Abstract Background: Pre-operative stereotactic breast irradiation (SPBI) improves targeting precision and has the potential to decrease treatment-related toxicity while downstaging or eliminating cancer. Accurate pCR prediction may allow omission of surgery. We are reporting initial interim results of the performance of multiparametric breast MRI in identifying pathologic response rate after single-fraction pre-operative SPBI in early stage estrogen receptor-positive (ER+), HER-2 negative breast cancer. Methods: In an ongoing single center prospective single-arm trial 22 patients with ER+/HER2-negative, cN0, unifocal, invasive breast cancer ≤3 cm underwent single-dose ablative SPBI, followed by definitive surgery per standard of care. Residual disease vs complete imaging response to SPBI was assessed on baseline and post-SPBI/pre-operative breast MRI. Pathologic response was categorized using MDACC Residual Cancer Burden (RCB) Categories. Wilcoxon rank sum test was used to assess the correlation of pathologic complete response (pCR) or RCB status on final path with MRI lesion size, volume, diffusion weighted imaging apparent diffusion coefficient (ADC) value, and when available, presence of residual enhancement on pre-operative MRI. MRI volume and ADC values were obtained using a dedicated CAD software. Area under the receiver operating Curve(AUC) was constructed to assess the diagnostic performance of pre-operative MRI on predicting pathologic response. Results Mean patient age was 64.9 (SD±7.3) years, tumor diameter 1.2(± 0.6) cm, pre-SPBI volume 6.4 ± 9.1 cm3, post SPBI volume 1.8 ± 5.6 cm3, ADC 0.91 ×10-3 mm2/s. At surgical pathology, 6(27.2%) achieved pCR/RCB-0, 9(41%) RCB-I, 7(31.8%) RCB-II. Mean baseline MRI size (1.2 ± 0.7 vs 1.3 ± 0.6, p=0.88), enhancement volumes (6.0±6.2 vs 7.9±9.9 cm3, p=0.59), DWI ADC values (1.1 ± 0.3 vs 0.9 ± 0.2 mm2/s, p-0.48) were not significantly different between tumors that showed pCR vs RCB I-II at surgical pathology. Time elapsed from SBRT to surgery were significantly longer in patients with pCR (264.3 ± 74.2 vs 183.7 ± 68.3 days, p=0.04) On pre-op MRI, mean enhancement volume was smaller in cancers that had pCR, however it did not reach significance (0.1 ± 0.1 vs 2.1 ± 6.0 cm3, p=0.2). Of 18 patients with available pre-operative MRI, residual enhancement was present in 7, of these 6/7(87%) had RCB I-II, and 1/7(13%) RCB-0 at surgical pathology. Conversely, in 11 patients with no residual enhancement on MRI, pathology showed pCR/RCB-0 in 5(45.5%), while 6(54.5%) had RCB I or II. Presence of residual enhancement on pre-operative MRI had an AUC of 0.68 (95%CI 0.43; 0.93, p=0.2) in predicting residual cancer after SPBI. Conclusion: Detecting residual enhancement on pre-operative MRI is a strong indicator of residual disease after SPBI, while lack of enhancement is not a reliable predictor of pCR. Our preliminary results do not indicate an association between baseline MRI enhancement volume, DWI ADC and pathologic response. Citation Format: Baṣak Dogan, Sunati Sahoo, Marilyn Leitch, Prasanna Alluri, Robert Timmerman, Deborah Farr, Asal Rahimi. MRI response Assessment of Single Fraction Pre-operative Stereotactic Radiotherapy: Preliminary results [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 PO2-22-06.