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Episomal and integrated human papillomavirus type 16 loads and anal intraepithelial neoplasia in HIV-seropositive men

Alvarez, Jennifera,b; Pokomandy, Alexandra DEc,d; Rouleau, Daniellea,b; Ghattas, Georged,e; Vézina, Sylvief; Coté, Pierreg; Allaire, Guya; Hadjeres, Rachida; Franco, Eduardo Lc; Coutlée, Françoisa,b,cfor the HIPVIRG Study Group

doi: 10.1097/QAD.0b013e32833db9ea
Clinical Science

Objectives: To assess levels of episomal and integrated human papillomavirus type 16 (HPV-16) loads in HIV-seropositive men who have sex with men (MSM) in anal infection and to study the association between episomal and integrated HPV-16 loads and anal intraepithelial neoplasia (AIN).

Study design: A cohort study of 247 HIV-positive MSM followed each 6 months for 3 years. Overall, 135 (54.7%) men provided 665 HPV-16-positive anal samples.

Methods: Episomal and integrated HPV-16 loads were measured with quantitative real-time PCR assays. HPV-16 integration was confirmed in samples with a HPV-16 E6/E2 of 1.5 or more with PCR sequencing to demonstrate the presence of viral–cellular junctions.

Results: The HPV-16 DNA forms in anal samples were characterized as episomal only in 627 samples (94.3%), mixed in 22 samples (3.3%) and integrated only in nine samples (1.4%). HPV-16 episomal load [odds ratio (OR) = 1.5, 95% confidence interval (CI) 1.1–2.1], number of HPV types (OR = 1.4, 95% CI 1.1–1.8) and current smoking (OR = 4.8, 95% CI 1.3–18.6) were associated with high-grade AIN (AIN-2,3) after adjusting for age and CD4 cell counts. Integrated HPV-16 load was not associated with AIN-2,3 (OR = 0.7, 95% CI 0.4–1.1). Considering men with AIN-1 at baseline, four (16.7%) of the 24 men who progressed to AIN-2,3 had at least one sample with integrated HPV-16 DNA compared with three (23.1%) of 13 men who did not progress (OR = 0.7, 95% CI 0.2–3.8; P = 0.64). Integration was detected in similar proportions in samples from men without AIN, with AIN-1 or AIN-2,3.

Conclusion: High episomal HPV-16 load but not HPV-16 integration load measured by real-time PCR was associated with AIN-2,3.

aDépartements de Microbiologie et Infectiologie, Pathologie, et Laboratoire de Virologie Moléculaire, Centre Hospitalier de l'Université de Montréal, Canada

bDépartements de Microbiologie et Immunologie, Université de Montréal, Canada

cDivision of Cancer Epidemiology, McGill University, Canada

dDepartment of Medicine and Immunodeficiency Clinic, McGill University Health Center, Canada

eDepartment of Medicine, McGill University, Canada

fClinique Médicale l'Actuel, Canada

gClinique Médicale du Quartier Latin, Montreal, Quebec, Canada.

*Members of the HIPVIRG Study group are listed in the Acknowledgements.

Received 30 April, 2010

Revised 20 June, 2010

Accepted 29 June, 2010

Correspondence to François Coutlée, Hôpital Notre-Dame du Centre Hospitalier de l'Université de Montréal, 1560 Sherbrooke est, Montreal, QC H2L 4M1, Canada. Tel: +1 514 890 8000/25162; fax: +1 514 412 7512; e-mail:

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High-grade anal intraepithelial neoplasia (AIN-2,3), the precursor lesion for anal cancer, can be detected over a 4-year period in nearly half of HIV-seropositive MSM [1–4]. Human papillomavirus (HPV) infection causes AIN and anal cancer [5]. More than 95% of HIV-seropositive MSM are infected in the anal canal by HPV [6–8]. Prevalent AIN-2,3 and progression to higher AIN grades have been associated with HPV-16, HPV-18 or HPV-31 infection, multiple HPV types, HPV-16 E6 polymorphism and lower blood CD4 T-cell counts [1,4,6,7,9–15]. HPV-16 is the most common genotype in high-grade anogenital intraepithelial neoplasia and invasive cancer [8,16,17]. We have previously shown in HIV-seropositive women that HPV-16 DNA viral load was associated with HPV-16 persistence and high-grade cervical intraepithelial neoplasia (CIN-2,3), a disease sharing clinical and histological similarities with AIN [5,18,19]. A recent study on HIV-seropositive men also reported that high HPV-16 viral loads were associated with AIN-2,3 [6].

HPV-16 DNA can be detected in genital samples as an episome or as integrated forms into the host cellular DNA. Integration of high-risk HPV types into cellular DNA is considered to be a key event in the progression of intraepithelial neoplasia to invasive cancer [20,21]. HPV-16 integration results in the disruption of the E2 gene and overexpression of HPV oncogenes. The contribution of episomal and integrated HPV-16 forms to the role of HPV-16 load in AIN-2,3 as well as the longitudinal assessment of HPV-16 load and integration in the natural history of HPV infection have not been well defined.

The objectives of the current study were to describe the episomal and integrated forms of HPV-16 loads in the course of anal infection in HIV-seropositive men and assess whether they were associated with prevalent AIN-2,3 or progression to low-grade AIN (AIN-1) and AIN-2,3.

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Patients and methods

Study protocol

Participants for the current report were selected from individuals participating in the Human Immunodeficiency and Papilloma Virus Research Group (HIPVIRG) cohort, a longitudinal study on the natural history of HPV infection and AIN in HIV-seropositive MSM described previously [10]. Briefly, 18–65-year-old MSM treated or expected to be treated with HAART within the next 6 months were enrolled from January 2002 through January 2005 after giving written informed consent. The study was approved by institutional review boards at all sites. For the current analysis, we included only participants who tested positive for anal HPV-16 DNA at least once in the study.

Participants were evaluated at a baseline visit and every 6 months for 3 years with a self-administered questionnaire, blood CD4 T-cell counts and plasma HIV RNA load [10]. Epithelial cells from the anal canal were collected with a Dacron swab and resuspended in 1.5 ml of PreservCyt (Cytyc Corporation, Boxborough, Massachusetts, USA), as previously described [10]. High-resolution anoscopy (HRA) with biopsies of aceto-white areas were obtained at baseline and yearly thereafter for those without AIN or with AIN-1, and every 6 months for individuals with AIN-2,3 [10]. Histological slides were reviewed by two pathologists and graded using the criteria used for CIN into normal, AIN-1, AIN-2 and AIN-3 [22].

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Real-time PCR assays for human papillomavirus type 16 E6 and E2 loads

Sample DNA from anal cells in PreservCyt was extracted with Master pure (Epicentre, Madison, Wisconsin, USA) [8]. β-Globin DNA was detected by amplification with PC04 and GH20 primers [8]. β-Globin-positive samples were amplified for HPV DNA with PGMY primers and genotyped with the reverse Line-blot detection system for 36 genital types, as previously described [8,10]. HPV-16-positive samples were screened for the presence of inhibitors by amplification of internal controls for β-globin and HPV-16 E6 in a Light Cycler PCR and detection system (Roche Molecular Systems, Laval, Quebec, Canada), as reported previously [19,23]. The presence of PCR inhibitors was suspected when 1000 copies of one or both internal controls generated a signal corresponding to less than 700 copies [19]. All samples tested were free of inhibitors. Two microliters of purified sample was tested in duplicate in each real-time PCR assay according to published protocols for quantification of HPV-16 E6, HPV-16 E2 DNA, and β-globin DNA to estimate the cell content of samples [19,23]. As HPV-16 integration often disrupts the HPV-16 E2 gene [24], the presence of integrated HPV-16 DNA was suspected for specimens with ratios of HPV-16 E6 and E2 (HPV-16 E6/E2) of 2 or more, as previously demonstrated for HPV-16 and HPV-33 [19,23,25,26]. Integrated viral loads were obtained by subtracting the number of HPV-16 E2 copies (episomal form) from the number of HPV-16 E6 copies (episomal and integrated forms).

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Detection of integrated papillomavirus sequences with PCR

Rather than restricting confirmation of integration to specimens with HPV-16 E6/E2 of 2.0 or more, the physical state of HPV-16 DNA for samples with an E6/E2 ratio of 1.5 or more was confirmed with detection of integrated papillomavirus sequences with PCR (DIPS-PCR), as described by Luft et al. [27]. Due to limitations in the amount of sample available, 0.6 μg of extracted DNA was digested with 10 units Sau3A I but not with Taq1. Briefly, digested DNA was ligated with the enzyme-specific adaptor AL1-AS1 overnight with T4 DNA ligase and then amplified with 0.2 μmol/l of each of the HPV-16 primers described by Luft et al. [27] and 16E2–3 [23]. The exponential PCR reaction was performed with each nested HPV-16 primers and adaptor-specific primer AP1 on products from the first PCR run [27]. To determine whether they contained both viral and cellular DNA, amplicons of unexpected size were sequenced using the fluorescent cycle-sequencing method (BigDye terminator ready reaction kit; Perkin-Elmer, Montreal, Canada) on an ABI PRISM 3100 (Applied Biosystems). Sequences were analyzed with the BLAST software to determine the presence of HPV-16 and human sequences ( A control reaction amplifying a chromosome 21 locus was also performed [27].

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Statistical analysis

Factors associated with AIN-2,3 or AIN progression were first assessed by univariate analysis with a Fisher's exact test for categorical variables and a Mann–Whitney test for continuous (CD4 cell counts, HIV RNA loads, HPV-16 viral loads, and age) and numerical (number of HPV types per sample) variables. The magnitude of the association between HPV-16 episomal and integrated loads and AIN was assessed by calculating odds ratios (ORs) and respective 95% confidence intervals (CIs) by unconditional multiple logistic regression while controlling for factors significant in univariate analysis. Correlations were measured with the Spearman rank correlation coefficient. Cox proportional hazard regression was used to assess the association between HPV-16 integration and time to progression from AIN-1 to AIN-2,3, controlling for potential confounders. Statistical analyses were performed with STATISTICA version 6 software (StatSoft, Tulsa, Oklahoma, USA). All statistical tests were two-sided and considered to be statistically significant at P less than 0.05.

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Study population at base line

The baseline characteristics of the 247 participants in the HIPVIRG cohort have been described recently [10]. The current analysis only included participants infected with HPV-16 at least once. The demographic and clinical characteristics of these 135 participants are provided in Table 1. Episomal and integrated HPV-16 loads measured on the first HPV-16-positive sample extended from 0.1 to 31 289.0 HPV-16 DNA copies per cell (median 22.3) and 0 to 31.7 HPV-16 DNA copies per cell (median 0), respectively. Episomal HPV-16 load correlated with CD4 cell counts (correlation coefficient R = −0.23, P = 0.007) and with HIV RNA load (R = 0.20, P = 0.02), but not with age (P = 0.90). There was no correlation between integrated HPV-16 load and CD4 cell counts or HIV RNA load (P ≥ 0.10).

Table 1

Table 1

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Risk factors for anal intraepithelial neoplasia 2 and 3

Considering the first HPV-16-positive visit with complete histological and virological results, 18.5, 37.8, and 42.9% of 135 men had no AIN, AIN-1, and AIN-2,3, respectively (Table 1). Logarithm-transformed episomal HPV-16 loads but not integrated HPV-16 loads were distributed normally. Compared with men without AIN, episomal HPV-16 loads were nearly two logs higher in anal samples from men with AIN-2,3 (P = 0.002) and one log higher in men with AIN-1 (P = 0.05) (Fig. 1a). The levels of episomal HPV-16 DNA in men without AIN overlapped substantially with those in men with AIN-1 or AIN-2,3. Of the 58 men with AIN-2,3 at the first HPV-16-positive visit, 24 (41.4%) had AIN-1 concurrently. The episomal HPV-16 load measured in the 24 men with AIN-2,3 and concurrent AIN-1 (median 25.1) was similar to that found in men with only AIN-2,3 (median 92.8, P = 0.10). Episomal HPV-16 loads were similar between men under a successful HAART for more than 6 months and those treated with another regimen (P = 0.61) or those failing HAART (P = 0.64).

Fig. 1

Fig. 1

In contrast, integrated HPV-16 loads were similar across all grades of anal lesions (Fig. 1b). Integrated HPV-16 defined as a HPV-16 E6/E2 of at least 2 was found on samples from three (12%, 95% CI 3.3–30.8) of 25 men without AIN, five (9.8%, 95% CI 3.8–21.4) of 51 with AIN-1, and two (3.5%, 95% CI 2.7–12.4) of 58 men with AIN-2,3. In univariate analysis, current smoking, a higher number of HPV types and a higher episomal HPV-16 but not integrated HPV-16 load increased the risk for AIN-2,3 (Table 2). These factors remained significant in multivariate analysis, with current smoking being the most significant factor (Table 2). In multivariate analysis, a higher number of HPV types and lower CD4 cell counts were significantly associated with AIN-1 (Table 3).

Table 2

Table 2

Table 3

Table 3

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Human papillomavirus type 16 integration and progression to anal intraepithelial neoplasia 2 and 3

We then investigated whether HPV-16 integration could identify men at higher risk of progression to higher grades of AIN over 3 years. Of the 62 HPV-positive men followed for at least three visits over 1 year or more, 16 men without AIN or with AIN-1 at accrual did not progress (designated as ‘nonprogressors’), including three AIN-1 that regressed, whereas 46 progressed (‘progressors’) from no AIN to AIN or from AIN-1 to AIN-2,3 (Table 1). Integrated HPV-16 forms were detected as frequently in progressors [eight (17.8%, 95% CI 9.0–31.6%) of 45] compared with nonprogressors [four (23.5%, 95% CI 9.1–47.8%) of 17, P = 0.72]. Episomal HPV-16 loads were higher in progressors (51.3 ± 17.8 HPV-16 copies per cell) compared with nonprogressors (12.0 ± 22.4 HPV-16 copies per cell), but the difference was not statistically significant (P = 0.12).

Of the 37 participants with AIN-1 at baseline, four (16.7%) of the 24 men who progressed to AIN-2,3 had at least one sample with integrated HPV-16 DNA compared to three (23.1%) of 13 men who did not progress (OR 0.7, 95% CI 0.2–3.8, P = 0.64). Taking into consideration the time to progression from AIN-1 to AIN-2,3 using proportional hazard regression, presence of integrated HPV-16 DNA, current smoking, and CD4 cell counts were not associated (P > 0.24 for each variable), but the number of HPV types (P = 0.045) was associated with progression to AIN-2,3. In depth evaluations of total viral loads of HPV-16 and other high-risk types and AIN progression overtime will be the subject of a future publication (manuscript in preparation).

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Integrated human papillomavirus type 16 forms in anal samples

Overall, 16 samples from 14 men had an HPV-16 E6/E2 ratio between 1.5 and 2.0 and 31 samples from 17 men had a ratio at least 2.0, suggesting HPV-16 integration. The latter 17 participants had a mean of 4.2 ± 0.5 (range 1–7) HPV-16-positive samples, of which a mean of 1.8 ± 0.4 (range 1–7) samples per participant generated an HPV-16 E6/E2 ratio of 2.0 or more. At the visit at which integration was first detected, four men did not have AIN, seven had AIN-1, and six had AIN-2,3. There was no pattern of HPV-16 integration during the course of HPV infection: integrated HPV-16 forms could be detected in the first HPV-16-positive sample only (n = 6), at the middle visit of many HPV-16-positive samples during follow-up (n = 6), at the last HPV-16-positive visit (n = 2), or in all samples (n = 3).

The correlates of having an HPV-16 E6/E2 ratio of 2.0 or more were then assessed. In univariate analysis, the number of HPV types in sample (OR = 0.8, 95% CI 0.6–0.9) and HPV-16 episomal load (OR = 0.5, 95% CI 0.3–0.7) reduced the risk of having a HPV-16 E6/E2 ratio of 2.0 or more, whereas HPV-16 persistence, AIN grade, age, current smoking, CD4 cell counts, and time on HAART were not significant (data not shown). In multivariate analysis, only episomal HPV-16 load remained statistically significant (OR = 0.5, 95% CI 0.3–0.8).

The presence of integration was further investigated with DIPS-PCR to study the presence of HPV-16–human DNA junctions. In the 31 samples from 17 men with HPV-16 E6/E2 ratios of at least 2.0, amplification could not be obtained with any primer pair for six samples, suggesting extensive viral deletions during integration, and amplification of viral and cellular junctions was obtained in 18 samples. As described by others, sequencing of amplicons of a size different than expected failed in six samples due to unknown reasons [28]. Finally, a small amount of DNA precluded the analysis by DIPS-PCR for one sample. Of the 16 samples obtained from 14 men with an E6/E2 ratio between 1.5 and 2.0, only one sample (A-7 from patient 019) generated a fragment of unexpected size that contained viral and human sequences.

Sites of integration in the human genome in these 19 samples are presented in Table 4. In the nine samples from three men in whom only integrated forms were detected by real-time PCR (+++ in the table), the presence of integrated forms was confirmed by DIPS-PCR. In three participants, HPV-16 integration occurred within cellular genes. In one sample, HPV-16 was integrated in the C1ORF24 gene on chromosome 1, a gene associated with carcinomas [29]. Three participants provided more than one sample containing integrated forms (three of five, three of three and six of seven visits): integration sites in the human genome varied from visit to visit for two participants, whereas the site of insertion in the genome could not be established with certainty for the third. Samples from the participant with invasive cancer did not contain enough DNA for DIPS-PCR.

Table 4

Table 4

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The findings of this study underscore the importance of episomal but not integrated HPV-16 loads to predict AIN-2,3, similarly to CIN-2,3 in HIV-seropositive women, as discussed below [23]. Previous work had demonstrated the association between total HPV-16 load and AIN-2,3 [6], but had not evaluated whether the quantity of episomal, integrated, or mixed forms increased with AIN grade. A greater quantity of episomal HPV-16 could result from greater viral replication and could expose epithelial cells to a greater quantity of HPV oncoproteins, increasing the risk for AIN-2,3. Current but not past smoking as well as a higher number of HPV types detected in a sample increased the risk of AIN-2,3 in our study. The association between smoking and AIN or anal cancer has been reported by others [30,31]. Counseling men to stop smoking could have an impact on the occurrence of AIN-2,3. Several studies have reported that a greater number of HPV types increased the likelihood of AIN-2,3 [3,6,8,13,30]. A higher number of HPV types may reflect a lower anti-HPV immunity, although there was no correlation between number of HPV types and CD4 cell counts. Multiple types could also act synergistically to induce transformation of epithelial cells or each type could carry its own risk for cancer development (additive risk).

Although the analytical sensitivity of real-time PCR assays for quantitation of HPV-16 E6 and E2 DNA utilized in this study is excellent [23], the clinical sensitivity and specificity of these assays to detect HPV integration have not been thoroughly assessed [19,25,32–34]. Reconstitution experiments demonstrated that HPV-16 integration is detected using a strict definition of integration when integrated HPV-16 forms are in 100-fold excess of episomal HPV-16 DNA [24]. Thus, integrated HPV-16 DNA may be undetected in the presence of abundant episomal HPV-16 DNA molecules [24]. Differentiation of transcriptionally active integrated forms could be a more sensitive method for detecting HPV-16 integration [35,36]. As preinvasive lesions often contain a mixture of episomal and integrated HPV-16 forms, E2 protein expressed from intact episomal HPV-16 DNA could repress integrated HPV-16 DNA, resulting in a false-negative transcription assay [37–39]. Theoretically, an HPV-16 E6/E2 ratio above 1.0 could suggest integration. However, previous works on HPV-6, HPV-16, and HPV-33 have demonstrated that HPV E6/E2 ratios below 2.0 can result from assay variability rather than true differences between E6 and E2 quantities [19,23,25,26]. In fact, only one sample with an HPV-16 E6/E2 below 2.0 but above 1.5 contained integrated forms. We may have also underestimated HPV-16 integration because of disruptions in genes other than E2 [24,35]. Nevertheless, HPV-16 E2 is the most frequently disrupted gene, with 78.4% of cancers harboring HPV-16 forms with rupture in the E2 hinge [24,40]. Moreover, disruption in other genes can result in the loss of E2 sequences as well if a segment of the HPV genome is deleted during integration.

HPV-16 integration often disrupts the E2 gene, resulting in unregulated expression of HPV oncogenes and activation of human telomerase [20]. This process causes deregulated cellular proliferation and increases genomic instability, both contributing to transform cells. Integrated HPV-16 loads and presence of integrated forms were similar across AIN grades in our study. The absence of difference across AIN grades may be related to small numbers of participants with integration detected. Nevertheless, our study suggests that integration occurs in normal mucosal cells and any grade of AIN. Integrated HPV-16 DNA has also been detected in women with CIN-1 and in those without CIN [19,24,41–44]. Results from various studies on the association between integration and CIN-2,3 in women have been inconsistent, but usually studies have reported that expression of transcripts from integrated forms but not integrated HPV-16 DNA was associated with CIN-2,3 [36,41,43–47]. With an optimized assay, integrated HPV-16 loads were similar between HIV-seropositive women without and with CIN-2,3 [23]. HPV-33 integration was also not associated with CIN-2,3 [25]. A recent study reported an association between HPV-16 integration and abnormal anal cytology, but did not use histopathology to grade lesions [48]. Criteria for defining HPV-16 integration were also not described in that study. Weak associations between integration and AIN-2,3 could have been missed in our study, considering that only 58 men had AIN-2,3.

One of the newly proposed models of HPV carcinogenesis suggests that HPV integration occurs initially at many sites in the human genome during persistent HPV infection, and that only some integrated HPV molecules will contribute to transformation eventually [49]. Transcriptional activity of integrated HPV-16 DNA can be suppressed by E2 proteins from episomal forms [20,37–39,50]. Considering that episomal loads increase with AIN grade, it is unlikely that AIN-2,3 results from uncontrolled expression of E6 and E7 from integrated forms. On the contrary, higher number of copies of episomal HPV-16 could result in increased expression of viral oncogenes. Moreover, integrated HPV-16 forms were inconsistently detected over time in our study, possibly because they are present at low levels or are a transient phenomenon. In vitro, high-level expression of HPV oncogenes from integrated HPV forms is preceded by the loss of episomal HPV [38,51]. If this is so, then integrated HPV may have a role in transformation after the loss of episomal HPV-16 that is the highest in AIN-2,3. Considering the important proportion of invasive anogenital cancer containing integrated HPV-16 forms, however, integration may be an important factor in progression from high-grade lesions to cancer [21,44]. Future studies should evaluate concurrently HPV-16 DNA integration and expression of HPV-16 E6 and E2 mRNA to determine in vivo the sequence of events and discriminate transcriptionally inactive from active integrated HPV-16 forms [20].

HPV-16 integration is believed to be favored by viral persistence, high viral loads, and genomic instability in transformed cells [20]. The only factor associated with HPV-16 integration in our study was a low episomal viral load, similarly to previous work on HPV-33 in CIN [25]. This may reflect the absence of replication of HPV-16 or occurrence of integration when HPV-16 replication was shut down by viral or host factors.

Despite the lack of sensitivity of real-time PCR assays to detect HPV-16 integration and the limited number of participants in the cohort with AIN-2,3, our study has several strengths. The presence of inhibitors of PCR was screened in our tests [19,23]. Moreover, the amounts of sample cellular DNA tested were below the levels that interfere with HPV-16 E6 quantitation [26]. The status of anal disease was well established by high-resolution anoscopy. The virology and pathology laboratories were blinded to the results of each other and to the anoscopy findings. Two pathologists examined independently anal biopsies to establish the presence and grade of AIN. The main limitation of our study was the relatively low number of cohort participants, although significant associations were still demonstrated between episomal HPV-16 loads and number of HPV types and AIN-2,3. Studies in larger cohorts could demonstrate a weaker effect of integrated HPV-16 loads on risk of progression.

In conclusion, our study confirms the association between HPV-16 viral load and AIN. This association is driven by episomal HPV-16 and not because of an increase in the quantity of integrated HPV-16 DNA as measured by real-time PCR. Integration of HPV-16 DNA can occur at all stages of AIN and even in the absence of AIN. HPV-16 integration seems to be a polyclonal and sometimes transient phenomenon initially before invasive cancer. Factors involved in the loss of episomal HPV-16 and clonal expansion of cells containing integrated HPV-16 forms still need to be resolved.

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This project was supported by a grant from the Canadian Institutes of Health Research (CIHR). We thank Roche Molecular Systems for reagents for PGMY Assays. We thank the Réseau FRSQ-SIDA Maladies Infectieuses for facilitating the recruitment of participants. The Canadian Cancer Society and National Cancer Institute of Canada support the HIPVIRG cohort. A CIHR Team grant supports the work of J.A. A.d.P. was supported by the Canadian HIV Trials Network postdoctoral fellowship. We would like to thank Jean-Marc Trépanier and Serge Coté for maintenance of database of the HIPVIRG study and sampling.

Members of the HIPVIRG Study group are (in alphabetical order): G. Allaire, J.G. Baril, M. Boissonnault, L. Charest, M.A. Charron, S. Coté, P. Coté, F. Coutlée, A. de Pokomandy, H. Dion, S. Dufresne, J. Falutz, C. Fortin, E. Franco, G. Ghattas, N. Gilmore, I. Gorska, R. Hadjeres, P. Junod, M. Klein, R. Lalonde, F. Laplante, R. Leblanc, D. Legault, B. Lessard, D. Longpré, J. McLeod, J.P. Maziade, D. Murphy, V.K. Nguyen, R. O'Brien, D. Phaneuf, D. Rouleau, J.P. Routy, J. Szabo, D. Tessier, R. Thomas, E. Toma, C. Tremblay, J.M. Trépanier, B. Trottier, C. Tsoukas, H. Turner, S. Vezina.

J.A. conducted the viral load and integration studies and interpretation of results. A.D.E.P. participated in the analysis and interpretation of results, D.R. was involved in study design and recruitment. G.G. performed high-resolution anoscopy and was involved in study design and interpretation of results. S.V. was involved in patient accrual and strategy for recruitment. P.C. was involved in patient accrual and study protocol. G.A. and R.H. reviewed the histology and interpreted the results. E.L.F. was involved in statistical analysis and grant support. F.C. is the principal investigator of the study having obtained funding and been involved at all levels of the study, high-resolution anoscopies, laboratory testing, interpretation, and writing of the manuscript. All authors have read the manuscript, made corrections, and accepted the final manuscript.

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anal intraepithelial neoplasia; HIV; human papillomavirus load; human papillomavirus natural history; human papillomavirus type 16; integration; MSM

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