Detection of Clinically Significant BRCA Large Genomic Rearrangements in FFPE Ovarian Cancer Samples: A Comparative NGS Study

Background: Copy number variations (CNVs), also referred to as large genomic rearrangements (LGRs), represent a crucial component of BRCA1/2 (BRCA) testing. Next-generation sequencing (NGS) has become an established approach for detecting LGRs by combining sequencing data with dedicated bioinformatics pipelines. However, CNV detection in formalin-fixed paraffin-embedded (FFPE) samples remains technically challenging, and it cannot always be guaranteed that such in-formation will be reliably obtained. Therefore, optimization is needed, and implementing a robust analysis strategy for routine clinical practice could provide significant advantages. Methods: This study evaluated 40 FFPE ovarian cancer (OC) samples from patients undergoing BRCA testing. The performance of the amplicon-based NGS Diatech Myriapod® NGS BRCA1/2 panel (Diatech Pharmacogenetics, Jesi, Italy) was assessed for its ability to detect BRCA CNVs, and results were compared to two hybrid capture-based reference assays. Results: Among the 40 analyzed samples (17 CNV-positive and 23 CNV-negative for BRCA genes), the Diatech pipeline showed high concordance with the reference methods. In a clinical di-agnostic setting, the evaluated method achieved an overall accuracy of about 96%, with a sensitivity of 94% and specificity of 96%. Despite one inconclusive result due to low sequencing quality and one sample with a somatic CNV in BRCA1 that was not detected, the Diatech Myriapod® NGS BRCA1/2 panel kit demonstrated strong potential for routine clinical application in CNV detection from FFPE tissue. Conclusions: These findings support the clinical utility of NGS-based CNV analysis in FFPE sam-ples when combined with appropriate bioinformatics tools. Integrating visual inspection of CNV plots with automated CNV calling improves the reliability of CNV detection and enhances the in-terpretation of results from tumor tissue. Accurate CNV detection directly from tumor tissue may reduce the need for reflex germline testing and improve turnaround times. Nevertheless, blood-based testing remains essential to determine whether detected BRCA CNVs are of germline or so-matic origin, particularly in cases with a strong clinical suspicion of a germline CNV.