Regulation of HPV tests
Chris JLM.Meijer Dept of Pathology
Vrije Universiteit Medical Center
Amsterdam
The Netherlands
cjlm.meijer@vumc.nl
Melbourne 21-02-2015
Role of HPV in cervical carcinogenesis
2-5years
15-25
years
12-20years
Part CIN2 and CIN3
CIN1, part CIN2
1. Persistent infection with hrHPV necessary for cervical carcinogenesis
2. No HPV, no cancer
3. 14 hrHPV types responsible for>99% of allCxCa:
HPV 16 and 18 cause ~70% of all CxCa
Phylogenetic tree of Papilloma virusses
Alpha 10 group: lrHPV 6,11
Alpha 9 group
hrHPV 16,31,33
Alpha7 group
hrHPV 18,45
Genital HPV types
8kb double stranded DNA viruses, absolutely host and tissue specific.
Can’t grow virus in tissue culture. Classified by genotype not serotype.
Alpha-papillomavirus group 7 and 9 contain most important high risk types
HPV detection
• Currently used HPV detection assays rely on
detection of viral nucleic acids (DNA, RNA)
– Principle: hybridisation with complementary
sequences
Some commercial HPV tests
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Hybrid Capture 2 Qiagen
Digene HPV genotyping RH kit Qiagen
Digene HPV genotyping LX kit, Qiagen
Amplicor HPV Test Roche
Cobas 4800 HPV Test Roche
Linear array Roche
SPF10/LiPa DDL
NucliSens EasyQ HPV Biomerieux
Aptima Gen-Probe
Cervista HPV HR Hologic
BIOPAP QTS HPV Kit Loxo
Reveal HPV Real-Time HPV Detection Kit GenoID
AID STD assay GenID
AID HPV screening kit GenID
AID HPV typing kit GenID
Linear ArrayExtra HPV Genotyping Kit Innogenetics
PCR Human Papillomavirus Detection Set Takara Mirus Bio
HPV DNA Chip Biomedlab
Array Papillomavirus Genomica
ProDect Chip HPV typing Bcs Biotech S.P.A
PapType Genera Biosystems
LCD Array HPV 3.5 Chipron
Abbott RT HRHPV Test
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AnyplexII HPV28 Seegene®
HPV riskTest Self-Screen
Viroactiv Virofem
HPV OncoTest Invirion Diagnostics
Genpoint Tm HPV test Dako-Oxoid
Abbott RealTime High Risk HPV
Abbott
Luminex HPV Genotyping
Multimetrix/Progen
Papillocheck Greiner BioOne
PreTect HPV Proofer Norchip
GP5+/6+-PCR Diassay
BD Onclarity HPV assay
HPV Test principles
HPV testprinciples
 Signal amplification following hybridization
• Hybrid capture: Hybrid capture 2 (HC2; Qiagen): RNA probe cocktail
(commercial)
• Invader technology: Cervista HPV-HR (Hologic): invader probe
(commercial)
• In situ hybridisation (ISH): in situ hybridisation of DNA with probe cocktail
(commercial)
 Target amplification followed by detection
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Broad spectrum PCR
Isothermal E6/E7 RNA amplification methods
Commonly used read-out systems for PCR
assays
 Enzyme immuno assays
 Cocktail of oligoprobes per well (no genotyping)
eg. GP5+/6+-PCR, SPF10, Roche Amplicor
 Reverse hybridization assays (genotyping)
 Type-specific oligoprobes immobilized on:
 Strips/filters
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Linear Array (PGMY; Gravitt et al. 1998)
LiPA (SPF10; Kleter et al., 1999)
RLB (GP5+/6+-PCR; van de Brule et al., 2002)
Consensus HR HPV genotyping strip (GP5+/6+-PCR, PGMY, Amplicor; de
Koning et al., 2006)
• Most HPV test used in clinical practice
detect pooled hrHPV of 13-15 hrHPV types.
• Always HPV 16/18 included and often
separately reported
IARC classification of hrHPV types adopted by WHO
Based on phylogenetic and epidemiologic evidence
• Category 1: Carcinogenic:
HPVs of alpha 9 group: 16, 31, 33, 35, 52, 58
HPV of alpha 7 group:18, 39, 45, 59
HPV of alpha 5 group: 51
HPV of alpha 6 group: 56,66
• Category 2A: possible carcinogenic: HPV 68, 70
• Category 2B: possibly carcinogenic based on phylogenetic
relationship with 1 or 2A:
HPV of alpha 5 group: 26, 29,82
HPV of alpha 6 group: 30,53,
HPV of alpha 7 group: 85
HPV of alpha 9 group: 67
Schematic representation of primer target regions of different type. and broad-spectrum DNA-PCR-based assays. The
specific
approximate fragment lengths (in nucleotides) are shown in
parenthesis. The reference genome of HPV16 is used
Tests for pooled hrHPV detection (1)
• Signal amplification
– Hybrid capture 2
– Cervista HPV HR test
• PCR amplification and enzyme immuno-assays
– GP5+/6+ PCR-EIA
– SPF10-PCR-DEIA
– Amplicor human Papilloma virus
•
Quantitative PCR amplification with partial genotyping
(HPV 16/18)
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COBAS 4800 HPV test
Abbott RealTime High Risk HPV test
Self-Screen HPV-Risk assay
BD Onclarity HPV Assay
Cervista HPV 16/18 Test
Tests for pooled HPV detection (2)
• HPV DNA full genotyping tests using broad-spectrum
PCR
– With reverse hybridization:LiPA25 version 1 , INNO-LiPA HPV
Genotyping Extra , LINEAR ARRAY HPV Genotyping Test, The
digene HPV Genotyping RH Test , digene HPV Genotyping LQ Test ,
Greiner PapilloCheck HPV-Screening
• HPV DNA full genotyping tests using multiplex type
specific PCR
– MPTS12 and MPTS123 PCR (labo biomedical products Rijswijk,N)
• HPV DNA full genotyping with quantitative Type specific
pCR
– TS HPV qPCR assays (Merck)
– AnyplexII HPV28 (seegene)
• HPV mRNA detection tests with pooled HPV detection
– Aptima HPV assay
Some commercial HPV tests
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Hybrid Capture 2 Qiagen
Digene HPV genotyping RH kit Qiagen
Digene HPV genotyping LX kit, Qiagen
Amplicor HPV Test Roche
Cobas 4800 HPV Test Roche
Linear array Roche
SPF10/LiPa DDL
NucliSens EasyQ HPV Biomerieux
Aptima Gen-Probe
Cervista HPV HR Hologic
BIOPAP QTS HPV Kit Loxo
Reveal HPV Real-Time HPV Detection Kit GenoID
AID STD assay GenID
AID HPV screening kit GenID
AID HPV typing kit GenID
Linear ArrayExtra HPV Genotyping Kit Innogenetics
PCR Human Papillomavirus Detection Set Takara Mirus Bio
HPV DNA Chip Biomedlab
Array Papillomavirus Genomica
ProDect Chip HPV typing Bcs Biotech S.P.A
PapType Genera Biosystems
LCD Array HPV 3.5 Chipron
Abbott RT HRHPV Test
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•
•
•
•
•
•
•
•
•
AnyplexII HPV28 Seegene®
HPV riskTest Self-Screen
Viroactiv Virofem
HPV OncoTest Invirion Diagnostics
Genpoint Tm HPV test Dako-Oxoid
Abbott RealTime High Risk HPV
Abbott
Luminex HPV Genotyping
Multimetrix/Progen
Papillocheck Greiner BioOne
PreTect HPV Proofer Norchip
GP5+/6+-PCR Diassay
BD Onclarity HPV assay
Q: Which HPV test to use
A: Dependent of intended use
HPV tests vary in their property to detect
the various types of HPV infections
Important distinctions:
• Analytical sensitivity and specificity
Detect all hrHPV infections: both transient
(irrelevant) and transforming infections
• Clinical sensitivity and specificity
Detect mainly HPV infections associated with
CIN2/CIN/CxCa(clinically relevant hrHPV
infections):
Choice of HPV DNA test should be matched
with its intended use.
• HPV tests with high analytical sensitivity
– Epidemiologic studies e.g. Disease association studies
– Vaccine efficacy trials
– Surveillance of HPV prevalence
• Clinically validated HPV tests detecting HPV
infections associated with clinical meaningfull disease
i.e CIN2, CIN3 or CxCa
– Primary cervical screening
– Triage of smears with equivocal cytology
– Test of cure (monitoring women for post-treatment CIN2+
For HPV testing in cervical screening
clinical validation is necessary
For screening purposes it is imperative to detect
transforming HPV infections associated with
(pre)cancer i.e CIN2,CIN3,CxCa and ignore the
other types of HPV infections (i.e transient HPV
infections)
Otherwise too many women without lesions enter
into diagnostic evaluation. Increase COSTS!
 Clinical validation of HPV tests obligatory!
 International guidelines have been formulated
hrHPV testing for cervical screening
Women 30-60year
• Recognized clinically validated HPV assays used successfully
in large screening trials (Artistic, NTCC,POBASCAM,
Swedescreen, VUSA-SCREEN, Kaiser Permanente study):
HC2 and GP5+/6+-PCR:
• Clinical sensitivity for CIN2+ range from : 94.3%-97.7%%
– pooled sensitivity HC2 for CIN2+: 97.9% (95%CI: 95.9%–99.9%)
 High negative predictive value
• Clinical specificity for CIN2+ range from 93%-97%
– Pooled specificity of HC2 :91.3% (95% CI:89.5–93.1%)
 Limit number of false HPV test positives to be followed
 Increase in analytical sensitivity compared to HC2 or
GP5+/6+-PCR will result in at maximum a small gain in
clinical sensitivity but a major decrease in clinical specificity
(i.e. more false positives)
Example: Case-control study: women with CIN3 vs women with normal
cytology (30 years) and no CIN2+ in next 2 years
Screening cohort
90
Cases: CIN3
80
%
Controls: CIN1
70
60
50
p<0.001
Clinically Validated
test: GP5+/6+PCR
40
clinically nonvalidated test: SPF10
30
20
10
0
Cases
Controls
N=25
N=193
Clinically validated:
HC2 and GP5+/6+
In women with normal cytology false positivity rate of a clinically nonvalidated test was significantly higher than that of a clinically validated test;
true positive CIN3+ rate is similar
Result: Unnecessary F-up, expensive, harmful, and overtreatment of
Hesselink et al., 2008
women
Example: Case-control study: women with CIN3 vs women with normal
cytology (30 years) and no CIN2+ in next 2 years
Screening cohort
90
Cases: CIN3
80
%
Controls: CIN1
70
60
50
p<0.001
Clinically Validated
test: GP5+/6+PCR
40
clinically nonvalidated test: SPF10
30
20
10
0
Cases
Controls
N=25
N=193
Clinically validated:
HC2 and GP5+/6+
In women with normal cytology false positivity rate of a clinically nonvalidated test was significantly higher than that of a clinically validated test;
true positive CIN3+ rate is similar
Result: Unnecessary F-up, expensive, harmful, and overtreatment of
Hesselink et al., 2008
women
Viral load analysis in concordant vs
discordant SPF10/GP5+/6+-PCR samples
Type-specific real time PCR
9
HPV 16
HPV 18
HPV 52
p<0.001
p<0.001
p=0.006
log [HPV copies/scrape]
8
7
6
5
4
3
GP-/
SPF+
(n=13)
GP+/
SPF+
(n=6)
GP-/
SPF+
(n=14)
GP+/
SPF+
(n=4)
GP-/
SPF+
(n=15)
GP+/
SPF+
(n=4)
HPV type
Samples negative by GP5+/6+-PCR but positive with SPF10 had significantly lower
viral loads
low viral loads point to clinically irrelevant (transient) infections
Clinical validation of other HPV assays
• In order to become validated for use in cervical
screening candidate HPV assays should prove:
– their value in large prospective screening studies
or
– non-inferiority to validated reference assays (HC2
or GP5+/6+-PCR) in cross-sectional clinical
equivalence studies
• Consensus guidelines for test requirements have
been developed by an international consortium
•
(Meijer et al. : Int J Cancer, 2009)
International guidelines for HPV test requirements for
primary cervical screening (formulated relative to HC2)
Candidate test should:
•
Have a clinical sensitivity for CIN2+ not less than 90% of
that of HC2 (women ≥ 30 years of screening population,
pooled sensitivity HC2:97.9% (95%CI: 95.9%–99.9%)
 to be tested on at least 60 samples of women with CIN2+,detected
by both cytology and/or HPV test: High NPV
• Have a clinical specificity for CIN2+ not less than 98% of
that of HC2 (women ≥ 30 years of screening population
Pooled specificity:94.1% (95%CI 93.4–94.8%)
 to be tested on at least 800 samples of women without CIN2+
consecutively sampled from a screening poplation:
low nr of false HPV Test positives without disease
Meijer CJ, et al. Int. J Cancer 2009
International guidelines for HPV test requirements for
primary cervical screening (formulated relative to HC2)
Display intra-laboratory reproducibility and
inter-laboratory agreement with a lower confidence
bound 87%
 to be tested on at least 500 samples of which 1/3 is positive
with validated HPV test
Meijer CJ, et al. Int. J Cancer 2009
Clinical performance of HPV assays can be
different on self-collected vaginal samples
than on cervical scrapings
Comparison GP5+/6+-PCR and HC2 on LBC versus vaginal
brush/FTA cartridge samples (outpatient population)
Cervical LBC
samples
FTA cartridge selfsamples
100
%
CIN2/3: n=13
90
80
70
60
50
GP5+/6+-PCR
40
HC2
30
20
10
0
Sens CIN2+
Sens CIN2+
Particularly sensitivity of HC2 for CIN2+ is severely compromised
when applied to FTA cartridge samples
De Bie et al. J Mol Diagn, 2011
Comparison Cervista and PCR/MALDI-TOF on LBC
versus vaginal self-samples
Cervical LBC
samples
%
Vaginal selfsamples
CIN3+: n=141
Sensitivity for CIN3+ of Cervista but not PCR/MALDI-TOF significantly
lower when applied to vaginal self- versus physician- samples (p=0.0001)
Belinson et al. IJC, 2011
Conclusions
• International guidelines for HPV test performance
are useful for the approval of HPV tests by public
health authorities and clinical use in cervical
cancer screening
• For self-samples, combinations of HPV test/selfsampling device need separate validation analysis
to ensure sufficient clinical accuracy of HPV selfsampling
Clinically validated HPV assays for cervical
screening
Avaliable HPV
detection assays
Many (>40)
- Hybrid Capture 2
- Diassay (GP5+/6+PCR)
- COBAS4800
- APTIMA
- HPV RealTime
- SPF10
- Amplicor
- Cervista
- PapilloCheck
- PGMY
- … (and so on)
HPV tests validated
for cervical screening
(cervical scrapings)
- Hybrid Capture 2*
- Diassay (GP5+/6+-PCR)*
- COBAS4800**
- HPV RealTime**
- PapilloCheck**
HPV tests validated
for cervical vaginal
lavages (Delphiscreener) and brush
- Diassay (GP5+/6+PCR)
- HPV-Risk assay
- APTIMA**#
- HPV-Risk assay**
- BD Onclarity HPV
assay**
Abbott real time PCR**
*Based on longitudinal studies
**Based on equivalence analysis according to guidelines
# Provided that data of long term NPV of mRNA testing become available
How to choose the hrHPV test in NL
• Tender for : which hrHPV test with a suitable
platform for high throughput. HPV test should
also be applicable on selfcollected c/v specimen
• Tender for: liquid based cytology medium. All
samples collected in LBC. Cytology triage on
Physician taken cervical smears directly on left
overs
• Genotyping not included
www.negometrix.nl
Conclusions
• In NL hrHPV test selected
– on international validation guidelines
– and on additional requirements as high throughput and performing similar
as on c/v self-samples
• No genotyping readout required
• Triage done by cytology.
Cytology should be performed on
-LBC left-over from HPVpos. physician taken cervical
smear
-or on extra cervical smear from an HPV self-sample pos
women taken by a physician
Acknowledgements
Department of Pathology VUMC
•
P.Snijders
•
N. Fransen
•
D. Heideman
•
M. Verkuyten
•
L. Rozendaal
•
D. Boon
•
M. Gök
•
M. Lettink
•
B. Hesselink
•
F. Topal
•
R. Steenbergen
•
D. Buma
•
S. Wilting
•
M. Bogaarts
•
D.Rijkaart
•
R. van Andel
•
V. Verhoef
•
R. Pol
•
M.Uijterwaal
•
M. Doeleman
•
M.Dijkstra
Screenings organisations
Midden-West,Oost and zuid
RIVM
• Nynke van der Veen
Dept of Pathology Erasmus mc
• F. van Kemenade
Department of clinical epidemiology and
biostatistics VUMC
• H. Berkhof
• B.witte
Gynaecologic Oncology VUMC
• G. Kenter
Gynaecologic Oncology Erasmusmc
• Theo Helmerhorst
Gynaecologic Oncology UUMC
•
Rene Verheijen
EEC consortia
• PreHDICT
• CoHeaHr
ERC advanced Grant
• Mass-care
Dutch Cancer foundation
ZON-MW
Thanks to HPV Team VUmc 2004-2015
From L to R: Peter,Sigrid, Feja, Aletta,
Folkert,
Nicole, Chris,
Antoinette,Bart,Saskia,Maaike
Coöperating Gynaecologists
Rene Verheijen,
Theo Helmerhorst,
Gemma kenter
From L to R: Dorien,Daniëlle,Bart,Maaike,
Renske, Chris,Mariëlle, Peter,
Jacqueline, Murat
430 participating general
Practitioners and more than
350.000 women