SUMMARYTesticular germ cell tumors (TGCT) are the most common malignant neoplasm in young men. DNA mismatch repair deficiency can lead to microsatellite instability (MSI), an important mechanism of genetic instability. A mutation of the BRAF gene has been implicated in the pathogenesis of several solid tumors and has recently become an important therapeutic target. The role of MSI and BRAF gene mutation in TGCT, particularly in refractory disease, is poorly understood and reported findings are controversial. In this study, we aimed to determine the frequency and clinical impact of MSI status and BRAF mutations in TGCT. DNA was isolated from formalin-fixed paraffin embedded (FFPE) tissue from 150 TGCT cases. The MSI phenotype was evaluated using multiplex PCR for five quasimonomorphic mononucleotide repeat markers. Exon 15 of the BRAF oncogene (V600E) was analyzed by PCR, followed by direct sequencing. Sixteen percent of cases were considered to have refractory disease. In a small subset of cases (17 for MSI and 18 for BRAF), the quantity and quality of DNA recovery were poor and therefore, were unable to be analyzed. The remaining 133 TGCT cases showed a complete absence of MSI. Of the 132 cases successfully evaluated for BRAF mutations, all were V600E wild-type. In conclusion, despite a distinct response of testicular germ cell tumors to therapy, microsatellite instability, and the BRAF V600E mutation were absent in all testicular germ cell tumors tested in this study.

Interactions between gut microbes and disease modifying antirheumatic drugs (DMARDs) have been proposed. The aim of the present study was to evaluate the presence of some specific bacteria in stool samples from Brazilian RA patients receiving DMARDs and correlate these data with diet, clinical parameters, and cytokines. Stool samples were used for gut bacteria evalutation by qPCR. Serum samples were used to quantify IL-4 and IL-10 by flow cytometer. Statistics were performed by Pearson chi-square, Mann–Whitney U test, and Spearman’s correlation. The study included 20 RA patients and 30 healthy controls. There were no significant differences (p > 0.05) in dietary habits between RA patients and controls. Concerning gut bacteria, we observed an increase in relative expression units (REU) of Bacteroides and Prevotella species in stool samples from patients, and a decrease in REU of Clostridium leptum when compared with healthy controls. Positive correlation between Prevotella and rheumatoid factor was detected. The IL-4 and IL-10 concentrations were increased in patients when compared with controls. We concluded that gut bacteria are different between RA patients receiving DMARDs and healthy controls. Further studies are necessary to determine the real role of gut microbes and their metabolities in clinical response to different DMARDs in RA patients.

Osteosarcomas commonly arise during the bone growth and remodeling in puberty, making it plausible to infer the involvement of epigenetic alterations in their development. We investigated DNA methylation and related genetic variants in 28 primary osteosarcomas aiming to identify deregulated driver pathways. Methylation and genomic data was obtained using the Illumina HM450K beadchips and the TruSight One sequencing panel, respectively. Aberrant DNA methylation was spread throughout the osteosarcomas genomes. We identified 3,146 differentially methylated CpGs comparing osteosarcomas and bone tissue samples, with high methylation heterogeneity, global hypomethylation and focal hypermethylation at CpG islands. Differentially methylated regions (DMR) were detected in 585 loci (319 hypomethylated and 266 hypermethylated), mapped to the promoter regions of 350 genes. These DMR-genes were enriched for biological processes related to skeletal system morphogenesis, proliferation, inflammatory response and signal transduction. Six tumor suppressor genes harbored deletions or promoter hypermethylation (DLEC1, GJB2, HIC1, MIR149, PAX6, WNT5A), and four oncogenes presented gains or hypomethylation (ASPSCR1, NOTCH4, PRDM16, RUNX3). Our analysis also revealed hypomethylation at 6p22, a region that contains several histone genes. DNMT3B gain was found to be a recurrent copy number change in osteosarcomas, providing a possible explanation for the observed phenotype of CpG island hypermethylation. While the detected open-sea hypomethylation likely contributes to the well-known osteosarcoma genomic instability, enriched CpG island hypermethylation suggests an underlying mechanism possibly driven by overexpression of DNMT3B likely resulting in silencing of tumor suppressors and DNA repair genes.