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HPV

HPV. Carcinoma of the Cervix. 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. Human Papillomavirus (HPV).

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HPV

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  1. HPV

  2. Carcinoma of the Cervix • 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.

  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. E2 E5 L1 E6 L2 E7 E1 E4 8Kbp 0 HUMAN PAPILLOMAVIRUS • 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. E2 E5 L1 E6 L2 E7 E1 E4 Disruption of HPV genome during integration • disruption of E1 to E2 of variable sizes • integration occurs at chromosome ”fragile sites”

  8. Experimental evidence ofHPV 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. HPV analysis • Hybrid Capture II • Liquid based system. • Low and high risk type analysis. • No information in relation to integration. • Indirect load information but NOT quantitative.

  16. Denature NA Hybridise Capture hybrids Label for detection Detect Schematic of Hybrid Capture II

  17. HPV analysis • Hybrid Capture II • Recommended cut-off for the HC-II test is 1 pg viral DNA perml of buffer, equivalent to about 5000 viral genomes. • This cut-off value has been reduced to 0.2 pg/ml but with theintroduction 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 in13% using the recommended cut-off of 1 pg/ml, and in 22.1%using a cut-off of 0.2 pg/ml.

  18. HPV analysis -PCR • PCR generic / consensus • GP 5 and 6 • PGYM • MY09/11 • SPF10 • GP5 and 6 + SYBR green

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

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

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

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

  23. In-situ hybridisation: detection of HPV

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