Prostate stem cell antigen (PSCA) has been suggested as biomarker and therapeutic target for prostate cancer. Recent advances showed that PSCA is up-regulated in other cancer entities, such as bladder or pancreatic cancer. However, the clinical relevance of PSCA-expression in breast cancer patients has not yet been established and is therefore addressed by the current study.PSCA-protein expression was assessed in 405 breast cancer patients, using immunohistochemistry (PSCA antibody MB1) and tissue microarrays.PSCA-expression was detected in 94/405 patients (23%) and correlated with unfavorable histopathological grade (p=0.011) and increased Ki67 proliferation index (p=0.006). We observed a strong positive correlation between PSCA-protein expression and HER2/neu receptor status (p<0.001). PSCA did not provide prognostic information in the analyzed cohort. Interestingly, the distribution of PSCA-expression among triple negative patients was comparable to the total population.We identified a subgroup of PSCA-positive breast cancer patients, which could be amenable for a PSCA-targeted therapy. Moreover, given that we found a strong positive correlation between PSCA- and HER/neu expression, targeting PSCA may provide an alternative therapeutic option in case of trastuzumab resistance. Breast cancer is the most common cancer in women worldwide . There aProstate stem cell antigen (PSCA) is located on chromosome 8q24.2, encodes for a 123 amino acid glycosylphosphatidylinositol (GPI)-anchored cell surface protein and belongs to the Thy-1/Ly-6 family. It was originally defined as an upregulated gene in a human prostate cancer LAPC-4 xenograft model . InteresPSCA shares 30% homology with the stem cell antigen type 2 (SCA-2), which is an immature lymphocyte cell surface marker. However, considering this rather weak homology to SCA-2, PSCA was inaccurately named, since it is actually neither a marker for a stem cell population, nor is PSCA exclusively expressed in prostatic tissue . SubsequCurrently, for breast cancer patients, there is limited data on PSCA. Some of these investigations have an epidemiological focus and report on genetic variation of the PSCA gene and its relation to breast cancer development. In this context, it was reported, for instance, that PSCA single nucleotide polymorphims (e.g. rs2294008 or rs2978974) are associated with increased risk of developing breast cancer . MoreoveIn order to exclude a heterogeneous PSCA-expression as well as to assess expression pattern and compartmental distribution of PSCA-protein in breast cancer patients, we performed immunohistochemical PSCA-staining in conventional tissue sections from primary breast tumors. PSCA-staining, which was positive in 4 of 18 samples (22%), appeared homogeneously and was observed with membranous and occasionally cytoplasmic PSCA-reactivity Figure . PSCA-stGiven the spatially homogeneous staining in PSCA-positive breast cancer tissues, we subsequently performed PSCA-tissue microarray analysis in a comprehensive set of clinically documented breast cancer patients (n=405). PSCA-expression was observed with a positivity rate of 23% (94/405 patients). Low PSCA-expression was detected in 16% (66/405 patients) and a high expression was observed in 7%  or OS  HER2/neu overexpressing patients  or ii) patients with intermediate risk, according to the criteria of St. Gallen 2007  n=236 f and 6.In summary, PSCA does not provide prognostic significance in our breast cancer cohort, neither in the overall study population nor in clinically relevant breast cancer subgroups.In the present study, we demonstrate clinical relevance of PSCA-expression in breast cancer patients. We observed homogenous distribution of PSCA-expressing tumor cells in breast cancer tissue and expected that a TMA-analysis of PSCA-expression will allow conclusions on PSCA-expression across a large number of patients. This assumption was corroborated by the fact that PSCA-positivity rate in conventional breast cancer tissue sections almost exactly corresponded to our TMA-analysis 22% vs. 23%). Although this positivity rate is smaller than in other cancer entities, such as prostate or pancreatic cancer, in which PSCA is up-regulated in 60- 80% , 13, we %. AlthouWe did not find an association of PSCA-expression and the patients’ survival, not even in consideration of clinically relevant breast cancer subgroups. This is in accordance to the aforementioned study on breast cancer, which also failed to show any statistically significant associations between PSCA mRNA expression and the patient's clinico-pathological data  and coulNAALADase” and folate hydrolase activity). PSMA is not only strongly expressed in prostate cancer, it is also upregulated in the neovasculature of solid tumors [Little is known about the function of PSCA. PSCA could possibly interact with other membranous proteins in order to activate downstream signalling pathways. Alternatively, PSCA could be shed from the cellular membrane through the cleavage of its GPI-anchor by phospholipase C and may act as a secreted ligand, which activates other receptor-mediated pathways . Recent d tumors . Neverthd tumors –37. Thisd tumors . Thus, tPSCA has attracted some attention as a therapeutic target. Early clinical trials using full-length antibodies against PSCA in prostate and pancreatic cancer have failed to show signs of efficacy , 39. ConConclusively, we provide the first comprehensive analysis of PSCA-expression and its clinical relevance in breast cancer. Our results suggest that PSCA-based immunotherapeutic and theragnostic approaches may become valuable additions to breast cancer therapy, especially for patients resistant to therapies targeting HER2/neu and to the subset of triple negative patients with PSCA-positive tumors.We analyzed formalin fixed paraffin embedded (FFPE) cancer tissue of 405 primary breast cancers patients, treated according to uniform guidelines in the Regional Breast Cancer Center Dresden between 2003 and 2011. This study was approved by the ethics committee of the University Hospital of Dresden, Germany (EK59032007) and was performed, according to the Declaration of Helsinki. Since this study was performed retrospectively on long term archived FFPE breast cancer tissue without interventions, no patient's consent was required by the guidelines of ethics committee. From each breast cancer, at least two cores were included in the tissue microarrays, resulting in a total number of five microarrays. In order to increase statistical power of comparison of breast cancers with different biological features, two of the tissue microarrays were enriched with triple negative and HER2/neu positive breast cancers cases. In order to exclude heterogeneous expression as well as to assess expression pattern and compartmental distribution of PSCA-expression in breast cancer patients, we performed immunohistochemical PSCA-staining in conventional tissue sections from primary breast tumors (n=18). To check the PSCA-expression in normal breast tissue, the tumor free cancer surrounding tissue in the conventional sections and two breast reduction specimens were analyzed. Table The expression of PSCA, estrogen and progesterone receptor, HER2/neu and Ki67 was analysed by immunohistochemistry. All staining results were scored semi-quantitatively considering the percentage of positive cells and the staining intensity. The staining result for PSCA was subdivided in negative (no positive cells), low expression (1 to 9% of tumor cells positive) and high expression (>9% positive tumor cells). Table Statistical analysis was performed by Chi-squared test for comparing the categorical clinico-pathological markers and PSCA-expression. The Cochrane-Armitage-trend test was performed for comparing ordered categorical variables and Kaplan-Meier estimation (log rank test) was used for survival analyses. Overall survival (OS) was defined from day of surgery until death. Progression-free survival (PFS) was defined from day of surgery until disease progression. OS and PFS for patients who did not die (for OS), respective suffer from progression/death (for PFS) during the observational period, were censored at the end of follow-up. Median follow up time was estimated using the reverse Kaplan-Meier-method. The significance threshold was p<0.05 for all tests. Because of the exploratory character of the analyses, no adjustment for multiple testing was performed.This study was approved by the ethics committee of the University Hospital of Dresden, Germany (EK59032007) and was performed, according to the Declaration of Helsinki. Since this study was performed retrospectively on long term archived FFPE breast cancer tissue, no patient's consent was required.


Physical inactivity is highly prevalent after stroke, increasing the risk of poor health outcomes including recurrent stroke. Tailoring of exercise programs to individual preferences can improve adherence, but no tools exist for this purpose in stroke.We identified potential questionnaire items for establishing exercise preferences via: (i) our preliminary Exercise Preference Questionnaire in stroke, (ii) similar tools used in other conditions, and (iii) expert panel consultations. The resulting 35-item questionnaire (SEPI-35) was administered to stroke survivors, along with measures of disability, depression, anxiety, fatigue and self-reported physical activity. Exploratory factor analysis was used to identify a factor structure in exercise preferences, providing a framework for item reduction. Associations between exercise preferences and personal characteristics were analysed using multivariable regression.Supervision-support, Confidence-challenge, Health-wellbeing, Exercise context, Home-alone, Similar others, Music-TV). Item reduction processes yielded a 13-item version (SEPI-13); in analysis of this version, the original factor structure was maintained. Lower scores on Confidence-challenge were significantly associated with disability (p = 0.002), depression (p = 0.001) and fatigue (p = 0.001). Self-reported barriers to exercise were particularly prevalent in those experiencing fatigue and anxiety.A group of 134 community-dwelling stroke survivors  participated. Analysis of the SEPI-35 identified 7 exercise preference factors (The SEPI-13 is a brief instrument that allows assessment of exercise preferences and barriers in the stroke population. This new tool can be employed by health professionals to inform the development of individually tailored exercise interventions. Physical activity is essential to post-stroke recovery, with evidence that exercise training improves functional capacity, increases quality of life and reduces the risk of subsequent cardiovascular events. The beneEvidence indicates that individual tailoring is a feature of effective interventions for increasing physical activity, both in general and stroAt present, no instruments exist for assessing exercise preferences in stroke survivors. Our primary aim was to develop a new questionnaire, the Stroke Exercise Preference Inventory (SEPI), to evaluate exercise preferences and barriers after stroke. A secondary aim was to determine the relationship between key personal characteristics  and self-reported exercise preferences and barriers, in order to evaluate whether these characteristics could account for individual differences on the SEPI.The Stroke Exercise Preference Inventory (SEPI) was developed in two stages: content development and content refinement. Stage 1 involved identifying a wide range of questionnaire items that covered meaningful aspects of exercise preferences after stroke. Once these items were finalised, Stage 2 involved administering them to a sample of stroke survivors and analysing the data to refine the questionnaire to a core set of items.To begin the development process, we built a list of potentially relevant questionnaire items. These items were drawn from multiple sources, including our preliminary Exercise Preference Questionnaire, a reviewTo further develop and ratify items that were relevant, easily comprehended and unambiguous, we convened an expert panel. Members Community-dwelling stroke survivors were included if they were aged ≥18 years and had sufficient English language comprehension. There was no limit placed on time since stroke. Participants with transient ischemic attack (TIA) were excluded. To maximise generalisability, participants were not excluded on the basis of disability severity or co-morbid health conditions. Participants were recruited via multiple settings, including a hospital stroke outpatient clinic , a rehabilitation hospital , community-based stroke support groups from around Australia, and through the National Stroke Foundation of Australia website. The study was approved by the Human Research Ethics Committee of Austin Health, and all participants provided written informed consent.Participants completed the SEPI-35 and other assessments in a single session, either face-to-face at the point of recruitment or remotely via mailed out questionnaire packs. For the remote completers, phone contact was always made to ensure data integrity and completeness. No data were recorded on participant response rates, as numerous questionnaire packs were supplied to interstate support groups, making it difficult to accurately track the number of potential responders.A 1-page questionnaire featuring demographic  and stroke-related  questions was administered. The primary outcome was the SEPI-35, which consisted of 35 items on preferences for exercise and 9 items on barriers to exercise participation. Examples of preference items included: ‘I like to exercise outdoors’ and ‘I like a trained instructor to supervise my exercises’. Participants were asked to indicate their level of agreement with each item by choosing a number between 0%  and 100% . The modified Rankin Scale (mRS) is a 7-point scale assessing disability, ranging from 0 (no symptoms) to 6 (death). It is widely used in stroke research, with good predictive validity and inter-rater reliability. To scoreThere is no consensus on a minimum sample size for valid exploratory factor analysis, although there is agreement that the larger the N and N:item ratio the better. Several Exploratory Factor Analysis using Principal Components Analysis was employed to identify the factor structure of the SEPI-35, with eigenvalues >1 extracted. Bartlett’s test of sphericity and Kaiser-Meyer-Olkin sampling adequacy were used to assess the reliability of the factor structure. In order to identify the most logical data structure, several factor rotations were examined, including varimax, direct oblimin, quartimax, equamax and promax.Item removal from the SEPI-35 was based on 4 guiding principles, informed by statistical evidence. The first principle was strength of factor loading. Items were removed if they did not load above 0.50 on any factor. Items with higher factor loadings were prioritised for selection in the reduced item pool. The second principle was strength of internal reliability. Items were favoured if they improved the internal reliability of a factor, as assessed using the Cronbach’s alpha and ‘Cronbach’s alpha if item deleted’ statistics for each item in a factor. The third principle was conceptual similarity. Within each factor, items deemed too conceptually similar were considered for removal. These judgements were weighed against the second principle, as conceptually similar items often had strong scale reliability properties. The fourth principle was discrimination strength. As per Item Response Theory, items that were better at discriminating between high and low scorers on a given factor were favoured for selection. To quantOnce the reduced pool of SEPI items had been finalised . First, we established whether age and sex were associated with scores on each factor using multivariable linear regressions. Additional regressions, adjusting for age and sex, were used to determine the association between each exercise preference factor score (as the dependent variable) and disability, depression, anxiety and fatigue (as the independent variable). For the purpose of representing the data in spider plots, participants were dichotomised on each of these variables: disabled (mRS≥3) or not, depressed (PHQ-9≥10) or not, anxious (GAD-7≥5) or not, fatigued (FAS≥25) or not.Data from the exercise barrier items, not our main focus here, were summarised using descriptive statistics. The relationship between personal characteristics  and the exercise barrier items was examined by computing Spearman correlation coefficients.Data on demographics, stroke details and other outcome measures for the 134 participants are presented in 2 = 2499.5, df = 595, p<0.01) and Kaiser-Meyer-Olkin sampling adequacy (0.85) was above the necessary level (0.60), indicating a strong likelihood of producing a distinct and reliable factor structure. Evaluated against our a priori estimation of factor groupings, the varimax rotation method produced the most logical factor structure. Factor analysis yielded 7 factors with eigenvalues >1, explaining 64% of total variance –with varimax rotation and number of factors fixed at 7 –showed that the original factor structure from the SEPI-35 was replicated  and Kaiser-Meyer-Olkin sampling adequacy was strong (0.75). Unweighted factor scores (means and standard deviations) are shown in Item reduction processes resulted in a set of 13 items, 2 each for the first 6 factors, and 1 for the 7ated see . The 7 fSimilar others , Home-alone  and lower scores on Music-TV . Female sex was related to higher z-scores on Home-alone .Older age was associated with higher factor z-scores on Supervision-support  and scored lower on Confidence-challenge . People with more depressive symptoms had a significantly higher preference for Music-TV  and scored lower on Confidence-challenge . People with more anxiety symptoms had a (non-significantly) stronger preference to exercise with Similar others . People with more fatigue had significantly lower scores on the Confidence-challenge factor .The following multivariable regressions were adjusted for age and sex. People with more disability had a significantly higher preference for The associations between personal characteristics  and each of the exercise preference factors  are visually represented in Means and standard deviations for each barrier item are presented in We developed a comprehensive 35-item exercise preference inventory (SEPI-35) through expert consultation, and identified 7 discernible exercise preference factors relevant to the stroke population. The factor structure of the SEPI-35 was maintained in a refined 13-item version (SEPI-13), providing a parsimonious measure for assessing key exercise preferences in the stroke population see . In addiSupervision-support, 2) Confidence-challenge, 3) Health-wellbeing, 4) Similar others, 5) Exercise context, 6) Home-alone, and 7) Music-TV. For some, receiving professional supervision and support can engender feelings of control during exercise and reduce safety-related fears.[Confidence-challenge factor assesses self-confidence in initiating and maintaining exercise, and the desire to be challenged. These two concepts are both closely related to exercise self-efficacy.[Confidence-challenge were linked to disability, depression and fatigue. This suggests that optimal challenge in an exercise program should be tailored on the basis of both physical and psychological considerations. The Similar others factor evaluates the desire to exercise with people who share important characteristics, such as medical condition or age. Such contact may be valued as a means of obtaining social support, exchanging information, or minimising the risk of negative evaluation.[Home-alone factor has clear implications for evaluating appropriateness of a home-based exercise program. Older age was found to be related to a preference for home-based exercise, which is consistent with previous findings.[Music-TV factor was based on a single item. Participants with more depressive symptoms expressed a greater preference for music or TV during exercise. This may function as a source of pleasurable entertainment to boost positive affect and reduce negative affect during exercise,[The 7 exercise preference factors were: 1) ed fears. For otheed fears. In our cefficacy. Making ealuation. We identfindings. The Musiexercise, or be vaexercise,Health-wellbeing . For the other 6 factors, level of agreement fell in the middle of the 0–100% scale , with substantial variability . With such large individual differences in exercise preferences, it is unsurprising that one-size-fits-all exercise programs suffer from problems with uptake and adherence.There was marked individual variability across all exercise preference factors, with the exception of eing see . For theRegarding exercise barriers, finding it hard to get started was the most commonly reported issue. This item had a particularly strong correlation with fatigue, which we know is prevalent after stroke. Few partThe present study has limitations. We cannot exclude the possibility of error, particularly related to overfitting of the data; this possibility would be minimised in a larger sample. A larger sample size would also have allowed more robust forms of validation of the factor structure . Our data were collected both face-to-face and remotely, and it is possible that responses differed by mode of collection. Despite sample characteristics indicating good population representation with respect to demographic, stroke, and physical and mental health characteristics, we did have disproportionately high recruitment from community-based stroke support groups (73%) and this may have contributed to selection bias. There was also a relatively high percentage of people (67%) reporting exercise beyond daily activities; this sample may have been more active than a typical chronic stroke group. We need to confirm the stability of the tool when used in patients much earlier in the stroke recovery process, when the development of exercise habits may play an important role in recovery and prevention of further stroke.The SEPI-13 is a brief new instrument which can be used to assess the exercise preferences of stroke survivors. We have developed it for use in physical rehabilitation and exercise counselling settings as a tool to promote dialogue about exercise and inform program design. The SEPI-13, which can be administered without prior training, is likely to be most relevant to exercise physiologists, physiotherapists and exercise counsellors. It may assist in identifying meaningful facets of exercise experience that could otherwise go unmentioned. Regarding implementation, we are currently evaluating the feasibility and usefulness of the SEPI-13 in an inpatient rehabilitation setting. The SEPI-13 also has research applications, and could be used to improve our understanding of the relationship between individual exercise preferences and adherence to exercise programs after stroke.S1 Appendix(DOC)Click here for additional data file.S1 Data(SAV)Click here for additional data file.


We present a methodology which can be utilized to select proton or photon radiotherapy in prostate cancer patients. Four state-of-the-art competing treatment modalities were compared  for a cohort of 25 prostate cancer patients, with and without correction strategies for prostate displacements. Metrics measured from clinical image guidance systems were used. Three correction strategies were investigated; no-correction, extended-no-action-limit, and online-correction. Clinical efficacy was estimated via radiobiological models incorporating robustness (how probable a given treatment plan was delivered) and stability (the consistency between the probable best and worst delivered treatments at the 95% confidence limit). The results obtained at the cohort level enabled the determination of a threshold for likely clinical benefit at the individual level. Depending on the imaging system and correction strategy; 24%, 32% and 44% of patients were identified as suitable candidates for proton therapy. For the constraints of this study: Intensity-modulated proton therapy with online-correction was on average the most effective modality. Irrespective of the imaging system, each treatment modality is similar in terms of robustness, with and without the correction strategies. Conversely, there is substantial variation in stability between the treatment modalities, which is greatly reduced by correction strategies. This study provides a ‘proof-of-concept’ methodology to enable the prospective identification of individual patients that will most likely (above a certain threshold) benefit from proton therapy. Prostate cancer is the most common cancer diagnosis and the third-leading cause of cancer death . When prIn silico trials offer a potential solution to this dilemma as they allow multiple-simulation of virtual randomized clinical trials for different treatment modalities with different treatment strategies for the same patients, facilitating direct like-for-like quantitative comparisons of probable clinical outcomes via radiobiological models ,7,8. ROCNotwithstanding the many positive attributes of trials, there is an increasing belief that equating all evidence-based medicine with trials is an undue simplification  and as aThis study, within the context of prostate cancer patients, addresses technical issues related to delivering scientific evidence for the application of precision radiation oncology , in effect providing an innovative methodology for utilization in a clinical decision support system (CDSS) for prostate cancer patients.(1)IMRT plans were generated at MAASTRO Clinic, the Netherlands, through seven treatment fields  optimized on the planning target volume (PTV) with leaf positions and 6MV photons, planned with RayStation .(2)VMAT plans were generated at MAASTRO Clinic, the Netherlands, through dual anterior arcs  with 91 control points per arc, optimized on the PTV with leaf positions and 6MV photons, planned with RayStation.(3)PSPT plans were generated at UPenn RPTC, USA, through two treatment fields (90°–270°) optimized on the clinical target volume (CTV) with individualized beam apertures, range compensators, and an assumed constant relative biological effectiveness (RBE) of 1.1. Dose smearing and aperture expansion of 10 mm as well as 3.5% range uncertainty distally and proximally were allowed for, planned with XiO (Elekta).(4)IMPT plans were generated by MAASTRO Clinic, the Netherlands, using two treatment fields (90°–270°) optimized on the CTV through pencil beam scanning and an assumed constant RBE of 1.1. Range uncertainty margins of 3.0% + 1.5 mm were allowed for distally and proximally, planned with RayStation.We considered four state-of-the-art competing X-EBRT and P-EBRT techniques. The planned dose distributions for an example patient for each modality are depicted in Treatment planning (TP) was based upon international commission on radiation units and measurements (ICRU) dose prescription criteria ,25. ExacOrgan at risk (OAR) sparing was prioritized in the following order: rectal-wall, sigmoid-colon, bladder, small intestines, femoral heads and skin. The TP constraints used for these OARs followed the published recommendations of quantitative analyses of clinical normal tissue effects criteria (QUANTEC) ,26. DoseThe conformity index (COIN)  providesThe radiobiological models and parameter assumptions used in this study have been extensively described previously ,7,8 and Displacement probability distributions were provided by 3DUS which measured inter-fraction prostate displacements for 56 patients treated at University Hospital Galway—Saolta University Health Care Group, Ireland. Published data for CBCT imaging  was alsoThe no-correction strategy relies entirely on the plan/PTV to ensure that the prescription dose is delivered to the CTV during treatment.The extended-no-action-limit-(eNAL)-correction strategy  involvesThe online-correction strategy consists of daily target localization, and isocentre realignment if necessary, to ensure that the prescription dose is delivered to the CTV during treatment. Online-correction is widespread in X-EBRT clinical practice; however, it is not universally utilized in P-EBRT clinical practice, although the need is recognized .The metrics in http://www.raysearchlabs.com/automated-treatment-planning/#scripting) was used to simulate the displacement of the prostate for P-EBRT .Dose recalculation is typically not required for X-EBRT treatment of the prostate ; conversThe conventional definition of robustness is the confidence that a plan metric will be delivered within limits with a high probability, and has been fully described previously . In thisplan and NTCPplan are obtained from the DVHs for the CTV and rectal-wall respectively for the planned treatment. TCPmedian is obtained from the median DVH for the CTV, produced from the 100 simulated treatments. Robustness indicates how likely a given treatment plan was executed in terms of TCP with respect to the plan. Stability indicates the range of TCP for the simulated treatments at the 95% confidence interval double-sided. The NTCPplan provides a control metric for the initial treatment plan. Score combines all these quantities into a single figure of merit ranging from −1  to +1 . The philosophy of the score metric is based upon the following: TCP provides the probability of tumor control, while NTCP provides the probability of toxicity of normal tissue, the maximum difference between the two is known as the ideal therapeutic index. This constitutes the first aspect of the score metric. However, the probability of the therapeutic index must be placed in the context of the second aspect of the score metric, robustness and stability. Robustness provides the probability of achieving the most probable therapeutic index. Stability provides the probability of convergence between the best probable and worst probable therapeutic indices.TCPThe workflow for this study is depicted in For all treatment modalities; satisfactory CTV and PTV coverage was achieved and OAR constraints were met for all patient plans. To illustrate the effect of inter-fraction prostate displacement on the CTV dose to be delivered, an example patient to be treated by IMRT, VMAT, PSPT and IMPT is presented in Each modality was evaluated based upon prostate displacement probability distributions provided by 3DUS and CBCT, with and without correction strategies. The mean, standard deviation and range for all the assessment metrics are listed in plan was highest for IMPT 56 ± 11% (range: 30–70%) and lowest for VMAT 49 ± 13% (range: 14–67%). NTCPplan was lowest for IMPT 10 ± 3 (range: 5–17%) and highest for IMRT 12 ± 3% (range: 7–17%). IMRT3DUS (no-correction) was least robust 97 ± 3% (range: 89–100%) and VMATCBCT (online-correction) was most robust 101 ± 0% (range: 100–103%). Improved conformity resulted in less stable treatment. The correction strategies greatly improve stability; by a maximum of 19% (eNAL-correction) to 24% (online-correction) for IMPTCBCT and a minimum of 11% (eNAL-correction) to 18% (online-correction) for PSPTCBCT. However, the correction strategies have limited influence on robustness; a maximum improvement of 0% (eNAL-correction) to 3% (online-correction) for IMPTCBCT and a minimum of 0% (eNAL-correction) to 0% (online-correction) for IMPT3DUS.At the cohort level: TCPThe results obtained at the cohort level reported in Image-guided radiotherapy (IGRT) is associated with an improvement in biochemical tumor control among high-risk patients  and therIntra-fraction motion was not simulated in this study. Analysis of intra-fraction motion of the prostate for a large dataset of patients revealed that on average the prostate was displaced >3 and >5 mm for 13.2% and 3.1% of the total treatment time per fraction, respectively . The med95, defined as the difference in dose delivered before and after realignment to 95% of the target volume, was reported to be restored to 0% for isocenter realignments of ≤11 mm in all planes of motion. It is rational to expect similar values for VMAT.Previous studies have shown that geometrical realignment in the case of the prostate reasonably well retrieves the planned dose distribution for IMRT  and PSPT78, defined as the volume percentage receiving the prescribed dose of 78 Gy, was reported to be completely restored for isocenter realignments ≤10 mm in the AP and SI planes of motion. It is reasonable to assume that the same would be true for IMPT with appropriate margins [For PSPT  optimize margins . To confRadiobiological response is dependent upon physical factors such as total dose, fractional dose, LET (linear energy transfer), and biological factors such as radiosensitivity, hypoxia status, and RBE. The TCP model utilized here provided excellent correlation between predicted and reported 5-year clinical outcomes in prostate cancer patients treated by IMRT, VMAT, PSPT and IMPT. The NTCP model is based upon 3D-conformal radiotherapy escalation studies of early-stage prostate cancer . It is pplan and NTCPplan data to be normally distributed for each modality; this supports the assumption that our patient cohort is representative of the overall population.Increased patient sample size is always desirable from a statistical viewpoint. Twenty-five patients are on the lower end of what is appropriate to draw firm conclusions and is a limiting factor of this study. However, the patient sample size is similar to previous in silico published studies ,43,44,45In silico trials enable prospective identification of patients who will likely benefit, in this instance through evaluation of technologies (both imaging and treatment). They can improve efficiency (patient throughput), efficacy (outcomes), and economy (cost-effectiveness) by providing streamlined, consistent, and knowledge-driven tools to support clinical decision making. However, such approaches are only as strong as the models/data upon which they are founded ,47.With regard to endorectal balloons (ERB), intra-fraction prostate displacement is significantly reduced and inter-fraction prostate displacement is not . TherefoThe results obtained at the cohort level reported in The results derived through the 25 patients analyzed in this study appear reasonable, representative and actionable. However, the results of this study should be replicated in similar or larger cohorts to mitigate the effect of unaccounted influencing factors, improving the statistics and reducing uncertainty, therefore increasing confidence in both the methodology and the results. Beyond that, to provide the highest possible level of evidence for the utility of such a clinical decision support system it should be validated in a prospective clinical trial. Additionally, application of the methodology presented here to the scenario of Carbon-ion therapy, as well as extending the modelling to incorporate cost-effectiveness, would be worthy contributions.The results of this study predict that for these clinical conditions, planning criteria, radiobiological models, and simulations parameters: IMPT with image guidance is predicted to be the most effective treatment modality. Irrespective of the imaging system, each treatment modality is similar in terms of robustness, with and without the correction strategies. Conversely, there is substantial variation in stability between the treatment modalities, which is greatly reduced with the correction strategies. Correction strategies, preferably online-correction, for inter-fraction prostate displacement appear essential to maintain normal tissue sparing whilst ensuring that the target receives the prescribed dose. However, image guidance is not routinely employed at present in most P-EBRT centers. We recommend the application of appropriate image guidance correction strategies in PSPT and IMPT. Finally, in circumstances where IGRT is not feasible, plan stability and robustness coupled with prostate immobilization techniques must also be seriously considered. This in silico trial provides a methodology which can be utilized in a clinical decision support system (CDSS) to justify the selection of proton (P-EBRT) or photon (X-EBRT) external beam radiotherapy in prostate cancer patients, by way of an in silico trial.


Background: Allopurinol-induced severe cutaneous adverse reactions (SCARs), including drug rash with eosinophilia and systemic symptoms (DRESS), Stevens-Johnson syndrome (SJS) and toxic epidermal necrosis (TEN), are life-threatening autoimmune reactions. Evidence is growing that epigenetic variation, particularly DNA methylation, is associated with autoimmune diseases. However, the potential role of aberrant DNA methylation in allopurinol-SCARs is largely unknown.Objective: To address the knowledge gap between allopurinol-SCARs and DNA methylation, we studied the DNA methylation profiles in peripheral blood cells from allopurinol-SCARs and allopurinol-tolerant subjects.Methods: A genome-scale DNA methylation profiling was conducted using the Illumina Infinium HumanMethylation450 (HM450) platform on 15 patients with allopurinol-SCARs  and 20 age- and gender-matched allopurinol-tolerant controls at disease onset. Pyrosequencing was used to validate the candidate CpG (cytosine-guanine dinucleotide) sites in an independent cohort of 40 allopurinol-SCARs and 48 allopurinol-tolerants.Results: After bioinformatics analysis of methylation data obtained from HM450 BeadChip, we identified 41 differentially methylated CpG loci (P < 0.05) annotated to 26 genes showing altered DNA methylation between allopurinol-SCARs and allopurinol-tolerants. Among these genes, significant hypomethylation of PSORS1C1 (cg24926791) was further validated in a larger sample cohort, showing significant difference between DRESS and controls (P = 0.00127), ST (SJS and TEN) and controls (P = 3.75 × 10−13), and SCARs and controls (P = 5.93 × 10−15).Conclusions: Our data identified differentially methylated genes between allopurinol-SCARs and allopurinol-tolerant controls and showed that PSORS1C1 hypomethylation was associated with allopurinol-SCARs  during disease onset, suggesting that aberrant DNA methylation may be a mechanism of allopurinol-SCARs.Limitations: Firstly, the data come from whole blood samples known to possess epigenetic heterogeneity, i. e., blood samples comprise a heterogeneous cell population with varying proportions of distinct cell-types with different DNA methylation patterns. Consequently, the interpretation of DNA methylation results should be performed with great caution due to the heterogeneous nature of the sample. Secondly, whether the identified disease-associated changes of epigenome precede disease onset, or result from the disease progression, needs further investigation. Comparing the methylation status before patients develop allopurinol-SCARs and after may help examine methylation levels from disease onset to disease progression. Allopurinol, an uric acid-decreasing drug widely used for hyperuricemia and its complications, can cause severe cutaneous adverse reactions (SCARs), which includes drug rash with eosinophilia and systemic symptoms (DRESS), Stevens-Johnson syndrome (SJS) and toxic epidermal necrosis (TEN) , allele HLA-B*5801. In Asian populations, allopurinol-SCARs has been shown to occur almost exclusively in HLA-B*5801 positive individuals in Han Chinese of Taiwan, Hong Kong, and mainland China     was reported to be associated with global T cell hypomethylation  platform. Unraveling epigenetic factors in allopurinol-SCARs will not only benefit our understandings of genetic and epigenetic interaction in drug-hypersensitivity, but may also lead to new therapies or discovery of epigenetic biomarkers for the disease.http://www.chictr.org.cn/showprojen.aspx?proj=6628) and was approved by the Independent Ethics Committee of Institute of Clinical Pharmacology, Central South University (CTXY-110011-2). Patients who had taken allopurinol and were diagnosed with SJS/TEN  or DRESS by at least two dermatologists according to the consensus diagnostic criteria  and 20 allopurinol-tolerant subjects matched for age and gender were recruited to the study for DNA methylation profiling. Validation of candidate sites was performed in another sample cohort of 40 allopurinol-SCARs and 48 allopurinol-tolerants using pyrosequencing. The blood samples were taken on the first day when allopurinol-SCARs patients were admitted to the hospitals where the treatment was initiated. The rash happened a few days to around 3 weeks before reporting to the hospitals. The samples of allopurinol-tolerant patients were collected during their routine visit to the rheumatology clinics. A flowchart showing the design of the study is presented in Supplementary Figure Genomic DNA was extracted from 2 ml venous blood of each participant followed by centrifugation at 2,500 rpm for 15 min using Wizard® Genomic DNA Purification Kit . The extracted DNA underwent bisulfite conversion by the EZ DNA Methylation™ Kit (Zymo Research) according to the manufacturer's instructions. Converted DNA was then applied to the Illumina Infinium HM450 BeadChip arrays  and hybridized on BeadChips. Each chip was scanned by a HiScan 2000 (Illumina). The array measured methylation at >480,000 CpG sites in 99% of RefSeq genes. The array incorporated 484,660 probes in total, including the quality control probes.t-test analysis in the package of limma. The P-value mentioned herein is the adjusted P-value after multiple testing adjustment by false discovery rate (FDR)-controlling procedure. Probes with P < 0.05 were considered statistically different between the tested groups. All genes measured by the HM450 were used as the reference set in this analysis.The raw data of 484,660 probe signal intensities were obtained through the Genome Studio software. After comparing with the different probe florescence and probe types, the raw data were filtered, normalized and corrected. Then the probes were subjected to a second round of filtering by excluding probes targeting the X and Y chromosomes, probes overlapping with single nucleotide polymorphisms (SNPs), probes containing repeat sequences of ≥10 bp. Finally, 360,012 probes were left for differentially expressed methylation comparison through empirical Bayes moderated P-values of < 0.05 were deemed significantly different from background. We also included an additional filter requiring the magnitude of mean β-value difference ≥ 0.2 between groups for all group comparisons. Data analysis was performed by the statistical programming language R (version 3.1.0) (www.r-project.org) and packages from the Bioconductor project  terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway using the Gene Annotation Tool to Help Explain Relationships (GATHER) webtool  and unmethylated (U) signal intensities for each probe for each sample using the formula (β = M/M+U+100). To evaluate the exactitude of the obtained β value and remove noise, probes with detection 00. To evArray data were validated by bisulfite pyrosequencing, which was performed by a PyroMark Q24 ID pyrosequencing instrument according to the manufacturer's instructions (Qiagen). To increase template quantity for pyrosequencing assays, 200 ng of genomic DNA was treated with EZ DNA Methylation™ Kit. Thereafter, amplicons containing CpGs of interest were prepared by PCR using the forward, reverse and sequencing primers, which were designed by PyroMark Assay Design software (Qiagen), specific for bisulfite-converted DNA . All the numerical variables in this article were presented as mean ± standard deviation (SD). Independent-samples student's P < 0.05 and a minimum median β-value difference of 20%, we identified 20 probes (11 genes) with significant hypomethylation and 21 probes (15 genes) with significant hypermethylation in allopurinol-SCARs  in TEN, 339 methylated probes (231 genes) in SJS/TEN overlap, and 54 methylated probes (34 genes) in SJS, respectively  of methylated genes in different phenotypes of allopurinol-SCARs, among which psoriasis susceptibility 1 candidate 1 , protein kinase C zeta  and dual specificity phosphatase 22  were hypomethylated in each phenotype of allopurinol-SCARs.We next conducted a locus-by-locus differential DNA methylation analysis in each phenotype of allopurinol-SCARs under the condition of y Figure . We founP < 0.05, results obtained from choosing “inter from network”). The top 10 GO annotations are shown in Figure P < 0.05), with 6 or 3 genes in each pathway.In order to understand the features of these epigenetic regulated genes, a GATHER functional annotation analysis was performed. It indicated that the differentially methylated set of genes was significantly enriched in GO biological processes including regulation of physiological process, lymphocyte differentiation, histone deacetylation, protein amino acid deacetylation, anti-apoptosis, regulation of isotype switching, regulation of biological process, and negative regulation of apoptosis  of each gene, which are more likely to interfere with the function of promoters. Assay primers were provided in Supplementary Table To ensure that our findings were not dependent on the specific population analyzed, we performed gene-specific DNA methylation analysis on an independent cohort of 40 allopurinol-SCARs and 48 allopurinol-tolerant controls , CpG 1 and CpG2. CpG 1 and CpG2 were unintentionally found locus after the array site , which imply that DRESS is less in association with PSORS1C1 hypomethylation. The results suggest that DRESS and SJS/TEN are parts of the same spectrum of diseases but with less severity. In parallel to the testing cohort, we combined the data of SJS, SJS/TEN overlap and TEN, and found the same tendency of methylation changes compared to the allopurinol-tolerant controls . As expected, we observed that all the cases manifested hypomethylation in the validation samples compared to the controls . Patients with PSORS1C1 methylation level lower than 90% had 30.22-fold  higher risk to develop allopurinol-SCARs than allopurinol-tolerants .We also quantitatively measured methylation at P < 0.05 for differential methylation. Since DNA methylation studies in complex diseases did not obtain the same broad range of variation as cancer studies  synovial tissues, indicating that PSORS1C1 might play an important role in the development of RA  and CBZ-SJS (Amstutz et al., PSORS1C1 is a pre-existing trait that increases susceptibility to allopurinol-SCARs. If this is the case, PSORS1C1 hypomethylation is the risk factor that can be utilized to predict the susceptible patients before they are admitted to the hospital. But compared with PSORS1C1 hypomethylation, it is obvious that HLA-B*5801 is a much more reliable susceptibility marker for allopurinol-SCARs, as it has a much higher positive predictive value, negative predictive value and significance. Moreover, as a genetic factor, allele HLA-B*5801 is easier to detect via genotyping of DNA isolated from whole blood, with no need for biopsy of the susceptible tissue. It is however possible that the observed epigenomic alterations may be the result of disease progression, which dynamically change along with the severity of the disease. Thus, it could be a predictor performed in the affected tissue while therapy for the disease is still in progress to know the severity and prognosis of the disease. This test could be done during the onset of the disease to monitor possible adverse reactions and the treatment effect. In any case, all the patients are selected during disease onset period in our study, demonstrating that allopurinol-SCARs are associated with aberrantly methylated genes. Aberrant DNA methylation indicates that such changes may contribute to the disease and may help to understand possible underlying mechanisms. Further studies could measure the methylation levels before, during and after the disease to examine changes during disease onset and progression.An important issue that needs to be addressed in our study is whether the identified disease-related epigenome changes precede disease onset, or result from the disease progression. The blood samples of allopurinol-SCARs patients were taken after developing adverse reactions. The aim of our study was, in the first instance, to retrospectively review their epigenomic traits that may make patients susceptible to develop drug hypersensitivity. However, we can only know who the patients are after onset of the disease and when they are admitted to the hospital. There are a few days of delay between onset of the adverse reactions and admission to the hospital. So, we presume that the differential methylation of Our study has limitations. We only investigated peripheral blood for ascertaining DNA methylation, since it is difficult to obtain skin tissue samples from patients with allopurinol-SCARs. Blood may not be an ideal surrogate tissue, despite recent researches support the utilization of peripheral blood lymphocyte DNA to detect epigenetic biomarkers of organ-specific diseases (Anglim et al., PSORS1C1 was significantly associated with the development of allopurinol-SCARs, suggesting that aberrant DNA methylation may be a mechanism of allopurinol-SCARs.In conclusion, our study demonstrated a genome-scale difference in peripheral blood DNA methylation profiles between allopurinol-SCARs and allopurinol-tolerant controls when the disease occurred. Our results demonstrated that hypomethylation of BS and LC performed lab experiments, analyzed the data and wrote the paper; JL and HX collected clinical patient's information; YX, LH, ZL, MZ, FH and XY collected DNA samples and made records; XiaopingC, HZ, ZL, XiangC, and WZ designed the study and revised the manuscript. All the authors contributed to the final paper.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 reviewer TS and handling editor declared their shared affiliation.


Due to age dependent organ manifestation, diagnosis of Marfan syndrome (MFS) is a challenge, especially in childhood. It is important to identify children at risk of MFS as soon as possible to direct those to appropriate treatment but also to avoid stigmatization due to false diagnosis. We published the Kid-Short Marfan Score (Kid-SMS) in 2012 to stratify the pre-test probability of MFS in childhood. Hence we now evaluate the predictive performance of Kid-SMS in a new cohort of children. We prospectively investigated 106 patients who were suspected of having MFS. At baseline, children were examined according to Kid-SMS. At baseline and follow-up visit, diagnosis of MFS was established or rejected using standard current diagnostic criteria according to the revised Ghent Criteria (Ghent-2). At baseline 43 patients were identified with a risk of MFS according to Kid-SMS whereas 21 patients had Ghent-2 diagnosis of MFS. Sensitivity was 100%, specificity 77%, negative predictive value 100% and Likelihood ratio of Kid-SMS 4.3. During follow-up period, three other patients with a stratified risk for MFS were diagnosed according to Ghent-2. We confirm very good predictive performance of Kid-SMS with excellent sensitivity and negative predictive value but restricted specificity. Kid-SMS avoids stigmatization due to diagnosis of MFS and thus restriction to quality of life. Especially outpatient pediatricians and pediatric cardiologists can use it for primary assessment. Marfan syndrome (MFS) is an inherited connective tissue disorder with multifaceted phenotype involving cardiac, ophthalmologic and skeletal symptoms as well as skin, lung and dura abnormalities ,2. Due tFBN1 mutation [et al. published revised Ghent Criteria (Ghent-2)   in the Marfan clinic at the University Heart Center Hamburg. All patients had complete assessment of manifestations of MFS with echocardiography and clinical examination. If necessary and reasonable we also accomplished magnetic resonance imaging (MRI) and genetic analysis.At baseline, children were examined according to Kid-SMS. At baseline and follow-up visit, diagnosis of MFS was established or rejected using current standard diagnostic criteria according to Ghent-2.Roman et al. [We performed echocardiography with General Electric Vivid 7 with 10, 5 and 3 MHz probes. To obtain measurement of aortic root diameters we operated in parasternal long axis view on 2D and M-Mode images using leading edge to leading edge technique at end diastole. We evaluated dilatation of sinus of valsalvae according to n et al. . For then et al. . We estiWe collected data using Filemaker software V.10 pro advanced. To perform statistical analysis, we used SPSS V16.0 and for tables and figures we used Microsoft Excel 2003 and SPSS V16.0.p values < 0.05 as significant. We compared qualitative data by Fisher’s exact test. We also determined positive likelihood ratio for Kid-SMS. Likelihood ratio larger than ten was considered as extremely reliable to identify patients with MFS, whereas values between three and ten were useful and values below three were not useful. Finally, we used Kaplan Meier analysis and log-rank test to display age of diagnosis with Kid-SMS and Ghent-2 [p values < 0.05 as significant.We expressed quantitative variables as means with standard deviation and qualitative data as numbers. For comparison of quantitative data between groups we used unpaired t test and considered  Ghent-2 . Age of To assess clinical data and samples, we obtained informed consent of patients or parents of patients. The study was approved by the Hamburg ethical board (Project identification code: PV 4005).Mean age of first presentation to Marfan clinic was 12.18 ± 5.49 years  .FBN1 mutation without Ghent-2 diagnosis of MFS, whereas Kid-SMS identified two at a risk of MFS. Besides, 14 patients were identified at risk of MFS without current diagnosis according to Ghent-2 or FBN1 mutation .Sensitivity of Kid-SMS was 100% (CI 95% 0.86 to 1.00), whereas specificity was 77% (CI 95% 0.66 to 0.85). Positive predictive value was 56% (CI 95% 0.40 to 0.71) and negative predictive value 100% (CI 95% 0.94 to 1.00). Likelihood ratio was 4.3, which reveals good predictive performance of Kid-SMS. Fisher’s exact test showed statistic significance   and age of declared risk stratification with Kid-SMS, which may be very high risk, high risk or moderate risk (continuous line). Analysis did not show significant difference concerning age of diagnosis or risk stratification  .In this study, all patients with diagnosed MFS according to Ghent-2 were stratified for risk of MFS with Kid-SMS, which is shown by perfect negative predictive value. Kid-SMS also defines a group where diagnosis cannot be assured yet but Kid-SMS recommends a safe follow-up regime. This avoids stigmatization and unnecessary restrictions in quality of life due to false diagnosis of chronic disease. Especially children should be protected from stigmatization to support psychosocial development und encourage setting of resources for psychosocial coping in life.FBN1 mutation without diagnosis with Ghent-2. Others (14) were neither diagnosed with Ghent-2 nor showed FBN1 mutation.At first presentation (baseline), Kid-SMS identified a distinctly larger group of patients with a risk of MFS than patients with diagnosis according to Ghent-2. During follow-up period, three patients were diagnosed according to Ghent-2, whereas Kid-SMS already stratified a risk of MFS in those patients at baseline. Nevertheless, there is a group of children with stratified risk of MFS where Ghent-2 was negative at final visit. Some of those (2) showed FBN1 mutation were at a moderate risk of MFS due to positive family history of MFS. Those patients were aged 1.1 and 8.4 years at final presentation. Due to age dependent organ manifestation in MFS symptoms may not be present especially in early childhood. Those patients need a strict follow-up regime to evaluate upcoming manifestations of MFS later on. They should be treated as patients with MFS and may subsequently be diagnosed according to Ghent-2.Both patients with FBN1 mutation, Kid-SMS did not predict a risk of MFS. One showed FBN1 mutation with isolated mitral and tricuspid valve prolapse. Thus, this patient would of course be followed up by cardiological evaluation. Consequently, in case of aortic enlargement or other cardiac pathologies, further diagnostic tools and necessary treatment would be induced. Both of the others showed a FBN1 mutation with a positive systemic score and no vascular involvement aged 15.8 and 17.6 years. Those children had no vascular symptoms, which are life-limiting in MFS. Even though MFS is unlikely in those patients they may have another connective-tissue disorders like MASS phenotype associated with FBN1 mutation.In three patients with Prediction of risk of MFS with Kid-SMS in patients without diagnosis according to Ghent-2 or genetic analysis was established in two patients who showed dilatation of sinus of valsalvae in combination with skeletal symptoms.Another five patients were at moderate risk of MFS because of dilatation of sinus of valsalvae. Even though there were no other symptoms of MFS those patients will be followed up in a specialized center and may be diagnosed later on with another disease involving thoracic aortic aneurysm . Seven sAlthough Kid-SMS also predicts a risk of MFS in some patients without disease, it will probably cover patients with other syndromes, including thoracic aortic aneurysms like Loeys-Dietz syndrome or Shprintzen-Goldberg syndrome, that also need regular follow-up. The main criteria in Kid-SMS are cardiac and vascular involvement of MFS. Dilatation of sinus of valsalvae is the most common feature in children with MFS . And it We were not able to examine larger groups of children with other syndromes, including thoracic aortic aneurysms, to analyze exact power of discrimination of Kid-SMS. Although improvement of differential diagnosis was one reason to revise the Ghent criteria, this is important for Kid-SMS but not the main focus . ConsideIn total, at risk stratification for MFS with Kid-SMS, children were younger at diagnosis than according to Ghent-2. Early identification of patients with a risk of MFS enables their inclusion in a safe follow-up regime and can identify the possible need for prophylactic medical treatment. Indeed, first presentation to a Marfan clinic is not the time of incidence of symptoms and thus not the time of detection of Kid-SMS, which may be the reason for absent significance. To evaluate a better reliable age of pre-test probability with Kid-SMS use of the score in outpatient children with reasonable suspicion of MFS is necessary. In our experience we see many children where Kid-SMS is the first diagnostic tool that correctly supports or excludes diagnosis of MFS.According to level of risk stratification with Kid-SMS, further examinations are recommended. Patients with very high risk of MFS, which is equivalent to diagnosis according Ghent-2, must be presented to a specialized center of MFS. In patients with a high risk or moderate risk with dilatation of SV, full examination concerning Ghent-2 has to be completed urgently, even though risk of MFS is lower in moderate risk patients. In areas where access to MRI and genetic analysis is limited, further examinations can be delayed as long as regular echocardiographic follow-up is assured. Also, performance of MRI may not be possible sometimes due to low age of patients where quality of MRI data is reduced due to fast heart rate and children would need anesthesia for examination. Patients with moderate risk of MFS due to positive family history further examinations are recommended to include or exclude them in a follow-up regime. Due to age dependent onset of organ manifestation, many children of our study group changed level of risk stratification during follow-up, which represents flexibility of the score. Except the change from negative to positive risk stratification, we only comment on first level of risk stratification of MFS in results and discussion. A restriction of the application of Kid-SMS, especially in outpatient care, is the performance of echocardiography, which requires expert experience. Nevertheless, this is much more easily available than a specialized pediatric center for MFS.Unfortunately, this study does not represent the use of Kid-SMS in outpatient children, which is indeed one of the most important skills of the score. To evaluate use of Kid-SMS in those children, further analysis and studies are necessary. But even though our clinic is a specialized center, we used Kid-SMS to give parents first advice for probability of MFS and to decide how urgent further investigations were. Kid-SMS involves symptoms that are also examined to assure diagnosis according to Ghent-2. Thus, performance of the score is not time-consuming and does not require additional examinations, which would limit its usefulness, especially in outpatient care. In summary, practice of Kid-SMS in this patient group was valuable for children with assumed MFS. Kid-SMS showed very good sensitivity and restricted specificity. Likelihood ratio shows good predictive performance of stratifying risk of MFS with Kid-SMS. It was easily executable and not time-consuming. After all, Kid-SMS is not supposed to replace Ghent-2 diagnosis of MFS. Instead, it should be used in addition to Ghent-2 to improve diagnosis in childhood and assess early pre-test probability of MFS. Patients without a definite diagnosis or exclusion of disease are thus not stigmatized with the diagnosis of MFS but in a safe follow-up regime.Especially pediatricians and pediatric cardiologists not working in specialized centers can use it for a risk assessment and to estimate whether or not a patient requires transfer to a specialized center. Particularly for them, Kid-SMS is an easily executable diagnostic tool for first screening of MFS in childhood.Whereas diagnosis of MFS is sophisticated, Kid-SMS is a useful tool to assess early pre-test probability of MFS in childhood, especially for pediatricians and pediatric cardiologists in out-patient care.


Plasmodium falciparum transmission to mosquitoes. We aimed to assess the efficacy and safety of primaquine and methylene blue in preventing human to mosquito transmission of P falciparum among glucose-6-phosphate dehydrogenase (G6PD)-normal, gametocytaemic male participants.Primaquine and methylene blue are gametocytocidal compounds that could prevent P falciparum malaria. G6PD-normal participants with gametocytes detected by blood smear were randomised 1:1:1:1 in block sizes of eight, using a sealed-envelope design, to receive either sulfadoxine-pyrimethamine and amodiaquine, sulfadoxine-pyrimethamine and amodiaquine plus a single dose of 0·25 mg/kg primaquine, dihydroartemisinin-piperaquine, or dihydroartemisinin-piperaquine plus 15 mg/kg per day methylene blue for 3 days. Laboratory staff, investigators, and insectary technicians were masked to the treatment group and gametocyte density of study participants. The study pharmacist and treating physician were not masked. Participants could request unmasking. The primary efficacy endpoint, analysed in all infected patients with at least one infectivity measure before and after treatment, was median within-person percentage change in mosquito infectivity 2 and 7 days after treatment, assessed by membrane feeding. This study is registered with ClinicalTrials.gov, number NCT02831023.This was a phase 2, single-blind, randomised controlled trial done at the Clinical Research Centre of the Malaria Research and Training Centre (MRTC) of the University of Bamako . We enrolled male participants aged 5–50 years with asymptomatic Between June 27, 2016, and Nov 1, 2016, 80 participants were enrolled and assigned to the sulfadoxine-pyrimethamine and amodiaquine (n=20), sulfadoxine-pyrimethamine and amodiaquine plus primaquine (n=20), dihydroartemisinin-piperaquine (n=20), or dihydroartemisinin-piperaquine plus methylene blue (n=20) groups. Among participants infectious at baseline (54 [68%] of 80), those in the sulfadoxine-pyrimethamine and amodiaquine plus primaquine group (n=19) had a median 100% (IQR 100 to 100) within-person reduction in mosquito infectivity on day 2, a larger reduction than was noted with sulfadoxine-pyrimethamine and amodiaquine alone . The dihydroartemisinin-piperaquine plus methylene blue (n=11) group had a median 100% (IQR 100 to 100) within-person reduction in mosquito infectivity on day 2, a larger reduction than was noted with dihydroartemisinin-piperaquine alone . Haemoglobin changes were similar between gametocytocidal arms and their respective controls. After exclusion of blue urine, adverse events were similar across all groups .P falciparum transmission. Both primaquine and methylene blue were well tolerated.Adding a single dose of 0·25 mg/kg primaquine to sulfadoxine-pyrimethamine and amodiaquine or 3 days of 15 mg/kg per day methylene blue to dihydroartemisinin-piperaquine was highly efficacious for preventing Bill & Melinda Gates Foundation, European Research Council. Since 2000, scale-up of interventions has led to substantial gains in malaria control across sub-Saharan Africa.Plasmodium falciparum in areas of low malaria transmission and emerging antimalarial drug resistance.Several malaria control and elimination strategies target the plasmodium parasite in human beings, including case management, seasonal malaria chemo-prevention, and mass drug administration. Drug combinations used in these strategies do not target the gametocyte stage of the parasite, allowing continued human to mosquito transmission despite clearance of the asexual blood stage infection. WHO recommends addition of a single 0·25 mg/kg dose of primaquine (single low-dose primaquine) to standard artemisinin-based combination therapy to reduce onward transmission of Evidence before the studyClinicalTrials.gov and PubMed between Sept 18, 2015, and Sept 15, 2017, using the search terms “primaquine” OR “methylene blue” AND “malaria” AND “falciparum”, OR “primaquine” OR “methylene blue” AND “transmission”, OR “primaquine” OR “methylene blue” AND “gametocyte”. No language restrictions were used in the search. Although numerous studies, including randomised controlled trials, systematic reviews, and meta-analyses, have reported on the efficacy and safety of single low-dose primaquine as a Plasmodium falciparum gametocytocidal drug, only four efficacy studies used membrane feeding assays, the gold standard for infectivity assays, to assess the transmission-blocking effect of primaquine. None of these studies assessed the efficacy of primaquine combined with sulfadoxine-pyrimethamine plus amodiaquine. One study in Colombia assessed the efficacy of adding a single dose of 0·75 mg/kg primaquine to sulfadoxine-pyrimethamine plus amodiaquine on P falciparum gametocyte clearance. Using blood smear microscopy, authors found lower gametocytaemia on days 4 and 8, but no clearance of gametocytes by day 8. Since no direct measurements of membrane feeding assays were done, it is unclear if the persisting gametocytes would have been infectious to mosquitoes. Eight published trials of methylene blue were identified. Two studies assessed the effect of methylene blue with either artesunate, amodiaquine, or artesunate-amodiaquine on gametocytes. Groups receiving methylene blue had lower gametocytaemia by at least day 7. Neither of these studies assessed transmission using membrane feeding assays, nor was methylene blue paired with dihydroartemisinin-piperaquine, one of the major drugs used in community drug interventions, known for its long prophylactic period.We searched Added value of this studyP falciparum. Our findings show that antimalarial combinations of sulfadoxine-pyrimethamine plus amodiaquine with 0·25 mg/kg primaquine and dihydroartemisinin-piperaquine with a 3-day treatment course of 15 mg/kg methylene blue are safe and block human to mosquito transmission by day 2 post-treatment.To our knowledge, this is the first study to use the membrane feeding assay to directly quantify the effect of primaquine (partnered with sulfadoxine-pyrimethamine plus amodiaquine) and methylene blue (partnered with dihydroartemisinin-piperaquine) on human to mosquito transmission of Implications of all the available evidencePotent gametocytocidal drugs primaquine and methylene blue are two promising transmission-blocking tools that can be used for falciparum malaria control and elimination. Future studies should focus on assessment of the community-level effect of adding gametocytocidal drugs to first-line treatments and large-scale, drug-based interventions, including seasonal malaria chemoprophylaxis (sulfadoxine-pyrimethamine plus amodiaquine) and mass drug administration (dihydroartemisinin-piperaquine).P falciparum transmission is unclear. The antimalarial combination sulfadoxine-pyrimethamine and amodiaquine has strong treatment and chemoprophylactic effects. Sulfadoxine-pyrimethamine and amodiaquine is the standard drug combination for seasonal malaria chemoprevention and has been highly effective in reducing malaria morbidity and mortality in the Sahel and sub-Sahel regions of Africa, where seasonal malaria chemoprevention is widely implemented.First, whether addition of single low-dose primaquine to non-artemisinin-based combination therapy can prevent P falciparum gametocytes, resulting in partial reductions in human to mosquito transmission.14The second question is whether addition of methylene blue  to artemisinin-based combination therapy reduces post-treatment transmission potential. Methylene blue has highly potent gametocytocidal effectsP falciparum gametocytes (the parasite stage infectious to mosquitoes), the addition of gametocytocidal drugs to these regimens might prevent transmission shortly after treatment and limit the spread of drug-resistant parasites.Since neither sulfadoxine-pyrimethamine and amodiaquine or dihydroartemisinin-piperaquine target the mature stage of The aim of this study was to establish the safety and efficacy of the addition of single low-dose primaquine to sulfadoxine-pyrimethamine and amodiaquine, and the addition of methylene blue to dihydroartemisinin-piperaquine in reducing human to mosquito transmission among glucose 6-phosphate dehydrogenase (G6PD)-normal male participants in Mali.This was a phase 2, single-blind, randomised controlled trial done at the Clinical Research Centre of the Malaria Research and Training Centre (MRTC) of the University of Bamako .P falciparum malaria at enrolment, had at least two P falciparum gametocytes per 500 white blood cells on thick film microscopy , and had an absence of other non-P falciparum species on blood film. Female participants were excluded to mitigate the risk of haemolysis through incorrect classification of G6PD-deficient female heterozygotes. Participants were also excluded if they had a serious or chronic illness , weighed 80 kg or more, reported antimalarial use within 7 days of screening, or reported allergies to study drugs.Study participants were recruited from the town of Ouélessébougou, Mali, and its surrounding villages. Malaria transmission in this region is hyperendemic and highly seasonal with peaks from July to November. A study team, comprised of clinicians and lab technicians, met with community leaders, village health workers, and heads of households from each village to explain the study and obtained approval to conduct the study. Before participant screening, village health workers used a door-to-door approach to inform households of the date and location where consenting and screening would take place. Participants were enrolled if they were male, aged 5–50 years, G6PD-normal by Carestart G6PD rapid diagnostic test , had a haemoglobin concentration of at least 10 g/dL, had asymptomatic Participants aged at least 18 years provided written informed consent before screening and enrolment. Parental consent was obtained for participants younger than 18 years, and assent was obtained from children aged 12–17 years.Ethical approval was granted by the Ethics Committee of the Faculty of Medicine, Pharmacy, and Dentistry of the University of Science, Techniques, and Technologies of Bamako , the Committee on Human Research at the University of California San Francisco , and the Research Ethics Committee of the London School of Hygiene & Tropical Medicine . For the full study protocol see the Patients were individually randomised in a 1:1:1:1 ratio, in block sizes of eight, to receive either sulfadoxine-pyrimethamine and amodiaquine, sulfadoxine-pyrimethamine and amodiaquine with single low-dose primaquine, dihydroartemisinin-piperaquine, or dihydroartemisinin-piperaquine with methylene blue. A UCSF investigator (JMB) randomly generated the treatment assignment using Excel , which was linked to a pre-specified, unique participant ID number. The investigator assembled sealed, opaque envelopes with the participant ID number on the outside and treatment assignment contained within. The site pharmacist in Mali (AM) opened the envelopes and provided treatment. The study assignment was masked to investigators and staff involved in assessing all laboratory outcomes. Insectary technicians were masked to the treatment group and gametocyte density of study participants. The study pharmacist and treating physician were not masked. Participants could ask the study physician which treatment they received.Upon enrolment and collection of day 0 samples, treatment regimens were administered to participants after a fatty snack (biscuits) given to prevent gastrointestinal side-effects and vomiting , 2, and 7. Venous blood was collected in heparin tubes stored at 37°C and placed in a membrane feeding system within 1 min of collection. For the first three enrolled participants, about 70 Pfs25) and male  gametocyte mRNA, 100 μL of blood was collected in EDTA and transferred into 500 μL of RNAprotect Cell Reagent . Samples were stored at MRTC at −80°C until shipment on dry ice at a controlled temperature (−80°C to −70°C). Total nucleic acids were extracted by MagNAPure LC automated extractor . qRT-PCR was done as previously described using sex-specific trendlines of cultured gametocytes.Measurements of gametocyte density were taken before treatment (day 0), and on days 1, 2, 7, 14, 28, and 42 after enrolment. Gametocyte density was measured using blood smear microscopy and by real-time reverse transcription-PCR (qRT-PCR). Blood slides were stained with Giesma and independently read by expert research microscopists over 500 fields for quantification of gametocytes and asexual parasites. For qRT-PCR quantification of female  if at least one dissected mosquito had at least one oocyst present.Secondary outcomes included the prevalence, density, circulation time (days), and sex ratios of female and male gametocytes. Estimates of gametocyte sex ratio were restricted to samples with a total gametocyte density greater than 0·2 gametocytes per μL (20 gametocytes per 100 μL of blood sample).Safety outcomes assessed included the number of adverse events and the mean within-person change in haemoglobin concentration per group after treatment, the proportion of participants who had a greater than 25% decrease in haemoglobin, and the proportion of participants with 8 g/dL or less haemoglobin at any point during follow-up. Adverse events were assessed at each follow-up visit and monitored passively by medical staff. All adverse events were graded by severity  according to the study's standard operating procedures and based on the Division of Microbiology and Infectious Diseases (DMID) toxicity tables. Adverse events were further categorised based on whether they were considered serious by the treating physician, and categorised by their causal relationship to the study drugs . Adverse events were considered causal if they were categorised as either possibly, probably, or definitely related to study drug.Based on preliminary data from a similar infectivity trial,vs treatment group). Our analysis of the change in mosquito infectivity included all randomised participants who had an infectivity measurement before treatment and at least one infectivity measurement after treatment. Additionally, we did an analysis of change in infectivity among the subset of participants who infected at least one mosquito after treatment.For the primary efficacy outcome, we calculated the median within-person percentage change in infectivity and accompanying IQRs for each group. Positive percentages indicated reductions in infectivity. We used a one-sided non-parametric Wilcoxon rank-sum test for pairwise comparisons of the change in infectivity after treatment .vs sulfadoxine-pyrimethamine and amodiaquine alone; and dihydroartemisinin-piperaquine plus methylene blue vs dihydroartemisinin-piperaquine alone), unless otherwise stated. Statistical analyses were done using Stata version 14.0 and SAS version 9.4.All other comparisons between proportions were done using two-sided tests, and we used Fisher's exact test for between-group comparisons and McNemar's test for within-group comparisons. Wilcoxon's signed rank tests were used for within-group comparisons and Wilcoxon rank-sum tests were used for between-group comparisons. All analyses compared treatment with or without gametocytocidal compounds .The trial was overseen by an independent data safety monitoring committee, monitored by an external clinical trials monitoring group , and registered with The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit the publication.Between June 27, 2016, and Nov 1, 2016, 1749 male participants were screened for eligibility. 80 participants were enrolled and randomly assigned to receive either sulfadoxine-pyrimethamine and amodiaquine plus primaquine (n=20), dihydroartemisinin-piperaquine plus methylene blue (n=20), and their comparator groups, sulfadoxine-pyrimethamine and amodiaquine alone (n=20), and dihydroartemisinin-piperaquine alone n=20; . 72 90%.Figure 180 membrane feeding assays were done before treatment, 80 on day 2 after treatment, and 76 on day 7 after treatment. 20 599 mosquitoes were used, 18 026 (88%) of which survived to the day of dissection. Before treatment, 54 (68%) of 80 individuals infected at least one mosquito , and 908Before treatment, all study participants with blood samples available for molecular analysis (n=78) had detectable concentrations of both female and male gametocytes. Gametocyte sex ratios were female biased at enrolment, with no differences in gametocyte prevalence or density between study arms . The preThe mean circulation time of female gametocytes was longer than that of male gametocytes for all treatment arms . The sulCorrelations were found between qRT-PCR gametocyte density and mosquito infectivity rates . Fairly Absolute and within-person percentage change in mean haemoglobin concentration did not differ between the sulfadoxine-pyrimethamine and amodiaquine plus primaquine and sulfadoxine-pyrimethamine and amodiaquine alone groups or the dihydroartemisinin-piperaquine plus methylene blue and dihydro-artemisinin-piperaquine alone groups at any point during follow-up . One parOverall, 59 (74%) of 80 participants reported a total of 162 adverse events during follow-up, with 19 of these events being bluish colouration of urine . The numThis study shows the pronounced transmission-blocking effects of addition of gametocytocidal drugs to standard antimalarial treatments. Addition of single low-dose primaquine to sulfadoxine-pyrimethamine and amodiaquine or methylene blue to dihydroartemisinin-piperaquine resulted in almost complete blockage of human to mosquito transmission by day 2, and a larger reduction in transmission than that offered by sulfadoxine-pyrimethamine and amodiaquine or dihydroartemisinin-piperaquine alone. No differences in adverse events (excluding blue urine) or haemolysis were observed between groups, suggesting primaquine and methylene blue are safe in this population.P falciparum infection to prevent formation of mature gametocytes is crucial for malaria control.We examined gametocyte dynamics and infectivity to mosquitoes following treatment with sulfadoxine-pyrimethamine and amodiaquine plus primaquine or dihydroartemisinin-piperaquine plus methylene blue, two drug regimens that have important roles in malaria control and elimination efforts. The importance of our direct assessments of infectivity is illustrated by the observation that the transmission-blocking effect of gametocytocidal drugs precedes gametocyte clearance.P falciparum gametocyte carriers using mosquito infectivity assays, confirming the potency of methylene blue as suggested by previous in-vitroTo our knowledge, this study is the first to formally show the potent transmission-blocking effect of methylene blue in P falciparum gametocyte carriage and infectivity. Sulfadoxine-pyrimethamine and amodiaquine alone did not have an effect on reducing human to mosquito transmission in the week following treatment. Most individuals treated with sulfadoxine-pyrimethamine and amodiaquine carried gametocytes for at least 1 month following treatment with no reduction in mosquito infection rates achieved by day 7 after treatment. Sulfadoxine-pyrimethamine and amodiaquine is recommended by WHO for use in seasonal malaria chemoprevention. Although the primary objective of seasonal malaria chemoprevention is to reduce malaria morbidity and mortality and not to directly reduce transmission, adding single low-dose primaquine to seasonal malaria chemoprevention might confer indirect community benefits for malaria transmission by reducing post-treatment transmission potential. Transmission reduction by seasonal malaria chemoprevention could be further improved by expanding the age range to include adolescents because gametocyte carriage is high in this populationWhen combined with sulfadoxine-pyrimethamine and amodiaquine,Countries moving towards malaria elimination could consider adding a transmission-blocking drug to first-line treatments or community-wide mass drug administration to rapidly reduce population-level malaria transmission. Modelling studies suggest that, given adequate coverage and multiple rounds of mass drug administration, addition of gametocytocidal drugs  to antimalarial combinations with a long prophylactic period  might result in larger reductions in transmission than with dihydroartemisinin-piperaquine treatment alone.Our study revealed that gametocytocidal drugs might have different effects on male and female gametocytes. The strongly reduced circulation time of male gametocytes following methylene blue treatment supports hypotheses generated from in-vitro assessments that methylene blue disproportionally affects male gametocytes.P falciparum gametocyte densities. Therefore, our findings might not represent the effect of single low-dose primaquine and methylene blue in other malaria-infected populations, particularly those with lower gametocyte densities who have lower pre-treatment and post-treatment transmission potential and G6PD-deficient populations. Although studies in Burkina Faso showed that methylene blue is unlikely to be responsible for clinically relevant haemolysis in individuals with or without G6PD-deficiency,Our study has several limitations. First, the study population analysed was not as per intention to treat, as one patient was lost to follow-up and seven patients withdrew. This factor was unlikely to cause attribution bias since the reasons given for withdrawals were unrelated to study drug. Second, our study endpoint was the proportion of oocyst-positive mosquitoes, based on the assumption that even low oocyst densities are likely to render mosquitoes infective.P falciparum transmission in G6PD-normal male participants. Further work is needed to understand what effect these findings could have on malaria control and elimination strategies, using mathematical models and community trials to assess costs, risks, and benefits of adding single low-dose primaquine or methylene blue to existing regimens.Adding a single dose of 0·25 mg/kg primaquine to sulfadoxine-pyrimethamine and amodiaquine or 3 days of 15 mg/kg per day methylene blue to dihydroartemisinin-piperaquine were safe and highly efficacious methods for preventing human to host Division of Microbiology and Infectious Diseases toxicity tables see https://www.niaid.nih.gov/research/dmid-safetyreporting-pharmacovigilanceFor


The present study aimed to systematically evaluate the effectiveness and safety of the intrafascial and interfascial nerve sparing (ITR-NS and ITE-NS) radical prostatectomy. PubMed, Embase, and Cochrane Library databases were searched for eligible studies. Meta-analysis with random-effects model was performed. Six comparative trials were selected and embraced in this research, including one randomized controlled trial, three prospective comparative trials, and two retrospective comparative trials. With regard to perioperative parameters, no significant association of operative time, blood loss, transfusion rates, duration of catheterization, and hospital stay existed between ITR-NS and ITE-NS. With respect to the functional results, ITR-NS had advantages in terms of both continence and potency recovery compared with ITE-NS. In reference to the oncologic results, the ITR-NS showed lower overall positive surgical margin (PSM) compared with ITE-NS but pT2 PSM and biochemical recurrence free rates were similar to the two surgical types. This study demonstrates that ITR-NS has better continence at 6 mo and 36 mo and better potency recovery at 6 mo and 12 mo postoperatively, regardless of the surgical technique. The cancer control of ITR-NS was also better than that of ITE-NS. This may be explained by the fact that patients in ITE-NS group present higher risk cancer than patients in ITR-NS group. Radical prostatectomy (RP) is the standard surgical treatment for localized prostate cancer. However, the postoperative impotence and especially the urinary incontinence following RP are still a matter of trouble for patients5.Prostate cancer is the most common nonskin malignancy in western men and the second leading cause of cancer-related death among men in United Stateset al.6 performed a systematic review and meta-analysis that summarized the existing evidence on the influence of the preservation of the NVBs on continence after RP. Their study suggested that early urinary continence rate (at 6 mo time point) was improved for patients undergoing nerve-sparing RP compared with patients undergoing non-nerve-sparing RP. In recent years, certain urologists have compared intrafascial nerve-sparing (ITR-NS) with interfascial nerve-sparing (ITE-NS) RP, and the results are inconclusive12. The ITR-NS technique is considered a dissection that follows a plane on the prostate capsule and it allows a whole-thickness preservation of the NVBs13. Reeves and colleagues did not assess the specific nerve-sparing technique6.Many approaches, such as open retropubic RP, laparoscopy RP, and robot-assisted RP, have been applied in RP. Recently, Reeves To answer this important question, we carried out the present systematic review and meta-analysis to summarize the current existing evidence for clinical practice. In this study, we comprehensively evaluated the functional outcomes, oncologic outcomes, and perioperative parameters of ITR-NS and ITE-NS.14. The study protocol of this systematic review and meta-analysis was published in the PROSPERO register (registration number: CRD42016038687).This systematic review and meta-analysis was performed according to the Preferred Reporting Items of Systematic Reviews and Meta-analysis (PRISMA) statementA literature search of the PubMed, Embase, and Cochrane Library was conducted up to March 2016 (updated to July 2017) to identify potentially relevant trials. The following terms were searched: (“prostatic neoplasms” OR “prostate” OR “prostate cancer”) AND  AND . The study language was restricted to English. In addition, reference lists in the recent reviews, meta-analysis, and included articles were checked for identifying any potentially relevant studies. The detailed search strategy for each database is presented in Supplementary Table The inclusion criteria were established according to PICOS  principle as presented in Table Two authors independently screened titles and abstracts of all search results. Studies were selected based on the pre-specified inclusion and exclusion criteria. Any discrepancy was resolved by discussion. Two authors independently extracted the following data from each identified study: study details , methods , patients , intervention , and outcome .17. The Cochrane collaboration’s tool assesses risk of bias in six domains: (1) selection bias; (2) performance bias; (3) detection bias; (4) attribution bias; (5) reporting bias; and (6) other bias15. The Newcastle-Ottawa Scale assesses risk of bias in three domains: (1) selection of the study population; (2) comparability of groups; and (3) ascertainment of outcome16. We evaluated that follow-up was adequate if the maximum follow-up was more than 2 yr (i.e. 24 mo). The quality of evidence was assessed according to the GRADE system using GRADEpro GDT software.We used the Cochrane collaboration’s tool for risk of bias assessment of RCTs and the Newcastle-Ottawa Scale for risk of bias assessment of observational studiesp ≤ 0.1) test and I2 metric. All statistical analysis was performed using RevMan 5.3 . A two-sided p value less than 0.05 represented a statistically significant difference, except for heterogeneity test. Publication bias was detected using funnel plot if the included studies were more than five for each outcome.Meta-analysis was performed to aggregate the results if studies were sufficiently similar. Due to the clinical heterogeneity implicated in the included studies, random-effects model was applied to estimate summary risk ratios (RRs) and corresponding 95% confidence intervals (CIs). Sensitivity analysis was conducted through sequentially excluding retrospective studies. Subgroup analysis according to timing of outcome measurement was performed if sufficient data was available. Heterogeneity was tested using chi-square . Patients in one study8 who underwent RP were relatively younger than those in other studies included in this meta-analysis. Furthermore, the surgical techniques used in the included studies were different. These factors would introduce some selection bias and clinical heterogeneity.Tables 12 reported the postoperative urinary continence recovery rate at 3 mo; two studies9 reported it at 6 mo; five studies12 reported it at 12 mo; one study12 reported it at 36 mo . The results of meta-analysis with random-effects model showed that patients undergoing ITR-NS had significantly better continence outcomes reported on 6 mo  and 36 mo  compared with those undergoing ITE-NS  and 12 mo   Fig. . Sensiti14) Fig. . The qua10 reported the postoperative potency recovery rate at 3 mo; two studies9 reported it at 6 mo; four studies10 reported it at 12 mo . The results of meta-analysis with random-effects model showed that patients undergoing ITR-NS had significantly better potency outcomes reported on 6 mo  and 12 mo  compared with those suffering from ITE-NS   Fig. . Sensiti06) Fig. . The qua11 reported the PSM rate after the RP . The results of meta-analysis with random-effects model showed that patients undergoing ITR-NS had significantly lower PSM rate compared with those experiencing ITE-NS . The results of meta-analysis with random-effects model showed that patients receiving ITR-NS had similar pT2 PSM rate compared with those undergoing ITE-NS . The results of meta-analysis with random-effects model showed that patients undergoing ITR-NS had similar BCR free rate compared with those experiencing ITE-NS  and oncologic outcome  compared with ITE-NS. These findings were supported by sensitivity analyses which took the prospective studies into consideration alone. The results suggested that there was a difference in continence between techniques at 6 months and 36 months but not at 12 months. This might be caused by the various procedures of different techniques or it was a spurious result.20 , the PSM and perioperative complication rates of primary surgical treatments21, the primary surgical treatments for prostate cancer26, transperitoneal and extraperitoneal robot-assisted RP27, and the efficacy and safety of conventional laparoscopic RP with a transperitoneal approach versus that of an extraperitoneal approach28. Therefore, none of these studies focused on the surgical technique of RP. In 2015, Reeves et al.6 systematically reviewed the association of NVBs sparing in RP with postoperative urinary continence outcomes. They found that avoiding damage to the nerve activity surrounding the prostate promotes urinary control in the first 6 mo after nerve sparing RP. In addition, from theoretically, the ITR-NS had better function than ITE-NS in functional outcomes as we mentioned in introduction. The result of our systematic review and meta-analysis was supported by the Reeves’s study6.To our knowledge, this systematic review and meta-analysis is the first study to comprehensively evaluate this topic. The previous reviews or systematic reviews or meta-analysis evaluated the techniques of RPet al.29 proposed a grading system based on four grades of dissection according to veins surrounding the prostate. Schatloff et al.30 proposed a grading system based on five grades of dissection according to arterial periprostatic vasculature. The grade 1 of Tewari’s approach and grade 5 of Schatloff’s approach was equal to ITR-NS. As we acknowledged that cancer control is the most important goal of RP. The different dissection planes concept aims for an incremental security margin of prostate, instead of true incremental nerves sparing13. In this review, we found that ITR-NS was not significantly presented with risk of PSM, pT2 PSM and BCR free rate compared with ITR-NS. This might be due to restricted patient selection of the included studies. Therefore, the choice of surgical technique or dissection plane should be made based on the specific situation of patients in clinical practice, such as clinical examination, biopsy results, and imaging results6.Tewari 9 used laparoscopic RP; two studies11 used robot-assisted RP; and two studies12 used open retropubic RP. These six comparative trials included one RCT7, three prospective comparative trials12, and two retrospective comparative trials11. We performed sensitivity analysis through excluding retrospective studies. The summary result of PSM rate was changed while excluding the retrospective studies. Therefore, the robustness of the result is weak. Third, we only included studies published in English. In addition, grey literature was not included. Hence, language bias might occur in this study. Fourth, due to limited number of included studies, we did not fully detect the publication bias. Of course, the publication bias is inevitable because we included studies published in English and excluded grey literature. The publication bias might decrease the reliability and credibility of this meta-analysis and systematic review. Moreover, the sample size and statistical power were relatively insufficient to identify the true difference of the two surgical techniques. Ultimately, the meta-analysis is a secondary analysis and its quality is based on the included studies. Our meta-analysis included studies with a RCT with high risk of bias and five longitudinal studies with moderate risk of bias. Therefore, the quality of the evidence was consequentially degraded.Although we used a systematic method to perform the meta-analysis, certain limitations also should be taken into consideration. First, our systematic review only identified one RCT, and the absence of high quality RCT might weaken the reliability of the meta-analysis. Second, low to moderate between-study heterogeneity was detected, which might be attributed to different surgical techniques, study design, selection bias, and surgeon experience. Selection bias between the two techniques was a major bias in the present meta-analysis, which implied that higher risk patients tended to undergo interfascial technique and lower risk patients tended to an intrafascial technique. However, patients with Gleason score more than 8 were only in two trials and the PSA levels were all similar as presented in Table With respect to further researches, multi-center clinical trials, if possible, RCTs should be performed to evaluate the effectiveness and safety of ITR-NS and ITE-NS. In addition, further studies also should elucidate the functional anatomy of urinary continence and erectile function.In conclusion, this systematic review and meta-analysis demonstrates that ITR-NS has better continence at 6 mo and 36 mo and better potency recovery at 6 mo and 12 mo postoperatively, regardless of the surgical technique. This finding might be due to more nerves were saved and less damage of the periprostatic tissue in ITR-NS compared with ITE-NS. The cancer control of ITR-NS was also better than that of ITE-NS. This may be explained by the fact that patients in ITE-NS group present higher risk cancer than patients in ITR-NS group. Further studies are needed to verify the conclusion in future.Supplementary tables