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Purpose: Our main goal was to evaluate a panel of molecular markers for the detection of cancer cells in serous effusions and to determine their value as an adjunctive reverse transcription-PCR (RT-PCR) test to cytologic examination., Experimental Design: One hundred fourteen serous effusions from 71 patients with tumors and 43 patients with benign diseases were subjected to RT-PCR for expression of carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (Ep-CAM), E-cadherin, mammaglobin, mucin 1 (MUC1) isoforms MUC1/REP, MUC1/Y, and MUC1/Z, calretinin, and Wilms' tumor 1 susceptibility gene., Results: CEA, Ep-CAM, E-cadherin, and mammaglobin were specifically expressed in malignant effusions. The sensitivity of RT-PCR in cytologically negative malignant effusions was 63.1% combining CEA and Ep-CAM (with 100% specificity) and reached 78.9% adding MUC1/Y or MUC1/Z (with 93% specificity). In the whole population of effusions, the combination of cytology with RT-PCR of CEA and Ep-CAM yielded a 90.1% sensitivity, a specificity and a positive predictive value of 100%, and a 86% negative predictive value for malignancy. Adding MUC1/Y or MUC1/Z to the panel, the sensitivity was 94.5% with 93% specificity, 95.7% PPV, and 90.9% negative predictive value. Moreover, CEA and mammaglobin were specifically expressed in epithelial malignancies, and mammaglobin was mainly expressed in effusions from breast carcinoma (97.3% of specificity)., Conclusions: A combination of cytology and RT-PCR analysis of CEA and Ep-CAM significantly improved the detection sensitivity of tumor cells in serous effusions. RT-PCR analysis of CEA, Ep-CAM, and mammaglobin in serous effusions could be a beneficial adjunct to cytology for the diagnosis of malignancy.

Purpose: We examined the feasibility of using molecular characterization of circulating tumor cells as a method for early detection of breast cancer., Research Design: Women without a prior history of cancer who had a breast abnormality detected on imaging followed by a breast biopsy were enrolled in this study. Density gradient centrifugation and immunomagnetic capture were used to enrich for epithelial cells from approximately 20 mL of blood. Real-time reverse transcription-PCR was used to quantitate the expression levels of the highly breast-specific genes, mammaglobin, gamma-aminobutyric acid type A receptor pi subunit (GABA A(pi)), B305D-C, and B726P in the epithelial cell-enriched samples., Results: The assay was technically feasible in 154 of 199 accrued patients. From their clinical assessment, 100 patients had benign breast disease, 10 patients had ductal carcinoma in situ, and 44 patients had invasive breast cancer. We constructed a diagnostic test that classified patients with mammaglobin levels of at least 32.2 copies/pg beta-actin (units) in their circulating epithelial cells as positive for invasive breast cancer. This resulted in a sensitivity and specificity of 63.3% and 75.0%, respectively. A diagnostic test that classified patients as positive for invasive breast cancer when either mammaglobin levels were >46.3 units or B305D-C levels were >11.6 units increased the sensitivity and specificity to 70.5% and 81.0%, respectively. In the latter test, 12 of the 14 node-positive breast cancer patients were correctly identified. Including GABA A(pi) and B726P in the test did not increase its diagnostic potential., Conclusions: These results suggest that molecular characterization of circulating epithelial cells using mammaglobin and B305D-C offers potential for early detection of invasive breast cancer.

There is increasing evidence that molecular detection of micrometastatic breast cancer in the axillary lymph nodes (ALN) of breast cancer patients can improve staging. Molecular analyses of samples obtained from the Minimally Invasive Molecular Staging of Breast Cancer Trial (n = 489 patients) indicate that whereas the majority of molecular markers are informative for the detection of metastatic breast cancer (significant disease burden), only a few are sensitive for the detection of micrometastatic disease (limited disease burden). Frequency distribution and linear regression analyses reveal that relative levels of gene expression are highly correlated with apparent sensitivity for the detection of micrometastic breast cancer (P < 0.05). These data provides statistical validation of the concept that the most informative markers for detection of micrometastatic disease are those that are most highly expressed in metastatic disease. To test this hypothesis, we developed an innovative microarray strategy. RNA from a metastatic breast cancer ALN was diluted into RNA from a normal lymph node and analyzed using Affymetrix microarrays. Expression analysis indicated that only two genes [mammaglobin (mam) and trefoil factor 1 (TFF1)] were significantly overexpressed at a dilution of 1:50. Real-time reverse transcription-PCR analysis of pathology-negative ALN (n = 72) confirm that of all the markers tested, mam and TFF1 have the highest apparent sensitivity for detection of micrometastatic breast cancer. We conclude that a dilutional microarray approach is a simple and reliable method for the identification of informative molecular markers for the detection of micrometastatic cancer.

The presence of regional lymph node metastasis is one of the most significant poor-prognosis factors in patients with biliary tract carcinoma. To establish a sensitive reverse transcription (RT)-PCR assay to detect micrometastases in lymph nodes of biliary tract carcinoma, we first investigated the optimal markers in biliary tract carcinoma. The expressions of the six candidates for a suitable RT-PCR marker [mammaglobin B, carcinoembryonic antigen (CEA), cytokeratin (CK) 20, prostate-specific antigen, and melanoma antigens (MAGE-1 and MAGE-3)] were evaluated in two bile duct cancer cell lines and human biliary tract carcinoma tissues. Of 32 carcinoma tissues, mammaglobin B, CEA, prostate-specific antigen, MAGE-1, MAGE-3, and CK 20 were expressed in 28 (88%), 26 (81%), 4 (13%), 5 (16%), 7 (22%), and 9 (28%), respectively. Mammaglobin B and CEA were considered to be good markers of the six candidates. We then examined 209 lymph nodes obtained from 15 patients with biliary tract carcinoma by RT-PCR assay using both mammaglobin B and CEA and compared the results with those of histological examination. All of 20 histologically positive lymph nodes for metastasis displayed the PCR product(s) of marker genes. Of 189 histologically negative nodes, 24 (13%) nodes expressed mammaglobin B and/or CEA mRNA, suggesting the presence of micrometastasis. Our findings suggest that mammaglobin B and CEA could be useful RT-PCR markers for the detection of lymph node micrometastases in biliary tract carcinomas. Our RT-PCR assay allows accurate clinical staging necessary for patient stratification with respect to adjuvant therapy after surgery.