Numerous studies have indicated an increased risk of invasive anal cancer in HIV-infected patients in the precombination antiretroviral therapy (cART) era [1–4] and that the risk of anal cancer remained increased despite the use of cART [5–10]. The incidence of invasive anal cancer was reported to be up to hundred-fold higher in HIV-infected men who have sex with men (MSM) as compared with the general population . Anal cancer shares several biological characteristics with cervical cancer, and particularly the association with human papillomavirus (HPV) infection and the existence of squamous intra-epithelial lesions (SIL) that are precursor lesions of invasive cancer.
The prevalence and incidence of HPV-related anogenital SIL in HIV-infected MSM have been extensively studied in the pre-cART era and shown to be higher in HIV-positive than in HIV-negative MSM [12–17]. The natural history of anal HPV infection and SIL in the cART era is, however, not fully understood. Antiretroviral treatment is associated with suppression of HIV replication and immune restoration that has resulted in a dramatic decrease in HIV-related mortality and morbidity [18–21]. If cART, despite an increase in CD4 T cells and immune restoration to several opportunistic pathogens [22–24] did not result in enhanced control of HPV, one could expect a progression of HPV disease and an increased incidence of anogenital cancer. At the present time, there are only few available data on the impact of cART-mediated immune restoration on the prevalence and incidence of anal HPV infection and anal SIL in HIV-infected individuals.
Studies, which evaluated the association between exposure to cART and anal HPV infection or anal SIL have shown conflicting results [25–29] probably explained by differences in study designs and lack of accurate assessment of correlations between HPV-related anal disease and immunologic and virologic responses under cART. The aim of this prospective study was to assess the impact of cART-associated immune restoration on anal SIL in a cohort of HIV-infected MSM starting a first-line cART regimen.
Between May 2006 and October 2007, 94 HIV-1 seropositive MSM naive of cART were included in a longitudinal cohort study before starting a first-line regimen of cART.
HIV-1-infected individuals were eligible for the study if they had acquired HIV through homo/bisexual contact, were over 18 years of age, were naive of cART and were planned to start a first line of cART. The patients were recruited in seven hospitals in Paris, France. The study was approved by the Ethics Review Board of Hôpital Pitié-Salpetrière, Paris. Patients provided signed written consent before inclusion in the study. All men were interviewed using a standardized comprehensive self-administered questionnaire that included questions on age, tobacco use, medical history, history of sexually transmitted infection (STI), age at first intercourse, total number of sexual partners, and total number of receptive and insertive anal intercourse. Samples were collected within 3 months before starting the first line of cART and at month 12 and month 24 of cART.
Cytologic and histologic analyses
An anal examination was conducted that included insertion of a Dacron swab (Bio Mérieux, Marcy l’étoile, France) used for anal cytology and virological analyses. The swab was immediately agitated vigorously and rinsed in a methanol-based fixative vial (PreservCyt; Hologic Corporation, Boxborough, Massachusetts, USA). Each vial was used for ThinPrep cytology (Hologic Corporation). Slides were prepared from the vial using the ThinPrep 2000 processor (Hologic Corporation). After collection of cytologic sample, patients underwent conventional anoscopic examination or high-resolution anoscopy  depending on the recruiting site. During high-resolution anoscopy, 3% acetic acid solution and Lugol solution were successively applied on the anal mucosa to better visualize the lesions. In case of visible lesion under anoscopy examination, a biopsy was performed. Biopsy specimens were fixed in Bouin fixative for routine histopathologic examination. Anal cytology and histology were evaluated independently of each other without knowledge of clinical status or HPV results. Anal cytology was classified as normal, atypical squamous cell of undetermined significance (ASC-US), atypical squamous cell – cannot exclude high-grade SIL (ASC-H), low-grade SIL (LSIL), or high-grade SIL (HSIL) using the 2001 Bethesda system criteria for evaluation of cervical cytology . One pathologist examined all biopsy specimens and classified them. If both cytologic and histologic results were available for analysis, the more severe result was considered.
Detection of anal human papillomavirus DNA
Virological samples were immediately stored in a transport medium vial (Copan Italia, Brescia, Italy). Anal HPV DNA was detected by real time PCR and Linear Array HPV genotyping test (Roche Diagnostics) performed in a blinded fashion. Samples exhibiting the HPV band were genotyped by the Roche linear array assay, which distinguishes 27 HPV genotypes (oncogene and nononcogene) commonly found in the female genital tract.
CD4 cell count and plasma HIV RNA
The CD4 cell counts and plasma HIV-RNA levels (detection limit of 50 copies/ml), closest to the period of the anal examination and within 2 months, were collected.
Data were analyzed using the SAS 9.1 software (SAS Institute, Inc, Cary, North Carolina, USA). Variables were summarized using the SAS procedures FREQ for categorical variables and the MEANS for continuous variables. Association between regression or progression of lesions at week 24 and either the baseline CD4 cell count or CD4 change between baseline and week 24, or viral load below 50 copies at week 12 or 24 was assessed using nonparametric Mann–Whitney tests or exact chi-square test. All hypothesis tests were two-sided and were tested at the α = 0.05 level of significance.
Ninety-four HIV-infected patients were recruited in the study. Seventy-six patients had results of cytology available for at least two visits (68 at baseline and month 12 and 69 at baseline and month 24), including 54 individuals for which results were available at all three visits. The characteristics of the study population are presented in Table 1. Only 5% of the patients had presented a prior AIDS-defining event and the median CD4 cell count was 301 cells/μl [interquartile range (IQR): 242–339]. Fifty-three percent of the patients started cART with a protease inhibitor-containing regimen.
A significant increase in CD4 cell count was observed from 301 cells/μl at baseline to 545 cells/μl at month 24; 93% of the patients exhibited plasma HIV viral load below 50 copies/ml at month 24.
Anal squamous intraepithelial lesions
At baseline, an abnormal result was observed in 45 of 76 patients (59%) with prevalent LSIL in 27 patients (36%) and HSIL in seven patients (9%). Abnormal cytology was found in 40 of 68 patients assessed at month 12 (59%) with LSIL in 23 patients (34%) and HSIL in 10 patients (15%). Abnormal result was observed in 36 of 69 patients assessed at month 24 (52%) with LSIL in 23 patients (33%) and HSIL in six patients (9%). At baseline, the prevalence of any lesion was 63% in patients with HPV-16 infection (n = 38) and 53% in those with other oncogene HPV genotypes (n = 32) (P = 0.469). Prevalence of HSIL was 18% in patients with HPV-16 infection versus 0% in patients with other oncogene HPV genotypes (P = 0.013).
The anal disease status at month 12 and month 24 according to baseline diagnosis is presented in Fig. 1. Among patients with no lesion at baseline, 10 patients (35.7%) exhibited a SIL at month 12 (LSIL in seven patients and HSIL in three patients). Six patients with no lesion at baseline exhibited SIL at month 24 (LSIL in five patients and HSIL in one patient). Regression of anal lesions was observed, without specific therapeutic intervention, in all five patients with HSIL at baseline (LSIL in four patients and normal in one patient at month 12; ASC-H in two patients, LSIL in two patients and normal in one patient at month 24).
Among the patients exhibiting LSIL at baseline, HSIL was found in only two patients at month 12 and no progression to HSIL was observed at month 24. The persistence of LSIL was observed in 15 patients (65%), whereas a regression to normal was observed in 22% of the patients.
Regression of the severity of the lesions or occurrence of a new lesion between baseline and 24 months of cART was neither significantly associated with baseline CD4 cell counts, change in CD4 cell counts at months 12 and 24, nor with exhibiting a viral load below 50 copies/ml at those time-points.
Overall, regression of the severity of lesions was observed at month 24 in 18 of 41 patients with a lesion at baseline (44%), whereas lesion occurrence was observed in 10 of 27 patients without lesion at baseline (37%).
To our knowledge, the present prospective cohort study is the first to enroll HIV-infected MSM when starting a first-line cART regimen in order to assess the impact of immune restoration on anal HPV infection and anal SIL. We found a high prevalence (59%) of anal SIL at baseline in cART-naive HIV-infected MSM, which was maintained up to month 24 of effective cART (59% at month 12 and 52% at month 24). The rate of acquisition and disappearance of anal lesions were both high (37 and 44%, respectively). As no patient underwent specific treatment for anal SIL during the follow-up, these results reflected the natural history of anal SIL in HIV-infected patients receiving cART.
When considering the subgroup of patients exhibiting anal HPV-16 infection at baseline, prevalence and incidence of HSIL were higher as compared with the group of patients exhibiting other high-risk HPV anal infection. These data are in accordance with previous studies indicating that HPV-16 is highly prevalent in MSM with anal lesions and often associated with HSIL [29,32]. The high prevalence of anal SIL at baseline in cART naive HIV-infected MSM found here, independently of the presence or absence of HPV-16, was similar to that previously reported in ART-naive patients . The longitudinal design of our study allowed us to show that this high prevalence of anal SIL was maintained up to 2 years of effective cART. We previously reported on a high prevalence of anal SIL in a cross-sectional study of 45 HIV-infected MSM receiving cART for a median of 32 months . Palefsky et al. also assessed anal HPV infection and anal SIL 6 months prior to and 6 months after the initiation of cART in a cohort of 98 HIV-infected MSM. Among the patients exhibiting ASCUS or LSIL prior to cART, 18% progressed and 21% regressed under cART. In more recent studies, the same group reported on a high prevalence of anal SIL in a cohort of 357 cART-treated MSM .
Only a marginally significant lower risk of anal intraepithelial neoplasia (AIN)-2,3 in patients receiving cART for more than 4 years was recently reported in a cohort of 247 HIV-infected MSM receiving or initiating cART followed up for 3 years . This is in accordance with a recent report indicating that the rates of anal cancer for HIV-infected individuals increased after the early ART era and then stabilized in more recent years 
Taken together, the present data emphasized that cART exhibited limited or no effect on the course of anal SIL, findings in agreement with the increase in the incidence of anal invasive cancer despite cART [5–10]. The need for systematic screening for anal lesions in HIV-infected patients is currently underestimated in clinical practice. Further studies are needed to provide a practical screening tool for anal HPV disease and to demonstrate the cost effectiveness of a systematic screening of HIV-infected MSM.
VALPARAISO study group: Hôpital Européen Georges Pompidou, Paris (L.W., C.P., D. Tisne-Dessus); Hôpital Saint Antoine, Paris (P-M.G., B.L., J-F.C.); Hôpital Pitié Salpétrière, Paris (C. Katlama, R.T., A. Simon, B. Mory); Hôpital Henri Mondor, Creteil (Y. Levy); Hôpital Necker, Paris (J-P Viard); Hôpital Bichat, Paris (P. Yeni, R. Landman, L Abramowitz); Hôpital Lariboisière, Paris, France (A Rami).
VALPARAISO Study Scientific committee: C.P., D.C., A.S.M., E.T., E.L., B.C-P., C. Rouzioux, J-P.V., S.T., M. Leruez, L.W..
The patients enrolled in the VALPARAISO Study.
C.P., A.S.M., L.W. and D.C. were involved in the design of the study. C.P., L.W., R.T. and P.M.G. recruited the patients and collected the samples. B.C.P. was responsible for the cytological and histologic analyses. A.S.M. coordinated the virologic studies. E.L. performed statistical analyses. S.T. was involved in the monitoring of the study. D.C. coordinated the methodology and supervised the statistical analyses. C.P., L.W. and D.C. wrote the article, receiving contributions from, B.C.P., E.L. and A.S.M.
This study was funded by Association pour la Recherche en Immunopathologie (ARI), Paris and Sidaction-Ensemble contre le Sida.
Conflicts of interest
There are no conflicts of interest.
1. Melbye M, Cote TR, Kessler L, Gail M, Biggar RJ. High incidence of anal cancer among AIDS patients. The AIDS/Cancer Working Group
2. Melbye M, Rabkin C, Frisch M, Biggar RJ. Changing patterns of anal cancer incidence in the United States, 1940–1989
. Am J Epidemiol
3. Goedert JJ. The epidemiology of acquired immunodeficiency syndrome malignancies
. Semin Oncol
4. Frisch M, Biggar RJ, Engels EA, Goedert JJ. Association of cancer with AIDS-related immunosuppression in adults
5. Piketty C, Selinger-Leneman H, Grabar S, Duvivier C, Bonmarchand M, Abramowitz L, et al. Marked increase in the incidence of invasive anal cancer among HIV-infected patients despite treatment with combination antiretroviral therapy
6. Patel P, Hanson DL, Sullivan PS, Novak RM, Moorman AC, Tong TC, et al. Incidence of types of cancer among HIV-infected persons compared with the general population in the United States, 1992-2003
. Ann Intern Med
7. Hessol NA, Pipkin S, Schwarcz S, Cress RD, Bacchetti P, Scheer S. The impact of highly active antiretroviral therapy on non-AIDS-Defining cancers among adults with AIDS
. Am J Epidemiol
8. Engels EA, Pfeiffer RM, Goedert JJ, Virgo P, McNeel TS, Scoppa SM, Biggar RJ. Trends in cancer risk among people with AIDS in the United States 1980–2002
9. Diamond C, Taylor TH, Aboumrad T, Bringman D, Anton-Culver H. Increased incidence of squamous cell anal cancer among men with AIDS in the era of highly active antiretroviral therapy
. Sex Transm Dis
10. Clifford GM, Polesel J, Rickenbach M, Dal Maso L, Keiser O, Kofler A, et al. Cancer risk in the Swiss HIV Cohort Study: associations with immunodeficiency, smoking, and highly active antiretroviral therapy
. J Natl Cancer Inst
11. Bower M, Powles T, Newsom-Davis T, Thirlwell C, Stebbing J, Mandalia S, et al. HIV-associated anal cancer: has highly active antiretroviral therapy reduced the incidence or improved the outcome?
. J Acquir Immune Defic Syndr
12. Critchlow CW, Holmes KK, Wood R, Krueger L, Dunphy C, Vernon DA, et al. Association of human immunodeficiency virus and anal human papillomavirus infection among homosexual men
. Arch Intern Med
13. 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
14. Critchlow CW, Hawes SE, Kuypers JM, Goldbaum GM, Holmes KK, Surawicz CM, Kiviat NB. Effect of HIV infection on the natural history of anal human papillomavirus infection
15. Palefsky JM. Human papillomavirus infection and anogenital neoplasia in human immunodeficiency virus-positive men and women
. J Natl Cancer Inst Monogr
16. 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
17. Kiviat NB, Critchlow CW, Hawes SE, Kuypers J, Surawicz C, Goldbaum G, et al. Determinants of human immunodeficiency virus DNA and RNA shedding in the anal-rectal canal of homosexual men
. J Infect Dis
18. Lee LM, Karon JM, Selik R, Neal JJ, Fleming PL. Survival after AIDS diagnosis in adolescents and adults during the treatment era, United States, 1984–1997
19. Lewden C, Raffi F, Chene G, Sobel A, Leport C. Mortality in a cohort of HIV-infected adults started on a protease inhibitor-containing therapy: standardization to the general population
. J Acquir Immune Defic Syndr
20. Mocroft A, Ledergerber B, Katlama C, Kirk O, Reiss P, d’Arminio Monforte A, et al. Decline in the AIDS and death rates in the EuroSIDA study: an observational study
21. Murphy EL, Collier AC, Kalish LA, Assmann SF, Para MF, Flanigan TP, et al. Highly active antiretroviral therapy decreases mortality and morbidity in patients with advanced HIV disease
. Ann Intern Med
22. Autran B, Carcelain G, Li TS, Blanc C, Mathez D, Tubiana R, et al. Positive effects of combined antiretroviral therapy on CD4+ T cell homeostasis and function in advanced HIV disease
23. Havlir DV, Schrier RD, Torriani FJ, Chervenak K, Hwang JY, Boom WH. Effect of potent antiretroviral therapy on immune responses to Mycobacterium avium in human immunodeficiency virus-infected subjects
. J Infect Dis
24. Komanduri KV, Viswanathan MN, Wieder ED, Schmidt DK, Bredt BM, Jacobson MA, McCune JM. Restoration of cytomegalovirus-specific CD4+ T-lymphocyte responses after ganciclovir and highly active antiretroviral therapy in individuals infected with HIV-1
. Nat Med
25. Palefsky JM, Holly EA, Efirdc JT, Da Costa M, Jay N, Berry JM, Darragh TM. Anal intraepithelial neoplasia in the highly active antiretroviral therapy era among HIV-positive men who have sex with men
26. Palefsky JM, Holly EA, Ralston ML, Da Costa M, Bonner H, Jay N, et al. Effect of highly active antiretroviral therapy on the natural history of anal squamous intraepithelial lesions and anal human papillomavirus infection
. J Acquir Immune Defic Syndr
27. Piketty C, Darragh TM, Heard I, Da Costa M, Bruneval P, Kazatchkine MD, Palefsky JM. High prevalence of anal squamous intraepithelial lesions in HIV-positive men despite use of highly active antiretroviral therapy
. Sex Transm Dis
28. 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
29. de Pokomandy A, Rouleau D, Ghattas G, Trottier H, Vezina S, Cote P, et al. HAART and progression to high-grade anal intraepithelial neoplasia in men who have sex with men and are infected with HIV
. Clin Infect Dis
30. Jay N, Berry JM, Hogeboom CJ, Holly EA, Darragh TM, Palefsky JM. Colposcopic appearance of anal squamous intraepithelial lesions
. Dis Colon Rectum
31. Solomon D, Davey D, Kurman R, Moriarty A, O’Connor D, Prey M, et al. The 2001 Bethesda System: terminology for reporting results of cervical cytology
32. Poizot-Martin I, Henry M, Benhaim S, Obry-Roguet V, Figarella D, Tamalet C. High level of HPV 16 and 18 DNA load in anal swabs from male and female HIV-1 infected patients
. J Clin Virol
33. Silverberg MJ, Lau B, Justice AC, Engels E, Gill MJ, Goedert JJ, et al. Risk of Anal Cancer in HIV-Infected and HIV-Uninfected Individuals in North America
. Clin Infect Dis