HCV genotype 3 and squamous cell carcinoma antigen (SCCA)-IgM are independently associated with histological features of NASH in HCV-infected patients.
Martini A1, Fattovich G2,3, Guido M1, Bugianesi E4, Biasiolo A1, Ieluzzi D2, Gallotta A5, Fassina G5, Merkel C1, Gatta A1, Negro F6,7, Pontisso P1.
1Department of Medicine, University of Padua, Padua, Italy; 2Division of Gastroenterology and Endoscopy, Azienda Ospedaliera Universitaria Integrata, Verona, Italy; 3Department of Medicine, University of Verona, Verona, Italy; 4Division of Gastroenterology and Hepatology, Department of Medical Sciences, University of Torino, Torino, Italy; 5Xeptagen, VEGA Park, Venice, Italy; 6Division of Clinical Pathology, University Hospitals of Geneva, Geneva, Switzerland; and 7Division Gastroenterology and Hepatology, University Hospitals of Geneva, Geneva, Switzerland Abstract
Nonalcoholic steatohepatitis (NASH) enhances the risk of progressive liver disease. In chronic hepatitis C (CHC), liver steatosis is frequent, especially in genotype 3, but its clinical significance is debated. As squamous cell carcinoma antigen (SCCA)-IgM has been associated with advanced liver disease and risk of tumour development, we evaluated its occurrence in CHC and the possible relation with NASH at liver biopsy. Using a validated ELISA, serum SCCA-IgM was measured in 91 patients with CHC at the time of liver biopsy performed before antiviral treatment, at the end of treatment and 6 months thereafter, and in 93 HCV-negative patients with histological diagnosis of nonalcoholic fatty liver disease, as controls. SCCA-IgM was detected in 33% of CHC patients and in 4% of controls. This biomarker was found more elevated in CHC patients with histological NASH, and at multivariate analysis, SCCA-IgM and HCV genotype 3 were independently associated with NASH [OR (95% CI): 6.94 (1.21-40) and 27.02 (4.44-166.6)]. As predictors of NASH, HCV genotype 3 and SCCA-IgM had a specificity and a sensitivity of 97% and 44%, and of 95% and 27%, respectively. PPV and NPV were 80% and 86% for HCV genotype 3 vs 73% and 72% for SCCA-IgM. In patients with sustained virologic response to therapy, SCCA-IgM levels decreased significantly, while these remained unchanged in nonresponders. In conclusion, SCCA-IgM is detectable in one-third of patients with CHC and significantly correlates with histological NASH.
INTRODUCTION
Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are increasingly relevant public health issues owing to their close association with the worldwide epidemics of diabetes and obesity [1,2]. Rapidly growing literature indicates that NAFLD also contributes to noncirrhotic hepatocellular carcinoma (HCC) [2–4]. The distinction between simple steatosis and NASH, that is the true factor increasing the risk of progressive liver disease and of HCC [2], remains a challenge because a simple, reliable and noninvasive diagnostic tool has not been identified yet [5–7]. At present, histopathology is considered the reference standard [8], although liver biopsy has several drawbacks, including interobserver variability and invasiveness, which limits its usefulness especially in the follow-up of NAFLD [9].
Several studies have reported variable success using a combination of clinical and laboratory tests to distinguish NASH from NAFLD [2,10–13], but they have not been fully validated in different populations or gained widespread acceptance for clinical use [12,14,15]. Fatty liver can be induced also by hepatitis C virus (HCV) [16] and it is more frequent and severe in patients infected with HCV genotype 3 [17], where the degree of steatosis correlates with viral load and with virus behaviour in response to antiviral therapy, with reported regression of steatosis in patients with persistent viral clearance [18]. Hepatitis C virus infection appears to interfere with some aspects of lipid and glucose metabolism, but it is not clear yet which event occurs first [19]. This is an
important issue to resolve, because there is evidence that HCV infection also accelerates the development of type 2 diabetes mellitus in predisposed individuals [20]. Eradication of HCV infection is associated with reduced incidence of glucose metabolism disturbances after treatment, independently of other predisposing factors [21,22]. Liver steatosis is detectable histologically in up to 70% of the HCVinfected patients, depending on viral genotype [23], but the clinical significance of this finding is still debated. Some authors reported that hepatic steatosis significantly increases the risk of relapse in patients with genotype 3 [24], while several clinical studies suggest that hepatic steatosis is an independent risk factor of HCC development [25–27]. It has not been proven yet that viral steatosis can contribute to fibrogenesis, and if an association exists, it seems to occur in patients with metabolic steatosis [28,29]. Squamous cell carcinoma antigen (SCCA) is a serine protease inhibitor detectable in normal squamous epithelium but also expressed by neoplastic epithelial cells [30]. Overexpression of SCCA isoforms has been described in HCC tissue but not in normal liver [31], and a recent study has reported their expression in liver stem/progenitor cells [32].
In addition, SCCA has been found more expressed in highgrade dysplastic nodules and HCC than in large regenerative nodules, suggesting a role in hepatocarcinogenesis [33]. SCCA-1 isoform (now termed SERPINB3) renders cells more resistant to apoptotic cell death [34,35] and inhibits
migration of NK cells [36]. It is also capable to induce epithelial–mesenchymal transition, with increased invasiveness potential [37], and to induce TGF-beta [38]. A recent study in a mouse model of acute liver failure has demonstrated that the presence of SERPINB3 determines an enhanced inflammatory background, mainly mediated by higher levels of Th1 proinflammatory cytokines [39]. At the serological level, high or increasing levels of SCCA-IgM immune complexes were associated with more advanced liver disease and increased risk of HCC development [40–44]. The purpose of this study was to evaluate the occurrence of SCCA-IgM in serum of CHC patients and its possible relation with histological features of potential disease progression, in particular with the presence of NASH at liver biopsy.
PATIENTS AND METHODS
Patients
The study population included 91 patients with CHC referred consecutively to the Division of Gastroenterology and Endoscopy, Azienda Ospedaliera Universitaria Integrata of Verona, and who fulfilled the following inclusion criteria: histologically proven CHC with HCV-RNA detectable in the serum by polymerase chain reaction (PCR), not previously treated with antiviral agents; Caucasian race; age between 18 and 65 years; absence of coexisting hepatitis B virus or human immunodeficiency virus infection; absence of decompensated cirrhosis; absence of relevant concurrent medical conditions, including liver disease of aetiologies other than HCV infection; alcohol intake <25 g/day in the 6 months preceding liver biopsy; availability of the results of routine laboratory studies including glucose, insulin, ferritin, total cholesterol and triglycerides; and availability of serum samples taken the same day of liver biopsy (or within the preceding or the following week) before antiviral treatment, at the end of treatment and 6 months thereafter stored at _20 °C and never thawed previously. The homeostasis model assessment of insulin resistance (HOMA-IR) score was calculated using the following equation: HOMA-IR = fasting insulin (mIU/L) 9 fasting glucose (mmol/L) /22.5. Body mass index (BMI) was calculated as weight divided by the square of the height (kg/m2). Patients were treated according to international guidelines; therapy consisted of peginterferon-a2a (180 lg/week) or peginterferon-a2b (1.5
lg/kg/week) in combination with ribavirin (800–1200 mg/day) for an intended duration of 48 weeks for HCV genotypes 1 or 4 and of 24 weeks for HCV genotypes 2 or 3. A sustained virologic response (SVR) to therapy was defined as HCV-RNA negative 24 weeks after stopping therapy. Sera of 93 consecutive HCV-negative patients with histological diagnosis of NAFLD
were also included as controls. Demographic, metabolic and biochemical characteristics of the patients included in the study are reported in Table S1. Serum samples were collected after obtaining informed consent, as approved by the institutional ethics committee, according to the principles of the Declaration of Helsinki. SCCA-IgM determination Squamous cell carcinoma antigen-IgM immune complex levels were measured in serum by ELISA (Hepa-IC, Xeptagen, Venice, Italy), according to the manufacturer’s instructions. The amount of SCCA-IgM was expressed in arbitrary units/mL (AU/mL), and the value of 200 AU/mL was used as cut-off [44].
Liver histology
All liver biopsy samples were formalin-fixed, paraffin embedded and routinely stained with haematoxylin–eosin and van Gieson for collagen. Grading and staging of chronic hepatitis were performed using the METAVIR scoring system. Steatosis was separately assessed in a
semiquantitative way, on a scale from 0 to 3: 0, absent: <5%; 1, mild: 6–33%; 2, moderate: 34– 66%; and 3, severe: >66%. Predominant zonal distribution of steatosis was also recorded. In HCV-positive patients, final diagnoses were chronic hepatitis C (CHC) alone, CHC and steatosis with ‘metabolic’ pattern, defined as severe and/or central zone predominant steatosis, and CHC and NASH, defined by the presence of centrilobular hepatocyte ballooning and sinusoidal fibrosis [16]. In 8 HCV-positive cases, selected on the basis of high or low SCCA-IgM levels in serum,
immunohistochemistry for SCCA-1 was carried out as previously described [33], using an anti-SCCA-1 monoclonal antibody (1:20, anti-SCCA-1 clone 8H11, Santa Cruz Biotechnology, Santa Cruz, CA, USA) as primary antibody. All liver biopsies were coded and scored by a single pathologist (M. G. at the University of Padua, Italy) who was blinded to HCV genotype, clinical and biological data.
Statistical analysis
Continuous variables were expressed as means _ standard deviation (SD) or medians (range) and categorical variables as frequency and percentage. Comparison between groups was carried out using the Student’s t-test and unpaired t-test with Welch’s correction or the Mann–Whitney U-test for continuous variables, and the chi-squared-test or Fisher’s exact probability test for categorical data, when appropriate. Two-sided P < 0.05 were considered statistically significant. For univariate logistic regression analysis, SCCA-IgM, HOMA-IR and HCV genotype 3 were in addition selected as the variables more likely associated with NASH. For multivariate analysis, only variables
achieving a significant result in univariate analysis were included. All analyses were performed using GraphPad Prism 5 software (San Diego, CA, USA).
RESULTS
Body mass index, lipidic profile and ferritin levels, typically associated with the metabolic syndrome, were significantly higher among NAFLD compared to HCV-infected patients (Table S1). Circulating SCCA-IgM immunocomplexes were more frequently detected in CHC patients than in HCV-negative patients (34.9% vs 4.3%, P < 0.0001). Due to the low frequency of positivity of SCCA-IgM in the HCV-negative population, further analyses of the biomarker behaviour
were carried out only in HCV-positive patients. Behaviour of SCCA-IgM in relation to histological diagnosis of NASH Patients with more severe grades of steatosis had significantly higher SCCA-IgM values, as shown in Fig. 1a. In addition, this biomarker was found more elevated in patients with histological diagnosis of HCV and NASH, compared to the remaining patients (Fig. 1b). The immunohistochemistry results, carried out in a selection of representative cases, supported the close correlation between the extent of SCCA-1 in the liver and SCCA-IgM in serum. Indeed, in cases negative for SCCA-IgM in serum, SCCA-1 was barely detectable in the corresponding liver biopsy,
even in the presence of steatosis (Figure S1B). On the contrary, this serpin was remarkably expressed in patients with high SCCA-IgM values in serum (Figure S2). The characteristics of patients with histological diagnosis of HCV and NASH compared to the remaining patients are described in detail in Table 1, showing that genotype 3 and HOMA-IR were the variables with the highest statistical difference in relation to the presence of NASH. The clinical characteristics of HCV-negative patients in relation to NASH are described in Table S2. The reported results indicate that in these patients, the variables with the highest statistical difference were BMI, HOMA-IR and ferritin. In HCV-positive patients, univariate logistic regression analysis was carried out, and among the variables described in Table 2, SCCA-IgM and HCV genotype 3 showed a significant
association with the presence of NASH that was maintained also at multivariate analysis, while HOMA-IR and ferritin did not reach statistical significance. SCCA-IgM and HCV genotype
Distribution of SCCA-IgM was not significantly different in HCV genotypes, in terms of both level of reactivity and level of frequency, although patients with genotype 3 tended to be more frequently positive for this biomarker (Figure S3). As NASH was found significantly associated with HCV genotype 3, beside SCCA-IgM, to better define the characteristics of patients with and without genotype 3, biochemical and histological parameters were analysed in the two groups. Patients with HCV genotype non-3 showed higher BMI and total cholesterol, while patients with HCV genotype 3 showed more severe degree of steatosis (>33%) and were more frequently positive for
histological diagnosis of NASH (Table S3). When all patients were analysed in relation to the presence or absence of SCCA-IgM (Table 3), the only significant differences were observed at the histological level, as those with SCCA-IgM had a more severe grade of steatosis and a higher frequency of NASH. In addition, patients with HCV genotype 3 and SCCAIgM had more insulin resistance, more severe steatosis (>33%) and presented more frequently with NASH compared to patients without SCCA-IgM (Table 3).
Thus, we assessed the diagnostic performance of HCV genotype 3 and of SCCA-IgM positivity for the identification of histological NASH (Table 4). Both genotype 3 and SCCA-IgM showed a specificity higher that 95%, while sensitivity was higher for genotype 3 than for SCCA-IgM (44.4% vs 26.7%). Positive and negative predictive values were ≥80% for genotype 3 and >70% for SCCA-IgM. SCCA-IgM behaviour in relation to antiviral response Figure 2 shows the behaviour of SCCA-IgM in relation to antiviral therapy. In patients with SVR, the levels of the
immunocomplexes decreased significantly after 6 months of treatment and remained persistently low even at 6 months after the end of therapy. In nonresponders, SCCA-IgM values showed an insignificant drop at 6 months of treatment, reaching almost initial values at 6 months after therapy withdrawal.
DISCUSSION
Steatosis and NASH occur frequently in HCV infection [1,16], and their role in the progression of chronic liver disease is debated. NASH has been recognized as a progressively increasing risk factor of liver disease worsening and of HCC development [2]. To date, several noninvasive methods for the identification of the patients with these histological signs are emerging, but the identification of specific serological biomarkers remains still elusive [23]. The circulating immune complex SCCA-IgM has been proposed as a marker of liver fibrosis progression in untreated patients with chronic hepatitis of different aetiologies [43]. In this setting, SCCA-IgM complexes were detectable in about one-third of the patients and monitoring their behaviour over time
revealed that levels increased in the majority of the patients with progression of liver fibrosis stage ≥2, while values remained substantially stable or slightly decreased in most of the patients without disease progression during a median time frame of 6 years [43]. In agreement with these findings, a significant decrease in SCCA-IgM levels was observed in sera of patients with CHC and a persistent virologic response to antiviral therapy [45,46].
The present study was addressed to evaluate the potential usefulness of SCCA-IgM for the
the patients with histological features of NASH. The results of the study have shown that this biomarker was detectable in about one-third of CHC patients, while it was rarely detected in HCV-negative patients with NAFLD. In CHC patients, the positivity of SCCA-IgM was significantly associated with the presence of NASH at the histological level, especially in patients infected with genotype 3. The probability of histological diagnosis of NASH was higher for HCV genotype 3 patients in comparison with SCCA-IgM positivity, but the selective distribution of HCV genotype 3 in patients with chronic HCV infection [47] may limit the usefulness of this parameter to detect patients with histological NASH. Moreover, in our population, HOMA-IR, according to a previous study [16], was not a predictor of NASH at multivariate analysis, although additional studies reported that insulin resistance is associated with NASH in patients undergoing bariatric surgery [48] and that the hepatic insulin signalling is impaired in NASH and HCV patients [49]. As SCCA-IgM was found as an independent variable of NASH at multivariate analysis, this biomarker may be useful to identify HCV-infected patients with histological features of potential disease progression and HCC development. In the liver of HCV-positive patients, the expression of SCCA-1 was found to correlate with the levels of circulating SCCA-IgM in serum of the corresponding patients. It is known that SCCA isoforms are not detectable in normal hepatocytes, but chronically damaged hepatocytes start to synthesize these molecules [40], and the highest levels are achieved in preneoplastic and neoplastic liver lesions [31,33,50]. This defence mechanism to oxidative stress can determine initially a higher resistance to apoptotic cell death, but also a higher oncogenic potential over time. The recent finding that EpCAM-positive liver progenitor cell expresses this molecule and its detection in periportal areas of ductular reaction in cirrhotic livers [32] further support this hypothesis. Circulating levels of free SCCA isoforms in serum are very low, and the search for them could be improved when bound to IgM [40,51], considered as one of the most important players of the innate immunity [52]. The fact that in serum SCCA was detected mainly in HCVpositive, compared to HCV-negative patients, may be explained as a) as a specific feature of HCV infection or b) as a feature associated with the presence of NASH, independently of the aetiology, but detectable mainly in HCV infection, due to its easier IgM production (that amplifies the ELISA signal), as a result of a lower threshold required for B-cell activation after the
engagement of CD81 by the HCVE2 protein [53]. The hypothesis that SCCA-IgM may reflect HCV-induced liver damage was further supported by the finding that sustained antiviral response was able to induce a significant reduction in the levels of this biomarker, confirming previous findings [45,46]. It is interesting to note that among the few patients with HCV clearance but persistent detection of this biomarker, HOMA-IR remained elevated. The slight decrease in serum SCCA-IgM detected during treatment in nonresponder patients may be ascribed to the suppressive effect on SCCA expression exerted by interferon, as recently described in in vitro models [54]. In conclusion, the results of the present study indicate that SCCA-IgM is detectable in about one-third of CHC patients and its presence is significantly associated with histological NASH.
ACKNOWLEDGEMENTS
The authors are deeply grateful to Vincenza Guzzardo for providing immunohistochemistry data. This work was supported in part by the following research grants: National Ministry of Health (Project Code: RF-2009-1492312), University of Padova [Project No. CPDA110795] and Associazione Italiana per la Ricerca sul Cancro (AIRC Project No.10235).
CONFLICT OF INTERESTS
Giorgio Fassina (Chief Executive Officer) and Andrea Gallotta (Senior Scientist) are employees of Xeptagen S.p.A.
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Additional Supporting Information may be found in the online version of this article: Table S1. Demographic, metabolic and biochemical characteristics of the
patients included in the study.
Table S2. Characteristics of HCV negative patients according to the presence of histologic NASH. Table S3. Characteristics of HCV positive patients according to HCV
