Laboratory Considerations
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1 Laboratory Considerations In Support of HPV Vaccine Programs Elizabeth R. Unger PhD, MD Team Leader HPV Laboratory Centers for Disease Prevention and Control Towards Comprehensive Cervical Cancer Control: Region of the Americas May 2008, Mexico City Disclaimer: The findings and conclusions in this presentation have not been formally disseminated by the Centers for Disease Control and Prevention and should not be construed to represent any agency determination or policy.
2 Outline Introduction to HPV Laboratory Methods Description of WHO HPV LabNet Role of Laboratory in HPV Vaccine Implementation and Monitoring Laboratory Initiatives to Improve HPV Surveillance
3 Unique Feature of HPV No simple in vitro culture methods Antibody methods lack sensitivity Natural exposure results in <70% seroconversion Studies of HPV require detection of HPV genetic information Requires a cellular sample from the site of infection Only current infections detected
4 Paradox of HPV Testing HPV infection is not treated Treatment is directed to HPV-associated cervical neoplasia HPV tests detect viral DNA not disease, not even infection Analytic performance = HPV detection Clinical performance = Disease detection
5 HPV: A family of more than 100 viruses HPV62 MM7 LVX82 CP8304 LVX100 CP6108 MM8 HPV61 HPV54CP0861 HPV57 HPV27 HPV2A HPV68 LVX160 HPV39 HPV59 HPV45 HPV18 HPV43 HPV40 HPV7 HPV67 HPV58 HPV33 HPV35 HPV52 HPV31 MM9 HPV16 HPV28 HPV3 HPV10 HPV29 HPV64 HPV34 HPV26 HPV69 HPV51 ISO39 MM4 HPV30 HPV53 HPV56 HPV66 HPV32 HPV42 HPV6b HPV11 HPV13 HPV44
6 HPV Laboratory Methods Infection monitored by DNA detection Sample and assay frame view of disease Complicates definition of latent, occult, persistent or recurrent infection
7 Importance of Sampling Biopsies provide direct correlation between pathology and virus Includes basal layer of epithelium Limited area sampled Not suitable for screening Exfoliated cervical cytology samples provide noninvasive approach for screening women Quality dependent on collection method and anatomic site Appropriate sample in men is less clear
8 HPV DNA Assays Multiple HPV types complicate assays Sensitivity and type-specificity vary Inter-assay comparisons difficult Direct hybridization Southern blot, dot blot, in situ, HybridCapture Amplification (PCR) Type specific, versus consensus
9 HPV Hybrid Capture Assay Liquid hybridization, chemiluminescent detection analogous to ELISA format Probe mix: HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 No control for amount or quality of DNA Good inter-laboratory comparisons Basis for PATH s low cost point of care test (Care HPV)
10 The Digene Hybrid Capture 2 HPV DNA Test Denature DNA in clinical samples Detect signal by chemiluminescence. Raw data is reported as RLU (relative light units). Results are reported as a ratio: Relative Light Units/Cutoff Value. (RLU/CO 1.0 is considered positive) Add sample to an RNA probe pool specific for either low-risk types or high-risk types of HPV Add antibodies conjugated with alkaline phosphatase. (Signal is amplified fold) Capture RNA:DNA hybrids on the surface of wells coated with antibodies specific for the hybrids Note: Illustrations are from the Digene Corporation website:
11 HPV PCR Assays Consensus assays Most target L1 region Type(s) determined by type specific hybridization or other method PGMY09/11 (450 bp) GP5+/6+ (150 bp) SPF-PCR (65 bp) Type specific assays Most target E6/E7 region Variants can cause false negative results capsid proteins L2 L1 URR E5 P97 E4 E6 late cytoplasmic protein transformation E2 E7 transformation E1 episome replication transcriptional regulator
12
13 HPV Serology: L1-VLP Assays Antibodies are made to conformational epitopes Requires L1-VLPs VLP production not standardized Different expression systems, preparative methods, QC approach ELISA-based detection Reaction indicates past or current infection No gold-standard for setting threshold for positive result (being addressed by WHO standard sera)
14 WHO HPV LabNet Mission To contribute to improving quality of laboratory services for effective surveillance and monitoring of HPV vaccination impact, through enhanced, state-of-the-art laboratory support
15 Harmonization of HPV Laboratory Testing Procedures Allow inter-laboratory comparisons Standardize epidemiologic studies of HPV Facilitate assessment of HPV vaccine efficacy Implement validated, standardized laboratory procedures (including sample collection) Develop quality assurance system and proficiency testing Train personnel and supply equipment if required Provide a network for surveillance
16 Call for applications 2006 First Meeting January 2008 Call for National and Local Reference Laboratories not yet opened
17 HPV LabNet Differs From Other WHO LabNetworks HPV LabNet is formed, but disease target is primarily cervical cancer not HPV Impact on disease is long-range goal of vaccination Requires >10 years after population vaccination Problem is chronic rather than acute No immediate threat to world from outbreaks No treatment for infection High cost of vaccination, screening, testing High test volume required
18 HPV Surveillance Goals Must Be Established First Surveillance needs currently vary widely across the world Country-specific needs depend on: Knowledge of disease burden Type- and age-specific prevalence of HPV Cervical cancer prevalence Cancer registries Infrastructure for cervical cancer screening and treatment
19 HPV Surveillance Requires Integration of Efforts HPV LabNet will be one part of solution WHO HPV LabNet Education Vaccination Screening Academic Public Health Industry NGO s
20 Possible Goals of Surveillance Before Vaccine Introduction Type- and age-specific HPV prevalence in population May be required to validate vaccine types match country s needs Could direct age of vaccination Provide baseline to monitor impact of vaccine Age specific cervical cancer incidence and HPV type distribution
21 HPV Vaccine as Impetus to Address Cervical Cancer Problem is cervical cancer control Dual approach likely to be adopted in countries without screening Screening programs based on See and Treat / HPV testing Vaccination Is it necessary to distinguish contribution of vaccination versus screening?
22 Possible Goals of Surveillance After Vaccine Introduction Vaccine Efficacy Cervical Cancer Incidence Desired goal, but realistic only in long-range Intermediate endpoints Difficulties discussed in following slides Vaccine Uptake Estimates of vaccine uptake in population Serology could be marker Vaccine Registry Focused outcome correlation Safety
23 Intermediate End-Points for Post-Vaccine Surveillance Cervical cancer precursors CIN 2/3 Requires infrastructure for cervical cancer screening, colposcopy, biopsy/leep and histopathology laboratories Denominator difficult to determine in absence of systematic screening Imprecision in case ascertainment Change in screening/intervention guidelines could impact detection Poor inter-observer agreement on histology Type-specific prevalence in population High cost of sampling and testing makes long-term ongoing surveillance difficult
24 Role of HPV LabNet Before Vaccination Provide expertise on sampling and testing Estimate of HPV prevalence Serology DNA testing type-specific assays HPV type-specific prevalence in cervical cancer Training Build capacity for HPV testing in regional and national laboratories Train local laboratories to integrate hrhpv detection and cervical cancer screening
25 Possible Role of HPV LabNet After Vaccination Basically same as before vaccine introduction Main difference serology cannot be used to estimate burden of exposure; shifts to measure of vaccine coverage Provide expertise on sampling and testing Estimate HPV prevalence in population DNA testing type-specific assays HPV prevalence in cervical cancer and CIN Histopathology review via virtual slides Serology testing to monitor vaccine uptake Continue training to build testing capacity
26 What prevents HPV testing from being widely available? Trained personnel Will benefit other public health problems Laboratory facilities HPV testing labs could provide start of infrastructure to improve all medical testing Affordable testing reagents VLPs Standardized typing reagents Standards and proficiency testing
27 Laboratory Initiatives to Improve Surveillance ( faster cheaper better ) Validate non-invasive sampling methods to monitor vaccine uptake (oral sample) Standardize and validate self-sampling methods for HPV DNA testing (dry swab, paper smear) Improve high-throughput methods for extraction and type-specific detection Determine type-specific reproducibility of commercially available assays
28 Acknowledgements CDC HPV Laboratory and CVDB Lauri Markowitz and CDC HPV Workgroup Tiequn Zhou and WHO HPV LabNet
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