A Pyrosequencing Assay for the Detection of EZH2Y641 Mutations in Diffuse Large B cell Lymphoma and Follicular Lymphoma - Abstract
Aims: Enhancer of Zeste Homolog 2 (EZH2) is a critical enzymatic subunit of Polycomb Repressive Complex 2 (PRC2), which provides inhibitory regulation of gene transcription through trimethylation of histone H3 on lysine 27 (H3K27me3). The somatic mutation EZH2 Y641 in its highly conserved catalytic SET domain has been reported in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) by methods of Sanger sequencing and whole exome sequencing. To develop a clinically applicable assay uses pyrosequencing method for accurate detection of EZH2Y641 mutations in formalin- fixed-paraffin-embedded (FFPE) tissue.
Methods: We validated the pyrosequencing method for the detection of EZH2Y641 mutation in DLBCL cell lines. We also screened a cohort of 43 DLBCL and 12 FL cases by pyrosequencing, and correlated with the results of Sanger sequencing of EZH2 exon 15.
Results: We detected EZH2 Y641 mutations in 3/22 (13.6%) germinal center B cell-like DLBCL, 2/12 (16.7%) FL and none of the 21 activated B cell-like DLBCL. The mutations detected by pyrosequencing were in complete agreement with Sanger sequencing. We have identified the most common 4 types of mutations of EZH2 Y641.
Conclusion: The rapid and accurate detection of the EZH2 mutation fromFFPE tissue sections in the clinical laboratory will facilitate screening patients with DLBCL and FL for enrollment of clinical trials using EZH2 inhibitors.