In vitro expansion of erythroid progenitors from polycythemia vera patients leads to decrease in JAK2 allele

Abstract

Objectives-A G>T transversion in a tyrosine kinase JAK2 (V617F) was reported in over 80% of patients with polycythemia vera (PV). Current evidence suggests that JAK2 V617F somatic mutation is involved in the pathogenesis of PV, since it confers erythropoietin independent proliferation to erythroid progenitor cells. However, several unanswered questions regarding the essential role of JAK2 V617F arose as a) it is not a dominant mutation, b) it is not PV specific since it is found in several myeloproliferative disorders and c) some (~20%) PV patients lack the JAK2 V617F mutation. We set up to investigate the relative frequency of JAK2 V617F in in vitro expanded PV progenitors.Methods-In vitro expansion of erythroid progenitors from mononuclear cells was optimized. Frequency of JAK2 V617F allele was measured by using allele-specific real-time PCR. Clonality was performed using established procedure.Results-In vitro expansion of PV erythroid progenitors and differentiated dendritic cells resulted in a decrease of the frequency of JAK2 V617F allele compared to granulocytes or CD235 + erythroid progenitors. Clonality analysis demonstrated that while granulocytes of these PV patients were clonal, expanded erythroid cells were polyclonal. However, in vitro expanded PV erythroid progenitors still had ~two fold increased proliferative capacity in comparison to erythroid progenitors from healthy individuals. Erythropoietin favors the cells without JAK2 V617F allele. Dendritic cells in one out of three patients remained clonal. Conclusion-JAK2V617F mutation does not provide a proliferative/survival advantage to the PV clone during in vitro expansion. These data suggest that the JAK2 V617F mutation plays an important role in the biology of PV, yet it may not be the PV-initiating event.

Sequential treatment of CD34+ cells from patients with primary myelofibrosis with chromatin-modifying agents eliminate JAK2V617F-positive NOD/SCID marrow repopulating cells

“…This observation is likely a consequence of the loss of the proliferative advantage of JAK2V617F-positive HSCs/HPCs due to the addition of cytokines to in vitro cultures which have been reported by several groups. 16,28 Since the ability of primitive human hematopoietic cells to engraft sublethally irradiated immunodeficient mice is the standard surrogate in vivo assay for human HSCs, our findings suggest that in PMF both short-term HSCs and long-term HSCs are involved by the malignant process and can be eliminated by in vitro treatment with CMAs.…”

The phenotype of polycythemia due to Croatian homozygous VHL (571C>G:H191D) mutation is different from that of Chuvash polycythemia (VHL 598C>T:R200W)

“…24 Briefly, 1x10 6 cells/mL were cultured in StemSpan ™ Serum-Free Expansion Medium (StemCell Technologies) containing different cytokine cocktails from day 1-7 (100 ng/mL of fetal liver tyrosine kinase 3 ligand, 100 ng/mL of thrombopoietin, and 100 ng/mL of stem cell factor), day 8-14 (50 ng/mL of stem cell factor, 50 ng/mL of insulin-like growth factor-1, and 3 U/mL of erythropoietin), and day 15-21 (50 ng/mL of insulin-like growth factor-1, and 3 U/mL of erythropoietin). All cytokines were a kind gift from Amgen (Thousand Oaks, CA, USA).…”

JAK2V617F-positive polycythemia vera and Philadelphia chromosome-positive chronic myeloid leukemia: one patient with two distinct myeloproliferative disorders

“…To note that a specific time-and dose-dependant effect of IM on FDCP cell line expressing the JAK2V617F mutant has been reported. 11 In this case, we did not increase the dose of IM to reduce the hematocrit, but IM therapy was ineffective on the PV clone, as described previously. …”