Mutations of EZH2 In Myeloproliferative Neoplasms with Myelofibrosis: Correlation with Molecular and Clinical Data

Although an increasing number of gene mutations provide new insights into the pathogenesis of myeloproliferative neoplasms (MPN), the biological origins of myelofibrosis (MF) remain poorly understood. Most likely, to date unknown genetic lesions contribute to disease phenotype and influence clinical outcome. Recently, mutations of EZH2 (enhancer of zeste 2) in 7q36.1 encoding the catalytic subunit of the histone methyltransferase PRC2 (polycomb repressive complex 2) have been identified in myeloid malignancies including MF. Functional studies indicate that EZH2 plays an important role in stem cell renewal by epigenetic repression of target genes. Mutations of EZH2 are considered to result in loss of tumor suppressor activity. These data indicate that EZH2 mutations may be important in the pathogenesis of myeloid malignancies such as MF. Aim: To explore the frequency of EZH2 mutations in a cohort of genetically and clinically well-characterized MPN patients with MF and acute myeloid leukemia (AML) secondary to MPN with 7q alteration.

All coding exons of EZH2 (2-20) were analyzed for mutations in 89 MPN patients using DNA sequencing: primary MF (PMF), n=62; secondary MF (SMF), n=21; AML secondary to MPN (sAML) with 7q alteration, n=6. Data on the mutation status of IDH1/IDH2 (Exon 4), JAK2 (V617F), and MPL (W515L) were available in all cases. In addition, all cases were analyzed on Affymetrix 250K (n=66) or 6.0 (n=23) SNP-arrays allowing for genome-wide screening of copy-number alterations (CNA) and uniparental disomies (UPD) at high resolution.

In total, 10 EZH2 mutations were identified in 8 patients (PMF, n=6; SMF, n=1; sAML, n=1) resulting in an overall mutation frequency of 9% (8/89); two PMF cases showed two EZH2 mutations each. The mutation frequency was not significantly different between PMF (6/62, 10%) and SMF (1/21, 5%) patients (p=0.67). EZH2 alterations identified in our study consisted of 8 missense mutations in exons 3, 9, 14–17, and 19, one insertion in exon 17 (c.1957_1958insT), and one deletion in exon 10 (c.1160_1161delAA). Eight of the 10 mutations were heterozygous; in one PMF and one post-PMF AML patient homozygous missense mutations were detected in exon 15 (c.G1720C) and 17 (c.A1947C), respectively; only the mutation in the latter patient was associated with UPD in 7q. None of the remaining 9 patients (PMF, n=4; sAML, n=5) with UPD (n=3) or deletion (n=6) in 7q had EZH2 mutations. EHZ2 mutations occurred in JAK2 mutated and unmutated patients (n=4, each) and none of the cases harbored additional mutations in IDH1/IDH2 or MPL. Furthermore, there was no correlation of the EZH2 mutation status with distinct CNA and/or UPD identified by SNP-arrays: in 5/8 patients, CNA such as trisomy 8, gains in 3q (29 Mb) and 6p (28 Mb), as well as loss in 13q (63 Mb), 18p (13.5 Mb), and 20q (22 Mb) were found. Four patients had large UPD (28-101 Mb) in 7q (n=1), 9p (n=2), or 11q (n=1). In total, only 2 of 8 cases lacked genomic aberrations; with regard to clinical characteristics, EZH2 mutations were associated with marked splenomegaly (20-28 cm) and leukocytosis (median white blood cell counts: 13.8 × 10⁹/L vs. 7.3 × 10⁹/L; p<0.0001).

Our study on 89 MF/sAML patients revealed EZH2 mutations as a frequent genetic lesion in these disease entities. However, EZH2 mutations were not associated with specific genetic or genomic aberrations. Further analyses comprising larger patient cohorts are necessary to explore the clinical role of EZH2 in MF.

No relevant conflicts of interest to declare.