NovelBRCA1andBRCA2Tumor 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.

Potent immunogenicity in BRCA1‐mutated patients with high‐grade serous ovarian carcinoma

“…Several next‐generation sequencing techniques have been developed to detect BRCA1 and BRCA2 mutations and thanks to the clinical application of PARP, an effective and reliable detection of these mutations in formalin‐fixed and paraffin embedded (FFPE) tissue samples has recently been developed . With the development of mutation detecting techniques, the clinical affirmation of our research will be possible although a large cohort will be needed because of the low mutation rate of BRCA in the whole heterogenic colony of ovarian cancer.…”

Missed therapeutic and prevention opportunities in women with BRCA‐mutated epithelial ovarian cancer and their families due to low referral rates for genetic counseling and BRCA testing: A review of the literature

“…The momentum for tumor testing has come because of the availability of the PARP inhibitors . Tumor testing will identify germline BRCA mutations, somatic BRCA mutations, and also homologous repair deficiency (HRD) (see the section below on PARP inhibitors), from either phenotypic DNA patterns or specific gene profiles, all of which predict for a greater PARP inhibitor effect .…”

“…Expert pathologic help (ie, access to pathologists) is needed to identify the best areas for sampling. In addition, the fixation process can lead to denaturing, and these degraded DNA products can lead to false‐positive reporting as mutations in the absence of the correct sequencing technology . Validated testing panels are needed to minimize the chance of false‐negative results, and updated bioinformatics are needed for variant calling .…”

“…Weren et al used a combined technique of single molecular inversion probe‐based next‐generation sequencing combined with multiplex ligation‐dependent probe amplification. Those authors achieved a 99.998% accuracy rate, with a false‐negative reading of nucleotides of only 1 in 1 million . Finally, a stringent validation process is needed for laboratory accreditation, ie, use of next‐generation sequencing, use of proven gene testing panels, and the availability and ability to use the bioinformatics needed for variant calling …”

A BRCA1 és BRCA2 gének tumorevolúcióban szerepet játszó mutációinak vizsgálata

“…A fragmentáltság miatt az FFPE DNS mintákból csak limitált hosszúságú fragmentek amplifikálhatóak, a gyenge minőségű és mennyiségű DNS templát pedig nagymértékben megnövelheti a PCR és szekvenálási hibák arányát [106]. Az FFPE mintákból végzett mutáció-analízis tehát sok szempontból kihívást jelent, többek között ez az oka annak, hogy kevés publikáció számol be FFPE mintából történő, szomatikus mutációk kimutatására is alkalmas BRCA1-2 diagnosztikai módszerek kifejlesztéséről [107]. Ismert olyan tanulmány is, amely az FFPE minta helyett fagyasztott szövetből izolált DNS-en végzett mutáció-analízist javasol a fent említett problémák kiküszöbölésére [108].…”