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.

Cross-Kingdom Interaction of miRNAs and Gut Microbiota with Non-Invasive Diagnostic and Therapeutic Implications in Colorectal Cancer.

Colorectal cancer (CRC) has one of the highest incidences among all types of malignant diseases, affecting millions of people worldwide. It shows slow progression, making it preventable. However, this is not the case due to shortcomings in its diagnostic and management procedure and a lack of effective non-invasive biomarkers for screening. Here, we discuss CRC-associated microRNAs (miRNAs) and gut microbial species with potential as CRC diagnostic and therapy biomarkers. We provide rich evidence of cross-kingdom miRNA-mediated interactions between the host and gut microbiome. miRNAs have emerged with the ability to shape the composition and dynamics of gut microbiota. Intestinal microbes can uptake miRNAs, which in turn influence microbial growth and provide the ability to regulate the abundance of various microbial species. In the context of CRC, targeting miRNAs could aid in manipulating the balance of the microbiota. Our findings suggest the need for correlation analysis between the composition of the gut microbiome and the miRNA expression profile.

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.

Microsatellite instability assessment is instrumental for Predictive, Preventive and Personalised Medicine: status quo and outlook.

A form of genomic alteration called microsatellite instability (MSI) occurs in a class of tandem repeats (TRs) called microsatellites (MSs) or short tandem repeats (STRs) due to the failure of a post-replicative DNA mismatch repair (MMR) system. Traditionally, the strategies for determining MSI events have been low-throughput procedures that typically require assessment of tumours as well as healthy samples. On the other hand, recent large-scale pan-tumour studies have consistently highlighted the potential of massively parallel sequencing (MPS) on the MSI scale. As a result of recent innovations, minimally invasive methods show a high potential to be integrated into the clinical routine and delivery of adapted medical care to all patients. Along with advances in sequencing technologies and their ever-increasing cost-effectiveness, they may bring about a new era of Predictive, Preventive and Personalised Medicine (3PM). In this paper, we offered a comprehensive analysis of high-throughput strategies and computational tools for the calling and assessment of MSI events, including whole-genome, whole-exome and targeted sequencing approaches. We also discussed in detail the detection of MSI status by current MPS blood-based methods and we hypothesised how they may contribute to the shift from conventional medicine to predictive diagnosis, targeted prevention and personalised medical services. Increasing the efficacy of patient stratification based on MSI status is crucial for tailored decision-making. Contextually, this paper highlights drawbacks both at the technical level and those embedded deeper in cellular/molecular processes and future applications in routine clinical testing.

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.

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.

DNA copy number variation: Main characteristics, evolutionary significance, and pathological aspects.

Copy number variants (CNVs) were the subject of extensive research in the past years. They are common features of the human genome that play an important role in evolution, contribute to population diversity, development of certain diseases, and influence host-microbiome interactions. CNVs have found application in the molecular diagnosis of many diseases and in non-invasive prenatal care, but their full potential is only emerging. CNVs are expected to have a tremendous impact on screening, diagnosis, prognosis, and monitoring of several disorders, including cancer and cardiovascular disease. Here, we comprehensively review basic definitions of the term CNV, outline mechanisms and factors involved in CNV formation, and discuss their evolutionary and pathological aspects. We suggest a need for better defined distinguishing criteria and boundaries between known types of CNVs.

miRNA Profiling of Hungarian Regressive Wilms' Tumor Formalin-Fixed Paraffin-Embedded (FFPE) Samples by Quantitative Real-Time Polymerase Chain Reaction (RT-PCR).

BACKGROUND Wilms' tumor is a common renal malignancy of early childhood with a generally favorable prognosis depending upon histological subtype. It is becoming increasingly clear that differences in miRNA (microRNA) expression signature represent important clues helping us predict a tumor's response to chemotherapy. In our study, we aimed to reveal miRNAs deregulated in regressive Wilms' tumors from FFPE (formalin-fixed, paraffin-embedded) samples, also showing whether such samples are reliable miRNA sources in Wilms' tumor. MATERIAL AND METHODS Samples from 8 Hungarian patients (3 males, 5 females, aged 1 to 7 years) were analyzed by qRT-PCR (quantitative real-time PCR). A PCR array was used in a pilot experiment, and selected miRNAs (miR-128-3p, miR-184, miR-194-5p, miR-203a) were studied in the rest of the samples using individual primers. RESULTS miR-194-5p was underexpressed in all tumor samples. miR-184 and miR-203a were underexpressed in 7 cases, the exception being a case with a high ratio of necrotic blastemal tissue. Results obtained with miR-128-3p are difficult to interpret due to varying directions of expression changes. CONCLUSIONS We conclude that a downregulation of miR-184, miR-194-5p, and miR-203a expression is observed in both regressive and blastemal tumors, but larger-scale studies are needed to confirm whether the degree of their underexpression correlates with the number of blastemal elements in a sample. In most of our FFPE samples aged up to 9 years, RNA extraction provided miRNA with quantity and quality sufficient for qRT-PCR-based analysis, emphasizing the relevance of pathological archives as miRNA sources in future studies.

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.

Quantitative RT-PCR-based miRNA profiling of blastemal Wilms' tumors from formalin-fixed paraffin-embedded samples.

Blastemal Wilms' tumors are associated with poor chemo-responsiveness and an adverse prognosis. Our aim was to contribute to the miRNA profiling of the disease, while demonstrating the value of archived formalin-fixed, paraffin-embedded (FFPE) samples as miRNA sources. MiRNA was extracted from tumor and normal tissues of 8 patients diagnosed with blastemal Wilms' tumor in Hungary. A quantitative real-time PCR-based protocol was used to identify miRNAs of interest and study the expression of selected miRNAs in all samples. Profiling of miRNA expression from FFPE samples turned out to be cost-effective in Wilms' tumor, as most miRNAs (including miRNA-194-5p, which was studied in all patients) showed expression alterations similar to the ones reported in the literature. MiR-184 expression was found to be lower than in previous studies, while the downregulation of miR-203a is a novel finding. MiR-184 may be downregulated in a subset of blastemal and other Wilms' tumors. A loss of miR-203a may or may not be specific to blastemal cells, but available evidence hints at its importance in the pathogenesis of Wilms' tumor. It should be considered for inclusion in future studies of miRNA expression.

The Wilms' tumour 1 gene as a factor in non-syndromic hypospadias: evidence and controversy.

Hypospadias is one of the most frequent congenital anomalies of the male external genitalia. Its pathogenesis is due to largely unknown or poorly understood genetic factors and is further complicated by environmental-intrauterine-risk factors. One of the genes currently in focus by molecular biologists and clinicians studying syndromic forms of hypospadias is the Wilms' tumour 1 (WT1) gene. There is controversy over whether WT1 defects are also responsible for isolated hypospadias. In this review, we briefly cover the role of WT1 as a transcription factor and discuss proposed pathogenic pathways leading to hypospadias, outlining possible directions for research. We assess available evidence on the gene's mutations and polymorphisms recently suggested in the background of the disease, and examine the putative role of WT1-associated proteins. We also review relevant aspects of genome-wide association studies carried out so far, and raise some points to consider in future efforts.

Nucleotide Transition 390C-T in the Wilms' Tumor 1 Gene: A Risk Factor of Hypospadias?

INTRODUCTION: The gene Wilms' tumor 1 (WT1) encodes a unique transcription factor. Its defects are known to cause a wide range of complex genitourinary malformations and may contribute to non-syndromic forms of hypospadias. MATERIALS AND METHODS: We performed WT1 mutation analysis and copy number analysis of WT1-interacting protein in 13 Hungarian patients diagnosed with isolated hypospadias. RESULTS: Sequencing of WT1 revealed a high frequency of heterozygosity for transition 390C-T (5 heterozygotes out of 13 patients, including 2 brothers). WT1-interacting protein had a normal copy number in all patients. CONCLUSION: Nucleotide substitution 390C-T may play a role in the pathogenesis of non-syndromic hypospadias. The genotype-phenotype correlation should be confirmed by a larger-scale analysis.

WT1 Expression in Adult Acute Myeloid Leukemia: Assessing its Presence, Magnitude and Temporal Changes as Prognostic Factors.

Expression of the gene Wilms tumor 1 (WT1) has been suggested as a marker of minimal residual disease in acute myeloid leukemia (AML), but literature data are not without controversy. Our aim was to assess the presence, magnitude and temporal changes of WT1 expression as prognostic factors. 60 AML patients were followed until death or the end of the 6-year observation period. Blood samples were taken at diagnosis, post-induction, during remission and in case of a relapse. Using quantitative real-time PCR, we determined WT1 expression from each sample, normalized it against the endogenous control gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and classified samples as negative, moderately positive or highly positive. We divided the patients into groups based on detected WT1 expression values, illustrated overall and disease-free survival on Kaplan-Meier curves, and compared differences between each group by the logrank test. Disappearance of WT1-positivity during chemotherapy had a favorable effect on survival. Interestingly, no difference was seen between the survivals of WT1-positive subgroups that expressed moderate or high levels of WT1 mRNA. A 1-log decrease in WT1 expression without becoming negative did not affect prognosis, either. Our results suggest that defining a cut-off value for WT1-positivity, rather than just using logarithmic figures of changes in gene expression, might have prognostic use in post-induction AML patients. We encourage further, larger-scale studies.

WT1 overexpression affecting clinical outcome in non-hodgkin lymphomas and adult acute lymphoblastic leukemia.

The Wilms tumor 1 (WT1) gene has a complex role as a transcriptional regulator, acting as tumor suppressor or oncogene in different malignancies. The prognostic role of its overexpression has been well-studied in leukemias, especially acute myeloid leukemia (AML), but not in lymphomas. For the first time to our knowledge, we present a study demonstrating the correlation of WT1 expression and survival in various non-Hodgkin lymphomas. We also studied the prognostic implications of WT1 overexpression in adult acute lymphoblastic leukemia (ALL). In our sample of 53 patients--25 with diffuse large B-cell lymphoma (DLBCL), 8 with mantle cell lymphoma (MCL), 9 with peripheral T-cell lymphoma (PTCL), 2 with Burkitt's lymphoma, 2 with mucosa-associated lymphoid tissue (MALT) lymphoma, and 7 with B-cell ALL--, we measured WT1 mRNA from blood samples by quantitative RT-PCR, and divided the patients into subgroups based on the level of expression. Kaplan-Meier survival curves were drawn and compared using the logrank test. In the sample of DLBCL patients, the difference in overall and disease-free survival between WT1-positive and negative subgroups was significant (p = 0.0475 and p = 0.0004, respectively), and in a few observed cases, a sudden increase in WT1 expression signified a relapse soon followed by death. Disease-free survival curves in MCL and ALL were similarly suggestive of a potential role played by WT1. In PTCL, though WT1-positivity was detected in 4 out of 9 cases, it did not seem to affect survival. The few cases of MALT and Burkitt's lymphoma all proved to be WT1-negative.

WT1 microdeletion and slowly progressing focal glomerulosclerosis in a patient with male pseudohermaphroditism, childhood leukemia, Wilms tumor and cerebellar angioblastoma.

The Wilms tumor 1 (WT1) gene is currently in focus by pediatric nephrologists as its mutations are associated with nephrotic syndrome, especially as part of complex clinical entities like Denys-Drash or Frasier syndrome. Renal failure may also develop in young WAGR patients, whose condition is attributed to a deletion at chromosomal region 11p13. However, only limited data exist on WT1 microdeletions. A 30-year-old male patient, with a history of genital malformations, a Wilms tumor manifested during the treatment of acute lymphoid leukemia (ALL) at the age of 4, and a cerebellar angioblastoma, was referred with proteinuria and a reduced glomerular filtration rate (GFR). Kidney biopsy revealed FSGS. Although all WT1 exons were amplified with polymerase chain reaction (PCR) and sequenced, none of them showed a mutation. However, an formalin-fixed, paraffin- embedded (FFPE) tissue sample of the patient's childhood Wilms tumor showed WT1- positivity restricted to the renal tumor cells, so the WT1 gene was investigated further. Using quantitative reverse transcription PCR (qRT-PCR), the gene was found to be present in only one copy in the patient's genomic DNA sample, while both copies were detected in both parents. In the patient's sister, the proximal region of WT1 was shown to have an extra copy. Evidence suggests that a heterozygous microdeletion of the gene WT1 is responsible for the patient's disease. It seems reasonable to assume a possible abnormality affecting meiotic crossing over at the WT1 locus in one of the parents.