Clinical Significance of MPL Mutations in Essential Thrombocythemia: Analysis of the PT-1 Cohort.
Authors: Beer Philip , Campbell Peter , Erber Wendy , Scott Linda , Bench Anthony , Bareford David , Bridget Wilkins , Wheatley Keith , Buck Georgina , Harrison Claire , Green Anthony 
Published: October 2019

Abstract
Activating mutations in MPL, the thrombopoietin receptor, were originally described in idiopathic myelofibrosis (IMF) and subsequently in a small minority of patients with essential thrombocythemia (ET). The prevalence and clinical significance of MPL mutations in ET has not been fully characterised. Using prospectively acquired diagnostic and follow-up data from the large MRC PT-1 study, we describe the clinical characteristics, complications and laboratory features of these mutations. Sequencing the entire MPL cDNA from 19 JAK2 wild-type ET patients did not reveal any mutations outside exon 10. Sequencing MPL exon 10 DNA in 88 ET and 112 IMF patients revealed MPLW515L (n=11), MPLW515K (n=3) and MPLS505N (n=2) mutations. The MPLS505N mutation, previously associated with inherited thrombocythemia, was clearly an acquired mutation in both patients (1 ET and 1 IMF). Sensitive PCR-based methods were then developed for all three mutant MPL alleles and used to genotype 776 patients with ET from the PT-1 trial. MPL mutations were detected in 4% of all patients, comprising 9% of JAK2 wild-type patients. MPL mutant patients were older at diagnosis than both JAK2 wild-type (p=0.0001) and JAK2V617F patients (p=0.09). Compared to JAK2V617F patients, MPL mutant patients had lower hemoglobin and higher platelet levels at diagnosis (p=0.0001 and p=0.006 respectively). There were no differences in diagnostic blood counts between MPL mutant and JAK2 wild-type patients. Diagnostic trephine histology for JAK2V617F (n=168), JAK2 wild-type (n=130) and MPL mutant patients (n=13) was reviewed by three independent histopathologists blinded to mutation status. MPL mutant samples were less cellular than JAK2V617F and JAK2 wild-type samples (p=0.0001 and p=0.003 respectively). Compared to both JAK2V617F and JAK2 wild-type samples, MPL mutant samples had lower erythroid cellularity (p=0.0006 and p=0.005 respectively) and granulocyte cellularity (p=0.006 and p=0.05 respectively). There were no differences in megakaryocyte cellularity or reticulin grade between the three groups. MPL mutant patients had an increased risk of venous thrombosis after trial entry when compared to JAK2 wild-type patients (p=0.02); the rate of venous thrombosis in the MPL mutant group was comparable to the JAK2V617F group. The mutant allele burden (assessed quantitatively by pyrosequencing) was significantly higher in patients with MPLW515K compared to patients with MPLW515L mutations (p=0.0001), suggesting biological differences in the transforming abilities of the two mutations. In colony assays using patient mononuclear cells the MPLW515L mutation was associated with cytokine-independent growth of megakaryocyte colonies but not with erythropoietin-independent erythroid colony growth (4/4 patients). Of 48 MPL mutant patients, only one also carried the JAK2V617F mutation by allele-specific PCR. Taken together, our results describe the prevalence, clinical features and laboratory manifestations of MPL mutations in the context of a large prospective study of ET.