Squamous cell anal cancer is infrequent but has been increasing in the general population; currently, the rate is approximately one per 100 000 . It occurs at much higher rates in HIV-positive men who have sex with men (MSM) (approximately 60–160/100 000) [2–5] and in other immunocompromised persons such as transplantation recipients . Rates of anal cancer among HIV-infected MSM are comparable to rates of cervical cancer in women prior to the initiation of routine screening for cervical dysplasia . Combination antiretroviral therapy (cART) has markedly reduced HIV-related opportunistic infections and mortality  but malignancies are now a major cause of morbidity in HIV-infected individuals ; these include AIDS-defining malignancies such as non-Hodgkin lymphoma and non-AIDS-defining malignancies such as anal cancer [2,4,10].
Anal cancer shares features with cervical cancer. Both are human papillomavirus (HPV)-associated squamous cell cancers, which occur at a squamocolumnar transition zone [11,12], and both are believed to arise from precancerous dysplastic lesions, which are nearly identical histologically [13,14]. These precancerous changes can be detected by cytologic sampling using Papanicolaou smears and by colposcopy or the analogous procedure in the anal canal, high-resolution anoscopy (HRA) with directed biopsy [14,15].
The use of these methods to detect cervical cancer and its precursors has been studied extensively  and are well recognized by healthcare practitioners. Although less well known, these methodologies have also been applied to the anus [17–20]. In high-risk populations, such as HIV-infected MSM, screening to prevent anal cancer may be warranted. However large, well designed, methodologically sound studies are required in order to determine the optimal screening strategy. In the current study, we investigate the test characteristics of anal cytology and HPV testing for the detection of histologically confirmed high-grade anal intraepithelial neoplasia (AIN 2+), as this is an immediate anal cancer precursor that likely requires treatment.
In this cross-sectional study, anal cytology, HPV testing and HRA were performed at the initial visit. In addition, participants completed a questionnaire on demographics, symptoms and risk behaviors at this visit. The test characteristics of anal cytology, HPV testing for the detection of AIN 2+ at HRA or both were determined using the data from the initial visit.
This study is registered at http://www.clinicaltrials.gov #NCT00188292.
Participants were part of the Toronto Research for Anal Cancer Evaluation (TRACE) anal cancer screening study  and were enrolled between December 2001 and November 2005. All were HIV-infected men aged 18 years or more with a history of anal receptive intercourse and who were attending tertiary care hospital HIV clinics or primary care clinics in Toronto, Canada. Patients were excluded if they had a history of anal cancer. At entry, none of the participants had known previous anal cytologic studies or anal biopsies, so no prior results were available to us. The presence or absence of any symptoms, including anal symptoms, did not affect eligibility.
At the initial visit, participants had concomitant anal cytology (Pap smears), HPV testing and HRA with directed biopsies. Additional visits, examinations and treatments were arranged for participants who had high-grade histologic or cytologic abnormalities. Two anoscopists (I.E.S. and J.T.) had received formal training in HRA and they examined all the participants. The biopsies were processed in a routine manner and three deeper levels were cut from each paraffin-embedded specimen. Evaluation of anal cytology and detection of oncogenic HPV by Hybrid Capture 2 Assay (Digene Corporation, Gaithersburg, Maryland, USA) was performed on a specimen that was collected by vigorously rotating a Dacron-tipped swab in the anal canal and that was then placed in liquid-based ThinPrep (Cytyc, Boxborough, Massachusetts, USA). The results from the Hybrid Capture 2 Assay were used to determine the test characteristics of HPV detection. Another anal swab was tested for HPV genotypes using the Linear Array HPV Genotyping Test (Roche Diagnostics, Indianapolis, Indiana, USA).
Cytology and biopsy specimens were assigned computer-generated random numbers and were independently assessed by two gynecologic pathologists (A.L. and W.C.) each with over 20 years of pathology experience. The pathologists were masked to each other's interpretations, to the corresponding cytology specimen of the biopsy being examined, to the corresponding histology of the cytology being examined and to clinical and other laboratory information. After all specimens were reported, the pathologists met at a multiheaded microscope and reached consensus on any specimen for which there had not been agreement in the independent review.
The cytology was classified according to the modified Bethesda system for cervical cytology . Biopsies were classified as normal, AIN 1, AIN 2/3 or invasive carcinoma. Perianal lesions were excluded from the analysis.
HRA was done using a plastic anoscope with visualization of the anal squamocolumnar junction using 6–15× magnification after sequential mucosal staining with 3% acetic and Lugol's iodine . Biopsies were taken by endoscopic forceps from areas that appeared abnormal . Abnormal areas were those that were acetowhite, had abnormal-looking vessels or were Lugol's negative.
Demographic characteristics, symptoms in the previous 12 months, risk behaviors and duration of HIV (number of years since the first positive antibody test) were obtained from the questionnaire, which was completed by each participant without assistance from study personnel. The most recent CD4 cell count, viral load and previous medical history including current antiretroviral regimen were obtained from the participant's clinical record.
Demographic characteristics were summarized for the study population using frequency and percentage for categorical variables and median and interquartile range for continuous variables. We calculated sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of cytology and presence of oncogenic HPV on Hybrid Capture testing to detect histologically confirmed AIN 2+ on a per patient basis. If the anal canal at HRA appeared normal, no biopsy was taken and the anal canal for that patient was designated as ‘Normal’. If biopsies were taken, patient status was designated by the consensus diagnosis of the most severe histologic lesion in that patient.
The predetermined primary outcome was the sensitivity of any anal cytologic abnormality to detect histologically confirmed intraanal AIN 2, AIN 3 or invasive squamous cell carcinoma (AIN 2+). The cytologic test was defined as positive if at least atypical squamous cells of undetermined significance (ASCUS) was present, that is, if either ASCUS, low-grade squamous intraepithelial lesion (LSIL) or high-grade squamous intraepithelial lesion (HSIL) was present. On the basis of this primary outcome and assuming a sensitivity of 70%, we estimated that we would need a sample size of 100 patients with AIN 2+ to be able to determine the sensitivity with a 95% confidence interval (CI) of ±10%. As the prevalence of AIN 2+ was estimated to be 25% in this population, we planned to enroll 400 patients in order to obtain 100 patients with AIN 2+.
The study was approved by the Research Ethics Board of the University Health Network and was carried out in accordance with the Declaration of Helsinki of 1975, as revised in 1983.
We enrolled 401 patients. The median age was 44 years with a median CD4 cell count of 390 cells/μl, median viral load less than 50 copies/ml, 76% were on cART and they had been HIV positive for a median of 14.0 years. Only 31.0% had an AIDS-defining illness and 18.0% had a CD4 cell count of less than 200 cells/μl. Ninety-two percent reported at least one gastrointestinal symptom, most commonly mild bowel irregularity or blood noted on wiping (Table 1).
Prevalence of human papillomavirus, cytologic or histologic abnormalities
At their initial visit, all patients had anal cytology, HPV testing and anal biopsy if indicated by the HRA appearance. Cytology was abnormal in 67% of patients: HSIL, 12%; LSIL, 43% and ASCUS, 12% (Table 2). The biopsies from the first visit were abnormal in 68% of patients and the worst grade of lesion was AIN 2+, 25% and AIN 1, 43%. No invasive cancers were initially identified. In order to assess whether there might have been a learning effect whereby the anoscopists would have detected more AIN 2+ later in the study, we examined that trend over time. The rate of AIN 2+ detection was 25 per 100 (25%) for the first 100 patients and 73 per 301 (24%) for the remaining patients.
HPV was detected in 93% of patients with multiple HPV types in 92%. Oncogenic HPV types were found in 352 of 400 (88%): HPV 16 (38%), HPV 18 (20%), HPV 31(16%), HPV 33 (15%), HPV 53 (20%), HPV 52 (18%), HPV 68 (12%) and HPV 56 (12%).
Test characteristics of oncogenic human papillomavirus and cytology at the initial visit
Using a threshold for abnormal of at least ASCUS (≥ASCUS), abnormal cytology detected histologically confirmed intraanal AIN 2+ with a high sensitivity but with low specificity (Table 3). Using this definition, abnormal cytology failed to detect only 16 of 98 (16%) AIN 2+, whereas 186 of 268 (69%) of abnormal cytologic results were not associated with AIN 2+. If abnormal cytology was defined as HSIL, the sensitivity of cytology dropped, whereas the specificity markedly increased. HSIL on cytology missed 77 of 98 (79%) of histologic AIN 2+, whereas 26 of 47 (55%) of cytologic HSIL results were unassociated with presence of AIN 2+ histologically. Furthermore, the test characteristics of abnormal anal cytology did not differ if we excluded the first 100 patients (data not shown). As these rates did not differ, we used the data for the whole group in all analyses.
All patients with AIN 2+ were positive for oncogenic HPV; conversely, none of the patients who were negative for oncogenic HPV had AIN 2+. If we used a combination of any abnormal cytology and oncogenic HPV as a predictor of AIN 2+, there was minimal improvement in the test characteristics compared with using the cytology alone (sensitivity 84 vs. 84% and specificity 43 vs. 39%).
Forty-nine men had anal cytology which showed ASCUS and 42 (86%) of them were test positive for oncogenic HPV. Nine men with ASCUS cytology had biopsy-proven AIN 2+, and all nine were oncogenic HPV positive. Therefore, in patients with ASCUS on cytology, oncogenic HPV testing had a sensitivity of 100% (95% CI 70–100) and a specificity of 17.5% (95% CI 8.7–32.0) for high-grade AIN.
In the current study, we have analyzed anal cytology (Pap testing) and HPV detection in MSM as screening methods for detection of high-grade intraepithelial neoplasia (AIN 2+), as these lesions are most likely to lead to anal cancer and treatment of these lesions may potentially prevent cancer. We found a high prevalence (24%) of histologic AIN 2+ in our high-risk population at initial screening. However, anal cytology only showed HSIL in 12%, suggesting that it is an insensitive test (if done once) for detection of high-grade histologic disease. High-risk (oncogenic) HPV types were present in almost 90% of our patients; as most of the patients denied recent sexual activity, many of these are likely long-standing persistent infections. Persistent oncogenic cervical HPV infection is a strong risk factor for the occurrence of high-grade cervical intraepithelial neoplasia (CIN) and progression to invasive cancer  and this situation is likely similar for anal cancer in MSM.
Anal cancer rates are increasing in the general population in both men and women and rates are particularly high in HIV-positive MSM [1–5] likely due to exposure to HPV through multiple partners  and prolonged immune deficiency, which permits persistence of HPV. In the era of cART, survival in persons with HIV has dramatically improved, as deaths from opportunistic infections and AIDS-defining malignancies are markedly diminished . Despite its undisputed efficacy in preventing many HIV-related complications, there is mounting evidence that cART not only fails to protect against anal precancers and cancers [2–4,23,24] but also that the incidence of anal cancer is increasing [1,3,25,26]. Because of this increasing burden of anal cancer, there is an urgent need to find effective ways of screening to prevent anal cancer in HIV-infected MSM.
In immunocompetent persons, high-grade anal dysplasia progresses to invasive malignancy in an important proportion of cases (14% over a period of 8 years) . In anal and cervical cancers, precancerous lesions appear to transition through similar cellular stages that can be detected by anal or cervical cytology (Pap tests) or biopsy. Although there are no randomized controlled trials to support screening to prevent cervical cancer, ecological studies have shown that cervical Pap testing and colposcopy with treatment of abnormalities has been associated with a marked reduction in cervical cancer  and countries without effective screening have high rates of cervical cancer .
HIV-positive MSM have a high prevalence of anal dysplasia (AIN) and it seems likely that these lesions are anal cancer precursors. Given the strong parallels with cervical cancer, it would seem intuitive that detection and removal of these high-grade anal lesions (AIN 2+), particularly in HIV-infected MSM, might reduce anal cancer rates even though this is unproven. In HIV-uninfected persons, anal cancer detection at an earlier stage improves survival [28,29] so early cancer detection in HIV-infected patients may also be beneficial [30,31]. Anal cancer screening seemed to prolong quality-adjusted life expectancy as demonstrated in a state-transition Markov model and it should be done every 12–24 months depending on the prevalence of anal dysplasia . Even though a number of centers do screen for anal cancer and precancer in high-risk MSM, the optimal methods and strategies are uncertain.
Potential screening tests in MSM such as anal cytology or anal HPV detection are simple to do, much cheaper, less time-consuming and require less expertise than HRA. Cervical Pap testing for women, at the threshold of ASCUS or greater to detect CIN 2 or greater, only has a sensitivity of 50%  but it is a simple strategy that can be done repeatedly and it has been responsible for the dramatic decrease in cervical cancer incidence . The incremental benefit of repeated anal Pap testing in HIV-positive MSM is not known.
Cervical oncogenic HPV detection in women is more sensitive but may be less specific than cervical Pap testing for detecting CIN 2/3 . In our population, we found similar test characteristics for anal oncogenic HPV testing in MSM. Although the absence of oncogenic HPV completely predicted that AIN 2+ was not present (NPV = 100%), the usefulness of this test is limited because of the extremely high prevalence of oncogenic HPV infection in this population.
In women who show cervical ASCUS cytology on Pap smear, oncogenic HPV testing is helpful to identify those who should undergo colposcopic examination . We found that among HIV-positive MSM, testing for anal oncogenic HPV in those with ASCUS had very low specificity for detection of AIN 2+; HPV testing in this context is unlikely to be useful, as the HPV prevalence is so high. Indeed, in women, cervical oncogenic HPV testing is also not recommended when cytology shows LSIL, as the HPV prevalence in these women is also over 80% .
In the current study, anal Pap testing using ASCUS as the threshold was sensitive in detecting AIN 2+ but there were so many abnormal Pap tests that only a small number were excluded from requiring HRA. If we used HSIL as a threshold to predict AIN 2+, specificity was increased but substantial numbers of men with histological AIN 2+ were missed as a result of the very poor sensitivity of the Pap test. Because of the high prevalence of AIN 2+ in HIV-positive MSM and because of the suboptimal test characteristics of one-time cytological and HPV testing, it can be argued that HRA should be a first-line screening strategy in this high-risk population; however, anal cytology may provide complementary information . Clinicians interested in screening their HIV-positive MSM must also take costs, availability of local expertise in doing HRA and the willingness of patients to have this procedure into consideration when selecting a screening strategy.
The strengths of the current study are: we could effectively compare liquid-based anal cytology, HPV detection and HRA, as these were done simultaneously on all patients and the results reported independently to avoid bias; histologically proven AIN 2+ was used as the reference standard; over 400 patients were enrolled and followed prospectively and cytologic and histologic results were independently assessed by two pathologists. We focused on AIN 2+ detection because CIN 2+ constitutes the threshold for treatment in the cervix .
The implementation of anal cancer screening programs is hampered by the lack of data from randomized control trials. Nevertheless, the data presented here on the detection of AIN will be useful in evaluating the utility of early anal cancer screening in HIV-positive MSM. Future studies should focus on treatment and the effectiveness of screening in the prevention of anal cancer, including the cost-effectiveness, in other high-risk groups such as women with previous gynecologic malignancy and transplant recipients.
Ontario HIV Treatment Network (grant #ROGB184) and the Canadian Cancer Society (grant #18164) funded the capital costs, research assistant and operating costs. J.S. was supported by a Career Scientist Award from the Ontario HIV Treatment Network.
I.S. and J.T. did all the anoscopic examinations; M.S. organized the anal cancer screening treatments and database; A.L. and W.C. assessed all the pathology specimens; J.R. and C.D. did statistical analyses; J.B.M. and S.C. performed HPV analyses; study conception and design was done by I.S., A.L., J.R., J.B.M., W.C. and J.T.
There are no conflicts of interest.
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