A multi-analyte cell-free DNA-based blood test for early detection of hepatocellular carcinoma.

The limited performance of guideline-recommended abdominal ultrasound and serum alpha-fetoprotein (AFP) highlights the urgent, unmet need for new biomarkers for more accurate detection of early hepatocellular carcinoma (HCC). To this end, we have conducted a prospective clinical validation study to evaluate the performance of the HelioLiver Test, a multi-analyte blood test combining cell-free DNA methylation patterns, clinical variables, and protein tumor markers. A blinded, multicenter validation study was performed with 247 subjects, including 122 subjects with HCC and 125 control subjects with chronic liver disease. The performance of the HelioLiver Test was compared with AFP and the GALAD score as established HCC surveillance blood tests. The performance of the HelioLiver Test (area under the receiver operating characteristic curve [AUROC] = 0.944) was superior to both AFP (AUROC = 0.851; p < 0.0001) and GALAD (AUROC = 0.899; p < 0.0001). Using a prespecified diagnostic algorithm, the HelioLiver Test showed sensitivities of 85% (95% confidence interval [CI], 78%-90%) for HCC of any stage and 76% (95% CI, 60%-87%) for early stage (American Joint Committee on Cancer [AJCC] I and II) HCC. In contrast, AFP (≥20 ng/mL) alone and the GALAD score (≥-0.63) showed lower sensitivities of 62% (95% CI, 54%-70%) and 75% (95% CI, 67%-82%) for HCC overall, and 57% (95% CI, 40%-71%) and 65% (95% CI, 49%-79%) for early stage (AJCC I and II) HCC, respectively. The specificities of the HelioLiver Test (91%; 95% CI, 85%-95%), AFP (97%; 95% CI, 92%-99%), and the GALAD score (94%; 95% CI, 88%-97%) were similar for control subjects. The HelioLiver Test showed superior performance for HCC detection compared to with both AFP and the GALAD score and warrants further evaluation in HCC surveillance settings.

Prospective multicenter study to evaluate capsule endoscopy competency using a validated assessment tool.

BACKGROUND AND AIMS: Capsule endoscopy (CE) is an established, noninvasive modality for examining the small bowel. Minimum training requirements are based primarily on guidelines and expert opinion. A validated tool to assess the competence of CE is lacking. In this prospective, multicenter study, we determined the minimum number of CE procedures required to achieve competence during gastroenterology fellowship; validated a capsule competency test (CapCT); and evaluated any correlation between CE competence and endoscopy experience. METHODS: We included second- and third-year gastroenterology fellows from 3 institutions between 2013 and 2018 in a structured CE training program with supervised CE interpretation. Fellows completed the CapCT with a maximal score of 100. For comparison, expert faculty completed the same CapCT. Trainee competence was defined as a score ≥90% compared with the mean expert score. Fellows were tested after 15, 25, and 35 supervised CE interpretations. CapCT was validated using expert consensus and item analysis. Data were collected on the number of previous endoscopies. RESULTS: A total of 68 trainees completed 102 CapCTs. Fourteen CE experts completed the CapCT with a mean score of 94. Mean scores for fellows after 15, 25, and 35 cases were 83, 86, and 87, respectively. Fellows with at least 25 interpretations achieved a mean score ≥84 in all 3 institutions. CapCT item analysis showed high interobserver agreement among expert faculty (k = 0.85). There was no correlation between the scores and the number of endoscopies performed. CONCLUSION: After a structured CE training program, gastroenterology fellows should complete a minimum of 25 supervised CE interpretations before assessing competence using the validated CapCT, regardless of endoscopy experience.

Patient-oriented education and visual-aid intervention are inadequate to identify patients with potential capsule retention: a prospective randomized study.

Background/aims: The key procedure-related risk with video capsule endoscopy (VCE) is capsule retention, which should be suspected in patients who have not reported capsule passage. The study aims were to determine the frequency of capsule passage visualization and the difference in self-reporting of capsule passage between patients who receive patient-oriented education (POE) and patients who receive POE and a visual aid intervention in the form of a wrist band (WB). Methods: This was a prospective randomized study that enrolled patients undergoing VCE. Patients were randomly assigned to a POE group versus a POE and WB group. POE consisted of verbal education and an information booklet. Both groups received instructions to notify the study team regarding capsule passage. Results: Sixty patients (mean age 57 ± 18 years; 61% female) were included. A total of 57 patients were included in the analysis (3 lost to follow-up; 28 in POE group; 29 in WB group). Capsule passage status was reported by 68% without significant difference between POE and WB groups (72% vs. 64%; p = .51). Capsule passage status was obtained from all 57 patients with the addition of a proactive follow-up. Only 56% (n = 32) reported visualizing capsule passage. Of the remaining patients who did not visualize capsule passage, 60% (n = 15) reported on this without significant difference between the POE and WB groups (p = .23). Conclusions: Lack of visualization of capsule passage is a poor indicator of retention. Self-reporting of VCE passage status is suboptimal and the addition of a visual aid did not improve this parameter.

Small bowel bleeding in patients with left ventricular assist device: outcomes of conservative therapy versus balloon-assisted enteroscopy.

BACKGROUND: Small bowel bleeding (SBB) accounts for 30% of gastrointestinal bleeding (GIB) episodes in patients with a left ventricular assist device (LVAD). The aim of this study was to determine the outcomes of conservative therapy (CT) compared to balloon-assisted enteroscopy (BAE) in the management of SBB in LVAD patients. METHODS: A retrospective review was performed of a prospectively maintained LVAD database from January 2003 to July 2015. LVAD patients with SBB were classified into a BAE group or a CT group according to whether they did or did not undergo BAE. RESULTS: Forty-two patients (22 BAE, 20 CT) with mean age 66±9.3 years (79% male) were included. The yield of BAE was 64% without reported complications. Overt re-bleeding occurred in 40% of the BAE group compared to 22% of the CT group. The BAE group had a higher mean number of GIB hospitalizations per month compared to the CT group (0.07 vs. 0.03; incidence rate ratio [IRR] 2.72, 95% CI 1.06-6.98; P=0.04). There was no significant difference between the BAE and the CT groups in the number of packed red blood cell (pRBC) transfusions per month (0.42 vs. 0.18; IRR 2.31, 95% CI 0.88-6.04; P=0.09) or all-cause mortality (61% in the CT group and 42% in the BAE group; P=0.90). CONCLUSION: BAE is safe in LVAD patients and has a moderate therapeutic yield. In our cohort of patients, BAE did not appear to improve re-bleeding rate, GIB-related hospitalizations, pRBC transfusions or mortality compared to CT. However, future prospective trials with larger sample sizes are needed to confirm these findings.

Outcomes of repeat balloon assisted enteroscopy in small-bowel bleeding.

BACKGROUND/AIMS: The rate of recurrent small-bowel bleeding (SBB) remains high despite the advent of balloon assisted enteroscopy (BAE). The study aims were to determine: (1) the diagnostic and therapeutic yields, and adverse event rate of repeat BAE in SBB, and (2) the predictors of a positive repeat BAE. METHODS: A retrospective review of a BAE database was conducted. Patients who had > 1 BAE for SBB were included. Primary outcomes were diagnostic yield, therapeutic yield, and adverse events of repeat BAE. Secondary outcomes were predictors of a positive repeat BAE. RESULTS: A total of 175 patients (55 % men; mean age 64.1 ±â€Š16.3 years) were included. The diagnostic and therapeutic yields of repeat BAE were 55 % and 42 %, respectively. Repeat BAE adverse events occurred in 5 % with self-limited abdominal pain being most common. Patients with a positive repeat BAE were significantly older than the negative group (68.6 ±â€Š13.9 vs. 60.9 ±â€Š17.1; P  = 0.001) and were more likely to have cardiac comorbidities (OR 2.4, 95 %CI: 1.3 - 4.6; P  = 0.01), chronic kidney disease (OR 2.3, 95 %CI: 1.1 - 4.9; P  = 0.04), chronic obstructive pulmonary disease (OR 3.3, 95 %CI: 1.3 - 8.1; P  = 0.01), positive initial BAE (OR 3.6, 95 %CI: 1.9 - 6.8; P  < 0.001), and antegrade procedure (OR 3.3, 95 %CI: 1.7 - 6.1; P  < 0.001). On multivariate analysis, a positive initial BAE and antegrade route were the only significant predictive factors. CONCLUSIONS: Performing a repeat BAE for SBB appears safe and provided modest yields. A positive initial BAE and antegrade route were predictive of a positive repeat BAE.

Comparative quality assessment of esophageal examination with transnasal and sedated endoscopy.

Background Unsedated transnasal esophagoscopy (TNE) may offer a less expensive, mobile alternative to sedated esophagogastroduodenoscopy (EGD) for evaluations of reflux related complications. Comparisons of imaging quality by these methods are lacking. Methods Two reviewers evaluated videos of TNE and EGD procedures, performed during a community randomized study comparing endoscopic techniques. Subjects were randomized to EGD, TNE in endoscopy suite, or TNE in mobile research unit. Endoscopic quality was assessed using a validated scoring tool. Results In total, 115 videos (58 EGD, 28 endoscopy suite TNE, and 29 mobile TNE) were reviewed. Overall quality scores for TNE and EGD were excellent without a statistically significant difference (P = 0.30). There were no differences in gastroesophageal junction (GEJ) visualization scores, though EGD scored higher in esophageal passage (P < 0.05) and TNE scored higher in esophageal intubation (P < 0.05). There was no significant difference in any quality score between mobile TNE and gastrointestinal suite TNE. Conclusion Esophageal assessment with TNE or EGD was comparable in overall quality and GEJ visualization. TNE quality was not affected by procedure location. TNE is a feasible option for endoscopic assessment of reflux complications.

Changes in the Prevalence of Hepatitis C Virus Infection, Nonalcoholic Steatohepatitis, and Alcoholic Liver Disease Among Patients With Cirrhosis or Liver Failure on the Waitlist for Liver Transplantation.

BACKGROUND & AIMS: Concurrent to development of more effective drugs for treatment of hepatitis C virus (HCV) infection, there has been an increase in the incidence of nonalcoholic fatty liver disease. Data indicate that liver transplantation prolongs survival times of patient with acute hepatitis associated with alcoholic liver disease (ALD). We compared data on disease prevalence in the population with data from liver transplantation waitlists to evaluate changes in the burden of liver disease in the United States. METHODS: We collected data on the prevalence of HCV from the 2010 and 2013-2014 cycles of the National Health and Nutrition Examination Survey. We also collected data from the HealthCore Integrated Research Database on patients with cirrhosis and chronic liver failure (CLF) from 2006 through 2014, and data on patients who received transplants from the United Network for Organ Sharing from 2003 through 2015. We determined percentages of new waitlist members and transplant recipients with HCV infection, stratified by indication for transplantation, modeling each calendar year as a continuous variable using the Spearman rank correlation, nonparametric test of trends, and linear regression models. RESULTS: In an analysis of data from the National Health and Nutrition Examination Survey (2013-2014), we found that the proportion of patients with a positive HCV antibody who had a positive HCV RNA was 0.5 (95% confidence interval, 0.42-0.55); this value was significantly lower than in 2010 (0.64; 95% confidence interval, 0.59-0.73) (P = .03). Data from the HealthCore database revealed significant changes (P < .05 for all) over time in percentages of patients with compensated cirrhosis (decreases in percentages of patients with cirrhosis from HCV or ALD, but increase in percentages of patients with cirrhosis from nonalcoholic steatohepatitis [NASH]), CLF (decreases in percentages of patients with CLF from HCV or ALD, with an almost 3-fold increase in percentage of patients with CLF from NASH), and hepatocellular carcinoma (HCC) (decreases in percentages of patients with HCC from HCV or ALD and a small increase in HCC among persons with NASH). Data from the United Network for Organ Sharing revealed that among patients new to the liver transplant waitlist, or undergoing liver transplantation, for CLF, there was a significant decrease in the percentage with HCV infection and increases in percentages of patients with nonalcoholic fatty liver disease or ALD. Among patients new to the liver transplant waitlist or undergoing liver transplantation for HCC, proportions of those with HCV infection, nonalcoholic fatty liver disease, or ALD did not change between 2003 and 2015. CONCLUSIONS: In an analysis of 3 different databases (National Health and Nutrition Examination Survey, HealthCore, and United Network for Organ Sharing), we found the proportion of patients on the liver transplant waitlist or undergoing liver transplantation for chronic HCV infection to be decreasing and fewer patients to have cirrhosis or CLF. However, the percentages of patients on the waitlist or receiving liver transplants for NASH or ALD are increasing, despite different relative burdens of disease among the entire population of patients with cirrhosis.

Effects of carbon dioxide insufflation during direct cholangioscopy on biliary pressures and vital parameters: a pilot study in porcine models.

BACKGROUND AND AIMS: Direct per-oral cholangioscopy allows endoscopic visualization of the biliary tract. Insufflation with carbon dioxide (CO2) is an alternative to saline solution irrigation during direct cholangioscopy. There are no data on maximal CO2 insufflation in direct cholangioscopy without causing biliary barotrauma or fatal gas embolism. We aimed to evaluate the safety of increasing CO2 insufflation in direct cholangioscopy without causing biliary barotrauma or fatal gas embolism. METHODS: This was an in vivo animal study. Four domestic pigs, under general endotracheal anesthesia, were used. The first animal was used to validate the feasibility of direct cholangioscopy and biliary pressure measurements, after which all animals underwent laparotomy, insertion of a pressure transducer in the cystic duct, and direct transpapillary placement of the cholangioscope. The common bile duct (CBD) and cystic duct were ligated to contain the instilled gas and exclusively expose the biliary tree. Insufflation of CO2 started at 200 mL/min and was continuously increased until there was evidence of bile duct rupture (as measured by a drop in intraductal pressures) or instability of vital signs (hypotension, bradycardia, bradypnea, O2 desaturation). Necropsy was performed on all animals to assess the liver and biliary system for evidence of barotrauma. RESULTS: CO2 was insufflated up to 8 L/min without causing bile duct rupture or instability in vital signs despite increasing CBD pressure with insufflation. There was significant correlation between CO2 flow with partial pressure of CO2 in arterial blood (PaCO2) (coefficient, 0.96-1.00; P < .01) and end tidal expired CO2 (EtCO2) (coefficient, 0.94-1.00; P < .01). However, the pulse rate, respiratory rate, arterial blood pressure, and O2 did not correlate with the amount of CO2 flow. There was no evidence of hepatic or biliary barotrauma on necropsy. CONCLUSIONS: This pilot experience in porcine models suggests that CO2 insufflation is safe for direct cholangioscopy and does not result in biliary barotrauma or vital signs instability.

Outcomes of double-balloon enteroscopy-assisted direct percutaneous endoscopic jejunostomy tube placement.

BACKGROUND AND STUDY AIMS: In a large series, conventional direct percutaneous endoscopic jejunostomy (DPEJ) tube placement with push endoscopes failed in approximately one-third of patients. In a pilot study, double-balloon enteroscopy (DBE)-assisted DPEJ tube placement was successful in all patients in whom attempted conventional DPEJ had failed. The study aim was to assess the technical success of and adverse events related to DBE-DPEJ tube placement in a large cohort of patients. PATIENTS AND METHODS: The medical records of all patients who underwent DBE-DPEJ tube placement between July 2010 and November 2013 were reviewed using a prospectively maintained electronic database. Data were abstracted for patient demographics, indications for DPEJ, gut anatomy, technical success rate, causes of failure, and adverse events. RESULTS: The study comprised a total of 94 patients (39 men; mean age 56 years; body mass index [BMI] 23 ±â€Š6.4 kg/m(2)). The most common indication for DPEJ was gastroparesis (n = 29). Altered gut anatomy was present in 36 patients (38 %). DBE-DPEJ tube placement was technically successful in 87 patients (93 %). The mean procedure duration was 33 minutes (range 15 - 88). DBE-DPEJ tube placement failed in seven patients (7 %), primarily because of limited instrument advancement in the setting of presumed surgical adhesions. Post-procedural adverse events occurred in eight patients (9 %), with one serious adverse event, which was a gastric interposition requiring surgical repair. CONCLUSIONS: Compared with the published outcomes of DPEJ by conventional endoscopy, DBE-DPEJ tube placement was technically successful in a high proportion of patients (93 %) and with a relatively low rate of significant adverse events.

Outcomes and Predictors of Rebleeding After 2-Octyl Cyanoacrylate Injection in Acute Gastric Variceal Hemorrhage.

BACKGROUND AND AIM: Endoscopic injection of 2-octyl cyanoacrylate (2-OCA) is used on an off-label basis for gastric variceal hemorrhage (GVH) in the United States. We assessed the efficacy, safety, and predictors of rebleeding after gastric variceal obturation (GVO) with 2-OCA in patients with acute GVH. MATERIALS AND METHODS: A retrospective analysis was performed of patients with GVH who underwent 2-OCA injection for GVO over a 15-year period. Rates of acute hemostasis, predictors of rebleeding, and cyanoacrylate-related adverse events were assessed. RESULTS: A total of 95 patients (63 males, median age 59±14 y) were analyzed. Gastric varices were categorized as GOV-1 (3%), GOV-2 (61%), and isolated gastric varices type 1 (36%) per Sarin classification. Initial hemostasis was achieved in all patients. Successful GVO, defined as sustained hemostasis within a month after injection, was achieved in 87 (92%) patients. Failed GVO with in-hospital rebleeding was observed in 8 (8%) patients. On univariate analysis, only the model for end-stage liver disease score was associated with an increased risk of rebleeding (odds ratio 1.2; 95% confidence interval, 1.1-1.4; P<0.01). Glue-related adverse events consisted of pulmonary emboli in 2 patients (2.1%), resulting in death in 1 patient. All cause in-hospital mortality was 13% due to uncontrolled gastric variceal rebleeding (n=3), renal failure (n=6), metastatic hepatocellular carcinoma (n=1), hemorrhagic stroke (n=1), and pulmonary embolism (n=1). CONCLUSIONS: Injection of 2-OCA was effective at achieving hemostasis in a high proportion of patients (92%) admitted for acute GVH. The risk of glue-related pulmonary embolism approximated 2% in our patient cohort, including 1 fatality.

Comparative diagnostic performance of volumetric laser endomicroscopy and confocal laser endomicroscopy in the detection of dysplasia associated with Barrett's esophagus.

BACKGROUND AND AIMS: Probe-based confocal laser endomicroscopy (pCLE) and volumetric laser endomicroscopy (VLE) (also known as frequency domain optical coherence tomography) are advanced endoscopic imaging modalities that may be useful in the diagnosis of dysplasia associated with Barrett's esophagus (BE). We performed pCLE examination in ex-vivo EMR specimens and compared the diagnostic performance of using the current VLE scoring index (previously established as OCT-SI) and a novel VLE diagnostic algorithm (VLE-DA) for the detection of dysplasia. METHODS: A total of 27 patients with BE enrolled in a surveillance program at a tertiary-care center underwent 50 clinically indicated EMRs that were imaged with VLE and pCLE and classified into neoplastic (N = 34; high-grade dysplasia, intramucosal adenocarcinoma) and nonneoplastic (N = 16; low-grade dysplasia, nondysplastic BE), based on histology. Image datasets (VLE, N = 50; pCLE, N = 50) were rated by 3 gastroenterologists trained in the established diagnostic criteria for each imaging modality as well as a new diagnostic algorithm for VLE derived from a training set that demonstrated association of specific VLE features with neoplasia. Sensitivity, specificity, and diagnostic accuracy were assessed for each imaging modality and diagnostic criteria. RESULTS: The sensitivity, specificity, and diagnostic accuracy of pCLE for detection of BE dysplasia was 76% (95% confidence interval [CI], 59-88), 79% (95% CI, 53-92), and 77% (95% CI, 72-82), respectively. The optimal diagnostic performance of OCT-SI showed a sensitivity of 70% (95% CI, 52-84), specificity of 60% (95% CI, 36-79), and diagnostic accuracy of 67%; (95% CI, 58-78). The use of the novel VLE-DA showed a sensitivity of 86% (95% CI, 69-96), specificity of 88% (95% CI, 60-99), and diagnostic accuracy of 87% (95% CI, 86-88). The diagnostic accuracy of using the new VLE-DA criteria was significantly superior to the current OCT-SI (P < .01). CONCLUSION: The use of a new VLE-DA showed enhanced diagnostic performance for detecting BE dysplasia ex vivo compared with the current OCT-SI. Further validation of this algorithm in vivo is warranted.

Prediction of response to endoscopic therapy of Barrett's dysplasia by using genetic biomarkers.

BACKGROUND: Endoscopic therapy for the treatment of high-grade dysplasia (HGD) and intramucosal cancer (IMC) in Barrett's esophagus (BE) may not always result in complete remission of dysplasia (CRD). OBJECTIVE: To determine whether genetic alterations in the Barrett's mucosa can predict response to endoscopic therapy. DESIGN: Retrospective cohort study. SETTING: Tertiary-care institution. PATIENTS: Selected patients who underwent endoscopic therapy for BE containing HGD/IMC between 2003 and 2010. INTERVENTIONS: Endoscopic therapy combining mucosal resection and different ablation modalities was performed based on patient characteristics, endoscopic findings, and technique evolution. Fluorescence in situ hybridization was used to evaluate genetic alterations on baseline endoscopic cytology brushings by using probes directed to loci 8q24 (MYC), 9p21 (CDKN2A; alias P16), 17q12 (ERBB2; alias Her-2/neu), and 20q13.2 (ZNF217). MAIN OUTCOME MEASUREMENTS: Genetic biomarkers predicting achievement of CRD after endoscopic therapy. RESULTS: A total of 181 patients were included (145 men; 66 ± 10 years of age). There were 130 patients (72%) who responded to endoscopic therapy with CRD. Multiple gains detected by fluorescence in situ hybridization was found to be a negative predictor (hazard ratio 0.57; 95% confidence interval, 0.40-0.82) after adjusting for potential clinical confounders. Similar results were found when analyses were restricted to patients (n = 66) undergoing radiofrequency ablation (hazard ratio 0.58; 95% confidence interval, 0.31-1.09). LIMITATIONS: Retrospective study, heterogeneity of treatment modalities. CONCLUSION: Patients with multiple gains detected by brush cytology specimens may have a lower response rate to endoscopic therapy. The presence of multiple gains can be an adjunct to standard histology in prognosticating BE patients with HGD/IMC undergoing endoscopic therapy.

Application of confocal laser endomicroscopy in the diagnosis and management of Barrett's esophagus.

Confocal laser endomicroscopy is an advanced endoscopic imaging modality that can be used for the diagnosis of early mucosal dysplasia in various gastrointestinal conditions. It provides histology-like images at 1000-fold magnification. The technology offers potential advantages in the diagnosis of Barrett's esophagus and early esophageal cancer due to the low yield of the current practice of surveillance endoscopy with biopsies. Confocal laser endomicroscopy has the potential to eliminate the need for biopsy, establish diagnosis and facilitate application of endoscopic therapy during the time of actual endoscopy. There are several studies that have demonstrated reasonable diagnostic accuracy in patients undergoing surveillance for Barrett's esophagus from tertiary academic medical centers. However, the application of confocal laser endomicroscopy in routine clinical endoscopy is still in the process of refinement. Its role in the diagnosis and treatment of Barrett's-associated dysplasia will continue to evolve with improvement in technology, criteria for diagnosis and experience among endoscopists in interpreting confocal imaging.

Obstructive sleep apnea is a risk factor for Barrett's esophagus.

BACKGROUND & AIMS: Common risk factors for obstructive sleep apnea (OSA) and Barrett's esophagus (BE) include obesity and gastroesophageal reflux disease (GERD). The aims of this study were to assess the association between OSA and BE and to determine whether the association is independent of GERD and body mass index (BMI). METHODS: Patients who had undergone a diagnostic polysomnogram and esophagogastroduodenoscopy were identified by using Mayo Clinic (Rochester, Minnesota) databases from January 2000-November 2011. They were randomly matched for age, sex, and BMI at time of polysomnogram into the following groups: BE but no OSA (n = 36), OSA but no BE (n = 78), both (n = 74), or neither (n = 74). Clinical and demographic variables were abstracted from medical records. The association between OSA and BE was assessed by using a multiple variable logistic model that incorporated age, sex, BMI, clinical diagnosis of GERD, and smoking history. RESULTS: Subjects with OSA had an 80% increased risk for BE compared with subjects without OSA (odds ratio, 1.8; 95% confidence interval, 1.1-3.2; P = .03). These findings were independent of age, sex, BMI, GERD, and smoking history. Increasing severity of OSA, measured by using the apnea-hypopnea index, was associated with an increased risk of BE (odds ratio, 1.2 per 10-unit increase in apnea-hypopnea index; 95% confidence interval, 1.0-1.3; P = .03). CONCLUSIONS: In this case-control study, OSA was associated with an increased risk of BE, potentially through BMI and GERD independent mechanisms. Patients with OSA may benefit from evaluation for BE.