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More sensitive hepatitis C virus RNA detection: What for?

2010; Elsevier BV; Volume: 52; Issue: 6 Linguagem: Inglês

10.1016/j.jhep.2010.02.007

1600-0641

Jean‐Michel Pawlotsky,

Liver Disease Diagnosis and Treatment

Definition of rapid virologic response with a highly sensitive real-time PCR-based HCV RNA assay in peginterferon alfa-2a plus ribavirin response-guided therapyJournal of HepatologyVol. 52Issue 6PreviewAssessing hepatitis C virus (HCV)-RNA levels is integral to response-guided therapy. Rules for early discontinuation and determination of treatment duration were mainly established with HCV-RNA assays with a detection limit of ⩽50 IU/ml (COBAS Amplicor™ HCV [CA]). The currently used real-time PCR-based COBAS Ampliprep™/COBAS-TaqMan™ HCV (CAP-CTM) test has a detection limit of approximately 10 IU/ml. It is unknown whether shortening of treatment duration to 16/24 weeks in patients with rapid virological response at week 4 (RVR) and viral loads between 10 and 50 IU/ml is possible. Full-Text PDF Hepatitis C virus (HCV) RNA quantification was made possible by means of standardized, commercial assays in the early to mid-1990s [[1]Pawlotsky J.M. Measuring hepatitis C viremia in clinical samples: can we trust the assays?.Hepatology. 1997; 26: 1-4Crossref PubMed Scopus (77) Google Scholar]. These assays were broadly used in both retrospective and prospective cohort studies and clinical trials. They showed that, in contrast to, for instance, human immunodeficiency virus infection, the HCV RNA level has no prognostic value on the natural outcome of HCV-related liver disease. By contrast, monitoring of HCV RNA levels was shown to be crucial to assess the response to antiviral therapy and tailor treatment to it [[2]National Institutes of Health Consensus Development Conference Statement: Management of Hepatitis C, June 10–12, 2002. Hepatology 2002;36:S3–20.Google Scholar]. The concept of “response-guided therapy” emerged when it was demonstrated that the early on-treatment viral kinetics were more strongly related to the final outcome of therapy than any pre-therapeutic parameter. Different time points were considered key to assess the on-treatment virological response to pegylated interferon (IFN)-α and ribavirin therapy. Initially, week 12 was used as a decision time point to stop therapy in patients who did not achieve a 2-log decrease or more in HCV RNA level relative to baseline, or an undetectable HCV RNA at this time point. Indeed, the likelihood of a sustained virological response (SVR) in these patients, if treatment was continued, was less than 2% [3Davis G.L. Wong J.B. McHutchison J.G. Manns M.P. Harvey J. Albrecht J. Early virologic response to treatment with peginterferon alfa-2b plus ribavirin in patients with chronic hepatitis C.Hepatology. 2003; 38: 645-652Crossref PubMed Scopus (786) Google Scholar, 4Ferenci P. Fried M.W. Shiffman M.L. Smith C.I. Marinos G. Goncales Jr., F.L. et al.Predicting sustained virological responses in chronic hepatitis C patients treated with peginterferon alfa-2a (40 KD)/ribavirin.J Hepatol. 2005; 43: 425-433Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar]. More recently, week 4 was used to classify the patients who responded by a more than a 2-log drop at week 12 into three groups: (1) patients with undetectable HCV RNA at week 4 (rapid virological response, RVR) (2) patients with detectable HCV RNA at week 4 but undetectable HCV RNA at week 12 (early virological response [EVR] for some, complete early virological response [cEVR] for others) (3) patients with detectable HCV RNA at week 12 in spite of a more than 2-log drop at this time point (slow virological response for some, partial early virological response [pEVR] for others). Week 24 is sometimes used to further subclassify slow virological responders into those who achieve undetectable HCV RNA at this time point, and those who do not. Common guidelines, for response-guided HCV therapy with pegylated IFN-α and ribavirin, recommend the treatment of rapid virological responders for 24 weeks if they have a low baseline HCV RNA level (<400,000-800,000 IU/ml according to the study); early (or complete early) virological responders for 48 weeks; and slow (or partial early) virological responders for 72 weeks if their HCV RNA is undetectable at week 24 [5Zeuzem S. Buti M. Ferenci P. Sperl J. Horsmans Y. Cianciara J. et al.Efficacy of 24 weeks treatment with peginterferon alfa-2b plus ribavirin in patients with chronic hepatitis C infected with genotype 1 and low pretreatment viremia.J Hepatol. 2006; 44: 97-103Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 6Moreno C. Deltenre P. Pawlotsky J.M. Henrion J. Adler M. Mathurin P. Shortened treatment duration in treatment-naive genotype 1 HCV patients with rapid virological response: a meta-analysis.J Hepatol. 2010; 52: 25-31Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 7Berg T. von Wagner M. Nasser S. Sarrazin C. Heintges T. Gerlach T. et al.Extended treatment duration for hepatitis C virus type 1: comparing 48 versus 72 weeks of peginterferon-alfa-2a plus ribavirin.Gastroenterology. 2006; 130: 1086-1097Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar, 8Pearlman B.L. Ehleben C. Saifee S. Treatment extension to 72 weeks of peginterferon and ribavirin in hepatitis C genotype 1-infected slow responders.Hepatology. 2007; 46: 1688-1694Crossref PubMed Scopus (223) Google Scholar]. These recommendations most likely apply whatever the HCV genotype. Shorter than 24 week therapy, in patients infected with genotypes 2 and 3 who achieve an RVR, remains controversial, as conflicting results have been reported [9Mangia A. Santoro R. Minerva N. Ricci G.L. Carretta V. Persico M. et al.Peginterferon alfa-2b and ribavirin for 12 vs 24 weeks in HCV genotype 2 or 3.N Engl J Med. 2005; 352: 2609-2617Crossref PubMed Scopus (598) Google Scholar, 10Shiffman M.L. Suter F. Bacon B.R. Nelson D. Harley H. Sola R. et al.Peginterferon alfa-2a and ribavirin for 16 or 24 weeks in HCV genotype 2 or 3.N Engl J Med. 2007; 357: 124-134Crossref PubMed Scopus (509) Google Scholar, 11Lagging M. Langeland N. Pedersen C. Farkkila M. Buhl M.R. Morch K. et al.Randomized comparison of 12 or 24 weeks of peginterferon alpha-2a and ribavirin in chronic hepatitis C virus genotype 2/3 infection.Hepatology. 2008; 47: 1837-1845Crossref PubMed Scopus (155) Google Scholar]. As most of these treatment decisions are based on undetectable HCV RNA levels at different time points, the lower limit of detection of commercial HCV RNA assays has become critical. Early quantitative assays, based on the branched DNA technology or classical competitive polymerase chain reaction (PCR), were poorly sensitive. Thus, non-quantitative, qualitative assays had to be used to detect small amounts of HCV RNA, and viremia was considered undetectable below the lower limit of detection of these assays [[12]Pawlotsky J.M. Molecular diagnosis of viral hepatitis.Gastroenterology. 2002; 122: 1554-1568Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar]. The Cobas Amplicor™ assay (Roche Molecular Systems, Pleasanton, California) dominated the market for many years and, after standardization of HCV RNA quantification units was achieved, its lower limit of detection was found to be 50 international units (IU)/ml. Therefore, as this assay was used in most, if not all of the clinical trials run with pegylated IFN-α and ribavirin, an “undetectable HCV RNA” in fact corresponded to an HCV RNA level below 50 IU/ml at the different time points. Major progress has been made in the technology used to quantify viral genomes. Nowadays, most laboratories use real-time PCR-based methods. In theory, these methods provide better analytical sensitivity, broader dynamic ranges of quantification, improved specificity, precision and reproducibility, and offer opportunities for high throughput and automation [[13]Chevaliez S. Pawlotsky J.M. Diagnosis and management of chronic viral hepatitis: antigens, antibodies and viral genomes.Best Pract Res Clin Gastroenterol. 2008; 22: 1031-1048Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar]. Two real-time PCR platforms are currently available for detection and quantification of HCV RNA: the Cobas Taqman® (CTM) platform, which can be used together with automated sample preparation with the Cobas AmpliPrep® device (CAP–CTM, Roche Molecular Systems); and the Abbott platform (Abbott Molecular, Des Plaines, Illinois), which uses the m2000RT amplification platform together with the m2000SP device for sample preparation. CAP–CTM has been reported to underestimate HCV RNA levels in approximately 15% of HCV genotype 2 and 30% of HCV genotype 4 samples, essentially due to nucleotide mismatches with the primers and probes used [14Chevaliez S. Bouvier-Alias M. Brillet R. Pawlotsky J.M. Overestimation and underestimation of hepatitis C virus RNA levels in a widely used real-time polymerase chain reaction-based method.Hepatology. 2007; 46: 22-31Crossref PubMed Scopus (122) Google Scholar, 15Chevaliez S. Bouvier-Alias M. Castera L. Pawlotsky J.M. The Cobas AmpliPrep–Cobas TaqMan real-time polymerase chain reaction assay fails to detect hepatitis C virus RNA in highly viremic genotype 4 clinical samples.Hepatology. 2009; 49: 1397-1398Crossref PubMed Scopus (37) Google Scholar]. A newer, improved version of this assay will be released soon. In contrast, the current version of the Abbott assay accurately quantifies HCV RNA whatever the genotype [16Chevaliez S. Bouvier-Alias M. Pawlotsky J.M. Performance of the Abbott real-time PCR assay using m2000SP and m2000RT for hepatitis C virus RNA quantification.J Clin Microbiol. 2009; 47: 1726-1732Crossref PubMed Scopus (63) Google Scholar, 17Vermehren J. Kau A. Gartner B.C. Gobel R. Zeuzem S. Sarrazin C. Differences between two real-time PCR-based hepatitis C virus (HCV) assays (RealTime HCV and Cobas AmpliPrep/Cobas TaqMan) and one signal amplification assay (Versant HCV RNA 3.0) for RNA detection and quantification.J Clin Microbiol. 2008; 46: 3880-3891Crossref PubMed Scopus (97) Google Scholar]. Another assay, developed by Siemens Medical Solutions Diagnostics (Tarrytown, New York) will become available soon. Real-time PCR assays can detect and quantify minute amounts of circulating HCV RNA. The Abbott assay has a lower limit of detection and quantification of 12 IU/ml. The first-generation CAP–CTM assay reports different types of results in the low HCV RNA level range, including “undetectable” (undetermined cutoff), “detectable but not quantifiable” ( 15 IU/ml). Importantly, HCV RNA levels between 15 and 43 IU/ml fall out of the linear range of quantification of the assay, and the manufacturer recommends interpreting the absolute value with caution. Thus, the definition for “undetectable HCV RNA” has changed with the implementation of real-time PCR assays and this could have consequences in the way patients are categorized for treatment decisions. In the current issue of the Journal, Sarrazin et al. [[18]Sarrazin C. Shiffman M.L. Hadziyannis S.J. Lin A. Colucci G. Ishida H. et al.Definition of rapid virologic response with a highly sensitive real-time PCR-based HCV RNA assay in peginterferon alfa-2a plus ribavirin response-guided therapy.J Hepatol. 2010; 52: 832-838Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar] reanalyzed stored serum samples from patients included in two large international trials with the CAP–CTM assay. The patients had been treated with pegylated IFN-α and various doses of ribavirin for various durations. Originally, undetectable HCV RNA had been defined as an HCV RNA level below 50 IU/ml in the qualitative, non-quantitative Cobas Amplicor™ assay. The authors could retest baseline, week 4, week 12, end-of-treatment, and end-of-follow-up samples from 164 out of the 1311 genotype 1-infected patients included in the trial by Hadziyannis et al. [[19]Hadziyannis S.J. Sette Jr., H. Morgan T.R. Balan V. Diago M. Marcellin P. et al.Peginterferon-alpha2a and ribavirin combination therapy in chronic hepatitis C: a randomized study of treatment duration and ribavirin dose.Ann Intern Med. 2004; 140: 346-355Crossref PubMed Scopus (2733) Google Scholar], and week 4 samples from 135 out of the 1469 genotype 2- and 3-infected patients included in the trial by Shiffman et al. [[10]Shiffman M.L. Suter F. Bacon B.R. Nelson D. Harley H. Sola R. et al.Peginterferon alfa-2a and ribavirin for 16 or 24 weeks in HCV genotype 2 or 3.N Engl J Med. 2007; 357: 124-134Crossref PubMed Scopus (509) Google Scholar]. We can conclude the following from their study: (1) at weeks 4, 12, and at the end of treatment, the vast majority of patients with undetectable HCV RNA with the Cobas Amplicor™ assay (<50 IU/ml) also have an HCV RNA level below the lower limit of quantification of the CAP–CTM assay (<15 IU/ml); (2) the lower limit of quantification in real-time PCR (15 IU/ml) can be used as a decision threshold to define the rapid or (complete) early virological response and make a decision to shorten therapy to 24 or 16 weeks in genotype 1 or genotype 2–3 rapid virological responders, respectively; (3) the patients with detectable but unquantifiable (<15 IU/ml) HCV RNA at week 4 or 12 in CAP–CTM relapse more often than those with truly undetectable HCV RNA at these time points. These results, although based on a relatively small number of patients and retrospective, tend to indicate that, with pegylated IFN-α and ribavirin therapy, the lower limits of quantification of the real-time PCR assays can be used for response-guided therapy without altering the recommendations based on clinical trials in which a threshold of 50 IU/ml was used. These findings are not truly surprising if one realizes that the difference between 15 and 50 IU/ml is only 0.5 Log10 IU/ml, and given the lack of accuracy of quantification between 15 and 43 IU/ml in the version of the CAP–CTM assay used in the study. They question the meaning of the lower limit of detection of the CAP–CTM assay relative to its lower limit of quantification. The fact that a substantial number of patients with detectable but unquantifiable HCV RNA at weeks 4 and 12 relapsed, whereas those with undetectable HCV RNA in CAP–CTM did not, suggests that the latter group has HCV RNA levels far below the lower limit of detection of the assay at the time of determination. It is likely that these patients became HCV RNA undetectable before week 4. In the absence of available samples before this time point, the study does not answer the question as to when the patients who achieved an SVR lost detectable HCV RNA with the CAP–CTM assay. Chronic hepatitis C therapy is evolving and the current standard-of-care will soon be just a memory. The triple combination of pegylated IFN-α, ribavirin, and a direct acting antiviral molecule, initially a specific HCV protease inhibitor, will likely become standard for treatment-naïve and non-responder patients within a couple of years. The addition of a potent protease inhibitor considerably accelerates the first slope of viral decline during the first few days of therapy [20Reesink H.W. Zeuzem S. Weegink C.J. Forestier N. van Vliet A. de Rooij J. et al.Rapid decline of viral RNA in hepatitis C patients treated with VX-950: a phase Ib, placebo-controlled, randomized study.Gastroenterology. 2006; 131: 997-1002Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 21Sarrazin C. Rouzier R. Wagner F. Forestier N. Larrey D. Gupta S.K. et al.SCH 503034, a novel hepatitis C virus protease inhibitor, plus pegylated interferon alpha-2b for genotype 1 nonresponders.Gastroenterology. 2007; 132: 1270-1278Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 22Forestier N. Reesink H.W. Weegink C.J. McNair L. Kieffer T.L. Chu H.M. et al.Antiviral activity of telaprevir (VX-950) and peginterferon alfa-2a in patients with hepatitis C.Hepatology. 2007; 46: 640-648Crossref PubMed Scopus (234) Google Scholar]. It might also impact the second slope of viral decline, but this has not yet been witnessed, as the majority of patients lose their HCV RNA early on therapy. Phase II trials with pegylated IFN-α, ribavirin and telaprevir or boceprevir have shown higher breakthrough and relapse rates in patients who did not achieve an RVR at week 4, as assessed by real-time PCR [23Hezode C. Forestier N. Dusheiko G. Ferenci P. Pol S. Goeser T. et al.Telaprevir and peginterferon with or without ribavirin for chronic HCV infection.N Engl J Med. 2009; 360: 1839-1850Crossref PubMed Scopus (967) Google Scholar, 24McHutchison J.G. Everson G.T. Gordon S.C. Jacobson I.M. Sulkowski M. Kauffman R. et al.Telaprevir with peginterferon and ribavirin for chronic HCV genotype 1 infection.N Engl J Med. 2009; 360: 1827-1838Crossref PubMed Scopus (994) Google Scholar, 25Kwo P. Lawitz E. McCone J. Schiff E. Vierling J. Pound D. et al.HCV SPRINT-1 final results: SVR 24 from a phase 2 study of boceprevir plus PegIntron (peginterferon alfa-2b)/ribavirin in treatment-naive subjects with genotype 1 chronic hepatitis C.J Hepatol. 2009; 50: S4Abstract Full Text PDF PubMed Google Scholar]. As a result, response-guided therapy has been adopted in the design of the ongoing Phase III trials and no patient with detectable HCV RNA at week 4 of therapy will receive less than 48 weeks of therapy. However, a recent study with telaprevir has shown that these patients represent less than 20% of those who are treated [[26]Marcellin P. Forns X. Goeser T. Ferenci P. Nevens F. Carosi G. et al.Virological analysis of patients receiving telaprevir administered q8h or q12h with peginterferon alfa-2a or alfa-2b, ribavirin in treatment-naive patients with genotype 1 hepatitis C: study C208.Hepatology. 2009; 50: 395AGoogle Scholar]. Thus, the vast majority of patients receiving the triple combination achieve an RVR and the ongoing trials will not establish the ideal treatment duration in this group. The key question with these new therapies will thus not be whether or not the patients achieve an RVR at week 4, but when they lose HCV RNA on treatment, and how this information can be used to tailor the dose and duration of therapy. Unfortunately, the question will not be answered by the ongoing Phase III trials. Thus, as soon as the new drugs reach the market, it will be essential to conduct new prospective trials aimed at determining the ideal time points for assessment of HCV RNA loss, assessing the respective predictive values of on-treatment viral kinetics, and pretreatment parameters (including recently identified genetic markers of IFN response), and directing future procedures for response-guided therapy. Because of their intrinsic characteristics, in particular their analytical sensitivity, real-time PCR assays are the ideal tools to address this issue. In this respect, the study by Sarrazin et al. is just a prologue to a new season of an “HCV virology in therapy” series.