- Email: [email protected]
Anal-Rectal Cytology and Anal Cancer Screening
Anal-Rectal Cytology and Anal Cancer Screening Mark Lane Welton, MD,†,§,* Barbara Winkler, MD,†,§,* and Teresa M. Darragh, MD†,§,* Over the past few decades there has been an alarming increase in the incidence of squamous cell carcinoma of the anus in both men and women. Immunocompromised patients and men who have sex with men are at especially high risk. Since its introduction by Dr. George Papanicolaou, cervical-vaginal cytology has been an extremely effective tool in decreasing the numbers of deaths in women from cervical cancer and has proven to be one of the great successes in preventative medicine. This article explores the efficacy of the use of analogous cytologic methods for the detection of precancers and cancers of the squamous epithelium in the region of the anal canal. The techniques for obtaining analrectal cytology are detailed with emphasis on the preparation and interpretation of cytologic samples. Criteria for specimen adequacy are discussed in the context of the Bethesda 2001 System. The triage of abnormal anal-rectal cytology and its correlation with anoscopic examination is also presented. Effective clinical-pathologic correlation requires close cooperation and communication between clinicians and pathologists. Clinicians and pathologists need to familiarize themselves with the technique of anal-rectal cytology to maximize the close surveillance and expectant management that are necessary for the prevention of anal carcinoma in high-risk patient populations. Semin Colon Rectal Surg 15:196-200 © 2004 Elsevier Inc. All rights reserved. KEYWORDS anal-rectal cytology, HPV, anal carcinoma, anal dysplasia
T
he incidence of anal cancer is increasing steadily. The American Cancer Society reported 4010 new cases for 2004, which is roughly equivalent to a rate of 1 case/100,000 in the general population.1 In contrast, in 2000 only 3400 cases of anal cancer were reported.2 The rate is higher in patients considered at high risk—the immunocompromised, men who have sex with men (MSM), and women with a history of cervical precancers or cancer.3-6 Early detection using cytologic screening has been an extremely effective method in the reduction of cervical cancer deaths. Because of numerous similarities between the tissues of the uterine cervix and the distal rectum and anus, analrectal cytology (ARC) is being proposed as a method for the early detection and prevention of anal cancer. Both anal and cervical cancers are squamous cell carcinomas primarily caused by the human papilloma virus (HPV), in particular HPV 16. Anal and cervical cancer arise within a defined zone
†From the Department of Surgery, Stanford University, School of Medicine, Stanford, CA. §Quest Diagnostics, Teterboro, NJ. *Departments of Pathology and OB/Gyn, University of California, San Francisco, San Francisco, CA. Address reprint requests to: Teresa M. Darragh, MD, UCSF/Mt. Zion Medical Center, Dept. of Pathology, 1600 Divisadero Street, Room B221, San Francisco, CA 94115. E-mail: [email protected].
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of squamous metaplasia, the transformation zone in the cervix, and a similar region in the distal rectum and anus, often referred to as the anal transition zone. These zones of immature squamous metaplasia in the cervix and anus are hypothesized to be at particular risk for infection with the HPV. The normal tissue trauma and repair in these transformation zones, as occurs with changes in vaginal pH or during sexual penetration, may facilitate HPV infection. In both the cervix and the anus, the development of cancer is preceded by highgrade squamous intraepithelial precursors that are essentially morphologically and biologically identical. In the perianal skin and anal mucosa, these squamous cancer precursors have been variably labeled as anal intraepithelial neoplasia (AIN) 2/3, moderate/severe dysplasia, carcinoma in situ, high-grade squamous intraepithelial lesions (HSIL), Bowenoid papulosis, and Bowen’s disease. A similar proliferation of diagnostic terms exists for histologically identical lesions of the vulva, vagina, and cervix. This multiplicity of diagnostic terms originates from the various disciplines of expertise in clinical medicine and pathology. However, the variety of diagnostic terminology should not lead to the misunderstanding that these are distinct disease states that should be treated differently or have differing etiologies— this is not the case. Despite the confusion in reporting terminology, the same cytologic and histologic criteria should be
Anal-rectal cytology and anal cancer screening applied when evaluating these tissues. All HPV-related lesions of the lower genital tract are morphologically similar; their appearance is more dependent on whether the lesions arise on a mucosal or skin surface. Familiarity with interpretation of cervical-vaginal cytology and histology should allow for interpretation of ARC. Despite all the similarities described above, anal cancer and cervical cancer stand in stark contrast in one regard, incidence. Cervical cancer is decreasing in incidence, while the incidence of anal cancer is increasing unchecked. The reduction in cervical cancer is due in large part to the introduction of the Papanicolaou (Pap) test about 50 years ago. Before the introduction of the Pap test in the United States, the incidence of cervical cancer was approximately 40/100,000 women. Cervical Pap smears and colposcopicdirected ablation of targeted lesions has resulted in a 75% decrease in incidence of cervical cancer to 8 to 10/100,000 cases per year. Anal cancer, in contrast, has increased 96% in men and 39% in women over the last 30 years. In HIVnegative and HIV-positive MSM the rates are especially high, 35/100,000 and 70/100,000, respectively.7,8 Given the impact of the Pap test on cervical cancer, several groups have been interested in the potential benefit of ARC in the identification and treatment of the premalignant anal lesions; low- and high-grade squamous intraepithelial lesions (LSIL and HSIL). A number of studies have demonstrated the efficacy and accuracy of screening with ARC for anal squamous intraepithelial lesions (ASIL) in MSM.9,10 The sensitivity of cytology for the detection of biopsy-proven SIL was found to be 69 and 47% in HIV-negative and HIV-positive MSM, respectively. With repeat ARC, the sensitivity improved to 81 and 50% for these groups.11 These results are similar to those seen in the uterine cervix. These data have led some to pursue studies of ARC in other patient populations. Early findings suggest that all patients with HIV infection are at substantial increased risk for anogenital infection with HPV and should benefit from screening.12,13 The clinical use of ARC is increasing in frequency in select centers but most clinicians and pathologists are largely unfamiliar with the methods used to obtain and interpret ARC samples. The following clinical protocol is a practical approach. Patients should be asked to refrain from anoreceptive intercourse for 24 hours before an anal cytologic examination. Lubricants should not be used by the care provider before obtaining a cytologic sample because the lubricant may interfere with the processing and interpretation of the cytologic sample. The sample may be obtained with the patient placed in the lateral decubitus, prone-jackknife, or lithotomy positions. A tap-water-moistened Dacron swab is then inserted approximately 5 to 6 cm into the anal canal and distal rectal vault. The swab handle is rotated 360° while firm lateral pressure is applied to the handle and it is slowly withdrawn from the anus. The lateral pressure insures that the mucosal surface, rather than rectal contents, is sampled. Inserting the tip of the Dacron swab above the dentate line insures adequate sampling of the distal rectal mucosa and metaplastic epithelium of the anal transition zone, the area at highest risk for infection by oncogenic HPV. A cytology sam-
197 ple that does not include cells from above the dentate line is not adequate for representation of the anorectal transformation zone; a sample of the perianal skin is not adequate. The cellular sample is either placed in a vial of liquid preservative, for liquid-based cytology, or smeared directly onto a glass slide and fixed as with conventional Pap smears. ARC sampling tends to be more difficult than cervical sampling because, while the cervix is directly visualized during Pap collection by using a speculum, the anal canal mucosa and distal rectum are not directly visualized when the cells are collected. With experience with the technique and feedback from the laboratory as to the adequacy of the sample, the specimen adequacy, and cellularity of ARC can equal that of cervical samples. Typically the pathologist finds a combination of rectal columnar cells, squamous metaplastic cells, nucleated squamous cells, and anucleated squames representing all tissues types encountered as the swab passes from proximal to distal in the anal canal. Liquid-based cytology is utilized with increasing frequency because of its ease of use and superior specimen preparation. Advantages of liquid-based cytology include increased cellular yield, improved cell preservation, decreased contamination with feces and bacteria, and decreased airdrying and mechanical artifact. Familiarity with the technique used by the laboratory is critical so that the clinical care provider prepares the sample correctly. Samples to be evaluated with ThinPrep should be placed in either CytoLyt or PreservCyt, depending on the laboratory’s preference. In the laboratory, the sample may be either processed as a nongynecologic specimen or processed similar to gynecologic samples. If processed as a nongynecologic specimen, the sample is placed in CytoLyt and the laboratory uses a blue filter and runs the sample on sequence No. 1 for superficial cells or sequence No. 3 for fluids/fine-needle aspirations. One of the authors (T.D.) prefers to place the anal sample in PreservCyt and processes the sample in a manner identical to cervical Pap tests (using the white filter and the gynecologic program, sequence No. 4 for gynecologic samples); this is an “off-label” use. If the specimen is to be analyzed with the TriPath PrepStain System, the sample must be submitted in CytoRich Red Fixative and processed using standard nongynecologic sample procedures. Labeling of the specimens with the correct CPT codes is critical to compensate the laboratory because third-party payers currently will not reimburse for ARC as a screening test or if the gynecologic codes are chosen. The CPT codes for nongynecologic samples are 88108 for liquid-based studies, 88104 for conventional samples (direct smears), and 88160 for anorectal smears with a Dacron sponge. Interpretation of ARC is guided by criteria established in the 2001 Bethesda System guidelines modified for anal-rectal samples. These morphologic criteria used in the interpretation of ARC are essentially similar to those used in the evaluation of gynecologic samples. The Bethesda System Atlas, published in 2004, provides guidelines for sample adequacy specific for ARC with a minimum cellularity established as 2000 to 3000 nucleated squamous cells for conventional smears or one to two per high power field (hpf) for ThinPreps
M.L. Welton, B. Winkler, and T.M. Darragh
198
Figure 1 LSIL (anal-rectal cytology, high power). (Color version of figure is available online.)
(diameter of 20 mm) and three to six nucleated cells/hpf for TriPath’s PrepStain System (diameter 13 mm) with a ⫻40 objective.14 The presence of metaplastic squamous cells or rectal columnar cells indicates that the anal transformation zone has been sampled and is reported as such. These criteria for specimen adequacy are similar to those used for gynecologic samples. LSIL are characterized by koilocytosis, and by the presence of superficial and high-intermediate squamous cells with an atypical nucleus (Fig. 1). Nuclear changes may include binucleation, enlargement, irregularities of the nuclear membrane, chromatin clumping, and hyperchromasia. Atypical parakeratotic cells are common and “empty halos” or degenerating koilocytes are frequently identified. HSIL have abnormal squamous cells that are of the lowintermediate or immature squamous metaplastic type; these cells are typically smaller than superficial and high-intermediate cells. There is a high nucleus to cytoplasmic (N/C) ratio with nuclear enlargement, although this enlargement can be subtle, and is paired with a wrinkled or irregular nuclear membrane (Fig. 2). The chromatin may be clumped or there may be chromatin margination and clearing in some nuclei. Nucleoli are inconspicuous. The high-grade cells may be in clusters or seen as single cells. They are not usually present in sheets or syncytia. These findings may be present in nonkeratinizing and keratinizing lesions. In the latter, atypical surface parakeratotic cells are seen. In our experience, invasive squamous cell carcinoma of the anus is difficult to prospectively diagnose on ARC; it may be difficult to accurately differentiate from a high-grade keratinizing lesion. Here, pairing ARC with the clinical examination is essential. The cytologic diagnosis of invasive squamous cell carci-
noma is more problematic in ARC than in cervical-vaginal samples because the typical tumor diathesis of invasion may be subtle or absent. The presence of bizarre atypical squamous cells, nucleoli, or profound nuclear chromatin changes provides clues to the possibility of invasion. Atypical parakeratosis may be a more prominent cellular component of the invasive ARC. Clinicians and pathologists should recognize that the prospective diagnosis of invasion may be unreliable with ARC and that clinically suspicious lesions should be extensively sampled to rule out invasion, regardless of the cytologic result. Reactive and reparative changes are less commonly seen in ARC than in gynecologic samples unless an infection that results in inflammation and ulceration, such as herpes simplex virus, is present. Samples with cells that do not meet the criteria for SIL but have atypical features such as nuclear enlargement without chromatin abnormalities are classified as atypical squamous cells— undetermined significance (ASC-US) or atypical squamous cells— cannot exclude HSIL (ASC-H). The major morphologic difference between these two categories of atypical squamous cells is cell size, cell maturation, and N/C ratio. The ASC-US cells are of intermediate or superficial type, while the ASC-H are smaller, metaplastic-type cells of low-intermediate or basal type with a higher N/C ratio. HPV testing has not been FDA approved for ARC and its role is yet to be defined in the triage of atypical ARC. The patient populations that are targeted for ARC screening have very high rates of anal HPV infection, potentially minimizing the utility of HPV testing for triage to highresolution anoscopy. All patients with ASC-US or higher on ARC should be referred for digital rectal examination and high-resolution
Anal-rectal cytology and anal cancer screening
199
Figure 2 HSIL (anal-rectal cytology, high power). (Color version of figure is available online.)
anoscopy (HRA) or, at the least, digital rectal examination and anoscopy with acetic acid.15 HRA is essentially equivalent to cervical colposcopy, with modifications for use in the distal rectum and anus.16 A cotton gauze pad is wrapped around the swab of a scopette and is soaked in acetic acid (3 to 5% may be used but 3% is preferred because the anus is somewhat acid-sensitive) and inserted into the anus (an anoscope may be used to facilitate this). The vinegar-soaked, gauze-covered scopette is allowed to saturate the distal rectal and anal mucosa for at least 1 minute. After the gauze is withdrawn, an anoscope is inserted and the anus is visualized through a magnification system such as a colposcope, operating microscope, or loops. A colposcope or operating microscope is preferred because loops have a lower resolution that may compromise identification of more subtle, yet important changes, that assist in the distinction between LSIL and HSIL. Both LSIL and HSIL have well-demarcated regions of the mucosa that whiten after the application of acetic acid, known as acetowhite epithelium (AWE). High-grade disease may be distinguished from low-grade lesions by architectural, morphologic, and vascular criteria. Low-grade lesions tend to be more architecturally exuberant with topographic changes that present as papillary or macular-papular growths or with cauliflower-like surface contours.16 High-grade lesions tend to be flat with characteristic vascular changes associated with the AWE. These vascular changes are characterized as punctate, mosaic, or “streaky.” Exophytic lesions may harbor HSIL deep within the lesion in areas that cannot be well-visualized and should be thoroughly sampled by biopsy. An exophytic lesion with vessel changes suggestive of HSIL should be considered suspicious for malignancy. Lesions with central ulceration and firmness or induration on
digital examination are also suspicious for invasive carcinoma. Deep biopsy is necessary for more definitive classification. The keratinized perianal skin may also be examined under magnification after the application of acetic acid. This procedure is especially useful in the patient with apparent circumferential acetowhitening as may be seen with “Bowen’s disease.” Identification of the regions most worrisome for HSIL allows targeting of those tissues for directed biopsy and treatment, reducing the number of biopsies and destruction of regions that harbor the less worrisome, yet still acetowhite, LSIL. The controversy surrounding the methods for treatment or monitoring of the HSIL are discussed elsewhere in this publication. We prefer HRA-targeted destruction of biopsy proven intra-anal HSIL and targeted destruction or excision of peri-anal HSIL to prevent the development of anal or perianal cancers. Since recurrence of HSIL is common, particularly in the HIV-positive population, close monitoring with HRA is useful to detect recurrence or the development of early invasion lesions. Early detection increases the chances for less morbid interventions. Patients with larger anal cancers or those who are node-positive have a poorer prognosis than those with less advanced disease. In particular, immunocompromised patients tolerate the standard treatment for advanced anal cancer less well, with increased short-term and long-term complication rates, poor response rates, and poorer survival after salvage therapies. Close surveillance and a high index of suspicion are crucial to expectant management in this population. HPV-DNA testing has not been FDA approved for screening for precancers of the anorectal junction and its role is yet
200 to be defined in the triage of atypical ARC. However, HPVDNA testing may prove to be as useful an adjunctive technology for the diagnosis and follow-up of anal SIL as it has for cervical SIL. Preliminary data suggest that the negative predictive value of HPV-DNA testing is superior to that of ARC, especially in HIV⫹ MSM.17 Further investigation is needed to define the role of HPV-DNA testing in squamous pathology of the anorectal junction because it may provide practical information for the clinical triage of high-risk patients and patients with abnormal cytology and for posttreatment management. In conclusion, similar to the cervical Pap test, ARC is an effective screening tool for the early detection of premalignant squamous lesions of the anal canal, especially in patients considered at risk for anal squamous cell carcinoma. Again, similar to the cervical cancer model, early detection and treatment of anal HSIL and anal cancer should lead to a significant reduction in the morbidity and mortality of anal cancer. More clinicians and pathologists need to recognize the serious impact of the changing epidemiology of anal squamous cancer and should familiarize themselves with the diagnosis and management of HPV-related squamous diseases of the anorectal junction.
References 1. Jemal A, Tiwari RC, Murray T, et al: Cancer statistics, 2004. CA Cancer J Clin 54(1):8-29, 2004 2. Greenlee RT, Murray T, Bolden S, et al: Cancer statistics, 2000. CA Cancer J Clin 50(1):7-33, 2000 3. Melbye M, Sprogel P: Aetiological parallel between anal cancer and cervical cancer. Lancet 338(8768):657-659, 1991 4. Cress RD, Holly EA: Incidence of anal cancer in California: increased incidence among men in San Francisco, 1973-1999. Prev Med 36(5): 555-560, 2003 5. Penn I: Cancers of the anogenital region in renal transplant recipients. Analysis of 65 cases. Cancer 58(3):611-616, 1986
M.L. Welton, B. Winkler, and T.M. Darragh 6. Penn I: Cancer in the immunosuppressed organ recipient. Transplant Proc 23(2):1771-1772, 1991 7. Daling JR, Weiss NS, Klopfenstein LL, et al: Correlates of homosexual behavior and the incidence of anal cancer. JAMA 247(14):1988-1990, 1982 8. Palefsky JM: Anal human papillomavirus infection and anal cancer in HIV-positive individuals: an emerging problem. AIDS 8(3):283-295, 1994 9. Goldie SJ, Kuntz KM, Weinstein MC, et al: Cost-effectiveness of screening for anal squamous intraepithelial lesions and anal cancer in human immunodeficiency virus-negative homosexual and bisexual men. Am J Med 108(8):634-641, 2000 10. Goldie SJ, Kuntz KM, Weinstein MC, et al: The clinical effectiveness and cost-effectiveness of screening for anal squamous intraepithelial lesions in homosexual and bisexual HIV-positive men. JAMA 281(19): 1822-1829, 1999 11. Palefsky JM, Holly EA, Hogeboom CJ, et al: Anal cytology as a screening tool for anal squamous intraepithelial lesions. J Acquir Immune Defic Syndr Hum Retrovirol 14(5):415-422, 1997 12. Holly EA, Ralston ML, Darragh TM, et al: Prevalence and risk factors for anal squamous intraepithelial lesions in women. J Natl Cancer Inst 93(11):843-849, 2001 13. Piketty C, Darragh TM, DaCosta M, et al: High prevalence of anal human papillomavirus infection and anal cancer precursors among HIV-infected persons in the absence of anal intercourse. Ann Intern Med 138(6):453-459, 2003 14. Darragh TM, Birdsong GG, Luff RD, et al: Anal-rectal cytology, In Solomon D and Nayar R (eds): The Bethesda System for Reporting Cervical Cytology: definitions, criteria, and explanatory notes. New York, NY, Springer-Verlag, 2004 15. Chin-Hong PV, Palefsky JM: Natural history and clinical management of anal human papillomavirus disease in men and women infected with human immunodeficiency virus. Clin Infect Dis 35(9):1127-1134, 2002 16. Jay N, Berry JM, Hogeboon CJ, et al: Colposcopic appearance of anal squamous intraepithelial lesions: relationship to histopathology. Dis Colon Rectum 40(8):919-928, 1997 17. Winkler B, Goldstone SE, Huang X, et al: Human Papillomavirus testing for the detection of precancerous lesions of the anus, in International Papillomavirus Meeting, 2005. Vancouver, Canada.
T
he incidence of anal cancer is increasing steadily. The American Cancer Society reported 4010 new cases for 2004, which is roughly equivalent to a rate of 1 case/100,000 in the general population.1 In contrast, in 2000 only 3400 cases of anal cancer were reported.2 The rate is higher in patients considered at high risk—the immunocompromised, men who have sex with men (MSM), and women with a history of cervical precancers or cancer.3-6 Early detection using cytologic screening has been an extremely effective method in the reduction of cervical cancer deaths. Because of numerous similarities between the tissues of the uterine cervix and the distal rectum and anus, analrectal cytology (ARC) is being proposed as a method for the early detection and prevention of anal cancer. Both anal and cervical cancers are squamous cell carcinomas primarily caused by the human papilloma virus (HPV), in particular HPV 16. Anal and cervical cancer arise within a defined zone
†From the Department of Surgery, Stanford University, School of Medicine, Stanford, CA. §Quest Diagnostics, Teterboro, NJ. *Departments of Pathology and OB/Gyn, University of California, San Francisco, San Francisco, CA. Address reprint requests to: Teresa M. Darragh, MD, UCSF/Mt. Zion Medical Center, Dept. of Pathology, 1600 Divisadero Street, Room B221, San Francisco, CA 94115. E-mail: [email protected].
196
1043-1489/04/$-see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1053/j.scrs.2005.04.001
of squamous metaplasia, the transformation zone in the cervix, and a similar region in the distal rectum and anus, often referred to as the anal transition zone. These zones of immature squamous metaplasia in the cervix and anus are hypothesized to be at particular risk for infection with the HPV. The normal tissue trauma and repair in these transformation zones, as occurs with changes in vaginal pH or during sexual penetration, may facilitate HPV infection. In both the cervix and the anus, the development of cancer is preceded by highgrade squamous intraepithelial precursors that are essentially morphologically and biologically identical. In the perianal skin and anal mucosa, these squamous cancer precursors have been variably labeled as anal intraepithelial neoplasia (AIN) 2/3, moderate/severe dysplasia, carcinoma in situ, high-grade squamous intraepithelial lesions (HSIL), Bowenoid papulosis, and Bowen’s disease. A similar proliferation of diagnostic terms exists for histologically identical lesions of the vulva, vagina, and cervix. This multiplicity of diagnostic terms originates from the various disciplines of expertise in clinical medicine and pathology. However, the variety of diagnostic terminology should not lead to the misunderstanding that these are distinct disease states that should be treated differently or have differing etiologies— this is not the case. Despite the confusion in reporting terminology, the same cytologic and histologic criteria should be
Anal-rectal cytology and anal cancer screening applied when evaluating these tissues. All HPV-related lesions of the lower genital tract are morphologically similar; their appearance is more dependent on whether the lesions arise on a mucosal or skin surface. Familiarity with interpretation of cervical-vaginal cytology and histology should allow for interpretation of ARC. Despite all the similarities described above, anal cancer and cervical cancer stand in stark contrast in one regard, incidence. Cervical cancer is decreasing in incidence, while the incidence of anal cancer is increasing unchecked. The reduction in cervical cancer is due in large part to the introduction of the Papanicolaou (Pap) test about 50 years ago. Before the introduction of the Pap test in the United States, the incidence of cervical cancer was approximately 40/100,000 women. Cervical Pap smears and colposcopicdirected ablation of targeted lesions has resulted in a 75% decrease in incidence of cervical cancer to 8 to 10/100,000 cases per year. Anal cancer, in contrast, has increased 96% in men and 39% in women over the last 30 years. In HIVnegative and HIV-positive MSM the rates are especially high, 35/100,000 and 70/100,000, respectively.7,8 Given the impact of the Pap test on cervical cancer, several groups have been interested in the potential benefit of ARC in the identification and treatment of the premalignant anal lesions; low- and high-grade squamous intraepithelial lesions (LSIL and HSIL). A number of studies have demonstrated the efficacy and accuracy of screening with ARC for anal squamous intraepithelial lesions (ASIL) in MSM.9,10 The sensitivity of cytology for the detection of biopsy-proven SIL was found to be 69 and 47% in HIV-negative and HIV-positive MSM, respectively. With repeat ARC, the sensitivity improved to 81 and 50% for these groups.11 These results are similar to those seen in the uterine cervix. These data have led some to pursue studies of ARC in other patient populations. Early findings suggest that all patients with HIV infection are at substantial increased risk for anogenital infection with HPV and should benefit from screening.12,13 The clinical use of ARC is increasing in frequency in select centers but most clinicians and pathologists are largely unfamiliar with the methods used to obtain and interpret ARC samples. The following clinical protocol is a practical approach. Patients should be asked to refrain from anoreceptive intercourse for 24 hours before an anal cytologic examination. Lubricants should not be used by the care provider before obtaining a cytologic sample because the lubricant may interfere with the processing and interpretation of the cytologic sample. The sample may be obtained with the patient placed in the lateral decubitus, prone-jackknife, or lithotomy positions. A tap-water-moistened Dacron swab is then inserted approximately 5 to 6 cm into the anal canal and distal rectal vault. The swab handle is rotated 360° while firm lateral pressure is applied to the handle and it is slowly withdrawn from the anus. The lateral pressure insures that the mucosal surface, rather than rectal contents, is sampled. Inserting the tip of the Dacron swab above the dentate line insures adequate sampling of the distal rectal mucosa and metaplastic epithelium of the anal transition zone, the area at highest risk for infection by oncogenic HPV. A cytology sam-
197 ple that does not include cells from above the dentate line is not adequate for representation of the anorectal transformation zone; a sample of the perianal skin is not adequate. The cellular sample is either placed in a vial of liquid preservative, for liquid-based cytology, or smeared directly onto a glass slide and fixed as with conventional Pap smears. ARC sampling tends to be more difficult than cervical sampling because, while the cervix is directly visualized during Pap collection by using a speculum, the anal canal mucosa and distal rectum are not directly visualized when the cells are collected. With experience with the technique and feedback from the laboratory as to the adequacy of the sample, the specimen adequacy, and cellularity of ARC can equal that of cervical samples. Typically the pathologist finds a combination of rectal columnar cells, squamous metaplastic cells, nucleated squamous cells, and anucleated squames representing all tissues types encountered as the swab passes from proximal to distal in the anal canal. Liquid-based cytology is utilized with increasing frequency because of its ease of use and superior specimen preparation. Advantages of liquid-based cytology include increased cellular yield, improved cell preservation, decreased contamination with feces and bacteria, and decreased airdrying and mechanical artifact. Familiarity with the technique used by the laboratory is critical so that the clinical care provider prepares the sample correctly. Samples to be evaluated with ThinPrep should be placed in either CytoLyt or PreservCyt, depending on the laboratory’s preference. In the laboratory, the sample may be either processed as a nongynecologic specimen or processed similar to gynecologic samples. If processed as a nongynecologic specimen, the sample is placed in CytoLyt and the laboratory uses a blue filter and runs the sample on sequence No. 1 for superficial cells or sequence No. 3 for fluids/fine-needle aspirations. One of the authors (T.D.) prefers to place the anal sample in PreservCyt and processes the sample in a manner identical to cervical Pap tests (using the white filter and the gynecologic program, sequence No. 4 for gynecologic samples); this is an “off-label” use. If the specimen is to be analyzed with the TriPath PrepStain System, the sample must be submitted in CytoRich Red Fixative and processed using standard nongynecologic sample procedures. Labeling of the specimens with the correct CPT codes is critical to compensate the laboratory because third-party payers currently will not reimburse for ARC as a screening test or if the gynecologic codes are chosen. The CPT codes for nongynecologic samples are 88108 for liquid-based studies, 88104 for conventional samples (direct smears), and 88160 for anorectal smears with a Dacron sponge. Interpretation of ARC is guided by criteria established in the 2001 Bethesda System guidelines modified for anal-rectal samples. These morphologic criteria used in the interpretation of ARC are essentially similar to those used in the evaluation of gynecologic samples. The Bethesda System Atlas, published in 2004, provides guidelines for sample adequacy specific for ARC with a minimum cellularity established as 2000 to 3000 nucleated squamous cells for conventional smears or one to two per high power field (hpf) for ThinPreps
M.L. Welton, B. Winkler, and T.M. Darragh
198
Figure 1 LSIL (anal-rectal cytology, high power). (Color version of figure is available online.)
(diameter of 20 mm) and three to six nucleated cells/hpf for TriPath’s PrepStain System (diameter 13 mm) with a ⫻40 objective.14 The presence of metaplastic squamous cells or rectal columnar cells indicates that the anal transformation zone has been sampled and is reported as such. These criteria for specimen adequacy are similar to those used for gynecologic samples. LSIL are characterized by koilocytosis, and by the presence of superficial and high-intermediate squamous cells with an atypical nucleus (Fig. 1). Nuclear changes may include binucleation, enlargement, irregularities of the nuclear membrane, chromatin clumping, and hyperchromasia. Atypical parakeratotic cells are common and “empty halos” or degenerating koilocytes are frequently identified. HSIL have abnormal squamous cells that are of the lowintermediate or immature squamous metaplastic type; these cells are typically smaller than superficial and high-intermediate cells. There is a high nucleus to cytoplasmic (N/C) ratio with nuclear enlargement, although this enlargement can be subtle, and is paired with a wrinkled or irregular nuclear membrane (Fig. 2). The chromatin may be clumped or there may be chromatin margination and clearing in some nuclei. Nucleoli are inconspicuous. The high-grade cells may be in clusters or seen as single cells. They are not usually present in sheets or syncytia. These findings may be present in nonkeratinizing and keratinizing lesions. In the latter, atypical surface parakeratotic cells are seen. In our experience, invasive squamous cell carcinoma of the anus is difficult to prospectively diagnose on ARC; it may be difficult to accurately differentiate from a high-grade keratinizing lesion. Here, pairing ARC with the clinical examination is essential. The cytologic diagnosis of invasive squamous cell carci-
noma is more problematic in ARC than in cervical-vaginal samples because the typical tumor diathesis of invasion may be subtle or absent. The presence of bizarre atypical squamous cells, nucleoli, or profound nuclear chromatin changes provides clues to the possibility of invasion. Atypical parakeratosis may be a more prominent cellular component of the invasive ARC. Clinicians and pathologists should recognize that the prospective diagnosis of invasion may be unreliable with ARC and that clinically suspicious lesions should be extensively sampled to rule out invasion, regardless of the cytologic result. Reactive and reparative changes are less commonly seen in ARC than in gynecologic samples unless an infection that results in inflammation and ulceration, such as herpes simplex virus, is present. Samples with cells that do not meet the criteria for SIL but have atypical features such as nuclear enlargement without chromatin abnormalities are classified as atypical squamous cells— undetermined significance (ASC-US) or atypical squamous cells— cannot exclude HSIL (ASC-H). The major morphologic difference between these two categories of atypical squamous cells is cell size, cell maturation, and N/C ratio. The ASC-US cells are of intermediate or superficial type, while the ASC-H are smaller, metaplastic-type cells of low-intermediate or basal type with a higher N/C ratio. HPV testing has not been FDA approved for ARC and its role is yet to be defined in the triage of atypical ARC. The patient populations that are targeted for ARC screening have very high rates of anal HPV infection, potentially minimizing the utility of HPV testing for triage to highresolution anoscopy. All patients with ASC-US or higher on ARC should be referred for digital rectal examination and high-resolution
Anal-rectal cytology and anal cancer screening
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Figure 2 HSIL (anal-rectal cytology, high power). (Color version of figure is available online.)
anoscopy (HRA) or, at the least, digital rectal examination and anoscopy with acetic acid.15 HRA is essentially equivalent to cervical colposcopy, with modifications for use in the distal rectum and anus.16 A cotton gauze pad is wrapped around the swab of a scopette and is soaked in acetic acid (3 to 5% may be used but 3% is preferred because the anus is somewhat acid-sensitive) and inserted into the anus (an anoscope may be used to facilitate this). The vinegar-soaked, gauze-covered scopette is allowed to saturate the distal rectal and anal mucosa for at least 1 minute. After the gauze is withdrawn, an anoscope is inserted and the anus is visualized through a magnification system such as a colposcope, operating microscope, or loops. A colposcope or operating microscope is preferred because loops have a lower resolution that may compromise identification of more subtle, yet important changes, that assist in the distinction between LSIL and HSIL. Both LSIL and HSIL have well-demarcated regions of the mucosa that whiten after the application of acetic acid, known as acetowhite epithelium (AWE). High-grade disease may be distinguished from low-grade lesions by architectural, morphologic, and vascular criteria. Low-grade lesions tend to be more architecturally exuberant with topographic changes that present as papillary or macular-papular growths or with cauliflower-like surface contours.16 High-grade lesions tend to be flat with characteristic vascular changes associated with the AWE. These vascular changes are characterized as punctate, mosaic, or “streaky.” Exophytic lesions may harbor HSIL deep within the lesion in areas that cannot be well-visualized and should be thoroughly sampled by biopsy. An exophytic lesion with vessel changes suggestive of HSIL should be considered suspicious for malignancy. Lesions with central ulceration and firmness or induration on
digital examination are also suspicious for invasive carcinoma. Deep biopsy is necessary for more definitive classification. The keratinized perianal skin may also be examined under magnification after the application of acetic acid. This procedure is especially useful in the patient with apparent circumferential acetowhitening as may be seen with “Bowen’s disease.” Identification of the regions most worrisome for HSIL allows targeting of those tissues for directed biopsy and treatment, reducing the number of biopsies and destruction of regions that harbor the less worrisome, yet still acetowhite, LSIL. The controversy surrounding the methods for treatment or monitoring of the HSIL are discussed elsewhere in this publication. We prefer HRA-targeted destruction of biopsy proven intra-anal HSIL and targeted destruction or excision of peri-anal HSIL to prevent the development of anal or perianal cancers. Since recurrence of HSIL is common, particularly in the HIV-positive population, close monitoring with HRA is useful to detect recurrence or the development of early invasion lesions. Early detection increases the chances for less morbid interventions. Patients with larger anal cancers or those who are node-positive have a poorer prognosis than those with less advanced disease. In particular, immunocompromised patients tolerate the standard treatment for advanced anal cancer less well, with increased short-term and long-term complication rates, poor response rates, and poorer survival after salvage therapies. Close surveillance and a high index of suspicion are crucial to expectant management in this population. HPV-DNA testing has not been FDA approved for screening for precancers of the anorectal junction and its role is yet
200 to be defined in the triage of atypical ARC. However, HPVDNA testing may prove to be as useful an adjunctive technology for the diagnosis and follow-up of anal SIL as it has for cervical SIL. Preliminary data suggest that the negative predictive value of HPV-DNA testing is superior to that of ARC, especially in HIV⫹ MSM.17 Further investigation is needed to define the role of HPV-DNA testing in squamous pathology of the anorectal junction because it may provide practical information for the clinical triage of high-risk patients and patients with abnormal cytology and for posttreatment management. In conclusion, similar to the cervical Pap test, ARC is an effective screening tool for the early detection of premalignant squamous lesions of the anal canal, especially in patients considered at risk for anal squamous cell carcinoma. Again, similar to the cervical cancer model, early detection and treatment of anal HSIL and anal cancer should lead to a significant reduction in the morbidity and mortality of anal cancer. More clinicians and pathologists need to recognize the serious impact of the changing epidemiology of anal squamous cancer and should familiarize themselves with the diagnosis and management of HPV-related squamous diseases of the anorectal junction.
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