LAUSR

5-aminolevulinic acid (5-ALA) fluorescence-guided resection increases the percentage of complete CNS tumor resections and improves the progression-free survival of IDH-wildtype glioblastoma patients. A small subset of IDH-wildtype glioblastoma show no 5-ALA fluorescence. An explanation for these challenging cases is missing. In this study, we used DNA methylation profiling to characterise non-fluorescent glioblastomas. Patients with newly diagnosed and recurrent IDH-wildtype glioblastoma that underwent surgery were analysed. The intensity of intraoperative 5-ALA fluorescence was categorized as non-visible or visible. DNA was extracted from tumors and genome-wide DNA methylation patterns were analyzed using the Illumina EPIC (850k) arrays. Furthermore, 5-ALA intensity was measured by flow cytometry on human gliomasphere lines (BT112 and BT145). Of 74 included patients, 12 (16.2%) patients had a non-fluorescent glioblastoma, which were compared to 62 glioblastomas with 5-ALA fluorescence. Clinical characteristics were equally distributed between both groups. We did not find significant differences between DNA methylation subclasses and 5-ALA fluorescence (p = 0.24). The distribution of cells of the tumor microenvironment was not significantly different between the non-fluorescent and fluorescent tumors. Copy number variations in EGFR and simultaneous EGFRvIII expression were strongly associated with 5-ALA fluorescence since all non-fluorescent glioblastomas were EGFR-amplified (p < 0.01). This finding was also demonstrated in recurrent tumors. Similarly, EGFR-amplified glioblastoma cell lines showed no 5-ALA fluorescence after 24 hours of incubation. Our study demonstrates an association between non-fluorescent IDH-wildtype glioblastomas and EGFR gene amplification which should be taken into consideration for recurrent surgery and future studies investigating EGFR-amplified gliomas.