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Follicular lymphoma (FL) is the second most common non-Hodgkin lymphoma in the Western world. FL cell-intrinsic and cell-extrinsic factors influence FL biology and clinical outcome. To further our understanding of the genetic basis of FL, we performed whole-exome sequencing of 23 highly purified FL cases and 1 transformed FL case and expanded findings to a combined total of 114 FLs. We report recurrent mutations in the transcription factor STAT6 in 11% of FLs and identified the STAT6 amino acid residue 419 as a novel STAT6 mutation hotspot (p.419D/G, p.419D/A, and p.419D/H). FL-associated STAT6 mutations were activating, as evidenced by increased transactivation in HEK293T cell-based transfection/luciferase reporter assays, heightened interleukin-4 (IL-4) -induced activation of target genes in stable STAT6 transfected lymphoma cell lines, and elevated baseline expression levels of STAT6 target genes in primary FL B cells harboring mutant STAT6. Mechanistically, FL-associated STAT6 mutations facilitated nuclear residency of STAT6, independent of IL-4-induced STAT6-Y641 phosphorylation. Structural modeling of STAT6 based on the structure of the STAT1-DNA complex revealed that most FL-associated STAT6 mutants locate to the STAT6-DNA interface, potentially facilitating heightened interactions. The genetic and functional data combined strengthen the recognition of the IL-4/JAK/STAT6 axis as a driver of FL pathogenesis.

Follicular lymphoma (FL) constitutes the second most common non-Hodgkin lymphoma in the western world. FL carries characteristic recurrent structural genomic aberrations. However, information regarding the coding genome in FL is still evolving. Here, we describe the results of massively parallel exome sequencing and single nucleotide polymorphism 6.0 array genomic profiling of 11 highly purified FL cases, and 1 transformed FL case and the validation of selected mutations in 102 FL cases. We report the identification of 15 novel recurrently mutated genes in FL. These include frequent mutations in the linker histone genes HIST1H1 B-E (27%) and mutations in OCT2 (also known as POU2F2; 8%), IRF8 (6%), and ARID1A (11%). A subset of the mutations in HIST1H1 B-E affected binding to DNMT3B, and mutations in HIST1H1 B-E and in EZH2 or ARID1A were largely mutually exclusive, implicating HIST1H1 B-E in epigenetic deregulation in FL. Mutations in OCT2 (POU2F2) affected its transcriptional and functional properties as measured through luciferase assays, the biological analysis of stably transduced cell lines, and global expression profiling. Finally, multiple novel mutated genes located within regions of acquired uniparental disomy in FL are identified. In aggregate, these data substantially broaden our understanding of the genomic pathogenesis of FL.

Introduction Follicular lymphoma (FL) constitutes the second most common non-Hodgkin’s lymphoma in the Western world. FL carries characteristic recurrent structural genomic aberrations. However despite recent advances, knowledge regarding the coding genome in FL is still evolving and is currently incomplete. Methods To further our understanding of the genetic basis of follicular lymphoma (FL), we used solution exon capture of sheared and processed genomic DNA isolated from FACS-sorted lymphomatous B-cells and paired CD3+ T-cells isolated from twenty three cases of FL and one case of DLBCL (which was transformed from prior FL), followed by paired-end (96-101 base pair read length per side) massively parallel sequencing. The sequence data were characterized by a mean depth of coverage of 41, and 90% of bases in the target region were covered by at least 10 reads. Bioinformatics pipelines developed by our bioinformatics core served as the primary data source to nominate candidate mutated genes in downstream data analysis. Results The bioinformatics pipeline nominated 711 distinct candidate mutations in 24 FL cases. Sanger sequence validation confirmed 39 recurrently (≥ 2 FL cases) mutated genes. Genes with confirmed mutations in ≥ 2 FL cases in the discovery cohort were subsequently selectively expanded into a combined FL validation cohort of 114 cases. In addition to frequent mutations in MLL2, CREBBP, BCL2, TNFRSF14, EZH2, OCT2, ARID1A, IRF8 and MEF2B, we here report novel mutations in STAT6 in FL. STAT6 mutations were identified in 11% (12/114) of FL and predominantly affected the DNA binding domain (DBD; comprising STAT6 amino acids 268-430). Two FL cases each carried two distinct STAT6 mutations, presumably targeting both alleles. Of interest, the majority of FL-associated STAT6 mutations affected a single amino acid codon (codon 419), resulting in the STAT6 mutants p.419D>D/G or p.419D>D/H. These FL-associated STAT6 mutations are distinct from mutations previously described in primary mediastinal B-cell lymphoma (PMBCL). Given the involvement of STAT6 in signal transduction pathways activated by multiple cell surface receptors, as well as the recently described involvement of STAT6 in antiviral innate immunity (involving an interaction between the STAT6 DBD and the protein STING), we are currently exploring functional consequences of the novel STAT6 mutations in FL and cell line models. Conclusion We report identification of somatic mutations in STAT6 in 11% of FL. These mutations predominantly affected the STAT6 DNA binding domain. We identify a novel STAT6 mutation hotspot in STAT6 codon 419 (p.419D>D/G or p.419D>D/H). Disclosures: Lebovic: Genentech: Speakers Bureau; Allos/Spectrum: Speakers Bureau; Celgene: Speakers Bureau; Onyx: Speakers Bureau.

Abstract 147 Introduction: Follicular lymphoma (FL) constitutes the second most common non-Hodgkin's lymphoma in the Western world. FL carries characteristic recurrent structural genomic aberrations. However despite recent advances, knowledge regarding the coding genome in FL is still evolving and currently incomplete. Methods: To further our understanding of the genetic basis of Follicular Lymphoma (FL), we used solution exon capture of sheared and processed genomic DNA isolated from FACS-sorted lymphomatous B-cells and paired CD3+ T-cells isolated from eleven cases of FL and one case of DLBCL transformed from prior FL followed by paired-end (96 base pair read length per side) massively parallel sequencing. Data from three independent HiSeq2000-based runs were pooled to maximize coverage. The sequence data were characterized by excellent technical QC parameters including a depth of coverage range of 43–73 and with 90% of bases in the target region covered by at least 10 reads. Data were subsequently analyzed using a validated bioinformatics pipelines (Personal Genome Diagnostics Inc., Baltimore, Maryland) serving as the primary data source to nominate candidate mutated genes. To complement the next gen sequencing data, we analyzed the 12 FL cases that constituted the discovery cohort for next generation sequencing for acquired genomic copy number aberrations (aCNA/LOH) and acquired uniparental disomy (aUPD) using SNP 6.0 array profiling. Data for paired DNA samples (sorted FL B-cells versus CD3 cells) were analyzed using dChip-based visual and algorithmic data analysis methods. Results: The bioinformatics pipeline nominated between 13 and 86 (mean of 47) somatically mutated genes per FL case; of these, 480 represented distinct genes. Importantly, 32 genes were nominated to be mutated in ≥2 out of 12 cases and all these candidate recurrent gene mutations and various other genes for a total of N=122 genes were subjected to Sanger sequence validation. Overall, we validated 68 genes as mutated in at least 1/12 FL discovery set cases. These included frequent mutations in MLL2 (8/12), CREBBP (7/12) and BCL2 (5/12). In addition, we identified 19 novel recurrently mutated genes (≥2 out of 12 FL cases with mutations). From these 19 genes and functionally related genes we selected 10 genes for a complete mutation analysis in a total 57 FL cases. Within the group of recurrently mutated genes we have identified a gene involved in apoptosis threshold regulation as well as multiple novel genes involved in B-cell transcriptional control. Further, we identify frequent mutations in the linker Histone genes HIST1H1 B-E. Finally, through incorporation of SNP array profiling data, we identify multiple candidate target genes for frequent aUPD in FL and further a list of single mutated genes (PTEN, A20, ARID1A, VAV1, TLR2 and TLR8 and others) that can be directly implicated in the pathogenesis of afflicted FL cases. Conclusion: This large genomic profiling study identifies 19 novel recurrently mutated genes in FL, including an apoptosis regulator, transcription factors and linker histones, thereby substantially broadening our understanding of the genetic basis of FL. Disclosures: Lebovic: Genentech: Speakers Bureau.