Abstract Background: An IRB-approved single arm prospective multi-institution trial was designed to determine the efficacy and outcome of single fraction 20 Gy intra-operative radiation therapy (IORT) using disposable balloon electronic brachytherapy at the time of breast conserving surgery for early-stage breast cancer (women at least 40 years old, infiltrating ductal carcinoma [IDC] or ductal carcinoma in situ [DCIS], single lesion no larger than 3 cm, pN0). Ipsilateral breast tumor recurrences (IBTR) at median 5-year follow-up, the primary protocol endpoint of the trial, would be analyzed with outcomes compared to reported whole breast radiation therapy (WBRT) results. Methods: Between May 2012–July 2018,1199 enrolled breast cancer patients at twenty-six national and international institutions were successfully treated per protocol with lumpectomy plus single 20 Gy fraction IORT using disposable balloon electronic brachytherapy. Data collection and retrospective chart review included demographics, treatment, histopathology, toxicity, IBTR (defined as recurrence in the lumpectomy cavity/index quadrant), and survival. Results: All subjects were successfully treated with a single 20 Gy fraction of IORT. IORT cohort characteristics of the treated subjects are summarized in Table 1. Sixty-six (5.5%) patients received subsequent unplanned risk-adjusted WBRT. At median 5.0-year follow-up (range 0.5 – 9 years), there were 42 (3.50%) IBTR. The original mean tumor size of patients with IBTR was 13.6mm (range 0.03 - 30mm). The mean time to IBTR was 47.6 months (range 12 – 98 months). There were 30 IBTR in patients originally diagnosed with IDC, and 12 IBTR among patients originally diagnosed with DCIS. Three patients originally diagnosed with IDC recurred as DCIS, while 4 patients with DCIS recurred as IDC. The remainder recurred with the same pathology as their original diagnoses. Sorting by 2017 ASTRO accelerated partial breast irradiation (APBI) criteria, there were 25 IBTR in patients categorized immediately following lumpectomy plus IORT as Suitable, 12 IBTR among patients categorized immediately following lumpectomy plus IORT as Cautionary, and 5 IBTR in patients categorized immediately following lumpectomy plus IORT as Unsuitable. Nine (0.75%) patients experienced new ipsilateral primary breast cancers, with 8 classified as Suitable and one as Cautionary using post-lumpectomy plus IORT ASTRO APBI criteria. Seventeen (1.4%) patients experienced acute serious adverse events (SAEs). These included 5 wound infections, 4 hematomas, 2 skin ulcerations, 2 cellulitis, 2 seromas, 1 skin necrosis, and 1 wound dehiscence. All SAEs resolved within 6 months of IORT. One patient died of breast cancer metastases at 3-year follow-up. One patient who was diagnosed with IBTR at 3-year follow-up died 3 years later from dementia. There were 45 unrelated patient deaths. Conclusions: At median 5.0-year follow-up, the 1199 early-stage breast cancer patients successfully treated in this multi-institution trial with a single 20 Gy fraction of IORT to the lumpectomy cavity at the time of partial mastectomy experienced an IBTR of 3.50%. This recurrence rate is acceptable when compared to the 0.9% - 2.5% IBTR reported for WBRT, given the benefits of IORT (convenience, decreased exposure to XRT, better cosmetic outcome, patient preference). Table 1. IORT cohort characteristics Citation Format: Barbara Schwartzberg, AM Nisar Syed, Ajay Bhatnagar, Sophia Rahman, Veronica Jones, Albert Chang, Todd Cockerham, Virginia Osborn, Robert Cohen, Charles Hodge, Christina Lopez-Penalver, Bapsi Chakravarthy, William Dooley, Chika Madu, Atsuko Okabe, Maen Farha, Andrea Madrigrano, Christopher Morrison, Geoffrey Neuner, Craig Wengler, Steven David, Katayoon Toosie, Bryon Stephens. Median Five-Year Follow-Up Results from the Multi-Institution Trial for the Treatment of Early-Stage Breast Cancer Using Intra-Operative Electronic Brachytherapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-22-04.