Improving performance of liver biopsy in fibrosis assessment
Authors: Tarik Asselah, Patrick Marcellin, Pierre Bedossa
Journal: Journal of Hepatology (2014)

qFibrosis: A fully-quantitative innovative method incorporating histological features to facilitate accurate fibrosis scoring in animal model and chronic hepatitis B patientsJournal of HepatologyVol. 61Issue 2PreviewThere is increasing need for accurate assessment of liver fibrosis/cirrhosis. We aimed to develop qFibrosis, a fully-automated assessment method combining quantification of histopathological architectural features, to address unmet needs in core biopsy evaluation of fibrosis in chronic hepatitis B (CHB) patients. Full-Text PDF Open Access It is important to stage fibrosis in chronic hepatitis for 2 purposes: to select candidates for therapy and to follow fibrosis progression/regression on treatment. Chronic viral hepatitis is a prolonged inflammatory disease of the liver that may lead to the progressive development of fibrosis. Because fibrosis and its end-point cirrhosis are the main causes of morbidity and mortality, fibrosis assessment is considered as the most relevant information for the evaluation of the severity of the disease and as a useful indicator for prognosis and treatment decision. Because fibrosis is a morphological damage, liver biopsy has come to be the natural gold standard for staging the disease. However, the high prevalence of chronic hepatitis B in addition to the morbidity and the cost generated by this procedure have triggered an intensive search for alternative methods for fibrosis evaluation. A number of non-invasive techniques have been developed which can be used instead of liver biopsy to assess liver disease severity, including serum markers and transient elastography [1Marcellin P. Ziol M. Bedossa P. Douvin C. Poupon R. de Lédinghen V. et al.Non-invasive assessment of liver fibrosis by stiffness measurement in patients with chronic hepatitis B.Liver Int. 2009; 29: 242-247Crossref PubMed Scopus (429) Google Scholar, 2Martinot-Peignoux M. Carvalho-Filho R. Lapalus M. Netto-Cardoso A.C. Lada O. Batrla R. et al.Hepatitis B surface antigen serum level is associated with fibrosis severity in treatment-naïve, e antigen-positive patients.J Hepatol. 2013; 58: 1089-1095Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar]. However, most non-invasive tests fail to differentiate between adjacent stages of fibrosis, and are generally only accurate when distinguishing cirrhosis from no or minimal fibrosis [[3]Estrabaud E. Vidaud M. Marcellin P. Asselah T. Genomics and HCV infection: progression of fibrosis and treatment response.J Hepatol. 2012; 57: 1110-1125Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar]. Therefore, despite sampling error, liver biopsy remains the gold standard for determining histologic grade and stage. Specifically, in HBV infection, patients for whom antiviral therapy is being considered, are candidates for liver biopsy to assess fibrosis stage. Indeed, clinical guidelines on the management of chronic HBV infection recommend liver biopsy as the reference method for evaluation of liver disease for both diagnosis and assessment of eligibility for treatment [[4]European Association for the Study of the Liver EASL clinical practice guidelines: management of chronic hepatitis B virus infection.J Hepatol. 2012; 57: 167-185Abstract Full Text Full Text PDF PubMed Scopus (2720) Google Scholar]. Furthermore, it has been demonstrated that regression of fibrosis is usually obtained with maintained on-treatment HBV inhibition during long-term therapy with nucleos(t)ide analogues such as entecavir or tenofovir [5Marcellin P. Asselah T. Long-term therapy for chronic hepatitis B: hepatitis B virus DNA suppression leading to cirrhosis reversal.J Gastroenterol Hepatol. 2013; 28: 912-923Crossref PubMed Scopus (42) Google Scholar, 6Chang T.T. Liaw Y.F. Wu S.S. Schiff E. Han K.H. Lai C.L. et al.Long-term entecavir therapy results in the reversal of fibrosis/cirrhosis and continued histological improvement in patients with chronic hepatitis B.Hepatology. 2010; 52: 886-893Crossref PubMed Scopus (816) Google Scholar, 7Marcellin P. Gane E. Buti M. Afdhal N. Sievert W. Jacobson I.M. et al.Regression of cirrhosis during tenofovir disoproxil fumarate treatment for chronic hepatitis B.Lancet. 2013; 381: 468-475Abstract Full Text Full Text PDF PubMed Scopus (1356) Google Scholar]. Also, there is now strong evidence that not only fibrosis but also cirrhosis can also be reversed in a significant proportion of patients when HBV replication is controlled. Since cirrhosis reversion may change prognosis and significantly reduce the risk of end-stage liver disease-related complications, it might be clinically relevant to look for cirrhosis reversal after long-term HBV oral therapy. Semiquantitative scoring systems are part of histological assessment (METAVIR, Knodell, Ishak score) (Table 1) [8Metavir cooperative group Inter- and intra-observer variation in the assessment of liver biopsy of chronic hepatitis C.Hepatology. 1994; 20: 15-20Crossref PubMed Scopus (1884) Google Scholar, 9Bedossa P. Dargère D. Paradis V. Sampling variability of liver fibrosis in chronic hepatitis C.Hepatology. 2003; 38: 1449-1457Crossref PubMed Scopus (2054) Google Scholar]. Fibrosis is scored in stages while necroinflammation is evaluated by grade. Staging fibrosis is an assessment of the combination of the amount of fibrosis and architectural disorganization. These semiquantitative histological scores are used in both clinical trials and for individual evaluation.Table 1Liver biopsy in viral hepatitis – scoring systems in liver fibrosis.P-P, portal to portal; P-C, portal to central. Open table in a new tab P-P, portal to portal; P-C, portal to central. A more quantitative approach for fibrosis assessment is morphometry, a technique based on image analysis of digitalized tissue sections. Liver sections had to be stained with sirius red, a stain that binds to all connective tissue, but primarily to collagen as proven by a correlation between the amount of bound stain and the chemically determined collagen content [[10]Jimenez W. Parés A. Caballería J. Heredia D. Bruguera M. Torres M. et al.Measurement of fibrosis in needle liver biopsies: evaluation of a colorimetric method.Hepatology. 1985; 5: 815-818Crossref PubMed Scopus (124) Google Scholar]. The quantification of total fibrosis is usually expressed as a percentage, the ratio of fibrous tissue area stained with sirius red upon the total tissue surface (Collagen Proportional area or CPA). Fibrosis progression (or regression) can be defined as any increase (or reduction) in fibrosis amount when comparing paired liver biopsies. In this issue of Journal of Hepatology, Xu et al. developed qFibrosis: a fully-automated method incorporating both collagen amount quantitation and information about spatial location and organisation of fibrous tissue through a sophisticated automated process with the use of a proprietary algorithm. This approach was tested in an animal model of fibrosis and liver biopsies of chronic hepatitis B patients [[11]Xu S. Wang Y. Tai D.C.S. Wang S. Cheng C.L. Peng Q. et al.qFibrosis: a fully-quantitative innovative method incorporating histological features to facilitate accurate fibrosis scoring in animal model and chronic hepatitis B patients.J Hepatol. 2014; 61: 260-269Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar]. The main fibrosis patterns, namely, portal collagen (portal expansion), septal collagen (bridging fibrosis), and fibrillar collagen (fine collagen distributed in the pericellular/perisinusoidal space of Disse) were identified through image acquisition and processing, and translated into quantitative parameters to build up qFibrosis indices. qFibrosis relies on a special microscope imaging technique that allows efficient collection of fibrous architectural information, the non-linear optical second harmonic generation (SHG)/two-photon excitation fluorescence (TPEF) microscopy that was previously reported [12Tai D.C. Tan N. Xu S. Kang C.H. Chia S.M. Cheng C.L. et al.Fibro-C-Index: comprehensive, morphology-based quantification of liver fibrosis using second harmonic generation and two-photon microscopy.J Biomed Opt. 2009; 14: 044013Crossref Scopus (73) Google Scholar, 13Gailhouste L. Le Grand Y. Odin C. Guyader D. Turlin B. Ezan F. et al.Fibrillar collagen scoring by second harmonic microscopy: a new tool in the assessment of liver fibrosis.J Hepatol. 2010; 52: 398-406Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar]. SHG/TPEF can analyse and quantify collagen fibers in 2D and 3D formats because of the intrinsic optical properties of fibrillar collagen molecules in stain-free biopsy sections [[14]Zipfel W.R. Williams R.M. Webb W.W. Nonlinear magic: multiphoton microscopy in the biosciences.Nat Biotechnol. 2003; 21: 1369-1377Crossref PubMed Scopus (3284) Google Scholar]. In the rat model, qFibrosis values increased with fibrosis progression and showed significant differences between all semiquantitative stages of fibrosis (p <0.001) while CPA showed drastic changes only in advanced stages fibrosis and was unable to differentiate between early stages. In the chronic hepatitis B biopsies, qFibrosis values, obtained from 69 biopsies longer than 15 mm, successfully differentiated between all METAVIR stages (p <0.05). In comparison, CPA could only differentiate between stages 3 and 4. The authors reported that qFibrosis was less sensitive to sampling error. Also qFibrosis can aid in correction of sampling error-mediated intraobserver variation. In an independent cohort of chronic hepatitis B liver biopsies, qFibrosis indices faithfully replicated Metavir fibrosis scoring, with better differentiation ability between stages than with CPA measurements. The AUC values of qFibrosis for detection of different stages were from 0.90 to 0.95, while the AUC values of CPA were smaller (0.84–0.92). Thus, this piece of work demonstrates the accuracy and reproducibility of qFibrosis as a new parameter in quantitative evaluation of liver fibrosis. Nevertheless, and before claiming its wide diffusion, this needs further studies. First, it remains to demonstrate the additional value of this sophisticated technique which is costly and time-consuming by comparison to the usual semiquantitative scoring systems. Although semiquantitative scores have been proved to be clinically relevant, the clinical relevance in assessment of such a level of accuracy needs to be demonstrated. To date, and despite significant technical development in the past 30 years, the place of quantitative image analysis of liver fibrosis is still limited. CPA is used only in evaluation of fibrosis in clinical trials although a recent study suggest this approach might be relevant for prognosis. Furthermore, the pattern of fibrosis changes according to etiology of the underlying chronic liver disease. Indeed, while in viral hepatitis fibrosis spread from periportal area, fibrosis is mainly perisinusoidal and located in zone 3 in NAFLDs. Since qFibrosis includes pattern of fibrosis into the quantification algorithm, there is strong supposition that one algorithm will not fit for all diseases. And what about associated comorbidities such as hepatitis C and NAFLD? Although there is an urgent need to validate new non-invasive markers, qFibrosis will still need to sample the liver. Finally, this study convincingly demonstrates the accuracy and reproducibility of qFibrosis as a new tool in quantitative evaluation of liver fibrosis. This innovative approach has still to find its way but it may enhance the information provided by the liver biopsy especially in the context of clinical research in chronic liver disease. The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.