Soluble CD163, a Macrophage Activation Marker, Is Independently Associated With Fibrosis in Patients With Chronic Viral Hepatitis B and C Konstantin Kazankov,1 Francisco Barrera,2 Holger Jon Møller,3 Bo Martin Bibby,4 Hendrik Vilstrup,1 Jacob George,2 and Henning Grønbæk1 Macrophages are involved in inflammation and liver fibrosis and soluble (s)CD163 is a specific marker of activated macrophages. We investigated associations between sCD163 and biochemical and histological parameters of inflammatory activity and fibrosis in 551 patients with chronic hepatitis C virus (HCV) and 203 patients with chronic hepatitis B virus (HBV) before antiviral treatment. Scheuer histological scores of activity and fibrosis were obtained. Clinical, biochemical, and metabolic parameters were recorded. We measured sCD163 by enzyme-linked immunosorbent assay (ELISA). Soluble CD163 was higher in patients with HCV compared to HBV (3.6 [interquartile range (IQR) 2.55.4] versus 2.4 [IQR 1.8-3.6] mg/L, P < 0.001). sCD163 was associated with fibrosis stages for both HCV (odds ratio [OR] 1.49, 95% confidence interval [CI]: 1.38-1.61) and HBV (OR 1.32, 95% CI: 1.17-1.49) patients, with highest levels in patients with advanced fibrosis and cirrhosis. sCD163 was a marker of fibrosis independent of other biochemical parameters and known risk factors. We created two novel sCD163-based fibrosis scores, CD163-HCV-FS and CD163-HBV-FS, which showed areas under the receiver operating characteristics curve (AUROC) of 0.79 (95% CI: 0.74-0.83) and 0.71 (95% CI: 0.62-0.79), respectively, for significant fibrosis. Compared to existing fibrosis scores, CD163-HCV-FS was significantly superior to the aspartate aminotransferase (AST) to platelet ratio index (APRI) for all fibrosis stages and to FIB-4 for significant fibrosis, but CD163-HBV-FS was not. Conclusion: sCD163 levels are increased in patients with chronic viral hepatitis, reflecting macrophage activation. Increased sCD163 is associated with the severity of disease and predicts fibrosis. A sCD163-based fibrosis score, CD163-HCV-FS, is superior to APRI and FIB-4 for the diagnosis of significant fibrosis in patients with HCV infection. (HEPATOLOGY 2014;60:521-530)
T
here is increasing evidence that macrophages are involved in inflammation and fibrosis in liver diseases. In the liver, macrophages are observed in close proximity with cells involved in fibrosis formation and produce a number of growth factors and proinflammatory substances involved in
this process.1,2 The mechanism of action of monocytes and resident liver macrophages (Kupffer cells) in inflammation and fibrogenesis in chronic viral hepatitis is not fully understood, but studies suggest that macrophages function differently in the presence of hepatitis C (HCV) and hepatitis B (HBV) virus.3-5 Further, a
Abbreviations: ALP, alkaline phosphatase; ALT, alanine transaminase; ANOVA, analysis of variance; APRI, AST to platelet ratio index; AST, aspartate transaminase; AUROC, area under the receiver operating characteristics curve; BMI, body mass index; CD163-HBV-FS, sCD163-based fibrosis score for HBV patients; CD163-HCV-FS, sCD163-based fibrosis score for HCV patients; ELISA, enzyme-linked immunosorbent assay; GGT, gamma-glutamyltransferase; HBV, hepatitis B virus; HCV, hepatitis C virus; HOMA-IR, homeostatic model assessment of insulin resistance; IL-6, interleukin-6; INR, international normalized ratio; IQR, interquartile range; MELD, Model for Endstage Liver Disease; NPV, negative predictive value; PCR, polymerase chain reaction; PPV, positive predictive value; sCD163, soluble CD163; TGF-b, transforming growth factor-b; TLR, toll-like receptor; TNF-a, tumor necrosis factor-a. From the 1Department of Hepatology and Gastroenterology, Aarhus University Hospital, Aarhus, Denmark; 2Storr Liver Unit, University of Sydney and Westmead Hospital, Westmead, Australia; 3Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark; 4Department of Biostatistics, Aarhus University, Aarhus, Denmark. Received May 14, 2013; accepted March 11, 2014. Supported by the NOVO Nordisk Foundation, the Danish Strategic Research Council (10-092797), and grants from the National Health and Medical Research Council of Australia (632630, 1049857, 1053206) and the Robert W. Storr Bequest to the Sydney Medical Foundation of the University of Sydney. 521
522
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pathway involving activation of monocytes and macrophages by endotoxin with subsequent up-regulation of proinflammatory cytokines, such as tumor necrosis factor (TNF)-a and interleukin (IL)-6, has been suggested.6 Moreover, activated Kupffer cells secrete transforming growth factor (TGF)-b, which activates hepatic stellate cells and induces fibrosis.7 Thus, Kupffer cells play an important role in liver inflammation and fibrosis and macrophage-specific markers may function as specific fibrosis and/or inflammatory markers. Furthermore, future therapies may target Kupffer cells specifically.8 CD163 is a lineage-specific hemoglobin-haptoglobin scavenger receptor expressed exclusively on monocytes and macrophages and up-regulated in conditions with macrophage activation.9-11 CD163 is shed from the macrophage surface into the circulation upon activation of cell surface Toll-like receptors (TLRs) and is found in the blood as soluble CD163 (sCD163).12 sCD163 has been investigated as a biochemical marker of macrophage activation.13 We have shown close associations between sCD163 levels, liver dysfunction, and portal hypertension in patients with cirrhosis.14,15 In these reports, we primarily studied sCD163 levels in patients with alcoholic liver disease, but Kupffer cells are also known to be activated in chronic viral hepatitis. Hiraoka et al.16,17 studied cell surface and soluble CD163 in patients with acute and chronic hepatitis and found that the frequencies of macrophages expressing CD163 and levels of sCD163 were higher in acute viral hepatitis compared to chronic viral hepatitis. In line with this, we demonstrated very high sCD163 levels in patients with acute liver failure from viral hepatitis.18 We undertook this study in a large cohort of treatment-na€ıve chronic HCV- and HBV-infected patients with histological data for disease activity and fibrosis. We assessed macrophage activation by means of sCD163 and related our findings to biochemical and histological parameters of liver disease. We hypothesized that sCD163 levels were elevated in chronic viral hepatitis; that the levels correlated with the severity of liver disease; and that sCD163 might serve as an independent marker of advanced disease. To do this, we examined sCD163 in HCV and HBV patients for its association with fibrosis stage. Finally,
HEPATOLOGY, August 2014
new sCD163-based fibrosis scores, CD163-HCV-FS and CD163-HBV-FS, were developed and compared to the aspartate aminotransferase (AST) to platelet ratio index (APRI) and the FIB-4 score.
Materials and Methods We performed a cross-sectional study in 551 patients with chronic HCV and 203 patients with chronic HBV infection who were referred to the Storr Liver Unit, Westmead Hospital, Westmead, Australia, between July 1991 and August 2010 for evaluation of chronic viral hepatitis. The diagnosis of chronic HCV infection was confirmed by the presence of anti-HCV antibodies (Monolisa anti-HCV; Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) and viral RNA as detected by polymerase chain reaction (PCR) (Amplicor HCV; Roche Diagnostics, Branchburg, NJ). Hepatitis C virus genotyping was performed with a second-generation reverse-hybridization line probe assay (Inno-Lipa HCV II; Innogenetics, Zwijndrecht, Belgium). The diagnosis of chronic hepatitis B was confirmed by the presence of hepatitis B surface antigen in the blood for more than 6 months, hepatitis B core antibodies and HBV-DNA detection by signal amplification hybridization microplate assay (Digene HBV Test using Hybrid Capture 2, Digene) with a lower limit of detection of 0.5 pg/mL (1.42 3 105 virus copies/mL), or by real-time PCR. Patients dually infected with HCV and HBV (n 5 9), or coinfected with human immunodeficiency virus (n 5 1) were excluded. None of the patients had antiviral treatment prior to inclusion. Liver biopsy was performed as part of the workup for assessment of severity of steatosis, inflammation, and fibrosis. The stained biopsies were examined by experienced pathologists and scored according to the Scheuer scoring system.19 Steatosis was graded as described by Brunt et al.20 All biopsies had a minimum of 11 portal tracts, and inadequate biopsies were excluded. Consequently, histological data were missing in 38 patients with HCV and three patients with HBV infection. At the time of the liver biopsy, basic demographic and clinical data were obtained, including gender, age, ethnicity, height, weight, and waist circumference. Alcohol consumption was assessed by
Address reprint requests to: Konstantin Kazankov, M.D., Department of Hepatology and Gastroenterology, Aarhus University Hospital, Norrebrogade 44, DK8000 Aarhus C, Denmark. E-mail: [email protected]; fax: 10045 78 46 28 60. C 2014 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27129 Potential conflict of interest: Nothing to report.
HEPATOLOGY, Vol. 60, No. 2, 2014
two separate interviews with the patient and close family members. Body mass index (BMI) was calculated from height and weight. At the same time, a fasting blood sample was drawn and routine biochemical tests were performed as described below. Additional blood samples were taken and frozen at 280 C for future research. All patients signed an informed consent form in accordance with the Helsinki Declaration; the acquisition, storage, and use of the blood samples was approved by the Sydney West Area Health Service Ethics Committee. Biochemical Analyses. The concentrations of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), gammaglutamyltransferase (GGT), total bilirubin, prothrombin time, international normalized ratio (INR), fasting glucose and insulin, serum albumin, hemoglobin, platelets, leukocytes, triglycerides, and cholesterol and its components were determined prior to the liver biopsy using standard assays and methods. Homeostatic model assessment of insulin resistance (HOMA-IR) was calculated from fasting glucose and insulin.21 The Model for Endstage Liver Disease (MELD) score was calculated using bilirubin, creatinine, and INR.22 The AST to platelet ratio index (APRI) was calculated according to the established formula: (AST(IU/L)/ upper normal limit) 3 100/platelets(109/L).23 The FIB-4 index was calculated as follows: age(years) 3 1 AST(IU/L)/(platelets(109/L) 3 (ALT(IU/L)) =2).24 The plasma concentration of sCD163 was determined in duplicate in samples that had been frozen at 280 C by an in-house sandwich enzyme-linked immunosorbent assay (ELISA) using a BEP-2000 ELISA-analyzer (Dade Behring) as previously described.25 Control samples and serum standards with concentrations that ranged from 6.25 to 200 lg/ L were included in each run. The interassay coefficient of variation in the current project (n 5 20) was 3.56.0% at a level of 1.31 mg/L and 6-10% at a level of 3.59 mg/L. The limit of detection (lowest standard) was 6.25 lg/L. Soluble CD163 is resistant to repeated freezing and thawing.25 Statistical Methods. One-way analysis of variance (ANOVA) was used for the comparison of multiple groups and Student t test to study differences of normally distributed variables between the groups. For the nonnormally distributed data, Kruskal-Wallis and Mann-Whitney tests, respectively, were used. The relationships between sCD163 and other continuous variables were analyzed by simple linear regression (after transformation using natural logarithm [log]) or Spearman’s rank correlation. Spearman’s rank test was used
KAZANKOV ET AL.
523
to study associations between sCD163 and histological scores. To study the differences in sCD163 between patients with HBV and HCV infection with the same histological scores of fibrosis and inflammation, we used two-way ANOVA with post-hoc t tests. To assess differences in proportions, v2-test or Fisher’s exact test were used. We performed multiple ordered logistic regression analysis with Scheuer fibrosis score as the dependent and sCD163 as the explanatory variable in two different models. These models provided odds ratios (OR) for a given fibrosis stage corresponding to specific increases in sCD163. We chose to present the results corresponding to a 25% increase in sCD163 based on the actual median difference of 28% in sCD163 levels between patients who differed in Scheuer fibrosis score by 1 (Fig. 1C). In Model 1, we aimed to determine whether sCD163 was associated with the fibrosis score directly or through its relationship with inflammation scores and known fibrosis risk factors. Age, gender, BMI, ethnicity, alcohol consumption, and presence of genotype 1 (HCV only) were identified as risk factors for liver fibrosis in chronic viral hepatitis and included in Model 1. Scheuer scores for lobular and portal inflammation were also included. In Model 2, our goal was to investigate whether sCD163 was a marker of fibrosis when adjusted for demographic, clinical, and biochemical parameters shown to be associated with fibrosis in previous studies.26 Thus, we included age, gender, BMI, ethnicity, genotype 1, alcohol consumption, albumin, platelets, ALT, AST, INR, and HOMA-IR in Model 2. All continuous variables were logarithmically transformed. To identify candidate variables for the new sCD163-based fibrosis scores (CD163-HCV-FS and CD163-HBVFS) we performed backward elimination based on the likelihood ratio test with significance limit of 0.1 including all variables in Model 2. Then all possible subsets of these candidate variables were examined using nonparametric receiver operating characteristics (ROC) analyses for the presence of liver cirrhosis (defined by Scheuer fibrosis score of 4), advanced (F3) and significant fibrosis (F2). The subsets of variables providing the highest areas under the ROCcurves (AUROCs) in patients with HCV and HBV infection were chosen for the new scores. These new scores were compared to APRI and FIB-4 using the test of equality of ROC areas. Sensitivity, specificity, positive and negative predictive values (PPV and NPV) were determined for appropriate cutoff values of CD613-HCV-FS and CD163-HBV-FS, based on the ROC-curves.
524
KAZANKOV ET AL.
HEPATOLOGY, August 2014
Fig. 1. Soluble CD163 in histological scores of inflammatory activity and fibrosis in patients with HCV and HBV infection. (A) sCD163 and Scheuer lobular inflammation score (0-4). HCV: Spearman’s rho 5 0.31, P < 0.001; HBV: rho 5 0.31, P < 0.001 (B) sCD163 and Scheuer portal inflammation score (0-4). HCV: rho 5 0.39, P < 0.001; HBV: rho 5 0.42, P < 0.001 (C) sCD163 and Scheuer fibrosis score (0-4). HCV: rho 5 0.45, P < 0.001; HBV: rho 5 0.32, P < 0.001. Boxes represent interquartile ranges with medians; whiskers show adjacent values (the highest value lower or equal to: 75% quartile 11.5 3 interquartile range; the lowest value higher or equal to: 25% quartile 21.5 3 interquartile range). Punctured lines represent reference interval (0.89-3.95 mg/L). *0.01 < P < 0.05 between patients with HCV and HBV infection; **P < 0.01 between patients with HCV and HBV infection.
All data are expressed as medians with interquartile ranges (IQR) or proportions, and P 0.05 were considered statistically significant. STATA v. 12.0 (StataCorp) was used for data analysis.
Results Patient Characteristics. Basic demographic, clinical, and biochemical data for the HCV and HBV patients are presented in Table 1. The two groups did not differ significantly in terms of gender, the majority of the patients being male. Patients with HCV infection were slightly, but significantly older than HBV patients. BMI and HOMA-IR were higher in HCV patients; however, with no significant difference in the prevalence of diabetes between the groups. There was a significant difference in the consumption of alcohol between HCV and HBV patients (Table 1). We observed higher ALT, AST, and GGT levels in HCV patients compared to HBV. Albumin was slightly but
significantly lower in patients with HCV infection. INR was significantly lower in HCV patients, although the two groups had the same median values. A trend toward lower platelets in HCV patients was observed. Soluble CD163 levels were significantly higher in HCV patients (3.6(2.5-5.4) mg/L) compared to those with HBV infection (2.4(1.8-3.6) mg/L), P < 0.001. Histological Scores of Activity and Fibrosis in HCV and HBV Patients. The patients with HCV infection had more advanced disease with higher scores for Scheuer fibrosis (P 5 0.001) compared to HBV patients (Table 1). None of the patients with biopsy-verified HCV or HBV cirrhosis had decompensated cirrhosis. In addition, HCV patients had higher scores for Scheuer portal inflammation (P < 0.001) and steatosis (P < 0.001). There was no significant difference in the score for Scheuer lobular inflammation between the two groups (P 5 0.45). The full distribution of histological scores in patients with HCV and HBV infection is presented in Supporting Table 1.
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KAZANKOV ET AL.
525
Table 1. Demographic, Clinical, and Biochemical Parameters in Patients With HCV and HBV Infection HCV n 5 551
HBV n 5 203
43 (36-49) 348 (63%) : 203 (37%)
41 (33-49) 130 (64%) : 73 (36%)
446 (82.6%) 41 (7.6%) 34 (6.3%) 10 (1.8%) 9 (1.7%) 11
152 (74.9%) 28 (13.8%) 17 (8.3%) 6 (3.0%) 0 (%) 0
265 (51.7%) 248 (48.3%) 99 (19.3%) 45 (8.8%) 38 7.5 (6.4-8.4) 26 (23-30) 25 (4.6%) 82 (52-144) 62 (43-100) 11 (8-14) 77 (66-96) 50 (29-93) 1 (0.9-1) 43 (41-45) 2.22 (1.43-3.78) 4.5 (3.9-5.1) 2.6 (2-3.2) 1.3 (1-1.6) 0.98 (0.75-1.36) 151 (140-160) 6.8 (5.6-8.4) 225 (186-276)
139 (69.5%) 61 (30.5%) 21 (10.5%) 4 (2.0%) 3 8.5 (6.4-8.8) 24 (21-27) 4 (2.2%) 49 (29-78) 46 (37-62) 10 (8-14) 80 (67-99) 26 (19-47) 1 (1-1.1) 45 (43-48) 1.43 (0.67-2.35) 4.9 (4.4-5.6) 2.9 (2.4-3.5) 1.3 (1.2-1.7) 0.98 (0.72-1.37) 146 (137-157) 5.4 (4.6-6.5) 220 (190-257)
Parameter
Age (years) Gender (Male : Female) Alcohol consumption (n) < 10 g/day 10-19 g/day 20-39 g/day 40-59 g/day 60 g/day Missing record Fibrosis stage (n) No/mild fibrosis (F0-1) Significant fibrosis or higher (F2) Advanced fibrosis or higher (F3) Cirrhosis (F4) Missing histology MELD BMI (kg/m2) Diabetes (n) ALT (IU/L, males
T
here is increasing evidence that macrophages are involved in inflammation and fibrosis in liver diseases. In the liver, macrophages are observed in close proximity with cells involved in fibrosis formation and produce a number of growth factors and proinflammatory substances involved in
this process.1,2 The mechanism of action of monocytes and resident liver macrophages (Kupffer cells) in inflammation and fibrogenesis in chronic viral hepatitis is not fully understood, but studies suggest that macrophages function differently in the presence of hepatitis C (HCV) and hepatitis B (HBV) virus.3-5 Further, a
Abbreviations: ALP, alkaline phosphatase; ALT, alanine transaminase; ANOVA, analysis of variance; APRI, AST to platelet ratio index; AST, aspartate transaminase; AUROC, area under the receiver operating characteristics curve; BMI, body mass index; CD163-HBV-FS, sCD163-based fibrosis score for HBV patients; CD163-HCV-FS, sCD163-based fibrosis score for HCV patients; ELISA, enzyme-linked immunosorbent assay; GGT, gamma-glutamyltransferase; HBV, hepatitis B virus; HCV, hepatitis C virus; HOMA-IR, homeostatic model assessment of insulin resistance; IL-6, interleukin-6; INR, international normalized ratio; IQR, interquartile range; MELD, Model for Endstage Liver Disease; NPV, negative predictive value; PCR, polymerase chain reaction; PPV, positive predictive value; sCD163, soluble CD163; TGF-b, transforming growth factor-b; TLR, toll-like receptor; TNF-a, tumor necrosis factor-a. From the 1Department of Hepatology and Gastroenterology, Aarhus University Hospital, Aarhus, Denmark; 2Storr Liver Unit, University of Sydney and Westmead Hospital, Westmead, Australia; 3Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark; 4Department of Biostatistics, Aarhus University, Aarhus, Denmark. Received May 14, 2013; accepted March 11, 2014. Supported by the NOVO Nordisk Foundation, the Danish Strategic Research Council (10-092797), and grants from the National Health and Medical Research Council of Australia (632630, 1049857, 1053206) and the Robert W. Storr Bequest to the Sydney Medical Foundation of the University of Sydney. 521
522
KAZANKOV ET AL.
pathway involving activation of monocytes and macrophages by endotoxin with subsequent up-regulation of proinflammatory cytokines, such as tumor necrosis factor (TNF)-a and interleukin (IL)-6, has been suggested.6 Moreover, activated Kupffer cells secrete transforming growth factor (TGF)-b, which activates hepatic stellate cells and induces fibrosis.7 Thus, Kupffer cells play an important role in liver inflammation and fibrosis and macrophage-specific markers may function as specific fibrosis and/or inflammatory markers. Furthermore, future therapies may target Kupffer cells specifically.8 CD163 is a lineage-specific hemoglobin-haptoglobin scavenger receptor expressed exclusively on monocytes and macrophages and up-regulated in conditions with macrophage activation.9-11 CD163 is shed from the macrophage surface into the circulation upon activation of cell surface Toll-like receptors (TLRs) and is found in the blood as soluble CD163 (sCD163).12 sCD163 has been investigated as a biochemical marker of macrophage activation.13 We have shown close associations between sCD163 levels, liver dysfunction, and portal hypertension in patients with cirrhosis.14,15 In these reports, we primarily studied sCD163 levels in patients with alcoholic liver disease, but Kupffer cells are also known to be activated in chronic viral hepatitis. Hiraoka et al.16,17 studied cell surface and soluble CD163 in patients with acute and chronic hepatitis and found that the frequencies of macrophages expressing CD163 and levels of sCD163 were higher in acute viral hepatitis compared to chronic viral hepatitis. In line with this, we demonstrated very high sCD163 levels in patients with acute liver failure from viral hepatitis.18 We undertook this study in a large cohort of treatment-na€ıve chronic HCV- and HBV-infected patients with histological data for disease activity and fibrosis. We assessed macrophage activation by means of sCD163 and related our findings to biochemical and histological parameters of liver disease. We hypothesized that sCD163 levels were elevated in chronic viral hepatitis; that the levels correlated with the severity of liver disease; and that sCD163 might serve as an independent marker of advanced disease. To do this, we examined sCD163 in HCV and HBV patients for its association with fibrosis stage. Finally,
HEPATOLOGY, August 2014
new sCD163-based fibrosis scores, CD163-HCV-FS and CD163-HBV-FS, were developed and compared to the aspartate aminotransferase (AST) to platelet ratio index (APRI) and the FIB-4 score.
Materials and Methods We performed a cross-sectional study in 551 patients with chronic HCV and 203 patients with chronic HBV infection who were referred to the Storr Liver Unit, Westmead Hospital, Westmead, Australia, between July 1991 and August 2010 for evaluation of chronic viral hepatitis. The diagnosis of chronic HCV infection was confirmed by the presence of anti-HCV antibodies (Monolisa anti-HCV; Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) and viral RNA as detected by polymerase chain reaction (PCR) (Amplicor HCV; Roche Diagnostics, Branchburg, NJ). Hepatitis C virus genotyping was performed with a second-generation reverse-hybridization line probe assay (Inno-Lipa HCV II; Innogenetics, Zwijndrecht, Belgium). The diagnosis of chronic hepatitis B was confirmed by the presence of hepatitis B surface antigen in the blood for more than 6 months, hepatitis B core antibodies and HBV-DNA detection by signal amplification hybridization microplate assay (Digene HBV Test using Hybrid Capture 2, Digene) with a lower limit of detection of 0.5 pg/mL (1.42 3 105 virus copies/mL), or by real-time PCR. Patients dually infected with HCV and HBV (n 5 9), or coinfected with human immunodeficiency virus (n 5 1) were excluded. None of the patients had antiviral treatment prior to inclusion. Liver biopsy was performed as part of the workup for assessment of severity of steatosis, inflammation, and fibrosis. The stained biopsies were examined by experienced pathologists and scored according to the Scheuer scoring system.19 Steatosis was graded as described by Brunt et al.20 All biopsies had a minimum of 11 portal tracts, and inadequate biopsies were excluded. Consequently, histological data were missing in 38 patients with HCV and three patients with HBV infection. At the time of the liver biopsy, basic demographic and clinical data were obtained, including gender, age, ethnicity, height, weight, and waist circumference. Alcohol consumption was assessed by
Address reprint requests to: Konstantin Kazankov, M.D., Department of Hepatology and Gastroenterology, Aarhus University Hospital, Norrebrogade 44, DK8000 Aarhus C, Denmark. E-mail: [email protected]; fax: 10045 78 46 28 60. C 2014 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27129 Potential conflict of interest: Nothing to report.
HEPATOLOGY, Vol. 60, No. 2, 2014
two separate interviews with the patient and close family members. Body mass index (BMI) was calculated from height and weight. At the same time, a fasting blood sample was drawn and routine biochemical tests were performed as described below. Additional blood samples were taken and frozen at 280 C for future research. All patients signed an informed consent form in accordance with the Helsinki Declaration; the acquisition, storage, and use of the blood samples was approved by the Sydney West Area Health Service Ethics Committee. Biochemical Analyses. The concentrations of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), gammaglutamyltransferase (GGT), total bilirubin, prothrombin time, international normalized ratio (INR), fasting glucose and insulin, serum albumin, hemoglobin, platelets, leukocytes, triglycerides, and cholesterol and its components were determined prior to the liver biopsy using standard assays and methods. Homeostatic model assessment of insulin resistance (HOMA-IR) was calculated from fasting glucose and insulin.21 The Model for Endstage Liver Disease (MELD) score was calculated using bilirubin, creatinine, and INR.22 The AST to platelet ratio index (APRI) was calculated according to the established formula: (AST(IU/L)/ upper normal limit) 3 100/platelets(109/L).23 The FIB-4 index was calculated as follows: age(years) 3 1 AST(IU/L)/(platelets(109/L) 3 (ALT(IU/L)) =2).24 The plasma concentration of sCD163 was determined in duplicate in samples that had been frozen at 280 C by an in-house sandwich enzyme-linked immunosorbent assay (ELISA) using a BEP-2000 ELISA-analyzer (Dade Behring) as previously described.25 Control samples and serum standards with concentrations that ranged from 6.25 to 200 lg/ L were included in each run. The interassay coefficient of variation in the current project (n 5 20) was 3.56.0% at a level of 1.31 mg/L and 6-10% at a level of 3.59 mg/L. The limit of detection (lowest standard) was 6.25 lg/L. Soluble CD163 is resistant to repeated freezing and thawing.25 Statistical Methods. One-way analysis of variance (ANOVA) was used for the comparison of multiple groups and Student t test to study differences of normally distributed variables between the groups. For the nonnormally distributed data, Kruskal-Wallis and Mann-Whitney tests, respectively, were used. The relationships between sCD163 and other continuous variables were analyzed by simple linear regression (after transformation using natural logarithm [log]) or Spearman’s rank correlation. Spearman’s rank test was used
KAZANKOV ET AL.
523
to study associations between sCD163 and histological scores. To study the differences in sCD163 between patients with HBV and HCV infection with the same histological scores of fibrosis and inflammation, we used two-way ANOVA with post-hoc t tests. To assess differences in proportions, v2-test or Fisher’s exact test were used. We performed multiple ordered logistic regression analysis with Scheuer fibrosis score as the dependent and sCD163 as the explanatory variable in two different models. These models provided odds ratios (OR) for a given fibrosis stage corresponding to specific increases in sCD163. We chose to present the results corresponding to a 25% increase in sCD163 based on the actual median difference of 28% in sCD163 levels between patients who differed in Scheuer fibrosis score by 1 (Fig. 1C). In Model 1, we aimed to determine whether sCD163 was associated with the fibrosis score directly or through its relationship with inflammation scores and known fibrosis risk factors. Age, gender, BMI, ethnicity, alcohol consumption, and presence of genotype 1 (HCV only) were identified as risk factors for liver fibrosis in chronic viral hepatitis and included in Model 1. Scheuer scores for lobular and portal inflammation were also included. In Model 2, our goal was to investigate whether sCD163 was a marker of fibrosis when adjusted for demographic, clinical, and biochemical parameters shown to be associated with fibrosis in previous studies.26 Thus, we included age, gender, BMI, ethnicity, genotype 1, alcohol consumption, albumin, platelets, ALT, AST, INR, and HOMA-IR in Model 2. All continuous variables were logarithmically transformed. To identify candidate variables for the new sCD163-based fibrosis scores (CD163-HCV-FS and CD163-HBVFS) we performed backward elimination based on the likelihood ratio test with significance limit of 0.1 including all variables in Model 2. Then all possible subsets of these candidate variables were examined using nonparametric receiver operating characteristics (ROC) analyses for the presence of liver cirrhosis (defined by Scheuer fibrosis score of 4), advanced (F3) and significant fibrosis (F2). The subsets of variables providing the highest areas under the ROCcurves (AUROCs) in patients with HCV and HBV infection were chosen for the new scores. These new scores were compared to APRI and FIB-4 using the test of equality of ROC areas. Sensitivity, specificity, positive and negative predictive values (PPV and NPV) were determined for appropriate cutoff values of CD613-HCV-FS and CD163-HBV-FS, based on the ROC-curves.
524
KAZANKOV ET AL.
HEPATOLOGY, August 2014
Fig. 1. Soluble CD163 in histological scores of inflammatory activity and fibrosis in patients with HCV and HBV infection. (A) sCD163 and Scheuer lobular inflammation score (0-4). HCV: Spearman’s rho 5 0.31, P < 0.001; HBV: rho 5 0.31, P < 0.001 (B) sCD163 and Scheuer portal inflammation score (0-4). HCV: rho 5 0.39, P < 0.001; HBV: rho 5 0.42, P < 0.001 (C) sCD163 and Scheuer fibrosis score (0-4). HCV: rho 5 0.45, P < 0.001; HBV: rho 5 0.32, P < 0.001. Boxes represent interquartile ranges with medians; whiskers show adjacent values (the highest value lower or equal to: 75% quartile 11.5 3 interquartile range; the lowest value higher or equal to: 25% quartile 21.5 3 interquartile range). Punctured lines represent reference interval (0.89-3.95 mg/L). *0.01 < P < 0.05 between patients with HCV and HBV infection; **P < 0.01 between patients with HCV and HBV infection.
All data are expressed as medians with interquartile ranges (IQR) or proportions, and P 0.05 were considered statistically significant. STATA v. 12.0 (StataCorp) was used for data analysis.
Results Patient Characteristics. Basic demographic, clinical, and biochemical data for the HCV and HBV patients are presented in Table 1. The two groups did not differ significantly in terms of gender, the majority of the patients being male. Patients with HCV infection were slightly, but significantly older than HBV patients. BMI and HOMA-IR were higher in HCV patients; however, with no significant difference in the prevalence of diabetes between the groups. There was a significant difference in the consumption of alcohol between HCV and HBV patients (Table 1). We observed higher ALT, AST, and GGT levels in HCV patients compared to HBV. Albumin was slightly but
significantly lower in patients with HCV infection. INR was significantly lower in HCV patients, although the two groups had the same median values. A trend toward lower platelets in HCV patients was observed. Soluble CD163 levels were significantly higher in HCV patients (3.6(2.5-5.4) mg/L) compared to those with HBV infection (2.4(1.8-3.6) mg/L), P < 0.001. Histological Scores of Activity and Fibrosis in HCV and HBV Patients. The patients with HCV infection had more advanced disease with higher scores for Scheuer fibrosis (P 5 0.001) compared to HBV patients (Table 1). None of the patients with biopsy-verified HCV or HBV cirrhosis had decompensated cirrhosis. In addition, HCV patients had higher scores for Scheuer portal inflammation (P < 0.001) and steatosis (P < 0.001). There was no significant difference in the score for Scheuer lobular inflammation between the two groups (P 5 0.45). The full distribution of histological scores in patients with HCV and HBV infection is presented in Supporting Table 1.
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KAZANKOV ET AL.
525
Table 1. Demographic, Clinical, and Biochemical Parameters in Patients With HCV and HBV Infection HCV n 5 551
HBV n 5 203
43 (36-49) 348 (63%) : 203 (37%)
41 (33-49) 130 (64%) : 73 (36%)
446 (82.6%) 41 (7.6%) 34 (6.3%) 10 (1.8%) 9 (1.7%) 11
152 (74.9%) 28 (13.8%) 17 (8.3%) 6 (3.0%) 0 (%) 0
265 (51.7%) 248 (48.3%) 99 (19.3%) 45 (8.8%) 38 7.5 (6.4-8.4) 26 (23-30) 25 (4.6%) 82 (52-144) 62 (43-100) 11 (8-14) 77 (66-96) 50 (29-93) 1 (0.9-1) 43 (41-45) 2.22 (1.43-3.78) 4.5 (3.9-5.1) 2.6 (2-3.2) 1.3 (1-1.6) 0.98 (0.75-1.36) 151 (140-160) 6.8 (5.6-8.4) 225 (186-276)
139 (69.5%) 61 (30.5%) 21 (10.5%) 4 (2.0%) 3 8.5 (6.4-8.8) 24 (21-27) 4 (2.2%) 49 (29-78) 46 (37-62) 10 (8-14) 80 (67-99) 26 (19-47) 1 (1-1.1) 45 (43-48) 1.43 (0.67-2.35) 4.9 (4.4-5.6) 2.9 (2.4-3.5) 1.3 (1.2-1.7) 0.98 (0.72-1.37) 146 (137-157) 5.4 (4.6-6.5) 220 (190-257)
Parameter
Age (years) Gender (Male : Female) Alcohol consumption (n) < 10 g/day 10-19 g/day 20-39 g/day 40-59 g/day 60 g/day Missing record Fibrosis stage (n) No/mild fibrosis (F0-1) Significant fibrosis or higher (F2) Advanced fibrosis or higher (F3) Cirrhosis (F4) Missing histology MELD BMI (kg/m2) Diabetes (n) ALT (IU/L, males
