Evaluation of a novel diagnostic kit using the semi-dry dot-blot method with an automated reader for detecting breast cancer metastases in sentinel lymph nodes: A combined analysis of two multicenter prospective studies in Japan.

Abstract

586 Background: The semi-dry dot-blot (SDB) method is a diagnostic procedure for detecting lymph node (LN) metastases using an anti-cytokeratin (CK) antibody, based on the premise that epithelial components such as CK protein are not present in normal LNs. Thus, the presence of CK protein in the lavage fluid of sectioned LNs indicates metastasis. Because this method does not involve loss of LN tissue, it can be used in parallel with conventional histopathological diagnosis. We developed a novel diagnostic kit utilizing the SDB method and a newly developed anti-CK19 antibody to detect macrometastases over 2.0 mm in diameter. Two multicenter prospective studies were conducted to evaluate the diagnostic performance of this kit, and we herein report a combined analysis of the two studies. Methods: A total of 825 sentinel LNs were dissected between January and December 2021, and 909 LNs between August 2023 and November 2024, at seven institutions in Japan from 880 patients with breast cancer. Patients who received neoadjuvant chemotherapy or endocrine therapy were excluded. LNs were sliced at 2-mm intervals and washed with phosphate-buffered saline. Cells in the lavage fluid were centrifuged and lysed, and the extracted proteins were applied to the SDB kit. Absorbance was measured using an automated reader to determine the presence of metastases. The washed LNs underwent blinded intraoperative and permanent histopathological examination. Diagnostic performance of the SDB kit was compared with permanent histology, with primary endpoints being sensitivity, specificity, and overall concordance for detecting macrometastases. Results: Of the 1,734 LNs analyzed, 199 were diagnosed as macrometastases and 1,535 as non-macrometastases, including 64 micrometastases measuring more than 0.2 mm and not up to 2.0 mm in diameter, based on permanent histopathology. Using a CK absorbance cutoff of 11.9, the SDB kit correctly identified 185 of the 199 macrometastatic LNs and 1,496 of the 1,535 non-macrometastatic LNs. This corresponded to a sensitivity of 93.0%, specificity of 97.5%, and overall concordance of 96.9%. Additionally, the kit enabled diagnosis within approximately 20 minutes, at a cost of under $30 per test and less than $3,000 for the reader device. Conclusions: The novel SDB kit with an automated reader demonstrated high accuracy, rapid turnaround, and cost-effectiveness in detecting breast cancer LN metastases, while preserving LN tissue. It proved particularly effective in identifying macrometastases. Commercial availability of the kit and reader is planned in the near future.