Mutated ASXL1 and number of somatic mutations as possible indicators of progression to chronic myelomonocytic leukemia of myelodysplastic syndromes with single or multilineage dysplasia

Myelodysplastic syndromes (MDS) are heterogeneous clonal disorders characterized by ineffective hematopoiesis and transformation in acute myeloid leukemia (AML).1 However, disease progression is not always to overt AML; MDS may develop myeloproliferative features and move to overlap-diseases [MDS/myeloproliferative neoplasms (MPN)], such as chronic myelomonocytic leukemia (CMML).

Some authors reported unexpected transformation of de novo MDS to CMML independently of the presence of relative monocytosis (>10%) at initial diagnosis of MDS.32 In these reports, apart from an evident increase in total white blood cell count (WBC) and monocytosis, alterations of other clinical parameters and/or cytogenetic and molecular abnormalities were not associated directly with progression.3 Deep sequencing demonstrated the high frequency of somatic mutations in MDS as well as the acquisition of additional mutations during disease history.54 Some somatic mutations are often shared by MDS and MDS/MPN and contribute in different ways to the prognosis of these disorders.86

Padron et al.4 identified a similar pattern of mutations at diagnosis and in the myeloproliferative stage of MDS patients that transformed to CMML, with maintained variant allele frequency (VAF). The acquisition of new mutations was nevertheless evident at progression (ETV6, NRAS, CBL, and RUNX1) and differently to this, but in the same line of multi-step clonal evolution, it was observed that MDS progressing to AML acquired mutations of RUNX1, ETV6, PHF6, NRAS and KRAS.5

Here, on a molecular level, we describe a cohort of lower risk MDS patients who evolved to CMML. All consecutive cases diagnosed at Azienda Ospedaliero Universitaria Careggi (Florence, Italy) as MDS in accordance with World Health Organization (WHO) 2008 criteria between April 2010 and September 2016 were revised. Out of 360 cases analyzed, we identified 9 patients reclassified with revised WHO 20169 presenting a diagnosis of MDS with single lineage dysplasia (MDS-SLD) or MDS with multilineage dysplasia (MDS-MLD) who progressed to overt CMML after variable follow-up periods (referred as Group 1) (Table 1). In all patients, absolute and relative monocytosis was absent during the entire duration of MDS and the median time to CMML transformation was 21 months (range 10–41) (Table 1). Paired DNA samples of the MDS and CMML phases were isolated in all 9 cases. Bone marrow (BM) mononuclear cells were collected and cryopreserved at diagnosis (MDS phase) and at transformation (CMML phase). Informed consent was provided in accordance with the Declaration of Helsinki and local ethics committee approval. Twenty-two candidate genes were selected for sequencing analysis (See Online Supplementary Appendix for methods), referred to in literature as those most frequently mutated in MDS and CMML:1210 ASXL1, EZH2, TP53, SF3B1, U2AF1, SRSF2, TET2, DNMT3A, ETV6, RUNX1, NPM1, FLT3, CBL, SETBP1, CSF3R, CEBPA, IDH1, IDH2, JAK2, MPL, CARL, NRAS. Patients’ characteristics are presented in Table 1. In brief, 5 were males, and the median age at diagnosis was 67 years (range 56–76 years). At diagnosis, 4 patients had MDS-SLD and 5 patients had MDS-MLD. At transformation, 3 patients presented a CMML-0, 4 patients a CMML-1, and 2 patients a CMML-2 according to the WHO 2016 classification. WBC, absolute monocyte count (AMC) and blast count in CMML phase were statistically higher than in MDS phase (mean WBC 3.64×10/L vs. 13×10/L, P=0.001; AMC 0.21×10/L vs. 1.6×10/L, P=0.004; blast count 0% vs. 6%, P=0.0001). There was no significant statistical difference in hemoglobin level (10.85 g/dL vs. 9.7 g/dL, P=0.42) or platelet count (147×10/L vs. 86×10/L, P=0.11) between the MDS and CMML phases. After the initial diagnosis of MDS, all patients were treated exclusively with erythropoiesis-stimulating agents, and all patients with and without stable disease were followed every three months. In both phases, 5 patients presented a normal karyotype, one patient a monosomy 7, one patient a trisomy 8, and one patient a trisomy 14. Only case #1 presented a new trisomy 8 at progression (Table 1). After sequencing, coverage across targeted regions was 2500X (minimum 316 and maximum 9877). In the MDS phase, somatic mutations were identified in 9 genes, resulting in 29 variants. All mutations were heterozygous, and comprised 10 missense, 10 nonsense, and 9 frameshift mutations. Eight of the 9 CMML-progressing cases presented three mutations or more (median number of mutations: 3, range 2–5) (Online Supplementary Figure S1). The mutational analysis of the CMML phase confirmed the presence of all the 29 variants found at diagnosis (Figure 1). In general, mutation VAF did not change significantly, while some mutated clones were marginally expanded during progression. An acquisition of two missense variants was presented in the CMML samples: case #3 with a new variant of the DNMT3A gene and case #7 with a new variant of the gene NRAS (Figure 1). In our cohort of patients, the acquisition of new somatic mutations was not closely correlated with progression, as 7 of the 9 cases studied did not acquire new mutations. We then compared the mutational profile of Group 1 with a second cohort of lower risk MDS patients that did not present CMML transformation or progression to higher risk MDS or AML (referred as Group 2). Clinical data of Group 2 are summarized in Online Supplementary Table S1. Groups 1 and 2 were matched by age, sex and International Prognostic Scoring System (IPSS) risk; Group 2 had a longer OS than Group 1 (67 vs. 38 months) (Online Supplementary Figure S2). After sequencing of Group 2, coverage across the targeted regions of 3441X (minimum 410 and maximum 11401) was obtained. Thirteen cases (28%) did not present any mutation in the 22 genes analyzed, and 33 cases (72%) presented at least one mutation (median number of mutations: 1, range 1–3). The first important observation is that Group 1 cases presented with a higher number of mutated genes: 88% cases of Group 1 showed 3 or more mutations compared with 2 cases (4%) of Group 2 (Figure 2 and Online Supplementary Figure S1). The second observation is that the most frequently mutated genes in Group 1 were ASXL1 and TET2 that were mutated in all cases and in 6 of 9 cases, respectively (Figure 2A). Consistent with reports in literature, TET2, SF3B1 and DNMT3A were the most frequently mutated genes in Group 2 cases (19%, 24% and 28%, respectively),1110 while ASXL1 was present in only 3 cases (7%): in 2 cases as an isolated mutation and in one case in combination with TET2 and SRSF2 (Figure 2B). The latter case has been censored because the patient received an early hematopoietic stem cell transplant, and therefore it was not possible to evaluate progression. Group 1 cases shared the constant presence of ASXL1 mutations. Based on average sequencing depth versus VAF, ASXL1 mutations in Group 1 appear both as clonal and subclonal. Moreover, VAF of ASXL1 in cases #3 and #8 did not vary significantly while other mutations presented in the same samples increased in size (Figure 1). It seems that, together with the presence of ASXL1 mutations, the increase in size of other clones could be related to the disease progression. An ASXL1 mutation is present in 45% of CMML cases and has an adverse prognostic impact on OS.1312 TET2 mutations, the second most frequent in Group 1, are widely prevalent in CMML (46%) and their prognostic relevance remains unclear;14 in our series TET2 mutations were not present in any of our cases that progressed from MDS to CMML. Co-mutation of ASXL1 and TET2 is associated with very poor survival in CMML.14 In order to evaluate the weight of ASXL1 and TET2 mutation positive clones in this peculiar MDS-to-CMML progression, we compared VAF data of the MDS and CMML phases using the paired sign test. VAF of ASXL1 was significantly higher in CMML than in the corresponding MDS phase (42% vs. 29%, respectively, P=0.039) while VAF of TET2 was not significantly varied in either phase (55% vs. 46%, respectively, P=0.38) (Online Supplementary Figure S3A and B), suggesting that early presence and increase in the burden of mutated ASXL1 during progression of the disease to myeloproliferative features have a prominent and determining role. It should be stressed that the presence of ASXL1 mutation was a rare event (7%) in matched lower risk MDS cases of Group 2 without any kind of progression. Although our study was limited by the number of cases evaluated, no significant changes were observed in the mutation profile during progression to CMML. Although the presence of acquired mutations in other genes not included in this study cannot be excluded, nevertheless, our observations strongly suggest that a “pre-CMML state” could possibly be identified. All “pre-CMML” cases were characterized by worsening anemia that required treatment, in line with the role of ASXL1 in erythropoiesis.15 These very particular “pre-CMML”-MDS cases seem to be well characterized by enhanced genetic instability, justifying multiple co-mutations, and by the constant feature of early ASXL1 mutation. To note, none of the patients with a “pre-CMML”-MDS presented TP53 mutations, which is very rarely found in CMML. This suggests that the presence of ASXL1 mutations and TP53 wild type could be part of this definition of “pre-CMML” state. Although this kind of progression MDS-to-CMML is quite rare, further studies involving larger numbers of these patients should be carried out in order to confirm whether this kind of evolution is predictable and would, therefore, justify an earlier start to therapy in lower risk MDS with ASXL1 mutations.

Table 1.Clinical and cytogenetic characteristics of myelodysplastic syndrome patients that progress to chronic myelomonocytic leukemia.

Figure 1.Representation of mutations and variant allele frequency (VAF) (%) in the paired DNA samples of the myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) phases in the Group 1 of patients. Each graph represents cases #1 to #9 of Group 1. Genes are color coded and VAFs (y-axis) and protein change (x-axis) are represented for each variant. Our panel gene was as follows (only the first nine genes were mutated in Group 1 of patients at MDS diagnosis): ASXL1, EZH2, RUNX1, SETBP1, SF3B1, U2AF1, SRSF2, TET2, DNMT3A, TP53, ETV6, NPM1, FLT3, CBL, CSF3R, CEBPA, IDH1, IDH2, JAK2, MPL, CARL.

Figure 2.Mutated genes in patients included in Group 1 (A) and Group 2 (B). (A) All 9 patients of Group 1 presented at least two mutations. (B) In Group 2, 13 cases (28%) did not present any mutation and are all represented in the last gray column. Genes are color coded and every case is represented in a column.