bromochloroacetic-acid and Colonic-Neoplasms

Topics: Adult; Antineoplastic Combined Chemotherapy Protocols; Chemotherapy, Adjuvant; Colectomy; Colon; Colonic Neoplasms; Colonic Polyps; Colonoscopy; Diagnosis, Differential; Doxorubicin; Gastrointestinal Hemorrhage; Humans; Ifosfamide; Immunohistochemistry; Intestinal Mucosa; Keratins; Lymphoma, Non-Hodgkin; Male; Rectum; Tomography, X-Ray Computed; Treatment Outcome

Sentinel lymph node (SLN) is a concept but also a technical possibility that can be studied and applied to almost all organs with cancer. For colorectal cancer surgery, some possibilities of using the SLN are possible, other implausible and some completely new especially aware of possible analysis of SLN by a molecular biology technique. The orientation of dissection or "lymph road mapping" can be designed for this case or the surgeon may want to limit his actions, particularly in patients with a history of colonic surgical resection, to keep the digestive function in maintaining vascular axes considered not involved in the metastatic process. The use of the single analysis of SLN to determine the positive or negative status of the cleaning has failed because of the frequency of false negatives in part to the size of colic advanced cancers at diagnosis. The use of "ultra-stading" by multiple section or exhaustion of the block, can lead to reconsider a stage N0 to N1 as a point, if the analysis technique remains in HES. Unlike the "ultra-stading" by RT- PCR or immunohistochemistry was even more discussed and seems not equivalent in terms of prognosis and therefore no giving formally justification for adjuvant therapy. Currently, a new technique for molecular biology, named "OSNA", allows an analysis of all the SLN in less than 45 minutes. It is therefore possible to obtain during surgery analysis of a node with the same level of information than traditional analysis using HES. If this node is positive and if the strategy in case of positive lymph nodes was determined prior for this patient, it is possible to anticipate this strategy and place after colectomy during the same anesthesia, venous access quickly to start postoperative chemotherapy. This new technique for analyzing lymph applied to the SLN opens a new potential application of this concept in digestive oncology.

Topics: Colonic Neoplasms; Colorectal Neoplasms; Coloring Agents; Humans; Keratins; Lymph Nodes; Neoplasm Staging; Nucleic Acid Amplification Techniques; RNA, Messenger; Sentinel Lymph Node Biopsy

We report a case of microcystic/reticular schwannoma of the proximal sigmoid colon in a 61-year-old man. A 12-mm polyp was detected while the patient was undergoing screening for colorectal neoplasm. This rare variant of schwannoma was initially described in 2008 and shows a predilection for the visceral organs, predominantly the gastrointestinal tract. We also review 11 other reported cases of microcystic/reticular schwannomas in the gastrointestinal tract. Unlike conventional gastrointestinal schwannomas, which are more common in the stomach, this variant appears to be more common in the large intestine. Histologic examination of this polyp showed predominant lipoblast-like vacuolated cells within a myxoid stroma with focal spindle cell areas. Features suggestive of malignancy, like nuclear pleomorphism, mitosis, or necrosis, were absent. Immunohistochemistry for S100 protein showed strong nuclear and cytoplasmic positivity, whereas cytokeratin and CD117 stains were negative. It is important to entertain microcystic/reticular schwannoma in the differential diagnosis of a signet ring cell adenocarcinoma or a myxoid gastrointestinal stromal tumor, particularly on small biopsy specimens.

Topics: Calgranulin A; Carcinoma, Signet Ring Cell; Colonic Neoplasms; Diagnosis, Differential; Gastrointestinal Stromal Tumors; Humans; Immunohistochemistry; Keratins; Male; Microscopy, Electron, Transmission; Middle Aged; Neurilemmoma; Proto-Oncogene Proteins c-kit

The differential diagnosis of ovarian carcinomas, including secondary tumors, remains a challenging task. Mucinous carcinomas of the ovary are rare and can be easily confused with metastatic mucinous carcinomas that may present clinically as a primary ovarian tumor. Most of these originate in the gastrointestinal tract and pancreas. International Federation of Gynecology and Obstetrics (FIGO) stage is the single most important prognostic factor, and stage I carcinomas have an excellent prognosis; FIGO stage is largely related to the histologic features of the ovarian tumors. Infiltrative stromal invasion proved to be biologically more aggressive than expansile invasion. Metastatic colon cancer is frequent and often simulates ovarian endometrioid adenocarcinoma. Although immunostains for cytokeratins 7 and 20 can be helpful in the differential diagnosis, they should always be interpreted in the light of all clinical information. Occasionally, endometrioid carcinomas may exhibit a microglandular pattern simulating sex cord-stromal tumors. However, typical endometrioid glands, squamous differentiation, or an adenofibroma component are each present in 75% of these tumors whereas immunostains for calretinin and alpha-inhibin are negative. Endometrioid carcinoma of the ovary is associated in 15-20% of the cases with carcinoma of the endometrium. Most of these tumors have a favorable outcome and they most likely represent independent primary carcinomas arising as a result of a Mullerian field effect. Although the criteria for distinguishing metastatic from independent primary carcinomas rely mainly upon conventional clinicopathologic findings, loss of heterozygosity and gene mutation analyses can be helpful. Transitional cell carcinomas are distinguished from undifferentiated carcinomas by the presence of thick, undulating papillae with smooth luminal borders, microspaces, and tumor cells with distinctive 'urothelial' appearance. Krukenberg tumors are metastatic adenocarcinomas traditionally perceived as composed of mucin-filled signet-ring cells associated with a striking proliferation of the ovarian stroma but many variations on this pattern occur.

Topics: Base Sequence; beta Catenin; Colonic Neoplasms; Cytoskeletal Proteins; Diagnosis, Differential; Female; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Mutation; Ovarian Neoplasms; Prognosis; ras Proteins; Trans-Activators

Colonic carcinoma metastasis to the thyroid is rare. Here the authors present the case of an 81-year-old lady who presented with metastatic colonic adenocarcinoma in her thyroid gland. This case is unique as it is the first to demonstrate metastasis from the colon to the thyroid with no other site involvement. The use of cytokeratin immunohistochemical staining is reviewed along with the current perspectives on the concept of skip metastasis.

Topics: Adenocarcinoma; Aged, 80 and over; Biomarkers, Tumor; Colonic Neoplasms; Female; Humans; Keratins; Neoplasm Proteins; Thyroid Neoplasms

Micrometastasis are defined by the existence of cells or groups of cells in target organs. In the particular cas of colon cancers, although lymph node involvement is frequent, metastatic medullary involvement (while rarely at the origin of identified metastasis) can also be observed. Furthermore, micrometastatics cells can be identified in the circulating blood. This research relies on recent technics of immunocytochemistry with image analysis or molecular biology technics (generally PCR or RT-PCR). It is essential to have a specific reliable marker of metastatic cells. The prognostic value of identifying micrometastasis in organs also remains to be defined.

Topics: Biomarkers, Tumor; Bone Marrow Examination; Carcinoembryonic Antigen; Colonic Neoplasms; DNA, Neoplasm; Guanylate Cyclase; Humans; Immunohistochemistry; Keratins; Mutation; Neoplastic Cells, Circulating; Polymerase Chain Reaction; Predictive Value of Tests; Prognosis; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Reproducibility of Results

Clinically evident metastases of carcinomas to the thyroid gland are rare, particularly from a colorectal primary tumor. We present a case of colonic adenocarcinoma metastatic to the thyroid gland with histopathologic and immunohistochemical findings. A 68-year-old woman with a history of Dukes' stage B colon carcinoma presented a mass in the thyroid gland. The tumor was confirmed to be metastatic adenocarcinoma from the colon. The immunohistochemical findings demonstrated positive staining for cytokeratin 20, low-molecular-weight cytokeratin, villin and carcinoembryonic antigen, but stains were negative for cytokeratin 7 and thyroglobulin.

Topics: Adenocarcinoma; Aged; Biomarkers, Tumor; Carcinoembryonic Antigen; Carrier Proteins; Colonic Neoplasms; Female; Humans; Immunoenzyme Techniques; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Microfilament Proteins; Thyroid Neoplasms; Thyroid Nodule; Tomography, X-Ray Computed

Differentiation of intestinal epithelial cells involves a complex process of establishment of cell polarity, commitment to cell lineage, and inhibition of cell division. Polarized epithelial cells are characterized by specific junctional complexes and cytoskeletal proteins which produce specific membrane domains. Intestinal cytoskeletal proteins are often preserved in neoplastic colonic tissues, and can be used to identify the cell of origin of poorly differentiated cancers. In this context, these proteins are markers of organ-specific differentiation. In addition, since loss of cytoskeletal polarity commonly occurs in transformed cells, aberrant expression of these proteins may be used as a marker of neoplasia in the colon. Normal polarization of basolateral proteins (secretory component) and apical proteins such as brush border hydrolases, cytoskeletal proteins (villin, fodrin), and carcinoembryonic antigen can become disrupted in adenomas and cancers. Cytoskeletal intermediate filaments (cytokeratins) demonstrate increased immunoreactivity in villous adenomas and cancers compared with normal colonic crypts. Altered actin bundles are found in preneoplastic mucosa such as colon from patients with familial polyposis coli. Molecular mechanisms responsible for altered cytoskeletal structures remain unclear; however, altered protein phosphorylation most likely plays a role. For example, the phosphorylation status of cytoskeletal and junctional complex proteins appears to influence their solubility and interactive properties, which may result in altered cell polarity. Markers of altered cytoskeletal structure and polarity can identify neoplastic colonocytes; however, the extent to which they can be used as intermediate markers of colonic neoplasia remains to be determined.

Topics: Actins; Animals; Biomarkers; Cell Differentiation; Colon; Colonic Neoplasms; Cytoskeletal Proteins; Cytoskeleton; Epithelial Cells; Epithelium; Humans; Keratins; Microfilament Proteins; Precancerous Conditions

1. CEA, a well established marker for benign and malignant colonic tumors is widely used for tissue staining and body fluid measurement. Highly specific monoclonal antibodies are now available. It is likely that CEA gene(s) will be available soon. 2. Monoclonal antibodies to blood group precursor antigens, especially extended LeX and LeY are also available and are known to detect premalignant lesions. 3. The demonstration that LeY is expressed in purified CEA specimens suggests a complementary relationship between the two markers of possible clinical utility. 4. Systematic comparison of both families of marker is timely. 5. Experienced pathologists have classified and standardized the histology of adenomatous polyps and their premalignant counterparts. The use of serial sections of identical tissues will permit comparison of several candidate markers in the same lesion. Selection of lesions which contain benign, malignant, and invasive components in the same section will provide best control, minimizing the need for exhaustive studies. 6. Laminin staining gives useful indication of early invasion through the basement membrane. It will complement morphologic and marker evidence for early malignancy and invasion. 7. It is unnecessary to investigate all polyps. Focus should be on high risk patients with (1) large polyps, severe dysplasia and advanced villous change, (2) synchronous polyps and invasive cancer, and (3) familial and other multiple polyposis disorders. 8. A plethora of other candidate markers is available, only a few of which are mentioned.

Topics: Antibodies, Monoclonal; Biomarkers, Tumor; Carcinoembryonic Antigen; Cell Line; Colonic Neoplasms; Humans; Immunohistochemistry; Intestinal Polyps; Keratins; Lewis Blood Group Antigens; Neoplasm Invasiveness; Precancerous Conditions

The relationship between primary colon cancer and occult nodal metastases (OMs) detected by cytokeratin immunohistochemistry (CK-IHC) is unknown. We sought to investigate the correlation of clinicopathologic features of colon cancer with OMs and to identify predictors of OM.. Patients with colon cancer from 5 tertiary referral cancer centers enrolled in a prospective trial of staging had standard pathologic analysis performed on all resected lymph nodes (using hematoxylin and eosin staining [H&E]). Nodes negative on H&E underwent CK-IHC to detect OMs, which were defined as micrometastases (N1mic) or isolated tumor cells (N0i+). Patients who were negative on both H&E and CK-IHC were defined as node negative (NN), and those positive on H&E were node positive (NP). The relationships between tumor characteristics and OMs were analyzed using the Kruskal-Wallis and the Fisher exact test.. OMs were identified in 23.4% (25/107) of patients. No significant differences were found in demographics, tumor location, tumor size, and number of nodes examined between groups. Compared with the NN group, patients with OMs had more tumors that were T3/T4 (72% vs 57%; P < .001), had tumors of higher grade (28% vs 12%; P = .022), and had tumors with lymphovascular invasion (16% vs 3%; P < .001).. Adverse primary pathologic colon cancer characteristics correlate with OMs. In patients with negative nodes on H&E and stage T3/T4 colon cancer, lymphovascular invasion, or high tumor grade, consideration should be given to performing CK-IHC. The detection of OMs in this subset may influence decisions regarding adjuvant chemotherapy and risk stratification.

Topics: Aged; Aged, 80 and over; Colonic Neoplasms; Feasibility Studies; Female; Humans; Keratins; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Predictive Value of Tests; Prospective Studies; Sentinel Lymph Node Biopsy; Tumor Burden

The principal role of sentinel lymph node (SLN) sampling and ultrastaging in colon cancer is enhanced staging accuracy. The utility of this technique for patients with colon cancer remains controversial.. This multicenter randomized trial was conducted to determine if focused assessment of the SLN with step sectioning and immunohistochemistry (IHC) enhances the ability to stage the regional nodal basin over conventional histopathology in patients with resectable colon cancer.. Between August 2002 and April 2006 we randomly assigned 161 patients with stage I-III colon cancer to standard histopathologic evaluation or SLN mapping (ex vivo, subserosal, peritumoral, 1% isosulfan blue dye) and ultrastaging with pan-cytokeratin IHC in conjunction with standard histopathology. SLN-positive disease was defined as individual tumor cells or cell aggregates identified by hematoxylin and eosin (H&E) and/or IHC. Primary end point was the rate of nodal upstaging.. Significant nodal upstaging was identified with SLN ultrastaging (Control vs. SLN: 38.7% vs. 57.3%, P = 0.019). When SLNs with cell aggregates < or =0.2 mm in size were excluded, no statistically significant difference in node-positive rate was apparent between the control and SLN arms (38.7% vs. 39.0%, P = 0.97). However, a 10.7% (6/56) nodal upstaging was identified by evaluation of H&E stained step sections of SLNs among study arm patients who would have otherwise been staged node-negative (N0) by conventional pathologic assessment alone.. SLN mapping, step sectioning, and immunohistochemistry (IHC) identifies small volume nodal disease and improves staging in patients with resectable colon cancer. A prospective trial is ongoing to determine the clinical significance of colon cancer micrometastasis in sentinel lymph nodes.

Topics: Aged; Chi-Square Distribution; Colonic Neoplasms; Coloring Agents; Female; Humans; Immunoenzyme Techniques; Keratins; Logistic Models; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Military Personnel; Neoplasm Staging; Prognosis; Prospective Studies; Rosaniline Dyes; Sentinel Lymph Node Biopsy; Treatment Outcome; United States

The 25% rate of recurrence after complete resection of stage II colon cancer (CC) suggests the presence of occult nodal metastases not identified by hematoxylin and eosin staining (H&E). Interim data from our ongoing prospective multicenter trial of sentinel node (SN) biopsy indicate a 29.6% rate of micrometastases (MM) identified by immunohistochemical staining (IHC) of H&E-negative SNs in CC. We hypothesized that these MM have prognostic importance.. Between March 2001 and August 2006, 152 patients with resectable colorectal cancer were enrolled in the trial. IHC and quantitative RT-PCR (qRT) assay were performed on H&E-negative SNs. Results were correlated with disease-free survival.. The sensitivity of lymphatic mapping was significantly better in CC (75%) than rectal cancer (36%), P<0.05. Of 92 node-negative CC patients 7 (8%) were upstaged to N1 and 18 (22%) had IHC MM. Four patients negative by H&E and IHC were positive by qRT. At a mean follow-up of 25 months, 15 patients had died from noncancer-related causes, 12 had developed recurrence, 5 had died of CC (2 with macrometastases, 3 with MM), and 7 were alive with disease. The 12 recurrences included 4 patients with SN macrometastases and 6 with SN MM (2 by IHC, 4 by qRT). One of the 2 SN-negative recurrences had other positive lymph nodes by H&E. All patients with CC recurrences had a positive SN by either H&E/IHC or qRT. No CC patient with a negative SN by H&E and qRT has recurred (P=0.002).. This is the first prospective evaluation of the prognostic impact of MM in colorectal cancer. These results indicate that the detection of MM may be clinically relevant in CC and may improve the selection of patients for adjuvant systemic chemotherapy. Patients with CC who are node negative by cumulative detection methods (H&E/IHC and qRT) are likely to be cured by surgery alone.

Topics: Aged; Colectomy; Colonic Neoplasms; Coloring Agents; Disease-Free Survival; Female; Fluorescent Dyes; Follow-Up Studies; Humans; Immunohistochemistry; Keratins; Lymphatic Metastasis; Male; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; Prospective Studies; Rectal Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Rosaniline Dyes; Sentinel Lymph Node Biopsy; Survival Rate

To determine whether sentinel lymph node (LN) sampling (SLNS) could reduce the number of nodes required to characterize micrometastatic disease (MMD) in patients with potentially curable colon cancer.. Cancer and Leukemia Group B 80001 was a study to determine whether SLNS could identify a subset of LNs that predicted the status of the nodal basin for resectable colon cancer and, therefore, could be extensively evaluated for the presence of micrometastases. Patients enrolled onto this study underwent SLNS after injection of 1% isosulfan blue, and both sentinel nodes (SNs) and non-SNs obtained during primary tumor resection were sectioned at multiple levels and stained using anti-carcinoembryonic antigen and anticytokeratin antibodies.. Using standard histopathology, SNs failed to predict the presence of nodal disease in 13 (54%) of 24 node-positive patients. Immunostains were performed for patients whose LNs were negative by standard histopathology. Depending on the immunohistochemical criteria used to assign LN positivity, SN examination resulted in either an unacceptably high false-positive rate (20%) or a low sensitivity for detection of MMD (40%).. By examining both SNs and non-SNs, this multi-institutional study showed that SNs did not accurately predict the presence of either conventionally defined nodal metastases or MMD. As a result, SLNS is not a useful technique for the study of MMD in patients with colon cancer.

Topics: Biomarkers, Tumor; Carcinoembryonic Antigen; Colonic Neoplasms; Humans; Immunohistochemistry; Keratins; Lymph Nodes; Lymphatic Metastasis; Neoplasm Staging; Predictive Value of Tests; Sentinel Lymph Node Biopsy

The detection of isolated tumor cells (ITC) in the bone marrow of patients with epithelial malignancies is an independant prognostic factor for several entities as breast cancer, colorectal cancer or non-small lung cancer. However, with conventional immunocytology using Ficoll density gradient and APAAP staining, only a small proportion of the bone marrow samples can be scanned for cytokeratin-positive (CK+) cells. To improve detection rates, we evaluated the enrichment of ITC by magnetic activated cell sorting (MACS) compared to regularly stained cytospins. Recovery experiments with a CK+ breast cancer cell line (SKBR3) were performed to calculate the MACS enrichment rate. Bone marrow was obtained by aspiration from 20 patients with carcinomas of epithelial origin and from 17 controls. ITC were enriched and stained with magnetically labeled CAM 5.2 antibodies directed to cytokeratin 7 and 8. MACS of SKBR3 seeded in peripheral blood revealed average recovery rates of 62% and 48% and average enrichment factors of 104-fold and 8139-fold of the CK+ cells after one and after two separations, respectively. After immunomagnetic enrichment, CK+ cells were detected in 16 of 20 (80%) cancer patients, whereas only 7 (35%) patients showed CK+ cells without magnetic enrichment (P = 0.002). Ten of twelve (83%) patients with metastatic disease (stage M1) and six of eight (75%) patients without any overt metastases (M0) had CK+ cells in their bone marrow. None of the negative controls showed any CK+ cells. Enrichment with magnetically labeled anti cytokeratin antibodies increases the detection rate of epithelial cells in bone marrow of cancer patients compared to immunocytology.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Biomarkers, Tumor; Bone Marrow Neoplasms; Breast Neoplasms; Carcinoma; Carcinoma, Non-Small-Cell Lung; Colonic Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Lung Neoplasms; Magnetics; Male; Middle Aged; Prospective Studies; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Lymph node (LN) metastasis is one of the most significant prognostic factor in colorectal cancer. In fact, therapeutic decisions are based on LN status. However, multiple studies have reported on the limitations of the conventional pathological LN examination techniques, and therefore, the actual number of patients with LN positive colorectal cancer is probably underestimated. We assume that lymphatic tumor dissemination follows an orderly sequential route. We report here a simple and harmless coloration technique that was recently elaborated, and that allows us to identify the sentinel LN(s) (SLN) or first relay LNs in colorectal cancer patients. The main endpoint of this clinical trial is the feasibility of the technique.. Twenty patients treated by surgery for a colic cancer were admitted in this protocol. A subserosal peritumoral injection of lymphazurin 1% was performed 10 min before completing the colic resection. A pathologist immediately examined the specimens, harvested the colored SLN, and examined them by serial cuts (200 microm) with H&E staining, followed by immunohistochemical staining (AE1-AE3 cytokeratin markers), when serial sections were classified as cancer free.. The preoperative identification of the SLN was impossible in at least 50 of the cases, however, SLNs were identified by the pathologist in 90% of cases. In two patients (10%) SLN was never identified. The average number of SLN was 3.9. Immunohistochemical analysis of the SLN has potentially changed the initial staging (from Dukes B to Dukes C) for 5 of the 20 patients (25%). On the other hand, there was one patient (5%) with hepatic metastasis from adenocarcinoma for whom SLN pathology was negative for metastasis (skip metastasis).. SLN biopsy is readily feasible with identification of SLN in at least 90% of patients with colorectal cancers. Our results indicate that 45% of patients initially staged as Dukes B had tumor cells identified in their SLN when these were subjected to our protocol. This represented a 25% upgrading rate when our complete study population is considered. However, controversy persist about the clinical significance and metastatic potential of these often very small clusters of tumor cells.

Topics: Biomarkers, Tumor; Carcinoma; Colonic Neoplasms; Humans; Immunohistochemistry; Keratins; Lymph Node Excision; Lymphatic Metastasis; Patient Care Planning; Preoperative Care; Rosaniline Dyes; Sentinel Lymph Node Biopsy

Tumour budding (TB) is an established prognostic feature in multiple cancers but is not routinely assessed in pathology practice. Efforts to standardise and automate assessment have shifted from haematoxylin and eosin (H&E)-stained images towards cytokeratin immunohistochemistry. The aim of this study was to compare manual H&E and cytokeratin assessment methods with a semi-automated approach built within QuPath open-source software.. TB was assessed in cores from the advancing tumour edge in a cohort of stage II/III colon cancers (n = 186). The total numbers of buds detected with each method were as follows: manual H&E, n = 503; manual cytokeratin, n = 2290; and semi-automated, n = 5138. More than four times the number of buds were identified manually with cytokeratin assessment than with H&E assessment. One thousand seven hundred and thirty-four individual buds were identified with both manual and semi-automated assessments applied to cytokeratin images, representing 75.7% of the buds identified manually (n = 2290) and 33.7% of the buds detected with the semi-automated method (n = 5138). Higher semi-automated TB scores were due to any discrete area of cytokeratin immunopositivity within an accepted area range being identified as a bud, regardless of shape or crispness of definition, and to the inclusion of tumour cell clusters within glandular lumina ('luminal pseudobuds'). Although absolute numbers differed, semi-automated and manual bud counts were strongly correlated across cores (ρ = 0.81, P < 0.0001). All methods of TB assessment demonstrated poorer survival associated with higher TB scores.. We present a new QuPath-based approach to TB assessment, which compares favourably with established methods and offers a freely available, rapid and transparent tool that is also applicable to whole slide images.

Topics: Aged; Biomarkers, Tumor; Cohort Studies; Colonic Neoplasms; Colorectal Neoplasms; Eosine Yellowish-(YS); Female; Hematoxylin; Humans; Immunohistochemistry; Keratins; Machine Learning; Male; Prognosis; Staining and Labeling

Circulating tumor cells (CTCs) in the blood of cancer patients are of high clinical relevance. Since detection and isolation of CTCs often rely on cell dimensions, knowledge of their size is key. We analyzed the median CTC size in a large cohort of breast (BC), prostate (PC), colorectal (CRC), and bladder (BLC) cancer patients. Images of patient-derived CTCs acquired on cartridges of the FDA-cleared CellSearch

Topics: Breast Neoplasms; Cell Count; Cell Line, Tumor; Cell Nucleus; Cell Size; Cohort Studies; Colonic Neoplasms; Female; Humans; Indoles; Keratins; Male; Neoplastic Cells, Circulating; Prostatic Neoplasms; Urinary Bladder Neoplasms

Using digital scans of all tumour slides/case, we analysed CD8. In rectal cancer, tumour budding has clinical relevance in both primarily surgically treated patients and in those with neoadjuvantly treated patients, where it characterises highly aggressive residual disease. CD8

Topics: CD8 Antigens; CD8-Positive T-Lymphocytes; Colonic Neoplasms; Drug Therapy; Female; Humans; Immunohistochemistry; Keratins; Male; Neoadjuvant Therapy; Neoplasm Staging; Prognosis; Rectal Neoplasms

Histone deacetylases, HDACs, have been demonstrated to play a critical role in epigenetic signaling and were found to be overexpressed in several type of cancers; therefore, they represent valuable targets for anticancer therapy. 9-Hydroxystearic acid has been shown to bind the catalytic site of HDAC1, inducing G0/G1 phase cell cycle arrest and activation of the p21

Topics: Adenocarcinoma; Albumins; Cell Membrane; Cell Proliferation; Cell Survival; Chemistry Techniques, Synthetic; Colonic Neoplasms; Drug Discovery; HCT116 Cells; Histone Deacetylase 1; HT29 Cells; Humans; Keratins; Nanoparticles; Reactive Oxygen Species; S Phase Cell Cycle Checkpoints; Signal Transduction; Solubility; Stearic Acids

The complexity of tumor histomorphology reflects underlying tumor biology impacting on natural course and response to treatment. This study presents a method of computer-aided analysis of tissue sections, relying on multifractal (MF) analyses, of cytokeratin-stained tumor sections which quantitatively evaluates of the morphological complexity of the tumor-stroma interface. This approach was applied to colon cancer collection, from an adjuvant treatment randomized study. Metrics obtained with the method acted as independent markers for natural course of the disease, and for benefit of adjuvant treatment. Comparative analyses demonstrated that MF metrics out-performed standard histomorphological features such as tumor grade, budding and configuration of invasive front. Notably, the MF analyses-derived "α

Topics: Aged; Chemotherapy, Adjuvant; Colonic Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Keratins; Male; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis

The characterization of circulating tumor cells (CTCs) could substantially improve the management of cancer patients. However, their study is still a matter of debate, often due to lymphocyte contamination. In the present paper, an investigation of CTCs was carried out for the first time using DEPArray, a dielectrophoresis-based platform able to detect and sort pure CTCs. Analyses were conducted on peripheral blood (PB) samples from patients with metastatic colon cancer. After 100% pure cell recovery and whole genome amplification, KRAS gene mutation of CTCs was screened and compared to gene status in the primary tumor tissue. CTCs were found in 21 colon cancer patients (52.5%), with more than three tumor cells per 7.5 ml. KRAS gene mutation analysis, showed a mutational concordance between CTCs and primary tumor in 50% of matched cases. The present study demonstrates for the first time the feasibility of analyzing at the molecular level pure CTCs avoiding lymphocyte contamination using an innovative instrumentation, and a KRAS discordance between CTCs and primary tissue. Our results present dielectrophoresis-based procedures as a new standard in single cell analysis and recovery and invite careful reflection on the value of CTCs characterization.

Topics: Aged; Case-Control Studies; Cell Line, Tumor; Cell Separation; Centrifugation, Density Gradient; Colonic Neoplasms; Electrophoresis; Female; Humans; Keratins; Male; Middle Aged; Mutation, Missense; Neoplastic Cells, Circulating; Prospective Studies; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Sensitivity and Specificity; Sequence Analysis, DNA

Since circulating tumor cells (CTCs) have metastatic potential, their genetic and phenotypic characteristics could provide crucial information to establish the most effective therapy. We assessed the clinical utility of a methodology that allows the simultaneous analysis of CTC phenotype and genotype in colon cancer patients and, in addition, whether this methodology could provide complementary information to that obtained by the primary tumor biopsy. Thirty-three non-metastatic (stages 0-III) colon cancer patients and 9 healthy donor samples were evaluated. All peripheral blood samples (10 ml) were analyzed by cytokeratin immunomagnetic enrichment. Eight samples were analyzed by immunocytochemistry and 25 samples were analyzed by FICTION technique for simultaneous cytokeratin expression and chromosome 17 and ERBB2 gene status. A further study was carried out in one patient who showed CTC heterogeneity in chromosomal abnormalities. We analyzed HER2 protein expression on CTCs and FISH and HER2 protein expression in primary tumor of this patient. Our results show that 9.09% of patients had cytokeratin-positive CTCs (CK+/CTCs in peripheral blood). One of the patients showed heterogeneity in chromosomal 17 abnormalities and two different CK expression patterns on CTCs: one CK+/CTCs and one CK-/CTCs. Furthermore, 63.33% of these CTCs overexpressed HER2 protein while the primary tumor of this patient was diploid and did not express HER2 protein. We describe a methodology that allows the simultaneous genetic and phenotypic analysis of CTCs in colon cancer patients, which may provide essential information to select patients who might benefit from specific therapy.

Topics: Adenocarcinoma; Cell Separation; Chromosomes, Human, Pair 17; Colonic Neoplasms; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Genes, erbB-2; Genotype; Humans; Immunohistochemistry; Immunophenotyping; In Situ Hybridization, Fluorescence; Keratins; Male; Neoplasm Grading; Neoplasm Staging; Neoplastic Cells, Circulating; Phenotype

Determining the fraction of tumor cells in colon carcinoma samples analyzed for KRAS mutation status is important for choosing the proper testing modality and accurately interpreting the results. However, when asked to determine the tumor cell fraction in tissue samples, different pathologists give considerably different estimations, possibly leading to erroneous interpretation of KRAS mutation analysis results and poor treatment choices. To address this issue, we developed a free, easy-to-use computer program that estimates the tumor cell fraction on colon carcinoma slides that are immune-stained with anti-cytokeratin antibody. The program differentiates between the tumor area and the surrounding stroma on the basis of the immunostaining. Sixty such samples were evaluated by the program. In addition, the actual tumor cell fraction in these samples was measured. The tumor cell fraction estimated by the computer program showed a highly significant correlation with the actual measurements (R=0.64, P<0.001). In addition, we found that a short calibration step before beginning the computer estimation increased the accuracy of the results. In 4 cases (7%), there was some discrepancy between the computer estimation and the actual measurements; however, this was attributed to lower-quality immunohistochemical staining, indicating the importance of this phase in the analysis. In conclusion, we believe that this program can be used for standardizing the evaluation of the tumor cell fraction in colon carcinoma and that its use might aid in making better diagnosis and treatment choices for these patients.

Topics: Antibodies, Monoclonal; Colonic Neoplasms; Early Detection of Cancer; Humans; Image Cytometry; Image Processing, Computer-Assisted; Immunohistochemistry; Keratins; Mutation; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Software

The short chain fatty acids (SCFAs) are inhibitors of histone deacetylases (HDACi); they are produced naturally in the colon by fermentation. They affect cellular processes at a molecular and transcriptional level, the mechanisms of which may involve large numbers of proteins and integrated pathways. Butyrate is the most biologically potent of the SCFAs in colon epithelial cells, inhibiting human colon carcinoma cell proliferation and inducing apoptosis in vitro. In order to investigate the hypothesis that propionate and valerate possess unique and independent actions from butyrate, we combined proteomic and cellomic approaches for large-scale comparative analysis. Proteomic evaluation was undertaken using an iTRAQ tandem mass-spectrometry workflow and high-throughput High-content Analysis microscopy (HCA) was applied to generate cellomic information on the cell cycle and the cytoskeletal structure. Our results show that these SCFAs possess specific effects. Butyrate was shown to have more pronounced effects on the keratins and intermediate filaments (IFs); while valerate altered the β-tubulin isotypes' expression and the microtubules (MTs); propionate was involved in both mechanisms, displaying intermediate effects. These data suggest distinct physiological roles for SCFAs in colon epithelial function, offering new possibilities for cancer therapeutics.

Topics: Apoptosis; Butyrates; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Epithelial Cells; Fatty Acids, Volatile; Histone Deacetylases; Humans; Intermediate Filaments; Keratins; Microtubules; Propionates; Proteome; Proteomics; Tubulin; Valerates

A 92-year-old male presented for routine endoscopic surveillance of his gastrointestinal (GI) tract. He did not have any GI symptoms currently, and the patient had undergone a right nephrectomy for renal cell carcinoma 17 years previously. A lower GI endoscopy revealed polyps in the ascending colon, hepatic flexure, and sigmoid colon (2 polyps). All the polyps were snared and removed in toto. Histological evaluation of all 4 polyps showed similar features. There was expansion of the lamina propria by sheets of clear cells arranged in a nested pattern with a rich vascular network. Immunohistochemistry showed the tumor to be positive for low-molecular-weight cytokeratin, CD10, and vimentin. The features were morphologically and immunophenotypically that of clear cell renal cell carcinoma. This case highlights an extremely unusual presentation of recurrent renal cell carcinoma as multiple, separate colonic polyps 17 years after resection of the primary tumor.

Topics: Aged, 80 and over; Carcinoma, Renal Cell; Colonic Neoplasms; Colonic Polyps; Diagnosis, Differential; Humans; Keratins; Kidney Neoplasms; Male; Molecular Weight; Neoplasms, Multiple Primary; Neprilysin; Vimentin

Topics: Adenocarcinoma; Aged; Colonic Neoplasms; Head; Histocytochemistry; Humans; Immunohistochemistry; Keratins; Magnetic Resonance Imaging; Male; Microscopy; Orbital Neoplasms; Tomography, X-Ray Computed; Whole Body Imaging

The study aimed to prove the prognostic meaning of micrometastases blood circulation during liver resections for cancer lesions. 33 patients took part in the study. Circulating micrometastases were detected in blood using immunocytological method with pancytoceratine antibodies KL-1 and CAM 5.2. The majority of patients had colon cancer liver metastases (72,7%). Blood was sampled once in 8 patients, the rest 25 patients had double sampling: before and after liver mobilization. Patients with multiple liver metastases demonstrated tumor cells circulation more often. Of 58 tests, 25 were positive for tumor cells. 3-year survival in those patients was 45,7 ± 13,1%, 5-year survival was 24,4 ± 13,3%. Survival rates for patients with no circulating tumor cells detected were significantly higher.

Topics: Colonic Neoplasms; Cryosurgery; Enterohepatic Circulation; Flow Cytometry; Hepatectomy; Humans; Hyperthermia, Induced; Immunohistochemistry; Keratins; Liver; Liver Neoplasms; Neoplastic Cells, Circulating; Polymerase Chain Reaction; Predictive Value of Tests; Prognosis; Survival Rate; Vascular Surgical Procedures

A 53-year-old woman had a tumor in the ascending colon. CT revealed tumor invasion to the surrounding tissue and also showed multiple swollen lymph nodes, liver metastases and ascites. Colonic tumor with severe stenosis was diagnosed by colonoscopy and the obtained biopsy specimen revealed poorly differentiated adenocarcinoma. Immunohistochemically, the tumor was positive for CEA, CK7, MUC2, MUC5AC·MUC6 (spotty) and negative for CK20, CDX2, TTF-1, GCDFP-15. Cytology of ascites also showed malignant cells. Although these protein expressions were specific for not primary colonic cancer but metastasis from ovarian cancer, the case was clinically and pathologically diagnosed as poorly differentiated adenocarcinoma of the colon with peritoneal metastases composed of micropapillary carcinoma. MLH1 and MSH2 protein expressions were normal. Even though modified FOLFOX6 chemotherapy was administered, the patient rapidly worsened due to pulmonary carcinomatous lymphangiosis and died a month after diagnosis. To determine the high-risk group of metastases, it seems necessary to require the accumulation of further cases evaluated by a precise immunohistochemistrical approach.

Topics: Adenocarcinoma; Colonic Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Middle Aged; Peritoneal Neoplasms

Topics: Biomarkers; Biomarkers, Tumor; Colonic Neoplasms; Humans; Immunohistochemistry; Keratin-18; Keratin-8; Keratins; Neoplasm Metastasis; Neoplasm Staging; Sentinel Lymph Node Biopsy

Stable cell lines obtained by spontaneous immortalization might represent early stages of malignant transformation and be useful experimental models for studies of mechanisms of cancer development. The FHC (fetal human cells) cell line has been established from normal fetal colonic mucosa. Detailed characterization of this cell line and mechanism of spontaneously acquired immortality have not been described yet. Therefore, we characterized the FHC cell line in terms of its tumorigenicity, cytogenetics, and TP53 gene mutation analysis. FHC cells displayed capability for anchorage-independent growth in semisolid media in vitro and formed solid tumors after transplantation into SCID (severe combined immunodeficiency) mice. This tumorigenic phenotype was associated with hypotriploidy and chromosome number ranging from 66 to 69. Results of comparative genetic hybridization arrays showed that most chromosomes included regions of copy number gains or losses. Region 8q23 approximately 8q24.3 (containing, e.g., MYC proto-oncogene) was present in more than 20 copies per nucleus. Moreover, we identified mutation of TP53 gene in codon 273; triplet CGT coding Arg was changed to CAG coding His. Expression of Pro codon 72 polymorphic variant of p53 was also detected. Mutation of TP53 gene was associated with abolished induction of p21(Waf1/Cip1) and MDM-2 proteins and resistance to apoptosis after genotoxic treatment. Because of their origin from normal fetal colon and their relative resistance to the induction of apoptosis, FHC cells can be considered a valuable experimental model for various studies.

Topics: Animals; Apoptosis; Carcinoembryonic Antigen; Cell Adhesion; Cell Growth Processes; Cell Line, Transformed; Cell Transformation, Neoplastic; Colon; Colonic Neoplasms; Comparative Genomic Hybridization; Cytogenetic Analysis; DNA Damage; DNA Mutational Analysis; Female; Fetus; Genes, p53; HCT116 Cells; Humans; In Situ Hybridization, Fluorescence; Karyotyping; Keratins; Mice; Mice, SCID; Neoplasm Transplantation; Phenotype; Proto-Oncogene Mas; Signal Transduction

A study is made of the clinical repercussions of occult metastases-micrometastases (MMs+)-or isolated tumour cells (ITCs+) in the lymph nodes of patients with stage IIA and IIB colon adenocarcinoma initially considered as corresponding to N0.. A retrospective study of 39 patients with stage IIA and IIB (T3-T4 N0 M0) colon adenocarcinoma, subjected to similar surgical and adjuvant chemotherapy treatment, with long and careful follow-up (minimum: 5 years, mean: 81.7 months) was performed on their previously resected lymph nodes, with the aid of new histological and immunohistochemical (cytokeratin) sections, in order to detect MMs or ITCs. Disease-free survival (DFS) and global survival (GS) in the two groups (patients with MMs+ or ITCs+ vs. patients without MMs or ITCs) were compared at 5 years based on the corresponding Kaplan-Meier survival curves, with the Breslow test.. A total of 382 lymph nodes from the 39 patients (mean: 9.8; standard deviation: 6.09) were revised. MMs+ were detected in 2 cases and ITCs+ in 2 more cases on the Cytokeratin study. GS of the whole series at 5 years was 89.74% (35 patients alive) with a DFS at 5 years of 79.49% (31 patients free of disease), but the 2 cases with MMs+ were dead at 5 years, with high statistical differences between both groups (MMs+/MMs-) (p<0.0001). When comparing the group of MMs+/ITCs+ patients and the group of MM-/ITCs- patients, the DFS and GS times at 5 years were higher in the MMs-/ITCs- group (p=0.0692 and p=0.006 respectively).. Although the incidence of MMs+ or ITCs+ in the examined lymph nodes was low, the presence of MMs is related to a dramatic reduction in GS and DFS at 5 years. We encourage a detailed histological study of lymph nodes resected in patients with deep penetrating colon tumours in order to assure a pN0 status.

Topics: Adenocarcinoma; Adenocarcinoma, Mucinous; Adult; Aged; Chemotherapy, Adjuvant; Colonic Neoplasms; Female; Follow-Up Studies; Humans; Immunoenzyme Techniques; Keratins; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging; Prognosis; Retrospective Studies; Survival Rate

Topics: Adenocarcinoma; Biomarkers; CA-125 Antigen; Colon, Sigmoid; Colonic Diseases; Colonic Neoplasms; Colonoscopy; Diagnosis, Differential; Endometriosis; Female; Humans; Keratins; Middle Aged; Receptors, Estrogen; Tomography, X-Ray Computed

Jaundice, a common feature of advanced colon cancer, is usually due to liver parenchyma metastasis, but it can sometimes be caused by extrahepatic biliary obstruction. This rare event is related to metastasis to the lymph nodes placed behind the duodenum, along the choledochus or the vena porta, extrinsically compressing the common duct. Stenosis of the common bile duct secondary to parietal metastatic involvement is extremely rare. We report on a case of colon carcinoma metastasis to the intrapancreatic tract of the common bile duct, with a review of the literature.

Topics: Aged; Bile Duct Neoplasms; CDX2 Transcription Factor; Colonic Neoplasms; Combined Modality Therapy; Female; Homeodomain Proteins; Humans; Immunohistochemistry; Keratins

Rabbit antibodies may have favorable properties compared to mouse antibodies, including high affinities and better antigen recognition. We used a biochemical and reverse immunologic approach to generate and characterize rabbit anti-phospho-keratin and anti-keratin monoclonal antibodies (MAb). Human keratins 8 and 18 (K8/K18) were used as immunogens after isolation from cells pretreated with okadaic acid or pervanadate to promote Ser/Thr or Tyr hyperphosphorylation, respectively. Selected rabbit MAb were tested by immunofluorescence staining, immunoprecipitation, and 2-dimensional gels. Keratin phospho and non-phospho-mutants were used for detailed characterization of two unique antibodies. One antibody recognizes a K8 G61-containing epitope, an important epitope given that K8 G61C is a frequent mutation in human liver diseases. This antibody binds K8 that is not phosphorylated on S73, but its binding is ablated by G61 but not S73 mutation. The second antibody is bispecific in that it simultaneously recognizes two epitopes: one phospho (K8 pS431) conformation-independent and one non-phospho conformation-dependent, with both epitopes residing in the K8 tail domain. Therefore, a reverse immunologic and biochemical approach is a viable tool for generating versatile rabbit MAb for a variety of cell biologic applications including the potential identification of physiologic phosphorylation sites.

Topics: Animals; Antibodies, Bispecific; Antibodies, Monoclonal; Brain; Brain Chemistry; Cell Line; Colonic Neoplasms; Cricetinae; Enzyme Inhibitors; Epitopes; HT29 Cells; Humans; Immunoblotting; Immunohistochemistry; Keratin-18; Keratin-8; Keratins; Kidney; Liver; Liver Diseases; Mice; Mutation; Okadaic Acid; Phosphorylation; Rabbits; Serine; Stomach Neoplasms; Transfection; Vaccination; Vanadates

Distinction of intestinal-type sinonasal adenocarcinoma (ITAC) from adenocarcinoma of intestinal origin metastatic to the sinonasal cavity may be extremely difficult on histologic grounds alone. We studied the role of cytokeratin (CK) and mucin (MUC) expression in differentiating ITAC, metastatic adenocarcinoma of intestinal origin, and non-intestinal-type sinonasal adenocarcinoma (non-ITAC).. We stained specimens from 5 cases of ITAC and 4 cases of non-ITAC, along with 4 colonic and 3 duodenal adenocarcinoma controls, with CK7 and CK20, MUC2 and MUC5, neuron-specific enolase (NSE), chromogranin (CHR), and carcinoembryonic antigen (CEA) in order to examine the possible combinations of markers that best aid in the diagnosis of these lesions. We also performed a retrospective review of our clinical experience with these rare lesions.. CK7 staining was positive in all ITAC and non-ITAC cases, whereas all cases displaying gastrointestinal-type differentiation (ITAC and metastatic intestinal cases) stained positive for both CK20 and MUC2. Staining for MUC5, NSE, CHR, and CEA was variable.. Tumors with the CK7+, CK20+, MUC2+ immunophenotype are likely primary sinonasal lesions, whereas tumors with the CK7-, CK20+, MUC2+ profile warrant further clinical evaluation to exclude metastatic disease from the gastrointestinal tract. Complete surgical resection of ITAC remains the mainstay of therapy.

Topics: Adenocarcinoma; Adult; Aged; Biomarkers, Tumor; Colonic Neoplasms; Diagnosis, Differential; Duodenal Neoplasms; Female; Humans; Immunohistochemistry; Keratin-20; Keratin-7; Keratins; Male; Middle Aged; Mucin-2; Mucins; Paranasal Sinus Neoplasms; Retrospective Studies

A 63-year-old man who had undergone resection of colon cancer 15 years previously was found to have a right hilar mass on chest X-ray, and an elevated serum carcinoembryonic antigen level. The hilar lymph nodes were resected with the right upper lobe, and the initial diagnosis was colon cancer metastasis to the right hilar lymph nodes. Although the resection was incomplete, and no additional treatment was given, the patient remained free of recurrence for 10 years. This prompted us to reconsider our diagnosis using immunohistochemistry. The resected lymph nodes were found to be positive for thyroid transcription factor 1 (TTF-1) and cytokeratin 7, and negative for surfactant apoprotein (SAP), cytokeratin 20, and napsin A. The neuroendocrine markers and thyroglobulin were also negative. These findings led us to diagnose T0N1 lung cancer. There are reports of patients with clinical T0N1,2 lung cancer having exceptionally good prognoses despite noncurative treatment; however, to our knowledge, this is the first case of a patient with T0N1 lung cancer diagnosed by immunohistochemistry, with a good prognosis despite incomplete resection. In this case, TTF-1 and cytokeratin staining was particularly helpful in the differential diagnosis.

Topics: Adenocarcinoma; Apoproteins; Aspartic Acid Endopeptidases; Colonic Neoplasms; Humans; Immunohistochemistry; Keratin-20; Keratin-7; Keratins; Lung Neoplasms; Lymph Nodes; Male; Middle Aged; Neoplasm Staging; Nuclear Proteins; Prognosis; Pulmonary Surfactant-Associated Proteins; Thyroid Nuclear Factor 1; Time Factors; Transcription Factors

To compare gene expression profiles of pancreatic adenocarcinoma tissue specimens, human pancreatic and colon adenocarcinoma and leukemia cell lines and normal pancreas samples in order to distinguish differentially expressed genes and to validate the differential expression of a subset of genes by quantitative real-time RT-PCR (RT-QPCR) in endoscopic ultrasound-guided fine needle aspiration (EUS-guided FNA) specimens.. Commercially dedicated cancer cDNA macroarrays (Atlas Human Cancer 1.2) containing 1176 genes were used. Different statistical approaches (hierarchical clustering, principal component analysis (PCA) and SAM) were used to analyze the expression data. RT-QPCR and immunohistochemical studies were used for validation of results.. RT-QPCR validated the increased expression of LCN2 (lipocalin 2) and for the first time PLAT (tissue-type plasminogen activator or tPA) in malignant pancreas as compared with normal pancreas. Immunohistochemical analysis confirmed the increased expression of LCN2 protein localized in epithelial cells of ducts invaded by carcinoma. The analysis of PLAT and LCN2 transcripts in 12 samples obtained through EUS-guided FNA from patients with pancreatic adenocarcinoma showed significantly increased expression levels in comparison with those found in normal tissues, indicating that a sufficient amount of high quality RNA can be obtained with this technique.. Expression profiling is a useful method to identify biomarkers and potential target genes. Molecular analysis of EUS-guided FNA samples in pancreatic cancer appears as a valuable strategy for the diagnosis of pancreatic adenocarcinomas.

Topics: Acute-Phase Proteins; Adenocarcinoma; Biomarkers, Tumor; Biopsy, Fine-Needle; Cell Line, Tumor; Colonic Neoplasms; Endosonography; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Humans; Keratin-7; Keratins; Leukemia; Lipocalin-2; Lipocalins; Pancreatic Neoplasms; Prognosis; Proto-Oncogene Proteins; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Tissue Plasminogen Activator

Sentinel lymph node (SLN) mapping has become a cornerstone of oncologic surgery because it is a proven method for identifying nodal disease in melanoma and breast cancer. In addition, it can ameliorate the surgical morbidity secondary to lymphadenectomy. However, experience with SLN mapping for carcinoma of the colon and other visceral malignancies is limited. This study represents an update to our initial pilot experience with SLN mapping for carcinoma of the colon. Consenting patients over the age of 18 diagnosed with adenocarcinoma of the colon were included in this study. At the time of operation, 1 to 2 mL of isosulfan blue was injected with a 25-gauge needle into the subserosa at 4 sites around the edge of the palpable tumor. The SLN was identified visually and excised followed by a standard lymphadenectomy and surgical resection. SLNs were evaluated by standard hematoxylin and eosin (H&E) evaluation as well as immunohistochemical (IHC) techniques for carcinoembryonic antigen and cytokeratin if the H&E was negative. Sixty-nine patients underwent SLN mapping. A SLN was identified in 93 per cent (64 of 69) of patients. Nodal metastases were identified in 38 per cent (26 of 69) of patients overall. In 5 patients, the only positive node identified was the SLN, 2 of which were positive by IHC criteria alone. Therefore, 3 per cent (2 of 69) of patients were upstaged by SLN mapping. This technique was 100 per cent specific while being 46 per cent sensitive. Fourteen patients had false-negative SLNs. Metastasis to regional lymph nodes remains the key prognostic factor for colon cancer. SLN mapping is feasible for colon cancer and can identify a subset of patients who could benefit from adjuvant chemotherapy. Although SLN mapping did not alter the surgical management of colon cancer, it does make possible a more focused and cost-effective pathologic evaluation of nodal disease. We do not suggest routine utilization of SLN mapping for colon cancer, but we believe that the data supports proceeding with a national trial.

Topics: Adenocarcinoma; Aged; Body Mass Index; Carcinoembryonic Antigen; Colectomy; Colonic Neoplasms; Coloring Agents; False Negative Reactions; Feasibility Studies; Female; Fluorescent Dyes; Humans; Keratins; Lymph Node Excision; Lymphatic Metastasis; Male; Neoplasm Staging; Pilot Projects; Rosaniline Dyes; Sensitivity and Specificity; Sentinel Lymph Node Biopsy

Lymph node status is the most important predictive factor in the treatment of colorectal cancer. As sentinel lymph node (SLN) biopsy might upstage stage II colon cancer, it could have therapeutic consequences in the future. We investigated the feasibility of in vivo SLN detection with Patent Blue V dye and evaluated nodal microstaging and ultrastaging using cytokeratin immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR).. In 30 consecutive patients operated on for colon cancer, subserosal injection with Patent Blue dye was used for SLN detection in four different hospitals under the supervision of one regional coordinator. In searching for occult micrometastases, each SLN was examined at three levels. In tumor-negative SLNs at routine hematoxylin-eosin (H&E) examination (pN0) we performed CK8/CK18 immunohistochemistry (IHC) and RT-PCR for carcinoembryonic antigen (CEA).. The procedure was successful in 29 out of 30 patients (97%). The SLN was negative in 18 patients detected by H&E and IHC. In 16 patients the non-SLN was also negative, leading to a negative predictive value of 89% and an accuracy of 93%. Upstaging occurred in 10 patients (33%) - 7 by IHC and 3 by RT-PCR. Aberrant lymphatic drainage was seen in 3 patients (10%).. The SLN concept in colon carcinoma using Patent Blue V is feasible and accurate. It leads to upstaging of nodal status in 33% of patients when IHC and PCR techniques are combined. Therefore, the clinical value of SLN should be the subject of further studies.

Topics: Aged; Aged, 80 and over; Carcinoembryonic Antigen; Colonic Neoplasms; Coloring Agents; DNA, Neoplasm; Feasibility Studies; Female; Follow-Up Studies; Humans; Immunohistochemistry; Keratins; Lymph Nodes; Male; Middle Aged; Neoplasm Staging; Reverse Transcriptase Polymerase Chain Reaction; Rosaniline Dyes; Sentinel Lymph Node Biopsy

The efficacy of screening for colorectal cancer using a simple blood-based assay for the detection of tumor cells disseminated in the circulation at an early stage of the disease is gaining positive feedback from several lines of research. This method seems able to reduce colorectal cancer mortality and may replace colonoscopy as the most effective means of detecting colonic lesions.. In this work, we present a new microarray-based high-throughput screening method to identifying candidate marker mRNAs for the early detection of epithelial cells diluted in peripheral blood cells. This method includes 1. direct comparison of different samples of colonic mucosa and of blood cells to identify consistent epithelial-specific mRNAs from among 20,000 cDNA assayed by microarray slides; 2. identification of candidate marker mRNAs by data analysis, which allowed selection of only 10 putative differentially expressed genes; 3. Selection of some of the most suitable mRNAs (TMEM69, RANBP3 and PRSS22) that were assayed in blood samples from normal subjects and patients with colon cancer as possible markers for the presence of epithelial cells in the blood, using reverse transcription-polymerase chain reaction (RT-PCR).. Our present results seem to provide an indication, for the first time obtained by genome-scale screening, that a suitable and consistent colon epithelium mRNA marker may be difficult to identify.. The design of new approaches to identify such markers is warranted.

Topics: Adult; Aged; Automation; Colonic Neoplasms; Epithelial Cells; Female; Gene Expression Profiling; Humans; Keratin-20; Keratins; Male; Membrane Proteins; Middle Aged; Neoplastic Cells, Circulating; Nuclear Proteins; Nucleocytoplasmic Transport Proteins; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Serine Endopeptidases

Six cases of an unusual variant of primary pulmonary adenocarcinoma resembling colorectal and sinonasal adenocarcinoma are presented. Pulmonary intestinal-type adenocarcinoma occurs in elderly Caucasians and is associated with a histology characteristic of colorectal/enteric adenocarcinoma: a garland-like architecture with a 'gland in gland' periphery, central 'dirty' necrosis, and elongated stratified columnar cells, lacking significant goblet or signet ring differentiation. While a resemblance to intestinal adenocarcinoma by light microscopy is present, immunohistochemical studies comparing these carcinomas with metastatic colorectal adenocarcinoma clearly show a respiratory phenotype with the neoplastic cells expressing thyroid transcription factor-1 and cytokeratin 7 to the exclusion of cytokeratin 20, and failing to express CDX-2. Stains for a variety of epithelial mucins (MUC1, MUC2, MUC5AC) also support this observation. The differential diagnosis with other pulmonary adenocarcinomas, especially those with mucinous differentiation, is discussed.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; CDX2 Transcription Factor; Cell Differentiation; Colonic Neoplasms; Female; Homeodomain Proteins; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Lung Neoplasms; Male; Middle Aged; Mucin 5AC; Mucin-1; Mucin-2; Mucins; Nuclear Proteins; Thyroid Nuclear Factor 1; Trans-Activators; Transcription Factors

High fruit and vegetable intake is known to reduce the risk of colon cancer. To improve understanding of this phenomenon the action of different phytochemicals on colon cells has been examined. One such compound is quercetin that belongs to the group known as flavonoids. The purpose of this study was to determine the influence of quercetin on the proteome of the SW480 human colon adenocarcinoma cell line, specifically to identify proteins that could be the molecular targets of quercetin in its amelioration of the progression of colon cancer. To this end, two-dimensional gel electrophoresis and mass spectrometry were used to identify proteins that underwent a change in expression following treatment of the cells with 20 muM quercetin. This could elucidate how quercetin may reduce the progression of colon cancer.. Quercetin treatment of the SW480 human colon cancer cells was found to result in the decreased expression of three proteins and the increased expression of one protein. The identified proteins with decreased expression were type II cytoskeletal 8 keratin and NADH dehydrogenase Fe-S protein 3. The other protein with decreased expression was not identified. The protein with increased expression belonged to the annexin family.. Several proteins were determined to have altered expression following treatment with quercetin. Such changes in the levels of these particular proteins could underlie the chemo-protective action of quercetin towards colon cancer.

Topics: Annexins; Cell Line, Tumor; Colonic Neoplasms; Electrophoresis, Gel, Two-Dimensional; Humans; Hydrogen-Ion Concentration; Iron-Sulfur Proteins; Keratin-8; Keratins; Mass Spectrometry; NADH Dehydrogenase; Proteomics; Quercetin

Metastasis-the spread of tumour cells from a primary lesion to distant organs-is the main cause of cancer-related death, and bone marrow (BM) is a frequent site for the settlement of disseminated tumour cells. Many BM samples harbour isolated tumour cells, whereas tumour cell clusters, as the potential precursors of solid distant metastases, are rarely detected after current enrichment procedures. We have analysed BM samples from 43 patients with carcinomas of the breast, colon and ovaries; 41 of these patients had no clinical signs of overt metastases (stage M0). Tumour cells in BM were enriched with immunomagnetic beads coupled to monoclonal antibodies against both EpCAM and HER2/neu. After enrichment, tumour cells were identified by immunostaining with the anti-cytokeratin antibody A45-B/B3. In total, 886 CK-positive cells were detected in 16 (35%) samples after immunomagnetic enrichment as compared to 34 cells in 9 (21%) samples using Ficoll density centrifugation previously used as the standard enrichment technique. Most remarkably, clusters of 2 to 10 CK-positive cells were found in 75% of CK-positive samples enriched by immunobeads, whereas no CK-positive cell clusters were detected after Ficoll enrichment. The method described offers an excellent tool for the enrichment of micrometastatic tumour cell clusters; these clusters may represent the initial stage of development from a single disseminated tumour cell towards an overt metastasis.

Topics: Bone Marrow Neoplasms; Breast Neoplasms; Cell Separation; Centrifugation, Density Gradient; Colonic Neoplasms; Female; Ficoll; Humans; Immunohistochemistry; Immunomagnetic Separation; Keratins; Male; Neoplasm Metastasis; Neoplasm Staging; Ovarian Neoplasms

A 62-year-old man had been followed because of an elevated serum level of carcinoembryonic antigen without the detection of any cancer lesions. However, there was a sudden increase in the serum level of carcinoembryonic antigen, and abdominal imagings showed a hepatic tumor with peripheral intrahepatic bile duct dilatation, and a submucosal tumor at the sigmoid colon with intact mucosa. Histopathological findings showed that the hepatic tumor had perineural invasion, suggesting an intrahepatic cholangiocarcinoma, and that the colon tumor infiltrated the submucosa, while its mucosa was intact. Both tumors showed similar pathological features and were positive for cytokeratin 20 and 7. These findings suggested intrahepatic cholangiocarcinoma with metastatic sigmoid colon cancer.

Topics: Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Carcinoembryonic Antigen; Cholangiocarcinoma; Colonic Neoplasms; Humans; Immunohistochemistry; Keratin-20; Keratin-7; Keratins; Male; Middle Aged; Sigmoid Neoplasms

Histologic differentiation of primary ovarian carcinoma from colonic carcinoma metastatic to the ovary may be difficult. Cytokeratin 7 (CK7) and cytokeratin 20 (CK20) immunostaining is usually used, but these markers lack specificity for ovarian and intestinal epithelium, and overlapping results have been reported. Cdx2 is a transcription factor whose expression in normal tissues is limited to the intestinal epithelium. It is also expressed in the vast majority of colonic carcinomas and in a sizeable proportion of cases of gastric, pancreatobiliary, and ovarian mucinous carcinomas. We evaluated Cdx2, CK7, and CK20 expression in 50 ovarian carcinomas (15 serous, 20 mucinous, and 15 endometrioid), 15 colonic carcinomas metastatic to the ovaries, and 20 primary colonic carcinomas. The extent (1-25%/1+, 26-75%/2+, >75%/3+) and intensity (weak/1+, strong/2+) of staining were recorded semiquantitatively. All primary and metastatic colonic carcinomas had diffuse (3+) strong Cdx2 reactivity. All serous and endometrioid tumors were Cdx2 negative, whereas mucinous carcinomas had 1+ or 2+ immunoreactivity. All ovarian carcinomas had strong diffuse CK7 staining, whereas all colonic carcinomas were negative for CK7. CK20 stained diffusely and strongly all primary and metastatic colonic carcinomas and was 1+ or 2+ in all mucinous carcinomas, in 67% of serous carcinomas, and in 33% of endometrioid carcinomas. In conclusion, 1) Cdx2 is a highly sensitive (100%) marker for colonic carcinoma metastatic to the ovary; 2) Cdx2 is more specific than CK20 as it is not expressed by serous and endometrioid carcinomas; and 3) a limited panel of Cdx2 and CK7 helps in distinguishing colonic carcinomas metastatic to the ovaries (Cdx2+/CK7-) from primary ovarian serous (Cdx2-/CK7+), endometrioid (Cdx2-/CK7+), and mucinous (Cdx2+/CK7+) carcinomas.

Topics: Biomarkers, Tumor; Carcinoma; CDX2 Transcription Factor; Colonic Neoplasms; Diagnosis, Differential; Female; Homeodomain Proteins; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Ovarian Neoplasms; Sensitivity and Specificity; Trans-Activators

Two-dimensional difference gel electrophoresis (2-D DIGE) coupled with mass spectrometry (MS) was used to investigate tumor-specific changes in the proteome of human colorectal cancers and adjacent normal mucosa. For each of six patients with different stages of colon cancer, Cy5-labeled proteins isolated from tumor tissue were combined with Cy3-labeled proteins isolated from neighboring normal mucosa and separated on the same 2-D gel along with a Cy2-labeled mixture of all 12 normal/tumor samples as an internal standard. Over 1500 protein spot-features were analyzed in each paired normal/tumor comparison, and using DIGE technology with the mixed-sample internal standard, statistically significant quantitative comparisons of each protein abundance change could be made across multiple samples simultaneously without interference due to gel-to-gel variation. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and tandem (TOF/TOF) MS provided sensitive and accurate mass spectral data for database interrogation, resulting in the identification of 52 unique proteins (including redundancies due to proteolysis and post-translationally modified isoforms) that were changing in abundance across the cohort. Without the benefit of the Cy2-labeled 12 sample mixture internal standard, 42 of these proteins would have been overlooked due to the large degree of variation inherent between normal and tumor samples.

Topics: Adult; Aged; Aged, 80 and over; Colonic Neoplasms; Databases as Topic; Electrophoresis, Gel, Two-Dimensional; Female; Humans; Image Processing, Computer-Assisted; Intestinal Mucosa; Keratin-3; Keratins; Male; Mass Spectrometry; Middle Aged; Protein Isoforms; Proteome; Proteomics; Sensitivity and Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

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

Although the utility of lymphatic mapping (LM) and sentinel lymph node (SLN) biopsy in patients with melanoma and breast carcinoma has been well documented, this same is not true for patients with colon carcinoma. The authors previously reported a high false-negative rate for SLN biopsy in patients with colon carcinoma using isosulfan blue dye alone. The objective of the current study was to determine whether radiocolloid would increase the sensitivity of LM/SLN biopsy in patients with colon carcinoma.. The authors performed LM on 57 patients with colon carcinoma using both isosulfan blue dye and radiocolloid. The SLN(s) were identified by either their blue color or by increased radioactivity. The SLNs then underwent both routine histologic sectioning and immunohistochemical (IHC) staining for cytokeratins.. An SLN was identified in 56 patients (98%). Radiocolloid was able to identify only 1 additional positive SLN (9%). Overall, it was found that the disease had metastasized to the lymph nodes in 22 patients, even though there was no evidence of disease in the SLN(s) in 11 of those 22 patients on routine histologic sectioning (false-negative rate, 50%; sensitivity, 50%). In five patients, IHC of the SLN was the only indicator of metastatic disease. The inclusion of IHC-positive SLNs in these calculations would decrease the false-negative rate to 17% and would increase the sensitivity of SLN biopsy to 83%.. In the current study, the addition of radiocolloid did not increase the sensitivity of detection of positive SLN(s) compared with the use of isosulfan blue dye alone. IHC of the SLN potentially may increase the sensitivity of LM and reduce the false-negative rate. However, the long-term prognostic significance of IHC in patients with colon carcinoma remains controversial.

Topics: Adult; Aged; Aged, 80 and over; Colonic Neoplasms; Female; Humans; Immunoenzyme Techniques; Keratins; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Prognosis; Prospective Studies; Radionuclide Imaging; Radiopharmaceuticals; Rosaniline Dyes; Sensitivity and Specificity; Sentinel Lymph Node Biopsy; Technetium Tc 99m Sulfur Colloid

Topics: Adenocarcinoma, Mucinous; Aged; Carcinoembryonic Antigen; Colonic Neoplasms; Female; Fluorouracil; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratins; Ki-67 Antigen; Neoplasms, Radiation-Induced; Time Factors

Resistance to anticancer drugs such as the widely used antimetabolite 5-fluorouracil (FU) is one of the most important obstacles to cancer chemotherapy. Using GeneChip arrays, we compared the expression profile of different stages of FU resistance in colon cancer cells after in vitro selection of low-, intermediate- and high-resistance phenotypes. Drug resistance was associated with significant changes in expression of 330 genes, mainly during early or intermediate stage. Functional annotation revealed a majority of genes involved in signal transduction, cell adhesion and cytoskeleton with subsequent alterations in apoptotic response, cell cycle control, drug transport, fluoropyrimidine metabolism and DNA repair. A set of 33 genes distinguished all resistant subclones from sensitive progenitor cells. In the early stage, downregulation of collagens and keratins, together with upregulation of profilin 2 and ICAM-2, suggested cytoskeletal changes and cell adhesion remodeling. Interestingly, 6 members of the S100 calcium-binding protein family were suppressed. Acquisition of the intermediate-resistance phenotype included upregulation of the well-known drug resistance gene ABCC6 (ATP-binding cassette subfamily C member 6). The very small number of genes affected during transition to high resistance included the primary FU target thymidylate synthase. Although limited to an in vitro model, our data suggest that resistance to FU cannot be explained by known mechanisms alone and substantially involves a wide molecular repertoire. This study emphasizes the understanding of resistance as a time-depending process: the cell is particularly challenged at the beginning of this process, while acquisition of the high-resistance phenotype seems to be less demanding.

Topics: Antimetabolites, Antineoplastic; Calcium-Binding Proteins; Cell Adhesion; Collagen; Colonic Neoplasms; Down-Regulation; Drug Resistance, Neoplasm; Fluorouracil; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Keratins; Oligonucleotide Array Sequence Analysis; Phenotype; Tumor Cells, Cultured; Up-Regulation

Carcinosarcoma is a rare tumor that shows both epithelial and stromal malignant differentiation. Most reported cases of carcinosarcoma affect the female genital tract (and are called malignant mixed müllerian tumors), but there are also some isolated reports of cases affecting the lung and the head and neck area. Carcinosarcomas only rarely affect the gastrointestinal tract, mainly the esophagus. To the best of our knowledge, only eight cases of carcinosarcoma of the colon have been reported to date. For some lesions, the term 'sarcomatoid carcinoma' is preferred to 'carcinosarcoma', as both stromal and epithelial cells have shown cytokeratin expression on immunohistochemistry. The expression 'carcinosarcoma' should be applied only to those lesions, the stromal elements of which do not express epithelial markers. We report a new case of carcinosarcoma affecting the left colon. The most unique feature of this tumor is that it shows chondro-and osteosarcomatous differentiation, a feature that has been described previously in only one colonic carcinosarcoma. We discuss the histopathological and immunohistochemical features of this lesion as well as its possible histogenesis.

Topics: Aged; Aged, 80 and over; Carcinosarcoma; Colonic Neoplasms; Humans; Immunohistochemistry; Keratins; Male; Vimentin

Molecular structural changes in hair, skin and breast associated with breast cancer have been observed using synchrotron fibre diffraction. These results raised the possibility that such hair studies might be used as a non-invasive diagnostic marker for breast cancer. A series of double-blinded studies was undertaken to establish the specificity of such a tool. Hair samples from controls, patients with cancers of the breast and other sites including the colon were studied. An associated study of collagenous colon tissue was also undertaken.. Single hairs were used for the fibre diffraction study. Collagenous colon samples were dissected near the tumour and from the ends of the colon section. The protocols followed exactly the associated breast cancer studies.. A ring of radius 4.53+/- 0.03 nm, superimposed on the patterns obtained for the controls, was observed in the diffraction patterns for the hair of all colon cancer patients. This radius is different from that observed for breast cancer. In the collagen tissue study, two discrete sets of additional rings are superimposed on the normal collagenous colon pattern, one for samples adjacent to the tumour and one for samples distal from the tumour. The latter revealed that tissue at the point of dissection was not always normal.. Here we show that, since the sensitivity and specificity for the difference observed in the diffraction patterns of hair in colon cancer are both 100%, a fibre diffraction analysis could be a simple, non-invasive screening test for colon cancer.

Topics: Biomarkers, Tumor; Breast Neoplasms; Colonic Neoplasms; Diagnostic Tests, Routine; Double-Blind Method; Female; Hair; Humans; Intermediate Filaments; Keratins; Mass Screening; Models, Molecular; Predictive Value of Tests; Sensitivity and Specificity; X-Ray Diffraction

Expression of Fas ligand (FasL/CD95L) may help to maintain colon cancers in a state of immune privilege by inducing apoptosis of antitumor immune effector cells. Colon tumor-derived cell lines appear to be relatively insensitive to apoptosis mediated by their own or exogenous FasL in vitro, despite expression of cell surface Fas. In our present study, we sought to investigate if FasL upregulated in human colon cancers leads to any increase in apoptosis of the tumor cells in vivo. FasL and Fas receptor (APO-1/CD95) expression by tumor cells were detected immunohistochemically. Apoptotic tumor cell death was detected by immunohistochemistry for caspase-cleaved cytokeratin-18. FasL expression did not correlate with the extent of apoptosis of tumor cells. There was no significant local difference in the frequency of apoptosis of tumor cells between tumor nests that expressed FasL (mean = 2.4%) relative to those that did not (mean = 2.6%) (p = 0.625, n = 10; Wilcoxon signed rank). FasL expressed by the tumor cells appeared to be functional, since FasL expression in tumor nests correlated with diminished infiltration of tumor-infiltrating lymphocytes (TILs). TILs were detected using immunohistochemistry for CD45. Expression of FasL by tumor nests was associated with a mean 4-fold fewer TILs relative to FasL-negative nests (range 2.4-33-fold, n = 10, p < 0.003). Together, our results indicate that colon tumors are insensitive to FasL-mediated apoptosis in vivo.

Topics: Adenocarcinoma; Apoptosis; Colonic Neoplasms; Fas Ligand Protein; fas Receptor; Female; Humans; Immunoenzyme Techniques; In Situ Nick-End Labeling; Keratins; Leukocyte Common Antigens; Ligands; Lymphocyte Depletion; Lymphocytes, Tumor-Infiltrating; Male; Membrane Glycoproteins; Up-Regulation

Cdx-2 is expressed in normal colonic epithelia and in most colorectal adenocarcinomas. No data exist on Cdx-2 expression in primary cervical adenocarcinoma with colonic differentiation.. To ascertain the utility of Cdx-2 and a limited immunohistochemical panel in differentiating between primary cervical adenocarcinoma with intestinal differentiation and secondary (colonic) cervical adenocarcinoma, which call for different surgical and chemotherapeutic treatment protocols.. We examined cervical tract adenocarcinomas in women with previously negative medical histories for neoplastic disease and in women with colonic carcinoma. An immunohistochemical panel consisting of cytokeratin 7, cytokeratin 20, carcinoembryonic antigen, and a new marker, Cdx-2, was evaluated in all cases. The clinical data, the morphologic features, and the immunohistochemical staining patterns were compared.. Of the tumors diagnosed as metastatic intestinal adenocarcinoma of the cervix, based on clinical data and hematoxylin-eosin-stained sections, all were Cdx-2 positive, whereas Cdx-2 was not expressed in any of our cases of primary cervical adenocarcinoma with colonic differentiation. Carcinoembryonic antigen was expressed both in primary cervical tumor and in secondary (intestinal) cervical adenocarcinoma. Cytokeratin 20 was not expressed in our cases of cervical adenocarcinoma, and it was not expressed in 7.15% of cervical metastases from intestinal carcinoma. Immunostaining with cytokeratin 7 was positive in cervical adenocarcinoma, but was negative in secondary (intestinal) cervical adenocarcinoma.. Our immunohistochemical analysis shows that Cdx-2 has good specificity and would be a good marker to use in a limited panel of immunohistochemical markers, such as cytokeratin 7, cytokeratin 20, and carcinoembryonic antigen, to distinguish primary cervical adenocarcinoma from intestinal metastases to the cervix.

Topics: Adenocarcinoma; Carcinoembryonic Antigen; Carcinoma; CDX2 Transcription Factor; Cell Differentiation; Colon; Colonic Neoplasms; Diagnosis, Differential; Female; Homeodomain Proteins; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Trans-Activators; Uterine Cervical Neoplasms

Adenocarcinomas of nonsalivary origin represent approximately 10% to 20% of all sinonasal malignancies and are characterized by varying histopathologic features and uncertain histogenesis. To better understand the histogenesis and phenotypic heterogeneity of these tumors, we performed immunohistochemical analyses for cytokeratin (CK) 7 and CK20 on 12 primary sinonasal adenocarcinomas (SNACs) representing the histopathologic spectrum of these tumors, adjacent normal mucosa, and 2 metastatic adenocarcinomas from colonic primaries. The demographic and clinicopathologic characteristics of our cohort were similar to those in previously published series. Our results indicate that histologically normal respiratory-type epithelium and submucosal seromucous glands show restricted reactivity to CK7. Epithelial metaplasia of surface epithelium associated with enteric SNACs was accompanied by a conversion from CK7 positivity to CK20 positivity. All primary enteric-type carcinomas and the 2 colonic metastases were reactive to CK20, but all nonenteric-type tumors were negative for CK20 (P=0.003) and positive for CK7. In some of the enteric types, coexpression of CK7 and CK20 was noted. We conclude that (1) nonenteric-type (seromucinous) adenocarcinoma may originate directly from surface respiratory-type epithelium or from seromucous glands, (2) metaplastic transformation of surface respiratory to enteric-type epithelium precedes the development of enteric adenocarcinoma, and (3) coordinate analyses of CK7 and CK20 reactivity may aid the differential diagnosis of adenocarcinoma in the sinonasal tract.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Biomarkers, Tumor; Cell Transformation, Neoplastic; Colonic Neoplasms; Diagnosis, Differential; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Male; Middle Aged; Nose Neoplasms; Paranasal Sinus Diseases; Phenotype; Respiratory Mucosa

Colonic mucosa typically expresses cytokeratin (CK) 20 but not CK7. This CK20+/CK7- profile has been used to distinguish colonic adenocarcinoma from others arising in the lung, breast, or genitourinary tract. CK7 expression in colorectal adenocarcinoma has been reported to be rare, and when present, a metastatic origin needs to be excluded. However, we have observed a higher frequency of CK7 positivity in rectal adenocarcinomas. Paraffin sections of 42 rectal tumors (7 adenomas and 35 adenocarcinomas), 11 colonic adenocarcinomas proximal to the rectum, and 18 nonneoplastic rectoanal mucosa were randomly selected and immunostained for CK7 and CK20 with a standard avidin-biotin complex method. Immunoreactivity was recorded semiquantitatively. Cytoplasmic CK7 immunoreactivity was noted in 29 of 42 (69%) rectal glandular neoplasms (3 of 7 adenomas [43%] and 26 of 35 adenocarcinomas [74%]) and 9 of 18 (50%) nonneoplastic anorectal mucosal samples. In contrast, only 3 of the 11 (27%) colonic adenocarcinomas proximal to rectum were CK7 positive. Because of the relatively higher frequency of CK7 expression in rectal epithelial neoplasms, when CK7 is applied to distinguish primary colorectal versus metastatic origin, its reactivity should be interpreted with caution and should not be used as the sole evidence for excluding a rectal primary, particularly in tumors involving the rectal or pelvic region.

Topics: Adenocarcinoma; Antigens, Neoplasm; Colonic Neoplasms; Diagnosis, Differential; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Neoplasm Metastasis; Rectal Neoplasms; Sensitivity and Specificity

We are investigating the mechanism responsible for the overexpression of the keratin 18 (K18) gene in tumorigenic clones from the SW613-S human colon carcinoma cell line, as compared with non-tumorigenic clones. We have previously shown that this mechanism affects the minimal K18 promoter (TATA box and initiation site). We report here that treatment of the cells with histone deacetylase inhibitors stimulates the activity of the promoter in non-tumorigenic cells but has no effect in tumorigenic cells, resulting in a comparable activity of the promoter in both cell types. The adenovirus E1A protein inhibits the activity of the K18 promoter specifically in tumorigenic cells. This inhibition can be reversed by an excess of CBP protein. The conserved region 1 (CR1) of E1A, which is involved in the interaction with the CBP/p300 co-activators, is necessary to the inhibitory capacity of E1A. A 79 amino acid long N-terminal fragment of E1A, encompassing the two domains of E1A necessary and sufficient for binding to CBP (N-terminus and CR1), has the same differential inhibitory capacity on the K18 promoter as wild-type E1A. Forced recruitment of GAL4-CBP fusion proteins to the K18 promoter results in a greater stimulation of its activity in non-tumorigenic than in tumorigenic cells. The histone acetyltransferase activity of CBP is essential for this differential stimulation and the presence of the CBP2 domain greatly augments the activation capacity of the fusion protein. Chromatin immunoprecipitation experiments carried out with anti-acetylated histone antibodies showed no difference in the level of histone acetylation in the region of the K18 promoter between the two cell types. The structure of chromatin in the promoter region is similar in tumorigenic and non-tumorigenic cells, as determined by mapping of DNase I hypersensitive sites and probing the accessibility of the DNA to restriction endonucleases. From all these results we conclude that alteration of an acetylation mechanism involving the CBP (or p300) protein and acting on a non-histone substrate is responsible for the higher activity of the K18 promoter in tumorigenic cells of the SW613-S cell line.

Topics: Acetylation; Acetyltransferases; Adenovirus E1A Proteins; Carcinoma; Carrier Proteins; Cell Cycle Proteins; Chromatin; Clone Cells; Colonic Neoplasms; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Histone Acetyltransferases; Histone Deacetylase Inhibitors; Histones; Humans; Keratins; p300-CBP Transcription Factors; Promoter Regions, Genetic; Protein Structure, Tertiary; Trans-Activators; Transcription Factors; Transcriptional Activation; Tumor Cells, Cultured

Mucinous bronchioloalveolar carcinomas (BACs) can closely mimic metastatic adenocarcinoma to the lung both clinically and morphologically. Several studies have demonstrated that the differential expression of cytokeratin 7 (CK7) and cytokeratin 20 (CK20) is a valuable diagnostic tool in differentiating primary pulmonary adenocarcinomas (PPAs) (usually CK7 positive/CK20 negative) from metastatic colonic adenocarcinoma (usually CK7 negative/CK20 positive). The present study is designed to correlate the histologic subtypes of PPA with expression of 7 and 20. A total of 113 cases of bonafide PPA were selected and classified according to the 1999 World Health Organization criteria as adenocarcinoma, NOS (n = 80), nonmucinous BAC (n = 14), and mucinous BAC (n = 19). Representive sections of all the tumors were immunohistochemically analyzed for CK7 and CK20 expression. To evaluate the diagnostic utility of CK7 and CK20 expression, 6 cases of colonic adenocarcinoma metastatic to the lung were tested with the same antibodies and compared with mucinous BAC. Results were expressed in a semiquantitative fashion based on the percentage of positive tumor cells: <10%, focal; 10% to 25%, 1+; 26% to 75%, 2+; > or =76%, 3+. All 113 PPAs exhibited strong, diffuse CK7 expression. With respect to CK20 expression, 17 of the 19 cases (89.4%) of mucinous BAC showed moderate to strong expression of this protein, whereas only 10 cases of conventional adenocarcinomas and 4 cases of nonmucinous BAC exhibited expression. All 6 examples of metastatic colonic adenocarcinomas were negative for CK7 and strongly positive for CK20. In summary, mucinous BAC is distinct from other PPAs by virtue of its CK20 expression. Although the CK7/CK20 immunoprofile is a valuable diagnostic marker for differentiating primary lung adenocarcinoma from metastatic colonic adenocarcinoma, caution should be exercised when dealing with mucinous BAC.

Topics: Adenocarcinoma; Adenocarcinoma, Bronchiolo-Alveolar; Adenocarcinoma, Mucinous; Biomarkers, Tumor; Colonic Neoplasms; Diagnosis, Differential; Humans; Immunoenzyme Techniques; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Lung Neoplasms; Retrospective Studies

Ordered cell cycle progression requires the expression and activation of several cyclins and cyclin-dependent kinases (Cdks). Hyperosmotic stress causes growth arrest possibly via proteasome-mediated degradation of cyclin D1. We studied the effect of hyposmotic conditions on three colonic (Caco2, HRT18, HT29) and two pancreatic (AsPC-1 and PaCa-2) cell lines. Hyposmosis caused reversible cell growth arrest of the five cell lines in a cell cycle-independent fashion, although some cell lines accumulated at the G(1)/S interface. Growth arrest was followed by apoptosis or by formation of multinucleated giant cells, which is consistent with cell cycle catastrophe. Hyposmosis dramatically decreased Cdc2, Cdk2, Cdk4, cyclin B1, and cyclin D3 expression in a time-dependent fashion, in association with an overall decrease in cellular protein synthesis. However, some protein levels remained unaltered, including cyclin E and keratin 8. Selective proteasome inhibition prevented Cdk and cyclin degradation and reversed hyposmotic stress-induced growth arrest, whereas calpain and lysosome enzyme inhibitors had no measurable effect on cell cycle protein degradation. Therefore, hyposmotic stress inhibits cell growth and, depending on the cell type, causes cell cycle catastrophe with or without apoptosis. The growth arrest is due to decreased protein synthesis and proteasome activation, with subsequent degradation of several cyclins and Cdks.

Topics: Apoptosis; Calpain; CDC2 Protein Kinase; CDC2-CDC28 Kinases; Cell Survival; Colonic Neoplasms; Cyclin B; Cyclin B1; Cyclin D1; Cyclin D3; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Cyclins; Cysteine Endopeptidases; DNA Fragmentation; Flow Cytometry; G1 Phase; Giant Cells; Humans; Keratins; Lysosomes; Microscopy, Electron; Models, Biological; Multienzyme Complexes; Osmosis; Osmotic Pressure; Pancreatic Neoplasms; Proteasome Endopeptidase Complex; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; S Phase; Time Factors; Tumor Cells, Cultured

Topics: Adenoma; Adult; Biopsy; Colonic Diseases; Colonic Neoplasms; Colonoscopy; Diagnosis, Differential; Endometriosis; Female; Humans; Keratins; Magnetic Resonance Imaging

The clinicopathologic features of a case of endometrioid adenocarcinoma arising from colonic endometriosis that clinically and histologically mimicked a primary colonic carcinoma are reported. The differential diagnostic features of the tumor leading to the correct diagnosis included associated endometriosis, a minor mucosal component, focal squamous differentiation, absence of dirty necrosis, low nuclear grade, absence of a colonic adenoma, and a CK7+/CK20-/CEA- immunophenotype.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Carcinoembryonic Antigen; Carcinoma, Endometrioid; Colonic Diseases; Colonic Neoplasms; Diagnosis, Differential; Endometriosis; Female; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins

The regulation of apoptotic cell death may have a profound effect on the pathogenesis and progression of colon cancer. Survivin, a member of the inhibitor of apoptosis gene family, has been detected in fetal tissue and in a variety of human malignancies. In the current study, we investigated survivin expression by an immunohistochemical approach in benign, hyperplastic, premalignant, and malignant lesions of the colon. Survivin was detected in all cases of normal colonic mucosa (20/20), hyperplastic polyps (20/20), adenomatous polyps (20/20), and in both well differentiated and moderately differentiated colonic adenocarcinomas (20/20). In the normal colonic mucosa, survivin expression was mostly restricted to the base of the colonic crypts. All epithelial cells showed uniformly intense staining for survivin in hyperplastic polyps. By contrast, adenomas and adenocarcinomas showed a heterogeneous staining pattern with cell-to-cell, gland-to-gland, and regional variability in the intensity of survivin staining. In contrast to the basal preponderance of staining in normal colonic mucosa, numerous survivin positive cells were present at the luminal surface of hyperplastic polyps, adenomatous polyps, and adenocarcinomas. In conclusion, the expression of survivin is not a specific marker of adenocarcinoma of the colon but does show characteristic and reproducible patterns of expression in non-neoplastic proliferative lesions and in normal colonic mucosa.

Topics: Colon; Colonic Neoplasms; HeLa Cells; Humans; Hyperplasia; Immunohistochemistry; Inhibitor of Apoptosis Proteins; Intestinal Mucosa; Keratins; Ki-67 Antigen; Microtubule-Associated Proteins; Neoplasm Proteins; Protein Biosynthesis; Survivin

histological features of metastasis are not always specific of the origin. The usefulness of cytokeratin 7 and 20 (CK7 and CK20) in cerebral metastases from an adenocarcinoma was studied in a series of 78 patients.. metastases from lung adenocarcinoma showed a CK7+/CK20- pattern in 79% of cases, CK7+/CK20+ in 19% and CK7-/CK20- in 2%. When observed, positivity for cytokeratin 20 was generally focal or weak. Breast adenocarcinoma metastases were CK7+/CK20- in 71% of cases and CK7-/CK20- in 29%. Less differentiated tumours were usually negative for both cytokeratins. Metastases by colonic adenocarcinoma were CK7-/CK20+ in 100% of cases. Cytokeratins 7 and 20 are useful to distinguish lung from colonic metastatic carcinoma. In case of double positivity, a more intense CK7 expression is rather suggestive of a lung origin.. these results might modify paraclinic investigations in search of the primitive tumor.

Topics: Adenocarcinoma; Brain Neoplasms; Breast Neoplasms; Colonic Neoplasms; Diagnosis, Differential; Humans; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Lung Neoplasms

We present a case of synchronous breast and colon carcinoma in a pleural effusion, to our knowledge the first such reported case in the English-language literature. The patient was a 55-yr-old white female with known metastatic breast and colon carcinoma who developed a malignant pleural effusion which demonstrated two strikingly different populations of malignant cells by immunohistochemical study of cell block material. One cell population demonstrated a cytokeratin (CK)7+/CK20-/ER+ phenotype, while the other demonstrated a CK7-/CK20+/ER- phenotype, consistent with breast and colon origin, respectively. An immunohistochemical survey of archival breast and colon primary and metastatic carcinomas confirmed the established CK7+/CK20- phenotype of breast and CK7-/CK20+ phenotype of colon primary carcinomas, and the maintenance of this phenotype in metastases thereof. A survey of benign and malignant mesothelial lesions confirmed the absence of staining for estrogen receptor, but showed 6/10 cases weakly positive for CK20, which has not been described in other published series. This unusual case graphically illustrates the utility of cytokeratin subset immunohistochemistry in effusion cytology.

Topics: Adenocarcinoma; Breast Neoplasms; Carcinoma, Ductal, Breast; Colonic Neoplasms; Fatal Outcome; Female; Humans; Immunoenzyme Techniques; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Middle Aged; Pleural Effusion, Malignant

A large percentage of cases of perianal Paget disease are associated with an internal cancer, most commonly rectal adenocarcinoma. Immunostains for cytokeratin 7, cytokeratin 20, and gross cystic disease fluid protein 15 are useful in identifying cases associated with rectal adenocarcinoma. The Paget cells and rectal adenocarcinoma cells of these lesions typically have a cytokeratin 7(+)/cytokeratin 20(+)/gross cystic disease fluid protein 15(-) immunophenotype. It is not known whether rectal adenocarcinoma unassociated with perianal Paget disease has the same cytokeratin profile as rectal adenocarcinoma associated with perianal Paget disease.. To evaluate the immunohistochemical cytokeratin 7 and 20 profile of resected rectal adenocarcinoma unassociated with perianal Paget disease as well as that of normal anal glands from hemorrhoidectomy specimens.. We performed immunohistochemistry for cytokeratins 7 and 20 on tissues from 30 cases of rectal adenocarcinoma unassociated with perianal Paget disease and 12 hemorrhoidectomy specimens from 12 cases with normal anal glands. We defined positive staining as any immunoreactivity within the neoplastic cells.. Twenty-six of 30 cases of rectal adenocarcinoma (87%) had a cytokeratin 7(-)/cytokeratin 20(+) immunophenotype, similar to the immunophenotype of cases of nonrectal large intestine adenocarcinoma. In 4 cases (13%), neoplastic cells coexpressed cytokeratins 7 and 20. Anal glands stained strongly for cytokeratin 7 but were negative for cytokeratin 20 in all cases, and the anal transitional zone mucosa had a similar immunophenotype.. Rectal adenocarcinoma unassociated with perianal Paget disease has a cytokeratin profile similar to that of nonrectal large intestine adenocarcinoma. These data suggest that rectal adenocarcinoma unassociated with perianal Paget disease has a different cytokeratin profile than rectal adenocarcinoma associated with perianal Paget disease.

Topics: Adenocarcinoma; Anal Canal; Colonic Neoplasms; Hemorrhoids; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Paget Disease, Extramammary; Rectal Neoplasms

We studied 14 mucinous and 26 nonmucinous bronchioloalveolar adenocarcinomas (BACs) with thyroid transcription factor (TTF), cytokeratin (CK) 7, CK20, and villin to characterize their staining patterns with these antibodies and identify staining differences between the neoplasms. We also stained 11 mucinous colon adenocarcinomas with the same antibodies to compare their reaction patterns with mucinous BACs. All pulmonary neoplasms were confirmed pulmonary primary BACs. Three (21%) of 14 mucinous neoplasms had weak TTF reactivity in fewer than 25% of neoplastic cell nuclei, and the other 11 (79%) were nonreactive. In contrast, 24 (92%) of 26 nonmucinonus BACs were strongly TTF reactive. Eleven mucinous BACs (79%) had CK20 reactivity in more than 25% of neoplastic cells, whereas only 1 nonmucinous BAC (4%) had reactivity in fewer than 50% of the cells. One mucinous BAC (7%) had villin reactivity in approximately 10% of the neoplastic cells. All mucinous colon adenocarcinomas were diffusely reactive with CK20 and villin. Mucinous and nonmucinous BACs have disparate staining patterns with TTF and CK20. Mucinous BACs are usually TTF nonreactive and CK20 reactive, but nonreactive with villin, which distinguishes them from mucinous colon adenocarcinomas.

Topics: Adenocarcinoma, Bronchiolo-Alveolar; Adenocarcinoma, Mucinous; Biomarkers, Tumor; Carrier Proteins; Cell Nucleus; Colonic Neoplasms; Female; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Lung Neoplasms; Male; Microfilament Proteins; Nuclear Proteins; Staining and Labeling; Thyroid Nuclear Factor 1; Transcription Factors

Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are important regulators of endothelial cell (EC) survival. Current models suggest that an increase in Ang-2 expression in ECs leads to the initiation of angiogenesis. The authors hypothesized that the imbalance of Ang-1 and Ang-2 activities in colon carcinoma leads to a net gain in Ang-2 function.. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses and immunofluorescent double-staining were performed to examine human colon carcinoma cell lines, surgical specimens, normal mucosa, and liver metastases for the expression of Ang-1 and Ang-2.. RT-PCR analyses revealed that 7 of 18 colon carcinoma cell lines expressed Ang-1, and 14 of 18 colon carcinoma cell lines expressed Ang-2 (P < 0.05). Of the surgical specimens from patients with colon carcinoma, 6 of 11 specimens expressed Ang-1, and 11 of 11 specimens expressed Ang-2 (P < 0.05). However, Ang-1 and Ang-2 were expressed with relative equal frequency in normal mucosa (P = 0.62). Immunofluorescent staining (n = 20 specimens) revealed the presence of Ang-2 protein in normal mucosa and tumor epithelium, but Ang-1 was expressed only in normal mucosa. A similar pattern was found for hepatic colorectal metastases. Double staining for Ang-1 or Ang-2 and cytokeratin-22 (an epithelial marker) demonstrated that Ang-1 was produced by uninvolved, normal colonic epithelium, whereas Ang-2 was produced by normal and malignant colonic epithelium.. In patients with colon carcinoma, Ang-2 is expressed ubiquitously in tumor epithelium, whereas expression of Ang-1 in tumor epithelium is rare. The net gain of Ang-2 activity is possibly an initiating factor for tumor angiogenesis.

Topics: Actins; Angiopoietin-1; Angiopoietin-2; Colonic Neoplasms; Gene Expression; Humans; Immunohistochemistry; Keratins; Membrane Glycoproteins; Neovascularization, Pathologic; Platelet Endothelial Cell Adhesion Molecule-1; Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured

Although the sentinel node (SN) concept has been validated in malignant melanoma and breast cancer, the application of this concept for other solid tumors, including gastrointestinal (GI) cancer, is still controversial. We have demonstrated the feasibility of radioguided SN mapping during laparotomy in patients with esophageal, gastric, and colorectal cancers. In 188 patients, the SNs identified by this technique had an overall diagnostic accuracy of 96% for regional lymph node metastasis. Aberrant drainage sites that have been called skip metastasis from the primary lesion were detectable using this method. More recently, we have undertaken SN mapping during laparoscopic surgery. A combination of radiotracer and blue dye optimized the identification of SNs that drained GI cancers. Our preliminary data indicate that laparoscopic mapping of the SN is a sensitive intraoperative technique for identifying lymph node micrometastasis, and we believe that it will become an important component of a minimally invasive approach to early-stage GI cancers.

Topics: Colonic Neoplasms; Coloring Agents; Esophageal Neoplasms; Gastrointestinal Neoplasms; Humans; Keratins; Laparoscopy; Lymphatic Metastasis; Radionuclide Imaging; Sentinel Lymph Node Biopsy; Stomach Neoplasms; Technetium Tc 99m Aggregated Albumin

Recently several reverse transcription-PCR techniques have been proven to be useful for the detection of circulating micrometastases. However, this way intact cell clusters that were found in animal experiments of prognostic value could not be detected. In this study, evaluation and modification of a commercial, cytokeratin-based, immunomagnetic cell separation method was performed for the detection of intact cell clusters in colorectal carcinoma patients.. Thirty-two colon cancer patients (6 were in Dukes stage B, 13 in stage C, and 13 in stage D) and 20 healthy donor samples were evaluated. Immunomagnetic cell separation was performed from the buffy coat of peripheral blood samples (20 ml) using the Carcinoma Cell Enrichment Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), avoiding any filtering steps. The enriched cell fraction was cytocentrifuged and immunocytochemically labeled using a pancytokeratin antibody (MNF116; Dako).. Of 20 healthy samples, 2 contained one cytokeratin-positive cell. Of 32 single samples from malignant cases, 24 showed cytokeratin-positive cells. Tumor cell clusters, mixed-cell doublets (one cytokeratin-positive and -negative cell), and mixed-cell clusters were detected in 22 of 24 patients. In six cases, cytokeratin-positive dendritic-like cells were detected. Follow-up data indicate that chemotherapy cannot destroy all of the circulating tumor cell clusters.. Using the methods presented, we could detect circulating colon cancer cells and cell clusters in colon carcinoma patients. Similar cellular structures were described previously only in rats. Present data prove that such structures are present in human colorectal cancer, too.

Topics: Cell Adhesion; Cell Separation; Colonic Neoplasms; Colorectal Neoplasms; Humans; Keratin-7; Keratins; Neoplasm Staging; Tumor Cells, Cultured

The value of lymphatic mapping and sentinel lymph node biopsy in the treatment of colon cancer is controversial. The purpose of this study was to determine the accuracy of lymphatic mapping in patients with colon cancer.. Forty-eight patients with colon cancer underwent lymphatic mapping and sentinel lymph node biopsy using isosulfan blue dye followed by standard surgical resection. The sentinel lymph nodes underwent thin sectioning as will as immunohistochemical staining for cytokeratin, in addition to standard hematoxylin and eosin staining.. In 47 (98%) patients, a sentinel lymph node was identified. Sixteen patients had lymph nodes containing metastatic disease, and in 6 patients the sentinel lymph node was positive for disease. In no patient was the sentinel lymph node the only site of metastatic disease. In 10 patients the sentinel lymph node was negative for disease, whereas the nonsentinel lymph nodes contained metastatic disease (false negative rate = 38%).. The role of lymphatic mapping and sentinel lymph node biopsy in colon cancer is not as clear as its role in other tumors. Further large prospective studies are needed to evaluate the accuracy and potential benefit of this procedure in patients with colon cancer.

Topics: Aged; Aged, 80 and over; Colonic Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Sentinel Lymph Node Biopsy

To study the distinctive clinicopathologic and immunohistochemical difference between ovarian metastatic carcinomas and primary ovarian carcinomas.. The clinical and pathological features of 27 cases of ovarian metastatic carcinomas (gastric carcinomas 12 cases, colon carcinomas 11 cases, others 4 cases) obtained from our department were reviewed. Immunostainings for CK (AE1/AE3), CK7, CK20, CEA, vimentin, nm23 were performed with SP staining methods.. On gross examination, metastasis from gastric adenocarcinoma were usually bilateral, while solid (11/12) and metastases from colonic adenocarcinoma were more often unilateral and cystic (7/11). Microscopically, metastases from gastric adenocarcinoma revealed signet ring cells or poorly differentiated adenocarcinomas (12/12), whereas metastases from colonic adenocarcinomas showed similar morphology of endometrioid adenocarcinoma (8/11). The majority of ovarian metastases of gastric carcinoma (7/12) and colon carcinoma (8/11) were CK20 positive. In particular, CK20 was invariably expressed in colon cancer metastases. Most of the ovarian metastatic carcinomas from the gastrointestinal tract failed to react with immunostaining of CK7. A combined use of CEA, vimentin and nm23 had made a correct classification for 11/12 cases of the gastric carcinoma, 10/11 cases of the colonic cancer.. CK7 and CK20 have been proved to be useful antibodies in distinguishing between metastatic carcinomas and primary carcinomas of the ovary. Combined use of a panel of antibodies can give more significant results.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Breast Neoplasms; Carcinoembryonic Antigen; Carcinoma, Endometrioid; Carcinoma, Signet Ring Cell; Colonic Neoplasms; Diagnosis, Differential; Female; Follow-Up Studies; Genes, Tumor Suppressor; Humans; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Middle Aged; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Ovarian Neoplasms; Stomach Neoplasms; Vimentin

Epidemiological evidence suggests that dietary calcium and vitamin D intake are inversely related to incidence of colon cancer. Previous studies have demonstrated that supplementation of the diet with calcium in the form of calcium tablets or low-fat dairy foods alters colonic epithelial cell proliferation from a higher- to a lower-risk pattern. The present study compared relative effects of administration of calcium carbonate at approximately 900 mg/day (calcium) with those of a low-fat dairy food diet providing about the same amount of calcium (dairy) in a cross-over "head-to-head" study of 40 subjects at risk for colonic neoplasia. Dietary intake of macronutrients was similar in the two study periods, except for a slight increase in protein intake during dairy calcium supplementation. Rectal epithelial cell proliferation was studied in flat endoscopically normal-appearing mucosa at baseline and at the end of each of the two study periods and showed a significant reduction in epithelial crypt cell labeling index from 12.5% to 9.1% (calcium) or 9.3% (dairy) as well as in proliferating cells in the upper 40% of the crypt from 0.09 to 0.03 in the calcium- and low-fat dairy-supplemented intervention groups. No significant changes in two epithelial cell differentiation markers, cytokeratin AE1 and acidic mucins, were found. Furthermore, there were no differences in epithelial cell apoptosis or expression of the proapoptotic gene product BAK. These data indicate that increased dietary calcium given as supplements or in the diet in low-fat dairy foods lowers epithelial cell proliferation indexes from a higher- to a lower-risk pattern. Because supplemental calcium has been shown to reduce the recurrence of colonic adenomatous polyps in patients at increased risk for colonic neoplasia, our data suggest that supplemental low-fat dairy foods may also be effective.

Topics: Adenomatous Polyposis Coli; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; Biopsy; Calcium Carbonate; Calcium, Dietary; Cell Differentiation; Cell Division; Colon; Colonic Neoplasms; Dairy Products; Diet; Diet, Fat-Restricted; Dietary Supplements; Epithelial Cells; Female; Humans; Keratins; Male; Membrane Proteins; Middle Aged; Mucins; Racial Groups; Rectum

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

Patients with metastatic renal and colon carcinoma have a very poor prognosis. In many cases, the tumor recurs after surgical excision and chemotherapy. Therefore, it might be beneficial for cancer patients to induce an immune attack against the tumor by inserting a cytokine gene into the tumor cells. Here, two different techniques for isolation of single tumor cells were compared. An enzymatic solution was superior to an EDTA/DTT isolation solution for establishing tumor primary cultures. In total, 18 primary cell cultures could be established from 68 patients with colon and renal cell carcinoma. Cells were further characterized concerning fibroblast contamination, cell proliferation and HLA-typing. These primary tumor cells might be of value for cytokine gene transfer and in vaccination protocols for cancer patients.

Topics: Adenocarcinoma; Antigens, Neoplasm; Carcinoma, Renal Cell; Cell Culture Techniques; Cell Division; Cell Separation; Clone Cells; Colonic Neoplasms; Cytokines; Electroporation; HLA Antigens; Humans; Keratins; Kidney Neoplasms; Neoplasm Metastasis; Recombinant Fusion Proteins; Transfection; Tumor Cells, Cultured

Calretinin (CR) is a Ca(2+)-binding protein (CaBP) of the EF-hand family expressed in a cell-type-specific manner and thought to act as a Ca(2+) buffer. Based upon previous studies, CR can undergo Ca(2+)-induced conformational changes, suggesting that it may also belong to the subfamily of Ca(2+)-sensor proteins that are characterized by their ability to interact with target ligands. To elucidate the role of CR, we used the undifferentiated colon adenocarcinoma cell line WiDr, which expresses significant amounts of CR. It has been shown previously that combined treatment with an inducer of differentiation sodium butyrate (NaBt) and a cell growth inhibitor hexamethylene bisacetamide (HMBA) or treatment with CR antisense oligonucleotides is down-regulating CR in parallel with a decrease of cell growth, suggesting a possible involvement of CR in maintaining the undifferentiated phenotype of WiDr cells. Furthermore, CR is absent from normal colon cells and from well-differentiated colon adenocarcinoma cell lines (e.g., Caco-2). Since members of the EF-hand family of proteins are interacting with cytoskeletal components, we investigated the possible association of CR with the cytoskeleton in WiDr cells. With double immunofluorescence stainings and immunoprecipitation experiments, we show close association of CR with intermediate filaments or microtubules in WiDr cells. Treatment with NaBt either disrupted or strongly diminished this interaction, respectively. The same effect was observed after elevation of [Ca(2+)](i) by applying the ionophore A-23187. These data suggest that CR may contribute to the transformation of enterocytes by interfering with the differentiation process, i.e., acting at both levels: cell shape dynamics and mitosis.

Topics: Adenocarcinoma; Butyrates; Calbindin 2; Calcimycin; Colonic Neoplasms; Humans; Intermediate Filaments; Ionophores; Keratins; Microtubules; S100 Calcium Binding Protein G; Tubulin; Tumor Cells, Cultured

Colon signet ring cell adenocarcinomas are uncommon, high-grade neoplasms. Given their rarity, the question of primary colon or metastatic gastric adenocarcinoma frequently arises when signet ring cell carcinoma is seen in a colonoscopic biopsy or in biopsies procured from other regions of the body. A second related question regarding colon and gastric signet ring cell carcinomas is their immunophenotypic similarities with the glandular form of adenocarcinoma in each site. We studied the immunohistochemical phenotype of 14 colonic signet ring cell adenocarcinomas and compared them with immunophenotype of 27 gastric signet ring cell adenocarcinomas. We also compared the immunophenotype of the 27 gastric signet ring cell with the immunophenotype of 19 gastric gland-forming adenocarcinomas, and the immunophenotype of the 14 colonic signet ring cell adenocarcinomas to the immunophenotype of 20 colonic gland-forming adenocarcinomas to identify staining differences in the neoplastic cells of the two architectures. Antibodies studied were cytokeratins 7, 17, 19, and 20, CA 19-9, CA-125. estrogen receptor, and gross cystic disease fluid protein 15. Sixty-four percent of colon signet ring cell adenocarcinomas had either no staining or focal staining with cytokeratin 7 compared with diffuse staining in 63% of gastric signet ring cell adenocarcinomas (P = 0.016). Seventy-two percent of colon signet ring cell adenocarcinomas had diffuse staining with cytokeratin 20 compared with no or focal staining in 50% of gastric signet ring cell adenocarcinomas (P = 0.019). Fifty-seven percent of the colon signet ring cell adenocarcinomas had a cytokeratin 7 (-)/cytokeratin 20 (+) staining pattern compared with 11% of gastric signet ring cell adenocarcinomas (P = 0.004). Forty-four percent of gastric signet ring cell adenocarcinomas had a cytokeratin 7 (+)/cytokeratin 20 (-) pattern, compared with none of the colon signet ring cell adenocarcinomas (P = 0.004). The staining distribution of the antibody battery was similar in colon signet ring cell and colon glandular adenocarcinoma and gastric signet ring cell and gastric glandular adenocarcinomas. When signet ring cell adenocarcinoma is encountered in a colon biopsy, a colon primary is supported if the neoplastic cells have a cytokeratin 7 (-)/cytokeratin 20 (+) staining pattern, and a gastric primary is supported if they have a cytokeratin 7 (+)/cytokeratin 20 (-) staining pattern. The signet ring morphology at each site had an

Topics: Adenocarcinoma; Apolipoproteins; Apolipoproteins D; CA-125 Antigen; CA-19-9 Antigen; Carcinoma, Signet Ring Cell; Carrier Proteins; Colonic Neoplasms; Glycoproteins; Humans; Immunohistochemistry; Immunophenotyping; Keratins; Membrane Transport Proteins; Receptors, Estrogen; Stomach Neoplasms

Over one third of patients with stage II colonic adenocarcinoma experience tumor recurrence. Because effective adjuvant therapy is now available, it is important to identify subsets of patients at higher risk for relapse who may benefit from early treatment. Immunohistochemistry has been used to detect microscopic metastases in histologically uninvolved mesenteric lymph nodes, but the prognostic significance of minimal nodal involvement has not been established.. Hematoxylin and eosin (H&E)-stained recuts of 900 mesenteric lymph nodes from 55 patients (range, 2-47; mean, 16.4 nodes per case) with resected pT3 or pT4, N0, M0 (TNM stage II) colonic adenocarcinomas were re-examined for the presence of metastases and then stained immunohistochemically for keratin using the AE1:AE3 antibody. Twenty-seven patients did not experience recurrence of tumor within 5 years following resection (no evidence of disease [NED]); 28 patients relapsed during the same time frame. Lymph nodes from 10 patients having colonic resections for nonneoplastic disorders also were stained as controls. Keratin-positive cells and cell clusters were quantified in the lymph nodes, and comparisons were made between patients with and without tumor relapse.. In the relapse group, four patients had positive nodes already identified on the H&E-stained recuts and had to be excluded from further analysis. Sixteen additional patients had keratin-positive cells; thus, 16 of 24 (67%) had micrometastases. In the NED group, one patient had a positive node on H&E staining and 22 additional patients had keratin-positive cells, so 22 of 26 (84%) patients had micrometastases. In the patients who had micrometastases, there was a mean of 3.5 and 4.6 positive nodes in the relapse and NED groups, respectively, and a mean of 11.3 and 12.4 keratin-positive cells or clusters in the relapse and NED groups, respectively. No keratin-positive cells were found in the 1 to 21 (mean, 9.1) nodes per case studied in the control patients.. Micrometastases to histologically uninvolved mesenteric lymph nodes commonly are detected in patients with pT3 or pT4 colonic adenocarcinomas on recuts stained immunohistochemically for keratin. Nodal micrometastases detected by immunohistochemical staining are not useful for identifying stage II patients at higher risk for relapse.

Topics: Adenocarcinoma; Colonic Neoplasms; Coloring Agents; Disease Progression; Eosine Yellowish-(YS); Female; Hematoxylin; Humans; Immunohistochemistry; Keratins; Lymphatic Metastasis; Male; Mesentery; Neoplasm Recurrence, Local; Neoplasm Staging; Recurrence; Risk Factors

Despite the use of radical locoregional therapeutic methods and although conventional methods of diagnosis give no indication of metastases at the time of operation, distant metastases develop in approximately 50% of carcinoma patients within 5 years. While local relapses after the R0 resection of solid tumors are mainly a matter of concern for the surgeon, distant metastases can be traced back to the systemic dissemination of tumor cells at the time of operation.. A prospective study is presented in which 145 patients suffering from colon carcinoma were analyzed for the prognostic relevance of isolated disseminated tumor cells detected in the bone marrow (IDT BM). The patients were operated on between 1993 and 1997 and subsequently observed until 1999.. The monoclonal antibody A45-B/B3 was used with the immunocytochemical standard method for detecting IDT BM. For the purpose of cell cultivation, the cells were marked with the HEA-125 antibody and separated by means of magnetic cell sorting (MACS).. In this investigation the presence of isolated disseminated tumor cells, as indicated by the A45-B/B3 antibody, proved to be an independent prognostic factor for survival time. The risk of an earlier death increased in node-negative and metastases-free patients with the detection of IDT BM by a factor of 12.60. The detection of IDT BM also represented an independent prognostic factor for the time until advancement of the tumor. The risk of an earlier relapse increased with the detection of disseminated tumor cells in the bone marrow containing the A45-B/B3 antibody by a factor of 18.02. A generally acknowledged standardization of the method is desirable. Due to the importance of the independent prognostic IDT BM factor, this method of ascertaining the pathological stage should be established at institutions of higher learning.

Topics: Aged; Antibodies, Monoclonal; Biomarkers, Tumor; Bone Marrow; Carcinoma; Colonic Neoplasms; Female; Follow-Up Studies; Humans; Immunohistochemistry; Keratins; Male; Middle Aged; Neoplasm Seeding; Prognosis; Prospective Studies; Survival Rate

The keratin 18 (K18) gene is overexpressed in cells of tumorigenic clones isolated from the SW613-S human colon carcinoma cell line, compared to cells of nontumorigenic clones. The isolated minimal promoter (TATA box and initiation site) of the K18 gene has by itself a differential activity in tumorigenic and nontumorigenic cells. An Sp1 binding site located upstream of the TATA box contributes to the high level of expression of the gene in tumorigenic cells. We report here that the Sp1 gene is not differentially expressed between the two cell types and that this is also the case for genes coding for factors of the preinitiation complex known to directly interact with the Sp1 protein. Further, DNase I footprinting experiments and mutagenesis analysis indicated that the mechanism responsible for the differential activity of the minimal K18 promoter apparently does not involve the binding of a factor to a specific sequence. During the course of these experiments, it was found that the initiation site of the K18 promoter is actually located 11 bp upstream of the +1 position previously reported and that the TATA box is the only essential element of the minimal promoter. Treatment of the cells with histone deacetylase inhibitors was more efficient at stimulating the activity of the K18 promoter in nontumorigenic cells than in tumorigenic cells. We propose that overexpression of the K18 gene in tumorigenic cells could result from of a high level of acetylation of histones and/or of factors controlling the activity of the transcription complex.

Topics: Base Sequence; Binding Sites; Butyrates; Colonic Neoplasms; Deoxyribonuclease I; DNA Footprinting; DNA, Complementary; Gene Expression Regulation; Humans; Keratins; Molecular Sequence Data; Mutagenesis; Promoter Regions, Genetic; Sp1 Transcription Factor; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Topics: Cell Separation; Colonic Neoplasms; DNA, Neoplasm; Epithelial Cells; Genes, p53; Globins; Humans; Keratins; Kidney Glomerulus; Loss of Heterozygosity; Male; Molecular Biology; Nucleic Acids; Prostate

Hemidesmosomes (HDs) are cellular junctions that anchor epithelial cells to the extracellular matrix (ECM) and are associated morphologically with the cytoskeleton. Hemidesmosomal molecular components include two proteins involved in linking intermediate filaments, HD1/plectin and BP230, and two transmembrane proteins, BP180 and the alpha6beta4 integrin, a laminin receptor. In cells lacking BP230 and BP180, HD1/plectin still associates with alpha6beta4 integrin, forming HD-like structures, called type II HDs. In the present study, we used an intestinal epithelial cell line that expresses HD1/plectin and the alpha6beta4 integrin to investigate the regulation of assembly of these proteins in type II HDs. These compounds were found to be clustered at sites of cell-ECM contact and their polarized localization was influenced by either cell confluency or extracellular matrix deposition. Conventional and immunoelectron microscopy showed that HD1/plectin and the beta4 integrin subunit are colocalized in an adhesion structure. Using cytoskeleton-disrupting drugs and confocal microscopy, we demonstrated that type II HDs are made up of numerous individual plaques whose assembly into a cluster requires actin filaments, but not microtubules.

Topics: Actin Cytoskeleton; Actins; Antigens, Surface; Cell Adhesion; Cell Count; Cell Size; Colonic Neoplasms; Cytochalasin B; Cytoskeleton; Desmosomes; Epithelial Cells; Extracellular Matrix; Humans; Integrin alpha6beta4; Integrins; Intermediate Filament Proteins; Intestinal Mucosa; Keratins; Microscopy, Confocal; Microscopy, Electron; Microtubules; Plectin; Pseudopodia; Tumor Cells, Cultured; Vinblastine; Vinculin

In order to standardize dual-fluorescence DNA flow cytometry using cytokeratin (CK) antibodies, normal colonic mucosa and tumor tissue were sampled from 308 colorectal surgical specimens. Fresh colon specimens were processed directly and stored frozen until dissociation. The samples were divided into aliquots for manual dissociation with tweezers and scalpel, and parallel dissociation with an automated disaggregation device (Medimachine, DAKO Diagnostika GmbH, Hamburg, Germany). An indirect immunofluorescence method with anti-cytokeratin antibodies and propidiumiodide was applied and measured on a single-laser flow cytometer (FACScan, Becton Dickinson [BDI], Heidelberg, Germany). Evaluation with CellFit (BDI) or MultiPlus (Phoenix Flow Systems, San Diego, CA) showed that dual-parameter fluorescence propidiumiodide (DNA staining) and fluorescein-isothiocyanate (cytokeratin labeling) provides a reasonable staining method for DNA analysis of epithelial cells. No significant differences in coefficient of variation in CK-gated versus ungated cells could be observed. Normal colon mucosa served as a reliable internal, diploid DNA control. Medimachine dissociation led to a significantly higher gain of cytokeratin-positive cells compared to percentage of cytokeratin-positive cells after manual tissue disaggregation. Cytokeratin gating led to a clear-cut separation of S-phase fractions within the respective ploidy groups, irrespective of manual or automated dissociation. The S-phase fraction increased significantly from normal tissue to diploid and nondiploid tumors. In general, automated tissue preparation with the Medimachine allows simple cell-isolation for dual DNA/CK-flow cytometric measurement, improving the gain of CK-positive cells, and facilitating a standardized DNA analysis.

Topics: Carcinoma; Colonic Neoplasms; DNA, Neoplasm; Flow Cytometry; Humans; Keratins; Prognosis; Staining and Labeling

To discriminate between adenocarcinomas that are primary to the ovary and metastatic to the ovary, especially of colonic and breast origin, by immunohistochemistry, using stepwise discriminant analysis or a decision tree.. 312 routinely processed, formalin fixed tissue specimens were used. The tumours were divided into a learning set (n = 159), composed of primary tumours of ovary, breast, and colon, and a test set, comprising 134 metastases from these sites and an additional 19 primary ovarian carcinomas. The immunohistochemical panel was composed of antibodies against cytokeratin 7 (CK7) and 20 (CK20), CA125, vimentin, carcinoembryonic antigen (CEA), gross cystic disease fluid protein-15 (GCDFP-15), and the oestrogen receptor (ER). The staining results of the tumours were expressed as the product of the staining intensity and the percentage of positive tumour cells. Analyses were first performed on the learning set and then evaluated on the test set.. Although the immunostaining patterns showed a considerable overlap between the three types of adenocarcinoma, the breast carcinomas were typically positive for GCDFP-15 and often for ER, and negative for vimentin. Ovarian carcinomas were always positive for CK7 and to a lesser extent for CA125. Colonic carcinomas showed prominent positivity for CEA and CK20, while no staining was seen for ER and vimentin. In discriminant analysis, six antibodies (alpha CK7, alpha CK20, alpha CA125, alpha CEA, alpha ER, and alpha GCDFP-15) appeared to be necessary for optimal classification: 89% of the learning set and 82% of the test set were classified correctly. In the decision tree, only four antibodies (alpha CK7, alpha CEA, alpha ER, and alpha GCDFP-15) were used to obtain a correct classification score of 89% for the learning set and 84% for the test set.. Using a semiquantitative assessment of the immunostaining results by a restricted panel of six antibodies with stepwise discriminant analysis, 80-90% of the adenocarcinomas of colon, breast, and ovary can be correctly classified. Discriminant analysis is computer aided and therefore an easy method and for each case a probability value of the classification result is obtained. The intuitive decision tree method provides a slightly better result, requires only four antibodies, and offers a more practical method for the surgical pathologist.

Topics: Adenocarcinoma; Apolipoproteins; Apolipoproteins D; Biomarkers, Tumor; Breast Neoplasms; CA-125 Antigen; Carcinoembryonic Antigen; Carrier Proteins; Colonic Neoplasms; Decision Trees; Diagnosis, Differential; Discriminant Analysis; Female; Glycoproteins; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Membrane Transport Proteins; Ovarian Neoplasms; Receptors, Estrogen; Vimentin

Tumor-associated antigens may be expressed as surface glycoproteins. These molecules undergo qualitative and quantitative modifications during cell differentiation and malignant transformation. During malignant transformation, incomplete glycosylation is common, and certain glycosylation pathways are preferred. These antigens might help distinguish between ovarian and colonic adenocarcinomas in the primary and metastatic lesions. Different cytokeratins have been proposed as relatively organ-specific antigens.. We used monoclonal antibodies against T1, Tn, sialosyl-Tn, B72.3, CA125, carcinoembryonic antigen, and cytokeratins 7 and 20 to detect tumor-associated glycoproteins and keratin proteins in ovarian and colonic carcinomas.. CA125, carcinoembryonic antigen, and cytokeratins 7 and 20 can distinguish between colonic and serous or endometrioid adenocarcinomas of the ovary in both primary and metastatic lesions. Mucinous ovarian adenocarcinomas differed in that they express carcinoembryonic antigen and cytokeratins 7 and 20 and weakly express CA125. The other glycoprotein antigens were equally expressed by ovarian and colonic adenocarcinomas and therefore were of no use in distinguishing between these 2 entities.. A panel of monoclonal antibodies against cytokeratins 7 and 20 antigens, CA125, and carcinoembryonic antigen is useful in differentiating serous and endometrioid adenocarcinomas of the ovary from colonic adenocarcinomas. Mucinous ovarian adenocarcinomas cannot be distinguished from colonic adenocarcinomas using immunohistochemistry.

Topics: Adenocarcinoma; Antibodies, Monoclonal; CA-125 Antigen; Carcinoembryonic Antigen; Colonic Neoplasms; Diagnosis, Differential; Female; Humans; Immunoenzyme Techniques; Keratins; Ovarian Neoplasms

Cytokeratin (CK) immunohistochemistry revealed changes in the CK19 immunoreactivity in human gastrointestinal epithelium during embryonic and fetal development. These changes were particularly marked in the jejunum and ileum. CK19 immunoreactivity was strong up to the 11th week of pregnancy, but was absent between weeks 12 and 17, and reappeared weakly from week 18 to week 24. This temporal pattern correlated with that of cell proliferation investigated by immunohistochemical detection of proliferating cell nuclear antigen. Marked CK expression was associated with a low proliferative rate and vice versa. To test whether these results were relevant to the assessment of intestinal metaplasia and the risk of malignant transformation with poor cell differentiation, adenomas and adenocarcinomas of the colon, intestinal metaplasia of the stomach, and two types of gastric carcinoma were also examined by CK19 immunohistochemistry. Substantial CK19 immunoreactivity was found in well-differentiated cancers and low-grade dysplasias with low cell proliferation, whereas only weak CK19 immunoreactivity was found in poorly differentiated carcinomas and high-grade dysplasias with a high proliferation rate.

Topics: Adenocarcinoma; Aging; Cell Differentiation; Cell Division; Colonic Neoplasms; Gastric Mucosa; Gastrointestinal Neoplasms; Humans; Ileum; Immunohistochemistry; Jejunum; Keratins; Proliferating Cell Nuclear Antigen; Reference Values; Stomach; Stomach Neoplasms

The keratin 18 (K18) gene is expressed at a normal level in cells of nontumorigenic clones derived from the SW613-S human colon carcinoma cell line, but is overexpressed in cells of tumorigenic clones. A high level of expression was also found in the cells from 10 of 15 other human colon carcinoma cell lines. The expression of the gene is downregulated in differentiating Caco-2 cells, resulting in a normal expression level. Determination of K18 mRNA half-life in growing and confluent Caco-2 cells indicated that this downregulation does not take place at a posttranscriptional level. The density of RNA polymerase molecules on the K18 gene, as measured in nuclear run-on experiments, is the same in growing and confluent Caco-2 cells, but the rate of synthesis of K18 transcripts in confluent Caco-2 cells, as determined by in vivo pulse-labeling, is 35% of that in growing cells. Nuclear run-on experiments carried out with nuclei prepared from growing or confluent Caco-2 cells treated with 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB) indicated that a reduction in both the initiation and elongation rates of RNA polymerase molecules occurs on the K18 gene in confluent Caco-2 cells. This leads to a decreased rate of K18 transcript production with no reduction in the polymerase density on the gene. Evidence is provided that the mechanisms responsible for the differential expression of the K18 gene between tumorigenic and nontumorigenic SW613-S cells and between growing and differentiating Caco-2 cells share some similarities.

Topics: Blotting, Northern; Caco-2 Cells; Cell Differentiation; Colonic Neoplasms; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Keratins; Plasmids; RNA, Messenger

Thirty-five chordomas and more than 100 other tumors that have to be considered in the differential diagnosis, were immunohistochemically analyzed using a panel of antibodies including those to subsets of keratins (K), HBME-1, a monoclonal antibody recognizing an unknown antigen on mesothelial cells, and neuroendocrine markers. The patterns of immunoreactivities in chordoma were compared with those in renal cell carcinoma, colorectal mucinous adenocarcinoma, pituitary adenoma, skeletal chondrosarcoma, and extraskeletal myxoid chondrosarcoma (ESMC). Chordomas were consistently positive for keratin cocktail AE1/AE3, and for the individual keratins K8 and K19, and nearly always positive for K5, but they showed negative or only sporadic reactivity for K7 and K20. The keratin K8 and K19 reactivity was retained in those chordomas showing solid sheets of epithelioid, spindle cells, or cartilaginous metaplasia, and in one of two cases showing overtly sarcomatous transformation. In comparison, keratins were never present in skeletal chondrosarcoma, although K8 and to a lesser extent K19 were seen in occasional cases of ESMC with chordoid features. HBME-1 reacted strongly with chordoma and skeletal chondrosarcoma but was almost never positive in renal or colorectal carcinoma. These carcinomas lacked K5-reactivity, in contrast to chordoma. Chordomas were also consistently positive for neuron-specific enolase and occasionally focally for synaptophysin, but never for chromogranin. In contrast, pituitary adenomas regularly expressed the full spectrum of neuroendocrine markers and differed from chordoma by having a narrower repertoire of keratins, often showing negative or focal keratin 8- or AE1/AE3 reactivity and being almost always K19-negative. These findings indicate that chordoma can be immunohistochemically separated from tumors that can resemble it. Immunohistochemistry is especially useful in the diagnosis of small biopsy specimens that offer limited material for morphological observation.

Topics: Adenocarcinoma, Mucinous; Adenoma; Antibodies, Monoclonal; Antigens, Neoplasm; Biomarkers, Tumor; Bone Neoplasms; Carcinoma; Chondrosarcoma; Chordoma; Colonic Neoplasms; Diagnosis, Differential; Humans; Immunohistochemistry; Keratins; Kidney Neoplasms; Pituitary Neoplasms

Villin (V) is a glycoprotein of microvilli associated with rootlet formation. Most colonic adenocarcinomas have a V positive (+), cytokeratin (CK) 20 (+), CK7-negative (-) immunophenotype; most lung adenocarcinomas have a CK20(-), CK7(+) immunophenotype. The reports of villin immunoreactivity in lung adenocarcinoma range from 6% to 68% in studies using various fixations and varied anti-villin antibodies. Some lung adenocarcinomas have microvilli with rootlets leading to possible diagnostic confusion with metastatic colonic adenocarcinoma to lung. Nine primary lung adenocarcinomas with rootlets on ultrastructure (including four bronchioloalveolar carcinomas [BAC]), four metastatic lung adenocarcinomas with rootlets, nine metastatic colon adenocarcinomas to lung, and 10 randomly selected lung adenocarcinomas without rootlets (including five BAC), were immunostained with monoclonal antibodies to villin (1D2C3), CK7 (OV-TL12/30), and CK20 (Ks20.8) using a streptavidin peroxidase technique with heat-induced epitope retrieval. All primary lung adenocarcinomas with rootlets were CK7(+) CK20(-), and six of nine (67%) were V(+). Cytoplasmic villin positivity occurred in a diffuse--five of nine (56%), focal--two of nine (22%), or brush border pattern--two of nine (22%). Two of four metastatic lung adenocarcinomas with rootlets were V(+). One metastatic lung adenocarcinoma had a CK7(+), CK20(+), V(-) phenotype. All metastatic colonic adenocarcinomas were V(+), CK20(+), CK7(-), and 1 of 10 (10%) lung adenocarcinomas without rootlets was V(+), and all 10 were CK20(-), and CK7(+). In summary, villin positivity is more common in lung adenocarcinoma with rootlets (67%) than those without rootlets (10%). AU primary lung adenocarcinomas were CK7(+), CK20(-). The combination of villin, CK 7, and CK 20 is helpful in differentiating metastatic colon adenocarcinoma from lung adenocarcinoma with rootlets.

Topics: Adenocarcinoma; Biomarkers, Tumor; Calcium-Binding Proteins; Carrier Proteins; Colonic Neoplasms; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Lung Neoplasms; Microfilament Proteins; Microscopy, Electron; Microvilli

We describe three cases of benign signet-ring cell aggregates in the colon associated with pseudomembranous colitis, adenomatous polyp of the colon and ulcerated mucosa of the gallbladder excised for gallstones. In all cases, we found loose, benign signet-ring cell aggregates overlying the ulcerated mucosa surface, simulating signet ring-cell carcinoma. The most important sign of the benign signet-ring cell aggregates is that they are always confined to the surface of the mucosa of the intestine or gallbladder mucosa or crypts of the intestinal epithelium. In no case did we see an invasion of these cells into the lamina propria of the mucosa. In all cases, the benign signet-ring cell aggregates were immunohistochemically positive with antibodies to cytokeratins. The occurrence of benign signet-ring cell aggregates is a rare and very misleading diagnostic pitfall which must be differentiated from signet-ring cell carcinoma of the colon and gallbladder.

Topics: Adenomatous Polyps; Carcinoma, Signet Ring Cell; Colonic Neoplasms; Diagnosis, Differential; Enterocolitis, Pseudomembranous; Female; Gallbladder Neoplasms; Humans; Immunohistochemistry; Intestinal Mucosa; Keratins; Male; Middle Aged

To discriminate adenocarcinoma metastases originating from either colon or ovary, a panel of immunohistochemical markers was evaluated. For this purpose, paraffin sections from 157 primary and metastatic colonic and ovarian carcinomas were immunostained. These cases were divided into a learning group of 46 colonic and 54 ovarian carcinomas and a test group of 29 colonic and 28 ovarian carcinomas, including all metastatic tumors, among which were five with unknown primary site at the time of testing. The sections were immunostained with antibodies against carcinoembryonic antigen (CEA), cytokeratin 7 (CK7), cytokeratin 20 (CK20), CA125, vimentin, and CA19.9. Staining results were expressed as the product of staining intensity and percentage of positive tumor cells. Stepwise discriminant analysis was applied on the learning set to obtain a classification function for both tumors. The validity of the classification function was evaluated using the test set. There was considerable overlap in immunostaining for both tumor types, but colonic carcinomas were typically positive for CEA and CK20 and negative for CK7 and CA125. Ovarian carcinomas were typically positive for CK7 and CA125 and negative for CEA and CK20. In discriminant analysis, the best combination of markers appeared to be CK7 and CEA. Only one sample of the test group (2%) was misclassified. Taking learning and test groups together, 136 of the 157 samples (87%) were correctly classified with high posterior probability (PP > .8). However, from the 28 mucinous ovarian carcinomas, only 19 (68%) could correctly be classified with high PP. When excluding the nonmucinous ovarian carcinomas from the analysis, overall 87 of 103 (84.5%) of the samples were correctly classified (PP > .8) with a combination of CEA, CK7, and also vimentin. From the 28 mucinous ovarian carcinomas, only two (7%) were misclassified, and four could not be classified with sufficient certainty. In neither analysis did CK20, CA125, or CA19.9 emerge as discriminatory parameters. Based on the same data, an intuitive flow chart was constructed with which 129 of 157 cases could be classified (only one falsely) without further statistical analysis. The five metastases with an at first unknown primary could, according to the follow-up, all be classified correctly with high PP. Most ovarian carcinomas, including the mucinous ones, can be discriminated with high probability from colonic carcinomas using a panel of three antibodies directed

Topics: Adenocarcinoma; Biomarkers, Tumor; CA-125 Antigen; CA-19-9 Antigen; Carcinoembryonic Antigen; Cell Count; Colonic Neoplasms; Decision Trees; Diagnosis, Differential; Discriminant Analysis; Female; Humans; Immunoenzyme Techniques; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Neoplasms, Unknown Primary; Ovarian Neoplasms; Retrospective Studies; Vimentin

Somatic mutations of the K-ras oncogene play an important role in colorectal carcinogenesis. We determined whether rat colon epithelial cells could be transformed by introducing retroviruses carrying the activated human K-ras oncogene alone. Primary epithelial cells from the rat distal colon were infected with retroviruses carrying wild-type and two types of activated K-ras (asp and val at codon 12) cDNAs. Cells infected with the wild-type K-ras virus showed no change in morphology and died within 3 weeks, whereas the activated K-ras virus-infected cells underwent morphological changes within 3 days and continued to proliferate. From these cells, several cell lines were subsequently established. Epithelial cells transformed by activated K-ras formed colonies in soft agar culture and tumors in athymic nude mice. Multiple copies of human K-ras genes and large amounts of K-ras mRNAs and proteins were found in the transformed cells. These data suggest that overexpression of activated K-ras transforms rat colon epithelial cells.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Colon; Colonic Neoplasms; Epithelial Cells; Female; Genes, ras; Humans; Keratins; Mice; Mice, Nude; Neoplasm Transplantation; ras Proteins; Rats; Rats, Inbred F344; Retroviridae; RNA, Messenger; Transfection

The aim was to investigate the significance of lymph node micrometastases in Dukes Stages A and B colorectal cancer.. Archival specimens were examined from 147 patients (96 colon, 51 rectum; 44 Stage A, 103 Stage B) who had surgery between 1987 and 1994. One lymph node section from each node (colon, 1-11; median, 4; rectum, 1-15; median, 3) was examined with use of an anticytokeratin antibody.. Forty-seven (32 percent) patients had micrometastases. At follow-up in June 1996, 23 patients had died of cancer or with known tumor relapse, after a median time of 28 (range, 5-67) months; 8 of 47 (17 percent) patients had micrometastases, 15 of 100 (15 percent) did not. No statistically significant differences were observed according to micrometastases when the results were analyzed with respect to Dukes stage or survival time. The median survival time of living patients with micrometastases was 48 (range, 18-97) months, and for patients without micrometastases, 48 (range, 19-111) months. Six of 96 living patients had a tumor relapse; three of these displayed micrometastases.. Lymph node micrometastases are not a useful prognostic marker in Dukes Stages A and B and do not imply different strategies for additional therapy or follow-up.

Topics: Adult; Aged; Aged, 80 and over; Colonic Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging; Rectal Neoplasms; Survival Analysis

Hemidesmosomes (HDs) mediate adhesion of epithelial cells to the extracellular matrix and have morphological associations with intermediate-size filaments (IFs). Hemidesmosomal molecular components including HD1, the two bullous pemphigoid antigens, and the integrin alpha 6 beta 4 have been identified in HDs of stratified and complex epithelium. In this study, we report that HT29-Fu cells, a human colonic tumor cell line, express two hemidesmosomal components (HD1, alpha 6 beta 4) associated in an adhesion structure termed type II HDs. Immunofluorescence studies showed a colocalization of HD1 and alpha 6 beta 4 in basal patches between actin stress fibers. Using cytochalasin B or vinblastine, two drugs which disrupt the cytoskeleton, we demonstrate that the redistribution of HD1 was probably induced by the reorganization of the basal cytokeratin network. We also show that in vitro HD1 binds to polymerized cytokeratin intermediate filaments; this suggests that HD1 in intestinal epithelial cells functions as a linker protein connecting cytokeratin filaments to the basal plasma membrane, probably through the beta 4 subunit of the integrin alpha 6 beta 4.

Topics: Actins; Adenocarcinoma; Cell Adhesion; Cell Differentiation; Cell Polarity; Colonic Neoplasms; Cytochalasin B; Cytoskeleton; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Intermediate Filament Proteins; Intermediate Filaments; Keratins; Neoplasm Proteins; Organelles; Tumor Cells, Cultured; Vinblastine

Topics: Aged; Biomarkers; Biopsy; Carcinoma; Carcinoma, Giant Cell; Colonic Neoplasms; Diagnosis, Differential; Female; Humans; Keratins

Previously, we have reported on successful imaging of colon, rectal, and pancreatic carcinomas in patients by using a radiolabeled all-human monoclonal antibody, COU-1, directed against modified cytokeratin. To further develop this antibody for use as an immunoconjugate, COU-1 was cloned by phage display selection and the human Fab fragment was expressed in bacteria. Analysis by confocal laser scanning microscopy demonstrated that COU-1 bound in a uniform punctate pattern to the surface of viable carcinoma cells stained at 4 degrees C, and binding increased significantly when cells were cultured on fibronectin, laminin, or collagen IV. In the case of fibronectin, COU-1 staining was particularly enhanced at intercellular junctions. When carcinoma cells were cultured with COU-1 at 37 degrees C for 6 hr, the antibody was found in large perinuclear vesicles and the punctate surface staining was significantly reduced. Similar results were obtained using intact IgM COU-1 and the recombinant Fab fragment. Immunohistological studies indicated that COU-1, in contrast to murine monoclonal antibodies against normal cytokeratin 8 and 18, could differentiate between malignant and normal colon epithelia, and between colon cancer metastasis in the liver and surrounding normal hepatocytes. Within biopsies of malignant tissue, COU-1 exhibited membrane-associated staining of proliferating cells, while resting cells had a filamentous pattern. Thus, modified cytokeratin at the surface of carcinoma cells may represent a new target for immunoconjugates and may explain the promising results of the phase I/II clinical study.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Bacteriophages; Binding Sites, Antibody; Cell Line; Colon; Colonic Neoplasms; Endocytosis; Humans; Immunoglobulin Fab Fragments; Intestinal Mucosa; Keratins; Molecular Sequence Data; Recombinant Proteins

Carcinosarcoma is a rare neoplasm that displays morphological features of both an adenocarcinoma and a sarcoma. The question is whether two tumors co-exist or whether the two morphological aspects represent sequential steps in tumor progression. We report a case of carcinosarcoma of the caecum in a young female. To characterize the two tumor cell populations and to gain insight into the pathogenesis of the lesion, we conducted immunohistochemical and ultrastructural analyses of the tumor. The biphasic aspect of the tumor showed an admixture of carcinoma and spindle-cell sarcomatoid areas. Both adenocarcinoma and sarcomatous cells were positive for cytokeratins. Vimentin was undetectable in the epithelial portion, but many of the sarcomatous cells stained for vimentin. Electron microscopic analyses of the sarcomatous portion revealed budding of "retroviral particles" from the rough endoplasmic reticulum cisternae. Our data support the contention that "carcinosarcoma" is a part of a single clinicopathological continuum with "spindle-cell carcinoma", the former being the biphasic expression of the neoplasia, the latter the monophasic expression; the presence of productive retroviral infection in the sarcomatous cells could constitute one of the additional support in tumor progression from the carcinomatous to the sarcomatous phase.

Topics: Adenocarcinoma; Adult; Biomarkers, Tumor; Carcinosarcoma; Cell Transformation, Neoplastic; Colonic Neoplasms; Disease Progression; Endoplasmic Reticulum, Rough; Fatal Outcome; Female; Humans; Keratins; Neoplasm Proteins; Organelles; Retroviridae; Sarcoma; Vimentin

This study used a co-culture system with Transwell tissue-culture inserts to investigate the role of primary cultures of rat peritoneal mesothelial cells on the proliferation of rat colon-carcinoma cells (CC531 cells). Mesothelial cells significantly inhibited the growth of CC531 cells, while, conversely, CC531 cells stimulated the growth of mesothelial cells. Receptor-binding studies demonstrated the presence of high-affinity IGF-I receptors on the mesothelial and CC531 cells. Both cell types also produced IGF-I, as measured by radioimmunoassay. IGF-I stimulated DNA synthesis in mesothelial cells, but had no effect on the growth of CC531 cells. In co-culture, it was found that IGF-I potentiated the inhibitory effect of mesothelial cells on CC531 cells. The effect of IGF-I on mesothelial-cell proliferation was additive to the stimulatory effect of CC531 cells. TGF-beta had no effect on the growth of the CC531 cells, suggesting that this growth (-inhibitory) factor is not involved in the inhibitory effect of mesothelial cells on CC531 cell growth. The study provides evidence for the existence of a paracrine loop between mesothelial and colon-carcinoma cells, giving more insight into the basic cellular mechanisms that may modulate the growth of intraperitoneal colon carcinoma. Inhibition of CC531-cell proliferation by rat mesothelial cells might explain the earlier finding that tumour cells grow poorly in a surgically uncompromised abdomen.

Topics: Animals; Cell Division; Cells, Cultured; Coculture Techniques; Colonic Neoplasms; Culture Media, Conditioned; Disease Models, Animal; Dose-Response Relationship, Drug; Epithelial Cells; Immunohistochemistry; Insulin-Like Growth Factor I; Keratins; Male; Paracrine Communication; Rats; Rats, Inbred Strains; Receptor, IGF Type 1; Transforming Growth Factor beta; Tumor Cells, Cultured; Vimentin; von Willebrand Factor

Carcinomas metastatic to the ovary are often difficult to distinguish from primary ovarian carcinomas. Adenocarcinoma of the colon may simulate both primary endometrioid and mucinous ovarian tumors. The histologic finding of "dirty" necrosis in association with a "garland" or cribriform pattern has been suggested as a useful feature in distinguishing metastatic colonic carcinomas from primary endometrioid ovarian carcinomas. This study was performed to determine the use of "dirty" necrosis in distinguishing primary ovarian carcinoma from metastatic colonic carcinoma by studying 71 of the former and 10 of the latter. At least focal dirty necrosis was found in 68% of primary ovarian epithelial cancers, including 92% of the endometrioid subtype, and in 100% of the metastatic colonic carcinomas. A subgroup of cases was evaluated immunohistochemically using cytokeratin (CK) 7, CK 20 and carcinoembryonic antigen (CEA). The phenotype of CK 7 +/CK 20-/CEA-was present in 92% of primary ovarian carcinomas studied, whereas, 90% of metastatic colonic carcinomas were CK 7-/CK 20 +/CEA+. The finding of dirty necrosis is not specific for metastatic colonic cancer, and differential cytokeratin immunostaining is a useful adjunct in this differential diagnosis.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoembryonic Antigen; Carcinoma; Colonic Neoplasms; Female; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratins; Middle Aged; Necrosis; Ovarian Neoplasms

Eight cases of a distinctive histological variant of bowel cancer characterized by an anaplastic morphology were identified from 2,650 colonic malignancies (0.3%). The tumors were histologically composed of sheets of anaplastic tumor cells with frequent atypical mitoses, absence of gland formation, and mucicarmine and periodic acid-Schiff (PAS) negativity. Positive immunostaining for cytokeratin and vimentin was observed in eight cases and for epithelial membrane antigen in three; whereas carcinoembryonic antigen, alpha-fetoprotein, S-100 protein, HMB-45 antimelanoma antigen, leukocyte common antigen, and neuroendocrine markers were uniformly negative. Ultrastructural examination demonstrated intercellular tight junctions, focal surface microvilli, and apical terminal webs or long rootlets of microfilaments supporting a colonic derivation. At the time of diagnosis, metastases to regional lymph nodes were found in seven cases and to the liver in six. All patients in this study died of tumor within 9 months. This report emphasizes a poorly recognized variant of colonic carcinoma, characterized by a high degree of anaplasia and malignant behavior. The differential diagnosis for these lesions is discussed.

Topics: Adenocarcinoma; Aged; Biomarkers, Tumor; Carcinoma; Carcinoma, Large Cell; Colonic Neoplasms; Diagnosis, Differential; Humans; Immunoenzyme Techniques; Keratins; Liver Neoplasms; Lymphatic Metastasis; Male; Microvilli; Middle Aged; Mucin-1; Survival Analysis; Tight Junctions; Vimentin

The detection of locally-disseminated disease is one of the principal goals of oncologic surgery. For this study, a hand-held, gamma-detecting probe was used intraoperatively to assess the extent of colorectal carcinoma in patients previously injected with radiolabeled antibody to the TAG-72 antigen (CC49); this technique is known as Radioimmunoguided Surgery (RIGS) (Neoprobe Corporation, Dublin, OH). RIGS-positive areas (i.e. those with increased signal over background) have previously been shown to contain carcinoma in a high proportion of cases. However, some RIGS-positive areas had no tumor detectable by clinical examination or routine histopathologic analysis. This study was undertaken to determine if the presence of occult metastases might account for this disparity.. A total of 57 regional lymph nodes (LN), resected from 16 patients with primary (9) or recurrent (7) colorectal carcinoma, were studied. The patients were injected with 125I labeled CC49 murine monoclonal antibody approximately 3 weeks prior to surgery. After routine histologic evaluation, the LN were analyzed for occult metastases; paraffin sections were cut at 5 levels (50 micron apart) and were examined by histology (hematoxylin and eosin stain [H & E]) and by immunohistochemistry (IHC) with a cocktail of monoclonal antibodies to cytokeratins.. Fifty-seven LN were included in this study; 17 were H & E-positive (i.e., contained tumor by routine histologic examination [overt tumor]), while 40 LN were H & E-negative (i.e., no evidence of tumor after routine histologic examination). Thirty-nine LN were RIGS-positive, but only 14 of these were H & E-positive. Of the 25 RIGS-positive/H & E-negative LN, 10 (40%) demonstrated the presence of occult metastases after serial section/IHC analysis. Thus, a total of 27 LN contained metastatic carcinoma (17 overt, 10 occult); routine histologic analysis was able to identify tumor in only 17 of these 27 LN (63%), while the probe signaled the presence of tumor in 24 of these LN (89%). None of the RIGS-negative/H & E-negative LN were found to have occult metastases (0/15). Specific immunoreactivity with CC49 antibody was observed in 5 of 15 RIGS-positive/H & E-negative LN in which no tumor could be identified by any method (histopathology or IHC. CC49 immunoreactivity was not observed in 15 RIGS-negative/H & E-negative LN.. The finding of a RIGS-positive LN had a significant association with the presence of tumor cells (P < 0.05). In this study, the RIGS procedure was more sensitive than clinical or histopathologic examination in detecting the regional spread of a tumor. Furthermore, in LN that showed no evidence of tumor by routine histopathologic examination, a positive RIGS reading was significantly associated with the presence of occult LN metastases (P < 0.01). This study is the first to demonstrate the detection of histologically occult tumor by a remote imaging device. RIGS assessment is a highly sensitive method for detecting occult tumor deposits, and may guide therapeutic intervention in patients with colorectal carcinoma.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Chi-Square Distribution; Colonic Neoplasms; Colorectal Neoplasms; Gamma Cameras; Glycoproteins; Humans; Immunohistochemistry; Iodine Radioisotopes; Keratins; Lymph Nodes; Lymphatic Metastasis; Neoplasm Metastasis; Potassium Iodide; Probability; Radiography; Radioimmunodetection; Recurrence; Reproducibility of Results

Recent studies have shown that various tumor cells accumulate ubiquitin (Ub)-conjugated proteins, the profiles of which differ from those of normal cells. To identify the Ub-conjugated proteins accumulated specifically by human carcinoma cells, a two-dimensional immunoblot analysis of 31 surgically resected human primary colorectal carcinoma tissues was performed using an anti-Ub monoclonal antibody, KM691. Two distinct Mr 42,000 and 45,000 proteins in the Triton X-insoluble fractions of carcinoma tissues reacted with this antibody, whereas only one Mr 45,000 protein reacted in normal tissues. The Mr 42,000 Ub-conjugated proteins were specific to carcinoma tissues from 25 patients (80.6%). One of the purified Mr 42,000 proteins was digested with Achromobacter protease I. This protein was identified as a cytokeratin 8 (CK 8) fragment based on both molecular mass determination and molecular mass searching of Achromobacter protease I-digested fragments of proteins registered in a protein sequence data base. Two-dimensional immunoblot analysis with an anti-CK 8 antibody confirmed that all of the Mr 42,000 proteins were CK 8 degradation products. These results demonstrate that human colorectal carcinomas specifically accumulate Mr 42,000 Ub-conjugated CK 8 fragments. This accumulation was observed frequently not only in advanced (18/22, 81.8%), but also in early stage cases (7/9, 77.8%), suggesting that it occurs even in the early stages of colorectal carcinoma progression.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Antibody Specificity; Base Sequence; Colonic Neoplasms; Female; Humans; Immunoblotting; Keratins; Male; Middle Aged; Molecular Sequence Data; Molecular Weight; Neoplasm Proteins; Rectal Neoplasms; Sigmoid Neoplasms; Ubiquitins

The histologic distinction between adenocarcinoma primary to the ovary and metastatic to the ovary can be difficult. In an effort to facilitate this distinction, we have evaluated the use of immunohistochemical techniques with antibodies to cytokeratins 7 and 20, along with antibodies to carcinoembryonic antigen. We studied 24 primary ovarian adenocarcinomas, 11 colonic adenocarcinomas metastatic to the ovary, and 10 primary adenocarcinomas of the colon. Four ovarian adenocarcinomas metastatic to the colon were also studied. All but one of the primary and metastatic colonic carcinomas were negative for cytokeratin 7, whereas all the primary and metastatic ovarian carcinomas were positive for cytokeratin 7. The tumors metastatic to the ovary were all positive for cytokeratin 20 and carcinoembryonic antigen. Among the primary ovarian carcinomas, none of six serous tumors, three of seven endometrioid tumors, and three of 11 mucinous tumors were positive for cytokeratin 20. Ten of the primary ovarian tumors were negative for carcinoembryonic antigen using both monoclonal and polyclonal antibodies. One of the ovarian tumors was negative for carcinoembryonic antigen with the monoclonal antibody but positive using the polyclonal antibody. Cytokeratin 7 is the most helpful marker in the distinction between primary ovarian carcinoma and colonic carcinoma metastatic to the ovary.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Biomarkers, Tumor; Carcinoembryonic Antigen; Colonic Neoplasms; Female; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratins; Ovarian Neoplasms

In this study, we demonstrated the clonal origin of trisomy for chromosome 7 in epithelial cells of colon neoplasia. By using the double-target fluorescence in situ hybridization (FISH) technique in frozen tissue sections that were also immunostained for keratin and vimentin, ratio analysis of FISH signals for chromosomes 7 and 17 could be performed in epithelial (cytokeratin-positive) or stromal (vimentin-positive) areas. The data demonstrated that trisomy for chromosome 7 is found exclusively in the epithelial compartments and not in the stroma of colon adenocarcinoma. We then demonstrated the occurrence of trisomy for chromosome 7 in the different types of epithelial neoplastic cells, i.e., columnar and goblet cells, which were isolated from frozen tissue sections by mechanical disaggregation of colon tissue and mild lysis of the cells while protease activity was inhibited. In these cell suspensions, the columnar cells were detected with an antibody to villin, and the goblet cells were stained for mucin, whereas all cells were subsequently subjected to FISH for chromosome 7. For analysis of neuroendocrine cells, which are present in a very low frequency in colon neoplasia, frozen tissue sections that were immunostained for Chromogranin A could be used. Individual neuroendocrine cells could be distinguished in these thin frozen tissue sections. The presence of trisomy for chromosome 7 in all three different epithelial cell types strengthens our suggestion that this chromosomal aberration is found in the epithelial stem cell compartment of colon neoplasia.

Topics: Adenoma; Carcinoma; Cell Line; Chromosome Aberrations; Chromosomes, Human, Pair 7; Clone Cells; Colonic Neoplasms; Epithelium; Humans; In Situ Hybridization, Fluorescence; Keratins; Trisomy; Vimentin

Serum CYFRA21-1 levels were studied in 127 cases of colorectal cancer. The positive rates for serum CYFRA21-1 were 34.6% in primary colorectal cancer. There was a significant correlation between the positive rates of serum CYFRA21-1 and liver metastases, peritoneal dissemination, lymph node metastases, or clinical stage. The survival rate for patients in the CYFRA21-1 positive group was lower than those with CYFRA21-1 negative group. Among patients who underwent curative operation, patients is the CYFRA21-1 positive group gave a recurrence rate of 26.6%, against 9.4% in the CYFRA21-1 negative group. There was no correlation between serum CYFRA21-1 levels and serum CEA levels. These findings suggest that Serum CYFRA21-1 levels may be a useful indicator in estimating the prognosis for colorectal cancer.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Biomarkers, Tumor; Colonic Neoplasms; Humans; Keratins; Liver Neoplasms; Lymphatic Metastasis; Neoplasm Invasiveness; Prognosis; Rectal Neoplasms; Survival Rate

The differentiation of ovarian metastases from colonic carcinoma and primary ovarian carcinoma can be difficult. To assess the utility of cytokeratin 7 and cytokeratin 20 immunostains in this setting, we studied routinely processed, formalin-fixed tissue from 165 ovarian tumors, including 45 serous carcinomas, 40 mucinous carcinomas, 64 endometrioid carcinomas, and 16 metastatic colonic adenocarcinomas with an avidin-biotin immunohistochemical technique. A cytokeratin 7+/cytokeratin 20- immunophenotype was seen in 86% of the endometrioid carcinomas, 27% of the mucinous carcinomas, 40% of the serous carcinomas, and none of the metastatic colorectal carcinomas. Conversely, a cytokeratin 7-/cytokeratin 20+ immunophenotype was seen in 94% of the metastatic colonic tumors, 5% of the mucinous carcinomas, and none of the endometrioid or serous tumors. We concluded that cytokeratin immunostains can be helpful in distinguishing metastatic colonic adenocarcinoma from primary ovarian carcinomas, particularly endometrioid carcinomas. Rare ovarian mucinous carcinomas may show the same immunophenotype as metastatic colonic carcinomas.

Topics: Adenocarcinoma; Biomarkers, Tumor; Carcinoma, Endometrioid; Colonic Neoplasms; Diagnosis, Differential; Female; Humans; Immunoenzyme Techniques; Keratins; Ovarian Neoplasms

Circulating cancer cells in the blood play a central role in the metastatic process. Their number can be very small and techniques for their detection need to be both sensitive and specific. Polymerase chain reaction (PCR) has been successfully used to detect small numbers of tumour cells in haematological cancer in which abnormalities in DNA are sufficiently consistent to make this possible. For most solid tumours this not yet feasible. However, we have found that reverse transcriptase (RT)-PRC for tissue-specific gene expression is a useful technique for identifying small numbers of circulating cells in melanoma and neuroblastoma patients. In this report we describe detection of colon carcinoma cells by RT-PCR using CK 20 mRNA as a marker. Unlike other cytokeratin genes examined (CK 8 and CK 19), CK 20 was not transcribed in normal haematopoietic cells. This suggests a role for RT-PCR in the detection of colon carcinoma metastasis in blood and bone marrow, using CK 20 as the target gene. Future analysis of clinical material will determine the clinical significance of this technique.

Topics: Adenocarcinoma; Base Sequence; Biomarkers, Tumor; Bone Marrow; Breast Neoplasms; Carcinoma, Transitional Cell; Colonic Neoplasms; Epithelium; Humans; Keratins; Molecular Sequence Data; Neoplasm Metastasis; Neoplasm Proteins; Neoplastic Cells, Circulating; Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Sensitivity and Specificity; Tumor Cells, Cultured; Urinary Bladder Neoplasms

We report on six patients with tumours of visceral organs (three patients with endometrial adenocarcinoma with squamous cell differentiation, one patient with atypical hyperplasia of endometrium, and two patients with adenocarcinoma of the colon with squamous cell differentiation), where unquestionable differentiation into shadow cells was observed. In all six cases the shadow cells were found mostly in the morules of immature squamous cells. The shadow cells were morphologically identical, on the light microscopical and ultrastructural level, to similar cells found in pilomatricomas. They were often accompanied by granulomatous giant cell reaction and calcification.

Topics: Adenocarcinoma; Adult; Carcinoma, Squamous Cell; Cell Differentiation; Colonic Neoplasms; Endometrial Neoplasms; Endometrium; Female; Humans; Hyperplasia; Keratins; Male; Microscopy, Electron; Middle Aged; Uterine Neoplasms; Uterus

Two human ovarian (OV-MZ-10, OV-MZ-15) and two colon cancer cell lines (CO-MZ-5, CO-MZ-6) were newly established in permanent cell culture. These cell lines have been maintained in vitro for 5-6 years, the passage number varying from 25 to 228. They were established from ascites or solid tumours at the time of primary surgery. By clinical and histopathological judgement alone all four cell lines would have been interpreted as ovarian cancer cell lines. Morphological criteria or the expression of the tumour-associated antigens CA-125 and CEA allowed no differential diagnosis. Only the analysis of the expression of different cytokeratins and vimentin enabled us to verify the different origin of the cell lines. Ovarian cancer cell lines, in contrast to the colon cancer cell lines, are positive for the expression of cytokeratin (CK) 7 and for vimentin. CK 20 proved to be the marker with the best discrimination. CK 20 was found exclusively in the colon carcinoma cell lines, but not in the ovarian carcinoma cell lines. The evaluation of cytokeratin expression is a helpful diagnostic modality in differentiating between adenocarcinoma cell lines derived from ovarian and colon tumours.

Topics: Adult; Aged; Animals; Antigens, Tumor-Associated, Carbohydrate; Carcinoembryonic Antigen; Cell Division; Cell Line; Colonic Neoplasms; Culture Techniques; Female; Humans; Keratins; Male; Mice; Mice, Nude; Microscopy, Electron; Microscopy, Electron, Scanning; Middle Aged; Ovarian Neoplasms; Transplantation, Heterologous; Tumor Cells, Cultured

The filament forming ability and post-translational modifications of the human intermediate filaments, keratin polypeptides 8 and 18 (K8/18), were studied in recombinant baculovirus-infected insect (Spodoptera frugiperda, Sf9) cells. No change in cell morphology was noted after high levels of K8/18 were expressed in Sf9 cells coinfected with recombinant virus-containing human K8 and K18. Immunofluorescence staining showed that K8/18 expressed in Sf9 cells formed somewhat what disorganized and rope-like filaments, in contrast with K8 or K18 expression alone, which did not result in filament formation. K8/18 expressed in Sf9 cells were glycosylated (O-linked N-acetylglucosamine) and phosphorylated, and each modification occurred on different molecules of K8 and K18, as previously found in human HT29 cells. The glycosylation and phosphorylation of K18 in human and insect cells were very similar as determined by tryptic peptide mapping and localization to the head and proximal rod domains. In contrast, differences were noted in the relative intensity of the tryptic phospho- and glycopeptides of K8 expressed in human and insect cells and in the ratio of K8 to K18 phosphorylation in human and insect cells. Our results show that although quantitative differences exist, the post-translational modification of K8/18 expressed in insect cells is quite similar to its mammalian counterpart, especially for K18. Baculovirus expressed K8/18 should prove useful for mapping phosphorylation and glycosylation sites and for studying factors involved in organized filament assembly in mammalian cells.

Topics: Adenocarcinoma; Amino Acid Sequence; Animals; Cell Line; Colonic Neoplasms; Female; Fluorescent Antibody Technique; Gene Expression Regulation; Genes, Insect; Glycosylation; Humans; Keratins; Molecular Sequence Data; Moths; Ovary; Peptide Mapping; Phosphorylation; Protein Processing, Post-Translational; Tumor Cells, Cultured

Keratin is a member of the intermediate filament family in epithelial cells. Two-dimensional gel electrophoresis of different epithelial cells has shown 20 different keratin polypeptides. Therefore, mapping of the keratin polypeptides can be used to define a specific tissue.. Cytokeratin expression was investigated by using monoclonal antibodies in human surgical specimens and autopsy material of pancreatic, gastric, liver, and colon carcinomas and cholangiocarcinomas, and their metastasis to lymph nodes and liver was examined. In addition, rat acinar cell carcinomas were used to compare cytokeratin expression in ductal vs. acinar cell pancreatic carcinomas.. Human pancreatic ductal carcinomas expressed keratins 7, 8, 18, and 19, whereas the majority of rat acinar carcinomas did not express keratins typical for ducts in rat pancreas. The keratin patterns of gastric and colon carcinomas were identical with keratins 8, 18, and 19. In contrast, hepatocellular carcinomas expressed the same keratin pattern as pancreatic acinar carcinomas with keratins 8 and 18, whereas cholangiocarcinomas expressed keratin 7, 8, 18, and 19, similar to pancreatic ductal carcinomas. Metastasis of pancreatic ductal and colon carcinomas retained their keratin patterns.. Keratin polypeptide typing of unknown malignant cells can be a useful tool for cell identification.

Topics: Animals; Azaserine; Carcinoma, Acinar Cell; Carcinoma, Ductal, Breast; Colonic Neoplasms; Epithelium; Gastrointestinal Neoplasms; Humans; Immunohistochemistry; Intermediate Filaments; Keratins; Male; Neoplasms, Experimental; Pancreatic Neoplasms; Rats; Rats, Inbred Lew; Stomach Neoplasms

To obtain a better understanding of the cellular and hormonal mechanisms responsible for the malignant bone resorption associated with metastatic carcinoma, we sought to identify whether tumor cells or tumor infiltrating macrophages were capable of lacunar bone resorption.. Tumor cells and tumor-infiltrating macrophages (TIMS), (nonspecific esterase and F4/80 positive: cytokeratin and tartrate-resistant acid phosphatase and calcitonin response negative), were isolated from carcinomas that developed after subcutaneous implantation of human breast, colon, and cervical carcinoma cell lines into MFI athymic nude mice. These cells were cultured alone or with stromal cells on bone slices and evidence of lacunar resorption sought by scanning electron microscopy.. After 7 to 14 days in co-culture with UMR106 osteoblast-like cells in the presence of 1,25-dihydroxy vitamin D3, only cells of the TIM population differentiated into osteoclast-like cells (nonspecific esterase-negative: tartrate-resistant acid phosphatase-positive) capable of extensive lacunar bone resorption.. Cells within the TIM population but not tumor cells are capable of differentiation into osteoclast-like cells which can resorb bone extensively. Both 1,25 dihydroxyvitamin D3 and bone stromal cells are necessary for this to occur. TIM differentiation into cells capable of lacunar resorption could account for a component of the extensive osteolysis associated with carcinomatous skeletal metastases.

Topics: Adenocarcinoma; Animals; Bone Resorption; Breast Neoplasms; Calcitonin; Calcitriol; Carcinoma, Squamous Cell; Cell Differentiation; Cells, Cultured; Colonic Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Macrophages; Mice; Mice, Nude; Microscopy, Electron, Scanning; Neoplasm Transplantation; Osteoblasts; Osteoclasts; Time Factors; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Adenocarcinomas of uncertain origin are a frequent problem for surgical pathologists. To determine the utility of immunostaining for cytokeratin 7 and cytokeratin 20 in the separation of pulmonary adenocarcinomas from colonic adenocarcinomas, we studied routinely processed, formalin-fixed tissue from 151 of these tumors using commercially available monoclonal antibodies and an avidin-biotin immunohistochemical technique. Used alone, neither cytokeratin 7 immunostaining or cytokeratin 20 immunostaining reliably separated these tumors. However, the immunophenotype of cytokeratin 7 positive/cytokeratin 20 negative was seen in 86% of the pulmonary adenocarcinomas, and in 0% of the colonic adenocarcinomas. Conversely, the cytokeratin 7-negative/cytokeratin 20-positive immunophenotype was seen in 77% of the colonic carcinomas, and in 0% of the pulmonary tumors. In conclusion, cytokeratin 7/cytokeratin 20 immunostaining patterns may be helpful in separating pulmonary adenocarcinomas from colonic adenocarcinomas.

Topics: Adenocarcinoma; Colonic Neoplasms; Diagnosis, Differential; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratins; Lung Neoplasms

DNA flow cytometry measurements were performed using cytokeratin as a second parameter to identify epithelial cells selectively in fresh and in archival paraffin samples of normal and adenocarcinoma tissues from breast and colon. Fresh specimens consisted of 22 adenocarcinomas of breast, 20 adenocarcinomas of colon, 16 control breast samples, and 13 control colon samples. Paraffin block specimens consisted of 22 adenocarcinomas of breast (the same as fresh samples), 20 adenocarcinomas of colon (the same as fresh samples), 37 control breast samples and 34 control colon samples. The average proportion of cytokeratin-positive cells per group ranged from 31 to 55% for fresh samples and from 14 to 34% for paraffin samples. For aneuploid cells populations of adenocarcinomas, which consist only of epithelial cells, the average percentage of cytokeratin-positive cells ranged from 60 to 72%. The technique gave satisfactory measurements of ploidy and of cell cycle data in both types of samples. Cell cycle measurements were less accurate than ploidy measurements in both types of samples, and multiple sampling will be required for adequate accuracy. The average S-phase fraction of cytokeratin-positive cells ranged from 6 to 15% for fresh specimens and from 11 to 20% for paraffin samples. Similar data were obtained for the proliferative index (G1 + S + G2 + M phases). The coefficients of variation were smaller for proliferative index than for S-phase fraction data, indicating greater accuracy. Paraffin data give higher cycling cell measurements than corresponding fresh data, so separate standardization of measurements may be required for fresh and for paraffin data.

Topics: Adenocarcinoma; Breast Neoplasms; Colonic Neoplasms; DNA, Neoplasm; Epithelium; Flow Cytometry; Humans; Keratins; Paraffin Embedding; Staining and Labeling

Mucinous adenocarcinomas of the ovary were studied immunohistochemically for cytokeratins 7 and 18, either to determine whether the ovarian tumor was primary or a metastasis or to establish the histogenetic origin of the tumor. Primary ovarian tumors were strongly positive for both cytokeratins, while ovarian metastases from colonic cancers were negative for cytokeratin 7, as were the colonic cancers. Three of four ovarian tumors complicated by pseudomyxoma peritonei were negative for cytokeratin 7, indicating appendiceal origin. Two of seven mucinous tumors associated with dermoid cysts were negative for cytokeratin 7, suggesting gastrointestinal origin.

Topics: Adenocarcinoma, Mucinous; Adult; Aged; Colonic Neoplasms; Diagnosis, Differential; Female; Humans; Immunohistochemistry; Keratins; Middle Aged; Ovarian Neoplasms; Pseudomyxoma Peritonei

Cryosections of normal colon (NC), tubular and villous adenomas (TA, VA), and variably differentiated colon adenocarcinomas (CA) were immunostained with monoclonal antibodies to alpha 1-6 and alpha v, and beta 1-4 integrin subunits; select samples were stained for cytokeratin (Ck) 20 and villin. In NC, alpha 2 staining was strongest in crypt cells; alpha 1,3 and alpha v, and beta 1,3 and beta 4, and Ck 20 and villin predominated in superficial enterocytes. In TA and VA, monolayered glands showed integrin, Ck 20 and villin patterns that differed slightly from both crypt and superficial enterocytes. Complex glands in VA showed decreased integrin staining and basal polarization; Ck 20 and villin were strong only in luminal cells. CA showed overall weaker integrin staining than adenomas. Regardless of invasion depth, well formed malignant glands mimicked TA; pleomorphic glands mimicked VA with focal basal integrin polarization and solid clusters displayed scanty integrins, uneven Ck 20, and villin in occasional cells. Diverse integrins in crypt compared with superficial enterocytes reflect changing adhesive requirements as cells migrate and terminally differentiate. Decreasing expression and altered distribution of integrins, Ck 20 and villin noted in TA, VA, and in CA of increasing grade indicate that certain adhesive and cytoskeletal features more closely relate to glandular architecture than to depth of invasion.

Topics: Adenocarcinoma; Adenoma; Antibodies, Monoclonal; Carcinoma; Carrier Proteins; Cell Differentiation; Colon; Colonic Neoplasms; Epithelium; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; Integrins; Keratins; Microfilament Proteins; Tissue Distribution

Immunocytochemistry was used in parallel with conventional cytology to detect circulating malignant epithelial cells in 42 patients undergoing resection for colorectal cancer. Preoperative peripheral and peroperative mesenteric venous blood samples were taken. Tumour cells were isolated on a density gradient and cytospins prepared. Slides were stained by conventional cytology (May-Grünwald-Giemsa) and by an indirect immunoperoxidase technique with the anticytokeratin antibody KG8.13. Using conventional cytology, definite morphological evidence of malignancy was observed in three patients and suspicious features in a further seven. Immunocytochemistry confirmed these findings in all three of the malignant but in only one of the suspicious cases. Counts of immunostained cytospins showed the concentration of tumour cells in blood samples from these four patients to be in the range 0-954 cells/ml. This study supports the use of immunological markers to detect and enumerate malignant cells. This method provides a powerful tool to investigate one aspect of the metastatic process.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Cell Count; Colonic Neoplasms; Female; Humans; Immunoenzyme Techniques; Intraoperative Period; Keratins; Male; Mesenteric Veins; Middle Aged; Neoplastic Cells, Circulating; Rectal Neoplasms

Inositol hexaphosphate (InsP6 or phytic acid) has been shown to have antineoplastic action in in vivo models of colon carcinogenesis. We therefore investigated its effect on proliferation and differentiation of the human colon cancer cell line HT-29 in vitro. Proliferation was evaluated by neutral red incorporation assay, and differentiation was assessed by expression of the markers, cytokeratin, carcinoembryonic antigen (CEA) and beta-D-galactose-[1-->3]-N-acetyl-galactosamine (Gal-GalNAc). InsP6 in the culture media (0.66-10 mM) inhibited cell proliferation in a dose-dependent manner (P < 0.001), while inositol or inositol hexasulfate used as controls or media without InsP6 did not show any suppressive effect. The expression of the tumor marker, Gal-GalNac, was augmented (100.7% increase) by low dose (0.66 mM) of InsP6 but was subsequently suppressed with higher concentrations of InsP6. The expression of cytokeratin and CEA were both augmented by either InsP6 or inositol at all concentrations tested, although the degree of augmentation was milder with inositol than with InsP6. The combination of InsP6 and inositol (both 0.66 mm) resulted in augmentation (P < 0.001) of cytokeratin expression, while that of CEA remained unchanged. The inhibitory effect of InsP6 on cell proliferation was not altered by combination with additional inositol at any concentrations tested. Our results show that InsP6 inhibits cell proliferation and concomitantly increases differentiation and is therefore a candidate chemopreventive and chemotherapeutic agent for human large intestinal cancer.

Topics: Adenocarcinoma; Carcinoembryonic Antigen; Cell Differentiation; Cell Division; Colonic Neoplasms; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Edetic Acid; Galactosamine; Humans; Keratins; Phytic Acid; Tumor Cells, Cultured

We studied the solubility of keratin polypeptides 8 and 18 (K8/18), which are the predominant intermediate filaments in the human colonic epithelial cell line HT29. We find that asynchronously growing cells (G0/G1 stage of the cell cycle) have a substantial pool of soluble keratin that constitutes approx. 5% of total cellular keratin. This soluble keratin pool was observed after immunoprecipitation of K8/18 from the cytosolic fraction of cells disrupted using three detergent-free methods. Several other cell lines showed a similar significant soluble cytosolic K8/18 pool. Arrest of HT29 cells in G2/M stage of the cell cycle was associated with a concurrent increase in keratin solubility. Comparison of K8/18 obtained from the soluble cytosolic fraction and the insoluble high-speed pellet fraction showed similar levels of phosphorylation and glycosylation and similar tryptic radiolabeled phospho- and glycopeptide patterns. Soluble K8/18 can form characteristic 10 nm filaments in vitro as determined by electron microscopy. Cross-linking of soluble K8/18 followed by immunoprecipitation resulted in dimeric and tetrameric forms, based on migration in SDS-polyacrylamide gels. In addition, cross-linked and native soluble K8/18 showed similar migration on nondenaturing gels and similar sedimentation after sucrose density gradient centrifugation. Our results indicate that simple epithelial keratins are appreciably more soluble than previously recognized. The soluble keratin form is assembly competent and appears to be primarily tetrameric. Although K8/18 solubility was found to increase during mitotic arrest, glycosylation and phosphorylation did not play an obvious role in generating the soluble fraction, suggesting an alternate mechanism for keratin solubility.

Topics: Adenocarcinoma; Animals; Cell Line; Colonic Neoplasms; Epithelium; Glycosylation; HeLa Cells; Humans; Intermediate Filaments; Interphase; Keratins; Kidney; Macropodidae; Metaphase; Neoplasm Proteins; Phosphorylation; Protein Processing, Post-Translational; Solubility; Tumor Cells, Cultured

Keratins are a subgroup of cytoskeletal intermediate filament proteins found in most epithelial cells. Some reports have suggested that keratins may be found on the cell surface as well as their well-accepted cytoskeletal location. A major part of the evidence in the interpretation of cell surface expression of keratins is cell surface radioiodination. Here we show that lactoperoxidase-catalyzed iodination of colonic and breast tissue culture cells results in radiolabeling of the keratins when cells are manipulated. No labeling of keratins is detected when cells are labeled directly on the tissue culture dish. A similar result was obtained when intact cells were biotinylated using water-soluble sulfo-NHS-biotin. Partitioning of the keratins to a soluble and an insoluble pool after "cell surface" 125I-labeling showed that both pools became iodinated. Indirect immunofluorescence showed that binding of a panel of anti-keratin antibodies to intact epithelial cells occurs only on the cells that are more adherent, which are the cells that require longer manipulation to remove from the tissue culture dish. Taken together, our results indicate that the reported expression of cell surface keratins in some cells likely reflects intracellular keratins. In addition, the method of epithelial cell handling can dramatically alter the leakiness of cell surface iodination techniques.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Biotin; Breast Neoplasms; Cell Membrane; Cell Membrane Permeability; Colonic Neoplasms; Cytoskeleton; Fluorescent Antibody Technique; Humans; Intermediate Filaments; Iodine Radioisotopes; Keratins; Methods; Precipitin Tests; Specimen Handling; Tumor Cells, Cultured

The distribution pattern of cytokeratin (CK) subtypes, an intermediate filament of cytoskeleton, was examined in adenomas and carcinomas of the colon and rectum. For the detection of the cytokeratin subtypes, monoclonal antibodies to the 54 Kd keratin polypeptide (CK No. 7 according to Moll's classification), 52.5 Kd (CK No. 8), 45 Kd (CK No. 18), and 40 Kd (CK No. 19) were used for immunohistochemical observation. Although No. 7 was positive in normal mucosa and adenoma with mild to moderate atypia, it could not be recognized in carcinoma. On the other hand the expression of No. 18 was confirmed in carcinoma, adenoma, and normal mucosa, and there were some differences in its distribution pattern in those with or without glandular formation and in areas showing infiltration of tumor cells. No. 18 expression was on the luminal side of normal colonic mucosa, adenoma, and well-differentiated adenocarcinoma; in the infiltrating area its reactivity was localized diffusely in the cytoplasm of tumor cells showing moderately or poorly differentiated adenocarcinoma cells. As to No. 8 and No. 19, they were recognized in normal mucosa, adenoma, and carcinoma. These results suggested the intimate relationship between expression of CK subtypes, cellular differentiation, and structural differentiation of colorectal carcinoma.

Topics: Adenocarcinoma; Adenoma; Colon; Colonic Neoplasms; Humans; Immunoenzyme Techniques; Intestinal Mucosa; Keratins; Rectal Neoplasms; Rectum

Cytokeratin-type intermediate filaments are a polygenic family of insoluble proteins that vary according to cell of origin and have been proposed as potentially useful markers of differentiation in epithelial malignancies. Because gastrointestinal malignancies resemble each other in their expression of many soluble antigens, we compared the cytokeratins of seven colonic, three gastric, six pancreatic, and one duodenal carcinoma cell lines, and one colon villous adenoma cell line. Cytokeratins were characterized by one- and two-dimensional gel electrophoresis, immunoblotting, and immunocytochemistry. These cell lines expressed combinations of cytokeratins 7, 8, 18, and 19, which are typical of the "simple" epithelial pattern found in normal ductal and glandular tissues of the gastrointestinal tract. However, pancreatic carcinoma cell lines expressed additional cytokeratins that are normally found in stratified squamous epithelium and epidermoid (squamous cell) carcinomas. These additional cytokeratins consisted of cytokeratin 16 in all six cell lines and cytokeratins 4, 13, and 16 in one cell line. These results suggest that cytokeratin patterns represent stable markers that may aid in distinguishing gastrointestinal malignancies.

Topics: Adenocarcinoma; Animals; Cell Line; Colonic Neoplasms; Diagnosis, Differential; Humans; Keratins; Mice; Pancreatic Neoplasms; Stomach Neoplasms; Tumor Cells, Cultured

Short-term maintenance of fetal rat colonic tissue in vitro has been demonstrated using a collagen matrix organ culture system. The introduction of single (v-myc, v-rasH, v-src) oncogenes or combinations of oncogenes (v-myc/rasH, v-myc/src) into normal colon mucosal elements was established using retroviral vectors, resulting in enhanced proliferation and migration of epithelial cells from the lumen of tissue implants. Expression of a single oncogene in normal colon epithelium did not result in the establishment of cell lines. In contrast, expression of cooperating oncogenic elements resulted in cell lines in greater than 80% of experiments, revealing different morphological characteristics dependent upon the oncogene combination used. Confirmation of the expression of viral transcripts was determined using Northern blot analysis and viral oncoprotein expression using Western blot analysis (p21) and an immunoprecipitation kinase assay (src). Expression of keratin filaments was lost following passaging of cell lines but could be induced by the myc/ras transformants by growth on Rat-1 feeder layers. This induction phenomenon was not observed with myc/src lines, and although these expressed high levels of sucrase isomaltase the epithelial origin of these cells is unclear. Karyotypic analysis performed on three myc/ras-transformed cell lines revealed a normal chromosome complement associated with transformation. In this report we describe a novel in vitro transformation system for normal rat colonic epithelium mediated by the introduction of oncogene elements using different retroviral vector constructs. The potential to generate cell lines representing different stages of neoplastic progression using relevant genetic components presents significant advantages for the study of cellular and molecular interactions underlying colon neoplastic progression.

Topics: Animals; Blotting, Northern; Blotting, Western; Carrier Proteins; Cell Transformation, Neoplastic; Colon; Colonic Neoplasms; CSK Tyrosine-Protein Kinase; Fluorescent Antibody Technique; Gene Expression; Genes, myc; Genes, ras; Genes, src; Karyotyping; Keratins; Membrane Glycoproteins; Microfilament Proteins; Oncogenes; Organ Culture Techniques; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-myc; Proto-Oncogene Proteins p21(ras); Proto-Oncogene Proteins pp60(c-src); Rats; Rats, Inbred WF; RNA; src-Family Kinases; Sucrase-Isomaltase Complex

In the present study we investigated the cytokeratin (CK) polypeptide expression in gastric and colonic adenocarcinomas. A battery of monoclonal anti-cytokeratin-specific antibodies and anti-vimentin were used. While the majority of cases displayed simple epithelial characteristics, in three of 17 cases of gastric adenocarcinomas and in one of 20 cases of colonic adenocarcinomas, CK polypeptides 13 (54 kd) and 16 (48 kd) were occasionally detected. These CK polypeptides, characteristic of squamous nonkeratinizing epithelia, were found in cases in which no evidence of squamous differentiation could be demonstrated by histologic examination. We believe that the presence of these unique CK polypeptides points to the squamous differentiation potential of the tumor cells.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Colonic Neoplasms; Fluorescent Antibody Technique; Humans; Keratins; Peptides; Stomach Neoplasms

Subclones of the SW 613-S human colon carcinoma cell line differ by their ability to induce tumors in nude mice and by their level of amplification of the c-myc gene. Clones with a high level of amplification are tumorigenic in nude mice whereas those with a low level are not. Genes overexpressed in the tumorigenic clones as compared to the nontumorigenic ones were searched by differential screening of a cDNA library. Two cDNA clones corresponding to cytokeratin K18 and ferritin-H chain were isolated. The steady state level of the corresponding mRNAs is higher in cells of all tumorigenic clones. The level of cytokeratin K8 mRNA, the specific partner of cytokeratin K18 in intermediate filaments of epithelial cells, is also elevated in these cells. For all three genes, this is mainly due to an increase in the transcription rate, as shown by a nuclear run-on assay. Immunoblotting experiments showed that cytokeratins K8, K18, and K19 are more abundant in cells of tumorigenic clones. The mRNA of the other subunit of apo-ferritin (ferritin-L chain) is expressed at the same level in both types of clones. The mRNAs of cytokeratins K18 and K8 and of ferritin-H chain are also overexpressed in cells of nontumorigenic clones which have acquired a tumorigenic phenotype after transfection of c-myc gene copies.

Topics: Base Sequence; Clone Cells; Colonic Neoplasms; Ferritins; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Keratins; Molecular Sequence Data; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured

A total of 113 patients with carcinomas of breast, testis, stomach and colon were examined for lymph node metastases by means of an exact case-by-case comparison by conventional histology and by immunohistochemistry using the anticytokeratin monoclonal antibody A45-B/B3. Among 891 examined lymph nodes, 90% of metastases were recognized by both methods, about 2% by histology alone, and more than 10% by immunohistochemistry alone. The method can be applied for intraoperative frozen section diagnosis.

Topics: Breast Neoplasms; Colonic Neoplasms; Female; Frozen Sections; Humans; Immunohistochemistry; Keratins; Lymph Nodes; Lymphatic Metastasis; Male; Paraffin; Stomach Neoplasms; Testicular Neoplasms

We report a modification of the avidin-biotin-peroxidase complex (ABC) technique for the light and electron microscopic detection of antigens in tissue sections. An immunological approach was used instead of the DAB reaction to reveal ABC bound to antigen-antibody complexes. Affinity-purified polyclonal antibodies against horseradish peroxidase were complexed to particles of colloidal gold and applied for reaction with the horseradish peroxidase molecules of the ABC. For light microscopic immunolabeling, the signal produced by the anti-horseradish peroxidase antibody-gold complex required silver intensification. The ABC immunogold reaction as compared with the standard ABC technique, in particular with silver intensification of the DAB reaction product, provided superior resolution in paraffin sections. Furthermore, section pre-treatment to block endogenous peroxidase activity could be omitted and no potentially hazardous substrate was used. The ABC immunogold reaction was successfully applied for electron microscopic immunolabeling on Lowicryl K4M thin sections. We propose that the ABC immunogold reaction is a useful alternative to the standard ABC technique and can be equally well applied to light and electron microscopy.

Topics: Adenocarcinoma; Carcinoembryonic Antigen; Colon; Colonic Neoplasms; Horseradish Peroxidase; Humans; Immunoenzyme Techniques; Immunohistochemistry; Keratins

Mouse monoclonal anticytokeratin antibodies (BA 16; BA 17; CO 8.2; C18.2; C 10; LE 41) were used for examination of 5 patients with large bowel carcinoma ("left colon") using the indirect immunoperoxidase technique. For each case cryostat sections were examined from three localizations: primary tumour of large bowel, mucosa in the vicinity of the tumour, and mucosa of distant part from the primary tumour. The antibodies BA 16 and BA 17 produced strongly positive results. Antibody C 18.2 seems to be less suitable for detection of cytokeratin expression in this part of large bowel. The obtained results indicate that no significant differences were found in expression of cytokeratins in the three localizations by histologically well or moderately differentiated adenocarcinoma.

Topics: Adult; Aged; Antibodies, Monoclonal; Colonic Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Male; Middle Aged

Three patients are described who presented with a large colonic adenoma in which a solid, undifferentiated carcinomatous component was found on microscopic examination. Despite small size (1.0 and 1.5 cm) and submucosal location in two cases, the tumours had metastasized to regional lymph nodes and the liver and death ensued at 4, 11 and 18 weeks after surgery. Immunocytochemistry was positive for carcino-embryonic antigen, low molecular weight cytokeratins and neuron specific enolase in all three cases and scanty dense core granules of neurosecretory type were found in one of two examined by electron microscopy. These 'neuroendocrine' carcinomas are compared with 'pure' adenomas and 'ordinary' poorly differentiated adenocarcinomas of the colon from which they differ, mainly by lack of glandular differentiation and mucus secretion, although two adenocarcinomas also showed patchy reactivity for neuron specific enolase. The term 'neuroendocrine' may be disputed but is now well established to describe a tumour that runs a uniquely aggressive course and for which radical surgery alone cannot provide a cure.

Topics: Adenocarcinoma; Adenoma; Aged; Aged, 80 and over; Carcinoma, Small Cell; Cell Differentiation; Colonic Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Male; Middle Aged; Phosphopyruvate Hydratase

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

We have previously characterized the diversity of cellular responses to transforming growth factor (TGF)-beta 1 from human colon carcinoma cells. We now show that morphological alteration and part of the growth-inhibitory responses elicited by growth factor (GF) are associated with the secondary effect of the induction of synthesis of extracellular matrix (ECM) glycoproteins. Specifically, morphological alteration is associated with the ECM glycoprotein laminin, and growth inhibition is associated with both laminin and fibronectin. Both TGF-beta 1 and TGF-beta 2 down-modulate the expression of nucleolar protein B23 (also known as numatrin or nucleophosmin, a positive regulator of cell proliferation). With one exception, the biological effects of both TGF-beta 1 and TGF-beta 2 on these human colon carcinoma cell lines are identical. Both GFs up-modulate the expression of carcinoembryonic antigen (CEA) and CEA-related gene products. However, some of these products are differentially regulated by TGF-beta 1 and TGF-beta 2. The differences in the profile of the induction of these CEA and CEA-related gene products, in the responsive cells, functionally distinguish TGF-beta 1 from TGF-beta 2. Finally, we identified and characterized some of the cellular proteins, the expression of which is up-modulated by GF. These proteins are epithelium-associated, differentiation-related cytokeratins. Both TGF-beta 1 and TGF-beta 2 up-modulate expression of the acidic and basic subtypes of human keratins in the responsive human colon carcinoma cells. Both the responsive and unresponsive cells, however, possess receptors for GFs.

Topics: Carcinoembryonic Antigen; Cell Differentiation; Cell Division; Colonic Neoplasms; Cross Reactions; Humans; Immunoblotting; Keratins; Microscopy, Phase-Contrast; Nuclear Proteins; Nucleophosmin; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Transforming Growth Factors; Tumor Cells, Cultured

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

Topics: Adenocarcinoma; Colonic Neoplasms; Humans; Immunohistochemistry; Keratins; Lymph Nodes; Plasma Cells; Plasmacytoma

We present two cases of small-bowel adenocarcinoma and dysplasia in patients with longstanding Crohn's disease. In one case, the dysplasia and cancer were exclusively located in the terminal ileum, whereas in the other case, several cancers were found from the ileum toward the transverse colon. In both cases, we found a clinically unsuspected Dukes C1 mucinous adenocarcinoma together with large foci of polypoid villous dysplasia or with multifocal high-grade dysplasia and intramucosal carcinoma. Immunohistochemical staining for carcinoembryonic antigen (CEA) revealed a different staining pattern in various diseased areas. The intensity of CEA staining paralleled the histologic degrees of dysplasia and neoplasia. Cytokeratin expression was disturbed in inflamed mucosa, and it was more pronounced in high-grade dysplasia and invasive carcinoma. We conclude that the presence of dysplasia in an intestinal biopsy of a patient with Crohn's disease should arouse the pathologist's suspicion of carcinoma and force him or her to take multiple sections from strictures and polypoid lesions, especially since the clinical symptoms of a carcinoma may be obscured by the symptoms of inflammatory bowel disease. Immunohistochemical staining with CEA and cytokeratin are useful in the objectivation of dysplasia.

Topics: Adenocarcinoma; Adult; Carcinoembryonic Antigen; Colitis; Colonic Neoplasms; Crohn Disease; Female; Humans; Ileal Neoplasms; Ileitis; Intestinal Mucosa; Keratins; Male; Middle Aged; Neoplasm Invasiveness

Immunoperoxidase stains were performed on normal and neoplastic tissue from prostate, colon, thyroid, lung, nerve, uterus, and placenta embedded in both plastic (glycolmethacrylate [GMA]) and paraffin. Positive results in plastic section were obtained for carcinoembryonic antigen (CEA), keratin, epithelial membrane antigen (EMA), thyroglobulins, S-100, prostate-specific antigen, human chorionic gonadotrophin (HCG), and beta-HCG. More delicate staining with more precise localization of antigens is noted. Superior (paraformaldehyde) fixation and cold processing followed by GMA polymerization (4 degrees C) allow for optimum antigen survival. After fixation, tissue processing involves a series of 0.1 mol/L phosphate buffer rinses with sucrose and ammonium chloride in a conventional dip-and-dunk processor placed in a 4 degrees C cold room. Acetone dehydrations are used before GMA infiltration, cold polymerization, and sectioning. Before immunoperoxidase staining, the plastic section is digested in .25% bovine trypsin for ten minutes. The immunoperoxidase methods described can be useful when small biopsies are routinely embedded in plastic to obtain improved histologic (hematoxylin-eosin) sections. There may also be research applications in quantifying antigen expression in benign, dysplastic, and neoplastic tissues by examining the stains under high power.

Topics: Adenocarcinoma; Antigens; Carcinoembryonic Antigen; Colonic Neoplasms; Humans; Immunoenzyme Techniques; Keratins; Male; Membrane Glycoproteins; Methacrylates; Mucin-1; Paraffin; Placenta; Prostatic Hyperplasia; Thyroglobulin; Thyroid Gland

An immuno-histological study of metastatic adenocarcinoma has revealed the following results. Metastatic adenocarcinomas of the lymph-node of pulmonary and colonic origin were positive for CEA and negative for lysozymes, and those from gastric, pancreatic, and gallbladder tumors were positive CEA and lysozymes, and those from gastric and pancreatic tumors were positive for the secretory component. The prostate specific antigen was exclusively positive for metastatic prostatic adenocarcinoma with a low frequency and prostate acid phosphatase had many false positive results. Thyroglobulin was found to be positive only to colloid. Lactalbumin showed no specificity to metastatic breast adenocarcinoma. For achieving the final diagnosis of a primary tumor, its location in lymph nodes, the clinical history and the results of other examinations must also be taken into consideration.

Topics: Adenocarcinoma; Biomarkers, Tumor; Carcinoembryonic Antigen; Colonic Neoplasms; Humans; Keratins; Lactalbumin; Lung Neoplasms; Lymphatic Metastasis; Muramidase; Neoplasms, Unknown Primary; Secretory Component

Eighty colon carcinomas reflecting the histologic spectrum were studied immunohistochemically; their epithelial characteristics had been established by demonstrating cytokeratin polypeptides. Paraffin sections were immunostained with monoclonal antibody (Mab) A-80 that recognizes a mucin-like glycoprotein related to exocrine differentiation. Sequential sections were immunostained with neuroendocrine (NE) differentiation antibodies: NSE, human chromogranin A, serotonin, somatostatin, substance P and VIP. Twenty-one/80 carcinomas immunoreacted exclusively with Mab A-80; these included adenocarcinomas with variably defined glands, colloid, "solid", and linitits plastica carcinomas. Eleven/80 carcinomas immunoreacted only with antibodies to NE markers. Twenty-nine/80 carcinomas of histologically variable patterns expressed both exocrine and NE antigens. A notable group of 19 adenocarcinomas immunostaining with Mab A-870 included a minority NE cell subpopulation. We tentatively conclude that given a limited battery of immunoprobes, colon carcinomas comprise 4 groups: 1) pure exocrine carcinomas, 2) pure NE carcinomas, 3) mixed exocrine and NE carcinomas, and 4) exocrine carcinomas with occasional NE cells. Thus, phenotypically mixed exocrine and NE carcinomas comprise the largest group while the second largest group exhibited exclusively features of exocrine phenotype. Preliminary clinical correlative data indicate that pure NE colon carcinomas behave more aggressively than their exocrine counterparts; moreover, colon carcinomas containing a NE subpopulation, even if small, also seem to behave worse than their counterparts without an NE subpopulation.

Topics: Antibodies, Monoclonal; Antigens, Tumor-Associated, Carbohydrate; Carcinoma; Cell Differentiation; Chromogranins; Colonic Neoplasms; Humans; Immunoenzyme Techniques; Keratins; Microscopy, Electron; Phosphopyruvate Hydratase; Serotonin; Somatostatin; Substance P

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 detection of early micrometastasis or disseminated single tumor cells poses a problem for conventional diagnosis procedures. Using a panel of monoclonal antibodies against cytokeratin and the 17-1A epithelial antigen we identified immunocytochemically tumor cells in bone marrow of patients with breast cancer (n = 155) and colorectal cancer (n = 57) at the time of surgery of the primary tumor. Monoclonal antibody CK2, recognizing the human cytokeratin component 18 in simple epithelia, appeared to be the most suitable reagent because of its negative reaction with bone marrow samples of the noncarcinoma patients (n = 75). Its specificity was further demonstrated in a double-marker staining procedure using an anti-leukocyte common antigen monoclonal antibody (T200) as counterstain. A comparative analysis showed that immunocytology was clearly superior to conventional cytology (n = 212) and histology (n = 39). In 9.5-20.5% of patients without distant metastasis, tumor cells could be detected in bone marrow. We found a significant correlation between tumor cells in bone marrow and conventional risk factors, such as distant metastasis or lymph node involvement. In a first approach toward immunotherapy we demonstrated in 3 patients that infused monoclonal antibody 17-1A can label single tumor cells in bone marrow in vivo. We then used this approach to follow up 7 patients undergoing 17-1A therapy in an adjuvant clinical trial.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Bone Marrow; Breast Neoplasms; Colonic Neoplasms; Epithelium; Humans; Immunoenzyme Techniques; Keratins; Neoplasm Metastasis; Rectal Neoplasms

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

A unique case of carcinosarcoma of the colon is reported. The tumor invaded the bowel wall deeply, metastasized widely, resisted multi-agent chemotherapy, and caused the patient's death 4 years later. The tumor was composed of adenosquamous carcinoma admixed with sarcoma showing osseous, cartilaginous, and nonspecific spindle-cell differentiation. Although carcinoembryonic antigen appeared limited to carcinoma cells, cytokeratin immunoreactivity was observed in both carcinoma and sarcoma cells. Like carcinosarcomas at other body sites, the finding of cytokeratin in sarcoma cells supports partial epithelial differentiation in this component, likely retained from carcinoma precursor cells.

Topics: Aged; Carcinoembryonic Antigen; Carcinosarcoma; Chondrosarcoma; Colonic Neoplasms; Histocytochemistry; Humans; Immunochemistry; Keratins; Male; Osteosarcoma; S100 Proteins

Cells of the normal colonic mucosa express several types of cytokeratins, the characteristic intermediate filament proteins of epithelial cells. An immunohistochemical study was designed to examine the expression of two distinct groups of cytokeratins, recognized by monoclonal antibodies AE1 and AE3, in the colonic mucosa and to compare the findings with those obtained with a large number of polypoid lesions (adenomatous and hyperplastic) and carcinomas of the colon. AE1 and AE3 immunostaining was found in the surface epithelium and upper portions of the crypts of Lieberkühn (functional zone) of normal colonic mucosa, whereas the lower portions of the crypts (proliferative compartment) were unreactive with both AE1 and AE3. Polypoid lesions of the colonic mucosa can be placed into two categories based on their patterns of cytokeratin expression. Solitary tubular adenomas and hyperplastic polyps are composed of AE1 and AE3 nonexpressing cells with only few, patchy areas of AE1 and AE3 expressing cells present within glands and in the surface epithelium. In contrast, villous adenomas show strong AE1 and AE3 reactivity throughout the glands. Furthermore, tubular and villous adenomas, and even histologically normal mucosa in patients with familial polyposis, show AE1/AE3 expression throughout the glands and surface epithelium. Colonic carcinomas show a predominance of AE1/AE3 expressing cells. Thus, cytokeratins recognized by monoclonal antibodies AE1 and AE3 represent molecular markers of cellular maturation in the normal colonic mucosa, that are expressed in colonic carcinomas and, in addition, serve as markers that distinguish colonic mucosa and adenomas with a high risk for development of cancers from those with a lower risk.

Topics: Adenocarcinoma; Adenoma; Adenomatous Polyposis Coli; Antibodies, Monoclonal; Cell Differentiation; Cell Transformation, Neoplastic; Colon; Colonic Neoplasms; Epithelium; Humans; Hyperplasia; Intestinal Mucosa; Keratins

The search for tumour markers was intensified with the advent of monoclonal antibody technology. To date no tumour specific markers have been found. Despite this, monoclonal antibodies have helped to identify cells in terms of their origin and function and therefore added a different dimension to studies of both benign and malignant disease processes. Advances in molecular biology have made cooperation between scientists and clinicians in all branches of medicine essential in order to piece together a more complete picture of any disease. This article describes the production and characterisation of two epithelial specific monoclonal antibodies (CAM5.2 and CAM17.1) with potential clinical value by a surgeon temporarily transposed to a laboratory environment.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Antigens, Surface; Colonic Neoplasms; Female; Humans; Immunoenzyme Techniques; Keratins; Lymphatic Metastasis; Rectal Neoplasms; Uterine Cervical Neoplasms

Sera of human colonic carcinoma xenografted rnu/nu rats were used to immunize rnu/+rats in order to obtain an immune response against circulating human tumor-associated components. After fusion of rat spleen cells with mouse myeloma cells monoclonal antibody MAB 108 could be established which reacted with two 40 and 45 kD cytokeratins as well as with vimentin, with a soluble 37 kD protein apparently derived from the 45 kD protein and with a 37 kD protein released by tumor cells. The MAB 108-specific epitope was also detected in tissue polypeptide antigen (TPA), a human tumor-associated antigen originally described by Björklund et al. (22).

Topics: Animals; Antibodies, Monoclonal; Cell Line; Colonic Neoplasms; Electrophoresis, Polyacrylamide Gel; Epitopes; Humans; Keratins; Mice; Molecular Weight; Neoplasm Transplantation; Peptides; Rats; Tissue Polypeptide Antigen

Carcinomas of different origin have been tested in immunofluorescence microscopy with the monoclonal murine antibodies CK1-CK4, which recognize a single cytokeratin polypeptide (human cytokeratin No. 18) present in simple but not in stratified squamous epithelia, and with the monoclonal antibody KG8.13 and guinea pig kerA antibodies, both of which recognize a variety of cytokeratins common to almost all epithelial cell types. Tumors derived from simple epithelia, including adenocarcinomas and some other tumors such as ductal breast carcinomas, were strongly stained by all three antibodies. So was a transitional carcinoma of the bladder. In contrast, basal cell epithelioma, cloacogenic carcinoma, and squamous cell carcinoma of skin, tongue, and esophagus appeared negative with CK1-CK4 but positive with the other two antibodies. Other squamous cell carcinomas derived from epiglottis and cervix uteri showed a mixture of positive and negative cells when tested with CK1-CK4, although all tumor cells were positive when tested with KG8.13 and with kerA. Thus, use of an appropriate collection of cytokeratin antibodies with different specificities not only allows tumors of epithelial origin to be distinguished from other tumor types but, in addition, allows a further subdivision of carcinomas in relation to their histologic origin.

Topics: Adenocarcinoma; Animals; Antibodies, Monoclonal; Antibody Specificity; Carcinoma; Carcinoma, Transitional Cell; Colonic Neoplasms; Fluorescent Antibody Technique; Guinea Pigs; Histocytochemistry; Humans; Intermediate Filament Proteins; Keratins; Mice; Urinary Bladder Neoplasms

Five thousand seven hundred seventy-eight adenomas or adenomas containing carcinoma from 3215 patients were examined by routine histologic methods for the presence of epithelial metaplasias. Three forms of epithelial metaplasia were encountered: squamous cell metaplasia (0.44%), Paneth cell metaplasia (0.20%), and melanocytic metaplasia (0.017%). In several instances multiple forms of metaplasia were encountered in the same polyp. In those cases in which the paraffin blocks were available, a Grimelius stain was performed. Grimelius-positive cells were present in 63% of the adenomas containing a metaplastic cell type. All cases with Paneth cell differentiation were immunoreactive for lysozyme; all lesions containing areas of squamous differentiation were immunoreactive for keratin except 2. The histopathologic features of these cases are discussed, and it is concluded that rather than representing a true metaplastic process, Paneth cell, squamous cell, and melanocyte differentiation represent the full range of cellular differentiation that endodermally derived tissues can exhibit, particularly when they undergo neoplastic alterations.

Topics: Adenoma; Adult; Age Factors; Aged; Cell Differentiation; Colonic Neoplasms; Female; Histocytochemistry; Humans; Intestinal Polyps; Intestine, Large; Keratins; Male; Melanocytes; Metaplasia; Middle Aged; Muramidase; Rectal Neoplasms; Retrospective Studies; Sex Factors

CAM 5.2 is a murine monoclonal antibody, raised against the colon carcinoma cell line HT29, which recognises lower molecular weight intracellular cytokeratin proteins within secretory epithelia. Extensive indirect immunohistochemical studies have confirmed that this antibody stains formalin fixed (and freshly frozen) normal and malignant human tissue in a consistent manner. Reliable staining of conventionally processed pathological tissues provides more accurate identification and staging of human malignant epithelial diseases.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neoplasm; Cell Line; Colonic Neoplasms; Fixatives; Fluorescent Antibody Technique; Humans; Hybridomas; Immunoenzyme Techniques; Immunoglobulin G; Keratins; Mice; Mice, Inbred BALB C; Staining and Labeling

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

The light microscopic, electron microscopic and histochemical features of a highly malignant colonic tumor resected from a 39 year old man are presented. The tumor was composed predominantly of undifferentiated cells with focally admixed neuroendocrine, exocrine and squamous cells, occasionally arranged in an organoid manner. Histochemically the tumor contained argyrophilic cells as well as cells that reacted positively with the antibodies to alpha-1-antitrypsin, alpha-1-antichymotrypsin, carcinoembryonic antigen and lysozyme. The term "stem cell carcinoma of the intestine" is proposed for this highly malignant tumor composed of undifferentiated cells exhibiting only focally their multidirectional developmental capacity.

Topics: Adult; alpha 1-Antichymotrypsin; alpha 1-Antitrypsin; Carcinoembryonic Antigen; Carcinoma; Chymotrypsin; Colonic Neoplasms; Histocytochemistry; Humans; Immunologic Techniques; Keratins; Male; Microscopy, Electron; Muramidase; Staining and Labeling

An ultrastructural analysis of the organisation of 10 nm intermediate filaments during mitosis, in normal and malignant human colonic epithelial cells present in resected material, was carried out. Similar changes in the integrity of the 10 nm filaments were seen both in normal mitotic cells (which have a highly specialised filamentous network during interphase), and malignant cells (which show a disorganised network during interphase). The 10 nm filaments were seen to shorten and 'fray' in early mitosis. This was associated with the formation of spheroidal aggregates of thin filaments and small granules, both free in the cytoplasm and associated with surface desmosomes. At mid-mitosis cells were essentially devoid of obvious filamentous material, (including the spheroidal aggregates). The filaments reformed in daughter cells after cytokinesis was completed.

Topics: Colon; Colonic Neoplasms; Cytoskeleton; Epithelium; Fluorescent Antibody Technique; Humans; Keratins; Mitosis

Twenty-five primary gastrointestinal carcinomas have been studied using immunofluorescence microscopy with affinity-purified antibodies to prekeratin and to vimentin. The tissues were alcohol fixed and paraffin embedded before use. In all cases (i.e., one case of esophagal carcinoma, seven stomach carcinomas, and 17 large bowel carcinomas) the tumor cells are stained by antibodies to prekeratin. In cases in which only very few tumor cells are present, such as signet ring carcinoma, immunofluorescence with prekeratin antibody provides an easy way to visualize single tumor cells. When the same specimens were tested with antibodies to vimentin, the tumor cells were unstained, and only the fibroblasts and vessels of the stroma were decorated. Four of the tumors were also negative when tested with antibodies specific for either desmin, or glial fibrillary acidic protein or neurofilaments. Three metastases to the abdominal region from tumors originating in the ovary, stomach, and large bowel were like the primary tumors in that the tumor cells were positive when stained with antibodies to prekeratin and negative when tested with the antibodies to vimentin.

Topics: Adenocarcinoma; Antibodies; Carcinoma, Squamous Cell; Colonic Neoplasms; Cytoskeleton; Esophageal Neoplasms; Fluorescent Antibody Technique; Gastrointestinal Neoplasms; Humans; Keratins; Muscle Proteins; Neoplasm Metastasis; Protein Precursors; Stomach Neoplasms; Vimentin

The distribution of intermediate - sized filaments in human colon mucosa as well as in adenomas and carcinomas of the colon was studied by means of both immunohistology and electron microscopy. The epithelial cells of the colonic mucosa are definitely labelled with antibodies against prekeratin (cytokeratin). Interwoven filaments of the prekeratin type are present in the basal compartments of the epithelial cells; they surround the nuclei and mucus droplets and form an apical skeletal disc. Pericryptal connective tissue is prekeratin negative and vimentin positive. Benign hyperplastic polyps have a high content of prekeratin. The potential precursors of colonic carcinoma, i.e., the tubular and villous adenomas, also show an increase in intermediate-sized filaments of the prekeratin type. Correspondingly, electron microscopy reveals elongated bundles of intermediate-sized filaments arising from the desmosomes of the lateral and basal cell membranes. The prekeratin content is particularly high in adenocarcinomas and highest in mucinous carcinomas. As expected, the stroma of all neoplasms studied is prekeratin-negative, but distinctly vimentin-positive. In one moderately differentiated adenocarcinoma there was evidence of "vimentin-positive" tumor cells. These changes may be caused by binding of cytokeratins with an unknown substance in vimentin antisera, as observed similarly by Moll et al. (1982) in a transitional cloacogenic carcinoma.

Topics: Adenocarcinoma; Adenocarcinoma, Mucinous; Adenoma; Colon; Colonic Neoplasms; Cytoskeleton; Humans; Intermediate Filament Proteins; Intestinal Mucosa; Keratins; Microscopy, Electron; Protein Precursors; Vimentin