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.

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

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.

BackgroundThe primary goal of preoperative systemic treatment (PST) in patients with breast cancer is downsizing of tumors to enhance the rate of breast conserving surgery. Additionally, preoperative systemic treatment offers the possibility to assess for chemosensitivity of early stage disease. In various cancers the prognostic value of neutrophil/lymphocyte ratio (NLR) was demonstrated, indicating that high NLR determines worse prognosis of the patients. The goal of our study was to evaluate the predictive and prognostic value of NLR in early stage breast cancer patients undergoing PST.Methods247 female patients with histologically proven breast cancer were analysed in this retrospective analysis. The NLR before the initiation of PST was documented. Histopathological response in surgically removed specimens was evaluated using a modified Sinn regression score and the pCR defined as no invasive tumor in primary tumor and lymph nodes. NLR was correlated with response to PST and disease free survival.ResultsPST was categorized into five groups (anthracycline containing, anthracycline and taxane containing, taxane containing, hormone treatment and other chemotherapies). pCR rate was defined as no invasive rest of tumor either in primary tumor or (ypT0 = Sinn) or in primary tumor and in lymph nodes (ypT0isypN0). Median NLR in patients without any invasive tumor rest was significantly higher than in patients either with some invasive tumor rest or not responding to chemotherapy. Despite this primary difference, the results were not stable across the analysed treatment groups particularly in the group with highest pCR rates (taxane and anthracycline treatment). Further, no association with disease free survival could be observed.ConclusionsAlthough there was a reverse trend with the higher NLR prior to systemic treatment in patients who achieved pCR, we could not demonstrate predictive or prognostic value of NLR in the cohort of early stage breast cancer patients treated with PST.

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Archival paraffin-embedded tumor specimens offer a wealth of information for both cancer research and for routine clinical applications. However, the use of formalin-fixed, paraffin-embedded specimens for quantitative real-time PCR is not yet a standard diagnostic method in many laboratories, in particular for the quantification of human papillomavirus (HPV). Particularly high-risk HPV types are involved in almost 100% of the carcinogenesis of cervical cancer. We compared the diagnostic applicability and sensitivity of real-time PCR to that of chromogenic tyramide-signal-amplified in situ hybridization and conventional PCR for the detection of HPV from archival tissue in 164 cases of carcinoma in situ and cervical cancer. Furthermore, we examined whether the viral load of HPV is of prognostic relevance. Our findings indicate that patients in tumor stage I with a lower viral load of HPV type 16 (HPV16; up to 1,000 copies/ng of DNA) had a significantly better survival than HPV 16-negative patients (P ‫؍‬ 0.037). We observed a greater sensitivity of both real-time PCR and conventional PCR for the detection of HPV16 and -18 compared to signal amplified in situ hybridization. We found a considerable concordance between HPV16 ( ‫؍‬ 0.661) and HPV18 ( ‫؍‬ 0.781) status as measured by real-time PCR and conventional PCR, indicating similar sensitivities. We recognized an inhibitory effect of formalin fixation and paraffin embedding on the evaluation of real-time PCR quantification.Formalin-fixed, paraffin-embedded (FFPE) tissues retained in pathology archives are an important resource for molecular pathology studies and for diagnostic applications. The use of in situ hybridization (ISH) and PCR assays have both been reported for DNA analysis in formalin-fixed archival tissues (6,7,25,40,42,48). Both assays have the advantage that only small amounts of tissue are required, and both tolerate the degradation of target nucleic acids due to the formalin fixation and paraffin-embedding procedures. Each assay per se has its own advantages and disadvantages.For a pathologist, the retention of morphology and the histological context of a tumor is very important. Especially in the case of cervical carcinoma, the phenomenon of koilocytosis indicates an infection with human papillomavirus (HPV) (4) that can only be detected by microscopic analysis of tissue sections. Moreover, episomal and integrated viral DNA can only be differentiated by ISH due to the staining pattern (8). With the use of chromogenic tyramide-signal-amplified ISH (CISH) it is even possible to detect and localize single or very few HPV copies within infected nuclei (15,36,49). However, this technique is rather cost-intensive and quite involved for routine applications.Conventional PCR is held to be the most sensitive detection method available (9,16,20,27,37,41). Although this is probably true, it has some limitations, as does ISH, in that it cannot quantify viral load in a prognostic-diagnostic significant manner. This restriction can be circumvented by the use o...