Investigating structural variants of BRCA1/2 as a novel biomarker of homologous recombination deficiency

Abstract

Homologous recombination deficiency (HRD) represents a cancer vulnerability that has been successfully exploited through the use of PARP inhibitors by synthetic lethality. In high grade serous ovarian cancer (HGSOC), PARP inhibitors, such as olaparib, have revolutionised treatments outcomes for patients and are now routinely clinically used. PARP inhibitors are also approved for breast, pancreatic and prostate cancer, but their use is largely restricted to patients with a BRCA1/2 mutation. Though BRCA1/2 mutations are the archetypal event resulting in HRD, there is increasing evidence that there are patients with an HRD phenotype beyond those with a BRCA1/2 mutation. This therefore represents an opportunity to broaden the use of PARP inhibitors, including their use in other tumour types. Structural variants (SVs) are increasingly recognised as key cancer driver events. However, their importance has been underappreciated due to limited access to whole genome sequencing (WGS). Previously, SVs of BRCA1/2 were found to occur in ~15% of HGSOC cases, and were associated with reduced BRCA1/2 gene expression and higher genomic HRD scores. Furthermore, BRCA1/2 SVs have been shown to occur in a range of other tumour types, at a lower frequency. Despite the existing evidence supporting the importance of BRCA1/2 SVs in tumourigenesis, their functional impact has not been directly studied in vitro, including via functional homologous recombination (HR) assays. Moreover, the phenotype associated with this defect has not been explored in a range of different tumour types. Through in silico analysis, a provisional panel of candidate cell lines with BRCA1 or BRCA2 copy number loss were identified. These underwent detailed characterisation of existing genomic data, along with validation of copy number loss by quantitative PCR (qPCR). This resulted in a high confidence panel of candidate cell lines (n=11) from multiple tumour sites (ovary, breast, soft tissue sarcoma and bone sarcoma) with copy number loss of BRCA1 or BRCA2, which are likely to have BRCA1/2 SV deletions. These were investigated alongside known HR deficient comparator lines and an HR proficient cell line. The phenotype of these candidate cell lines was assessed, which included BRCA1/2 gene expression, BRCA1/2 protein expression and HR status by RAD51 immunofluorescence following irradiation. Drug sensitivity to chemotherapeutics and PARP inhibitors (olaparib, novel PARP1 selective PARP inhibitor AZD5305) was also performed via proliferation assay and colony formation assay. In the cell lines with BRCA1 SVs, two of the seven cell lines demonstrated a low BRCA1 gene expression (comparable to the BRCA1 mutant comparator), HR deficiency by RAD51 immunofluorescence and PARP inhibitor sensitivity. These were both Ewing’s sarcoma cell lines. There were a further two cell lines with BRCA1 SVs, originating from a breast and an ovarian tumour, that were functionally HR deficient, but did not demonstrate PARP inhibitor sensitivity. Four of the five cell lines with BRCA2 SVs had reduced BRCA2 gene expression (similar to that of a known BRCA2 mutant cell line). Furthermore, four of the five cell lines with BRCA2 SVs were functionally HR deficient and three of these cell lines were PARP inhibitor sensitive. These cell lines originated from varying tumour sites including ovary and two histological types of soft tissue sarcoma (leiomyosarcoma, rhabdomyosarcoma). These data suggest SVs, particularly in BRCA2, may represent a novel biomarker of HRD, which could be therapeutically exploitable with PARP inhibitors. Given that PARP inhibitors are not used routinely in sarcomas, this could represent a new therapeutic indication. This in vitro work therefore suggested that the accurate identification of BRCA1/2 SVs via copy number loss could have high clinical utility in identifying patients suitable for PARP inhibitors. With this in mind, the final chapter of this body of work aimed to identify a simple costeffective laboratory test to identify these defects. It was hypothesised that qPCR could be an efficient laboratory biomarker assay for detecting SVs. This work assessed the sensitivity and specificity of qPCR in detecting copy number variation of BRCA1/2, in two HGSOC clinical cohorts (cohort 1 n=355, cohort 2 n=89), in conjunction with matched next generation sequencing (cohort 1 panel sequencing, cohort 2 WGS). This work demonstrated qPCR had poor sensitivity for detecting BRCA1/2 SVs, with copy number variation at the reference probe erroneously leading to BRCA1/2 copy number loss detection. Overall, this suggests that next generation sequencing, such as WGS, remains the optimal method for accurately detecting these events