Use Of 46/1 Haplotype Permits To Follow JAK2^V617F Clonal Architecture In PV Patients: Clonal Evolution and Impact Of IFNα Treatment

Somatic V617F oncogenic mutation of the protein kinase JAK2 is the most prevalent genetic abnormality in the three myeloproliferative neoplasms (MPNs), namely polycythemia vera (PV, 95%), essential thrombocythemia (ET, 55%) and myelofibrosis (MF, 50%). About 30% of PV patients are homozygous for this mutation due to mitotic homologous recombination (HR). JAK2 46/1 haplotype is strongly associated with the cis-aquisition of JAK2^V617F mutation. Since, HR involves most of 46/1 haplotype, JAK2^V617F and 46/1 tagging SNPs are also reduced to homozygosity. We hypothesized that 46/1 tagging SNPs, which are in complete linkage disequilibrium with JAK2, can serve as a measure of JAK2^V617F homozygosity. 46/1 allele burden (X%) can be used to calculate the HR (HR%) that is a measure of JAK2^V617F/V617F clones [(X%-50%)x2]. JAK2^V617F/V617F frequency and JAK2^V617F allele burden (Y%) can be then exploited to calculate the frequency of JAK2^V617F/WT [(Y%-RH%)x2] and JAK2^WT [100-(JAK2^V617F/WT%+ JAK2^V617F/V617F)] clones. The purpose of this study was to calculate the clonal frequency of WT, JAK2^V617F/WT and JAK2^V617F/V617F in progenitors compartments of PV patients.

Here, we have dissected the JAK2^V617F clonal architecture in 9 PV patients heterozygous for the 46/1 haplotype. 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/WT and JAK2^V617F/V617F clones. Next, we compared thees results with the experimental clonal frequency of WT, JAK2^V617F/WT and JAK2^V617F/V617F clones in individual colonies derived from the CD34⁺CD38⁺ compartment. This algorithm was validated in majority of patients. Moreover, we have exploited this formula to the terminally differentiated polynuclear neutrophils (PNN) and found that JAK2^V617F/V617Fclones acquire strong amplification during differentiation.

Finally, we used this model to assess the therapeutic potential of IFNα in a cohort of 15 PV patients. IFNα exposure resulted in more impressive and rapid decrease of JAK2^V617F allele burden of pure JAK2^V617F homozygous patients than in pure JAK2^V617F heterozygous patients. Calculations revealed this decrease in JAK2^V617F is due to preferential targeting of JAK2^V617F/V617Fclones in responding patients.

These results demonstrate that JAK2 46/1 haplotype can be used to estimate JAK2^V617F clonal architecture in PV patients. This simple modeling can be useful to follow the efficacy and specificity of treatment on JAK2^V617F clones in MPNs, without needing exploration at the unicellular level. In addition, it suggests that IFNα treatment more specifically targets the JAK2^V617F/V617F clone in responding patients.