EZH2 codon 641 mutations are common in BCL2-rearranged germinal center B cell lymphomas

Abstract

Mutations at codon 641 of EZH2 are recurrent in germinal center B cell lymphomas, and the most common variants lead to altered EZH2 enzymatic activity and enhanced tri-methylation of histone H3 at lysine 27, a repressive chromatin modification. As an initial step toward screening patients for cancer genotype-directed therapy, we developed a screening assay for EZH2 codon 641 mutations amenable for testing formalin-fixed clinical specimens, based on the sensitive SNaPshot single nucleotide extension technology. We detected EZH2 mutations in 12/55 (22%) follicular lymphomas (FL), 5/35 (14%) diffuse large B cell lymphomas with a germinal center immunophenotype (GCB-DLBCL), and 2/11 (18%) high grade B cell lymphomas with concurrent rearrangements of BCL2 and MYC. No EZH2 mutations were detected in cases of Burkitt lymphoma (0/23). EZH2 mutations were frequently associated with the presence of BCL2 rearrangement (BCL2-R) in both the FL (28% of BCL-R cases versus 0% of BCL2-WT cases, p<0.05) and GCB-DLBCL groups (33% of BCL2-R cases versus 4% of BCL2-WT cases, p<0.04), and across all lymphoma types excluding BL (27% of BCL2-R cases versus 3% of BCL2-WT cases, p<0.003). We confirmed gain-of-function activity for all previously reported EZH2 codon 641 mutation variants. Our findings suggest that EZH2 mutations constitute an additional genetic "hit" in many BCL2-rearranged germinal center B cell lymphomas. Our work may be helpful in the selection of lymphoma patients for future trials of pharmacologic agents targeting EZH2 and EZH2-regulated pathways.

Figure 1. SNaPshot detection of EZH2 codon 641 mutations.

A: Representative electropherograms for SNaPshot genotyping of EZH2 in B cell lymphomas. Five representative tracings are shown, representing lymphomas with wild-type EZH2 codon 641 and each of the four distinct missense substitutions detected in this study. Two complementary mutant peaks (arrows) appear in each mutant sample, corresponding to detection of the missense substitution by both the forward and reverse primers at the same nucleotide position. B: Distribution of EZH2 codon 641 missense mutations detected by SNaPshot.

Figure 2. Frequency of EZH2 mutations in germinal center lymphomas by BCL2 status.

Within each class of germinal center lymphoma, EZH2 mutations were significantly more frequent in cases with rearrangements of BCL2. Burkitt lymphoma (not shown) universally lacked both aberrations. The two total columns at right include all lymphomas in the study (fourth column) or all lymphomas except BL (fifth column) – see table 1 for subtype distribution. Abbreviations: FL = follicular lymphoma, GCB-DLBCL = germinal center B-cell phenotype diffuse large B-cell lymphoma, DHL = “double hit” high-grade B-cell lymphoma (MYC and BCL2 rearranged), BL = Burkitt lymphoma.

Figure 3. Western blot of mutant EZH2 expression in NIH-3T3 fibroblasts.

Transgenic expression of vectors encoding lymphoma-associated EZH2 codon 641 mutants, but not wild-type or SET domain-truncated (ΔSET) EZH2, leads to a consistent increase in global trimethylation of H3K27 compared to vector control. Western blot antibodies are listed on the right. Ponceau S depicts total protein at ∼15 kDa on acid-extract blots.