Abstract Illumina’s TruSight™ Oncology 500 (TSO500) ctDNA v2 assay, enables comprehensive genomic profiling from plasma, allowing non-invasive, repeatable monitoring of tumor evolution during clinical studies. The assay covers a broad panel of 523 clinically relevant cancer genes and provides sensitive detection of key biomarkers such as SNVs, indels, CNVs, gene fusions, MSI, and TMB, all from a simple blood draw. Therefore, this RUO assay is well-suited for exploratory biomarker discovery, response monitoring, and mechanistic insights during immunotherapy and targeted therapy development without the need for fresh tissue biopsies. A validation study was performed to characterize the analytical performance of TSO500 ctDNA v2 assay on the NovaSeq™ X Plus platform for comprehensive genomic profiling of 523 cancer-related genes in ctDNA. The assay was evaluated at two CellCarta laboratories (Antwerp, Belgium and Naperville, USA). Accuracy was assessed using reference materials with a variant allele frequency (VAF) threshold of 0.5% for SNVs and indels. Across both sites, the assay demonstrated 95% overall percent agreement for SNV, indel, CNV, MSI, and TMB results, with positive percent agreement 93.5% and negative percent agreement 95% for SNV, indel, and CNV (no negative reference materials were available for MSI and TMB). Precision assessments showed 97% concordance for intra-run, inter-run, and inter-operator comparisons across all variant classes. Limit of detection verification confirmed 100% detection of SNVs down to 0.25% VAF, indels down to 0.5% VAF, and CNVs at 1.0 fold change. The optimal input for the assay was determined to be 20 ng ctDNA; however, evaluation of lower inputs (10 ng and 5 ng) demonstrated that while these samples did not consistently meet QC thresholds, SNV and indel detection remained robust, with 98% concordance for SNVs and 95% for indels. Lower input material may therefore be used for exploratory analyses, with the understanding that reduced coverage may limit detection sensitivity (potential false negatives) but does not introduce false-positive variant calls. These results confirm that the TSO500 ctDNA v2 assay delivers reproducible, high-resolution genomic profiling across sites, supporting its applicability for exploratory biomarker analyses and longitudinal response monitoring in clinical studies. Citation Format: Gabriela Edwards Faret, Emmanuel Rivière, Silvie Franck, Bart Tegenbos, Lisa Van den Bossche, Joke Verbist, Lien Heyrman, Magdalena Lesnicki, Rachel Raz, Brian Chapman, Eric Harness, Jan Van de Velde, Dirk Goossens, Jurgen Del Favero. Scaling liquid biopsy biomarker discovery: Multi-center, analytical validation of high resolution ctDNA profiling using TSO500 v2 on NovaSeq X Plus abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3245.
Faret et al. (Fri,) studied this question.
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