human papillomavirus (hpv) diagnosis
TRANSCRIPT
Human Papillomavirus (HPV) Diagnosis
Sepehr N. TabriziThe Royal Women’s Hospital
WHO Regional Reference Laboratory for HPV Diagnosis and Control
C l CCervical Cancer
• 2nd most common cancer of2 most common cancer of women worldwide
½ million new cases annually (80% in– ~ ½ million new cases annually (80% in developing countries)Cancer usually appears in people aged– Cancer usually appears in people aged >35
Burden of cervical disease in Australiaf
227 deaths
~ 700 cervical cancer cases- 13th
14 500 high grade abnormalities
16 500 low grade abnormalities
100 000 abnormal Pap smears
~ 2 million women screened
* Annual events. 1. NHMRC. 2005. 2. AIHW, 2006. 3. Blomfield et al, 2005
Cervical Cancer and H ll (HPV)Human papillomavirus (HPV)
– ~ 99% of ICC cases have detectable HPV DNA
– Molecular & in vitroepidemiological association– epidemiological association
– HPV 16 and 18 are recognized icarcinogen
Risk of Cervical Cancer by HPV Typef y yp
NegativeNegative16184531523358355951
0 1 10 100 1000 10 000
516
110.1 10 100 1000 10,000
*CI = confidence intervalMuñoz N, Bosch FX, de Sanjosé S, et al. N Engl J Med. 2003;348:518–527.
Odds Ratio (95% CI*)
Cancer Relative Risk
Lung Cancer- smoking 10
Liver Cancer- HCV 20
Liver Cancer HBV 50 100Liver Cancer- HBV 50-100
Cervical Cancer- HPV 300-500Cervical Cancer HPV 300 500
O h HPV fOther HPV infection
• Vulvar Cancer (50%)• Recurrent Respiratory Papillomatosis• Recurrent Respiratory Papillomatosis
[RRP] (100%)H d & k (28%)• Head & neck cancers (28%)
• Anal cancer (85%)• Penile cancer (80%)
P f H ld HProfessor Harald zur Hausen
Joint Nobel Prize winner in Physiology /Medicine 2008in Physiology /Medicine 2008
P llPapillomaviruses• Family: Papillomavirusy• Non-enveloped dsDNA
viruses– 55-nm spherical capsid coat
• Tropism for squamous p qepithelium
• Widely distributed in higher y gvertebrates– Tight species specificity
H llHuman papillomavirus• DS ~8kb , static (recombination and (
mutations are rare events)• Early region
– Six ORF-• E6 and E7 transforming oncoprotein• Rest are required for viral replication
• Late regiong– Encodes for 2 viral capsid proteins
• L1 ORF - most conserved and used for identification of new PV types
• Upstream regulatory region– Sequences that control
transcription of the viral genomep g
H P llHuman Papillomavirus• Cannot be routinely propagated in-
vitro • Detection and typing is based on• Detection and typing is based on
nucleic acid sequence– >10% sequence variation = new type; q yp ;
2-10% = subtype, <2% = variant • Types assigned sequential number
based on order of discoverybased on order of discovery– No relation to phylogeny
• Heterogeneous group of ~ 200Heterogeneous group of 200genotypes
• Two major branches, differing affinities for site of infection
• Cutaneous: Keratinized squamous epithelium
• Mucosal: Non-Mucosal: Nonkeratinized squamous epitheliumepithelium
7016
6016
40
50Neg
30 18%
10
20
0
10
Normal CIN1 CIN2 CIN3 >CIN3
HPV prevalence in ICC biopsies(n = 191)
ICC52.9
50
60
ysed
30
40
opsi
es a
naly
18.320
30
cent
age
of b
io
1.6 1.6 1.6 1.62.63.16.3
0
10
16 18 26 31 33 35 39 45 51 52 55 56 58 59 68 82 83 73 6 11 40 42 53 54 57 66 84
Perc
Predominant types: 16, 18, 45, 39 and 73
HR HPV LR HPV
Geographical variations: cervical cancer
0 20 60 411.4 4 4
0.8
0.5
0.5
13.5
20.8
54.9
0.20.60.4
3.33.93.2
56
17.2
4.4
2.94.2
43.4
3.4
5.4
4.2
1
1 4
1618
22.115.3
4.5
2
3.4
50.23.1
1.6
1.6
1.417.5
3 3
213
18453133
14.17.9
2.6
51.77
4
2.9
3.3585235Others
1.01.6
1.610.51.6
10.6
5.5Others
In contrast regional distribution of HPV type prevalence in cervical cancer shows 16, 18 most common worldwide;
52.9
18.3
0.56.3
Clifford GM, et al., (2003) HPV types in invasive cervical cancer worldwide: a meta-analysis. Br J Cancer 88: 63–73 Stevens M, Tabrizi S, Garland SM. HPV genotyping using a modified Linear Array detection protocol. J Virol Methods 2006
thereafter geographical variation.
Temporal trends of HPV types in i l (1920 2005)
100%
cervical cancer (1920–2005)
80
100
HPV 1660
40OTHER TYPES
40
20HPV 18
0HPV 18
PREVALENCE ESTIMATE
95% CIBeijing IPV 2007, abstract 4B-05
N l HNatural History
40% 20% 1%SpontaneousSpontaneous 40% 20% 1%pRegression Rate
pRegression Rate
Pic.exe
C l CCervical Cancer• Highly preventable disease!
Screening & early detection– Screening & early detection• Implemented early to detect (Pap smear) / treat cancer
precursors.• Availability of molecular tests to increase sensitivity of• Availability of molecular tests to increase sensitivity of
detection.
– VaccinesVaccines• Two vaccines available• ↓ incidence of HPV-16 infection and related dysplasia• Prevents HPV residing in genital tract (↓ transmission)Prevents HPV residing in genital tract (↓ transmission)
Detection by microscopy
• Electron Microscopy
• ImmunohistochemistryImmunohistochemistry (group specific antigen)
D b MDetection by Microscopy• Pap smear - treat cancer precursors.
• Histology
P P i i i f CIN2+ Hi lPap Positivity for CIN2+ Histology
HANNOVERFRENCH_PRIVFRENCH_PUBL
HAMMERSMITHJENA
TUEBINGEN
SEATTLECANADA
HART
Proportion0 .1 .3 .5 .7 .9 1
Combined
Cuzick J et al Int J Cancer 2006 119 European Nth American studies HPV primary screening
Sensitivity of Pap 53% [95% CI wide]
HPV Positivity for CIN 2+ Histology
FRENCH PRIVFRENCH_PUBL
JENATUEBINGENHANNOVER
FRENCH_PRIV
HARTCANADA
HAMMERSMITHJENA
C bi d
SEATTLEHART
Proportion0 .1 .3 .5 .7 .9 1
Combined
Cuzick J et al Int J Cancer 2006 119 European Nth American studies HPV primary screening
Sensitivity of HPV DNA 96 % [95% CI tight]
Impact of Pap screening
13.2
6.9
1 9
4.0
1.9
Source: AIHW (Australian Institute of Health and Welfare) & AACR (Australasian Association of Cancer Registries) 2008. Cancer in Australia: an overview, 2008. Cancer series no. 46. Cat. no. CAN 42.Canberra: AIHW.
D b S lDetection by Serology
• Not yet diagnostic• Virus Like Particles
– non-infectious analogs of a pathogenic virus– VLP- L1 assemble in vitro
VLP d ti t t d di d– VLP production not standardized– Different expression systems, preparative methods,
QC approach – Formats vary (direct vs. indirect)– No gold-standard for setting threshold for positive
resultresult– Few inter-laboratory comparisons
Detection by molecular biologyDetection by molecular biology
• HPV DNA/mRNA- different assaysdifferent assays- different sensitivities
(clinical diagnosis vs surveillance)(clinical diagnosis vs surveillance)
Commercial HPV tests (1)( )• Hybrid Capture 2 Qiagen• Care HPV Qiagen• Care HPV Qiagen• Luminex HPV assay Qiagen• Consensus HPV typing kit Qiagen
A li HPV T t R h• Amplicor HPV Test Roche• COBAS 4800 HPV test Roche• NucliSENS EasyQ HPV Biomerieuxy• Aptima Gen-Probe• Cervista Hologic (Third Wave Tech)• BIOPAP QTS HPV Kit Loxo• BIOPAP QTS HPV Kit Loxo• Abbott RealTime High Risk HPV , Abbott• AID STD assay GenID
AID HPV i kit G ID• AID HPV screening kit GenID• AID HPV typing kit GenID
Commercial HPV tests (2)( )• Linear ArrayExtra HPV Genotyping Kit Innogenetics• PCR Human Papillomavirus Detection Set TakaraPCR Human Papillomavirus Detection Set Takara
Mirus Bio• Array Papillomavirus Genomica• ProDect Chip HPV typing Bcs Biotech S P AProDect Chip HPV typing Bcs Biotech S.P.A• PapType Genera Biosystems• LCD Array HPV 3.5 Chipron
S l HPV G t i S• Seeplex HPV Genotyping Seegene• Viroactiv Virofem• HPV OncoTest Invirion Diagnostics• Genpoint Tm HPV test Dako-Oxoid• Reveal HPV Real-Time HPV Detection Kit GenoID• Luminex HPV Genotyping Multimetrix/ProgenLuminex HPV Genotyping, Multimetrix/Progen • Papillocheck, Greiner BioOne Etc
P l Ch RPolymerase Chain Reaction
• Very sensitive about 1-10 copies /100,000 cells• Allows testing of various sample types• Allows testing of archival samples with poorer
quality DNAC• Consensus assays – generally target L1 region
• conserved• primers vary:
MY09/MY11, SPF1/SPF2PGMY09/PGMY11, GP5+/GP6+PGMY09/PGMY11, GP5 /GP6
• detection systems vary:ELISA, line blot
40%
50%
GP5+ / 6+
Ranking of HPV types in Hong Kong
10%
20%
30%
0%HPV16 HPV18 HPV31 HPV33 HPV35 HPV52 HPV58
40%
50%
MY09 / 11
10%
20%
30%
0%HPV16 HPV18 HPV31 HPV33 HPV35 HPV52 HPV58
40%
50%
PGMY09 /11
10%
20%
30%
0%HPV16 HPV18 HPV31 HPV33 HPV35 HPV52 HPV58
HPV 16 HPV 18 HPV 31 HPV33 HPV 35 HPV 52 HPV 58
P.Chan et al, Int J Cancer. 2005 Jul 19
Why QA for HPV? •Diverse range assays
•Diff t iti it d ifi it•Different sensitivity and specificity
•DemandDemand• ↑ requests for testing• ↑ labs performing testing• ↑ new tests for HPV testing
•QAP tl il bl i A t li th h RCPA•QAP currently available in Australia through RCPA
WHO HPV Lab Network Centres for surveillance/monitoring in areas matching ambitious vaccine introduction
Selected 9 centres for strengthening lab facilities for HPV-DNA and antibody detection
What test to use?CLINICAL:• screening• triage of equivocal/ borderline Paps• triage of equivocal/ borderline Paps• monitor post dysplasia treatment
EPIDEMIOLOGY:• surveillance geographical areas
- HPV prevalence- HPV prevalence- HPV genotype prevalence
VACCINE:• pre and post vaccine implementation
- vaccine efficacy trialsvaccine efficacy trials- different regions worldwide
V L k P lVirus Like Particles
• Described in 1990• Resemble the virus physically and p y y
immunologically
• DS DNA
• No growth cell cultureC.Laverty
HPV prophylactic vaccines:L1 protein self assembles into VLPs1–4protein self-assembles into VLPs1 4
1. Berzofsky JA, et al. J Clin Invest. 2004;114:450–462.2. Kirnbauer R, et al. Proc Natl Acad Sci USA. 1992;89:12180–12184.3. Modis Y, et al. EMBO J. 2002;21:4754–4762.
•• highly immunogenic• elicit type-specific antibodies that
are neutralizing
Currently licensed prophylactic HPV i d h i i
BIVALENT QUADRIVALENT
vaccines: product characteristicsBIVALENTCervarixTM1
QUADRIVALENTGardasil®2
Antigen VLPs of HPV 16 & 18 VLPs of HPV 16 18 6 & 11Antigen VLPs of HPV 16 & 18 VLPs of HPV 16, 18, 6 & 11
Adjuvant AS04 (Al(OH)3 + MPL) AHHS
Expression system
Baculovirus expression vector Yeast
Administration 0, 1 & 6 months by intramuscular injection
0, 2 & 6 months by intramuscular injectionby intramuscular injection by intramuscular injection
1. CervarixTM. European Summary of Product Characteristics, 2007;2. Gardasil®. European Summary of Product Characteristics, 2008.
Potential changes to screening program
• Reduction of HPV 16/18 which will reduce HSIL
• Reduction of HSIL will lead to reduced exposure for reading Pap slides for p g ptraining purposes
• Cost effectiveness and effectiveness of cervical screening is likely to change over time
Some potential possible changes to screening program
• Primary HPV DNA testing– Use of validated assays in screening populationy g p p– Use of HPV typing (16/18 positive) or other
progression markers– Allows further extension of pap interval– Cytology triage
Use of ne c tolog technolog• Use of new cytology technology– Automated image analysis with LBC
Potential Screening AlgorithmWomen aged 25-64 y
HPV Test
Normal 5 Cytology
positiveNegative
Year RecallCytology
Normal, Borderline
of mild
≥ moderate
ColposcopyHPV 16/18 typing
mRNA, p16
Colposcopy
at 6-12 months
3- 5 Year
Negativepositive
3- 5 Year Recall Colposcopy
Cuzick
Implications of vaccines for HPV ti i l tiprevention in population
• vaccinating could prevent > 70% of invasive• vaccinating could prevent > 70% of invasive cervical cancers and HPV-related diseaseI ti f di b d d• Issues:proportion of disease burden caused by various genotypes in different regions?
• Replacement as vaccine types eliminated?
Research Plan
1. To estimate the prevalence of type specific genital human papillomavirus (HPV) infection prior vaccine:
- in the Australian female population- by age group- Indigenous statusIndigenous status- cervical (Papanicolaou or "Pap") smear status- region of residence
2. To assess the potential impact of an HPV population vaccination strategy through the use of disease modellingmodelling
Percentage of women positive for HRHPV g pby Indigenous status and age group
60%
40%
50%
HPV
non Indigenous %
30%
% p
ositi
ve H
Indigenous %
10%
20%
0%15-19 20-24 25-29 30-34 35-39 40-44 45-49 50-54 55+
Age group (years)g g p (y )
None of the differences by age group are statistically significant
HPV 16 and 18 positivity HPV 16 and 18 positivity by Indigenous status and age group
25%
20%
ve
non Indigenous HPV 16
non Indigenous HPV 18
15%
tage
pos
itiv
Indigenous HPV16
Indigenous HPV18
5%
10%
Perc
en
0%15-19 20-24 25-29 30-34 35-39 40+15 19 20 24 25 29 30 34 35 39 40+
Age group (years)