Comparison of the Roche AMPLICOR® Human Papillomavirus (HPV)

Test with the GP5+/GP6+ PCR HPV assay in cervical samples with
known cytology and histology
Adriaan JC van den Brule, Elna Moerland, Peter Wiering, Joost van Toren, Paul JM Klinkhamer
Laboratory for Pathology, PAMM Institute, Eindhoven, The Netherlands

Introduction AMPLICOR HPV Test

● Human papillomavirus (HPV) DNA testing has become an important component of the GP5+/GP6+ PCR EIA
100
clinical management of cervical premalignant lesions and cervical cancer screening programs1–6
● The Digene Hybrid Capture® 2 (hc2) test and in-house general primer-based PCR 80

Prevalence (%)
systems, such as GP5+/GP6+, are most commonly used
● Unlike hc2 testing, PCR DNA testing can be readily applied to archival paraffin-embedded 60
specimens
40
● PCR-based HPV DNA tests, such as the GP5+/GP6+ system7 and the PGMY system,8 are
clinically validated and have been used for research purposes, but there has been a lack of 20
commercially available, standardized kits for use in clinical practice
● Evaluation of the analytical and clinical sensitivity of new HPV tests is needed before their 0
CIN0 CIN1 CIN2 CIN3 Condyloma
implementation in clinical practice and cervical cancer screening programs n = 35 n = 28 n = 30 n = 28 acuminata
● The new Roche AMPLICOR® HPV Test9 was recently introduced by Roche, and detects Grade of dysplasia n = 11
13 high-risk (HR) HPV types Both methods identified the same CIN2 and CIN3 samples

Figure 2. High-risk HPV prevalence in archival specimens (n = 132)

Objective
● To compare the AMPLICOR HPV Test and the GP5+/GP6+ PCR enzyme immunoassay
AMPLICOR HPV Test
(EIA) in cervical samples with known cytology and histology 100
GP5+/GP6+ PCR EIA

Materials and methods Prevalence (%)

80
● A total of 132 samples were obtained as archival paraffin-embedded tissue sections from
women with diagnoses ranging from normal to CIN3 histology 60
● A total of 266 cervical scrapes were collected in PreservCyt® preservative solution (Pap1,
Pap2, Pap3A) and stored at room temperature 40

● DNA was extracted using the High Pure PCR Template Preparation procedure for the
cervical scrapes; paraffin-embedded tissues were pretreated with proteinase K and boiling 20

● AMPLICOR HPV Test:

0
– Multiplex PCR for 13 HR HPV types and the human β-globin gene as an internal
Pap1 Pap2 Pap3a Pap3b
control n = 31 n = 139 n = 99 n=2
– Detects PCR products in a microwell plate (MWP) format, using a cocktail of
13 oligoprobes Figure 3. High-risk HPV prevalence in cervical scrapes (n = 266)
● GP5+/GP6+ PCR EIA:7
– One set of general primers, detecting a broad range of HPV genotypes
– Detects PCR products in an MWP, using either a HR or low-risk cocktail of probes Conclusions
– β-globin PCR performed separately for quality control of samples
● In general, both the AMPLICOR HPV Test and the GP5+/GP6+ PCR EIA could
● Discrepant samples were re-tested
be successfully performed on very small aliquots of samples in liquid cytology
X 5 AMPLICOR primers media, or on samples obtained from archival paraffin-embedded tissue
AMPLICOR 165 bp ● Both methods identified the same CIN2/CIN3 samples, with the exception
X 7 AMPLICOR primers of two cases (one AMPLICOR-negative and one GP5+/GP6+-negative)

HPV L1 gene ● Discrepant cases (mostly Pap 1/2 cytology and condyloma acuminata
CIN0 and CIN1 samples) may be reflecting low copy-number samples of
GP5+/GP6+ primers
HR HPV, and it will be interesting to determine whether these are clinically
GP5+/GP6+ 150 bp
relevant HPV infections (i.e. leading to progressive cervical disease)10
GP5+/GP6+ primers
● Although the analytical sensitivity of the AMPLICOR HPV Test appears to
Figure 1. AMPLICOR and GP5+/GP6+ primers be higher than that of the GP5+/GP6+ PCR EIA, clinical sensitivity has yet

Results to be studied

Table 1. Target genotypes

References
AMPLICOR HPV Test GP5+/GP6+ PCR EIA
1. Meijer CJ, Snijders PJ, van den Brule AJ. CMAJ 2000;163:535–8
HR HPV types: HR HPV types: 2. Nobbenhuis MA, Walboomers JM, Helmerhorst TJ et al. Lancet 1999;354:20–5
16, 18, 31, 33, 35, 39, 45, 16, 18, 31, 33, 35, 39, 45, 3. Lorincz AT, Richart RM. Arch Pathol Lab Med 2003;127:959–68
51, 52, 56, 58, 51, 52, 56, 58,
4. Schiffman M, Herrero R, Hildesheim A et al. JAMA 2000;283:87–93
59, 68 59, 66, 68
5. Petry K, Menton S, Menton M et al. Br J Cancer 2003;88:1570–7
LR HPV types: 6. Munoz N, Bosch FX, de Sanjose S et al. N Engl J Med 2003:348:518–27
6, 11, 26, 32, 34, 40, 42, 43, 44, 53, 54, 55, 57, 61, 67, 70, 7. Van den Brule AJ, Pol R, Fransen-Daalmeijer N et al. J Clin Microbiol 2002;40:779–87
71 (CP 8061), 72, 73, 81 (CP 8304), 82/MM4, 82/IS39,
8. Coutlee F, Gravitt P, Kornegay J et al. J Clin Microbiol 2002;40:902–7
83 (MM7), 84 (MM8), 85, 86, CP 6108, JC 9710
9. Kornegay JR, Shepard AP, Hankins C et al. J Clin Microbiol 2001;39:3530–6
HR: High-risk; LR: Low-risk 10. Snijders PJ, van den Brule AJ, Meijer CJ. J Pathol 2003;201:1–6