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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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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.
HUMAN PAPILLOMAVIRUS
E2
E6
E7
E5
L1
L2
E1
E4
8Kbp
0
 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
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.
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.
Disruption of HPV genome
during integration
E2
E6
E7
E5
L1
L2
E1
E4
- disruption of E1 to E2 of variable sizes
- integration occurs at chromosome ”fragile sites”
Experimental evidence of
HPV transforming capacity
RAFT culture experiments with wild type
and mutant E6/E7 constructs
E6 mutant: in RAFT culture
HPV
Cells infected with oncogenic HPV types
Immortalisation
Uncontrolled cell proliferation
Carcinoma of the cervix
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MOLECULAR ONCOLOGY
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over 95% of cervical SCCs associated with high risk HPV types
(16,18,31,33,45); 40-70% of adenocarcinomas.
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HPVs also found in CIN:
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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.
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
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.
How do we screen?
• HPV analysis
– Type
– Load
– Viral integration
HPV analysis
• Technologies available
– Hybrid Capture II
– PCR generic, (incl. PGYM, GP5 and 6,
green)
– Type specific DNA PCR
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Solution phase PCR
TaqMan PCR
NASBA (HPV proofer)
In-situ hybridisation (ISH)
Sequence genotyping
In-cell PCR
– ICC
SYBR
HPV analysis
• Hybrid Capture II
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Liquid based system.
Low and high risk type analysis.
No information in relation to integration.
Indirect load information but NOT quantitative.
Denature NA
Hybridise
Label for detection
Capture hybrids
Detect
Schematic of Hybrid Capture II
HPV analysis
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 -PCR
• PCR generic / consensus
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GP 5 and 6
PGYM
MY09/11
SPF10
GP5 and 6 + SYBR green
Computer-generated amplification plot from a SYBR-green HPV run
Detection sensitivity 5-10 copies/reaction
HPV analysis
• Type specific PCR
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Solution phase PCR
Taq Man q(PCR)
NASBA (HPV proofer)
In-situ hybridisation
HPV genotyping
Taq Man PCR
HPV
Beta actin
Detection sensitivity = 1-2 copies per reaction
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
In-situ hybridisation: detection of HPV