Ovarian cancer: in search of better marker systems based on DNA repair defects

Abstract

Ovarian cancer is the fifth most common female cancer in the Western world, and the deadliest gynecological malignancy. The overall poor prognosis for ovarian cancer patients is a consequence of aggressive biological behavior and a lack of adequate diagnostic tools for early detection. In fact, approximately 70% of all patients with epithelial ovarian cancer are diagnosed at advanced tumor stages. These facts highlight a significant clinical need for reliable and accurate detection methods for ovarian cancer, especially for patients at high risk. Because CA125 has not achieved satisfactory sensitivity and specificity in detecting ovarian cancer, numerous efforts, including those based on single and combined molecule detection and "omics" approaches, have been made to identify new biomarkers. Intriguingly, more than 10% of all ovarian cancer cases are of familial origin. BRCA1 and BRCA2 germline mutations are the most common genetic defects underlying hereditary ovarian cancer, which is why ovarian cancer risk assessment in developed countries, aside from pedigree analysis, relies on genetic testing of BRCA1 and BRCA2. Because not only BRCA1 and BRCA2 but also other susceptibility genes are tightly linked with ovarian cancer-specific DNA repair defects, another possible approach for defining susceptibility might be patient cell-based functional testing, a concept for which support came from a recent case-control study. This principle would be applicable to risk assessment and the prediction of responsiveness to conventional regimens involving platinum-based drugs and targeted therapies involving poly (ADP-ribose) polymerase (PARP) inhibitors.

Figure 1

Schematic overview of susceptibility genes for familial ovarian cancer. Ten to fifteen percent of ovarian cancer cases are of familial origin. Until now, 16 susceptibility genes causing at least six cancer susceptibility syndromes have been identified [12,13]. However, approximately 80 to 90% of the hereditary ovarian cancer cases can be explained by mutations in BRCA1, BRCA2, RAD51C, and RAD51D, which cause Hereditary Breast and Ovarian Cancer Syndrome.

Figure 2

Interactome of ovarian cancer susceptibility gene products summarizing DNA damage response activities and assays for the detection of functional defects. Functional and physical interactions between DNA repair-related ovarian cancer susceptibility gene products are schematically drawn and their role in different DNA repair mechanisms and checkpoint responses during the cell cycle are indicated [–35]. Various readouts for DNA repair failure that have been assayed as potential biomarkers for ovarian cancer risk are positioned next to the corresponding mechanisms, as discussed in the text [,,–39]. One-headed arrow, recruitment or activation; two-headed arrow, physical interaction; stippled arrow, transcriptional regulation; encircled “P”, phosphorylation; blocked line, inhibition; blue-circled protein names, ovarian carcinoma susceptibility gene product; red letters, processes with relevance for genome stability; vaulted black arrow, detection of a repair defect. Note that breaks may also occur in cell cycle phases other than G1/S phase.

Figure 3

Detection of error-prone DSB repair activities in peripheral blood lymphocytes. Peripheral blood lymphocytes from high-risk breast and ovarian cancer individuals, breast cancer patients, and healthy control individuals were comparatively analyzed using the EGFP-based DSB repair assay system [39]. Peripheral blood lymphocytes were isolated from blood samples by Ficoll gradient centrifugation, brought into ex vivo culture, and pathway-specific substrates nucleofected (amaxa) into these lymphocytes together with the expression plasmid for the endonuclease I-SceI. The schematic drawing outlines the representative DNA substrates for measurements of error-prone DSB repair activities, specifically NHEJ and SSA. These substrates are composed of an I-SceI recognition sequence within a mutated EGFP gene, which allows the targeted introduction of a DSB. After NHEJ and SSA, the wild-type EGFP gene is reconstituted, and the fraction of EGFP-positive cells is quantified flow cytometrically by FACS analysis.