Association between DNA methylation and shortened survival in patients with advanced colorectal cancer treated with 5-fluorouracil based chemotherapy

Abstract

Purpose: There are no good genomic markers of survival in patients with advanced colorectal cancer. The CpG island methylator phenotype (CIMP) marks a distinctive pathway in colorectal cancer. We sought to determine the prognostic significance of CIMP in advanced colorectal cancer patients treated with 5-fluorouracil (5-FU) in an Eastern Cooperative Oncology Group clinical trial.

Experimental design: We studied 188 patients enrolled on protocol E2290, a five-arm trial comparing 5-FU, 5-FU in combination with N-phosphonoacetyl-l-aspartic acid, oral leucovorin, i.v. leucovorin, or IFNalpha-2a in patients with advanced colorectal cancer. Methylation of MINT1, MINT31, hMLH1, p14ARF, and p16INK4a in DNA extracted from formalin-fixed paraffin-embedded specimens was evaluated by combined bisulfite restriction analysis, and methylation of MINT2 was studied by methylation-specific PCR.

Results: Methylation frequencies were 21% for MINT1, 23% for MINT2, 24% for MINT31, 4% for hMLH1, 11% for p14ARF, and 17% for p16INK4a. Methylation of MINT1, MINT31, p14ARF, and p16INK4a were correlated, as expected. There was no association between methylation and clinicopathologic factors or response to therapy. Methylation of MINT1, MINT31, p14ARF, or p16INK4a was associated individually with shortened overall survival. Hazard ratios were 1.51 (P = 0.05) for MINT1, 1.70 (P = 0.006) for MINT31, 2.22 (P = 0.001) for p14ARF, and 1.51 (P = 0.05) for p16INK4a. Concurrent methylation of two or more genes of the CIMP-associated subset (MINT1, MINT31, p14ARF and p16INK4a) defined a group of cases with markedly reduced overall survival and hazard ratio was 3.22 (P < 0.0001 in multivariate analyses).

Conclusions: CIMP is associated with poor survival in advanced colorectal cancer patients.

Fig. 1

Representative examples of methylation analysis by COBRA (top) or by MSP (bottom). For COBRA, the arrows point to band shifts after restriction enzyme digestion, which indicate the presence of methylation. For MSP, amplification using M primers represents methylation. P1 to P13, 13 different cancers; PC, positive control (DNA from the RKO or SW48 cell lines).

Fig. 2

Kaplan-Meier curves of survival by methylation of individual genes. A, MINT1; B, MINT31; C, p16^INK4a; D, p14^ARF; solid line, unmethylated; stippled line, methylated.

Fig. 3

Kaplan-Meier curve of survival by methylation of multiple genes. Solid line, methylation of 0 to 1 gene; stippled line, methylation of >1 gene.