New Possible Ways to Use Exosomes in Diagnostics and Therapy via JAK/STAT Pathways.

Exosomes have the potential to be the future of personalized diagnostics and therapy. They are nano-sized particles between 30 and 100 nm flowing in the extracellular milieu, where they mediate cell-cell communication and participate in immune system regulation. Tumor-derived exosomes (TDEs) secreted from different types of cancer cells are the key regulators of the tumor microenvironment. With their immune suppressive cargo, TDEs prevent the antitumor immune response, leading to reduced effectiveness of cancer treatment by promoting a pro-tumorigenic microenvironment. Involved signaling pathways take part in the regulation of tumor proliferation, differentiation, apoptosis, and angiogenesis. Signal transducers and activators of transcription factors (STATs) and Janus kinase (JAK) signaling pathways are crucial in malignancies and autoimmune diseases alike, and their potential to be manipulated is currently the focus of interest. In this review, we aim to discuss exosomes, TDEs, and the JAK/STAT pathways, along with mediators like interleukins, tripartite motif proteins, and interferons.

Alterations of miRNA Expression in Diffuse Hyperplastic Perilobar Nephroblastomatosis: Mapping the Way to Understanding Wilms' Tumor Development and Differential Diagnosis.

Wilms' tumor (WT) is the most common renal malignancy in children. In diffuse hyperplastic perilobar nephroblastomatosis (DHPLN), nephrogenic rests result in a bulky enlargement of the kidney, a condition considered as a premalignant state before WT. Despite relevant clinical differences between WT and DHPLN, they are often challenging to distinguish based on histology. Molecular markers would improve differential diagnosis, but none are available at present. In our study, we investigated the potential of microRNAs (miRNAs) as such biomarkers, also aiming to shed light on the chronological order of expression changes. Formalin-fixed, paraffin-embedded (FFPE) samples from four DHPLN cases and adjacent healthy tissues were tested using a PCR array containing primers for 84 miRNAs implicated in genitourinary cancer. Expression in DHPLN was compared to WT data available in dbDEMC. Let-7, miR-135, miR-146a-5p, miR-182-5p, miR-183-5p, miR-20b-3p, miR-29b-3p, miR-195-5p and miR-17-5p showed potential to be used as biomarkers to distinguish WT and DHPLN in cases when traditional differential diagnosis is inconclusive. Our study also revealed miRNAs which may play a role in the initial steps of the pathogenesis (at a precancerous stage) and ones which become deregulated later in WT. More experiments are needed to confirm our observations and find new candidate markers.

Mitochondrial DNA copy number changes, heteroplasmy, and mutations in plasma-derived exosomes and brain tissue of glioblastoma patients.

Glioblastoma is the most common malignant tumor of the central nervous system (CNS) in adults. Glioblastoma cells show increased glucose consumption associated with poor prognosis. Since mitochondria play a crucial role in energy metabolism, mutations and copy number changes of mitochondrial DNA may serve as biomarkers. As the brain is difficult to access, analysis of mitochondria directly from the brain tissue represents a challenge. Exosome analysis is an alternative (still poorly explored) approach to investigate molecular changes in CNS tumors. We analyzed brain tissue DNA and plasma-derived exosomal DNA (exoDNA) of 44 glioblastoma patients and 40 control individuals. Quantitative real-time PCR was performed to determine mtDNA copy numbers and the Kruskal-Wallis and Mann-Whitney U test were used for statistical analysis of data. Subsequently, sequencing libraries were prepared and sequenced on the MiSeq platform to identify mtDNA point mutations. Tissue mtDNA copy number was different among controls and patients in multiple comparisons. A similar tendency was detected in exosomes. Based on NGS analysis, several mtDNA point mutations showed slightly different frequencies between cases and controls, but the clinical relevance of these observations is difficult to assess and likely less than that of overall mtDNA copy number changes. Allele frequencies of variants were used to determine the level of heteroplasmy (found to be higher in exo-mtDNA of control individuals). Despite the suggested potential, the use of such biomarkers for the screening and/or diagnosis of glioblastomas is still limited, thus further studies are needed.

Prognostic Modelling Studies of Coronary Heart Disease-A Systematic Review of Conventional and Genetic Risk Factor Studies.

This study aims to provide an overview of multivariable prognostic modelling studies developed for coronary heart disease (CHD) in the general population and to explore the optimal prognostic model by comparing the models' performance. A systematic review was performed using Embase, PubMed, Cochrane, Web of Science, and Scopus databases until 30 November 2019. In this work, only prognostic studies describing conventional risk factors alone or a combination of conventional and genomic risk factors, being developmental and/or validation prognostic studies of a multivariable model, were included. A total of 4021 records were screened by titles and abstracts, and 72 articles were eligible. All the relevant studies were checked by comparing the discrimination, reclassification, and calibration measures. Most of the models were developed in the United States and Canada and targeted the general population. The models included a set of similar predictors, such as age, sex, smoking, cholesterol level, blood pressure, BMI, and diabetes mellitus. In this study, many articles were identified and screened for consistency and reliability using CHARM and GRIPS statements. However, the usefulness of most prognostic models was not demonstrated; only a limited number of these models supported clinical evidence. Unfortunately, substantial heterogeneity was recognized in the definition and outcome of CHD events. The inclusion of genetic risk scores in addition to conventional risk factors might help in predicting the incidence of CHDs; however, the generalizability of the existing prognostic models remains open. Validation studies for the existing developmental models are needed to ensure generalizability, improve the research quality, and increase the transparency of the study.

Extracellular Nucleic Acids in the Diagnosis and Progression of Colorectal Cancer.

Colorectal cancer (CRC) is the 3rd most common malignant neoplasm worldwide, with more than two million new cases diagnosed yearly. Despite increasing efforts in screening, many cases are still diagnosed at a late stage, when mortality is high. This paper briefly reviews known genetic causes of CRC (distinguishing between sporadic and familial forms) and discusses potential and confirmed nucleic acid biomarkers obtainable from liquid biopsies, classified by their molecular features, focusing on clinical relevance. We comment on advantageous aspects such as better patient compliance due to blood sampling being minimally invasive, the possibility to monitor mutation characteristics of sporadic and hereditary CRC in a disease showing genetic heterogeneity, and using up- or down-regulated circulating RNA markers to reveal metastasis or disease recurrence. Current difficulties and thoughts on some possible future directions are also discussed. We explore current evidence in the field pointing towards the introduction of personalized CRC management.

Liquid Biopsy as a Source of Nucleic Acid Biomarkers in the Diagnosis and Management of Lynch Syndrome.

Lynch syndrome (LS) is an autosomal dominant inherited cancer predisposition disorder, which may manifest as colorectal cancer (CRC), endometrial cancer (EC) or other malignancies of the gastrointestinal and genitourinary tract as well as the skin and brain. Its genetic cause is a defect in one of the four key DNA mismatch repair (MMR) loci. Testing of patients at risk is currently based on the absence of MMR protein staining and detection of mutations in cancer tissue and the germline, microsatellite instability (MSI) and the hypermethylated state of the MLH1 promoter. If LS is shown to have caused CRC, lifetime follow-up with regular screening (most importantly, colonoscopy) is required. In recent years, DNA and RNA markers extracted from liquid biopsies have found some use in the clinical diagnosis of LS. They have the potential to greatly enhance the efficiency of the follow-up process by making it minimally invasive, reproducible, and time effective. Here, we review markers reported in the literature and their current clinical applications, and we comment on possible future directions.

The Role of Exosomes in Cancer Progression.

Early detection, characterization and monitoring of cancer are possible by using extracellular vesicles (EVs) isolated from non-invasively obtained liquid biopsy samples. They play a role in intercellular communication contributing to cell growth, differentiation and survival, thereby affecting the formation of tumor microenvironments and causing metastases. EVs were discovered more than seventy years ago. They have been tested recently as tools of drug delivery to treat cancer. Here we give a brief review on extracellular vesicles, exosomes, microvesicles and apoptotic bodies. Exosomes play an important role by carrying extracellular nucleic acids (DNA, RNA) in cell-to-cell communication causing tumor and metastasis development. We discuss the role of extracellular vesicles in the pathogenesis of cancer and their practical application in the early diagnosis, follow up, and next-generation treatment of cancer patients.

Circulating Cell-Free Nucleic Acids: Main Characteristics and Clinical Application.

Liquid biopsy recently became a very promising diagnostic method that has several advantages over conventional invasive methods. Liquid biopsy may serve as a source of several important biomarkers including cell-free nucleic acids (cf-NAs). Cf-DNA is widely used in prenatal testing in order to characterize fetal genetic disorders. Analysis of cf-DNA may provide information about the mutation profile of tumor cells, while cell-free non-coding RNAs are promising biomarker candidates in the diagnosis and prognosis of cancer. Many of these markers have the potential to help clinicians in therapy selection and in the follow-up of patients. Thus, cf-NA-based diagnostics represent a new path in personalized medicine. Although several reviews are available in the field, most of them focus on a limited number of cf-NA types. In this review, we give an overview about all known cf-NAs including cf-DNA, cf-mtDNA and cell-free non-coding RNA (miRNA, lncRNA, circRNA, piRNA, YRNA, and vtRNA) by discussing their biogenesis, biological function and potential as biomarker candidates in liquid biopsy. We also outline possible future directions in the field.

Recurrent, Severe Aphthous Stomatitis and Mucosal Ulcers as Primary Manifestations of a Novel STAT1 Gain-of-Function Mutation.

Chronic mucocutaneous candidiasis (CMC) characterized by persistent and recurrent Candida infection of the skin, nails, and the mucosa membranes has been proposed as the major infectious phenotype in patients with gain-of-function mutation of signal transducer and activator of transcription 1 (STAT1) 1. However, viral infections caused mostly by herpesviruses, and a broad range of autoimmune disorders may also be part of the clinical phenotype. We report here on a 31 years old female patient suffering from severe mucosal aphthous mucositis and ulcers and recurrent herpes simplex for decades. We found a previously unknown heterozygous sequence variant in STAT1 (c.1219C>G; L407V) affecting the DNA-binding domain of the protein in the patient and her 4 years old daughter. We found this mutation gain-of-function (GOF) by using immunoblot and luciferase assays. We detected low proportion of IL-17A-producing CD4+ T cell lymphocytes by using intracellular staining and flow cytometry. Candida-induced secretion of IL-17A and IL-22 by mononuclear cells from the patient was markedly decreased compared to controls. These data suggest that the novel mutant allele may result in impaired differentiation of CD4+ T cells to CD4+/IL-17+ cells. The clinical phenotype of the disease in this patient was unique as it was dominated primarily by severe aphthous stomatitis and ulcerative esophagitis and only partly by typical CMC resulting in diagnostic delay. We suggest that patients with severe recurrent aphthous stomatitis and esophagitis should be evaluated for STAT1 GOF mutation. Based on the broad clinical spectrum of the disease, we also suggest that CMC and CMC disease may not be an appropriate term to define clinically STAT1 GOF mutation.

Quantification of mtDNA in extracellular vesicles.

The liquid biopsy preserves a noninvasive technique to analyze promising biomarkers in cell-free bodyfluids, mainly in cell-free plasma. The most cells secrete extracellular vesicles into the extracellular place which can be isolated, analyzed easily due to the wide range of different protocols and commercial kits. The mitochondrial DNA isolated from biofluids can serve as new view in early diagnosis of various diseases (e.g. cancers, cardiovascular diseases). In this chapter, possible protocols of mitochondrial DNA copy number quantification are discussed presenting some ways to determine the mtDNA level of extracellular vesicles in different diseases.

Circulating miRNA Profiling in Plasma Samples of Ovarian Cancer Patients.

Ovarian cancer is one of the most common cancer types in women characterized by a high mortality rate due to lack of early diagnosis. Circulating miRNAs besides being important regulators of cancer development could be potential biomarkers to aid diagnosis. We performed the circulating miRNA expression analysis in plasma samples obtained from ovarian cancer patients stratified into FIGO I, FIGO III, and FIGO IV stages and from healthy females using the NanoString quantitative assay. Forty-five miRNAs were differentially expressed, out of these 17 miRNAs showed significantly different expression between controls and patients, 28 were expressed only in patients, among them 19 were expressed only in FIGO I patients. Differentially expressed miRNAs were ranked by the network-based analysis to assess their importance. Target genes of the differentially expressed miRNAs were identified then functional annotation of the target genes by the GO and KEGG-based enrichment analysis was carried out. A general and an ovary-specific protein-protein interaction network was constructed from target genes. Results of our network and the functional enrichment analysis suggest that besides HSP90AA1, MYC, SP1, BRCA1, RB1, CFTR, STAT3, E2F1, ERBB2, EZH2, and MET genes, additional genes which are enriched in cell cycle regulation, FOXO, TP53, PI-3AKT, AMPK, TGFß, ERBB signaling pathways and in the regulation of gene expression, proliferation, cellular response to hypoxia, and negative regulation of the apoptotic process, the GO terms have central importance in ovarian cancer development. The aberrantly expressed miRNAs might be considered as potential biomarkers for the diagnosis of ovarian cancer after validation of these results in a larger cohort of ovarian cancer patients.

Aberrant Methylation Status of Tumour Suppressor Genes in Ovarian Cancer Tissue and Paired Plasma Samples.

Ovarian cancer is a highly heterogeneous disease and its formation is affected by many epidemiological factors. It has typical lack of early signs and symptoms, and almost 70% of ovarian cancers are diagnosed in advanced stages. Robust, early and non-invasive ovarian cancer diagnosis will certainly be beneficial. Herein we analysed the regulatory sequence methylation profiles of the RASSF1, PTEN, CDH1 and PAX1 tumour suppressor genes by pyrosequencing in healthy, benign and malignant ovarian tissues, and corresponding plasma samples. We recorded statistically significant higher methylation levels (p < 0.05) in the CDH1 and PAX1 genes in malignant tissues than in controls (39.06 ± 18.78 versus 24.22 ± 6.93; 13.55 ± 10.65 versus 5.73 ± 2.19). Higher values in the CDH1 gene were also found in plasma samples (22.25 ± 14.13 versus 46.42 ± 20.91). A similar methylation pattern with positive correlation between plasma and benign lesions was noted in the CDH1 gene (r = 0.886, p = 0.019) and malignant lesions in the PAX1 gene (r = 0.771, p < 0.001). The random forest algorithm combining methylation indices of all four genes and age determined 0.932 AUC (area under the receiver operating characteristic (ROC) curve) prediction power in the model classifying malignant lesions and controls. Our study results indicate the effects of methylation changes in ovarian cancer development and suggest that the CDH1 gene is a potential candidate for non-invasive diagnosis of ovarian cancer.

Expression of CD24 in plasma, exosome and ovarian tissue samples of serous ovarian cancer patients.

CD24 is a small molecular weight cell-surface protein and an independent marker for poor prognosis in the different type of cancers. We aimed to determine the expression of CD24 in plasma, exosomes and ovarian tissue samples of serous ovarian cancer patients. We collected tissue and blood samples from 21 cases of serous ovarian cancer and eight healthy controls. We used silica adsorption method for isolation of RNA. The cDNA was synthesized using quantitative real-time PCR. We used beta-globin as a housekeeping gene for the normalization of the data. Protein-protein and miRNA networking were analyzed. There was a significant difference in the expression of CD24 in ovarian tissue between controls and patients (0.16 ± 0.32 vs. 44.97 ± 68.06; p < 0.01), while CD24 did not show expression in each plasma and exosome samples. There was a correlation in the expression of CD24 and FIGO grading between controls and patients. CD24 expression was detected in exosomes in 38.1% of patients, mainly with FIGO III, and in their plasma in 9.5% of cases. Our network analysis shows LYN, SELP, FGR, and NPM1 proteins are interacting with CD24. Our study demonstrates higher expression of CD24 in ovarian cancer patients' tissue samples, and there is an association with FIGO classification. However, CD24 showed expression only in some cell-free plasma and exosome samples.

Circulating epithelial-mesenchymal transition-associated miRNAs are promising biomarkers in ovarian cancer.

Ovarian cancer is the fifth most common cause of cancer death among women that is mostly due to the difficulty of early diagnosis. Circulating miRNAs proved to be reliable biomarkers in various cancers. We screened 9 miRNAs, which are involved in epithelial-mesenchymal transition, in the plasma samples of patients with malignant (n = 28) or non-malignant (n = 12) ovarian tumors and disease-free healthy volunteers (n = 60) by qRT-PCR. The expression levels of miR200a, miR200b, miR200c, miR141, miR429, miR203a, miR34b (p < 0.001) and miR34a (p < 0.01) were significantly higher in the malignant samples than in healthy controls. MiR203a, miR141 (p < 0.01), miR200a and miR429 (p < 0.05) levels were also higher in malignant compared to non-malignant samples. ROC-AUC was the highest in the case of miR200c: 0.861 (95%CI = 0.776-0.947). Spearman's rank correlation analysis revealed positive correlation between the plasma levels of the studied miRNAs that was the highest between miR200b and miR200c (rs = 0.774; p < 0.001). Target analysis also suggested tight interaction between these miRNAs in the regulation of cancer development. The agreement of diagnostic tests based on miRNA levels and the standard CA125 or HE4 was weak according to Cohen's kappa values. We conclude that miR200 family members, miR34b and miR203a might be promising complementary biomarkers in ovarian cancer.

Detection of cell-free, exosomal and whole blood mitochondrial DNA copy number in plasma or whole blood of patients with serous epithelial ovarian cancer.

Ovarian tumor is one of the leading causes of cancer among women. Patients are diagnosed at an advanced stage, usually. There is a need for new specific and sensitive biomarkers. Mitochondrial DNA copy number change was observed in various cancers. Our aim was to detect mitochondrial DNA copy number in whole blood (wb-mtDNA) and in plasma (cell-free and exosome encapsulated mtDNA) in patients with serous epithelial ovarian tumor. DNA was isolated from EDTA blood and plasma obtained from 24 patients and 24 healthy controls. Exosomes were isolated from cell-free plasma, and exosome encapsulated DNA (exoDNA) was extracted. Quantitative-real-time PCR was performed with Human Mitochondrial DNA (mtDNA) Monitoring Primer Set. Kruskall­Wallis and Mann­Whitney U test were used for data analysis. Wb-mtDNA copy number was significantly different among healthy controls and patients in multiple comparison (p = 0.0090 considering FIGO stage independently, and p = 0.0048 considering early- and late-stage cancers). There was a significant decrease among early-stage, all advanced stage and all cancer patients (FIGO I: 32.5 ± 8.3, p = 0.0061; FIGO III + IV: 37.2 ± 13.7 p = 0.0139; FIGO I + III + IV: 35.6 ± 12.2, p = 0.0017) or FIGO III patients alone (32.8 ± 5.6, p = 0.00089) compared to healthy controls. We found significant increase in copy number in exosomal mtDNA in cancer patients (236.0 ± 499.0, p = 0.0155), advanced-stage cancer patients (333.0 ± 575.0, p = 0.0095), of FIGO III (362.0 ± 609.2, p = 0.0494), and FIGO IV (304.0 ± 585.0, p = 0.0393) patients alone but not in samples of FIGO I patients (10.0 ± 3.5, p = 0.3907). In multiple comparison the increase was significant considering early- and late-stage cancers (p = 0.0253). Cell-free mtDNA copy numbers were not increased significantly. We found the highest copy number of mtDNA in exosomes, followed by plasma and peripheral blood in late-stage cancer patients. We observed significant difference in wb-mtDNA copy number between healthy controls and both early- and late-stage cancer patients.

Identification of miR-146a and miR-196a-2 single nucleotide polymorphisms at patients with high-grade serous ovarian cancer.

MicroRNAs play an essential role in the regulation of gene expression and tumor development. Single nucleotide polymorphism (SNP) can be observed in miRNAs and could influence gene expression. We aimed to identify miR-146a rs2910164 and miR-196a-2 rs11614913 polymorphisms in ovarian cancer patients and controls. 75 patients and 75 controls were involved. DNA was isolated from blood samples. MiR-146a rs2910164 and miR-196a-2 rs11614913 were determined by LightSnip kit. We used melting curve analysis for allele classification. Network analysis was made to find common target genes. We detected 72.67% G allele frequency of miR-146a rs2910164 in controls and 82.00% in patients group (p = 0,053). GG, GC and CC genotypes occurred with 53.33%, 38.67% and 8.00% among controls, with 65.33%, 33.33% and 1.33% among patients, (p = 0.0917). Allele C of miR-196a-2 rs11614913 occurred in 59.33% of controls and in 67.33% of patients (p = 0.15). CC, CT and TT genotypes occurred with 37.33%, 44.00%, and 18.67% frequency in controls, with 46.67%; 41.33% and 12.00% in patients (p = 0.3815). Network analysis found ATG9A, LBR, MBD4 and RUFY2 genes to be targets for both miRNAs. SNPs of miR-146a and miR-196a-2 showed no significant differences between patients and controls. More investigations are required to clarify the exact role of these SNPs in ovarian cancer.