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
Monika Brüggemann
(1, 2)
,
Michaela Kotrova
(3, 2)
,
Henrik Knecht
(4)
,
Jack Bartram
(2)
,
Myriam Boudjogrha
(5)
,
Vojtech Bystry
(6)
,
Grazia Fazio
(7)
,
Eva Froňková
(3)
,
Mathieu Giraud
(8)
,
Andrea Grioni
(7)
,
Jeremy Hancock
(9)
,
Dietrich Herrmann
(4)
,
Cristina Jimenez
(10)
,
Adam Krejci
(6)
,
John Moppett
(11)
,
Tomas Reigl
(6)
,
Mikaël Salson
(12, 8)
,
Blanca Scheijen
(13)
,
Martin Schwarz
(14, 4)
,
Simona Songia
(7)
,
Michael Svaton
(3)
,
Jacques J. M. van Dongen
(15)
,
Patrick Villarese
(16, 17)
,
Stephanie Wakeman
(9)
,
Gary Wright
(2)
,
Giovanni Cazzaniga
(7)
,
Frédéric Davi
(5)
,
Ramón García-Sanz
(18, 10)
,
David Davi
(5)
,
Patricia Groenen
(19)
,
Michael Hummel
(20)
,
Elizabeth Macintyre
(21)
,
Kostas C Stamatopoulos
(22)
,
Christiane Pott
(23, 4)
,
Jan Trka
(24, 3)
,
Nikos Darzentas
(6, 4)
,
Anton Langerak
(25)
,
David Gonzalez
(26)
1
UKSH -
University Medical Center of Schleswig–Holstein = Universitätsklinikum Schleswig-Holstein
2 Department of Paediatric Haematology
3 CLIP - Childhood Leukaemia Investigation Prague
4 Department of Hematology
5 CHU Pitié-Salpêtrière [AP-HP]
6 CEITEC MU - Central European Institute of Technology [Brno]
7 Centro Ricerca Tettamanti
8 CRIStAL - Centre de Recherche en Informatique, Signal et Automatique de Lille - UMR 9189
9 Bristol Genetics Laboratory (Southmead Hospital)
10 IBSAL - Instituto de Investigación Biomédica de Salamanca
11 Department of Pediatric Haematology
12 BONSAI - Bioinformatics and Sequence Analysis
13 Department of Pathology [Nijmegen]
14 Liebherr-Werk Nenzing GmbH
15 Department of Immunology
16 ERL 8254 - Equipe Inserm U1163 - Laboratory of molecular mechanisms of hematologic disorders and therapeutic implications
17 CHU Necker - Enfants Malades [AP-HP] - Unité d'Immunologie et d'Hématologie Pédiatrique
18 Haematology Department
19 Department of Pathology, Radboud University Medical Center, Nijmegen, The Netherlands.
20 Charité - UniversitätsMedizin = Charité - University Hospital [Berlin]
21 CHU Necker - Enfants Malades [AP-HP]
22 Institute of Applied Biosciences, Thessaloniki, Greece.
23 University Hospital Schleswig–Holstein
24 Department of Paediatric Haematology/Oncology
25 Erasmus MC - Erasmus University Medical Center [Rotterdam]
26 Centre for Cancer Research and Cell Biology
2 Department of Paediatric Haematology
3 CLIP - Childhood Leukaemia Investigation Prague
4 Department of Hematology
5 CHU Pitié-Salpêtrière [AP-HP]
6 CEITEC MU - Central European Institute of Technology [Brno]
7 Centro Ricerca Tettamanti
8 CRIStAL - Centre de Recherche en Informatique, Signal et Automatique de Lille - UMR 9189
9 Bristol Genetics Laboratory (Southmead Hospital)
10 IBSAL - Instituto de Investigación Biomédica de Salamanca
11 Department of Pediatric Haematology
12 BONSAI - Bioinformatics and Sequence Analysis
13 Department of Pathology [Nijmegen]
14 Liebherr-Werk Nenzing GmbH
15 Department of Immunology
16 ERL 8254 - Equipe Inserm U1163 - Laboratory of molecular mechanisms of hematologic disorders and therapeutic implications
17 CHU Necker - Enfants Malades [AP-HP] - Unité d'Immunologie et d'Hématologie Pédiatrique
18 Haematology Department
19 Department of Pathology, Radboud University Medical Center, Nijmegen, The Netherlands.
20 Charité - UniversitätsMedizin = Charité - University Hospital [Berlin]
21 CHU Necker - Enfants Malades [AP-HP]
22 Institute of Applied Biosciences, Thessaloniki, Greece.
23 University Hospital Schleswig–Holstein
24 Department of Paediatric Haematology/Oncology
25 Erasmus MC - Erasmus University Medical Center [Rotterdam]
26 Centre for Cancer Research and Cell Biology
Mathieu Giraud
- Function : Author
- PersonId : 279
- IdHAL : magiraud
- ORCID : 0000-0003-2741-8047
- IdRef : 094640610
Martin Schwarz
- Function : Author
- PersonId : 1030678
Ramón García-Sanz
- Function : Author
- PersonId : 763389
- ORCID : 0000-0003-4120-2787
Elizabeth Macintyre
- Function : Author
- PersonId : 762165
- ORCID : 0000-0003-0520-0493
Jan Trka
- Function : Author
- PersonId : 891577
Anton Langerak
- Function : Author
- PersonId : 784443
- ORCID : 0000-0002-2078-3220
Abstract
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.