Abstract P4-02-04: Evaluation of novel diagnostic kits for detecting metastatic lymph nodes in breast cancer patients using the semi-dry dot-blot method combined with an automatic reader
Authors: Otsubo Ryota , Yano Hiroshi , Matsumoto Megumi , Fukushima Ayako , Yoon Han-Seung , Hirakawa Hiroshi , Oikawa Masahiro , Matsuda Katsuya , Nakashima Masahiro , Nagayasu Takeshi 
Published: March 2020

Abstract
Background: The semi-dry dot-blot (SDB) method is a diagnostic procedure for detecting lymph node (LN) metastases. Metastases are confirmed by the presence of cytokeratin (CK) in the lavage fluid of sectioned LNs using anti-CK antibody on the basis of the theory that epithelial components such as CK are not found in normal LNs. We evaluated novel SDB kits that use a newly developed anti-CK19 antibody and an automatic reader for diagnosing LN metastases in breast cancer patients. Methods: We obtained 127 LNs dissected from 78 breast cancer patients between January 2017 and April 2019 at Nagasaki University Hospital, including 18 dissected axillary LNs and 109 sentinel LNs. These were sliced at 2-mm intervals and washed with phosphate-buffered saline. The suspended cells in the lavage fluid of sliced LNs were centrifuged and lysed to extract protein. The extracted protein was applied to the SDB kit to diagnose LN metastasis using an automatic reader that evaluates absorbance. The washed LNs were blindly diagnosed by pathologists using hematoxylin and eosin (H&E) staining and immunohistochemical (IHC) analysis with anti-CK19 antibody. Diagnoses based on absorbance were also compared with CK19 protein concentration detected by enzyme-linked immunosorbent assay (ELISA).Results: Of the 127 LNs, 52 were assessed as positive and 75 as negative by histological examination with H&E and CK19 IHC. With a borderline CK19 absorbance of seven milli-absorbance (mAbs) for detecting LN metastases excluding isolated tumor cells, the sensitivity, specificity, and accuracy of the SDB kit were 92.3%, 100%, and 96.9%, respectively. In four false-negative cases, there were two CK-19-negative breast cancers and two micrometastases with obvious heat denaturation. With a borderline CK19 absorbance of 50 mAbs for distinguishing macrometastases from micrometastases, the sensitivity, specificity and accuracy of the SDB kit were 81.8%, 97.6%, and 92.1%, respectively. Diagnoses based on the kits and CK19 protein concentration, as determined by ELISA, were well correlated (r=0.90). Furthermore, diagnosis was achieved in approximately 20 min using the kits, at a cost of less than 30 USD. Conclusions: The kits used in our study with an automatic reader were accurate, quick, and cost-effective in diagnosing LN metastases without the loss of LN tissue. However, measures should be taken for clinical use in CK19-negative breast cancer and regarding heat denaturation during surgery.
Citation Format: Ryota Otsubo, Hiroshi Yano, Megumi Matsumoto, Ayako Fukushima, Han-Seung Yoon, Hiroshi Hirakawa, Masahiro Oikawa, Katsuya Matsuda, Masahiro Nakashima, Takeshi Nagayasu. Evaluation of novel diagnostic kits for detecting metastatic lymph nodes in breast cancer patients using the semi-dry dot-blot method combined with an automatic reader [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-02-04.