Identification of Functionally Primitive and Immunophenotypically Distinct Subpopulations in Secondary Acute Myeloid Leukemia By Mass Cytometry
Authors: Bandyopadhyay Shovik , Yu Liyang , Fisher Daniel A.C. , Malkova Olga , Oh Stephen T. 
Published: October 2019

Abstract
Introduction: Mass cytometry is a powerful tool for analyzing cellular networks, with the ability to generate massive data sets encompassing > 40 parameters measured simultaneously at the single cell level. Various groups have created a variety of platforms to analyze this high dimensional data in unique and efficient ways. These tools have a range of applications: from using phenotypic similarities to cluster cells, stratifying unique signaling subpopulations based on observed stimulation responses, mapping the developmental trajectory of cell types, among many others. We have previously utilized mass cytometry to characterize NFkB hyperactivation in myeloproliferative neoplasms. Here we applied mass cytometric analysis to a cohort of patients with secondary acute myeloid leukemia (sAML) following a history of chronic MPN. The objective of this work was to identify populations of functionally primitive leukemic cells, relying not only on traditional immunophenotypic designations (which can vary considerably in sAML), but also by inferring functional status based on the presence or absence of cytokine hypersensitivity and constitutively active signaling in specific cell populations.
Results: Dimensionality reduction and clustering analysis by viSNE and SPADE identified multiple cell subsets outside the hematopoietic stem/progenitor cell (HSPC) compartment that exhibited overt thrombopoietin (TPO) sensitivity, while healthy controls had highly localized responses largely restricted to the HSPC compartment. Using Phenograph, ten sAML metaclusters were identified containing cells from six sAML patients analyzed. One of these metaclusters represented a distinct subpopulation of CD61+ CD34- CD38- CD45lo cells with variable CD90 and CD11b expression. This subpopulation of CD61+ cells was not identified by manual gating, and exhibited significantly greater STAT3/STAT5 phosphorylation in response to TPO than did lineage-negative CD34+ CD38- cells in five out of six (83%) AML patients examined. In addition, substantially elevated basal STAT3 phosphorylation in this population was hypersensitive to TPO and largely resistant to ex vivo ruxolitnib. The classify function of Phenograph was utilized to determine whether the cytokine hypersensitivity observed in the viSNE and SPADE analysis could be entirely accounted for by the aforementioned CD61+ CD34- CD38- CD45lo population. The signaling responses highly predictive of specific cell types were identified, which were used to assess the functional status of sAML cells compared to healthy Lin- CD34+ CD38- cells. By this approach, sAML cells were found to exhibit significant incongruity between surface cell type designation and functional designation. Furthermore, functionally primitive cells displayed a spectrum of myeloid surface markers, suggesting that restricting analysis to a subset of strictly surface-defined cells would potentially obscure populations of interest.
Conclusions: Our analysis revealed a distinct, previously undescribed population of CD61+ CD34- CD38- CD45lo cells in sAML. While the biological relevance of this population requires validation by functional assays, this result demonstrates that immunophenotypic changes in traditional surface-marker-defined populations may conceal important cell populations. These cells, and other functionally primitive but mature-designated cells could be relevant to studying sAML disease pathogenesis, progression, and/or response to therapy. This study further demonstrates the potential for mass cytometry to elucidate rare leukemic subpopulations in highly heterogeneous tumors.

Disclosures
Oh: Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Research Funding; CTI: Research Funding.