Screening for MPL mutations in Essential Thrombocythemia and Primary Myelofibrosis: Normal Mpl Expression and Absence of Constitutive STAT3 and STAT5 Activation in MPLW515L-Positive Platelets.

Abstract 3910

Poster Board III-846

The identification of exon 10 MPL mutations has begun to unravel the pathogenesis of JAK2V617F-negative essential thrombocythemia (ET) and primary myelofibrosis (PMF). Overall frequency of MPL mutations ranges from 1 to 4% for ET and 5 to 11% for PMF patients. Overexpression of these mutant alleles in cell lines and animal models lead to constitutive receptor activation, activation of downstream signaling pathways and hypersensitivity to thrombopoietin (TPO). However, the precise functional effects of these mutations in signaling and TPO-response in patient samples have not been investigated. Decreased Mpl expression is a molecular hallmark of myeloproliferative neoplasms (MPN). This defect is more frequent in patients positive for JAK2V617F, suggesting a biologic relationship between Mpl expression and the underlying molecular pathogenesis. The pattern of Mpl expression in patients with exon 10 MPL mutations has not been explored. The aim of this study was to analyze the frequency of MPLW515L, MPLW515K and MPLS505N mutations in a cohort of patients with ET and PMF and to determine whether MPLW515L leads to impaired Mpl expression, constitutive STAT3 and STAT5 activation and enhanced response to thrombopoietin (TPO) in patient samples. One in one hundred (1%) patients with ET and 1 in 11 with PMF were positive for MPLW515L by allele-specific PCR and sequencing in platelet and/or leukocyte samples, while none harboured the MPLW515K and MPLS505N mutations; both MPL515L-positive patients were JAK2V617F-negative. Platelet surface Mpl expression in the MPLW515L-positive ET patient by flow cytometry did not differ from a normal control, Mpl/isotype ratio was 3.6 vs 3.2, respectively, and normal total Mpl content was found by Western blot, Mpl/B3 integrin ratio was 99% of controls (n=5), while plasma TPO levels were mildly elevated by ELISA, 45.8 pg/mL vs 0 (0-32) pg/mL in controls (n=20). MPL transcripts by real-time RT-PCR in platelets from both MPLW515L-positive patients were similar to values found for this ET cohort (n=20), which did not differ significantly from normal controls (n=10), MPL/GAPDH ratio was 0.25 and 0.26, for MPL-positive patients, 0.24 (0.12-0.97) for MPL-negative patients, and 0.39 (0.21-0.78) for controls, p= 0.1. Constitutive STAT3 and STAT5 phosphorylation was not detected by immunoblotting and phosphorylation in response to increasing concentrations of TPO did not differ from controls. The low frequency of MPL mutations in this cohort is in agreement with previous studies, highlighting the need for identifying additional molecular defects in JAK2V617F-negative patients. The finding of normal Mpl levels in MPLW515L-positive platelets indicates this mutation does not lead to dysregulated Mpl expression, as frequently shown for MPN patients. Therefore, although impaired Mpl expression can arise from a molecular mechanism different from JAK2V617F, this phenotypic abnormality seems not to be linked to MPLW515L. The lack of spontaneous STAT3 and STAT5 activation and the normal response to TPO is unexpected as MPLW515L leads to constitutive receptor activation and hypersensitivity to TPO in experimental models.