Abstract Background: Although the role of the 21-gene Breast Recurrence Score® assay is well established to predict response to adjuvant chemotherapy in the setting of node-negative hormone receptor (HR)-positive, HER2-negative breast cancers (BC), fewer studies have evaluated the assay in the neoadjuvant setting. Due to the correlation between a high Recurrence Score® (RS) result and pathological complete response (pCR), the Breast Recurrence Score assay has been used to aid in selecting between chemotherapy (CT) or endocrine therapy. We wanted to further understand the impact of the assay upon physician treatment recommendations and the use of chemotherapy in this patient cohort. Methods: We conducted a multicenter, prospective, observational study in patients with clinically node-negative HR-positive, HER2-negative BC with T2-T3 disease being considered for neoadjuvant therapy. Physicians were required to complete two questionnaires indicating treatment choice, including CT, endocrine therapy, or surgery, prior to and post availability of RS result. Patients were followed up for 6 months after commencement of neoadjuvant therapy. The primary objective was to evaluate the change in the physician’s recommendation for neoadjuvant CT prior to and post assay results. As a secondary objective, we also evaluated the impact of the RS result on physician’s expressed level of confidence. Results: A total of 70 patients were enrolled between April 2018 and November 2021 at five hospital centers, as part of the McPeak Sirois Group of Quebec. The median age of the cohort was 60 years (range, 30 to 79 years). 24.3 % (n=17) of the cohort consisted of patients aged < 50 years, and 75.7% (n=53) were ≥ to 50 years. 29.0% (n=20) of the patients had a RS < 16, 39.1% (n=27) had a RS between 16-25, and 31.9% (n=22) had a RS > 25. For the entire cohort, the RS result led to a net reduction in chemotherapy recommendation by 33.3% (OR (odds of having CT post-RS recommendation versus pre-RS recommendation) = 0.23 [95% CI: 0.12-0.44]; P< 0.0001), and 39.2% net reduction in the use of chemotherapy at 6-month follow-up (OR = 0.18 [95% CI: 0.09-0.35]; P< 0.0001). Furthermore, the RS result led to a 35.3% net reduction in physician recommendation of CT for patients < 50 years (OR = 0.19 [95% CI: 0.04-0.83]; P=0.027) and a 32.7% net reduction for patients ≥ 50 years (OR = 0.24 [95% CI: 0.11-0.50]; P=0.0001). For patients with a RS < 16, there was a reduction in CT recommendation by 75.0%, and by 44.4% for patients with a RS between 16 - 25 (OR = 0.15 [95% CI: 0.06-0.38]; P< 0.0001). Moreover, RS results led to an increase in confidence in physician treatment decisions for 59.4% of patients (OR = 12.53 [95% CI: 5.46-28.78]; P< 0.0001). Conclusion: We determined that the 21-gene Breast Recurrence Score assay altered neoadjuvant treatment decisions, leading to a reduction in the use of chemotherapy by about one-third, regardless of age. Additionally, the assay increased physician confidence in their treatment recommendation for about 60% of patients. This demonstrates the potential clinical utility of the assay to decrease the use of CT in the neoadjuvant setting amongst HR-positive, node-negative BC patients in Quebec. Citation Format: Mariya Yordanova, Lucas Sideris, Pierre Dubé, Jean-Francois Boileau, Julie Lemieux, Catalin Mihalcioiu, Sylvie Levesque, Marie-Claude Guertin, Erica Patocskai, Rami Younan, André Robidoux, Saima Hassan. The impact of the 21-gene Recurrence Score® assay upon physician treatment recommendations in the neoadjuvant setting in lymph node-negative breast cancer patients in a multicenter prospective study in Quebec [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-01-39.