Hepatitis B Biomarkers: Clinical Significance of the Old and the New

Hepatitis B Biomarkers: Clinical Significance of the Oldand the New

Andrés Duarte-Rojo & Jordan J. Feld

Published online: 21 August 2010# Springer Science+Business Media, LLC 2010

Abstract Chronic hepatitis B is a dynamic disease with avery variable outcome ranging from mild, asymptomatic,nonprogressive disease to end-stage liver disease and/orhepatocellular carcinoma. Clinically significant endpointstake years or decades to develop and thus variousbiomarkers—ranging from clinical data to sophisticatedvirologic diagnostics—are used as surrogates for diseaseprogression. Liver biopsy remains a robust indicator ofdisease severity; however, noninvasive markers may offer auseful alternative with some advantages. Clinical decisionsare often made using alanine transaminase; however, itlacks adequate specificity to be used in isolation. Serologicmarkers (hepatitis B early antigen and hepatitis B surfaceantigen) provide information about the degree of immunecontrol of viral replication, and thus remain importanttherapeutic indices. Sensitive measurement of serum hepa-titis B virus DNA level is an indispensable biomarker. Itssuppression is important, but only because it represents abridge to more permanent stages of disease resolution.Newer assays, including hepatitis B surface antigen titersand hepatitis B genotyping, have therapeutic implicationsand are becoming more widely available. Clinicians caringfor patients with chronic hepatitis B must be aware of theutility and limitations of available surrogate biomarkers.

Keywords Chronic hepatitis B . Biomarkers . Cirrhosis .

Hepatocellular carcinoma . HBeAg . HBsAg . Viral load .

Genotype . Transient elastography

Introduction

Hepatitis B virus (HBV) infection has a wide array ofpresentations with an unpredictable prognosis. Rarelypresenting as acute or fulminant hepatitis, either leading todeath or resolving without sequelae, it more commonlycauses a largely asymptomatic infection persisting aschronic hepatitis B (CHB). Those who develop CHB mayexperience an uncomplicated course with inactive disease,or progress to cirrhosis, end-stage liver disease (ESLD), andhepatocellular carcinoma (HCC). The outcome is influ-enced by multiple host and virologic factors.

However, 25% to 40% of patients with CHB will progressto an adverse outcome, and do so after several years todecades of infection. The long and variable course of thedisease makes it difficult to determine how an individualpatient will fare. The natural history of CHB is divided intophases of immune tolerance, immune activation/clearance,immune control, and hepatitis B early antigen (HBeAg)–negative CHB. Each stage defines the behavior and generalprognosis of the disease (Fig. 1). In many cases, however, aclear-cut differentiation between stages cannot be made, andsome flip-flopping between them may occur. Furthermore,even for individuals in the same stage, the long-termprognosis may well be different. Defining precisely wherean individual is and where they are headed within thespectrum of HBV progression is difficult. A variety ofbiomarkers have been identified to stratify patients into riskcategories and help determine who should be treated, withwhat, and for how long. Ranging from clinical data toadvanced virologic diagnostics, these biomarkers are clearlyhelpful; however, it is critical to understand their limitations,most notably that they are all only imperfect surrogatemarkers to help predict the important long-term clinicalconsequences of a very complex disease.

A. Duarte-Rojo : J. J. Feld (*)Toronto Western Hospital Liver Centre, University Health Network,University of Toronto,399 Bathurst Street, 6B Fell Pavilion, Room 158,Toronto, Ontario, M5T 2S8, Canadae-mail: [email protected]

Curr Hepatitis Rep (2010) 9:187–196DOI 10.1007/s11901-010-0053-3

Clinical Data

Symptoms in CHB are often mild (if present), extremelynonspecific, and therefore unreliable markers of the disease.Male gender, older age, family history of liver cancer, andperhaps cigarette smoking can help identify patients atincreased risk for HCC [1]. Commonly used clinical scores(eg, Child-Turcotte-Pugh or the Model for End-Stage LiverDisease [MELD]) are useful in the right clinical setting, butare insensitive for the great majority of patients who havenot progressed to advanced cirrhosis.

Liver Biopsy

Historically, liver biopsy has been considered the mostrobust evaluation of disease status. Fibrosis semiquantifica-tion provides useful prognostic information, but liver biopsyhas major drawbacks. The invasive nature of the procedureand the risk of complications make repeated assessmentimpractical, which is not ideal in such a dynamic disease.

Furthermore, important sampling variation occurs in up to25% of biopsies and imperfect interobserver agreement (k!0.70), particularly if biopsies are smaller than 20 to 25 mm!1 mm, and contain fewer than 11 complete portal tracts [2].

Even with adequate liver histology, interpretation of theresults is not entirely straightforward. Once cirrhosis ispresent, the risks for HCC and the complications of ESLDare greatly increased. However, it is less clear whatsignificance progression between the pre-cirrhotic stagesof fibrosis carries. Intuitively, the more distant a patient isfrom the cirrhotic stage, the better the outcome; however,only minimal data show this to be true. Although reportednumerically, the histologic scoring systems are all, at best,semi-quantitative and the distance (and thus the risk)between two adjacent stages is unknown, but certainly notlinear. Once a patient develops cirrhosis, histology becomesless useful. Attempts have been made to grade the severityof cirrhosis; however, quantification of fibrosis correlatespoorly with clinical outcomes.

An added consideration is the role of regression offibrosis. Clear evidence now exists that with prolonged

Fig. 1 Phases comprising the natural history of chronic hepatitis B(CHB) according to biomarkers. The immune active phases, whetherhepatitis B early antigen (HBeAg)+ or HBeAg-, are associated with anincreased risk of disease progression and adverse outcomes (topright). As a patient moves down toward the inactive phase, withincreased immune control, the risk for an adverse outcome progres-sively decreases. However, increasing age and previously accumulated

hepatic damage are important risk factors, particularly of hepatocel-lular carcinoma. As a result, all patients require continued follow-up.Reversion to CHB after hepatitis B surface antigen (HBsAg)seroconversion is extremely rare in the absence of immunosuppres-sion. ALT—alanine transaminase; HBV—hepatitis B virus; ULN—upper limit of normal

188 Curr Hepatitis Rep (2010) 9:187–196

inactive disease, established fibrosis may regress [3]. Is apatient who progressed from F1 to F2 at higher or lowerrisk of complications than a patient who regressed from F4to F2? To date, no study has addressed the clinicalsignificance of the architectural distortion that persists afterregression of fibrosis and particularly whether this may be arisk factor for HCC [3]. Liver biopsy still clearly has a rolein the management of CHB; however, the development ofnew noninvasive markers of fibrosis may offer someadvantages.

Noninvasive Markers of Hepatic Fibrosis

Blood Markers

Fibrogenesis is a dynamic process. Therefore, it is useful toget serial measurements of fibrosis, a challenging task withliver biopsy. To overcome the limitations of biopsy, severalserum markers of fibrosis have been validated (Table 1). Ingeneral, these blood-based tests measure a combination ofindices: collagen deposition (hyaluronic acid), architecturalchange (!-glutamyl transpeptidase, aspartate transaminase),liver synthetic function (bilirubin), and portal hypertension(platelet count). Combinations of these markers correlatefairly well with histologic evaluation of fibrosis. Becausethe gold standard—liver biopsy—is itself imperfect, it is

possible that the performance of these tests has beenunderestimated. The fact that the correlation improves inparallel with the size of the liver biopsy suggests this maybe the case [4]. Serum markers of fibrosis may reflect amore global assessment of hepatic fibrosis than a smallliver biopsy sample. It is important to recognize that someof the serum-based tests can be influenced by conditionsother than fibrosis (eg, hemolysis, Gilbert’s syndrome, andeven active inflammation). The most popular panel is theFibrotest (BioPredictive, Paris, France), which includes fiveblood tests along with patient age and gender to predictdegree of fibrosis. Importantly, beyond fibrosis, Fibrotestscores have been shown to predict CHB-derived complica-tions and survival [5, 6]. Other noninvasive markers thathave been less well validated in CHB include FIB-4(patient age, aspartate aminotransferase, alanine amino-transferase, and platelets) and APRI (aspartate aminotrans-ferase to platelet ratio index), both of which predict liverfibrosis based on routine blood work with fairly goodaccuracy [7]. The real benefits of these tests in CHB will bethe use of longitudinal measurements and correlation withclinical outcomes in patients with cirrhosis.

Transient Elastography

Measurement of liver stiffness by transient elastography(TE) is an alternative noninvasive approach for assessing

Table 1 Noninvasive markers of fibrosis

Study and predictor Estimation N AUC (95% CI) CUV Sensitivity Specificity

Halfon et al. [5]; Fibrotest F2-4 vs F0-1 1580 0.800 NR NR NR(0.770–0.840)

Kim et al. [7]; FIB-4a F2-4 vs F0-1 668 0.865 NR NR NR

F4 vs F0-3 (0.835–0.895) 3.6 30% 98%

0.926(0.907–0.945)

Kim et al. [7]; ASPRIb F4 vs F0-3 668 0.937 NR NR NR(0.919–0.954)

Kim et al. [7]; SPRIc F4 vs F0-3 668 0.882 NR NR NR(0.856–0.909)

Kim et al. [7]; APRId F4 vs F0-3 668 0.731 NR NR NR(0.691–0.770)

Wong et al. [15]; Fornse F3-4 vs F0-2 156 0.700 5.2 99% 26%(0.620–0.780)

a FIB-4: (age [years]!AST [U/L])/(platelet [109 /L] ! ALT [U/L]1/2 )b ASPRI=sum age in years to SPRI as follows: < 30=0; 30–69=consider 1!decade; " 70=5c SPRI=(spleen size [cm]/platelet count [109 /L])!100d APRI=([AST/ULN]/platelet [109 /L])!100e Forns=7.811 - 1.131!ln(platelet count)+0.781 ! ln(GGT)+3.467!ln(age) - 0.014!(cholesterol)

ALT alanine transaminase, APRI AST to platelet ratio, ASPRI age, spleen size, platelet ratio, AST aspartate transaminase, AUC area under thereceiver operating characteristic curve, CI confidence interval, CUV cut-off value, F fibrosis stage (range: 0–4), FIB-4 fibrosis 4, GGT !-glutamyltranspeptidase, NR not reported, SPRI spleen size, platelet ratio, ULN upper limit of normal

Curr Hepatitis Rep (2010) 9:187–196 189

fibrosis. TE avoids many of the pitfalls of liver biopsy, andthe technique evaluates 100 times the amount of liver tissueseen in a liver biopsy sample [8]. It is noninvasive and canbe performed quite rapidly, making it useful in the busyoutpatient setting. Various stiffness thresholds have beenused to define different levels of fibrosis, ranging from lessthan 7.7 kPa to exclude fibrosis (F#2) to 15.1 kPa orgreater to identify cirrhosis. These data include patientswith all etiologies of liver disease, although for patientswith CHB the cut-off points were on average somewhathigher [9•]. An important consideration is that alaninetransaminase (ALT) flares in CHB may increase TEmeasurements, with a subsequent reduction upon ALTnormalization [10, 11]. When ALT is above the upper limitof normal (ULN), there is an increase of 1.5 to 5 kPa foreach stage of fibrosis, compared to TE readings in thepresence of normal ALT levels. This increase is larger inadvanced stages of fibrosis, and as a result different cut-offvalues were proposed for patients with normal or increasedlevels of ALT. Thus, if ALT is normal, a TE reading greaterthan 10.1 to 12 kPa can establish cirrhosis with a specificityof 88% to 95%, whereas in the context of ALT elevation,specificity for cirrhosis is not good until the TE is greaterthan 13.4 to 15.5 kPa [12•, 13]. In contrast, to excludefibrosis, values below 5 to 6 KPa have greater than 90%sensitivity [12•, 13]. Unfortunately, many patients fallwithin a “gray zone” of TE readings, with values suggest-ing some, but not advanced, fibrosis. Whether a liverbiopsy or other noninvasive markers would clarify thesituation remains unclear. In chronic hepatitis C infection,TE was shown to predict the development of ascites andesophageal varices [14]. If this proves true in CHB as well,TE may have significant advantages over liver biopsy inmany clinical scenarios.

A Rational Approach for Noninvasive Markers

In the few studies that addressed the usefulness of bothserum markers and TE, the latter proved to be moreaccurate [14, 15]. However, whenever noninvasive testsare used, any single method or single cut-off point maynot suffice. Combining two methods may be a usefulapproach. If serum tests and TE agree, a liver biopsy isusually concordant, whereas disagreement between thetwo approaches will necessitate a liver biopsy forclarification. The cut-off values must also be rationallyused according to the clinical scenario. For example,confirmation of cirrhosis is relevant for deciding to starttreatment or hepatoma surveillance, and thus cut-offvalues with high specificity are more useful in thisscenario. On the contrary, in a patient with presumablyinactive CHB, the main concern would be the exclusion ofsignificant fibrosis (" F2), to define the degree of follow-

up needed. In this situation, cut-off values maximizingsensitivity would be preferable.

Serum Aminotransferase Levels

ALT levels increase in blood whenever damage occurs tothe hepatocyte, and thus are often used as a measure ofinflammatory activity and ongoing hepatic damage. Animportant issue in evaluation of CHB is what constitutes anormal ALT. Although 40 U/L in males and 31 U/L infemales were traditionally considered the ULN (based onthe 95th percentile of a healthy population), at least twostudies found that these values may be too high. Prati et al.[16] identified 30 and 19 U/L as the 95th percentile inmales and females, and showed that these thresholdsimproved the ability to identify patients with active diseasein hepatitis C infection. A Korean study of insurance datafrom more than 90,000 men found that ALT values above30 U/L were associated with increased liver-related mor-tality, and the increased risk was observed even with ALTvalues between 20 and 29 U/L [17]. In the United States,data from the National Health and Nutrition ExaminationSurvey (NHANES) III survey confirmed increased liver-related mortality of the two sets of thresholds (40 and 31 U/L, 30 and 19 U/L) with a stronger risk when the lower onewas used [18•]. Based on this evidence, American Associ-ation for the Study of Liver Diseases (AASLD) practiceguidelines for CHB suggest using ALT cut-offs of 30 U/L formen and 19 U/L for women [19].

In CHB, several studies have identified high ALT levelsas being associated with increased risk of progression [1,20]; however, high levels also predict a better response totreatment [21•, 22••]. Although most clinicians take noteof high ALT values as indication for investigation and/ortherapy, it is less clear what to do in the face of normal ornear-normal values. Yuen et al. [23] showed that ALTvalues between 0.5 and 1 times ULN were associated withan increased risk of complications from cirrhosis, whencompared to patients with levels less than 0.5 ULN.Another study found that 33% of patients with CHBwhose ALT levels were less than the ULN had significantfibrosis [24].

Although these data are useful, there are two importantcaveats. First, all studies evaluated ALT at baseline withoutlongitudinal assessment. Perhaps more importantly, in bothhealthy and CHB populations, the absolute risk in thosewith ALT values between the low and the traditionalthresholds [16, 17, 18•, 23, 24] albeit relatively increased,was still very low. This emphasizes the point that usinglower thresholds increases sensitivity but reduces positivepredictive value in both diseased and healthy populations.Taken together, these data suggest that the lower ALT


thresholds may have reasonable sensitivity for excluding asignificant risk of progression, at least in the short term;however, in patients with indeterminate ALT values, otherHBV biomarkers are needed and longitudinal follow-up isrequired.

Hepatitis B Early Antigen

HBeAg is a soluble protein produced by HBV that is notrequired in the viral lifecycle. It is thought to play a role intolerizing the immune system to the virus, particularly inthe setting of neonatal infection, and its presence generallyindicates a high level of ongoing viral replication. Earlynatural history studies documented that after spontaneousHBeAg loss, the prognosis for patients was greatlyimproved, with lower rates of progression to cirrhosis anddecreased incidence of liver cancer [1, 20, 25–27]. As aconsequence, HBeAg loss with or without seroconversion(development of anti-HBe) was, and remains, an importanttherapeutic endpoint.

Unfortunately, loss of HBeAg does not necessarilyportend a better prognosis. Some patients revert to HBeAgpositivity, whereas others develop HBeAg-negative CHB[28, 29] because of mutations in the open-reading frame(precore) or promoter (basic core promoter) of the HBeAgprotein [30]. Reactivation of CHB occurs at an estimated rateof 2% to 3% per year [29], leading to HBeAg - CHB in asmany as 20% to 40% of patients [31••, 32••]. Fattovich et al.[31••] showed that over 25 years of follow-up, patients whoexperienced HBeAg- CHB or HBeAg reversion had a similarrisk for liver-related death as those who never lost HBeAg,when compared to patients with a sustained remission(persistently normal ALT and suppressed HBV DNA).

Recent data from Taiwan have shown that the age atHBeAg seroconversion may be the most relevant factorassociated with prognosis. Patients who lost HBeAg beforeage 30 had very low rates of cirrhosis and HCC in 15 yearsof follow-up, compared to those who did not clear HBeAguntil after age 40 (Fig. 2). By multivariable analysis, foreach decade delay in seroconversion, there was a 45%increase in the risk for HBeAg- CHB, and a 2.5-foldincrease in the risk of cirrhosis (Fig. 2) [32••].

It is unknown if treatment-induced HBeAg loss has thesame significance as spontaneous HBeAg clearance. How-ever, a recent meta-analysis with more than 2000 patientsshowed that interferon-associated HBeAg loss reduced therisk of HCC by 70% and cirrhosis by 54%, when comparedto untreated patients [33]. Whether the same benefit willresult from nucleos(t)ide-associated HBeAg loss remains tobe confirmed.

Although HBeAg is clearly important, like otherbiomarkers of HBV, it cannot be considered in isolation.

The presence of HBeAg is almost always associated withhigh levels of viral replication. It is therefore difficult todetermine if the negative outcomes associated with persis-tence of HBeAg are caused by the protein itself, theassociated high viral loads, or a combination thereof. TheHBV genotype is also very important. The secondarystructure of the pregenomic RNA (the template for HBVreplication) in genotype D HBV is stabilized by thepresence of the precore mutation. As a result, HBeAgseroconversion occurs much earlier in patients withgenotype D; however, the consequence is that patients withgenotype D are much more likely to have precore mutantHBV and thus develop HBeAg- CHB. The strong correla-tions between the various biomarkers necessitates that theynot be considered in isolation.

Hepatitis B Surface Antigen

The presence of hepatitis B surface antigen (HBsAg) in theblood indicates HBV infection. With HBsAg clearance, theimmune system has achieved control of viral replicationeven though HBV DNA likely remains in the liver ofinfected individuals as covalently closed circular DNA(cccDNA). Natural history studies have documented spon-taneous HBsAg loss at rates of 0.5% to 2% per year, withhigher rates occurring in areas of low endemicity [24, 26,31••, 34, 35••]. In general, spontaneous HBsAg loss orseroconversion carries an excellent prognosis, with somewell-designed prospective studies supporting this concept[24, 35••, 36]. Recently, Simonetti et al. [35••] reported 20-year follow-up data in an Alaskan cohort. They found thatafter spontaneous HBsAg clearance, the risk for HCCdecreased by more than fivefold. Similarly, Chen et al.

Fig. 2 Age at hepatitis B early antigen (HBeAg) seroconversion andthe rate of adverse outcomes after 15 years of follow-up. HBeAg-

chronic hepatitis B (white bars), cirrhosis (gray bars), and hepatocel-lular caricinoma (black bars). CHB—chronic hepatitis B


[37•] found that the risk for HCC or liver-related death was4.6-fold and 2.1-fold higher among inactive HBsAg carriersthan their HBsAg- counterparts. However, some patientsmay still be at risk for developing adverse outcomes. In aclinic-based study, Yuen et al. [38•] followed 298 patientswith HBsAg clearance for a median of 9 years, and couldidentify seven cases of HCC and five with complications ofESLD. Interestingly, although based on a small number ofpatients, they found no negative outcomes in patientswithout pre-existing cirrhosis who cleared HBsAg before50 years of age [38•]. It will be important to confirm thisage threshold in other studies to guide follow-up of patientsin clinical practice.

The impressive data on the prognostic importance ofHBsAg loss make it an attractive therapeutic target.Importantly, similar to spontaneous HBsAg loss, Moucariet al. [39] recently showed that treatment-associated HBsAgseroconversion eliminated the risk for HCC or mortality,and fibrosis regressed in most patients. Although relapsesof HBsAg positivity after HBsAg clearance have beenreported [21•, 22••], seroreversion is rare in the absence ofimmunosuppression.

Because of its important prognostic significance, interesthas increased in trying to identify patients who willultimately lose HBsAg. Recently, serum titer of HBsAgwas shown to be predictive of future HBsAg loss. InHBeAg+ CHB, continuous decline in HBsAg titer duringpeginterferon therapy is predictive of eventual HBsAgclearance [39]. Similarly, in HBeAg- disease, low pretreat-ment values or on-treatment decline of HBsAg can predictsustained HBsAg loss in peginterferon-treated patients. AnHBsAg level less than 10 IU/mL after 48 weeks oftreatment with peginterferon, or an on-treatment decline ofgreater than 1 log10IU/mL, predicts HBsAg clearancewithin 3 years of completing treatment [40•]. HBsAg titeralso seems to correlate with levels of cccDNA in liverbiopsy specimens, suggesting that it may be a marker of theamount of replication-competent HBV in the liver. Ongoingstudies may further clarify the utility of HBsAg titers formonitoring and defining the duration of treatment, whichwill ultimately improve the utility of what is likely the mostimportant prognostic biomarker in CHB.

HBV DNA

The progressive improvement in the direct measurement ofserum HBV DNA levels over the past few decades has madeHBV DNA an increasingly important biomarker for CHB.The first-generation assays were based on signal amplifica-tion using hybridization or branched-DNA technologies,with sensitivities down to 4 log10IU/mL. Newer targetamplification techniques based on polymerase chain reaction

(PCR) go well below this cut-off, with reproducible detectiondown to 1.1 log10IU/mL with real time-PCR/TaqMan(Roche Molecular Diagnostics, Indianapolis, IN) techniques[41, 42]. The improvement in sensitivity makes it difficult tocompare current data with that from older studies, particu-larly in terms of defining rates of HBV DNA suppression toundetectable levels. However, it is also important to considerthat the true significance of the low levels of HBV DNA thatcan now be quantified is somewhat unclear.

A seminal study demonstrating the importance of HBVDNA levels was the REVEAL-HBV (Risk Evaluation ofViral Load Elevation and Associated Liver Disease/Cancerin HBV) study from Taiwan. The investigators were able todemonstrate a biologic gradient of increasing HCC riskwith increasing baseline serum HBV DNA levels (Table 2).Notably, even among HBeAg- patients with normal ALTand no cirrhosis, HCC risk still correlated with HBV DNAtiter. Importantly, patients were not treated in this study.Therefore, it is not clear whether suppressing HBV DNAlevels in those with high baseline values would havederived the same benefit as starting at a lower HBV titer.However, it is reassuring that in the subgroup of patientswith a baseline determination of 3.3 log10IU/mL or greater,a decreasing HBV DNA level at follow-up was associatedwith a decreased risk of HCC [1]. Whether the HBV DNAlevel itself is causative, however, remains unclear because itmay be the immune mechanisms controlling viral replica-tion that also reduce the risk of HCC. In another publicationfrom the same cohort, the authors reported that increasingbaseline HBV DNA level was also associated with anincreased risk for the development of cirrhosis (Table 2).One important caveat was that this study used ultrasonog-raphy, a relatively insensitive method, to diagnose cirrhosis.[20]. Once cirrhosis is established, it seems that the level ofHBV DNA is less relevant to the development ofcomplications [43, 44].

HBV DNA level was also shown to be a marker andpredictor of active liver disease in patients with HBeAg-

CHB. In a prospective study of Asian and Caucasianpatients (N=74) HBV DNA levels predicted future ALTelevations (> 40 U/L), whereas other factors such asgender, age, ethnicity, genotype, or mutations did not. Athreshold value of 3.3 log10IU/mL was best for predictingfuture ALT elevations [45]. This study provided supportfor prior retrospective findings suggesting that the HBVDNA threshold for predicting active disease in HBeAg-

CHB should be decreased from 4.4 to 3.3 log10IU/mL[46]. Importantly, however, this study and many othersshowed that HBV DNA measurement—independent of itsinaccuracies—is a constantly changing variable needingserial intermittent assessment, and there is no absolutethreshold that can faithfully define disease activity in allpatients at all times.


The recognition of the relevance of HBV DNA titer inpredicting CHB prognosis has led to the widely heldcontention that suppressing HBV DNA to undetectablelevels is a relevant end-point of therapy. This concept ofvirologic response has been adopted by the AASLD,European Association for the Study of the Liver (EASL),and Asian Pacific Association for the Study of the Liver(APASL) in their guidelines [19, 47, 48]. Althoughvirologic response is now often considered the most criticalassessment of treatment, it is important to recognize thatthere are no robust outcome studies showing the utility of avirologic response on its own. Virologic response is abridge to more permanent stages of disease control: HBeAgand HBsAg loss/seroconversion. HBV DNA levels are alsomeasured to monitor ongoing efficacy of therapy and toscreen for viral resistance. Low on-treatment levels of HBVDNA predict HBeAg seroconversion [22••, 39], whereashigh HBV DNA levels at baseline or at the time of HBeAgseroconversion predict a higher rate of seroreversion aftertherapy is stopped [49, 50].

In HBeAg- disease, HBsAg loss/seroconversion is a rareevent, leaving HBV DNA suppression as the main measureof treatment efficacy. The high relapse rate with treatmentdiscontinuation has led to the consideration of treating untilHBsAg loss, a prospect that may take many years, if notdecades. Whether sustained DNA suppression in the absenceof HBsAg loss will lead to clear clinical benefits remains tobe seen. Clearly, HBV DNA is a critical determinant forCHB management, and like the other biomarkers in thisdisease, it must be considered in the correct context withrelation to the other parameters of CHB activity.

Genotypes and Mutations

Eight HBV genotypes have been identified (A–H), eachone differing from the others by at least 8% of the entiregenome. Studies assessing the relevance of hepatitis Bgenotypes have been difficult to perform because of

genotypic geographic distributions. Reported patterns in-clude genotype A in Northern Europe, North America,Africa, and India; D in Eastern Europe, South Asia,Mediterranean, and Middle Eastern countries; B and C inEast Asia and Pacific Islands; E in central Africa; F inindigenous Amerindians; G and H are rarer and without acharacteristic distribution. Most studies have comparedonly genotypes with overlapping distributions, particularlygenotype B versus C and A versus D. When compared togenotype B, genotype C has been associated with laterHBeAg seroconversion, a higher rate of seroreversion, andincreased risk of HCC [24, 51, 52•]. In the Alaskanpopulation, where all but genotype E have been described,genotype C was associated with late HBeAg seroconversion,the F variant was most notably associated with HCC, and alower risk was found in D compared to A [53]. Genotypesmay also predict response to treatment with interferon,particularly in HBeAg+ disease. Buster et al. [54•] publisheda multinational experience of peginterferon in 721 patientswith HBeAg+ disease, showing a decreased probability ofachieving a sustained response from genotypes A through D(37%, 25%, 20%, and 8%, respectively). In addition toHBeAg clearance, a long-term follow-up study showed thatthe loss of HBsAg was also higher in patients with genotypeA, followed by genotype B [54•], reinforcing the betterprognosis of these variants. Although less data exist aboutHBeAg- patients, there seems to be a higher HBsAgclearance in patients with genotype A than in non-Agenotypes [21•]. Currently, the AASLD and EASL guide-lines recommend that in patients with genotype A, peginter-feron should be entertained as first-line therapy [19, 47]. Inaddition to the differences between viral genotypes, it isimportant to consider that apparent differences in outcomemay also relate to host genetics that differ by ethnicity, giventhe marked regional variation. As genome-wide associationstudies become more widely available, the viral and hostcomponents hopefully will be clarified.

Mutations in the HBV genome have been evaluated inrelation to the risk of HCC. A recent meta-analysis including

Table 2 Risks for hepatocellular carcinoma and cirrhosis in relation to HBV-DNA level according to the REVEAL-HBV study

HBV-DNA level (log10IU/mL) Risk for HCC Risk for cirrhosis

N OR (95% CI) N OR (95% CI)

<1.78 874 Reference group 869 Reference group

" 1.78 to<3.3 1,161 1.1 (0.2–2.3) 1,150 1.4 (0.9–2.2)

" 3.3 to<4.3 643 2.3 (1.1–4.9) 628 2.5 (1.6–3.8)

" 4.3 to<5.3 349 6.6 (3.3–13.1) 333 5.6 (3.7–8.5)

" 5.3 627 6.1 (2.9–12.7) 602 6.5 (4.1–10.2)

95% CI 95% confidence interval, HBV hepatitis B virus, HCC hepatocellular carcinoma, OR odds ratio, REVEAL-HBV study Risk Evaluation ofViral Load Elevation and Associated Liver Disease/Cancer in HBV


11,582 patients, mostly bearing genotypes B and C, showedthat PreS mutations and C1653T, T1753V, A1762T/G1764Asubstitutions (Enhancer II/BCP/Precore regions) were associ-ated with an increased risk of HCC, after controlling forconfounders. Notably, the mutations were more common inpatients with more advanced disease, ranging from inactiveCHB to cirrhosis. It is hoped that future work will clarify therole of HBV mutations in hepatic carcinogenesis [55•]. Thelimited availability of access to assess these mutations makesit difficult to consider HBV genome mutation analysis auseful biomarker for CHB currently, but it is foreseeable thatin the future it may prove clinically useful.

Conclusions

CHB is a dynamic disease that evolves over decades withextremely variable clinical outcomes. Identifying whichpatients will develop progressive disease and which patientswill remain inactive remains a major clinical challenge. Noneof the currently available biomarkers, in isolation, is adequatefor risk stratification. Furthermore, the significance of eachmarker greatly depends on the clinical scenario. A high HBVDNA level in an immunotolerant patient is likely of noconcern, whereas a much lower HBVDNA titer in an HBeAg-

patient may be an indication for therapy. Refining the utilityof the standard biomarkers (ALT, HBeAg, HBsAg) invarious clinical contexts, and clarifying the importance ofnewer biomarkers (HBsAg titer, HBV DNA, noninvasivemarkers of fibrosis), continues to be a priority and hopefullywill allow us eventually to prognosticate for our patientswith a much greater degree of certainty, ultimately leading toimproved therapeutic strategies.

Acknowledgment Andrés Duarte-Rojo receives funding for aClinical Fellowship from the Instituto de Ciencia y Tecnología delDistrito Federal, ICyTDF (México), and The American College ofGastroenterology, ACG: International Training Grant 2009 (USA).

Disclosure Dr. Feld has served on national advisory boards forHoffman-Laroche and Merck (Schering), received speaking honorariafromHoffman-Laroche, and received a travel grant fromGilead Sciences.No other potential conflict of interest relevant to this article was reported.

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Hepatitis B Biomarkers: Clinical Significance of the Old and the New

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