The Relation Between EZH2 Expression and Clinical Outcomes in Colorectal Cancer Patients Treated with anti-EGFR Therapeutics

THE RELATION BETWEEN EZH2 EXPRESSION AND CLINICAL OUTCOMES IN COLORECTAL CANCER PATIENTS TREATED WITH ANTIEGFR THERAPEUTICS Itaru Yamamoto, Katsuhiko Nosho, Hiroyoshi Kurihara, Hisayoshi Igarashi, Shinichi Kanno, Keisuke Ishigami, Hideyuki Koide, Kei Mitsuhashi, Yasutaka Sukawa, Kenji Okita, Ichiro Takemasa, Hiroyuki Yamamoto, Yasuhisa Shinomura, Hiroshi Nakase Background & Aims: The polycomb group protein enhancer of zeste homolog 2 (EZH2) is a methyltransferase that suppresses microRNA-31 (miR-31) in various human malignancies including colorectal cancer. We recently suggested that miR-31 regulates the signaling pathway downstream of epidermal growth factor receptor (EGFR) in colorectal cancer. Therefore, we conducted this study to assess the relation between EZH2 expression and clinical outcomes in metastatic colorectal cancer patients treated with anti-EGFR therapeutics. Methods: We evaluated EZH2 expression using immunohistochemistry and assessed miR31 and gene mutations [KRAS (codon 61/146), NRAS (codon 12/13/61), and BRAF (codon 600)] in 113 colorectal cancer patients harboring KRAS (codon 12/13) wild-type who were treated with anti-EGFR therapeutics. Progression-free survival (PFS) and overall survival (OS) were evaluated. Results: During the follow-up study of the 113 patients with colorectal cancer treated with anti-EGFR therapeutics who were eligible for survival analysis, 66 patients died (all deaths were confirmed to be attributed to colorectal cancer). The median followup periods for PFS and OS were 6.1 and 46 months, respectively. EZH2 expression scores of 0 (negative), 1 (weak), 2 (moderate), and 3 (strong) were observed in 11%, 21%, 18%, and 50% of the colorectal cancer tissues. We made EZH2 into a dichotomous expression variable, defining scores of 2-3 as high expression and those of 0-1 as low-expression. EZH2 expression low-expression group was significantly associated with shorter PFS (log-rank test: P = 0.021) and OS (log-rank test: P = 0.034) in colorectal cancer patients treated with anti-EGFR therapy. In low miR-31 expression group and KRAS (codon 61/146), NRAS, and BRAF wild-type group, significantly shorter PFS (log-rank test: P = 0.022, P = 0.037, P = 0.033, and P = 0.023, respectively) were observed in EZH2 low-expression groups compared with high-expression groups. In the multivariate Cox regression analysis for PFS, low EZH2 expression was associated with a shorter PFS (HR: 1.75; 95% CI: 1.02-2.93; P = 0.045), independent of the mutational status and miR-31. Conclusion: Our data showed that EZH2 expression was associated with survival in patients with metastatic colorectal cancer who underwent surgical treatment and chemotherapy with anti-EGFR antibodies. Moreover, low EZH2 expression was associated with a shorter PFS in patients with colorectal cancer, independent of gene mutations in the downstream part of the EGFR pathway and miR-31 expression, suggesting that EZH2 expression is a useful and additional prognostic biomarker for anti-EGFR therapy.

(A) The expression of dysregulated lncRNA, miRNA and mRNA identified from GEO databases; (B) The relationships between lncRNA, miRNA and mRNA were shown in the ceRNA network.

Tu1986 ROLE OF CDK9 INHIBITION AS A SENSITIZER TO RADIATION IN ESOPHAGEAL ADENOCARCINOMA: IN VITRO AND IN VIVO EFFICACY STUDY Omkara L. Veeranki Preoperative chemoradiation in neoadjuvant setting is the standard of care for loco-regional esophageal adenocarcinoma (EAC). However, less than 30% of patients develop complete pathological response indicating need for newer therapeutic strategies. Cyclin dependent kinase 9 (CDK9) is found to be over expressed in EAC compared to Barrett's esophagus. Our previous studies demonstrated strong antitumor effects on inhibition of CDK9 in EAC both in vitro and in vivo. Here we report augmented tumor regression in irradiated xenografts on combination with Flavopiridol, a well-established clinically used CDK inhibitor, compared to single modality treatments. In vitro studies indicate that Flavopiridol could radiosensitize FLO-1 and SKGT4 EAC cells with sustained 53BP1 foci especially in SKGT4 cells. Flavopiridol and BAY1143572, a more selective inhibitor of CDK9, could radiosensitize additional EAC radiation sensitive and resistant cell lines (SKGT4-R, OE-33 and OE-33-R) as analyzed by MTT assay, apoptosis, formation of 53BP1 foci and clonogenic assay. Flavopiridol and BAY1143572 showed a radio-synergistic action by downregulating MCL-1 and Axl. In conclusion, the radio-sensitizing capacities of CDK9 inhibitors presented here suggest that their adjuvant administration might improve EAC therapy. Percent tumor necrosis/rate of growth (mm/day)

Tu1988 POTENTIAL LINK BETWEEN FUSOBACTERIUM ENRICHMENT AND DNA METHYLATION ACCUMULATION IN THE INFLAMMATORY COLONIC MUCOSA IN ULCERATIVE COLITIS Tomomitsu Tahara, Ichiro Hirata, Mitsuo Nagasaka, Yoshihito Nakagawa, Tomoyuki Shibata, Naoki Ohmiya BACKGROUND AND AIM: Fusobacterium enrichment has been associated with colorectal cancer development. Ulcerative colitis (UC) associated tumorigenesis is characterized as high degree of methylation accumulation through continuous colonic inflammation. The aim of this study was to investigate a potential link between Fusobacterium enrichment and DNA methylation accumulation in the inflammatory colonic mucosa in UC. METHODS: In the candidate analysis, inflamed colonic mucosa from 84 UC patients were characterized the methylation status of colorectal cancer related 24 panels of genes. In the genome-wide analysis, an Infinium HumanMethylation450 BeadChip array was utilized to characterize the methylation status of >450,000 CpG sites for fourteen UC patients. Results were correlated with Fusobacterium status. RESULTS: UC with Fusobacterium enrichment (FB high) was characterized as high degree of type C (for cancer-specific) methylation compared to other (FB low/neg) samples (P<0.01). Genes hyper methylated in FB high samples included well known type C genes in colorectal cancer, such as MINT2 and 31, P16 and NEULOG1. Multivariate analysis demonstrated that the FB high status held an increased likelihood for mean Z score hypermethylation of type C genes (odds ratio: 16.18, 95% confidence interval: 1.94-135.2, P=0.01). Genome-wide methylation analysis demonstrated a unique methylome signature of FB high cases irrespective of promoter, outside promoter, CpG and non-CpG sites. Group of promoter CpG sites that were exclusively hypermethylated in FB high cases significantly codified the genes related to the catalytic activity ( P=0.039). CONCLUSION: Our findings suggest that Fusobacterium accelerates DNA methylation in specific groups of genes in the inflammatory colonic mucosa in UC.

Tu1989 MOLECULAR SUBTYPES COMBINING CPG ISLAND METHYLATOR PHENOTYPE (CIMP) AND TP53 HOT SPOT MUTATION STATUS PROVIDE DISTINCT CLINICOPATHOLOGICAL FEATURES IN GASTRIC CANCER Tomomitsu Tahara, Masaaki Okubo, Tomohiko Kawamura, Noriyuki Horiguchi, Takamitsu Ishizuka, Mitsuo Nagasaka, Yoshihito Nakagawa, Tomoyuki Shibata, Naoki Ohmiya Background/ Aim: Accumulation of both genetic and epigenetic abnormalities plays an important role in gastric cancer (GC) development. We investigated whether combining methylation phenotype with genetic anomalies such as TP53 and KRAS mutation status would predict prognosis of GC patients. Patients/Methods: CpG island methylator phenotype (CIMP) using conventional five panels of markers (MINT1, 2, 12, 25 and 31), MLH1 methylation, TP53 and KRAS mutation status were characterized in 214 GCs in relation to their clinicopathological features and prognosis. Results: We showed that the molecular subtypes based on the CIMP and TP53 hot spot mutation status (R175, G245, R248, R273,

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S-1027

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Tu1987