Background

Squamous cell carcinoma (SCC) compromises more than 70% of all anal cancer malignancies, and represents 1.5 to 3% of cancers of the gastrointestinal tract [1, 2]. Persistent infection with high-risk types of human papillomavirus (HR-HPV) is responsible for >80% of cases. SCC mainly affects women over the age of 50 years; among men, SCC predominantly affects individuals in the age range of 20 to 49 years [3]. There are few epidemiological data for anal human papillomavirus (HPV) infection in Brazil. According to Instituto Nacional de Câncer (INCA), in Brazil, anal cancer accounts for 1 to 2% of all colon tumors and 2 to 4% of all cancers that affect the large intestine [4]. This rate rises to alarming proportions in specific risk groups, such as in homosexual men and in HIV-positive patients. Palesfky et al. [5] reported that the incidence of anal HPV was 61 and 93% in HIV-seronegative and HIV-seropositive homosexual male patients, respectively. In women, the corresponding values were 42 and 76%, respectively.

Although HPV infection is essential for the development of anal SCC, the exact frequency of this disease is not known. In Brazil, the data regarding this type of cancer are included in the statistics for colorectal cancer. Therefore, epidemiological data representative of a specific region is essential for implementing particular guidelines for treatment and prevention, e.g. an effective vaccination schedule, especially in high-risk groups. HPV infection, a sexually transmitted disease (STD) with potentially malignant development, that affects both men and women, is of global concern. There is a lack of studies about anal cancer in the Northeast region of Brazil, where social-economic conditions are precarious. Therefore, a thorough study of the prevalence and genotyping of HPV in patients diagnosed with anal cancer in São Luís, a city of the Northeast region, is potentially important for the prevention and early diagnosis of this disease. In addition, the understanding of HPV prevalence and knowledge of the viral subtype distribution constitute important epidemiological information that can assist the development of local or regional public policies to prevent HPV and of new vaccines.

The aims of this study were to detect and assess genotype-specific HPV prevalence in patients with anal SCC and to examine the association between viral presence and clinicopathological aspects of disease.

Methods

Enrollment

Retrospective study performed in paraffin-embedded anal tumor tissue samples collected from two public reference hospitals in Maranhão, a city in the Northeast region of Brazil. The 27 samples included in the study were from patients diagnosed with anal cancer between the years of 2001 and 2011. Patient information and data on histopathological characteristics of tumors were obtained from medical records. As the samples were obtained from paraffin-embedded tumors, written informed consent was not required from the patients. The identity of the patients was not disclosed in this study. This work was approved by the Ethics in Research Committee of the University Hospital of the Federal University of Maranhão (n° 348.397).

Inclusion criteria:

Paraffin blocks and histological slides of anal tumors as a result of biopsy or surgical treatment at any follow–up in the archives of the Pathology Services. Exclusion criteria: Patients in which reports did not provide complete information.

HPV detection

The histopathological characteristics of the samples were reviewed by the pathologist, and blocks with tumor representativeness (over 50% of the total area of the fragment) were selected. After microtomy, the sections were deparaffinized and stored at 4°C for DNA extraction. The extraction of genomic DNA from the samples was performed using the QIAamp DNA FFPE Tissue Purification Kit (QIAGEN®) according to the extraction protocol suggested by the manufacturer.

The amplification and HPV genotyping was performed using the INNO-LiPA HPV Genotyping Extra kit (INNOGENETICS), which is capable of detecting 28 different HR types of HPV (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82), a number of low-risk HPV genotypes (6, 11, 40, 43, 44, 54,and 70), and the additional types 69, 71, and 74. Hybridized band signals corresponding to HPV types were visually interpreted according to the manufacturer’s instructions.

Statistical analysis

The data was tabulated using the Microsoft Office® Excel 2010 application for calculation of frequencies, mean, median, standard deviation, minimum, and maximum of the variables used in the EpiInfo 7 program. The descriptive statistics for categorical variables were represented as a distribution of frequencies. Means, medians, standard deviation, minimum, and maximum values, and confidence intervals were used to represent numerical variables. Tables and graphs were constructed following the descriptive analysis. In order to evaluate the relationship between epidemiological and clinical data and the presence of HPV, the chi-square test and Fisher’s exact test were used, and the results were considered statistically significant at 5% probability and for p < 0.05.

Results

Anal tumor biopsies were evaluated in 27 patients aged 32–85 years, with a mean age of 54.96 years and standard deviation of ± 15.81, presenting a higher prevalence between 42 and 52 years (Table 1). Among the patients involved in the study, 74.07% (20/27) were female. The demographic and distribution of clinical is shown on Table 1.

Table 1 Age and clinical presentation of 27 patients diagnosed with anal cancer
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With regard to the location of the lesions, the anal canal was the most affected area. Vegetating and ulcerative lesions, observed in 22.22% of the cases, were the most commonly occurring type of lesion. Most lesions (55.56%) were between 2.1 and 5.0 cm in size.

Table 2 demonstrate the pathological characteristics of the anal tumors analyzed. According to UJCC/TNM staging, most tumors were grade III (33.33%) and moderately differentiated (70.38%). The findings indicate that invasion was present in 33.33% of the tumors examined, and that infiltration occurred in at least one of the structures (anal canal wall, perianal region, perirectal fat, vagina and uterine cervix, stroma, muscle layer, perineural and vulvar). Lymph node involvement was found in 25.93% of the tumors examined.

Table 2 Pathologic characteristics of anal tumors from 27 patients diagnosed with anal cancer
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HPV DNA was detected in 81.48% (22/27) of the anal tumor samples. All tumors (100%) were found to be infected with high-risk viral types; HPV-16 was present in all samples (Fig. 1). Among the high-risk HPV types, types 18, 29, 39, 45, 52, 59, 66, and 68 were also present. HPV-11 was the only low-risk genotype detected. In addition, in 50.09% (12/22) of the samples, only one viral type (simple infection) was detected. HPV-16 infection, which is associated with high oncogenic risk, was found in all cases studied. The prevalence of multiple infections in the samples studied was 45.45% (10/22) (Table 3).

Fig. 1
figure 1

The graphic shows the number of samples infected with the different HPV types. All positive samples were infected with HPV-16. HPV-52 was present in 7 samples; HPV-11 and HPV-18 in 6 samples; HPV-69 in 3 samples; HPV-29, HPV-39, HPV-59, and HPV-66 in 2 samples each; and HPV-45 in 1 sample

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Table 3 HPV prevalence and distribution according to oncogenic risk in 27 patients diagnosed with anal cancer
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No association was found (p < 0.05) between HPV infection and histopathological and clinical variables (Table 4).

Table 4 Association between clinical presentation and pathological characteristics in HPV infected patients with anal cancer
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Discussion

The results of the present study indicate that most patients with anal SCC was 54.96 years constituting a sample younger than that reported in the literature. According to several researches, the peak incidence of anal cancer is between the ages of 58 and 64 years [6,7,8,9,10]. The mean age of patients with anal cancer may had vary among studies, possibly because of the type of population studied and the presence of risk factors such as sexual orientation, immunosuppression (patients with HIV+ infection and transplant recipients), tobacco smoking, and alcoholism [6,7,8,9,10].

We did not evaluated the social economic status of our population but the study was developed on a very poor area of Brazil, with one of the lowest human development indexes. According to National Cancer Institute, cervical cancer and penile cancer are the second more incident type of cancer among women and men, respectively, in Maranhão [4]. Both cancer, as well as anal SCC, are associated with HPV infection and with poor social-economic status, health conditions and hygiene practices of the population. This condition may explain the younger status of our studied population.

Our samples presented a high prevalence of HPV, which are consistent with those reported in the literature. Alemany and coauthors applied a retrospective approach (1986–2011) to evaluate 496 cases of invasive anal cancer for DNA-HPV presence in a multi-center study involving patients from Europe, North America, Latin America, Africa, and Asia. The prevalence of HPV infection for each continent was 87.6, 95.8, 90.4, 61.9, and 81.1%, respectively [10]. In Brazil, Aguiar and co-authors conducted a study of patients with anal carcinomas in Goiânia-Goiás, and found that 76.3% of the samples collected were positive for HPV [9]. The results of our study also corroborate the findings of Varnai and colleagues [11], Ramamoorthy and colleagues [12], de Vuyst and colleagues [13], and Rodel and colleagues [14], who reported the detection of HPV-DNA presence in 60.6, 80, 73, and 95.8% of anal cancer samples examined, respectively.

Torres Neto and colleagues [15] performed a retrospective study about mains demographics characteristics of anal cancer patients in Sergipe, another city of Northeast State of Brazil. However, they did not performed any HPV analysis.

Infection with oncogenic, or high-risk, HPV-16 was detected in all cases. Other studies also report that HPV-16 was the main HPV type found in over 80% of carcinomas [10,17,, 16–18].

Female patients constituted the majority of the cases on our work. Alemany and co-authors [10] found that female patients constituted the majority of the cases of SCC (66.3%). Ouhoummanea and co-authors [8] found that the majority of incidence of anal cancer (60%) was in women. Aguiar and colleagues found that 62.8% of patients with anal cancer were women [9]. The results of a study of Czech patients with anal cancer by Tachezy et al. [6], in which the same number of patients was analyzed as in our study, revealed that 52.4% of the patients were female. Two other studies, conducted in Brazil, found that the occurrence of anal cancer was 62.8–78.8% among the female patients studied [19, 20].

The prevalence of anal SCC in women may be explained by the anatomical proximity between the vaginal opening and the anus. A study by Giraldo and co-authors [21] of 184 Brazilian women demonstrated a significant association between cervical intraepithelial lesions and anal intraepithelial lesions. Anal lesions were found to be present in 17.4% of patients with genital lesions, but only in 3.2% of patients without genital lesions. HPV infection in women may also be facilitated by non-sexual practices and self-inoculation (via vaginal secretions, transfer of fomites, and digital transfer), in view of the anatomical proximity between the vaginal opening and the anus [11, 22].

In what concerns the pathologic characteristics of the anal SCC samples, the most common morphology type observed on our work was the ulcerative form, presented a size of 2.1–5.0 cm, came from the anal canal region and moderately differentiated. However, the majority did not present invasion, infiltration, metastasis, and lymph node involvement. These clinical pathological characteristics are in accordance with others findings. Salati and Kadi [23] reported that the mean size of anal tumors investigated in their study was 3.9 cm. They report that spread of anal cancer is mainly local and regional. Usually, the tumor spreads into the ischiorectal fossae, the prostatic urethra and bladder in men, and the vagina in women. Anal cancer may spread via the lymphatic vessels (10–15%). Hematogenous spread develops in fewer than 10% of cases [23]. Studies have shown that the incidence of nodal metastasis in anal cancer is low (approximately 10%) [17, 24]. Ouhoummanea and co-authors [8] reported that, of the tumors studied, 43% were moderately differentiated.

No statistically significant association (p < 0.05) was found between HPV infection and clinical and histopathological variables. Other reports have also concluded that anal tumors are highly likely to be positive for infection with HR-HPV, regardless of the morphology of the tumor [6].

The understanding of HPV prevalence and knowledge of the viral subtype distribution constitute important epidemiological information that can assist the development of local or regional public policies to prevent HPV and of new vaccines. The present study, in agreement with other epidemiological and molecular studies, demonstrates that HPV infection is an important etiological agent of anogenital cancer [21, 25].

Conclusion

HPV DNA was found in 22 of the 27 analyzed samples (81.48%). The high-risk type HPV 16 was observed in 100% of the lesions while HPV-11 was the only low-risk genotype detected. The average age of the patients in the study was 54.96 years old. Prevalent lesions were larger than 2 cm, grade III (33.33%) and moderately differentiated (70.38%). The clinical and histopathological variables did not tend to have an association with infection by the HPV virus.