Background: Immune cell analysis by next-generation sequencing (NGS) has shown utility in the fields of hematology-oncology and immuno-oncology research. In hemato-oncology research, advantages provided by NGS-based techniques include a lower limit-of-detection and simpler paths to standardization compared to flow-based methods, as well as eliminate sample-specific primer design often required for qPCR-based methods. Owing to primer-primer interactions and incompatibility of reaction conditions, current multiplex PCR immune cell assays require separate reactions to survey each receptor (TCRB, TCRG, IGH, IGK, IGL,⋯), depleting samples and leading to a longer time-to-answer. Two assays for immune receptor analysis based on Ion AmpliSeq technology have been developed to circumvent these issues, allowing for the effective use of up to thousands of primers in a single reaction. These two highly multiplexed NGS assays provide for efficient single-reaction library preparation for detection of IGH, IGK, and IGL chains and the TCRB and TCRG chains, respectively.
Methods: Primer panels target the variable gene and joining gene regions of the individual loci (IGH, IGK, IGL and TCRB/TCRG) for all alleles found within the IMGT database, which allows readout of the complementary-determining region 3 (CDR3) sequence of each receptor. The B cell assay includes primers to amplify IGK loci rearrangements involving the kappa deletion and constant region intronic elements. To evaluate the performance of each assay, clonality detection assessment and limit-of-detection testing used gDNA from a total of 65 research samples representing common B and T cell malignancies. Sequencing was performed using the Ion GeneStudio S5 System and analysis was completed using Ion Reporter 5.16.
Results: Clonality assessments carried out using gDNA collected from both cell line and clinical research samples (CLL, B-ALL, Multiple Myeloma, Burkitt's Lymphoma, NHL, DLBCL, and T-ALL) show a >90% overall positive detection rate. Measurements of linearity-of-response and limit-of-detection were carried out using T and B cell lines and clinical research samples diluted in PBL gDNA to between 10-2 and 10-6 by mass. Both the TCR and BCR multi-receptor assays perform as expected, with linear response of detection to the sample dilution, including the ability to detect clones of interest at 10-6.
Conclusions: These results demonstrate the robustness of newly developed Ion AmpliSeq assays for multi-receptor B and T cell analysis. These assays will cut the time-to-answer for translational research studies as well as simplify clonality assessment and rare clone detection in hematology research.
For research use only.
Citation Format: Geoffrey Marc Lowman, Michelle Toro, Loni Pickle, Stephanie Ostresh, Shrutii Sarda, Chenchen Yang. NGS characterization of multiple immune receptors from a single multiplex PCR reaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2779.