{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,5]],"date-time":"2026-03-05T23:26:51Z","timestamp":1772753211707,"version":"3.50.1"},"reference-count":38,"publisher":"MDPI AG","issue":"24","license":[{"start":{"date-parts":[[2025,12,17]],"date-time":"2025-12-17T00:00:00Z","timestamp":1765929600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Diagnostics"],"abstract":"<jats:p>Background and Clinical Significance: DNA methylation profiling has revolutionized the classification of central nervous system (CNS) tumors, providing insights into tumor prognosis, recurrence, and personalized treatments. However, a significant challenge remains in classifying rare or molecularly undefined high-grade gliomas (HGGs) that fail to match existing methylation reference classes. This study evaluates the clinical, histopathological, and molecular characteristics of three unclassifiable cases through a retrospective analysis. Methylation profiling was performed by the National Institute of Health based on the 11b6 and 12b6 of the Heidelberg classifier, as well as the National Cancer Institute\/Bethesda classifier. The cases were evaluated for histopathological features, molecular markers, and clinical outcomes. Case Presentation: We present three adult patients with histologically confirmed HGGs that were unclassifiable by standard DNA methylation profiling. All patients presented with diverse clinical and radiographic findings. Histopathological examination confirmed high-grade glial neoplasms in each case. However, methylation profiling failed to yield clear matches for any known class. Instead, profiling suggested indeterminate IDH-wildtype neoplasms with aggressive clinical courses. Following treatment, one patient experienced disease progression and died, while the other two remained without evidence of recurrence at follow-up. Conclusions: These cases underscore the persistent diagnostic challenges posed by CNS tumors that are unclassifiable by current DNA methylation, highlighting the urgent need for expanded reference datasets. While methylation profiling has transformed the field of tumor diagnostics, its limitations still exist. Enhanced collaboration to broaden diagnostic categories is essential to broaden diagnostic classifiers. Until these tools are refined, integration of clinical, histological, and molecular findings is imperative to optimize patient management, improve classification accuracy, and optimize therapeutic outcomes. Unclassifiable HGGs represent a critical gap in CNS tumor diagnostics. Addressing this requires global collaboration to enrich methylation databases. In the interim, a multimodal diagnostic strategy remains essential for the management of these challenging tumors.<\/jats:p>","DOI":"10.3390\/diagnostics15243225","type":"journal-article","created":{"date-parts":[[2025,12,17]],"date-time":"2025-12-17T09:43:46Z","timestamp":1765964626000},"page":"3225","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Limitations of DNA Methylation Profiling in High-Grade Gliomas: Case Series"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9495-3181","authenticated-orcid":false,"given":"Marcus N.","family":"Milani","sequence":"first","affiliation":[{"name":"Medical School, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-8275-6625","authenticated-orcid":false,"given":"Constance P.","family":"Chen","sequence":"additional","affiliation":[{"name":"Medical School, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lindsey","family":"Sloan","sequence":"additional","affiliation":[{"name":"Department of Radiation Oncology, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-6830-5308","authenticated-orcid":false,"given":"Elizabeth C.","family":"Neil","sequence":"additional","affiliation":[{"name":"Department of Neurology, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aundeep","family":"Yekula","sequence":"additional","affiliation":[{"name":"Department of Neurosurgery, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Garret","family":"Fitzpatrick","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Liam","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"746","DOI":"10.1007\/s11102-021-01151-6","article-title":"Identification of mutant K-RAS in pituitary macroadenoma","volume":"24","author":"Aran","year":"2021","journal-title":"Pituitary"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"469","DOI":"10.1038\/nature26000","article-title":"DNA methylation-based classification of central nervous system tumours","volume":"555","author":"Capper","year":"2018","journal-title":"Nature"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1186\/s40478-024-01728-9","article-title":"Unclassifiable CNS tumors in DNA methylation-based classification: Clinical challenges and prognostic impact","volume":"12","author":"Drexler","year":"2024","journal-title":"Acta Neuropathol. Commun."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"vdad076","DOI":"10.1093\/noajnl\/vdad076","article-title":"Clinical utility of whole-genome DNA methylation profiling as a primary molecular diagnostic assay for central nervous system tumors\u2014A prospective study and guidelines for clinical testing","volume":"5","author":"Galbraith","year":"2023","journal-title":"Neuro-Oncol. Adv."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"924","DOI":"10.1016\/j.jmoldx.2022.04.009","article-title":"Validation of Whole Genome Methylation Profiling Classifier for Central Nervous System Tumors","volume":"24","author":"Kam","year":"2022","journal-title":"J. Mol. Diagn. JMD"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"530","DOI":"10.1097\/CCO.0000000000001089","article-title":"When do I ask for a DNA methylation array for primary brain tumor diagnosis?","volume":"36","author":"Barresi","year":"2024","journal-title":"Curr. Opin. Oncol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"181","DOI":"10.1007\/s00428-023-03632-4","article-title":"Molecular neuropathology: An essential and evolving toolbox for the diagnosis and clinical management of central nervous system tumors","volume":"484","author":"Bertero","year":"2024","journal-title":"Virchows Arch. Int. J. Pathol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"e13233","DOI":"10.1111\/bpa.13233","article-title":"Refinement of prognostication for IDH-mutant astrocytomas using DNA methylation-based classification","volume":"34","author":"Kling","year":"2024","journal-title":"Brain Pathol. Zur. Switz."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"478","DOI":"10.1111\/nan.12610","article-title":"Brain tumour diagnostics using a DNA methylation-based classifier as a diagnostic support tool","volume":"46","author":"Boldt","year":"2020","journal-title":"Neuropathol. Appl. Neurobiol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"571","DOI":"10.1093\/neuonc\/noab227","article-title":"Impact of the methylation classifier and ancillary methods on CNS tumor diagnostics","volume":"24","author":"Wu","year":"2022","journal-title":"Neuro-Oncology"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1093\/neuonc\/noac177","article-title":"DNA methylation subclasses predict the benefit from gross total tumor resection in IDH-wildtype glioblastoma patients","volume":"25","author":"Drexler","year":"2023","journal-title":"Neuro-Oncology"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Morgacheva, D., Ryzhova, M., Zheludkova, O., Belogurova, M., and Dinikina, Y. (2023). DNA methylation-based diagnosis confirmation in a pediatric patient with low-grade glioma: A case report. Front. Pediatr., 11.","DOI":"10.3389\/fped.2023.1256876"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1231","DOI":"10.1093\/neuonc\/noab106","article-title":"The 2021 WHO Classification of Tumors of the Central Nervous System: A summary","volume":"23","author":"Louis","year":"2021","journal-title":"Neuro-Oncology"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1001\/jamaoncol.2024.5506","article-title":"Molecular Testing for the World Health Organization Classification of Central Nervous System Tumors: A Review","volume":"11","author":"Horbinski","year":"2025","journal-title":"JAMA Oncol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"728","DOI":"10.1016\/j.ccell.2015.04.002","article-title":"Molecular Classification of Ependymal Tumors across All CNS Compartments, Histopathological Grades, and Age Groups","volume":"27","author":"Pajtler","year":"2015","journal-title":"Cancer Cell"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"273","DOI":"10.1007\/s00401-018-1837-8","article-title":"Anaplastic astrocytoma with piloid features, a novel molecular class of IDH wildtype glioma with recurrent MAPK pathway, CDKN2A\/B and ATRX alterations","volume":"136","author":"Reinhardt","year":"2018","journal-title":"Acta Neuropathol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"682","DOI":"10.1016\/S1470-2045(17)30155-9","article-title":"DNA methylation-based classification and grading system for meningioma: A multicentre, retrospective analysis","volume":"18","author":"Sahm","year":"2017","journal-title":"Lancet Oncol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"518","DOI":"10.1093\/nop\/npad040","article-title":"Current status of DNA methylation profiling in neuro-oncology as a diagnostic support tool: A review","volume":"10","author":"Singh","year":"2023","journal-title":"Neuro-Oncol. Pract."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"917","DOI":"10.1038\/s41591-023-02255-1","article-title":"Multiomic neuropathology improves diagnostic accuracy in pediatric neuro-oncology","volume":"29","author":"Sturm","year":"2023","journal-title":"Nat. Med."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Tam, O.C.H., Ho, R.S.L., Chan, S., Li, K.K.W., Lam, T.-L., Cheung, E.T.Y., Cheung, O.-Y., Ho, W.W.S., Cheng, K.K.F., and Shing, M.M.K. (2023). Genome-Wide DNA Methylation Profiling as Frontline Diagnostics for Central Nervous System Embryonal Tumors in Hong Kong. Cancers, 15.","DOI":"10.3390\/cancers15194880"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Wenger, A., and Car\u00e9n, H. (2022). Methylation Profiling in Diffuse Gliomas: Diagnostic Value and Considerations. Cancers, 14.","DOI":"10.3390\/cancers14225679"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"S16","DOI":"10.1093\/neuonc\/noab143","article-title":"DNA methylation profiling as a model for discovery and precision diagnostics in neuro-oncology","volume":"23","author":"Pratt","year":"2021","journal-title":"Neuro-Oncology"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1111\/pin.13402","article-title":"Decoding the DNA methylome of central nervous system tumors: An emerging modality for integrated diagnosis","volume":"74","author":"Satomi","year":"2024","journal-title":"Pathol. Int."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1016\/j.semcancer.2017.11.010","article-title":"Glioma epigenetics: From subclassification to novel treatment options","volume":"51","author":"Gusyatiner","year":"2018","journal-title":"Semin. Cancer Biol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"vdaa155","DOI":"10.1093\/noajnl\/vdaa155","article-title":"Near haploidization is a genomic hallmark which defines a molecular subgroup of giant cell glioblastoma","volume":"2","author":"Baker","year":"2020","journal-title":"Neuro-Oncol. Adv."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1111\/bpa.12874","article-title":"Biology and grading of pleomorphic xanthoastrocytoma-what have we learned about it?","volume":"31","author":"Vaubel","year":"2021","journal-title":"Brain Pathol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"82","DOI":"10.1186\/s40478-025-01987-0","article-title":"High-grade astrocytoma with piloid features: A single-institution case series and literature review","volume":"13","author":"Goethe","year":"2025","journal-title":"Acta Neuropathol. Commun."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2857","DOI":"10.1038\/s41598-025-87079-4","article-title":"Diagnostic impact of DNA methylation classification in adult and pediatric CNS tumors","volume":"15","author":"Lebrun","year":"2025","journal-title":"Sci. Rep."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"214","DOI":"10.1002\/(SICI)1097-0215(19990412)81:2<214::AID-IJC8>3.0.CO;2-L","article-title":"Cancer risk in mutation carriers of DNA-mismatch-repair genes","volume":"81","author":"Aarnio","year":"1999","journal-title":"Int. J. Cancer"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1111\/j.1399-0004.2008.01125.x","article-title":"Cumulative lifetime incidence of extracolonic cancers in Lynch syndrome: A report of 121 families with proven mutations","volume":"75","author":"Barrow","year":"2009","journal-title":"Clin. Genet."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"4074","DOI":"10.1200\/JCO.2001.19.20.4074","article-title":"MSH2 mutation carriers are at higher risk of cancer than MLH1 mutation carriers: A study of hereditary nonpolyposis colorectal cancer families","volume":"19","author":"Vasen","year":"2001","journal-title":"J. Clin. Oncol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"717","DOI":"10.1111\/ene.12647","article-title":"Glioblastomas, astrocytomas and oligodendrogliomas linked to Lynch syndrome","volume":"22","author":"Therkildsen","year":"2015","journal-title":"Eur. J. Neurol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Bhargav, A.G., Domino, J.S., Alvarado, A.M., Tuchek, C.A., Akhavan, D., and Camarata, P.J. (2023). Advances in computational and translational approaches for malignant glioma. Front. Physiol., 14.","DOI":"10.3389\/fphys.2023.1219291"},{"key":"ref_34","first-page":"177","article-title":"Machine learning applications to neuroimaging for glioma detection and classification: An artificial intelligence augmented systematic review","volume":"89","author":"Buchlak","year":"2021","journal-title":"J. Clin. Neurosci. Off. J. Neurosurg. Soc. Australas."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"110","DOI":"10.1016\/j.semcancer.2023.03.006","article-title":"Emerging role of artificial intelligence in diagnosis, classification and clinical management of glioma","volume":"91","author":"Luo","year":"2023","journal-title":"Semin. Cancer Biol."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Morello, G., La Cognata, V., Guarnaccia, M., Gentile, G., and Cavallaro, S. (2025). Artificial Intelligence-Driven Multi-Omics Approaches in Glioblastoma. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms26199362"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"36756","DOI":"10.1038\/s41598-025-20711-5","article-title":"Integrating deep learning and radiomics for preoperative glioma grading using multi-center MRI data","volume":"15","author":"Yin","year":"2025","journal-title":"Sci. Rep."},{"key":"ref_38","unstructured":"Milani, M., Chen, C., and Chen, L. (2025, January 19\u201322). Methylation Profiling Limitations for High Grade Brain Tumors. Proceedings of the 2025 101st AANP Annual Meeting, Minneapolis, MN, USA."}],"container-title":["Diagnostics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2075-4418\/15\/24\/3225\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,17]],"date-time":"2025-12-17T09:54:49Z","timestamp":1765965289000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2075-4418\/15\/24\/3225"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,17]]},"references-count":38,"journal-issue":{"issue":"24","published-online":{"date-parts":[[2025,12]]}},"alternative-id":["diagnostics15243225"],"URL":"https:\/\/doi.org\/10.3390\/diagnostics15243225","relation":{},"ISSN":["2075-4418"],"issn-type":[{"value":"2075-4418","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,12,17]]}}}