Background: Monitoring of circulating tumor cells (CTCs) in blood of carcinoma patients treated with novel compounds may be a measurement of treatment effectiveness. Before it can be used clinically, a reliably method is needed to enumerate CTCs. We compared two methods for CTC enumeration, OnkoQuick and the CellSearch system. Methods: We drew 22.5 ml of blood into three CellSave tubes from 15 healthy donors and 61 patients with metastatic carcinoma. After pooling, 15 ml was processed with OncoQuick and 7.5 ml with CellSearch.Results: With both methods no CTCs were found in healthy donors. At least one CTC was detected in 14 of 61 patients (23%) with OncoQuick and 33 of 61 patients (54%) with CellSearch (P < 0.0001). The number of CTCs detected was larger for CellSearch (mean 20 CTCs/7.5 ml of blood) than for OncoQuick (3 CTCs/7.5 ml; P < 0.0001).Conclusion: The CellSearch system is a more accurate and sensitive method to enumerate CTCs. Further studies are warranted to evaluate CTC enumeration by the CellSearch system as a monitoring tool for the evaluation of the efficacy of novel anticancer agents. q 2005 International Society for Analytical Cytology

BRAF V600E is the predominantly occurring mutation of the cytoplasmic kinase BRAF , and , in colorectal cancer , its determination provides a diagnostic exclusion criterion for hereditary nonpolyposis colorectal cancer. The aim of our study was to develop a sensitive BRAF V600E high resolution melting (HRM) assay. We first established and optimized the BRAF HRM assay using a cell line dilution model , enabling us to detect 1% mutant DNA in a background of wild-type DNA. In a comparison , DNA sequencing and real-time allele-specific PCR in the cell line dilution model HRM assay proved to be more sensitive than DNA sequencing and denaturing high performance liquid chromatography , retaining the same sensitivity as real-time allele-specific PCR. In a learning set of 13 patients with known BRAF V600 status , the mutation was detected with high concordance by all four methods. Finally , we validated the HRM assay on 60 formalinfixed , paraffin-embedded colorectal cancer samples. Although all mutated samples were correctly identified by HRM , the detection limit of the HRM assay decreased when using low-quality DNA derived from formalin-fixed , paraffin-embedded samples. In conclusion , HRM analysis is a powerful diagnostic tool for detection of BRAF V600E mutation with a high sensitivity and high-throughput capability. Despite the expected decrease in sensitivity , HRM can reliably be applied in archival formalin-fixed , paraffin-embedded samples tissues.

SUMMARY:The high incidence of HER2 overexpression on the cell surface of breast cancer cells and the recognized prognostic and potentially predictive value of HER2 render this cell surface receptor a novel and important therapeutic target. Although immunohistochemistry (IHC; HercepTest) and fluorescence in situ hybridization (FISH; PathVysion and INFORM)-both approved by the Food and Drug Administration-have emerged as the most viable assays for evaluation of HER2 status in routine clinical practice, there is still no consensus on which is the best method for assessing HER2 status. Therefore, our specific objective was to establish a chromogenic in situ hybridization (CISH) assay for the detection of HER2 amplification on a cohort of 173 archival invasive breast carcinomas. Results were compared with HercepTest, which is the most frequently used method for detecting HER2 alteration. Additionally, HER2 gene copy number was investigated using differential PCR (dPCR) as a testing system. HER2 overexpression was found by IHC in 24.3%; HER2 amplification was found by CISH in 19.1% and by dPCR in 9.2% of the tumors. The overall concordance rate was 95.9% between CISH and IHC and 85.0% between dPCR and IHC. Kappa statistics revealed an excellent agreement between IHC and CISH ( ϭ 0.878), but only a moderate agreement was found between IHC and dPCR ( ϭ 0.482). Discrepant cases between CISH and HercepTest and all IHC-positive cases (ϩ2 and ϩ3), a total of 42 cases, were analyzed with the FISH PathVysion (Vysis) assay. Among 25 HercepTest-positive cases (score ϩ3), 2 showed no gene amplification by FISH or CISH. Four of 13 tumors with weak HER2 overexpression (score ϩ2) were negative with both FISH and CISH. Concordance between CISH and FISH was 100% for the 38 cases analyzed. The current study showed that CISH represents a practical and simple assay for evaluating HER2 gene amplification in archival material, offering a promising alternative to IHC or FISH for the routine diagnostic setting. (Lab Invest 2002, 82:1007-1014.

Loss of epithelial morphology and the acquisition of mesenchymal characteristics are typical for carcinoma cells in tumour progression. In human breast carcinomas, up-regulation of tenascin-C (TN-C) and vimentin (Vim) is frequently observed in cancer cells and correlates with increased malignancy. Thus, it is possible that TN-C is co-expressed with Vim, representing cancer cells that have undergone epithelial-mesenchymal transition (EMT). This study examined 128 breast carcinomas using immunohistochemical techniques to demonstrate that mammary cancer cells are a prominent source of both TN-C and Vim. Statistical analysis revealed a significant association between TN-C and Vim expression in cancer cells. TN-C expression also correlated positively with overexpression of c-erbB-2 oncoprotein and down-regulation of oestrogen receptors (ERs). Eleven human mammary cancer cell lines and two 'normal' cell lines were examined by western blotting and immunohistochemistry. Co-expression of TN-C and Vim was detected in the carcinosarcoma cell line HS 578T, SK-BR-3 (B), fibroblast-like MDA-MB-231 cells, and the myoepithelial cell line HBL 100. These findings suggest that TN-C and Vim, when co-expressed in mammary carcinoma cells, represent regulator genes likely to be involved in EMT during mammary carcinogenesis.

The purpose of this study was to investigate the influence of androgen receptor (AR) expression in tumour tissue on survival of patients with metastatic breast cancer. Tumour specimens from 232 patients with metastatic breast cancer were examined for presence of AR by immunohistochemistry. According to the extent of immunostaining, AR expression was classified as score 0, 1+, 2+, or 3+. AR positivity was observed in 164 (70.7%) tumours. The median survival after disease recurrence (SAR) of patients with AR-expressing tumours was significantly longer compared to that of patients with AR-negative tumours (21.89 months, 95% CI 17.23-26.55 vs 11.99 months, 95% CI 9.36-14.62; log-rank test 0.0282). In addition, patients with AR score 3+ had a significantly longer disease-free survival (DFS) compared to patients with AR score 0, 1+, and 2+, (24.67 months, 95% CI 13.72-35.62 vs 16.36 months, 95% CI 13.18-19.54, log-rank test 0.0043). Multivariate Cox analyses showed no statistically significant influence of AR expression on DFS or SAR. In conclusion, SAR is significantly longer in patients with AR-expressing breast carcinoma. However, AR expression is not an independent prognostic factor for SAR in metastatic breast cancer.