Insights into IGH clonal evolution in BCP-ALL: frequency, mechanisms, associations, and diagnostic implications.

Introduction: The malignant transformation leading to a maturation arrest in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) occurs early in B-cell development, in a pro-B or pre-B cell, when somatic recombination of variable (V), diversity (D), and joining (J) segment immunoglobulin (IG) genes and the B-cell rescue mechanism of VH replacement might be ongoing or fully active, driving clonal evolution. In this study of newly diagnosed BCP-ALL, we sought to understand the mechanistic details of oligoclonal composition of the leukemia at diagnosis, clonal evolution during follow-up, and clonal distribution in different hematopoietic compartments. Methods: Utilizing high-throughput sequencing assays and bespoke bioinformatics we identified BCP-ALL-derived clonally-related IGH sequences by their shared 'DNJ-stem'. Results: We introduce the concept of 'marker DNJ-stem' to cover the entirety of, even lowly abundant, clonally-related family members. In a cohort of 280 adult patients with BCP-ALL, IGH clonal evolution at diagnosis was identified in one-third of patients. The phenomenon was linked to contemporaneous recombinant and editing activity driven by aberrant ongoing DH/VH-DJH recombination and VH replacement, and we share insights and examples for both. Furthermore, in a subset of 167 patients with molecular subtype allocation, high prevalence and high degree of clonal evolution driven by ongoing DH/VH-DJH recombination were associated with the presence of KMT2A gene rearrangements, while VH replacements occurred more frequently in Ph-like and DUX4 BCP-ALL. Analysis of 46 matched diagnostic bone marrow and peripheral blood samples showed a comparable clonal and clonotypic distribution in both hematopoietic compartments, but the clonotypic composition markedly changed in longitudinal follow-up analysis in select cases. Thus, finally, we present cases where the specific dynamics of clonal evolution have implications for both the initial marker identification and the MRD monitoring in follow-up samples. Discussion: Consequently, we suggest to follow the marker DNJ-stem (capturing all family members) rather than specific clonotypes as the MRD target, as well as to follow both VDJH and DJH family members since their respective kinetics are not always parallel. Our study further highlights the intricacy, importance, and present and future challenges of IGH clonal evolution in BCP-ALL.

Isocitrate dehydrogenase 1 mutation drives leukemogenesis by PDGFRA activation due to insulator disruption in acute myeloid leukemia (AML).

Acute myeloid leukemia (AML) is characterized by complex molecular alterations and driver mutations. Elderly patients show increased frequencies of IDH mutations with high chemoresistance and relapse rates despite recent therapeutic advances. Besides being associated with global promoter hypermethylation, IDH1 mutation facilitated changes in 3D DNA-conformation by CTCF-anchor methylation and upregulated oncogene expression in glioma, correlating with poor prognosis. Here, we investigated the role of IDH1 p.R132H mutation in altering 3D DNA-architecture and subsequent oncogene activation in AML. Using public RNA-Seq data, we identified upregulation of tyrosine kinase PDGFRA in IDH1-mutant patients, correlating with poor prognosis. DNA methylation analysis identified CpG hypermethylation within a CTCF-anchor upstream of PDGFRA in IDH1-mutant patients. Increased PDGFRA expression, PDGFRA-CTCF methylation and decreased CTCF binding were confirmed in AML CRISPR cells with heterozygous IDH1 p.R132H mutation and upon exogenous 2-HG treatment. IDH1-mutant cells showed higher sensitivity to tyrosine kinase inhibitor dasatinib, which was supported by reduced blast count in a patient with refractory IDH1-mutant AML after dasatinib treatment. Our data illustrate that IDH1 p.R132H mutation leads to CTCF hypermethylation, disrupting DNA-looping and insulation of PDGFRA, resulting in PDGFRA upregulation in IDH1-mutant AML. Treatment with dasatinib may offer a novel treatment strategy for IDH1-mutant AML.

Mesenchymal stromal cells of myelodysplastic syndrome and acute myeloid leukemia patients have distinct genetic abnormalities compared with leukemic blasts.

Mesenchymal stromal cells (MSCs) are an essential cell type of the hematopoietic microenvironment. Concerns have been raised about the possibility that MSCs undergo malignant transformation. Several studies, including one from our own group, have shown the presence of cytogenetic abnormalities in MSCs from leukemia patients. The aim of the present study was to compare genetic aberrations in hematopoietic cells (HCs) and MSCs of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) patients. Cytogenetic aberrations were detected in HCs from 25 of 51 AML patients (49%) and 16 of 43 MDS patients (37%). Mutations of the FLT3 and NPM1 genes were detected in leukemic blasts in 12 (23%) and 8 (16%) AML patients, respectively. Chromosomal aberrations in MSCs were detected in 15 of 94 MDS/AML patients (16%). No chromosomal abnormalities were identified in MSCs of 36 healthy subjects. We demonstrate herein that MSCs have distinct genetic abnormalities compared with leukemic blasts. We also analyzed the main characteristics of patients with MSCs carrying chromosomal aberrations. In view of these data, the genetic alterations in MSCs may constitute a particular mechanism of leukemogenesis.

Chromosomal aberrations in bone marrow mesenchymal stroma cells from patients with myelodysplastic syndrome and acute myeloblastic leukemia.

OBJECTIVE: Bone marrow mesenchymal stroma cells (BMSC) are key components of the hematopoietic microenvironment. The question of whether BMSC from patients with hematological disorders have cytogenetic abnormalities is discussed controversially, some studies indicating that they are cytogenetically normal and others providing evidence of their aberrations. PATIENTS AND METHODS: We performed standard and molecular cytogenetic analyses of both hematopoietic cells and BMSC from 31 patients with myelodysplastic syndrome (MDS, n = 18) and acute myeloid leukemia (AML, n = 13) and 7 healthy individuals. Mononuclear cells were isolated from fresh bone marrow aspirates at the time of initial diagnosis for cytogenetic analysis of hematopoietic cells (HC) and selection of BMSC. RESULTS: Clonal cytogenetic aberrations were observed in HC from 8 (44%) MDS and 8 (61%) AML patients. Cytogenetic analyses of BMSC were successfully performed in 27 of the 31 cases. Structural chromosomal aberrations, including t(1;7), t(4;7), t(7;9), t(7;10), t(7;19), t(15;17), and others, were detectable in BMSC from 7 of 16 (44%) MDS and 6 of 11 (54%) AML patients. The breakpoints of chromosomes in BMSC were typical for leukemia aberrations. Two patients showed clonal chromosomal markers. CONCLUSIONS: BMSC from MDS and AML patients show chromosomal abnormalities. Although the majority of cytogenetic aberrations in BMSC were not clonal and differed from chromosomal markers in HC from the same individual, detection of typical chromosomal changes in BMSC suggests enhanced genetic susceptibility of these cells in MDS/AML. This may indicate potential involvement of BMSC in the pathophysiology of MDS/AML.