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.

Comparison of point-shear wave elastography (ElastPQ) and transient elastography (FibroScan) for liver fibrosis staging in patients with non-alcoholic fatty liver disease.

BACKGROUND AND AIMS: ElastPQ is a point shear wave elastography technique used to non-invasively assess liver fibrosis. We compared liver stiffness measurements (LSM) by ElastPQ and fibroscan transient elastography (F-TE) in a cohort of patients with non-alcoholic fatty liver disease (NAFLD). We further evaluated the performance of ElastPQ in a subgroup of patients with available liver histology. MATERIALS AND METHODS: We included patients with NAFLD who presented in a dedicated multidisciplinary clinic. Anthropometric parameters, blood tests and elastography measurements were obtained using F-TE and ElastPQ as part of routine clinical care. RESULTS: We enrolled 671 patients with NAFLD, mean age 55.8 ± 13 years, body mass index (BMI) 31.5 ± 5.7 kg/m2 , 56.6% males, 41% diabetes, 53.7% hypertension, 68% dyslipidaemia. ElastPQ showed an excellent correlation with F-TE (Spearman's r = 0.80, p < .001), which was better for mild/moderate stages of fibrosis. Independent predictors of a >2 kPa discrepancy between the two techniques were a larger waist circumference and F-TE ≥10 kPa. In the subgroup of 159 patients with available histology, ElastPQ showed similar diagnostic accuracy with F-TE in staging liver fibrosis (ElastPQ area under the curves 0.84, 0.83, 0.86 and 0.95, for F ≥ 1, F ≥ 2, F ≥ 3 and F = 4 respectively). Optimal cut-off values of ElastPQ for individual fibrosis stages were lower than those of F-TE. CONCLUSIONS: ElastPQ shows an excellent correlation with F-TE in patients with NAFLD, which was better for lower LSM. The optimal cut-off values of ElastPQ are lower than those of F-TE for individual stages of fibrosis. ElastPQ has similar diagnostic accuracy to F-TE for all stages of fibrosis.

Establishing Reliability Criteria for Liver ElastPQ Shear Wave Elastography (ElastPQ-SWE): Comparison Between 10, 5 and 3 Measurements.

PURPOSE: ElastPQ is a new elastography technique for non-invasive liver fibrosis staging. However, it does not have validated reliability criteria. We tested the reliability of a different number of measurements in patients with chronic liver disease and explored whether the application of quality criteria improves the diagnostic performance. MATERIALS AND METHODS: All patients underwent liver stiffness assessment (LSM) with ElastPQ and Fibroscan (F-TE). The mean, median, standard deviation (SD) and interquartile range (IQR) of 10, 5 and 3 measurements were retrospectively collected for each patient and compared to each other. Liver histology was available in a subset of patients. RESULTS: Overall, 400 patients met the inclusion criteria. Non-alcoholic fatty liver disease (NAFLD) was the most represented etiology (75 %), followed by primary sclerosing cholangitis (PSC), primary biliary cholangitis (PBC) and autoimmune hepatitis (AIH). The correlation of medians was significantly better between 10 and 5 measurements than between 10 and 3. The difference of medians was significant only in the comparison between 10 and 3 measurements. The correlation between ElastPQ and F-TE was equally good for 10 and 5 measurements and significantly improved after an IQR/median ≤ 30 % was applied. The diagnostic performance of ElastPQ was better with the median value of 10 and 5 measurements and improved if LSM values were obtained with IQR/M ≤ 30 %. CONCLUSION: The median value of 5 valid LSMs suffices for the reliable estimation of liver stiffness using ElastPQ. The quality criterion of IQR/M ≤ 30 % should also be followed when using this technique.

Collagen proportionate area predicts clinical outcomes in patients with alcohol-related liver disease.

BACKGROUND: No prognostic tools are established for alcohol-related liver disease (ALD). Collagen proportionate area (CPA) measurement is a technique that quantifies fibrous tissue in liver biopsies using digital image analysis. AIM: To assess the predictive value of CPA on hepatic decompensation and liver-related mortality in ALD METHODS: In a multicentre cohort study, we included 386 patients with biopsy-verified ALD and with long-term follow-up. In the development cohort of 276 patients, we assessed the predictors of hepatic decompensation and liver-related death in standard and competing risk multivariable Cox regression analyses. The results were validated in an independent prospective cohort of 110 patients, where CPA was also correlated with liver stiffness measurement (LSM). RESULTS: In the development cohort, 231 (84%) patients had early/compensated ALD (non-cirrhotic or compensated cirrhosis) and 45 (16%) had decompensated cirrhosis. In the validation cohort, all patients had early/compensated ALD. Independent predictors of liver-related mortality were higher CPA values (HR = 1.04, 95% CI 1.02-1.04) and advanced fibrosis (HR = 2.80, 95% CI 1.29-6.05) with similar results in standard and competing risk multivariable Cox regression analysis. In early/compensated ALD, CPA was the only independent predictor of hepatic decompensation and liver-related death (HR = 1.08, 95% CI 1.06-1.11). In the prospective cohort, we validated that CPA independently predicts hepatic decompensation in early/compensated ALD. The predictive power of CPA and LSM was equally strong. CONCLUSIONS: CPA predicts liver-related mortality in ALD and hepatic decompensation and/or liver-related death in early/compensated ALD. Traditional histological assessment may benefit from the addition of CPA to the evaluation of ALD.

Collagen proportionate area is an independent predictor of long-term outcome in patients with non-alcoholic fatty liver disease.

BACKGROUND: Collagen proportionate area (CPA) measurement is a technique that quantifies fibrous tissue in liver biopsies by measuring the amount of collagen deposition as a proportion of the total biopsy area. CPA predicts clinical outcomes in patients with HCV and can sub-classify cirrhosis. AIM: To test the ability of CPA to quantify fibrosis and predict clinical outcomes in patients with NAFLD. METHODS: We assessed consecutive patients with biopsy-proven NAFLD from three European centres. Clinical and laboratory data were collected at baseline and at the time of the last clinical follow-up or death. CPA was performed at two different objective magnifications, whole biopsy macro and ×4 objective magnification, named standard (SM) and high (HM) magnification respectively. The correlation between CPA and liver stiffness was assessed in a sub-group of patients. RESULTS: Of 437 patients, 32 (7.3%) decompensated and/or died from liver-related causes during a median follow-up of 103 months. CPA correlated with liver stiffness and liver fibrosis stage across the whole spectrum of fibrosis. HM CPA was significantly higher than SM CPA in stages F0-F3 but similar in cirrhosis, reflecting a higher ability to capture pericellular/perisinusoidal fibrosis at early stages. Age at baseline (HR: 1.04, 95% CI: 1.01-1.08), HM CPA (HR: 1.04 per 1% increase, 95% CI: 1.01-1.08) and presence of advanced fibrosis (HR: 15.4, 95% CI: 5.02-47.84) were independent predictors of liver-related clinical outcomes at standard and competing risk multivariate Cox-regression analysis. CONCLUSIONS: CPA accurately measures fibrosis and is an independent predictor of clinical outcomes in NAFLD; hence it merits further evaluation as a surrogate endpoint in clinical trials.

Maintenance immunosuppression for adults undergoing liver transplantation: a network meta-analysis.

BACKGROUND: As part of liver transplantation, immunosuppression (suppressing the host immunity) is given to prevent graft rejections resulting from the immune response of the body against transplanted organ or tissues from a different person whose tissue antigens are not compatible with those of the recipient. The optimal maintenance immunosuppressive regimen after liver transplantation remains uncertain. OBJECTIVES: To assess the comparative benefits and harms of different maintenance immunosuppressive regimens in adults undergoing liver transplantation through a network meta-analysis and to generate rankings of the different immunosuppressive regimens according to their safety and efficacy. SEARCH METHODS: We searched CENTRAL, MEDLINE, Embase, Science Citation Index Expanded, World Health Organization International Clinical Trials Registry Platform, and trials registers until October 2016 to identify randomised clinical trials on immunosuppression for liver transplantation. SELECTION CRITERIA: We included only randomised clinical trials (irrespective of language, blinding, or publication status) in adult participants undergoing liver transplantation (or liver retransplantation) for any reason. We excluded trials in which participants had undergone multivisceral transplantation or participants with established graft rejections. We considered any of the various maintenance immunosuppressive regimens compared with each other. DATA COLLECTION AND ANALYSIS: We performed a network meta-analysis with OpenBUGS using Bayesian methods and calculated the odds ratio, rate ratio, and hazard ratio (HR) with 95% credible intervals (CrI) based on an available-case analysis, according to National Institute of Health and Care Excellence Decision Support Unit guidance. MAIN RESULTS: We included a total of 26 trials (3842 participants) in the review, and 23 trials (3693 participants) were included in one or more outcomes in the review. The vast majority of the participants underwent primary liver transplantation. All of the trials were at high risk of bias, and all of the evidence was of low or very low quality. In addition, because of sparse data involving trials at high risk of bias, it is not possible to entirely rely on the results of the network meta-analysis. The trials included mainly participants undergoing primary liver transplantation of varied aetiologies. The follow-up in the trials ranged from 3 to 144 months. The most common maintenance immunosuppression used as a control was tacrolimus. There was no evidence of difference in mortality (21 trials; 3492 participants) or graft loss (15 trials; 2961 participants) at maximal follow-up between the different maintenance immunosuppressive regimens based on the network meta-analysis. In the direct comparison, based on a single trial including 222 participants, tacrolimus plus sirolimus had increased mortality (HR 2.76, 95% CrI 1.30 to 6.69) and graft loss (HR 2.34, 95% CrI 1.28 to 4.61) at maximal follow-up compared with tacrolimus. There was no evidence of differences in the proportion of people with serious adverse events (1 trial; 719 participants), proportion of people with any adverse events (2 trials; 940 participants), renal impairment (8 trials; 2233 participants), chronic kidney disease (1 trial; 100 participants), graft rejections (any) (16 trials; 2726 participants), and graft rejections requiring treatment (5 trials; 1025 participants) between the different immunosuppressive regimens. The network meta-analysis showed that the number of adverse events was lower with cyclosporine A than with many other immunosuppressive regimens (12 trials; 1748 participants), and the risk of retransplantation (13 trials; 1994 participants) was higher with cyclosporine A than with tacrolimus (HR 3.08, 95% CrI 1.13 to 9.90). None of the trials reported number of serious adverse events, health-related quality of life, or costs. FUNDING: 14 trials were funded by pharmaceutical companies who would benefit from the results of the trial; two trials were funded by parties who had no vested interest in the results of the trial; and 10 trials did not report the source of funding. AUTHORS' CONCLUSIONS: Based on low-quality evidence from a single small trial from direct comparison, tacrolimus plus sirolimus increases mortality and graft loss at maximal follow-up compared with tacrolimus. Based on very low-quality evidence from network meta-analysis, we found no evidence of difference between different immunosuppressive regimens. We found very low-quality evidence from network meta-analysis and low-quality evidence from direct comparison that cyclosporine A causes more retransplantation compared with tacrolimus. Future randomised clinical trials should be adequately powered; performed in people who are generally seen in the clinic rather than in highly selected participants; employ blinding; avoid postrandomisation dropouts or planned cross-overs; and use clinically important outcomes such as mortality, graft loss, renal impairment, chronic kidney disease, and retransplantation. Such trials should use tacrolimus as one of the control groups. Moreover, such trials ought to be designed in such a way as to ensure low risk of bias and low risks of random errors.

Everolimus is safe within the first month after liver transplantation.

BACKGROUND & AIMS: Sirolimus should not be started within the first month after liver transplantation (LT) because of an increased risk of adverse outcomes. The evidence regarding everolimus is lacking but the manufacturer transposed the same warning. We aimed to evaluate the safety of everolimus started within the first month after LT. METHODS: A consecutive cohort 187 LT patients (2009-2013) with a tacrolimus-based immunosuppression was evaluated. Patients starting everolimus within the first month after LT (n = 33; 17.6%) were compared with those starting everolimus thereafter (n = 25; 13.4%) or not receiving everolimus (n = 129; 69%). The median follow-up after LT was 21 months (IQR 7-36). Prospective outcomes were evaluated by using Kaplan-Meier curves and Cox's regression. RESULTS: The incidence of hepatic artery thrombosis was not significantly different in patients early treated with everolimus when compared with the remaining cohort (0% vs 9.1%; p = 0.12). Other vascular complications occurred in 9.1% of patients with early everolimus vs 7.3% in the remaining cohort (p = 0.72). No wound healing complications were detected with early everolimus. There were similar rates of incisional hernia (p = 0.31), infections (p = 0.15), renal impairment (p = 0.37), and histologically-proven acute rejection (p = 0.24) between groups. The rates of hyperlipidemia were increased with early everolimus (29.9% vs 16.5% at 3 years; p = 0.018). Graft loss and mortality rates were similar between groups (p = 0.34 and p = 0.94 respectively), after adjusting for possible confounding factors. CONCLUSIONS: Everolimus combined with reduced tacrolimus proved to be safe within the first month after LT. Future trials may be allowed to implement everolimus early after LT.

Strategies to improve outcome of patients with hepatocellular carcinoma receiving a liver transplantation.

Liver transplantation is the only therapeutic option which allows to treat both, the hepatocellular carcinoma and the underlying liver disease. Indeed, liver transplantation is considered the standard of care for a subset of patients with cirrhosis and hepatocellular carcinoma. However, tumour recurrence rates are as high as 20%, and once the recurrence is established the therapeutic options are scarce and with little impact on prognosis. Strategies to minimize tumour recurrence and thus to improve outcome may be classified into 3 groups: (1) An adequate selection of candidates for liver transplantation by using the Milan criteria; (2) An optimized management within waiting list including prioritization of patients at high risk of tumour progression, and the implementation of bridging therapies, particularly when the expected length within the waiting list is longer than 6 mo; and (3) Tailored immunosuppression comprising reduced exposure to calcineurin inhibitors, particularly early after liver transplantation, and the addition of mammalian target of rapamycin inhibitors. In the present manuscript the available scientific evidence supporting these strategies is comprehensively reviewed, and future directions are provided for novel research approaches, which may contribute to the final target: to cure more patients with hepatocellular carcinoma and with an improved long term outcome.

Lack of agreement for defining 'clinical suspicion of rejection' in liver transplantation: a model to select candidates for liver biopsy.

The gold standard to diagnose acute cellular rejection (ACR) after liver transplantation (LT) is histological evaluation, but there is no consensus to select patients for liver biopsy. We aimed to evaluate the agreement among clinicians to select candidates for liver biopsy early after LT. From a protocol biopsy population (n = 690), we randomly selected 100 LT patients in whom the biopsy was taken 7-10 days after LT. The clinical information between LT and protocol biopsy was given to nine clinicians from three transplant centres who decided whether a liver biopsy was needed. The agreement among clinicians to select candidates for liver biopsy was poor: κ = 0.06-0.62, being κ < 0.40 in 76% of comparisons. The concordance between indication for liver biopsy and moderate-severe ACR in the protocol biopsy was κ < 0.30 in all cases. A multivariate model based on the product age-by-MELD (OR = 0.81; P = 0.013), delta eosinophils (OR = 1.5; P = 0.002) and mean tacrolimus trough concentrations <6 ng/ml within the prior 4 days (OR = 11.4; P = 0.047) had an AUROC = 0.84 to diagnose moderate-severe histological ACR. In conclusion, the agreement among clinicians to select patients for liver biopsy is very poor. If further validated the proposed model would provide an objective method to select candidates for liver biopsy after LT.