Digital pathology: accurate technique for quantitative assessment of histological features in metabolic-associated fatty liver disease
Corresponding Author
David Marti-Aguado
Valencia, Spain
Correspondence
David Martí-Aguado, Department of Gastroenterology and Hepatology, Clinic University Hospital, INCLIVA Health Research Institute, Valencia, Spain; Rio Hortega, Instituto Salud Carlos III, Madrid, Spain.
Email: [email protected]
Search for more papers by this authorCorresponding Author
David Marti-Aguado
Valencia, Spain
Correspondence
David Martí-Aguado, Department of Gastroenterology and Hepatology, Clinic University Hospital, INCLIVA Health Research Institute, Valencia, Spain; Rio Hortega, Instituto Salud Carlos III, Madrid, Spain.
Email: [email protected]
Search for more papers by this authorThe complete list of authors' affiliation are listed in Appendix 1.
The Handling Editor for this article was Professor Jonathan Rhodes, and it was accepted for publication after full peer-review.
Funding information
This study was funded by the Spanish Ministry of Science and Innovation, Instituto de Salud Carlos III (PI19/0380) and GILEAD Sciences (Grant Number: GLD19/00050). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
Summary
Background
Histological evaluation of metabolic-associated fatty liver disease (MAFLD) biopsies is subjective, descriptive and with interobserver variability.
Aims
To examine the relationship between different histological features (fibrosis, steatosis, inflammation and iron) measured with automated whole-slide quantitative digital pathology and corresponding semiquantitative scoring systems, and the distribution of digital pathology measurements across Fatty Liver Inhibition of Progression (FLIP) algorithm and Steatosis, Activity and Fibrosis (SAF) scoring system
Methods
We prospectively included 136 consecutive patients who underwent liver biopsy for MAFLD at three Spanish centres (January 2017-January 2020). Biopsies were scored by two blinded pathologists according to the Non-alcoholic Steatohepatitis (NASH) Clinical Research Network system for fibrosis staging, the FLIP/SAF classification for steatosis and inflammation grading and Deugnier score for iron grading. Proportionate areas of collagen, fat, inflammatory cells and iron deposits were measured with computer-assisted digital image analysis. A test-retest experiment was performed for precision repeatability evaluation.
Results
Digital pathology showed strong correlation with fibrosis (r = 0.79; P < 0.001), steatosis (r = 0.85; P < 0.001) and iron (r = 0.70; P < 0.001). Performance was lower when assessing the degree of inflammation (r = 0.35; P < 0.001). NASH cases had a higher proportion of collagen and fat compared to non-NASH cases (P < 0.005), whereas inflammation and iron quantification did not show significant differences between categories. Repeatability evaluation showed that all the coefficients of variation were ≤1.1% and all intraclass correlation coefficient values were ≥0.99, except those of collagen.
Conclusion
Digital pathology allows an automated, precise, objective and quantitative assessment of MAFLD histological features. Digital analysis measurements show good concordance with pathologists´ scores.
Open Research
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Supporting Information
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| apt16100-sup-0001-Supinfo.docxWord document, 36.4 MB | Supplementary Material |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
REFERENCES
- 1Younossi Z, Anstee QM, Marietti M, et al. Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention. Nat Rev Gastroenterol Hepatol. 2018; 15: 11-20.
- 2Dulai PS, Singh S, Patel J, et al. Increased risk of mortality by fibrosis stage in nonalcoholic fatty liver disease: systematic review and meta-analysis. Hepatology. 2017; 65: 1557-1565.
- 3 European Association for the Study of the Liver (EASL); European Association for the Study of Diabetes (EASD); European Association for the Study of Obesity (EASO). EASL-EASD-EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease. J Hepatol. 2016; 64: 1388-1402.
- 4Burt AD, Lackner C, Tiniakos DG. Diagnosis and assessment of NAFLD: definitions and histopathological classification. Semin Liver Dis. 2015; 35: 207-220.
- 5Bedossa P, Carrat F. Liver biopsy: the best, not the gold standard. J Hepatol. 2009; 50: 1-3.
- 6Ratziu V, Charlotte F, Heurtier A, et al. Sampling variability of liver biopsy in nonalcoholic fatty liver disease. Gastroenterology. 2005; 128: 1898-1906.
- 7Poynard T, Lenaour G, Vaillant JC, et al. Liver biopsy analysis has a low level of performance for diagnosis of intermediate stages of fibrosis. Clin Gastroenterol Hepatol. 2012; 10: 657-663.e7.
- 8Davison BA, Harrison SA, Cotter G, et al. Suboptimal reliability of liver biopsy evaluation has implications for randomized clinical trials. J Hepatol. 2020:S0168–8278(20)30399–8. https://doi.org/10.1016/j.jhep.2020.06.025. [Epub ahead of print].
- 9Kleiner DE, Brunt EM, Van Natta M, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005; 41: 1313-1321.
- 10Brunt EM, Janney CG, Di Bisceglie AM, Neuschwander-Tetri BA, Bacon BR. Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions. Am J Gastroenterol. 1999; 94: 2467-2474.
- 11Bedossa P, Poitou C, Veyrie N, et al. Histopathological algorithm and scoring system for evaluation of liver lesions in morbidly obese patients. Hepatology. 2012; 56: 1751-1759.
- 12Ratziu V. A critical review of endpoints for non-cirrhotic NASH therapeutic trials. J Hepatol. 2018; 68: 353-361.
- 13Rinella ME, Tacke F, Sanyal AJ, Anstee QM; Participants of the AASLD/EASL Workshop. Report on the AASLD/EASL joint workshop on clinical trial endpoints in NAFLD. Hepatology. 2019; 70: 1424-1436.
- 14Melo RCN, Raas MWD, Palazzi C, Neves VH, Malta KK, Silva TP. Whole slide imaging and its applications to histopathological studies of liver disorders. Front Med (Lausanne). 2020; 6:310.
- 15Paradis V, Quaglia A. Digital pathology, what is the future? J Hepatol. 2019; 70: 1016-1018.
- 16Stasi C, Tsochatzis EA, Hall A, et al. Comparison and correlation of fibrosis stage assessment by collagen proportionate area (CPA) and the ELF panel in patients with chronic liver disease. Dig Liver Dis. 2019; 51: 1001-1007.
- 17Jedrzkiewicz J, Bronner MP, Salama ME, et al. Liver fibrosis quantification by digital whole slide imaging and two photon microscopy with second harmonic generation. Int J Pathol Clin Res. 2018; 4:078.
- 18Mendes LC, Ferreira PA, Miotto N, et al. Elastogram quality assessment score in vibration-controlled transient elastography: Diagnostic performance compared to digital morphometric analysis of liver biopsy in chronic hepatitis C. J Viral Hepat. 2018; 25: 335-343.
- 19Campos CFF, Paiva DD, Perazzo H, et al. An inexpensive and worldwide available digital image analysis technique for histological fibrosis quantification in chronic hepatitis C. J Viral Hepat. 2014; 21: 216-222.
- 20Huang Y, de Boer WB, Adams LA, MacQuillan G, Bulsara MK, Jeffrey GP. Image analysis of liver biopsy samples measures fibrosis and predicts clinical outcome. J Hepatol. 2014; 61: 22-27.
- 21Abe T, Hashiguchi A, Yamazaki K, et al. Quantification of collagen and elastic fibers using whole-slide images of liver biopsy specimens. Pathol Int. 2013; 63: 305-310.
- 22Calvaruso V, Burroughs AK, Standish R, et al. Computer-assisted image analysis of liver collagen: relationship to Ishak scoring and hepatic venous pressure gradient. Hepatology. 2009; 49: 1236-1244.
- 23Masugi Y, Abe T, Tsujikawa H, et al. Quantitative assessment of liver fibrosis reveals a nonlinear association with fibrosis stage in nonalcoholic fatty liver disease. Hepatol Commun. 2017; 2: 58-68.
- 24Buzzetti E, Hall A, Ekstedt M, et al. Collagen proportionate area is an independent predictor of long-term outcome in patients with non-alcoholic fatty liver disease. Aliment Pharmacol Ther. 2019; 49: 1214-1222.
- 25Eslam M, Newsome PN, Sarin SK, et al. A new definition for metabolic dysfunction-associated fatty liver disease: an international expert consensus statement. J Hepatol. 2020; 73: 202-209.
- 26Grundy SM, Brewer HB Jr, Cleeman JI, Smith SC Jr, Lenfant C. Definition of metabolic syndrome: report of the National Heart, Lung, and Blood Institute/American Heart Association conference on scientific issues related to definition. Circulation. 2004; 109: 433-438.
- 27Torlakovic EE, Naresh K, Kremer M, van der Walt J, Hyjek E, Porwit A. Call for a European programme in external quality assurance for bone marrow immunohistochemistry; report of a European Bone Marrow Working Group pilot study. J Clin Pathol. 2009; 62: 547-551.
- 28Turlin B, Ramm GA, Purdie DM, et al. Assessment of hepatic steatosis: comparison of quantitative and semiquantitative methods in 108 liver biopsies. Liver Int. 2009; 29: 530-535.
- 29Hall AR, Dhillon AP, Green AC, et al. Hepatic steatosis estimated microscopically versus digital image analysis. Liver Int. 2013; 33: 926-935.
- 30Straub BK, Stoeffel P, Heid H, Zimbelmann R, Schirmacher P. Differential pattern of lipid droplet-associated proteins and de novo perilipin expression in hepatocyte steatogenesis. Hepatology. 2008; 47: 1936-1946.
- 31Deugnier Y, Turlin B. Pathology of hepatic iron overload. World J Gastroenterol. 2007; 13: 4755-4760.
- 32Reinhard E, Adhikhmin M, Gooch B, Shirley P. Color transfer between images. IEEE Comput Graphics Appl. 2001; 21: 34-41.
- 33Yegin EG, Yegin K, Ozdogan OC. Digital image analysis in liver fibrosis: basic requirements and clinical implementation. Biotechnol Biotechnol Equip. 2016; 30: 653-660.
10.1080/13102818.2016.1181989 Google Scholar
- 34 European Society of Radiology (ESR). ESR statement on the validation of imaging biomarkers. Insights Imaging. 2020; 11: 76.
- 35Eddowes PJ, Sasso M, Allison M, et al. Accuracy of FibroScan controlled attenuation parameter and liver stiffness measurement in assessing steatosis and fibrosis in patients with nonalcoholic fatty liver disease. Gastroenterology. 2019; 156: 1717-1730.
- 36van der Poorten D, Samer CF, Ramezani-Moghadam M, et al. Hepatic fat loss in advanced nonalcoholic steatohepatitis: are alterations in serum adiponectin the cause? Hepatology. 2013; 57: 2180-2188.
- 37Pelusi S, Cespiati A, Rametta R, et al. Prevalence and risk factors of significant fibrosis in patients with nonalcoholic fatty liver without steatohepatitis. Clin Gastroenterol Hepatol. 2019; 17: 2310-2319.
- 38Rosselli M, MacNaughtan J, Jalan R, Pinzani M. Beyond scoring: a modern interpretation of disease progression in chronic liver disease. Gut. 2013; 62: 1234-1241.
- 39Standish RA, Cholongitas E, Dhillon A, Burroughs AK, Dhillon AP. An appraisal of the histopathological assessment of liver fibrosis. Gut. 2006; 55: 569-578.
- 40Huang YI, de Boer WB, Adams LA, et al. Image analysis of liver collagen using sirius red is more accurate and correlates better with serum fibrosis markers than trichrome. Liver Int. 2013; 33: 1249-1256.
- 41Pais R, Charlotte F, Fedchuk L, et al. A systematic review of follow-up biopsies reveals disease progression in patients with non-alcoholic fatty liver. J Hepatol. 2013; 59: 550-556.
- 42Tsochatzis E, Bruno S, Isgro G, et al. Collagen proportionate area is superior to other histological methods for sub-classifying cirrhosis and determining prognosis. J Hepatol. 2014; 60: 948-954.
- 43Bowlus CL, Pockros PJ, Kremer AE, et al. Long-term obeticholic acid therapy improves histological endpoints in patients with primary biliary cholangitis. Clin Gastroenterol Hepatol. 2020; 18: 1170-1178.
- 44Nasr P, Fredrikson M, Ekstedt M, Kechagias S. The amount of liver fat predicts mortality and development of type 2 diabetes in non-alcoholic fatty liver disease. Liver Int. 2020; 40: 1069-1078.
- 45Munsterman ID, van Erp M, Weijers G, et al. A novel automatic digital algorithm that accurately quantifies steatosis in NAFLD on histopathological whole-slide images. Cytometry B Clin Cytom. 2019; 96: 521-528.
- 46Mendes LC, Ferreira PA, Miotto N, et al. Controlled attenuation parameter for steatosis grading in chronic hepatitis C compared with digital morphometric analysis of liver biopsy: impact of individual elastography measurement quality. Eur J Gastroenterol Hepatol. 2018; 30: 959-966.
- 47Bedossa P; FLIP Pathology Consortium. Utility and appropriateness of the fatty liver inhibition of progression (FLIP) algorithm and steatosis, activity, and fibrosis (SAF) score in the evaluation of biopsies of nonalcoholic fatty liver disease. Hepatology. 2014; 60: 565-575.
- 48Pai RK, Kleiner DE, Hart J, et al. Standardising the interpretation of liver biopsies in non-alcoholic fatty liver disease clinical trials. Aliment Pharmacol Ther. 2019; 50: 1100-1111.
- 49Fujii H, Ikura Y, Arimoto J, et al. Expression of perilipin and adipophilin in nonalcoholic fatty liver disease; relevance to oxidative injury and hepatocyte ballooning. J Atheroscler Thromb. 2009; 16: 893-901.
- 50Dey A, Allen J, Hankey-Giblin PA. Ontogeny and polarization of macrophages in inflammation: blood monocytes versus tissue macrophages. Front Immunol. 2015; 5:683.
- 51Brunt EM, Kleiner DE, Wilson LA, et al. Portal chronic inflammation in nonalcoholic fatty liver disease (NAFLD): a histologic marker of advanced NAFLD-Clinicopathologic correlations from the nonalcoholic steatohepatitis clinical research network. Hepatology. 2009; 49: 809-820.
- 52Younossi ZM, Stepanova M, Rafiq N, et al. Pathologic criteria for nonalcoholic steatohepatitis: interprotocol agreement and ability to predict liver-related mortality. Hepatology. 2011; 53: 1874-1882.
- 53Gadd VL, Skoien R, Powell EE, et al. The portal inflammatory infiltrate and ductular reaction in human nonalcoholic fatty liver disease. Hepatology. 2014; 59: 1393-1405.
- 54Schwenger K, Chen L, Chelliah A, et al. Markers of activated inflammatory cells are associated with disease severity and intestinal microbiota in adults with non-alcoholic fatty liver disease. Int J Mol Med. 2018; 42: 2229-2237.
- 55Estep M, Mehta R, Bratthauer G, et al. Hepatic sonic hedgehog protein expression measured by computer assisted morphometry significantly correlates with features of non-alcoholic steatohepatitis. BMC Gastroenterol. 2019; 19: 27.
- 56Lackner C, Gogg-Kamerer M, Zatloukal K, Stumptner C, Brunt EM, Denk H. Ballooned hepatocytes in steatohepatitis: the value of keratin immunohistochemistry for diagnosis. J Hepatol. 2008; 48: 821-828.
- 57Bugianesi E, Manzini P, D'Antico S, et al. Relative contribution of iron burden, HFE mutations, and insulin resistance to fibrosis in nonalcoholic fatty liver. Hepatology. 2004; 39: 179-187.
- 58George DK, Goldwurm S, Macdonald GA, et al. Increased hepatic iron concentration in nonalcoholic steatohepatitis is associated with increased fibrosis. Gastroenterology. 1998; 114: 311-318.
- 59Ganne-Carrié N, Christidis C, Chastang C, et al. Liver iron is predictive of death in alcoholic cirrhosis: a multivariate study of 229 consecutive patients with alcoholic and/or hepatitis C virus cirrhosis: a prospective follow up study. Gut. 2000; 46: 277-282.
- 60Nelson JE, Klintworth H, Kowdley KV. Iron metabolism in nonalcoholic fatty liver disease. Curr Gastroenterol Rep. 2012; 14: 8-16.
- 61Nelson JE, Wilson L, Brunt EM, et al.; Nonalcoholic Steatohepatitis Clinical Research Network. Relationship between the pattern of hepatic iron deposition and histological severity in nonalcoholic fatty liver disease. Hepatology. 2011; 53: 448-457.
- 62Valenti L, Fracanzani AL, Bugianesi E, et al. HFE genotype, parenchymal iron accumulation, and liver fibrosis in patients with nonalcoholic fatty liver disease. Gastroenterology. 2010; 138: 905-912.
- 63Liu F, Goh GB, Tiniakos D, et al. qFIBS: a novel automated technique for quantitative evaluation of fibrosis, inflammation, ballooning, and steatosis in patients with nonalcoholic steatohepatitis. Hepatology. 2020; 71: 1953-1966.
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