Adjuvant chemotherapy after radical surgery is the standard treatment for patients with node-positive colorectal cancer (CRC; Stage III) and has been shown to improve survival. However, its use in patients with node-negative CRC (Stage II), which are approximately one-third of all cases, has been controversial and is not routinely recommended. Yet, up to 30% of Stage II patients relapse within 5 years of surgery.1 To date, there are no recommended pathological or molecular markers for predicting which Stage II patients are likely to relapse.2
For this reason, we have read with utmost interest the article by Andersen et al.3 The authors investigated the association between genomic alterations (in terms of copy-number variations) and risk of recurrence in patients with Stages II and III CRC and identified a copy-number loss on chromosome 16 (16p13.2) that was predictive of metastatic recurrence in microsatellite-stable (MSS) Stages II and III CRCs.3
We would like to contribute on the subject of CRC prognostic biomarkers by briefly reporting on our studies, in which we assessed the possibility of predicting recurrence for the most critical group of CRC patients, i.e., Stage II, by investigating the prognostic implications of promoter CpG island hypermethylation status, a common epigenetic alteration in sporadic CRC.4 The subset of sporadic cancers with widespread promoter methylation constitutes a distinct phenotype, often referred to as the CpG island methylator phenotype (CIMP or CIMP high),4 which has been associated with proximal colon location, microsatellite instability (MSI) and high frequency of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations.5 However, more than half of the tumors with high CpG island methylation are MSS, and it is still unclear whether these MSS-methylated tumors share the improved prognosis of MSI cancers.6 A marked controversy surrounds the prognostic implications of CIMP as some studies suggest an adverse effect of CIMP on survival, whereas others seem to downplay the prognostic value of CIMP or even question its favorable significance.7 These different views might be explained by differences in the populations studied or in the methodologies and in the marker panels used to determine CIMP status.
To evaluate whether changes in the methylation status of CpG islands of specific marker genes in MSS node-negative CRCs can predict occurrence of relapse, the methylation status of 42 early stage patients was investigated by examining promoter methylation of five CIMP-related markers.8 All the patients underwent surgical resection at Fondazione IRCCS Istituto Nazionale dei Tumori di Milano in the period April 1998 to February 2007, and they either relapsed (with local or distant metastasis) within 5 years (23 patients, median time to relapse: 18 months, range 3–38) or remained relapse-free for at least 5 years after surgery (19 patients, median relapse-free time: 96 months, range 60–135). All the patients were MSS. Written informed consent was obtained from all patients, and the study was approved by the Institutional Review Board. The age at surgery ranged between 47 and 83 (median, 67) in relapsed patients, and between 30 and 83 in relapse-free patients (median, 65). Genomic DNA was extracted from fresh frozen specimens using the QIAamp DNA Mini Kit-Tissue Protocol (Qiagen, Chatsworth, CA). DNA was then subjected to bisulfite modification and methylation-specific polymerase chain reaction amplification; methylation changes were examined using the five classical CIMP-related markers, p16, MINT1, MINT2, MINT31 and hMLH1, as described in other studies.8
Methylation levels were stratified into three classes by the number of observed methylated loci (Nmet): low methylation (for Nmet less than or equal to 2), intermediate methylation (for Nmet equal to 3) and full methylation (for Nmet greater than 3). A comparison of methylation levels in relapsed and relapse-free patients was performed with the SAS software by using a logistic regression model adjusted by age (Version 9.2.; SAS Institute, Cary, NC). Our results suggest a trend of increasing number of methylated loci in relapse-free patients that was not observed in relapsed patients (Table 1). The predictive capability of this variable was rather satisfactory showing an area under the receiver operating characteristic curve of 0.73 (95% confidence interval: 0.58–0.88). Our finding is consistent with the observed better outcome for CIMP-high tumors across all the stages (I–IV)9 and with the recent finding that DNA methylation is lower in recurrent Stage III proximal CRC.10 These results emphasize the potential association of methylation levels at five CIMP-related loci with disease recurrence in MSS Stage II CRC. Methylation at these loci could be further investigated for its possible use as a prognostic variable in Stage II CRC to identify which patients are more likely to relapse and might benefit from adjuvant chemotherapy. As this analysis was based on a small number of cases, this observation should be taken with caution and needs to be confirmed in larger sample sets.