Abstract 2778: Validation of a next generation sequencing clinical assay for detection of immunoglobulin heavy chain clonality and somatic hypermutation in chronic lymphocytic leukemia
Authors: Tan Xin-Xing , Peters Tricia , Berry Meredith , Jeter Kristyn , Lovell Brigitte , Venegas Victor , Mou Yanglong , Thomas Brad , Funari Vincent , William Josette 
Published: June 2022

Abstract
Next-generation sequencing (NGS) provides a powerful high-throughput approach to identify and track B-cell immunoglobulin (IG) heavy chain clonality and to assess somatic hypermutation status in a massively parallel manner. The NGS-based clonality/SHM testing demonstrated superior performance over the conventional capillary electrophoresis (CE) methods in characterization of B-cell neoplasms. However, its broad utilization in clinical diagnostics requires extensive validation of assays as well as standardization of data interpretation. We present here an NGS assay designed and validated specifically for IG clonality/SHM characterization of B-cell malignancies. The assay was designed to simultaneously target the Leader, FR1, FR2, and FR3 regions of the IGH gene to identify clonal IGH VH-JH rearrangement as well as the IGK gene to identify clonal IGK VK-JK, VK-Kde, and INTR-Kde rearrangement. The validation was performed to assess assay accuracy, specificity, sensitivity, repeatability, and reproducibility, with both pre-characterized reference controls and prospectively collected clinic peripheral blood and bone marrow aspirate specimen included. Triplicates were included and testing was performed at different times and by different operators to assess the assay precision. Over 60 clinic peripheral blood and bone marrow aspirate specimens were collected, including patients with B-cell malignancies such as CLL, B-ALL, and DLBCL as well as heathy donors, processed within 7 days for DNA extraction, and assessed by the NGS assay for IGH clonality and SHM assessment. Assay sensitivity as low as 2.5% for baseline clonality and as low as 0.001% for tracking MRD were observed. Near-perfect assay specificity and precision were observed at these sensitivity levels. The validated NGS assay was further qualified by ERIC (the European Research Initiative on CLL) with a certificate granted to standardize the data interpretation of this assay for testing in chronic lymphocytic leukemia. In conclusion, a NGS IG clonality/SHM assay was analytically and clinically validated under medical oversight, with a demonstrated rigor of the test by its high sensitivity/specificity and robust reproducibility. The clinical diagnostic testing results with this assay is interpreted in accordance with ERIC standards for reliable clinical reporting.
Citation Format: Xin-Xing Tan, Tricia Peters, Meredith Berry, Kristyn Jeter, Brigitte Lovell, Victor Venegas, Yanglong Mou, Brad Thomas, Vincent Funari, Josette William. Validation of a next generation sequencing clinical assay for detection of immunoglobulin heavy chain clonality and somatic hypermutation in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2778.