Complicated urinary tract infections (c-UTIs) are among the most common nosocomial infections and a substantial part of the antimicrobial agents used in hospitals is for the treatment of c-UTIs. Data from surveillance can be used to guide the empirical treatment choices of clinicians when treating c-UTIs. We therefore used nation-wide surveillance data to evaluate antimicrobial coverage of agents for the treatment of c-UTI in the Netherlands.We included the first isolate per patient of urine samples of hospitalised patients collected by the Infectious Disease Surveillance Information System for Antibiotic Resistance (ISIS-AR) in 2012, and determined the probability of inadequate coverage for antimicrobial agents based on species distribution and susceptibility. Analyses were repeated for various patient groups and hospital settings.Escherichia coli (47%), Enterococcus spp. (14%), Proteus mirabilis (8%), and Klebsiella pneumoniae (7%). For all species combined, the probability of inadequate coverage was <5% for amoxicillin or amoxicillin-clavulanic acid combined with gentamicin and the carbapenems. When including gram-negative bacteria only, the probability of inadequate coverage was 4.0%, 2.7%, 2.3% and 1.7%, respectively, for amoxicillin, amoxicillin-clavulanic acid, a second or a third generation cephalosporin in combination with gentamicin, and the carbapenems (0.4%). There were only small variations in results among different patient groups and hospital settings.The most prevalent bacteria in 27,922 isolates of 23,357 patients were Enterococcus spp., considered as less virulent, and the carbapenems, considered as last-resort drugs, empirical treatment for c-UTI with the best chance of adequate coverage are one of the studied beta-lactam-gentamicin combinations. This study demonstrates the applicability of routine surveillance data for up-to-date clinical practice guidelines on empirical antimicrobial therapy, essential in patient care given the evolving bacterial susceptibility.When excluding  Urinary tract infections (UTIs) are among the most common nosocomial infections and a substantial part of the antimicrobial agents used in hospitals is for the treatment of UTIs www.swab.nl). The previous SWAB guideline for the antimicrobial treatment of complicated UTI (c-UTI) dates from 2006 Another important predictor of treatment failure is antimicrobial resistance Data from surveillance can be used to guide the empirical treatment choices of clinicians Escherichia coli is the most common causative pathogen in UTIs worldwide ISIS-AR collects interpretations of antimicrobial susceptibility , including underlying MIC values and disk zone diameters (if available), and patient data  of all routinely cultured bacterial species of participating medical microbiology laboratories located in various regions of the Netherlands ISIS-AR lacks clinical data and only collects antimicrobial susceptibility data of bacterial isolates with limited patient background data. We therefore defined a c-UTI as a positive urine sample /ml since ISIS-AR does not collect information on CFU/ml) from a hospitalised patient since uncomplicated infections are rare in the hospital setting We included the first isolate per patient of urine samples of patients aged > = 18 years hospitalised from January to December 2012. Isolates from patients admitted at Intensive Care Units (ICU) were excluded since ICU-patients are usually more ill, receive more antimicrobials and often have urinary catheters. Additionally, rates of antimicrobial resistance at ICUs are higher than at non-ICU hospital departments www.eucast.org). For gram-positive bacteria, MIC values were unavailable for the majority of isolates. We therefore used antimicrobial susceptibility interpretations as reported by the participating laboratories. For the most common uropathogens, we determined to proportion of isolates non-susceptible for each antimicrobial agent separately, for the third generation cephalosporins as a group , for the carbapenems as a group , and for some specific antimicrobial combinations used for empirical treatment .For Enterobacteriaceae and other gram-negative bacteria, we reinterpreted the available MIC values of isolates for amoxicillin, amoxicillin- clavulanic acid, piperacillin-tazobactam, cefuroxime, ceftazidime, cefotaxime/ceftriaxone, ciprofloxacin, imipenem, meropenem, trimethoprim-sulphamethoxazole, nitrofurantoin and gentamicin as susceptible or non-susceptible using the European Committee for Antimicrobial Susceptibility Testing (EUCAST) 2012 (version 2.0) guidelines , resulting in 6,394 resistant E. coli); of the 2,361 Proteus mirabilis, 22.9% is resistant to agent A  resulting in 541 resistant P. mirabilis. The weighted average of resistance of these two pathogens to agent A would be  6,935/16,383 = 42.3%, implying that in 42.3% of patients, the antimicrobial coverage of agent A is expected to be inadequate. When assessing the probability of inadequate coverage for a specific antimicrobial agent, we adjusted for pathogens that are considered intrinsically resistant to that agent , according to the EUCAST expert rules To assess the probability of inadequate antimicrobial coverage, we calculated a weighted average of non-susceptibility for all uropathogens combined and for the gram-negative bacteria only. A weighted average implies that the distribution of species in urine samples was taken into account. For example, of the 14,022 Proportions of non-susceptibility and probability of inadequate coverage were calculated as described as above. Fleiss Quadratic Approximation was used for the calculation of 95% confidence intervals (95% CI). To assess the generalizability of or our results we performed similar analyses for various patient groups and urine sample types, namely 1) community-onset versus hospital-associated c-UTI; 2) spontaneously passed midstream urine versus urine originating from catheters; 3) male patients versus female patients; 4) urine samples from all patients versus urine samples from patients with a blood sample with an identical species submitted within 7 days of the urine sample with that species .Finally, similar analyses were performed for different hospital settings, namely 1) community hospitals; 2) teaching hospitals; 3) university hospitals/tertiary referral centres. All three settings have their own specific patient population with university hospitals usually having the highest rates of antimicrobial resistance due to the more complicated nature of its patients. To assess regional differences, hospital settings were defined for each participating laboratory and analysis were repeated for each participating laboratory separately.All data analyses were performed using SAS/STAT software, SAS System for Windows 9.3, SAS Institute Inc., Cary, NC, USA.The data of the bacterial isolates and their susceptibility results used in this study belong to the microbiological laboratories participating in ISIS-AR and was obtained as part of routine clinical care in the past. Written or verbal consent of patients was therefore not obtained. Furthermore, all patient identifiers had been previously removed and data were analysed anonymously. According to the Dutch Medical Research Involving Human Subjects Act (WMO) this study was considered exempt from review by an Institutional Review Board.E. coli , Enterococcus spp. , P. mirabilis , Klebsiella. pneumoniae , Pseudomonas aeruginosa , β-haemolytic streptococci group B , Staphylococcus aureus , K. oxytoca , Enterobacter cloacae , and Morganella morganii . The remaining 2.113 (7.6%) isolates were uncommon pathogens each accounting for less than 1% of the total number of isolates.We included 27,922 isolates from 23,357 patients. The most predominantly found pathogens were: E. coli were less frequently identified in hospital-associated c-UTI than in community-onset c-UTI , while Enterococci, P. mirabilis, P. aeruginosa, and E.cloacae were more frequently found in hospital-associated c-UTI than in community-onset c-UTI. In urine samples from patients representing urosepsis , the top three pathogens was substantially different from samples of patients considered to have a c-UTI without a bloodstream infection. In samples from patients with urosepsis, E. coli was far more frequently isolated , while Enterococcus spp. were only isolated in 0.8% (95%CI 0.4–1.3%) versus 16.3% (95%CI 15.8–16.8%) urine samples.Since gram-negative bacteria represent the majority of causative pathogens, in particular in patients representing urosepsis , we asseThere were variations in the probability of inadequate coverage between the different hospital settings  and diffThis study provides current information regarding the distribution of pathogens and their antimicrobial susceptibility patterns in urine samples from hospitalised patients in the Netherlands. Furthermore, we show that routinely collected surveillance data on antimicrobial resistance are useful for developing guidelines on antimicrobial therapy.E. coli in 72% of urine samples of female general practice patients P. aeruginosa and Enterobacter spp. However, when considering a percentage of 5% as the upper limit for inadequate coverage that is often used for decision making on empiric therapy for life-threatening infections Enterococcus spp since they are intrinsically resistant. This resulted in a higher probability of inadequate coverage with cefuroxime or 3GC in combination with gentamicin when compared to amoxicillin-clavulanic acid in combination with gentamicin. However, when only gram-negative uropahogens are considered there are no clinically relevant differences in probability of inadequate coverage for amoxicillin-clavulanic acid in combination with gentamicin and the cephalosporins in combination with gentamicin.The large amount of patient data in our study, enabling sub-analyses for different patients groups, distinguishes this study from previous studies on UTI in the Netherlands that found E. coli as the major pathogen and susceptibility patterns found in this group of patients identify the same antimicrobial agents with a probability of inadequate coverage below 5% as found in other patient groups. Additionally, it is not routine practice to collect urine samples for microbiological testing in the case of asymptomatic bacteriuria in the Netherlands Although our study benefits from the large amount of data, it is limited by the lack of clinical information on actual infections and patient treatment. However, we performed sub analysis on data of patients with a blood sample and a simultaneous urine sample both with identical species that are likely to represent life-threatening infections with the urinary tract as the source of infection. Results from these sub analysis identified Enterococcus spp., that have a low prevalence in serious c-UTI such as urosepsis, the most suitable empirical treatment for c-UTI in hospitalized patients should be intravenous therapy with amoxicillin, depending on the local resistance patterns with clavulanic acid, or a second or third generation cephalosporin, all combined with an aminoglycoside that have a useful additive role in the treatment of serious infections by gram-negative bacteria, such as c-UTI When considering routine surveillance data of urine samples and when excluding Although we found differences in the pathogen distribution and antimicrobial coverage between men and women, we do not recommend separate guidelines for the empirical treatment of c-UTI in these two patient groups since no major differences were found among those antimicrobial agents with a probability of inadequate treatment below 10%. Additionally, we do not recommend last-resort agents, such as the carbapenems for empirical treatment since broad-spectrum agents are associated with the selection of drug-resistant organisms The results from this study are in line with the recommendations of the recently revised SWAB guideline for empirical treatment of c-UTI


Few studies are available evaluating the impact of rapid-acting insulin analogues on long-term diabetes outcomes. Our aim was to compare the use of rapid-acting insulin analogues versus human regular insulin in relation to the occurrence of diabetic complications in a cohort of diabetic patients through the analysis of administrative databases.A population-based cohort study was conducted using administrative data from four local health authorities in the Abruzzo Region . Diabetic patients free of macrovascular disease at baseline and treated either with human regular insulin or rapid-acting insulin analogues were followed for a maximum of 3 years. The incidence of diabetic complications was ascertained by hospital discharge claims. Hazard ratios (HRs) and 95% CIs of any diabetic complication and macrovascular, microvascular and metabolic complications were estimated separately using Cox proportional hazard models adjusted for patients’ characteristics and anti-diabetic drug use. Propensity score matching was also used to adjust for significant difference in the baseline characteristics between the two treatment groups.A total of 2,286 patients were included: 914 receiving human regular insulin and 1,372 rapid-acting insulin analogues. During the follow-up, 286 (31.3%) incident events occurred in the human regular insulin group and 235 (17.1%) in the rapid-acting insulin analogue group. After propensity score-based matched-pair analyses, rapid-acting insulin analogues users had a HR of 0.73 (0.58–0.92) for any diabetes-related complication and HRs of 0.73 (0.55–0.93) and 0.55 (0.32–0.96) for macrovascular and metabolic complications respectively, as compared with human regular insulin users. No difference between the two groups was found for microvascular complications.Our findings suggest that the use of rapid-acting insulin analogues is associated with a lower risk of cardiovascular and metabolic complications compared with human regular insulin use. The major challenge in diabetes management is preventing or delaying the progression of diabetes-related complications. The landmark trials in type 1  and typeRecently an increasing body of evidence, not yet fully supported by randomized clinical trials, has suggested that postprandial hyperglycemia is directly implicated in cardiovascular complications -7. The hThe use of rapid-acting insulin analogues  provides lower postprandial glucose excursions in people with diabetes -11. NeveA recent observational study showed that the rapid-acting insulin analog gluslisine was associated with a reduced incidence of macro- and microvascular events in patients with type 2 diabetes in comparison with human regular insulin . HoweverThe aim of this study is therefore to compare the use of rapid-acting insulin analogues and human regular insulin in relation to the first occurrence of any diabetes-related complication in a cohort of diabetic patients without macrovascular disease at baseline through the analysis of administrative databases. We conducted a retrospective, population-based cohort study using administrative data from four local health authorities in the Abruzzo Region of Central Italy, that comprise about 900,000 inhabitants . Details regarding data sources and study design have been published previously . BrieflyA flow chart of the study population selection is depicted in At baseline, we excluded patients if they had (i) at least one prescription of antiplatelets (excluding low-dose aspirin), pentoxifylline or nitrates, which are commonly used for the treatment of ischemic cardiovascular diseases, or a diagnosis of macrovascular disease in hospital discharge or procedure codes reported on prescriptions for services use ; (ii) noEach individual accumulated person-years of follow-up from January 1, 2006 until the date of hospital admission for any diabetic complications, censoring (death or emigration), or December 31, 2008 (i.e. the end of the follow-up), whichever occurred first. For each patient the following potential confounders were assessed at baseline: age, sex, metabolic and microvascular complications, and concomitant drugs. The algorithm developed for identifying the microvascular and metabolic complications at baseline (reasonable proxy measure of severity and duration of diabetes) has been described elsewhere . PrescriFinally, we also assessed the number of hemoglobin A1C tests performed by patients during the follow-up.The primary outcome of this study was the first occurrence of any diabetes-related complication during the follow-up. The secondary outcome was the first occurrence of specific clinical conditions including macrovascular, microvascular and metabolic complications . To evalThe chi-square test  or unpaired t-test (continuous variable) for numerical variables were used to determine whether there were differences between baseline characteristics of patients treated with human regular insulin or rapid-acting insulin analogues.Two statistical methods were used to evaluate the risk associated with bolus insulin treatment. First, we constructed Cox proportional hazards models to derive hazard ratios (HRs) and 95% confidence intervals (CIs), using the human regular insulin group as the reference group. Estimates were adjusted for age, gender, previous microvascular and metabolic complications, number of HbA1c tests, use of low-dose aspirin, antiarrhythmics agents, antihypertensive and lipid-lowering agents and mean daily dosage of anti-diabetic drugs in quartiles. Second, we performed a propensity-score (PS) matched Cox proportional hazard analysis. Specifically, we used PS analysis to identify subsamples of the two treatment groups  that are “balanced” on all covariates listed in Kaplan-Meier estimates were used to generate time to event curves for the primary- and secondary outcomes. In the unmatched sample, survival curves were compared across the two treatment groups by the log-rank test. In the matched sample, survival curves were constructed using paired Kaplan-Meier estimates and compared using the Klein-Moeschberger test.http://www.R-project.org) and SPSS software version 17.1 for Windows . All analyses were performed using R  in the human regular insulin group and 235 (17.1%) in the rapid-acting insulin analogue group, with a difference between the two treatment groups in the cumulative HR for any diabetic complications that increased over time (p<0.0001 by the log-rank test) . After multivariable adjustments, using human regular insulin group as the reference group, the HR was 0.56 (95% CI: 0.45-0.69) for any diabetic complications in the rapid-acting insulin analogue group. Regardless of secondary outcomes, we found 203 macrovascular events (22.2%) in the human regular insulin group and 147 (10.7%) in the rapid-acting insulin analogue group, with a significant reduction in macrovascular complications associated with the use of rapid-acting insulin analogues  . Among mThe PS matched Cox models supported these results . SpecifiBecause we have previously shown that the use of insulin glargine was associated with a lower risk of diabetic complications than intermediate- and long-acting human insulins , we repeFor all endpoints, results were similar to those presented for the entire study population. In particular in the unmatched subcohorts, the adjusted HR estimates were 0.57 (95% CI: 0.46-0.72) for any diabetic complication, 0.57 (95% CI: 0.43-0.76) for macrovascular complications and 0.39 (95% CI: 0.22-0.69) for metabolic complications in rapid-acting insulin users as compared with those on human regular insulin; no statistical significant difference was found between the two subgroups in relation to microvascular event rates . All resThe major finding of our study is that patients given rapid-acting insulin analogues developed fewer diabetic complications  than those given human regular insulin. Due to the lack of randomization, significant differences in baseline characteristics exist between the two treatment study group. Patients on human regular insulin are expected to be at higher risk of developing cardiovascular complications than those on rapid-acting insulin analogue, because they were older and more likely to use low-dose aspirin and antiarrhythmic and antihypertensive agents. On the other hand, patients treated with bolus insulin analogues were more often men, reported a higher number of HbA1c tests and a higher mean daily dose of all anti-diabetic agents than patients receiving human regular insulin. Despite these differences between the two study cohorts, the reduction of both primary and secondary outcomes remained significant even after adjusting for these and other potential confounders evaluated in this study. These results were also supported by PS matched-pair analyses performed on the two balanced subsamples.Moreover, based on our previous finding that a lower risk of any diabetic complication was associated with the use of insulin glargine  we carriOur findings were in line with a previous observation suggesting that the use of rapid-acting insulin analogue glulisine could be associated with a risk reduction of 24% for macrovascular events in patients with type 2 diabetes as compared with the use of human regular insulin . In thatNevertheless, we acknowledge that an observational study using administrative data is prone to potential biases. In particular, our findings may be subject to confounding by indication due to the lack of randomization. In fact the allocation of treatment is based on the clinical judgment of physicians and may be related to a different cardiovascular risk profile between the two groups. However, all PS matching resulted in full balance of all patients characteristics and is likely to have minimized confounding by indication enhancing the comparability of patients. In addition, Cox regression analyses were conducted with macrovascular complications as the endpoint after stratification for gender or age (<65 years versus ≥65 years); the results remained essentially unchanged when the association between study treatment and macrovascular events was examined in the different subgroups .The presence of unrecognized confounders could lead us to over-estimate the magnitude of the association between exposure and outcome as compared with the results of randomized clinical trials. The analysis of confounders that were included in the Cox-models was limited to variables collected by administrative data. A good deal of information on the degree of metabolic control, severity and duration of diabetes and on lifestyle attitudes, which are known to influence the likelihood of developing complications , is not Another limitation of our study is that our database does not allow us to ascertain with confidence the type of diabetes as a potential confounder. The higher proportion of younger patients in the rapid-acting insulin analogue group suggests the possibility of a higher prevalence of patients with type 1 diabetes. Therefore, we attempted to explore the effect of this variable on our results by using the following criteria for identifying type 1 diabetes cases: no records of oral hypoglycemic agents, specific ICD9 code for type 1 diabetes (250.x1 or 250.x3), diabetic ketoacidosis episode (250.x1), age<40 year . AccordiOur findings could be explained by a better quality of care in the group treated with bolus insulin analogues that could be associated with a more favorable prognosis in that group . HoweverAdditionally, our study population included diabetic patients free of macrovascular diseases at baseline, who may have been misclassified because of medical errors in the medication and diagnosis codes or the short baseline period (1 year). However, this type of error does not seem to be very high because the cohort of diabetic patients with macrovascular disease at baseline represented about 30% of the overall sample, which is consistent with results from previous landmark studies ,3. FinalDespite these intrinsic limitations of observational data, several speculative explanations for our findings should be considered. In particular there are three demonstrated clinical effects of the rapid-acting insulin analogues that could explain why they may be more effective than the human regular insulin in reducing the risk of diabetic complications.Overall glycemic control may be better with rapid-acting insulin analogues than with human regular insulin. These agents can be injected immediately before the meals, whereas human regular insulin should be injected 30-45 min before the meals . In realAnother plausible account might be that the greater beneficial effect of bolus insulin analogues on the reduction of diabetic complications (particularly macrovascular events), might be also attributable to their ability to better control PPG excursions. In line with those results, the Diabetes Intervention Study (DIS)  and The Finally, several studies have also shown a lower occurrence of hypoglycemia with the use of rapid-acting insulin analogues ,29. In oThis analysis from a real-world setting suggests that the use of rapid-acting insulin analogues, in comparison with human regular insulin, is associated with a lower risk of diabetic complications and particularly macrovascular events. Future trials may be required to determine the potential long-term clinical benefits of bolus insulin analogues in preventing cardiovascular diseases in diabetic patients.Figure S1Flow chart for creating and validating propensity score-matched pairs.(PDF)Click here for additional data file.Figure S2Absolule standardized differences in covariates between the two study treatment groups, before and after propensity score matching.(PDF)Click here for additional data file.Figure S3Cumulative hazard according to the two treatment group matched for propensity score of (A) Any diabetes-related complications , (8) Macrovascular complications , (C) Microvascular complications  and (D) Metabolic complications .(PDF)Click here for additional data file.Table S1Baseline characteristics and propensity score matched baseline characteristics of study sub-cohorts without prescription of insulin glargine treated with human regular insulin or a rapid-acting insulin analogues.(PDF)Click here for additional data file.Table S2Cumulative incidence and Cox Proportional Hazard Ratios (HRs) of diabetes-related complications in patients without prescription of insulin glargine and treated with human regular insulin or a rapid-acting insulin analogue.(PDF)Click here for additional data file.Table S3Cumulative incidence and Cox Proportional Hazard Ratios (HRs) of Macrovascular complications in patients treated with human regular insulin or a rapid-acting insulin analogue stratified by Gender or Age.(PDF)Click here for additional data file.


Ovarian cancer, like most solid tumors, is in dire need of effective therapies. The significance of this trial lies in its promise to spearhead the development of combination immunotherapy and to introduce novel approaches to therapeutic immunomodulation, which could enable otherwise ineffective vaccines to achieve clinical efficacy.Tumor-infiltrating T cells have been associated with improved outcome in ovarian cancer, suggesting that activation of antitumor immunity will improve survival. However, molecularly defined vaccines have been generally disappointing. Cancer vaccines elicit a modest frequency of low-to-moderate avidity tumor-specific T-cells, but powerful tumor barriers dampen the engraftment, expansion and function of these effector T-cells in the tumor, thus preventing them from reaching their full therapeutic potential. Our work has identified two important barriers in the tumor microenvironment: the blood-tumor barrier, which prevents homing of effector T cells, and T regulatory cells, which inactivate effector T cells. We hypothesize that cancer vaccine therapy will benefit from combinations that attenuate these two barrier mechanisms.We propose a three-cohort sequential study to investigate a combinatorial approach of a new dendritic cell (DC) vaccine pulsed with autologous whole tumor oxidized lysate, in combination with antiangiogenesis therapy (bevacizumab) and metronomic cyclophosphamide, which impacts Treg cells.This study uses a novel autologous tumor vaccine developed with 4-day DCs pulsed with oxidized lysate to elicit antitumor response. Furthermore, the combination of bevacizumab with a whole tumor antigen vaccine has not been tested in the clinic. Finally the combination of bevacizumab and metronomic cyclophosphamide in immunotherapy is novel. Ovarian cancer cells are antigenic and express a multitude of known tumor-associated antigens (TAAs) including Her-2/neu -3, p53 , NY-ESO-bona fide rejection antigens in the clinic [+ cytotoxic T-cells (CTLs) as well as CD4+ T helper (Th) cells, a possibly necessary condition to ensure tumor homing of low affinity CD8+ cells [P < 0.0001) [Therapeutic cancer vaccines have the potential to break immune tolerance and induce long-term immune response against cancer cells. However, molecularly defined vaccines directed towards known TAAs have either failed to produce clinical responses or have yielded transient responses in ovarian cancer patients to date, as none of the above antigens, except for NY-ESO-1, have been proven to be e clinic ,45,46. Ae clinic -49. The e clinic . Tumor c8+ cells -53. Whol8+ cells . Some of 0.0001) . Althoug 0.0001) -58, some 0.0001) -61. In t 0.0001) .+ and CD8+ T-cell responses in vitro [A widely used and straightforward method of whole tumor cell preparation already used in clinical trials is necrotic whole tumor cell lysate. The efficacy of the necrotic cell lysate can be further enhanced by oxidative modification using hypochlorous acid (HOCl) treatment . It has in vitro -66. The in vitro ,68. The in vitro -71. Secoin vitro . Third, in vitro -75, leadin vitro ,76. In pin vitro . This wiDCs loaded with whole tumor lysate have been investigated in several clinical trials for their ability to induce anti-tumor T-cell responses ,77-80. BWe developed a faster, four-day protocol for DC preparation, using GM-CSF, IL-4 and serum-free AIM-V media that is suitable for clinical use. We showed that Day-4 DCs generated with this protocol are similar to “classic” Day-7 DCs, in terms of phenotype and phagocytic capability, and have a higher capacity than Day-7 DCs to produce IL-12p70 following LPS and IFN-γ stimulation. In addition, these Day-4 DCs were highly immunogenic, and efficiently primed ovarian tumor-specific T-cells in vitro in peripheral blood lymphocytes from healthy volunteers and ovarian cancer patients .+CD25+ T regulatory (Treg) cells when administered in combination with a GM-CSF secreting tumor vaccine, resulting in increased CTL induction and improved vaccine efficacy [It has been shown that vascular endothelial growth factor (VEGF) suppresses tumor antigen presentation through blockade of myeloid DC differentiation and maturation, leading to tumor immune tolerance -95, a prefficacy ,103. Simefficacy . A singlefficacy .BR), which is up regulated in tumor endothelium and deregulates endothelial ICAM-1 expression and T-cell adhesion. This blood-tumor endothelial barrier can be disrupted by ETBR antagonists, resulting in dramatic increase of T-cell homing in tumors and significant efficacy of otherwise ineffective vaccine therapy in the mouse, thus calling for human experimentation [BR is upregulated by VEGF. In vitro, ET-1 signaling through ETAR in cancer cell lines leads to de novo production of VEGF, a process regulated by HIF-1-alpha [2 production [+ TILs were detected in ID8-VEGF tumors. In agreement with others [+ TILs in ID8-VEGF tumors, while DMSO vehicle had no effect on TILs (unpublished data). Normalization of tumor vasculature through disruption of the VEGF/VEGFR-2 axis was also shown to increase extravasation of adoptively transferred T-cells into the tumor and improve adoptive cell transfer immunotherapy in a murine cancer model [neu-induced breast cancer, it was demonstrated that this combination treatment accelerated tumor regression augmenting the lytic activity of CD8+ cytotoxic T-cells [Work by us and others have demonstrated that angiogenesis mechanisms also impair the effector arm of antitumor immunity by blocking homing of effector T-cells into tumors. This is in part mediated through the endothelin B receptor , given three days prior to vaccination with autologous melanoma cells admixed with Bacillus Calmette-Guerin (BCG), to treat patients with melanoma [+ suppressor T-cells.Although, the traditional view has been that chemotherapy may neutralize antitumor immune response generated through vaccine therapy, emergent data indicate that chemotherapy can be combined safely with immunotherapy with possibly additive or synergistic effects that are dose and schedule dependent. The most extensively investigated drug to enhance vaccine potency is cyclophosphamide, a drug previously used in ovarian cancer as standard of care in combination with cisplatin. One of the first observation was made by Berd and colleagues who used a regimen of low-dose cyclophosphamide  induced a decrease the number and the relative frequency of circulating regulatory T-cells, and that the dose of 250 mg/m2 was able to impair the suppressor function of regulatory T-cells. It was also shown in a phase I trial of pancreatic cancer patients that inhibition of regulatory T-cells (when using 250 mg/m2 intravenous cyclophosphamide) resulted in recruitment of high-avidity effector T-cells to tumors, leading to prolonged progression-free survival and overall survival [Cyclophosphamide has been used also in humans to augment cancer immunotherapy ,124. In 200 mg/m2. Single-survival .It has been hypothesized that combining VEGF blockade with low-dose (metronomic) chemotherapy may have positive antiangiogenic or antitumor effects . In a muWe propose a phase I, three-cohort, single-center study to establish the safety and proof of concept of Oxidized tumor Cell pulsed DC (OCDC) vaccine administered intranodally, alone  or in combination with either intravenous bevacizumab  or intravenous bevacizumab and intravenous cyclophosphamide  in subjects with recurrent ovarian, fallopian tube or primary peritoneal cancer  for DC manufacturing. Patients will receive OCDC tumor vaccine in combination with other agents in a design that escalates combinatorial complexity. OCDC is prepared at the Cell and Vaccine Production Facility of the University of Pennsylvania. It will be released in sterile syringes containing ~2.5-5 × 106 dendritic cells (OCDC vaccine) intranodally, while subjects in Cohort 2 will receive the same five doses of vaccine in combination with intravenous bevacizumab (10 mg/kg) given every two weeks on days 0, 14, 28, 42 and 56. Subjects in Cohort 3 will receive the same five doses of vaccine on days 0, 14, 28, 42 and 56, while bevacizumab (10 mg/kg) plus cyclophosphamide (200 mg/m2) will be given the day before each vaccination.Subjects in Cohort 1 will receive five doses of 5–10 × 10Subjects will be offered to undergo apheresis within two weeks after vaccine #4 or one to three weeks after vaccine #5 to collect vaccine primed PBL for use in a follow-on study of adoptive T-cell therapy. Each treatment cohort will be evaluated separately, to detect any side effects that may be due to the vaccine in combination with other biological agents. Patients in Cohort 2 will be taken off bevacizumab, if they experience severe adverse events (SAEs) at least likely related to bevacizumab, but will retain the option of continuing OCDC vaccination on the study. Subjects in Cohort 3 will be taken off bevacizumab/cyclophosphamide, if they experience severe adverse events (SAEs) at least likely related to bevacizumab or cyclophosphamide, but will retain the option of continuing OCDC vaccination on the study. Subjects will be taken off the study completely if they experience SAEs, which are possibly, probably or definitely related to OCDC. Termination of enrollment for each cohort will be triggered with ≥2 DLTs  in the first 5 subjects and ≥3 DLTs at any time. The rules for early stopping for toxicity do not depend on the availability of immune response data; any subject that gets at least one vaccination injection is included. For this study, 25 evaluable subjects will be treated, and we estimate that up to 30 subjects may need to be enrolled, assuming a 20% failure rate for generating the dendritic cell vaccine.Subjects will be followed daily for the first 5 days (starting at Day 0), and then biweekly until week 8 (End of Study). At screening and 30 days following the fifth vaccine dose (day 86), subjects will undergo immune assessment. Immune monitoring will be performed on blood samples from all participating patients to assess the vaccine induced antitumor immune response and the composition of circulating T-cell subpopulations. This study obtained approval from national health agencies and from the Institutional Review Board of the University of Pennsylvania and is performed in accordance with the Helsinki Declaration, the International Conference of Harmonization Good Clinical Practice guidelines, and local regulatory requirements. Written informed consent will be obtained from each patient.The primary objective of the study is to determine the feasibility and safety of administering OCDC intranodally alone, in combination with intravenous bevacizumab, or in combination with intravenous bevacizumab plus cyclophosphamide in subjects with recurrent ovarian, fallopian tube or primary peritoneal cancer. The secondary objectives of the study are to obtain pilot data on immunogenicity on OCDC administered intranodally in subjects, to assess the effect of the proposed treatments on peripheral blood regulatory T-cells and on the tumor microenvironment, as well as to evaluate clinical responses up to 114 days.Toxicity will be graded by NCI Common Toxicity Criteria (NCI-CTC) Version 4.0 and will be tabled by treatment cohort. Immune response will be evaluated by descriptive statistics, scatter plots of pre- and post-vaccine values of the various immune parameters, and relevant fold changes by treatment cohort. Exploratory longitudinal analyses (repeated measures ANOVA or linear mixed effects models) will be used to examine time trends , test for differences between baseline and post-vaccine time points within cohorts and discern differences among cohorts. Clinical responses scored by RECIST criteria will be tabulated by treatment cohort.The proposed first-in- human study is innovative in many ways. First, it translates novel concepts of combinatorial immunomodulation of the tumor microenvironment from the laboratory to the clinic. The notion of blocking VEGF in combination with the whole tumor cancer vaccine is novel and has not been tested in the clinic. Preclinical data in solid tumor mouse models show that blocking VEGF enhances de novo T-cell infiltration into the tumor  and, wheWe propose a new combinatorial therapy approach to mobilize antitumor immunity against ovarian cancer. The strong association between the presence of intraepithelial T-cells along with other biomarkers of immune activation in tumors and improved clinical outcome suggests that mobilization of antitumor immunity should yield clinical benefit in many patients with EOC, a notion preliminarily supported by many published pilot studies. Our group and other groups have however, revealed the existence of numerous and overlapping mechanisms of immune dysfunction in ovarian cancer, which will have to be abated in order to effectively mobilize antitumor immunity. The combinatorial approach proposed herein is a first attempt to utilize readily available therapeutic tools with known clinical and biological behavior to address some of the tumor barriers.+ cells can enhance tumor engraftment and persistence of low affinity anti-tumor CD8+ cells [Given the paucity of reliable tumor rejection antigens in EOC , we propose to use a whole tumor lysate vaccine. Given the easy accessibility of primary but also recurrent tumors in the peritoneal cavity, and the general acceptance of primary or secondary surgical cytoreductive surgery as the standard of care for these patients, autologous tumor lysate is a feasible approach in this population. Autologous tumor lysates provide a convenient and personalized source of multiple tumor antigens, possibly encompassing all the relevant class I and II epitopes against which antitumor immune response can be mounted, including private and mutated epitopes. Incorporation of class II epitopes could be especially important, since the coexistence of tumor-reactive CD48+ cells . The cho8+ cells ,134.+ PBMC cells in as little as 40 hours [There is no strictly defined standard duration of culture to generate human PBMC-derived DCs. To date, most clinical studies have used a 7-day culture with GM-CSF and IL-4. However, data from Czerniecki and colleagues have shown that fully functional antigen-presenting cells (APC) can be rapidly developed from CD1440 hours ,136. Alt40 hours . These 440 hours . We sele40 hours . IL-12 i40 hours -140 demo40 hours . Okada eIn a murine tumor model, DC pulsed with tumor lysate and injected intranodally resulted in greater sensitization of T-cells and improved anti-tumor responses . In a raAdoptive immunotherapy has frequently resulted in tumor rejection in the human, suggesting that a critical number of high-avidity tumor-reactive T-cells are probably required to effectively overcome barriers in the tumor microenvironment. On the contrary, cancer vaccines have most commonly failed to induce overwhelming tumor responses in patients. Preclinical models show that if one can defeat immune barriers in the tumor microenvironment, one can enable low-avidity/low-frequency antitumor immune responses induced by vaccines to become clinically effective. Work from our lab and from other labs shows that among tumor microenvironment barriers preventing the engraftment, expansion and function of antitumor effector T-cells, two can be readily targeted in ovarian cancer: a) angiogenesis driven by VEGF, and b) Treg cells.Vascular endothelial growth factor is highly expressed and plays an important role in tumor progression of ovarian carcinoma. Positive immunostaining for VEGF was observed in 97% (68 out of 70) of ovarian carcinomas  and high2, which can inhibit effector lymphocyte function and/or DC maturation and function. It is quite possible that some of these tumor endothelial immunomodulatory mechanisms may not be directly mediated by VEGF. For example, we previoulsy found that many of the specific markers of tumor endothelium in ovarian cancer are induced not by VEGF but rather by a combination of hypoxia and inflammatory mediators [Therefore, VEGF blockade can provide a) important antiangiogenic therapeutic effects, b) attenuate the endothelial blood-tumor barrier Figure  and c) iediators . ImportaVEGF plays an important role in suppression of DC maturation. It has been demonstrated that DCs from cancer patients are functionally impaired; furthermore, the increase of immature dendritic cells in the periphery was closely correlated with serum VEGF levels but not with TGF-β, IL-6 or GM-CSF . Gabrilo+ CD25+ FOXP3+ human Treg cells suppress tumor-specific T-cell immunity and contribute to growth of human tumors in vivo in a study of 104 individuals affected with ovarian carcinoma. They found that there was a significant correlation between tumor Treg cell content and survival in the group as a, and also for individuals in stage II, , III or IV disease [+ CD25+ T-cells, delays tumor growth, and improves cure rates when followed by non-curative immunotherapy [2 can transiently decrease Treg frequencies and enhance tumor-specific immune response in breast cancer patients in combination with a cancer vaccine [Treg cells have been shown to be present in ovarian cancer ,157. Cur disease . Presentotherapy . The samotherapy . Jaffee  vaccine ,160.2 administered intravenously, which conveniently matches the schedule of bevacizumab and vaccine. Based on reported effects, this schedule and dose of cyclophosphamide should reduce Treg cells. The concomitant administration of intravenous low-dose cyclophosphamide and bevacizumab has never been tested before. Importantly, Treg cells and VEGF are interconnected; hypoxia (which drives expression of VEGF) induces also accumulation of CCR10+ Treg cells in ovarian tumors, while Treg cells can in turn reprogram the tumor microenvironment towards angiogenesis [We recently reported on a pilot study (UPCC-11807), where we administered oral metronomic cyclophosphamide at 50 mg daily every other week to patients with EOC in combination with bevacizumab. We saw no significant effect of this dose of cyclophosphamide on peripheral blood Treg. Thus, in the present study we chose the dose of 200 mg/mogenesis . Thus, iogenesis . We propIn summary, this study responds to the urgent need created by the above observations to test in the clinic a combinatorial regimen that administers cancer vaccine in combination with Treg and VEGF blockade. The present trial will enable us to take the first step in this clinical development endeavor, testing the feasibility and safety of such an approach while we collect pilot biological data from the periphery and the tumor microenvironment. Upon completion of this phase I study, we will be in a position to dissect the individual contribution of bevacizumab or cyclophosphamide to vaccine therapy through rationally designed and adequately powered phase II randomized studies, based on the results of the present study.APC: Antigen-presenting cell; CTL: Cytotoxic T lymphocyte; CVPF: Cell and vaccine production facility; DC: Dendritic cell; DLT: Dose-limiting toxicity; DTH: Delayed type hypersensitivity; ECOG: Eastern Cooperative Oncology Group; EOC: Epithelial ovarian cancer; EOS: End of study; GM-CSF: Granulocyte/macrophage colony-stimulating factor; HOCL: Hypochlorous acid; KLH: Keyhole Limpet hemocyanin; LFTU: Long-term follow-up; OCDC: Oxidized tumor cell pulsed dendritic cell (vaccine); PBMC: Peripheral blood mononuclear cell; SAE: Serious adverse event; TAA: Tumor-associated antigen; TIL: Tumor-infiltrating lymphocytes.Authors declare that they do not have competing interest to disclose.LK contributed to the conception of the study, the clinical trial design, overseeing the clinical trial and writing the manuscript, CC contributed to the preclinical data. JT is the PI of the study, GM contributed to the preclinical data, KB contributed to writing the manuscript, RM contributed to the clinical trial design and the reviewing of the manuscript. GC contributed to the conception of the study, the clinical trial design, overseeing the clinical trial and reviewing the manuscript. All authors read and approved the final manuscript.


Objective. The role of single preoperative serum CA-125 levels in predicting pelvic or paraaortic lymph node metastasis in patients operated for epithelial ovarian cancer has been investigated.  Methods. 176 patients diagnosed with epithelial ovarian carcinoma after staging laparotomy between January 2002 and May 2010 were evaluated retrospectively.  Results. The mean, geometric mean, and median of preoperative serum CA-125 levels were 632,6, 200,29, and 191,5 U/mL, respectively. The cut-off value predicting lymph node metastases in the ROC curve was 71,92 U/mL, which is significant in logistic regression analysis (P = 0.005). The preoperative log CA-125 levels were also statistically significant in predicting lymph node metastasis in logistic regression analysis (P = 0.008).  Conclusions. The tumor marker CA-125, which increases with grade independent of the effect of stage in EOC, is predictive of lymph node metastasis with a high rate of false positivity in Turkish population. The high false positive rate may obscure the predictive value of CA-125. Epithelial ovarian cancer (EOC) is the sixth most frequent cause of cancer death in women and is the leading cause of mortality resulting from overall gynecologic malignancies in the United States and Europe . Over thMost patients with ovarian cancer have widespread disease at the time of diagnosis due to asymptomatic nature of early stage disease . While tLymph node metastasis has been proven to be a prognostic factor in EOC , 9. LateCarbohydrate antigen 125 (CA-125) is a high molecular-weight glycoprotein expressed by epithelial ovarian tumors as well as on the surface of cells of mesothelial origin . CurrentThe purpose of the current study was to investigate single preoperative serum CA-125 level in predicting lymph node metastases in Turkish patients with EOC.The data of 225 patients who were diagnosed with EOC cancer after staging laparotomy at the Department of Gynecologic Oncology of Kanuni Sultan Suleyman Research and Teaching Hospital from January 2002 to May 2010 were retrospectively reviewed. Informed consent from the patients was not obtained because of the retrospective nature of the study. The study was approved by the ethics committee of the hospital .The inclusion criteria were as follows: patients with histological confirmation of EOC; those who underwent pelvic or para-aortic lymphadenectomy during staging laparotomy; and those with preoperative imaging studies (CT and MRI) for the evaluation of lymph node involvement.12 patients with endometrioid type EOC who had synchronous endometrioid type endometrial adenocarcinoma, 3 patients who also had tubal cancer, 5 patients who had metastases to ovaries from other genital organs or other organs, 2 patients who also had endometrial hyperplasia that can affect CA-125 levels, 8 patients operated for indications other than ovarian cancer and had no preoperative CA-125 levels or imaging studies, 10 patients who had staging laparotomy in another hospital and applied for secondary cytoreductive surgery, and 9 patients who were operated after neoadjuvant chemotherapy were excluded from the study. Finally there were 176 patients fulfilling the criteria for enrollment in the study.Optimal debulking surgery was defined as a residual tumor ≤1 cm in maximal diameter. The tumors were staged according to the 2009 FIGO staging system and histologically defined according to World Health Organization (WHO) classification. All patients were referred to medical oncology for chemotherapy treatment after surgery.P = 0.008). Statistical analyses were performed using MedCalc statistical software . A P value <0.05 was considered as statistical level of significance.Imaging studies were performed within one week before staging laparotomy. Preoperative serum CA-125 levels were measured before staging laparotomy using a radioimmunoassay kit (Roche F170 Moduler system). The upper normal value of serum CA-125 level was 35 U/mL. The cut-off value of single serum CA-125 level for the detecting of lymph node metastasis preoperatively was calculated using the receiver operating characteristic (ROC) curve and its significance was determined by logistic regression analysis with the odds ratio (OR) and its 95% confidence interval (CI). The logarithmic transformation of preoperative CA-125 (log CA-125) levels was also evaluated for predicting lymph node metastasis using logistic regression analysis  were in menopausal state. The demographic features of the study population are shown in Histologically, 46,6% of the tumors (82 cases) were serous cystadenocarcinoma, 34,1% (60 cases) were mucinous cystadenocarcinoma, 9,7% (17 cases) were endometrioid carcinoma, and 5,1% (9 cases) were clear cell carcinoma. 8 of the cases were diagnosed as undifferentiated tumors. The pathological specimens that could not be discerned if they are serous or mucinous were accepted as undifferentiated. The distribution of histologic subtypes of epithelial ovarian cancers in our study is shown in In 47 cases , lymph node metastasis was proven by pathological evaluation. Pelvic lymph node metastasis was observed in 35 patients  and para-aortic lymph node metastasis was observed in 23 (13%). The mean number of positive lymph nodes acquired in pelvic or para-aortic lymphadenectomies was 7,8. The mean number of positive pelvic and para-aortic lymph nodes was 12,9 and 2,6, respectively.P = 0.0003. Two-way analysis of variance (ANOVA) showed that both grade and FIGO stage have significant effect on serum CA-125 levels, although grade's effect and stage's effect are not dependent on each other as shown by tests of between-subjects effects.When the cases were stratified for grade, the mean and median CA-125 levels in grade 1, grade 2, and grade 3 groups were 350,7 and 76; 526,7 and 140; and 641 and 239,5, respectively, and this distribution had a P = 0.003) , and lymph node metastasis in imaging studies was found as a significant factor in detecting lymph node involvement. When log CA-125 as a continuous variable was evaluated for lymph node metastasis by logistic regression with the same clinical factors, it was also statistically significant ). According to this equation, two optimal cut-off values with maximal Youden indices were further studied. The value with the highest index was 71,92 U/mL (area under curve (AUC) = 0,636 ± 0,05; = 0.003) . This cunificant .While treating ovarian cancer, it is imperative to be prepared for an extended procedure preceding the operation. The tumor marker CA-125, the most extensively studied molecule for ovarian cancer in the literature, seems to be the most promising biomarker to predict the stage in a given patient –18. We aIn our study, we found that a single preoperative serum CA-125 level can predict lymph node metastasis. However, the significant cut-off values we have ended up with are relatively low levels. For a serum CA-125 level of more than 72 and 123, the false positive ratio is 67,4% and 55%, respectively. While these values may be helpful in guiding clinical management, the false positive ratios are too high to use as a screening tool for predicting lymph node metastasis. Log CA-125 levels were also found statistically significant for predicting lymph node metastasis. On the other hand, logarithmic transformation of laboratory results in clinical settings is not always feasible. However, there are some studies that have shown preoperative CA-125 employable in detecting lymph node metastases. The study of Kim et al. on 99 Asian women with epithelial ovarian cancer has found the significant cut-off value of ≥535 U/mL which had a sensitivity and specificity in predicting lymph node metastasis similar to imaging studies . AnotherThe cut-off value of 72 U/mL and the geometric mean serum CA-125 of 200,29 U/mL for all the patients enrolled in the study are relatively much lower than those values found in ovarian cancer studies mentioned above. The possible reasons of our lower values may stem from the fact that the number of early stage ovarian cancer patients is higher in our study population and a subset of patients who had lymph node metastasis and high CA-125 levels but were found eligible for neoadjuvant therapy were excluded during the enrollment process. However, serum CA-125 levels may rise above normal because of the tumor extension and metastasis while tumor mass is another overlapping source of high serum CA-125 levels. Namely, high serum CA-125 levels that may relate to lymph node metastasis may also be encountered in women with advanced stage disease. Our exclusion of advanced stage cases was supposed to eliminate this effect; meanwhile it lowered our CA-125 values in general. However, it has also been shown that peritoneal implants outside the pelvis may occur in lower serum CA-125 values than values observed in patients with retroperitoneal lymph node metastasis .In a recent meta-analysis of imaging studies in the detection of lymph node involvement in ovarian cancer by Yuan et al., CT and MRI did not have optimal performance in detecting lymph node metastasis  while PET or PET/CT was a more accurate modality  . AlthougIn our study, serum CA-125 levels, behaving typically as other tumor markers, increased gradually with increasing grade of the lesions independent of the effect of the stage. This was shown to be statistically significant, as seen in the literature .Many tumor markers have been studied for screening and tumor progression in ovarian cancer. The cancer cell or the immune response against the tumor through the extracellular matrix, the vasculature, and the patient's body fluids has been investigated thoroughly with many approaches from transcripts to posttranslational modifications, producing ample markers for research in the diagnosis, screening, and prognosis of ovarian cancer , 19. HowRace, irregular periods, oral contraceptive use, bilateral oophorectomy, and history of infertility treatment were also reported to be altering baseline CA-125 levels, suggesting CA-125 cut-off could be tailored for individuals in the future if prospective studies to be conducted support these findings . CA-125 There are some limitations present in our study, which stem from the retrospective nature of the design of the study, relatively small number of subjects, and the uneven distribution of ovarian cancer stages among these subjects that may obscure the statistical significance of CA-125. Furthermore, solely a single preoperative value of CA-125 level was investigated while the rate of increase of CA-125 preoperatively or comparison of preoperative levels with baseline levels may be analyzed for further research in predicting lymph node metastasis. Postoperative serum CA-125 levels have also been demonstrated as prognostic factors in ovarian cancer .In conclusion, many physiologic processes and racial and genetic variations may contribute to the versatility of the tumor marker CA-125, which increases with grade independent of the effect of stage in EOC and is predictive of lymph node metastasis with a high rate of false positivity in Turkish population. The high false positive rate may obscure the predictive value of CA-125 and the role of CA-125 in this issue needs to be settled. Further studies on preoperative CA-125 provide a vast area to be explored in detecting lymph node metastasis or advanced stage disease in EOC.


Most meta-analyses include data from one or more small studies that, individually, do not have power to detect an intervention effect. The relative influence of adequately powered and underpowered studies in published meta-analyses has not previously been explored. We examine the distribution of power available in studies within meta-analyses published in Cochrane reviews, and investigate the impact of underpowered studies on meta-analysis results.For 14,886 meta-analyses of binary outcomes from 1,991 Cochrane reviews, we calculated power per study within each meta-analysis. We defined adequate power as ≥50% power to detect a 30% relative risk reduction. In a subset of 1,107 meta-analyses including 5 or more studies with at least two adequately powered and at least one underpowered, results were compared with and without underpowered studies. In 10,492 (70%) of 14,886 meta-analyses, all included studies were underpowered; only 2,588 (17%) included at least two adequately powered studies. 34% of the meta-analyses themselves were adequately powered. The median of summary relative risks was 0.75 across all meta-analyses (inter-quartile range 0.55 to 0.89). In the subset examined, odds ratios in underpowered studies were 15% lower  than in adequately powered studies, in meta-analyses of controlled pharmacological trials; and 12% lower  in meta-analyses of controlled non-pharmacological trials. The standard error of the intervention effect increased by a median of 11% (inter-quartile range −1% to 35%) when underpowered studies were omitted; and between-study heterogeneity tended to decrease.When at least two adequately powered studies are available in meta-analyses reported by Cochrane reviews, underpowered studies often contribute little information, and could be left out if a rapid review of the evidence is required. However, underpowered studies made up the entirety of the evidence in most Cochrane reviews. Systematic reviews of intervention studies aim to synthesise all available evidence meeting pre-specified eligibility criteria. Such criteria seldom address sample size. Meta-analyses may therefore include data from one or more small studies which, individually, do not have power to detect a modest intervention effect. Small studies tend to report greater intervention effects than larger studies Some researchers argue for excluding small studies from meta-analyses. Specifically to reduce the effects of publication bias, Stanley suggested discarding 90% of the study estimates, so that conclusions are based on only the most precise 10% of studies Arguments for including small studies in meta-analyses uphold that evidence synthesis is best informed by all reasonably unbiased evidence and that no such evidence should be discarded lightly. Cut-offs based on study size, although scientifically cost-efficient, introduce an extra element of subjectivity and might not ameliorate bias if the remaining large studies are insufficiently critiqued In this paper, we explore the levels of power available in studies included in published meta-analyses, and examine the relative influence of adequately powered and underpowered studies on these meta-analyses.Cochrane Database of Systematic Reviews , which was provided by the Nordic Cochrane Centre. Each meta-analysis was categorized by type of outcome, types of intervention compared, and medical specialty to which the research question related, as described elsewhere To examine power per study within meta-analyses and to explore whether this varies across different settings, we use evidence from the j, power was calculated with respect to a fixed baseline event rate, i within meta-analysis j . The power of meta-analysis j to detect a 30% relative risk reduction or equivalently a log relative risk of The focus of this paper is on the power of primary studies within meta-analyses, but it is interesting also to examine the power of the meta-analyses themselves. In each meta-analysis CDSR meta-analyses that include five or more studies, with at least two adequately powered  of the 14,886 CDSR meta-analyses, all studies were underpowered (RRR30). In many settings, a 20% relative risk reduction would be more realistic, and 85% of the meta-analyses included no studies powered to detect RRR20. Only 2,588 (17%) meta-analyses included at least two studies powered at 50% or more to detect RRR30, and only 1,291 (9%) included at least two studies powered at 80% or more. Median power within CDSR meta-analyses was low for RRR30 at 13% power, with an inter-quartile range (iqr) of 7% to 31% power. Some studies were generously powered, with 2,571/77,237 (3.3%) having at least 98% power for RRR30 (or RRR30 .iqr 0.55 to 0.89).Power of studies to detect the summary relative risk in their meta-analysis was also low: 11,422 (77%) meta-analyses included no studies with ≥50% power and only 2,236 (15%) meta-analyses included at least two studies with ≥50% power. The median of summary relative risks was 0.75 across all meta-analyses  consisted entirely of underpowered studies. However, median power within cancer meta-analyses remained low at 24% power (iqr 10% to 57% power).By outcome, we expected power to be lower for events that are typically rare. Power was indeed somewhat lower for meta-analyses reporting all-cause mortality and cause-specific mortality/major morbidity event/composite , and somewhat higher for meta-analyses relating to resource use, signs/symptoms reflecting continuation/end of disease or a mixture of subjective outcomes see .RRR30 was 22%, with a further 12% powered at 50–80% to detect RRR30, but 66% were underpowered. At 34%, the proportion of adequately powered meta-analyses was substantially larger than the proportion of meta-analyses including at least two adequately powered studies, but remains low.iqr 11% to 72% power). There was some variation across medical areas; in cancer, 51% of meta-analyses were powered at 50% or more. Differences in meta-analytic power across medical areas, outcome and intervention-comparison types were largely in the same direction as differences in meta-analysis summaries of study power  were eligible for inclusion in subset A. The impact of excluding the underpowered trials on the results of these meta-analyses is summarised in Of the 14,886 Across the 1,107 meta-analyses, there was a broad spread of ratios representing changes to the summary log odds ratio. The median ratio was 0.96 for the fixed-effect model and 0.94 for the random-effects model. The results correspond to a slight shift towards the null value when underpowered studies were removed, more so under the random-effects model in which small studies have greater influence.iqr −96% to +18%).Under the random-effects model, it is possible for precision to be gained  when studies are removed, if the heterogeneity estimate is sufficiently reduced. The non-zero between-study heterogeneity in 851 meta-analyses decreased by a median of 21% when underpowered studies were removed , and only 17% of meta-analyses included at least two studies with at least 50% power for RRR30. There was some variation across medical areas and outcome types, but individual studies’ power was low across all types of meta-analyses.Underpowered studies made up the entirety of the evidence in most meta-analyses reported by Cochrane reviews: in 70% of In a meta-epidemiological analysis of 1,107 meta-analyses, we found that odds ratios in underpowered studies were on average 10% lower  than those in adequately powered studies. This should be regarded as a lower limit on the difference, since the database contains treatment comparisons that have underlying relative risks either side of 1. Indeed, the difference was larger among comparisons involving a control or placebo group , in which we might expect the direction of effect to be more consistent across meta-analyses. In meta-analyses in which at least two adequately powered studies are available, underpowered studies often had relatively little impact on the summary estimate of the odds ratio. The summary estimate shifted slightly toward the null when underpowered studies were removed, under both fixed-effect and random-effects models. The extent to which precision was lost when underpowered studies were excluded varied across meta-analyses. Some meta-analyses included a few very large studies, which dominated their results, while in other meta-analyses all studies were similarly sized and exclusion of underpowered studies led to greater losses in precision.increased when underpowered studies were removed, in settings where, for example, the largest studies in the meta-analysis had produced extremely different results.On average, the between-study heterogeneity estimate decreased when underpowered studies were excluded from meta-analyses, which may be expected since underpowered studies tend to observe more extreme effect estimates. Within the subset of 1,107 meta-analyses examined, the heterogeneity estimate sometimes decreased substantially when underpowered studies were removed. However, we also found examples where the heterogeneity CDSR meta-analyses had at least 50% power to detect RRR30. Elsewhere, in the setting of cumulative meta-analysis in particular, the information size required for a meta-analysis to detect a particular effect size has been used to examine whether meta-analyses contain enough information to be conclusive RRR30 is comparable with, but much more precise than, the 39% of 174 meta-analyses from the Cochrane Neonatal Group, which were found to meet the information size criterion for RRR30 with 80% power by Brok et al. The meta-analyses themselves were better powered than the primary studies within meta-analyses, as we would expect: overall, 34% of Our work is limited to meta-analyses from Cochrane reviews, which may not be representative of meta-analyses in general. In particular, the differences observed between medical areas may reflect differing advice or editorial policies between Cochrane Review Groups which oversee different medical areas rather than disease-specific differences.CDSR database, and so we were unable to look at the association between study age and power. It is possible that studies carried out in more recent years were more generously powered. However, the reasons for lack of power in completed studies include over-enthusiasm of researchers for the effectiveness of a new intervention, problems with recruitment to the study, and inaccurate sample size calculations Although underpowered when included in meta-analyses, some original studies may have been adequately powered for their own primary outcomes, since the results extracted for meta-analysis might have related to secondary outcomes. For example, a study designed to detect a difference in measures of depression would be unlikely to be adequately powered for all-cause mortality. We do not therefore intend to criticise authors of primary studies for the very low levels of power in these meta-analyses. Publication dates of primary studies were not always available in the CDSR data set are consistent with previous findings, but offer much greater precision. For example, in a combined analysis of 13 meta-analyses evaluating effects on pain in patients with osteoarthritis, Nüesch et al. It is well known that small studies included in a meta-analysis tend to show more extreme treatment effects than larger studies. The differences observed between underpowered and adequately powered studies in the The practical implications of our findings for systematic reviews and meta-analyses vary according to review purpose and the research time available. Systematic reviews commissioned to inform public health policy decisions, by the National Institute for Health and Clinical Excellence (NICE) for example, are often carried out to tight deadlines In conclusion, we found that underpowered studies play a very substantial role in meta-analyses reported by Cochrane reviews, since the majority of meta-analyses include no adequately powered studies. In meta-analyses including two or more adequately powered studies, the remaining underpowered studies often contributed little information to the combined results, and could be left out if a rapid review of the evidence is required.Appendix S1Detailed exploration of the role of underpowered studies.(DOC)Click here for additional data file.


Amyotrophic Lateral Sclerosis (ALS) is an adult onset neurodegenerative disease, which is universally fatal. While the causes of this devastating disease are poorly understood, recent advances have implicated RNA-binding proteins (RBPs) that contain predicted prion domains as a major culprit. Specifically, mutations in the RBPs TDP-43 and FUS can cause ALS. Cytoplasmic mislocalization and inclusion formation are common pathological features of TDP-43 and FUS proteinopathies. Though these RBPs share striking pathological and structural similarities, considerable evidence suggests that the ALS-linked mutations in TDP-43 and FUS can cause disease by disparate mechanisms. In a recent study, Couthouis et al. screened for protein candidates that were also involved in RNA processing, contained a predicted prion domain, shared other phenotypic similarities with TDP-43 and FUS, and identified TAF15 as a putative ALS gene. Subsequent sequencing of ALS patients successfully identified ALS-linked mutations in TAF15 that were largely absent in control populations. This study underscores the important role that perturbations in RNA metabolism might play in neurodegeneration, and it raises the possibility that future studies will identify other RBPs with critical roles in neurodegenerative disease. Amyotrophic Lateral Sclerosis (ALS) is an adult-onset neurodegenerative disease that is typically fatal within 2–5 years of disease onset. Loss of upper and lower motor neurons leads to paralysis and is usually the cause of death. Most cases are sporadic with no family history, but ~10% of cases are familial [SOD1 underlie ~20% of familial ALS (fALS) cases and ~1% of sporadic ALS (sALS) cases [Efforts to understand the causes of ALS and methods to mitigate its pathogenesis have been severely limited by an inability to identify ALS genes. For the past two decades, most ALS research has focused on the ubiquitously expressed copper/zinc superoxide dismutase 1 (SOD1). Mutations in S) cases –7. TheseTDP43 were found in sALS and fALS cases, but not in control cases [ The identification of a novel ALS disease protein in 2006 dramatically altered our understanding of this disease and brought to light the potential role RNA-binding proteins (RBPs) play in neurodegeneration. TDP-43 was found in cytoplasmic inclusions in diseased brain [ol cases . This fiTDP-43 is a highly conserved, ubiquitously expressed protein that has various roles in RNA metabolism , 9. TDP- The identification of the RBP, TDP-43, as a causative agent of ALS-directed research towards the possibility that other RBPs might also play a role in ALS. This led to the contemporaneous discovery by two groups of the ALS disease protein Fused in Sarcoma, or FUS . Like TDFUS contains an N-terminal QGSY-rich domain, followed by a Glycine-rich domain, an RRM, and two RGG-rich domains , 10. Lik Prions are infectious proteins that are prone to misfold and aggregate and can spread from organism to organism in a DNA/RNA-independent manner –17. TheyTDP-43 and FUS both contain a predicted prion domain similar to those found in yeast prion proteins like Sup35 and Ure2 , 18–20. To test the prediction that these RBPs are intrinsically aggregation-prone, a cell-free system was established to monitor the propensity of pure protein for spontaneous aggregation , 22, 25.Saccharomyces cerevisiae has been extensively used to gain insight into many neurodegenerative processes [ The baker's yeast rocesses . Furtherrocesses . Importarocesses , 27–34.  Though TDP-43 and FUS share many striking similarities , severalMost of the TDP-43 ALS-linked mutations tested to date accelerate aggregation in vitro, whereas the FUS mutations have no effect , 22. Q33 Given the largely overlapping functions and protein domains of TDP-43 and FUS, it is likely that many other RBPs play a crucial role not only in ALS but also in neurodegeneration more generally , 18, 25.First, the entire human genome was surveyed for proteins that contained RRMs . Of the Interestingly, TAF15 shares many similarities with TDP-43 and FUS . Its dom The identification of TDP-43 as an ALS gene altered the genetic landscape of neurodegenerative disease. The importance of altered RNA processing in disease pathogenesis is now much more widely appreciated. Furthermore, the identification of other RBPs that all contain RRMs and prion domains is unlikely to be mere coincidence , 18. IndThough much progress has been made in the identification and characterization of the roles that RBPs play in ALS, much is still beyond our current understanding. For instance, it is still not known whether these proteins cause disease by a loss of function or gain of function or both , 16–18.


Klebsiella (60%) was the most common organism producing ESBL followed by Escherichia coli (30%) and Pseudomonas (10%). Eighty percent of the ESBL-producing organisms were sensitive to piperacillin-tazobactam. Risk factors found significant by univariate analysis (P < 0.05) were preterm, low birthweight, perinatal asphyxia, respiratory distress syndrome, anaemia, metabolic acidosis, prolonged mechanical ventilation (>7 days), length of hospitalization, length of level 3 stay, prior antibiotic use, central venous catheter duration, peripherally inserted central venous catheter duration, and total parenteral nutrition duration. Factors that retained significance in the logistic regression model were duration of hospital stay  and gestational age . There was no significant difference in the mortality between the two groups.This prospective cohort study was conducted to determine the frequency of infections caused by extended-spectrum beta-lactamase- (ESBL-) producing organisms, various bacteria producing ESBL, antibiotic susceptibility of these organisms, and the risk factors associated with these infections in a neonatal intensive care unit in a tertiary care hospital in North India. Of the 150 neonates enrolled in the study, 47 culture-positive neonates were included in the study cohort and were divided into two groups: ESBL-positive (8 neonates) and ESBL-negative (39 neonates) cohorts. Various organisms were isolated from 72 culture samples in these 47 neonates. Of these, 10 culture samples grew ESBL-positive organisms and 62 samples grew ESBL-negative organisms. The frequency of ESBL-producing organisms was found to be 5.3%. ESBL infection incidence densities were found to be 3.4 per 1000 patient-days.  Klebsiella species were found among the clinical isolates and the mechanism of this resistance was the production of extended-spectrum beta-lactamase (ESBL) [Broad spectrum penicillins and first-generation cephalosporins remained the first line of defense for nearly 20 years, before resistance to them by beta-lactamases produced by gram-negative bacilli was found to be a serious threat to the common infections prevalent in community and hospital settings . In a feKlebsiella species and Escherichia coli, but they have also been detected in Enterobacter species, Salmonella species, Morganella morganii, Proteus mirabilis, Serratia marcescens, and Pseudomonas aeruginosa [The first ESBL isolates were discovered in Western Europe in the mid-1980s . ESBLs aruginosa . Escherichia coli as two of the six drug-resistant microbes to which new therapies are urgently needed [Escherichia coli in the community, clinicians should be aware of the potential of treatment failures associated with serious infections caused by these bacteria [E. coli infections in nonhospitalized patients has been recently described in several countries [Major risk factors for infection with ESBL-producing organisms are widespread use of third-generation cephalosporins, prolonged intensive care unit (ICU) or hospital stay, instrumentation and catheterization . Patienty needed . Due to bacteria . The emeountries –11.pneumoniae is an important cause of nosocomial infections [In neonates (0–28 days) ESBL-producing Klebsiella fections . HoweverThis prospective cohort study was conducted in the tertiary-level referral and inborn neonatal units of the Department of Pediatrics and Neonatology and the Department of Microbiology at Postgraduate Institute of Medical Education and Research and associated Dr. Ram Manohar Lohia Hospital, New Delhi, India, during December 2009–November 2010. Written informed consent was obtained for each subject from the parents. The study was approved by the institute's Ethics Committee.http://dx.doi.org/10.1155/2013/756209),neonates with two or more risk factors , 13, seeneonates with clinical features suggestive of sepsis , see alsneonates with positive sepsis screen , 16, seeAll patients with suspected neonatal sepsis admitted to the referral and inborn neonatal units of Dr. Ram Manohar Lohia Hospital, New Delhi, were included in the study. Suspected neonatal sepsis was defined as the presence of two or more of the following:Neonates in whom consent was denied and neonates in whom bacterial culture grew a mixture of organisms were excluded from the study.The following clinical samples were obtained from the suspected cases of neonatal sepsis during the study period as directed by their clinical condition as and where relevant: blood/urine/cerebrospinal fluid/stool/pus/peripheral long line catheter tip/central venous catheter tip/endotracheal tube tip/tracheal aspirate/bronchoalveolar lavage/pleural tap fluid/pericardial tap fluid/ascitic tap fluid. The samples were subjected to standard microbiological methods to isolate and identify the organism, to find the antibiotic susceptibility patterns, and to detect the ESBL-producing organisms.Information regarding the patient, symptoms, signs, diagnosis, antibiotic usage, interventions, ICU admissions, and outcome was obtained in a semistructured pro forma. Both ESBL-positive and ESBL-negative cohorts were followed up till one of the following end points: discharge, death, or left against medical adviceAntimicrobial susceptibility testing of all isolates was performed by Kirby Bauer disk diffusion method. In this method, the inoculums were adjusted to the turbidity of a 0.5 McFarland standard and swabbed onto the surface of a Muller-Hinton agar plate. After putting the disks onto the inoculated plates, the plates were incubated at 37°C for 24 hours. Antibiotic potency of the discs was standardized against the reference strain. All susceptibility results were interpreted according to the CLSI . The following antimicrobial agents were used for susceptibility testing: ampicillin (A), amikacin (Ak), aztreonam (Ao), ceftazidime (Ca), ceftazidime-clavulanic acid (Cac), cefotaxime (Ce), ceftriaxone (Ci), ciprofloxacin (Cf), cotrimoxazole (Co), cefepime (Cpm), chloramphenicol (C), gentamicin (G), meropenem (Mr), nalidixic acid (Na), nitrofurantoin (Nf), netilmicin (Nt), norfloxacin (Nx), ofloxacin (Of), and piperacillin-tazobactam (Pt). For detection of ESBL production, modified double-disk test was performed as a screening test. ESBL production and susceptibility to antimicrobial agents were detected on the same plate. Susceptibility testing was performed as previously described. Disks containing ceftazidime alone and a combination of clavulanic acid and ceftazidime were placed in a distance of 25 mm (centre to centre). The zones of inhibition for ceftazidime alone and ceftazidime plus clavulanic acid were compared. An increase in zone diameter of 5 mm in the presence of clavulanic acid indicated the presence of ESBL in the test organisms. Automated identification system  was used for reconfirmation of ESBL production.incidence densities were reported as the number of infections per 1000 patient days. Categorical variables were reported as numbers with proportion. Continuous variables were reported as mean with standard deviation or median with interquartile range. The association of the study variables with infections caused by ESBL-producing and non-ESBL-producing organisms was tested by univariate analysis. Continuous variables that were normally distributed were analyzed using Student's t-test, and the continuous variables that were not normally distributed were analyzed using Wilcoxon rank sum test. The Chi-square or Fisher exact test was used to compare categorical variables. All variables that were found to be statistically significant , as a risk factor for infections produced by ESBL-producing organisms, by univariate analysis, were further analyzed by using a multivariate logistic regression model. Statistical software package STATA 11  was used for the data analysis. Assuming the percentage of neonates with ESBL infections to be 15% in suspected cases of neonatal sepsis admitted to this hospital, with power of 80% with a confidence level of 90%, the minimum sample size needed was 118 cases and with a confidence level of 95%, the minimum sample size needed was 142. A total of 150 cases were enrolled in the study during the study period. The frequency of infections caused by ESBL-producing organisms was reported as the number of infections per 100 neonates with suspect sepsis. ESBL infection A total of 150 neonates with suspect sepsis were enrolled in the study during the period of December 2009–November 2010. Culture-positive gram-negative organisms were found in 59 neonates. Of these mixture of organisms was found in 12 neonates and was excluded from the study. Hence, a total of 47 culture-positive neonates were included in the study cohort. The study cohort was further divided into two cohorts—8 neonates with ESBL-positive cultures and 39 neonates with ESBL-negative cultures . The freKlebsiella (60%) was the most common organism producing ESBL followed by Escherichia coli (30%) and Pseudomonas (10%). The sources of various organisms producing ESBL and the various organisms producing ESBL are listed in Tables Various organisms were isolated from 72 culture samples in 47 neonates. Of these, 10 culture samples grew ESBL-positive organisms in 8 neonates and 62 samples grew ESBL-negative organisms in 39 neonates. P value = 0.011). Eighty percent of ESBL-producing organisms were sensitive to piperacillin-tazobactam, fifty percent to meropenem, and ten percent to aztreonam, amikacin, and gentamicin. The antimicrobial resistance patterns of the ESBL-producing and non-ESBL-producing organisms are given in Prior antibiotic use  was found to be a significant risk factor associated with ESBL-positive infections  and gestational age . Possible risk factors for infection with ESBL-producing organisms are listed in Among the ESBL-positive cohort, five neonates (62.5 percent) improved, one neonate (12.5 percent) was taken against medical advice, and two neonates (25 percent) expired. Among the ESBL-negative neonates, 32 neonates (82 percent) improved, 1 neonate was taken against medical advice, and 6 neonates (15.4 percent) expired.E. coli and Klebsiella, respectively, in neonatal infections [E. coli and K. pneumonia to be 22% [Infections caused by ESBL-producing organisms are a significant cause of neonatal morbidity and mortality all over the world mainly attributed to the widespread use of broad-spectrum antibiotics. The incidence of the infections caused by ESBL-producing organisms varies considerably in different geographical situations, from 37% in Latin America, 7% in the United States , to 5–56fections . Anothero be 22% . The incKlebsiella (60%) was the most common organism producing ESBL followed by E. coli (30%) and Pseudomonas (10%). The bacterial spectrum in the present study is comparable to that in other studies [Enterobacter and Acinetobacter species, we did not find ESBL production in these organisms [ studies –26. Thourganisms . These irganisms , 29. Klebsiella spp. and ciprofloxacin resistance are closely associated [E. coli isolates resistant to fluoroquinolones in India [The antimicrobial resistance patterns of both ESBL-producing and non-ESBL-producing organisms were comparable with those of other studies , 30. Ampsociated . Recentlin India . The onlin India . Though in India .Preterm and low-birthweight neonates were more prone to infections with ESBL-producing organisms which was consistent with other studies , 28, 35,In the present study, respiratory distress syndrome was found to be the major diagnosis in the ESBL-positive neonates. It was the major diagnosis in Huang et al. study, but association was not studied. This association may be attributed to the fact that these neonates are mostly preterm low-birthweight neonates and require interventional procedures and mechanical ventilation .In the present study, the presence of central venous catheters and peripherally inserted central venous catheters (PICC) was significantly associated with ESBL-positive infections which was not a consistent finding and was different in various settings , 35–37. Endotracheal intubation  and prolNot only the length of hospitalization but also the length of level 3 stay was found to be a risk factor for ESBL infections in neonates which was an additional finding in this study compared to other studies where only the length of hospitalization was found to be a risk factor , 28. ThiMortality rates in ESBL-positive infections were higher as compared to the non-ESBL infections , 28. In During the multivariate logistic regression analysis, the length of hospitalization and gestational age were found to be independent risk factors associated with ESBL-positive infections, which is in harmony with other studies , 28. ESBL test should be routinely done in all culture-positive samples growing gram-negative organisms as the infections by ESBL-producing organisms are a significant problem in neonates. Judicious prescription of antibiotics is recommended as prior use of antibiotics is a significant risk factor for ESBL production. Strict aseptic precautions should be maintained in handling the neonates especially the preterm and low-birthweight neonates. Interventions and duration of hospital stay should be minimized as far as possible. Longitudinal surveillance of the microbial flora and their antibiotic sensitivity pattern should be done in every hospital periodically to know the existing flora and for appropriate management of the infection by these organisms.Table-1: Risk factors for early onset sepsisTable-2.1: Clinical features suggestive of sepsisTable-2.2 :Specific featuresTable-3: Sepsis ScreenClick here for additional data file.