Human Papillomavirus EQA
Pilot Program
(QCMD HPVDNA09/10/11/12)
Ed Schuuring
Pathology, UMCG, Groningen, NL
Scientific expert and advisor for QCMD HPV Program
Lid SKML-beleid, sector Pathologie
Presentatie SKMLdeelnemersvergadering
5 februari 2013
HPV in scrapings of (pre)malignant cervical lesions
~4% high-risk HPV-positive
~85% high-risk HPV-positive
Normal cervix
CIN III
100% high-risk HPV-positive
Nieuwe praktijkrichtlijn BVO cervixcytologie:
indicatie HPV-onderzoek bij Pap2/3A1
Pap 1
97.1%
Retour BVO 5 jaar
HPV-
Pap 2
Pap 3A1
2.1%
Herhalen
6 maanden
+ HPV
Pap 3A2 >
0,6%
Bulkmans, the Lancet, 2007
Bais, Int J Cancer, 2005
Rebolj, Int J Cancer, 2007
Pap 1
70%
Pap 2
Pap 3A1
25%
Pap 3A2>
5%
HPV+
HPV49%
Herhalen 12 maanden
HPV+
51%
Gynaecoloog
J Eijsink
The early detection of cervical cancer in scraping
population-based screening programs worldwide
1) morphocytology only
•
The classical approach
2) primary morphocytology and HPV reflex testing
•
Presently used commonly (eg Dutch guidelines)
3) morphocytology/HPV co-testing
•
New guideline in USA (Saslow 2012)
4) primary HPV testing and reflex morphocytology
•
New guideline in NL (under review with minister) in 2013 ?
Commercial HPV tests
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Hybrid Capture 2 Qiagen
Digene HPV genotyping RH kit Qiagen
Digene HPV genotyping LX kit, Qiagen
Roche Amplicor HPV Test
Roche Linear array HPV Genotyping test
Innogenetics INNO-LiPA HPV Genotyping test
NucliSens EasyQ HPV Biomerieux
Aptima Gen-Probe
Human Papilloma Virus reagents Third Wave
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
Seeplex HPV Genotyping Seegene
Viroactiv Virofem
HPV OncoTest Invirion Diagnostics
Genpoint Tm HPV test Dako-Oxoid
Abbott RealTime High Risk HPV Abbott
Luminex HPV Genotyping, Multimetrix/Progen
Greiner PapilloCheck HPV Screening
PreTect HPV Proofer Norchip
………
Analytical and clinical sensitivity of HPV-detection assays
Clinical sensitivity of HC2 is
5000 HPV copies
Analytical sensitivity of PCRbased methods detecting
<10 HPV copies
Adapted from Snijders et al. Journal of Pathology 2003; 201:1-6
Clinical validated HPV-tests (in the Netherlands)
Digene HC2 HPV test (Qiagen)
GP5+/GP6+ PCR EIA (Vumc/Qiagen)
Cobas 4800 HPV test (Roche)
Cervista hrHPV test (Hologic)
GenProbe-Aptima hrHPV assay (Hologic)
Abbott realtime HR HPV assay (Abbott)
Kwantitatieve multiplex RT HPV test (PON)
FDA-approved clinical HPV tests
Digene HC2 HPV test (Qiagen)
Cervista HPV HR test (Hologic) > internal control
Cervista HPV 16/18 test (Hologic) > internal control
GenProbe-Aptima hrHPV assay (Hologic) > based on RNA
Cobas 4800 HPV test (Roche) > HPV16/18 separate and internal control
available HPV EQA platforms
1) QCMD HPVDNA:
• using established cell lines in LBC (4 HPV types)
2) WHO HPV panel:
• Plasmid DNA spiked into cell line DNA (>30-45 types)
3) NEQAS UK:
• Patient samples (~4 samples)
Fagan, JClinVirol2010
QCMD 2009-2012 Human Papillomavirus DNA
QCMD-HPV EQA Pilot Program
Internationale rondzending:
* Analytische sensitiviteit
* Klinische sensitiviteit
UMCG: Ed Schuuring (Scientific Advisory Board)
UMCG: Lorian Slagter-Menkema (preparation/validation)
Reference-labs: UMCG, UMCU, VUMC, UMCRadboud
QCMD: Paul Wallace, Catherina di Lorenze
QCMD = Quality Control for Molecular Diagnostics (Scotland)
QCMD 2009/10/11/12 Human Papillomavirus DNA EQA Pilot Program
doel
Primaire doelstelling:
•
Aanbieden van een panel om de klinische-relevante sensitiviteit van de in
lab gebruikte test te valideren
Secondaire doelstellingen:
•
Aanbieden van een panel om de analytische sensitiviteit van de in lab
gebruikte test te valideren
•
Aanbieden van een panel om de accuraatheid van de genotypering van de
in lab gebruikte test te valideren
• Door simulatie van klinische samples mbv “established”
BMKH-cellijnen in dunne-laag-cytologie
QCMD 2009/10/11/12 Human Papillomavirus DNA EQA Pilot Program
participation
HPVDNA09
HPVDNA10
HPVDNA11
HPVDNA12
(not finished)
Participants
108
155
167
171
136
149
153
Responders
98
Countries
26
26
27
31
Deelnemers in
Nederland
14
21
27
?
Datasets:
- Analytical
- Clinical
- Genotyping
113
113
66
44
77
84
88
133
114
91
144
115
QCMD 2009 Human Papillomavirus DNA EQA Pilot Study
panel composition of first EQA
Clinical testing
• 10 samples in original PreservCyt tubes
• 7 dilutions of cervical cancer cell lines with approx 400 HPV16 copies/cells:
HC2 was used to determine the clinical-relevant threshold
• 2 positive controls (HPV18 and HPV67) and single HPV-negative control
• all samples tested in a second reference-lab (VuMC, Amsterdam, Peter Snijders)
QCMD 2011 Human Papillomavirus DNA EQA Pilot Study
panel composition of first EQA
Clinical testing
• 9 samples in original PreservCyt tubes
• 5 dilutions of cervical cancer cell lines with approx 400 HPV16 copies/cells
HC2 was used to determine the clinical-relevant threshold
• 2 positive controls (HPV18 and HPV45) and two HPV-negative controls
• all samples pre-tested in reference-labs by PCR (1x), HC2 (2x), cobas (1x)
HPVDNA11 – Clinical results
1: All participants
HPVDNA11 – Clinical results
1: All participants
2: The Netherlands (8x HC2 en 15 PCR-based HPV tests)
Rapportage van lab dat clinical testing uitvoert
performance-score
Rapportage van lab dat clinical testing uitvoert
(Cumulative %) performance of clinical interpretation
Clinical performance of all versus Dutch participants
QCMD-HPVDNA2011
Performance of
all participants
Performance of
Dutch participants
QCMD 2011 Human Papillomavirus DNA EQA Pilot Study
overall clinical performance
Overall performance poor
Expected to be below the HC2=1.0 threshold
(representing analytical-pos/clin-neg sampeles
Performance clicinal test poor
(should be negative in all 100%)
Performance analytical test is poor
(should be negative in clinical setting (100%)
QCMD 2011 Human Papillomavirus DNA EQA Pilot Study
panel composition of first EQA
Analytical testing
• 9 samples in original PreservCyt tubes
• 5 dilutions of cervical cancer cell lines with approx 400 HPV16 copies/cells
HC2 was used to determine the clinical-relevant threshold
• 2 positive controls (HPV18 and HPV45) and two HPV-negative controls
• all samples pre-tested in reference-labs by PCR (1x), HC2 (2x), cobas (1x)
HPVDNA11 – Analytical results
1: All participants
2: The Netherlands
Analytical performance of all versus Dutch participants
QCMD-HPVDNA2011
Performance of
all participants
Performance of
Dutch participants
QCMD 2009/10/11 Human Papillomavirus DNA EQA Pilot Study
overall performance
2009
2010
2011
Analytical 100%
Analytical 1x incorrect
Analytical FP
nd
nd
5.1%
40.6%
77.0%
14.1%
59.1%
81.8%
4.5%
Clinical 100%
Clinical 1x incorrect
Clinical FP
nd
nd
5.1%
45.5%
67.6%
13.0%
5.3%
27.9%
4.5%
1) High FP rate > 2/10 samples are true negative controls
2) Very low overall clinical performance > only high positive (Caski) with 50-500 HPV16copies/cell) and very low copy numbers (SiHa with ~2-3 HPV16-copies/cell
Pre-test threshold van klinisch-relevante HPV assays
Clinical HPV-tests:
HC2: 1 pg/ml = ~5000 kopieën
Abbott: = ~5000 kopieën
Cobas = ~300 kopieën (afhankelijk van HPV-type)
Dus eigenlijk kunnen we geen panel definiëren met een
klinische threshold omdat HC2 niet meer de standaard is
QCMD HPV testen vanaf 2013 (planning)
Vanaf 2013 alleen toetsing performance van analytische interpretatie
HPV-positieve, klinische relevante samples alleen als educatief samples in
panel (eigen interpretatie op basis van performance van andere met
vergelijkbare testen)
Vanaf 2013 verwoording mbt klinische interpretatie verbeteren
Kleinere panels en meer rondzendingen/jaar
Andere matrices (SurePath, DNA, freeze-dry)
Andere HPV-types, dubbel-/triple-infecties
Ontwikkelen van referentie/calibratie-sets
Advisory board is aangevuld met internationale assessors
Dank voor uw aandacht
Vragen ?
e.schuuring@umcg.nl