Abstract
Introduction: Polycythemia vera (PV) is a clonal hematologic malignancy characterized by overproduction of mature erythrocytes that exhibits a propensity for transformation to myelofibrosis (MF) and/or secondary acute myeloid leukemia (sAML). Like the majority of myeloproliferative neoplasms (MPN), the defining genetic feature of PV is the JAK2 V617F mutation, occurring in >95% of patients, resulting in overactive JAK-STAT signaling which drives myeloproliferation and can contribute to inflammation. We have demonstrated that aberrant NFkB pathway signaling plays an integral role in maintaining monocyte-derived inflammatory cytokine production in MF (Fisher et al, Leukemia 2017 & 2019). We have recently shown that inflammation is a hallmark of PV as well, but the signaling pathways and cell populations responsible have not been elucidated (Fowles et al, Leukemia 2019). Mass cytometry (CyTOF) enables the simultaneous measurement of 40+ markers with single cell resolution, thereby allowing comprehensive analysis of intracellular signaling networks in defined hematopoietic cell populations. We utilized mass cytometry to investigate cell populations and inflammatory signaling pathways in a cohort of PV patients including those who transformed to MF or secondary acute myeloid leukemia (sAML). Methods: Plasma cytokine levels were measured with the U-PLEX Human Cytokine 10plex assay (MSD) from 20 normal donors, 22 PV, 92 MF and 6 sAML patients, as well as serial samples from PV patients transformed to MF/sAML. Cryopreserved cells bone marrow (BM) and/or peripheral blood (PB) from 15 PV patients and 12 normal donors from were incubated with or without TPO or TNF and then stained with surface and intracellular antibodies prior to being subjected to mass cytometric analysis. Cell population frequency and intracellular signaling were identified and calculated through viSNE analysis and manual gating in Cytobank. Results: Plasma levels of inflammatory cytokines IL-6, IL-8, MIP-1a, and TNF increased with disease severity from PV to MF and sAML. For serial samples, an increased number of elevated cytokines was observed in post-transformation samples, suggesting a role for inflammation in MPN disease progression. Mass cytometry analysis revealed expansion of CD14+CD16+ intermediate monocytes and CD16+CD14-CD33+ non-classical monocytes in PV PB compared to both normal BM and PB. A reduction in CD14+CD16- classical monocytes was observed in PV compared to normal in both BM and PB, suggesting a shift in pro-inflammatory subsets in PV. Heightened NFKB activation in response to TNF stimulation was observed in lineage negative CD34+ cells in the majority of PV PB and BM samples tested compared to normal. Similarly, abnormally elevated pSTAT3/5 levels were frequently observed in response to TPO stimulation. In monocyte populations, basal elevations in several signaling markers including pNFkB, pSTAT1, and pMAPKAP2 were identified. Notably, in serial samples from patients transformed to MF or sAML, evidence of progressive NFkB hyperactivation associated with disease evolution was observed. Conclusions: Inflammatory cytokines are known to be elevated in MPN in part through constitutive JAK-STAT signaling, and recently we identified NFkB signaling as an important contributor in MF. To our knowledge NFkB has not previously been investigated in PV. This study suggests that NFkB signaling is similarly important to PV and might play a role in disease transformation. Our plasma cytokine data identified a correlation with higher cytokine levels associated with disease severity from PV to MF and sAML. This is consistent with prior studies associating IL-6 and IL-8 with leukemic transformation of MF. The observed expansion of CD14+CD16+ monocytes in PV is notable because studies have associated this population with increased endothelial cell adhesion and atherosclerosis. These associations have also been reported in JAK2 V617F-mutant red blood cells and neutrophils, raising the question of how the JAK2 mutation affects the function of pro-inflammatory monocyte subsets. Integration of this study with molecular and gene expression data to further investigate the role of inflammation in MPN progression are ongoing. Disclosures Oh: Novartis: Consultancy; Incyte: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees.
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