Digestive and Liver Disease

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1 Digestive and Liver Disease 45S (2013) S323 S331 Contents lists available at ScienceDirect Digestive and Liver Disease journal homepage: Review article The importance of HCV RNA measurement for tailoring treatment duration Kai-Henrik Peiffer, Christoph Sarrazin Klinikum der J. W. Goethe-Universität, Medizinische Klinik 1, Theodor-Stern-Kai 7, Frankfurt am Main, Germany article info abstract Article history: Available online 30 September 2013 Keywords: Triple-therapy Realtime-PCR TMA bdna LLOD LLOQ The introduction of telaprevir and boceprevir in the treatment of chronically HCV genotype 1 infected patients has led to substantially improved sustained virologic response rates and shorter treatment duration for a growing group of patients. Management and monitoring of patients receiving protease inhibitor-based triple therapy is of major importance and has become more complicated. Close monitoring of HCV RNA levels for patients on protease inhibitor-based therapy to identify subjects who are eligible for shortening of treatment duration, are virological non-responders or are in danger of experiencing a viral breakthrough is strongly recommended. Several virological tools including qualitative and quantitative HCV RNA assays for detection and quantification of HCV RNA are commercially available. We review these methods and their implications for HCV therapy as well as current sustained virologic response definition, stopping rules and recommendations for protease inhibitor-based treatment durations Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. 1. Introduction With the approval of the two NS3/4A serine protease inhibitors telaprevir (Incivek; Vertex Pharmaceuticals, Inc., Cambridge, MA, USA) and boceprevir (Victrelis; Merck & Co., Inc., Whitehouse Station, NJ, USA) a new era of treatment in genotype 1 HCV infected patients has begun. The addition of one of these direct-acting antiviral (DAA) agents to peginterferon (PegIFN) and ribavirin (RBV) leads to substantially improved sustained virologic response (SVR) rates in treatment-naïve and experienced patients [1 5]. Although triple therapy offers higher SVR rates and in several cases shorter treatment durations, management and monitoring of patients receiving antiviral therapy has become more complicated. National and international guidelines recommend close monitoring of HCV RNA levels for patients on protease inhibitor-based therapy to identify subjects who are eligible for shortening of treatment duration, are virological non-responders or are in danger of a viral breakthrough due to the emergence of resistant mutants [6 8]. Several virological methods are available to detect and quantify viral RNA and to identify relevant viral mutations potentially associated with resistance to antiviral drugs. Methods for HCV RNA detection after reverse transcription include target endpoint Corresponding author at: Klinikum der J. W. Goethe-Universität, Medizinische Klinik 1, Theodor-Stern-Kai 7, Frankfurt am Main, Germany. Tel.: ; fax: address: sarrazin@em.uni-frankfurt.de (C. Sarrazin). polymerase chain reaction (PCR), transcription mediated amplification (TMA) and the recently developed, widely used real-time target amplification systems for conserved regions of the HCV genome (real-time-pcr). Because conventional PCR and TMA assays detect and quantify HCV RNA after possible saturated enzymatic amplification reactions they have a narrow range of quantification [9]. In some cases high viral loads are not accurately quantified while low levels of HCV RNA are sometimes not detected. In contrast real-time target techniques quantify HCV RNA during the exponential phase of amplification allowing detection of minute amounts of RNA (down to approx. 10 International Units (IU)/ml) and accurate quantification up to approximately 10 8 IU/ml [10 12]. Because of their dynamic range real-time-pcr based methods are recommended as suitable for the clinical needs for diagnosis and monitoring of HCV infection [9,13]. Response guided therapy (RGT) with PegIFN and RBV has been the recommended treatment for HCV genotype 1 infected patients for more than 10 years and is still the recommended treatment for genotype 2 6 infected patients. However, only a small group (10 20%) of genotype 1 naïve patients treated with PegIFN/RBV met the criteria for shortening of treatment duration with a low baseline viral load and achievement of a rapid virological response (RVR: HCV RNA undetectable at week 4 of therapy). With the introduction of the two potent protease inhibitors 40 60% of treatment naïve genotype 1 infected patients treated with a DAA based triple therapy are now eligible for shorter treatment durations (24 28 weeks) [2,3]. Therefore, the detection and report of low levels of viraemia has become of major importance for the characterization /$ Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

2 S324 K.-H. Peiffer, C. Sarrazin / Digestive and Liver Disease 45S (2013) S323 S331 Fig. 1. Results of repeated testing of serial diluted samples from different HCV genotypes and different media (serum/plasma) for determination of an overall limit of detection (LOD) of the Cobas HPS/CobasTaqMan v2.0 (A), CobasAmpliprep/TaqMan v.2.0 (B) and the Abbott RealTime HCV Assay (C) according to the EU and US package inserts, Colucci et al. (2007) and Zitzer et al. (2013) [27,28,55 60]. of virological response and treatment decisions. Currently only few data are available of how the use of different HCV RNA assays affects treatment decisions and clinical outcome in times of triple therapy. Here, we summarize different available HCV RNA assays with a focus on real-time-pcr based techniques and their implications for modern HCV therapy as well as current SVR definition, stopping rules and recommendations for protease inhibitor-based treatment durations. 2. Different qualitative and quantitative HCV RNA assays, LLOD vs. LLOQ The detection of HCV RNA in serum or plasma samples is the actual gold standard for diagnosis of an active HCV infection and for monitoring of antiviral treatment [13,14]. A variety of different qualitative and quantitative HCV RNA assays are commercially available. Widely used methods include target endpoint PCRs, TMA, branched DNA (bdna) methods and real-time target amplification systems (real-time-pcr). HCV RNA can be detected depending on the assay in serum and plasma samples and is reported if quantifiable in IU referring to the HCV RNA standard established by the WHO in 1997 [15,16]. Quantitative HCV RNA assays have a lower limit of quantification (LLOQ) and a lower limit of detection (LLOD). Whereas the LLOQ is the lowest HCV RNA level where the quantification of the test is still accurate, the LLOD is a statistically calculated value that represents the lowest HCV RNA concentration that is reliably detected by the assay. Actually, the LLOD and the LLOQ of the HCV RNA assays that are most commonly used in clinical routine (the real-time-pcr-based Roche CobasAmpliprep/CobasTaqMan (CAP/CTM) and Abbott RealTime HCV assays) are identical (15 IU/ml and 12 IU/ml respectively). Consequently, there are three different levels of HCV RNA reported by the test: (1) HCV RNA not detected, (2) HCV RNA detected but below the limit of quantification (LLOQ) and (3) HCV quantifiable detected (value LLOQ). A test result reported as HCV RNA detected but below the limit of quantification (LLOQ) means that residual HCV viraemia was detected but cannot be quantified. For determination of the LLOD of a HCV RNA assay serial dilutions of HCV RNA samples are performed and are measured by the assay multiple times. Based on the resulting data the LLOD is statistically calculated as the value that represents the lowest HCV RNA concentration that is detected by the assay in 95% of the cases. Therefore, samples with HCV RNA concentrations

3 K.-H. Peiffer, C. Sarrazin / Digestive and Liver Disease 45S (2013) S323 S331 S325 Assay/ Trade name Distributor Technology Market status Qualitative assays CobasAmplicor HCV 2.0 PCR FDA, CE Versant HCV (TMA) Siemens TMA FDA, CE Quantitative assays CobasAmplicor HCV Monitor 2.0 PCR FDA, CE HCV SuperQuant National Genetics Institute PCR none Versant HCV RNA 3.0 (bdna) Siemens bdna FDA, CE High Pure System and CobasAmpliprep/ CobasTaqManv2.0 Realtime PCR FDA, CE System Abbott RealTime HCV Artus Hepatitis C QS-RGQ Versant HCV RNA 1.0 (kpcr) Abbott Diagnostics Realtime PCR FDA, CE Qiagen Realtime PCR CE Siemens Realtime PCR CE Fig. 2. Overview of different commercially available diagnostic HCV RNA assays with market status in the US and EU. below this LLOD can also often be measured as detectable (Fig. 1a c). Several HCV RNA assays are approved by the European Union (CE label) and the FDA and are currently commercially available (Fig. 2). In times of availability of highly sensitive and quantitative real-time PCR-based assays, with sensitivities of IU/ml, there is no medical need for qualitative assays PCR, bdna and TMA In conventional PCR-based systems the HCV RNA in the tested sample acts as a template in a reverse-transcription enzymatic reaction generating cdna. In the next step the generated cdna is amplified in a defined number of heating-cycles to amplicons in another enzymatic reaction using a DNA polymerase [17].

4 S326 K.-H. Peiffer, C. Sarrazin / Digestive and Liver Disease 45S (2013) S323 S331 Assay/ Trade name Distributor Input volume[ml] RNA extraction technique RNA extraction procedure Amplification LLOD [IU/ml] LLOQ [IU/ml] CobasAmpliprep/ CobasTaqMan v2.0 0,65 Silica based capture technique (CobasAmpliprep) (CobasTaqMan) Manual with high Cobas HPS/ CobasTaqMan v2.0 0,65 Silica based capture technique pure system viral nucleic acid kit (CobasTaqMan) (HPS) Abbott RealTime HCV Abbott Diagnostics 0,5 or 0,2 Magnetic particle technology (m2000sp) (m2000rt) 12 (0,5ml)/ 30 (0,2ml) 12 (0,5ml)/ 30 (0,2ml) Artus Hepatitis C QS-RGQ Qiagen 1,2 Magnetic particle technology (QIAsymphony SP) Rotor-Gene Q Versant HCV RNA 1.0 (kpcr) Siemens 0,5 silica-coated magnetic particle technique (VersantkPCRMol ecular System) (VersantkPCRMole cular System) Fig. 3. Overview and features of different commercially available real-time PCR-based HCV RNA assays. This technique allows a qualitative, and depending on the assay, quantitative, HCV RNA measurement. The amplicons are detected after the amplification process by hybridization to immobilized nucleotides with chemiluminescent labels that are complimentary to the amplicon. By adding a known amount of a synthetic standard, creating a competitive reaction, a quantification of the amplicons is possible by comparison to a standard curve. An example for a qualitative classic PCR based assay is the COBAS Amplicor HCV v2.0 (, Pleasanton, CA, USA) with a LLOD of 50 IU/ml for plasma and 60 IU/ml for serum samples [18]. The FDA and CE approved COBAS Amplicor HCV Monitor v2.0 (, Pleasanton, CA, USA) is an example for a quantitative conventional PCR based assay with a LLOD of 500 IU/ml and a linear range of quantification up to IU/ml [19,20]. The bdna assay differs from conventional PCR-techniques in amplification of a signal instead of the cdna template. After reverse transcription of the HCV RNA, the resulting DNA strands bind to immobilized oligonucleotides with a complementary sequence to a conserved region of the HCV genome. In the next step labelled oligonucleotides bind to the free ends of the bound DNA allowing detection by a chemiluminescent reaction. The FDA and CE labelled Versant HCV RNA 3.0 quantitative assay (Siemens Healthcare Diagnostics, Tarrytown, NY, USA) is a system based on this technique. With a LLOD of 615 IU/ml a linear quantification is ensured between 615 and IU/ml [21]. TMA based techniques have a different approach than the conventional PCR reaction. After preparation of the sample and RNA isolation, so called capture oligonucleotides bind to highly conserved regions of the HCV RNA and are captured by magnetic micro particles separating the viral RNA from the plasma. In the next step a reverse-transcription enzymatic reaction generates a double stranded DNA including a T7-promotor, used as a template for generation of multiple run-off transcripts by a RNA polymerase. Detection of RNA amplicons is based on hybridization to acridium ester labelled DNA-probes and a subsequent hybridization protection assay. However, TMA based techniques are more sensitive for the detection of low levels of viraemia than conventional PCR based methods [22]. An example of a qualitative TMA assay approved by the EU and the FDA is the Versant HCV RNA Qualitative Assay (Siemens Healthcare Diagnostics, Tarrytown, NY, USA) with a LLOD of approximately 5 IU/ml [14,23]. A quantitative isothermal realtime-tma based assay is currently under development (Hologic Gen-Probe Inc., San Diego, CA, USA). Isothermal amplification procedures used by the TMA method may be advantageous for parallel testing of different viruses. Real-time PCR techniques for HCV RNA measurement have been recently developed and have been already established in the majority of labs performing HCV diagnostics (Fig. 3). These assays have a very low limit of detection and ensure a broad dynamic range of quantification [24]. Whereas in the conventional PCR-, TMA- and bdna-based systems the detection of the amplicons takes place after the amplification process, the real-time PCR based systems allow a simultaneous amplification and detection. After RNA preparation and cdna synthesis HCV specific oligonucleotides linked to a reporter and a quenched molecule bind to the resulting cdna between the two primer binding sites. During primer initiated, polymerase mediated, amplification these molecules are degraded or released by the enzyme, emitting a fluorescent signal. This signal is measured simultaneously to amplification, is intensified by each cycle and is proportional to the HCV RNA amount in the sample of interest. Because of their specificity, sensitivity, accuracy and broad dynamic range real-time PCR quantification assays have been widely used for measurement of many viruses [13]. The FDA and CE labelled CTM HCV Test (, Pleasanton, CA, USA) is a real-time-pcr based HCV quantification assay that is produced to quantify serum and plasma samples either in combination with the fully automated CAP system extracting HCV RNA via magnetic particles or with the manual high pure system (HPS) viral nucleic acid extraction kit. The first CAP/CTM version detected HCV RNA of 15 IU/ml in 95% of the cases (LLOD) and ensured a dynamic linear range from 43 IU/ml (LLOQ) up to at least IU/ml. The first version was shown

5 K.-H. Peiffer, C. Sarrazin / Digestive and Liver Disease 45S (2013) S323 S331 S327 to underestimate viral loads of patients infected with genotype 4, and sometimes of patients infected with genotype 2 [24,25]. Besides frequent underestimations in genotype 4 samples, due to mutations in the 5 UTR region of the HCV genome this version even failed to detect HCV RNA at all in a few highly viraemic genotype 4 infected patients [10,26]. This inaccuracy was solved with the development of the second CAP/CTM version (CAP/CTM HCV quantitative test, v2.0) [27]. A second version of the CTM assay combined with manual nucleic acid extraction (HPS/CTM) was introduced already a few years ago. With a reduced sample input volume this system was shown to accurately quantify HCV RNA samples independent of the genotype [27 29]. However, this HPS/CTM v2.0 assay has different features (LLOD and LLOQ 25 IU/ml) compared to the recently introduced CAP/CTM v2.0 assay (LLOD and LLOQ 15 IU/ml). Whereas today the CAP/CTM v2.0 and the Abbott RealTime HCV assays are the most commonly used commercial HCV tests in daily clinical practice worldwide, the HPS/CTM v2.0 assay was used for all phase 3 clinical studies for the approval of telaprevir and boceprevir [1 4]. For these trials HCV RNA concentrations below the LLOD/LLOQ are reported as HCV RNA detected, less than 25 IU/ml. The 95% probability of detecting HCV RNA in HCV genotype 1 infected patients in serum and plasma is 8.8 and 9.3 IU/ml, respectively. The Abbott RealTime HCV assay (Abbott Molecular, Des Plaines, IL, USA) for use with the m2000 platform is another FDA approved and CE labelled commercially available real-time-pcr based assay for the quantification of HCV RNA. This assay allows semiautomatic testing of plasma and serum samples and shows a specific HCV RNA quantification with a linear range from 12 IU/ml up to 100 million IU/ml and a LLOD/LLOQ of 12 IU/ml [12]. As for the other assays the quantification is accurate in all known genotypes [12,27,30,31]. The Artus Hepatitis C QS-RGQ assay (Qiagen, Hilden, Germany) is another commercially available CE labelled real-time-pcr-based system that has so far not been approved by the FDA. In comparison to the CTM assay a slightly lower sensitivity was observed for this assay [32]. The real-time-pcr-based Versant HCV RNA 1.0 assay (Siemens Healthcare Diagnostics, Tarrytown, NY, USA) has been developed for the replacement of the qualitative TMA-based and quantitative bdna-based Versant assays and is now commercially available and CE labelled. The manufacturer guarantees a LLOD of 15 IU/ml and a dynamic range of quantification from 15 up to 100 million IU/ml. However, no published data is available for the performance of this assay in comparison to other real-time-based tests. Even though all HCV RNA assays are calibrated to the international standard established by the WHO and are referred to IU there may be significant differences in sensitivities in detecting low amounts of HCV RNA and in the absolute quantification. Detection limits depend on many different factors, which include input volume and material, nucleic acid extraction procedures, design of primers and probe, internal vs. external control and cycling conditions. Differences in HCV RNA concentrations can be explained by the fact that different versions of the WHO standard are used for calibration of a particular assay. Furthermore, tolerable deviations to the WHO standard (±0.2 log 10) may be in contrary directions for different assays. For example, quantification with the Abbott RealTime HCV Assay showed a slight underestimation ( 0.2 to 0.3 log 10 IU/ml), whereas quantification with the Roche CAP/CTM showed a slight overestimation (+0.2 to +0.3 log 10 IU/ml) of the WHO Standard (Fig. 4) [10]. Although differences for both assays in comparison to the WHO standard are acceptable in clinical practice, HCV RNA baseline concentrations measured by the Roche CAP/CTM assay were found to be 0.5 log 10 IU/ml higher (approx. 3-fold) in comparison to the Abbott RealTime HCV test [10,25,33]. With regard to the newly introduced stopping rules based on absolute HCV RNA concentrations (cut-offs at 100 and IU/ml for Boceprevir and Telaprevir triple therapy, respectively) it is currently unknown whether they precisely apply to the use of different HCV RNA assays. 3. Definition of SVR, response-guided therapy (RGT) and current stopping rules in times of DAAs Definition of sustained virological response (SVR) for patients treated with a dual combination of PegIFN and RBV is based on undetectable HCV RNA level (<50 IU/ml) according to the 2011 EASL guidelines and as an absence of HCV RNA from serum by a sensitive PCR-based assay according to the 2009 AASLD guidelines 24 weeks after end of treatment (EOT). In all phase III clinical trials for the approval of telaprevir and boceprevir the second version of the CTM HCV test in combination with the high pure system (HPS/CTM HCV test, v2.0) was used. The use of this second generation, real-time- PCR based system led to a different definition of SVR by the FDA using the LLOQ of the test (undetectable HCV RNA level (<25 IU/ml) 24 weeks after EOT) [1 5,13,14]. The LLOQ of the assay was chosen for definition of SVR because of suspected false positive HCV RNA results 24 weeks after the end-of-treatment that were reported as detectable but lower than the LLOQ for post-treatment follow-up samples. However, further follow-up tests revealed negative HCV RNA without any evidence for a virological relapse. In a number of studies with dual combination of PegIFN and RBV the possibility of shortening treatment duration to 24 weeks without loss of efficacy in treatment-naïve HCV genotype 1 infected patients with low baseline viral load (< IU/ml) and rapid virologic response (RVR, defined as undetectable HCV RNA at week 4 of treatment by various assays) was established [34 36]. For identification of patients who are eligible for response-guided triple-therapy (RGT) with shortening to 24, 28 or 36 weeks the LLOD (HCV RNA < 25 IU/ml undetectable by the HPS/CTM assay) of the test at predefined time points during treatment was used in the phase 3 boceprevir and telaprevir trials. In the SPRINT-2 trial treatment-naïve patients in the RGT arm with an extended rapid virological response (ervr: undetectable HCV RNA at study week 8 until week 24) were eligible for shorter treatment duration of 28 weeks [2]. Based on equal SVR rates in comparison to ervr patients who were treated for 48 weeks RGT was approved in treatment-naïve non-cirrhotic patients in the EU and US [8]. In the boceprevir approval study for relapsers and partial non-responders (RESPOND-2) [1] RGT was performed in one study arm comparing 36 vs. 48 weeks of treatment in patients who achieved an ervr defined as being HCV RNA undetectable at weeks 8 and 12. Due to comparable efficacy shortening to 36 weeks was approved in non-cirrhotic patients in the US [8]. In the telaprevir approval study for RGT (ILLUMINATE) patients with ervr defined as HCV RNA undetectable at weeks 4 and 12 were randomized to 24 or 48 weeks of total treatment duration. Shortening to 24 weeks in treatment-naïve, non-cirrhotic patients who achieved ervr was approved in the EU and US. Although there are no phase 3 data for RGT in treatment experienced patients based on phase 2 study results, shortening of treatment to 24 weeks was also approved for relapsers receiving telaprevir-based triple therapy [6,7,14,37]. To avoid emergence of resistant mutants, which is associated with virological failure (i.e. breakthrough and relapse), the adherence to stopping rules for the DAA treatment is strongly recommended. Because of a low barrier to resistance, monotherapy with boceprevir or telaprevir leads to viral breakthrough due to selection of resistant mutants within the first few days of therapy [38 41] The likelihood of resistance is increased in patients with

6 S328 K.-H. Peiffer, C. Sarrazin / Digestive and Liver Disease 45S (2013) S323 S331 WHO Standard Abbott RealTime HCV Roche CAP/CTM v1.0 Nominal input IU/ml (log 10 ) Mean (range) IU/ml Mean (range) log 10 Mean difference to WHO Standard Mean (range) IU/ml Mean (range) log 10 Mean difference to WHO Standard ( ) 3.0 (2,8-3,0) - 0, ( ) 3,5 (3,5-3,5) +0, ( ) 4,1 (4,0-4,2) - 0, ( ) 4,6 (4,3-4,8) +0,2 Fig. 4. Quantification of Abbott RealTime HCV Assay and Roche CAP/TaqMan v1.0 in comparison to the WHO standard [10]. a poor response to PegIFN and RBV as well as patients infected with the 1a subtype due to lower barrier to resistance in this subtype [2,42]. In addition, protease inhibitor resistant virus strains are typically associated with a lower replicational fitness leading to a low viral turnover. Hence, if stopping rules are not respected, compensatory mutations may enhance the fitness of the viral protease and increase the ability to replicate leading to development of long-time persistent resistant mutants that may affect response to future re-treatments [42,43]. Therefore patients on protease inhibitor-based triple therapy should undergo close HCV RNA monitoring, ideally with the same HCV RNA assay in the same lab, and the protease inhibitor treatment should be discontinued if virological breakthrough occurs (>1 log increase in serum HCV RNA above nadir or >100 IU/ml if tested undetectable previously) [8]. Furthermore, based on a 100% negative predictive value all patients receiving a telaprevir-based triple therapy should end treatment if HCV RNA is >1000 IU/ml at week 4 or 12 or is detectable at week 24. Patients receiving a boceprevir-based triple therapy should stop treatment if the HCV RNA level is 100 IU/ml at week 12 or detectable at week 24 [42 44]. 4. Implication of HCV RNA measurements and different assays While for the dual combination therapy with PegIFN and RBV only 10 20% of treatment-naïve patients achieved a RVR with undetectable HCV RNA levels at week 4 of treatment ervr rates achieved during triple therapies with boceprevir and telaprevir are 44% and 57 65%, respectively. Thus, the majority of triple therapy patients benefit from shortened treatment duration and SVR rates in this subgroup are extremely high (89 96%) [2,3]. Thus, given the long duration, side effects and costs, a maximum of patients should benefit from shortened treatment duration. However, few HCV RNA assays have been used for establishment of virological rules for RGT in pivotal trials. These assays either have been replaced by realtime PCR based tests (Amplicor HCV Qualitative and TMA assays) or have been used in approval studies but are not widely used in clinical practice (HPS/CTM assay). Currently, few data are available of how the use of different cutoffs (i.e. LLOD vs. LLOQ) or even different HCV RNA assays affects RGT decision rules. For dual therapy with PegIFN/RBV decisions for shorter treatment durations based on the LLOQ of real-time-pcr based HCV quantification seemed to be sufficient [45]. For example, in one large study week 4 samples of genotype 1 3 infected patients treated with a response-guided dual therapy in which treatment decisions were based on HCV RNA measurement with a conventional qualitative PCR-based HCV RNA assay (CobasAmplicor HCV Qualitative assay, LOD 50 IU/ml) were re-analyzed with a realtime-pcr based assay (CAP/CTM assay version 1). RVR rates were similar with both assays if for the CAP/CTM assay a cut-off of 15 IU/ml was used. When samples with detectable HCV RNA but below 15 IU/ml were included a significant number of patients had still detectable residual viraemia while testing undetectable with the conventional-pcr. However, SVR rates did not differ after shortened treatment (16/24 weeks) in patients who were tested undetectable using conventional-pcr compared to those who had undetectable or detectable HCV RNA but below 15 IU/ml with the real-time-pcr-based assay [45]. In another study, samples of 136 genotype 1 infected patients receiving dual RGT (based on undetectable HCV RNA at week 4 analyzed by the TMA assay) were reanalyzed with the CAP/TaqMan v2.0 and the Abbott RealTime HCV assays (Fig. 5) [46]. Using the LLOD of the tests 19 58% of the patients were tested HCV RNA positive with the real-time PCR based assays while tested negative with the TMA at week 4, thus leading to a different determination of RVR rates. Nevertheless, SVR rates did not differ and no subsequent relapses were observed in patients with residual viraemia at week 4 of treatment by the real-time PCR-based assays, despite shortening of treatment duration [46]. In phase 2 and 3 boceprevir and telaprevir studies the realtime PCR-based HPS/CTM v2.0 assay was used. In phase 2 studies treatment duration was randomized to 24/28 vs. 48 weeks independent of initial virologic response [42,47,48]. A retrospective analysis of SVR rates depending on virologic response at week 4 of triple-therapy showed significantly lower SVR rates in patients with shortened treatment duration (24/28 weeks) with residual viraemia (detectable HCV RNA but <25 IU/ml) in comparison to those with completely undetectable HCV RNA (Fig. 6) [49]. As in phase 3 studies shortened treatment duration was allowed only for patients with undetectable HCV RNA at treatment decision time points, no data are available for analysis of the importance of residual viraemia in RGT. Based on the results of phase 2 studies however, shortening of treatment duration was only approved for patients with completely undetectable HCV RNA at week 4 of triple therapy and decision time points thereafter. However, it remains unclear if the use of different real-time-pcr based assays with slightly different sensitivities had an impact on treatment duration and outcome. In a first study a large number of samples from

7 K.-H. Peiffer, C. Sarrazin / Digestive and Liver Disease 45S (2013) S323 S331 S329 Roche Roche CAP/TaqMan Abbott RealTime TMA neg. CAP/TaqMan Abbott RealTime v.2.0 pos. HCV pos. v.2.0 HCV & subsequent & subsequent pos. pos. relapse relapse TW (19%) 15 (58%) 0/6 (0%) 0/15 (0%) TW (13%) 11 (16%) 3/9 (33%) 3/11 (27%) EOT (1%) 4 (4%) 0/1 (0%) 2/4 (50%) FU (0%) 2 (2,2%) 0/0 (0%) 0/2 (0%) Fig. 5. Reanalysis with Abbott RealTime HCV Assay and Roche CAP/TaqMan v.2.0 of samples obtained from genotype 1 infected patients receiving RGT with peginterferon alfa and ribavirin based on HCV RNA undetectability at week 4 (TMA) [46]. Fig. 6. Probability of relapse for patients with residual viraemia in response guided triple therapy with boceprevir and telaprevir in phase 2 trials [49]. Abbott RealTime HCV not detected <12 IU/mL detected 12 IU/mL Roche CAP/CTM v2.0 not detected <15 IU/mL detected 15 IU/mL Fig. 7. HCV RNA quantification with Abbott RealTime HCV Assay and Roche CAP/TaqMan v.2.0 of week 4 samples obtained from genotype 1 infected patients receiving telaprevir based triple therapy [46]. before and during conventional triple therapy with the NS3 pro- tease inhibitor Simeprevir (PILLAR study) which were analyzed by

8 S330 K.-H. Peiffer, C. Sarrazin / Digestive and Liver Disease 45S (2013) S323 S331 the HPS/CTM v2.0 assay were retested with the Abbott RealTime HCV assay [50]. As expected, HCV RNA concentrations at baseline measured by the Abbott RealTime HCV assay were generally lower compared to the values obtained by the HPS/CTM test (difference approx. 0.4 log 10). However, while the Abbott RealTime assay yielded high HCV RNA samples generally lower than the HPS/CTM, lower HCV RNA samples tested generally higher with the Abbott RealTime HCV Assay than when assessed with the TaqMan, resulting in longer time to HCV RNA undetectability when quantified with the Abbott RealTime assay. Interestingly, measurement by the Abbott RealTime assay showed a significantly lower rate of undetectable HCV RNA (<LLOD) than measurements with the Taq- Man assay, leading to a different determination of undetectable HCV RNA concentrations at week 4 of treatment in approx. 26% of cases. Overall SVR rates in patients with shortened treatment duration in this study however, were 85 96%. Another study that was presented at the 2012 AASLD meeting revealed similar results for the comparison of the Abbott RealTime assay and CAP/CTM v2.0 in samples obtained from patients on telaprevir based triple-therapy (Fig. 7) [46]. The use of the Abbott RealTime assay instead of the TaqMan assay led to a different determination of RVR in 35% of the examined cases. Thus, if the Abbott RealTime assay together with undetectable HCV RNA at week 4 of triple therapy had been used in these studies, a significantly smaller proportion of patients would have benefitted from shortened treatment duration. An interim analysis of another study that was presented at the recent International Liver Congress (EASL 2013) revealed similar results for the comparison of the Abbott RealTime assay and CAP/CTM v2.0 in samples obtained from patients on telaprevir based triple-therapy [51]. The use of the Abbott RealTime assay instead of the TaqMan assay led to a different determination of RVR in 27% of the examined cases, and 59% of samples at week 4 of triple therapy had undetectable HCV RNA by the CAP/CTM v2.0 but detectable HCV RNA by the Abbott RealTime assay. However, also in patients with shortened treatment duration with undetectable HCV RNA by the CAP/CTM v2.0 but detectable HCV RNA by the Abbott RealTime assay no virologic relapse has been observed so far. Therefore, the use of a different cut-off for the Abbott RealTime assay at treatment decision time points (i.e. RNA undetectable or detectable but below the LLOQ of 12 IU/ml) might be sufficient for telaprevir and boceprevir based triple therapy. Another study also presented at this year s EASL compared the CAP/CTM v2.0, and the HPS/CTM v2.0 in samples obtained from patients receiving protease inhibitor-based triple therapy originally tested with the CAP/CTM v1.0 during RGT treatment [52]. Only 31% of the samples tested HCV RNA undetectable with the CAP/CTM v1.0 were also tested negative with the two other assays. None of these patients experienced a virological failure. A greater likelihood of treatment failure was observed when a detectable HCV RNA result was found with the other assays, as this obviously indicates the presence of significant residual viraemia by repeated testing. Response-guided triple therapy with next generation protease inhibitors will be approved soon. Interestingly, in the underlying phase 3 studies different ervr definitions have been used. Treatment naïve patients on faldaprevir or simeprevir based triple therapies were eligible for shortened treatment duration with RNA undetectable or detectable but below the LLOQ of 25 IU/ml at week 4 and undetectable at week 8 or 12, respectively. In the phase 3 STARTVerso-1 study a faldaprevir-based triple therapy was investigated in 652 treatment-naïve genotype 1 infected patients [53]. RGT was based on achieving an early treatment success (ETS), which was defined as HCV RNA below the limit of quantification (LLOQ) at week 4 and undetectable RNA at week % of the patients were eligible for shorter treatment duration and 86 89% of these patients achieved SVR12. However, SVR rates after shortened treatment in the subgroup of patients with residual viraemia at week 4 ( 25 IU/ml detectable) were 72 77% while those patients with undetectable HCV RNA at week 4 ( 25 IU/ml undetectable) achieved SVR in 91 94% of cases. Similar results have been obtained in the phase 3 QUEST-1 and QUEST-2 trials with large numbers of genotype 1 treatment-naïve patients treated with a simeprevir-based conventional triple therapy [54]. These data confirm that residual viraemia at week 4 of triple therapy, obtained by the HPS/CTM assay, indicates a higher likeliness of relapse after shortened treatment duration. Additional predictors may help to determine the subset of patients with no enhanced risk of virologic relapse after 24 weeks of overall treatment duration despite residual viraemia at week 4. Furthermore, these data also confirm the need for the determination of cut-offs for other assays for the proper identification of patients which are suitable for short treatment. 5. Conclusions With the introduction of telaprevir and boceprevir, close HCV RNA monitoring has become of major importance for optimizing treatment durations and virological response. Several virological tools for HCV RNA quantification are currently widely available including classic-pcr-, TMA, bdna and real-time-pcr based systems. Because of their specificity, sensitivity, accuracy and broad dynamic range the use of real-time PCR quantification assays for management of DAA based triple therapy is strongly recommended and most widely used. Current data recommend the use of the LLOD instead of the LLOQ at treatment decision points for identifying patients eligible for RGT. Treatment-naïve patients without cirrhosis on boceprevir based triple therapy may be considered for RGT if HCV RNA is undetectable at treatment week 8 and 12 (ervr). Treatment-naïve patients and relapsers without cirrhosis on telaprevir based triple therapy may be considered for RGT if HCV RNA is undetectable at treatment week 4 and 12 (ervr). To avoid emergence of resistant mutants the adherence to stopping rules for the DAA treatment is strongly recommended. If virological breakthrough (>1 log increase in serum HCV RNA above nadir or >100 IU/ml if tested undetectable previously) occurs, DAA treatment should be immediately stopped. Few data are available on the implications of the use of different HCV RNA assays on treatment duration and outcome. Although first results show significantly different determinations of ervr when different assays were used, further studies are needed to evaluate the impact of different assays on RGT rules. Conflict of interest KH Peiffer: nothing to declare. C Sarrazin: Speakers bureau, grant support and advisory boards for Roche, Abbott, Siemens, Qiagen, Vertex, Janssen, MSD. This article is part of a supplement supported by an unrestricted educational grant from Janssen Pharmaceutica NV. Janssen has had no editorial control or involvement in the content of this article. The views and opinions within this supplement are those of the authors and not necessarily those of Janssen. References [1] Bacon BR, Gordon SC, Lawitz E, et al. Boceprevir for previously treated chronic HCV genotype 1 infection. New England Journal of Medicine 2011;364: [2] Poordad F, McCone Jr J, Bacon BR, et al. Boceprevir for untreated chronic HCV genotype 1 infection. New England Journal of Medicine 2011;364: [3] Jacobson IM, McHutchison JG, Dusheiko G, et al. Telaprevir for previously untreated chronic hepatitis C virus infection. New England Journal of Medicine 2011;364: [4] Zeuzem S, Andreone P, Pol S, et al. Telaprevir for retreatment of HCV infection. New England Journal of Medicine 2011;364: [5] Sherman KE, Flamm SL, Afdhal NH, et al. Response-guided telaprevir combination treatment for hepatitis C virus infection. New England Journal of Medicine 2011;365:

9 K.-H. Peiffer, C. Sarrazin / Digestive and Liver Disease 45S (2013) S323 S331 S331 [6] Leroy V, Serfaty L, Bourliere M, et al. Protease inhibitor-based triple therapy in chronic hepatitis C: guidelines by the French Association for the Study of the Liver. Liver International 2012;32: [7] Sarrazin C, Berg T, Cornberg M, et al. Expert opinion on boceprevir- and telaprevir-based triple therapies of chronic hepatitis C. Zeitschrift fur Gastroenterologie 2012;50: [8] Ghany MG, Nelson DR, Strader DB, et al. An update on treatment of genotype 1 chronic hepatitis C virus infection: 2011 practice guideline by the American Association for the Study of Liver Diseases. Hepatology 2011;54: [9] Chevaliez S, Rodriguez C, Pawlotsky JM. New virologic tools for management of chronic hepatitis B and C. Gastroenterology 2012;142:1303 e1 13 e1. [10] Vermehren J, Kau A, Gartner BC, et al. Differences between two realtime 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. Journal of Clinical Microbiology 2008;46: [11] Chevaliez S, Bouvier-Alias M, Pawlotsky JM. Performance of the Abbott realtime PCR assay using m2000sp and m2000rt for hepatitis C virus RNA quantification. Journal of Clinical Microbiology 2009;47: [12] Fytili P, Tiemann C, Wang C, et al. Frequency of very low HCV viremia detected by a highly sensitive HCV-RNA assay. Journal of Clinical Virology 2007;39: [13] EASL Clinical Practice Guidelines: management of hepatitis C virus infection. Journal of Hepatology 2011;55: [14] Ghany MG, Strader DB, Thomas DL, et al. Diagnosis, management, and treatment of hepatitis C: an update. Hepatology 2009;49: [15] Saldanha J, Lelie N, Heath A. Establishment of the first international standard for nucleic acid amplification technology (NAT) assays for HCV RNA, WHO Collaborative Study Group. Vox Sanguinis 1999;76: [16] Saldanha J, Heath A, Aberham C, et al. World Health Organization collaborative study to establish a replacement WHO international standard for hepatitis C virus RNA nucleic acid amplification technology assays. Vox Sanguinis 2005;88: [17] Pawlotsky JM. Molecular diagnosis of viral hepatitis. Gastroenterology 2002;122: [18] COBAS AMPLICOR HCV MONITOR Test v. Package insert. Volume Revision 10.0; [19] Konnick EQ, Erali M, Ashwood ER, et al. Performance characteristics of the COBAS Amplicor Hepatitis C Virus (HCV) Monitor, Version 2.0. International Unit assay and the National Genetics Institute HCV Superquant assay. Journal of Clinical Microbiology 2002;40: [20] Lee SC, Antony A, Lee N, et al. Improved version 2.0 qualitative and quantitative AMPLICOR reverse transcription-pcr tests for hepatitis C virus RNA: calibration to international units, enhanced genotype reactivity, and performance characteristics. Journal of Clinical Microbiology 2000;38: [21] Morishima C, Chung M, Ng KW, et al. Strengths and limitations of commercial tests for hepatitis C virus RNA quantification. Journal of Clinical Microbiology 2004;42: [22] Morishima C, Morgan TR, Everhart JE, et al. HCV RNA detection by TMA during the hepatitis C antiviral long-term treatment against cirrhosis (Halt-C) trial. Hepatology 2006;44: [23] Gorrin G, Friesenhahn M, Lin P, et al. Performance evaluation of the VERSANT HCV RNA qualitative assay by using transcription-mediated amplification. Journal of Clinical Microbiology 2003;41: [24] Sarrazin C, Gartner BC, Sizmann D, et al. Comparison of conventional PCR with real-time PCR and branched DNA-based assays for hepatitis C virus RNA quantification and clinical significance for genotypes 1 5. Journal of Clinical Microbiology 2006;44: [25] Chevaliez S, Bouvier-Alias M, Brillet R, et al. Overestimation and underestimation of hepatitis C virus RNA levels in a widely used real-time polymerase chain reaction-based method. Hepatology 2007;46: [26] Chevaliez S, Bouvier-Alias M, Castera L, et al. 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: [27] Zitzer H, Heilek G, Truchon K, et al. Second-generation Cobas AmpliPrep/Cobas TaqMan HCV quantitative test for viral load monitoring: a novel dual-probe assay design. Journal of Clinical Microbiology 2013;51: [28] Colucci G, Ferguson J, Harkleroad C, et al. Improved COBAS TaqMan hepatitis C virus test (Version 2.0) for use with the High Pure system: enhanced genotype inclusivity and performance characteristics in a multisite study. Journal of Clinical Microbiology 2007;45: [29] Vermehren J, Colucci G, Gohl P, et al. Development of a second version of the Cobas AmpliPrep/Cobas TaqMan hepatitis C virus quantitative test with improved genotype inclusivity. Journal of Clinical Microbiology 2011;49: [30] Elkady A, Tanaka Y, Kurbanov F, et al. Performance of two Real-Time RT-PCR assays for quantitation of hepatitis C virus RNA: evaluation on HCV genotypes 1-4. Journal of Medical Virology 2010;82: [31] LaRue H, Rigali L, Balada-Llasat JM, et al. Performance of the Abbott RealTime and Roche Cobas TaqMan hepatitis C virus (HCV) assays for quantification of HCV genotypes. Journal of Clinical Microbiology 2012;50: [32] Paba P, Fabeni L, Perno CF, et al. Performance evaluation of the Artus hepatitis C virus QS-RGQ assay. Journal of Virological Methods 2012;179: [33] Sizmann D, Boeck C, Boelter J, et al. Fully automated quantification of hepatitis C virus (HCV) RNA in human plasma and human serum by the COBAS AmpliPrep/COBAS TaqMan system. Journal of Clinical Virology 2007;38: [34] Hadziyannis SJ, Sette Jr H, Morgan TR, et al. Peginterferon-alpha2a and ribavirin combination therapy in chronic hepatitis C: a randomized study of treatment duration and ribavirin dose. Annals of Internal Medicine 2004;140: [35] Jensen DM, Morgan TR, Marcellin P, et al. Early identification of HCV genotype 1 patients responding to 24 weeks peginterferon alpha-2a (40 kd)/ribavirin therapy. Hepatology 2006;43: [36] Zeuzem S, Poordad F. Pegylated-interferon plus ribavirin therapy in the treatment of CHC: individualization of treatment duration according to on-treatment virologic response. Current Medical Research and Opinion 2010;26: [37] Muir AJ, Poordad FF, McHutchison JG, et al. Retreatment with telaprevir combination therapy in hepatitis C patients with well-characterized prior treatment response. Hepatology 2011;54: [38] Susser S, Welsch C, Wang Y, et al. Characterization of resistance to the protease inhibitor boceprevir in hepatitis C virus-infected patients. Hepatology 2009;50: [39] Reesink HW, Zeuzem S, Weegink CJ, 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: [40] Sarrazin C, Kieffer TL, Bartels D, et al. Dynamic hepatitis C virus genotypic and phenotypic changes in patients treated with the protease inhibitor telaprevir. Gastroenterology 2007;132: [41] Sarrazin C, Rouzier R, Wagner F, et al. SCH , a novel hepatitis C virus protease inhibitor, plus pegylated interferon alpha-2b for genotype 1 nonresponders. Gastroenterology 2007;132: [42] McHutchison JG, Everson GT, Gordon SC, et al. Telaprevir with peginterferon and ribavirin for chronic HCV genotype 1 infection. New England Journal of Medicine 2009;360: [43] Kieffer TL, Sarrazin C, Miller JS, et al. Telaprevir and pegylated interferon-alpha- 2a inhibit wild-type and resistant genotype 1 hepatitis C virus replication in patients. Hepatology 2007;46: [44] Bartels DJ, Zhou Y, Zhang EZ, et al. Natural prevalence of hepatitis C virus variants with decreased sensitivity to NS3.4A protease inhibitors in treatmentnaive subjects. Journal of Infectious Diseases 2008;198: [45] Sarrazin C, Shiffman ML, Hadziyannis SJ, 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. Journal of Hepatology 2010;52: [46] Peiffer K-H, Gerber L, Susser S, et al. Significant differences between different assays for assessment of HCV RNA undetectability during response guided therapy in patients treated with PEG-Interferon. Ribavirin with or without a protease inhibitor. Hepatology 2012;56:560A. [47] Foster G, Hezode C, Bronowicki J, et al. Activity of telaprevir alone or in combination with peginterferon alpha-2a and ribavirin in treatment naive genotype 2 and 3 hepatitis-c patients: interim results of study C209. Journal of Hepatology 2009;50(Suppl. 1):S22. [48] Kwo PY, Lawitz EJ, McCone J, et al. Efficacy of boceprevir, an NS3 protease inhibitor, in combination with peginterferon alfa-2b and ribavirin in treatment-naive patients with genotype 1 hepatitis C infection (SPRINT-1): an open-label, randomised, multicentre phase 2 trial. Lancet 2010;376: [49] Harrington PR, Zeng W, Naeger LK. Clinical relevance of detectable but not quantifiable hepatitis C virus RNA during boceprevir or telaprevir treatment. Hepatology 2012;55: [50] Fevery B, Susser S, Lenz O, et al. Comparison of two quantitative HCV RNA assays in samples from patients treated with a protease inhibitor-based therapy: Implications for response guided therapy. Journal of Hepatology 2012;56:S26. [51] Vermehren J, Aghemo A, Falconer K, et al. Undetectable HCV-RNA in telaprevirtreated patients: low concordance between two highly sensitive real-time PCR assays. Journal of Hepatology 2013;58(Suppl.) [Abstract 506]. [52] Maasoumy B, Cobb B, Halfon P, et al. A clinical trial evaluating low HCV RNA viremia in patients treated with triple therapy regimens: implications for patient management using different assys in clinical pratice. Journal of Hepatology 2013;58 [Abstract 855]. [53] Ferenci P, Asselah T, Foster G, et al. Faldaprevir plus pegylated Interferon alfa-2a and ribavirin in chronic HCV genotype-1 treatment-naive patients: final results from Startverso1, a randomised, double-blind, placebo-controlle phase III trial. Journal of Hepatology 2013;58 [Abstract 1416]. [54] Manns M, Marcellin P, Poordad F. Simeprevir (TMC435) with PegInterferon/Ribavirin for treatment of chronic HCV genotype-1 infection in treatment-naive patients: results from QUEST-2, a phase III trial. Journal of Hepatology 2013;58 [Abstract 1413]. [55] COBAS AmpliPrep/COBAS TaqMan HCV Quantitative Test. Version 2.0: EU Package insert. Doc Rev. 1.0; [56] COBAS AmpliPrep/COBAS TaqMan HCV Test. v2.0: US package insert Doc Rev 1.0; [57] COBAS TaqMan HCV Test v2.0 For Use With The High Pure System. EU package insert. Doc Rev. 9.0; [58] COBAS TaqMan HCV Test v2.0 For Use With The High Pure System. US package insert. Doc Rev. 2.0; [59] Abbott RealTime HCV. EU package insert; [60] Abbott RealTime HCV. US package insert; 2011.