Analytical Validation of a Pan-Cancer NGS Assay for In-House Liquid Biopsy Testing: An International Multicenter Study

: Liquid biopsy (LBx) assays are transforming precision oncology by providing a less invasive alternative to tissue biopsies. These assays screen for tumour genetic alterations in circulating free DNA (cfDNA), which typically requires detecting variants at low allele frequencies and therefore a high sensitivity and specificity. This international, multicenter analytical performance study evaluated the Hedera Profiling 2 ctDNA test panel (HP2) (Hedera Dx, Epalinges, Switzerland), a hybrid capture-based NGS assay for the detection of somatic alterations in cfDNA. Covering 32 genes, HP2 enables the detection of SNVs, Indels, Fusions, CNVs, and MSI status from a single DNA-only workflow. The analytical performance was assessed using reference standards and a diverse cohort of 137 clinical samples pre-characterized by orthogonal methods. In reference standards with variants spiked-in at 0.5% allele frequency, sensitivity and specificity were 96.92% and 99.67% respectively for SNVs/Indels and 100% for Fusions. In clinical samples, SNVs/Indels detection showed high concordance (94% for ESCAT Level I variants) with orthogonal methods. Evidence for solid sensitivity was also found for CNV detection and MSI status determination. Overall, the HP2 assay showed significant potential as a sensitive and efficient Pan-Cancer test for LBx testing in a decentralized molecular pathology laboratory setting.