Abstract P2-01-23: Validation of diagnostic procedure for metastatic lymph nodes in breast cancer using a semi-dry dot-blot method and novel anti-cytokeratin 18+19 antibodies
Authors: Otsubo Ryota , Oikawa Masahiro , Hirakawa Hiroshi , Yano Hiroshi , Nagayasu Takeshi 
Published: May 2015

Introduction: Sentinel lymph node (SLN) biopsy is a common diagnostic procedure for breast cancer. However, because of a shortage of pathology specialists in Japan and discordance between intra-operative and final pathological diagnoses of SLN metastasis, new diagnostic modalities are desperately required. We previously reported a novel method of detecting metastasis in SLNs by a semi-dry dot-blot (SDB) method with 93.3% sensitivity, 96.9% specificity and 96.6% accuracy. Here, we evaluated the efficacy of the SDB method using novel anti-cytokeratin (CK) 18+19 antibodies to diagnose lymph node metastases in breast cancer.

Materials and methods: We obtained 73 lymph nodes dissected from 43 patients with breast cancer from July 2013 to May 2014 at Nagasaki University Hospital and the Japanese Red Cross Nagasaki Genbaku Hospital, including 55 sentinel lymph nodes and 18 dissected axillary lymph nodes, which were sliced at 2-mm intervals and washed with phosphate-buffered saline. This lavage fluid was used to diagnose lymph node metastasis by the SDB method; whereas the washed lymph nodes were blindly diagnosed by pathologists using with hematoxylin and eosin (H&E) stain. Suspended cells in the lavage fluid were centrifuged; the cell pellet was lysed with lysis buffer to extract protein, which was then challenged and visualized with anti-cytokeratin 18 and 19 antibodies, each at 1μg/ml and 0.1μg/ml to distinguish between micrometastases or isolated tumor cells (ITC) and macrometastases; and with chromogen on a dot-blot membrane. Diagnoses based on the SDB method were compared with their H&E counterparts. When the SDB method and H&E-based examinations did not agree, we examined specimens immunohistochemically with anti-cytokeratin18+19 antibodies.

Results: Of the 73 lymph nodes, 25 were assessed as positive and 48 as negative by the permanent pathological examination with H&E. The SDB method made correct diagnoses in all positive cases and 42 of the 48 pathologically negative cases. We found four micrometastases and one ITC in the positive cases, which were difficult to diagnose as positive at the lower antibody concentration, but were clearly positive at the higher concentration. When the 6 discrepant cases were examined immunohistochemically, we found two cases of ITC and one of micrometastasis. Sensitivity, specificity and accuracy of the SDB method in detecting cancer cells were 100% (95% confidence interval [95% CI]: 100–100%), 93.8% (95% CI: 86–101%) and 95.9% (95% CI: 91–100%), respectively.

Conclusions: The SDB method using anti-CK18+19 antibodies is simple and accurate for diagnosing lymph node metastases; estimating metastatic amount may be possible with different antibody concentrations. We are producing an SDB kit that uses these antibodies.

Citation Format: Ryota Otsubo, Masahiro Oikawa, Hiroshi Hirakawa, Hiroshi Yano, Takeshi Nagayasu. Validation of diagnostic procedure for metastatic lymph nodes in breast cancer using a semi-dry dot-blot method and novel anti-cytokeratin 18+19 antibodies [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-01-23.