Abstract Background: Many guidelines recommended multi-gene assays to determine an indication of postoperative chemotherapy for estrogen receptor-positive (ER+)/HER2-negative (HER2−) breast cancer patients. The 21-gene signature assay (Oncotype DX) is one such multi-gene assay and is recommended by the National Comprehensive Cancer Network (NCCN) Guidelines for Breast Cancer. The TAILORx trial determined that chemotherapy was indicated for patients with a high risk of recurrence score (high RS; ≧26) on the 21-gene signature assay. Although chemotherapy improves the prognosis of patients in the high-RS group, there is a subgroup of these patients who relapse after chemotherapy. It is expected that survival of this subgroup would improve with additional treatment, such as with CDK4/6 inhibitors. However, factors that predict relapse after chemotherapy in the high-risk patients are unknown. The aim of this study was to identify clinical factors that might predict relapse in the high-RS patients who underwent postoperative chemotherapy. To this end, we focused on tumor infiltrating lymphocytes (TILs) and neutrophil-lymphocyte ratio (NLR) in peripheral blood, and whether these factors are useful in predicting prognosis. Methods: A retrospective analysis of 48 patients who had the 21-gene signature assay performed postoperatively in our institute was performed. Of the 48 patients, 38 were in the low-RS group (1-25), and 10 were in the high-RS group (≧ 26). The cutoff values for TILs and NLR were set to 10% and 2.72, respectively, and the correlations with disease-free survival (DFS) were examined. Results: During the follow-up period (median, 1614 days), six out of 48 patients had distant metastases. Of these, four were in the low-RS and two were in the high-RS group. There was a significant correlation between TILs and RS (median and range of RS, 17.5, 6-47, for TILs-low; and 24, 15-66, for TILs-high; p = 0.0032), and TILs were higher in the high-RS group than in others. There was no correlation between NLR and RS (p = 0.215). In the RS-high patients, DFS was marginally (p = 0.0701) and significantly (p = 0.0253) shorter for TILs-low and NLR-high subgroups, respectively. In the high-RS group, none of six patients with high TILs relapsed, and two of four patients with low TILs relapsed. Conclusions: A subgroup of high-RS ER+/HER2− breast cancer patients with low TILs or high NLR may have a poorer prognosis. TILs are known to correlate with the effect of chemotherapy, and chemotherapy may be less effective in the low-TILs group, even with high-RS scores. TILs and NLR may be useful in determining the need for additional treatment in the high-RS group. Citation Format: Haruka Kanaoka, Masayuki Nagahashi, Eri Ishikawa, Ayako Bun, Reiko Fukui, Hiromi Ozawa, Tomoko Higuchi, Keiko Natori, Michiko Imamura, Yuichi Takatsuka, Yasuo Miyoshi. Clinical impact of tumor infiltrating lymphocytes and neutrophil-lymphocyte ratio in estrogen receptor-positive/HER2-negative breast cancer patients with high 21-gene signature recurrence scores [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 P4-07-26.