Abstract 3615: Analysis of microRNAs in cerebrospinal fluid of brain tumor patients
Authors: Sana Jiri , Kopkova Alena , Vecera Marek , Juracek Jaroslav , Machackova Tana , Ahmad Parwez , Gablo Natalia Anna , Fadrus Pavel , Svoboda Marek , Slaby Ondrej 
Published: August 2018

Abstract
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&lt;0.01).Conclusion: We have demonstrated that CSF of various brain tumors (GBM, LGG, meningioma, and brain metastasis) is characterized by specific miRNA signature. Moreover, we found miRNA signature with ability to differentiate LGG and GBM from CSF. Our results suggest, that after independent validations, CSF miRNAs could serve as promising biomarkers in brain tumors. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-34553A, 15-33158A, 15-31627A, 15-34678A, 16-31314A, 16-31765A and by grant of Czech Grant Agency nr. 16-18257S, 17-17636S.
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.