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<0.001). A classifier model was developed based on the expression of 6 miRNAs (hsa-miR-141-3p, hsa-miR-141-5p, hsa-miR-146a-5p, hsa-miR-194-5p, hsa-miR-200b-3p and hsa-miR-365b-5p) with the ability to correctly classify 91.5% of samples. Subsequent validation confirmed both significantly different expression of selected miRNAs in 5 BM groups as well as their diagnostic potential. CONCLUSION: To date, our study is the first to analyze miRNA expression in various types of BMs using small RNA sequencing to develop a diagnostic classifier and, thus, to help stratify BMs of unknown primary. The presented results confirm the importance of studying the dysregulated expression of miRNAs in BMs and the diagnostic potential of the validated 6-miRNA signature.

1st Department of Pathology St Anne's University Hospital Brno Czech Republic

Central European Institute of Technology Masaryk University Brno Czech Republic

Central European Institute of Technology Masaryk University Brno Czech Republic;

Department of Biology Faculty of Medicine Masaryk University Brno Czech Republic

Department of Comprehensive Cancer Care Masaryk Memorial Cancer Institute Brno Czech Republic

Department of Neurosurgery St Anne's University Hospital Brno Czech Republic

Department of Neurosurgery University Hospital Brno Brno Czech Republic

Department of Neurosurgery University Hospital Brno Brno Czech Republic;

Department of Pathology University Hospital Brno Brno Czech Republic

Department of Radiation Oncology Masaryk Memorial Cancer Institute Brno Czech Republic

Faculty of Medicine Masaryk University Brno Czech Republic

The Fingerland Department of Pathology University Hospital Hradec Kralove Hradec Kralove Czech Republic

Zobrazit více v PubMed

Ostrom QT, Wright CH, Barnholtz-Sloan JS. Brain metastases: epidemiology. Handb Clin Neurol. 2018;149:27–42. doi: 10.1016/B978-0-12-811161-1.00002-5. PubMed DOI

Sperduto PW, Mesko S, Li J, Cagney D, Aizer A, Lin NU, Nesbit E, Kruser TJ, Chan J, Braunstein S, Lee J, Kirkpatrick JP, Breen W, Brown PD, Shi D, Shih HA, Soliman H, Sahgal A, Shanley R, Sperduto WA, Lou E, Everett A, Boggs DH, Masucci L, Roberge D, Remick J, Plichta K, Buatti JM, Jain S, Gaspar LE, Wu CC, Wang TJC, Bryant J, Chuong M, An Y, Chiang V, Nakano T, Aoyama H, Mehta MP. Survival in patients with brain metastases: summary report on the updated diagnosis-specific graded prognostic assessment and definition of the eligibility quotient. J Clin Oncol. 2020;38(32):3773–3784. doi: 10.1200/JCO.20.01255. PubMed DOI PMC

Pentheroudakis G, Golfinopoulos V, Pavlidis N. Switching benchmarks in cancer of unknown primary: from autopsy to microarray. Eur J Cancer. 2007;43(14):2026–2036. doi: 10.1016/j.ejca.2007.06.023. PubMed DOI

Lamba N, Wen PY, Aizer AA. Epidemiology of brain metastases and leptomeningeal disease. Neuro Oncol. 2021;23(9):1447–1456. doi: 10.1093/neuonc/noab101. PubMed DOI PMC

Barnholtz-Sloan JS, Sloan AE, Davis FG, Vigneau FD, Lai P, Sawaya RE. Incidence proportions of brain metastases in patients diagnosed (1973 to 2001) in the Metropolitan Detroit Cancer Surveillance System. J Clin Oncol. 2004;22(14):2865–2872. doi: 10.1200/JCO.2004.12.149. PubMed DOI

Berghoff AS, Schur S, Füreder LM, Gatterbauer B, Dieckmann K, Widhalm G, Hainfellner J, Zielinski CC, Birner P, Bartsch R, Preusser M. Descriptive statistical analysis of a real life cohort of 2419 patients with brain metastases of solid cancers. ESMO Open. 2016;1(2):e000024. doi: 10.1136/esmoopen-2015-000024. PubMed DOI PMC

Nayak L, Lee EQ, Wen PY. Epidemiology of brain metastases. Curr Oncol Rep. 2012;14(1):48–54. doi: 10.1007/s11912-011-0203-y. PubMed DOI

Sperduto PW, Chao ST, Sneed PK, Luo X, Suh J, Roberge D, Bhatt A, Jensen AW, Brown PD, Shih H, Kirkpatrick J, Schwer A, Gaspar LE, Fiveash JB, Chiang V, Knisely J, Sperduto CM, Mehta M. Diagnosis-specific prognostic factors, indexes, and treatment outcomes for patients with newly diagnosed brain metastases: a multi-institutional analysis of 4,259 patients. Int J Radiat Oncol Biol Phys. 2010;77(3):655–661. doi: 10.1016/j.ijrobp.2009.08.025. PubMed DOI

Nieder C, Spanne O, Mehta MP, Grosu AL, Geinitz H. Presentation, patterns of care, and survival in patients with brain metastases: what has changed in the last 20 years. Cancer. 2011;117(11):2505–2512. doi: 10.1002/cncr.25707. PubMed DOI

Balestrino R, Rudà R, Soffietti R. Brain metastasis from unknown primary tumour: moving from old retrospective studies to clinical trials on targeted agents. Cancers (Basel) 2020;12(11):3350. doi: 10.3390/cancers12113350. PubMed DOI PMC

Pavlidis N, Pentheroudakis G. Cancer of unknown primary site. Lancet. 2012;379(9824):1428–1435. doi: 10.1016/S0140-6736(11)61178-1. PubMed DOI

Kakimoto Y, Tanaka M, Kamiguchi H, Ochiai E, Osawa M. MicroRNA stability in FFPE tissue samples: Dependence on GC content. PLoS One. 2016;11(9):e0163125. doi: 10.1371/journal.pone.0163125. PubMed DOI PMC

Siegl F, Vecera M, Roskova I, Smrcka M, Jancalek R, Kazda T, Slaby O, Sana J. The significance of microRNAs in the molecular pathology of brain metastases. Cancers (Basel) 2022;14(14):3386. doi: 10.3390/cancers14143386. PubMed DOI PMC

Illumina: NextSeq System Denature and Dilute Libraries Guide. Available at: https://support.illumina.com/downloads/nextseq-500-denaturing-diluting-libraries-15048776.html. [Last accessed on October 25, 2022]

Andrews S. FastQC: a quality control tool for high throughput sequence data. Available at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc. [Last accessed on October 25, 2022]

Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. journal. 2014;17(1):10. doi: 10.14806/ej.17.1.200. DOI

Hannon GJ. FASTX-Toolkit. . Available at: http://hannonlab.cshl.edu/fastx_toolkit. [Last accessed on October 25, 2022]

Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright AJ. miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res. 2006;34(Database issue):D140–D144. doi: 10.1093/nar/gkj112. PubMed DOI PMC

Pantano L, Estivill X, Martí E. SeqBuster, a bioinformatic tool for the processing and analysis of small RNAs datasets, reveals ubiquitous miRNA modifications in human embryonic cells. Nucleic Acids Res. 2010;38(5):e34. doi: 10.1093/nar/gkp1127. PubMed DOI PMC

Ewels P, Magnusson M, Lundin S, Käller M. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016;32(19):3047–3048. doi: 10.1093/bioinformatics/btw354. PubMed DOI PMC

Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, Smyth GK. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43(7):e47. doi: 10.1093/nar/gkv007. PubMed DOI PMC

Kursa M, Rudnicki W. Feature selection with the Boruta package. Journal of Statistical Software. 2015;36(11):1–13. doi: 10.18637/jss.v036.i11. DOI

Bonneau E, Neveu B, Kostantin E, Tsongalis GJ, De Guire V. How close are miRNAs from clinical practice? A perspective on the diagnostic and therapeutic market. EJIFCC. 2019;30(2):114–127. PubMed PMC

Korpal M, Kang Y. The emerging role of miR-200 family of microRNAs in epithelial-mesenchymal transition and cancer metastasis. RNA Biol. 2008;5(3):115–119. doi: 10.4161/rna.5.3.6558. PubMed DOI PMC

van Roy F, Berx G. The cell-cell adhesion molecule E-cadherin. Cell Mol Life Sci. 2008;65(23):3756–3788. doi: 10.1007/s00018-008-8281-1. PubMed DOI PMC

Korpal M, Lee ES, Hu G, Kang Y. The miR-200 family inhibits epithelial-mesenchymal transition and cancer cell migration by direct targeting of E-cadherin transcriptional repressors ZEB1 and ZEB2. J Biol Chem. 2008;283(22):14910–14914. doi: 10.1074/jbc.C800074200. PubMed DOI PMC

Dykxhoorn DM, Wu Y, Xie H, Yu F, Lal A, Petrocca F, Martinvalet D, Song E, Lim B, Lieberman J. miR-200 enhances mouse breast cancer cell colonization to form distant metastases. PLoS One. 2009;4(9):e7181. doi: 10.1371/journal.pone.0007181. PubMed DOI PMC

Nicholson AG, Tsao MS, Beasley MB, Borczuk AC, Brambilla E, Cooper WA, Dacic S, Jain D, Kerr KM, Lantuejoul S, Noguchi M, Papotti M, Rekhtman N, Scagliotti G, van Schil P, Sholl L, Yatabe Y, Yoshida A, Travis WD. The 2021 WHO classification of lung tumors: Impact of advances since 2015. J Thorac Oncol. 2022;17(3):362–387. doi: 10.1016/j.jtho.2021.11.003. PubMed DOI

Alexander M, Kim SY, Cheng H. Update 2020: Management of non-small cell lung cancer. Lung. 2020;198(6):897–907. doi: 10.1007/s00408-020-00407-5. PubMed DOI PMC

Hiddinga BI, Raskin J, Janssens A, Pauwels P, Van Meerbeeck JP. Recent developments in the treatment of small cell lung cancer. Eur Respir Rev. 2021;30(161):210079. doi: 10.1183/16000617.0079-2021. PubMed DOI PMC

Ferracin M, Pedriali M, Veronese A, Zagatti B, Gafà R, Magri E, Lunardi M, Munerato G, Querzoli G, Maestri I, Ulazzi L, Nenci I, Croce CM, Lanza G, Querzoli P, Negrini M. MicroRNA profiling for the identification of cancers with unknown primary tissue-of-origin. J Pathol. 2011;225(1):43–53. doi: 10.1002/path.2915. PubMed DOI PMC

Rosenfeld N, Aharonov R, Meiri E, Rosenwald S, Spector Y, Zepeniuk M, Benjamin H, Shabes N, Tabak S, Levy A, Lebanony D, Goren Y, Silberschein E, Targan N, Ben-Ari A, Gilad S, Sion-Vardy N, Tobar A, Feinmesser M, Kharenko O, Nativ O, Nass D, Perelman M, Yosepovich A, Shalmon B, Polak-Charcon S, Fridman E, Avniel A, Bentwich I, Bentwich Z, Cohen D, Chajut A, Barshack I. MicroRNAs accurately identify cancer tissue origin. Nat Biotechnol. 2008;26(4):462–469. doi: 10.1038/nbt1392. PubMed DOI

Rosenwald S, Gilad S, Benjamin S, Lebanony D, Dromi N, Faerman A, Benjamin H, Tamir R, Ezagouri M, Goren E, Barshack I, Nass D, Tobar A, Feinmesser M, Rosenfeld N, Leizerman I, Ashkenazi K, Spector Y, Chajut A, Aharonov R. Validation of a microRNA-based qRT-PCR test for accurate identification of tumor tissue origin. Mod Pathol. 2010;23(6):814–823. doi: 10.1038/modpathol.2010.57. PubMed DOI

Varadhachary GR, Spector Y, Abbruzzese JL, Rosenwald S, Wang H, Aharonov R, Carlson HR, Cohen D, Karanth S, Macinskas J, Lenzi R, Chajut A, Edmonston TB, Raber MN. Prospective gene signature study using microRNA to identify the tissue of origin in patients with carcinoma of unknown primary. Clin Cancer Res. 2011;17(12):4063–4070. doi: 10.1158/1078-0432.CCR-10-2599. PubMed DOI

Meiri E, Mueller WC, Rosenwald S, Zepeniuk M, Klinke E, Edmonston TB, Werner M, Lass U, Barshack I, Feinmesser M, Huszar M, Fogt F, Ashkenazi K, Sanden M, Goren E, Dromi N, Zion O, Burnstein I, Chajut A, Spector Y, Aharonov R. A second-generation microRNA-based assay for diagnosing tumor tissue origin. Oncologist. 2012;17(6):801–812. doi: 10.1634/theoncologist.2011-0466. PubMed DOI PMC

Laprovitera N, Riefolo M, Porcellini E, Durante G, Garajova I, Vasuri F, Aigelsreiter A, Dandachi N, Benvenuto G, Agostinis F, Sabbioni S, Berindan Neagoe I, Romualdi C, Ardizzoni A, Trerè D, Pichler M, D’Errico A, Ferracin M. MicroRNA expression profiling with a droplet digital PCR assay enables molecular diagnosis and prognosis of cancers of unknown primary. Mol Oncol. 2021;15(10):2732–2751. doi: 10.1002/1878-0261.13026. PubMed DOI PMC