title: A reliable method for the detection of BRCA1 and BRCA2 mutations in fixed tumour tissue utilising multiplex PCR-based targeted next generation sequencing.
abstract: BACKGROUND
  Germline mutations in BRCA1 or BRCA2 lead to a high lifetime probability of developing ovarian or breast cancer. These genes can also be involved in the development of non-hereditary tumours as somatic BRCA1/2 pathogenic variants are found in some of these cancers. Since patients with somatic BRCA pathogenic variants may benefit from treatment with poly ADP ribose polymerase inhibitors, it is important to be able to test for somatic changes in routinely available tumour samples. Such samples are typically formalin-fixed paraffin-embedded (FFPE) tissue, where the extracted DNA tends to be highly fragmented and of limited quantity, making analysis of large genes such as BRCA1 and BRCA2 challenging. This is made more difficult as somatic changes may be evident in only part of the sample, due to the presence of normal tissue.
  
  
  METHODS
  We examined the feasibility of analysing DNA extracted from FFPE ovarian and breast tumour tissue to identify significant DNA variants in BRCA1/ BRCA2 using next generation sequencing methods that were sensitive enough to detect low level mutations, multiplexed to reduce the amount of DNA required and had short amplicon design. The utility of two GeneRead DNAseq Targeted Exon Enrichment Panels with different designs targeting only BRCA1/2 exons, and the Ion AmpliSeq BRCA community panel, followed by library preparation and adaptor ligation using the TruSeq DNA PCR-Free HT Sample Preparation Kit and NGS analysis on the MiSeq were investigated.
  
  
  RESULTS
  Using the GeneRead method, we successfully analysed over 76% of samples, with >95% coverage of BRCA1/2 coding regions and a mean average read depth of >1000-fold. All mutations identified were confirmed where possible by Sanger sequencing or replication to eliminate the risk of false positive results due to artefacts within FFPE material. Admixture experiments demonstrated that BRCA1/2 variants could be detected if present in >10% of the sample. A sample subset was evaluated using the Ion AmpliSeq BRCA panel, achieving >99% coverage and sufficient read depth for a proportion of the samples.
  
  
  CONCLUSIONS
  Detection of BRCA1/2 variants in fixed tissue is feasible, and could be performed prospectively to facilitate optimum treatment decisions for ovarian or breast cancer patients.
authors: Personalised HealthCare and Biomarkers, Innovative Medicines and Early Development, AstraZeneca, Alderley Park, Macclesfield, SK10 4TG UK.
authors: Gillian
authors: G
authors: Ellison
authors: Genomic Diagnostics Laboratory, Manchester Centre for Genomic Medicine, Central Manchester University Hospitals NHS Foundation Trust, Saint Mary's Hospital, Oxford Road, Manchester, M13 9WL UK.
authors: Shuwen
authors: S
authors: Huang
authors: Translational Science, Oncology Innovative Medicines Unit, AstraZeneca, Alderley Park, Macclesfield, SK10 4TG UK.
authors: Hedley
authors: H
authors: Carr
authors: Genomic Diagnostics Laboratory, Manchester Centre for Genomic Medicine, Central Manchester University Hospitals NHS Foundation Trust, Saint Mary's Hospital, Oxford Road, Manchester, M13 9WL UK.
authors: Andrew
authors: A
authors: Wallace
authors: R&D Information, AstraZeneca, Alderley Park, Macclesfield, SK10 4TG UK.
authors: Miika
authors: M
authors: Ahdesmaki
authors: Genomic Diagnostics Laboratory, Manchester Centre for Genomic Medicine, Central Manchester University Hospitals NHS Foundation Trust, Saint Mary's Hospital, Oxford Road, Manchester, M13 9WL UK.
authors: Sanjeev
authors: S
authors: Bhaskar
authors: Personalised HealthCare and Biomarkers, Innovative Medicines and Early Development, AstraZeneca, Alderley Park, Macclesfield, SK10 4TG UK.
authors: John
authors: J
authors: Mills