Valerio Gaetano Vellone

Implementing NGS-based BRCA tumour tissue testing in FFPE ovarian carcinoma specimens: hints from a real-life experience within the framework of expert recommendations

Aims Next Generation Sequencing (NGS)-based BRCA tumour tissue testing poses several challenges. As a first step of its implementation within a regional health service network, an in-house validation study was compared with published recommendations. Methods Epithelial ovarian cancer (EOC) formalin-fixed paraffin-embedded specimens stored in the archives of the eight regional pathology units were selected from a consecutive series of patients with known BRCA germline status. Two expert pathologists evaluated tumour cell content for manual macrodissection. DNA extraction, library preparation and NGS analyses were performed blinded to the germinal status. Parameters used in the study were confronted with guidelines for the validation of NGS-based oncology panels and for BRCA tumour tissue testing. Results NGS analyses were successful in 66 of 67 EOC specimens, with good quality metrics and high reproducibility among different runs. In all, 19 BRCA pathogenic variants were identified: 12 were germline and 7 were somatic. A 100% concordance with blood tests was detected for germline variants. A BRCA1 variant showed a controversial classification. In different areas of two early stage EOCs showing somatic variants, intratumour heterogeneity not relevant for test results (variant allele frequency >5%) was observed. Compared with expert recommendations, main limitations of the study were absence of controls with known somatic BRCA status and exclusion from the validation of BRCA copy number variations (CNV). Conclusions A close collaboration between pathology and genetics units provides advantages in the implementation of BRCA tumour tissue testing. The development of tools for designing and interpreting complex testing in-house validation could improve process quality.