Identification of Tyrosine Kinase Mutations by Large-Scale DNA Sequencing in Patients with Chronic Myelomonocytic Leukemia/Atypical Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is caused by Bcr-Abl, a constitutively active tyrosine kinase. Chronic myelomonocytic leukemia (CMML) and atypical chronic myeloid leukemia (aCML) both represent diseases with clinical features resembling CML, suggesting that their pathogenesis may be similar. Consistent with this, activating mutations of the platelet-derived growth factor receptor (PDGFR) and other tyrosine kinases are found in isolated patients, but the pathogenesis remains unclear in the majority. Elucidation of mechanistically important targets in the pathogenesis of CMML/aCML would allow for the design of targeted therapies for these disorders, perhaps repeating the success of imatinib in CML. To that end, we undertook a large-scale DNA sequencing screen aimed at identifying clinically important mutations in CMML.

Methods: We assessed the genotypic status of 298 exons comprising the activation loop, juxtamembrane domain, and pseudokinase domain (where applicable) of all members of the tyrosine kinase family as well as exon 2 of the small GTPase, K-RAS. Ninety-six percent of exons were successfully amplified and sequenced in a 32-patient cohort with CMML/aCML. Sequence data was analyzed in comparison to wild type, and all mutations were screened against the database of human single-nucleotide polymorphisms (SNPs) for elimination of previously determined, clinically non-relevant sequence variations. Non-annotated amino acid exchanges were counterchecked by sequencing the respective exons in 96 healthy controls.

Results: DNA sequence analysis of patients with CMML revealed that 19% of patients harbored the K-RASG13D mutation previously reported in acute myelogenous leukemia (AML) and CMML/aCML. In addition, 9% of patients harbored the JAK2V617F mutation that has been implicated in various myeloproliferative disorders. However, no other previously described, activating mutation was found in this DNA sequence screen. Notably, activating mutations in FLT3 and c-KIT, as detected in AML patients, were not found. We did, however, identify 73 different sequence variations. After exclusion of published SNPs and silent mutations, we identified thirty-one novel amino acid-changing mutations in 27 different exons. Thus far, comparison with normal controls has been completed for 20 exons and is ongoing in the remainder. Ten potential mutations were identified as previously undescribed SNPs, while 17 may represent true mutations. These include the tyrosine kinases FRK, FLT4, and EPHA8 among others. Studies are ongoing to determine the functional relevance of these novel mutations.

Conclusions: Less than one-third of patients with CMML have previously described, activating tyrosine kinase or K-RAS mutations. Hence, as many as 70% of patients suffer from disease of unknown molecular origin. DNA sequence analysis has revealed several candidate mutations that may, upon further investigation, lead to molecular targets for therapeutic intervention in patients with CMML/aCML.