A comparison of the Digene Hybrid Capture® 2 assay and the Roche AMPLICOR®

Human Papillomavirus (HPV) Test for the detection of ‘high-risk’ HPV genotypes
in DNA extracts from liquid-based cytology samples collected from women
whose cytology was graded ‘borderline’
Alexandra Sargent1, Andrew Bailey1, Paula Wheeler2, Henry Kitchener2, Gerald Corbitt1, Julian Peto3
on behalf of the ARTISTIC Trial Study Group
1
Clinical Virology, The Royal Infirmary, Manchester, UK; 2University Department of Obstetrics & Gynaecology, St Mary’s Hospital, Manchester, UK;
3Institute of Cancer Research, Sutton, Surrey, UK

Introduction Table 1. Additional genotypes identified by Roche line blot assay analysis

● Persistent infection with high-risk (HR) types of the human papillomavirus (HPV) has an
High-risk HPV types detected by the Additional types detected by the
essential role in the etiology of cervical cancer1–2
AMPLICOR HPV and hc2 tests (n = 13) Roche line blot assay (n = 24)
● HPV testing may therefore have implications for the management of women with cervical
16, 18, 31, 33, 35, 39, 45, 51, 6, 11, 26, 40, 42, 53, 54, 55, 61, 62,
lesions, and for primary cervical screening
52, 56, 58, 59, 68 64, 66, 67, 69, 70, 71, 72, 73, 81, 82,
● In many women, samples demonstrate borderline cytology; HPV status may provide a IS39, 83, 84, 89 (CP6108)
method of determining which of these women are at higher risk of underlying high-grade
cervical intraepithelial neoplasia
● The ARTISTIC trial is assessing 28,000 women aged 20–64 years, to determine whether the Results
addition of HPV testing to cytology would increase both the effectiveness and
Comparison of the AMPLICOR HPV Test and the hc2 test
cost-effectiveness of primary cervical cancer screening
● The target HR genotypes for the AMPLICOR HPV Test and the hc2 test are shown in
– The trial is currently in the third year of screening, in an estimated 5–6-year study period Table 1
● If HPV testing is to be incorporated into cervical cancer screening programs, it is ● 1434 samples with borderline cytology have been tested (Table 2a):
important to be able to offer women the most accurate test available – 817 samples gave AMPLICOR-negative, hc2-negative concordant results
● Here, we compare two methods for detecting the 13 most common HR HPV genotypes, and – 387 samples gave AMPLICOR-positive, hc2-positive concordant results
present data showing the sensitivity and specificity of the PCR-based Roche AMPLICOR® – 163 samples were AMPLICOR-positive and hc2-negative
HPV Test – 67 samples were AMPLICOR-negative and hc2-positive
– An improved line blot assay (LINEAR ARRAY® HPV Test) analysis of samples is ongoing
Objective
● 420 samples with normal cytology have been tested (Table 2b):
● To compare the AMPLICOR HPV Test and the Digene Hybrid Capture® 2 (hc2) test, using
– 358 samples gave AMPLICOR-negative, hc2-negative concordant results
clinical samples from the UK-based ARTISTIC trial
– 19 samples gave AMPLICOR-positive, hc2-positive concordant results
Materials and methods – 20 samples were AMPLICOR-positive and hc2-negative
– 23 samples were AMPLICOR-negative and hc2-positive
● Liquid-based cytology samples (PreservCyt® preservative solution) were collected during
the ARTISTIC trial after patient consent, for cytological evaluation and HPV testing: – An improved line blot assay (LINEAR ARRAY® HPV Test) analysis of samples is ongoing

– Trial arms are stratified by age, to provide similar expected numbers of abnormal entry
Table 2a. Concordance analysis: Borderline samples
smears in each of four age groups (20–29, 30–39, 40–49 and 50–64 years)
AMPLICOR HPV Test
– Women are randomized 3:1 to the study arm (HPV-revealed) or control arm
+ –
(HPV-concealed); smears are performed by liquid-based cytology (ThinPrep®), and HPV
+ 387 67
hc2

testing by hc2
– 163 817
● 1700 borderline and 400 negative cytology samples were obtained
Total 550 884
● DNA was extracted from 50-µL aliquots of the stored material using the Roche Total
Nucleic Acid MagNA Pure extraction protocol, resuspended in a final volume of 100 µL
Table 2b. Concordance analysis: Normal samples
and stored at –70°C; aliquots (50 µL) were tested using the AMPLICOR HPV Test
(Figure 1) AMPLICOR HPV Test
+ –
● The Roche prototype line blot assay was used to identify genotypes, in order to clarify
discrepant analyses (Table 1) + 19 23
hc2

– 20 358
Total 39 381
Native HPV
genome UNG Denature and
digestion hybridize
Wash, add
avidin-HRP
conjugate
Conclusions
Biotinylated PCR
primers
● The final determination of diagnostic outcomes will provide a basis for
HPV
probe
demonstrating actual and relative sensitivities of both tests in clinical
Hybridization
practice

● Genotyping is currently ongoing, in order to determine comparative test

Oxidized Oxidized
TMB TMB
characteristics
TMB TMB
Add stop solution,
read absorbance

HRP produces color upon Immobilized hybrids react

References
addition of TMB substrate with avidin-HRP
1. Walboomers JM, Jacobs MV, Manos MM et al. J Pathol 1999;189:12–9
HPV: Human papillomavirus; UNG: Uracil-N-glycosylase; PCR: Polymerase chain reaction; HRP: Horseradish peroxidase; TMB: 3,3',5,5'-tetramethylbenzidene
2. Remmink AJ, Walboomers JM, Helmerhorst TJ et al. Int J Cancer 1995;61:306–11
The ARTISTIC Trial Study Group: HC Kitchener, J Peto, S Moss, G Corbitt, M Desai, P Maguire, C Roberts,
Figure 1. The AMPLICOR HPV Test R Dowie, P Wheeler