Abstract Background: Despite the advances by targeted therapies, particularly in HER2-positive and hormone receptor (HR)-positive metastatic breast cancer (MBC), chemotherapy remains a mainstay of the treatment in all breast cancer subtypes. Response probabilities to chemotherapy seem to decrease with increasing lines of treatment, but little is known about the response dependency on the previous therapy line. Here, we present data from the MBC registry of the Austrian Study Group for Medical Tumor Therapy (AGMT-MBC-Registry). Methods: The AGMT-MBC-Registry is an ongoing multicenter registry for MBC patients in Austria. Patients with available hormone receptor and negative HER2 status, at least two consecutive lines of chemotherapy and sufficient progression-free survival (PFS) data were included in this analysis. Multivariable adjusted hazard ratios (HR) for PFS were estimated by Cox regression models and included the following parameters: age, HR status, disease-free survival (de novo metastatic or ≥ 24 months vs. < 24 months), visceral disease, number of involved metastatic sites at diagnosis of MBC, chemotherapy treatment line, and response in previous treatment line. Response was defined as PFS > 4 month and non-response as a PFS ≤ 4 month. Cox model was extended as proposed by Andersen and Gill due to possibility of multiple events per patient and thereby resulting dependencies (Sousa-Ferreira & Abreu, 2019). Results: As of 15/04/2021, 2024 patients were included in the registry. Out of them, 405 patients (20.0%) had documented HER2 negative disease with at least two lines of consecutive chemotherapy. Median PFS for first-, second-, third-, and forth-line chemotherapy were 6.0 months (95% CI 5.5-6.5), 3.7 months (95% CI 3.1-4.3), 2.9 months (95% CI 2.5-3.2), and 2.8 months (95% CI 2.6-3.2), respectively. Median overall survival was 31.8 months (95% CI 27.2-34.8). Median number of chemotherapy lines was 3 (range 2-8). The most commonly used cytotoxic agents were paclitaxel and capecitabine in first- and second-line, and eribulin in third-, and fourth-line chemotherapy. Combinations of more than one cytotoxic drug were given in 36%, 29%, 28% in first-, second-, and third/fourth-line. Patients who did not respond to the previous therapy line, had a response in 43% (95% CI 33-53), 34% (95% CI 25-43), and 30% (95% CI 21-39) to second-, third-, and fourth-line. In those patients, investigator assessed overall response rate (partial remission or complete remission) was 27% (95% CI 13-40), 23% (95% CI 9-37), and 33% (95% CI 16-51), respectively. A response to third-line chemotherapy in patients who did not respond to first- and second-line therapy, was seen in 16.7% of patients. Ratios between PFS of interest and PFS of the previous treatment line were 0.63 (95% CI 0.55-0.68), 0.57 (95% CI 0.41-0.62), and 0.76 (95% CI 0.58-0.9) in second-, third-, and forth-line therapy. In multivariable analysis, responses in second- to fourth-line therapy were statistically significant associated with disease-free survival (HR 0.75, P=0.022), and treatment line (HR 1.17 per increasing treatment line, P=0.004), but not for the response to the previous treatment line (HR 0.85, P=0.068). Conclusion: A continuously decreasing PFS for cytotoxic drugs from treatment line to treatment line was confirmed in our analysis. However, in multivariable analysis, response, defined as PFS > 4 months, was not statistically associated with response to the previous treatment line. Therefore, a lack of response to first, second-, - or third-line chemotherapy should not trigger discontinuation of anticancer therapy in case of further available treatment options. Citation Format: Gabriel Rinnerthaler, Simon P Gampenrieder, Christoph Tinchon, Andreas Petzer Petzer, Marija Balic, Heibl Sonja, August F Zabernigg, Daniel Egle, Margit Sandholzer, Florian Roitner, Johannes Andel, Petra Pichler, Christophe Hager, Michael Hubalek, Michael Knauer, Christian F Singer, Richard Greil. Response pattern to chemotherapy in metastatic breast cancer (MBC): Real-word data from the Austrian AGMT_MBC-Registry [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-16-03.