
==== Front
Acta Neuropathol
Acta Neuropathol
Acta Neuropathologica
0001-6322
1432-0533
Springer Berlin Heidelberg Berlin/Heidelberg

39225924
2792
10.1007/s00401-024-02792-0
Correspondence
Glioblastoma, IDH-wildtype with primarily leptomeningeal localization diagnosed by nanopore sequencing of cell-free DNA from cerebrospinal fluid
Sol Nik 1
Kooi Evert-Jan 2
Pagès-Gallego Marc 34
Brandsma Dieta 1
Bugiani Marianna 2
de Ridder Jeroen 34
Wesseling Pieter 25
http://orcid.org/0000-0003-0412-5313
Vermeulen Carlo c.vermeulen-2@umcutrecht.nl

34
1 https://ror.org/03xqtf034 grid.430814.a 0000 0001 0674 1393 Netherlands Cancer Institute - Antoni Van Leeuwenhoek Hospital, Dept. of Neuro-Oncology, Amsterdam, The Netherlands
2 https://ror.org/05grdyy37 grid.509540.d 0000 0004 6880 3010 Amsterdam University Medical Centers/VUmc, Dept. of Pathology, Amsterdam, The Netherlands
3 grid.7692.a 0000000090126352 Center for Molecular Medicine, Utrecht University Medical Center, Utrecht, The Netherlands
4 https://ror.org/01n92vv28 grid.499559.d Oncode Institute, Utrecht, The Netherlands
5 grid.487647.e Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands
3 9 2024
3 9 2024
2024
148 1 354 7 2024
19 8 2024
19 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
http://dx.doi.org/10.13039/501100021821 Oncode Institute P2022-0045 de Ridder Jeroen http://dx.doi.org/10.13039/501100023452 Hanarth Fonds issue-copyright-statement© Springer-Verlag GmbH Germany, part of Springer Nature 2024
==== Body
pmcA 74-year old male presented with a headache, hand tremors, and gait and cognitive disturbances, which progressively worsened over the previous two months. He had a history of prostate cancer for which he was treated with radiotherapy 5 years earlier and a neuroendocrine tumor (NET) grade 1 in his terminal ileum 2 months prior. MRI of the brain revealed leptomeningeal contrast enhancement around the brainstem, cerebellum, and surrounding the cranial nerves in the absence of intraparenchymal lesions (Fig. 1a-c). The differential diagnosis at that time included a ‘neuroinflammatory disease’ and metastatic disease (also acknowledging that leptomeningeal metastasis from prostate cancer and grade 1 NET can occur [10]). Apart from the NET in the ileum, gallium-dotate and fluorodeoxyglucose (FDG) PET-CT scan revealed no extracranial primary tumor or metastatic disease. Cerebrospinal fluid (CSF) obtained by lumbar puncture showed a slightly increased leukocyte number (14 cells/µl), normal glucose (2.9 mmol/l), and an increased total protein levels (8.75 g/l), consistent with leptomeningeal metastatic disease. Cytology and circulating tumor cell analysis of CSF [8] remained negative, even at a third lumbar puncture. Next-generation sequencing of cell-free DNA (cfDNA), however, revealed a telomerase reverse transcriptase (TERT) promoter mutation, strengthening the hypothesis that the patient actually had a leptomeningeal malignancy [3].Fig. 1 Clinical diagnostics. a Sagittal and axial post-contrast T1-weighted MRI images show contrast enhancement around the folia of the cerebellum and the pons (indicated arrows in I, II). Axial post-contrast T1-weighted image shows contrast enhancement of cranial nerve VII and VIII on both sides (arrows in III). b Size distribution of the sequence reads obtained from CSF DNA sequencing. c CNV profile obtained from nanopore sequencing. d Sturgeon classification with a confident (> 0.95) classification as Glioblastoma—MID

Recently, nanopore sequencing for cfDNA from CSF was reported [1], showing promising results in some but not all cases. As a biopsy was considered too burdensome and with a fair chance of not yielding a representative tumor sample, we opted for performing nanopore sequencing of CSF. Approximately 200 ng of DNA was extracted from 2 mL of CSF. Using an adjusted library prep protocol (see Supplementary information) sequencing of 100 ng of the sample resulted in 18 million reads. Mapped read lengths show the typical ~ 150 basepair periodicity expected for cfDNA (Fig. 1b), with the majority of fragments in the 143–175 basepair range. The copy-number variation (CNV) profile (Fig. 1c) revealed several alterations, including PDGFRA and MDM4 gain and CDKN2A/B loss, the high deviation indicating a high tumor fraction. DNA methylation analysis using the Sturgeon classifier [9] on the full or subsampled dataset yielded a high confidence score for Glioblastoma, subtype Midline (GBM—MID) (Fig. 1d) (Supplementary Table 1). Later on, DNA methylation analysis using the Illumina Infinium EPIC methylation array and the Heidelberg classifier version 11b4 [2] confirmed the classification as GBM—MID (calibrated score > 0.99) as well as the alterations in the CNV profile. In contrast, sequencing DNA extracted from the cell pellet obtained from CSF yielded a flat copy-number profile and indicated with a high confidence ‘inflammatory tissue’, likely due to the predominance of inflammatory cells in the pellet (Supplementary Fig. 1).

Given the widespread intracranial disease and the clinical deterioration, best supportive care was provided. The patient's condition rapidly worsened and he soon succumbed. Autopsy revealed dispersed thickening and opacity of the leptomeninges especially around the frontal lobes (Fig. 2a), the cerebellum and brainstem. Macroscopy did not reveal a tumor mass within the brain parenchyma. Microscopically widespread infiltration of tumor cells throughout the leptomeninges was present (Fig. 2b). Intraparenchymal growth was subtly evident on microscopic evaluation showing diffuse infiltrative growth beneath the ependymal lining of the ventricles (Fig. 2c,d) and subpially in the mesencephalon (Supplementary Fig. 2). The tumor cells exhibited astroglial morphology and highly atypical nuclei with coarse chromatin (Fig. 2d). Immunohistochemistry confirmed the glial nature of the tumor (Olig2; Fig. 2e). Occasional mitotic figures were observed, but necrosis and florid microvascular proliferation were absent. Notably, tumor cell proliferation as evidenced by Ki-67 staining was moderate to high (~ 20–30%; Fig. 2f). These post-mortem findings in combination with identification of the TERT promoter mutation allowed for a histomolecular diagnosis of glioblastoma, IDH-wildtype [5], completely in line with the diagnosis suggested by nanopore sequencing of CSF during the life of the patient.Fig. 2 Autopsy findings. A coronal brain section showed leptomeningeal opacification and thickening (a; white arrowheads), the parasagittal part with a rough surface partly due to the presence of granulations of Pacchioni. Microscopically, atypical cells are diffusely spread throughout the leptomeningeal compartment (b). In several regions, such cells were also (but relatively subtly) present in the subependymal/periventricular region, e.g., near the hippocampus (c, d; square box), with more dense accumulation of these cells in an adjacent, deeply invaginating cerebral sulcus (*). Immunohistochemical staining for Olig2 (e) and Ki-67 (f) confirmed the glial nature of the tumor and its moderate proliferative activity, respectively. L: leptomeninges; CTX: molecular layer of the cerebral cortex; HE: hematoxylin and eosin staining

In conclusion, we present a case of a glioblastoma, IDH-wildtype, growing primarily in the leptomeninges. Such cases have rarely been reported before [4, 6, 7] and are very challenging to diagnose. DNA methylation profiling using nanopore sequencing of cfDNA from CSF and the Sturgeon classifier offered in this case a fast and minimally invasive means to reach a straightforward suggestion for a diagnosis. We hypothesize that the high amount of tumor DNA recovered from CSF is a result of the large contact surface between tumor cells and the CSF. In small and circumscribed tumors, cfDNA recovery and sequencing is likely more challenging. The finding of a TERT promoter mutation, the alterations in the CNV profile, and a high confidence classification gave sufficient evidence for the diagnosis high-grade glioma/glioblastoma. This diagnosis was very helpful for the patient and his family, as it served as the basis to continue with palliative care.

Supplementary Information

Below is the link to the electronic supplementary material.Supplementary file1 Cell pellet sequencing. The cell pellet obtained from the same CSF used for cfDNA sequencing was also processed and sequenced. a. Size distribution density plot of the sequence read length, showing a much larger read size than in the cfDNA. b. Copy-number profile, showing a flat profile with few clear variations. c. Sturgeon result showing a high confidence score for inflammatory tissue. (PDF 1493 KB)

Supplementary file2 Autopsy findings continued. Examination of the mesencephalon at the level of the substantia nigra (SN) revealed no clear macroscopic abnormalities (a). Microscopic examination (b; black arrows) revealed the presence of glioma subpially along the outer contour of the mesencephalon (c; high power image corresponding to the square box with dotted line indicated in b) as well as in the leptomeninges (not shown). Additionally, tumor growth was observed in the leptomeningeal compartment surrounding the oculomotor nerve (n. III) (d; high power image corresponding to the uninterrupted lined box square in b). Abbreviations: n.III: oculomotor nerve; HE: hematoxylin and eosin (H&E) staining; SN: Substantia nigra (PDF 6301 KB)

Funding

This work was funded by Oncode Institute, P2022-0045, and an unrestricted grant of Stichting Hanarth Fonds, The Netherlands, to Jeroen de Ridder.

Data availability

Data is available upon reasonable request.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Nik Sol and Evert-Jan Kooi have contributed equally to this work.
==== Refs
References

1. Afflerbach A-K Rohrandt C Brändl B Sönksen M Hench J Frank S Classification of brain tumors by nanopore sequencing of cell-free DNA from cerebrospinal fluid Clin Chem 2024 70 250 260 10.1093/clinchem/hvad115 37624932
Afflerbach A-K, Rohrandt C, Brändl B, Sönksen M, Hench J, Frank S et al (2024) Classification of brain tumors by nanopore sequencing of cell-free DNA from cerebrospinal fluid. Clin Chem 70:250–26037624932 10.1093/clinchem/hvad115
2. Capper D Jones DTW Sill M Hovestadt V Schrimpf D Sturm D DNA methylation-based classification of central nervous system tumours Nature 2018 555 469 474 10.1038/nature26000 29539639
Capper D, Jones DTW, Sill M, Hovestadt V, Schrimpf D, Sturm D et al (2018) DNA methylation-based classification of central nervous system tumours. Nature 555:469–47429539639 10.1038/nature26000
3. El Zarif T Machaalani M Nawfal R Nassar AH Xie W Choueiri TK TERT promoter mutations frequency across race, sex, and cancer type Oncologist 2024 29 1 8 14 10.1093/oncolo/oyad208 37462445
El Zarif T, Machaalani M, Nawfal R, Nassar AH, Xie W, Choueiri TK et al (2024) TERT promoter mutations frequency across race, sex, and cancer type. Oncologist 29(1):8–1437462445 10.1093/oncolo/oyad208
4. Katsuhara T Moro N Ohta T Homma T Yoshino A Solitary primary intracranial leptomeningeal glioblastoma invading the normal cortex: case report Mol Clin Oncol 2018 8 466 470 29599982
Katsuhara T, Moro N, Ohta T, Homma T, Yoshino A (2018) Solitary primary intracranial leptomeningeal glioblastoma invading the normal cortex: case report. Mol Clin Oncol 8:466–47029599982
5. Louis DN Perry A Wesseling P Brat DJ Cree IA Figarella-Branger D The 2021 WHO classification of tumors of the central nervous system: a summary Neuro Oncol 2021 23 1231 1251 10.1093/neuonc/noab106 34185076
Louis DN, Perry A, Wesseling P, Brat DJ, Cree IA, Figarella-Branger D et al (2021) The 2021 WHO classification of tumors of the central nervous system: a summary. Neuro Oncol 23:1231–125134185076 10.1093/neuonc/noab106
6. Riva M Bacigaluppi S Galli C Citterio A Collice M Primary leptomeningeal gliomatosis: case report and review of the literature Neurol Sci 2005 26 129 134 10.1007/s10072-005-0446-1 15995830
Riva M, Bacigaluppi S, Galli C, Citterio A, Collice M (2005) Primary leptomeningeal gliomatosis: case report and review of the literature. Neurol Sci 26:129–13415995830 10.1007/s10072-005-0446-1
7. Shuangshoti S Shuangshoti S Primary diffuse leptomeningeal glioblastoma multiforme of brainstem and spinal cord clinically mimicking meningitis: case report and review of literature J Med Assoc Thai 1996 79 403 408 8855617
Shuangshoti S, Shuangshoti S (1996) Primary diffuse leptomeningeal glioblastoma multiforme of brainstem and spinal cord clinically mimicking meningitis: case report and review of literature. J Med Assoc Thai 79:403–4088855617
8. van Bussel MTJ Pluim D Milojkovic KB Bol M Sikorska K Linders DTC Circulating epithelial tumor cell analysis in CSF in patients with leptomeningeal metastases Neurology 2020 94 5 e521 e528 10.1212/WNL.0000000000008751 31907288
van Bussel MTJ, Pluim D, Milojkovic KB, Bol M, Sikorska K, Linders DTC et al (2020) Circulating epithelial tumor cell analysis in CSF in patients with leptomeningeal metastases. Neurology 94(5):e521–e528. 10.1212/WNL.000000000000875131907288 10.1212/WNL.0000000000008751
9. Vermeulen C Pagès-Gallego M Kester L Kranendonk MEG Wesseling P Verburg N Ultra-fast deep-learned CNS tumour classification during surgery Nature 2023 622 842 849 10.1038/s41586-023-06615-2 37821699
Vermeulen C, Pagès-Gallego M, Kester L, Kranendonk MEG, Wesseling P, Verburg N et al (2023) Ultra-fast deep-learned CNS tumour classification during surgery. Nature 622:842–84937821699 10.1038/s41586-023-06615-2
10. Versluis JM Brandsma D van den Berg JG Tesselaar M Leptomeningeal metastases of a well-differentiated neuroendocrine tumour: a rare entity BMJ Case Rep 2018 10.1136/bcr-2018-226557 30391925
Versluis JM, Brandsma D, van den Berg JG, Tesselaar M (2018) Leptomeningeal metastases of a well-differentiated neuroendocrine tumour: a rare entity. BMJ Case Rep. 10.1136/bcr-2018-22655730391925 10.1136/bcr-2018-226557
