Hierarchy in somatic mutations arising during genomic evolution and progression of follicular lymphoma

Abstract

Key Points• Analysis of coding genomes of FL tumor subpopulations reveals striking clonal diversity at diagnosis and progression.• Within a hierarchy of somatic evolution of FL coding genomes, many recurrent mutations are subclonal at diagnosis.Follicular lymphoma (FL) is currently incurable using conventional chemotherapy or immunotherapy regimes, compelling new strategies. Advances in high-throughput sequencing technologies that can reveal oncogenic pathways have stimulated interest in tailoring therapies toward actionable somatic mutations. However, for mutation-directed therapies to be most effective, the mutations must be uniformly present in evolved tumor cells as well as in the self-renewing tumor-cell precursors. Here, we show striking intratumoral clonal diversity within FL tumors in the representation of mutations in the majority of genes as revealed by whole exome sequencing of subpopulations. This diversity captures a clonal hierarchy, resolved using immunoglobulin somatic mutations and IGH-BCL2 translocations as a frame of reference and by comparing diagnosis and relapse tumor pairs, allowing us to distinguish early versus late genetic eventsduring lymphomagenesis. We provide evidence that IGH-BCL2 translocations and CREBBP mutations are early events, whereas MLL2 and TNFRSF14 mutations probably represent late events during disease evolution. These observations provide insight into which of the genetic lesions represent suitable candidates for targeted therapies. (Blood. 2013;121(9):1604-1611) IntroductionFollicular lymphoma (FL) is a common form of non-Hodgkin lymphoma (NHL) arising from mature B cells. FL tumor cells share identical immunoglobulin (Ig) gene rearrangements, indicating that the transforming founder mutation(s) occurs subsequent to VDJ recombination. These cells express markers of mature B-lineage including surface-Ig and CD19, and germinal center B-cell markers such as LMO2, CD10, and BCL6. 1 The B-cell marker CD20, which is also the target of the anti-lymphoma therapy Rituximab, 2,3 is expressed to a variable degree on FL tumors. 4,5 CD20 levels can predict primary responsiveness of diverse lymphomas to Rituximab,6 and changes in CD20 expression contribute to Rituximab resistance and relapse. 7,8 For instance, CD20 expression levels are associated with survival in patients with aggressive lymphomas treated with or without Rituximab. 9 These studies each found CD20 to be variably expressed on cells within the same tumor, suggesting that this may be a marker of underlying clonal diversity.The genetic hallmark of FL is the t(14;18)(q32;q21) translocation that places the antiapoptotic BCL2 oncogene under control of the Ig heavy-chain enhancer. 10 This lesion is present in Ͼ 90% of FL cases, 11 but is also detectable in the majority of older healthy adults suggesting that it is not sufficient to induce clinical disease. 12 Recently, mutation of a histone methyltransferase gene, MLL2, was identified in 89% of FL cases, indicating that this may also be a founder mutation. 13 Genes encodin...

Mutations in early follicular lymphoma progenitors are associated with suppressed antigen presentation

“…S8B). This is in line with our prior observations that allelic frequencies within the bulk tumor are poorly predictive of clonal origin (12). The average mutational burden of the EIP was 221 mutations (range, 59-447 mutations).…”

“…We performed exome sequencing on 65 tumors from 28 patients. Ten tumors from eight patients, including two patients with paired diagnosis and relapse, have been described previously (12). For the remaining 55 tumors from 20 patients, 11 patients had paired diagnosis and a single relapse, six patients had paired diagnosis and two relapses, and three patients had paired diagnosis and three relapses.…”

“…The t(14;18)(q32;q21) translocation was detected using a nested PCR assay with multiplexed primers specific for the Ig heavy-chain J-region, major break region, minor cluster region, and internal cluster region (12). PCR products were visualized on a 2% (wt/vol) agarose gel, and size of the product used to determine the translocation breakpoint (SI Appendix, Fig.…”

“…S10G). To ensure this effect was not the result of cosegregation between CREBBP mutations and deletions of the MHC class II locus, we interrogated somatic copy number data from our exome series and additional publicly available high-resolution data (12,13,(22)(23)(24). Although we detected deletions of the MHC class II locus in a subset of patients (SI Appendix, Fig.…”

“…This and other evidence therefore suggests that BCL2 translocations are not sufficient for lymphomagenesis and may be harbored in FL precursors, and that secondary genetic alterations are needed to drive clinical disease (4, 9, 10). Next-generation sequencing studies of FL have identified frequent mutation of chromatin-modifying genes (CMGs) (11)(12)(13)(14)(15). These include inactivating mutations of genes that apply activating euchromatin-associated marks [lysine-specific methyltransferase 2D (KTM2D), CREB binding protein (CREBBP), and E1A binding protein p300 (EP300)] and activating mutations within a gene that applies a repressive heterochromatin-associated mark (enhancer of zeste homolog 2, EZH2).…”