HPV

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

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.

HUMAN PAPILLOMAVIRUS

E6

E7 E1

E2 E5

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

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.

E6

E7 E1

E2 E5

E4

L2

L1

Disruption of HPV genome during integration

- disruption of E1 to E2 of variable sizes- integration occurs at chromosome ”fragile sites”

Experimental evidence ofHPV transforming capacity

RAFT culture experiments with wild typeand mutant E6/E7 constructs

E6 mutant: in RAFT culture

HPV

Cells infected with oncogenic HPV types

Immortalisation

Uncontrolled cell proliferation

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.

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, SYBR

green)– Type specific DNA PCR

• Solution phase PCR• TaqMan PCR• NASBA (HPV proofer)• In-situ hybridisation (ISH)• Sequence genotyping• In-cell PCR

– ICC

• 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

Schematic of Hybrid Capture II

Hybridise Capture hybridsDenature NA

Label for detection Detect

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 generic / consensus– GP 5 and 6– PGYM– MY09/11

– SPF10

– GP5 and 6 + SYBR green

HPV analysis -PCR

Computer-generated amplification plot from a SYBR-green HPV runDetection sensitivity 5-10 copies/reaction

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

HPV analysis

Taq Man PCR

Detection sensitivity = 1-2 copies per reaction

HPV

Beta actin

• 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

In-situ hybridisation: detection of HPV