46/1 Haplotype Permits to Follow JAK2 Homologous Recombination: Modeling JAK2^V617F clonal Architecture in PV Patients

Abstract 1757

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell (HSC) disorders characterized by excess proliferation of one or several myeloid lineages. More than 95% polycythemia vera (PV) and 50–60% essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients harbor a somatic 1849 G>T mutation in JAK2 gene. Moreover about 30% of PV patients are homozygous for this mutation due to a loss of heterozygosity after a mitotic homologous recombination (HR). Among 92 haplotypes of chromosome 9p, 46/1 haplotype is strongly associated with the cis-aquisition of JAK2^V617F mutation. The purpose of this study was to estimate the clonal frequency of WT, JAK2^V617F/+ and JAK2^V617F/V617F in progenitors compartments. Here, we have modeled the JAK2^V617F clonal architecture in 9 PV patients heterozygous for the 46/1 haplotype by using the level of JAK2 and the 46/1 haplotype as a marker to follow HR. First we measured the global JAK2^V617F and 46/1 allele burden in CD34⁺ cells either by allele-specific PCR or by Ion Torrent sequencing in order to calculate the expected WT, JAK2^V617F/+ and JAK2^V617F/V617F clones. Next, we compared the results with the experimental clonal frequency of WT, JAK2^V617F/+ and JAK2^V617F/V617F cells in individual colonies derived from the CD34⁺CD38⁺ compartment. In majority of patients, the observed values corresponded to the expected values suggesting that JAK2 46/1 haplotype can be used to estimate JAK2^V617F clonal structure in PV patients. In three JAK2 46/1 heterozygous hemochromatosis patients used as controls, no JAK2 46/1 homozygous clone was observed showing that 46/1 haplotype itself was not responsible for HR. Furthermore, we have studied the proliferative advantage of the mutated clones in patients. No proliferative advantage of JAK2^V617F clone has been observed in between CD34⁺CD38⁻ and CD34⁺CD38⁺ progenitors stages whereas strong amplification of JAK2^V617F clone was found in terminally differentiated polynuclear neutrophils (PNN). Moreover, during evolution of MPN in one patient, we observed an amplification of the JAK2^V617F/V617F clone in both the CD34⁺CD38⁻ and CD34⁺CD38⁺cell compartments suggesting acquisition of a proliferative advantage of the homozygous clone over time.

This simple modeling could help to understand the effect of treatments on the JAK2^V617F clonal structure without working at the unicellular level.