Recurrent Somatic Genomic Alterations in Follicular NHL (FL) Revealed By Exome and Custom-Capture Next Generation Sequencing

Background: Follicular lymphoma (FL) is the most common indolent NHL (iNHL), exhibits a variable clinical course, and remains largely incurable. The pathogenesis of FL is complex and involves over expression of Bcl2 via t(14;18) translocation, as well as copy number alterations, recurrent somatic mutations, and changes in the tumor microenvironment. In line with recent publications, we hypothesized that recurrent somatic genomic mutations in FL will be present and may impact FL development, progression, transformation, and clinical outcomes.

Methods: To address this, we performed exome sequencing (NimbleGen SeqCap EZ V2.0) of tumor and normal frozen tissue pairs from 24 patients in a discovery cohort with untreated FL (12), relapsed FL (6), or transformed FL/iNHL (6). We developed a custom capture assay (NimbleGen) that targets 7.05 MB corresponding to the coding, 5’ and 3’ UTR regions of 1717 genes. The custom capture genes included somatic mutations identified in our exome discovery cohort (898 genes) or somatic mutations previously published to be recurrently mutated in B cell NHL (819 genes). Instrument data from the discovery cohort exome and re-sequenced custom capture were combined and analyzed using the McDonnell Genome Institute (MGI) somatic caller pipeline (5 SNV callers, 3 indel callers), filtered (minimum 20X coverage, minimum 2.5% VAF, maximum 10% normal VAF) and manually reviewed. Additionally, the 1717 custom capture strategy was used to sequence an extension cohort consisting of FFPE tumor samples from 80 patients with FL, achieving >20x coverage for >75% of the targeted region.  All discovery and extension samples have clinical annotations that include FLIPI prognostic score, treatment, and clinical outcomes.

Results: Combined analysis of exome and custom capture data for the discovery cohort yielded a robust data set with good sequence coverage of >78% of the targeted regions with at least 20x depth in all samples and a mean depth of 89x.  Based upon somatic mutations identified and manually reviewed using this approach, we conservatively estimate 0.98 mutations per MB in FL.  23 genes were recurrently mutated in 3 or more cases, and an additional 75 genes recurrently mutated in 2 cases in the discovery cohort. Consistent with recent publications (Li H et. al., Blood, 2014; Green MR, PNAS, 2015; Yildiz M et al, Blood, 2015) we confirmed a number of genes that were highly recurrently mutated in FL [TNFRSF14 (50%), Bcl2 (25%), IRF8 (13%), TP53 (13%)] including chromatin modifying genes consisting of histone methyl transferases [KMT2D/MLL2 (58%), EZH2 (13%)], histone acetyltransferases [CREBBP (42%), EP300 (17%)], histone linkers [HIST1H1C (13%), HIST1H1E (8%), HIST1H2BO (8%), HIST1H3G (8%), HIST2H2AC (8%); collectively 42%].  We also confirmed (ATP6V1B2, 13%) and found unreported (ATP6AP2, 8%; ATP6V0A1, 4%; ATP6V1F, 4%) mutations in vacuolar ATPase proton pump genes and P5 or Ca++ ATPase genes (ATP13A2, 4%; ATP13A4, 4%, ATP2B4, 4%;). We confirmed (CD79B, 13%; BCL10, 8%) and found unreported (CD22, 13%) mutations in components of the B cell receptor signaling pathway. The previously unreported recurrent mutations in CD22 were consistent with loss-of function (2 missense, 1 nonsense, 1 frame shift deletion). As a negative regulator of BCR signaling, mutation of CD22 may represent a strategy of to enhance BCR signals in malignant germinal center B cells.  We also identified members of the SWI/SNF complex mutated in 33% of this FL cohort: ARID1B (8%), BCL11A (4%), SMARCB1 (4%) in addition to previously reported members BCL7A (12%), SMARCA4 (8%), ARID1A (4%).  Somatic mutations were also identified in the Notch pathway: DTX1 (29%), Notch2 (4%), Notch3 (4%), Notch4 (4%). We identified several genes that have not been reported as highly recurrent in FL CXCR4 (42%, mutation calls primarily in RNA), DMD (13%), DNAH9 (13%), FLG (13%), GON4L (13%), PCDH7 (13%), RLTPR (13%), SCN7A (13%), ST6GAL1 (13%).

Conclusions: FL genomes harbor a large number of recurrent mutations, consistent with a role in the development and progression of this malignancy.  Analysis of the extension cohort and association of recurrently mutated genes and pathways with clinical outcomes is ongoing and will be presented.