Non-cirrhotic Liver Research Articles

Serum cytokeratin-18 is a non-invasive biomarker for evaluating disease severity in patients with liver cirrhosis and hepatocellular carcinoma

Background: To investigate the dynamic changes in cytokeratin-18 (CK18) levels in patients with liver cirrhosis and hepatocellular carcinoma (HCC) undergoing surgery, and to evaluate its diagnostic value for HCC and acute liver injury. Methods: Serum levels of total CK18 and CK18 fragments were measured using specific enzyme-linked immunosorbent assays in patients with liver cirrhosis and HCC, and control patients with liver cavernous hemangioma. Clinical parameters were measured 1 day before and 1, 3, and 5 days after surgery. Results: CK18 levels were markedly elevated in patients with liver cirrhosis and HCC compared with control patients with non-cirrhotic liver disease (total CK18: 262.9±130.0 U/L, control 158.7±44.5 U/L, P=0.038; CK18 fragments: 145.1±69.6 U/L, control 81.6±15.2 U/L, P=0.005), but fell to near preoperative levels at postoperative day (POD) 5. CK18 levels paralleled perioperative changes in alanine transaminase (ALT), reaching peak values on POD 1, and then decreasing gradually to near preoperative levels by POD 5. The correlation coefficients for the relationships between serum CK18 released by hepatocyte necrosis and ALT levels were 0.659 in the cancer group and 0.922 in the control group. Serum ALT levels remained above preoperative levels at POD 5. The levels of total CK18 and CK18 fragment released by hepatocyte necrosis were remarkably higher on POD 1 than the levels before operation. Conclusions: Serum levels of total CK18 and CK18 fragments were higher in HCC patients with chronic hepatitis B-related liver cirrhosis compared with levels in patients with liver cavernous hemangioma. Liver cancer may have a greater impact on serum CK18 levels than cirrhosis. Surgical injury can increase serum CK18 levels, and CK18 may be a more sensitive marker of liver injury than ALT.

Abstract IA24: The scourge of hepatocellular cancer in Africa

Abstract Worldwide hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths with 80% of cases occurring in poor countries. In Africa HCC is the most common cause of cancer-related deaths in men and the third most common in women. Although HCC is prevalent in all Sub-Saharan African countries where data exist, the highest prevalences have been reported in countries in West Africa with annual incidences as high as 20 per 100 000 inhabitants. Historically viral hepatitis, in particular hepatitis B virus (HBV), aflatoxin and dietary iron overload have been implicated as the main etiological factors, but both alcoholic liver disease and metabolic syndrome-associated non-alcoholic fatty liver disease (NAFLD) seem to play an increasingly important role. There are striking differences in the clinical presentation, clinical course and prognosis of HCC in African patients, compared to developed countries. In African populations, in particular in rural areas, the disease occurs at a younger age with a mean age at presentation as low as 34.7 years having been reported. Furthermore, a larger percentage of HCCs present in non-cirrhotic livers. In well-resourced countries the diagnosis of cirrhosis would prompt tumour-directed investigations and screening for HCC with potentially earlier detection of tumours. This partially explains the fact that African patients present with larger tumours and more frequently with advanced disease in terms of metastatic burden. African patients are therefore less likely to undergo curative-intended intervention with resection rates as low as 1% being reported in rural African populations, compared to up to 37% in well-resourced Western countries. Whereas improved survival has been reported for patients undergoing curative-intended intervention in a number of well-resourced countries, the prognosis for African patients in general remains dismal with annual mortality rates approaching the annual incidence. The continent faces tremendous challenges in managing HCC. With virus-related HCC being responsible for approximately 90% of HCC cases, primary prevention with HBV immunization will have an immense impact on the occurrence of HCC. In spite of immunization having been shown to be cost-effective in low and middle income countries and a WHO recommendation that the HBV vaccine should be incorporated into the expanded program of immunization, the roll-out in Africa has been hampered by regulatory, financial, logistical, social and cultural barriers. Diagnostic capabilities, universally very limited in African countries, are typically concentrated in urban facilities, out of reach of rural patients. Access to treatment, whether curative- or palliative-intended is limited. It is a perverse coincidence that in the recently published report of The Lancet Commission on Global Surgery Western, Central and Eastern Sub-Saharan Africa were identified respectively as the regions with the highest, second highest and third highest rates of surgical need per population in the world. One can question whether there, on a global scale, is sufficient focus on the unique and often desperate fate of African patients. With some exceptions, mainstream HCC initiatives are exclusive, or are perceived as being exclusive of African institutions. This is ominously evident in the fact that less than 1% of currently ongoing clinical HCC trials are conducted on the continent, in spite of Africa being one of the hotbeds of HCC. Citation Format: Eduard Jonas. The scourge of hepatocellular cancer in Africa [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr IA24.

Transporter Expression in Noncancerous and Cancerous Liver Tissue from Donors with Hepatocellular Carcinoma and Chronic Hepatitis C Infection Quantified by LC-MS/MS Proteomics.

Protein expression of major hepatobiliary drug transporters (NTCP, OATPs, OCT1, BSEP, BCRP, MATE1, MRPs, and P-gp) in cancerous (C, n = 8) and adjacent noncancerous (NC, n = 33) liver tissues obtained from patients with chronic hepatitis C with hepatocellular carcinoma (HCV-HCC) were quantified by LC-MS/MS proteomics. Herein, we compare our results with our previous data from noninfected, noncirrhotic (control, n = 36) and HCV-cirrhotic (n = 30) livers. The amount of membrane protein yielded from NC and C HCV-HCC tissues decreased (31%, 67%) relative to control livers. In comparison with control livers, with the exception of NTCP, MRP2, and MATE1, transporter expression decreased in NC (38%-76%) and C (56%-96%) HCV-HCC tissues. In NC HCV-HCC tissues, NTCP expression increased (113%), MATE1 expression decreased (58%), and MRP2 expression was unchanged relative to control livers. In C HCV-HCC tissues, NTCP and MRP2 expression decreased (63%, 56%) and MATE1 expression was unchanged relative to control livers. Compared with HCV-cirrhotic livers, aside from NTCP, OCT1, BSEP, and MRP2, transporter expression decreased in NC (41%-71%) and C (54%-89%) HCV-HCC tissues. In NC HCV-HCC tissues, NTCP and MRP2 expression increased (362%, 142%), whereas OCT1 and BSEP expression was unchanged. In C HCV-HCC tissues, OCT1 and BSEP expression decreased (90%, 80%) relative to HCV-cirrhotic livers, whereas NTCP and MRP2 expression was unchanged. Expression of OATP2B1, BSEP, MRP2, and MRP3 decreased (56%-72%) in C HCV-HCC tissues in comparison with matched NC tissues (n = 8), but the expression of other transporters was unchanged. These data will be helpful in the future to predict transporter-mediated hepatocellular drug concentrations in patients with HCV-HCC.

Clinicopathological and prognostic significance of PKM2 protein expression in cirrhotic hepatocellular carcinoma and non-cirrhotic hepatocellular carcinoma

Pyruvate kinase M2 (PKM2), a key protein in glucose and lipid metabolism, has been reported to be related to carcinogenesis in various malignancies. However, its roles in hepatocellular carcinoma with cirrhotic liver (CL) and hepatocellular carcinoma with non-cirrhoticliver (NCL) haves not been investigated. In our study western bloting, qRT-PCR and immunohistochemistry were performed to evaluate the clinical significance of PKM2 protein expression in CL and NCL. The results revealed that PKM2 protein expression was significantly higher in HCC tissues than in their adjacent non-tumour tissues. The high expression rates of PKM2 were more frequently noted in CL (45. 6%) than in NCL (31. 9%) tissues. High PKM2 expression in CL and NCL tissues was significantly associated with vascular invasion (P = 0.002 and P = 0.004, respectively) and intrahepatic metastasis (P < 0.001 and P = 0.019, respectively). Importantly, Kaplan–Meier survival analysis showed that the disease-specific survival (DSS) and recurrence-free survival (RFS) were lower in CL with high PKM2 expression than in NCL with high PKM2 expression (P = 0.003 and P = 0.003, respectively). Overall, high PKM2 expression was more frequently found in CL than in NCL, and PKM2 overexpression was associated with poor survival rates in patients with CL and NCL.

Liver and systemic hemodynamics in children with cirrhosis: Impact on the surgical management in pediatric living donor liver transplantation.

Cirrhosis in adults is associated with modifications of systemic and liver hemodynamics, whereas little is known about the pediatric population. The aim of this work was to investigate whether alterations of hepatic and systemic hemodynamics were correlated with cirrhosis severity in children. The impact of hemodynamic findings on surgical management in pediatric living donor liver transplantation (LT) was evaluated. Liver and systemic hemodynamics were studied prospectively in 52 children (median age, 1 year; 33 with biliary atresia [BA]). The hemodynamics of native liver were studied preoperatively by Doppler ultrasound and intraoperatively using invasive flowmetry. Portosystemic gradient was invasively measured. Systemic hemodynamics were studied preoperatively by Doppler transthoracic echocardiography and intraoperatively by using transpulmonary thermodilution. Hemodynamic parameters were correlated with Pediatric End-Stage Liver Disease (PELD) score and the histological degree of fibrosis (collagen proportionate area [CPA]). Cirrhosis was associated with a 60% reduction of pretransplant total liver flow (n = 46; median, 36 mL/minute/100 g of liver) compared with noncirrhotic livers (n = 6; median, 86 mL/minute/100 g; P = 0.002). Total blood flow into the native liver was negatively correlated with PELD (P < 0.001) and liver CPA (P = 0.005). Median portosystemic gradient was 14.5 mm Hg in children with cirrhosis and positively correlated with PELD (P < 0.001). Portal vein (PV) hypoplasia was observed mainly in children with BA (P = 0.02). Systemic hemodynamics were not altered in our children with cirrhosis. Twenty-one children met the intraoperative criteria for PV reconstruction using a portoplasty technique during the LT procedure and had a smaller PV diameter at pretransplant Doppler ultrasound (median = 3.4 mm; P < 0.001). Cirrhosis in children appears also as a hemodynamic disease of the liver, correlated with cirrhosis severity. Surgical technique for PV reconstruction during LT was adapted accordingly. Liver Transplantation 23 1440-1450 2017 AASLD.

Etiology and mode of presentation of chronic liver diseases in India: A multi centric study.

There is a paucity of health policy relevant data for chronic liver disease from India, impeding formulation of an interventional strategy to address the issue. A prospective, multicentric study to delineate the etiology and clinical profile of chronic liver disease in India is reported here. A centrally coordinated and monitored web-based data repository was developed (Feb, 2010 to Jan, 2013) and analyzed. Eleven hospitals from different parts of India participated. Data were uploaded into a web based proforma and monitored by a single centre according to a standardized protocol. 1.28% (n = 266621) of all patients (n = 20701383) attending the eleven participating hospitals of India had liver disease. 65807 (24·68%) were diagnosed for the first time (new cases). Of these, 13014 (19·77%, median age 43 years, 73% males) cases of chronic liver disease were finally analyzed. 33.9% presented with decompensated cirrhosis. Alcoholism (34·3% of 4413) was the commonest cause of cirrhosis while Hepatitis B (33·3%) was predominant cause of chronic liver disease in general and non-cirrhotic chronic liver disease (40·8% out of 8163). There was significant interregional differences (hepatitis C in North, hepatitis B in East and South, alcohol in North-east, Non-alcoholic Fatty Liver Disease in West) in the predominant cause of chronic liver disease. Hepatitis B (46·8% of 438 cases) was the commonest cause of hepatocellular Cancer.11·7% had diabetes. Observations of our study will help guide a contextually relevant liver care policy for India and could serve as a framework for similar endeavor in other developing countries as well.

Diagnostic Value of Glypican3, Heat Shock Protein 70 and Glutamine Synthetase in Hepatocellular Carcinoma Arising in Cirrhotic and Non-Cirrhotic Livers

Histopathological distinction of various nodular lesions in liver with sufficient sensitivity and specificity is a challenge even in an expert set up. The panel of immunohistochemical markers composed of glutamine synthetase (GS), Glypican3 (GPC3) and heat shock protein 70 (HSP70) was recommended by the International Consensus Group for Hepatocellular Neoplasia group for the differentiation of high grade dysplastic nodule and early hepatocellular carcinoma (HCC). The panel has been extensively validated in the western population. This study aims to test this panel on Indian population on resected, explanted and autopsy cirrhotic and non-cirrhotic liver specimens of HCC. This study was conducted on 39 such liver specimens (12 cirrhotic, 12 pre-cirrhotic and 11 non-cirrhotic, non-fibrotic livers), including 35 cases of HCC over a period of 12 years. Immunohistochemistry was performed with antibodies against GS, GPC3 and HSP70 on the sections containing both malignant and dysplastic nodules. The diagnostic yield depended upon the nature of background liver pathology and was found to be high for only those HCCs arising in cirrhotic background, when positivity of any two markers was taken to be in favor of HCC (sensitivity-58.33%; specificity-100%). GS had a sensitivity and Negative predictive value of 100% for HCCs arising in cirrhotic livers. Strong positivity for GS is a highly sensitive marker for HCC in a cirrhotic background regardless of the differentiation of the tumor in Indian population. This may be due to preferential activation of Wnt pathway in Indian patients with cirrhosis. The sensitivity of the panel was too low for detecting HCCs arising in non-cirrhotic livers, even in the pre-cirrhotic chronically inflamed livers, even though the specificity was high. GPC3 and HSP70 appear to be useful as individual markers for HCCs arising in non-cirrhotic livers.

Spontaneous Rupture of a Fibrolamellar Variant Hepatocellular Carcinoma in an Innocent Liver

Introduction: Fibrolamellar hepatocellular carcinoma (FL-HCC) is a unique variant of hepatocellular carcinoma. The majority of cases present with nonspecific symptoms like vague abdominal pain, weight loss and fatigue. Ruptured FL-HCC occurs rarely and mortality in the acute phase is very high. We report a rare case of a ruptured FL-HCC successfully managed with transarterial embolization for hemostasis. Case: A 37-year-old male previously in good health presented with a severe, sharp epigastric pain that started one hour prior to the presentation. He denied trauma, fever, nausea, vomiting, diarrhea. Tenderness in the epigastrium was noted, with no palpable masses, guarding or rigidity. His blood pressure and pulse were 159/105 mmHg and 105 beats/min. Platelets and coagulation parameters were within normal limits, while hemoglobin of 12 g/dL, wbc of 11 k/mL and elevated transaminases (AST of 214 IU/L, ALT of 138 IU/L, ALP 98 IU/L, T.Billi 1.5 mg/dL) were noted. Abdominal CT scan with contrast revealed an 8 cm lobulated mass with central hypodensity in the left hepatic lobe with perilesional blood and free pelvic fluid, indicating tumor rupture (figure 1). CT angiography showed tumor neovascularization from a branch of the left hepatic artery which was embolized using transarterial gelfoam. Liver MRI (figure 2) and biopsy (figure 3) were consistent with fibrolamellar variant hepatocellular carcinoma. Hepatitis serologies and AFP levels were unremarkable. After 4 days, as the symptoms resolved and the lab results were stable, patient was discharged and underwent a left hepatectomy 3 weeks later. Discussion: FL-HCC occurs commonly in the left lobe of a young and non-cirrhotic liver. Typically cross sectional imaging reveals a lobulated mass with well-defined margins, areas of hypervascularity and a central calcified scar. Histologic appearance is characterized by eosinophilic polygonal shaped cells separated by lamellar fibrosis. Surgical resection is the treatment of choice with better outcome when compared to conventional HCC. Disease recurrence after complete surgical resection is however high in the first 5 years. Tumors > 5 cm in size are at high risk for rupture with high mortality and recurrence rates secondary to significant spillage of tumor. While an emergency hepatectomy is preferred in unstable patients, those that are hemodynamically stable can undergo radiologic transarterial embolization for hemostasis followed by staged hepatectomy.Figure: Axial section of CT Abdomen and Pelvis with IV contrast: (on the left) An 8 x 7 cm heterogeneous lobulated mass with central hypo density in the left hepatic lobe with arrow pointing perilesional blood concerning for tumor rupture. (on the right) An arrow pointing hyperdensity fluid surrounding urinary bladder likely blood from tumor rupture.Figure: MRI of the abdomen with liver protocol post embolization showing a 7.7 x 6.6 cm well circumscribed expansile lesion in left lobe of liver with global hypo T2 signal intensity with areas of hyper T2 signal intensities as well as central stellate shaped scar of low T2 signal intensity.Figure: Histopathologic examination of haematoxylin and eosin stained liver biopsy specimen (on the left) under high power magnification showing tumor cells with hepatocellular appearance: large polygonal cells, ample eosinophilic cytoplasm, with focal bile pigment, marked nuclear atypia, and Intranuclear pseudoinclusions. (on the right) under low power magnification showing tumor cells separated by hyaline collagen.

Hepatocellular Carcinoma in Non-Cirrhotic Liver Patients: Is it Possible?

Cirrhosis is a major risk factor for developing HCC (hepatocellular carcinoma), therefore cirrhotic patients should still be included in surveillance HCC programs even after sustained virological response (SVR). To date, there are few studies on non-cirrhotic patients and development of HCC. A 64-year-old man with history of end-stage renal disease, diabetes mellitus type 2 and hepatitis C, treated unsuccessfully with interferon and DAA (direct acting antiviral) agent, was being evaluated for kidney transplant, and found to have new liver lesions. Laboratory work up was significant for alpha-fetoprotein (AFP) 6.2, hepatitis C ribonucleic acid (HCV RNA) 475,514 IU/mL with normal transaminases, bilirubin, albumin and INR. Computed tomography (CT) of the abdomen showed two hypodense liver lesions, more apparent on delayed imaging. First one was an ill-defined 2.4 x 1.7 cm lesion at the segment VIII, with irregular nodular enhancement. Second one was a 1.7 x 1.7cm lesion on segment IV/VII. These radiological findings were suspicious for hepatocellular carcinoma. Positron Emission Tomography (PET) CT scan showed increased uptake in the liver lesion on segment VIII. Of note, an ultrasonography of the abdomen done 6 months prior to the CT as part of surveillance for HCC was negative for hepatic lesions. Fibroscan (Transient Elastography) showed mild disease, with a reading of 6.1 kPa. Liver biopsy was consistent with mild portal inflammation/mild fibrosis and moderately differentiated hepatocellular carcinoma. Patient was referred to Interventional Radiology for Y90 radio embolization. This case illustrates that even patients with no bridging fibrosis or cirrhosis can develop HCC. This stands out from our current understanding of HCC development and its relationship with final stages of liver fibrosis. The risk of recurrent HCC after being cured is 0.33 percent per year. Therefore, patients with SVR and established cirrhosis are included in the screening HCC program. Age 65 or more, and presence of cirrhosis at the time of SVR were associated with annual HCC risk of 0.9% and 1.55%, respectively. With our case report, we raise the question that non-cirrhotic patients with chronic hepatitis C infection who were treated with DAA should be part of the screening program of HCC. Their inclusion in the program warrants prospective future trials and detailed information regarding timing and imaging studies.Figure

Vitamin A status and its determinants in patients with cystic fibrosis.

Routine administration of vitamin A, recommended in CF patients, can help to prevent its deficiency. However, high vitamin A supplementation may lead to its excessive level and possible toxicity. Therefore, the aim of the present study was to assess the status of vitamin A and the determinants of its body resources in CF patients. In 196 CF patients aged from 4 months to 47 years, the following parameters were analysed: nutritional status (standardized body weight and height, serum albumin concentration) and clinical expression of disease (lung function - spirometry; biochemical markers of liver function - ALT, AST, GGT; respiratory tract colonization by Pseudomonas aeruginosa; diabetes; cirrhosis, non-cirrhotic liver disease; exocrine pancreatic function - fecal elastase-1 concentration; blood clotting - INR and vitamin A supplementation). Median vitamin A concentration in the study group was 383.0 ng/ml (1st-3rd quartile: 316.5-457.0). Vitamin A deficiency was found in 32 (16.3%) subjects studied. Vitamin A concentrations above the reference range were observed only in 3 (1.5%) CF patients. CF patients with vitamin A deficiency were significantly older and had lower values of FEV1 compared to CF subjects with normal vitamin A status. Moreover, vitamin A deficiency occurred more frequently in CF patients with diabetes, Pseudomonas aeruginosa colo- nization, worse lung function and in those without vitamin A supplementation. However, in multiple linear regression analyses, none of the independent variables was documented to be important for predicting vita- min A status. Vitamin A body resources in CF patients are mostly normal. Moreover, there are no good de- terminants of vitamin A status in these patients. Further studies targeted at exploring potential toxicity and deficiencies of vitamin A in CF patients are needed.

Temporal trends, clinical patterns and outcomes of NAFLD-related HCC in patients undergoing liver resection over a 20-year period.

Non-alcoholic fatty liver disease (NAFLD) is an increasing cause of hepatocellular carcinoma (HCC) worldwide. NAFLD-HCC often occurs in noncirrhotic liver raising important surveillance issues. To determine the temporal trends for prevalence, clinical characteristics and outcomes of NAFLD-HCC in patients undergoing liver resection. Consecutive patients with histologically confirmed HCC who underwent liver resection over a 20-year period (1995-2014). NAFLD was diagnosed based on past or present exposure to obesity or diabetes without other causes of chronic liver disease. A total of 323 HCC patients were included, 12% with NAFLD. From 1995-1999 to 2010-2014, the prevalence of NAFLD-HCC increased from 2.6% to 19.5%, respectively, P=.003, and followed the temporal trends in the prevalence of metabolic risk factors (28% vs 52%, P=.017), while hepatitis C-HCC decreased (from 43.6% to 19.5%, P=.003). NAFLD-HCC occurred more frequently in the absence of bridging fibrosis/cirrhosis (63% of cases, P<.001 compared to other aetiologies). Within the NAFLD group, tumour characteristics were similar between F0-F2 and F3-F4 patients, except for a higher proportion of single nodules (95% vs 54%, P<.01). A total of 53% patients had tumour recurrence and 40% died. NAFLD-HCC had similar time to recurrence and survival as HCCs of other aetiologies. Satellite nodules, tumour size, microvascular invasion and male sex but not the aetiology were independently associated with recurrence. Non-alcoholic fatty liver disease increased substantially over the past 20years among resectable HCCs. It is now the leading cause of HCC occuring without/or with only minimal fibrosis. NAFLD patients are older, with larger tumours while survival and recurrence rates are as severe as in other aetiologies.

Factores epidemiológicos relacionados al Carcinoma Hepatocelular.

Introducción: En la etiología del carcinoma hepatocelular (CHC) se ha relacionado a la Hepatitis B, C, diabetes mellitus tipo 2, obesidad y el sexo masculino. Aunque en la mitad de los casos se presente en un hígado no cirrótico. El objetivo de este trabajo fue es demostrar las características epidemiológicas asociadas al CHC en un centro único de estudio. Métodos: El presente estudio observacional retrospectivo fue realizado en pacientes masculinos diagnosticados histológicamente de CHC entre 2009-2013 en el Hospital de Solca-Guayaquil. Se midieron variables, edad, procedencia, ocupación, etiología, presencia de cirrosis y tipo de tratamiento realizado. Se analizaron los casos comparando los pacientes con y sin cirrosis. Resultados: Ingresaron al estudio 67 casos de pacientes con CHC con una media de 64.6 años. El 43.3 % de los pacientes pertenecientes a la provincia del Guayas, 32.8 % obreros de profesión. En el grupo cirrosis (n=38) el Virus de la Hepatitis B con 13 casos (19.4 %) en el grupo sin cirrosis (n=29) fueron 6 casos (9 %) P=0.22. DM + Obesidad en el grupo de cirrosis 1 caso (2.6 %) en el grupo sin cirrosis 14 casos (20.9 %) Odds Ratio 0.029 (IC 95% 0.0035 a 0.24) P&lt;0.001. Hábito alcohólico 5 casos (7.5 % ) en el grupo de Cirrosis y 0 casos en el grupo sin Cirrosis RR 1.88 (IC 95 % 1.49 a 2.37) P=0.04. Conclusión: La hepatitis B es el principal factor prevalente en pacientes con CHC, seguido de la presencia de Diabetes Mellitus tipo juntamente con besidad. En pacientes sin cirrosis la primera explicación de la etiología de CHC es la Diabetes Mellitus tipo 2 juntamente con la presencia de un IMC &gt;30 kg/m2. El hábito alcohólico es un factor de riesgo estadísticamente significativo en el grupo de pacientes que presentó cirrosis hepática y CHC.

A small population of liver endothelial cells undergoes endothelial-to-mesenchymal transition in response to chronic liver injury.

Rising evidence points to endothelial-to-mesenchymal transition (EndMT) as a significant source of the mesenchymal cell population in fibrotic diseases. In this context, we hypothesized that liver endothelial cells undergo EndMT during fibrosis progression. Cirrhosis in mice was induced by CCl4 A transgenic mouse expressing a red fluorescent protein reporter under the control of Tie2 promoter (Tie2-tdTomato) was used to trace the acquisition of EndMT. Sinusoidal vascular connectivity was evaluated by intravital microscopy and high-resolution three-dimensional confocal microscopy. A modest but significant fraction of liver endothelial cells from both cirrhotic patients and CCl4-treated Tie2-tdTomato mice acquired an EndMT phenotype characterized by the coexpression of CD31 and α-smooth muscle actin, compared with noncirrhotic livers. Bone morphogenetic protein-7 (BMP-7) inhibited the acquisition of EndMT induced by transforming growth factor-β1 (TGF-β1) treatment in cultured primary mouse liver endothelial cells from control mice. EndMT was also reduced significantly in vivo in cirrhotic Tie2-tdTomato mice treated intraperitoneally with BMP-7 compared with untreated mice (1.9 ± 0.2 vs. 3.8 ± 0.3%, respectively; P < 0.05). The decrease of EndMT in cirrhotic livers correlated with a significant decrease in liver fibrosis (P < 0.05) and an improvement in the vascular disorganization rate (P < 0.05). We demonstrated the acquisition of the EndMT phenotype by a subpopulation of endothelial cells from cirrhotic livers in both animal models and patients. BMP-7 treatment decreases the occurrence of the EndMT phenotype and has a positive impact on the severity of disease by reducing fibrosis and sinusoidal vascular disorganization.NEW & NOTEWORTHY A subpopulation of liver endothelial cells from cirrhotic patients and mice with liver fibrosis undergoes endothelial-to-mesenchymal transition. Liver endothelial cells from healthy mice could transition into a mesenchymal phenotype in culture in response to TGF-β1 treatment. Fibrotic livers treated chronically with BMP-7 showed lower EndMT acquisition, reduced fibrosis, and improved vascular organization.

Rate of hepatitis C viral clearance by human livers in human patients: Liver transplantation modeling primary infection and implications for studying entry inhibition.

To better understand the dynamics of early hepatitis C virus (HCV) infection, we determined how rapidly non-cirrhotic HCV-uninfected liver allografts clear HCV from the circulation of cirrhotic HCV-infected patients at the time of transplantation but before administration of immunosuppression. Specifically, we characterized serum HCV kinetics during the first 90 min of reperfusion for 19 chronically HCV-infected patients transplanted with an HCV-uninfected liver by measuring serum viral load immediately prior to reperfusion (t = 0) and then every 15 min for a total of 90 min (t = 90). Immunosuppression was withheld until all samples were taken to better model primary infection. During this period, rates of viral clearance varied more than 20-fold with a median rate constant of 0.0357 1/min, range 0.0089–0.2169; half-life (minutes) median 19.4, range 3.2–77.8. The majority of viral clearance occurred within the first 60 min. The amount of blood transfused during this 90-min period (a potential confounding variable of this human liver transplant model of primary infection) accounted for 53% and 59% of k (r = 0.53, p = 0.05) and half-life (r = 0.59, p = 0.03) variability, respectively. No other clinical variables tested (age, allograft weight, and degree of reperfusion injury as assessed by peak postoperative ALT or AST) accounted for the remaining variability (p>0.05). Conclusion: In a human liver transplant model of primary infection, HCV rapidly clears the bloodstream. With approximately 90% of clearance occurring in the first 90 minutes of reperfusion, studies of HCV entry inhibition could utilize rate of clearance during this early period as an outcome measure.

A cost-effectiveness analysis of the diagnostic strategies for differentiating focal nodular hyperplasia from hepatocellular adenoma.

We evaluated the cost-effectiveness of a gadoxetic acid-enhanced MRI (EOB-MRI) strategy compared with conventional MRI strategy and biopsy to differentiate focal nodular hyperplasia (FNH) from hepatocellular adenoma (HCA). A decision tree model was constructed to compare the cost-effectiveness of EOB-MRI, conventional MRI with extracellular contrast agents, and biopsy as the initial diagnostic modality in patients with incidentally detected focal liver lesions suspected of being FNH or HCA. We analysed the cost and effectiveness, i.e. probability of successful diagnosis of each strategy. Costs were based on utilisation rates and Medicare reimbursements in the USA and South Korea. In the base case analysis of our decision tree model, the effectiveness of the three strategies was similar. The cost of the EOB-MRI strategy ($1283 in USA, $813 in South Korea) was lowest compared with the biopsy strategy ($1725 in USA, $847 in South Korea) and the conventional MRI strategy ($1750 in USA, $962 in South Korea). One-way, two-way and probabilistic sensitivity analysis showed unchanged results over an acceptable range. EOB-MRI strategy is the most cost-effective strategy for differentiating FNH from HCA in patients with incidentally detected focal liver lesions in a non-cirrhotic liver. • The effectiveness of the three strategies was similar. • The cost of the EOB-MRI strategy was lowest. • EOB-MRI strategy is the most cost-effective for differentiating FNH from HCA.

Abstract 624: Characterization of PD-L1 expression and the immune cell microenvironment in hepatocellular carcinoma (HCC) and non-cirrhotic liver tissue adjacent to HCC

Abstract Still-emerging evidence suggests that HCC is at least moderately responsive to therapeutic agents that target tumor immune suppression pathways, specifically the programmed death 1/programmed death ligand 1 (PD-L1) pathway. Prior studies raise the possibility that PD-L1 is a prognostic biomarker in HCC, where its expression levels have been reported to correlate with tumor aggressiveness and recurrence following surgical resection. The purpose of this study was to evaluate the expression of PD-L1 in non-cirrhotic liver tissue adjacent to HCC and HCC itself in 68 procured tissue samples, as well as elucidate the immune cell composition of the HCC tumor microenvironment. All cases showed the typical morphology of HCC and were classified as low- to high-grade trabecular, pseudoglandular, or solid with the common cytoplasmic features. Immunohistochemical (IHC) staining for PD-L1 in tumor-free liver (TFL) revealed PD-L1 staining in sinusoidal lining cells (macrophages [Mϕ] and endothelial cells [EC], as confirmed by double labeling for CD68 and CD31), although there was considerable heterogeneity in the extent of PD-L1 staining. More specifically, in TFL, some but not all CD68+ Mϕ were also PD-L1+, whereas most CD31+ EC were also PD-L1+. Furthermore, we evaluated PD-L1 staining qualitatively and semiquantitatively in HCC. Only few tumor cells displayed membranous PD-L1 staining, and 62/68 of the cases (91.2%) were categorized as PD-L1- vs 6/68 (8.8%) PD-L1+ (≥1% and &amp;lt;25% = PD-L1+); instead, PD-L1 expression within the tumor microenvironment predominantly emanated from immune cell infiltrates (as confirmed by PD-L1/pan-cytokeratin double labeling). Then we assessed the immune milieu in HCC tissue specimens using quantitative IHC. We found considerable interspecimen variation in the number of CD68+ Mϕ, CD8+ cytotoxic T lymphocytes, and FoxP3+ regulatory T lymphocytes. Comparing the number of immune cells in different tumor compartments showed that the prevalence of CD68+ cells (p=.0005), CD8+ cells (p=.0004), and FoxP3+ cells (p=.05) was significantly higher within the invasive margin vs the center of the tumor. Quantitative analysis of the immune cell content also showed that there was no correlation between T lymphocyte infiltration (CD8+/FoxP3+) and Mϕ infiltration in any compartment. Taken together, our findings confirm the intertumoral heterogeneity of the immune cell microenvironment in HCC; however, future studies are needed to correlate these findings to the clinical setting with immunooncologic treatments. Citation Format: Christian Ihling, Sienna Yoast, Yue Zhang, Bartholomew Naughton, Miriam Urban, P. Alexander Rolfe, Eveline Frick-Krieger, Isabelle Dussault. Characterization of PD-L1 expression and the immune cell microenvironment in hepatocellular carcinoma (HCC) and non-cirrhotic liver tissue adjacent to HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 624. doi:10.1158/1538-7445.AM2017-624

The occurrence of hepatocellular carcinoma in different risk stratifications of clinically noncirrhotic nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) may be a cause of hepatocellular carcinoma (HCC), but its high prevalence challenges current surveillance strategies. We aimed to evaluate HCC incidences in different risk stratifications for noncirrhotic NAFLD. Using Taiwan's National Health Insurance Research Database, we located 31,571 patients with NAFLD between the years 1998 and 2012. After excluding other causes of hepatitis, underlying cirrhosis or malignancy, 18,080 patients were recruited for final analysis. Cumulative incidences of HCC were analyzed after adjusting for competing mortality. With a median follow-up duration of 6.32 years in the study cohort, the 10-year cumulative incidence of HCC was 2.73% [95% confidence interval (CI): 1.69-3.76%]. Hepatoprotectant was used as a surrogate marker for elevated serum alanine transaminase (ALT). After adjusting for age, gender, hypertension, hypercholesterolemia, diabetes mellitus, gout, statin use, metformin use and aspirin use, elevated ALT was independently associated with an increased HCC risk [hazard ratio (HR) 6.80, 95% CI: 3.00-15.42; p < 0.001]. Multivariate stratified analysis verified this association in all subgroups (HR> 1.0). Moreover, increased age (HR 1.08 per year, 95% CI: 1.05-1.11) and statin use (HR 0.29, 95% CI: 0.12-0.68) were also identified as independent risk factors. The 10-year cumulative HCC incidence was highest in older (age >55 years) patients with ALT elevation (12.41%, 95% CI: 5.99-18.83%), but lowest in younger patients without ALT elevation (0.36%, 95% CI: 0-1.08%). The incidence of HCC was relatively low in patients with clinically noncirrhotic NAFLD, however, HCC risk was significantly increased in older patients experiencing an elevated serum ALT.

MiRNA Signature of Hepatocellular Carcinoma Vascularization: How the Controls Can Influence the Signature.

miRNA deregulation and vascular modifications constitute promising predictors in the study of hepatocellular carcinoma (HCC). In the literature, the relative miRNA abundance in HCC is usually determined using as control non-matched tumoral tissue, healthy liver, or cirrhotic liver. However, a common standard RNA control for the normalization toward the tissue gene expression was not settled yet. To assess the differences existing in the quantitative miRNA gene expression in HCC on tissue according to two different liver controls. A wide array of miRNAs was analyzed on 22 HCCs arisen in cirrhotic and non-cirrhotic livers by means of microfluidic cards. Control samples included total RNA extracted from healthy and cirrhotic livers. Immunohistochemistry for CD34 and Nestin was performed to assess the pattern of intratumoral vascular modifications. Six miRNAs were deregulated in HCCs using either controls: miR-532, miR-34a, miR-93, miR-149#, miR-7f-2#, and miR-30a-5p. Notably, the miRNA expression changed significantly between HCCs arisen in cirrhotic and non-cirrhotic livers, according to the control used for normalization. Different miRNA profiles were found also in HCCs with different vascular patterns, according to the control used for normalization. Our data confirm that the choice of the methodology, and particularly the control used for normalization, represents the main concern in miRNA evaluation, particularly in a heterogeneous model such as liver pathology. Still we observed the deregulation of some common miRNAs as promising in HCC cancerogenesis and progression. A standardized control will be a crucial achievement to compare miRNA expression among different laboratories.

Noncirrhotic hepatocellular carcinoma: etiology and occult hepatitis B virus infection in a hepatitis B virus-endemic area.

Background:Although hepatocellular carcinoma (HCC) usually develops in cirrhotic livers, a minority of cases occur in noncirrhotic livers (NCLs). We investigated etiology, clinicopathological features, and occult hepatitis B virus (HBV) infection (OBI) in patients with NCL HCC in an HBV-endemic area.Methods:A total of 710 patients who underwent resection or transplantation for HCC at the National Cancer Center (NCC), Korea, were enrolled. HCC and fibrosis stage were diagnosed pathologically.Results:A total of 178 patients (25%) did not have cirrhosis (NCL group). The main cause of HCC was HBV infection (77.2%), followed by cryptogenic disease (11.0%). The prevalence of NCL was 19.2%, 32.5%, 50.0%, and 48.7% among patients with HBV, hepatitis C virus (HCV), alcoholic, and cryptogenic disease, respectively (p < 0.05); corresponding nonfibrosis rates were 8.1%, 0%, 19.0%, and 24.3%, respectively. The NCL group was significantly older, with a larger tumor size, smaller tumor number, lower tumor stage, and more frequent non-HBV etiology. Among non-HBV HCC cases, 130 (80.2%) had antibodies against HBV core (HBc) and 55 (38.5%) had OBI. OBI-positive rates of 0%, 31.8%, and 52.6% were detected among HCV, alcoholic, and cryptogenic HCC cases, respectively. OBI did not correlate with advanced fibrosis. The NCL and liver cirrhosis (LC) groups did not differ in median overall survival.Conclusion:Regardless of etiology, a significant number of HCC patients, including half of nonviral cases, did not have LC. Half of cryptogenic HCC cases had OBI. This study promotes an understanding of fibrosis and OBI among patients with HCC in an HBV-endemic area.

Molecular landscape of fibrolamellar carcinoma (FLC): Beyond the DNAJB1-PRKACA chimera.

e15668 Background: FLC is a rare cancer arising in non-cirrhotic liver of young adults. A functional chimeric transcript resulting from the in-frame fusion of DNAJB1 with PRKACA genes on chromosome 19 is thought to be universal and unique in FLC. This chimera has possible role in pathogenesis, yet with no determined prognostic or therapeutic value. Methods: Archival FFPE samples from FLC patients who prospectively consented to an IRB-approved protocol from 2014 onward were analyzed using the MSK-IMPACT platform, a hybridization capture based next generation sequencing assay for targeted deep sequencing of all exons and selected introns of &gt; 400 key cancer genes. Tumor and matched normal libraries were sequenced on an Illumina HiSeq 2500. Sequencing output was processed using a custom analysis pipeline. Archer targeted RNAseq was performed in select cases. Demographic, treatment, and outcome data was prospectively collected. Fisher’s exact tests were performed to look for associations between clinical characteristics and genetic alterations. Results: 18 patients were identified. Median age at diagnosis was 18 years and 61% were women. 17 (94%) underwent upfront surgery, and median number of therapies was 5. The DNAJB1-PRKACA fusion transcript was detected in 100% of cases. 7 samples were negative via MSK-IMPACT, (2 false-negatives, and 5 due to an older panel not including the fusion transcript) and were confirmed positive on Archer targeted RNAseq. The second most common genetic mutation was TERT, detected in 4 patients, whereas CIC, NOTCH1, ERBB3, EPHAS, MCL1, PNRC1, NCOR, INPP4B, ARID1A, CHECK1, NKX3-1, TET1, RAD52, DIS3, SF3B1, MDM2, MED12, MPL, KRAS, PAK7, BCOR, HGF, ROS1, MYCL1, CTNNB1, and YAP1 were detected in 1 patient each. 3-year overall survival (OS) was 98%, median OS was not reached. Conclusions: This limited cohort of patients with FLC exhibited universally the DNABJ1-PRKACA chimera, and demonstrated a favorable outcome compared to a 3-year OS approaching 70% previously reported by the Fibrolamellar Consortium. The fusion transcript did not imply a prognostic value considering its expression among all patients with FLC. Other drivers or the lack of may be responsible for the improved outcome in this small cohort