Functional Dissection of Age-Related Differences in Disease: Phenotype in Polycythemia Vera

Stephen Oh, M.D., Ph.D.

Project Overview:
Lay Summary: Polycythemia vera is a chronic blood cancer that can cause severe complications and early death. Differences in clinical features and outcome have been observed in younger (age ≤ 45) versus older (age ≥ 65) patients with PV. We seek to understand the basis for these differences, and propose a series of genetic and functional studies to address these issues. The long term goal of this work is to leverage the findings from this study to improve longevity for patients with PV.

Full Summary: Polycythemia vera (PV) is a chronic myeloproliferative neoplasm (MPN) that is defined by erythrocytosis and associated with thrombotic complications and a propensity for transformation to myelofibrosis (MF) or secondary acute myeloid leukemia (sAML). Overall survival for patients with PV is substantially impaired compared to age-matched controls. Recent studies have identified age-related differences in disease characteristics and clinical outcome in PV, including distinct JAK2 V617F allele burdens, differences in white blood cell counts, and varying sites of thrombotic complications. Despite these differences, younger and older PV patients face a similar risk of transformation to MF and sAML. The biological explanation for these differences is not well understood. The overall objective of this study is to utilize phenotypic and functional genomics tools to delineate age-related differences in disease phenotype associated with PV. We hypothesize that intrinsic differences in the composition of genetic changes in the malignant clone dictate these age-related clinical features.

We propose to perform genomic analyses (exome sequencing) on a cohort of ten younger (age ≤ 45) and ten older (age ≥ 65) patients with PV, to delineate the constellation of genetic alterations that define these two subgroups of patients with distinct clinical characteristics. To characterize phenotypic and functional differences between these two subgroups, we will generate and functionally characterize patient-derived induced pluripotent stem cells (iPSCs) via hematopoietic development and self-renewal assays, as well as intracellular signaling analyses via mass cytometry. In addition, CRISPR site-directed gene editing will be utilized to revert JAK2 V617F and other driver mutations in patient-derived iPSCs from younger and older patients. Taken together, these studies will identify intrinsic genetic, phenotypic, and functional differences responsible for age-associated clinical characteristics in PV. A deeper understanding of the underlying biology that drives disease phenotype will have important ramifications for PV prognostication and development of improved therapeutic approaches.

Progress Report

Over the past year, our efforts have focused on assembling a cohort of ten younger (age ≤ 45) and ten older (age ≥ 65) patients with polycythemia vera. Cryopreserved samples from these 20 patients were retrieved, and individual samples were stained and flow sorted to isolate myeloid cells (tumor) and T cells (normal control). Genomic DNA isolated from these samples was then prepared for exome sequencing performed in conjunction with the McDonnell Genome Institute. Sequencing is in progress with data analysis to begin imminently. These studies will delineate the constellation of genetic alterations that define these two subgroups of patients with distinct clinical characteristics. Order of mutation acquisition and clonal hierarchy of putative driver mutations will be inferred based on individual mutation frequencies. To confirm these findings, single-cell derived colonies will be genotyped for specific mutations. These studies will distinguish the genetic drivers responsible for age-associated clinical phenotypes in polycythemia vera. Based on the findings from this work, subsequent experiments will focus on the generation and functional characterization of patient-derived induced pluripotent stem cells (iPSCs) from these two subgroups.