Comprehensive characterization of mutations, gene expression, and immune repertoires in malignant lymphoma by anchored multiplex PCR and next-generation sequencing

Background: Malignant lymphoma can be driven by a diversity of mutation types and can be classified into subtypes based on characteristic mutations or gene expression profiles. Furthermore, the spectrum of antigen receptors, or the immune repertoire, provides a means to identify malignant clones, enabling disease monitoring in response to various interventions. However, comprehensive characterization of mutations, gene expression, and immune repertoires is challenging, as specific assays are required for each. We developed targeted NGS assays based on Anchored Multiplex PCR (AMP) that enable comprehensive mutation detection, gene expression analysis, and characterization of the expressed immune repertoire. Methods: AMP uses molecular barcodes (MBCs) ligated to cDNA ends and gene-specific primers for amplification, enabling open-ended sequencing of target regions to identify both known and unknown mutations, including novel gene fusions. MBC adapters ligated to RNA fragments prior to amplification enable relative gene expression analysis. Furthermore, open-ended amplification enables immune chain mRNA interrogation from a single side, eliminating the need for opposing primers that bind within the highly variable V-segment and thus preventing clone dropout due to somatic hypermutation. This also facilitates CDR3 sequence capture from highly fragmented RNA inputs. Results: We developed primer sets to detect known and unknown mutations and analyze relative gene expression levels in lymphoma. Here, we show the ability of this assay to detect characteristic IGH fusions as well as detect partner-gene expression levels in diffuse large B-cell lymphoma (DLBCL). Furthermore, this assay enabled NGS-based expression profiling for identification of DLBCL subtypes in a small cohort of samples. We also developed the Archer Immunoverse assay for sequencing of the expressed B-cell and T-cell repertoire. We validated the quantitative reproducibility and sensitivity of the AMP-based IGH assay using mRNA isolated from peripheral blood leukocytes of healthy and B-cell chronic lymphocytic leukemia (B-CLL) donors. Our data showed high reproducibility between replicates and quantitative clone tracking down to 0.01%, with the ability to determine IGHV mutational status. Furthermore, we identify a high-frequency B-cell clones in FFPE DLBCL samples as well as hypermutation status. Conclusions: AMP-based NGS enables detection of mutations, gene expression, and the expressed immune repertoire for comprehensive characterization and monitoring of malignant lymphoma. Keywords: gene expression profile (GEP); gene rearrangement; immunophenotype