P05.01 Comparative analysis of RNA versus DNA as input material for IGH repertoire sequencing panels for immuno-oncology applications and rare clone detection

BackgroundRecent progress in tumor immunotherapies have shown the importance of next generation sequencing (NGS) T cell repertoire profiling to characterize T cell immune response to treatment. Understanding the role of the B cell repertoire upon stimulation of the immune system by checkpoint blockade is paramount for immunotherapeutic approaches in treatment of B cell malignancies, as well as understanding B cell function within traditional I/O strategies. The ability to detect low frequency B cell clones enables numerous hematology/oncology research applications, including identification of potential biomarkers and minimal residual disease (MRD) research. Historically, efforts to track the frequency of malignant B cells by IGH chain sequencing have utilized DNA input given potential challenges in accurately quantifying template copy number from RNA data owing to B cell subtype specific variation in the expression of the B cell receptor. Hypothetically, however, RNA input based monitoring could be advantageous both owing to reduced input requirements and superior ability to detect B call malignancies of plasmablast and plasma cell origin, where the BCR is robustly expressed. Here we compared the ability of RNA and DNA based IGH chain sequencing to detect Burkitt’s Lymphoma cell lines and Chronic Lymphocytic Leukemia samples at a frequency of 10-6 from peripheral blood.Materials and MethodsHere we present performance for rare clone detection utilizing the Ion OncomineTM BCR IGH-SR assay and the Ion OncomineTM BCR IGH-LR assay. These assays use multiplex primers targeting all known IGH germline variable genes in the framework 1 (FR1) or framework 3 (FR3) regions of the B cell receptor using either DNA or RNA as input. To evaluate detection sensitivity of the IGH-SR assay we utilized DNA or RNA from Burkitt’s lymphoma cell lines as well as clinical chronic lymphocytic leukemia (CLL) samples controllably added to a background of peripheral blood leukocytes (PBL) by mass ratio to create specimens with a known target B cell frequency. Automated downsampling analysis was used to confirm libraries were sequenced to saturation. Library preparation and analysis was performed in replicate to quantify sensitivity of detection.ResultsFor each cell line, we prepared and sequenced (1) 30 libraries derived from amplification of 2ug gDNA spiked with 2pg cell line gDNA and (2) 10 libraries derived from amplification of 100ng RNA spiked with 0.1pg cell line total RNA. The Burkitt’s lymphoma cell line and CLL samples were detected in 10/30 and 8/30 libraries respectively, consistent with the performance of orthologous DNA-based sequencing approaches. For RNA libraries, the Burkitt’s lymphoma and CLL samples were detected in each library (10/10 and 10/10, respectively).ConclusionsHere we demonstrate the ability to detect B cell clones down to 10-6 from gDNA and RNA inputs utilizing the Ion OncomineTM BCR IGH-SR assay. Feasibility for rare clone detection is shown in gDNA or RNA enabling B cell minimal residual disease research, and high sensitivity characterization the B cell role in response to checkpoint blockade within the tumor microenvironment. Importantly, we find that RNA based IGH sequencing may significantly reduce input requirements for rare clone detection, potentially enabling routine detection of clones at 10-6 frequency from a single library.Disclosure InformationG.M. Lowman: A. Employment (full or part-time); Significant; ThermoFisher Scientific. L. Pickle: A. Employment (full or part-time); Significant; ThermoFisher Scientific. M. Toro: A. Employment (full or part-time); Significant; ThermoFisher Scientific. J. Chang: A. Employment (full or part-time); Significant; ThermoFisher Scientific. D. Topacio-Hall: A. Employment (full or part-time); Significant; ThermoFisher Scientific. T. Looney: A. Employment (full or part-time); Significant; ThermoFisher Scientific.