Analysis of nonleukemic cellular subcompartments reconstructs clonal evolution of acute myeloid leukemia and identifies therapy‐resistant preleukemic clones

Abstract

To acquire a better understanding of clonal evolution of acute myeloid leukemia (AML) and to identify the clone(s) responsible for disease recurrence, we have comparatively studied leukemia‐specific mutations by whole‐exome‐sequencing (WES) of both the leukemia and the nonleukemia compartments derived from the bone marrow of AML patients. The T‐lymphocytes, B‐lymphocytes and the functionally normal hematopoietic stem cells (HSC), that is, CD34+/CD38−/ALDH+ cells for AML with rare‐ALDH+ blasts (<1.9% ALDH+ cells) were defined as the nonleukemia compartments. WES identified 62 point‐mutations in the leukemia compartment derived from 12 AML‐patients at the time of diagnosis and 73 mutations in 3 matched relapse cases. Most patients (8/12) showed 4 to 6 point‐mutations per sample at diagnosis. Other than the mutations in the recurrently mutated genes such as DNMT3A, NRAS and KIT, we were able to identify novel point‐mutations that have not yet been described in AML. Some leukemia‐specific mutations and cytogenetic abnormalities including DNMT3A(R882H), EZH2(I146T) and inversion(16) were also detectable in the respective T‐lymphocytes, B‐lymphocytes and HSC in 5/12 patients, suggesting that preleukemia HSC might represent the source of leukemogenesis for these cases. The leukemic evolution was reconstructed for five cases with detectable preleukemia clones, which were tracked in follow‐up and relapse samples. Four of the five patients with detectable preleukemic mutations developed relapse. The presence of leukemia‐specific mutations in these nonleukemia compartments, especially after chemotherapy or after allogeneic stem cell transplantation, is highly relevant, as these could be responsible for relapse. This discovery may facilitate the identification of novel targets for long‐term cure.

Single-Cell DNA Sequencing and Immunophenotypic Profiling to Track Clonal Evolution in an Acute Myeloid Leukemia Patient

“…Although our report is limited to a single case, these data support the concept that mutations in genes implied in epigenetic regulation are initial events in clonal evolution and they arose from preleukemic progenitor cells, being early founder hits prior to leukemogenic events [3,4]. Furthermore, our data showed that NPM1 mutation represent a secondary hit in agreement with previous published reports that considered it a leukemogenic event, driving progression to AML [3,4,28]. The presence of both DNMT3A and NPM1 mutations in the granulocytic population indicates their appearance in early stages of hematopoiesis, giving rise to hematopoietic clones that persist over time and survive to different therapies [3,4].…”

“…The present report shows the FLT3-ITD as the late event after DNMT3A and NPM1 mutations, according to other papers that described proliferative mutations in genes involved in signaling as final events in leukemogenesis [28,30]. Therefore, the most likely scenario, confirmed by the fishplot prediction, is that mutations were acquired sequentially in an ancestral clone giving rise to new clonal populations of cells that would coexist with the surviving parental clones.…”

“…The presence of both DNMT3A and NPM1 mutations in the granulocytic population indicates their appearance in early stages of hematopoiesis, giving rise to hematopoietic clones that persist over time and survive to different therapies [3,4]. This hypothesis would be supported by the persistence of copies for mutant NPM1 after allo-HSCT despite successful BM engraftment with a complete donor chimerism, suggesting that the preleukemic clone still exists after allo-HSCT [28,29]. The dysplastic granulocytic immunophenotype observed by MFC supported this genetic data, and could be explained by the presence of high number of these cells after separation by density gradient, because the dysplasia could modify the cell density.…”

Put in a “Ca2+ll” to Acute Myeloid Leukemia

“…Acute myeloid leukemia arises either de novo, in which the earliest mutation triggers the development of the disease, or may expand from other hematological malignancies following the stepwise increase of chromosomal and/or genetic/epigenetic abnormalities [55][56][57][58][59]. Until now, the mechanisms supporting the regulation and clinical significance of the Ca 2+ /mitochondria pathways have remained mostly unidentified in LSCs.…”

Leukemic Stem Cell: A Mini-Review on Clinical Perspectives

“…These mutations alone are incapable of inducing leukemia and appear as precursor events to late mutations that transform pre-LSCs into LSCs, which is shown in Figure 1. Therefore, the genetic alterations present in LSCs are related to proliferation and active signaling (26)(27)(28)(29)(30)(31)(32)(33).…”