Metayer, Catherine

Abstract Little is known about the interaction between constitutive and somatic genetic variation in carcinogenesis. Genome-wide association studies (GWAS) of childhood acute lymphoblastic leukemia (ALL) have identified 6 genetic associations, including the single nucleotide polymorphism (SNP) rs3731217, located between the CDKN2A and CDKN2B genes. Somatic deletions of this chromosome 9p21.3 region are common in cancer, and occur in ~30% of childhood ALL. We replicated the rs3731217 association with childhood ALL in Hispanics (P = 0.021) via a GWAS of 323 childhood ALL cases and 454 controls, using the Illumina OmniExpress SNP array. SNP imputation analysis across chromosome 9p21.3 revealed a stronger association at SNP rs3731249 (P = 6.4x10-4; OR = 2.77), a low-frequency missense variant in exon 2 (p.Ala148Thr) of CDKN2A (MAFcontrols = 2.1%). These associations were replicated in an additional 378 ALL cases and 536 controls using Taqman assays, where the missense variant again had a greater magnitude of effect (Prs3731249 = 6.5x10-3; OR rs3731249 = 2.00 versus Prs3731217 = 0.029; OR rs3731217= 1.38). We assessed the effect of rs3731249 on risk of other cancers with known chr9p21.3 GWAS associations, but observed no association between this variant and either adult glioblastoma or melanoma risk, suggesting an ALL-specific role for rs3731249. We hypothesized that cases heterozygous for rs3731249 may show allelic imbalance, with preferential loss of the wildtype (WT) allele when hemizygous chr9p21.3 deletions occur in leukemic cells. To test this, we developed a novel method termed “SMART-ddPCR” (Somatic Mutation Allelic Ratio Test using Droplet Digital PCR). Concentration of risk and WT alleles was measured by ddPCR using the rs3731249 Taqman assay in diagnostic bone marrow (i.e. tumor) DNA from 35 known heterozygotes. Allelic imbalance due to somatic CDKN2A alterations was identified in 17 cases, of which 14 had a higher risk:WT allele ratio (P = 0.006, 1-sided binomial significance test), thus demonstrating preferential loss of WT rs3731249 and retention of the missense allele. Subclonal heterogeneity in CDKN2A copy number was also evident, suggesting that CDKN2A alterations are secondary events in leukemogenesis. Preferential allelic imbalance towards the rs3731249 risk allele in ALL tumor DNA provides strong evidence of an important role for this missense variant in leukemogenesis. We show, for the first time, a direct relationship between constitutive and somatic genetic variants underlying ALL development, suggesting that such constitutive variants can provide an additional substrate for selection during tumor evolution. Disclosures No relevant conflicts of interest to declare.

Abstract Genome-wide association studies (GWAS) have identified SNPs in six genes that are associated with childhood acute lymphoblastic leukemia (ALL). A lead SNP was found to occur on chromosome 9p21.3, a region that is deleted in 30% of childhood ALLs, suggesting the presence of causal polymorphisms linked to ALL risk. We used SNP genotyping and imputation-based fine-mapping of a multiethnic ALL case–control population (Ncases = 1,464, Ncontrols = 3,279) to identify variants of large effect within 9p21.3. We identified a CDKN2A missense variant (rs3731249) with 2% allele frequency in controls that confers three-fold increased risk of ALL in children of European ancestry (OR, 2.99; P = 1.51 × 10−9) and Hispanic children (OR, 2.77; P = 3.78 × 10−4). Moreover, of 17 patients whose tumors displayed allelic imbalance at CDKN2A, 14 preferentially retained the risk allele and lost the protective allele (PBinomial = 0.006), suggesting that the risk allele provides a selective advantage during tumor growth. Notably, the CDKN2A variant was not significantly associated with melanoma, glioblastoma, or pancreatic cancer risk, implying that this polymorphism specifically confers ALL risk but not general cancer risk. Taken together, our findings demonstrate that coding polymorphisms of large effect can underlie GWAS “hits” and that inherited polymorphisms may undergo directional selection during clonal expansion of tumors. Cancer Res; 75(22); 4884–94. ©2015 AACR.