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

Abstract

AimsNext 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.MethodsEpithelial 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.ResultsNGS 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).ConclusionsA 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.

Tumor BRCA Testing in Epithelial Ovarian Cancers: Past and Future—Five-Years’ Single-Institution Experience of 762 Consecutive Patients

“…We confirmed the feasibility of tumor BRCA testing in the routine clinical setting, with a very low failure rate (less than 1%) and a turnaround time consistent with clinical need (median TAT less than two weeks), strengthening our previous results [ 21 ] and in line with the recently reported experience of other Italian groups [ 19 , 23 , 29 ]. However, both the studies of Marchetti [ 23 ] and Turchiano [ 29 ] were based on a fresh-frozen tissue approach, which was a very promising strategy to overcome the degradation and chemical modification of DNA resulting from formalin fixation and paraffin embedding, but it required framework and facilities (i.e., biobanking) not always available in hospitals.…”

“…Thus, different scientific Societies, including the American Society of Clinical Oncology (ASCO), the National Cancer Comprehensive Network (NCCN), the Society of Gynecologic Oncology (SGO), the European Society of Medical Oncology (ESMO) and the AssociazioneItaliana di Oncologia Medica (AIOM) have recommended BRCA1/2 testing at the time of EOC diagnosis [ 11 , 12 , 13 , 14 ]. Guidelines for Next Generation Sequencing (NGS)-based BRCA test implementation have been proposed [ 15 , 16 , 17 , 18 ] and the feasibility of tumor BRCA assessment has been recently documented [ 19 , 20 , 21 , 22 , 23 ]. However, some critical issues still need to be addressed.…”

Systematic Review of Olaparib in the Treatment of Recurrent Platinum Sensitive Ovarian Cancer

“…The BRCA gene has been shown to be a good predictor of ovarian cancer prognosis. Claudia Marchetti et al (17) and Daniela Rivera et al (18) showed that the IMPLEMENTATION of NGS based BRCA tumor tissue detection in FFPE ovarian cancer specimens confirmed that BRCA gene could predict the prognosis of patients. Genetic testing can be developed for patients through collaboration between pathology and genetics.…”

Probability of detecting germline BRCA1/2 pathogenic variants in histological subtypes of ovarian carcinoma. A meta-analysis

“…Despite this uncertainty in the estimate proportion, germline BRCA1/2 PVs were detected in all histological subtypes of OC. Also, in seven studies that incorporated an extra round of pathological revision in their study design, germline BRCA1/2 PVs were identified in all histological subtypes [1,28,31,34,42,47,48].…”