Acta Neuropathologica

, Volume 137, Issue 2, pp 297–306 | Cite as

Molecular profiling of tumors of the brainstem by sequencing of CSF-derived circulating tumor DNA

  • Changcun Pan
  • Bill H. Diplas
  • Xin Chen
  • Yuliang Wu
  • Xiong Xiao
  • Liping Jiang
  • Yibo Geng
  • Cheng Xu
  • Yu Sun
  • Peng Zhang
  • Wenhao Wu
  • Yu Wang
  • Zhen Wu
  • Junting Zhang
  • Yuchen Jiao
  • Hai YanEmail author
  • Liwei ZhangEmail author
Original Paper


Brainstem gliomas are molecularly heterogeneous diseases, many of which are difficult to safely surgically resect and have limited treatment options due to their eloquent location. These constraints pose challenges to biopsy, which limits the use of routine molecular profiling and identification of personalized therapies. Here, we explored the potential of sequencing of circulating tumor DNA (ctDNA) isolated from the cerebrospinal fluid (CSF) of brainstem glioma patients as a less invasive approach for tumor molecular profiling. CSF was obtained from patients either intraoperatively (91.2%, 52/57), from ventricular-peritoneal shunt (3.5%, 2/57), or by lumbar puncture (5.3%, 3/57), all prior to surgical manipulation of the tumor. Deep sequencing of glioma-associated genes was performed on CSF-derived ctDNA and, where available, matched blood and tumor DNA from 57 patients, including nine medullary and 23 diffuse intrinsic pontine gliomas (DIPG). At least one tumor-specific mutation was detected in over 82.5% of CSF ctDNA samples (47/57). In cases with primary tumors harboring at least one mutation, alterations were identified in the CSF ctDNA of 97.3% of cases (36/37). In over 83% (31/37) of cases, all primary tumor alterations were detected in the CSF, and in 91.9% (34/37) of cases, at least half of the alterations were identified. Among ten patients found to have primary tumors negative for mutations, 30% (3/10) had detectable somatic alterations in the CSF. Finally, mutation detection using plasma ctDNA was less sensitive than sequencing the CSF ctDNA (38% vs. 100%, respectively). Our study indicates that deep sequencing of CSF ctDNA is a reliable technique for detecting tumor-specific alterations in brainstem tumors. This approach may offer an alternative approach to stereotactic biopsy for molecular profiling of brainstem tumors.


Liquid biopsy ctDNA DIPG Brainstem H3K27M mutation 



We thank Lin Qiao and Xuefeng Guo for their help in collecting samples. We would like to thank Honglin Zhu and Yufei Yang for their helpful advices in data analysis. The authors thank Nancy Chu Ji, who was hired to assist in the illustration for Fig. 1, which was inspired by Wang et al. [37].

Author contributions

LZ, HY, and BHD designed the study. CP, XC, YW, XX, YG, PZ, WW, and YW collected the samples, CP, and LJ performed the experiments and collected the data. CP, LJ, YJ, and LZ analyzed the results. CP, LJ, BHD, HY, and LZ wrote the manuscript. CX, YS, ZW, JZ, and YJ, gave conceptual advice.


Financial support was provided by the National Key Technology Research and Development Program of the Ministry of Science and Technology of China (Grant nos. 2014BAI04B01 and 2015BAI12B04), Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support (Grant no. ZYLX201608), and Grant for CP from Beijing Municipal Bureau of Human Resources and Social Security (Grant no. 2017-ZZ-117).

Compliance with ethical standards

Conflict of interest

HY is a founder of Genetron Health and receives royalties from Personal Genome Diagnostics (PGDX) and Agios. YJ is a co-founder of and Scientific Advisor for Genetron Health. BHD serves as a scientific consultant for Genetron Health.

Supplementary material

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Supplementary material 1 (XLSX 11 kb)
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Supplementary material 6 (XLSX 25 kb)
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Supplementary material 7 Detection of brain tumor-associated alterations in tumor tissue DNA and CSF-derived ctDNA. Comparing the portions of patients with detectable somatic alterations in 47 patients with available matched CSF ctDNA and tumor tissue DNA reveals that the CSF shows similar patterns to tumor DNA across different a tumor types, b tumor locations and c tumor volumes. (TIFF 20275 kb)


  1. 1.
    Bettegowda C, Sausen M, Leary RJ, Kinde I, Wang Y, Agrawal N et al (2014) Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med 6:224. CrossRefGoogle Scholar
  2. 2.
    Buczkowicz P, Hoeman C, Rakopoulos P, Pajovic S, Letourneau L, Dzamba M et al (2014) Genomic analysis of diffuse intrinsic pontine gliomas identifies three molecular subgroups and recurrent activating ACVR1 mutations. Nat Genet 46:451–456. CrossRefGoogle Scholar
  3. 3.
    Bugiani M, Veldhuijzen van Zanten SEM, Caretti V, Schellen P, Aronica E, Noske DP et al (2017) Deceptive morphologic and epigenetic heterogeneity in diffuse intrinsic pontine glioma. Oncotarget 8:60447–60452. CrossRefGoogle Scholar
  4. 4.
    Castel D, Philippe C, Calmon R, Le Dret L, Truffaux N, Boddaert N et al (2015) Histone H3F3A and HIST1H3B K27M mutations define two subgroups of diffuse intrinsic pontine gliomas with different prognosis and phenotypes. Acta Neuropathol 130:815–827CrossRefGoogle Scholar
  5. 5.
    Cracolici V, Mujacic I, Kadri S, Alikhan M, Niu N, Segal JP et al (2018) Synchronous and metastatic papillary and follicular thyroid carcinomas with unique molecular signatures. Endocr Pathol 29:9–14. CrossRefGoogle Scholar
  6. 6.
    De Mattos-Arruda L, Mayor R, Ng CK, Weigelt B, Martinez-Ricarte F, Torrejon D et al (2015) Cerebrospinal fluid-derived circulating tumour DNA better represents the genomic alterations of brain tumours than plasma. Nat Commun 6:8839. CrossRefGoogle Scholar
  7. 7.
    Diaz LA Jr, Bardelli A (2014) Liquid biopsies: genotyping circulating tumor DNA. J Clin Oncol 32:579–586. CrossRefGoogle Scholar
  8. 8.
    Fedorov A, Beichel R, Kalpathy-Cramer J, Finet J, Fillion-Robin JC, Pujol S et al (2012) 3D Slicer as an image computing platform for the quantitative imaging network. Magn Reson Imaging 30:1323–1341. CrossRefGoogle Scholar
  9. 9.
    Fisher BJ, Hu C, Macdonald DR, Lesser GJ, Coons SW, Brachman DG et al (2015) Phase 2 study of temozolomide-based chemoradiation therapy for high-risk low-grade gliomas: preliminary results of Radiation Therapy Oncology Group 0424. Int J Radiat Oncol Biol Phys 91:497–504. CrossRefGoogle Scholar
  10. 10.
    Fontebasso AM, Papillon-Cavanagh S, Schwartzentruber J, Nikbakht H, Gerges N, Fiset PO et al (2014) Recurrent somatic mutations in ACVR1 in pediatric midline high-grade astrocytoma. Nat Genet 46:462–466. CrossRefGoogle Scholar
  11. 11.
    Frazier JL, Lee J, Thomale UW, Noggle JC, Cohen KJ, Jallo GI (2009) Treatment of diffuse intrinsic brainstem gliomas: failed approaches and future strategies: a review. J Neurosurg Pediatr 3:259–269. CrossRefGoogle Scholar
  12. 12.
    Gerasimidis K, Bertz M, Quince C, Brunner K, Bruce A, Combet E et al (2016) The effect of DNA extraction methodology on gut microbiota research applications. BMC Res Notes 9:365. CrossRefGoogle Scholar
  13. 13.
    Hargrove D, Bartels U, Bouffet E (2006) Diffuse brainstem glioma in children: critical review of clinical trials. Lancet Oncol 7:241–248. CrossRefGoogle Scholar
  14. 14.
    Hoffman LM, DeWire M, Ryall S, Buczkowicz P, Leach J, Miles L et al (2016) Spatial genomic heterogeneity in diffuse intrinsic pontine and midline high-grade glioma: implications for diagnostic biopsy and targeted therapeutics. Acta Neuropathol Commun 4:1. CrossRefGoogle Scholar
  15. 15.
    Huang TY, Piunti A, Lulla RR, Qi J, Horbinski CM, Tomita T et al (2017) Detection of histone H3 mutations in cerebrospinal fluid-derived tumor DNA from children with diffuse midline glioma. Acta Neuropathol Commun 5:28. CrossRefGoogle Scholar
  16. 16.
    Jansen MH, van Zanten SEV, Aliaga ES, Heymans MW, Warmuth-Metz M, Hargrave D et al (2015) Survival prediction model of children with diffuse intrinsic pontine glioma based on clinical and radiological criteria. Neuro Oncol 17:160–166CrossRefGoogle Scholar
  17. 17.
    Jiang T, Li X, Wang J, Su C, Han W, Zhao C et al (2017) Mutational landscape of cfDNA identifies distinct molecular features associated with therapeutic response to first-line platinum-based doublet chemotherapy in patients with advanced NSCLC. Theranostics 7:4753–4762. CrossRefGoogle Scholar
  18. 18.
    Johung TB, Monje M (2017) Diffuse intrinsic pontine glioma: new pathophysiological insights and emerging therapeutic targets. Curr Neuropharmacol 15:88–97CrossRefGoogle Scholar
  19. 19.
    Kadri S, Long BC, Mujacic I, Zhen CJ, Wurst MN, Sharma S et al (2017) Clinical validation of a next-generation sequencing genomic oncology panel via cross-platform benchmarking against established amplicon sequencing assays. J Mol Diagn 19:43–56. CrossRefGoogle Scholar
  20. 20.
    Kieran MW, Goumnerova LC, Prados M, Gupta N (2016) Biopsy for diffuse intrinsic pontine glioma: a reappraisal. J Neurosurg Pediatr 18:390–391. CrossRefGoogle Scholar
  21. 21.
    Klimo P, Panandiker ASP, Thompson CJ, Boop FA, Qaddoumi I, Gajjar A et al (2013) Management and outcome of focal low-grade brainstem tumors in pediatric patients: the St. Jude experience. J Neurosurg Pediatr 11:274–281. CrossRefGoogle Scholar
  22. 22.
    Long W, Yi Y, Chen S, Cao Q, Zhao W, Liu Q (2017) Potential new therapies for pediatric diffuse intrinsic pontine glioma. Front Pharmacol 8:495. CrossRefGoogle Scholar
  23. 23.
    Mackay A, Burford A, Carvalho D, Izquierdo E, Fazal-Salom J, Taylor KR et al (2017) Integrated molecular meta-analysis of 1,000 pediatric high-grade and diffuse intrinsic pontine glioma. Cancer Cell 32(520–537):e525. Google Scholar
  24. 24.
    Mandell LR, Kadota R, Freeman C, Douglass EC, Fontanesi J, Cohen ME et al (1999) There is no role for hyperfractionated radiotherapy in the management of children with newly diagnosed diffuse intrinsic brainstem tumors: results of a Pediatric Oncology Group phase III trial comparing conventional vs. hyperfractionated radiotherapy. Int J Radiat Oncol Biol Phys 43:959–964CrossRefGoogle Scholar
  25. 25.
    Martinez-Ricarte F, Mayor R, Martinez-Saez E, Rubio-Perez C, Pineda E, Cordero E et al (2018) Molecular diagnosis of diffuse gliomas through sequencing of cell-free circulating tumour DNA from cerebrospinal fluid. Clin Cancer Res. Google Scholar
  26. 26.
    Nikbakht H, Panditharatna E, Mikael LG, Li R, Gayden T, Osmond M et al (2016) Spatial and temporal homogeneity of driver mutations in diffuse intrinsic pontine glioma. Nat Commun 7:11185. CrossRefGoogle Scholar
  27. 27.
    Olar A, Wani KM, Alfaro-Munoz KD, Heathcock LE, van Thuijl HF, Gilbert MR et al (2015) IDH mutation status and role of WHO grade and mitotic index in overall survival in grade II-III diffuse gliomas. Acta Neuropathol 129:585–596. CrossRefGoogle Scholar
  28. 28.
    Pan CC, Chen X, Xu C, Wu WH, Zhang P, Wang Y et al (2016) Brainstem gangliogliomas: prognostic factors, surgical indications and functional outcomes. J Neurooncol 128:445–453. CrossRefGoogle Scholar
  29. 29.
    Pentsova EI, Shah RH, Tang J, Boire A, You D, Briggs S et al (2016) Evaluating cancer of the central nervous system through next-generation sequencing of cerebrospinal fluid. J Clin Oncol 34:2404–2415. CrossRefGoogle Scholar
  30. 30.
    Puget S, Beccaria K, Blauwblomme T, Roujeau T, James S, Grill J et al (2015) Biopsy in a series of 130 pediatric diffuse intrinsic pontine gliomas. Childs Nerv Syst 31:1773–1780. CrossRefGoogle Scholar
  31. 31.
    Puget S, Philippe C, Bax DA, Job B, Varlet P, Junier MP et al (2012) Mesenchymal transition and PDGFRA amplification/mutation are key distinct oncogenic events in pediatric diffuse intrinsic pontine gliomas. PLoS One 7:e30313. CrossRefGoogle Scholar
  32. 32.
    Roujeau T, Di Rocco F, Dufour C, Bourdeaut F, Puget S, Sainte Rose C et al (2011) Shall we treat hydrocephalus associated to brain stem glioma in children? Childs Nerv Syst 27:1735–1739. CrossRefGoogle Scholar
  33. 33.
    Saratsis AM, Kambhampati M, Snyder K, Yadavilli S, Devaney JM, Harmon B et al (2014) Comparative multidimensional molecular analyses of pediatric diffuse intrinsic pontine glioma reveals distinct molecular subtypes. Acta Neuropathol 127:881–895. CrossRefGoogle Scholar
  34. 34.
    Taylor KR, Mackay A, Truffaux N, Butterfield Y, Morozova O, Philippe C et al (2014) Recurrent activating ACVR1 mutations in diffuse intrinsic pontine glioma. Nat Genet 46:457–461. CrossRefGoogle Scholar
  35. 35.
    Vanan MI, Eisenstat DD (2015) DIPG in children—what can we learn from the past? Front Oncol 5:237. CrossRefGoogle Scholar
  36. 36.
    Veldhuijzen van Zanten SEM, Lane A, Heymans MW, Baugh J, Chaney B, Hoffman LM et al (2017) External validation of the diffuse intrinsic pontine glioma survival prediction model: a collaborative report from the International DIPG Registry and the SIOPE DIPG Registry. J Neurooncol. Google Scholar
  37. 37.
    Wang Y, Springer S, Zhang M, McMahon KW, Kinde I, Dobbyn L et al (2015) Detection of tumor-derived DNA in cerebrospinal fluid of patients with primary tumors of the brain and spinal cord. Proc Natl Acad Sci USA 112:9704–9709. CrossRefGoogle Scholar
  38. 38.
    Wu G, Broniscer A, McEachron TA, Lu C, Paugh BS, Becksfort J et al (2012) Somatic histone H3 alterations in pediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas. Nat Genet 44:251–253. CrossRefGoogle Scholar
  39. 39.
    Wu G, Diaz AK, Paugh BS, Rankin SL, Ju B, Li Y et al (2014) The genomic landscape of diffuse intrinsic pontine glioma and pediatric non-brainstem high-grade glioma. Nat Genet 46:444–450. CrossRefGoogle Scholar
  40. 40.
    Zhang L, Chen LH, Wan H, Yang R, Wang Z, Feng J et al (2014) Exome sequencing identifies somatic gain-of-function PPM1D mutations in brainstem gliomas. Nat Genet 46:726–730. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of NeurosurgeryBeijing Tiantan Hospital, Capital Medical UniversityBeijingChina
  2. 2.Department of PathologyDuke University Medical Center, The Preston Robert Tisch Brain Tumor CenterDurhamUSA
  3. 3.Genetron Health (Beijing) Co. Ltd.BeijingChina
  4. 4.State Key Lab of Molecular Oncology, Laboratory of Cell and Molecular Biology, National Cancer Center/Cancer HospitalChinese Academy of Medical Sciences and Peking Union Medical College, Collaborative Innovation Center for Cancer MedicineBeijingChina
  5. 5.China National Clinical Research Center for Neurological DiseaseBeijingChina

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