van der Snoek, Eric M. MD, PhD*; van der Ende, M. E. MD, PhD†; den Hollander, J. C. MD‡; Schutten, M. PhD§; Neumann, H. A. M. MD, PhD*; van Doornum, G. J. J. MD, PhD§
Anal cancer is an uncommon malignancy, with an estimated incidence in the general population between 0.8 and 1.4 cases per 100,000 person-years.1 Numbers of patients with anal cancer and preneoplastic anal lesions (anal intraepithelial neoplasia [AIN]) in western European countries and the United States have increased in recent decades.2–4 Recent studies conclude that the incidence of anal cancer ranges from 42 to 137 cases per 100,000 person-years of observation among HIV-positive men and women.5 The relative risk of invasive anal cancer among men with AIDS compared with men without known HIV was up to 352 in the era of highly active antiretroviral therapy (HAART).6
Anal cancer may be preceded by AIN. Several investigators have reported a high and varying occurrence of AIN in men who have sex with men (MSM). Percentages up to 43% of high-grade AIN have been reported in both cross-sectional and longitudinal studies.7–11 Reported percentages vary considerably partly depending on interpretation of the histology and the skills of the anoscopist in identifying clinical aspects suspicious for AIN and in taking representative biopsies of theses lesions.
Persistent high-risk human papillomavirus (HPV) infection is an important risk factor for AIN and anal cancer because of its ability to trigger carcinogenic development. HIV-infected MSM are at increased risk of persistent HPV infection.12 There is evidence that the HPV types that are causally linked to cervical cancer are also linked to anal cancer.13 HPV-associated malignancies have a latency of 5 years to several decades in immunocompetent individuals, immunosuppression probably accelerates the development of malignancies.6 Low CD4 cell counts have been reported to be associated with an increased risk of AIN 2 or 3 in both longitudinal and prospective, cross-sectional studies.14–16
Prevalences of HPV infection in MSM are high. A recent cohort study in Montreal detected HPV DNA in 97.9% of all 247 MSM tested. Median number of concomitant HPV types was 5. Most common types were HPV-16 and HPV-6. High prevalence of anal HPV has been reported not to decline with age.17
HAART suppresses HIV viral replication and partly restores cellular immunity. Whether HAART has any effect on the clinical course of an HPV infection, HPV persistence, or the incidence of AIN is still under debate.6,18–23 HAART increases longevity, and thus the time at risk for the development of anal premalignancies and cancer.6 HAART may also induce clearing of HPV infection, regression, and prevention of development of anal cancer. In women, HAART has been reported to associate with increased cervical HPV clearance but not with decrease of cervical intraepithelial neoplasia (CIN) in longitudinal cohort studies.24,25
This cross-sectional study was performed to determine the prevalence of AIN in a group of 250 HIV-positive MSM in Rotterdam and to investigate the values of several predictors of AIN in the context of duration and response to HAART.
MATERIALS AND METHODS
Patients and Study Design
From February 2007 to March 2009, 250 individuals were included in a Rotterdam study on the screening of HPV-related anal premalignancies in HIV-positive MSM. All patients were recruited from the outpatient clinics of 4 hospitals. Participants gave written informed consent before start of the study. The institutional review board of our hospital approved the study.
Highly Active Antiretroviral Therapy
Antiretroviral therapy was started in patients with CD4 cell counts below 300 × 106 cells/L or in case of symptoms consistent with the diagnosis of AIDS. This has been standard treatment in The Netherlands for more than 5 years now.
HIV Viral Load Measurement
HIV RNA was in the majority of cases assessed quantitatively with the Cobas Ampliprep/Cobas Amplicor version 1.5 (lower limit of detection: 50 copies/mL; Roche Molecular Systems, Penzberg, Germany).
Behavioral data collected from all participants by means of a questionnaire included age, current smoking habits, sexual orientation, number of sex partners, including practice of anal sex and earlier diagnosis of anogenital warts. From the medical records, the following data were collected: length for known HIV positivity, use and duration of HAART, recent CD4 cell count, nadir CD4 cell count, HIV viral load, and the previous (lifetime) occurrence of an AIDS-defining event. All participants were asked to report complaints in the anal region, such as intermittent itch, pain, and bloody or purulent discharge.
Anal examination was performed at the department of dermatology by the same experienced dermatologist in all patients. The examination consisted of visual inspection of the perianal and intra-anal area before and after acetic acid (5%) application. Acetic acid application increases visibility of HPV-related intraepithelial lesions. Inspection of the anal canal took place using high-resolution anoscopy. An HPV-related intraepithelial lesion was suspected in case of either local atypical aceto-whitening or (exophytic) elevated lesions easily distinguishable from the surrounding normal mucosa. Biopsies were taken from the centre of suspicious lesions detected during examination.
All biopsies were examined by the same pathologist using paraffin-embedded sections of formalin-fixed tissue stained with hematoxylin and eosin. AIN grade 1 or mild dysplasia was defined as lower third architectural disruption. In AIN grade 2 or moderate dysplasia, architectural disruption was seen in both lower and middle third of the squamous epithelium. AIN grade 3 or severe dysplasia was based on the finding of severe architectural disruption throughout the epithelium without disruption of the basal membrane.
HPV DNA Sample Collection
Specimens for assessment of HPV DNA were collected using a non-Dacron cotton swab moistened with sodium chloride 0.9% (Medical Wire & Equipment Co. (Bath) Ltd. Corsham, Wiltshire, United Kingdom), swabbing both the perianal and intra-anal area. The swabs were immediately placed into standard collecting tubes without transport medium and stored at −20°C before being sent to the Department of Virology for further processing. Total nucleic acids were isolated at the MagnaPureLC Isolation Station (Roche Applied Science).
Detection and Typing of HPV
Detection and typing of HPV DNA was performed using the INNO-LiPa HPV Genotyping Extra assay (Innogenetics, Ghent, Belgium). The INNO-LiPA HPV Genotyping Extra assay is a polymerase chain reaction-based line hybridization assay that utilizes several biotinylated consensus primers (SPF10) to amplify a region of the L1 gene of HPV types.26 The assay covers all currently known high-risk HPV (HR-HPV) genotypes and probable HR-HPV genotypes (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82), as well as a number of low-risk HPV (LR-HPV) genotypes (6, 11, 40, 43, 44, 54, 70) and some additional types (69, 71, 74). Fully automated processing of the strips was executed by using Auto-LiPA, and automated interpretation of the strips was performed with LiRAS for LiPA HPV (Innogenetics).26
We used the epidemiologic classification according to Munoz et al27 and Miyashita et al,28 which groups HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 as high-risk and considered types 26, 53, 66, 70, 69 or 71, 74, and “unclassified” (X) as “unknown” with regard to risk.
Data were compared in order to assess statistically significant differences in several characteristics (Tables 1 and 2), including the prevalence of HPV (Table 3) between the groups with and without dysplasia. Prevalence was calculated as the number of positive tests per 100 tested individuals. For testing differences between the groups, the exact χ2 test was used, after all explanatory variables had been dichotomized. Age, number of lifetime sexual partners, length for known HIV positivity, and CD4 cell count and viral load count were tested as continuous variables. Statistical significance was defined as a P value of less than 0.05.
Next, the variables age, length for known HIV positivity, and use of HAART were compared between groups (Table 2). To adjust for confounding variables, logistic regression analysis was used. The primary selection of covariables for entering in the model, along with group, was based on univariable analysis in 2 by 2 tables; an exact P value below 0.05 was used. Analyses were done using SPSS 18.0.
Table 3-b. Results H...Image Tools
A total of 250 males were included in this study, 210 without and 40 with AIN. Patients' characteristics are summarized in Table 1. The median age of all males included was 46.5 years (interquartile range [IQR]: 40.0–53.3). The median length of known HIV positivity was 8.0 years (IQR: 4.0–13.0). Of the cohort, 201 patients (80.4%) were on HAART. The median length of use of HAART was 7.0 years (IQR: 3.0–10.0). The median CD4 cell count was 490.0 × 106 cells/L (IQR: 357.5–640.0), and the median nadir CD4 cell count was 229.0 × 106 cells/L (IQR: 120.0–310.0). Of all patients, 67.1% had HIV RNA below the limit of 50 copies/mL. The median number of copies of those with detectable (>50 copies/mL) HIV RNA was 6285.0 (IQR: 322.5–38125.0).
Of all males, 247 (98.8%) had had sex with men only, in the preceding 6 months. Other patients were bisexual. A majority of 69.6% reported to have had receptive anal sex in the preceding 12 months. In all, 111 patients (44.4%) reported a previous history of anogenital warts. Ninety patients (36.0%) were current smokers.
Males using HAART did not significantly differ from the ones without HAART with regard to circumcision before age of 10 years, age at sexual debut, sexual orientation, number of lifetime sexual partners, (recent) practice of receptive anal intercourse, complaints in the anal area, or smoking habit. Those who used HAART were significantly older (median, 48 vs. 39 years; P < 0.0005) and were longer known as being HIV-positive (median, 10 vs. 4 years; P < 0.0005).
Among all 250 patients, 108 (43.2%) had lesions suspicious for HPV-related intraepithelial lesion. A total of 122 biopsies were taken. Of all these biopsies, 60 (49.2%) were taken from the perianal area and 62 (50.8%) from the intra-anal area. In 14 patients, both perianal and intra-anal biopsies were taken. Histologic analyses showed AIN 1 in 24 patients (22.2%), AIN 2 in 6 patients (5.6%), and AIN 3 in 10 patients (9.3%). AIN 3 was observed equally often at the perianal and intra-anal area. Of 10 males with AIN 3, 4 had clinically bowenoid AIN 3. Bowenoid lesions are characterized by pigmented papules, which are histologically similar to Bowen disease (i.e., severe dysplasia) and may have a less aggressive nature than other AIN 3 in HIV-positive MSM. These males were significantly younger than the patients with non-bowenoid clinical expression of their AIN 3 (median age, 27.0 vs. 50.0 years; P = 0.019). The results of all biopsies taken are summarized in Table 4.
In univariable analysis, age (P = 0.011), length for known HIV positivity (P = 0.040), and use of HAART (P = 0.004) were significantly associated with AIN. Neither CD4 cell count, nadir CD4 cell count nor HIV viral load were associated with the prevalence of AIN. All other characteristics in Table 1 were not related with AIN.
In multivariable analyses, the use of HAART was associated with the absence of AIN (Table 2). This was the case when age, length for known HIV positivity, and use of HAART were used in the analyses.
Intermittent anal itch was reported by 59 (23.6%) patients and pain in the anal region on a regular basis by 34 (13.6%) patients. Sporadic bloody or purulent discharge was reported by 51 (20.4%) patients. None of these complaints were significantly more frequent in those diagnosed with AIN.
HPV types were identified in 224 of 247 samples (90.7%). Data are summarized in Table 3. In only 3 specimens (1.2%), no detection could be performed. A median number of 3 HPV types were detected in positive specimens (IQR: 1–4; maximum number: 13). Of those males with HPV, 171 of 224 (76.3%) had one or more HR-HPV type (). LR-HPV was detected in only 27 of 224 (12.1%) positive specimens. HPV types most often detected were HPV-52 (43.8%; HR-HPV), HPV-39 (24.1%; HR-HPV), HPV-74 (21.9%; unknown risk), HPV-54 (21.0%; LR-HPV), and HPV-51 (18.3%; HR-HPV). Well-known HR-HPV types 16 and 18 were detected in 17.4% and 10.7% of positive specimens, respectively. The LR-HPV types 6 and 11 were detected in 17.4% and 14.3% of positive specimens, respectively.
In MSM with AIN, the HPV types 6 and 16 were detected significantly more often compared with those with no dysplasia (P = 0.009 and 0.009)P in the sentence “In MSM with AIN, the HPV…” are OK as given.--. No differences were found regarding high-risk types, number of types, or multiple infections.
In MSM with HAART, the absence of any HPV infection was found significantly more often compared with those who did not use HAART (11.5% vs. 0%; P = 0.010). Multiple infections were detected more often in those who did not use HAART (80.9% vs. 64.5%; P = 0.037).
In this large cross-sectional study in a group of 250 HIV-positive MSM, the prevalence of AIN was 16.0%, which is in line with data from German and French studies.7–9 Our study supports the data of the longitudinal cohort study by De Pokomandy et al29 that usage of HAART may lower the risk on AIN.
Data on the impact of HAART on the prevalence of AIN are scarce. Earlier studies did not report this association. This maybe due to small numbers of patients18,22 or due to recent introduction of HAART.21 In a more recent longitudinal study in a cohort of 357 HIV seropositive gay men in San Francisco, Palefsky et al did not assess the effect of antiretroviral therapy possibly because some of the patients in his study were still not using a full HAART regimen.23 A single longitudinal cohort study on the effect of HAART on CIN showed that women on HAART were 40% more likely to demonstrate regression and less likely to demonstrate progression.19 Another group confirmed a higher regression rate of CIN in HAART-treated women in a prospective longitudinal study.30 In contrast, in a recent longitudinal cohort study in women with HIV and at-risk women without HIV by Paramsothy et al, HAART was associated with enhanced cervical HPV clearance, but not with Pap test regression.24 In this study, only 20% of the women on HAART had HIV RNA <500 copies/mL due to low adherence, which may have underestimated the effects associated with HAART.
Data of the longitudinal cohort study by De Pokomandy et al29 suggest that receiving HAART for more than 4 years may contribute to some benefit against AIN 2 or 3.
The prevalence of AIN in this study was not associated with duration of known HIV infection, (nadir) CD4 counts, HIV viral load, or the previous occurrence of AIDS-defining events. This may be explained by the high percentage of patients successfully using HAART, with normal median recent CD4 cell counts in both groups.
HPV infection, the etiologic agent of AIN, was detected significantly more often in treatment-naïve patients compared with those who used HAART (100% vs. 88.5%; P < 0.010). Differences regarding prevalence of HPV infections in both groups are in agreement with data from several previous studies.1,20,31,32 Although this difference in our study is significant, a percentage of 88.5%, it is still a high rate of (probably persistent) HPV infections.
Despite potent anti-HIV therapy, with suppression of HIV replication and an increase of CD4 cell counts, this may not be sufficient to reduce HPV persistence but it may be helpful to induce regression of newly acquired acute HPV infection. Our cross-sectional study cannot answer that question. Longitudinal studies on the effect of HAART on HPV persistence in women indeed do report significantly enhanced clearance of HPV after starting HAART.24,33 One may hypothesize that longer duration of successful HAART could eventually diminish the risk of AIN.
In this study, the prevalence of AIN was significantly related with the prevalence of both HPV types 16 and 6. HPV type 16 has been linked to different types of anogenital cancers. There was no association between prevalence of AIN and other HR-HPV types or the number of high-risk types. This can be explained by the high prevalence (90.7%) of HPV in this group of patients, of which 76.3% had at least one high-risk type. The prevalence of at least one high-risk type was detected equally often in those with and without AIN.
In contrast to other studies, we did not find an association with nadir CD4 cell count. The nadir CD4 counts in our study were relatively high. This may explain the lower prevalence of AIN 2 or 3 in this study. In contrast to the study by De Pokomandy et al,29 duration of HAART regimen was not related with the prevalence of AIN.
The strength of this study is the combination of the assessment of AIN and HPV in a group of patients with a high rate of successful HAART, with detailed clinical and laboratory parameters, such as the previous occurrence of AIDS-defining events, recent and nadir CD4 counts, and HIV viral load. Furthermore, the same physician evaluated all patients.
This study was limited by the cross-sectional design. No anal cytology has been used as screening method for the detection of AIN. This limitation might explain the lower percentage of AIN found in this group of HIV-positive MSM compared with other groups where both histology and cytology were used. Due to these small numbers of AIN, it was not possible to separately examine data of AIN 1 versus AIN 2/3. Finally, HPV samples in this study were done on exfoliated anal cells and not from the biopsies taken. Although these superficial specimens are extremely sensitive, this might have influenced the outcomes of HPV infections.
In conclusion, in this cross-sectional study in 250 HIV-positive MSM, the use of HAART was associated with a significantly reduced prevalence of AIN (OR = 2.28; P = 0.045) and a significantly lower prevalence of HPV (P = 0.010). AIN was associated with HPV types 6 and 16. This association between the prevalence of AIN and the absence of HAART may contribute to the current debate on when to start HAART in HIV-infected individuals. Current European guidelines advice initiation of HAART in case of CD4 cell counts between 350 and 500 × 106 cells/L. In patients coinfected with hepatitis B or C, HAART should be initiated even when CD4 cell counts are 500 × 106 cells/L or above. Because HPV infection can be considered coinfection, earlier introduction in these patients may be useful, as it may influence its clinical course and related burden.
1. Chin-Hong PV, Palefsky JM. Natural history and clinical management of anal human papillomavirus disease in men and women infected with human immunodeficiency virus. Clin Infect Dis 2002; 35:1127–1134.
2. Goldstone SE, Winkler B, Ufford LJ, et al.. High prevalence of anal squamous intraepithelial lesions and squamous-cell carcinoma in men who have sex with men as seen in a surgical practice. Dis Colon Rectum 2001; 44:690–698.
3. Drobacheff C, Dupont P, Mougin C, et al.. Anal human papillomavirus DNA screening by Hybrid Capture II in human immunodeficiency virus-positive patients with or without anal intercourse. Eur J Dermatol 2003; 13:367–371.
4. Johnson LG, Madeleine MM, Newcomer LM, et al.. Anal cancer incidence and survival: The surveillance, epidemiology, and end results experience, 1973–2000. Cancer 2004; 101:281–288.
5. Park IU, Palefsky JM. Evaluation and management of anal intraepithelial neoplasia in HIV-negative and HIV-positive men who have sex with men. Curr Infect Dis Rep 2010; 12:126–133.
6. Diamond C, Taylor TH, Aboumrad T, et al.. Increased incidence of squamous cell anal cancer among men with AIDS in the era of highly active antiretroviral therapy. Sex Transm Dis 2005; 32:314–320.
7. Kreuter A, Brockmeyer NH, Hochdorfer B, et al.. Clinical spectrum and virologic characteristics of anal intraepithelial neoplasia in HIV infection. J Am Acad Dermatol 2005; 52:603–608.
8. Kreuter A, Brockmeyer NH, Weissenborn SJ, et al.. German Competence Network HIV/AIDS. Penile intraepithelial neoplasia is frequent in HIV-positive men with anal dysplasia. J Invest Dermatol 2008; 128:2316–2324.
9. Abramowitz L, Benabderrahmane D, Ravaud P, et al.. Anal squamous intraepithelial lesions and condyloma in HIV-infected heterosexual men, homosexual men and women: Prevalence and associated factors. AIDS 2007; 21:1457–1465.
10. Salit IE, Tinmouth J, Chong S, et al.. Screening for HIV-associated anal cancer: Correlation of HPV genotypes, p16, and E6 transcripts with anal pathology. Cancer Epidemiol Biomarkers Prev 2009; 18:1986–1992.
11. Schlecht HP, Fugelso DK, Murphy RK, et al.. Frequency of occult high-grade squamous intraepithelial neoplasia and invasive cancer within anal condylomata in men who have sex with men. Clin Infect Dis 2010; 51:107–110.
12. Del Mistro A, Chieco Bianchi L. HPV-related neoplasias in HIV-infected individuals. Eur J Cancer 2001; 37:1227–1235.
13. Giuliano AR, Tortolero-Luna G, Ferrer E, et al.. Epidemiology of human papillomavirus infection in men, cancers other than cervical and benign conditions. Vaccine 2008; 26:K17–K28.
14. Elfgren K, Kalantari M, Moberger B, et al.. A population-based five-year follow-up study of cervical human papillomavirus infection. Am J Obstet Gynecol 2000; 183:561–567.
15. Ferenczy A, Coutlée F, Franco E, et al.. Human papillomavirus and HIV coinfection and the risk of neoplasias of the lower genital tract: A review of recent developments. CMAJ 2003; 169:431–434.
16. Piketty C, Darragh TM, Da Costa M, et al.. High prevalence of anal human papillomavirus infection and anal cancer precursors among HIV-infected persons in the absence of anal intercourse. Ann Intern Med 2003; 6:453–459.
17. de Pokomandy A, Rouleau D, Ghattas G, et al.. HIPVIRG Study Group. Prevalence, clearance, and incidence of anal human papillomavirus infection in HIV-infected men: The HIPVIRG cohort study. J Infect Dis 2009; 199:965–973.
18. Piketty C, Darragh TM, Heard I, et al.. High prevalence of anal squamous intraepithelial lesions in HIV-positive men despite the use of highly active antiretroviral therapy. Sex Transm Dis 2004; 31:96–99.
19. 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.
20. 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.
21. Palefsky JM, Holly EA, Ralston ML, et al.. Effect of highly active antiretroviral therapy on the natural history of anal squamous intra-epithelial lesions and anal human papillomavirus infection. J AIDS 2001; 28:422–428.
22. Fox P, Stebbing J, Portsmouth S, et al.. Lack of response of anal intra-epithelial neoplasia to highly active antiretroviral therapy. AIDS 2003; 17:279–280.
23. Palefsky JM, Holly EA, Efirdc JT, 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.
24. Paramsothy P, Jamieson DJ, Heilig CM, et al.. The effect of highly active antiretroviral therapy on human papillomavirus clearance and cervical cytology. Obstet Gynecol 2009; 113:26–31.
25. Minkoff H, Zhong Y, Burk RD, et al.. Influence of adherent and effective antiretroviral therapy use on human papillomavirus infection and squamous intraepithelial lesions in human immunodeficiency virus-positive women. J Infect Dis 2010; 201:681–690.
26. Seme K, Lepej SZ, Lunar MM, et al.. Digene HPV Genotyping RH Test RUO: Comparative evaluation with INNO-LiPA HPV Genotyping Extra Test for detection of 18 high-risk and probable high-risk human papillomavirus genotypes. J Clin Virol 2009; 46:176–179.
27. Munoz N, Bosch FX, De sanjosé S, et al.. Epidemiologic classification of Human papillomavirus types associated with cervical cancer. N Engl J Med 2003; 348:518–527.
28. Miyashita M, Agdamag DM, Sasagawa T, et al.. High-risk HPV types in lesions of the uterine cervix of female commercial sex workers in the Philippines. J Med Virol 2009; 81:545–551.
29. de Pokomandy A, Rouleau D, Ghattas G, 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 2011; 52:1174–1181.
30. Heard I, Tassie JM, Kazatchkine MD, et al.. Highly active antiretroviral therapy enhances regression of cervical intraepithelial neoplasia in HIV-seropositive women. AIDS 2002; 16:1799–1802.
31. Six C, Heard I, Bergeron C, et al.. Comparative prevalence, incidence and short-term prognosis of cervical squamous intraepithelial lesions amongst HIV-positive and HIV-negative women. AIDS 1998; 12:1047–1056.
32. de Sanjosé S, Palefsky J. Cervical and anal HPV infections in HIV positive women and men. Virus Res 2002; 89:201–211.
33. Fife KH, Wu JW, Squires KE, et al.. Prevalence and persistence of cervical human papillomavirus infection in HIV-positive women initiating highly active antiretroviral therapy. J Acquir Immun Defic Syndr 2009; 51:274–282.