We conducted this study to examine prognostic effect of CDKN2A
(p16) promoter methylation and loss of CDKN2A (p16) independent of the CpG island methylator phenotype (CIMP), utilizing 902 clinically and molecularly-annotated colorectal cancers. CDKN2A
methylation has been associated with CIMP, which has been associated with patient prognosis.21, 31, 32
However, none of the previous studies14-30
has examined prognostic effect of CDKN2A
methylation or loss of CDKN2A (p16) in colorectal cancer independent of CIMP status. Furthermore, sample sizes were relatively small (N<200) in all but one study (N=404).16
Our resource of the 902 colorectal cancers identified in the two prospective cohort studies has enabled us to precisely estimate the frequency of colorectal cancers with a specific molecular feature (CDKN2A
methylation, CIMP-high, etc.). The large number of cases has also provided a sufficient power in our multivariate logistic regression analysis and survival analysis. In our current study, we have found that CDKN2A
methylation or loss of CDKN2A (p16) is not independently associated with prognosis of colorectal cancer patients. On the other hand, CDKN2A
methylation is independently associated with CIMP, inactive PTGS2 (COX-2) and BRA
F mutation. This study provides robust and useful data on CDKN2A
promoter methylation and loss of expression in colorectal cancer.
Prognostic significance of CDKN2A
promoter methylation or loss of CDKN2A (p16) in colorectal cancer14-30
or normal-appearing mucosa adjacent to colorectal cancer50
has been studied (). Without analysis of CIMP that is a potential confounder, some studies reported that CDKN2A
methylation was significantly associated with worse prognosis in multivariate analysis;17, 23
however, those studies were limited by small sample sizes (N=90 and N=151, respectively). In contrast, a study on 182 stage IV cases in a five-arm trial21
or another study (N=104)19
did not show an independent prognostic effect of CDKN2A
methylation, which is in agreement with our current data.
With regard to loss of CDKN2A (p16) expression, one study (N=117) reported an association between CDKN2A (p16) loss and high mortality,26
whereas two larger studies by Lam et al.29
(N=194) and by Norrie et al.16
(total N=404; N=161 for immunohistochemistry) found no prognostic role of p16 loss, in agreement with our current data. Essentially, there has been no definitive evidence to support an independent prognostic role of CDKN2A
methylation or loss of expression in colorectal cancer.
It should be noted that small studies are more prone to “publication bias” than large studies. This phenomenon of publication bias happens because studies with null findings have a higher likelihood of being unwritten and unpublished compared to those with significant results. Large studies with adequate statistical power are less prone to publication bias even with null data. Therefore, we should weigh more on large-scale studies when we evaluate the published literature on prognostic significance of any biomarker.
Examining molecular changes or prognostic factors is important in cancer research.51-57
A molecular classification based on MSI and CIMP status is increasingly important, because MSI and CIMP reflect global genomic and epigenomic aberrations in tumor cells.58
LINE-1 methylation also reflects global DNA methylation level. MSI, CIMP, LINE-1 methylation, and related molecular events such as BRAF
mutation, are important potential confounders, because these molecular features have been associated with prognosis in large-scale studies.21, 31, 32, 59
Our current study is the only one study that assessed the prognostic significance of CDKN2A
methylation and loss of CDKN2A (p16) independent of these potential confounders.
As expected from the likely causal link, CIMP-high is associated with CDKN2A
promoter methylation. According to our previous study,32
CIMP-high appears to be an independent predictor of low colon cancer-specific mortality. It has been well known that two features which are associated with each other do not always demonstrate similar independent prognostic effects. This is well exemplified by BRAF
mutation and MSI in colon cancer. Studies have shown that BRAF
mutation is associated with MSI-high.9, 52, 58 BRAF
mutation in colon cancer has been associated with poor prognosis,32, 52, 60
whereas MSI-high has been associated with good prognosis.61
Therefore, it is not surprising to find the significant prognostic role of CIMP-high and no independent prognostic role of CDKN2A
There are limitations in this study. For example, data on cancer treatment were limited. Nonetheless, it is unlikely that chemotherapy use substantially differed according to CDKN2A methylation or loss of CDKN2A (p16) in tumor, since such data were not available for treatment decision making. In addition, beyond cause of mortality, data on cancer recurrences were not available in these cohorts. Nonetheless, given the median survival for metastatic colon cancer was approximately 10-12 months during much of the time period of this study, colorectal cancer-specific survival may be a reasonable surrogate of colorectal cancer-specific outcome.
There are advantages in utilizing the database of the two prospective cohort studies, the Nurses' Health Study and the Health Professionals Follow-up Study to examine prognostic significance of CDKN2A
methylation or loss of CDKN2A (p16). Anthropometric measurements, family history, other clinical information, pathologic and tumor staging data, and tumoral molecular features were prospectively collected, blinded to patient outcome. For each immunohistochemical marker, an agreement study on a proportion of cases from a large number of cases was used to assess the interobserver reproducibility of scoring. Cohort participants who developed cancer were treated at hospitals throughout the U.S., and thus more representative colorectal cancers in the general U.S. population than patients in one to several academic hospitals. There were no demographic difference between cases with tumor tissue analyzed and those without tumor tissue analyzed.34
Finally, our rich tumor database enabled us to simultaneously assess pathologic and tumoral molecular features and control for confounding by a number of tumoral molecular alterations.
In conclusion, in our large database of colorectal cancers, CDKN2A methylation or loss of CDKN2A (p16) is not independently associated with prognosis. On the other hand, CDKN2A methylation is independently associated with CIMP status, low PTGS2 expression. Further studies are needed to elucidate the exact mechanisms by which CDKN2A methylation or loss of CDKN2A (p16) leads to colorectal cancer development.