Liver stiffness and non-invasive tests predict overall survival in chronic hepatitis C. However, in patients chronically infected with hepatitis B virus (HBV), only the association between liver stiffness and the risk of hepatocellular carcinoma has been published.
To evaluate the 5-year prognostic value of liver stiffness, non-invasive tests of liver fibrosis, and liver biopsy, to predict overall survival in chronic hepatitis B.
In a consecutive cohort, we prospectively assessed fibrosis, with liver stiffness, FibroTest, APRI, FIB-4 and liver biopsy (if indicated). We examined death and liver transplantation during a 5-year follow-up, and factors associated with overall survival.
A total of 600 patients (men 64%, mean age 42 years, inactive carriers 36%) with chronic hepatitis B were included. At 5 years, 25 patients were dead (13 liver-related deaths) and four patients had liver transplantation. Overall survival was 94.1% and survival without liver-related death 96.3%. No liver-related death was observed in inactive carriers. Survival was significantly decreased in patients diagnosed with severe fibrosis, whatever the non-invasive method used (P < 0.0001), or liver biopsy (P = 0.02). Patients' prognosis decreased as liver stiffness and FibroTest increased. In multivariate analysis, FibroTest and liver stiffness had the highest hazard ratio with survival. The association persisted after adjustment on age, necro-inflammatory histological activity presumed by ActiTest and treatment.
Liver stiffness measurement or FibroTest can predict survival in chronic HBV infection. Thus, these tools may help physicians to early assess prognosis and discuss specific treatments, such as liver transplantation.
The evaluation of liver fibrosis is a key step to manage a chronic liver disease and to assess its prognosis, as complications mainly occur in patients with advanced stages. Portal hypertension, ascites or hepatocellular carcinoma are associated with a shorter survival. Early assessment of the risk of bad prognosis helps the physician to manage patients with cirrhosis and to decide for a liver transplantation. Known risk factors for disease progression in chronic hepatitis B can be broadly divided into host, and viral factors. Host factors include age, gender, genetic susceptibility, family history, obesity and immune status. Viral factors can include HBeAg status, hepatitis B virus (HBV) DNA level, genotype, mutants and co-infection.
Liver biopsy does not satisfy quality criteria as a surrogate endpoint marker because of its complication rate, sampling error, intra- and interobserver variability, expense and patient reluctance to undergo serial monitoring.[1, 2]
Different scores such as the Child-Pugh score or the MELD score (model of end stage liver disease) are correlated with the prognosis of advanced cirrhosis and help the physicians to take care of cirrhotic patients.[3, 4] In the past decade, non-invasive markers have been developed as an alternative for liver biopsy, to evaluate the severity of chronic liver diseases, such as FibroTest (Biopredictive, Paris, France; FibroSure-Labcorp, Burlington, NC, USA). It has a high degree of accuracy and reproducibility in predicting bridging fibrosis and cirrhosis in patients with chronic liver diseases. In chronic hepatitis B, its performance is highly significant, with area under the ROCs (AUROCs) of 0.84–0.90 and 0.85–0.87 for the diagnosis of significant fibrosis and cirrhosis respectively.[6, 7] FibroTest has a 5-year prognostic value similar to that of liver biopsy for the prediction of cirrhosis decompensation and survival in patients with chronic hepatitis C virus (HCV),[8-10] chronic hepatitis B and alcoholic liver disease. The aspartate aminotransferase (AST) to platelet ratio index (APRI), and the FIB-4 score, have significant diagnostic performance for fibrosis and cirrhosis, and are significantly associated with mortality in patients with chronic HCV infection. To our knowledge, no study has evaluated the association of APRI or FIB-4 score with the long-term survival in HBV.
Liver stiffness measurement (LSM) using M probe of FibroScan (Echosens, Paris, France) is a non-invasive method for the diagnosis of liver fibrosis. It has a high degree of accuracy and reproducibility in predicting bridging fibrosis and cirrhosis in patients with chronic liver diseases.[15-20] In chronic hepatitis B, its performance is highly significant, with AUROCs of 0.81–0.87 and 0.85–0.93 for the diagnosis of significant fibrosis and cirrhosis respectively.[7, 21-23] There is a relationship between the value of liver stiffness and various complications of cirrhosis, such as oesophageal varices, variceal bleeding, portal hypertension, ascites and hepatocellular carcinoma. Moreover, LSM can predict survival in chronic hepatitis C. In HBV patients, the risks of hepatocellular carcinoma development, liver-related events and liver-related mortality increase with LSM.[25-27] To our knowledge, there is no study evaluating a relationship between 5-year overall survival and liver stiffness in patients with chronic hepatitis B.
The aim of this prospective study was to evaluate the 5-year prognostic value of liver stiffness, FibroTest, APRI, FIB-4 and liver biopsy for predicting survival in patients with chronic hepatitis B.
Patients and methods
From April 2003 to December 2010, we prospectively collected data on a large cohort of consecutive patients addressed to our centre for chronic hepatitis B. At baseline, we noted the clinical, biological, endoscopic and radiological parameters, and any liver decompensation in the past. During the follow-up, we collected the natural history events, such as liver complications, liver transplantation or death. Study patients belonged to a prospective hospital-based cohort of the Hepatology Unit of Haut-Lévêque Hospital (University Hospital of Bordeaux, Pessac, France). In this cohort study, we targeted follow-up consultations and exams every 6 or 12 months or closer. The end of the follow-up was 31 March 2011. Final analysis of the data was done in February 2012.
We included all consecutive patients with an age over 18 and chronic hepatitis B of any severity. The determination of chronic hepatitis B was made using standard diagnostic criteria: positive HBs antigen for more than 6 months. Inactive carriers were defined as patients with negative HBe antigen, normal alanine aminotransferase (ALT) level (at least four times a year), and HBV-DNA <20 000 IU/mL (at least twice in the past 5 years). Exclusion criteria were all other causes of chronic liver disease. Patients with HIV infection or ascites were excluded. HBV treatment was given according to EASL guidelines.
The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki. Patients were enrolled after written informed consent was obtained.
Clinical and biological parameters
For all patients, the following parameters were determined at the time of liver stiffness. Clinical parameters included weight, height, past history of ascites or bleeding varices and hepatocellular carcinoma. Biological parameters included AST, ALT, γ-glutamyl-transpeptidase (GGT), total bilirubin, platelet count, prothrombin time and different scores: FibroTest, APRI and FIB-4. In patients with cirrhosis, oesophageal varices were examined by upper gastrointestinal endoscopy. Since ascites is a physical limitation to the technique because elastic waves do not propagate through liquids, patients with ascites were excluded.
FibroTest and ActiTest scores were computed on the Biopredictive website (www.biopredictive.com). APRI index was calculated as follows: aspartate transaminase (× upper limit of normal) × 100/platelet count (giga/L). FIB-4 score was calculated as follows: [age (in years) × AST (U/L)]/[platelet count (giga/L) × ALT (U/L)1/2]. For each non-invasive test, we used the most usual published cut-off for severe fibrosis or cirrhosis (F3F4 or F4 according to Metavir score).
Liver stiffness measurement
Liver stiffness measurement was performed with the patient lying in dorsal decubitus with the right arm in maximal abduction, on the right lobe of the liver, through intercostal spaces. The operator, assisted by a time-motion ultrasound image, located a liver portion at least 6 cm thick and free of large vascular structures. When the target area had been located, he pressed the probe button to commence the measurements. The measurement depth was between 25 and 65 mm using the M probe of FibroScan. Ten validated measurements were performed on each patient. The results were expressed in kilopascals (kPa). Only procedures with at least 10 validated measurements and an interquartile range (IQR) inferior to 30% of the median value were considered reliable.[17, 30] Measurement of liver stiffness was performed in our unit by specialised nurses.
Patients with possible indication of treatment or with discordance between FibroTest and LSM (less than 10% of cases) had a liver biopsy, which was processed with standard techniques. One experienced pathologist (Brigitte Le Bail), unaware of the biochemical markers, evaluated the stage of fibrosis and grade of activity according to the METAVIR scoring system.
If needed, patients had a conventional treatment of their diseases during the follow-up (oral nucleoside/nucleotide therapy). The overall 5-year survival (whatever the cause of death, and including liver transplantation) was the ‘a priori’ main end-point used to compare the prognostic value of the different methods. Survival time was calculated from the date of liver stiffness to the endpoint date (March 31, 2011). This interval was censored at the time of last follow-up. Each year, for patients who had not been seen at our hospital in the previous 12 months, we found out whether they were living and, if not, the date and the cause of death. For patients who were still alive, we either interviewed the patients or obtained information through their physicians. For patients who died outside our hospital, we obtained information about the date and cause of death from their physicians or family. The survival without liver-related death (including death related to liver disease and liver transplantation) was also assessed.
Tests were used as follows: chi-squared test for qualitative comparisons, Mann–Whitney test for quantitative comparisons, AUROC curves for the diagnostic and univariate prognostic analyses. The prognostic AUROCs were estimated by the empirical (nonparametric) method of Delong et al., or by binomial approach when events were less than 29 cases, and compared using the paired method. Comparisons between diagnostic AUROCs were performed using the Obuchowski method to take into account the risk of multiple testing and the spectrum effect.[34-36]
The Obuchowski measure allows to compare two biomarkers with a single test, avoiding appropriate correction for the type I error when comparing two biomarkers for different stages or grades. This measure is a multinomial version of the AUROC. With N (=5) categories of the gold standard outcome (histological fibrosis stage) and AUROCst, the estimate of the AUROC of diagnostic tests for differentiating between categories s and t, the Obuchowski measure, is a weighted average of the N(N–1)/2 (=10) different AUROCst corresponding to all the pairwise comparisons between two of the N categories. Each pairwise comparison has been weighted to take into account the distance between activity grades (i.e. the number of units on the ordinal scale). A penalty function proportional to the difference in METAVIR units between grades was defined: the penalty function was 0.25 when the difference between stages was 1, 0.50 when the difference was 2, 0.75 when the difference was 3 and 1 when the difference was 4. The Obuchowski measure can be interpreted as the probability that the non-invasive index will correctly rank two randomly chosen patient samples from different activity grades according to the weighting scheme, with a penalty for misclassifying patients. The overall Obuchowski measure is not equivalent to a usual AUROC curve as the measurements are weighted according to the distance between stages.
For survival analyses, time-dependent Kaplan–Meier analysis for survival curves, the log-rank test for univariate comparisons and the Cox proportional hazard model for multivariate analysis were performed. We also used the Cox model including the two best prognostic factors (FibroTest and LSM) to assess their performance by AUROCs. Because of the limited number of events, only overall 5-year survival was assessed. Because of possible colinearity effect between FibroTest and age, and known association between stiffness age and necro-inflammatory activity, a multivariate analysis was performed adjusted on age and ActiTest. We used liver stiffness values expressed in log values to perform statistical analysis and comparisons.
We used two-sided statistical tests for all analyses. Due to the number of biomarkers comparisons (n = 4) a P value <0.01 was considered as significant, between 0.01 and 0.05 as borderline, greater than 0.05 as not significant. Statistical analyses were performed using Number Cruncher Statistical Systems software and R software [library(nonbinROC)and library(ROCR)].
The predetermined factors associated with prognosis were fibrosis as the main factor, and three covariates: HBV treatment, age and necro-inflammatory grade at baseline presumed by ActiTest. Treatment was defined as at least one treatment given during the follow-up.
As biopsy was performed in 214 subjects only, a sensitivity analysis of survival was performed including biopsy stage, FibroTest and LSM.
All the variables have been introduced initially in the multivariate analysis as continuous variables for continuous data: age and biomarkers. If using the continuous quantitative value in the Cox regression model, the maximum likelihood procedure cannot reach convergence (no completion), a semi-quantitative value of the continuous data was used in the multivariate model.
Characteristics of patients
A total of 650 patients were recruited (Figure 1). Among these patients, 23 (3.5%) were excluded for not interpretable LSM: liver stiffness failure (no valid shot) in eight patients (1.2%), less than 10 valid measurements in seven patients (1.1%), IQR/liver stiffness >0.30 in eight patients (1.2%). Moreover, eight patients (1.3%) had no FibroTest (two missing and six not applicable due to high ALT level). At least, 19 patients had no follow-up (3.1%). Therefore, 600 patients were evaluated. APRI and FIB4 were missing for 12 patients out of these 600 patients (platelet count or AST or ALT level missing).
Median follow-up was 49.7 months (IQR 32.2–63.2). Clinical and biological characteristics of the patients are indicated in Table 1. A majority of them were men (64%), and mean age was 42.5 ± 15.2 years. Most of patients (56.5%) had normal ALT level (<40 IU/L in women, <50 IU/L in men). Only 13% of patients had ALT level >2 × ULN. Ninety-four patients (15.7%) had liver stiffness >9 kPa (cut-off value for severe fibrosis or cirrhosis in HBV patients with normal ALT level), 46 patients (7.7%) had FibroTest >0.74, 10 patients (1.7%) had APRI >2, and 27 patients (4.6%) had FIB-4 score >3.25. Liver biopsy was available in 214 patients (35.7%) and showed severe fibrosis or cirrhosis in 86 cases (40.2%). Median liver biopsy samples length was 22 mm.[16-30]
Table 1. Characteristics of the 600 patients with chronic hepatitis B infection
Accuracy of biomarkers for the diagnosis of fibrosis stage
As indicated in Table 2, liver stiffness and FibroTest had comparable diagnostic accuracy for the diagnosis of fibrosis stage, but higher accuracy in comparison with other biomarkers, when biopsy was taken as the reference.
Table 2. Overall accuracy (weighted AUROCs) of biomarkers for the diagnosis of fibrosis stages in patients with chronic hepatitis B infection
Comparison: Z-test (P significance)
LSM, liver stiffness measurement.
A total of 209 of 600 patients had biopsy and all the four biomarkers performed.
The Obuchowski measure estimates the overall accuracy (weighted AUROC) that is the weighted mean of the 10 pairwise comparisons stage by stage (F0 vs. F1, F0 vs. F2, F0 vs. F3, F0 vs. F4, F1 vs. F2, F1 vs. F3, F1 vs. F4, F2 vs. F3, F2 vs. F4, and F3 vs. F4).
At the beginning of the study, 11 patients (1.8%) had a hepatocellular carcinoma. At the end of follow-up, six of them were still alive. The overall number of death/transplantation was 29 (4.8%) of 600, 17 liver-related and 12 not liver-related deaths. Among these 29 patients, 25 patients were men, and 13 patients (44.8%) had LSM ≤9 kPa at inclusion. All transplanted patients received liver transplantation for HCC.
At inclusion, 209 patients (36.3%) were inactive carriers, 272 patients (47.4%) had chronic hepatitis with negative HBe antigen, and 94 patients (16.3%) had chronic hepatitis with positive HBe antigen. During follow-up, 452 patients (75.3%) did not receive any treatment for HBV infection.
Among the 94 patients with liver stiffness >9 kPa, 53 patients (56.4%) did not receive any treatment (six deaths in this group), and 41 patients received treatment (10 deaths in this group). Among the 506 patients with liver stiffness ≤9 kPa, 399 patients (78.8%) did not receive any treatment (eight deaths in this group), and 107 patients received treatment (five deaths in this group).
Details of the causes of deaths are given in Table 3. In the overall population (600 patients), the 5-year overall survival was 0.94 (95% CI 0.91–0.96), and the 5-year survival without liver-related death or transplantation was 0.96 (95% CI 0.94–0.98).
Table 3. Causes of the 29 deaths or transplantations during the follow-up of 600 patients with chronic hepatitis B infection
Liver-related deaths (n = 17)
Deaths unrelated to liver disease (n = 12)
Hepatocellular carcinoma (n = 7)
Non liver cancer (n = 6)
Hepatic failure (n = 4)
Cardiovascular disease (n = 2)
Variceal bleeding (n = 1)
Infection (n = 1)
Hepatorenal syndrome (n = 1)
Unknown (n = 3)
Liver transplantation (n = 4)
Only three deaths were observed in the 209 inactive carriers. All these deaths were unrelated to the liver (lung and pancreas cancers).
Prediction of survival
The survival probability of patients classified according to liver stiffness, FibroTest, APRI, FIB-4 and liver biopsy is detailed in Figure 2 and their specific association with survival in Table 4.
Table 4. Prognostic value of biopsy and biomarkers for predicting mortality or liver transplantation in patients with chronic hepatitis B
For FibroTest and liver stiffness, two cut-offs were evaluated for predicting survival: 0.73 and 0.85 for Fibrotest, 9 and 20 kPa for liver stiffness, according to previous published results. As indicated in Figure 2A, B, overall survival was decreased according to liver stiffness and FibroTest values. The 5-year overall survival was 97.1% in patients with liver stiffness <9 kPa, and 61.5% in patients with liver stiffness >20 kPa, and 96.8% for FibroTest ≤0.73, and 49.2% in patients with FibroTest >0.85.
In univariate and multivariate analysis, FibroTest and liver stiffness had the higher prognostic values. The association persisted after adjustment on age, necro-inflammatory histological activity presumed by ActiTest, HBV-DNA and treatment.
When the prognostic performances were expressed using AUROC (Figure 3), there was high significance for Fibrotest 0.82 (95% CI 0.71–0.89), and liver stiffness 0.80 (0.70–0.87) without significant difference between these biomarkers.
To our knowledge, no studies reported on the association between liver histology, APRI, FIB-4 scores, liver stiffness and survival in HBV patients. Only FibroTest has been shown to be associated with survival in chronic hepatitis B.[40, 41] Our study is the first showing that liver stiffness has a prognostic value at 5 years for overall survival in patients with chronic HBV infection. Moreover, to our knowledge, this is the first study evaluating the prognosis value of different non-invasive tests for overall survival in patients with chronic HBV infection. The present study shows that liver stiffness and FibroTest can predict similar survival in HBV patients.
The second main finding that liver stiffness and FibroTest had better prognosis values than liver biopsy, the classical gold standard for staging, was expected as the prognosis gets worse as these quantitative fibrosis estimates increased. This cannot be the case for the METAVIR cirrhosis as there is a unique stage ‘F4’ for cirrhosis. A better prognostic value of FibroTest vs. biopsy had been already observed by Ngo et al.[8, 9] and Vergniol et al.[8, 9] for HCV patients.
The third main finding is the ranking of non-invasive methods for prognosis purpose. From the results, liver stiffness and FibroTest had better prognostic values than FIB4 and APRI score. The result confirms previous results published in HCV patients. We have not evaluated other biomarkers such as Fibrometer, ELF and Hepascore. However, we cannot compare liver stiffness and FibroTest since liver biopsy was missing in a large number of patients.
Since ascites is a physical limitation to the technique because elastic waves do not propagate through liquids, patients with ascites at baseline were excluded. The prognosis of those patients is known to be bad, and our results may have been underestimated comparing to the reality. Moreover, we used only M probe of FibroScan for the assessment of liver stiffness. New methods as ARFI or Aixplorer need to be evaluated as prognosis factors. Another limitation of this study is that our centre is a tertiary centre. Therefore, patients may not be representative of a larger HBV patient population. However, in this study, 23% of biopsied patients had cirrhosis and a majority of patients had F0F1F2 fibrosis.
HBe Ag status was available in more than 95% of subjects, but we did not collect HBV-DNA in all cases at inclusion (60 missing data). The association of non-invasive markers and HBV-DNA as prognosis model should be of interest. Moreover, the relationship between Fibrotest or liver stiffness and clinical events related to cirrhosis (hepatocellular carcinoma, ascites, etc.) needs to be further evaluated.
The ALT and AST are surrogates of hepatic necro-inflammation, which tend to fluctuate significantly in chronic hepatitis B and might not reflect the fibrosis staging as accurately as in other liver diseases. Therefore, in patients with ALT level >2 or 3 × ULN, our results need to be further evaluated.
In our study, we decided to use published FibroTest and liver stiffness cut-offs for severe fibrosis and cirrhosis. To test clinically relevant cut-offs, we chose the previously published values for severe fibrosis and cirrhosis,[19, 22, 42] doing the hypothesis they may be associated with a worse prognosis. To have a sensitive test, we decided to evaluate a cut-off for severe fibrosis instead of a cut-off for cirrhosis. Therefore, we hypothesise that all patients with cirrhosis were included in the analysis. We also studied the prognosis value of a liver stiffness higher than 20 kPa and FibroTest >0.85, known to be associated with liver-related complications.[5, 8, 24] Our results show that, in the group of patients with cirrhosis, an increasing liver stiffness or FibroTest value is associated with a worse prognosis, emphasising the concept of non-invasive prediction of survival in cirrhotic patients with liver stiffness or FibroTest. The evolution of liver stiffness or FibroTest values over time is of major importance. However, this point was not evaluated in our study and needs further evaluation.
In our study, we decided not to test the Child-Pugh score as a prognosis factor. Indeed, the prognosis value of Child-Pugh score is well established in cirrhotic patients. Natural history of HBV patients is related to the amount of fibrosis, we therefore chose to test only non-invasive techniques for the diagnosis of fibrosis.
Few data exist showing that liver stiffness and FibroTest may decrease under treatment,[43, 44] suggesting it may be correlated with an improved survival. In our study, we found that liver stiffness and FibroTest were accurate prognostic factors independently of treatment. Only 25% of our patients received any treatment. Therefore, we could not evaluate the effect of treatment on survival. The interaction between treatment, liver stiffness and survival needs further evaluation.
Death and liver transplantation are sudden and objective criteria that can be precisely assessed. Since a large majority of the patients were treated in our centre, and almost all the events occurred in the southwest area of France, this allows us a sharp evaluation of survival using the Kaplan–Meier method. Our weakness of the present study was that the included population was not a random, community-based population. Another limitation is the absence of repeated measurements of standardised HBV-DNA, which has been associated with survival and occurrence of hepatocellular carcinoma. We have therefore not separated our population according to the standard classification using ‘active’ and ‘inactive carriers’. We acknowledge that new studies should confirm the previously observed greater prognostic value of fibrosis biomarkers in comparison with viral load. In our population, HBV-DNA had no independent prognostic value (HBV-DNA was available in 540 cases). This could be due to a lack of power, but another rational explanation is that the REVEAL study had not assessed the baseline fibrosis stage using validated biomarker. At least, although non-invasive tests are important as prognostic indications of fibrosis, we need to keep in mind that hepatocellular carcinoma can develop in HBV independently of the presence of cirrhosis.
In this prospective study, we confirmed the prognostic value of liver stiffness and FibroTest on survival. This information is of a major importance, helping us to sharpen our various tools for the follow-up of our patients. Liver stiffness, as a good predictive factor of survival, may help the physician to evaluate earlier the severity of those diseases, to decide with stronger arguments of a liver transplantation or a portosystemic shunt, and to evaluate more precisely the surgical risk of our cirrhotic patients. It may also be used as a complement to the ‘score foie’ to decide of the emergency of a liver transplantation in a cirrhotic patient. LSM and/or FibroTest could replace liver biopsy for the evaluation of the disease, whatever the stage of the disease.
In our clinical practice, liver stiffness and FibroTest are already strong evidence to give information to patients and their family on the severity of the liver disease. Using these predictive data, it should help us in the future to caution our patients and their family of the importance of total alcohol abstinence or on the need for serious compliance with anti-viral therapy, and to plan the timing of possible liver transplantation.
Guarantor of the article: None.
Author contributions: Julien Vergniol: study concept and design, analysis and interpretation of results. Juliette Foucher: acquisition of data, technical support. Faiza Chermak and Charline Barthe: acquisition of data. Pierre-Henri Bernard: acquisition of data. Brigitte Le Bail: acquisition of data, analysis and interpretation. Wassil Merrouche: acquisition of data, technical support. Victor de Lédinghen: study concept and design, drafting of the manuscript, statistical analysis, study supervision. All authors approved the final version of the manuscript.
Declaration of personal interests: Juliette Foucher received funding from Roche, Janssen-Cilag, Gilead and Bristol-Myers Squibb. Victor de Lédinghen received funding from Merck, Roche, Bristol-Myers Squibb, Janssen-Cilag, Boehringer-Ingelheim, Gilead and Echosens.