Abstract 233: Inter-site performance comparison of a next generation sequencing (NGS) assay for minimal residual disease detection research in diffuse large B-Cell lymphoma (DLBCL) using plasma circulating tumor DNA (ctDNA)
Authors: Woestmann Corinna , Lin Hai , McNamara Sylvie , Shin Maria , Bermejo Clara , Hammer Sabrina , Heitzer Ellen , Blombery Piers , Yaung Stephanie , Su Bowdoin 
Published: April 2023

Abstract
Background: Sequencing of ctDNA holds tremendous potential in oncology for disease monitoring to identify patients who relapse or become refractory. This amounts to approximately 35-40% of patients with DLBCL who fail treatment with standard of care immunochemotherapy. We have extended and modified a plasma-based NGS workflow for DLBCL. To evaluate inter-site robustness of our workflow, we conducted a pilot study to measure performance of the assay at three laboratories.
Materials and Methods: A total of 98 samples were available for analysis. Given the requirement for sequencing multiple replicates, contrived samples were used in this pilot study. All labs performed targeted next generation sequencing (NGS) on all samples using a modified version of the AVENIO ctDNA analysis workflow (Roche) which is based on the previously described CAPP-Seq workflow. [1-3] The targeted NGS panel was designed to cover ~314 kb of regions in or near 466 genes relevant for minimal residual disease and cell-of-origin detection in DLBCL. QC metrics were compared on 6 healthy donor plasma samples. For analyzing the sensitivity of single nucleotide variant (SNV) calling, 3 plasma samples were blended so that expected variants were titrated to 1% allelic frequency (AF), and 3 samples to 0.5% AF to mimic low pretreatment ctDNA levels in DLBCL. For analyzing performance of MRD detection, the SNVs in 5 samples were titrated down to 0.05%, 0.01% or 0.005% AF to represent on-treatment samples. MRD detection was evaluated by Monte Carlo sampling comparing ctDNA level against background. [3]
Results: All replicates of the 6 healthy donor samples had comparable extracted cfDNA within 30% between sites. On-target rates ranged between 67.2 and 74.8% (median: 71.0%) across all sites. Deduplicated sequencing depth was within 20% between sites and ranged from 1321-7346 (median: 2845), depending on the sample. Observed error rates were below 0.005% for all samples. Testing sites achieved 98.6-100% (median: 100%) sensitivity for SNVs at 1% AF blends, and 87.5-100% (median: 96.9%) for 0.5% AF blends. All external sites consistently detected MRD with p &lt; 0.005 at AF levels of 0.05% and 0.01%, and p &lt; 0.05 at 0.005% AF level.
Conclusion: QC metrics were highly consistent for the same samples prepared at different testing sites. Sensitivity of SNV calling was high, and MRD results were consistent across replicates and sites. We demonstrate that the AVENIO ctDNA NHL assay for plasma and NGS applications provides robust performance for MRD monitoring research. AVENIO ctDNA NHL assay is for Research Use Only; not for use in diagnostic procedures. 1. Scherer et al. Sci Transl Med. 2016; 8(364). 2. Kurtz et al. J Clin Oncol. 2018; 36(28):2845-2853. 3. Newman et al. Nat Bioech. 2016.
Citation Format: Corinna Woestmann, Hai Lin, Sylvie McNamara, Maria Shin, Clara Bermejo, Sabrina Hammer, Ellen Heitzer, Piers Blombery, Stephanie Yaung, Bowdoin Su. Inter-site performance comparison of a next generation sequencing (NGS) assay for minimal residual disease detection research in diffuse large B-Cell lymphoma (DLBCL) using plasma circulating tumor DNA (ctDNA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 233.