Multistep pathogenesis of chronic myelomonocytic leukemia in patients.

BACKGROUND: A multistep pathogenesis of myeloid leukemia including mutations in epigenetic, spliceosome, and signaling genes has been recently demonstrated in a preclinical model but is poorly validated in patients. METHODS: Clinical, phenotypic, and biologic features were compared between three distinct molecularly defined CMML cohorts including TET2 monomutated patients (T, n = 10), TET2/SRSF2 bimutated patients (TS, n = 19), and patients who had NRAS mutations in addition to TET2/SRSF2 comutations (TSN, n = 14). RESULTS: Median survival was 90, 45, and 9 months, respectively (p = .001). Whereas no patient in the T and TS group transformed into acute myeloid leukemia (AML), 6/14 patients in the TSN group had AML at study entry or transformed during follow-up. Leukocyte counts, blast cell counts, and LDH levels were significantly higher in TSN vs. TS and T, respectively, whereas hemoglobin and platelet values were not significantly different. Increased growth factor-independent myeloid colony formation was restricted to TSN but not found in T and TS, respectively. The proportion of patients showing in vitro myelomonocytic skewing in T, TS, and TSN was 0%, 56%, and 100%, respectively (p = .010). CONCLUSION: Our results demonstrate that the model of multistep pathogenesis in CMML can be recapitulated in patients regarding clinical, phenotypic, and biologic features.

Myelomonocytic skewing in chronic myelomonocytic leukemia: phenotypic, molecular and biologic features and impact on survival.

BACKGROUND: Myelomonocytic skewing is considered as a key pathophysiologic phenomenon in chronic myelomonocytic leukemia (CMML), but its prevalence and potential correlation with phenotypic, genotypic, and clinical features are poorly defined. METHODS: Skewed differentiation toward the myelomonocytic over erythroid commitment as indicated by an inverse ratio of myelomonocytic/erythroid colonies was investigated in 146 patients with CMML by semisolid in vitro cultures. RESULTS: There was a high prevalence of myelomonocytic skewing in patients with CMML (120/146, 82%); whereas, this phenomenon was rare in normal individuals (1/98, 1%). Patients with CMML with myelomonocytic skewing had higher white blood cell and peripheral blast cell counts, and lower platelet values. The number of mutations in genes of the epigenetic and/or splicing category was higher in CMML patients with as compared with patients without skewing. Patients with myelomonocytic skewing had more frequently mutations in RASopathy genes and higher growth factor independent myeloid colony formation. Interestingly, the lack of myelomonocytic skewing discriminated patients with CMML with a particularly favorable prognosis (60 vs 19 months, P = .003) and a minimal risk of transformation. CONCLUSION: Myelomonocytic skewing as determined by semisolid cultures can discriminate subgroups of patients with CMML with a different phenotype, a different genotype, and a different prognosis.

Impact of age on the cumulative risk of transformation in patients with chronic myelomonocytic leukaemia.

In older patients with chronic myelomonocytic leukaemia (CMML) and limited life expectancy due to age and or comorbidities, it is particularly important to consider the risk of transformation for individualised treatment decisions. There is limited information on potential differences between younger and older CMML patients regarding the cumulative risk of transformation as well as haematological, molecular and biologic characteristics. We analysed data from the Austrian Biodatabase for CMML (ABCMML) to compare these parameters in 518 CMML patients. Categorisation of patients into 3 age-related groups: <60 years, 60-79 years and ≥80 years, showed a significantly lower risk of transformation at higher age by competing risk analysis, with a 4-year risk of 39%, 23% and 13%, respectively (P < .0001). The lower probability of transformation was associated with a lower percentage of blast cells in the peripheral blood (PB) of older patients. Furthermore, we provide a simple score based on age, PB blasts and platelet counts that allowed us to define subgroups of CMML patients with a different cumulative transformation risk, including a low-risk group with a transformation risk of only 5%. Our findings may facilitate reasonable treatment decisions in elderly patients with CMML.

Molecular Basis and Clinical Application of Growth-Factor-Independent In Vitro Myeloid Colony Formation in Chronic Myelomonocytic Leukemia.

We have originally reported that colony-forming units granulocyte/macrophage (CFU-GM) formation is an in vitro feature of chronic myelomonocytic leukemia (CMML) and a strong predictor for short survival. Elucidation of the molecular basis underlying this in vitro phenomenon could be helpful to define molecular features that predict inferior outcome in patients. We studied the correlation between the mutational landscape and spontaneous colony formation in 164 samples from 125 CMML patients. As compared to wildtype samples, spontaneous in vitro CFU-GM formation was significantly increased in samples containing mutations in NRAS, CBL and EZH2 that were confirmed as independent stimulatory factors by multiple regression analysis. Inducible expression of mutated RAS but not JAK2 was able to induce growth factor independence of Ba/F3 cells. Whereas high colony CFU-GM growth was a strong unfavorable parameter for survival (p < 0.00001) and time to transformation (p = 0.01390), no single mutated gene had the power to significantly predict for both outcome parameters. A composite molecular parameter including NRAS/CBL/EZH2, however, was predictive for inferior survival (p = 0.00059) as well as for increased risk of transformation (p = 0.01429). In conclusion, we show that the composite molecular profile NRAS/CBL/EZH2 derived from its impact on spontaneous in vitro myeloid colony formation improves the predictive power over single molecular parameters in patients with CMML.

Correlation of RAS-Pathway Mutations and Spontaneous Myeloid Colony Growth with Progression and Transformation in Chronic Myelomonocytic Leukemia-A Retrospective Analysis in 337 Patients.

Although the RAS-pathway has been implicated as an important driver in the pathogenesis of chronic myelomonocytic leukemia (CMML) a comprehensive study including molecular and functional analyses in patients with progression and transformation has not been performed. A close correlation between RASopathy gene mutations and spontaneous in vitro myeloid colony (CFU-GM) growth in CMML has been described. Molecular and/or functional analyses were performed in three cohorts of 337 CMML patients: in patients without (A, n = 236) and with (B, n = 61) progression/transformation during follow-up, and in patients already transformed at the time of sampling (C, n = 40 + 26 who were before in B). The frequencies of RAS-pathway mutations (variant allele frequency ≥ 20%) in cohorts A, B, and C were 30%, 47%, and 71% (p < 0.0001), and of high colony growth (≥20/105 peripheral blood mononuclear cells) 31%, 44%, and 80% (p < 0.0001), respectively. Increases in allele burden of RAS-pathway mutations and in numbers of spontaneously formed CFU-GM before and after transformation could be shown in individual patients. Finally, the presence of mutations in RASopathy genes as well as the presence of high colony growth prior to transformation was significantly associated with an increased risk of acute myeloid leukemia (AML) development. Together, RAS-pathway mutations in CMML correlate with an augmented autonomous expansion of neoplastic precursor cells and indicate an increased risk of AML development which may be relevant for targeted treatment strategies.

RAS mutations drive proliferative chronic myelomonocytic leukemia via a KMT2A-PLK1 axis.

Proliferative chronic myelomonocytic leukemia (pCMML), an aggressive CMML subtype, is associated with dismal outcomes. RAS pathway mutations, mainly NRASG12D, define the pCMML phenotype as demonstrated by our exome sequencing, progenitor colony assays and a Vav-Cre-NrasG12D mouse model. Further, these mutations promote CMML transformation to acute myeloid leukemia. Using a multiomics platform and biochemical and molecular studies we show that in pCMML RAS pathway mutations are associated with a unique gene expression profile enriched in mitotic kinases such as polo-like kinase 1 (PLK1). PLK1 transcript levels are shown to be regulated by an unmutated lysine methyl-transferase (KMT2A) resulting in increased promoter monomethylation of lysine 4 of histone 3. Pharmacologic inhibition of PLK1 in RAS mutant patient-derived xenografts, demonstrates the utility of personalized biomarker-driven therapeutics in pCMML.

The Austrian biodatabase for chronic myelomonocytic leukemia (ABCMML) : A representative and useful real-life data source for further biomedical research.

In the Austrian biodatabase for chronic myelomonocytic leukemia (ABCMML) clinicolaboratory real-life data have been captured from 606 CMML patients from 14 different hospitals over the last 30 years. It is the only large biodatabase worldwide in which functional methods such as semisolid in vitro cultures complement modern molecular methods such as next generation sequencing. This provides the possibility to comprehensively study the biology of CMML. The aim of this study was to compare patient characteristics with published CMML cohorts and to validate established prognostic parameters in order to examine if this real-life database can serve as a representative and useful data source for further research. After exclusion of patients in transformation characteristics of 531 patients were compared with published CMML cohorts. Median values for age, leukocytes, hemoglobin, platelets, lactate dehydrogenase (LDH) and circulating blasts were within the ranges of reported CMML series. Established prognostic parameters including leukocytes, hemoglobin, blasts and adverse cytogenetics were able to discriminate patients with different outcome. Myeloproliferative (MP) as compared to myelodysplastic (MD)-CMML patients had higher values for circulating blasts, LDH, RAS-pathway mutations and for spontaneous myelomonocytic colony growth in vitro as well as more often splenomegaly. This study demonstrates that the patient cohort of the ABCMML shares clinicolaboratory characteristics with reported CMML cohorts from other countries and confirms phenotypic and genotypic differences between MP-CMML and MD-CMML. Therefore, results obtained from molecular and biological analyses using material from the national cohort will also be applicable to other CMML series and thus may have a more general significance.