Immunoreactivity of p16 in Anal Cytology Specimens. BACKGROUND. Cytology has been proposed as a potential screening tool in the

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1 66 CANCER CYTOPATHOLOGY Immunoreactivity of p16 in Anal Cytology Specimens Histologic Correlation Farbod Darvishian, M.D. 1 Elizabeth A. Stier, M.D. 2 Robert A. Soslow, M.D. 1 Oscar Lin, M.D. 1 1 Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York. 2 Department of Surgery, Gynecology Service, Memorial Sloan-Kettering Cancer Center, New York, New York. BACKGROUND. Cytology has been proposed as a potential screening tool in the evaluation of squamous anorectal disease in view of the morphologic similarities between anal and cervical squamous lesions. Previous studies have demonstrated that p16 overexpression correlates with the degree of dysplasia in the uterine cervix with promising results. Due to potential diagnostic pitfalls in anal cytology, p16 overexpression in these specimens was studied. METHODS. Patients with anorectal cytology who underwent follow-up biopsy within 1 year were selected. Forty-three anorectal cytologic specimens from 29 patients were selected. One slide of each case was destained. Avidin-biotin immunocytochemical studies with the monoclonal antibody CINtec p16 INK4a were performed. The results of the p16 immunostaining were correlated with the histologic findings. RESULTS. Twenty-eight of the 43 cases demonstrated the presence of squamous cells immunoreactive for p16 in cytology specimens. The p16-positive cells were identified in cases of low-grade squamous intraepithelial lesion (LSIL) (n 3 cases), high-grade squamous intraepithelial lesion (HSIL) (n 22 cases), and invasive squamous carcinoma (n 1 case), and in 2 cases with negative follow-up biopsies. No cell immunoreactive for p16 was found in 15 cases (5 benign cases and 10 cases with either LSIL or HSIL). The sensitivity and specificity of p16 immunoreactivity in the detection of anal intraepithelial neoplasia or carcinoma were 72% and 71%, respectively. The positive and negative predictive values were 93% and 33%, respectively. CONCLUSIONS. The presence of p16 immunoreactivity is a good predictor of dysplasia in anal specimens. However, the sensitivity and specificity of this marker are not high. Cancer (Cancer Cytopathol) 2006;108: American Cancer Society. KEYWORDS: anus, cytology, p16, immunocytochemistry. Address for reprints: Oscar Lin, M.D., Ph.D., Cytology Service, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., C-518, New York, NY 10021; Fax: (212) ; lino@ mskcc.org Received May ; revision received July ; accepted July Anorectal squamous disease is a common finding in human immunodeficiency virus (HIV)-infected patients. 1 7 Anal squamous cell carcinoma (SQC) is a rare tumor in the general population but is the fourth most commonly reported malignancy in men with HIV infection. 8 The exact pathogenesis of anal dysplasia and SQC is still unknown, although it is most likely similar to cervicovaginal lesions in women. Anal SQC has comparable histologic features with cervical SQC; it is frequently associated with squamous intraepithelial neoplasia and has been shown to have a strong association with human papillomavirus (HPV). 9 In fact, it has been reported that approximately 93% of HIV-positive men and 76% of HIV-positive women have anal HPV infection. 10,11 Overexpression of p16 has been proposed as a useful surrogate biomarker in the identification of squamous lesions harboring HPV 2006 American Cancer Society DOI /cncr Published online 10 January 2006 in Wiley InterScience (

2 p16 Immunoreactivity in Anal Cytology/Darvishian et al. 67 DNA. A previous study has demonstrated that the degree of p16 overexpression correlates with the degree of dysplasia in the uterine cervix. 12 In cervical carcinogenesis, the E6 and E7 oncogenes of HPV are reported to cause inactivation of the tumor suppressor gene protein products p53 and Rb. 13 In cervical lesions, the overexpression of p16 most likely is caused by increased levels of the transcription factor E2F-1. E2F-1 is released from the Rb protein after binding to the oncogenic E7 protein rather than Rb protein phosphorylation by cyclin-dependent kinases Cytology has been proposed as a potential screening tool in the evaluation of squamous anorectal disease because of the morphologic similarities between anal and cervical SQC and intraepithelial neoplasias. 17,18 Friedlander et al. 19 reported that anorectal cytology had a sensitivity of 92% in the detection of anorectal squamous dysplasia. Their findings further support the use of anorectal cytology in the evaluation of anal dysplasia, particularly in high-risk individuals. Despite its relatively high sensitivity and similarities, the same group has shown that diagnostic pitfalls exist. To address these potential pitfalls, we proposed to study p16 overexpression, a promising marker that has been previously studied in the uterine cervix. MATERIALS AND METHODS The patient population was selected from the files of the Cytology Service in the Department of Pathology at Memorial Sloan-Kettering Cancer Center over a period of 2 years. The criteria for selection were the following: patients with anorectal cytology who had undergone follow-up biopsy within 1 year. Forty-three anorectal cytologic specimens from 29 patients were selected. Twenty of the 29 patients were positive for HIV, represented by 17 male and 12 female patients. All HIV-positive females also had a history of gynecologic (vulvar, vaginal, or cervical) intraepithelial neoplasia. The patient ranged in age from years (mean, 44.8 yrs). Each of the 43 cytology specimens was comprised of 2 ThinPrep (Cytic Corporation, Marlborough, MA) slides stained with the Papanicolaou stain. One slide of each case was destained. Avidinbiotin immunocytochemical studies with the monoclonal antibody CINtec p16 INK4a (Dako Corporation, Carpinteria, CA), clone E6H4, were performed on destained slides. Immunocytochemical staining enhancement with heat epitope retrieval was performed using a regular vegetable steamer. The sections were placed in a solution of citrate buffer solution (ph 6.0), steamed for 30 minutes, and then cooled before immunocytochemical staining. The antigen antibody reaction was observed using 3,3 -diaminobenzidine as the chromogen. Known positive tissues were used as controls. The specimens were evaluated for the presence of cells with cytoplasmic and/or nuclear staining, which was considered a positive result. The results of the p16 immunostaining were correlated with the histologic findings. The histologic sections were classified as benign, low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesion (HSIL), and invasive SQC. RESULTS Twenty-eight of 43 cases showed the presence of squamous cells immunoreactive for p16 in the destained cytology specimens. The p16-positive cells were identified in 3 cases of LSIL (Fig. 1), 22 cases of HSIL (Fig. 2), 1 case of invasive SQC, and 2 cases with negative follow-up biopsies. The staining varied from weak to strong, with no correlation noted between the degree of dysplasia and the intensity of staining. No cells immunoreactive for p16 were found in 15 cases represented by 5 benign cases and 10 cases with either LSIL (Fig. 3) or HSIL. A summary of the results including the number of cases in each category are shown in Table 1. The presence of p16-immunoreactive cells in the cytology specimens demonstrated a sensitivity and specificity in the detection of anal intraepithelial neoplasia or carcinoma of 72% and 71%, respectively. The positive and negative predictive values (PPV and NPV) were 93% and 33%, respectively. DISCUSSION Anorectal cytology has been advocated as a screening tool for anal lesions in high-risk populations, such as HIV-positive men. The rationale for this test is based on the similarities to cervical disease and cost-effectiveness of the test. 9,10 Previous studies have described the cytomorphologic features and diagnostic limitations associated with this relatively new specimen type ThinPrep slide preparations appear to be more effective than conventional preparations. 19,21 Sherman et al. 21 reported that ThinPrep specimens detected nearly eight times as many SIL cases when compared with conventional smears. Darragh et al. 20 and Friedlander et al. 19 also demonstrated that Thin- Prep preparations containing a t-zone component detected anal intraepithelial neoplasia (AIN) more frequently than those that completely lacked a t-zone element. Previous studies have shown that the main diagnostic problem in anal cytology was the presence of atypical keratinized squamous cells. 19,21,23 Atypical keratinized cells are usually associated with a high suspicion for an abnormal keratinized lesion or SQC in cervicovaginal specimens, but in anal specimens such cells should be interpreted with care. The appearance

3 68 CANCER (CANCER CYTOPATHOLOGY) February 25, 2006 / Volume 108 / Number 1 FIGURE 1. Low-grade intraepithelial lesion. (A) ThinPrep specimen (Papanicolaou stain). (B) p16 immunoreactivity. (C) Histologic section (H & E). Original magnification 400 (A,B); 200 (C). FIGURE 2. High-grade intraepithelial lesion. (A) ThinPrep specimen (Papanicolaou stain). (B) p16 immunoreactivity. (C) Histologic section (H & E). Original magnification 400 (A,B); 200 (C)

4 p16 Immunoreactivity in Anal Cytology/Darvishian et al. 69 TABLE 1 Correlation of p16 Immunoreactivity in Cytology Specimens and Follow-Up Biopsies Histology p16 immunoreactivity Benign LSIL HSIL SQC Negative Positive LSIL: low-grade squamous intraepithelial lesion; HSIL: high-grade squamous intraepithelial lesion; SQC: squamous cell carcinoma. FIGURE 3. Low-grade intraepithelial lesion. (A) ThinPrep specimen (Papanicolaou stain). (B) Absent p16 immunoreactivity. (C) Histologic section (H&E). Original magnification 400 (A,B); 200 (C) of these keratinized cells can vary from benign to markedly atypical, and a false-positive diagnosis of squamous carcinoma can be made easily. Another potential pitfall is the association between reactive epithelial changes and SQC, especially in HIV-positive patients, who are prone to multiple infectious diseases such as herpes infection. Several authors have demonstrated that increased expression of high-risk HPV oncogenes in dysplasias of the female genital tract lead to an overexpression of p16 and its overexpression can be reliably studied by immunohistochemical studies. 12,24 33 Based on the same premise, the role of p16 as an adjunct marker also has been evaluated in cytology specimens, particularly liquid-based specimens The sensitivity and specificity of p16 overexpression in the detection of dysplastic lesions of the cervix have been reported to be as high as 99.9% and 100%, respectively. 40 Based on these findings, we proposed to study the role of p16 in anal cytology specimens. Our results demonstrated that p16 in anal cytology specimens had a high PPV in the detection of anal dysplasias, especially high-grade lesions, but a low NPV. Therefore, the presence of p16-immunoreactive cells was considered to be highly associated with dysplasia. The problem was that the absence of cells immunoreactive for p16 was not as diagnostically useful because there were several cases of dysplasia in which no cell immunoreactive for p16 was found. The current study results also demonstrated that p16 immunoreactivity was not restricted to HSIL because cells immunoreactive for p16 were found in three of six cases of LSIL and two cases with negative biopsies. It is noteworth that one of the cases with negative follow-up underwent a perianal biopsy showing HSIL approximately 1 month before the cytology specimen was taken. This patient most likey was infected with high-risk HPV and residual dysplasia at the time of the cytology sampling that resolved afterward. The lower sensitivity of p16 in anorectal cytology for the detection of dysplasias might be related to

5 70 CANCER (CANCER CYTOPATHOLOGY) February 25, 2006 / Volume 108 / Number 1 sampling because small lesions might be easily missed. Also, the lower sensitivity in the current study also might be related to the antibody used. The antibody used in this study was an antibody designed for use in cervical cytology specimens as described in their package insert and not anal specimens. Also, most prior studies in cervical cytology specimens employed antibodies from other manufacturers. 34,35,37,40 To our knowledge, the use of Hybrid Capture 2 (Digene, Gaithersburg, MD) HPV testing is not as well established in anal specimens as in cervical specimens in our institution. Therefore, we were unable to evaluate the HPV status in our patients and assess the correlation between p16 overexpression. Nonetheless, similar to cervical carcinoma, HPV infection has been shown to play a significant role in the development of anal condyloma, anal intraepithelial neoplasia, and anal carcinoma. 2,3,9,41 44 Lu et al. 45 already demonstrated the presence of p16 overexpression and the presence of HPV infection in all their cases of anal invasive squamous carcinoma. Varnai et al. 46 also reported a high incidence of HPV infection in anal squamous carcinoma and AIN using a polymerase chain reaction (PCR) methodology. The same group proposes PCR as the method of choice in the evaluation of HPV infection in anal specimens. The presence of p16 immunoreactivity is a good predictor of dysplasia in anal specimens. However, the sensitivity and specificity of this marker is not high. Additional studies are required to reach a definite conclusion regarding the role of p16 in anal cytology. REFERENCES 1. Palefsky JM, Holly EA, Ralston ML, et al. 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