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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.


Monika Brüggemann, Michaela Kotrová, Henrik Knecht, Jack Bartram, Myriam Boudjogrha, Vojtech Bystry, Grazia Fazio, Eva Froňková, Mathieu Giraud, Andrea Grioni, Jeremy Hancock, Dietrich Herrmann, Cristina Jiménez, Adam Krejci, John Moppett, Tomas Reigl, Mikael Salson, Blanca Scheijen, Martin Schwarz, Simona Songia, Michael Svaton, Jacques J M van Dongen, Patrick Villarese, Stephanie Wakeman, Gary Wright, Giovanni Cazzaniga, Frédéric Davi, Ramón García-Sanz, David Gonzalez, Patricia J T A Groenen, Michael Hummel, Elizabeth A Macintyre, Kostas Stamatopoulos, Christiane Pott, Jan Trka, Nikos Darzentas, Anton W Langerak, EuroClonality-NGS working group. Standardized next-generation sequencing of immunoglobulin and T-cell receptor gene recombinations for MRD marker identification in acute lymphoblastic leukaemia; a EuroClonality-NGS validation study. Leukemia. 2019 Sep;33(9):2241-2253

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PMID: 31243313

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