Mechanisms of MAFG Dysregulation in Cholestatic Liver Injury and Development of Liver Cancer.

BACKGROUND & AIMS: MAF bZIP transcription factor G (MAFG) is activated by the farnesoid X receptor to repress bile acid synthesis. However, expression of MAFG increases during cholestatic liver injury in mice and in cholangiocarcinomas. MAFG interacts directly with methionine adenosyltransferase α1 (MATα1) and other transcription factors at the E-box element to repress transcription. We studied mechanisms of MAFG up-regulation in cholestatic tissues and the pathways by which S-adenosylmethionine (SAMe) and ursodeoxycholic acid (UDCA) prevent the increase in MAFG expression. We also investigated whether obeticholic acid (OCA), an farnesoid X receptor agonist, affects MAFG expression and how it contributes to tumor growth in mice. METHODS: We obtained 7 human cholangiocarcinoma specimens and adjacent non-tumor tissues from patients that underwent surgical resection in California and 113 hepatocellular carcinoma (HCC) specimens and adjacent non-tumor tissues from China, along with clinical data from patients. Tissues were analyzed by immunohistochemistry. MAT1A, MAT2A, c-MYC, and MAFG were overexpressed or knocked down with small interfering RNAs in MzChA-1, KMCH, Hep3B, and HepG2 cells; some cells were incubated with lithocholic acid (LCA, which causes the same changes in gene expression observed during chronic cholestatic liver injury in mice), SAMe, UDCA (100 µM), or farnesoid X receptor agonists. MAFG expression and promoter activity were measured using real-time polymerase chain reaction, immunoblot, and transient transfection. We performed electrophoretic mobility shift, and chromatin immunoprecipitation assays to study proteins that occupy promoter regions. We studied mice with bile-duct ligation, orthotopic cholangiocarcinomas, cholestasis-induced cholangiocarcinoma, diethylnitrosamine-induced liver tumors, and xenograft tumors. RESULTS: LCA activated expression of MAFG in HepG2 and MzChA-1 cells, which required the activator protein-1, nuclear factor-κB, and E-box sites in the MAFG promoter. LCA reduced expression of MAT1A but increased expression of MAT2A in cells. Overexpression of MAT2A increased activity of the MAFG promoter, whereas knockdown of MAT2A reduced it. MAT1A and MAT2A had opposite effects on the activator protein-1, nuclear factor-κB, and E-box-mediated promoter activity. Expression of MAFG and MAT2A increased, and expression of MAT1A decreased, in diethylnitrosamine-induced liver tumors in mice. SAMe and UDCA had shared and distinct mechanisms of preventing LCA-mediated increased expression of MAFG. OCA increased expression of MAFG, MAT2A, and c-MYC, but reduced expression of MAT1A. Incubation of human liver and biliary cancer cells lines with OCA promoted their proliferation; in nude mice given OCA, xenograft tumors were larger than in mice given vehicle. Levels of MAFG were increased in human HCC and cholangiocarcinoma tissues compared with non-tumor tissues. High levels of MAFG in HCC samples correlated with hepatitis B, vascular invasion, and shorter survival times of patients. CONCLUSIONS: Expression of MAFG increases in cells and tissues with cholestasis, as well as in human cholangiocarcinoma and HCC specimens; high expression levels correlate with tumor progression and reduced survival time. SAMe and UDCA reduce expression of MAFG in response to cholestasis, by shared and distinct mechanisms. OCA induces MAFG expression, cancer cell proliferation, and growth of xenograft tumors in mice.

Fabrication of microporous layer - free hierarchical gas diffusion electrode as a low Pt-loading PEMFC cathode by direct growth of helical carbon nanofibers.

Improving interfacial contact between each component in the proton exchange membrane fuel cell (PEMFC) can lead to a significant increase in power density and Pt utilization. In this work, the junction between the catalyst layer and gas diffusion layer (GDL) is greatly enhanced through direct attachment of helical carbon nanofibers, giving rise to a hierarchical structure within the electrical interconnections. The alternative novel GDL is produced by spraying a thin layer of Pd2C60 precursor on commercial carbon paper, followed by chemical vapor deposition growth resulting in a surface morphology of well-attached nanofibers surrounding the microfibers present in the commercial carbon paper. Subsequent solvothermal deposition of platinum nanoparticles allowed evaluation of its suitability as gas diffusion electrode in cathodic H2/O2 PEMFC environment. A combination of lowered charge transfer resistance and enhanced Pt-utilization is attributed to its unique wire-like appearance and its robust properties. The fabricated microporous layer - free GDL is suitable for relatively aggressive membrane electrode assembly fabrication procedures and is produced by industrially favorable techniques, rendering it capable of efficiently supporting small amounts of precious metal catalyst nanoparticles in various PEM applications.

Prohibitin 1 suppresses liver cancer tumorigenesis in mice and human hepatocellular and cholangiocarcinoma cells.

Prohibitin 1 (PHB1) is best known as a mitochondrial chaperone, and its role in cancer is conflicting. Mice lacking methionine adenosyltransferase α1 (MATα1) have lower PHB1 expression, and we reported that c-MYC interacts directly with both proteins. Furthermore, c-MYC and MATα1 exert opposing effects on liver cancer growth, prompting us to examine the interplay between PHB1, MATα1, and c-MYC and PHB1's role in liver tumorigenesis. We found that PHB1 is highly expressed in normal hepatocytes and bile duct epithelial cells and down-regulated in most human hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). In HCC and CCA cells, PHB1 expression correlates inversely with growth. PHB1 and MAT1A positively regulate each other's expression, whereas PHB1 negatively regulates the expression of c-MYC, MAFG, and c-MAF. Both PHB1 and MATα1 heterodimerize with MAX, bind to the E-box element, and repress E-box promoter activity. PHB1 promoter contains a repressive E-box element and is occupied mainly by MAX, MNT, and MATα1 in nonmalignant cholangiocytes and noncancerous tissues that switched to c-MYC, c-MAF, and MAFG in cancer cells and human HCC/CCA. All 8-month-old liver-specific Phb1 knockout mice developed HCC, and one developed CCA. Five-month-old Phb1 heterozygotes, but not Phb1 flox mice, developed aberrant bile duct proliferation; and one developed CCA 3.5 months after left and median bile duct ligation. Phb1 heterozygotes had a more profound fall in the expression of glutathione synthetic enzymes and higher hepatic oxidative stress following left and median bile duct ligation. CONCLUSION: We have identified that PHB1, down-regulated in most human HCC and CCA, heterodimerizes with MAX to repress the E-box and positively regulates MAT1A while suppressing c-MYC, MAFG, and c-MAF expression; in mice, reduced PHB1 expression predisposes to the development of cholestasis-induced CCA. (Hepatology 2017;65:1249-1266).

Peribiliary hepatic cysts presenting as hilar cholangiocarcinoma in a patient with end-stage liver disease.

Peribiliary cysts are cystic dilatations of peribiliary glands in the liver. They are present in ~50% of cirrhotic patients, but are underrecognized because they are usually asymptomatic and rarely present as obstructive jaundice. A 63-year-old male with hepatitis C cirrhosis, awaiting liver transplantation, had a new finding of intrahepatic dilatation on magnetic resonance imaging. This was initially concerning for cholangiocarcinoma, but was ultimately diagnosed as peribiliary cysts. Peribiliary cysts can imitate cholangiocarcinoma on imaging. Therefore, awareness of this condition is essential because misdiagnosis may lead to inappropriate delay or denial for liver transplantation. The ideal imaging modalities to identify peribiliary cysts are magnetic resonance cholangiography and drip infusion cholangiographic computed tomography, though hepatic dysfunction may limit the usefulness of the latter. Peribiliary cysts should be considered in cirrhotic patients with cholestasis, biliary dilatations and negative biopsy of the biliary system for malignancy.

Targeting mTOR in Pancreatic Ductal Adenocarcinoma.

Treatment options for advanced pancreatic ductal adenocarcinoma (PDAC) are limited; however, new therapies targeting specific tumor-related molecular characteristics may help certain patient cohorts. Emerging preclinical data have shown that inhibition of mammalian target of rapamycin (mTOR) in specific KRAS-dependent PDAC subtypes leads to inhibition of tumorigenesis in vitro and in vivo. Early phase II studies of mono-mTOR inhibition have not shown promise. However, studies have shown that combined inhibition of multiple steps along the mTOR signaling pathway may lead to sustained responses by targeting mechanisms of tumor resistance. Coordinated inhibition of mTOR along with specific KRAS-dependent mutations in molecularly defined PDAC subpopulations may offer a viable alternative for treatment in the future.

Impact of margin status and lymphadenectomy on clinical outcomes in resected pancreatic adenocarcinoma: implications for adjuvant radiotherapy.

BACKGROUND: Adjuvant chemoradiotherapy (CRT) in the treatment of pancreatic ductal adenocarcinoma (PDA) is controversial. Minimal data exists regarding the clinical significance of margin clearance distance and lymph node (LN) parameters, such as extent of dissection and LN ratio. We assessed the impact of these variables on clinical outcomes to more clearly define the subset of patients who may benefit from adjuvant radiotherapy (RT). METHODS: We identified 106 patients with resected stage 1-3 PDA from 2007-2013. Resection margins were categorized as positive (tumor at ink), ≤1, or >1 mm. LN evaluation included total number examined (NE), number of positive nodes (NP), ratio of NP to NE (NR), extent of dissection, and positive periportal LNs. The impact of these variables was assessed on disease-free survival (DFS) and overall survival (OS) using multivariate cox proportional hazards modeling. RESULTS: In patients receiving adjuvant chemotherapy (CT) alone, greater margin clearance led to improved DFS (P=0.0412, HR =0.51). Range of NE was 4-37, with a mean of 19. NE was not associated with DFS or OS, yet absolute NP of 5 or more was associated with a significantly worse DFS (P=0.005). Whereas periportal lymphadenectomy did not result in improved DFS or OS, patients with positive periportal LN had worse clinical outcomes (DFS, P=0.0052; OS, P=0.023). The use of adjuvant CRT was associated with improved OS (P=0.049; HR=0.29). CONCLUSIONS: In patients receiving adjuvant CT alone, there was a clinically significant benefit to clearing the surgical margin beyond tumor at ink. Having ≥5 NP and positive periportal LN led to significantly worse clinical outcomes. The addition of adjuvant RT to CT in resected PDA improved OS. A comprehensive evaluation of resection margin distance and LN parameters may identify more patients at risk for locoregional failure who may benefit from adjuvant CRT.

Cultured circulating tumor cells and their derived xenografts for personalized oncology.

Recent cancer research has demonstrated the existence of circulating tumor cells (CTCs) in cancer patient's blood. Once identified, CTC biomarkers will be invaluable tools for clinical diagnosis, prognosis and treatment. In this review, we propose ex vivo culture as a rational strategy for large scale amplification of the limited numbers of CTCs from a patient sample, to derive enough CTCs for accurate and reproducible characterization of the biophysical, biochemical, gene expressional and behavioral properties of the harvested cells. Because of tumor cell heterogeneity, it is important to amplify all the CTCs in a blood sample for a comprehensive understanding of their role in cancer metastasis. By analyzing critical steps and technical issues in ex vivo CTC culture, we developed a cost-effective and reproducible protocol directly culturing whole peripheral blood mononuclear cells, relying on an assumed survival advantage in CTCs and CTC-like cells over the normal cells to amplify this specified cluster of cancer cells.

Profile of Inflammation-associated genes during Hepatic Differentiation of Human Pluripotent Stem Cells.

Expression of genes associated with inflammation was analyzed during differentiation of human pluripotent stem cells (PSCs) to hepatic cells. Messenger RNA transcript profiles of differentiated endoderm (day 5), hepatoblast (day 15) and hepatocyte-like cells (day 21) were obtained by RNA sequencing analysis. When compared to endoderm cells an immature cell type, the hepatic cells (days 15 and 21) had significantly higher expression of acute phase protein genes including complement factors, coagulation factors, serum amyloid A and serpins. Furthermore, hepatic phase of cells expressed proinflammatory cytokines IL18 and IL32 as well as cytokine receptors IL18R1, IL1R1, IL1RAP, IL2RG, IL6R, IL6ST and IL10RB. These cells also produced CCL14, CCL15, and CXCL- 1, 2, 3, 16 and 17 chemokines. Endoderm cells had higher levels of chemokine receptors, CXCR4 and CXCR7, than that of hepatic cells. Sirtuin family of genes involved in aging, inflammation and metabolism were differentially regulated in endoderm and hepatic phase cells. Ligands and receptors of the tumor necrosis factor (TNF) family as well as downstream signaling factors TRAF2, TRAF4, FADD, NFKB1 and NFKBIB were differentially expressed during hepatic differentiation.

Hemodynamic Consequences of Laparoscopy for Patients on Mechanical Circulatory Support.

BACKGROUND: Technologic advances and superior survival with mechanical circulatory support (MCS) have led to an expanding population that develops intraabdominal conditions requiring intervention. Whether laparoscopy can be performed without detrimental effects on hemodynamics and device function is not well described. MATERIALS AND METHODS: Effects of laparoscopy performed on MCS were retrospectively assessed. Intraoperative hemodynamics and device function were compared with the same time interval 24 hours prior to surgery using intrapatient paired t tests. Outcomes included survival, transfusion, thromboembolic events, and infection. RESULTS: Twelve patients with ventricular assist devices or total artificial hearts underwent laparoscopy from 2012 to 2014. Median follow-up was 116 days. Operations included cholecystectomy, diagnostic laparoscopy, gastrojejunostomy, and gastrostomy. There were no differences between preoperative and intraoperative mean arterial pressure, heart rate, and inotrope or vasopressor requirements (P > .05). Device fill volume, flow, rate, and power were unchanged (P > .05), whereas pulsatility index decreased by 0.2 (95% confidence interval, 0.03, 0.36) with laparoscopy (P = .03). All intraoperative fluctuations in hemodynamics and device function improved with reduction of pneumoperitoneum, adjusting device speed, or pharmacologic support. There were no operative mortalities. Thirty-day survival and survival to discharge were 75% and 50%, respectively. Despite antiplatelet therapy and preoperative international normalization ratio of 2.2 ± 0.9, there were no re-operations for bleeding, and 50% did not require transfusion. Two patients with recent cardiac surgery had thromboembolic events: one stroke and one device thrombus. None had postoperative bacteremia or driveline infection. CONCLUSIONS: Laparoscopy can be performed on MCS with low morbidity and mortality and minimal perturbations in hemodynamics and device function.

Outcomes of vascular resection in pancreaticoduodenectomy: single-surgeon experience.

Extension of pancreatic adenocarcinoma into adjacent vasculature often necessitates resection of the portal vein (PV) and/or superior mesenteric vein (SMV) during pancreaticoduodenectomy (PD). The vein is reconstructed primarily by end-to-end anastomosis of vein remnants or venoplasty or by use of autologous or synthetic vein grafts. The objective of this study was to review outcomes in patients undergoing PD for pancreatic adenocarcinoma, specifically comparing the short- and long-term outcomes between the patients undergoing vascular resection and those undergoing standard PD. All patients undergoing PD for pancreatic adenocarcinoma by a single surgeon between 2007 and 2012 were reviewed. Of the 61 patients identified, 18 patients underwent vascular resection of the PV (four patients), SMV (10 patients), or both (four patients). The remaining 43 patients had standard PD. Demographic, perioperative, pathological, and long-term outcomes data were collected and both vascular and standard groups were compared. Both groups had similar demographics. The vascular group had significantly longer operative times (529 vs 406 minutes; P < 0.01) with a trend to greater estimated blood loss (0.64 vs 0.53 L; P = 0.06). Pathological analysis showed no difference between the two groups with regard to lymph node status/ratio and rate of R0 resection (94 vs 91%; P = 0.57); however, the size of the tumor was significantly greater in the vascular group (4.2 vs 3 cm; P < 0.01). Short-term outcomes were similar in the vascular group and standard group, respectively, with no difference in pancreatic fistula rate (6 vs 7%; P = 1.0), transfusion rate (44 vs 35%; P = 0.57), and median length of stay (8 vs 7 days; P = 0.10), and there was no 30-day mortality in either group. Based on Kaplan-Meier methods, the median recurrence-free survival was 18 versus 23 months (P = 0.37) in the vascular and standard groups, respectively, and the overall survival was almost identical in both groups, each with a median of 31 months (P = 0.91). In our experience, mesenteric and PV resection during PD was performed safely and without compromise of short- or longer-term outcomes. It can be performed safely and patients have no significant difference in perioperative outcomes or overall survival.

A simple algorithm for drain management after pancreaticoduodenectomy.

Pancreatic fistula (PF) continues to be the Achilles' heel of pancreaticoduodenectomy (PD) with both morbidity and mortality linked to its occurrence. The optimal drain management strategy after PD remains unclear. We evaluated drain amylase (DA) levels on postoperative Day (POD) 0 to 5 in 76 consecutive patients undergoing PD to determine the patterns associated with PF. Of these 76 patients, eight patients (11%) developed Grade A, B, or C PF by International Study Group of Pancreatic Fistula criteria. POD 1 DA levels correlated closely with PF rates when high (greater than 5000 U/L, 100% PF rate) and low (less than 100 U/L, 2% PF rate). In patients with intermediate POD 1 DA (100 to 5000 U/L), 42 and 74 per cent had low DA levels on POD 3 and 5, respectively, and the PF rate was four of 31 (13%). Overall, the temporal pattern of decreasing DA levels after PD correlates closely with the risk of PF, and only two patients (5%) developed PF after early DA levels had normalized. Based on these data, we propose an algorithm of monitoring DA daily with drain removal when the level is less than 100 U/L. In our patient group drain removal would have occurred on a mean of 1.8 days and median 1 day after surgery.

Team play in surgical education: a simulation-based study.

BACKGROUND: Simulation-based training provides a low-stress learning environment where real-life emergencies can be practiced. Simulation can improve surgical education and patient care in crisis situations through a team approach emphasizing interpersonal and communication skills. OBJECTIVE: This study assessed the effects of simulation-based training in the context of trauma resuscitation in teams of trainees. METHODS: In a New York State-certified level I trauma center, trauma alerts were assessed by a standardized video review process. Simulation training was provided in various trauma situations followed by a debriefing period. The outcomes measured included the number of healthcare workers involved in the resuscitation, the percentage of healthcare workers in role position, time to intubation, time to intubation from paralysis, time to obtain first imaging study, time to leave trauma bay for computed tomography scan or the operating room, presence of team leader, and presence of spinal stabilization. Thirty cases were video analyzed presimulation and postsimulation training. The two data sets were compared via a 1-sided t test for significance (p < 0.05). Nominal data were analyzed using the Fischer exact test. RESULTS: The data were compared presimulation and postsimulation. The number of healthcare workers involved in the resuscitation decreased from 8.5 to 5.7 postsimulation (p < 0.001). The percentage of people in role positions increased from 57.8% to 83.6% (p = 0.46). The time to intubation from paralysis decreased from 3.9 to 2.8 minutes (p < 0.05). The presence of a definitive team leader increased from 64% to 90% (p < 0.05). The rate of spine stabilization increased from 82% to 100% (p < 0.08). After simulation, training adherence to the advanced trauma life support algorithm improved from 56% to 83%. CONCLUSIONS: High-stress situations simulated in a low-stress environment can improve team interaction and educational competencies. Providing simulation training as a tool for surgical education may enhance patient care.

Recurrent hepatocellular carcinoma after liver transplant: identifying the high-risk patient.

BACKGROUND: Recurrence of hepatocellular carcinoma (HCC) after liver transplantation (LT) is rarely curable. However, in view of the advent of new treatments, it is critical that patients at high risk for recurrence are identified. METHODS: Patients undergoing LT for HCC at a single centre between 2002 and 2010 were reviewed and data on clinical parameters and explant pathology were analysed to determine factors associated with HCC recurrence. All necrotic and viable tumour nodules were included in explant staging. All patients underwent LT according to the United Network for Organ Sharing (UNOS) Model for End-stage Liver Disease (MELD) tumour exception policies. RESULTS: Liver transplantation was performed in 122 patients with HCC during this period. Rates of recurrence-free survival in the entire cohort at 1 year and 3 years were 95% and 89%, respectively. Thirteen patients developed HCC recurrence at a median of 14 months post-LT. In univariate analysis the factors associated with HCC recurrence were bilobar tumours, vascular invasion, and stage exceeding either Milan or University of California San Francisco (UCSF) Criteria. Multivariate analysis showed pathology outside UCSF Criteria was the major predictor of recurrence; when pathology outside UCSF Criteria was found in combination with vascular invasion, the predicted 3-year recurrence-free survival was only 26%. CONCLUSIONS: Explant pathology can be used to predict the risk for recurrent HCC after LT, which may allow for improved adjuvant and management strategies.

An unexpected finding of a bronchogenic cyst presenting as a thyroid mass.

Herein we present a case of a bronchogenic cyst masquerading as a thyroid mass. Bronchogenic cysts are rare congenital malformations that result from an abnormal development of the ventral foregut during organogenesis. They are commonly asymptomatic lesions rarely found in the neck. In our case, a young male presented with complaints of neck discomfort over a long period presumed to be secondary to a thyroid mass based on imaging studies. Fine needle aspiration was inconclusive. Post-operative pathological sectioning revealed evidence of a bronchogenic cyst. These lesions have a low incidence of malignancy, and complete surgical excision has been recommended.