A comprehensive analysis of penile cancer in the region with the highest worldwide incidence reveals new insights into the disease.

BACKGROUND: Although penile cancer (PC) is uncommon in developed countries, it is widespread in developing countries. The state of Maranhão (Northeast, Brazil) has the highest global incidence recorded for PC, and, despite its socioeconomic vulnerability, it has been attributed to human papillomavirus (HPV) infection. This study aimed to determine the histopathological features, the prevalence of HPV infection, and the immunohistochemical profile of PC in Maranhão. METHODS: A retrospective cohort of 200 PC cases were evaluated. HPV detection was performed using nested-PCR followed by direct sequencing for genotyping. Immunohistochemistry (IHC) was performed using monoclonal antibodies anti-p16INK4a, p53, and ki-67. RESULTS: Our data revealed a delay of 17 months in diagnosis, a high rate of penile amputation (96.5%), and HPV infection (80.5%) in patients from Maranhão (Molecular detection). We demonstrated the high rate of HPV in PC also by histopathological and IHC analysis. Most patients presented koilocytosis (75.5%), which was associated with those reporting more than 10 different sexual partners during their lifetime (p = 0.001). IHC revealed frequent p16INK4a overexpression (26.0%) associated with basaloid (p < 0.001) and high-grade tumors (p = 0.008). Interestingly, p16 appears not to be a better prognostic factor in our disease-free survival analysis, as previously reported. We also demonstrated high ki-67 and p53 expression in a subset of cases, which was related to worse prognostic factors such as high-grade tumors, angiolymphatic and perineural invasion, and lymph node metastasis. We found a significant impact of high ki-67 (p = 0.002, log-rank) and p53 (p = 0.032, log-rank) expression on decreasing patients' survival, as well as grade, pT, stage, pattern, and depth of invasion (p < 0.05, log-rank). CONCLUSIONS: Our data reaffirmed the high incidence of HPV infection in PC cases from Maranhão and offer new insights into potential factors that may contribute to the high PC incidence in the region. We highlighted the possible association of HPV with worse clinical prognosis factors, differently from what was observed in other regions. Furthermore, our IHC analysis reinforces p16, ki-67, and p53 expression as important diagnosis and/or prognosis biomarkers, potentially used in the clinical setting in emerging countries such as Brazil.

Downregulation of miR-145 is associated with perineural invasion in penile carcinoma.

BACKGROUND: Human papillomavirus (HPV) infection is a risk factor for penile cancer (PC). The miR-145 expression has been correlated to this virus genomic amplification. In this context, this work aims to determine the expression level of miR-145 in penile tumors infected by high-risk HPV and correlate it with the clinicopathological characteristics of the tumor and protein expression of p53. METHODS: Formalin-fixed paraffin-embedded from 52 patients with PC, at diagnosis and prior to any cancer treatment, were obtained. HPV identification was performed by nested type PCR, and miR-145 expression was obtained by qRT-PCR. Immunohistochemical analysis of p53 and Ki-67 was performed. RESULTS: Tumoral miR-145 expression was significantly lower compared to adjacent tissue. Additionally, there was a significant reduction of miR-145 expression in invasion perineural, histological associated HPV, and absence of p53 expression in positive HPV cases. HPV infection was detected in 86.5%, the most frequent HPV16. Reduced disease-free survival was observed in patients with low expression of miR-145. CONCLUSIONS: Our data suggest that the underexpression of miR-145 may be triggered by HPV action, decreasing protein expression of p53, and being correlated with perineural invasion. Therefore, the deregulation of miR-145 provides clues as to the potential role in penile carcinogenesis and is also a potential candidate for validation in noninvasive samples.

DNA damage in an estuarine fish inhabiting the vicinity of a major Brazilian port.

The Itaqui Port Complex (northeastern Brazil) is one of the largest Brazilian port facilities, whose effluents and waste are dumped directly into the estuarine waters. Although environmental monitoring has been a concern around this site, there has been no toxicogenetics study on organisms living in this environment. Thus, we assessed the toxicogenetics potential of the estuarine waters surrounding Itaqui, using the native catfish Sciades herzbergii as a biomonitor. We found a significantly higher frequency of genetic damage and mutations in the animals collected near to Itaqui in both seasons compared to the reference site (distant from Itaqui with no port activities). We also quantified chemical elements in the surface water and sediments near the port and found that clorine, phosphorus, zinc, and boron were above the limits set by the Brazilian legislation. We suggest that such contaminants are involved in the origin of DNA damage. Moreover, we recommend including toxicogenetics assays in the environmental monitoring of pollutants, as well as in the definition of their allowable limits, as they could be used as law enforcement tools and help to predict large-scale contamination events associated with port activities.

Increased Glycated Hemoglobin Levels in Patients With Helicobacter pylori Infection Are Associated With the Grading of Chronic Gastritis.

Background: Recent studies have found an association between Helicobacter pylori infection and prediabetes. Whether H. pylori per se or host factors are involved in the disturbance of glycated hemoglobin needs further investigation. The aim of this study was to determine the association of glycated hemoglobin levels with endoscopic diagnosis and the inflammatory response in H. pylori infection. Methods: A cross-sectional study was carried out in 88 dyspeptic non-diabetic adults who underwent esophagogastroduodenoscopy. The diagnosis of H. pylori infection was performed through urease test and histopathological exam. Cases were initially distributed into two groups: control (without H. pylori infection, n = 22) and HP (patients with H. pylori infection, n = 66). HbA1c was measured to determine prediabetes status according to the American Diabetes Association criteria, and then the groups were subdivided into non-prediabetic (n = 14), prediabetic (n = 8), non-prediabetic HP (n = 26) and prediabetic HP (n = 40) groups. Gastric mucosa was histologically evaluated to determine H. pylori density and inflammatory activity according to Sydney System. To investigate the balance of anti-inflammatory and pro-inflammatory cytokines we measured interleukin 10 (anti-inflammatory) and Tumor Necrosis Factor-a (pro-inflammatory) in the plasma or in the gastric mucosa. Results: Patients with H. pylori infection had higher mean HbA1c levels than those without H. pylori infection. However, increased HbA1c levels were not associated with H. pylori-related factors but with the bacterial density, the intensity of inflammation and the activity of the chronic gastritis. In addition, H. pylori infection per se did not alter IL-10 and TNF-α neither in the plasma nor in the gastric mucosa, but the bacterial density was negatively correlated with systemic and local IL-10 expression. Although no correlation was found between systemic cytokines and HbA1c levels, local anti-inflammatory cytokine was correlated with HbA1c levels. Conclusion: Long-term H. pylori infection is associated with prediabetes. This association is not related to the presence of H. pylori per se but depends on the extent of bacterial colonization and the degree of both local inflammation and activity of the chronic gastritis.

Genomic profiling reveals the pivotal role of hrHPV driving copy number and gene expression alterations, including mRNA downregulation of TP53 and RB1 in penile cancer.

The incidence of penile cancer (PeCa) is increasing worldwide, however, the highest rates are reported in underdeveloped countries. The molecular mechanisms that underly the onset and progression of these tumors are still unclear. Therefore, our goal was to determine the genome-wide copy number alterations and the involvement of human papiloma virus (HPV) (TP53 and RB1), inflammatory (COX2 and EGFR), and PI3K/AKT pathway (AKT1, AKT2, EGFR, ERBB3, ERBB4, PIK3CA, and PTEN) associated genes in patients with PeCa from a high incidence region in Brazil (Maranhão). HPV genotyping was performed by nest-PCR and genome sequencing, copy number alterations (CNAs) by array comparative genomic hybridization and gene copy number status, gene, and protein expression by quantitative polymerase chain reaction, reverse transcriptase-quantitative polymerase chain reaction, and immunohistochemistry, respectively. HPV genotyping revealed one of the highest frequencies of HPV reported in PeCa, affecting 96.4% of the cases. The most common CNAs observed were located at the HPV integration sites, such as 2p12-p11.2 and 14q32.33, where ADAM 6, KIAA0125, LINC00226, LINC00221, and miR7641-2, are mapped. Increased copy number of ERBB3 and EGFR genes were observed in association with COX2 and EGFR overexpression, reinforcing the role of the inflammatory pathway in PeCa, and suggesting anti-EGFR and anti-COX2 inhibitors as promising therapies for PeCa. Additionally, TP53 and RB1 messenger RNA downregulation was observed, suggesting the occurrence of other mechanisms for repression of these oncoproteins, in addition to the canonical HPV/TP53/RB1 signaling pathway. Our data reinforce the role of epigenetic events in abnormal gene expression in HPV-associated carcinomas and suggest the pivotal role of HPV driving CNAs and controlling gene expression in PeCa.

Angiolymphatic invasion and absence of koilocytosis predict lymph node metastasis in penile cancer patients and might justify prophylactic lymphadenectomy.

To analyze possible clinical-pathological parameters and predictors of lymph node metastasis and evaluate the impact of lymphadenectomy in the survival of these patients.A retrospective study of patients diagnosed with penile cancer and submitted to regional lymphadenectomy at two reference hospitals in Maranhão, Northeast, Brazil, an area where the disease has a high incidence. We described here clinical and histopathological characteristics of patients diagnosed between January 2009 and September 2017.Fifty-five patients with an average age of 55.4 years (range: 25-84 years) were analyzed, with 24.4 months being the average time between the onset of symptoms and start of treatment. Among patients without palpable lymph nodes at the first examination, 51% were affected by inguinal metastasis. In the multivariate analysis, the presence of angiolymphatic invasion (P = .029) and absence of koilocytosis (P = .001) were found to be predictive factors for lymph node metastasis. Patients submitted to prophylactic lymphadenectomy presented with a disease-free period of 25.4 months (±5.81), whereas those who underwent therapeutic lymphadenectomy presented with a disease-free period of 19.9 months (±3.12).Angiolymphatic invasion and absence of koilocytosis appeared to be predictive factors for lymph node metastasis. Therefore, the submission of patients with metastatic risk to prophylactic lymphadenectomy may improve their survival. Thus, prophylactic lymphadenectomy in patients at risk for inguinal metastasis may create a positive impact in survival rates.

MIR-107, MIR-223-3P and MIR-21-5P Reveals Potential Biomarkers in Penile Cancer.

BACKGROUND: Inguinal lymph node involvement is the main prognostic factor in patients with penile cancer. However, there is a lack of marker/s for lymph node metastasis. microRNAs have been investigated as potential markers for prognosis of various types of cancer. Taking this into consideration, our main goal was to determine the association of miR-223-3p, miR-107, and miR-21-5p expression with clinicopathological characteristics, as well as presence of lymph node metastasis in patients with penile cancer. METHODS: Formalin-fixed paraffin-embedded penile squamous cell carcinoma specimens from 50 patients, at diagnosis and prior to any cancer treatment, were obtained. Tissue samples comprising at least 70% malignant cells and adjacent non-tumor tissues were evaluated by using qRT-PCR for expression level of miR-223-3p, miR-107 and miR-21-5p. Additionally, molecular identification of HPV was performed by PCR, and the expression levels of PTEN were analyzed by immunohistochemistry. RESULTS: Penile squamous cell carcinoma primary tumors presented higher expression of miR-223-3p, miR-107, and miR-21-5p when compared to non-tumor adjacent tissues. Upregulation of miR-223-3p was associated lymph node metastasis. Higher expression of miR-107 was associated with worsening of prognosis (as observed by histological grade II and III, tumors bigger than 2.0 cm, stage III and IV, and lower disease-free survival). In addition, higher expression of miR-107 and miR-21-5p was correlated to the absence of PTEN protein expression. CONCLUSIONS: Our data demonstrate that higher expression of miR-223-3p, miR-107, and miR-21-5p is correlated with poor prognosis in penile cancer. The upregulation of these microRNAs potentially affect critical cancer pathways and may be important for the prognosis and response to therapy in penile cancer.

The antileishmanial drug miltefosine (Impavido(®)) causes oxidation of DNA bases, apoptosis, and necrosis in mammalian cells.

Miltefosine was developed to treat skin cancer; further studies showed that the drug also has activity against Leishmania. Miltefosine is the first oral agent for treating leishmaniasis. However, its mechanism of action is not completely understood. We have evaluated the induction of DNA damage by miltefosine. Cytotoxicity and genotoxicity (comet assay) tests were performed on human leukocytes exposed to the drug in vitro. Apoptosis and necrosis were also evaluated. In vivo tests were conducted in Swiss male mice (Mus musculus) treated orally with miltefosine. Oxidation of DNA bases in peripheral blood cells was measured using the comet assay followed by digestion with formamidopyrimidine glycosylase (FPG), which removes oxidized guanine bases. The micronucleus test was performed on bone marrow erythrocytes. Miltefosine caused DNA damage, apoptosis, and necrosis in vitro. Mice treated with miltefosine showed an increase in the DNA damage score, which was further increased following FPG digestion. The micronucleus test was also positive.