Background: Depatuxizumab mafodotin (depatux-m, formerly ABT-414) is an EGFR-directed antibody-drug conjugate being developed for treatment of EGFR-amplified glioblastoma (GBM). As therapeutic pressure engenders tumor adaptations, it is important to understand the stability of biomarkers targeted by precision medicine approaches such as depatux-m. Therefore, we assessed EGFR amplification (amp) and expression in longitudinally-sampled GBMs from patients (pts) treated +/- depatux-m to explore biomarker stability. Methods: Formalin-fixed, paraffin embedded GBM tumor tissue was analyzed from 68 patients who underwent at least 2 surgeries; EGFR amp was detected by in situ hybridization (e.g., FISH) or next-generation sequencing in all samples. Fifty-six pts did not receive depatux-m; among 12 pts who did, EGFR expression was also evaluated by RNA sequencing. Results: Of 56 pts who did not receive depatux-m, 31 (55%) had tumors harboring EGFR amp at 1^st surgery (initial diagnosis); among those, EGFR amp was maintained at re-operation in 27 (87%), and not maintained in 4 (13%). None of the 25 cases without baseline EGFR amp acquired it at the 2^nd surgery. Of 12 pts treated with depatux-m between surgeries, 9 cases harbored EGFR amp at baseline which was maintained in 4 (44%) at the 2^nd surgery, all 4 of which had the highest levels of EGFR expression at baseline. Of the 3 cases without EGFR amp at baseline, none acquired amplification at the 2^nd surgery, and all 3 had the lowest levels of EGFR expression at study start. Conclusions: The presence of EGFR amp in GBM tissue at baseline was maintained at 2^nd surgery in 87% of pts who received treatment other than depatux-m, and in 44% following depatux-m exposure. Therefore, depatux-m exposure appears to reduce EGFR amp maintenance (p = 0.0159 by Fisher’s exact test); accordingly, the therapeutic approach may influence EGFR status. In no case was EGFR amp acquired at recurrence, regardless of depatux-m therapy. Ongoing analyses of additional tumor samples will increase power and further examine concordance among EGFR amp assays.