m6A RNA modification and its writer/reader VIRMA/YTHDF3 in testicular germ cell tumors: a role in seminoma phenotype maintenance.

BACKGROUND: Covalent RNA modifications, such as N-6-methyladenosine (m6A), have been associated with various biological processes, but their role in cancer remains largely unexplored. m6A dynamics depends on specific enzymes whose deregulation may also impact in tumorigenesis. Herein, we assessed the differential abundance of m6A, its writer VIRMA and its reader YTHDF3, in testicular germ cell tumors (TGCTs), looking for clinicopathological correlates. METHODS: In silico analysis of TCGA data disclosed altered expression of VIRMA (52%) and YTHDF3 (48%), prompting subsequent validation. Formalin-fixed paraffin-embedded tissues from 122 TGCTs (2005-2016) were selected. RNA extraction, cDNA synthesis and real-time qPCR (Taqman assays) for VIRMA and YTHDF3 were performed, as well as immunohistochemistry for VIRMA, YTHDF3 and m6A, for staining intensity assessment. Associations between categorical variables were assessed using Chi square and Fisher's exact test. Distribution of continuous variables between groups was compared using the nonparametric Mann-Whitney and Kruskal-Wallis tests. Biomarker performance was assessed through receiver operating characteristics (ROC) curve construction and a cut-off was established by Youden's index method. Statistical significance was set at p < 0.05. RESULTS: In our cohort, VIRMA and YTHDF3 mRNA expression levels differed among TGCT subtypes, with Seminomas (SEs) depicting higher levels than Non-Seminomatous tumors (NSTs) (p < 0.01 for both). A positive correlation was found between VIRMA and YTHDF3 expression levels. VIRMA discriminated SEs from NSTs with AUC = 0.85 (Sensitivity 77.3%, Specificity 81.1%, PPV 71.6%, NPV 85.3%, Accuracy 79.7%). Immunohistochemistry paralleled transcript findings, as patients with strong m6A immunostaining intensity depicted significantly higher VIRMA mRNA expression levels and stronger VIRMA immunoexpression intensity (p < 0.001 and p < 0.01, respectively). CONCLUSION: Abundance of m6A and expression of VIRMA/YTHDF3 were different among TGCT subtypes, with higher levels in SEs, suggesting a contribution to SE phenotype maintenance. VIRMA and YTHDF3 might cooperate in m6A establishment in TGCTs, and their transcript levels accurately discriminate between SEs and NSTs, constituting novel candidate biomarkers for patient management.

Dengue fever as a potential cause of sickle cell intrahepatic cholestasis: A report of two cases.

Sickle cell intrahepatic cholestasis is a potentially fatal syndrome characterized by jaundice, painful hepatomegaly, and organ dysfunction. Two cases of sickle cell intrahepatic cholestasis associated with dengue fever were described. Endothelial damage/dysfunction is a mechanism involved in severe hepatobiliary complications related to sickle cell diseases. However, the reasons for the lack of increase in the admission of patients with sickle cell disease having severe acute hepatobiliary complications triggered by endothelial damage/dysfunction due to dengue fever remain unknown. This study describes the first association between sickle cell intrahepatic cholestasis and dengue fever.

Germ cell tumour subtypes display differential expression of microRNA371a-3p.

Testicular germ cell tumours (TGCTs) are a heterogeneous group of neoplasms, mostly affecting young men. Curability rates are high and adequate treatment relies on careful and accurate pathological and clinical assessment. Indeed, TGCTs' histopathological subtyping is critical for adequate therapeutic decision. Considering the limitation of currently available serum biomarkers, novel candidates have been proposed, most notably miR-371a-3p, which outperformed classical serum markers, but no detailed information concerning TGCT subtype was available. Thus, we carried out evaluation of miR-371a-3p expression levels among TGCT subtypes using a consecutive cohort of tissue samples. MiR-371a-3p discriminated TGCTs from control tissues with high sensitivity and specificity (AUC = 0.99). Furthermore, seminomas displayed higher miR-371a-3p expression levels compared to non-seminomatous TGCTs, which also showed significant differences among them. Nonetheless, prepubertal TGCTs depicted lower miR-371a-3p expression levels than postpubertal TGCTs. Globally, miR-371a-3p expression levels decreased in parallel with progressive cell differentiation. We concluded that miR-371a-3p is TGCTs-specific and it might be clinically useful for early detection and disease monitoring.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

Testicular germ cell tumors: revisiting a series in light of the new WHO classification and AJCC staging systems, focusing on challenges for pathologists.

Testicular germ cell tumors (TGCTs) are strikingly heterogeneous, reflecting a complex tumor model, posing serious challenges for pathologists. Accurate classification and staging, according to most recent systems, is fundamental. We aimed to revise a series of consecutively diagnosed TGCTs (2005-2016) in light of the new World Health Organization (WHO) classification and American Joint Committee on Cancer (AJCC) staging systems, discussing dilemmas imposed to pathologists. All 164 patients' clinical files/histological slides were reviewed. Follow-up was last updated on November 2017. Statistical analysis was performed with SPSS (v24). P < 0.05 was considered significant. Non-seminomatous tumors (NSTs) showed more frequently cysts, necrosis, hemorrhage, lymphovascular invasion (LVI) and higher stage than seminomas (SEs) (P < .001, P = .015, P < .001, P = .001, P = .007). Embryonal carcinoma (EC), yolk sac tumor (YST) and teratoma (TE) were the most frequent components in mixed tumors (82.5%, 82.5% and 80.7%). SEs with "atypical features" showed more LVI, higher mitotic count and more extensive necrosis (P = .030, P < .001, P = .016). LVI and >50%EC component, but not rete testis invasion, were associated with higher stage (P < .001, P = .009). Regarding SEs, there was an association between tumor size and both stage (P = .004) and LVI (P < .001). Only four patients disclosed altered stage group when AJCC 8th Edition was employed. Disease recurrence/progression occurred in 5.4% of cases. In two cases, tumor components in metastasectomy specimens were not present in the primary TGCT. Overall survival at 5 years was 98.6%. TGCTs are challenging neoplasms, and pathologists and clinicians alike must be aware of recent updates in classification and staging for adequately tailoring treatment strategies.

The epigenetics of testicular germ cell tumors: looking for novel disease biomarkers.

Testicular germ cell tumors (TGCT) are a group of heterogeneous, biologically diverse and clinically challenging neoplasms. Despite the relatively low incidence and mortality rates, a subgroup of patients with disseminated disease relapse after conventional therapy and have a dismal prognosis. Moreover, TGCT afflict mostly young men and have therapeutic peculiarities, with some patients showing resistance to cisplatin-based treatments and others being troubled by irreversible side effects, such as infertility. Most TGCT share a common tumorigenic pathway and are cytogenetically similar, making room for Epigenetics to explain its heterogeneity at pathological and clinical level. In this review, we summarize the foremost epigenetic alterations among TGCT focusing on their clinical potential as diagnostic, prognostic and predictive biomarkers.

Association between solitary keratoacanthoma and cigarette smoking: a case-control study.

Solitary keratoacanthoma (KA) is a common benign epithelial tumor of the skin characterized by rapid growth and a tendency toward spontaneous regression. The exact etiology and classification of KA are a matter of debate. Smokers also seem to be more affected than persons who never smoke. The objective of this study was to evaluate the association between solitary KA and smoking habit. A case-control study involving 78 patients diagnosed with KA and 199 controls from the related community was performed to evaluate the association between cigarette smoking and KA. A higher smoking prevalence was noted in cases (69.2 %) than controls (21.6 %) and the odds ratio adjusted for sex and age was 9.1 (95 % CI 4.9 to 17.1, p< 0.01). The mean tumoral diameter at surgery and the site of involvement was not statistically related to smoking. These findings suggest that cigarette smoking is associated with the development of KA.

Dengue fever as a potential cause of sickle cell intrahepatic cholestasis: A report of two cases

Abstract Sickle cell intrahepatic cholestasis is a potentially fatal syndrome characterized by jaundice, painful hepatomegaly, and organ dysfunction. Two cases of sickle cell intrahepatic cholestasis associated with dengue fever were described. Endothelial damage/dysfunction is a mechanism involved in severe hepatobiliary complications related to sickle cell diseases. However, the reasons for the lack of increase in the admission of patients with sickle cell disease having severe acute hepatobiliary complications triggered by endothelial damage/dysfunction due to dengue fever remain unknown. This study describes the first association between sickle cell intrahepatic cholestasis and dengue fever.

Detecção e genotipagem de papilomavírus humano em lesões de queratoacantoma solitário de pacientes imunocompetentes / Detection and genotyping of human papillomavirus in solitary keratoacanthoma lesions of immunocompetent patients

FUNDAMENTOS: O queratoacantoma é neoplasia cutânea benigna que incide preferencialmente em indivíduos de pele clara, faixa etária elevada, acometendo áreas fotoexpostas. Além da exposição à radiação ultravioleta, sua etiologia é relacionada a diversos carcinógenos, entre eles a infecção pelo papilomavírus humano (HPV). OBJETIVOS: Avaliar a prevalência do DNA do HPV, bem como seus genótipos, em lesões de queratoacantoma solitário de pacientes imunocompetentes. MÉTODOS: Foram estudados queratoacantomas de pacientes sem evidências de imunocomprometimento, excisados entre 1996 e 2000 em hospital universitário. Realizaram-se cortes histológicos, desparafinização e extração de DNA desses fragmentos. Os espécimes positivos para DNA de HPV foram submetidos ao seqüenciamento gênico, para determinação do genótipo. RESULTADOS: Foram estudados 58 pacientes com idade média de 64,5±13,8 anos. A proporção entre os sexos foi semelhante, e as localizações mais comuns foram os membros superiores (50 por cento) e a face (27,6 por cento). Detectou-se DNA de HPV em 48 (82,7 por cento) fragmentos de queratoacantomas, sendo os genótipos 6, 11 e 16 os prevalentes. CONCLUSÕES: A alta prevalência do achado de DNA de HPV em lesões de queratoacantoma solitário pode sugerir a participação viral em sua oncogênese.

BACKGROUND - Keratoacanthoma is a benign cutaneous neoplasm that preferentially affects fair skin individuals of older age groups and involves sun-exposed areas. Apart from ultraviolet radiation exposure, its etiology is related to several carcinogens, including human papillomavirus (HPV) infection. OBJECTIVES: To assess the prevalence of HPV DNA and its genotypes in solitary keratoacanthoma lesions of immunocompetent patients. Methods: Keratoacanthoma lesions of patients with no evidence of immunological involvement, excised from 1996 to 2000, at a university hospital, were assessed. Histological aspects, deparafinization and DNA extraction of these lesions were evaluated. The specimens positive for HPV DNA were submitted to gene sequencing to determine the genotype. RESULTS: Fifty-eight patients were studied, mean age of 64.5±13.8 years. Both sexes were similarly affected, and the most common sites were upper limbs (50 percent) and face (27.6 percent). HPV DNA was detected in 48 (82.7 percent) keratoacanthoma fragments, and the genotypes 6, 11 and 16 were the most prevalent. CONCLUSIONS: The high prevalence of HPV DNA in solitary keratoacanthoma lesions may suggest viral participation in its oncogenesis.

Avaliação do efeito antiparasitário do omeprazol na prevenção do desenvolvimento de lesões cutâneas em hamsters infectados por Leishmania brasiliensis / Evaluation of omeprazole's antiparasitary effect preventing the development of cutaneous lesions due to Leishmania brasiliensis in hamsters

FUNDAMENTOS: A leishmaniose tegumentar americana permanece doença endêmica em diversas regiões brasileiras. A sobrevivência do parasita no interior dos macrófagos se deve, em parte, pela atividade de uma K+/H+-ATPase de membrana que pode ser inibida pelo omeprazol. OBJETIVOS: Avaliar a eficácia do omeprazol na prevenção do desenvolvimento de lesões de leishmaniose em hamsters. MÉTODOS: Empregaram-se 18 hamsters, divididos em três grupos: o grupo L recebeu apenas a inoculação de L. brasiliensis na pata anterior direita, o grupo O recebeu apenas doses diárias de 0,4mg de omeprazol subcutâneo, e o grupo L+O recebeu o inóculo de leishmanias e o tratamento com omeprazol desde o dia da inoculação. O estudo foi conduzido por 42 dias, realizaram-se medidas dos diâmetros das patas semanalmente, e, ao final do estudo, foram realizados esfregaços das lesões para verificação dos parasitas. RESULTADOS: Os hamsters dos grupos L e L+O desenvolveram lesões de leishmaniose tegumentar havendo ulceração em duas patas do grupo L e uma do grupo L+O. Ao final do estudo, a mobilidade e vitalidade no grupo L foram menores que em L+O, e estas menores que no grupo O. Os diâmetros das patas inoculadas nos grupos L e L+O foram significativamente maiores que no início do estudo (p<0.05). Não houve diferença significativa entre os diâmetros das patas dos grupos L e L+O ao final do estudo (p0,05), sendo detectados parasitas no esfregaço das lesões dos dois grupos. CONCLUSÕES: Omeprazol, no protocolo utilizado, não evitou o desenvolvimento de lesões de leishmaniose tegumentar em hamsters.

BACKGROUND: Cutaneous leishmaniasis remains an endemic disease in several brazilian regions. The parasite survival in macrophages is due to a membrane K+/H+-ATPase that can be inhibited by omeprazole. OBJECTIVES: Evaluate omeprazoles efficacy preventing the development of cutaneous leishmaniasis in hamsters. METHODS: Eighteen hamsters were divided in 3 groups: the L group received an inoculation of L. brasiliensis on right paw, the O group received daily 0,4mg omeprazole subcutaneously, and L+O group received both omeprazole and the inocule. The study was performed in 42 days, and the measurements of the diameter of paws were done weekly. At the end of the study was carried out a smear to verify the presence of parasites. RESULTS: Hamsters fitted in L and L+O groups have developed cutaneous leishmaniasis lesions, there were ulcerations in 2 hamsters from L group and 1 from L+O group. At the end of the study, the vitality and mobility of animals from L group were less prominent than L+O and O groups. Diameters of inoculated paws in L and L+O groups were significantly larger in comparison to begin of the study (p<0,05). There were no statistical difference between diameters of the paws from L and L+O groups at the end of study (p0,05). Parasites were detected at microscopic smears of lesions from both groups. CONCLUSIONS: HamstersÆ cutaneous lesions due to Leishmania sp. inoculation couldnÆt be prevented by omeprazole, using this protocol.