Abstract Background: Although the 2009 AJCC incorporated the internal mammary sentinel lymph node biopsy (IM-SLNB) concept, there has been little change in surgical practice patterns due to the low internal mammary sentinel lymph nodes (IM-SLNs) visualization rate (VR) with the traditional radiotracer injection technique (average 13%, 0-37%). In this study, various injection techniques were evaluated in term of the IM-SLNs VR in clinically axilla lymph nodes (ALNs) negative patients, and the impact of IM-SLNB on the diagnostic and prognostic value were analyzed both in clinically ALNs negative (NCT01642511) and positive patients (NCT01668914). Methods: 340 patients with T1-2 invasive breast cancer were enrolled. Clinically ALNs negative patients (n=293) were divided into 3 groups according to the study period and radiotracer injection technique. Group A: traditional technique (peritumoral intraparenchymal injection) for the initial 58 cases; Group B: periareolar intraparenchymal injection under the ultrasonography guidance in the latter 235 cases, the injection sites were chosen at the 6 and 12 o’clock positions 0.5-1.0 cm from areola (about 2.0-4.0 cm from the nipple). Group B was then separated into 2 groups according to the radiotracer injection volume: Group B1, low volume (<0.5ml/point, n=41); Group B2, high volume (≥0.5ml/point, n=194). Clinically ALNs positive patients (n=47) were managed as group B2. IM-SLNB was performed for patients with IM-SLNs visualized on preoperative lymphoscintigraphy and/or detected by intraoperative gamma probe. Results: Clinically ALNs-negative: The IM-SLNs VR was significantly higher by lymphoscintigraphy in Group B than that in Group A (63.0% vs. 13.8%, P<0.001), while the axillary VR was similar in these two groups (84.3% vs. 77.6%, P=0.227). The VR of sentinel lymph nodes was improved by the intraoperative gamma probe compared to lymphoscintigraphy: Group A (axilla: 77.6%→98.3%, P=0.001; internal mammary: 13.8%→15.5%, P=0.794) and Group B (axilla: 84.3%→98.7%, P<0.001; internal mammary: 63.0%→70.6%, P=0.078). Group B2 was found to have the highest IM-SLNs VR (74.2% vs. 53.7% Group B1, P=0.009). The successful rate of IM-SLNB was 95.8%, and arrived 100% after 20 cases learning curve. In the patients who underwent IM-SLNB, the IM-SLNs metastases rate was 8.7%, systemic treatment was changed only in 4.3%; however, radiotherapy treatment was changed in 8.7%. For the patients with upper inner quadrant tumor, systemic and radiotherapy treatment was changed in 8.8% and 20.6%. Clinically ALNs-positive: IM-SLNB was performed in all patients with drainage to IM-SLNs (VR: 70.2%, 33/47); the successful rate was 100 % (33/33). The IM-SLNs was positive in 21.2% patients, and the internal mammary radiotherapy could be guided with this IM-SLNB results. Conclusions: The modified injection technique (periareolar intraparenchymal, high volume and ultrasonography guidance) significantly improved the IM-SLNs VR, making the routine IM-SLNB possible in daily practice. IM-SLNB could provide individual staging, prognosis, and decision-making of the internal mammary radiotherapy for breast cancer patients, especially in positive ALNs and high-risk negative ALNs patients. Citation Format: Peng-Fei Qiu, Bin-bin Cong, Rong-Rong Zhao, Yan-Bing Liu, Guo-Ren Yang, Peng Chen, Yong-Sheng Wang. Internal mammary sentinel lymph node biopsy with modified injection technique: High visualization rate and accurate staging [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-01-22.