Comparison and correlation of fibrosis stage assessment by collagen proportionate area (CPA) and the ELF panel in patients with chronic liver disease.

BACKGROUND: Fibrosis progression is the common consequence of most chronic liver diseases. AIMS: To evaluate the performance of Collagen Proportionate Area (CPA) and ELF using Ishak's score in patients with chronic liver diseases. METHODS: Retrospective analysis of medical data from patients on whom a liver biopsy was performed as part of the diagnostic assessment. CPA was calculated by using digital image analysis and then compared with Ishak and ELF scores. RESULTS: 143 patients (84 men (59%); mean age 48.8 ±â€¯12.8 years) were evaluated. Patients were mainly affected by viral hepatitis (92 HCV and 8 HBV). CPA and ELF values increased with worsening Ishak stage (P < 0.001) and their median values were significantly different among Ishak stages (P < 0.001). There was a significant correlation between CPA and ELF (r = 0.5). In AUROC analysis, CPA and ELF had similar diagnostic accuracy in identifying cirrhosis, but CPA had higher diagnostic accuracy than ELF in identifying significant or absent fibrosis. High ELF scores were observed in non-cirrhotic patients who suffered non-liver related deaths. CONCLUSIONS: This study demonstrated that CPA and ELF values successfully identified patients with advanced fibrosis or cirrhosis, thus confirming the role of ELF as a clinical method for non-invasive assessment of fibrosis stage in chronic hepatitis.

Transaminase abnormalities and adaptations of the liver lobule manifest at specific cut-offs of steatosis.

There is little documented evidence suggesting that liver fat is responsible for liver injury in the absence of other disease processes. We investigated the relationships between liver fat, aminotransferases and hepatic architecture in liver biopsies with simple steatosis. We identified 136 biopsies with simple steatosis from the Royal Free Hospital Archives with both clinical data and sufficient material. Digital image analysis was employed to measure fat proportionate area (mFPA). Hepatocyte area (HA) and lobule radius (LR) were also measured. There were significant increases in ALT (p < 0.001) and AST (p = 0.013) with increased fat content and evidence to suggest both 5% and 20% mFPA as a cut-off for raised ALT. In liver with increased fat content there were significant increases in HA (p < 0.001). LR also increased as mFPA increased to 10% (p < 0.001), at which point the lobule ceased to expand further and was counterbalanced with a decrease in the number of hepatocytes per lobule (p = 0.029). Consequently there are mechanisms of adaption in the liver architecture to accommodate the accumulation of fat and these are accompanied by significant increases in transaminases. These results support the generally accepted cut-off of 5% fat for steatosis and indicate 20% as a threshold of more severe liver injury.

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.

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.

The relationship between transient elastography and histological collagen proportionate area for assessing fibrosis in chronic viral hepatitis.

BACKGROUND: Collagen proportionate area (CPA) has a better correlation with hepatic venous pressure gradient (HVPG) than with Ishak stage. Liver stiffness measurement (LSM) is proposed as non invasive marker of portal hypertension/disease progression. Our aim was to compare LSM and CPA with Ishak staging in chronic viral hepatitis, and HVPG in HCV hepatitis after transplantation. METHODS: One hundred and sixty-nine consecutive patients with chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infections pre/post liver transplantation (LT), had a liver biopsy combined with LSM (transient elastography), CPA (biopsies stained with Sirius Red and evaluated by digital image analysis and expressed as CPA) and HVPG (measured contemporaneously with transjugular biopsies in LT HCV patients). RESULTS: LSM was dependent on CPA in HBV (r (2) = 0.61, p < 0.0001), HCV (r (2) = 0.59, p < 0.0001) and LT groups (r (2) = 0.64, p < 0.0001). In all three groups, CPA and Ishak were predictors of LSM, but multivariately CPA was better related to LSM (HBV: r (2) = 0.61, p < 0.0001; HCV: r (2) = 0.59, p < 0.0001; post-LT: r (2) = 0.68, p < 0.0001) than Ishak stage. In the LT group, multiple regression analysis including HVPG, LSM, aspartate aminotransferase to platelet ratio index (APRI) and Ishak stage/grade, showed that only CPA was related to HVPG (r (2) = 0.41, p = 0.01), both for HVPG ≥6 mmHg (OR 1.34, 95 % CI 1.14-1.58; p < 0.0001) or ≥10 mmHg (OR 1.25, 95 % CI 1.06-1.47; p = 0.007). CONCLUSION: CPA was related to LSM in HBV or HCV hepatitis pre/post-LT. CPA was better related to LSM than Ishak stage. In the LT HCV group, CPA was better related to HVPG than Ishak stage/grade, LSM or APRI. CPA may represent a better comparative histological index for LSM, rather than histological stages.

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.

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.

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.

Predicting severity and clinical course of acute rejection after liver transplantation using blood eosinophil count.

Acute cellular rejection remains an important source of morbidity after liver transplantation, particularly if rejection is moderate or severe, as this usually is treated. Currently liver biopsies are seldom performed, so diagnostic noninvasive markers would be useful. We evaluated 690 consecutive first liver transplant patients to assess whether peripheral eosinophilia could predict moderate-severe rejection and its course. A protocol biopsy was performed 6 ± 2.5 days after transplant. A second biopsy was taken 6.1 ± 2 days after the first in 487 patients to assess histological improvement. Liver function tests, peripheral eosinophil count and changes between first and second biopsy, were evaluated using logistic regression. Histological rejection was present in 532 patients (77.1%), with moderate (30.6%) and severe rejection (3.9%). Peripheral eosinophil count was strongly associated with moderate-severe rejection (OR = 2.15; P = 0.007), although the area under ROC curve (AUROC) was 0.58. On second biopsy, rejection improved in 119 (24.4%) patients. The delta in eosinophil count between the first and second biopsies was the only independent predictor of histological improvement (OR = 3.12; P = 0.001), irrespective of whether bolus steroids were used (OR = 2.77; P = 0.004); AUROC was 0.72. Peripheral eosinophilia is not sufficiently predictive of moderate-severe histological rejection. However the changes in eosinophil count over time can accurately predict the histological resolution of rejection.

Liver collagen proportionate area predicts decompensation in patients with recurrent hepatitis C virus cirrhosis after liver transplantation.

BACKGROUND AND AIMS: Current histological scoring systems do not subclassify cirrhosis. Computer-assisted digital image analysis (DIA) of Sirius Red-stained sections measures fibrosis morphologically producing a fibrosis ratio (collagen proportionate area [CPA]). CPA could have prognostic value within a disease stage, such as cirrhosis. The aim of the present study was to evaluate CPA in patients with recurrent hepatitis C virus (HCV) allograft cirrhosis and assess its relationship with hepatic venous pressure gradient (HVPG). METHODS: In 121 consecutively-transplanted HCV patients with HVPG, measured contemporaneously with transjugular liver biopsies, 65 had Ishak stage 5 or 6 disease (43 with HVPG measurement). Biopsies were stained with Sirius Red for DIA, and the collagen content was expressed as a CPA. In three cases, a tissue for Sirius Red staining was not obtained, and the patients were excluded. RESULTS: Sixty-two patients were analyzed. The median HVPG was 8 mmHg (interquartile range [IQR]: 5-10). Portal hypertension (HVPG ≥ 6 < 10 mmHg) was present in 30 (69.8%), and HVPG ≥ 10 mmHg in 13 (30.2%). The median CPA was 16% (IQR 10.75-23.25). Median Child-Pugh score and HVPG were not significantly different between Ishak fibrosis stage 5 or 6, whereas CPA was statistically different: 13% in stage 5 (IQR 8.3-12.4) versus 23% in stage 6 (IQR 17-33.7, P < 0.001). In the multivariate analysis, CPA was the only variable significantly associated with clinically-significant portal hypertension (HVPG ≥ 10 mmHg, odds ratio: 1.085, confidence interval: 1.004-1.172, P = 0.040). A CPA of 14% was the best cut-off value for clinically-significant portal hypertension (CSPH) and liver decompensation, which occurred in 24 patients. Event-free survival was significantly shorter in patients with CSPH or with a CPA value ≥ 14%, or with a combination of both. CONCLUSION: In Ishak stages 5 and 6, CPA correlated with HVPG, but had a wider range of values, suggesting a greater sensitivity for distinguishing "early" from "late" severe fibrosis/cirrhosis. CPA was a unique, independent predictor of HVPG ≥ 10 mmHg. CPA can be used to subclassify cirrhosis and for prognostic stratification.

Computer-assisted image analysis of liver collagen: relationship to Ishak scoring and hepatic venous pressure gradient.

UNLABELLED: Histopathological scoring of disease stage uses descriptive categories without measuring the amount of fibrosis. Collagen, the major component of fibrous tissue, can be quantified by computer-assisted digital image analysis (DIA) using histological sections. We determined relationships between DIA, Ishak stage, and hepatic venous pressure gradient (HVPG) reflecting severity of fibrosis. One hundred fifteen patients with hepatitis C virus (HCV) who had undergone transplantation had 250 consecutive transjugular liver biopsies combined with HVPG (median length, 22 mm; median total portal tracts, 12), evaluated using the Ishak system and stained with Sirus red for DIA. Liver collagen was expressed as collagen proportionate area (CPA). Median CPA was 6% (0.2-45), correlating with Ishak stage (stage 6 range, 13%-45%), and with HVPG (r = 0.62; P < 0.001). Median CPA was 4.1% when HVPG was less than 6 mm Hg and 13.8% when HVPG was 6 mm Hg or more (P < 0.0001) and 6% when HVPG was less than 10 mm Hg and 17.3% when HVPG was 10 mm Hg or higher (P < 0.0001). Only CPA, not Ishak stage/grade, was independently associated by logistic regression, with HVPG of 6 mm Hg or more [odds ratio, 1.206; 95% confidence interval (CI), 1.094-1.331; P < 0.001], or HVPG of 10 mm Hg or more (odds ratio, 1.105; 95% CI, 1.026-1.191; P = 0.009). CPA increased by 50% (3.6%) compared with 20% in HVPG (1 mm Hg) in 38 patients with repeated biopsies. CONCLUSION: CPA assessed by DIA correlated with Ishak stage scores and HVPG measured contemporaneously. CPA was a better histological correlate with HVPG than Ishak stage, had a greater numerical change when HVPG was low, and resulted in further quantitation of fibrosis in cirrhosis.

Cure of Acanthamoeba cerebral abscess in a liver transplant patient.

Acanthamoeba-related cerebral abscess and encephalitis are rare but usually fatal, being caused by free-living amoebic infections usually occurring in immunocompromised patients. In patients receiving transplants, a literature review showed that the infection is universally fatal. The diagnosis is often missed despite appropriate investigations including lumbar puncture, computerized tomography, and brain biopsy. We present the first reported liver transplant patient with Acanthamoeba cerebral abscess. The diagnosis was made in brain tissue removed at decompressive frontal lobectomy. He was successfully treated with a 3-month course of co-trimoxazole and rifampicin. There was no recurrence of the disease after 11 years of follow-up.

Autoimmune hepatitis associated with Kikuchi-Fujimoto's disease.

We describe a 20-year-old woman with autoimmune hepatitis (AIH) with cirrhosis who developed Kikuchi-Fujimoto's disease (KFD) and de novo minor features of systemic lupus erythematosus (SLE). This is the first report of a patient with histologically confirmed AIH developing KFD (histiocytic necrotizing lymphadenitis). One previous case described KFD after AIH (diagnosed clinically but without biopsy). KFD is a rare condition of unknown aetiology, first described in 1972, characterized by fever and cervical adenopathy and has a self-limiting course. KFD is associated with SLE, and SLE in turn can be associated with abnormal liver function tests, which in a minority of cases may be due to AIH. The association of AIH, KFD, and SLE in our patient suggests an autoimmune pathogenesis of KFD.

Efficacy of loco-regional ablation therapy of HCC in a population of liver transplanted patients.

BACKGROUND AND OBJECTIVE: Loco-ablation therapy (LAT) has become standard treatment for patients with HCC who are candidates for liver transplantation (LT). The aim of this study was: to evaluate if LAT was able to induce complete necrosis of tumour mass; to determine the tumour recurrence rate after LT and factors associated with recurrence. PATIENTS: The percentage and the distribution of necrosis in 116 HCC nodules of 61 patients with (26 patients) and without (35 patients) previous types of LAT were examined in explanted livers. RESULTS: Total necrosis was found only in 7% of treated nodules, and 42% of these showed absence of necrosis. The amount of necrosis was significantly related to the gross appearance of HCC: a single nodule with smaller adjacent satellite nodules showed a higher percentage of necrosis. No relation was found between the amount of necrosis and the type of LAT. Recurrence was observed in 11.5% and 8.5% of patients with and without previous LAT, respectively (P = ns). CONCLUSIONS: LAT very rarely induces complete necrosis; the amount of necrosis seems to depend on the growth pattern of the tumour and not on the type of previous LAT; the tumour size, measured at explant, is the only variable significantly related to recurrence.

Hepatic venous pressure gradient to assess fibrosis and its progression after liver transplantation for HCV cirrhosis.

Progression of fibrosis following recurrent hepatitis C virus (HCV) infection is frequent after liver transplantation (LT). Histology remains the gold standard to assess fibrosis, but the value of hepatic venous pressure gradient (HVPG) is being explored. We evaluated patients with recurrent HCV infection after LT to assess whether HVPG correlates with liver histology, particularly fibrosis. A total of 90 consecutive patients underwent 170 HVPG measurements concomitant with transjugular liver biopsy (TJB), with 31.5 (range, 6-156) months of follow up. Median biopsy length was 22 mm and total portal tract count was 12 (complete 6, partial 6). Median HVPG was 4 mmHg: 38% of patients > or =6 mmHg (portal hypertension, PHT), 13% > or =10 mmHg. HVPG correlated with Ishak stage (r = 0.73, P < 0.001) for mild (0-3) and severe fibrosis (4-6), and grade score (r = 0.47, P < 0.001), but neither correlated with interval from LT nor biopsy length. HVPG was > or =10 mmHg in 15 patients: 12 had stage 5 or 6, and 3 severe portal expansion. HVPG was repeated in 49, between 7 and 60 months with weak correlation to fibrosis score (r = 0.30, P = 0.045). A total of 12 patients with HVPG > or =6 mmHg had fibrosis score < or =3, while 8 patients had normal HVPG but fibrosis stage > or =4. These discrepancies were mostly associated with specific histological features such as perisinusoidal fibrosis rather than errors in measuring HVPG. In 29 with HVPG <6 mmHg at 1 yr, none decompensated compared to 4 of 13 (31%) with PHT. In conclusion, HVPG correlates with fibrosis and its progression, due to recurrent HCV infection, assessed in TJB.

Sustained, spontaneous disappearance of serum HCV-RNA under immunosuppression after liver transplantation for HCV cirrhosis.

Immunosuppression is a main determinant for the increased Hepatitis C Virus (HCV) replication after liver transplantation and the accelerated course of recurrent HCV liver disease. We present two patients both with diabetes, renal dysfunction with proteinuria converted to sirolimus therapy, who cleared serum HCV RNA without antiviral treatment. This is a potentially important observation that should stimulate study into factors that may help viral clearance from blood.

The histopathology of regeneration in massive hepatic necrosis.

Massive hepatic necrosis (MHN) is a condition that offers an opportunity to study the remarkable ability of the liver to become repopulated with hepatocytes. A maximal regenerative stimulus is expected in cases of MHN (Roskams et al. APMIS Suppl 1991;23:32-39). Sequential chronological observations, after a severe degree of liver cell loss, permit study of the human equivalent of the situation in animal models in which circulating and bone marrow-derived stem and liver progenitor cells are recruited to the hepatopoietic process. To date, the bone marrow and circulating precursors have not been identified morphologically in human material. We present data that suggest that the circulating liver progenitor could have a lymphoblastoid morphological appearance. Similar cells are seen among the cellular infiltrate of MHN. We have found that combinations of markers, such as CD117/CD133 positive CD45/tryptase negative are useful to isolate these cells using cell-sorting technology. This may facilitate their expansion in vitro and the development of their use for therapeutic purposes. In MHN, the residual portal tracts and ductular reaction with the associated lymphoid infiltrate (some of which are probably liver cell progenitors derived from the circulation) constitute the fundamental regenerative community unit in which hepatopoiesis takes place. Defining the hepatopoietic process is hindered by the lack of morphological transitional forms in the period between the progenitors within the circulation and when they assume recognizable hepatocytic form as "metaplastic" hepatocytes associated with the ductular reaction. By achieving a better comprehension of these processes of liver cell restoration, we will be better placed to accelerate liver recovery in MHN, for example by the administration of granulocyte colony stimulating factor (GCSF). Thus, more patients will be able to restore their own livers and avoid liver transplantation.