Surrogate Markers of Efficacy For Medical Treatment of Viral Hepatitis

Waiting for signs of disease progression is impractical when sizing up the effectiveness of therapy for hepatitis B and C. Timothy M. Block, PhD, and W. Thomas London, MD, discuss alternate means of evaluation.

Abstract

As a chronic illness, hepatitis presents a challenge for clinical research, regulatory bodies, and clinicians treating patients — how can one adequately assess the effectiveness of antiviral therapy after years or decades of undiagnosed infection? Because the outcome of infection with hepatitis B or C (HBV, HCV) may follow years or decades of infection, the clinical benefit of a therapeutic may not be recognized during the short time frames of most human clinical trials. This is in clear contrast to many other therapeutics, where the outcome and efficacy can be evaluated in few weeks or months. In HBV and HCV, it is impractical to wait until the disease has abated to determine a drug’s benefit, so surrogate markers that can predict disease outcome are essential to developing effective therapies. This review will highlight the ability of surrogate markers to detect early disease and to evaluate the effectiveness of HBV and HCV therapies.

INTRODUCTION

In chronic hepatitis B virus (HBV) or hepatitis C virus (HCV), the therapeutic goal is to reduce the incidence of severe or fatal liver diseases, such as advanced hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) (Block 2003, Seef 2002). Because these diseases may not manifest until decades after infection, we must rely partly on surrogate indicators to determine the effectiveness of a therapeutic regimen for chronic HBV or HCV infections.

Infections from HBV and HCV in humans characteristically result in either a clinically apparent, acute, self-limiting disease or a long-term infection that is unresolved and persistent (Block 2003). Acute infections in adults are usually mild, and life-threatening hepatitis develops in fewer than 1 percent of cases (Lok 2001b). Long-term persistent infections present the greatest challenge relative to the evaluation of drug efficacy.

The long period of hepatitis progression is illustrated in Figure 1, which depicts an idealized time line, with years of infection displayed in the X axis and general clinical categories shown as boxes of varying lengths. Varying values of the markers are represented on the Y axis. Serological markers are described only for HBV. Other markers could apply to HBV or HCV. Viremia (viral DNA for HBV, RNA for HCV) and alanine aminotransferase (ALT) levels are thought to correspond with disease status, but are not absolute and, in the case of HBV, DNA levels often decline in HCC. This variability is emphasized by the varying line height. All values in Figure 1 are idealized and intended for illustration purposes only, and are not based on any study in particular.

FIGURE 1.

Clinical stages of chronic viral hepatitis and common surrogate markers of disease

AH=Acute hepatitis, ALT=alanine aminotransferase, HBeAg=hepatitis B e antigen, HCC=hepatocellular carcinoma.

Cirrhosis and primary liver cancer may result from many years of unresolved, persistent infection. Every year, between 1 and 6 percent of individuals who are chronically infected with HBV or HCV and have developed cirrhosis also develop HCC (El-Serag 1999, 2001; Di Bisceglie 1988; Lok 2001a). It is generally accepted that the clinical disease course depends partly on viremia. It’s uncertain, however, the extent to which the quality, quantity, and duration of viremia influence disease outcome.

“Quality” includes virus genetics and viral DNA versus protein in serum; “quantity” relates to the titer of the virus. The outcome may vary depending on the individual infected and the viral strain (Block 2003, Tang 2004). Nevertheless, virological and biochemical markers generally correspond to clinical disease as shown in Figure 1. They are so variable, though, that we cannot rely on any one marker for clinical judgment. There are few rules governing the behavior of a virological or biochemical marker in liver disease and viral hepatitis. Levels of hepatitis B viral genome may decline over the course of disease (less information is available for HCV), and HCC may occur in individuals with low or high viremia. ALT levels often rise as hepatitis and cirrhosis become more severe, but these levels are poor predictors of the occurrence of HCC.

Chronic HBV and HCV

Although there are similarities between the natural histories of chronic hepatitis due to HBV and HCV, they are two highly distinct viruses. HBV is the prototype member of the hepadnaviridae virus family. It has a small, partially double-stranded DNA genome (∼3.5 kb) (Seeger 2000). HCV is a hepacivirus; it contains a single-stranded RNA genome (∼9 kb) that is not reverse-transcribed into DNA (Bartenschlager 2000).

As shown in Table 1 (page 44) and reviewed in Chen (2002) and Conjeevaram (2003), there currently are two pharmaceuticals approved by the U.S. Food and Drug Administration for treating HCV in the United States — interferon alone or in combination with ribavirin. Interferon may be used in an unconjugated or pegylated form. For treating HBV, more options are available: interferon, unconjugated or pegylated, and two different viral DNA polymerase inhibitors (lamivudine and adefovir dipivoxil) (Conjeevaram 2003, Lok 2001b).

TABLE 1.

FDA-approved medications for chronic viral hepatitis and generally accepted virological end points

Chronic hepatitis B virus	Chronic hepatitis C virus
Interferons Lamivudine Adefovir	Interferons, pegylated or unconjugated Interferons, pegylated or unconjugated, with ribavirin

SOURCES: CHEN 2002, CONJEEVARAM 2003

Can disease biomarkers be end points of efficacy?

The goals of therapy are reductions in mortality and morbidity, but the success of antivirals is increasingly evaluated on the basis of short-term virological and histological milestones, despite remaining uncertainties regarding the use of biomarkers to track disease status. The current surrogate goals of therapy are summarized in Table 2.

TABLE 2.

Goals of current antiviral therapy for hepatitis B and C

Hepatitis C virus	Hepatitis B virus
Loss of detectable viral RNA in serum 6 months after Rx	Sustained reductions in viral DNA in serum HBeAg sero-conversion, if HBeAg positive at entry

HBeAg=Hepatitis B e antigen.

SOURCES: DAVIS 2002, CONJEEVARAM 2003

Viremia levels and histological scoring, which are based on baseline and end-of-treatment liver biopsies, are the accepted standards. Clinicians are generally most comfortable relying on histological changes in the liver. Tests for liver damage, such as tests for ALT and AST (aspartate aminotransferase), are dependent variables of disease, but these tests do not predict clinical outcomes effectively.

Sustained virological responses (SVR), however, have been linked to improvements in inflammation and fibrosis. This fact that supports the validity of virological markers as end points (Davis 2002, Marcellin 2002, McHutchinson 2001, Poynard 2000, Santantonio 2000, Trimoulet 2004).

Windows on disease and predictors of disease outcome

Liver biopsy has the appeal of being a direct report on the disease, and an improvement indicated by a biopsy certainly would indicate a favorable outcome. Yet a biopsy is relatively expensive, can sometimes be painful, and carries small but certain risks of bleeding and (rarely) death. It is always an inconvenience to the patient, often requiring leave from work for 1 or more days. Multiple biopsies from an individual usually are impractical. Moreover, a biopsy represents only a small sample, not the entire liver.

Despite the relationship between a sustained virological response and histological improvement, there has been a reluctance to rely on virological markers as sole end points of efficacy, either for the clinical evaluation of a drug or for making treatment decisions.

Treatment with antivirals is not necessarily lifelong, and interferon therapy rarely lasts longer than a year, so a key question is whether a reduction in viremia due to an antiviral agent will last beyond the end of treatment. Curiously, an extremely early virological response (after weeks of therapy) to interferons correlates with a sustained benefit beyond treatment in individuals infected with HCV but not HBV (Santantonio 2000, Trimoulet 2004) — that is, a greater than 10-fold reduction in serum viral genome levels within the first weeks of therapy generally correlates to a sustained reduction of viral load in patients with chronic hepatitis C; this is not the case in patients with chronic hepatitis B.

Unfortunately, this correlation in HCV-infected patients was not sufficient for clinical decision-making. Also, for analyzing this correlation, multiple blood samples had to be taken from the same individual during the first week of treatment to obtain multiple data points, limiting the usefulness of this approach.

An extremely early virological response, measured after a few weeks of therapy should not be confused with an early virological response, measured after 12 weeks of therapy. There is general agreement that a reduction in viremia after 12 weeks of interferon therapy correlates with outcome in HCV patients (again, not in HBV patients), while a lack of reduction is a negative predictor of long-term response. On the other hand, a positive response does not ensure sustained benefit (Davis 2002).

Extremely early or early virological responses (after 2 or 12 weeks of therapy, respectively) may not predict sustained response in HBV patients. But reductions in viremia of at least 100-fold at the end of treatment have been associated with the achievement of other clinical end points, such as sustained virological response beyond treatment, and serological markers hepatitis B early antigen (HBeAg) loss, hepatitis B e antibody (HBeAb) gain (Mommeja-Marin 2003, Santantonio 2000).

Combined analysis of many studies suggested that there was no greater clinical benefit after 100-fold or larger reductions of viremia (Mommeja-Marin 2003). This is illustrated in Figure 4. The leveling of results could be due to the relatively small number of studies or of variations in study design and performance. Nevertheless, these analyses raise the possibility that there is a threshold for reduction of viral load necessary for antiviral benefit as well as a limit to what an antiviral alone can achieve.

The search continues for alternatives to histology and complements to the current serum markers. Platelet counts, combined with other markers, were shown to correlate with cirrhosis and fibrosis (Wai 2003). FibroTest and ActiTest are two examples of blood tests that have shown some promise; these tests measure five and six markers, respectively, and generate a score of fibrosis activity (Poynard 1997). A multimarker score would seem to have advantages over a single marker test. Yet these correlations have not been sufficient to gain general acceptance by hepatologists.

Detection of hepatocellular carcinoma

The most devastating outcome of viral hepatitis B and C is HCC (Block 2003, Seef 2002). Virological and serological markers, which are useful in determining whether an antiviral is effective in the short term, are not useful in determining the long-term risk of HCC. HCC can occur in individuals without cirrhosis and with low or undetectable levels of HBV DNA (Block 2003, Brechot 1987). Therefore, reduced levels of viral DNA and the absence of cirrhosis, determined at a single time point, cannot exclude the development of HCC (Evans 1998, Yao 1997).

Disease status usually is monitored by physical assessment, ultrasound imaging of the liver, and analysis of serum for a panel of markers (Lok 2001b) Many of the constituents of the serum panel (e.g., ALT levels) vary throughout the course of chronic hepatitis and are of limited use in early detection of HCC. Ultrasound detection requires a 2- to 3-cm tumor mass to be present and often occurs at a stage when the prognosis is already extremely poor (Brechot 1987, Hoofnagle 1997, Ikeda 2003, Sherman 1995). The positive predictive value of a small hypo- or hyperechoic mass being an HCC is only about 15 percent (Sherman 2001). Given that early surgical intervention is the best hope for patient survival, much more accurate detection of tumors smaller than 3 cm is needed (Berchot 1987, Di Bisceglie 1998, Ikeda 2003, Sherman 1995).

The level of alphafetoprotein (AFP) correlates significantly with a diagnosis of HCC (Aoyagi 1988, Buamah 1984, Lok 2001a). AFP as a sole indicator of HCC is of limited value; however, it is often present in the absence of serious disease (Aoyagi 1988). For example, in a prospective study of 1,069 chronic carriers of HBV in Canada, AFP elevations of greater than 20 ng/mL had a positive predictive value of only 9 percent (Sherman 2001). Nevertheless, even the limited association of AFP elevation with HCC underscores the potential of serum as a source of biomarkers for liver disease.

Less attention has been given to serum markers other than AFP for the early detection of HCC. Protooncogene mutations (p53, p16, fas, cMyc, others) and even routinely performed laboratory tests and serum polypeptides (osteopontins, desgamma (abnormal) carboxyprothrombin [DCP], autoantibodies, prothrombins, other polypeptides) have been correlated with HCC in some studies (Block 2003, Marrero 2003, Mita 1998). The degree of usefulness, if any, for detecting early HCC and for evaluating antiviral efficacy has not been demonstrated.

We have been searching for surrogate markers of disease using genetic and proteomic approaches. We performed a systematic analysis of the serum proteomes from individuals chronically infected with HBV or HCV compared with their disease status (Communale 2004, Steel 2001, Steel 2003). Our research showed that a resident Golgi polypeptide (GP73) can be detected in the circulation in amounts that correlate with the presence of HCC (Block in preparation). The detection of a polypeptide that increases in the presence of HCC is important and unusual.

Other proteomic work is preliminary, but a pattern is unfolding suggesting that various liver-derived polypeptides decline as disease progresses. These include complements, apolipoprotein isoforms, and haptoglobins (Steel, work in progress). Some of these are components of FibroTest, mentioned previously.

Finally, we have determined that human urine contains DNA that is derived, in part, from circulation (Su 2004). Therefore, we are attempting to develop assays for the detection of cancer-derived DNA in the urine, in the hope that a simple urine test might permit early detection of liver disease or cancer — and perhaps other cancers — from sites that are distal to the urinary tract (Wang 2004).

CONCLUSION

The goal of antiviral therapy for hepatitis B and C is to prevent cirrhosis and HCC and to maintain patient quality of life. Given the long interval between an initial infection and significant liver disease, it is impractical to use clinical states as end points to evaluate a new drug. Histology is the preferred end point, but it necessitates a biopsy procedure. In much the same way that reductions in serum cholesterol and blood pressure serve as surrogate end points in the evaluation of statins and antihypertension agents, reductions in viral load or changes in serum biomarkers could be used as end points in the evaluation of anti-hepatitis agents.

Different therapeutics may require different surrogate markers. For example, treatments that reduce inflammation or enhance immune recognition may have little effect on viremia. We hope that the identification of these surrogate markers will expedite the discovery of new drugs, reduce the cost of drug discovery, and reduce risks to patients in clinical trials.

FIGURE 2.

Correlation between reductions in hepatitis B virus viremia and improvements in histologically assessed disease in chronic hepatitis B patients undergoing lamivudine or adefovir therapy

SOURCE: MOMMEJA-MARIN 2003