UICC HPV and CERVICAL CANCER CURRICULUM

Transcription

1 UICC HPV and CERVICAL CANCER CURRICULUM

2 01 Chapter 2.d. Screening and diagnosis - HPV analysis and typing Prof. Suzanne Garland MD Director of Microbiological Research, Head of Clinical Microbiology and Infectious Diseases The Royal Women's Hospital Melbourne, Australia Associate Prof. Sepehr Tabrizi Senior Research Scientist, Department of Microbiology The Royal Women's Hospital Melbourne, Australia

3 02 Virus-like particles (VLPs) Non-infectious analogs of a pathogenic virus VLP-L1 assembles in vitro into empty capsids or VLPs VLP production not standardised - Different expression systems, preparative methods, quality control approaches - Formats of identification vary (direct vs. indirect) - No gold-standard for setting threshold for positive result - Few inter-laboratory comparisons

4 03 Virus-like particles (VLPs) VLPs can be analysed using electron microscopy techniques W

5 04 HPV antibody detection Antibodies - Presence represents past or present infection - Can be generated against HPV early (E) proteins or late (L) proteins - L1 neutralizing antibodies correlate with vaccine-initiated protection

6 05 HPV antibody detection Various assays developed to detect antibodies in sera/mucosa: - Enzyme linking immunosorbent assay (ELISA): measures total Ig, both neutralising and non-neutralising - Competitive binding assays, e.g. competitive Luminex assay (clia): measures neutralising Ig to one epitope only, but all Ig classes - Pseudovirion assay: measures total neutralising Ig, all subclasses - Western blot - Radio-immunoprecipitation

7 06 HPV DNA detection by microscopy in tissue Immunohistochemistry (group-specific antigen) Pap smear: detect cancer precursor cells

8 07 Molecular Detection Techniques for HPV DNA Amplification methods Signal amplification - Hybrid Capture (HC2) Target amplification - Polymerase chain reaction PCR HPV DNA - HPV mrna

9 08 Hybrid Capture 2 assay Current FDA approved test tube format; 1999 micro-titre format Designed to work with exfoliated cervical cell sample Recommended collection kit includes brush and sample transport medium - Collects endo- and ectocervical cells

10 09 Hybrid Capture 2 assay Sensitivity of 5000 genomes/sample Low-risk probe mix - HPV types 6, 11, 42, 43, 44 High-risk probe mix - HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 Results not type-specific No control for input number of cells Good interlaboratory comparison

11 10 Polymerase Chain Reaction Very sensitive about 1-10 copies /100,000 cells Allows testing of various sample types Allows testing of archival samples with poorer quality DNA Consensus assays: generally target L1 region - Conserved - Primers vary: MY09/MY11 SPF1/SPF2 PGMY09/PGMY11 GP5+/GP6+ - Detection systems vary: ELISA dot blot

12 11 Different PCR methodologies AMPLICOR HPV Test LINEAR ARRAY HPV Test INNO-LiPA Genotyping Test - HPV genotyping on paraffin-embedded sections - Tissue sections can be quite old and subject to DNA degradation (including during extraction)

13 12 Different PCR methodologies HPV DNA Chip format HPV Microarray Flow Cytometry Array

14 13 Other methodologies to detect HPV types Luminex s xmap Technology Norchip - HPV Proofer

15 14 Limitations and challenges Diverse range assays Different sensitivity and specificity Lack of readily available controls, QA & QC

16 15 Practical use of HPV DNA testing methods Clinical Screening Triage of equivocal / borderline Pap smears Monitor post-dysplasia treatment Epidemiology Surveillance geographical areas - HPV prevalence - HPV genotype prevalence Vaccine Pre- and post-vaccine implementation - Vaccine efficacy trials - Different regions worldwide

17 16 Thank you This presentation is available at