Background and Objective: Galectin-9  is one of the galectin family members which are known as proteins with β-galactoside-binding affinity. Accumulative evidence suggest that Gal-9 plays multifaceted roles in tumor biology. However, the prognostic significance of Gal-9 in solid cancer patients remains controversial. The objective of the study was to clarify the prognostic significance of Gal-9 in solid tumors via meta-analysis.Methods: We searched PubMed, Embase and the Cochrane library for studies that report the correlation between Gal-9 expression and prognosis or clinicopathological parameters in solid cancer patients from inception to October 2017, with no language restriction. We calculated pooled hazard ratio (HR) and 95% confidence interval (CI) to investigate the prognostic significance of Gal-9 expression in solid tumors. We also calculated Odds ratio (OR) to explore the association between Gal-9 expression and clinicopathological features.Results: We included Fourteen studies with 2326 patients in our meta-analysis. The synthetic results revealed that high Gal-9 expression indicated improved overall survival  but had no correlation with disease-free survival (DFS)/recurrence-free survival (RFS)  in solid tumors. In stratified analyses, high Gal-9 expression was significantly correlated with improved OS in hepatocellular carcinoma and colon cancer and with improved DFS/RFS in gastric cancer and non-small cell lung cancer. In addition, ethnicity and the method of data extraction didn’t affect the positive prognostic values of high Gal-9 expression. Moreover, high Gal-9 expression was significantly correlated with a smaller depth of invasion , an earlier histopathological stage , negative lymph node metastasis  and negative distal tumor metastasis .Conclusion: Gal-9 expression indicates beneficial outcome in patients with solid tumors and is correlated with the pathogenesis of solid tumors. Gal-9 may serve as a prognostic biomarker and an emerging therapeutic target against solid tumors. Cancer is one of the leading causes of death worldwide. Surgery, radiation and chemotherapy are the commonly recommended first-line therapies for most cancer cases. Nevertheless, these treatments are not beneficial to all patients, and this lack of benefit may be due to tumor heterogeneity . Moreovein vivo study, in which Gal-9 induced apoptosis and inhibited the growth of hepatocellular carcinoma (HCC) cells  cells . MoreoveC) cells . ParadoxC) cells . InitialC) cells . A subseC) cells . Gal-9 aC) cells . ConsistC) cells . RecentlC) cells . The divRecently, increasing attention has been given to the prognostic value of Gal-9 in cancer patients. However, whether Gal-9 has prognostic value in patients with solid tumors remains unclear. Some evidence indicates that high expression of Gal-9 contributes to a better outcome for various solid tumors . NeverthHence, a systematic analysis of the correlation between Gal-9 expression and the prognosis of solid cancer patients by means of a meta-analysis of existing available data is necessary. Herein, we assessed the correlation between Gal-9 expression and survival by pooling data from published publications. Consequently, we found that Gal-9 was a positive indicator in solid cancer patients. To the best of our knowledge, we report the first meta-analysis to clarify the prognostic implication of Gal-9 expression among solid cancer patients.This systematic review and meta-analysis was implemented following the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines .We systematically searched PubMed, Embase and the Cochrane library for literatures published up to October 2017, without restrictions on language or region. The following keywords were used to carry out the search:  AND . We also referred to the reference and citation of the retrieved publications.The following inclusion criteria were used in the meta-analysis: (1) the studies published as original articles; (2) the papers that investigated the correlation between Gal-9 expression and prognosis of solid cancer patients, such as overall survival (OS), disease-free survival (DFS), and recurrence-free survival (RFS) or clinicopathological features; (3) As to prognostic data, there were enough available data for directly extraction or indirectly estimate for hazard ratios (HR) and 95% confidence interval (CI); (3) the sample size of every study is more than fifty individuals; Studies were excluded if they failed to meet all criteria. For multiple publications reporting the same study or based on identical datasets, we used the most informative or most recent publication to avoid duplication. To ensure the reliability of the search results, two authors (XZ and LS) independently carried out the retrieval and screening in accordance with the standardized approach, and the consensus was reached by discussion.1 (Two authors (XZ and LS) independently reviewed all included studies and extracted the data with a standardized protocol. If the authors couldn’t reach a consensus, a third researcher made the final decision. Following are the extracted context: the clinicopathological characteristic; first author’s name; publication year; ethnicity of the patients; number of patients; cancer type; trial design; median age; cutoff value of high Gal-9 expression; median follow-up time and the range; use of multivariate or univariate model; outcome; method used to detect Gal-9 expression; specimen type and localization of Gal-9. If there was no available HR and 95%CI for direct extraction, we extract the survival data from Kaplan–Meier curves by the software Engauge Digitizer version 4.11 . If bothAlso, two authors (XZ and LS) independently evaluated the quality of the included studies according to the Newcastle-Ottawa scale (NOS) criteria, a widely used tool for the quality assessment of observational or non-randomized studies . Each st2 test and I2 statistic to evaluate the heterogeneity across studies  was used to evaluate the correlation between Gal-9 expression and clinicopathological parameters. We used χ studies . If P <  applied . If not, applied . We perf applied . For allFigure 1.We identified 351 studies by systematic literature search. After reviewing the title and abstract, 215 were excluded for the reason that they were irrelevant to the topic. Finally, we included 14 from the remaining 29 studies. . FollowiTable 1. These studies were published between 2005 and 2017. Twelve of the included studies reported a correlation between OS and Gal-9 expression, and six of the included studies further reported a relationship between DFS/RFS and Gal-9 expression. In addition, nine studies presented a correlation between clinicopathological features and Gal-9. These studies were carried out in 5 countries . Eight studies were prospective cohort trials, and six were retrospective cohort trials. Patients in all studies were reported to received resections. Regarding cancer type, studies related to HCC (n = 4) constituted the largest proportion among all included publications, followed by breast cancer (n = 2), gastric cancer (n = 2), non-small cell lung cancer (NSCLC) (n = 2), urinary tumors (n = 2), colon cancer (n = 1) and melanoma (n = 1). Most studies reported a localization of Gal-9 in cytoplasm, together with several studies reported an additional localization in nucleus or cell membrane or both. High Gal-9 expression levels across studies was defined by heterogeneous cutoff values, and the most common strategy was to regard the median score as the cutoff point. All studies comprised a median follow-up time ranging from 24.4 to 118 months. The most commonly used test method for Gal-9 was immunohistochemistry (IHC), with one study using qRT-PCR and another study using immunofluorescence staining (IFS). The collected HR were calculated with a multivariate model in nine studies and with a univariate model in five studies. Ten studies detected the Gal-9 expression with a Paraffin embedded block, one study utilized frozen tissue and four studies didn’t report their specimens specifically. The scores of all included studies were above five according to Newcastle-Ottawa scale (NOS), indicating that all studies were of high methodological quality (Table 2).We included data from 2326 patients in this meta-analysis. The main features of the 14 studies were summed up in P = 0.006) . This pooled meta-analysis was carried out by the random effects model on account of significant heterogeneity . To further explore the potential sources of heterogeneity, we utilized subgroup analyses, which is summarized in Figure 2B.Twelve studies comprising 2208 patients reported OS. This meta-analysis revealed that high Gal-9 expression was associated with improved OS among patients with various solid tumors . Nevertheless, no significant association was evident between Gal-9 expression and OS among patients with gastric cancer , NSCLC , urinary tumors . By the way, we couldn’t get a pooled HR from colon cancer or melanoma because there’s only a single study reporting the prognostic data for OS.Subgroup analyses regarding cancer type clarified that increased Gal-9 expression was significantly correlated with longer OS for patients with HCC , along with no heterogeneity  and might be less reliable. The results showed that high Gal-9 expression was associated with improved OS in studies extracting data directly , which was in line with the data extracting indirectly . Nevertheless, the pooled results were not significant in studies applying other detection method . Considering that IHC was the most widely used method in clinical practice, our results still made sense . In addition, pooled results were not significant in studies applying retrospective trial design , with significant heterogeneity . To further explore the difference between trial design in homogeneous population. We performed subgroup analyses by trial design in HCC with fixed-effects model. The results displayed that pooled HR in two subgroups were both significant  , and no significant heterogeneity was observed .Further, we performed subgroup analyses by ethnicity. The results revealed that Gal-9 predicted improved OS in Eastern Asian populations  and in Caucasian populations . We also performed subgroup analyses regarding the data extraction method, for the reason that the HRs and 95% P = 0.527) . Because significant heterogeneity was observed , a random effects model was applied to determine the summary of DFS/RFS.The HRs for DFS/RFS were available in six studies comprising a total of 814 patients. Unlike OS, Gal-9 expression had no significant correlation with DFS/RFS  or urinary tumors . We couldn’t get a pooled HR from gastric cancer or NSCLC because there’s only a single study reporting the prognostic data for OS.With respect to the stratified analysis by cancer type, no significant association was evident between Gal-9 expression and DFS/RFS for breast cancer  and trial design  had different effects on DFS/RFS. Moreover, no evident correlation was observed between Gal-9 expression level and DFS/RFS in any subgroup divided by ethnicity and the method of data extraction .Subgroup analyses revealed that the method of detection .In order to investigate the trends of the results, we also performed cumulative meta-analysis by publication year. The results indicated that the significant association between Gal-9 expression and OS became increasingly stable and that the CI narrowed since the first study performed by Figure 5B).As to DFS/RFS, the results of cumulative meta-analysis revealed that the association between Gal-9 expression and DFS/RFS became inconclusive since the Fu’s research. After two subsequent studies (Liu’s research and Grosset’s research) were reported, the CI narrowed, but the results remained inconclusive (P < 0.001), histopathological stage , metastasis of lymph node  and distal tumor metastasis  but not with sex, vascular invasion or tumor number (Table 3).To comprehensively analyze the role of Gal-9 expression as a biomarker in solid tumors, we investigated the association between Gal-9 expression and clinicopathological characteristics. Nine studies were included , from whFigure 6). In addition, Egger’s test  and Begg’s test  further confirmed no existence of publication bias.We combined funnel plots, Egger’s tests and Begg’s tests to assess whether a publication exist. Visual estimation of the funnel plots didn’t display evident asymmetry , indicating that this outlier is the source of statistical heterogeneity. After excluding Fu’s study, the remaining homogenous studies displayed a more significant correlation between high Gal-9 expression and better OS in solid tumors  , which further confirms the reliability of the results for OS.All studies were successively omitted to judge the impact of individual study on the combined results. As shown in Recently, multiple published studies have examined the prognostic implication of Gal-9 expression among patients with solid tumors. However, there exist controversies between the results of these studies. In the present meta-analysis, we showed that high Gal-9 expression was associated with better OS in solid cancer patients, while had no association between high Gal-9 expression and DFS/RFS. We found that in different cancer types, Gal-9 did not have a consistent effect on prognosis. High Gal-9 expression was significantly associated with better OS in HCC and colon cancer and better DFS/RFS in gastric cancer and NSCLC; however, the association remained inconclusive for other cancer types. Moreover, high Gal-9 expression was significantly correlated with a smaller depth of invasion, an earlier histopathological stage, negative lymph node metastasis and negative distal tumor metastasis. Overall, high Gal-9 expression in the tumor might improve prognosis, prevent recurrences of solid tumors, and be an emerging therapeutic target against solid tumors.Although Gal-9 has been investigated for over 20 years, the mechanism of its role involved in improving cancer patients’ survival remains unknown. One possible explanation is its role of enhancing cell aggregation. Accumulative evidence showed that endogenous expression or exogenous administration of Gal-9 in various tumor cells could enhance cell aggregation and adhesion . Meanwhiin vivo, therefore whether elevated Gal-9 expression in patients would prevent tumor metastasis in such a manner remains to be determined.Another possible explanation involves Gal-9 and its capability of inducing apoptosis. It is reported that addition of recombinant Gal-9 induced apoptosis of various malignant cells, such as hematological malignant cells , 2015, mMoreover, the anti-tumor immune response of Gal-9 is also a feasible explanation. Gal-9 has been reported to strengthen antitumor immunity of CD8+ T cells cooperated with DC cells through Gal-9-Tim-3 pathway . A similRegarding the cancer type, high expression of Gal-9 predicts favorable OS in patients with HCC and colon cancer. However, no significant correlation between Gal-9 and OS was observed for NSCLC, gastric cancer, urinary tumors and melanoma. Our finding that Gal-9 does not have the same prognostic value for different types of tumors indicates that Gal-9 might play different roles in different tumors depending on the type of cancer. For example, Gal-9 expression implies a trend toward a poor clinical outcome in urinary tumors, for both OS and DFS/RFS. This discrepant effect may be ascribed to the roles of different Gal-9 variants. As for the subgroup analyses, high Gal-9 expression had a positive impact on prognosis in prospective studies, with concordant results for both OS and DFS/RFS. In contrast, as to retrospective studies, high Gal-9 expression showed no significant association with OS or DFS/RFS. Given that the results contained a significant heterogeneity in retrospective studies, we were cautious about the results and further performed a subgroup analysis for trial design in homogeneous population of patients with HCC. Consequently, we found that in HCC, the correlation between Gal-9 expression and OS was both significant.We found that the prognostic value of Gal-9 for OS and DFS/RFS was not consistent. The results of a cumulative meta-analysis may provide a clue toward understanding the inconsistent results between OS and DFS/RFS. In contrast to OS, only two additional studies reporting DFS/RFS were reported since Fu’s study, and these reports narrowed the CI, but the results remained inconclusive. Therefore, we presume that as more high-quality studies reporting the correlation between Gal-9 expression and the survival of solid cancer patients become available, the data for the OS and DFS/RFS of solid tumor patients may consistently show a correlation with Gal-9 expression. In addition, as OS is well recognized as a more meaningful parameter than DFS/RFS in the survival analysis of cancer patients, we believe our study provides meaningful statistical evidence supporting the important prognostic value of Gal-9 as a favorable predictor in solid cancer patients.In the last few decades, therapeutic strategies, such as the administration of recombinant protease-resistant Gal-9, have been implemented in preclinical studies. The results show that the administration of recombinant Gal-9 can prevent tumor progression by inducing the apoptosis of cells from various malignancies in murine models . Our metIn the meantime, this meta-analysis is limited by several aspects. First, the results of the meta-analysis of the OS and RFS/PFS data show heterogeneity, which cannot be eliminated. We found that Fu’s study was the source of the statistical heterogeneity. Because Fu’s study investigated clear-cell renal cell carcinoma, which was distinct from other included studies. We prefer that the heterogeneity was mainly result from cancer type, probably because of the different localization of Gal-9 and different Gal-9 variants in various cancer type. In addition, there might be many other reasons for potential heterogeneity, such as the method used, sample size, follow-up time, specimen type and cutoff value, which varied among studies. However, we attempted to reduce the impact of heterogeneity through subgroup analyses. By investigating different groups of patients with homogeneous characteristics for different cancers, the results would be translatable to the clinic. Second, this meta-analysis only includes studies from Southeast Asia, North America, and New Zealand, and therefore the prognostic values of Gal-9 may not be generally applied worldwide. Finally, studies that report negative results may not have been published, which may potentially exaggerate the significance of the association between Gal-9 expression and improved prognosis. Thus, further high-quality studies regarding Gal-9 are required to resolve the abovementioned limitations.Despite these limitations, this meta-analysis is of great significance in that it demonstrates the association between high Gal-9 expression and a better prognosis for solid tumor patients and indicates Gal-9 as a promising target against cancer therapy.XZ designed the study, retrieved the relevant papers and data, performed the data analyses, and wrote the manuscript. LS designed the study, performed the data analyses, and participated in the writing of manuscript. DJ designed the study, assisted with the data analyses, and participated in the writing of manuscript. GX assisted with the data analyses and participated in the writing of manuscript. JmZ designed the study and retrieved the relevant papers and data. LL and JjZ designed the study and retrieved the relevant papers and data. ZY retrieved the relevant papers and data and assisted with the data analyses. HL designed the study, assisted with the writing of manuscript, and supervised the study. The final approval of all authors was reached.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The recent advances in identification of the molecular mechanisms related to tumorigenesis and angiogenesis, along with the understanding of molecular alterations involved in renal cell carcinoma (RCC) pathogenesis, has allowed the development of several new drugs which have revolutionized the treatment of metastatic renal cell carcinoma (mRCC).This process has resulted in clinically significant improvements in median overall survival and an increasing number of patients undergoes two or even three lines of therapy. Therefore, it is necessary a long-term perspective of the treatment: planning a sequential and personalized therapeutic strategy to improve clinical outcome, the potential to achieve long-term response, and to preserve quality of life (QOL), minimizing treatment-related toxicity and transforming mRCC into a chronically treatable condition.Because of the challenges still encountered to draw an optimal therapeutic sequence, the main focus of this article will be to propose the optimal sequencing of existing, approved, oral targeted agents for the treatment of mRCC using evidence-based data along with the knowledge available on the tumor behavior and mechanisms of resistance to anti-angiogenic treatment to provide complementary information and to help the clinicians to maximize the effectiveness of targeted agents in the treatment of mRCC. Renal cell carcinoma (RCC) has seen in last years a rapid and continuous increase of new cases . ApproxiThe treatment of metastatic renal cell carcinoma (mRCC) has been revolutionized over the past 10 years, with the development of several new drugs, whose use has resulted in clinically significant improvements in median overall survival compared with the previous treatment options, limited to cytokines such as interleukin-2 and interferon α (IFN-α) . These iThe recent advances in identification of the molecular mechanisms related to tumorigenesis, angiogenesis, cell growth and proliferation, along with the understanding of molecular alterations involved in RCC pathogenesis -12, alloBased on these known signaling pathways, targeted systemic treatments have been specifically designed and approved: the humanized anti-VEGF monoclonal antibody bevacizumab , 14 in cThe introduction of these several new targeted agents raises many questions on how to use them in order to maximize their effectiveness and offer to the patient a greater number of therapeutic opportunities, in the most appropriate setting -18: thesThe data available often derived from studies planned to obtain drug registration, where different agents are compared with placebo or IFN-α, with little indirect differences among them in terms of progression-free survival (PFS) and overall survival (OS) .Furthermore, although targeted agents have demonstrated PFS benefit, durable responses are rare and most patients with mRCC eventually experience disease progression , 22.Therefore, in the new era of targeted therapies for the treatment of mRCC, it would be appropriate to plan a sequential and personalized therapeutic strategy to improve clinical outcome, to potentially achieve long-term response, and to preserve quality of life (QOL), minimizing treatment-related toxicity and transforming mRCC into a chronically treatable condition.The main focus of this article will be to propose the optimal sequencing of existing, approved, oral targeted agents for the treatment of metastatic renal clear cell carcinoma, the most frequent subtype of sporadic RCC (70-85%) , althougIn the first-line treatment of mRCC population VEGF-targeted agents have improved patient outcomes compared with the previous cytokines-based standard of care.Current evidence-based guidelines recommend three drugs as first-line treatment for patients with good-intermediate risk: bevacizumab (combined with IFN-α), sunitinib and pazopanib .All three agents have demonstrated PFS prolongation, in phase III trials over either IFN-α or placebo -32.First-line temsirolimus has demonstred activity in patients with poor prognosis .Sunitinib, a multitarget oral TKI that inhibits the VEGF and PDGF receptors, was compared to IFN-α in a randomized trial of untreated patients, showing improved median PFS (11 vs 5 months) and median OS (26 vs 21 months) and a response rate over 40% . These rPazopanib, another oral multitarget TKI with inhibitory activity against VEGF and PDGF receptor, was compared, head-to-head, with sunitinib, in a randomized non-inferiority trial in first-line setting (COMPARZ): the pazopanib and sunitinib groups had similar median PFS  .There were no significant differences in response rates and OS between the two agents  .The analyses of QOL were in favor of pazopanib.The improved safety profile and QOL of pazopanib were confirmed by “The Preference Study of Pazopanib Versus Sunitinib in Advanced or Metastatic Kidney Cancer” (PISCES) which evaluated patients' preference for pazopanib or sunitinib in treatment-naive patients with mRCC: patients were randomized to pazopanib for 10 weeks, and after 2 weeks of washout, to sunitinib for 10 weeks, or to the opposite sequence.At 22 weeks, patients completed a questionnaire assessing their preferences: significantly more patients preferred Pazopanib (70%) over Sunitinib (22%); 8% did not prefer any agent .These findings have shown Sunitinib and Pazopanib are agents with similar efficacy in first-line treatment, but the use of Pazopanib as first line therapy, in patient with good-intermediate risk mRCC, seems more convenient for his better safety profile.Most patients initially treated with an anti-VEGF therapy in first-line setting, eventually develop resistance and subsequent disease progression. , 38Current post first-line therapies for mRCC, target the VEGFr and mTOR pathways. Approved treatment options include sorafenib, axitinib and everolimus. Sunitinib is also an option if it was not previously administered .Sorafenib is a multikinase inhibitor of multiple growth factor receptors as VEGFr, PDGFr, Flt-3 and c-Kit and Raf-1, a member of RAF/MEK/ERK signaling pathway ; axitiniThe choice of a second-line treatment represents a controversy about the optimal sequence to be proposed: in the VEGFr TKI-resistant setting, is it better to continue treatment with a different VEGFr-TKI or to overcome cross-resistance by switching to a drug with a different mechanism of action? (i.e. a mTORi)? , 44, 45.Although switching to a different signalling pathway appears to be rational, current data support the use of both mTORi and TKI: they are associated with similar median progression-free survival (PFS) of about 4-5 months.However, while axitinib is compared with another TKI in the AXIS trial, everolimus, in the RECORD-1 trial, is compared with placebo.The AXIS trial is a phase III RCT study comparing axitinib and sorafenib as second line treatment in patients with advanced clear cell RCC, after a prior systemic regimen including sunitinib (54%), cytokines (35%), bevacizumab (8%) or temsirolimus (3%). Although in the overall population, patients treated with axitinib experienced a significantly longer PFS than those on sorafenib , in the patients subgroup with disease progression on sunitinib, PFS was shorter with both axitinib and sorafenib  than in patients pretreated with cytokines  .No OS differences in either axitinib and sorafenib group was detected , 48.As regards mTORi after the VEGFr inhibition-based therapy, data are provided by the RECORD-1 phase trial. In this study patients progressing on first line therapy were randomized to receive everolimus or placebo.One or two previous VEGFr inhibition based therapies were permitted, including bevacizumab and cytokines. For this reason the results should be interpreted with caution because only 21% of study population were pure second-line therapy progressing after first-line treatment with sunitinib. In this subgroup median PFS was 4.6 with everolimus vs 1.8 months with placebo. 53% of patients received one TKI and cytokine (PFS 5.2 vs 1.8 months) and 26% were third line after two TKIs (PFS 4 vs 1.8 months) -52.Since number and type of previous treatments are crucial, the subsequent RECORD-4 was planned, a phase II trial of only second-line everolimus, which enrolled patients into 3 subgroups based on prior first-line therapy: sunitinib, other anti-VEGF agents  or cytokines. Median PFS was 5.7 months (3.7-11.3) with prior sunitinib, 7.8 months (5.7-11.0) with prior other anti-VEGFs and 12.9 months (2.6-NE) with prior cytokines (preliminary data) .To date there is no direct comparison between the two drugs, axitinib and everolimus, in the post-VEGFr-TKI second-line setting.Making an indirect comparison between the two mentioned studies in order to identify a drug supported by further evidences, we can summarize that:- The median PFS with axitinib and everolimus in patients with disease progression after sunitinib in first line setting is similar (4.8 months axitinib vs 4.6 months everolimus); but in the AXIS trial axitinib has an active comparator (sorafenib), while everolimus is compared with placebo;- In the AXIS trial all population of patients treated with axitinib or sorafenib were pure second-line; in RECORD-1 trial patients were previously treated with one or also two lines of therapy. The final data from RECORD-4, with only second-line everolimus, are still expected.- Patients randomized to the axitinib arm in the AXIS trial, in absence of adverse events, could have undergone an increase in dose , while patients in Everolimus arm in RECORD-1 trial did not undergo this dose escalation.- No OS difference in AXIS trial was detected. In RECORD-1, patients in treatment with placebo were allowed to cross over to everolimus upon disease progression. Accordingly, OS data are conditioned by the possibility of cross-over.The first trial that provides a direct comparison between agents with different mechanisms of action is the INTORSECT study; in this trial mRCC patients who had progressed on first-line sunitinib, were randomized to temsirolimus, an mTORi, or sorafenib, a VEGFr TKI.p = 0.014) [The difference in median PFS was no statistically significant; median OS was in favor of sorafenib  .This could suggest an advantage for the VEGFr TKI-VEGFr TKI sequence compared to VEGFr TKI-mTORi sequence, but it really does not clarify the controversy since it compares two treatments that do not represent the best option in the second-line setting.A study investigating sequential therapies is the RECORD-3 trial. In this phase 2 study, patients were randomized to first line everolimus followed by second-line sunitinib or the opposite sequence, sunitinib followed by everolimus. Results of final analysis do not lead to any change in the standard sequence of sunitinib on first-line followed in second-line by everolimus at disease progression ; RECORD-1 and preliminary RECORD-4 results show the clinical benefit of everolimus in the second line setting but they haven't an active comparator; results of INTORSECT trial that compared a VEGF-TKI with an mTORi as second-line (TKI-mTORi sequence), are not directly transferable in clinical practice.We can conclude that both everolimus and axitinib are effective options after first-line VEGFr-TKI failure, but the absence of head-to-head comparisons, doesn't solve the controversy for the choice of treatment at present.Sorafenib might be still considered as an alternative option.Most data related to third-line treatments result from retrospective cohort studies and subgroup analysis: comparative retrospective assessment of the sequence TKI-TKI-mTORi versus TKI-mTORi-TKI suggests superiority of TKI-TKI-mTORi ; subgroup = 0.063). Interim OS analysis was also similar in the two arms  [Treatment in the third-line setting was assessed for the first time in the GOLD trial. In this phase 3 study, patients who received one previous VEGF-TKI inhibitor and one previous mTORi were randomly assigned to receive dovitinib  or sorafenib: PFS difference between sorafenib and dovitinib was not statistically significant  .This study support the re-treatment with VEGF TKI in third-line, after one previous TKI-mTORi sequence (TKI-mTORi-TKI sequence).There is also evidence that in patients who have progressed on prior targeted therapy with sunitinib and another TKI or mTORi, the “re-challenge” with sunitinib seems to have a clinical benefit, although with shorter progression-free survival with respect to the first-line treatment -63.These data support the hypothesis that resistance to targeted therapy could be transient.The optimal sequence of target agent for the treatment of mRCC is not well defined.Assessing the evidence from latest trial, using currently approved oral VEGFR-TKI and mTORi for mRCC, we developed an algorithm, highlighting the strengths of each agents to support his eventual choice Figure .At the time of the choice of first-line therapy we must primarily take into account primarily the potential efficacy of the drug for the individual patient or the subsequent treatment options?Given the increased survival of patients with advanced disease and that an increasing number of patient is able to undergoes two or even three lines of therapy, it is necessary a long-term vision and, therefore, planning is better than improvising.Basing on current drug labels, we can choose between two sequential strategies, which is the best: TKI/TKI/mTORi sequence or TKI/mTORi/TKI sequence?Unfortunately, to date there are no consistent clinical data to support a clear and strong a sequence rather than another.Considering the individual drugs, with reference to the first line of treatment, the oral options available today are sunitinib and pazopanib. Both of them have shown equal effectiveness, so it could be use of pazopanib for its better tolerability.In second line both everolimus and axitinib are associated with similar median PFS: everolimus as a second and third-line therapy show an increase of PFS versus placebo in the soubgroup analysis within RECORD-1 trial.However the data from AXIS and INTORSECT trials suggested that patients treated with previous TKI may respond to another TKI in second line and cross-resistance may not appear.To underline that axitinib has an active comparator (sorafenib), as opposed to everolimus (compared with placebo).Unfortunately it is not possible today to administer today axitinib after pazopanib and this precludes the possibility of using a sequence potentially very effective. Given the similarity between sunitinib and pazopanib, it would be desirable that this may become feasible in the near future.Treatment for third-line setting depends largely on the choices made previously Figure .In these TKI-refractory patients it would be appropriate to draw clinical trials with direct comparisons between the drugs, comparing a TKI such as axitinib with a mTORi such as everolimus. This would add important data to choose the best therapeutic sequence.However, for the decision-making process, should the clinicians consider only evidences arising from individual clinical trials or should they consider also other factors, such as those addressing resistance mechanisms and tumor behavior?Because of the challenges still encountered to draw an optimal therapeutic sequence, the integration of evidence-based data with knowledge available about tumor biology could provide complementary information and help clinicians to maximize the effectiveness of targeted agents in the treatment of mRCC.We have tried to do it starting from the central role of angiogenesis in the pathogenesis of renal tumor. RCC is a hyper-vascular tumor and VEGF have a key role as mediator of angiogenesis in this tumor , 65.Anti-angiogenic drugs typically have transitory efficacy: they produce more or less durable responses, followed by disease progression due to the development of resistance to therapy , 38.From a clinical point of view, indeed, in same patients anti-angiogenic drugs can achieve a good control of the disease for long periods, conversely in other patients there is a very rapid disease progression , 67.We know from literature that resistance to anti-angiogenic treatment is mainly caused by the onset of adaptive mechanisms of cancer cells.As a response to the inhibition of angiogenesis by VEGF pathway inhibitors, tumor cells promote the transcription of pro-angiogenic factors , 69, theThese resistance mechanism may be mediated by the activation of angiogenesis-related pathways independent of VEGFR and PDGFR, which are inhibited by first-line anti-angiogenic drugs , 77.For instance the slow progression under the treatment with sunitinib or pazopanib could be associated with a low capacity to develop adaptive mechanism.We suppose that in patients with slow progression tumor is still VEGF-dependent.Therefore we might overcome this transitory resistance by a wider spectrum of receptor inhibition  or by a stronger inhibition through more potent drugs .Since AXIS trial provided strong data about the superiority of axitinib over sorafenib in this setting of patients, axitinib should be the best option as second-line treatment for patients experiencing slow progression during firs-line sunitinib or pazopanib.The patients who received a TKI and subsequently a different TKI, could undergo a third-line treatment with a further TKI or the mTORi everolimus.However the use of a third-line TKI (i.e. sunitinib or sorafenib) after two previous lines of TKIs is not supported by strong evidence-based data. Conversely the use of everolimus after TKIs in first and second-line treatment met favorable outcomes in RECORD-1 trial.Patients who develop a rapid progression during firs-line TKI may have an intrinsic primary resistance to anti-angiogenic drugs, so that the resistance mechanism would not be adaptive. Thus in these patients we should address a different pathway.mTOR inhibition by everolimus could be the most valid option as second-line treatment.Accordingly for third-line treatment in this subset of patients a change of mechanism of action appears more reasonable. So after first-line TKI and second-line mTORi, a different TKI could be chosen as third-line treatment. Sunitinib is supported by retrospective studies about rechallange. As an alternative option sorafenib is sustained by data from the GOLD study Figure .Ongoing trials and other recently concluded -83 contiRecently, Immunoncology, represent a new and promising frontier for many malignancies, such as RCC.The aim of Immunotherapy is to improve the organism's competence to direct the immune system against cancer cells.The normal immune system requires T cell activation for its activity; some immunomodulatory molecules such as cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and programmed death-1 (PD-1), are involved in the mechanism for T cells activation , 85.They are inhibitory molecules, dysregulated in several cancer, resulting in defective ability of immune system to react against tumor cells.As a consequence, checkpoint inhibitors have been developed and are under evaluation -92.Nivolumab, a fully human IgG4 monoconal antibody directed against PD1, demonstrated effectiveness in patients with metastatic melanoma and non-small cell lung cancer.In a phase II trial three nivolumab doses  have been assessed in mRCC patients pretreated with VEGFR TKIs, mTORi and immunotherapy. No dose-response relationship was detected for PFS; median OS was 18.2 months by the 0.3 mg/kg dose and was not reached by other doses .Basing on data from CheckMate 016 trial, a phase I study of nivolumab + ipilimumab (a human CTLA-4-blocking antibody) in patients with mRCC , 95, a pRecently the results of two pivotal, randomized, phase 3 trial, about nivolumab and cabozantinib, respectively, compared with everolimus have been published: both provide substantial benefit in patients treated with previous VEGF targeted therapy.The first, CheckMate 025, an open-label phase 3 trial, investigates nivolumab versus everolimus in patients who have failed one or more anti-VEGF therapy.The median PFS was similar (4.6 months with nivolumab and 4.4 months with everolimus) while Nivolumab showed considerable OS advantage and tolerability over everolimus (OS 25.0 months with nivolumab and 19.6 months with everolimus) .The second randomised phase 3 trial (METEOR) compared everolimus with cabozantinib, a small-molecule tyrosine kinase inhibitor of MET, VEGFR2 and AXL. The setting tested was the second-line after failure of one line or more of VEGF targeted therapy.Cabozantinib showed superior PFS (7.4 mo) versus everolimus (3.8 mo). Grade 3 or 4 adverse events were more numerous in cabozantinib sub-group (68%) than everolimus-group (58%).In the interim analysis is evident a trend of cabozantinib toward an OS advantage but the final planned mature OS data is expected in 2016 .Both these new promising drugs, nivolumab and cabozantinib, not yet be used in the clinical practice. With their next introduction, a promising therapeutic algorithm will be available in the near future Figure .Physicians now have many more tools to improve outcomes of mRCC.Planning the appropriate and personalized sequence of targeted agents may help to achieve the best clinical outcome in these patients by the maximum number of available targeted agents.Given the short differences in median PFS observed between treatments, in the absence of clinical trials with direct comparisons between the drugs, the optimal therapeutic sequence is still under investigation.Our opinion is that, in addition to evidences from clinical trials, other factors must be also considered for decision making such as the safety profile of drugs , 99, comTo integrate evidence-based data from trials with clinical observations about tumor biology could provide complementary information and help clinicians to individualize the treatment and to generate the optimal sequence for each patient.New and promising agents, such as immune based therapies, continue to be explored; data from ongoing trial will help to develop more effective therapeutic strategies and new algorithms.


Mycobacterium tuberculosis (SAT-TB assay) was designed to diagnose rapidly pulmonary tuberculosis (PTB). Unfortunately, the diagnostic advantage is unclear from previous small sample studies. In the current inquiry, a large sample size was used to reevaluate the clinical accuracy of the SAT-TB assay using sputum specimens.The AmpSure simultaneous amplification and testing method for the detection of 2 = 1327.437; P = 0.000), wheresa significantly lower than that of sputum culture (89.0%) . The specificity of SAT-TB was significantly higher than that of sputum smears (96.3%) , whereas no significant difference was found compared with sputum cultures (99.6%) .A total of 3608 patients with suspected PTB were enrolled prospectively for diagnosis from sputum specimens using the SAT-TB assay. Of these, 2457 had a definite diagnosis of PTB confirmed by positive microbiology, or pathologic findings of TB in the lung, or clinical diagnosis of active PTB following anti-TB treatment with a favorable response. The sensitivity, specificity and accuracy of the SAT-TB assay were 75.8%, 100%, and 80.2%, respectively. The sensitivity of SAT-TB was significantly higher than that of the sputum smear (23.8%) (XPositive results in the SAT-TB assay using sputum specimens indicates that active PTB is present and anti-TB treatment is strongly recommended regardless of smear and culture test results. Simultaneous amplification and testing method for detection of Mycobacterium tuberculosis is an accurate, cheap, and rapid method for PTB diagnosis. The Chinese government is making strenuous efforts to provide rapid diagnosis and treatment facilities through national support and project grants. At present, the diagnosis of pulmonary TB is routinely made by bacteriologic verification from sputum or bronchial alveolar lavage fluid specimens.5 The acid-fast bacilli (AFB) smear test is the most commonly used method and offers rapid results, but has the disadvantage of poor sensitivity and does not discriminate TB from nontuberculous mycobacterial (NTM) infections.6 Mycobacterium culture is the gold standard for confirming TB but can take up to 6 to 8 weeks to obtain the results, rendering this diagnostic method inconvenient in routine clinical practice. Therefore, diagnostic capacity remains limited, especially in TB endemic countries like China but the development and adoption of new diagnostic tools will help to accelerate the diagnosis of TB.Tuberculosis (TB) is a major public health problem worldwide. According to data from the World Health Organization (WHO), approximately 9 million people were newly diagnosed with active TB in 2013 and 1.5 million people died from the disease.7 One previous study of SAT-TB assay focused on its accuracy with limited samples and demonstrated that its overall sensitivity for the diagnosis of PTB was 67.7%.8 Sensitivities for smear negative specimens were reported to be 39.2% and 93%.9 Although these studies were all carried out under controlled, standardized conditions, the results showed marked differences making it necessary to reevaluate the SAT-TB assay sensitivity with many more samples.The AmpSure simultaneous amplification and testing method for detection of Mycobacterium tuberculosis complex by using isothermal RNA amplification and real-time fluorescence detection has recently been introduced into a small number of Chinese hospitals for the early diagnosis of TB. It combines the technologies of nucleic acid isolation, simultaneous amplification, and testing with fluorescence-labeled hybridization probes. It is faster than conventional bacteriological methods and, importantly, it has excellent reproducibility.Thus, the current large sample study using sputum from suspected active PTB and HIV-negative adult patients was undertaken to assess the sensitivity, specificity, and accuracy of SAT-TB assays in real life situations in high TB burdened regions of China.The Ethics Committee of Shanghai Pulmonary Hospital approved this prospective study and written informed consent was obtained from each participant before enrollment. All adult patients admitted to the Shanghai Pulmonary Hospital were screened between January 2014 and April 2015. Inclusion criteria were: suspected active PTB; adults (≥17 years); no previous history of anti-TB treatment; negative HIV status. Exclusion criteria were: inability to provide sputum for examinations; no finalized diagnosis after examination and treatment (obscure diagnosis). A standard questionnaire was completed by each patient before enrollment, including basic demographic data, history of TB contacts, previous TB, current TB symptoms, anti-TB treatments as well as underlying diseases and concurrent therapies. All patients who were suspected of having active TB were tested using the SAT-TB assay, acid-fast bacilli AFB smear, and culture tests at enrollment, in addition to the T-SPOT. Interferon-gamma release assay as well as physical, pathologic, and radiographic examinations.Bacteriologic diagnosis based on positive MTB in culturesPathologic diagnosis of PTB based on analyses of resected lung tissuesActive PTB was confirmed when radiographic or chest computed tomography (CT) manifestations were in accordance with PTB pattern and accompanied by one of the following criteria at enrollment or later during the study period:If the patients did not meet one of the above 2 criteria, clinical diagnosis was confirmed by meeting all of the following categories: Patients with signs or symptoms of PTB and typical manifestation of TB on chest computed tomography; Patients received anti-TB medication for 2 months with a favorable response, based on improved signs or symptoms and chest CT results. For these cases chest CT was reviewed every 2 months until the patients were treated for 6 months. The clinical experts decided the final diagnosis of PTB, and they were blinded to the results of the SAT-TB assays; no evidence of non-TB lung disease based on laboratory and radiographic data; positive T-SPOT. Interferon-gamma release assays.We excluded patients who had failed to meet the above 4 categories (obscure diagnosis). An evidence-based strategy was used to ensure that individuals with PTB were correctly diagnosed and had access to appropriate treatment as soon as possible, and also to ensure the quality of every step in the diagnostic process.Clinical information, including treatment processes and the discharge diagnosis, were recorded from patients’ medical charts by investigators. For patients whose diagnosis was not established during hospitalization, an outpatient interview was conducted once a month after discharge to obtain the final diagnosis. At the end of the monthly follow-ups for 6 months, each patient was classified into one of 3 predefined clinical categories, namely active PTB, non-TB pulmonary disease or without a final diagnosis.10 The detection threshold of a positive sputum smear was smear positive grade 1. All tests were performed at the tuberculosis reference laboratory in Shanghai Pulmonary Hospital, and quality control was routinely performed.Morning sputum specimens were obtained before treatment. Sputum samples were routinely tested by smear fluorescence microscopy and by culture strain identification Lowenstein–Jensen medium, as well as the Bactec MGIT 960 System  according to World Health Organization guidelines.Simultaneous amplification and testing method for detection of Mycobacterium tuberculosis was performed on sputum samples according to the manufacturer's instructions . Sputum specimens were collected and transported to the laboratory, and then processed after being dissolved in sodium hydroxide. During the SAT- TB assay, the target 16S rRNA from MTB combined with T7 promoter fused primers was reverse transcribed into cDNA by Moloney murine leukemia virus reverse transcriptase. Then, multiple RNA copies were generated from the cDNA copy using T7 RNA polymerase, which were again transcribed into the T7 promoter, including cDNAs. The accumulated cDNAs were detected by fluorescence-labeled specific probes.9 a sample size of 100 patients was required to conduct a power test at the 95% level. Sensitivity, specificity, positive and negative predictive values, as well as accuracy rates of the SAT-TB assay, sputum smear and cultures were calculated. Numerical variables are shown as the mean ± standard deviation. The categorical variables were analyzed using Fisher exact or Pearson χ2 tests where appropriate and 2-tailed tests were used. The concordance of agreement between SAT-TB assays and smear and culture data was assessed using Cohen kappa test . P values ≤0.05 were considered to be statistically significant.Statistical analysis was conducted using SPSS ver. 18.0 software . Assuming a positive rate of 93% for the SAT-TB assay in bacteriologically and clinically confirmed PTB patients,A total of 3608 hospitalized patients with suspected active PTB were prospectively enrolled into the study.A total of 589 patients were excluded because of obscure diagnosis, 12 patients were excluded because of previously diagnosed PTB and 7 patients were excluded because of lost to follow up. When patients with obscure diagnosis had been treated for 2 months with anti-TB agents without improved chest CT results, the anti-TB agent applications were stopped. In these 589 patients, no individual was SAT-TB positive and no sputum smear AFB and mycobacterial culture test was positive. Finally, the data from 3000 patients were analyzed. According to comprehensive evaluations and follow-ups, 2457 patients (81.9%) were diagnosed with PTB and 543 patients (18.1%) with non-TB pulmonary disease. Among the 2457 active PTB patients, 2187 (89.0%) were confirmed by microbiology, 174 subjects (7.1%) by clinical diagnosis, and 96 subjects (3.9%) by pathologic diagnosis , whereas significantly lower than that of sputum cultures (89.0%) . The specificity of SAT-TB (100%) for the diagnosis of PTB was significantly higher than that of sputum smears (96.3%) , whereas there was no significant difference with sputum cultures (99.6%)  (Tables The sensitivity of SAT-TB (75.8%) for the diagnosis of PTB was significantly higher than that of sputum smears 23.8%) . Of the 1872 smear-negative PTB patients, the sensitivity of the SAT-TB assay was 72.3%, which was significantly lower than that of sputum cultures .For the 585 smear-positive PTB patients, the sensitivity of the SAT-TB assay was 86.8% and was significantly lower than that of sputum cultures . In the 552 subjects whose sputum smears and cultures were both positive, the SAT-TB positivity rate was 91.5% (505/552) and in the entire culture positive group, SAT-TB detected 1848 of 2187 patients (84.5%). In the 33 solely smear positive cases, only 3 (9.1%) were also deemed positive with the SAT-TB test. Notably, SAT-TB detected 11 PTB cases, which were negative for both sputum smears and culture tests  was found between the SAT-TB assay and sputum cultures , indicating suboptimal agreement between the 2 tests.A moderate level of agreement  assay was introduced recently in China, but the high cost makes it unaffordable as a routine test,14 because the price is $100 in Shanghai public markets and not covered by the public medical insurance, whereas the cost for a SAT-TB assay is $30 and under medical reimbursement coverage. The SAT-TB assay provides another unique advantage over PCR, as the detection target is rRNA. RNA is much more labile outside the reaction tube than DNA amplification products. Because PCR detection commonly experiences contamination problems, the SAT-TB assay can reduce the risk of laboratory contamination and false-positive results. The sensitivity and negative predictive value of SAT-TB assay were lower than that of other TB RNA PCR tests available on the market .15 The MTD test, however, requires expensive specialized detection equipment, which prevents the assay from being widely adopted in resource-limited settings. The SAT-TB assay can be performed on real-time PCR instruments, which can be found in most clinical laboratories.Many studies have demonstrated that PCR tests are routinely used for molecular diagnostic, for its rapidity and relatively high sensitivity and specificity, compared with conventional smear or culture tests. False-positive and false-negative results, however, are unavoidable with the current technology.8 but in their report the culture sensitivity was only 61.7%. In contrast, Fan et al9 reported sensitivities of 90% for bronchial washing fluid and 93% for sputum samples, with only slight differences to culture tests, and the sensitivity of the SAT-TB assay in smear-negative patients in their study was much higher than that in the current study (93.0% versus 72.3%). There are considerable sensitivity differences of SAT-TB tests, but it should be noted that the culture sensitivities differed in all studies using the same culture test method (Bactec MGIT 960). Fan et al tried to explain the SAT-TB sensitivity limitation with peripheral localized lesions, which would also explain the different results of the culture detection method. On the contrary, for nucleic acid amplification methods the presence of inhibitors of enzymatic amplification, a low number of mycobacteria, and/or an unequal distribution in the test suspension, are proposed reasons for sensitivity restrictions,16 which might also be the case for the SAT-TB method.The sensitivity of the SAT-TB test in our study was 75.8% versus 89% for culture detection, which was somewhat higher than the 67.6% reported by Cui et al,Our research differs from previous work in terms of its sample size and design. The 3000-cohort size was much larger than that of previous studies , so the bulk of our data is likely to be more objective and accurate. In addition, on repeated tests of sputum smears, the SAT-TB assay was performed once without knowing the status of the smear results, which often occurs in clinical practice.Twenty patients with NTM lung disease were identified as false-positives by sputum smears but were negative according to the SAT-TB assay results, which showed a 0.743 agreement with culture methods. The current findings suggest that the SAT-TB assay is not only more sensitive for detecting MTB in sputum than a sputum smear test but also might alert physicians to NTM lung diseases in patients with a positive smear and a negative SAT-TB assay result. As SAT-TB assay results can be accomplished within 120 minutes, when a sputum sample is both SAT-TB assay and smear positive, it is recommended that anti-TB treatment be started in clinically suspected PTB without waiting for culture results (6–8 weeks). If a sputum sample is SAT-TB assay positive and smear negative, anti-TB treatment is still strongly recommended regardless of the results of smear and culture tests. If a sputum sample is SAT-TB assay negative and smear positive, tuberculosis could be a possible result of the diagnosis and anti-TB drugs are recommended as the first treatment. Subsequently, when the sputum culture results identify the disease as NTM, the treatment should be changed to anti-NTM medications. Conversely, cases in which both the SAT-TB assay and smear test results are negative, other pulmonary diseases should be considered first.Our study is the first large cohort study (3000 patients) that has evaluated the SAT-TB assay in clinical practice. We found that the SAT-TB assay of sputum specimens could increase the TB diagnostic yield significantly, and is more useful than a sputum smear test. It is an accurate diagnostic method for PTB, especially in developing countries because of its low cost and rapidity. On the contrary, its low negative predictive value limits its usability to exclude the diagnosis of PTB and therefore SAT-TB-negative specimens still require conventional culture tests (solid or liquid) and specification. In addition, currently available technologies are not mutually exclusive.As various PCR methods for the diagnosis of TB are introduced, verification and comparison will be necessary. Further multicenter studies are needed to confirm unequivocally the findings of the current study.


Dual renin angiotensin system (RAS) blockade using angiotensin-receptor blockers (ARBs) in combination with angiotensin converting enzyme inhibitors (ACEIs) is reported to improve proteinuria in both diabetic and non-diabetic patients. However, its renoprotective effect and safety remain uncertain in patients with advanced chronic kidney disease (CKD). From January 1, 2000 through June 30, 2009, we enrolled 14,117 pre-dialytic stage 5 CKD patients with serum creatinine >6mg/dL and hematocrit <28% under the treatment with erythropoiesis stimulating agents and RAS blockade. We used Cox proportional hazards regression models to estimate the hazard ratios (HRs) against the commencement of long-term dialysis and all-cause mortality for ACEI/ARB users. Over a median follow-up of 7 months, 9,867 patients (69.9%) required long-term dialysis and 2,805 (19.9%) died before progression to end-stage renal disease requiring dialysis. In comparison with the ARB-only users, dual blockade with ACEIs and ARBs was associated with a significantly higher risk of (1) death in all CKD patients  and in diabetic subgroup ; (2) composite endpoint of long-term dialysis or death in diabetic subgroup ; (3) hyperkalemia-associated hospitalization in non-diabetic subgroup . However, ACEIs users were associated with higher mortality than ARBs users in all CKD patients  and in diabetic subgroup . Monotherapy of RAS blockade, especially ARB, is more effective and safer than dual RAS blockade in pre-dialytic stage 5 CKD patients. Several investigations have documented its benefit for renal protection to the patients with early CKD (serum creatinine level: 1.5–3.0 mg/dl) and non-0 mg/dl). Thus, thvs monotherapy) with the risk of long-term dialysis and/or death in this nationwide, large cohort of patients with pre-dialytic stage-5 CKD who had hypertension and anemia, and were treated with ESAs.Previous investigations have disclosed that dual renin angiotensin system (RAS) blockade (combination therapy with an ACEI and an ARB) is more effective in proteinuria reduction, which may provide additional cardiovascular or renoprotective benefit, than either drug alone in renal disease. However,The present study used data from Taiwan’s National Health Insurance (NHI) Research Database which was launched in 1995, managed and released to the public by the National Health Research Institute of Taiwan, and up to the present covers more than 99%, approximating 23 million, of the residents in Taiwan. This mandatory universal program offers all their medical records, including date of birth, sex, diagnostic codes, medical procedure and prescription of drugs. Diseases are coded according to the 2001 International Classification of Diseases, ninth revision, Clinical Modification (ICD-9-CM). Any information that would expose the identities of individual patients is de-identified. Having been taken as the primary source for several published studies, NHIRD has also had the accuracy of diagnoses be repeatedly validated). This study was approved by the Institutional Review Board of Taipei Veterans General Hospital.This nationwide, retrospective cohort study was performed in Taiwan to determine the association between ACEI/ARB usage and the prognosis of advanced CKD. Patients with a primary diagnosis of CKD  subjected to ESA treatment combined with RAS blockade agent (ACEI or ARB) from January 1, 2000 through June 30, 2009 were enrolled into this study. The National Health Insurance has set the level of serum creatinine >6 mg/dl and hematocrit <28% as the indication for the use of ESA. According to the national report of the Department of Health, 85% of the patients with stage 5 CKD were subjected to ESA treatment if they were not on the list requiring renal replacement therapy. Since the median hematocrit level reported at the initiation of dialysis was 24.4% in an interquartile range from 20.6% to 27.5% in Taiwan, the cohort of the subjects selected in the current study should therefore be the most representative of patients with pre-dialytic stage 5 CKD. The use of ACEI/ARB was defined as prescription within 90 days after the first ESA treatment. To prevent immortal date bias, the 91th day after the first ESA treatment was re-defined as the index date. Patients who did not receive ACEI/ARB, who were younger than 20 years or older than 100 years of age, or who had received renal replacement therapy  before ESA treatment were excluded from this study.Totally 24,765 patients were included in the current study. The subjects who had received ACEI only, ARB only, or the combined therapy with ACEI plus ARB within 90 days after the first ESA prescription were defined as ACEI group, ARB group, or ACEI +ARB group accordingly. The remaining patients who took both ACEI and ARB without combination during this period were defined as ACEI/ARB group. Analyses were all conducted on an intention-to-treat basis according to the patients’ initial assignment regardless any subsequent changes in their ACEI/ARB regimen.Baseline demographic characteristics, including age, gender, comorbidity, geographic location, nephrologist visits and medications were extracted and recorded. Nephrologist visits and comorbidities, including diabetes mellitus, stroke sequela, coronary artery disease and cancer, were defined as record within 3 years before the index date. Charlson comorbidity index (CCI) scores were used to determine overall systemic health. Each increase in the CCI score was associated with a stepwise increase of cumulative mortality. Anti-hypertensive agents other than ACEI/ARB analyzed in this study included diuretics, α-blockers, β-blockers, and calcium channel blockers. Medications that may potentially influence potassium balance were also excluded, e.g. Calcium polystyrene sulfonate, Sodium polystyrene sulfonate and sodium bicarbonate.The observation period started from the index date till the patients died or began long-term dialysis, or December 31, 2009, whichever occurred earlier. The renal outcome was defined as the status when the patients entered long-term dialysis. The composite outcome of long-term dialysis or death referred to either the starting date of long-term dialysis or death, whichever came first. The first event of hospitalization associated with a diagnosis of hyperkalemia (ICD-9 code 276.7) during observation period was defined as hyperkalemia-associated hospitalization.Baseline characteristics were compared by one-way ANOVA for continuous variables and Chi-Square test for categorical variables. In the multivariate Cox proportional hazards regression models, the effects of ACEI, ARB, ACEI+ARB, or ACEIs/ARBs were further adjusted for age, sex, Charlson comorbidity index (CCI), diabetes mellitus, coronary artery disease, stroke, cancer, frequency of visits to nephrologists within 3 years before the index date , geographic location , and types of non-ACEI/non-ARB anti-hypertensive agents used. Study entry was defined as the index date. Results were expressed as hazard ratios (HRs) compared with ARB user. The proportional hazards assumption, the constant HR over time, was evaluated by comparing the estimated log-log survival curves for all time-independent covariates. All the log-log survival plots assessed graphically showed 2 parallel lines, indicating no violation of the assumption. Adjusted HRs for long-term dialysis and the composite outcome associated with ACEI/ARB use were further analyzed among subgroups based on participants’ characteristics (see below). The cumulative hazards of long-term dialysis and the composite outcome over time were compared among the ACEI, ARB, ACEI/ARB and ACEI + ARB groups by using the Nelson-Aalen method to adjust covariates adopted in the Cox proportional hazards regression models. All P values were 2-sided, and the level of significance was set at .05. Analyses were performed using commercially available software .We identified 24,765 pre-dialytic advanced CKD patients between January 1, 2000 and January 31, 2009 who met the inclusion criteria. Among them, 10,648 patients died or commenced chronic dialysis within 90 days after the index date. The remaining 14,117 patients were enrolled into the study and classified into four groups as ARB only , ACEI only , ACEI/ARB  and concurrent ACEI+ARB use  according to their drug prescriptions . The meaIn a median follow-up of 7 months, 9,867 patients (69.9%) required long-term dialysis and 2,805 (19.9%) died before progression to end-stage renal disease requiring dialysis. The incidence rates of long-term dialysis, death or the composite outcome of dialysis or death are listed in When patients were stratified into diabetic and non-diabetic subgroups by taking the status of the patients treated with ARB only as reference index, dual blockade with concurrent use of ACEI and ARB was associated with a significantly higher risk of the composite outcome of long-term dialysis or death only in diabetic subgroup . , Fig 2D 2.[Our study demonstrated that dual blockade therapy with ACEIs and ARBs in predialysis CKD patients was not significantly associated with long-term dialysis or death. Dual RAS blockade has been thought to be more renoprotective than monotherapy because of its effect on proteinuria reduction in the short-term treatment. However,2. To our kIn this study, the subgroup analysis disclosed a 10% higher risk of long-term dialysis or death among diabetic patients. The different effect of dual RAS blockade and monotherapy between diabetic and non-diabetic patient has also been reported by Kim et al. in their small population crossover trial. not in tFew studies were focused on the effect of dual RAS blockade to non-diabetic CKD patient group. A former Clinical trial on Aliskiren in Type 2 Diabetes and Cardiorenal End Points (ALTITUDE) choosing the direct renin inhibitor Aliskiren, instead of ACEI, as the second RAS blockade added on ARB in diabetic patients was terminated early because of increasing adverse events including stroke, hyperkalemia and renal complications. On the oOur finding of increased risk of death not only in dual blockade users but also in ACEI users as compared with ARB users was supported by the report of Chan et al. who compared the relative effectiveness of newly add-on ACEIs, ARBs and dual blockades in reducing cardiovascular mortality in about 9,300 chronic hemodialysis (HD) patients over 6 years. After adThe reason why ACEIs may be associated with higher risk of death could be explained by the following mechanisms. First, the anti-inflammatory effect of ACEIs may be less potent than ARBs according to the report by Gamboa et al., who conThe inflammatory status in the period after dialysis should be more severe than that before dialysis because of less excretion of pro-inflammatory cytokines due to lower residual renal function. Furthermore, DM patients are also associated with higher inflammatory status than non-DM patients. Thus, the weaker anti-inflammatory effect by ACEI (vs. ARB) may be associated with a higher risk of death in either study period  or patient subgroup (DM vs. non-DM) with more severe inflammation.Hyperkalemia was one of the most concerned adverse effects of RAS blockade, either ACEIs or ARBs. The combination therapy with ACEI plus ARB exhibits even more complete blockade of RAS capable to elevate serum potassium level to a much higher level than ACEI or ARB monotherapy can in CKD patients. A previoThe major strengths of our study are the large sample size and its nationally representative nature, which rely on a comprehensive medical utilization claim data base to include all the CKD patients as possible. Bias on the date of immortality was eliminated as best as can by selecting patients who survived beyond 90 days after ESA prescription and could be observed thereafter. However,Our study demonstrates the difference in the effectiveness and safety between ACEIs, ARBs and dual RAS blockade therapy (ACEI plus ARB) in pre-dialytic stage 5 CKD patients. As compared with ARBs, dual blockade with ACEIs and ARBs shows a higher risk of death and composite endpoint of long-term dialysis or death in diabetic subgroup as well as a higher rate of hyperkalemia-associated hospitalization in non-diabetic subgroup, and ACEIs are associated with a higher risk of death, especially in diabetic subgroup. Thus, monotherapy of RAS blockade, especially ARB, is more effective and safer than dual RAS blockade in pre-dialytic stage 5 CKD patients.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


The recoding of genetic information through RNA editing contributes to proteomic diversity, but the extent and significance of RNA editing in disease is poorly understood. In particular, few studies have investigated the relationship between RNA editing and disease at a genome-wide level. Here, we developed a framework for the genome-wide detection of RNA sites that are differentially edited in disease. Using RNA-sequencing data from 100 hippocampi from mice with epilepsy  and 100 healthy control hippocampi, we identified 256 RNA sites (overlapping with 87 genes) that were significantly differentially edited between epileptic cases and controls. The degree of differential RNA editing in epileptic mice correlated with frequency of seizures, and the set of genes differentially RNA-edited between case and control mice were enriched for functional terms highly relevant to epilepsy, including “neuron projection” and “seizures.” Genes with differential RNA editing were preferentially enriched for genes with a genetic association to epilepsy. Indeed, we found that they are significantly enriched for genes that harbor nonsynonymous de novo mutations in patients with epileptic encephalopathy and for common susceptibility variants associated with generalized epilepsy. These analyses reveal a functional convergence between genes that are differentially RNA-edited in acquired symptomatic epilepsy and those that contribute risk for genetic epilepsy. Taken together, our results suggest a potential role for RNA editing in the epileptic hippocampus in the occurrence and severity of epileptic seizures. Epilepsy is a common, serious disease characterized by unprovoked and spontaneously recurring epileptic seizures. Approximately 2% of the world's population is affected by epilepsy at some time in their lives . EpilepsHTR2C gene transcripts, five RE sites have been reported in close proximity to each other, which produce a diverse repertoire of 28 mRNAs and 20 protein isoforms  where inosine is translated as if it were guanosine , and the conversion of cytosine to uracil (C-to-U) where uracil is translated as if it were thymine (C-to-T). These base-specific changes to RNA result from site-specific deamination of nucleotides catalyzed by ADARs (adenosine deaminases that act on RNA) and APOBEC1, the latter belonging to the APOBEC cytidine deaminases . RE is aisoforms . RE may isoforms .The A-to-I type of RE has been reported as the most abundant conversion, with over 16,000 RNA-edited sites in whole brain tissue described  and withIn this study, we performed a genome-wide differential RE association study (GWRAS) of epilepsy using a mouse model of acquired symptomatic epilepsy. Animal models of epilepsy have played a fundamental role in advancing our understanding of the basic mechanisms underlying ictogenesis and epileptogenesis  and offen = 200) were initially used for the prediction of RNA-editing events, and the identified RE events were then further analyzed in epileptic and control mice to identify differential RE (DRE) between cases and controls (Methods).The study design used for the genome-wide differential RE association analysis of epilepsy was a case control analysis using a mouse model of acquired symptomatic TLE A. High-tP-value < 0.05, using REDItools)  and for which there were no systematic differences in the mapping, base quality, and read coverage between epileptic and control hippocampi .To maximize the likelihood of identifying true RE events, we carried out genome-wide prediction of RE events using RNA sequencing data from all 200 mice hippocampi. After removal of known single nucleotide variants (SNVs) and using stringent filters for base  and mapping quality  (see Methods), we identified 201,322 unique mRNA sites with an alternate allele significantly different from its corresponding DNA sequence in at least one sample . Similar analysis of the 9277 nonclustered RE sites , identified 256 DRE sites  . Overall, A-to-I DRE was observed in 56 genes for all DRE and 31 for the clustered set, while C-to-U editing was observed in 31 and 10 genes for all and clustered DRE sites, respectively  using a binomial distribution and the nonparametric Wilcoxon signed-rank (WSR) test to identify RNA-edited sites that are significantly differentially edited between cases and controls. Since parametric and nonparametric approaches have their respective advantages and disadvantages, to identify differential RE sites here, we took the conservative route of taking the overlap of the results obtained by both approaches (see Methods). Using this strategy, considering the set of 4780 clustered RE sites, we identified 134 RE sites (in 39 unique protein coding genes) as differentially edited  between epileptic and control hippocampi . Additionally, we identified 10 transcripts with 68 C-to-U sites DRE between epileptic and control mouse hippocampus, which also included four C-to-U sites which had been previously reported as RNA-editing events in macrophages  belonged to the two most prevalent known classes of editing  . We found that when ∼20%–25% of the sites are “noise,” the estimate of the false-positive rate for DRE is ∼10% for all RE sites and ∼5% for “clustered” RE sites, respectively.Among the sites DRE between epilepsy cases and controls, the percentages of noncanonical sites were 7.8% and 4.5% for all and clustered data sets, respectively. Noncanonical sites are often considered to be an indication of a potential false-positive rate of detection of RE events . In our Supplemental Table S2). In addition, we observed that 75% of the DRE sites have higher mean RE levels in epileptic hippocampi compared to controls  or clustered DRE sites . However, we found that DRE sites were significantly enriched in highly expressed genes .The difference between mean editing levels for sites found to be significantly DRE between epileptic and control hippocampus ranged between −15% and 15%, with the majority (72%) of the sites having small RE differences (<5%)  . This significant overrepresentation of previously reported RE sites provides an independent line of evidence supporting the validity of the predicted RE sites. Second, we undertook Sanger sequencing in 22 RE sites which consisted of 14 clustered and six nonclustered sites predicted to be DRE between cases and controls, as well as two sites which were removed as false-positive predictions under our stringent filtering criteria. The predicted DRE sites consisted of fourteen A-to-I, four C-to-U, and two noncanonical RE sites. Using 10 mouse hippocampus samples (five cases and five controls), the percentage of RE was estimated from Sanger sequencing using ab1 Peak Reporter Tool , “synapse” , and “seizures”  .To investigate whether genes DRE between epilepsy cases and controls are involved in functional processes and pathways relevant to epilepsy, we carried out functional enrichment analysis taking the conservative approach of using the set of genes that are expressed in mouse hippocampus as the background (see Methods). We observed significant enrichment for processes with Gene Ontology (GO) or phenotype terms relevant for epilepsy and neuronal excitability among the genes impacted by DRE, including “neuron projection” (BH-adjusted n = 10) of samples . Next, we tested whether these previously reported RE sites were differentially edited between epileptic and control hippocampi (as previously described), resulting in the identification of 114 DRE sites (in 70 genes) . The majority of these DRE sites resided in the intronic and/or exonic regions of the genes . In addition to the expected A-to-I transitions, we also observed five DRE sites with C-to-U editing. The 70 genes impacted by DRE in epileptic mouse hippocampus had significant enrichment for GO term “kainate selective glutamate receptor activity”  . The two databases DARNED and RADAR were built from the union of RE sites identified across heterogeneous tissue types including brain. The identification of functionally related GO terms from DRE analysis of RE sites previously reported in DARNED and RADAR and the set of RE events identified in this study provide an independent level of evidence supporting our predictions and suggests we would have missed many DRE sites potentially relevant to epilepsy had we restricted our analysis to previously known sites.To further validate our findings, we also considered RE sites that had been previously reported with high confidence in the DARNED and RADAR databases . Union oSupplemental Fig. S6A; Supplemental Table S4). Here, we observed little overlap between GO terms enriched among genes DRE in epilepsy and those enriched among genes differentially expressed in epilepsy, consistent with our earlier observation that DRE sites are not enriched for genes differentially expressed in epilepsy (above). These results suggest that differential gene expression alone does not account for the observed differences in DRE between epilepsy cases and controls. While some functional terms enriched among genes highly expressed in hippocampi overlapped with those enriched among genes DRE in epilepsy , these terms were very similar for both epileptic cases and nonepileptic controls .To further investigate whether the observed functional overrepresentation in transcripts impacted by DRE A is not Supplemental Table S5). Consistent with previous observations .We used the variant effect predictor from Ensembl to characterize the effects of edited sites on the function of genes affected by DRE . C-to-U edited genes were most strongly enriched for microglia and oligodendrocytes, while A-to-I edited genes were enriched for astrocytes as well as interneurons. Overall, we found no enrichment for endothelial, mural, and ependymal cell types. Interestingly, A-to-I and C-to-U genes with DRE were almost mutually exclusive to each other in terms of their cell-type specificity. This is keeping with previous data showing that C-to-U editing is highly expressed and active in macrophages/microglial cell types (To provide further insights into the cellular origin of each RE type (A-to-I and C-to-U), we used a published set of cell-type marker genes obtained by single-cell RNA-seq analysis of mouse hippocampus . The identified motif was significantly similar   to the previously described sequence motif shown to be associated with C-to-U edited sites . Taken together, the enrichment of microglia marker genes among C-to-U DRE sites, their frequent occurrence in clusters , their occurrence within AT-rich regions and the significant differential expression of genes encoding enzymes responsible for C-to-U editing suggests that the C-to-U DRE sites could potentially be catalyzed by the APOBEC family of enzymes. Similarly, ADAR and ADARB1, key enzymes for facilitating A-to-I editing, are significantly differentially expressed as well as highly expressed in both cases and controls . However, this observation alone is unlikely to explain the full spectrum of observed A-to-I DRE in our study where A-to-I editing represents a mixture of high (73%) and low (27%) edited sites with respect to control hippocampus, suggesting potential involvement of cofactors.Previous studies have shown that C-to-U editing has been associated with a nucleotide motif found ±5 bp to the edited base . We thered sites . In addi−3 for both clustered and all DRE sites)   (P = 0.06).To further investigate the role of DRE in epilepsy, we tested the association between the level of DRE and frequency of seizures observed in the epileptic mice. Here, seizure frequency (SF) was assessed by two weeks of continuous video monitoring of behavioral seizures in 100 epileptic mice (see Methods). First, for each of the RE sites identified in the mouse hippocampus (n = 9277), we calculated the (nonparametric) Spearman correlation between the RE percentage and the total number of seizures observed in each epileptic mouse. These Spearman correlations were normally distributed, with mean correlation close to zero, suggesting that the majority of edited sites are not associated with epilepsy A. In conE sites) A. We alsall DRE) B. Intere× 10−16) B, while n = 356), autism spectrum disorder , schizophrenia , and intellectual disability  (see Methods for cohort references). For controls, we used 1891 nonneurological control samples as previously reported  enriched for association to generalized epilepsy  identified in neurodevelopmental whole-exome sequencing (WES) studies that shared similar sequencing technologies, coverage criteria, and variant calling methodology . Collectreported . The genreported A,B. No eepilepsy A,B. We aepilepsy  and obseepilepsy A,B. ThesSupplemental Fig. S8; Supplemental Table S7). These results highlight that a subset of genes DRE in the mouse epileptic hippocampus are also RNA-edited in the human epileptic hippocampus, but further analysis will be required to determine if DRE in these genes is associated with human epilepsy.To explore the hypothesis that the DRE identified in the epileptic mouse hippocampus might be relevant to human epilepsy, we analyzed RNA sequencing data from dentate gyrus samples ascertained from six patients with mesial temporal lobe epilepsy together with matched whole-exome sequence data . ConsideRecent intense scrutiny of RNA editing has led to a better appreciation of the potential sources of bias and error associated with the identification of RE . ConsequIn this study, we present a comprehensive framework for testing differential RNA-editing events in disease. We aimed to detect RNA-edited sites in an accurate manner and in an adequately powered sample set, and therefore we started from the analysis of a large sample of 200 mouse hippocampi. We then used a stringent data filtering strategy that included detection of the edited site in at least 10 samples and removal of all known single nucleotide variants and indels, estimated to constitute >95% of SNPs in outbred mice and with novel variant rate of 0.8% in the NMRI mice strain (used in this study) . We furtOur genome-wide DRE analysis yielded a set of 256 sites (in 87 unique genes) significantly differentially edited between epileptic and control hippocampi, of which a subset of 134 sites (in 39 unique genes) were clustered . Genes with DRE  were specifically enriched for functional terms highly relevant to neural processes and epilepsy. Notably, 70% of exonic DRE sites are predicted to be deleterious. These results suggested a potential relationship between DRE and the occurrence of seizures and epilepsy. To investigate the potential role of transcripts impacted by DRE in epilepsy, we tested whether transcripts significantly differentially edited in the mouse model of epilepsy were enriched for genes with a genetic relationship to epilepsy. Considering both all and clustered DRE, we found genes impacted by DRE are enriched for genes impacted by de novo mutations in patients with epileptic encephalopathy, as well as significantly enriched for association to common forms of generalized epilepsy. These enrichments suggest that RE events in the mouse epileptic hippocampus are nonrandomly occurring and are potentially disrupting important genes that modulate specific pathways and processes relevant for human epilepsy. In keeping with this, we found that RNA editing is also associated with seizure frequency in epileptic mice and that fold change of DRE was significantly and positively correlated with seizures. Given the high expression of the genes with differential RE editing and their known functional relevance to epilepsy, it is possible that even a relatively small change in RNA editing might have a biological role, in particular when multiple genes with differential editing are impacting the same pathways and biological processes in the hippocampus.In summary, our genome-wide analysis of DRE in epilepsy identified hundreds of DRE sites which are enriched for genes associated with epilepsy and which are significantly correlated with seizures in the mouse epilepsy model. Analysis of the cell types and molecular processes associated with the epilepsy-associated RNA editing reveals cell-type–specific RNA editing and a potential involvement of both microglial and neuronal processes . These rStatus epilepticus was induced in male NMRI mice (weighting 28–32 g at the beginning of the study) by a single injection of pilocarpine as previously described . Brieflyn = 200; 100 controls and 100 pilocarpine-treated mice). Sample preparation for RNA sequencing (RNA-seq) was performed according to the protocols recommended by the manufacturers . Sequencing was done using an Illumina HiSeq 2000 sequencer, with paired-end 75-bp nucleotide reads according to the protocol recommended by the vendor. Raw reads were mapped to the reference mouse genome (mm10) using TopHat version 2.0.8 -FR outbred mouse colony (on 2.0.8 .Rigorously Annotated Database of A-to-I RNA editing (RADAR) (DAtabase of RNA EDiting (DARNED) (Known RNA-edited sites were curated using publicly available databases: (1)  (RADAR) ; and (2)(DARNED) . DARNED (DARNED) . These n(DARNED) . We filtP-value < 0.05 for the overrepresentation of alternate allele in each site meeting the above-mentioned criteria. Initially, a site was considered to be edited if at least one sample was observed to have a significant enrichment of alternate allele . Identified edited sites were also filtered for all known mouse single nucleotide variations available from Ensembl mouse SNP database version 137. Additional biases such as strand and variant distance bias were removed as described in P-value > 0.05 was used to filter the sites/samples. Further, we removed read sequences with very high similarity (>95%) with other genomic regions and filtered out sites that resided within mouse regions harboring genome duplication events and the sites within 4 bp of an exon-intron boundary .P-value > 0.05). In addition, for the clustered RE data set, we filtered RE sites that were not within 50 bp of another RE site at the transcript level.Finally, we filtered all the sites that were supported by less than 10 samples with at least five reads supporting an alternate nucleotide , and the identified sites should not be in Hardy-Weinberg equilibrium (http://www-huber.embl.de/users/anders/HTSeq)  . Read cos/HTSeq) , and a cE is the probability of the RNA-edited site to be differentially edited between cases (pilocarpine-treated mice) and controls (naïve mice), CC is the case control status, and ε is the random effect.To identify differentially RNA-edited sites, we used two statistical methods in parallel and then integrated the results to synthesize the list of DRE sites identified by each method as follows. First, for each site, we build a generalized linear mixed model based on binomial distribution to model the probability of RNA editing as a function of case control status (fixed effect) and individual (random effect):P-values obtained from the test using R version 3.02  and controls (naïve mice). The Benjamini and Hochberg method was used to correct the ion 3.02 .P-values) were combined by means of the sdef   with the purpose of finding common features, in this case, differential RNA-editing sites commonly identified by the GLMM and Wilcoxon rank test. The overall significance of maximum overlap was tested using 105 Monte Carlo permutations, and the significance level is reported as a Monte Carlo P-value.Finally, the results of the GLMM and Wilcoxon rank test   package 5 permutations to estimate the significance of enrichment. The −log10  of differential RNA editing was used as a metric to rank RNA-edited sites, and gene sets were defined as the set of known RNA-edited sites available from public databases: (1) RADAR  RADAR , and (2)1) RADAR . DARNED 1) RADAR . In addi2[1] FPKM value in 5% of the samples). Enrichments obtained from Gene Ontology and KEGG pathways were considered as repositories for functional terms.If the edited site was within annotated gene boundaries (TSS and 3′ end), the gene was linked with a differential RNA-editing site. Genes enclosing RNA-edited sites were searched for enrichment in functional terms using the WEB-based GEneSeT AnaLysis Toolkit  , where tA manual literature search was performed using PubMed in order to check if genes have been previously associated with epilepsy, seizures, or neuropsychiatric disorders.The Ensembl variant effect predictor (VEP) part of Ensembl tool release version-75 was used to functionally annotate the DRE sites. To predict the consequence of the DRE sites overlapping with exonic regions, VEP uses the SIFT (sorts intolerant from tolerant amino acid substitutions) method  to inferP-values were FDR-corrected.We tested genes with DRE for overlap with genes that were previously associated with epilepsy using the DisGeNET database . We perfn = 356) (n = 4186) (n = 1004) (n = 192)  , autism  = 4186) , schizop = 1004) , and intn = 192) . For conn = 192) .To integrate these data across their variable sources and coverage, we assumed each gene has 100% of its consensus CDS covered across all trios, as previously described . For eacP-value) controlled for the number of SNPs in each gene and the LD between those SNPs. In all analyses, gene-based P-values were calculated using VEGAS2 and the top 10% option with 100,000 iterations and a gene window consisting of the transcriptional start and stop position of each gene. For the International League Against Epilepsy (ILAE) Consortium on Complex Epilepsies  using the Z-test-based bootstrapping method   to generilepsies , the defg method   in 10 samples. The RNA-editing percentage varied from 5% to 90% D. The peTo show the conservation in human epilepsy, we have extracted reads from the combined list of DRE sites from all three approaches. RNA-seq reads were extracted from the hippocampi of six epileptic patients . RNA-seqhttp://www.ebi.ac.uk/ena) under accession number PRJEB18790. The catalog of identified RNA-edited sites are included in Supplemental Table S8 and their corresponding RNA-seq reads are publicly available on Figshare.com and can be accessed from https://dx.doi.org/10.6084/m9.figshare.4476323 or from Imperial College London's secure server (http://goo.gl/HoOEuT). Sanger sequencing traces have been submitted to the NCBI Trace Archive (https://trace.ncbi.nlm.nih.gov/Traces/trace.cgi) under TI numbers 2344112081-2344112279.The RNA-seq data generated in this study have been submitted to the European Nucleotide Archive (ENA;