Analytical and clinical validation of a novel CE-IVD kit for hotspot mutations in liquid biopsy samples.

mutations have been implicated in the prognosis and therapeutic response in HER2-positive early breast cancer, with variants located in exon 9 and exon 20 shown to modulate sensitivity to neoadjuvant chemotherapy. Several molecular diagnostic platforms based on PCR or NGS have been developed for the detection of mutations in both tumor tissues and plasma-derived cfDNA, and some commercially available assays have already received FDA approval. The aim of this study was to evaluate the analytical and clinical performance of a novel CE-IVD molecular assay (Oncolipsy, kit, Pharmassist, Greece) for hotspot PIK3CA mutations (p.E542K, p.E545K, p.E545Q, p.H1047R) in liquid biopsy (LB) specimens. Analytical validation included assessment of sensitivity, specificity and reproducibility using certified liquid biopsy reference standards. Subsequently, the clinical utility of the assay was evaluated by analyzing plasma-cfDNA, CTC-derived gDNA and primary tumor samples for hotspot mutations. A total of 55 peripheral blood samples from breast cancer (BrCa) patients and 30 primary tumors (FFPEs) were examined. The same samples were also tested with two commercially available assays, the cobas® Mutation Test (Roche Diagnostics) and the ddPCR mutation test (BioRad), and the results were directly compared. Our findings demonstrate that the Oncolipsy kit exhibits high analytical detectability and excellent specificity for detecting hotspot mutations, at low variant allele frequencies. Clinical evaluation confirmed its robustness for liquid biopsy applications, with p.H1047R identified as the most frequent mutation. Concordance with both commercially available assays was high, with minor discrepancies attributable to differences in mutation coverage or detection thresholds. In conclusion, the CE-IVD Oncolipsy kit represents a highly detectability, specific and cost-effective real-time PCR-based solution for high throughput detection of four clinically relevant hotspot mutations in liquid biopsy samples.