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

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.

Erratum: Mina, R.; et al. Minimal Residual Disease in Multiple Myeloma: State of the Art and Future Perspectives. J. Clin. Med. 2020, 9, 2142

“…Other non-commercial NGS technologies are under investigation: the LymphoTrack ® assay (Invitrogen, US-MA) has been recently validated in a phase II study [2], and the EuroClonality-NGS Consortium (an international group of 21 academic laboratories experienced in NGS) has recently validated IG/TR NGS assays and a bioinformatic tool for an academic study on MRD [3].…”

“…Martinez-Lopez et al validated an "in-house deep-sequencing" method in a phase II study by using standardized primers developed by the Biomed-2 Concerted Action, in order to amplify all IGH or IGK sequences [2]. Furthermore, the EuroClonality-NGS Consortium (an international group of 21 academic laboratories experienced in NGS) has recently validated IG/TR NGS assays and a bioinformatic tool for an academic study on MRD [3].…”

Reading the B-cell receptor immunome in chronic lymphocytic leukemia: revelations and applications

“…To tackle these challenges, to ensure stringent external quality control, and to develop standardized protocols, guidelines, and a pipeline for NGS-MRD quantification, the EuroClonality−NGS Working Group has been established [58]. Steps in the development of such a pipeline have recently been published, including validated primer sets [65], the development of spiked in-tube quality control to correct for amplification biases [66], and the ARReST/Interrogate bioinformatics platform for repertoire analysis [67].…”

Monitoring minimal residual disease in the bone marrow using next generation sequencing

“…We have implemented LymphoTrack as standard of care in the pathology laboratory at Memorial Sloan Kettering Cancer Center and have excellent experiences using both assays (24,28,29,31). As an non-commercial alternative, a new set of NGS-based MRD assays were recently published by the EuroClonality/BIOMED-2 consortium (32,33). Although the research efforts of the BIOMED-2 consortium is primarily acute lymphoblastic leukemia, their assays have an excellent track-record, and the previous version has been applied successfully to multiple myeloma (34).…”