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How Should TMA Be Used-- Hepatitis C minimal residual viremia (MRV) detected by TMA at the end of Peg-IFN plus ribavirin therapy predicts post-treatment relapse
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Journal of Hepatology Jan 2006
Martina Gerottoa, Francesca Dal Peroa, Gladis Bortolettoab, Alessia Ferrarib, Roberta Pistisb, Giada Sebastianib, Stefano Fagiuolic, Stefano Realdonac, Alfredo Albertiab
a VIMM-Venetian Institute of Molecular Medicine, via Orus 2, Padova, Italy
b Department of Clinical and Experimental Medicine, University of Padova, Via Giustiniani 2, 35129 Padova, Italy
c Department of Gastroenterology, University of Padova, Padova, Italy
NOTE from Jules Levin: abstract for an oral presentation at AASLD Nov 2005 on the utility of TMA follows this published study below)
Hepatitis C virus (HCV) infection is the main cause of chronic liver disease, cirrhosis and liver cancer in the Western word [1]. Interferon alpha (IFN-a) in combination with ribavirin is the treatment of choice for both naive patients and for patients who have previously failed to respond to interferon monotherapy. The primary goal of therapy is to reach sustained virological response defined as undetectable HCV RNA 24 weeks after the end of therapy. The recent introduction of pegylated forms of interferon (Peg-IFN) has significantly improved the success of therapy and sustained virological response can now be achieved in 42-51% of patients with genotypes 1 and 4 and in up to 70-90% of patients with genotypes 2 or 3 [2-5]. Nevertheless, despite the increased rates of sustained virological responders, still from 15 to 25% of the cases who have become HCV-RNA negative by conventional PCR during therapy, experience viral relapse after treatment withdrawal. The exact mechanism at the basis of these virological relapses has still to be defined and there are no identified on-treatment markers able to predict whether the patient will develop a sustained virological response or will relapse. Such a marker would be undoubtfully most useful for prognostic assessment at the end of therapy as well as for identifying patients who may need further prolongation of treatment. Recently, it has been reported that low levels of viremia, undetectable by conventional PCR methods, might be identified at the end of therapy by transcription-mediated amplification assay in patients who relapse after therapy withdrawal. These data were obtained by retrospective analysis of selected groups of patients treated with conventional, not pegylated interferon used alone or with ribavirin and, more recently in patients treated with Peg-IFN monotherapy [6-9]. We have here analyzed prospectively a large cohort of consecutive patients treated for chronic hepatitis C with a standard schedule of Peg-IFN and ribavirin with the aim to assess whether minimal residual viremia (MRV) detected by TMA at the end of therapy in PCR negative cases could reliably predict relapse after therapy withdrawal.
".....12.5% of patients with undetectable HCV RNA by PCR at the End of Treatment had MRV (minimal residual viremia) using the sensitive TMA test......TMA could identify a subgroup of patients who might benefit from a more prolonged treatment period...... Minimal residual viremia detected by TMA was highly predictive of post-treatment relapse that, overall was seen in 25/26 (96%) TMA positive patients compared to only 14% of the TMA negative patients.... Moreover, ET-MRV was detected mainly in HCV-1 patients treated with Peg-IFN plus ribavirin for 48 weeks and in HCV-3 infected cases treated for 24 weeks, while was extremely rare in HCV-2 patients treated for 24 weeks. Recent studies have shown that treatment for 48 or 24 weeks might be sub-optimal for a subgroup of patients with HCV-1 or HCV-3, respectively. Longer duration of therapy in patients showing end-of-therapy MRV might, therefore, be worthwhile to be explored....
Around 15-25% of chronic hepatitis C patients treated with Peg-IFN plus ribavirin become HCV-RNA negative by PCR during therapy but relapse after its withdrawal. We investigated whether minimal residual viremia (MRV) might be detected in these cases by Transcription-Mediated Amplification (TMA).
ABSTRACT
Methods: Two hundred and ninety-two consecutive patients (143 HCV-1, 82 HCV-2, 56 HCV-3 and 11 HCV-4) were prospectively treated with a standard schedule of Peg-IFNa 2b plus ribavirin combination and end-of-therapy response was assessed by conventional PCR using 2 protocol serum samples obtained 6-8h before the last two scheduled weekly injections of Peg-IFN. PCR negative samples were re-tested by TMA and the results were then correlated with the virological outcome after therapy withdrawal.
Results: Among 208 patients who were repeatedly HCV-RNA negative by PCR at the end-of-therapy, 26 (12.5%) were found HCV-RNA positive by TMA. Twenty-two of them, (96%) were PCR-relapsers after therapy withdrawal, compared to only 14% of the 182 TMA negative patients (P<0.0001). This virological profile was more frequent in HCV-1 and HCV-3 infected patients and correlated with a slower virological response during therapy.
Conclusions: At the end of Peg-IFN plus ribavirin therapy, TMA is superior to PCR in identifying patients with sustained HCV-RNA clearance.
Discussion
The rate of successful HCV eradication in chronically infected patients has significantly increased following the introduction of Peg-IFN plus Ribavirin combination therapy. Nevertheless, there are still patients who become HCV-RNA negative by PCR during and at the end of therapy but then show virological and biochemical relapse soon after treatment withdrawal. Such virological relapse was observed in 25% of our 208 consecutive patients who had been treated with standard doses of Peg-IFNa 2b plus ribavirin and were found HCV-RNA negative by PCR at the end of therapy. When the PCR negative samples obtained from these patients at the end of therapy were tested by TMA, minimal residual viremia (MRV) could be detected in 12.5% of them. MRV was highly predictive of virological and biochemical relapse after therapy that was observed in 96% of ET-TMA positive patients compared to only 14% of ET-TMA negative cases. Thus, TMA had an excellent positive predictive value (PPV=96%) and specificity (99%) for virological relapse while its negative predictive value (NPV) was 86% and the sensitivity only around 50%, due to the presence of a number of patients who relapsed despite being TMA negative at the end of treatment. This finding suggest that even with a highly sensitive technique for serum HCV-RNA such as the TMA assay, MRV may be undetectable while most likely present, indicating the need for more sensitive detection methods, as also reported in patients treated with Peg-IFNa 2a monotherapy [8]. Nevertheless, our study, represents the first exploring the prognostic value of ET-MRV detected by TMA with a prospective design in patients treated with Peg-IFN plus ribavirin and, supports the clinical utility of the TMA assay in identification of patients who will relapse after treatment withdrawal. These conclusions are in agreement with the results obtained previously in smaller series of patients treated with standard interferon [6,7]. In our study, MRV detected by TMA at end of therapy was associated with slower virological response during the early phase of treatment suggesting that ET-MRV could identify a subgroup of patients who might benefit from a more prolonged treatment period. Moreover, ET-MRV was detected mainly in HCV-1 patients treated with Peg-IFN plus ribavirin for 48 weeks and in HCV-3 infected cases treated for 24 weeks, while was extremely rare in HCV-2 patients treated for 24 weeks. Recent studies [14-17] have shown that treatment for 48 or 24 weeks might be sub-optimal for a subgroup of patients with HCV-1 or HCV-3, respectively. Longer duration of therapy in patients showing end-of-therapy MRV might, therefore, be worthwhile to be explored. Since, the conclusion of this study we have given three additional months of Peg-IFN plus ribavirin treatment to ten patients (all HCV-1) showing MRV by TMA after 48 weeks of treatment. In only one of them negativization of TMA was achieved while the remaining nine patients remained positive up to week 60 and all relapsed after therapy withdrawal (unpublished). These results would suggest that either three additional months were insufficient to achieve complete eradication of HCV or MRV could reflect virus stains or/and virus compartmentalization that are particularly resistant to the effects of Peg-IFN plus ribavirin combination therapy. Molecular characterization of MRV-associated HCV is ongoing in our unit and will possibly help us clarifying these issues.
In conclusions, our findings obtained with a prospective analysis, confirm that the TMA assay performed with a standardized sampling protocol at the end Peg-IFNa 2b plus ribavirin combination therapy is superior to conventional PCR for the correct identification of patients who have cleared HCV during therapy.
Results
End of therapy response (ETR) by qualitative PCR
Of the 292 patients included in this study 68 (23%) stopped therapy prematurely due to either non-response or to adverse events. Of the remaining 224 patients who completed the full-scheduled period of treatment, 208 (93%) were HCV RNA negative by PCR at the end of therapy (71.2% by intent-to-treat analysis). All these patients were PCR negative in both serum samples tested 1 week apart and included 86 out of 143 (60%) HCV-1 infected patients, 72 out of 82 (88%) with HCV-2, 45 out of 56 (80%) with HCV-3, five out of 11 (45%) with HCV-4. HCV-RNA was negative by PCR at the end of therapy in 150 out of 211 (71%) patients who were receiving their first course of antiviral therapy and in 58 out of 81 (72%) of those who were re-treated. When these 208 PCR negative patients were tested by TMA, 182 (88%) were confirmed negative in both samples also by TMA while the remaining 26 were TMA positive in at least one of the two samples, including 18/143 (13%) HCV-1, 2/82 (2%) HCV-2 and, 6/55 (11%) HCV-3 infected patients. A PCR negative/TMA positive profile at the end of therapy was more frequently seen in re-treated (14/81; 17%) compared to naive (12/211; 6%) patients (P=0.0005).
End of therapy comparison of PCR negative/TMA positive versus PCR negative/TMA negative patients
Table 2 describes the comparison between the 182 patients who were PCR negative/TMA negative (ETR TMA neg) and the 26 who were PCR negative/TMA positive (ETR TMA pos) at the end of therapy. A positive TMA test at the end of therapy was significantly more frequent in patients with HCV-1, in those receiving re-treatment and in the presence of a slow virologic response during therapy as testified by an HCV-RNA test still positive at 12 weeks. On the other hand, infection with HCV-2 was associated with a significantly reduced probability of being TMA positive at the end of therapy.
Minimal residual viremia detected by TMA was highly predictive of post-treatment relapse that, overall was seen in 25/26 (96%) TMA positive patients compared to only 14% of the TMA negative patients. This difference was independent of HCV genotype. As shown in Table 3, in 19 of the 26 patients with MRV at the end of therapy the virological relapse after therapy was associated with biochemical relapse while in the remaining seven patients ALT were still normal 6 months after therapy withdrawal.
Patients and methods
Patients and study design
Two hundred and ninety two consecutive patients with chronic hepatitis C treated with Peg-IFNa 2b plus ribavirin combination therapy were included. Informed consent to participate was obtained from each patient and the study protocol was conform to the ethical guidelines of the 1975 Declaration of Helsinki. The baseline patients characteristics are described in Table 1. All cases received standardized doses of Peg-IFNa 2b (1.5mg/kg/week) and of ribavirin (1000-1200mg/daily according to body weight). Patients infected by HCV-1 or HCV-4 were assessed for virological response after 12 weeks of treatment and continue on therapy only if a ≥2 log reduction in viremia level had occurred [10]. These patients were treated for 48 weeks while, therapy was stopped at 12 weeks in non-responders. Patients with HCV-2 or HCV-3 were treated for 24 weeks independently of the early virological response. The Peg-IFNa 2b and ribavirin doses were reduced during treatment whenever needed for side effects, and these dose reductions were done according to the labeling. All patients with an initial response who completed the assigned treatment period were tested for HCV-RNA at the end of therapy (week 48 for genotypes 1 and 4; week 24 for genotypes 2 and 3) to define the virological response at these time-points (ETR=end of therapy response). For this purpose, two separate serum samples obtained 6-8h before the last two scheduled weekly injections of Peg-IFN were tested by qualitative PCR (Cobas Amplicor HCV RNA test-Roche Molecular System, Branchburg, NJ, USA) with a limit of detection of 50-100IU/mL. Samples found negative by PCR were tested for minimal residual viremia (MRV) using the Versant HCV RNA Qualitative Assay (TMA) (Bayer Health Care, Diagnostics Division, Tarrytown, NY, USA) as described below. Therapy was then stopped in all patients at the scheduled time point (week 48 for HCV.1 and HCV-4; week 24 for HCV-2 and HCV-3) independently of the PCR and TMA findings.
Post treatment follow-up consisted of a minimum of 6 months. After this period, patients were tested for HCV-RNA by qualitative PCR (Cobas Amplicor HCV RNA test-Roche Molecular System) to defined sustained virologic response (SVR) according to standard criteria [10].
HCV RNA testing
HCV RNA was measured quantitatively at baseline and at week 12 by Versant HCV RNA 3.0 (Versant HCV RNA 3.0 Assay (bDNA), Bayer Diagnostics, Emeryville, Calif.), and was also tested qualitatively at week 12, 24, 48 during treatment and again 6 months after therapy withdrawal by PCR (COBAS Amplicor HCV RNA test v. 2.0; Roche Molecular Systems). End-of-therapy specimens that were found HCV RNA negative by PCR were analyzed using the TMA assay following the manufacturer's instructions. Briefly, 500ml of serum were added to 400ml of lysis buffer containing an RNA internal control (IC) and magnetic beads linked to oligonucleotides to which the 5'UTR of the HCV RNA and the IC can bind. This specific binding to the beads allows to concentrate the sample for the following amplification step. During this phase RNA amplicons are synthesized in an isothermal reaction. Amplification products were then revealed by a luminometer after adding to the sample a mix of hybridization probes having sequence and fluorescent dye specific either for the HCV RNA or the IC. The TMA assay has been validated to have a sensitivity of 5-10IU/ml or 25-50copies/ml [11,12], whereas the detection limit of the Roche Cobas Amplicor HCV version 2.0 is 50-100IU/ml [13].
Statistical analysis
All analyses to determine the statistical significance of the estimated differences, expressed as percentage, identified in patients with different treatment outcome were calculated by Fischer exact probability test. P is significant when < 0.05.
AASLD Nov 2005 Abstract
UTILITY OF TMA TESTING DURING ANTIVIRAL TREATMENT OF ADVANCED HEPATITIS C
Chihiro Morishima, University of Washington, Seattle, WA; Timothy R.
Morgan, University of California-Irvine, VA Medical Center, Long
Beach, CA; David R. Gretch, University of Washington, Seattle, WA;
Elizabeth C. Wright, New England Research Institutes, Watertown,
MA; James E. Everhart, National Institute of Diabetes and Digestive
and Kidney Diseases, Bethesda, MD; for the HALT-C Trial Group,
University of Washington, Seattle, WA
Background: Qualitative HCV RNA assays with improved analytical sensitivity are available, but their added clinical benefit is not clear. The Bayer VERSANT HCV RNA Qualitative (TMA) Assay* (lower limit of detection (LOD) 10 IU/mL) was evaluated for its ability to predict sustained virologic response (SVR) using stored serum from the HALT-C trial.
Methods: Patients with chronic hepatitis C, prior non-response to interferon, and advanced liver disease were treated with peginterferon alfa-2a and ribavirin for 24 weeks.
Patients with undetectable HCV RNA using the Roche COBAS Amplicor HCV Test, v. 2.0 assay (LOD 100 IU/mL in serum) at week 20 (W20) continued therapy for a total of 48 weeks and were assessed for SVR (absence of serum HCV RNA by the Amplicor test at W72).
Serum aliquots from 1070 subjects at W20 and in fewer numbers at other weeks were tested by TMA.
Results: 1044 of 1045 Amplicor-positive samples from different timepoints were also positive by TMA. The single discrepant sample had additional testing that indicated HCV RNA negativity. Among 1293 Amplicor-negative samples, 279 (21.6%) were TMA-positive, with the rate of TMA positivity decreasing during therapy.
The majority of subjects with Amplicor-negative/ TMA-positive discordant results subsequently developed treatment breakthrough or relapse (defined using Amplicor results):
84% from W20, 87% from W24, and 89% from W48.
Patients who were TMA-negative at W12, W20, or both were more likely to
achieve SVR (74%, 62% and 82%, respectively) than were patients
who were Amplicor-negative at these timepoints (58%, 48%, 61%,
respectively).
The added benefit of TMA testing to the early virologic response (EVR, defined as at least a 2 log drop of HCV RNA level at W12) was evaluated in subjects with EVR who were Amplicor-negative at W20 and received 48 weeks of treatment. Of 48 patients with two consecutive TMA-positive results at W20 and W24, none achieved SVR (95% CI: 0-7.4%). By contrast, 67% of subjects with consecutive TMA-negative results at W20 and W24
did achieve SVR.
Conclusions: Among patients treated with combination therapy for chronic hepatitis C, the TMA test detects HCV RNA in all specimens that are Amplicor-positive, as well as an additional 21.6% that are Amplicor-negative. The increased sensitivity of the TMA test can be helpful in identifying patients with low levels of HCV RNA who are likely to relapse when therapy is stopped. Furthermore, among patients with EVR and a negative Amplicor test at W20, persistent detection of HCV with the TMA test during therapy predicts failure to achieve SVR.
*FDA-approved for detection of HCV RNA as evidence of active
infection.
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