Noninvasive biomarkers FibroTest and ActiTest versus liver biopsy in chronic hepatitis C patients: the Middle East experience

Introduction

Liver injury due to chronic hepatitis C (CHC) infection seems to be responsible for induction of fibrogenesis. Assessment of fibrosis is important 1) to estimate the prognosis; 2) for decision on antiviral therapy; and 3) for surveillance of hepatocellular carcinoma [1,2]. Liver biopsy (LB) is widely regarded as the gold standard for assessing hepatitis C virus (HCV)-related fibrosis but the sampling variability (40% for fibrosis staging), a high intra- and inter-pathologist variability, and adverse events limit its utility. In addition, a LB samples only 1/50,000th of the liver parenchyma [3-5].

There are numerous histological scoring systems proposed for grading of inflammation and the staging of fibrosis (Metavir, Ishak, and Scheuer) [6]. Two panels of simple biochemical markers have emerged as an alternative to LB in patients with CHC and other forms of chronic liver disease; FibroTest (FT) and ActiTest (AT) [7,8]. The fibrosis index (FT) consists of an algorithm of five fibrosis markers, α-2 macroglobulin (A2M), apolipoprotein A1, haptoglobin, total bilirubin, and γ-glutamyl transpeptidase (GGT), and the necroinflamatory activity index (AT) combines the same 5 markers, plus alanine aminotransferase (ALT). These panels demonstrated high predictive values for significant disease in patients with CHC [9]. Recent studies strongly suggest that due to the limitation and risk of LB, as well as, the improvement of the diagnostic accuracy of biochemical markers, LB need not be considered mandatory in all patients with CHC [10]. Since September 2002 these tests (FT and AT) have been used in several countries as an alternative to LB. A recent study compared percutaneous biopsy with laparoscopic biopsy, demonstrating that cirrhosis was missed in almost 30% of cases by percutaneous biopsy. These studies indicate that the potential for error in staging disease can be as high as 40% and that even cirrhosis can be missed in up to 30% of patients. Clearly LB is not the gold standard [5].

The aims of this study were: 1) to estimate the observed diagnostic value of FT by area under the receiver operating characteristic curve (AUROC) and the standardized AUROC for advanced fibrosis and cirrhosis, using LB as reference; 2) to estimate the diagnostic value (AUROC) for AT for moderate to severe and severe necroinflammatory activity using LB as reference; and 3) to evaluate the concordance between biomarkers and histology on LB.

Patients and methods

This is prospective study done in a tertiary care hospital in Doha, Qatar, according to the regulations of the research and ethics committee. A total of 110 patients referred to our center were studied. The inclusion criteria were: reactive to anti HCV antibody test (i.e. HCV-Ab positive); and detectable HCV RNA for the last 6 months. Exclusion criteria included: alcohol consumption regardless its quantity; concomitant liver diseases (presence of HBs antigen, autoimmune hepatitis, hemochromatosis diagnosed by genetic markers, Wilson’s disease, and α1 anti-trypsin deficiency); and HIV-Ab positivity.

Signed informed consent was obtained from all patients before their inclusion. LB and biochemical markers, abdominal ultrasound, and coagulation profile were performed on the same day. All LBs were done bedside by a hepatologist using Menghini aspiration needle, 16 G. LB was analyzed by a single histopathologist. A total of 111 voluntary blood donors were prospectively included in the control group. The aim of analyzing seromarkers in this group was to determine the specificity of FT in controls.

Results

110 patients and 111 controls were included in the study. 3/110 (2.7%) patients were excluded (1 had no LB, 2 were excluded using security algorithms provided with FT calculation for unusual deviation with the value of haptoglobin in FT results). 5/111 (4.5%) controls were excluded using security algorithms provided with FT calculations (1 control for unusual deviation with the median value of haptoglobin, 4 controls for unusual deviation with the median value of A2M). Finally, 107 patients and 106 controls were included.

Mean age for patients was 46.1 years and for controls was 46.7 years, with male ratios being 66% and 48% respectively (P=0.07) (Table 1). The study group had predominantly genotype 4 infection (90%) followed by genotype 1 (10%). The fibrosis stages in LB were: 36% no or minimal fibrosis (F0, F1); 56% bridging fibrosis (F2, F3); and 7.5% liver cirrhosis (F4). However, the fibrosis stages as estimated by FT were: no or minimal fibrosis in 52% patients and in 95% controls; bridging fibrosis in 22% patients and in 4% controls; cirrhosis in 25% patients and in 1% controls (P<0.0001) (Table 2). The activity grades in LB showed that 46% had no or minimal activity (A0, A1) while 54% showed moderate to severe activity (A2, A3). However, AT showed no or minimal activity in 67% patients and in 100% controls (P<0.0001); moderate to severe activity in 33% patients and in 0% controls (P<0.0001). The mean values for biochemical markers, as expected, showed significant differences between patients and controls (Table 3).

Table 1 Comparison of the baseline and study characteristics of the patient and control groups

Table 2 The distribution of fibrosis and activity scores in the patients’ group fibrosis Metavir stage

Table 3 Serum biochemical markers in the study groups

Mean FT value was significantly higher in patients than controls [0.49±0.03 vs. 0.18±0.01, P<0.0001]. Prevalence of fibrosis stages according to FT in patients versus controls were, F0-F1 52% vs. 95% (P=0.005), F2-F3 24% versus 4% (P=0.0004), cirrhosis (F4) 25% vs. 1% (P<0.0001), respectively. Mean value of AT was significantly higher in patients vs. controls [0.45±0.02 vs. 0.10±0.01, P<0.0001]. Prevalence of activity grades according to AT in patients versus controls were, A0-A1 67% vs. 100% (P<0.0001) and A2-A3 33% vs 0%, (P<0.0001), respectively. FT-observed AUROC (95%CI) for advanced fibrosis (F3F4) was 0.73 (0.62-0.81)(P<0.0001)(Fig. 1). For advanced fibrosis, the adjusted AUROC (95%CI) according to the observed DANA of the study (=1.841) was 0.80 (0.69-0.88). FT-observed AUROC (95%CI) for cirrhosis (F4) was 0.94 (0.86-0.98) (P<0.0001) (Fig. 1). AT-observed AUROC (95%) for moderate and severe activity (A2A3) was 0.72(0.61-0.81) (P<0.0001) and for severe activity (A3) was 0.88 (0.78-0.93).

Figure 1 Area under the receiver operating characteristic curve of FibroTest (A) for advanced fibrosis (F3F4 Metavir) = 0.80 (0.69-0.88), and (B) for cirrhosis = 0.94 (0.86-0.98)

Discussion

Biomarkers are now recommended as alternative to LB by European [14] and Canadian guidelines [15]. The reported AUROC of FT for predicting ≥F2 fibrosis varies from 0.74 to 0.84 and for predicting F4 fibrosis it varies from 0.71 to 0.87 [16-21]. However there is only limited data showing the efficacy of FT in a cohort with predominantly genotype 4 infection [16]. Halfon et al reported high diagnostic accuracy for AT (AUROC of 0.73, CI=0.69-0.77) for predicting A2-A3 necroinflammation [21]. Our study showed that fibrosis estimated by FT was significantly higher in patients compared with controls (mean 0.49 vs. 0.18), P<0.0001. In the patients, the AUROC for advanced fibrosis (F3, F4) was 0.73 (0.62-0.81), but, when adjusted according to the observed DANA, it was 0.80 (0.69-0.88); AUROC for cirrhosis (F4) was 0.94 (0.86-0.98). The AUROC of AT for moderate and severe activity (A2A3) was 0.72 (0.61-0.81); AUROC for severe activity (A3) was 0.88 (0.78-0.93).

The discordance in two classes (advanced versus non-advanced fibrosis) between FT and LB (according to the international definition) was only 18/107 (17%). Likewise, for cirrhosis versus non-cirrhosis the discordance was only 7/107 (6.5%). The activity assessment by AT as compared to LB (no/minimal versus moderate/severe activity) showed a discordance of only 11/107 (10.3%) and for severe versus not severe activity the discordance was 10/107 (9.3%). The concordance rates for FT and AT were much higher for the subgroup of patients with good quality LB. ALT is an intracellular liver enzyme and a good indicator for liver cell necrosis, a common pathway for many hepatotoxic insults; when it is taken as an indicator of activity the discordance analysis suggested that LB could have false-positive and false-negative results for the activity estimation.

Different studies assessing F0-F1 fibrosis vs. F2-F3-F4 in patients with chronic HCV using different seromarkers had comparable results when assessed by AUROC. Imbert-Bismut et al showed FT AUROC of 0.8 [19]; Forns et al showed FT AUROC of 0.86 [22]; Adams et al showed Hepascore (bilirubin, GGT, hyaluronate, A2M, age, sex) AUROC of 0.85 [23]; Patel et al showed Fibrospect (TIMP-1, A2M, hyaluronate) AUROC of 0.83 [24]; other studies comparing F0-F1-F2-F3 vs. F4 (cirrhosis) showed FT AUROC of 0.92 [32], and Hepascore AUROC of 0.94 [25], so the results of this study had comparable results to most of other studies.

The combination of non-invasive markers in assessing liver fibrosis showed that performing FT, APRI, and Forns index with LB improves the diagnostic accuracy for liver fibrosis in CHC patients. However, with even in the “best” scenario an AUROC >0.90 can rarely be achieved even for a perfect marker [26]. Newer protocols combining biochemical and imaging (transient elastography) has shown promise in increasing the accuracy [27].

FT is not only good predictive for liver fibrosis, but it can be useful in estimation the degree of portal hypertension. It has significant correlation with hepatic venous pressure gradient (HVPG). In a study by Thabut et al, there was a significant correlation between FT and HVPG. In cirrhotic patients, FT was significantly higher when there was severe portal hypertension (0.87±0.15 vs. 0.73±0.14, respectively, P=0.02) [28].

FT is especially useful for evaluating fibrosis progression over time and monitoring the outcomes of therapy, since few patients are eager to undergo frequent repeat biopsies. The limitation of this study was that the number of patients who fulfilled the criteria of good quality LB was limited.

In conclusion, FT-AT had excellent observed and standardized diagnostic values for fibrosis and activity. The concordance between FT and LB was fair, with discordance of two or more stages in less than 20% for advanced fibrosis and less than 10% for cirrhosis. Similar results were obtained for the AT. Sensitivity analysis showed higher diagnostic values for FT and AT when LB samples were longer than 15 mm and not fragmented. Discordant results between LB and biomarkers decreased in the group of good quality biopsy. In our cohort of patients with chronic hepatitis C (predominantly genotype 4) infection, FiboTest and ActiTest appears to be a good non invasive marker for assessment of liver fibrosis and inflammatory activity.