Adaptation Of A Clinical Multiparameter Flow Cytometry Strategy For The Enrichment Of Measurable Residual Disease Cells In Acute Myeloid Leukemia

Abstract

Acute myeloid leukemia (AML) is a heterogenous blood cancer originating in the bone marrow and arising from the abnormal growth and lack of differentiation of myeloid cells. Even after intense treatment, disease recurrence is a major problem in AML and is attributed to cells that remain following treatment. We are now able to detect these residual cells after treatment with sensitive assays. This is known as measurable residual disease cells (MRD). The detection of MRD is possible using multiparameter flow cytometry assays and quantitative PCR (qPCR). The detection of MRD has been shown to be predictive of survival. However, there is very limited understanding of the biology of MRD as it is currently not possible to isolate MRD cells and study them in functional assay or by gene expression assays. Our research now aimed to modify methods used for MRD detection to allow for the isolation and characterization of MRD cells from remission samples. To facilitate this, a recently published a multiparameter flow cytometry (MFC) panel based on the ELN recommendations was established and modified for use on frozen samples. This panel allows to the identification of early myeloid progenitor cells and the asynchronous expression of maturation markers as well as the aberrant expression of lineage markers. To identify patient samples with MRD, we used sensitive clinical qPCR assays for the detection of different NPM1 mutations as well as RUNX1-RUNX1T1. More than 160 samples from 86 patients were collected and samples with MRD identified from 3 patients. Based on our MFC panel we sorted these cells from the follow up samples through the use of either early myeloid progenitor markers, or aberrant marker expression. Using this approach, we could enrich for the level of MRD in these samples. This method is now available and could be a first step in the prospective isolation of MRD cells for functional studies and gene expression profiling.