Julien Chaigneau

FibroMeters: a family of blood tests for liver fibrosis

FibroMeters are blood tests for liver fibrosis with several specificities: two main diagnostic targets (fibrosis stage and area of fibrosis); adaptation to specific causes; and results confirmed by an expert system. Thus, FibroMeters comprise six different tests: one for staging and one for quantitation of liver fibrosis in each of the three main causes of chronic liver disease-chronic viral hepatitis, alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD). FibroMeters display a high overall diagnostic accuracy and are the only tests to correctly classify 100% of HCV patients without fibrosis or with cirrhosis. They have 90% predictive values in a higher proportion of patients than with other usual blood tests. A 90% correct classification is available in 100% of HCV patients with the following reliable diagnostic intervals: F0/1, F1/2, F2+/-1, F3+/-1. In real-life conditions, the reproducibility of FibroMeters is higher than that of liver biopsy or ultrasonographic elastometry. FibroMeters are robust tests with the most stable diagnostic performance across different centers. Optional tests are also available, such as a specific one for cirrhosis, which has a diagnostic accuracy of 93.0% (AUROC: 0.92) and a 100% positive predictive value for diagnosis of HCV cirrhosis. Determination by FibroMeters of the area of fibrosis - the only direct, non-invasive, quantitative measurement of liver fibrosis - are especially useful for following-up cirrhosis as it correlates well with clinical events. FibroMeters are also very accurate in HVB or HIV-HCV co-infected patients. The tests specific for ALD and NAFLD also have a high diagnostic accuracy (AUROCs: 0.96 and 0.94, respectively, for significant fibrosis).

Diagnosis of different liver fibrosis characteristics by blood tests in non-alcoholic fatty liver disease.

Aims: Our aim was to develop an accurate, non‐invasive, blood‐test‐based method for identifying the main characteristics of liver fibrosis in non‐alcoholic fatty liver disease (NAFLD).

Quantification of portal–bridging fibrosis area more accurately reflects fibrosis stage and liver stiffness than whole fibrosis or perisinusoidal fibrosis areas in chronic hepatitis C

Morphometry provides an objective evaluation of fibrosis in liver diseases. We developed an image analysis algorithm using automated thresholding and segmentation to separately quantify the areas and the fractal dimensions of portal–bridging fibrosis and perisinusoidal fibrosis in chronic hepatitis C liver biopsies. We studied 427 digitized liver biopsies and compared the automated measures of the different fibrosis compartments with (1) the Metavir F (fibrosis) and A (activity) histological scores, (2) the digitally assessed area of steatosis, and (3) the liver stiffness measured by elastography (Fibroscan). The perisinusoidal fibrosis area was higher than that of portal fibrosis in stages ≤F2; it reached its highest value in F2 stage and stabilized thereafter. The F3 stage was characterized by equal proportions of portal–bridging and perisinusoidal fibrosis, whereas portal–bridging area was predominant in cirrhosis. Measurement of portal–bridging fibrosis showed highly significantly different values between contiguous F stages; the ratio of portal–bridging fibrosis/perisinusoidal fibrosis displayed less overlap between Metavir stages than did the whole fibrosis area values. Fractal dimension showed that portal–bridging fibrosis tended to display a homogeneous surface-like spatial organization, whereas perisinusoidal fibrosis appeared more heterogeneous according to stage and curvilinear. The portal–bridging fibrosis area was low in cases with low Metavir activity and little steatosis, and became predominant with increasing activity and steatosis. Using stepwise multiple linear regression analysis, the liver stiffness was independently correlated to the portal–bridging fibrosis area (first step, P<0.001), the steatosis area (second step, P<0.001), and the Metavir A grade (third step, P=0.001), but not to the perisinusoidal fibrosis area. Automated quantification in a large cohort of chronic hepatitis C showed that perisinusoidal fibrosis progressively grew in early fibrosis stages but did not increase in septal or cirrhotic stages and that the portal–bridging fibrosis area appeared as a more accurate tool to assess fibrosis progression than the whole fibrosis area.

Steatosis degree, measured by morphometry, is linked to other liver lesions and metabolic syndrome components in patients with NAFLD.

Background and aim We carried out morphometric measurements of steatosis to evaluate relationships between steatosis degree and other liver lesions or metabolic syndrome components in nonalcoholic fatty liver disease (NAFLD). Patients and methods We developed an algorithm to measure steatosis area. Two hundred and fourteen patients with NAFLD were included in derivation (10) and validation (204) groups. Controls consisted of patients who were steatosis-free (12), patients with chronic hepatitis C (188), and patients with alcoholic chronic liver disease (94). Results Accuracy of steatosis area was considered as good or very good in at least 72% of cases by three pathologists. Steatosis areas were as follows: NAFLD=10.3±9.7%, virus=2.4±3.1%, alcohol=7.8±8.2% (P<0.0001). Steatosis area was closely related to steatosis grades in NAFLD (P<0.0001 for linear trend). Steatosis area increased from the fibrosis stage F0 to the fibrosis state F2, then decreased in the stages F3 and F4 (cirrhosis) (P<0.0001 for quadratic trend). Fibrosis was present in an average steatosis area of approximately 4% (defining significant steatosis) and in nonalcoholic steatohepatitis by approximately 8% (defining severe steatosis). Steatosis and fibrosis area increased symmetrically until approximately 10%, then steatosis area decreased to null as average fibrosis area reached 32%. Average fasting glycemia (approximately 92 mg/dl) or triglycerides and BMI plateaued before a steatosis area of approximately 4%, then increased thereafter. Significant steatosis was present in 61.3% of NAFLD versus 20.2% of viral hepatitis (P<0.0001) and in 58.7% of alcoholic liver diseases (P=0.674). Conclusions The average threshold of steatosis area is 4% for the development of fibrosis or metabolic syndrome components and 8% for nonalcoholic steatohepatitis. Steatosis area may contribute to defining the normal range and clinical course of metabolic components.

Treatment of liver fibrosis: Clinical aspects

The main objective of antifibrotic treatment is to avoid the complications of chronic liver disease where its cause cannot be treated. Three main therapeutic endpoints can be targeted: cause; comorbidity; and fibrosis. Antifibrotic treatment is any intervention independent of cause that is intended to modify the course and/or level of fibrosis through direct action on the mechanisms of fibrosis. Several modalities are here considered: reduction of fibrosis course; reversion of fibrosis; and reversion of cirrhosis. Semiquantitative histological staging and morphometry are complementary techniques for monitoring fibrosis. The degree of fibrosis should preferentially be estimated by fibrosis progression based on measurements taken at baseline and during treatment, rather than by raw static measurements. Surrogate markers are the only tools for assessing drug efficacy in clinical practice, and are especially useful for checking compliance and identifying poor or non-responders. We propose to define non-response as no decrease in fibrosis progression. The renin-angiotensin system is a good candidate target for antifibrotic treatment, and angiotensin-II type-1 receptor blockers, such as sartans, are probably effective. Clinical trials are currently ongoing using marketed drugs, while new multitargeted drugs are likely to emerge from basic research.

Fibrosis progression under maintenance interferon in hepatitis C is better detected by blood test than liver morphometry.

Summary.  We evaluated whether quantitative measurements of liver fibrosis with recently developed diagnostics outperform histological staging in detecting natural or interferon‐induced changes. We compared Metavir staging, morphometry (area and fractal dimension) and six blood tests in 157 patients with chronic hepatitis C from two trials testing maintenance interferon for 96 weeks. Paired liver biopsies and blood tests were available for 101 patients, and there was a significant improvement in Metavir activity and a significant increase in blood tests reflecting fibrosis quantity in patients treated with interferon when compared with controls – all per cent changes in histological fibrosis measures were significantly increased in F1 vs F2–4 stages only in the interferon group. For the whole population studied between weeks 0 and 96, there was significant progression only in the area of fibrosis (AOF) (P = 0.026), FibroMeter (P = 0.020) and CirrhoMeter (P = 0.003). With regards to dynamic reproducibility, agreement was good (ric ≥ 0.72) only for Metavir fibrosis score, FibroMeter and CirrhoMeter. The per cent change in AOF was significantly higher than that of fractal dimension (P = 0.003) or Metavir fibrosis score (P = 0.015). CirrhoMeter was the only blood test with a change significantly higher than that of AOF (P = 0.039). AOF and two blood tests, reflecting fibrosis quantity, have high sensitivity and/or reproducibility permitting the detection of a small progression in liver fibrosis over two years. A blood test reflecting fibrosis quantity is more sensitive and reproducible than morphometry. The study also shows that maintenance interferon does not improve fibrosis, whatever its stage.

1045 SEVERAL PATHOLOGICAL LESIONS INFLUENCE NON-INVASIVE TESTS OF LIVER FIBROSIS

Methods: Ten patients with unresectable multinodular unilobar HCC and and a target main lesion larger than 3 cm underwent CEUS before, 2 days after, and 30 to 40 days after combined single step therapy. The schedule consisted of percutaneous RFA of the target lesion followed by lobar TACE. The percentage of necrosis was calculated at the sonographic section that depicted the largest diameter of the tumor. Differences in the extent of early (2 days after treatment) and delayed (30 to 40 days after treatment) necrosis were quantitatively and subjectively assessed. Results: Early post-treatment tumor necrosis ranged from 50% to 96% (mean 82.8±11%) and was significantly higher than delayed tumor necrosis which ranged (p < 0.001). Concordance between early and delayed CEUS on the presence of residual vascularization was obtained in four patients. In the remaining six patients the results were discordant: in four cases tumor necrosis was complete in the early evaluation and disappeared on the follow-up, whereas a better rate of delayed response was found in 2 patients. Conclusion: Short term CEUS cannot be considered a reliable modality for the evaluation of the real extent of necrosis. In particular, the absence of enhancement two days after procedure does not always indicate a complete ablation and cannot exclude the presence of viable tumoral tissue.

1141 IMPACT OF LIVER BIOPSY FRAGMENT LENGTH AND LOCATION ON AUTOMATED QUANTITATIVE MEASURES OF FIBROSIS AND STEATOSIS

responses that protect chimpanzees from chronic infection. Since pre-existing anti-vector immunity may limit vaccine efficacy we performed a phase-I clinical trial using adenoviral vectors found at low sero-prevalence to assess the safety and immunogenicity of this approach in humans. Methods: Replicative-defective human Ad6 and a novel simian AdCh3 vector that encode 1985 amino-acids derived from the NS3–5 region of a genotype-1b strain were administered (i.m.) in a double prime, heterologous boost strategy to 27 healthy volunteers. Volunteers were primed with two doses of the same Adenovirus vector 4-weeks apart in successive cohorts, at 3 vaccine doses (5×108vp, 5×109vp, 2.5×1010vp) and boosted 24 weeks later with the alternative vector at 2.5×1010vp. Immunogenicity was assessed by ex-vivo IFNg-ELISpot (using 6 peptide pools spanning the HCV immunogen), thymidine incorporation proliferation and ICS assays. Results: Both vectors were found to be highly immunogenic after a single vaccination. Following priming vaccinations with AdHu6 the average peak HCV specific ELISpot response was 1863 SFU/10 PBMC and with AdCh3 2186 SFU/10 PBMC at the higher vaccine dose (2.5×1010vp). All vaccinees responded to the highest vaccine dose of both vectors and at this dose HCV specific responses were always multi-specific with 7/10 individuals responding to 4 or more peptide pools. Proliferative responses to multiple NS proteins were readily detected. Preferential T-cell boosting was observed with the AdHu6/AdCh3 prime/boost regimen; here the average peak postboost T-cell response was 1080 SFU/10 PBMC. ICS showed that both polyfunctional CD4+ and CD8+ HCV specific T-cell responses are induced. Importantly T-cell responses were maintained to the last time point assessed-52 weeks post prime. Vaccination was very well tolerated with mild/moderate local and systemic reactions and no serious adverse advents. Conclusions: We have generated a novel T-cell vaccine based on adenovirus vectors from rare serotypes, that is safe in man, induces polyfunctional CD4+ and CD8+ T cells, is highly immunogenic against multiple antigenic targets, and which is durable to at least a year.