ECCMID2005HPV(4c)

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

INTRODUCTIONHuman 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 signifi cantly 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 eff ort 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.

RESULTSSeventy-four retrospective cervical swab samples collected in Cytyc Th inPrep

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. Th e remaining 20 samples yielded

inconclusive results with the HC2 assay.

DNA was isolated by a modifi ed Qiagen DNA extraction protocol (Qiagen

RNA/DNA Mini kit) or with the Epicentre MasterPure DNA purifi cation

kit. Four microliters of DNA was used for each 50 microliter amplifi cation

reaction.

HPV-positive samples were identifi ed by Ct values and dissociation profi les.

HPV PCR-positive samples were sequenced with a SBDP protocol and conven-

tional four-color sequencing. HPV types were identifi ed by BLAST (v2.2.9)

analysis using four color sequencing data. BLAST scores above 450 were

considered signifi cant to determine a type. SBDP data were analyzed by visual

pattern matching to four color plasmid sequences.

HR HPV types were identifi ed 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 identifi ed 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 identifi ed among the 20 samples unresolved

with HC2 assay (Table 3).

Table 3. HR HPV infections identifi ed by the CDx prototype assay in samples unresolved with HC2 assay

Number

Reference lab

“in-house” PCR

result

CDx prototype

assay resultComments

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

Profi ling method can be used for

high-throughput detection of cervical

samples for the presence of high risk

HPV types.

OBJECTIVETo 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

[email protected]

METHODSSamples

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 Amplifi cation – 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

purifi cation 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 fl ow 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 amplifi ed to generate

450 bp amplicons. One nanogram of plasmid DNA* was used for

each amplifi cation 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 amplifi cation Ct values and

dissociation profi les (Tm) as shown in Figure 2 and 3.

Figure 2. Amplifi cation curves of HR HPV-containing plasmids

Figure 3. Dissociation profi les of HR HPV types

* Plasmids were amplifi ed using TempliPhi™ (Amersham Biosciences)

prior to PCR to produce single-stranded DNA

SBDP Profi ling 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 confi rmed 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” profi les 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

DISCUSSIONWe have recently developed a real-time probe-less PCR assay for

preferential amplifi cation of HR HPV types in cervical samples. Th is

assay utilizes a Single Base Dye Primer Profi ling 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 amplifi cation, 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. Th ese fi ndings demonstrate the potential of

the prototype assay to detect HR HPV in samples undetermined by

the HC assay.

References

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