BACKGROUND: The current World Health Organisation (WHO) classification of central nervous system tumors comprises over 100 entities. Most of these are defined by purely histological criteria, with varying and sometimes overlapping spectra. Histological diagnosis is often challenging, however, especially in cases with limited or non-representative biopsy material. Thus, molecular technologies that can complement standard pathology testing have the potential to greatly enhance diagnostic precision and improve clinical decision-making. DNA methylation profiling, acting as a 'fingerprint' of cellular origin and molecular alterations, is one such promising technology. METHODS: We have assembled a reference dataset of more than 2,000 methylation profiles using the Illumina HumanMethylation450 (450k) array, currently representing over 50 brain tumor entities or subgroups. The array platform is suitable for both frozen and paraffin-embedded material, with minimal DNA input required. Each new diagnostic case receives an entity prediction with an associated probability score as a confidence measure. Genome-wide copy number profiles (e.g. for scoring 1p/19q loss or gene amplifications) and target gene methylation data (e.g. MGMT) generated from the array provide important additional information. RESULTS: In addition to the reference cohort, more than 500 diagnostic samples from Heidelberg University Hospital and external institutions have been processed. Approximately 5-10% of cases displayed a discrepancy between histological and molecular diagnoses. Careful re-examination of these often resulted in refinement of the original diagnosis, and improved patient care.Furthermore, samples collected for the reference cohort have led to significant improvements in our understanding of the biology of several tumor types, including the identification of further subgroups for several entities and associations with recurrent copy number changes and/or mutations. CONCLUSIONS: Our understanding of the molecular alterations underlying brain tumors has grown enormously in recent years, and it is crucial that this is translated into the clinic promptly. DNA methylation profiling is one tool with the potential to become an important part of the diagnostic armoury of neuropathologists. This relatively inexpensive and robust method is well suited to complement standard histopathologic techniques and improve diagnostic accuracy, thereby optimising patient management. We are currently expanding our pipeline to include additional diagnostic centres, allowing for further refinement and validation as well as broader international access. SECONDARY CATEGORY: Tumor Biology.