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.

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

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

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.

Acute-on-chronic liver failure 2018: a need for (urgent) liver biopsy?

INTRODUCTION: 'Acute-on-Chronic-Liver Failure (ACLF)' entered hepatology practice by the end of the 20th century. Although we lack precise and universally agreed definitions, acute decompensation of chronic liver disease with jaundice and deranged clotting, multi-organ failure and high, short-term mortality are hallmarks of the syndrome. Timely recognition and and treatment, including urgent liver transplantation, may save the life of certain patients. The diagnosis and management are mostly based on clinical features, but some have suggested to incorporate histopathology (liver biopsy). This may add to the differentiation between acute and chronic disease, primary and concomitant etiologies, and identify prognostic determinants. Areas covered: A review of the literature on ACLF and the outcome of the discussions at a topical international meeting on specific histopathological aspects of diagnosis and prognosis of the syndrome. Expert commentary: There is a lack of standardized descriptions of histopathological features and there is limited prospective experience with the role of pathology of ACLF. It is important for the clinical hepatologist to understand the potential and limitations of (transjugular) liver biopsy in ACLF and for the pathologist to help address the clinical question and recognise the histopathological features that help to characterize ACLF, both in terms of diagnosis and prognosis.

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.

Decellularized human liver as a natural 3D-scaffold for liver bioengineering and transplantation.

Liver synthetic and metabolic function can only be optimised by the growth of cells within a supportive liver matrix. This can be achieved by the utilisation of decellularised human liver tissue. Here we demonstrate complete decellularization of whole human liver and lobes to form an extracellular matrix scaffold with a preserved architecture. Decellularized human liver cubic scaffolds were repopulated for up to 21 days using human cell lines hepatic stellate cells (LX2), hepatocellular carcinoma (Sk-Hep-1) and hepatoblastoma (HepG2), with excellent viability, motility and proliferation and remodelling of the extracellular matrix. Biocompatibility was demonstrated by either omental or subcutaneous xenotransplantation of liver scaffold cubes (5 × 5 × 5 mm) into immune competent mice resulting in absent foreign body responses. We demonstrate decellularization of human liver and repopulation with derived human liver cells. This is a key advance in bioartificial liver development.

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.

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.

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.

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.

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.

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.

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.

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.

Spectroscopic photoacoustic imaging of lipid-rich plaques in the human aorta in the 740 to 1400 nm wavelength range.

Spectroscopic photoacoustic imaging has the potential to discriminate between normal and lipid-rich atheromatous areas of arterial tissue by exploiting the differences in the absorption spectra of lipids and normal arterial tissue in the 740 to 1400 nm wavelength range. Identification of regions of high lipid concentration would be useful to identify plaques that are likely to rupture (vulnerable plaques). To demonstrate the feasibility of visualizing lipid-rich plaques, samples of human aortas were imaged in forward mode, at wavelengths of 970 and 1210 nm. It was shown that the structure of the arterial wall and the boundaries of lipid-rich plaques obtained from the photoacoustic images were in good agreement with histology. The presence of lipids was also confirmed by comparing the photoacoustic spectra (740 to 1400 nm) obtained in a region within the plaque to the spectral signature of lipids. Furthermore, a lipid-rich plaque was successfully imaged while illuminating the sample through 2.8 mm of blood demonstrating the possibility of implementing the photoacoustic technique in vivo.

Fibrosis distribution in explanted cirrhotic livers.

AIMS: Little information is available regarding the distribution of fibrosis within cirrhotic livers. We measured collagen in cirrhotic explants to determine if fibrosis differs (i) between left (L) and right (R) lobes, and (ii) between different aetiologies. METHODS AND RESULTS: Ten cases each of common aetiologies of cirrhosis were studied: alcoholic liver disease (ALD), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), autoimmune hepatitis (AIH), hepatitis C virus (HCV) and hepatitis B virus (HBV). A total of 120 tissue blocks (one block each from L and R lobes) were studied. Collagen was measured as collagen proportionate area (CPA), i.e. the proportion of the tissue sections stained by picro-Sirius red. L and R lobes contained similar amounts of fibrosis (r = 0.788; P < 0.0001) with good agreement between L and R lobes (Bland-Altman analysis, R lobe bias = 1.35%). Median CPA across all aetiologies (R plus L lobes) was 21.5%, (L = 8-40%, R = 10-47%). There was more fibrosis in ALD (30%, 15-47%) than PBC (23.5%, 16-34%) and PSC (22.5%, 8-33%), which in turn showed more than AIH (18.5%, 10-40%), HCV (17%, 13-31%) and HBV (16.5%, 8-30%). CONCLUSIONS: At the time of transplantation cirrhotic livers have different ranges of collagen proportionate area, according to aetiology. R lobe fibrosis corresponds with L lobe fibrosis. The range of fibrosis within each aetiological group could be useful for prognostic subclassification.

Beyond "cirrhosis": a proposal from the International Liver Pathology Study Group.

"Cirrhosis" is a morphologic term that has been used for almost 200 years to denote the end stage of a variety of chronic liver diseases. The term implies a condition with adverse prognosis due to the well-known complications of portal hypertension, hepatocellular carcinoma, and liver failure. However, recent advances in the diagnosis and treatment of chronic liver diseases have changed the natural history of cirrhosis significantly. This consensus document by the International Liver Pathology Study Group challenges the usefulness of the word cirrhosis in modern medicine and suggests that this is an appropriate time to consider discontinuing the use of this term. The role of pathologists should evolve to the diagnosis of advanced stage of chronic liver disease, with emphasis on etiology, grade of activity, features suggestive of progression or regression, presence of other diseases, and risk factors for malignancy, within the perspective of an integrated clinicopathologic assessment.

Serum ferritin is a discriminant marker for both fibrosis and inflammation in histologically proven non-alcoholic fatty liver disease patients.

INTRODUCTION: Differentiation between steatosis and non-alcoholic steatohepatitis (NASH) in non-alcoholic fatty liver disease (NAFLD) is important as NASH progress to cirrhosis. No specific laboratory/imaging technique exists either to diagnose NASH or to select patients for liver biopsy. PATIENTS AND METHODS: We evaluated serum ferritin and the features of metabolic syndrome with respect to histological inflammation and/or fibrosis in NAFLD patients. The Kleiner scoring system was used to classify NAFLD in consecutive liver biopsies. One hundred and eleven patients: median age 52.6, 64 males, obesity 62, diabetes mellitus (DM) 58, arterial hypertension 26 and hyperlipidaemia 40%. RESULTS: Histologically, 40.7 had fatty liver, 30.6% had borderline NASH, 28.7% had NASH and 11% had cirrhosis. Multivariate regression showed that diabetes, serum ferritin concentrations, body mass index (BMI) and AST were independently associated with NASH: together, the areas under the receiver operating characteristic (AUROC) was 0.91 (95% confidence interval 0.86-0.96); fibrosis was associated with ferritin concentrations and BMI: AUROC 0.87, portal inflammation with ferritin and DM: AUROC 0.82, while lobular inflammation was associated with BMI, DM and ferritin: AUROC 0.85. CONCLUSION: Serum ferritin concentrations and BMI are strongly associated with fibrosis, portal and lobular inflammation in NAFLD patients. Both ferritin and BMI are potential discriminant markers to select patients for liver biopsy and are associated with inflammation and fibrosis.