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One-step Nucleic Acid Amplification for Intraoperative Detection of Lymph Node Metastasis in Breast Cancer Patients

Authors' Affiliations: Departments of ¹ Pathology and ² Surgery, Osaka Police Hospital, ³ Department of Breast and Endocrine Surgery, Osaka University Graduate School of Medicine, Departments of ⁴ Pathology, ⁵ Clinical Laboratory, and ⁶ Surgery, Osaka Medical Center for Cancer and Cardiovascular Diseases, and ⁷ Department of Molecular Pathology, Osaka University Graduate School of Medicine and Health Science, Osaka, Japan; Departments of ⁸ Pathology and ⁹ Breast Surgery, Cancer Institute of the Japanese Foundation for Cancer Research, and Departments of ¹⁰ Pathology and ¹¹ Breast Surgical Oncology, St. Luke's International Hospital, Tokyo, Japan; Departments of ¹² Pathology II and ¹³ Surgery I, National Defense Medical College, Saitama, Japan; and ¹⁴ Central Research Laboratories, Sysmex Corp., Kobe, Japan

Requests for reprints: Nariaki Matsuura, Department of Molecular Pathology, Graduate School of Medicine and Health Science, Osaka University, 1-7 Yamadaoka, Suita, Osaka 565-0817, Japan. E-mail: matsuura{at}sahs.med.osaka-u.ac.jp.

Purpose: Detection of sentinel lymph node (SLN) metastasis in breast cancer patients has conventionally been determined by intraoperative histopathologic examination of frozen sections followed by definitive postoperative examination of permanent sections. The purpose of this study is to develop a more efficient method for intraoperative detection of lymph node metastasis.

Experimental Design: Cutoff values to distinguish macrometastasis, micrometastasis, and nonmetastasis were determined by measuring cytokeratin 19 (CK19) mRNA in histopathologically positive and negative lymph nodes using one-step nucleic acid amplification (OSNA). In an intraoperative clinical study involving six facilities, 325 lymph nodes (101 patients), including 81 SLNs, were divided into four blocks. Alternate blocks were used for the OSNA assay with CK19 mRNA, and the remaining blocks were used for H&E and CK19 immunohistochemistry–based three-level histopathologic examination. The results from the two methods were then compared.

Results: We established CK19 mRNA cutoff values of 2.5 x 10² and 5 x 10³ copies/µL. In the clinical study, an overall concordance rate between the OSNA assay and the three-level histopathology was 98.2%. Similar results were obtained with 81 SLNs. The OSNA assay discriminated macrometastasis from micrometastasis. No false positive was observed in the OSNA assay of 144 histopathologically negative lymph nodes from pN0 patients, indicating an extremely low false positive for the OSNA assay.

Conclusion: The OSNA assay of half of a lymph node provided results similar to those of three-level histopathology. Clinical results indicate that the OSNA assay provides a useful intraoperative detection method of lymph node metastasis in breast cancer patients.