Fernanda N Carestiato

Prevalence of human papillomavirus infection in the genital tract determined by hybrid capture assay

Human Papillomavirus (HPV) infection is the most prevalent sexually-transmitted virus worldwide. It is known to be the etiological agent of cervical cancer and cervical intraepithelial neoplasia (CIN). Consequently, there is strong motivation to evaluate HPV testing in cervical cancer screening. Recently developed, the second generation of the hybrid capture test (HCA II) is a non-radioactive, relatively rapid, hybridization assay, designed to detect 18 HPV types divided into high and low-risk groups. We evaluated 7,314 patients (5,833 women and 1,481 men) for HPV infection by HCA II. Among them, 3,008 (41.1%) presented HPV infection: 430 (14.2%) had HPV DNA of low risk for cancer, 1,631 (54.2%) had high risk HPV types and 947 (31.5%) had both types. The prevalence in females was 44.9%. The prevalence of HPV DNA in the group for which cytological results were available was slightly higher: 55.3% (1007/1824). Significant differences were detected in the frequency of HPV infection of the cervix between normal cases and those with high-grade squamous-intraepithelial lesions (HSIL)(P<0.0001). Among males, the prevalence was 26.2%, composed of 9.1% in Group A, 9.7% in Group B and 7.4% with multiple infections. We observed that male prevalence was lower and that low-risk types were more frequent than in females. HPV viral load was significantly greater in SILs than in normal or inflammatory cases (P<0.0001), suggesting an association between high viral load values and risk of SIL. Because of high costs, the HCA II test cannot be recommended for routine mass screening for cervical infection in poor countries. Nevertheless, it was found to be a useful tool, when combined with cytology, discovering high-risk infections in apparently normal tissues and revealing silent infections that may be responsible for the maintenance of HPV in the general population. These findings point to the need for close and careful management of patients, thereby reducing overtreatment, allowing analysis of both sexual partners and finally contributing to the control of genital infections associated with a risk for cancer.

Human papillomavirus infection in Rio de Janeiro, Brazil: a retrospective study

There is considerable data to support a central role for human papillomavirus (HPV) in the etiology of cervical cancer. More than a 100 HPV types have been described, and 40 have been isolated from benign and malignant genital lesions. Consequently, there is strong motivation to evaluate HPV testing for cervical cancer screening. Few studies concerning the natural history of HPV infection have been conducted in the state of Rio de Janeiro. We determined the prevalence of HPV types in female genital lesions by using Hybrid Capture Assay (HCA) and we retrospectively analyzed the course of HPV infection. Our sample included 788 women attended at Laboratórios Sérgio Franco. The average age of the participants was 29.6 years. HPV prevalence and cytological diagnosis were determined. The overall prevalence of HPV DNA in the study group was 50.1% (395/788), ranging from 25% (NORMAL) to 100% in high-grade intraepithelial lesions (HSIL). High risk HPV was found in 12% inflammatory, 58.3% HPV, 63.2% LSIL and 100% HSIL. A retrospective analysis of 78 patients showed that 22 presented persistent lesions, 2 had progressive lesions, 4 had regressive lesions, 13 showed latent infections, 18 were transiently infected and 19 were submitted to curative treatment. No cases of cancer were registered in this population, which can afford private medical care and regular follow-up exams. We suggest that HCA be used in specific cases involving persistent and recurrent lesions.

AN UPWARD TREND IN DNA P16INK4A METHYLATION PATTERN AND HIGH RISK HPV INFECTION ACCORDING TO THE SEVERITY OF THE CERVICAL LESION

SUMMARY High-risk human papillomavirus (hr-HPV) infection is necessary but not sufficient for cervical cancer development. Recently, P16INK4A gene silencing through hypermethylation has been proposed as an important cofactor in cervical carcinogenesis due to its tumor suppressor function. We aimed to investigate P16INK4A methylation status in normal and neoplastic epithelia and evaluate an association with HPV infection and genotype. This cross-sectional study was performed with 141 cervical samples from patients attending Hospital Moncorvo Filho, Rio de Janeiro. HPV detection and genotyping were performed through PCR and P16INK4A methylation by nested-methylation specific PCR (MSP). HPV frequency was 62.4% (88/141). The most common HPV were HPV16 (37%), HPV18 (16.3%) and HPV33/45(15.2%). An upward trend was observed concerning P16INK4A methylation and lesion degree: normal epithelia (10.7%), low grade lesions (22.9%), high grade (57.1%) and carcinoma (93.1%) (p < 0.0001). A multivariate analysis was performed to evaluate an association between methylation, age, tobacco exposure, HPV infection and genotyping. A correlation was found concerning methylation with HPV infection (p < 0.0001), hr-HPV (p = 0.01), HSIL (p < 0.0007) and malignant lesions (p < 0.0001). Since viral infection and epigenetic alterations are related to cervical carcinoma, we suggest that P16INK4A methylation profile maybe thoroughly investigated as a biomarker to identify patients at risk of cancer.

Human papillomavirus infection among sexual partners attending a Sexually Transmitted Disease Clinic in Rio de Janeiro, Brazil

Cervical cancer is a major source of illness and death among women worldwide and genital infection with oncogenic human papillomavirus (HPV) its principal cause. There is evidence of the influence of the male factor in the development of cervical neoplasia. Nevertheless, the pathogenic processes of HPV in men are still poorly understood. It has been observed that different HPV types can be found among couples. The objective of the present study was to investigate HPV infections in female patients (n = 60 females/group) as well as in their sexual partners and to identify the concordance of HPV genotypes among them. By using the polymerase chain reaction, we detected a 95% prevalence of HPV DNA in women with cervical intraepithelial neoplasia (CIN) compared to 18.3% in women with normal cervical epithelium, with a statistically significant difference (P < 0.001). The HPV DNA prevalence was 50% in male partners of women with CIN and 16.6% in partners of healthy women. In the control group (healthy women), only 9 couples were simultaneously infected with HPV, and only 22.2% of them had the same virus type, showing a weak agreement rate (kappa index = 0.2). Finally, we observed that HPV DNA was present in both partners in 30 couples if the women had CIN, and among them, 53.3% shared the same HPV type, showing moderate agreement, with a kappa index of 0.5. This finding supports the idea of circulation and recirculation of HPV among couples, perpetuating HPV in the sexually active population, rather than true recurrences of latent infections.

Analysis of molecular biology techniques for the diagnosis of human papillomavirus infection and cervical cancer prevention

The objective of the present study was to evaluate the usefulness of molecular methodologies to access human papillomavirus genome in the genital tract. Samples from 136 women aged 17 to 52 years old obtained from the Dr. Sérgio Franco Laboratories between 2000 and 2001, were analyzed by the hybrid capture assay and amplified by PCR with generic primers MY09/MY11 and specific primers for types 16, 18, 31, 33, 35, 58. Viral genome was detected in 71.3% of the samples by hybrid capture and 75% by amplification. When cytopathology was used as a reference method for screening lesions, hybrid capture (p=0) and amplification (p=0.002) presented positive association. The 3 methods showed absolute agreement when cytopathology confirmed papillomavirus infection and high grade intraepithelial lesion. Disagreements occurred for 10 cases: seven inflammatory cases positive by PCR and negative for hybrid capture and 3 low squamous intraepithelial lesions positive for hybrid capture but negative for amplification. In conclusion, hybrid capture was shown to be sensitive and specific enough for use in clinical routines. Moreover, the evaluation of viral load values obtained by this method were shown to be related to the severity of the lesion and merit further studies to analyze the possible association with risk of progression to malignancy.

Human papillomavirus, Epstein‐Barr virus, and methylation status of p16ink4a in penile cancer

Little is known about penile carcinogenesis. The aim of this study was to evaluate the prevalence of HPV and EBV, and the methylation status of p16ink4a in penile cancer samples, and to contribute to the understanding of the mechanisms responsible for penile cancer development. HPV DNA was detected in 63.6% of 122 cases, with HPV16 being the most prevalent type. EBV DNA was detected in 47.7%, with EBV‐1 being the most prevalent type. HPV/EBV co‐infections were found in 27.3% of the cases. Hypermethylation in p16ink4a was detected in 64.5% of 110 tested cases. An association between the absence of HPV absence and p16ink4a hypermethylation was also found. Death and/or progressive disease was associated with grade (P = 0.001), T stage (P < 0.0001), and N stage (P < 0.0001). In the multivariable model, grade and N stage were independent risk factors for disease‐free survival (P = 0.008 and P < 0.001, respectively). Patients without viral infection had a median age significantly lower than that of the HPV‐infected patients. We suggest at least two pathways for penile carcinogenesis, one HPV‐independent linked to epigenetic events, probably via p16ink4a inactivation; and another, dependent on HPV infection.

P3.236 Study of genital cancer aetiology: association of human papilomavirus (HPV) and merkel cell polyomavyrus (MCPYV)

Introduction The genital infection by the HPV is among the most frequent sexually transmitted diseases (STD) worldwide, and it may result in lesions that can lead to the carcinogenesis of the genital tract. However, other factors may be associated with the onset or progression of the tissue malignancy process, such as the MCPyV, which may present oncogenic profile in the epithelial tissue. This study aims to investigate the presence of MCPyV and HPV in malignant lesions of the male and female genital tract, in order to contribute to the elucidation of the role of these viruses in the cellular malignancy process and to the epidemiological knowledge regarding the prevalence of both viruses in neoplastic lesions. Methods This is a cross-sectional study evaluating the prevalence of HPV and MCPyV infection in samples of cervical carcinoma and penile cancer. To date, we have obtained 112 samples of penile carcinoma and 31 samples of cervical carcinoma. So, we aim to detect the presence of HPV DNA by the polymerase chain reaction (PCR) technique using the generic primers MY09/MY11; to genotype HPVs by specific PCR to the E6 gene; to detect and quantify DNA of the MCPyV by the Nested PCR technique and real-time PCR; to investigate the presence of truncation mutations in the major T antigen of MCPyV. Results Results are partial. To date, all the male samples were analysed. We verified the presence of HPV in 54 (48.2%) of these samples, in which the most prevalent type was the HPV16 (66%). The cervical carcinoma samples are still under analysis. Conclusion The collection of cervical neoplasia samples is still being performed. In 2015, our research group found a case of multiple infection by HPV, MCPyV and Epstein-Barr virus in a case of squamous cell carcinoma of the penis in Rio de Janeiro. This was the first report of the presence of MCPyV in this type of penile lesion. Thus, we look forward to find results that contribute to the presence of MCPyV in genital malignant lesions and to elucidate its role in the oncogenic pathway of malignant lesions.

P3.207 Human papillomavirus infection in oral and anogenital sites: prevalence and rates of concordance

Introduction HPV infection causes cancer at several anatomical sites. However, the natural history of the infection in non-cervical sites have been understudied, especially at the oral epithelium. Methods In our study, we investigated 351 samples from three different sites of 117 patients, searching for HPV By generic and specific PCR and Microarray, and related risk factors. Results HPV DNA prevalence was 89.5% (105/117) in the genital lesions, 53.8% (63/117) in oral samples and 59% (69/117) in anal samples. Regarding the risk factors associated with HPV in the genital lesions, we found statistically significant rates for oral (p=0.039) and anal sex practices (p=0.0000012). For oral samples, we observed a relevant correlation concerning oral contraceptive use (p=0.039), tobacco smoking (p=0.036) and alcohol use (p=0.0075) while in anal samples, we found higher risk for HPV infection in patients relating non-exclusive sexual partners (p=0.013). The presence of viral DNA in all the three sites was observed in 36.8% of the cases (43/117). Among them, 18% (21/117) presented concordant HPV genotypes, diverging from the literature, corroborating that there is still much to learn about HPV natural history, since different biological behaviours are expected within different populations. Conclusion In our study we also evaluated if the detection of oral HPV would suggest an infection in the anogenital tract. Nevertheless, our results showed only 36.8% of correlation pointing out that it is not suitable as a an auxiliary biomarker for HPV anogenital infections.