Analytical Performance Evaluation of a 523-Gene Circulating Tumor DNA Assay for Next-Generation Sequencing-Based...

Area of Science:

• Oncology
• Genomics
• Molecular Diagnostics

Background:

• Liquid biopsies offer a minimally invasive approach for tumor profiling.
• Comprehensive genomic profiling is crucial for heterogeneous and metastatic cancers.
• Next-generation sequencing (NGS) enables detailed analysis of tumor DNA from blood.

Purpose of the Study:

• To evaluate the analytical performance of the TruSight Oncology 500 (TSO500) ctDNA assay.
• To assess the assay's sensitivity, specificity, and reproducibility for detecting various genetic variants.
• To determine the optimal cell-free DNA (cfDNA) input requirements for reliable tumor profiling.

Main Methods:

• Analytical performance evaluation using reference and patient samples.
• Testing the TSO500 assay, a 523-gene NGS panel, with varying cfDNA input amounts (30 ng, 20 ng, 15 ng, 5 ng).
• Detection of single-nucleotide variants, insertions/deletions, and fusions down to 0.5% variant allele frequency (VAF).

Main Results:

• The TSO500 assay demonstrated high sensitivity and low variant detection variability at 30 ng cfDNA input, detecting variants down to 0.5% VAF.
• Concordance with digital droplet PCR (ddPCR) results was observed in patient samples.
• Reduced cfDNA input (≤20 ng) and lower VAFs (<0.5%) led to decreased sensitivity and increased variability.

Conclusions:

• The TSO500 ctDNA assay provides convincing analytical performance for comprehensive tumor profiling.
• The assay is comparable to other NGS-based ctDNA assays with smaller panels.
• It holds significant value for screening a broad range of tumor-associated genes, especially when tissue biopsy is challenging.