Original article HCV RNA assay sensitivity impacts the management of patients treated with direct-acting antivirals

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1 Antiviral Therapy 2015; 20: (doi: /IMP2810) Original article HCV RNA assay sensitivity impacts the management of patients treated with direct-acting antivirals Gavin Cloherty 1 *, Daniel Cohen 2, Christoph Sarrazin 3, Heiner Wedemeyer 4, Stephane Chevaliez 5,6, Christine Herman 1, Barry Bernstein 2, Jean Michel Pawlotsky 5,6 1 Abbott Molecular, Des Plaines, IL, USA 2 AbbVie, North Chicago, IL, USA 3 Goethe University, Frankfurt, Germany 4 Hannover Medical School, Hannover, Germany 5 National Reference Center for Viral Hepatitis B, C and D, Department of Virology, Henri Mondor Hospital, University of Paris-Est, Créteil, France 6 INSERM U955, Créteil, France *Corresponding author gavin.cloherty@abbott.com Background: Application of response-guided therapy (RGT) rules to the treatment of HCV infection with pegylated interferon-a2a and ribavirin, and direct-acting antivirals (DAAs) such as the NS3/4A protease inhibitors (PIs) boceprevir and telaprevir, relies on the determination of viral genotype and on-treatment HCV RNA level. Currently there are few data available regarding the clinical impact of the analytical differences that exist between different HCV RNA quantification assays on treatment decisions such as those involved in RGT. Methods: We sought to ascertain the concordance between two HCV RNA quantification assays, the Roche/ High-Pure-System COBAS TaqMan (CTM) version 2 and Abbott RealTime HCV (ART), and to understand the impact of different assay characteristics on treatment decisions. We evaluated 1,336 specimens collected from 74 patients enrolled in the Phase II CHAMPION-2 study of the investigational DAAs ABT-450 (an acylsulfonamide NS3/4A PI), ABT-072 and ABT-333 (both non-nucleoside NS5B polymerase inhibitors). Results: HCV RNA level results were highly correlated, but CTM values were higher than those from ART by an average of 0.46 log. Use of ART HCV RNA level results led to a higher positive predictive value of week 4 viral load for the achievement of a sustained virological response 24 weeks after the end of treatment (100% versus 87% using the lower limit of detection as the threshold). Conclusions: This study suggests that HCV viral load assay performance characteristics need to be taken into consideration when managing HCV patients with RGT. Further studies are required to determine whether a consensus HCV RNA level threshold can be established or whether HCV viral load assays with greater sensitivity can increase cure rates with RGT. Introduction HCV is one of the most common blood-borne pathogens in the world and a major source of morbidity globally [1]. The long term impact of HCV infection is highly variable, ranging from minimal effects to chronic hepatitis, advanced fibrosis, cirrhosis and hepatocellular carcinoma [2]. Worldwide it is estimated that million people are chronically infected with HCV, with 3 4 million new infections per year and over 350,000 deaths due to HCV-related liver disease each year [3]. The goal of therapy is to eradicate HCV infection in order to prevent complications associated with HCVrelated liver disease [2]. HCV has been treated for 20 years with interferon-based therapies, such as pegylated interferon-a2a or -2b and ribavirin (PEG-IFN/RBV). IFN-based therapies have substantial side-effect profiles and only moderate efficacy as measured by sustained virological response (SVR; defined as undetectable HCV RNA 24 weeks after cessation of treatment). A personalized approach to the treatment of HCV with PEG-IFN/RBV, known as response-guided therapy (RGT), which relies on the determination of viral genotype and close monitoring of on-treatment viral kinetics to guide treatment duration from weeks, is now the standard of care [4 6]. Although a validated RGT 2015 International Medical Press (print) (online) 177

2 G Cloherty et al. approach may make patient management more complex, it ultimately results in a personalized treatment regimen that is optimized for safety and efficacy [7]. In 2011 two new direct-acting antivirals (DAAs), the NS3/4A protease inhibitors (PIs) boceprevir and telaprevir, were approved for the treatment of HCV genotype 1 infection in treatment-naive patients in combination with PEG-IFN/RBV [8,9]. This triple therapy has become the new standard of care for the treatment of HCV genotype 1 infection. The possibility of using RGT with these two drugs was evaluated in the pivotal clinical trials ADVANCE (for telaprevir/ PEG-IFN/RBV) and SPRINT-2 (for boceprevir/peg- IFN/RBV) in treatment-naive patients. In both of these trials, HCV RNA was quantified and RGT rules were determined using viral load data generated using the manual Roche High-Pure-System/COBAS TaqMan (CTM) assay, version 2 (Roche Molecular, Pleasanton, CA, USA) [10,11]. Other HCV RNA tests are available and each has different analytical performance characteristics [12]. There are currently no data available which demonstrate the clinical implications of these analytical differences on treatment decisions. It is important to ascertain the concordance between different commercially available assays of comparable sensitivity for samples with low-level viraemia, and to understand the impact of different assay characteristics on treatment outcomes. Several new DAAs are currently being evaluated in clinical trials, both with and without PEG-IFN/ RBV [13,14]. ABT-450 is an investigational acylsulfonamide NS3/4A PI identified by AbbVie and Enanta, with nanomolar potency against genotype 1 HCV in subgenomic replicon systems [15]; it is co-dosed with low-dose ritonavir (denoted ABT-450/r), to achieve therapeutic exposures at once-daily dosing frequency. ABT-072 and ABT-333 are both non-nucleoside NS5B polymerase inhibitors [16]. Each of these drugs was studied in combination with PEG-IFN/RBV in the Phase II CHAMPION-2 study [16]. We used specimens collected during this study to investigate the potential for differences in clinical utility between two viral load assays, CTM and Abbott RealTime HCV (ART; Abbott Molecular, Des Plaines, IL, USA), with respect to prediction of therapy outcomes. Methods The Phase II CHAMPION-2 study (ClinicalTrials.gov identifier NTC , M11-602) was a blinded, randomized, placebo-controlled, dose-ranging study of ABT-450/r, ABT-072 or ABT-333 to evaluate safety, tolerability, pharmacokinetics and antiviral activity in treatment-naive subjects with genotype 1 chronic HCV infection [16]. Study drugs were administered at two (ABT-333) or three (ABT-072 and ABT-450/r) different doses as monotherapy for 3 days followed by addition of PEG-IFN and RBV for 12 weeks (8 treatment groups total, 6 to 10 participants per drug/dose, plus 11 placebo subjects). At week 12 the investigational DAAs were discontinued and subjects received PEG-IFN/RBV without a DAA for a total of 24 to 48 weeks, depending on the kinetics of RNA decrease in the first 12 weeks (see below). Clinical trial details and outcomes have been presented elsewhere [15,16]. A total of 2,252 serial plasma samples from the 74 subjects who were enrolled in the study were tested prospectively using both ART and CTM. Each HCV RNA assay was performed according to the manufacturer s instructions. No result was obtained for one or both assays for 31 specimens. Overall concordance, correlation and Bland Altman analyses were performed using all results that were within the dynamic range of quantification of both tests (that is, over the lower limit of quantitation [LLOQ]) using Prism 6 (GraphPad Software, La Jolla, CA, USA). Results from 57 subjects who completed a study visit 24 weeks after the end of treatment (51 subjects received PEG-IFN/RBV+DAA, 6 subjects received PEG-IFN/RBV+ placebo) were included in the assessment of the predictive value of rapid virological response (RVR) and early virological response (EVR), evaluated at weeks 4 and 12, respectively, for achieving SVR. Management of patients during the study based on RVR and EVR were determined using the CTM viral load result and the lower limit of detection (LLOD; 10 ). Subjects who achieved RVR and maintained undetectable viral load at all subsequent visits (extended RVR) were eligible to stop PEG-IFN/RBV on or after week 24. SVR was defined as undetectable (<LLOD) HCV RNA at the last follow-up visit after treatment cessation [17]; in most cases this was 24 weeks post-treatment. Using a less stringent criterion of HCV RNA level below the LLOQ would have resulted in one additional patient meeting an SVR definition; this patient had detectable but unquantifiable HCV RNA according to both viral load assays used. The positive predictive value (PPV) and negative predictive value (NPV) of RVR and EVR, respectively, for achieving SVR were determined using different threshold viral levels (<LLOQ or LLOD). PPV was defined as the number of patients with week 4 HCV RNA level below LLOQ or LLOD who achieved SVR divided by the total number of patients with week 4 viral load below the LLOQ or LLOD. NPV based on week 12 viral load was defined as the number of patients with detectable or quantifiable viral load who failed to achieve SVR divided by the total number of patients with detectable or quantifiable viral load at week International Medical Press

3 HCV RNA assay sensitivity impacts DAA-treated patient management Results A total of 2,221 paired HCV RNA level results from the 74 patients enrolled in the study were obtained; 764 (34.4%) were <LLOD for both assays, 54 (2.4%) were <LLOD for CTM but >LLOD for ART, 16 (0.7%) were <LLOD for ART but >LLOD for CTM, and 1,387 (62.4%) were >LLOD for both assays. There were 1,336 specimens with detectable HCV RNA results that were within the dynamic range for both tests; significant correlation between the two measurements was observed (R 2 =0.98; Figure 1). However, HCV RNA levels from the CTM assay tended to be higher than the corresponding result from ART by an average of 0.46 log 10 (95% CI 0.03, 0.89 log 10 ; Figure 2). The CTM value was higher than ART in 98% of comparisons. In 38% of comparisons, the CTM result was greater than 0.5 log 10 higher than ART. There was a tendency for the difference between results (CTM-ART) to be higher when viral loads were high compared to when they were low: 65% of specimens with a difference below the lower limit of the 95% CI had average viral loads below 4 log 10, whereas only 17% of those with a difference over the upper limit did (Figure 2). Clinical treatment decisions are usually made based on the week 4 or week 12 HCV RNA level result being above or below a specific threshold, with or without respect to the assay used. We evaluated the concordance of HCV RNA results generated by the ART and CTM assays using assay-specific LLOD or LLOQ cutoffs as well as a consensus threshold of 25 (Tables 1, 2 and 3). Using the LLOD as the threshold, amongst all patients with a viral load result, at week 4 and 12, the Figure 1. Correlation between viral load results measured with ART or CTM R 2 =0.98 n=1,336 ART viral load, log CTM viral load, log 10 All pairs of results in the dynamic range (> limit of quantification) are shown. The regression line is shown as a solid diagonal line. Theoretical identity is shown as a dashed line. ART, Abbott RealTime HCV assay; CTM, Roche/High-Pure-System COBAS TaqMan assay. Antiviral Therapy

4 G Cloherty et al. Figure 2. Bland Altmann plot comparing viral load results measured with ART or CTM Difference (CTM-ART), log Average viral load, log 10 The average bias (0.46 log 10 ) is shown as a dotted line and the 95% limits of agreement (0.029, 0.89 log 10 ) as thin dashed lines. ART, Abbott RealTime HCV assay; CTM, Roche/High-Pure-System COBAS TaqMan assay. Table 1. HCV RNA assay concordance by LOD as cutoff Week 4 (concordance 87%) a Week 12 (concordance 92%) b CTM<LOD CTM>LOD CTM<LOD CTM>LOD ART<LOD ART>LOD Values are n. a n=69. b n=59. ART, Abbott RealTime HCV assay; CTM, Roche/High- Pure-System COBAS TaqMan assay; LOD, limit of detection. Table 2. HCV RNA assay concordance by LOQ as cutoff Week 4 (concordance 93%) a Week 12 (concordance 97%) b CTM<LOQ CTM>LOQ CTM<LOQ CTM>LOQ ART<LOQ ART>LOQ Values are n. a n=69. b n=59. ART, Abbott RealTime HCV assay; CTM, Roche/High- Pure-System COBAS TaqMan assay; LOQ, limit of quantification. level of concordance based on RGT decision criteria was 87% (60 of 69) and 92% (54 of 59). Using the LLOQ as the threshold, at week 4 and 12, the level of concordance based on RGT decision criteria increased to 93% (64 of 69) and 97% (57 of 59). Using 25 as the threshold, at week 4 and 12, had no incremental impact on the level of concordance based on RGT decision criteria, 91% (63 of 69) and 97% (57 of 59), respectively. Of the 57 subjects who completed the trial (51 of whom received a DAA), 38 achieved SVR (37 DAA-treated). The number of patients achieving SVR was the same using either HCV RNA quantification assay. The PPV of RVR, defined using viral load below each assay s LLOD, LLOQ or 25 at week 4, was evaluated based on viral load results from each assay (Table 4). Using the LLOD as the threshold to define RVR, 20 of the 23 patients with undetectable HCV RNA by CTM achieved SVR (PPV 87%); for ART, all 15 RVR patients with undetectable HCV RNA achieved International Medical Press

5 HCV RNA assay sensitivity impacts DAA-treated patient management Table 3. HCV RNA assay concordance using 25 as cutoff SVR (PPV 100%). Using the LLOQ as the threshold (<25 for CTM, <12 for ART), 28 of the 31 patients with an RVR based on CTM also reached SVR (PPV 90%) whereas 27 of the 29 patients with week 4 HCV RNA undetectable or detectable but <LLOD using ART also achieved SVR (PPV 93%). Using a threshold of 25 for both assays, 28 of the 31 patients with an RVR based on CTM also reached SVR (PPV 90%) whereas 29 of the 32 patients with week 4 HCV RNA<25 using ART also achieved SVR (PPV 91%). The NPV of not having an EVR (viral load above each assay s LLOD, LLOQ or 25 at week 12) was also evaluated based on viral load results from each assay (Table 4). Using the LLOD as the threshold, 10 of the 11 patients failing to reach EVR according to CTM results also did not achieve SVR (NPV 91%); for ART, 11 of the 13 non-evr (HCV RNA detectable) patients failed to reach SVR (NPV 85%). Using the LLOQ as the threshold ( 25 for CTM, 12 for ART), all of the nine patients not reaching EVR based on CTM also failed to achieve SVR (NPV 100%), while for ART all seven patients not reaching HCV RNA<LLOQ also failed to reach SVR (NPV 100%). The outcomes of individual patients with discordant viral load results at week 4 or 12 are informative to consider. A total of eight patients had week 4 HCV RNA detectable by ART but not CTM; three of these patients did not achieve SVR. Two of these three patients received shortened duration of therapy with the DAA (ABT-450/r in both cases) based on the undetectable CTM result. At this same time point, there were no patients with HCV RNA detectable by CTM but not ART. At week 12, three patients had HCV RNA detectable by ART but not CTM; two of them achieved SVR. One patient had HCV RNA detectable by CTM but not ART; this patient achieved SVR. Discussion Week 4 (concordance 91%) a Week 12 (concordance 97%) b CTM CTM CTM CTM <25 >25 <25 >25 ART< ART> Values are n. a n=69. b n=59. ART, Abbott RealTime HCV assay; CTM, Roche/High- Pure-System COBAS TaqMan assay. Based on parallel testing of over 1,300 specimens from 74 patients, we observed an average difference of 0.46 log 10 between the CTM and ART, with CTM Table 4. PPV and NPV for SVR using different viral load cutoffs Week 4 PPV a (n=56) Week 12 NPV b (n=49) <LOD <LOQ <25 >LOD >LOQ >25 CTM 20/23 28/31 28/31 10/11 9/9 9/9 (87%) (90%) (90%) (91%) (100%) (100%) ART 15/15 27/29 29/32 11/13 7/7 7/7 (100%) (93%) (91%) (85%) (100%) (100%) a For week 4 positive predictive value (PPV), values are number of patients with viral load below the cutoff with sustained virological response (SVR; numerator) versus total number with viral load below the cutoff (denominator). b For week 12 negative predictive value (NPV), values are number of patients with viral load above the cutoff without SVR (numerator) versus total number with viral load above the cutoff (denominator). ART, Abbott RealTime HCV assay; CTM, Roche/High-Pure-System COBAS TaqMan assay; LOD, limit of detection; LOQ, limit of quantification. reporting the higher result in 98% of comparisons, as previously reported [18 25]. This observed difference could have a meaningful impact on RGT, which relies on specific quantitative thresholds close to LLOD or LLOQ. Interestingly, while the majority of CTM results were higher than ART in all viral load ranges, the number of specimens with higher ART viral load results (that is, difference versus CTM<0) was relatively greater at low viral loads (<10,000 ) than at higher viral loads (Figure 2). This overestimation of viral loads by CTM at the upper end and underestimation at the low end of the dynamic range is in keeping with the conclusions made by Chevaliez et al. [26], who proposed that this might be the effect of an interaction with an unidentified blood component which was less evident at the low end of the linear range of the CTM assay. It should also be noted that in this study the ART test is a highly automated method and the CTM method used manual nucleic acid extraction which may introduce some variability to the results, particularly at the low end. CTM uses an internal quantitation standard (IQS) to calibrate viral load results. The IQS cannot adjust for variances at both the upper and lower end of the dynamic range. Other studies have described that CTM results are not linear across the entire dynamic range and require the use of a third order polynomial linear regression (y=ax 3 +bx 2 +cx) with ±0.2 log 10 allowable maximum difference from linearity [27,28]. In contrast, the ART assay employs a simple linear function (y=ax+b) using a two point external calibration strategy to quantitate viral load. The ART assay has been shown in studies to be linear across the entire dynamic range of the test. Therefore, it is possible that a contributing factor to the observed discordance is the calibration strategy employed by each assay. Using the assay LLOD as the threshold, the ART method had a higher PPV at week 4 than CTM (100% versus 87%) and identified three Antiviral Therapy

6 G Cloherty et al. CTM-negative, ART-positive subjects who ultimately failed to achieve an SVR at week 24. Thus, had ART been used to prospectively inform RGT, 2/3 of these patients would have been maintained on PEG-IFN/ RBV for a full 48 weeks, potentially providing them with a higher likelihood of achieving a SVR. With different thresholds, such as the assay-specific LLOQ or 25, the differences in PPV between assays are fewer. There was very little or no difference in NPV based on week 12 viral load results. This supports the use of a common threshold for decisionmaking across clinical studies. This study, if confirmed with larger numbers of patients, suggests that performance differences between HCV viral load assays need to be taken into consideration when managing HCV patients with RGT. If a threshold higher than the LLOD is used, such as the LLOQ or a consensus threshold such as 25, some subjects who may benefit from extended therapy may not receive it. Further studies are required to determine whether a consensus HCV viral load threshold can be established, whether these findings translate to IFN-free DAA regimens and/or whether HCV viral load assays with greater sensitivity can increase cure rates with RGT. Acknowledgements We would like to thank the patients and investigators involved in CHAMPION-2 (NTC ); for their participation in the trial and Neil Parkin, Data First Consulting, Inc. (Menlo Park, CA, USA), for assistance with manuscript preparation. Disclosure statement GC and CH are employees of Abbott Molecular; DC and BB are employees of AbbVie. HW has received honoraria for consulting or speaking engagements from Abbott, AbbVie, Biolex, Bristol Myers Squibb, Boehringer Ingelheim, Eiger Pharmaceuticals, Falk Foundation, Gilead Sciences, ITS, JJ/Janssen-Cilag/ JanssenTE, Medgenics, Merck/Schering Plough, Novartis, Novira, Roche, Roche Diagnostics, Siemens, Transgene and Viiv. 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