Inhibition of TLR7 and TLR9 Reduces Human Cholangiocarcinoma Cell Proliferation and Tumor Development.

BACKGROUND: Toll-like receptors (TLRs) are key players in innate immunity and modulation of TLR signaling has been demonstrated to profoundly affect proliferation and growth in different types of cancer. However, the role of TLRs in human intrahepatic cholangiocarcinoma (ICC) pathogenesis remains largely unexplored. AIMS: We set out to determine if TLRs play any role in ICCs which could potentially make them useful treatment targets. METHODS: Tissue microarrays containing samples from 9 human ICCs and normal livers were examined immunohistochemically for TLR4, TLR7, and TLR9 expression. Proliferation of human ICC cell line HuCCT1 was measured by MTS assay following treatment with CpG-ODN (TLR9 agonist), imiquimod (TLR7 agonist), chloroquine (TLR7 and TLR9 inhibitor) and IRS-954 (TLR7 and TLR9 antagonist). The in vivo effects of CQ and IRS-954 on tumor development were also examined in a NOD-SCID mouse xenograft model of human ICC. RESULTS: TLR4 was expressed in all normal human bile duct epithelium but absent in the majority (60%) of ICCs. TLR7 and TLR9 were expressed in 80% of human ICCs. However, TLR7 was absent in all cases of normal human bile duct epithelium and only one was TLR9 positive. HuCCT1 cell proliferation in vitro significantly increased following IMQ or CpG-ODN treatment (P < 0.03 and P < 0.002, respectively) but decreased with CQ (P < 0.02). In the mouse xenograft model there was significant reduction in size of tumors from CQ and IRS-954 treated mice compared to untreated controls. CONCLUSION: TLR7 and TLR9 should be further explored for their potential as actionable targets in the treatment of ICC.

Histological sub-classification of cirrhosis using collagen proportionate area in patients with chronic hepatitis C.

Collagen proportionate area (CPA, %) is used to quantify liver fibrosis. Here, we assessed CPA performance to sub-classify cirrhosis. CPA was measured in explanted livers from consecutively transplanted patients for hepatitis C virus-related cirrhosis. Model for end-stage liver disease (MELD), Child-Pugh score and decompensating events (ascites, variceal bleeding, non-obstructive jaundice and encephalopathy) were recorded at the time of liver transplant. Of the 154 patients, 24%, 12%, 35%, 24% and 5% had zero, one, two, three and four previous decompensating events. Patients with decompensation had significantly higher CPA than those without (25.1 ± 8.4 vs 15.8 ± 5.5, P < .001). Decompensation was independently associated with CPA, bilirubin and albumin or with CPA and MELD score. CPA did not differ between patients with one, two, three or four decompensating events (22.2 ± 6.3 vs 26.6 ± 8.9 vs 24.5 ± 7.7 vs 24.4 ± 10.9, P = .242). Overall, CPA correlates with the clinical severity of cirrhosis until the advent of decompensation but not with subsequent decompensating events.

Histology of portal vascular changes associated with idiopathic non-cirrhotic portal hypertension: nomenclature and definition.

Idiopathic non-cirrhotic portal hypertension (INCPH) is a rare vascular liver disease that has attracted new interest in recent years. It is characterised by clinical signs of portal hypertension in the absence of cirrhosis or severe fibrosis and any known cause of portal hypertension. As much uncertainty exists about INCPH pathophysiology, and no definite diagnostic tests are available, liver biopsy is an essential tool for achieving a definite diagnosis. Unfortunately, the histological diagnosis of INCPH is not always straightforward, as the characteristic lesions are unevenly distributed, vary greatly in their severity, are often very subtle, and are not all necessarily present in a single case. Furthermore, specifically for the characteristic portal vessel changes observed in INCPH, the terminology and definition are ambiguous, which adds complexity to the already complex clinicopathological scenario. An international study group of liver pathologists and hepatologists pursued a consensus on nomenclature for the portal vascular lesions of INCPH. Such standardisation may assist pathologists in the recognition of such lesions, and will possibly facilitate further advancement in this field.

Role of aetiology in the progression, regression, and parenchymal remodelling of liver disease: implications for liver biopsy interpretation.

Clinicopathological concepts on acute and chronic liver disease have evolved rapidly during the last few years, with advances in general and specific treatment options and improved patient outcomes. The old paradigm of 'irreversibility' of cirrhosis had been challenged in major ways, and the validity of the usage of the term 'cirrhosis' has come into question. This paper addresses aetiology-based clinicopathological concepts and features that may deserve attention because they may determine disease outcome and, specifically, patterns of regression and remodelling. A variety of therapeutic interventions may influence remaining disease features after elimination of damaging agents (virus, alcohol, etc.), and determine the final clinical outcome including the risk of hepatocellular carcinoma (HCC). New concepts create new responsibilities and opportunities for the pathologist to contribute to the understanding of liver pathology and communicate this with clinical colleagues and researchers.

Antibodies to human serum amyloid P component eliminate visceral amyloid deposits.

Accumulation of amyloid fibrils in the viscera and connective tissues causes systemic amyloidosis, which is responsible for about one in a thousand deaths in developed countries. Localized amyloid can also have serious consequences; for example, cerebral amyloid angiopathy is an important cause of haemorrhagic stroke. The clinical presentations of amyloidosis are extremely diverse and the diagnosis is rarely made before significant organ damage is present. There is therefore a major unmet need for therapy that safely promotes the clearance of established amyloid deposits. Over 20 different amyloid fibril proteins are responsible for different forms of clinically significant amyloidosis and treatments that substantially reduce the abundance of the respective amyloid fibril precursor proteins can arrest amyloid accumulation. Unfortunately, control of fibril-protein production is not possible in some forms of amyloidosis and in others it is often slow and hazardous. There is no therapy that directly targets amyloid deposits for enhanced clearance. However, all amyloid deposits contain the normal, non-fibrillar plasma glycoprotein, serum amyloid P component (SAP). Here we show that administration of anti-human-SAP antibodies to mice with amyloid deposits containing human SAP triggers a potent, complement-dependent, macrophage-derived giant cell reaction that swiftly removes massive visceral amyloid deposits without adverse effects. Anti-SAP-antibody treatment is clinically feasible because circulating human SAP can be depleted in patients by the bis-d-proline compound CPHPC, thereby enabling injected anti-SAP antibodies to reach residual SAP in the amyloid deposits. The unprecedented capacity of this novel combined therapy to eliminate amyloid deposits should be applicable to all forms of systemic and local amyloidosis.

Equilibrium contrast-enhanced CT imaging to evaluate hepatic fibrosis: initial validation by comparison with histopathologic sampling.

PURPOSE: To prospectively evaluate hepatic extracellular volume (ECV) fraction measurement at equilibrium computed tomographic (CT) imaging compared with both fibrosis quantified with histologic analysis and the enhanced liver fibrosis panel (ELF) in a cohort of patients with chronic hepatitis. MATERIALS AND METHODS: This prospective study was approved by the regional ethics committee. All patients gave fully informed written consent. Forty patients with a clinical indication for liver biopsy were prospectively recruited for liver ECV quantitation at equilibrium CT imaging. Biopsy samples underwent digital image analysis and assessment of collagen content expressed as the collagen-proportionate area (CPA). Spearman correlation was used to evaluate association between ECV, ELF, and CPA. Multiple regression analysis was used to test prediction of CPA by a model that combined ECV and ELF. ECV, ELF score, and CPA were compared with Ishak stage by using the Kruskal-Wallis test. RESULTS: Complete ECV, ELF, and CPA were available in 33 patients. Liver ECV, CPA, and ELF had a median of 0.26 (interquartile range [IQR], 0.24-0.29), 5.0 (IQR, 3.0-15.0), and 9.71 (IQR, 8.14-10.92), respectively. Hepatic ECV demonstrated good association with CPA (r = 0.64; P < .001) and ELF score (r = 0.38; P < .027), with no significant difference in strength of correlation (P = .177). The regression model that combined ELF and ECV achieved good prediction of CPA (R(2) = 0.67; P < .001). Significant variation in ECV and ELF was seen between fibrosis stage groups. CONCLUSION: Hepatic ECV measured with equilibrium CT imaging is associated with biopsy-derived CPA and serum ELF-validated markers of liver fibrosis. This suggests that equilibrium CT imaging can quantify diffuse fibrosis in chronic liver disease.

Mechanism of cell death in acute-on-chronic liver failure: a clinico-pathologic-biomarker study.

BACKGROUND & AIMS: Mortality of patients who develop acute-on-chronic liver failure (ACLF) is unacceptably high but the predominant mode of cell death is unknown. The aim of this study was to evaluate whether plasma levels of caspase-cleaved cytokeratin M30 (marker of apoptosis) and uncleaved cytokeratin M65 (marker of total cell death) are altered in ACLF patients and relate this to liver histology. METHODS: Twenty-seven patients with acute decompensation of liver disease were divided into two groups: no-ACLF (n = 11) or ACLF (n-16). Healthy controls (n = 8) and acute liver failure (ALF) patients (n = 10) were also enrolled. Cell death was assessed in plasma using an ELISA kit (M30 and M65). Simultaneous biopsy samples were analysed for M30 and caspase-3 staining. RESULTS: Plasma M30 value was significantly elevated in ACLF patients compared with healthy volunteers (P = 0.0001), it was also significantly higher in ACLF patients compared with no-ACLF patients (P = 0.002). M65 levels were higher in ALF compared with ACLF patients (P = 0.002) but the apoptotic index defined by M30/M65 ratio was significantly higher in ACLF patients. Patients with extra-hepatic failure had higher M30 levels compared with patients without organ failure (P = 0.03). M30 staining in liver was more marked in the patients with ACLF and was observed in all the patients that died. CONCLUSIONS: The results of this study suggest that hepatocyte apoptosis is the predominant mode of cell death in ACLF, which can be identified in the peripheral blood. Further studies are required to validate our findings and to determine whether M30 can be used as a biomarker of apoptosis or as a target for therapy.

Effect of toll-like receptor 7 and 9 targeted therapy to prevent the development of hepatocellular carcinoma.

BACKGROUND & AIMS: Chronic liver disease is a predisposing factor for development of hepatocellular carcinoma (HCC). Toll-like receptors play a crucial role in immunity against microbial pathogens and recent evidence suggests that they may also be important in pathogenesis of chronic liver disease. The purpose of this study was to determine whether TLR7 and TLR9 are potential targets for prevention and progression of HCC. METHODS: Tissue microarrays containing liver samples from patients with cirrhosis, viral hepatitis and HCC were examined for expression of TLR7 and TLR9 and the data obtained was validated in liver specimens from the hospital archives. Proliferation of human HCC cell lines was studied following stimulation of TLR7 and TLR9 using agonists (imiquimod and CpG-ODN respectively) and inhibition with a specific antagonist (IRS-954) or chloroquine. The effect of these interventions was confirmed in a xenograft model and diethylnitrosamine (DEN)/nitrosomorpholine (NMOR)-induced model of HCC. RESULTS: TLR7 and TLR9 expression was up-regulated in human HCC tissue. Proliferation of HuH7 cells in vitro increased significantly in response to stimulation of TLR7. TLR7 and TLR9 inhibition using IRS-954 or chloroquine significantly reduced HuH7 cell proliferation in vitro and inhibited tumour growth in the mouse xenograft model. HCC development in the DEN/NMOR rat model was also significantly inhibited by chloroquine (P < 0.001). CONCLUSION: The data suggest that inhibiting TLR7 and TLR9 with IRS-954 or chloroquine could potentially be used as a novel therapeutic approach for preventing HCC development and/or progression in susceptible patients.

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.

Collagen proportionate area is superior to other histological methods for sub-classifying cirrhosis and determining prognosis.

BACKGROUND & AIMS: One-year survival in cirrhosis ranges from 1 to 57% depending on the clinical stage. Accurate sub-classification has important prognostic implications but there is no stage beyond cirrhosis using current qualitative histological systems. We compared the performance of all histological semi-quantitative and quantitative methods specifically developed for sub-classifying cirrhosis that have been described to date, with collagen proportionate area (CPA), to evaluate how well they distinguish patients with and without hepatic clinical decompensation at presentation, and in predicting future decompensating events. METHODS: We included consecutive patients with a histological diagnosis of cirrhosis that had a suitable liver biopsy between 2003 and 2007. We used semi-quantitative histological scoring systems proposed by Laennec, Kumar, and Nagula. We also measured quantitatively nodule size, septal width and fibrous tissue expressed in CPA. RESULTS: Sixty-nine patients, mean age 52.3±11years, mean MELD 11.8±5.8, median follow-up 56months. Main aetiologies were alcohol (38%) and hepatitis C (27.5%). Twenty-four patients (34.8%) had had a previous episode of clinical decompensation. Amongst the 45 patients who were compensated, 11 (24%) decompensated on follow-up. In Cox regression, amongst all histological parameters, CPA was the only variable independently associated with clinical decompensation up to the time of biopsy, with an odds ratio that ranged from 1.245 to 1.292. Furthermore, only CPA was significantly associated with future decompensation (OR: 1.117, 95% CI 1.020-1.223; p=0.017). CONCLUSIONS: Cirrhosis can be accurately sub-classified using quantification of fibrosis with CPA, and furthermore CPA is the only independent predictor of clinical decompensation amongst all other histological sub-classification systems described to date.

Inflammation-based scores do not predict post-transplant recurrence of hepatocellular carcinoma in patients within Milan criteria.

Increased preoperative inflammation scores, such as neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and inflammation-based index (IBI) have been related to post-transplant HCC recurrence. We evaluated the association between inflammation-based scores (NLR, PLR, IBI) and post-LT HCC recurrence as well as tumor necrosis after transarterial embolization. 150 consecutive patients who underwent transplantation for HCC within the Milan criteria between 1996 and 2010 were included; data regarding inflammatory markers, patient and tumor characteristics were analyzed. NLR, PLR, and IBI were not significantly associated with post-LT HCC recurrence or worse overall survival. Increased NLR and PLR were associated with complete tumor necrosis in the subset of patients who received preoperative transarterial embolization (P < 0.05). Cox regression analysis revealed that absence of neoadjuvant transarterial therapy (OR = 4.33, 95% CI = 1.28-14.64; P = 0.02) and no fulfillment of the Milan criteria in the explanted liver (OR = 3.34, 95% CI = 1.08-10.35; P = 0.04) were independently associated with post-LT HCC recurrence inflammation-based scores did not predict HCC recurrence post-LT in our group of patients. NLR and PLR were associated with better response to TAE, as this was recorded histologically in the explanted liver. Histological fulfillment of the Milan criteria and absence of neoadjuvant transarterial treatment were significantly associated with post-LT HCC recurrence.

HBsAg quantification for identification of liver disease in chronic hepatitis B virus carriers.

BACKGROUND & AIMS: Quantification of hepatitis B surface antigen (HBsAg) has been proposed as a useful diagnostic marker for clinical staging (identification of inactive carrier state) and prognosis of chronic hepatitis B virus (HBV) infection. The aim of this study was to investigate the correlation between HBsAg levels in serum and histological liver damage in patients with chronic infection. METHODS: HBsAg levels in serum (by Abbott Architect) were related to HBV DNA, ALT and histological score (n=160) and covalently closed circular DNA (cccDNA) (n=84). RESULTS: HBsAg levels correlated with cccDNA, serum HBV DNA, ALT and high inflammation scores (P<0.001). Among HBeAg-negative patients, an HBsAg level below 3.0 log10 IU/ml identified minimal liver damage (normal ALT and mild inflammation) with a predictive value of 92% (alone) or 96% (in combination with HBV DNA<4.0 log10 copies/ml), whereas an HBsAg level above 3.5 log10 IU/ml identified severe inflammation with a predictive value of 16% (alone) or 33% (in combination with HBV DNA>5.0 log10 copies/ml). CONCLUSIONS: HBsAg levels reflect clinical stage and liver disease, and a combined quantification of HBsAg and HBV DNA may improve clinical staging.

The use of guideline images to improve histological estimation of hepatic steatosis.

BACKGROUND & AIMS: Guideline images of specific fat proportionate area (FPA) percentages have recently been published to aid the histological assessment of liver steatosis as subjective estimates of FPA are usually overestimated. To assess, (i) the effect of guideline images on accuracy and concordance of estimated FPA (eFPA), (ii) experience of steatosis grading systems on eFPA, (iii) the effect of magnification on assessment of FPA (iv) and produce a range of guideline images at x4 objective magnification (OM). METHODS: Two circulations of sample images (C1 and C2) were circulated to UK liver external quality assessment histopathology scheme members who were asked to independently evaluate steatosis. Each circulation consisted of 15 images taken at both x20 and x4OM representing the full range of steatosis. C1 was distributed first, then C2 with guideline images of FPA 6 weeks later. RESULTS: Participants overestimated FPA in C1. In C2, there was significant improvement in accuracy (P < 0.001) of eFPA for sample images with mFPA >5%. Concordance of x4OM eFPA was substantial in both circulations (C1 K = 0.878, C2 K = 0.724). CONCLUSION: The tendency to overestimate eFPA has been corroborated and can be largely corrected with the use of guideline images (without needing digital image analysis). There is a need to redefine steatosis grades that are clinically significant and validated using an accurate quantification of steatosis.

Early tacrolimus exposure after liver transplantation: relationship with moderate/severe acute rejection and long-term outcome.

BACKGROUND & AIMS: Liver transplant (LT) patients might be overimmunosuppressed as recommendations for tacrolimus trough concentrations (TC) within 4-6 weeks after liver transplantation are set too high (10-15 ng/ml). Early tacrolimus exposure was evaluated in relation to acute rejection and long-term outcomes. METHODS: Four hundred and ninety-three consecutive LT patients receiving tacrolimus as primary immunosuppression (1995-2008) were analyzed. Acute rejection was diagnosed using protocol biopsies at day 6.1 ± 2.5. Median follow-up was 7.3 years (IQR 3.9-10.5). Early tacrolimus exposure (<15 days) was evaluated against moderate/severe acute rejection, chronic rejection, graft loss, chronic renal impairment and mortality using multiple logistic and Cox regression. RESULTS: Maintenance immunosuppression was tacrolimus monotherapy (48.1%), double therapy combination with antimetabolites or steroids (18%), or triple therapy combination with antimetabolites and steroids (33.9%). Histological grade of acute rejection was moderate in 157 cases (31.8%) and severe in 19 cases (3.9%). Tacrolimus TC>7 ng/ml on the day of protocol biopsy was associated with less moderate/severe rejection (23.8%) compared with<7 ng/ml (41.2%) (p = 0.004). Mean tacrolimus TC 7-10 ng/ml within 15 days after LT were associated with reduced risk of graft loss (RR = 0.46; p = 0.014) compared to TC 10-15 ng/ml. A peak TC>20 ng/ml within this period was independently related to higher mortality (RR = 1.67; p = 0.005), particularly due to cardiovascular events, infections and malignancy (RR = 2.15; p = 0.001). Early tacrolimus exposure did not influence chronic rejection (p = 0.58), or chronic renal impairment (p = 0.25). CONCLUSIONS: During the first 2 weeks after LT, tacrolimus TC between 7 and 10 ng/ml are safe in terms of acute rejection and are associated with longer graft survival.

Digital image analysis of collagen assessment of progression of fibrosis in recurrent HCV after liver transplantation.

BACKGROUND & AIMS: Histological assessment of fibrosis progression is currently performed by staging systems which are not continuous quantitative measurements. We aimed at assessing a quantitative measurement of fibrosis collagen proportionate area (CPA), to evaluate fibrosis progression and compare it to Ishak stage progression. METHODS: We studied a consecutive cohort of 155 patients with recurrent HCV hepatitis after liver transplantation (LT), who had liver biopsies at one year and were subsequently evaluated for progression of fibrosis using CPA and Ishak staging, and correlated with clinical decompensation. The upper quartile of distribution of fibrosis rates (difference in CPA or Ishak stage between paired biopsies) defined fast fibrosers. RESULTS: Patients had 610 biopsies and a median follow-up of 116 (18-252) months. Decompensation occurred in 29 (18%) patients. Median Ishak stage progression rate was 0.42 units/year: (24 (15%) fast fibrosers). Median CPA fibrosis progression rate was 0.71%/year (36 (23%) fast fibrosers). Clinical decompensation was independently associated by Cox regression only with CPA (p=0.007), with AUROCs of 0.81 (95% CI 0.71-0.91) compared to 0.68 (95% CI 0.56-0.81) for Ishak stage. Fast fibrosis defined by CPA progression was independently associated with histological de novo hepatitis (OR: 3.77), older donor age (OR: 1.03) and non-use/discontinuation of azathioprine before 1 year post-LT (OR: 3.85), whereas when defined by Ishak progression, fast fibrosers was only associated with histological de novo hepatitis. CONCLUSIONS: CPA fibrosis progression rate is a better predictor of clinical outcome than progression by Ishak stage. Histological de novo hepatitis, older donor age and non-use/discontinuation of azathioprine are associated with rapid fibrosis progression in recurrent HCV chronic hepatitis after liver transplantation.

A systematic review of microvascular invasion in hepatocellular carcinoma: diagnostic and prognostic variability.

Selected patients with hepatocellular carcinoma are candidates to receive potentially curative treatments, such as hepatic resection or liver transplantation, but nevertheless there is a high risk of tumor recurrence. Microvascular invasion is a histological feature of hepatocellular carcinoma related to aggressive biological behavior. We systematically reviewed 20 observational studies that addressed the prognostic impact of microvascular invasion, either after liver transplantation or resection. Outcomes were disease-free survival and overall survival. In liver transplantation, the presence of microvascular invasion shortened disease-free survival at 3 years (relative risk (RR)=3.41 [2.05-5.7]; five studies, n=651) and overall survival both at 3 years (RR=2.41 [1.72-3.37]; five studies, n=1,938) and 5 years (RR=2.29 [1.85-2.83]; six studies, n=2,003). After liver resection, microvascular invasion impacted disease-free survival at 3 and 5 years (RR=1.82 [1.61-2.07] and RR=1.51 [1.29-1.77]; four studies, n=1,501 for both comparisons). However inter/intraobserver variability in reporting and the lack of definition and grading of microvascular invasion has led to great heterogeneity in evaluating this histological feature in hepatocellular carcinoma. Thus, there is an urgent need to clarify this issue, because determining prognosis and response to therapy have become important in the current management of hepatocellular carcinoma. In this systematic review, we summarize the diagnostic and prognostic data concerning microvascular invasion in hepatocellular carcinoma and present a basis for consensus on its definition.

Sample size requirement for digital image analysis of collagen proportionate area in cirrhotic livers.

AIMS: The requirements for adequate cirrhotic liver biopsy size have not been established for quantitative fibrosis measurements (collagen proportionate area: CPA). We evaluated the CPA of virtual biopsies in cirrhosis to elucidate (i) the amount of tissue required to achieve reliable CPA measurements and (ii) the effect of aetiology on sample size requirements. METHOD AND RESULTS: A total of 120 cirrhotic tissue blocks (six aetiologies) were studied. A representative 100 mm(2) region was selected from each block and a reference CPA measured. Each image (n = 120) was divided into 100 × 1 mm(2) images; CPA was measured for each 1 mm(2) and virtual biopsies of different sizes were created from the 1 mm(2) components. For each virtual biopsy size the probability that the virtual biopsy CPA would be within 5% of the reference CPA was calculated. There were 441 000 virtual biopsies. Biopsy size versus probability plots indicated that, for 90% probability that the virtual biopsy CPA can be expected to be within 5% of the reference CPA, 22-28 mm(2) of analysable tissue is required depending on liver disease aetiology; and that a 75% probability level requires a biopsy with 12-15 mm(2) of analysable tissue. CONCLUSION: The sample size required for a given probability level depends on the aetiology of cirrhosis, and this should be taken into account when judging the reliability of cirrhotic liver biopsy CPA.

Transarterial embolization as neo-adjuvant therapy pretransplantation in patients with hepatocellular carcinoma.

BACKGROUND & AIMS: Neo-adjuvant transarterial therapies are commonly used for patients with HCC in the waiting list for liver transplantation (LT) to delay tumour progression, however, their effectiveness is not well-established. We studied the effect of pre-LT transarterial therapies on post-LT HCC recurrence, using the explanted liver histology to assess therapeutic efficacy and the predictors of response to these therapies. METHODS: We included 150 consecutive patients from our prospectively compiled database, listed for liver transplantation using the Milan criteria. Transarterial embolization without chemotherapeutic agents was the transarterial therapy used as standard of care. PVA particles were the embolizing agent of choice. RESULTS: Sixty-seven (45%) patients had TAE as bridging therapy to liver transplantation, of which 60 were transplanted after 2001. The majority of patients (36, 54%) had partial tumour necrosis after transarterial therapy, whereas 22 (33%) had complete tumour necrosis and 9 (13%) had no necrosis. HCC post-transplant recurrence was independently associated with no neo-adjuvant transarterial therapy (OR 5.395, 95% CI 1.289-22.577; P = 0.021) and the total radiological size of HCC nodules (OR 1.037, 95% CI 1.006-1.069; P = 0.020). CONCLUSIONS: Pre-transplant TAE with the more permanently occluding PVA particles significantly reduces post-transplant HCC recurrence in patients within the Milan criteria.

Hepatic steatosis estimated microscopically versus digital image analysis.

BACKGROUND & AIMS: Evaluate in liver biopsies: (i) interobserver agreement of estimates of fat proportionate area (eFPA) and steatosis grading, (ii) the relationship between steatosis grades and measured fat proportionate area (mFPA, digital image analysis), (iii) the accuracy of eFPA, (iv) to present images to aid standardization and accuracy of eFPA. METHODS: Twenty-one liver biopsies were selected from the Royal Free Hospital (RFH) histopathology archive to represent the full range of histopathological steatosis severity. As many non-overlapping fields of parenchyma as possible were photographed at ×20 objective magnification from the biopsies (n = 651). A total of 15 sample images were selected to represent the range of steatosis seen. Twelve hepatopathologists from 11 sites worldwide independently evaluated the sample images for steatosis grade [normal (none)/mild/moderate/severe], and eFPA (% area of liver parenchyma occupied by fat). RESULTS: The hepatopathologists had good linear correlation between eFPA and mFPA for sample images (r = 0.924, P < .001) and excellent concordance (kappa = 0.91, P < 0.001). Interobserver concordance of steatosis grade showed 'substantial agreement' (kappa = 0.64). There was significant difference between eFPA and mFPA in the sample images for mild, moderate and severe steatosis (P = 0.024, P < 0.001, P < 0.001 respectively): the observers consistently over-estimated the eFPA. CONCLUSION: Hepatopathologists showed 'excellent' interobserver agreement in eFPA and 'substantial' agreement in assigning steatosis grade (precision was high). However, compared with mFPA, eFPA was inaccurate. eFPA systematically exceeds mFPA; generally the overestimation increases with severity of steatosis. Considering that non-invasive technologies for estimating liver fat utilize histopathology as reference, such assessments would benefit from quantitative validation of visually estimated microscopic liver fat percentages.

Mucosal addressin cell adhesion molecule (MAdCAM-1) expression is upregulated in the cirrhotic liver and immunolocalises to the peribiliary plexus and lymphoid aggregates.

BACKGROUND: Enhanced cell expression of MAdCAM-1 is critical in tissue recruitment of lymphocytes in response to stimuli expressing the α4ß7 integrin. MAdCAM-1 is well characterized in gut mucosa with emerging evidence of hepatic expression. AIMS: (i) Compare quantitative/semi-quantitatively MAdCAM-1 expression in relation to early and advanced liver diseases (ii) Define the fine structure of vascular plexuses/lymphatics in the portal tract on which MAdCAM-1 is expressed. METHODS: Using alkaline phosphatase anti-alkaline phosphatase methodology on paraffin embedded tissue sections (n=28) from cirrhotic individuals who underwent orthotopic liver transplant, we evaluated MAdCAM-1 expression and compared with pre-cirrhotic, fulminant hepatitis B, and non-cirrhotic portal hypertension tissue sections. The positive controls included normal colon tissue with negative controls without primary antibody and isotype-matched purified IgG. We developed a real time PCR to quantify levels of MAdCAM-1 mRNA in our samples. RESULTS: MAdCAM-1 was expressed in 27/28 of the cirrhotic sections, localized primarily to septal areas within (i) endothelium of the peribiliary vascular plexus (PBP) (ii) lymphoid aggregates, with absence from normal, non-cirrhotic portal hypertension and pre-cirrhotic livers. There was significant upregulation of MAdCAM-1 mRNA in cirrhosis (p<0.011), consistent with immunohistochemical analysis. CONCLUSIONS: MAdCAM-1 is up-regulated in cirrhosis with expression on PBP and lymphoid aggregates. MAdCAM-1 is likely to contribute to the localization and recruitment of α4ß7 lymphocytes during the pathogenesis of cirrhosis. MAdCAM-1 could be a useful marker of advanced liver disease. Further studies with respect to the expression of MAdCAM-1 in the presence of reversible and non-reversible stages of liver disease may be of merit.