Clinical Therapeutics/Volume 31, Number 2,2009

Case Report

Evolution of Hepatitis B Virus Polymerase Mutations in aPatient With HBeAg-Positive Chronic Hepatitis B VirusTreated With Sequential Monotherapy and Add-OnNucleoside/Nucleotide Analogues

Feifei Wang, M01,2; Honghai Wang, Ph02; Hongbo Shen, Ph02; Chengyan Meng, MOl;Xinhua Weng, MOl; and Wenhong Zhang, M01,2

1Department ofInfectious Disease, Huashan Hospital, Fudan University, Shanghai, People's Republic ofChina; and 2State Key Laboratory ofGenetic Engineering, Institute ofGenetics, School ofLife Science,Fudan University, Shanghai, People's Republic ofChina

ABSTRACTBackground: Nucleoside/nucleotide analogues are a

fundamental tool for the treatment of chronic hepatitisB virus (HBV). Sequential anti-HBV treatment mightlead to the selection of mutations.

Objective: This report aimed to analyze the geneticevolution of the reverse-transcriptase (RT) gene ofviral quasispecies in a patient with hepatitis B e anti­gen (HBeAg)-positive chronic HBV who received,sequentially, lamivudine (LAM), adefovir dipivoxil(AOV), and AOV + telbivudine (LOT) combinationtreatment over a total of 108 weeks.

Methods: A 20-year-old Chinese man presentedto Huashan Hospital, Fudan University, Shanghai,People's Republic of China, with hepatitis B surfaceantigen-positive and HBeAg-positive chronic HBVand was sequentially treated with LAM 100 mg/d for18 weeks,ADV 10 mg/d for 68 weeks,andADV 10 mg/d+ LOT 600 mg/d combination treatment for 22 weeks.Compliance was monitored every 4 weeks using a pillcount. For genotypic analysis, the RT region of thepolymerase gene from the serum of this patient wasamplified, cloned, and sequenced. Fifty clones withHBV insert were selected for sequencing at weeks 0(baseline), 18,22,60, 70, 86, and 108.

Results: The rtM204V1L LAM-resistance mutationwas detected in 4.4% (2/45) of clones prior to LAMtreatment. At week 18 during LAM treatment, thertM2041 mutation became predominant, being pres­ent in 79.5% (35/44) of clones. The rtM2041 mutationwas associated with compensatory mutations (rtL180Mand rtT184L). A total of 9.1 % (4/44) of the clones

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harbored the rtL180M + rtT184L + rtM2041 muta­tions. Two new mutations, rtL229V and rtV1911,were detected in 75.0% (33/44) and 11.4% (5/44) ofclones, respectively. At week 22 during AOV treat­ment, LAM-resistance mutations (rtL180M, rtT184L,rtM2041, rtV1911, and rtL229V) were not detected.At week 86 during AOV therapy, the rtN236T AOV­resistance mutation was detected in 58.8% (20/34) ofclones. A total of 20.6% (7/34) of the clones harboredthe rtK212T + rtM250L mutation, and rtA181 V wasfound in 2.9% (1/34) of the clones. At week 108, afterthe patient had been receiving AOV + LOT combina­tion therapy for 22 weeks, rtS202G and rt1269T hademerged, representing 28.9% (13/45) and 8.9% (4/45),respectively, of the viral population during AOV +LOT combination treatment. We also detected severalpolymorphic sites, including rtF221 y, rtS223A, rt1224V,rtN238H, rtL267Q, and rtQ271M, during the se­quential treatment. After 22 weeks of combinationtreatment, HBV ONA count was decreased to lessthan the lower limit of quantitation (<200 copies/mL).

Conclusions: This report identified HBV mutationsthat escaped the antiviral pressure of LAM, AOV, andAOV + LOT in this patient and provided insight intothe process of mutation selection through genotypic

Data in this article were presented in poster form at the HongKong-Shanghai International Liver Congress 2008; June 12-1 S, 2008;Hong Kong.

Accepted for publication October 31,2008.doi:1 0.1 016/j.c1inthera.2009.02.0160149-2918/$ - see front matter

© 2009 Excerpta Medica Inc. All rights reserved.

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analysis during antiviral treatment. Mutations selectedunder sequential treatments of LAM, ADV, and ADV +LDT can lead to a series of compensatory mutations,which partially restore the level of viral replication.ADV administered in combination with LDT ap­peared to be effective in this selected case with clinicalor virologic resistance to sequential treatment withLAM and ADV. (Clin Ther. 2009;31:360-366)© 2009 Excerpta Medica Inc.

Key words: hepatitis B virus, nucleoside/nucleotideanalogues, drug resistance, mutations.

INTRODUCTIONChronic hepatitis B virus (HBV) is an importantcause of morbidity and mortality worldwide, affecting>400 million people (-S % of the world's population).1

The goals of treatment in patients with chronic HBVare to prevent disease progression and avoid end-stageliver disease and hepatocellular carcinoma.2,3 In re­cent years, treatment of chronic HBV has been im­proved, with the availability of nucleoside/nucleotideanalogues (NAs), such as lamivudine (LAM), adefovirdipivoxil (ADV), telbivudine (LDT), and entecavir(ETV).4,5 NAs target the HBV reverse transcriptase(RT), thus inhibiting viral replication and leading tovirologic, biochemical, and histologic improvement inmost patients.6However, the emergence of drug-resistantmutations has become an increasing problem duringtreatment with NAs.7,8 The viral quasispecies mayundergo changes under the selective pressure of anti­viral treatment.9 Drug resistance has been associatedwith the emergence of polymerase gene mutations,followed by viral breakthrough, subsequent increasein alanine aminotransferase (ALT) levels, and, in someinstances, liver failure. 10-13

Mutations conferring resistance to LAM (rtM204V/I)have been mapped in the conserved tyrosine, methio­nine, aspartate, aspartate (YMDD) motif within theC domain of the viral RT.14,15 rtL180M and rtV173Lin the B domain of HBV polymerase act as compen­satory mutations that partially restore the replica­tive capacity of YMDD mutation strains in vitro. 16,17In addition to LAM, ADV is an oral prodrug ofthe acyclic nucleotide monophosphate analogue(9-[2-(phosphonomethoxy)ethyl))-adenine without cross­resistance with LAM.18 The important mutationsthat have been confirmed to confer resistance includertN236T within the D domain of the viral RT and

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F. Wang et a1.

rtA181 V/T in the B domain of the RT.19,20 A novelrtI233V mutation has been reported to be associatedwith primary resistance to ADV. 21 ,22 A recently devel­oped NA-LDT-is an L-nucleoside that is structur­ally related to LAM and was approved by the US Foodand Drug Administration in late 2006 for the treat­ment of active chronic HBV infection in patients aged~16 years.23 ,24 Until now, 1 mutation, rtM204I, hasbeen observed in patients treated with LDT.25,26

This report describes a case of a patient who re­ceived, successively, LAM monotherapy, ADV mono­therapy, and ADV + LDT combination treatment. Adetailed clonal HBV genome analysis was conductedon sequential serum samples throughout the course oftreatment. The evolution of viral quasispecies was in­vestigated, and whether these mutations exist on thesame or different viral genomes was determined.

CASE REPORTA 20-year-old Chinese man with a IS-year history

of hepatitis B surface antigen (HBsAg)-positive, hepa­titis B e antigen (HBeAg)-positive chronic HBV pre­sented to Huashan Hospital, Fudan University, Shang­hai, People's Republic of China. The patient had notpreviously received antiviral treatment and was start­ed on LAM monotherapy (100 mg/d) (Figure). Whenviral breakthrough emerged at week 19, treat­ment with LAM was discontinued and the patientwas switched to ADV monotherapy (10 mg/d). At76 weeks of ADV treatment, HBV DNA was de­creased to 2.36 x 103 copies/mL. However, 86 weekslater, HBV DNA level was increased to 6.30 x103copies/mL, and LDT (600 mg/d) was added to theongoing treatment regimen.

This case was conducted in agreement with the in­stitutional review board at the hospital. The patientprovided written informed consent before treatmentwas given.

Blood Collection and Extraction ofSerum Hepatitis B Viral DNA

Sera were collected prospectively at weeks 0 and 18(during LAM treatment); 22, 60, 70, and 86 (duringADV treatment); and 108 (during ADV + LDT combi­nation treatment). HBV DNA was extracted from200 pL of serum using a commercial kit (QIAampDNA Mini kit, Qiagen GmbH, Hilden, Germany).The extracted DNA was used for amplification ofHBV DNA and clonal analysis.

361

Clinical Therapeutics

.......C.......r

'-'"

20

40

60

180

160

ALT

ADV >(10 mg/d)----

(60~~g/d)D

,t I It I 'I I I' IIt

I 'I I It I )!, 0;, ;, ;, ;, ;, ;,

10 18 22 31 60 70 76 86 108

Time (wk)

6

....... 5

...JEVI.~ 4Cl..0v

0

t>O 3..Q'-'"

«z 20

~I

00

Figure. Hepatitis Bviral (HBV) DNA counts and alanine aminotransferase (ALT) concentrations during treat­ment with nucleoside/nucleotide analogues (Iamivudine [LAM] monotherapy, adefovir dipivoxil [ADV]monotherapy, and ADV + telbivudine [LOT] combination treatment) in a 20-year-old Chinese manwith a 15-year history of hepatitis B surface antigen-positive, hepatitis B e antigen-positive chronichepatitis B virus who had not previously received antiviral treatment.

laboratory TestsRoutine complete blood count and a biochemical

study, including measurements of ALT and bilirubin andcreatinine tests, were performed. HBsAgianti-HBs andHBeAgianti-HBe were detected using microparticleenzyme immune assay (Axsym, Abbott Diagnostics,Abbott Park, lllinois). Serum HBV DNA was quanti­fied using a real-time polymerase chain reaction (PCR)assay (LightCycler-FastStart DNA Master, Roche Diag­nostics, Mannheim, Germany). The lower limit of de­tection of this PCR technique was 200 copies/mL. HBVgenotype was assessed using direct sequencing.

Nested Polymerase Chain Reaction andCloning of Hepatitis B Viral Polymerase Gene

The first-round PCR was performed with 2 pL of DNAextract in a 25-pL reaction mixture containing 10 x PCRbuffer including 100-mM tris(hydroxymethyl)amino-

methane (pH, 8.3), 500-mM potassium chloride,15-mM magnesium chloride, 2.5-mM deoxyribo­nucleotide triphosphate, 2.5 U Taq polymerase (TaKaRaBio, Inc., Tokyo, Japan), and the 20-pM outer primersPI (5'-CAAGGTATGTTGCCCGTTTGTCCTC-3')and P2 (5'-AGGCAGGATAGCCACATT-3'). PCR wasperformed as follows: 94°C for 5 minutes, 94°C for30 seconds, 55°C for 30 seconds, noc for 1 minutefor 35 cycles, and noc for 10 minutes. The first­round PCR products (2 pL) served as the templatesfor the second round of PCR using primers P3(5'-ACTGCACTTGTATTCCCATCCCAT-3') and P4(5'-ATAGGTCTATTTACAGGCAG-3'), and the ampli­fication protocol was the same as above. The second­round PCR products were purified using the QIAquickPCR Purification Kit (Qiagen GmbH).

To determine whether the various drug resistance­associated mutations existed on the same HBV ge-

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nomes, PCR-purified HBV DNA was cloned intoPCR4-TOPO vector (Invitrogen Corporation, Carls­bad, California). The colonies were grown over­night at 37°C in Luria-Bertani plates mixed withampicillin (100 pg/mL). Fifty clones with HBV insertwere selected at each time point and sent to Invitro­gen for DNA sequencing. The sequences of all ofthe clones from each sample were compared withnucleic acid sequences in GenBank (GI:3551339) us­ing MegAlign software (DNASTAR, Inc., Madison,Wisconsin).

RESULTSViral and Biochemical Reaction inResponse to Treatment

During LAM, ADV, and ADV + LDT treatment inthis patient, HBV DNA counts and ALT concentra­tions were determined and are shown in detail in thetable. After LAM treatment, serum HBV DNA leveldecreased to 2.86 x 104 copies/mL from 7.97 x 104

copies/mL at week 10 but fluctuated thereafter, to5.48 x 104 copies/mL at week 18. At week 22, theHBV DNA level rose to 9.93 x 104 copies/mL, whichwas increased from the initial level (7.97 x 104 copies/mL). After starting ADV treatment, HBV DNA levelwas decreased to 2.36 x 103 copies/mL at week 76 butrose slowly to 6.30 x 103 copies/mL at week 86. There­after, LDT was added to the ongoing treatment withADV, and again the serum HBV DNA level wasdecreased to less than the lower limit of detection«200 copies/mL). The fluctuation pattern of HBVDNA and ALT levels during treatment with NAs isshown in the figure.

Evolution of the Viral Quasispecies DuringLamivudine Monotherapy

The rtM204V1L LAM-resistance mutation wasdetected in 4.4% (2/45) of HBV clones prior to LAMtreatment. At week 18 (during LAM treatment), thertM2041 mutation in the YMDD motif became pre­dominant, being present in 79.5% (35/44) of theclones. The rtM2041 mutation was associated withcompensatory mutations in the conserved B domain(rtL180M and rtT184L). A total of 9.1% (4/44)of HBV clones harbored the rtL180M + rtT184L +rtM2041 mutations. In addition, 2 new muta­tions, rtL229V and rtV1911, were detected in75.0% (33/44) and 11.4% (5/44) of the clones,respectively.

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F. Wang et a1.

Evolution of the Viral Quasispecies DuringAdefovir Monotherapy

After viral breakthrough at 18 weeks, the patientwas switched to ADV treatment. After 4 weeks, theLAM-resistance mutations (rtL180M, rtT184L, rt­M2041, rtV1911, and rtL229V) were not detected. Atweek 86 of ADV treatment, the rtA181 V and rtN236TADV-resistance mutations were detected in 2.9%(1/34) and 58.8% (20/34) of the clones, respectively.In addition, 20.6% (7/34) of the clones harbored thertK212T + rtM250L mutation.

Evolution of the Viral Quasispecies DuringAdefovir + Lamivudine Combination Therapy

After adding LDT to the ongoing ADV treatment,the viral quasispecies evolved with the extension ofthe treatment. Two novel mutations, rtS202G andrt1269T, were detected in 28.9% (13/45) and 8.9%(4/45) of the clones, respectively.

Additional changes in the nucleic acid sequence ofthe polymerase RT gene DNA sequence analysis showedadditional mutations other than those in the YMDDmotif, including a previously reported mutation,rtF221 Y, and several novel mutations, such as rtS223A,rt1224Y, rtN238H, rtL267Q, and rtQ271M, at poly­morphic sites.

DISCUSSIONIn the present report, the genetic mutations of HBVpolymerase RT domain and the changes in HBV DNAcounts and ALT concentrations were examined con­tinuously during LAM monotherapy, ADV mono­therapy, and ADV + LDT combination treatment.

Sequence analysis of HBV clones isolated from thepatient's sera during treatment revealed the evolutionof the viral quasispecies. In this report, the rtM204V1LLAM-resistance mutation was found in 4.4% (2/45)of clones prior to LAM treatment. With regard to thedevelopment pattern of the YMDD motif mutations,it has been reported that the rtM204V mutation wasdetected in >90% of LAM-resistant cases in Westerncountries. IS However, in Asia, the rtM2041 mutationwas detected in -60% of cases resistant to LAM.Genotypic resistance was found in -20% of LAM­treated patients per year. In the present report, theprevalence of the rtM2041 mutation was consistentwith those in previous studies.27,28 At week 18 (duringLAM treatment), the rtM2041 mutation became pre­dominant, being present in 79.5% (35/44) of the

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Clinical Therapeutics

Table. Evolution ofdrug-resistant mutations on clonal analysis ofserial samples. Values are relative rates (%)of clones.

LAM AOV AOV + LOT(Weeks 0-18) (Weeks 19-86) (Weeks 87-108)

Week 0 Week 18 Week 22 Week 60 Week 70 Week 86 Week 108(n = 45 (n = 44 (n = 38 (n = 35 (n = 30 (n = 34 (n = 45

Mutation Clones) Clones) Clones) Clones) Clones) Clones) Clones)

T2135 38 (84.4) 41 (93.2) 35 (92.1) 25 (71.4) 22 (73.3) 6 (17.6) 45 (100)T222A 24 (53.3) 22 (57.9) 18 (51.4) 19 (63.3) 45 (100)K212T 17 (37.8) 12(31.6) 9 (25.7)M204L 1 (2.2)M204V 1 (2.2)L229V 33 (75.0)M204/ 31 (70.5)5246P 21 (47.7)/269L 11 (25.0)5246H 8 (18.2)5256C 8 (18.2)/233T 5 (11.4)V1911 5 (11.4)L180M

+ T184L+ M204/ 4 (9.1)

N2485 3 (6.8)T213Y 3 (6.8)K275R 2 (4.5)5246L 2 (4.5)T184A 2 (5.3)T213P 22 (73.3)N236T 20 (58.8)K212T

+ M250L 7 (20.6)A181V 1 (2.9)5202G 13 (28.9)/269T 4 (8.9)

LAM - lamivudine 100 mg/d; AOV - adefovir dipivoxil10 mg/d; LOT - telbivudine 600 mg/d.

clones. As expected, the rtL180M + rtM2041 combi­nation mutation observed in patients29,30 who failedLAM was also detected in the present study. ThertL180M and rtM204V mutations are associated witha third mutation at codon position 184 or 200.31 Wealso found that 9.1 % (4/44) of the clones harboredthe rtL180M + rtT184L + rtM2041 combination mu­tation. These mutations were progressively selected to

364

become the major variant species, with a viral loadrebound from 2.86 x 104 copies/mL at week 10 to5.48 x 104 copies/mL at week 18 (during LAM mono­therapy). However, the rtV173L mutation that hadbeen described in previous reports30,32 was not foundduring LAM treatment in the present study.

When the patient was switched to ADV treatmentafter viral breakthrough emerged at week 18, as ex-

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pected, sequence analysis of HBV clones isolated atweek 22 found no LAM-resistance mutations (rtL180M,rtT184L, rtM204I, rtV1911, and rtL229V). Themultidrug-resistance phenomenon was not observedin this patient who received AOV after LAM failure,suggesting that wild-type virus rapidly replaced themutations after LAM treatment was discontinued.Until week 86 (during AOV treatment), the rtA181Vand rtN236T mutations associated with AOV resis­tance were detected in 2.9% (1/34) and 58.8%(20/34) of the clones, respectively.

After HBV ONA viral load rebounded to 6.30 x103 copies/mL from 2.36 x 103 copies/mL duringAOV treatment, the patient was switched to AOV +LOT. Prior to this case, only 1 mutation, rtM204I, wasreported to be associated with LOT resistance. 25 Inthe present report, the emergence of a mutation har­boring the rtS202G substitutions, representing 28.9%(13/45) of the viral population during AOV + LOTcombination treatment, was unexpected. The rtS202Gmutation had been described only in patients withtreatment failure with ETV. A literature search foundno previously published descriptions of the emergenceof the rtS202G mutation during AOV + LOT combi­nation treatment; however, the finding of rtS202G inthe present report suggests potential cross-resistancebetween ETV and AOV + LOT.

We reported the selection and evolution of muta­tions with increased replication fitness or maximal viralresistance during sequential antiviral pressure of LAM,ADV, and AOV + LOT. These data provide insight intothe selection process of escape mutations during se­quential NA treatment. We also described that thecombination of the 2 antiviral agents (AOV + LOT),which have not been found to have cross-resistance,can prevent the selection of these variants. However,the emergence of some new variants, especially thertS202G mutation, suggests the possibility of the se­lection of a new compensatory mutation during com­bination antiviral treatment, which has been associ­ated with cross-resistance to ETV.30 Thus, theprevention of drug resistance should be evaluated inclinical practice, although this case was treated effec­tively with the combination of AOV + LOT.

CONCLUSIONSThis case of an HBsAg-positive, HBeAg-positive pa­tient treated with LAM, AOV, and AOV + LOT pro­vided insight into the process of mutation selection

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F. Wang et a1.

during antiviral treatment. Mutations selected undersequential treatments of LAM, AOV, and AOV + LOTmight lead to a series of compensatory mutations thatpartially restore viral replication. AOV administratedin combination with LOT appeared to be effective inthis selected case with clinical or virologic resistanceto the sequential treatment of LAM and AOV, al­though new compensatory mutations emerged duringthe treatment.

ACKNOWLEDGMENTThis research and its publication was financially sup­ported by the National Basic Research Program ofChina (973 Program 2005CB523102).

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