Abstract P1-01-27: Novel diagnostic procedure of metastasis to the sentinel lymph node of breast cancer using a semi-dry dot-blot method
Authors: Otsubo R , Oikawa M , Shibata K , Hirakawa H , Yano H , Matsumoto M , Hatachi T , Nakao K , Hayashi T , Abe K , Kinoshita N , Nakashima M , Taniguchi H , Omagari T , Itoyanagi N , Nagayasu T 
Published: August 2014

Abstract
Background: Sentinel lymph node (SLN) biopsy is a common surgical procedure. However, novel diagnostic modalities are required because of a shortage of pathology specialists in Japan and discordance between the intraoperative and final pathological diagnosis of SLN metastasis. Efficient methods that use molecular markers for detecting SLN metastasis, such as one-step nucleic acid amplification, are commercially available, but special equipment (e.g., thermal cycler) is costly and the problem of false-negatives for CK19 non-expressing cells remains unresolved. Recently, we developed an easy, quick and cost-effective method for detection of cancer cells in lymph nodes by applying dot-blot analysis technology, called the “semi-dry dot-blot method (SDB method)”. The SDB method visualizes the presence of cancer cells with washing of sectioned lymph nodes by anti-pancytokeratin antibody (AE1/AE3) and chromogen on a dot-blot membrane. This method can detect 0.01 mg/mL protein extracted from cancer tissue and 20 suspension cells (MCF-7) in approximately 20 minutes. The current study evaluated the efficacy of our SDB method in the diagnosis of SLN metastasis in breast cancer.
Methods: (I) One hundred eighty dissected lymph nodes from 29 cases, including breast, lung, gastric and colorectal cancer, were analyzed. Each lymph node was sliced at the maximum diameter and washed by phosphate-buffered saline (PBS), and this lavage fluid (PBS) was used for diagnosis of LN metastasis by the SDB method, and washed lymph node was sent to pathological section for pathological diagnosis. The sensitivity, specificity and accuracy of the SDB method were determined to compare with the final pathology report.
(II) A multicenter prospective clinical trial was conducted, where 132 SLN samples from 78 cases of clinically node-negative breast cancer were analyzed. Each SLN was sliced at 2-mm intervals and washed by PBS. Lavage fluid of sliced SLNs was used for the diagnosis of SLN metastasis by the SDB method in a blinded manner. The sensitivity, specificity, accuracy and the time required for the SDB method were determined and compared with the intra-operative pathology report.
Results: (I) Lymph node metastasis was detected in 35 lymph nodes (19.4%). Comparison of the results between the final pathology and the SDB method showed complete concordance (accuracy: 100%).
(II) Of the 132 lymph nodes eligible for analysis, 13 (10.0%) were assessed as positive and 114 as negative by the SDB method and intra-operative pathological examination, with an accuracy of 96.2%. All pathologically positive nodes, which included one micro metastasis, were also detected by the SDB method; (sensitivity: 100%). One hundred fourteen of the 119 pathologically negative nodes were assessed as negative with the SDB method (specificity: 95.8%). The mean required times of 16 cases for intra-operative pathological diagnosis and simultaneous SDB method were 43.5 and 42.2 minutes, respectively.
Conclusions: The SDB method is simple, fast, accurate and cost-effective for the intra-operative diagnosis of SLN metastasis. Because there is no loss of lymph node tissue, the SDB method and pathological investigation can be performed simultaneously.
Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-01-27.