Tsai, Tsen-Fang MD*; Kuo, Guan-Tin MD†; Kuo, Lu-Ting MD, PhD‡; Hsiao, Cheng-Hsiang MD†§
SQUAMOUS PROLIFERATIVE LESIONS, SUCH as condyloma, high-grade squamous intraepithelial neoplasm, and squamous cell carcinoma (SCC), are the most common neoplasms in the anal region.1
Histologically, the anus is similar to the lower female genital tract. In both the anal canal and uterine cervix, there is a transformation zone between the epithelia of the external squamous lining and the internal columnar cell lining. Biologically, these squamo-columnar junctions are especially susceptible to infection by the human papilloma virus (HPV).2 HPV is a ubiquitous virus with more than 100 types of the virus been identified worldwide.3 Genotypically, HPV can be divided into 2 groups according to the transforming activity of the virus. The low-risk groups are associated with condylomata acuminata and low-grade squamous intraepithelial lesion. The high-risk groups are usually found in high-grade squamous intraepithelial lesion and invasive cancer.3 Furthermore, type prevalence may vary with different races.4
Most HPV infection in the anorectal region gets through anal intercourse,5 whereas there are patients with anal wart or HPV infection but without sexual exposure.2 In their review of patients with anal cancer, Daling et al.6 found that men with anal cancer were more likely to have engaged in homosexual activity. Homosexual activity is also a risk factor for human immunodeficiency virus (HIV) infection, thus anal cancer should be more prevalent in patients with AIDS. Melbye et al.7 confirmed that AIDS patients in the United States incurred an 84-fold increase in relative risk for developing anal cancer compared with the general population. In addition, anal cancer occurs earlier in HIV-positive white men compared with HIV-negative white analogs (mean age 37 and 63 years, respectively). However, all the studies of anal cancer were from Western countries.6,8,9 The culture of Asian countries is quite different from that of Western countries, and the sexual attitude and prevalence of HIV infection in Asian community has been confirmed to be different from other ethnic population in the United States.10,11 It is important, therefore, to understand the status of anal squamous proliferative lesion (ASPL) in Asian country and to find out the prevalence of HIV infection in patients with ASPL.
Materials and Methods
Medical records of the patients were identified and retrieved from the archives of National Taiwan University Hospital (NTUH) from 1991 to 2005. NTUH is a major medical center in northern Taiwan. About 2.5% to 3.2% of total patients in Taiwan were admitted to NTUH each year. During the 15 years, 123 patients were diagnosed to have condyloma by histologic examination. Among them, 41 patients had lesions in the anal and perineal region and the other patients had condyloma in the genital organs. Anal high-grade squamous intraepithelial lesion (AHSIL) was diagnosed in 12 patients including 2 patients with coexistent anal condyloma. Forty-six patients with anal cancer were diagnosed in the same period and 22 of them had SCC. Of the 22 patients, 9 women with anal SCC also had advanced cervical cancer. Because the anal SCC in these 9 individuals was more likely secondary to the direct invasion of the cervical cancer, they were excluded in the following study. In the end, a total of 64 patients constituted the following study. Ten hemorrhoid specimens from patients with matched age and sex distribution were also collected as controls.
Formalin-fixed and paraffin-embedded (FFPE) surgical specimens of the 64 enrolled patients and the 10 control patients with hemorrhoid were retrieved from the archive of the department of pathology. Tissue DNA was extracted from the FFPE blocks. Briefly, two 10-μm slices of tissue were deparaffinized with 1000-μL xylene, which was then removed using alcohol before the precipitate was vacuum dried. The DNA pellet was extracted using a QIAamp DNA mini kit (Qiagen, Valencia, CA) according to the manufacturer's instruction. The adequacy of the extracted DNA was first evaluated by polymerase chain reaction (PCR) analysis of the β-actin gene. Samples with adequate DNA were subjected to further analysis. Where condyloma and AHSIL lesions were coexistent, microdissection of the various lesions was performed before DNA extraction.
HPV Gene Amplification by Nested PCR
PCR was used for amplification of the HPV gene with L1 consensus primers. Before amplification, the reaction mixture was treated with 1 IU of uracil-N glycosylase to prevent DNA carry-over, and with 0.2 g of TaqStart antibody (Clontech, Franklin Lakes, NJ) for hot-start PCR. Each PCR was performed in a Geneamp PCR System 9700 (Applied Biosystems, Foster City, CA) with the first denaturation step at 94°C for 3 minutes and a 5-minute final extension at 72°C. The conditions for, and number of, denaturation-annealing-extension cycles were dependent on the various primer sets. General consensus primers MY09/GP6+ were used for the first round PCR to amplify the corresponding part of the HPV L1 gene. Nested-PCR was then carried out with the primers GP5+/GP6+.12
Gene Chip HPV Genotyping
A HPV gene chip (Easychip HPV Genotyping Array, King Car, Taipei, Taiwan) was used to hybridize 15 μL of the amplified product, with revert-blot hybridization to detect 39 types of HPV DNA (6, 11, 16, 18, 26, 31, 32, 33, 35, 37, 39, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 72, 74, 82, CP8061, CP8304, L1AE5, MM4, MM7, and MM8) in a single reaction, as reported by Lai et al.13 Two independent PCR products of each specimen were hybridized using a gene chip. The 2 gene chip results were compared and cross-checked with the results of DNA sequencing analysis. Type-specific PCRs were performed to resolve any discrepancies between 2 gene chip results.
Evaluation of E2/E6 Ratio by qRT-PCR
Real-time PCR was performed using the ABI Prism 7700 Sequence Detection System and the TaqMan Universal PCR Master Mix (Applied Biosystems). Primer Express software (version 1.0b6; PE Applied Biosystems) was used to design the primers and probe for specific amplification of the E2 and E6 HPV open reading frames in this study. The E6 probe was labeled with 6-carboxyfluorescein at the 5′ end, and Dark Quencher (Scandinavian Gene Synthesis AB, Koping, Sweden) at the 3′ end. The E2 probe was labeled with BODIPY R6G (BODIPY dyes licensed to Scandinavian Gene Synthesis by Molecular Probes) at the 5′ end and Dark Quencher at the 3′ end.
The E2 and E6 amplimers were 76 and 81 bp, respectively. The final primer and probe concentrations (total volume 50 μL), were 0.3 and 0.1 μmol/L, respectively. Fifty nanograms of target DNA from biopsy specimens was applied to the reaction mixture. Two standard curves were obtained by amplification of a dilution series (50 million to 500 copies) of a HPV16 clone plotted against HPV copy number.
We analyzed the records and pathologic specimens of 64 patients: 41 with condyloma, 12 with AHSIL, and 13 with anal SCC. Among the 41 patients with anal condyloma, only 5 were women, and the remaining 36 patients were men (mean age, F: 40 and M: 36 years, respectively) and 2 of them were HIV-positive.
Twelve patients had AHSIL (M/F 8:4). Three of the 8 male patients were HIV-positive with a mean age 36 years and they all developed HIV in the last 5 years (2001–2005). The mean age of the remaining 5 HIV-negative males was 55 years. Of the 3 HIV-positive male patients, 1 had coexistent AHSIL and condyloma. The 4 female patients with AHSIL were all HIV-negative (mean age 71 years). All but 1 patient with condyloma or AHSIL had survived at the time of writing. Only 1 patient died because of pneumonia with sepsis.
Of the 13 patients with anal squamous carcinoma, 8 were men and 5 were women (mean age 68 and 66 years, respectively). Ten of the 13 patients died of complications of advanced anal cancer, such as sepsis, intestinal obstruction, tumor bleeding, etc, within 2 years after operation.
During the 15-year study period, the number of patients with anal condyloma increased. Only 2 patients were diagnosed with anal condyloma in the initial 5 years; however, there were 17 and 22 such cases in 1996–2000 and 2001–2005, respectively. By contrast, 2, 3, and 7 patients were diagnosed with AHSIL during the corresponding periods. The number of cases involving anal SCC also increased, but the rise was less significant by comparison with the other 2 diseases.
Tissue DNA was extracted from the FFPE blocks of 64 patients with ASPL and 10 control patients with hemorrhoids. In 7 cases of condyloma and 1 case of SCC, the DNA sample was inadequate for further analysis because of the absence of the β-actin gene in the extracted DNA by PCR analysis. The DNA samples of the remaining 56 patients were adequate for further HPV typing. Low-risk HPV group (including types 6, 11, and 54) were present in all but 1 of the 34 specimens of condyloma. Types 6 (n = 23) and 11 (n = 13) were the 2 major low-risk HPV types in the condylomatous lesions. High-risk HPV group (including types 16, 58, 31, and 18) were found in the lesions of AHSIL and anal SCC. HPV type 16, the most common high-risk group, was found in 7 of 12 and 10 of 13 of AHSIL and anal cancer lesions, respectively. HPV type 58 was the second frequent high-risk HPV type infecting 4 patients (2 AHSIL and 2 anal SCC). Multiple HPV infection was identified in 11 patients with condyloma (11 of 34) and 4 patients with ASHIL (4 of 12) including the 2 patients with coexistent condyloma and ASHIL. Only 1 of 13 patients with anal SCC had mixed HPV infection. One HIV-positive male patient with AHSIL had multiple high-risk HPV infection (HPV58/51/62). Two control hemorrhoid specimens also had HPV infection and they were infected with HPV11 and 31, respectively. No squamous proliferative lesion could be identified in the 2 control specimens after scrutinized the tissue sections. Detailed results of the HPV study are listed in Table 1.
HPV-16 integration status was evaluated by qRT-PCR of the 18 paraffin-embedded samples containing HPV-16; however, only 8 contained sufficient and adequate DNA for analysis. Integration was defined by absence of the E2 signal or E2/E6 ratios in the range 0.001 to 0.003. An E2/E6 ratio in the range 0.004 to 0.99 indicated the presence of both integrated and episomal forms, whereas ratios more than 1 indicated a predominance of episomal forms.14 Of the 8 samples in our study, only 1 patient had no E2 signal, indicating the presence of exclusively integrated form, whereas the other 7 patients had mixed forms with the E2/E6 ratio ranged from 0.08 to 0.98 and suggesting the presence of both the episomal and integrated variants. The detailed clinical information and HPV16 integration status for each patient are provided in Table 2.
It is well established that HPV is implicated in anogenital squamous cell neoplasia and more than 100 types of HPV has been identified. Genotypically, HPV can be divided into 2 groups according to the transforming activity of the virus. Low-risk types, such as type 6, 11, 42, 43, 44, 53, 54, and 62 are usually associated with condyloma. All but 1 condyloma in our study was infected with low-risk HPV; 23 cases with type 6 and 13 cases with type 11. The high-risk group, including type 16, 18, 31,33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 67, 68, and 70 are frequently found in high-grade intraepithelial neoplasia and invasive cancer.3,15 In our study, all the AHSIL and SCC specimens contained high-risk HPV genes. HPV type 16 was the most common high-risk type (65%) followed by HPV type 58 (17%). Other high-risk HPVs, including type 18, 31, 33, and 51, were identified occasionally. The high prevalence of HPV58 in our study is consistent with the study of cervical swab in Taiwanese women.13 HPV58 prevalence is not unique to Taiwan, it has also been reported in other Asian countries, such as Japan, China, and Hong-Kong.16,17
Multiple HPV infection was found in 14 patients, and it was most frequent in AHSIL (4 of 12) and condyloma (11 of 34) but least common in invasive cancer (1 of 12). This observation suggests that selection for high-risk HPV clones may occur during disease progression of anal cancer. Two of 10 hemorrhoid specimens in our study also had HPV DNA in their tissue extracts. The prevalence rate is similar to the general population of Taiwan.13
The development of HPV associated SCC should be due to persistent infection of high-risk HPV. Persistent infection with these HPV types favors integration of viral DNA into the host cells.18 Previous studies suggest that benign cervical HPV lesions mostly contain the viral DNA in episomal forms. In contrast, cervical cancer usually has integrated viral DNA into the host genome. Integration of viral DNA will disrupt E1 and E2 HPV16 ORF and decrease E2 protein expression but up-regulate the expression of the E6 and E7 oncoproteins. The overexpression of E6 and E7 oncoproteins may interfere with the normal cell cycle by targeting the p53 and pRb tumor-suppressor proteins, leading to cell proliferation.3 Thus, the ratio of E2/E6 may reflect the physical status of HPV16 and is closely associated with disease progression.14,19 In integration status, the E2/E6 ratio is usually less than 0.003. An E2/E6 ratio in the range 0.004 to 0.99 indicates the presence of both integrated and episomal forms, whereas ratios more than 1 implies a predominance of episomal forms.14 In our study, only 8 samples with HPV16 infection had enough tissue DNA for quantitative real-time PCR: 5 with AHSIL lesion and 3 with anal SCC. Only 1 anal SCC lesion contained exclusively integrated HPV16, whereas the other 7 samples had coexistent episomal and integrated forms of HPV16 DNA. However, the ratio of E2/E6 of these 7 specimens is not correlated with the severity of the dysplasia.
In the study, 4 male patients were HIV-positive and they all were diagnosed in the last 5 years between 2001 and 2005. Three of them developed AHSIL. The mean age of these HIV-positive men with AHSIL was much younger than that of HIV-negative analogs (36 vs. 55 years), and about the same as age of condyloma onset (36 years). Moreover, 3 of the 4 HIV-positive patients also had multiple HPV infection. The high prevalence of multiple HPV infection, early onset and high incidence of AHSIL in HIV-positive men, indicates that immunosuppressive status in HIV-positive patients may not only increase multiple HPV infection but it may also hasten the progression to AHSIL lesion. HSIL is a precancerous lesion; quite a few cases will progress to invasive SCC within a mean duration of 15 years.3 However, all the anal SCC in the study developed in the 6th or 7th decades similar to the era before onset of the HIV epidemic in Western countries. Increasing HIV-positive AHSIL in recent years in our study suggests that we should do more efforts to promote sexual safety in men who have sex with men. In addition, anal cytology screening or HPV vaccine in HIV-positive men who have sex with men may be helpful in early detection of AHSIL and prevention of HPV infection in the future.20
We also noted a substantial increase in the number of cases of anal cancer or precancerous lesions in NTUH between 1991 and 2005, this finding probably suggests increasing incidence in the population. However, many other factors, such as increasing patient's awareness and improving economic status, could contribute to the increasing number of persons with anal disease. We could not distinguish rising incidence from other possibilities by the retrospective study and more epidemiologic researches are indicated to understand these trends.
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