A Single-Step Immunocapture Assay to Quantify HCC Exosomes Using the Highly Sensitive Fluorescence Nanoparticle-Tracking Analysis.

Introduction: Extracellular vesicles could serve as a non-invasive biomarker for early cancer detection. However, limited methods to quantitate cancer-derived vesicles in the native state remain a significant barrier to clinical translation. Aim: This research aims to develop a rapid, one-step immunoaffinity approach to quantify HCC exosomes directly from a small serum volume. Methods: HCC-derived exosomes in the serum were captured using fluorescent phycoerythrin (PE)-conjugated antibodies targeted to GPC3 and alpha-fetoprotein (AFP). Total and HCC-specific exosomes were then quantified in culture supernatant or patient-derived serums using fluorescence nanoparticle tracking analysis (F-NTA). The performance of HCC exosome quantification in the serum was compared with the tumor size determined by MRI. Results: Initially we tested the detection limits of the F-NTA using synthetic fluorescent and non-fluorescent beads. The assay showed an acceptable sensitivity with a detection range of 104-108 particles/mL. Additionally, the combination of immunocapture followed by size-exclusion column purification allows the isolation of smaller-size EVs and quantification by F-NTA. Our assay demonstrated that HCC cell culture releases a significantly higher quantity of GPC3 or GPC3+AFP positive EVs (100-200 particles/cell) compared to non-HCC culture (10-40 particles/cell) (p<0.01 and p<0.05 respectively). The F-NTA enables absolute counting of HCC-specific exosomes in the clinical samples with preserved biological immunoreactivity. The performance of F-NTA was clinically validated in serum from patients ± cirrhosis and with confirmed HCC. F-NTA quantification data show selective enrichment of AFP and GPC3 positive EVs in HCC serum compared to malignancy-free cirrhosis (AUC values for GPC3, AFP, and GPC3/AFP were found 0.79, 0.71, and 0.72 respectively). The MRI-confirmed patient cohort indicated that there was a positive correlation between total tumor size and GPC3-positive exosome concentration (r:0.78 and p<0.001). Conclusion: We developed an immunocapture assay that can be used for simultaneous isolation and quantification of HCC-derived exosomes from a small serum volume with high accuracy.

The number of hepatocellular carcinoma foci as predictor of poor response to tumor-directed therapies in patients awaiting liver transplantation: a prospective cohort study.

BACKGROUND AND AIMS: Tumor-directed therapies (TDTs) are a constitutive part of hepatocellular carcinoma (HCC) treatment in patients awaiting liver transplantation (LT). While most patients benefit from TDTs as a bridge to LT, some patients drop out from the waiting list due to tumor progression. The study aimed to determine the risk factors for poor treatment outcome following TDTs among patients with HCC awaiting LT. METHODS: A total of 123 patients with HCC were evaluated with 92 patients meeting Milan Criteria enrolled in the prospective cohort study. Tumor response was evaluated using the modified Response Evaluation Criteria for Solid Tumors for HCC 1 month after the procedure. The risk factors for progressive disease (PD) and dropout were evaluated. RESULTS: After TDT, 55 patients (59.8%) achieved complete or partial response (44.6% and 15.2% respectively), 17 patients (18.5%) had stable disease, and 20 patients (21.7%) were assessed as PD. Multivariate analysis revealed a significant and independent association between the number of HCC foci and PD ( P  = 0.03, OR = 2.68). There was no statistically significant association between treatment response and demographics, MELDNa score, pre-and post-treatment alpha-fetoprotein (AFP), cumulative tumor burden the largest tumor size, or TDT modality. PD was the major cause of dropout in our cohort. Pre-treatment AFP levels ≥200 ng/ml had a strong association with dropout after TDTs ( P  = 0.0005). CONCLUSION: This study demonstrated the presence of multifocal HCC is the sole prognostic factor for PD following TDTs in HCC patients awaiting LT. We recommend prioritizing patients with multifocal HCC within Milan criteria by exception points for LT to improve the dropout rate.

Impaired Autophagy Response in Hepatocellular Carcinomas Enriches Glypican-3 in Exosomes, Not in the Microvesicles.

Background and Aim: HCC development in liver cirrhosis is associated with impaired autophagy leading to increased production of extracellular vesicles (EVs) including exosomes and microvesicles. The goal of the study is to determine which of these particles is primarily involved in releasing of HCC-specific biomarker glypican-3 (GPC3) when autophagy is impaired. Methods: Streptavidin-coated magnetic beads were coupled with either biotinylated CD63 or Annexin A1 antibodies. Coupled beads were incubated with EVs isolated from either HCC culture or serum. EVs captured by immuno-magnetic beads were then stained with FITC or PE fluorescent-conjugated antibodies targeting exosomes (CD81), and microvesicles (ARF6). The percentage of GPC3 enrichment in the microvesicles and exosomes was quantified by flow cytometry. The impact of autophagy modulation on GPC3 enrichment in exosomes and microvesicles was assessed by treating cells with Torin 1 and Bafilomycin A1. For clinical validation, GPC3 content was quantified in microvesicles, and exosomes were isolated from the serum of patients with a recent HCC diagnosis. Results: The immune-magnetic bead assay distinguishes membrane-derived microvesicles from endosome-derived exosomes. The GPC3 expression was only seen in the CD63 beads group but not in the Annexin A1 beads group, confirming that in HCC, GPC3 is preferentially released through exosomes. Furthermore, we found that autophagy induction by Torin1 decreased GPC3-positive exosome secretion and decreased microvesicle release. Conversely, autophagy inhibition by Bafilomycin A1 increased the secretion of GPC3-positive exosomes. Serum analysis showed CD81+ve EVs were detected in exosomes and ARF6+ve vesicles were detected in microvesicles, suggesting that immunoaffinity assay is specific. The exosomal GPC3 enrichment was confirmed in isolated EVs from the serum of patients with HCC. The frequency of GPC3-positive exosomes was higher in patients with HCC (12.4%) compared to exosomes isolated from non-cirrhotic and healthy controls (3.7% and 1.3% respectively, p<0.001). Conclusion: Our results show that GPC3 is enriched in the endolysosomal compartment and released in exosome fractions when autophagy is impaired.

Interferon-alpha-induced hepatitis C virus clearance restores p53 tumor suppressor more than direct-acting antivirals.

The mechanism why hepatitis C virus (HCV) clearance by direct-acting antivirals (DAAs) does not eliminate the risk of hepatocellular carcinoma (HCC) among patients with advanced cirrhosis is unclear. Many viral and bacterial infections degrade p53 in favor of cell survival to adapt an endoplasmic reticulum (ER)-stress response. In this study, we examined whether HCV clearance by interferon-alpha or DAAs normalizes the ER stress and restores the expression of p53 tumor suppressor in cell culture. We found that HCV infection induces chronic ER stress and unfolded protein response in untransformed primary human hepatocytes. The unfolded protein response induces chaperone-mediated autophagy (CMA) in infected primary human hepatocytes and Huh-7.5 cells that results in degradation of p53 and induced expression of mouse double minute 2 (Mdm2). Inhibition of p53/Mdm2 interactions by small molecule (nutlin-3) or silencing Mdm2 did not rescue the p53 degradation, indicating that HCV infection induces degradation of p53 independent of the Mdm2 pathway. Interestingly, we found that HCV infection degrades p53 in a lysosome-dependent mechanism because lysosome-associated membrane protein 2A silencing restored p53 degradation. Our results show that HCV clearance induced by interferon-alpha-based antiviral therapies normalizes the ER-stress response and restores p53, whereas HCV clearance by DAAs does neither. We show that decreased expression of p53 in HCV-infected cirrhotic liver is associated with expression of chaperones associated with ER stress and the CMA response. Conclusion: HCV-induced ER stress and CMA promote p53 degradation in advanced liver cirrhosis. HCV clearance by DAAs does not restore p53, which provides a potential explanation for why a viral cure by DAAs does not eliminate the HCC risk among patients with advanced liver disease. We propose that resolving the ER-stress response is an alternative approach to reducing HCC risk among patients with cirrhosis after viral cure. (Hepatology Communications 2017;1:256-269).

Cutaneous Metastases from Primary Hepatobiliary Tumors as the First Sign of Tumor Recurrence following Liver Transplantation.

Cutaneous metastasis from hepatobiliary tumors is a rare event, especially following liver transplantation. We report our experience with two cases of cutaneous metastases from both hepatocellular carcinoma and mixed hepatocellular/cholangiocarcinoma following liver transplantation, along with a review of the literature.

Methotrexate in ulcerative colitis: a nationwide retrospective cohort from the Veterans Affairs Health Care System.

BACKGROUND: There are paucity of data regarding the utility of methotrexate (MTX) in the management of ulcerative colitis (UC). The aim of this study was to describe the efficacy of MTX in achieving steroid-free remission. METHODS: A retrospective cohort study was conducted using the nationwide Veterans Affairs database to identify steroid-dependent patients with UC using MTX for the period 2001 to 2011. Patients were followed up for 15 months after MTX initiation by tracking their prednisone, MTX, thiopurines, and infliximab dispense. Endpoints were: (1) successful remission, defined as cessation of prednisone filling activity while continuing MTX; (2) failure with continuance, failure to be weaned off steroids while continuing MTX; (3) failure with discontinuance, cessation of MTX while continuing steroids. RESULTS: We included 91 patients with UC with mean age 59 years. The average weekly dose for oral and parenteral MTX was 14 and 25 mg/week, respectively. The average daily dose for prednisone within the oral MTX and parenteral MTX groups was 12 and 25 mg/day, respectively. By the 12th month of follow-up, 37% and 30% of patients on oral and parenteral MTX, respectively, were able to discontinue steroid. There was a nonsignificant trend toward dose reduction of steroids in those who were concomitantly taking oral MTX and steroids. CONCLUSIONS: Our study represents the largest cohort of patients with MTX and UC reported to date and suggests that approximately one-third of patients were successfully weaned off steroids with MTX therapy. MTX should be considered in the long-term management of patients with UC on steroids.

Absence of Hepatitis B Resistance Mutants before Introduction of Oral Antiviral Therapy.

Introduction. The aim of this study was to assess whether hepatitis B virus drug resistant mutations antedated the widespread use of nucleos(t)ide analogues in treatment naïve patients. A number of reports have suggested that drug resistant mutants can be detected in apparently treatment naïve patients. Study. Fifty deidentified serum samples collected from 1986 to 1992 from patients with replicative chronic HBV infection at the University of Miami were genotyped and tested for resistance mutations using a line probe assay InnoLiPA HBV DR v2/v3. Serum HBV DNA was measured. All patients had documented chronic HBV infection with a detectable viral load, HBeAg seropositivity, and absence of HIV infection. Results. Of the 50 individuals included, 86% were male, mean age was 40 ± 12 years, and mostly genotype A. The mean HBV DNA was 126 pg/mL (range 6.4 to 557.0). No mutations were identified. Conclusions. The absence of drug induced mutations in these sera collected several years prior to the introduction of oral antiviral therapy suggests that these mutations do not occur in treatment naïve populations. Detection of drug resistance in an apparently treatment naïve subject suggests either unrecognized prior antiviral therapy or infection by an inoculum from a treatment experienced patient.

A case of a ruptured sclerosing liver hemangioma.

Hemangiomas are the most common benign tumors found in the liver, typically asymptomatic, solitary, and incidentally discovered. Although vascular in nature, they rarely bleed. We report a case of a 52-year-old woman with a previously stable hemangioma who presented to our hospital with signs and symptoms indicative of spontaneous rupture. We review the literature, focusing on diagnosis and management of liver hemangiomas.

Interstitial pneumonitis in the transplant patient: consider sirolimus-associated pulmonary toxicity.

Interstitial pneumonia in a transplant patient can have a varied etiology. Sirolimus (Rapamycin; Rapamune) is a popularly used immunosuppressant in solid organ transplantation that has anecdotally been associated with pulmonary toxicity. Sirolimus-induced pulmonary toxicity consists of a range of syndromes that is characterized by the presence of organizing pneumonia, interstitial pneumonitis, pulmonary alveolar proteinosis, focal fibrosis, or by the presence of alveolar hemorrhage. Diagnosis can be challenging and is usually made by exclusion of other etiologies. In this report we present two cases of sirolimus-associated pulmonary toxicity with a review of the literature.