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

Abstract

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.

Keywords: AML; CFU-GM; CMML; NGS; in vitro cultures; prognosis.

Figure 1

Overall survival (a) and time to acute myeloid leukemia transformation (b) in chronic myelomonocytic leukemia (CMML) patients stratified by the presence or absence of high (≥100/10⁵ mononuclear cells (MNC)) spontaneous in vitro myeloid colony formation.

Figure 2

Ba/F3 cells with inducible expression of JAK2 V617F (a) or RAS G12V cDNA (b) were cultured with or without doxycycline (to induce oncogene expression) in the presence of IL-3. Then, cells were washed and incubated in various concentrations of recombinant murine IL-3 (0–1000 pg/mL) for 48 h. After incubation, proliferation was determined by ³H uptake. Results are expressed as percent of control (³H-thymidine uptake in the presence of the maximum IL-3 concentration) and represent the mean ± SD of three independent experiments.

Figure 3

Overall survival (a) and time to AML transformation (b) in CMML patients stratified by the presence or absence of molecular aberrations. MT—mutated; WT—wildtype; COMP—composite molecular profile including mutations in NRAS, CBL or EZH2.