Abstract P5-01-03: Evaluation of Novel Diagnostic Kits Using the Semi-Dry Dot-Blot Method Combined with an Automatic Reader for Detecting Metastases in Sentinel Lymph Nodes of Patients with Breast Cancer: a Multi-Center Prospective Study

Abstract Background: The semi-dry dot-blot (SDB) method, a diagnostic procedure for detecting lymph node (LN) metastases using an anti-cytokeratin (CK) antibody, is based on the theory that epithelial components, such as CK, are not found in normal LNs. Thus, metastases are diagnosed according to the presence of CK in the lavage fluid of sectioned LNs. We prospectively evaluated novel SDB kits that use a newly developed anti-CK19 antibody and an automatic reader for diagnosing sentinel LN metastases in patients with breast cancer as a multi-center study. Methods: We obtained 924 sentinel LNs dissected from 405 patients with breast cancer between January 2021 and December 2021 at six institutes in Japan. We excluded patients who underwent neoadjuvant chemotherapy and neoadjuvant endocrine therapy. LNs were sectioned at 2-mm intervals and washed with phosphate-buffered saline. Cells suspended in the lavage fluid of sectioned LNs were centrifuged and lysed to extract protein. The extracted protein was applied to the SDB kit to diagnose LN metastasis using an automatic absorbance reader. Hematoxylin and eosin (H&E) stained and washed LNs were blindly examined by pathologists using intraoperative and permanent histological examination. Diagnoses based on SDB kit and automatic reader findings were compared with diagnoses made by permanent histological examination of paraffin-embedded H&E-stained sections of LNs. Primary endpoints were the sensitivity, specificity, and overall agreement of the SDB kit for distinguishing macrometastases from non-macrometastases. Results: Ninety-four of the 924 LNs were assessed as macrometastases, 40 as micrometastases, and 790 as isolated tumor cells by histological examination. Compared with patients with non-macrometastases, those with macrometastases had significantly younger age (p< 0.01), larger primary tumor (p< 0.01), higher nuclear grade (p=0.04), increased lymphatic invasion (p< 0.01), and increased venous invasion (p=0.01). Using a borderline CK19 absorbance of 11.9 milli-absorbance for detecting macrometastases with an area under the curve of 0.989, the sensitivity, specificity, and overall agreement of the SDB kit were 94.7%, 98.3%, and 97.9%, respectively. Moreover, the sensitivity, specificity, and overall agreement of the intraoperative histological examination compared with permanent histological examination for distinguishing macrometastases from non-macrometastases were 91.4%, 99.1%, and 98.3%, respectively. Furthermore, the kits and automatic reader yielded diagnoses within approximately 20 min at a cost of < 30 USD for the SDB kit and < 3,000 USD for the automatic reader. Conclusions: The kits with an automatic reader used in our study were accurate, quick, and cost-effective in diagnosing LN metastases without the loss of LN tissue and were particularly useful for distinguishing macrometastases. We plan to make the SDB kit and automatic reader commercially available worldwide soon. Citation Format: Megumi Matsumoto, Masaaki Baba, Aya Tanaka, Sayaka Kuba, Michi Morita, Fujiko Kaseida, Hiroko Hayashi, Shigeto Maeda, Hiroshi Yano, Eiko Inamasu, Akiko Ogiya, Kenta Sekiya, Masahiro Nakashima, Hideki Ikari, Shinji Ohno, Hiroyuki Takei, Katsunori Yanagihara, Susumu Eguchi, Takeshi Nagayasu, Ryota Otsubo. Evaluation of novel diagnostic kits using the semi-dry dot-blot method combined with an automatic reader for detecting metastases in sentinel lymph nodes of patients with breast cancer: a multi-center prospective study [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 P5-01-03.