andreaskaufmanncharitede Clinic for Gynecology CharitéUniversitätsmedizin Berlin Germany Screening Methods for HPV and Dysplasia Detection Prevention of Cervical Cancer THE ROLE OF HPV INFECTION ID: 776674
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Andreas M. Kaufmann, PhDandreas.kaufmann@charite.deClinic for Gynecology, Charité-Universitätsmedizin Berlin, Germany
Screening Methods for HPV and Dysplasia Detection
Prevention
of Cervical Cancer: THE ROLE OF HPV INFECTIONVilnius, 26.4.2017
Slide2The most important measure to
prevent cervical cancer in future is
HPV VACCINATION
Slide3Cervical
cancer – Prevention in GermanyTrends in Incidence and Mortality
Most
effective
cancer
prevention
programme
where
screening
established
Slide4© AMK
Progression from Infection to Cervical Cancer
7-30
years
Infection
CIN 1
CIN 2/3
Invasive
CxCa
Ca. 50%
8-18
months
infection
Virus
production
transformation
CxCa
invasion
DNA
Detection
E6/E7 RNA/Protein
detection
!
Cytology
Screening
Methods
:
Slide5abnormal cells in
micropscope
CxCa
Prevention (primary screening)
When
positive
triage
,
diagnosis, therapy
1) PAP smear
2) HPV Test
High-
risk
HPV16,18,31,33,…
Low-
risk
HPV6, 11, 42,…..
Slide6Primary Screening: HPV Detection vs Cytology
Cuzick et al., Int J Cancer 2008
HPV Test: high
Sensitivity
But
Cytology
:
low
Sensitivity / high Specificity
Internationally
shift from cytology to HPV Test2) WHO: „countries that do notyet have cytology established should start directly with HPV Testing“
3)
Maybe
use
cytology
as
triage
method
4)
Potentially
better
molecular
triage
tests
possible
Slide7© AMK
Parameter of Test Quality
Cytology HPV DNASensitivity (CIN2+) 50% (80%) >95%Specificity 98% 80-90%Positive Predictive Value >20-40% <10%Negative Predictive Value 50% >98%Post Test Probability ??? >10%
Depending on frame work conditions: Age, Interval, Test characteristics…
Which
conditions
adequate
for
the
program
?
Slide8© AMK
Cuzick et al., BJC 108 (2013)
@
Comparison of 6 HPV Tests in a Screening Population
Slide9© AMK
176 464 women aged 20–64 years were randomly assigned to HPV-based (experimental arm) or cytology-based (control arm) screening in Sweden (Swedescreen), The Netherlands (POBASCAM), England (ARTISTIC), and Italy (NTCC). Followed up for a median of 6.5 years (1 214 415 person-years) and identified 107 invasive cervical carcinomas
Invasive CxCa after initial Screening in RCTs in 2nd Round
HPV
HPV
Cytology
Cytology
-60-70%
-45%
Higher
sensitivity
for
CIN3 and therapy => less CxCa incidence in follow up
Ronco et al., Lancet 2014; 383: 524–32
missed
CIN3/CxCa in 1st round
new
CxCa in 2nd round
Slide10Elfgren
et al., 2016
Persistence
>12 month
CIN2+ Development after HPV Persistence
Genotype-specific persistence
unknown persistence
HPV negative / genotype change
Slide11© AMK
Quality Control for HPV Testing
Smear
quality
(
sampling
during
colposcopy
?)
Lab Infrastructure (
contamination
risk
, PCR
problem
)
validated
Test
Internal
control
for
cellular
DNA!
Monitoring
of
processes
Intra
/
inter
Lab
reproducibility
Proficiency
Testing
of
reference
laboratory
(e.g.,
Equalis
)
Slide12Targets for HPV Detection
E6-E7: Oncogenes
E1-E5: regulatory genes
L1-L2: Capsid
L1
L2
E4
E6
E7
URR
E5
E1
E2
HPV 16
7905
bp
GP-PCR
Systems:
MY09/11
GP5+/6+
SPF10
450
bp
150
bp
65
bp
Generic
primer
amplify
all HPV
types
L1
is
most
heterogenous
gene
(
selection
pressure
)
Multiplex PCR
mRNA
Oncoproteins
Multiplex PCR
Probe-
based
Systems
E6/E7
are
never
deleted
!
Slide13>220 commercially available HPV test systems
§ Guidelines recommend only HR-HPV testing as LR-HPV testing has no clinical use in CxCa screening or triage of positive cytology.
probes
DNA, PCR
RNA
Protein
Slide14© AMK
14 HR-HPV genotypes includedclinically validated versus Hybrid Capture 2 (HC2) and/or GP5+/GP6+ PCR-(EIA) (Diassay)HC2 und Diassay validated in large prospective clinical studies (women >30 years)Shows clin. Sensitivity for CIN2+ of 95%Shows clin. Specificity for CIN2+ of 90.7 to 94.1%New HPV test formats should have relative:90% of HC2 sensitivity98% of HC2 specificity
Guideline criteria for validation of HPV Test Formates
Meijer et al., J Clin Virol. 2009
Slide15Validation, ApprovalTarget sequenceType spectrumGenotypingPracticabilityCostTriage potential
Parameters for Choice
Adequacy
for
a
given
system
Slide16Commercial PCR-based HPV tests
TestTime to first resultReleased**TargetHPV types*ReportedQIA hc24.5-5 hours (6-7 hrs. total)March 2000Whole genome13 HRHR screenAbbott rtHPV4.5 hoursJan 2009***L113 HR+ HPV66HR screen16/18 includedCervista~3 hours (6-7 hrs. total)March 2009 (HR and 16/18)L113 HR + HPV66HR screen16/18 separate Cobas 48004.5 hoursApril 2011L1DNA13 HR+ HPV66HR screen16/18 includedAPTIMA 3.5 hrsOctober 2011E6/E7mRNA13 HR+ HPV66HR screen16,18/45 separate Anyplex II HPV HR – Seegene4.5 – 5 hrsJune 2012***L1DNA13 HR+ HPV66All genotypedBD Onclarity HPV3.5 hrsFeb 2014***E6/E7 DNA13 HR+ HPV66HR screen16, 18, 31, 45, 51, 52 includedXpert® HPV< 60 minutesApril 2014***E6/E7DNA13 HR + HPV66HR screen16, 18/45 included
* 13 HR types: 16, 18,45, 31, 33, 35, 39, 51, 52, 56, 58, 59 and 68
** USA FDA approval date unless otherwise indicated
*** CE-IVD
Slide17Partial
genotypingFDA approvalautomationMarket power
Cobas
(Roche)
Slide18Athena Studie: „Addressing the need for advanced screening“
47.000 Frauen, US multi-center Studie, doppel-verblindetCobas HPV Test (Roche) 12 hrHPV und HPV16/18 Genotypisierung93,5% Sensitivität, 69,3% Spezifität für CIN3, 0,3% falsch negativ
Identifizierung der Frauen mit höchstem absoluten/relativen Risiko einer CIN3+10% HPV16 und/oder 18 positive bei unauffälliger Zytologie hatten eine CIN3+ 16% HPV16 und/oder 18 positive mit ASC-US Zytologie hatten eine CIN3+
Wright TC et al., Am J Clin Pathol. 2011 Oct;136(4):578-586
PCR basierter
Cobas
Test:
Genotypisierung
und Sensitivität
Slide19CIN 3 Risk
when HPV16/18+ Cobas Roche HPV-Test
All HPV 16/18 positives (
as
of
age
25)
immediate
colposcopy
…?
Clinically
and
ethically
adequate
?
Slide20Genotyping more than 16 and 18 indicator for future disease:Genotyping HPV16/18 recomended by guidelines. Genotype 31 and 33 higher progression risk!>90% of Adenocarcinoma is 16, 18 and 45 associated. Typ 45 to be added to16/18, Detection type-specific persistence in Screening and follow up for risk assessment. Vaccinated populations: less genotypes 16/18 => new needs for Screening
Onclarity (BD): Extended Genotyping
Slide21BD Onclarity™ HPV Assay1 step for: 6 individual genotypes:16, 18, 31, 45, 51, 523 distinct groups: 33, 5856, 59, 6635, 39, 68
Extended genotyping: OnclARITY (BD) tEST
IC
16
18
45
IC
33,58
31
56,59,66
IC
51
52
35,39,68
Lower risk for disease
Basis for 77% invasive SCC and 94% of
AdCa
Regionale
Variance & Importance
Slide22Cepheid:
Xpert HPV – Test characteristics
3 simple steps:
Thinprep sampleRoutine LBC cytology
1 ml into Cartouche
Insert
Cartouche
start
automated
PCR
analysis
Result
in 60 min
Slide23Result – Xpert HPV (Cepheid)
Der
GeneXpert
®
HPV (Human Papillomavirus) Assay
qualitative
in vitro-Test from
ThinPrep
Pap-Test PreservCyt-LBC
.
Real-Time-PCR (
RT-PCR
) on target DNA (
On^cogene
E6/E7
) of
14 high-risk HPV-Types in a single reaction
Differentiation of :
HPV 16
HPV 18 and 45 in combination
11 other high-risk Types in a group result
(31, 33, 35, 39, 51, 52, 56, 58, 59, 66 und 68)
internal validity control
represents by 3 different curves P3, P4 and P5
Slide24Result presentation – Xpert HPV
Xpert
HPV Test uses 6 Fluorochrome channels
HMBS = Hydroxymethylbilan-Synthase (Housekeeping-Gen)
No
high
throughput
, simple, expensive
=>
adequate
?
Slide25Advantage by full genotyping
Multiple Types differentiatedHR-HPV differentiatedPersistence identical HPV type vs change in typeTest of cure – dysplasia-causing type eliminiert, reinfection with other type?HPV vaccine types identified (loss of protection?)
HR-HPV: 16, 18 (31, 45)
10-15%
70-85%
HR-HPV: 33, 35, 39, 51, 52, 56, 58, 59, 68, 73
70-85%
20-35%
Infection
CINIII/CxCa
Slide26Full HPV genotyping testing „HPV Array“ (AID/GenID)
Multiplex PCR
E1
gene
18 HR-HPV
11 LR-HPV
GAPDH
Read out by ELISA type stainingELISpot reader96 samples, high throughputAutomatic objective reading, evaluation and reporting in 10 minSimple handlingSimple PCR/reader equipment
CS PP 02.32
Slide27EliSpot
Reader and AiDot analysis software used for HPV Easy readout
10 min readout for
96 well plateEvaluation of HPV typesRelated to DNA contentGAPDH internal control
Available in many african countries
for CD4 characterization in HIV+
Slide28Full Genotyping: HPV Multiplexed Genotyping (WHO Reference test)
Schmitt M. et al., JOURNAL OF CLINICAL MICROBIOLOGY, 2006 und 2008
GP5+/6+- L1 PCR
PCR product
bonds on
beads
Biotinylated primer
high
analytical
sensitivity
=>
Epidemiology
Slide29Nr.HPV 6HPV 11HPV 16HPV 18HPV 26HPV 31HPV 33HPV 35HPV 39HPV 42HPV 43HPV 45HPV 51HPV 52HPV 53HPV 54HPV 56HPV 57HPV 58HPV 59HPV 66HPV 68HPV 70HPV 72HPV 73HPV 82HPV resultß-ControlPK mix7515411742824241121910471.551211963104.5365754424692410ok NK MM11611032223232225435.5256.51.536ok 111512132.52223223224361.545135NN1234ok21117751222.52223223323436.526523516 602ok321111215322.5323323463.55.53562134.5731782ok4118.501.513222.53233223437266245NN 763ok511101214323323332252.56266235NN 908ok61181145331332380432.5253.5626524431, 52 845ok792191114312322322253626633561835ok811911143242.5233223436265225NN 948ok9118.50.52133123233222426275245NN1487ok10112739111.5421.523232333547256234161204ok111190213.53122242333536265235NN 630ok12119121.53323333222.53436266235NN 7 DNA EXTR
Result Multiplexed Genotyping Gyn Lab Charite
Full
Genotypising
HPV Typ
Number
MFI
value
relative
to
ß-Globin internal
control
Ca. Virus load
Multiple
Types
Non-
sufficient DNA amount
Slide30New Test Systems
and Molecular Triage
Reflex cytology? (co-testing!)All colposcopy?additional biomarker?
Molecular
Triage?
Biomarker
Methylation
marker
HPV
oncoprotein
expression
strength
Slide31© AMK
Slide32Arbor Vita E6 Cervical Test(Biomarker for disease)Triage test!
Simple lateral flow Immunoassay Test Detection of HPV16 and 18 (typing), 5 additional HPV types in development9 samples in <3 h (hands on time appx. 2 h)Sensitivity for CIN3+ 53.5% for CervixCa 91.7%Specificity for CIN3+ 98.9%PPV for CIN3+ 40.8%NPV for CIN3+ 99.37%
Zhao et al., 2013
High
specificity
for
disease
Simple, robust
technology
Low
resource
settings
Slide33Simplicity of Oncoprotein E6 Cervical Test (Workstation Setup)
Slide34© AMK
Positivity according to disease stage
Slide35© AMK
Onco
E6TM positive test indicates high risk for future disease
Risk prediction: Follow-Up Study, 1 year, 5 years
1 year risk for CIN2+/CIN3+ after positive Test in NILM Pathology patients
30
fold
risk after 12 months88 fold risk after 5 years.
44% after 5
years
!
Slide36Screening results and disease burden
AVE6HR-HPVscreening positive2.1% (41/1999)37.6% (752/2000)triaging85% (35/41) colpo38.4% (298/752) cytotreatment referral 28.5% (10/35) w/changes5.5% (16/298) ASCUSdisease found100% (10/10)6.25% (1/16)
We
need
a
test
with
high
sensitivity
and
even
more
specificity
!
Also AVE6
allows
risk
assessment
.
Slide37Biomarker
for high-grade and progressive dysplasia
© AMK
E6 Oncoprotein Expression
CIN2/3
E6 neg.
50%?
E6 pos. 50%?
CxCa
E6 pos.
100%
Only truely progressive dysplasia expresses E6 in detectable amount
CIN1
E6 neg. 0.5%
E6 pos. 14,5%
Slide38Summary
Primary HPV
testing
can
improve
CxCa
screening
due
to
higher
sensitivity
Specificity
problem
to
be
solved
by
biomarkers
Adequacy
of
the
test
for
a
given
situation
Genotyping
can
be
important
New
assay
formats
can
improve
existing
possibilities
and
add
a
prognostic
information
Slide39