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Background/Aim: Brain metastases (BMs) are the most frequent intracranial tumors in adults and one of the greatest challenges for modern oncology. Most are derived from lung, breast, renal cell, and colorectal carcinomas and melanomas. Up to 14% of patients are diagnosed with BMs of unknown primary, which are commonly characterized by an early and aggressive metastatic spread. It is important to discover novel biomarkers for early identification of BM origin, allowing better management of patients with this disease. Our study focused on microRNAs (miRNAs), which are very stable in frozen native and FFPE tissues and have been shown to be sensitive and specific diagnostic biomarkers of cancer. We aimed to identify miRNAs with significantly different expression in the five most frequent groups of BMs and develop a diagnostic classifier capable of sensitive and specific classification of BMs. Materials and Methods: Total RNA enriched for miRNAs was isolated using the mirVana miRNA Isolation Kit from 71 fresh-frozen histopathologically confirmed BM tissues originating in 5 cancer types. Sequencing libraries were prepared using the QIAseq miRNA Library Kit and sequenced on the NextSeq 500 platform. MiRNA expression was further validated by RT-qPCR. Results: Differential analysis identified 373 miRNAs with significantly different expression between 5 BM groups (p

Brain metastases are the most frequent intracranial tumors in adults and the cause of death in almost one-fourth of cases. The incidence of brain metastases is steadily increasing. The main reason for this increase could be the introduction of new and more efficient therapeutic strategies that lead to longer survival but, at the same time, cause a higher risk of brain parenchyma infiltration. In addition, the advances in imaging methodology, which provide earlier identification of brain metastases, may also be a reason for the higher recorded number of patients with these tumors. Metastasis is a complex biological process that is still largely unexplored, influenced by many factors and involving many molecules. A deeper understanding of the process will allow the discovery of more effective diagnostic and therapeutic approaches that could improve the quality and length of patient survival. Recent studies have shown that microRNAs (miRNAs) are essential molecules that are involved in specific steps of the metastatic cascade. MiRNAs are endogenously expressed small non-coding RNAs that act as post-transcriptional regulators of gene expression and thus regulate most cellular processes. The dysregulation of these molecules has been implicated in many cancers, including brain metastases. Therefore, miRNAs represent promising diagnostic molecules and therapeutic targets in brain metastases. This review summarizes the current knowledge on the importance of miRNAs in brain metastasis, focusing on their involvement in the metastatic cascade and their potential clinical implications.

Introduction: Glioblastoma (GBM) is the most frequent primary astrocytoma characterized by an aggressive and diffuse growth and a poor prognosis. The median survival of patients with GBM is only 12 to 16 months from diagnosis despite conventional therapy. It is, therefore, important to identify new therapeutic targets, which would allow a more effective treatment. In our present study, we focus on long non-coding RNAs (lncRNAs), the regulators of gene expression in both physiological conditions and GBM pathology, and their potential as targets for future therapy. Material and Methods: Our study included 219 GBM patients and 29 intractable epilepsy patients as controls who signed an informed consent prior to treatment. Previously, cDNA libraries were prepared from 77 rRNA-depleted RNA samples and sequenced, and the expression of 11 significantly dysregulated lncRNAs was validated in 188 specimens by RT-qPCR. Results were newly validated in an independent TCGA-GBM cohort. Moreover, LINC00634 expression was upregulated in U251 and T98G cells. Cells transfected with an empty vector served as negative controls (NCs). The effect of upregulated LINC00634 expression on viability, migration, and clonogenicity was studied in vitro, and, in the case of U251-derived cell line, in vivo in immunodeficient mice with the focus on GBM growth and diffuse character. Results: Out of the 538 significantly dysregulated lncRNAs (P < 0.001) identified by transcriptome sequencing, a panel of 10 downregulated lncRNAs (SNAI3-AS1, LINC00882, RFPL1S, MIR137HG, TTLL7-IT1, PWAR6, LINC00634, LINC00632, DGCR5, LINC00982; logFC ≤ -2; P < 0.001) and 1 upregulated lncRNA (BTN2A3P; logFC ≥ 2; P < 0.001) in GBM was validated previously by qPCR in another cohort (P < 0.0001 for all lncRNAs) and newly in an independent TCGA-GBM dataset. The upregulated LINC00634 expression in U251 and T98G cells was confirmed by qPCR to be approximately 11,000-fold and 600-fold, respectively, compared to the expression in NCs. Although no significant effect on viability, migration and clonogenicity was observed in vivo, a less diffuse growth pattern was observed in U251-derived GBMs with upregulated expression of LINC00634 in vivo compared with NCs. Conclusion: A significant dysregulation of lncRNAs was previously observed in GBM compared to non-tumor controls using both transcriptome sequencing and RT-qPCR and was newly confirmed also by analyzing the TCGA-GBM dataset. We also showed possible effect of LINC00634 upregulation on the growth pattern of U251-derived GBM tumors in vivo. Our study shows that lncRNAs are dysregulated in GBM and could, therefore, serve as promising diagnostic biomarkers in GBM as well as potential therapeutic targets. The research was supported by the project National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102, funded by the European Union - Next Generation EU). Citation Format: Marek Vecera, Lenka Radova, Radim Lipina, Stefan Reguli, Martin Smrcka, Radim Jancalek, Michal Filip, Marketa Hermanova, Leos Kren, Dagmar Al Tukmachi, Tana Machackova, Petra Pokorna, Jiri Sana, Ondrej Slaby. Long non-coding RNAs are dysregulated in glioblastoma and LINC00634 may affect the diffuse growth of U251-derived tumors in vivo. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3765.

Brain metastases (BMs) comprise a heterogeneous group of the most frequent intracranial tumors in adults, most originating in lung, breast, renal cell, and colorectal carcinomas and melanomas. Despite the recent improvements in imaging methodology resulting in earlier BM identification and advancements in treatment strategies, BMs are still a significant cause of patient morbidity. Furthermore, BMs frequency increases due to more prolonged survival of cancer patients and population aging. Since the most widely used prognostic scoring systems for BMs require prior knowledge of the primary origin and up to 14% of BMs are classified as BMs of unknown primary, there is an urgent unmet need for accurate biomarkers for identification of BM origin. MiRNAs are non-coding RNAs with an approximate length of 22 nucleotides, functioning as post-transcriptional regulators of gene expression. Dysregulated miRNA expression profile has been observed in many pathological processes, including the complex and not fully understood metastatic cascade. These molecules are very stable and present not only in tissues but also in human body fluids, including blood plasma and cerebrospinal fluid (CSF). Based on these facts, both tissue and circulating miRNAs are extensively studied as potential diagnostic biomarkers. Specific miRNA signatures of BMs were obtained using high-throughput miRNA profiling (Illumina small RNA sequencing) on 3 types of samples (metastatic tissue, blood plasma, CSF) from a cohort of 30 patients with BMs originating in the 5 tumor types – lung, breast, renal cell and colorectal carcinomas and melanomas (6 patients per group, 87 samples in total, only 3 CSF samples from RCC patients available). We identified significantly differentially expressed miRNAs in BM tissues with the ability to differentiate between primary origins. Tissue miRNAs could identify BMs originating from breast, colorectal and renal cell carcinomas and melanomas with high specificity and sensitivity. Interestingly, the heterogeneity of lung carcinomas was also characteristic for the corresponding BMs, making it challenging to distinguish accurately from other BMs. Even though the tissue-specific miRNA signature was the most precise, our results suggest a significant diagnostic potential of circulating miRNAs from CSF for BM patients. Therefore, these short and stable molecules could potentially help identify the origin of BMs of unknown primary. The research is supported by project National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union - Next Generation EU. Citation Format: Dagmar Al Tukmachi, Michaela Ruckova, Marek Vecera, Tana Machackova, Petra Pokorna, Marketa Hermanova, Michal Hendrych, Leos Kren, Ivana Roskova, Vaclav Vybihal, Hana Valekova, Radim Jancalek, Jiri Sana, Martin Smrcka, Ondrej Slaby. Tumor tissue and cerebrospinal fluid microRNA profiles enable the classification of brain metastasis accordingly to their origin. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3758.

BACKGROUND Brain metastases (BMs) are intracranial tumors that frequently occur in adult cancer patients. Because the prognosis of patients with BM individually varies, it would be useful to improve prognostic scoring tools by including new high-performance biomarkers. MicroRNAs (miRNAs) appear to be promising in this regard as they are highly stable and, thus, suitable for both next-generation sequencing (RNA-Seq) and retrospective analyses in formalin-fixed and paraffin-embedded (FFPE) tissues.Material and METHODS Total RNA enriched for miRNAs was isolated from 71 freshly frozen histopathologically confirmed BMs with origin in 5 tumor types (ca lung - 37%, melanoma - 23%, ca breast - 18%, RCC - 15%, CRC - 7%) using mirVana miRNA Isolation Kit (Thermo Fisher Scientific). Sequencing libraries were prepared from RNA using the QIAseq miRNA Library Kit (Qiagen) and sequenced using the NextSeq 500 platform (Illumina). The miRNA molecules were subsequently transcribed from total RNA samples isolated from a retrospective set of 119 FFPE tissues using the TaqMan Advanced miRNA cDNA Synthesis Kit, and the expression of selected miRNAs was validated in a pilot experiment by qPCR using TaqMan Fast Advanced Master Mix and appropriate TaqMan MicroRNA Assays (all from Thermo Fisher Scientific). RESULTS The differential analysis identified 373 miRNAs with significantly different expression among the 5 BMs groups (p< 0.001). A molecular classifier based on the expression of 32 miRNAs was able to classify all samples correctly. Out of these, seven, including miR-122-5p, miR-141-3p, miR-146a-5p, miR-194-5p, miR-200c-3p, miR-211-3p, and miR-215-5p, were chosen for subsequent validation and their significantly different expression in 5 BMs groups was validated. CONCLUSIONS Presented results confirm the importance of studying dysregulated miRNA expression in BM and the diagnostic potential of validated miRNAs. The study was prepared with the grant support of the Ministry of Health of the Czech Republic - grant No. NV18-03-00398.

INTRODUCTION Modern comprehensive local treatment of BM provides the possibility of adequate local control while maintaining a satisfactory quality of life. METHODS Patients after metastasectomy and postoperative radiotherapy treated between 2007-2020 were evaluated. Local control was assessed separately at the site of the operated MM (localPFS) and separately in other parts of the brain (distalPFS). RESULTS A total of 118 patients were enrolled (70 with WBRT), 54% were women, the median age was 60 years, the median performance status Karnofsky 80%. The most common primary tumor was lung carcinoma (39/118, 33%). Targeted stereotactic radiotherapy was significantly more often indicated in operations performed after 2016 (p < 0.001). In the period 2013-2019, gross total resection was achieved significantly more often (83 vs 64%; p = 0.061). The median follow-up is 49 months. The median overall survival (OS) is 9 months (6.2 - 12), the median localPFS 22 months (14 - unattainable), median distPFS 11 months (6.8 - 27 months), median extracranialPFS 11 months (5.9 - 15). Stratification of patients according to the prognostic index brainmetgpa.com led to significant separation of patients for OS (p = 0.00017). In a multivariate analysis, stereotactic radiotherapy was statistically significant positive prognostic factor for OS compared to WBRT (median OS 17 vs. 5.6 months, HR 0.59, p = 0.018) with no effect on localPFS. CONCLUSION Our results of comprehensive local treatment of BM are comparable with the results presented by important foreign studies from prestigious oncology centers. We described targeted stereotactic postoperative radiotherapy as an independent positive prognostic factor after brain metastasectomy. The study was supported by the programme project of the Ministry of Health of the Czech Republic with reg. no. NV18-03-00398, NV19-03-00501 and NV19-03-00559 and Supported by Ministry of Health, Czech Republic-conceptual development of research organization (FNBr, 65269705).

MicroRNAs (miRNAs) are a well-known subclass of short non-coding RNAs responsible for posttranscriptional gene silencing and have been described as dysregulated in many cancers. They have also been shown to be both specific diagnostic, prognostic, and predictive biomarkers as well as therapeutic targets. Therefore, specific miRNA expression patterns of BMs of various origins could serve as a promising diagnostic tool for determining both the original tumor and the prognosis in patients with BMs of unknown origin. For identifying significantly dysregulated miRNAs among BMs (n=90) with various origin and non-tumor brain tissues (n=12), small RNAseq analyses were used. cDNA libraries were prepared using QIAseq miRNA Library Kit and purified by Qiaseq beads. The final sequencing analyses were performed by Next 500/550 High Output v2 Kit-75 cycles using the NextSeq 500 instrument. For miRNA mapping and analysis, Miraligner and MirBase were used. Bioinformatic analysis of obtained sequencing data identified 472 significantly dysregulated miRNAs (logFc >2, adj.p-value< 0.05) between BM and non-tumor samples. The comparison of BMs origin from lung BMs (n = 26) with other BMs revealed 132 significantly dysregulated miRNAs, mainly miR-4662a-5p, miR-1179, miR-211-5p, miR-146a-5p, and miR-194-5p. The most significantly dysregulated miRNAs in breast BMs were miR-4728-3p, miR-211-5p, miR-184, miR-365b-5p, and miR-2115-3p. In BMs originating from melanoma, miR-200c-3p, miR-141-5p, miR-200b-5p, miR-514a-3p, and miR-200b-3p showed the most aberrant expression. We have demonstrated that miRNA profiling could be a potent tool for the partition of brain metastases based on their origin. We found that miRNA signatures corresponding to particular origins are rather distinct from the profiles of the rest of BMs. Our results suggest that after validation, miRNA profiling can be used to identify the origin of brain metastases and potentially for the refinement of the diagnosis. Supported by the Ministry of Health of the Czech Republic, grant nr. NV18-03-00398.

Background/Aim: Glioblastoma (GBM) is one of the deadliest human cancers responding very poorly to therapy. Although the central nervous system has been traditionally considered an immunologically privileged site with an enhanced immune response, GBM appears to benefit from this immunosuppressive milieu. Immunomodulatory molecules play an important role in immune tumor-host interactions. Non-classical human leukocyte antigens (HLA) class Ib molecules HLA-E, HLA-F, and HLA-G have been previously described to be involved in protecting semi-allogeneic fetal allografts from the maternal immune response and in transplant tolerance as well as tumoral immune escape. Unfortunately, their role in GBM remains poorly understood. Our study, therefore, aimed to characterize the relationship between the expression of these molecules in GBM on the transcriptional level and clinico-pathological and molecular features of GBM as well as the effect of ionizing radiation. Materials and Methods: We performed the analysis of HLA-E, HLA-F, and HLA-G mRNA expression in 69 GBM tissue samples and 21 non-tumor brain tissue samples (controls) by reverse transcription polymerase chain reaction. Furthermore, two primary GBM cell cultures had been irradiated to identify the effect of ionizing radiation on the expression of non-classical HLA molecules. Results: Analyses revealed that both HLA-E and HLA-F are significantly up-regulated in GBM samples. Subsequent survival analysis showed a significant association between low expression of HLA-E and shorter survival of GBM patients. The dysregulated expression of both molecules was also observed between patients with methylated and unmethylated O-6-methylguanine-DNA methyltransferase (MGMT) promoter. Finally, we showed that ionizing radiation increased HLA-E expression level in GBM cells in vitro. Conclusion: HLA-E and HLA-F play an important role in GBM biology and could be used as diagnostic biomarkers, and in the case of HLA-E also as a prognostic biomarker.

Introduction: Glioblastoma (GBM) is the most common malignancy affecting the CNS. Despite radical therapy, recurrence of GBM is a frequent and relatively early event in the course of the disease. One of the putative mechanisms of early recurrence of GBM is the existence of glioblastoma stem cells (GSCs) that can resist therapy and subsequently establish new GBM foci. PIWI-interacting RNAs (piRNAs), responsible for maintaining genome stability, may play a key role not only in the biology of germ and stem cells but also GSCs since their dysregulated expression has been described in several cancers, including GBM. Thus, the identification of specific piRNAs in GSCs could allow the distinction of these cells from other GBM cells and their eventual targeting. Some piRNAs could also be promising biomarkers useful for prognosis and prediction of disease progression. Material and Methods: Native GBM tissues dissociated using the Papain Dissociation System were used for culturing of paired cell primary cultures. Cells were divided into two aliquots with different culture conditions. GSCs were cultured in DMEM/F12 medium supplemented with bFGF and EGF growth factors. DMEM medium containing 10% fetal bovine serum (FBS) was used to culture non-stem GBM cells. Subsequently, the presence of neural stem cell markers CD133 and SOX2, the ability of GSCs to establish tumors in immunodeficient mice, and the ability to differentiate into more mature cell types were analyzed. RNA isolated from the paired primary cultures was used for small-RNA sequencing (RNA-seq). QIAseq miRNA Library Kit was used for the preparation of cDNA libraries. Sequencing analysis was performed using the NextSeq 500/550 High Output v2 Kit (75 cycles) and the NextSeq 500 sequencer. Results: Twelve paired primary cultures were prepared to global piRNA expression profiling. Small RNA-seq bioinformatics analysis identified between 3.45 and 5.51% of unique mapped piRNA reads in all samples; GSCs expressing higher levels of piRNA molecules in comparison with paired GBM cells. Subsequently, we identified a set of significantly differentially expressed piRNAs in GSCs. Expression of selected piRNAs was then artificially regulated in primary GSCs in vitro, and the effect of these molecules on neurosphere formation, viability, and cell genome stability was analyzed. Finally, we also observed an association of piR-23231 with overall survival of GBM patients. All results will be presented at the AACR Annual Meeting 2022. Conclusion: Based on the results so far, piRNAs seem to play an important role in GSCs as well as GBM biology and their targeting may be a promising tool for therapy of GBM patients. The study was supported by the programme project of the Ministry of Health of the Czech Republic with reg. no. NV19-03-00501 and NV19-03-00559. Citation Format: Jiri Sana, Frantisek Siegl, Marek Vecera, Pavel Fadrus, Jaroslav Juracek, Petra Pokorna, Karolina Trachtova, Tomas Kazda, Ondrej Slaby. Small RNA-seq analysis of PIWI-interacting RNAs in glioblastoma stem cells: Identification of new therapeutic targets in glioblastoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 908.

Central nervous system (CNS) malignancies include primary tumors that originate within the CNS as well as secondary tumors that develop as a result of metastatic spread. Circulating microRNAs (miRNAs) were found in almost all human body fluids including cerebrospinal fluid (CSF), and they seem to be highly stable and resistant to even extreme conditions. The overall aim of our study was to identify specific CSF miRNA patterns that could differentiate among brain tumors. These new biomarkers could potentially aid borderline or uncertain imaging results onto diagnosis of CNS malignancies, avoiding most invasive procedures such as stereotactic biopsy or biopsy. In total, 175 brain tumor patients (glioblastomas, low-grade gliomas, meningiomas and brain metastases), and 40 non-tumor patients with hydrocephalus as controls were included in this prospective monocentric study. Firstly, we performed high-throughput miRNA profiling (Illumina small RNA sequencing) on a discovery cohort of 70 patients and 19 controls and identified specific miRNA signatures of all brain tumor types tested. Secondly, validation of 9 candidate miRNAs was carried out on an independent cohort of 105 brain tumor patients and 21 controls using qRT-PCR. Based on the successful results of validation and various combination patterns of only 5 miRNA levels (miR-30e, miR-140, let-7b, mR-10a and miR-21-3p) we proposed CSF-diagnostic scores for each tumor type which enabled to distinguish them from healthy donors and other tumor types tested. In addition to this primary diagnostic tool, we described the prognostic potential of the combination of miR-10b and miR-196b levels in CSF of glioblastoma patients. In conclusion, we performed the largest study so far focused on CSF miRNA profiling in patients with brain tumors, and we believe that this new class of biomarkers have a strong potential as a diagnostic and prognostic tool in these patients.

MicroRNAs (miRNAs) are a well-known subclass of short non-coding RNAs responsible for posttranscriptional gene silencing and have been described as dysregulated in many cancers. They have also been shown to be both specific diagnostic, prognostic, and predictive biomarkers as well as therapeutic targets. Therefore, specific miRNA expression patterns of BMs of various origins could serve as a promising diagnostic tool for determining both the original tumor and the prognosis in patients with BMs of unknown origin. For identifying significantly dysregulated miRNAs among BMs (n = 90) with various origin and non-tumor brain tissues (n = 12), small RNAseq analyses were used. cDNA libraries were prepared using QIAseq miRNA Library Kit and purified by Qiaseq beads. The final sequencing analyses were performed by Next 500/550 High Output v2 Kit-75 cycles using the NextSeq 500 instrument. For miRNA mapping and analysis, Miraligner and MirBase were used. Bioinformatic analysis of obtained sequencing data identified 472 significantly dysregulated miRNAs (logFc>2, adj.p-value We have demonstrated that miRNA profiling could be a potent tool for the partition of brain metastases based on their origin. We found that miRNA signatures corresponding to particular origins are rather distinct from the profiles of the rest of BMs. Our results suggest that after validation, miRNA profiling can be used to identify the origin of brain metastases and potentially for the refinement of the diagnosis. Supported by the Ministry of Health of the Czech Republic, grant nr. NV18-03-00398.

Glioblastoma stem-like cells (GSCs) are critical for the aggressiveness and progression of glioblastoma (GBM) and contribute to its resistance to adjuvant treatment. MicroRNAs (miRNAs) are small, non-coding RNAs controlling gene expression at the post-transcriptional level, which are known to be important regulators of the stem-like features. Moreover, miRNAs have been previously proved to be promising diagnostic biomarkers in several cancers including GBM. Using global expression analysis of miRNAs in 10 paired in-vitro as well as in-vivo characterized primary GSC and non-stem glioblastoma cultures, we identified a miRNA signature associated with the stem-like phenotype in GBM. 51 most deregulated miRNAs classified the cell cultures into GSC and non-stem cell clusters and identified a subgroup of GSC cultures with more pronounced stem-cell characteristics. The importance of the identified miRNA signature was further supported by demonstrating that a Risk Score based on the expression of seven miRNAs overexpressed in GSC predicted overall survival in GBM patients in the TCGA dataset independently of the IDH1 status. In summary, we identified miRNAs differentially expressed in GSCs and described their association with GBM patient survival. We propose that these miRNAs participate on GSC features and could represent helpful prognostic markers and potential therapeutic targets in GBM.

Cerebrospinal fluid (CSF) baths extracellular environment of the central nervous system, and thus, it is ideal source of tumor diagnostic biomarkers like microRNAs (miRNAs), short non-coding RNAs involved in the pathogenesis of many cancers. As dysregulated levels of brain tumor specific miRNAs have been already observed in CSF, analysis of CSF miRNAs in brain tumor patients might help to develop new diagnostic platform. Next-Generation sequencing (NGS) was performed for analysis of small RNAs in 89 CSF samples taken from 32 glioblastomas (GBM), 14 low-grade gliomas (LGG), 11 meningiomas, 13 brain metastases and 19 non-tumor donors. Subsequently, according to NGS results levels of 10 miRNAs were measured in independent set of CSF samples (41 GBM, 44 meningiomas, 12 brain metastases and 20 non-tumor donors) using TaqMan Advanced miRNA Assays. NGS analysis revealed 22, 12 and 35 CSF miRNAs with significantly different levels in GBM, meningiomas, and brain metastases (adj.p < 0.0005, adj.p < 0.01, and adj.p < 0.005) respectively, in comparison with non-tumor CSF samples. Subsequent validation of selected CSF miRNAs has confirmed different levels of 7 miRNAs in GBM, 2 in meningiomas, and 2 in brain metastases compared to non-tumors. Panel of miR-30e-5p and miR-140-5p was able to distinguish brain metastases with 65% sensitivity and 100% specificity compared to non-tumor samples (AUC = 0.8167); panel of miR-21-3p and miR-196-5p classified metastatic patients with 78% sensitivity and 92 % specificity in comparison to GBM (AUC = 0.90854) and with 75% sensitivity and 83% specificity compared to meningiomas (AUC = 0.84848). We have observed that CSFs from patients with various primary brain tumors and metastases are characterized by specific miRNA signatures. This work was supported by the Ministry of Health, Czech Republic grant nr. NV18-03-00398 and the Ministry of Education, Youth and Sports, Czech Republic under the project CEITEC 2020 (LQ1601).

Introduction. Incidence rate of primary brain tumors and brain metastases counts yearly around 40 patients per 100 000 persons in the world and is still growing. Prognosis and therapy differs between brain tumor types and, therefore, accurate and early diagnosis might improve survival and life quality of patients. However, current diagnostic approaches are limited by localization and tissue heterogeneity of brain tumors. Cerebrospinal fluid (CSF) bathes central nervous system (CNS) and thus is supposed to reflect all pathological conditions. From this perspective, CSF looks as ideal source of diagnostic biomarkers of brain tumors. MicroRNAs, short non-coding RNAs involved in the pathogenesis of many cancers including brain tumors, might represent group of new biomarkers. In addition, dysregulated levels of brain tumor specific miRNAs have been already observed also in CSF. Following these facts, analysis of CSF miRNAs in brain tumor patients promises a new diagnostic approach enabling more accurate diagnosis. Material and methods. Next-generation sequencing was performed for analysis of small RNAs in 89 CSF samples taken from 32 GBM, 14 low-grade glioma (LGG), 11 meningioma, 13 brain metastasis patients and 19 non-tumor donors. CleanTag Small RNA Library Prep Kit (TriLink BioTechnologies) was used for cDNA library preparation. NextSeq 500 instrument together with Next 500/550 High Output v2 Kit - 75 cycles (both Illumina) were used for final sequencing analysis. Subsequently, according to NGS results we measured levels of 13 miRNAs in independent set of CSF samples (35 GBM, 42 meningiomas, 8 metastasis patients and 19 control) using TaqMan Advanced miRNA Assays (ThermoFisher Scientific). Results. NGS analysis revealed 25, 3, 2 and 14 CSF miRNAs significantly differently expressed in GBM, meningiomas, LGG and metastasis patients (p < 0.001, for LGG p < 0.05), respectively, in comparison with control CSF samples. In addition, 6 miRNAs showed different levels between GBM and LGG (p < 0.05). Subsequent validation of selected CSF miRNAs has confirmed different levels of 7 miRNAs in GBM (miR-10a, miR-196a, miR-196b, miR-30e, miR-30c, miR-7b, miR-7c), 3 in meningioma (miR-140, miR-21, miR-30e) and 2 in brain metastasis (miR-30e and miR-7c) compared to control CSF samples (p < 0.05). Validation of miRNA levels in LGG CSF samples is in progress and will be part of the poster presentation. Conclusion. We have observed that CSF from patients with various brain tumors (GBM, LGG, meningioma, and brain metastasis) is characterized by specific miRNA signature. Our results suggest potential of CSF miRNAs to be useful biomarkers in brain tumors. This work was supported by Ministry of Health of the Czech Republic grant nr. NV18-03-00398, grant of Czech Grant Agency nr. 17-17636S and by the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601). Citation Format: Alena Kopkova, Jiri Sana, Marek Vecera, Tana Machackova, Pavel Fadrus, Julia Kovacova, Ondrej Slaby. Global analysis and validation of miRNA levels in cerebrospinal fluid of brain tumor patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1806.

Introduction: Glioblastoma multiforme (GBM) is the most frequent primary brain malignancy of astrocytic origin. The prognosis remains very poor with the median overall survival (OS) being between 12 and 16 months from diagnosis despite early use of conventional medical therapy. Identifying new therapeutic targets, as well as prognostic and predictive biomarkers for accurate stratification of patients is therefore of utmost importance. Long non-coding RNAs (lncRNAs) are regulators of gene expression having critical impact on both physiological processes and the molecular pathology of GBM, indicating their potential as biomarkers and therapeutic targets. Material and Methods: Our study included 219 GBM patients and 29 patients with non-dominant anterior temporal cortexes resected during surgery for intractable epilepsy. Informed consent approved by the local Ethical Commission was obtained from each patient. RNA (RIN > 8) from 77 specimens was used for next-generation RNA sequencing (RNAseq). rRNA depletion and cDNA library preparation were done with RiboCop rRNA Depletion Kit V1.2 (Lexogen) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB), respectively. RNAseq was performed using NextSeq 500 High Output Kit and NextSeq 500 instrument (both Illumina). 8,414 lncRNAs and their sequential variants with non-zero RPKM at least in one sample were statistically evaluated. The alignment and target counts were performed with CLC genomic workbench. Selected significantly dysregulated lncRNAs between GBM and non-tumor controls were analyzed in a larger cohort of 188 specimens by qRT-PCR and the expression data normalized to PPIA was then evaluated by Mann-Whitney U test. Results: Statistical analysis revealed 538 (P < 0.001) dysregulated lncRNAs in GBMs compared to non-tumor brain tissue samples. The expression of top 10 downregulated lncRNAs (SNAI3-AS1, LINC00882, RFPL1S, MIR137HG, TTLL7-IT1, PWAR6, LINC00634, LINC00632, DGCR5, LINC00982; logFC ≤ -2; P < 0.001) and 1 upregulated lncRNA (BTN2A3P; logFC ≥ 2; P < 0.001) in GBM and non-tumor controls was successfully validated by qRT-PCR (P < 0.0001). Moreover, the statistical analysis revealed 22 lncRNAs significantly dysregulated between patients with OS less than 12 months and those with OS equal or more than 12 months (P < 0.01). Conclusion: We observed significant dysregulation of lncRNAs in GBM tissues compared to non-tumor controls based on the results of both RNASeq and qRT-PCR. We also found 22 lncRNAs to be dysregulated in relation to overall survival. Our study indicates that lncRNAs could serve as promising diagnostic and prognostic biomarkers in GBM. This work was supported by Ministry of Health of the Czech Republic grant nr. NV18-03-00398, grant of Czech Grant Agency nr. 17-17636S, and by the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601). Citation Format: Marek Vecera, Jan Oppelt, Lenka Radova, Radim Lipina, Stefan Reguli, Martin Smrcka, Radim Jancalek, Michal Filip, Marketa Hermanova, Leos Kren, Jiri Sana, Alena Kopkova, Julia Kovacova, Ondrej Slaby. Dysregulated expression of lncRNAs in glioblastoma multiforme and their association with overall survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3575.

Glioblastoma multiforme ( GBM) is the most aggressive intracranial tumor characterized with infaust prognosis. Despite advances in neurosurgical and radiotherapeutic techniques and chemotherapy, the median overall survival ranges between 12-15 months from diagnosis. The main cause of treatment failure is considered the presence of tumor cells resistant to conventional therapy, mainly radiotherapy. MicroRNAs (miRNAs) are small, non-coding RNAs that function as post-transcriptional regulators of gene expression and have been repeatedly proven to play important roles in pathogenesis and biological features of many cancers, including GBM and its radioresistant phenotype. In our study, we established radioresistant cells from the commonly used human GBM cell lines T98G, U87MG and U251. Consequently, we performed global miRNA expression profiling in both radioresistant and parental cell lines and identified 113 miRNAs with significantly different expression (p < 0.05) between these two groups (73 miRNAs were up-regulated, 40 miRNAs were down-regulated). Some of these miRNAs have been previously described in relation to ionizing radiation, and others were herein identified for the first time. We believe that after deeper functional investigation of identified miRNAs in relation to radioresistance, these miRNAs present potential predictive biomarkers or therapeutic targets in GBM.

Gliomas are the most common malignancies of the central nervous system. Because of tumor localization and the biological behavior of tumor cells, gliomas are characterized by very poor prognosis. Despite significant efforts that have gone into glioma research in recent years, the therapeutic efficacy of available treatment options is still limited, and only a few clinically usable diagnostic biomarkers are available. More and more studies suggest non-coding RNAs to be promising diagnostic biomarkers and therapeutic targets in many cancers, including gliomas. One of the largest groups of these molecules is long non-coding RNAs (lncRNAs). LncRNAs show promising potential because of their unique tissue expression patterns and regulatory functions in cancer cells. Understanding the role of lncRNAs in gliomas may lead to discovery of the novel molecular mechanisms behind glioma biological features. It may also enable development of new solutions to overcome the greatest obstacles in therapy of glioma patients. In this review, we summarize the current knowledge about lncRNAs and their involvement in the molecular pathology of gliomas. A conclusion follows that these RNAs show great potential to serve as powerful diagnostic, prognostic, and predictive biomarkers as well as therapeutic targets.

Introduction: Head and neck cancers are the sixth most common cancers worldwide mainly represented by the squamous cell carcinoma (HNSCC). Very important treatment modality in HNSCC is radiotherapy (RT). Closed as well as ongoing clinical trials are evaluating especially potential for reduced-dose RT in less aggressive radiosensitive HNSCC defined by human papillomavirus (HPV) positivity, with promise of less acute and late toxicity. To this end, variety of different biomarkers with promising predictive value is currently investigated in HNSCC. MicroRNAs (miRNAs) are short endogenous RNAs that post-transcriptionally modulate gene expression and their deregulated expression has been observed in many cancers including HNSCC. Specific expression patterns of miRNAs have been also shown to predict prognosis and therapeutic response in HNSCC. Aim of our study was to identify tumor tissue miRNAs enabling of predict locoregional control (LRC) in HNSCC patients who underwent intensity-modulated RT. Materials and methods: We have analyzed global miRNA expression profiles in 43 FFPE tumor biopsies collected from HNSCC patients treated with intensity-modulated radiation therapy, who were divided into two groups according to their LRC as follows: short LRC [n = 22; median 5.1 months (min 1.3, max 18.6)] vs. long LRC [n = 21; 60.4 (46.8, 98.8)]. This analysis has been performed using the hybridization Affymetrix GeneChip miRNA 4.0 array. Validation of miRNA candidates was performed in independent cohort of 64 HNSCC patients. MiRNA determination was carried out by RT qPCR technology using the miRNA-specific RT stem-loop primers according to the Taq-Man MicroRNA Assay protocol (Thermo Fisher Scientific). Results: We identified 24 miRNAs with significantly different expression between both examined groups (p < 0.05; average log(Fold Change) = 0.42). Based on pre-defined critera, 12 miRNAs were selected for independent validation, from which miR-421 and miR-1228 were differentially expressed between groups of patients with short and long LRC. Moreover, risk-score based on combination of these 2 miRNAs expression and CD44 status enabled to predict 3-years LRC with 82% sensitivity and 82% specificity (AUC = 0.798). Conclusion: Our results suggest that miR-421 and miR-1228 are promising predictive biomarkers in HNSCC patients treated with intensity-modulated RT. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-34553A, 15-33158A, 15-31627A, 15-34678A, 16-31314A and 16-31765A. Citation Format: Parwez Ahmad, Jiri Sana, Marek Vecera, Jaroslav Juracek, Tana Machackova, Natalia Anna Gablo, Marek Svoboda, Marketa Hermanova, Marek Slavik, Pavel Slampa, Pavel Smilek, Ondrej Slaby. Predictive value of tumor microRNAs in head and neck cancer patients treated with intensity-modulated radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5406.

Introduction: Glioblastoma (GBM) is the most frequent primary brain tumor of astrocytic origin. The prognosis is unfavourable with the median overall survival (OS) being between 12 and 15 months from diagnosis. Identification of new therapeutic targets, as well as new prognostic and predictive biomarkers for more accurate stratification of patients presents significant unmet medical needs. Long non-coding RNAs (lncRNAs) are regulators of gene expression playing important roles in the molecular pathology of GBM, indicating their potential as biomarkers and therapeutics targets. Material and Methods: Our study included 80 GBM patients treated with Stupp protocol and 16 patients with non-dominant anterior temporal cortexes resected during surgery for intractable epilepsy. Informed consent approved by the local Ethical Commission was obtained from each patient. IDH1 mutations and MGMT methylation status were evaluated in all GBMs. RNA (RIN > 8) from 96 specimens was used for next-generation RNA sequencing (RNAseq). rRNA depletion and cDNA library preparation were performed with RiboCop rRNA Depletion Kit V1.2 (Lexogen) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB), respectively. RNAseq was done using NextSeq 500 High Output Kit and NextSeq 500 instrument (both Illumina). 24,087 protein-coding and 8,414 non-coding RNAs and their sequential variants with non-zero RPKM at least in one sample underwent statistical evaluation. CLC genomic workbench was used for the alignment and target counts. Results: Statistical analysis revealed 84 (P < 0.001) dysregulated lncRNAs in GBMs compared to non-tumor brain tissue samples. The results also showed 485 dysregulated protein-coding RNAs with P < 0.001 and 24 protein-coding RNAs with P < 0.000001. 35 lncRNAs showed significant dysregulation when lncRNA profiles of GBM tissues with methylated MGMT promoter (≥ 25% methylation) were compared to those with unmethylated promoter (P < 0.01). When lncRNA patterns of GBM samples with mutated IDH1 were compared to those with wild-type IDH1, 60 lncRNAs were found to be significantly dysregulated (P < 0.001). Correlating lncRNA expression patterns with OS uncovered 6 lncRNA signature which enabled identifying patients with significantly worse prognosis (OS < 6 months). Conclusion: We described significant dysregulation of lncRNAs and protein-coding RNAs in GBM tissue compared to non-tumor brain tissue and specific lncRNA patterns linked to MGMT methylation and IDH1 mutation status. We also identified 6 lncRNA signature allowing sensitive prognostic stratification of GBM patients. Our study indicates that lncRNAs could serve as promising diagnostic and prognostic biomarkers in GBM. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-33158A, 15-34553A, 15-31627A, 15-34678A, 16-31314A, 16-31765A and by grant of Czech Grant Agency nr. 16-18257S. Citation Format: Marek Vecera, Romana Butova, Radim Lipina, Stefan Reguli, Martin Smrcka, Radim Jancalek, Michal Filip, Marketa Hermanova, Leos Kren, Pavol Mojak, Jaroslav Juracek, Tana Machackova, Natalia A. Gablo, Jiri Sana, Ondrej Slaby. Clinicopathological subgroups of glioblastoma patients are characterized by specific lncRNA expression patterns [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2459.

Introduction: The majority of primary or metastatic liver tumors are unresectable (because of tumor size, location, poor performance status or multifocality), therefore other therapeutic modalities as radiofrequency ablation (RFA) and transarterial chemoembolization (TACE) are applied. RFA is a localized thermal treatment technique designed to produce tumor destruction by heating tumor tissue, while TACE combines cytotoxic effect of particle based tumor ischemia and locoregional chemotherapy. Both methods cause characteristic changes in liver tissue (inflammation, hypoxia, elevated temperature, tissue destruction) accompanied by targeted systemic secretion of microRNA into the bloodstream. Since RFA and TACE differ in the dynamics with which they affects the tumor tissue, we aimed to investigate whether the expression level of circulating microRNAs related to hypoxia (miR-21 and miR-210), liver injury (miR-122) and epithelial-mesenchymal transition (miR-200a) could reflect such changes. Material and methods: This study consisted of 14 patients diagnosed with primary hepatocellular carcinoma (HCC) (median age 73; TACE) and 20 patients diagnosed with liver metastases of colorectal cancer (median age 63; 17 patients - RFA, 3 patients - TACE). RFA was performed using the rf/mw generator (AngioDynamics). For TACE drug eluting beads (Biocompatibles Ltd.) loaded with irinotecan for mCRC patients and doxorubicin for HCC patients were used. The concentrations of miRNA were determined for all patients in series of blood plasma from 4 time points (before intervention, immediately after intervention, 24 hours after intervention, 1 week after intervention) using miRNA-specific TaqMan assays and qRT-PCR method. Results: In RFA cases we observed significant increase of investigated miRNA concentrations immediately after intervention (miR-122, FC = 15, P = 0.0002; miR-200a, FC = 1.9, P = 0.015). In TACE we observed delayed increase in circulating miRNA concentrations at time point 24 hours after intervention (miR-21, FC = 10.4, P < 0.0001; miR-210, FC = 9.0, P = 0.03; miR-122, FC = 27, P = 0.0004; miR-200a, FC = 4.0, P = 0.0098). In both methods, the initial increase was followed by a steady decline of miRNA levels. Identified dynamic changes in circulating miRNA levels were in accordance with the nature of RFA and TACE biologic effects. In selected cases, we observed specific dynamic miRNA patterns to be linked to the course of the disease (e.g. necessity of additional intervention). Conclusions: Our preliminary data indicates potential usage of circulating miRNAs for monitoring of the systemic effects of RFA and TACE therapy and their ability to reflect efficacy of intervention procedures. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-33158A, 15-34553A, 15-31627A, 16-31314A, and 15-34678A. Citation Format: Jaroslav Juracek, Tomas Andrasina, Barbora Cechova, Petra Vesela, Jan Zavadil, Tana Machackova, Jiri Sana, Marek Vecera, Natalia Gablo, Marek Svoboda, Nahum Goldberg, Ondrej Slaby. Dynamic measurements of circulating microRNAs reflect different biological effects of radiofrequency ablation and transarterial chemoembolisation in liver cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 521.

Introduction: Cerebrospinal fluid (CSF) is a body fluid with many important functions that is in direct contact with the extracellular environment of the central nervous system (CSN). Therefore, CSF serves inter alia as a both communication channel allowing the distribution of various substances among CNS cells as well as a reservoir of waste products that these cells released. For these reasons, CSF is a potential source of diagnostic biomarkers of many neurological diseases including brain tumors. Recent studies have revealed that CSF contains also circulating microRNAs (miRNAs), short non-coding RNAs, which have been described as biofluid diagnostic markers in many cancers. The analysis of CSF miRNAs in patients affected by brain tumors could enable more precise diagnosis and patient stratification.Material and methods: We performed next-generation sequencing analysis of small RNAs (small RNAseq) in 89 CSF samples taken from 35 GBM, 14 low-grade glioma (LGG), 14 meningioma, and 8 brain metastasis patients as well as 18 non-tumor donors. Informed consent approved by the local Ethical Commission was obtained from each patient before the lumbar puncture. cDNA libraries were prepared using CleanTaq Small RNA Library Prep Kit (TriLink BioTechnologies) and, subsequently, purified by Agencourt AMPure XP (Beckman Coulter). The final sequencing analysis was performed by Next 500/550 High Output v2 Kit - 75 cycles using the NextSeq 500 instrument (both Illumina). For miRNA mapping and analysis, an online tool Chimira was used. Obtained data were subsequently statistically evaluated in the environment of statistical language R using the Bioconductor edgeR and DESeq2 package.Results: We observed 211 miRNAs to be expressed in more than 60 CSF samples with 1 395 normalized counts in average (min 3, max 77 284; miR-486-5p was excluded from analysis due to its artificial expression). From these miRNAs, 63 showed different levels in CSF of GBM, 16 in LGG, 24 in meningioma, and 31 in brain metastases patients in comparison with CSF of non-tumor donors. MiR-10a-5p and miR-196a-5p have significantly highest levels in CSF of GBM samples. Moreover, 6 miRNAs (miR-30c-5p, miR-30e-5p, miR-145-5p, miR-320c, miR-3960, and miR-6131) indicated significantly different levels between GBM and LGG (P Citation Format: Jiri Sana, Alena Kopkova, Marek Vecera, Jaroslav Juracek, Tana Machackova, Parwez Ahmad, Natalia Anna Gablo, Pavel Fadrus, Marek Svoboda, Ondrej Slaby. Analysis of microRNAs in cerebrospinal fluid of brain tumor patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3615.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the most common and aggressive type of pancreatic cancer, associated with limited treatment options and high mortality rates. Radical resection remains the most effective curative approach for patients in early stage of disease. However, there is a high risk of surgical morbidity/mortality, and therefore development of prognostic assays to identify those patients who will not benefit from surgical resection, present important unmet medical need. miRNAs are small, non-coding RNAs, that post-transcriptionally regulate gene expression. miRNAs are frequently deregulated under pathological conditions including PDAC and therefore present promising candidates for new prognostic biomarkers in PDAC. Materials and methods: We analyzed preoperative plasma samples obtained from 45 PDAC patients who underwent radical tumor resection. Patients were divided into two prognostic groups: 23 patients with poor prognosis (median overall survival (OS) = 10 months) and 22 patients with good prognosis (median OS = 25 months). cDNA libraries were prepared using CleanTaq Small RNA Library Prep Kit (TriLink). The sequencing analysis were performed by Next 500/550 High Output v2 Kit - 75 cycles using the NextSeq 500 instrument (both Illumina). For miRNA mapping and analysis, an online tool Chimira was used. Obtained data were statistically evaluated using the Bioconductor edgeR and DESeq2 package. Results: When miRNA expression profiles of the patients from good and poor prognostic group were compared, 41 miRNAs were identified to have significantly different plasma levels between the two groups (P Conclusion: Our findings suggest, that there is a blood plasma miRNAs signature associated with the prognosis of PDAC patients, and after independent validation, miRNAs from this signature may allow for a prognostic stratification of PDAC patients. This work was supported by Ministry of Health of the Czech Republic, grant nr.15-33158A, 15-34553A, 15-31627A, 15-34678A, 16-31314A, 16-31765A and by grant of Czech Grant Agency nr. 16-18257S. Citation Format: Natalia Gablo, Vladimir Prochazka, Tana Machackova, Eva Vencovska, Zdenek Kala, Jiri Sana, Parwez Ahmad, Jaroslav Juracek, Marek Vecera, Marek Svoboda, Ondrej Slaby. Small RNA sequencing of preoperative blood plasma identifies microRNA signature enabling to find pancreatic cancer patients who will not benefit from surgical resection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4976.

Introduction: Rectal cancer accounts for approximately one third of all colorectal cancers (CRC), which belong among leading causes of cancer deaths worldwide. Standard treatment for locally advanced rectal cancer (cT3/4 and/or cN+) includes neoadjuvant chemoradiotherapy (CHRT) with fluoropyrimidines followed by radical surgical resection. MicroRNAs (miRNAs) are small non-coding RNAs playing significant roles in the pathogenesis of many cancers including rectal cancer. MiRNAs could present the new predictive biomarkers of response to CHRT in rectal cancer patients. Materials and Methods: In prospective one-center study, 48 patients diagnosed with rectal cancer who underwent neoadjuvant chemoradiotherapy followed by surgical treatment were included. Blood plasma samples were collected before the neoadjuvant chemoradiotherapy (in 24 cases) and after the therapy at the time of clinical restaging (in 48 cases). Total RNA was isolated from 72 blood plasma samples of 48 patients. cDNA libraries were prepared using CleanTaq Small RNA Library Prep Kit (TriLink BioTechnologies). The final sequencing analyses were performed by Next 500/550 High Output v2 Kit - 75 cycles using the NextSeq 500 instrument (both Illumina). For miRNA mapping and analysis, an online tool Chimira was used. Obtained data were statistically evaluated using the Bioconductor edgeR and DESeq2 package. Results: When miRNA profiles of samples collected before and after therapy were compared, 10 miRNAs showed higher levels and of 2 miRNAs lower levels in pre-treatment specimens (P Citation Format: Tana Machackova, Dominika Brchnelova, Zdenek Kala, Vladimir Prochazka, Tomas Grolich, Lukas Fiala, Beata Hemmelova, Jaroslav Juracek, Marek Vecera, Jiri Sana, Natalia Gablo, Parwez Ahmad, Marek Svoboda, Ondrej Slaby. Deep sequencing of circulating microRNAs in rectal cancer patients undergoing neoadjuvant chemoradiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5409.

Introduction: Glioblastoma multiforme (GBM) is the most frequent primary brain tumor of astrocytic origin characterized by very poor prognosis. Despite conventional therapeutic protocol the prognosis of GBM patients is very poor with median of overall survival ranging between 12 and 15 months from diagnosis. Therefore, many financial charges and lot of effort is spent in research of new therapeutic approaches that could prolong the survival of GBM patients. Long non-coding RNAs (lncRNAs) are a relatively new class of noncoding gene regulators playing critical roles in tumor biology, including GBM. From this perspective, lncRNAs seem to be promising therapeutic targets in GBM patients. Material and Methods: We performed next-generation sequencing analysis of fresh-frozen histopatologically confirmed 45 GBM tissues and 5 non-tumor brain tissues obtained from non-dominant anterior temporal cortexes resected during surgery for intractable epilepsy. Informed consent approved by the local Ethical Commission was obtained from each patient before the treatment. rRNA depletion and cDNA library preparation were performed with GeneRead rRNA Depletion Kit (Qiagen) and NEXTflex Rapid Directional qRNA-Seq Kit (Bioo Scientific), respectively. Sequencing was held using NextSeq 500 High Output Kit and NextSeq 500 instrument (both Illumina). Statistical analysis evaluated 24 087 protein-coding and 8 414 non-coding RNAs and their sequential variants with non-zerou RPKM (Reads Per Kilobase of transcript per Million mapped reads) at least in one sample. We used CLC genomic workbench for the alignment and target counts. Targeted regulation of ZFAS1 level have been carried out by the transient transfection of specific siRNA in GBM stable cell lines (A172, T98G, U87MG, U251). Viability and migration were analyzed in vitro using MTT and scratch wound healing assay, respectively. Results: Statistical analysis has revealed 274 (P < 0.01) deregulated lncRNAs in GBMs in comparison with non-tumor brain samples. Moreover, the results have showed also 489 deregulated proten-coding RNAs with P value less than 0.001 and 26 protein-coding RNAs with P value less than 0.000001. For subsequent in vitro functional analyses was chosen one of the most upregulated lncRNAs in GBM samples ZFAS1. Targeted downregulation of this molecule led to the significant reduction of viability in all examined GBM cell lines. Decreasing of proliferation potential was observed only in A172 a U251 cell lines. Conclusion: We have demonstrated a deregulation of many lncRNAs and protein-coding RNAs in GBM tissue in comparison with non-tumor brain tissue. Moreover, ZFAS1, one of the most upregulated lncRNAs in GBM tissue, is involved in regulation of viability and migration of GBM cell lines in vitro. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-33158A, 15-34553A, 15-31627A, 15-34678A, 16-31314A, 16-31765A and by grant of Czech Grant Agency nr. 16-18257S. Note: This abstract was not presented at the meeting. Citation Format: Jiri Sana, Marek Vecera, Romana Butova, Jaroslav Juracek, Tana Machackova, Parwez Ahmad, Natalia Anna Gablo, Kamila Souckova, Leos Kren, Radim Lipina, Martin Smrcka, Ondrej Slaby. ZFAS1 is upregulated in GBM tissue and affects viability and migration of GBM cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3445. doi:10.1158/1538-7445.AM2017-3445

Growing evidence suggests that microRNAs (miRNAs) are involved in the development and progression of colorectal cancer (CRC). In the present study, deregulation and functioning of tumor suppressive miR-215-5p was evaluated in CRC. In total, 448 tumor tissues and 325 paired adjacent healthy tissues have been used for miRNA expression analyses. We proved that miR-215-5p is significantly down-regulated in tumor tissues compared to non-tumor adjacent tissues and its decreased levels correlate with the presence of lymph node metastases, tumor stage, and shorter overall survival in CRC patients. To identify specific cellular processes affected by ectopic expression of miR-215-5p, a series of in vitro experiments have been performed using transient transfection of miR-215-5p mimics into four CRC cell lines. Increased levels of miR-215-5p significantly reduced proliferation, clonogenicity, and migration of CRC cells, lead to cell cycle arrest in G2/M phase and p53-dependent induction of apoptosis. The ability of miR-215-5p to inhibit tumor growth was confirmed in vivo by use of NSG mice model and stable cell line over-expressing miR-215-5p. Finally, we proved epiregulin and HOXB9 to be the direct targets of miR-215-5p by luciferase assay and western blot analyses. Since epiregulin is EGFR ligand and HOXB9, is its transcriptional inducer, we suggest, that the main molecular link between miR-215-5p and CRC cells phenotypes presents the EGFR signaling pathway, which is one of the canonical pathogenic pathways in CRC. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-33158A, 15-34553A, 15-31627A, 15-34678A, 16-31314A, 16-31765A and by grant of Czech Grant Agency nr. 16-18257S. Citation Format: Petra Faltejskova-Vychytilova, Jana Merhautova, Pablo Conesa-Zamora, Katerina Slaba, Tana Machackova, Marek Svoboda, Marek Vecera, Jitka Mlcochova, Jaroslav Juracek, Jiri Sana, Parwez Ahmad, Natalia Gablo, Ondrej Slaby. MiR-215-5p is a tumor suppressor in colorectal cancer targeting EGFR ligand epiregulin and its transcriptional inducer HOXB9 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3425. doi:10.1158/1538-7445.AM2017-3425

Bladder cancer is the most common cancer of the urinary tract. More than 90% of bladder cancers are urothelial carcinoma, which are divided into non-muscle-invasive and muscle-invasive forms. Non-muscle-invasive tumors frequently recur (50-70%) and can also progress to invasion form (10-15%). These patients are monitored by cystoscopy and may have multiple resections over many years. Improved monitoring method is needed, ideally via urine analysis, which could reduce the morbidity and costs associated with long follow up. Currently there are no molecular biomarkers which could diagnose or accurately predict disease progression. We aimed to develop a clinically applicable, specific and sensitive panel of urine microRNAs enabling detect bladder cancer and predict risk of progression to muscle-invasive form.Within the exploratory phase of study we have analyzed expression profiles of 1733 miRNAs in urine supernatant of 16 bladder cancer patients (6 muscle invasive, 5 high-grade muscle non-invasive, 5 low-grade muscle non-invasive), 17 controls, 10 RCC patients and 4 urinary tract infections (UTI) using Affymetrix miRNA microarrays. Diagnostic and prognostic potential of selected microRNAs was further validated on independent samples in training phase (50 bladder cancer patients, 15 controls) and validation phase (100 bladder cancer patients, 55 controls, 45 renal cancer patients) using specific TaqMan assays and qRT-PCR method.Global expression profiling identified set of 76 miRNAs able distinguish bladder cancer patients from healthy controls (P < 0,01), thereof 64 highly up-regulated and 12 down-regulated. Moreover 23 miRNAs were able distinguish invasive and non-invasive forms of UCUB (P < 0,01) and 18 miRNAs high-grade and low-grad non-invasive (p < 0,01). Set of 12 miRNAs with highest expression level and statistical significance was validated in training phase of study. Based on the results the panel of three miRNAs (miR-31, miR-93, miR-191) was profiled. In validation phase we confirmed diagnostic potential and ability of this urine miRNA-based panel to diagnose patients with bladder cancer with high sensitivity and specificity (AUC = 0,8794, sensitivity = 82%, specificity = 80%).Our data have shown that urinary microRNAs could serve as sensitive and specific biomarkers of urinary bladder cancer and could be useful tool to increase sensitivity of standard cytological examination and reduce costs associated with long-term follow-up of bladder cancer patients. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-33158A, 15-34553A, 15-31627A, 15-34678A, 16-31314A, 16-31765A and by grant of Czech Grant Agency nr. 16-18257S.. All rights reserved. Citation Format: Jaroslav Juracek, Tana Machackova, Marek Vecera, Kamila Souckova, Jiri Sana, Parwez Ahmad, Natalia Anna Gablo, Ondrej Slaby, Michal Stanik, Jan Dolezel. Panel of urinary cell-free microRNAs in detection of urinary bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5451. doi:10.1158/1538-7445.AM2017-5451

Introduction: Glioblastoma multiforme (GBM) is the most frequent intracranial malignity of astrocytic origin in adults. This tumor is characterized by infaust prognosis, which is primarily caused by resistance to the therapy and early relapses relate to the presence of glioblastoma stem cells (GSCs). These cells form neurospheres in vitro and express markers of stemness such as Sox-2, Oct-4, and nestin. Targeting of GSCs could be a novel promising therapeutic approach leading to the overcome of therapy resistance and better prognosis of GBM patients. One of the approaches how to successfully regulate GSC is a targeted regulation of microRNAs (miRNAs). These small non-coding RNA molecules post-transcriptionally regulate an expression of more than 2/3 of all human genes that are also involved in stem cell associated signaling pathways. Moreover, deregulated expression of some miRNAs has been observed in many cancers including GBM. Cell lines and Methods: We have prepared eleven Sox-2 positive and negative paired primary GBM cell lines, which have been cultured under DMEM/F12 medium supplemented with bFGF, EGF, and B12 supplement and DMEM medium supplemented with 10% FBS, respectively. The global miRNA expression analysis was performed using GeneChip miRNA 4.0 Array (Affymetrix). Targeted regulation of miRNA levels have been carried out by the transient transfection of specific anti-miRNAs or miRNA mimics in GSC cell lines NCH 601 acquired from Interdisciplinary Center For Neurosciences (Heidelberg, Germany). The sphere formation assay was analyzed using IncuCyte ZOOM instrument (Essen BioScience). Sox-2 and nestin expressions were analyzed on both protein and transcriptional levels using combination of PAGE with Western blotting and real-time PCR, respectively. Results: Analysis of Sox-2 positive and negative paired GBM cell lines revealed 29 differentially expressed miRNAs, from which miR-93-3p, miR-95-5p, miR-106b-5p, miR-22-3p, and miR-3195 showed high significance (adjust. P value < 0.05) and association with both Sox-2 (Spearman R; p < 0.002) and nestin (Spearman R; p < 0.02) expressions. MiR-22-3p and miR-3195 showed lower expression whereas other miRNAs higher expression in Sox-2 positive GBM cells. The in vitro analyses also suggest that miR-22-3p and miR-106b-5p affect the sphere formation potential and Sox-2 expression in GSC cells. Conclusion: Taken together, our data suggest that miR-22-3p and miR-106b-5p are probably involved in GSC biology and, thus, should be promising molecular targets to overcome GBM therapeutic resistance. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-33158A, 15-34553A, 15-31627A and 15-34678A. All rights reserved. Citation Format: Jiri Sana, Marek Vecera, Romana Butova, Pavel Fadrus, Leos Kren, Jaroslav Juracek, Robert Illiev, Jitka Mlcochova, Zuzana Ozanova, Petra Vychytilova, Ondrej Slaby. Global microRNA expression analysis of Sox-2 positive and negative glioblastoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1959.