- Email: [email protected]
Comparison of the sensitivities of three commercial assays for detection of the high risk HPV types 16, 18 and 45
G Model VIRMET 12156 1–4
ARTICLE IN PRESS Journal of Virological Methods xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
1
Comparison of the sensitivities of three commercial assays for detection of the high risk HPV types 16, 18 and 45
2
3
4
Q1
Kate Cuschieri a,∗ , Alison Hardie b , Siri Hovland c , Bente Hoaas c , Frank Karlsen d , Heather Cubie e
5
Q2
a
6 7 8 9
Scottish HPV Reference Laboratory, Royal Infirmary of Edinburgh, EH16 4SA, United Kingdom Specialist Virology Centre, Royal Infirmary of Edinburgh, EH16 4SA, United Kingdom NorChip AS, Industriveien 8, N-3490 Klokkarstua, Norway d Norwegian Center of Expertise on Micro- and Nanotechnology Vestfold University College, 103 Tønsberg, Norway e HPV Research Group, Queens Medical Research Institute, EH16 4TJ, United Kingdom b c
10
a b s t r a c t
11 12 13 14 15 16
Article history: Received 4 November 2012 Received in revised form 8 May 2013 Accepted 16 May 2013 Available online xxx
17 18 19 20 21 22 23 24 25
26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
Keywords: HPV hc2 PreTect HPV-Proofer APTIMA Q3 DNA mRNA Sensitivity
The relative and analytical sensitivity of the APTIMA HPV test (AHPV, broad-spectrum, target amplification) and the PreTect HPV-Proofer (type-specific, target amplification) for the detection of HPV mRNA in various cell lines was compared. Equivalent relative sensitivity for the HPV 16-containing cell lines (2.5 cells/ml with both CaSki and SiHa) was observed for the mRNA assays – and similar sensitivities were observed for the detection of HPV 18 (HeLa) and 45 (MS751); ranging from 2.5 cells/ml (Proofer) to 25 cells/ml (APTIMA). In relation to analytical sensitivity, again, the mRNA assays showed similar sensitivities to each other, ranging from 0.1 to 1 cell per reaction for APTIMA and 0.1 to 10 cells per reaction for PreTect HPV-Proofer (depending on cell line). Both mRNA assays consistently achieved a higher analytical sensitivity than a DNA based comparator – the Hybrid Capture 2 High-Risk HPV DNA test (hc2). This study indicates that mRNA tests had high analytical sensitivity, higher than a well established DNA-test based when using cell lines as target. Implications for clinical application are discussed. © 2013 Published by Elsevier B.V.
Human papillomavirus detection is used increasingly for cervical disease management across the world. Comparison studies have shown that differences exist with respect to the clinical sensitivity and specificity of various assays. However, when considering the separate contexts in which HPV testing is relevant (primary screening, triage of low grade disease, test of cure), having tests which differ in their performance could allow judicious application according to such contexts and their requirements. With respect to commercially available HPV assays, manufacturers’ instructions provide some information on assay sensitivities although the extent of this varies according to assay – and sensitivity according to HPV type is not detailed for all. This short report describes a comparison of the relative and analytical sensitivity of two commercially available mRNA HPV detection assays, the APTIMA HPV test (Genprobe, San Diego, CA) and the PreTect HPV-Proofer assay (NorChip AS, Klokkarstua, Norway) using HPV-containing cell-lines spiked into cytological preservative fluid – PreservCyt (Hologic, Bedford, USA). Additionally, the analytical sensitivity of the two mRNA assays was compared with a HPV DNA-based assay, the Hybrid Capture 2 HPV DNA Test (hc2 Qiagen, Gaithersburg, MD).
∗ Corresponding author. Tel.: +44 131 242 6039; fax: +44 131 242 6008. E-mail address: [email protected] (K. Cuschieri).
The PreTect HPV-Proofer assay was performed at two separate sites, specifically the Royal Infirmary of Edinburgh and at NorChip headquarters, Norway. Sites were blinded to the results of the other until completion of testing. For more information on the characteristics of the assays used refer to Table 1 The study involved the use of human cervical cancer cell lines (CaSki, SiHa, HeLa and MS751). CaSki and SiHa are HPV 16 positive cell lines. In CaSki cells the HPV genome is present in as integrated concatemers including 500–600 copies that are hyper methylated with one copy being active transcriptionally at promoter p97 (Kalantari et al., 2004; Van Tine et al., 2004). In addition, some of the HPV 16 genomes present in the CaSki cells exist as truncated episomes (Smotkin and Wettstein, 1986; Spence et al., 1988). The SiHa cell line consists of 1–2 copies/cell of HPV 16 (Sherman et al., 1992; Chardonnet et al., 1995). HeLa is an HPV 18 positive human cell line and containing around 100 copies of integrated HPV 18 genomes (Ambros and Karlic, 1987; Mincheva et al., 1987). MS751 is an HPV 45 positive human cell line containing integrated-only genomes (McLaughlinDrubin et al., 2003; Jesudasan et al., 1995). All cell-lines were cultivated at NorChip and then shipped to the Royal Infirmary of Edinburgh in stock solution (500,000 cells/ml within PreservCyt). Assays were performed according to the manufacturers’ instructions. PreTect HPV-Proofer analyses were only performed for the relevant HPV-multiplex mix, according to the
0166-0934/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jviromet.2013.05.013
Please cite this article in press as: Cuschieri, K., et al., Comparison of the sensitivities of three commercial assays for detection of the high risk HPV types 16, 18 and 45. J. Virol. Methods (2013), http://dx.doi.org/10.1016/j.jviromet.2013.05.013
47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71
ARTICLE IN PRESS
G Model VIRMET 12156 1–4
K. Cuschieri et al. / Journal of Virological Methods xxx (2013) xxx–xxx
2 Table 1 Test principles and properties of assays.
PreTect HPV-Proofer
APTIMA
hc2
Cells-lysed before target mRNA is isolated from the specimen by use of capture oligomers via target capture that utilizes magnetic microparticles Target-TMA
No extraction performed. Hybridization to target DNA. RNA:DNA hybrids are captured onto the surface of a microplate well coated with antibodies specific for RNA:DNA hybrids.
Amplification type
Cells-lysed prior to complete NA extraction by Boom’s method, including heterogeneous silica particles optimizing the isolation of small amounts RNA followed by high purification including several wash steps Target-NASBA
HPV target HPV-types included in the assay
E6/E7 mRNA 16-18-31-33-45
Primers-/and probes
Primer sets designed for each specific HPV-type followed by the detection using Molecular Beacon-probes. Two targets multiplexed (U1A/HPV16, HPV18/31, HPV33/45) Yes Yes 5
E6/E7 mRNA 16-18-31-33-35-39-45-51-5256-58-59-66-68 5 group specific primer-set (groups A1, A2, C1, C2, D)
Sample-preparation
Individual typing Performance control Volume (ml) sample input
78
specific cell-line (U1A/HPV16 mix for CaSki and SiHa; HPV18/31 mix for HeLa; HPV33/45 mix for MS751). Sensitivity was assessed by two different approaches (A and B – described below) and determined as the lowest concentration yielding 100% positivity over 3 parallels. Percentage hit rate (N of 10 parallels) was calculated for the subsequent dilution of the cell lines.
79
(A) Relative sensitivity
72 73 74 75 76 77
107
Assessment of PreTect HPV-Proofer and APTIMA. Approach A allowed for the influence of the different assaydriven sample preparation steps to be assessed. By processing samples according to the manufacturer’s instructions, a different number of cells within each assay reaction were obtained, mimicking the scenario for a clinical sample For the APTIMA assay, cell lines were titrated in five, 10-fold dilutions of each of the cell-lines: CaSki (HPV 16), SiHa (HPV 16), HeLa (HPV 18) and MS751 (HPV 45) from 25,000 cells/ml to 2.5 cells/ml. Three to ten 1 ml withdrawals (for calculation of percentage hit rate) of each dilution were added to 4 ml of lysis buffer from which 400 l was used as input for the assay. This approach led to a final concentration range of 2000 cells/reaction (least dilute) to 0.2 cells/reaction (most dilute) being tested. Preparation of dilutions and assay-runs were performed by the same operator For the Proofer assay, cell lines were titrated in five, 10-fold dilutions of each of the cell lines (from 25,000 cells/ml as above). However, for this assay three to ten withdrawals (for calculation of percentage hit rate) of 5 ml were made for extraction purposes generating an extract of 50 l of which 5 l was used as input for the assay. This lead to a final concentration range of 12,500 cells per/reaction (least dilute) to 125 cells/reaction (most dilute). All withdrawals and extractions for PreTect HPV-Proofer were performed at NorChip, by the same operator and using the same instrument, in order to minimize the possibility of between-site variation in extraction. Aliquots of extracts from all dilutions were then shipped to the Royal Infirmary of Edinburgh for testing.
108
(B) Analytical sensitivity
80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106
109 110 111 112 113
Assessment of APTIMA, PreTect HPV-Proofer and hc2. Approach B allowed us to compensate for the different input requirements of the assays through the preparation of separate cellline dilutions designed to provide the same number of cells per final reaction for each assay.
No Yes 1
Combination of antibody capture and chemiluminescent signal detection L1 DNA 16-18-31-33-35-39-45-51-52-56-58-59–68 HPV RNA full genome probes to prevent false negatives caused by gene deletions
No No 4
Consequently, for APTIMA, PreTect HPV-Proofer and hc2, adjusted dilutions of the cell lines CaSki (HPV 16), SiHa (HPV 16), HeLa (HPV 18) and MS751 (HPV 45) resulting in the same final number of cells within each assay reaction, and ranging from 10,000 cells per reaction to 0.1 cells per reaction were prepared as follows. For APTIMA, six 10-fold dilutions (125,000 cells/ml to 125 cells/ml) were prepared. One withdrawal of 1 ml was made and added to 4 ml of lysis buffer from which 400 l was used as input for the assay. This leads to a final concentration range of 10,000–0.1 cells/reaction. For hc2, six 10-fold dilutions (5000–0.05 cells/ml) were prepared. One withdrawal of 4 ml was made which was concentrated into a 150 l pellet, of which half was used (75 l) as input for the assay. This leads to a final concentration range of 10,000–0.1 cells/reaction. For Proofer, nucleic acid extracted from 100,000 cells (2000 cells/l extract) was diluted in six 10-fold dilutions (2000 cells/l extract to 0.02 cells/l extract), of which 5 l was used as input for the assay. Again, this leads to a final concentration range of 10,000–0.1 cells/reaction. As per approach A, withdrawals and extractions for Prooferanalyses were performed at NorChip. In all three assays, 3–10 parallels were prepared from each dilution for calculation of percentage hit rate. Assessment of the mRNA assays by approach A showed that their sensitivities were similar with equivalent results for the HPV 16 containing cell lines (2.5 cells/ml for 100% hit rate). Small differences between the two other HPV cell lines containing HPV 18 and 45 were observed. For the PreTect HPV-Proofer and APTIMA, 100% hit rate for both HPV 18 and 45 containing cell lines was achieved at 2.5 cells/ml and 25 cells/ml, respectively (Table 2). According to interrogation by approach B, both mRNA assays showed a higher analytical sensitivity than hc2 for all four cell lines tested (Table 3). Although all individual assays showed some variation in sensitivity according to cell line, the greatest variation was exhibited with hc2 which ranged from a sensitivity of 10 cells/reaction with CaSki (HPV 16) to 100,000 for MS751 (HPV 45). The mRNA assays showed similar sensitivities to each other, ranging from 0.1 to 1 cell per reaction for APTIMA and 0.1 to 10 cells per reaction for PreTect HPV-Proofer, depending on cell line. Slightly lower sensitivity of the Proofer (compared to the APTIMA) was observed for 3 of the 4 cell lines (CaSki, SiHa and MS751) when the Proofer assay was performed in Edinburgh – this difference was only observed for one cell line (CaSki) when the Proofer was performed at NorChip. With respect to any diagnostic
Please cite this article in press as: Cuschieri, K., et al., Comparison of the sensitivities of three commercial assays for detection of the high risk HPV types 16, 18 and 45. J. Virol. Methods (2013), http://dx.doi.org/10.1016/j.jviromet.2013.05.013
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3
Table 2 Relative sensitivity of two HPV mRNA detection assays via approach A simulating a clinical setting by dilutions of HPV containing cell lines. Assay
Concentration: number of cells per ml sample (hit rate %) CaSki (HPV16)
PreTect HPV-Proofer (RIE) PreTect HPV-Proofer (NorChip) APTIMA
2.5 (100%) 2.5 (100%) 2.5 (100%)
SiHa (HPV16) 0.25 (40%) 0.25 (40%) 0.25 (40%)
2.5 (100%) 2.5 (100%) 2.5 (100%)
HeLa (HPV18) 0.25 (10%) 0.25 (40%) 0.25 (40%)
2.5 (100%) 2.5 (100%) 25 (100%)
MS751 (HPV45) 0.25 (60%) 0.25 (80%) 2.5 (70%)
2.5 (100%) 2.5 (100%) 25 (100%)
0.25 (60%) 0.25 (60%) 2.5 (80%)
Table 3 Analytical sensitivity of 3 HPV assays, via approach B adjusting the concentration of HPV containing cell lines to represent the same final number of cells in each reaction. Assay
Concentration: number of cells per reaction (hit rate%) CaSki (HPV16)
PreTect HPV-Proofer (RIE) PreTect HPV-Proofer (NorChip) APTIMA hc2
160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206
10 (100%) 1 (100%) 0.1 (100%) 10 (100%)
SiHa (HPV16) 1 (80%) 0.1 (20%) 0.01 (0%) 1 (0%)
10 (100%) 1 (100%) 1 (100%) 10,000 (100%)
assay, inter-laboratory variation does occur and it is perhaps unsurprising that a higher sensitivity was observed in the industrial lab where the technology was developed. As the RNA extraction occurred at the one site, different extraction protocols cannot account for the difference although it is conceivable that RNA transit and short term storage could have affected RNA quality. Whether these inter-lab variations would have a significant impact on clinical performance is moot, it would have been of interest for an additional independent laboratory to have performed the experiments described. This technical study shows that the two mRNA assays have a higher analytical sensitivity compared to the hc2 (between 10-fold and 106 -fold difference depending on cell line/target/testing site). For E6/E7 mRNA tests, high analytical sensitivity may not present the same issues that it would for DNA based tests which target a structural gene, given that E6/E7 oncoproteins are essential for malignancy (zur Hausen, 2002). Indeed, data are available which suggest that the APTIMA assay which has the same type range as hc2 (plus 1 additional type) shows equivalent clinical sensitivity to the hc2 and confers a higher clinical specificity for the detection of high-grade lesions, as demonstrated in both colposcopy referral populations and routinely screened populations (Monsonego et al., 2010; Reuschenbach et al., 2010; Ratnam et al., 2011). The PreTect HPV-Proofer assay has also shown higher specificity than DNA based assays in referral and screening populations (Molden et al., 2005; Szarewski et al., 2012) although this could be driven by the smaller type-range of the assay, coupled with the preferential targeting of E6/E7 mRNA transcripts. Extrapolation of our findings could suggest that the higher specificity of the APTIMA and PreTect HPV Proofer assays observed in clinical studies, when compared to hc2, may not be attributable to the intrinsic analytical sensitivity of these tests which appear to be higher than hc2 when cell lines are assessed. Another interesting finding was that the sensitivity of hc2 for the detection of the HPV 45 cancer cell line was much lower than for HPV 16 or 18. This is at variance with what is stated in the hc2 product insert which purports a similar analytical sensitivity for HPV 16, 18 and 45 using plasmid material. However, the plasmids may contain gene sequences alternative to those available within cell lines used in this study. There is also evidence that cells with integrated HPV genomes can lack L1 genes due to integrationrelated deletion, which may explain this lower sensitivity (Geisbill et al., 1997). The number of invasive cervical cancers caused by HPV 45 is relatively small compared to the number caused by HPV 16 and 18, but as the prevalence of HPV 16 and 18 decreases in immunized cohorts, robust detection of “other” high-risk types will have increasing relevance.
HeLa (HPV18) 1 (70%) 0.1 (10%) 0.1 (10%) 1000 (0%)
0.1 (100%) 0.1 (100%) 0.1 (100%) 1000 (100%)
MS751 (HPV45) 0.01 (30%) 0.01 (70%) 0.01 (50%) 100 (0%)
10 (100%) 0.1 (100%) 0.1 (100%) 100,000 (100%)
1 (90%) 0.01 (20%) 0.01 (10%) 10,000 (0%)
There are limitations to this study – while we can report on the performance of commercial assays when the same variable is assessed (i.e. limiting dilutions of cell line material), none of the assays provide precise quantification of target. Application of a suitable quantification method for RNA could have provided further, complimentary information by relating levels of E6/E7 transcripts within these cell lines to the observations derived from the dilution approach. This could become a focus for future work given our existing knowledge gaps regarding what levels of transcription are relevant (and therefore diagnostic) for transformation. To conclude, in this technical study, commercial mRNA assays had a high analytical sensitivity for the detection of HPV in cell line material – higher than that observed with hc2; the relevance of this finding for clinical applications should be explored further. Conflict of interest statement K. Cuschieri has sat on the advisory board for Genprobe and her institution has received research funding from both Genprobe and NorChip. Role of the funding source NorChip supplied the materials/equipment required to undertake this work. Analysis of the results was performed independently. References Ambros, P.F., Karlic, H.I., 1987. Chromosomal insertion of human papillomavirus 18 sequences in HeLa cells detected by nonisotopic in situ hybridization and reflection contrast microscopy. Hum. Genet. 77, 251–254. Chardonnet, Y., Lizard, G., Chignol, M.C., Schmitt, D., 1995. Analytical methods for evaluation on whole cells of human papillomavirus infection. Bull. Cancer 82, 107–113. Geisbill, J., Osmers, U., Dürst, M., 1997. Detection and characterization of human papillomavirus type 45 DNA in the cervica carcinoma cell line MS751. J. Gen. Virol. 78, 655–658. Jesudasan, R.A., Rahman, R.A., Chandrashekharappa, S., Evans, G.A., Srivatsan, E.S., 1995. Deletion and translocation of chromosome 11q13 sequences in cervical carcinoma cell lines. Am. J. Hum. Genet. 56, 705–715. Kalantari, M., Calleja-Macias, I.E., Tewari, D., Hagmar, B., Lie, K., Barrera-Saldana, H.A., Wiley, D.J., Bernard, H.U., 2004. Conserved methylation patterns of human papillomavirus type 16 DNA in asymptomatic infection and cervical neoplasia. J. Virol. 78, 12762–12772. McLaughlin-Drubin, M.E., Wilson, S., Mullikin, B., Suzich, J., Meyers, C., 2003. Human papillomavirus type 45 propagation, infection, and neutralization. Virology 312, 1–7. Mincheva, A., Gissmann, L., zur Hausen, H., 1987. Chromosomal integration sites of human papillomavirus DNA in three cervical cancer cell lines mapped by in situ hybridization. Med. Microbiol. Immunol. 176, 245–256.
Please cite this article in press as: Cuschieri, K., et al., Comparison of the sensitivities of three commercial assays for detection of the high risk HPV types 16, 18 and 45. J. Virol. Methods (2013), http://dx.doi.org/10.1016/j.jviromet.2013.05.013
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Molden, T., Kraus, I., Karlsen, F., Skomedal, H., Nygård, J.F., Hagmar, B., 2005. Comparison of human papillomavirus messenger RNA and DNA detection: a cross-sectional study of 4,136 women >30 years of age with a 2-year follow-up of high-grade squamous intraepithelial lesion. Cancer Epidemiol. Biomarkers Prev. 2, 367–372. Monsonego, J., Hudgens, M.G., Zerat, L., Zerat, J.C., Syrjänen, K., Halfon, P., Ruiz, F., Smith, J.S., 2010. Evaluation of oncogenic human papillomavirus RNA and DNA tests with liquid-based cytology in primary cervical cancer screening: the FASE study. Int. J. Cancer 29, 691–701. Ratnam, S., Coutlee, F., Fontaine, D., Bentley, J., Escott, N., Ghatage, P., Gadag, V., Holloway, G., Bartellas, E., Kum, N., Giede, C., Lear, A., 2011. Aptima HPV E6/E7 mRNA test is as sensitive as hybrid capture 2 assay but more specific at detecting cervical precancer and cancer. J. Clin. Microbiol. 49, 557–564. Reuschenbach, M., Clad, A., von Knebel Doeberitz, C., Wentzensen, N., Rahmsdorf, J., Schaffrath, Griesser, H., Freudenberg, N., von Knebel Doeberitz, M., 2010. Performance of p16INK4a-cytology, HPV mRNA, and HPV DNA testing to identify high grade cervical dysplasia in women with abnormal screening results. Gynecol. Oncol. 119, 98–105. Sherman, L., Alloul, N., Golan, I., Durst, M., Baram, A., 1992. Expression and splicing patterns of human papillomavirus type-16 mRNAs in pre-cancerous
lesions and carcinomas of the cervix, in human keratinocytes immortalized by HPV 16, and in cell lines established from cervical cancers. Int. J. Cancer 50, 356–364. Smotkin, D., Wettstein, F.O., 1986. Transcription of human papillomavirus type 16 early genes in a cervical cancer and a cancer-derived cell line and identification of the E7 protein. Proc. Natl. Acad. Sci. U.S.A. 83, 4680–4684. Spence, R.P., Murray, A., Banks, L., Kelland, L.R., Crawford, L., 1988. Analysis of human papillomavirus sequences in cell lines recently derived from cervical cancers. Cancer Res. 48, 324–328. Szarewski, A., Mesher, D., Cadman, L., Austin, J., Ashdown-Barr, L., Ho, L., Terry, G., Liddle, S., Young, M., Stoler, M., McCarthy, J., Wright, C., Bergeron, C., Soutter, W.P., Lyons, D., Cuzick, J., 2012. Comparison of seven tests for high-grade cervical intraepithelial neoplasia in women with abnormal smears: the predictors 2 study. J. Clin. Microbiol. 50, 1867–1873. Van Tine, B.A., Kappes, J.C., Banerjee, N.S., Knops, J., Lai, L., Steenbergen, R.D., Meijer, C.L., Snijders, P.J., Chatis, P., Broker, T.R., Moen Jr., P.T., Chow, L.T., 2004. Clonal selection for transcriptionally active viral oncogenes during progression to cancer. J. Virol. 78, 11172–11186. zur Hausen, H., 2002. Papillomaviruses and cancer: from basic studies to clinical application. Nat. Rev. Cancer 2, 342–350.
Please cite this article in press as: Cuschieri, K., et al., Comparison of the sensitivities of three commercial assays for detection of the high risk HPV types 16, 18 and 45. J. Virol. Methods (2013), http://dx.doi.org/10.1016/j.jviromet.2013.05.013
271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290
ARTICLE IN PRESS Journal of Virological Methods xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
1
Comparison of the sensitivities of three commercial assays for detection of the high risk HPV types 16, 18 and 45
2
3
4
Q1
Kate Cuschieri a,∗ , Alison Hardie b , Siri Hovland c , Bente Hoaas c , Frank Karlsen d , Heather Cubie e
5
Q2
a
6 7 8 9
Scottish HPV Reference Laboratory, Royal Infirmary of Edinburgh, EH16 4SA, United Kingdom Specialist Virology Centre, Royal Infirmary of Edinburgh, EH16 4SA, United Kingdom NorChip AS, Industriveien 8, N-3490 Klokkarstua, Norway d Norwegian Center of Expertise on Micro- and Nanotechnology Vestfold University College, 103 Tønsberg, Norway e HPV Research Group, Queens Medical Research Institute, EH16 4TJ, United Kingdom b c
10
a b s t r a c t
11 12 13 14 15 16
Article history: Received 4 November 2012 Received in revised form 8 May 2013 Accepted 16 May 2013 Available online xxx
17 18 19 20 21 22 23 24 25
26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
Keywords: HPV hc2 PreTect HPV-Proofer APTIMA Q3 DNA mRNA Sensitivity
The relative and analytical sensitivity of the APTIMA HPV test (AHPV, broad-spectrum, target amplification) and the PreTect HPV-Proofer (type-specific, target amplification) for the detection of HPV mRNA in various cell lines was compared. Equivalent relative sensitivity for the HPV 16-containing cell lines (2.5 cells/ml with both CaSki and SiHa) was observed for the mRNA assays – and similar sensitivities were observed for the detection of HPV 18 (HeLa) and 45 (MS751); ranging from 2.5 cells/ml (Proofer) to 25 cells/ml (APTIMA). In relation to analytical sensitivity, again, the mRNA assays showed similar sensitivities to each other, ranging from 0.1 to 1 cell per reaction for APTIMA and 0.1 to 10 cells per reaction for PreTect HPV-Proofer (depending on cell line). Both mRNA assays consistently achieved a higher analytical sensitivity than a DNA based comparator – the Hybrid Capture 2 High-Risk HPV DNA test (hc2). This study indicates that mRNA tests had high analytical sensitivity, higher than a well established DNA-test based when using cell lines as target. Implications for clinical application are discussed. © 2013 Published by Elsevier B.V.
Human papillomavirus detection is used increasingly for cervical disease management across the world. Comparison studies have shown that differences exist with respect to the clinical sensitivity and specificity of various assays. However, when considering the separate contexts in which HPV testing is relevant (primary screening, triage of low grade disease, test of cure), having tests which differ in their performance could allow judicious application according to such contexts and their requirements. With respect to commercially available HPV assays, manufacturers’ instructions provide some information on assay sensitivities although the extent of this varies according to assay – and sensitivity according to HPV type is not detailed for all. This short report describes a comparison of the relative and analytical sensitivity of two commercially available mRNA HPV detection assays, the APTIMA HPV test (Genprobe, San Diego, CA) and the PreTect HPV-Proofer assay (NorChip AS, Klokkarstua, Norway) using HPV-containing cell-lines spiked into cytological preservative fluid – PreservCyt (Hologic, Bedford, USA). Additionally, the analytical sensitivity of the two mRNA assays was compared with a HPV DNA-based assay, the Hybrid Capture 2 HPV DNA Test (hc2 Qiagen, Gaithersburg, MD).
∗ Corresponding author. Tel.: +44 131 242 6039; fax: +44 131 242 6008. E-mail address: [email protected] (K. Cuschieri).
The PreTect HPV-Proofer assay was performed at two separate sites, specifically the Royal Infirmary of Edinburgh and at NorChip headquarters, Norway. Sites were blinded to the results of the other until completion of testing. For more information on the characteristics of the assays used refer to Table 1 The study involved the use of human cervical cancer cell lines (CaSki, SiHa, HeLa and MS751). CaSki and SiHa are HPV 16 positive cell lines. In CaSki cells the HPV genome is present in as integrated concatemers including 500–600 copies that are hyper methylated with one copy being active transcriptionally at promoter p97 (Kalantari et al., 2004; Van Tine et al., 2004). In addition, some of the HPV 16 genomes present in the CaSki cells exist as truncated episomes (Smotkin and Wettstein, 1986; Spence et al., 1988). The SiHa cell line consists of 1–2 copies/cell of HPV 16 (Sherman et al., 1992; Chardonnet et al., 1995). HeLa is an HPV 18 positive human cell line and containing around 100 copies of integrated HPV 18 genomes (Ambros and Karlic, 1987; Mincheva et al., 1987). MS751 is an HPV 45 positive human cell line containing integrated-only genomes (McLaughlinDrubin et al., 2003; Jesudasan et al., 1995). All cell-lines were cultivated at NorChip and then shipped to the Royal Infirmary of Edinburgh in stock solution (500,000 cells/ml within PreservCyt). Assays were performed according to the manufacturers’ instructions. PreTect HPV-Proofer analyses were only performed for the relevant HPV-multiplex mix, according to the
0166-0934/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jviromet.2013.05.013
Please cite this article in press as: Cuschieri, K., et al., Comparison of the sensitivities of three commercial assays for detection of the high risk HPV types 16, 18 and 45. J. Virol. Methods (2013), http://dx.doi.org/10.1016/j.jviromet.2013.05.013
47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71
ARTICLE IN PRESS
G Model VIRMET 12156 1–4
K. Cuschieri et al. / Journal of Virological Methods xxx (2013) xxx–xxx
2 Table 1 Test principles and properties of assays.
PreTect HPV-Proofer
APTIMA
hc2
Cells-lysed before target mRNA is isolated from the specimen by use of capture oligomers via target capture that utilizes magnetic microparticles Target-TMA
No extraction performed. Hybridization to target DNA. RNA:DNA hybrids are captured onto the surface of a microplate well coated with antibodies specific for RNA:DNA hybrids.
Amplification type
Cells-lysed prior to complete NA extraction by Boom’s method, including heterogeneous silica particles optimizing the isolation of small amounts RNA followed by high purification including several wash steps Target-NASBA
HPV target HPV-types included in the assay
E6/E7 mRNA 16-18-31-33-45
Primers-/and probes
Primer sets designed for each specific HPV-type followed by the detection using Molecular Beacon-probes. Two targets multiplexed (U1A/HPV16, HPV18/31, HPV33/45) Yes Yes 5
E6/E7 mRNA 16-18-31-33-35-39-45-51-5256-58-59-66-68 5 group specific primer-set (groups A1, A2, C1, C2, D)
Sample-preparation
Individual typing Performance control Volume (ml) sample input
78
specific cell-line (U1A/HPV16 mix for CaSki and SiHa; HPV18/31 mix for HeLa; HPV33/45 mix for MS751). Sensitivity was assessed by two different approaches (A and B – described below) and determined as the lowest concentration yielding 100% positivity over 3 parallels. Percentage hit rate (N of 10 parallels) was calculated for the subsequent dilution of the cell lines.
79
(A) Relative sensitivity
72 73 74 75 76 77
107
Assessment of PreTect HPV-Proofer and APTIMA. Approach A allowed for the influence of the different assaydriven sample preparation steps to be assessed. By processing samples according to the manufacturer’s instructions, a different number of cells within each assay reaction were obtained, mimicking the scenario for a clinical sample For the APTIMA assay, cell lines were titrated in five, 10-fold dilutions of each of the cell-lines: CaSki (HPV 16), SiHa (HPV 16), HeLa (HPV 18) and MS751 (HPV 45) from 25,000 cells/ml to 2.5 cells/ml. Three to ten 1 ml withdrawals (for calculation of percentage hit rate) of each dilution were added to 4 ml of lysis buffer from which 400 l was used as input for the assay. This approach led to a final concentration range of 2000 cells/reaction (least dilute) to 0.2 cells/reaction (most dilute) being tested. Preparation of dilutions and assay-runs were performed by the same operator For the Proofer assay, cell lines were titrated in five, 10-fold dilutions of each of the cell lines (from 25,000 cells/ml as above). However, for this assay three to ten withdrawals (for calculation of percentage hit rate) of 5 ml were made for extraction purposes generating an extract of 50 l of which 5 l was used as input for the assay. This lead to a final concentration range of 12,500 cells per/reaction (least dilute) to 125 cells/reaction (most dilute). All withdrawals and extractions for PreTect HPV-Proofer were performed at NorChip, by the same operator and using the same instrument, in order to minimize the possibility of between-site variation in extraction. Aliquots of extracts from all dilutions were then shipped to the Royal Infirmary of Edinburgh for testing.
108
(B) Analytical sensitivity
80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106
109 110 111 112 113
Assessment of APTIMA, PreTect HPV-Proofer and hc2. Approach B allowed us to compensate for the different input requirements of the assays through the preparation of separate cellline dilutions designed to provide the same number of cells per final reaction for each assay.
No Yes 1
Combination of antibody capture and chemiluminescent signal detection L1 DNA 16-18-31-33-35-39-45-51-52-56-58-59–68 HPV RNA full genome probes to prevent false negatives caused by gene deletions
No No 4
Consequently, for APTIMA, PreTect HPV-Proofer and hc2, adjusted dilutions of the cell lines CaSki (HPV 16), SiHa (HPV 16), HeLa (HPV 18) and MS751 (HPV 45) resulting in the same final number of cells within each assay reaction, and ranging from 10,000 cells per reaction to 0.1 cells per reaction were prepared as follows. For APTIMA, six 10-fold dilutions (125,000 cells/ml to 125 cells/ml) were prepared. One withdrawal of 1 ml was made and added to 4 ml of lysis buffer from which 400 l was used as input for the assay. This leads to a final concentration range of 10,000–0.1 cells/reaction. For hc2, six 10-fold dilutions (5000–0.05 cells/ml) were prepared. One withdrawal of 4 ml was made which was concentrated into a 150 l pellet, of which half was used (75 l) as input for the assay. This leads to a final concentration range of 10,000–0.1 cells/reaction. For Proofer, nucleic acid extracted from 100,000 cells (2000 cells/l extract) was diluted in six 10-fold dilutions (2000 cells/l extract to 0.02 cells/l extract), of which 5 l was used as input for the assay. Again, this leads to a final concentration range of 10,000–0.1 cells/reaction. As per approach A, withdrawals and extractions for Prooferanalyses were performed at NorChip. In all three assays, 3–10 parallels were prepared from each dilution for calculation of percentage hit rate. Assessment of the mRNA assays by approach A showed that their sensitivities were similar with equivalent results for the HPV 16 containing cell lines (2.5 cells/ml for 100% hit rate). Small differences between the two other HPV cell lines containing HPV 18 and 45 were observed. For the PreTect HPV-Proofer and APTIMA, 100% hit rate for both HPV 18 and 45 containing cell lines was achieved at 2.5 cells/ml and 25 cells/ml, respectively (Table 2). According to interrogation by approach B, both mRNA assays showed a higher analytical sensitivity than hc2 for all four cell lines tested (Table 3). Although all individual assays showed some variation in sensitivity according to cell line, the greatest variation was exhibited with hc2 which ranged from a sensitivity of 10 cells/reaction with CaSki (HPV 16) to 100,000 for MS751 (HPV 45). The mRNA assays showed similar sensitivities to each other, ranging from 0.1 to 1 cell per reaction for APTIMA and 0.1 to 10 cells per reaction for PreTect HPV-Proofer, depending on cell line. Slightly lower sensitivity of the Proofer (compared to the APTIMA) was observed for 3 of the 4 cell lines (CaSki, SiHa and MS751) when the Proofer assay was performed in Edinburgh – this difference was only observed for one cell line (CaSki) when the Proofer was performed at NorChip. With respect to any diagnostic
Please cite this article in press as: Cuschieri, K., et al., Comparison of the sensitivities of three commercial assays for detection of the high risk HPV types 16, 18 and 45. J. Virol. Methods (2013), http://dx.doi.org/10.1016/j.jviromet.2013.05.013
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G Model VIRMET 12156 1–4
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3
Table 2 Relative sensitivity of two HPV mRNA detection assays via approach A simulating a clinical setting by dilutions of HPV containing cell lines. Assay
Concentration: number of cells per ml sample (hit rate %) CaSki (HPV16)
PreTect HPV-Proofer (RIE) PreTect HPV-Proofer (NorChip) APTIMA
2.5 (100%) 2.5 (100%) 2.5 (100%)
SiHa (HPV16) 0.25 (40%) 0.25 (40%) 0.25 (40%)
2.5 (100%) 2.5 (100%) 2.5 (100%)
HeLa (HPV18) 0.25 (10%) 0.25 (40%) 0.25 (40%)
2.5 (100%) 2.5 (100%) 25 (100%)
MS751 (HPV45) 0.25 (60%) 0.25 (80%) 2.5 (70%)
2.5 (100%) 2.5 (100%) 25 (100%)
0.25 (60%) 0.25 (60%) 2.5 (80%)
Table 3 Analytical sensitivity of 3 HPV assays, via approach B adjusting the concentration of HPV containing cell lines to represent the same final number of cells in each reaction. Assay
Concentration: number of cells per reaction (hit rate%) CaSki (HPV16)
PreTect HPV-Proofer (RIE) PreTect HPV-Proofer (NorChip) APTIMA hc2
160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206
10 (100%) 1 (100%) 0.1 (100%) 10 (100%)
SiHa (HPV16) 1 (80%) 0.1 (20%) 0.01 (0%) 1 (0%)
10 (100%) 1 (100%) 1 (100%) 10,000 (100%)
assay, inter-laboratory variation does occur and it is perhaps unsurprising that a higher sensitivity was observed in the industrial lab where the technology was developed. As the RNA extraction occurred at the one site, different extraction protocols cannot account for the difference although it is conceivable that RNA transit and short term storage could have affected RNA quality. Whether these inter-lab variations would have a significant impact on clinical performance is moot, it would have been of interest for an additional independent laboratory to have performed the experiments described. This technical study shows that the two mRNA assays have a higher analytical sensitivity compared to the hc2 (between 10-fold and 106 -fold difference depending on cell line/target/testing site). For E6/E7 mRNA tests, high analytical sensitivity may not present the same issues that it would for DNA based tests which target a structural gene, given that E6/E7 oncoproteins are essential for malignancy (zur Hausen, 2002). Indeed, data are available which suggest that the APTIMA assay which has the same type range as hc2 (plus 1 additional type) shows equivalent clinical sensitivity to the hc2 and confers a higher clinical specificity for the detection of high-grade lesions, as demonstrated in both colposcopy referral populations and routinely screened populations (Monsonego et al., 2010; Reuschenbach et al., 2010; Ratnam et al., 2011). The PreTect HPV-Proofer assay has also shown higher specificity than DNA based assays in referral and screening populations (Molden et al., 2005; Szarewski et al., 2012) although this could be driven by the smaller type-range of the assay, coupled with the preferential targeting of E6/E7 mRNA transcripts. Extrapolation of our findings could suggest that the higher specificity of the APTIMA and PreTect HPV Proofer assays observed in clinical studies, when compared to hc2, may not be attributable to the intrinsic analytical sensitivity of these tests which appear to be higher than hc2 when cell lines are assessed. Another interesting finding was that the sensitivity of hc2 for the detection of the HPV 45 cancer cell line was much lower than for HPV 16 or 18. This is at variance with what is stated in the hc2 product insert which purports a similar analytical sensitivity for HPV 16, 18 and 45 using plasmid material. However, the plasmids may contain gene sequences alternative to those available within cell lines used in this study. There is also evidence that cells with integrated HPV genomes can lack L1 genes due to integrationrelated deletion, which may explain this lower sensitivity (Geisbill et al., 1997). The number of invasive cervical cancers caused by HPV 45 is relatively small compared to the number caused by HPV 16 and 18, but as the prevalence of HPV 16 and 18 decreases in immunized cohorts, robust detection of “other” high-risk types will have increasing relevance.
HeLa (HPV18) 1 (70%) 0.1 (10%) 0.1 (10%) 1000 (0%)
0.1 (100%) 0.1 (100%) 0.1 (100%) 1000 (100%)
MS751 (HPV45) 0.01 (30%) 0.01 (70%) 0.01 (50%) 100 (0%)
10 (100%) 0.1 (100%) 0.1 (100%) 100,000 (100%)
1 (90%) 0.01 (20%) 0.01 (10%) 10,000 (0%)
There are limitations to this study – while we can report on the performance of commercial assays when the same variable is assessed (i.e. limiting dilutions of cell line material), none of the assays provide precise quantification of target. Application of a suitable quantification method for RNA could have provided further, complimentary information by relating levels of E6/E7 transcripts within these cell lines to the observations derived from the dilution approach. This could become a focus for future work given our existing knowledge gaps regarding what levels of transcription are relevant (and therefore diagnostic) for transformation. To conclude, in this technical study, commercial mRNA assays had a high analytical sensitivity for the detection of HPV in cell line material – higher than that observed with hc2; the relevance of this finding for clinical applications should be explored further. Conflict of interest statement K. Cuschieri has sat on the advisory board for Genprobe and her institution has received research funding from both Genprobe and NorChip. Role of the funding source NorChip supplied the materials/equipment required to undertake this work. Analysis of the results was performed independently. References Ambros, P.F., Karlic, H.I., 1987. Chromosomal insertion of human papillomavirus 18 sequences in HeLa cells detected by nonisotopic in situ hybridization and reflection contrast microscopy. Hum. Genet. 77, 251–254. Chardonnet, Y., Lizard, G., Chignol, M.C., Schmitt, D., 1995. Analytical methods for evaluation on whole cells of human papillomavirus infection. Bull. Cancer 82, 107–113. Geisbill, J., Osmers, U., Dürst, M., 1997. Detection and characterization of human papillomavirus type 45 DNA in the cervica carcinoma cell line MS751. J. Gen. Virol. 78, 655–658. Jesudasan, R.A., Rahman, R.A., Chandrashekharappa, S., Evans, G.A., Srivatsan, E.S., 1995. Deletion and translocation of chromosome 11q13 sequences in cervical carcinoma cell lines. Am. J. Hum. Genet. 56, 705–715. Kalantari, M., Calleja-Macias, I.E., Tewari, D., Hagmar, B., Lie, K., Barrera-Saldana, H.A., Wiley, D.J., Bernard, H.U., 2004. Conserved methylation patterns of human papillomavirus type 16 DNA in asymptomatic infection and cervical neoplasia. J. Virol. 78, 12762–12772. McLaughlin-Drubin, M.E., Wilson, S., Mullikin, B., Suzich, J., Meyers, C., 2003. Human papillomavirus type 45 propagation, infection, and neutralization. Virology 312, 1–7. Mincheva, A., Gissmann, L., zur Hausen, H., 1987. Chromosomal integration sites of human papillomavirus DNA in three cervical cancer cell lines mapped by in situ hybridization. Med. Microbiol. Immunol. 176, 245–256.
Please cite this article in press as: Cuschieri, K., et al., Comparison of the sensitivities of three commercial assays for detection of the high risk HPV types 16, 18 and 45. J. Virol. Methods (2013), http://dx.doi.org/10.1016/j.jviromet.2013.05.013
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lesions and carcinomas of the cervix, in human keratinocytes immortalized by HPV 16, and in cell lines established from cervical cancers. Int. J. Cancer 50, 356–364. Smotkin, D., Wettstein, F.O., 1986. Transcription of human papillomavirus type 16 early genes in a cervical cancer and a cancer-derived cell line and identification of the E7 protein. Proc. Natl. Acad. Sci. U.S.A. 83, 4680–4684. Spence, R.P., Murray, A., Banks, L., Kelland, L.R., Crawford, L., 1988. Analysis of human papillomavirus sequences in cell lines recently derived from cervical cancers. Cancer Res. 48, 324–328. Szarewski, A., Mesher, D., Cadman, L., Austin, J., Ashdown-Barr, L., Ho, L., Terry, G., Liddle, S., Young, M., Stoler, M., McCarthy, J., Wright, C., Bergeron, C., Soutter, W.P., Lyons, D., Cuzick, J., 2012. Comparison of seven tests for high-grade cervical intraepithelial neoplasia in women with abnormal smears: the predictors 2 study. J. Clin. Microbiol. 50, 1867–1873. Van Tine, B.A., Kappes, J.C., Banerjee, N.S., Knops, J., Lai, L., Steenbergen, R.D., Meijer, C.L., Snijders, P.J., Chatis, P., Broker, T.R., Moen Jr., P.T., Chow, L.T., 2004. Clonal selection for transcriptionally active viral oncogenes during progression to cancer. J. Virol. 78, 11172–11186. zur Hausen, H., 2002. Papillomaviruses and cancer: from basic studies to clinical application. Nat. Rev. Cancer 2, 342–350.
Please cite this article in press as: Cuschieri, K., et al., Comparison of the sensitivities of three commercial assays for detection of the high risk HPV types 16, 18 and 45. J. Virol. Methods (2013), http://dx.doi.org/10.1016/j.jviromet.2013.05.013
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