Microbiome Sex-Related Diversity in Non-Muscle-Invasive Urothelial Bladder Cancer.

Sex-specific discrepancies in bladder cancer (BCa) are reported, and new studies imply that microbiome may partially explain the diversity. We aim to provide characterization of the bladder microbiome in both sexes diagnosed with non-muscle-invasive BCa with specific insight into cancer grade. In our study, 16S rRNA next-generation sequencing was performed on midstream urine, bladder tumor sample, and healthy-appearing bladder mucosa. Bacterial DNA was isolated using QIAamp Viral RNA Mini Kit. Metagenomic analysis was performed using hypervariable fragments of the 16S rRNA gene on Ion Torrent Personal Genome Machine platform. Of 41 sample triplets, 2153 taxa were discovered: 1739 in tumor samples, 1801 in healthy-appearing bladder mucosa and 1370 in midstream urine. Women were found to have smaller taxa richness in Chao1 index than men (p = 0.03). In comparison to low-grade tumors, patients with high-grade lesions had lower bacterial diversity and richness in urine. Significant differences between sexes in relative abundance of communities at family level were only observed in high-grade tumors.

Effects of Soluble Dextrin Fiber from Potato Starch on Body Weight and Associated Gut Dysbiosis Are Evident in Western Diet-Fed Mice but Not in Overweight/Obese Children.

BACKGROUND: The study investigated the impact of starch degradation products (SDexF) as prebiotics on obesity management in mice and overweight/obese children. METHODS: A total of 48 mice on a normal diet (ND) and 48 on a Western diet (WD) were divided into subgroups with or without 5% SDexF supplementation for 28 weeks. In a human study, 100 overweight/obese children were randomly assigned to prebiotic and control groups, consuming fruit and vegetable mousse with or without 10 g of SDexF for 24 weeks. Stool samples were analyzed for microbiota using 16S rRNA gene sequencing, and short-chain fatty acids (SCFA) and amino acids (AA) were assessed. RESULTS: Results showed SDexF slowed weight gain in female mice on both diets but only temporarily in males. It altered bacterial diversity and specific taxa abundances in mouse feces. In humans, SDexF did not influence weight loss or gut microbiota composition, showing minimal changes in individual taxa. The anti-obesity effect observed in mice with WD-induced obesity was not replicated in children undergoing a weight-loss program. CONCLUSIONS: SDexF exhibited sex-specific effects in mice but did not impact weight loss or microbiota composition in overweight/obese children.

Cervical microbiota dysbiosis associated with high-risk Human Papillomavirus infection.

High-risk Human Papillomavirus (HR-HPV) genotypes, specifically HPV16 and HPV18, pose a significant risk for the development of cervical intraepithelial neoplasia and cervical cancer. In the multifaceted cervical microenvironment, consisting of immune cells and diverse microbiota, Lactobacillus emerges as a pivotal factor, wielding significant influence in both stabilizing and disrupting the microbiome of the reproductive tract. To analyze the distinction between the cervical microbiota and Lactobacillus-dominant/non-dominant status of HR-HPV and non-infected healthy women, sixty-nine cervical swab samples were analyzed, included 44 with HR-HPV infection and healthy controls. All samples were recruited from Human Papillomavirus-based cervical cancer screening program and subjected to 16s rRNA sequencing analysis. Alpha and beta diversity analyses reveal no significant differences in the cervical microbiota of HR-HPV-infected women, including 16 and 18 HPV genotypes, and those with squamous intraepithelial lesion (SIL), compared to a control group. In this study we identified significantly lower abundance of Lactobacillus mucosae in women with HR-HPV infection compared to the control group. Furthermore, changes in bacterial diversity were noted in Lactobacillus non-dominant (LND) samples compared to Lactobacillus-dominant (LD) in both HR-HPV-infected and control groups. LND samples in HR-HPV-infected women exhibited a cervical dysbiotic state, characterized by Lactobacillus deficiency. In turn, the LD HR-HPV group showed an overrepresentation of Lactobacillus helveticus. In summary, our study highlighted the distinctive roles of L. mucosae and L. helveticus in HR-HPV infections, signaling a need for further research to demonstrate potential clinical implications of cervical microbiota dysbiosis.

Breast cancer but not the menopausal status is associated with small changes of the gut microbiota.

Background: Possible relationships between gut dysbiosis and breast cancer (BC) development and progression have been previously reported. However, the results of these metagenomics studies are inconsistent. Our study involved 88 patients diagnosed with breast cancer and 86 cancer-free control women. Participants were divided into groups based on their menopausal status. Fecal samples were collected from 47 and 41 pre- and postmenopausal newly diagnosed breast cancer patients and 51 and 35 pre- and postmenopausal controls, respectively. In this study, we performed shotgun metagenomic analyses to compare the gut microbial community between pre- and postmenopausal BC patients and the corresponding controls. Results: Firstly, we identified 12, 64, 158, and 455 bacterial taxa on the taxonomy level of phyla, families, genera, and species, respectively. Insignificant differences of the Shannon index and ß-diversity were found at the genus and species levels between pre- and postmenopausal controls; the differences concerned only the Chao index at the species level. No differences in α-diversity indexes were found between pre- and postmenopausal BC patients, although ß-diversity differed these subgroups at the genus and species levels. Consistently, only the abundance of single taxa differed between pre- and postmenopausal controls and cases, while the abundances of 14 and 23 taxa differed or tended to differ between premenopausal cases and controls, and between postmenopausal cases and controls, respectively. There were similar differences in the distribution of enterotypes. Of 460 bacterial MetaCyc pathways discovered, no pathways differentiated pre- and postmenopausal controls or BC patients, while two and one pathways differentiated cases from controls in the pre- and postmenopausal subgroups, respectively. Conclusion: While our findings did not reveal an association of changes in the overall microbiota composition and selected taxa with the menopausal status in cases and controls, they confirmed differences of the gut microbiota between pre- and postmenopausal BC patients and the corresponding controls. However, these differences were less extensive than those described previously.

Human Papillomavirus Infections and the Role Played by Cervical and Cervico-Vaginal Microbiota-Evidence from Next-Generation Sequencing Studies.

This comprehensive review encompasses studies examining changes in the cervical and cervico-vaginal microbiota (CM and CVM) in relation to human papillomavirus (HPV) using next-generation sequencing (NGS) technology. HPV infection remains a prominent global health concern, with a spectrum of manifestations, from benign lesions to life-threatening cervical cancers. The CM and CVM, a unique collection of microorganisms inhabiting the cervix/vagina, has emerged as a critical player in cervical health. Recent research has indicated that disruptions in the CM and CVM, characterized by a decrease in Lactobacillus and the overgrowth of other bacteria, might increase the risk of HPV persistence and the progression of cervical abnormalities. This alteration in the CM or CVM has been linked to a higher likelihood of HPV infection and cervical dysplasia. NGS technology has revolutionized the study of the cervical microbiome, providing insights into microbial diversity, dynamics, and taxonomic classifications. Bacterial 16S rRNA gene sequencing, has proven invaluable in characterizing the cervical microbiome, shedding light on its role in HPV infections and paving the way for more tailored strategies to combat cervical diseases. NGS-based studies offer personalized insights into an individual's cervical microbiome. This knowledge holds promise for the development of novel diagnostic tools, targeted therapies, and preventive interventions for cervix-related conditions, including cervical cancer.

Association of Mitochondrial Variants with the Joint Occurrence of Polycystic Ovary Syndrome and Hashimoto's Thyroiditis.

BACKGROUND: The prevalence of Hashimoto's thyroiditis (HT) among women with polycystic ovary syndrome (PCOS) is higher than in the general female population, but the factors predisposing to the coexistence of these disorders remain unclear. This study employed whole genome sequencing of mitochondrial DNA to identify genetic variants potentially associated with the development of PCOS and HT and predisposing to their joint occurrence. RESULTS: A total of 84 women participated, including patients with PCOS, HT, coexisting PCOS and HT (PCOS + HT) and healthy women. Both Fisher's exact and Mann-Whitney U statistical analyses were performed to compare the frequency of variants between groups. Ten differentiating variants were common to both analyses in PCOS + HT vs. PCOS, one in PCOS + HT vs. HT, and six in PCOS + HT vs. control. Several variants differentiating the PCOS + HT group from PCOS and controls were identified, located both in the mitochondrial genes (including the MT-CYB, MT-ND1, MT-ND2, MT-ND4, MT-ND6, MT-CO1, MT-CO3) and the D-loop region. Only two variants differentiated PCOS + HT and HT groups. One variant (13237a in MT-ND5) was common for all three comparisons and underrepresented in the PCOS + HT group. Functional enrichment analysis showed 10 pathways that were unique for the comparison of PCOS + HT and PCOS groups, especially related to ATP production and oxidative phosphorylation, and one pathway, the NADH-quinone oxidoreductase, chain M/4, that was unique for the comparison of PCOS + HT and control groups. Notably, nine pathways shared commonality between PCOS + HT vs. PCOS and PCOS + HT vs. control, related to the biogenesis and assembly of Complex I. CONCLUSION: This study provides novel insights into the genetic variants associated with oxidative stress in women with coexisting PCOS and HT. Mitochondrial dysfunction and oxidative stress appear to play a role in the pathogenesis of both conditions. However, more mitochondrial variants were found to differentiate women with both PCOS and HT from those with PCOS alone than from those with HT alone.

MCPIP1 functions as a safeguard of early embryonic development.

Monocyte chemoattractant protein-induced protein 1 (MCPIP1), also called Regnase-1, is an RNase that has been described as a key negative modulator of inflammation. MCPIP1 also controls numerous tumor-related processes, such as proliferation, apoptosis and differentiation. In this study, we utilized a zebrafish model to investigate the role of Mcpip1 during embryogenic development. Our results demonstrated that during embryogenesis, the expression of the zc3h12a gene encoding Mcpip1 undergoes dynamic changes. Its transcript levels gradually increase from the 2-cell stage to the spherical stage and then decrease rapidly. We further found that ectopic overexpression of wild-type Mcpip1 but not the catalytically inactive mutant form resulted in an embryonic lethal phenotype in zebrafish embryos (24 hpf). At the molecular level, transcriptomic profiling revealed extensive changes in the expression of genes encoding proteins important in the endoplasmic reticulum stress response and in protein folding as well as involved in the formation of primary germ layer, mesendoderm and endoderm development, heart morphogenesis and cell migration. Altogether, our results demonstrate that the expression of zc3h12a must be tightly controlled during the first cell divisions of zebrafish embryos and that a rapid decrease in its mRNA expression is an important factor promoting proper embryo development.

Diarrheal-associated gut dysbiosis in cancer and inflammatory bowel disease patients is exacerbated by Clostridioides difficile infection.

Introduction: Low diversity gut dysbiosis can take different forms depending on the disease context. In this study, we used shotgun metagenomic sequencing and gas chromatography-mass spectrometry (GC-MS) to compared the metagenomic and metabolomic profiles of Clostridioides (Clostridium) difficile diarrheal cancer and inflammatory bowel disease (IBD) patients and defined the additive effect of C. difficile infection (CDI) on intestinal dysbiosis. Results: The study cohort consisted of 138 case-mix cancer patients, 43 IBD patients, and 45 healthy control individuals. Thirty-three patients were also infected with C. difficile. In the control group, three well-known enterotypes were identified, while the other groups presented with an additional Escherichia-driven enterotype. Bacterial diversity was significantly lower in all groups than in healthy controls, while the highest level of bacterial species richness was observed in cancer patients. Fifty-six bacterial species had abundance levels that differentiated diarrheal patient groups from the control group. Of these species, 52 and 4 (Bacteroides fragilis, Escherichia coli, Klebsiella pneumoniae, and Ruminococcus gnavus) were under-represented and over-represented, respectively, in all diarrheal patient groups. The relative abundances of propionate and butyrate were significantly lower in fecal samples from IBD and CDI patients than in control samples. Isobutyrate, propanate, and butyrate concentrations were lower in cancer, IBD, and CDI samples, respectively. Glycine and valine amino acids were over- represented in diarrheal patients. Conclusion: Our data indicate that different external and internal factors drive comparable profiles of low diversity dysbiosis. While diarrheal-related low diversity dysbiosis may be a consequence of systemic cancer therapy, a similar phenotype is observed in cases of moderate to severe IBD, and in both cases, dysbiosis is exacerbated by incidence of CDI.

The role of microbiota in the pathophysiology of uterine fibroids - a systematic review.

For a long time, the uterus had been considered a sterile organ, meaning that under physiological conditions the uterus would not be colonized by bacteria. Based on available data, it may be concluded that the gut and uterine microbiome are related, and that the role of this microbiome is greater than expected. Despite being the most common pelvic neoplasms in women of reproductive age, uterine fibroids (UFs) are still poorly understood tumors whose etiology has not been fully determined. This systematic review presents the relationship between intestinal and uterine dysbiosis and uterine fibroids. A systematic review of three medical databases was carried out: the MEDLINE/PubMed, Scopus and Cochrane. In this study, 195 titles and abstracts were reviewed, including only original articles and clinical trials of uterine microbiome criteria. Finally, 16 studies were included to the analysis. In recent years, researchers dealing with reproduction in a broad sense have focused on the microbiome in various locations to study its role in the pathogenesis and, consequently, the prevention and treatment of diseases of the genital organ. Conventional microbial detection methods are not suitable for identifying bacteria, which are difficult to culture. Next-generation sequencing (NGS) provides an easier and faster and more informative analysis of bacterial populations. It seems that gut microbiota dysbiosis has the potential to be a risk factor for uterine fibroids or affect the disease process. Some changes were shown in many types of bacteria, such as Firmicutes, Proteobacteria, Actinobacteria and Verrucomicrobia detected in fecal samples in patients with uterine fibroids. In view of the few results on the link between the microbiome and uterine fibroids, further intensive studies in humans and animal models are necessary, including the possible use of different microbiome modulations in the prevention or treatment of uterine fibroids.

The interplay between gingival crevicular fluid microbiome and metabolomic profile in intensively treated people with type 1 diabetes - a combined metagenomic/metabolomic approach cross-sectional study.

Aims: This study aimed to assess the gingival crevicular fluid (GCF) microbiome and metabolome of adults with type 1 diabetes (T1D) treated with continuous subcutaneous insulin infusion (CSII). Methods: In this cross-sectional study, the GCF of adults with T1D treated with CSII and non-diabetic controls were sampled, and metagenomic/metabolomic analyses were performed. Results: In total, 65 participants with T1D and 45 healthy controls with a mean age of 27.05 ± 5.95 years were investigated. There were 22 cases of mild gingivitis (G) in the T1D group. There were no differences considering the Shannon and Chao indices and ß-diversity between people with T1D and G, with T1D without G, and healthy controls. Differential taxa were identified, which were mainly enriched in people with T1D and G. Acetic acid concentration was higher in people with T1D, regardless of the presence of G, than in healthy controls. Propionic acid was higher in people with T1D and G than in healthy controls. Isobutyric and isovaleric acid levels were higher in individuals with T1D and G than in the other two subgroups. The concentration of valeric acid was lower and that of caproic acid was higher in people with T1D (regardless of gingival status) than in healthy controls. Conclusions: The identification of early changes in periodontal tissues by targeting the microbiome and metabolome could potentially enable effective prevention and initial treatment of periodontal disease in people with T1D.

Gene Expression-Based Functional Differences between the Bladder Body and Trigonal Urothelium in Adolescent Female Patients with Micturition Dysfunction.

The aim of this study is to determine the molecular differences between the urothelial transcriptomes of the bladder body and trigone. The transcriptomes of the bladder body and trigonal epithelia were analyzed by massive sequencing of total epithelial RNA. The profiles of urothelial and urinal microbiomes were assessed by amplicon sequencing of bacterial 16S rRNA genes in 17 adolescent females with pain and micturition dysfunction and control female subjects. The RNA sequencing identified 10,261 differentially expressed genes (DEGs) in the urothelia of the bladder body and trigone, with the top 1000 DEGs at these locations annotated to 36 and 77 of the Reactome-related pathways in the bladder body and trigone, respectively. These pathways represented 11 categories enriched in the bladder body urothelium, including extracellular matrix organization, the neuronal system, and 15 categories enriched in the trigonal epithelium, including RHO GTPase effectors, cornified envelope formation, and neutrophil degranulation. Five bacterial taxa in urine differed significantly in patients and healthy adolescent controls. The evaluation of their transcriptomes indicated that the bladder body and trigonal urothelia were functionally different tissues. The molecular differences between the body and trigonal urothelia responsible for clinical symptoms in adolescents with bladder pain syndrome/interstitial cystitis remain unclear.

Combination of HLA-DQ2/-DQ8 Haplotypes and a Single MSH5 Gene Variant in a Polish Population of Patients with Type 1 Diabetes as a First Line Screening for Celiac Disease?

Patients with type 1 diabetes (T1D) are at increased risk for developing celiac disease (CD). The aim of the study was to assess the usefulness of celiac-specific human leukocyte antigen (HLA) haplotype and the rs3130484 variant of MSH5 gene, a previously described non-HLA variant associated with CD in the Polish population as a first-line screening for CD in T1D pediatric patients. Serological CD screening performed in the T1D group (n = 248) and healthy controls (n = 551) allowed for CD recognition in 20 patients (8.1%) with T1D (T1D + CD group). HLA-DQ2, HLA-DQ8 and the rs3130484 variant were genotyped with TaqMan SNP Genotyping Assays. The T1D + CD group presented a higher, but not statistically significant, frequency of HLA-DQ2 in comparison with T1D subjects. Combining the rs3130484 with HLA-DQ2/HLA-DQ8 typing significantly increased the sensitivity of HLA testing from 32.7% to 68.7%, and the accuracy of estimating CD prediction from 51.7% to 86.4% but decreased the specificity from 100% to 78.2%. The receiver operating characteristic curve analysis confirmed the best discrimination for the combination of both genetic tests with an area under curve reaching 0.735 (95% CI: 0.700-0.7690) in comparison with 0.664 (95% CI: 0.632-0.696) for HLA typing alone. Results show the low utility of HLA-DQ2/HLA-DQ8 typing for CD screening in T1D pediatric patients. Combination of the rs3130484 variant of the MSH5 gene and HLA testing increases both the sensitivity and the predictive value of the test accuracy, but still, the obtained values are not satisfactory for recommending such testing as the first-line screening for CD in T1D patients.

ESCRT-I fuels lysosomal degradation to restrict TFEB/TFE3 signaling via the Rag-mTORC1 pathway.

Within the endolysosomal pathway in mammalian cells, ESCRT complexes facilitate degradation of proteins residing in endosomal membranes. Here, we show that mammalian ESCRT-I restricts the size of lysosomes and promotes degradation of proteins from lysosomal membranes, including MCOLN1, a Ca2+ channel protein. The altered lysosome morphology upon ESCRT-I depletion coincided with elevated expression of genes annotated to biogenesis of lysosomes due to prolonged activation of TFEB/TFE3 transcription factors. Lack of ESCRT-I also induced transcription of cholesterol biosynthesis genes, in response to inefficient delivery of cholesterol from endolysosomal compartments. Among factors that could possibly activate TFEB/TFE3 signaling upon ESCRT-I deficiency, we excluded lysosomal cholesterol accumulation and Ca2+-mediated dephosphorylation of TFEB/TFE3. However, we discovered that this activation occurs due to the inhibition of Rag GTPase-dependent mTORC1 pathway that specifically reduced phosphorylation of TFEB at S112. Constitutive activation of the Rag GTPase complex in cells lacking ESCRT-I restored S112 phosphorylation and prevented TFEB/TFE3 activation. Our results indicate that ESCRT-I deficiency evokes a homeostatic response to counteract lysosomal nutrient starvation, that is, improper supply of nutrients derived from lysosomal degradation.

A Set of 17 microRNAs Common for Brain and Cerebrospinal Fluid Differentiates Primary Central Nervous System Lymphoma from Non-Malignant Brain Tumors.

The diagnosis of primary central nervous system (CNS) lymphoma, which is predominantly of the diffuse large B-cell lymphoma type (CNS DLBCL), is challenging. MicroRNAs (miRs) are gene expression-regulating non-coding RNAs that are potential biomarkers. We aimed to distinguish miR expression patterns differentiating CNS DLBCL and non-malignant CNS diseases with tumor presentation (n-ML). Next generation sequencing-based miR profiling of cerebrospinal fluids (CSFs) and brain tumors was performed. Sample source-specific (CSF vs. brain tumor) miR patterns were revealed. Even so, a set of 17 miRs differentiating CNS DLBCL from n-ML, no matter if assessed in CSF or in a tumor, was identified. Along with the results of pathway analyses, this suggests their pathogenic role in CNS DLBCL. A combination of just four of those miRs (miR-16-5p, miR-21-5p, miR-92a-3p, and miR-423-5p), assessed in CSFs, discriminated CNS DLBCL from n-ML samples with 100% specificity and 67.0% sensitivity. Analyses of paired CSF-tumor samples from patients with CNS DLBCL showed significantly lower CSF levels of miR-26a, and higher CSF levels of miR-15a-5p, miR-15b-5p, miR-19a-3p, miR-106b-3p, miR-221-3p, and miR-423-5p. Noteworthy, the same miRs belonged to the abovementioned set differentiating CNS DLBCL from non-malignant CNS diseases. Our results not only add to the basic knowledge, but also hold significant translational potential.

Peripheral Blood Cells from Patients with Hodgkin's and Diffuse Large B Cell Lymphomas May Be a Better Source of Candidate Diagnostic miRNAs Than Circulating miRNAs.

Hodgkin lymphoma (HL) and diffuse large B cell lymphoma (DLBCL) represent 15% and 20%, respectively, of all lymphoma types. The aim of this study was to identify and compare circulating serum miRNA (c-miRNA) and peripheral whole blood miRNA (wb-miRNA) profiles in patients with these lymphomas. Serum samples (20 HL, 21 DLBCL, and 30 healthy controls) and whole blood samples (21 HL, 17 DLBCL patients, and 30 healthy controls) were collected at the time of diagnosis. Serum and whole blood were also collected from 18 HL/17 DLBCL and eight HL/nine DLBCL patients, respectively, after treatment. Pairwise comparisons identified 125 c-miRNAs (adjusted P value < 0.05) showing significant dysregulation between 30 healthy controls and patients; of these, 47 and 55 differentiated controls from pretherapeutic HL and DLBCL patients, respectively. In addition, 60 and 16 c-miRNAs differentiated controls from posttherapeutic HL and DLBCL, respectively. Pairwise comparisons identified 292 wb-miRNAs (adjusted P value < 0.05) showing significant dysregulation between 30 controls and patients; of these, 103 and 169 differentiated controls from pretherapeutic HL and DLBCL, respectively, and 142 and 151 wb-miRNAs differentiated controls from posttherapeutic HL and DLBCL, respectively. Thus, lymphoma-associated miRNAs may be a better source of noninvasive candidate biomarkers than miRNAs in serum. It is unclear whether miRNA alterations in lymphoma cells are similar to those observed in white blood cells.

The Search of miRNA Related to Invasive Growth of Nonfunctioning Gonadotropic Pituitary Tumors.

PURPOSE: Nonfunctioning gonadotropic pituitary neuroendocrine tumors (PitNETs) are among the most frequent neoplasms of pituitary gland. Although PitNETs are commonly considered benign, a notable part of patients suffer from tumor recurrence after treatment. Invasive growth of pituitary tumor is among the most important prognostic factors. Since molecular features of invasiveness are of potential clinical usefulness, this study was aimed to verify whether invasive and noninvasive nonfunctioning gonadotropic PitNETs differ in the miRNA expression profile and whether the differences could provide a possible molecular classifier. METHODS: miRNA profiles were determined in 20 patients (11 invasive and 9 noninvasive tumors) using next-generation sequencing. The expression of selected miRNAs was assessed in the independent cohort of 80 patients with qRT-PCR. RESULTS: When miRNA profiles of invasive and noninvasive tumors were compared, 29 miRNAs were found differentially expressed. Hsa-miR-184, hsa-miR-181a-2-3p, hsa-miR-93-3p, hsa-miR-574-5p, hsa-miR-185-5p, and hsa-miR-3200-5p showed a potential clinical value according to ROC curve analysis. Unfortunately, differential expression of only hsa-miR-185-5p was confirmed in the validation cohort, with AUG at 0.654. CONCLUSION: Differences in miRNAs expression profiles in invasive and noninvasive gonadotropic PitNETs are slight and the level of miRNA expression seems not to be applicable as useful classifier of tumor invasiveness.

DNA Methylation Influences miRNA Expression in Gonadotroph Pituitary Tumors.

microRNAs are involved in pathogenesis of cancer. DNA methylation plays a role in transcription of miRNA-encoding genes and may contribute to changed miRNA expression in tumors. This issue was not investigated in pituitary neuroendocrine tumors (PitNETs) previously. DNA methylation patterns, assessed with HumanMethylation450K arrays in 34 PitNETs and five normal pituitaries, were used to determine differentially methylated CpGs located at miRNA genes. It showed aberrant methylation in regions encoding for 131 miRNAs. DNA methylation data and matched miRNA expression profiles, determined with next-generation sequencing (NGS) of small RNAs, were correlated in 15 PitNETs. This showed relationship between methylation and expression levels for 12 miRNAs. DNA methylation and expression levels of three of them (MIR145, MIR21, and MIR184) were determined in the independent group of 80 tumors with pyrosequencing and qRT-PCR and results confirmed both aberrant methylation in PitNETs and correlation between methylation and expression. Additionally, in silico target prediction was combined with analysis of established miRNA profiles and matched mRNA expression pattern, assessed with amplicon-based NGS to indicate putative target genes of epigenetically deregulated miRNAs. This study reveals aberrant DNA methylation in miRNA-encoding genes in gonadotroph PitNETs. Methylation changes affect expression level of miRNAs that regulate putative target genes with tumorigenesis-relevant functions.

Signatures of circulating microRNA in four sarcoma subtypes.

Background: Sarcomas are rare malignant tumors of mesenchymal origin. The discovery of circulating biomarkers with high diagnostic value could supplement diagnosis of this heterogenous group of tumors. The aim of this study was to identify the profiles of circulating miRNA (c-miRNAs) in four groups of common bone and soft tissue sarcomas. Methods: At the time of diagnosis, blood samples were collected from 86 patients: 36 with locally advanced/unresectable/metastatic gastrointestinal stromal tumor (GIST) who received first-line treatment with imatinib; 16 with locally advanced osteosarcoma (OS); 26 with locally advanced synovial sarcoma (SS); and eight with locally advanced Ewing sarcoma (ES). In addition, samples were collected from 30 healthy controls. C-miRNAs were isolated using a miRCURY RNA Isolation Kit, followed by preparation of cDNA libraries and sequencing on the Ion Proton platform. Results: Pair-wise comparisons identified 156 unique c-miRNAs (adjusted P-value < 0.05) showing significant dysregulation between controls and patients; of these, 24, 36, 42, and 99 differentiated controls from pretherapeutic OS, SS, ES, and GIST, respectively. Ten c-miRNAs were commonly altered in at least three sarcoma types. Receiver operating characteristic curves and area under the curve (ROC-AUC) analyses revealed that a four-miRNA diagnostic classifier was able to differentiate controls from ES, GIST, OS, and SS, with AUC-ROC values of 1, 0.97, 0.95, and 0.94, respectively. Conclusions: Aberrant miRNA expression signatures were identified in serum from patients with four different sarcoma subtypes. Differences in miRNA expression profiles between sarcoma patients and healthy volunteers suggest that miRNAs may play a role in sarcoma development.

Mutational Mosaics of Cell-Free DNA from Pancreatic Cyst Fluids.

BACKGROUND: Pancreatic cyst fluids (PCFs) enriched in tumor-derived DNA are a potential source of new biomarkers. The study aimed to analyze germinal variants and mutational profiles of cell-free (cf)DNA shed into the cavity of pancreatic cysts. METHODS: The study cohort consisted of 71 patients who underwent endoscopic ultrasound fine-needle aspiration of PCF. Five malignant cysts, 19 intraductal papillary mucinous neoplasms (IPMNs), 11 mucinous cystic neoplasms (MCNs), eight serous cystic neoplasms (SCNs), and 28 pseudocysts were identified. The sequencing of 409 genes included in Comprehensive Cancer Panel was performed using Ion Proton System. The mutation rate of the KRAS and GNAS canonical loci was additionally determined using digital PCR. RESULTS: The number of mutations detected with NGS varied from 0 to 22 per gene, and genes with the most mutations were: TP53, KRAS, PIK3CA, GNAS, ADGRA2, and APC. The frequencies of the majority of mutations did not differ between non-malignant cystic neoplasms and pseudocysts. NGS detected KRAS mutations in malignant cysts (60%), IPMNs (32%), MCNs (64%), SCNs (13%), and pseudocysts (14%), with GNAS mutations in 20%, 26%, 27%, 13%, and 21% of samples, respectively. Digital PCR-based testing increased KRAS (68%) and GNAS (52%) mutations detection level in IPMNs, but not other cyst types. CONCLUSIONS: We demonstrate relatively high rates of somatic mutations of cancer-related genes, including KRAS and GNAS, in cfDNA isolated from PCFs irrespectively of the pancreatic cyst type. Further studies on molecular mechanisms of pancreatic cysts malignant transformation in relation to their mutational profiles are required.