Clonal Leukemic Evolution In Myelodysplastic Syndromes With ASXL1 and EZH2 mutations: A Comparative Analysis Of 58 Paired Samples

ASXL1 and EZH2 are histone modifiers. Mutations of ASXL1 and EZH2 genes have been described in both myelodyspolastic syndromes (MDS) and acute myeloid leukemia (AML). The role of ASXL1 or EZH2 mutations in the progression from MDS to secondary AML (sAML) is unclear. We aimed to determine the clinical relevance of ASXL1 and EZH2 mutations in patients with MDS and to investigate the role of ASXL1 or EZH2 mutation with its cooperating mutated genes in sAML progression.

ASXL1 and EZH2 mutations were analyzed on bone marrow samples from 126 patients with de novo MDS (45 RAEB1, 53 RAEB2, 24 RCMD and 4 RARS). Paired matched MDS/sAML samples were available for ASXL1 mutational analysis in 58 patients and for EZH2 in 54 patients. Mutational analysis of ASXL1 exon 12 was performed by PCR assays followed by direct sequencing, EZH2 mutations were first screened with denaturing high-performance liquid chromatography on amplified PCR fragments covering the whole coding sequencing from exons 2 to 20 of EZH2 gene followed by sequencing of the abnormal profile. Additional 20 known gene mutations in myeloid neoplasms were also examined in patients carrying ASXL1 or EZH2 mutations. Relative allele frequency was determined by pyrosequencing.

Among the 126 patients, ASXL1 mutations were detected in 18 (14.3%) patients and EZH2 in 11 patients (8.7%), 3 of them had both mutations. Taken together, 20.6% of patients carried mutations of ASXL1 and/or EZH2. ASXL1-mutated patients had male predominance (17 out of 18 patients, P=0.012) and fewer circulating blasts (P=0.007). ASXL1-mutated and -unmutated patients had no difference in hemoglobin levels, white blood cell counts, platelet counts, cytogenetics, WHO subtypes, bone marrow blasts, IPSS-R or risk to sAML. ASXL1 mutations had no impact on overall survival (P=0.765) or time to sAML transformation (P=0.605). No significant difference was observed between EZH2 mutation status and clinicohematologic features or outcomes. Of the 58 paired samples, ASXL1 mutations were detected in 8 cases at diagnosis of MDS, all were also present at the time of sAML progression with no difference in the mutant allele burden (P=0.614), 2 patients acquired ASXL1 mutations. Five had EZH2 mutations at both phases of disease which exhibited a similar allele frequency (P= 0.434), none lost and one acquired EZH2 mutation at sAML phase. Progression to sAML was accompanied by additional gene mutations including RUNX1 (n=4), TET2 (n=3), CEBPα (n=2), PTPN11 (n=2), and one each for FLT3-ITD, CBL, MLL-PTD, DNMT3A, IDH2 and EZH2 in ASXL1-mutated patients; TET2 (n=4), RUNX1 (n=3), N-RAS (n=2) and single cases for DNMT3A, IDH1, and ASXL1 in EZH2-mutated patients. Cooperating mutations were either detected in both MDS and sAML samples or newly appeared in sAML samples except one who lost JAK2V617F mutation while acquired EZH2 and N-RAS mutations during sAML progression.

Our study on a large cohort of paired MDS and sAML samples demonstrated that ASXL1 or EZH2 mutations remained stable at both phases of disease in most patients. Clonal evolution can occur and cooperation of additional gene mutations is frequently detected in patients harboring ASXL1 or EZH2 mutations in the progression of MDS to sAML.

This work was supported by NHRI-EX102-10003NI and DOH102-TD-C-111-006, Taiwan.

No relevant conflicts of interest to declare.