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Background: Classification of colorectal cancer (CRC) according to gene expression profiling remains controversial with regards to their ability to stratify patients for precision therapy. Several studies have suggested that ‘stem cell like’ or ‘epithelial-mesenchymal transition (EMT)’ signature associates with poor prognosis. Meanwhile CRCs with BRAF V600E mutation have shown poor outcomes in several clinical trials, but the biological roles of BRAF mutation have not been fully elucidated. Materials and Methods: To evaluate biological roles of BRAF mutations in CRC, we performed mRNA microarray analysis in the following three subsets; eight BRAF-mutant CRC tissues without MSI, six BRAF-mutant CRCs with MSI and five BRAF-wild type CRCs with MSI. Pathway analyses were performed by Gene Set Enrichment Analysis (GSEA). Following identification of candidate biomarkers that influence poor outcomes and associate with BRAF V600E mutation, we examined these candidate biomarkers in a cohort of 1068 CRC patients who underwent surgical resection of their primary tumor and/or metastatic lesions from 1994 to 2015 at the Okayama University Hospital. Results: By the GSEA, prominent signatures enriched in CRCs with BRAF V600E mutation vs. wild-type were EMT-related processes (EMT and myogenesis). Additionally, CRC with BRAF V600E mutation strongly associated with inactivation of Wnt signaling and intestinal differentiation-related genes. Among the differentially expressed genes between CRCs with BRAF V600E mutation versus wild-type, Wnt-antagonist, Secreted frizzled-related proteins (SFRPs) were significantly upregulated in CRCs with BRAF V600E mutation. As SFRPs are well known to be inactivated in CRCs by promoter hypermethylation, and up to 80% of CRCs are methylated in the promoter region of the SFRP2 gene, we focused and analyzed SFRP2 promoter methylation status in 782 stage II-IV CRCs of our cohort by a modified highly sensitive assay for bisulfite DNA followed by fluorescence-based PCR, as we previously reported (JNCI 2009). SFRP2 methylation was observed in 498 (64.2%) stage II-IV CRCs. Interestingly, SFRP2 unmethylated CRCs with genetic mutation in the KRAS/BRAF genes demonstrated significantly poorer outcome in RFS compared with SFRP2 methylated CRCs with genetic mutations in the KRAS/BRAF genes (5-years RFS rate were 46.8% vs 72.1%; P = 0.015, Hazard ratio 2.06 [95% CI, 1.12 to 3.69]; P = 0.021). Conclusion: Our results suggest that SFRP2 methylation status could be a potential prognostic biomarker for stage II-IV CRCs, especially with genetic mutations in the KRAS/BRAF genes. Citation Format: Kazuya Yasui, Takeshi Nagasaka, Toshiaki Toshima, Takashi Kawai, Kunitoshi Shigeyasu, Yoshiko Mori, Junko Haraga, Keiichiro Nakamura, Yuzo Umeda, Hiroshi Tazawa, Ajay Goel, Toshiyoshi Fujiwara. Gene expression profiles in BRAF V600E mutant colorectal cancer and association with SFRP2 methylation status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5711. doi:10.1158/1538-7445.AM2017-5711

Background: Endometrial cancer accounts for the second percentage of Lynch syndrome related tumors following colorectal cancer. Inactivation of MSH2 was frequently observed in endometrial cancer with microsatellite instability (MSI) or mismatch repair complex deficiency (dMMR). With respect to MSH2 deficiency (dMSH2), not like MLH1 deficiency, most of dMSH2 were caused by germline mutations in the MSH2 gene or germline EpCAM deletions. Meanwhile, heritable germline epimutations in MSH2 have been reported in a few Lynch syndrome families that lacked germline mutations in the MSH2 gene. We previously provided evidence for frequent MSH2 hypermethylation in Lynch syndrome colorectal tumors with dMSH2 and MSH2 methylation may serve as the “second hit” at the wild-type allele. Materials and Methods: In this study, we analyzed MSH2 promoter methylation status, as well as MLH1 methylation status, and expression status of the mismatch repair proteins (MLH1, MSH2, PMS2, and MSH6) by immunohistochemistry in a cohort of 138 endometrial cancer tissues surgically resected at Okayama University Hospital. DNA was extracted from formalin-fixed, paraffin-embedded tissue, analyzed MSI status by four mononucleotide markers and both MLH1 and MSH2 promoter methylation status by a fluorescent quantitative bisulfite PCR assay. Results: Endometrial cancers displaying MSI or dMMR were observed in 40 (29.0%) or 41 cases (29.7%), respectively. Endometrial cancers with MSH2 deficiency were observed in eight (5.8%) of 138 tumors (19.5% of dMMR). MSH2 promoter methylation was present in 7 cases (5.1% in 138 tumors), and significantly correlated with dMSH2 (P=0.0041, Fisher's exact probability test). Then, we also examined the family history of first-degree relatives retrospectively. In this cohort, although patients with MMR deficiency were significantly associated with family history of Lynch syndrome related tumor (P Conclusions: In this study, we demonstrated that MSH2 methylation significantly correlated with dMSH2 and may have strong relation with family history of Lynch syndrome related tumor, taking a role as “second hit” to the MSH2 gene. Citation Format: Junko Haraga, Takeshi Nagasaka, Keiichirou Nakamura, Tomoko Haruma, Takeshi Nishida, Akihiro Nyuya, Kazuya Yasui, Hisashi Masuyama, Toshiyoshi Fujiwara, Yuji Hiramatsu. Clinicopathological significance of endometrial cancer with MSH2 deficiency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4277. doi:10.1158/1538-7445.AM2017-4277