Correction: Safety and utility of image-guided research biopsies in relapsed high-grade serous ovarian carcinoma-experience of the BriTROC consortium.

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Safety and utility of image-guided research biopsies in relapsed high-grade serous ovarian carcinoma-experience of the BriTROC consortium.

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.

A randomised, phase II trial of the DNA-hypomethylating agent 5-aza-2'-deoxycytidine (decitabine) in combination with carboplatin vs carboplatin alone in patients with recurrent, partially platinum-sensitive ovarian cancer.

BACKGROUND: Our previous laboratory and clinical data suggested that one mechanism underlying the development of platinum resistance in ovarian cancer is the acquisition of DNA methylation. We therefore tested the hypothesis that the DNA hypomethylating agent 5-aza-2'-deoxycytodine (decitabine) can reverse resistance to carboplatin in women with relapsed ovarian cancer. METHODS: Patients progressing 6-12 months after previous platinum therapy were randomised to decitabine on day 1 and carboplatin (AUC 6) on day 8, every 28 days or carboplatin alone. The primary objective was response rate in patients with methylated hMLH1 tumour DNA in plasma. RESULTS: After a pre-defined interim analysis, the study closed due to lack of efficacy and poor treatment deliverability in 15 patients treated with the combination. Responses by GCIG criteria were 9 out of 14 vs 3 out of 15 and by RECIST were 6 out of 13 vs 1 out of 12 for carboplatin and carboplatin/decitabine, respectively. Grade 3/4 neutropenia was more common with the combination (60% vs 15.4%) as was G2/3 carboplatin hypersensitivity (47% vs 21%). CONCLUSIONS: With this schedule, the addition of decitabine appears to reduce rather than increase the efficacy of carboplatin in partially platinum-sensitive ovarian cancer and is difficult to deliver. Patient-selection strategies, different schedules and other demethylating agents should be considered in future combination studies.

Lymphokine-activated killer and dendritic cell carriage enhances oncolytic reovirus therapy for ovarian cancer by overcoming antibody neutralization in ascites.

Reovirus is an oncolytic virus (OV), which acts by both direct tumor cell killing and priming of antitumor immunity. A major obstacle for effective oncolytic virotherapy is effective delivery of OV to tumor cells. Ovarian cancer is often confined to the peritoneal cavity and therefore i.p. delivery of reovirus may provide the ideal locoregional delivery, avoiding systemic dissemination. However, ovarian cancer is associated with an accumulation of ascitic fluid, which may interfere with oncolytic viral therapy. Here, we investigated the effect of ascites on reovirus-induced oncolysis against primary ovarian cancer cells and ovarian cancer cell lines. In the absence of ascites, reovirus was cytotoxic against ovarian cancer cells; however, cytotoxicity was abrogated in the presence of ascitic fluid. Neutralizing antibodies (NAb) were identified as the cause of this inhibition. Loading OV onto cell carriers may facilitate virus delivery in the presence of NAb and immune cells which have their own antitumor effector activity are particularly appealing. Immature dendritic cells (iDC), Lymphokine-activated killer (LAK) cells and LAKDC cocultures were tested as potential carriers for reovirus for tumor cell killing and immune cell priming. Reovirus-loaded LAKDC, and to a lesser degree iDC, were able to: (i) protect from NAb and hand-off reovirus for tumor cell killing; (ii) induce a proinflammatory cytokine milieu (IFNÉ£, IL-12, IFNα and TNFα) and (iii) generate an innate and specific antitumor adaptive immune response. Hence, LAKDC pulsed with reovirus represent a novel, clinically practical treatment for ovarian cancer to maximise both direct and innate/adaptive immune-mediated tumor cell killing.

Adenovirus-mediated hypoxia-targeted gene therapy using HSV thymidine kinase and bacterial nitroreductase prodrug-activating genes in vitro and in vivo.

Hypoxia is an important factor in tumor growth. It is associated with resistance to conventional anticancer treatments. Gene therapy targeting hypoxic tumor cells therefore has the potential to enhance the efficacy of treatment of solid tumors. Transfection of a panel of tumor cell lines with plasmid constructs containing hypoxia-responsive promoter elements from the genes, vascular endothelial growth factor (VEGF) and erythropoietin, linked to the minimal cytomegalovirus (mCMV) or minimal interleukin-2 (mIL-2) promoters showed optimum hypoxia-inducible luciferase reporter gene expression with five repeats of VEGF hypoxic-response element linked to the mCMV promoter. Adenoviral vectors using this hypoxia-inducible promoter to drive therapeutic transgenes produced hypoxia-specific cell kill of HT1080 and HCT116 cells in the presence of prodrug with both herpes simplex virus thymidine kinase/ganciclovir and nitroreductase (NTR)/CB1954 prodrug-activating systems. Significant cytotoxic effects were also observed in patient-derived human ovarian cancer cells. The NTR/CB1954 system provided more readily controllable transgene expression and so was used for in vivo experiments of human HCT116 xenografts in nude mice. Subjects treated intratumorally with Ad-VEGFmCMV-NTR and intraperitoneal injection of CB1954 demonstrated a statistically significant reduction in tumor growth. Immunohistochemistry of treated xenografts showed a good correlation between transgene expression and hypoxic areas. Further investigation of these hypoxia-inducible adenoviral vectors, alone or in combination with existing modalities of cancer therapy, may aid in the future development of successful Gene-Directed Enzyme Prodrug Therapy systems, which are much needed for targeting solid tumors.

ASPM and microcephalin expression in epithelial ovarian cancer correlates with tumour grade and survival.

BACKGROUND: The clinico-pathological and molecular heterogeneity of epithelial ovarian cancer (EOC) complicates its early diagnosis and successful treatment. Highly aneuploid tumours and the presence of ascitic fluids are hallmarks of EOC. Two microcephaly-associated proteins, abnormal spindle-like microcephaly-associated protein (ASPM) and microcephalin, are involved in mitosis and DNA damage repair. Their expression is deregulated at the RNA level in EOC. Here, ASPM and microcephalin protein expression in primary cultures established from the ascites of patients with EOC was determined and correlated with clinical data to assess their suitability as biomarkers. METHODS: Five established ovarian cancer cell lines, cells derived from two benign ovarian ascites samples and 40 primary cultures of EOC derived from ovarian ascites samples were analysed by protein slot blotting and/or immunofluorescence to determine ASPM and microcephalin protein levels and their cellular localisation. Results were correlated with clinico-pathological data. RESULTS: A statistically significant correlation was identified for ASPM localisation and tumour grade, with high levels of cytoplasmic ASPM correlating with grade 1 tumours. Conversely, cytoplasmic microcephalin was only identified in high-grade tumours. Furthermore, low levels of nuclear microcephalin correlated with reduced patient survival. CONCLUSION: Our results suggest that ASPM and microcephalin have the potential to be biomarkers in ovarian cancer.

Role of cell surface molecules and autologous ascitic fluid in determining efficiency of adenoviral transduction of ovarian cancer cells.

Adenovirus is the most frequently used virus in gene therapy clinical trials. There have been conflicting reports on the ability of adenovirus to transduce primary ovarian cancer samples and the expression of relevant cell surface molecules. These factors were examined using primary ovarian cancer cells cultured from ascites and solid tumor to gain insights into the clinical use of adenovirus in ovarian cancer. The level of transduction of primary cultures was much higher than uncultured cells and established cell lines, and correlated with higher levels of coxsackie-adenovirus receptor (CAR) and integrin expression. Growth of primary cultures in autologous ascitic fluid prevented an increase in CAR expression and inhibited transduction compared with cells treated in supplemented RPMI. Cells at the periphery of solid tumor samples were transduced using a replication-incompetent virus and correlated with CAR expression. However, transduction was abolished by autologous ascitic fluid, despite the expression of CAR. We conclude that the use of adenoviruses for ovarian cancer gene therapy will require testing in the presence of inhibitory factors in ascitic fluid. The clinical use of adenoviral vectors may require circumvention of such inhibitory factors and the use of replication competent adenovirus to enable efficient viral penetration of the cancer.

Retargeted adenoviral cancer gene therapy for tumour cells overexpressing epidermal growth factor receptor or urokinase-type plasminogen activator receptor.

We have assessed the ability of bispecific fusion proteins to improve adenovirus-mediated transfer of therapeutic and marker transgenes. We constructed an expression vector that can be easily modified to synthesize a variety of fusion proteins for retargeting adenoviral gene therapy vectors to cell surface markers, which are differentially expressed between normal and cancer cells. Adenoviral transduction can be improved in a number of tumour cell lines which overexpress EGFR (epidermal growth factor receptor) or uPAR (urokinase-type plasminogen activator receptor), but which have only low levels of endogenous hCAR (human coxsackie B and adenovirus receptor) expression. Up to 40-fold improvement in beta-galactosidase transgene expression was seen using an EGFR retargeting protein, and up to 16-fold using a second fusion protein targeting uPAR. In vitro, our uPAR retargeting fusion protein improved the sensitivity to adenoviral herpes simplex virus thymidine kinase/ganciclovir by an order of magnitude, whereas in vivo, our EGFR retargeting protein is able to significantly delay tumour growth in rodent animal models in a dose-dependent manner. The 'cassette' design of our fusion protein constructs offers a flexible method for the straightforward synthesis of multiple adenoviral retargeting proteins, directed against a variety of tumour-associated antigens, for use in clinical trials.

Women with peritoneal carcinomatosis of unknown origin: Efficacy of image-guided biopsy to determine site-specific diagnosis.

OBJECTIVES: To evaluate the use of image-guided biopsy (IGB) in routine clinical practice to obtain site-specific diagnoses in women presenting with peritoneal carcinomatosis (PC). STUDY DESIGN: Retrospective case study. SETTING: Tertiary referral centre. POPULATION: A total of 149 consecutive women with PC who underwent IGB. METHODS: Biopsy was performed in women considered unsuitable for primary surgery because of poor performance status or disease unlikely to be optimally debulked, with a prior history of malignancy or where there was clinicoradiological uncertainty about primary tumour site. Standard haematoxylin-eosin histological analysis was supplemented with immunohistochemistry. MAIN OUTCOME MEASURES: The rate of site-specific diagnosis. RESULTS: A total of 149 women underwent IGB using computed tomography or ultrasound over a 6-year period. The only complication was one rectus sheath haematoma. In 138 (93%) women, a site-specific cancer diagnosis was made on the IGB (including 111 müllerian tract, 8 gastrointestinal tract, 4 breast and 3 lymphoma); in ten women, a repeat biopsy was necessary, giving an overall failure rate of 7%. In a further six women, malignancy was confirmed but a site-specific diagnosis could not be made, and in four women, biopsy showed benign tissue. A site-specific diagnosis was obtained in 29 of the 32 women (94%) with previous malignancy, of which 18/32 (56%) showed a new primary cancer. CONCLUSIONS: IGB is a safe and accurate technique for providing site-specific diagnoses in women with PC in routine clinical practice, including those with a previous relevant malignancy. IGB can replace laparoscopic or open biopsy in defining primary therapeutic options. The data would suggest that the biopsy should be performed with ultrasound where feasible.

Image-guided biopsy in women with breast cancer presenting with peritoneal carcinomatosis.

When women with a history of breast cancer present with peritoneal carcinomatosis, the differential diagnosis lies between recurrent breast cancer or a new primary tumor. This scenario is of particular relevance to women with a BRCA gene mutation, who have a genetic predisposition to develop second primary tumors of the ovary, fallopian tube, and peritoneum. We describe the use of image-guided core biopsy as an alternative to laparoscopy or exploratory laparotomy in providing minimally invasive diagnosis in this increasingly common clinical dilemma.

Novel urothelium specific gene expression identified by differential display reverse transcriptase-polymerase chain reaction.

PURPOSE: Understanding the molecular basis of differential gene expression among different tissues at various developmental stages and in neoplastic transformation is an important biological goal. The potential clinical applications of this improved understanding are more precise diagnosis of disease, prediction of prognosis, novel targeted therapies and prediction of response to therapy. MATERIALS AND METHODS: Differential display reverse transcriptase-polymerase chain reaction was used to compare gene expression in bovine urothelium to that in autologous lung, esophagus, liver and spleen. Products that appeared to have urothelial specific expression were sequenced and assessed for homology with known sequences. Ribonuclease protection assays were used to further confirm the expression pattern. RESULTS: A total of 32 discrete cDNAs were identified, including 3 products from genes known to be urothelium specific in their expression, 16 with significant homology to bovine, human or mouse expressed sequence tags and 5 with no sequence homology to any currently available sequence. Urothelium specific mRNA expression was confirmed for 3 genes by ribonuclease protection assays and one (Udd06) was further characterized as a urea transporter. CONCLUSIONS: The use of differential display reverse transcriptase-polymerase chain reaction and other complementary techniques for parallel gene expression analysis will permit the complete characterization of the urothelial transcriptome and help identify potential molecular targets for rationally targeted therapy.

Transcriptional control of the human urothelial-specific gene, uroplakin Ia.

The transcriptional control elements of tissue-specific genes may be exploited in the design of therapeutic constructs for use in human gene therapy. The uroplakins are a family of four proteins which form the asymmetric unit membrane of the urothelium. We have cloned the human uroplakin Ia gene and defined its genomic structure and transcriptional start site. Using quantitative RT-PCR in an extended panel of normal tissues, we have demonstrated highly urothelial-specific expression of this gene. A Dual-Luciferase assay was used to assess the transcriptional activity of a variety of promoter fragments of the human uroplakin Ia gene. A highly specific promoter fragment (consisting of 2147 bp of 5'-flanking sequence, intron 1 and the 5' UTR) was identified which regulated urothelial-specific expression in vitro. The human uroplakin Ia promoter identified has potential use in future gene therapy strategies to restrict transgene expression to the urothelium.

Maintenance treatment with interferon for advanced ovarian cancer: results of the Northern and Yorkshire gynaecology group randomised phase III study.

A randomised phase III trial was conducted to assess the role of interferon-alpha (INFalpha) 2a as maintenance therapy following surgery and/or chemotherapy in patients with epithelial ovarian carcinoma. Patients were randomised following initial surgery/chemotherapy to interferon-alpha 2a as 4.5 mega-units subcutaneously 3 days per week or to no further treatment. A total of 300 patients were randomised within the study between February 1990 and July 1997. No benefit for interferon maintenance was seen in terms of either overall or clinical event-free survival. We conclude that INF-alpha is not effective as a maintenance therapy in the management of women with ovarian cancer. The need for novel therapeutics or strategies to prevent the almost inevitable relapse of patients despite increasingly effective surgery and chemotherapy remains.

Cytogenetic alterations in ovarian clear cell carcinoma detected by comparative genomic hybridisation.

Ovarian clear cell carcinoma (OCCC) accounts for a small but significant proportion of all ovarian cancers and is a distinct clinical and pathological entity. It tends to be associated with poorer response rates to chemotherapy and with a worse prognosis. Little is known about possible underlying genetic changes. DNA extracted from paraffin-embedded samples of 18 pure OCCC cases was analysed for genetic imbalances using comparative genomic hybridisation (CGH). All of the 18 cases showed genomic alterations. The mean number of alterations detected by CGH was 6 (range 1-15) indicating a moderate level of genetic instability. Chromosome deletions were more common than amplifications. The most prominent change involved chromosome 9 deletions in 10 cases (55%). This correlates with changes seen in other epithelial ovarian cancers. This deletion was confirmed using microsatellite markers to assess loss of heterozygosity (LOH) at four separate loci on chromosome 9. The most distinct region of loss detected was around the IFNA marker at 9p21 with 41% (11 out of 27 cases) LOH. Other frequent deletions involved 1p (five out of 18; 28%); 11q (four out of 18; 22%) and 16 (five out of 18; 28%). Amplification was most common at chromosome 3 (six out of 18; 33%); 13q (four out of 18; 22%) and 15 (three out of 18; 17%). No high-level amplifications were identified. These features may serve as useful prognostic indicators in the management of OCCC.

Adenovirus-mediated gene therapy for bladder cancer: efficient gene delivery to normal and malignant human urothelial cells in vitro and ex vivo.

Existing local therapies for superficial transitional cell carcinoma (TCC) of the bladder have limited success in preventing progression to life-threatening, muscle-invasive disease, and novel therapies are needed. Recent studies have raised doubts concerning the feasibility of adenovirus-mediated gene therapy for bladder cancer. We have therefore investigated adenoviral transduction of normal and malignant human urothelial cells, both as primary cultures and in intact epithelium. All 15 primary normal human urothelial cell lines tested were transduced in vitro by Adv-cmv-beta-gal at high efficiency, and better than most human TCC cell lines. Eight primary human TCC explants were also successfully transduced. In contrast, in intact normal urothelium, transduction efficiency was lower, and occurred only in superficial epithelial layers. Expression of the hCAR adenovirus receptor, however, occurred throughout the full thickness of urothelium. Transduction of human TCC biopsy specimens was at least as efficient as intact normal urothelium.We demonstrate for the first time that adenoviral transduction of both normal and malignant human urothelial cells is feasible. A physical barrier, rather than hCAR status, may be the main determinant of transduction of intact epithelium. Clinical trials of adenovirus-mediated gene therapy for superficial bladder cancer are warranted.

Uroplakin gene expression by normal and neoplastic human urothelium.

cDNA sequences for human uroplakins UPIa, UPIb, UPII, and UPIII were cloned and used to investigate uroplakin transcription by normal and neoplastic urothelial cells. Normal urothelium expressed mRNA for all four uroplakins, although UPIII could be detected only by ribonuclease protection assay. By in situ hybridization, UPIa and UPII were confined to superficial cells and UPIb was also expressed by intermediate cells. Cultured normal human urothelial cells showed a proliferative basal/intermediate cell phenotype and constitutive expression of UPIb only. Uroplakin expression by transitional cell carcinoma cell lines was related to their differentiated phenotype in vitro. RT4 cells expressed all uroplakins, VM-CUB-3 expressed three uroplakins, RT112 and HT1376 cells expressed only UPIb in high abundance, and COLO232, KK47, and EJ cells had no detectable expression. These results correlated with patterns of uroplakin expression in tumors. UPIa and UPII were detected superficially only in well differentiated transitional cell carcinoma papillae. UPIb was positive in seven of nine and overexpressed in five of nine noninvasive transitional cell carcinomas and was also present in four of eight invasive transitional cell carcinomas. Lymph node metastases retained the same pattern of UPIb expression as the primary tumor. Unlike the three differentiation-regulated uroplakins, UPIb may have an alternative role in urothelial cell/tissue processes.

A phase II study of interferon-alpha, interleukin-2 and 5-fluorouracil in advanced renal carcinoma: clinical data and laboratory evidence of protease activation.

OBJECTIVE: To confirm the activity and evaluate the toxicity of the combination of subcutaneous interferon-alpha (IFN-alpha) and interleukin-2 (IL-2) with intravenous 5-fluorouracil (5-FU) in patients with advanced and recurrent renal carcinoma and of performance status 0-2. Additionally, to examine protease, complement and neutrophil activation as potential mediators of IL-2 toxicity. PATIENTS AND METHODS: Fifty-five patients were treated in an 8-week cycle with IFN-alpha (6 MU/m2 on day 1 in weeks 1 and 4 and thrice weekly in weeks 2-3, and 9 MU/m2 thrice weekly in weeks 5-8) IL-2 (20 MU/m2 on days 3-5 in weeks 1 and 4 and 5 MU/m2 thrice weekly in weeks 2-3) and 5-FU (750 mg/m2 on day 1 of weeks 5-8). Patients responding to the first cycle were eligible to continue with further cycles. Toxicity and effects on quality of life were assessed using World Health Organization criteria and the Rotterdam Symptom Checklist and Hospital Anxiety and Depression Scale. Serum levels of C3a, prekallikrein and elastase-alpha 1 proteinase inhibitor (elastase-alpha 1-antitrypsin) were assayed in a subset of patients before, during and after the administration of high-dose IL-2 in week 1. RESULTS: There were partial remissions in nine patients, with responses in 24% (95% CI 10-38%) of evaluable patients and 16% of all patients. Amongst 25 evaluable patients who had undergone nephrectomy, the response rate was 32% (95% CI 14-50%), whereas there was only one response amongst 22 patients who had not undergone nephrectomy. The median survival for patients with stable disease or partial remission exceeded 22 months. Outcome and survival were related to performance status, number of sites of metastases and nephrectomy. This group of patients was of relatively poor performance status and 18 patients (36%) failed to complete one 8-week treatment cycle. Cardiovascular and renal toxicities were less than those seen with intravenous IL-2 schedules but 44% of patients experienced at least one grade III toxicity and only 14% reported less than two grade II toxicities. Plasma levels of elastase-alpha 1 proteinase inhibitor exceeded the normal range in three of seven patients tested before treatment and increased in all seven patients after treatment with IL-2. The same three patients had raised levels of C3a before treatment and in all patients examined, C3a increased after treatment with IL-2. In contrast, plasma prekallikrein concentrations were below normal before treatment and decreased further afterwards. CONCLUSIONS: This study confirms the activity of this regimen in patients of good performance status, with limited sites of disease and in those who are fit for nephrectomy, but also showed that treatment was associated with considerable toxicity. The administration of IL-2 is associated with protease activation which may be a suitable target for pharmacological intervention in attempts to ameliorate toxicity.

Metastatic breast carcinoma involving the gastric antrum and duodenum: computed tomography appearances.

We describe the computed tomography (CT) appearances of three cases of antroduodenal linitis plastica metastases from breast carcinoma. Two of the three cases had biliary obstruction as a consequence and required endoscopic stenting. Antroduodenal linitis plastica should be considered as a possible cause for jaundice in patients with breast carcinoma.

A comparison of nerve grafting and tissue expansion techniques in the rat.

This study compares nerve repair following tissue expansion with nerve repair using an interposed graft in the rat. Group I had expansion conducted over 2 weeks at 40 mmHg. A 4 mm segment was excised from the lengthened nerve and repaired primarily. Group II had a 4 mm segment of nerve excised and then replaced as an interposition graft. Group III was sham-operated controls. Thirteen weeks postoperatively, all animals were evaluated using walking track analysis. Thirty-five rats finished the study: Eleven in group I, 10 in group II, and 14 in group III. The Sciatic Functional Index (SFI) was calculated for each group as follows: group I, -57 +/- 11 (mean +/- standard deviation); group II, -59 +/- 25; group III, -13 +/- 6.5. The control group was significantly better than either experimental group (P < 0.01). The two experimental groups were not statistically different. Nerve repair following expansion allowed only one coaptation to be used. Functional results were the same as with interposition grafting. Repair by the expansion technique would eliminate the need to harvest a nerve graft, and the subsequent donor defect.