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Objective: To determine the rates of germline BRCA1 and BRCA2 mutations in Scottish patients with ovarian cancer, before and after a change in testing policy., Design: Retrospective cohort study., Setting: Four cancer/genetics centres in Scotland., Population: Patients with ovarian cancer undergoing germline BRCA1 and BRCA2 (gBRCA1/2) sequencing before 2013 (under the 'old criteria', with selection based solely on family history), after 2013 (under the 'new criteria', with sequencing offered to newly presenting patients with non-mucinous ovarian cancer), and in the 'prevalent population' (who presented before 2013, but were not eligible for sequencing under the old criteria but were sequenced under the new criteria)., Methods: Clinicopathological and sequence data were collected before and for 18 months after this change in selection criteria., Main Outcome Measures: Frequency of germline BRCA1, BRCA2, RAD51C, and RAD51D mutations., Results: Of 599 patients sequenced, 205, 236, and 158 were in the 'old criteria', 'new criteria', and 'prevalent' populations, respectively. The frequency of gBRCA1/2 mutations was 30.7, 13.1, and 12.7%, respectively. The annual rate of gBRCA1/2 mutation detection was 4.2 before and 20.7 after the policy change. A total of 48% (15/31) 'new criteria' patients with gBRCA1/2 mutations had a Manchester score of <15 and would not have been offered sequencing based on family history criteria. In addition, 20 patients with gBRCA1/2 were identified in the prevalent population. The prevalence of gBRCA1/2 mutations in patients aged >70 years was 8.2%., Conclusions: Sequencing all patients with non-mucinous ovarian cancer gives a much higher annual gBRCA1/2 mutation detection rate, with the frequency of positive tests still exceeding the 10% threshold upon which many family history-based models operate., Tweetable Abstract: BRCA sequencing all non-mucinous cancer patients increases mutation detection five fold., (© 2018 Royal College of Obstetricians and Gynaecologists.)

Background: Investigating tumour evolution and acquired chemotherapy resistance requires analysis of sequential tumour material. We describe the feasibility of obtaining research biopsies in women with relapsed ovarian high-grade serous carcinoma (HGSC)., Methods: Women with relapsed ovarian HGSC underwent either image-guided biopsy or intra-operative biopsy during secondary debulking, and samples were fixed in methanol-based fixative. Tagged-amplicon sequencing was performed on biopsy DNA., Results: We screened 519 patients in order to enrol 220. Two hundred and two patients underwent successful biopsy, 118 of which were image-guided. There were 22 study-related adverse events (AE) in the image-guided biopsies, all grades 1 and 2; pain was the commonest AE. There were pre-specified significant AE in 3/118 biopsies (2.5%). 87% biopsies were fit-for-purpose for genomic analyses. Median DNA yield was 2.87 μg, and was higher in biopsies utilising 14 G or 16 G needles compared to 18 G. TP53 mutations were identified in 94.4% patients., Conclusions: Obtaining tumour biopsies for research in relapsed HGSC is safe and feasible. Adverse events are rare. The large majority of biopsies yield sufficient DNA for genomic analyses-we recommend use of larger gauge needles and methanol fixation for such biopsies, as DNA yields are higher but with no increase in AEs.

Abnormalities in the control and execution of apoptosis are seen in many malignancies, including ovarian carcinoma. Many of these abnormalities involve the mitochondrial pathway of apoptosis, including overexpression of BIR-containing inhibitor of apoptosis protein (IAP) family proteins as well as dysregulated apoptosome function. We sought to stimulate the mitochondrial pathway of apoptosis by constructing a recombinant adenovirus encoding mature, processed Smac/DIABLO (Ad CMV tSmac), the second mitochondrial activator of caspases. Transfection of ovarian carcinoma cells with Ad CMV tSmac leads to increasing apoptosis in a dose-dependent manner. By contrast, transfection of IOSE397 immortalized normal ovarian surface epithelial cells does not cause apoptosis. We also show that the processed form of Smac is primarily expressed in the cytosol of ovarian carcinoma cells. Smac co-immunoprecipitates with both survivin and XIAP and stimulates survivin, but not XIAP, down-regulation. This down-regulation does not result from transcriptional changes, as determined by quantitative real-time PCR, but cycloheximide treatment indicates that survivin half-life is reduced from 6 to 2 h, which is secondary to ubiquitination and proteasomal degradation. RNA interference, however, suggests that survivin does not act to inhibit Smac-mediated apoptosis, which is confirmed by cotransfection with the phosphorylation mutant, survivin T34A. Finally, intraperitoneal delivery of Ad CMV tSmac increases median survival of mice bearing human ovarian carcinoma xenografts. We believe that expression of Smac/DIABLO can stimulate the intrinsic pathway of apoptosis in ovarian carcinoma without damaging normal ovarian tissue and therefore has therapeutic potential.

We have constructed Ad CMV-Smac, a recombinant adenovirus encoding Smac/DIABLO, the recently described second mitochondrial activator of caspases. Transfection of ovarian carcinoma cells with Ad CMV-Smac at multiplicities of infection of 3-60 pfu/cell leads to increasing apoptosis in a dose-dependent manner. Western blot analysis confirms that Smac-induced apoptosis proceeds via a pathway mediated primarily by caspase-9 that can be inhibited by zLEHD-fmk and overexpression of the X-linked inhibitor of apoptosis protein (XIAP). In contrast, there is no cleavage of either caspase-8 or caspase-12. Ad CMV-Smac appears to induce apoptosis independently of cytochrome c release from mitochondria and is not inhibited by overexpression of Bcl-2. Ad CMV-Smac can combine with other proapoptotic factors, such as cisplatin, paclitaxel, and procaspase-3, to produce greater levels of apoptosis in transfected cells.

The enzyme nitroreductase from E. coli can reduce the weak, monofunctional alkylating agent 5-(aziridin-1-yl)-2, 4-dinitrobenzamide (CB1954) to a potent cytotoxic species that generates interstrand crosslinks in DNA. Nitroreductase therefore has potential as a "suicide enzyme" for cancer gene therapy, as cells that express nitroreductase become selectively sensitive to the prodrug CB1954. We have incorporated a nitroreductase expression cassette into a replication-defective adenovirus vector (Ad-CMV-ntr), which allowed efficient gene transfer to SK-OV-3 or IGROV-1 ovarian carcinoma cells. Nitroreductase levels increased in line with multiplicity of infection, and this was reflected in increasing sensitisation of the cells to CB1954, reaching an optimum (approx. 2, 000-fold sensitisation) with 25-50 p.f.u. per cell. Similar Ad-CMV-ntr-dependent sensitisation to CB1954 was seen in 3 of 6 low-passage primary ovarian tumour lines. Cells grown at low-serum concentration to inhibit proliferation remained equally susceptible to the Ad-CMV-ntr-dependent cytotoxicity of CB1954, indicating a distinct advantage over retroviral gene delivery and other popular enzyme-prodrug systems for human tumours with a low rate of cell proliferation. Additionally, cisplatin-resistant cells were sensitised towards CB1954 by Ad-CMV-ntr as efficiently as the parental cells, indicating that the system could be effective in patients with cisplatin-resistant tumours. In a murine xenograft model for disseminated peritoneal carcinomatosis with ascites, treatment of nude mice bearing intraperitoneal SUIT2 tumours with Ad-CMV-ntr and CB1954 almost doubled the median survival from 14 to 26 days (p < 0.0001)., (Copyright 2000 Wiley-Liss, Inc.)