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[Introduction] In recent years, tumor cells have immune escape mechanism and immune checkpoint inhibitor therapy (anti PD-1/ PD-L1 antibody) has shown benefit in various cancers. Somatic mutations have the potential to encode ‘’non-self’’immunogenic antigens and lymphocytes infiltrate tumor cells in Microsatellite-instable (MSI) endometrial cancers. Therefore, immune checkpoint inhibitor therapy might be effective in MSI endometrial cancers. [Method] Mismatch repair protein (MLH1, PMS2, MSH2, and MSH6), tumor-infiltrating lymphocytes (CD8), and PD-1/PD-L1 expression were assessed by immunohistochemistry in 60 patients with endometrial cancer. We examined whether MSI status have enhanced immune microenvironment and become the therapeutic effect predictor of PD-1/PD-L1 immunotherapy in endometrial cancer. [Results] Loss of mismatch repair protein (MSI group) was identified in 8 (13.3 %) of 60 patients with endometrial cancer. Expression of tumor-infiltrating lymphocytes (CD8) and PD-L1/PD-1 were significantly higher in MSI group compared to MSS group (p=0.001, p=0.044 and p=0.013). [Conclusion]These results suggested that immune checkpoint inhibitor (anti PD-1/PD-L1 antibody) is effective in endometrial cancers with MSI. MSI testing is likely to be a biomarker for PD-1/PD-L1 immunotherapy in endometrial cancer. Citation Format: Hitomi Yamashita, Kentaro Nakayama, Noriyoshi Ishikawa, Toshiko Minamoto, Tomoka Ishibashi, Kohei Nakamura, Kaori Sanuki, Ruriko Ono, Masako Ishikawa, Takeshi Isobe, Satoru Kyo. Microsatellite instability is a potential biomarker for immune checkpoint inhibitor in endometrial cancer [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 2621. doi:10.1158/1538-7445.AM2017-2621

Purpose: Recent studies suggest that fallopian tube secretory epithelial cells (FTSECs) are potential cells-of-origin for high-grade serous ovarian carcinoma (HGSOC). Several genetic alterations are involved in HGSOC carcinogenesis, but the minimal requirement for tumor initiation remains unclear. Therefore, we sought to identify oncogenic mutations indispensable for HGSOC carcinogenesis in a stepwise model using immortalized FTSECs. Experimental Design: We established an in vitro stepwise carcinogenesis model using immortalized FTSECs overexpressing cyclin D1, CDK4R24C, and hTERT, and mimicked select genetic abnormalities identified as gene alterations essential for carcinogenesis, including p53, c-Myc, or RAS/PI3K pathway mutations. Results: Our analyses revealed two distinct patterns of gene alterations essential for HGSOC carcinogenesis: p53/KRAS/AKT and p53/KRAS/c-Myc. Dominant-negative p53 expression alone or in combination with oncogenic KRAS (KRASV12), constitutively active AKT (CA-AKT), or c-Myc in immortalized cells failed to induce a tumorigenic phenotype; however, overexpression of either CA-AKT or c-Myc in combination with dominant-negative p53 and KRASV12 was sufficient to confer tumorigenic potential. Importantly, all transformed FTSECs formed tumors in xenograft mice, which were grossly, histologically, and immunohistochemically similar to human HGSOC. Interestingly, mice harboring tumors with c-Myc amplifications displayed extensive metastases, consistent with the increased dissemination observed in their human counterparts. C-Myc is associated with cell proliferation in vitro; therefore, this genetic abnormality may promote HGSOC progression. Conclusions: Collectively, our data showed that aberrant p53/KRASV12/c-Myc or p53/KRASV12/PI3K-AKT signaling is the minimal requirement for FTSEC carcinogenesis. Moreover, the model generated with this evidence will likely facilitate analysis of early events in HGSOC carcinogenesis. Citation Format: Kohei Nakamura, Kentaro Nakayama, Tohru Kiyono, Noriyoshi Ishikawa, Toshiko Minamoto, Tomoka Ishibashi, Kaori Sanuki, Hitomi Yamashita, Ruriko Ono, Kouji Iida, Hiroki Sasamori, Razia Sultana, Mohammad Mahmud Hossain, Masako Ishikawa, Satoru Kyo. In vitro reconstitution of high-grade serous ovarian carcinoma from primary fallopian tube secretory epithelial cells [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 5755. doi:10.1158/1538-7445.AM2017-5755

High-grade serous ovarian carcinoma (HGSOC) is the most lethal gynecologic malignancy in the world. Recent studies suggest that fallopian tubal secretory epithelial cells (FTSECs) are candidates for the origin of HGSOC. Several genetic alterations involved in the carcinogenesis have been reported in HGSOC, but their minimal requirement and the specific combinations remained unclear. To clarify these points, we established an in vitro stepwise carcinogenesis model using immortalized FTSECs with overexpressed cyclinD1/cdk4 and hTERT, in which we additionally mimicked selected genetic abnormalities frequently observed in clinical samples. Mutational analysis using clinical samples of HGSOCs identified frequent p53 mutations (96%). Furthermore, RAS/PI3K pathway is important signaling pathway and was deregulated in 45% of HGSOCs. PI3K/AKT pathway is also important pathway in HGSOCs. Thus, we introduced dominant negative form of p53 alone or in combination with oncogenic mutant KRAS allele into immortalized cells, but both failed to exhibit tumorigenic phenotypes. Additional introduction of genetic factors were attempted, and overexpression of c-Myc or phosphorylated Akt (p-Akt) were independently found to confer tumorigenic phenotypes. Importantly, all transformed FTSECs exhibited high-grade serous carcinomas that were grossly, histologically, and immunohistochemically similar to human HGSOCs. Thus, p53/KRAS/c-Myc or p53/KRAS/PI3K-AKT pathways were determined to be minimal required for carcinogenesis. The strength of our study is the use of immortalized FTSECs that have original characteristics of fallopian tube as well as the introduction of genetic mutations clinically observed in human HGSOCs. This experimental model provides a foundation for future studies of early events in carcinogenesis of HGSOCs and the identification of candidate targets for drug development and secreted biomarkers for early detection. It is also likely that a similar model can be broadly applied to studying the normal biology of other epithelial cells where precursor lesions and malignant counterparts are not entirely understood. Citation Format: Kohei Nakamura, Kentaro Nakayama, Tohru Kiyono, Noriyoshi Ishikawa, Masako Ishikawa, Hiroshi Katagiri, Toshiko Minamoto, Tomoka Ishibashi, Emi Sato, Kaori Sanuki, Hitomi Yamashita, Kouji Iida, Razia Sultana, Satoru Kyo. Establishment of in vitro carcinogenic model of high-grade serous ovarian carcinoma using immortalized fallopian tubal secretory epithelial cell. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5160.