Search

Background The development of efficient strategies for managing high-risk human papillomavirus (HR-HPV)-positive women is a major challenge when human papillomavirus-based primary screening is being performed. The objectives of this study were to evaluate the comparative effectiveness of HR-HPV testing based on self-collection (SC) and HR-HPV testing based on collection by a health professional (HP) and to assess the potential usefulness of HR-HPV testing combined with testing with the biomarkers p16/Ki-67, α-mannosidase, and superoxide dismutase 2 (SOD2). Methods This was a cross-sectional study of 232 women admitted for colposcopy because of an abnormal Papanicolaou smear. The collected material underwent liquid-based cytology, HR-HPV detection, and immunocytochemical testing (p16/Ki-67, α-mannosidase, and SOD2). The gold standard was the histopathological result; the positive reference was CIN2+. Results The overall accuracy of HR-HPV testing was 76.6%; the results for the SC group (78.1%) and the HP group (75.2%) were similar. The positive predictive values (HP, 76.5%; SC, 80.0%), the negative predictive values (HP, 66.7%; SC, 64.3%), the positive likelihood values (HP, 1.35; SC, 1.36), and the negative likelihood values (HP, 0.21; SC, 0.19) were also similar. p16/Ki-67 showed higher sensitivity than the other 2 biomarkers: 78.1% versus 45.8% for α-mannosidase and 44.5% for SOD2. The specificities of the biomarkers were equivalent: 71.4% for p16/Ki-67, 77.8% for α-mannosidase, and 71.2% for SOD2. In the HP group, accuracy also leaned more heavily toward the final score (using α-mannosidase and SOD2) without statistical significance (80.8% vs 77.9%). The contrast with the SC group yielded the same level of accuracy. Conclusions SC, when associated with testing with biomarkers, is as accurate as collection by HPs in the detection of women at risk for cervical cancer.

It is suggested that HPV-18 variants from the A lineage have higher oncogenic potential compared to B variants. Some studies show uneven distribution of HPV-18 variants in cervical adenocarcinomas and squamous cell carcinomas. Regarding HPV-18 variants’ functions, the few studies reported focus on E6, and none were performed using natural host cells. Here, we immortalized primary human keratinocytes (PHKs) with E6/E7 of HPV-18 A1 and B1 sublineages and functionally characterized these cells. PHK18A1 reached immortalization significantly faster than PHK18B1 and formed a higher number of colonies in monolayer and 3D cultures. Moreover, PHK18A1 showed greater invasion ability and higher resistance to apoptosis induced by actinomycin-D. Nevertheless, no differences were observed regarding morphology, proliferation after immortalization, migration, or epithelial development in raft cultures. Noteworthy, our study highlights qualitative differences among HPV-18 A1 and B1 immortalized PHKs: in contrast to PHK18A1, which formed more compact colonies and spheroids of firmly grouped cells and tended to invade and migrate as clustered cells, morphologically, PHK18B1 colonies and spheroids were looser, and migration and invasion of single cells were observed. Although these observations may be relevant for the association of these variants with cervical cancer of different histological subtypes, further studies are warranted to elucidate the mechanisms behind these findings.

Introduction: The main risk factor for the development of cervical cancer (CC) is persistent infection by human papillomavirus (HPV) oncogenic types. In order to persist, HPV exhibits a plethora of immune evasion mechanisms. PI3/Elafin (Peptidase Inhibitor 3) is an endogenous serine protease inhibitor involved in epithelial protection against pathogens. PI3/Elafin’s role in CC is still poorly understood. Materials and Methods: In the present study, we addressed PI3/Elafin protein detection in 123 CC samples by immunohistochemistry and mRNA expression in several datasets available at Gene Expression Omnibus and The Cancer Genome Atlas platforms. Results: We observed that PI3/Elafin is consistently downregulated in CC samples when compared to normal tissue. Most of PI3/Elafin-positive samples exhibited this protein at the plasma membrane. Besides, high PI3/Elafin expression at the cellular membrane was more frequent in in situ stages I + II than in invasive cervical tumor stages III + IV. This indicates that PI3/Elafin expression is gradually lost during the CC progression. Of note, advanced stages of CC were more frequently associated with a more intense PI3/Elafin reaction in the nuclei and cytoplasm. Conclusion: Our results suggest that PI3/Elafin levels and subcellular localization may be used as a biomarker for CC severity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 19 (COVID-19), was declared pandemic by the World Health Organization in March 2020. SARS-CoV-2 binds its host cell receptor, angiotensin-converting enzyme 2 (ACE2), through the viral spike (S) protein. The mortality related to severe acute respiratory distress syndrome (ARDS) and multi-organ failure in COVID-19 patients has been suggested to be connected with cytokine storm syndrome (CSS), an excessive immune response that severely damages healthy lung tissue. In addition, cardiac symptoms, including fulminant myocarditis, are frequent in patients in a severe state of illness. Diacerein (DAR) is an anthraquinone derivative drug whose active metabolite is rhein. Different studies have shown that this compound inhibits the IL-1, IL-2, IL-6, IL-8, IL-12, IL-18, TNF-α, NF-κB and NALP3 inflammasome pathways. The antiviral activity of rhein has also been documented. This metabolite prevents hepatitis B virus (HBV) replication and influenza A virus (IAV) adsorption and replication through mechanisms involving regulation of oxidative stress and alterations of the TLR4, Akt, MAPK, and NF-κB signalling pathways. Importantly, rhein inhibits the interaction between the SARS-CoV S protein and ACE2 in a dose-dependent manner, suggesting rhein as a potential therapeutic agent for the treatment of SARS-CoV infection. Based on these findings, we hypothesize that DAR is a multi-target drug useful for COVID-19 treatment. This anthraquinone may control hyperinflammatory conditions by multi-faceted cytokine inhibition and by reducing viral infection.

Alterations in specific DNA damage repair mechanisms in the presence of human papillomavirus (HPV) infection have been described in different experimental models. However, the global effect of HPV on the expression of genes involved in these pathways has not been analyzed in detail. In the present study, we compared the expression profile of 135 genes involved in DNA damage repair among primary human keratinocytes (PHK), HPV-positive (SiHa and HeLa) and HPV-negative (C33A) cervical cancer derived cell lines. We identified 9 genes which expression pattern distinguishes HPV-positive tumor cell lines from C33A. Moreover, we observed that Three Prime Repair Exonuclease 1 (TREX1) expression is upregulated exclusively in HPV-transformed cell lines and PHK expressing HPV16 E6 and E7 oncogenes. We demonstrated that TREX1 silencing greatly affects tumor cells clonogenic and anchorage independent growth potential. We showed that this effect is associated with p53 upregulation, accumulation of subG1 cells, and requires the expression of E7 from high-risk HPV types. Finally, we observed an increase in TREX1 levels in precancerous lesions, squamous carcinomas and adenocarcinomas clinical samples. Altogether, our results indicate that TREX1 upregulation is important for cervical tumor cells growth and may contribute with tumor establishment and progression., This research was supported by grants of FAPESP (2010/20002-0; 2008/57889-1) and CNPq (480552/2011-8); and fellowships for BP (CNPq 573799/2008-3; CAPES 1524553), WSA (FAPESP 2012/16512-8), MM (FAPESP 2011/14416-9), SH (2013/27006-9).

Persistent infection with high-risk human papilloma virus (HR-HPV) is the main risk factor for the development of invasive cervical cancer although is not sufficient to cause cervical cancer. Several host and environmental factors play a key role in cancer initiation/progression, including cytokines and other immune-response mediators. Here, we characterized the response to the individual and combined action of the pro-inflammatory cytokines tumor necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL) on HPV-transformed cells and human keratinocytes ectopically expressing E6 and E7 early proteins from different HPV types. We showed that keratinocytes expressing HPV early proteins exhibited global alterations in the expression of proteins involved in apoptosis regulation/execution, including TNF and TRAIL receptors. Besides, we provided evidence that TNF receptor 1 (TNFR1) was down-regulated and may be retained in the cytoplasm of keratinocytes expressing HPV16 oncoproteins. Finally, fluorescence analysis demonstrated that cytokine treatment induced the production and release of reactive oxygen and nitrogen species (ROS/RNS) in cells expressing HPV oncogenes. Alterations in ROS/RNS production and apoptosis regulatory factors expression in response to inflammatory mediators may favor the accumulation of genetic alterations in HPV-infected cells. Altogether, our results suggested that these events may contribute to lesion progression and cancer onset.

In vitro culture of primary or established cell lines is one of the leading techniques in many areas of basic biological research. The use of pure or highly enriched cultures of specific cell types obtained from different tissues and genetics backgrounds has greatly contributed to our current understanding of normal and pathological cellular processes. Cells in culture are easily propagated generating an almost endless source of material for experimentation. Besides, they can be manipulated to achieve gene silencing, gene overexpression and genome editing turning possible the dissection of specific gene functions and signaling pathways. However, monolayer and suspension cultures of cells do not reproduce the cell type diversity, cell-cell contacts, cell-matrix interactions and differentiation pathways typical of the three-dimensional environment of tissues and organs from where they were originated. Therefore, different experimental animal models have been developed and applied to address these and other complex issues in vivo. However, these systems are costly and time consuming. Most importantly the use of animals in scientific research poses moral and ethical concerns facing a steadily increasing opposition from different sectors of the society. Therefore, there is an urgent need for the development of alternative in vitro experimental models that accurately reproduce the events observed in vivo to reduce the use of animals. Organotypic cultures combine the flexibility of traditional culture systems with the possibility of culturing different cell types in a 3D environment that reproduces both the structure and the physiology of the parental organ. Here we present a summarized description of the use of epithelial organotypic for the study of skin physiology, human papillomavirus biology and associated tumorigenesis.

Cervical cancer, caused by high oncogenic risk Human Papillomavirus (HPV) infection, continues to be a public health problem, mainly in developing countries. Using peptide phage display as a tool to identify potential molecular targets in HPV associated tumors, we identified α-mannosidase, among other enriched sequences. This enzyme is expressed in both tumor and inflammatory compartment of the tumor microenvironment. Several studies in experimental models have shown that its inhibition by swainsonine (SW) led to inhibition of tumor growth and metastasis directly and indirectly, through activation of macrophages and NK cells, promoting anti-tumor activity. Therefore, the aim of this work was to test if swainsonine treatment could modulate anti-tumor immune responses and therefore interfere in HPV associated tumor growth. Validation of our biopanning results showed that cervical tumors, both tumor cells and leukocytes, expressed α-mannosidase. Ex vivo experiments with tumor associated macrophages showed that SW could partially modulate macrophage phenotype, decreasing CCL2 secretion and impairing IL-10 and IL-6 upregulation, which prompted us to proceed to in vivo tests. However, in vivo, SW treatment increased tumor growth. Investigation of the mechanisms leading to this result showed that SW treatment significantly induced the accumulation of myeloid derived suppressor cells in the spleen of tumor bearing mice, which inhibited T cell activation. Our results suggested that SW contributes to cervical cancer progression by favoring proliferation and accumulation of myeloid cells in the spleen, thus exacerbating these tumors systemic effects on the immune system, therefore facilitating tumor growth.

Oxidative stress reflects an imbalance in the maintenance of the intracellular redox state resulting in the accumulation of oxidant species that may contribute to tumorigenesis. The superoxide dismutase 2 protein (SOD2) contributes to cell homeostasis by catalyzing the dismutation of superoxide anion radicals in oxygen and hydrogen peroxide, preventing the direct inactivation of biomolecules. SOD2 protein can be induced by the activation of the NF-kB pathway in several cell types following treatment with interleukin 1β (IL-1β) and tumor necrosis factor alpha (TNFα), both proinflammatory cytokines. In addition, previous studies from the group have shown a direct correlation between increased levels of SOD2 protein and severity of uterine cervix lesions. The aim of this study is to elucidate the involvement of SOD2 in the pathogenesis associated with HPV and in the effect of proinflammatory cytokines. Methods: Primary human keratinocytes transduced with HPV16 oncogenes and cervical cancer derived cell lines were treated with the proinflammatory cytokines TNFα, and IL-1beta. The expression of SOD2 protein involved in IL-1 β and TNFα signaling pathways was determined by Western blot and ELISA. Results: Preliminary results suggest the presence of higher levels of SOD2 expression in uterine cervix-derived tumor cell lines when compared to normal primary keratinocytes. Treatment with IL-1β and TNFα induces SOD2 protein accumulation in in the HPV16 positive cervical-derived tumor line, SiHa. On the other hand, no SOD2 induction was observed in the HPV-negative cervical cancer derived cell line C33. Further replications and additional experiments are needed for conclusions. Citation Format: Gabriela Ávila Fernandes Silva, Rafaella Almeida Lima Nunes, Enrique Boccardo, Luisa Lina Villa, Lara Termini. Study of superoxide dismutase-2 protein in HPV-mediated cell transformation [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B69.

"Manuscript", BACKGROUND: Matrix metalloproteinases (MMP) are important enzymes in the tumor microenvironment associated with progression of cervical intraepithelial neoplasia (CIN) toward squamous cell carcinoma (SCC) of the cervix. However, the role of MMPs in the inflammatory process associated with Chlamydia trachomatis infection concomitant with the carcinogenic process driven by HPV has not yet been addressed. In the present study, we analyzed the state of the MMP-9-RECK axis in cervical carcinogenesis. METHODS: The levels of MMP-9 and RECK expression were analyzed by immunocytochemistry in liquid-based cytology samples from 136 women with high-grade cervical lesions (CIN2/CIN3) and cervical SCC diagnosed by LLETZ, and in 196 women without cervical neoplasia or CIN1. Real-time qPCR was performed to analyze expression of MMP-9 and RECK in 15 cervical samples. The presence of HPV-DNA and other genital pathogens was evaluated by PCR. RESULTS: We found a higher expression of MMP-9 [OR, 4.2; 95% confidence interval (CI), 2.2-7.8] and lower expression of RECK (OR, 0.4; 95% CI, 0.2-0.7) in women with CIN2/CIN3/SCC when compared with women from the control group (no neoplasia/CIN1). A statistically significant association was also found between MMP-9/RECK imbalance and infection by alpha-9 HPV and C. trachomatis. The prevalence of C. trachomatis infection was significantly higher in women with high-grade cervical disease (OR, 3.7; 95% CI, 1.3-11.3). CONCLUSIONS: MMP-9/RECK imbalance in cervical smears is significantly associated with high-grade cervical diseases and infection by alpha-9 HPV and C. trachomatis. IMPACT: MMP-9/RECK imbalance during cervical inflammation induced by C. trachomatis might play a role in HPV-mediated cervical carcinogenesis., This work was supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), numbers 2008/03232-1 (to L.L. Villa) and 2012/09746-2 (to M.G. Discacciati and S.S. Maria-Engler) and National Counsel of Technological and Scientific Development pharmaceutical innovation (CNPQ-INCT-if; to S.S. Maria-Engler).

Using cDNA fragments from the FAPESP/lICR Cancer Genome Project, we constructed a cDNA array having 4512 elements and determined gene expression in six normal and six tumor gastric tissues. Using t-statistics, we identified 80 cDNAs whose expression in normal and tumor samples differed more than 3.5 sample standard deviations. Using Self-Organizing Map, the expression profile of these cDNAs allowed perfect separation of malignant and non-malignant samples. Using the supervised learning procedure Support Vector Machine, we identified trios of cDNAs that could be used to classify samples as normal or tumor, based on single-array analysis. Finally, we identified genes with altered linear correlation when their expression in normal and tumor samples were compared. Further investigation concerning the function of these genes could contribute to the understanding of gastric carcinogenesis and may prove useful in molecular diagnostics.

Polymorphism in the Toll-like receptor (TLR) 9 gene has been shown to have a significant role in some diseases; however, little is known about its possible role in the natural history of human papillomavirus (HPV) infections. We investigated the association between a single-nucleotide polymorphism (SNP) (rs5743836) in the promoter region of TLR9 (T1237C) and type-specific HPV infections. Specimens were derived from a cohort of 2462 women enrolled in the Ludwig–McGill Cohort Study. We randomly selected 500 women who had a cervical HPV infection detected at least once during the study as cases. We defined two control groups: (i) a random sample of 300 women who always tested HPV negative, and (ii) a sample of 234 women who were always HPV negative but had a minimum of ten visits during the study. TLR9 genotyping was performed using bidirectional PCR amplification of specific alleles. Irrespective of group, the WT homozygous TLR9 genotype (TT) was the most common form, followed by the heterozygous (TC) and the mutant homozygous (CC) forms. There were no consistent associations between polymorphism and infection risk, either overall or by type or species. Likewise, there were no consistently significant associations between polymorphism and HPV clearance or persistence. We concluded that this polymorphism in the promoter region of TLR9 gene does not seem to have a mediating role in the natural history of the HPV infection.

We thank Dr. Alda Wakamatsu, Dr. Venâncio Avancini and Dr. Alexandre Ab'Saber from Faculty of Medicine, University of São Paulo, for technical assistance and selection of tissues samples, and João Simão Pereira Sobrinho for help in protein sample processing., Cervical cancer is the third most common cancer in women worldwide. Persistent infection with high-risk HPV types, principally HPV16 and 18 is the main risk factor for the development of this malignancy. However, the onset of invasive tumor occurs many years after initial exposure in a minority of infected women. This suggests that other factors beyond viral infection are necessary for tumor establishment and progression. Tumor progression is characterized by an increase in secretion and activation of matrix metalloproteinases (MMPs) produced by either the tumor cells themselves or tumor-associated fibroblasts or macrophages. Increased MMPs expression, including MMP-2, MMP-9 and MT1-MMP, has been observed during cervical carcinoma progression. These proteins have been associated with degradation of ECM components, tumor invasion, metastasis and recurrence. However, few studies have evaluated the interplay between HPV infection and the expression and activity of MMPs and their regulators in cervical cancer. We analyzed the effect of HPV16 oncoproteins on the expression and activity of MMP-2, MMP-9, MT1-MMP, and their inhibitors TIMP-2 and RECK in cultures of human keratinocytes. We observed that E7 expression is associated with increased pro-MMP-9 activity in the epithelial component of organotypic cultures, while E6 and E7 oncoproteins co-expression down-regulates RECK and TIMP-2 levels in organotypic and monolayers cultures. Finally, a study conducted in human cervical tissues showed a decrease in RECK expression levels in precancer and cancer lesions. Our results indicate that HPV oncoproteins promote MMPs/RECK-TIMP-2 imbalance which may be involved in HPV-associated lesions outcome., This research was funded by FAPESP-São Paulo Research Foundation: 2005/58885-1 (to LBSC), 2008/58817-4 (to SSME) and 2008/03232-1 (to LLV); and by PRPG-USP: Pró-Reitoria de Pós-Graduação da Universidade de São Paulo and CNPq: Conselho Nacional de Pesquisa e Desenvolvimento (to SSME).

Overexpression of kallikrein 7, a proteolytic enzyme important for epithelial cell shedding, may be causally involved in carcinogenesis, particularly in tumor metastasis and invasion. In this study, we have evaluated hK7 (human kallikrein 7) protein levels by immunohistochemistry in 367 cervical histological samples including 35 cases of cervicitis, 31 low-grade cervical intraepithelial neoplasia, 51 high-grade cervical intraepithelial neoplasia (H-SIL), 197 squamous cervical carcinomas (SCC) and 53 cervical adenocarcinomas. We have observed that hK7 staining increased with the severity of cervical disease. Intense hK7 staining was found in 15.2% of cervicitis samples, in contrast to 55% of H-SIL and 68% of SCC. Moreover, 92.5% of adenocarcinomas also exhibited intense hK7 staining. Differences in the expression of hK7 could potentially be used as a biomarker for the characterization of different stages of cervical disease., Grant sponsor: Fundacao de Amparo a Pesquisa do Estado de Sao Paulo; Grant number: 05/57274-9

Background Persistent infection by high risk HPV types (e.g. HPV-16, -18, -31, and -45) is the main risk factor for development of cervical intraepithelial neoplasia and cervical cancer. Tumor necrosis factor (TNF) is a key mediator of epithelial cell inflammatory response and exerts a potent cytostatic effect on normal or HPV16, but not on HPV18 immortalized keratinocytes. Moreover, several cervical carcinoma-derived cell lines are resistant to TNF anti-proliferative effect suggesting that the acquisition of TNF-resistance may constitute an important step in HPV-mediated carcinogenesis. In the present study, we compared the gene expression profiles of normal and HPV16 or 18 immortalized human keratinocytes before and after treatment with TNF for 3 or 60 hours. Methods In this study, we determined the transcriptional changes 3 and 60 hours after TNF treatment of normal, HPV16 and HPV18 immortalized keratinocytes by microarray analysis. The expression pattern of two genes observed by microarray was confirmed by Northern Blot. NF-κB activation was also determined by electrophoretic mobility shift assay (EMSA) using specific oligonucleotides and nuclear protein extracts. Results We observed the differential expression of a common set of genes in two TNF-sensitive cell lines that differs from those modulated in TNF-resistant ones. This information was used to define genes whose differential expression could be associated with the differential response to TNF, such as: KLK7 (kallikrein 7), SOD2 (superoxide dismutase 2), 100P (S100 calcium binding protein P), PI3 (protease inhibitor 3, skin-derived), CSTA (cystatin A), RARRES1 (retinoic acid receptor responder 1), and LXN (latexin). The differential expression of the KLK7 and SOD2 transcripts was confirmed by Northern blot. Moreover, we observed that SOD2 expression correlates with the differential NF-κB activation exhibited by TNF-sensitive and TNF-resistant cells. Conclusion This is the first in depth analysis of the differential effect of TNF on normal and HPV16 or HPV18 immortalized keratinocytes. Our findings may be useful for the identification of genes involved in TNF resistance acquisition and candidate genes which deregulated expression may be associated with cervical disease establishment and/or progression.

BACKGROUND: Penile squamous cell carcinoma (PSCC) is a rare tumor. In specific regions of developing countries, however, it may represent one the most frequent male tumors. Presence of lymph node metastasis (LNM) is the main factor significantly associated with reduced survival, and molecular markers to predict it have been extensively pursued. HER2 is an oncogene part of the EGFR family, often overexpressed in PSCC. HER2 overexpression is associated with poor prognosis and presence of LNM in breast tumors, and it is generally coamplified with topoisomerase IIα (TOP2A), a neighboring gene that is a target for anthracyclines. MATERIAL AND METHODS: We evaluated the frequency of HPV infection in 84 PSCC samples.The presence or absence of HPV DNA was established using generic primers (GP5+/GP6+) specific for the L1 gene of several HPVs. Genotyping of positive samples was done by dot blot hybridization using radioactive probes. Expression of HER2 and TOP2a was evaluated through immunohistochemistry and automatedly quantified using ScanScope XT, Aperio. Sixty cases were also submitted to a triple-color fluorescence in situ hybridization (FISH) assay using probes for the genes of HER2 and TOP2a, and also for the chromosome 17 centromere. Cases showing two signals of each probe were considered non-altered, those showing more than two signals of each probe were considered polysomy and those showing more HER2 or TOP2a signals compared to centromere signals were considered amplified. RESULTS: HPV infection, characterized by detection of viral DNA on the samples, was observed in 22.6% (19) of the cases. Among the positive HPV samples, 51.3% were infected by HPV16, 21.6% by HPV18, and 2.7% by both subtypes concomitantly. In agreement with studies that showed better prognosis of tumors with HPV infection, 5-year and 10-year survival of HPV-positive patients was, respectively, 79.1% and 70.8%, whereas of HPV-negative patients was 60.2% and 38.7% (p=0,018). Immunohistochemistry showed nuclear positivity for TOP2a in 74.2% (106) cases and cytoplasmic expression of HER2 was seen in 20,3% (29) of cases, which was associated with higher histologic grade (p Citation Format: Alice M. Silva, Lara Termini, Jose Ivanildo Neves, Isabela Cunha, Ademar Lopes, Gustavo Guimaraes, Fernando A. Soares. Evaluation of HER2 and topoisomera II alpha (TOP2a) expression and gene number in penile carcinoma according to HPV status. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5296. doi:10.1158/1538-7445.AM2013-5296

INTRODUCTION: Penile carcinoma is an infrequent tumor. However, in developing countries it is a common disease and in specific regions it may represents the most frequent tumor among men. The most important prognostic clinical factor is the presence of lymph node metastasis, which is associated with significant reduced survival to the penile carcinoma patients. Nevertheless, until the present date it was not possible to establish clinical or molecular prognostic markers. Various factors are associated with increased risk for de development of this disease, such as poor hygienic habits, lack of circumcision and HPV infection. HPV DNA can be detected in 20% to 80% of penile squamous cell carcinomas (PSCC). However, the role of HPV on patients’ prognosis is still uncertain. METHODS: We evaluated the frequency of HPV infection in 189 PSCC samples. The presence or absence of HPV DNA was established using generic primers (GP5+/GP6+) specific for the L1 gene of several HPVs. Specimens positive for HPV DNA were genotyped by dot blot hybridization using radioactive probes. We also evaluated the expression levels of the proteins p16 and MGMT through immunohistochemistry. These two proteins show aberrant levels in a wide range of tumors. Protein expression was quantified using an automated image system (ScanScope XT - Aperio), which determined the percentage and intensity of positive cells. The quantification was used to determine if there was loss of expression or normal (positive) expression. RESULTS: HPV infection, characterized by detection of viral DNA on the samples, was observed in 19.6% (37) of the cases. Among the positive HPV samples, 51.3% were infected by HPV16, 21.6% by HPV18 and 2.7% by both HPV16 and 18 concomitantly. The presence of HPV infection was more frequent among tumors with thickness lower than 5mm, suggesting that PSCC with HPV are less aggressive. This finding is in agreement with some studies that showed better prognosis of tumors with HPV infection. The immunohistochemistry assays showed reduced p16 expression in 35.4% (63) and loss of MGMT in 90.1% (146) cases. None association was observed between HPV infection and p16 protein expression. It is well established that the presence of HPV is associated with p16 positive expression. One interesting observation was that MGMT positive expression was associated with positive viral infection. Until this moment, there is no study describing this association in PSCC neither in other tumor types. It was observed an association between p16 reduced levels and poor differentiated tumors, this pathologic variable is associated with bad prognosis. MGMT loss of expression was associated with the presence of lymph node metastasis, suggesting that expression status of this protein can be a molecular marker for PSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1165. doi:1538-7445.AM2012-1165

Penile carcinoma is an uncommon disease and the most frequent subtype is squamous cell carcinoma. It represents less than 1% of all cases in developed countries. However, in developing countries it may represent 20% of all male cancers. The main risk factors associated to this tumor are poor hygienic habits, phimosis, HPV infections and high number of sexual partners. Very little is known about penile carcinoma pathogenesis, etiology and molecular biology features. In this study, we evaluated the hypermethylation frequency of two genes that present high frequencies of methylation in various tumor types: CDKN2A e MGMT. CDKN2A plays an important role in cell cycle control, encoding a protein (p16) that inhibits pRB phosphorilation through inactivation of the CDK-cyclin D complex, so blocking cell cycle progression. MGMT encodes a DNA repair protein that removes alkyl and methyl groups from guanines without DNA double strand breaks. Hypermethylation of these genes leads to transcriptional repression and has an important role in tumoregenesis. Unfortunately, scarce data is available about the role of hypermethylation in penile carcinomas. We selected 128 patients with penile squamous cell carcinoma surgically treated in a Brazilian cancer center. DNA from the formalin fixed paraffin embedded samples was obtained using a phenol-chloroform standard protocol. The DNA samples were submitted to bisulfite conversion, amplification and sequencing by the pyrosequencing approach, a quantitative methodology that allows the evaluation of the methylation levels in each CpG site of the target sequence. We also analyzed the expression levels by immunohistochemistry of the proteins p16 and MGMT to verify if the presence of hypermethylation is leading to gene silencing. We observed absence of CDKN2A hypermethylation in all samples and MGMT was hypermethylated in 57.4% of the cases, with levels of methylation varying from 16% to 86%. It was observed p16 positive expression in 66.8% of samples and Mgmt was present in only 10.9%. The decrease in MGMT expression was observed more frequent in tumors thicker than 5 millimeters and in patients with palpable nodes, suggesting a more aggressive tumor behavior in the absence of this protein. MGMT hypermethylation was not associated with reduced protein expression. Among samples with loss of MGMT expression 55.7% were methylated suggesting that only the presence of methyl radicals is not enough to induce gene silencing, it is probable that other mechanisms such as chromatin condensation are also required to repress gene transcription. The high frequency of p16 positivity could be explained by the presence of HPV infection, detected by others in about 30% of penile cancers. It also suggests that other members of the pRb pathway would be altered, such as cyclin D and Cdk amplifications or pRb loss. It cannot be discarded that other pathways of cell cycle control may also be deregulated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C17.