Good Old Aspirin Might Protect Against Liver Fibrosis

Medscape Medical News > Conference News

Neil Osterweil
November 10, 2014

BOSTON — Will wonder drugs never cease? Aspirin, already touted for its cardiovascular and anti-inflammatory prowess, seems to be flexing its muscles against liver fibrosis, particularly in people at risk for chronic liver disease, according to new research.

"Clinical equipoise is emerging that may justify prospective randomized trials of aspirin and, potentially, other antiplatelet drugs such as antifibrotic agents," Gordon Jiang, MD, a gastroenterology fellow at the Beth Israel Deaconess Medical Center in Boston, and colleagues state in a scientific poster here at The Liver Meeting 2014.

In a population-based cross-sectional study of more than 14,000 adults, there was "a consistent association between aspirin use and less liver fibrosis," Dr. Jiang and colleagues report.

The team drew on the National Health and Nutrition Examination Survey (NHANES) III to look at the association between aspirin, ibuprofen, and liver fibrosis. Fibrosis was measured with four validated noninvasive indices: Fibrosis-4, the nonalcoholic fatty liver disease fibrosis score, the aspartate aminotransferase/platelet ratio index, and the Forns Index.

To see if the association between aspirin and fibrosis protection is stronger in patients most at risk for fibrosis, they did additional analyses in patients with viral hepatitis, heavy drinkers, and patients with fatty liver disease.

On the four measures, the use of aspirin was consistently associated with lower stages of liver fibrosis. In contrast, there was virtually no link between ibuprofen use and liver fibrosis.

Similarly, in an analysis of patients with and without chronic liver disease (hepatitis B or C infection, more than 5 alcoholic drinks per day, or suspected nonalcoholic steatohepatitis), aspirin but not ibuprofen was consistently associated with lower stages of fibrosis.

For patients with or at risk for liver disease, compared with those without risk factors, there was about a 5-fold increase in the negative coefficient for the interaction between aspirin use and liver fibrosis. This suggests that the protective effect of aspirin is much larger in patients with chronic liver disease, said researcher Yury Popov, MD, PhD, assistant professor of medicine at the Beth Israel Deaconess Medical Center.

The researchers acknowledge that the study was limited by the observational design, and by that fact that the NHANES III data were limited to 1 month of drug use, "whereas the protection against liver fibrosis likely requires long-term use."

They speculate that the antiplatelet activity of aspirin, rather than its anti-inflammatory properties, likely account for its positive effects on fibrosis.

"Our observations support emerging experimental and clinical evidence for a pathologic link between platelet activation and liver fibrosis," they write.

In a separate study, also presented here, another group of researchers report that "platelets drive liver fibrosis through the direct activation of hepatic stellate cells in chronically injured liver."

"We also found that aspirin in the long term, at the low dose — the antiplatelet dose — is reducing — well actually, preventing — fibrosis in the mouse model," Dr. Popov, who was involved in both studies, told Medscape Medical News.

More potent antiplatelet agents — such as clopidogrel (Plavix), prasugrel (Effient), and others currently in the pipeline — could have an even stronger effect against fibrosis, said Athan Kuliopulos, MD, PhD, professor of medicine at the Tufts University Sackler School of Biomedical Sciences in Boston, who was not involved with the study.

"And this is just liver fibrosis," he told Medscape Medical News. "What does it mean for outcome?"

He said that if, as he and his colleagues theorize, platelet activation or action is a primary cause of fibrosis, it might be possible to "bypass the platelet entirely," and target the factor Xa protease.

Dr. Jiang has disclosed no relevant financial relationships. Dr. Popov reports consulting for and receiving research grants from Gilead Sciences. Dr. Kuliopulos is the CEO of Oasis Pharmaceuticals.

The Liver Meeting 2014: American Association for the Study of Liver Diseases (AASLD). Abstract 449, presented November 8, 2014; abstract 778, presented November 9, 2014.

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Traditional Chinese Medicine (TCM) for fibrotic liver disease: Hope and hype

Journal of Hepatology
Volume 61, Issue 1, Pages 166–168, July 2014

Lijun Zhang, Detlef Schuppan
Institute of Translational Immunology, University Medical Center, Johannes Gutenberg University Mainz, Germany

Received: January 15, 2014; Received in revised form: February 24, 2014; Accepted: March 6, 2014; Published Online: April 26, 2014

DOI: http://dx.doi.org/10.1016/j.jhep.2014.03.009

Therapies that specifically retard progression or induce regression of fibrotic liver disease have entered the clinical stage[[1], [2]]. This is remarkable, since clinical validation of antifibrotic efficacy remains difficult and since the mere reduction of excess scarring, without an improvement of hepatic angio-architecture and synthetic function is a disputed primary clinical endpoint [3]. Importantly, as in cancer therapies, drugs that address more than a single pathogenic pathway are usually more efficient than single highly specific pathway modulators [1]. Moreover, the high costs and risks of drug development have opened the field for drug repurposing, i.e., the validation and use of defined agents that were already tested clinically for diseases other than fibrosis. These insights have further sparked an interest in a more holistic pharmacological approach to fibrosis, which includes reevaluation of the herbs of Traditional Chinese Medicine (TCM).

The recorded history of TCM can be traced back over 2000 years, although it is common belief that its origins range as far back as Chinese civilization, i.e., >5000 years. TCM derived its theoretical foundations, including diagnostic and treatment methodologies, from ancient Chinese philosophy. It virtually dragged the Chinese nation through periods of poverty and chaos as an exclusive medical way of health care, before Western medicine was introduced after a series of revolutionary campaigns in the 20th century.

Unlike Western medicine, TCM strictly relies on the two therapeutic pillars of holism and syndrome differentiation. In this system, the human body is not simply regarded as an entity in which different parts work interdependently but also a portion of the universe in which it is embedded and with which it interacts. Sickness is triggered by the imbalance of Yin and Yang, a concept of contrary but complementary forces in nature, resulting in various symptoms with a dynamic trend in the course of disease. Here, holistic but individualized approaches that use herbal remedies are targeted at the complex syndrome, to help the body regain balance and harmony.

Chronic liver diseases (CLD) have never been described as such in written documentations of TCM. Therefore, it has been difficult to establish a precise description of CLD in modern TCM, and symptomatic terms such as abdominal swelling, yellow skin and right upper abdominal pain were included only in the 1970s [4]. Thereafter, the definition of CLD in TCM was gradually refined to encompass: (1) Weakening of the Qi (biological substances/activities that preserve life); (2) blockage of meridians (circulation channels of Qi) by blood stasis; (3) generation of dampness and heat (inflammatory pathogens) [5]. Only then studies began to systematically examine the effect of TCM on defined CLD [6].Major aims were the eradication or suppression of the underlying cause of CLD, such as HBV or HCV infection, and treatment of its hard endpoints, cirrhosis and HCC, with a major focus on fibrosis. However, there remained problems to reconcile TCM, which traditionally valued empiricism and holistic philosophy, with the Western approach to CLD, such as a reproducible standardization of herbs using quantifiable lead compounds (biologically active ingredients), the frequent lack of rigorous stratification of patients or absence of a double-blind, randomized, placebo-controlled clinical trial design. Moreover, mechanistic preclinical validation of TCM drugs is still in its infancy, with a focus on select cultures of liver cells and cell lines, and a relatively narrow spectrum of in vivo rodent models of liver inflammation and fibrosis. Finally, some TCM drugs have been accused of negligent safety evaluation, based on case reports of hepatotoxicity or nephrotoxicity [7],largely due to contamination with heavy metals or toxic alkaloids. However, the Chinese government has begun to initiate a national safety plan in 2011, investing in the modernization of TCM ($100 million from the National Natural Science Foundation in 2012), to promote research on lead compound identification and mechanisms of action, on a better standardization and well controlled clinical trials.

Thus numerous clinical trials, some of them NIH-registered, were initiated and several formulations received approval by the China Food and Drug Administration. Approved and broadly marketed anti-fibrotic formulations are: Compound 861, an aqueous extract of 10 herbs targeting blood stasis and liver stagnation [8]; Biejiaruangan Compound (CBJRGC), a prescription containing Carapax trionycis (turtle shell) and 10 herbs to soften the liver [9]; Fuzheng Huayu (FZHY), consisting of six herbs, to resolve blood stasis and nourish the liver [10]. Both CBJRBC and FZHY reached sales >$30 million during 2012 in China, and an antifibrotic effect of FZHY has been suggested in a recently completed US FDA-approved trial in patients with chronic hepatitis C [11]. Table 1 lists major published studies on TCM for the treatment of liver fibrosis.

Only the 3 major ingredients are listed. Compound 861: Salvia miltiorrhiza, Radix Astragali, Cordyceps; Biejiaruangan Compound: Carapax trionycis, Panax pseudoginseng, Radix Paeoniae Rubra; Fuzhenghuayu Capsule: Salviae miltiorrhizae, Cordyceps, Semen Persicae; Qiang gan ruan jian Tang: Salvia miltiorrhiza, Radix Astragali, Carapax trionycis; Qiang gan ruan jian Fang: Codonopsis pilosula, Angelica sinensis, Carapax trionycis; Qiang gan ruan jian Wan: Radix Astragali, Carapax trionycis, Manis pentadactyla; Qianggan Capsule: Salvia miltiorrhiza, Radix Astragali, Codonopsis pilosula; Yi gan kang: Angelica sinensis, Radix Astragali, Salvia miltiorrhiza; Jian pi bu shen Fang: Radix Astragali, Salvia miltiorrhiza, Atractylodes macrocephala.

RC, randomized controlled; CHB, chronic hepatitis B; CHC, chronic hepatitis C; TG, triglycerides; TC, total cholesterol; HDL-C, high density lipoprotein-cholesterol.

Predictably, TCM should provide a rich resource for developing anti-inflammatory, anti-infectious, and anti-fibrotic drug candidates. A major challenge is its reconciliation with Western medicine, requiring translation of the TCM codes into a more scientific language, and identification of effective lead compounds in the heterogeneous herbal mixtures or extracts. The latter has already been achieved in some cases, revealing, e.g., interesting plant-derived polyphenols, flavonoids or alkaloids that serve as antivirals, antioxidants or anti-fibrotics, often affecting different liver cells and signaling pathways(Fig. 1). However, identification of single active components partly contradicts the holistic theory of TCM which postulates that active ingredients and herbs will not work in isolation, a paradigm that reemerges even in Western medicine. An example is the appreciation of, e.g., the combined effect of nutrition, the intestinal microbiome, and physical exercise on metabolic, liver, and cardiovascular health [12]. Nonetheless, a valid compromise is to test individual compounds in isolation and then recombine agents with proven efficacy.

Fig. 1 The possibility of translating traditional Chinese medicine (TCM) into therapy of chronic liver diseases (CLD). The merger of holistic TCM with the more particularistic and complementary views of Western medicine holds promise for the development of novel drugs for CLD including liver fibrosis. Implementation of cutting-edge technologies and rigorous study design should finally create innovation and synergism. Shown are examples of TCM drugs and their cellular activities relevant for liver inflammation and fibrosis. Implementation of cutting-edge technologies and rigorous study design should finally create innovation and synergism. MΦ: macrophage; HSC, hepatic stellate cell; HEP: hepatocyte.

Controversies how to further develop TCM are ongoing in China. One group deplores the modernization of TCM as submission to Western rules, the other group demands its continuing overhaul. There is also a concern of deteriorating drug quality, including contaminants accumulating in cultured herbs. Moreover, many young practitioners feel that adherence to TCM will compromise their career, and patients, especially in the cities, are increasingly skeptical about the efficacy of TCM. Still there is an overwhelming consensus that implementation of methodological improvements and rigorous scientific testing along the principles of evidence-based medicine will help to exploit the vast potential of TCM.

Financial support

DS received funding from the NIH, European Union, the State of Rhino-Palatinate, the German Research Foundation, and the German Ministry of Education and Research.

Conflict of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

References

[1] Schuppan, D. and Kim, Y.O. Evolving therapies for liver fibrosis. J Clin Invest. 2013; 123: 1887–1901 View in Article

[2] Friedman, S.L., Sheppard, D., Duffield, J.S., and Violette, S. Therapy for fibrotic diseases: nearing the starting line. Sci Transl Med. 2013; 5: 167sr161 View in Article

[3] Schuppan, D. and Pinzani, M. Anti-fibrotic therapy: lost in translation?. J Hepatol. 2012; 56: S66–S74 View in Article

[4] Liu, C.H., Liu, P., Hu, Y.Y., Xu, L.M., and Liu, C. Progress of clinical and basic research on liver fibrosis with Traditional Chinese Medicine. World Sci Technol. 2007; 9: 112–119 View in Article

[5] Liu, P. Treatment of cirrhosis and liver fibrosis by traditional Chinese medicine. Chin Hepatol. 2002; 7: 33–35 View in Article

[6] Luk, J.M., Wang, X., Liu, P., Wong, K.F., Chan, K.L., Tong, Y. et al. Traditional Chinese herbal medicines for treatment of liver fibrosis and cancer: from laboratory discovery to clinical evaluation. Liver Int. 2007; 27: 879–890 View in Article

[7] Schuppan, D., Jia, J.D., Brinkhaus, B., and Hahn, E.G. Herbal products for liver diseases: a therapeutic challenge for the new millennium. Hepatology. 1999; 30: 1099–1104 View in Article

[8] Yin, S.S., Wang, B.E., Wang, T.L., Jia, J.D., and Qian, L.X. The effect of Cpd 861 on chronic hepatitis B related fibrosis and early cirrhosis: a randomized, double blind, placebo controlled clinical trial. Zhonghua Gan Zang Bing Za Zhi. 2004; 12: 467–470 View in Article

[9] Guo, S.G., Zhang, W., Jiang, T., Dai, M., Zhang, L.F., Meng, Y.C. et al. Influence of serum collected from rat perfused with compound Biejiaruangan drug on hepatic stellate cells. World J Gastroenterol. 2004; 10: 1487–1494 View in Article

[10] Liu, P., Hu, Y.Y., Liu, C., Xu, L.M., Liu, C.H., Sun, K.W. et al. Multicenter clinical study on Fuzhenghuayu capsule against liver fibrosis due to chronic hepatitis B. World J Gastroenterol. 2005; 11: 2892–2899 View in Article

[11] Liu Zhihua. One step closer for US to recognize TCM. China daily [Internet] 2013 Nov 27 [cited 2014 Jan 10]. Available from: http://www.chinadaily.com.cn/culture/2013-11/27/content_17133333.htm.  View in Article

[12] Schuppan, D. and Schattenberg, J.M. Non-alcoholic steatohepatitis: pathogenesis and novel therapeutic approaches. J Gastroenterol Hepatol. 2013; 28: 68–76 View in Article

© 2014 European Association for the Study of the Liver. Published by Elsevier Inc. User rights governed by an Open Access license.

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Study Provides More Evidence that Statins Help Slow Liver Fibrosis in Hepatitis C

Provided by HPCLive

By Marcia Frellick | May 05, 2014

New research from the Hepatitis C Antiviral Long-term Treatment Against Cirrhosis (HALT-C) trial cohort indicates that continuous statin use can significantly reduce liver fibrosis progression in patients with advanced chronic hepatitis C infection.

Study results, released Sunday at Digestive Disease Week 2014 in Chicago, IL, add to evidence demonstrating that statins have anti-proliferative, anti-angiogenic, and anti-inflammatory effects on hepatic cells.
Although animal models have demonstrated that statins, or 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, effectively prevent the progression of liver fibrosis, little human data was available.

Tracey G. Simon, MD, an internist in the Gastrointestinal Unit at Massachusetts General Hospital, and Brigham and Women's Hospital in Boston, MA, and colleagues studied 547 non-cirrhotic patients with chronic hepatitis C who previously had not responded to standard interferon therapy.
The patients had Ishak Fibrosis Staging scores ≥ 3 and underwent serial liver biopsies at baseline, 1.5 years, and 3.5 years after the trial began. Inflammation was graded on an 18-point histology activity index.

Patients reported statin use as part of the comprehensive medical history taken prior to enrollment and at each follow-up visit over the length of the study. Statin users were more likely to be African American, to have lower baseline alanine transferase (ALT) levels, and to be diabetic when compared to patient who reported no statin use.

The mean change in Ishak score over the study period for those who used statins was -0.34 during 3.5 years of observation, while the mean change in the Ishak score among those in the non-statin group was +0.42 [(SE 0.07), p= 0.006] after adjustment for baseline fibrosis score.

Continuous statin use was linked with a significant decrease in time to histological progression even after adjusting for known predictors of histological outcome, including diabetes, body mass index, platelets, and hepatic steatosis (HR 0.31, 95% CI 0.10 - 0.97).

Therapies to reduce the progression of liver scarring are critical as the damage keeps the liver from performing essential functions. Slowing the progression can slow hepatic decompensation and help patients live longer. However, some clinicians have been reluctant to initiate statin therapy along with treatment for hepatitis C without more evidence that they are safe and effective for this purpose.

The HALT-C researchers said further prospective studies with a large proportion of statin users are needed to define the optimal timing for starting statins, the ideal length of therapy, and the impact on those with less severe fibrosis or other etiologies of liver disease.

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ProMetic's PBI-4050 Delivers New Positive Preclinical Results in Liver Fibrosis and Liver Cancer

LAVAL, QUEBEC, CANADA, - April 10, 2014 - ProMetic Life Sciences Inc. (TSX: PLI) (OTCQX: PFSCF), ("ProMetic" or the "Corporation") presented new pre-clinical data at the 2014 annual meeting of the European Association for the Study of the Liver ("EASL") currently being held in London, UK. The new data supports the claim that PBI-4050's anti-fibrotic activity could also address various liver conditions such as non-alcoholic steatohepatitis ("NASH"), a condition affecting 2% to 5% of Americans, as well as liver cancer.

PBI-4050's favorable effect in reducing the progression of fibrosis in the liver was demonstrated in a gold standard animal model where liver fibrosis is induced by chronic administration of carbon tetrachloride ("CCL₄"), a chemical which at high chronic doses, causes irreversible damages to the liver and kidneys.  Animals treated with PBI-4050 displayed a significant reduction of liver lesions as evidenced by histology and relevant biomarkers results. Following prolonged exposure to CCL₄, a significant number of the non-treated animals also developed hepatocellular carcinoma unlike those treated with PBI-4050.

"These results clearly indicate that PBI-4050's anti-fibrotic activity is at the core of the fibrosis regulation pathway in the liver", stated Dr. Lyne Gagnon, Head of Biology & Immunology at ProMetic.

Dr. John Moran, Chief Medical Officer at ProMetic stated "The progression from fibrosis to cirrhosis to hepatocellular carcinoma is well defined in humans. The positive effects observed with PBI-4050 in multiple challenging animal models bodes well for its potential use to treat various medical conditions involving and or leading to fibrosis." 

The presentation at the EASL annual conference is available on the ProMetic website at: http://www.prometic.com/en/therapeutics/conferences.php

More on NASH:         

Non-alcoholic steatohepatitis or NASH is a common, often "silent" liver disease. It resembles
alcoholic liver disease, but occurs in people who drink little or no alcohol. The major feature in NASH is fat in the liver, along with inflammation and damage. Most people with NASH feel well and are not aware that they have a liver problem. Nevertheless, NASH can be severe and can lead to cirrhosis, in which the liver is permanently damaged and scarred and no longer able to work properly. NASH affects 2 to 5 percent of Americans

More on Hepatocellular Carcinoma

Hepatic fibrosis is an outcome of many chronic liver diseases, including hepatitis B virus, hepatitis
C virus, alcoholic liver disease and non-alcoholic steatohepatitis. Liver fibrosis is characterized by the excess accumulation and alteration of extracellular matrix molecules, including collagen, in the tissue. Liver fibrosis can progress to liver cirrhosis, liver failure, portal hypertension and hepatocellular carcinoma. Liver transplantation is the only treatment available for patients with advanced stage of fibrosis.

About ProMetic Life Sciences Inc.

ProMetic Life Sciences Inc. (www.prometic.com) is a long established biopharmaceutical company with globally recognized expertise in bioseparations, plasma-derived therapeutics and small-molecule drug development.  ProMetic offers its state of the art technologies for large-scale purification of biologics, drug development, proteomics and the elimination of pathogens to a growing base of industry leaders and uses its own affinity technology that provides for highly efficient extraction and purification of therapeutic proteins from human plasma in order to develop best-in-class therapeutics and orphan drugs. ProMetic is also active in developing its own novel small-molecule therapeutic products targeting unmet medical needs in the field of fibrosis, cancer and autoimmune diseases/inflammation. Headquartered in Laval (Canada), ProMetic has R&D facilities in the UK, the U.S. and Canada, manufacturing facilities in the UK and business development activities in the U.S., Europe and Asia.

Forward Looking Statements

This press release contains forward-looking statements about ProMetic's objectives, strategies and businesses that involve risks and uncertainties. These statements are "forward-looking" because they are based on our current expectations about the markets we operate in and on various estimates and assumptions. Actual events or results may differ materially from those anticipated in these forward-looking statements if known or unknown risks affect our business, or if our estimates or assumptions turn out to be inaccurate. Such risks and assumptions include, but are not limited to, ProMetic's ability to develop, manufacture, and successfully commercialize value-added pharmaceutical products, the availability of funds and resources to pursue R&D projects, the successful and timely completion of clinical studies, the ability of ProMetic to take advantage of business opportunities in the pharmaceutical industry, uncertainties related to the regulatory process and general changes in economic conditions. You will find a more detailed assessment of the risks that could cause actual events or results to materially differ from our current expectations in ProMetic's Annual Information Form for the year ended December 31, 2013, under the heading "Risk and Uncertainties related to ProMetic's business".  As a result, we cannot guarantee that any forward-looking statement will materialize. We assume no obligation to update any forward-looking statement even if new information becomes available, as a result of future events or for any other reason, unless required by applicable securities laws and regulations.  All amounts are in Canadian dollars unless indicated otherwise.

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Computed tomography findings in liver fibrosis and cirrhosis

19 February 2014, doi:10.4414/smw.2014.13923
Cite this as: Swiss Med Wkly. 2014;144:w13923

Huber Adrian^a, Ebner Lukas^a, Montani Matteo^b, Semmo Nasser^c, Roy Choudhury Kingshuk^d, Heverhagen Johannes^a, Christe Andreas^a

^a Institute of Radiology, University Hospital Inselspital, Bern, Switzerland
^b Institute of Pathology, University of Bern, Switzerland
^c Institute of Hepatology, University Hospital Inselspital, Bern, Switzerland
^d Statistics Department, University College Cork, Ireland

Summary

PRINCIPLES: Computed tomography (CT) is inferior to the fibroscan and laboratory testing in the noninvasive diagnosis of liver fibrosis. On the other hand, CT is a frequently used diagnostic tool in modern medicine. The auxiliary finding of clinically occult liver fibrosis in CT scans could result in an earlier diagnosis. The aim of this study was to analyse quantifiable direct signs of liver remodelling in CT scans to depict liver fibrosis in a precirrhotic stage.

METHODS: Retrospective review of 148 abdominal CT scans (80 liver cirrhosis, 35 precirrhotic fibrosis and 33 control patients). Fibrosis and cirrhosis were histologically proven. The diameters of the three main hepatic veins were measured 1–2 cm before their aperture into the inferior caval vein. The width of the caudate and the right hepatic lobe were divided, and measured horizontally at the level of the first bifurcation of the right portal vein in axial planes (caudate-right-lobe ratio). A combination of both (sum of liver vein diameters divided by the caudate-right lobe ratio) was defined as the ld/crl ratio. These metrics were analysed for the detection of liver fibrosis and cirrhosis.

RESULTS: An ld/crl-r <24 showed a sensitivity of 83% and a specificity of 76% for precirrhotic liver fibrosis. Liver cirrhosis could be detected with a sensitivity of 88% and a specificity of 82% if ld/crl-r <20.

CONCLUSION: An ld/crl-r <24 justifies laboratory testing and a fibroscan. This could bring forward the diagnosis and patients would profit from early treatment in a potentially reversible stage of disease.

Key words: liver fibrosis and cirrhosis; abdominal computed tomography; hepatic vein diameter; caudate right lobe ratio

Introduction

Liver cirrhosis is the final consequence of all chronic liver diseases [1]. Most common causes are alcoholic fatty liver disease (AFLD), nonalcoholic fatty liver disease (NAFLD) and viral hepatitis [2, 3]. Chronic inflammation leads to potentially reversible liver fibrosis and ends in irreversible cirrhosis with cross-linked collagen and regenerative nodules [4]. Early diagnosis improves the benefit of therapeutic strategies before the development of irreversible and potentially lethal complications such as loss of liver function, oesophageal variceal bleeding, hepatic encephalopathy and hepatocellular carcinoma [5, 6].

The noninvasive diagnosis of liver fibrosis and cirrhosis is built on laboratory testing and the well-established fibroscan [7]. Recently, new sensitive methods using magnetic resonance imaging (MRI) have been described, such as MR-elastography [8], double contrast-enhanced MRI [9] and diffusion weighted MRI [10]). Computed tomography (CT) is useful for imaging liver cirrhosis complications, such as portosystemic collaterals with bleeding or hepatocellular carcinoma (HCC). However, this is not an appropriate method for the primary diagnosis of liver fibrosis, because of the radiation dose and inferior accuracy compared to the fibroscan. On the other hand, clinically occult liver fibrosis as an auxiliary finding in routine abdominal CT scans is underdiagnosed. Even liver cirrhosis has a mediocre sensitivity (77.1%–84.3%) and specificity (52.9%–67.6%) in CT [11]. However, since CT is an important and frequently used diagnostic tool in modern medicine, an accurate method to detect liver fibrosis in CT scans could bring forward the diagnosis and enable treatment in an early stage of fibrosis before its clinical appearance.

We hypothesise that indirect findings of liver remodelling occur rather late when chronic portal hypertension has already been established (e.g. splenomegaly, gastrointestinal wall thickening, portosystemic collaterals, recanalisation of the umbilical vein and ascites [12–14]).

Qualitative direct signs of liver remodelling (atrophy of the right liver lobe with a notch between right and caudate lobe, heterogeneity of liver parenchyma, nodular surface, blunt liver edge and enlarged gall bladder fossa [12–14]) are limited parameters as a result of subjective reader impression and experience.

Thus, we propose the use of quantifiable direct signs of hepatic remodelling which are assessable in axial planes without the need for time-consuming image reconstructions.

There are two interesting metrics for direct liver remodelling: the caudate-right lobe ratio (crl-r) [18], which describes the width of the caudate lobe in proportion to the width of the right hepatic lobe, and measurement of the hepatic vein diameters [19]. We hypothesise that these metrics correlate with early liver fibrosis in a precirrhotic stage and can be used as quantifiable markers to depict liver fibrosis in abdominal CT scans. An analysis of these metrics alone and in combination for the detection of liver fibrosis was performed, as was a comparison with other qualitative and quantitative imaging findings.

Patients and methods

Patient population:

A total of 148 patients (108 male/40 female) were retrospectively included between January 2009 and March 2012 at our hospital, including 80 patients with histologically proven liver cirrhosis (fibrosis stage 4), 35 with histologically proven precirrhotic liver fibrosis stage 1–3 and a control group of 33 trauma patients without known liver pathology. The mean age of all selected patients was 57.3 years (range: 32–75 years). Informed consent was not required owing to the retrospective nature of this study.

The 80 patients (59 male/21 female) with liver cirrhosis (29 Child A, 31 Child B, 30 Child C) and the 35 patients with precirrhotic stage of liver fibrosis (6 fibrosis grade 1, 10 fibrosis grade 2 and 19 fibrosis grade 3) were included if they had undergone a CT scan with portal venous phase in the radiological information system (Centricity RISi 4.1, GE Healthcare) of our hospital. Liver fibrosis and cirrhosis was histologically proven by intercostal percutaneous biopsy from the right liver lobe with the “Menghini-technique” with pre- and post-procedural sonographic checks. Patients who had undergone an earlier partial liver resection or liver transplantation or those who had a transjugular portosystemic shunt (TIPS) were excluded.

Reasons for the abdominal CT scans were as follows (cirrhosis group/precirrhotic fibrosis group): HCC (40/18), tumour other than HCC (9/6), portal vein thrombosis (8/0), abscess (6/4), bleeding (7/0), acute abdomen (4/2), pancreatitis (3/3), trauma (2/1), abdominal hernia (1/0) and portal vein thrombosis (0/1).

The control group consisted of 33 consecutively selected trauma patients (23 male/10 female) with a mean age of 58.4 years (range: 51–70 years) who were examined with a portal venous phase abdominal CT scan. Patients with liver laceration, known liver fibrosis or cancer, and patients receiving potentially hepatotoxic medication were excluded. A summary of the patient population is shown in figure 1.

Figure 1 Patient population. Abdominal computed tomography scans of 148 patients were retrospectively analysed. Included were 80 patients with liver cirrhosis, 35 patients with earlier liver fibrosis and 33 control patients without known liver disease.

The clinical records of all patients in the fibrosis/cirrhosis group were surveyed. The aetiology of fibrosis was as follows: AFLD in 42 patients (37%), viral hepatitis in 45 patients (39%), NAFLD in 11 patients (10%), haemochromatosis in 5 patients (4%) and alpha-1–antitrypsin deficiency (A1AD) in 2 patients (2%). Aetiology of the fibrosis was unknown in 10 patients (9%).

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Image analysis of liver biopsy samples measures fibrosis and predicts clinical outcome

Journal of Hepatology

Article in Press

Yi Huang, W. Bastiaan de Boer, Leon A. Adams, Gerry MacQuillan, Max K. Bulsara, Gary P. Jeffrey 

Received 19 September 2013; received in revised form 20 January 2014; accepted 22 February 2014. published online 07 March 2014.
Accepted Manuscript

Abstract

Background & Aims

Histopathological scoring of liver fibrosis mainly measures architectural abnormalities and requires a minimum biopsy size (greater than or equal to 10mm). Liver collagen quantification may allow use of small size biopsies and improve the prediction of clinical outcomes. This study evaluated the ability of the collagen proportional area (CPA) measurement to predict clinical outcomes.

Methods

Clinical outcomes were determined using population based data-linkage for chronic hepatitis C (CHC) patients from 1992-2012. Quantitative digital image analysis of liver biopsies was used for CPA measurement.

Results

533 patients with a biopsy size greater than or equal to 5 mm were included. Median follow up was 10.5 years. 26 developed hepatocellular carcinoma (HCC), 39 developed liver decompensation and 33 had liver related death. 453 had Metavir F0-F2 and 80 had F3-F4. CPA ranged from 1.3%-44.6%. CPA and Metavir stage were independently associated with liver related death. Metavir stage, CPA stage and age were independently associated with HCC. CPA stage (C1: 0%-5%, C2: 5%-10%, C3: 10%-20%, C4: >20%) stratified risk and a significant difference in outcomes was present between all CPA stages for HCC and between C2-C3 and C3-C4 for decompensation and liver related death. The 15 year composite endpoint-free survival was 97% for C1, 89% for C2, 60% for C3, 7% for C4. C4 had significantly worse survival than ⩽C3 (p<0.001) in cirrhotic patients.

Conclusions

CPA stage gave additional information regarding risk stratification for adverse clinical outcomes independent of Metavir stage.

Abbreviations: CPA, collagen proportional area, CHC, chronic hepatitis C, HCC, hepatocellular carcinoma, HCV, hepatitis C virus, AUROC, area under receiver operating characteristic curves, HR, hazard ratio

Keywords: Collagen proportional area, Chronic hepatitis C, Histological stage, Liver complication, Liver related death

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An Inexpensive and Worldwide Available Digital Image Analysis Technique for Histological Fibrosis Quantification in Chronic Hepatitis C

Journal of Viral Hepatitis

C. F. F. Campos, D. D. Paiva, H. Perazzo, P. S. Moreira, L. F. F. Areco, C. Terra, R. Perez, F. A. F. Figueiredo

J Viral Hepat. 2014;21(3):216-222.

Abstract and Introduction

Abstract

Hepatic fibrosis staging is based on semiquantitative scores. Digital imaging analysis (DIA) appears more accurate because fibrosis is quantified in a continuous scale. However, high cost, lack of standardization and worldwide unavailability restrict its use in clinical practice. We developed an inexpensive and widely available DIA technique for fibrosis quantification in hepatitis C, and here, we evaluate its reproducibility and correlation with semiquantitative scores, and determine the fibrosis percentage associated with septal fibrosis and cirrhosis. 282 needle biopsies staged by Ishak and METAVIR scores were included. Images of trichrome-stained sections were captured and processed using Adobe® Photoshop® CS3 and Adobe® Bridge® softwares. The percentage of fibrosis (fibrosis index) was determined by the ratio between the fibrosis area and the total sample area, expressed in pixels calculated in an automated way. An excellent correlation between DIA fibrosis index and Ishak and METAVIR scores was observed (Spearman's r = 0.95 and 0.92; P < 0.001, respectively). Excellent intra-observer reproducibility was observed in a randomly chosen subset of 39 biopsies with an intraclass correlation index of 0.99 (95% CI, 0.95–0.99). The best cut-offs associated with septal fibrosis and cirrhosis were 6% (AUROC 0.97, 95% CI, 0.95–0.99) and 27% (AUROC 1.0, 95% CI, 0.99–1), respectively. This new DIA technique had high correlation with semiquantitative scores in hepatitis C. This method is reproducible, inexpensive and available worldwide allowing its use in clinical practice. The incorporation of DIA technique provides a more complete evaluation of fibrosis adding the quantification to architectural patterns.

Introduction

Chronic hepatitis C is a major public health problem and continues to be a leading cause of chronic liver disease and cirrhosis worldwide.^[1] Liver fibrosis staging is still essential in management of these patients. The accurate assessment of hepatic fibrosis plays a critical role determining antiviral treatment, screening strategies and prognosis.^[1,2] Furthermore, patients with advanced fibrosis and cirrhosis have a poor prognosis, presenting lower survival rate than mild fibrosis patients.^[3–8]

Currently, liver biopsy is still considered the gold standard for fibrosis staging.^[9] Traditional histological assessment is based on semiquantitative scoring systems (Ishak and METAVIR score).^[10,11] Although frequently used, these scoring systems do not allow a precise quantification of liver fibrosis. Moreover, they are subjective measurements with high rates of intra- and interobserver variability.^[12]

During the last years, methods to quantify liver fibrosis through computer software, called digital image analysis (DIA), have been developed.^[13–22] DIA allows quantitative assessment of fibrosis using pixel counting to estimate fibrosis area. The great advantage of this method over semiquantitative scores is that DIA is a truly quantitative method, not influenced by subjective visual interpretation of the observer.^[23] This new technology has been applied to estimate liver fibrosis in chronic viral hepatitis.^[21,24] However, the use of different softwares, high cost, lack of standardization of this method and worldwide unavailability restrained its use to a few numbers of specialized centres.^[25]

The primary aim of this study was to develop an inexpensive and worldwide available DIA technique for fibrosis quantification in liver biopsies of patients with chronic hepatitis C. Secondary aims were (i) to compare METAVIR and Ishak scoring systems with this new quantitative assessment of fibrosis, (ii) to determine the intra-observer reproducibility of the fibrosis index and (iii) to establish the most accurately cut-off associated to septal fibrosis and cirrhosis.

Material and Methods

This prospective observational study included liver core needle biopsies obtained from 282 patients with chronic hepatitis C at the Pathology Department, University of the State of Rio de Janeiro, Brazil. Hepatitis C infection was characterized by the presence of HCV-RNA in blood serum, and liver biopsy was performed according to routine clinical practice.

The study protocol was conducted in accordance with the ethical principles, the guidance of the Helsinki Declaration and the Good Clinical Practice Guidelines. The study was approved by the local Ethics Committee and all patients signed the informed consent.

Histological Analysis

All samples were obtained with 14G Menghini needles, fixed in a 10% neutral-buffered formalin solution and cut in 5-mm-thick sections. Routinely, haematoxylin and eosin, Masson's trichrome and reticulin stains were performed. The inclusion criteria of the liver sample were at least five complete portal tracts and size of 15 mm.

A single experienced pathologist (P.S.M.) evaluated all samples, blinded to clinical data and DIA results. Staging was carried out using the Ishak and METAVIR scores.^[10,11] Based on these two semiquantitative scores, two clinical scenarios were considered as follows: septal fibrosis (Ishak score ≥ 3 or METAVIR ≥ 2), indicative of antiviral treatment and cirrhosis (Ishak score 6 or METAVIR 4), indicative of varices and hepatocellular carcinoma surveillance.

Digital Image Analysis

The digital imaging acquisition system consisted of an Olympus E-330 7.5 megapixels camera (Olympus Corporation, Tokyo, Japan) attached by an adapter to a trinocular Olympus BX 41 microscope (Olympus Corporation). The camera was connected to an analogue Sony PVM-14N5U Trinitron monitor (Olympus Corporation), to facilitate the visualization of the microscopic fields. The overall costs of this assemble were approximately US$ 5650.

Each sample was digitalized in a sequential way, respecting the linear distribution of microscopic fields of the core needle biopsies, using a 40× magnification objective. The images were captured using automatic adjustments for white and luminosity of the camera, with maximal resolution (3748 × 2736 pixels), and saved as 24 bits RGB images in the Joint Photographic Experts Group (JPEG) format. Depending on the size of the sample, six to eighteen images were captured and saved in folders identified with each protocol number, in a personal computer (PC). Using Adobe® Bridge® (Adobe Corporation, San Jose, CA, USA), running in a Windows® 7 64 bits environment, the folder containing the captured images of each individual biopsy was identified. The average cost of the software was U$ 1200.

The images were selected and the following command line was taken as follows: 'Tools', 'Photoshop' and 'Photomerge'. Following this command line, the Adobe® Photoshop® program started automatically and a menu box containing the instructions with the selected files was displayed. In the checkboxes, the options 'Auto' in the 'Layout' space and 'Blend images together' in 'Source files' were, by default, selected. Afterwards, running the command, the program automatically mixed the digitalized microscopic fields and a panoramic wide field image representing the entire area of the biopsy stained with Masson's trichrome was constructed. This wide field panoramic image was saved in the JPEG format, with maximal resolution, representing, each one, a single, totally digitalized virtual image of the biopsy.

The following actions were taken for individual adjustments of the wide field biopsy images: extraction of the background and undesired elements like fragments of the capsule, soft tissues and thick vessels walls, using the command 'Extract' in the 'Filters' tool menu. Using the 'Auto levels' action, in the 'Image', 'Adjustments' menu, lightness and brightness were automatically adjusted for each wide field image. Afterwards, following the technique described by Dahab et al.,^[20] using the 'Selective Color' command presented under the 'Image' drop-down menu, the red, magenta, cyan and blue colours were adjusted after checking the 'Relative' option in the 'Selective Color' dialogue box. From the colour table, the red and magenta colours were chosen sequentially and their cyan component was reduced to -100% and the magenta component expanded to +100%. The cyan and blue colours were selected, their cyan component expanded to +100% and their magenta component reduced to –100%.

For the automatic calculation of the pixels corresponding to the total area of the sample, using the 'Magic Wand Tool', from the tools palette, the extracted background was selected and, using the command 'Select Inverse' of the 'Magic Wand Tool', the area corresponding to the biopsy tissue stained in cyan/blue, red/magenta and grey was, then, selected. The histogram command serves as an internal measurement of tonal distribution as the basis for automated image manipulation. The histogram of the selected area, which represented the totality of pixels of the sample counted the total area of the digitalized biopsy in pixels. Next, the 'Magic Wand Tool' and the 'Similar' command were applied again and all the cyan/blue area of the sample, corresponding to the fibrous tissue, was selected. The histogram of the selected area represented the totality of fibrous tissue in the sample.

The mean time spent in the process of acquisition and image analysis was around 15 min. Smaller samples (consisting of six images before the generation of the wide field image) took around 10 min from the capture process until the last steps of image analysis, while larger samples (consisting of eighteen images before the generation of the wide field image) consumed around 20 min.

The result of this process is shown in Fig. 1. The fibrosis index (FI) was the total area of fibrosis divided by the total area of the section multiplied by 100, as showed by Dahab et al.:^[20]

Figure 1. Wide field image representing the entire biopsy (Ishak score 1, METAVIR F1). Fibrosis area = 2800 pixels. Total area = 122495 pixels. Fibrosis index = 2800 × 100/122495 = 2.2%.

Intra-observer reproducibility was assessed in a randomly chosen a subset of 39 biopsies. The pathologist was blinded to the initial measurements and to the Ishak and METAVIR scores.

Statistical Analysis

Statistical analyses were performed using SPSS (v.17.0.0) software (SPSS Inc., Chicago, IL, USA) and MedCalc software package version 12.2 (MedCalc Software, Mariakerke, Belgium). In all analyses, significance was determined when P < 0.05 assuming two-tailed tests. Spearman's correlation index was used to evaluate the correlation of the DIA technique with METAVIR and Ishak stages. The intraclass correlation coefficient was used to measure the intra-observer reproducibility. The receiver operating characteristic (ROC) curve and area under the ROC (AUROC) curve were applied to establish the fibrosis index associated with septal fibrosis and cirrhosis.

Results

This study showed that was possible to develop an inexpensive and worldwide DIA technique for liver fibrosis quantification using hardware and software easily found in the market. The total cost of the system was less than US$ 8000.

Correlation Between DIA Technique and Semiquantitative Scores

The fibrosis indexes obtained by DIA according to Ishak and METAVIR scores are shown in Table 1 and in Figs 2 & 3, respectively. An excellent correlation between FI obtained by DIA and Ishak and METAVIR semiquantitative scoring systems was observed (Spearman's r = 0.95 and 0.92, respectively, P < 0.001).

Table 1.  Fibrosis index according to staging by Ishak and METAVIR scores

Ishak score		METAVIR score
Stage (n)	Fibrosis index (%)	Stage (n)	Fibrosis index (%)
0 (5)	0.8 ± 0.05	0 (5)	0.8 ± 0.05
1 (41)	2.5 ± 0.7	1 (99)	3.9 ± 1.7
2 (58)	4.9 ± 1.5	2 (79)	7.4 ± 1.5
3 (79)	4.1 ± 1.5	3 (77)	20.4 ± 5
4 (28)	15.3 ± 3.5	4 (22)	34.6 ± 1.6
5 (49)	23.3 ± 2.9
6 (22)	34.6 ± 1.6

Figure 2. Distribution of mean (± confidence internal (CI)) fibrosis percentage data by Ishak scores.

Figure 3. Distribution of mean (± confidence internal (CI)) fibrosis percentage data by METAVIR scores.

Assessment of Intra-observer Reproducibility

To evaluate the reproducibility of this new technique, a subset of 39 randomly specimens were re-evaluated by the same observer. These samples were representative of all stages of fibrosis, and the observer was blinded to clinical data and first measurements results. Excellent intra-observer reproducibility was observed, resulting in an intraclass correlation index of 0.99 (95% CI 0.95–0.99).

Diagnosis of Septal Fibrosis and Cirrhosis

ROC analysis for fibrosis index yielded an optimum cut-off of 6% with an AUROC of 0.97 (95% CI, 0.95–0.99) as the best power distinction for septal fibrosis according to both semiquantitative scoring systems providing a sensitivity of 91% (95% CI, 86–95%) and specificity of 99% (95% CI, 95–100%). Furthermore, the most accurate cut-off for diagnosis of cirrhosis was 27% with an AUROC of 1.0 (95% CI, 0.99–1) according to both scores classification, presenting a sensitivity of 100% (95% CI, 85–100%) and specificity of 100% (95% CI, 99–100%).

Discussion

Liver fibrosis is a major parameter guiding the diagnosis and prognosis of chronic liver disease.^[3] Studies based on liver biopsies to evaluate fibrosis in chronic hepatitis C usually relies on categorical scoring systems rather than direct measurement of the amount of fibrous tissue.^[22] Several DIA techniques for liver fibrosis quantification have been developed.^[13–22] It is considered as a promising tool because it provides results on a continuous scale, rather than merely five or seven qualitative stages.^[22] In this article, we described an inexpensive and worldwide available DIA technique that provides objective quantification of fibrosis in liver biopsies without the need of expensive digital glass-scanning devices and third party software.

Adobe® Photoshop® is largely used in the biomedical literature and considered 'inexpensive and commonly available' imaging software.^{[19,20,26–29]} Two previous studies described DIA techniques for liver fibrosis quantification using older versions of this software.^[19,20] The newer versions can be easily acquired from the World Wide Web. This software upgrade combined with the improvement of hardware performance allowed the development of very powerful image analysis tools. These make possible the whole sample digitalization (virtual large field images of the biopsies) without needing expensive glass-scanning devices.

Although Sirius Red is known as one of the best techniques for collagen histochemistry quantification of liver fibrosis,^[30] the authors preferred to use Masson's trichrome stain. This staining method is worldwide available in most pathology laboratories, and thus, more suitable to the purposes of the present study. A perfect contrast between fibrous tissue stained in blue and parenchyma stained in red was obtained following the 'Selective color' procedure described in the methodology, using the Masson's trichrome stain. We reinforce that similar results can be reached in a liver biopsy slide stained with Sirius Red by adjusting different colour pallets: reds and yellows. In samples stained by this histochemical method, red and yellow colours will be representative of fibrous tissue and parenchyma, respectively.

Our DIA technique had an excellent correlation with the traditional semiquantitative Ishak score and METAVIR classification (r = 0.95 and r = 0.92, respectively). Other studies have already observed a high correlation between DIA techniques and staging.^[18,21] It demonstrated a high capacity of DIA techniques in discriminating the different stages of fibrosis. Our study includes a larger sample and evaluated a cheaper method. Another advantage of the DIA method described in this study is its reproducibility with high rates of intra-observer concordance. We showed an intraclass correlation index of 0.99 (95% CI 0.95–0.99). This reinforces its reliability. Further studies are welcome to define interobserver variability and intercenter reproducibility. A small amount of training time and minimal knowledge in informatics are enough to accurately reproduce the method described. We anticipate that an easy learning curve, low cost and use of worldwide available technology will permit the application of this method in most pathology laboratories.

Important clinical cut-off points for septal fibrosis and for cirrhosis were established in this study. Patients with fibrosis index higher than 6% would have indication for antiviral therapy, while patients with fibrosis index higher than 27% would have indication for endoscopy and hepatocellular carcinoma surveillance. The role of these cut-off points in clinical practice should be better investigated in further studies.

The strengths of our DIA method were that images were processed on colour RGB model, the area of fibrosis was not manually manipulated, the software has adjustments of colours allowing a greater contrast between fibrosis and normal hepatic tissue, and the hardware/software packet is inexpensive and worldwide available. It also should be highlighted that all the fibrosis stages were well represented in this large sample size. Thus, this DIA method can be apply in current clinical practice with a very low cost.

In our methodology, the quantification of fibrosis automatically selects all blue sample allowing to map fibrous tissue including the delicate perisinusoidal fibrosis, which is usually undetected by examiner's eye. The precisely quantification of perisinusoidal fibrosis might be an advantage of this method in quantitative assessment of liver fibrosis in patients with others chronic liver disease than CHC, especially nonalcoholic fatty liver disease (NAFLD). Digital quantification of fibrosis by this method can be a future tool to ease the differentiation between patients with simple steatosis from those with nonalcoholic steatohepatitis (NASH). It can also contribute for a more precise staging in the last condition.

The histological diagnosis based on semiquantitative architectural changes in association with DIA techniques may express more precisely the actual fibrosis state. This combination allows evaluating not only the fibrosis distribution but also the fibrosis amount. We agree with the idea that DIA techniques are not suitable for replacing traditional qualitative and semiquantitative liver biopsy evaluation.^[31] More important, it should be considered as a complementary tool for the traditional histological methods.

Some potential applications for DIA techniques as complementary tool could be expected. In patients undergoing sequential liver biopsies showing the same stage, the variation in fibrosis index could estimate more precisely the progression of fibrosis. Another potential application could be in clinical trials for measuring the effect of novel antifibrogenic therapies when more objective and broader scale information is needed. This would be important for the validation of new noninvasive imaging techniques and indirect serum markers of fibrosis.^[32,33] It could also be useful in case of discordance of staging among different pathologists.

In summary, the DIA technique for liver fibrosis quantification described in this article is inexpensive and was developed using worldwide available technologies. The process is simple, reproducible and showed a high correlation with semiquantitative scores. It provides a more complete evaluation of fibrosis adding the quantification to the architectural patterns.

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Source

The evolution of non-invasive tests of liver fibrosis is associated with prognosis in patients with chronic hepatitis C

Hepatology

Accepted Article (Accepted, unedited articles published online and citable. The final edited and typeset version of record will appear in future.)

Original Article

J. Vergniol¹, J. Boursier^2,4, C. Coutzac¹,  S. Bertrais⁴, J. Foucher¹, C. Angel², F. Chermak¹, I. Fouchard Hubert^2,4, W. Merrouche¹, F. Oberti^2,4, V. de Lédinghen^1,3, P. Calès^2,4

DOI: 10.1002/hep.27069

Copyright © 2014 American Association for the Study of Liver Diseases

Publication History
Accepted manuscript online: 12 FEB 2014 05:58AM EST
Manuscript Accepted: 6 FEB 2014
Manuscript Revised: 25 JAN 2014
Manuscript Received: 29 SEP 2013

Keywords: hepatitis C;  survival;  prognosis;  fibrosis;  cirrhosis;  liver stiffness;  FibroScan;  blood fibrosis test

Abstract

Introduction: No data are available about the prediction of long-term survival using repeated non-invasive tests of liver fibrosis in chronic hepatitis C (CHC). We aimed to assess the prognostic value of 3-year liver stiffness measurement (LSM), APRI, and FIB-4 evolution in CHC.

Patients and methods: CHC patients with two LSM (1000-1500 days interval) were prospectively included. Blood fibrosis tests APRI and FIB-4 were calculated the day of baseline (bLSM) and follow-up (fLSM) LSM. Evolution of fibrosis tests was expressed as delta: (follow-up-baseline results)/duration. Date and cause of death were recorded during follow-up that started the day of fLSM.

Results: 1025 patients were included. Median follow-up after fLSM was 38.0 months (IQR: 27.7-46.1) during which 35 patients died (14 liver-related death) and 7 had liver transplantation. Prognostic accuracy (Harrell C-index) of multivariate models including baseline and delta results was not significantly different between LSM and FIB-4 (p≥0.24) whereas FIB-4 provided more accurate prognostic models than APRI (p=0.03). By multivariate analysis including LSM variables, overall survival was independently predicted by bLSM, delta (dLSM), and SVR. Prognosis was excellent in patients having bLSM <7 kPa, SVR, or no increase (<1 kPa/year) in 7-14 kPa bLSM. Prognosis was significantly impaired in patients with increase (≥1 kPa/year) in 7-14 kPa bLSM, or decrease (≤0 kPa/year) in ≥14 kPa bLSM (p=0.949 between these two groups). Patients with increase (>0 kPa/year) in ≥14 kPa bLSM had the worst prognosis. Baseline and delta FIB-4 also identified patient subgroups with significant different prognosis.

Conclusion: Three-year evolution of non-invasive tests of liver fibrosis has a strong prognostic value in CHC patients. These tests should be repeated to monitor patients and predict their outcome. (Hepatology 2014;)

Source

Fibrosis progression in human immunodeficiency virus/hepatitis C virus coinfected adults: Prospective analysis of 435 liver biopsy pairs - 'HIV/HCV Coinfected's Liver Disease Can Progress Quickly'

Provided by NATAP

Download The PDF Here

Hepatology
Jan 16 2014
Early View (Online Version of Record published before inclusion in an issue)

\Monica A. Konerman,1 Shruti H. Mehta,2 Catherine G. Sutcliffe,2 Trang Vu,1 Yvonne Higgins,1 Michael S. Torbenson,3 Richard D. Moore,1,2 David L. Thomas,1,2 and Mark S. Sulkowski1

From the 1Johns Hopkins Hospital/University School of Medicine, Baltimore, MD; 2Johns Hopkins Bloomberg School of Public Health, Baltimore, MD; 3Johns Hopkins Hospital Department of Pathology, Baltimore, MD.

-----------------------------

HIV/HCV Coinfected's Liver Disease Can Progress Quickly: "(34%) had progression of one or more METAVIR stages between biopsies and are referred to as "progressors......Measures of obesity (BMI, P = 0.02), diabetes (P = 0.01), and hepatic steatosis (P = 0.01) at the time of the first biopsy were associated with progression of fibrosis on the subsequent liver biopsy"....genotype 1 is identified in Table 4 below as being associated with fibrosis progression\

from Jules: many of you may recall this was presented initially as an abstractat a major conference several years ago.

"our data related to the incidence and correlates of progressive hepatic fibrosis among 282 HIV/HCV coinfected adults who underwent serial fibrosis staging resulting in 435 paired liver biopsies provide several insights into liver disease progression in HIV/HCV coinfected patients."

"In conclusion, approximately one-third of HIV/HCV coinfected patients experienced fibrosis progression of at least one METAVIR stage over a relatively short period of time, including patients with no or minimal fibrosis on first biopsy and those taking ART. Patients with persistent liver enzyme elevation, particularly of serum AST, were more likely to progress, suggesting that this simple measurement may be useful in identifying coinfected patients at greater risk for HCV disease progression. The association of obesity and its related complications with fibrosis progression underscores the potential importance of this modifiable risk factor. However, our limited ability to accurately predict progression in most patients, underscores the need for additional research to understand the basis for variable HCV disease progression in HIV-infected patients."

"The majority of coinfected patients in our prospective cohort had minimal fibrosis on initial liver biopsy. Nonetheless, over a median follow-up time of 2.5 years we observed fibrosis progression (≥1 METAVIR stage) in one-third of individual patients between the first and second liver biopsy (n = 282) and among one-third of all biopsy pairs (n = 435). While the majority of histologic change was limited to one METAVIR stage, progression of two or more stages was found in ~9% of biopsy pairs. Further,nearly 45% of patients with no evidence of hepatic fibrosis on the initial biopsy had at least stage 1 fibrosis on subsequent liver biopsy. This observed incidence of fibrosis progression over a relatively short time interval is consistent with our earlier observations and those reported in other HIV/HCV coinfected patient cohorts in which progressive disease was noted in 17% to 50% of paired histologic evaluations"

"Our finding of steatosis as a predictor of fibrosis progression is in concordance with recent investigations on this topic that have found the presence of steatosis to be strongly associated with advanced fibrosis"

"Among the 435 biopsy pairs, 149 (34%) had progression of one or more METAVIR stages between biopsies and are referred to as "progressors"(Table 3). Markers of increased hepatic inflammation measured by median AST and ALT as well as noninvasive measures of liver disease (AST-platelet ratio index [APRI] and FIB4 index) were associated with subsequent fibrosis progression.Measures of obesity (BMI, P = 0.02), diabetes (P = 0.01), and hepatic steatosis (P = 0.01) at the time of the first biopsy were associated with progression of fibrosis on the subsequent liver biopsy. Compared to nonobese patients, those with BMI >30 at the time of the initial biopsy had higher AST levels, higher prevalence of steatosis, higher histological activity indexes, and were more likely to be male "

"we did not detect an association of ART, HIV RNA suppression, or CD4 cell count with fibrosis progression. In contrast, we recently observed that the receipt of ART was independently associated with a 66% reduction in the risk of HCV-related clinical outcomes including end-stage liver disease, hepatocellular carcinoma, or liver-related death.[25] Taken together, these findings suggest that while the treatment or prevention of HIV disease may reduce liver inflammation and clinical outcomes, ART alone is not sufficient to prevent fibrosis progression in coinfected patients."

"Similar to baseline correlates, measures of hepatic inflammation were significantly different between progressors and nonprogressors between biopsies. The median AST and ALT levels between biopsies were significantly higher among biopsy pairs with fibrosis progression compared to those without fibrosis progression (P = 0.0004 for ALT and P < 0.0001 for AST). In addition, between biopsies, pairs with fibrosis progression had a significantly greater proportion of ALT and AST values >2.5 times the upper limit of normal compared with those without progression (P = 0.0002 for ALT and P < 0.0001 for AST). Time between biopsies was not associated with progression. After adjustment for baseline and between biopsy factors in multivariate analysis, the proportion of AST level >100 IU/mL between biopsies was independently associated with subsequent fibrosis progression for all biopsy pairs and for biopsy pairs restricted to those with minimal fibrosis (stage 0 or 1) (Table 4)"

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Fibrosis progression in human immunodeficiency virus/hepatitis C virus coinfected adults: Prospective analysis of 435 liver biopsy pairs

Monica A. Konerman,1 Shruti H. Mehta,2 Catherine G. Sutcliffe,2 Trang Vu,1 Yvonne Higgins,1 Michael S. Torbenson,3 Richard D. Moore,1,2 David L. Thomas,1,2 and Mark S. Sulkowski1 From the 1Johns Hopkins Hospital/University School of Medicine, Baltimore, MD; 2Johns Hopkins Bloomberg School of Public Health, Baltimore, MD; 3Johns Hopkins Hospital Department of Pathology, Baltimore, MD.

ABSTRACT:

Human immunodeficiency virus (HIV)/hepatitis C virus (HCV) coinfection is associated with progressive liver disease. However, the rate of progression is variable and the ability to differentiate patients with stable versus progressive HCV disease is limited. The objective of this study was to assess the incidence of and risk factors for fibrosis progression in a prospective cohort of coinfected patients. Overall, 435 liver biopsy pairs from 282 patients without cirrhosis were analyzed. Biopsies were scored according to the METAVIR system by a single pathologist blind to biopsy sequence. Fibrosis progression was defined as an increase of at least one METAVIR fibrosis stage between paired biopsies. The majority of patients were African American (84.8%), male (67.7%), and infected with HCV genotype 1 (93.4%). On initial biopsy, no or minimal fibrosis was identified in 243 patients (86%). The median interval between biopsies was 2.5 years. Fibrosis progression was observed in 97 of 282 (34%) patients and 149 of 435 (34%) biopsy pairs. After adjustment, greater body mass index (adjusted odds ratio [aOR]: 1.04 per 1 unit increase), diabetes (aOR: 1.56), and hepatic steatosis (aOR: 1.78) at the time of initial biopsy were marginally associated with subsequent fibrosis progression. Between biopsies, elevated serum aspartate and alanine aminotransferase (AST, ALT) (aOR AST: 3.34, ALT: 2.18 for >25% values >100 U/L versus <25% values >100 U/L) were strongly associated with fibrosis progression. Conclusion: Fibrosis progression is common among HIV/HCV coinfected patients; these data suggest that progression can be rapid. Persistent elevations in serum transaminase levels may serve as important noninvasive markers to identify subsets of patients who are more likely to progress and thus warrant closer monitoring and consideration of HCV treatment.

Due to shared modes of transmission, 15%-30% of individuals with human immunodeficiency virus (HIV) infection are coinfected with hepatitis C virus (HCV).[1, 2] In the era of antiretroviral therapy (ART), chronic HCV infection leads to progressive liver disease, resulting in end-stage liver disease, hepatocellular carcinoma, and death in some, but not all coinfected patients.[3-5] While the variable progression of HCV disease is well recognized, the rate and risk factors for progressive liver disease in HIV/HCV coinfected patients are incompletely understood. Several studies conducted shortly after the availability of highly active ART suggest that effective treatment of HIV may be associated with decreased risk of liver disease progression.[6-10] However, the contributions of other potentially modifiable (e.g., obesity) and unmodifiable (e.g., age) factors to the worsening of hepatic fibrosis have not been determined. Greater understanding of such factors may have important implications for the clinical management of HIV/HCV coinfected patients. For example, current HCV treatment guidelines for HIV-infected patients recommend treatment of those patients at the greatest risk for developing liver disease.

Some, but not all, expert guidelines recommend HCV treatment for HIV-infected patients independent of biopsy stage based on an assumption of rapidly progressive disease in this population.[11-17] The identification of factors associated with progression may help to refine clinical decision-making as well as identify potentially modifiable exposures. Accordingly, the objective of this study was to determine the incidence of and risk factors for fibrosis progression in a prospective cohort of coinfected adults who underwent serial liver biopsy with the aim of identifying coinfected patients with no or minimal fibrosis who are at risk for progressive liver disease over a relatively short period of time.

Patients and Methods

Study Population

This prospective cohort study evaluated 289 HIV/HCV coinfected adults who received medical care in an urban HIV clinic in Baltimore, Maryland, from July 1993 until December 2008. Treatment for HIV and/or HCV was provided by healthcare providers according to published practice guidelines.[18, 19]Individuals with at least two liver biopsies as part of their medical care were included in the study. A total of 282 patients had an initial noncirrhotic biopsy and were assessed. Of these individuals, 124 had more than two liver biopsies including 97 patients with three biopsies, 25 with four biopsies, and two with five biopsies. In total, these 282 patients contributed 435 liver biopsy pairs to the analysis. For all patients, demographic, clinical, and laboratory data were abstracted from patient charts and a laboratory database by trained personnel. Data on injection drug use and alcohol abuse were ascertained based on physician diagnosis, chart review, and self-reports.

Laboratory Evaluations

All subjects had standard laboratory assessments performed by licensed clinical laboratories, including a complete blood cell count, serum chemistry panels, alanine aminotransferase (ALT) levels, aspartate aminotransferase (AST) levels, CD4 cell count, and plasma HIV-RNA level. HCV antibody testing was performed using a sensitive and specific enzyme immunoassay. HCV RNA and genotype testing were performed using reverse-transcriptase polymerase chain reaction.

Liver Histology

A transcutaneous liver biopsy was performed using an 18G needle. Liver tissue was fixed in 10% formalin, and paraffin-embedded sections were stained with hematoxylin-eosin and trichome stains. Biopsies were scored according to the METAVIR and the modified histological activity index scoring system by a single pathologist (M.T.) blind to biopsy sequence. The scale to classify fibrosis was as follows: F0 = no fibrosis; F1 = portal fibrosis without septa; F2 = portal fibrosis with few septa; F3 = numerous septa without cirrhosis; F4 = cirrhosis. Steatosis was scored based on the percentage of hepatocytes affected according to a 5-point scale as follows: Grade 0: none; 1: <5% fat; 2: 5%-<30% fat; 3: 30%-60% fat; 4: >60% fat.[20] All biopsies were deemed adequate for inclusion based on expert opinion by the hepatopathologist (M.T.) including assessment of size and number of portal tracts. The median length of the first biopsy of a pair was 12.0 mm (interquartile range [IQR] 10.0, 14.0 mm), while the median length of the second biopsy of a pair was 13.0 mm (IQR 10.0, 15.0 mm). The median number of portal tracts for the first biopsy of a pair was 10 (IQR 8, 13), and for the second biopsy of a pair was 11 (IQR 9, 14).

Statistical Analysis

Significant fibrosis progression was defined as an increase of at least one METAVIR stage between the biopsies. The proportion of patients who had fibrosis progression was equivalent when analyzed using the Ishak scoring system (Supporting Table 1). Subsequently, the remaining analysis was done using the METAVIR scoring system alone. To characterize changes over time, we used 435 biopsy pairs contributed by 282 individuals such that the unit of analysis was the biopsy pair and not the individual (e.g., for a patient with three biopsies, biopsy 1 -> 2 was analyzed as one pair and biopsy 2 -> 3 as a second pair).

Univariate and multivariate logistic regression with generalized estimating equation were used to assess determinants of fibrosis progression in order to account for the correlation of biopsy pairs from within the same individuals. Variables associated with fibrosis progression in univariate analysis with P < 0.15 were considered for multivariate models. Time between biopsies was included as a covariate in the models as a categorical variable (<2, 2-2.9, 3-3.9, 4+ years). Further variables that had been previously identified as predictors of progression (gender, race, and age) were forced into models regardless of statistical significance. A series of models were built, first including fixed covariates and covariates at first biopsy and then including covariates between serial biopsies. Due to colinearity, separate models were built for laboratory values measured between serial biopsies.

Predictors of interest included fixed characteristics, characteristics at the time of the first biopsy, and characteristics between the serial biopsies. Fixed characteristics included demographics, HCV genotype, and history of alcohol or injection drug use. Characteristics at the time of the first biopsy (within six months) included diabetes, body mass index (BMI), CD4 count, HIV-RNA level, ever and cumulative ART exposure up to the first biopsy, HCV-RNA level, HCV treatment before initial biopsy, cumulative HCV treatment, duration of HCV infection (estimated by age at first injection), median AST and ALT levels, hepatic steatosis, histological necroinflammatory activity, and METAVIR score. BMI was categorized according to the standard classification system as follows: normal 18.5-24.99, overweight 25-29.99, obese ≥30. The presence of diabetes was determined by clinical diagnosis.

Predictors between biopsies included any ART use between biopsies, cumulative ART use between biopsies, change in CD4 cell count and HIV viral load, HCV treatment, change in AST and ALT levels, and change in BMI. HIV viral load and CD4 cell count between biopsies were analyzed as the proportion of CD4 cell counts that were <200 cells/μL and the proportion of HIV RNA measurements that were undetectable (<400 copies/mL). ALT and AST were examined as the cumulative proportion of ALT and AST levels more than 2.5 times the upper limit of normal reference range (AST 37 U/L; ALT 40 U/L). BMI change was defined as greater than a one unit increase or decrease in BMI.

Analyses were performed using SAS v. 9.1 software (SAS Institute, Cary, NC). Approval
This study was approved by the Johns Hopkins Medicine Institutional Review Boards and written informed consent was obtained for all participants.

Results

Study Population

The demographic and clinical characteristics of the study population at initial biopsy are shown in Table 1. The median age was 44.5 years (IQR 40.5, 48.7). The majority of individuals were African American (84.8%), male (67.7%), and infected with HCV genotype 1 (93.4%). A history of injection drug use (76.6%) and alcohol abuse (48%) were frequently reported. The median BMI was 25.4 (IQR 22.5, 29.2). At the time of first biopsy most patients were receiving ART (69.2%) and had been for a median duration of 1.9 years (IQR 0, 4.3). Only 28% of patients had never received ART. The median CD4 cell count was 386 cells/μL and 15.9% had CD4 cell counts <200 cells/μL. The majority of patients (55.9%) had an HIV RNA level below the limit of detection. The median ALT and AST were 47 U/L (IQR 31, 75) and 46 U/L (IQR 33, 71), respectively. AST levels exceeding 100 U/L were observed in 14.7% (40 of 272) of patients at the time of the first biopsy and were associated with clinical history of alcohol abuse and the absence of ART (Supporting Table 2). The median HCV-RNA level was 700,000 IU/mL (IQR 500,000-1,530,000). Most patients (279 of 282, 99%) had not received HCV treatment before initial biopsy.

On initial biopsy, no or minimal fibrosis (METAVIR stage 0 or 1) was identified in 243 patients (86%), whereas 31 patients (11%) had METAVIR stage 2, and eight patients (2.8%) had METAVIR stage 3. With regard to necroinflammatory activity, 173 patients (65.8%) had a score <5 with 90 patients (34.2%) having a score of 5 or greater. Hepatic steatosis (any grade) was observed in 50 patients (12.8%).

Incidence of Fibrosis Progression

The median interval between biopsies was 2.5 years (IQR 2-3.2 years). Fibrosis progression was observed in 97 of 282 (34%) patients between their first and second liver biopsy. Among the 435 biopsy pairs, fibrosis progression was observed in 149 (34%), with 39 biopsy pairs (8.9%) demonstrating an increase of two or more METAVIR fibrosis stages. Notably, fibrosis progression was detected in 45% of 179 pairs in which the initial biopsy of the pair revealed no fibrosis (METAVIR stage 0; Table 2). While the majority of those with progression had stage 1 fibrosis on the second biopsy, 14 biopsy pairs (7.8%) had progression of two or more METAVIR fibrosis stages.

Correlates of Progression at Baseline

Among the 435 biopsy pairs, 149 (34%) had progression of one or more METAVIR stages between biopsies and are referred to as "progressors" (Table 3). Markers of increased hepatic inflammation measured by median AST and ALT as well as noninvasive measures of liver disease (AST-platelet ratio index [APRI] and FIB4 index) were associated with subsequent fibrosis progression. Measures of obesity (BMI, P = 0.02), diabetes (P = 0.01), and hepatic steatosis (P = 0.01) at the time of the first biopsy were associated with progression of fibrosis on the subsequent liver biopsy. Compared to nonobese patients, those with BMI >30 at the time of the initial biopsy had higher AST levels, higher prevalence of steatosis, higher histological activity indexes, and were more likely to be male (Supporting Table 3). The modified histological activity index was not related to fibrosis progression. Measures of biopsy quality including the length of the specimen were not associated with fibrosis progression. The median length was 12 mm among both nonprogressors (IQR 10, 14) and progressors (IQR 9, 14) with a Wilcoxon rank sum test P value of 0.13. Similarly, measures of HIV disease (CD4 cell count, HIV-RNA, ART exposure) at the time of initial biopsy were not significantly different among progressors and nonprogressors. After adjustment for baseline factors in multivariate analysis, AST level >100 IU/mL at the time of the first liver biopsy was independently associated with subsequent fibrosis progression for all biopsy pairs (adjusted odds ratio [aOR] 2.12, 95% confidence interval [CI] 1.06-4.26) but not for biopsy pairs restricted to those with minimal fibrosis (stage 0 or 1) at first biopsy (aOR 1.54, 95% CI 0.63-3.72) (Table 4).

Among pairs METAVIR stage 0 or 1 (n = 371) on initial biopsy, we also characterized the accuracy of elevated AST level (>100 U/L) at the time of the initial liver biopsy in prediction of fibrosis progression. The sensitivity of this threshold was low (13%), with a specificity of 93%. The positive predictive value of AST >100 U/L at baseline was 53%, whereas the negative predictive value was 68%.

Correlates of Progression Between Serial Biopsies

The univariate correlations of exposures between biopsies and progression were similar to those measures at baseline (Table 3). Antiretroviral therapy and suppression of HIV replication between biopsies were not associated with fibrosis progression. The change in CD4 cell count and the proportion of measured HIV-RNA values <400 copies/mL median IQR were also not statistically different between the two groups. Treatment for HCV infection with interferon plus ribavirin was prescribed in between 90 biopsy pairs, 58 (20.5%) of the nonprogressors, and 32 (21.5%) of the progressors (P = 0.81). Of the 90 biopsy pairs, HCV treatment resulted in durable or transient viral response in 17 instances (three in sustained virologic response [SVR] and 14 with relapse). None of the treated progressors achieved SVR, whereas SVR was achieved in three out of 58 treated nonprogressors (P = 0.19).

Similar to baseline correlates, measures of hepatic inflammation were significantly different between progressors and nonprogressors between biopsies. The median AST and ALT levels between biopsies were significantly higher among biopsy pairs with fibrosis progression compared to those without fibrosis progression (P = 0.0004 for ALT and P < 0.0001 for AST). In addition, between biopsies, pairs with fibrosis progression had a significantly greater proportion of ALT and AST values >2.5 times the upper limit of normal compared with those without progression (P = 0.0002 for ALT and P < 0.0001 for AST). Time between biopsies was not associated with progression. After adjustment for baseline and between biopsy factors in multivariate analysis, the proportion of AST level >100 IU/mL between biopsies was independently associated with subsequent fibrosis progression for all biopsy pairs and for biopsy pairs restricted to those with minimal fibrosis (stage 0 or 1) (Table 4). Additional sensitivity analyses were performed to assess the impact of restricting analysis to only the first biopsy in the pair, a change of at least two METAVIR stages, biopsy length, and number of portal tracts and the results were not significantly changed (Supporting Table 4).

Discussion

Effective ART has substantially reduced the incidence of acquired immune deficiency syndrome (AIDS)-related death among HIV-infected adults; among those coinfected with HCV, liver disease has emerged as an important cause of morbidity and mortality. While HIV infection has been consistently associated with more rapid progression of hepatic fibrosis, the mechanisms underlying this association are incompletely understood. In this context, our data related to the incidence and correlates of progressive hepatic fibrosis among 282 HIV/HCV coinfected adults who underwent serial fibrosis staging resulting in 435 paired liver biopsies provide several insights into liver disease progression in HIV/HCV coinfected patients.

The majority of coinfected patients in our prospective cohort had minimal fibrosis on initial liver biopsy. Nonetheless, over a median follow-up time of 2.5 years we observed fibrosis progression (≥1 METAVIR stage) in one-third of individual patients between the first and second liver biopsy (n = 282) and among one-third of all biopsy pairs (n = 435). While the majority of histologic change was limited to one METAVIR stage, progression of two or more stages was found in ~9% of biopsy pairs. Further, nearly 45% of patients with no evidence of hepatic fibrosis on the initial biopsy had at least stage 1 fibrosis on subsequent liver biopsy. This observed incidence of fibrosis progression over a relatively short time interval is consistent with our earlier observations and those reported in other HIV/HCV coinfected patient cohorts in which progressive disease was noted in 17% to 50% of paired histologic evaluations.[3, 9, 10, 21, 22] However, the precision of these prior estimates of progression was limited by small sample size.[9, 21, 22] For example, Schiavini et al.[9]calculated a rate of fibrosis progression of 50% based on analysis of 36 paired liver biopsies. Taken together, these data indicate that progression of HCV disease can occur in HIV/HCV coinfected persons with minimal fibrosis on initial staging. Since most patients had been HCV-infected for many years prior to this first biopsy, this observation suggests that fibrosis progression may be nonlinear in this patient population and underscores the need for serial monitoring in such patients (Supporting Figure 1). Importantly, among persons with minimal disease on first biopsy, the progression was generally limited to one METAVIR stage; as such, serial monitoring allows for the detection of individuals with progressive disease prior to the onset of clinical liver disease such as hepatocellular carcinoma or end-stage liver disease.

We also identified baseline and time-varying factors associated with fibrosis progression between biopsy pairs. Interestingly, HCV genotype 1 was associated with an increased risk of progression in some models. While it is possible that this reflects biological differences in disease related to HCV diversity, our cohort was relatively homogeneous with respect to patient (largely African American) and viral (largely genotype 1) characteristics. As such, this finding requires validation in other settings. Our data confirm the relationship of chronically elevated serum liver enzyme levels, namely AST and ALT levels, and fibrosis progression. While the biologic mechanism underlying the observed relationship of AST level and disease was not directly measured, elevated serum AST levels >100 U/L in our cohort were associated with alcohol abuse and failure to be on ART and may reflect the impact of these factors on disease progression. Similar to our prior study, elevated serum AST at baseline and between histologic assessment were independently associated with progression; HIV/HCV coinfected patients for whom measured AST levels were always <100 U/L were significantly less likely to have evidence of fibrosis progression on the next liver biopsy. Thus, AST level may represent an inexpensive, routinely obtained biomarker to identify persons at greater risk of progressive disease. Interestingly, we found that higher AST levels were associated with clinical history of alcohol abuse and the lack of treatment with antiretrovirals.

Although alcohol is clearly related to HCV disease pathogenesis, accurate assessment of alcohol intake in clinical cohorts may be challenging due to underreporting by patients. The observation that patients with elevated AST levels are at greater risk of progression may represent the effect of undetected alcohol exposure; novel alcohol biomarkers such as phosphatidylethanol or carbohydrate-deficient transferrin may be useful to further assess the contribution of underreported alcohol exposure.[23, 24] Despite the observation that ART exposure was associated with a lower likelihood of having high AST levels, we did not detect an association of ART, HIV RNA suppression, or CD4 cell count with fibrosis progression. In contrast, we recently observed that the receipt of ART was independently associated with a 66% reduction in the risk of HCV-related clinical outcomes including end-stage liver disease, hepatocellular carcinoma, or liver-related death.[25] Taken together, these findings suggest that while the treatment or prevention of HIV disease may reduce liver inflammation and clinical outcomes, ART alone is not sufficient to prevent fibrosis progression in coinfected patients.

We also found that markers of metabolic derangement at initial biopsy were associated with fibrosis progression, although the statistical significance did not persist in all models after multivariate analysis. The impact of obesity and its associated complications, namely hepatic steatosis and diabetes, have appropriately become focal points of investigation in the ART era during which time the metabolic profile of HIV-infected patients has shifted.[26] Our finding of steatosis as a predictor of fibrosis progression is in concordance with recent investigations on this topic that have found the presence of steatosis to be strongly associated with advanced fibrosis.[27-30] In fact, Gaslightwala and Bin[27] found in their investigation of 154 coinfected patients that fibrosis progression rates increased in a linear fashion with the grade of hepatic steatosis. Similarly, diabetes and insulin resistance are additional complications of obesity that have been identified as independent predictors of cirrhosis.[31, 32] Prospective studies are needed to investigate strategies to modify obesity and to assess the impact on the risk of fibrosis progression in HIV/HCV coinfected patients. In the absence of such prospective data, our findings suggest that measures to facilitate weight loss should be a priority in obese coinfected patients and those with a normal BMI should strive to maintain this.

While the major strength of our study is the prospective assessment of histologic disease progression in a large sample of coinfected patients, there are several limitations to our findings. First, our cohort consists primarily of African American patients infected with HCV genotype 1; our findings may not be generalizable to more diverse patient populations including those infected with other HCV genotypes. Second, our patients who underwent serial liver biopsies were engaged in medical care and were willing to undergo multiple liver biopsies; this patient population may differ from coinfected patients who were not referred for care. Noninvasive methods of fibrosis assessment such as liver elastography may be a useful tool to overcome this potential bias. Third, few HCV/HIV coinfected patients who were successfully treated for HCV underwent serial liver biopsy; while not unexpected, this limits our ability to assess the impact of HCV eradication on disease progression. Finally, studies based on liver biopsy are subject to sampling error and misclassification. To limit misclassification, biopsies were read as pairs by a single expert hepatopathologist who was blind to biopsy sequence. The criteria for adequacy of the biopsy specimen used in our cohort may also represent a limitation since we did not apply specific criteria for adequacy based on length or number of portal tracts. However, sensitivity analysis in which such criteria were applied did not change our findings. Finally, while it is possible that some patients with apparent fibrosis progression reflect sampling error, only 33 of 256 biopsy pairs with fibrosis on the first biopsy of the pair had evidence of fibrosis regression (12.8%), suggesting that misclassification was not common.

In conclusion, approximately one-third of HIV/HCV coinfected patients experienced fibrosis progression of at least one METAVIR stage over a relatively short period of time, including patients with no or minimal fibrosis on first biopsy and those taking ART. Patients with persistent liver enzyme elevation, particularly of serum AST, were more likely to progress, suggesting that this simple measurement may be useful in identifying coinfected patients at greater risk for HCV disease progression. The association of obesity and its related complications with fibrosis progression underscores the potential importance of this modifiable risk factor. However, our limited ability to accurately predict progression in most patients, underscores the need for additional research to understand the basis for variable HCV disease progression in HIV-infected patients.

Source