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Implementing NGS-based BRCA tumour tissue testing in FFPE ovarian carcinoma specimens: hints from a real-life experience within the framework of expert recommendations
  1. Daniela Rivera1,
  2. Michele Paudice2,
  3. Viviana Gismondi1,
  4. Giorgia Anselmi3,
  5. Valerio Gaetano Vellone2,3,
  6. Liliana Varesco1
  7. on behalf of the Ligurian BRCA Working Group
    1. 1 Hereditary Cancer Unit, IRCCS Ospedale Policlinico San Martino, Genova, Liguria, Italy
    2. 2 Department of Surgical Sciences and Integrated Diagnostics (DISC), Univeristy of Genoa, Genova, Liguria, Italy
    3. 3 Anatomic Pathology University Unit, IRCCS Ospedale Policlinico San Martino, Genova, Liguria, Italy
    1. Correspondence to Dr Valerio Gaetano Vellone, Department of Surgical and Diagnostic Sciences, University of Genoa, Genova 16126, Italy; valerio.vellone{at}


    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.

    • ovarian neoplasms
    • oncogenes
    • pathology
    • molecular

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    • Handling editor Runjan Chetty.

    • DR, MP, VGV and LV contributed equally.

    • Collaborators Ligurian BRCA Working Group: Paola Baccini (Anatomic Pathology Unit, ASL 4, Sestri Levante, Italy); Rodolfo Brizio (Anatomic Pathology Unit, ASL 1, Sanremo, Italy); Paolo Dessanti (Anatomic Pathology Unit, ASL 5, La Spezia, Italy); Ezio Fulcheri (Fetal, Placental & Gynecologic Pathology Unit, IRCCS Istituto Giannina Gaslini, Genoa, Italy); Marina Gualco (Anatomic Pathology Unit, ASL 3, Genoa, Italy); Eugenio Marinaro (Anatomic Pathology Unit, Ospedali Galliera, Genoa, Italy); Mariangela Rutigliani (Anatomic Pathology Unit, Ospedali Galliera, Genoa, Italy); Ezio Venturino (Anatomic Pathology Unit, ASL 2, Savona, Italy).

    • Contributors LV and VGV conceived and designed the study. GA contributed to the technical part relating to the preanalytical phase. DR, MP and VG performed the experiments and analysed the data. All authors wrote the paper and approved the final version.

    • Funding This project was supported by AstraZeneca under liberal donation to the University of Genoa (VGV).

    • Disclaimer The funding organisation had no influence on the conduct of the project and the evaluation of the data.

    • Competing interests None declared.

    • Patient consent for publication Not required.

    • Ethics approval The study has been approved by the Ligurian Ethical Committee (472REG2015).

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • Data availability statement All data relevant to the study are included in the article or uploaded as supplementary information. There are no additional unpublished data from the study.

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