Methylation of WNT target genes AXIN2 and DKK1 as robust biomarkers for recurrence prediction in stage II colon cancer

R Kandimalla,R Jover,X Llor,J P Medema,M Andreu,A Goel,J F Linnekamp,A Castells,S Van Hooff

doi:10.1038/oncsis.2017.9

Abstract

Stage II colon cancer (CC) still remains a clinical challenge with patient stratification for adjuvant therapy (AT) largely relying on clinical parameters. Prognostic biomarkers are urgently needed for better stratification. Previously, we have shown that WNT target genes AXIN2, DKK1, APCDD1, ASCL2 and LGR5 are silenced by DNA methylation and could serve as prognostic markers in stage II CC patients using methylation-specific PCR. Here, we have extended our discovery cohort AMC90-AJCC-II (N=65) and methylation was analyzed by quantitative pyrosequencing. Subsequently, we validated the results in an independent EPICOLON1 CC cohort (N=79). Methylation of WNT target genes is negatively correlated to mRNA expression. A combination of AXIN2 and DKK1 methylation significantly predicted recurrences in univariate (area under the curve (AUC)=0.83, confidence interval (CI): 0.72–0.94, P<0.0001) analysis in stage II microsatellite stable (MSS) CC patients. This two marker combination showed an AUC of 0.80 (CI: 0.68–0.91, P<0.0001) in the EPICOLON1 validation cohort. Multivariate analysis in the Academic Medical Center (AMC) cohort revealed that both WNT target gene methylation and consensus molecular subtype 4 (CMS4) are significantly associated with poor recurrence-free survival (hazard ratio (HR)methylation: 3.84, 95% CI: 1.14–12.43; HRCMS4: 3.73, 95% CI: 1.22–11.48). CMS4 subtype tumors with WNT target methylation showed worse prognosis. Combining WNT target gene methylation and CMS4 subtype lead to an AUC of 0.89 (0.791–0.982, P<0.0001) for recurrence prediction. Notably, we observed that methylation of DKK1 is high in BRAF mutant and CIMP (CpG island methylator phenotype)-positive cancers, whereas AXIN2 methylation appears to be associated with CMS4. Methylation of AXIN2 and DKK1 were found to be robust markers for recurrence prediction in stage II MSS CC patients. Further validation of these findings in a randomized and prospective manner could pave a way to identify poor prognosis patients of stage II CC for AT.

Highlights

Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide
There is an urgent need for molecular biomarkers for a better stratification of patients who will be benefitted by the adjuvant chemotherapy, especially in microsatellite stable (MSS) stage II CRC patients
Methylation of AXIN2, DKK1, APCDD1, ASCL2 and LGR5 was determined by methylation-specific PCR (MSP) in our previous

Summary

Introduction

Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide. In 2016, there were estimated 95 270 newly diagnosed colon cancer (CC) cases, and 49 190 deaths in the United States. Survival of patients is closely related with the tumor stage at the time of diagnosis as 5-year relative survival rates range from 65% for all stages, 90% local, 71% regional and 13% in distant disease (SEER Cancer Statistics Review, 1975–2010; http://seer.cancer.gov/ csr/1975_2010). The prognosis of low-risk stage II patients who have undergone curative surgery is relatively good as relapse occurs only in a small fraction of them.. The prognosis of low-risk stage II patients who have undergone curative surgery is relatively good as relapse occurs only in a small fraction of them.3,4 Identifying these relapse-prone patients could significantly contribute to the optimization of treatment selection. Based on the presented clinical risk factors, patients are divided into low or high risk and the AT is advised in the latter case. This may lead to the under or over treatment of low- and high-risk groups. The association of CpG island methylator phenotype (CIMP), microsatellite instability (MSI) and mutations in BRAF and KRAS with the prognosis has been studied extensively and it has been found that MSI patients have a good prognosis and should not receive AT in stage II. there is an urgent need for molecular biomarkers for a better stratification of patients who will be benefitted by the adjuvant chemotherapy, especially in microsatellite stable (MSS) stage II CRC patients

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