Exploring the role of cerebrospinal fluid miRNA in brain tumour diagnostics

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The National Institute of Health estimates that approximately 6 in 100 000 individuals develop brain or central nervous system-related cancers each year1. Current approaches in the diagnosis of brain cancer include CT scans, MRI and tissue biopsy. However, such techniques can be limited by tumour location and heterogeneity, and in the case of tissue biopsy, its highly invasive nature. “Liquid biopsies” have become an increasingly prevalent tool in the development of cancer monitoring techniques during the past decade, with focus on levels of circulating miRNA species. Such liquid biopsies include samples of easy-to-obtain bodily fluids including blood, urine, and cerebrospinal fluid (CSF). CSF, which is in direct contact with the central nervous system, is a particularly promising liquid biopsy fluid for monitoring brain tumor progression.

Researchers at Masaryk University and University Hospital Brno in the Czech Republic undertook the largest study so far investigating CSF miRNA profiles in patients with brain tumours. Their comprehensive study was broken into two phases, beginning with a “discovery phase” in which researchers took a broad look at patient’s miRNA profiles, and a “validation phase” in which researchers narrowed down specific candidate miRNA biomarkers.

In the discovery phase, 89 CSF samples were taken from a group of subjects including both healthy controls and patients with various brain tumour diagnoses. RNA was extracted from the CSF samples using Norgen's Urine microRNA Purification Kit. Following library preparation, samples were sequenced on the NextSeq 500 instrument. Sequencing analysis illustrated significant differences in miRNA profiles between patients with various categories of brain tumours and the control group. Based on these results, nine miRNAs were selected to be evaluated in the second phase of the study.

Figure 1 A) Glioblastoma vs. control, B) Low-grade gilomas vs. control, C) Meningioma vs. control, and D) Brain metastasis vs. control

Cancers 2019, 11, 1546

Figure 1: Hierarchical clustering based on cerebrospinal fluid (CSF) miRNA expression profiles of glioblastomas and controls (A); low-grade gliomas and controls; (B) meningiomas and controls (C); and brain metastases and controls (D). Blue color always indicates CSF specimen collected from controlindividual. A gradient of green and red colors is used in the heatmap (green color indicates lower expression whereas red color indicates higher expression of individual miRNAs in analyzed samples).

Early identification of brain tumour type and origin can help identify the best treatment options to improve patients’ quality of life. During the validation phase, researchers attempted to pinpoint specific miRNAs that could be used to identify and distinguish categories of brain tumours. Using an independent cohort of patients and controls, researchers isolated RNA from 126 CSF samples (using Norgen's Urine microRNA Purification Kit) and ran triplicate PCRs on the nine aforementioned candidate miRNAs. In conclusion, it was determined that the levels of five miRNAs in particular could be used to distinguish between either the presence of a brain tumour or the type of tumour present. Furthermore, two miRNAs in particular provided promising results for determining the long-term prognosis of glioblastoma patients. 

Figure 2: A) let-7b, B) let-7c, C) miR-10a, D) miR-10b, E) miR-21-3p, F) miR-30e, G) miR-140, H) miR-196a, I) miR-196b

Cancers 2019, 11, 1546

Figure 2: Validation of candidate cerebrospinal fluid miRNA biomarkers (A let-7b, B let-7c, C miR-10a, D miR-10b, E miR-21-3p, F miR-30e, G miR-140, H miR-196a, I miR-196b). In controls, patients with glioblastoma (GBM), meningioma, brain metastasis, and low-grade glioma (LGG).

Figure 3. A) brain timor patients; and B) glioblastoma, meningioma and brain metastasis patients based on detection of selected miRNAs in CSF

Cancers 2019, 11, 1546

Figure 3: Diagnostic schemas for brain tumor patients stratification of (A) brain tumor patients; and (B) glioblastoma, meningioma and brain metastasis patients based on detection of selected miRNAs in CSF. DS = Diagnostic Score; AUC = Area Under Curve; CSF = Cerebrospinal fluid.

Specific microRNAs examined in this study, such as miR-10b, have also been investigated as biomarkers for brain and other tumours in previous research2,3. Further research elucidating the role of specific miRNAs in cancer progression could help not only in the detection of cancer, but also in development of novel therapeutic targets for cancer treatment.

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  1. Howlader N, Noone AM, Krapcho M, Miller D, Brest A, Yu M, Ruhl J, Tatalovich Z, Mariotto A, Lewis DR, Chen HS, Feuer EJ, Cronin KA (eds). SEER Cancer Statistics Review, 1975-2016, National Cancer Institute. Bethesda, MD, https://seer.cancer.gov/csr/1975_2016/, based on November 2018 SEER data submission, posted to the SEER web site, April 2019.

  2. Teplyuk, Nadiya M., et al. “MicroRNAs in cerebrospinal fluid identify glioblastoma and metastatic brain cancers and reflect disease activity.” Neuro-oncology 14.6 (2012): 689-700.3.

  3. Khalighfard, Solmaz, et al. “Plasma miR-21, miR-155, miR-10b, and Let-7a as the potential biomarkers for the monitoring of breast cancer patients.” Scientific reports 8.1 (2018): 17981.

  4. PUBLICATION Kopkova, A., Sana, J., Machackova, T., Vecera, M., Radova, L., Trachtova, K., … & Fadrus, P. (2019). Cerebrospinal Fluid MicroRNA Signatures as Diagnostic Biomarkers in Brain Tumors. Cancers, 11(10), 1546. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6826583/

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