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FW: NATAP: Sensitive TMA HCV RNA Test-what is its role?

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  • alleypat
    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
    Message 1 of 1 , Dec 27, 2005
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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

      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-N1) in combination with ribavirin is the treatment of choice for
      both naC/ve 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.

      bb&..12.5% of patients with undetectable HCV RNA by PCR at the End of
      Treatment had MRV (minimal residual viremia) using the sensitive TMA
      testb&b&TMA could identify a subgroup of patients who might benefit from a
      more prolonged treatment periodb&b& 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
      patientsb&. 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 exploredb&.


      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-IFNN1 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-IFNN1 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-IFNN1 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-IFNN1 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 naC/ve (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-IFNN1 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-IFNN1 2b (1.5N<g/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 b %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-IFNN1 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 (VersantB. 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, 500N<l of
      serum were added to 400N<l of lysis buffer containing an RNA internal
      control (IC) and magnetic beads linked to oligonucleotides to which the
      5b2UTR 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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