Onel, Kenan

Abstract Purpose: DROSHA, DGCR8, and DICER1 regulate miRNA biogenesis and are commonly mutated in cancer. Although DGCR8 and DICER1 germline pathogenic variants (GPV) cause autosomal dominant tumor predisposition, no association between DROSHA GPVs and clinical phenotypes has been reported. Experimental Design: After obtaining informed consent, sequencing was performed on germline and tumor samples from all patients. The occurrence of germline DROSHA GPVs was investigated in large pediatric and adult cancer datasets. The population prevalence of DROSHA GPVs was investigated in the UK Biobank and Geisinger DiscovEHR cohorts. Results: We describe nine children from eight families with heterozygous DROSHA GPVs and a diagnosis of pineoblastoma (n = 8) or Wilms tumor (n = 1). A somatic second hit in DROSHA was detected in all eight tumors analyzed. All pineoblastoma tumors analyzed were classified as miRNA processing–altered 1 subtype. We estimate the population prevalence of germline DROSHA loss-of-function variants to be 1:3,875 to 1:4,843 but find no evidence for increased adult cancer risk. Conclusions: This is the first report of DROSHA-related tumor predisposition. As pineoblastoma and Wilms tumor are also associated with DICER1 GPVs, our results suggest that the tissues of origin for these tumors are uniquely tolerant of general miRNA loss. The miRNA processing–altered 1 pineoblastoma subtype is associated with older age of diagnosis and better outcomes than other subtypes, suggesting DROSHA GPV status may have important clinical and prognostic significance. We suggest that genetic testing for DROSHA GPVs be considered for patients with pineoblastoma, Wilms tumor, or other DICER1-/DGCR8-related conditions and propose surveillance recommendations through research studies for individuals with DROSHA GPVs.

Abstract Background Recent genome wide association studies (GWAS) of Hodgkin lymphoma (HL) have identified several associations at both HLA and non-HLA loci. However, much of HL heritability remains unexplained. Methods To identify novel risk loci, we performed a meta-analysis of 3 HL GWAS including a total of 1,810 cases and 7,879 controls. Results were replicated in an independent set of 1,163 cases and 2,580 controls, for a total of 3,097 and 11,097 cases and controls combined, respectively. participants in discovery and replication stages were of European descent. quality control and imputation we conducted a meta-analysis addressing 1,004,829 variants (λ= 1.10, λ1000= 1.03). Associations between SNP genotypes and HL risk were evaluated under a log-additive model of inheritance adjusting for sex, study center and significant principal components to control for population stratification. We performed an analysis with all HL cases and then conducted stratified analyses by histological subtype (classical, nodular sclerosis and mixed cellularity), age at diagnosis (nodular sclerosis among those diagnosed at 15- 35 years in all studies, and those diagnosed at 35 and older years in the European Study only) and EBV tumor status (negative and positive). We then used a bioinformatic approach (FunciSNP) to identify potential functional variants associated with HD risk correlated with risk loci of interest. We extracted publically available ENCODE data on biofeatures to identify potential functional motifs associated with the index SNP or correlated SNPs. Finally, we measured expression levels of the two alternative mRNA transcripts in lymphoblastoid cell lines (LCLs) from 49 post-therapy HL patients and 25 unaffected controls. RT-PCR was carried out in triplicate. Relative expression levels were calculated relative to TBP as housekeeping gene. Linear models were used to assess correlation between genotype and TCF3expression levels. Results The meta-analysis identified a novel susceptibility variant at chromosome 19p13.3 (rs1860661) associated with HL risk (Odds Ratio [OR]= 0.78, P=2.0*10-8, I2=0%). variant is located in intron 2 of TCF3 (also known as E2A), a regulator of B- and T-cell lineage commitment. was also significantly associated with HL (OR= 0.85, P=0.002) in the replication series of 1,281 cases and 3,218 controls. the combined analysis consisting of the discovery and replication sets, rs1860661was strongly associated with HL (OR=0.81,=3.5*10-10), with no evidence of heterogeneity between contributing studies (Phom=0.41, I2=0%). The number of G alleles defined by rs1860661 was significantly associated with a reduced risk of each HL subtype except EBV positive HL. rs1860661 and two correlated SNPs, rs10413888 (r2=0.90) and rs8103453 (r2=0.89) identified by FunciSNP analysis map in or near marks of open chromatin and in DNAse hypersensitivity sites in TCF3 in CD20+ B cell lines., the protective minor alleles of these SNPs as defined by the G-G-G haplotype map to the binding sites for ZBTB7a (rs10413888 and rs1860661) and (rs8103453) transcription factors, likely improving the binding efficiency to the sites which may result in increased transcription rates of TCF3. TCF3 is encoded by two alternative transcripts (E12 and E47). Higher expression levels of TCF3-E47, whose transcription start site is located close to rs1860661, was associated with the rs1860661-G allele in controls (P=0.02), but not in HL patients (P=0.22). Conclusion/Discussion TCF3 is essential for the commitment of lymphoid progenitors to both B-cell and T-cell lineage development. A molecular and phenotypic hallmark of classical HL is the loss of the B-cell phenotype in HRS cells, including lack of demonstrable B-cell receptor and most B-cell specific markers such as CD19 or CD20. HRS cells have a low level of TCF3, particularly homodimers of the isoform E47, due to expression of the ABF-1 and ID2 inhibitors that bind to TCF3. Thus, higher TCF3 levels in HRS precursor cells may lead to enhanced retention of the B cell phenotype, thereby conferring a protective effect. These data suggest a link between the 19p13.3 locus including TCF3 and HL risk, indicating that TCF3 could be relevant to HL etiology and pathogenesis. Disclosures: Link: Genentech: Consultancy; Millenium: Consultancy; Pharmacyclics: Consultancy; Spectrum: Consultancy.

Abstract The first two authors and last two authors contributed equally. Genome-wide association studies (GWAS) have identified risk loci for Acute Lymphoblastic Leukemia (ALL), Chronic Lymphoblastic Leukemia (CLL) and Non-Hodgkin Lymphoma, however an Acute Myeloid Leukemia (AML) GWAS has not been published to date. We performed a GWAS to identify AML and Myelodysplastic Syndrome (MDS) risk loci using a nested case-control study design in the DISCOVeRY-BMT cohorts which includes almost 2000 AML and MDS patients as cases and 2813 unrelated donors as controls. Genotyping was performed using the Illumina Human OmniExpress BeadChip and imputed using the Haplotype Reference Consortium, yielding > 8 million high-quality variants for statistical analysis. Logistic regression models with AML (de novo AML with normal cytogenetics, de novo AML with abnormal cytogenetics and therapy-related AML) and MDS cases and European American healthy donor controls adjusted for age and sex were used to test the association of each SNP with disease status. To identify the strongest association signal with disease we conducted a summary statistic SNP-based association analysis (ASSET) using non-overlapping AML and MDS cases implemented in R statistical software. ASSET uses an exhaustive search for SNPs with small but common pleiotropic effects across groups of traits while accounting for the multiple tests required by the subset search, as well as any shared controls between groups. This approach allowed us to further investigate the heterogeneity within AML subtypes and to gain increased power by pooling subtypes that show pleiotropic effects. ASSET genome wide (GW) significance is defined as P<5.0x10-8, however, we present results of each subset analysis. ASSET analyses identified an association of T allele at rs12203592, Interferon Regulatory Factor 4 (IRF4), with increased risk of de novo AML and MDS (Figure 1) (OR=1.37; 95% CI, 1.25-1.51, Pmeta=1.59x10-10). The variant has been reported significant in GWAS of skin pigmentation, hair color, non-melanoma skin cancer, squamous cell carcinoma, actinic keratosis, and childhood ALL; GW case-control studies of neuroblastoma and breast cancer show suggestive association signals (P<5 x 10-6). IRF4 belongs to the IRF family of transcription factors and is a key regulator of differentiation stages in hematopoiesis. The T allele at rs12203592 is associated with significantly increased expression of IRF4 in whole blood and lung tissue and in vitro mouse studies have shown the T allele to be associated with higher levels of IRF4 transcription. rs12203592 is <80bp from an IRF4 transcription start site and in an important position within NF-κB motifs in multiple blood and immune cell lines, supporting the hypothesis that this SNP modulates NF-κB repression of IRF4 expression. Another variant in the IRF4 regulatory region, rs62389423 (A allele), showed a putative association with subsets of de novo AML and MDS (OR=1.36; 95% CI,1.21-1.52, Pmeta=1.2x10-7). Although not in linkage disequilibrium with rs12203592, the A allele has also been previously associated with susceptibility to skin cancer and melanoma in multiple GWAS. Analysis of 596 UKbiobank CLL cases and >300,000 controls, shows the A allele correlates with a 50% increased risk of CLL (95% CI, 1.32-1.73, P= 2.4x10-9).Several other GWAS also show an association between additional IRF4 variants and CLL. Analyses in individual subtypes also revealed an intronic variant, rs10098598, in RAD21 to be associated with de novo AML (OR=2.18; 95% CI, 1.60-2.97; Pmeta=8.9x10-7). RAD21, on8q24, a region known to be associated with multiple cancer types, is involved in DNA double-stranded break repair and in chromatid cohesion in mitotic cells. Loss of function would theoretically lead to chromosomal instability and tumorigenesis. Components of the cohesion complex (including RAD21) are somatically mutated in ~12% of de novo AML and MDS patients, where it is frequently an early event. We provide the first GW evidence of association between a common variant and AML susceptibility. This SNP has been shown to be associated with multiple phenotypes, suggesting that there are pleiotropic effects at work. Our RAD21 finding is consistent with the role of cohesion in leukemogenesis and provides some evidence of its role in de novo AML susceptibility. Replication and further subset analyses with genome-wide data in >2000 AML cases are ongoing. Disclosures Griffiths: Alexion Inc.: Honoraria, Research Funding; Astex/Otsuka Pharmaceuticals: Honoraria, Research Funding; Celgene, Inc: Honoraria, Research Funding; Novartis, Inc.: Research Funding; Pfizer, Inc.: Research Funding. McCarthy:Bristol Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria.

Abstract Both genome wide association studies (GWAS) of common variation and exome wide association studies (EXWAS) of rare variation have successfully identified disease susceptibility variants for a variety of diseases. One GWAS of inherited susceptibility to Acute Myeloid Leukemia (AML) has been conducted, but no EXWAS have been performed to measure risk of AML attributable to low-frequency constitutional genetic variation. We performed the first EXWAS of risk of AML as a nested case-control study in the DISCOVeRY-BMT (Determining the Influence of Susceptibility Conveying Variants Related to one-Year mortality after BMT) cohorts. The DISCOVeRY-BMT parent study examined transplant-related mortality in leukemia patients undergoing unrelated donor allogeneic BMT. To identify low frequency variants and genes contributing to increased susceptibility to AML we used genotype data from the Illumina HumanExome BeadChip typed in the DISCOVeRY-BMT cohorts; the HumanExome BeadChip contains 242,901 variants, which are mainly protein-coding variants. The optimal sequence kernel association test (SKAT-O) was used to analyze gene-level associations with risk of AML. These gene-based tests evaluate the cumulative effects of multiple single gene variants on risk of AML. Analyses were performed in all European American AML cases and two subtypes: 1) de novo AML, 2) de novo AML with normal cytogenetics. Models were adjusted for age at transplant and principal components to control for population stratification. For gene-based tests at least 2 variants with minor allele frequency (MAF) ≤ 5%, were required to be present in the gene. This yielded a total of 13,687 genes tested, and a Bonferroni corrected significance level of P<3.65 x 10-6. Association tests were performed in 1,189 AML cases reported to CIBMTR 2000-08 (Cohort 1) and 327 AML cases reported to CIBMTR from 2009-11 (Cohort 2). Controls in Cohorts 1 (n=1,986) and 2 (n= 515) were 10/10 HLA-matched unrelated donors who passed a comprehensive medical exam and deemed healthy. We used metaSKAT to combine Cohorts 1 and 2 and obtain p-values of association with AML. We present the results of gene-level tests significant in both cohorts. The likely pathogenicity of these variants was determined in silico using SIFT, PolyPhen and MutationTaster. Patient characteristics are in Table 1. DNMT3A, on chromosome 2, was associated in the gene-based test with risk of AML (Pmeta=1.70x10-9, Table 2). Three missense variants at MAF <1% comprise both overall AML and de novo AML gene-based association: exm177559 (Asn->Ser), exm177507 (Arg->His), and exm177543 (Arg->Trp). Normal cytogenetics de novo AML gene-based assocations consisted of only 2 of these variants: exm177559 and exm177507 (Table 2). While prevalence of exm177507 is <1% for all AML cases, in de novo AML with normal cytogenetics the MAF was higher at 3%. The other 2 variants had a MAF<1% irrespective of subtype. Somatically, DNMT3A is most frequently mutated in hematologic malignancies, with >30% of de novo AML cases with a normal karyotype and >10% of MDS patients having DNMT3A mutations. Although these are germline gene associations all three of the variants found have been reported somatically in hematologic malignancies. In 200 AML cases from The Cancer Genome Atlas (TCGA) p.R882H (represented as exm177507 on the exome chip) was a frequent somatic mutation (25%). Exm177543 (p.R635W) and exm177559 (p.N501S) are reported in the Catalogue of Somatic Mutations in Cancer (COSMIC) as somatic mutations involved in hematopoietic and lymphoid tissue in both cell lines and humans. Exm177507 and exm177543 show evidence of pathogenicity in all three in silico tools, while exm177559 was reported as deleterious and disease causing by Sift and MutationTaster, respectively. Our results show that multiple potentially pathogenic missense germline variants in DNMT3A comprise the gene-based association with AML, specifically de novo AML with normal cytogenetics. Given the functional nature of these variants it is possible germline risk stratification could be informative in determining AML risk, and subsequently development of AML harboring DNMT3A mutations. Confirmation of these findings in additional cohorts could have implications for individualized risk screening, prediction and prognosis. Additional cytogenetic subgroup analyses, including treatment-related AML, are underway. Disclosures Hahn: Novartis: Equity Ownership; NIH: Research Funding. McCarthy:Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; The Binding Site: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gamida Cell: Honoraria, Membership on an entity's Board of Directors or advisory committees. Sucheston-Campbell:NIH/NCI: Research Funding.

Abstract #the authors contributed equally to this work While survival outcomes after HLA-matched unrelated donor (URD) blood and marrow transplant (BMT) have significantly improved over the last two decades, about 40% of patients die before 1-year post URD BMT. Previously we performed a genome-wide association study (GWAS) named DISCOVeRY-BMT (Determining the Influence of Susceptibility COnveying Variants Related to one-Year mortality after BMT) to investigate the contributions of common genetic variants on survival outcomes. To address the specific contributions of low frequency (≤ 1%) exonic variants on survival outcomes, we used the DISCOVeRY-BMT cohorts typed with the Illumina HumanExome BeadChip containing 242,901 variants, which are mainly protein-coding variants. We used a gene-based test (the optimal sequence kernel association test (SKAT-O)) to evaluate the cumulative effects of multiple genetic variants within a gene on overall survival (OS), transplant-related mortality (TRM), and disease-related mortality (DRM) 1 year after URD BMT. SKAT-O measures the overall significance of the gene and allows the variants to have different directions and magnitude of effects. We used a 2-stage study design: SKAT-O analysis was carried out in Cohort 1 consisting of 1,972 AML, ALL or MDS patients reported to the Center for International Blood and Marrow Transplant Research from 2000 to 2008 and their 2,006 10/10 HLA-matched URD. Genes with a suggestive association with each outcome (P < 1×10-3) entered the final meta-analysis including Cohort 1 and an independent cohort (Cohort 2), consisting of 503 recipients and 520 10/10 HLA-matched URD from 2009 to 2011. The analysis included recipients and URD of European descent due to the low frequency of other populations. All association models included age at BMT, diagnosis (AML, ALL, MDS), disease status at BMT, cell source (peripheral blood, marrow), year of BMT, and principal components from EIGENSTRAT to control for population stratification. Only coding variants (missense and nonsense) with minor allele frequency ≤ 1% were included in our analysis, which resulted in > 12,000 genes tested in our gene-based analysis in recipients and donors. Exome-wide significance was set at Pmeta< 4.10×10-6 after Bonferroni correction for the total number of genes tested. The likely pathogenicity of these variants was determined in silico using SIFT and PolyPhen. In recipients, we identified OR51D1, a member of the olfactory receptor gene family, as significantly associated with both OS and TRM. The OS association with ORD51D1 is driven by TRM as the same six missense variants contribute to both TRM and OS, but not to DRM. In donors, four (ALPP, EMID1, SLC44A5, LRP1), one (HHAT), and two (LYZL4, NT5E) genes were significantly associated with OS, TRM, and DRM, respectively (Table). ALPP encodes an alkaline phosphatase. SLC44A5 is a member of the solute carrier gene family. LRP1 (low density lipoprotein receptor-related protein 1) encodes an endocytic receptor involved in several cellular processes, including intracellular signaling, lipid homeostasis, and clearance of apoptotic cells. HHAT encodes an enzyme that acts within the hedgehog secretory pathway, which is involved in hematologic malignancy. LYZL4 is a member of a family of lysozyme-like genes, which have a protective role in host defense. The encoded protein of NT5E, CD270, is used as a determinant of lymphocyte differentiation. The likely pathogenicity of several of the functional variants in all significant genes identified was predicted to be both (possibly or probably) damaging and deleterious (Table), thus indicating that the amino acid substitution of the variant may not be well tolerated. One damaging variant in LRP1 has been identified as a somatic mutation in hematopoietic and lymphoid tissue. Our study is the first analysis of the low-frequency coding variant contribution to URD BMT survival outcomes. We found that the missense variants contributing to many of these significant gene associations shows evidence of pathogenicity and thus it is biologically plausible these variants are contributing to survival outcomes. Further confirmation of these findings, and studies of the functional consequences of protein-coding changes in these genes, may provide more individualized risk prediction and prognosis as well as alternative donor selection strategies. Table. Table. Disclosures McCarthy: Gamida Cell: Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; The Binding Site: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hahn:Novartis: Equity Ownership; NIH: Research Funding. Sucheston-Campbell:NIH/NCI: Research Funding.

Key PointsIKZF1 associations with high-risk B-ALL may differ by age and sex. A novel variant on chromosome 14, rs189434316, is associated with over a 3.5-fold risk of normal cytogenetic B-ALL.

AbstractEpidemiologic studies show an increased risk of non‐Hodgkin lymphoma (NHL) in patients with autoimmune disease (AD), due to a combination of shared environmental factors and/or genetic factors, or a causative cascade: chronic inflammation/antigen‐stimulation in one disease leads to another. Here we assess shared genetic risk in genome‐wide‐association‐studies (GWAS).Secondary analysis of GWAS of NHL subtypes (chronic lymphocytic leukemia, diffuse large B‐cell lymphoma, follicular lymphoma, and marginal zone lymphoma) and ADs (rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis). Shared genetic risk was assessed by (a) description of regional genetic of overlap, (b) polygenic risk score (PRS), (c)"diseasome", (d)meta‐analysis.Descriptive analysis revealed few shared genetic factors between each AD and each NHL subtype. The PRS of ADs were not increased in NHL patients (nor vice versa). In the diseasome, NHLs shared more genetic etiology with ADs than solid cancers (p = .0041). A meta‐analysis (combing AD with NHL) implicated genes of apoptosis and telomere length.This GWAS‐based analysis four NHL subtypes and three ADs revealed few weakly‐associated shared loci, explaining little total risk. This suggests common genetic variation, as assessed by GWAS in these sample sizes, may not be the primary explanation for the link between these ADs and NHLs.

Key Points We conducted the first exome-wide association study between germ line variant genotype and survival outcomes after unrelated-donor BMT. A number of novel genes were found to significantly affect survival outcomes.

Key PointsCandidate SNP associations with survival outcomes after URD transplant are most likely false-positive findings. Over 85% of candidate SNPs are not linked to a biochemical function; of those that are, about half are not linked to the candidate gene.

Nodular sclerosing Hodgkin lymphoma (NSHL) is a distinct, highly heritable Hodgkin lymphoma subtype. We undertook a genome-wide meta-analysis of 393 European-origin adolescent/young adult NSHL patients and 3315 controls using the Illumina Human610-Quad Beadchip and Affymetrix Genome-Wide Human SNP Array 6.0. We identified 3 single nucleotide polymorphisms (SNPs) on chromosome 6p21.32 that were significantly associated with NSHL risk: rs9268542 (P = 5.35 × 10−10), rs204999 (P = 1.44 × 10−9), and rs2858870 (P = 1.69 × 10−8). We also confirmed a previously reported association in the same region, rs6903608 (P = 3.52 × 10−10). rs204999 and rs2858870 were weakly correlated (r2 = 0.257), and the remaining pairs of SNPs were not correlated (r2 < 0.1). In an independent set of 113 NSHL cases and 214 controls, 2 SNPs were significantly associated with NSHL and a third showed a comparable odds ratio (OR). These SNPs are found on 2 haplotypes associated with NSHL risk (rs204999-rs9268528-rs9268542-rs6903608-rs2858870; AGGCT, OR = 1.7, P = 1.71 × 10−6; GAATC, OR = 0.4, P = 1.16 × 10−4). All individuals with the GAATC haplotype also carried the HLA class II DRB1*0701 allele. In a separate analysis, the DRB1*0701 allele was associated with a decreased risk of NSHL (OR = 0.5, 95% confidence interval = 0.4, 0.7). These data support the importance of the HLA class II region in NSHL etiology.