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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

for Pathology, PAMM Institute, Eindhoven, The Netherlands Introduction ● Human papillomavirus (HPV) DNA testing has

Introduction

Human papillomavirus (HPV) DNA testing has become an important component of the clinical management of cervical premalignant lesions and cervical cancer screening programs 16

The Digene Hybrid Capture® 2 (hc2) test and in-house general primer-based PCR systems, such as GP5+/GP6+, are most commonly used

Unlike hc2 testing, PCR DNA testing can be readily applied to archival paraffin-embedded specimens

PCR-based HPV DNA tests, such as the GP5+/GP6+ system 7 and the PGMY system, 8 are clinically validated and have been used for research purposes, but there has been a lack of commercially available, standardized kits for use in clinical practice

Evaluation of the analytical and clinical sensitivity of new HPV tests is needed before their implementation in clinical practice and cervical cancer screening programs

The new Roche AMPLICOR® HPV Test 9 was recently introduced by Roche, and detects 13 high-risk (HR) HPV types

Objective

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

Materials and methods

A total of 132 samples were obtained as archival paraffin-embedded tissue sections from women with diagnoses ranging from normal to CIN3 histology

A total of 266 cervical scrapes were collected in PreservCyt® preservative solution (Pap1, Pap2, Pap3A) and stored at room temperature

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

AMPLICOR HPV Test:

Multiplex PCR for 13 HR HPV types and the human β-globin gene as an internal control

Detects PCR products in a microwell plate (MWP) format, using a cocktail of 13 oligoprobes

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

β-globin PCR performed separately for quality control of samples

Discrepant samples were re-tested

X 5 AMPLICOR primers AMPLICOR 165 bp X 7 AMPLICOR primers HPV L1 gene GP5+/GP6+
X 5 AMPLICOR primers
AMPLICOR 165 bp
X 7 AMPLICOR primers
HPV L1 gene
GP5+/GP6+ primers
GP5+/GP6+ 150 bp
GP5+/GP6+ primers

Figure 1. AMPLICOR and GP5+/GP6+ primers

Results

Table 1. Target genotypes

AMPLICOR HPV TestGP5+/GP6+ PCR EIA 100 80 60 40 20 0 CIN0 CIN1 CIN2 CIN3 n =

GP5+/GP6+ PCR EIA 100 80 60 40 20 0 CIN0 CIN1 CIN2 CIN3 n =
GP5+/GP6+ PCR EIA
100
80
60
40
20
0
CIN0
CIN1
CIN2
CIN3
n = 35
n = 28
n = 30
n =
28
Grade of dysplasia
Condyloma
acuminata
n = 11
Prevalence (%)

Both methods identified the same CIN2 and CIN3 samples

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

AMPLICOR HPV Test 100 GP5+/GP6+ PCR EIA 80 60 40 20 0 Pap1 Pap2 Pap3a
AMPLICOR HPV Test
100
GP5+/GP6+ PCR EIA
80
60
40
20
0
Pap1
Pap2
Pap3a
Pap3b
n = 31
n = 139
n =
99
n = 2
Prevalence (%)

Figure 3. High-risk HPV prevalence in cervical scrapes (n = 266)

Conclusions

In general, both the AMPLICOR HPV Test and the GP5+/GP6+ PCR EIA could

be successfully performed on very small aliquots of samples in liquid cytology

media, or on samples obtained from archival paraffin-embedded tissue

Both methods identified the same CIN2/CIN3 samples, with the exception

of two cases (one AMPLICOR-negative and one GP5+/GP6+-negative)

Discrepant cases (mostly Pap 1/2 cytology and condyloma acuminata

CIN0 and CIN1 samples) may be reflecting low copy-number samples of

HR HPV, and it will be interesting to determine whether these are clinically

relevant HPV infections (i.e. leading to progressive cervical disease) 10

Although the analytical sensitivity of the AMPLICOR HPV Test appears to

be higher than that of the GP5+/GP6+ PCR EIA, clinical sensitivity has yet

to be studied

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, 51, 52, 56, 58, 59, 68

16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68

LR HPV types:

6, 11, 26, 32, 34, 40, 42, 43, 44, 53, 54, 55, 57, 61, 67, 70,

71

(CP 8061), 72, 73, 81 (CP 8304), 82/MM4, 82/IS39,

83

(MM7), 84 (MM8), 85, 86, CP 6108, JC 9710

HR: High-risk; LR: Low-risk

3. Lorincz AT, Richart RM. Arch Pathol Lab Med 2003;127:959–68

4. Schiffman M, Herrero R, Hildesheim A et al. JAMA 2000;283:87–93

5. Petry K, Menton S, Menton M et al. Br J Cancer 2003;88:1570–7

6. Munoz N, Bosch FX, de Sanjose S et al. N Engl J Med 2003:348:518–27

7. Van den Brule AJ, Pol R, Fransen-Daalmeijer N et al. J Clin Microbiol 2002;40:779–87

8. Coutlee F, Gravitt P, Kornegay J et al. J Clin Microbiol 2002;40:902–7

9. Kornegay JR, Shepard AP, Hankins C et al. J Clin Microbiol 2001;39:3530–6

10. Snijders PJ, van den Brule AJ, Meijer CJ. J Pathol 2003;201:1–6