A Case of Chronic Myelomonocytic Leukemia Progressing from Dysplastic to Proliferative Phenotype after Acquisition of a RAS Point Mutation.

Chronic myelomonocytic leukemia (CMML) is a myeloid malignancy characterized by high heterogeneity of clinical and hematological features, classified into a new category of myelodysplastic/myeloproliferative diseases by the last World Health Organization. Poor data are available on the biology of CMML and molecular mechanisms related to disease development and progression. Among hematological malignancies, CMML has the highest frequency of point mutations of the RAS gene family (20% to 35% of patients). Patients with proliferative variant of the disease (MP-CMML, WBC > 12 x 10⁹/L) have significantly higher frequency of RAS point mutations than patients with dysplastic variant (MD-CMML, WBC < 12 x 10⁹/L). In fact RAS mutations have been shown to associate with features of cell proliferation and monocytosis rather than dysplasia. However, the exact role of RAS mutations in the pathophysiology of CMML is unknown. Ras family members are small G-proteins that regulate cellular proliferation, differentiation and apoptosis. Switching from the active GTP- to the inactive GDP-bound state is associated with a conformational change of the switch I (residues 30–38) and switch II (60–76) regions. Activating point mutations of codons 12, 13, 59, 61 and 63 have been shown to decrease the weak intrinsic GTPase activity of Ras, leading to its constitutive activation. In April 2003 a patient with diagnosis of MD-CMML (WBC = 6,5 x 10⁹/L) was admitted at our hospital. Genomic DNA extracted from peripheral blood mononuclear cells was amplified with primers for exons 1 and 2 of N- and K-RAS by PCR, and subsequently sequenced. At this stage of the disease, no mutation was detected. In January 2005 the patient experienced a rapid progression to MP-subtype, with WBC counts constantly over 30 x 10⁹/L. Molecular analysis was repeated and a mutation GGA → GAA in codon 60 of N-RAS was found, leading to the substitution of Glycine with Glutamate in the corresponding aminoacidic position. PCR amplification and direct sequencing of both forward and reverse strands from the genomic sample were repeated twice and the presence of mutation confirmed. Gly⁶⁰ is highly conserved among Ras family members; it is localized in the switch II region which is involved in the binding to GTPase-activating proteins (GAPs) and in the catalytic mechanism. Preliminary data, obtained from a computational model of the Gly⁶⁰ →Glu mutant protein, show that the negative charge of Glutamate replacing the neutral aminoacid Glycine may exert a highly destabilizing effect by interfering with electrostatic interactions of the “arginine finger” of GAPs, thus altering the conformational changes necessary for Ras activation. Mutations affecting codon 60 of Ras proteins were rarely reported in human cancer and consequently little is known about their transforming potential; however, detection of Gly⁶⁰ →Glu substitution in a CMML patient after progression from MD- to MP- phenotype suggests a pathogenetic event. To the best of our knowledge this is the first time that a RAS point mutation is identified concomitantly with progression of CMML from dysplastic to proliferative variant. Based on these results, further experiments are on going to characterize the novel mutant protein and thus to define whether N-Ras activation may be effectively involved in the progression of CMML.