CHFR METHYLATION AS AN EPIGENETIC MARKER FOR RECURRENCE OF COLON CANCER M. D. Anderson Cancer Center, Houston, Texas Motofumi Tanaka, Salil Sethi, Donghui Li, Dipen Maru, Gail Bland, James L. Abbruzzese, Cathy Eng

Background Epigenetic inactivation of tumor suppressor genes by promoter hypermethylation has been implicated in carcinogenesis (1,2). Prior studies have indicated the predictive and prognostic utility of these methylated genes in primary colorectal tumors (3,4). However, it is unclear whether the methylation profile of tumor suppressor genes can serve as a potential biomarker for tumor recurrence following curative surgical resection.

Background(contd.) The recurrence rate of colon cancer after curative surgical resection is reported to be 20%-30% following adjuvant chemotherapy (5). The identification of prognostic indicators for recurrent disease is clearly warranted and may have an impact on the routine colon cancer surveillance. Promoter hypermethylation of the CHFR (checkpoint with forkhead- associated and RING finger domains), ID4 (inhibitor of DNA binding), and RECK (reversion-inducing cysteine rich protein with Kazal motifs) genes have been associated with reduced mRNA and/or protein expression in colorectal cancer.

Objective To identify potential DNA methylation biomarkers to predict recurrence - free survival (RFS) and overall survival (OS) in locally advanced colon cancer patients following curative surgical resection.

Methods Study Design and Population 72 patients with the AJCC stage II (T 3-4 N 0 M 0 ) and III (T X N 1-3 M 0 ) colon cancer, who had undergone surgical resection at M.D. Anderson from , were included. A retrospective chart review was conducted to collect demographic and clinical information. A waiver of informed consent was provided. Patients with a family history of HNPCC were excluded.

Methods(contd.) DNA methylation DNA samples were extracted from the fresh frozen colon cancer tumor tissues and were subjected to a treatment with with bisulfite. Pyrosequencing method (Biotage Co.) was used to quantitatively detect methylation status. Methylation status was evaluated in promoter regions within 300 base pairs from the transcription start site (TSS) of CHFR, ID4, and RECK genes, and also in the MINT1 loci. Mean methylated rate of multiple CpG sites was used for analysis. We defined 30% in methylated rate as methylation-negative, -low, and -high, respectively.

Methods(contd.) Statistical Analysis Survival outcomes were determined by Kaplan-Meier plot, log-rank test for univariate analysis and Cox’s proportional hazard model for multivariate analysis. The correlation between clinicopathological features and methylation status was analyzed by χ 2 test.

Results The median follow-up period was 56.6 months. 17 (23.6%) patients developed recurrence during the follow up, and 9 (12.5%) patients have died. Methylation rates of CHFR, MINT1, ID4, and RECK genes were 61.1%, 31.9%, 48.6%, and 8.3%, respectively. The CHFR methylation-high (43%) group was found to have a lower RFS (P=.009) and OS (P=.018%) when compared with the CHFR methylation- negative (39%) and -low (18%) group (Fig 1). Methylation status of MINT1, ID4, and RECK 23 was not associated with RFS and OS. CHFR methylation-high group was more frequently found to have a N 2 disease (P=.042) with a higher propensity to affect the right-side (P=.004). Multivariate analysis indicated that T 4 disease and the CHFR methylation- high were significant predictors for tumor recurrence.

Patient Demographics VariableNumber of Number of p* Number of p* patients Death Recurrence Age (years)  >70 Sex Male Female Tumor site Right colon Left colon AJCC tumor (T) T3 T4 AJCC lymph node (N) N0 N1 N2 AJCC Stage II III Histological grade well/ moderate poor/mucinous Adjuvant chemotherapy Yes No * Log rank test

Methylation status and survival Variable No. of No. of p* No. of p* patients Deaths Recurrence CHFR methylation Negative (<15 %) Positive (≥15 %) Negative or Low (< 30%) High (≥ 30%) MINT1 methylation Negative (<15 %) Positive (≥15 %) Negative or Low (< 30%) High (≥ 30%) ID4 methylation Negative (<15 %) Positive (≥15 %) Negative or Low (< 30%) High (≥ 30%) RECK methylation Negative (<15 %) Positive (≥15 %) Negative or Low (< 30%) High (≥ 30%) * Log rank test

Clinicopathological characteristics stratified by CHFR methylation VariableNegative or Low High p* (< 30%) (≥ 30%) Tumor site Right colon Left colon AJCC lymph node (N) N0/N1 N2 AJCC primary tumor (T) T3 T4 Histological grade Well/moderate Poor/undifferentiated (39) 23 (61) 25 (74) 9 (26) (61) 22 (39) 5 (33) 10 (67) (57) 27 (43) 4 (44) 5 (56) (51) 27 (49) 12 (71) 5 (29) *X 2 test

Kaplan-Meier survival and CHFR methylation CHFR < 30% CHFR > 30% P = 0.009

Kaplan-Meier survival and CHFR methylation CHFR < 30% CHFR > 30% P = 0.023

Multivariate analysis of Recurrence- free survival CovariateHazard ratio* (95% CI) p-value AJCC primary tumor (T) [T3-T4] AJCC lymph node (N) [N0-N1] [N1-N2] Histological grade [poorly/undifferentiated] CHFR methylation status[≥30%] 3.99 ( ) (0.58–6.34) (0.59 –10.6) (0.65–10.6) (1.12–13.5) *HR adjusted for age, sex, tumor site, and adjuvant chemotherapy

Discussion CHFR is a check-point protein in the cell cycle that delays the entry into metaphase in response to mitotic stress (6). Hypermethylation of CHFR is closely associated with a loss of gene expression and a mitotic check-point dysfunction, which in turn sensitizes the affected cells to microtubule inhibitors (7). Decreased CHFR expression was reported to be associated with malignant progression i.e. accelerated growth rate, higher mitotic index, enhanced invasiveness, and increased motility, when studied in vitro, (8).

Discussion(cont’d.) Our analysis indicates that the epigenetic inactivation of CHFR is associated with lymph node metastasis which is a poor prognostic indicator of recurrence and survival. Increased methylation of the CHFR promoter is a potential epigenetic marker for colon cancer recurrence and overall survival. Further analysis with a greater sample size will be conducted for validation. If validated, CHFR methylation may have an impact on the current recommendations for colon cancer surveillance.

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