hpv
DESCRIPTION
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). - PowerPoint PPT PresentationTRANSCRIPT
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HPV
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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
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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.
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HUMAN PAPILLOMAVIRUS
E6E7 E1
E2 E5
E4
L2L1
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
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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.
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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.
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E6E7 E1
E2 E5
E4
L2L1
Disruption of HPV genome during integration
- disruption of E1 to E2 of variable sizes- integration occurs at chromosome ”fragile sites”
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Experimental evidence ofHPV transforming capacity
RAFT culture experiments with wild typeand mutant E6/E7 constructs
E6 mutant: in RAFT culture
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HPV
Cells infected with oncogenic HPV types
ImmortalisationUncontrolled cell proliferation
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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.
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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
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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.
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How do we screen?
• HPV analysis– Type– Load– Viral integration
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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
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• 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
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Schematic of Hybrid Capture II
Hybridise Capture hybridsDenature NA
Label for detection Detect
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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
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• PCR generic / consensus– GP 5 and 6– PGYM– MY09/11– SPF10
– GP5 and 6 + SYBR green
HPV analysis -PCR
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Computer-generated amplification plot from a SYBR-green HPV runDetection sensitivity 5-10 copies/reaction
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• Type specific PCR– Solution phase PCR– Taq Man q(PCR)– NASBA (HPV proofer)– In-situ hybridisation– HPV genotyping
HPV analysis
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Taq Man PCR
Detection sensitivity = 1-2 copies per reaction
HPV
Beta actin
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• 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
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In-situ hybridisation: detection of HPV