Analytical Validation of a Next-Generation Sequencing Assay for Detecting Minimal Residual Disease in Lymphoid Malignancies

• Published in: Blood 2024-11, Vol.144, p.4972-4972
• Main Authors: Zhou, Yiwei, Ma, Zhiyuan, Huang, Shiang, Li, Zhenhui, Lei, Beining, Zou, Fang, Li, Qijiao, Liang, Liang, Sun, Li, Zhang, Jie
• Format: Article
• Language: English
• ISSN: 0006-4971
• DOI: 10.1182/blood-2024-199029

Introduction: Traditionally, patients who have achieved a complete response (CR) determined by multiparameter flow cytometry (MFC) or polymerase chain reaction (PCR)-based assays with a detection threshold of 10^-4 to 10^-5, subsequently experienced relapse, are believed to have residual tumor cells. Rearranged immune genes encoding B-cell receptors (BCR) and T-cell receptors (TCR) constitute valuable molecular markers for tracking residual leukemic cells in patients with lymphoid malignancies. To improve the sensitivity of minimal residual disease (MRD) monitoring, the method based on next-generation sequencing (NGS) for detecting TCR and BCR gene rearrangements has been established. In this study, we have developed the immune-gene sequencing assay in lymphoid Malignancies which included IGH(VDJ), IGH(DJ), IGK, IGL, TRB (VDJ), TRB (DJ), TRD (VDJ), TRD (DJ) and TRG rearrangements detections, extending tumor clonotypes identification and enhancing the detection limitation of MRD. Methods: To validate the linearity, limit of detection (LOD), and precision of immune-gene sequencing assay, we employed genomic DNA (gDNA) from cell lines and bone marrow samples from patients diagnosed with acute lymphoblastic leukemia (ALL), multiple myeloma (MM), and chronic lymphocytic leukemia (CLL), as well as gDNA from malignant tissues and circulating cell-free DNA (cfDNA) from peripheral blood (PB) of lymphoma patients. The DNA samples were subjected to a two-step multiplex PCR amplification process. In the first PCR step, we employed primers that specifically targeting CDR3 regions of the immunoglobulin genes as well as regions of the TCR genes. The second PCR step incorporated a unique barcode sequence and Illumina adapter to each amplified product. Subsequently, all libraries were sequenced using an Illumina NovaSeq X Plus system. Results: Basically, the detection rates of lymphoid hematological diseases originating from B cells is higher than 90% while of T-ALL is higher than 70% under immune-gene sequencing assay. In a small cohort of CLL and B-Lymphoma patients, the detection rates achieved 100%. Although lymphoid hematological diseases are predominantly of monoclonal origin, the rearrangements of immune genes display considerable diversity. Most patients exhibit more than one dominated clonotype, and it is believed that the incompletely rearranged clonotypes contributes to the positive detection of patients. Linearity of BCR and TCR rearrangements detection exhibit