Abstract P3-01-18: Validation of a novel diagnostic kit using the semi-dry dot-blot method for detecting metastatic lymph nodes in breast cancer

Abstract

Abstract Background: The semi-dry dot-blot (SDB) method is a diagnostic procedure for detecting lymph node (LN) metastases. The metastases are visualized by the presence of cytokeratin (CK) with lavage fluid of sectioned LNs by anti-pancytokeratin antibody, based on the theory that epithelial components such as CK are not found in normal LNs. We previously reported 93.3% sensitivity, 96.9% specificity, and 96.6% accuracy for this method in detecting metastasis in sentinel LNs, compared with permanent pathological diagnosis in breast cancer. In this study, we evaluated a novel kit that applies the SDB method using the newly developed anti-CK19 antibody for diagnosing LN metastases in breast cancer. Methods: We obtained 141 LNs dissected from 81 breast cancer patients from July 2013 to April 2015 at Nagasaki University Hospital and the Japanese Red Cross Nagasaki Genbaku Hospital, including 33 dissected axillary LNs and 108 sentinel LNs, which were sliced at 2-mm intervals and washed with phosphate-buffered saline. The suspended cells in the lavage fluid of sliced LNs were centrifuged to collect the cell pellet and lysed with lysis buffer to extract protein. This extracted protein was used with the kit to diagnose LN metastasis. The washed LNs were blindly diagnosed by pathologists using hematoxylin and eosin (H&E) stain. Diagnoses based on the kit were compared with their H&E counterparts. Results: Of the 141 LNs, 57 were assessed as positive and 84 as negative by permanent pathological examination with H&E. Use of the kit resulted in correct diagnoses in 46 of the 57 pathologically positive cases and all of pathologically negative cases. Sensitivity, specificity and accuracy of the kit in detecting LN metastases were 80.7% (95% confidence interval [95% CI]: 75.6–80.7%), 100% (95% CI: 96.5–100%), and 92.2% (95% CI: 88.1–92.2%), respectively. In 11 false-negative cases, there were 9 micrometastases; therefore, sensitivity was 95.5% (95% CI: 90.1–95.5%) in cases of macrometastases. Diagnosis was achieved in approximately 20 min using the kit, reducing the diagnostic time by half compared with the original SDB method. The cost of this kit was within 8 USD, and we are currently developing an improved kit for the detection of smaller metastases. Conclusions: The kit in our study is accurate, quick, and cost-effective in diagnosing LN metastases without the loss of LN tissue. Its sensitivity in detecting macrometastases is excellent, which is important in clinical practice. Citation Format: Otsubo R, Hirakawa H, Oikawa M, Shibata K, Tanaka A, Matsumoto M, Yano H, Nagayasu T. Validation of a novel diagnostic kit using the semi-dry dot-blot method for detecting metastatic lymph nodes in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-01-18.