BJD. Summary. British Journal of Dermatology CLINICAL AND LABORATORY INVESTIGATIONS

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1 CLINICAL AND LABORATORY INVESTIGATIONS BJD British Journal of Dermatology Anal carcinoma in human immunodeficiency virus-positive men: results of a prospective study from Germany A. Kreuter, A. Potthoff, N.H. Brockmeyer, T. Gambichler, J. Swoboda,* M. Stücker, M. Schmitt, H. Pfister,à U. Wieland and German Competence Network HIV AIDSà Department of Dermatology, Ruhr University Bochum, Gudrunstrasse 56, D Bochum, Germany *Institute of Cytology, Bad Godesberg, Bonn, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany àinstitute of Virology, National Reference Center for Papilloma- and Polyomaviruses, University of Cologne, Cologne, Germany Summary Correspondence Alexander Kreuter. a.kreuter@derma.de Accepted for publication 16 February 2010 Key words anal carcinoma, anal intraepithelial neoplasia, human immunodeficiency virus, human papillomavirus, men who have sex with men Conflicts of interest ClinicalTrials.gov identifier: NCT Group information: A list of the Competence Network HIV AIDS members appears at DOI /j x Background Anal intraepithelial neoplasia (AIN), a human papillomavirus (HPV)- associated potential precursor lesion of anal cancer, is frequent among human immunodeficiency virus (HIV)-positive men who have sex with men (MSM). There is a paucity of data published on the progression of high-grade AIN to invasive cancer as well as on clinical and virological characteristics comparing anal margin and anal canal carcinoma. Objectives To search for anal carcinoma and AIN in a large series of HIV-positive MSM, to assess treatment response of anal carcinoma, and to analyse lesional HPV spectrum of anal cancers. Methods Detection of anal carcinoma and AIN was performed using cytology, high-resolution anoscopy, and histology in case of abnormal findings. Additionally, HPV analyses for 36 high- and low-risk a-hpv types were performed in patients with anal carcinoma. Results In total, 446 German HIV-positive MSM were examined within an observation period of 5 years and 10 months. Of these, 116 (26Æ0%) patients had normal findings, 163 (36Æ5%) had low-grade AIN, 156 (35Æ0%) had high-grade AIN, and 11 (2Æ5%) had anal carcinoma as evidenced by the highest grade of cytology histology. Five patients with anal cancer, who had refused treatment of their precancerous lesions, had progressed from high-grade AIN to invasive cancer within a median time of 8Æ6 months. All anal cancers carried high-risk a-hpv types. All five squamous cell carcinomas (SCCs) of the anal canal were HPV16 positive. In contrast, only one of the four anal margin SCCs were HPV16 positive (HPV31, HPV33 and HPV33 + HPV68 were found in the other three anal margin SCCs). HPV59 was found in two adenocarcinomas, one of which additionally carried HPV33. In contrast to the cancer biopsies, a broad spectrum of surface high- and low-risk HPV types was found in anal swabs of the patients. Surgical excision resulted in long-term disease control of all anal margin carcinomas, whereas combined chemoradiotherapy in carcinomas of the anal canal was associated with high recurrence rates, high toxicity, and high mortality. Conclusions Anal carcinoma and AIN are frequent in HIV-positive men, even in patients participating in anal cancer prevention programmes. High-grade dysplasia in these patients can progress to invasive cancer within a short period of time. Anal margin carcinoma and anal canal carcinoma differ substantially in their lesional HPV spectrum, prognosis and treatment response. Anal carcinoma is a rare neoplasm that accounts for approximately 1% of all gastrointestinal tumours. In the general population, anal carcinoma mostly affects middle-aged adults, with a female predominance of up to three to six times seen in some European treatment centres. 1 Its annual incidence is around one per individuals, and about 3500 new 1

2 2 Anal carcinoma in HIV-positive men, A. Kreuter et al. cases are diagnosed each year in the U.S.A. 1 The incidence of anal cancer has increased over the past two decades, particularly in special population subgroups. Men who have sex with men (MSM), especially MSM with human immunodeficiency virus (HIV) infection, are at high risk for anal cancer development. 2 While HIV-negative MSM have an estimated incidence rate of 35 per person-years which is similar to the incidence of cervical cancer before the introduction of cervical Pap screening, anal cancer incidence rates in HIV-positive MSM are much higher (about per personyears). 3,4 A recent study analysed the incidence of various cancer types in HIV-positive individuals from two large prospective cohorts in the U.S.A. The highest incidence rates of all non-aids-defining malignancies in this study were observed for anal cancer, continuously increasing from 1992 to In contrast to several virus-associated diseases such as human herpesvirus-8-associated Kaposi sarcoma or cytomegalovirus-associated retinitis, highly active antiretroviral therapy (HAART) seems to have no beneficial effects on anal cancer. 6 As HAART significantly prolongs life expectancy in HIV-positive individuals, anal cancer could become one of the major medical problems of this population in the future. Anal carcinoma and cervical carcinoma share several similarities, including the same risk factors (e.g. sexual behaviour, smoking and immunosuppression) and their causative association to infections with high-risk human papillomaviruses (HPVs). 7,8 The tumours arise from precursor lesions called anal intraepithelial neoplasia (AIN) or cervical intraepithelial neoplasia (CIN). Facing the biological similarities of CIN and AIN, anal cytology in at-risk populations has been recommended by several research groups according to the principles of cervical screening. 9 However, others have refuted this suggestion as there is currently no conclusive evidence of the effectiveness of screening in reducing anal cancer morbidity and mortality. 10 Although several studies have been published on AIN incidences and prevalences in HIV-positive MSM, there is a paucity of data addressing the progression of anal dysplasia to anal cancer, differences in lesional HPV types comparing anal margin and anal canal carcinoma, and differences in treatment outcome of anal cancer depending on the localization of disease. In 2003, a screening and treatment programme for anal dysplasia was established at the Department of Dermatology at Ruhr University Bochum in order to prevent anal cancer development. The aim of this study was to evaluate the cumulative prevalence of AIN and anal carcinoma in a large number of German HIV-positive MSM during an observation period of 5 years and 10 months. Moreover, we determined type-specific HPV infection in patients in whom a diagnosis of anal carcinoma was made, and assessed the treatment response of anal carcinoma. Materials and methods Patients and clinical examination HIV-positive MSM older than 18 years were recruited from the infectious diseases unit of the Department of Dermatology at Ruhr University Bochum and from four cooperating specialists for internal medicine (located in Cologne, Düsseldorf and Dortmund) providing health care for individuals with HIV. Both asymptomatic patients and patients with clinical, cytological or histological evidence for HPV-associated anal lesions were included in this study. The U.S. Centers for Disease Control and Prevention (CDC) classification system was used to assess the severity of HIV disease by CD4 cell counts and by the presence of specific HIVrelated conditions (see cm-105_disease). All patients were interviewed regarding medical history, lifetime use of HAART, and behavioural factors including lifetime history of smoking (cigarettes per day, numbers of years smoking). All participants underwent a standardized clinical examination including digital rectal examination (DRE) and high-resolution anoscopy (HRA) as previously described. 11,12 In brief, a wooden cotton swab soaked in 3% acetic acid was placed into the anal canal and kept there for about 2 min. A disposable plastic anoscope (Cooper Surgical, Trumbull, CT, U.S.A.) was then inserted into the anal canal. While looking through the binoculars of a conventional colposcope (Kolposkop 150 FC; Zeiss, Oberkochen, Germany) at a distance of about 30 cm, the entire circumference of the distal rectum and anal epithelium, squamous-columnar transitional zone and perianal area were visualized at 30 magnification. Visible intra-anal lesions were biopsied with an endoscopic forceps (Hildyard post nasal forceps 3Æ5 mm; Gyrus ENT LCC, Bartlett, TN, U.S.A.), and conventional punch biopsies were obtained in perianal lesions. The protocol of the study was approved by the ethics review board of the Ruhr University Bochum and the study was conducted according to Declaration of Helsinki principles. Patients gave their written informed consent to participate in the study. Anal cytology and anal histopathology Anal swabs for cytology and HPV testing were obtained before HRA. Swabs were moistened in water, inserted into the anal canal until resistance was met at the distal rectum wall, carefully rotated in a circular motion, and cells were placed on a glass slide for cytopathological evaluation. Cytology results were classified according to the Bethesda system as normal, atypical squamous cells of undetermined significance (ASC-US), low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL) or invasive cancer. 13 Anal dysplasia was histologically divided into three grades [confined to the lower third (AIN1), lower two-thirds (AIN2) or full thickness of the epithelium (AIN3)] as previously described. 14 LSIL and HSIL correspond to AIN1 and AIN2 3, respectively. A composite diagnosis was made based on anal cytology and histopathology, as described before. 15 If both cytological and histological results were available, the more severe diagnosis was used for data analyses. 6 The highest grade of anal dysplasia of each individual found within the

3 Anal carcinoma in HIV-positive men, A. Kreuter et al. 3 Table 1 Results of anal examination in 446 human immunodeficiency virus-positive men who have sex with men Dysplasia grade Number of patients a (%) HPV positive (%) High-risk HPV-positive b (%) HPV16 positive (%) Normal (26Æ0) (84) (73) (36) Low-grade anal dysplasia c (36Æ5) (90) (82) (36) High-grade anal dysplasia d (35Æ0) (98) (95) (68) Anal carcinoma (2Æ5) (100) (100) 6 11 (55) a In 303 patients several anal samples were collected within the study period. In those patients the highest dysplasia grade found within the study period is given in the table. Twenty-four (15%) of the 156 high-grade anal dysplasias were detected only by high-resolution anoscopy-guided histology [anal intraepithelial neoplasia (AIN) 2 or AIN3], and had normal (n = 3), atypical squamous cells of undetermined significance (ASC-US; n = 3) or low-grade squamous intraepithelial lesion (LSIL; n = 18) cytology results. b Human papillomavirus (HPV) types 16, 18, 26, 31, 33, 35, 39, 45, 51 53, 56, 58, 59, 66, 68, 73 and 82 were considered high-risk a-hpv types. 20 c Cytological LSIL and histological AIN1 are considered as low-grade anal dysplasia. d Cytological HSIL and histological AIN2 or AIN3 are considered as high-grade anal dysplasia. observation period was used for final data analysis as shown in Table 1. Participants were considered as normal if anal cytology was normal and no clinical lesions were present on HRA. Human papillomavirus analyses DNA isolation and HPV DNA analyses were performed as described before Briefly, DNA was isolated using the QIAamp DNA mini-kit (Qiagen, Hilden, Germany), and b-globin gene polymerase chain reaction (PCR) was carried out to demonstrate that the samples contained adequate DNA and were free of substances inhibitory to PCR. 16 A5 A10 nested PCR was used for the detection of a-hpv DNA. 18 For HPV typing, internal, biotinylated A6 A8 PCR products were hybridized with 36 type-specific digoxigenin-labelled oligonucleotide probes in an enzyme immunoassay. 19 Anal cancer biopsies were additionally HPV typed using a bead-based multiplex genotyping assay as described before. 17 HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51 53, 56, 58, 59, 66, 68, 73 and 82 were considered as high-risk a-hpv types. HPV types 6, 11, 34, 40, 42 44, 54, 55, 57, 61, 70 72, 81, 83, 84 and 89 were considered as low-risk a-hpv types. 20 Treatment of anal dysplasia and anal carcinoma All visible clinical lesions that histologically displayed AIN were treated, irrespective of the histopathological grade. Patients with intra-anal dysplasia were treated with electrocautery followed by 85% trichloroacetic acid or imiquimod, and perianal lesions were treated with either imiquimod or electrocautery, as previously reported. 12,21,22 An algorithm for the diagnosis, treatment and follow up of participating patients based on anal cytology histology and HRA results used in this study is depicted in Figure 1. Surgical excision was performed in carcinomas of the anal margin (Fig. 2), whereas carcinomas of the anal canal were treated with combined chemoradiotherapy (CRT) including 5-fluorouracil and mitomycin, as previously reported (Fig. 3). 23 Data analysis Baseline examination (HRA and cytological examination) Normal cytology and normal HRA findings ASC-US and normal HRA findings LSIL / HSIL HRA and cytological examination (repeat every year) Next HRA and cytological examination after 6 months Treatment* (and next HRA and cytological examination after 6 months) Biopsy (histology) in case of AIN: treatment* if normal histology: next HRA and cytological examination (with rebiopsy) after 3 months Biopsy and treatment* (and next HRA and cytological examination after 3 months) Repeat the smear Repeat HRA Data analysis was performed using the statistical package Med- Calc Software (Mariakerke, Belgium). CD4 cell counts and LSIL and normal clinical findings in HRA LSIL and abnormal findings in HRA HSIL and normal clinical findings in HRA HSIL and abnormal findings in HRA Fig 1. Algorithm for the diagnosis, treatment and follow up of human immunodeficiency virus-positive patients participating in this study based on anal cytology histology and high-resolution anoscopy (HRA) results. AIN, anal intraepithelial neoplasia; ASC-US, atypical squamous cells of undetermined significance; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion. *Patients with intra-anal dysplasia were treated with electrocautery (single treatment or multiple treatments at monthly intervals) followed by 85% trichloroacetic acid (single treatment or multiple treatments at monthly intervals) or imiquimod (three times weekly for a total of 16 weeks). Perianal lesions were treated with either imiquimod (three times weekly for a total of 16 weeks) or electrocautery (single treatment or multiple treatments at monthly intervals).

4 4 Anal carcinoma in HIV-positive men, A. Kreuter et al. (a) Fig 2. Clinical presentation of a carcinoma of the anal margin. An erythematous tumour, 2 2 cm in diameter, is present at the perianal skin of patient 7. Moreover, numerous verrucous and brown papules (histologically confirmed high-grade anal dysplasia) are located on the entire circumference of the perianal area. (b) HIV-1 RNA loads are expressed as median and interquartile range (IQR). Results Population characteristics From 1 October 2003 to 31 July 2009, 446 HIV-positive MSM were examined for HPV-related anal disease [benign lesions (anal condylomata acuminata), anal dysplasia (AIN1 3) and anal cancer]. Twenty-seven of all 473 recruited patients declined to participate in the study. At study entry patients age ranged between 18 and 72 years (median 41). Median CD4 cell counts were 470 mm )3 (IQR 67Æ3) and median HIV-1 RNA loads were 53 copies ml )1 (IQR 195Æ4). One hundred and forty-six patients were in CDC stage A, 121 in stage B, 126 in stage C, and in 53 patients the CDC stage was unknown. Three hundred and one (67%) of the 446 patients had received HAART at study entry. Two hundred and twentynine patients were smokers (51%). The mean follow-up time was 20Æ5 months [range 0 (single visit) 67]. The results on the prevalence of anal cancer and anal dysplasia are depicted in Table 1. One hundred and sixteen patients (26%) had a normal clinical status, 163 (36Æ5%) patients had low-grade anal dysplasia, 156 (35%) patients had high-grade anal dysplasia, and 11 (2Æ5%) patients had invasive anal carcinoma as evidenced by the highest grade of cytology histology within the study period. In contrast to anal cancer, none of the highgrade anal dysplasias was detectable by DRE. Twenty-four of the 156 (15%) high-grade dysplasias were detected only by HRA-guided histology (AIN2 3), and not by cytology. Characteristics of human immunodeficiency virus infection in patients with anal carcinoma The characteristics of the HIV status of the 11 patients with anal carcinoma are detailed in Table 2. Mean patient age at (c) Fig 3. Clinical presentation of an advanced carcinoma of the anal canal before and after combined chemoradiotherapy. (a) A large ulcerated tumour that arose from the transformation zone (linea dentata) is present in patient 1. (b) Complete clearance of former anal carcinoma following combined chemoradiotherapy. (c) Widespread recurrence with secondary bacterial infection 18 months after combined chemoradiotherapy. anal cancer diagnosis was 50Æ6 years (range 41 69), mean duration of HIV infection at anal cancer diagnosis was 13Æ6 years (range 6 21), and mean time on HAART was 8Æ5 years (range 5 13). Eight patients were in CDC stage C3 and three were in stage B3. The most common opportunistic diseases found in the eight patients in CDC stage C3 were candida oesophagitis (CO) and non-hodgkin lymphoma (NHL) (three patients with CO, two patients with NHL and two

5 Anal carcinoma in HIV-positive men, A. Kreuter et al. 5 Table 2 Characteristics of human immunodeficiency virus (HIV) infection in patients with anal carcinoma Patient Age (years) Duration of HIV infection (years) a Duration of HAART (years) a CDC stage b CD4 cell nadir (cells mm )3 ) Present CD4 cell count (cells mm )3 ) a Present HIV-1 RNA load (copies ml )1 ) a Previous opportunistic infections and AIDS-defining diseases Other non-aidsdefining neoplasms C CO, CMV, HIV-E, NHL C < 40 CO, CMV, PCP B < 40 Laryngeal carcinoma C NHL BCC C CO, HIV-E, KS C NHL, PCP B < C < 40 CO, CMV, NHL C < 40 KS, PCP C < 40 CO B Colon carcinoma a At time of anal cancer diagnosis. b An explanation of the CDC staging system appears at HAART, highly active antiretroviral therapy; CDC, U.S. Centers for Disease Control and Prevention; CO, candida oesophagitis; CMV, cytomegalovirus infection; HIV-E, HIV-associated encephalitis; NHL, non-hodgkin lymphoma; PCP, Pneumocystis carinii pneumonia; KS, Kaposi sarcoma; BCC, basal cell carcinoma. patients with both). Median CD4 cell nadir and CD4 cell count at anal cancer diagnosis were 98 cells mm )3 (IQR 143) and 290 cells mm )3 (IQR 257), respectively. Nine of the 11 (82%) patients were heavy cigarette smokers (more than 20 cigarettes daily for at least 20 years) at the time of anal cancer diagnosis, one patient (patient 8) had quit smoking 10 years before anal cancer diagnosis, and one patient (patient 11) had never smoked. All patients with anal carcinoma had a history of recurrent anal condylomata acuminata. In all 11 patients with anal cancer, HRA yielded pathological findings. However, all anal cancers were also detectable by simple diagnostic procedures such as DRE or clinical inspection (Table 3). Human papillomavirus analysis in anal carcinoma Nine of the 11 patients with invasive anal carcinoma had anal squamous cell carcinoma (ASCC), and two patients had primary adenocarcinoma (AC) of the anus. Six of the 11 carcinomas were located in the anal canal (five ASCC and one AC), and five were located at the anal margin (four ASCC and one AC). The results of HPV analysis are shown in Table 3. All 11 anal carcinomas (100%) carried lesional high-risk HPV types, and HPV16 was the most common HPV type (55%). In nine of the cancers, only a single high-risk HPV type was present; two high-risk HPV types were found in an ASCC and in an AC, respectively. Five (83%) of the six anal canal carcinomas and all five (100%) ASCCs located in the anal canal were HPV16 positive. In contrast, only one of the five (20%) carcinomas of the anal margin was HPV16 positive (HPV31, HPV33, HPV33 + HPV59 and HPV33 + HPV68 were found in the four other anal margin carcinomas, respectively). In two patients with primary anal AC, HPV59 (anal canal AC) and HPV33 + HPV59 (anal margin AC) were found. In contrast to the cancer biopsies, most patients with anal cancer carried a broad spectrum of additional high- and low-risk HPV types on the surface of the anal mucosa, as determined by analysis of anal swabs (Table 3). The mean number of surface HPV types (high- and low-risk types) found per patient within the study period was 8Æ2 (range 1 18), the mean number of surface high-risk HPV types was 4Æ7 (range 1 10), and the mean number of surface low-risk HPV types was 3Æ5 (range 0 8). The high-risk HPV types found in the anal cancers were also present in the patients anal surface swabs (Table 3). Treatment response in patients with anal carcinoma In five of the 11 (45%) patients with anal carcinoma, highgrade anal dysplasia was previously diagnosed (Table 3), but these patients refused subsequent treatment of anal dysplasia and developed invasive anal carcinoma. Mean time from highgrade AIN to progression to anal carcinoma was 8Æ6 months. The clinical course and outcome following treatment of anal carcinoma is depicted in Table 3. Surgical excision was performed in all five patients with anal margin carcinoma, and one of these patients (patient 9) additionally received radiation therapy (50 Gy cumulative dose). None of the patients had a local relapse of anal margin carcinoma. Two of the patients died because of reasons not associated with anal margin carcinoma (myocardial infarction in patient 5 and metastatic laryngeal carcinoma in patient 3). Five of the six patients with carcinoma of the anal canal were treated with CRT (patient 8 refused CRT and later committed suicide). One patient (patient 11) had long-term disease control following CRT, two patients had recurrent disease, and two patients died because of adverse events related to CRT (Table 3).

6 6 Anal carcinoma in HIV-positive men, A. Kreuter et al. Table 3 Clinical and virological characteristics of patients with anal carcinoma Patient Type of anal cancer Previous HG-AIN Localization of (months) a carcinoma DRE Clinical inspection b Lesional HPV types c Surface anal high-risk HPV types d Surface anal low-risk HPV types d Treatment of anal carcinoma Clinical course Outcome 1 ASCC Canal , 18, 31, 45, 52 54, 83 CRT Recurrence after CRT, followed by radical surgery 2 ASCC Yes (9) Canal ND ND CRT Recurrence after CRT, metastatic disease 3 ASCC Margin Neg , 33, 58 SE NR Dead (unrelated to ASCC) SE NR Alive 4 ASCC Yes (4) Margin Neg. + 33, 68 16, 18, 26, 33, 35, 45, 51, 52, 58, 68 5 ASCC Yes (6) Margin Neg , 26, 18, 31, 35, 52, 59 6 ASCC Yes (17) Canal , 31, 45, 52, 53, 58, 82 6, 34, 43, 44, 54, 83, 84, 89 Alive Dead 54, 61, 83, 84, 89 SE NR Dead (unrelated to ASCC) 11, 34, 40, 44, 70, 81, 83 CRT SAE of CRT (pneumonitis) Dead 7 ASCC Margin Neg SE NR Alive 8 ASCC Yes (7) Canal + Neg , 31, 58 6, 42 None Refused CRT Dead (suicide) 9 AC Margin Neg. + 33, 59 16, 31, 33, 56, 59 6, 34, 40, 42, 44 SE, R NR Alive 10 AC Canal + Neg , 35, 53, 59, 66 40, 44, 70, 81 CRT SAE of CRT (septicaemia) Dead 11 ASCC Canal + Neg , 72 CRT NR Alive HG-AIN, high-grade anal intraepithelial neoplasia; DRE, digital rectal examination; HPV, human papillomavirus; ASCC, anal squamous cell carcinoma; CRT, chemoradiotherapy; ND, not determined; SE, surgical excision; NR, no relapse; SAE, severe adverse event; AC, adenocarcinoma; R, radiotherapy. +, positive result of DRE (palpable mass) or clinical inspection; Neg., negative result (no pathological findings) of DRE or clinical inspection. a Months between diagnosis of HG-AIN and progression to anal cancer. b Clinical inspection (with the naked eye) of the perianal area and of the external part of the anal canal (after spreading the buttocks). c Lesional HPV types found in anal cancer biopsies. d Surface anal HPV types found in anal swabs of the respective patients within the study period. HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51 53, 56, 58, 59, 66, 68, 73 and 82 were considered high-risk a-hpv types. HPV types 6, 11, 34, 40, 42 44, 54, 55, 57, 61, 70 72, 81, 83, 84 and 89 were considered low-risk a-hpv types. 20

7 Anal carcinoma in HIV-positive men, A. Kreuter et al. 7 Except for the five patients with anal cancer who refused treatment of their high-grade AIN lesions, none of the patients with anal dysplasia treated within the observation period progressed to anal carcinoma. Discussion HIV-associated immunosuppression seems to play a major role in the pathogenesis of anal dysplasia in HIV-positive men. Several mechanisms of interaction between HIV and HPV have been postulated, including an attenuation of systemic cell-mediated immune responses to HPV antigens, aberrant expression of cytokines (e.g. interleukin-6) known to modulate HPV gene expression, increased expression of growth factors, and direct effects of HIV-1 Tat that potentiate the expression of the HPV oncogenes E6 and E7. 24 In line with this, the relative risk for AIN and the 4-year incidences of high-grade AIN (49% vs. 17%) are significantly higher in HIV-positive MSM as compared with HIV-negative MSM. 25 There is evidence for the lack of beneficial effects of HAART to reduce AIN and anal cancer. 2,6 Actually, prolonged immunodeficiency seems to provide an optimal environment for the development of HPV-associated anogenital dysplasia. 22 Experts therefore recommend screening for AIN in high-risk populations using anal cytology followed by HRA in case of abnormal findings. 22 The calculated sensitivity (69 93%) and specificity (32 59%) of anal cytology to identify AIN is similar to those reported for cervical cancer screening. 9 This study demonstrated that a high proportion of German HIV-positive MSM have anal dysplasia, and that anal cancer in these patients is frequent in the post-haart era. To our best knowledge, this is the first prospective study on AIN and anal cancer incidences performed in Germany, and is one of the largest studies that have been published so far. Our findings are in line with previous investigations on anal dysplasia in HIV-positive MSM. In a pre-haart era study from San Francisco, CA, U.S.A., published more than 10 years ago, 106 of 277 (38%) HIV-positive MSM developed high-grade AIN in a 4-year observation period. 25 A more recent study from the same investigators performed in the post-haart era has shown that 81% of 357 HIV-positive MSM had anal dysplasia of any grade, and 52% had high-grade AIN. 6 Our results of 72% (319 of 446 patients) AIN of any grade and 35% (156 of 446 patients) high-grade anal dysplasia are in concordance with these studies. So far, only few data are available on the progression of anal dysplasia to invasive anal cancer. In a study that compared AIN in 29 immunocompetent individuals and six patients on immunosuppressive therapy, three of the six immunosuppressed individuals had a progression from highgrade AIN to invasive cancer within a median time of 5 years. 26 Another study on 72 patients with AIN revealed that eight (13%) cases of high-grade anal dysplasia progressed to ASCC within a median time of 42 months. 27 However, the proportion of immunosuppressed or HIV-positive individuals that progressed to anal cancer remains unknown. We have observed that five patients with anal cancer who refused treatment of anal dysplasia progressed from high-grade AIN to invasive cancer within a much shorter period of time (8Æ6 months), and that none of the patients with AIN of any grade treated in the observation period developed anal cancer. This observation strongly argues for regular clinical examination and early treatment of high-grade AIN, particularly in HIV-positive MSM. Although there are many studies on anal dysplasia, relatively few data are available on anal cancer in HIV-positive MSM in the post-haart era. A recent analysis from the French Hospital Database on HIV, a large study in HIV-positive MSM including 132 anal cancers, revealed results similar to our observations. 28 HIV-positive patients developed anal cancer at a young age (median 42Æ8 years), had low CD4 cell count nadirs (median 75 cells mm )3 ), and had long-term usage of HAART (median duration 37Æ1 months). Thus, cumulative lifetime immunosuppression may play an important role in anal cancer development. Anal carcinoma is causally linked to HPV infection. Two recent meta-analyses have shown that 73Æ4% and 65Æ6% of all anal cancers are caused by HPV16. 29,30 In the study that found an HPV16 prevalence of 65Æ6% in invasive anal cancer, anal cancers of men were less frequently HPV16 positive than those of women (60Æ0 vs. 73Æ7%). 30 In our HIV-positive male patients, 55% of anal cancers were HPV16 positive. However, comparing the localization of disease, 83% of anal canal carcinomas (and 100% of canal ASCCs) but only 20% of anal margin carcinomas were HPV16 positive. HPV vaccination is highly effective in preventing HPV16 infection and HPV16- associated CIN in HPV-naive women. If HPV vaccination is also shown to be efficacious in preventing HPV16 infection in men, a high proportion of anal canal cancers, especially anal ASCCs, would be preventable. However, HPV types found in our study in cancers at the anal margin would not be covered by the currently available HPV vaccines, although some crossprotection has been reported for HPV31. Interestingly, the vaccine-included high-risk type HPV18 was found in 43 of 156 (28%) high-grade anal dysplasias, but not in any of the 11 anal cancers in our study. Although high-risk HPV types 16, 18 and 45 account for about 90% of AC of the cervix, none of these types was detected in the two ACs analysed here. Primary treatment of anal margin carcinoma is wide surgical excision. Prognosis in these patients is generally good, with a 5-year survival of 80%. 31 In line with this, none of our patients with anal margin cancer experienced local recurrence or distant disease. Standard therapy of anal canal cancer is CRT including 5-fluorouracil and mitomycin. Although there is evidence that HIV-positive and HIV-negative patients have similar rates of complete response following CRT, relapse-free survival and local disease control are significantly shorter, and side-effects related to CRT are significantly higher, in HIV-positive individuals. 32 Accordingly, two of our five patients with anal canal cancer had recurrent

8 8 Anal carcinoma in HIV-positive men, A. Kreuter et al. disease after CRT, and two of them died because of severe CRT-related side-effects. Tobacco smoking is a well-established risk factor for HPV-associated anogenital cancer. It was recently shown that smokers with anal cancer are more likely to die from recurrence than nonsmokers, with 45% of smokers being dead compared with only 20% of nonsmokers by 5 years after treatment. 33 In line with this, only one patient with canal ASCC had long-term response to CRT, and this patient had never smoked. Therefore, patients should be cautioned about tobacco smoking once anal carcinoma is diagnosed in an attempt to improve their long-term outcome. The results reported herein should be interpreted in light of the limitations of the study. We have performed an open study including both asymptomatic and symptomatic individuals, and treatment was offered to all patients with AIN of any grade. An observation-only arm would have provided important additional information on the progression rate of high-grade AIN to invasive cancer. A randomized controlled trial to demonstrate the efficacy of AIN treatment in reducing anal cancer morbidity and mortality has so far not been performed. There is increasing agreement in the clinical community that such a randomized trial should be performed, because a successful outcome could lead to the implementation of routine screening for at-risk patients. 10,22 Nevertheless, an observation-only arm could raise ethical concerns. We cannot exclude a bias towards an over-representation of anal dysplasia and anal cancer in a referral clinic specialized in HPV-associated disease in our study. Nevertheless, our data on anal dysplasia are very similar to those reported in the literature. 6,15 Finally, our observation period of 5 years and 10 months is probably too short to obtain valid insights into the natural history of HPV-associated malignant anal lesions in HIV-positive MSM. In conclusion, this study demonstrated that anal margin carcinoma and anal canal carcinoma differ substantially in their lesional HPV spectrum, prognosis and treatment response. Anal dysplasia is present in a high proportion of HIV-positive MSM, and high-grade AIN in these patients can progress to invasive cancer within a short period of time. There is increasing evidence that anal cancer is preventable by early treatment of its precursor lesions. Cytological AIN screening, ideally based on the results of randomized controlled studies, should be adopted in high-risk populations, including HRA in case of pathological findings. What s already known about this topic? Anal intraepithelial neoplasia (AIN) and anal cancer are frequent in HIV-positive men who have sex with men. Only few data exist on the progression of high-grade AIN to invasive cancer as well as on clinical and virological characteristics comparing anal margin and anal canal carcinoma. What does this study add? This prospective study demonstrated that high-grade anal dysplasia in HIV-positive men can progress to invasive cancer within a short period of time. Anal margin carcinoma and anal canal carcinoma differ substantially in their lesional human papillomavirus spectrum, prognosis and treatment response. Acknowledgments We thank Sabine Richter, Nicole Scholz, Tanja Blome, Monika Junk and Nabila Ristow for their excellent technical assistance. The Federal Ministry of Education and Research (grant no. 01 KI 0771, TP7), the German Network of Competence HIV AIDS (grant no. 01 KI 0501) and FoRUM (Forschungsförderung Ruhr University Bochum Medizinische Fakultät, project F ) funded this work. References 1 Clark MA, Hartley A, Geh JI. Cancer of the anal canal. Lancet Oncol 2004; 5: Kreuter A, Wieland U. Human papillomavirus-associated diseases in HIV-infected men who have sex with men. Curr Opin Infect Dis 2009; 22: Daling JR, Weiss NS, Hislop TG et al. Sexual practices, sexually transmitted diseases, and the incidence of anal cancer. N Engl J Med 1987; 317: 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 2002; 35: Patel P, Hanson DL, Sullivan PS et al. 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