Myeloproliferative Neoplasm (MPN) Blastic Transformation Occurs at the Level of Hematopoietic Stem Cells
Authors: Wang Xiaoli , Hu Cing Siang , Tripodi Joseph , Najfeld Vesna , Petersen Bruce , Rampal Raajit K. , Farnoud Noushin , Famulare Christopher , Mascarenhas John , Hoffman Ronald 
Published: October 2019

Myeloproliferative neoplasm-blast phase (MPN-BP) and de novo acute myeloid leukemia (AML) each have distinct mutational patterns and clinical courses. MPN-BP patients have a particularly dismal prognosis with a median survival of less than 6 months with currently available therapies. So far, the cellular hierarchy that characterizes MPN-BP and the evolution of various leukemia-initiating clones (LIC) in MPN-BP have not been well delineated. We therefore established an in vivo MPN-BP xenograft model to address these questions.

Among the 22 patients with MPN-BP studied 11 were cytogenetically normal while the remainder had multiple chromosomal abnormalities including del(5), del(20q), del(14), +1q, del(17p). 86% of the patients had at least 2 myeloid malignancy gene mutations including JAK2, ASXL1,TET2, MPL, SF3B1, RUNX1, U2AF1, PTPN11, IDH1/2, SRSF2 and TP53. These findings indicate that MPN-BP is characterized by multiple mutational events and cytogenetic abnormalities. T cell-depleted mononuclear cells from 8 of 14 patients engrafted in NSG mice {>0.5% hCD45+ cells in bone marrow (BM)}. Among them, samples from 6 patients resulted in a high degree of hCD45+ cell chimerism (34.6±6.4% in BM) and recapitulated numerous aspects of MPN-BP within 4 months, including the presence of at least 20% hCD45dimCD33+ cells or hCD34+ cells, or at least 20% blasts as detected by morphological examination of the marrow and leukemia cell dissemination to the spleen and PB. These mice had a 2.8±0.6- fold increase in splenic weight as compared to mice receiving PBS alone. The leukemic mice were characterized by reduced blood counts, suggesting that MPN-BP cells suppressed normal murine hematopoiesis, or led to cytopenias due to hyper-splenism. Moreover, the greater degrees of blast cell chimerism and the higher frequency of leukemia initiating cells as determined by limiting dilution analyses correlated with a shorter time to leukemia initiation and an inferior clinical outcome of the transplanted NSG mice. Grafts from each of these 6 MPN-BP patients produced a large number of donor-derived myeloid cells and a smaller number of lymphoid cells (mostly CD3+ and few CD19+). Cells belonging to each of these lineages and leukemic cells in primary recipients produced from Pts 4, 5, 6 and 11 had an identical proportion of chromosomally abnormal and mutated cells as primary cells [Pt 4: JAK2V617F, TET2 and PHF6; Pt 5 and 11: Del (20q), +8; Pt 6: +1q, del(17p)], except that a small proportion of T cells from Pts 5 and 11 lacked chromosomal abnormalities. Furthermore, the degree of MPN-BP engraftment and leukemic cell burden increased with the subsequent 3 serial transplantations even when the recipients received progressively smaller numbers of MPN-BP cells from the prior recipient. Primary Pt 6 originally had a JAK2V617F+ PV but lost JAK2V617F at the time the MPN-BP occurred at which time there were two clonal cell populations, one with +1q (12%) and the other del(17p) (80%), the site of the TP53 gene, as well as normal cells (8%). In the primary recipient NSG the donor derived cells were JAK2V617F- but contained +1q (1%) and del(17p) (98.5%) and cytogenetically normal (0.5%). +1q and JAK2V617F were not observed, while cells containing the TP53 deletion alone were detected in donor derived leukemic cells, mature myeloid and T cells in the secondary and subsequent serial recipients. Furthermore, del(17p) was found in phenotypically isolated HSCs, MPPs, MLPs, CMPs, GMPs, MEPs, and mature T cells within the CD33- cell fraction as well as CD45dimCD33+ AML blasts selected from primary MPN-BP cells from Pt6. However, +1q was found exclusively in purified MLPs and MEP. These observations establish that cytogenetic and mutational events that lead to MPN-BP occur at different stages along the developmental HSC hierarchy and that a small population of normal HSCs persist. Furthermore, in JAK2V617F+ MPNs that develop MPN-BP and lose JAK2V617F, additional cytogenetic events occur at different stages along the JAK2V617F- MPN-BP-stem cell hierarchy. Our ability to serially transplant the LIC from these patients has allowed us to create the first MPN-BP PDX model that will not only extend our understanding of MPN-BP stem cell biology but might also prove useful for screening drugs to treat MPN-BP.

Disclosures

Rampal: Jazz: Consultancy, Honoraria; Incyte: Honoraria, Research Funding; Stemline: Research Funding; Constellation: Research Funding; Celgene: Honoraria. Mascarenhas:Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Roche: Research Funding; Novartis: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Promedior: Research Funding; Janssen: Research Funding. Hoffman:Formation Biologics: Research Funding; Summer Road: Research Funding; Merus: Research Funding; Incyte: Research Funding; Janssen: Research Funding.