LAUSR

In myeloproliferative neoplasms (MPN), somatic mutations in genes recurrently mutated in myeloid leukemia, apart from known MPN driver mutations (JAK2, MPL, CALR), are common and can increase the risk of transformation to acute leukemia, also called blast phase (BP). However, when mutations in these genes are acquired, the nature of the cells bearing these mutations, and how each mutation cooperates to promote blast transformation remains largely unknown. We therefore examined serially collected blood and bone marrow samples from patients with myelofibrosis (MF) who progressed to BP to further elucidate the genetic basis of blast transformation. The aims of our study were to determine the temporal acquisition of mutations during chronic phase (CP) that contribute to blast transformation and to define the cellular origins within the hematopoietic hierarchy of the clone fated to progress to BP (termed BP-fated clone). Our cohort included 9 MF patients (8 JAK2V617Fpositiveand 1 MPLW515Lpositive) who progressed to BP and for whom CP and BP samples were previously collected and banked. The time interval between CP and BP collections ranged between 1.5 to 6.6 years. In 4 patients, additional CP samples were available from intervening time points. Whole genome sequencing (WGS) of leukemic blasts (BP sample), the MPN clone (CP sample), and germline control (T-cells or buccal DNA) was performed to identify somatic mutations. We detected somatic mutations at BP in 19 genes recurrently mutated in myeloid leukemia (ML) with an average of 5.3 (range 2-8) genes mutated per patient. Genes that were mutated in 2 or more BP samples include SRSF2 (n=5), ASXL1 (n=4), TET2 (n=4), IDH1/2 (n=4), RUNX1 (n=4), NRAS (n=4), KRAS (n=2), U2AF1 (n=2), PHF6 (n=2), and STAG2 (n=2). Notably, 73% of ML gene mutations identified at BP were already acquired and present at ≥5% variant allele frequency in CP. The remaining (27%) ML gene mutations were not detected at CP and were, thus, termed BP-specific mutations. To achieve our study aims, we sorted hematopoietic stem and progenitor cell (HSPC) populations (HSC, MPP, LMPP, CMP, MEP, GMP), as well as, mature cell populations (myeloid, erythroid, T-cell, B-cell and NK) from CP samples and performed DNA whole genome amplification. We then interrogated sorted cell populations for BP-specific mutations by droplet digital PCR (ddPCR) to identify low frequency mutations. Our results revealed that BP-specific ML gene mutations could be detected at low frequencies (range between 0.2-5%) in one or more cell populations several years (1.5-3 years) prior to BP diagnosis. Importantly, we detected these low frequency mutations within the HSC population from several patients, indicating that BP-fated clones were derived from an HSC. This finding is being verified in all patients by targeted sequencing of additional BP-specific mutations that were identified by WGS (average of 300 variants per patient, range 37 to 659). In one patient analyzed to date, additional low frequency BP-specific mutations have been detected within the HSC population, and thereby confirm the BP cell of origin as an HSC in this individual. Generalization of this finding will be confirmed by targeted sequencing of sorted populations from the remaining patients. In summary, BP-fated clones often appear several years prior to blast transformation and can be traced back to HSCs. Identification of BP-fated clones that remain dormant strongly suggests that mechanisms beyond the acquisition of somatic mutations in ML genes (including but not limited to epigenetic alterations, acquisition of non-coding mutations, inflammation) are necessary to effectively promote full leukemic transformation. McNamara: Novartis Pharmaceutical Canada Inc.: Consultancy. Maze:Pfizer Inc: Consultancy; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Tsui:Novartis: Consultancy, Honoraria. Minden:Trillium Therapetuics: Other: licensing agreement. Gupta:Incyte: Honoraria, Research Funding; Sierra Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Research Funding; Sierra Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees.