Changes in the Liver Transplant Waiting List after Expanding to the 'Up-to-Seven' Criteria for Hepatocellular Carcinoma.

We aimed to assess changes in the composition of the waiting list for liver transplantation (LT) after expanding from Milan to "up-to-seven" criteria in patients with hepatocellular carcinoma (HCC). A consecutive cohort of 255 LT candidates was stratified in a pre-expansion era (2016-2018; n = 149) and a post-expansion era (2019-2021; n = 106). The most frequent indication for LT was HCC in both groups (47.7% vs. 43.4%; p = 0.5). The proportion of patients exceeding the Milan criteria in the explanted liver was nearly doubled after expansion (12.5% vs. 21.1%; p = 0.25). Expanding criteria had no effect in drop-out (12.3% vs. 20.4%; p = 0.23) or microvascular invasion rates (37.8% vs. 38.7%; p = 0.93). The length on the waiting list did not increase after the expansion (172 days [IQR 74-282] vs. 118 days [IQR 67-251]; p = 0.135) and was even shortened in the post-expansion HCC subcohort (181 days [IQR 125-232] vs. 116 days [IQR 74-224]; p = 0.04). Tumor recurrence rates were reduced in the post-expansion cohort (15.4% vs. 0%; p = 0.012). In conclusion, expanding from Milan to up-to-seven criteria for LT in patients with HCC had no meaningful impact on the waiting list length and composition, thus offering the opportunity for the adoption of more liberal policies in the future.

Helicobacter Species and Hepato-Biliary Tract Malignancies: A Systematic Review and Meta-Analysis.

Helicobacter species may cause chronic inflammation of the biliary tract, but its relationship with cancer is controversial. We performed a systematic review and meta-analysis to evaluate the association between Helicobacter species and hepatobiliary tract malignancies. Twenty-six studies (4083 patients) were included in qualitative synthesis, and 18 studies (n = 1895 qualified for meta-analysis. All studies were at high-intermediate risk of bias. Most studies combined several direct microbiological methods, mostly PCR (23 studies), culture (8 studies), and/or CLOtest (5 studies). Different specimens alone or in combination were investigated, most frequently bile (16 studies), serum (7 studies), liver/biliary tissue (8 studies), and gastric tissue (3 studies). Patients with Helicobacter species infection had an increased risk of hepatobiliary tract malignancies (OR = 3.61 [95% CI 2.18-6.00]; p < 0.0001), with high heterogeneity in the analysis (I2 = 61%; p = 0.0003). This effect was consistent when Helicobacter was assessed in bile (OR = 3.57 [95% CI 1.73-7.39]; p = 0.0006), gastric tissue (OR = 42.63 [95% CI 5.25-346.24]; p = 0.0004), liver/biliary tissue (OR = 4.92 [95% CI 1.90-12.76]; p = 0.001) and serum (OR = 1.38 [95% CI 1.00-1.90]; p = 0.05). Heterogeneity was reduced in these sub-analyses (I2 = 0-27%; p = ns), except for liver/biliary tissue (I2 = 57%; p = 0.02). In conclusion, based on low-certainty data, Helicobacter species chronic infection is associated with a tripled risk of hepatobiliary tract malignancy. Prospective studies are required to delineate public health interventions.

Enumeration and Characterization of Circulating Tumor Cells in Patients with Hepatocellular Carcinoma Undergoing Transarterial Chemoembolization.

Circulating tumor cells (CTCs), and particularly circulating cancer stem cells (cCSC), are prognostic biomarkers for different malignancies and may be detected using liquid biopsies. The ex vivo culture of cCSCs would provide valuable information regarding biological aggressiveness and would allow monitoring the adaptive changes acquired by the tumor in real time. In this prospective pilot study, we analyzed the presence of EpCAM+ CTCs using the IsoFlux system in the peripheral blood of 37 patients with hepatocellular carcinoma undergoing transarterial chemoembolization (TACE). The average patient age was 63.5 ± 7.9 years and 91.9% of the patients were men. All patients had detectable CTCs at baseline and 20 patients (54.1%) showed CTC aggregates or clusters in their peripheral blood. The increased total tumor diameter (OR: 2.5 (95% CI: 1.3-4.8), p = 0.006) and the absence of clusters of CTCs at baseline (OR: 0.2 (95% CI: 0.0-1.0), p = 0.049) were independent predictors of a diminished response to TACE. Culture of cCSC was successful in five out of thirty-three patients, mostly using negative enrichment of CD45- cells, ultra-low adherence, high glucose, and a short period of hypoxia followed by normoxia. In conclusion, the identification of clusters of CTCs before TACE and the implementation of standardized approaches for cCSC culture could aid to predict outcomes and to define the optimal adjuvant therapeutic strategy for a true personalized medicine in hepatocellular carcinoma.

PRPF8 increases the aggressiveness of hepatocellular carcinoma by regulating FAK/AKT pathway via fibronectin 1 splicing.

Hepatocellular carcinoma (HCC) pathogenesis is associated with alterations in splicing machinery components (spliceosome and splicing factors) and aberrant expression of oncogenic splice variants. We aimed to analyze the expression and potential role of the spliceosome component PRPF8 (pre-mRNA processing factor 8) in HCC. PRPF8 expression (mRNA/protein) was analyzed in a retrospective cohort of HCC patients (n = 172 HCC and nontumor tissues) and validated in two in silico cohorts (TCGA and CPTAC). PRPF8 expression was silenced in liver cancer cell lines and in xenograft tumors to understand the functional and mechanistic consequences. In silico RNAseq and CLIPseq data were also analyzed. Our results indicate that PRPF8 is overexpressed in HCC and associated with increased tumor aggressiveness (patient survival, etc.), expression of HCC-related splice variants, and modulation of critical genes implicated in cancer-related pathways. PRPF8 silencing ameliorated aggressiveness in vitro and decreased tumor growth in vivo. Analysis of in silico CLIPseq data in HepG2 cells demonstrated that PRPF8 binds preferentially to exons of protein-coding genes, and RNAseq analysis showed that PRPF8 silencing alters splicing events in multiple genes. Integrated and in vitro analyses revealed that PRPF8 silencing modulates fibronectin (FN1) splicing, promoting the exclusion of exon 40.2, which is paramount for binding to integrins. Consistent with this finding, PRPF8 silencing reduced FAK/AKT phosphorylation and blunted stress fiber formation. Indeed, HepG2 and Hep3B cells exhibited a lower invasive capacity in membranes treated with conditioned medium from PRPF8-silenced cells compared to medium from scramble-treated cells. This study demonstrates that PRPF8 is overexpressed and associated with aggressiveness in HCC and plays important roles in hepatocarcinogenesis by altering FN1 splicing, FAK/AKT activation and stress fiber formation.

Spliceosomal profiling identifies EIF4A3 as a novel oncogene in hepatocellular carcinoma acting through the modulation of FGFR4 splicing.

INTRODUCTION: Altered splicing landscape is an emerging cancer hallmark; however, the dysregulation and implication of the cellular machinery controlling this process (spliceosome components and splicing factors) in hepatocellular carcinoma (HCC) is poorly known. This study aimed to comprehensively characterize the spliceosomal profile and explore its role in HCC. METHODS: Expression levels of 70 selected spliceosome components and splicing factors and clinical implications were evaluated in two retrospective and six in silico HCC cohorts. Functional, molecular and mechanistic studies were implemented in three cell lines (HepG2, Hep3B and SNU-387) and preclinical Hep3B-induced xenograft tumours. RESULTS: Spliceosomal dysregulations were consistently found in retrospective and in silico cohorts. EIF4A3, RBM3, ESRP2 and SRPK1 were the most dysregulated spliceosome elements in HCC. EIF4A3 expression was associated with decreased survival and greater recurrence. Plasma EIF4A3 levels were significantly elevated in HCC patients. In vitro EIF4A3-silencing (or pharmacological inhibition) resulted in reduced aggressiveness, and hindered xenograft-tumours growth in vivo, whereas EIF4A3 overexpression increased tumour aggressiveness. EIF4A3-silencing altered the expression and splicing of key HCC-related genes, specially FGFR4. EIF4A3-silencing blocked the cellular response to the natural ligand of FGFR4, FGF19. Functional consequences of EIF4A3-silencing were mediated by FGFR4 splicing as the restoration of non-spliced FGFR4 full-length version blunted these effects, and FGFR4 inhibition did not exert further effects in EIF4A3-silenced cells. CONCLUSIONS: Splicing machinery is strongly dysregulated in HCC, providing a source of new diagnostic, prognostic and therapeutic options in HCC. EIF4A3 is consistently elevated in HCC patients and associated with tumour aggressiveness and mortality, through the modulation of FGFR4 splicing.

Endoscopic findings unrelated to portal hypertension in patients with liver cirrhosis undergoing a varicose vein screening programme. / Hallazgos endoscópicos no relacionados con hipertensión portal en pacientes con cirrosis hepática sometidos a un programa de cribado de varices.

AIM: To determine the prevalence of endoscopic lesions unrelated with portal hypertension in patients with cirrhosis. PATIENTS AND METHODS: Cross-sectional study including a consecutive cohort of patients with liver cirrhosis enrolled in a screening program of oesophageal varices who underwent an upper gastrointestinal endoscopy from November, 2013, to November, 2018. Clinical predictors of endoscopic lesions unrelated to portal hypertension were analyzed by univariate and multivariate logistic regression. RESULTS: A total of 379 patients were included. The most frequent aetiology of liver disease was alcohol consumption (60.4%). The prevalence of endoscopic lesions unrelated with portal hypertension was 39.6% (n=150). Among 96 patients with peptic lesions, urease was obtained in 56.2% of patients (positive in 44.4% of them). The prevalence of endoscopic lesions unrelated to portal hypertension was not associated with age, gender, liver function or ultrasound findings of portal hypertension. The prevalence of endoscopic lesions unrelated to portal hypertension was not associated with age, gender, liver function or ultrasound findings of portal hypertension. Smokers had a trend to increased prevalence of endoscopic lesions unrelated to portal hypertension (43.2% vs. 34.6%; p=0.09), particularly peptic ulcer (6.4% vs. 0.6%; p=0.05) and peptic duodenitis (17.3% vs. 6.3%; p=0.002). Active smoking was the only independent predictor of peptic ulcer or duodenitis (OR=2.56; p=0.017). CONCLUSION: Active smoking is a risk factor for endoscopic lesions unrelated to portal hypertension. This finding should be further investigated to reassess endoscopic screening programs in cirrhotic smokers.

Circulating Tumor Cells in Hepatocellular Carcinoma: A Comprehensive Review and Critical Appraisal.

Hepatocellular carcinoma (HCC) is the fifth most common neoplasm and a major cause of cancer-related death worldwide. There is no ideal biomarker allowing early diagnosis of HCC and tumor surveillance in patients receiving therapy. Liquid biopsy, and particularly circulating tumor cells (CTCs), have emerged as a useful tool for diagnosis and monitoring therapeutic responses in different tumors. In the present manuscript, we evaluate the current evidence supporting the quantitative and qualitative assessment of CTCs as potential biomarkers of HCC, as well as technical aspects related to isolation, identification, and classification of CTCs. Although the dynamic assessment of CTCs in patients with HCC may aid the decision-making process, there are still many uncertainties and technical caveats to be solved before this methodology has a true impact on clinical practice guidelines. More studies are needed to identify the optimal combination of surface markers, to increase the efficiency of ex-vivo expansion of CTCs, or even to target CTCs as a potential therapeutic strategy to prevent HCC recurrence after surgery or to hamper tumor progression and extrahepatic spreading.

Clearance of Circulating Tumor Cells in Patients with Hepatocellular Carcinoma Undergoing Surgical Resection or Liver Transplantation.

BACKGROUND: In patients with hepatocellular carcinoma (HCC), a complete clearance of circulating tumor cells (CTCs) early after liver transplantation (LT) or surgical resection (LR) could prevent tumor recurrence. METHODS: prospective pilot study including patients with HCC who underwent LR or LT from September 2017 to May 2020. Enumeration of CTCs was performed in peripheral blood samples (7 mL) using the Isoflux® system (Fluxion Biosciences) immediately before surgery, at post-operative day 5 and at day 30. A clinically relevant number of CTCs was defined as >30 CTCs/sample. RESULTS: 41 HCC patients were included (mean age 58.7 ± 6.3; 82.9% male). LR was performed in 10 patients (24.4%) and 31 patients (75.6%) underwent LT. The main etiology of liver disease was chronic hepatitis C (31.7%). Patients undergoing LR and LT were similar in terms of preoperative CTC count (p = 0.99), but clearance of CTCs within the first month was more pronounced in the LT group. Clusters of CTCs at baseline were associated with incomplete clearance of CTCs at day 30 (54.2% vs. 11.8%, p = 0.005), which in turn impacted negatively on survival (p = 0.038). CONCLUSION: Incomplete clearance of CTCs after surgery could be a surrogate marker of HCC aggressiveness.

Diagnosis and treatment of hepatocellular carcinoma. Update of the consensus document of the AEEH, AEC, SEOM, SERAM, SERVEI, and SETH. / Diagnóstico y tratamiento del carcinoma hepatocelular. Actualización del documento de consenso de la AEEH, AEC, SEOM, SERAM, SERVEI y SETH.

Hepatocellular carcinoma (HCC) is the most common primary liver neoplasm and one of the most common causes of death in patients with cirrhosis of the liver. In parallel, with recognition of the clinical relevance of this cancer, major new developments have recently appeared in its diagnosis, prognostic assessment and in particular, in its treatment. Therefore, the Spanish Association for the Study of the Liver (AEEH) has driven the need to update the clinical practice guidelines, once again inviting all the societies involved in the diagnosis and treatment of this disease to participate in the drafting and approval of the document: Spanish Society for Liver Transplantation (SETH), Spanish Society of Diagnostic Radiology (SERAM), Spanish Society of Vascular and Interventional Radiology (SERVEI), Spanish Association of Surgeons (AEC) and Spanish Society of Medical Oncology (SEOM). The clinical practice guidelines published in 2016 and accepted as National Health System Clinical Practice Guidelines were taken as the reference documents, incorporating the most important recent advances. The scientific evidence and the strength of the recommendation is based on the GRADE system.

Splicing factor SF3B1 is overexpressed and implicated in the aggressiveness and survival of hepatocellular carcinoma.

Splicing alterations represent an actionable cancer hallmark. Splicing factor 3B subunit 1 (SF3B1) is a crucial splicing factor that can be targeted pharmacologically (e.g. pladienolide-B). Here, we show that SF3B1 is overexpressed (RNA/protein) in hepatocellular carcinoma (HCC) in two retrospective (n = 154 and n = 172 samples) and in five in silico cohorts (n > 900 samples, including TCGA) and that its expression is associated with tumor aggressiveness, oncogenic splicing variants expression (KLF6-SV1, BCL-XL) and decreased overall survival. In vitro, SF3B1 silencing reduced cell viability, proliferation and migration and its pharmacological blockade with pladienolide-B inhibited proliferation, migration, and formation of tumorspheres and colonies in liver cancer cell lines (HepG2, Hep3B, SNU-387), whereas its effects on normal-like hepatocyte-derived THLE-2 proliferation were negligible. Pladienolide-B also reduced the in vivo growth and the expression of tumor-markers in Hep3B-induced xenograft tumors. Moreover, SF3B1 silencing and/or blockade markedly modulated the activation of key signaling pathways (PDK1, GSK3b, ERK, JNK, AMPK) and the expression of cancer-associated genes (CDK4, CD24) and oncogenic SVs (KLF6-SV1). Therefore, the genetic and/or pharmacological inhibition of SF3B1 may represent a promising novel therapeutic strategy worth to be explored through randomized controlled trials.

Tacrolimus as an Effective and Durable Second-Line Treatment for Chronic Autoimmune Hepatitis: A Multicentric Study.

BACKGROUND: Autoimmune hepatitis (AIH) is a chronic liver disease able to progress to acute liver failure, cirrhosis, and liver cancer. A significant proportion of patients fail to first-line therapy or develop severe toxicity. AIMS: To assess safety and effectiveness of tacrolimus as a second-line therapy in AIH patients. METHODS: Multicentric retrospective study of AIH patients treated with tacrolimus for at least 3 months as a second-line therapy. Effectiveness was defined as complete normalization of transaminases and IgG. RESULTS: A total of 23 AIH patients were included in the final analysis. In 13% of patients tacrolimus was initiated because of toxicity to previous first-line treatments and the rest were switched because of previous non-efficacy. Tacrolimus was effective in 18 patients (78%; 95%CI: 55.20-91.92%). The median time receiving tacrolimus was 16 months (IQR 20). There was a sustained response with a significant improvement in all liver enzymes and IgG on last follow-up. Only one patient discontinued tacrolimus at the third month because of severe neuropathy, and ototoxicity. Responders were significantly older at diagnosis of AIH (41 ± 13 vs. 27 ± 10 years old; p = 0.0496). CONCLUSION: Tacrolimus is effective and well tolerated as a second-line therapy in patients with AIH.

Activation of mTOR Signaling Pathway in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer and occurs mainly in patients with liver cirrhosis. The mammalian target of rapamycin (mTOR) signaling pathway is involved in many hallmarks of cancer including cell growth, metabolism re-programming, proliferation and inhibition of apoptosis. The mTOR pathway is upregulated in HCC tissue samples as compared with the surrounding liver cirrhotic tissue. In addition, the activation of mTOR is more intense in the tumor edge, thus reinforcing its role in HCC proliferation and spreading. The inhibition of the mTOR pathway by currently available pharmacological compounds (i.e., sirolimus or everolimus) is able to hamper tumor progression both in vitro and in animal models. The use of mTOR inhibitors alone or in combination with other therapies is a very attractive approach, which has been extensively investigated in humans. However, results are contradictory and there is no solid evidence suggesting a true benefit in clinical practice. As a result, neither sirolimus nor everolimus are currently approved to treat HCC or to prevent tumor recurrence after curative surgery. In the present comprehensive review, we analyzed the most recent scientific evidence while providing some insights to understand the gap between experimental and clinical studies.

Bevacizumab Allows Preservation of Liver Function and its Regenerative Capacity after Major Hepatectomy.

BACKGROUND: Parallel to the safety of liver resections, new chemotherapy drugs have emerged for the control of liver metastases. However, there is unclear evidence about the combination of intensive BVZ-therapy and extended resections. The main aim was to analyse the impact of Bevacizumab (BVZ) in terms of liver safety and tolerability in two experimental models: a basal-toxicity situation and after major hepatectomy. METHODS: Eighty male-Wistar rats were grouped as toxicity analysis (sham-operated rats-OS-) and regeneration after- surgery analysis (hepatectomy rats-H-). Eight further subgroups were created according to sacrifice (6- hours-6h- or 24-hours-24h-) and dose (µg) of BVZ (none, 100, 200, 400). Several measurements were performed, including biochemical serum samples, histopathological analysis, cytokines (IL-6, TNF-α, TGF-ß), oxidative-stress (GSH/GSSG, ATP), lipid-peroxidation (TBARS) and epidermal and vascular endothelium growth-factors (EGF and VEGF). RESULTS: In the toxicity analysis, safe results with BVZ were observed, with no significant differences among the groups. A trend towards a lower oxidative status was observed in the OS 6 h-100, -200 and -400 versus the OS 6 h-none group. Similar results were observed in the hepatectomy model, with stable oxidative-stress-index and IL-6, TNF- α, and TGF- ß levels. Despite higher lipid peroxidation status, overall regeneration was preserved. As expected, VEGF was almost undetectable in BVZ-treated groups after resection, but not in the non-resection group. CONCLUSION: It was concluded that liver status was not impaired by BVZ even at the high-dose. Similarly, liver regeneration after extended hepatectomy in BVZ-treated animals was well-preserved. Extended liver resections may be encouraged in BVZ-treated patients due to its excellent tolerability and good liver regeneration status.

Dysregulation of the Splicing Machinery Is Associated to the Development of Nonalcoholic Fatty Liver Disease.

CONTEXT: Nonalcoholic fatty liver disease (NAFLD) is a common obesity-associated pathology characterized by hepatic fat accumulation, which can progress to fibrosis, cirrhosis, and hepatocellular carcinoma. Obesity is associated with profound changes in gene-expression patterns of the liver, which could contribute to the onset of comorbidities. OBJECTIVE: As these alterations might be linked to a dysregulation of the splicing process, we aimed to determine whether the dysregulation in the expression of splicing machinery components could be associated with NAFLD. PARTICIPANTS: We collected 41 liver biopsies from nonalcoholic individuals with obesity, with or without hepatic steatosis, who underwent bariatric surgery. INTERVENTIONS: The expression pattern of splicing machinery components was determined using a microfluidic quantitative PCR-based array. An in vitro approximation to determine lipid accumulation using HepG2 cells was also implemented. RESULTS: The liver of patients with obesity and steatosis exhibited a severe dysregulation of certain splicing machinery components compared with patients with obesity without steatosis. Nonsupervised clustering analysis allowed the identification of three molecular phenotypes of NAFLD with a unique fingerprint of alterations in splicing machinery components, which also presented distinctive hepatic and clinical-metabolic alterations and a differential response to bariatric surgery after 1 year. In addition, in vitro silencing of certain splicing machinery components (i.e., PTBP1, RBM45, SND1) reduced fat accumulation and modulated the expression of key de novo lipogenesis enzymes, whereas conversely, fat accumulation did not alter spliceosome components expression. CONCLUSION: There is a close relationship between splicing machinery dysregulation and NAFLD development, which should be further investigated to identify alternative therapeutic targets.

mTOR Expression in Liver Transplant Candidates with Hepatocellular Carcinoma: Impact on Histological Features and Tumour Recurrence.

(1) Background: The mammalian target of rapamycin (mTOR) pathway activation is critical for hepatocellular carcinoma (HCC) progression. We aimed to evaluate the mTOR tissue expression in liver transplant (LT) patients and to analyse its influence on post-LT outcomes. (2) Methods: Prospective study including a cohort of HCC patients who underwent LT (2012⁻2015). MTOR pathway expression was evaluated in the explanted liver by using the "PathScan Intracellular Signalling Array Kit" (Cell Signalling). Kaplan-Meier and Cox regression analyses were performed to evaluate post-LT HCC recurrence. (3) Results: Forty-nine patients were included (average age 56.4 ± 6, 14.3% females). Phospho-mTOR (Ser2448) was over-expressed in peritumoral tissue as compared with tumoral tissue (ΔSignal 22.2%; p < 0.001). The mTOR activators were also increased in peritumoral tissue (phospho-Akt (Thr308) ΔSignal 18.2%, p = 0.004; phospho-AMPKa (Thr172) ΔSignal 56.3%, p < 0.001), as they were the downstream effectors responsible for cell growth/survival (phospho-p70S6K (Thr389) ΔSignal 33.3%, p < 0.001 and phospho-S6RP (Ser235/236) ΔSignal 54.6%, p < 0.001). MTOR expression was increased in patients with multinodular HCC (tumoral p = 0.01; peritumoral p = 0.001). Increased phospho-mTOR in tumoral tissue was associated with higher HCC recurrence rates after LT (23.8% vs. 5.9% at 24 months, p = 0.04). (4) Conclusion: mTOR pathway is over-expressed in patients with multinodular HCC and is it associated with increased post-LT tumour recurrence rates.

Selecting patients with hepatocellular carcinoma for liver transplantation: incorporating tumor biology criteria.

Liver transplantation (LT) is the optimal therapeutic option for patients with liver cirrhosis and hepatocellular carcinoma (HCC). Due to universal donor shortage, only the patients with limited tumor burden (under the so-called Milan criteria) are considered as potential candidates for LT in most institutions. It is expected that in the near future, more liver grafts will be available for patients with HCC due to the implementation of new direct antivirals against hepatitis C, leaving a prone scenario to consider expanding Milan criteria. A moderate expansion of Milan criteria could be implemented without increasing the risk of tumor recurrence if patients with favorable biological behavior are carefully selected. Incorporating information regarding tumor biology in the decision-making algorithm would result in a more rational use of LT in patients with HCC. In the present review, surrogate markers of tumor biology are critically evaluated as potential tools to be combined with existing radiological criteria. In addition, the current state of liquid biopsy is discussed, as this cutting-edge technology may reshape the management of HCC in the upcoming years.

Molecular predictors of prevention of recurrence in HCC with sorafenib as adjuvant treatment and prognostic factors in the phase 3 STORM trial.

OBJECTIVE: Sorafenib is the standard systemic therapy for advanced hepatocellular carcinoma (HCC). Survival benefits of resection/local ablation for early HCC are compromised by 70% 5-year recurrence rates. The phase 3 STORM trial comparing sorafenib with placebo as adjuvant treatment did not achieve its primary endpoint of improving recurrence-free survival (RFS). The biomarker companion study BIOSTORM aims to define (A) predictors of recurrence prevention with sorafenib and (B) prognostic factors with B level of evidence. DESIGN: Tumour tissue from 188 patients randomised to receive sorafenib (83) or placebo (105) in the STORM trial was collected. Analyses included gene expression profiling, targeted exome sequencing (19 known oncodrivers), immunohistochemistry (pERK, pVEGFR2, Ki67), fluorescence in situ hybridisation (VEGFA) and immunome. A gene signature capturing improved RFS in sorafenib-treated patients was generated. All 70 RFS events were recurrences, thus time to recurrence equalled RFS. Predictive and prognostic value was assessed using Cox regression models and interaction test. RESULTS: BIOSTORM recapitulates clinicopathological characteristics of STORM. None of the biomarkers tested (related to angiogenesis and proliferation) or previously proposed gene signatures, or mutations predicted sorafenib benefit or recurrence. A newly generated 146-gene signature identifying 30% of patients captured benefit to sorafenib in terms of RFS (p of interaction=0.04). These sorafenib RFS responders were significantly enriched in CD4+ T, B and cytolytic natural killer cells, and lacked activated adaptive immune components. Hepatocytic pERK (HR=2.41; p=0.012) and microvascular invasion (HR=2.09; p=0.017) were independent prognostic factors. CONCLUSION: In BIOSTORM, only hepatocytic pERK and microvascular invasion predicted poor RFS. No mutation, gene amplification or previously proposed gene signatures predicted sorafenib benefit. A newly generated multigene signature associated with improved RFS on sorafenib warrants further validation. TRIAL REGISTRATION NUMBER: NCT00692770.

Preliminary results from the use of intraoperative real-time biliary oxygen monitoring in liver transplantation.

BACKGROUND AND AIMS: Biliary anastomosis is a frequent area of complications after liver transplantation (LT) and a potential area of "microangiopathy". The concept of a "marginal bile duct" is unexplored. The main aim was to make a preliminary evaluation of the utility of an innovative real-time oxygen microtension (pO2mt) testing device for the assessment of bile duct viability during LT and to correlate these pO2mt values with microvascular tissue quality by histopathology and outcomes. PATIENTS AND METHODS: Observational prospective cohort study with 23 patients. Oxygen microtension measurements were made placing a micropO2 probe in different areas of recipient and donor's bile duct intraoperative. RESULTS: Mean pO2mt in the graft bile duct at the level of the anastomosis 103.82 (31-157) mm Hg, being 121.52 (55-174) mm Hg 1.5 cm proximal to the hilar plate (P < 0.001). Mean pO2mt in the recipient's bile duct was 117.87 (62-185) mm Hg, while a value of 137.30 (81-198) mm Hg was observed 1.5 cm distal to the anastomosis (P < 0.001). Cystic duct resection (12 cases) was also related with higher pO2mt values at anastomosis [117.8 (93-157) vs 88.54 (31-124) mm Hg] and distal to anastomosis [135.6 (111-174) vs 106.2 (55-133) mm Hg; P < 0.001]. Patients with 1-, 3-, and 12-month biliary complications had significantly lower pO2mt in the intraoperative measurements. CONCLUSION: Our preliminary results show that distal borders of donor and recipient bile ducts may be low-vascularized areas. Tissue pO2mt is significantly higher in areas close to the hilar plate and to the duodenum in donor and recipient's sides, respectively. Bile duct injury and biliary complications are associated with worse tissue pO2mt.

Regulation of the oxidative balance with coenzyme Q10 sensitizes human glioblastoma cells to radiation and temozolomide.

OBJECTIVES: To investigate how the modulation of the oxidative balance affects cytotoxic therapies in glioblastoma, in vitro. MATERIAL AND METHODS: Human glioblastoma U251 and T98 cells and normal astrocytes C8D1A were loaded with coenzyme Q10 (CoQ). Mitochondrial superoxide ion (O2-) and H2O2 were measured by fluorescence microscopy. OXPHOS performance was assessed in U251 cells with an oxytherm Clark-type electrode. Radio- and chemotherapy cytotoxicity was assessed by immunostaining of γH2AX (24 h), annexin V and nuclei morphology, at short (72 h) and long (15 d) time. Hif-1α, SOD1, SOD2 and NQO1 were determined by immunolabeling. Catalase activity was measured by classic enzymatic assay. Glutathione levels and total antioxidant capacity were quantified using commercial kits. RESULTS: CoQ did not affect oxygen consumption but reduced the level of O2- and H2O2 while shifted to a pro-oxidant cell status mainly due to a decrease in catalase activity and SOD2 level. Hif-1α was dampened, echoed by a decrease lactate and several key metabolites involved in glutathione synthesis. CoQ-treated cells were twofold more sensitive than control to radiation-induced DNA damage and apoptosis in short and long-term clonogenic assays, potentiating TMZ-induced cytotoxicity, without affecting non-transformed astrocytes. CONCLUSIONS: CoQ acts as sensitizer for cytotoxic therapies, disarming GBM cells, but not normal astrocytes, against further pro-oxidant injuries, being potentially useful in clinical practice for this fatal pathology.