Amplicon-based next-generation sequencing (NGS) of immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements for clonality assessment, marker identification and quantification of minimal residual disease (MRD) in lymphoid neoplasms has been the focus of intense research, development and application. However, standardization and validation in a scientifically controlled multicentre setting is still lacking. Therefore, IG/TR assay development and design, including bioinformatics, was performed within the EuroClonality-NGS working group and validated for MRD marker identification in acute lymphoblastic leukaemia (ALL). Five EuroMRD ALL reference laboratories performed IG/TR NGS in 50 diagnostic ALL samples, and compared results with those generated through routine IG/TR Sanger sequencing. A central polytarget quality control (cPT-QC) was used to monitor primer performance, and a central in-tube quality control (cIT-QC) was spiked into each sample as a library-specific quality control and calibrator. NGS identified 259 (average 5.2/sample, range 0-14) clonal sequences vs. Sanger-sequencing 248 (average 5.0/sample, range 0-14). NGS primers covered possible IG/TR rearrangement types more completely compared with local multiplex PCR sets and enabled sequencing of bi-allelic rearrangements and weak PCR products. The cPT-QC showed high reproducibility across all laboratories. These validated and reproducible quality-controlled EuroClonality-NGS assays can be used for standardized NGS-based identification of IG/TR markers in lymphoid malignancies.
- MeSH
- akutní lymfatická leukemie genetika MeSH
- genetické markery genetika MeSH
- genová přestavba T-lymfocytů genetika MeSH
- geny pro imunoglobuliny genetika MeSH
- geny TcR genetika MeSH
- imunoglobuliny genetika MeSH
- lidé MeSH
- receptory antigenů T-buněk genetika MeSH
- referenční standardy MeSH
- rekombinace genetická genetika MeSH
- reprodukovatelnost výsledků MeSH
- reziduální nádor genetika 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
The B cell receptor immunoglobulin (Ig) gene repertoires of marginal zone (MZ) lymphoproliferations were analyzed in order to obtain insight into their ontogenetic relationships. Our cohort included cases with MZ lymphomas (n = 488), i.e. splenic (SMZL), nodal (NMZL) and extranodal (ENMZL), as well as provisional entities (n = 76), according to the WHO classification. The most striking Ig gene repertoire skewing was observed in SMZL. However, restrictions were also identified in all other MZ lymphomas studied, particularly ENMZL, with significantly different Ig gene distributions depending on the primary site of involvement. Cross-entity comparisons of the MZ Ig sequence dataset with a large dataset of Ig sequences (MZ-related or not; n = 65 837) revealed four major clusters of cases sharing homologous ('public') heavy variable complementarity-determining region 3. These clusters included rearrangements from SMZL, ENMZL (gastric, salivary gland, ocular adnexa), chronic lymphocytic leukemia, but also rheumatoid factors and non-malignant splenic MZ cells. In conclusion, different MZ lymphomas display biased immunogenetic signatures indicating distinct antigen exposure histories. The existence of rare public stereotypes raises the intriguing possibility that common, pathogen-triggered, immune-mediated mechanisms may result in diverse B lymphoproliferations due to targeting versatile progenitor B cells and/or operating in particular microenvironments. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
- Klíčová slova
- antigen, immunoglobulin gene, marginal zone lymphoma, ontogeny,
- MeSH
- genová přestavba B-lymfocytů genetika MeSH
- geny pro imunoglobuliny genetika MeSH
- geny pro těžké řetězce imunoglobulinů genetika MeSH
- hypervariabilní oblasti genetika MeSH
- lidé MeSH
- lymfom z B-buněk marginální zóny genetika MeSH
- mutace genetika MeSH
- nádorové mikroprostředí MeSH
- receptory antigenů B-buněk genetika MeSH
- variabilní oblast imunoglobulinu genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- hypervariabilní oblasti MeSH
- receptory antigenů B-buněk MeSH
- variabilní oblast imunoglobulinu MeSH
Minimal residual disease (MRD) monitoring is an essential tool for risk group stratification in current treatment protocols for childhood acute lymphoblastic leukaemia (ALL). Although quantitative detection of clonal immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements is currently considered to be the standard method, leukaemia fusion genes provide other possible targets for MRD follow-up, as already demonstrated in TEL/AML1-positive ALLs. We analysed and compared MRD levels quantified by BCR/ABL transcript detection and by the standard Ig/TCR-based method in 218 bone marrow specimens from 17 children with BCR/ABL-positive ALL. We found only a limited overall correlation of MRD levels as assessed by the two methods (correlation coefficient R(2)=0.64). The correlation varied among patients from excellent (R(2)=0.99) to very poor (R(2)=0.17). Despite identical sensitivity of the approaches, 20% of the samples were negative by the Ig/TCR approach whereas positive by the BCR/ABL method. We show that multilineage involvement is at least partly responsible for the discrepancy. Moreover, our data demonstrate that BCR/ABL monitoring enables better and earlier prediction of relapse compared to the standard Ig/TCR methodology. We conclude that BCR/ABL-based MRD monitoring of childhood ALL is a clinically relevant tool and should be performed in parallel with the standard Ig/TCR follow-up.
- MeSH
- akutní lymfatická leukemie klasifikace genetika MeSH
- bcr-abl fúzní proteiny genetika MeSH
- dítě MeSH
- genová přestavba T-lymfocytů genetika MeSH
- geny pro imunoglobuliny genetika MeSH
- indukce remise MeSH
- kultivované buňky MeSH
- lidé MeSH
- lokální recidiva nádoru genetika MeSH
- messenger RNA genetika metabolismus MeSH
- mladiství MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- předškolní dítě MeSH
- prekurzorové B-lymfoidní buňky metabolismus patologie MeSH
- prognóza MeSH
- retrospektivní studie MeSH
- reziduální nádor genetika MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bcr-abl fúzní proteiny MeSH
- messenger RNA MeSH
Many structures and molecules closely related to those involved in the specific process of immunoglobulin (Ig) hypermutation existed before the appearance of primordial Ig genes. Consequently, these structures can be found even in animals and organisms distinct from vertebrates; likewise, homologues of hypermutation enzymes are present in a broad range of species, from bacteria to mammals. Our analysis, based predominantly on primary structure, demonstrates the existence of molecules similar to Ig domains, variable Ig domains (IGv), and antigen receptors (AR) in unicellular organisms, nonvertebrate metazoans, and nonvertebrate Coelomata, respectively. In addition, we deal here with some important structural properties of CDR1-like segments of the selected sponge adhesion molecule GCSAMS exhibiting chimerical Ig domain similarities, and demonstrate the occurrence of conserved regions corresponding to Ohno's modern intact primordial building block in the C-terminal part of IGv-related segments of nonvertebrate origin. The results of our analysis are also discussed with respect to the possible phylogeny of molecules preceding the hypothetical common antigen receptor ancestor.
- MeSH
- fylogeneze * MeSH
- geny pro imunoglobuliny genetika MeSH
- lidé MeSH
- molekulární evoluce MeSH
- molekulární sekvence - údaje MeSH
- mutace * MeSH
- mutační analýza DNA metody MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- výpočetní biologie metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Diagnostic approach to non-Hodgkin's lymphomas requires a combination of laboratory methods. Methods used in morphology constitute the basis of the diagnostics; in many instances it is necessary to combine them with methods of molecular genetics. The latter method plays a key role in the detection of B cell clonality using identification of the rearrangement of IGH and/or IGK genes and in detection of the chromosomal translocations specific for some lymphomas. METHODS AND RESULTS: Using PCR we investigated 113 patients with malignant B cell lymphomas of different types (follicular--FL, mantle cell--MCL, small cell--CLL/SLL, diffuse large cell--DLBCL). We established the IGH gene clonal rearrangement in 85% of the cases (96/113), and the clonal rearrangement of the IGK gene in 58.3% patients (42/72). Combination of both approaches (IGH and IGK) revealed a positive result in 90.3% (102/113). The highest yield was rendered in patients with CLL/SLL and with MCL (100%), and it was 86 and 87% in cases with FL and DLBCL. CONCLUSIONS: The detection of clonality in lymphomas helps to distinguish a malignant disease from polyclonal hyperplastic and lymphoproliferative disorders of B cells. The recognition of clonal rearrangements of the IGH and IGK genes serves for a long term monitoring of the disease activity in cases in which there are no other molecular markers available. The demonstration of lymphoma characteristic translocations is relatively specific and useful but at present its usefulness is reduced in cases with variable breakpoint regions.
- MeSH
- B-buněčný lymfom diagnóza genetika MeSH
- buněčné klony MeSH
- diagnostické techniky molekulární * MeSH
- genová přestavba MeSH
- geny pro imunoglobuliny genetika MeSH
- lidé MeSH
- polymerázová řetězová reakce MeSH
- translokace genetická MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- anglický abstrakt MeSH
- časopisecké články MeSH
- práce podpořená grantem MeSH
A unique case of ALL in three monozygotic triplets diagnosed at the age of 24, 27 and 37 months is described. Archived bone marrow smears were available for molecular analysis of immunoglobulin heavy chain (IGH) and IGK genes and T-cell receptor (TCR)-delta and gamma gene rearrangements. A shared IGH rearrangement was found in triplets "A" and "B", and an identical rearrangement of TCR-delta in triplets "B" and "C". These data suggest a common, monoclonal initiation of ALL in one of these three triplets, followed by dissemination of clonal progeny to the other twins via vascular anastomoses within the single, monochorionic placenta that they shared in utero. Differences in IGH rearrangements in diagnostic samples also indicates divergent subclonal evolution of the original "pre-leukaemic" clone.
- MeSH
- akutní lymfatická leukemie genetika imunologie MeSH
- buněčný rodokmen MeSH
- dospělí MeSH
- dvojčata monozygotní MeSH
- genová přestavba - gama řetězec receptoru antigenů T-buněk MeSH
- genová přestavba T-lymfocytů * MeSH
- genová přestavba * MeSH
- geny pro imunoglobuliny genetika MeSH
- geny TcR delta genetika MeSH
- geny TcR gama genetika MeSH
- imunoglobuliny genetika MeSH
- klonování DNA MeSH
- kostní dřeň metabolismus MeSH
- lidé MeSH
- polymerázová řetězová reakce MeSH
- předškolní dítě MeSH
- sekvenční analýza DNA MeSH
- trojčata MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- práce podpořená grantem MeSH
- Názvy látek
- IgK MeSH Prohlížeč
- imunoglobuliny MeSH
