Objective
The objective of the study was to assess the incidence of, and risk factors for, abnormal anal cytology and anal intraepithelial neoplasia (AIN) 2-3 in human immunodeficiency virus (HIV)–infected women.
Study Design
This prospective study assessed 100 HIV-infected women with anal and cervical specimens for cytology and high-risk human papillomavirus (HPV) testing over 3 semiannual visits.
Results
Thirty-three women were diagnosed with an anal cytologic abnormality at least once. Anal cytology abnormality was associated with current CD4 count less than 200 cells/mm 3 , anal HPV infection, and a history of other sexually transmitted infections (STIs). Twelve subjects were diagnosed with AIN2-3: 4 after AIN1 diagnosis and 4 after 1 or more negative anal cytology. AIN2-3 trended toward an association with history of cervical cytologic abnormality and history of STI.
Conclusion
Repeated annual anal cytology screening for HIV-infected women, particularly for those with increased immunosuppression, anal and/or cervical HPV, a history of other STIs, or abnormal cervical cytology, will increase the likelihood of detecting AIN2-3.
Anal cancer rates in human immunodeficiency virus (HIV)–infected individuals have continued to increase over the past decade despite the widespread use of highly active antiretroviral therapy (HAART). Among HIV-infected women, the risk for anal cancer is approximately 14 times higher than the risk among HIV-uninfected women. Squamous cell carcinoma of the anus shares biologic similarities with cervical cancer, including detectable precancerous lesions and persistent human papillomavirus (HPV) infection. It is likely that the pathogenesis of anal cancer is similar to that of cervical cancer: that is, anal HPV infection, in conjunction with other yet-to-be-determined factors, leads to the development of anal intraepithelial neoplasia (AIN) 2-3, a likely precursor to anal cancer. Much like cervical cytology is used to screen for cervical cancer, anal cytology can be used to screen for anal cancer. Individuals with abnormal anal cytology are referred for high-resolution anoscopy (HRA) (colposcopic evaluation of the anus) with directed biopsy for histologic diagnosis.
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There are limited longitudinal data describing anal HPV infection and intraepithelial neoplasia among HIV-infected women; in comparison, there have been numerous cohort studies providing extensive information on cervical HPV infection and cervical disease in HIV-infected women. The data on anal HPV infection and abnormal anal cytology in HIV-infected women have primarily focused on prevalent abnormalities. Among the studies in which HRA and biopsy were performed, histology results were available in only a small percentage of the study participants. Additionally, only 1 longitudinal study describing the incidence of abnormal anal cytology, which was conducted in the pre-HAART/early HAART era, has been published.
We have previously published the baseline data from our prospective cohort of 100 HIV-infected women assessing the prevalence of anal cytologic abnormalities and anal HPV infection as well as their relationship to abnormal cervical cytology and cervical HPV infections. At baseline, the prevalence of anal cytologic abnormalities was 17% and anal HPV was 16% in our cohort. Abnormal anal cytology was associated with cervical and anal HPV positivity, cervical cytologic abnormality, current and nadir CD4 count less than 200 cells/mm 3 , history of sexually transmitted infection (STI) (other than cervical/anal HPV), and alcohol use. Fourteen women (of 19 referred) underwent HRA, and AIN2-3 was detected in 3 (21%). We now present our longitudinal findings on the incidence and prevalence of anal cytologic abnormalities and AIN2-3 as well as the factors associated with abnormal anal cytology and AIN2-3 results.
Materials and Methods
This prospective, observational pilot study was conducted at the Center for Infectious Diseases (CID), the primary site of HIV care at Boston Medical Center (BMC), an inner-city safety net hospital in Boston, MA. English-speaking, HIV-infected women between the ages of 18 and 64 years who had not had a cervical or anal cytology test, colposcopy, or HRA in the 6 months prior to enrollment were eligible to participate.
The exclusion criteria included pregnancy, use of chronic anticoagulation medication, and life expectancy of less than 1 year. The study participants were recruited by the nurse practitioner in the CID from HIV-infected women scheduled for gynecology care (including cervical cytology). The protocol for the study was approved by the BMC Institutional Review Board.
After obtaining written informed consent, each subject provided a detailed history on routine gynecological health care and risk factors for the development of anal cytological abnormalities. The details of this questionnaire have been published previously. History of STI other than cervical or anal HPV was recorded based on patient self-report and included gonorrhea, Chlamydia, trichomonas, herpes, syphilis, and genital warts. Additional medical and laboratory data were collected from the electronic medical record including the most recent cervical cytology results prior to enrollment, CD4 T-cell count, and HIV viral load performed within the prior 6 months.
Following the questionnaire and history, a visual examination of the lower genital tract was performed, and samples were collected for cervical and anal cytology and HPV testing with Hybrid Capture 2 (HC2) (QIAGEN Corp, Gaithersburg, MD). Cervical cytology specimens were collected in the standard fashion and processed using the BD SurePath preservative vial (Becton, Dickinson and Co, Franklin Lakes, NJ). The anal cytology specimens were collected with a small polyester swab soaked in tap water and gently inserted until resistance from the wall of the rectum was met (approximately 4.5 cm). The swab was then withdrawn with lateral pressure, using a spiral motion to sample the entire circumference of the anal canal. The swab was processed using the BD SurePath preservative vial (Becton, Dickinson and Co).
After the cytology testing at the BMC Pathology Laboratory was performed, the residual from the cytology specimen was run for HC2. The HC2 assay assesses the presence of 13 types of high-risk associated HPV (HR-HPV) infection (HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68). HC2, however, has not been validated for the detection of HR-HPV in the anus.
Follow-up was determined based on the anal and cervical results. Women with cervical cytological abnormalities: low-grade squamous intraepithelial lesion (LSIL) and high-grade squamous intraepithelial lesion (HSIL) with or without detection of HR-HPV were referred for colposcopic examination, which involved microscopic visualization of cervical lesions with biopsy. Women with a cytology test showing atypical squamous cells of unknown significance (ASCUS) and an HR-HPV were also referred for colposcopic examination. Women with cervical HR-HPV and normal cytology were not assessed with colposcopy but were recommended for repeat cytology and HR-HPV testing in 6 months.
Subjects with any grade of anal cytological abnormality or anal HR-HPV were referred for examination with HRA. (Because the utility of screening for anal intraepithelial neoplasia based on anal HPV alone in HIV-infected women is unknown, the protocol was amended during the study to refer participants with anal HR-HPV detected by HC2 for HRA, regardless of the anal cytology result. Only 4 women with anal HPV diagnosed at the baseline visit were not referred for HRA prior to this change in protocol.)
HRA was performed using a lubricated plastic disposable anoscope and applying 5% acetic acid to the anal mucosa and anoderm. An examination was then performed under colposcopic visualization of the anal canal from the dentate line to the anal verge. Identified lesions (with acetowhite changes, punctuation, abnormal vascularity, and masses) were biopsied under direct visualization with a mini-Tischler biopsy forceps after application of local anesthetic. All colposcopy and HRA procedures were performed by a gynecologist (E.A.S.) with expertise in colposcopy and HRA.
Participants identified with high-grade intraepithelial neoplasia of the anus (AIN2-3) cervix (CIN2-3), vagina (VaIN2-3), or vulva (VIN2-3) were referred for treatment as recommended clinically. Subjects with no evidence of AIN2-3 or CIN2-3 were scheduled to be followed every 6 months with cervical and anal evaluations for cytology and HPV for a total of 3 visits, with follow-up colposcopy or HRA as determined by the study protocol. The 6-month follow-up interval was selected based on prior published studies of anal cytologic abnormalities in HIV-infected women.
It was estimated that a sample size of 100 women would be feasible for this pilot study to complete enrollment over 6 months. Because the objective of the analysis was to compare women who were diagnosed with abnormal anal cytology at any study visit with women who had normal anal cytology at every visit attended, we performed 2 sets of univariate and multivariate logistic regression analyses comparing women with abnormal anal cytology at any visit with the following: (1) women with normal anal cytology at every visit attended (which included women lost to follow-up after the first or second study visit) and (2) those who had normal anal cytology at all 3 study visits (not lost to follow-up). Because the results were similar, regardless of the number of visits, the analysis including all subjects with normal anal cytology at every visit attended, regardless of number of visits, is presented to preserve statistical power.
Categorical variables were assessed using a Fisher exact test, and continuous variables were assessed with a Student t test for independent samples. Multiple logistic regression analyses were performed using stepwise forward selection with a cutoff P < .10 for inclusion in the final model. Predictors of interest were chosen based on a cutoff P < .10 in univariate modeling and included current CD4 count, nadir CD4 count, current HAART use, history of STI (other than cervical/anal HPV), cigarette smoking, and cervical or anal HR-HPV positive test result. Because cervical HPV and cervical cytologic abnormalities were closely related, only cervical HR-HPV was included in the multivariate model because cervical HR-HPV was more strongly associated with anal cytologic abnormalities in univariate results.
We also assessed factors associated with the AIN2-3 diagnosis using unadjusted odds ratios. Because only women with abnormal anal cytology and/or anal HPV were eligible for HRA in this study, we felt the associations between HRA and abnormal anal cytology and anal HPV would be biased and thus did not include these 2 factors in this analysis. All analyses were performed in SAS version 9.1 (SAS Institute, Inc, Cary, NC). Final results are interpreted using an α= 0.05 level of significance.
Results
One hundred women were enrolled in the study between October 2006 and May 2007 and followed up for completion of HRA through April 2010: 87 were followed up for at least 2 visits, and 71 took part in all 3 study visits. Approximately 150 women were seen for a gynecologic visit with the nurse practitioner during the period of enrollment, and about 17% of those patients spoke a language other than English for a recruitment of approximately 80% of potentially eligible women.
For the 87 women followed up for more than 1 visit, the median length of follow-up, including time to final HRA if performed, was 704 days (range, 73–1154 days). Thirteen of 87 participants with at least 2 study visits (15%) took more than 1 year to return for study visit 2. Sixty-three of 71 participants with 3 visits (89%) took more than 1 year to return for study visit 3. The study population has been described in detail elsewhere. In brief, 78% of subjects were black, 56% were born outside the United States, and the median age was 40 years (range, 22–57 years). Eleven had a baseline CD4 count of 200 cells/mm 3 or less, 62% had an undetectable HIV viral load, and 79% were on HAART defined as 3 or more active HIV drugs. Only 5 women reported having undergone a prior anal cytology test. Twenty-two percent reported a history of receptive anal intercourse. Ten percent of women had a prior history of VIN and 27% had a history of treatment for CIN. Twenty-three percent of the women were active cigarette smokers, whereas 15% had a history of intravenous drug use.
Thirty-three of 99 women (33%) were diagnosed with an anal cytologic abnormality at least once. One subject had inadequate anal cytology for analysis at the initial visit and was lost to follow-up; therefore, 99 subjects are included in the analysis. The anal cytology results at each study visit, censored after initial abnormal anal cytology diagnosis, are shown in the Figure . HPV results for women with concurrent normal cytology are shown in the Figure . Anal HPV was detected in 22 of the 33 women (67%) with abnormal anal cytology at some time point during follow-up. The highest grade of anal cytologic abnormality was ASCUS in 20 (61%) and LSIL in 13 (39%). There were no HSIL anal cytology results.
Among the 70 women (of 82) with a normal baseline anal cytology result who were followed up for more than 1 visit, 16 (23%) had an abnormality diagnosed on a repeat cytology test, 9 of whom (56%) had more than 1 prior negative anal cytology result. The incidence of anal cytology abnormality was 13.1 cases per 100 person-years of follow-up. Thirty-six of 99 women had a positive anal HPV test at 1 or more time points and 14 of those women (39%) had no anal cytologic abnormalities during the study. One individual had an AIN2 diagnosis after positive anal HPV testing and then had abnormal anal cytology testing after HRA. Forty-eight of 100 women had a positive cervical HPV test at any point during study follow-up (24 of 99 at baseline), and 30 (63%) had an abnormal cervical cytology result at least once during the study (21 of 100 at baseline).
Subject characteristics stratified by abnormal anal cytology diagnosis are shown in Table 1 . In an unadjusted analysis, anal cytology abnormality was associated with current or nadir CD4 count less than 200 cells/mm 3 , current HAART, cigarette smoking, a history of treatment for CIN, cervical cytology abnormality, and HPV detected in the cervix and/or anus ( Table 1 ). History of a STI other than cervical/anal HPV and nadir CD4 count less than 200 cells/mm 3 trended toward an association with anal cytologic abnormality.
| Variable | Abnormal anal cytology, n = 33 n (%) | No abnormal cytology, n = 66 n (%) a | Univariate OR (95% CI) | Multivariate OR (95% CI) |
|---|---|---|---|---|
| Age (y), mean years ± SD | 42.3 ± 8.5 | 40.0 ± 7.8 | 1.04 (0.98–1.1) | |
| Race | ||||
| Black | 26 (79) | 52 (79) | Reference | |
| White | 6 (18) | 11 (17) | 1.1 (0.4–3.3) | |
| Hispanic | 1 (3) | 3 (5) | 0.7 (0.1–6.7) | |
| United States born | 17 (52) | 26 (39) | 1.6 (0.7–3.8) | |
| HIV diagnosed ≥5 years before enrollment | 24 (73) | 39 (59) | 1.8 (0.7–4.6) | |
| Current CD4 count <200 cells/mm 3 | 9 (27) | 2 (3) | 12.0 (2.4–59.6) | 12.8 (2.0–82.0) |
| Nadir CD4 <200 cells/mm 3 (n = 97) | 17 (52) | 20 (31) | 2.3 (1.0–5.5) | NS |
| Viral load <75 copies/mL | 20 (61) | 42 (64) | 0.9 (0.4–2.1) | |
| Current HAART | 31 (94) | 47 (71) | 6.3 (1.4–28.8) | NS |
| Age first coitus ≤15 y | 15 (45) | 19 (29) | 2.1 (0.9–4.9) | |
| ≥6 lifetime partners | 16 (48) | 29 (44) | 1.2 (0.5–2.8) | |
| History of anal sex | 9 (27) | 13 (20) | 1.5 (0.6–4.1) | |
| History of STI (other than cervical or anal HPV) | 24 (73) | 35 (53) | 2.4 (0.95–5.8) | 3.6 (1.1–11.5) |
| Cigarette smoking | 12 (36) | 11 (17) | 2.9 (1.1–7.5) | NS |
| Alcohol use | 11 (33) | 14 (21) | 1.9 (0.7–4.7) | |
| IVDU | 7 (21) | 7 (11) | 2.3 (0.7–7.1) | |
| Last cervical Papanicolaou prior to study (n = 83) | ||||
| NILM | 18 (72) | 52 (90) | Reference | |
| ASCUS | 1 (4) | 2 (3) | 1.4 (0.1–16.9) | |
| LSIL | 5 (20) | 4 (7) | 3.6 (0.9–14.9) | |
| HSIL | 1 (4) | 0 (0) | — | |
| History of treatment of cervical dysplasia | 14 (42) | 13 (20) | 3.0 (1.2–7.5) | NS |
| Any cervical abnormality at any visit (HPV or cytology) | 25 (76) | 31 (47) | 3.5 (1.4–9.0) | b |
| Cervical HPV positive at any visit | 24 (73) | 24 (36) | 4.7 (1.9–11.7) | NS |
| Cervical cytological abnormality at any visit | 19 (58) | 19 (29) | 3.4 (1.4–8.0) | b |
| Anal HPV positive any visit | 22 (67) | 14 (21) | 7.4 (2.9–18.9) | 6.2 (2.2–16.9) |
a One observation had inadequate anal cytology and was excluded;
b Because cervical HPV and cervical cytologic abnormalities were closely related, only cervical HPV positivity was included in the multivariate model based on univariate results.
In multivariate modeling, anal cytologic abnormality was associated with current CD4 count less than 200 cells/mm 3 (odds ratio [OR], 12.8; 95% confidence interval [CI], 2.0–82.0), anal HPV infection (OR, 6.2; 95% CI, 2.2–16.9), and a history of an STI other than cervical/anal HPV (OR, 3.6; 95% CI, 1.1–11.5) ( Table 1 ). A cervical HPV positive test result, cigarette smoking, current HAART, nadir CD4 count less than 200 cells/mm 3 , and a history of treatment for cervical dysplasia were not significant and therefore were not included in the final multivariate model.
Forty-seven women were diagnosed with abnormal anal cytology or anal HPV infection at any study visit, and 36 of those women (77%) underwent high-resolution anoscopy. Twelve of 36 women who underwent HRA were diagnosed with AIN2-3 (33%). Cytology and histology results of those diagnosed with AIN2-3 are summarized in Table 2 .
| # | Visit 1 | Visit 2 | Visit 3 | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Anal cytology result | Anal HPV | HRA result | Days to HRA result a | Anal cytology result | Anal HPV | HRA result | Days to HRA result a | Anal cytology result | Anal HPV | HRA result | Days to HRA result a | |
| 1 | LSIL | – | AIN2 | 90 | ||||||||
| 2 | LSIL | + | AIN3 | 41 | ||||||||
| 3 | LSIL | + | AIN3 | 125 | ||||||||
| 4 | NILM | – | N/A | N/A | NILM | + | AIN2 | 363 | ||||
| 5 | ASCUS | + | AIN1 | 52 | ASCUS | + | AIN2 | 283 | ||||
| 6 | ASCUS | + | Refused | N/A | LSIL | + | AIN2 | 615 | ||||
| 7 | LSIL | – | AIN1 | 30 | LSIL | – | AIN2 | 219 | ||||
| 8 | LSIL | – | AIN1 | 30 | ASCUS | + | AIN2 | 240 | ||||
| 9 | ASCUS | + | Refused | N/A | ASCUS | + | AIN3 | 346 | ||||
| 10 | NILM | – | N/A | N/A | LSIL | – | AIN3 | 265 | ||||
| 11 | ASCUS | – | AIN1 | 125 | NILM | – | N/A | N/A | ASCUS | – | AIN3 | 895 |
| 12 | NILM | + | Not referred b | N/A | ASCUS | + | NILM | 419 | LSIL | + | AIN3 | 1154 |
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