Key Points• FL carries mutations in linker histone H1 B, C, D, and E genes in 27% of cases.• FL carries recurrent mutations in OCT2 (POU2F2), IRF8, and ARID1A.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.

• FL-associated STAT6 mutations hyperactivate the IL-4/JAK/STAT6 axis.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. (Blood. 2015;125(4):668-679)

Purpose: Chronic lymphocytic leukemia (CLL)-associated gene mutations that influence CLL cell fitness and chemotherapy resistance should increase in clonal representation when measured before therapy and at relapse.Experimental Design: To uncover mutations associated with CLL relapse, we have performed whole-exome sequencing in a discovery cohort of 61 relapsed CLL patients identifying 86 recurrently mutated genes. The variant allele fractions (VAF) of 19 genes with mutations in !3 of 61 cases were measured in 53 paired pre-and posttreatment CLL samples sorted to purity using panel-based deep resequencing or by droplet digital PCR.Results: We identify mutations in TP53 as the dominant subclonal gene driver of relapsed CLL often demonstrating substantial increases in VAFs. Subclonal mutations in SAMHD1 also recurrently demonstrated increased VAFs at relapse. Mutations in ATP10A, FAT3, FAM50A, and MGA, although infrequent, demonstrated enrichment in !2 cases each. In contrast, mutations in NOTCH1, SF3B1, POT1, FBXW7, MYD88, NXF1, XPO1, ZMYM3, or CHD2 were predominantly already clonal prior to therapy indicative of a pretreatment pathogenetic driver role in CLL. Quantitative analyses of clonal dynamics uncover rising, stable, and falling clones and subclones without clear evidence that gene mutations other than in TP53 and possibly SAMHD1 are frequently selected for at CLL relapse.Conclusions: Data in aggregate support a provisional categorization of CLL-associated recurrently mutated genes into three classes (i) often subclonal before therapy and strongly enriched after therapy, or, (ii) mostly clonal before therapy or without further enrichments at relapse, or, (iii) subclonal before and after therapy and enriching only in sporadic cases.

Purpose The identification of gene mutations and structural genomic aberrations that are critically involved in CLL pathogenesis is still evolving. One may postulate that genomic driver lesions with effects on CLL cell proliferation, apoptosis thresholds or chemotherapy resistance should increase in frequency over time when measured sequentially in a large CLL cohort. Experimental Design We sequentially sampled a large well-characterized CLL cohort at a mean of 4 years between samplings and measured acquired copy number aberrations (aCNA) and LOH using SNP 6.0 array profiling and the mutational state of TP53, NOTCH1 and SF3B1 using Sanger sequencing. The paired analysis included 156 patients, of whom 114 remained untreated and 42 received intercurrent therapies, predominantly potent chemo-immunotherapy, during the sampling interval. Results We identify a strong effect of intercurrent therapies on the frequency of acquisition of aCNAs in CLL. Importantly, the spectrum of acquired genomic changes was largely similar in patients that did or did not receive intercurrent therapies; therefore, various genomic changes that become part of the dominant clones are often already present in CLL cell populations prior to therapy. Further, we provide evidence that therapy of CLL with pre-existing TP53 mutations results in outgrowth of genomically very complex clones which dominate at relapse. Conclusions Using complementary technologies directed at the detection of genomic events that are present in substantial proportions of the clinically relevant CLL disease bulk, we capture aspects of genomic evolution in CLL over time, including increases in the frequency of genomic complexity, specific recurrent aCNAs and TP53 mutations.