Session: 803. Emerging Tools, Techniques, and Artificial Intelligence in Hematology: Poster III
Hematology Disease Topics & Pathways:
Lymphoid Leukemias, ALL, CLL, Lymphomas, Non-Hodgkin lymphoma, Diseases, Lymphoid Malignancies, Technology and Procedures, Measurable Residual Disease , Molecular testing
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 exhibited excellent from 1 to 10^-6 in a gradient of cell lines and clinical samples. We estimated the limit of detection (LOD) and precision for BCR and TCR in clinical samples, suggested that 1 to 2 tumor cells in mixed samples can be reliably detected by the assay. And the assay showed excellent precision with a coefficient of variation (CV) of less than 5%. Comparing the MRD values of clinical samples assessed by MFC and the immune-gene sequencing assay, the results indicated that NGS of the immune repertoire exhibited significantly higher sensitivity in both B-ALL and T-ALL. Meanwhile, the 100% predictive positive agreement (PPA) between MFC and NGS indicates the reliability of immune-gene sequencing assay.
In applications exploration, immune-gene sequencing assay revealed that cfDNA in PB of diffuse large B-cell lymphoma (DLBCL) patients contained identical clonotypes of those found in tissues, providing the potential tool for MRD tracking in DLBCL. And we also performed the assay for tracking residual tumor cells of patients at the timepoint prior to relapse to validate the predictive effect of the NGS assay on B-ALL relapse. In total, the immune-gene sequencing assay has provided a robust method for predicting B-ALL relapse 1 to 5 months in advance.
Conclusion:
In this study, we provided an assay based on immune gene rearrangement and established an analytic system to valid the reliability and sensitivity of immune-gene sequencing assay. We suggested the assay included light chain and incomplete rearrangements detection, can enhance the primary detection and MRD tracking capabilities. Most importantly, we have not only confirmed the immune-gene sequencing assay possesses significantly higher sensitivity, capable of achieving a deeper MRD-negative diagnosis, but also demonstrated its substantial potential for predicting relapses in B-ALL patients well in advance.
Disclosures: No relevant conflicts of interest to declare.
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