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Mutations in the BRCA1/2 genes were recently shown to be associated with an increased risk for colorectal cancer. We characterized the largest cohort available of BRCA1/2 mutation carriers with colorectal cancer. We analyzed 32 patients with lower gastrointestinal cancers and germline BRCA1/2 mutations from two large academic hospital registries; 91% of patients were of Ashkenazi ancestry, 78% were women, and 62.5% were carriers of BRCA1 gene mutations. A high percentage of colorectal tumors (34.5%) had a mucinous histology and were located atypically in the left colon. Two patients had anal cancer with unusual histology and an additional patient had mucinous small bowel carcinoma. Gene expression analysis showed significant correlation between the gene signatures of left mucinous colorectal cancer and basal-like breast cancer. Our results imply that Ashkenazi BRCA1/2 mutation carriers with colorectal cancer might have unique characteristics with a high rate of left-sided, mucinous histology colorectal cancer, and possibly anal carcinoma. This report suggests a phenotypic influence of defects in DNA repair genes on colorectal tumors.

Introduction Heparanase, the sole heparan sulfate degrading enzyme, has a role in cellular invasion. Accordingly, a large number of studies have demonstrated an association between heparanase expression and tumor stage and patients' prognosis. In colon carcinoma, heparanase shows increased expression in tumor compared to normal tissue and its expression correlates with the presence of metastasis. One of the regulatory mechanisms of heparanase expression is de-methylation on its promoter. In the present study we evaluated the role of heparanase promoter methylation in colon carcinoma. Material and methods Analysis of heparanase promoter methylation was done on 32 samples of colon carcinoma as well as 30 samples of normal colonic mucosa. DNA was extracted from FFPE tissue and subjected to bisulfite conversion. The relative fraction of methylated and unmethylated DNA was evaluated using quantitative real-time PCR. Results The fraction of methylated DNA was 1 ± 3.4% in the colon carcinoma group, and 2.5 ± 3.3% in the normal colon group (P = 0.11). Only one case in the normal group and one case in the tumor group showed more than 10% methylation in the heparanase promoter. Conclusion We did not find any significant difference in heparanase promoter methylation between colon carcinoma and normal colonic mucosa, suggesting that heparanase overexpression in colon carcinoma is mediated by other mechanisms.

Introduction: KRAS mutations in colon carcinomas are associated with lack of response to anti-EGFR monoclonal antibody treatment. Therefore, patients must undergo genetic testing to be eligible for treatment. Several methods for KRAS mutation analysis exist, but many are not sensitive enough to detect a mutation in samples with low fraction of malignant cells. In the present study, we developed a KRAS mutations detection method that is both simple and sensitive. Methods: Usingalockednucleicacid(LNA)containingoligonucleotide, wedevelopedaPCRclampingmethodthatpreferentially amplifies the mutated over wild type KRAS. We evaluated the sensitivity of this method using serial dilutions of plasmids containing wild-type and mutated KRAS fragments. Additionally, KRAS mutation status was evaluated on 60 archived tissue samples of colon carcinoma, and compared to direct sequencing and high resolution melting (HRM) methods. Results: The PCR clamping method could detect as little as 1% mutated DNA in the sample analyzed. Of the 29KRAS mutations identified by the PCR clamping method, only 23 (79%) were identified by standard direct sequencing. The results of PCR clamping correlated with HRM results. Conclusions: LNA based PCR clamping method is a simple and highly sensitive method for the detection of KRAS mutations.

KRAS mutation status has a significant role determining anti-epidermal growth factor receptor (anti-EGFR) treatment response in colon carcinoma patients. Malignant transformation is a dynamic process and therefore, it is conceivable that, at a certain point, the tumor cells’ mass might be heterogeneous for particular mutations. Therefore, the fraction of tumor cells carrying a particular mutation may be more relevant for treatment than the simple determination of presence or absence of mutation. The purpose of this study is to assess whether or not KRAS mutation status is heterogeneous and, if so, to what extent in colon carcinoma samples. Deoxyribonucleic acid was extracted from formalin-fixed paraffin-embedded samples of colon carcinoma and analyzed for the presence of KRAS mutation. The relative fraction of mutated versus wild-type KRAS alleles was evaluated by real-time polymerase chain reaction. Additionally, the relative fraction of cancer cells in the tissue sample was evaluated using computer assisted morphometric analysis. Using this data, we calculated the fraction of mutation containing cells in the samples. Colon carcinoma (169 cases) were analyzed, and a KRAS mutation was found in 75 cases (44%), of which 42 were available for morphometric analysis. In 41 (97.6%) of these cases, the fraction of mutation containing tumor cells was 50% or higher, indicating the absence of significant KRAS mutation status heterogeneity. There was a strong positive correlation (R = 0.66, P < 0.0001) between the fraction of mutated KRAS alleles and the fraction of cancer cells in the samples. The strong positive correlation between the fraction of mutated KRAS alleles and the fraction of cancer cells in the samples indicate homogeneity of KRAS mutation status in colorectal carcinoma.