Long-term outcomes after bladder-preserving tri-modality therapy for patients with muscle-invasive bladder cancer.

PURPOSE: To evaluate trimodal conservative treatment as an alternative to radical surgery for urothelial muscle-invasive bladder cancer (MIBC). PATIENTS AND METHODS: This retrospective study reported the carcinologic and functional results of patients (pts) presenting a cT2/T3 N0M0 operable MIBC and fit for surgery, treated by a conservative strategy. Treatment consisted of a transurethral resection (TURB) followed by concomitant bi-fractionated split-course radiochemotherapy (RCT) with 5FU-Cisplatine. A control cystoscopy was performed six weeks after the induction RCT (eq45Gy) with systematic biopsies. Patients with complete histologic response achieved RCT protocol. Salvage surgery was proposed to pts with persistent tumor. RESULTS: 313 pts (83% cT2 and 17% cT3) treated between 1988 and 2013 were included, with a median follow-up of 59 months and 67-year mean age. After the induction RCT, the histologic response rate was 83%. After five years, overall, disease-free, and functional bladder-intact survival rates were respectively 69%, 61%, and 69%, significantly better for pts in complete response after induction RCT. Late urinary and digestive toxicities were limited, with respective rates of 4% and 1.5% of grade 3 toxicity. CONCLUSION: Trimodal strategy with RCT after TURB showed interesting functional and oncologic results and should be considered as an alternative to surgery in well-selected pts.

Low-phospholipid-associated cholelithiasis syndrome: Prevalence, clinical features, and comorbidities.

BACKGROUND & AIMS: Low-phospholipid-associated cholelithiasis (LPAC) syndrome, a rare genetic form of intrahepatic cholelithiasis in adults, is still poorly understood. We report the results of the largest-ever case-control study of patients with LPAC syndrome aiming to assess the prevalence, clinical features, and comorbidities of the disease. METHODS: We included all LPAC cases diagnosed between 2001 and 2016 in 11 French centres. Controls consisted of all patients who underwent a cholecystectomy for common gallstone disease in a single non-academic centre over 1 year. A logistic regression analysis was used to identify the clinical features associated with LPAC syndrome across several patient strata with increasing levels of diagnostic confidence. The ratio between the incident cases of LPAC syndrome and the total number of cholecystectomies for gallstones was used to assess the relative prevalence of the disease. RESULTS: In this study, 308 cases and 206 controls were included. LPAC syndrome accounted for 0.5-1.9% of all patients admitted with symptomatic gallstone disease. Age at first symptoms <40 years, absence of overweight, persistence of symptoms after cholecystectomy, intrahepatic micro- or macrolithiasis, common bile duct (CBD) lithiasis, and no history of cholecystitis were independently associated with LPAC diagnosis. ATP-binding cassette subfamily B member 4 (ABCB4) variants, present in 46% of cases, were associated with CBD lithiasis, chronic elevation of gamma-glutamyltransferase (GGT), and personal or family history of hepato-biliary cancer. CONCLUSIONS: In this case-control study, LPAC syndrome accounted for approximately 1% of symptomatic cholelithiasis in adults. In addition to pre-established diagnostic criteria, normal weight, CBD lithiasis, and no history of cholecystitis were significantly associated with the syndrome. ABCB4 gene variations in patients with LPAC were associated with CBD lithiasis, chronic cholestasis, and a personal or family history of hepato-biliary cancer. LAY SUMMARY: In the largest case-control study ever conducted in patients with LPAC syndrome, a rare genetic form of intrahepatic cholelithiasis in young adults, LPAC syndrome was found in approximately 1% of all patients admitted to the hospital for symptomatic gallstones and, in addition to the pre-established characteristics of the syndrome (age at first symptoms <40 years, recurrence of symptoms after cholecystectomy, and/or imaging evidence of intrahepatic microlithiasis), was associated with lower BMI, higher prevalence of common bile duct stones, and lower incidence of acute cholecystitis. ABCB4 gene variants, which were detected in about half of cases, were associated with common bile duct stones and a personal or family history of hepato-biliary cancer.

Novel defatting strategies reduce lipid accumulation in primary human culture models of liver steatosis.

Normothermic perfusion provides a means to rescue steatotic liver grafts, including by pharmacological defatting. In this study, we tested the potential of new drug combinations to trigger defatting in three human culture models, primary hepatocytes with induced steatosis, primary hepatocytes isolated from steatotic liver, and precision-cut liver slices (PCLS) of steatotic liver. Forskolin, L-carnitine and a PPARα agonist were all combined with rapamycin, an immunosuppressant that induces autophagy, in a D-FAT cocktail. D-FAT was tested alone or in combination with necrosulfonamide, an inhibitor of mixed lineage kinase domain like pseudokinase involved in necroptosis. Within 24 h, in all three models, D-FAT induced a decrease in triglyceride content by 30%, attributable to an upregulation of genes involved in free fatty acid ß-oxidation and autophagy, and a downregulation of those involved in lipogenesis. Defatting was accompanied by a decrease in endoplasmic reticulum stress and in the production of reactive oxygen species. The addition of necrosulfonamide increased the efficacy of defatting by 8%-12% in PCLS, with a trend towards increased autophagy. In conclusion, culture models, notably PCLS, are insightful to design strategies for liver graft rescue. Defatting can be rapidly achieved by combinations of drugs targeting mitochondrial oxidative metabolism, macro-autophagy and lipogenesis.

An updated evaluation of serum sHER2, CA15.3, and CEA levels as biomarkers for the response of patients with metastatic breast cancer to trastuzumab-based therapies.

BACKGROUND: The transmembrane receptor tyrosine kinase HER2 is overexpressed in approximately 15% of breast tumors and correlates with poor clinical prognosis. Several treatments that target HER2 are approved for treatment of HER2-positive metastatic breast cancer. The serum biomarkers most widely used to monitor anti-HER2 therapies in patients with HER2-positive metastatic breast cancer currently are CA15.3 and CEA. Nevertheless, their clinical utility in patients with breast cancer remains a subject of discussion and controversy; thus, additional markers may prove useful in monitoring the therapeutic responses of these patients. The extracellular domain of HER2 can be shed by proteolytic cleavage into the circulation and this shed form, sHER2, is reported to be augmented during metastasis of HER2-positive breast tumors. Here, we studied the clinical usefulness of sHER2, CA15.3, and CEA for monitoring treatment for breast cancer. METHODS: We measured prospectively pretreatment and post-treatment serum levels (day 1, 30, 60 and 90) of these three biomarkers in 47 HER2-positive, metastatic breast cancer patients treated with trastuzumab in combination with paclitaxel. Evaluation of the disease was performed according to the Response Evaluation Criteria in Solid Tumor (RECIST) at day 90. RESULTS: Patients with progressive disease at day 90 had smaller relative changes between day 1 and day 30 than those with complete, partial or stable responses at day 90: -9% versus -38% for sHER2 (P = 0.02), +23% versus -17% for CA15.3 (P = 0.005) and +29% versus -26% for CEA (P = 0.02). Patients with progressive disease at day 90 were less likely than the other patients to have a relative decrease of > 20% in their biomarker levels at day 30: 6% vs 33% for sHER2 (P = 0.03), 0% vs 27% for CA15.3 (P = 0.03), 4% vs 29% for CEA (P = 0.04). No patient with progressive disease at day 90 had > 20% reduction of the average combined biomarker levels at day 30 whereas 63% of the other patients had (P = 0.003). Moreover, when we analyzed a > 10% reduction of the average biomarker levels no patient with progressive disease at day 90 had a decrease > 10% at day 30 whereas 78% of other patients had (P<0.001, Se = 100%, Sp = 78%). CONCLUSION: We show that regular measurement of sHER2, CA15.3, and CEA levels is useful for predicting the therapeutic response and for monitoring HER2-targeted therapy in patients with HER2-positive metastatic breast cancer. The average decrease of the three biomarkers with a threshold of > 10% appears to be the best parameter to distinguish patients who go on to have progressive disease from those who will have a complete, partial or stable response.

Genetic contribution of ABCC2 to Dubin-Johnson syndrome and inherited cholestatic disorders.

BACKGROUND AND AIMS: The ABCC2 gene is implicated in Dubin-Johnson syndrome (DJS), a rare autosomal recessive liver disorder. The primary aim of this study was to determine the diagnostic value of ABCC2 genetic testing in the largest cohort of DJS reported to date. The high number of patients with cholestatic manifestations in this series prompted us to evaluate the genetic contribution of rare, potentially pathogenic ABCC2 variants to other inherited cholestatic disorders. METHODS: The cohort study included 32 patients with clinical DJS diagnosis, and 372 patients referred for the following disorders: low phospholipid-associated cholelithiasis (LPAC) syndrome, intrahepatic cholestasis of pregnancy (ICP) and benign recurrent intrahepatic cholestasis (BRIC). ABCC2 was screened by next-generation sequencing. RESULTS: Most patients with clinical DJS had positive genetic diagnosis (n = 30; 94%), with a great diversity of point mutations and copy number variations in ABCC2. Strikingly, eight (27%) of these patients showed transient cholestatic features at presentation: four neonatal cholestasis, two ICP, one contraceptive-induced cholestasis and one sporadic cholestasis. Conversely, the frequency of rare, heterozygous, potentially pathogenic ABCC2 variants in patients with LPAC, ICP or BRIC did not differ significantly from that of the general population. CONCLUSIONS: This large series reveals that DJS is a highly homogeneous Mendelian disorder involving a large spectrum of ABCC2 variants. Genetic testing is crucial to establish early DJS diagnosis in patients with atypical presentations, such as neonatal cholestasis. This study also provides no evidence for the contribution of rare, potentially pathogenic ABCC2 variants to other inherited cholestatic disorders.

The Complementary Value of Magnetic Resonance Imaging and Vibration-Controlled Transient Elastography for Risk Stratification in Primary Sclerosing Cholangitis.

OBJECTIVES: Magnetic resonance (MR) risk scores and liver stiffness (LS) have individually been shown to predict clinical outcomes in primary sclerosing cholangitis (PSC). The aim of this study was to assess their complementary prognostic value. METHODS: Patients with PSC from 3 European centers with a 3-dimensional MR cholangiography available for central reviewing and a valid LS measurement assessed by vibration-controlled transient elastography by FibroScan performed within a 6-month interval were included in a longitudinal retrospective study. The MR score (Anali) without gadolinium (Gd) was calculated according to the formula: (1 × dilatation of intrahepatic bile ducts) + (2 × dysmorphy) + (1 × portal hypertension). The primary end point was survival without liver transplantation or cirrhosis decompensation. The prognostic values of LS and Anali score without Gd were assessed using Cox proportional hazard models. RESULTS: One hundred sixty-two patients were included. Over a total follow-up of 753 patient-years, 40 patients experienced an adverse outcome (4 liver transplantations, 6 liver-related deaths, and 30 cirrhosis decompensations). LS and Anali score without Gd were significantly correlated (ρ = 0.51, P < 0.001) and were independently associated with the occurrence of an adverse outcome. Optimal prognostic thresholds were 10.5 kPa for LS and 2 for the Anali score without Gd. Hazard ratios (95% confidence interval) were 2.07 (1.06-4.06) and 3.78 (1.67-8.59), respectively. The use in combination of these 2 thresholds allowed us to separate patients into low-, medium-, and high-risk groups for developing adverse outcomes. The 5-year cumulative rates of adverse outcome in these 3 groups were 8%, 16%, and 38% (P < 0.001), respectively. DISCUSSION: The combined use of MRI and vibration-controlled transient elastography permits easy risk stratification of patients with PSC.

Postoperative Imaging After Lobectomy: Predicting the Displacement and Change in Orientation of Nonresected Lung Nodules.

OBJECTIVES: The objective of this study was to determine the effect of a lobectomy to the location and orientation of nonresected lung nodule and its corresponding airway. METHODS: We reviewed preoperative and postoperative computed tomography of patients who underwent lobectomies and have a separate nonresected nodule in the ipsilateral lung. Displacement of the nonresected nodule and angulation of its corresponding segmental bronchus were measured. RESULTS: Fifty nodules from 40 patients (30 females, 10 male; mean ± SD age, 67 ± 7 years) were assessed. Nodules are displaced clockwise after right upper, right middle, and left lower lobectomies and counterclockwise after right lower and left upper lobectomies. Displacement of the remaining nodules was greater in the craniocaudal plane, followed by anteroposterior and transverses planes (mean, 3.7, 2.5, and 1.9 cm, respectively). CONCLUSIONS: Remaining ipsilateral nodules and their associated segmental airways are displaced in a predictable fashion after lobectomy. This may help in the assessment of follow-up imaging.

Insulin receptor isoform A favors tumor progression in human hepatocellular carcinoma by increasing stem/progenitor cell features.

Hepatocellular carcinoma (HCC) is one of the most common and deadly neoplasms. Insulin receptor (IR) exists in two isoforms, IR-A and IR-B, the latter being predominantly expressed in normal adult hepatocytes while IR-A is overexpressed in HCC to the detriment of IR-B. This study evaluated the biological functions associated with IR-A overexpression in HCC in relation to expression of its ligand IGF-II. The value of INSRA:INSRB ratio which was increased in ˜70% of 85 HCC was associated with stem/progenitor cell features such as cytokeratin-19 and α-fetoprotein and correlated with shorter patient survival. IGF2 mRNA upregulation was observed in 9.4% of HCC and was not associated with higher INSRA:INSRB ratios. Ectopic overexpression of IR-A in two HCC cell lines presenting a strong autocrine IGF-II secretion loop or not stimulated cell migration and invasion. In cells cultured as spheroids, IR-A overexpression promoted gene programs related to stemness, inflammation and cell movement. IR-A also increased cell line tumorigenicity in vivo after injection to immunosuppressed mice and the sphere-forming cells made a significant contribution to this effect. Altogether, these results demonstrate that IR-A is a novel player in HCC progression.

Simple Magnetic Resonance Scores Associate With Outcomes of Patients With Primary Sclerosing Cholangitis.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) has a variable, often progressive, course. Magnetic resonance cholangiography (MRC) is used in the diagnosis of PSC. Magnetic resonance risk scoring systems, called Anali without and with gadolinium, are used to predict disease progression, determined by radiologic factors. We aimed to assess the prognostic value of Anali scores in patients with PSC and validate our findings in a separate cohort. METHODS: We performed a retrospective study of patients with large-duct PSC (internal cohort, 119 patients in France; external cohort, 119 patients in Canada, Italy, and the United Kingdom). All the first-available MRC results were reviewed by 2 radiologists and the Anali scores were calculated as follows: Anali without gadolinium = (1× dilatation of intrahepatic bile ducts) + (2× dysmorphy) + (1× portal hypertension); Anali with gadolinium = (1× dysmorphy) + (1× parenchymal enhancement heterogeneity). The primary end point was survival without liver transplantation or cirrhosis decompensation. The prognostic value of Anali scores was assessed by Cox regression modeling. RESULTS: During a total of 549 patient-years for the internal cohort and 497 patient-years for the external cohort, we recorded 2 and 8 liver transplantations, 4 and 3 liver-related deaths, and 26 and 25 cirrhosis decompensations, respectively. In the univariate analysis, factors associated with survival without liver transplantation or cirrhosis decompensation in the internal cohort were as follows: serum levels of bilirubin, aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transferase, alkaline phosphatase, albumin, and Anali scores. Anali scores without and with gadolinium identified patients' survival without liver transplantation or cirrhosis decompensation with a c-statistic of 0.89 (95% CI, 0.84-0.95) and 0.75 (95% CI, 0.64-0.87), respectively. Independent prognostic factors identified by multivariate analysis were Anali scores and bilirubinemia. The prognostic value of Anali scores was confirmed in the external cohort. CONCLUSIONS: In internal and external cohorts, we found that Anali scores, determined from MRC, were associated with outcomes of patients with PSC. These scores might be used as prognostic factors.

Diet-Induced Dysbiosis and Genetic Background Synergize With Cystic Fibrosis Transmembrane Conductance Regulator Deficiency to Promote Cholangiopathy in Mice.

The most typical expression of cystic fibrosis (CF)-related liver disease is a cholangiopathy that can progress to cirrhosis. We aimed to determine the potential impact of environmental and genetic factors on the development of CF-related cholangiopathy in mice. Cystic fibrosis transmembrane conductance regulator (Cftr)-/- mice and Cftr +/+ littermates in a congenic C57BL/6J background were fed a high medium-chain triglyceride (MCT) diet. Liver histopathology, fecal microbiota, intestinal inflammation and barrier function, bile acid homeostasis, and liver transcriptome were analyzed in 3-month-old males. Subsequently, MCT diet was changed for chow with polyethylene glycol (PEG) and the genetic background for a mixed C57BL/6J;129/Ola background (resulting from three backcrosses), to test their effect on phenotype. C57BL/6J Cftr -/- mice on an MCT diet developed cholangiopathy features that were associated with dysbiosis, primarily Escherichia coli enrichment, and low-grade intestinal inflammation. Compared with Cftr +/+ littermates, they displayed increased intestinal permeability and a lack of secondary bile acids together with a low expression of ileal bile acid transporters. Dietary-induced (chow with PEG) changes in gut microbiota composition largely prevented the development of cholangiopathy in Cftr -/- mice. Regardless of Cftr status, mice in a mixed C57BL/6J;129/Ola background developed fatty liver under an MCT diet. The Cftr -/- mice in the mixed background showed no cholangiopathy, which was not explained by a difference in gut microbiota or intestinal permeability, compared with congenic mice. Transcriptomic analysis of the liver revealed differential expression, notably of immune-related genes, in mice of the congenic versus mixed background. In conclusion, our findings suggest that CFTR deficiency causes abnormal intestinal permeability, which, combined with diet-induced dysbiosis and immune-related genetic susceptibility, promotes CF-related cholangiopathy.

Thoracic Skeletal Muscle Is Associated With Adverse Outcomes After Lobectomy for Lung Cancer.

BACKGROUND: Assessment of risk associated with lung cancer resection is primarily based on evaluation of cardiopulmonary function and remains imprecise. We investigated the relationship between thoracic muscle and early outcomes after lobectomy. METHODS: Cross-sectional area of skeletal muscle was measured at the level of the fifth thoracic vertebra on computed tomography in 135 consecutive patients before lobectomy for lung cancer. Patients were stratified into low and high muscle groups using the sex-specific muscle median. Primary outcome was a composite of any postoperative complication as per The Society of Thoracic Surgeons General Thoracic Surgical Database. Secondary outcomes included postoperative respiratory complications, postoperative intensive care unit admission, hospital length of stay, and hospital readmission within 30 days of hospital discharge. The χ2 test, adjusted multivariable regression analysis, and likelihood ratio test were performed. RESULTS: Patients with low muscle were significantly more likely to have any postoperative complication and respiratory postoperative complications. Although postoperative intensive care unit admission was similar for low muscle and high muscle groups, low muscle patients had longer hospital length of stay and a higher rate of hospital readmission. Adjusted multivariable regression revealed the independent association of thoracic muscle with all outcomes. The likelihood ratio test suggested that thoracic muscle adds predictive capability to information captured by preoperative pulmonary function testing. CONCLUSIONS: Low thoracic muscle is independently associated with increased postoperative complications and health care utilization among patients undergoing lobectomy for lung cancer. Evaluation of thoracic muscle may enhance risk prediction models.

Quantifying the effect of slice thickness, intravenous contrast and tube current on muscle segmentation: Implications for body composition analysis.

OBJECTIVES: To quantify the effect of IV contrast, tube current and slice thickness on skeletal muscle cross-sectional area (CSA) and density (SMD) on routine CT. METHODS: CSA and SMD were computed on 216 axial CT images obtained at the L3 level in 72 patients with variations in IV contrast, slice thickness and tube current. Intra-patient mean difference (MD), 95 % CI and limits of agreement were calculated using the Bland-Altman approach. Inter- and intra-analyst agreement was evaluated. RESULTS: IV contrast significantly increased CSA by 1.88 % (MD 2.33 cm2; 95 % CI 1.76-2.89) and SMD by 5.99 % (p<0.0001). Five mm slice thickness significantly increased mean CSA by 1.11 % compared to 2 mm images (1.32 cm2; 0.78-1.85) and significantly decreased SMD by 11.64 % (p<0.0001). Low tube current significantly decreased mean CSA by 4.79 % (6.44 cm2; 3.78-9.10) and significantly increased SMD by 46.46 % (p<0.0001). Inter- and intra-analyst agreement was excellent. CONCLUSIONS: IV contrast, slice thickness and tube current significantly affect CSA and SMD. Investigators designing and analysing clinical trials using CT for body composition analysis should report CT acquisition parameters and consider the effect of slice thickness, IV contrast and tube current on myometric data. KEY POINTS: • Intravenous contrast, slice thickness and tube current significantly affect myometric data. • Image acquisition parameter variations may obscure intrapatient muscle differences on serial measurements. • Investigators using CT for body composition analysis should report CT acquisition parameters.

Lumbar skeletal muscle index derived from routine computed tomography exams predict adverse post-extubation outcomes in critically ill patients.

PURPOSE: To evaluate the effect of a skeletal muscle index derived from a routine CT image at the level of vertebral body L3 (L3SMI) on outcomes of extubated patients in the surgical intensive care unit. MATERIALS AND METHODS: 231 patients of a prospective observational trial (NCT01967056) who had undergone CT within 5days of extubation were included. L3SMI was computed using semi-automated segmentation. Primary outcomes were pneumonia within 30days of extubation, adverse discharge disposition and 30-day mortality. Secondary outcomes included re-intubation within 72h, total hospital costs, ICU length of stay (LOS), post-extubation LOS and total hospital LOS. Outcomes were analyzed using multivariable regression models with a priori-defined covariates height, gender, age, APACHE II score and Charlson Comorbidity Index. RESULTS: L3SMI was an independent predictor of pneumonia (aOR 0.96; 95% CI 0.941-0.986; P=0.002), adverse discharge disposition (aOR 0.98; 95% CI 0.957-0.999; P=0.044) and 30-day mortality (aOR 0.94; 95% CI 0.890-0.995; P=0.033). L3SMI was significantly lower in re-intubated patients (P=0.024). Secondary analyses suggest that L3SMI is associated with total hospital costs (P=0.043) and LOS post-extubation (P=0.048). CONCLUSION: The lumbar skeletal muscle index, derived from routine abdominal CT, is an objective prognostic tool at the time of extubation.

Heregulin-1ß and HER3 in hepatocellular carcinoma: status and regulation by insulin.

BACKGROUND: The heregulin-1ß/HER3-driven pathway is implicated in several epithelial malignancies and its blockade is currently undergoing clinical investigation. Paradoxically, the status and the regulation of this pathway is poorly known in hepatocellular carcinoma (HCC). METHODS: Using 85 HCC obtained after tumour resection, heregulin-1ß and HER3 expression was evaluated by real-time RT-PCR, ELISA and/or immunohistochemistry. Statistics were performed to analyze associations between gene expression and clinicopathological parameters. The effects of insulin on the heregulin-1ß/HER3 pathway was investigated in four HCC cell lines. RESULTS: HER3 mRNA was upregulated in 52 % of tumours, while heregulin-1ß mRNA was downregulated in 82 %. Hepatitis B and C viral infections were respectively associated with high and low HER3 mRNA expression. No association was seen between neither HER3 or heregulin-1ß mRNA and prognostic factors, survival or recurrence. Immunohistochemistry showed predominant cytoplasmic staining of HER3 in tumours but the staining was nonreproducible. HER3 mRNA and protein levels were not correlated in liver tissues. In HCC cells, insulin promoted HER3 proteasomal degradation and inhibited heregulin-1ß stimulation of cell migration. HER3 and insulin receptor co-immunoprecipitated in these cells. The loss of insulin receptor expression by RNA interference sensitized cells to heregulin-1ß-induced AKT phosphorylation. CONCLUSIONS: Autocrine heregulin-1ß loop is uncommon in HCC and HER3 mRNA expression is differentially influenced by hepatitis viruses. Insulin is a negative regulator of HER3 protein expression and function in HCC cells. Altogether these data may explain why HER3 and heregulin-1ß expression have no prognostic value and suggest that HCC patients are unlikely to derive benefit from HER3-targeted monotherapies.

Loss of ezrin in human intrahepatic cholangiocarcinoma is associated with ectopic expression of E-cadherin.

AIMS: Ezrin connects proteins from the plasma membrane to the subcortical cytoskeleton, and contributes to epithelial integrity by interacting with the cell-cell adhesion molecule E-cadherin. In the liver, ezrin is restricted to cholangiocytes, where it regulates biliary secretory functions. During carcinogenesis, ezrin expression is impaired and associated with enhancement of cell migratory activity in cancer cells; therefore, we aimed to analyse ezrin in cholangiocarcinogenesis. METHODS AND RESULTS: Ezrin expression was evaluated by immunohistochemistry on tissue microarrays from 94 surgical specimens of intrahepatic cholangiocarcinoma (CCA), and correlated with clinicopathological factors and E-cadherin expression. Ezrin function was also analysed in human CCA cell lines. In CCA, ezrin was negative/weakly expressed in 49 cases (52%) and moderately/strongly expressed in 45 cases (48%), mostly in cell cytoplasm. The negative/weak expression of ezrin was more frequent in peripheral than in perihilar CCA (P = 0.002), and was associated with high tumour size (P = 0.001), low mucus secretion (P = 0.042), the presence of satellite nodules (P = 0.024), and ectopic cytoplasmic expression of E-cadherin (P = 0.005). In vitro, silencing of ezrin in CCA cells caused internalization of E-cadherin and favoured cell migration. CONCLUSIONS: Ezrin is down-regulated during cholangiocarcinogenesis, and its loss results in a more aggressive phenotype.

EGF/EGFR axis contributes to the progression of cholangiocarcinoma through the induction of an epithelial-mesenchymal transition.

BACKGROUND & AIMS: Epithelial-mesenchymal transition (EMT) is a cellular process involved in cancer progression. The first step of EMT consists in the disruption of E-cadherin-mediated adherens junctions. Cholangiocarcinoma (CCA), a cancer with a poor prognosis due to local invasion and metastasis, displays EMT features. EGFR, a receptor tyrosine kinase, plays a major role in CCA progression. The aim of the study was to determine if EMT is induced by EGFR in CCA cells. METHODS: In vivo, the expression of E-cadherin was analysed in CCA tumours of 100 patients and correlated with pathological features and EGFR expression, and in a xenograft model in mice treated with gefitinib, an inhibitor of EGFR. In vitro, the regulation of EMT by EGFR was investigated in CCA cell lines. RESULTS: In human CCA, a cytoplasmic localization of E-cadherin occurred in 50% of the tumours was associated with the peripheral type of CCA, tumour size, the presence of satellite nodules and EGFR overexpression. In xenografted tumours, E-cadherin displayed a cytoplasmic pattern whereas the treatment of mice with gefitinib restored the membranous expression of E-cadherin. In vitro, EGF induced scattering of CCA cells that resulted from the disruption of adherens junctions. Internalization and decreased expression of E-cadherin, as well as nuclear translocation of ß-catenin, were observed in EGF-treated CCA cells. In these cells, EMT-transcription factors (i.e., Slug and Zeb-1) and mesenchymal markers (i.e., N-cadherin and α-SMA) were induced, favoring cell invasiveness through cytoskeleton remodeling. All these effects were inhibited by gefitinib. CONCLUSIONS: The EGF/EGFR axis triggers EMT in CCA cells highlighting the key role of this pathway in CCA progression.

Mitogenic insulin receptor-A is overexpressed in human hepatocellular carcinoma due to EGFR-mediated dysregulation of RNA splicing factors.

Insulin receptor (IR) exists as two isoforms resulting from the alternative splicing of IR pre-mRNA. IR-B promotes the metabolic effects of insulin, whereas IR-A rather signals proliferative effects. IR-B is predominantly expressed in the adult liver. Here, we show that the alternative splicing of IR pre-mRNA is dysregulated in a panel of 85 human hepatocellular carcinoma (HCC) while being normal in adjacent nontumor liver tissue. An IR-B to IR-A switch is frequently observed in HCC tumors regardless of tumor etiology. Using pharmacologic and siRNA approaches, we show that the autocrine or paracrine activation of the EGF receptor (EGFR)/mitogen-activated protein/extracellular signal-regulated kinase pathway increases the IR-A:IR-B ratio in HCC cell lines, but not in normal hepatocytes, by upregulating the expression of the splicing factors CUGBP1, hnRNPH, hnRNPA1, hnRNPA2B1, and SF2/ASF. In HCC tumors, there is a significant correlation between the expression of IR-A and that of splicing factors. Dysregulation of IR pre-mRNA splicing was confirmed in a chemically induced model of HCC in rat but not in regenerating livers after partial hepatectomy. This study identifies a mechanism responsible for the generation of mitogenic IR-A and provides a novel interplay between IR and EGFR pathways in HCC. Increased expression of IR-A during neoplastic transformation of hepatocytes could mediate some of the adverse effects of hyperinsulinemia on HCC.

Genotype-phenotype relationships in the low-phospholipid-associated cholelithiasis syndrome: a study of 156 consecutive patients.

UNLABELLED: The low-phospholipid-associated cholelithiasis syndrome (LPAC; OMIM 171060) is a peculiar form of intrahepatic cholelithiasis occurring in young adults, associated with ABCB4/MDR3 gene sequence variations. Our aim was to determine the genotype-phenotype relationships in 156 consecutive patients with the criteria of LPAC syndrome. A variant was detected in 79 (61 missense and 18 truncating sequence variants), 63 being monoallelic. The clinical features (age at onset, high prevalence in women, frequency and severity of acute and chronic complications, intrahepatic cholestasis of pregnancy [ICP]) were similar in the patients with or without ABCB4 gene sequence variation. Truncating variations were associated with an earlier onset of symptoms both in women and men. Acute and chronic biliary complications were variant-independent. Half of the women who had pregnancy developed ICP. The frequency of ICP and fetal complications were similar in patients with missense and truncating variants. CONCLUSION: The LPAC syndrome is more frequent in women and highly associated with ICP. Half of the patients harbored missense or truncating variants of the ABCB4 gene. The characteristics of the patients without detectable variant are similar to those with variant, indicating that yet unexplored regions of the ABCB4 and other genes may be involved.

Impact of online education on intern behaviour around joint commission national patient safety goals: a randomised trial.

PURPOSE: To compare the effectiveness of two types of online learning methodologies for improving the patient-safety behaviours mandated in the Joint Commission National Patient Safety Goals (NPSG). METHODS: This randomised controlled trial was conducted in 2010 at Massachusetts General Hospital and Brigham and Women's Hospital (BWH) in Boston USA. Incoming interns were randomised to either receive an online Spaced Education (SE) programme consisting of cases and questions that reinforce over time, or a programme consisting of an online slide show followed by a quiz (SQ). The outcome measures included NPSG-knowledge improvement, NPSG-compliant behaviours in a simulation scenario, self-reported confidence in safety and quality, programme acceptability and programme relevance. RESULTS: Both online learning programmes improved knowledge retention. On four out of seven survey items measuring satisfaction and self-reported confidence, the proportion of SE interns responding positively was significantly higher (p<0.05) than the fraction of SQ interns. SE interns demonstrated a mean 4.79 (36.6%) NPSG-compliant behaviours (out of 13 total), while SQ interns completed a mean 4.17 (32.0%) (p=0.09). Among those in surgical fields, SE interns demonstrated a mean 5.67 (43.6%) NPSG-compliant behaviours, while SQ interns completed a mean 2.33 (17.9%) (p=0.015). Focus group data indicates that SE was more contextually relevant than SQ, and significantly more engaging. CONCLUSION: While both online methodologies improved knowledge surrounding the NPSG, SE was more contextually relevant to trainees and was engaging. SE impacted more significantly on both self-reported confidence and the behaviour of surgical residents in a simulated scenario.