Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

Thomas Boyer; Fanny Gonzales; Adriana Plesa; Pauline Peyrouze; Adeline Barthelemy; Soizic Guihard; Bruno Quesnel; Christophe Roumier; Claude Preudhomme; Meyling Cheok

doi:10.3791/56976

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Method Article

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

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DOI:

10.3791/56976

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March 26th, 2018

Thomas Boyer¹^,² , Fanny Gonzales²^,³ , Adriana Plesa⁴ , Pauline Peyrouze² , Adeline Barthelemy² , Soizic Guihard² , Bruno Quesnel²^,⁵ , Christophe Roumier¹^,² , Claude Preudhomme¹^,² , Meyling Cheok²

¹Laboratoire d’hématologie, CHU Lille, ²Univ. Lille, Inserm, CHU Lille, UMR-S 1172 - JPArc - Centre de Recherche Jean-Pierre AUBERT Neurosciences et Cancer, ³Service d’hématologie pédiatrique, hôpital Jeanne de Flandre, CHU Lille, ⁴Laboratoire d’hématologie CH Lyon Sud, ⁵Service des Maladies du Sang, hôpital Claude Huriez, CHU Lille

Summary

We outline a protocol to detect simultaneous expression of leukemia stem cell markers on primary acute myeloid leukemia cells by flow cytometry. We show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts.

Abstract

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in adults. Initially, AML is a disease of hematopoietic stem cells (HSC) characterized by arrest of differentiation, subsequent accumulation of leukemia blast cells, and reduced production of functional hematopoietic elements. Heterogeneity extends to the presence of leukemia stem cells (LSC), with this dynamic cell compartment evolving to overcome various selection pressures imposed upon during leukemia progression and treatment. To further define the LSC population, the addition of CD90 and CD45RA allows the discrimination of normal HSCs and multipotent progenitors within the CD34+CD38- cell compartment. Here, we outline a protocol to detect simultaneous expression of several putative LSC markers (CD34, CD38, CD45RA, CD90) on primary blast cells of human AML by multiparametric flow cytometry. Furthermore, we show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts. This method of detection and quantification of putative LSC may be used for clinical follow-up of chemotherapy response (i.e., minimal residual disease), as residual LSC may cause AML relapse.

Introduction

Acute myeloid leukemia (AML) is the most common cause of leukemia-associated mortality. Initially, AML is a clonal disease of hematopoietic stem cells (HSC) characterized by arrest of differentiation, subsequent accumulation of blast cells, and reduced production of functional hematopoietic elements. Recently, clonal heterogeneity of the blast cell population has been established essentially by using next generation sequencing (NGS) strategies and showing the existence of intra-clonal evolution of tumor cells¹.

Heterogeneity extends to the presence of leukemic stem cells (LSC). It is hypothesized that this dynamic ce....