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

Abstract

IntroductionThe 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.MethodsUtilizing high-throughput sequencing assays and bespoke bioinformatics we identified BCP-ALL-derived clonally-related IGH sequences by their shared ‘DNJ-stem’.ResultsWe 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.DiscussionConsequently, 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.

Proximally biased V(D)J recombination in the clonal evolution of IGH alleles in KMT2A::AFF1 BCP‐ALL of all age classes

“…We observed more DNJ H stems in KMT2A-r samples as compared to other types of BCP-ALL, and the CDR3 regions in KMT2A-r samples are shorter. This is in line with the observations reported by Darzentas et al, 19,20 who reported a detailed analysis of clonal evolution in adult BCP-ALL with different contributions of de novo V (D)J recombination and V H replacement in different BCP-ALL subtypes. V H replacement can occur repeatedly leading to prolonged CDR3 regions due to footprint nucleotides left behind at each V H replacement step located between the canonical and the cryptic recombination signal sequence.…”

“…In addition to classical V(D)J recombination, 13 B‐cell receptors (BCRs) are known to be modified by direct V H to V H DJ H recombination (V H replacement) 14,15 and this process is known to occur also in BCP‐ALL cells 16–18 . Whether the relative contribution of V(D)J recombination and V H replacement to clonal evolution of BCRs displays BCP‐ALL subtype specificity is currently a matter of investigation 19,20 …”

“…[16][17][18] Whether the relative contribution of V(D)J recombination and V H replacement to clonal evolution of BCRs displays BCP-ALL subtype specificity is currently a matter of investigation. 19,20 During the analysis of BCR rearrangements in RNA-sequencing (RNA-Seq) data from BCP-ALL samples, we noticed an unusual abundance of rearrangements involving IGHV6-1 in samples carrying KMT2A-r fusion genes. To quantify this observation, we employed MiXCR analysis of RNA-Seq reads (Figure 1).…”

“… 16 , 17 , 18 Whether the relative contribution of V(D)J recombination and V H replacement to clonal evolution of BCRs displays BCP‐ALL subtype specificity is currently a matter of investigation. 19 , 20 …”

Flow Sorting, Whole Genome Amplification and Next-Generation Sequencing as Combined Tools to Study Heterogeneous Acute Lymphoblastic Leukemia

“…However, a recent study indicated that many IGH gene rearrangements, with shared stem D H -N additions-J H, are detectable in BCP-ALL samples below the 5% threshold. Allegedly, these sequences represent gene rearrangements that underwent continued D H /V H -DJ H recombination and V H replacement, [32]. Hence, whilst a high threshold might guarantee specificity in the overall assignment of clonotypes to ALL, it comes with a risk of overlooking a plethora of less abundant, potentially therapy-resistant subclones that could give rise to relapse.…”