Introduction: Pediatric brain tumours (PBT) are one of the most common malignancies during childhood, with variable severity according to the location and histological type. Certain types of gliomas, such a glioblastoma and diffuse intrinsic pontine glioma (DIPG), have a much higher mortality than ependymoma and medulloblastoma. Early detection of PBT is essential for diagnosis and therapeutic interventions. Liquid biopsies have been demonstrated using cerebrospinal fluid (CSF), mostly restricted to cell free DNA, which display limitations of quantity and integrity. In this pilot study, we sought to demonstrate the detectability and robustness of cell free histones in the CSF. Methods: We collected CSF samples from a pilot cohort of 8 children with brain tumours including DIPG, medulloblastoma, glioblastoma, ependymoma and others. As controls, we collected CSF samples from nine children with unrelated blood malignancies and without brain tumours. We applied a multichannel flow imaging approach on ImageStream(X) to image indiviual histone or histone complexes on different channels. Results: Single histones (H2A, macroH2A1.1, macroH2A1.2 H2B, H3, H4 and histone H3 bearing the H3K27M mutation), and histone complexes are specifically detectable in the CSF of PBT patients. H2A and its variants macroH2A1.1/macroH2A1/2 displayed the strongest signal and abundance, together with disease associated H3K27M. In contrast, mostly H4 is detectable in the CSF of pediatric patients with blood malignancies. Discussion: In conclusion, free histones and histone complexes are detectable with a strong signal in the CSF of children affected by brain tumours, using ImageStream(X) technology and may provide additive diagnostic and predictive information.

Department of Adaptive Biotechnologies Global Change Research Institute CAS Brno Czechia

Department of Medical Genetics Medical University of Varna Varna Bulgaria

Department of Pediatric Oncology University Hospital Brno and Faculty of Medicine Masaryk University Brno Czechia

Department of Stem Cell Biology and Transplantology Research Institute of the Medical University of Varna Varna Bulgaria

Faculty of Health Liverpool John Moores University Liverpool United Kingdom

International Clinical Research Center St Anne's University Hospital Brno Czechia

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Andersson D., Fagman H., Dalin M. G., Stahlberg A. (2020). Circulating cell-free tumor DNA analysis in pediatric cancers. Mol. Asp. Med. 72, 100819. 10.1016/j.mam.2019.09.003 PubMed DOI

Azad T. D., Jin M. C., Bernhardt L. J., Bettegowda C. (2020). Liquid biopsy for pediatric diffuse midline glioma: a review of circulating tumor DNA and cerebrospinal fluid tumor DNA. Neurosurg. Focus 48 (1), E9. 10.3171/2019.9.FOCUS19699 PubMed DOI PMC

Baade P. D., Youlden D. R., Valery P. C., Hassall T., Ward L., Green A. C., et al. (2010). Trends in incidence of childhood cancer in Australia, 1983-2006. Br. J. Cancer 102 (3), 620–626. 10.1038/sj.bjc.6605503 PubMed DOI PMC

Babicki S., Arndt D., Marcu A., Liang Y., Grant J. R., Maciejewski A., et al. (2016). Heatmapper: web-enabled heat mapping for all. Nucleic Acids Res. 44 (W1), W147–W153. 10.1093/nar/gkw419 PubMed DOI PMC

Bauden M., Pamart D., Ansari D., Herzog M., Eccleston M., Micallef J., et al. (2015). Circulating nucleosomes as epigenetic biomarkers in pancreatic cancer. Clin. Epigenetics 7, 106. 10.1186/s13148-015-0139-4 PubMed DOI PMC

Bereshchenko O., Lo Re O., Nikulenkov F., Flamini S., Kotaskova J., Mazza T., et al. (2019). Deficiency and haploinsufficiency of histone macroH2A1.1 in mice recapitulate hematopoietic defects of human myelodysplastic syndrome. Clin. Epigenetics 11 (1), 121. 10.1186/s13148-019-0724-z PubMed DOI PMC

Bonner E. R., Bornhorst M., Packer R. J., Nazarian J. (2018). Liquid biopsy for pediatric central nervous system tumors. NPJ Precis. Oncol. 2, 29. 10.1038/s41698-018-0072-z PubMed DOI PMC

Borghesan M., Fusilli C., Rappa F., Panebianco C., Rizzo G., Oben J. A., et al. (2016). DNA hypomethylation and histone variant macroH2A1 synergistically attenuate chemotherapy-induced senescence to promote hepatocellular carcinoma progression. Cancer Res. 76 (3), 594–606. 10.1158/0008-5472.CAN-15-1336 PubMed DOI PMC

Bounajem M. T., Karsy M., Jensen R. L. (2020). Liquid biopsies for the diagnosis and surveillance of primary pediatric central nervous system tumors: a review for practicing neurosurgeons. Neurosurg. Focus 48 (1), E8. 10.3171/2019.9.FOCUS19712 PubMed DOI

Brinkmann V., Reichard U., Goosmann C., Fauler B., Uhlemann Y., Weiss D. S., et al. (2004). Neutrophil extracellular traps kill bacteria. Science 303 (5663), 1532–1535. 10.1126/science.1092385 PubMed DOI

Buschbeck M., Hake S. B. (2017). Variants of core histones and their roles in cell fate decisions, development and cancer. Nat. Rev. Mol. Cell. Biol. 18 (5), 299–314. 10.1038/nrm.2016.166 PubMed DOI

Buzova D., Braghini M. R., Bianco S. D., Lo Re O., Raffaele M., Frohlich J., et al. (2022). Profiling of cell-free DNA methylation and histone signatures in pediatric NAFLD: a pilot study. Hepatol. Commun. 6 (12), 3311–3323. 10.1002/hep4.2082 PubMed DOI PMC

Buzova D., Maugeri A., Liguori A., Napodano C., Lo Re O., Oben J., et al. (2020). Circulating histone signature of human lean metabolic-associated fatty liver disease (MAFLD). Clin. Epigenetics 12 (1), 126. 10.1186/s13148-020-00917-2 PubMed DOI PMC

Demagny J., Roussel C., Le Guyader M., Guiheneuf E., Harrivel V., Boyer T., et al. (2022). Combining imaging flow cytometry and machine learning for high-throughput schistocyte quantification: a SVM classifier development and external validation cohort. EBioMedicine 83, 104209. 10.1016/j.ebiom.2022.104209 PubMed DOI PMC

Di Meo A., Bartlett J., Cheng Y., Pasic M. D., Yousef G. M. (2017). Liquid biopsy: a step forward towards precision medicine in urologic malignancies. Mol. Cancer 16 (1), 80. 10.1186/s12943-017-0644-5 PubMed DOI PMC

Dominical V., Samsel L., McCoy J. P., Jr. (2017). Masks in imaging flow cytometry. Methods 112, 9–17. 10.1016/j.ymeth.2016.07.013 PubMed DOI PMC

Gojo J., Pavelka Z., Zapletalova D., Schmook M. T., Mayr L., Madlener S., et al. (2019). Personalized treatment of H3K27m-mutant pediatric diffuse gliomas provides improved therapeutic opportunities. Front. Oncol. 9, 1436. 10.3389/fonc.2019.01436 PubMed DOI PMC

Hargrave D. R., Zacharoulis S. (2007). Pediatric CNS tumors: current treatment and future directions. Expert Rev. Neurother. 7 (8), 1029–1042. 10.1586/14737175.7.8.1029 PubMed DOI

Holdenrieder S., Stieber P., von Pawel J., Raith H., Nagel D., Feldmann K., et al. (2004). Circulating nucleosomes predict the response to chemotherapy in patients with advanced non-small cell lung cancer. Clin. Cancer Res. 10 (181), 5981–5987. 10.1158/1078-0432.CCR-04-0625 PubMed DOI

Humphries J. M., Penno M. A., Weiland F., Klingler-Hoffmann M., Zuber A., Boussioutas A., et al. (2014). Identification and validation of novel candidate protein biomarkers for the detection of human gastric cancer. Biochim. Biophys. Acta 1844 (5), 1051–1058. 10.1016/j.bbapap.2014.01.018 PubMed DOI

Jessa S., Blanchet-Cohen A., Krug B., Vladoiu M., Coutelier M., Faury D., et al. (2019). Stalled developmental programs at the root of pediatric brain tumors. Nat. Genet. 51 (12), 1702–1713. 10.1038/s41588-019-0531-7 PubMed DOI PMC

Johnson K. J., Cullen J., Barnholtz-Sloan J. S., Ostrom Q. T., Langer C. E., Turner M. C., et al. (2014). Childhood brain tumor epidemiology: a brain tumor epidemiology consortium review. Cancer Epidemiol. Biomarkers Prev. 23 (12), 2716–2736. 10.1158/1055-9965.EPI-14-0207 PubMed DOI PMC

Li D., Bonner E. R., Wierzbicki K., Panditharatna E., Huang T., Lulla R., et al. (2021). Standardization of the liquid biopsy for pediatric diffuse midline glioma using ddPCR. Sci. Rep. 11 (1), 5098. 10.1038/s41598-021-84513-1 PubMed DOI PMC

Liu A. P., Northcott P. A., Robinson G. W., Gajjar A. (2022). Circulating tumor DNA profiling for childhood brain tumors: technical challenges and evidence for utility. Lab. Invest. 102 (2), 134–142. 10.1038/s41374-021-00719-x PubMed DOI

Lo Re O., Douet J., Buschbeck M., Fusilli C., Pazienza V., Panebianco C., et al. (2018a). Histone variant macroH2A1 rewires carbohydrate and lipid metabolism of hepatocellular carcinoma cells towards cancer stem cells. Epigenetics 13 (8), 829–845. 10.1080/15592294.2018.1514239 PubMed DOI PMC

Lo Re O., Fusilli C., Rappa F., Van Haele M., Douet J., Pindjakova J., et al. (2018b). Induction of cancer cell stemness by depletion of macrohistone H2A1 in hepatocellular carcinoma. Hepatology 67 (2), 636–650. 10.1002/hep.29519 PubMed DOI

Lo Re O., Maugeri A., Hruskova J., Jakubik J., Kucera J., Bienertova-Vasku J., et al. (2019). Obesity-induced nucleosome release predicts poor cardio-metabolic health. Clin. Epigenetics 12 (1), 2. 10.1186/s13148-019-0797-8 PubMed DOI PMC

Lo Re O., Mazza T., Giallongo S., Sanna P., Rappa F., Vinh Luong T., et al. (2020). Loss of histone macroH2A1 in hepatocellular carcinoma cells promotes paracrine-mediated chemoresistance and CD4(+)CD25(+)FoxP3(+) regulatory T cells activation. Theranostics 10 (2), 910–924. 10.7150/thno.35045 PubMed DOI PMC

Louis D. N., Perry A., Reifenberger G., von Deimling A., Figarella-Branger D., Cavenee W. K., et al. (2016). The 2016 world health organization classification of tumors of the central nervous system: a summary. Acta Neuropathol. 131 (6), 803–820. 10.1007/s00401-016-1545-1 PubMed DOI

Lu V. M., Alvi M. A., McDonald K. L., Daniels D. J. (2018). Impact of the H3K27M mutation on survival in pediatric high-grade glioma: a systematic review and meta-analysis. J. Neurosurg. Pediatr. 23 (3), 308–316. 10.3171/2018.9.PEDS18419 PubMed DOI

Luger K., Mader A. W., Richmond R. K., Sargent D. F., Richmond T. J. (1997). Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389 (6648), 251–260. 10.1038/38444 PubMed DOI

Manda-Handzlik A., Demkow U. (2019). The brain entangled: the contribution of neutrophil extracellular traps to the diseases of the central nervous system. Cells 8 (12), 1477. 10.3390/cells8121477 PubMed DOI PMC

McEwen A. E., Leary S. E. S., Lockwood C. M. (2020). Beyond the blood: CSF-derived cfDNA for diagnosis and characterization of CNS tumors. Front. Cell. Dev. Biol. 8, 45. 10.3389/fcell.2020.00045 PubMed DOI PMC

Mehrotra M., Singh R. R., Loghavi S., Duose D. Y., Barkoh B. A., Behrens C., et al. (2018). Detection of somatic mutations in cell-free DNA in plasma and correlation with overall survival in patients with solid tumors. Oncotarget 9 (12), 10259–10271. 10.18632/oncotarget.21982 PubMed DOI PMC

Miller D. C. (2023). The world health organization classification of tumors of the central nervous system, fifth edition, 2021: a critical analysis. Adv. Tech. Stand Neurosurg. 46, 1–21. 10.1007/978-3-031-28202-7_1 PubMed DOI

Northcott P. A., Buchhalter I., Morrissy A. S., Hovestadt V., Weischenfeldt J., Ehrenberger T., et al. (2017). The whole-genome landscape of medulloblastoma subtypes. Nature 547 (7663), 311–317. 10.1038/nature22973 PubMed DOI PMC

Ogle L. F., Orr J. G., Willoughby C. E., Hutton C., McPherson S., Plummer R., et al. (2016). Imagestream detection and characterisation of circulating tumour cells - a liquid biopsy for hepatocellular carcinoma? J. Hepatol. 65 (2), 305–313. 10.1016/j.jhep.2016.04.014 PubMed DOI

Panditharatna E., Kilburn L. B., Aboian M. S., Kambhampati M., Gordish-Dressman H., Magge S. N., et al. (2018). Clinically relevant and minimally invasive tumor surveillance of pediatric diffuse midline gliomas using patient-derived liquid biopsy. Clin. Cancer Res. 24 (23), 5850–5859. 10.1158/1078-0432.CCR-18-1345 PubMed DOI PMC

Parpart-Li S., Bartlett B., Popoli M., Adleff V., Tucker L., Steinberg R., et al. (2017). The effect of preservative and temperature on the analysis of circulating tumor DNA. Clin. Cancer Res. 23 (10), 2471–2477. 10.1158/1078-0432.CCR-16-1691 PubMed DOI

Parsons D. W., Li M., Zhang X., Jones S., Leary R. J., Lin J. C., et al. (2011). The genetic landscape of the childhood cancer medulloblastoma. Science 331 (6016), 435–439. 10.1126/science.1198056 PubMed DOI PMC

Paugh B. S., Qu C., Jones C., Liu Z., Adamowicz-Brice M., Zhang J., et al. (2010). Integrated molecular genetic profiling of pediatric high-grade gliomas reveals key differences with the adult disease. J. Clin. Oncol. 28 (18), 3061–3068. 10.1200/JCO.2009.26.7252 PubMed DOI PMC

Pazienza V., Borghesan M., Mazza T., Sheedfar F., Panebianco C., Williams R., et al. (2014). SIRT1-metabolite binding histone macroH2A1.1 protects hepatocytes against lipid accumulation. Aging (Albany NY) 6 (1), 35–47. 10.18632/aging.100632 PubMed DOI PMC

Rahier J. F., Druez A., Faugeras L., Martinet J. P., Gehenot M., Josseaux E., et al. (2017). Circulating nucleosomes as new blood-based biomarkers for detection of colorectal cancer. Clin. Epigenetics 9, 53. 10.1186/s13148-017-0351-5 PubMed DOI PMC

Rees P., Summers H. D., Filby A., Carpenter A. E., Doan M. (2022). Imaging flow cytometry: a primer. Nat. Rev. Methods Prim. 2, 86. 10.1038/s43586-022-00167-x PubMed DOI PMC

Salloum R., McConechy M. K., Mikael L. G., Fuller C., Drissi R., DeWire M., et al. (2017). Characterizing temporal genomic heterogeneity in pediatric high-grade gliomas. Acta Neuropathol. Commun. 5 (1), 78. 10.1186/s40478-017-0479-8 PubMed DOI PMC

Saratsis A. M., Yadavilli S., Magge S., Rood B. R., Perez J., Hill D. A., et al. (2012). Insights into pediatric diffuse intrinsic pontine glioma through proteomic analysis of cerebrospinal fluid. Neuro Oncol. 14 (5), 547–560. 10.1093/neuonc/nos067 PubMed DOI PMC

Schilde L. M., Kosters S., Steinbach S., Schork K., Eisenacher M., Galozzi S., et al. (2018). Protein variability in cerebrospinal fluid and its possible implications for neurological protein biomarker research. PLoS One 13 (11), e0206478. 10.1371/journal.pone.0206478 PubMed DOI PMC

Seoane J., De Mattos-Arruda L., Le Rhun E., Bardelli A., Weller M. (2019). Cerebrospinal fluid cell-free tumour DNA as a liquid biopsy for primary brain tumours and central nervous system metastases. Ann. Oncol. 30 (2), 211–218. 10.1093/annonc/mdy544 PubMed DOI

Sexton-Oates A., MacGregor D., Dodgshun A., Saffery R. (2015). The potential for epigenetic analysis of paediatric CNS tumours to improve diagnosis, treatment and prognosis. Ann. Oncol. 26 (7), 1314–1324. 10.1093/annonc/mdv024 PubMed DOI

Siddiq M. M., Hannila S. S., Zorina Y., Nikulina E., Rabinovich V., Hou J., et al. (2021). Extracellular histones, a new class of inhibitory molecules of CNS axonal regeneration. Brain Commun. 3 (4), fcab271. 10.1093/braincomms/fcab271 PubMed DOI PMC

Sperling R., Bustin M. (1976). Histone dimers: a fundamental unit in histone assembly. Nucleic Acids Res. 3 (5), 1263–1275. 10.1093/nar/3.5.1263 PubMed DOI PMC

Stallard S., Savelieff M. G., Wierzbicki K., Mullan B., Miklja Z., Bruzek A., et al. (2018). CSF H3F3A K27M circulating tumor DNA copy number quantifies tumor growth and in vitro treatment response. Acta Neuropathol. Commun. 6 (1), 80. 10.1186/s40478-018-0580-7 PubMed DOI PMC

Sun Y., Li M., Ren S., Liu Y., Zhang J., Li S., et al. (2021). Exploring genetic alterations in circulating tumor DNA from cerebrospinal fluid of pediatric medulloblastoma. Sci. Rep. 11 (1), 5638. 10.1038/s41598-021-85178-6 PubMed DOI PMC

Taylor M. D., Northcott P. A., Korshunov A., Remke M., Cho Y. J., Clifford S. C., et al. (2012). Molecular subgroups of medulloblastoma: the current consensus. Acta Neuropathol. 123 (4), 465–472. 10.1007/s00401-011-0922-z PubMed DOI PMC

Thomas J. O., Kornberg R. D. (1975). An octamer of histones in chromatin and free in solution. Proc. Natl. Acad. Sci. U. S. A. 72 (7), 2626–2630. 10.1073/pnas.72.7.2626 PubMed DOI PMC

Tsoneva D. K., Ivanov M. N., Conev N. V., Manev R., Stoyanov D. S., Vinciguerra M. (2023). Circulating histones to detect and monitor the progression of cancer. Int. J. Mol. Sci. 24 (2), 942. 10.3390/ijms24020942 PubMed DOI PMC

Wu D., Ingram A., Lahti J. H., Mazza B., Grenet J., Kapoor A., et al. (2002). Apoptotic release of histones from nucleosomes. J. Biol. Chem. 277 (14), 12001–12008. 10.1074/jbc.M109219200 PubMed DOI

Extracellular Histones Profiles of Pediatric H3K27-Altered Diffuse Midline Glioma