EZH2 mutations and impact on clinical outcome: an analysis in 1,604 patients with newly diagnosed acute myeloid leukemia

The enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase and functional core subunit of the polycomb repressive complex 2 (PRC2), which is a key epigenetic regulator involved in embryonic development and transcriptional repression of genes by catalyzing the methylation of histone H3 at lysine 27 (H3K27me2/3). EZH2 overexpression has been associated with oncogenic activity and worse progression-free survival in several types of cancer including lymphoma, melanoma, and prostate and breast cancer. Moreover, the detection of recurrent EZH2 mutations, both gain-of-function in lymphomas and loss-of-function, e.g. in medulloblastoma, and bladder and renal cancers, point to a mutual role of EZH2 for disease pathology depending on the distinct type of cancer and indicate the potential of EZH2 as a therapeutic target. In myeloid malignancies such as myelodysplastic syndromes (MDS), loss of EZH2 activity by inactivating mutations is associated with poor prognosis. More recently, EZH2 inactivation by post translational modification was shown to induce chemoresistance in acute myeloid leukemia (AML). However, data on the frequency and prognostic role of EZH2 mutations in AML are still scarce and mostly confined to smaller cohorts. To investigate the prevalence and prognostic impact of this alteration in more detail, we analyzed a large cohort of AML patients (n=1,604) for EZH2 mutations. All patients had newly diagnosed AML, were registered for trials investigating intensive induction chemotherapy of the Study Alliance Leukemia (SAL) (AML96, AML2003 or AML60+, SORAML), and had available material at diagnosis. All analyses were carried out under the auspices of the SAL-bioregistry. Screening for EZH2 mutations and associated alterations was performed using Next-Generation Sequencing (NGS) (TruSight Myeloid Sequencing Panel, Illumina) on an Illumina MiSeq-system using bone marrow (BM) or peripheral blood (PB) (Online Supplementary Appendix). The myeloid gene panel (Illumina) targets 54 genes associated with myeloid neoplasms including full coding exons of EZH2, encoded on the long arm of chromosome 7 (7q36.1). Detection was conducted with a defined cutoff of 5% variant allele frequency (VAF) (median coverage 3,076; range 824-30,565). Patients’ clinical characteristics and co-mutations were analyzed according to the mutational status. Furthermore, multivariate analysis was used to identify the impact of EZH2 mutations on outcome. In addition, a more detailed subgroup analysis of EZH2-mut patients was conducted to integrate clinical outcome with the allelic state of mutations, affected functional domains (CXC-SET), and predicted effects of EZH2 mutations on protein expression. EZH2 mutations were found in 63 of 1,604 (4%) patients, which is in the range of prevalence (2-13%) typically observed for EZH2mutations in myeloid malignancies. A detailed list of all detected EZH2 variants is provided in Online Supplementary Table S1. In total, 50 of 63 patients (79%) harbored one single mutation in EZH2, while 13 individuals carried two different EZH2 variants (double mutated). Mutations were detected within several exons (2-6; 8-12; 14-20) and functional domains (D1, D2, CXC and SET), respectively (Figure 1A). In line with previous findings, most EZH2 variants were detected in exons 17 and 18 (28%), comprising the highly conserved SET domain, important for the catalytic activity of the EZH2 protein. The majority of detected mutations (67% missense and 33% nonsense/frameshift) were single nucleotide variants (SNV) (86%), followed by small indel mutations. All frameshift and nonsense mutations resulting in a premature stop of transcription were predicted to be “inactivating” for EZH2 protein expression (Figure 1B). One patient harbored a known EZH2 gain-of-function variant at position A682 (A677) recurrently found in large B-cell and non-Hodgkin lymphomas. These contrasting observations support recent findings that point to a potential stage-specific role of EZH2 in AML, exerting a tumor suppressor function during early AML induction and an oncogenic function during tumor maintenance. Other pathogenic SNV found in two or more patients were detected at residues R25 (COSM53003), G159 (COSM96480), R288 (COSM1000721), R313 (COSM6916439), R502 (COSM87274), N673 (COSM87276), R690 (COSM52980), and E745 (COSM1087033) (highlighted in Figure 1A). The p.Glu745Lys mutation (detected in 2 patients) was previously associated with acute lymphoblastic leukemia and lymphoma in Weaver Syndrome patients. Mutations were detected with a median allele burden of 42% (range 6-97%) (Figure 1B). The majority of patients (n=39) were heterozygous for their EZH2 variant, likely representing clonal events. Subclonal EZH2 variants (with significantly lower allelic ratio compared to all other co-mutated driver genes) were detected in nine patients. Patients with EZH2 allelic ratios >70% (n=15) were considered homozygous. EZH2 mutations were previously detected in both monoallelic and biallelic states in MDS/myeloproliferative neoplasms (MPN). Descriptive statistics of clinical parameters and associ-