A three-way comparison of GP5+/6+ PCR, MWP PCR and

Hybrid Capture II within a population-based cohort study

Cecilia Wahlström, Thomas Iftner, Joakim Dillner, Lena Dillner
The Swedescreen Study Group

Introduction ● In addition, the AMPLICOR HPV Test was used on 88 samples

with CIN ≥ 2 histology, using two different DNA extraction GP5+/6+ PCR EIA +ve GP5+/6+ PCR EIA -ve Gray zone
(n = 358) (n = 100) (n = 104)
● Persistent infection with high-risk (HR) types of human methods:
papillomavirus (HPV) is a major factor in the development of
– Freeze/thaw/boiling (with SDS/proteinase K digestion to Prevalence Prevalence Prevalence
cervical carcinoma1 AMPLICOR 6.4% AMPLICOR 1.8% AMPLICOR 0.43%
remove inhibitors, if required)
● HPV DNA testing of cervical specimens is becoming an
important component of organized cervical cancer screening – QIAGEN QIAamp MinElute™
programs:2
– For improved management of women with low-grade
cytology results3
Results Total population prevalence
AMPLICOR 8.6%

– In combination with cytology as primary screening4 ● Results are currently available for 765 samples from 642 women Figure 2. Population-based prevalence at an AMPLCOR OD450
cut-off of 2.2, calculated from the representative
● For integration into clinical screening, an HPV test should (Table 2), 562 of whom represent an unbiased selection from the
cohort (n =562)
detect multiple HR HPV types and be: intervention arm (population-based representative cohort
– Easy to perform [PBRC]): ● The method of DNA extraction did not substantially affect the
results of the AMPLICOR HPV Test (Table 3)
– Highly reproducible, with high analytical specificity and – 83 women developed CIN ≥ 2, 75 of whom were rep-
clinical sensitivity resented in the PBRC Table 3. AMPLICOR HPV Test results in CIN ≥ 2 samples, using
– Amenable for high-throughput analysis and automation two different DNA extraction methods
● All women who subsequently developed CIN ≥ 2/cancer within
● We compared three HPV DNA testing methods (Table 1): 4 years of baseline (n=83) were positive by the AMPLICOR AMPLICOR- Beta-globin- Beta-globin-
– GP5+/6+ PCR enzyme-linked immunosorbent assay (EIA; HPV Test (100% sensitivity), and 82 were positive by the positive positive negative
n n n
cut-off point: three times the mean optical density [OD] of GP5+/6+ PCR EIA (99% sensitivity); sensitivity was 93% for the
two negative controls) hc2 test Freeze/thaw/ 83 83a 0
– The PCR-based Roche AMPLICOR® HPV Test (cut-off boiling
point: OD450 ≥ 0.2) QIAGEN 83 82 1
Table 2. AMPLICOR HPV Test and hc2 test results in
– The liquid hybridization-based Digene Hybrid Capture® 2 population-based representative cohorts (PBRCs) MinElute™
(hc2) test (cut-off point: relative light unit > 1) aOne
PBRC stratum AMPLICOR- hc2- sample was negative after freeze/thaw/boiling, but became positive
after SDS/proteinase K digestion; CIN: Cervical intraepithelial neoplasia
positive positive
Table 1. Target HPV types n (%) n (%) ● Of 15 samples that contained type 66 by RDBH:
Assay Types detected GP5+/6+ PCR EIA- 328 (92) 269 (75) – 12 tested hc2-positive

GP5+/6+ PCR EIA 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, positive (n = 358) – Five tested AMPLICOR-positive. Due to the higher
58, 59, 66, 68 Gray-zone samples 38 (37) 9 (9) sensitivity of the AMPLICOR HPV Test (vs GP5+/6+), the
AMPLICOR HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, (n = 104)a samples may be co-infected with another low-level HR
Test 58, 59, 68
GP5+/6+ PCR EIA- 10 (10) 5 (5) HPV type; samples will be further evaluated
hc2 test 16, 18, 31, 33, 35, 39, 45, 51, 52, 56,
58, 59, 68 negative (n = 100)

aODs
of gray-zone samples were considered negative
Conclusions
Objective
● Population-based positivity rates were 7.1% for GP5+/6+ PCR ● The AMPLICOR HPV Test is a highly sensitive
● To evaluate three different HPV DNA tests for longitudinal EIA (433/6089), 16% for the AMPLICOR HPV Test and 10% for method (100%) for detecting histopath-
specificity and sensitivity for the subsequent development of hc2 (Figure 1) ologically confirmed high-grade CIN or cancer
cervical intraepithelial neoplasia (CIN) or cancer, within
population-based, organized screening ● The clinical significance of the 16%
● All women who had subsequent CIN ≥ 2/cancer had an
population-based positivity obtained with the
AMPLICOR HPV Test OD450 > 2.2 (recommended cut-off, 0.2):
AMPLICOR HPV Test remains to be
Materials and methods – The estimated population prevalence of such strong
determined
AMPLICOR HPV Test positivity was 9% (Figure 2)
● A prospective cohort of 12,527 women attending population- ● Results from the AMPLICOR HPV Test were
based, organized screening was followed-up on a comprehensive comparable for DNA samples extracted by a
registry basis for 4 years, to assess CIN ≥ 2/cancer development
rapid and simple freeze/thaw/boiling tech-
GP5+/6+ PCR EIA +ve GP5+/6+ PCR EIA -ve Gray zone
● Women were randomized 1:1 to either intervention (colposcopy (n = 358) (n = 100) (n = 104) nique and those extracted by QIAGEN
of women with HPV persistence) or control (no action taken methodology
after tests, but a matched number of random colposcopies) Prevalence Prevalence Prevalence
groups AMPLICOR 6.5% AMPLICOR 9.1% AMPLICOR 0.62%
hc2 5.3% hc2 4.6% hc2 0.15%

● DNA was extracted by freeze/thaw/boiling, and HPV detection

was performed by GP5+/6+ PCR EIA.5 Positivity was
confirmed, and genotyping results obtained, by reverse dot-blot
hybridization (RDBH)
● 7.1% of women with valid tests (433/6089) were positive
● Comparative HPV DNA testing using the AMPLICOR HPV Test
and the hc2 test was subsequently performed on enrollment
Figure 1. Population-based prevalence calculated from the
samples from all HPV-positive samples (n= 514)
References
Total population prevalence
1. Walboomers JM, Jacobs MV, Manos MM et al. J Pathol 1999;189:12–9
AMPLICOR 16%
hc2 10% 2. Iftner T, Villa LL. J Natl Cancer Inst Monogr 2003:80–8
3. ALTS Group. J Natl Cancer Inst 2000;92:397–402
4. Cuzick J, Szarewski A, Cubie H et al. Lancet 2003;362:1871–6
representative cohort (n =562) 5. Jacobs MV, Snijders PJ, Voorhorst FJ et al. J Clin Pathol 1999;52:498–503