Acute Erythroid Leukemias Have a Distinct Molecular Hierarchy from Non-Erythroid Acute Myeloid Leukemias

In 2016 the World Health Organization (WHO) has reclassified the acute erythroid/myeloid type of acute myeloid leukemia (AML) (thereafter designated M6AML), formerly described as M6a-AML by the FrenchAmerican-British cooperative group, in either AML or myelodysplastic syndrome (MDS) depending on the number of blasts. We previously proposed a molecular classification of M6-AML in adult patients using targeted next generation sequencing (tNGS) and array comparative genomic hybridization (aCGH).We showed that the adult M6-AML share the same molecular profile as the other AML and were mainly distributed in four major classes with mutations in either NPM1, transcription factors (e.g. RUNX1), splicing factors and/or chromatin modifiers (e.g. ASXL1, SRSF2, U2AF1), or TP53. This was confirmed and refined in a recent, more comprehensive study. Based on these results and on the 2016 WHO classification we revisited the gene mutations and prognosis of our series of M6-AML. No molecular differences in the number of mutations or in the molecular classes were found between the M6-AML regardless of whether they were reclassified as AML or as myelodysplastic syndromes (AEL-MDS). Thus, in our series, the WHO 2016 had no impact on the prognosis whereas molecular stratification did. M6-AML seem to be AML in their own right: although they may have some particularities such as frequent TP53 bi-allelic alteration or rare gene fusions, globally they do not have specific molecular profiles and their prognosis is similar or close to non-M6AML and better assessed by mutations. This led us to surmise that an important part of the difference between M6-AML and non-M6-AML could be due to a different leukemic cell-of-origin. To test this hypothesis, we compared the mutations present in different compartments of the cellular hierarchy of seven M6-AML and five non-M6-AML. The 12 patients were selected according to their whole bone marrow mutational status and to the availability of live cells in our biobank. In the M6-AML group, three NPM1-mutated patients, three TP53-mutated patients and one patient mutated in U2AF1 (splicing factors and/or chromatin modifiers class) were studied. The nonM6-AML group comprised three NPM1-mutated and two TP53-mutated patients. The main clinical, biological and molecular characteristics are presented in the Online Supplementary Table S1. We used multi-parameter-fluorescence-activated cell sorting (FACS) to isolate the different cell compartments of the hematopoietic hierarchy and Sanger-sequencing to establish the molecular status of each isolated subpopulation. After obtaining the written consent of the patients according to our ethical committee regulations and biobank procedures, we did a CD34 enrichment from the 12 bone marrow samples collected at diagnosis and obtained from the IPC/CRCM biobank, which operates under authorization # AC-2007-33 granted by the French Ministry of Research. Cell separation was done by using immunomagnetic CD34 beads from Milteny Biotec. To isolate the different immature subpopulations according to the classical hierarchical model of hematopoiesis, CD34 cells were subsequently costained with a mixture of eight monoclonal antibodies and then sorted using a FACSARIAIII (BD Biosciences). In parallel, the CD34 population was costained with a mixture of seven monoclonal antibodies. Cell sorting is detailed in the Online