Patient Derived Xenotransplantation Model of Atypical Chronic Myeloid Leukemia (aCML)

Background:

Atypical chronic myeloid leukemia (aCML) is a rare disorder classified as one of the MPN/MDS overlap syndromes. aCML usually presents like CML but lacks the pathognomonic BCR-ABL fusion found in CML. Most patients progress to acute myeloid leukemia (AML) with a median time to AML of 11.2 months and have a median overall survival of only 12.4 months (Wang et al. Blood 2014). Recurrently mutated genes found in aCML patients include SETBP1 , CSF3R, NRAS, EZH2, ASXL1, ETNK1, and U2AF1.

The pathogenesis of aCML is poorly understood and neither specific nor effective treatments besides hematopoietic stem cell transplantation are available. We therefore aimed at developing a patient derived xenotransplantation model that allows serial transplantation and expansion of human leukemic cells and evaluation of novel treatments and drugs in vivo.

Patient and Methods:

Bone marrow cells were harvested from a patient diagnosed with atypical CML based on persistent leukocytosis, immature circulating myeloid precursors (16% metamyelocytes, 8% myelocytes, 9% blasts), marked dysgranulopoiesis, minimal monocytosis and basophilia, hypercellular bone marrow with high myeloid/erythroid ratio and 6% myeloid blasts, dysplasia in megakaryocytes and erythroid progenitors, and absence of BCR-ABL and mutated JAK2. The patient had moderate anemia and normal platelet counts and cytogenetic analysis showed a normal karyotype.

Eight hundred thousand bone marrow cells were injected intravenously in NOD/SCID IL-2 receptor γ (NSG) deficient mice. We monitored these mice for human cell engraftment by regular eye bleeds every 4 weeks. Bone marrow and spleen cells from engrafted mice were retransplanted in secondary and tertiary mice of the NSG strain transgenic for SCF, IL3 and GM-CSF (NSGS). Patient cells were analyzed for mutations in fifty four genes by next generation sequencing and mutations were confirmed by Sanger sequencing in primary patient cells and cells from tertiary mice.

Results: Human CD45+ cells from the aCML patient showed increasing engraftment over time in the NSG mouse reaching 16% in peripheral blood and 35% in spleen at 26 weeks after transplantation. In secondary (n=2) and tertiary (n=4) mice we used NSGS recipient mice and observed considerably accelerated engraftment kinetics leading to 19, 21 and 73% human cells in peripheral blood, spleen and bone marrow, respectively, between 12 and 15 weeks after transplantation. The myeloid marker CD33 was expressed in 86% of human bone marrow cells, while lymphoid markers CD3 and CD19 were absent. The stem and progenitor phenotype CD34+CD38- was found in 11% of human cells. The progenitor marker CD123 was expressed in 42% of cells, while the myeloid marker CD14 was expressed in 6% of cells. Hemoglobin levels and platelet counts were considerably lower in secondary and tertiary recipients of aCML cells compared to control animals. Spleens were enlarged at time of sacrifice with an average spleen weight of 150 mg. Morphological evaluation of bone marrow cells in tertiary recipients revealed a characteristic picture for aCML with 39% neutrophils, 8% blasts and 53% myeloid progenitors and monocytes. Molecular analysis identified mutations in ASXL1, RUNX1 and EZH2 with variant allele frequencies of 49, 48 and 46 percent, respectively that were confirmed in human cells from tertiary recipient mice. Thus, we show that primary aCML cells can be expanded and serially transplanted in immunodeficient mice and suggest clonal stability of this model.

Conclusion:

We provide the first patient derived xenotransplantation model for atypical CML, which preserves the phenotypic and molecular characteristics of the primary disease and allows serial transplantation and expansion of aCML cells. This model will serve to better understand the pathogenesis of aCML and to test urgently needed novel treatment approaches.