• MeSH
• Precursor Cell Lymphoblastic Leukemia-Lymphoma genetics   metabolism   pathology   MeSH
• Immunogenetics * MeSH
• Humans MeSH
• Biomarkers, Tumor genetics   MeSH
• Prognosis MeSH
• Neoplasm, Residual genetics   metabolism   pathology   MeSH
• Whole Genome Sequencing standards   MeSH
• RNA-Seq standards   MeSH
• Transcriptome * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Letter MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Biomarkers, Tumor 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
• Precursor Cell Lymphoblastic Leukemia-Lymphoma genetics   MeSH
• Genetic Markers genetics   MeSH
• Gene Rearrangement, T-Lymphocyte genetics   MeSH
• Genes, Immunoglobulin genetics   MeSH
• Genes, T-Cell Receptor genetics   MeSH
• Immunoglobulins genetics   MeSH
• Humans MeSH
• Receptors, Antigen, T-Cell genetics   MeSH
• Reference Standards MeSH
• Recombination, Genetic genetics   MeSH
• Reproducibility of Results MeSH
• Neoplasm, Residual genetics   MeSH
• Computational Biology methods   MeSH
• High-Throughput Nucleotide Sequencing methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Genetic Markers MeSH
• Immunoglobulins MeSH
• Receptors, Antigen, T-Cell 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
• Genetic Markers genetics   MeSH
• Gene Rearrangement genetics   MeSH
• Immunoglobulins genetics   MeSH
• Humans MeSH
• Receptors, Antigen, T-Cell genetics   MeSH
• Reproducibility of Results MeSH
• Neoplasm, Residual genetics   MeSH
• Quality Control MeSH
• Computational Biology methods   MeSH
• High-Throughput Nucleotide Sequencing methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Genetic Markers MeSH
• Immunoglobulins MeSH
• Receptors, Antigen, T-Cell MeSH