Abstract 1391: Single cell analysis of Lin-CD34-CD45- cells from primary AML samples reveals leukemia clones with stem cell-like properties distinct from CD34+CD38-CD123+ LSC
Authors: Benton Christopher B. , Al Rawi Ahmed , Min Taejin , Wang Rui-Yu , Schober Wendy , Wang Zhiqiang , Zeng Zhihong , Hokanson Jeffrey , Zhu Zhu , Su Xiaoping , Zheng Xiaofeng , Lu Karen , Carter Bing , Davis Richard E. , Kantarjian Hagop , Konopleva Marina , Andreeff Michael 
Published: August 2015

Abstract
Phenotypically characterized Lin-CD34+CD38-CD123+ AML cells (leukemia stem cells, LSC) are believed to contain leukemia initiating cells (LIC). In the normal hematopoietic developmental hierarchy, CD34- cells are primitive hematopoietic stem cells that occupy the apex of the hematopoietic tree (Cell Stem Cell. 2013 Aug 1;13(2):161-74). A subpopulation of cells from normal individuals that lack cell surface markers (Lin-CD34-CD45- phenotype) were previously shown to have hematopoietic (and endothelial) potential (Blood. 2011 Aug 25;118(8):2105-15). Lin-CD34-CD45- cells from AML patient samples have also been shown to have LIC capacity in limited experiments (J Clin Invest. 2011 Jan;121(1):384-95). We investigated samples from 40 AML patients with active disease to determine the prevalence and frequency of Lin-CD34-CD45- cells, and determined that Lin-CD34-CD45- cells comprise 0.18 ± 0.07% of mononuclear cells in AML patients, comparable to the frequency found in normal samples (0.35 ± 0.20%, P = 0.32, n = 10). In 15 AML samples with informative cytogenetics, Lin-CD34-CD45- subpopulation contained high percentages of leukemia cells as determined by FISH (58.5 ± 8.5%). Relapsed patients with ≥ five prior different lines of therapy had significantly higher percentages of relative leukemic Lin-CD34-CD45- cells (84.6 ± 5.4% vs. 51.0 ± 11.3%, P = 0.015), and the number of prior therapies positively correlated with the fraction of leukemic Lin-CD34-CD45- cells (r = 0.55, P = 0.032). Using conventional flow cytometry and mass cytometry we sought to determine whether individual Lin-CD34-CD45- cells contained any identifying cell surface markers. The Lin-CD34-CD45- population was heterogeneous among and within patients, however some samples contained Lin-CD34-CD45- cells that expressed stem cell markers including CD38, CD47, CD99, CD117, CD146, or CD184. We further evaluated the Lin-CD34-CD45- population, LSC, and bulk AML by gene expression arrays using three primary AML samples. Gene set enrichment revealed that in comparison to LSC, Lin-CD34-CD45- cells have unique expression patterns similar to chemoresistant stem cells characterized by increased proliferation and oxidative phosphorylation. Single cell RNA-sequencing of Lin-CD34-CD45- cells, LSC, and bulk leukemia from one FLT3+ DNMT3A-mutated sample confirmed that individual Lin-CD34-CD45- cells had leukemia gene expression patterns. While some individual Lin-CD34-CD45- cells had gene expression similar to LSC, others possessed unique gene expression profiles. Results suggest that Lin-CD34-CD45- cells represent a putative stem cell state distinct from classically defined LSC. Investigations into alternate stem cell subpopulations that contribute to relapse and therapy resistance are warranted, and may reveal targets for eliminating LIC.
Citation Format: Christopher B. Benton, Ahmed Al Rawi, Taejin Min, Rui-Yu Wang, Wendy Schober, Zhiqiang Wang, Zhihong Zeng, Jeffrey Hokanson, Zhu Zhu, Xiaoping Su, Xiaofeng Zheng, Karen Lu, Bing Carter, Richard E. Davis, Hagop Kantarjian, Marina Konopleva, Michael Andreeff. Single cell analysis of Lin-CD34-CD45- cells from primary AML samples reveals leukemia clones with stem cell-like properties distinct from CD34+CD38-CD123+ LSC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1391. doi:10.1158/1538-7445.AM2015-1391