Transcript Document

BD Onclarity™ HPV Assay:
Design and Applications within
a Health Technology in Change
LMC congress 2013, Cape Town
Dr. Martin Ryser
BD Diagnostics
Outline
• Overview on HPV test technologies
• The BD HPV solution: BD Viper™ LT and
BD Onclarity™ HPV Assay
BD Totalys™ System
Application of HPV testing in
cervical cancer prevention
• Primary screening with Cytology. Triage of low
grade cytology (ASCUS or LSIL) with HPV
testing
• Primary Screening with HPV testing. Triage of
HPV positive women with cytology.
• Co-testing (widely used in the US): Primary
screening with both cytology and HPV.
• HPV testing for test of cure/treatment follow up
after conization.
BD Totalys™ System
HPV testing: Molecular target selection
E6/E7 Region
L1 Region
- Coding for major viral
Capsid protein
- Used in Vaccination
- Strongly conserved amongst
High rísk HPV types
- Consensus primer sets
Binding sites: SPF10,
GP5+/6+, MY09/11
BD Totalys™ System
- Coding region for
viral oncogenes
- Target for mRNA
detection
- Less conserved
- Type specific
primer sets needed
HPV-DNA Detection via Signal Amplification
Qiagen Hybrid Capture II
• Full genome probes
• Based on the number of
scientific proof sources over 15
years HCII is still an important
comparator assay
• Cross reactivity with certain LR
types
• No cellularity control
• No genotyping
• Requires large amount of
sample
• Limited automation
BD Totalys™ System
HPV-DNA Detection Via Consensus Primer PCR
Roche cobas, Abbott RealTime
• Real-time PCR
• Modified L1 consensus primers
• Cellularity control
• Can do 16/18 genotyping in a
single well with primary reaction
• Clinical sensitivity can be lower
than benchmark due to L1
deletion in cancer cases
BD Totalys™ System
HPV-mRNA Detection
Gen-Probe Aptima
• Detects E6/E7 mRNA
targets via transcription
mediated amplication
• High documented CIN2+
sensitivity >90% (Aptima)
• Improved clinical specificity
to detects CIN2+
• No cellularity control
• Genotyping (16, 18/45) only
available as separate assay
• mRNA based technology
with lacking proof of
longterm NPV and
undetermined optimal
screening intervals
BD Totalys™ System
Genotyping Via PCR and Hybridization
Roche Linear Array, LIPA
• L1 consensus primers
• PCR amplification
• Hybridization
• Provides full HPV
genotyping of high risk and
low risk types
• Manual procedure for low
throughput
• Open system with
contamination risk
• No clinical systems
BD Totalys™ System
The BD HPV Solution
Overview:
• The BD Solution: BD ViperTM LT and BD
Onclarity™ HPV
• Rationale for BD Onclarity™ HPV test design
• Performance data
• Application of BD Onclarity™ HPV in different
screening algorithms
•
BD Totalys™ System
BD VoC outcome: The Need for Flexibility and
Adaptability in HPV Testing
“Diagnostic accuracy is key. I can
not afford any false negatives”
BD Totalys™ System
“HPV testing will be run in a lab with
limited experience in molecular
testing. Therefore, we need a closed
system with simple operation and
minimal contamination risk "
The Instrument: BD Viper™ LT System
• Dual iso-thermal (SDA) and
thermalcycling (real time PCR)
reader for classic VIPER assays
(GC/CT, HSV) and HPV detection
• Fully automated specimen
processing and molecular testing
• Ready to use reagents stored at
room temperature
• Tabletop Platform.
• Flexible sample collection
media
– BD SurePathTM Liquid Cytology
– BD Co-collection Tube
– Hologic ThinPrep® Liquid Cytology
• Up to 120 patient specimens
processed per 8 hour shift for
HPV testing
BD Totalys™ System
The Assay: BD Onclarity™ HPV
• Real-time PCR Technology
• Four optical channels: ROX,
FAM, HEX, Cy5
• Unique molecular targets:
type specific primer sets
detecting HPV E6/E7 DNA
• Cellularity control in each PCR
• Flexible genotyping readouts
available at the moment of
screening
BD Totalys™ System
BD Onclarity™ HPV: The E6/E7 DNA-Target
Why E6 E7 instead of L1?
• In the course of cellular
transformation HPV often
integrates into the host genome.
During integration viral
sequences like the E1 or L1
regions can be deleted, while
the oncogenic regions E6/E7
are almost always preserved.
• It has been demonstrated that
around 5 to 20% of cervical
cancers show L1 deletions and
might be missed when using L1
consensus primer PCR.
BD Totalys™ System
L1 versus E6/E7 cancer
Cancer
Type
Primary L1 HR
Positive (%)
L1 Missed Reflex E6/E7
HR Positive (%)
Number of Inferred
L1
deletions (% Total)
Reference
7 (9.1%)
Zielinski et al.,
J Pathol 2003;
201: 535–543
(HPV type missed by
L1)
Adeno
Carcinoma
65/77 (84.4%)
BD Totalys™ System
7/12 (58.3%)
(HPV 16, 18, 45)
L1 versus E6/E7 cancer
Cancer
Type
Primary L1 HR
Positive (%)
L1 Missed Reflex E6/E7
HR Positive (%)
Number of Inferred
L1
deletions (% Total)
Reference
16 (22.5%)
Hagmar et al.,
Medical Oncol.
and Tumor
Pharmacother.
1992; 9(3): 113117
114 (29.2%)
Karlsen et al.,
J. Clin.
Microbiol.
1996; 34: 20952100
(HPV type missed by
L1)
L1 Primer
Detection (%)
E6/E7 Type Specific
Primer Detection (%)
32/71 (45%)
48/71 (67.6%)
Invasive
Squamous
Carcinoma
(HPV 16, 18, 31, 33)
Invasive
Cervical
Carcinoma
217/391 (55.5%)
BD Totalys™ System
331/391 (84.7%)
BD Onclarity™ HPV : The E6/E7 DNA-Target
Why DNA instead of mRNA?
• DNA based assays have demonstrated outstanding longterm NPV in numerous studies,
justifying screening intervals of up to 10 years after a negative HPV screening result.
Mark Schiffman
IPV 2010
•
E6/E7 RNA based assays show high sensitivity and specificity in cross sectional studies,
however longitudinal outcome data is not available
BD Totalys™ System
BD™ HPV–GT: The E6/E7 DNA-Target
Why DNA instead of mRNA?
• DNA based assays have demonstrated outstanding lonterm NPV in numerous studies,
justifying screening intervals of up to 10 years after a negative HPV screening result.
Mark
Schiffman
„...HrHPV tests that do not focus on the detection of DNA (e.g.
those
targeting mRNA)
do not comply with the guidelines as their NPV is not known,
which
means that the
IPV
2010
optimal screening interval is also unknown. In such cases, largescale longitudinal
studies are needed...“
(Dutch Health Council, advisory report Population screening for cervical cancer)
„...the available knowledge on the low-risk period after a negative HPV DNA test
(that defines the appropriate screening interval) cannot be automatically applied to
non-HPV DNA tests (such as HPV viral oncogene mRNA tests or other Biomarkers)
and longitudinal data are needed for the latter...”
(Ronco et al. Health Technology Assessment report, Epidemiologia & Prevenzione
2012)
• E6/E7 RNA based assays show high sensitivity and
specificity in cross sectional studies, however
™ System
BD Totalys
longitudinal
outcome data is limited
BD ViperTM LT and BD Onclarity™ HPV Assay:
Harmonized Workflow
Specimen Input:
0.5 mL
Step 1: Pre-warm
Specimens
BD SurePath™
BD SurePath™
+ 1.7 mL
HPV
diluent
Step 2: BD FOX™
DNA Extraction
Step 3: PCR Amplification
Integrated
real time PCR
Load and Go
G1
G2
G3
IC
IC
IC
16
33_58
51
Iron Oxide dissolvable film
18
31
52
BD FOXTM Extraction:
1. Bind DNA
2. Wash
3. Elute
4. Neutralize
45
59,
56_66
35,
39_68
0.8 mL
ThinPrep®
2.2 mL
HPV
diluent
BD
BDCo-collection
Totalys™ System
Device
Lysis and
Homogenization
Real-time PCR
Detection and Results
Output for all 3 GT
Tubes
BD Onclarity™ HPV Performance Data:
Study on 473 archived referral samples in comparison to
HCII and Linear Array
Diagnostic performance for detection of CIN3+
Sensitivity
Specificity
PPV
NPV
BD HPV 90.0%
43.9%
12.9%
97.9%
HCII
41.8%
12.2%
97.3%
87.5%
Good concordance for typing results with Roche Linear Array
(average kappa 0.65) and Line Blot assay (average kappa 0.82)
BD Totalys™ System
“…we demonstrated that the clinical performance of the BD assay
was clinically comparable to HC2, a well-validated clinical test and
benchmark for clinical carcinogenic HPV DNA detection….”
BD Totalys™ System
BD Onclarity™ HPV : Detection of
Mixed Infections
HPV plasmid targets extracted from SurePath on BD Viper LT at
challenging level (~ 3X C95).
GT Tube**
Individual Target
~ 3X C95
Competing Targets
at 1 x 106 Copies (each)
Individual Target
Detected
16
18, 45
Yes
18
16, 45
Yes
45
16, 18
Yes
31
33, 56, 58, 59, 66
Yes
58*
31, 56, 59, 66
Yes
66*
31, 33, 58
Yes
51
52, 35, 39, 68
Yes
52
51, 35, 39, 68
Yes
35*
51, 52
Yes
G1
G2
G3
* Representative for each paired assay was chosen.
** Human Cellular Background Present. (C33a cells at 1.4 x 105 per Sample)
• BD Onclarity™ HPV shows no interference of competing
targets that are added in 1000 to 10000 fold excess, providing
highly accurate results in mulitple infections
• Cross reactivity with low risk types is absent
BD Totalys™ System
SurePath sample stability for BD
Onclarity™ HPV testing
45 BD SurePath™ and 45 Hologic PreservCyt® specimens originally tested in the Fall of 2009
were retested with BD Onclarity™ HPV again in the Spring of 2012 (median age 2.6 years, stored
at 2-8°C) demonstrating very consistent results between fresh and archived samples.
Vaughan et al. IPV congress 2012
• Results demonstrate that HPV DNA can be efficiently extracted from
archived SurePath samples on BD VIPER LT
• Preliminary data suggests that HPV-DNA can be efficiently extracted from
FFPE histology sections on BD VIPER LT
BD Totalys™ System
HPV Genotypes Associated with Cervical Cancer in Africa
% of cervical cancers with indicated HPV types in Africa
60%
50%
48%
40%
30%
23%
20%
10%
10%
5%
3%
2%
2%
1%
52
31
51
33
0%
16
18
45
35
HPV types
de Sanjose et al. 2010 Lancet Oncology
Current HPV vaccines cover HPV 16 and HPV 18, which account for only 71%
of all cancers, which is not sufficient stop screening in vaccinated populations.
BD Totalys™ System
BD Onclarity™ HPV : Genotyping Beyond 16 and 18
BD Totalys™ System
The BD Onclarity™ HPV in Various Test Algorithms
Assess hrHPV status
Triage of abnormal cytology
Test of cure
HPV screening with cytology triage
Qiagen, Gen-Probe, Roche, Abbott, BD Onclarity™ HPV
Result is positive or negative. No Genotyping
Asses hrHPV status and
type for HPV 16 or 18
Co-testing with 16 18 risk stratification
(see scheme below)
US ASCCP guidelines
Danish check-out test algorithm
Roche, Abbott, BD Onclarity™ HPV
Result is positive or negative with limited Genotyping
Wentzensen 2010 at the Lancet
Oncology congress
BD Totalys™ System
The BD Onclarity™ HPV in Various Test Algorithms
Assess hrHPV status and
extend genotypíng beyond 16 18
Direct risk stratification for types 16 18 45
Test for type persistence in screening
Or test of cure after treatment
BD Onclarity™ HPV
Genotyping results provided for 6 individual types
(represents >90% of all cervical cancers) plus additional
3 sub groupings
• In total 10% of cervical cancers are associated with type
45 in Africa. Most of the type 45 associated cancers are
Adenocarcinoma that are often missed in cytology.
• HPV 45 should be added to 16/18 to rule out type 45
related adenocarcinoma. Negativity in 16, 18 and 45 helps
to increase cytology triage efficiency.
BD Totalys™ System
BD Onclarity™ HPV in Vaccination Surveillance
• Provides extended genotyping on a population base,
which can not be matched with small scale genotyping
surveillance studies that are conducted in reference
centers only.
• Typing beyond 16 and 18 is necessary to monitor:
– HPV type incidence and type persistence in a
vaccinated population
– Determine efficiency und durability of Vaccine
cross protection
– Monitor possible type replacement
– BD™ HPV-GT Design prevents biased
amplification for correct assessment of vaccine
effects.
BD Totalys™ System
The BD™ Totalys System Overview
BD VIPER
Table
SingleLT:
Vial
for top model,
BDMiddleware
Onclarity™ solution
HPV: Safe
and adaptable
provides
integrating
extraction
Cytology
and with Real
HPVbidirectional
technologycommunication
based the unique E6 E7
time Molecular
PCR. Ready
to use reagents
testing
DNA target
into LIS system
aiding in
stored at room temperature
sample traceability, risk
stratification, and lab
accreditation
Fully Integrated Automation
NEW BD SurePath
Collection Vial
2D All
barcodes
“Not
LBC’sand
areinstrument
the same”
functionality
provide
fullUnsats
chain ofand
BD SurePath
provides
lowest
custody
from
sample
collection
Highest Disease detection. to
results reporting
C-Tube Rack
BD HPV
BD CT/GC
Cytology
Cell Pellet
Monarch
Middleware
BD SurePath
BD Totalys™ System
28
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Thank You!
BD Totalys™ System