The document evaluates the performance of a prototype HPV assay developed by Celera Diagnostics for detecting high-risk HPV strains in cervical samples. Seventy-four cervical samples previously tested with another assay were analyzed. The prototype assay detected high-risk HPV in 28 samples, showing high concordance with previous results for most samples. Some samples identified as mixed infections or different types than the previous assay. The prototype assay has potential for high-throughput detection of HPV in clinical samples and identification of mixed infections.
ỨNG DỤNG CHẨN ĐOÁN PHÂN TỬ TRONG NHÓM BỆNH UNG THƯ
ECCMID2005HPV(4c)
1. Evaluation of the Celera Diagnostics Prototype HPV Assay for Detection of
High Risk HPV Strains in Patient Samples
Natalia Marlowe, Robert Bruce, Michelle Owens, Thomas White and Michael Zoccoli
Celera Diagnostics, Alameda, CA, USA
INTRODUCTION
Human papillomavirus is one of the most common causes of
sexually transmitted diseases resulting in an estimated 288,000
deaths annually from cervical cancer worldwide. An estimated 20
million Americans, or 15% of the population, are infected with
HPV. Of those, 50–75% are infected by high-risk (HR) subtypes
which are strongly associated with the development of invasive
cervical cancer (ICC).
Of more than 30 subtypes found in the anogenital tract, at least
13 are considered HR, as they are significantly associated with
progression to ICC. Subtypes HPV16 and HPV18 are the most
prevalent having been found in 67.2% of ICC cases. Most of the
other HR types are phylogenetically related to HPV16 (31, 33,
35, 52, 58) and HPV18 (39, 45, 68). Infection with multiple types
occurs in approximately 5–30% of infected women.
HPV testing is conducted with a variety of methods including
in situ hybridization, monoclonal and polyclonal antibodies,
hybrid capture, PCR, and mRNA detection. In an effort to
accommodate high throughput detection for HR HPV types, we
recently developed a prototype HPV assay using a Single Base Dye
Primer (SBDP) approach. In the present study, we evaluate assay
performance on 74 patient samples.
RESULTS
Seventy-four retrospective cervical swab samples collected in Cytyc ThinPrep
solution were obtained from a US reference lab. Fifty-four of them were
previously successfully typed with Digene Hybrid Capture® 2 (HC 2) assay:
32 as HR and 22 as negative for HR types. The remaining 20 samples yielded
inconclusive results with the HC2 assay.
DNA was isolated by a modified Qiagen DNA extraction protocol (Qiagen
RNA/DNA Mini kit) or with the Epicentre MasterPure DNA purification
kit. Four microliters of DNA was used for each 50 microliter amplification
reaction.
HPV-positive samples were identified by Ct values and dissociation profiles.
HPV PCR-positive samples were sequenced with a SBDP protocol and conven-
tional four-color sequencing. HPV types were identified by BLAST (v2.2.9)
analysis using four color sequencing data. BLAST scores above 450 were
considered significant to determine a type. SBDP data were analyzed by visual
pattern matching to four color plasmid sequences.
HR HPV types were identified in 28/74 clinical samples using the Celera
Diagnostics prototype assay.
Of the 54 cervical samples previously successfully typed by HC2 we detected
the presence of HR types in 24 samples (Table 1).
Table 1. HR HPV Types identified by Celera Diagnostics prototype
assay
Sample ID
Single Base
Dye Primer
Sequencing
Four Color
Dye Primer
Sequencing
BigDye
Terminator
Sequencing
321 56 56 56
327 16 16 16
330 51 51 51
333 16/31 16/31 16/31
337 51 51 51
338 45 45 45
339 59 59 59
340 52 52 52
349 31 31 31
350 52 52 52
351 31 31 31
352 16 16 16
354 52 52 52
355 59 59 59
356 68 68 68
359 16 16 16
360 35 35 35
362 56 56 56
363 16 16 16
364 59 59 59
367 58 58 58
369 16 16 16
371 18 18 18
374 58 58 58
HR HPV type determinations for these samples were concordant with HC2
HR determinations. Of the 8 discrepant samples, three typed as 66 by the
prototype assay, one sample typed as 6, two samples were indeterminate
mixtures and the remaining two were negative (Table 2, Figure 7).
Table 2. Discrepant samples between HC2 and Celera Diagnostics
prototype assay
Sample ID HC2 Assay Call CDx Call
322 HR 6*
329 HR Neg**
334 HR Neg**
335 HR Pos***
336 HR 66
358 HR 66
370 HR 66
376 HR Pos***
* Sample may contain another HPV type as well (mixtures)
** Low DNA yield from extraction
* * * Indeteminate mixture
Figure 7. Discordant typing results for sample 370(HC2 call HR, CDx
call LR type 66)
In addition, four HR samples were identified among the 20 samples unresolved
with HC2 assay (Table 3).
Table 3. HR HPV infections identified by the CDx prototype assay in
samples unresolved with HC2 assay
Number
Reference lab
“in-house” PCR
result
CDx prototype
assay result
Comments
1 6 59/? Mixture
2 Neg Neg
3 16 16
4 Neg Neg
5 Neg Neg
6 31 66/? Mixture
7 6 6
8 Neg Neg
9 16 16
10 16 Neg
11 Neg Neg
12 Neg Neg
13 6 16/? Mixture
14 Neg 58/? Mixture
15 6 6
16 18 18/45 Mixture
17 Neg 66
18 Neg Neg
19 18 18
20 Neg Neg
Five samples were determined to be HPV mixed infections, containing at
least one HR type. Detection of mixed infection in clinical sample is shown
in Figure 8.
Figure 8. Detection of mixed HPV infection in clinical sample
CONCLUSIONS
This feasibility study demonstrates
that a rapid Single Base Dye Primer
Profiling method can be used for
high-throughput detection of cervical
samples for the presence of high risk
HPV types.
OBJECTIVE
To evaluate the prototype assay performance for the detection of HR HPV strains in cervical samples.
Natalia Marlowe PhD
Celera Diagnostics
1401 Harbor Bay Parkway, Alameda, CA 94502
natalia.marlowe@celeradiagnostics.com
METHODS
Samples
Seventy four retrospective cervical samples previously typed with
Digene Hybrid Capture® 2 (HC2) were received from a US reference
lab.
Twenty nine HPV-containing plasmids (including 13 HR oncogenic
types: HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68) were
received from Abbott Laboratories
Prototype HPV Genotyping Assay Concept
l Total DNA extraction (manual or automated)
l PCR Amplification – 450 bp in L1 region of HPV
Determine presence or absence of HPV sequences with real-time
PCR in the presence of SYBR® Green
l Sequence HPV amplicon with Single Base Dye Primer without
purification or separation steps
l Rapid sequencing protocol on ABI PRISM® 3100 Genetic
Analyzer
l Determine HPV subtype by pattern matching
l Based on HPV subtype detected, infer HPV cancer risk
Figure 1. Prototype HPV assay flow chart
Real-Time PCR with SYBR®
Green for Detection of
HR Oncogenic HPV-Types
Consensus PCR primers in the conserved region of the L1 gene (ref 1)
were selected to preferentially amplify HR HPV types.
Twenty nine plasmids with HPV sequences were amplified to generate
450 bp amplicons. One nanogram of plasmid DNA* was used for
each amplification reaction. PCR reactions were run for 40 cycles on
the ABI PRISM® 7000 Sequence Detection System. A dissociation
protocol (from 60 degrees to 95) was performed after PCR completion.
PCR products were analyzed based on the amplification Ct values and
dissociation profiles (Tm) as shown in Figure 2 and 3.
Figure 2. Amplification curves of HR HPV-containing
plasmids
Figure 3. Dissociation profiles of HR HPV types
* Plasmids were amplified using TempliPhi™
(Amersham Biosciences)
prior to PCR to produce single-stranded DNA
SBDP Profiling for Detection of HR Oncogenic HPV-
Types on Plasmid DNA
PCR primers containing an 18-nucleotide tail, with no homology
to HPV or human sequences were used to generate tailed amplicon
for subsequent downstream sequencing. PCR positive samples
were sequenced with a SBDP protocol and conventional four-color
sequencing methodologies: ABI PRISM BigDye® Terminator and
BigDye® Primer. SBDP sequence reactions were performed with a
BigDye® Primer Cycle Sequencing Ready Reaction kit using 4 µl of
C-premix and 1 µl of PCR product. Cycle sequencing was carried out
according to the standard protocol for BigDye® Primer and BigDye®
Terminator Cycle Sequencing on GeneAmp PCR System 9700®.
Sequencing data were analyzed on the ABI PRISM® 3100 Genetic
Analyzer. HPV HR oncogenic types were confirmed by NCBI
BLAST (v2.2.9) analysis using four color sequencing data (Figure
4). SBDP data were analyzed by visual pattern matching to four color
plasmid sequences (Figure 5).
Figure 4. Comparison between single base dye primer
and four color BDT and BDP (displayed with three colors
removed) of HPV 16
Figure 5. Single base “C” profiles of four HR HPV types
Detection of Mixed Infection with a SBDP Method
To demonstrate the feasibility of detection of HPV mixtures present in
a sample we tested 50:50 plasmid mixtures containing two HPV types.
An example of plasmid mixture detection is shown in Figure 6.
Figure 6. Example of mixed infection of HPV 16 and HPV 68
DISCUSSION
We have recently developed a real-time probe-less PCR assay for
preferential amplification of HR HPV types in cervical samples. This
assay utilizes a Single Base Dye Primer Profiling approach to determine
HR HPV type. In this feasibility study we evaluated prototype assay
performance on a set of cervical samples previously typed with the
Digene HC2 test. As shown in Table 1, a high correlation was observed
between HC2 and the prototype assay for 24 HR HPV infected
samples. Of the remaining 8 HR HPV samples (according to the HC2),
two samples showed no amplification, two samples were indeterminate
mixtures and four samples were typed as low risk HPV by the prototype
assay (Table 2, Figure 7).
In the twenty samples unresolved by the HC2 assay we detected the
presence of HR HPV in 4 samples and the presence of mixed HPV
infection in 5 samples. These findings demonstrate the potential of
the prototype assay to detect HR HPV in samples undetermined by
the HC assay.
References
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