structural relationship with the human acidic, 40-kD cytokeratin.

Topics: Animals; Antibodies, Monoclonal; Cattle; Cell Line; Cross Reactions; Cytoskeletal Proteins; Desmoplakins; Epitopes; Female; Fluorescent Antibody Technique; Humans; Keratins; Membrane Proteins; Molecular Weight; Neoplasms

The monoclonal antibodies BA16 and BA17, reacting specifically with human keratin 19 (40 kD) have been tested by immunohistochemical staining methods for their reaction with a wide range of human tumours and cultured cells. Primary adenocarcinomas and their metastases showed a homogeneously positive reaction with greater than 95% of the tumour cells staining. Non-epithelial tumours, basaliomas and squamous cell carcinomas were unstained, while benign breast lesions and a thyroid adenoma show a mosaic pattern of stained and unstained (5-40%) cells. These three staining patterns were also seen in cultured cells. Positive homogeneous staining was seen in all breast cancer cell lines examined with the exception of PMC42, which exhibits stem cell characteristics, and which showed the heterogeneous pattern of staining seen in milk cell cultures. Non-epithelial lines and strains, two cell lines from cervical carcinomas and three SV40 transformed breast epithelial lines were unstained. The antibodies BA16 and 17 are potentially useful reagents for distinguishing adenocarcinomas (and their metastases) from non-epithelial tumours and from squamous carcinomas. They may also discriminate between benign and malignant breast lesions, and identify a specific differentiation phenotype in the secretory cell lineage.

Topics: Antibodies, Monoclonal; Breast Neoplasms; Cell Line; Cells, Cultured; Epitopes; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; Keratins; Male; Neoplasms; Testicular Neoplasms

Topics: Humans; Keratins; Neoplasms; Thymus Gland; Thymus Neoplasms

We examined the immunological cross-reactivity between tissue polypeptide antigen (TPA) and keratin protein because of the reported sequence homology between these two proteins. TPA showed positive immuno-reactivity against both polyclonal and monoclonal antibodies for keratin. The binding of [125I]TPA with anti-keratin could be displaced dose-dependently by unlabeled keratin. However, no cross-reaction between keratin and anti-TPA was found. TPA also showed the positive immuno-reactivity with antibodies for blood group antigens (A, B and Lewis substances), but keratin did not. A standard solution of Lewis substance reacted with neither anti-TPA nor with anti-keratin. These data strongly suggest that TPA has an immunological similarity with both keratin protein and blood group antigens. When [125I]TPA and [125I]keratin were gel-filtered on a Sephadex G-200 column, the radioactivities of TPA and keratin were found mainly in the void volume fraction (MW greater than 200 000) and the MW of approximately 60 000, respectively. Chromatography on Sepharose 6B suggested that the MW of [125I]TPA was 320 000. When sera of cancer patients were gel-filtered on a Sephadex G-200 column, TPA activity was distributed mainly in the void volume fraction in all tested cases. This experiment suggests that TPA may be a glycoprotein (MW is 320 000) with both keratin-like and blood group antigen-like determinants.

Topics: Antibody Specificity; Blood Group Antigens; Chromatography, Gel; Epitopes; Humans; Immunochemistry; Keratins; Neoplasms; Peptides; Radioimmunoassay; Tissue Polypeptide Antigen

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neoplasm; Cell Differentiation; Cytoskeleton; Humans; Keratins; Neoplasms; Rats

A large number of human neoplasms were tested for their keratin expression in routinely processed tissues by a simple, three-stage immunoperoxidase method using a broadly reactive monoclonal anti-keratin antibody AE1, which recognizes a number of keratin polypeptides distributed in a wide variety of epithelia. All carcinomas, with the exception of hepatocellular, adrenocortical, and basal cell carcinomas and occasional renal cell, pulmonary small-cell, and pulmonary large-cell anaplastic carcinomas, reacted with this antibody irrespective of differentiation, in most instances displaying staining of strong or moderate intensity in the majority of tumor cells. Equivocal results were obtained in some seminomas and dysgerminomas. Malignant melanoma, large-cell lymphoma, Hodgkin's disease, malignant histiocytosis, and stromal mesenchymal elements in all tumors did not show any reactivity with AE1. Even after routine processing, the determinant detected by AE1 is conserved and restricted to epithelial neoplasms. This suggests that AE1 would be valuable in the diagnostic distinction of anaplastic carcinoma from lymphoma and melanoma in routinely processed tissues.

Topics: Antibodies, Monoclonal; Antibody Specificity; Carcinoma; Epithelium; Immunoenzyme Techniques; Keratins; Neoplasm Proteins; Neoplasms

Topics: Antibodies, Monoclonal; Diagnosis, Differential; Humans; Immunoenzyme Techniques; Keratins; Neoplasms

Twenty-one anaplastic tumors were studied by light microscopy (LM), immunoperoxidase staining using anti-epidermal cytokeratin (ECK) and anti-Mallory body cytokeratin (MBCK) antibodies, and electron microscopy (EM), to determine whether an epithelial origin could be confirmed. The tumors were derived from lung, stomach, colon, breast, uterus, kidney, bladder, and mesothelium. By LM, the tumors consisted of either large and polygonal, spindle or small, round cells. With immunoperoxidase staining, 11 (52%) of the anaplastic tumors were positive for ECK, positivity being either absent or only weak in the main tumor mass, but marked in areas of infiltration and metastases. In contrast, all of the anaplastic tumors were positive for MBCK in the main tumor mass, infiltrating areas, and metastases. In the case of adenocarcinomas, staining was either web-like or diffuse throughout the cytoplasm with concentration occurring at the cell surface, whereas in mesotheliomas, the staining was either diffuse or showed focal perinuclear accentuation. Twelve of 13 anaplastic tumors examined by EM showed epithelial features (desmosomes, tonofilaments, lumina, and/or microvilli). As controls, 21 non-epithelial tumors (five melanomas, eight sarcomas, and eight lymphomas) showed no reactivity with either cytokeratin antibody. These studies show that the epithelial nature of undifferentiated and poorly differentiated tumors can be confirmed by immunohistochemistry using anti-cytokeratin antibodies.

Topics: Epithelium; Humans; Immunoenzyme Techniques; Keratins; Kidney Neoplasms; Lymphoma; Melanoma; Neoplasms; Sarcoma; Tissue Distribution

The expression of specific keratin polypeptides in human neoplasms was investigated by the immunoblot technique using monoclonal anti-keratin antibodies. Mr 50,000 and 58,000 keratins, recognized by AE1 and AE3 antibodies, respectively, were detected only in carcinomas of stratified epithelial origin, but not in carcinomas derived from simple epithelia. No keratin was detected in nonepithelial tumors including melanoma, lymphoma, neurofibroma, and sarcoma. The results suggest that the Mr 50,000 and 58,000 keratins provide useful molecular markers for identifying neoplasms of stratified squamous epithelial origin.

Topics: Antibodies, Monoclonal; Clinical Laboratory Techniques; Female; Humans; Keratins; Molecular Weight; Neoplasms

Fifty primary carcinomas, nineteen nonmuscular sarcomas, ten rhabdomyosarcomas, one malignant ependymoblastoma and seventeen sympathetic derived tumors have been studied to determine what type of intermediate filaments they express, using affinity purified antibodies to prekeratin, vimentin, desmin, glial fibrillary acidic protein (GFA) and to the different neurofilament proteins. The tissue was ethanol fixed and paraffin embedded before use. In all carcinoma cases the tumor cells are stained by antibodies to prekeratin, whereas tumor cells in nonmuscular sarcomas could only be labeled by antibodies to vimentin. In the cases of rhabdomyosarcomas tumor cells are clearly decorated by antibodies to desmin. The one case of malignant ependymoblastoma was positive when tested with antibodies directed against glial fibrillary acidic protein (GFA). While most of the sympathetic derived tumors react positively with sera directed against the different neurofilament proteins, three neuroblastomas did not react with sera against any of the known intermediate filament types. Wider use of such sera would seem particularly useful in cases such as neuroblastomas, undifferentiated carcinomas, lymphomas and rhabdomyosarcomas.

Topics: Antibodies, Neoplasm; Cytoskeleton; Desmin; Female; Fluorescent Antibody Technique; Glial Fibrillary Acidic Protein; Humans; Intermediate Filament Proteins; Keratins; Neoplasms; Protein Precursors; Vimentin

Purified type beta transforming growth factor from human platelets (TGF beta) radioiodinated with 125I-labeled Bolton and Hunter reagent was found to bind to a variety of cultured cells of both epithelial and mesenchymal origin, including normal human fibroblasts and keratinocytes. TGF beta binding sites have also been found on three mouse embryo-derived fibroblast-like cell lines with lower levels of TGF beta binding on the chemically transformed derivatives of these cell lines. A variety of human tumor cell lines was shown to have an inverse correlation between their level of TGF beta binding and their ability to form colonies in soft agar. The mouse embryo-derived AKR-2B (clone 84A) cells reached maximal binding of 125I-labeled TGF beta after 2 hr at 22 degrees C. Scatchard analysis of the equilibrium binding of TGF beta to AKR-2B (clone 84A) cells gives a Kd of 33 pM with approximately equal to 10,500 binding sites per cell. This Kd for TGF beta binding to AKR-2B (clone 84A) cells agreed well with the ED50 of 40 pM for stimulation of colony formation of these cells by TGF beta. The TGF beta binding sites on the AKR-2B cells were shown to be specific for TGF beta with no significant competition with epidermal growth factor, fibroblast growth factor, or insulin and only a small level of competition with high concentrations of platelet-derived growth factor. Partially purified preparations with TGF beta-like activity from mouse embryos and medium conditioned by mouse embryo-derived cells competed effectively for binding to the TGF beta receptor.

Topics: Animals; Binding, Competitive; Blood Platelets; Cell Line; Embryo, Mammalian; Epithelium; ErbB Receptors; Fibroblasts; Growth Substances; Humans; Keratins; Kinetics; Mesoderm; Mice; Neoplasms; Peptides; Receptors, Cell Surface; Transforming Growth Factors

Immunoperoxidase staining for human keratin proteins (hKP) was performed cytochemically in samples from 79 cancer patients, and histochemically in samples from 134 cancer patients. Immunohistochemically, hKP was present in almost all patients with squamous cell carcinoma (lung), transitional cell carcinoma (urinary bladder), adenocarcinoma (lung, stomach, breast, ovary), bronchiole-alveolar carcinoma (lung), and large cell carcinoma (lung). It was detected in 40% of the patients with small cell carcinoma (lung). Histochemically, hKP was present in patients with squamous cell carcinoma (lung, uterine body), adenocarcinoma (lung, stomach, colon, gall bladder, thyroid, uterine body), adenosquamous cell carcinoma (gall bladder, uterine body), Signet-ring cell carcinoma (stomach), clear cell carcinoma (uterine body) and undifferentiated carcinoma (uterine body). However, it was not detected in patients with brochiole ++-alveola ++ carcinoma (lung) and mucinous carcinoma (gall bladder).

Topics: Breast Neoplasms; Colonic Neoplasms; Female; Histocytochemistry; Humans; Keratins; Lung Neoplasms; Male; Neoplasms; Stomach Neoplasms; Urinary Bladder Neoplasms; Uterine Neoplasms

Because of the broad clinical interest which tissue polypeptide antigen (TPA) has attracted as a tumor marker, human cell lines and human tissues have been analyzed for TPA expression using immunofluorescence microscopy. Epithelial cell lines including HeLa, MCF-7, and A-431 are recognized by TPA antibodies whereas human lines of non-epithelial origin are not. The positive staining patterns coincide with keratin-type intermediate filaments of the cytoskeleton. On tissue sections a subset of epithelial cells including uterine epithelium, bile duct cells in liver and tumor cells in breast carcinoma are strongly positive; cells of the squamous epithelia of skin and tongue as well as cells of non-epithelial origin are negative. In immunoblots of human epidermis, human tongue mucosa, human hair follicles, Detroit 562 cells, HeLa cells, MCF-7 and RT-4 cells, only keratins 8, 18 and 19 show TPA antigenicity. Conversely a TPA preparation is recognized by various antibodies known to react with keratins, including alpha-IFA, KG 8.13.2 and two antibodies which recognize keratins 18 (CK2) and 19, respectively. Our results thus relate TPA to human keratins 8, 18 and 19 which are known cytoskeletal components in both normal and malignant epithelial cells of simple and non-squamous origin. We speculate that the elevated levels of circulating TPA antigenicity present in the sera of patients with carcinoma, which are often used to monitor tumor progression, correspond to soluble proteolytic fragments originating from this particular keratin subgroup.

Topics: Animals; Antibodies; Antigen-Antibody Complex; Antigens; Antigens, Neoplasm; Cell Line; Clinical Laboratory Techniques; Dipodomys; Female; Fluorescent Antibody Technique; Humans; Keratins; Neoplasms; Peptides; Structure-Activity Relationship; Tissue Polypeptide Antigen

Forty-six tumors in children were examined using light microscopy and subsequently frozen sections were stained with antiprekeratin and antivimentin antisera, so that the tumors could be classified by tissue of origin. Except for two adrenal cortical carcinomas and four liver tumors, most epithelial neoplasms continued to produce prekeratin filaments, a characteristic of normal epithelial cells. Tumors and cells of epithelial origin did not produce vimentin filaments, whereas normal and neoplastic mesenchymal cells did. Tumors with both epithelial and mesenchymal components produced vimentin filaments in mesenchymal areas and prekeratin in epithelial areas. Tumors of lymphoid origin showed variable production of vimentin filaments, depending on the amount of cell cytoplasm, but did not contain prekeratin filaments. Of the neuroectodermal tumors, only the ganglioneuroma contained vimentin filaments and none contained prekeratin filaments. Thus, antibodies to both prekeratin and vimentin filaments are useful in diagnosing childhood neoplasms and studying their histogenesis.

Topics: Child; Cytoskeleton; Fluorescent Antibody Technique; Humans; Immune Sera; Intermediate Filament Proteins; Keratins; Neoplasms; Protein Precursors; Staining and Labeling; Vimentin

Topics: Female; Humans; Joint Diseases; Keratins; Middle Aged; Neoplasms; Sarcoma; Synovial Membrane

The presence of intracellular keratin was examined in 230 human neoplasms using indirect immunofluorescence on fresh frozen, acetone-fixed sections. The use of antikeratin antibodies raised in rabbits against human callus and purified by affinity chromatography proved to be a rapid, sensitive, and reliable method of demonstrating keratin. Epithelial tissues and epithelial-derived neoplasms were found to contain keratin, whereas tissues and neoplasms of mesenchymal, lymphoreticular, or neural crest origin did not contain intracellular keratin. This technic is a useful adjunct for the surgical pathologist in the diagnosis of poorly differentiated neoplasms. Its application either confirmed, modified, or in several instances, changed the original light microscopic impression. The modified or changed diagnoses were confirmed by transmission electron microscopy.

Topics: Adult; Aged; Antibodies; Breast Neoplasms; Carcinoma, Basal Cell; Female; Fluorescent Antibody Technique; Humans; Keratins; Lymphoma, Large B-Cell, Diffuse; Male; Mediastinal Neoplasms; Middle Aged; Neoplasms; Skin Neoplasms; Thymoma

Metastatic tumor cells of epithelial origin present in effusions from human serous cavity fluids (ascites or pleural fluid) were examined for their intermediate-sized filament types by using antibodies to keratin, vimentin, and desmin in the indirect immunofluorescence technique. Solid epithelial tumors (both primary carcinomas and their metastases) contain keratin intermediate-sized filaments exclusively. However, when these cells are present in ascitic or pleural fluid, they also express vimentin, which occurs in a fibrillar organization. The possible effects of this additional, but temporary, cytoskeleton on metastatic growth or aggressiveness (or both) are discussed.

Topics: Carcinoma; Cytoskeleton; Fluorescent Antibody Technique; Humans; Intermediate Filament Proteins; Keratins; Neoplasm Metastasis; Neoplasms; Vimentin

Human epithelial cells contain, intermediate-sized filaments formed by polypeptides related to epidermal alpha-keratin ("cytokeratins") which are expressed in different combinations in different epithelia. Using cytoskeletal proteins from human biopsies and autopsies we have examined, by two-dimensional gel electrophoresis and immunoblotting experiments, the cytokeratin polypeptide patterns of diverse primary and metastatic carcinomas and have compared them with those of corresponding normal epithelial tissues and cultured cells. Five groups of carcinoma cytokeratin patterns can be discriminated. (1) Cytokeratins typical of simple epithelia (polypeptides Nos. 7, 8, 18, 19) are expressed, in various combinations, by many adenocarcinomas, for example those of gastrointestinal tract. (2) Cytokeratins typical of stratified epithelia (Nos. 1, 5, 6, 10, 11, 14-17) are found, in various combinations, in squamous cell carcinomas of skin and tongue. (3) Complex patterns showing polypeptides Nos. 7, 8, 18, 19, and one basic component (No. 5 or 6) are detected in certain carcinomas of the respiratory tract and the breast. (4) Complex patterns containing cytokeratins widespread in stratified epithelia (Nos. 4-6, 14-17) as well as components Nos. 8 and 19 occur in diverse squamous cell carcinomas derived from non-cornified stratified epithelia, with or without additional small amounts of cytokeratin No. 18. (5) Patterns of unusually high complexity can be found in some rare tumors as is shown for a cloacogenic carcinoma. No significant qualitative changes of expression of cytokeratins were found when primary tumors and metastases were compared. When compared with cytokeratin patterns of normal epithelia, carcinomas of the first type usually display a high degree of relatedness to the tissue of origin. Other carcinomas do not express some of the cytokeratins present in the tissue of their origin and, vice versa, certain components which are minor or apparently absent in normal tissue are major cytokeratins in the corresponding tumor. These differences may be explained by cell type selection during carcinogenesis, but changes of expression during tumor development cannot be categorically excluded. The possibility of cell type heterogeneity within a given tumor is also discussed. Similarly complex patterns of cytokeratin polypeptides have been noted in certain cultured human carcinoma cell lines (e.g., A-431, RPMI 2650, Detroit 562, A-549) and can also be observed in cel

Topics: Breast Neoplasms; Carcinoma, Squamous Cell; Cell Line; Cytoskeleton; Digestive System Neoplasms; Electrophoresis, Polyacrylamide Gel; Epithelium; Humans; Keratins; Liver Neoplasms; Lymphatic Metastasis; Neoplasms; Peptides; Rectal Neoplasms; Respiratory Tract Neoplasms; Urinary Bladder Neoplasms

A panel of seven monoclonal antibodies was applied to smears of cell deposit from 70 pleural and peritoneal fluids, using an immunoalkaline phosphatase (IAP) procedure. The cases were chosen to show typical cytological patterns, both benign and malignant, and in this way the diagnostic value of the method could be assessed. The antibodies used were 2D1 (anti-leucocyte), Ca 1, HMFG-2 (anti-milk fat globule membrane), LE61 and M73 (both anti-intermediate filament antibodies), anti-CEA, and K92 (anti-keratin). The anti-leucocyte antibody was found useful for distinguishing lymphoma from carcinoma. Anti-CEA gave positive reactions in 80% of carcinoma cases and was not seen to react with any other cell types. Ca 1 was positive with some cells in 95% of carcinoma cases, but mesothelial cells reacted with it in two cases. A strong reaction with the anti-milk fat globule membrane antibody was very constant in carcinoma but was also seen in mesothelial cells in 30% of benign effusions. The anti-keratin reacted with malignant cells in only a small proportion of cases. The antibodies against epithelial intermediate filaments reacted equally strongly with benign mesothelial cells and carcinoma cells, but gave negative reactions with lymphoma cells. It is concluded that a suitably chosen panel of monoclonal antibodies can be of great value in identifying neoplastic cells in serous effusions.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Ascitic Fluid; Carcinoembryonic Antigen; Cytoskeleton; Female; Humans; Immunoenzyme Techniques; Keratins; Leukocytes; Male; Membrane Proteins; Mucin-1; Neoplasms; Pleural Effusion; Staining and Labeling

Topics: Histocytochemistry; Humans; Immunologic Techniques; Keratins; Molecular Weight; Neoplasms

Topics: Animals; Antibodies; Biopsy, Needle; Carcinoma; Cytoskeleton; Fluorescent Antibody Technique; Humans; Keratins; Muscle Proteins; Neoplasm Metastasis; Neoplasms; Neoplasms, Experimental; Sarcoma; Vimentin

Topics: Animals; Cricetinae; Cytoskeleton; Humans; Keratins; Mitosis; Muscle Proteins; Neoplasms; Vimentin

Topics: Aging; Cell Division; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Cyclic AMP; Epithelial Cells; Gene Expression Regulation; Humans; In Vitro Techniques; Keratins; Neoplasms

Fifty primary gastrointestinal and breast carcinomas, four embryonal rhabdomyosarcomas, six nonmuscular mesenchymal malignant tumors and one mesothelioma have been studied to determine what type of intermediate filaments they express, using affinity purified antibodies to prekeratin, vimentin and desmin and FITC or peroxidase labeled second antibodies. The tissues were alcohol fixed and paraffin embedded before use. In all carcinoma cases the tumor cells are stained by antibodies to prekeratin, while the vimentin antibody only decorates the stroma. Prekeratin positive tumor cells are not only seen in well differentiated tumors, but also in signet ring cell carcinomas. In the case of rhabdomyosarcoma the tumor cells clearly were decorated by antibodies to desmin, while the vimentin antibody only stained very few tumor cells. In cases of nonmuscular mesenchymal tumors, the tumor cells could only be labeled by antibodies to vimentin and not by antibodies to prekeratin or desmin. In biphasic tumors like mesothelioma, different parts of the tumor were separated by antibodies to prekeratin and vimentin.

Topics: Adenocarcinoma, Mucinous; Breast Neoplasms; Carcinoma; Child; Cytoskeleton; Desmin; Female; Gastrointestinal Neoplasms; Humans; Intermediate Filament Proteins; Keratins; Mesothelioma; Neoplasms; Neoplasms, Germ Cell and Embryonal; Protein Precursors; Rhabdomyosarcoma; Sarcoma; Vimentin

Fibrillar proteins with a role in cellular shape and motility are present in the cytoplasm of most animal cells. They vary greatly in size, organization, and reactivity according to cell type and can be separately identified by the use of recently developed monospecific antisera and indirect immunofluorescence staining. Prekeratin, a structural protein in the form of intermediate sized filaments is present exclusively in cells of epithelial origin. In the present study 41 human tumors of various organs and their normal tissue counterparts were reacted with prekeratin antiserum and examined by immunofluorescence staining in paraffin embedded sections. Prekeratin was identified in all epithelial cells of the squamous type, which gave the strongest staining reaction, and in smaller amounts in epithelial cells of other histologic types. Cells of lymphoid, melanic, neural, and connective tissue origin were not stained. Thus, combining the specificity of antiprekeratin sera with the selectivity of immunofluorescence staining resulted in a new method of identifying tissues that is applicable to the differential diagnosis of tumors.

Topics: Diagnosis, Differential; Epithelium; Fluorescent Antibody Technique; Humans; Keratins; Neoplasms; Protein Precursors

Forty-three tumors were investigated by means of immunofluorescence with the use of antibodies against the following different classes of intermediate-sized (10 nm) filament proteins: 1) cytokeratins, 2) vimentin, and 3) desmin. In general, the immunologic features of tumor-cell intermediate filaments are those present in their tissue of origin. It can be seen, therefore, that, during neoplastic transformation, there are no major changes in the synthesis of the type of intermediate filament proteins when compared to normal tissues. Immunologic identification of these proteins furnishes the surgical pathologist with a quick and clear-cut way to differentiate tumors of mesenchymal origin from epithelial neoplasms, and in particular to distinguish between malignant lymphomas and lymph node metastases of undifferentiated carcinomas.

Topics: Adult; Animals; Antibodies; Breast Neoplasms; Carcinoma; Cytoskeleton; Desmin; Female; Guinea Pigs; Humans; Keratins; Male; Mice; Microtubules; Middle Aged; Muscle Proteins; Neoplasms; Protein Precursors; Sarcoma; Skin Neoplasms; Vimentin

The distribution of intracellular keratin was studied in a variety of human tumors using a previously described immunoperoxidase technique employing antikeratin antibodies. Squamous cell carcinomas, transitional cell tumors, and mesotheliomas exhibited strong reactivity with antikeratin antibodies. Mammary adenocarcinomas were either negative or weakly positive. In the lung, an organ which can give rise to several morphologically distinct forms of carcinoma, only the squamous cell type stained strongly for keratin; undifferentiated lung carcinomas were negative, and adenocarcinomas were either negative or weakly positive. Colonic, renal, and prostatic adenocarcinomas were negative. Sarcomas, lymphomas, and neural tumors were uniformly negative. The analysis of intracellular keratin by the immunoperoxidase technique appears helpful in establishing the epithelial nature of primary or metastatic poorly differentiated neoplasms.

Topics: Adenocarcinoma; Carcinoma, Squamous Cell; Carcinoma, Transitional Cell; Humans; Immunoenzyme Techniques; Keratins; Mesothelioma; Neoplasm Metastasis; Neoplasms

Topics: Animals; Animals, Zoo; Buffaloes; Female; Keratins; Neoplasms; Rumen

Rare intracytoplasmic membranous structures were observed in keratinocytes, histiocytes, Langerhans cells and in metastatic cells of several dermatoses. The morphological study is not conclusive of their functional activity. They seem related to the cellular activity, and with no diagnostic value.

Topics: Cell Membrane; Endoplasmic Reticulum; Histiocytes; Humans; Keratins; Langerhans Cells; Neoplasms; Skin Diseases

Topics: Electrons; Female; Humans; Keratins; Microscopy; Microscopy, Electron; Neoplasms; Pathology; Uterine Cervical Neoplasms

Topics: Craniopharyngioma; Humans; Keratins; Neoplasms; Pituitary Neoplasms

Topics: Dermoid Cyst; Female; Granuloma; Humans; Keratins; Laparoscopy; Medical Records; Neoplasms; Neoplasms, Germ Cell and Embryonal; Ovarian Neoplasms; Peritoneum; Teratoma

Topics: Craniopharyngioma; Humans; Keratins; Neoplasms; Pituitary Neoplasms

Topics: Carcinogens; Humans; Keratins; Neoplasms

Topics: Animals; Carcinoma; Keratins; Neoplasms; Papilloma; Rabbits; Tumor Virus Infections