Background: There remains an unmet need to identify prognostic and predictive molecular biomarkers in advance Ewing sarcoma (ES). We sought to assess the influence of MGMT promoter methylation status on response rate, time to progression (TTP), and overall survival (OS) following salvage irinotecan and temozolomide (IT) chemotherapy. Methods: Data of advanced ES patients, treated with IT chemotherapy from Jan, 2014 to Jan, 2020 were retrospectively collected. Patients were required to have previously received and progressed after vincristine, doxorubicin, and cyclophosphamide alternating with ifosfamide and etoposide (VDC-IE). MGMT promoter methylation status was assessed by Methylation Sensitive Restriction Enzyme quantitative-PCR (MSRE-qPCR) on non-decalcified Formalin-fixed paraffin embedded (FFPE) tissue using internally developed primers and the OneStep qMethyl Kit (ZYMO RESEARCH CORP). Responses were assessed by response evaluation criteria in solid tumors (RECIST v. 1.1). TTP and OS were assessed by the Kaplan-Meier method. Survival comparisons were performed by the Log-rank test. Results: Herein, we present data of the preliminary analysis of the first 18 patients who underwent MGMT promoter methylation testing. Patients had a median age of 18 years (range: 5-34 years), and were predominantly male (n=11; 61%). The primary tumor was located in the pelvis in 9 patients (50%), femur in 3 (17%), tibia in 2 (11%), kidney in 2 (11%), chest wall in one (6%), and scapula in one patient (6%). IT was given in a second (n=15) or third-line setting (n=3). At the time of initiation of IT, 13 patients (72%) had distant metastasis, and 5 (28%) had unresectable local progression in the pelvis (n=4) or chest wall (n=1). The mean percentage of MGMT promoter methylation was 29% (range: 3-98%). Five patients (28%) had methylated MGMT promoter, whereas the remaining had partially methylated (n=6; 33%) or unmethylated (n=7;39%) promoter. Five patients (28%) had objective response, with no observed difference according to MGMT promoter methylation (p=0.58 for comparison between methylated and unmethylated/ partially methylated). Median TTP was 4.9 and 2.2 months for patients with methylated and partially methylated/ unmethylated MGMT respectively; p=0.76. The corresponding median OS was 69.4 and 14.3 months in favor of the methylated group; p=0.3. Conclusions: This preliminary data suggests a possible prognostic role for MGMT promoter methylation, with a markedly extended median OS for the methylated group. Nevertheless, the median OS difference did not reach statistical significance in this preliminary analysis. We plan to report data of the final analysis after finalizing MGMT testing for the rest of our study patients.