Abstract Background: As a first-echelon nodal drainage site of breast cancer, the status of axillary lymph nodes (ALN) and internal mammary lymph nodes (IMLN) is both valuable for regional staging and treatment choice. The internal mammary sentinel lymph node biopsy (IM-SLNB) may provide minimally invasive staging, and guide individual IMLN radiation. Modified technique (periareolar intraparenchymal, high volume and ultrasound guidance) got a high internal mammary sentinel lymph nodes (IM-SLN) visualization rate of 71.1% in single center, and the prospective multicenter study was designed to verify its repeatability (CBCSG026, NCT03541278). High visualization rate and low false negative rate are prerequisites for the widespread of IM-SLNB. The question arises as to whether IM-SLN detected with the modified technique should be considered as “true” IM-SLN. The prospective, multicenter, clinical validation study of IM-SLNB followed by internal mammary lymph node dissection (IM-LND) was designed to verify the accuracy of IM-SLNB in patients with ALN positive breast cancer (CBCSG027, NCT03024463). Methods: While CBCSG026 trial enrolled patients with both axillary negative and positive breast cancer, CBCSG027 trial only enrolled axillary positive patients receiving mastectomy (either biopsy proving cN+ disease or cN0 with positive axillary SLN). The 1st to 3rd intercostal IM-LND was performed immediately after IM-SLNB to verify its accuracy in the CBCSG027 trial. Result: From May 2018 to June 2022, 600 and 264 patients were enrolled in the CBCSG026 and CBCSG027 trial from seven centers in China, respectively. Among the 600 recruited patients in the CBCSG026, the IM-SLN visualization rate was 65.0% (390/600), which was significantly related to patient’s age, body mass index, radiotracer intensity and interval time between injection and IM-SLN identification (all P< 0.05). The IM-SLNB successful rate was 97.4% (380/390), and the complication was 6.9%. The median number of IM-SLN was 1. The overall IM-SLN metastases rate was 18.9% (72/380), with 33.0% (65/195) and 3.8% (7/185) in ALN positive and negative patients, respectively. Multivariate analysis showed that the tumor size (P=0.028), the tumor location (P< 0.001) and the number of positive ALNs (P< 0.001) were independent predictors of IM-SLN metastasis. Those variables were included in a novel nomogram (Table 1), which was significantly better than the probability based on the number of metastatic ALNs alone according to the current guidelines (area under the curve: 0.860 vs. 0.804, P< 0.001). Of the 264 patients enrolled in the CBCSG027 trial, 185 patients (70.1%) had IM-SLN visualization (included 107 with cN+ disease and 78 with positive axillary SLN). The median number of IM-SLN and IM-nSLN was 2 (1~4) and 3 (1~9), respectively. The positive rate of IMLN and IM-SLN was 37.8% (70/185) and 36.8% (68/185), respectively, yielding the false negative rate of IM-SLNB 2.9% (2/70), the accuracy of 98.9% (183/185) and the sensitivity of 97.1% (68/70). The false negative rate of patients with cN+ disease and patients with positive axillary SLN was 4.8% (2/42) and 0, respectively. The positive IM-SLNs were the only positive IMLNs identified in 51.4% (36/70) patients. IM-SLNB can change the pN stages of 37.2% (68/183) patients. IMLN irradiation could be avoided in 72.7% (80/110) patients with axillary pN1 and 46.7% (35/75) with pN2/N3 disease in the study. Conclusions: The modified technique of radiotracer injection (periareolar intraparenchymal, high volume, and ultrasound guidance) can significantly improve the detection rate of IM-SLN with very low false-negative rate with the prospective, multicenter validation results, providing minimally invasive staging and guiding individual IMLN radiation. When there is no IM-SLN visualization, the nomogram can predict the risk of IMLN metastasis and guide IMLN radiation. The nomogram which can predict the risk of IMLN metastasis The nomogram which can predict the risk of IMLN metastasis Citation Format: Yong-Sheng Wang, Qing Lu, Shi-Guang Zhu, Wen-He Zhao, Guang-Lun Yang, Yuan-Xi Huang, Hong Zhong, Xiao Sun, Pengfei Qiu. Prospective, multicenter, clinical validation study of the repeatability and accuracy of internal mammary sentinel lymph node biopsy with modified injection technique (CBCSG026/CBCSG027) [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 P2-14-06.