Characterization of Cytogenetic Abnormalities and Mutations in ASXL1, SRSF2, CBL and JAK2 Genes in Chronic Myelomonocytic Leukemia

Chronic myelomonocytic leukemia (CMML) is a clonal haematopoietic malignancy characterized by features from both myelodisplastic syndromes and myeloproloferative neoplasms. CMML median overall survival is 20 months and 15–30% of cases progress to acute myeloid leukemia (AML). Molecular biology of CMML is poorly understood. Clonal cytogenetic abnormalities are found in 20–30% of cases. Recently, some recurrent gene mutations have been reported. The most frequent mutated genes are TET2 (30–50%), ASXL1 (35–56%), SRSF2 (28–47%), RUNX1 (8–37%), CBL (10–22%) and K/NRAS (2–22%). We sought to characterize type, frequency and prognostic implication of cytogenetic alterations and genetic mutations (ASXL1, CBL, JAK2 and SRSF2) in a cohort of 20 patients with CMML.

A retrospective study was performed on 20 CMML cases (11 CMML-1, 9 CMML-2) for whom clinical and biological data were available. Median age at diagnosis was 66 years (range: 49 – 84 years), with a male predominance of 1.9:1 and 42% frequency of progression to AML. Conventional cytogenetic analysis was performed in bone marrow (BM) samples obtained at diagnosis (n=19) and at progression to AML (n=8). For mutation analysis, DNA was extracted from BM samples at diagnosis (n= 20). Sanger sequencing was performed to study mutations in ASXL1 (exon 12), SRSF2 (exon 1) and CBL (exons 8 and 9) genes, while JAK2 V617F mutation was analyzed by endpoint genotyping. Survival analysis was performed using Kaplan-Meier estimate and log-rank tests were used for comparisons. The χ2 and Fisher's exact tests were used to analyze differences in the distribution of variables among patient subsets.

Aberrant karyotypes were seen in 5/19 (26%) cases at diagnosis and 6/8 (83%) cases at progression to AML. Alterations observed at diagnosis were typical from CMML (trisomy 8, 7q deletions and 12p structural alterations), while those observed at progression were more common in AML (Table 1). Heterozygous somatic ASXL1 mutations were detected in 8/19 (42%) cases. A total of 6 different mutations were seen in these 8 cases, being the recurrent mutation p.G646WfsX12 (g.76303dupG) the only one seen in more than one patient (4/8, 50%). In addition, 4 cases harboured two point mutations located at 3' of exon 12 (g.79017A>G and g.78128C>T). Somatic SRSF2 mutations were detected in 4/20 (20%) cases. All of them were heterozygous, missense mutations located at hotspot P95. In 3 cases P95 changed to P95H (g.535 C>A), while in 1 case it changed to P95L (g.535 C>T). In addition, 1 silence mutation was detected in 1 patient at the beginning of exon 1 (g.395 C>T) of SRSF2. No mutations were seen in CBL and JAK2 genes. ASXL1 and SRSF2 mutations did not correlate with either CMML-1/CMML-2 subtypes or myelodysplastic/myeloproliferative variants. Survival analysis revealed that the only adverse prognostic factor was CMML-2 subtype. Compared to CMML-1, patients with CMML-2 had worse overall survival (median OS at 2 years: 18% vs 21%, p=0.05) and progression-free survival (median PFS at 2 years: 21% vs 57%, p=0.002). Mutations in ASXL1 had no prognostic impact, while a trend towards higher PFS was seen in cases with SRSF2 mutations, although it was not statistically significant.

Cytogenetic analysis revealed that the percentage of aberrant karyotypes in CMML increases considerably at progression to AML. Mutational analysis showed that ASXL1 and SRSF2 are frequently mutated in CMML. There are multiple ASXL1 mutations throughout the exon 12, while missense SRSF2 mutations located at the hotspot P95. CMML-2 is still the only prognostic factor associated to worse OS and PFS.

No relevant conflicts of interest to declare.