Cell cycle arrest and p53 prevent ON-target megabase-scale rearrangements induced by CRISPR-Cas9.

The CRISPR-Cas9 system has revolutionized our ability to precisely modify the genome and has led to gene editing in clinical applications. Comprehensive analysis of gene editing products at the targeted cut-site has revealed a complex spectrum of outcomes. ON-target genotoxicity is underestimated with standard PCR-based methods and necessitates appropriate and more sensitive detection methods. Here, we present two complementary Fluorescence-Assisted Megabase-scale Rearrangements Detection (FAMReD) systems that enable the detection, quantification, and cell sorting of edited cells with megabase-scale loss of heterozygosity (LOH). These tools reveal rare complex chromosomal rearrangements caused by Cas9-nuclease and show that LOH frequency depends on cell division rate during editing and p53 status. Cell cycle arrest during editing suppresses the occurrence of LOH without compromising editing. These data are confirmed in human stem/progenitor cells, suggesting that clinical trials should consider p53 status and cell proliferation rate during editing to limit this risk by designing safer protocols.

CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells.

CRISPR-Cas9 is a promising technology for gene therapy. However, the ON-target genotoxicity of CRISPR-Cas9 nuclease due to DNA double-strand breaks has received little attention and is probably underestimated. Here we report that genome editing targeting globin genes induces megabase-scale losses of heterozygosity (LOH) from the globin CRISPR-Cas9 cut-site to the telomere (5.2 Mb). In established lines, CRISPR-Cas9 nuclease induces frequent terminal chromosome 11p truncations and rare copy-neutral LOH. In primary hematopoietic progenitor/stem cells, we detect 1.1% of clones (7/648) with acquired megabase LOH induced by CRISPR-Cas9. In-depth analysis by SNP-array reveals the presence of copy-neutral LOH. This leads to 11p15.5 partial uniparental disomy, comprising two Chr11p15.5 imprinting centers (H19/IGF2:IG-DMR/IC1 and KCNQ1OT1:TSS-DMR/IC2) and impacting H19 and IGF2 expression. While this genotoxicity is a safety concern for CRISPR clinical trials, it is also an opportunity to model copy-neutral-LOH for genetic diseases and cancers.

Ultrasound-induced Cavitation enhances the efficacy of Chemotherapy in a 3D Model of Pancreatic Ductal Adenocarcinoma with its microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is supported by a complex microenvironment whose physical contribution to chemoresistance could be overcome by ultrasound (US) therapy. This study aims to investigate the ability of US-induced inertial cavitation in association with chemotherapy to alter tumor cell viability via microenvironment disruption. For this purpose, we used a 3D-coculture PDAC model partially mimicking the tumor and its microenvironment. Coculture spheroids combining DT66066 cells isolated from KPC-transgenic mice and murine embryonic fibroblasts (iMEF) were obtained by using a magnetic nanoshuttle method. Spheroids were exposed to US with incremental inertial cavitation indexes. Conditions studied included control, gemcitabine, US-cavitation and US-cavitation + gemcitabine. Spheroid viability was assessed by the reduction of resazurin and flow cytometry. The 3D-coculture spheroid model incorporated activated fibroblasts and produced type 1-collagen, thus providing a partial miniature representation of tumors with their microenvironment. Main findings were: (a) Gemcitabine (5 µM) was significantly less cytotoxic in the presence of KPC/iMEFs spheroids compared with KPC (fibroblast-free) spheroids; (b) US-induced inertial cavitation combined with Gemcitabine significantly decreased spheroid viability compared to Gemcitabine alone; (c) both cavitation and chemotherapy affected KPC cell viability but not that of fibroblasts, confirming the protective role of the latter vis-à-vis tumor cells. Gemcitabine toxicity is enhanced when cocultured spheroids of KPC and iMEF are exposed to US-cavitation. Although the model used is only a partial representation of PDAC, this experience supports the hypothesis that US-inertial cavitation can enhance drug penetration and cytotoxicity by disrupting PDAC microenvironment.

Outcomes of endoscopic ultrasound-guided biliary drainage: A systematic review and meta-analysis.

Background: Success and event rates of endoscopic ultrasound (EUS)-guided biliary drainage vary with techniques, and results from different studies remain inconsistent. Objective: We conducted a proportion meta-analysis to evaluate the efficacy and safety of EUS-guided biliary drainage and compare the outcomes of current procedures. Methods: We searched MEDLINE, Embase, Cochrane and Web of knowledge to identify studies reporting technical success, clinical success and complication rates of EUS-guided biliary drainage techniques to estimate their clinical and technical efficacy and safety. Results: We identified 17 studies including a total of 686 patients. The overall clinical success and technical success rates were respectively 84% confidence interval (CI) 95% (80-88) and 96% CI 95% (93-98) for hepaticogastrostomy, and respectively 87% CI 95% (82-91) and 95% CI 95 (91-97) for choledochoduodenostomy. Reported adverse event rates were significantly higher (p = 0.01) for hepaticogastrostomy (29% CI 95% (24-34)) compared to choledochoduodenostomy (20% CI 95% (16-25)). Compared with hepaticogastrostomy, the pooled odds ratio for the complication rate of choledochoduodenostomy was 2.01 (1.25; 3.24) (p = 0.0042), suggesting that choledochoduodenostomy might be safer than hepaticogastrostomy. Conclusion: The available literature suggests choledochoduodenostomy may be a safer approach compared to hepaticogastrostomy. Randomized controlled trials with sufficiently large cohorts are needed to compare techniques and confirm these findings.

Self-assembling peptide matrix for the prevention of esophageal stricture after endoscopic resection: a randomized controlled trial in a porcine model.

Esophageal stricture formation after extensive endoscopic resection remains a major limitation of endoscopic therapy for early esophageal neoplasia. This study assessed a recently developed self-assembling peptide (SAP) matrix as a wound dressing after endoscopic resection for the prevention of esophageal stricture. Ten pigs were randomly assigned to the SAP or the control group after undergoing a 5-cm-long circumferential endoscopic submucosal dissection of the lower esophagus. Esophageal diameter on endoscopy and esophagogram, weight variation, and histological measurements of fibrosis, granulation tissue, and neoepithelium were assessed in each animal. The rate of esophageal stricture at day 14 was 40% in the SAP-treated group versus 100% in the control group (P = 0.2). Median interquartile range (IQR) esophageal diameter at day 14 was 8 mm (2.5-9) in the SAP-treated group versus 4 mm (3-4) in the control group (P = 0.13). The median (IQR) stricture indexes on esophagograms at day 14 were 0.32 (0.14-0.48) and 0.26 (0.14-0.33) in the SAP-treated and control groups, respectively (P = 0.42). Median (IQR) weight variation during the study was +0.2 (-7.4; +1.8) and -3.8 (-5.4; +0.6) in the SAP-treated and control groups, respectively (P = 0.9). Fibrosis, granulation tissue, and neoepithelium were not significantly different between the groups. The application of SAP matrix on esophageal wounds after a circumferential endoscopic submucosal dissection delayed the onset of esophageal stricture in a porcine model.

Antireflux versus conventional self-expanding metallic Stents (SEMS) for distal esophageal cancer: results of a multicenter randomized trial.

INTRODUCTION: Self-expanding metal stents (SEMS) are commonly used in the palliation of dysphagia in patients with inoperable esophageal carcinoma. However, they predispose to gastroesophageal reflux when deployed across the gastroesophageal junction. The aims of this study were to: 1) assess the influence of the antireflux valve on trans-prosthetic reflux (primary outcome); and 2) compare the results of SEMS with and without antireflux valve in terms of reflux symptoms, quality of life (QOL), improvement of dysphagia and adverse events (secondary outcomes). PATIENTS AND METHODS: Thirty-eight patients were enrolled in nine centers. Carcinomas were locally advanced (47 %) or metastatic. After randomization, patients received either a covered SEMS with antireflux valve (n = 20) or a similar type of SEMS with no antireflux device but assigned to standard proton pump inhibitor therapy and postural advice (n = 18). Trans-prosthetic reflux was assessed at day 2 using a radiological score based on barium esophagography performed after Trendelenburg maneuver and graded from 0 (no reflux) to 12 (maximum). Monthly telephone interviews were conducted for Organisation Mondiale de la Santé (OMS) scoring from 0 (excellent) to 5 (poor), QOL assessment (based on the Reflux-Qual Simplifié scoring system) from 0 (poor) to 100 (excellent), dysphagia scoring from 0 (no dysphagia) to 5 (complete dysphagia) and regurgitation scoring from 0 (no regurgitation) to 16 (maximum). RESULTS: No difference was noted in terms of age, sex, size of lesion, prosthesis length or need for dilation prior to SEMS placement. No difficulty in placing SEMS nor complications were noted. Radiological scores of reflux were found to be significantly lower in patients with an antireflux stent compared to the conventional stent and associated measures. The regurgitation scores were significantly decreased in patients with antireflux stents during the first 2 months after stent placement and thereafter, they were similar in the two groups. QOL and dysphagia were improved in both groups. Survival rates were comparable in the two groups. CONCLUSIONS: No difference was observed between the two types of SEMS regarding the palliation of dysphagia and improvement of QOL. However, SEMS with an antireflux valve were more effective in preventing trans-prosthetic gastroesophageal reflux but at the cost of an increased likehood of minor adverse events (migrations and/or obstruction of the SEMS).

Safety and feasibility of endoscopic biliary radiofrequency ablation treatment of extrahepatic cholangiocarcinoma.

BACKGROUND: Biliary bipolar radiofrequency ablation (RFA) is a new treatment for extrahepatic cholangiocarcinoma (CCA) currently under evaluation. The purpose of this study was to evaluate the safety, particularly biliary fistula occurrence, and the feasibility of biliary RFA in a homogeneous group of patients treated using the same RFA protocol. METHODS: Twelve patients with inoperable or unresectable CCA were included in a bicentric case series study. After removal of biliary plastic stents, a radiofrequency treatment with a new bipolar probe (Habib™ EndoHBP) was applied. The energy was delivered by a RFA generator (VIO 200 D), supplying electrical energy at 350 kHz and 10 W for 90 s. At the end of the procedure, one or more biliary stents were left in place. Adverse events were assessed per-procedure and during follow-up visits. RESULTS: CCA was confirmed in all patients by histology (66%), locoregional evolution or metastatic evolution. The types of CCA were Bismuth I stage (N = 4), Bismuth II stage (N = 3), Bismuth III stage (N = 2) and Bismuth IV stage (N = 3). No serious adverse events occurred within 30 days following endoscopic treatment: One patient had a sepsis due to bacterial translocation on day 1 and another had an acute cholangitis on day 12 due to early stent migration. No immediate or delayed biliary fistula was reported. The ergonomics of the probe made treatment easy in 100 % of cases. Mean survival was 12.3 months. CONCLUSION: Endoscopic radiofrequency treatment of inoperable CCA appears without major risks and is feasible. No major adverse events or biliary fistula were identified.

Topical hemostatic powder promotes reepithelialization and reduces scar formation after extensive esophageal mucosal resection.

The development of techniques for endoscopic resection has provided new strategies for radical conservative treatment of superficial esophageal neoplasms, even those that are circumferential, such as Barrett's neoplasia. However, it is necessary to prevent the formation of scar tissue that can be responsible for esophageal strictures following circumferential resection. Preliminary data have suggested the possible efficacy of a hemostatic powder in the promotion of wound healing. The study aims to assess the effectiveness of Hemospray (Cook Medical) in a swine model of post-endoscopic esophageal stricture. Our prospective controlled study included 21 pigs. A 6-cm circumferential submucosal dissection of the esophagus (CESD) was performed in each pig. Group 1 (n = 11) only underwent CESD and Group 2 (n = 10) had repeated Hemospray applications after CESD. Clinical, endoscopic, and radiological monitoring were performed, blood levels of four inflammatory or pro-fibrotic cytokines were assessed, and histological analysis was performed. Median esophageal diameter was greater in the group treated with Hemospray (2 mm [1-3] vs. 3 mm [2-4], P = 0.01), and the rate of symptomatic esophageal stricture was 100% and 60% in Groups 1 and 2, respectively (P = 0.09). The thicknesses of esophageal fibrosis and inflammatory cell infiltrate were significantly lower in Group 2 than in Group 1 (P = 0.002 and 0.0003, respectively). The length of the neoepithelium was greater in Group 2 than in Group 1 (P = 0.0004). Transforming growth factor-ß levels were significantly lower in Group 2 than in Group 1 (P = 0.01). The application of Hemospray after esophageal CESD reduces scar tissue formation and promotes reepithelialization, and therefore is a promising therapeutic approach in the prevention of post-endoscopic esophageal stricture.

Systematic review: the prevention of oesophageal stricture after endoscopic resection.

BACKGROUND: Extensive endoscopic resections for the treatment of early oesophageal neoplasia can result in fibro-inflammatory strictures that require repeated interventions, which significantly alter the patients' quality of life. AIMS: To review current evidence about the prevention of oesophageal strictures following endoscopic resections. METHODS: Systematic search of PubMed and Embase from inception to March 2015 using appropriate keywords. All original publications in English were included, and articles on the treatment of oesophageal stricture were excluded. RESULTS: Of the 461 hits, 62 studies were included in the analysis. Among the wound-protective strategies, polyglycolic acid sheets showed the most convincing evidence with a 37.5% stricture rate and excellent safety. Regenerative medicine, using cell sheets of autologous keratinocytes, resulted in a 25% stricture rate, although with cost and availability concerns. Among anti-proliferative treatment modalities, steroid treatment, either endoscopically injected triamcinolone in the resection wound or orally administered prednisolone, proved effective with an overall stricture rate of 13.5%, with safety concerns regarding late oesophageal perforations and infectious morbidity. Among mechanical treatment options, poorly effective and high-risk preventive balloon dilation tend to be replaced by prophylactic covered stent, with 18-28% stricture rates. CONCLUSIONS: Although oral or locally injected steroids are promising options, no currently available technique is sufficiently efficient and devoid of significant safety concerns to recommend its routine use for the prevention of strictures after extensive endoscopic resection. Improving our knowledge in the mechanisms of oesophageal wound healing will guide the development of novel methods for stricture prevention.

An observational study of driving distractions on urban roads in Spain.

The present research investigated the prevalence of driver engagement in secondary tasks and whether there were any differences by age and gender, as well as day of the week and time of the day. Two independent researchers observed 6578 drivers at nine randomly selected urban locations in Girona, Spain. Nearly 20% of the drivers observed were engaged in some type of secondary task, with the most common being: conversing with a passenger (11.1%), smoking (3.7%) and talking on a handheld mobile phone (1.3%). Surprisingly there were no differences by gender, but there were age-related differences with younger drivers being more frequently observed engaged in a number of different types of secondary tasks while driving (i.e. drinking, talking on a handheld mobile phone, and texting or keying numbers). Logistic regression showed that younger drivers, and to a lesser extent middle-age drivers, were significantly more likely to be observed engaged in a technological distraction than older drivers. Conversely, non-technological distractions were significantly predicted by day of the week, time of the day and location. A substantial number of the drivers observed in this study were putting themselves at an increased risk of becoming involved in a crash by engaging in non-driving related tasks at the same time as driving. Furthermore, the higher crash rate among young drivers may be partially accounted for by their more frequent engagement in some types of secondary tasks while driving.

Self-expanding plastic stent removed after radiochemotherapy for advanced esophageal cancer.

Endoscopic evaluation after chemoradiotherapy (CR) is impossible with an esophageal stent in place. The main study objective was to evaluate self-expanding plastic stent (SEPS) removal post-CR. Secondary end-points were the improvement of dysphagia and patients' quality of life. From October 2008 to March 2011, 20 dysphagic patients who suffered from advanced esophageal cancer were enrolled in a multicenter, prospective study. SEPS was inserted prior to CR and then removed endoscopically. SEPS efficiency (dysphagia score) and tolerance, as well as the patients' quality of life (European Organization for the Research and Treatment of Cancer Quality of Life Questionnaire validated for the esophagus), were monitored. Continuous variables were compared using a paired t-test analysis for matched data. A P-value of less than 0.05 was considered statistically significant. Twenty patients (15 men and 5 women), aged 61.5 years (±9.88) (range 43-82 years), with adenocarcinoma (n = 12) and squamous cell carcinoma (n = 8), were enrolled. SEPS were successfully inserted in all patients (100%). There was one perforation and three episodes of migration. All of these complications were medically treated. The mean dysphagia score at the time of stent placement was 2.79 (0.6). Mean dysphagia scores obtained on day 1 and day 30 post-SEPS placement were 0.7 (0.9) (P < 0.0001) and 0.45 (0.8) (P < 0.0001), respectively. Quality of Life Questionnaire validated for the esophagus score showed an improvement in dysphagia (P = 0.01) and quality of oral feeding (P = 0.003). All SEPS were removed endoscopically without complications. In two patients, the stent was left in place due to metastatic disease. SEPS are extractable after CR of esophageal cancer. Early stenting by SEPS prior to and during CR may reduce dysphagia and improve quality of oral alimentation.

Stages of change and decisional balance for condom use with a romantic partner.

This article examines the relationship between decisional balance and stages of change for consistent condom use with a current romantic heterosexual partner. The sample consisted of 619 undergraduate students (35.1% males; mean of age = 20.31). The results show that the pros and cons of using condoms were significantly related to the stages of change for condom use, but that the pros were more strongly related. Therefore, campaigns for preventing the transmission of the HIV virus and other sexually transmitted diseases should attempt to maximize the perceived advantages of condom use, rather than refuting the cons.

Endoscopic submucosal dissection in a European setting. A multi-institutional report of a technique in development.

BACKGROUND AND STUDY AIMS: Endoscopic submucosal dissection (ESD) is a technique for "en bloc" resection of superficial tumors of the gastrointestinal tract. In France, experience with this technique is still limited. We wanted to assess the development of ESD in France, with special attention to short term outcomes. PATIENTS AND METHODS: Members of the Société Française d'Endoscopie Digestive (SFED) who declared performing ESD reported their cases prospectively on a voluntary basis. Demographic, clinical, and technical data, and the results of immediate complications were collected. Case reports were completed prospectively by each investigator before pooled analysis. RESULTS: A total of 188 consecutive case reports were collected from 16 centers. The median case mix per center was 6 patients (range 1-43). The lesion sites treated by ESD were the stomach (n = 75), esophagus (n = 27), duodenum (n = 1), cecum (n = 2), right colon (n = 3), transverse colon (n = 5), sigmoid (n = 3), and rectum (n = 72). The median size of the lesions was 26 mm (range 2-150 mm). En bloc resection was achieved in 77.1% of cases, with complete R0 resection in 72.9%. Histopathology results showed high grade dysplasia or superficial cancer in 71.2%. The median duration of ESD was 105 minutes (range 20-450 minutes). The short term morbidity was 29.2% including 34 cases of perforation (18.1%), and 21 hemorrhages (11.2%) during the 24 hours following ESD, 89% of which were managed conservatively or endoscopically. CONCLUSION: In this early experience, the feasibility of ESD appeared to be good but R0 resection and complication rates did not match those reported by Japanese authors and must be improved by an extended practice.

Long-term follow-up after biliary stent placement for postcholecystectomy bile duct strictures: a multicenter study.

BACKGROUND AND STUDY AIMS: Endoscopic stenting is a recognized treatment of postcholecystectomy biliary strictures. Large multicenter reports of its long-term efficacy are lacking. Our aim was to analyze the long-term outcomes after stenting in this patient population, based on a large experience from several centers in France. METHODS: Members of the French Society of Digestive Endoscopy were asked to identify patients treated for a common bile duct postcholecystectomy stricture. Patients with successful stenting and follow-up after removal of stent(s) were subsequently included and analyzed. Main outcome measures were long-term success of endoscopic stenting and related predictors for recurrence (after one stenting period) or failure (at the end of follow-up). RESULTS: A total of 96 patients were eligible for inclusion. The mean number of stents inserted at the same time was 1.9±0.89 (range 1-4). Stent-related morbidity was 22.9% (n=22). The median duration of stenting was 12 months (range 2-96 months). After a mean follow-up of 6.4±3.8 years (range 0-20.3 years) the overall success rate was 66.7% (n=64) after one period of stenting and 82.3% (n=79) after additional treatments. The mean time to recurrence was 19.7±36.6 months. The most significant independent predictor of both recurrence and failure was a pathological cholangiography at the time of stent removal. CONCLUSION: Endoscopic stenting helps to avoid surgery in more than 80% of patients bearing postcholecystectomy common bile duct strictures. However, a persistent anomaly on cholangiography at the time of stent removal is a strong predictor of recurrence and may lead to consideration of surgery.

Application of ANAES guidelines for colonoscopy in France: a practical survey.

OBJECTIVES: In 2004, the French health authorities published guidelines on the indications for colonoscopy. However, no study has evaluated the awareness of healthcare practitioners of these guidelines. The aim of this study was to determine the level of awareness of the ANAES guidelines among French gastroenterologists. PATIENTS AND METHODS: A questionnaire comprising 20 multiple choice questions (MCQ) was presented to a group of 79 gastroenterologists between February and June in 2008. The questions covered screening tests for colon cancer (one question), endoscopic mucosal resection (two questions) and the ANAES guidelines (17 questions). According to the number of colonoscopies performed per year (less than 100, 100-500, more than 500), the answers to these questions were analyzed separately. RESULTS: Among the practitioners carrying out less than 100, 100-500 and more than 500 colonoscopies per year, the guidelines for colon cancer screening were known by 33, 50 and 56%, respectively, the quality criteria for endoscopic mucosal resection by 0, 0 and 3.7%, respectively, and the ANAES guideline indications for colonoscopy by 34.3, 51.2 and 48.9%, respectively (P<0.001). The ANAES guidelines were significantly better known by practitioners who were performing more than 100 colonoscopies per year, while the indications for control colonoscopy were less often correctly anticipated. No differences were found concerning postponed indications. CONCLUSION: The ANAES guidelines consists of the following elements: (1) awareness of the ANAES guidelines is poor, with control colonoscopy being correctly anticipated in just over a third of the gastroenterologists; (2) performing more than 100 colonoscopies per year improves knowledge of the ANAES guidelines; and (3) the ANAES guidelines need to be simplified and should be covered by continuing medical education.

Contribution of endoscopic ultrasound-guided fine-needle aspiration in the workup of mediastinal lymph nodes.

BACKGROUND AND AIM: The aim of this retrospective study was to assess endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA), a minimally invasive technique, to determine its diagnostic accuracy and morbidity in the etiological diagnoses of mediastinal lymph nodes. METHODS: A total of 84 patients underwent EUS-FNA biopsy of the mediastinal lymph nodes, and were classified as either malignant disease, inflammatory disease or incidental diagnosis, according to the suspected clinical condition. To evaluate the diagnostic accuracy of EUS-FNA in each group, a comparison of the cytological results obtained with 19- and 22-gauge needles was performed. RESULTS: All 84 procedures were carried out between January 2004 and June 2008. Six patients were excluded because of non-contributory results. On analyzing the results of EUS-FNA in the malignant group (n=41), the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were 93, 100, 100 and 85%, respectively. In the inflammatory (n=20) and incidental diagnosis (n=17) groups, the sensitivity, specificity, PPV and NPV were 85, 100, 100 and 75%, and 80, 100, 100 and 77%, respectively. There was no significant difference between the FNA results with the 19-gauge (20/78 patients) versus 22-gauge (58/78 patients) needles (P=0.55). Also, no complications were reported either during the procedure or after the 6-month follow-up. CONCLUSION: EUS-FNA is a safe and accurate diagnostic procedure for the study of mediastinal lymph nodes not only for malignancy, but also for inflammatory diseases and incidental diagnoses. In addition, there is no difference in diagnostic accuracy whether FNA is carried out with a 19-gauge or 22-gauge needle.