Crouch, Simon

Abstract We previously identified five single nucleotide polymorphisms (SNPs) at four susceptibility loci for diffuse large B-cell lymphoma (DLBCL) in individuals of European ancestry through a large genome-wide association study (GWAS). To further elucidate genetic susceptibility to DLBCL, we sought to validate two loci at 3q13.33 and 3p24.1 that were suggestive in the original GWAS with additional genotyping. In the meta-analysis (5662 cases and 9237 controls) of the four original GWAS discovery scans and three replication studies, the 3q13.33 locus (rs9831894; minor allele frequency [MAF] = 0.40) was associated with DLBCL risk [odds ratio (OR) = 0.83, P = 3.62 × 10−13]. rs9831894 is in linkage disequilibrium (LD) with additional variants that are part of a super-enhancer that physically interacts with promoters of CD86 and ILDR1. In the meta-analysis (5510 cases and 12 817 controls) of the four GWAS discovery scans and four replication studies, the 3p24.1 locus (rs6773363; MAF = 0.45) was also associated with DLBCL risk (OR = 1.20, P = 2.31 × 10−12). This SNP is 29 426-bp upstream of the nearest gene EOMES and in LD with additional SNPs that are part of a highly lineage-specific and tumor-acquired super-enhancer that shows long-range interaction with AZI2 promoter. These loci provide additional evidence for the role of immune function in the etiology of DLBCL, the most common lymphoma subtype.

Abstract Background: We previously identified 5 SNPs at 4 susceptibility loci for diffuse large B-cell lymphoma (DLBCL) in individuals of European ancestry through a large genome-wide association study (Cerhan et al, Nat Gen 2014;46:1233-8); however, much of the heritability remains unexplained. To further elucidate genetic susceptibility to DLBCL, we sought to validate 2 loci at 3q13.33 and 3p24.1 that were suggestive in the original report. Methods: We selected two SNPs rs9831894 (3q13.33) and rs6773363 (3p24.1) for de novo replication from studies at the Mayo Clinic, MD Anderson, and Memorial Sloan Kettering. Logistic regression was used to estimate odds ratios (ORs), using a log-additive model, and adjusting for age, gender and significant eigenvectors for each genotyping center separately. For both SNPs, we then conducted a meta-analysis of all replication studies (n=3) and our original GWAS studies (n=4), encompassing in total 5662 cases and 9237 controls for rs9831894, and 5510 cases and 12,817 controls for rs6773363. Meta-analysis was conducted using the fixed-effects inverse variance method based on the β estimates and standard errors from each study. Results: In a meta-analysis of the 4 original GWAS scans (3856 cases and 7666 controls), rs9831894 (MAF=0.40) was associated with DLBCL risk (OR=0.84, P=4.52 x 10-9) and this replicated in a meta-analysis of the 3 studies with de novo genotyping (OR=0.80, P=4.17 x 10-5); the overall meta-analysis showed a strong association with DLBCL risk (OR=0.83, P=3.62 x 10-13). This locus maps near a plausible candidate gene, CD86, a protein coding gene and a member of the immunoglobulin superfamily that encodes a type I membrane protein. Binding of CD86 with cytotoxic T-lymphocyte-associated protein 4 negatively regulates T-cell activation and diminishes the immune response, while binding of CD86 with CD28 antigen is a costimulatory signal for activation of the T-cell. In a meta-analysis of the same 4 GWAS scans, rs6773363 (MAF=0.45) was tentatively associated with DLBCL risk (OR=1.17, P= 3.68 x 10-7) and this replicated in a meta-analysis of the studies with de novo genotyping (OR=1.27, P=3.78 x 10-7); the overall meta-analysis showed a strong association with DLBCL risk (OR=1.20, P=2.31 x 10-12). This locus also maps near a plausible candidate gene, eomesodermin (EOMES), a transcription factor crucial for embryonic development of the central nervous system and also putatively involved in T-cell differentiation in viral infection defense. Conclusion: In this follow-up analysis of our initial GWAS, we have identified two additional loci associated with risk of DLBCL, the most common lymphoma subtype. These loci provide additional evidence for the role of immune function in the etiology of DLBCL. Citation Format: Geffen Kleinstern, Michelle Hildebrand, Vijai Joseph, Sonja I. Berndt, Hervé Ghesquières, James McKay, Sophia S. Wang, Alexandra Nieters, David Cox, Alain Monnereau, Angela R. Brooks-Wilson, Qing Lan, Mads Melbye, Rebecca D. Jackson, Lauren R. Teras, Mark P. Purdue, Claire M. Vajdic, Demetrius Albanes, Anne Zeleniuch-Jacquotte, Simon Crouch, Yawei Zhang, Susan L. Slager, Xifeng Wu, Karin E. Smedby, Gilles Salles, Christine F. Skibola, Nathaniel Rothman, Stephen J. Chanock, James R. Cerhan. Inherited variants at 3q13.33 and 3p24.1 influences risk of diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 221.

Objective Determinants of the increased risk of diffuse large B-cell lymphoma (DLBCL) in SLE are unclear. Using data from a recent lymphoma genome-wide association study (GWAS), we assessed whether certain lupus-related single nucleotide polymorphisms (SNPs) were also associated with DLBCL. Methods GWAS data on European Caucasians from the International Lymphoma Epidemiology Consortium (InterLymph) provided a total of 3857 DLBCL cases and 7666 general-population controls. Data were pooled in a random-effects meta-analysis. Results Among the 28 SLE-related SNPs investigated, the two most convincingly associated with risk of DLBCL included the CD40 SLE risk allele rs4810485 on chromosome 20q13 (OR per risk allele=1.09, 95% CI 1.02 to 1.16, p=0.0134), and the HLA SLE risk allele rs1270942 on chromosome 6p21.33 (OR per risk allele=1.17, 95% CI 1.01 to 1.36, p=0.0362). Of additional possible interest were rs2205960 and rs12537284. The rs2205960 SNP, related to a cytokine of the tumour necrosis factor superfamily TNFSF4, was associated with an OR per risk allele of 1.07, 95% CI 1.00 to 1.16, p=0.0549. The OR for the rs12537284 (chromosome 7q32, IRF5 gene) risk allele was 1.08, 95% CI 0.99 to 1.18, p=0.0765. Conclusions These data suggest several plausible genetic links between DLBCL and SLE.

Abstract Background: Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype and is clinically aggressive. Relatives of DLBCL patients are at elevated risk for DLBCL. In small genome-wide association studies (GWAS) in which all NHL subtypes were combined, no conclusive loci for DLBCL have been previously identified in individuals of European background, whereas a recent study conducted in East Asia identified a locus at 3q27. Methods: To identify genetic susceptibility loci for DLBCL, we conducted a meta-analysis of three new GWAS and one prior scan, totaling 3,857 DLBCL cases and 7,666 controls of European ancestry. Imputation was used to combine all the data using the 1000 Genomes Project release v3 and IMPUTE2. Based on these findings, we selected 6 promising SNPs for genotyping in an additional 202 cases and 3,431 controls, which were then included in a second meta-analysis. SNPTEST was used to estimate odds ratios (ORs), using the additive model and adjusting for age, gender and significant eigenvectors. Results: In our multi-stage analysis, five independent SNPs in four loci achieved genome-wide significance marked by rs116446171 at 6p25.3 (EXOC2; OR=2.33, P=1.60x10-20), rs13255292 and rs4733601 at 8q24.21 (PVT1; OR=1.23, P=4.86x10-12 and OR=1.18, P=1.63x10-8, respectively), rs2523607 at 6p21.33 (HLA-B; OR=1.34, 3.35x10-9) and rs2681416 at 3q13.33 (CD86; OR=1.20, P=6.04x10-9). The two 8q24.21 SNPs displayed minimal linkage disequilibrium (r2=0.03) and conditional analysis supported their independence. We did not observe a notable signal for a locus previously reported for DLBCL on 3q27 in East Asia, rs6773854 (OR=1.06, P=0.81). We estimated that common SNPs, including but not limited to the loci discovered in this study, explain approximately 16% of the variance for DLBCL risk overall. Discussion: The susceptibility locus at 6p25.3 maps near a plausible DLBCL candidate gene, EXOC2 (exocyst complex component 2), which interacts with Ral proteins, and disruption of the Ral-exocyst regulatory node impacts cellular and developmental processes associated with malignant transformation and progression. The two 8q24.21 variants are approximately 1Mb telomeric to the 8q24 region linked with multiple cancers including CLL. Both variants are positioned in close proximity to PVT1, which is a non-coding RNA implicated in the MYC activation, and MYC is known to be deregulated in some DLBCLs. The locus at 3q13.33 maps to CD86 (B7-2), a B-7 family member that along with CD80 (B7-1) is essential in cognate interactions between T- and B-lymphocytes; both play key roles in immunodeficiency, autoimmune diseases, and in anti-tumor immunity. Finally, we had multiple signals in the HLA region, the strongest of which was at 6p21.33 near HLA-B. HLA-B encodes the HLA class I heavy chain paralogue, which heterodimerizes with a light chain (β2 microglobulin) and plays a central role in presenting intracellularly processed self or foreign antigens to CD8+ cytotoxic T lymphocytes. Class I molecules have been extensively linked to the risk of a variety of immune-mediated diseases and cancers including DLBCL. Conclusions: These data provide substantial new evidence for genetic susceptibility to this B-cell malignancy, and point towards pathways involved in immune recognition and immune function in the pathogenesis of DLBCL. Citation Format: James R. Cerhan, Sonja I. Berndt, Joseph Vijai, Hervé Ghesquières, James McKay, Sophia S. Wang, Zhaoming Wang, Meredith Yeager, Alexandra Nieters, David Cox, Alain Monnereau, Christopher Flowers, Anneclaire J. De Roos, Angela R. Brooks-Wilson, Qing Lan, Gianluca Severi, Mads Melbye, Rebecca D. Jackson, Lauren R. Teras, Mark Purdue, Claire Vajdic, Demetrius Albanes, Kimberly A. Bertrand, Anne Zeleniuch-Jacquotte, Simon Crouch, Yawei Zhang, Paolo Vineis, Susan L. Slager, Karin E. Smedby, Gilles Salles, Christine F. Skibola, Nathaniel Rothman, Stephen J. Chanock, on behalf of the NHL GWAS Project. Genome-wide association study identifies multiple susceptibility loci for diffuse large B-cell lymphoma. [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 LB-272. doi:10.1158/1538-7445.AM2014-LB-272

Abstract Background. Follicular lymphoma (FL) is an indolent B-cell malignancy with a variable clinical course that may transform to more aggressive forms of lymphoma. Previous modestly powered GWAS have reported multiple FL susceptibility loci in the HLA class I and II regions at 6p21.32-33, but novel non-HLA susceptibility loci and the genetic architecture of FL associations in the HLA region remain to be elucidated. Methods. We conducted the largest GWAS of FL to date consisting of 2,142 cases and 6,221 controls of European ancestry from 22 studies scanned on the Illumina OmniExpress. After imputation of common SNPs using IMPUTE2 and 1,000 Genomes Project v3 data, we conducted a meta-analysis of these data with two previous FL GWAS (n=586 cases, 1,537 controls) and replicated top hits in an additional 629 cases and 4,283 controls. Expression quantitative trait loci (eQTL) analyses were performed to evaluate the effects of associated variants on gene expression. Classical HLA allele imputations and stepwise regression analyses are underway to further characterize the HLA associations. Results. In the joint meta-analysis, the strongest association with FL risk was observed in the HLA region, where a large number of SNPs showed genome-wide significance (P<5x10-8 to P= 1.84x10-84), particularly in HLA class II at 6p21.32 where several HLA eQTLs were identified (P < 0.05). Outside HLA, we identified four novel loci associated with FL at 11q23.3 (CXCR5, rs4938573; P=3.14x10-15), 11q24.3 (ETS1, rs4937362; P=3.33x10-9), 3q13.33 (CD86, rs2681416; P=9.79x10-11) and 3q28 (LPP, rs6444305, P=2.55x10-8). These novel susceptibility loci were located in four biologically relevant genes involved in immune regulation: CXCR5 and its ligand, CXCL13, are involved in guiding B-cells into the B-cell zones of secondary lymphoid organs as well as T-cell migration. ETS1 is expressed in lymphoid cells and regulates immune cell function including differentiation, survival, and proliferation. CD86 and CD80 are classic members of the B7 costimulatory pathway that is important in maintaining immune function, suppression of autoimmunity and antitumor surveillance. LPP encodes a LIM domain-containing protein of the zyxin family and participates in cell adhesion, cell migration, proliferation and transcription dynamics. Conclusions. This large GWAS provides further support for the important role of common genetic variation in non-HLA genes and further evidence of the key role that HLA immune-regulatory genes play in the pathogenesis of FL. Citation Format: Christine F. Skibola, Sonja I. Berndt, James R. Cerhan, Zhaoming Wang, Joseph Vijai, Lucia Conde, Paul de Bakker, Sophia S. Wang, Claire M. Vajdic, Brenda M. Birmann, Susan L. Slager, James McKay, Paige M. Bracci, Alexandra Nieters, Qing Lan, Angela R. Brooks-Wilson, Martha S. Linet, Demetrius Albanes, John J. Spinelli, Roel C.H. Vermeulen, Mark P. Purdue, Meredith Yaeger, Lauren R. Teras, Silvia de Sanjose, Alain Monnereau, Simon Crouch, Jia Nee Foo, Henrik Hjalgrim, Gianluca Severi, Brian K. Link, Kimberly A. Bertrand, Yawei Zhang, Karin E. Smedby, Stephen J. Chanock, Nathaniel Rothman, NHL GWAS Consortium. Meta-analysis of genome-wide association studies identifies novel susceptibility loci for follicular lymphoma. [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 5072. doi:10.1158/1538-7445.AM2014-5072