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).
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 . 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.
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 . Previous work had demonstrated the association between total HPV-16 load and AIN-2,3 , 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 , 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 . Thus, integrated HPV-16 DNA may be undetected in the presence of abundant episomal HPV-16 DNA molecules . 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 . 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 . HPV-33 integration was also not associated with CIN-2,3 . A recent study reported an association between HPV-16 integration and abnormal anal cytology, but did not use histopathology to grade lesions . 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 . 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 .
HPV-16 integration is believed to be favored by viral persistence, high viral loads, and genomic instability in transformed cells . 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 . 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 . 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.
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.
1. Palefsky JM, Holly EA, Ralston ML, Jay N, Berry JM, Darragh TM. High incidence of anal high-grade squamous intra-epithelial lesions among HIV-positive and HIV-negative homosexual and bisexual men. AIDS 1998; 12:495–503.
2. Sobhani I, Walker F, Roudot-Thoraval F, Abramowitz L, Johanet H, Hénin D, et al
. Anal carcinoma: incidence and effect of cumulative infections. AIDS 2004; 18:1561–1569.
3. Palefsky JM, Holly EA, Efirdc JT, Da-Costa M, Jay N, Berry JM, et al
. Anal intraepithelial neoplasia in the highly active antiretroviral therapy era among HIV-positive men who have sex with men. AIDS 2005; 19:1407–1414.
4. Critchlow CW, Surawicz CM, Holmes KK, Kuypers J, Daling JR, Hawes SE, et al
. Prospective study of high grade anal squamous intraepithelial neoplasia in a cohort of homosexual men: influence of HIV infection, immunosuppression and human papillomavirus infection. AIDS 1995; 9:1255–1262.
5. Zbar AP, Fenger C, Efron J, Beer-Gabel M, Wexner SD. The pathology and molecular biology of anal intraepithelial neoplasia: comparisons with cervical and vulvar intraepithelial carcinoma. Int J Colorectal Dis 2002; 17:203–215.
6. Salit I, Tinmouth J, Chong S, Raboud J, Diong C, Su D. Screening for HIV-associated anal cancer: correlation of HPV genotypes, p16, and E6 transcripts with anal pathology. Cancer Epidemiol Biom Prev 2009; 18:1986–1992.
7. Palefsky JM, Holly EA, Ralston ML, Jay N. Prevalence and risk factors for human papillomavirus infection of the anal canal in human immunodeficiency virus (HIV)-positive and HIV-negative homosexual men. J Infect Dis 1998; 177:361–367.
8. Gohy L, Gorska I, de Pokomandy A, Rouleau D, Ghattas G, Allaire G, et al
. Genotyping of human papillomavirus in anal biopsies and anal swabs collected from HIV-seropositive men with anal dysplasia. J Acquir Immune Def Syndr 2008; 49:32–39.
9. Kiviat NB, Critchlow CW, Holmes KK, Kuypers J, Sayer J, Dunphy C, et al
. Association of anal dysplasia and human papillomavirus with immunosuppression and HIV infection among homosexual men. AIDS 1993; 7:43–49.
10. de Pokomandy A, Rouleau D, Ghattas G, Vezina S, Cote P, Macleod J, et al
. Prevalence, clearance and incidence of anal human papillomavirus infection in HIV-infected men: the HIPVIRG cohort study. J Infect Dis 2009; 199:965–973.
11. Rowhani Rahbar A, Hawes SE, Sow PS, Toure P, Feng Q, Dem A, et al
. The impact of HIV status and type on the clearance of human papillomavirus infection among Senegalese women. J Infect Dis 2007; 196:887–894.
12. Palefsky JM, Holly EA, Gonzales J, Lamborn K, Hollander H. Natural history of anal cytologic abnormalities and papillomavirus infection among homosexual men with group IV HIV disease. J Acquir Immune Def Syndr 1992; 5:1258–1265.
13. Palefsky JM, Holly EA, Hogeboom CJ, Ralston ML, Da Costa MM, Botts R, et al
. Virologic, immunologic, and clinical parameters in the incidence and progression of anal squamous intraepithelial lesions in HIV-positive and HIV-negative homosexual men. J Acquir Immune Defic Syndr 1998; 17:314–319.
14. Wilkin TJ, Palmer S, Brudney KF, Chiasson MA, Wright TC. Anal intraepithelial neoplasia in heterosexual and homosexual HIV-positive men with access to antiretroviral therapy. J Infect Dis 2004; 190:1685–1691.
15. Da Costa MM, Hogeboom CJ, Holly EA, Palefsky JM. Increased risk of high-grade anal neoplasia associated with a human papillomavirus type 16 E6 sequence variant. J Infect Dis 2002; 185:1229–1237.
16. Snijders PJ, Steenbergen RDM, Heideman DAM, Meijer CJLM. HPV-mediated cervical carcinogenesis: current concepts and clinical implications. J Pathol 2006; 208:152–164.
17. Varnai AD, Bollmann M, Griefingholt H, Speich N, Schmitt C, Bollmann R, Decker D. HPV in anal squamous cell carcinoma and anal intraepithelial neoplasia (AIN). Impact of HPV analysis of anal lesions on diagnosis and prognosis. Int J Colorectal Dis 2006; 21:135–142.
18. Fontaine J, Hankins C, Money D, Rachlis A, Pourreaux K, Ferenczy A, Coutlee F. Human papillomavirus type 16 (HPV-16) viral load and persistence of HPV-16 infection in women infected or at risk for HIV. J Clin Virol 2008; 43:307–312.
19. Fontaine J, Hankins C, Mayrand MH, Lefevre J, Money D, Gagnon S, et al
. High levels of episomal and integrated HPV-16 DNA are associated with high-grade cervical lesions in women at risk or infected with HIV. AIDS 2005; 19:785–794.
20. Pett M, Coleman N. Integration of high-risk human papillomavirus: a key event in cervical carcinogenesis? J Pathol 2007; 212:356–367.
21. Hopman AHN, Smedts F, Dignef W, Ummulen M, Sonke G, Mravunac M, et al
. Transition to high-grade cervical intraepithelial neoiplasia to micro-invasive carcinoma is characterized by integration of HPV16/18 and numerical chromosome abnormalities. J Pathol 2004; 202:23–33.
22. Lytwyn A, Salit IE, Raboud J, Chapman W, Darragh T, Winkler B, et al
. Interobserver agreement in the interpretation of anal intraepithelial neoplasia. Cancer 2005; 103:1447–1456.
23. Azizi N, Brazete J, Hankins C, Money D, Fontaine J, Koushik A, et al
. Influence of HPV-16 E2 polymorphism on quantitation of HPV-16 episomal and integrated DNA in cervicovaginal lavages from women with cervical intraepithelial neoplasia. J Gen Virol 2008; 89:1716–1728.
24. Arias-Pulido H, Peyton CL, Joste NE, Vargas H, Wheeler CM. Human papillomavirus type 16 integration in cervical carcinoma in situ and in invasive cervical cancer. J Clin Microbiol 2006; 44:1755–1762.
25. Khouadri S, Villa LL, Gagnon S, Koushik A, Richardson H, Ferenczy A, et al
. Viral load of episomal and integrated forms of Human papillomavirus type 33 in high-grade squamous intraepithelial lesions of the uterine cervix. Int J Cancer 2007; 121:2674–2681.
26. Ruutu MP, Kulmala SM, Peitsaro P, Syrjanen SM. The performance of the HPV16 real-time PCR integration assay. Clin Cancer Res 2008; 41:423–428.
27. Luft F, Klaes R, Nees M, Durst M, Heilmann V, Melsheimer P, von-Knebel-Doeberitz M. Detection of integrated papillomavirus sequences by ligation-mediated PCR (DIPS-PCR) and molecular characterization in cervical cancer cells. Int J Cancer 2001; 92:9–17.
28. Matovina M, Sabol I, Grubisic G, Gasperov NM, Grce M. Identification of human papillomavirus type 16 integration sites in high-grade precancerous cervical lesions. Gynecol Oncol 2009; 113:120–127.
29. Maciel RM, Kimura ET, Cerutti JM. Pathogenesis of differentiated thyroid cancer (papillary and follicular). Arq Bra Endocrinol Metabol 2005; 49:691–700.
30. Palefsky JM, Holly EA, Ralston ML, Arthur SP, Hogeboom CJ, Darragh TM. Anal cytological abnormalities and anal HPV infection in men with Centers for Disease Control group IV HIV disease. Genitourin Med 1997; 73:174–180.
31. Daling JR, Madeleine MM, Johnson LG, Schwartz SM, Shera KA, Wurscher MA, et al
. Human papillomavirus, smoking, and sexual practices in the etiology of anal cancer. Cancer 2004; 101:270–280.
32. De Marco L, Gillio-Tos A, Bonello L, Ghisetti V, Ronco G, Merletti F. Detection of human papillomavirus type 16 integration in preneoplastic cervical lesions and confirmation by DIPS-PCR and sequencing. J Clin Virol 2007; 38:7–13.
33. Fujii T, Masumoto N, Saito M, Hirao N, Niimi S, Mukai M, et al
. Comparison between in situ hybridization and real-time PCR technique as a means of detecting the integrated form of human papillomavirus 16 in cervical neoplasia. Diagn Mol Pathol 2005; 14:103–108.
34. Nagao S, Yoshinouchi M, Miyagi Y, Hongo A, Kodama J, Itoh S, Kudo T. Rapid and sensitive detection of physical status of human papillomavirus type 16 DNA by quantitative real-time PCR. J Clin Microbiol 2002; 40:863–867.
35. Ziegert C, Wentzensen N, Vinokurova S, Kisseljov F, Einenkel J, Hoeckel M, et al
. A comprehensive analysis of HPV integration loci in anogenital lesions combining transcript and genome-based amplification techniques. Oncogene 2003; 22:3977–3984.
36. Klaes R, Woerner SM, Ridder R, Wentzensen N, Duerst M, Schneider A, et al
. Detection of high-risk cervical intraepithelial neoplasia and cervical cancer by amplification of transcripts derived from integrated papillomavirus oncogenes. Cancer Res 1999; 59:6132–6136.
37. Herdman MT, Pett MR, Roberts I, Alazawi WO, Teschendorff AE, Zhang XY, et al
. Interferon-beta treatment of cervical keratinocytes naturally infected with human papillomavirus 16 episomes promotes rapid reduction in episome numbers and emergence of latent integrants. Carcinogen 2006; 27:2341–2353.
38. Pett MR, Herdman MT, Palmer RD, Yeo GS, Shivji MK, Stanley MA, Coleman N. Selection of cervical keratinocytes containing integrated HPV16 associates with episome loss and an endogenous antiviral response. Proc Natl Acad Sci U S A 2006; 103:3822–3827.
39. Hafner N, Driesch C, Gajda M, Jansen L, Kirchmayr R, Runnebaum IB, Durst M. Integration of the HPV16 genome does not invariably result in high levels of viral oncogene transcripts. Oncogene 2008; 27:1610–1617.
40. Kalantari M, Karlsen F, Kristensen G, Holm R, Hagmar B, Johansson B. Disruption of the E1 and E2 reading frames of HPV 16 in cervical carcinoma is associated with poor prognosis. Int J Gynecol Pathol 1998; 17:146–153.
41. Huang LW, Chao SL, Lee BH. Integration of human papillomavirus type-16 and type-18 is a very early event in cervical carcinogenesis. J Clin Pathol 2008; 61:627–631.
42. Peitsaro P, Johansson B, Syrjanen S. Integrated human papillomavirus type 16 is frequently found in cervical cancer precursors as demonstrated by a novel quantitative real-time PCR technique. J Clin Microbiol 2002; 40:886–891.
43. Cheung JL, Lo KW, Cheung TH, Tang JW, Chan PK. Viral load, E2 gene disruption status, and lineage of human papillomavirus type 16 infection in cervical neoplasia. J Infect Dis 2006; 194:1706–1712.
44. Guo M, Sneige N, Silva EG, Jan YJ, Cogdell DE, Lin E, et al
. Distribution and viral load of eight oncogenic types of human papillomavirus (HPV) and HPV 16 integration status in cervical intraepithelial neoplasia and carcinoma. Mod Pathol 2007; 20:256–266.
45. Hudelist G, Manavi M, Pischinger KI, Watkins-Riedel T, Singer CF, Kubista E, Czerwenka KF. Physical state and expression of HPV DNA in benign and dysplastic cervical tissue: different levels of viral integration are correlated with lesion grade. Gynecol Oncol 2004; 92:873–880.
46. Kulmala S-MA, Syrjanen SM, Gyllensten UB, Shabalova IP, Petrovichev N, Tosi PT, et al
. Early integration of high copy HPV-16 detectable in women with normal and low grade cervical cytology and histology. J Clin Pathol 2007; 59:513–517.
47. Briolat J, Dalstein V, Saunier M, Joseph K, Caudroy S, Pretet JL, et al
. HPV prevalence, viral load and physical state of HPV-16 in cervical smears of patients with different grades of CIN. Int J Cancer 2007; 121:2198–2204.
48. Canadas MP, Darwich L, Sirera G, Bofill M, Pinol M, Garcia Cuyas F, et al
. Human papillomavirus 16 integration and risk factors associated in anal samples of HIV-1 infected men. Sex Transm Dis 2010; 37:311–315.
49. Yu T, Ferber MJ, Cheung TH, Chung TK, Wong YF, Smith DI. The role of viral integration in the development of cervical cancer. Cancer Genet Cytogenet 2005; 158:27–34.
50. Bechtold V, Beard P, Raj K. Human papillomavirus type 16 E2 protein has no effect on transcription from episomal viral DNA. J Virol 2003; 77:2021–2028.
51. Peitsaro P, Hietanen S, Johansson B, Lakkala T, Syrjanen S. Single copy heterozygote integration of HPV 33 in chromosomal band 5p14 is found in an epithelial cell clone with selective growth advantage. Carcinogenesis 2002; 23:1057–1064.