Abstract Background The probability of axillary lymph node converting from positive to negative after neoadjuvant systemic therapy (NST) for breast cancer patients is approximately 40%. A variety of axillary lymph node biopsy surgeries based on sentinel lymph node biopsy (SLNB) can help to omit axillary lymph node dissection (ALND). The common disadvantages, however, are mainly the restrictions, such as the need for strict regulation of the use of radiotracers, the high number of retrieved lymph nodes required, and the need to purchase ancillary equipment. The more restrictions there are, the more they limit the spread and availability of the technique and, more importantly, reduce the number of patients who will benefit. In fact, for patients with metastatic lymph nodes, the sentinel lymph nodes must be included in the lymph nodes that have metastasized. If these metastatic lymph nodes can be accurately marked and precisely retrieved after NST, it is possible to assess whether the axillary lymph nodes are converted from positive to negative without the need for SLNB. Materials and methods Breast cancer patients with pathologically confirmed axillary lymph node metastases who were receiving NST were recruited for this study. Before NST, carbon nanoparticles suspension injections were injected into the cortex of the pathologically confirmed metastatic lymph node to stain the region where the metastatic lymph node was located. The lymph nodes in the stained region were retrieved by stained region lymph node biopsy (SrLNB) during the axillary surgery, followed by ALND, without the use of SLNB. Finally, the identification rate and false-negative rate (FNR) of SrLNB were statistically analyzed according to pathology data. Results A total of 159 patients were successfully enrolled in this study between September 24, 2020 and December 2, 2022, with an axillary pCR rate of 40.9% (65/159). The identification rate of SrLNB was 100% (159/159), with an overall FNR of 5.3% (5/94). Subgroup analysis revealed that in the subgroups of 1, 2, 3 and ≥4 SrLN, the FNR was 0%, 0%, 0% and 8.9% (5/56), respectively, whereas in the cN1, cN2 and cN3 subgroups the FNR was 7.7% (3/39), 2.6% (1/38) and 5.9% (1/17), respectively. The FNR indexes were within the safety threshold of 10% in all subgroups. Conclusion SrLNB has a high identification rate and a low FNR and is useful for screening patients with positive to negative axillary lymph nodes for omitting ALND. srLNB requires only the use of an affordable and radiation-free carbon nanoparticles suspension injection for marking, making the whole technique simpler and easier to disseminate. The data from this study also suggest that SLNB may not be mandatory for axillary biopsy in patients with positive axillary lymph nodes undergoing NST. Table 1. General characteristics of enrolled patients. Table 2. Analysis of factors affecting axillary pCR rates. Table 3. Analysis for the FNR of SrLNB. Citation Format: Jue Wang, Rui Chen, Cuiying Li, Hailing Zha, Qiannan Zhu, Lifeng Huang, Xiaoming Zha. Clinical study of stained region lymph node biopsy (SrLNB) in axillary surgery after neoadjuvant systemic therapy in patients with axillary lymph node positive breast cancer [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 PO2-23-06.