Ig gene (IG) clonality analysis has an important role in the distinction of benign and malignant B-cell lymphoid proliferations and is mostly performed with the conventional EuroClonality/BIOMED-2 multiplex PCR protocol and GeneScan fragment size analysis. Recently, the EuroClonality-NGS Working Group developed a method for next-generation sequencing (NGS)-based IG clonality analysis. Herein, we report the results of an international multicenter biological validation of this novel method compared with the gold standard EuroClonality/BIOMED-2 protocol, based on 209 specimens of reactive and neoplastic lymphoproliferations. NGS-based IG clonality analysis showed a high interlaboratory concordance (99%) and high concordance with conventional clonality analysis (98%) for the molecular conclusion. Detailed analysis of the individual IG heavy chain and kappa light chain targets showed that NGS-based clonality analysis was more often able to detect a clonal rearrangement or yield an interpretable result. NGS-based and conventional clonality analysis detected a clone in 96% and 95% of B-cell neoplasms, respectively, and all but one of the reactive cases were scored polyclonal. We conclude that NGS-based IG clonality analysis performs comparable to conventional clonality analysis. We provide critical parameters for interpretation and discuss a first step toward a quantitative scoring approach for NGS clonality results. Considering the advantages of NGS-based clonality analysis, including its high sensitivity and possibilities for accurate clonal comparison, this supports implementation in diagnostic practice.

• MeSH
• B-buněčný lymfom genetika   MeSH
• B-lymfocyty imunologie   MeSH
• buněčné klony imunologie   MeSH
• fenotyp MeSH
• folikulární lymfom genetika   MeSH
• genová přestavba * MeSH
• geny pro imunoglobuliny * MeSH
• imunoglobuliny - kappa-řetězce genetika   MeSH
• lidé MeSH
• multiplexová polymerázová řetězová reakce metody   MeSH
• senzitivita a specificita MeSH
• správnost dat MeSH
• těžké řetězce imunoglobulinů genetika   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• validační studie 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

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

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

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