EZH2 mutations are frequent and represent an early event in follicular lymphoma

Abstract

Key Points• EZH2 mutations occur in more than 25% of follicular lymphoma patients.• Mutations predominantly represent an early/clonal event in the pathogenesis of the disease.Gain of function mutations in the H3K27 methyltransferase EZH2 represent a promising therapeutic target in germinal center lymphomas. In this study, we assessed the frequency and distribution of EZH2 mutations in a large cohort of patients with follicular lymphoma (FL) (n 5 366) and performed a longitudinal analysis of mutation during the disease progression from FL to transformed FL (tFL) (n 5 33). Mutations were detected at 3 recurrent mutation hot spots (Y646, A682, and A692) in 27% of FL cases with variant allele frequencies (VAF) ranging from 2% to 61%. By comparing VAF of EZH2 with other mutation targets (CREBBP, MLL2, TNFRSF14, and MEF2B), we were able to distinguish patients harboring clonal EZH2 mutation from rarer cases with subclonal mutations. Overall, the high incidence of EZH2 mutations in FL and their stability during disease progression makes FL an appropriate disease to evaluate EZH2 targeted therapy. (Blood. 2013; 122(18):3165-3168) Introduction Next-generation sequencing (NGS) studies have shown frequent mutations in epigenetic regulators in almost all cases of follicular lymphoma (FL).1,2 These include EZH2, the catalytic subunit of PRC2, which catalyzes trimethylation of lysine 27 on histone H3 (H3K27me3), a repressive chromatin mark.3 Somatic gain-of-function mutations of EZH2 at codon Y646 (previously Y641) were identified in 7% to 22% of FLs and germinal center B-cell type diffuse large B-cell lymphomas leading to elevated H3K27 trimethylation [4][5][6][7][8] with mutations at codons A682 and A692 described in isolated cases of diffuse large B-cell lymphomas. 2,9-11 As highly selective EZH2 inhibitors have now been developed, [12][13][14] we set out to assess EZH2 mutation status, the effect of mutations on global gene expression, and the clonal representation of EZH2 mutations as the disease progresses. Study design Patient samplesGenomic DNA from 181 diagnostic FL patients with accompanying clinical and gene expression data 15 were obtained through the Lymphoma/Leukemia Molecular Profiling Project consortium. DNA from 185 additional FL patients (56 obtained at diagnosis and 129 at relapse) and 33 paired FL and transformed FL (tFL) samples were sourced from the tissue archive at the Barts Cancer The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.The publisher or recipient acknowledges right of the US government to retain a nonexclusive, royalty-free license in and to any copyright covering the article.BLOOD, 31 OCTOBER 2013 x VOLUME 122, NUMBER 18 3165For personal use only. on May 9, 2018. by guest www.bloodjournal.org From Institute. The study was approved by the London Research Ethical Committee (05/Q0605/140) and was conducted in accordance with the...

Somatic Mutation Screening Using Archival Formalin-Fixed, Paraffin-Embedded Tissues by Fluidigm Multiplex PCR and Illumina Sequencing

“…Nearly one-third of the mutations (45/159 Z 28%) detected by Fluidigm PCR and Illumina MiSeq sequencing were missed by original PCR and Sanger sequencing, albeit confirmed by further Sanger sequencing or cloning and sequencing of the PCR products. Consistent with our findings, Bodor et al 27 also found an improvement of 39% in mutation detection by amplicon-based NGS in comparison with conventional PCR and Sanger sequencing. A proportion of the somatic mutations additionally detected by Fluidigm/MiSeq sequencing may represent subclonal genetic changes.…”

The European Hematology Association Roadmap for European Hematology Research: a consensus document

“…Because there does not appear to be a single target that can be applied to all cases, a combination of novel biomarkers (based on genomic, proteomic, transcriptomic, and metabolomic analyses of biopsies) will be required. It will be important to identify those molecular events involved in early development of the disease 90,91 and those involved in progression and transformation. 88 The prognosis and clinical course of FL appears to be highly dependent on the tumor microenvironment, and immunohistochemical methods are being assessed to address this.…”

“…Where possible, consent should be obtained for the procurement and storage of use of excess tissue from lymph node biopsies and normal tissue at the time of presentation and at each subsequent disease relapse for research purposes in order to investigate the molecular biology of these diseases. [90][91][92] and the processes involved in transformation, should be a common goal. 88 A key issue would be performing both genetic and microenvironment analyses on longitudinal and/or paired FL biopsies to obtain an integrated view on bidirectional dependency.…”

Prognostic relevance of CD163 and CD8 combined with EZH2 and gain of chromosome 18 in follicular lymphoma: a study by the Lunenburg Lymphoma Biomarker Consortium

“…The pertinent question is whether these findings on the prognostic value of microenvironment populations and genomic alterations can be translated to application in daily clinical practice. The mutations in EZH2 are largely clustered in codon Y646 (Online Supplementary Figure S1) and are therefore technically very easily amenable to simple polymerase chain reaction (PCR) techniques, 55 while chromosomal gains can be monitored using fluorescence in situ hybridization (FISH) or NGS. 56,57 This signifies that EZH2 mutation status and chromosome 18 gain have a high potential for clinical implementation, in contrast to the microenvironment population markers CD8 and CD163.…”