Significance of aberrant DNA methylation for cancer diagnostics and therapy.

BACKGROUND: Epigenetics is a scientific field that covers changes in gene expression that are not caused by the alteration of the nucleotide sequence in the DNA strand. Together with sequential changes, epigenetic reprogramming is a recognized cancer hallmark driving carcinogenesis. The underlying mechanisms of epigenetically-driven gene expression changes are diverse. However, one of the most extensively studied mechanisms is a change in DNA methylation. Under physiological conditions, DNA methylation ensures tissue-specific gene silencing and helps to maintain genome stability. With malignant transformation, genomic DNA undergoes global hypomethylation as well as locus-specific hypermethylation in promoters of tumor suppressor genes. In the last few decades, specific aberrant DNA methylation changes have emerged as both cancer-associated biomarkers and therapeutic targets and prompted ongoing efforts to enhance both diagnostic and therapeutic means in oncology. PURPOSE: The main purpose of this review is to introduce both established and emerging DNA methylation-based biomarkers for cancer diagnostics with a focus on biomarkers that are either routinely used or have been developed as commercial tests with certification for their use within in vitro diagnostics. Furthermore, therapeutic options for targeting aberrant DNA methylation are described, including both approved compounds and newly developed agents undergoing clinical investigation.

Molecular testing of endometrial carcinoma in real-world clinical practice.

BACKGROUND: Molecular classification has brought significant changes in the management of endometrial cancer (EC). In this article, we aim to analyze our first experience with an implementation of molecular testing into daily clinical practice. MATERIALS AND METHODS: In all newly diagnosed EC, the status of mismatch repair (MMR) and p53 proteins has been evaluated immunohistochemically as a part of the routine histopathological examination since May 2021. In tumors that do not meet clinical criteria for a low risk and those with MMR deficiency or p53 mutation, the molecular genetic testing of the POLE gene is performed as well. Recommendations for adjuvant treatment or follow-up are subsequently made based on the risk of recurrence. Genetic counselling is proposed to all patients with MMR-deficient tumors or family history of cancer. RESULTS: A total of 85 patients with newly diagnosed EC between May 2021 and May 2022 were enrolled in the analysis. The median age was 66 years. The results of molecular testing were as follows: 22 (26%) MMR-deficient, 8 (9%) p53-mutated and none POLE-ultramutated of those 40 tumors with performed POLE sequencing. A total of 46 (51%) patient had a low risk, 2 (2%) intermediate, 14 (16%) high-intermediate and 20 (24%) patients had a high risk of recurrence. Advanced or metastatic diseases were diagnosed in 6 (7%) patients. The median time between surgery and multidisciplinary tumor board decision was 21 days (8-36). A total of 76 (90%) patients underwent the whole treatment plan according to the recurrence risk. At the time of analysis, the results of genetic testing were available in 18 patients and revealed 4 (22%) carriers of a pathogenic variant in any of the genes associated with Lynch syndrome. CONCLUSION: Molecular testing combining immunohistochemical analyses of MMR and p53 proteins in all newly diagnosed EC patients with sequencing analysis of POLE in those with non-low-risk disease is feasible and does not prolong the time needed for treatment decision.

Biomarkers as prognostic and predictive factors in patients with hepatocellular carcinoma undergoing radiological oncological interventions.

BACKGROUND: Hepatocellular carcinoma is the most common malignant liver tumor in adults and thermal ablation and transarterial embolization are important methods of therapy. Thermal ablation can be used in early stages. Methods based on the transarterial approach, especially transarterial chemoembolization, play an important role in intermediate stage diseases. The success of procedures depends not only on the biological nature and the size of the tumor, on the technical design of the procedure and on the patient's response to treatment, but also on the molecular changes associated with these procedures. In addition to classic predictive and prognostic factors including age, patient comorbidities, Child-Pugh score, tumor characteristics, presence of large surrounding vessels, and portal vein thrombosis, molecular prognostic and predictive factors (serum biomarkers) are often mentioned in studies. Currently, only a-fetoprotein is routinely used as a prognostic biomarker; however, there are studies referring to new serum biomarkers that can potentially help to classical markers and imaging methods to determine the cancer prognosis and predict the success of therapy. These biomarkers most often include g-glutamyltranspeptidase, des- g-carboxyprothrombin, some types of microRNAs, inflammatory and hypoxic substances, whose serum levels are changed by the intervention therapies. Evaluation of these molecules could lead to the optimization of the medical intervention (choice of therapy method, timing of treatment) or change the management of patient follow-up after interventions. Although several biomarkers have shown promising results, most serum biomarkers still require validation in phase III studies. PURPOSE: The aim of this work is to present a comprehensive overview of classical and molecular biomarkers that could potentially help in the prognostic stratification of patients and better predict the success and effect of radiological intervention methods.

Swim bladder as a primary site of mycobacterial infection in Nothobranchius 'belly sliders'.

The swim bladder inflates early after fish hatching via its interconnection with the digestive tract (ductus pneumaticus). This interconnection may serve as a portal to foreign particles, including bacteria, causing deficiencies in primary swim bladder inflation. We histologically examined 134 African annual killifish (genus Nothobranchius) with secondary loss of swim bladder function ('belly sliders'). We demonstrate that these fish lost the ability of air regulation in their swim bladders likely due to Mycobacterium spp. infection at an individual-specific age. Nearly all examined belly sliders had thickened swim bladder walls, and their swim bladder was filled with material containing mycobacteria, cell debris, young monocytic cells and phagocyting macrophages. Mycobacterial infection was restricted to the swim bladder in juveniles, where mycobacteria likely enter the host through the ductus pneumaticus. Infection in adults was systemic and mycobacteria were present in all examined organs. Presence of mycobacteria in the epithelial lining and submucosal layers of the digestive tract of adults suggests that it may also serve as the entrance site of infection. We suspect 2 sources of Mycobacterium contamination: dietary (with bloodworms) and/or contaminated hatching substrate. These sources of contamination may be eliminated by use of laboratory dry feed and egg disinfection prior to hatching.

Barriers in systemic delivery and preclinical testing of synthetic microRNAs in animal models: an experimental study on miR-215-5p mimic.

Mus musculus is the most commonly used animal model in microRNA research; however, little is known about the endogenous miRNome of the animals used in the miRNA-targeting preclinical studies with the human xenografts. In the presented study, we evaluated the NOD/SCID gamma mouse model for the preclinical study of systemic miR-215-5p substitution with a semitelechelic poly[N-(2-hydroxypropyl)-methacrylamide]-based carrier conjugated with miR-215-5p-mimic via a reductively degradable disulfide bond. Murine mmu-miR-215-5p and human hsa-miR-215-5p have a high homology of mature sequences with only one nucleotide substitution. Due to the high homology of hsa-miR-215-5p and mmu-hsa-miR-215-5p, a similar expression in human and NOD/SCID gamma mice was expected. Expression of mmu-miR-215 in murine organs did not indicate tissue-specific expression and was highly expressed in all examined tissues. All animals included in the study showed a significantly higher concentration of miR-215-5p in the blood plasma compared to human blood plasma, where miR-215-5p is on the verge of a reliable detection limit. However, circulating mmu-miR-215-5p did not enter the human xenograft tumors generated with colorectal cancer cell lines since the levels of miR-215-5p in control tumors remained notably lower compared to those originally transfected with miR-215-5p. Finally, the systemic administration of polymer-miR-215-5p-mimic conjugate to the tail vein did not increase miR-215-5p in NOD/SCID gamma mouse blood plasma, organs, and subcutaneous tumors. It was impossible to distinguish hsa-miR-215-5p and mmu-miR-215-5p in the murine blood and organs due to the high expression of endogenous mmu-miR-215-5p. In conclusion, the examination of endogenous tissue and circulating miRNome of an experimental animal model of choice might be necessary for future miRNA studies focused on the systemic delivery of miRNA-based drugs conducted in the animal models.

Integrated dia-gnostics of diffuse gliomas.

Recently, the World Health Organization (WHO) classification of tumours of the central nervous system (CNS) has brought essential changes. The currently valid revised WHO 2016 classification of CNS tumours introduced the concept of integrated dia-gnostics, which incorporated not only histopathological morphological finding and immunophenotype but also molecular-genetic characteristics of the tumour. Thus, the final integrated dia-gnosis comprises the traditional morphological and growth pattern characteristics of a tumour including histopathological grade and also specific molecular bio-markers. The classification of tumour based on a combination of both tumour phenotype and genotype enables more precise prognostic stratification, increases the objectivity of dia-gnostics and prediction of response to treatment. In 2017, an international platform, The Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy - not official WHO (cIMPACT-NOW), was established to create and formulate practical recommendations for integrated dia-gnostics of CNS tumours and upcoming WHO classification. The incorporation of molecular bio-markers into the integrated dia-gnostics radically changed the classification of diffuse gliomas, which include entities with different morphological characteristics, genetic alterations and bio-logical behaviour. This review article summarizes essential morphological, immunophenotypical and molecular genetic characteristics of diffuse gliomas within the scope of integrated dia-gnostics according to the valid WHO classification of tumours of the CNS and subsequent recommendations of dia-gnostic approaches. This work was supported by grant of the Ministry of Health of the Czech Republic - Conceptual Development of a Research Organization (MMCI 00209805) and Grant Agency of Masaryk University (MUNI/A/1562/2018). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.

Rare Hereditary Burden associated with a Hypercalcemic Small-Cell Carcinoma of Cervix in a Young Female Patient.

BACKGROUND: Oncological diseases have, in most cases, a multifactorial etiology, composed of a combination of external and internal environmental factors. Hereditary tumorous syndromes are mostly autosomal dominant diseases with incomplete but very high penetrance. OBSERVATION: The patient, an 18-year-old virgin female, consulted a gynecologist in June 2018 because of metrorrhagia. Magnetic resonance imaging revealed a cervical tumor with the dimensions 80 × 90 × 80 mm. Histological analysis confirmed the presence of a very rare hypercalcemic type of small-cell carcinoma of the cervix. Further investigation of the germinal exom of the patient showed pathological variations in genes PALB2 and BRCA2, presented with recommendation of detailed examination by medical genetics. CONCLUSION: Clinical experience with this type of tumor is very limited, but it still comes with some useful outcome. Small cell carcinomas of the gynecologic tract are very rare, aggressive diseases, with very poor prognosis, affecting mainly young women. Their origin is most often the ovaries, based on most clinical data, but these tumor also localize to the endometrium, cervix, vagina and vulva. It is an extremely rare type of cancer, for which clinical data is scant due to the extremely low number of reported cases. In this patient, the carcinoma had an unusual genetical mutation burden, which she inherited from her parents. In the light of these findings, we recommend that patients suspected of having a small-cell of the gynecologic tract provide a detailed family history, and that genetic testing be considered in similar cases. This work was supported by MH CR grant 16-33209A and research program of Charles University Progress Q40/06. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 10. 6. 2019 Accepted: 9. 9. 2019.

Utilization of Next Generation Sequencing in Analysis of Circulating MicroRNAs as Predictive Biomarkers for Patients with Locally Advanced Rectal Carcinoma.

BACKGROUND: MicroRNAs (miRNA) are short non-coding RNAs involved in post-transcriptional regulation of gene expression. MiRNAs are essential regulators of both physiological processes as of pathogeneses of many diseases, and their dysregulation was observed in many malignancies including rectal cancer. Circulating miRNAs presented in blood plasma could be potential candidates for non-invasive predictive biomarkers of the response of patients with locally advanced rectal cancer to chemoradiotherapy. Presented study aims to evaluate the potential of next-generation sequencing in the analysis of circulating miRNAs. MATERIAL AND METHODS: MiRNA expression profiles were done using samples of RNA isolated from blood plasma collected during TNM restaging and paired samples collected before initiation of neoadjuvant chemoradiotherapy. Sequencing libraries were prepared using kit which implements universal molecular indices that help to sensitively filter biological bias during data analysis. Sequencing data were processed by multidimensional biostatistical approaches. CONCLUSION: We identified specific miRNA profile enabling to distinguish the patients accordingly to their response to chemoradiotherapy. This work was supported by the Czech Ministry of Health grant No. 16-31765A. The authors declare they have no potential confl icts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 22. 2. 2019 Accepted: 27. 2. 2019.

Dynamic Changes in Circulating MicroRNA Levels in Liver Cancer Patients Undergoing Thermal Ablation and Transarterial Chemoembolization.

BACKGROUND: Hepatic cancer patients who cannot undergo surgical resection of tumour are candidates for methods of interventional radiology - transarterial chemoembolization (TACE) or thermal ablative (TA) therapy. Both methods are causing characteristic changes in liver tissue (inflammatory immune response, hypoxia, elevated temperature, tissue destruction) which are accompanied with systemic secretion of cytokines or microRNAs (miRNAs). The aim of our study was to investigate whether the level of circulating miRNAs related to hypoxia (miR-21 and miR-210), liver injury (miR-122) and epithelial-mesenchymal transition (miR-200a) could reflect systemic effect of these intervention techniques. MATERIALS AND METHODS: Study consisted of 10 primary hepatocellular carcinoma patients treated with TACE and 10 patients with liver metastases of colorectal cancer treated with TA. Thermal ablation was performed using the radiofrequency or microwave generator (RITA, Microsulis, AngioDynamics,Inc), for TACE drug eluting beads (DCBeads, Biocompatibles Ltd.) were used. Tumours were evaluated using RECIST (Response Evaluation Criteria in Solid Tumours), mRECIST (modified RECIST) criterion and volumetry. For all patients we determined concentrations of miRNA in blood plasma samples from four time points (before intervention, immediately after intervention, 24 hours after intervention, 1 week after intervention) using TaqMan® Assays and quantitative real time polymerase chain reaction method. RESULTS: After both intervention techniques we observed changes in circulating miRNA levels. In TA cases we observed significant increase of miR-122 and miR-200a concentrations immediately after intervention, on the contrary in TACE we observed increase in miRNA concentration at time point 24 hours after intervention (miR-21, miR-210, miR-122, miR-200a). Increased concentration of circulating miRNA was followed by subsequent decline to initial level. These changes were consistent with presumed biological effect of TA and TACE on tumour tissue. CONCLUSION: Data of this pilot study show potential usage of circulating miRNA for monitoring of systemic effect of thermal ablative and intraarterial therapies. This work was created at Masaryk University as part of the project MUNI/A/1574/2018 and it was supported by Czech Ministry of Health grants No. 15-32484A, 16-31765A and 16-31314A. The authors declare they have no potential confl icts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 1. 3. 2019 Accepted: 4. 3. 2019.

Molecular Classification of Medullobastomas by Whole Genome Expression Profiling.

BACKGROUND: Medulloblastoma (MB) is the most common malignant tumour of the central nervous system in children. MB is considered to be high risk tumour propensity to metastasize. In the Czech Republic, approximately 10-12 children are affected annually by this tumour. Recent progress in molecular diagnostics helps to refine the diagnosis and estimate clinical prognosis of the disease. Currently, MBs are subclassified into WNT-activated, SHH-activated, group 3, and 4 based on molecular pathways that drive their tumorigenesis. Each subtype differs in its histopathology, clinical features, genomic changes and gene expressions. The aim of our study is to classify patients MBs into four basic molecular groups and compare our results with published data. MATERIAL AND METHODS: In our study we analysed expression profiles using Affymetrix GeneChip Human Gene 1.0. ST Array (Thermo Fisher Scientific, MA, USA). As input material RNA extracted from the fresh frozen tissue was used. Molecular classification based on the method established by P. Northcott in 2011 was performed. RESULTS: From April 2015 to February 2019, 21 patients with MBs were included in our study. Median age of the patients at the time of diagnosis was 6 years, 14 boys and 7 girls were enrolled. Gene expression profiling and molecular classification of MBs was performed. Based on this methodology, we found the most frequently represented subgroup of MB was group 4 (9 patients, 43%), followed by group 3 (5 patients, 24%), SHH-activated MB (4 patients, 19%) and the least represented subgroup was WNT-activated MB (3 patients, 14%). Results of molecular subgroup classification of MBs were successfully correlated with histopathological findings and other molecular-genetic examinations. CONCLUSION: Molecular classification of MBs has been established in our institution allowing better understanding of this heterogeneous disease and helping clinicians in therapeutic planning in affected patients. This work was supported by the Czech Ministry of Health grant No. 16-33209A. All rights reserved.  he authors declare they have no potential confl icts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 1. 3. 2019 Accepted: 4. 3. 2019.

Analysis of Blood Plasma MicroRNAs to Enable Identification of Patients with Pancreatic Ductal Adenocarcinoma Who Will Benefit from Surgical Resection

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is the most common malignancy of pancreas, characterized by extremely poor prognosis largely due to problem with early diagnosis and lack of progress in personalization of therapy. Of all available treatment strategies, radical surgical resection of the tumour in its early stage remains the only possibility how to reach long-term survival. However, even a technically perfect surgical resection may still not provide a survival benefit for all PDAC patients. Appropriate selection of patients for surgical resection is one the important medical needs in management of PDAC patients. MATERIAL AND METHODS: To this study we enrolled 24 PDAC patients who underwent surgical resection and preoperatively collected their blood plasma specimends. Patients were divided into to two prognostic groups according to their overall survival - 12 patients with poor prognosis (median overall survival 10 months) and 12 patients with good prognosis (median overall survival 25 months). Small RNA sequencing technology was applied to screen for microRNAs (miRNA) with differential levels between both PDAC patients group. cDNA libraries were prepared using QIAseq miRNA Library Kit (Qiaqen) and sequencing by NextSeq500 instrument (Illumina). RESULTS: When miRNA expression profiles of the PDAC patients from good and poor prognostic groups were compared, 61 miRNAs were identified to have significantly different plasma levels between the two groups (p < 0.05). A total of 21 miRNAs showed increased expression and 40 miRNAs showed decreased expression in a group of patients with poor prognosis compared to patients with good prognosis. CONCLUSION: This study demonstrated differences in miRNA expression profiles in preoperative plasma specimens of PDAC patients with short and long overall survival. Our observations indicate that after independent validations plasma miRNAs might become useful biomarkers for identification of PDAC patients having clinical benefit from surgical resection of the tumour. This work was supported by Czech Ministry of Health, grant No. 16-31314A. All rights reserved. The authors declare they have no potential confl icts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 8. 3. 2019 Accepted: 9. 3. 2019.

Effect of Tumor Size and p16 Status on Treatment Outcomes - Achievement of Complete Remission in Prospectively Followed Patients with Oropharyngeal Tumors.

BACKGROUND: Oropharyngeal squamous cell tumors associated with human papillomavirus infection (p16 positive tumors) have better prognosis than p16 negative tumors regardless of the more advanced stage of the disease. Tumor volume (GTVt+n) is generally an important factor affecting treatment results of ionizing radiation. The aim of this prospective non-randomized study is to evaluate the effect of tumor volume on the (chemo)radiation treatment results in a group of patients with p16 negative and p16 positive oropharyngeal tumors. PATIENTS AND METHODS: Patients with confirmed squamous cell tumor of the oropharynx of stages III and IV, according to the 7th version of the TNM (tumor-nodes-metastases) classification, were eligible for this study. The main exclusion criteria were palliative treatment, neoadjuvant chemotherapy or planned concomitant therapy with cetuximab. Patients were treated according to standardized protocols with curative intent. Primary tumor volume (GTVt) and involved nodes volume (GTVn) were obtained from radiotherapy planning system for further statistical analysis. The differences in tumor volumes between the groups according to p16 expression were assessed with subsequent testing of probability to achieve complete remission (CR) of the disease in both groups. RESULTS: In total, 49 patients - 84% men, median age 60.5 years, 25 (51%) patients p16 positive, 40 (82%) underwent concomitant chemoradiotherapy. Median of GTVt in the whole patients group is 40.2 ccm, GTVn 11.78 ccm and median volume of the whole tumor burden (GTVt+n) 70.21 ccm (range 11.05-249). Median of GTVn was greater in the p16 positive cohort (p = 0.041). In the entire group, the median time to reach CR was 91 days (95% CI 86-107 days) from the end of radiotherapy. In the group of p16 negative patients, 14 achieved CR (61%) out of 23 patients, in p16 positive group 20 (80%) out of 25 patients (p = 0.111). P16 negative patients had a longer time to CR (p = 0.196, HR 1.58, 95% CI 0.79-3.18). None of the independently assessed volumetric parameters of the tumor (GTVt, GTVn, GTVt+n) affected CR in the p16 positive patients group, while there was a significant impact of the whole tumor burden (GTVt+n) in the p16 negative cohort (median 58.1 ccm in CR patients vs. 101.9 ccm, p = 0.018). CONCLUSION: We have showed less GTVt+n dependence to achieve CR in p16 positive tumors in comparison with p16 negative tumors. Thus, p16 positive oropharyngeal squamous cell cancers should not be withdrawn from the curative treatment intent based on the greater GTVt+n. Key words oropharyngeal neoplasms - p16 status - treatment outcome - tumor burden - complete remission This work was supported by grant of the Ministry of Health of the Czech Republic AZV 15-31627A and by grant of the Ministry of Health of the Czech Republic - Conceptual development of a research organization (MMCI 00209805). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 2. 11. 2018 Accepted: 11. 11. 2018.

Effective Immunotherapy of Glioblastoma in an Adolescent with Constitutional Mismatch Repair-Deficiency Syndrome.

BACKGROUND: Individuals with constitutional mismatch repair-deficiency syndrome (CMMR-D) are characterised by early occurrence of colon cancer, haematological malignancies, and brain tumors (malignant gliomas, high-grade gliomas) in childhood, adolescence, and early adulthood. High mutational tumor burden is typical of glioblastoma in CMMR-D patients and could be a reason why this type of glioblastoma responds well to immunotherapies, including those that employ checkpoint inhibitors. OBSERVATION: We describe a case of an adolescent with CMMR-D that had been genetically proven by whole exome sequencing (c.2T>A/p.M1K and c.2521delT/p.W841fs PMS2 gene mutation). The patient presented successively with colon cancer and glioblastoma with a high mutational burden. The individualized glioblastoma therapy was based on the biological tumor profile and included immunotherapy with a combination of vaccination with autologous dendritic cells producing IL-12 and nivolumab, in addition to radiotherapy with metronomic temozolomide. The patient is still alive 21 months after the initial glioblastoma diagnosis and shows a complete therapeutic response documented by repeated magnetic resonance examinations. CONCLUSION: Individuals with CMMR-D should be regularly examined using established algorithms. Whole body magnetic resonance imaging can play a key role, because it enables the early diagnosis of malignancy during the asymptomatic period. Malignancies in CMMR-D patients usually exhibit a hypermutated genotype and respond to immunotherapy. Conventional glioblastoma therapy is only palliative. Patients can benefit from an individualized therapeutic plan based on the tumor biological profile. Extensive molecular analysis of the tumor tissue is necessary. Key words hereditary cancer predisposition syndromes - glioblastoma - whole exome sequencing - immunotherapy - vaccines - checkpoint inhibitors This study was supported by the research project of the Czech Ministry of Health AZV 16-33209A (Next generation sequencing and express profiling as diagnostic tools for personalized therapeutic plans in children with solid tumors). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 26. 9. 2018 Accepted: 18. 11. 2018.

[MicroRNA Analysis for Extramedullary Multiple Myeloma Relapse]. / Analýza mikroRNA u extramedulárního relapsu mnohocetného myelomu.

INTRODUCTION AND AIMS: Multiple myeloma (MM) is the second most common hematooncological disease. Patient survival has been greatly improved by the introduction of new drugs into clinical practice, but survival is negatively affected by the so-called extramedullary relapse (EM), caused by the loss of plasma cell dependence on the bone marrow microenvironment and their migration out of the bone marrow. The nature and causes of this process are currently unclear. MicroRNAs (miRNAs) are short, non-coding RNA molecules involved in many physiological and pathological processes. Their significance in the pathogenesis of MM has been demonstrated by several studies. We assume that they are also involved in the development of the EM. The aim of this study was to analyze different miRNA expression between MM and EM patients. MATERIAL AND METHODS: Using next generation sequencing, we analyzed 39 samples of bone marrow cells from MM patients at diagnosis and 9 bone marrow plasma samples of EM patients. RESULTS: In total, 2,278 miRNA were sequenced, but only 658 miRNAs were analyzed as they were expressed in all samples and had at least 20 reads. Expression data were generated using the Chimira tool from fastq data. All sequences were mapped using miRBase v20. Further analyses were performed using the R/Bioconductor package. The Bayesian procedure was used for normalization of expression. P values were adjusted using the Benjamini-Hochberg method. Analysis found 10 miRNA (p < 0.0005) that are statistically significantly expressed in EM vs. MM patients - these are miR-26a-5p, miR-26b-5p, miR-30e-5p, miR-424-3p, miR-503-5p, miR-767-5p, miR-105-5p, miR-5695-5p, miR-450b-5p and miR-92b-3p. These miRNAs will be further verified by qPCR method on a larger set of MM and EM patients. CONCLUSION: Our pilot study has shown that there are differentially expressed miRNAs between MM and EM patients.Key words: multiple myeloma - microRNA - carcinogenesis - next generation sequencing The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papersThis work was supported by grant MZ CR AZV 17- 29343A. Submitted: 17. 3. 2018Accepted: 20. 3. 2018.

[Pilot Study on MicroRNAs as Biomarkers of Response to Sunitinib Treatment in Patients with Metastatic Renall Cell Carcinoma]. / MikroRNA jako biomarkery odpovedi na lécbu sunitinibem u pacientu s metastatickým karcinomem ledvin - pilotní studie.

BACKGROUND: Renal cell carcinoma (RCC) accounts for 2-3% of all malignant tumours. Metastatic RCC (mRCC) is commonly treated with tyrosine kinase inhibitors (TKI). Effective TKIs administration can be achieved only by accurate prediction of therapeutical response. Therefore, the aim of this study was to analyse papers concerning predictive potential of microRNA (miRNA). MATERIAL AND METHODS: We chose seven candidate miRNAs and analysed their expression on 44 patients divided into cohort with poor and good response to sunitinib treatment. Patients were divided into two groups according to progression-free survival. RNA from tissue samples was isolated and expression of selected miRNAs was measured using quantitative PCR with miRNA-specific TaqMan probes. RESULTS: We successfully validated two miRNAs to be differentially expressed in responding and non-responding patients to sunitinib treatment. Other analysed miRNAs have not shown predictive potential. CONCLUSION: From miRNAs studied so far, two miRNAs had predictive value according to present study.Key words: microRNA - renal cell carcinoma - sunitib The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Supported by Ministry of Health of the Czech Republic, grant No. 15-34678A. All rights reserved.Submitted: 19. 3. 2018Accepted: 20. 3. 2018.

[Dysregulation of Long Non-coding RNAs in Glioblastoma Multiforme and Their Study Through Use of Modern Molecular-Genetic Approaches]. / Dysregulace dlouhých nekódujících RNA u multiformního glioblastomu a jejich studium s vyuzitím moderních molekulárne-genetických prístupu.

BACKGROUND: Glioblastoma (GBM) is the most frequent primary brain tumor characterized by an unfavourable prognosis despite multimodal therapy. Therefore, a lot of efforts and financial resources are dedicated to the research of new therapeutic targets and prognostic or predictive biomarkers. Long non-coding RNAs (lncRNAs) are regulators of gene expression which play a significant role in GBM pathology and, thus, present promising candidates. MATERIAL AND METHODS: Our study included 14 patients with GBM and 8 patients with intractable epilepsy from whom we acquired brain tissues during surgical intervention. Ribosomal RNA depleted RNA was used for sequencing by NextSeq 500 instrument (Illumina). Statistical analysis evaluated 24,087 protein-coding and 8,414 non-coding RNAs and their sequential variants with non-zero reads per kilobase per million mapped reads (RPKM) at least in one sample. CLC Genomic Workbench was used for the alignment and target counts. Targeted downregulation of up-regulated ZFAS1, one of the identified lncRNA, level has been carried out by the transient transfection of specific small interfering RNA (siRNA) in GBM stable cell lines (A172, U87MG, T98G). The success of transfection and viability were analyzed in vitro using quantitative real time polymerase chain reaction and MTT assay, resp. RESULTS: Statistical analysis has revealed 274 (p < 0.01) dysregulated lncRNAs in GBMs in comparison with non-tumor brain tissues. Moreover, the results have showed 489 dysregulated mRNAs (p < 0.0001) and 26 mRNAs (p < 0.000001). Transfection of ZFAS1 inhibitor led to successful downregulation of ZFAS1 expression level, although it did not have a significant effect on proliferation of GBM cells. CONCLUSION: We described a significant dysregulation of lncRNAs and mRNAs in GBM tissue in comparison with non-tumor tissue. We also succesfully decreased expression level of ZFAS1, which in turn, however, had no impact on the viability of GBM cell lines.Key words: glioblastoma - long non-coding RNA - next-generation sequencing The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. This tudy was supported by Ministry of Health of the Czech Republic, grant No. 15-33158A. All rights reserved.Submitted: 19. 3. 2018Accepted: 10. 4. 2018.

[MicroRNAs in Prediction of Response to Radiotherapy in Head and Neck Cancer Patients - Pilot Study]. / MikroRNA v predikci odpovedi na radioterapii u pacientu s nádory hlavy a krku - pilotní studie.

BACKGROUND: Radiotherapy plays a key role in the treatment of squamous cell head and neck cancers (HNSCC). The effectivity of radiation therapy is often limited by radioresistance of these tumours. microRNAs (miRNAs) are endogenous, evolutionary conserved, small non-coding RNAs involved in regulation of cellular processes associated with radioresistance. The objective of this study was to identify miRNA profile enabling to predict the radiation treatment outcomes in HNSCC patients. MATERIAL AND METHODS: The retrospective study included HNSCC patients who underwent a definitive radiotherapy. Patients were divided into two groups according to loco-regional control (LRC) as follows - short LRC (n = 22; median 5.1 months (min. 1.3, max, 18.6)) vs. long LRC (n = 21; 60.4 (min. 46.8, max. 98.8)) group. Global miRNA expression profiles were obtained by use of Affymetrix microarray technology (GeneChip miRNA 4.0 Array). RESULTS: We identified 24 miRNAs to be significantly associated with LRC (p < 0.05), all of these miRNAs were upregulated in patients with short LRC. Out of these miRNAs, 12 miRNAs with p < 0.025 and 4 miRNAs with p < 0.01 have been identified. CONCLUSION: miRNAs seems to be promising as potential biomarkers predicting radiotherapy treatment outcomes in patients with HNSCC.Key words: microRNAs - radiotherapy - head and neck cancer The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Supported by Ministry of Health of the Czech Republic, grant No. 15-31627A. All rights reserved.Submitted: 19. 3. 2018Accepted: 20. 3. 2018.

[Usage of Cerebrospinal Fluid for microRNA Analysis]. / Vyuzití mozkomísního moku pro analýzu mikroRNA.

BACKGROUNDS: Deregulated levels of miRNAs, short noncoding RNAs associated with pathogenesis of many diseases, have been observed in cerebrospinal fluid (CSF). Therefore, the analysis of CSF miRNAs in patients affected by tumors of central nervous system (CNS) might help to develop new diagnostic platform enabling more precise diagnosis. Thus, in our study we tried to optimize methodical approaches to be used for miRNA detection as RNA isolation and selection of suitable technology for global high-throughput miRNA profiling. MATERIAL AND METHODS: In the optimization phase of RNA isolation from CSF, various commercially available kits with different protocol modifications were compared. Two quantitative polymerase chain reaction panels and Next Generation Sequencing method were tested for selection of the most suitable method for miRNA comprehensive profiling. RESULTS: The Urine miRNA Purification kit (Norgen) and Next Generation Sequencing was selected as the most suitable kit for RNA extraction from CSF and method for miRNA comprehensive profiling, resp. CONCLUSION: We established a protocol for RNA isolation and miRNA comprehensive profiling in CSF clinical specimens.Key words: brain neoplasm - cerebrospinal fluid - microRNA The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. This study was supported by Ministry of Health of the Czech Republic, grant No. 15-34553A. All rights reserved.Submitted: 19. 3. 2018Accepted: 10. 4. 2018.

[Urinary MicroRNAs as Potential Biomarkers of Bladder Cancer]. / Mocové mikroRNA jako potenciální biomarkery karcinomu mocového mechýre.

BACKGROUND: Currently, there are no urinary-based tumour markers with sufficient sensitivity and specificity to replace cystoscopy in the detection of bladder cancer (BCA). Urinary microRNAs are emerging as clinically useful class of biomarkers for early and non-invasive detection of urologic malignancies. PATIENTS AND METHODS: In this study, 155 patients with BCA and 83 healthy controls were enrolled. Expression profiles of urinary miRNAs were obtained using Affymetrix miRNA microarrays and candidate miRNAs further validated in independent cohort using specific TaqMan assays and quantitative real-time polymerase chain reaction method. RESULTS: Whole-genome profiling identified miRNA signature with significantly different concentrations in urine of BCA compared to controls (p < 0.01). In the independent validation phase of the study, three miRNAs were confirmed to have significantly higher levels in urine of patients with BCA in comparison with control groups (p < 0.0001). In addition, we observed significant decrease in two miRNAs (p < 0.01) concentrations in the urinary samples collected 3 months after surgery compared to pre-operative samples. CONCLUSION: We identified and validated miRNAs to have significantly higher concentrations in urine of patients with BCA in comparison with controls. Our data have shown that urinary miRNAs could serve as sensitive and specific biomarkers enabling non-invasive detection of BCA.Key words: urinary microRNAs - biomarkers - bladder cancer The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. This study was supported by Ministry of Health of the Czech Republic, grant No. 15-31071A. All rights reserved.Submitted: 19. 3. 2018Accepted: 20. 3. 2018.