Utility of Multiplex Mutation Analysis in the Diagnosis of Chronic Myelomonocytic Leukemia

Chronic myelomonocytic leukemia (CMML) is a myeloid neoplasm characterized by both myeloproliferative and myelodysplastic features in addition to persistent peripheral blood monocytosis (>1×109/L) that is required for the diagnosis. Clonal cytogenetic abnormalities are identified in only 20%-30% of CMML patients and it can be diagnostically challenging to exclude reactive monocytosis in some cases. Several gene mutations have recently been implicated in the pathogenesis of CMML that involve tyrosine kinase-signaling pathways, transcriptional regulation, metabolism, splicing, and epigenetic regulatory mechanisms. This study was designed to assess recurrent mutations in CMML using a multiplex mass spectrometry based approach, and to determine the utility of mutation screening in CMML, particularly in cytogenetically normal cases. The Oregon Health and Science University (OHSU) surgical pathology database was searched from 2010-2012 to identify consecutive CMML cases fulfilling WHO diagnostic criteria. Cytogenetic analyses and molecular studies were performed on the diagnostic bone marrow specimens. DNA extracts were screened for point mutations using a multiplex PCR panel with mass-spectroscopy read out that covers 370 point mutations across 31 genes associated with leukemia. Of the 48 CMML cases identified in the OHSU files, 43 had available cytogenetic studies. Of these, 10/43 cases (23%) had cytogenetic abnormalities including: trisomy 8 (n=4), trisomy 21 (n=2), deletion 7q (n=1), del 13q (n=1), complex karyotype (n=1) and t (3;3) (n=1). Of the cases with cytogenetic data, 22 had available DNA for mutation analysis, and 11 of these genotyped cases (50%) had detectable mutations in the following genes: CBL (n=3), CKIT, JAK2, KRAS (n=2), NRAS (n=3) and NPM1. Nine cases with detected mutations had normal cytogenetics. Concomitant molecular and cytogenetic abnormalities were seen in 2 cases: one case with trisomy 8 and CBL C384Y and one case with trisomy 21 and JAK2 V617F. In the 22 cases with available cytogenetic and molecular data, performing routine multiplex molecular testing in addition to cytogenetic studies in CMML patients increased the detection of genetic abnormalities from 23% (5/22) to 64% (14/22), with frequent CBL and RAS mutations in our cohort. This study confirms that gene mutations are common events in CMML, and multiplex mutation analysis can be applied in the clinical setting to assist in diagnosis and may identify actionable mutations for targeted therapy.