For HIV-infected people, anal cancer is becoming an increasingly major problem. In HIV-infected MSM, the incidence of anal cancer ranges from 65 to 130 cases per 100 000 person-years, a rate 30–100 times higher than that of the general population [1,2]. Today, it is one of the most common non-AIDS-defining cancers in this at-risk population [1,2].
Anal cancer has many epidemiologic, virologic, and histologic similarities to cervical cancer, including the precursor of a high-grade squamous intraepithelial lesion (HSIL) [3,4]. Such similarities have fostered the introduction of screening programs striving to replicate the public health successes of cervical dysplasia screening, which has significantly reduced the burden of this disease [5–8].
Like cervical dysplasia screening, anal cytology is commonly used as the first step of screening algorithm for premalignant anal lesions. Patients with abnormal anal cytology results are typically referred for high-resolution anoscopy (HRA) to obtain histological samples of the lesions. HRA with directed biopsy, a technique similar to cervical colposcopy, is currently accepted as the gold standard tool for anal dysplasia diagnosis [5–8]. Nevertheless, there are some anatomic differences between anal and cervical dysplasias that likely account for some of the differences in the performance of the diagnostic operating tools. Thus, anal cytology is collected ‘blindly’, without direct visualization of the squamocolumnar junction. This perhaps explains the low reproducibility and lower sensitivity of anal cytology as compared with cervical cytology in detecting high-grade lesions [9–11].
Human papillomavirus (HPV) testing is also a major tool in screening for cervical disorder and, in recent years, has been incorporated into cotesting with cytology, or even replacing this as the singular, first diagnostic technique in different screening algorithms [12,13]. However, its usefulness in the screening of anal dysplasia, mainly in the risk group of HIV-infected MSM in whom the prevalence of oncogenic HPV is high, is not clear.
In this study's facilities, anal dysplasia screening includes systematically performing anal liquid cytology, HPV testing, and HRA during the same visit. This differs from the majority of screening programs in which HRA is only performed after abnormal cytology findings. Thus, this protocol provides a unique opportunity to assess the contribution of anal cytology and HPV testing in the detection of high-grade anal intraepithelial neoplasia (HGAIN).
The objective of this study is, therefore, to evaluate the utility of oncogenic HPV testing alone, and the cotesting of HPV testing and anal cytology for detecting HGAIN in a cohort of HIV-infected MSM.
The Screening and Treatment of Anal Dysplasia Unit was created in May 2009 in the University Hospital Vall d’Hebron (Barcelona, Spain). All outpatients MSM with HIV infection attending the HIV Units from this hospital and the University Hospital of Mar (Barcelona, Spain) were informed about the screening program and participation was advised. All HIV-infected patients included gave informed consent to use the information available in the database and medical records. The study was approved by the Commission of Medical Ethics of Hospital Vall d’Hebron.
Study variables and data collection
From each patient, the following variables were recorded: sociodemographic data (age, current tobacco smoking, country of origin); HIV-infection-related data (date of HIV diagnosis, AIDS-defining illnesses, hepatitis C virus antibodies, nadir and current CD4+cell count, zenith and actual HIV viral load, HAART, and time on HAART); sexual behavior data (number of sexual partners, age at which individuals became sexually active, stable sexual partner, use of condom, and history of sexually transmitted diseases); and results of the screening program (anal cytology, HPV detection, HRA, and histology results).
Anal dysplasia screening procedure
Our institution's anal dysplasia screening program includes anal liquid cytology and HPV testing at each visit, followed by a digital anorectal examination and HRA. The anal cytology, HPV testing, and HRA are performed simultaneously; thus, the results of each intervention are not known at the time of performance. For this study, cytology was obtained by introducing a Dacron swab 3–5 cm into the anal canal and softly rotating it for 30 s. The swab was then introduced into 20 ml of PreservCyt/ThinPrep Pap test solution (Cytyc Iberia SL, Barcelona, Spain) and shaken for 30 s. This sample was used to perform the cytological analysis and HPV testing. Cytology results were classified as normal, atypical squamous cells of uncertain significance (ASCUS), HSIL, atypical squamous cells – cannot exclude HSIL (ASC-H), low-grade squamous intraepithelial lesion (LSIL), or squamous-cell carcinoma (SCC), in accordance with the Bethesda classification system . HRA was performed by using a technique described previously , including the topical application of acetic acid and Lugol's solution in the anal canal, in accordance with the IANS International Guidelines . Moreover, the entire circumference of the squamocolumnar junction was carefully visualized. The distal anal canal was examined as the anoscope was being withdrawn, and then the perianal area was examined after additional acetic acid was applied. Anal biopsies were performed on lesions suspicious for HGAIN as revealed by HRA. Suspicious lesions were defined as those having an abnormal surface configuration (flat leukoplakia, hyperkeratotic leukoplakia), abnormal tinctorial characteristics (acetowhite or Lugol negative), or an atypical vascular profile (punctuation, mosaicism, and atypical vessels). If no changes suggestive of high-grade lesions were noted based on HRA, but ASC-H or HSIL cytology suggested the presence of a lesion, a new HRA was conducted to perform random biopsies of the squamocolumnar junction. This procedure is in accordance with IANS recommendations . Following the histology examination, anal samples were classified as normal, low-grade anal intraepithelial neoplasia (LGAIN), HGAIN, or SCC .
The follow-up of patients included visits (with anal cytology, HPV determination, and HRA examinations) at 6–12 month intervals depending on prior results.
Human papillomavirus detection and typing
DNA was extracted from cell suspensions (in PreservCyt/ThinPrep solution) by using the ‘QIAsymphony DSP Virus/Pathogen Kit’ (QIAGEN, Hilden, Germany). The quality of DNA was tested for amplification of an actin gene, and specific sequences of papillomavirus were amplified by using ‘CLART Genomica HPV2’ (Madrid, Spain), in accordance with the manufacturer's instructions. This kit enables the detection of 35 HPV types (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 68, 68, 70, 71, 72, 73, 81, 82, 83, 84, 85, and 89).
To assess the utility of oncogenic HPV testing, anal cytology, and cotesting of HPV and anal cytology for detecting HGAIN, sensitivity, specificity, positive, and negative predictive values were calculated by using the histological results of HRA-guided biopsy as the reference test for HGAIN diagnosis. If multiple biopsies were taken, screening status was designated by the consensual diagnosis of the most severe histological lesion observed. If HRA results normal and cytology appeared normal, LSIL or ASCUS, no biopsy was taken and the patient's status was considered to be ‘free of HGAIN’. However, if the visual HRA was described as normal but ASC-H or HSIL cytology was obtained, a subsequent visit was conducted to perform HRA-guided random biopsies of the squamocolumnar junction.
An anal cytologic test result was defined as abnormal if ASCUS, ASC-H, LSIL, or HSIL was present. Oncogenic HPV testing results were considered positive if some of the HPV genotypes associated with the highest risk of malignancy were detected (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82) . Cotesting (combination of oncogenic HPV determination and anal cytology) results were considered positive if at least one of these test results were positive.
Analysis was performed at first screening visit as well as including all visits (first and follow-up visits) of patients participating in the screening program. In this second analysis, we did not consider the results of previous tests, so there is not an analysis of repeated screenings. We also assessed the utility of oncogenic HPV determination to detect HGAIN in patients with specific (ASCUS or LSIL) cytology.
Categorical variables are described as numbers (percentages) and continuous variables as median (interquartile range, IQR) unless otherwise specified.
IBM SPSS statistics software for Windows (Version 19.0; IBM, Armonk, New York, USA) was used for the statistical analyses.
From May 2009 to August 2016, 692 HIV-infected MSM underwent a total of 1827 anal cytologies, 1841 HRA examinations, and 1607 HPV testings. Anal cytology and HPV testing were not performed because of problems in sample obtaining and/or processing in 0.8 and 12.7% of screening visits, respectively. The median age of patients was 40.8 (IQR: 33.7–47.2) years, with a median time since HIV infection diagnosis of 5 years; 86.5% were receiving HAART. The median CD4+ cell count was 600 cells/μl, and 76.4% of patients had an HIV load below the level of detection.
With regard to sexual habits, 50.3% of study participants had a current, stable sexual partner, the median number of different lifetime sexual partners was 150 (IQR: 50–400), and 63% reported previous diagnosis of a sexually transmitted disease other than HIV. The characteristics are summarized in Table 1.
Prevalence of abnormal cytology, human papillomavirus infection, and histological dysplasia
At the first screening visit, anal cytology results were normal in 267 patients (38.6%), LSIL in 204 (29.5%), ASCUS in 187 (27%), and ASC-H or HSIL in 27 (3.9%); seven (1%) cytologies were not evaluable. HPV genotypes were detected in 561 (92.6%) of 606 patients who underwent HPV testing, with multiple HPV types in 47.7% and median of two genotypes (IQR: 1–3). Oncogenic HPV genotypes were found in 482 patients (79.5%), HPV 16 in 176 (33.3%), and HPV 18 in 62 (11.7%). Of the 692 patients, in 198 of them anal biopsies were performed at the first visit. The biopsies were abnormal in 65.6% of cases, with LGAIN in 23.7%, and HGAIN in 41.9%. No invasive cancers were identified.
Of patients with confirmed HGAIN at first visit, nine of 83 (10.8%) had normal cytology results, seven of 77 (9.1%) had negative oncogenic HPV test results, and two of 70 (2.9%) had normal cytology and negative oncogenic HPV test results. In the group of patients with normal cytology results, 69.9% had amplification of oncogenic HPV types; therefore, only 12.4% of all patients had negative results for both tests (cytology without dysplasic changes and no oncogenic HPV detection).
Test characteristics of screening strategies
Among HIV-infected MSM at baseline, abnormal anal cytology had a sensitivity of 89.2% and specificity of 44.2% for detecting HGAIN, whereas oncogenic HPV testing showed a sensitivity of 90.9% and specificity of 24.4%. The sensitivity and specificity of HPV 16 and/or 18 detection were 56 and 63%, respectively. When a combination of any abnormal cytology findings and/or oncogenic HPV test results were used as a predictor of HGAIN, the sensitivity was 97.4% and specificity was 14% (Table 2). The test characteristics from different techniques did not differ when all screening visits were included (Table 3).
The usefulness of HPV testing in patients with a specific cytology at baseline was as follows: 161 men had anal cytology results that showed ASCUS and underwent HPV testing; 74.5% of these patients tested positive for oncogenic HPV. Altogether, 23 men with ASCUS cytology had biopsy-proven HGAIN, and 21 of these were oncogenic HPV-positive. Therefore, in patients with ASCUS cytology, oncogenic HPV testing showed a sensitivity of 91.3% and specificity of 28.3% in detecting HGAIN (Table 2).
Similarly, of the 117 patients with LSIL cytology who underwent HPV testing, 32 had HGAIN and 30 had oncogenic HPV. Thus, the sensitivity of oncogenic HPV testing was 93.7% and specificity was 9.4% (Table 2). These data did not differ when we considered results from all visits, not only those from the first visit, in the analyses (Table 3).
There is some debate about the optimal screening strategy to detect HGAIN in high-risk patients. Although there are no worldwide recommendations made, the most accepted strategy suggests a sequential process beginning with an anal cytology examination and followed by HRA for patients found to have abnormal cytology results [5–8]. The potential usefulness of HPV testing in these screening strategies has not been adequately studied; therefore, here we have evaluated the utility of anal liquid cytology, HPV testing, and the two tests combined, for detecting HGAIN in HIV-infected MSM.
In our study, abnormal anal cytology showed a sensitivity of 89.2% and specificity of 44% for detecting HGAIN. Similarly, Salit et al. observed a sensitivity of 84% and specificity of 39%. A recent meta-analysis confirms these results, reporting a sensitivity for abnormal cytology of 90% and specificity of 33% for HGAIN diagnosis [11,19]. Anal cytology has some important advantages over HRA: it is simpler to perform, cheaper, less time-consuming, and requires less expertise. Indeed, it seems to be useful for screening of high-risk individuals, especially when performed on yearly basis, as the sensitivity of cytology improves when it is performed repeatedly during the follow-up of these patients. However, the lower reproducibility and lesser sensitivity than those of cervical cytology, as well as the common discrepancies observed with histologic findings, explain why alternative techniques are being investigated to improve the diagnostic accuracy of anal dysplasia [8–11].
Data from our study showed that amplification of oncogenic HPV as a primary test has a sensitivity of 90.9% and specificity of 24.4% for diagnosis of high-grade anal disease. Salit et al. found a sensitivity of 100% (all patients with HGAIN had oncogenic HPV), but a specificity of 16%. Other studies on HIV-infected MSM found that oncogenic HPV testing had a sensitivity range from 80.4 to 88.8% and a specificity from 21.8 to 44% [20–23]. Taking these data together, it appears that oncogenic HPV testing has no greater diagnostic accuracy compared with anal cytology in these high-risk patients.
If we consider that some specific cytology results can provide more information than HPV testing (e.g., HSIL cytology results have shown higher specificity than those of HPV 16 and/or 18 testing for diagnosing HGAIN), the utility of anal cytology is shown to be even greater.
When used HPV testing as an adjunct test to anal cytology, we observed a sensitivity of 97.4% and specificity of 14% for the detection of HGAIN. Hidalgo-Tenorio et al. observed that combined use of the two tests had a sensitivity of 100% and specificity of 16.2% in HIV-infected MSM. In another study, Berry et al. observed similar results. Overall, whereas cotesting with anal cytology and HPV testing has shown a high sensitivity for detection of anal dysplasia, its specificity has been low. As only 12% of the patients have negative results for both tests, in clinical practice, clinicians should refer most patients for HRA. Thus, cotesting does not provide benefits with respect to the protocol proposed by some authors to directly refer all patients for HRA without previous screening tests [24–26]. Moreover, performing the two tests substantially increases costs .
It has been postulated that using HPV testing for patients with ASCUS cytology could be beneficial in reducing the need for HRA [3,27] in cases in which patients have ASCUS cytology but negative oncogenic HPV test results. Thus, for HPV testing to be useful, it would be necessary for a significant number of patients to have negative oncogenic HPV test results and for the incidence of false negative tests to be low (high negative predictive value). One large study of 300 HIV-infected MSM with ASCUS cytology reported a 72% prevalence of oncogenic HPV genotypes and an 88% negative predictive value of HPV testing for HGAIN detection . In a study by Salit et al., 42 of 59 HIV-infected MSM with ASCUS cytology had positive oncogenic HPV test results, whereas all HGAIN patients had positive oncogenic HPV test results (negative predictive value of 100%). In our study, the prevalence of oncogenic HPV infection in patients with ASCUS cytology was 74.5%, and the negative predictive value was 95.1%. Therefore, the percentage of overall patients who could have potentially avoided HRA (as they had ASCUS cytology and had negative oncogenic HPV test results) was low, around 6–8%. Moreover, the usefulness of HPV determination in patients with LSIL was even less, as the test had a lower negative predictive value.
We believe our study shows that oncogenic HPV testing in HIV-infected MSM has limited utility for anal dysplasia detection, and, although it does not substantially improve the accuracy of anal cytology as a screening technique, it does increase costs. The high prevalence of HPV infection and the great diversity of HPV genotypes that can cause anal dysplastic lesions in these high-risk individuals explains the low utility of oncogenic HPV testing in the screening of anal precancerous lesions. However, in other at-risk populations, such as in HIV-infected women or other immunocompromised patients, HPV testing could yield different results and might, therefore, have utility.
The major strengths of the current study are that it included a large number of patients, and that all patients had HRA testing performed and anal biopsies of lesions found. Moreover, HRA was performed without knowledge of the cytology and HPV testing results. The study was, however, subject to some limitations, including the fact that data were collected from a single center; thus, we cannot determine whether our findings could be generalized to other settings. Moreover, anal biopsies were only collected from patients with either abnormal HRA results or ASC-H/HSIL cytology. In an optimally designed study, anal biopsies (guided-HRA in cases of abnormal HRA results or random biopsies if normal HRA results) would be performed on all patients; however, it might be difficult to justify the collection of biopsies for research purposes in the absence of suspected lesions . In addition, factors that could influence the diagnostic value of different tests such as CD4+ cell count or HGAIN size should also be taken into account . Finally, the cost effectiveness of the different techniques was not evaluated.
In summary, our study assessed the value of different screening tests to detect HGAIN in HIV-infected MSM. Abnormal anal cytology and positive oncogenic HPV test showed similar sensitivity and specificity for HGAIN detection; however, the two tests used together showed improved sensitivity but lowered specificity, thus requiring referral of a majority of patients to HRA. By using oncogenic HPV test results in patients with ASCUS, cytology allowed reducing the number of patients referred to HRA, but only in a low number of patients. Therefore, in our opinion, HPV testing does not improve HGAIN detection and, therefore, should not replace anal cytology as a screening technique in HIV-infected MSM. These results contribute to the knowledge regarding screening methods and might add valuable information to the discussion of guidelines for screening anal dysplastic lesions. Further studies are needed, however, to clarify the cost-effectiveness of these screening strategies.
Role of authors: J.B. and A.C. had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
Author contributions were as follows: Design and conduct of the study: J.B. Collection, management of the data: J.B. and A.C. Analysis and interpretation of data: J.B., A.C., and V.F. Preparation of the article: J.B., A.C., J.H.-L., S.L., A.G., MªC.D., J.V., J.N., and V.F. Review and approval of the article: J.B., A.C., J.H.-L., S.L., A.G., MªC.D., J.V., J.N., E.R., and V.F. Decision to submit the article for publication: J.B., A.C., J.H.-L., S.L., A.G., MªC.D., J.V., J.N., E.R., and V.F. All authors approved the final version of the article.
No competing financial interests exist. This work was institutionally supported by SPANISH AIDS Research Network RD16/0025/007 - ISCIII - FEDER.
Conflicts of interest
There are no conflicts of interest.
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Keywords:Copyright © 2017 Wolters Kluwer Health, Inc.
anal cytology; high-grade anal intraepithelial neoplasia; high-resolution anoscopy; human papillomavirus