JAK2 R1063H Variant Enhances V617F Constitutive Signaling and Favors Development of Essential Thrombocythemia with Increased Hemoglobin and Neutrophils

The clinical consequences of the highly prevalent JAK2 V617F mutation in Ph-negative myeloproliferative neoplasms (MPNs) are well studied. However, the impact on the MPN phenotype of co-occurring JAK2 variants is less well characterized. In this study, we focused on JAK2 R1063H, a variant previously reported in a pediatric case of hereditary erythrocytosis (Kapralova et al, Blood 2016) and in 3 out of 93 polycythemia vera patients that were JAK2 V617F-positive (Levine et al, Cancer Cell 2005). It has been described as a very rare polymorphism in population (frequency 0.004377). In order to gain insight into the clinical and functional relevance of coexisting JAK2 mutations, we tested a cohort of 390 JAK2 V617F-positive MPN patients for JAK2 R1063H and identified 14 carriers of both mutations. From the clinical perspective, the double-positive patients exhibited predominantly an essential thrombocythemia phenotype that was accompanied by significantly higher neutrophil granulocyte counts and hemoglobin values, when compared to those harboring only JAK2 V617F mutation. By employing targeted NGS for genes known to be involved in myeloid malignancies we found that the mutational profile and the number of additional somatic mutations in double-mutant patients seemed to be comparable to previous studies of JAK2 V617F-positive MPNs. Next, we used digital droplet PCR (ddPCR) for JAK2 R1063H and JAK2 V617F allele burden assessment. Quantification of JAK2 R1063H allele indicated that in 8 patients the variant was heterozygous, likely inherited (the percentage of the mutant allele being around 50%). In 3 patients with a high JAK2 V617F allelic burden a nearly homozygous status for R1063H was identified (a fractional abundance >80%) suggesting that one R1063H allele was inherited and the second one was acquired by uniparental disomy, as a result of mitotic recombination. In 3 other patients, the JAK2 R1063H mutation was most likely acquired due to the low percentage of the mutant allele (between 20.7% and 31.5%). For cis/trans configuration analysis of JAK2 V617F and R1063H mutations, we performed single-colony sequencing of subcloned JAK2 cDNA from 9 out of 14 patients. A cis configuration of the mutations was detected in 7 and a trans configuration in 2 cases. In one patient, the cis configuration of JAK2 mutations combined with results obtained by ddPCR (the allele burdens for R1063H and V617F were 31.5% and 52.8%, respectively) suggested that R1063H was acquired,