E-cadherin expression is a sensitive and specific method for detection of carcinoma cells in fluid specimens.

The distinction between reactive mesothelial cells and carcinoma in pleural, peritoneal, and pericardial fluids is often difficult. We and others previously showed that E-cadherin, an epithelial-specific adhesion protein, can be useful for this distinction. In this study we tested the sensitivity and specificity of E-cadherin compared to, and in combination with, conventional cytology for assessment of carcinoma in fluids. Cytyc ThinPreptrade mark slides (Marlborough, MA) from 102 sequential fluids were evaluated for E-cadherin expression by routine immunologic techniques. No evidence of E-cadherin staining was seen in 71 cases, while 31 showed unequivocally positive staining. Sensitivity and specificity were independently determined for E-cadherin alone (72% and 97%, respectively), cytomorphology alone (62% and 100%, respectively), and both together (92% and 100%, respectively). We conclude that assessment of E-cadherin expression has sensitivity and specificity comparable to, or better than, conventional cytomorphology. If both cytomorphology and E-cadherin are used together, a definitive and correct diagnosis could have been made on nearly every case in this study.

Frequent mutation and nuclear localization of beta-catenin in anaplastic thyroid carcinoma.

Beta-catenin is an ubiquitously expressed cytoplasmic protein that has a crucial role in both E-cadherin-mediated cell-cell adhesion and as a downstream signaling molecule in the wingless pathway. Stabilization of beta-catenin followed by nuclear translocation and subsequent T-cell factor/lymphoid-enhancing factor-mediated transcriptional activation has been proposed as an important step in oncogenesis. Stabilization may occur through activating mutations in exon-3 at the phosphorylation sites for ubiquitination and degradation of beta-catenin. Immunohistochemical subcellular localization of beta-catenin and mutational analysis of exon-3 of the beta-catenin gene by single-strand conformational polymorphism followed by DNA sequencing was performed on 37 samples from 31 patients with anaplastic thyroid carcinoma. Immunofluorescent staining showed nuclear localization in 15 (42%) of the 36 samples examined. Nucleotide sequencing of mobility shifts detected by single-strand conformational polymorphism revealed somatic alterations in 19 (61%) of the 31 patients analyzed. We conclude that mutations in beta-catenin are common in anaplastic thyroid cancer and that they may activate transcription, as illustrated by frequent nuclear localization of the protein. These findings support the idea that beta-catenin acts as an oncogene and contributes to the highly aggressive behavior of this tumor.

Expression of c-met is a strong independent prognostic factor in breast carcinoma.

BACKGROUND: The c-met protooncogene encodes the met protein, the receptor for scatter factor/hepatocyte growth factor, a growth factor that modulates the motility and stable interaction of the epithelial cells. This study assesses the expression of met receptor in breast carcinoma and its prognostic value with respect to survival. METHODS: Immunofluorescence was used to evaluate 91 archival breast carcinoma specimens using a polyclonal antibody to the cytoplasmic domain of the receptor. Cases were scored by two pathologists on a percentage basis and then converted to binary scores (positive or negative) on the basis of a bimodal distribution. RESULTS: Strong expression of met was found in 20 invasive ductal breast tumor specimens (22%). The 5-year survival of patients whose tumors showed decreased met expression was 89%, in contrast to a 52% 5-year survival rate in patients whose tumors expressed met (P = 0.008). This trend also was observed in patients without lymph node metastases at presentation, in whom met negative patients had a 95% 5-year survival compared with only 62% for met positive patients (P = 0.006) Multivariate analysis using the Cox proportional hazards model showed met expression to be an independent predictor of survival, with a predictive value nearly equivalent to that associated with lymph node status. CONCLUSIONS: The authors conclude that expression of met in patients with invasive ductal carcinoma of the breast is a strong, independent predictor of decreased survival and may be a useful prognostic marker with which to identify a subset of patients with more aggressive disease.

The expression of p120ctn protein in breast cancer is independent of alpha- and beta-catenin and E-cadherin.

Several studies have reported loss or alteration of expression of E-cadherin in breast cancer and more recently changes in levels of expression of the catenins. We used immunofluorescence to examine E-cadherin, alpha-catenin, beta-catenin, and p120ctn (formerly p120CAS) expression in 91 cases of invasive ductal carcinoma. As expected, all four proteins co-localize to the junctional regions of the cells. Although nuclear localization has been described for beta-catenin in colonic polyps, no examples were found in these breast cancer cases. We found that, although alteration is common in the catenins and E-cadherin, complete loss, as exemplified by E-cadherin in lobular carcinoma (where E-cadherin is frequently mutated), is rarely seen. In contrast, the catenin-related protein p120ctn shows an expression pattern that is significantly unrelated to the other catenins (or E-cadherin), including complete loss of expression in approximately 10% of the cases. No statistically significant correlations with traditional prognostic indicators were observed with any of these proteins. We conclude 1) that expression of E-cadherin and alpha- and beta-catenin are generally retained at the membrane although frequently reduced or altered, 2) that complete loss of p120ctn expression is seen in approximately 10% of the cases, and 3) that there is a significant correlation in the expression of E-cadherin and the catenins but no correlation between these molecules and p120ctn, suggesting an absence of coordinate regulation.

The cell adhesion molecule, E-cadherin, distinguishes mesothelial cells from carcinoma cells in fluids.

BACKGROUND: The distinction between benign reactive mesothelial cells and well differentiated carcinoma can be difficult in pleural, peritoneal, and especially pericardial fluids. E-cadherin is an adhesion protein that is specifically expressed in cells of epithelial lineage. In this study, anti-E-cadherin antibodies were used to identify and distinguish carcinoma cells from reactive mesothelial cells. METHODS: Pleural, peritoneal, and pericardial fluids were prepared using the Cytyc Thin Prep processor. The specimens were comprised of a mix of 45 cases that were diagnosed as carcinoma, suspicious, or reactive by Papanicolaou staining of routine material seen by the authors' service. Routine immunologic techniques were used with a commercially available E-cadherin antibody. RESULTS: In most cases of carcinoma, tumor cells showed a strong positive membranous reaction product (32 of 37). This included four cases that were not cytomorphologically diagnosed as malignant, but subsequently proved to be malignant. E-cadherin staining was not observed in five tumors, two of which were not expected to express this protein. One benign case showed cells staining for E-cadherin, although the cells were not malignant by morphologic criteria. Because this case was a surgical pelvic washing, these cells more likely were epithelial contaminants than true false-positives. CONCLUSIONS: The epithelial specific cell-cell adhesion marker E-cadherin reliably distinguishes reactive mesothelial cells from carcinoma and is a useful adjunctive test to distinguish benign reactive mesothelial cells from well differentiated carcinoma cells in fluid specimens.

Molecular cloning and characterization of Na,K-ATPase from Hydra vulgaris: implications for enzyme evolution and ouabain sensitivity.

We have used molecular and biochemical techniques to analyze Na,K-ATPase from a simple metazoan, Hydra vulgaris. First we isolated and characterized cDNA clones encoding the Na,K-ATPase alpha subunit from a Hydra lambda gt11 cDNA library. The open reading frame predicts a protein of 1031 amino acids that bears a high degree of primary sequence and secondary structure similarity to mammalian, avian, and arthropod alpha subunits. The predicted Hydra alpha subunit contains charged residues at the termini of the H1-H2 extracellular domain, suggesting that the Hydra alpha subunit may be resistant to cardiac glycoside inhibition. Biochemical analysis of partially purified Hydra Na,K-ATPase reveals both high- and low-affinity components of ouabain-inhibitable ATPase activity. Our results suggest that the evolutionary ancestor of all metazoans possessed a Na,K-ATPase alpha subunit that was highly conserved with respect to its vertebrate counterparts. Further, expression of a ouabain-resistant Na,K-ATPase activity in Hydra suggests that cardiac glycoside resistance arose randomly during evolution of the Na,K-ATPase.

ELISA analysis of BACTEC bottles for the earlier diagnosis of tuberculosis.

Enzyme-linked immunosorbent assays (ELISA) have enabled earlier identification of Mycobacterium tuberculosis (TB) in clinical settings by utilizing both TB antibody and antigen detection. We studied the sensitivity and specificity of ELISA detection of TB antigen by using a commercially available anti-BCG antibody in conjunction with BACTEC 7H12B culture bottles. We compared these results against those obtained with cultures of Mycobacterium avium-intracellulare and Mycobacterium kansasii. All BACTEC bottles were inoculated with known concentrations of organisms. TB antigen was detected by ELISA in BACTEC culture bottles of TB 12 days before the BACTEC system could itself identify the species. A growth index of greater than or equal to 10 reliably indicated that tuberculosis antigen was detectable by ELISA. The correlation between growth index and antigen concentration was extremely high (r = 0.95 and p less than 0.001). There was insignificant cross-reactivity with the other atypical mycobacteria at the levels of growth tested. This technique potentially offers a sensitive and specific means for the early identification of TB in BACTEC culture bottles.