Background: Depatux-M is a tumor-specific antibody-drug-conjugate consisting of an antibody (ABT-806) bound to the toxin monomethylauristatin-F. In the primary analysis on EORTC 1410 we reported a trend (p = 0.06) towards improved overall survival (OS) in patients with EGFR-amplified (amp) recurrent glioblastoma treated with Depatux-M in combination with TMZ. Methods: Eligible were patients with centrally confirmed EGFRamp glioblastoma at 1st recurrence after TMZ chemo-irradiation, occurring ≥3 months after radiotherapy. Patients were randomized to either a) Depatux-M 1.0 mg/kg every 2 weeks intravenously, or b) the same treatment combined with TMZ 150-200 mg/m² day 1-5 every 4 weeks, or c) either LOM or TMZ (TMZ/LOM) depending on the time of relapse. Primary endpoint was OS. Pharmacokinetic (PK) sampling was done on day (d) 1 before and after dosing, d 4-7, d 1 course 2 before and after dosing, d 5-7 course 4, d 1 course 3, and then every 2 cycles. All available PK samples were used to calculate the Depatux-M average concentration during course 1 (CavgC1). The level of EGFRamp was determined using both qPCR, next generation sequencing and FISH. Results: An updated OS comparison of Depatux-M in combination with TMZ versus TMZ/LOM with longer follow-up, performed after 220 observed deaths using log-rank test and cox models stratified by stratification factors at randomization showed a HR of 0.68 (95%CI [0.48, 0.95]; p = 0.024) and 1-year OS rates of 40% versus 28%. In multivariate analysis including performance status, MGMT, surgery for the recurrence, time from last TMZ to relapse and lesion diameter, CavgC1 was a significant predictor for OS (HR 0.96, 95% CI [0.93, 0.98], p = 0.0013). In Depatux-M treated patients, EGFR status (high vs low level amplification) did not correlate with OS. Conclusions: This updated OS analysis of Depatux-M in combination with TMZ confirmed the OS improvement in EGFRamp recurrent glioblastoma. In Depatux-M treated patients, higher drug levels during course 1 were associated with improved OS but high levels of EGFR amplification at first diagnosis were not. Clinical trial information: NCT02343406