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Integrative Study of EZH2 Mutational Status, Copy Number, Protein

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Integrative Study of EZH2 Mutational Status, Copy Number, Protein Stomper et al. Clin Epigenet (2021) 13:77 https://doi.org/10.1186/s13148-021-01052-2 RESEARCH Open Access Integrative study of EZH2 mutational status, copy number, protein expression and H3K27 trimethylation in AML/MDS patients Julia Stomper1†, Ruth Meier1†, Tobias Ma1, Dietmar Pfeifer1, Gabriele Ihorst2, Nadja Blagitko‑Dorfs3, Gabriele Greve1, Dennis Zimmer1, Uwe Platzbecker4,5, Anne Hagemeijer6, Ingrid Schmitt‑Graef7 and Michael Lübbert1,8,9* Abstract Background: Mutations in the EZH2 gene are recurrently found in patients with myeloid neoplasms and are associ‑ ated with a poor prognosis. We aimed to characterize genetic and epigenetic alterations of EZH2 in 58 patients (51 with acute myeloid leukemia and 7 with myelodysplastic or myeloproliferative neoplasms) by integrating data on EZH2 mutational status, co‑occurring mutations, and EZH2 copy number status with EZH2 protein expression, histone H3K27 trimethylation, and EZH2 promoter methylation. Results: EZH2 was mutated in 6/51 acute myeloid leukemia patients (12%) and 7/7 patients with other myeloid neoplasms. EZH2 mutations were not overrepresented in patients with chromosome 7q deletions or losses. In acute myeloid leukemia patients, EZH2 mutations frequently co‑occurred with CEBPA (67%), ASXL1 (50%), TET2 and RAD21 mutations (33% each). In EZH2‑mutated patients with myelodysplastic or myeloproliferative neoplasms, the most common co‑mutations were in ASXL1 (100%), NRAS, RUNX1, and STAG2 (29% each). EZH2 mutations were associated with a signifcant decrease in EZH2 expression (p 0.0002), which was similar in patients with chromosome 7 aberra‑ tions and patients with intact chromosome 7. An= association between EZH2 protein expression and H3K27 trimeth‑ ylation was observed in EZH2‑unmutated patients (R2 0.2, p 0.01). The monoallelic state of EZH2 was not associ‑ ated with EZH2 promoter hypermethylation. In multivariable= analyses,= EZH2 mutations were associated with a trend towards an increased risk of death (hazard ratio 2.51 [95% confdence interval 0.87–7.25], p 0.09); similarly, low EZH2 expression was associated with elevated risk (hazard ratio 2.54 [95% confdence interval 1.07–6.04],= p 0.04). = Conclusions: Perturbations of EZH2 activity in AML/MDS occur on diferent, genetic and non‑genetic levels. Both low EZH2 protein expression and, by trend, EZH2 gene mutations predicted inferior overall survival of AML patients receiving standard chemotherapy. Keywords: Acute myeloid leukemia, EZH2, Mutations, Protein expression, H3K27 trimethylation, Promoter methylation, Survival Background *Correspondence: michael.luebbert@uniklinik‑freiburg.de Enhancer of Zeste Homolog 2 (EZH2), the catalytic † Julia Stomper and Ruth Meier contributed equally to this work. domain of Polycomb Repressive Complex 2 (PRC2), cata- 1 Department of Medicine I (Hematology, Oncology and Stem Cell Transplantation), Medical Center – University of Freiburg, Freiburg, lyzes the repressive mark of trimethylation of histone Germany H3 lysine 27 (H3K27me3) [1]. Te EZH2 gene is located Full list of author information is available at the end of the article on chromosome 7q36.1, a region frequently deleted in © The Author(s) 2021, corrected publication 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Stomper et al. Clin Epigenet (2021) 13:77 Page 2 of 14 acute myeloid leukemia (AML), myelodysplastic syn- degradation in order to overcome chemoresistance to dromes (MDS), and myeloproliferative neoplasms (MPN) inhibiting the PRC2 components EZH1 and -2 to reduce [2]. MDS and AML patients with monosomy 7 or 7q quiescent leukemia stem cells [4, 17]. (-7/del(7q)) were found to have reduced EZH2 mRNA In the present study, we analyzed a cohort of 58 patients expression levels compared to patients without these (mostly AML). Since potential mechanisms of EZH2 per- alterations [3, 4]. Te role of EZH2 in tumorigenesis turbation involve mutations, allelic loss, and promoter appears to be context-dependent, since both EZH2 over- hypermethylation, we sought to determine those param- expression and loss of function are associated with difer- eters, their relationship, and prognostic impact, confrm- ent types of cancer [5]. In patients with MDS or MDS/ ing the overall worse outcome in the presence of EZH2 MPN, recurrent loss-of-function EZH2 mutations [6–8] mutations and decreased EZH2 expression. are associated with a poor prognosis [9], and EZH2 is considered by many to act as a bona fde tumor suppres- Results sor gene [10, 11]. In AML, the prevalence of EZH2 muta- EZH2 mutational status, co‑occurring mutations, tions is lower and less well studied [12]. Regarding the and chromosome 7 loss in AML/MDS patients transformation of MDS and MPN to AML, loss of EZH2 In the cohort of 58 selected patients with myeloid neo- function also appears to play an ambiguous role, being plasms (see Table 1, 2, and Additional File 1: Table S1 able to attenuate and promote leukemic transformation for patient characteristics), the EZH2 gene showed depending on the disease context and cooperating muta- mutations (mostly missense or nonsense) in 6/51 AML tions [11, 13, 14]. While EZH2 inhibitors are already patients (12%) and 7/7 non-AML patients (selected being evaluated in phase 1 and 2 clinical trials in diferent specifcally for this genotype, hence 100%, Table 2), solid tumors and B cell lymphoma [15], not only in MDS and was unmutated in 45/51 AML patients (88%). [16] but also in AML, EZH2 is increasingly under study Te EZH2 mutation rate in the diferent AML sub- as a potential therapeutic target. Yet, due to its complex groups was 12% (3/25) in de novo AML, 9% (2/22) in function, thus far the mechanisms of action of proposed AML with myelodysplasia-related changes (MRC), and applications difer widely, ranging from preventing EZH2 25% (1/4) in therapy-related AML (t-AML). Two of 6 Table 1 Patient characteristics AML Myeloid Neoplasms (Non‑AML) Total 51 7 Subtype (%) AML with recurrent MDS‑MLD: 2 genetic abnormalities: 20 (39) MDS‑EB‑2: 1 AML‑MRC: 22 (43) CMML‑2: 1 t‑AML: 4 (8) MDS/MPN: 2 AML, NOS: 5 (10) CNL: 1 Age (years; median and range) 63 (20–86) 69 (42–84) Sex Male 25 4 Female 26 3 EZH2‑mutated patients (%) 6 (12) 7 (100) Mutations per patient (median and range) 3 (0–7) 4 (3–7) Del(7q) or ‑7 (%) 13 (25) 2 (29) WBC ( ­109/L; median and range) 10.4 (0.6–192.6) 18.4 (1.6–126.5) × BM blasts (%; median and range) 63 (10–100) 4 (1–16) Treatment Induction chemotherapy (%) 31 (61) 0 HSCT (%) 27 (53) 4 (57) Non‑intensive therapy (%) 13 (25) 3 (43) AML-MRC acute myeloid leukemia with myelodysplasia-related changes, BM bone marrow, CMML chronic myelomonocytic leukemia, CNL chronic neutrophilic leukemia, HSCT hematopoietic stem cell transplantation, MDS-EB myelodysplastic syndrome with excess blasts, MDS-MLD MDS with multilineage dysplasia, MPN myeloproliferative neoplasm, NOS not otherwise specifed, WBC white blood cells, t-AML therapy-related AML Stomper et al. Clin Epigenet (2021) 13:77 Page 3 of 14 Table 2 Clinical characteristics of EZH2‑mutated patients Patient Sex Age Disease Karyotype Del(7q) % WBC PB blasts Treatment # (years) subtype or ‑7 monosomy ( ­109/L) (%) 7 cells (FISH) × AML patients 14 m 29 AML with 46,XY [22] No 0 16.4 64 Induction recurrent chemo, genetic HSCT abnormali‑ ties 35 f 84 AML‑MRC (his‑ 48,XX,der(1)t(1;5) No 0 2.66 2 LDAC, tranyl‑ tory of MDS) (p36;?), der(1) cypromine, t(1;5)(p36;?),der(5)+ ATRA t(5;11)(p15;q23)t(1;5) (p36;q15),der(11) t(5;11)(?;q23),del(12) (p12p13), 19 [5]/49,idem,+ 8 [9]/46,XX [6]+ 50 m 71 AML, NOS 46,XY [21] No 0 101.73 26 Induction chemo, HSCT 5 f 74 AML with 46,XX [20] No 0 31.23 49 Induction recurrent chemo, genetic HSCT abnormali‑ ties 21 m 71 t‑AML 46,XY,del(12)(p12p13) [30] No 0 4.22 54 DAC 27 m 68 AML‑MRC 45,XY,inv(3)(q21.3q26.2),‑7 Yes 40 2.01 5 DAC, HSCT [20] 59 m 75 AML, NOS 47,XY, 8 [2] No 0 3.6 5† DAC (history of + MPN) 60 m 61 AML‑MRC (his‑ 46,XY [20] No 0 9.5 24† DAC, ATRA tory of MDS) MDS/MPN patients 51 m 81 MDS‑MLD 45,XY,‑7,der(17)t(1;17) Yes 54 1.61 0 AZA (p36;p12) [6]/46,XY [14] 49 f 42 MDS‑MLD 45,XX,‑7 [9]/46,XX [13] Yes 16 2.0 3 Upfront HSCT 25 m 82 MDS/MPN 46,XY,del(17p13),i(17q11) No 0 39.72 10 HU, AZA (progression (40%)* to AML) 22 f 61 CNL 46,XX No 0 71.14 0 HU, HSCT 33 m 84 CMML‑2 47,XY, 8 [19]/46,XY [1] No 0 18.4 2 DAC + 40 f 69 MDS/MPN 47,XX, 8 [11] No 0 126.45 6 HU, HSCT + 10 m 68 MDS‑EB‑2 47,XY, 8 [12]/46,XY [8] No 0 1.67 0 AZA, HSCT + AML acute myeloid leukemia, ATRA all-trans retinoic acid, AZA azacitidine, CMML chronic myelomonocytic leukemia, CNL chronic neutrophilic leukemia, DAC decitabine, FISH fuorescence in situ hybridization, HSCT hematopoietic stem cell transplantation, HU hydroxyurea, LDAC low dose cytarabine, MDS-EB myelodysplastic syndrome with excess blasts, MDS-MLD MDS with multilineage dysplasia, MPN myeloproliferative neoplasm, MRC myelodysplasia-related changes, NOS not otherwise specifed, PB peripheral blood, t-AML therapy-related AML, WBC white blood cells *FISH analysis † Bone marrow blasts EZH2-mutated (mut) AML patients and 5/7 MDS/ Both EZH2-mut AML and MDS/MPN patients had a MPN patients had 2 mutations within the EZH2 gene median of 4 mutations (Fig.
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