CORRESPONDENCE

Ethnic Differences in Viral Dominance Patterns in Patients with Hepatitis B Virus and Hepatitis C VirusDual Infection

To the Editor:

We read with great interest the article entitled ‘‘Ethnic Differen-ces in Viral Dominance Patterns in Patients with Hepatitis B Virusand Hepatitis C Virus Dual Infection,’’ recently published byNguyen et al. in this journal.1

The study was designed to evaluate and compare the demo-graphic, clinical, and viral characteristics of multiethnic patientsinfected by hepatitis virus admitted at two large liver centers in theUnited States. The investigators defined the study design as amatched case-control study, in which the case group includedpatients with hepatitis B virus (HBV) and hepatitis C virus (HCV)dual infection, and the control group comprised patients with HBVmonoinfection.

However, in our opinion, there is an important methodologicalissue in the reported data: The main conclusion of the article wasnot about the comparison between cases and controls, but rather itunderscored a secondary analysis with cases only in which ethnicdifferences were examined in terms of viral dominance patternsamong HBV and HCV dual-infected patients. It is our opinionthat the investigators deviated from the scope of the case-controlstudy originally proposed, because it is clear that the investigationwas not delineated to test the hypothesis that ended up becomingthe core conclusion, including the title of the article. Therefore,the investigators’ conclusions regarding viral dominance were notconsistent with the a priori study aims proposed.

GABRIELE ROCKENBACH, M.Sc.ALEXANDRE JOSE DE MELO NETO, M.D.NEMORA TREGNAGO BARCELLOS, M.D., Ph.D.FERNANDO HERZ WOLFF, M.D., Ph.D.Postgraduate Studies in Epidemiology

School of MedicineUniversidade Federal do Rio Grande do SulRamiro Barcelos, 2600, Rio Grande do Sul, Brazil

Reference1. Nguyen LH, Ko S, Wong SS, Tran PS, Trinh HN, Garcia RT, et al.

Ethnic differences in viral dominance patterns in patients with hepatitisB virus and hepatitis C virus dual infection. HEPATOLOGY 2011;53:1839-1845.

CopyrightVC 2012 by the American Association for the Study of Liver Diseases.View this article online at wileyonlinelibrary.com.DOI 10.1002/hep.24533Potential conflict of interest: Nothing to report.

Reply:

We appreciate Ronkenback et al.’s interest in our study.1 The aimof the study was to further characterize hepatitis B virus/hepatitis Cvirus (HBV-HCV) dual infection, including viral dominance pat-tern, which is one of the most important topics in the study of HBV-HCV dual-infected patients. In addition to examining the clinicalcharacteristics of HBV-HCV dual-infected cases, in comparison withHBV monoinfected controls, an analysis of viral dominance was suit-ably part of our main aims, even if it did not require the comparisonof HBV-HCV cases and HBV monoinfected controls. The majorfindings and primary conclusions of the study are that HBV-HCVdual-infected and HBV monoinfected patients generally had similarclinical characteristics, Asian ethnicity is a major independent predic-tor of HBV-dominant disease, and HCV dominance with undetect-able HBV DNA is more common in non-Asians, all of which werewithin the scope of our major aims.

LONG H. NGUYEN, B.A.MINDIE NGUYEN, M.D., M.A.S.Stanford University

School of MedicineStanford, CA

Reference1. Nguyen LH, Ko S, Wong SS, Tran PS, Trinh HN, Garcia RT, et al. Eth-

nic differences in viral dominance patterns in patients with hepatitis B vi-rus and hepatitis C virus dual infection. HEPATOLOGY 2011;53:1839–1845.

CopyrightVC 2012 by the American Association for the Study of Liver Diseases.View this article online at wileyonlinelibrary.com.DOI 10.1002/hep.25719Potential conflict of interest: Nothing to report.

Identification of Endogenous Normalizers for Serum MicroRNAs by Microarray Profiling:U6 Small Nuclear RNA Is Not a Reliable Normalizer

To the Editor:

We read with great interest the results that the expression ofcirculating microRNA (miRNA) miR-122 was substantiallyhigher in acetaminophen-induced acute liver injury (APAP-ALI)patients, compared to healthy controls, using quantitative real-time polymerase chain reaction (PCR) assays with U6 small nu-clear RNA (snRNA) as an internal control, as reported by StarkeyLewis et al.1 However, serum miRNA expression profiles gener-ated from a large number of human samples by our laboratoryindicate that circulating U6 snRNA is not a reliable internalnormalizer.

The accuracy of circulating miRNA expression analysis criticallydepends on proper normalization of the data. Endogenous normal-izer specific for circulating miRNAs have not yet been well defined.Although some cell/tissue miRNA normalizers, including U6 andmiR-16, have been used in circulating miRNA data analyses, recentstudies suggest that cell/tissue normalizers may not serve as circulat-ing normalizers.2 To identify the miRNAs with the most stableexpression in human serum, we have evaluated 117 serum miRNAexpression profiles from young, aging, and different disease condi-tions using a real-time PCR array system, based on a global expres-sion mean normalization strategy.3 Of 332 miRNAs detected in se-rum, 58 displayed consistent expression across all samples (Fig. 1A).

1640

Our criteria to identify ideal circulating miRNA normalizers includes(1) no statistical difference among all groups, (2) the smallest varia-tion across all samples (standard deviation of |�DCT| < 1); and (3)relative high expression, closest to the global mean expression.3 ThreemiRNAs, miR-374a, miR-374b, and let-7d, met all criteria (Fig. 1B),which well resemble the mean expression value and are stablyexpressed in all groups. Therefore, they could serve as ideal endoge-nous normalizers for circulating miRNAs. However, U6 and miR-16expression was significantly different among the four groups (Fig.1C), further supporting the previous notion that they are not reliableinternal normalizers. Most important, U6 was differentially expressedbetween healthy young and aging groups (Fig. 1D). Thus, cautiousinterpretation of the data reported by Starkey Lewis et al. is war-ranted. Different normalization methods should be further employedto make sure that findings are robust, irrespective of the way ofstandardization.

We are convinced that these three miRNAs identified here arethe best circulating endogenous controls reported thus far,although more validation in different conditions may still beneeded. We recommend that suitable endogenous controls shouldbe selected in light of the study design and research conditions,and that the use of two to three endogenous normalizers togetherresembling the mean expression value may additionally reducebias and variation.

RUIQUN QI, M.S.1,2,3

MATTHEW WEILAND, M.S.1,2

XING-HUA GAO, M.D., PH.D.3

LI ZHOU, M.D.1,2,4

QING-SHENG MI, M.D., PH.D.1,2,41Henry Ford Immunology Program2Department of Dermatology

Henry Ford Health System, Detroit, MI3Department of Dermatology

No. 1 Hospital of China Medical UniversityShenyang, China

4Department of Internal MedicineHenry Ford Health System, Detroit, MI

References1. Starkey Lewis PJ, Dear J, Platt V, Simpson KJ, Craig DG, Antonie DJ,

et al. Circulating microRNAs as potential markers of human drug-induced liver injury. HEPATOLOGY 2011;54:1767-1776.

2. Chen X, Ba Y, Ma L, Cai X, Yin Y, Wang K, et al. Characterization ofmicroRNAs in serum: a novel class of biomarkers for diagnosis of can-cer and other diseases. Cell Res 2008;18:997-1006.

Fig. 1. Identification of suitable internal controls by microarray profiling. (A) Serum miRNA expression profiles. Serum miRNA expressions from117 human samples, including healthy controls at different ages (young group [n ¼ 25; age, 27.4 6 10.9 years]; aging group [n ¼ 27; age,58.3 6 10.5 years]) as well as different disease conditions, including autoimmune type 1 diabetes (n ¼ 31; age, 23.5 6 12.1 years) and ma-lignant melanoma (n ¼ 34; age, 60.1 6 16.7 years), were profiled with TaqMan Human MicroRNA array card A (v2.1), performed on a 7900HTFast Real-Time PCR System (Applied Biosystems, Foster City, CA), using the manufacturer’s recommended protocol. The cycle threshold (Ct) val-ues were obtained with SDS 2.3 and RQ manager 1.2 software (Applied Biosystems) and then data were analyzed with Real-Time StatMiner 4.2software (Integromics, Inc., Granada, Spain). Of 332 expressed miRNAs, 58 miRNAs were consistently expressed across all samples and werechosen to determine a global mean for further global normalization. The �DCt [�(Ct-global mean)] was calculated, and heatmap analysis wasperformed with hierarchical clustering. (B) Internal normalizers for serum miRNA analysis. Pairwise comparisons among the four groups by a non-parametric Wilcoxon test identified three miRNAs (miR-374a, miR-374b, and let-7d), showed no significant differences among the four groups (P> 0.1), small variations (SD ¼ 0.86, 0.78, and 0.82, respectively), and was very close to the global mean expression value. (C) Expression ofmiR-16 and U6 showed significant differences among the four groups (P < 0.01-0.001) with a large variation (SD of 1.46 and 2.85, respec-tively). (D) Expression of U6 was significantly different between the healthy young and aging groups. SD, standard deviation.

HEPATOLOGY, Vol. 55, No. 5, 2012 CORRESPONDENCE 1641

3. Mestdagh P, Van Vlierberghe P, De Weer A, Muth D, Westermann F,Speleman F, et al. A novel and universal method for microRNA RT-qPCR data normalization. Genome Biol 2009;10:R64.

Fig. 1. Profiling endogenous microRNA normalizers in healthy andALI patient sera. (A-D) Individual serum miRNAs were assayed from15 randomly selected healthy controls and ALI patients from ourstudy using TaqMan human microRNA assays (Applied Biosystems,Foster City, CA). To serve as an exogenous control species, 5 lL of a10 fM solution of cel-lin-4-5p was added to each denatured serumsample during RNA extraction. The Ct values of hsa-let-7d-5p (A),hsa-miR-374a-3p (B), U6 snRNA (C), and cel-lin-4-5p (D) are shownin panels in grouped healthy controls and ALI patients. Each openspot represents an individual Ct value. The ranges of Ct values(%CV) in healthy controls are 31.2-33.3 (1.74%), 36.4-46.2(7.58%), 31.0-35.2 (3.55%), and 34.7-36.4 (1.51%) for hsa-let7d-5p, hsa-miR-374a-3p, U6 snRNA, and cel-lin-4-5p, respectively. Theranges of Ct values (%CV) in ALI patients are 32.5-36.7 (2.99%),37.6-undetected (7.21%), 34.9-40.9 (4.69%), and 34.1-36.6(1.88%) for hsa-let7d-5p, hsa-miR-374a-3p, U6 snRNA, and cel-lin-4-5p, respectively. The expression of hsa-let-7d-5p and U6 snRNA issignificantly different between groups. ***P < 0.001 (Student ttest). (E-H). Circulating liver-enriched miR-122 is significantly higherin ALI patients regardless of the normalization strategy applied. Pan-els show the relative dynamic range of circulating miR-122 ingrouped controls (n ¼ 15) and ALI patients (n ¼ 15) when normal-ized with hsa-let-7d-5p (E), hsa-miR-374a-3p (F), U6 snRNA (G),and cel-lin-4-5p (H). The DDCt miR-122 group medians (25th, 75thpercentiles) in healthy controls versus ALI patients are 22.3 (19.0,38.9) versus 1764 (907, 6724), 2906 (1184, 25268) versus199358 (89889, 4155000), 11.2 (6.89, 33.1) versus 923 (343,4211), and 294 (178, 420) versus 4689 (2410, 15662) when nor-malized against hsa-let-7d-5p, hsa-miR-374a-3p, U6 snRNA, andcel-lin-4-5p, respectively. The expression of serum miR-122 was sig-nificantly higher in ALI patients compared to healthy controls irre-spective of the normalization strategy applied. ***P < 0.001, **P< 0.01 (Mann-Whitney U test). (I) Nonnormalized serum miR-122 Ctvalues show a highly significant difference between healthy controlsand ALI patients. The variation in Ct values of serum miR-122 inhealthy controls and ALI patients are shown in groups. The median(25th, 75th percentile) miR-122 Ct values are 27.3 (27.1, 27.5)and 23.8 (21.1, 24.6) for controls and ALI patients, respectively.The range of miR-122 Ct values (%CV) are 26.8-28.2 (1.4%) and17.6-30.4 (12.4%) for controls and ALI patients, respectively. ***P< 0.001 (Mann-Whitney U test).

CopyrightVC 2012 by the American Association for the Study of Liver Diseases.View this article online at wileyonlinelibrary.com.DOI 10.1002/hep.25558Potential conflict of interest: Nothing to report.This work was supported, in part, by a grant from Henry Ford Health SystemStart-up Grant for the Immunology Program T71016 (to Q.S.M.), HenryFord Stubnitz Grant J80005 (to Q.S.M.), Changjiang Scholars by ChineseMinistry of Education IRT0760 (to X.G.), and the Melanoma Research Alli-ance (to L.Z.).

Reply

We read the letter from Qi et al. regarding the use of endoge-nous normalizers for serum microRNAs with much interest. Theauthors describe a panel of serum miRNAs (miR-374a, miR-374b,and let-7d) that appear to be expressed at consistent levels betweenhealthy and chronic disease groups. These microRNAs may besuperior normalizers over currently used reference species.

This is a young and evolving field, and this contribution is wel-comed as it may help define parameters for the most accurate quantifi-cation of human circulating microRNAs. There is no single universallyaccepted reference species used in circulatory microRNA studies. Infact, the use of such normalizers employed in this field has beendiverse, and include a range of exogenous and endogenous species.1–4

In response to the findings by the authors, we have now profiledendogenous human let-7d-5p and miR-374a-3p (the closest availablespecies using the TaqMan platform) alongside U6 small nuclearRNA (snRNA) and exogenous Caenorhabditis elegans-specific lin-4in 15 randomly selected healthy controls and acute liver injury (ALI)patients from our study. We found that let-7d has low variationwithin healthy controls (coefficient of variation [CV] 1.7%) and ALIpatients (CV 3.0%) (Fig. 1A). However, in common with U6snRNA, we observed a modest but statistically significant difference(P < 0.001) in let-7d levels between healthy controls and ALIpatients, which supports our notion that many reference moleculesmay be perturbed in these very sick, acutely injured patients. The se-rum profile of miR-374a-3p was less promising, with high variationin controls (CV 7.58%) and ALI patients (CV 7.21%) and lowmean abundance (Ct ¼ 40.1) (Fig. 1B). Furthermore, miR-374a-3pwas not detected in six ALI patients. It would be interesting to profilethe related miR-374a-5p in light of the authors’ findings. The

1642 CORRESPONDENCE HEPATOLOGY, May 2012

exogenous species, cel-lin-4, provided the least variation in controls(CV 1.5%) and ALI patients (CV 1.9%), while exhibiting no differ-ence between groups (Fig. 1D). Importantly, we find that miR-122is increased in ALI patients regardless of which normalizer we use(Fig. 1E-1H). Furthermore, as we report in our study (Supportingfig. 4A), serum miR-122 is increased in ALI patients when weremove normalization (Fig. 1I). These data support our findings andsuggest that let-7d-5p in particular may add value as an endogenousnormalizer, alongside or instead of U6 snRNA and other referencespecies in circulating microRNA studies.

PHILIP STARKEY LEWIS, B.SC., M.RES.JAMES DEAR, PH.D., M.R.C.P.VIVIEN PLATT, B.SC., M.SC.JONATHAN MOGGS, B.SC., PH.D.CHRIS GOLDRING, B.SC., PH.D.B. KEVIN PARK, B.SC., PH.D.Department of Pharmacology and Therapeutics

School of Biomedical SciencesUniversity of LiverpoolLiverpool, UK

References1. Mahn R, Heukamp LC, Rogenhofer S, Von Ruecker A, Muller SC,

Ellinger J. Circulating microRNAs (miRNA) in serum of patients withprostate cancer. Urology 2011;77:1265.e9-1265.e16.

2. Zhang Y, Jia Y, Zheng R, Guo Y, Wang Y, Guo H, et al. PlasmamicroRNA-122 as a biomarker for viral-, alcohol-, and chemical-relatedhepatic diseases. Clin Chem 2010;56:1830-1838.

3. Wang G, Tam LS, Li E, Kwan B, Chow KM, Luk C, et al. Serum andurinary free microRNA level in patients with systemic lupus erythema-tosus. Lupus 2011;20:493-500.

4. Wang F, Zheng Z, Guo J, Ding X. Correlation and quantitation ofmicroRNA aberrant expression in tissues and sera from patients withbreast tumor. Gynecol Oncol 2010;119:586-593.

CopyrightVC 2012 by the American Association for the Study of Liver Diseases.View this article online at wileyonlinelibrary.com.DOI 10.1002/hep.25565Potential conflict of interest: Dr. Moggs is an employee of Novartis.

Hepatitis E Virus-Specific T-Cell Response After Transplantation

To the Editor:

We read with interest the article by Suneetha et al.1 They suggestthat hepatitis E virus (HEV)-specific T-cell proliferative responses aredecreased in transplant patients, particularly in those with chronichepatitis.1 Some important points need to be addressed.

The investigators suggest that patients with detectable T-cellresponses may not necessarily require antiviral treatment, but mightbe observed for spontaneous viral clearance.1 However, they pro-vided insufficient data to support this conclusion. In the transplantresolved-hepatitis group, apart from patient KTxR1, in whomT-cell response was studied during acute infection, very fewpatients had any T-cell response. Until further data are available,ribavirin therapy should be used in patients with chronic HEVinfection who do not achieve viral clearance subsequent to immu-nosuppressive dose reduction.2

The investigators suggest that a Th2-type response was onlyfound in patients with chronic hepatitis. However, only 2 patientsfrom the chronic group and 2 others from the resolving group hadinterleukin-10 secretion; hence, no conclusion can be drawn.

To understand T-cell responses in HEV infection, it is impor-tant to have appropriate controls. We wonder whether a controlgroup of transplant patients with and without previous exposure toHEV should have been included. We note that the controls weresignificantly different to study patients in terms of age and sex,and previous exposure was defined using an insensitive assay.

Interestingly, Suneetha et al. reported that cluster of differentia-tion (CD)4þ and CD8þ T-cell responses against HEV peptides,which were undetectable when patients were viremic, becamedetectable soon after HEV clearance when treated with ribavirintherapy (n ¼ 3) or when immunosuppressive therapy wasdecreased (n ¼ 2).1 Although decreasing immunosuppression mayallow T-cell responses, the explanation for the increased T-cellresponse in patients who cleared HEV subsequent to ribavirin ther-apy is unclear. Important data are absent from the article, includ-ing duration of ribavirin therapy and the temporal relationship toT-cell testing as well as changes in immunosuppressive regime. IfT-cell response was assessed in patients that were still receiving rib-avirin, one can speculate that its beneficial effect on HEV infectioncould be related to its immunomodulation. Conversely, in cases

where T-cell response was studied after ribavirin therapy andwithout modifying the immunosuppressive regimen, how do theinvestigators explain the increased T-cell response? These above-mentioned details are mandatory to understand the mechanism ofaction of ribavirin in treating HEV infection.

Finally, additional data would be of interest Were blood samplesobtained systematically before intake of immunosuppressants? If not,this may dramatically influence the analysis of T-cell response. Isthere a correlation between HEV viral load and T-cell response?

In conclusion, this study is a first step for the understanding ofHEV infection in immunosuppressed patients. Additional studiesare required.

NASSIM KAMAR, M.D., PH.D.1,2,3

FLORENCE LEGRAND-ABRAVANEL, PHARM.D., PH.D.2,3,4

HARRY R. DALTON, D.PHIL.5

JACQUES IZOPET, PHARM.D., PH.D.2,3,41Department of Nephrology, Dialysis, and Organ Transplantation

CHU Rangueil, Toulouse, France2INSERM U1043, IFR–BMT, CHU Purpan, Toulouse, France3Universit�e Paul Sabatier, Toulouse, France4Department of Virology, CHU Purpan, Toulouse, France5European Center for Environment and Human Health

Peninsula College of Medicine and DentistryTruro, UK

References1. Suneetha PV, Pischke S, Schlaphoff V, Grabowski J, Fytili P, Gronert

AA, et al. HEV-specific T-cell responses are associated with control ofHEV infection. HEPATOLOGY 2011 Oct 17. doi: 10.1002/hep.24738.

2. Kamar N, Rostaing L, Abravanel F, Garrouste C, Lhomme S, Esposito L,et al. Ribavirin therapy inhibits viral replication in patients with chronichepatitis E virus infection. Gastroenterology 2010;139:1612-1618.

CopyrightVC 2012 by the American Association for the Study of Liver Diseases.View this article online at wileyonlinelibrary.com.DOI 10.1002/hep.25578Potential conflict of interest: Nothing to report.

HEPATOLOGY, Vol. 55, No. 5, 2012 CORRESPONDENCE 1643

Reply:

We thank Kamar et al. for their interest in our article. We haveto stress that the primary aim of our study was to address basicprinciples of immune control in hepatitis E virus (HEV) infectionand, possibly, to generate additional hypotheses. As in any otherstudy describing, for the first time, an association between a diseasecondition and a distinct function of immune cells, potential clini-cal implications obviously need to be confirmed in independentstudies. We would therefore be happy to support any other groupin testing HEV-specific T-cell responses.

Ribavirin, a cheap and easy-to-apply drug, is fortunately effec-tive in most (but not all!) patients with persistent HEV infection.However, ribavirin treatment is not always uncomplicated,because side effects and comorbidities may prevent optimal dos-ing of ribavirin in single patients. Among several other—maybemore important—findings, one potential novel hypothesis thatemerged from our study was that antiviral therapy may bedelayed in patients with detectable HEV-specific T-cell responses.The association between strength, frequency, and quality (i.e.,interferon-gamma versus interleukin-10) of T-cell responses andHEV RNA status is striking and should be self-explanatory. Weare surprised that Kamar et al.1 neglect the importance of theincluded control groups in this type of study. Of note, all but 3HEV RNA-negative transplant patients had acquired HEV aftertransplantation and thus cleared HEV spontaneously and devel-oped HEV-specific T-cell responses. On the contrary, a correla-tion between the level of HEV viremia and strength of T-cellresponses could not be established because immune responseswere very weak or absent in this group.

Similar to hepatitis C, we currently do not know the detailedmode of action how ribavirin suppresses HEV replication. Modula-tion of immune responses could potentially contribute to the clear-ance of HEV infection. It might indeed be interesting to study, in

more detail, the temporal relationship between ribavirin therapyand the evolution of HEV-specific T-cell responses, which wasunfortunately not possible here because of limitations in sampleavailability.

We are just in the beginning of understanding potential inter-actions between HEV and the host’s immune system. This topicis of particular interest, because HEV did not evolve as a virusleading to prolonged, or even persisting, infections. The keyobservations of our study were that we describe, for the first time,the weakness of HEV-specific T-cell responses in chronic hepatitisE, whereas strong, multispecific responses can be observed inresolved patients. The finding that the blocking of certain costi-mulatory pathways can restore some levels of HEV-specific im-munity has implications for understanding immunological mecha-nisms of chronicity. We invite our colleagues in Toulouse toaddress similar research questions in their cohort of individualswith chronic HEV infection. However, because the number ofchronic hepatitis E patients is still limited, only collaborativeprojects across centers will be able to answer many of the above-discussed issues.

POTHAKAMURI VENKATA SUNEETHA, PH.D.SVEN PISCHKE, M.D.HEINER WEDEMEYER, M.D.Department of Gastroenterology, Hepatology, and Endocrinology

Hannover Medical SchoolHannover, Germany

CopyrightVC 2012 by the American Association for the Study of Liver Diseases.View this article online at wileyonlinelibrary.com.DOI 10.1002/hep.25577Potential conflict of interest: Nothing to report.

Prioritizing Treatment Experienced Patients with Hepatitis C Infection for Treatmentwith Telaprevir: a Number Needed to Treat Approach

To the Editor:

We read with interest the evaluation of the efficacy of telapre-vir in patients with well-characterized interferon responses.1 Thisstudy adds to the accumulating body of evidence supporting theuse of telaprevir for a wide range of patients with hepatitis C vi-rus (HCV) genotype 1 infection. It remains unclear whichpatients should be prioritized for retreatment with triple therapyand if this decision is likely to have an effect on patientmortality.

We used a number needed to treat (NNT) analysis todetermine the benefit of telaprevir-containing triple therapy inpreventing liver-related mortality. The NNT approach allowsthe calculation of a clinically meaningful summary of theeffect of a particular treatment2 and is sensitive to the efficacyof telaprevir and also the underlying risk of mortality byincorporation of the absolute risk reduction (ARR) in itscalculation.

A meta-analysis of treatment-experienced patients with HCVestimated the annual liver-related mortality rate at 0.81%.3 Inpatients with sustained virological response (SVR), that risk isreduced to 0.19%, an ARR of 0.62%. In the study reported byMuir et al., the overall SVR rate was 59%.1 Thus, in this selected

population, the NNT to prevent 1 death per year is 278. Becausethe mortality estimate from the meta-analysis included studieswith follow-up of approximately 5 years, the NNT can be reason-ably extrapolated to a 5-year NNT of 56.

In treatment-experienced patients with advanced fibrosis, theannual liver-related mortality rate is significantly higher at 2.73%.3

This is reduced in patients with SVR to 0.52%, an ARR of2.21%. However, the likelihood of SVR is lower in this groupand, although few patients with bridging fibrosis or cirrhosis wereincluded, is likely to be in the region of 40%.1 Taking these data,the 1-year NNT for patients with advanced fibrosis is 113, and the5-year NNT is 23.

These findings suggest that to have the maximal effect onmortality, the focus should be on treating patients withadvanced disease in the first instance. These patients have themost to gain from treatment with triple therapy, and althoughthere are concerns regarding viral resistance,4 this substantialimprovement in outcome should be included in clinical decisionmaking. This analysis cautions against interpretation of SVRrates without consideration of the underlying mortality risksassociated with HCV infection and provides clinically useful in-formation for the caring physician when counseling patientsregarding retreatment.

1644 CORRESPONDENCE HEPATOLOGY, May 2012

IAN A. ROWE, B.SC., M.B., CH.B., M.R.C.P.(UK)1,2,3

MATTHEW J. ARMSTRONG, M.B., CH.B., M.R.C.P.2,3

DIARMAID D. HOULIHAN, M.B., CH.B.2,31Hepatitis C Virus Research Group

University of BirminghamBirmingham, UK

2Center for Liver Research and NHR Biomedical Research UnitUniversity of BirminghamBirmingham, UK

3Liver and Hepatobiliary UnitQueen Elizabeth Hospital BirminghamBirmingham, UK

References1. Muir AJ, Poordad FF, McHutchison JG, Shiffman ML, Berg T, Ferenci

P, et al. Retreatment with telaprevir combination therapy in hepatitis C

patients with well-characterized prior treatment response. HEPATOLOGY

2011;54:1538-1546.2. Smeeth L, Haines A, Ebrahim S. Numbers needed to treat derived

from meta-analyses—sometimes informative, usually misleading. BMJ1999;318:1548-1551.

3. Singal AG, Volk ML, Jensen D, Di Bisceglie AM, Schoenfeld PS. Asustained viral response is associated with reduced liver-related morbid-ity and mortality in patients with hepatitis C virus. Clin GastroenterolHepatol 2010;8:280-288, 288.e1.

4. Pawlotsky JM. Treatment failure and resistance with direct-actingantiviral drugs against hepatitis C virus. HEPATOLOGY 2011;53:1742-1751.

CopyrightVC 2012 by the American Association for the Study of Liver Diseases.View this article online at wileyonlinelibrary.com.DOI 10.1002/hep.25553Potential conflict of interest: Nothing to report.

Effect of Interleukin-28B on Treatment Outcome in Hepatitis C Virus G1/4 Patients ReceivingResponse-Guided Therapy With Pegylated Interferon Alpha-2a/Ribavirin

To the Editor:

We read with interest that Scherzer et al. demonstrated thatslow-responder patients with an interleukin-28B (IL-28B)rs12979860 T allele benefited from therapy extension. The investi-gators state that to ‘‘…(their) knowledge, such clear evidence of anassociation between relapse and rs12979860 genotype has not beenreported previously.’’1 However, we published similar findings 3months before, from a U.S. trial of slow responders to pegylatedinterferon (Peg-IFN) alpha-2b and ribavirin (RBV).2,3

After institutional review board approval, 90 patients partici-pated by providing additional informed consent for genetic testing.These patients represented 89% of our slow-responding patients

from our original trial.2 The findings are shown below.In short, slow-responding patients to Peg-IFN/RBV benefit

from treatment extension to 72 weeks, by virtue of diminishedrates of relapse, if they harbor any non-CC genotype (i.e., IL-28Bmajor mutation).

We believe the investigators were inadvertently unaware of ourfindings because of nearly concurrent submission times. However,we are writing to inform your readers that the HEPATOLOGY dataare confirmatory, which have now been demonstrated, albeit retro-spectively, in two disparate slow-responding populations.

BRIAN L. PEARLMAN, M.D., F.A.C.P.1,2,3

CAROLE EHLEBEN, ED.D.21Center for Hepatitis C, Atlanta Medical Center, Atlanta GA2Medical College of Georgia, Augusta GA3Emory School of Medicine, Atlanta GA

References1. Scherzer TM, Stattermayer AF, Strasser M, Laferl H, Maieron A,

Stauber R, et al. Impact of IL28B on treatment outcome in hepatitis Cvirus G1/4 patients receiving response-guided therapy with peginter-feron alpha-2a/ribavirin. HEPATOLOGY 2011;54:1518-1526.

2. Pearlman BL, Ehleben C, Saifee S. Treatment extension to 72 weeks ofpeginterferon and ribavirin in hepatitis C genotype 1-infected slow res-ponders. HEPATOLOGY 2007;46:1688-1694.

3. Pearlman BL, Ehleben C. The IL-28B genotype predicts which slow-responding hepatitis C-infected patients will benefit from treatmentextension. Am J Gastroenterol 2011;106:1370-1371.

CopyrightVC 2012 by the American Association for the Study of Liver Diseases.View this article online at wileyonlinelibrary.com.DOI 10.1002/hep.25552Potential conflict of interest: Dr. Pearlman advises and is on the speakers’ bu-reau of Merck.

48 weeks 72 weeks P Value

SVR (T/T or C/T) 10% (3/31) 41% (13/32) 0.005

Relapse rate

(T/T or C/T) 61% (19/31) 13% (4/32) 0.0002

SVR (C/C) 38% (5/13) 36% (5/10) 0.58

Relapse rate

(C/C) 54% (7/13) 50% (7/14) 0.54

HEPATOLOGY, Vol. 55, No. 5, 2012 CORRESPONDENCE 1645