Introduction:

Assessment of homologous recombination (HR) function, by RAD51 focus formation, in primary cultures of ascites cells from patients with ovarian cancer is an important predictive and prognostic marker in ovarian cancer [1]. However, this assay is labor-intensive, time-consuming and is technically-challenging so not suitable for routine clinical practice. We therefore correlated methylation and mutation status of BRCA1/2 genes with HR function in a panel of ovarian cancer samples.

Methods:

Matched samples of ascites and tumor tissue were taken from patients undergoing primary surgery for epithelial ovarian cancer. Tumor samples were formalin fixed and paraffin embedded whilst the ascites samples were used to generate primary cultures. HR status was determined in the primary cultures as previously described [2]. DNA was extracted from the FFPE tissue and the pellets of primary culture cells. Methylation of BRCA1 was measured using a real time PCR based duplex assay for BRCA1 and the reference gene ACTB. A next generation sequencing based assay was used to detect mutations and other genomic alterations in a large panel of cancer-associated genes, including BRCA1/2.

Results:

To date we have collected paired samples from 39 patients which have been characterised for HR function. Molecular analysis of the first 17 samples (9 HR defective, 8 HR competent) revealed that all mutations and other genomic alterations detected in ascitic derived primary cultures were also found in matched FFPE tumor tissues. All 13 tumors with serous histology contained p53 mutations, whilst the remaining 4 tumors without p53 mutations were non-serous in histology (2 mucinous and 2 endometrioid). Deleterious BRCA2 mutations were identified in 3 of 17 tumors, all of which were HR defective. While no deleterious BRCA1 mutation was detected, methylation of BRCA1 was identified in 3 of 17 tumors, two of which were HR defective. Mutation of BRCA2 was mutually exclusive to methylation of BRCA1. The remaining 4 of 9 HR defective tumors do not have BRCA1/2 mutations or BRCA1 methylation, suggesting other mechanisms can also result in a HR defective phenotype.Conclusions

As expected, deleterious BRCA2 mutations conferred a HR defective phenotype in cells but methylation of BRCA1 was not universally associated with HR dysfunction. This may be as a result of only partial silencing of the gene by methylation and further work is required to identify thresholds of methylation which may predict HR status. The use of BRCA1/2 mutation testing alone is unlikely to identify the majority of HR defective ovarian tumors.

References

1. Mukhopadhyay, A., et al., Cancer Research, 2012.

2. Mukhopadhyay, A., et al., Clinical Cancer

Research, 2010. 16(8): p. 2344-2351.

Citation Format: Richard J. Edmondson, Asima Mukhopadhyay, Leander Van Neste, Wim Van Criekinge, Nicola Curtin. Molecular characterization of matched ovarian ascites and tumors suggests multiple mechanisms for a homologous recombination defective phenotype. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5608. doi:10.1158/1538-7445.AM2014-5608