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High Prevalence of Anal Squamous Intraepithelial Lesions in HIV-Positive Men Despite the Use of Highly Active Antiretroviral Therapy

Piketty, Christophe MD, PhD*; Darragh, Teresa M. MD†; Heard, Isabelle MD*; Da Costa, Maria MSc‡; Bruneval, Patrick MD§; Kazatchkine, Michel D. MD, PhD*; Palefsky, Joel M. MD‡

Sexually Transmitted Diseases: February 2004 - Volume 31 - Issue 2 - pp 96-99
Article

Background: The impact of highly active antiretroviral therapy (HAART) on the natural history of HPV infection and anal squamous intraepithelial lesions (SIL) in HIV-infected men who have sex with men (MSM) is poorly documented.

Goal: The goal of this study was to evaluate the prevalence of anal HPV infection and SIL inpatients under HAART.

Study Design: Forty-five HIV-infected protease inhibitor-experienced MSM were enrolled in a cross-sectional study. Each patient provided anal samples for anal cytology, histology, and human papillomavirus (HPV) DNA testing.

Results: The patients had previously received HAART for a median of 32 months. Anal cytology was abnormal in 32 of 45 (71%) patients, including high-grade SIL in 10 patients (22%), low-grade SIL in 19 patients (42%), and atypical squamous cells of undetermined significance in 3 patients (7%). HPV DNA was detected 36/45 men (80%). The prevalence of anal SIL and HPV infection were similar in patients exhibiting a significant increase in CD4+ cell count after HAART initiation compared with those who did not.

Conclusion: Our results demonstrate a high prevalence of anal SIL, including high-grade SIL, and anal HPV infection in HIV-infected MSM despite immune restoration under HAART.

ANAL CANCER IS ASSOCIATED with human papillomavirus (HPV) infection. 1,2 The prevalence and incidence of anal HPV infection and anal squamous intraepithelial lesions (ASIL) in HIV-positive men who have sex with men (MSM) have been studied in the pre-HAART era and both are more common in HIV-positive MSM than in HIV-negative MSM. 3–5 The prevalence and incidence of anal HPV infection and ASIL in HIV-positive MSM have also been found to increase with decreased CD4+ cell count. 3,5 Recent studies estimated the incidence of anal cancer to be 2-fold higher in HIV-infected MSM compared with HIV-negative MSM, 6 and the relative risk of developing anal cancer among HIV-positive MSM was shown to be 37-fold higher than in the general population. 7 To date, the impact of highly active antiretroviral therapy (HAART) on the natural history of HPV infection and ASIL is poorly documented. We report on the high prevalence of HPV infection and ASIL in a cohort of HIV-infected MSM receiving HAART despite a significant increase in CD4+ cell count since HAART initiation.

A study of 45 HIV-infected men who have sex with men found a high prevalence of anal squamous intraepithelial lesions, including high-grade SIL, and anal HPV infection despite immune restoration under highly active antiretroviral therapy.

*Department of Immunology, INSERM U 430 and Université Pierre et Marie Curie, Hôpital Européen Georges Pompidou, Paris, France; the Departments of Pathology and Medicine, University of California, San Francisco, California; and the §Department of Pathology, Hôpital Européen Georges Pompidou, Paris, France

The authors thank Drs. Laurence Weiss, Gustavo Gonzalez-Canali, Dominique Batisse, Marina Karmochkine, Martin Buisson, and Didier Jayle for their help in enrolling patients in the study, and Helena Bonner and Daniel Felmlee for their technical assistance.

This work was supported by SIDACTION and Cytyc Corporation, France, and in part funds provided by the Division of Research Resources 5 mol/l01-RR-00079, U.S. Public Health Service.

Correspondence: Christophe Piketty, MD, PhD, Hôpital Européen Georges Pompidou, 20 rue Leblanc 75015 Paris, France. E-mail: Christophe.piketty@egp.ap-hop-paris.fr

Received for publication June 12, 2003,

revised September 23, 2003, and accepted September 29, 2003.

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Methods

Patients

Between June 1999 and October 2000, 120 HIV-seropositive MSM attending the outpatient clinic of Hôpital Européen Georges Pompidou, Paris, were enrolled in a cross-sectional study of anal HPV infection and ASIL in HIV-seropositive men. 8 Among this cohort, all the HIV-infected MSM receiving an antiretroviral regimen, including a protease inhibitor, for at least 6 months were analyzed in a substudy to analyze patients with significant increase in CD4 cell count under HAART as previously described. 9 The protocol and written informed consent documents were approved by the Ethics Review Board of Hôpital Pitié-Salpetrière, Paris. Patients provided signed written consent before inclusion in the study.

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Cytologic and Histologic Analyses

A thorough anal examination was conducted that included insertion of 2 Dacron swabs for anal cytology and HPV testing. The swabs were immediately rinsed in a vial of PreservCyt fixative fluid (Cytyc Corp., Boxborough, MA). Each vial was used for HPV testing and ThinPrep cytology. An aliquot was taken from the vial for HPV testing; slides were then prepared from the vial using the ThinPrep 2000 processor (Cytyc Corp.). In case of abnormal cytology, consenting subjects underwent anoscopic examination and biopsy. Biopsy specimens were fixed in 10% formalin for routine histopathologic examination. Anal cytology and anal histology were evaluated independently of each other. Anal cytology was classified as normal, atypical squamous cell of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LSIL), or high-grade squamous intraepithelial lesion (HSIL) using the Bethesda system criteria for evaluation of cervical cytology.

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Detection of Anal Human Papillomavirus DNA

Polymerase chain reaction (PCR) for anal HPV DNA detection was performed. HPV DNA was amplified with the use of the MY09/MY11 consensus HPV L1 primers as well as primers for amplification of the human β-globin gene as an indicator of specimen adequacy. 10 After 30 amplification cycles, specimens were probed with a biotin-labeled HPV L1 consensus probe mixture. A separate membrane was probed with the biotin-labeled probes for the human β-globin gene.

Type-specific probing was performed for the following HPV types individually: 6, 11, 16, 18, 26, 31, 32, 33, 35, 39, 40, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 66, 68, 69, 70, and 73, Pap 155, Pap 291, AE2, and a mix containing 2, 13, 34, 42, 57, 62, 64, 67, 72, and W13B.

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CD4+ Cell Count and Plasma HIV RNA

The CD4+ cell counts and plasma HIV RNA closest to the period within 2 months of the anal examination were obtained. The nadir CD4+ level was defined as the lowest CD4+ cell count observed during the previous follow up of the patients. All the patients were followed at the clinic because they were known to be HIV-positive and the nadir of CD4+ cell count represented the true nadir before receiving HAART. Absolute numbers of CD4+ T cells were determined by standard-flow cytometry. Plasma HIV RNA levels were determined by the branched chain DNA signal amplification assay (Quantiplex HIV-RNA; Chiron, Emeryville, CA).

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Statistical Analysis

Data were analyzed using the Statview 5 package (SAS Institute, Inc., Cary, NC). Differences across groups were tested with the Fisher exact test (categorical variables) and the nonparametric Mann Whitney U test for continuous variables. If both cytologic and histologic results were available for analysis, a subject’s diagnosis was categorized as the more severe result like in our previous study. 5 For the purpose of the analysis, we chose the median increase in CD4+ cell count and the median nadir of CD4+ cell count as stratification points.

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Results

The study included 45 HIV-1-seropositive MSM with a median age of 38 years (range, 27–62 years). Sixteen patients (36%) reported at least one AIDS-defining event before study enrollment. Patients had previously received antiretroviral treatment for a median time of 53 months (range, 11–149 months) and protease inhibitors for a median time of 32 months (range, 11–53 months). The median nadir of CD4+ cell count at time of HAART initiation was 113 × 106 cells/L (range, 4–385). Baseline median values of CD4+ cell counts and plasma HIV RNA were 275 × 106 cells/L (range, 8–500) and 2.9 log copies/mL (range, 1.7–5.7), respectively. The median increase in CD4+ cell count from time to HAART initiation to time of study was 167 × 106 cells/L (range, 0–466).

Of the 45 anal cytologic samples of the study population, 32 were abnormal (71%), including 4 ASCUS, 22 LSIL, and 6 HSIL. An anoscopy and anal biopsy of a visible lesion were performed in 27 of the 32 patients with abnormal cytology.

Anal histology and/or cytology was abnormal in 32 of 45 (71%) patients, including ASCUS in 3 patients (7%), LSIL in 19 patients (42%), and HSIL in 10 patients (22%). HPV DNA was detected in 36 of 45 (80%) patients, including high-risk genotypes in 22 of 36 (61%) patients. The median number of HPV types was 2 (range, 1–6). Multiple HPV-type infection was found in 20 of 36 patients (56%). HPV type 16 was found in 8 of 36 HPV-infected patients (22%). HPV 18, 33, and 45 were found in 7 (19%), 6 (17%), and 4(11%) HPV-infected patients, respectively. The low-risk HPV types 6 and 11 were found in 12 (33%) and 7 (19%) HPV-infected patients, respectively.

The prevalence of HPV infection and ASIL were not significantly different between patients with CD4+ cell count at study enrollment above or below 250 × 106 cells/L (Table 1). Among patients with a nadir CD4+ cell count less than 100 × 106 cells/L, the prevalence of anal HPV infection and ASIL did not differ between patients with a HAART-associated increase in CD4+ cell count greater than or equal to 150 × 106 cells/L and those with an increase in CD4+ cell count less than 150 × 106 cells/L (Table 2). Similarly, no differences were seen between these groups among those who had a nadir CD4+ level greater than or equal to 100 × 106 cells/L (Table 2). Among patients with a HAART-associated increase in CD4+ cell count greater than or equal to 150 × 106 cells/L, the prevalence of anal HPV infection and ASIL did not differ between patients with a nadir CD4+ cell count greater than or equal to 100 × 106 cells/L and those with a nadir CD4+ cell count less than 100 × 106 cells/L (data not shown).

No significant difference was observed between patients with and without anal HPV infection or between patients with and without abnormal cytology with regard to median CD4+ cell count, median nadir of CD4+ count, median increase in CD4+ cell count, median HIV RNA level, and median duration of previous protease inhibitor regimen.

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Discussion

The prevalence of anal HPV infection and ASIL observed in our cohort receiving HAART for a median duration of 32 months is within the range observed in previous studies performed during the pre-HAART area. 4,5,11 Although the sample size is small, no significant difference in the prevalence of anal HPV infection and ASIL was observed between patients exhibiting low nadir CD4+ cell count or not and increased CD4+ cell count greater than 150 × 106 cells/L or not after a median time of 32 months under HAART. Our data suggest that immune restoration under HAART is not associated with a decrease of the prevalence of anal HPV infection and ASIL. However, our ability to detect these associations could have been limited as a result of the small sample size of this study. Consistent with our results, a recently published prospective study showed no significant regression of ASIL after 6 months of follow up under HAART and little effect on the detection of HPV using polymerase chain reaction and the Hybrid Capture methods. 12 Six months after starting HAART, anal HSIL, LSIL, and ASCUS were found in 19 of 76 (25%), 30 of 76 (40%), and 10 of 76 (13%) patients, respectively.

Taken together, these results contrast with some reports regarding cervical SIL in HIV-infected women receiving HAART. 13,14 The latter studies suggest that HAART altered the course of cervical SIL in HIV-infected women, reducing progression and increasing regression in some but not all of the study population. However, the effect of HAART on cervical SIL remains unclear. No significant reduction in the prevalence of cervical SIL was observed after a mean duration of 15 months under HAART in another study. 15 In addition, as observed in anal HPV infection, HAART did not reduce the high prevalence of cervical HPV infection. 15,16

Our data suggest that all HIV-positive MSM remain at risk of ASIL despite a HAART-associated increase in CD4+ level and should be considered for anal cytology screening. 17 One could speculate that if HAART exhibits no or little effect on the restoration of specific immunity against HPV, there would be more time for HSIL to progress to cancer. This could in turn lead to an increase in anogenital cancer among men and women receiving HAART because they would be living longer. Further prospective studies are needed to better understand the impact of HAART on anal HPV infection and ASIL.

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References

1. Melbye M, Sprogel P. Aetiological parallel between anal cancer and cervical cancer. Lancet 1991; 338: 657–659.
2. Palefsky JM, Holly EA, Gonzales J, Berline J, Ahn DK, Greenspan JS. Detection of human papillomavirus DNA in anal intraepithelial neoplasia and anal cancer. Cancer Res 1991; 51: 1014–1019.
3. Critchlow CW, Hawes SE, Kuypers JM, et al. Effect of HIV infection on the natural history of anal human papillomavirus infection. AIDS 1998; 12: 1177–1184.
4. Kiviat NB, Critchlow CW, Holmes KK, et al. Association of anal dysplasia and human papillomavirus with immunosuppression and HIV infection among homosexual men. AIDS 1993; 7: 43–49.
5. 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.
6. Goedert JJ, Cote TR, Virgo P, et al. Spectrum of AIDS-associated malignant disorders. Lancet 1998; 351: 1833–1839.
7. Frisch M, Biggar RJ, Goedert JJ. Human papillomavirus-associated cancers in patients with human immunodeficiency virus infection and acquired immunodeficiency syndrome. J Natl Cancer Inst 2000; 92: 1500–1510.
8. Piketty C, Darragh TM, Da Costa M, et al. High prevalence of anal HPV infection and anal cancer precursors among HIV-infected individuals in the absence of anal intercourse. Ann Intern Med 2003; 183: 453–459.
9. Piketty C, Weiss L, Thomas F, Mohamed AS, Belec L, Kazatchkine MD. Long-term clinical outcome of human immunodeficiency virus-infected patients with discordant immunologic and virologic responses to a protease inhibitor-containing regimen. J Infect Dis 2001; 183: 1328–1335.
10. 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.
11. Palefsky JM. Human papillomavirus infection and anogenital neoplasia in human immunodeficiency virus-positive men and women. J Natl Cancer Inst Monogr 1998; 23: 15–20.
12. Palefsky JM, Holly EA, Ralston ML, et al. Effect of highly active antiretroviral therapy on the natural history of anal squamous intraepithelial lesions and anal human papillomavirus infection. J Acquir Immun Defic Syndr 2001; 28: 422–428.
13. Minkoff H, Ahdieh L, Massad LS, et al. The effect of highly active antiretroviral therapy on cervical cytologic changes associated with oncogenic HPV among HIV-infected women. AIDS 2001; 15: 2157–2164.
14. Heard I, Tassie JM, Kazatchkine MD, Orth G. Highly active antiretroviral therapy enhances regression of cervical intraepithelial neoplasia in HIV-seropositive women. AIDS 2002; 16: 1799–1802.
15. Lillo FB, Ferrari D, Veglia F, et al. Human papillomavirus infection and associated cervical disease in human immunodeficiency virus-infected women: effect of highly active antiretroviral therapy. J Infect Dis 2001; 184: 547–551.
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