Chronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia are characterized by persistent monocytosis and combined dysplastic morphology with features of a myeloproliferative syndrome.1 Together with atypical chronic myelogenous leukemia (aCML) and the category of unclassifiable myelodysplastic/myeloproliferative diseases, they belong to the same WHO diagnostic group.1 Activation of the RAS signaling pathway is almost universal in juvenile myelomonocytic leukemia, albeit the result of diverse genetic lesions, including mutations of N-RAS, K-RAS, NF-1 and PTPN11 (reviewed in Lauchle et al.2). The genetics of CMML and aCML is yet more complex. Various studies reported activating mutations of N-RAS and K-RAS in 20 to almost 60% of patients, and mutations in PTPN11 in isolated cases.3, 4 NF-1 (neurofibromatosis 1) has not been studied extensively, but one study of patients with MDS (including CMML) and acute myeloid leukemia did not find major rearrangements at the genomic level.5 In contrast to juvenile myelomonocytic leukemia, activating mutations in tyrosine kinases, including PDGFRA/B, KIT, FGFR1, FLT3, CSF-1R and JAK2, have also been implicated in small subsets of patients.6, 7, 8, 9, 10, 11 Mutations in RAS or in tyrosine kinases primarily drive proliferation, and are collectively referred to as ‘type 1’ mutations. Type 1 mutations do not usually coexist within the same leukemic clone, although there are occasional exceptions.8, 12 The cumulative frequency of previously reported mutations in RAS or a tyrosine kinase in CMML and aCML is not known, as no comprehensive mutational analysis has been performed in such patients. However, given that most of the reported tyrosine kinase mutations are infrequent, it is likely that screening for established mutations in specific kinases and RAS would leave a considerable proportion of cases unexplained. We therefore hypothesized that previously unidentified tyrosine kinase mutations may be involved in the pathogenesis of CMML and aCML. To test this, we performed a comprehensive mutational screen of the tyrosine kinome in a cohort of such patients.
Thirty-two patients were included in this study. In 26 the disease was classified as CMML, in 5 as aCML and in 1 as an unclassifiable myelodysplastic/myeloproliferative disorder (Table 1). Karyotyping was unavailable for eight CMML patients, reflecting the fact that some samples were acquired up to 20 years ago. In five patients without available karyotype, BCR-ABL was excluded by fluorescence in situ hybridization (FISH). There was no evidence for a rearrangement of chromosome 5 in any of the patients with available cytogenetics. Primers were designed to amplify the activation loops and/or juxtamembrane domains of all 90 known human tyrosine kinases, the JH2 domains of JAK1, JAK2, JAK3 and TYK2, as well as the entire coding sequence of H-RAS, K-RAS and N-RAS. To exclude single nucleotide polymorphisms, all candidate mutations were compared with public databases and, where appropriate, with a panel of 96 samples of normal genomic DNA from individuals of similar ethnic background (for a list of primers and conditions, see Loriaux et al.13).
A total of 298 exons were sequenced, with good quality traces (Phred quality score of at least 2014) in 286 exons (96%). For 12 exons, we were unable to obtain good quality sequence, despite multiple attempts using a variety of primers for amplification and sequencing (Supplementary Table 1). We detected JAK2 V617F in three patients (9%). These patients have been reported previously.8 A number of novel sequence variations were identified. However, on comparison with a set of 96 DNA samples from normal individuals, these were all identified as single nucleotide polymorphisms with the exception of one sequence variation in EPHA8 (P607H) (Supplementary Table 2). This residue is highly promiscuous across the EPH kinase family and various species (Supplementary Figure 1) and therefore likely also represents a rare polymorphism or a ‘passenger mutation’. Thus, we did not detect novel or known mutations in any tyrosine kinase other than JAK2, including those in which mutations were previously reported in CMML (KIT, FLT3, CSF-1R) (Table 1). We detected RAS mutations in nine patients (22%), six in K-RAS and three in N-RAS, including two ‘non-canonical’ K-RAS mutations (T74P and A146T), which were found in one and two patients, respectively. Functional characterization of these mutations is reported elsewhere (manuscript under review) in the context of several novel RAS mutations associated with leukemia (Table 1).
Our results suggest that point mutations in established mutational hot spots in leukemia-associated tyrosine kinases, that is, the juxtamembrane domain and the activation loops, are rare in patients with CMML and aCML and that as yet unidentified genetic lesions must be responsible in those patients who do not exhibit mutations in RAS or in any of the other tyrosine kinases previously implicated. Such unidentified mutations could reside in tyrosine kinase domains not included in our sequence analysis (for example, the extracellular domain), in genes that regulate RAS or tyrosine kinases or they could lead to activation by mechanisms not detected by point mutation screening, such as translocations. Given the limited size of the cohort under study, it is also possible that we missed mutations that occur at a low or very low frequency. For example, assuming mutual exclusivity of type 1 mutations, the probability of detecting at least one mutant case in the 20 patients without RAS or JAK2 mutations would be 98.9% for a mutation occurring with an incidence of 20%. However, for a mutation with only 1% incidence, this probability falls to just 18.2%. The relative small size of our cohort may also explain the lack of detection of any patients with mutations of FLT3 (reported with a frequency of 3.1%) and CSF-1R (reported with a frequency of 8%).10 However, although it is clear that our study is not powered to detect one specific low-incidence mutation, low-frequency mutations in multiple kinases would not likely have escaped detection. Moreover, our data are consistent with two recent studies that failed to detect novel activating mutations in tyrosine kinases in large cohorts of patients with acute myeloid leukemia.13, 15 In contrast to these studies, the frequency of ‘passenger’ mutations was low (only N502S of DDR1 and P607H of EPHA8). Although the reason for this discrepancy is not known, one potential explanation is that CMML may exhibit a lesser degree of genomic instability compared with acute myeloid leukemia. In summary, we find no evidence that as yet unreported point mutations in the activation loops, juxtamembrane domains and pseudokinase domains of tyrosine kinases are a major pathogenetic mechanism in CMML and aCML.
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This study was supported in part by NHLBI Grant HL082978-01 (MWD), and the Leukemia and Lymphoma Society (MWD).
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Tyner, J., Loriaux, M., Erickson, H. et al. High-throughput mutational screen of the tyrosine kinome in chronic myelomonocytic leukemia. Leukemia 23, 406–409 (2009). https://doi.org/10.1038/leu.2008.187
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