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PDF file - 78K, Tissue sample collection.

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Purpose: Epithelial ovarian tumors (EOT) are among the most lethal of malignancies in women. We have previously identified ZIC2 as expressed at a higher level in samples of a malignant form (MAL) of EOT than in samples of a form with low malignant potential (LMP). We have now investigated the role of ZIC2 in driving tumor growth and its association with clinical outcomes.Experimental Design:ZIC2 expression levels were analyzed in two independent tumor tissue collections of LMP and MAL. In vitro experiments aimed to test the role of ZIC2 as a transforming gene. Cox models were used to correlate ZIC2 expression with clinical endpoints.Results:ZIC2 expression was about 40-fold in terms of mRNA and about 17-fold in terms of protein in MAL (n = 193) versus LMP (n = 39) tumors. ZIC2 mRNA levels were high in MAL cell lines but undetectable in LMP cell lines. Overexpression of ZIC2 was localized to the nucleus. ZIC2 overexpression increases the growth rate and foci formation of NIH3T3 cells and stimulates anchorage-independent colony formation; downregulation of ZIC2 decreases the growth rate of MAL cell lines. Zinc finger domains 1 and 2 are required for transforming activity. In stage I MAL, ZIC2 expression was significantly associated with overall survival in both univariate (P = 0.046) and multivariate model (P = 0.049).Conclusions:ZIC2, a transcription factor related to the sonic hedgehog pathway, is a strong discriminant between MAL and LMP tumors: it may be a major determinant of outcome of EOTs. Clin Cancer Res; 18(16); 4313–24. ©2012 AACR.

PDF file, 62K, A. List of primer pair sequences used for RT-qPCR analysis and respective annealing temperatures. B. List of siRNA sequences used. C. Details of deletion constructs (see Fig. 4A).

PDF file, 96K, Role in transformation of individual domains of ZIC2 protein assessed by deletion analysis.

PDF file, 198K, Forced over-expression of ZIC2 in NIH-3T3 cells produces increased cell proliferation and knockdown of ZIC2 protein inhibits proliferation of ovarian cancer cell lines.

Purpose: Gene expression profile was analyzed in 68 stage I and 15 borderline ovarian cancers to determine if different clinical features of stage I ovarian cancer such as histotype, grade, and survival are related to differential gene expression.Experimental Design: Tumors were obtained directly at surgery and immediately frozen in liquid nitrogen until analysis. Glass arrays containing 16,000 genes were used in a dual-color assay labeling protocol.Results: Unsupervised analysis identified eight major patient partitions, one of which was statistically associated to overall survival, grading, and histotype and another with grading and histotype. Supervised analysis allowed detection of gene profiles clearly associated to histotype or to degree of differentiation. No difference was found between borderline and grade 1 tumors. As to recurrence, a subset of genes able to differentiate relapsers from nonrelapsers was identified. Among these, cyclin E and minichromosome maintenance protein 5 were found particularly relevant, as their expression was inversely correlated to progression-free survival (P = 0.00033 and 0.017, respectively).Conclusions: Specific molecular signatures define different histotypes and prognosis of stage I ovarian cancer. Mucinous and clear cells histotypes can be distinguished from the others regardless of tumor grade. Cyclin E and minichromosome maintenance protein 5, whose expression was found previously to be related to a bad prognosis of advanced ovarian cancer, appear to be potential prognostic markers in stage I ovarian cancer too, independent of other pathologic and clinical variables.

Purpose: Epithelial ovarian cancer (EOC) is one of the most lethal gynecologic diseases, with survival rate virtually unchanged for the past 30 years. EOC comprises different histotypes with molecular and clinical heterogeneity, but up till now the present gold standard platinum-based treatment has been conducted without any patient stratification. The aim of the present study is to generate microRNA (miRNA) profiles characteristic of each stage I EOC histotype, to identify subtype-specific biomarkers to improve our understanding underlying the tumor mechanisms.Experimental Design: A collection of 257 snap-frozen stage I EOC tumor biopsies was gathered together from three tumor tissue collections and stratified into independent training (n = 183) and validation sets (n = 74). Microarray and quantitative real-time PCR (qRT-PCR) were used to generate and validate the histotype-specific markers. A novel dedicated resampling inferential strategy was developed and applied to identify the highest reproducible results. mRNA and miRNA profiles were integrated to identify novel regulatory circuits.Results: Robust miRNA markers for clear cell and mucinous histotypes were found. Specifically, the clear cell histotype is characterized by a five-fold (log scale) higher expression of miR-30a and miR-30a*, whereas mucinous histotype has five-fold (log scale) higher levels of miR-192/194. Furthermore, a mucinous-specific regulatory loop involving miR-192/194 cluster and a differential regulation of E2F3 in clear cell histotype were identified.Conclusions: Our findings showed that stage I EOC histotypes have their own characteristic miRNA expression and specific regulatory circuits. Clin Cancer Res; 19(15); 4114–23. ©2013 AACR.

Supplementary Data from Analysis of Gene Expression in Early-Stage Ovarian Cancer

PDF file, 82K, Univariate analysis. Linear regression analysis between histopathological features and ZIC2 expression. Units are 103 multiplied.

PDF file, 87K, Main characteristics of patients and tissues. A, clinical and histopathological parameters of the 232 EOT biopsies enrolled in the study and subdivided into tumor tissue collection A and B. UK unknown. n, number of patients enrolled. NA, not available.

PDF file, 61K, qRT-PCR and densitometric analysis of ZIC2 expression. R is the ratio of the median distribution of ZIC2 expression levels measured by real time qRT-PCR or Western Blot in the MAL biopsies compared to the LMP biopsies or between stage III versus stage I. For each dataset, the ZIC2 median and the 25-75th percentiles are reported (IQ-range). p is the level of significance according to the Mann-Whitney t test (p

Surgical staging in endometrial cancer has evolved and sentinel lymph node (SLN) mapping has replaced a full pelvic and paraaortic lymphadenectomy in several cases. An intraoperative evaluation of SLN might identify patients who could benefit the most from a full lymphadenectomy. The aim of this study is to evaluate the clinical relevance of frozen section of SLN. A retrospective analysis in patients with endometrial cancer who underwent SLN mapping with intraoperative evaluation at frozen section between February 2016 and September 2019 was performed. In case of metastatic involvement, a full lymphadenectomy was performed. Fifty-eight patients met the inclusion criteria. Clinical–pathologic characteristics of the patients and surgical data were analyzed. Overall, bilateral and unilateral detection rates were 100% (58/58), 89.7% (52/58), and 10.3% (6/58), respectively. Eight patients had a stage IIIC disease at permanent section. Frozen section detected SLN metastases in four of eight patients. Of these, two were micrometastases and two were macrometastases. At frozen section of the SLNs, no macrometastases were misdiagnosed. Sensitivity, specificity, accuracy, positive predictive value and negative predictive value of frozen section in detecting metastases was 50%, 100%, 93%, 100% and 92.6%, respectively. The intraoperative evaluation of SLN in endometrial cancer accurately identifies patients with macrometastases. This is the cohort that might benefit the most of a full lymphadenectomy for a higher risk of additional lymph node metastases.

Background The majority of prevalence studies on deep vein thrombosis (DVT) in severe COVID-19 patients are retrospective with DVT assessment based on clinical suspicion. Our aim was to prospectively and systematically estimate the occurrence of DVT in critically ill mechanically ventilated patients, and to identify potential risk factors for DVT occurrence and mortality. Methods All patients with COVID-19 admitted to our 45-beds intensive care unit (ICU) between March 6, 2020 and April 18, 2020 requiring invasive ventilatory support were daily screened for DVT with lower extremities and jugular veins ultrasonography. Univariate and multivariable logistic regression models were performed in order to identify predictors of DVT and mortality. Results Seventy-six patients were included in the final analysis (56 men, mean age 67 years, median SOFA 7 points, median SAPS II 41 points, median PaO2/Fi02 10.8 kPa). The period prevalence of DVT was 40.8%. Thirty-one DVTs were diagnosed. Twenty-five DVTs (80.6% of total DVTs) were catheter-related, mainly in the jugular veins. Twentysix DVTs (83.9%) occurred in patients receiving enhanced antithrombotic prophylaxis. No independent variable was predictive of DVT occurrence. Twenty-eight patients (36.8%) died during the ICU stay. Age and SOFA score were independently associated with mortality. Conclusions A high number of critically ill mechanically ventilated COVID-19 patients developed a DVT. The majority of DVTs were catheter-related and occurred under intensive prophylactic anticoagulation. Routine ultrasound of the jugular veins should be suggested in this patient population, and in particular in presence of a central venous catheter.

Background: During the Covid-19 pandemic, nurses experienced increased pressure. Consequently, ethical concerns and psychological distress emerged. This study aimed to assess nurses’ ethical conflict, resilience and psychological impact, and compare these variables between nurses who worked in Covid-19 wards and nurses who did not. Methods: Design—Multicentre online survey. Setting—Multi-site public hospital, all nursing staff were invited to participate. The survey included validated tools and a novel instrument to assess ethical conflict. Spearman’s rho coefficient was used to assess correlations between ethical conflict and psychological distress, logistic regressions to evaluate relationships between nurses’ characteristics and outcome variables, and the Mann–Whitney/t-test to compare groups. Results: 548 questionnaires out of 2039 were returned (275 = Covid-19, 273 = non-Covid-19). We found a low–moderate level of ethical conflict (median = 111.5 [76–152]), which emerged mostly for seeing patients dying alone. A moderate and significant positive correlation emerged between ethical conflict and psychological distress rs (546) = 0.453, p &lt, 0.001. Nurses working in Covid-19-ICUs (OR = 7.18, 95%CI = 3.96–13.01, p &lt, 0.001) and Covid-19 wards (OR = 5.85, 95%CI = 3.56–9.6, 0.001) showed higher ethical conflict. Resilience was a protective factor for ethical conflict. Conclusions: Ethical conflict was significantly linked to psychological distress, while a higher level of resilience was found to be a protective factor. These results can be informative for nursing management in future similar crises.

// Lara Paracchini 1, * , Laura Mannarino 1, * , Ilaria Craparotta 1 , Chiara Romualdi 2 , Robert Fruscio 3 , Tommaso Grassi 3 , Vittoria Fotia 4 , Giulia Caratti 1 , Patrizia Perego 5 , Enrica Calura 2 , Luca Clivio 1 , Maurizio D’Incalci 1 , Luca Beltrame 1, § , Sergio Marchini 1, § 1 Department of Oncology, IRCCS Istituto di Ricerche Farmacologiche “Mario Negri”, Milano, Italy 2 Department of Biology, University of Padova, Padova, Italy 3 Clinic of Obstetrics and Gynaecology, University of Milano-Bicocca, San Gerardo Hospital, Monza, Italy 4 PhD program in Experimental Medicine, University of Pavia, Pavia, Italy 5 Pathology Unit University of Milan-Bicocca, San Gerardo Hospital, Monza, Italy * both are first authors § both are last co-authors Correspondence to: Maurizio D’Incalci, email: maurizio.dincalci@marionegri.it Keywords: ovarian cancer, temporal heterogeneity, spatial heterogeneity, targeted next generation sequencing, drug resistance Received: March 01, 2016 Accepted: May 23, 2016 Published: July 09, 2016 ABSTRACT Stage III/IV epithelial ovarian cancer (EOC) is a systemic disease. The clonal relationship among different tumor lesions at diagnosis (spatial heterogeneity) and how tumor clonal architecture evolves over time (temporal heterogeneity) have not yet been defined. Such knowledge would help to develop new target-based strategies, as biomarkers which can adjudge the success of therapeutic intervention should be independent of spatial and temporal heterogeneity. The work described in this paper addresses spatial and temporal heterogeneity in a cohort of 71 tumor biopsies using targeted NGS technology. These samples were taken from twelve high grade serous (HGS) and seven non HSG-EOC, both at the time of primary surgery when the tumor was naive to chemotherapy and after chemotherapy. Matched tumor lesions growing in the ovary or at other anatomical sites show very different mutational landscapes with branched tumor evolution. Mutations in ATM, ATR, TGFB3, VCAM1 and COL3A1 genes were shared across all lesions. BRCA1 and BRCA2 genes were frequently mutated in synchronous lesions of non HGS-EOC. Relapsed disease seems to originate from resistant clones originally present at the time of primary surgery rather than from resistance acquired de novo during platinum based therapy. Overall the work suggests that EOC continues to evolve. More detailed mapping of genetic lesions is necessary to improve therapeutic strategies.

Purpose Triple-negative breast cancer (TNBC) represents a subtype of breast cancer which lacks the expression of oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2): TNBC accounts for approximately 20% of newly diagnosed breast cancers and is associated with younger age at diagnosis, greater recurrence risk and shorter survival time. Therapeutic options are very scarce. Aim of the present analysis is to provide further insights into the clinical activity of metronomic chemotherapy (mCHT), in a real-life setting. Methods We used data included in the VICTOR-6 study for the present analysis. VICTOR-6 is an Italian multicentre retrospective cohort study, which collected data of metastatic breast cancer (MBC) patients who have received mCHT between 2011 and 2016. Amongst the 584 patients included in the study, 97 were triple negative. In 40.2% of the TNBC patients, mCHT was the first chemotherapy treatment, whereas 32.9% had received 2 or more lines of treatment for the metastatic disease. 45.4% out of 97 TNBC patients received a vinorelbine (VRL)-based regimen, which resulted in the most used type of mCHT, followed by cyclophosphamide (CTX)-based regimens (30.9%) and capecitabine (CAPE)-based combinations (22.7%). Results Overall response rate (ORR) and disease control rate (DCR) were 17.5% and 64.9%, respectively. Median progression free survival (PFS) and overall survival (OS) were 6.0 months (95% CI: 4.9–7.2) and 12.1 months (95% CI: 9.6–16.7). Median PFS was 6.9 months for CAPE-based regimens (95% CI: 5.0–18.4), 6.1 months (95% CI: 4.0–8.9) for CTX-based and 5.3 months (95% CI: 4.1–9.5) for VRL-based ones. Median OS was 18.2 months (95% CI: 9.1-NE) for CAPE-based regimens and 11.8 months for VRL- (95% CI: 9.3–16.7 and CTX-based ones (95%CI: 8.7–52.8). Tumour response, PFS and OS decreased proportionally in later lines. Conclusion This analysis represents the largest series of TNBC patients treated with mCHT in a real-life setting and provides further insights into the advantages of using this strategy even in this poor prognosis subpopulation.

Background The epidemic phenomenon leading to a progressive increase in benzodiazepine prescriptions represents a challenge for healthcare systems. In the hospital setting, indicators of prescription variation and potential of overuse are lacking and are rarely monitored. Inter‐hospital monitoring/benchmarking, via peer‐pressure, can foster the motivation to change. The aim of this investigation was to analyse whether, the reduction in new benzodiazepine prescriptions obtained thanks to a Choosing Wisely campaign, also contributed to reducing inter‐hospital variation. Methods Secondary analysis of a multicentre longitudinal intervention in a network of five teaching hospitals in Switzerland. We set out to explore the effect, on inter‐hospital benzodiazepine prescription variation, of a continuous monitoring/benchmarking strategy, which was proven effective in reducing the intra‐hospital prescription rate. The variance was used to assess inter‐hospital variation. To investigate the impact of the intervention a segmented regression analysis of interrupted time series was performed. Results A total of 36 299 admissions over 42 months were analysed (1 July 2014 to 31 December 2017). Before the intervention a significant constant upward trend in inter‐hospital variability was found (+0.901; SE 0.441; P

This study was designed to investigate the mode of action of trabectedin in myelomonocytic leukemia cells by applying systems biology approaches to mine gene expression profiling data and pharmacological assessment of the cellular effects. Significant enrichment was found in regulons of target genes inferred for specific transcription factors, among which MAFB was the most upregulated after treatment and was central in the transcriptional network likely to be relevant for the specific therapeutic effects of trabectedin against myelomonocytic cells. Using the Connectivity Map, similarity among transcriptional signatures elicited by treatment with different compounds was investigated, showing a high degree of similarity between transcriptional signatures of trabectedin and those of the topoisomerase I inhibitor, irinotecan, and an anti-dopaminergic antagonist, thioridazine. The study highlights the potential importance of systems biology approaches to generate new hypotheses that are experimentally testable to define the specificity of the mechanism of action of drugs.

Background: Different studies suggest that fulvestrant 500 mg every 28 days (HD-FUL) could be an active treatment in HR+ advanced breast cancer (ABC) patients even treated with aromatase inhibitors in the adjuvant setting. The aim of this analysis is to describe the outcome of ABC patients treated with HD-FUL as first-line treatment in terms of median duration of treatment and the overall response rate in a real-world setting. Methods: For the purpose of the present analysis, we considered two data sets of HR+ ABC patients collected in Italy between 2012 and 2015 (EVA and GIM-13 AMBRA studies). Results: Eighty-one and 91 patients have been identified from the two data sets. The median age was 63 years (range 35–82) for the EVA and 57.8 years (range 35.0–82.3) for the AMBRA patients. ORRs were 23.5 and 24.3% in the whole population, 26.9% in the patients with bone only, and 21.8 and 21.4% in those with visceral metastases. The median duration of HD-FUL was 11.6 months (range 1–48) and 12.4 months (range 2.9–70.0) in the two data sets, respectively. Conclusion: These data suggest that HD-FUL should still continue to play a significant role as first-line therapy in HR+ ABC patients.

Summary Background Reducing unnecessary laboratory blood testing in the hospital setting represents a challenge to improve the adequacy of healthcare and a tricky task for teaching hospitals. Our hospital network actively participates in the Choosing Wisely Campaign and is engaged in avoiding unnecessary low value interventions and investigations. We aimed to study whether a multi‐level approach combining educational and web‐system based interventions, could be effective in reducing laboratory testing and related costs. Methods Multicenter, proof of concept, prospective, observational, before and after study, in a network of public hospitals in Switzerland. All patients admitted between 1 January 2015 and 31 December 2017 were analyzed. A multi‐level strategy based on online continuous monitor benchmarking and educational support was applied in the internal medicine services. The primary outcome was a significant reduction in the number of laboratory tests per patient and per day during the hospital stay. Secondary outcomes were reduction in the blood sample volume taken per patient and per day in laboratory costs. Results Over the 36 months of the study, 33 309 admissions were analyzed. A significant reduction of laboratory tests per patient and per day of hospitalisation was found:–11%, P‐value

Reducing the inappropriate benzodiazepine (BZD) prescriptions represents a challenge for health care systems worldwide. The 'Choosing Wisely' campaign recommends against the use of BZD in the elderly as the first choice for insomnia, agitation, or delirium. We aimed to determine whether a transparent monitoring-benchmarking together with educational interventions, on top of the internal publication of a targeted recommendation, could be effective in curbing BZD prescriptions.Multicenter before and after study in a network of five southern-Switzerland teaching hospitals. An intervention based on a transparent continuous monitoring-benchmarking system, called 'Reporting Wisely', able to collect, analyze, and report data on BZD prescriptions and educational interventions focused on themed meetings, audit, and feedback, was implemented. The intervention was limited to the Internal Medicine. The impact of the intervention on new BZD prescriptions and de-prescribing at hospital discharge, was assessed using segmented regression analyses of interrupted time-series and comparing Internal Medicine to Surgery.Between July 1The implementation of a dual intervention based on transparent monitoring-benchmarking and multidisciplinary education has proved useful in curbing new BZD prescriptions and in promoting BZD de-prescribing in the hospital setting.

Background The majority of patients with stage III–IV epithelial ovarian cancer (EOC) relapse after initially responding to platinum-based chemotherapy, and develop resistance. The genomic features involved in drug resistance are unknown. To unravel some of these features, we investigated the mutational profile of genes involved in pathways related to drug sensitivity in a cohort of matched tumors obtained at first surgery (Ft-S) and second surgery (Sd-S). Patients and methods Matched biopsies (33) taken at Ft-S and Sd-S were selected from the ‘Pandora’ tumor tissue collection. DNA libraries for 65 genes were generated using the TruSeq Custom Amplicon kit and sequenced on MiSeq (Illumina). Data were analyzed using a high-performance cluster computing platform (Cloud4CARE project) and independently validated. Results A total of 2270 somatic mutations were identified (89.85% base substitutions 8.19% indels, and 1.92% unknown). Homologous recombination (HR) genes and TP53 were mutated in the majority of Ft-S, while ATM, ATR, TOP2A and TOP2B were mutated in the entire dataset. Only 2% of mutations were conserved between matched Ft-S and Sd-S. Mutations detected at second surgery clustered patients in two groups characterized by different mutational profiles in genes associated with HR, PI3K, miRNA biogenesis and signal transduction. Conclusions There was a low level of concordance between Ft-S and Sd-S in terms of mutations in genes involved in key processes of tumor growth and drug resistance. This result suggests the importance of future longitudinal analyses to improve the clinical management of relapsed EOC.

[This corrects the article DOI: 10.18632/oncotarget.25874.].

ObjectivesWe examined major issues associated with sharing of individual clinical trial data and developed a consensus document on providing access to individual participant data from clinical trials, using a broad interdisciplinary approach.Design and methodsThis was a consensus-building process among the members of a multistakeholder task force, involving a wide range of experts (researchers, patient representatives, methodologists, information technology experts, and representatives from funders, infrastructures and standards development organisations). An independent facilitator supported the process using the nominal group technique. The consensus was reached in a series of three workshops held over 1 year, supported by exchange of documents and teleconferences within focused subgroups when needed. This work was set within the Horizon 2020-funded project CORBEL (Coordinated Research Infrastructures Building Enduring Life-science Services) and coordinated by the European Clinical Research Infrastructure Network. Thus, the focus was on non-commercial trials and the perspective mainly European.OutcomeWe developed principles and practical recommendations on how to share data from clinical trials.ResultsThe task force reached consensus on 10 principles and 50 recommendations, representing the fundamental requirements of any framework used for the sharing of clinical trials data. The document covers the following main areas: making data sharing a reality (eg, cultural change, academic incentives, funding), consent for data sharing, protection of trial participants (eg, de-identification), data standards, rights, types and management of access (eg, data request and access models), data management and repositories, discoverability, and metadata.ConclusionsThe adoption of the recommendations in this document would help to promote and support data sharing and reuse among researchers, adequately inform trial participants and protect their rights, and provide effective and efficient systems for preparing, storing and accessing data. The recommendations now need to be implemented and tested in practice. Further work needs to be done to integrate these proposals with those from other geographical areas and other academic domains.

e12552 Background: mCHT is the minimum biologically effective dose of a chemotherapeutic agent, given at regular dosing regimen with no prolonged drug free interval, that leads to anti-tumor activity. Old regimens included Cyclophosphamide-Methotrexate (CM), whereas in the last years new regimens, such as Vinorelbine (VRL) and Capecitabine (CAPE)-based have been developed. Aim of this observational retrospective ongoing study is to describe the use of mCHT in ABC pts across 5 years and the clinical characteristics of the pts together with efficacy of old (CM-like) vs new (VRL/CAPE-based) metronomic regimens in terms of response and disease control. Methods: We retrospectively identified from clinical records those HER2-ve ABC pts who have received any kind of mCHT in the years 2011-2015, alone, or in combination with a non-metronomic drug. Standard statistical approaches were used for describing the sample characteristics. Logistic and non proportional hazard analysis were used to identify factors associated with response, and time to treatment failure and survival, respectively. This preliminary analysis focuses on Response Rate (RR) and Disease Control Rate (DCR). Results: From June 2011 to December 2015, 267 pts have been identified till now and 233 are fully evaluable. Median age at mCHT start was 67 years. 81% was HR+ and 33% had non-visceral metastatic disease. 22% of the pts received CM, 55% VRL-based and 23% mCAPE-based regimens. mCHT use increased over the time from 15.0% (2011) to 30% (2015). As 1st-line treatment, CM was administered in 27% of compared with more than 48% of patients receiving CAPE/VRL-based regimens. Overall Response Rate (ORR) was 28% and Disease Control Rate (DCR) was 79%. Median duration of mCHT was 6.2 months. New generation metronomic regimens produced higher ORR in comparison to old ones (32% vs 13.5%), with similar duration of treatment (6.4 vs 5.4 months, respectively). Conclusions: The use of mCHT in the treatment of HER2-ve ABC pts has deeply changed across the last 5 years, being new generation regimens used in earlier lines of treatment, producing interesting results in terms of objective response and disease control.

Summary Background International Federation of Gynecology and Obstetrics stage I epithelial ovarian cancer (EOC) has a significantly better prognosis than stage III/IV EOC, with about 80% of patients surviving at 5 years (compared with about 20% of those with stage III/IV EOC). However, 20% of patients with stage I EOC relapse within 5 years. It is therefore crucial that the biological properties of stage I EOCs are further elucidated. MicroRNAs (miRNAs) have shown diagnostic and prognostic potential in stage III and IV EOCs, but the small number of patients diagnosed with stage I EOC has so far prevented an investigation of its molecular features. We profiled miRNA expression in stage I EOC tumours to assess whether there is a miRNA signature associated with overall and progression-free survival (PFS) in stage I EOC. Methods We analysed tumour samples from 144 patients (29 of whom relapsed) with stage I EOC gathered from two independent tumour tissue collections (A and B), both with a median follow-up of 9 years. 89 samples from tumour tissue collection A were stratified into a training set (51 samples, 15 of which were from patients who relapsed) for miRNA signature generation, and into a validation set (38 samples, seven of which were from patients who relapsed) for signature validation. Tumour tissue collection B (55 samples, seven of which were from patients who relapsed) was used as an independent test set. The Cox proportional hazards model and the log-rank test were used to assess the correlation of quantitative reverse transcription PCR (qRT-PCR)-validated miRNAs with overall survival and PFS. Findings A signature of 34 miRNAs associated with survival was generated by microarray analysis in the training set. In both the training set and validation set, qRT-PCR analysis confirmed that 11 miRNAs (miR-214, miR-199a-3p, miR-199a-5p, miR-145, miR-200b, miR-30a, miR-30a*, miR-30d, miR-200c, miR-20a, and miR-143) were expressed differently in relapsers compared with non-relapsers. Three of these miRNAs (miR-200c, miR-199a-3p, miR-199a-5p) were associated with PFS, overall survival, or both in multivariate analysis. qRT-PCR analysis in the test set confirmed the downregulation of miR-200c in relapsers compared with non-relapsers, but not the upregulation of miR-199a-3p and miR-199a-5p. Multivariate analysis confirmed that downregulation of miR-200c in the test set was associated with overall survival (HR 0·094, 95% CI 0·012–0·766, p=0·0272) and PFS (0·035, 0·004–0·311; p=0·0026), independent of clinical covariates. Interpretation miR-200c has potential as a predictor of survival, and is a biomarker of relapse, in stage I EOC. Funding Nerina and Mario Mattioli Foundation, Cariplo Foundation (Grant Number 2010-0744), and the Italian Association for Cancer Research.

e13076Background: mCHT is the minimum biologically effective dose of a CHT agent, given at regular dosing regimen with no prolonged drug free interval. Aim of this observational retrospective study...

Harnessing the power of ‘disruptive’ technologies in a peer-to-peer network is a cost-effective way for non-profit research organizations to manage their clinical trials data and provide decentralised control to ensure clinical trials are conducted for the benefit of patients rather than for any one of the collaborating laboratories. Sharing data between multiple centres is important in clinical research to reduce any selection bias of patients participating in a trial when the patients involved at any one centre may not be representative of the population as a whole and the number of patients needed for a statistically meaningful result is greater than one centre can provide. Regulation of data sharing in clinical trials is essential to avoid accidental loss of data and to maintain control of access to data. Regular backups and measures to restrict unauthorised access to the data are an essential part of quality control for regulatory compliance of clinical data collection tasks. FDA CFR 21 part 11, which is an important regulation for clinical trials, explicitly require this level of compliance. Avoiding accidental 'prior-disclosure' of data and preventing unauthorised or inappropriate use of it is important for the correct attribution of work done by clinical research scientists. Many aspects of clinical research rely on ‘virtual organizations’ because management of clinical research projects often spans institutional boundaries to avoid duplication of effort and to share resources in order to solve problems economically. Clinical researchers working in different laboratories collaborate and share their data. The laboratories involved need to develop trust relationships to share data and intellectual property. This requires agreement about data management: In particular, where to store the shared data, which will curate it, who has access to it and how to share the data. In this paper we discuss a cost-effective and decentralised network-based approach to management of clinical data that disrupts established practice of using a centralised web-based database for all aspects of managing clinical trials data.

Background Clinical and pathological parameters of patients with epithelial ovarian cancer (EOC) do not thoroughly predict patients' outcome. Despite the good outcome of stage I EOC compared with that of stages III and IV, the risk assessment and treatments are almost the same. However, only 20% of stage I EOC cases relapse and die, meaning that only a proportion of patients need intensive treatment and closer follow-up. Thus, the identification of cell mechanisms that could improve outcome prediction and rationalize therapeutic options is an urgent need in the clinical practice. Patients and methods We have gathered together 203 patients with stage I EOC diagnosis, from whom snap-frozen tumor biopsies were available at the time of primary surgery before any treatment. Patients, with a median follow-up of 7 years, were stratified into a training set and a validation set. Results and conclusions Integrated analysis of miRNA and gene expression profiles allowed to identify a prognostic cell pathway, composed of 16 miRNAs and 10 genes, wiring the cell cycle, ‘Activins/Inhibins’ and ‘Hedgehog’ signaling pathways. Once validated by an independent technique, all the elements of the circuit resulted associated with overall survival (OS) and progression-free survival (PFS), in both univariate and multivariate models. For each patient, the circuit expressions have been translated into an activation state index (integrated signature classifier, ISC), used to stratify patients into classes of risk. This prediction reaches the 89.7% of sensitivity and 96.6% of specificity for the detection of PFS events. The prognostic value was then confirmed in the external independent validation set in which the PFS events are predicted with 75% sensitivity and 94.7% specificity. Moreover, the ISC shows higher classification performance than conventional clinical classifiers. Thus, the identified circuit enhances the understanding of the molecular mechanisms lagging behind stage I EOC and the ISC improves our capabilities to assess, at the time of diagnosis, the patient risk of relapse.

Summary Choosing Wisely: from theory to practice “Choosing Wisely” is an innovative approach that the Network of Southern Switzerland Public Hospitals has decided to promote. Five standard diagnostic or therapeutic procedures have been chosen to explore the potential benefi t of the “Choosing Wisely” initiative: the prescripti on of benzodiazepines, proton pump inhibitors or antibiotics on discharge from hospital, exposure to ionising radiation in radiological imaging and the number of blood samples taken during hospitalisation. As a fi rst step we compared these variables in the medical and surgical departments of the four major public hospitals in Ticino. We observed signifi cant and unexpected practical differences between specialties and between the different institutions. These results were presented to all concerned healthcare stakeholders. The next steps are to develop continuous monitoring of these indicators and specific recommendations by involving patients in the consciousness-raising process.

«Choosing Wisely»: von der Theorie zur Praxis Luca Gabuttia, Michela Pironib, Roberto Della Brunac, Juergen Heinkeld, Luca Clivioe, Angela Grecoe a Division of Internal Medicine and Nephrology, Ospedale Regionale di Locarno, Switzerland, b Central Pharmacy Service, Clinical Pharmacy Division, Ente Ospedaliero Cantonale, Switzerland, c Department of Laboratory Medicine, Ospedale Regionale di Bellinzona e Valli, Bellinzona, Switzerland, d Division of Radiology, Ospedale Regionale di Locarno, Switzerland, e Quality and Patient Safety Service, Ospedale Regionale di Locarno, Switzerland

Growing use of cloud computing in clinical trials prompted the European Clinical Research Infrastructures Network, a European non-profit organisation established to support multinational clinical research, to organise a one-day workshop on the topic to clarify potential benefits and risks. The issues that arose in that workshop are summarised and include the following: the nature of cloud computing and the cloud computing industry; the risks in using cloud computing services now; the lack of explicit guidance on this subject, both generally and with reference to clinical trials; and some possible ways of reducing risks. There was particular interest in developing and using a European ‘community cloud’ specifically for academic clinical trial data. It was recognised that the day-long workshop was only the start of an ongoing process. Future discussion needs to include clarification of trial-specific regulatory requirements for cloud computing and involve representatives from the relevant regulatory bodies.

Clinical data management is a vital vehicle in medical research to ensure the integrity and quality of data used to take medical decisions. When applied to clinical trials or observational studies it must comply with several regulations and guidelines developed in recent years; these include general purpose national and international legislation for data privacy and data protection, good clinical practice (GCPs) for medical interventions, and specific guidelines for electronic data submission to regulatory agencies. The aim of this article is to briefly illustrate the above-mentioned guidelines and legislation from the data-acquisition perspective and to describe how this process can be improved through a proper and prudent application of current information technology. Furthermore, a practical application using the current state of the art and, in particular, the web-based technology, will be presented with two real case studies. Drug Dev. Res. 67:245-250, 2006. © 2006 Wiley-Liss, Inc.

Background The present study is aimed to identify genetic pathways correlated with chemoresistance in epithelial ovarian cancer (EOC). Methods We compared the molecular profiles of 23 tumour biopsies of stage III–IV (training set) at primary surgery, before chemotherapy, to the profile from the same patients at second surgery, after several lines of platinum (Pt)-based chemotherapy when the tumours were resistant. In the hypothesis that identified markers were related to Pt-resistance and to prognosis, we validated this signature in 52 EOC taken at primary surgery (validation set) selected to be either very sensitive to the first line therapy, i.e. not relapsing before one year from the end of therapy, or resistant, i.e. relapsing within 6months from the end of therapy. Results In the training set, we identified a resistance signature indicative of the activation of epithelial to mesenchymal transition (EMT) by transforming growth factor (TGF)-beta pathway. We then validated this signature in 52 EOC taken at primary surgery (validation set). Some genes involved in EMT, such as B MP and a ctivin m embrane- b ound i nhibitor (BAMBI), and mir-141 resulted in association with overall or progression free survival. Conclusion Some genes involved in EMT were associated to overall or progression free survival, suggesting EMT as vital to the resistance mechanisms.

Purpose: Epithelial ovarian cancer (EOC) is one of the most lethal gynecologic diseases, with survival rate virtually unchanged for the past 30 years. EOC comprises different histotypes with molecular and clinical heterogeneity, but up till now the present gold standard platinum-based treatment has been conducted without any patient stratification. The aim of the present study is to generate microRNA (miRNA) profiles characteristic of each stage I EOC histotype, to identify subtype-specific biomarkers to improve our understanding underlying the tumor mechanisms. Experimental Design: A collection of 257 snap-frozen stage I EOC tumor biopsies was gathered together from three tumor tissue collections and stratified into independent training (n = 183) and validation sets (n = 74). Microarray and quantitative real-time PCR (qRT-PCR) were used to generate and validate the histotype-specific markers. A novel dedicated resampling inferential strategy was developed and applied to identify the highest reproducible results. mRNA and miRNA profiles were integrated to identify novel regulatory circuits. Results: Robust miRNA markers for clear cell and mucinous histotypes were found. Specifically, the clear cell histotype is characterized by a five-fold (log scale) higher expression of miR-30a and miR-30a*, whereas mucinous histotype has five-fold (log scale) higher levels of miR-192/194. Furthermore, a mucinous-specific regulatory loop involving miR-192/194 cluster and a differential regulation of E2F3 in clear cell histotype were identified. Conclusions: Our findings showed that stage I EOC histotypes have their own characteristic miRNA expression and specific regulatory circuits. Clin Cancer Res; 19(15); 4114–23. ©2013 AACR.

Purpose: Epithelial ovarian tumors (EOT) are among the most lethal of malignancies in women. We have previously identified ZIC2 as expressed at a higher level in samples of a malignant form (MAL) of EOT than in samples of a form with low malignant potential (LMP). We have now investigated the role of ZIC2 in driving tumor growth and its association with clinical outcomes. Experimental Design: ZIC2 expression levels were analyzed in two independent tumor tissue collections of LMP and MAL. In vitro experiments aimed to test the role of ZIC2 as a transforming gene. Cox models were used to correlate ZIC2 expression with clinical endpoints. Results: ZIC2 expression was about 40-fold in terms of mRNA and about 17-fold in terms of protein in MAL (n = 193) versus LMP (n = 39) tumors. ZIC2 mRNA levels were high in MAL cell lines but undetectable in LMP cell lines. Overexpression of ZIC2 was localized to the nucleus. ZIC2 overexpression increases the growth rate and foci formation of NIH3T3 cells and stimulates anchorage-independent colony formation; downregulation of ZIC2 decreases the growth rate of MAL cell lines. Zinc finger domains 1 and 2 are required for transforming activity. In stage I MAL, ZIC2 expression was significantly associated with overall survival in both univariate (P = 0.046) and multivariate model (P = 0.049). Conclusions: ZIC2, a transcription factor related to the sonic hedgehog pathway, is a strong discriminant between MAL and LMP tumors: it may be a major determinant of outcome of EOTs. Clin Cancer Res; 18(16); 4313–24. ©2012 AACR.

Myxoid Liposarcomas (MLS), characterized by the expression of FUS-CHOP fusion gene are clinically very sensitive to the DNA binding antitumor agent, trabectedin. However, resistance eventually occurs, preventing disease eradication. To investigate the mechanisms of resistance, a trabectedin resistant cell line, 402-91/ET, was developed. The resistance to trabectedin was not related to the expression of MDR related proteins, uptake/efflux of trabectedin or GSH levels that were similar in parental and resistant cells. The 402-91/ET cells were hypersensitive to UV light because of a nucleotide excision repair defect: XPG complementation decreased sensitivity to UV rays, but only partially to trabectedin. 402-91/ET cells showed collateral sensitivity to temozolomide due to the lack of O(6) -methylguanine-DNA-methyltransferase (MGMT) activity, related to the hypermethylation of MGMT promoter. In 402-91 cells chromatin immunoprecipitation (ChIP) assays showed that FUS-CHOP was bound to the PTX3 and FN1 gene promoters, as previously described, and trabectedin caused FUS-CHOP detachment from DNA. Here we report that, in contrast, in 402-91/ET cells, FUS-CHOP was not bound to these promoters. Differences in the modulation of transcription of genes involved in different pathways including signal transduction, apoptosis and stress response between the two cell lines were found. Trabectedin activates the transcription of genes involved in the adipogenic-program such as c/EBPα and β, in 402-91 but not in 402-91/ET cell lines. The collateral sensitivity of 402-91/ET to temozolomide provides the rationale to investigate the potential use of methylating agents in MLS patients resistant to trabectedin.

Purpose: Gene expression profile was analyzed in 68 stage I and 15 borderline ovarian cancers to determine if different clinical features of stage I ovarian cancer such as histotype, grade, and survival are related to differential gene expression. Experimental Design: Tumors were obtained directly at surgery and immediately frozen in liquid nitrogen until analysis. Glass arrays containing 16,000 genes were used in a dual-color assay labeling protocol. Results: Unsupervised analysis identified eight major patient partitions, one of which was statistically associated to overall survival, grading, and histotype and another with grading and histotype. Supervised analysis allowed detection of gene profiles clearly associated to histotype or to degree of differentiation. No difference was found between borderline and grade 1 tumors. As to recurrence, a subset of genes able to differentiate relapsers from nonrelapsers was identified. Among these, cyclin E and minichromosome maintenance protein 5 were found particularly relevant, as their expression was inversely correlated to progression-free survival (P = 0.00033 and 0.017, respectively). Conclusions: Specific molecular signatures define different histotypes and prognosis of stage I ovarian cancer. Mucinous and clear cells histotypes can be distinguished from the others regardless of tumor grade. Cyclin E and minichromosome maintenance protein 5, whose expression was found previously to be related to a bad prognosis of advanced ovarian cancer, appear to be potential prognostic markers in stage I ovarian cancer too, independent of other pathologic and clinical variables.

age distribution at the time of MBC diagnosis: 41-57 years (n=3) and 76/79 years (n=2). Two patients had solitary metastatic lesions (bone, n=2; lung, n=1), one patient had extensive bone metastases, and one patient had multiple bony and pulmonary metastases. In only one of the five cases did the patient receive chemotherapy in the supposed palliative situation. Discussion: There are patients who, despite the diagnosis of distant metastases, may be considered cured of their disease; however, these cases are rare. The long-term survivors in our study cohort comprised a relatively heterogeneous group with regard to age at MBC diagnosis, therapy and metastatic sites. The factors contributing to the quite rare and felicitous case of long-term survival or even cure, can hardly be evaluated systematically and remain a black box. It appears, however, that aggressive chemotherapy-containing regimens, are not the key to solving the mystery of this black box.

Introduction: Trabectedin is a marine alkaloid originally isolated from Ecteinascidia turbinata approved in Europe for the second line of therapy in soft tissue sarcomas (STS). Here we characterize and compare the gene expression profile of 402-91/ET Res liposarcoma cells obtained after continuous exposure to trabectedin in vitro with that of their parental cell line 402-91. Methods: Drug sensitivity was measured by colony assay and Flow Cytometric analysis was used to analyze cell cycle perturbations. Microarray analysis between sensitive and 402-91/ET Res cells was performed using commercially available arrays onto which 44K oligos were pre-spotted. Gene expression analysis was performed with “R” package software. Pathway analysis was performed using Metacore software. qRT-PCR was used for data validation. Statistical analysis was performed using Graphpad software. Results: 402-91/ET Res cells were selected in vitro for their stable resistance to trabectedin by step-wise increase in the drug concentration (25 nM) lasting 14 months. Resistance to Trabectedin was stable over 22 months without compound exposure. Cells were cross resistant to melphalan, but more sensitive to taxanes, vinblastine, temozolomide and UV rays. Resistance was not associated to the expression of MDR proteins as well as different uptake/efflux. Cell cycle analysis revealed that trabectedin is able to induce a G2-M block in the sensitive cell line but not in the resistant one. The expression levels of the FUS-CHOP gene were comparable between the 402-91 and 402-91/ET Res cells but with a different trans-activation ability. Microarray analysis identified 511 probes set able to discriminate between sensitive and resistant cell lines. Pathway analysis revealed that most of the genes altered in the 402-91/ET Res cells were associated with liposarcoma phenotype and functionally involved in the CREB pathway as well as control of apoptosis. Conclusion: Data suggests that in the 402-91/ET Res cells FUS-CHOP trans-activation activity has been impaired. Studies are in progress to address whether these mechanisms are implicated in sensitivity of liposarcoma cell lines to trabectedin. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B64.

Background Studies undertaken in academic settings have shown that bupropion hydrochloride can double the odds of smoking cessation compared with placebo. To assess whether these results are applicable in primary care, we launched a double-blind, placebo-controlled, randomized trial to be conducted by general practitioners. Methods We assigned 593 healthy smokers to receive bupropion hydrochloride, 150 mg twice a day, or placebo daily for 7 weeks (hereinafter, bupropion group [n = 400] and placebo group [n = 193], respectively). After the baseline visit, 4 clinical visits and 3 telephone calls were scheduled over the 1-year period. The primary end points were biochemically confirmed continuous abstinence at week 7 and at week 52. Results Seventy-one Italian general practitioners enrolled participants from April 2004 to May 2005. Of the bupropion group, 41.0% were continuously abstinent from week 4 to week 7 compared with 22.3% of the placebo group (multivariate odds ratio, 2.37; 95% confidence interval, 1.60-3.53). The continuous abstinence rates from week 4 to week 52 were 25% in the bupropion group and 14% in the placebo group (odds ratio, 2.11; 95% confidence interval, 1.32-3.39). The mean weight gain was similar in both groups and among long-term abstainers was 3 kg in women and 4 kg in men. More participants in the bupropion group experienced an adverse event than those in the placebo group, but the percentage who discontinued use of the study medication was similar. Conclusions Bupropion more than doubled the odds of continuous abstinence from smoking. The adherence of general practitioners and participants to the protocol was excellent, making our findings robust and easy to generalize to the context of primary care.

Background In an effort to ensure that all physicians have access to valid and reliable evidence on drug effectiveness, the Italian Drug Agency sponsored a free-access e-learning system, based on Clinical Evidence, called ECCE. Doctors have access to an electronic version and related clinical vignettes. Correct answers to the interactive vignettes provide Continuing Medical Education credits. The aims of this trial are to establish whether the e-learning program (ECCE) increases physicians' basic knowledge about common clinical scenarios, and whether ECCE is superior to the passive diffusion of information through the printed version of Clinical Evidence. Design All Italian doctors naïve to ECCE will be randomised to three groups. Group one will have access to ECCE for Clinical Evidence chapters and vignettes lot A and will provide control data for Clinical Evidence chapters and vignettes lot B; group two vice versa; group three will receive the concise printed version of Clinical Evidence. There are in fact two designs: a before and after pragmatic trial utilising a two by two incomplete block design (group one versus group two) and a classical design (group one and two versus group three). The primary outcome will be the retention of Clinical Evidence contents assessed from the scores for clinical vignettes selected from ECCE at least six months after the intervention. To avoid test-retest effects, we will randomly select vignettes out of lot A and lot B, avoiding repetitions. In order to preserve the comparability of lots, we will select vignettes with similar, optimal psychometric characteristics. Trial registration ISRCTN27453314