1. HPV

2.  Many risk factors for development of cervical cancer. no routinely used positive predictive biological markers, which identify women at risk of developing high-grade lesions and ultimately invasive cancer. Carcinoma of the Cervix

3. Human Papillomavirus (HPV) Strong association with development of invasive cancer. >70 types of HPV. Low risk (6,11). High risk (16,18,31,33,35,39,45,51,52,56,58,59,66, 68). Exposure to HPV is followed by a serological response to viral capsid proteins (VLPs). Immune response is assoc. with persistent HPV infection and is type specific.

4. HUMAN PAPILLOMAVIRUS E6 E7 E1 E2E5 E4 L2 L1 0 8Kbp  small DNA viruses,8kb double stranded genome  a single host may be infected with different HPVs  Two forms of HPV infection of the Cervix –Episomal –Integrated

5. HPV Integration of HPV DNA into host  loss of E2 orf.  Transcription of E6 and E7 is unregulated.  Transformation events within the cell. Checkpoint for cell proliferation and transcription is lost.

6. HPV Expressed E6 and E7 proteins can then interact with other tumour suppressor genes including p53 and pRB  uncontrolled cellular proliferation and malignant transformation. 3 splice variants of E6 HPV 16 recognised: E6 I, II and III.

7. E6 E7 E1 E2E5 E4 L2 L1 Disruption of HPV genome during integration - disruption of E1 to E2 of variable sizes - integration occurs at chromosome ”fragile sites”

8. Experimental evidence of HPV transforming capacity RAFT culture experiments with wild type and mutant E6/E7 constructs E6 mutant: in RAFT culture

9. HPV Cells infected with oncogenic HPV types Immortalisation Uncontrolled cell proliferation

10. Carcinoma of the cervix  MOLECULAR ONCOLOGY  over 95% of cervical SCCs associated with high risk HPV types (16,18,31,33,45); 40-70% of adenocarcinomas.  HPVs also found in CIN: 4-6% of normal women HPV 6 and 11 positive. CIN 1: 10- 30% HPV 6 &11 positive. CIN 2- 3: 75- 80% HPV 16, 18, 31, 33 positive; 1- 5% HPV 6,11 positive.  HPV E6 and E7 regions can transform epithelial cells and increase cellular levels of cyclins A,B and p34-cdc 2 and cyclin E.

11. HPV analysis Who do we screen? –All Women? –HPV as a triage? How do we screen? Does HPV analysis give prognostic information? HPV and other novel biomarkers of disease

12. Future role for HPV screening Post introduction of HPV vaccine  vaccines being produced to target HPV 16 and 18 E6/E7 regions.  requirement to monitor HPV status pre and post-vaccination.  possibility of using recombinant  anti-sense PNAs to specifically  target HPV E6 and E6 splice variants.

13. How do we screen? HPV analysis –Type –Load –Viral integration

14. HPV analysis Technologies available –Hybrid Capture II –PCR generic, (incl. PGYM, GP5 and 6, SYBR green) –Type specific DNA PCR Solution phase PCR TaqMan PCR NASBA (HPV proofer) In-situ hybridisation (ISH) Sequence genotyping In-cell PCR –ICC

15. Hybrid Capture II –Liquid based system. –Low and high risk type analysis. –No information in relation to integration. –Indirect load information but NOT quantitative. HPV analysis

16. Schematic of Hybrid Capture II HybridiseCapture hybridsDenature NA Label for detection Detect

17. Hybrid Capture II  Recommended cut-off for the HC-II test is 1 pg viral DNA per ml of buffer, equivalent to about 5000 viral genomes.  This cut-off value has been reduced to 0.2 pg/ml but with the introduction of false positives (Peyton et al).  Data comparing PCR with HC-II found PCR identified HPV in 24.5% of samples, while HC-II detected HPV in 13% using the recommended cut-off of 1 pg/ml, and in 22.1% using a cut-off of 0.2 pg/ml. HPV analysis

18. PCR generic / consensus –GP 5 and 6 –PGYM –MY09/11 –SPF 10 –GP5 and 6 + SYBR green HPV analysis -PCR

19. Computer-generated amplification plot from a SYBR-green HPV run Detection sensitivity 5-10 copies/reaction

20. Type specific PCR –Solution phase PCR –Taq Man q(PCR) –NASBA (HPV proofer) –In-situ hybridisation –HPV genotyping HPV analysis

21. Taq Man PCR Detection sensitivity = 1-2 copies per reaction HPV Beta actin

22. In-situ hybridisation –Cloned HPV subtypes (Zur Hausen) –Automated platforms available. –Commercial probes: DAKO, Digene, Ventana, etc. HPV analysis Detection sensitivity = 1-5 copies per biopsy

23. In-situ hybridisation: detection of HPV