Abstract Objective(s): To explore the impact of pre-operative single fraction stereotactic ablative partial breast irradiation (SPBI) dose escalation (30, 34, or 38Gy) on toxicity and tumor response for early-stage hormone receptor (HR)+ breast cancer in an interim analysis of an expanded cohort phase I dose escalation study (NCT04040569). Methods: Eligible patients (pts) have < 3 cm, HR+, Her2 -, cN0 invasive breast carcinomas not requiring chemotherapy. Patients are treated on either MR LINAC, robotic radiosurgery, or cobalt stereotactic breast units. Endocrine therapy is started two weeks after SPBI. Surgery is completed 2-12 months after SPBI. The primary objective is to escalate single fraction SPBI to an ablative dose without exceeding maximum tolerable dose (MTD). Secondary endpoints include pathologic complete response (pCR), local control, toxicity, cosmesis, and distant disease-free survival. Near complete response (nCR) is defined as RCB 1 and Miller-Payne 4/5. Dose limiting toxicity (DLT) is defined as grade ≥3 toxicity or any grade 4/5 toxicity attributed to SPBI. Each dose cohort enrolls 7-15 pts. Dose escalation is permitted if 0/7, 2/9, ≤3/12, or ≤4/15 patients experienced a DLT within 90 days of SPBI. MTD is exceeded if more DLTs occur in any cohort. Results: From 12/2019 to 6/2023, 11 and 15 pts were treated with 30Gy and 34Gy, respectively. Rates of pCR/nCR are 37.5% for 30Gy versus 92.8% 34 Gy (p=0.01). At 30Gy, 8/11 pts (73%) underwent surgery with a median 4.3 (range 2.8-5.9) month interval from SPBI to surgery: 0/8 (0%) had a pCR and 3/8 (37.5%) had a nCR. At dose level 34Gy, 14/15 pts (93%) underwent surgery with a median 7.3 (range 5.9-12) month interval from SPBI to surgery: 6/14 (42.8%) had a pCR while 7/14 (50%) had a nCR. Of the 8 pts with a nCR, 50% had only 1-3mm of residual disease. The mean ki67 for the entire cohort was 12.0% +/- 6.9% at diagnosis and decreased to 1.4 +/-2.3% at surgery. 13/14 (92.8%) pts with residual disease had a ki67 < 3% after surgery and SPBI. There were 33 acute grade 1; 2 acute grade 2 (breast pain and dermatitis); and 10 late grade 1 [1 grade 2 (breast pain), and 1 grade 3 (slow healing wound) in an uncontrolled diabetic] toxicities. Conclusion: First study to show pre-operative SPBI up to 34Gy in a single fraction was safe and effective for early-stage HR+ breast cancer. Escalating the dose has achieved a dramatic improvement in pCR/nCR (92.8%) suggesting this is an exciting approach for potentially eliminating tumor with radiation/endocrine therapy alone in early stage breast cancer and potentially paving a path towards non-surgical management in highly selected patients. Citation Format: Asal Rahimi, A Marilyn Leitch, Baṣak Dogan, Prasanna Alluri, Deborah Farr, Mona Arbab, Stephen Seiler, Nathan Kim, Rachel Wooldridge, Nisha Unni, Chika Nwachukwu, Ina Patel, You Zhang, David Parsons, Anvy Nguyen, Howard E Morgan, Heather McArthur, Sunati Sahoo, Robert Timmerman. Early Results of a Phase I Pre-Operative Single Fraction Ablative Trial for Early Stage Breast Cancer [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 PS01-09.