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L.A, A.H, K.B.J. G.T and S.J.C supervised genotyping of samples.
L.A, P.K, R.Z.S, C.S.F, K.B.J, C.K, K.Y, S.J.C, P.H and R.N.H contributed to the design and execution of statistical analysis.
LA, S.J.C, P.H and R.N.H wrote the first draft of the manuscript.
R Z.S, C.S.F, G.M.P, A.A.A, H.B.B, M.G, K.H, E.J.J, A.L, W.Z, D.A, W.B, C.D.B, F.B, S.B, J.E.B, P.M.B, F.C, F.C-C, S.C, M.C, M.A, E.J.D, J.W.F, S.G, J.M.G, E.L.G, M.G, C.A.G, G.H, S.E.H, M.H, E.A.H, D.J.H, R.J, M.J, R.K, A.P.K, C.K, R.C.K, D.L, M.M, R.R.M, D.S.M, S.H.O, K.O, A.V.P, P.H.M.P, A.R, E.R, H.A.R, X.S, D.T, S.K.V.D.E, J.V, J.W, B.M.W, H.Y and A.Z conducted the epidemiologic studies and contributed samples to the PanScan GWAS and/or replication.
All authors contributed to the writing of the manuscript.
We conducted a two-stage genome-wide association study (GWAS) of pancreatic cancer, a cancer with one of the poorest survival rates worldwide. Initially, we genotyped 558,542 single nucleotide polymorphisms in 1,896 incident cases and 1,939 controls drawn from twelve prospective cohorts plus one hospital-based case-control study. In a combined analysis adjusted for study, sex, ancestry and five principal components that included an additional 2,457 cases and 2,654 controls from eight case-control studies, we identified an association between a locus on 9q34 and pancreatic cancer marked by the single nucleotide polymorphism, rs505922 (combined P=5.37 × 10-8; multiplicative per-allele odds ratio (OR) 1.20; 95% CI 1.12-1.28). This SNP maps to the first intron of the ABO blood group gene. Our results are consistent with earlier epidemiologic evidence suggesting that people with blood group O may have a lower risk of pancreatic cancer than those with groups A or B.
Pancreatic cancer shows amongst the highest mortality rates of any cancer, with a five year relative survival rate of less than 5%1,2. There is currently no effective screening test for the malignancy, and by the time of initial diagnosis, metastatic disease is commonly present. Established risk factors include a family history of pancreatic cancer, a medical history of diabetes type II and cigarette smoking3. Studies have also suggested an increased risk of pancreatic cancer within families with hereditary pancreatitis4,5. It has also been estimated that a small proportion of pancreatic cancers are due to highly penetrant germ-line mutations6. These studies suggested genetic contribution to pancreatic cancer, although there has been limited success in resolving common variants associated to this disease. We report here a genome-wide association study (GWAS) to resolve common variants associated to pancreatic cancer.
We conducted a GWAS in 1,896 cases and 1,939 controls of incident pancreatic cancer cases drawn from twelve prospective cohorts plus one hospital-based case-control study (American Cancer Society Cancer Prevention Study-II (CPS II)7 Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study (ATBC)8 European Prospective Investigation into Cancer and Nutrition Study (EPIC)9 CLUE II10 Health Professionals Follow-up Study (HPFS)11 New York University Women’s Health Study (NYUWHS)12 Nurses’ Health Study (NHS)11 Physicians’ Health Study I (PHS)11 Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO)13 Shanghai Men’s and Women’s Health Study (SMWHS)14,15 Women’s Health Initiative (WHI)16 the Women’s Health Study (WHS)17 and the Mayo Clinic Molecular Epidemiology of Pancreatic Cancer Study18; Supplemental Table 1). Eight case-control studies participated in the independent ‘Fast-Track’ replication phase of 2,457 cases and 2,654 controls (the University of Toronto19 University of California San Francisco20 Johns Hopkins University, MD Anderson Cancer Center21 PACIFIC Study of Group Health and Northern California Kaiser Permanente, Memorial Sloan-Kettering Cancer Center22 Yale University23 and the Mayo Clinic Molecular Epidemiology of Pancreatic Cancer Study18; Supplemental Table 2).
After quality control assessment of genotypes assayed using the HumanHap500 chip (Illumina, San Diego, CA), 558,542 SNPs were available for analysis. A logistic regression model was fit for genotype trend effects (1 d.f.) adjusting for study, age, sex, ancestry and the top five principal components of population stratification (Online Methods). The quantile-quantile plot (QQ plot) does not demonstrate a systematic deviation from the expected distribution, minimizing the likelihood of systematic genotype error or bias due to underlying population substructure (Supplemental Figure 1). The results of the GWAS are shown in Figure 1a. Because of the potential for survivor bias in case-control studies due to rapid mortality, we also analyzed the GWAS for cohort studies only as shown in Figure 1b (i.e., excluding Mayo subjects).
We conducted a rapid follow-up scan, or “Fast Track”, of SNPs from three regions in eight case-control studies (four hospital based and four population based). At least two SNPs per region were ranked among the lowest 25 p-values in the initial GWAS; 1.) chromosome 9q34, which includes the ABO gene (rs505922, rs495828, rs657152 and rs630014; ranked 2, 3, 8 and 17); 2.) chromosome 7q36, which includes Sonic Hedgehog (SHH), (rs167020, rs172310, and rs288746; ranked 6, 10 and 89); and 3.) a gene desert on chromosome 15q14 (rs8028529, rs4130461 and rs4459505, ranked 1, 18 and 26) (Table 1).
In a combined analysis of individuals of European background24 the strongest association with pancreatic cancer below the threshold for genome-wide significance25 was observed for a locus on chromosome 9q34, located within the first intron of ABO, a well-described blood group gene, marked by rs505922 (P=5.37 × 10−8; trend model; heterozygous odds ratio [ORHet] of 1.20; 95% CI 1.12-1.28 and homozygous odds ratio [ORHom] of 1.44; 95% CI 1.26-1.63). A comparable result was observed when all ethnic groups were included in the first stage (P=2.61 × 10−8; Supplemental Table 3). In the case-control replication set, we genotyped a second SNP, rs687621 (r2=1 with rs505922 in HapMap CEU and r2=0.91 in Stage 2 control individuals), located 12 kb centromeric in intron 2; the results provided confirmation of the locus (P=1.57×10−4 in the second-stage case-control studies only). In the combined analysis, a comparably strong signal was observed for rs630014 (P=1.58×10−7; ORHet 0.85, ORHom 0.71), which resides within 500 bp of rs505922 and is in linkage disequilibrium (r2=0.52 in HapMap CEU and 0.40 in PanScan GWAS European control individuals). After adjusting for rs505922, none of the remaining SNPs in ABO were significant at the P<0.01 level. The SNPs reside in a haplotype block that encompasses the proximal promoter and introns 1 and 2 (Figure 2).
Blood groups were first described by Karl Landsteiner in 1900 but the structure of the ABO antigens and their biosynthesis remained elusive until after 1950. The ABO gene encodes a glycosyltransferase that catalyzes the transfer of carbohydrates to the H antigen, forming the antigenic structure of the ABO blood groups. The proteins encoded by the A and B alleles of the ABO gene differ minimally in amino acid sequence but catalyze the transfer of different carbohydrates (N-acetylgalactosamine or galactose) onto the H antigen to form the A or B antigens. Individuals with the O blood group do not produce either the A or B antigens due to a single base deletion.
Our findings are notable because multiple studies, mainly from the 1950s and 1960s reported an association between ABO blood type and gastrointestinal cancers, strongest for gastric cancer but also for pancreatic cancer26,27. The protective allele (T) for rs505922 is in complete linkage disequilibrium (LD) (r2=1.0) with the O allele of the ABO locus, consistent with earlier reports showing increased risk of gastric and pancreatic cancer for individuals of the A and B blood groups. It is plausible that the single base deletion that generates the O blood group underlies the association signal but further mapping and laboratory work is required to determine which variant(s) account for the observed association.
Genetic variation in the first intron of the ABO gene has also been associated with circulating levels of serum tumor necrosis factor alpha (TNF-alpha) levels28 circulating soluble intracellular adhesion molecule 1 (sICAM-1)29 and plasma levels of alkaline phosphatase30. Although higher TNF-alpha levels are associated with the common allele of rs505922, protective for pancreatic cancer in our study, the data concerning the relationship between blood groups and TNF-alpha levels are inconsistent28. Furthermore, this region could be important for regulating circulating sICAM-1 levels as rs507666 and rs505922 (located 170 bps apart) were recently reported to be associated with circulating ICAM-1 levels29. Also, SNPs in the ABO locus, including rs657152, have been associated with plasma levels of liver derived alkaline phosphatase30. Lastly, altered ABO antigen expression has been observed in primary and metastatic pancreatic cancer as compared to normal pancreatic tissues31.
For rapidly fatal conditions, case-control studies are prone to distortion because they disproportionately include survivors. For variants unrelated to survival, case-control data are suitable for discovery and replication of risk-related markers. However, for variants related to survival, case-control studies yield biased estimates of the association with pancreatic cancer risk. ABO variants appear unrelated to survival and show strong and similar signals in both cohort and case-control data.
We observed an association at the genome-wide level (P=1.76 × 10−7) with SHH among cohorts that was not replicated in follow-up in case-control studies (P=0.12), raising three possibilities: the cohort finding is due to chance, SHH is related to both survival and to risk or that the SNPs failed to replicate because of chance (Table 1). Because there is substantial evidence that SHH plays a role in pancreatic carcinogenesis, further work is required to investigate this region32.
Pancreatic cancer is among the deadliest cancers with mortality rates approaching incidence rates1. Given there are few known risk factors, improved diagnostics and a finer understanding of the molecular pathogenesis are urgently needed. Our findings have identified the contribution of genetic variation in the ABO locus of 9q34 to pancreatic carcinogenesis, a finding that supports an epidemiologic observation first made half a century ago and recently confirmed33. We are currently conducting a GWAS in the eight studies of stage 2 in this study and anticipate that this will bring the identification of additional loci associated to pancreatic cancer. The discovery of additional genetic risk variants for this highly lethal cancer could contribute to novel risk stratification and improvements in prevention, early detection and therapeutic approaches to pancreatic cancer.
The authors gratefully acknowledge the energy and contribution of our late colleague, Robert Welch. Additional acknowledgements are in the Supplemental Note.