bromochloroacetic-acid and Colorectal-Neoplasms

The colon mucosa is lined with crypts of circa 300 cells, forming a continuous barrier whose roles include absorption of water, recovery of metabolic energy sources (notably short chain fatty acids), secretion of a protective mucus barrier, and physiological signalling. There is high turnover and replenishment of cells in the mucosa, disruption of this may lead to bowel pathologies including cancer and inflammatory bowel disease. Keratins have been implicated in the processes of cell death, epithelial integrity, response to inflammation and as a result are often described as guardians of the colonic epithelium. Keratin proteins carry extensive post-translational modifications, the cofactors for kinases, acetyl transferases and other modification-regulating enzymes are themselves products of metabolism. A cluster of studies has begun to reveal a bidirectional relationship between keratin form and function and metabolism. In this paper we hypothesise a mechanistic interaction between keratins and metabolism is governed through regulation of post-translational modifications and may contribute significantly to the normal functioning of the colon, placing keratins at the centre of a nutrition-metabolism-health triangle.

Topics: Colon; Colorectal Neoplasms; Humans; Inflammatory Bowel Diseases; Intestinal Mucosa; Keratins; Rectum

Keratin intermediate filament proteins are major cytoskeletal components of the mammalian simple layered columnar epithelium in the gastrointestinal tract. Human colon crypt epithelial cells express keratins 18, 19 and 20 as the major type I keratins, and keratin 8 as the type II keratin. Keratin expression patterns vary between species, and mouse colonocytes express keratin 7 as a second type II keratin. Colonic keratin patterns change during cell differentiation, such that K20 increases in the more differentiated crypt cells closer to the central lumen. Keratins provide a structural and mechanical scaffold to support cellular stability, integrity and stress protection in this rapidly regenerating tissue. They participate in central colonocyte processes including barrier function, ion transport, differentiation, proliferation and inflammatory signaling. The cell-specific keratin compositions in different epithelial tissues has allowed for the utilization of keratin-based diagnostic methods. Since the keratin expression pattern in tumors often resembles that in the primary tissue, it can be used to recognize metastases of colonic origin. This review focuses on recent findings on the biological functions of mammalian colon epithelial keratins obtained from pivotal in vivo models. We also discuss the diagnostic value of keratins in chronic colonic disease and known keratin alterations in colon pathologies. This review describes the biochemical properties of keratins and their molecular actions in colonic epithelial cells and highlights diagnostic data in colorectal cancer and inflammatory bowel disease patients, which may facilitate the recognition of disease subtypes and the establishment of personal therapies in the future.

Topics: Animals; Colon; Colorectal Neoplasms; Epithelium; Homeostasis; Humans; Keratins

Liver metastases emerge during the course of colorectal cancer (CRC) in 25-50% of patients. A small proportion of patients present intrabiliary growth. The absence of large series means that little is known about intrabiliary metastasis (IBM), its radiological diagnosis, the most suitable surgical techniques, and its prognostic implications.. A systematic search without limits was performed. The studies selected included patients with a diagnosis of CRC and associated IBM, either synchronous or metachronous.. Of 40 studies selected, 30 were case reports and 10 case series. The median time between diagnosis and IBM was 46.7 months (range 0-180). Most CRC metastases are CK7-/CK20+. Surgical treatment performed ranged from endoscopic resection to major hepatic resections combined with pancreatectomies. It seems that patients with IBM have a better survival than patients without this metastasis.. In a patient with a history of CRC presenting dilatation of the bile duct, IBM should be considered. More studies are needed to determine the most appropriate type of liver resection. It is also necessary to standardize the definition and terminology of this pathology, since the existing definitions may cause confusion and make it difficult to carry out case studies and case series.

Topics: Biliary Tract Neoplasms; Biomarkers, Tumor; Colorectal Neoplasms; Hepatectomy; Humans; Keratins; Prognosis

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

Tumor 'budding', loosely defined by the presence of individual cells and small clusters of tumor cells at the invasive front of carcinomas, has received much recent attention, particularly in the setting of colorectal carcinoma. It has been postulated to represent an epithelial-mesenchymal transition. Tumor budding is a well-established independent adverse prognostic factor in colorectal carcinoma that may allow for stratification of patients into risk categories more meaningful than those defined by TNM staging, and also potentially guide treatment decisions, especially in T1 and T3 N0 (Stage II, Dukes' B) colorectal carcinoma. Unfortunately, its universal acceptance as a reportable factor has been held back by a lack of definitional uniformity with respect to both qualitative and quantitative aspects of tumor budding. The purpose of this review is fourfold: (1) to describe the morphology of tumor budding and its relationship to other potentially important features of the invasive front; (2) to summarize current knowledge regarding the prognostic significance and potential clinical implications of this histomorphological feature; (3) to highlight the challenges posed by a lack of data to allow standardization with respect to the qualitative and quantitative criteria used to define budding; and (4) to present a practical approach to the assessment of tumor budding in everyday practice.

Topics: Adenocarcinoma; Biomarkers, Tumor; Cell Movement; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Humans; Keratins; Neoplasm Invasiveness; Neoplasm Staging; Prognosis

The role of adjuvant chemotherapy after resection of colorectal cancers (CRCs) is well understood for patients with stage-I or stage-III disease. Its efficacy for those with stage-II disease remains much less clear. Many investigators have sought to identify prognostic markers that might clarify which patients have the highest risk of recurrence and would, therefore, be most likely to benefit from chemotherapy. This systematic review examines evidence for the use of peripherally sampled, circulating tumour cells (CTCs) as such a prognostic marker.. A comprehensive literature search was used to identify studies reporting on the significance of CTCs in the postoperative blood of CRC patients.. Fourteen studies satisfied the inclusion criteria. Six of the nine studies that took blood samples 24 h or more postoperatively found detection of postoperative CTCs to be an independent predictor of cancer recurrence.. The presence of CTCs in peripheral blood at least 24 h after resection of CRCs is an independent prognostic marker of recurrence. Further studies are needed to clarify the optimal time point for blood sampling and determine the benefit of chemotherapy in CTC-positive patients with stage-II disease.

Topics: Aged; Carcinoembryonic Antigen; Chemotherapy, Adjuvant; Colorectal Neoplasms; Humans; Keratins; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Patient Selection; Postoperative Complications; Prognosis; Recurrence; Treatment Outcome

The prognostic relevance of lymphatic micrometastases in colorectal carcinoma is unclear. To determine the prognostic significance of micrometastases in colorectal cancer, a meta-analysis was performed on all studies, which reported 3-year disease-free survival (DFS) and overall survival (OS).. Published studies selected for meta-analysis contained sufficient data from which to extrapolate estimates of 3-year DFS and/or OS. From 1991-2003, 25 studies re-examined N0 lymph nodes by serial sectioning and immunohistochemical (IHC) staining or reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Eight studies (566 patients) with IHC detected micrometastases and three (173 patients) with RT-PCR micrometastases were used to determine DFS and OS. Weighted estimates of 3-year survival were combined across studies within each group, and the combined survival estimates were compared across groups using a binomial test.. Micrometastases were identified in all IHC studies; upstaging, including N1, N1mi and N0(i+), was achieved in 32% (179/566 patients). All RT-PCR studies identified micrometastases; upstaging to N0(mol+) was achieved in 37% (64/173 patients). There was a statistically significant difference in 3-year OS between RT-PCR positive N0(mol+) patients (77.8%) and those for whom micrometastases were not detected (96.6%) (P < .001).. The prognostic value of micrometastases detected retrospectively by RT-PCR is significant in AJCC stage II colorectal patients. Studies utilizing RT-PCR performed a more complete nodal analysis when compared to studies using IHC techniques. RT-PCR may also be more specific for the detection of clinically relevant micrometastases compared to IHC detected cytokeratins. Prospective studies are needed to evaluate the potential benefit of systemic chemotherapy in patients with molecular metastases.

Topics: Colorectal Neoplasms; Humans; Immunoenzyme Techniques; Keratins; Lymph Nodes; Lymphatic Metastasis; Neoplasm Staging; Prognosis; Retrospective Studies; Reverse Transcriptase Polymerase Chain Reaction; Survival Rate

Global gene expression profiling and molecular cytogenetics for single cells provide new opportunities to resolve important questions, such as the mechanisms of tumor cell selection during cancer progression. While gene expression analyses show that metastatic progression correlates with the deregulation of certain gene sets in the primary tumor, the direct analysis of disseminated cancer cells, the putative precursors of metachronous metastases, suggests that dissemination is a very early event in the genetic development of human cancers.

Topics: Animals; Bone Marrow; Breast Neoplasms; Chromosome Aberrations; Colorectal Neoplasms; Evolution, Molecular; Gene Expression Regulation, Neoplastic; Humans; Keratins; Models, Biological; Neoplasm Metastasis; Tumor Cells, Cultured

This review is concerned with the usefulness and the problem of biomarkers for cancer of digestive organs. Carcinoembryonic antigen (CEA) is a most popular and useful tumor marker for cancer of digestive organs. Squamous cell carcinoma (SCC) antigen and CYFRA have been reported as a useful tumor marker for esophageal cancer. CEA and CA 19-9 are a good prognostic factor in gastric cancer patients. The post-operative increase of serum CEA can be a predictive marker for the patients of colorectal cancer. Development of a radioimmunoassay for highly sensitive detection of tumor markers, they are considered to be useful for monitoring after treatment. But are not useful for the early diagnosis. The diagnosis of hepatocellular carcinoma (HCC) is based mainly on serological markers, such as alpha-fetoprotein and PIVKA-II. The two are useful complementary markers of HCC because they do not correlate with each other. But the problem of the false-positive rate for the patients with chronic hepatitis or liver cirrhosis is still remained. A typical marker of pancreatic and bile duct cancer is carbohydrate antigen, but the sensitivity of these markers is only 50%. Recent molecular biological analysis may be used as effective biomarkers in the diagnosis, prognosis, therapy, and risk assessment of digestive cancer.

Topics: alpha-Fetoproteins; Antigens, CD19; Antigens, Neoplasm; Biomarkers; Biomarkers, Tumor; Carcinoembryonic Antigen; Carcinoma, Hepatocellular; Carcinoma, Squamous Cell; Colorectal Neoplasms; Digestive System Neoplasms; Esophageal Neoplasms; Female; Humans; Keratin-19; Keratins; Lewis X Antigen; Liver Neoplasms; Pancreatic Neoplasms; Prognosis; Protein Precursors; Prothrombin; Stomach Neoplasms

CEA and CA19-9 are the two most common tumor markers for colorectal cancer that are currently utilized clinically. The positive rate of CEA is 40-60% and that of CA19-9 is 30-50%. Simultaneous use of the two markers is useful in evaluating the therapeutic effect and monitoring the recurrence of advanced colorectal cancer. Surgical specimens may also provide useful information for the appropriate treatment of patients. Using surgically resected lymph nodes, we examined micrometastasis to assess the spread of the cancer cells and the malignant potential of colorectal cancer. Immunohistochemical analysis using anti-cytokeratin antibody revealed no significant impact of micrometastasis on patient prognosis, while RT-PCR assay using CEA as a genetic marker suggested a positive value in predicting a rapid recurrence. Among various molecular markers, we found that CDC25B phosphatase was a powerful prognostic factor for colorectal cancer. Diagnosis of the existence and malignant potential of cancer cells, together with serum tumor marker levels, may help to construct a more useful system for the better treatment of colorectal cancer.

Topics: Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; CA-19-9 Antigen; Carcinoembryonic Antigen; cdc25 Phosphatases; Cell Adhesion Molecules; Cell Cycle Proteins; Colorectal Neoplasms; DCC Receptor; Female; Genes, src; Humans; Keratin-19; Keratins; Male; Oligosaccharides; Receptors, Cell Surface; Sialyl Lewis X Antigen; Tumor Suppressor Proteins

Topics: Bone Marrow; Bone Marrow Examination; Bone Marrow Neoplasms; Breast Neoplasms; Colorectal Neoplasms; Esophageal Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Lung Neoplasms; Lymphatic Metastasis; Neoplastic Cells, Circulating; Ovarian Neoplasms; Prognosis; Prospective Studies; Randomized Controlled Trials as Topic; Risk Factors; Stomach Neoplasms; Uterine Cervical Neoplasms

Topics: Adenocarcinoma; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Squamous Cell; Carcinoma, Transitional Cell; Cell Differentiation; Colorectal Neoplasms; Female; Humans; Keratins; Neoplasms, Glandular and Epithelial; Oropharyngeal Neoplasms; Pancreatic Neoplasms; Stomach Neoplasms; Urologic Neoplasms

Results of the few extant reports concerning the clinical significance of so-called "occult micrometastases" of lymph nodes of patients with Dukes A and B colorectal cancer have been variable. We examined the presumably negative nodes of a larger cohort of such patients who were enrolled in the National Surgical Adjuvant Breast and Bowel Project clinical trials R-01 and C-01 for the influence of what we preferably designate as nodal mini micrometastases on parameters of survival.. Mini micrometastases were detected by immunohistochemical staining of the original lymph node sections with anticytokeratin A1/A3 in a total of 241 Dukes A and B patients with rectal and 158 with colonic cancers. Their frequency, as well as that of nuclear and histologic grades, and an estimation of their relationship to relative risks were correlated with overall and recurrence-free survival by univariate and multivariate analyses.. Nodal mini micrometastases were detected in 73 of 399 (18.3 percent) patients of this cohort. They failed to exhibit any significant relationship to overall or recurrence-free survival. No association between the assessments of tumor differentiation and mini micrometastases was found. Nuclear and histologic grades also failed to further discriminate overall or recurrence-free survival in patients with A or B stages of colonic or rectal cancers in this cohort.. The immunohistochemical demonstration of nodal mini micrometastases failed to discriminate high- and low-risk groups of patients with colorectal cancer who were designated as being node-negative after routine pathologic examination.

Topics: Antigens, Neoplasm; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Lymph Nodes; Lymphatic Metastasis; Male; Prognosis; Prospective Studies; Risk Factors

Micrometastatic disease in bone marrow is of prognostic significance in colorectal cancer patients. However, detection rates of standard immunocytology are relatively low. We used magnetic activated cell sorting (MACS), a highly sensitive method, to increase detection rates and correlated the presence of cytokeratin (CK)-expressing cells with clinical parameters.. Bone marrow was obtained from 51 consecutive patients with newly diagnosed colorectal adenocarcinoma who underwent primary surgery and 18 control subjects. International Union Against Cancer (UICC) stage I disease was diagnosed in 11 patients, stage II disease was diagnosed in 14 patients, stage III disease was diagnosed in 12 patients, and stage IV disease was diagnosed in 14 patients. CK-positive cells were enriched and stained with magnetically labeled CAM 5.2 antibodies directed to CK 7 and 8.. CK-positive cells were found in 33 (65%) patients and were absent in 18 (35%). Four of 11 (36%) patients with UICC stage I disease, nine of 14 (64%) with stage II diease, eight of 12 (67%) with stage III disease, and 12 of 14 (86%) with stage IV disease were CK-positive. Epithelial cells were more frequently found in pT3/4 (72%) than in pT1/2 (36%) tumors (P =.026), but there was no difference for lymph node status. CK-positive patients had a higher chance for elevated carcinoembryonic antigen (85% v 15%, P = NS) and CA 19-9 levels (92% v 8%, P =.019). There were no significant differences in CA 72-4, sex, age, tumor grading, or tumor localization regarding the presence of CK-positive cells. All control subjects were CK-negative.. In searching for micrometastases in colorectal cancer patients, we have achieved high detection rates by using MACS. The presence of these cells correlated significantly with tumor stage, tumor extension, and the tumor marker CA 19-9.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Bone Marrow Neoplasms; Colorectal Neoplasms; Female; Humans; Immunomagnetic Separation; Keratins; Male; Middle Aged; Neoplasm Metastasis; Neoplasm Staging; Sensitivity and Specificity

Different opinions about flow cytometric estimates of DNA aneuploidy and/or S-phase fraction (SPF) as supplementary prognostic markers in colorectal cancer are to some degree associated with methodology. Using univariate DNA analysis, we have previously investigated the DNA ploidy in colorectal cancer, its heterogeneity within and between tumors and its relation to survival. To improve detection of DNA aneuploid subpopulations and particularly estimation of their SPF's we investigated a method for bivariate DNA/cytokeratin analysis on fine-needle aspirates of 728 frozen biopsies from 157 colorectal tumors. Unfixed aspirates were stained with propidium iodide and FITC-conjugated anti-cytokeratin antibody in a saponin-buffer. A significant association between SPF and debris was observed. There were no substantial difference in DNA ploidy patterns between univariate and bivariate measurements (concordance was 92-95%). No new DNA aneuploid subpopulations were detected in cytokeratin-gated compared to ungated or univariate histograms. Debris-adjusted SPF's of cytokeratin-gated histograms were significantly higher than of ungated histograms, also for subpopulations with DI>1.4 (p<0.0001). There was no significant association between SPF and survival.

Topics: Adult; Aged; Aged, 80 and over; Aneuploidy; Anti-Ulcer Agents; Biopsy; Colorectal Neoplasms; DNA, Neoplasm; Female; Flow Cytometry; Humans; Keratins; Male; Middle Aged; Prognosis; Prospective Studies; Ranitidine; Reproducibility of Results; S Phase; Survival Analysis

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

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

The clinical significance of detecting supposed tumour cell-derived mRNA transcripts in blood using the polymerase chain reaction (PCR) remains unclear. We have used a fully quantitative 5'-nuclease RT-PCR assay to screen for the expression of cytokeratins (ck) 19 and 20 and guanylyl cyclase C (GCC) in the peripheral blood of 21 healthy controls and 27 colorectal cancer patients. Expression of cytokeratin 19 and 20 mRNA was detected in 30% and 100% of samples, respectively, taken from healthy volunteers. There was no apparent difference in ck19 and ck20 mRNA transcription levels between controls and patients, or between patients with different Dukes' stages. While GCC mRNA was detected in only 1/21 control samples, it was expressed in approximately 80% of patients, although again there was no correlation between GCC levels and disease stage. Transcription levels of all three markers varied considerably between samples, even between samples taken from the same person at different times. We conclude that neither ck19 nor ck20 are reliable markers for the detection of colon epithelial cells in peripheral blood and that an evaluation of the usefulness of GCC awaits further longitudinal studies.

Topics: Adult; Aged; Biomarkers, Tumor; Cell Nucleus; Colorectal Neoplasms; Female; Guanylate Cyclase; Humans; Intermediate Filament Proteins; Keratin-20; Keratins; Male; Middle Aged; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Reference Standards; Reference Values; Reverse Transcriptase Polymerase Chain Reaction

The emerging clinical relevance of bone marrow micrometastasis has prompted several investigations, using a variety of immunocytochemical approaches. The present study was designed to evaluate some of the variables affecting the immunocytochemical detection of individual epithelial tumor cells in bone marrow. Using an alkaline phosphatase-antialkaline phosphatase staining technique, we evaluated bone marrow aspirates from 358 patients with primary carcinomas of the breast (n = 150), lung (n = 66), prostate (n = 42), or colorectum (n = 100). Individual tumor cells in cytological preparations were detected with monoclonal antibody (MAb) CK2 to the epithelial cytokeratin component 18 (CK18), which has been validated in extensive clinical studies. In addition, the utility of the broad-spectrum MAb A45-B/B3 was explored in this study. The high specificity of MAbs CK2 and A45-B/B3 was supported by analysis of bone marrow from 75 noncarcinoma control patients and by double-marker analysis with MAbs to mesenchymal marker proteins (CD45 and vimentin). In contrast, MAbs E29 and HMFG1, directed to mucin-like epithelial membrane proteins, cross-reacted with hematopoietic cells in 26.7-42.7% of all samples tested. The majority of the 154 positive samples (43.0%) from cancer patients displayed less than 10 CK18-positive cells per 8 x 10(5) marrow cells analyzed. The detection rate, however, was affected by blood contamination of the aspirate, the number of aspirates analyzed, and the number of marrow cells screened per aspiration site. Comparative immunostaining of bone marrow specimens with MAbs CK2 and A45-B/B3 indicated that downregulation of CK18 in micrometastatic carcinoma cells occurs in about 50% of the 172 samples analyzed, regardless of the primary tumor origin.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Alkaline Phosphatase; Antibodies, Monoclonal; Antibody Specificity; Antigens, Neoplasm; Bone Marrow; Breast Neoplasms; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Lung Neoplasms; Male; Membrane Glycoproteins; Mucin-1; Mucins; Neoplasm Metastasis; Prostatic Neoplasms; Sensitivity and Specificity

Topics: Colorectal Neoplasms; Humans; Immunohistochemistry; Keratins; Neoplasm Staging; Staining and Labeling

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

Cell-cell and cell-matrix adhesion proteins that have been implicated in colorectal epithelial integrity and epithelial-to-mesenchymal transition could be robust prognostic and potential predictive biomarkers for standard and novel therapies. We analyzed in situ protein expression of E-cadherin (ECAD), integrin β4 (ITGB4), zonula occludens 1 (ZO-1), and cytokeratins in a single-hospital series of Norwegian patients with colorectal cancer (CRC) stages I-IV (n = 922) using multiplex fluorescence-based immunohistochemistry (mfIHC) on tissue microarrays. Pharmacoproteomic associations were explored in 35 CRC cell lines annotated with drug sensitivity data on > 400 approved and investigational drugs. ECAD, ITGB4, and ZO-1 were positively associated with survival, while cytokeratins were negatively associated with survival. Only ECAD showed independent prognostic value in multivariable Cox models. Clinical and molecular associations for ECAD were technically validated on a different mfIHC platform, and the prognostic value was validated in another Norwegian series (n = 798). In preclinical models, low and high ECAD expression differentially associated with sensitivity to topoisomerase, aurora, and HSP90 inhibitors, and EGFR inhibitors. E-cadherin protein expression is a robust prognostic biomarker with potential clinical utility in CRC.

Topics: Antigens, CD; Biomarkers, Tumor; Cadherins; Cell Line, Tumor; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Humans; Immunohistochemistry; Keratins; Prognosis

Combined analysis of cytokeratin 7 (CK7) and cytokeratin 20 (CK20) is often used for assessing the origin of metastatic cancer. To evaluate the diagnostic utility of CK7 and CK20, tissue microarrays containing 15,424 samples from 120 different tumor types and subtypes and 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry. CK7 positivity was seen in 52% (8.7% weak, 5.9% moderate, 37% strong) and CK20 positivity in 23% (5.1% weak, 3.4% moderate, 15% strong) of interpretable tumors. Of 8390 positive tumors, 1181 (14%) showed positivity for CK7 and CK20, 5380 (64%) showed positivity for CK7 alone, and 1829 (22%) showed positivity for CK20 alone. CK20 predominated in gastrointestinal tract, urothelial and Merkel cell carcinomas. CK7 was usually negative in prostate cancer and colorectal cancer. Combined evaluation of CK7/CK20 revealed the best diagnostic utility in CK20 positive tumors, where CK7 negativity is often linked to colorectal origin while CK7 positivity argues for urothelial origin or mucinous ovarian cancer. Associations with unfavorable tumor features were found for cytokeratin 7 loss in breast cancer of no special type, urothelial and renal cell carcinomas, for CK7 overexpression in high-grade serous ovarian and gastric cancer, and for CK20 overexpression in urothelial carcinoma. CK20 loss was linked to MSI in gastric (p = 0.0291) and colorectal adenocarcinoma (p < 0.0001). These analyses provide comprehensive data on the frequency of CK7 and CK20 immunostaining - alone or in combination - in human cancers. These data facilitate interpretation of CK7/CK20 immunostaining in cancers.

Topics: Biomarkers, Tumor; Carcinoma, Transitional Cell; Colorectal Neoplasms; Humans; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Male; Urinary Bladder Neoplasms

The aim of this study was to investigate the relationship between tumor budding (TB) and clinicopathologic prognostic criteria in colorectal adenocarcinomas and to discuss the inclusion of the fourth group in the scoring system. A total of 131 cases were included in the study. TB was scored according to the classical 3-tiered scoring system and our proposed 4-tiered scoring system: BD0 (no buds), BD1* (1-4 buds), BD2 (5-9 buds), and BD3 (≥10 buds). Cytokeratin staining was applied to 80 randomly selected cases and TB scoring was re-evaluated. TB was not observed in 31 (23.7%) of 131 cases and was categorized as BD0. Patients with BD0 budding had lower pT category, AJCC stage, tumor grade, less lymph node metastasis, lymphovascular invasion, tumor deposits (p < 0.05), and longer overall survival than BD1* patients (log-Rank p: 0.018). There was significant compatibility between the evaluation of TB with H&E and cytokeratin (kappa: 0.727, p < 0.001). In conclusion, we think it is valuable to add the "BD0" category to the International Tumor Budding Consensus Conference (ITBCC) scores. However, more research with larger cohorts is needed for clinical applicability. H&E staining is sufficient for the assessment of budding, except in conditions such as increased inflammation where the tumor-stroma interface may be obscured.

Topics: Adenocarcinoma; Colorectal Neoplasms; Humans; Keratins; Lymphatic Metastasis; Neoplasm Staging; Prognosis

Although the primary origin of some carcinomas may be obscure to clinicians, its identification is crucial as it affects prognosis and treatment (especially novel targeted therapies). Immunohistochemistry (IHC) may be helpful in identifying the primary origin of carcinomas. This retrospective survey aimed to evaluate the frequency and accuracy of each IHC marker used to determine the origin of carcinomas.. The review of pathology department archives revealed 307 cases of cancer of unknown primary origin (CUP) between 2015 and 2020, which were accessible in the department archives. Demographic information, site of biopsy, clinical and pathologic diagnoses, and IHC results of the patients were collected.. The patients included 157 (51.15%) men and 150 (48.85%) women. The age of the patients ranged from 14 to 92 years, including 106 (34.5%) expired cases. In 27% of cases, the primary origin of carcinoma remained unknown. The agreement between pathologic and clinical diagnoses was 59%. The most common pattern of cytokeratin (CK) expression in CUP was CK7+/CK20- (55.3%), followed by CK7-/CK20- (19%), CK7+/CK20+ (15%), and CK7-/CK20+ (10.7%), respectively.. The IHC analysis may improve the diagnosis of CUPs. However, the origin of some cases remains unknown despite an IHC analysis, thereby necessitating the use of more diagnostic procedures or gene expression studies for reaching a definitive diagnosis.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Male; Middle Aged; Neoplasms, Unknown Primary; Retrospective Studies; Staining and Labeling; Young Adult

Colorectal carcinoma (CRC) is associated with significant morbidity and mortality worldwide. Cytokeratins (CKs) are widely expressed in various types of carcinomas, whereas in CRC it is usually CK7 - and CK20 + . A subset of CRCs is CK7 + . This study aims to determine the prevalence of CK7 expression in CRC and its impact on overall survival. We analyzed 300 randomly selected surgically treated CRC cases using paraffin embedded tumor tissue samples and evaluated CK7 and CK20 expression using the tissue microarray method. Tumors with positivity > 10% and > 25% of tumor cells were considered CK7 and CK20 positive, respectively. Expression of both CKs and several clinical-pathological variables (stage, grade, laterality, mismatch-repair/MMR status) were evaluated using patient follow up data (Kaplan-Meier analysis of cancer-specific survival (CSS)). Significant results include shorter CSS (restricted mean 4.98 vs. 7.74 years, P = 0.007) and 5-year survival (29.4% vs. 64.6%, P = 0.0221) in CK7 + tumors compared to CK7 - tumors, respectively; without significant association with grade, stage or right-sided location. These results were significant in a multivariate analysis. CK20 + tumors are more frequently MMR-proficient and left-sided. MMR-deficient tumors are more frequently right-sided and had longer survival. CK7 expression, right-sided location (rmean CSS 6.83 vs. 8.0 years, P = 0.043), MMR-proficiency (rmean CSS 7.41 vs. 9.32 years, P = 0.012), and UICC stages III + IV (rmean CSS 6.03 vs. 8.92 years, P < 0.001) of the tumor correlated with negative prognostic outcomes, whereas the most significant results concern stage and CK7 positivity. The result concerning negative prognostic role of CK7 differs from those obtained by several previous studies focused on this topic.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Colorectal Neoplasms; Diagnosis, Differential; Female; Humans; Intermediate Filament Proteins; Keratin-7; Keratins; Male; Middle Aged; Prognosis

Tumor budding is a promising and cost-effective biomarker with strong prognostic value in colorectal cancer. However, challenges related to interobserver variability persist. Such variability may be reduced by immunohistochemistry and computer-aided tumor bud selection. Development of computer algorithms for this purpose requires unequivocal examples of individual tumor buds. As such, we undertook a large-scale, international, and digital observer study on individual tumor bud assessment. From a pool of 46 colorectal cancer cases with tumor budding, 3000 tumor bud candidates were selected, largely based on digital image analysis algorithms. For each candidate bud, an image patch (size 256 × 256 µm) was extracted from a pan cytokeratin-stained whole-slide image. Members of an International Tumor Budding Consortium (n = 7) were asked to categorize each candidate as either (1) tumor bud, (2) poorly differentiated cluster, or (3) neither, based on current definitions. Agreement was assessed with Cohen's and Fleiss Kappa statistics. Fleiss Kappa showed moderate overall agreement between observers (0.42 and 0.51), while Cohen's Kappas ranged from 0.25 to 0.63. Complete agreement by all seven observers was present for only 34% of the 3000 tumor bud candidates, while 59% of the candidates were agreed on by at least five of the seven observers. Despite reports of moderate-to-substantial agreement with respect to tumor budding grade, agreement with respect to individual pan cytokeratin-stained tumor buds is moderate at most. A machine learning approach may prove especially useful for a more robust assessment of individual tumor buds.

Topics: Biomarkers, Tumor; Colorectal Neoplasms; Humans; Immunohistochemistry; Keratins; Machine Learning; Observer Variation

Prediction of lymph node metastasis (LNM) for early colorectal cancer (CRC) is critical for determining treatment strategies after endoscopic resection. Some histologic parameters for predicting LNM have been established, but evaluator error and inter-observer disagreement are unsolved issues. Here we describe an LNM prediction algorithm for submucosal invasive (T1) CRC based on machine learning.. We conducted a retrospective single-institution study of 397 T1 CRCs. Several morphologic parameters were extracted from whole slide images of cytokeratin immunohistochemistry using Image J. A random forest algorithm for a training dataset (n = 277) was executed and used to predict LNM for the test dataset (n = 120). The results were compared with conventional histologic evaluation of hematoxylin-eosin staining.. Machine learning showed better LNM predictive ability than the conventional method on some datasets. Cross validation revealed no significant difference between the methods. Machine learning resulted in fewer false-negative cases than the conventional method.. Machine learning on whole slide images is a potential alternative for determining treatment strategies for T1 CRC.

Topics: Aged; Algorithms; Colorectal Neoplasms; Endoscopy; False Negative Reactions; False Positive Reactions; Female; Humans; Immunohistochemistry; Keratins; Lymph Nodes; Lymphatic Metastasis; Machine Learning; Male; Middle Aged; Neoplasm Metastasis; Retrospective Studies; Risk Factors

Eradication of cancer stem-like cells (CSLCs) are becoming increasingly an important target for new cancer therapies. The ability to study their behavior in vitro will provide the opportunity for high-throughput testing of more effective treatments. In this study, spheroid-like structures' formation and enrichment of HT29 CSLCs were evaluated on a wool keratin-based substrate as a bio-mimic of natural extracellular matrix (ECM) proteins. The results indicated that culturing on keratin substrate increased spheroid formation ability and radio-/chemoresistance of HT29 cells. Moreover, cell surface expression of CD133 CSLCs' marker and the mRNA level of stemness genes such as Nanog, Oct4, and c-MYC were increased. These data suggest that keratin can potentially be used for spheroid-like structure formation and enrichment of HT29 CSLCs. In addition, it seems that the induction of stemness characteristics on keratin substrate is probably because of the activation of α

Topics: Biomarkers, Tumor; Colorectal Neoplasms; Extracellular Matrix; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Keratins; Neoplastic Stem Cells; Signal Transduction; Spheroids, Cellular

Tumour budding predicts survival of stage II colorectal cancer (CRC) and has been suggested to be associated with epithelial-to-mesenchymal transition (EMT). However, the underlying molecular changes of tumour budding remain poorly understood. Here, we performed multiplex immunohistochemistry (mIHC) to phenotypically profile tumours using known EMT-associated markers: E-cadherin (adherence junctions), integrin β4 (ITGB4; basement membrane), ZO-1 (tight junctions), and pan-cytokeratin. A subpopulation of patients showed high ITGB4 expression in tumour buds, and this coincided with a switch of ITGB4 localisation from the basal membrane of intact epithelium to the cytoplasm of budding cells. Digital image analysis demonstrated that tumour budding with high ITGB4 expression in tissue microarray (TMA) cores correlated with tumour budding assessed from haematoxylin and eosin (H&E) whole sections and independently predicted poor disease-specific survival in two independent stage II CRC cohorts (hazard ratio [HR] = 4.50 (95% confidence interval [CI] = 1.50-13.5), n = 232; HR = 3.52 (95% CI = 1.30-9.53), n = 72). Furthermore, digitally obtained ITGB4-high bud count in random TMA cores was better associated with survival outcome than visual tumour bud count in corresponding H&E-stained samples. In summary, the mIHC-based phenotypic profiling of human tumour tissue shows strong potential for the molecular characterisation of tumour biology and for the discovery of novel prognostic biomarkers.

Topics: Adult; Aged; Aged, 80 and over; Antigens, CD; Biomarkers, Tumor; Cadherins; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Female; Humans; Integrin beta4; Keratins; Male; Middle Aged; Neoplasm Staging; Prognosis; Tissue Array Analysis

High-grade tumor budding is an adverse prognostic factor for submucosal invasive (T1) colorectal cancer used to predict the risk for lymph node metastasis in endoscopically resected specimens. Cytokeratin immunohistochemistry is a potential option for evaluating tumor budding. The optimal cut-off value between low- and high-grade budding has not yet been determined, however, and the high inter-observer variability in selecting budding foci remains problematic. We explored the optimal cut-off value for predicting lymph node metastasis using cytokeratin immunohistochemistry, and developed a novel computer-assisted semiautomatic quantification method to reduce inter-observer variability. A retrospective single-institution study of 463 T1 colorectal cancer cases was conducted. Cases were split into derivation and validation datasets. Tumor budding foci were counted manually and semiautomatically using Image J software on cytokeratin immunohistochemistry-stained specimens. We determined the cut-off values and compared inter-observer variability among pathologists between the two methods. Univariate and multivariate analyses of the derivation dataset were performed to select the risk factors for lymph node metastasis. Predictive simulation for the validation dataset was conducted. The optimal cut-off values for the manual and semiautomatic methods were ≥10 and ≥12, respectively. For both methods, multivariate analyses revealed that venous invasion, lymphatic invasion, and high-grade tumor budding were independent risk factors for lymph node metastasis. The semiautomatic method provided significantly better inter-observer agreement. The predictive and observed lymph node metastasis frequencies were highly correlated in the validation dataset.

Topics: Aged; Automation, Laboratory; Biomarkers, Tumor; Cell Movement; Colorectal Neoplasms; Diagnosis, Computer-Assisted; Female; Humans; Image Interpretation, Computer-Assisted; Immunohistochemistry; Keratins; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Grading; Neoplasm Staging; Observer Variation; Predictive Value of Tests; Reproducibility of Results; Retrospective Studies; Risk Factors

The aim of this study was to clarify the quantitative and qualitative differences in tumour budding identification between haematoxylin and eosin (H&E) staining and immunohistochemical (IHC) staining for cytokeratin, and to estimate the respective clinical impacts in stage II colorectal cancer.. We retrospectively examined 314 surgically resected cases of stage II colorectal cancer, and assessed tumour budding on serial section slides with H&E staining and IHC staining for cytokeratin. Tumour budding counts based on cytokeratin-stained slides were strongly correlated with those based on H&E-stained slides, and had higher detection and reproducibility. On the basis of receiver operating characteristic analyses, the optimal cut-off values of budding counts for relapse-free survival (RFS) were 7 and 16 in a ×200 microscopic field with H&E and IHC staining, respectively. With these cut-off values, tumour budding based on H&E staining had a significant correlation with RFS (80.3% and 93.2% of 5-year RFS in the high-budding group and the low-budding group, respectively), and similar results were observed for IHC staining (79.9% and 91.7%, respectively). The Akaike Information Criterion value for RFS with H&E staining was favourable as compared with that with IHC staining.. Tumour budding counts based on cytokeratin-stained slides showed higher detection and better reproducibility, but did not have as satisfactory clinical impacts as those based on H&E staining.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma; Colorectal Neoplasms; Eosine Yellowish-(YS); Female; Humans; Immunohistochemistry; Keratins; Male; Middle Aged; Neoplasm Staging; Prognosis; Reproducibility of Results; Retrospective Studies; ROC Curve; Staining and Labeling

Tumor buds in colorectal cancer are hypothesized to undergo a (partial) epithelial-mesenchymal transition (EMT). If so, cytokeratin (CK) and vimentin (VIM) co-expression is expected. CK+/VIM+ can also be found in some stromal cells; however, their origin remains unclear. Here, we determine the frequency of CK+/VIM+ tumor cells and characterize the CK+/VIM+ stroma in colorectal cancer. Three cell populations (CK+, VIM+, CK+/VIM+) were sorted using DepArray and fluorescence-activated cell sorting (FACS). Tumor areas were selected to include tumor center, stroma and tumor budding. Fluorescence microscopy was used to visualize co-expressing cells on whole slides. A next-generation tissue microarray (ngTMA) of matched Pan-CK-positive and -negative stroma was constructed and stained for E-cadherin, VIM, Snail1, Twist1, Zeb1 and Zeb2, COL11A1, SPARC, CD90, α-SMA, FAP and WT1. CK+/VIM+ co-expressing tumor cells were detected using all three methods. With DepArray, only tumor budding areas contained CK+/VIM+ cells. The proportion of CK+/VIM+ tumor cells was low (1.5%-22%). CK+ stroma was associated with aggressive tumor features like distant metastasis (P = .0003), lymphatic invasion (P = .0009) and tumor budding (P = .0084). CK+/VIM+ stroma was characterized by positive WT1 (P < .001), ZEB2 (P < .001), TWIST1 (P = .009), and FAP (P = .003). Our data suggest that CK+/VIM+ tumor cells exist, albeit in low numbers and could represent a subgroup of tumor buds in partial EMT. CK+/VIM+ stroma may be of mesothelial origin and shows features of mesenchymal cells and cancer-associated fibroblasts. These results, together with the association with metastasis point to cells in mesothelial-mesenchymal transition (MMT). This atypical stroma may be a potential target for therapy.

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Female; Humans; Keratins; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Stromal Cells; Vimentin

Few data are available regarding the epithelial to mesenchymal transition (EMT) /mesenchymal to epitheilal transition (MET) in the liver metastasis of digestive cancers. The aim of this study was to establish EMT/MET metastatic tumor cell plasticity according to the histological growth pattern of liver metastases.. Biopsies from 25 patients with liver metastasis (desmoplastic, replacement and pushing type) were evaluated. Double immunostaining of E-cadherin/vimentin, keratin 8,18/vimentin and E-cadherin/ keratin 8,18 were performed.. The following cell types were noted: epithelial, mesenchymal, non-differentiated and differentiated hybrid mesenchymal/ epithelial and non-hybrid phenotype. All cases had mesenchymal/ epithelial phenotype cells. A significant correlation was found between the non-differentiated hybrid mesenchymal/ epithelial phenotype metastatic cells and histological growth pattern for gastric and colorectal cancer.. A MET-targeting strategy, in conjunction with conventional chemotherapy, may be useful for the treatment of liver metastases.

Topics: Antigens, CD; Cadherins; Cell Plasticity; Colorectal Neoplasms; Digestive System Neoplasms; Epithelial-Mesenchymal Transition; Humans; Immunohistochemistry; Keratins; Liver Neoplasms; Pancreatic Neoplasms; Stomach Neoplasms; Vimentin

Tumor budding is thought to represent a manifestation of epithelial-to-mesenchymal transition (EMT) and it has been correlated with poor patient outcomes in colorectal cancer (CRC). Our group recently demonstrated that human chorionic gonadotropin-β (hCGβ) modulates EMT in CRC. In the current study, based on the likely relationships between tumor budding and hCGβ expression, we examined their clinicopathologic significance in CRC. Twenty-eight of 80 (35.0%) CRC showed tumor budding. Tumor budding significantly correlated with lymph node metastasis (P < 0.01), pathologic stage (P < 0.01), lymphatic invasion (P = 0.044), and vascular invasion (P = 0.013). Thirteen of 80 (16.3%) CRC were hCGβ positive on immunohistochemistry. More tumor buds were present in the hCGβ-positive cases (P < 0.01), and tumor budding was significantly correlated with hCGβ positivity (P < 0.01). Cases with both tumor budding and hCGβ expression had the poorest prognosis compared with all other groups (P < 0.01). In conclusion, tumor budding and hCGβ expression are closely associated with EMT, and they are independent prognostic factors in CRC. They identify patients with an "EMT phenotype" who may respond to targeted molecular therapies.

Topics: Aged; Biomarkers, Tumor; Chorionic Gonadotropin, beta Subunit, Human; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Female; Humans; Immunohistochemistry; Keratins; Male; Middle Aged; Neoplasm Invasiveness; Prognosis; Proportional Hazards Models; Survival Analysis

Tumor budding is an independent prognostic factor in colorectal cancer. However, varying degrees of interobserver agreement and reproducibility challenges the use of tumor budding in diagnostics. Immunohistochemical staining of tumor slides with pan-cytokeratin visualizes the budding tumor cells and has been suggested to improve reproducibility. Here we demonstrate the methodology of tumor budding assessment using digital image analysis based on tumor slides stained for pan-cytokeratin, and investigate interobserver agreement, agreement between manual and digital assessment methods and digital reproducibility between users. Tumor slides from 126 patients with pT1/pT2 colorectal cancer were stained with pan-cytokeratin and tumor budding at the invasive tumor front was assessed by conventional manual microscopy. A digital image analysis algorithm for identification and quantification of budding tumor cells was developed and tested on the pan-cytokeratin stained slides. Manual assessment of tumor budding using pan-cytokeratin stained tumor slides exhibited high correlations (Spearman Rank 0.84-0.89, p < 0.001),excellent agreement between observers (Intra-class correlation coefficient (ICC): 0.86 -0.87) and 2.20 higher odds for regional metastases with increasing budding counts (p = 0.017). Digital image analysis correlated well to manual assessment (Spearman Rank 0.71-0.88) and agreement between the two methods was good (ICC 0.62-0.82). However, only a trend towards increased odds for metastatic progression was found for the adjusted digital estimates (p = 0.076). Digital estimates were higher than manual estimates, demonstrated by a systematic median difference of 3-4.5 buds. Image analysis was highly reproducible between users of the algorithm (ICC 0.98). In conclusion, assessment of tumor budding using pan-cytokeratin stained tumor slides is a method with high correlation and agreement between observers. Digital image analysis quantifies budding tumor cells in high agreement with manual estimates, but approval of the digital slides by a pathologist is mandatory. The method qualifies for further investigation.

Topics: Algorithms; Biomarkers, Tumor; Colorectal Neoplasms; Feasibility Studies; Humans; Image Interpretation, Computer-Assisted; Keratins; Observer Variation; Reproducibility of Results

Tumor budding, meaning a detachment of tumor cells at the invasion front of colorectal carcinoma (CRC) into single cells or clusters (<=5 tumor cells), has been shown to correlate to an inferior clinical outcome by several independent studies. Therefore, it has been discussed as a complementary prognostic factor to the TNM staging system, and it is already included in national guidelines as an additional prognostic parameter. However, its application by manual evaluation in routine pathology is hampered due to the use of several slightly different assessment systems, a time-consuming manual counting process and a high inter-observer variability. Hence, we established and validated an automatic image processing approach to reliably quantify tumor budding in immunohistochemically (IHC) stained sections of CRC samples.. This approach combines classical segmentation methods (like morphological operations) and machine learning techniques (k-means and hierarchical clustering, convolutional neural networks) to reliably detect tumor buds in colorectal carcinoma samples immunohistochemically stained for pan-cytokeratin. As a possible application, we tested it on whole-slide images as well as on tissue microarrays (TMA) from a clinically well-annotated CRC cohort.. Our automatic tumor budding evaluation tool detected the absolute number of tumor buds per image with a very good correlation to the manually segmented ground truth (R2 value of 0.86). Furthermore the automatic evaluation of whole-slide images from 20 CRC-patients, we found that neither the detected number of tumor buds at the invasion front nor the number in hotspots was associated with the nodal status. However, the number of spatial clusters of tumor buds (budding hotspots) significantly correlated to the nodal status (p-value = 0.003 for N0 vs. N1/N2). TMAs were not feasible for tumor budding evaluation, as the spatial relationship of tumor buds (especially hotspots) was not preserved.. Automatic image processing is a feasible and valid assessment tool for tumor budding in CRC on whole-slide images. Interestingly, only the spatial clustering of the tumor buds in hotspots (and especially the number of hotspots) and not the absolute number of tumor buds showed a clinically relevant correlation with patient outcome in our data.

Topics: Aged; Automation, Laboratory; Biomarkers, Tumor; Cell Adhesion; Cell Movement; Cluster Analysis; Colorectal Neoplasms; Female; Humans; Image Interpretation, Computer-Assisted; Immunohistochemistry; Keratins; Machine Learning; Male; Neoplasm Invasiveness; Neural Networks, Computer; Observer Variation; Predictive Value of Tests; Prognosis; Reproducibility of Results; Tissue Array Analysis

Background: Colorectal cancer (CRC) is a heterogeneous disease with a complex etiology. New prognostic factors\ need to be investigated. Our present focus is on histopathological significance and prognostic impact of tumor budding\ in CRC. Material and Methods: A total of 60 treatment-naive consecutive patients undergoing surgical resection of\ CRCs during the period of January 2011 to December 2013 were included in the study. Details of each related to their\ demographic and tumor profile were recorded. Hematoxylin and Eosin (H and E) and pan-cytokeratin details of each\ “case” immunohistochemically stained sections were examined for tumor budding assessment along with clinical\ features. Results: The most frequent site of involvement was the rectosigmoid and sigmoid colon (31.6%). The majority\ of the cases were moderately differentiated (75%), showed tumor invasion into the pericolic/subserosal fat (66.6%) and\ stage III (38.3%). Nodal involvement was present in 47%. Correlations between tumor budding and nodal involvement\ (p-value 0.039) and AJCC stage (p-value 0.021) were found to be statistically significant. Conclusion: Tumor budding\ is a promising and powerful predictor of lymph nodal metastasis and a higher stage of tumor and can be used as a\ marker for high-risk CRC. Routine H and E staining aided by cytokeratin immunostaining allows reproducible grading\ of tumor budding in CRC cases.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Colorectal Neoplasms; Eosine Yellowish-(YS); Female; Hematoxylin; Humans; Immunohistochemistry; Keratins; Lymphatic Metastasis; Male; Middle Aged; Prognosis; Staining and Labeling; Young Adult

Tumor budding is thought to reflect the epithelial-mesenchymal transition (EMT). However, the molecular mechanism linking tumor buds and the EMT remains unclear. Here, we examined the induction of tumor budding and EMT and their association with EMT-related proteins (ZEB1, TWIST, SNAIL, and SLUG) in colorectal cancer (CRC). Immunohistochemical expression of pan-cytokeratin was examined for identification of tumor budding in 101 CRCs. Grading of tumor budding was classified into low- and high-grade groups. Tissue microarray was conducted to identify tumor budding sites. The expression of E-cadherin, ZEB1, TWIST, SNAIL, and SLUG was examined in areas of tumor budding and the surrounding tumor stroma using a double-immunostaining method. Specifically, pan-cytokeratin and EMT-related proteins were assessed by double immunostaining. Low or no expression of E-cadherin was found in areas of tumor budding. Moreover, ZEB1, TWIST, SNAIL, and SLUG were not expressed in regions of tumor budding. However, the expression level of ZEB1 in the stromal cells surrounding tumor budding was significantly more frequent than that of TWIST, SNAI, and SLUG. In addition, the expression of EMT-related proteins in surrounding stromal cells was significantly greater in areas of high-grade tumor budding than in low-grade areas. Our present results suggest that EMT-related proteins play a minor role in forming tumor buds. In addition, our findings suggest the existence of subtypes of stromal cells in CRC with phenotypical and functional heterogeneity.

Topics: Adult; Aged; Aged, 80 and over; Antigens, CD; Biomarkers, Tumor; Cadherins; Cell Movement; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Female; Humans; Immunohistochemistry; Keratins; Male; Middle Aged; Neoplasm Grading; Neoplasm Invasiveness; Nuclear Proteins; Phenotype; Snail Family Transcription Factors; Stromal Cells; Tissue Array Analysis; Twist-Related Protein 1; Zinc Finger E-box-Binding Homeobox 1

Tumour budding in colorectal cancer (CRC) is a recognized prognostic parameter. The aim of this study was to address the use of cytokeratin immunostaining for the visualization and scoring of tumour buds.. Ten hotspots (0.238 mm. An OTB count in a single hotspot on cytokeratin-stained CRC tissue sections is a fast and reliable prognostic scoring system for the assessment of tumour budding. This approach should be considered in future studies.

Topics: Adult; Aged; Biomarkers, Tumor; Colorectal Neoplasms; Disease-Free Survival; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Keratins; Male; Middle Aged; Prognosis; Staining and Labeling

Tumor budding is a major risk factor for T1 colorectal cancer. Quality control of the pathological diagnosis of budding is crucial, irrespective of the pathologist's experience. This study examines the interobserver variability according to pathologists' experience and evaluates the influence of cytokeratin (CK) immunostaining in the assessment of budding. Hematoxylin-eosin (HE) and CK-immunostained slides of 40 cases with T1 primary colorectal cancer were examined. Budding grades were individually evaluated by 12 pathologists who we categorized into three groups by their experience (expert, with >10 years of experience (n = 4), senior, with 5-10 years (n = 4), and junior, < 5 years (n = 4)). The results revealed a tendency for the more experienced pathologists to assign higher budding grades compared to the less-experienced pathologists. In the junior group, the interobserver variability obtained with HE slides was poor, but it was markedly improved in the evaluation using CK-immunostained slides. The benefit of CK immunostaining was only slight in the expert group. CK immunostaining would be useful when a pathologist is not experienced enough or does not have enough confidence in the assessment of budding.

Topics: Colorectal Neoplasms; Humans; Immunohistochemistry; Keratins; Lymphatic Metastasis; Neoplasm Grading; Observer Variation; Reproducibility of Results

Tumor budding is recognized as an important risk factor for lymph node metastasis in pT1 colorectal cancer. Immunohistochemical staining for cytokeratin has the potential to improve the objective diagnosis of tumor budding over detection based on hematoxylin and eosin staining. However, it remains unclear whether tumor budding detected by immunohistochemical staining is a significant predictor of lymph node metastasis in pT1 colorectal cancer.. The purpose of this study was to clarify the clinical significance of tumor budding detected by immunohistochemical staining in comparison with that detected by hematoxylin and eosin staining.. This was a retrospective study.. The study was conducted at Niigata University Medical & Dental Hospital.. We enrolled 265 patients with pT1 colorectal cancer who underwent surgery with lymph node dissection.. Tumor budding was evaluated by both hematoxylin and eosin and immunohistochemical staining with the use of CAM5.2 antibody. Receiver operating characteristic curve analyses were conducted to determine the optimal cutoff values for tumor budding detected by hematoxylin and eosin and CAM5.2 staining. Univariate and multivariate analyses were performed to identify the significant factors for predicting lymph node metastasis.. Receiver operating characteristic curve analyses revealed that the cutoff values for tumor budding detected by hematoxylin and eosin and CAM5.2 staining for predicting lymph node metastases were 5 and 8. On multivariate analysis, histopathological differentiation (OR, 6.21; 95% CI, 1.16-33.33; p = 0.03) and tumor budding detected by hematoxylin and eosin staining (OR, 4.91; 95% CI, 1.64-14.66; p = 0.004) were significant predictors for lymph node metastasis; however, tumor budding detected by CAM5.2 staining was not a significant predictor.. This study was limited by potential selection bias because surgically resected specimens were collected instead of endoscopically resected specimens.. Tumor budding detected by CAM5.2 staining was not superior to hematoxylin and eosin staining for predicting lymph node metastasis in pT1 colorectal cancer.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Biomarkers; Biomarkers, Tumor; Colorectal Neoplasms; Coloring Agents; Eosine Yellowish-(YS); Female; Hematoxylin; Humans; Immunohistochemistry; Keratins; Logistic Models; Lymph Node Excision; Lymphatic Metastasis; Male; Middle Aged; Multivariate Analysis; Neoplasm Staging; Retrospective Studies; ROC Curve

Clusters of tumor cells are often observed in the blood of cancer patients. These structures have been described as malignant entities for more than 50 years, although their comprehensive characterization is lacking. Contrary to current consensus, we demonstrate that a discrete population of circulating cell clusters isolated from the blood of colorectal cancer patients are not cancerous but consist of tumor-derived endothelial cells. These clusters express both epithelial and mesenchymal markers, consistent with previous reports on circulating tumor cell (CTC) phenotyping. However, unlike CTCs, they do not mirror the genetic variations of matched tumors. Transcriptomic analysis of single clusters revealed that these structures exhibit an endothelial phenotype and can be traced back to the tumor endothelium. Further results show that tumor-derived endothelial clusters do not form by coagulation or by outgrowth of single circulating endothelial cells, supporting a direct release of clusters from the tumor vasculature. The isolation and enumeration of these benign clusters distinguished healthy volunteers from treatment-naïve as well as pathological early-stage (≤IIA) colorectal cancer patients with high accuracy, suggesting that tumor-derived circulating endothelial cell clusters could be used as a means of noninvasive screening for colorectal cancer. In contrast to CTCs, tumor-derived endothelial cell clusters may also provide important information about the underlying tumor vasculature at the time of diagnosis, during treatment, and throughout the course of the disease.

Topics: Cell Line; Colorectal Neoplasms; Humans; Keratins; Leukocyte Common Antigens; Multigene Family; Neoplastic Cells, Circulating; Prognosis; Tumor Cells, Cultured

Management of patients with stage II colorectal carcinomas remains challenging as 20 - 30% of them will develop recurrence. It is postulated that these patients may harbour nodal micrometastases which are imperceptible by routine histopathological evaluation. The aims of our study were to evaluate (1) the feasibility of multilevel sectioning method utilizing haematoxylin and eosin stain and immunohistochemistry technique with cytokeratin AE1/AE3, in detecting micrometastases in histologically-negative lymph nodes, and (2) correlation between nodal micrometastases with clinicopathological parameters. Sixty two stage I and II cases with a total of 635 lymph nodes were reviewed. Five-level haematoxylin and eosin staining and one-level cytokeratin AE1/AE3 immunostaining were performed on all lymph nodes retrieved. The findings were correlated with clinicopathological parameters. Two (3.2%) lymph nodes in two patients (one in each) were found to harbour micrometastases detected by both methods. With cytokeratin AE1/AE3, we successfully identified four (6.5%) patients with isolated tumour cells, but none through the multilevel sectioning method. Nodal micrometastases detected by both multilevel sectioning and immunohistochemistry methods were not associated with larger tumour size, higher depth of invasion, poorer tumour grade, disease recurrence or distant metastasis. We conclude that there is no difference between the two methods in detecting nodal micrometastases. Therefore it is opined that multilevel sectioning is a feasible and yet inexpensive method that may be incorporated into routine practice to detect nodal micrometastases in centres with limited resources.

Topics: Adult; Aged; Aged, 80 and over; Colorectal Neoplasms; Eosine Yellowish-(YS); Female; Hematoxylin; Humans; Immunohistochemistry; Keratins; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Micrometastasis; Neoplasm Recurrence, Local; Neoplasm Staging; Staining and Labeling

Tumor budding/sprouting has been shown to be an independent adverse prognostic factor in T1 and T3N0 colorectal carcinomas, however, its assessment could be improved by more accurate identification of budding carcinoma cells and consideration of budding areas. Moreover, tumor budding mechanisms are yet to be defined. In this study, we evaluated the identification of budding tumor cells by either H&E staining alone or H&E with immunohistochemistry and developed a scoring system based on budding grades and areas. We examined whether the budding score correlated with clinicopathologic features and prognosis and the association between tumor budding/sprouting and c-Met protein expression and phosphorylation and MET gene copy numbers because c-Met is known to play an important role in colorectal carcinoma tumorigenesis. Cytokeratin immunohistochemistry could identify tumors with shorter disease-free survival (DFS) from the low-grade budding group assessed with H&E alone. High budding scores based on budding grade and area were more significantly correlated with DFS than scores obtained using the budding grade alone. In tumors with a high budding score, c-Met expression and phosphorylation levels and MET gene copy numbers were significantly increased at the invasive front compared with those in superficial tumor portions. This study showed for the first time that high levels of phospho-c-Met at the invasive front were significantly associated with a high budding score and shorter DFS. In conclusion, a budding score assessed by budding grades and budding-positive areas correlates highly with clinicopathologic aggressive features of colorectal carcinoma.

Topics: Adult; Aged; Aged, 80 and over; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Keratins; Male; Middle Aged; Neoplasm Grading; Neoplasm Metastasis; Neoplasm Staging; Phosphorylation; Prognosis; Proto-Oncogene Proteins c-met; Retrospective Studies

The prognostic value of mucin expression has been reported in several studies. We examined the association between mucin expression and other previously reported prognostic factors, including infiltration of CD10⁺ myeloid cells, transforming growth factor-β1 (TGF-β1) expression, and tumor budding at invasion fronts. Immunohistochemical analysis of 206 colorectal samples was carried out to determine whether MUC1, MUC2, MUC4, and MUC5AC expression could predict the survival of colorectal cancer patients. Serial sections were stained for CD10, TGF-β1, and pan-cytokeratin in order to detect tumor budding. As per multivariate analyses, MUC1 expression appeared to be the most significant predictor of both recurrence-free survival and overall survival. MUC4 was only significant to predict recurrence-free survival, and MUC5AC could be a good marker in stage IV colorectal cancers that require additional chemotherapy. MUC1 (CD227) expression was associated with infiltration of CD10⁺ myeloid cells, TGF-β1 expression, and tumor budding grade. These findings suggest that MUC1 is indicative of poor prognoses that may be associated with immunosuppression and epithelial-mesenchymal transition. Furthermore, MUC1 expression appears to be a chemoattractant for CD10⁺ stromal cells.

Topics: Biomarkers, Tumor; Colorectal Neoplasms; Disease-Free Survival; Female; Humans; Keratins; Male; Middle Aged; Mucin-1; Myeloid Cells; Neprilysin; Transforming Growth Factor beta1

Keratin filaments form cytoskeletal networks in epithelial cells. Dynamic rearrangement of keratin filament networks is required for epithelial cells to perform cellular processes such as cell migration and polarization; however, the mechanism governing keratin filament rearrangement remains unclear. Here, we describe a novel mechanism of keratin cytoskeleton organization mediated by casein kinase Iα (CK-1α) and a newly identified keratin-associated protein, FAM83H. Knockdown of FAM83H induces keratin filament bundling, whereas overexpression of FAM83H disassembles keratin filaments, suggesting that FAM83H regulates the filamentous state of keratins. Intriguingly, keratin filament bundling is concomitant with the dissociation of CK-1α from keratin filaments, whereas aberrant speckle-like localization of CK-1α is observed concomitantly with keratin filament disassembly. Furthermore, CK-1α inhibition, similar to FAM83H knockdown, causes keratin filament bundling and reverses keratin filament disassembly induced by FAM83H overexpression, suggesting that CK-1α mediates FAM83H-dependent reorganization of keratin filaments. Because the N-terminal region of FAM83H interacts with CK-1α and the C-terminal region interacts with keratins, FAM83H might tether CK-1α to keratins. Colorectal cancer tissue also shows keratin filament disassembly accompanied with FAM83H overexpression and aberrant CK-1α localization, and FAM83H-overexpressing cancer cells exhibit loss or alteration of epithelial cell polarity. Importantly, knockdown of FAM83H inhibits cell migration accompanied by keratin cytoskeleton rearrangement in colorectal cancer cells. These results suggest that keratin cytoskeleton organization is regulated by FAM83H-mediated recruitment of CK-1α to keratins, and that keratin filament disassembly caused by overexpression of FAM83H and aberrant localization of CK-1α could contribute to the progression of colorectal cancer.

Topics: Casein Kinase Ialpha; Cell Line, Tumor; Colorectal Neoplasms; Cytoskeleton; HCT116 Cells; Humans; Keratins; Proteins; RNA, Small Interfering

Basaloid squamous cell carcinoma is a rare and aggressive variant of squamous cell carcinoma, which mostly occurs in the upper aerodigestive tracts. Basaloid squamous cell carcinoma also typically arises in the anal canal, but is extremely rare in the lower gastrointestinal tract. A 70-year-old man presented with loose stool and intermittent hematochezia 2 months ago. Colonoscopy showed an ulceroinfiltrative mass on the rectosigmoid colon from 16 cm to 18 cm above the anal verge. Conventional colonoscope could not pass through the lesion but it was possible with pediatric colonoscope. Abdominal CT scan showed 1.6 cm sized wall thickening with circumferential luminal narrowing in the rectosigmoid colon and multiple ill-defined low density masses in both lobes of the liver. Therefore, colon cancer with liver metastasis was suspected. However, basaloid cells were noted on histologic examination, and they were weakly positive for synaptophysin on immunohistochemical study. After palliative lower anterior resection, histologic examination of the resected specimen revealed basaloid differentiation with keratin pearls, and tumor cells were positively stained with high molecular weighted cytokeratin (34BE12) and CK 5/6. Thus, the patient was finally diagnosed with basaloid squamous cell carcinoma of rectosigmoid colon with distant metastases.

Topics: Aged; Carcinoma, Squamous Cell; Colonoscopy; Colorectal Neoplasms; Humans; Immunohistochemistry; Keratins; Liver Neoplasms; Lung Neoplasms; Male; Positron-Emission Tomography; Synaptophysin; Tomography, X-Ray Computed

Our aim was to examine cell transition events by detecting the frequency of intrapithelial α-smooth muscle actin (SMA)(+)/cytokeratin (CK)(+) cells during colorectal adenoma-carcinoma sequence, in relation to E-cadherin expression. Our further aim was to determine the proliferative activity of intraepithelial α-SMA(+) cells. Histologically healthy, adenoma, and colorectal cancer (CRC) biopsy samples were taken during routine colonoscopy and were included into tissue microarrays (TMAs). Slides immunostained for Ki-67, α-SMA, E-cadherin and pan-cytokeratin were digitalized and analyzed by using a digital microscope software. The proportion of α-SMA(+)/CK(+) cells was significantly higher in CRC samples (3.34 ± 1.01%) compared to healthy (1.94 ± 0.69%) or adenoma (1.62 ± 0.78%) samples (p < 0.01). E-cadherin expression negatively correlated with the number of α-SMA(+) cells. The majority of intraepithelial α-SMA(+) cells were in the proliferative phase. During tumor progression, the appearance of dot-like α-SMA staining in CK positive cells may indicate the initial phase of the epithelial-to-mesenchymal transition (EMT). The high proportion of intraepithelial α-SMA(+) proliferating cells may refer to their increased plasticity compared to differentiated cells. The negative correlation between E-cadherin and intraepithelial α-SMA expression suggests that EMT is facilitated by a loss of epithelial cell contact.

Topics: Actins; Adenoma; Biomarkers, Tumor; Cadherins; Cell Differentiation; Cell Transformation, Neoplastic; Colon; Colorectal Neoplasms; Disease Progression; Epithelial-Mesenchymal Transition; Follow-Up Studies; Humans; Immunoenzyme Techniques; Keratins; Longitudinal Studies; Neoplasm Staging; Prognosis; Rectum; Tissue Array Analysis

It is not clear if sentinel lymph node (SLN) mapping can improve outcomes in patients with colorectal cancers. The purpose of this study was to determine the prognostic values of ex vivo sentinel lymph node (SLN) mapping and immunohistochemical (IHC) detection of SLN micrometastasis in colorectal cancers.. Colorectal cancer specimens were obtained during radical resections and the SLN was identified by injecting a 1% isosulfan blue solution submucosally and circumferentially around the tumor within 30 min after surgery. The first node to stain blue was defined as the SLN. SLNs negative by hematoxylin and eosin (HE) staining were further examined for micrometastasis using cytokeratin IHC.. A total of 54 patients between 25 and 82 years of age were enrolled, including 32 males and 22 females. More than 70% of patients were T3 or above, about 86% of patients were stage II or III, and approximately 90% of patients had lesions grade II or above. Sentinel lymph nodes were detected in all 54 patients. There were 32 patients in whom no lymph node micrometastasis were detected by HE staining and 22 patients with positive lymph nodes micrometastasis detected by HE staining in non-SLNs. In contrast only 7 SLNs stained positive with HE. Using HE examination as the standard, the sensitivity, non-detection rate, and accuracy rate of SLN micrometastasis detection were 31.8% (7/22), 68.2% (15/22), and 72.2%, respectively. Micrometastasis were identified by ICH in 4 of the 32 patients with HE-negative stained lymph nodes, resulting in an upstaging rate 12.5% (4/32). The 4 patients who were upstaged consisted of 2 stage I patients and 2 stage II patients who were upstaged to stage III. Those without lymph node metastasis by HE staining who were upstaged by IHC detection of micrometastasis had a significantly poorer disease-free survival (p = 0.001) and overall survival (p = 0.004).. Ex vivo localization and immunohistochemical detection of sentinel lymph node micrometastasis in patients with colorectal cancer can upgrade tumor staging, and may become a factor affecting prognosis and guiding treatment.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Colorectal Neoplasms; Disease-Free Survival; Feasibility Studies; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Keratins; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Multivariate Analysis; Neoplasm Micrometastasis; Neoplasm Staging; Predictive Value of Tests; Prognosis; Proportional Hazards Models; Risk Assessment; Risk Factors; Sensitivity and Specificity; Sentinel Lymph Node Biopsy; Time Factors

The occurrence of adenocarcinoma after ileal pouch-anal anastomosis for ulcerative colitis (UC) is an infrequent but potentially lethal complication. Neither histomorphologic nor molecular features of pouch adenocarcinoma after ileal pouch-anal anastomosis have been fully investigated. We report the largest series of 12 pouch and peripouch adenocarcinomas and compared them with 58 randomly selected UC-associated adenocarcinomas. The mean age of patients with pouch/peripouch adenocarcinoma was 55.2 years (SD 14.8), which was not significantly different from that of controls (P=0.52). Pouch/peripouch adenocarcinoma and UC-associated adenocarcinoma had a comparable frequency of tumor-infiltrating lymphocytes, lack of dirty necrosis, mucin differentiation, signet ring cell differentiation, heterogeneity, and well differentiation (P>0.05 for all). Pouch/peripouch adenocarcinoma was more likely to show Crohn-like reaction compared with UC-associated adenocarcinoma (P=0.047). Loss of at least 1 mismatch repair protein was noted in 9% of pouch/peripouch adenocarcinomas and 9.6% of UC-related adenocarcinomas (P=1.0). There was no significant difference in the frequency of p53 overexpression (36.4% vs. 61.1%, P=0.184) or nuclear immunoreactivity for β-catenin (9% vs. 7.4%, P=0.99) in pouch/peripouch versus UC-associated adenocarcinomas, respectively. Pouch/peripouch and UC-associated adenocarcinoma had a comparable positive rate for CK7 (54.5% vs. 55.5%, P=0.99), CK20 (100% vs. 98.1%, P=0.99), and CDX2 (72.8% vs. 72.2%, P=0.99) by immunohistochemistry. In summary, pouch and peripouch adenocarcinoma can occur in patients without colorectal neoplasia and in those with idiopathic inflammatory bowel disease, can be potentially lethal, and has histomorphologic and molecular features similar to those of UC-associated adenocarcinoma.

Topics: Adenocarcinoma; Biomarkers, Tumor; CDX2 Transcription Factor; Colectomy; Colitis, Ulcerative; Colonic Pouches; Colorectal Neoplasms; DNA-Binding Proteins; Female; Homeodomain Proteins; Humans; Keratins; Lymphocytes, Tumor-Infiltrating; Male; Middle Aged; MutS Homolog 3 Protein; Neoplasm Staging; Postoperative Complications; Proctocolectomy, Restorative; Tissue Array Analysis

Lymph nodes from patients with colorectal cancer were immunohistochemically stained for cytokeratin to investigate the relationship between the presence of occult neoplastic cells (ONCs) and recurrence/metastasis. A total of 80 patients with stage III/Dukes' C colorectal cancer were divided into 16 patients who developed recurrence/metastasis (recurrence group) and 64 patients without recurrence (non-recurrence group). ONCs were compared between the two groups with respect to i) single cells (≥ 3 floating ONCs), ii) clusters of cells (1 or more floating aggregates of 2-20 ONCs) and iii) single cells + clusters. When single cells were detected, the sensitivity for recurrence was 87.5% (14/16, p = 0.002), the positive predictive value (PPV) was 32.6% (14/43), the specificity was 54.7% (35/64) and the negative predictive value (NPV) was 94.6% (35/37). For clusters, the sensitivity was 87.5% (14/16, p<0.001), PPV was 41.2% (14/34), specificity was 68.8% (44/64) and NPV 95.7% (44/46). With single cells + clusters, the values were 87.5% (14/16, p<0.001), 48.3% (14/29), 76.6% (49/64) and 96.1% (49/51), respectively. These results suggest that the detection of single cells + clusters is a sensitive indicator of a high risk of recurrence/ metastasis, while ONCs are useful for identifying the low-risk group of patients with stage III colorectal cancer.

Topics: Colorectal Neoplasms; Humans; Immunoenzyme Techniques; Keratins; Lymph Nodes; Lymphatic Metastasis; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; Sensitivity and Specificity

The study was performed to determine detection rate and prognostic relevance of disseminated tumour cells (DTC) in patients receiving curatively intended surgery for colorectal cancer (CRC).. The study population consisted of 235 patients with CRC prospectively recruited from five hospitals in the Oslo region. Bone marrow (BM) aspirates were collected at the time of surgery and the presence of DTC was determined by two immunological methods; immunomagnetic selection (using an anti-EpCAM antibody) and immunocytochemistry (using a pan-cytokeratin antibody). Associations between the presence of DTC and metastasis-free, disease-specific and overall survival were analysed using univariate and multivariate methods.. Disseminated tumour cells were detected in 41 (17%) and 28 (12%) of the 235 examined BM samples by immunomagnetic selection and immunocytochemistry, respectively, with only five samples being positive with both methods. The presence of DTC was associated with adverse outcome (metastasis-free, disease-specific and overall survival) in univariate and multivariate analyses.. The presence of DTC was associated with adverse prognosis in this cohort of patients curatively resected for CRC, suggesting that DTC detection still holds promise as a biomarker in CRC.

Topics: Adult; Aged; Aged, 80 and over; Analysis of Variance; Antigens, Neoplasm; Bone Marrow; Cell Adhesion Molecules; Colorectal Neoplasms; Disease-Free Survival; Epithelial Cell Adhesion Molecule; Female; Humans; Immunohistochemistry; Immunomagnetic Separation; Kaplan-Meier Estimate; Keratins; Male; Middle Aged; Neoplasm Staging; Neoplastic Cells, Circulating; Norway; Predictive Value of Tests; Prognosis; Prospective Studies

Although patients with Stage I colorectal cancer show an excellent prognosis, a few of them die of metastatic disease. In this subgroup of individuals, the search of occult metastasis might reveal that early dissemination of tumor cells could be the cause of cancer progression.. Through a Cancer Registry, we selected all patients with Stage I disease who died of metastatic tumor; a total of 32 patients were identified and in 25 of them paraffin-embedded material was available. The group was matched to 70 Stage I patients with favorable prognosis (controls). In cases and controls resected lymph nodes were cut, and micrometastases were searched using pan-cytokeratin antibodies.. Micrometastases were detected in 18 of 25 (72%) Stage I patients who died of the disease, while they were almost absent among controls (1 of 70, p < 0.001 by χ(2) test). Vascular invasion and tumor budding were more frequent among Stage I patients with an unfavorable prognosis than in controls. By regression analyses, micrometastases (HR 12.3, CI 4.8-32) and vascular invasion (HR 3.5, CI 1.4-8.5) maintained an independent association with prognosis (cancer-specific survival).. Micrometastasis in the lymph nodes can be revealed in the majority of patients with early colorectal cancer who die of tumor progression, while they appear extremely rare in Stage I individuals with good prognosis. The selection of patients through histology (vascular invasion) and search of occult metastatic cells might represent a way to identify individuals who might benefit from adjuvant chemotherapy.

Topics: Aged; Blood Vessels; Carcinoma; Case-Control Studies; Colorectal Neoplasms; Female; Humans; Keratins; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Invasiveness; Prognosis; Proportional Hazards Models; Regression Analysis

Angiogenesis is believed to be essential for the growth of metastatic tumors in the brain. We analyzed the vascularization of tumors formed by 4 epithelial cell lines (C38, ZR75, HT25, and H1650) and a fibrosarcoma (HT1080) cell line injected into the brains of mice. No peritumoral angiogenesis was observed. Tumors apparently acquired their vasculature by incorporation of native vessels. Vessel density was lower, but vessel diameter and vascular cell proliferation were higher within all tumors versus those in the peritumoral tissue. There was an inverse correlation between the number of incorporated vessels and vascular cell proliferation. Epithelial tumors with pushing growth patterns had lower vessel density and elevated vascular cell proliferation compared with invasive tumors. The incorporated vessels retained their normal structure, with the exception of astrocyte foot processes that were replaced by tumor cells. Attachment to the vascular basement membrane led to the differentiation of the ZR75 breast cancer cells. In the HT1080 metastases, there was intussusceptive angiogenesis, that is, the fibrosarcoma cells that were attached to the vessel caused lumen splitting and filled the developing pillars. Branching angiogenesis was not observed either in the tumors or in control cerebral wounds. These data suggest that sprouting angiogenesis is not needed for the incipient growth of cerebral metastases and that tumor growth in this model is a result of incorporation of host vessels.

Topics: Angiopoietin-1; Animals; Brain Neoplasms; Breast Neoplasms; Bromodeoxyuridine; Carcinoma; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Disease Models, Animal; DNA-Binding Proteins; Electron Microscope Tomography; Female; Glial Fibrillary Acidic Protein; Humans; Keratins; Lamins; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Neovascularization, Pathologic; Platelet Endothelial Cell Adhesion Molecule-1; Quinolines; Receptor, Platelet-Derived Growth Factor beta; Thiazoles; Vascular Endothelial Growth Factor A

Tumor budding (TB) is showing increasing promise as a colorectal cancer (CRC) prognosticator that is independent of TNM staging. β-Catenin is a component of the Wingless/Wnt signaling pathway that is bound to membrane-associated E-cadherin and is essential for its correct position and function.. This study was designed to detect TB in 44 resected primary CRC cases and also to compare β-catenin expression in the tumor budding sites (TBS) and in the tumor center. Tumor budding was assessed in both H&E and pankeratin immunostained sections. Agreement between TB scoring using pancytokeratin and H&E was tested. Also, typing of the tumor margin and determination of degree of cytoplasmic pseudo-fragmentation was done. Tumor budding, cytoplasmic pseudofragments and β-catenin expression were related to known CRC prognosticators.. Ten tumors (22.7%) showed low grade (LG) budding and 34 tumors (77.3%) showed high grade (HG) budding. The 34 HG budding tumors were further subdivided into moderate and severe (n=13, n=21, respectively) budding cancers. Twenty nine tumors (65.9%) showed LG cytoplasmic pseudofragments and 15 tumors (34.1%) showed HG pseudofragments. Scoring of TB on H&E and pankeratin stained sections revealed moderate agreement (Kappa=.558; p=<.000). A significant relation between TB and cytoplasmic pseudofragments was observed (p=.009). Both TB and cytoplasmic pseudofragments did not significantly associate with clinicopathologic parameters. Immunoreactivity of nuclear and cytoplasmic β-catenin was significantly higher at TBS compared to tumor center (p=.005, p=.000, respectively). In opposition, membranous β-catenin expression was significantly higher in the tumor center than in TBS (p=.001). Although, nuclear β-catenin accumulation at TBS was noted, yet, it did not relate significantly with both TB and cytoplasmic pseudofragments around TBS (p=.649; p=.675, respectively). Also, nuclear β-catenin immunoreactivity did not relate significantly with the various clinicopathological variables.. Pankeratin immunostaining facilitates typing of CRC invasive margin, and determination of the degree of TB and cytoplasmic pseudo-fragmentation. β-Catenin expression differs significantly between tumor center and TBS in CRC. Cut-offs for TB assessment should be unified and further studies are recommended to allow a better understanding of this process before establishing TB as a prognostic factor beyond the TNM staging in CRC.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; beta Catenin; Cell Nucleus; Colorectal Neoplasms; Female; Humans; Keratins; Male; Middle Aged; Neoplasm Invasiveness; Tumor Burden

Metastasis is a complex, multistep process that begins with the epithelial-mesenchymal transition (EMT). Circulating tumor cells (CTC) are believed to have undergone EMT and thus lack or express low levels of epithelial markers commonly used for enrichment and/or detection of such cells. However, most current CTC detection methods target only EpCAM and/or cytokeratin (CK) to enrich epithelial CTCs, resulting in failure to recognize other, perhaps more important, CTC phenotypes that lack expression of these markers. Here, we describe a population of complex aneuploid CTCs that do not express CK or CD45 antigen in patients with breast, ovarian, or colorectal cancer. These cells were not observed in healthy subjects. We show that the primary epithelial tumors were characterized by similar complex aneuploidy, indicating conversion to an EMT phenotype in the captured cells. Collectively, our study provides a new method for highly efficient capture of previously unrecognized populations of CTCs.. Current assays for CTC capture likely miss populations of cells that have undergone EMT. Capture and study of CTCs that have undergone EMT would allow a better understanding of the mechanisms driving metastasis.

Topics: Biomarkers, Tumor; Breast Neoplasms; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Female; Humans; Keratins; Neoplasm Metastasis; Neoplastic Cells, Circulating; Ovarian Neoplasms

The study aimed to evaluate the incidence of disseminated tumour cells (DTCs) in bone marrow (BM) preoperatively and during follow up and to correlate these with established risk factors in patients with colorectal cancer.. We prospectively studied BM in 57 patients using the anti-cytokeratin antibody A45-B/B3.. The overall detection rate of DTCs was 23% with a similar detection rate through all stages of the disease. No significant association was found between the presence of DTCs and clinicopathological parameters. After a median follow up of 35.4 months, no differences were found in relapse and overall survival between patients with and without DTC preoperatively. In 31 of 45 patients with local disease, we performed a follow-up BM examination after 1 year. In 26% of the patients, the BM status had changed as compared with the preoperative finding.. This is the first study to report the follow up of DTC in BM in colorectal cancer using the A45-B/B3 antibody. The presence of tumour cells in the preoperative BM had no impact on outcome. The BM status had changed after 12 months in a quarter of patients.

Topics: Aged; Aged, 80 and over; Antibodies, Monoclonal; Bone Marrow; Colorectal Neoplasms; Disease Progression; Disease-Free Survival; Female; Follow-Up Studies; Humans; Immunohistochemistry; Keratins; Male; Middle Aged; Neoplasm Metastasis; Prognosis; Prospective Studies; Statistics, Nonparametric; Time Factors

Most studies on the sentinel node (SN) procedure in patients with colorectal cancer include immunohistochemical analysis of the SN only. To evaluate the real diagnostic accuracy of the SN procedure with immunohistochemical analysis, the presence of occult tumour cells in all histologically negative lymph nodes was compared to the presence of these cells in SNs. Also the reproducibility of diagnosing occult tumour cells (OTC) and the sensitivity of three different antibodies was assessed.. Between November 2006 en July 2007, an ex vivo SN procedure was performed in 58 histologically N0 patients with colorectal cancer. All lymph nodes (n = 908, mean 15.7) were step-sectioned and immunohistochemistry was performed using two antibodies against cytokeratins (Cam5.2, and CK 20) and one antibody against BerEp-4.. OTC were identified in 19 of 58 patients, with micrometastases (0.2-2 mm) in 7 and isolated tumour cells (ITC)(<0.2 mm) in 12 patients. The overall agreement in diagnosing OTC between two independent pathologists was 86%. An SN was identified in 53 of 58 patients. All micrometastases were found in SNs. In two patients with negative SNs, ITC's were demonstrated in non-SNs (sensitivity 88%, and overall accuracy 96%).. Additional immunohistochemical analysis of histologically negative lymph nodes demonstrates occult tumour cells in 33% of the patients resulting in an upstaging rate of 12%. Occult tumour cells are predominantly found in the SN, therefore SN mapping has the potential to refine the staging system for patients with colorectal cancer.

Topics: Biomarkers; Biomarkers, Tumor; Chi-Square Distribution; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Keratin-20; Keratins; Lymphatic Metastasis; Male; Neoplasm Staging; Predictive Value of Tests; Reproducibility of Results; Sensitivity and Specificity; Sentinel Lymph Node Biopsy

Large number of patients with colorectal liver metastasis show recurrent disease after curative surgical resection. Identification of these high-risk patients may guide therapeutic strategies. The aim of this study was to evaluate whether the presence of disseminated tumor cells in bone marrow from patients undergoing surgical resection of colorectal liver metastases can predict clinical outcome.. Sixty patients with colorectal liver metastases were planned for a curative resection between 2001 and 2007. All patients underwent bone marrow aspiration before surgery. Detection of tumor cells was performed using immunocytochemical staining for cytokeratin (CK-ICC) combined with automated microscopy or indirectly using reverse transcription-polymerase chain reaction (RT-PCR).. Disseminated tumor cells were found in 15 of the 46 patients (33%) using CK-ICC and in 9 of 44 of the patients (20%) using RT-PCR. Patients with negative results for RT-PCR had a significant better disease-free survival after resection of their liver metastases (p = 0.02). This group also showed significant better overall survival (p = 0.002). CK-ICC did not predict a worse clinical outcome.. The presence of disseminated tumor cells in bone marrow detected using RT-PCR did predict a worse clinical outcome. The presence of cells detected with CK-ICC did not correlate with poor prognosis.

Topics: Aged; Biomarkers, Tumor; Biopsy, Needle; Bone Marrow; Bone Marrow Examination; Chemotherapy, Adjuvant; Colorectal Neoplasms; Disease-Free Survival; Female; Follow-Up Studies; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Keratins; Liver Neoplasms; Male; Middle Aged; Neoadjuvant Therapy; Neoplastic Cells, Circulating; Predictive Value of Tests; Proportional Hazards Models; Prospective Studies; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Treatment Outcome

Cytokeratins 8 and 18 have recently been identified as acetylated. The acetylation of other cytoskeletal proteins has been reported as linked to stabilility. As colorectal cells exist bathed in pharmacologically active levels of the HDACi butyrate, we sought to apply state-of-the-art High Content Analytical approaches to identify the effect of butyrate upon the cytoskeleton of colorectal cells. We observed butyrate caused an increase in acetylation at three distinct residues of cytokeratin 8 (K10, K471, and K482) and that the kinetics of altered acetylation were distinct, implying either separate HDACs, or a heirachy of acetylation. This change in acetylation was associated with a breakdown in the cytokeratin cytoskeleton, implying a functional role for cytokeratin acetylation.

Topics: Automation; Butyrates; Caco-2 Cells; Cell Line, Tumor; Cell Nucleus; Colorectal Neoplasms; Cytoskeleton; Dose-Response Relationship, Drug; Electronic Data Processing; Gene Expression Regulation, Neoplastic; Humans; Keratins; Models, Biological; Protein Processing, Post-Translational; Proteomics

To investigate the relationship between T lymphoma invasion and metastasis 1 (Tiam1) and epithelial-mesenchymal transition (EMT) in human colorectal carcinomas.. Tiam1, E-cadherin, CK, and vimentin expressions in normal colorectal epithelium, colorectal carcinomas (CRC) and CRC with lymphatic metastasis were determined by immunohistochemistry using a two-step method.. Tiam1 expression was significantly higher in CRC than in normal colorectal epithelium (P<0.01) in close correlation to the degree of tumor differentiation (P<0.05). Higher Tiam1 expression was detected in CRC with lymphatic metastasis than in primary CRC (P<0.05). The expressions of E-cadherin and CK in CRC tissues were significantly lowered in comparison with those in normal colorectal epithelium (P<0.01), showing a correlation to tumor differentiation (P<0.01) but not to lymphatic metastasis. Vimentin was significantly overexpressed in CRC (P<0.01) and correlated to tumor differentiation (P<0.01) but not to lymphatic metastasis. Tiam1 expression was inversely correlated to E-cadherin and CK, but positively to vimentin.. Tiam1 is related to the metastasis of colorectal carcinoma, and may induce EMT to promote CRC metastasis.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Cadherins; Cell Movement; Cell Transdifferentiation; Colorectal Neoplasms; Epithelial Cells; Female; Guanine Nucleotide Exchange Factors; Humans; Keratins; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; T-Lymphoma Invasion and Metastasis-inducing Protein 1; Young Adult

Medullary carcinoma and serrated adenocarcinoma are two variants of colon cancer which are associated to particular pathways. Medullary carcinoma is invariably associated with MSI while serrated adenocarcinoma is characterized by excess of methylation. TNM classification and the tumoral grade are still the most important prognostic factors. Several parameters including morphological criteria, molecular features or immunohistochemical markers seem to be relevant but none of them are today used in clinical practice. A more accurate approach of the evaluation of these additional parameters should improve their prognosis values. double dagger.

Topics: Adenocarcinoma; Carcinoma, Medullary; Colorectal Neoplasms; DNA Mismatch Repair; Humans; Inflammation; Keratins; Neoplasm Invasiveness; Prognosis

Sentinel lymph node mapping (SLNM) with multilevel sections (MLS) and cytokeratin immunohistochemistry (CK-IHC) of sentinel lymph nodes (SLNs) upstages 15-20% of patients (pts). False-positive SLNs occur in breast cancer due to mechanical transport of cells during mapping procedures, or to pre-existing benign cellular inclusions. Our prospective study evaluated whether colorectal mapping procedures alone caused false positives.. A total of 314 pts underwent SLNM with blue dye. Ninety of the pts underwent a second mapping in normal bowel away from the primary tumor. The first 1-5 blue nodes near the primary tumor were marked as SLNs; those near the second injection site were marked as nontumor SLNs (nt-SLNs). All SLNs and nt-SLNs were evaluated by MLS and CK-IHC.. Of 314 pts, 30 had benign tumor and 284 had invasive cancer. SLNM was successful in 274/284 (96.5%) invasive cancer pts, with 728 SLNs identified. Forty-six of the 274 pts (16.8%) had low-volume metastasis in 57 SLNs: 31 pts (11.3%) had 38 SLNs with micrometastasis (>0.2 mm,

Topics: Colorectal Neoplasms; False Positive Reactions; Hepatectomy; Humans; Keratins; Lymph Nodes; Lymphatic Metastasis; Neoplasm Staging; Prognosis; Prospective Studies; Sentinel Lymph Node Biopsy; Survival Rate; Treatment Outcome

Transforming growth factor-beta1 (TGF-beta1), a cytokine involved in various stages of cancer, is produced as a latent complex and requires processing to become active. We have determined total and active TGF-beta1 levels in homogenates of colorectal neoplasia. In contrast to total TGF-b levels, showing a stepwise increase in the mucosa-adenoma-carcinoma sequence, active TGF-beta1 levels are increased only in carcinomas but not in premalignant adenomas. Furthermore, solely active TGF-beta1 levels are associated with the stage of the carcinomas and worse patient prognosis. Active TGF-beta1 levels correlated significantly with plasminogen activator inhibitor (PAI)-1, alpha-smooth muscle actin (SMA) and several matrix-remodeling proteinases. Interestingly, SMA levels are also significantly increased in colorectal carcinomas but not in adenomas, suggesting that despite the enhanced total TGF-beta1 levels, myofibroblast accumulation is not (yet) occurring in these premalignant neoplasias. The correlation between active TGF-beta1 and SMA expression in tumors indicates that tumor-promoting myofibroblasts might arise as a result of increased TGF-beta1 activation. These data underline the significance of the interaction between malignant cells and (myo)-fibroblasts in the tumor microenvironment, modulating the biologic behavior of colorectal cancer.

Topics: Actins; Adenoma; Biomarkers, Tumor; Carcinoma; Colorectal Neoplasms; Desmin; Enzyme-Linked Immunosorbent Assay; Female; Fibroblasts; HT29 Cells; Humans; Immunohistochemistry; Keratins; Male; Muscle, Smooth; Plasminogen Activator Inhibitor 1; Reproducibility of Results; Sensitivity and Specificity; Smad2 Protein; Transforming Growth Factor beta1; Urokinase-Type Plasminogen Activator; Vimentin

Epithelial and stromal cells play an important role in the development of colorectal cancer (CRC). We aimed to determine the prognostic significance of both epithelial and stromal cell apoptosis in CRC.. Total apoptosis was determined by caspase-3 activity measurements in protein homogenates of CRC specimens and adjacent normal mucosa of 211 CRC patients. Epithelial apoptosis was determined by an ELISA specific for a caspase-3-degraded cytokeratin 18 product, the M30 antigen. Stromal apoptosis was determined from the ratio between total and epithelial apoptosis.. Epithelial and stromal apoptosis, as well as total apoptosis, were significantly higher in CRC compared with corresponding adjacent normal mucosa. Low total tumour apoptosis (< or = median caspase-3 activity) was associated with a significantly worse disease recurrence (hazard ratio (HR), 95% confidence interval (95% CI): 1.77 (1.05-3.01)), independent of clinocopathological parameters. Epithelial apoptosis was not associated with clinical outcome. In contrast, low stromal apoptosis (< or = median caspase-3/M30) was found to be an independent prognostic factor for overall survival, disease-free survival and disease recurrence, with HRs (95% CI) of 1.66 (1.17-2.35), 1.62 (1.15-2.29) and 1.69 (1.01-2.85), respectively.. Stromal apoptosis, in contrast to epithelial apoptosis, is an important factor with respect to survival and disease-recurrence in CRC.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Apoptosis; Caspase 3; Colorectal Neoplasms; Disease-Free Survival; Epithelial Cells; Female; Follow-Up Studies; Gastric Mucosa; Humans; Immunohistochemistry; Keratins; Male; Middle Aged; Neoplasm Recurrence, Local; Prognosis; Reproducibility of Results; Stromal Cells; Time Factors

Colorectal cancer (CRC) is the second most prevalent cause of cancer-related deaths in the Western world. 5-Fluorouracil (5-FU) is a standard chemotherapeutic drug to treat CRC. However, the response rate is less than 20% and patients who have responded to 5-FU may become resistant. Therefore there is an urgent need to examine the 5-FU response proteins so that patients with no response to 5-FU can change to other treatment strategies promptly. In this study, the proteomic expression profile in a CRC cell line SW480 before and after 5-FU treatment was examined using 2-dimensional electrophoresis technology. Fourteen proteins with differential expression were identified using mass spectrometry and 7 of them were validated using immunocytochemical (ICC) staining. Protein identification indicated that cyclophilin A, cytokeratin 19 (CK19), cytokeratin 8 (CK8), ras-related nuclear protein, heat shock protein 27 (hsp27) and peroxiredoxin 6 (Prx 6) were upregulated whereas heat shock protein 60 (hsp60), cytokeratin 18 (CK18), cytokeratin 9 (CK9), carbamoylphosphate synthetase I, alpha-enolase, heat shock protein 70 (hsp70), nm23 and beta-actin were down-regulated. Seven of the 14 proteins detected were validated by ICC staining, which showed that the expression of hsp27, Prx 6 and hsp70 correlated with that from proteomics profiling. Our results suggest that hsp27, Prx 6 and hsp70 are potential 5-FU response proteins and they may represent potential targets for further evaluation in other 5-FU-sensitive and -resistant CRC cell lines.

Topics: Antimetabolites, Antineoplastic; Cell Line, Tumor; Colorectal Neoplasms; Dose-Response Relationship, Drug; Electrophoresis, Gel, Two-Dimensional; Fluorouracil; Heat-Shock Proteins; HSP27 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Humans; Immunohistochemistry; Keratins; Molecular Chaperones; Neoplasm Proteins; NM23 Nucleoside Diphosphate Kinases; Nucleoside Diphosphate Kinase D; Proteomics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

To evaluate the differences that exist between peripheral and mesenteric serum levels of carcinoembryonic antigen (CEA) and cytokeratins in patients with colorectal adenocarcinoma.. One hundred and thirty-eight patients with colorectal adenocarcinoma who underwent surgery at Hospital Sao Paulo (Discipline of Surgical Gastroenterology of UNIFESP-EPM) between December 1993 and March 2000 were retrospectively analyzed. Differences between CEA and cytokeratin (TPA-M) levels in peripheral blood (P) and in mesenteric blood (M) were studied. Associations were investigated between peripheral and mesenteric levels and the staging and histopathological variables (degree of cell differentiation, macroscopic appearance, tumor dimensions and presence of lymphatic and venous invasion).. Differences were observed in the numerical values of the marker levels: CEA (M) (39.10 mg/L +/- 121.19 mg/L) vs CEA (P) (38.5 mg/L +/- 122.55 mg/L), P < 0.05; TPA-M (M) (325.06 U/L +/- 527.29 U/L) vs TPA-M (P) (279.48 U/L +/- 455.81 U/L), P < 0.01. The mesenteric CEA levels were higher in more advanced tumors (P < 0.01), in vegetating lesions (34.44 mg/L +/- 93.07 mg/L) (P < 0.01) and with venous invasion (48.41 mg/L +/- 129.86 mg/L) (P < 0.05). Peripheral CEA was higher with more advanced staging (P < 0.01) and in lesions with venous invasion (53.23 mg/L +/- 158.57 mg/L) (P < 0.05). The patients demonstrated increased mesenteric and peripheral TPA-M levels with more advanced tumors (P < 0.01 and P < 0.01) and in non-ulcerated lesions [530.45 U/L +/- 997.46 U/L (P < 0.05) and 457.95 U/L +/- 811.36 U/L (P < 0.01)].. The mesenteric levels of the tumor markers CEA and cytokeratins were higher than the peripheral levels in these colorectal adenocarcinoma patients. Higher levels of these biologic tumor markers are associated with an advanced state of cancerous dissemination.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Arteries; Biomarkers, Tumor; Carcinoembryonic Antigen; Colorectal Neoplasms; Female; Humans; Keratins; Male; Mesenteric Arteries; Middle Aged; Neoplasm Staging; Retrospective Studies

To compare the immunoprofiles of intrahepatic cholangiocarcinoma and metastatic colorectal adenocarcinoma for mucin glycoproteins (including MUC1, MUC2, MUC5AC and MUC6) and cytokeratins (including CK7, CK19 and CK20), and to assess their diagnostic value.. One hundred cases of intrahepatic cholangiocarcinoma and 21 cases of metastatic colorectal adenocarcinoma were enrolled into the study. Immunohistochemical study for MUC1, MUC2, MUC5AC, MUC6, CK7, CK19 and CK20 was carried out in all cases by EnVision method.. In intrahepatic cholangiocarcinoma, the expression rates of MUC1, MUC2, MUC5AC and MUC6 were 61.0%, 2.0%, 22.0% and 8.0% respectively, as compared to 57.1%, 47.6%, 19.0% and 23.8% respectively in metastatic colorectal adenocarcinoma. On the other hand, the expression rates of CK7, CK19 and CK20 in intrahepatic cholangiocarcinoma were 73.0%, 53.0% and 15.0% respectively, in contrast to 14.3%, 90.5% and 85.7% respectively in metastatic colorectal adenocarcinoma. The difference in expressions of MUC2, MUC6, CK7 and CK20 carried statistical significance.. The immunoprofile for mucin glycoproteins and cytokeratins provides important clues in distinguishing between intrahepatic cholangiocarcinoma and metastatic colorectal adenocarcinoma to liver. The immunophenotype of MUC2-/MUC6-/CK7+/CK20- indicates the diagnosis of intrahepatic cholangiocarcinoma, while MUC2+/MUC6+/CK7-/CK20+ suggests the possibility of metastatic colorectal adenocarcinoma.

Topics: Adenocarcinoma; Aged; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Biomarkers, Tumor; Cholangiocarcinoma; Colorectal Neoplasms; Female; Glycoproteins; Humans; Keratins; Male; Middle Aged; Mucins; Neoplasm Staging

Lymph node involvement is an important prognostic factor in colorectal cancer. Sentinel lymph node (SLN) evaluation for assessing lymph node status in colorectal cancer remains controversial. Here we evaluated the sensitivity, predictive value, and accuracy of SLN evaluation for determining lymph node status in resectable colon cancer.. A prospective phase 2 cohort study of SLN evaluation in colon cancer was conducted from September 1998 to April 2006. Patients underwent resection and SLN mapping with 1% isosulfan blue and (m99)Tc sulfur colloid injection. SLNs were evaluated by hematoxylin and eosin (HE) staining and, if findings were negative, by additional thin HE sections and immunohistochemical (IHC) staining for pancytokeratin and MOC31. Overall survival for patients with IHC-positive disease was evaluated by Kaplan-Meier analysis and the log rank test.. SLNs were identified in 119 (99%) of the 120 patients eligible for the study. Median number of SLNs identified was 4 (range, 0-13). Forty-nine patients (40%) had nodal metastases on HE. The SLN accurately identified nodal metastases in 29 (59%) of these 49 patients and was negative for metastases in 22 patients (41%). SLNs in eight patients (7%) were negative by HE but positive by IHC staining. Positive IHC status did not affect survival after a median follow-up of 33 months (P = .41).. The low sensitivity and high false-negative rate of SLN evaluation does not support this technique for improving the accuracy of nodal staging for patients with colon cancer. The significance of IHC-positive SLNs remains uncertain.

Topics: Adenocarcinoma, Mucinous; Adult; Aged; Aged, 80 and over; Colorectal Neoplasms; Female; Humans; Immunoenzyme Techniques; Keratins; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging; Prognosis; Prospective Studies; Rosaniline Dyes; Sensitivity and Specificity; Sentinel Lymph Node Biopsy

Early colorectal cancer (ECC) is curable by endoscopic local resection; however, 10% of patients with ECC exhibit lymph node (LN) metastasis. In the current study, accurate predictors for LN metastasis in patients with ECC were examined by using immunohistochemistry with the lymphatic endothelial hyaluronan receptor 1 (LYVE-1) antibody to discriminate between lymphatics and blood vessels.. Colorectal tissue specimens obtained from 71 patients with ECC, including 28 patients with regional LN metastasis, were immunostained with antibodies against LYVE-1, beta-catenin, claudin-3, claudin-4, and cytokeratin. The significance of the histopathologic variables for LN metastasis in ECC was investigated on the basis of specific histopathologic parameters.. Lymphatic invasion confirmed by LYVE-1 immunohistochemistry was observed mainly in the submucosal area around the primary tumor and rarely was observed in the tumor. Expression patterns of beta-catenin, claudin-3, and claudin-4 in cancer cells at the invasive front were irrelevant to LN status. Tumor size, depth of invasion, histologic tumor type, budding formation, and lymphatic invasion were statistically significant to LN status in univariate analysis; however, only 2 factors--lymphatic invasion and budding formation at the invasive front-were independent predictors of LN metastasis in ECC.. LYVE-1 immunohistochemistry appeared to be a useful method for detecting lymphatics invaded by cancer cells, and detailed examination of the submucosa around the tumor may be important for predicting LN metastasis. When lymphatic invasion and budding formation are observed histopathologically in patients with ECC, additional therapy, such as adjuvant chemotherapy or a curative resection of the regional LN, may be required.

Topics: Aged; Analysis of Variance; beta Catenin; Claudin-3; Claudin-4; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Lymphatic Metastasis; Male; Membrane Proteins; Middle Aged; Predictive Value of Tests; Vesicular Transport Proteins

Recent advances in immunohistochemical techniques have made it possible to identify micrometastasis using antibodies to cytokeratins (CK). The aim of the study was to determine the prevalence and prognostic significance of immunohistochemically detected micrometastasis (IHM) in patients with localised colorectal cancer (CRC) (Dukes' A and B). A further aim was to study the prognostic role of histopathological factors such as vascular invasion.. The original histology of 168 consecutive patients with Dukes' A or B tumours who had undergone curative resection was reviewed. Immunohistochemical staining was performed using CK antibodies, AE1/AE3 and MNF116 on all (n=898) lymph nodes. Survival analysis was performed on 105 cases that had been followed up until death or for at least 5 years.. IHM were detected in 17.3% of lymph nodes analysed. Adverse outcome (death/local recurrence) was recorded in 8/49 (16%) patients with IHD-positive nodes and in 10/56 (18%) patients negative for IHM. IHM was not associated with adverse outcome on either univariate (p=0.540) or multivariate analyses (p=0.673). There was no correlation of IHM with age, gender, site, size and grade of tumour, depth of tumour invasion or perineural and vascular invasion. Vascular invasion was the only independent prognostic factor identified.. We have shown that isolated CK-positive epithelioid cells are commonly found in morphologically benign pericolic lymph nodes of patients with localised (Dukes' A or B) CRC. These cells may represent occult micrometastasis but are not clinically significant. Vascular invasion identifies patients with localised CRC likely to develop recurrences or die of disease.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Biomarkers, Tumor; Colorectal Neoplasms; Female; Humans; Keratins; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; Survival Rate

Measure the association between the incidence of primary tumor staining and the identification of mediastinal lymph node (MLN) using cytokeratins, NM23, DCC-positive tumors, and vascular endothelial growth factor (VEGF) expression in T(2) and T(3)/N(0) colorectal cancers. The impact of MLN on both recurrence and survival was assessed.. There were 153 CORC patients (T(2), T(3)/N(0)) selected from a prospectively accrued database. All patients had been staged by routine histopathology after a curative resection and no patients received adjuvant chemotherapy. The primary tumors (PT) were assessed with a panel of immunohistochemical stains (cytokeratin, DCC, Nm23, and VEGF). If the PT was positive, the regional nodes were assessed with that marker(s). For any positive tumor marker, all lymph nodes (LNs, mean of 12.6+/-4.2) were stained for this marker.. Patient age ranged from 38 to 86 years with a mean age of 61.56+/-25.56 years. Mean follow-up was 72.1+/-32.4 months. Recurrence rate of the whole group was 19/153 (12.4%) and the mean time to recurrence was 37.6+/-23.6 months (15 to 77 months). Crude mortality was 39.9%, while the cancer specific mortality was 11.2% after the whole follow-up period. The relationship between PT staining and MLNs was: cytokeratin-PT 143 (93.5%)/MLN 9 (6.3%); NM23-PT 51 (33.3%)/MLN 3 (5.9%); DCC-PT 79 (53%)/MLN 3 (3.8%); and VEGF-PT 72 (47%)/MLN 4 (5.6%). Nineteen (12.4%) patients experienced tumor recurrence. No correlation exist between PT and/or MLN staining and either recurrence or survival. No patient with MLN with any stain experienced a recurrence. There was no advantage to using an individual stain or all four stains.. Immunohistochemical stains for PT and focused analysis of regional nodes did not improve prediction of survival or recurrence. Sentinel LN evaluation and the provision of adjuvant chemotherapy in node-negative patients should be questioned and not be utilized outside of a research protocol.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Colorectal Neoplasms; Follow-Up Studies; Humans; Immunohistochemistry; Keratins; Lymph Nodes; Lymphatic Metastasis; Middle Aged; Neoplasm Staging; Reproducibility of Results; Retrospective Studies; Time Factors

Lymphatic metastasis to the regional lymph nodes through the lymphatic vessels is an important indicator of poor prognosis in many types of malignant tumors. Recently, much attention has been paid to lymphangiogenesis for its possible role on tumor progression in various carcinomas. However, morphological evidence that lymphatic vessels actively proliferate in colorectal carcinoma has not been reported. Here, we first devised a triple immunostaining method to detect proliferating lymphatic vessels utilizing antibody to Ki-67 antigen as a marker of cell proliferation, antibody to cytokeratin as an epithelial cell marker, and antibody to podoplanin as a lymphatic vessel-specific marker. Ki-67/podoplanin-immunoreactivity enabled us to identify proliferating lymphatic vessels, while cytokeratin immunoreactivity allowed us to distinguish proliferating lymphatic vessels from Ki-67/cytokeratin-positive carcinoma cells in lymphatic lumens. Analyzing 64 colorectal carcinoma patients' samples using this technique, we showed that both lymphatic vessel density and proliferating activity of lymphatic vessels were significantly increased in colorectal carcinoma tissues compared with their normal counterparts. We then examined the correlation between the degree of lymphangiogenesis and patients' prognosis or clinicopathological variables, but no statistically significant differences were obtained in these analyses. Thus, these results combined together indicate that extensive lymphangiogenesis occurs in colorectal carcinoma, but that the degree of lymphangiogenesis alone is not an independent prognostic factor for this disease.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Cell Proliferation; Colorectal Neoplasms; Disease Progression; Female; Humans; Immunohistochemistry; Keratins; Ki-67 Antigen; Lymphangiogenesis; Lymphatic Vessels; Male; Membrane Glycoproteins; Middle Aged; Prognosis

Overexpression of thymosin beta-4 has been linked to malignant progression but the localization of this polypeptide within tumors is incompletely known. We therefore examined breast cancers for thymosin beta-4 using immunofluorescence. Reactive cells were identified with monoclonal cell marker antibodies. A very heterogeneous staining pattern for thymosin beta-4 was observed. Thus, while leukocytes and macrophages showed intense reactivity for this polypeptide, cancer cells, and endothelial cells showed a much more variable reactivity. A similar heterogeneous staining was observed also in colorectal carcinomas. The degree of staining of breast cancer cells for thymosin beta-4 correlated neither to histological grade nor to endothelial cell staining. However, there was a tendency toward correlation (P = 0.07) between staining of endothelial cells and histological grade. Treatment of cultured breast cancer cells (SK-BR-3) with 1-4 microg thymosin beta-4/mL significantly increased cell numbers, as determined by MTT-assays. These data reveal an unexpected cellular heterogeneity of thymosin beta-4 expression in breast and colonic carcinomas and suggest that local release of this polypeptide in the tumor microenvironment may modulate tumor behavior.

Topics: Breast Neoplasms; Cell Nucleus; Colorectal Neoplasms; Cytoplasm; Female; Fluorescent Antibody Technique; Humans; Keratins; Leukocytes; Thymosin

Although distinguishing metastatic colorectal adenocarcinoma from primary lung adenocarcinoma is often difficult, pre- or intra-operative identification is very important, as the resection areas for each diagnosis differ substantially. CDX2, a recently cloned homeobox gene, represents a highly specific and sensitive marker of colorectal adenocarcinoma. We evaluated CDX2 expression using pre- and intra-operative biopsy specimens. The study examined 50 consecutive colorectal adenocarcinoma metastases to the lung, including 20 biopsy specimens and 66 resected specimens, and 21 primary lung adenocarcinomas. All specimens were immunohistochemically stained for CDX2, cytokeratin (CK) 7, CK20 and thyroid transcription factor (TTF)-1, and scored in a semi-quantitative manner. Mean staining score in biopsy specimens was significantly higher for CDX2 than for CK20. Sensitivities for CDX2 and CK7-/20+ in biopsy specimens were 95.0 and 65.0%, respectively. If CDX2 immunostaining had not been performed, 8 biopsy specimens (40%), and 20 resected specimens (30.3%) might have been diagnosed as equivocal cases either as primary lung cancer or metastatic colorectal cancer, using other markers. These results suggest that positive CDX2 staining represents a highly sensitive and specific marker of metastatic colorectal carcinoma in both biopsy and resected specimens, and is superior to staining for the CK7-/20+ phenotype.

Topics: Adenocarcinoma; Biomarkers, Tumor; Biopsy; CDX2 Transcription Factor; Colorectal Neoplasms; Homeodomain Proteins; Humans; Immunoenzyme Techniques; Keratins; Lung Neoplasms; Male; Middle Aged; Phenotype; Preoperative Care; Sensitivity and Specificity

The histogenesis and biological behaviour of peripheral intrahepatic cholangiocarcinoma (peripheral CC) remain unclarified. The aim of this study was to examine the growth pattern of peripheral CC (24 cases) in comparison with hepatocellular carcinoma (HCC, 27 cases) and metastatic colorectal adenocarcinoma (MCA, 24 cases).. Tumour/surrounding liver borders were classified as: (i) fibrous encapsulation, (ii) compressive growth, and (iii) infiltrating replacement. Nineteen of 24 peripheral CCs showed (iii), whereas 23 of 27 HCCs showed (i) and 17 of 24 MCAs showed (ii). In (iii), carcinoma cells infiltrated the surrounding liver without compression, and hepatic supporting vascular structures such as portal tracts were secondarily incorporated into the tumour. In (i) and (ii), the surrounding liver was compressed and no or few portal tracts were incorporated within the tumour. Fifteen of 24 peripheral CCs were composed of carcinoma cells resembling reactive bile ductules and these cells were positive for neural cell adhesion molecule (NCAM), a marker of proliferating bile ductules. The remaining nine peripheral CCs were composed of ordinary adenocarcinoma and negative for NCAM.. A subgroup of peripheral CCs with an infiltrating replacement growth pattern resembles reactive bile ductules and expresses NCAM. 'Bile ductular carcinoma' may be a better term for this subgroup.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Autopsy; Bile Duct Neoplasms; Bile Ducts; Bile Ducts, Intrahepatic; Carcinoma, Hepatocellular; CD56 Antigen; Cholangiocarcinoma; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Liver; Liver Neoplasms; Male; Middle Aged; Neural Cell Adhesion Molecules; Organ Size; Vimentin

Although risk factors for histologically overt lymph node metastasis in patients with early-stage colorectal cancer have been clarified, the risk factors for occult lymph node metastasis are not clear. This study was designed to clarify risk factors for lymph node metastasis, including occult metastasis, in patients with colorectal cancer invading the submucosa and to determine the criteria for endoscopic resection of early colorectal cancer.. The risk factors for lymph node metastasis, including occult metastasis, were analyzed in 86 cases of surgically resected colorectal cancer invading the submucosa. The lymph nodes were assessed by immunohistochemistry with cytokeratin antibody CAM5.2.. The frequencies of overt and occult metastasis to the lymph nodes were 13 percent (11/86) and 13 percent (10/75), respectively. Multivariate analysis showed vascular invasion (P = 0.001) and tumor budding (P = 0.003) to be independent risk factors for lymph node metastasis, including occult metastasis. For tumors with submucosal invasion < or =1,000 microm, no lymph node metastasis was found. The frequencies of lymph node metastasis for tumors with submucosal invasion of 1,000 to 2,000 microm and >2,000 microm were 21 and 37 percent, respectively. In considering combinations of risk factors, there was no lymph node metastasis in tumors having neither vascular invasion nor tumor budding and submucosal invasion of < or =3,000 microm.. Vascular invasion, tumor budding, and the degree of submucosal invasion were significant risk factors for lymph node metastasis, including occult metastasis. These three factors can be used in combination to identify patients requiring additional surgery after endoscopic resection.

Topics: Aged; Biomarkers; Biomarkers, Tumor; Colonoscopy; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Intestinal Mucosa; Keratins; Lymph Nodes; Lymphatic Metastasis; Male; Neoplasm Staging; Prognosis; Risk Factors

In the past few years, tumour budding at the invasive margin has been reported as a new risk factor for lymph node metastasis in advanced colorectal cancers, but it is sometimes difficult to detect tumour budding in submucosal colorectal cancer by haematoxylin and eosin staining. We immunohistochemically examined tumour budding at the deepest invasive margin of 56 surgically resected submucosal colorectal carcinomas using anticytokeratin antibody CAM5.2, furthermore checked by AE1/AE3, and determined the relation between tumour budding and clinicopathological factors. Moreover, we used the monoclonal antibody D2-40 for immunohistochemistry to detect lymphatic involvement. Tumour budding was detected in 42 cases (75.0%), and the budding-positive group showed a significantly higher rate of lymph node metastasis (including isolated tumour cells) (16/42 vs 0/14; P=0.004) than the budding-negative group. The sensitivity and negative predictive value of tumour budding alone for lymph node metastasis were superior to those of lymphatic invasion alone. Furthermore, the specificity and positive predictive value of the combination of either lymphatic invasion or tumour budding were superior to those of lymphatic invasion alone. Tumour budding detected immunohistochemically by using CAM5.2 is a newly found risk factor for lymph node metastasis and may help to avoid oversurgery in the future.

Topics: Adenocarcinoma; Aged; Antibodies; Biomarkers; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Lymphatic Metastasis; Male; Middle Aged; Predictive Value of Tests; Risk Factors; Sensitivity and Specificity

Colorectal cancer patients diagnosed with stage I or II disease are not routinely offered adjuvant chemotherapy following resection of the primary tumor. However, up to 10% of stage I and 30% of stage II patients relapse within 5 years of surgery from recurrent or metastatic disease. The aim of this study was to determine if tumor-associated markers could detect disseminated malignant cells and so identify a subgroup of patients with early-stage colorectal cancer that were at risk of relapse.. We recruited consecutive patients undergoing curative resection for early-stage colorectal cancer. Immunobead reverse transcription-PCR of five tumor-associated markers (carcinoembryonic antigen, laminin gamma2, ephrin B4, matrilysin, and cytokeratin 20) was used to detect the presence of colon tumor cells in peripheral blood and within the peritoneal cavity of colon cancer patients perioperatively. Clinicopathologic variables were tested for their effect on survival outcomes in univariate analyses using the Kaplan-Meier method. A multivariate Cox proportional hazards regression analysis was done to determine whether detection of tumor cells was an independent prognostic marker for disease relapse.. Overall, 41 of 125 (32.8%) early-stage patients were positive for disseminated tumor cells. Patients who were marker positive for disseminated cells in post-resection lavage samples showed a significantly poorer prognosis (hazard ratio, 6.2; 95% confidence interval, 1.9-19.6; P = 0.002), and this was independent of other risk factors.. The markers used in this study identified a subgroup of early-stage patients at increased risk of relapse post-resection for primary colorectal cancer. This method may be considered as a new diagnostic tool to improve the staging and management of colorectal cancer.

Topics: Adult; Aged; Aged, 80 and over; Ascitic Fluid; Biomarkers, Tumor; Carcinoembryonic Antigen; Colorectal Neoplasms; Female; Humans; Keratin-20; Keratins; Laminin; Male; Matrix Metalloproteinase 7; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Peritoneal Lavage; Prognosis; Receptors, Eph Family; Reverse Transcriptase Polymerase Chain Reaction; Risk Factors; RNA, Messenger; Survival Rate

A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method for detection of cytokeratin 20-positive cells in blood characterized by two novel features was developed and tested on 99 patients with colorectal cancer, 110 with breast cancer, and 150 healthy subjects. To optimize the specificity and sensitivity of the method, two novel features were used. First, a primer overlapping two adjacent exons was generated to inhibit nonspecific amplification both in healthy donors and cancer patients; second, a non-end-point first-round amplification was used to increase sensitivity. The number of first-round cycles was chosen to reach the highest level of sensitivity while conserving quantitative characteristics. PCR efficiency increased from 88.9% in single-round RT-PCR to 99.0% in nested real-time RT-PCR. To establish sensitivity and specificity of the method, HT29 cells were serially diluted with normal blood. Detection limit improved from 100 HT29 cells (single-round RT-PCR) to 1 to 10 cells (nested real-time RT-PCR) per 3 ml of whole blood. None of the healthy subjects was positive, whereas 22 and 29% of all colorectal and breast cancer patients, respectively, had cytokeratin 20 cell equivalents in blood. The association between cytokeratin 20 cell equivalents and metastasis was statistically significant for breast (P = 0.026) but not colorectal cancer patients (P = 0.361). Negativity of all 150 healthy controls examined confers diagnostic potential to the method.

Topics: Biomarkers, Tumor; Breast Neoplasms; Carcinoma; Colorectal Neoplasms; Humans; Keratin-20; Keratins; Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Sensitivity and Specificity; Tumor Cells, Cultured

It is important to discriminate between primary and secondary lung cancer. However, often, the discriminating diagnosis of primary lung acinar adenocarcinoma and lung metastasis of colorectal cancer based on morphological and pathological findings is difficult. The purpose of this study was to evaluate the clinical usefulness of immunohistochemistry of beta-catenin, cytokeratin (CK) 7, and CK20 for the discriminating diagnosis of lung cancer.. We performed immunohistochemistry of beta-catenin, CK7, and CK20 in 19 lung metastasis of colorectal cancer samples, 10 corresponding primary colorectal cancer samples and 11 primary lung acinar adenocarcinoma samples and compared the levels of accuracy of the discriminating diagnosis by using antibodies against these antigens.. Positive staining of beta-catenin was observed in all the lung metastasis of colorectal cancer samples as well as in the primary colorectal cancer samples but in none of the primary lung acinar adenocarcinoma samples. Positive staining of CK7 was observed in 90.9% of the primary lung acinar adenocarcinoma samples and in 5.3% of the lung metastasis of colorectal cancer samples, but in none of the primary colorectal cancer samples. Positive staining of CK20 was observed in all the primary colorectal cancer samples and in 84.2% of the lung metastasis of colorectal cancer samples, but in none of the primary lung acinar adenocarcinoma samples.. Combined immunohistochemistry of beta-catenin, CK7, and CK20 is useful for making a discriminating diagnosis between lung metastasis of colorectal cancer and primary lung acinar adenocarcinoma. This method will enable accurate diagnosis of a lung tumor and will be useful for selecting appropriate therapeutic strategies, including chemotherapeutic agents and operation methods.

Topics: Adenocarcinoma; beta Catenin; Biomarkers, Tumor; Colorectal Neoplasms; Diagnosis, Differential; Gene Expression Profiling; Humans; Immunohistochemistry; Keratin-20; Keratin-7; Keratins; Lung Neoplasms; Retrospective Studies; Sensitivity and Specificity

The aim of this study was to investigate the immunohistochemical expression of p16, p53, and p63 proteins according to some pathologic parameters related to colorectal adenomas and adenocarcinomas such as grade of dysplasia and histologic type.. Immunohistochemistry with the antibodies p16, p53, and p63 was performed in tubular, tubular-villous, and villous adenomas (n = 30) and in well, moderately, and poorly differentiated adenocarcinomas (n = 30). The p63-positive cases were submitted to double immunolabeling with the cytokeratin 5 (CK5).. The p16 and p53 labelings were observed in some adenomas and adenocarcinomas but without any association with p63 expression, histologic type, or grade of differentiation of the neoplasm. P63 expression was found mainly in the villous adenomas and in the poorly differentiated adenocarcinomas. The poorly differentiated adenocarcinomas also exhibited coexpression of CK5 and p63.. Despite both p16 and p53 having been detected in colorectal neoplasms, they were not related to the different histologic variables nor to the expression of p63. However, p63 expression was closely associated with villous adenomas and poorly differentiated adenocarcinomas. Thus, p63 may represent a marker of poor differentiation in colorectal neoplasms. The coexpression of p63 and CK5 observed in this study could be related to divergent differentiation during the development of colorectal cancer, although further studies are warranted to refine the understanding of this process.

Topics: Adenocarcinoma; Adenoma; Biomarkers, Tumor; Colorectal Neoplasms; DNA-Binding Proteins; Genes, p16; Genes, p53; Genes, Tumor Suppressor; Humans; Immunohistochemistry; Keratins; Phosphoproteins; Trans-Activators; Transcription Factors; Tumor Suppressor Proteins

Different histogenetic pathways have been suggested between ulcerative colitis (UC)-associated neoplasia and sporadic colorectal neoplasia. Little is known about the cytokeratin (CK) and mucin expression in UC-associated neoplasms. To clarify the characteristics of UC-associated colorectal carcinogenesis, we examined the immunohistochemical expression of CK7, CK20, MUC2, MUC5AC and MUC6 in 90 colorectal neoplasms, including 22 UC-associated adenocarcinomas (colitic cancer; CC), ten high-grade dysplasias (HGD) in UC, nine low-grade dysplasias (LGD) in UC, 24 sporadic tubular adenomas (TA) and 25 adenocarcinomas (AC). CK7 was positive in most of UC-associated neoplasms: 59% of CC cases, 80% of HGD and 89% of LGD, respectively, whereas, in non-UC associated neoplasia, 21% of TA and 12% of AC. The frequency of MUC6 expression in UC-associated neoplasia was 32% in CC, 30% in HGD and 44% in LGD, respectively, whereas, in non-UC associated neoplasia, 4.2% in TA and 0% in AC. MUC5AC expression in UC-associated neoplasia was detectable in 73% of CC, 90% of HGD and 89% of LGD, respectively; in non-UC associated neoplasia 67% in AC and 20% in TA. There were obvious differences in the expression of CK7 and MUC6 between UC-associated neoplasms and sporadic tumors. The incidence of MUC5AC expression in UC-associated neoplasms was also higher than sporadic tumors. These results suggest that gastric-type mucins play an important role in the initial step of CC-tumorigenesis, and CK7 and gastric-type mucins may be useful in the differential diagnosis between UC-associated neoplasms and sporadic ones.

Topics: Adenocarcinoma; Adenoma; Biomarkers, Tumor; Colitis, Ulcerative; Colorectal Neoplasms; Fluorescent Antibody Technique, Direct; Humans; Immunoenzyme Techniques; Keratin-20; Keratin-7; Keratins; Mucins; Precancerous Conditions

To study lymph node micrometastases (LNMM), expression of nm23-H(1), MMP(9), TIMP(2) proteins, and their relationship and clinical significance in patients with stage Dukes B colorectal cancer.. Thirty patients with stage Dukes B colorectal cancer were studied. LNMM in these patients was detected by immunohistochemical anti-cytokeratin 20 (CK20) staining. The expression of nm23-H(1), MMP(9) and TIMP(2) proteins in primary tumors was examined by Strept-avidin-biotin complex method. Clinical-pathological data and survival of each patient were recorded and analyzed.. (1) The positive dyeing of CK20 was observed in 26.7% for cases and in 7.8% for lymph nodes of 30 patients with stage Dukes B colorectal cancer. (2) Different expression of nm23-H(1) and MMP(9) proteins in the patients between stage Dukes B and stage Dukes CD was observed (P < 0.05). The decreased nm23-H(1) expression, and/or the increased MMP(9) expression in primary stage Dukes B tumors were significantly associated with LNMM (P < 0.05). Sensitivity and specificity for detection of LNMM by using nm23-H(1) or MMP(9) were respectively 62.5% and 81.8% or 75.0% and 69.8%. If by combining nm23-H(1) with MMP(9), specificity for detection of LNMM became 90.9%. The expression of TIMP(2) protein was not related with stage Dukes and LNMM. (3) The percent of tumor recurrence and/or metastasis for the stage Dukes B patients with LNMM was significantly higher than that for the patients without LNMM (P < 0.05), but the survival percent for the patients with LNMM was significantly lower than that for the patients without LNMM. The outcome for the patients with nm23-H(1) (-) LNMM (+) or MMP(9) (+) LNMM (+) was significantly worse than that for patients with nm23-H(1) (+) LNMM (-) or MMP(9) (+) LNMM (-) (P < 0.05).. LNMM is detected by immunohistochemical anti-CK20 staining. The expression of nm23-H(1) and MMP(9) in primary stage Dukes B tumors was significantly associated with LNMM. The outcome in the LNMM patients with nm23-H(1) (-) and/or MMP(9) (+) were worse. Combining examination of CK20 for lymph nodes with expression of nm23-H(1) and MMP(9) for primary tumors is of important clinical significance for staging of Dukes, selection of adjuvant treatment and evaluation of prognosis in patients with colorectal cancer.

Topics: Colorectal Neoplasms; Humans; Keratins; Lymph Nodes; Lymphatic Metastasis; Matrix Metalloproteinase 9; Neoplasm Staging; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Prognosis; Tissue Inhibitor of Metalloproteinases

This study is aimed at establishing a sensitive approach to detect disseminated tumor cells in peripheral blood and evaluate its clinical significance. A total of 198 blood samples including 168 from colorectal carcinoma (CRC) patients and 30 from healthy volunteers were examined by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) to evaluate the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and cytokeratin 19 (CK19) mRNA. CEA mRNA was detected in 35.8% of patients and 3.3% of controls, CK20 mRNA in 28.3% of patients and 6.7% of controls, and CK19 mRNA in 41.9% of patients and 3.3% of controls. CEA and CK20 mRNA positive ratio increased with the advancing Dukes stages, but there was no significant difference in positive ratio between any two stages (P>0.05). Also, relatively high positive ratio of CEA, CK20 and CK19 mRNA expression was observed in some CRC patients with earlier Dukes stages. A higher positive ratio was obtained when two or three detection markers were combined compared to a single marker. Our study indicates that quantitative real-time RT-PCR detection for CEA, CK20 and CK19 mRNA in peripheral blood is a valuable tool for monitoring early stage dissemination of CRC cells in blood circulation.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoembryonic Antigen; Carcinoma; Colorectal Neoplasms; Female; Humans; Keratin-20; Keratins; Male; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sensitivity and Specificity

To evaluate the efficacy of postoperative serial assay of carcinoembryonic antigen (CEA) and cytokeratins for the detection of recurrent disease in patients with colorectal adenocarcinoma after radical surgery.. Between 1993 and 2000, 120 patients with colorectal adenocarcinoma underwent radical surgery in the Department of Surgical Gastroenterology, Federal University of Sao Paulo-Escola Paulista de Medicina, Sao Paulo, Brazil. Periodic postoperative evaluation was performed by assaying markers in peripheral serum, colonoscopy and imaging examination. Presence of CEA was detected using the Delfia method with 5 microg/L threshold, and cytokeratins using the LIA-mat TPA-M Prolifigen method with 72 U/L threshold.. In the first postoperative year, patients without recurrent disease had normal levels of CEA (1.5 +/- 0.9 microg/L) and monoclonal tissue polypeptide antigen-M (TPA-M, 64.4 +/- 47.8 U/L), while patients with recurrences had high levels of CEA (6.9 +/- 9.8 microg/L, P < 0.01) and TPA-M (192.2 +/- 328.8 U/L, P < 0.05). During the second postoperative year, patients without tumor recurrence had normal levels of CEA (2.0 +/- 1.8 microg/L) and TPA-M (50.8 +/- 38.4 U/L), while patients with recurrence had high levels of CEA (66.3 +/- 130.8 microg/L, P < 0.01) and TPA-M (442.7 +/- 652.8 U/L, P < 0.05). The mean follow-up time was 22.3 mo. There was recurrence in 23 cases. Five reoperations were performed without achieving radical excision. Rises in tumor marker levels preceded identification of recurrences: CEA in seven (30%) and TPA-M in eleven individuals (48%).. Intensive follow-up by serial assay of CEA and cytokeratins allows early detection of colorectal neoplasm recurrence.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoembryonic Antigen; Colorectal Neoplasms; Female; Follow-Up Studies; Humans; Keratins; Male; Middle Aged; Neoplasm Recurrence, Local; Predictive Value of Tests

Approximately 30% of patients with lymph node (LN)-negative colorectal carcinoma (CRC) die of tumor recurrence, which can be related to the presence of tumor cells in LNs not detected by conventional histopathologic analysis. However, the prognostic significance of occult cancer cells still remains uncertain. We evaluated the incidence and the prognostic significance of occult cancer cells in LNs from 395 consecutive patients with curatively resected stage IIA CRC using immunohistochemistry for cytokeratin 20. Immunostained tumor cells were categorized as micrometastases (MCMs) or isolated tumor cells (ITCs) according to the American Joint Committee on Cancer criteria. The detection rates were compared with the clinicopathologic characteristics of the patients and with cancer-specific survival. The median follow-up time was 128 months. Micrometastases were detected in 39 patients (9.9%), whereas ITCs were found in 112 (28.4%), for an overall frequency of 38.2%. None of the clinicopathologic parameters examined was correlated with the presence of occult cancer cells. Patients with ITCs and those with negative LNs showed a similar survival rate (77.7% and 78.3%, respectively), whereas patients with MCMs had a lower survival rate (64.1%). At the univariate analysis, MCMs, tumor growth pattern, extent of tumor spread, and Crohn's-like lymphoid reaction influenced the survival rate significantly. Nevertheless, at the multivariate analysis, only the pattern of tumor growth and the extent of tumor spread were independent prognostic factors. The detection of immunostained tumor cells in the LNs of patients with stage IIA CRC occurs relatively frequently but has no significant effect on prognosis.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Colorectal Neoplasms; Female; Fluorescent Antibody Technique, Direct; Humans; Immunoenzyme Techniques; Incidence; Keratin-20; Keratins; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging; Prognosis; Survival Rate

Recent cDNA expression profiling analyses indicate that within specific organ cancers Cytokeratins (CKs) dysregulation may identify subgroups with distinct biological phenotypes. Our objectives in this study were (1) to test whether cytokeratins were also distinct on the protein level, (2) to evaluate these biomarkers in a series of well-characterised CRCs, (3) to apply hierarchical cluster analysis to immunohistochemical data.. Tissue microarrays (TMA) comprising 468 CRC specimens from 203 patients were constructed to evaluate CK5, CK7, CK8, CK13, CK14, CK16, CK17, CK18, CK19 and CK20. In total, 2919 samples were analyzed.. Unsupervised hierarchical clustering discovered subgroups represented by reduced CK8 and CK20 expression, that differed by a shorter patients survival. The evaluation of the specific biomarkers by Kaplan-Meier analysis showed that reduced CK8 expression (p<0.01) was significantly associated with shorter patients' survival, but was not an independent factor correlated with tumour stage (pT), grading (G) and nodal stage (pN).. Reduced coexpression of CK8 and CK20 may indicate an epithelial-mesenchymal transition (EMT) representing an important step in the development of more aggressive CRCs. In addition, multiplex analysis of TMAs together with immunohistochemistry (IHC) supplemented by hierarchical clustering are a useful, promising and very powerful tool for the identification of tumour subgroups with diagnostic and prognostic signatures.

Topics: Biomarkers, Tumor; Cluster Analysis; Colorectal Neoplasms; Humans; Keratins; Tissue Array Analysis

Survival results of stage II colorectal cancer patients have led to major efforts to identify the subset of patients at risk for disease relapse and adjuvant therapies benefit. Immunohistochemistry is being explored to detect undetectable microscopic lymph node micrometastases.. A retrospective analysis of a 105 consecutive stage II colorectal cancer patients was performed. Two four-micres sections were obtained from each lymph node. These slides were stained with AE1-AE3 monoclonal antibodies against cytoskeleton using DAKO EnVision visualization system. Micrometastases were identified either as isolated cells or as well-defined glandular cell clusters with cytoplasm but not the nucleus stained with cytoskeleton antibodies.. 665 lymph nodes isolated from 105 patients were analyzed. Lymph nodes micrometastases were assessed in 26 out of the 105 patients. 42 (6.3%) out of 665 lymph nodes were infiltrated. Most of these metastases consisted of isolated cell cluster localized in marginal and interfollicular sinus of lymph nodes. The relapse rate was 23.1% among the patients with immunohistochemical detected lymph node micrometastes and 20.3% for the patients without lymph node involvement. This result lacked statistical significance (p = 0.759).. AE1/AE3 lymph node immunohistochemical staining in stage II colorectal cancer is an interesting biological phenomenon but it fails to identify patients at higher risk of relapse who deserve a more aggressive adjuvant attitude.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging; Prognosis; Retrospective Studies; Survival Analysis

Liver resection is the only potential cure for patients with colorectal liver metastasis. However, more than 30% of patients will develop tumour recurrence, probably caused by tumour cells disseminated before or during surgery. As prevention of cell dissemination is barely obtainable, alternative concepts have to be discussed.. The potential of leukocyte adhesion filters for the removal of cytokeratin positive cells (CK+) from blood was studied in 18 patients undergoing liver resection for colorectal liver metastasis. Blood sampling was done via a liver venous catheter during hepatic mobilisation. Filtration was done with an in-line WBF2 filter system. To define the relation between surgery and cell release we compared patients' pre-operative and intra-operative blood and bone marrow (BM) samples with their CK expression using immunochemical staining.. CK+ cells were detected in BM samples of nine of 14 patients before surgery, indicating early dissemination. In ten of 18 patients CK+ cells were detected in blood samples during hepatic mobilisation; all ten patients underwent major liver surgery (R0 resection). In those patients recurrent disease was observed more often (P < or = 0.05). In 17 of 18 patients CK+ cells were not detectable after filtration procedure, which indicated cell adhesion to the filter medium.. Liver resection due to metastasis leads to frequent intra-operative tumour cell shedding. As the detection of CK+ cells is correlated with disease recurrence, modification of surgical techniques to prevent cell dissemination, and additional therapeutic concepts such as advanced filtration technology, have to be discussed.

Topics: Bone Marrow; Catheterization; Cell Adhesion; Cell Count; Colorectal Neoplasms; Female; Filtration; Hepatectomy; Humans; Keratins; Liver Neoplasms; Male; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Seeding; Neoplastic Cells, Circulating; Survival Analysis

Isolated tumor cells are often found in the regional lymph nodes of colorectal cancer, although their prognostic significance has not been established yet. This study was performed to investigate the correlation between the presence of isolated tumor cells in lymph nodes and the histopathologic characteristics of colorectal cancers and, thus, to determine which factors are associated with isolated tumor cells.. We used immunohistochemistry with anticytokeratin antibody to examine 2,784 lymph nodes in 109 patients with node-negative colorectal cancers. The clinicopathologic features of the tumors with isolated tumor cells were compared with those without isolated tumor cells. The frequency, number, and level of the isolated tumor cells also were assessed.. Isolated tumor cells were detected in 335 lymph nodes (12 percent) from 71 patients (65.1 percent). Those tumors having isolated tumor cells in lymph nodes, compared with those not having isolated tumor cells, were characterized by large tumor size, high T stage (pT3 and pT4), angiolymphatic invasion, perineural invasion, absence of peritumoral lymphocytic response, microsatellite instability-negative phenotype, and tumor budding. Multivariate analysis showed that those factors independently associated with the presence of isolated tumor cells were high T stage, tumor budding, and microsatellite instability-negative phenotype. Among the 71 patients with high T stage and microsatellite instability-negative phenotype, tumors with isolated tumor cells were characterized by a high frequency of tumor budding compared with tumors without isolated tumor cells (85 vs. 36.4 percent). In a further study, the degree of budding, which was assessed by an immunohistochemical study of gamma2 chain of laminin-5, was closely related to the number and location of isolated tumor cells. Moreover, we found that most of the isolated tumor cells in the regional lymph nodes also expressed gamma2 chain of laminin-5.. Our results suggested that isolated tumor cells are derived from undifferentiated cancer cells or small clusters ("budding") at the invasive front. Thus, tumor budding may be used as an indicator of isolated tumor cells in lymph nodes with node-negative colorectal cancers.

Topics: Adult; Aged; Aged, 80 and over; Analysis of Variance; Carcinoma; Chi-Square Distribution; Colorectal Neoplasms; Humans; Immunohistochemistry; Keratins; Logistic Models; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging; Predictive Value of Tests; Prognosis; Sensitivity and Specificity

To evaluate the serum levels of cytokeratins and carcinoembryonic antigen (CEA) in diagnosis, staging and prognosis of patients with colorectal adenocarcinoma.. The sample consisted of 169 patients. One hundred blood donors formed the control group. Radical surgery was performed on 120 patients, with an average follow-up duration of 22.3 mo. Relapses occurred in 23 individuals after an average of 18.09 mo. CEA was assayed via the Delfia method with a limit of 5 ng/mL. Cytokeratins were assayed via the LIA-mat TPA-M Prolifigen method with a limit of 72 U/L.. In the diagnosis of patients with colorectal adenocarcinoma, CEA showed a sensitivity of 56%, a specificity of 95%, a positive predictive value of 94%, a negative predictive value of 50% and an accuracy of 76.8%. TPA-M had a sensitivity of 70%, a specificity of 96%, a positive predictive value of 97%, a negative predictive value of 66% and an accuracy of 93.6%. The elevation of one of the markers was shown to have a sensitivity of 76.9%, a specificity of 91%, a positive predictive value of 93.5%, a negative predictive value of 70% and an accuracy of 83.6%. There was no variation in the levels of the markers according to the degree of cell differentiation while there was an elevation in their concentrations in accordance with the increase in neoplastic dissemination. There was a statistically significant difference between the patients with stage IV lesions and those with stages I, II and III tumors. With regard to CEA, the average level was 14.2 ng/mL in patients with stage I lesions, 8.5 ng/mL in patients with stage II lesions, 8.0 ng/mL in patients with stage III lesions and 87.7 ng/mL in patients with stage IV lesions. In relation to TPA-M, the levels were 153.1 U/L in patients with stage I tumors, 106.5 U/L in patients with stage II tumors, 136.3 U/L in patients with stage III tumors and 464.3 U/L in patients with stage IV tumors. There was a statistical difference in patients with a high CEA level in relation to a shorter survival (P<0.05). However, there was no correlation between patients with high TPA-M levels and prognostic indices of patients undergoing radical surgery.. Cytokeratins demonstrate a greater sensitivity than CEA in the diagnosis of colorectal adenocarcinoma. There is an increase in the sensitivity of the markers with tumor dissemination. Cytokeratins cannot identify the worse prognosis in patients undergoing radical surgery. Cytokeratins constitute an advance in the direction of a perfect tumor marker in the treatment of patients with colorectal cancer.

Topics: Adenocarcinoma; Adolescent; Adult; Biomarkers, Tumor; Carcinoembryonic Antigen; Colorectal Neoplasms; Female; Follow-Up Studies; Humans; Keratins; Male; Middle Aged; Neoplasm Staging; Prognosis; Survival Rate

The ability to use archival tissue to test externally valid hypotheses of carcinogenesis is dependent on the availability of population-based samples of cancer tissue. Tissue microarrays (TMAs) provide an efficient format for developing population-based samples of tissue. A TMA was constructed consisting of archival tissue from patients diagnosed with invasive colorectal cancer in the state of Hawaii in 1995. The population representativeness of the TMA was evaluated by comparing patient and clinical characteristics of TMA cases to that of all cases of colorectal carcinoma diagnosed statewide in 1995. Cytokeratin 20 (CK20) and cytokeratin 7 (CK7) immunohistochemistry was used to validate the utility of the TMA, and the expression of these proteins was correlated with patient and tumor characteristics. The TMA comprised tissue specimens from 286 patients representing 47% of all invasive cases diagnosed statewide in 1995. TMA cases were comparable to all invasive colorectal cases statewide with respect to age, sex, race/ethnicity, anatomic site, and survival. There were some differences between TMA cases and all cases with respect to tumor stage, histological classification, and treatment. There were significant differences in the relative expression of CK20 and CK7 proteins between malignant and normal tissues and by tumor stage. Advanced cancers were more likely to have CK20+/cytokeratin 7+ (CK7+) profiles than early-stage cancers, which were predominantly CK20+/cytokeratin 7- (CK7-). CK7+ expression was not correlated with anatomic location of carcinomas. This well-characterized TMA offers a powerful tool for testing hypotheses regarding colorectal carcinogenesis, including the identification of potential markers of neoplastic development and progression.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Male; Middle Aged; Mortality; Neoplasm Invasiveness; Neoplasm Staging; Protein Array Analysis; Survival Rate

Lymph node infarction is a spontaneous coagulative necrosis of the affected lymph node and is frequently associated with concurrent and subsequent malignant lymphoma. However, this phenomenon appears to be rarely associated with metastatic carcinomas. Here, we report on the histopathologic and immunohistologic findings of three cases showing lymph node infarction in the regional lymph node associated with metastatic colorectal adenocarcinoma. Histologically, coagulative necrosis of metastatic carcinoma was surrounded by a thick rim of granuloma consisting of histiocytes with or without epithelioid features, foamy cells, and a small number of lymphocytes. The immunohistochemical study of the coagulative necrosis demonstrated that cytokeratins (AEI/AE3 and CAM5.2) and carcinoembryonic antigen (CEA) were well preserved in all three cases. However, compared with viable tumor tissues, only a few tumor cells were positive for epithelial membrane antigen. Using formalin-fixed and paraffin-embedded tissues, immunostaining for cytokeratins and CEA of the lymph node containing necrotic carcinoma may provide clinically valuable information.

Topics: Adenocarcinoma; Adult; Aged; Biomarkers, Tumor; Carcinoembryonic Antigen; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Infarction; Keratins; Lymph Nodes; Lymphatic Diseases; Lymphatic Metastasis; Middle Aged; Necrosis

The accuracy of DNA ploidy measurements of paraffin-embedded tissues is limited by the lack of resolution and the inability to identify the DNA diploid population unequivocally in bimodal DNA histograms. A multi-parameter DNA flow cytometric method has been developed that enables the simultaneous detection of neoplastic and stromal cells in samples from dewaxed 50 microm sections or 2 mm diameter punches of archival tissue blocks. The method combines heat pretreatment in sodium citrate buffer and subsequent enzymatic dissociation with a collagenase/dispase mixture. Cells were simultaneously stained for keratin (FITC), vimentin (R-PE), and DNA (PI) before flow cytometric analysis. The method was applied to 12 paraffin-embedded cervical carcinomas and four colorectal carcinomas. In all cervical cancers, distinct keratin-positive and vimentin-positive cell populations were observed. While the exclusive vimentin-positive cell fractions always yielded unimodal DNA content distributions, bimodal distributions were observed for the keratin-positive cell fractions in nine cervical carcinomas, whereas one cervical carcinoma showed three distinct G0G1 populations. Coefficients of variation of the G0G1 peaks ranged from 1.70% to 4.79%. Average background, aggregate, and debris values were 14.7% (vimentin-positive fraction) and 33.8% (keratin-positive fraction). Flow sorting confirmed that the exclusively vimentin-positive cell fractions represent different normal stromal and infiltrate cells that can serve as an internal ploidy reference enabling discrimination between DNA hypo-diploid and DNA hyper-diploid tumour cell subpopulations. The neoplastic origin of the keratin-vimentin co-expressing cells from two cervical carcinomas was confirmed by genotyping of flow-sorted samples revealing loss of heterozygosity (LOH) of 6p. This improved method obviates the need for fresh/frozen tumour tissue for high-resolution DNA ploidy measurements and enables the isolation of highly purified tumour subpopulations for subsequent genotyping.

Topics: Antibodies, Monoclonal; Colorectal Neoplasms; DNA, Neoplasm; Female; Flow Cytometry; Formaldehyde; Hot Temperature; Humans; Keratins; Loss of Heterozygosity; Neoplasm Proteins; Paraffin Embedding; Ploidies; Stromal Cells; Tissue Fixation; Uterine Cervical Neoplasms; Vimentin

The identification of specific protein markers for colorectal cancer would provide the basis for early diagnosis and detection, as well as clues for understanding the molecular mechanisms governing cancer progression. In this report, we describe the proteomic analysis of the samples of colorectal cancer corresponding to seven patients. We have used the highly sensitive two-dimensional differential gel electrophoresis (2-D DIGE) coupled with mass spectrometry (MS) for the identification of proteins differentially expressed in tumoral and neighboring normal mucosa. We have detected differences in abundance of 52 proteins with statistical variance of the tumor versus normal spot volume ratio within the 95th confidence level (Student's t-test; p < 0.05). Forty-one out of 52 analyzed proteins were unambiguously identified by matrix-assisted laser desorption/ionization-time of flight MS coupled with database interrogation as being differentially expressed in colorectal cancer. An ontology analysis of these proteins revealed that they were mainly involved in regulation of transcription (synovial sarcoma X5 protein, metastasis-associated protein 1), cellular reorganization and cytoskeleton (cytokeratins, vimentin, beta actin), cell communication and signal transduction (annexins IV and V, relaxin, APC), and protein synthesis and folding (heat shock protein 60, calreticulin, cathepsin D, RSP4) among others. Preliminary studies demonstrated that the differentially expressed proteins found by 2-D DIGE could be confirmed and validated by immunoblotting and immunohistochemistry analyses in those few cases where antibodies were available. We believe that the incorporation of more samples and new datasets will permit the definition of a collection of proteins with a potential interest as biomarkers for colorectal cancer.

Topics: Biopsy; Colorectal Neoplasms; Electrophoresis, Gel, Two-Dimensional; Humans; Intestinal Mucosa; Keratins; Neoplasm Proteins; Proteomics; Reference Values; Reproducibility of Results; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

To detect lymph nodes micrometastases and analyze its correlation with clinicopathological parameters in Dukes' A and B colorectal cancer patients.. One hundred and fourteen patients with colorectal cancer (Dukes' A 16; Dukes' B 98) undergoing curative operation without histological lymph nodes metastases were studied between 2001 and 2003. A total of 2,481 lymph nodes were analyzed using monoclonal cytokeratin antibody AE1/AE3 (DAKO, Carpinteria, CA) for immunohistochemistry.. In total, 33 (29%) patients were positive for cancer cell by immunohistochemistry. In 31 (94%) patients of them positive nodes showed single tumor cell or small groups of tumor cells; and tumor deposits measuring 0.2 and 0.37 mm in diameter in another 2 (6%) patients. Micrometastases were mainly located in the subcapsular sinus or paracortical sinus. There was no correlation between the positive lymph nodes and gender, age, tumor site, tumor size, histological type, histological grade, invasion depth, Dukes' staging and microsatellite instability (P>0.05).. Our findings suggest that immunohistochemical technique using monoclonal cytokeratin antibody AE1/AE3 may be a sensitive and reliable method for detecting lymph nodes micrometastases in Dukes' A and B colorectal cancer. The clinical significance of lymph nodes micrometastases is still not confirmed.

Topics: Antibodies; Colorectal Neoplasms; Humans; Immunohistochemistry; Keratins; Lymphatic Metastasis; Neoplasm Staging

The term tumour 'budding' has been coined for the detachment of tumour cells from the neoplastic glands of adenocarcinomas and is presumed to be an early step in the metastatic process. A limited number of studies have shown budding to be an adverse prognostic factor.. All primary single, non-metachronous TNM stage I/II colorectal carcinomas without neoadjuvant treatment resected in the years 1994-1999 were included (n = 186). Tumour buds were counted in pan-cytokeratin immunostains in a 0.785-mm2 field of vision (250 x). During follow-up 21 patients had distant metastases and 12 patients died of their disease. Budding was determined at 14 and 20.46, median and mean, respectively (range 0-120). A cut-off of 25 was found to be sensitive (0.76) and specific (0.739). Kaplan-Meier survival analysis showed high budding to be a strong adverse prognosticator. By Cox regression, high budding together with venous angioinvasion were independent prognostic factors.. This study confirms the prognostic value of budding in a contemporary series of colorectal carcinomas that by TNM were low risk. Technically easy, rapid and robust to determine, budding quantified in pan-cytokeratin stains significantly aids in the identification of high-risk patients and is recommended for more general use in surgical pathology.

Topics: Adult; Aged; Aged, 80 and over; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Male; Middle Aged; Multivariate Analysis; Neoplasm Metastasis; Neoplasm Staging; Prognosis; Survival Analysis; Survival Rate

Early diagnosis for colorectal cancer (CRC) by monitoring of gastrointestinal epithelial cells is a possible direct approach. Although human faeces form a heterogeneous mixture of gastrointestinal mucosal epithelial cells and other materials, we have developed a method to purify total RNA from human stool samples. The gene for faecal cytokeratin 19 (CK19) was highly expressed in stools from patients with metastatic CRC, but not from patients with non-metastatic CRC or from normal individuals. Thus, purified faecal RNA can be used for the detection of differentially expressed genes. This technique may help identifying meaningful faecal RNA markers for the non-invasive screening of patients with CRC.

Topics: Biomarkers, Tumor; Case-Control Studies; Colorectal Neoplasms; Diagnosis, Differential; Feces; Gene Expression Profiling; Humans; Keratins; Mass Screening; RNA, Neoplasm

Detection of circulating cancer cells is a useful indicator for the risk of recurrence of advanced carcinoma. The aim of the present study was to evaluate the potential value of a novel approach to detect the circulating cancer cells in patients with colorectal cancer. This method is based on a combination of isolation of epithelial cell by a combination of negative and positive immunomagnetic beads with detection of cytokeratin 20 (CK20) mRNA by reverse transcriptase-polymeras chain reaction (RT-PCR).. Peripheral blood samples were collected from 40 patients with colorectal carcinoma on the day before operation or chemical therapy. Mononuclear cells (MNC) were isolated by centrifugation through a Ficoll gradient. Each MNC sample was equally divided into three parts and then CD45 immunomagnetic beads and/or Ber-EP4 immunomagnetic beads were used to enrich colon cancer cells. Finally, the CK20 mRNA was detected by real-time quantitative RT-PCR. As a control, LS174T colon cancer cells were serially diluted with blood from healthy individuals.. When CD45 and Ber-EP4 immunomagnetic beads were used successively, a significant correlation between CK20 mRNA levels and the initial cell concentrations was found in the control recovery experiment. The sensitivity of the assay was one cancer cell in 1 mL healthy blood. In the patient group, CK20 mRNA was detected in 80.0%, 82.5% and 72.5% of patients when CD45, Ber-EP4, and CD45/Ber-EP4 immunomagnetic beads were used, respectively. The positive detection rates of patients with colorectal carcinoma at Dukes A, B, C, and D stage were 0.0% (0/2), 33.3% (3/9), 86.7% (13/15), and 92.9% (13/14), respectively. The CK20 mRNA positive detection rate in peripheral blood was significantly correlated with tumor diameter (P < 0.01, chi(2)), lymphatic metastasis (P < 0.05) and hepatic metastasis (P < 0.05), but not with the differentiation of tumor cells.. The combined use of negative and positive immunomagnetic beads followed by amplification of CK20 mRNA by means of RT-PCR is a non-invasive, sensitive, and specific assay for the detection of circulating colonic cancer cells.

Topics: Aged; Aged, 80 and over; beta 2-Microglobulin; Biomarkers, Tumor; Cell Line, Tumor; Colorectal Neoplasms; Female; Humans; Immunomagnetic Separation; Keratin-20; Keratins; Leukocyte Common Antigens; Male; Middle Aged; Neoplastic Cells, Circulating; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Distinguishing primary ovarian carcinoma from metastatic carcinoma to the ovary is often difficult by histologic examination alone. Recently an immunohistochemical marker CDX-2 was found to be of considerable diagnostic value in establishing the gastrointestinal origin of metastatic tumors. The aim of this study was to determine whether CDX-2 can distinguish between these malignancies. Paraffin-embedded tissue sections from 57 primary ovarian tumors and 40 metastatic tumors to the ovary were immunostained for CDX-2, and results were compared to the ancillary immunohistochemical results for CK7/CK20, CEA, CA125, and her-2/neu. CDX-2 immunoreactivity was observed in most of metastatic carcinomas with colorectal (91%) and appendiceal (100%) origin, however CDX-2 was negative in all primary ovarian carcinomas, except for the mucinous subtype. Almost all primary ovarian carcinomas including the mucinous subtype showed diffuse and strong immunoexpression for CK7. CEA and CA125 were mainly found in metastatic and primary ovarian carcinoma, respectively. Her-2/neu overexpression was only noted in a small proportion of primary and metastatic ovarian carcinomas. These results suggest that CDX-2 is very useful immunohistochemical marker for distinguishing metastatic colorectal carcinoma to the ovary from primary ovarian carcinoma, including the mucinous subtype. Furthermore, combination with CDX-2 and CK7 strengthen the differential diagnosis between these tumors.

Topics: CA-125 Antigen; Carcinoembryonic Antigen; CDX2 Transcription Factor; Colorectal Neoplasms; Diagnosis, Differential; Female; Homeodomain Proteins; Humans; Immunohistochemistry; Keratin-7; Keratins; Neoplasm Metastasis; Ovarian Neoplasms; Receptor, ErbB-2; Tissue Array Analysis; Trans-Activators

The aim of this study is to diagnose the evaluation of concentration of CEA and TPS in postoperative monitoring of patients with colorectal cancer.. We measured 178 consecutive patients with histopathologically confirmed colorectal cancer: 101 men and 78 women ages 22-86 (average age 54.7). Markers' CEA nad TPS concentration were evaluated before operation and every month after operation during the first 3 months and then every 3 months during 2 years. Relapse was detected in 47 patients.. In postoperative period in non-relapse group the mean (the average) concentration of CEA was 1.92+/-2.03 ng/ml and TPS 65.54+/-33.96 U/l and respectively in relapse group for CEA was 1.92+/-2.03 ng/ml and for TPS 65.54+/-33.96 U/l. The obtained results in investigated group show significantly statistical. The relapse was confirmed by using CEA concentration in 42 patients (89.4%). In case of TPS concentration relapse was confirmed in 38 patients (80.85%). The relapse was detected in 45 patients (95.74) if increase in CEA or TPS concentration was treated as a way of detecting relapse. TPS markers point out that the increase of TPS concentartion may be ahead of relapse symptoms at about 2-6 months.. TPS is a useful marker in postoperative monitoring of patients with colorectal cancer. The evaluation of TPS concentration allow to diagnose the recurrence of colorectal cancer earlier than by using burden markers--CEA. Common evaluation of TPS and CEA increase sensitivity in detection of relapse in patients with colorectal cancer.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoembryonic Antigen; Colorectal Neoplasms; Female; Follow-Up Studies; Humans; Keratins; Male; Middle Aged; Monitoring, Immunologic; Predictive Value of Tests; Recurrence; Sensitivity and Specificity

The entity of serrated adenoma of the colorectum was first proposed in 1990, and it was characterized as epithelial neoplasia combining the architectural features of a hyperplastic polyp with the cytological features of an adenoma. Over the past few years, various clinicopathological studies on serrated adenoma have been reported, but its histogenesis remains unclear. Recently the existence of a "serrated neoplasia pathway" leading to malignancy, which is different from the so-called adenoma-carcinoma sequence, has been discussed. Yao et al. reported that hyperplastic polyps and serrated adenomas share a common cell lineage with gastric differentiation. To clarify the existence of the serrated neoplasia pathway, we performed immunohistochemical staining of cytokeratin 7 (CK7) and cytokeratin 20 (CK20), which are commonly used to determine the primary site of a metastatic lesion, and we examined the pattern of CK7/CK20 expression in various colorectal lesions including 44 serrated adenomas, 25 hyperplastic polyps, 20 traditional adenomas, and 48 carcinomas. An obvious difference existed in the pattern of CK7/CK20 expression between the serrated lesions (hyperplastic polyps and serrated adenomas) and others. The majority of serrated adenomas and hyperplastic polyps presented a CK7+/CK20+ pattern, whereas most conventional adenomas and adenocarcinomas expressed CK7-/CK20+. Adenocarcinoma developing in serrated adenoma also presented a CK7+/CK20+ pattern. There are several reports that CK7 is a possible marker of transient dedifferentiation in the gastric carcinogenesis process. Taken together with the present results, a distinct pathway of colorectal carcinogenesis must exist, which is different from the adenoma-carcinoma sequence. CK7 is a possible marker for the serrated neoplasia pathway of colorectal carcinogenesis.

Topics: Adenocarcinoma; Adenoma; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Cell Transformation, Neoplastic; Colonic Polyps; Colorectal Neoplasms; Female; Gene Expression Profiling; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Male; Middle Aged

Small cell carcinoma is a rare malignant disease with high mortality, which is pathologically diagnosed by using routine neuroendocrinal markers, such as neuron-specific enolase (NSE), synaptophysin (SYN), chromogranin A (CgA). This study was to investigate the expression of CD56, a neural cell adhesion molecule (NCAM), in small cell carcinoma tissues, and to explore the possibility of CD56 as a diagnostic marker of small cell carcinoma.. Eighty samples of small cell carcinoma were collected, including 42 samples of small cell lung carcinoma (with 20 cases of lymph node metastases), 21 samples of small cell esophageal carcinoma, and 17 samples of small cell colorectal carcinoma. Thirty-eight samples of non-small cell lung cancer (with 28 cases of lymph node metastases), including 26 samples of squamous cell carcinoma and 12 samples of adenocarcinoma, were used as control. All samples were detected using markers of CD56, NSE, SYN, CgA, cytokeratin (CK), and epithelial membrane antigen (EMA) immunohistochemically.. Positive rate of CD56 was significantly higher in either small cell lung carcinoma or their metastatic lymph nodes than in either non-small cell lung carcinoma or their metastatic lymph nodes [90.5% (38/42) vs. 7.8% (3/38), 90.0% (18/20) vs. 3.5% (1/28); H=85.731, P<0.001]. Positive rate of CD56 in small cell carcinoma samples (86.3%, 69/80) were significantly higher than those of SYN (78.8%, 63/80), CgA (73.8%, 59/80), EMA (66.3%, 53/80), CK (61.3%, 49/80), and NSE (56.3%, 45/80) (H=38.871, P<0.001). Positive rate of CD56 in small cell lung carcinoma (90.5%, 38/42) was similar to those in small cell esophageal carcinoma (81.0%, 17/21) and small cell colorectal carcinoma (82.4%, 14/17) (H=1.651, P=0.438).. CD56 is highly expressed in either small cell carcinoma or their metastatic lymph nodes without organ-specificity. It could serve as a potential diagnostic marker of small cell carcinoma.

Topics: Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; CD56 Antigen; Chromogranin A; Colorectal Neoplasms; Diagnosis, Differential; Esophageal Neoplasms; Follow-Up Studies; Humans; Keratins; Lung Neoplasms; Lymphatic Metastasis; Mucin-1; Phosphopyruvate Hydratase; Synaptophysin

To evaluate the feasibility and utility of an ex vivo sentinel lymph node (SLN) identification and ultrastaging for colorectal cancer (CRC).. CRC patients undergoing resection of a primary colorectal cancer were considered for inclusion. Following resection, SLN identification was performed. The SLN was dissected from the mesentery and submitted separately for pathologic analysis. All lymph nodes were stained with HE. Blue lymph nodes, when negative by routine HE staining, were further analyzed.. A total of 62 tumors from 60 patients with colorectal cancer were studied. 95.2% (59/62) specimens was successfully identified. In these 59 specimens, a total of 1114 (18.9 per specimens) lymph nodes were examined; of these, 157 (14.9%) were designated as SLNs. The number of blue-stained lymph nodes removed ranged from 1 to 9, with a mean of 2.7 blue nodes identified. The sensitivity of a blue-stained lymph node identifying metastatic disease was 39.1%. The false-negative was 23.7%. In 4 specimens micrometastases were detected only by immunohistochemistry with cytokeratin.. Ex vivo sentinel lymph nodes mapping in colorectal cancer is feasible and can identify the SLNs with a very high success rate. Ex vivo SLN mapping improves pathologic staging of patients with CRC. The SLN evaluation should not replace attempts to harvest large number of nodes for standard processing. SLN mapping can help improving the number of nodes for pathological examination.

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

Hepatectomy and pancreatectomy are often associated with significant intraoperative blood loss leading to postoperative anemia, which has been demonstrated to lead to increased perioperative morbidity, a prolonged hospital stay, and decreased overall survival. Cancer has remained an absolute contraindication to autotransfusion because of the unproven concern about reinfusion of malignant cells. Thus, the aim of this study was to test for the presence of malignant cells in autotransfused filtered blood in patients undergoing major pancreatic and liver resection.. A prospective study of 20 consecutive patients evaluated the presence of malignant cells from autotransfusion filtered blood after resection by flow cytometric and immunohistochemical methods.. Ten patients underwent major hepatectomy for metastatic colorectal cancer, with a median blood loss of 500 mL (range, 200-700 mL). Three patients received a total of six units of packed red blood cells. Ten patients underwent pancreaticoduodenectomy for adenocarcinoma with a median blood loss of 400 mL (range, 200-1300 mL). Five patients received a total of nine units of packed red blood cells. Flow cytometry did not demonstrate the presence of any cytokeratin-positive carcinoma cells in filtered blood.. Intraoperative autotransfusion for major hepatectomy in metastatic colorectal cancer and pancreatectomy for adenocarcinoma is safe and should begin to be evaluated in a phase II study for efficacy.

Topics: Adenocarcinoma; Blood Loss, Surgical; Blood Transfusion, Autologous; Colorectal Neoplasms; Contraindications; Female; Flow Cytometry; Hemofiltration; Hepatectomy; Humans; Immunohistochemistry; Intraoperative Period; Keratins; Liver Neoplasms; Pancreatectomy; Pancreatic Neoplasms; Pancreaticoduodenectomy; Prospective Studies

We evaluated the usefulness of cytokeratin 20 (CK20) mRNA expression in the quantitative detection of circulating tumor cells in the blood of patients with colorectal cancer (CRC). Blood samples from healthy volunteers (HVs; n = 37), patients with localized (n = 42) and metastatic colorectal cancer (n = 40), and patients with chronic inflammatory bowel disease (CID; n = 15) were examined. After immunomagnetic enrichment using microbeads against human epithelial antigen, total RNA was extracted, reverse transcribed, and analyzed by real-time reverse transcriptase-polymerase chain reaction using the LightCycler instrument. CK20 expression in peripheral blood was found in 46 of 82 (56%) patients with CRC, 8 of 37 (22%) HVs, and 9 of 15 (60%) patients with CID. Levels of CK20 mRNA were significantly higher in blood samples from CRC patients (median 681) than in blood samples from HVs (median 0) (P = 0.001), whereas no difference could be detected between patients with CRC and CID. Although the present technique could not distinguish CRC from CID, the method warrants further efforts to improve sample preparation and tumor cell enrichment, which may render real-time CK20 reverse transcriptase-polymerase chain reaction a feasible technique in identifying circulating tumor cells in peripheral blood of cancer patients.

Topics: Adult; Aged; Aged, 80 and over; Case-Control Studies; Colorectal Neoplasms; Female; Health; HT29 Cells; Humans; Hydroxymethylbilane Synthase; Inflammatory Bowel Diseases; Keratin-20; Keratins; Male; Middle Aged; Reference Standards; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; ROC Curve; Sensitivity and Specificity

The diagnostic utility of aquaporin (AQP)-1 in liver tumors was tested and compared with other well-established markers. In 30 cholangiocarcinomas (CCs), 20 hepatocellular carcinomas (HCCs), and 10 metastatic colorectal carcinomas (MCCs) of the liver, expression of AQP-1, CD10, cytokeratin (CK) 7, CK20, and polyclonal carcinoembryonic antigen (pCEA) was tested. In addition, staining patterns of CD10 and pCEA were analyzed. To compare the selectivity of AQP-1 and CK7 as possible markers for differentiated cholangiocytes, liver biopsies of cholestatic disease were also analyzed. Aquaporin-1 expression was found in 93% of all CCs compared with 0% of HCC (P < .000001) and with 30% of MCC (P < .01). CD10 was positive in 16.7% of CC compared with 40% of HCC (P < .04) and to 20% of MCC (not significant). Cytokeratin 7 was positive in 90% of CC compared with 10% of HCC (P < .00001) and with 20% of MCC (P < .0001). Cytokeratin 20 was positive in 90% of MCC compared with 16.7% of CC (P < .0001) and with 20% of HCC (P < .00001). Canalicular staining patterns of CD10 and pCEA were observed in HCC (100% and 89.5%, respectively) but typically not in CC (0% and 6.7%, respectively) and never in MCC. In cholestatic disease, AQP-1 was expressed in differentiated epithelial cells of the bile ducts, whereas CK7-positive hepatocytes of Rappaport zone 1 did not show any AQP-1 reactivity. Therefore, AQP-1 seems to be a highly selective marker for differentiated cholangiocytes and can be very helpful in the differential diagnosis of liver tumors.

Topics: Adult; Aged; Aged, 80 and over; Aquaporin 1; Biomarkers, Tumor; Capillaries; Carcinoembryonic Antigen; Carcinoma, Hepatocellular; Cholangiocarcinoma; Cholestasis; Colorectal Neoplasms; Diagnosis, Differential; Female; Humans; Immunohistochemistry; Keratin-7; Keratins; Liver Neoplasms; Male; Middle Aged; Neprilysin

Distinguishing between primary adenocarcinomas and secondary colonic adenocarcinomas of the urinary bladder is often difficult because they appear morphologically similar but invariably require different treatment strategies. The aim of the study was to define the utility of a limited immunohistochemical panel consisting of CDX-2, cytokeratins 7 (CK7) and 20 (CK20), and carcinoembryonic antigen (CEA) in differentiating primary from secondary bladder adenocarcinomas. Formalin-fixed, paraffin-embedded tissues from 8 primary bladder adenocarcinomas and 23 colorectal adenocarcinomas involving the bladder were included in the study. Statistical analysis was performed using the Fisher exact test. The majority (87.5%) of primary bladder adenocarcinomas were CDX-2 negative, and only one case of primary bladder adenocarcinoma was positive, while CDX-2 was strongly expressed in the nucleus of all cases of secondary (colonic) bladder tumor (P < 0.0005). Five cases (62.5%) of primary bladder adenocarcinoma and one case (4.3%) of secondary bladder tumor showed positive staining for CK7 (P = 0.002), whereas CK20 showed positive staining in five cases (62.5%) of primary bladder adenocarcinoma and in all the secondary bladder tumors (P = 0.012). All 23 secondary bladder tumors and 7 primary bladder adenocarcinomas (87.5%) expressed CEA (P = 0.25). These data demonstrate that a restricted immunohistochemical panel consisting of CDX-2, CK7, CK20, and CEA may be of use in differentiating primary bladder adenocarcinoma from secondary adenocarcinoma of colorectal origin.

Topics: Adenocarcinoma; Aged; Biomarkers, Tumor; Carcinoembryonic Antigen; CDX2 Transcription Factor; Colorectal Neoplasms; Diagnosis, Differential; Female; Homeodomain Proteins; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Male; Trans-Activators; Urinary Bladder Neoplasms

To investigate the monoclonal antibody M30 for the assessment of apoptosis in colorectal tissues. Although Terminal deoxyribonucleotidyl transferase mediated nick end labelling (TUNEL) and in-situ end labelling (ISEL) are the methods most often used to demonstrate and quantify apoptosis in histological tissue sections, the interpretation and specificity of these techniques have been controversial. Immunohistochemistry using the monoclonal antibody M30 that recognizes caspase-cleaved cytokeratin 18 is considered to be a promising alternative but has yet to be validated against a generally accepted standard.. Paraffin sections of normal colonic mucosa (n = 30), normal mucosa obtained from resection margins from carcinomas (n = 30), colorectal adenomas (n = 84) and carcinomas (n = 40) were studied. Apoptosis of epithelial cells was assessed by M30 immunoreactivity and morphological criteria and expressed as a proportion of the total number of cells counted (apoptotic index). Mean apoptotic indices using M30 were 0.18 +/- 0.04% in normal mucosa, 0.42 +/- 0.04% in adenomas and 1.97 +/- 0.24% in carcinomas. Using morphological criteria, these indices were 0.23 +/- 0.03%, 0.62 +/- 0.06% and 1.78 +/- 0.19%, respectively. Apoptotic counts were higher in normal mucosa obtained from resection margins than in genuinely normal mucosa using the M30 antibody. Apoptotic indices obtained by M30 immunoreactivity and morphological criteria were positively correlated (r = 0.71, P < 0.01).. Assessment of apoptotic cells by M30 immunoreactivity correlates well with morphological criteria. Apoptotic indices increase in the course of the adenoma-carcinoma sequence. Apoptosis in normal mucosa obtained from resection margins differs from genuinely normal mucosa necessitating caution when interpreting studies of apoptosis in normal colonic mucosa. Our findings support the use of the M30 method in the study of apoptosis in colorectal tissues.

Topics: Adenocarcinoma; Adenoma; Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Apoptosis; Cell Count; Colon; Colorectal Neoplasms; Female; Humans; Immunoenzyme Techniques; In Situ Nick-End Labeling; Intestinal Mucosa; Keratins; Male; Middle Aged

Stool-based molecular techniques may improve strategies for colorectal cancer screening. Molecular methods have successfully been applied to detect tumour DNA in stool from patients diagnosed for colorectal carcinoma. In these assays human DNA has to be analyzed against a background of excess nucleic acids from bacteria and dietary waste products. More recently a different diagnostic approach has been described characterizing intact cells isolated from stool. In this study we combine both approaches preparing nucleic acids from isolated epithelial cells to evaluate if: a) tumour cell-specific RNA can be analyzed since cellular RNA molecules are prevented from early digestion by an intact cell membrane; and b) specificity or sensitivity of established DNA-based methods can be improved when epithelial cells are separated from other stool components. Comparing different protocols we found cell isolation using epithelium-specific antibodies to be more effective and reproducible than a technique using density gradient centrifugation. A detection limit of 10(4) cells per ml stool was determined when samples from healthy volunteers were spiked with epithelial cells. Amplification of human sequences from total stool DNA was more efficient than a correspondent amplification of DNA extracted from isolated cells, so that an improvement of DNA-based methods cannot be expected by introducing cell isolation procedures. RNA detection was successful in 1 of 5 patients with confirmed diagnosis of colorectal cancer. The authors suggest that low numbers of detectable cells might rather be a biological than an analytical problem limiting a routinely performed method for colorectal cancer diagnosis.

Topics: Base Sequence; Case-Control Studies; Colorectal Neoplasms; DNA, Neoplasm; Epithelial Cells; Feces; Genes, p53; Humans; Keratins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm

A case history is presented of a 53-year-old woman with an incidental finding of a breast lump, identified after having had chemotherapy for lung metastases from a rectal carcinoma. Clinical examination, ultrasound, mammography, fine needle aspiration and core biopsies could not prove definitively whether the breast lump represented a metastasis from colorectal carcinoma. Following local excision, the final diagnosis of metastatic colorectal carcinoma to the breast was based on the absence of any site of origin within the breast (i.e. no surrounding DCIS) and on the expression of cytokeratin CK7 and CK20 on immunohistochemistry. Postoperative chemotherapy was initiated. Four months later, although without local recurrence in the breast, the patient developed cutaneous metastatic deposits and active treatment was stopped. A review of other cases of breast metastases from extramammary sources is presented. Possible mechanisms for this rare and unusual phenomenon are discussed.

Topics: Adenocarcinoma; Biomarkers; Breast Neoplasms; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Lung Neoplasms; Middle Aged; Skin Neoplasms

Immunohistochemistry plays an important role in tracing the primary site in metastatic tumors of unknown origin. Therefore, determining the cytokeratin (CK) 20/CK7 pattern of metastases is one of the most helpful procedures as the CK20+/CK7- pattern is typical of colorectal adenocarcinomas. Expression of CDX2 protein is a new, highly specific and sensitive marker of the intestinal origin of adenocarcinomas. In the present study we compared the sensitivity and specificity of CDX2 expression and the CK20+/CK7- phenotype in predicting the colorectal origin of liver metastases.. The study was carried out on a consecutive series of 125 core-needle biopsies of metastatic adenocarcinomas of the liver. Most of the patients were followed up to death, and primary tumor localization could be established in 102 cases by a combination of clinical, radiological, histological and, in some cases, autopsy data. All the needle biopsies were immunohistochemically stained for CK7, CK20 and CDX2. CDX2 expression (at 10% and 50% cut-off levels) and the CK20/CK7 pattern of the metastases were correlated to the primary site established.. The CK20+/CK7- pattern showed a specificity of 98.7% in predicting colorectal primary localization, which was superior to that of CDX2 expression at both cut-off levels (90% and 95.3% respectively). The sensitivity of CDX2 expression in these circumstances was 84% at the 10% cut-off, somewhat higher than that of the CK20+/CK7- phenotype (79.5%), but lower at the 50% cut-off level (72.7%).. The CK20+/CK7- immunophenotype is more specific in predicting the colorectal origin of liver metastasis than CDX2 expression.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Antibodies; CDX2 Transcription Factor; Colorectal Neoplasms; Female; Homeodomain Proteins; Humans; Immunohistochemistry; Immunophenotyping; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Liver Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Phenotype; Sensitivity and Specificity; Trans-Activators

High levels of microsatellite instability (MSI-H) result from abnormal nucleotide mismatch repair in a subset of sporadic colorectal carcinomas (CRC) and in most CRC of hereditary non-polyposis colorectal cancer syndrome. CRC with MSI-H have distinctive clinical-pathologic features, but the immunophenotype has not been studied extensively. We evaluated immunohistochemical expression of cytokeratin 7 (CK7), cytokeratin 20 (CK20), and pancytokeratin (panCK) in 44 CRC from 22 paired MSI-H and microsatellite-stable (MSS) cases matched for clinical-pathologic characteristics. The mean percentage of CK20+ tumor cells was 84 +/- 6% in MSS CRC but only 37 +/- 8% in MSI-H CRC (P = 0.0007). Thirty-two percent (7/22, 95% confidence interval 14-55%) of MSI-H CRC were CK20-, as contrasted with 9% (2/22, 95% CI 1-29%, P = 0.13) of MSS CRC. CK20 expression was inversely correlated with levels of MSI (rs = -0.45, P = 0.006). CK7+ was infrequent (16%, 7/44, 95% CI 7-30%) and panCK+ was universal, with no significant differences between MSI-H and MSS CRC. Our study shows that decreased or even absent CK20 expression is a phenotypic characteristic of MSI-H CRC and that MSI-H explains much of the subset of CRC that lack CK20 expression. Our results also indicate that regulation of CK20 gene expression involves molecular pathways that are altered by MSI-H.

Topics: Colorectal Neoplasms; Female; Genomic Instability; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Male; Microsatellite Repeats; Middle Aged; Polymerase Chain Reaction; Retrospective Studies

Topics: Adenocarcinoma, Mucinous; Carcinoembryonic Antigen; Colorectal Neoplasms; Diagnosis, Differential; Female; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Ovarian Neoplasms

Substantial evidence supports that detailed analysis of the regional lymphatics will identify previously unrecognized micrometastatic disease in colorectal cancer. In order to determine whether the sentinel lymph node(s) (SLNs) harvested by ex vivo lymphatic mapping in node-negative colorectal cancer (CRC) are the most likely node(s) to harbor micrometastatic disease, we examined all nodes in CRC specimens in an identical fashion.. One hundred twenty-four specimens from patients with colorectal cancer were delivered to pathology in the fresh state and underwent ex vivo sentinel lymph node mapping. If negative by routine hematoxylin and eosin (H&E) analysis, the SLNs and non-SLNs were subjected to further analysis by level section H&E and immunohistochemical (IHC) analysis.. A mean of 30 nodes were harvested (range, 5-111). Fifty-one patients (41%) were found to be node-positive by routine H&E analysis. SLNs were identified in all but three specimens. A total of 2177 nodes were analyzed from the 66 H&E node-negative specimens (1883 non-SLNs and 294 SLNs). Overall, metastases were identified in 13 of 278 SLNs and in only 5 of 1829 non-SLNs (P <.001). Only 5 of 66 patients (7.5%) had evidence of metastatic disease in non-SLNs when the SLNs were negative. Thirteen apparently node-negative patients (19.3%) were upstaged by IHC analysis of the SLNs (P =.04).. If the SLN is negative by both H&E and IHC analysis, the probability of finding metastases in a non-SLN is remote. If microstaging is demonstrated to be prognostically relevant, focused examination should be of the SLN(s).

Topics: Adult; Aged; Aged, 80 and over; Carcinoma; Colorectal Neoplasms; Female; Hematoxylin; Humans; Immunohistochemistry; Keratins; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging

We studied the diagnostic value of Cdx2 to distinguish mucinous bronchioloalveolar carcinoma from mucinous colorectal adenocarcinoma metastatic to the lung. We retrieved 92 via the hospital computer system, including 30 mucinous bronchioloalveolar carcinomas, 32 nonmucinous bronchioloalveolar carcinomas, and 30 mucinous colorectal adenocarcinomas metastatic to the lung. All cases were confirmed by clinical history and surgical resection with occasional immunohistochemical studies. Cases were stained with antibodies against Cdx2, thyroid transcription factor-1 (TTF-1), cytokeratin (CK) 7, and CK20. Bronchioloalveolar carcinoma, mucinous type, showed positive staining for Cdx2, TTF-1, CK7, and CK20 in 0 (0%), 5 (17%), 30 (100%), and 18 (60%) of 30 cases, respectively; nonmucinous tumors were positive in 0 (0%), 30 (94%), 32 (100%), and 0 (0%) of 32 cases, respectively. For colorectal adenocarcinoma, the positive staining for Cdx-2, TTF-1, CK7, and CK20 was 29 (97%), 0 (0%), 7 (23%), and 29 (97%) of 30 cases, respectively. Our results demonstrated Cdx2 as a sensitive and specific marker for differentiating metastatic colorectal adenocarcinoma from mucinous bronchioloalveolar adenocarcinoma.

Topics: Adenocarcinoma; Adenocarcinoma, Bronchiolo-Alveolar; Avian Proteins; Biomarkers, Tumor; Colorectal Neoplasms; Diagnosis, Differential; Homeodomain Proteins; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Lung Neoplasms; Nuclear Proteins; Thyroid Nuclear Factor 1; Transcription Factors

Twenty-four cases of primary nonampullary small intestinal adenocarcinoma were immunohistochemically examined for the expression of cytokeratin (CK) 7 and CK20 and compared with 23 colorectal adenocarcinomas secondarily involving the small intestine by direct extension or metastasis. While normal small intestinal mucosa was diffusely positive for CK20 and completely negative for CK7 expression, all small intestinal adenocarcinomas (24 of 24) showed a variable degree of CK7 expression. Specifically, the CK7 staining pattern was diffuse in 13 cases (54%) and focal in the remaining cases. Sixteen small intestinal adenocarcinomas (67%) coexpressed CK7 and CK20, and 8 (33%) completely lost CK20 immunoreactivity when compared with adjacent non-neoplastic small intestinal mucosa. In the latter cases, the loss of CK20 immunoreactivity with a reciprocal emergence of CK7 expression was evident. This was in contrast to secondary colorectal adenocarcinomas where 22 cases (96%) expressed CK20, among which only 1 case showed focal CK7 expression. The remaining 1 case was negative for both CK7 and CK20. Interestingly, adenomatous epithelium associated with small intestinal adenocarcinomas identified in 18 cases also exhibited CK7 positivity with a sharp transition from CK7-negative normal-appearing epithelium. Taken together, these observations delineate an alteration of CK7 and CK20 expression profile that occurs early in small intestinal tumorigenesis. This unique pattern may be of diagnostic value in distinguishing primary small intestinal adenocarcinoma from secondary colorectal adenocarcinoma.

Topics: Adenocarcinoma; Adult; Aged; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Infant, Newborn; Intermediate Filament Proteins; Intestinal Neoplasms; Intestine, Small; Keratin-20; Keratin-7; Keratins; Male; Middle Aged; Retrospective Studies

It is know that bone marrow micrometastases are an indicator of poor prognosis in patients with epithelial tumors. At the same time little is known if they reflect minimal residual disease or cells distribution and marked metastatic potential. We aimed to perform a clinical study, which though with little gathered clinical material has a direction--investigation of bone marrow in patients with colorectal cancer by the time of surgery, carefully rendering an account of staging and performing curative resection. In intraoperatively done bone marrow biopsy with following investigation for cytoceratin 19 positive cells we found ol presence of metastatic cells in some the studied patients. Finding micrometastases in patients with colorectal cancer is a marker for more agressive course of the disease and for the need to change some of recent methods of treatment.

Topics: Biomarkers, Tumor; Biopsy, Fine-Needle; Bone Marrow Examination; Bone Marrow Neoplasms; Colorectal Neoplasms; Humans; Keratins; Neoplasm Metastasis; Neoplasm, Residual

In this study, 62 preoperative peripheral blood samples from patients with gastrointestinal carcinomas, 12 healthy volunteers, and ten patients with inflammatory gastrointestinal diseases were analyzed for the determination of CEA, CK19, and CK20 mRNA expression in peripheral blood, and its clinical significance was evaluated.. Nested reverse transcriptase-polymerase chain reaction (nested RT-PCR) was used to analyze CEA, CK19, and CK20 mRNA expression in peripheral blood. Fresh tumor tissues from patients with colorectal cancer (n=15) were used as a positive control.. Among 62 blood samples from patients with gastrointestinal cancer, the expression of CEA, CK19, and CK20 mRNA was 51.6% (32/62), 35.5% (22/62), and 48.4% (30/62), respectively. 74.2% (46/62) were positive for at least one marker. Fifteen tumor tissues were all positive for CEA, CK19, and CK20 mRNA. Only one blood sample from patients with no gastrointestinal carcinoma was positive for CEA mRNA.. Our results indicate that the molecular detection of circulating tumor cells in peripheral blood in patients with gastrointestinal carcinoma was significantly correlated with its malignant biological properties, and may be helpful in the selection of clinical treatment and judgment of prognosis.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoembryonic Antigen; Case-Control Studies; Colorectal Neoplasms; DNA Primers; Esophageal Neoplasms; Female; Gastrointestinal Neoplasms; Humans; Intermediate Filament Proteins; Keratin-20; Keratins; Male; Middle Aged; Neoplasm Staging; Neoplastic Cells, Circulating; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm

Nodal staging accuracy is important for prognosis and selection of patients for chemotherapy. Sentinel lymph node (SLN) mapping improves staging accuracy in breast cancer and melanoma and is being investigated for colorectal carcinoma.. To assess pathologic aspects of SLN staging for colon cancer.. Sentinel lymph nodes were identified with a dual surgeon-pathologist technique in 51 colorectal carcinomas and 12 adenomas. The frequency of cytokeratin (CK)-positive cells in mesenteric lymph nodes, both SLN and non-SLN, was determined along with their immunohistochemical characteristics.. The median number of SLNs was 3; the median number of total nodes was 14. The CK-positive cell clusters were detected in the SLNs of 10 (29%) of 34 SLN-negative patients. Adjusted per patient, SLNs were significantly more likely to contain CK-positive cells than non-SLNs (P <.001). Cell clusters, cytologic atypia, and/or coexpression of tumor and epithelial markers p53 and E-cadherin were supportive of carcinoma cells. Single CK-positive cells only, however, could not be definitively characterized as isolated tumor cells; these cells generally lacked malignant cytologic features and coexpression of tumor and epithelial markers and in 2 cases represented mesothelial cells with calretinin immunoreactivity. Colorectal adenomas were associated with a rare SLN CK-positive cell in 1 (8%) of 12 cases.. Sentinel lymph node staging with CK-immunohistochemical analysis for colorectal carcinomas is highly sensitive for detection of nodal tumor cells. Cohesive cell clusters can be reliably reported as isolated tumor cells. Single CK-positive cells should be interpreted with caution, because they may occasionally represent benign epithelial or mesothelial cells.

Topics: Adenoma; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Colectomy; Colorectal Neoplasms; Databases, Factual; Female; Humans; Immunohistochemistry; Keratins; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging; Sentinel Lymph Node Biopsy

M cells are found in intestinal follicle associated epithelium. Studies into the physiological and pathological roles of human M cells have been hampered by the lack of well-substantiated, specific markers for these cells. A critical literature review suggests the following molecules may potentially serve as such markers: CK7, FcaR (CD89), S100, CD1a, CD21, CD23, sialyl Lewis A, and cathepsin E. Normal ileum, appendix and colorectum were studied using paraffin-embedded, formalin-fixed tissue and immunohistochemistry for these 8 markers. Cathepsin E immunohistochemistry was also performed on cases of colorectal adenocarcinoma, colorectal adenoma, colorectal hyperplastic/metaplastic polyp, lymphocytic colitis, collagenous colitis, pseudomembranous colitis and active ulcerative colitis. Of the 8 markers tested, only cathepsin E appeared to be specific to follicle associated epithelium (expressed by cells with and without M cell morphology) and follicular crypt epithelium; this specificity was limited to the colorectum. Focal epithelial expression of cathepsin E was seen in adenocarcinoma, adenoma, hyperplastic/metaplastic polyp, ulcerative colitis and pseudomembranous colitis. In conclusion, cathepsin E is a specific marker of normal colorectal follicle associated epithelium and follicular crypt epithelium though is not specific to M cells within these compartments. None of the other 7 markers studied is exclusively expressed by human M cells.

Topics: Adenocarcinoma; Adenoma; Biomarkers; CA-19-9 Antigen; Cathepsin E; Colitis; Colorectal Neoplasms; Epithelial Cells; Humans; Immunoenzyme Techniques; Intestinal Mucosa; Keratin-7; Keratins; Lower Gastrointestinal Tract; Peyer's Patches

The main objectives of this study were to identify, by immunohistochemistry, possible micrometastasis in the regional lymph nodes previously considered free by conventional histopathological examination, and to assess their influence on the survival of patients with colorectal cancer that had been extirpated in a radical manner.. From 38 patients with Dukes B staging (Colorectal Carcinoma Stage II (T3 N0 M0 or T4 N0 M0)) colorectal carcinoma, 383 lymph nodes were studied in paraffin blocks that had previously been considered free by conventional histopathological examination. These were submitted to immunohistochemical study using AE1/AE3 anti-cytokeratin monoclonal antibodies to identify neoplastic epithelial cells.. Seven lymph nodes (1.82%) in six patients (15.78%) contained micrometastasis. The survival of the patients with extirpated colorectal carcinoma staged as Dukes B who had lymph node metastasis was less than in the group of patients without micrometastasis, although these values were not statistically significant.. This immunohistochemical method can be employed successfully in the detection of neoplastic cells in lymph nodes previously considered free. In this study, there was a trend towards lower survival in node-positive patients but this did not reach statistical significance.

Topics: Adult; Aged; Antibodies, Monoclonal; Biomarkers, Tumor; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Lymph Node Excision; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging; Survival Analysis

At present, reverse transcription (RT)-PCR against carcino-embryonic antigen mRNA is one of the few research tools for the detection of occult cells in histopathologically assessed negative lymph nodes from patients with colorectal cancer. The aim of this study was to investigate the suitability of supervised low-resolution image analysis of immunohistochemically stained sections as alternative.. Multiple sections (n = 50) of regional lymph nodes from patients with colorectal cancer were immunohistochemically stained and analyzed by applying low-resolution image analysis (flatbed scanning) for semiautomated detection of cytokeratin (CK)-positive stained cells. The sensitivity of this approach was demonstrated for 20 patients with stage II colorectal cancer and compared with RT-PCR regarding the detection of clinically assessed recurrence of disease within 10 years.. CK(+) cells were detected in all of the patients (n = 6; 100%) with recurrence, compared with five patients (83%) found positive by carcinoembryonic antigen RT-PCR. From patients (n = 14) who did not develop a recurrence, eight (57%) had positive lymph nodes. In all patients with recurrence, we visually identified at least one group of CK(+) cells (>/==" BORDER="0">2 cells).. Automated image analysis is a promising tool for the detection of occult cells in histopathologically negative nodes. It is potentially more sensitive but less specific for detecting recurrence of disease than conventional histopathology or RT-PCR and is particularly useful for the evaluation of sentinel nodes. Furthermore, it opens new ways for basic research of occult cells based on molecular profiling after laser-microdissection.

Topics: Automation; Biochemistry; Carcinoembryonic Antigen; Colorectal Neoplasms; Humans; Image Processing, Computer-Assisted; Immunohistochemistry; Keratins; Lymph Nodes; Lymphatic Metastasis; Neoplasms, Unknown Primary; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sensitivity and Specificity

Dissemination of lymph nodes is a known prognostic factor in colorectal carcinoma. Micrometastases in lymph nodes can be missed when studied by routine techniques. We analyzed 162 lymph nodes from 30 patients with colonic carcinoma and using routine techniques, they were classified as follows: two Dukes A; nineteen Dukes B; and nine Dukes C. A patient with benign colon disease served as negative control. Lymph nodes were all sectioned in halves, with one of the halves stored in liquid nitrogen for molecular biology examination by carcinoembryonic antigen expression. The other formalin-fixed and paraffin embedded halves were saved for both pathologic and immunohistochemical examination. For Dukes A and Dukes B tumors, reverse transcriptase-polymerase chain reaction (RT-PCR) had a 50% higher sensitivity in the detection of micrometastases. The expression of carcinoembryonic antigen (CEA) was detected in all Dukes C cases, which were considered as positive controls. These results showed that RT-PCR has a higher sensitivity in the detection of micrometastases than routine techniques, including immunohistochemistry.

Topics: Adult; Aged; Aged, 80 and over; Carcinoembryonic Antigen; Carcinoma; Colorectal Neoplasms; Electrophoresis, Polyacrylamide Gel; Female; Humans; Keratins; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging; Prospective Studies; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity

Liver metastasis is an important factor determining prognosis in colorectal cancer. The objective of this study was to assess whether colorectal cancer cells in the drainage veins can be detected by measuring telomerase activity and its detection is correlated with liver metastasis.. Telomeric repeat amplification protocol assay in combination with an immunomagnetic sorting was used for measuring telomerase activity of epithelial cells in blood samples collected from mesenteric (tumor-drainage) vein and peripheral vessels of 41 colorectal cancer patients. Telomerase activity was calculated as relative telomerase activity (RTA) against a control template and analyzed in terms of liver metastasis.. RTA of mesenteric blood samples was significantly higher in patients with liver metastasis (60.8%; n=7) than in those without metastasis (19.7%; n=34; P=.019). The RTA of peripheral blood sample was also higher in patients with liver metastasis (26.8%) than in those without metastasis (11.1%; p=.17). Moreover, 57% of cases with liver metastasis exhibited a positive telomerase activity in mesenteric blood sample, whereas it was 18% in cases without metastasis.. Our assay was proven to be a feasible method for detecting cancer cells in tumor-drainage veins. High telomerase activity of mesenteric blood samples reflected the existence of liver metastasis of colorectal cancer.

Topics: Adult; Aged; Aged, 80 and over; Carcinoembryonic Antigen; Colorectal Neoplasms; Female; Humans; Keratins; Liver Neoplasms; Male; Mesenteric Veins; Middle Aged; Neoplastic Cells, Circulating; Reverse Transcriptase Polymerase Chain Reaction; Telomerase

Although cytokeratin (CK) phenotyping of metastatic tumors is now routine in many laboratories, the clinical relevance of the procedure has seldom been addressed. We carried out a prospective clinical study of 134 consecutive cases of metastatic adenocarcinoma of the liver diagnosed by needle biopsies stained routinely for CK20 and CK7. The most probable localization of the primary tumor, deduced from this staining pattern, was stated in the original pathology report. The present study compared this assignment with the information available at the time of interpretation of the liver biopsy, to the results of the subsequent clinical investigation, and to the officially reported cause of death as outcome. As expected, the primary tumors were localized in the colon or in the rectum in 85% (34/40) of the CK20+/CK7- metastases. The definite diagnosis remained metastatic colorectal carcinoma in 83% (15/18) of the cases with diagnosed colorectal cancer before the liver biopsy. In the cases without a known primary tumor when the liver biopsy was interpreted, primary colorectal localization was accurately predicted in 86% (19/22) of the patients. Compared to the outcome, 77% (36/47) of the CK20+/CK7+ metastases had the expected pancreaticobiliary primary localization in 83% (30/36) without any primary tumor being known at the time of interpretation of the liver biopsy. In contrast, the majority of CK20- metastatic carcinomas had an unexpected primary localization, 50% (16/32) in the CK20-/CK7+ and 60% (9/15) in the CK20-/CK7- subgroup. In addition, the origin of the liver metastasis remained unknown in 37% (12/32) of CK20-/CK7+ cases. Thus, the CK20+/CK7- phenotype indicates a colorectal origin of the liver metastasis with considerable accuracy and independently of the available clinical information. The same is true for CK20+/CK7+ metastases, which indicate primary tumor localization in the pancreas or in the biliary tree. The results in the CK20- subgroups of the liver metastases are disappointing and cannot substantially help the clinical investigation.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Biliary Tract Neoplasms; Biomarkers; Biopsy, Needle; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Liver; Liver Neoplasms; Male; Middle Aged; Neoplasms, Unknown Primary; Pancreatic Neoplasms; Phenotype; Retrospective Studies

The distinction of metastatic mucinous carcinomas in the ovary from primary ovarian mucinous tumors (atypical proliferative/borderline and carcinoma) can be difficult because of similarities in morphology. We evaluated the immunohistochemical expression of cytokeratins 7 and 20 (CK 7, CK 20), Dpc4 (nuclear transcription factor inactivated in 55% of pancreatic carcinomas), and MUC5AC (a gastric mucin gene) in 57 primary ovarian mucinous tumors (41 atypical proliferative tumors and 16 carcinomas) and 46 metastatic mucinous carcinomas in the ovary. Primary ovarian mucinous tumors were virtually always diffusely positive for CK 7 (98%), Dpc4 (100%), and MUC5AC (98%) and often focally to diffusely positive for CK 20 (68%). Colorectal mucinous carcinomas were diffusely positive for CK 20 (100%) and Dpc4 (89%) and were distinguished from primary ovarian mucinous tumors by their frequent lack of expression of CK 7 and MUC5AC (67% were negative for each marker). Appendiceal carcinomas were diffusely positive for CK 20 (100%) and often negative for CK 7 (71%) but were often positive for MUC5AC (86%) and Dpc4 (100%). When primary ovarian and metastatic colorectal or appendiceal carcinomas shared expression of both CK 7 and CK 20, they could usually be distinguished by the pattern of positivity (diffuse CK 7 and patchy CK 20 in ovarian tumors and patchy CK 7 and diffuse CK 20 in colorectal and appendiceal tumors). Pancreatic carcinomas shared the same pattern of diffuse positivity for CK 7 (100%) and MUC5AC (92%) and focal to diffuse positivity for CK 20 (71%) as primary ovarian mucinous tumors but were negative for Dpc4 in 46%. Loss of Dpc4 expression is useful for distinguishing metastatic pancreatic carcinomas in the ovary from both primary ovarian mucinous tumors and metastatic mucinous carcinomas derived from other sites.

Topics: Adenocarcinoma, Mucinous; Appendiceal Neoplasms; Biomarkers, Tumor; Colorectal Neoplasms; Diagnosis, Differential; DNA-Binding Proteins; Female; Gastrointestinal Neoplasms; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Mucin 5AC; Mucins; Ovarian Neoplasms; Pancreatic Neoplasms; Smad4 Protein; Trans-Activators

We investigated the distribution and fate of apoptotic bodies during human development and in the adult, using an antibody (M30) that recognizes a neo-epitope formed early in the apoptotic cascade by caspase cleavage of cytokeratin 18. In the fetus, we found extensive accumulation of M30-positive, non-phagocytosed fragments in the red pulp of the spleen, subcutaneous and submucosal vessels, the interstitium of the lung, and the glomerular mesangium of the kidneys. In the liver, M30-immunoreactive fragments were found inside macrophages in the sinusoids. The number of these fragments and the intensity of the immunostaining increased with the gestational age of the fetus. In the adult, M30-positive fragments were barely detectable in normal tissues. However, many pathological situations, including both chronic degenerative processes and metastatic cancer, were associated with accumulation of M30-positive fragments in the red pulp of the spleen. In the liver and kidney, no fragments could be detected. Remarkably, 13 of the 16 patients with metastasized cancer showed pronounced accumulation of M30-positive fragments containing hematoxylin-reactive material in the red pulp of the spleen. In the non-cancerous cases, such DNA-containing fragments were only seen in 9 of 94 cases. The results show that when apoptotic activity is high, as during development in the fetus or during metastasis and other pathological processes in the adult, the phagocytic clearance of apoptotic bodies can be overloaded. These apoptotic fragments then accumulate in the spleen. The visual detection of apoptotic fragments is concluded to reflect increased cell turnover.

Topics: Adult; Apoptosis; Carcinoma; Colorectal Neoplasms; Embryo, Mammalian; Epithelium; Fetus; Humans; Immunoenzyme Techniques; In Situ Nick-End Labeling; Intestine, Small; Keratins; Liver; Macrophages; Phagocytosis; Spleen

Inconsistent conclusions have been drawn about the clinical significance of micrometastases in lymph nodes (LNs) of node-negative colorectal cancer (CRC) patients. We performed a comparative study of detection of micrometastases using immunohistochemistry (IHC) by anti-cytokeratin antibody and carcinoembryonic antigen (CEA)-specific reverse-transcriptase polymerase chain reaction (RT-PCR) in the same patients, in an attempt to move closer to their clinical application.. Sixty-four CRC patients, with RNA of good quality available from paraffin-embedded LN specimens, were selected from 84 stage II patients who underwent curative surgery between 1988 and 1996. We investigated associations between the presence of micrometastases by each method and prognosis.. Micrometastases were detected in 19 (29.6%) of 64 patients by RT-PCR and in 35 (54.7%) of 64 patients by IHC. By RT-PCR analysis, patients exhibiting a positive band for CEA mRNA had a significantly worse prognosis than those who were RT-PCR-negative, with respect to both disease-free and overall survival (P =.027 and.015, respectively). By IHC analysis, the presence of micrometastasis did not predict patient outcome in terms of either disease-free or overall survival. Infiltrating pattern of tumor growth characteristic was significantly associated with shorter disease-free survival among various clinical or pathologic factors. By multivariate Cox regression analysis, micrometastasis detected by RT-PCR and the Crohn's-like lymphoid reaction were both independent prognostic factors.. Micrometastases detected by RT-PCR, but not IHC, may be of clinical value in identifying patients who may be at high risk for recurrence of CRC and who are therefore likely to benefit from systemic adjuvant therapy.

Topics: Adenocarcinoma; Aged; Carcinoembryonic Antigen; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Analysis

Multidirectional differentiation in colorectal carcinomas is a rare phenomenon. Four cases are reported herein, and their clinical and pathologic characteristics are discussed. Two men and 2 women between the ages of 56 and 76 years who presented with abdominal symptoms are included in this report. Two tumors were located in the right colon, one in the splenic flexure, and one in the descending colon. Distant metastases were evident at presentation in 3 of 4 cases. Histologically, two tumors exhibited neuroendocrine and glandular differentiation; the third tumor was an adenocarcinoma with a sarcomatous component and the fourth tumor showed 3 lines of differentiation (glandular, squamous, and sarcomatoid). In all tumors evaluated, areas of adenocarcinomas were positive for low-molecular weight cytokeratin (CAM 5.2) and mucicarmine, but negative for high-molecular weight cytokeratin (AE3). The squamous cell component was AE3 positive and CAM 5.2 negative. The neuroendocrine component was highlighted by neuroendocrine markers and the sarcomatoid component revealed smooth muscle differentiation. All tumors (except one mucinous tumor) were negative for cytokeratin-20 staining. One patient was on supportive care for terminal metastatic carcinoma, and 2 patients were being treated with adjuvant chemotherapy at the time of this report. Colon carcinoma with multidirectional differentiation is a rare event and may originate from stem cells within the gastrointestinal mucosa, and/or represent the convergence of multiple tumors arising at the same site. This type of tumor should be considered in the differential diagnosis of a bowel biopsy with multiple histopathologic variants.

Topics: Adenocarcinoma; Aged; Carcinoma, Squamous Cell; Carmine; Cell Transformation, Neoplastic; Colorectal Neoplasms; Coloring Agents; Female; Humans; Immunohistochemistry; Keratins; Male; Middle Aged; Neoplasm Metastasis

The expressions of cytokeratin (CK) 7 and 20 have been studied in various primary and metastatic carcinomas, and their determination may help distinguish the site of origin of metastatic carcinomas. However, little is known about the factors that determine variations in their expression patterns in primary gastric and colorectal carcinomas. We investigated the expressions of CK7 and CK20 in 289 cases of gastric carcinoma and 225 cases of colorectal carcinoma using a tissue microarray. To evaluate CK7 and CK20 expression patterns of ovarian metastases from gastric or colorectal carcinomas, 54 cases of metastatic carcinomas to the ovary were examined. It was found that 71% (207 of 289) of the gastric carcinomas stained positively for CK7, whereas only 9% (21 of 225) of the colorectal carcinomas proved to be CK7 positive, and that 41% (117 of 289) of the gastric carcinomas and 73% (165 of 225) of the colorectal carcinomas were CK20 positive. The proportion of CK7+/CK20- was highest in the gastric carcinomas at 46% (132 of 289), and was independent of the histologic classification of Lauren (46% of the intestinal type, 45% of the diffuse type). The CK7 and CK20 expression patterns were different in colorectal carcinomas according to histologic grade and location of the tumor. CK7-/CK20+ had the greatest proportion (68%) in colorectal carcinomas, and this was dependent on the tumor's histologic grade (75% of low-grade versus 52% of high-grade) and location (46% of right-sided versus 76% of left-sided). Moreover, 42% (18 of 43) of gastric carcinomas metastatic to the ovary were CK7+/CK20-, whereas 19% (8 of 43) were CK7-/CK20+. All colorectal cancers metastatic to the ovary were CK7-/CK20+, except 1 case that was CK7-/CK20-. In conclusion, the CK7 and CK20 expression patterns in primary gastric carcinomas vary considerably, and those in colorectal carcinomas are associated with histologic grade and tumor location. The CK7-/CK20+ expression pattern is specific for metastatic colorectal carcinomas to the ovary, but has low predictability for colorectal origin in metastatic ovarian carcinoma.

Topics: Adenocarcinoma; Colorectal Neoplasms; Diagnosis, Differential; Female; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Ovarian Neoplasms; Stomach Neoplasms; Tissue Embedding

Systemically disseminated tumor cells have become the subject of intensive research as the presumed seminal precursors of later distant metastasis. We describe here a novel sensitive multimarker nested reverse transcription (RT)-PCR capable of detecting the individual expression of human MAGE-A genes MAGE-1, -2, -3/6, -4, and -12 by rare, disseminated tumor cells in bone marrow and blood of patients with many different types of cancer. We analyzed bone marrow aspirates from 106 patients with breast, lung, colorectal, and prostate cancer and with different sarcomas. Heterogeneous expression of the different MAGE genes was found frequently in all those kinds of malignancies, in sharp contrast to 30 bone marrow and 20 blood samples from healthy donors, which were completely MAGE negative. Expression of at least one MAGE gene in bone marrow was more frequent than cytokeratin-positive tumor cells detected by immunocytochemistry, although the results of both tests overlapped considerably. In 30 patients with clinically localized prostate cancer, analysis by the multimarker MAGE RT-PCR of bilateral bone marrow aspirates from the right and left iliac crest revealed a positivity rate of 60%, which was twice as high as that obtained with either an established prostate-specific antigen RT-PCR or by cytokeratin-based immunocytochemistry. Analysis of primary prostate cancer revealed MAGE expression patterns considerably concordant with those found in the corresponding bone marrow aspirates. Prostate cancer patients carrying an exceptionally high risk of metastatic relapse, as defined by clinical prognostic factors, were significantly more often MAGE positive than patients with a distinctly lower risk (P = 0.02, Fisher's exact test). More frequent MAGE expression in the peripheral blood of patients with metastatic prostate cancer compared with those with clinically localized disease added further evidence for the prognostic impact of the multimarker MAGE RT-PCR. Moreover, MAGE-positive bone marrow samples from a small group of seven sarcoma patients demonstrated the relevance of our multimarker RT-PCR in nonepithelial tumors. Because MAGE antigens can induce autologous cytolytic T lymphocytes in vivo, the determination of individual MAGE expression patterns in cancer patients may furthermore identify candidate vaccine targets for adjuvant immunotherapy.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Breast Neoplasms; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Lung Neoplasms; Male; Neoplasm Metastasis; Prostate-Specific Antigen; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Risk Factors; RNA, Messenger

Detection of cytokeratin-positive cells in bone marrow and peripheral blood may have prognostic significance in cancer patients. Furthermore, a correlation between uPAR expression on micrometastases and patient prognosis has been suggested. However, in patients with colorectal cancer, preoperative detection and characterization of tumour cells in bone marrow and peripheral blood, using an immunocytochemical approach, have not yet been substantiated as a prognostic tool.. Forty-one bone marrow aspirates and 38 peripheral blood aspirates, obtained preoperatively from patients with colorectal cancer, were immunocytochemically screened for cytokeratin-positive cells. Where cytokeratin-positive cells were observed, an additional microslide was double immunostained for simultaneous detection of cytokeratin and uPAR/CD87.. Cytokeratin-positive cells were observed in 4 out of 41 bone marrow aspirates (10%). None of the isolated cytokeratin-positive cells expressed uPAR. In peripheral blood, no cytokeratin-positive cells were observed.. The present study does not support the introduction of a routine immunocytochemical identification of cytokeratin-positive cells in preoperatively obtained bone marrow aspirates or peripheral blood from patients with colorectal cancer.

Topics: Adult; Aged; Biopsy; Bone Marrow; Colorectal Neoplasms; Humans; Immunohistochemistry; Keratins; Middle Aged; Preoperative Care

Lymph node metastasis in colorectal carcinoma is an important prognostic factor, yet the prognostic relevance of occult tumor cells in lymph nodes has not elucidated. This study was performed to investigate the correlation between isolated tumor cells in lymph nodes and malignancy potential in patients with Dukes B colorectal carcinoma and, thus, to determine whether presence of isolated tumor cells in lymph nodes has a prognostic significance.. To evaluate the incidence of isolated tumor cells in lymph nodes in patients with Dukes B colorectal carcinoma, 1,808 lymph nodes taken from 93 patients (19.4 per case) were assessed by immunohistochemical technique using a monoclonal antihuman cytokeratin (MNF 116). Clinicopathologic parameters and prognosis were compared between patients with and without isolated tumor cells.. Isolated tumor cells were identified in 54 lymph nodes from 29 patients (31.2 percent) by the immunostaining. No correlations were observed between the incidence of positive isolated tumor cells and various clinicopathologic parameters, including preoperative carcinoembryonic level, tumor site and size, histologic differentiation, pT stage, vascular invasion and lymphatic invasion, and perineural invasion. There was no difference in five-year survival estimated by Kaplan-Meier life-table method between positive and negative groups for isolated tumor cells (82.8 and 85.9 percent, respectively). Multivariate analyses showed that sex (P = 0.0236), serum carcinoembryonic level (>or= 5 ng/ml, P = 0.0002), and lymphatic vessel invasion (P = 0.0002) were significant factors in the survival time.. Immunohistochemical staining with an anticytokeratin antibody is useful in identifying isolated tumor cells in lymph nodes missed in routine hematoxylin-eosin staining, but clinically it seems to be of little prognostic value in patients with Dukes B colorectal carcinoma. Thus, this immunostaining technique does not offer a significant benefit of different strategies for additional therapy or follow-up during conventional pathologic staging using hematoxylin-eosin staining.

Topics: Adult; Aged; Antibodies, Monoclonal; Carcinoma; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Incidence; Keratins; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Invasiveness; Peritoneal Neoplasms; Prognosis; Retrospective Studies; Survival Analysis

The origin of metastatic adenocarcinoma lesions can sometimes be difficult to diagnose. The objectives of our study were to establish the cytokeratin staining pattern of primary and metastatic lung and colorectal adenocarcinomas, and to determine if this helps to identify the site of origin of metastatic lesions. We reviewed a total of 102 tissue samples from patients in our tumour registry, with either primary or metastatic lung or colorectal adenocarcinoma. Tissue sections were stained for cytokeratin 7 and 20 and read as positive or negative for staining. Clinical and radiologic information was reviewed from computerised charts. The cytokeratin 7+/cytokeratin 20- pattern characterised 96% (29 out of 30) of primary and 95% (21 out of 22) of metastatic lung adenocarcinomas. All the primary (26), and 88% (21 out of 24) of metastatic colorectal adenocarcinomas stained cytokeratin 7-/cytokeratin 20+. Samples from a variety of metastatic sites were evaluated for cytokeratin 7 and 20 staining. Out of the 102 samples, in 95% (97 out of 102) of the cases, the cytokeratin 7 and cytokeratin 20 staining pattern characterised and differentiated between lung and colorectal adenocarcinoma. Primary and metastatic lung adenocarcinomas show a cytokeratin 7+/cytokeratin 20- staining pattern, while colorectal adenocarcinomas stain cytokeratin 7-/cytokeratin 20+. Cytokeratin staining is helpful in the diagnostic differentiation of metastatic lesions from these two common primaries, and assists in determining the site of origin of metastatic lesions.

Topics: Adenocarcinoma; Biopsy; Colorectal Neoplasms; Diagnosis, Differential; Humans; Immunoenzyme Techniques; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Lung Neoplasms; Prognosis; Registries; Staining and Labeling

We prospectively analyzed the circulating tumor burden in colorectal cancer patients using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) for carcinoembryonic antigen (CEA) and cytokeratin 19 (CK19 ). We distinguished the mRNA levels in peripheral blood between 33 patients and 26 healthy controls with reference to SK-BR-3 cell line. We found CEA-mRNA in 88% of patients and 92% of controls, and CK19 mRNA in 64% of patients and 19% of controls. Our CK19 mRNA assay was sufficiently sensitive to detect one SK-BR-3 cell among 10(6) normal blood cells. The upper limit of CK19 mRNA among controls was exceeded by 14 patients, and 12 patients (86%) developed systemic metastases/recurrence. Significantly elevated CK19 mRNA levels appeared to originate from circulating malignant cells (P<0.0001). Of relevance, the CK19 mRNA level increased with advancing Dukes' stage and correlated directly with the serum CEA level (P=0.016). CK19 mRNA quantification may prove valuable for cancer staging and disease monitoring.

Topics: Adult; Aged; Aged, 80 and over; Carcinoembryonic Antigen; Colorectal Neoplasms; Female; Humans; Keratins; Male; Middle Aged; Neoplasm Staging; Neoplastic Cells, Circulating; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Fractalkine (CX3CL1) is synthesized as a type I transmembrane protein. Its unique CX(3)C chemokine domain is attached to a 241-amino acid mucin stalk, a 19-amino acid transmembrane domain, and a 37-amino acid intracellular domain of unknown function. A soluble form of fractalkine can be generated by proteolytic cleavage at the base of the mucin stalk. Novel monoclonal and polyclonal antibodies that specifically recognize only the amino- or carboxyl-terminal ends of the human fractalkine molecule have revealed that epithelial cells are the predominant cell type expressing transmembrane forms of fractalkine in human skin, the tonsil, and the large intestine. Using these specific anti-fractalkine reagents we do not detect high-level expression of fractalkine on endothelial cells in normal or inflamed colon samples obtained from patients with Crohn's disease or ulcerative colitis. In contrast to previous reports we do not detect fractalkine expression by Langerhans cells or immature dendritic cells in mucosal-associated lymphoid tissues in vivo. We show that the reagent used in previous studies, an anti-fractalkine N-terminal peptide antisera, cross-reacts with human CD84. Finally we discuss potential roles for fractalkine in constitutive leukocyte trafficking based on its observed pattern of expression in epithelia.

Topics: Adenocarcinoma; Animals; Antibodies; Antibody Specificity; Antigens, CD; Cell Line; Chemokine CX3CL1; Chemokines, CX3C; Chemokines, CXC; CHO Cells; Colon; Colorectal Neoplasms; Cricetinae; Cross Reactions; Epidermis; Epithelial Cells; Humans; Inflammatory Bowel Diseases; Keratins; Membrane Glycoproteins; Membrane Proteins; Palatine Tonsil; Peptides; Protein Isoforms; Signaling Lymphocytic Activation Molecule Family; Tumor Cells, Cultured

The goal was to investigate the prognostic value of various molecular markers like CEA, Cyclin D1, Bcl-2, c-Myc, p53, p21ras, Ki-67, CD44, Factor VIII-related antigen, cytokeratin-19, adenoma antigen, and prolactin in patients with Dukes B and Dukes C colorectal adenocarcinoma.. These molecular markers were localized immunohistochemically in nonmalignant (n = 36) and malignant (n = 98) diseases of the colorectum. Data were analyzed statistically using the SPSS software program. The patients with colorectal cancer were followed for a period of five years or their death within that period.. The expression of carcinoembryonic antigen, Cyclin D1, Bcl-2, CD44, cytokeratin-19 and prolactin was significantly higher in malignant diseases (P < 0.05), whereas, p21ras was found to be significantly higher in nonmalignant diseases (P = 0.002) as compared with their respective counterparts. Besides Dukes stage, multivariate analysis indicated a significantly reduced relapse-free survival in patients expressing CD44 and cytokeratin-19 (P < 0.005). Similarly, besides Dukes stage, multivariate analysis indicated a significantly poor overall survival in patients expressing CD44, cytokeratin-19 and prolactin (P < 0.01). In patients with Dukes B disease, only cytokeratin-19 and CD44 expression attained statistical significance (P < 0.05), whereas in patients with Dukes C disease, CD44, p21ras- and c-Myc expression attained statistical significance (P < 0.018). Also, a multivariate analysis in relation to treatment given was performed using CD44 and cytokeratin-19.. Besides Dukes stage, multivariate analysis of all the studied molecular markers showed that patients expressing CD44 and cytokeratin-19 had a significantly reduced relapse-free and poor overall survival. Moreover, patients expressing both these markers (CD44 and cytokeratin-19) had the lowest significant relative risk for developing recurrence than patients with both markers negative when treated with surgery followed by adjuvant chemotherapy as compared with patients treated with surgery alone. Thus, in patients with colorectal cancer, immunohistochemical localization of CD44 and cytokeratin-19 may be included as a part of routine pathologic evaluation along with conventional prognostic factors.

Topics: Adenocarcinoma; Adult; Biomarkers, Tumor; Colorectal Neoplasms; Female; Humans; Hyaluronan Receptors; Immunohistochemistry; Keratins; Male; Multivariate Analysis; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; Retrospective Studies; Survival Analysis

To detect the presence of cancer cells in peripheral and mesenteric blood and to examine their relationship to prognosis in colorectal cancer patients undergoing curative surgery, we examined cytokeratin 19 and 20 mRNA in peripheral and mesenteric venous blood. Using reverse transcriptase polymerase chain reaction, cytokeratin 19 and 20 mRNA was amplified in peripheral and mesenteric blood samples obtained from 35 colorectal cancer patients who underwent curative surgery. Cytokeratin 19 or 20 mRNA in peripheral or mesenteric blood samples was detected in 18 of 35 cases (51%). There was no significant difference in Dukes' staging between the positive and negative groups. The median follow-up period was 56 months. In the positive group six patients (33%) showed recurrences. One patient (6%) showed recurrence in the negative group. The recurrence rate was significantly higher in the positive group than in the negative group. Five-year disease-free survival was significantly better in the negative group than in the positive group (94% vs. 65%). The detection of cytokeratin 19 or 20 mRNA in peripheral or mesenteric blood is thus associated with the prognosis for colorectal cancer patients undergoing curative surgery. Although the presence of these mRNAs in blood samples is a prognostic marker, the clinical utility of this assay is questionable because of the low recurrence rate in the positive group.

Topics: Adult; Aged; Aged, 80 and over; Colorectal Neoplasms; Female; Humans; Intermediate Filament Proteins; Keratin-20; Keratins; Male; Middle Aged; Neoplasm Proteins; Reverse Transcriptase Polymerase Chain Reaction

To identify molecular alterations in the progression of colorectal carcinoma, we analyzed gene expression profiles of colon cancer cell lines derived from primary and metastatic tumors from a single patient. Of 2280 cDNAs investigated using our in-house microarray, the expression of 6 genes (tumor-associated antigen L6, L-plastin, the human homologue of yeast ribosomal protein S28, the B-cell translocation gene, mitochondrial aspartate-aminotransferase, and HLA-A) increased, while that of 2 genes (keratin 5 and phosphoglucomutase) decreased in metastatic-tumor-derived cells compared with primary-tumor-derived cells. Of these genes, we assessed the L-plastin gene, an actin-bundling protein, at the protein level using a tissue microarray consisting of 58 clinically stratified colorectal cancer specimens. Consistent with our microarray results, the expression of L-plastin was significantly correlated with the progression of cancer staging. Therefore, our results suggest that the L-plastin gene is a potential metastatic marker. In addition, combining cDNA microarrays and tissue arrays, as shown here, is thought to facilitate the rapid characterization of candidate biomarkers.

Topics: Antigens, Surface; Aspartate Aminotransferase, Mitochondrial; Base Sequence; Biomarkers, Tumor; Colorectal Neoplasms; DNA, Complementary; DNA, Neoplasm; Gene Expression Profiling; HLA-A Antigens; Humans; Keratin-5; Keratins; Membrane Glycoproteins; Microfilament Proteins; Neoplasm Proteins; Neoplasm Staging; Oligonucleotide Array Sequence Analysis; Phosphoglucomutase; Phosphoproteins; Receptors, Formyl Peptide; Receptors, Immunologic; Receptors, Peptide; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

The search continues to find methods to more effectively distinguish colorectal carcinoma patients who could be separated into high-risk and low-risk categories. Investigators have reported on the detection of occult micrometastases in bone marrow using antibodies to cytokeratin, which is a marker of epithelial cells but which has no tissue specificity, as opposed to villin, a cytoskeletal protein that is specifically involved in the formation of brush-border microvilli in the small intestine and colon epithelium. Specificity and sensitivity of antibody to villin (ID2C3) and antibody to cytokeratin (A45-B/B3) were first studied in normal bone marrow and in a test system in which cancer cell lines were mixed in normal bone marrow. In a preliminary study including 16 colorectal carcinoma patients, we compared the number of villin-positive cells with cytokeratin-presenting cells. As A45-B/B3, ID2C3 was determined to be sensitive enough to detect one cancer cell in 10(6) hematopoietic cells. Staining of hematopoietic cells with irrelevant antibody and a light staining of megakaryocytes with ID2C3 limited the specificity of the method. In colorectal carcinoma patients, correlation between ID2C3 and A45-B/B3 was 94%. Sensitivity and specificity of ID2C3 antibody to villin were satisfactory. Its clinical relevance must be investigated in further studies.

Topics: Aged; Aged, 80 and over; Antibodies, Monoclonal; Biomarkers, Tumor; Bone Marrow Cells; Bone Marrow Neoplasms; Carcinoma; Carrier Proteins; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Male; Microfilament Proteins; Middle Aged; Sensitivity and Specificity; Tumor Cells, Cultured

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

Despite the similarity in clinical appearance, there is a significant difference in the prognosis between primary extramammary Paget's disease (EPD) and the pagetoid spread of underlying regional internal malignancy (secondary EPD, pagetoid phenomenon). Fifteen cases of primary EPD (11 carcinoma in situ and four invasive carcinoma), seven cases of secondary EPD (five colorectal adenocarcinoma and two urothelial carcinoma), and six cases of anal canal carcinoma were retrieved and analysed immunohistochemically using six kinds of monoclonal anticytokeratin antibodies. No expression of cytokeratins 1, 5, 10, 13 and 14 was observed in any cases examined in this study. All 15 cases of primary EPD had the immunophenotype cytokeratin (CK)7+/CK20-. CK20 expression was diffusely positive in six cases of secondary pagetoid spread (two urothelial carcinoma and four colorectal adenocarcinoma), and focally in one case (a colorectal adenocarcinoma). In anal canal carcinoma, three of six cases showed CK20 diffuse expression and the remaining three cases expressed CK20 focally. CK7 expression was observed in three of six cases of anal canal carcinoma and in two of five cases of secondary EPD associated with colorectal adenocarcinoma. The combination of CK7 and CK20 demonstrates these to be useful markers in distinguishing 'primary' EPD from a pagetoid spread of extracutaneous malignancies. Namely, immunophenotypes other than CK7+/CK20- in Paget cells suggest underlying regional internal malignancy.

Topics: Adenocarcinoma; Anus Neoplasms; Biomarkers, Tumor; Carcinoma, Transitional Cell; Colorectal Neoplasms; Diagnosis, Differential; Humans; Immunoenzyme Techniques; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Neoplasm Proteins; Paget Disease, Extramammary

The objectives of this study were to examine the frequency of lymph node micrometastases detected by keratin immunohistochemistry and their relationship with survival behaviour.. A total of 133 consecutive patients staged as Duke's B, who had curative resection for colorectal cancer (CRC), comprised the study population. Patients who had died of a non-CRC-related cause or who became lost to follow-up were excluded, resulting in an amended population of 100. Study end-points were defined as disease-free survival of 5 years or CRC-related death. Paraffin-embedded lymph node sections were stained with a commercial cytokeratin antibody using a standard avidin-biotin technique.. One quarter of subjects had micrometastases. Fifty-six per cent of subjects with positive lymph nodes had an adverse outcome, compared with 11% of subjects with negative nodes. A highly significant association was found between lymph node cytokeratin expression and mortality in both the univariate (log rank P = 0.0001) and multivariate (Cox proportional hazards P = 0.0123) analysis.. Lymph node micrometastases detected by this inexpensive and simple technique are significantly associated with mortality in Duke's B CRC. This technique may be used to select patients for adjuvant chemotherapy.

Topics: Biomarkers, Tumor; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Lymph Nodes; Lymphatic Metastasis; Male; Prognosis; Proportional Hazards Models; Survival Analysis

In the liver, the immunostaining of cytokeratins (CK) 7 and 20 has been used to distinguish usual peripheral cholangiocarcinomas (CC) and colorectal carcinoma metastasis (CRM). However, other subtypes of CC are not infrequent and may be particularly difficult to distinguish from CRM by histology and even immunohistochemistry. Therefore, 48 CC from different locations, either peripheral (n = 19), or nonperipheral, that is, from the large intrahepatic bile ducts, the hilum, and the extrahepatic bile ducts (n = 29), and with different cytoarchitectural patterns were tested for CK7 and CK20 and compared with 31 CRM. CC were positive for CK7 and CK20 in 96% and 70%, respectively, whatever the architecture and differentiation of the tumor. The labeling index (LI) of CK7 in CC was always high, whereas it was low or moderate for CK20. CK20-positive phenotype was significantly more frequent in nonperipheral than in peripheral CC (82% vs 47%; p = 0.007). CRM were all positive for CK20 with a high LI, and mostly negative (81%) for CK7. In conclusion, (1) the CK immunoprofile of CC varies according to the location of the tumor in the biliary tract, peripheral CC being more often CK7+/CK20-, and nonperipheral ones CK7+/CK20+; and (2) a decision tree based on CK20 LI and CK7 positivity allows the distinction of CRM and CC, even for the nonperipheral type.

Topics: Adult; Aged; Aged, 80 and over; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Biomarkers, Tumor; Cholangiocarcinoma; Colorectal Neoplasms; Diagnosis, Differential; Female; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Male; Middle Aged; Predictive Value of Tests; Prognosis; Sensitivity and Specificity; Staining and Labeling

We evaluated the cytokeratin profile of intrahepatic cholangiocarcinoma with respect to its histological classification and intrahepatic location (peripheral vs. hilar), and compared its profile with that of a variety of metastatic adenocarcinomas in liver.. Expression of cytokeratins 7, 8, 18, 19 and 20 was immunohistochemically examined in intrahepatic cholangiocarcinoma (n = 77) and metastatic adenocarcinoma in liver (21 colorectal, 14 gastric, three gallbladder and three pancreatic cancers). Materials were autopsy or surgical specimens. Cytokeratins 7, 8, 18 and 19 were expressed in 75 (97%), 75 (97%), 59 (77%) and 71 (92%) cases of intrahepatic cholangiocarcinoma, respectively. Moderate and extensive expression of cytokeratin 18 was more frequent in the peripheral than in the hilar type. Moderate and extensive expression of cytokeratin 19 was seen in almost all cases of well-differentiated intrahepatic cholangiocarcinomas, while expression was decreased relatively in the moderately and decreased more in the poorly differentiated cases. While cytokeratin 20 was not found in non-neoplastic biliary epithelia or in well-differentiated intrahepatic cholangiocarcinomas, this cytokeratin was occasionally detectable in moderately and poorly differentiated intrahepatic cholangiocarcinomas and its expression was more frequent in the hilar type. Cytokeratin 20 expression was observed in 17 (81%) of metastatic adenocarcinomas in liver from colorectal regions, to a lesser degree in those from gastric regions, and was rare in those from gallbladder and pancreatic regions; cytokeratin 7 showed a reverse expression pattern in these metastatic adenocarcinomas in liver. The profile of cytokeratins 7 and 20 of metastatic colorectal and gastric carcinomas differed from that for intrahepatic cholangiocarcinomas, while that of metastatic gallbladder and pancreatic carcinoma was similar to that for intrahepatic cholangiocarcinomas. Moreover, cytokeratin 18 and 19 expression was significantly infrequent in metastatic gastric carcinomas than in intrahepatic cholangiocarcinomas and metastatic colorectal carcinomas.. The combined immunostaining of cytokeratins 7, 18, 19 and 20 is useful for the characterization of intrahepatic cholangiocarcinomas with respect to histological subtypes and intrahepatic location. It helps to differentiate intrahepatic cholangiocarcinoma from metastatic adenocarcinomas in liver and from colorectal and gastric regions; it also indicates the primary focus metastatic adenocarcinomas in livers.

Topics: Adenocarcinoma; Biomarkers; Cholangiocarcinoma; Colorectal Neoplasms; Gallbladder Neoplasms; Humans; Immunohistochemistry; Keratins; Liver Neoplasms; Pancreatic Neoplasms; Stomach Neoplasms

Metastatic signet ring cell carcinomas of unknown primary site can represent a clinical problem. Gastrointestinal signet ring cell carcinomas and invasive lobular carcinomas of the breast are the most common sources of these metastases. Immunohistochemical algorithms have been successfully used in the search for the unknown primary adenocarcinomas. In the present study a series of primary invasive lobular breast carcinomas (79 cases) and their metastases and a series of gastrointestinal signet ring cell carcinomas (22 primary and 13 metastases) were stained with monoclonal antibodies for cytokeratin (CK) 20 and CK7 and for estrogen receptors (ER). The staining was evaluated as negative (no staining), focally (less than 10% of the tumor cells stained) or diffusely positive. All the primary and metastatic gastrointestinal signet ring cell carcinomas proved to be CK20 positive, while only 2/79 (3%) of the primary and 1/21 metastatic lobular carcinomas (5%) stained positively for this CK. None of the gastrointestinal carcinomas and the majority of the lobular carcinomas expressed ER. The majority of the tumors were CK7+. Using CK20 alone, 33 of 34 metastases could be properly classified as gastrointestinal (CK20+) or mammary (CK20-). ER identified 31/34 of breast cancer metastases. By combining the results of CK20 and ER staining all the metastases could be properly classified as the CK20+/ER- pattern identified all the gastrointestinal tumors.

Topics: Antibodies, Monoclonal; Breast Neoplasms; Carcinoma, Signet Ring Cell; Colorectal Neoplasms; Diagnosis, Differential; Esophageal Neoplasms; Female; Gastrointestinal Neoplasms; Humans; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Lymphatic Metastasis; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms, Unknown Primary; Receptors, Estrogen; Stomach Neoplasms

In the absence of other metastatic disease, the presence of lymph node metastasis remains the most important determinant of survival in colorectal cancer (CRC). Cluster designation 44 variant 6 (CD44v6) over-expression is associated with worse outcome in all stages of CRC. The CD44v6 is believed to confer metastatic potential through its facilitation of migration, extravasation and proliferation, although the specific means by which it conveys an adverse prognosis in CRC is unknown. The aim of the present study was to determine if CD44v6 over-expression in Stage II CRC subjects was associated with the presence of lymph node micrometastases.. We assessed tumour CD44v6 expression in 43 randomly sampled subjects who had resections for Stage II CRC between 1984 and 1991 by using immunohistochemistry. Micrometastases were sought in corresponding lymph node (LN) sections using keratin immunohistochemistry.. There was a statistical trend between tumour CD44v6 over-expression and mortality (P = 0.09) and a significant relationship between LN cytokeratins and mortality (P = 0.01). There was no association between the detection of LN cytokeratins and tumour CD44v6 over-expression.. We conclude that the adverse survival effect of CD44v6 over-expression is not mediated though lymphatic spread and postulate that it may therefore facilitate haematogenous metastasis.

Topics: Colorectal Neoplasms; Glycoproteins; Humans; Hyaluronan Receptors; Immunohistochemistry; Keratins; Lymphatic Metastasis; Neoplasm Staging; Prognosis; Random Allocation; Survival Analysis

Expression of laminin-5 alpha3, beta3 and gamma2 protein subunits was investigated in colorectal adenocarcinomas using immunostaining and confocal microscopy. The laminin-5 heterotrimer was found in basement membranes and as extracellular deposits in tumor stroma. In contrast to the alpha3 subunit, which was under-expressed, the gamma2 and beta3 subunits were detected in the cytoplasm of carcinoma cells dissociating (budding) from neoplastic tubules, suggestive of focal alterations in laminin-5 assembly and secretion. Laminin-5 gamma2 or beta3 subunit-reactive budding carcinoma cells expressed cytokeratins but not vimentin; they did not proliferate and were not apoptotic. Furthermore, expression of laminin-5 gamma2 and beta3 subunits in budding cells was associated with focal under-expression of the E-cadherin-beta-catenin complex. Results from xenograft experiments showed that budding activity in colorectal adenocarcinomas could be suppressed when these tumors grew at ectopic s.c. sites in nude mice. In vitro, cultured colon carcinoma cells, but not adenoma-derived tumor cells, shared the laminin-5 phenotype expressed by carcinoma cells in vivo. Using colon carcinoma cell lines implanted orthotopically and invading the cecum of nude mice, the laminin-5-associated budding was restored, indicating that this phenotype is not only determined by tumor cell properties but also dependent on the tissue micro-environment. Our results indicate that both laminin-5 alpha3 subunit expression and cell-cell cohesiveness are altered in budding carcinoma cells, which we consider to be actively invading. We propose that the local tissue micro-environment contributes to these events.

Topics: Adenocarcinoma; Animals; beta Catenin; Cadherins; Cell Communication; Colorectal Neoplasms; Cytoskeletal Proteins; Disease Models, Animal; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; In Situ Nick-End Labeling; Keratins; Ki-67 Antigen; Laminin; Mice; Mice, Nude; Neoplasm Transplantation; Phenotype; RNA, Messenger; Stromal Cells; Trans-Activators; Tumor Cells, Cultured; Vimentin

Reverse transcriptase polymerase chain reaction (RT-PCR) is often used for sensitive detection of micrometastasis in peripheral blood, lymph nodes and bone marrow. While the utility of this method has been documented, it also has limitations in the detection of micrometastasis. The mRNA of target genes can be detected in healthy donors or in samples used for negative control, therefore the non-quantitativeness of conventional RT-PCR has been called into question. We analyzed the expression level of cytokeratin (CK) 18 mRNA in established esophageal and gastrointestinal carcinoma cell lines and non-epithelial cells, using quantitative RT-PCR, based on real time 'TaqMan TM' technology. CK 18 mRNA is more highly expressed in carcinoma cells than in non-epithelial cells. However, the expression level in non-epithelial cells was easily detected using conventional RT-PCR and agarose gel electrophoresis. In an analysis of CK 18 mRNA expression in peripheral venous blood in 13 healthy volunteers, we found that CK 18 mRNA was much less expressed than in cancer cell lines. However, the expression in all samples was at a level which was also detected using conventional RT-PCR. It would thus seem that not only qualitative, but also quantitative analysis, of the target mRNA is important to detect micrometastasis. Quantitative RT-PCR methods will make comparisons of the possible differences in expression levels of the target gene. For clinical applications, much further study is needed.

Topics: Adult; Colorectal Neoplasms; DNA Primers; Electrophoresis, Agar Gel; Esophageal Neoplasms; Genetic Vectors; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Keratins; Lung Neoplasms; Male; Reference Values; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sensitivity and Specificity; Stomach Neoplasms; Transcription, Genetic; Tumor Cells, Cultured

A prospective study is presented in which 293 patients suffering from breast cancer and colorectal carcinoma were analyzed for prognostic relevance of detected isolated disseminated tumor cells in bone marrow (IDTBM). The patients underwent surgery in the period from 1995 to 1997 and remained under observation until 1999. The monoclonal antibody A 45-B/B3 was used in the standard immuno-cytochemical method for detecting IDTBM, which represented an independent prognostic factor for survival time in patients with breast cancer or colorectal cancer. In breast cancer, when IDTBM were detected, the survival period was reduced by at least half. When disseminated tumor cells containing the A45-B/B3 antibody were detected in bone marrow, the risk of an earlier relapse of the tumor increased at least fourfold. In colorectal cancer, detection of IDTBM reduced survival time by a factor of 1.2-4.3. The risk of earlier relapse increased when disseminated tumor cells containing the A45-B/B3 antibody were detected in bone marrow by 2.8-8.1. Therefore, the use of IDTBM as an independent prognostic factor would provide an important method for determining the pathological stage of various cancers. Standardization of this technique into a generally accepted method would be especially desirable in treatment of patients with breast or colorectal cancer.

Topics: Actuarial Analysis; Aged; Bone Marrow; Breast Neoplasms; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Male; Multivariate Analysis; Neoplasm Proteins; Prognosis; Proportional Hazards Models; Prospective Studies; Recurrence; Survival Rate

Metastatic adenocarcinoma in the liver with an unidentified primary tumor site is a common clinical problem. Pathologists often are asked to identify the primary tumor site. The histologic picture itself usually is not helpful, because the histology may be similar in the metastases of tumors with different primary localizations. Immunohistochemistry can be helpful, but the previously recommended antibody panels are too complicated for everyday use.. A simple immunohistochemical algorithm with two monoclonal cytokeratin (CK) antibodies, CK20 and CK7, was tested on 93 autopsy cases of adenocarcinomas metastatic to the liver. Sections of the liver metastases were stained automatically and evaluated as negative (no staining), focally positive, or diffusely positive. Statistical comparison of the staining results for a single antibody was calculated as an odds ratio.. Thirty-six of 93 (39%) metastases proved to be CK20 positive (+). In this group, the CK20+/CK7 negative (-) pattern was highly characteristic for colorectal localization of the primary tumor, having been observed 17 of 21 of the cases (81%). The CK20+/CK7+ pattern of the metastatic liver adenocarcinomas was highly suggestive of primary localization in the pancreas or biliary tract (11 of 14 cases; 79%). Exclusion of the tumors originating in the stomach raised these values to 94% and 92%, respectively. The statistically calculated predicted probability of primary tumor site being in the colon or rectum for CK20+/CK7- metastasis was 78,41%, the probability of a primary tumor being located in the pancreas or biliary tract was 74,85%, if calculated for the whole study group.. The tested simple algorithm proved to be useful in CK20 positive (+) cases, predicting a primary tumor localization in the colon, rectum, pancreas, or biliary tract with high accuracy. The CK20- group was too heterogeneous to be classified adequately by these two antibodies.

Topics: Adenocarcinoma; Algorithms; Colorectal Neoplasms; Female; Gallbladder Neoplasms; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Liver Neoplasms; Male; Neoplasms, Unknown Primary; Pancreatic Neoplasms; Retrospective Studies; Stomach Neoplasms

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

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

Malignant tumors of the liver with intrabiliary growth are rare, except for some cholangiocarcinomas. Metastases with intrabiliary growth, whatever their origin, are rare. Moreover, colorectal metastasis can be particularly difficult to distinguish morphologically from some cholangiocarcinomas. We report 3 cases of late colorectal metastasis with intrabiliary growth, presenting as cholangiocarcinomas of the large ducts. Immunostaining with cytokeratins 7 and 20 attested the diagnostic and pointed out the spreading pattern of colorectal metastasis within biliary ducts. This study illustrates the capacity, probably underestimated, for colorectal metastasis to develop in the lumen of bile ducts and emphasizes the relevance of cytokeratin 7 and 20 immunostainings in such cases.

Topics: Aged; Bile Duct Neoplasms; Biomarkers, Tumor; Cholangiocarcinoma; Colorectal Neoplasms; Diagnosis, Differential; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratin-20; Keratin-7; Keratins; Liver Neoplasms; Male; Middle Aged

We report a series of 41 ovarian metastases from colorectal adenocarcinomas. The patients'mean age was 57.1 years at the time the metastasis was discovered, and 55.8 years at the time the primary carcinoma was found. The diagnosis of the primary tumour was anterior to the metastasis in 25 cases (mean interval 21 months), simultaneous in 13 and posterior in 3 others. The metastases formed cystic and solid masses with a mean weight of 330 g. The endometrioid architectural type was the most frequent, either pure (71%, 29/41) or associated with a mucinous component (17%, 7/41). Pure mucinous or other architectural types were rare. The endometrioid type was characterized by glands with a garland pattern, and intraluminal dirty tumoral necrosis. Immunohistochemistry helped to distinguish the metastases of endometrioid type from serous or endometrioid primary ovarian carcinoma; 71% of the former were CK7(-)/CK20(+), and 100% of the latter had the reverse profile CK7(+)/CK20(-).

Topics: Adenocarcinoma; Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Endometrioid; Colorectal Neoplasms; Diagnosis, Differential; Female; Follow-Up Studies; Humans; Keratins; Middle Aged; Ovarian Neoplasms; Time Factors

To study the relation between cancer cells in portal vein blood and liver metastasis of colorectal carcinoma.. The presence of CK20 mRNA was investigated by non-isotope RT-PCR in 54 cases.. The positive rate of CK2O mRNA in portal vein blood was 75.9%. The positive cases were significantly correlated with Dukes stage and live metastasis. There was a higher probability of liver metastasis in positive cases after operation.. The detection of CK20 in portal vein blood may improve the accuracy of clinical staging and diagnose liver micrometastasis of colorectal carcinoma.

Topics: Biomarkers, Tumor; Colorectal Neoplasms; Female; Humans; Keratin-20; Keratins; Liver Neoplasms; Male; Middle Aged; Neoplastic Cells, Circulating; Portal Vein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

We looked for disseminated tumour cells postoperatively in the bone marrow of 22 patients with gastric cancer and of 26 patients with colorectal cancer. On an average the follow-up was 6.5 months in the gastric cancer group and 9 months in the colorectal cancer group. In 27% of the gastric cancer group and in 30% of the colorectal cancer group follow-up aspirates were obtained. Micrometastases were detected in 96% of colorectal cancer patients and in 77% of gastric cancer patients. Both, postoperative and follow-up aspirations showed clear differences in the number of ck+ cells between the iliac crest sides. The majority of cells formed clusters from 2 to about 200 cells. In both groups no significant correlation was found between the number of tumour cells detected and the established risk factors (stage, tumour extension, lymph node involvement, distant metastasis) or the disease-free survival. In the gastric cancer group higher numbers of ck+ cells were seen in the low tumor stages I and II, as compared to stage III and IV. Besides, in this group significantly more ck+ cells were detected in younger patients (age under 63,5) than in the older group over 63,5 years. Colorectal cancer patients did not show this correlation. In follow-up aspirations the number of ck+ cells of the colorectal and also the gastric cancer group showed a tendency of rise in relapse and of fall in disease-free survival. There was a tendency of higher rates of ck+ cells in the colorectal cancer group when compared to gastric cancer patients.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Bone Marrow Cells; Bone Marrow Examination; Bone Marrow Neoplasms; Colorectal Neoplasms; Disease Progression; Female; Follow-Up Studies; Humans; Immunoenzyme Techniques; Keratins; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging; Prospective Studies; Risk Factors; Statistics as Topic; Stomach Neoplasms

Disseminated epithelial tumor cells have been detected in the bone marrow and blood of cancer patients by means of immunocytochemical or immunofluorescent staining of cytocentrifuge slides, multiparameter flow cytometry, and reverse transcriptase-polymerase chain reaction. However, it is hardly possible using such methods to detect tumor cells at a frequency below 10(-6). To increase the sensitivity of these detection techniques we have developed a new technology for the enrichment of disseminated epithelial tumor cells from hematopoietic cell samples by high-gradient magnetic cell sorting (MACS). Cells are permeabilized and fixed and carcinoma cells are magnetically labeled specifically with an anti-cytokeratin 8 monoclonal antibody (mAb) directly conjugated to superparamagnetic microbeads. Magnetically labeled cells are enriched on high-gradient magnetic columns. Tumor cells are detected in the enriched cell fraction by flow cytometry, fluorescence microscopy, or immunocytochemisty. In this study we demonstrated the method using a model system in which five to 5,000 cells from a breast cancer cell line were seeded into blood cell samples from a healthy donor containing 1.2 x 10(8) leukocytes. Tumor cells were 10,477+/-4242 (n=25)-fold magnetically enriched, and 57.7%+/-16.9% (n=33) of the initially seeded tumor cells were recovered. Applying the method to 20-40 mL blood samples from patients with advanced carcinomas of the breast, prostate, colon, rectum, or lung, we were able to detect between one and 6.8 x 10(4) cytokeratin-expressing tumor cells in 21 of 34 patients. This corresponds to frequencies of tumor cells between 6.8 x 10(-9) and 1.1 x 10(-3) among nucleated cells in the original sample. Enriched tumor cells were further analyzed for expression of tissue-specific and prognostic markers such as breast mucin glycoproteins, erbB2, and CD44v6 for additional characterization and to confirm their tumor origin. The technique described could become a valuable tool for the quantification and molecular characterization of metastatic carcinoma cells in hematopoietic tissue, and may ultimately prove useful in the diagnosis, prognosis, and monitoring of patients with carcinoma.

Topics: Adult; Aged; Breast Neoplasms; Carcinoma; Cell Separation; Colorectal Neoplasms; Epithelial Cells; Female; Humans; Hyaluronan Receptors; Immunohistochemistry; Immunologic Techniques; Keratins; Lung Neoplasms; Magnetics; Male; Middle Aged; Mucins; Neoplasm Metastasis; Prostatic Neoplasms; Receptor, ErbB-2; Tumor Cells, Cultured

Recent reports suggest that genetic examination of K-ras or p53 mutation is more sensitive for the detection of occult lymph node metastasis in colorectal carcinomas than conventional examination by haematoxylin and eosin (H & E) staining or immunohistochemistry for gene products. The aim of this study was, first, to define the microscopic characteristics of metastatic cancer cells in lymph nodes stained by the anti-cytokeratin antibody CAM5.2 for cytokeratins 8 and 18, and, second, to compare the detection rate of occult lymph node metastasis for immunohistochemical vs genetic methods.. K-ras mutations were first examined in primary tumours of seven cases which showed distant metastasis or local recurrence within 5 years of the initial surgery in spite of the original reporting of no lymph node metastasis by routine H & E staining. K-ras mutations were positive in three cases in primary tumours and lymph nodes, and the remaining four primary tumours were negative for p53 mutation as well as K-ras mutation. Therefore, genetic analysis of occult lymph node metastasis was uninformative, but occult metastasis was detected by cytokeratin staining in two of these four cases. Comparative study of cytokeratin-positive cells was performed on each of the 43 lymph nodes from three cases with K-ras mutations. Cancer cells were detected in 28 of the 43 lymph nodes (65.1%) by cytokeratin staining and in 10 of the 43 corresponding lymph nodes (23.3%) by genetic analysis. Artefactual contamination by cancer cells was present in eight of the 28 cytokeratin positive lymph nodes, and three of the eight nodes were genetically positive.. This study suggests that cytokeratin immunohistochemistry is more sensitive and specific for the detection of occult lymph node metastasis than genetic diagnosis by K-ras mutation in cases with genetic alterations as well as in cases without them.

Topics: Aged; Aged, 80 and over; Case-Control Studies; Colorectal Neoplasms; Female; Genes, p53; Genes, ras; Humans; Immunoenzyme Techniques; Keratins; Lymphatic Metastasis; Male; Middle Aged; Mutation; Prognosis; Sensitivity and Specificity

The purpose of this study was to assess the immunocytochemical status of bone marrow aspirates from patients with clinically isolated hepatic metastases to test the hypothesis that such findings would allow improved patient selection for liver-directed treatment.. All patients had biopsy-proven or presumed colorectal cancer metastatic to the liver and were scheduled for an operative procedure for hepatic resection or for hepatic artery catheter and chemotherapy pump implant. Immunocytochemical analysis of bone marrow aspirate smears was performed with a panel of monoclonal antibodies directed toward cytokeratins, Lewis Y antigen and A-33 colorectal epitopes.. Data from 80 patients indicated that bone marrow reactivity was present in 9.5 percent of those with resectable hepatic metastases and in 34 percent of those not resected (P = 0.03). No single monoclonal antibody or combination produced better discrimination.. Presence or absence of presumed occult colorectal cancer cells in the bone marrow of patients with isolated hepatic metastases is biologically interesting, but not useful in selecting or altering patient management.

Topics: Antibodies, Monoclonal; Biomarkers, Tumor; Bone Marrow; Bone Marrow Examination; Bone Marrow Neoplasms; Colorectal Neoplasms; Combined Modality Therapy; Humans; Immunoenzyme Techniques; Keratins; Lewis Blood Group Antigens; Liver; Liver Neoplasms; Prognosis

Topics: Antibodies, Monoclonal; Colorectal Neoplasms; DNA, Neoplasm; Eosine Yellowish-(YS); Flow Cytometry; Fluorescent Dyes; Follow-Up Studies; Hematoxylin; Humans; Immunohistochemistry; Keratins; Lymph Nodes; Lymphatic Metastasis; Neoplasm Staging; Ploidies; Prognosis; Retrospective Studies; Survival Rate

Among the members of the carcinoembryonic antigen (CEA) family, CD66a (human C-CAM) and CGM2 (CEA gene family member 2) mRNAs are frequently down-regulated in colorectal cancer. In contrast, nonspecific cross-reactive antigen (NCA) mRNA is overexpressed in the majority of these carcinomas. In animal models, the rodent homologues of CD66a have been shown to act as tumor suppressors, suggesting an important role in carcinogenesis. Here we investigate the mRNAs of CD66a, CGM2, and NCA in 22 human colorectal adenomas and the respective normal mucosa specimens by Northern blots. The expression of both CD66a and CGM2 changed in a concomitant fashion. Using oligonucleotides specific for the N-terminal domains, two CD66a transcripts 3.9 and 1.5 kb in size were identified. These showed a greater than 50% down-regulation in 20 of 22 and 18 of 22 adenomas, respectively. Reduction of the CGM2 message was observed in 21 of 22 cases. Complete or near-complete losses of the CD66a 3.9-kb mRNA and the CGM2 message were found in 13 of 22 and 15 of 22 of the tumors, respectively. The medians of CD66a and CGM2 expressions were between 0.3 and 0.0, respectively. The tumor:normal ratio of NCA mRNA expression was increased up to 2.4-fold in 11 of 22 adenomas. Altogether, these results compare well to the changes reported previously for colorectal carcinomas. The high frequency and early appearance of dysregulation of members of the carcinoembryonic antigen family during colorectal tumorigenesis suggests that these changes may be important for the development of the malignant phenotype.

Topics: Adenoma; Aged; Aged, 80 and over; Antigens, CD; Antigens, Differentiation; Antigens, Neoplasm; Blotting, Northern; Carcinoembryonic Antigen; Cell Adhesion Molecules; Colorectal Neoplasms; Female; GPI-Linked Proteins; Humans; Intestinal Mucosa; Keratins; Male; Membrane Glycoproteins; Middle Aged; RNA, Messenger

This study was undertaken to investigate the clinical implications of detection of genetic alterations in blood and lymph nodes in colorectal cancer patients.. The reverse transcriptase polymerase chain reaction product of the cytokeratin gene in blood was examined as a cancer cell-specific expression in 35 colorectal cancer patients. The K-ras or p53 gene mutations in the lymph nodes histopathologically negative for metastasis were studied by the mutant-allele-specific amplification method in 26 colorectal cancer patients.. The reverse transcriptase polymerase chain reaction assay was able to detect a cytokeratin reverse transcriptase polymerase chain reaction product at a concentration from a single to ten colon cancer cells per 10(6) normal peripheral blood mononuclear cells. Cytokeratin reverse transcriptase polymerase chain reaction products were detected in nine patients' blood samples, although none of the samples were cytologically detectable. The blood's cytokeratin positivity correlated with the invasive mode of the tumor (P < 0.05) and the presence of distant metastasis (P < 0.01). Two (50 percent) of four patients whose blood was positive for cytokeratin had recurrences. Of 17 patients with the K-ras or p53 gene mutation in primary tumors, 9 (53 percent) had the corresponding mutations in lymph nodes. Mutation positivity in lymph nodes correlated with the presence of lymphatic invasion of the primary tumor (P < 0.05). All patients with mutation-negative lymph nodes remained disease-free for more than two years after surgery.. Detection of cytokeratin reverse transcriptase polymerase chain reaction products in the blood and K-ras or p53 gene mutations in the lymph nodes histologically negative for metastasis may be applicable for clinical use, despite some limitations, and may serve as a useful clinical factor for stratifying patients who are at high or low risk for recurrence after surgery.

Topics: Biomarkers, Tumor; Colorectal Neoplasms; Female; Genes, p53; Genes, ras; Humans; Keratins; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Neoplastic Cells, Circulating; Polymerase Chain Reaction; RNA

The immunohistochemical diagnosis between epithelial mesothelioma and adenocarcinoma is currently based on the use of a panel of antibodies to adenocarcinoma-associated antigens and a few antibodies to mesothelial-associated antigens. Since the introduction of epitope retrieval methods, the sensitivity of many antibodies has been enhanced. Thus, a reevaluation of the mesothelioma/adenocarcinoma diagnostic panel becomes necessary. We studied 268 paraffin-embedded formalin-fixed tumor samples that included 57 epithelial mesotheliomas and 211 adenocarcinomas of various origins, comparing an extensive antibody panel with and without heat-induced epitope retrieval (HIER). Marked increase in the sensitivity of several antibodies, with no loss of specificity, was found when HIER was used. After statistical analysis, the antibodies to the epithelial glycoproteins carcinoembryonic antigen, BerEp4, and Bg8 emerged as the best discriminators between adenocarcinoma and epithelial mesothelioma within the entire panel. The mesothelium-associated antibodies, HBME-1, calretinin, and thrombomodulin were less sensitive and less specific than the former, although they were found to be useful on certain cases. Antibodies to cytokeratins and vimentin, although of minor diagnostic value in this context, may be helpful to evaluate the quality of antigen preservation. This study confirms the value of immunohistochemistry to accurately distinguish mesothelioma from adenocarcinoma when an antibody panel approach is used. The addition of heat-induced epitope retrieval methods increases the effectiveness of the procedure and is recommended for most of the antibody panel members.

Topics: Adenocarcinoma; Biomarkers, Tumor; Breast Neoplasms; Calbindin 2; Carcinoembryonic Antigen; Colorectal Neoplasms; Decision Trees; Diagnosis, Differential; Epitopes; Female; Hot Temperature; Humans; Immunohistochemistry; Keratins; Lung Neoplasms; Mesothelioma; Ovarian Neoplasms; Peritoneal Neoplasms; Pleural Neoplasms; Retrospective Studies; S100 Calcium Binding Protein G; Sensitivity and Specificity; Thrombomodulin; Vimentin

There is controversy about the prognostic significance of occult lymph node metastases detected by immunohistochemistry with the anti-cytokeratin antibody CAM 5.2. The aim of this study was to characterize occult lymph node metastases in colorectal carcinomas that might be associated with a higher risk of recurrence. Three hundred fifty-eight lymph nodes from 10 recurrent and 9 nonrecurrent cases of colorectal carcinoma were examined. All these patients had been reported originally as having no lymph node metastases by routine hematoxylin and eosin staining. Three 10-micron sections or ten 3-micron sections (30-micron total thickness) from each lymph node were stained with CAM 5.2 and examined for the presence of occult lymph node metastases. Occult metastases were detected in 67 of 175 lymph nodes from the recurrent cases, and in 23 of 183 lymph nodes from the nonrecurrent cases. The frequency of positive nodes was significantly higher in the recurrent cases. The recurrent cases had metastases in nodes more distant from the main tumor than did the nonrecurrent cases. Detection of occult lymph node metastases with cytokeratin immunohistochemistry may make it possible to identify patients with a higher risk of recurrence after the removal of a primary colorectal tumor.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Keratins; Liver Neoplasms; Lung Neoplasms; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Recurrence, Local; Platelet Endothelial Cell Adhesion Molecule-1; Prognosis

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

Tumor stage in bowel and breast cancer is the chief determinant of prognosis and the need for adjuvant therapy. The intermediate filament protein keratin 19 (K19) is expressed by epithelia and corresponding malignancies. Recently, the detection of K19 gene expression by RT-PCR has been reported as a sensitive tool for detecting occult metastasis to the lymph nodes and bone marrow of patients with primary epithelial tumors. Doubt has arisen, however, about the tissue specificity of K19 gene expression. In this study, we report a comparison of RT-PCR for K19 with another keratin, K20, in the lymph nodes and bone marrow of 15 patients with colorectal cancer, eight control patients who underwent bowel resection for benign disease, and four other control patients. K19 and K20 gene expression was detected in 84 and 26 of 109 lymph nodes and in 6 and 0 of 15 bone marrow aspirates, respectively, from colorectal cancer patients. K19 gene expression was also detected in 34 of 40 lymph nodes and in 5 of 12 bone marrow aspirates from control patients, whereas K20 gene expression was undetectable in any control sample. In conclusion, K19 RT-PCR lacks specificity as a marker of occult lymph node and bone marrow metastasis. In contrast, K20 RT-PCR was specific and should be evaluated further in future studies.

Topics: Colorectal Neoplasms; DNA, Complementary; Gene Amplification; Humans; Keratins; Lymphatic Metastasis; Microchemistry; Neoplasm Metastasis; Neoplasm Staging; Polymerase Chain Reaction

We performed immunohistochemical studies on 90 surgically resected liver tumors, including 30 tumors each from hepatocellular carcinoma (HCC), cholangiocarcinoma (CC), and metastatic colorectal adenocarcinoma (MCA), using monoclonal antibodies against cytokeratin (CK) 7, CK 19, and CK 20 to examine the differences in the CK expressions in primary and metastatic carcinomas of the liver. We also investigated the usefulness of such expression in the differential diagnosis in addition to existing markers such as alpha-fetoprotein, carcinoembryonic antigen, and carbohydrate antigen 19-9. For CK 7, all except for one (97%) of the CCs were diffusely positive, whereas only two (7%) HCCs and one (3%) MCAs were diffusely positive. For CK 19, 23 (77%) CCs and 19 (64%) MCAs were diffusely positive, whereas no HCCs were positive. For CK 20, 22 (74%) MCAs were diffusely positive, whereas no HCC and three (10%) CCs were diffusely positive. The findings concerning the expression of immunohistochemical CK are therefore considered to be useful in addition to the diagnostic criteria when making a differential diagnosis of primary and metastatic carcinomas of the liver.

Topics: Adenocarcinoma; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cholangiocarcinoma; Colorectal Neoplasms; Diagnosis, Differential; Humans; Immunoenzyme Techniques; Intermediate Filament Proteins; Keratin-20; Keratins; Liver Neoplasms

To evaluate the incidence and prognostic importance of micrometastatic disease in regional lymph nodes from Dukes' B colorectal carcinomas.. Retrospective study.. University hospital, Sweden.. 100 patients operated on for primary colorectal carcinoma, classified as Dukes' B lesions.. The regional lymph nodes were re-examined immunohistochemically using monoclonal antibodies against cytokeratin.. Incidence and prognostic importance of micrometastases.. Micrometastases were found in 39% (39/100) of the patients. The number of positive cells in the lymph nodes examined varied from 1 to over 100. They appeared as single cells or small clusters of cells located within the capsule or in the peripheral sinus of the lymph node. At least three sections from each of three lymph nodes had to be examined to identify 95% of the patients with lymph node micrometastases. The outcome of the patients with micrometastases was not significantly different from that of patients with no epithelial cells in the lymph nodes.. Micrometastases in regional lymph nodes are a interesting phenomenon but clinically seem to be of only weak prognostic value.

Topics: Aged; Colorectal Neoplasms; Disease-Free Survival; Female; Follow-Up Studies; Humans; Immunoenzyme Techniques; Incidence; Keratins; Lymph Nodes; Lymphatic Metastasis; Male; Prognosis; Proportional Hazards Models; Retrospective Studies; Time Factors

Immunohistochemical detection of bone marrow micrometastases has been reported as a prognostic marker in colorectal cancer. The aims of this study were to evaluate the potential advantage of flow cytometry as an objective method of identifying and quantifying micrometastatic deposits within bone marrow and to determine the prevalence and quantity of micrometastases in patients undergoing surgery for gastrointestinal cancers.. Flow cytometry was first validated by a controlled "spike" experiment in which varying numbers of neoplastic epithelial cells were added to bone marrow, and cytometry was performed in a blinded fashion. Three neoplastic cell lines (colonic and esophageal) with varying degrees of expression of cytokeratin-18 were used. Epithelial cells were detected by dual staining with fluorescence-labeled, monoclonal anti-cytokeratin, and propidium iodide.. Cytometry reproducibly detected the presence of > or = 10 neoplastic cells per 10(5) marrow cells. Micrometastases were found in 20%-30% of patients undergoing potentially curative resection of colorectal and gastroesophageal adenocarcinomas. There was a trend toward increasing positivity for marrow deposits with advanced Dukes' staging of colorectal cancer.. Flow cytometric assessment of bone marrow is a reliable, objective, and quantitative method of detecting micrometastatic deposits found in a substantial subset of patients undergoing surgery for gastrointestinal adenocarcinomas.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Bone Marrow Neoplasms; Carcinoma, Squamous Cell; Colorectal Neoplasms; Esophageal Neoplasms; Flow Cytometry; Gastrointestinal Neoplasms; Humans; Keratins; Prognosis; Prospective Studies; Stomach Neoplasms

Admixture of normal and neoplastic cells is a serious problem in the evaluation of tumor cell kinetic parameters by flow cytometry, in particular for DNA diploid tumors. The admixture of non-neoplastic cells, such as stromal cells and inflammatory cells, can disturb the estimation of the proliferative tumor fraction. This problem has been addressed in fresh tumor samples by applying bivariate flow cytometric analyses for DNA and cytokeratin. We have adapted this approach for formalin-fixed and paraffin-embedded tissue samples of colorectal carcinomas. After preparation of a single cell suspension from paraffin blocks by means of an enzymatic digestion step, the cells of epithelial origin were selectively stained with a panel of subtype specific cytokeratin antibodies. DNA analysis could thus be performed on the cytokeratin-positive cells. The proliferative fractions of the paraffin-embedded samples could be compared with those of the fresh tissue samples and a very good correlation was seen between DNA indices from fresh and paraffin-embedded material. As expected, after gating on the cytokeratin-positive cells an enrichment of the S-phase fraction was seen compared with the ungated cell population. However, this enrichment was more pronounced in the cell suspensions derived from the paraffin-embedded part of the tumor compared with the fresh disaggregated, ethanol-fixed part of the tumor.

Topics: Aneuploidy; Colorectal Neoplasms; DNA, Neoplasm; Flow Cytometry; Formaldehyde; G1 Phase; Humans; Immunohistochemistry; Keratins; Kinetics; Multivariate Analysis; Paraffin Embedding; Resting Phase, Cell Cycle; S Phase

Patients with transmurally invasive, lymph node negative colorectal carcinoma (Dukes' B) have a 5-year survival rate ranging from 53.9% to 84.9%. The authors postulate that patients with Dukes' B colon cancer who die of their disease have occult micrometastases in their pericolic lymph nodes at the time of original diagnosis. In an attempt to identify these occult micrometastases, pericolic lymph nodes from Dukes' B colon cancer resections were stained retrospectively with antibodies against cytokeratin (anti-keratin AE1/AE3, Boehringer Mannheim, Indianapolis, IN) and CC49 (a second-generation monoclonal antibody directed against TAG-72.. The authors reviewed all Dukes' B (transmurally invasive, lymph node negative) primary colorectal carcinoma resection specimens from the surgical pathology files of the Ohio State University Hospitals between 1984 and 1987. Survival data were obtained from the Tumor Registry of the Arthur G. James Cancer Hospital and Research Institute, Columbus, Ohio. The results were analyzed by univariate and multivariate analysis.. Fifty cases with 568 lymph nodes (11.3 per case) were examined with each antibody using standard immunoperoxidase techniques. Positive staining for cytokeratin was seen in 14 patients (33 lymph nodes), 6 of whom died of colon cancer within 66 months (43%). Only 1 of the 36 patients with cytokeratin-negative lymph nodes died of colon cancer over the same time period (3%, P = 0.0009 univariate, P = 0.0013 multivariate). There was no significant difference in survival between the CC49-positive and CC49-negative groups.. Immunoperoxidase techniques are capable of identifying micrometastatic disease in lymph nodes missed by routine hematoxylin and eosin staining. Further, the presence of cytokeratin-positive cells within lymph nodes correlated with a significantly poorer prognosis. Therefore, cytokeratin staining of pericolic lymph nodes in patients with Dukes' B colorectal cancer is recommended. Larger multicenter studies are needed, however, to confirm these results and to evaluate the appropriateness of adjuvant chemotherapy in patients whose disease is upstaged by immunohistochemical staining.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Colorectal Neoplasms; Glycoproteins; Humans; Immunohistochemistry; Keratins; Lymph Nodes; Lymphatic Metastasis; Prognosis; Retrospective Studies; Survival Rate

Micrometastases have been detected by immunocytochemical means in the lymph nodes of patients with otherwise node-negative cancer of the colon and rectum. This study examines the incidence and prognostic significance of nodal micrometastases in Dukes' B carcinoma. Five hundred and fifty-nine lymph nodes from 77 cases of Dukes' B carcinoma were examined for lymph node micrometastases by immunocytochemical staining for cytokeratin AE1:AE3. Micrometastases were detected in 19 cases (25 per cent). Cell clusters were present in ten cases, the remaining nine cases displaying only single cells. The presence of micrometastases was unrelated to age (P = 0.06), sex (P = 0.32), tumour site (P = 0.37), tumour size (P = 0.67), or tumour differentiation (P = 0.66). Ten-year survival estimates by the Kaplan-Meier lifetable method was 47 per cent in patients with and without micrometastases (chi 2 = 0.35 and 1 df, P = ns). The presence of nodal micrometastases detectable only by immunocytochemistry in patients with Dukes' B colorectal cancer does not justify reassignment to a more advanced disease stage.

Topics: Aged; Colorectal Neoplasms; Epithelium; Female; Humans; Immunohistochemistry; Incidence; Keratins; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging; Prognosis; Survival Rate

We examined the expression of carcinoembryonic antigen (CEA) and cytokeratin (CK) as well as the sialo- and sulphomucin content of 40 hyperplastic polyps (HPs), 6 mixed hyperplastic-adenomatous polyps, 30 adenomas and 40 adenocarcinomas of the colorectum. HPs showed a positive CEA expression in 95% of cases and a decreased sialo- and sulphomucin content compared with normal mucosa. Similar changes were observed in adenomas with low-grade dysplasia. The increase in CEA expression from HPs and adenomas to carcinomas was accompanied by a reduction of sialo- and sulphomucins with about three fourths of carcinomas being sialo- and sulphomucin negative. Oncofetal antigen expression concomitant with mucin changes observed in HPs may indicate impaired cellular maturation at a functional level before dysplastic changes become visible. CEA and CK positive macrophages were found in carcinomas predominantly at sites of tumor disruption and necrosis as well as within veins and lymphatic vessels. Our findings suggest that macrophages may play a role in CEA and CK release into the circulation and thus may be determinants of tumor marker serum levels.

Topics: Adenocarcinoma; Adenocarcinoma, Mucinous; Adenomatous Polyps; Biomarkers, Tumor; Carcinoembryonic Antigen; Cell Transformation, Neoplastic; Colon; Colonic Polyps; Colorectal Neoplasms; Humans; Hyperplasia; Keratins; Macrophages; Mucins; Necrosis; Neoplastic Cells, Circulating; Precancerous Conditions; Rectum

Biliary glycoprotein (BGP) is the human homologue of a cell adhesion molecule (CAM) of the rat designated Cell-CAM. The BGP gene is a member of the carcinoembryonic antigen gene family, which belongs to the immunoglobulin superfamily. BGP is expressed in cells of epithelial and myeloid origin. In granulocytes, BGP is a main antigen of the CD66 cluster of differentiation antigens that mediate the binding to endothelial E-selectin. Since BGP is a major human CAM, the expression of BGP was studied in 21 colorectal carcinoma tissue specimens and in the respective adjacent normal mucosae. As an internal control for epithelial mRNA, the expression of cytokeratin 18 was evaluated in parallel. In addition, the expression of carcinoembryonic antigen and nonspecific crossreacting antigen, which are highly homologous to BGP, was investigated. Two BGP mRNAs of 3.9 and 1.5 kilobases were detected in the normal colonic mucosa samples. The median of the tumor-to-normal ratios of mRNA expression was 0.2 for both BGP mRNAs. In contrast, the median was 1.2 for cytokeratin, 1.0 for carcinoembryonic antigen, and 1.4 for nonspecific crossreacting antigen. Relative to cytokeratin 18 expression, the expression of BGP was reduced to < or = 0.1 in half of the tumors and to < or = 0.4 in > 80% of the tumors. These findings indicate that the loss or reduced expression of the adhesion molecule BGP is a major event in colorectal carcinogenesis.

Topics: Antigens, CD; Antigens, Neoplasm; Carcinoembryonic Antigen; Carcinoma; Cell Adhesion Molecules; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Intestinal Mucosa; Keratins; Lymphatic Metastasis; Membrane Glycoproteins; RNA, Messenger; RNA, Neoplasm

The prognosis of colorectal carcinoma relies heavily on pathological staging which includes the metastatic state of lymph nodes. Colorectal resectates from 47 patients (41 with colorectal carcinoma and six with non-malignant disease) were entered into a study to assess the best method for detecting metastases in lymph nodes. The maximum number of lymph nodes was harvested at an initial careful examination of the specimen. Subsequently, the pericolic and perirectal fat was dissected out, dehydrated in alcohol, cleared in xylene and further lymph nodes were recovered. Both sets of lymph nodes were examined by the standard histological method and subsequently stained immunohistochemically for cytokeratins (CK). The mean number of lymph nodes recovered at the initial dissection from all 47 cases was 6.7, this was raised to 58.2 after xylene clearance, ie an average of 51.5 lymph nodes were not recovered by traditional methods. At the initial dissection no epithelial cells were detected in any of the lymph nodes from the nonmalignant cases or 25 of the malignant cases. In the other 16 cases, epithelial cells were detected by H&E in 38 lymph nodes. Thus the initial staging was 3 Dukes A, 22 Dukes B and 16 Dukes C. After immunohistochemistry, eight additional cases (originally staged Dukes B) showed epithelial cells in the lymph nodes, these were chiefly occult invasion, raising the involved lymph nodes number to 70. After xylene clearance and applying the CK staining, an additional 135 lymph nodes were found to be involved, thus the overall number of involved lymph nodes was increased to 205. The combined technique changed the Dukes staging in 12 out of 41 cases of colorectal carcinoma, resulting finally in 3 Dukes A, 10 Dukes B and 28 Dukes C, ie 55% of Dukes B become Dukes C.

Topics: Antibodies, Monoclonal; Colorectal Neoplasms; Humans; Immunohistochemistry; Keratins; Lymph Nodes; Lymphatic Metastasis; Neoplasm Staging; Xylenes

Much of the information recorded at the time of surgery for malignant disease has been used, alone or in combination, to indicate the outlook for that patient, including probability of metastasis. However, direct evidence of tumour seeding in distant organs at that time is not available. An immunocytochemical assay for the epithelial cytokeratin protein (CK18) may fill this gap since it is a feature of epithelial cells but would not normally be in bone marrow. We looked for disseminated tumour cells preoperatively in bone marrow from 88 patients with radically resected colorectal carcinomas. Smears for 28 (32%) patients were positive for cells of epithelial origin, which were absent from aspirates taken from 102 controls with non-malignant disease. The prognostic value was assessed in a follow-up study with a median observation time of 35 (12-58) months. Patients who had bone marrow tumour cells in the aspirate showed a significantly shorter disease-free survival than those without tumour cells (p = 0.0084). In a Cox regression model multivariate analysis demonstrated that the finding of tumour cells in bone marrow is an independent, significant (p = 0.0035) determinant of relapse. Although the skeleton is not a preferred site of overt metastasis in colorectal cancer the demonstration of tumour cells in bone marrow has to be taken as evidence of the general disseminative capability of an individual tumour.

Topics: Adenocarcinoma; Bone Marrow; Colorectal Neoplasms; Female; Follow-Up Studies; Humans; Keratins; Male; Middle Aged; Prognosis

The immunohistochemical localization of five antibodies against carcinoembryonic antigen (CEA), CA19-9, keratin, alpha-tubulin and secretory component (SC) was investigated in 14 lesions of adenocarcinoma (AC), 22 of adenoma with high-grade atypia (AH), 50 of adenoma with low-grade atypia (AL), and 15 of non-neoplastic mucosa (NNM) of the large intestine. The positive patterns for each staining were divided into three categories (patterns 1, 2, and 3). All neoplastic lesions (AC, AH and AL) were positive for CEA, while 85.7% of AC, 36.4% of AH and 6.0% of AL showed strongly positive staining (pattern 3). 78.6% of AC and 54.5% of AH were positive for CA19-9 in comparison to 20.0% of AL. For keratin, more than 95% of the neoplastic lesions were positive, while 78.6% of AC, 27.3% of AH and 22.0% of AL showed strongly positive staining (pattern 3). For alpha-tubulin, more than 85% of neoplastic lesions were positive, while 50.0% of AC, 36.3% of AH and 26.0% of AL showed strongly positive staining (pattern 3). For SC, in contrast, 42.9% of AC, 27.3% of AH and 8.0% of AL were negative, but 93.3% of NNM were positive. It was concluded that the positive staining rate, especially the rate of pattern 3 for each antibody correlated with the degree of atypia of the colorectal neoplastic lesions (AC, AH and AL).

Topics: Adenocarcinoma; Adenoma; Antibodies, Anti-Idiotypic; Antibodies, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Carcinoembryonic Antigen; Colorectal Neoplasms; Cytoskeleton; Humans; Keratins; Secretory Component; Staining and Labeling; Tubulin

The present study was performed to identify tumor cells in lymph nodes from colorectal adenocarcinomas considered free of disease by the classic hematoxylin-eosin stain, based on the detection of the carcinoembryonic antigen (CEA) and cytokeratins in neoplastic epithelial cells. For this purpose, 603 lymph nodes from 46 lesions were stained by the peroxidase-antiperoxidase technique. Tumor cells were detected in 22 nodes from 12 patients, mainly in the subcapsular sinuses, permitting a restaging of these patients into two groups: those now considered to have metastatic disease and those free of metastases. However, the 5-year follow-up showed no statistical differences in survival between the two groups.

Topics: Adenocarcinoma; Carcinoembryonic Antigen; Colorectal Neoplasms; Follow-Up Studies; Humans; Immunoenzyme Techniques; Keratins; Lymphatic Metastasis; Macrophages; Neoplasm Staging

Monoclonal antibodies (Mab) are potent probes to identify individual tumour cells or small tumour cell clusters in bone marrow. In the present study, various antibodies directed against either cell surface or intracytoplasmic antigens of epithelial cells were assessed for their ability to detect such cells in bone marrow of patients with breast, colorectal and gastric cancer. According to the presented data, monoclonal antibodies against intracellular cytokeratin (CK) components are superior in terms of specificity and sensitivity to antibodies reacting with epitopes of the cell membrane. Using a monoclonal antibody against the cytokeratin polypeptide 18 in connection with the alkaline phosphatase anti-alkaline phosphatase detection system (APAAP), we could detect tumour cells in bone marrow of 34 out of 97 patients with gastric cancer examined at the time of primary surgery. The incidence of positive findings was correlated to established risk factors, such as histological classification and locoregional lymph node involvement. Clinical follow-up studies on 38 patients demonstrated a significantly increased relapse rate in patients presenting with CK-positive cells in their bone marrow at the time of primary surgery. Thus the described technique may help to identify patients with gastric cancer carrying a high risk of early relapse.

Topics: Antibodies, Monoclonal; Antigens, Surface; Bone Neoplasms; Breast Neoplasms; Colorectal Neoplasms; Female; Humans; Keratins; Stomach Neoplasms

A monoclonal antibody (mAb) directed against the cytokeratin (CK) polypeptide no. 18 specifically expressed in cells derived from simple epithelia was used to detect epithelial tumor cells in bone marrow aspirates. Of 156 patients with colorectal carcinoma, 42 presented with cells at the time of primary surgery. The incidence of positive findings varied considerably with the size and the localization of the primary tumor, the involvement of regional lymph nodes, and the presence of clinically manifest metastases. Applying a sensitive double-staining procedure, we could demonstrate that epithelial cells in bone marrow showed a heterogeneic expression of receptors for epidermal growth factor (EGF-R) and transferrin (Tf-R) as well as of the proliferation-associated Ki67 antigen. Also human leukocyte antigen (HLA) class I antigens differed widely in their expression on the CK-positive cells. Clinical follow-up studies on 85 patients showed a significantly higher relapse rate in patients presenting with CK-positive cells in their bone marrow at the time of primary surgery. Twenty-three patients were monitored for the presence or absence of CK-positive cells in bone marrow over time. The majority of monitored patients (18 of 23) exhibited a constant pattern of immunocytochemical findings during the time of observation. Thus, the technique may be useful in identifying high-risk patients as well as in monitoring adjuvant therapeutic trials.

Topics: Antibodies, Monoclonal; Antigens, Surface; Bone Marrow; Carcinoma; Colorectal Neoplasms; ErbB Receptors; Histocompatibility Antigens Class I; Humans; Immunohistochemistry; Keratins; Ki-67 Antigen; Phenotype; Prognosis; Receptors, Transferrin; Recurrence; Risk Factors; Survival Rate

Four colon adenocarcinoma cell lines, CC-M2, CC-M3, CC-M4, and CC-M2NM, have been established from surgical specimens of 18 unselected patients without the use of "feeder" cells and additional growth factors (e.g., insulin, hydrocortisone, etc.) in the culture medium. The methods of primary cultivation of tissue explants are described. Studies of determination of morphology, growth curve, plating efficiency, chromosomal analysis, CEA and beta-HCG synthesis, and tumorigenicity, were done to characterize the cell lines. Significant variations have been found in one of the four cell lines, both in vitro and in vivo studies. There are distinct phenotypes in the established cell lines which may be useful in studying the cell differentiation and progression of colorectal cancer.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Animals; Carcinoembryonic Antigen; Chorionic Gonadotropin; Chorionic Gonadotropin, beta Subunit, Human; Chromosome Aberrations; Chromosome Disorders; Colorectal Neoplasms; Female; Humans; Karyotyping; Keratins; Male; Mice; Mice, Inbred BALB C; Middle Aged; Neoplasm Transplantation; Peptide Fragments; Tumor Cells, Cultured

Solid tumors contain heterogenous cell populations, resulting in flow cytometric (FCM) DNA quantitations of a mixture of tumor and host cells. Such mixed populations can result in dilution of the tumor cells by the host cells, in difficulty defining the diploid reference mean and in histogram peak overlap, precluding cell-cycle analysis. In this study, epithelial (tumor) cells and contaminating host cells in 100 consecutively accessioned human mammary and colorectal carcinomas were segregated in a multiparametric two-color FCM DNA analysis of intact, ethanol-fixed cells. These two carcinomas and bladder carcinomas contain a cytoskeleton of simple epithelium that is selectively stained with an FITC-labeled monoclonal antibody (MAb) to cytokeratin (CK: CAM 5.2-FITC). This MAb detects the CK 8, CK 18 and CK 19 consistently present in all layers of normal and neoplastic urothelium, colonic epithelium and mammary epithelium. Gating on CK in these tumors enables the nonstaining leukocytes, stromal fibroblasts and endothelial cells to be excluded from DNA analysis. A separate aliquot of each tumor evaluated was labeled with an MAb to leukocyte-common antigen (LCA-FITC) to serve as a patient-specific intrinsic diploid reference standard. Both the CK-labeled and LCA-labeled cells were then dual labeled for DNA with propidium iodide. This method (1) correctly identified the intrinsic diploid (LCA-positive) channel, allowing an accurate definition of normal cell DNA content for calculation of the DNA index; and (2) resulted in an increased sensitivity in the identification of both diploid and abnormal hyperdiploid tumor cell populations. It also (3) limited DNA cell cycle analysis to urothelial, colonic and mammary epithelial cells, the majority of which were neoplastic in carefully selected tumor samples. In addition, this method (4) clarified near-tetraploid populations that overlap the normal nonepithelial G2M region by diminishing the normal G2M peak and accentuating the aneuploid tetraploid G0G1 peak and (5) deconvoluted overlapping histograms composed of normal host and diploid-range or aneuploid tumor cells by gating on tissue-specific markers. This exclusion of host cells in both classes of tumors resulted in more accurate cell-cycle calculations in the former and allowed calculation of the S-phase fractions in the latter.

Topics: Antibodies, Monoclonal; Antigens, Differentiation; Breast Neoplasms; Calibration; Colorectal Neoplasms; DNA, Neoplasm; Female; Flow Cytometry; Histocompatibility Antigens; Humans; Keratins; Leukocyte Common Antigens; Staining and Labeling

Murine monoclonal antibodies directed against tumour associated antigens are potentially useful in tumour diagnosis and therapy. However, all the antigens they recognise may be heterogeneously expressed on tumours and this may allow escape of cells from therapy if a single monoclonal antibody is used. One approach is to use combinations of monoclonal antibodies recognising complementary cell surface antigens. A flow cytometric method which allows accurate quantitation of the intensity of staining and the percentage of fresh primary tumour cells binding a series of monoclonal antibodies has therefore been developed. This allows calculations as the number of drug molecules which could be potentially delivered by each monoclonal antibody and the optimal combination of antibodies which should be used. Monoclonal antibodies recognising Y hapten (C14), CEA (228, 161) and 791T-p72 antigen (791T/36) have been screened as a possible combination for colorectal cancer. There was inter-tumour variation in the binding of all the monoclonal antibodies although combinations could reduce or abrogate this problem. A combination of the monoclonal antibodies C14, 228, 791T/36 and 161 would recognise 100% of tumours. Sixty per cent of tumours bound all four antibodies, 78% any three, 90% any two and 100% any one antibody. There was also intra-tumour variation in the number of tumour cells per lesion that were recognised, the best monoclonal antibody, 161, stained a mean of 59% of cells per tumour whereas the anti-cytokeratin monoclonal antibody stained a mean of 74% of cells per tumour. An increased intensity of staining of tumour membranes was observed when a combination of C14 and 228 was used compared to binding of individual antibodies. Furthermore there was still no significant binding to normal colon membranes. Combinations of monoclonal antibodies which recognise a high percentage of tumours are likely to be necessary for monoclonal antibody drug targeting to prevent tumour recurrence and/or metastases.

Topics: Antibodies, Monoclonal; Antigen-Antibody Reactions; Antigens, Neoplasm; Antigens, Surface; Colorectal Neoplasms; Flow Cytometry; Humans; Keratins