Monitoring of minimal residual disease (MRD) after hematopoietic stem cell transplantation (HSCT) in patients with acute lymphoblastic leukemia (ALL) is vital for timely therapeutic intervention planning. However, interpreting low-positive results from the current standard method, quantitative PCR (qPCR) of immunoglobulin and T-cell receptor gene rearrangements (IG/TR), poses challenges due to the risk of false positivity caused by non-specific amplification. We aimed to improve MRD detection specificity using the next-generation amplicon sequencing (NGS) of IG/TR rearrangements for better relapse prediction. In pediatric and young adult ALL patients undergoing sequential post-HSCT MRD monitoring, we prospectively re-tested positive non-quantifiable qPCR results with NGS-MRD using the EuroClonality-NGS approach. We were able to confirm 13 out of 47 (27.7%) qPCR positive results using the more specific NGS-MRD method. Out of 10 patients with at least one MRD positivity confirmed by NGS, six relapsed (60%) 1-3.7 months after testing. Among 25 patients with all NGS-MRD results negative, two relapses occurred (8%) after 5.1 and 12.1 months. One-year RFS was 40% versus 96% and 3-year OS was 33.3% versus 94.4% for the NGS-positive and NGS-negative groups, respectively. The difference was not attributable to a varying rate of therapeutic interventions. Six patients out of 14 who had immunosuppressive treatment tapered or received donor lymphocyte infusion in response to MRD positivity developed significant graft versus host disease, leading to one fatality. This underscores the importance of enhancing the post-HSCT relapse risk prediction accuracy through NGS-MRD testing to avoid unnecessary interventions.

• Publikační typ
• časopisecké články MeSH

Can peripheral blood be used to detect residual disease in acute lymphoblastic leukaemia (ALL) when we increase the sensitivity of the method used? Bendig et al. found that a larger amount of material and the use of next-generation sequencing (NGS) detects MRD in peripheral blood in up to half of patients with B-cell precursor ALL (BCP-ALL) where routine examination was negative. However, a negative result does not exclude the presence of residual disease and thus still limits the use of peripheral blood. Commentary on: Bendig et al. Next-generation sequencing and high DNA input identify previously missed measurable residual disease in peripheral blood of B-cell precursor acute lymphoblastic leukaemia. Br J Haematol 2025; 206:353-356.

• Klíčová slova
• ALL, peripheral blood, residual disease,
• MeSH
• akutní lymfatická leukemie * krev   diagnóza   genetika   MeSH
• lidé MeSH
• reziduální nádor * diagnóza   krev   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Minimal/measurable residual disease (MRD) diagnostics using real-time quantitative PCR analysis of rearranged immunoglobulin and T-cell receptor gene rearrangements are nowadays implemented in most treatment protocols for patients with acute lymphoblastic leukemia (ALL). Within the EuroMRD Consortium, we aim to provide comparable, high-quality MRD diagnostics, allowing appropriate risk-group classification for patients and inter-protocol comparisons. To this end, we set up a quality assessment scheme, that was gradually optimized and updated over the last 20 years, and that now includes participants from around 70 laboratories worldwide. We here describe the design and analysis of our quality assessment scheme. In addition, we here report revised data interpretation guidelines, based on our newly generated data and extensive discussions between experts. The main novelty is the partial re-definition of the "positive below quantitative range" category by two new categories, "MRD low positive, below quantitative range" and "MRD of uncertain significance". The quality assessment program and revised guidelines will ensure reproducible and accurate MRD data for ALL patients. Within the Consortium, similar programs and guidelines have been introduced for other lymphoid diseases (e.g., B-cell lymphoma), for new technological platforms (e.g., digital droplet PCR or Next-Generation Sequencing), and for other patient-specific MRD PCR-based targets (e.g., fusion genes).

• MeSH
• akutní lymfatická leukemie genetika   diagnóza   MeSH
• genová přestavba * MeSH
• geny pro imunoglobuliny MeSH
• kvantitativní polymerázová řetězová reakce metody   normy   MeSH
• lidé MeSH
• reziduální nádor * genetika   diagnóza   MeSH
• směrnice pro lékařskou praxi jako téma normy   MeSH
• zajištění kvality zdravotní péče MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

T-cell receptor (TR) diversity of the variable domains is generated by recombination of both the alpha (TRA) and beta (TRB) chains. The textbook process of TRB chain production starts with TRBD and TRBJ gene rearrangement, followed by the rearrangement of a TRBV gene to the partially rearranged D-J gene. Unsuccessful V-D-J TRB rearrangements lead to apoptosis of the cell. Here, we performed deep sequencing of the poorly explored pool of partial TRBD1-TRBD2 rearrangements in T-cell genomic DNA. We reconstructed full repertoires of human partial TRBD1-TRBD2 rearrangements using novel sequencing and validated them by detecting V-D-J recombination-specific byproducts: excision circles containing the recombination signal (RS) joint 5'D2-RS - 3'D1-RS. Identified rearrangements were in compliance with the classical 12/23 rule, common for humans, rats, and mice and contained typical V-D-J recombination footprints. Interestingly, we detected a bimodal distribution of D-D junctions indicating two active recombination sites producing long and short D-D rearrangements. Long TRB D-D rearrangements with two D-regions are coding joints D1-D2 remaining classically on the chromosome. The short TRB D-D rearrangements with no D-region are signal joints, the coding joint D1-D2 being excised from the chromosome. They both contribute to the TRB V-(D)-J combinatorial diversity. Indeed, short D-D rearrangements may be followed by direct V-J2 recombination. Long D-D rearrangements may recombine further with J2 and V genes forming partial D1-D2-J2 and then complete V-D1-D2-J2 rearrangement. Productive TRB V-D1-D2-J2 chains are present and expressed in thousands of clones of human antigen-experienced memory T cells proving their capacity for antigen recognition and actual participation in the immune response.

• Klíčová slova
• NGS - next generation sequencing, T cell, TRB repertoire, Thymus, VDJ recombination,
• MeSH
• apoptóza * MeSH
• buněčné klony MeSH
• chromozomální aberace MeSH
• geny TcR beta * MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• myši MeSH
• paměťové T-buňky MeSH
• V(D)J rekombinace * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• Klíčová slova
• DNJ-stem, IGH rearrangements, VH replacement, acute lymphoblastic leukemia, clonal evolution, high-throughput sequencing, minimal residual disease,
• MeSH
• Burkittův lymfom * genetika   MeSH
• dospělí MeSH
• geny pro imunoglobuliny MeSH
• kostní dřeň patologie   MeSH
• lidé MeSH
• polymerázová řetězová reakce MeSH
• pre-B-buněčná leukemie * diagnóza   genetika   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We compared minimal/measurable residual disease (MRD) levels evaluated by routinely used real-time quantitative polymerase chain reaction (qPCR) patient-specific assays and by next-generation sequencing (NGS) approach in 780 immunoglobulin (IG) and T-cell receptor (TR) markers in 432 children with B-cell precursor acute lymphoblastic leukemia treated on the AIEOP-BFM ALL 2009 protocol. Our aim was to compare the MRD-based risk stratification at the end of induction. The results were concordant in 639 of 780 (81.9%) of these markers; 37 of 780 (4.7%) markers were detected only by NGS. In 104 of 780 (13.3%) markers positive only by qPCR, a large fraction (23/104; 22.1%) was detected also by NGS, however, owing to the presence of identical IG/TR rearrangements in unrelated samples, we classified those as nonspecific/false-positive. Risk group stratification based on the MRD results by qPCR and NGS at the end of induction was concordant in 76% of the patients; 19% of the patients would be assigned to a lower risk group by NGS, largely owing to the elimination of false-positive qPCR results, and 5% of patients would be assigned to a higher risk group by NGS. NGS MRD is highly concordant with qPCR while providing more specific results and can be an alternative in the front line of MRD evaluation in forthcoming MRD-based protocols.

• MeSH
• akutní lymfatická leukemie * diagnóza   genetika   terapie   MeSH
• dítě MeSH
• genová přestavba MeSH
• hodnocení rizik MeSH
• imunoglobuliny genetika   MeSH
• lidé MeSH
• pre-B-buněčná leukemie * diagnóza   genetika   terapie   MeSH
• receptory antigenů T-buněk genetika   MeSH
• reziduální nádor diagnóza   genetika   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• imunoglobuliny MeSH
• receptory antigenů T-buněk MeSH

Selection of the proper target is crucial for clinically relevant monitoring of minimal residual disease (MRD) in patients with acute lymphoblastic leukemia using the quantitation of clonal-specific immunoreceptor (immunoglobulin/T cell receptor) gene rearrangements. Consequently, correct interpretation of the results of the entire analysis is of utmost importance. Here we present an overview of the quality control measures that need to be implemented into the process of marker identification, selection, and subsequent quantitation of the MRD level.

• Klíčová slova
• Acute lymphoblastic leukemia, Minimal residual disease, Next-generation sequencing, PCR, Quality control,
• MeSH
• akutní lymfatická leukemie * diagnóza   genetika   MeSH
• biologické markery MeSH
• imunoglobuliny genetika   MeSH
• lidé MeSH
• reziduální nádor diagnóza   genetika   MeSH
• řízení kvality MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• biologické markery MeSH
• imunoglobuliny MeSH

• MeSH
• akutní lymfatická leukemie genetika   metabolismus   patologie   MeSH
• imunogenetika * MeSH
• lidé MeSH
• nádorové biomarkery genetika   MeSH
• prognóza MeSH
• reziduální nádor genetika   metabolismus   patologie   MeSH
• sekvenování celého genomu normy   MeSH
• sekvenování transkriptomu normy   MeSH
• transkriptom * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem MeSH
• Názvy látek
• nádorové biomarkery MeSH

Assessment of clonality, marker identification and measurement of minimal residual disease (MRD) of immunoglobulin (IG) and T cell receptor (TR) gene rearrangements in lymphoid neoplasms using next-generation sequencing (NGS) is currently under intensive development for use in clinical diagnostics. So far, however, there is a lack of suitable quality control (QC) options with regard to standardisation and quality metrics to ensure robust clinical application of such approaches. The EuroClonality-NGS Working Group has therefore established two types of QCs to accompany the NGS-based IG/TR assays. First, a central polytarget QC (cPT-QC) is used to monitor the primer performance of each of the EuroClonality multiplex NGS assays; second, a standardised human cell line-based DNA control is spiked into each patient DNA sample to work as a central in-tube QC and calibrator for MRD quantification (cIT-QC). Having integrated those two reference standards in the ARResT/Interrogate bioinformatic platform, EuroClonality-NGS provides a complete protocol for standardised IG/TR gene rearrangement analysis by NGS with high reproducibility, accuracy and precision for valid marker identification and quantification in diagnostics of lymphoid malignancies.

• MeSH
• genetické markery genetika   MeSH
• genová přestavba genetika   MeSH
• imunoglobuliny genetika   MeSH
• lidé MeSH
• receptory antigenů T-buněk genetika   MeSH
• reprodukovatelnost výsledků MeSH
• reziduální nádor genetika   MeSH
• řízení kvality MeSH
• výpočetní biologie metody   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• genetické markery MeSH
• imunoglobuliny MeSH
• receptory antigenů T-buněk MeSH

One of the hallmarks of B lymphoid malignancies is a B cell clone characterized by a unique footprint of clonal immunoglobulin (IG) gene rearrangements that serves as a diagnostic marker for clonality assessment. The EuroClonality/BIOMED-2 assay is currently the gold standard for analyzing IG heavy chain (IGH) and κ light chain (IGK) gene rearrangements of suspected B cell lymphomas. Here, the EuroClonality-NGS Working Group presents a multicentre technical feasibility study of a novel approach involving next-generation sequencing (NGS) of IGH and IGK loci rearrangements that is highly suitable for detecting IG gene rearrangements in frozen and formalin-fixed paraffin-embedded tissue specimens. By employing gene-specific primers for IGH and IGK amplifying smaller amplicon sizes in combination with deep sequencing technology, this NGS-based IG clonality analysis showed robust performance, even in DNA samples of suboptimal DNA integrity, and a high clinical sensitivity for the detection of clonal rearrangements. Bioinformatics analyses of the high-throughput sequencing data with ARResT/Interrogate, a platform developed within the EuroClonality-NGS Working Group, allowed accurate identification of clonotypes in both polyclonal cell populations and monoclonal lymphoproliferative disorders. This multicentre feasibility study is an important step towards implementation of NGS-based clonality assessment in clinical practice, which will eventually improve lymphoma diagnostics.

• MeSH
• B-buněčný lymfom genetika   MeSH
• genová přestavba genetika   MeSH
• geny pro imunoglobuliny genetika   MeSH
• imunoglobuliny - kappa-řetězce genetika   MeSH
• lidé MeSH
• lymfoproliferativní nemoci genetika   MeSH
• studie proveditelnosti MeSH
• těžké řetězce imunoglobulinů genetika   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• imunoglobuliny - kappa-řetězce MeSH
• těžké řetězce imunoglobulinů MeSH