Pleiotropic Analysis of Cancer Risk Loci on Esophageal Adenocarcinoma Risk

The incidence of esophageal adenocarcinoma (EA) has increased substantially over the past four decades in western countries (1). Most EA arises within Barrett's esophagus, a metaplastic transformation of the lower esophageal lining (1). Gastroesophageal reflux (GERD), high body mass index (BMI), and tobacco use are well-established risk factors for both EA and Barrett's esophagus, although with some variations in the magnitudes of these associations. These risk factors, together with low consumption of fruits and vegetables, may explain about 70% of the EA cases (see references in ref. 1). Genetic factors are thought to play an etiologic role based on familial clustering (1). In addition, results from the Barrett's and Esophageal Adenocarcinoma Genetic Susceptibility Study (BEAGESS) support a shared genetic susceptibility of EA and Barrett's esophagus (2). In further analysis of the BEAGESS data, an estimated 25% of the variation in EA risk is due to common genetic variants (1). However, from genome-wide association studies (GWAS) to date, only 7 loci have been identified to be associated with risk of EA or Barrett's esophagus (2, 3), and the effects of the individual genetic variants are small (2, 3). Motivated by studies investigating pleiotropic associations of GWAS-identified variants, some of which work has identified additional risk-associated loci for colorectal, endometrial, and pancreatic cancers (see references in ref. 4), we investigated in BEAGESS whether cancer-GWAS–identified variants could also influence the risk of EA and Barrett's esophagus.

Details of the design and methods of BEAGESS have been described elsewhere (2). In brief, this ancillary study included 1,504 EA and 2,384 Barrett's esophagus case patients and 2,163 control participants, all of European descent and for whom genome-wide genotyping and imputation were conducted and phenotype covariate data were available. We identified 407 SNPs that have previously been associated with one or more cancer sites at a P value < 5 × 10⁻⁸ in the National Human Genome Research Institute (NHGRI) GWAS catalog as of March 2014 (5). Genotyping was done using the Illumina Human Omni1-Quad platform. Imputation was performed using MaCH and Minimac software and a European reference panel from the 1000 Genomes project (2). This analysis included 387 of the 407 SNPs that had minor allele frequencies (MAF) >1% and passed genotyping and imputation quality control (e.g., imputation r² ≥ 0.3; ref. 2). We investigated associations with EA, Barrett's esophagus, and for these conditions combined (EA/Barrett's esophagus). ORs and 95% confidence intervals (CI) were calculated using unconditional logistic regression of case status on additive SNP genotype score, with adjustments for age, sex, and the first four principal components analysis eigenvectors (2). We performed stratum-specific analyses by smoking status (never, former, or current), smoking intensity (never, <30 pack-years, or ≥30 pack-years), BMI (<25, 25–<30, or ≥30 kg/m²), and history of reflux/heartburn (yes, no), and tested for the statistical significance of the interaction (product) term. Some SNPs included in the NHGRI catalog are in linkage disequilibrium with each other. Thus, we calculated “P values adjusted for correlated tests” to account for multiple testing of correlated SNPs (6). The adjusted P values (P_adj) < 0.05 are considered statistically significant. Analyses were conducted using SAS 9.2 (SAS Inc.) and R. All P values are two sided.

After correcting for multiple testing, none of the 387 SNPs were associated with risk of EA, Barrett's esophagus, or EA/Barrett's esophagus combined (Table 1 and Supplementary Table S1). No evidence was seen for effect modification by smoking status or intensity, BMI, or reflux/heartburn history (data not shown).

Studies investigating pleiotropic effects of GWAS-identified risk variants have revealed additional risk loci for endometrial, colorectal, and pancreatic cancer, but not for estrogen receptor (ER)–negative breast cancer (ER-positive cancers were not investigated), prostate cancer, or non-Hodgkin lymphoma (4, 7–9). Some studies, including those of non-Hodgkin lymphoma, endometrial cancer, and colorectal cancer, investigated cancer GWAS hits, whereas studies of pancreatic cancer, prostate cancer, and ER-negative breast cancer investigated cancer and noncancer GWAS hits (4, 7–9). Our investigation based on cancer GWAS SNPs is not supportive of shared genetic susceptibility between EA/Barrett's esophagus and other cancer sites. To our knowledge, this is the first study to investigate pleiotropic associations of cancer GWAS loci on risks of EA and Barrett's esophagus. Although our study sample size is moderate, the combined number of EA and Barrett's esophagus cases is larger than that of most previous studies on cancer pleiotropy (4, 7–9). For SNPs with MAF of 0.3 (the average MAF of all tested SNPs), we had 80% statistical power to detect an OR of 1.21 per minor allele for analyses on EA/Barrett's esophagus combined, with a Bonferroni-corrected type I error rate of 5%. Given that nearly one-half (47%) of the NHGRI-cataloged cancer-GWAS SNPs were from studies of more common cancers (Supplementary Table S1), it may be fruitful to repeat this analysis when additional GWAS loci identified from less common cancers become available.

No potential conflicts of interest were disclosed.

Conception and design: D.O. Stram, J. Lagergren, L. Bernstein, N.C. Bird, T.L. Vaughan, A.H. Wu

Development of methodology: D.O. Stram, L. Bernstein

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): W. Ye, J. Lagergren, N.J. Shaheen, L.J. Murray, L.J. Hardie, W.-H. Chow, H.A. Risch, D.A. Corley, D.C. Whiteman, L. Bernstein, N.C. Bird, T.L. Vaughan, A.H. Wu

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): E. Lee, D.O. Stram, W.E. Ek, S. MacGregor, P. Gharahkhani, L.J. Murray, H.A. Risch, D.A. Corley, D.M. Levine, A.H. Wu

Writing, review, and/or revision of the manuscript: E. Lee, D.O. Stram, W.E. Ek, L.E. Onstad, S. MacGregor, P. Gharahkhani, W. Ye, J. Lagergren, N.J. Shaheen, L.J. Hardie, M.D. Gammon, W.-H. Chow, H.A. Risch, D.A. Corley, D.M. Levine, D.C. Whiteman, L. Bernstein, N.C. Bird, T.L. Vaughan, A.H. Wu

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): L.E. Onstad, M.D. Gammon, T.L. Vaughan

Study supervision: L. Bernstein

The authors thank Dr. Yvonne Romero for her contributions to the BEACON consortium. They also thank Drs. Christian Abnet and Matthew Buas for their helpful discussion and suggestions regarding analyses.

This work was primarily funded by a U.S. NIH grant (R01CA136725) to T.L. Vaughan and D.C. Whiteman. E. Lee, D.O. Stram, W.E. Ek, L.E. Onstad, W. Ye, J. Lagergren, N.J. Shaheen, L.J. Murray, L.J. Hardie, M.D. Gammon, H.A. Risch, D.A. Corley, D.M. Levine, D.C. Whiteman, L. Bernstein, N.C. Bird, T.L. Vaughan, and A.H. Wu were supported by R01CA136725. T.L. Vaughan was also supported by grant K05CA124911. D.C. Whiteman was supported by a Research Fellowship from the National Health and Medical Research Council of Australia (NHMRC). S. MacGregor was supported by an Australian Research Council Future Fellowship. Additional funding sources for individual studies included in the BEACON GWAS, and for BEACON investigators, have been acknowledged.

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