Novel BRCA1 and BRCA2 Tumor Test as Basis for Treatment Decisions and Referral for Genetic Counselling of Patients with Ovarian Carcinomas

Abstract

With the recent introduction of Poly(ADP-ribose) polymerase inhibitors, a promising novel therapy has become available for ovarian carcinoma (OC) patients with inactivating BRCA1 or BRCA2 mutations in their tumor. To select patients who may benefit from these treatments, assessment of the mutation status of BRCA1 and BRCA2 in the tumor is required. For reliable evaluation of germline and somatic mutations in these genes in DNA derived from formalin-fixed, paraffin-embedded (FFPE) tissue, we have developed a single-molecule molecular inversion probe (smMIP)-based targeted next-generation sequencing (NGS) approach. Our smMIP-based NGS approach provides analysis of both strands of the open reading frame of BRCA1 and BRCA2, enabling the discrimination between real variants and formalin-induced artefacts. The single molecule tag enables compilation of unique reads leading to a high analytical sensitivity and enabling assessment of the reliability of mutation-negative results. Multiplex ligation-dependent probe amplification (MLPA) and Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) were used to detect exon deletions of BRCA1 and methylation of the BRCA1 promoter, respectively. Here, we show that this combined approach allows the rapid and reliable detection of both germline and somatic aberrations affecting BRCA1 and BRCA2 in DNA derived from FFPE OCs, enabling improved hereditary cancer risk assessment and clinical treatment of ovarian cancer patients.

Keywords: BRCA testing; BRCA1; BRCA2; PARP-inhibitor; cancer predisposition; ovarian cancer; personalized medicine; single molecule molecular inversion probes.

Figure 1

A: Average number of unique reads per smMIP. X‐axis: number of unique smMIPs included in our panel to sequence BRCA1 (n = 157 smMIPs) and BRCA2 (n = 260 smMIPs) (ordered for the genomic position of the target). Y‐axis: number of unique reads obtained per smMIP. The average and median number of unique reads per smMIP is 254 and 154, respectively. Numbers are based on sequencing results obtained from 107 FFPE samples (for details; see main text or Materials and Methods). B: Number of unique reads/smMIPs mapping to/targeting the plus or minus strand of the ORF of BRCA1 and BRCA2. smMIPs targeting plus/minus strand: total number of smMIPs targeting the plus/minus strand of the corresponding base‐pair position (range 0–3 smMIPs per strand per position). Average reads plus/minus strand: average number of unique reads mapping to the plus and minus strand based on 107 ovarian carcinoma samples. On average, the plus/minus strand were covered 356x/291x (BRCA1) and 334x/258x (BRCA2), respectively. Bars on the X‐axis represent the nucleotides located in the exons of BRCA1 and BRCA2, including the canonical splice sites. Red bars indicate regions (4 and 16 bp, respectively) without any smMIPs or mapped reads at the corresponding locus. Note that 100% of the ORF including the canonical splice sites is sequenced and that 99.8% is sequenced on both the plus and minus strand. C: Percentage of the ORF of BRCA1 and BRCA2 covered with at least 20 and 30 unique reads in 107 FFPE ovarian carcinoma samples. On average, 98.8% (median 99.8%, range 79.3%–100%) and 97.4% (median 99.6, range 60.9%–100%) of the ORF of BRCA1 and BRCA2, including the −20 and +20 intronic regions, was covered with a sequencing depth of at least 20x and 30x, respectively.

Figure 2

A: Pathogenic germline and somatic mutations in BRCA1 and BRCA2 detected using smMIP‐based targeted sequencing of FFPE tumor material. Lollipops above the bar: germline mutations detected in 47 FFPE ovarian carcinomas derived from 38 patients. Lollipops below the bar: somatic pathogenic mutations observed in seven FFPE ovarian carcinomas derived from five patients. B: Genomic location of 43 germline SNPs in BRCA1 and BRCA2 that were selected to determine the sensitivity of smMIP‐based next‐generation sequencing. Depicted base substitutions (lollipops) represent 15 and 28 benign germline variants in the ORF of BRCA1 and BRCA2, respectively. These variants were known to be present in the germline of a subset of the included ovarian carcinoma patients prior to smMIP‐based sequencing of BRCA1 and BRCA2 in the corresponding FFPE ovarian carcinomas. Numbers depict the total number of the corresponding germline variant observed in these ovarian carcinoma patients. All germline SNPs could successfully be detected using smMIP‐based targeted sequencing on DNA derived from the corresponding FFPE ovarian carcinoma sample.