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1 Laboratoire de Virologie Moléculaire, Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Montréal, Québec, Canada
2 Department of Epidemiology, Biostatistics and Occupational Health Medicine, McGill University, Montreal, Québec, Canada
3 Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, BC, Canada
4 Department of Medicine, Sunny Brook Health Science Centre, University of Toronto, Toronto, Ontario, Canada
5 Department of Pathology and Obstetrics & Gynecology, The Sir Mortimer B. Davis-Jewish General Hospital and McGill University, Québec, Canada
6 Departments of Oncology, Division of Epidemiology, McGill University, Montreal, Québec, Canada
7 Département de Microbiologie et Immunologie, Université de Montréal, Montréal, Québec, Canada
Correspondence
François Coutlée
francois.coutlee{at}ssss.gouv.qc.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Members listed in Acknowledgements. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). HPV-16 is the most oncogenic in terms of progression to CIN-2,3 and to cervical cancer (Clifford et al., 2003). Although most women are infected with HPV, only a minority of women will develop persistent HPV infection that may progress to high-grade CIN (CIN-2,3) and cancer (Ferenczy & Franco, 2002). Host and viral factors are involved in the progression of HPV-induced cervical lesions (Wang & Hildesheim, 2003). High HPV-16 viral loads have been associated with CIN-2,3 in case–control studies or with progression to CIN-2,3 or carcinoma in situ in prospective studies (Dalstein et al., 2003; Flores et al., 2006; Josefsson et al., 2000; Moberg et al., 2004; Schlecht et al., 2003). Although increased HPV-16 DNA viral load could help identify women infected with HPV-16 at greater risk for CIN-2,3, the substantial overlap of viral load values between women with and without CIN is a limitation (Cheung et al., 2006; Fontaine et al., 2005a; Guo et al., 2007).
HPV-16 integration is considered to be a key event in the progression of persistent HPV-16 infection to invasive cancer (Hopman et al., 2004; Kalantari et al., 1998), resulting in uncontrolled expression of HPV-16 E6 and E7 oncogenes. Quantification of viral load of integrated HPV-16 forms could prove to be a better biomarker for CIN-2,3 than HPV-16 viral load. Since HPV-16 integration often disrupts the E2 gene, current assays measuring HPV-16 integration are based on quantification with real-time PCR of HPV-16 E6 relative to E2 DNA (Arias-Pulido et al., 2006; Peitsaro et al., 2002). Detection of a greater quantity of HPV-16 E6 compared with HPV-16 E2 suggests the presence of integrated HPV-16 forms. We have shown recently with these assays that HPV-16 viral load of integrated forms was associated with CIN-2,3 in HIV-seropositive women (Fontaine et al., 2005a). Quantification of HPV-16 DNA can be influenced by HPV-16 polymorphism (Fontaine et al., 2005b). Only limited knowledge of HPV-16 E2 polymorphism was available when primers and probe were first selected for HPV-16 E2 quantification by real-time PCR (Nagao et al., 2002; Peitsaro et al., 2002).
The purpose of this study was to characterize HPV-16 E2 polymorphism. To investigate further the association between the viral load of HPV-16 integrated forms and CIN-2,3, we optimized a real-time PCR assay to quantify HPV-16 E2 targeting conserved sequences of HPV-16 E2. We then applied these real-time PCR assays for quantification of HPV-16 E6 and E2 on cervicovaginal lavages (CVL) collected during the course of a prospective cohort study on the natural history of persistent HPV infection in a population of HIV-seropositive and -seronegative women. We demonstrate here that HPV-16 E2 polymorphism influences the quantification of episomal and integrated HPV-16 of isolates from non-European lineages. Viral load of integrated HPV-16 forms or the presence of HPV-16 integrated forms, was not associated with CIN-2,3.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Study subjects were selected from women infected with HPV-16 participating in the Canadian Women's HIV Study between May 1993 and March 2002. The Canadian Women's HIV Study is a cross-sectional and cohort study that investigates the interplay between HPV and HIV infections on cervical pre-cancerous lesions. As described previously, 1055 women were enrolled in that study from sexually transmitted diseases, primary care and outpatient HIV care clinics from across Canada (Fontaine et al., 2005a; Hankins et al., 1999). Women were eligible to participate if they had provided written informed consent, were seropositive for HIV-1 or were seronegative for HIV, but were at risk for sexually transmitted diseases. The research protocol was approved by each local institutional Ethics committee. Since our cohort was initiated in 1993, very few HIV-seropositive women were under highly active anti-retroviral therapy.
A standardized questionnaire was administered upon study entry and at 6 month intervals. For all HIV-seropositive women, CD4 cell counts, Papanicolaou (Pap) smears and CVL were obtained at 6 month intervals (Coutlée et al., 1997; Hankins et al., 1999). For HIV-seronegative women, CVL and Pap smears were collected at 1 year intervals. Cytology smears were interpreted in one central pathology laboratory and confirmed by one pathologist. Colposcopy was performed systematically in participants with high-grade squamous intraepithelial lesion (HSIL) on cytology smears and was suggested to participants with smears showing low-grade SIL (LSIL). Of 732 HIV-seropositive and 323 HIV-seronegative women screened for HPV infection, 366 HPV-infected women (207 HIV-seropositive with a mean of follow-up of 27.3 months, 159 HIV-seronegative with a mean follow-up of 17.9 months) were followed prospectively.
Of the 1055 participants, 132 (12.5 %) had at least one CVL containing HPV-16 DNA at baseline or during follow-up visits. For this report, data were limited to 74 women (58 HIV-seropositive and 16 HIV-seronegative): 38 had colposcopy, 24 had three consecutive normal smears over a period of at least 12 months, seven had greater than two consecutive smears with LSIL, two had one smear with HSIL, three did not have colposcopy or cytology but were infected with African or Asian–American HPV-16 variants. The 58 HPV-16-positive women who were not included in the study because the diagnosis of CIN or SIL were uncertain or they had less than three normal smears, have been described elsewhere (Fontaine et al., 2005a).
Cervical samples from the Biomarkers of Cervical Cancer Risk (BCCR) study.
We also included 118 HPV-16-positive cervical samples from women recruited in the BCCR study. This hospital-based case–control study conducted in Montreal, Canada, investigates biomarkers for CIN-2,3 and cancer (Koushik et al., 2005). Controls were women with normal Pap smears, while cases were histologically confirmed as CIN-2,3-positive. Exfoliated cervical cells were collected with a cytobrush.
HPV DNA detection and HPV lineage assessment.
Cell suspensions from CVL collected in the Canadian Women's HIV Study were lysed with 0.8 % Tween 20 and digested with proteinase K (Coutlée et al., 1997). An aliquot of 5 µl from each processed sample was amplified for β-globin DNA with PC04-GH20 (Coutlée et al., 1997). Of the 5262 genital specimens collected in all study subjects at enrolment and follow-up visits (mean of 5.7 specimens per subject), 104 (2.0 %) failed to amplify β-globin. β-Globin-positive samples were tested for HPV DNA detection and typing with MY09-MY11-HMB01 consensus L1 PCR and type-specific probes (Coutlée et al., 1997). DNA was extracted from samples collected from the BCCR study using the Master pure kit as described previously (Koushik et al., 2005). β-Globin-positive samples were tested for HPV DNA with PGMY primers and typed with the Line blot assay (Roche). HPV-16 isolates were classified into European, African 1, African 2 or Asian–American lineages by direct PCR sequencing of the long control region (LCR) as described previously (Fontaine et al., 2005a).
HPV-16 E2 analysis by PCR sequencing.
Two fragments of HPV-16 E2 (nt 2654–3361 and 3200–3905) were amplified with primer pairs 16-E2-1 (5'-CGAAAATCCAGTGTATGAGC-3') and 16-E2-2 (5'-GTCGCTAAACACAGATGTAGGAC-3'); and 16-E2-3 (5'-GAGGGTCAAGTTGACTATTATGG-3') and 16-E2-4 (5'-AGCAAAGCAAAAAGCACG-3'). For each reaction, 2 µl of processed specimen was amplified in a 100 µl reaction volume containing 10 mmol Tris/HCl (pH 8.3) l–1, 50 mmol KCl l–1, 2.5 U AmpliTaq Gold enzyme (Roche Diagnostic Systems), 2.0 mmol MgCl2 l–1, 0.5 mmol each primer l–1 and 0.25 mmol dCTP, dTTP, dGTP and dATP l–1. HPV-16 E2 amplicons were generated by amplification in a 9600 thermal cycler (Perkin-Elmer) with an initial step of 9 min at 95 °C; 40 cycles for 60 s at 95 °C, 60 s at 55 °C and 60 s at 72 °C; and a final step for 7 min at 72 °C. Direct double-stranded PCR sequencing was performed on HPV-16 E2 DNA amplicons by using the fluorescent cycle sequencing method (BigDye terminator ready reaction kit; Perkin-Elmer) on 20 ng purified amplicons by using the same primers as above, with 25 cycles for 10 s at 96 °C, 5 s at 50 °C and 4 min at 62 °C. Sequence analysis was performed on an ABI Prism 3100 genetic analyser system at the DNA Sequencing Service of the Centre de Recherche du CHUM. Non-prototypic isolates were sequenced twice and, in all cases, confirmed the initial sequencing results. Sequences from HPV-16 E2 variants were aligned for classification with CLUSTAL W (version 1. 8) software.
Real-time PCR for HPV-16 viral load and integration.
HPV-16-positive CVL samples have been previously screened for the presence of PCR inhibitors with internal controls for HPV-16 E6, E2 and β-globin DNA (Fontaine et al., 2005a; Lefevre et al., 2004). Two microlitres of processed sample without inhibition were tested in duplicate in separate capillaries for quantification of HPV-16 E6 (RT-E6) and HPV-16 E2 (RT-E2-1) by using the real-time PCR assays for E6 and E2 described by Peitsaro et al. (2002) (Fontaine et al., 2005a), and for β-globin as described previously (Fontaine et al., 2005a; Lefevre et al., 2004). HPV-16 E6 DNA was also measured in all samples containing an African or Asian–American HPV-16 variant and in 40 samples randomly selected containing a European HPV-16 variant, with a second real-time PCR assay (HPV-16 E6 PG PCR) as described previously (Gravitt et al., 2003).
HPV-16 E2 was also quantified in duplicate by a novel real-time PCR, designated RT-E2-2, by using a 20 µl reaction mixture containing 1x DNA Master Hybridization Probe Mix with the Fast Start Taq DNA polymerase (Roche Molecular Biochemicals), 0.3 µM each HPV-16 primers NA-16-F and NA-16-R (Table 1), and 50 nM fluorogenic probe NA-16-P. Cycling parameters included an activation step at 95 °C for 10 min followed by 50 cycles at 95 °C for 15 s and 61 °C for 30 s. Cycle thresholds were compared to those of an HPV-16 titration curve obtained by 10-fold serial dilutions of an HPV-16 plasmid, kindly provided by Professor zur Hausen (Heideberg, Germany), in a fixed amount of 75 ng human genomic DNA (Roche Diagnostics) in 10 mM Tris/HCl (pH 8.2). HPV-16 E6 and E2 viral loads were expressed as the number of HPV-16 copies per cell equivalent. The integration status of HPV-16 was assessed by comparing the levels of HPV-16 E2 and E6 genes and was expressed as the E6/E2 ratio. The integrated HPV-16 viral load was calculated by subtracting the copy numbers of E2 (episomal) from the copy numbers of E6 (integrated and episomal) for specimens with an E6/E2 ratio >2.0, as discussed previously (Fontaine et al., 2005a; Khouadri et al., 2007).
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). All statistical tests were two-sided with statistical significance set at P<0.05. Statistical analyses were performed with STATISTICA version 6 software (StatSoft). |
RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Isolates from three Caucasian women identified as the Asian–American LCR variant (GenBank no. AY112662
[GenBank]
) were further classified into two E2 variants that had 20 and 21 variations compared with the E2 prototype. Isolates from six women (five of African origin and one Caucasian) identified as LCR variants T4, T8 (GenBank no. AF472508
[GenBank]
) and PF-18 of the African 1 lineage were classified into four E2 variants (median number of variations=17, range=16–19) compared with the E2 prototype. Three isolates identified as African 2 LCR variant (GenBank no. U34089
[GenBank]
) and collected from women of African origin were further classified into two E2 variants with 19 and 20 variations each compared with the E2 prototype. Fifty-six variation sites (median variations per variant=2, range=0–6) defined 32 HPV-16 E2 variants in the 118 European isolates (Fig. 2). Seventy-six of these isolates were HPV-16 E2 prototypes. All HPV-16 E2 variants shared the A2925G variation, as published in a correction to the reference HPV-16 sequence (Meissner, 1997; Swan et al., 2005).
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(Casas et al., 1999; Eriksson et al., 1999; Giannoudis et al., 2001; Graham & Herrington, 2000; Meissner, 1997; Sathish et al., 2004; Swan et al., 2005; Veress et al., 1999; Watts et al., 2002). Two primers and one internal probe were selected from conserved areas of E2 to detect the hinge region (Table 1
). The Tm of primers NA-16-F and NA-16-R selected for the RT-E2-2 assay were similar. RT-E2-2 and RT-E6 assays had a wide linear range that covered 10–106 copies of HPV-16 DNA, generated superimposed titration curves and reached the same end point of 10 HPV-16 DNA copies (data not shown). Amplification efficiencies for RT-E2-2 (median=94.8 %, range=93.9–95.1 %) and RT-E6 (median=94.4 %, range=93.5–95.1 %) were similar (P=0.81).
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To evaluate the variability between runs of calculating HPV-16 E6/E2 ratios, we tested 14 samples on three different runs with RT-E6-1 and RT-E2-2. Table 2 shows that estimation of HPV-16 E6/E2 was reproducible between runs. However, considering the initial and the triplicate estimations of E6/E2 ratio, two specimens with ratios above but near 2.0 falsely gave a ratio below two once out of three times (Table 2).
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). HPV-16 E6/E2 ratios greater than two were obtained for the six samples containing African 2 (n=3) or Asian–American (n=3) isolates when tested with RT-E2-1, but for only one of these samples when tested with RT-E2-2 (Table 3). Conversely, such a change in E6/E2 ratio with the use of RT-E2-2 was encountered with only one of 12 European isolates. With European isolates, HPV-16 E6/E2 ratios using RT-E2-1 (median=2.14, range=1.12–33.59) and RT-E2-2 (median=2.18, range=0.86–29.66) were similar. HPV-16 E6/E2 ratios calculated with African 1 isolates using RT-E2-1 (median=1.69, range=0.91–2.96) and RT-E2-2 (median=1.83, range=1.16–4.68) were also similar. In contrast, HPV-16 E6/E2 ratios obtained with African 2 isolates using RT-E2-1 (median=4.10, range=2.09–6.41) and RT-E2-2 (median=1.17, range=1.09–1.99) were different, similarly to ratios obtained with Asian–American isolates using RT-E2-1 (median=24.53, range=3.78–30.0) and RT-E2-2 (median=0.99, range=0.81–16.67). For each specimen, the ratio of number of HPV-16 DNA copies measured with RT-E2-2 and RT-E2-1 was calculated. The distribution of ratios of HPV-16 copies with RT-E2-2 and RT-E2-1 (RT-E2-2/RT-E2-1 in Table 3) obtained with European isolates (median=1.02, range=0.64–1.80) and with African 1 isolates (median=0.80, range=0.53–1.09) was similar (P=0.08), while ratios with African 2 (median=3.23, range=1.92–3.49) or Asian–American (median=3.78, range=1.47–37) isolates were different from that of European isolates (P<0.02 for each comparison).
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HPV-16 episomal and integrated viral loads and grade of cytology smears or CIN
Overall, 139 CVL (median=1 CVL per woman, range=1–8) collected from 74 women were analysed for HPV-16 viral load and integration by measuring HPV-16 E6 and E2 viral loads with the following assays. Samples were all tested with the RT-E2-2 assay to avoid underestimation of HPV-16 E2 viral load as demonstrated above. We had previously demonstrated that the RT-E6 assay could underestimate the quantity of HPV-16 E6 DNA for isolates belonging to the African phylogenetic branch (Fontaine et al., 2005b). Variations at nucleotide positions 109, 131, 143 and 145 of the E6 sequences compared with the European prototype in these isolates are located within probe and primer sequences used in the RT-E6 assay. These mismatches between RT-E6 reagents and HPV-16 E6 DNA sequence may explain the underestimation of viral loads of African isolates. In contrast, we obtained an excellent correlation between the two assays for European or Asian–American isolates. To avoid underestimating the amount of HPV-16 E6 in our cohort, eight samples from women infected with African 1 or African 2 isolates were tested with RT-E6 and HPV-16 E6 PG assays to investigate the influence of HPV-16 E6 polymorphism on quantification of HPV-16 DNA. The quantity of HPV-16 E6 DNA measured with HPV-16 E6 PG had a mean of 2.24±1.25-fold higher (range=1.37–5.26) than the quantity measured with RT-E6 (data not shown). In contrast, the ratio of HPV-16 DNA quantity measured with RT-E6 and HPV-16 E6 PG was of 1.08±0.47 for three isolates belonging to the Asian–American branch and of 0.98±0.25 for isolates from 40 samples belonging to the European branch. The following analyses used HPV-16 E6 copy numbers obtained with the HPV-16 PG assay for samples containing African variants and RT-E6 results for samples containing European or Asian–American variants.
We first measured HPV-16 viral loads in CVL samples collected from 69 women for whom Pap smears results were available (45 women with normal smears, three with ASCUS, 16 with LSIL and five with HSIL). Only one visit per participant with the highest grade of SIL was considered. Of these 70 women, 57 were infected with European variants, five by African 1 variants, three by African 2 variants, three by Asian–American variants and HPV-16 polymorphism was not assessed for two participants. As shown in Table 4, the distributions of HPV-16 total and episomal DNA viral loads were significantly different between women without SIL and those with LSIL or HSIL. Age, detection of high-risk HPV types other than 16, CD4 counts, race and HIV status were similar between women with various grades of SIL (P>0.10 for each comparison, data not shown). Controlling for age, CD4 counts and HIV infection, and the presence of non-European HPV-16 variants, HPV-16 total (OR=2.60, 95 % CI=1.01–6.92) or episomal (OR=1.69, 95 % CI=1.13–2.52) viral loads, but not integrated HPV-16 viral loads (OR=1.31, 95 % CI=0.93–1.88), were significantly associated with HSIL.
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). The latter 60 women (45 HIV-seropositive and 15 HIV-seronegative) had a median age of 29.1 years (range=18–63.3) and median CD4 counts of 287 cells µl–1 (range=6–960). Of these 60 women, 50 were infected with European HPV-16 variants, four by African 1 variants, two by African 2 variants, two by Asian–American variants and two samples could not be analysed. Total and episomal HPV-16 viral loads were significantly greater in women with CIN-2,3 compared with control women without CIN in univariate analysis (P=0.01). These associations remained significant in multivariate analysis controlling for age, HIV and CD4 count, and presence of non-European variant, for HPV-16 total viral load (OR=2.17, 95 % CI=1.11–4.23) and episomal viral load (OR=2.14, 95 % CI=1.09–4.19), but not for HPV-16 integrated viral load (OR=1.71, 95 % CI=0.90–3.26). There was a significant correlation between total and episomal viral loads (r=0.99, P<0.05). We then investigated if the presence of integrated forms irrespective of integrated viral load was associated with CIN-2,3. There was no difference in the proportion of women with a ratio 
2.0 in women without CIN (7 of 35) and with CIN-2,3 (5 of 11, P=0.24) or CIN-1 (5 of 14, P=0.50).
HPV-16 E6/E2 ratios 2 could also result from variability of quantification of HPV-16 E2 and E6. The variability of real-time PCR assays is maximal at very low target numbers as demonstrated by CVs calculated above and in the HPV-16 titration curves. There is thus an advantage of testing HPV-16 DNA copies in the range between 100 and 106 copies. Considering specimens from women without SIL and with HPV-16 E6/E2 >2, 20 samples were within this range with two additional samples being above 106 but below 5x106 copies. We also investigated the reproducibility of obtaining HPV-16 E6/E2 >2 in consecutive samples. Of the 29 women with more than one consecutive CVL (range=2–7) tested with real-time PCR, 26 had a cytological and/or histological diagnosis available. Similar HPV-16 E6/E2 ratios (<2 for 17 and >2 for two women) were obtained on all consecutive CVL tested for 19 women. One participant with CIN-2,3 provided three samples with HPV-16 E6/E2 ratios between 2.8 and 4.9, while another woman with CIN-1 provided samples with ratios of 2.4 and 4.2. Discordant HPV-16 E6/E2 ratios were obtained for 10 participants: six had only one of two to three samples with E6/E2 >2, one had two of three samples with E6/E2 >2 and three women had more than three samples tested, several of which had E6/E2 ratio >2 (one had five samples analysed, three with ratios from 2.1 to 3.4 and two with ratios from 0.7 to 0.9; one with CIN-1 had two consecutive samples with ratios of 2.1 and 3.4 and four subsequent samples with ratios from 1.2 to 1.9; one woman with CIN-1 had two samples with ratios of 2.3 and 2.6 and two samples with ratios of 0.5 and 1.8).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). HPV-16 E2 DNA polymorphism altered quantification of E2 in isolates from African 2 and Asian–American lineages, also affecting significantly the E6/E2 ratio measured with these samples. Overall, fewer specimens were considered to contain integrated HPV-16 forms with the novel HPV-16 E2 assay. Episomal and total HPV-16 viral loads, but not the presence of integrated forms, were associated with CIN-2,3 on biopsy or HSIL on cytology.
Most significant differences between RT-E2-1 and RT-E2-2 were encountered for samples containing Asian–American E2 variants. Sequence mismatches in primers and/or probe with viral sequences can introduce significant errors into quantitative PCR by impeding efficiency of amplification or hybridization reaction (Whiley & Sloots, 2005). Mismatches at the 3' end of primers introduce considerable error in quantification, with calculated copy numbers being 2–3 logs lower than the actual number of copies (Whiley & Sloots, 2005). HPV-16 viral load results from studies conducted on populations infected with non-European variants should be interpreted with caution if assays optimized with European variants are applied.
Differences between E6 and E2 viral loads could reflect technical limitations of the assays rather than true integration. Since integration is measured by subtracting E2 from E6, variability inherent to each assay will influence the calculated number of copies of integrated forms. Real-time PCR assays for HPV quantification have excellent intrarun and interrun reproducibility (Flores-Munguia et al., 2004; Fontaine et al., 2005a, b; Gravitt et al., 2003; Lefevre et al., 2003; Moberg et al., 2003). In our work, CVs obtained were less than 30 % for quantities of HPV-16 100 copies and above, as reported by others (Arias-Pulido et al., 2006; Fontaine et al., 2005b; Gravitt et al., 2003; Lefevre et al., 2003). Most samples analysed here were in the range of 100–106 copies, a range with less variability. Moreover, E6/E2 ratios measured for European isolates with two E2 assays were similar for most samples irrespective of initial quantities of HPV-16 DNA. Assay variability is thus unlikely to explain HPV-16 E6/E2 ratios over two. Some have reported in an American population that the hinge of E2 is the most frequent site of rupture opposed to the 3' end of E2 that was found to be the most frequently disrupted in a study conducted in China (Arias-Pulido et al., 2006; Cheung et al., 2006; Kalantari et al., 1998). The performance of real-time PCR assays to detect integration may be different between populations because of different HPV integration patterns.
Although the analytical sensitivity of real-time PCR for quantification of HPV-16 E6 and E2 DNA is excellent (Cheung et al., 2006; De Marco et al., 2007; Guo et al., 2007), the clinical sensitivity and specificity of these assays to detect HPV-16 integration against a gold standard method have not been thoroughly assessed (De Marco et al., 2007). Confirmation of the presence of integrated HPV-16 DNA forms detected by real-time PCR has been accomplished on a limited number of samples by various techniques including in situ hybridization (Fujii et al., 2005), Southern blot (Fontaine et al., 2005a; Nagao et al., 2002) and PCR sequencing methods identifying the presence of HPV-human DNA junctions (De Marco et al., 2007). One of the latter methods, the detection of integrated papillomavirus sequences-PCR, found integrated HPV-16 in all 11 samples that contained mixed or integrated HPV-16 forms by real-time PCR and tested negative for all 20 samples that did not contain integrated HPV-16 (De Marco et al., 2007). We also confirmed by real-time PCR the presence of integrated HPV-33 forms with restricted-site PCR (Khouadri et al., 2007). Complete concordance between real-time PCR and a combination of Southern blot and 2D-gel electrophoresis was obtained on eight specimens only containing episomal forms and four specimens with mixed HPV-16 forms (Nagao et al., 2002). Finally, a concordance rate of 86 % was obtained on 47 samples analysed with real-time PCR and in situ hybridization for the detection of HPV-16 integrated forms (Fujii et al., 2005). These publications suggest that real-time PCR assays are promising techniques for the detection of integrated HPV forms, but further studies on a greater number of specimens and using several techniques in parallel for the detection of integrated HPV-16 need to be conducted.
With optimized assays, our findings demonstrate that women infected with HIV or at risk for HIV infection, are infected with higher HPV-16 viral loads when they have CIN-2,3 than women without SIL or CIN. Recent case–control studies also reported that HPV-16 viral load was independently associated with CIN-2,3 (Flores et al., 2006; Fontaine et al., 2005a; Rajeevan et al., 2005; van Duin et al., 2002). In studies with prospective designs, high viral loads of HPV-16 DNA increased the risk of developing HSIL/CIN-3, carcinoma in situ or invasive cancer (Josefsson et al., 2000; Moberg et al., 2004, 2005; Schlecht et al., 2003; Ylitalo et al., 2000). In our study, integrated HPV-16 viral load was not associated with CIN-2,3 in multivariate analysis. Others reported a marginal association between the presence of integrated forms and CIN-2, while there was no significant association with CIN-3 or invasive cancer (Cheung et al., 2006) or between the presence of integration and cytology grade (Kulmala et al., 2006). One study reported a significant difference in the proportion of samples with HPV-16 integration between cervical cancer and CIN-2,3 (Guo et al., 2007). For specimens harbouring a mixture of episomal and integrated forms, the latter form accounts for >75 % of the total HPV-16 copy number (Cheung et al., 2006). We also found that in specimens with E6/E2 ratios >2, integrated HPV-16 accounted for 59–90 % of total HPV-16 DNA copies.
We recognize that our study has limitations. We assessed the association between grade of disease and HPV-16 viral loads by using cross-sectional analysis of cohort study data. We could rely on a high proportion of diagnoses based on histology and colposcopy results, reviewed by a dedicated pathologist. The prospective design of the Canadian Women's HIV Study allowed us to select the visit corresponding to the highest grade of SIL or CIN obtained on consecutive visits and also enabled us to evaluate the integration results over several visits. Most participants were infected or were at high risk for HIV infection. Our results cannot be extrapolated to the general population because participants in this study were at high risk for sexually transmitted diseases. The small number of participants with CIN or SIL restricted our analysis to test possible associations with potential mediating or confounding factors.
The difference between viral load values for women with CIN-2,3 and those lesion-free, although statistically significant, does not permit reliable data, classifying women into higher risk categories because of the important overlap of viral load values. More studies are needed to understand better the meaning of detecting integrated forms in normal women. The value of detecting integrated forms in pre-cancerous lesions should be investigated in cohort studies to monitor progression of such lesions. The presence of integrated forms should be investigated in normal women prospectively to assess if it is a determinant of progression to CIN, or progression from low-grade to high-grade CIN lesion. Although studies have suggested that integration could be a useful biomarker for CIN grade or progression, more studies need to be conducted with optimized assays and prospectively collected samples. Longitudinal studies with larger sample sizes are required to set the cut-off of E6/E2 ratios and viral load of integrated forms as predictive markers of CIN-2,3 or CIN progression and the utility of measuring integration with real-time PCR. The value of real-time PCR assays to detect integration needs to be established with standard confirmatory methods of integration.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 9 November 2007;
accepted 24 February 2008.
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