Introduction: High intake of alcohol and low intake of folate have been associated with gene promoter methylation. Increased promoter methylation of tumor suppressor genes plays an important role in renal cell cancer (RCC). On the other hand, however, alcohol intake is associated with a decreased risk of RCC risk in most prospective studies. We therefore investigated whether alcohol and dietary folate intakes were associated with gene promoter methylation in RCC.

Methods: The Netherlands Cohort Study (NLCS) with case-cohort design included 120,852 subjects aged 55-69 years in 1986. Diet was measured with a food-frequency questionnaire. After 20.3 years of follow-up, paraffin-embedded tumor blocks were collected from 454 incident RCC cases, of which 80% had clear cell (cc) histology. Methylation-specific PCR (MSP) was used to analyze promoter CpG island methylation in tumor DNA. Genes were selected that were known to be involved in ccRCC. Furthermore, genes were selected exhibiting 1) promoter CpG island methylation in ccRCC cell lines and not in normal kidney tissue in a genome-wide methylation screen (MBD-affinity massive parallel sequencing) and 2) ccRCC-specific down-regulation in the TCGA data. In total, we identified 14 genes (CDO1, CFTR, CSPG4, FST, FZD10, GAL, GRE1, LAD1, NEFH, NEURL, RASGFR2, SFN, SFRP1, VHL) as candidate markers. We computed a sum score of methylated genes and divided the ccRCC cases into those with low- (0-18% of the genes methylated), intermediate- (20-45%) and high- (46-100%) methylated tumors. Multivariable analyses were conducted using Cox regression analyses, stratified according to methylation sum score and included 4,439 subcohort members and 300 ccRCC cases.

Results: Prevalence of gene promoter methylation was on average 36%, but varied from 7.8% (VHL) to 96.5% (SFN).

Increasing intakes of alcohol were associated with decreased risk in all ccRCC cases, but not statistically significant: multivariable adjusted HR (95% CI) for ≥15g alcohol/day vs. 0 g/day 0.79 (0.54-1.16), and p for trend 0.44. In strata of ccRCC according to methylation sum score no significant heterogeneity was observed, although the HR for ≥ 15g alcohol/day versus 0 g/day was significantly decreased in ccRCC with a high methylation sum score: 0.36 (95% CI 0.14-0.93), p for trend, 0.14.

Dietary intake of folate was not associated with ccRCC risk: HR for the quintile of highest intake versus lowest intake: 1.07 (CI, 0.70-1.64) and p for trend, 0.59. Between strata of ccRCC cases according to methylation sum score no significant heterogeneity was observed. There was no effect-modification of alcohol and dietary folate intake on overall ccRCC risk, nor in strata according to methylation sum score.

Conclusion: Our findings show that alcohol and dietary folate intake are not associated with ccRCC risk, neither overall nor in strata according to methylation sum score.

Citation Format: Leo J. Schouten, Ivette A.G. Deckers, Piet A. van den Brandt, Patricia M.M.B. Soetekouw, Marcella M.M.L. Baldewijns, Manon van Engeland. Alcohol and dietary folate intake and gene promoter methylation in clear-cell renal cell cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1272. doi:10.1158/1538-7445.AM2014-1272