Genomic analysis of cellular hierarchy in acute myeloid leukemia using ultrasensitive LC-FACSeq

Caner Saygin,Alison Walker,Karilyn Larkin,Bhavana Bhatnagar,John C Byrd,Lynne V Abruzzo ,Deedra Nicolet,Pu Zhang,Jadwiga Labanowska,Gerard Lozanski,Jordan Skinner ,Gregory K Behbehani ,Steven Sher,Eileen Hu,Clara D Bloomfield,Tzyy-Jye Doong,Virginia M Goettl,Casey B Cempre ,Tierney Kauffman,Rosa Lapalombella,Nicole R Grieselhuber ,Shelley Orwick,Nyla A Heerema,Cecelia Miller ,Alice S Mims,Arletta Lozanski,James S Blachly

doi:10.1038/s41375-021-01295-1

Abstract

Hematopoiesis is hierarchical, and it has been postulated that acute myeloid leukemia (AML) is organized similarly with leukemia stem cells (LSCs) residing at the apex. Limited cells acquired by fluorescence activated cell sorting in tandem with targeted amplicon-based sequencing (LC-FACSeq) enables identification of mutations in small subpopulations of cells, such as LSCs. Leveraging this, we studied clonal compositions of immunophenotypically-defined compartments in AML through genomic and functional analyses at diagnosis, remission and relapse in 88 AML patients. Mutations involving DNA methylation pathways, transcription factors and spliceosomal machinery did not differ across compartments, while signaling pathway mutations were less frequent in putative LSCs. We also provide insights into TP53-mutated AML by demonstrating stepwise acquisition of mutations beginning from the preleukemic hematopoietic stem cell stage. In 10 analyzed cases, acquisition of additional mutations and del(17p) led to genetic and functional heterogeneity within the LSC pool with subclones harboring varying degrees of clonogenic potential. Finally, we use LC-FACSeq to track clonal evolution in serial samples, which can also be a powerful tool to direct targeted therapy against measurable residual disease. Therefore, studying clinically significant small subpopulations of cells can improve our understanding of AML biology and offers advantages over bulk sequencing to monitor the evolution of disease.

Highlights

It has been postulated that acute myeloid leukemia (AML) cells have a hierarchic organization, and leukemia stem cells (LSCs) are considered to be the origin of leukemogenesis, treatment failure, and relapse
Our results indicate that the clonal heterogeneity of cells within an individual AML patient may originate from a genetically heterogenous pool of LSCs
LSC frequency at diagnosis was not an independent predictor of patient survival, which suggests that the quantity of these cells at time of diagnosis does not have a direct correlation with their leukemogenic potential

Summary

1234567890();,: 1234567890();,: Introduction

Novel targeted therapies approved within the past 4 years improved outcomes in AML patients with select genomic aberrations. Limited cells acquired by fluorescence activated cell sorting in tandem with targeted amplicon-based sequencing (LC-FACSeq) technique enables accurate identification of mutations in immunophenotypically defined subpopulations of cells. This technique has been validated in both AML and chronic lymphocytic leukemia (CLL) [20]. Leveraging this novel method, we investigated the clonal composition of self-renewing and non-self-renewing cell populations in AML through genomic and functional analyses at diagnosis, remission and relapse

Materials and methods

Results

Discussion

Compliance with ethical standards