Human papillomavirus (HPV) has been associated with cancers of the cervix, vagina, vulva, anus, and oropharynx.1,21,2 It is a multifocal process affecting the anal and genital tract often simultaneously regardless of risk factors.3–53–53–5 Women with a history of cervical or vulvar dysplasia have an increased risk of developing anal cancer.6–86–86–8 Although screening for cervical cancer has long been considered the standard of care, little is known about anal cancer screening despite the growing number of new cases per year, the majority of these among women.9
Anal cancer and cervical cancer share biologic similarities leading to an interest in the use of cytology for anal cancer screening.10–1310–1310–1310–13 Although women with a history of HPV-related genital neoplasia are at risk for anal dysplasia and cancers, the role of screening remains unclear.4,7,8,144,7,8,144,7,8,144,7,8,14 The American Cancer Society released an anal cancer screening statement: “People at increased risk for anal intraepithelial neoplasia and cancer may benefit from screening. This includes…women who have had cervical cancer or vulvar cancer….”15
In a recent study, Lammé and colleagues16 found high-risk cervical HPV was associated with high-risk anal HPV and abnormal anal cytology, but they did not include women with a history of HPV-related genital cancers or vulvar dysplasia. The primary objective of this study was to assess the prevalence of abnormal anal cytology and high-risk anal HPV among women with a recent history of HPV-related genital neoplasia compared with women without a history of anogenital neoplasia.
MATERIALS AND METHODS
A cross-sectional cohort study was conducted at a large academic medical center in the Northeast. Women ages 18 years and older were recruited at the colposcopy clinic, gynecologic oncology office, and at routine gynecologic annual examination visits. Eligible participants were approached by a research assistant or clinician and offered entry into the study. To be considered eligible, participants had to be able to give informed consent in English or Spanish with interpreter assistance, as needed. Participants who opted for enrollment signed an informed consent in either English or Spanish. At study entry, participants were given a 10-question survey regarding their sexual behavior and risk factors for anal cancer. Demographics as well as relevant medical and surgical history were obtained from electronic medical charts. The study was approved by the Women & Infants Hospital institutional review board.
Women were considered to be high risk if they had: 1) a history within the prior 2 years of: atypical squamous cells of undetermined significance (ASC-US) cannot rule out high grade, low-grade squamous intraepithelial lesion cannot rule out high grade, or high-grade squamous intraepithelial lesion (HSIL) cervical cytology; 2) biopsy-proven cervical cancer or cervical intraepithelial lesion 2 or 3; 3) biopsy-proven vulvar cancer or vulvar intraepithelial lesion 2 or 3; or 4) biopsy-proven vaginal cancer or vaginal intraepithelial neoplasia 2 or 3. Women were considered low risk if they: 1) had a history of normal cervical cytology for the past 5 years; 2) never had cervical cancer or cervical intraepithelial lesion 2 or 3; 3) never had vulvar cancer or vulvar intraepithelial neoplasia 2 or 3; and 4) never had vaginal cancer or vaginal intraepithelial neoplasia 2 or 3 and were able to give informed consent with interpreter assistance as needed. Women were excluded if they were human immunodeficiency virus (HIV)–positive, were unable to give informed consent, or had a history of anal cancer or anal intraepithelial neoplasia.
All women had anal cytologic testing with thin-layer cytology. Specimens for anal cytopathologic examination were collected using a swab moistened with tap water and inserted 3–5 cm into the anus to the approximate depth of the squamocolumnar junction and then rotated while applying outward pressure and placed into a ThinPrep vial.11 All clinicians performing anal cytology had training consisting of a 15-minute PowerPoint presentation on anal cytology collection. A scheduled interim analysis one fourth of the way through recruitment was performed and we detected an abnormally high number of insufficient HPV results. At that time, it was noted that the swabs being used for anal cytopathologic examination were not Dacron but were cotton. All swabs were then switched to Dacron. All cytology samples were evaluated by two separate pathologists and the results entered in the electronic medical record. In the event of an abnormal anal cytology, participants were referred to a colorectal surgeon for high-resolution anoscopy regardless of cytologic abnormality. Anal biopsies were performed at the discretion of the colorectal surgeon.
Human papillomavirus typing was performed at the University of California, San Francisco laboratory using a complex multiplex real-time polymerase chain reaction test that simultaneously detects, types, and quantifies all 15 high-risk HPV types known to cause anogenital cancer. Human papillomavirus typing was run on the residual ThinPrep vial. The high-risk HPV subtypes tested were: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82. The test also detected and quantified the β-globin gene as an internal control, and this was used to determine the normalized viral load (viral load per cell) to eliminate sample variations. A standard curve was made with the amplified products for each of the 15 high-risk virus templates at the concentration range of 100–107. The crossing point fluorescence levels were plotted against the known standard concentration. Viral loads were determined based on the linear regression analysis of the standard curve. Multiple prior studies have shown this technique to have high reproducibility, sensitivity, and specificity.17–1917–1917–19 Anal HPV genotypes were collected for research purposes only.
Data analyses were performed using the statistical software package SAS 9.3. Categorical variables were compared by χ2 or Fisher's exact test. Continuous variables were compared by Student's t test or the nonparametric Wilcoxon rank-sum test. The Shapiro-Wilks test used to test for deviations from a normal distribution. Binomial 95% confidence intervals (CIs) were calculated for the prevalence of abnormal anal cytology and high-risk anal HPV. Crude and adjusted odds ratios (ORs) and 95% CIs were estimated by logistic regression. Variables that differed significantly between the high- and low-risk groups were included in the adjusted models. All P values presented were two-tailed with P<.05 considered statistically significant.
A sample size estimate was performed based on the primary outcome of abnormal anal cytology with a set power of 80% and an α (two-sided) of 5%. Assuming a prevalence of abnormal anal cytology in the high-risk group of 10%, based on prior studies and a very low prevalence of abnormal anal cytology in the general population, we determined an 8% difference would be clinically significant.7 This means we anticipated the low-risk group would have a 2% prevalence of abnormal anal cytology. To detect an absolute 8% difference, we determined that 348 women were needed in the high-risk group and 116 in the low-risk group.
Interim analyses were performed at 25%, 50%, and 75% recruitment. At the final interim analysis, the prevalence of abnormal anal cytology and the difference between groups was much larger than expected, so the research team stopped enrollment at that time. Three hundred three women were eligible and approached, 215 in the high-risk group and 88 in the low-risk group. Ninety percent of women approached consented to participate in the study, 190 women in the high-risk and 83 women in the low-risk group. Three women in the high-risk group were consented but did not have anal cytology or anal HPV testing performed and were excluded from the analyses.
As shown in Table 1, the median age was 47 years in the high-risk group and 57 years in the low-risk group (P<.001). The difference in age was in the younger than 30-year-old group; the low-risk group had fewer women younger than the age of 30 years. There was no difference by ethnicity between the two groups with the majority of women in both groups being non-Hispanic white. Women in the high-risk group were more likely to be current smokers (30.0%) than women in the low-risk group (12.1%) (P=.002), but there was no difference in having ever smoked between the two groups (P=.2). Twenty-seven percent of women in the high-risk group compared with 19% in the low-risk group had ever participated in anal intercourse (P=.3). There was no difference in the percent of women currently participating in anal intercourse between the two groups (P=.3). Among the high-risk group, 73.1% of women had a history of cervical cancer or dysplasia, 21.1% vulvar cancer or dysplasia, and 5.8% vaginal cancer or dysplasia (Table 1).
As shown in Table 2, 41.2% of women in the high-risk group had an abnormal anal cytology result compared with 21.7% in the low-risk group (P=.006). Approximately 10% of all samples were insufficient. The higher odds of abnormal anal cytology (compared with normal or insufficient) for the high-risk group (crude OR 2.50, 95% CI 1.37–4.56) remained statistically significant after adjustment for age and current smoking (adjusted OR 2.58, 95% CI 1.39–4.79). Nine women in the high-risk group had high-grade (HSIL or cannot rule out HSIL) cytology results compared with zero women in the low-risk group. Thirty-eight women (20.8%) in the high-risk group had high-risk HPV detected in the anal canal compared with only one woman (1.2%) in the low-risk group (crude OR 21.22, 95% CI 2.86–157.44, P<.001). Forty-six women (25.1%) in the high-risk group had insufficient high-risk HPV results compared with 28 (34.2%) in the low-risk group. Twenty-three women (12.6%) in the high-risk group had both an abnormal anal cytology result and high-risk HPV compared with zero women in the low-risk group (P<.001). In the high-risk group, of the 77 women with abnormal anal cytology, 38 tested negative for anal HPV, 15 had insufficient HPV results, and one was not completed. In the low-risk group, of the 18 women with abnormal anal cytology, 12 tested negative for HPV, five had insufficient HPV results, and one was not completed. Only eight women in the high-risk group and five women in the low-risk group had insufficient anal cytology and anal HPV testing.
All women with an abnormal anal cytology result were referred to anoscopy and 60.0% had anoscopy performed (64.9% in the high-risk group and 38.9% in the low-risk group). In the high-risk group, 28 (56.0%) women had biopsies performed at the time of anoscopy. Of these, three (10.7%) had normal anal biopsies and 25 (89.3%) had abnormal biopsies at the time of anoscopy. In the low-risk group, three (42.9%) had biopsies performed and none had dysplasia. The specific anal cytology and HPV results had no association with who went for anoscopy.
In the high-risk group, among those biopsied, 25 women had anal dysplasia or cancer identified compared with zero in the low-risk group (P=.03). Seventeen women in the high-risk group had anal intraepithelial neoplasia 1, five women had anal intraepithelial neoplasia 2, two women had anal intraepithelial neoplasia 3, and one cancer was identified. Eight women total had high-grade anal dysplasia (anal intraepithelial neoplasia 2 or 3) or cancer. Among these women, all had an abnormal anal cytology result and six also had high-risk positive HPV detected in the anal canal. The two women with negative anal high-risk HPV both had vulvar cancers. The anal cytology results varied among these eight women ranging from ASC-US to HSIL (three ASC-US, two low-grade squamous intraepithelial lesions, three HSIL). The one woman diagnosed with anal cancer had a history of vulvar cancer and an ASC-US anal cytology with negative anal high-risk HPV. Of the remaining seven women, two women had a history of vulvar cancer, one vulvar dysplasia, three cervical cancers, and one cervical high-grade dysplasia.
Women with a history of HPV-related genital neoplasia are at increased risk for anal dysplasia and cancers, but the role of anal cancer screening remains unclear.20,2120,21 We found a high percent of abnormal anal cytology and high-risk anal HPV among women with a history of genital neoplasia compared with women without a history of genital neoplasia. More importantly, of the high-risk women who underwent anoscopy, 4.2% had anal intraepithelial neoplasia 2 or worse detected, supporting the need for anal cancer screening in this population.
Women with a history of a genital neoplasia are at increased risk of developing anal cancer and may benefit from anal cancer screening. Similar to Ogunbiyi et al,14 we found a high rate of abnormal anal cytology (41.2%) among high-risk HIV-negative women compared with low-risk women (21.7%). Park et al and Lammé et al reported only 9% and 17.6% abnormal anal cytology, respectively, among women with high-grade lower genital tract intraepithelial neoplasia or cancer, but they both had lower risk populations.8,208,20 In addition, consistent with the literature, we found among our high-risk women who underwent high resolution anoscopy, 13.4% had anal dysplasia and eight (4.2%) had anal intraepithelial neoplasia 2 or greater.3,21,223,21,223,21,22 Although this is lower than that reported for other high-risk populations, it is similar to the 5% 5-year risk proposed by the American Society for Colposcopy and Cervical Pathology as a cutoff for referral to immediate cervical colposcopy.23
Human papillomavirus testing has been U.S. Food and Drug Administration–approved for primary screening in cervical cancer but has had limited use in anal cancer screening as a result of the high prevalence of HPV among at-risk populations.24–2624–2624–26 Recently, a systematic review reported the prevalence of anal high-risk HPV infection among HIV-negative women ranged from 4% to 36%.5 In our study, 20.8% of high-risk women had high-risk anal HPV detected compared with only 1.2% of low-risk women. Although 9.6% and 25.5% of our high-risk group had insufficient anal cytology and HPV testing, respectively, only 4.4% of women in this group had both insufficient anal cytology and HPV. Among those diagnosed with anal intraepithelial neoplasia 2 or worse, only two had negative high-risk HPV and both had vulvar cancers that were likely not HPV-related, suggesting a different method of screening among select women with vulvar cancer may be warranted. These findings suggest high-risk HPV testing plus anal cytology followed by referral to high-resolution anoscopy for all abnormal cytology results may be a useful anal cancer screening method. We are unable to determine the utility of referring women with a positive high-risk HPV test result and normal or insufficient anal cytology because we did not perform high-resolution anoscopy on these women in our study.
Anal cancer incidence among HIV-negative women has been increasing possibly as a result of increases in high-risk behaviors.27–2927–2927–29 However, similar to others, we found no difference in anal intercourse between groups supporting the HPV field effect concept.7,307,30 Other sexual behaviors including digital–anal sex have been associated with decreased anal HPV clearance rates and may not be captured in our study.26,2726,27
There were a number of study limitations. First, our study was an observational cohort study, and we cannot determine the sensitivity and specificity of anal cytology because only women with abnormal anal cytology were referred to anoscopy. Additionally, only 60% of those referred kept their appointment, leading to a selection bias. Second, we had a limited number of women younger the age of 30 years and this may be a lower risk group skewing the results.7 Another limitation was the high number of insufficient anal cytology and HPV results, which did not change with Dacron swabs. Although high numbers of insufficient anal cytology results were seen in other studies suggesting women may require repeat testing more frequently than with cervical cytology,10,1110,11 our high number of insufficient HPV results is possibly the result of performing HPV genotyping off of the ThinPrep vial cytology was performed on, so future studies may be improved by collecting separate samples. Finally, our study was conducted at a single institution, which limits the generalizability.
Our study is novel because it is one of the largest to date and included women with a history of cervical, vulvar, and vaginal dysplasia and cancer as well as had a low-risk comparison group. A recent study evaluated the prevalence of abnormal anal cytology and anal high-risk HPV among women with known cervical dysplasia, but this study did not include women with vulvar or vaginal dysplasia or women with a history of genital cancers, which is important because women with a history of vulvar neoplasia seem to be at highest risk for anal intraepithelial neoplasia 2 or greater.16
1. Bosch FX, Lorincz A, Muñoz N, Meijer CJ, Shah KV. The causal relation between human papillomavirus and cervical cancer. J Clin Pathol 2002;55:244–65.
2. Hampl M, Sarajuuri H, Wentzensen N, Bender HG, Kueppers V. Effect of human papillomavirus vaccines on vulvar, vaginal, and anal intraepithelial lesions and vulvar cancer. Obstet Gynecol 2006;108:1361–8.
3. Scholefield JH, Hickson WG, Smith JH, Rogers K, Sharp F. Anal intraepithelial neoplasia: part of a multifocal disease process. Lancet 1992;240:1271–3.
4. De Vuyst H, Clifford GM, Naciemento MC, Franceschi S. Prevalence and type distribution of human papillomavirus in carcinoma and intraepithelial neoplasia of the vulva, vagina and anus: a meta-analysis. Int J Cancer 2009;124:1626–36.
5. Stier EA, Sebring MC, Mendez AE, Ba FS, Trimble DD, Chiao EY. Prevalence of anal human papillomavirus infection and anal HPV-related disorders in women: a systematic review. Am J Obstet Gynecol 2015;213:278–309.
6. Edgren G, Sparen P. Risk of anogenital cancer after diagnosis of cervical intraepithelial neoplasia: a prospective population-based study. Lancet Oncol 2007;8:311–6.
7. Park IU, Ogilvie JW Jr, Anderson KE, Li ZZ, Darrah L, Madoff R, et al.. Anal human papillomavirus infection and abnormal anal cytology in women with genital neoplasia. Gynecol Oncol 2009;114:399–403.
8. Saleem AM, Paulus JK, Shapter AP, Baxter NN, Roberts PL, Ricciardi R. Risk of anal cancer in a cohort with human papillomavirus-related gynecologic neoplasm. Obstet Gynecol 2011;117:643–9.
10. Palefsky JM, Holly EA, Hogeboom CJ, Berry JM, Jay N, Darragh TM. Anal cytology as a screening tool for anal squamous intraepithelial lesions. J Acquir Immune Defic Syndr Hum Retrovirol 1997;14:415–22.
11. Kojic EM, Cu-Uvin S, Conley L, Bush T, Onyekwuluje J, Swan DC, et al.. Human papillomavirus infection and cytologic abnormalities of the anus and cervix among HIV-infected women in the study to understand the natural history of HIV/AIDS in the era of effective therapy (the SUN study). Sex Transm Dis 2011;38:253–9.
12. Sun XW, Kuhn L, Ellerbrock TV, Chiasson MA, Bush TJ, Wright TC Jr. Human papillomavirus infection in women infected with the human immunodeficiency virus. N Engl J Med 1997;337:1343–9.
13. Hillemanns P, Ellerbrock TV, McPhillips S, Dole P, Alperstein S, Johnson D, et al.. Prevalence of anal human papillomavirus infection and anal cytologic abnormalities in HIV-seropositive women. AIDS 1996;10:1641–7.
14. Ogunbiyi OA, Scholefield JH, Robertson G, Smith JH, Sharp F, Rogers K. Anal human papillomavirus infection and squamous neoplasia in patients with invasive vulvar cancer. Obstet Gynecol 1994;83:212–6.
16. Lammé J, Pattaratornkosohn T, Mercado-Abadie J, Alkhas A, Robinson A, Lanneau G. Concurrent anal human papillomavirus and abnormal anal cytology in women with known cervical dysplasia. Obstet Gynecol 2014;124:242–8.
17. Palefsky JM, Minkoff H, Kalish LA, Levine A, Sacks HS, Garcia P, et al.. Cervicovaginal human papillomavirus infection in human immunodeficiency virus-1 (HIV)-positive and high-risk HIV-negative women. J Natl Cancer Inst 1999;91:226–36.
18. Qu W, Jiang G, Cruz Y, Chang CJ, Ho GY, Klein RS, et al.. PCR detection of human papillomavirus: comparison between MY09/MY11 and GP5+/GP6+ primer systems. J Clin Microbiol 1997;35:1304–10.
19. Jiang G, Qu W, Ruan H, Burk RD. Elimination of false-positive signals in enhanced chemiluminescence (ECL) detection of amplified HPV DNA from clinical samples. Biotechniques 1995;19:566–8.
20. Cardinal LH, Carballo P, Lorenzo MC, García A, Suzuki V, Tatti S, et al.. Six-year experience with anal cytology in women with HPV in the lower genital tract: utility, limitations, and clinical correlation. Diagn Cytopathol 2014;42:396–401.
21. Santoso JT, Long M, Crigger M, Wan JY, Haefner HK. Anal intraepithelial neoplasia in women with genital intraepithelial neoplasia. Obstet Gynecol 2010;116:578–82.
22. Abramowitz L, Jacquard AC, Jaroud F, Haesebaert J, Siproudhis L, Pradat P, et al.. Human papillomavirus genotype distribution in anal cancer in France: the EDiTH V study. Int J Cancer 2011;129:433–9.
23. Massad LS, Einstein MH, Huh WK, Katki HA, Kinney WK, Schiffman M, et al.. 2012 updated consensus guidelines for the management of abnormal cervical cancer screening tests and cancer precursors. Obstet Gynecol 2013;121:829–46.
24. Palefsky JM. Anal cancer prevention in HIV-positive men and women. Curr Opin Oncol 2009;21:433–8.
25. Patel J, Salit IE, Berry MJ, de Pokomandy A, Nathan M, Fishman F, et al.. Environmental scan of anal cancer screening practices: worldwide survey results. Cancer Med 2014;3:1052–61.
26. Shvetsov YB, Hernandez BY, McDuffie K, Wilkens LR, Zhu X, Ning L, et al.. Duration and clearance of anal human papillomavirus (HPV) infection among women: the Hawaii HPV cohort study. Clin Infect Dis 2009;48:536–46.
27. Moscicki AB, Hills NK, Shiboski S, Darragh TM, Jay N, Powell K, et al.. Risk factors for abnormal anal cytology in young heterosexual women. Cancer Epidemiol Biomarkers Prev 1999;8:173–8.
28. Moscicki AB, Ma Y, Farhat S, Jay J, Hanson E, Benningfield S, et al.. Natural history of anal human papillomavirus infection in heterosexual women and risks associated with persistence. Clin Infect Dis 2014;58:804–11.
29. Howlader N, Noone AM, Krapcho M, Neyman N, Aminou R, Waldron W, et al.. SEER cancer statistics review, 1975–2009 (vintage 2009 populations). Bethesda (MD): National Cancer Institute. Available at: http://seer.cancer.gov/csr/1975_2009_pops09/
. Retrieved May 12, 2015.
30. Goodman MT, Shvetsov YB, McDuffie K, Wilkens LR, Zhu X, Thompson PJ, et al.. Sequential acquisition of human papillomavirus (HPV) infection of the anus and cervix: the Hawaii HPV Cohort Study. J Infect Dis 2010;201:1331–9.