Whole Exome Sequencing of Polycythemia Vera Reveals Novel Recurrent Somatic and Germline Variation

Abstract 705

Polycythemia vera (PV) is a clonally derived hematopoietic malignancy arising from a multipotent hematopoietic stem cell (HSC). Almost all patients have the acquired somatic mutation, JAK2^V617F. There is strong evidence suggesting that the JAK2^V617F mutation is not a PV-initiating genetic event in its pathogenesis. Thus, “pre- JAK2^V617F mutation(s)” account for clonal dominance before the acquisition of the JAK2^V617Fmutation. The existence of PV family clustering indicates that germline genetic variations contribute to PV genesis.

To explore possible PV-initiating events, we performed deep exome sequencing of 31 JAK2^V617F-positive PV patients. DNA from granulocytes (clonal cells) and their matched T-lymphocytes (polyclonal cells with germline DNA) were collected and exonic regions were sequenced to ∼130-fold coverage to identify somatic and germline base substitutions, insertions and deletions (indels), as well as copy number alterations (CNA). DNA from skin was also examined in 6 PV patients and was sequenced to ∼117-fold coverage in order to rescue any germline events that may have been missed in the T-lymphocytes.

Our findings confirm the somatic loss of heterozygosity (LOH) and trisomy at 9p. The proportion of JAK2^V617 as determined by the fraction of the reads harboring the mutations in the patient's granulocytes varied from almost undetectable to 100% of the reads. As previously reported, the degree of LOH at 9p correlated with the variant fraction of JAK2^V617F with stronger LOH associated with higher allelic burden of JAK2^V617F. We also observed single examples of LOH on 22q and 18p of which the latter is a novel finding in this study. In addition, we observed focal amplification at the PRSS1 locus in 75% of the PV granulocytes. Nine patients also harbor recurrent somatic mutations in genes with known roles in hematologic malignancies including four patients (13%) carrying IDH2 somatic mutations, 1 previously described and 2 novel. Additional somatic mutations were found in several other genes functioning in the growth signaling and chromatin remodeling processes.

Remarkably, four patients exhibited novel germline variation in JAK2 itself, including one canonical JAK2^V617F variant and 3 other missense variants. Moreover, nearly one half of the patients harbor novel germline variants in FAT2, a tumor suppressor essential for controlling cell proliferation. Interestingly, we also observed in 71% of patients, a germline common allele of EXO1, rs1047840, which has been linked to multiple cancers by genome-wide association studies (GWAS).

Our study, using whole exome sequencing, recapitulated all of the major findings of PV genomic alteration from earlier studies. Prior to this study JAK2^{V617 or K539L} and associated 9p LOH, were the only known somatic variation. We have extended previous observations by demonstrating recurrent germline and somatic variation that potentially could contribute to the initiation of the disease. Both CNA alterations and point mutations in important cancer genes were seen in a significant number of patients and germline variants were seen in almost all patients in the cohort. This study finds strong evidence in support of the hypothesis of a second hit contributing to PV onset and progression as well as germline predisposition of PV.