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Because cardiotoxicity is one of the leading causes of drug failure and attrition, the design of new protocols and technologies to assess proarrhythmic risks on cardiac cells is in continuous development by different laboratories. Current methodologies use electrical, intracellular Ca2+, or contractility assays to evaluate cardiotoxicity. Increasingly, the human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are the in vitro tissue model used in commercial assays because it is believed to recapitulate many aspects of human cardiac physiology. In this work, we demonstrate that the combination of a contractility and voltage measurements, using video-based imaging and fluorescence microscopy, on hiPSC-CMs allows the investigation of mechanistic links between electrical and mechanical effects in an assay design that can address medium throughput scales necessary for drug screening, offering a view of the mechanisms underlying drug toxicity. To assess the accuracy of this novel technique, 10 commercially available inotropic drugs were tested (5 positive and 5 negative). Included were drugs with simple and specific mechanisms, such as nifedipine, Bay K8644, and blebbistatin, and others with a more complex action such as isoproterenol, pimobendan, digoxin, and amrinone, among others. In addition, the results provide a mechanism for the toxicity of itraconazole in a human model, a drug with reported side effects on the heart. The data demonstrate a strong negative inotropic effect because of the blockade of L-type Ca2+ channels and additional action on the cardiac myofilaments. We can conclude that the combination of contractility and action potential measurements can provide wider mechanistic knowledge of drug cardiotoxicity for preclinical assays.

Aims Endurance training improves aerobic fitness and cardiac function in individuals with heart failure. However, the underlying mechanisms are not well characterized. Exercise training could therefore act as a tool to discover novel targets for heart failure treatment. We aimed to associate changes in Ca2+ handling and electrophysiology with micro-RNA (miRNA) profile in exercise trained heart failure rats to establish which miRNAs induce heart failure-like effects in Ca2+ handling and electrophysiology. Methods and results Post-myocardial infarction (MI) heart failure was induced in Sprague Dawley rats. Rats with MI were randomized to sedentary control (sed), moderate (mod)- or high-intensity (high) endurance training for 8 weeks. Exercise training improved cardiac function, Ca2+ handling and electrophysiology including reduced susceptibility to arrhythmia in an exercise intensity-dependent manner where high intensity gave a larger effect. Fifty-five miRNAs were significantly regulated (up or down) in MI-sed, of which 18 and 3 were changed towards Sham-sed in MI-high and MI-mod, respectively. Thereafter we experimentally altered expression of these “exercise-miRNAs” individually in human induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CM) in the same direction as they were changed in MI. Of the “exercise-miRNAs”, miR-214-3p prolonged AP duration, whereas miR-140 and miR-208a shortened AP duration. miR-497-5p prolonged Ca2+ release whereas miR-214-3p and miR-31a-5p prolonged Ca2+ decay. Conclusion Using exercise training as a tool, we discovered that miR-214-3p, miR-497-5p, miR-31a-5p contribute to heart-failure like behaviour in Ca2+ handling and electrophysiology and could be potential treatment targets.

Objective The clinical significance of anti-phosphatidylserine/prothrombin (aPS/PT) in antiphospholipid syndrome (APS) is still controversial. We assessed the prevalence of aPS/PT antibodies, their association with other anti-phospholipid antibodies (aPL) and with different APS clinical phenotypes. Methods We included 95 primary APS patients according to the Sydney classification criteria, and patients with thrombocytopenia and/or hemolytic anemia who also fulfilled the serological APS criteria. We tested aCL, anti-β2GP-I and aPS/PT antibodies (both IgG and IgM isotypes) and lupus anticoagulant (LA). We used χ2 test, Spearman's correlation coefficient, Mann-Whitney U test and logistic regression. Results Seventy-seven percent of patients had thrombosis, 50% hematologic involvement and 25% obstetric events (non-exclusive groups). Twenty patients had only hematologic features. The prevalence of IgG and IgM aPS/PT antibodies was 61% and 60%, respectively. Patients with LA+ had a higher prevalence and higher titers of IgG and IgM aPS/PT antibodies. aPS/PT antibodies correlated with aPL antibodies including LA. IgG aPS/PT antibodies were associated with thrombosis (OR 8.6 [95% CI 2.13–33.8, p = 0.002]) and pure hematologic features (OR 0.2, CI 95% 0.05–0.97, p = 0.004). IgM anti-β2GP-I antibodies conferred high risk for both hematologic (OR 7.9, 95% CI 1.88–34.61, p = 0.006) and thrombotic involvement (OR 7.4, 95% CI 1.76–31.12, p = 0.006). Conclusions aPS/PT antibodies were highly prevalent and correlated with other aPL antibodies. IgG aPTS/PT conferred a high risk for thrombosis, but not for pure hematologic involvement. aPS/PT antibodies may be a useful serological tool in the diagnosis and phenotypic characterization of APS patients.

Background The aim of this systematic review is to assess the impact of primary treatments with curative intention in patients with localized prostate cancer, measured with Patient-Reported Outcomes (PROs), and to examine differences among modalities within treatments. Methods We conducted a systematic literature search for January 2005-March 2017 following PRISMA guidelines, including longitudinal studies measuring disease-specific PROs in localized prostate cancer patients with a follow-up from pre- to post-treatment (≥1 year). Two reviewers independently extracted data and assessed risk of bias. The study is registered in PROSPERO: CRD42015019747. Results Of 148 identified studies, 60 were included in the meta-analyses. At the 1st year, radical prostatectomy patients showed small urinary irritative-obstructive improvement (0.37SD 95%CI 0.30, 0.45), but large deterioration for sexual function and incontinence with high heterogeneity (I2 = 77% and 93%). Moderate worsening in external radiotherapy patients for sexual function (−0.46SD 95%CI −0.55, −0.36), small urinary incontinence (−0.16SD 95%CI −0.23, −0.09) and bowel impairment (−0.31SD 95%CI −0.39, −0.23). Brachytherapy patients presented small deterioration in urinary incontinence (−0.29SD 95%CI −0.39, −0.19), irritative obstructive symptoms (−0.35SD 95%CI −0.47, −0.23), sexual function (−0.12SD 95%CI −0.24, −0.002), and bowel bother (−0.27SD 95%CI −0.42, −0.11). These patterns persisted up to the 5th year. High-intensity focused ultrasound and active surveillance only have results at 1st year, showing no statistically significant worsening. Conclusions No remarkable differences in PRO appeared between modalities within each treatment. Nowadays, available evidence supports brachytherapy as possible alternative to radical prostatectomy for patients seeking an attempted curative treatment limiting the risk for urinary incontinence and sexual dysfunction.

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) hold promise for assessment of drug-induced arrhythmias and are being considered for use under the comprehensive in vitro proarrhythmia assay (CiPA). We studied the effects of 26 drugs and 3 drug combinations on 2 commercially available iPSC-CM types using high-throughput voltage-sensitive dye and microelectrode-array assays being studied for the CiPA initiative and compared the results with clinical QT prolongation and torsade de pointes (TdP) risk. Concentration-dependent analysis comparing iPSC-CMs to clinical trial results demonstrated good correlation between drug-induced rate-corrected action potential duration and field potential duration (APDc and FPDc) prolongation and clinical trial QTc prolongation. Of 20 drugs studied that exhibit clinical QTc prolongation, 17 caused APDc prolongation (16 in Cor.4U and 13 in iCell cardiomyocytes) and 16 caused FPDc prolongation (16 in Cor.4U and 10 in iCell cardiomyocytes). Of 14 drugs that cause TdP, arrhythmias occurred with 10 drugs. Lack of arrhythmic beating in iPSC-CMs for the four remaining drugs could be due to differences in relative levels of expression of individual ion channels. iPSC-CMs responded consistently to human ether-a-go-go potassium channel blocking drugs (APD prolongation and arrhythmias) and calcium channel blocking drugs (APD shortening and prevention of arrhythmias), with a more variable response to late sodium current blocking drugs. Current results confirm the potential of iPSC-CMs for proarrhythmia prediction under CiPA, where iPSC-CM results would serve as a check to ion channel and in silico modeling prediction of proarrhythmic risk. A multi-site validation study is warranted.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and higher throughput platforms have emerged as potential tools to advance cardiac drug safety screening. This study evaluated the use of high bandwidth photometry applied to voltage-sensitive fluorescent dyes (VSDs) to assess drug-induced changes in action potential characteristics of spontaneously active hiPSC-CM. Human iPSC-CM from 2 commercial sources (Cor.4U and iCell Cardiomyocytes) were stained with the VSD di-4-ANEPPS and placed in a specialized photometry system that simultaneously monitors 2 wavebands of emitted fluorescence, allowing ratiometric measurement of membrane voltage. Signals were acquired at 10 kHz and analyzed using custom software. Action potential duration (APD) values were normally distributed in cardiomyocytes (CMC) from both sources though the mean and variance differed significantly (APD90: 229 ± 15 ms vs 427 ± 49 ms [mean ± SD, P < 0.01]; average spontaneous cycle length: 0.99 ± 0.02 s vs 1.47 ± 0.35 s [mean ± SD, P < 0.01], Cor.4U vs iCell CMC, respectively). The 10-90% rise time of the AP (Trise) was ∼6 ms and was normally distributed when expressed as 1/[Formula: see text] in both cell preparations. Both cell types showed a rate dependence analogous to that of adult human cardiac cells. Furthermore, nifedipine, ranolazine, and E4031 had similar effects on cardiomyocyte electrophysiology in both cell types. However, ranolazine and E4031 induced early after depolarization-like events and high intrinsic firing rates at lower concentrations in iCell CMC. These data show that VSDs provide a minimally invasive, quantitative, and accurate method to assess hiPSC-CM electrophysiology and detect subtle drug-induced effects for drug safety screening while highlighting a need to standardize experimental protocols across preparations.

Neuroendocrine tumors (NET) include a spectrum of malignancies arising from neuroendocrine cells throughout the body. The objective of this clinical investigation of retrospectively and prospectively collected data was to describe the prevalence, demographic data, clinical symptoms and methods of diagnosis of NET and the treatment and long-term follow-up of patients with NET. Data were provided by the participating centers and assessed for consistency by internal reviewers. All the cases were centrally evaluated (when necessary) by the pathologists in our group. The tissue samples were reviewed by hematoxylin and eosin and immunohistochemical staining techniques to confirm the diagnosis of NET. In total, 532 cases were documented: 461 gastroenteropancreatic-NET (GEP-NET) and 71 bronchial NET (BNET). All the tumors were immunohistochemically defined according to the World Health Organization (WHO) and European Neuroendocrine Tumor Society criteria. The most common initial symptoms in GEP-NET were abdominal pain, diarrhea, bowel obstruction, flushing, gastrointestinal bleeding and weight loss. The most common tumor types were carcinoid (58.0%), non-functional pancreatic tumor (23.0%), metastatic NET of unknown primary (16.0%) and functional pancreatic tumor (3.0%). Of the BNET, 89.0% were typical and 11.0% atypical carcinoids. Of the patients with GEP-NET, 59.2% had distant metastasis at diagnosis. The locations of the primary tumors in GEP-NET were the small bowel (26.9%), pancreas (25.2%), colon-rectum (12.4%), appendix (7.6%), stomach (6.9%), esophagus (2.8%), duodenum (2.0%) and unknown primary (16.3%). The histological subtypes based on the WHO classification were well-differentiated NET (20.1%), well-differentiated neuroendocrine carcinomas (66.5%) and poorly differentiated neuroendocrine carcinomas (10.3%). Overall, 67.3% of the patients underwent surgery, 41.2% with curative intent and 26.1% for palliative purposes. The 5-year survival rates were 65.1% (95% confidence interval, 58.0-71.4%) in GEP-NET and 100.0% in typical carcinoid of the lung. This observational, non-interventional, longitudinal study aimed to accumulate relevant information regarding the epidemiology, clinical presentation and current practices in the treatment of NET patients in Argentina, providing insight into regional differences and patterns of care in this heterogeneous disease.

Introduction:\ud \ud Voltage-sensitive optical (VSO) sensors offer a minimally invasive method to study the time course of repolarization of the cardiac action potential (AP). This Comprehensive in vitro Proarrhythmia Assay (CiPA) cross-platform study investigates protocol design and measurement variability of VSO sensors for preclinical cardiac electrophysiology assays.\ud \ud Methods:\ud \ud Three commercial and one academic laboratory completed a limited study of the effects of 8 blinded compounds on the electrophysiology of 2 commercial lines of human induced pluripotent stem-cell derived cardiomyocytes (hSC-CMs). Acquisition technologies included CMOS camera and photometry; fluorescent voltage sensors included di-4-ANEPPS, FluoVolt and genetically encoded QuasAr2. The experimental protocol was standardized with respect to cell lines, plating and maintenance media, blinded compounds, and action potential parameters measured. Serum-free media was used to study the action of drugs, but the exact composition and the protocols for cell preparation and drug additions varied among sites.\ud \ud Results:\ud \ud Baseline AP waveforms differed across platforms and between cell types. Despite these differences, the relative responses to four selective ion channel blockers (E-4031, nifedipine, mexiletine, and JNJ 303 blocking IKr, ICaL, INa, and IKs, respectively) were similar across all platforms and cell lines although the absolute changes differed. Similarly, four mixed ion channel blockers (flecainide, moxifloxacin, quinidine, and ranolazine) had comparable effects in all platforms. Differences in repolarisation time course and response to drugs could be attributed to cell type and experimental method differences such as composition of the assay media, stimulated versus spontaneous activity, and single versus cumulative compound addition.\ud \ud Discussion:\ud \ud In conclusion, VSOs represent a powerful and appropriate method to assess the electrophysiological effects of drugs on iPSC-CMs for the evaluation of proarrhythmic risk. Protocol considerations and recommendations are provided toward standardizing conditions to reduce variability of baseline AP waveform characteristics and drug responses.

Introduction Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) are emerging as new and human-relevant source in vitro model for cardiac safety assessment that allow us to investigate a set of 20 reference drugs for predicting cardiac arrhythmogenic liability using optical action potential (oAP) assay. Methods Here, we describe our examination of the oAP measurement using a voltage sensitive dye (Di-4-ANEPPS) to predict adverse compound effects using hiPS-CMs and 20 cardioactive reference compounds. Fluorescence signals were digitized at 10 kHz and the records subsequently analyzed off-line. Cells were exposed to 30 min incubation to vehicle or compound (n = 5/dose, 4 doses/compound) that were blinded to the investigating laboratory. Action potential parameters were measured, including rise time (Trise) of the optical action potential duration (oAPD). Results Significant effects on oAPD were sensitively detected with 11 QT-prolonging drugs, while oAPD shortening was observed with ICa-antagonists, IKr-activator or ATP-sensitive K+ channel (KATP)-opener. Additionally, the assay detected varied effects induced by 6 different sodium channel blockers. The detection threshold for these drug effects was at or below the published values of free effective therapeutic plasma levels or effective concentrations by other studies. Discussion The results of this blinded study indicate that OAP is a sensitive method to accurately detect drug-induced effects (i.e., duration/QT-prolongation, shortening, beat rate, and incidence of early after depolarizations) in hiPS-CMs; therefore, this technique will potentially be useful in predicting drug-induced arrhythmogenic liabilities in early de-risking within the drug discovery phase.

255 Background: Grade 2 (G2) Neuroendocrine tumors (NETs), of the digestive tract is a heterogeneous group of tumors. Several treatment options including chemotherapy and target therapy are available, but there is a lack of prospective trials assessing the role of pronostic factors in this population. Aim(s): to analyze prognostic factors and clinical characteristics in a population of patients with G2 GEP-NETs. To determine the role of ki 67 in the stratification of G2 population. Methods: Study population was obtained from our prospective database (Argentum Group). Survival was estimated using the Kaplan-Meier method and compared between Ki-67 quartiles using the log-rank test. Value of Ki-67 to discriminate mortality was assesed with a ROC curve analysis Results: 144 pts were evaluated. Mean age 54.9, 46.7% male. 102 (70.8%) with metastatic disease, mainly hepatic in 97 pts. (67.4%). 67.9 % underwent surgery. 34% received chemotherapy, and 10.9% target therapy. Median Ki-67 value was 6 (IQR 4-10), ROC curve=0.62 (95% CI 0.53 a 0.72 p=0.021. cut-off: 6.5 (sensitivity 62.2%, specificity 57.7%). Median survival was 97, 67, 51 and 27 months according stratification by quartile (p.001), 45 events (31.7%). Conclusions: Our results suggest that in the heterogenous G2 GEP-NETs there are significant differences in survival. This study was underpowered to detect differences between Ki-67 quartiles, we detected that chemotherapy was mostly used in the higher quartiles.

e14598 Background: Somatostatin receptor expression (SSTR), mainly subtypes 2 and 5, is a feature of well differentiated GEP-NETs. Such expression has a prognostic and predictive value for the use of somatostatin analogs.Ki 67, present during the cycle phases (G1, S, G2 y M) is a nuclear antigen associated with cell proliferation. The correlation between both clinical and pathological factors is under active investigation in patients with GEP-NETs. Methods: An analysis including 100 patients in the database of the Argentum Group was performed. Consecutive patients were included during the 2006-2007 period. In all cases, a centralized revision of the sample was conducted for diagnosis confirmation.The study of SSTR receptors in tissue and Ki67 was conducted using IHQ.The Kaplan-Meier method was used to analyse survival. Log-rank test was used for the comparative analysis of the variables of interest. Results: The expression of at least one of the SSTRs 2 (a) or 5 was found in 86% and 62% of cases respectivevly in this population. The univariate analysis showed significant differences in the expression of SSTR2 (a) but not in the expression of SSTR 5.The expression of the proliferative index (Ki 67) was associated with significant differences in relation to OS at 5 years, 84% (Ki 67 under or equal to 2%), 66% (Ki 67 between 3 and 15%) and 13% (Ki 67 over 15%). The OS at 5 years in patients with SSTR 2/5 positive and Ki 67 under 2% was 86%, 64% in pts. with SSTR 2/5 positive and Ki 67 over 2% and 20 % in pts. with SSTR 2/5 negative, independent Ki 67 (p .0023). Conclusions: The expression of at least one of the SSTRs 2 (a) or 5 was found in 86% and 62% of cases respectivevly in this population. In the population with GEP-NETs under study, a subgroup of patients with a better prognosis was identified in association with the expression of SSTR 2/5 and Ki67 below 2%. The assessment of SSTR 2(a) and 5 in tissues, together with the study of the proliferative index are a useful tool for prognosis assessment in these patients.