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.

Fig. 1

Schematic diagrams of rearrangements and primer sets. a Schematic diagrams of IGHV-IGHJ and IGHD-IGHJ rearrangements. The relative position of the VH family primers, DH family primers and consensus JH primers is given according to their most 5′ nucleotide upstream (−) or downstream (+) of the involved RSS. b Schematic diagrams of IGKV-IGKJ rearrangement and the two types of Kde rearrangements (V-Kde and intronRSS–Kde). The relative position of the IGKV, IGKJ, Kde, and intronRSS (INTR) primers is given according to their most 5′ nucleotide upstream (−) or downstream (+) of the involved RSS. c Schematic diagrams of TRBV-TRBJ rearrangement and TRBD-TRBJ rearrangement. The relative position of the TRBV family primers, TRBD primers and the TRBJ primers is given according to their most 5′nucleotide upstream (−) or downstream (+) of the involved RSS. d Schematic diagrams of TRGV–TRGJ rearrangement and the relative position of the TRGV and TRGJ primers. The relative position of the TRGV primers and the TRGJ primers is given according to their most 5′ nucleotide upstream (−) or downstream (+) of the involved RSS. e Schematic diagram of TRDV–TRDJ,TRDD–TRDJ, TRDD–TRDD, and TRDV–TRDD, TRDV-TRAJ29 rearrangements, showing the positioning of TRDV, TRDJ, TRDD, and TRAJ29 primers, all combined in a single tube. The relative position of the TRDV, TRDD, and TRDJ primers is indicated according to their most 50 nucleotides upstream (−) or downstream (+) of the involved RSS

Fig. 2

Schematic overview of the workflow for multicentre validation of IG/TR NGS assays for MRD marker identification in ALL. The IG and TR gene rearrangements are amplified in a two-step approach using multiplex PCR assays. Each of the participating laboratories performed NGS-based IG/TR MRD marker identification in 10 patients with ALL. A central polytarget control (cPT-QC) was used to monitor primer performance, and central in-tube controls (cIT-QC) were spiked to each sample as library-specific quality control and calibrator. Pipetting was performed in a 96-well format. The data analysis was performed using ARResT/Interrogate

Fig. 3

Results of multicentre validation of assays for MRD marker identification in ALL. Blue: Index sequences identified by Sanger sequencing. Red: Index sequences identified by NGS. Darkest blue/red are clonal sequences identified by both methods; lightest blue/red are sequences identified only by the respective method. Median blue/red are clonal sequences identified by both methods, but by NGS with an abundance of <5% after normalization

Fig. 4

Clonal evolution in a BCP-ALL patient. The dominant incomplete IGH rearrangement (IGHD6-13 - IGHJ4) was identified with an abundance of 89.4% together with three additional complete IGH rearrangements with lower abundance (1.21–1.55%) and the same DNJ sequence. Only the CDR3 region is shown for each sequence