Type-specific HPV E6/E7 mrna detection by real-time PCR improves

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1 JCM Accepts, published online ahead of print on 1 September 0 J. Clin. Microbiol. doi:./jcm.00- Copyright 0, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 Type-specific HPV E/E mrna detection by real-time PCR improves identification of cervical neoplasia Elin Andersson* 1, Cecilia Kärrberg, Thomas Rådberg, Lennart Blomqvist, Britt-Marie Zetterqvist, Walter Ryd, Magnus Lindh 1, Peter Horal 1. Departments of 1 Clinical Virology, Obstetrics and Gynecology and Pathology, Sahlgrenska Academy, University of Gothenburg, Sweden. Department of Gynecology and Obstetrics, Southern Älvsborg Hospital, Borås, Sweden. Division of Obstetrics and Gynecology, NU Hospital Organisation, Trollhättan, Sweden. *Corresponding author: Elin Andersson, Department of Clinical Virology, Guldhedsgatan B, 1 Gothenburg, Sweden Tel + 1 Mobile + 0 Fax elin.andersson@microbio.gu.se Downloaded from on September 1, 01 by guest 1

2 Abstract DNA-based HPV assays show high sensitivity but poor specificity in detecting high-grade cervical lesions. Assays detecting mrna of oncogenic E/E show higher specificity, but lack either detection of all high-risk HPV genotypes or the capacity to specify the detected genotypes. Therefore, a real-time PCR assay detecting type-specific E/E mrna was developed and the clinical performance evaluated. cervical LBC (liquid based cytology) samples from 0 women were analysed for HPV DNA and mrna with the in house realtime PCR as well as PreTect HPV-Proofer. The sensitivity of real-time PCR mrna-detection to detect histologically confirmed CIN+ (cervical intraepithelial neoplasia grade or higher) were 0.1, compared to 0. for DNA-analysis. The specificity was 0. compared to 0., and the positive predictive value (PPV) was higher for mrna (0. vs 0.) without any loss in negative predictive value (NPV). The sensitivity of the real-time PCR mrna-test was somewhat higher than for PreTect HPV-Proofer (0. vs 0.), when analysing for the same genotypes. The specificity was similar (0. vs 0.). When analysing for mrna of the eight most common genotypes in cervical cancer (HPV1, 1, 1,,,,, ), the sensitivity to detect CIN+ lesions was 0. and the specificity 0., with a PPV of 0.0. In conclusion, real-time PCR for detection of HPV E/E mrna transcripts can be a sensitive and specific tool in screening and investigation of cervical neoplasia. The composition of HPV-types in mrna-testing needs to be further investigated to optimize sensitivity and specificity. Downloaded from on September 1, 01 by guest

3 Introduction Cervical cancer is closely associated with infection of human papillomaviruses (HPV), but only a small proportion of these infections cause cancer. There are at least 1 oncogenic genotypes (HPV1, 1, 1,,,,, 1,,,, ) associated with high risk of cervical cancer (HR-HPV) and a number of genotypes that probably also have oncogenic properties (1). The viral proteins E and E are considered to be responsible for transformation of the infected epithelial cell, as well as the maintenance of the malignant phenotype. The proteins can affect many cellular proteins, such as the tumour suppressor proteins prb and p (reviewed by Ghittoni et al () and McLaughlin-Drubin and Munger (1)) in a manner that leads to extension of cellular life span (including resistance to apoptosis), DNA synthesis, genomic instability and interference with antiviral and antitumour immune responses. The mechanisms that regulate if an HPV infection will be cleared by the immune system or become persistent and cause transformation are not well understood. However, integration of the viral genome into the cellular genome seems to be an important event. Usually, the viral gene coding for E, a regulator of E/E transcription, is lost during integration. Thus, integration typically leads to overexpression of E/E, which may facilitate tumour progression (1). Moreover, common fragile sites are frequently targeted for viral integration, possibly causing genomic instability (). There are numerous commercial tests available for HR-HPV DNA detection, but only a few based on detection of oncogenic mrna. DNA detection tests are highly sensitive for detection of high-grade cervical intraepithelial neoplasia (CIN), and have been shown to be a valuable tool in triage of Atypical Squamous Cells of Uncertain Significance (ASCUS) and follow-up after treatment (). Furthermore, the use of HPV DNA tests in primary screening have in several studies been shown to be more sensitive than conventional cytology in detecting cervical cancer and severe pre-cancerous lesions, and may serve to prolong the screening interval (1,, 1). However, the specificities of HPV DNA tests for identification Downloaded from on September 1, 01 by guest

4 of cervical neoplasia are lower than for cytology especially among younger women (). Therefore, HPV-positive women need to be triaged before referral for further investigations, such as colposcopy, but the preferable triage is yet to be established. Cytology could be an alternative, as well as detection of HPV E/E mrna or cellular tumour markers such as p1 (). One commercially available mrna-test is the PreTect HPV-Proofer (NorChip, Klokkarstua, Norway), also called NucliSENS EasyQ (Biomérieux, Marcy l Etoile, France), which detects mrna of the five most common HPV-types, HPV1, 1, 1, and, based on nucleic acid sequence based amplification (NASBA) technique. The specificity of the test is higher than for DNA-tests (1, ), but the sensitivity is lower, and mainly due to the fact that it doesn t detect all HR-HPV, it can never be as sensitive as a DNA-test. The other commercially available mrna-test for HPV is APTIMA (Gen-Probe, San Diego, CA), which detects mrna of 1 HR-HPV as well as mrna of HPV and HPV, based on transcription-mediated amplification (TMA). However, APTIMA does not specify the individual detected HPV types. The test has similar sensitive as a DNA-test, but with higher specificity for detection of dysplasia (, 0, ). There is a difference in specificity between PreTect HPV-Proofer and APTIMA, the former being more specific. The reason for the higher sensitivity and lower specificity than observed with PreTect HPV-Proofer could be that APTIMA detects mrna of more genotypes, some more common in low-grade lesions. However, APTIMA also detects HPV DNA, even though it is more sensitive for mrna (). Similarly, there have been reports that the NASBA-technique can detect DNA (, ), causing false positive results. We have previously developed a real-time PCR test based on amplification of E/E DNA of 1 HR-HPV and LR-HPV (1). The performance of the method has been validated by showing agreement with the Linear Array assay (Roche) (1) and by a 0% proficiency when participating in the WHO LabNet proficiency panel study 00 (). We have adapted this assay to detect only mrna by adding a DNase-digesting step and a reverse transcription Downloaded from on September 1, 01 by guest

5 step. In this study we evaluate the clinical performance of this type-specific HPV mrna test, and correlate the results with the HPV DNA analysis (using the same primers and probes) and the mrna-test PreTect HPV-Proofer Materials and methods Samples Liquid based cytology (LBC) samples collected in PreservCyt media (Cytyc, Marlborough, MA, USA) from 0 women who were attending gynaecological screening (n=1, pregnant) or had been admitted to a referral center for investigation because of abnormalities in cervical cytology (n=1, pregnant) were included. The age of the women ranged between 1 and with a median and mean age of and years, respectively. Five of the women were sampled two or three times, resulting in samples. All women received information of the study design and provided written consent. Approval was obtained from the local ethics committee. Neoplasias were evaluated by colposcopy-directed biopsies and/or total excitional biopsies (conisation) and subsequent histological examination. An expert pathologist re-evaluated all histological samples. If the second diagnosis differed from the original diagnosis by more than one level of severity, the pathologist confirmed the diagnosis with another pathologist. DNA and RNA extraction DNA or total NA was extracted using a MagNA Pure LC instrument (Roche). For DNA analysis 0-00 µl of the LBC sample was used for extraction with the DNA I protocol. For mrna analysis - ml of the sample was briefly centrifuged and pelleted cells were resuspended in 1 ml of RLT lysis buffer (Qiagen, Hilden, Germany) for extraction with the totna LV protocol. To assure the quality of mrna, the LBC samples were not allowed a storage period longer than 0 days before resuspension in lysis buffer and total NA extraction. Downloaded from on September 1, 01 by guest

6 After lysis treatment, some samples were stored in -0 C before extraction. Prior to analysis, extractions were stored in -0 C. Real-time PCR The Taqman real-time PCR assay targets 1 high-risk (1, 1, 1,,,,, 1,,,, ) and two low-risk ( and ) types using E/E region primers and probes in a duplex format (1). Detection of the human gene betaglobin serves as a control of sample sufficiency. Briefly, µl of extracted DNA was added to master mix of µl Universal PCR master mix (Roche diagnostics, Branchburg, NJ) with 0. µm primers and 0. µm probes, supplemented with nuclease-free water to a final volume of 0 µl. After uracil DNA glycosylase activation at 0 C for min and initial denaturation at C for min, the PCR for DNA detection was run for cycles (1 s at C, 0 s at C) on an ABI 00 instrument (Applied Biosystems, Carlsbad, CA). The threshold cycle, Ct, value for each reaction was recorded (a low Ct-value indicates high amount of target). Only samples yielding a Ct value for betaglobin below were included in analysis. The modified method used for HPV mrna detection included a DNase digestion step, using the Ambion TURBO DNAfree kit (Applied Biosystems). Ten µl of the DNase-treated sample was added to a one-step RT-PCR mastermix containing 1 µl Ribonuclase inhibitor (RNase OUT) and 1 µl SuperScript (all Invitrogen, Carlsbad, CA), supplemented with nuclease-free water to a final reaction volume of 0 µl, including 0. µm primers and 0. µm probes. The PCR program was identical to that for DNA except for an initiating step of reverse transcription at C for 0 minutes. To ascertain that no remaining HPV DNA was present, the DNase-treated samples were also run with the DNA detection protocol, i.e. without the RT step. Any mrna detection was only accepted if the corresponding DNA was not detected or detected with a Ct value more than cycles above the Ct value for mrna. The E/E gene is transcribed into one full-length mrna-transcript coding for both proteins, but is also spliced to an E- Downloaded from on September 1, 01 by guest encoding transcript (HPV1 is the only genotype transcribed into two spliced transcripts as

7 well as a full-length) (). Our real-time PCR detects all transcripts, both full-length and spliced, except for genotypes,, and where only the full-length transcripts are detected. For HPV1, the shorter of the two spliced transcripts is not detected PreTect HPV-Proofer Detection of E/E mrna of genotypes 1, 1, 1, and was performed using the PreTect HPV-Proofer kit according to the manufacturer s guidelines. Briefly, the analysis is based on NASBA technique with isothermal amplification of mrna in a duplex format, measured in real-time. Five µl of total NA-extract was added to µl of a master mix with primers, molecular beacon probes and KCl. After incubation for min at C and min at 1 C, µl of enzyme was added and spinned down before amplification at 1 C. Analysis of the cellular U1A transcript was included in the test to determine the validity of the results. Statistics The sensitivity, specificity, positive predictive value and negative predictive value of each test algorithm were calculated with histologically confirmed CIN+ as gold standard, but calculations were also made for CIN+. Calculations of % confidence intervals (% CI) were based on the normal approximation to the binomial distribution as suggested by Harper and Reeves (1). Results Cytological and histological diagnoses For 1 (%) of the LBC samples, histological evaluations from biopsies and/or total excised specimens taken at the same time were available (Table 1). For samples with benign cytology (1 of them from women in screening) and two samples with ASCUS in cytology, no histological data was available. The histological diagnosis benign (n=) Downloaded from on September 1, 01 by guest

8 includes inflammation (n=), metaplasia (n=), ulcus (n=1) and HPV-infection without signs of CIN (n=). The diagnosis CIN (n=1) includes adenocarcinoma in situ (n=). Histological data were available for 0 cytologically benign samples, showing CIN1 or worse in cases (%) including six cases of CIN and four cases of cancer. These represent 1% of the women in this material undergoing investigation for dysplasia, mostly due to earlier atypical cytology. Overall, histological examination tended to upgrade the cytological diagnoses. HPV DNA and mrna type distribution Type-specific detection rates of HPV DNA and mrna according to histology are shown in Figure 1 (one sample with glandular dysplasia has been included in the group of CIN1). HPV1 was the most prevalent type in the samples histologically classified as CIN+ (n=, %), followed by HPV1 (n=1, 1%), HPV1 (n=1, 0%), HPV (n=1, 1%), HPV (n=1, 1%), HPV (n=, %) and HPV, HPV1 and HPV (n=, %). The five most common HPV-types in the samples with cancer were HPV1, HPV1, HPV, HPV and HPV1, in that order. When analysing for HPV mrna, the picture was similar. The most common genotype in CIN+ samples expressing E/E mrna was HPV1 (1, %), followed by HPV1 (1, 1%), HPV1 (1, 1%), HPV (, %), HPV (, %) and HPV (, %). Consequently, 0% of HPV-infections, % of HPV-infections and 0% of HPV- infections in CIN+ lesions showed expression of E/E mrna, in comparison to HPV (0%), HPV (0%), HPV1 (1%), HPV1 (%) and HPV1 (%). In % (/) of mrna-positive samples (% of them CIN+), mrna of multiple genotypes was present. The samples expressing mrna of two or more genotypes represent % of all CIN+ samples. Downloaded from on September 1, 01 by guest

9 In four CIN+ samples (three cancers), no HR-HPV mrna could be found. In two of these samples (both cancers), HR-HPV DNA was undetectable. (The samples tested positive for HPV or HPV0, respectively, with other methods). The two DNA-positive CIN+ samples with undetectable mrna were two single infections with HPV1 or HPV, respectively. Out of samples with benign histology, (1%) were HPV-positive and 1 (%) expressed E/E mrna. However, all these women had a history of dysplasia. When looking at a screening-cohort of 1 women (median age 1) with benign cytology (no histology available), the prevalence of HPV-infection was % () of which.% () showed expression of E/E mrna. Overall, there was a good agreement between DNA and mrna testing, and of all detected HPV types were identified by both DNA and mrna testing (%). As expected the DNA analysis had a higher detection rate, and identified HPV types that were not detected by mrna testing. Conversely, mrna was detected in samples in which the same genotype was not detected by the DNA assay. Sensitivity, specificity, PPV and NPV The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of detecting CIN+ or CIN+ lesions were calculated for analysis of HPV DNA, mrna and with PreTect HPV-Proofer. Furthermore, calculations were made for the two in house tests (DNA and mrna) when including only the five most common HPV-types in cervical cancer (HPV1, 1, 1,, () which are the five genotypes included in the PreTect HPV-Proofer assay). However, recent data suggest that in different parts of the world, the most common HPV-types in cervical cancer may vary (, 1). HPV1 and 1 are the most common world wide, and with a few exceptions, the most common genotypes after HPV1 and 1 are HPV1,,,, and, in varying order. We therefore made calculations for sensitivity, specificity, PPV and NPV also for these eight genotypes. Since it was not Downloaded from on September 1, 01 by guest

10 possible to differentiate between low and moderate grades of neoplasia in glandular cells, one sample with glandular neoplasia not reaching the level of adenocarcinoma in situ was included as CIN1 in the calculations tabulated in table. The sensitivity and specificity results are illustrated in Figure Discussion This study aimed to evaluate the clinical performance of a real-time PCR assay that detects mrna transcripts coding for the oncogenic proteins E and E of 1 high-risk HPV and low-risk, using the same primers and probes as described previously for HPV DNA. For LBC samples with various grades of cervical neoplasia, there was good agreement between HPV mrna and HPV DNA results although the detection rate was higher with the DNA assay, as expected. Our assay for mrna detection, which includes a step that verifies that the mrna signal is not due to detection of DNA, had a sensitivity of detection of CIN+ and CIN+ that was only slightly lower than for DNA-detection (0.1 vs 0. and 0. vs 0., respectively), but the NPV did not decrease compared to the DNA-test. Importantly, the specificity was higher for mrna than for DNA detection (0. vs 0. for CIN+ lesions and 0. vs 0. for CIN+ lesions). These results may be compared with the observations by Szarewski et al in which several HPV DNA and mrna tests were compared (). The sensitivity and specificity for CIN+ using the PreTect HPV-Proofer assay (detecting five HR-HPV) was 0. and 0. in their study as compared with 0. and 0. in our evaluation. When comparing our assay for mrna typing with PreTect HPV-Proofer, the sensitivity of real-time PCR was somewhat higher (0. vs 0. for CIN+ lesions and 0. vs 0.1 for CIN+ lesions, when analysing for the same five genotypes). This may reflect a higher analytical sensitivity by real-time PCR compared with NASBA (because samples negative with PreTect HPV-Proofer but positive with real-time PCR in general contained low amounts Downloaded from on September 1, 01 by guest

11 of virus, as indicated by high Ct values, data not shown). The genotype most commonly detected by real-time PCR but not PreTect HPV-Proofer was HPV1, and this is in agreement with calculations of analytical sensitivity of the NucliSENS EasyQ assay (based on the same platform as PreTect HPV-Proofer) showing that the sensitivity of detection of HPV1 mrna is -0 times lower than for the other types (1). Moreover, our in house real-time PCR detects not only full-length mrna, but also spliced mrna transcripts of most genotypes, in contrast to PreTect HPV-Proofer (1), which may increase the sensitivity of the real-time PCR assay. A high analytical sensitivity might confer a risk of detecting low amounts of HPV mrna not significant for disease, but the CIN+ specificity of the real-time PCR was equal to that for PreTect HPV-Proofer when analysing for the same genotypes. There have been suggestions that the high specificity of PreTect HPV-Proofer is mainly due to the fact that it analyzes the five most common genotypes, and that a DNA-test analysing these five genotypes might be just as specific (). However, this speculation is contradicted by our data, since specificity calculated for these five genotypes was higher for both CIN+ and CIN+ lesions using the mrna as compared with the DNA version of our real-time PCR (0. vs 0. and 0. vs 0.0, respectively), suggesting that presence of E/E transcripts is important for disease. The high specificity by mrna testing was illustrated by the finding that in a screening cohort of 1 women (median age 1) with normal cytology (but with no histology available), HPV mrna was detected in.% and HPV DNA in %. The PPV of detection of both CIN+ and CIN+ was therefore higher for mrna-detection compared to DNA-detection (0. vs 0. and 0. vs 0., respectively), suggesting that mrna testing may be a useful tool not only in triage, but also in primary screening of cervical neoplasias. One should bear in mind that not all CIN+ lesions will progress to cancer, and a hypothetical perfect test identifying only truly precancerous lesions would rate poorly in sensitivity with CIN+ in histology used as a golden standard (as in this and most other studies). Downloaded from on September 1, 01 by guest

12 The five most common genotypes present in CIN+ and CIN+ lesions were HPV1, followed by HPV1, 1, and (in that order). In LBC samples from our patients with cancer however, the five most common genotypes were HPV1, 1,, and 1. This may reflect that the oncogenic properties of the genotypes vary. This idea was supported by the observation that some genotypes expressed E/E mrna more often than others. The eight genotypes most prone to express mrna in CIN+ lesions were, in descending order, HPV,, 1, 1, 1,, 1, and, the same genotypes (except for HPV1) most commonly found in cervical cancer worldwide (, 1). Possibly, the association of these eight genotypes with cancers may be a consequence of their potential to express oncogenic mrna. Our finding encourages further and larger studies comparing mrna and DNA detection rates for different HPV types. We specifically evaluated the performance of the real-time PCR detection of mrna for only the above mentioned eight genotypes that are most commonly observed in cancer. With this limitation, the sensitivity of the assay increased somewhat compared to analysis of five genotypes, but the specificity did not substantially decrease and the PPV remained, suggesting that these eight might constitute a good balance between sensitivity and specificity. This was relevant also for HPV DNA testing, since analysing eight as compared to all genotypes resulted in a significant increase in specificity at the expense of only a small loss in sensitivity, however without decreasing the high NPV. Our data suggest that mrna-testing with real-time PCR may be a useful tool in investigation of as well as in primary screening for cervical neoplasias, and there might be an idea to consider which genotypes to include in further investigations to optimize sensitivity and specificity, especially in a post vaccine era when it may be necessary to reconsider HPV testing strategies. Acknowledgments Downloaded from on September 1, 01 by guest 1

13 We thank Monika Dohsé for technical assistance. This study was supported by grants from the Western region R&D Fund, ALF-funds, Capio Research foundation and Assar Gabrielsson foundation Legends to figures Figure 1. Type-specific detection (%) of HPV DNA and mrna, distributed by histology. Each HPV type in a multiple infection is counted, which may result in an accumulative percentage of more than 0. ADC: Adenocarcinoma, SCC: Squamous cervical carcinoma Figure. Clinical sensitivity and specificity (with % confidence intervals) of the different test algorithms for detection of A) CIN+ lesions and B) CIN+ lesions. References 1. Anttila, A., L. Kotaniemi-Talonen, M. Leinonen, M. Hakama, P. Laurila, J. Tarkkanen, N. Malila, and P. Nieminen. 0. Rate of cervical cancer, severe intraepithelial neoplasia, and adenocarcinoma in situ in primary HPV DNA screening with cytology triage: randomised study within organised screening programme. BMJ 0:c.. Bosch, F. X., A. N. Burchell, M. Schiffman, A. R. Giuliano, S. de Sanjose, L. Bruni, G. Tortolero-Luna, S. K. Kjaer, and N. Munoz. 00. Epidemiology and natural history of human papillomavirus infections and type-specific implications in cervical neoplasia. Vaccine Suppl :K1-1.. Boulet, G. A., I. M. Micalessi, C. A. Horvath, I. H. Benoy, C. E. Depuydt, and J. J. Bogers. Nucleic acid-sequence based amplification assay for HPV mrna detection and typing: evidence for DNA amplification. J Clin Microbiol. Downloaded from on September 1, 01 by guest 1

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16 Mesher, D., A. Szarewski, L. Cadman, H. Cubie, H. Kitchener, D. Luesley, U. Menon, G. Hulman, M. Desai, L. Ho, G. Terry, A. Williams, P. Sasieni, and J. Cuzick. 0. Long-term follow-up of cervical disease in women screened by cytology and HPV testing: results from the HART study. Br J Cancer :-. 1. Munoz, N., X. Castellsague, A. B. de Gonzalez, and L. Gissmann. 00. Chapter 1: HPV in the etiology of human cancer. Vaccine Suppl :S/ Pett, M., and N. Coleman. 00. Integration of high-risk human papillomavirus: a key event in cervical carcinogenesis? J Pathol 1:-. 0. Ratnam, S., F. Coutlee, D. Fontaine, J. Bentley, N. Escott, P. Ghatage, V. Gadag, G. Holloway, E. Bartellas, N. Kum, C. Giede, and A. Lear. 0. Aptima HPV E/E mrna test is as sensitive as Hybrid Capture Assay but more specific at detecting cervical precancer and cancer. J Clin Microbiol :-. 1. Ratnam, S., F. Coutlee, D. Fontaine, J. Bentley, N. Escott, P. Ghatage, V. Gadag, G. Holloway, E. Bartellas, N. Kum, C. Giede, and A. Lear. Clinical performance of the PreTect HPV-Proofer E/E mrna assay in comparison with that of the Hybrid Capture test for identification of women at risk of cervical cancer. J Clin Microbiol :-.. Rodriguez-Lazaro, D., J. Lloyd, J. Ikonomopoulos, M. Pla, and N. Cook. 00. Unexpected detection of DNA by nucleic acid sequence-based amplification technique. Mol Cell Probes 1:1-.. Sotlar, K., A. Stubner, D. Diemer, S. Menton, M. Menton, K. Dietz, D. Wallwiener, R. Kandolf, and B. Bultmann. 00. Detection of high-risk human papillomavirus E and E oncogene transcripts in cervical scrapes by nested RTpolymerase chain reaction. J Med Virol :-1.. Szarewski, A., L. Ambroisine, L. Cadman, J. Austin, L. Ho, G. Terry, S. Liddle, R. Dina, J. McCarthy, H. Buckley, C. Bergeron, P. Soutter, D. Lyons, and J. Downloaded from on September 1, 01 by guest 1

17 Cuzick. 00. Comparison of predictors for high-grade cervical intraepithelial neoplasia in women with abnormal smears. Cancer Epidemiol Biomarkers Prev 1:0-.. Thorland, E. C., S. L. Myers, B. S. Gostout, and D. I. Smith. 00. Common fragile sites are preferential targets for HPV1 integrations in cervical tumors. Oncogene :1-. Downloaded from on September 1, 01 by guest 1

18 Table 1. Cytological and histological diagnoses of LBC samples. Diagnosis Cytology, n= N (%) [median age, years] Histology, n=1 N (%) [median age, years] Benign (0) [1] () [] ASCUS (1) [0] - ASC-H (1.0) [1] - Glandular dysplasia (1.) [0] 1 (0.) [] CIN1 1 (.1) [1] (1) [1] CIN (.) [] (1) [] CIN 1 (.0) [] 1 (0) [1] Adenocarcinoma (1.) [] (.) [] Squamous cell carcinoma 1 (.) [] 1 (1) [] Histology not done on samples with median age 1 years. Downloaded from on September 1, 01 by guest

19 Table. Sensitivity, specificity, PPV and NPV of different test algorithms for the detection of histologically confirmed CIN+ or CIN+ lesions. Test Sensitivity (% CI) Specificity (% CI) PPV (% CI) NPV (% CI) HPV DNA CIN+ 0. ( ) 0. (0.-0.0) 0. (0.-0.) 0. ( ) CIN+ 0. ( ) 0. (0.0-0.) 0. (0.-0.0) 0. ( ) HPV DNA gt a CIN+ 0. ( ) 0. (0.-0.1) 0. (0.1-0.) 0. ( ) CIN+ 0. (0.-0.) 0.0 (0.1-0.) 0. (0.-0.) 0.0 (0.-0.) HPV DNA gt b CIN+ 0. (0.0-0.) 0.0 (0.-0.) 0.1 (0.-0.) 0.1 (0.-0.) CIN+ 0. (0.-0.) 0. (0.-0.) 0. (0.0-0.) 0. (0.-0.) HPV mrna CIN+ 0. ( ) 0. (0.0-0.) 0. (0.-0.) 0. ( ) CIN+ 0.1 (0.-0.) 0. (0.0-0.) 0. (0.-0.) 0.1 (0.-0.) HPV mrna gt CIN+ 0.0 (0.-0.) 0. (0.-0.1) 0. (0.0-0.) 0. (0.0-0.) CIN+ 0. (0.0-0.) 0. (0.-0.) 0.0 (0.-0.) 0. (0.-0.) HPV mrna gt CIN+ 0. (0.-0.) 0. (0.-0.) 0.0 ( ) 0. (0.-0.) CIN+ 0. (0.-0.1) 0. (0.-0.) 0.1 (0.-0.0) 0. (0.0-0.) PreTect HPV Downloaded from on September 1, 01 by guest Proofer CIN+ 0.1 ( ) 0.0 (0.-0.) 0.1 (0.1-0.) 0.1 (0.-0.) CIN+ 0. (0.-0.) 0. (0.0-0.) 0.0 (0.1-0.) 0.1 (0.-0.) a HPV1, 1, 1,,,,,, b HPV1, 1, 1,,.

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