PS2-05-20: Evaluation of novel diagnostic kits for detecting metastatic lymph nodes in breast cancer patients treated with neoadjuvant chemotherapy using the semi-dry dot-blot method: A single-center prospective study

Abstract Background: The semi-dry dot-blot (SDB) method is a diagnostic technique used to detect lymph node (LN) metastases by identifying cytokeratin (CK) proteins in the lavage fluid of sectioned LNs. This method is based on the principle that CK, an epithelial marker, is absent in normal lymphatic tissue. We prospectively evaluated novel SDB kits incorporating a newly developed anti-CK19 antibody and an automatic reader for diagnosing LN metastases in breast cancer patients. In a previous analysis of 1734 sentinel LNs (SLNs) from patients not receiving neoadjuvant chemotherapy (NAC), the kit demonstrated a sensitivity of 93.0%, specificity of 97.1%, and overall agreement of 96.7% for detecting macrometastases (>2.0 mm). However, the impact of NAC—especially in triple-negative and HER2-positive subtypes—on the diagnostic performance of the SDB method remains unclear, given that chemotherapy can cause cancer cell degeneration. This study aimed to evaluate whether the SDB kit could accurately detect macrometastases in patients undergoing NAC. Methods: A total of 127 LNs were collected from 74 breast cancer patients who underwent NAC followed by surgery between May 2022 and December 2024 at Nagasaki University Hospital. Both patients with and without axillary LN metastases were eligible. Eligible LNs were defined as all SLNs in patients who underwent SLN biopsy and two axillary LNs in patients who underwent axillary LN dissection. Each LN was sliced at 2-mm intervals and rinsed with phosphate-buffered saline. The resulting lavage fluid was centrifuged, and the cells were lysed to extract protein, which was then analyzed using the SDB kit and automatic absorbance reader. Pathologists, blinded to the SDB results, independently diagnosed LN metastasis using hematoxylin and eosin (H&E) staining. SDB-based diagnoses were compared with permanent histopathological diagnoses. Results: Among the 127 LNs, 56 were obtained via SLN biopsy and 71 via axillary LN dissection. Histological evaluation identified 16 macrometastases and 111 non-macrometastases (including 4 micrometastases and 1 case with isolated tumor cells). Using a CK19 absorbance cutoff of 11.9 milli-absorbance units (mAbs) for detecting macrometastases, the SDB kit correctly identified 15 of the 16 macrometastases and all 111 non-macrometastases. The resulting sensitivity, specificity, and overall agreement were 93.8%, 100%, and 99.2%, respectively. The single false-negative case involved a sparsely distributed residual tumor measuring 2.1 mm. The diagnostic process required approximately 20 minutes and cost less than USD 30 per kit. Conclusions: The novel SDB kits combined with an automatic reader provided rapid, accurate, and cost-effective detection of LN macrometastases without tissue loss, even after NAC. These findings support the potential utility of this method in clinical decision-making. A prospective multi-center study is planned to further validate clinical performance. Citation Format: R. Otsubo, Y. Hara, S. Urakawa, A. Tanaka, M. Akashi, S. Kuba, M. Matsumoto, R. Yamaguchi, K. Matsumoto. Evaluation of novel diagnostic kits for detecting metastatic lymph nodes in breast cancer patients treated with neoadjuvant chemotherapy using the semi-dry dot-blot method: A single-center prospective study [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-05-20.