Understanding Optimal Use and Interpretation of Assays in HCV This program is supported by educational grants from

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clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV Faculty Affiliation and Disclosure Paul Y. Kwo, MD Professor of Medicine Medical Director of Transplantation Division of Medicine/Gastroenterology/Hepatology Indiana University School of Medicine Indianapolis, Indiana Paul Y. Kwo, MD, has disclosed that he has received consulting fees from Abbott, Anadys, Boehringer Ingelheim, Bristol-Myers Squibb, Gilead Sciences, Johnson & Johnson, Merck, Novartis, and Vertex; has received fees for non-CME services from Bristol- Myers Squibb, Gilead Sciences, Merck, and Roche; and has contracted research with Abbott, Anadys, Bayer, Bristol-Myers Squibb, Conatus, GlaxoSmithKline, Merck, Novartis, Roche, and Vertex.

New Standard of Care for Patients With HCV

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV Patterns of Virologic Response Wks After Start of Therapy HCV RNA (log 10 IU/mL) [1] Undetectable RVREVREOTSVR Relapse Partial response Null response 1. Ghany MG, et al. Hepatology. 2009;49: McHutchison JG, et al. N Engl J Med. 2009;361: % chance of SVR with pegIFN/RBV [2]

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV Addition of TVR or BOC to PegIFN/RBV Improves SVR in Genotype 1 Patients  HCV NS3/4A protease inhibitors BOC and TVR approved by FDA, May 2011 [1,2] –Indicated in combination with pegIFN/RBV for treatment of genotype 1 HCV–infected patients who are previously untreated or who have failed previous therapy 1. Boceprevir [package insert]. May Telaprevir [package insert]. May Poordad F, et al. N Engl J Med. 2011;364: Jacobson IM, et al. N Engl J Med. 2011;364: Bacon BR, et al. N Engl J Med. 2011;364: Zeuzem S, et al. N Engl J Med. 2011;364: Vierling J, et al. AASLD Abstract SVR (%) Relapsers [5,6] Partial Responders [5,6] PegIFN + RBV Null Responders [6,7] BOC/TVR + pegIFN + RBV Treatment Naive [3,4]

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV Proper Use of HCV Assays Essential For Successful Management With HCV PIs  HCV RNA level important throughout treatment to determine –Eligibility for shortened therapy (response-guided therapy) –Discontinuation of therapy due to futility –Minimizes risk of resistance and unnecessary adverse events –Assessment of EOT response –Assessment of SVR  Additional genetic testing may help predict response to treatment

HCV RNA Assays in the Protease Inhibitor Era

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV Key Challenges Regarding Use of HCV RNA Assays in Protease Inhibitor Era  Package inserts for BOC and TVR specify different time points for monitoring HCV RNA  Available HCV RNA assays in practice have different quantifiable ranges  Different HCV RNA thresholds used for RGT determination vs SVR  Different HCV RNA thresholds used for defining treatment futility with BOC vs TVR

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV HCV RNA Assays: LLOD Is Distinct From LLOQ  LLOQ –Lowest HCV RNA concentration within linear range of assay –ie, smallest amount of HCV RNA that can be not only detected but also accurately quantified  LLOD –Lowest amount of HCV RNA concentration that can be detected with 95% probability to determine presence or absence  Commercially available quantitative assays may have differing LLOQ and LLOD levels

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV Adapted from Naeger LK, et al. Intl Workshop on Clinical Pharmacology of Hepatitis Therapy Abstract R-8. HCV RNA Levels and Relationship to LLOD and LLOQ Time Detectable/not quantifiable Viral RNA Titer SVR LLOQ LOD Detectable/ quantifiable Not quantifiable ± detectable Undetectable Goal of anti- HCV therapy HCV Treatment

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV FDA-Approved Qualitative HCV RNA Assays Assay (Manufacturer )MethodLLOD, IU/mLSetting Amplicor HCV v2.0 (Roche Molecular Systems) Manual RT-PCR50 Diagnosis and monitoring Cobas Amplicor HCV v2.0 (Roche Molecular Systems) Semiautomated RT-PCR50 Diagnosis and monitoring Ampliscreen (Roche Molecular Systems) Semiautomated RT-PCR< 50 Blood screening Versant HCV RNA Qualitative Assay (Siemens Healthcare Diagnostics) Semiautomated TMA10 Diagnosis and monitoring Procleix HIV-1/HCV Assay (Chiron Corporation) Manual TMA< 50 Blood screening Ghany MG, et al. Hepatology. 2009;49:  All assays report HCV RNA as detected/not detected

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV Quantitative HCV RNA Assays Assay (Manufacturer) [1] MethodDynamic Range, IU/mL (LLOQ-ULOQ) LLOD, IU/mL LLOQ = LLOD FDA Approved Amplicor HCV Monitor (Roche Molecular Systems) Manual RT-PCR ,000N/A Yes Cobas Amplicor HCV Monitor V2.0 (Roche Molecular Systems) Semiautomated RT-PCR , Yes Versant HCV RNA 3.0 Assay (bDNA) (Siemens Health Care Diagnostics) Semiautomated bDNA signal amplification 615-7,700, Yes LCx HCV RNA-Quantitative Assay (Abbott Diagnostics) Semiautomated RT-PCR25-2,630,00023 No SuperQuant (National Genetics Institute) Semiautomated RT-PCR30-1,470,00030 Yes No Cobas TaqMan HCV Test (Roche Molecular Systems) Semiautomated RT-PCR 43-69,000,00018 No Yes COBAS TaqMan HCV Test v2.0 for use with High Pure System (Roche Molecular Systems) Semiautomated RT-PCR ,000,00015 No Yes Abbott RealTime HCV Assay (Abbott Diagnostics) Semiautomated RT-PCR12-100,000,00012 Yes  Note that quantitative assays may have differing or identical LLOQ and LLOD levels  Phase III registration trials for both BOC and TVR used COBAS TaqMan HCV Test v2.0 for use with High Pure System (1.3% false-positive rate) [2] 1. Ghany MG, et al. Hepatology. 2009;49: Naeger LK, et al. Intl Workshop on Clinical Pharmacology of Hepatitis Therapy Abstract R-8.

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV SVR Rate by HCV RNA Status for BOC and TVR BOC/PR RGTT12/PR  SVR rate lower when HCV RNA not undetectable at key time points during therapy SVR (%) SVR (%) Undetectable Detectable/Below LLOQ Above LLOQ (> 25 IU/mL) Treatment Wk Naeger LK, et al. Intl Workshop on Clinical Pharmacology of Hepatitis Therapy Abstract R-8. Treatment Wk

Using HCV RNA Assays in Clinical Practice

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV / 314 Predictive Value of Baseline HCV RNA for Achieving SVR SVR (%) / / 82 > 800,000 IU/mL ≤ 800,000 IU/mL ADVANCE (TVR) [1] 1. Jacobson IM, et al. N Engl J Med. 2011;364: Poordad F, et al. N Engl J Med. 2011;364: SVR (%) SPRINT-2 (BOC) [2] / 54 45/ / 313 BOC/PR48 BOC/PR RGTT12PR arm n/N = ≥ 800,000 IU/mL < 800,000 IU/mL

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV Patients Responding Early Can Achieve High SVR Rates With Shortened Therapy  Response-guided therapy: patients who achieve optimal virologic response at early time points can receive abbreviated therapy without reducing their chance of SVR  Patients eligible for RGT –Boceprevir: noncirrhotic treatment-naive patients, previous relapsers, and previous partial responders [1,2] –RGT criterion: Must achieve undetectable HCV RNA at Wk 8 (ie, Wk 4 of triple therapy) and maintain it at Wk 24 –Telaprevir: noncirrhotic treatment-naive patients and previous relapsers* [2,3] –RGT criterion: Must achieve undetectable HCV RNA at Wk 4 of triple therapy and maintain it at Wk Boceprevir [package insert]. May Ghany MG, et al. Hepatology. 2011;54: Telaprevir [package insert]. May *AASLD guidelines state that RGT may be considered with TVR in previous partial responders.

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV Response-Guided Therapy Paradigm With BOC + PegIFN/RBV in Tx-Naive Patients BOC + PegIFN +  RBV PegIFN + RBV Early response stop at Wk 28; f/u 24 wks HCV RNA Undetectable Undetectable PegIFN + RBV 8 36 BOC + PegIFN +  RBV 24 HCV RNA Detectable Undetectable Slow response extend triple therapy to Wk 36; PR to Wk 48; f/u 24 wks < 100 IU/mL  Indicated for all noncirrhotic treatment-naive patients Boceprevir [package insert]. May Ghany MG, et al. Hepatology. 2011;54:

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV Response-Guided Therapy Paradigm With BOC + PegIFN/RBV in Tx-Exp Patients BOC + PegIFN +  RBV PegIFN + RBV HCV RNA Undetectable Undetectable PegIFN + RBV 8 36 BOC + PegIFN +  RBV 24 HCV RNA Detectable Undetectable < 100 IU/mL  Indicated for noncirrhotic previous relapsers or partial responders Boceprevir [package insert]. May Ghany MG, et al. Hepatology. 2011;54: Early response stop at Wk 36; f/u 24 wks Slow response PR to Wk 48; f/u 24 wks

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV Response-Guided Therapy Paradigm With TVR + PegIFN/RBV in Tx-Naive Patients TVR + PegIFN + RBV eRVR stop at Wk 24, f/u 24 wks PegIFN + RBV TVR + PegIFN + RBV PegIFN + RBV HCV RNA Undetectable Detectable (≤ 1000 IU/mL) Undetectable or detectable (≤ 1000 IU/mL) No eRVR extend pegIFN + RBV to Wk 48; f/u 24 wks HCV RNA  Indicated for all noncirrhotic treatment-naive patients Telaprevir [package insert]. May Ghany MG, et al. Hepatology. 2011;54: Undetectable

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV Response-Guided Therapy Paradigm With TVR + PegIFN/RBV in Tx-Exp Patients TVR + PegIFN + RBV PegIFN + RBV Detectable (≤ 1000 IU/mL) Undetectable/detectable (≤ 1000 IU/mL) No eRVR extend pegIFN + RBV to Week 48; f/u 24 wks HCV RNA 1. Telaprevir [package insert]. May Ghany MG, et al. Hepatology. 2011;54: *AASLD guidelines say RGT “may be considered” for prior partial responders [2] but package insert recommends 48 weeks of therapy [1]  Same as naives; indicated for noncirrhotic previous relapsers [1] * TVR + PegIFN + RBV eRVR stop at Wk 24, f/u 24 wks HCV RNA Undetectable 24 PegIFN + RBV Undetectable

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV HCV RNA Assay Characteristics for RGT With BOC or TVR  A quantitative assay with a LLOQ of ≤ 25 IU/mL and a LLOD of approximately IU/mL must be used  “Confirmed detectable but below limit of quantification HCV RNA result should not be considered equivalent to an undetectable HCV RNA result” Boceprevir [package insert]. May Telaprevir [package insert]. May Detectable/not quantifiable Viral RNA Titer SVR LLOQ LOD Detectable/ quantifiable Not quantifiable ± detectable Undetectable Goal of anti- HCV therapy

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV Predictive Value of Response to 4-Wk Lead-in Phase  ≥ 1 log 10 vs < 1 log 10 decline in HCV RNA following 4-wk lead-in phase with pegIFN/RBV strongly predicts SVR in patients receiving BOC-based therapy –Treatment-naive patients [1] –OR: 9.0; P <.001 –Treatment-experienced patients [2] –OR: 5.2; P < Poordad F, et al. N Engl J Med. 2011;364: Zeuzem S, et al. EASL Abstract 484.

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV Boceprevir [package insert]. May BOC + PegIFN +  RBV PegIFN + RBV 8 36 BOC + PegIFN +  RBV 24 Early response*; stop at Wk 28 or 36; f/u 24 wks F/u 24 wks *Undetectable HCV RNA at Wks 8 and 24 of therapy (Wk 4 of triple therapy). Wks Stop all treatment if HCV RNA ≥ 100 IU/mL Stop all treatment if HCV RNA detectable Use quantitative assay to determine if HCV RNA < or ≥ 100 IU/mL at Wk 12 Use assay with LLOD of IU/mL to determine if “target not detected” at Wk 24 Boceprevir Futility Rules: Wks 12 and 24 Key Time Points  Treatment-naive and treatment-experienced patients

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV Telaprevir Futility Rules: Wks 4, 12, and 24 Key Time Points  Treatment-naive and treatment-experienced patients TVR + PegIFN +  RBV Wks eRVR*; stop at Wk 24; f/u 24 wks PegIFN + RBV No eRVR; PegIFN + RBV Telaprevir [package insert]. May F/u 24 wks *Undetectable HCV RNA at Wks 4 and 12 of triple therapy. Use quantitative assay to determine if HCV RNA ≤ or > 1000 IU/mL at Wks 4 and 12 Use assay with LLOD of IU/mL to determine if “target not detected” at Wk 24 Stop all treatment if HCV RNA > 1000 IU/mL Stop all treatment if HCV RNA detectable

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV  EOT response defined as [1,2] –HCV RNA undetectable (or “target not detected”) at EOT –Using an assay with a sensitivity of IU/mL [1,2]  Detectable but < LLOQ values while on treatment predict lower SVR rates [3] 1. Boceprevir [package insert]. May Telaprevir [package insert]. May Naeger LK, et al. Intl Workshop on Clinical Pharmacology of Hepatitis Therapy Abstract R-8. HCV RNA Thresholds for EOT Response With BOC or TVR

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV Use of HCV RNA Assays to Assess SVR With BOC or TVR-Based Therapy  SVR to pegIFN/RBV previously defined as –Absence of detectable HCV RNA in serum using assay with sensitivity of at least 50 IU/mL 6 mos after EOT [1]  SVR defined by FDA in BOC and TVR package inserts as –HCV RNA < 25 IU/mL (LLOQ) 6 mos after EOT [2-3] 1. Lindsay KL, et al. Hepatology. 2002;36:S114-S Boceprevir [package insert]. May Telaprevir [package insert]. May 2011.

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV Summary: Use of HCV RNA Assays in Managing Patients Receiving BOC or TVR  A quantitative assay with an LLOQ of ≤ 25 IU/mL and an LLOD of approximately IU/mL must be used  HCV RNA < LLOQ not identical to HCV RNA undetectable –HCV RNA undetectable (HCV RNA target not detected) required to qualify for RGT –HCV RNA < LLOQ appropriate for assessing SVR –Carefully read HCV RNA assay report to ensure HCV RNA was undetected or “target not detected” prior to committing to truncated therapy Qualification/EndpointBOCTVR RGT HCV RNA undetectable at Wks 8 and 24 HCV RNA undetectable at Wks 4 and 12 Futility  HCV RNA ≥ 100 IU/mL at Wk 12  HCV RNA detectable at Wk 24  HCV RNA > 1000 IU/mL at Wk 4 or 12  HCV RNA detectable at Wk 24 EOT responseHCV RNA undetectable at EOT SVRHCV RNA < LLOQ 24 wks after EOT

Other Assays

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV P <.0001 P =.004 Thompson AJ, et al. Gastroenterol. 2010;139: IL28B Genotype the Strongest Baseline Predictor of SVR With PegIFN/RBV Metavir F0-2 White vs Black Fasting Serum Glucose < 5.6 mmol/L Hispanic vs Black HCV RNA ≤ 600,000 IU/mL CC vs Non-CC Odds Ratio (95% CI)

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV 1. Poordad F, et al. EASL Abstract Jacobson IM, et al. EASL Abstract SPRINT-2: BOC + PR48 [1] SVR (%) 44/ 55 82/ / 44 CC CT TT n/ N = ADVANCE*: T12PR [2] SVR (%) 45/ 50 48/ 68 16/ 22 CC CT TT n/ N = *IL28B testing in ADVANCE was in white pts only. IL28B Genotype Also Predicts Likelihood of Achieving SVR With BOC or TVR

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV IL28B Genotype Predicts Likelihood of Shortened Therapy With BOC or TVR 1. Poordad F, et al. EASL Abstract Jacobson IM, et al. EASL Abstract Eligibility for Shortened Therapy (%) 118/ / 304 CC CT/TT Eligibility for Shortened Therapy (%) 39/ 50 39/ 68 10/ SPRINT-2: BOC + PR [1] ADVANCE*: T12PR [2] *IL28B testing in ADVANCE was in white pts only n/ N = CC CT TT n/ N = 100

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV When to Consider IL28B Genotype Testing  IL28B genotype testing may be considered prior to therapy if more information about probability of response or treatment duration desired [1] –Commercially available tests  If patients have favorable CC genotype –Likelihood of SVR is high with pegIFN/RBV alone, but triple therapy may allow shorter therapy and, in one TVR study, higher SVR rates [2]  If patients have unfavorable CT/TT genotype –Likelihood of SVR is higher with triple therapy than with pegIFN/RBV [2,3]  Limited value of IL28B genotyping in treatment-experienced patients –Most have unfavorable TT or CT genotype 1. Ghany MG, et al. Hepatology. 2011;54: Jacobson IM, et al. EASL Abstract Poordad F, et al. EASL Abstract 12.

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV HCV Genotype and Subtype  HCV classified into 6 major genotypes (1-6) [1]  Genotype 1 (subtypes a and b) most common in United States (~ 75%) [2] –Subtype 1a more common than subtype 1b  Determining major genotype recommended for proper clinical management and predicting likelihood of response [3]  No current recommendations regarding HCV subtype testing 1. Simmonds P, et al. Hepatology. 2005;42: Zein N. Clin Microbiol Rev. 2000;13: Ghany MG, et al. Hepatology. 2009;49:

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV Higher SVR Rates With TVR in Patients With HCV Genotype 1b vs 1a Tx Naive [1] T12/PR SVR (%) Genotype 1a Genotype 1b Relapsers* [2] Null Responders* [2] Partial Responders* [2] 1. Jacobson IM, et al. N Engl J Med. 2011;364: Zeuzem S, et al. EASL Abstract *Pooled TVR arms.

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV Higher SVR Rates With BOC in Patients With HCV Genotype 1b vs 1a BOC RGT Genotype 1a Genotype 1b Treatment Naive [1] BOC/PR48 BOC RGT Treatment Experienced [2] 1. Poordad F, et al. N Engl J Med. 2011;364: Bacon BR, et al. N Engl J Med. 2011;364: SVR (%)

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV Genotype assayManufacturerMethod Trugene 5'NC HCV Genotyping kit SiemensDirect sequence analysis of the 5' noncoding region INNO-LiPa HCV IIInnogeneticsReverse hybridization analysis using genotype-specific oligonucleotide probes located in the 5' noncoding region Versant HCV Genotyping Assay 2.0 SiemensReverse hybridization analysis using genotype-specific oligonucleotide probes located in the 5' noncoding region Abbott RealTime HCV Genotype II AbbottGenotype-specific real-time PCR of the 5' noncoding region and NS5b  Incorrect typing among major genotypes rare (< 3%) Ghany MG, et al. Hepatology. 2009;49: Commercially Available HCV Genotype Assays

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV  Resistance-associated variants occur naturally [1] –Present in 5% to 7% of subject samples prior to treatment [2,3] –No apparent impact on likelihood of SVR –Selected for/enriched in patients failing PI-based therapy  Following treatment failure, resistance-associated variants decline over time after withdrawal of PI but may remain detectable for up to 2.5 yrs [4,5]  Lower genetic barrier to resistance (number of mutations required to overcome virologic activity of the regimen) with genotype 1a vs 1b with BOC/TVR–based regimens  Strict adherence to futility rules, ensuring patient adherence and tolerability of regimen essential to avoid resistance 1. Pawlotsky JM. Clin Liver Dis. 2003;7: Telaprevir [package insert]. May Boceprevir [package insert]. May Vierling JM, et al. EASL Abstract Sullivan JC, et al. EASL Abstract 8. HCV Resistance With TVR/BOC

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV HCV Resistance Testing  Commercial resistance test for HCV NS3/4 mutations now available –Provides genetic sequence for the nonstructural proteins NS3 and NS4A of HCV genotypes 1a and 1b  Role of resistance testing prior to treatment remains to be defined –No current recommendation to perform resistance testing for patients failing therapy

clinicaloptions.com/hepatitis Understanding the Optimal Use and Interpretation of Assays in HCV Summary: Use of Genotype and Resistance Assays With BOC/TVR  IL28B genotype testing –May be considered prior to therapy if more information about probability of response or treatment duration desired [1]  HCV subtype testing –No current recommendation to test prior to treatment –Patients with genotype 1b may be counseled that their chance of SVR is slightly higher than 1a patients  HCV resistance testing –No current recommendation regarding testing at baseline or upon treatment failure 1. Ghany MG, et al. Hepatology. 2011;54:

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