cMPL is a gene encoding for the thrombopoietin receptor that is essential for thrombopoiesis and contributes to pluripotent hematopoietic stem cell expansion. A gain of function cMPL mutation, MPLW515L, was identified in myeloid cells from patients with primary myelofibrosis (PMF). Subsequent studies identified a second gain of function mutation, MPLW515K, in PMF and essential thrombocytosis (ET). The prevalence of MPLW515L and MPLW515K mutations was 5% in PMF and 1% in ET. No mutant cMPL was detected in Polycythemia Vera (PV). The methods utilized in these assays were sensitive to mutant frequencies of >3–5%. We developed a rapid, sensitive, quantitative real time PCR assay based on a unique primer design wherein allelic discrimination was enhanced by the synergistic effect of a mismatch in the −1 position, and a locked nucleic acid nucleoside at the −2 position of the allele-specific primers. An assay of similar design can detect G1849T mutation of JAK2 in <0.1% mutant allele in peripheral blood granulocyte (
Nussenzveig Exp Hematol 2007 3:32
). We hypothesized that a similar high sensitivity assay would increase detection of mutant cMPL in Ph−MPDs. We analyzed genomic DNA from peripheral blood granulocytes of 197 MPD patients and found that 10/197 (5.1%) carried one of the two cMPL mutations. Further, 5 of these patients were also JAK2V617F positive. cMPL mutations were detected in 1/78 (1.3%) PV patients, 3/56 (5.4%) ET patients, 4/49 (8.2%) PMF patients, and 2/11 (18%) MPD-Unspecified patients. W515L accounted for 9/10 cases, with W515K accounting for only 1. Of the ten positive samples, five (including the patient with PV) had ≤1% mutant alleles. To confirm the validity of our assay, we tested DNA from 96 normal controls. Neither W515L nor W515K was detected (p=0.03 compared to samples from the Ph−MPD patients). Additionally, when DNA from megakaryocytic colonies from a patient with 0.70% mutant alleles was analyzed, 12.5% of colonies were found to be heterozygous for cMPLW515L. These studies demonstrate the sensitivity and accuracy of our assay and show that cMPL activating mutations are more common in ET than previously reported. Mutant allele frequency appears greater in megakaryocytic cultures perhaps indicating a proliferative advantage for the cMPL-mutant clone. That mutant cMPL and JAK2V617F can be found in the same patient demonstrates the molecular heterogeneity of Ph−MPDs and emphasizes the need for prospective studies designed to determine the relationship between genotype and clinical phenotype. Scott J. Samuelson and Sabina Swierczek contributed equally to this project.
