Paracrine Interaction of Cholangiocellular Carcinoma with Cancer-Associated Fibroblasts and Schwann Cells Impact Cell Migration.

Although the Mitogen-activated protein kinase (MAPK) pathway is enriched in cholangiocarcinoma (CCA), treatment with the multityrosine kinase-inhibitor Sorafenib is disappointing. While cancer-associated fibroblasts (CAF) are known to contribute to treatment resistance in CCA, knowledge is lacking for Schwann cells (SC). We investigated the impact of stromal cells on CCA cells and whether this is affected by Sorafenib. Immunohistochemistry revealed elevated expression of CAF and SC markers significantly correlating with reduced tumor-free survival. In co-culture with CAF, CCA cells mostly migrated, which could be diminished by Sorafenib, while in SC co-cultures, SC predominantly migrated towards CCA cells, unaffected by Sorafenib. Moreover, increased secretion of pro-inflammatory cytokines MCP-1, CXCL-1, IL-6 and IL-8 was determined in CAF mono- and co-cultures, which could be reduced by Sorafenib. Corresponding to migration results, an increased expression of phospho-AKT was measured in CAF co-cultured HuCCT-1 cells, although was unaffected by Sorafenib. Intriguingly, CAF co-cultured TFK-1 cells showed increased activation of STAT3, JNK, ERK and AKT pathways, which was partly reduced by Sorafenib. This study indicates that CAF and SC differentially impact CCA cells and Sorafenib partially reverts these stroma-mediated effects. These findings contribute to a better understanding of the paracrine interplay of CAF and SC with CCA cells.

Eso-Sponge® for anastomotic leakage after oesophageal resection or perforation: outcomes from a national, prospective multicentre registry.

BACKGROUND: Anastomotic leakage (AL) after oesophagectomy and oesophageal perforations are associated with significant morbidity and mortality. Minimally invasive endoscopy is often used as first-line treatment, particularly endoluminal vacuum therapy (EVT). The aim was to assess the performance of the first commercially available endoluminal vacuum device (Eso-Sponge®) in the management of AL and perforation of the upper gastrointestinal tract (GIT). METHODS: The Eso-Sponge® registry was designed in 2014 as a prospective, observational, national, multicentre registry. Patients were recruited with either AL or perforation within the upper GIT. Data were collected with a standardized form and transferred into a web-based platform. Twenty hospitals were enrolled at the beginning of the study (registration number NCT02662777; http://www.clinicaltrials.gov). The primary endpoint was successful closure of the oesophageal defect. RESULTS: Eleven out of 20 centres recruited patients. A total of 102 patients were included in this interim analysis; 69 patients with AL and 33 with a perforation were treated by EVT. In the AL group, a closure of 91 per cent was observed and 76 per cent was observed in the perforation group. The occurrence of mediastinitis (P = 0.002) and the location of the defect (P = 0.008) were identified as significant predictors of defect closure. CONCLUSIONS: The Eso-Sponge® registry offers the opportunity to collate data on EVT with a uniform, commercially available product to improve standardization. Our data show that EVT with the Eso-Sponge® is an option for the management of AL and perforation within the upper GIT.

Treatment of Complicated Anal Fistula by an Endofistular Polyurethane-Sponge Vacuum Therapy: A Pilot Study.

BACKGROUND: Treatment of supra- and transsphincteric anal fistulas remains a clinical challenge because current treatment results are variable and potentially endanger sphincter function. OBJECTIVE: Based on positive results of endoluminal polyurethane-sponge vacuum therapy in the upper and lower GI tract, a new system for endofistular vacuum therapy was developed for anal fistulas to utilize vacuum therapy to remove the endofistular pseudoepithelium and to induce granulation in the fistula tract. DESIGN: This study is based on a prospective case series. PATIENTS: Seven patients with complicated anal fistulas (3 associated with Crohn's disease and 4 of cryptoglandular origin) longer than 4 cm were treated. Initially, the fistula was curettaged and the first endofistular vacuum therapy sponge was positioned in the fistula tract. The inner fistula opening was closed by suture. A 125 mm Hg constant vacuum was applied to the sponge, and the endofistular vacuum therapy sponge was changed a median of 3 (3-5) times after each 48 to 72 hours of constant vacuum therapy. After final removal, the fistulas were reevaluated every other week for 3 months. MAIN OUTCOME MEASURE: The main outcome measured was the closure of the fistula. RESULTS: All patients tolerated the therapy well and no adverse events were observed. Fistula tract closure was demonstrated within 4 weeks after the termination of vacuum therapy. One patient with cryptoglandular fistula developed a recurrence within the follow-up of 3 months. LIMITATIONS: This was an observational study that had no control arm. CONCLUSION: In this pilot case series, the results are encouraging. Because endoluminal vacuum therapy would be a new and sphincter-sparing therapy, this concept warrants further investigation in controlled trials.

Evolutionary Distance Predicts Recurrence After Liver Transplantation in Multifocal Hepatocellular Carcinoma.

BACKGROUND: Liver transplantation (LTx) is a potentially curative treatment option for hepatocellular carcinoma (HCC) in cirrhosis. However, patients, where HCC is already a systemic disease, LTx may be individually harmful and has a negative impact on donor organ usage. Thus, there is a need for improved selection criteria beyond nodule morphology to select patients with a favorable outcome for LTx in multifocal HCC. Evolutionary distance measured from genome-wide single-nucleotide polymorphism data between tumor nodules and the cirrhotic liver may be a prognostic marker of survival after LTx for multifocal HCC. METHODS: In a retrospective multicenter study, clinical data and formalin-fixed paraffin-embedded specimens of the liver and 2 tumor nodules were obtained from explants of 30 patients in the discovery and 180 patients in the replication cohort. DNA was extracted from formalin-fixed paraffin-embedded specimens followed by genome wide single-nucleotide polymorphism genotyping. RESULTS: Genotype quality criteria allowed for analysis of 8 patients in the discovery and 17 patients in the replication set. DNA concentrations of a total of 25 patients fulfilled the quality criteria and were included in the analysis. Both, in the discovery (P = 0.04) and in the replication data sets (P = 0.01), evolutionary distance was associated with the risk of recurrence of HCC after transplantation (combined P = 0.0002). In a univariate analysis, evolutionary distance (P = 7.4 × 10) and microvascular invasion (P = 1.31 × 10) were significantly associated with survival in a Cox regression analysis. CONCLUSIONS: Evolutionary distance allows for the determination of a high-risk group of recurrence if preoperative liver biopsy is considered.

Long-term quality of life after endovac-therapy in anastomotic leakages after esophagectomy.

BACKGROUND: Endoluminal vacuum therapy (EVT) has been successfully established with promising survival rates in the treatment of anastomotic leakages after esophagectomy. It is still unclear how this therapy affects health related quality of life (HRQOL). METHODS: HRQOL was prospectively assessed using the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-C30 (EORTC QLQ-C30) questionnaire. Assessment was carried out prior to surgery, after discharge, 6 months and 12 months after surgery. We compared HRQOL after EVT (n=23) to patients without anastomotic leakages as a control group (n=50). Investigated parameters included age, sex, and localization of anastomosis, number of EVT sessions, length of ICU and hospital stay, therapy failure, anastomotic stricture, tumour stage, neoadjuvant and adjuvant treatment, sepsis. RESULTS: After esophagectomy HRQOL increased within 12 months. Compared to patients without leakages the EVT-group showed significantly better HRQOL-scores for pain, social and emotional functioning after discharge and 6 months after surgery. In the long-term follow up HRQOL was comparable between the groups. After EVT age, advanced tumour stage, tumour recurrence, anastomotic strictures, length of ICU and hospital stay and length of EVT had a significant influence on HRQOL. CONCLUSIONS: EVT is a promising therapeutic option in leakages after esophagectomy. In the long-term, HRQOL of EVT-treated patients is comparable to patients, who did not suffer from postsurgical leakages.

Cognitive Evaluation in Liver Transplant Patients Under Calcineurin Inhibitor Maintenance Therapy.

BACKGROUND: Neurological disorders due to calcineurin inhibitor (CNI) treatment pose a well-known problem after liver transplantation (LTx). In this study, the impact of CNIs on cognitive functioning during maintenance therapy was analyzed. A possible improvement of cognitive functioning, compliance and health-related quality of life (HRQoL) after conversion to a once-daily tacrolimus formulation was prospectively assessed. METHODS: In a cross-section analysis cognitive functioning of living donors (LD), waiting list patients and LTx patients was tested using a 4 times trail making test (4-TTMT). In a further investigator-initiated trial a possible improvement of cognitive functioning, HRQoL and compliance after conversion to the once-daily tacrolimus formulation was prospectively assessed over 1 year. HRQoL was assessed using an EORTC-QLQ C30 questionnaire and patient's compliance was assessed by the Basel Assessment of Compliance with Immunosuppressive Medication Scales questionnaire. Correlated data were sex, age, time after surgery, liver disease, model of end-stage liver disease score, creatinine, CNI type, and CNI trough levels. RESULTS: Two hundred eleven patients were included in this cross-section analysis. Twenty-seven patients agreed to participate in the investigator-initiated trial. LTx patients completed the 4-TTMT slower than living donor patients and faster than waiting list patients. Patients with twice daily cyclosporine A (CSA) formulation needed longer to finish the 4-TTMT than patients with the once-daily tacrolimus formulation. After drug conversion of a twice-daily CNI formulation to a once-daily tacrolimus formulation, CSA-treated patients needed longer to improve their cognitive functioning. HRQoL and compliance did not improve after drug conversion. CONCLUSIONS: Patients with once-daily tacrolimus formulation had a better psychomotor speed than CSA-treated patients. The conversion to once-daily tacrolimus formulation significantly improved cognitive functioning, but had no impact on HRQoL or compliance.

The Glasgow Prognostic Score and its variants predict mortality in living donor but not in deceased donor liver transplantation for hepatocellular carcinoma: A double-center validation study.

AIM: This study aimed to evaluate whether the Glasgow Prognostic Score (GPS) and its variants are able to predict mortality in live donor and deceased donor liver transplantation for hepatocellular carcinoma. METHODS: Data of 29 live donor and 319 deceased donor transplantations from two German transplant centers was analyzed. The GPS, modified GPS, hepatic GPS, and Abe score were investigated. Receiver operating characteristic (ROC) curve analysis was carried out to calculate the sensitivity, specificity, and overall model correctness of the investigated scores as a predictive model. Study end-points were 1-year, 3-year, and long-term mortality. RESULTS: A 1-year mortality of 19.1% (n = 61), 3-year mortality of 26.3% (n = 84), and overall mortality of 37.3% (n = 119) was observed. All investigated scores failed to predict outcome in deceased donor liver transplantation (areas under ROC curves <0.700), whereas GPS, hepatic GPS, modified GPS, and the Abe score reached areas under ROC curves >0.700 for the prediction of 1-year mortality in live donor transplantation. The GPS and Abe score were also able to predict 3-year mortality. None of the investigated scores was a reliable predictor of long-term mortality. CONCLUSION: Systemic inflammation-based scores have great prognostic potential in live donor transplantation. Abe score could be successfully externally validated in the current study for the first time. In deceased donor transplantation, none of the analyzed scores was able to allow reliable prediction for the investigated study end-points.

Limits of and Complications after Embolization of the Hepatic Artery and Portal Vein to Induce Segmental Hypertrophy of the Liver: A Large Mini-Pig Study.

BACKGROUND: The aim of this study was to compare arterial embolization (AE) with portal vein embolization (PVE) for the induction of segmental hypertrophy regarding procedural efficacy, safety and outcome. METHODS: A total of 29 mini pigs were subjected to PVE, AE or assigned to the sham (SO) group. Correspondingly, 75% of the hepatic artery or portal vein branches were embolized. Growth and atrophy of the liver lobes, calculating the liver-to-body weight index (LBWI), laboratory data, arteriography, portography, Doppler ultrasound (US) and histopathology were analyzed. RESULTS: After PVE, 2 animals had to be excluded due to technical problems. After AE, 4 animals had to be excluded because of technical problems and early sacrifice. Postprocedural US demonstrated effective AE and PVE of the respective lobes. Four weeks after PVE, portography showed a slow refilling of the embolized lobe by collateral portal venous vessels. Four weeks after AE, arteriography revealed a slight revascularization of the embolized lobes by arterial neovascularization. Segmental AE led to extensive necrotic and inflammatory alterations in the liver and bile duct parenchyma. Significant hypertrophy of the non-embolized lobe was only noted in the PVE group (LBWI: 0.91 ± 0.28%; p = 0.001). There was no increase in the non-embolized lobe in the AE (LBWI: 0.45 ± 0.087%) and SO group (LBWI: 0.45 ± 0.13%). CONCLUSION: PVE is safe and effective to induce segmental hypertrophy. Portal reperfusion by collateral vessels may limit hypertrophy. AE did not increase the segmental hepatic volume but carries the risk of extensive necrotic inflammatory damage.

Influence of mTOR-inhibitors and mycophenolic acid on human cholangiocellular carcinoma and cancer associated fibroblasts.

BACKGROUND: The incidence of Cholangiocellular Carcinoma (CCA) is increasing in the western world. The tumour has a high proportion of desmoplastic stroma and is correlated with a worse prognosis when cancer associated myofibroblasts (CAFs) are present. Recent studies showed promising results after liver transplantation (LTx) in non-resectable early stage CCA. Mycophenolic acid (MPA) and the mTor inhibitor Everolimus are used to prevent organ rejection but recently were shown to exhibit an antiproliferative effect on CCA-cells. Little is known about the influence of immunosuppressive drugs on tumour cell proliferation and migration after paracrine stimulation by CAFs. Moreover, it is still unknown, which signaling pathways are activated following these specific cell-cell interactions. METHODS: CCA cell lines HuCCT1 and TFK1 were utilized for the study. CAFs were derived from resected CCA cancer tissue. Cell viability was measured by the crystal violet assay and tumour cell invasion was quantified using a modified co-culture transmigration assay. Semiquantitative cytokine-expression was measured using a cytokine-array. Protein expression and phosphorylation of ERK, STAT3 and AKT was determined by Western-blot analysis. RESULTS: CCA cells treated with MPA exhibited a dose related decrease in cell viability in contrast to Cyclosporine A (CSA) treatment which had no effect on cell viability. Everolimus significantly inhibited proliferation at very low concentrations. The pro-invasive effect of CAFs in co-culture transmigration assay was significantly reduced by Everolimus at a concentration of 1nM (p = 0.047). In contrast, MPA and CSA showed no effect on tumour cell invasion. Treatment of CAFs with 1nM Everolimus showed a significant reduction in the expression of IL 8, IL 13, MCP1, MIF and Serpin E1. CCA-cells showed significant increases in phosphorylation of ERK, STAT3 and AKT under the influence of conditioned CAF-media. This effect was suppressed by Everolimus. CONCLUSIONS: The secretion of proinflammatory cytokines by CAFs may lead to increased activation of JAK/STAT3-, ERK- and AKT-signaling and increased migration of CCA-cells. Everolimus abrogates this effect and inhibits proliferation of CCA-cells even at low concentrations. LTx for non-resectable early stage CCA is currently performed in several clinical studies. Consistent with a role for common immunosuppressants in inhibiting tumour cell-proliferation and -invasion, our study indicates that a combination of standard therapies with Everolimus and MPA is a promising therapy option to treat CCA following LTx.

Mode of allocation and social demographic factors correlate with impaired quality of life after liver transplantation.

BACKGROUND: Health-related Quality of life (HRQoL) is a major goal of clinical management after liver transplantation (LTx). There is still disagreement on the effects of social-demographic factors and changes in the allocation system on HRQoL. The aim of this study was to evaluate the impact of social-demographic factors, mode of organ-allocation, waiting time and hepatocellular carcinoma (HCC) on HRQoL after LTx. METHODS: HRQoL was assessed using the EORTC-QLQ-C30 questionnaire, which was sent to 238 recipients. Investigated parameters included age, sex, distance to transplant center, follow-up at hospital, size of hometown, highest education, marital status, having children, background liver disease, waiting time, mode of allocation, HCC, hospitalization after LTx and diagnosis of malignancy after LTx. All evaluated parameters were entered into multivariate linear regression analysis. RESULTS: Completed questionnaire were returned by 73% of the recipients. After LTx, the HRQoL-function scales increased over time. Age, marital status, highest education, completed professional training, working status, job position, duration of waiting time to LTx, distance to transplant center, place offollow, HU-statuts, mode of organ allocation and duration of hospitalization were associated with significantly worse function- and significantly lower symptom scales. HCC as a primary disease did not affect HRQoL. CONCLUSIONS: Low HRQoL correlated significantly with MELD-based organ allocation, more than 28-day hospitalization, divorced status, lower education- and non-working status, higher distance to transplant center, follow up at transplant center, HU-status, shorter waiting time to LTx and younger age. Improvement of HRQoL after LTx may require clinical management of pain, psychotherapy and financial support.

Obesity accelerates epigenetic aging of human liver.

Because of the dearth of biomarkers of aging, it has been difficult to test the hypothesis that obesity increases tissue age. Here we use a novel epigenetic biomarker of aging (referred to as an "epigenetic clock") to study the relationship between high body mass index (BMI) and the DNA methylation ages of human blood, liver, muscle, and adipose tissue. A significant correlation between BMI and epigenetic age acceleration could only be observed for liver (r = 0.42, P = 6.8 × 10(-4) in dataset 1 and r = 0.42, P = 1.2 × 10(-4) in dataset 2). On average, epigenetic age increased by 3.3 y for each 10 BMI units. The detected age acceleration in liver is not associated with the Nonalcoholic Fatty Liver Disease Activity Score or any of its component traits after adjustment for BMI. The 279 genes that are underexpressed in older liver samples are highly enriched (1.2 × 10(-9)) with nuclear mitochondrial genes that play a role in oxidative phosphorylation and electron transport. The epigenetic age acceleration, which is not reversible in the short term after rapid weight loss induced by bariatric surgery, may play a role in liver-related comorbidities of obesity, such as insulin resistance and liver cancer.

Postdiagnosis body mass index and risk of mortality in colorectal cancer survivors: a prospective study and meta-analysis.

PURPOSE: Aim of this study was to investigate the association between postdiagnosis body mass index (BMI) and all-cause mortality in colorectal cancer (CRC) survivors in a prospective study and meta-analysis. METHODS: We conducted a prospective cohort study on 2,143 CRC survivors in Germany. Participants were recruited to the study on average 4 years after diagnosis, and postdiagnosis BMI was assessed at recruitment using a self-administered questionnaire. CRC survivors were followed up for a mean time of 3.5 years. The association between BMI and all-cause mortality was investigated using multivariable Cox proportional hazards models. Additionally, we performed a meta-analysis of studies on postdiagnosis BMI and all-cause mortality (n = 5, including this study) by applying random-effects models. RESULTS: In the prospective analysis, 349 participants died. BMI was not statistically significantly associated with all-cause mortality. Compared to normal weight survivors, the hazard ratios (HRs) [95% confidence interval (CI)] for all-cause mortality in underweight, overweight and obese survivors were 1.65 (0.79-3.45), 0.80 (0.62-1.03) and 0.84 (0.62-1.14), respectively. In the meta-analysis, individuals with underweight were at increased risk for all-cause mortality [HR (95% CI) 1.72 (1.18-2.49)], whereas individuals with overweight had a lower risk [HR (95% CI) 0.79 (0.71-0.88)], compared to normal weight subjects. For obesity, the risk of mortality was also reduced with only borderline significance [HR (95% CI) 0.88 (0.77-1.00)]. CONCLUSIONS: While the present study as well as single previously published studies showed that overweight was associated with a non-significant reduced risk for all-cause mortality, our meta-analysis indicated a decreased mortality risk among overweight CRC survivors.

Endoscopic endoluminal vacuum therapy in esophageal perforation.

BACKGROUND: Esophageal perforation is a serious disease with a high morbidity and mortality rate. Endoscopic vacuum therapy (EVT) is a new endoscopic treatment option, which is used to treat anastomotic leakages after rectal and esophageal resections. We report on 10 patients treated with EVT for esophageal perforation. METHODS: Clinical and therapy-related data such as age, sex, duration of intensive care stay, length of hospital stay, reasons for perforation, EVT-associated complications, mortality, need for alternative treatment options, and course of infectious variables were analyzed. RESULTS: Ten patients were treated with 54 vacuum sponges that were placed in upper gastrointestinal defects. Causes for perforation were iatrogenic, spontaneous, or foreign body-associated. Mean number of sponge insertions was 5.4 (range, 2 to 12) with a mean period of 19 ± 14.26 days. Successful therapy was achieved in 9 of 10 patients. After successful primary treatment, 1 patient died during therapy as a result of general failure of the cardiovascular system. In 1 patient, surgical resection was necessary after repeated Mallory-Weiss lesions and minor perforations during the course of immunosuppressive therapy. In a third patient an endoscopic stent was inserted in the clean wound cavity after primary EVT. CONCLUSIONS: In this small trial EVT has been shown to be a safe and feasible therapy option for perforations of the upper gastrointestinal tract. If necessary, EVT can be combined with operative revision for better control of the local septic focus or used as a bridging procedure for wound conditioning before aggressive surgical treatment.

Functional TLR5 genetic variants affect human colorectal cancer survival.

Toll-like receptors (TLR) are overexpressed on many types of cancer cells, including colorectal cancer cells, but little is known about the functional relevance of these immune regulatory molecules in malignant settings. Here, we report frequent single-nucleotide polymorphisms (SNP) in the flagellin receptor TLR5 and the TLR downstream effector molecules MyD88 and TIRAP that are associated with altered survival in a large cohort of Caucasian patients with colorectal cancer (n = 613). MYD88 rs4988453, a SNP that maps to a promoter region shared with the acetyl coenzyme-A acyl-transferase-1 (ACAA1), was associated with decreased survival of patients with colorectal cancer and altered transcriptional activity of the proximal genes. In the TLR5 gene, rs5744174/F616L was associated with increased survival, whereas rs2072493/N592S was associated with decreased survival. Both rs2072493/N592S and rs5744174/F616L modulated TLR5 signaling in response to flagellin or to different commensal and pathogenic intestinal bacteria. Notably, we observed a reduction in flagellin-induced p38 phosphorylation, CD62L shedding, and elevated expression of interleukin (IL)-6 and IL-1ß mRNA in human primary immune cells from TLR5 616LL homozygote carriers, as compared with 616FF carriers. This finding suggested that the well-documented effect of cytokines like IL-6 on colorectal cancer progression might be mediated by TLR5 genotype-dependent flagellin sensing. Our results establish an important link between TLR signaling and human colorectal cancer with relevance for biomarker and therapy development.