Detaileddistribution and frequencies of the EZH2 mutations. (A) Distribution and frequency of EZH2 SET domain mutations detected in 366 diagnostic and relapsed FL cases. Altogether, 106 mutations were detected in 101 FL patients. The most prevalent variants were the mutations resulting in replacement of the tyrosine at codon 646 (Y646), followed by A682 and A692 mutations. Of note, we also detected 3 previously unreported mutations in B-cell lymphomas: K634E, V637A, and V679M of unknown significance. (B) Comparison of VAF for EZH2, CREBBP, MLL2, TNFRSF14, and MEF2B mutations identified by NGS-based targeted resequencing demonstrating i, true clonal variants with similar VAFs (less than 20% difference between VAFs for EZH2 compared with the other genes excluding VAFs of >50%) across the genes analyzed (cases 1720 and 1839), ii, clonal variants with similar, but low VAFs across the mutation targets reflecting the low tumor content within these biopsy samples (cases 1087 and 1845), and iii, true subclonal EZH2 variants with lower EZH2 VAFs as compared with the other genes (cases 1088 and 465). (C) Hierarchical clustering and heatmap of 69 FL cases (51 wild type vs 18 EZH2 mutated) with estimated high tumor content (Δβ > 0.1657) and EZH2 VAF (>17%) showing the gene expression signature of 106 differentially expressed genes. (D) Clonal representation of EZH2 mutations during transformation of FL. Illustrated are the corrected VAFs observed in sequential FL and tFL samples. The EZH2 mutations were maintained during transformation in 6 cases with relatively stable VAFs, whereas it was restricted to the FL and tFL samples in single patients (cases 13 and 18). Case 23 harbored 2 EZH2 mutations (Y646F and K634E) in monoallelic configuration.