ADVANCED FIBROSIS

FIELD OF THE INVENTION:

The present invention relates to methods for assessing whether a patient suffering from chronic hepatitis B infection is associated with moderate or advanced fibrosis. BACKGROUND OF THE INVENTION:

Chronic viral infection such as hepatitis B (CHB) poses an important risk for the development of liver fibrosis. The clinical course and sequel of CHB varied among individuals. Most likely the pathogenesis of CHB is multifactorial as it involves both viral and host factors ¹ .

While HBsAg remains detectable for life, the titer of viral DNA decrease, HBeAg disappears from blood along with seroconversion to positivity to anti-HBe antibodies. The virus of such carriers, particularly in southern Europe and Asia, harbors mutations that prevent the production of HBeAg and thus impact the natural course of CHB. The most common naturally occurring HBV variants are those appearing in the Basal Core Promoter (BCP) and the pre-Core (PC) regions. Among these, a single mutation in the PC region (1896 ^A ), which regulates HBeAg synthesis ^2"3 and the double mutation in the BCP region (1762 ^T /1764 ^A ), which regulates HBeAg synthesis at transcriptional level ⁴ , are the most studied. Interestingly, it has been reported that PC mutation decreases the risk of HCC, whereas BCP mutations have been reported to be associated with advanced liver disease ^5"6 , indicating the distinct role of these mutants in the long-term outcome of CHB ⁷ . Studies have suggested that HBV genotypes may be associated with PC and BCP mutations ^8"12 . However, this association remains controversial ^13"15 . Asian studies suggest that genotype C is associated with advance fibrosis and genotype B with HCC occurrence ¹³ , while in Western countries no association between fibrosis and HBV genotypes are reported ^14"15 . These discrepancies might be due to the fact that in Asian studies only genotypes B and C are investigated ¹³ while in Western studies mainly investigated genotypes A and D ^14"15 .

Viral load has been reported to be the main actor of fibrosis progression ^16"20 . Inactive carriers (viral load < 2000 IU/ml) have no or minima liver lesion ²¹ , while high HBV-DNA level has been shown to significantly increase the incidence of cirrhosis and hepatocellular carcinoma (HCC) ⁶ ' ^19-20 . Recently, it has been suggested that HBsAg levels is correlated to fibrosis stages in HBeAg positive patients ^22"23 , and its combination with viral load important to define the minimal risk for the occurrence of HCC in HBeAg negative patients ^17"18 . Furthermore, HBsAg titer is a useful marker for monitoring natural history of HBV infection ²⁴ .

Genome-wide association studies showed that single nucleotide polymorphisms (SNPs) located within interferon lambda 3 (IFNL3), also known as IL28B, is associated with pegylated- interferon (PEG-IFN) plus ribavirin treatment induced hepatitis C clearance ^25"26 . Controversial results have been reported on the association between IFNL3 polymorphism and response to PEG-IFN therapy in CHB patients ^27-28 . More recently, studies suggested that IFNL3 polymorphism, in CHB patients, correlates with liver inflammation ²⁹ , increased susceptibility of HCC ³⁰ . However, these observations are controversial ³¹ .

Accurate assessment of liver fibrosis is essential for successful disease management in CHB patients. Liver biopsy is the gold standard. However it is invasive and can be subjected to variability and pathological interpretation. Significant progress has been made in noninvasive assessment of liver disease (serum biomarkers transient elastography) mainly reported in chronic hepatitis C ^32-34 . None of these technics take into account host and virological data, which are important confounders, such as ethnicity, ALT or etiology of liver disease.

SUMMARY OF THE INVENTION:

The present invention relates to methods for assessing whether a patient suffering from chronic hepatitis B infection is associated with moderate or advanced fibrosis. DETAILED DESCRIPTION OF THE INVENTION:

Fibrosis assessment is essential in patients with chronic hepatitis B (CHB). Little is known about the association between fibrosis, host factors and viral factors. The inventors investigated these relationships in a large cohort of consecutive, well-characterized, treatment-na ^' ive CHB patients. 406 patients with available liver biopsy, clinical and host characteristics, complete HBV virology (HBsAg and HBV-DNA levels, genotype, IFNL3 polymorphism, BCP/PC variants) determined the same day. Histological lesions were assessed using METAVIR score. Predictors of moderate to advance fibrosis (F2-F4) were evaluated. 101 HBeAg(+), 305 HBeAg(-) patients were included. Fibrosis stages FO-1, F2- F4 in 61% and 39% of the patients, respectively. In univariate analysis, advanced age (p<0.001), male (p=0.01), HBeAg(+) (p=0.06), ALT >2xN (<0.0001), HBV-DNA >4.3 logioIU/ml (p<0.0001), 1762 ^T /1764 ^A (<0.0001) and 1762 ^T /1764 ^A /1896 ^A (p=0.0001) variants, were associated with F2-F4. HBsAg >4.3 log ₁₀ IU/ml was associated with FO-Fl (p=0.04). In multivariate analysis, 1762 ^T /1764 ^A (p<0.0001), 1762 ^T /1764 ^A /1896 ^A (p=0.0001) variants (strongest predictors), HBV-DNA >4.3 logioIU/ml (p<0.0001), ALT >2xN (p=0.001), age (p=0.0002), were independently associated with F2-F4. The good discrimination to correctly identify F2-F4 versus FO-Fl obtained with this multivariate model (age + HBV-DNA + ALT + HBV variants) was evidenced by a c-statistic of 0.77 (95% CI, 0.72-0.82). Hosmer- Lemeshow test p-value of 0.7 showed no model misspecification. In conclusion, the main actors of fibrosis development are: age, ALT, HBV-DNA and HBV variants. They allow accurate identification of F2-F4 patients. Our results suggest that clinical as well as virological markers are relevant in predicting moderate to advanced fibrosis facilitating CHB patients monitoring.

Accordingly the present invention relates to a method for assessing whether a patient suffering from chronic hepatitis B infection is associated with moderate to advanced liver fibrosis comprising the steps of

(1) providing a blood sample from the patient

(2) determining the ALT level in the blood sample obtained from the patient and comparing said level with its predetermined reference value

(3) determining the HBV-DNA level in the blood sample obtained from the patient and comparing said level with its predetermined reference value

(4) detecting the presence or absence of the HBV 1762 ^T /1764 ^A variants in the blood sample obtained from the patient

(5) concluding that the patient has a high risk of having moderate to advanced liver fibrosis when the ALT level is higher than its predetermined reference value, the HBV-DNA level is higher than its predetermined reference value and the presence of the HBV 1762 ^T /1764 ^A variants is detected provided that the older the patient is, the higher the risk is. According to the invention, the patient is infected with HBV of any genotype and is typically infected with a HBV of genotype A, B, C, D; E...etc.

The term "liver fibrosis score" reflects the degree of progress of the liver fibrosis. The liver fibrosis score quantifies the liver tissue damage, in particular the nature, number and intensity of the fibrous lesions in the liver. Unless otherwise indicated, or unless the context dictates otherwise, the liver fibrosis scores indicated in the application are F scores established in accordance with the Metavir system (see Table 1), and the terms "score", "fibrotic score", "fibrosis score", "liver fibrosis score" and similar terms have the clinical significance of a Metavir F score, i.e. they qualify or even quantify the damage to the tissue, more particularly the lesions (or fibrosis) of a liver.

Table 1: correspondences between stages of liver fibrosis and Metavir F score.

As used herein, the term "moderate to advanced liver fibrosis" indicates that the patient has a score of F2, F3 or F4.

As used herein the term blood sample has its general meaning in the art and includes whole blood sample, serum sample and plasma sample.

Methods for determining the Alanine Aminotransferase (ALT) level in a blood sample are well known in the art and typically include those described in EXAMPLE. Typically a predetermined reference value for ALT is 2xN wherein N represents the ALT level measured in the healthy population as defined in Prati D, Taioli E, Zanella A, et al. Updated definitions of healthy ranges for serum alanine aminotransferase levels. Ann Intern Med. 2002;137: 1-10.

Methods for determining the HBV-DNA level in a blood sample are well known in the art and typically include those described in EXAMPLE. Typically, a predetermined reference value for HBV-DNA level is 4.3 log 10 IU/ml. Methods for determining the presence of the HBV 1762 ^T /1764 ^A variants are well known in the art and typically include those described in EXAMPLE.

In some embodiments, the method of the invention further comprises the step of detecting the presence or absence of the HBV 1762 ^T /1764 ^A /1896 ^A variants.

As used herein the term "high risk" means that the subject has at least 75% of risk of having moderate to advanced liver fibrosis. Accordingly the when the subject is at high risk of having moderate to advanced liver fibrosis, the probability is at least 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; 100%. Following characteristics are independently associated with moderate to advanced fibrosis (F2-F4, Metavir score system): older age (1.05 fold increase/per year); ALT > 2 times the upper limit of normal (ULN)(2.5 fold increase); HBV DNA > 4.3 logio IU/ml (2.7 fold increase), presence of 1762 ^T /1764 ^A variant (5.2 fold increase), presence of 1762 ^T /1896 ^A variant (4.5 fold increase). The ability to correctly discriminate F2-F4 from F0-F1 patients, was generated by a multivariate model (age + ALT + HBV-DNA + HBV variant) evidenced by a c-statistic of 0.77 (95% CI, 0.72-0.82).

The method of the present invention is clinically very useful. In particular, the method of the present invention is suitable for determining whether the patient is eligible or not to a liver biopsy. More particularly, the method of the present invention is particularly suitable for determining whether the patient is eligible or not to a treatment. For instance, when it is concluded that the patient has a moderate liver fibrosis; the clinician may elect not to administer treatment to the patient, judging, for example, that at this stage of the liver fibrosis, the risk/benefit ratio of the drug treatment which could be administered to the patient would not be favorable. On contrary when it is concluded that the patient has a high risk of having advanced liver fibrosis, the clinician will recommend the administration of a drug treatment to block or at least slow down the progress of this liver fibrosis, in order to reduce the risk of developing into cirrhosis. By being able to make these distinctions in a reliable manner, the method of the present invention can be used to administer, in good time, the drug treatments which are currently available to attempt to combat or at least alleviate a liver fibrosis. Since these drug treatments usually give rise to major side effects for the patient, the means of the invention provide very clear advantages as regards the general health of the patient. Standard treatment of CHB typically includes administration of the patient with a therapeutically effective amount of interferon. In the present invention, an interferon may be pegylated or non-pegylated and may include interferons such as: Intron-A®, interferon alfa-2b (Schering Corporation, Kenilworth, NJ); PEG-lntron®, peginteferon alfa-2b (Schering Corporation, Kenilworth, NJ); Roferon®, recombinant interferon alfa-2a (Hoffmann-La Roche, Nutley, NJ); Pegasys®, peginterferon alfa-2a (Hoffmann-La Roche, Nutley, NJ); Berefor®, interferon alfa 2 available (Boehringer lngelheim Pharmaceutical, Inc., Ridgefield, CT); Sumiferon®, a purified blend of natural alpha interferons (Sumitomo, Japan); Wellferon®, lymphoblastoid interferon alpha nl (GlaxoSmithKline); Infergen®, consensus alpha interferon (InterMune Pharmaceuticals, Inc., Brisbane, CA and Amgen, Inc., Newbury Park, CA); Alferon®, a mixture of natural alpha interferons (Interferon Sciences, and Purdue Frederick Co., CT); Viraferon®; and combinations of these interferons. Typically, the therapeutically effective amount of pegylated interferon alpha-2b is from 0.5 to 2.0 micrograms/kilogram per week on a weekly, three times a week, every other day or daily basis. The treatment regimen of the patient may further comprises administration of direct antiviral agents. Direct antiviral agent, is used herein to mean agents that interfere with specific steps in the hepatitis B virus (HBV) replication cycle. A direct antiviral agent that inhibits HBV replication may be for example any of the currently anti-HBV agents approved for the treatment of HBV, i.e. telbivudine, lamivudine, emtricitabine, entecavir, adefovir and tenofovir. The invention will be further illustrated by the following figures and examples.

However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

EXAMPLE: Non-invasive markers of fibrosis in HBeAg positive or negative chronic hepatitis B

Materials and methods:

Patients

Consecutive treatment-na ^' ive CHB patients were assessed at our center in Paris,

France, between 2000 and 2008. Inclusion criteria for patients were: HBsAg-positive (HBsAg), known HBeAg status; scheduled liver biopsy; and absence of HCV, HDV, or HIV co-infection. Serum samples and liver biopsies were obtained on the same day in all cases. Viral markers including HBeAg, anti-HBc, anti-HBs and anti-HBe were routinely determined in the serum of the patients using standard procedures (AxSYM system, ABBOTT, Rungis, France).

Histological Evaluation

The same pathologist examined histological preparations. Fibrosis stages were assessed using the METAVIR scoring system ³⁵ .

Serum HBsAg quantification

HBsAg levels were measured using the Elecsys ^® HBsAg II quant assay (Roche Diagnostics, Mannheim, Germany) and expressed as IU/mL or logioIU/mL.

Serum HBV-DNA quantification

Serum HBV-DNA levels were measured using the Cobas ^® Ampliprep/cobas TaqMan ^® assay (Roche Diagnostics, Mannheim, Germany) and expressed as IU/mL or logio IU/mL.

HBV genotyping and detection of BCP and PreC region mutations

HBV genotyping and detection of preC and BCP mutations in the HBV genome were carried out by reverse hybridization using INNO-LiPA HBVgenotyping assay and INNO- LiPA HBV precore assays, (Innogenetics, Ghent, Belgium).

IFNL3 polymorphism determination

IFNL3 genotyping for the SNP rs 12979860 was performed using direct sequencing (AmpliTaq gold ^® DNA polymerase and Big Dye ^® terminator vl,l cycle sequencing kit, Applied Biosystems, Warrington, UK). The PCR products were separated on an ABI 3130 sequencer and analyzed with SEQSCAPE 2,6 (Applied Biosystems, Warrington, UK) ²⁵ .

Statistical Analysis

Quantitative variables were expressed as mean ± standard deviation (SD). Statistical tests used for comparing HBeAg positive or HBeAg negative were the non-parametric Fisher's exact test for qualitative data and the non-parametric Wilcoxon signed-rank test for quantitative data. We search for markers of the severity of liver lesions (set into 2 steps F0-F1 and F2-F4). A logistic regression was used to determine whether the identified variables associated with fibrosis were independent risk factors. For the analysis serum ALT levels were set into 2 steps: <2xN and> 2xN. Variables with a p value <0.2 in univariate logistic regression analysis were introduced into the multivariate logistic regression analysis and were selected thereafter by using a backward selection method. First order interaction was tested for significant variables. The model discrimination was assessed by c-statistic and its 95% confidence interval, and model calibration was assessed by the Hosmer-Lemshow goodness- of-fit test. All statistical analyses were performed using SAS software version 9.3 (SAS Institute, Cary, NC, USA). A two-sided p value <0.05 was considered to be significant.

Results:

Study population

406 unselected, consecutive, treatment-na ^' ive CHB patients were included in the study: 101 patients (25%) were HBeAg (+) and 305 patients (75%) were HBeAg (-). Fibrosis stages F0-F1, F3, F3, and F4, were found in 61%, 23%, 8%, and 8%, respectively. Twenty nine patients were excluded from the statistical analysis: 25 with too low viral load to determine HBV genotypes, 2 infected with genotype F and 2 with genotype G, and thus the statistical analysis were performed on 377 patients (Table 1). There were more men than women, with 75% of patients in both HBeAg (+) and HBeAg (-) groups being male. Both the HBeAg (+) and HBeAg (-) groups were ethnically diverse: Asians were more prevalent in HBeAg (+) whereas African was more prevalent in HBeAg (-). Serum ALT levels >2xN were higher in HBe (+) than in HBe(-) patients (p<0.0001). HBV genotype C was more prevalent in HBeAg (+) whereas genotype D and E were more prevalent in HBeAg (-). HBeAg (+) patients were more frequently F3-F4, exhibited significantly high serum levels of both HBsAg (>4.3 log ₁₀ IU/ml) and HBV-DNA (>4.3 logio IU/ml). HBV wild type and the BCP mutation 1762 ^T /1764 ^A were found more frequently in HBeAg (+) while the BCP+preC mutations 1762 ^T /1764 ^A /1896 ^A and the preC mutation 1896 ^A found more frequently in HBeAg (-) patients. No associations were found between HBeAg status and gender or IFNL3 polymorphism (Table 1).

Tablel. Patient's characteristics according to HBeAg status.

* MeaniSD

** 29 patients were excluded from analysis: 25 indeterminate HBV genotypes due to low viral load, 2 infected with genotype F and 2 with genotype G.

Identification of Fibrosis markers: model development

Univariate analysis

In univariate analysis fibrosis stages F2-F4 were significantly associated with older age (per slice of 10 years) (p<0.0001), male gender (p=0.01), HBeAg (+) status (p=0.06), ALT >2xN (p<0.0001), high HBV-DNA (> 4.3 logioIU/ml) (p<0.0001), and 1762 ^T /1764 ^A (p<0.0001) and 1762 ^T /1764 ^A /1896 ^A variants (p<0.0001) (Table 2). Of note high HBsAg titer (>4.3 logioIU/ml) was associated with F0-F1 (p=0.04) (Table 2). No associations were found between fibrosis stages and ethnicity, HBV genotypes and IFNL3 polymorphism. Table 2. Predictors of the severity of liver disease (FO-Fl versus F2-F4) identified by univariate and multivariate analysis.

* Mean±SD ** logio Ul/ml. 29 patients were excluded from HBV genotype analysis: (25 indeterminate due to low viral load, 2 infected with genotype F and 2 with genotype G)

Multivariate analysis In the multivariate logistic regression analysis using a stepwise selection procedure, the independently associated predictors of F2-F4 were: 1762 ^T /1764 ^A (pO.0001) and 1762 ^T /1764 ^A /1896 ^A (p=0.0001) variants the strongest predictors followed, by age (p=0.0002), high HBV-DNA (>4.3 logioIU/ml) (p=0.005) and ALT >2xN (p<0.001). While gender, HBsAg titer and HBeAg status did not enter the model (Table 2). No significant interaction was found between variables of this model

Diagnostic performance of the model.

The good discrimination to correctly identify patients F0-F1 from F2-F4 patients obtained with this multivariate analysis model (age + HBV-DNA + ALT + HBV variants) was evidenced by a c-statistic of 0.77 (95% IC 0.72-0.82). The Hosmer-Lemeshow test p- value at 0.5 shows no model misspecification.

Discussion:

Several host and virological factors influence the course of the liver disease. Age, gender, high viral load, HBV genotypes C>B and D>A have been reported to be associated with more severe liver lesions. Although the literature is prolific with regards to studies evaluating factors associated with liver lesions, most of the reports investigated association between histological lesions with either virological or clinical data. Our study has an unique feature, it is to our knowledge the first investigating both demographical, ethnicity, clinical, host (IFNL3 polymorphism) and complete virological factors (HBsAg titer, HBV-DNA load, HBV genotypes, PC and BCP variants) in a sensitive analysis with the serum samples and patients data were collected the day of the liver biopsies and therefore represent an accurate snapshot of the stage of disease occurring at a specific time in unselected na ^' ive patients. Our main finding is that the likelihood at a determined time point for a patient to have moderate to advanced fibrosis (F2 to F4) is independently associated with: 1762 ^T /1764 ^A (5.2-fold increase) HBV variants the stronger predictor, 1762 ^T /1764 ^A /1896 ^A (4.5-fold increase) HBV variants, HBV-DNA (>4.3 logioIU/ml) (2.7-fold increase), ALT >2xN (2.5-fold increase), and older age (1.05-fold increase/per year), allowing to create an easy to use model differentiating F0-F1 from F2-F4 patients with high accuracy.

Asian studies have reported an association between IFNL3 polymorphism and active chronic hepatitis B ²⁹ or development of hepatocellular carcinoma ³⁰ . However, these studies lack of liver biopsy and furthermore, patients are mainly infected with HBV genotypes B and C. Our study is, so far, the first investigating the association between severity of the liver lesion and IFNL3 polymorphism in a well characterized hepatitis B patients population with an available liver biopsies and HBV genotypes A to E. We report that liver lesions are not associated with IFNL3 polymorphism similarly to hepatitis C patients ³⁶ .

1 18 19 22 HBV-DNA and HBsAg titers are associated with more severe fibrosis lesions ' ^" '

23 ' 37 ^" 38. High HBsAg level increases the risk of cirrhosis and hepatocellular carcinoma in patients with low HBV load ⁷ ' ^18~19 . More recently it has been reported in HBeAg positive patients a strong relationship between HBsAg titer and fibrosis stages, high HBsAg levels been associated with mild fibrosis ^22-23 . However, these studies were mostly performed in Asia and do not take into account other patients characteristics. Our study, performed in a patients population including characteristics such as: HBeAg status, ethnicity, age, gender, activity grades, ALT, HBV genotype, IFNL3 polymorphism, HBV variants, demonstrates an association between fibrosis stages with HBeAg (+) and low HBsAg titer only in univariate analsysis, reducing the direct implication of HBeAg status and HBsAg titers in the development of liver lesions. The prediction model shows that HBeAg (+) as well as HBsAg were no longer associated with moderate to advanced fibrosis, indicating that these two markers were dependent of other clinical and virological characteristics, such as age, ALT, HBV-DNA and HBV variants. Indeed, HBeAg and/or HBsAg levels have been reported to be associated with HBV-DNA levels disease activity ¹⁶ ' ¹⁹ ' ^38-40 age ¹⁶ ' ¹⁹ and precore mutations ⁶ .

Hepatitis B genotypes A and D are found in Western countries while genotypes B and C found in Asian countries. All have been reported to play a role in determining the clinical outcome of the liver disease ⁴¹ . Asian cross sectional studies found that patients with genotypes C have more severe disease outcome than genotypes B ¹³ ' ⁴² . A Western European study reports that genotype D is associated with more progressive liver disease than does genotype A ¹⁷ . However, these studies compared either genotypes B and C or A and D, none investigated the entire spectrum of HBV genotypes contrarily to our study including genotypes A to E. HBV variants have different distribution ^8"11 ' ⁴³ according to different genotypes. Similarly to HBV genotype the implication of HBV variants in the outcome of hepatitis B has been reported by either Asian studies involving genotypes B and C ⁷ ' ⁴⁴ or Western study involving genotypes A or D ⁸ . The high relative risk found in our model, independently from HBV genotypes, for BCP 1762 ^T /1764 ^A (>5-fold increase) and 1762 ^T /1764 ^A /1896 ^A (>4-fold increase) variants indicates that HBV variants are important in better identifying patients with moderate to advanced fibrosis than HBV genotypes (associated with fibrosis only in univaraite analysis), reinforce the clinical importance of HBV variants that have been reported to increase the risk for hepatocellular carcinoma ⁵ ' ^44-45 .

In conclusion, the main strength of the current study is the use of a large well characterized patients population (n=377) contributing to robust data giving an overview of the major epidemiological, clinical, host and complete virological factors occurring during the natural history of CHB and leading to the ability to propose non-invasive model identifying patients with moderate to severe fibrosis (F2-F4), without liver biopsy, suitable candidate for therapy. The ability to correctly identify F2-F4 versus FO-Fl obtained with this multivariate model (age + HBV-DNA + ALT + HBV variants) was evidenced by a c-statistic of 0.77 (95% CI, 0.72-0.82). The observation in our study that older age, HBV-DNA, increased ALT, and 1762 ^T /1764 ^A and 1762 ^T /1764 ^A /1896 ^A variants are associated with moderate to advanced fibrosis (F2-F4), suggests that they may be useful markers in facilitating patients monitoring.

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