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The Roche AMPLICOR® HPV Test as a primary screen for HPV

in cervical specimens collected in ThinPrep® and SurePath™


liquid cytology media
Jane Gibson, David Vinson, David Flynn, Rebecca Garcia, Dayanara Jacquin, Mark Kohler,
Mike Kratz, Michelle Moss, Rebecca Sneed, Dennis Anderson, Audrey Roberts
Department of Clinical Molecular Genetics, AmeriPath Center for Advanced Diagnostics, Orlando, Florida, USA

Introduction A Table 2. Genotypes identified in AMPLICOR-negative, hc2-


positive samples
HPV L1 gene
● Program-based screening using conventional cytology is
AMPLICOR-negative, hc2-positive
reducing cervical cancer mortality, but has limitations: PVUP/PVDN
x5 x7 (Genotypes detected in 20 samples)
— Each individual Pap smear has low sensitivity AMPLICOR
165 bp amplicon Type n
HPV Test
— Identification of abnormal cytology is subjective and
B 53 5
highly labor-intensive
HPV L1 gene
54 2
● Persistent human papillomavirus (HPV) infection is a 64 1
necessary cause of cervical carcinoma; testing for HPV PGMY 66 3
x5 x 13
has become an important component of screening 67 2
Roche LBA 450 bp amplicon 70 2
● Roche PCR-based testing offers an alternative to the 82 1
Digene Hybrid Capture® 2 (hc2) test 53 and 54 1
Figure 2. PCR primer positions and amplicon sizes for the
● The rapid and sensitive nature of amplification-based AMPLICOR HPV Test (A) and the Roche LBA (B) 53 and 62 1
technology (PCR), together with reduced specimen 53 and 70 1
66 and 70 1
requirements, provide an attractive method for HPV Results
testing for liquid cytology media
● Overall, HPV-positivity was 15.1% with the AMPLICOR
HPV Test and 9.3% with the hc2 test Discussion
Objectives ● 1650/2000 samples (83%) were AMPLICOR and hc2 ● HR HPV was detected by the Roche LBA in 45/138 (33%)
● To use the Roche AMPLICOR® HPV Test (Figure 1) as a concordant negative AMPLICOR-positive/hc2-negative specimens
primary screen for HPV in a cohort of 2000 randomly ● 151/2000 samples (8%) were concordant positive ● Roche LBA-negative samples are being analyzed by E7
selected cervical specimens collected in ThinPrep® and ● 199/2000 samples (9.9%) were discrepant: type-specific PCR to resolve discrepants further
SurePath™ media — 138 were AMPLICOR-positive, hc2-negative: ● All 34 AMPLICOR-negative/hc2-positive specimens
● 93 were Roche LBA-negative or low-risk (LR)- tested by the Roche LBA yielded either negative results
● To compare the results with those obtained using the hc2 (14/34 [41%]), or contained HPV types not intended to
positive (Table 1 shows Roche LBA results of the
test HR genotypes) be detected with the hc2 test (20/34 [59%])
— 34 were AMPLICOR-negative, hc2-positive: ● All 27 hc2-indeterminate samples were resolved using the
Native HPV
UNG Denature and ● 14 of these were Roche LBA-negative, and all AMPLICOR HPV Test or Roche LBA genotype analysis
genome hybridize
digestion
other genotypes were non-target types (Table 2)
None of these types is included in the probe spectra
Conclusions
Wash, add
avidin-HRP ●
PCR conjugate
Biotinylated
primers of the AMPLICOR HPV Test or the hc2 test
— 27 were hc2-indeterminate: ● These data suggest that the AMPLICOR HPV
HPV Test is a more sensitive method, compared
probe 12 of these were AMPLICOR-positive samples:

with hc2, for the detection of HR HPV in
Hybridization
— The Roche LBA identified 11 as HR-positive
cervical specimens collected in ThinPrep® and
Oxidized Oxidized
TMB TMB
(types 16 [n=4], 18 [n=3], 31 [n=1], 35 [n=1], SurePath™ liquid cytology media
59 [n=1] and 68 [n=1], and one as LR-positive
Add stop solution,
TMB TMB
● The presence of HR HPV was confirmed in
read absorbance (types 66 and 89 [CP6108])
33% of AMPLICOR-positive/hc2-negative
HRP produces color upon
addition of TMB substrate
Immobilized hybrids react
with avidin-HRP
● 15 were AMPLICOR-negative samples: specimens; negative findings in the remaining
— The Roche LBA was negative in all three cases
93 specimens may be due to the increased
HPV: Human papillomavirus; UNG: Uracil-N-glycosylase; PCR: Polymerase chain reaction; HRP: Horseradish peroxidase;
TMB: 3,3',5,5'-tetramethylbenzidene
tested; 12 additional AMPLICOR-negative sensitivity of the AMPLICOR HPV Test, relative
samples were not analyzed by the Roche LBA to the Roche LBA
Figure 1. AMPLICOR HPV Test Table 1. Genotypes identified in AMPLICOR-positive, hc2- ● E7 type-specific PCR testing is underway to
negative samples characterize these specimens further
No target HR HPV genotypes for the
Materials and methods AMPLICOR-positive, hc2-negative
(HR genotypes detected in 45 samples)

AMPLICOR HPV Test and the hc2 test were
● A total of 2000 randomly selected cervical cell specimens found in any of the 34 AMPLICOR-
Type n
in ThinPrep® or SurePath™ liquid cytology media (49% negative/hc2-positive specimens, suggesting
vs 51%, respectively) were included in the study: 16 4 false-positive hc2 results
18 7 ● Understanding and defining the clinical
— 79% of women were ≥ 30 years of age applications of a more sensitive HR HPV
31 2
— 94% had normal cytology 35 1 screening test will be an important component
of future HPV testing and cervical cancer
39 2
● Samples were analyzed in batches of approximately 250 screening
45 3
alternating ThinPrep® or SurePath™ stored samples ● A more sensitive HR HPV test, such as the
52 4
● HPV testing was performed using the FDA-updated hc2 AMPLICOR HPV Test, will contribute to our
56 2
test procedure and, following DNA extraction using overall understanding of the etiology of HPV
58 1
the QIAGEN QIAamp MinElute™ system, by the infections
59 2
AMPLICOR HPV Test; both of these assays detect 68 3
13 high-risk (HR) HPV genotypes Mixed: > 1 HR HPV; no LR 3 References
● DNA samples from discrepant specimens were genotyped Mixed: > 1 HR HPV; 2
with LR type(s) present 1. Gravitt PE, Peyton CL, Alessi TQ et al. J Clin Microbiol 2000;
using the Roche prototype line blot assay (LBA)1,2 38:357–61
Mixed: 1 HR HPV; with LR present 9 2. Gravitt PE, Peyton CL, Apple RJ et al. J Clin Microbiol 1998;
● The smaller size of the AMPLICOR HPV Test amplicon, 36:3020–7
High-risk (HR) types:3 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68
compared with that in the Roche LBA, lends itself to Possible high-risk types:3 23, 53, 66, 73, 82 3. Munoz M, Bosch FX, de Sanjose S et al. N Engl J Med 2003;
increased sensitivity (Figure 2) Low-risk (LR) types:3 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81 348:518–27