EZH2 Protein Expression Is Decreased in MDS and MDS/MPN and Correlated with EZH2 Mutation Status, Chromosomal 7 Abnormalities and Clinical Outcome
Authors: Mcgraw Kathy L , Nguyen Johnny , Alali Najla , Song Jinming , Sallman David , Padron Eric , Lancet Jeffrey E. , Qin Daihui , Epling-Burnette P.K. , Moscinski Lynn C. , Komrokji Rami S. , List Alan F , Zhang Ling 
Published: October 2019

Abstract
Background: The EZH2 gene (Enhancer of Zester homolog 2), located on chromosome (chr) 7q, is mutated in ~10% of patients with myelodysplastic syndromes (MDS) or MDS/myeloproliferative neoplasm (MDS/MPN). EZH2 gene mutations are associated with negative clinical outcomes; however, the role of EZH2 protein in MDS or MDS/MPN is largely unknown. In contrast to lymphomas or solid tumors in which expression is upregulated, decreased EZH2 mRNA expression was recently reported in 47% of MDS patients compared to normal controls and was more prevalent in patients with chr7 abnormalities. Furthermore, patients with decreased EZH2 expression lacking chr7 abnormalities had better overall survival (OS) (Cabrero M et al 2016). This study aims to explore EZH2 protein expression by immunohistochemistry (IHC) in MDS and MDS/MPN and the relationship to gene mutation and clinical outcome.
Methods: Retrospective analysis (Institutional Review Board approved) of MDS or MDS/MPN cases seen at Moffitt Cancer Center between 7/2013-5/2015 with myeloid targeted, next generation sequencing (NGS) panel was performed. Bone marrow biopsies corresponding to NGS date were used for IHC. Nuclear EZH2 expression was assessed semi-quantitatively using an IHC score calculated by multiplying signal strength (0-3+) with percent positivity in hematopoietic cells. OS was considered duration between date of diagnosis and date of death. P-values were determined using Chi-squared, student t-test, ANOVA, or log-rank analysis with p<0.05 considered statistically significant.
Results: EZH2 mutations were identified in 69/620 MDS and MDS/MPN patients (11.13%). Of these, IHC was performed on 44 patients with EZH2 mutations including 33 MDS and 11 MDS/MPN [median age 74 years (y), male to female (M: F) ratio 2:1], and on 25 EZH2 wild type (WT) patients (median age 70.5 y, M:F ratio 2.1:1, including 22 MDS and 3 MDS/MPN). While mean EZH2 IHC score was reduced in all MDS cases compared to hematologically normal, WT controls (n=5), more controls are required to reach statistical significance (MDS IHC score 77.60±8.09 control score 102.50±14.00, p=0.167). However, there was significantly decreased expression in EZH2 mutant compared to EZH2 WT cases (IHC score, 0.5716±0.09 vs 1.113±0.11, p<0.001). Reduced expression did not correlate with IPSS or R-IPSS (p=0.575 and p=0.906, respectively) when considering both mutant and WT cases, nor among mutated cases only (IPSS, p=0.589; R-IPSS, p=0.463). Mutated EZH2 had a significant adverse impact on OS. Median OS for WT EZH2 patients was not reached compared to 26.74 months for mutated patients (95% CI: 24.61-29.48) (p<0.001). However, decreased EZH2 expression in mutated cases did not alter OS with IHC cutoffs of 1.0 or 1.5 (p=0.436 and p=0.310, respectively), which may be attributed to low cohort numbers or variable reduction of EZH2 between patients. Within MDS cases, there was a trend towards lower expression with increasing variant allele frequency (VAF), however, a larger cohort is needed to reach significance (n=13, r= -0.461, p=0.11). Additional gene mutations (TET2, RUNX1 and ASXL1) did not impact OS (p=0.407, p=0.925, and p=0.902, respectively) when found in association with EZH2 mutations. We next assessed the relationship between EZH2 mutation and chr7 abnormalities determined by FISH. Indeed, EZH2 mutation was highly associated with chr7 abnormalities compared to WT cases (p=0.05). Furthermore, the EZH2 IHC score was significantly lower in patients with chr7 abnormalities compared to those without (0.461±0.11 and 0.943±0.09, respectively, p=0.02). In mutated cases, chr7 abnormalities were associated with significantly poorer OS than those without this cytogenetic abnormality (chr7 abnormality mean OS 13.41 months, 95% CI, 0.298-26.511; no chr7 abnormality, mean OS 61.18 months, 95% CI, 0.00-142.77, p=0.005). Notably, the mean EZH2 score was decreased in 5/7 patients with sequential sampling and disease progression (2 transformed to higher grade MDS, and 5 from MDS to AML).
Conclusion: EZH2 mutation is associated with reduced protein expression, chr7 abnormalities and inferior OS in MDS and MDS/MPN. Analysis of a larger cohort is warranted to explore the role of EZH2 protein in the pathogenesis and natural disease history of MDS and MDS/MPN.
Figure OS of patients with MDS and MDS/MPN with or without EZH2 mutation Figure. OS of patients with MDS and MDS/MPN with or without EZH2 mutation

Disclosures
Komrokji: Novartis: Consultancy, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.