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Volume 130, Issue 3, Pages 530-536 (September 2013) Detection of sentinel lymph node metastases in cervical cancer: Assessment of KRT19 mRNA in the one-step nucleic acid amplification (OSNA) method Satoshi Okamoto, Hitoshi Niikura, Kadzuki Nakabayashi, Kayo Hiyama, Maki Matoda, Nobuhiro Takeshima, Mika Watanabe, Satoru Nagase, Takeo Otsuki, Nobuo Yaegashi Gynecologic Oncology Volume 130, Issue 3, Pages 530-536 (September 2013) DOI: 10.1016/j.ygyno.2013.06.027 Copyright © 2013 The Authors Terms and Conditions

Fig. 1 (A) Protocol for determining optimal markers and cutoff values. Block a-prime was used for qRT-RCR or OSNA assays. Block c was used for histopathological examination. (B) Protocol for evaluating performance. SLNs were serially sectioned at two mm intervals perpendicular to the short axis. All cut surfaces were subjected to histopathological examination with H&E. Even numbered blocks were analyzed by OSNA. OSNA, one-step nucleic acid amplification. Gynecologic Oncology 2013 130, 530-536DOI: (10.1016/j.ygyno.2013.06.027) Copyright © 2013 The Authors Terms and Conditions

Fig. 2 (A) First selection results. Thirty-two mRNA markers were evaluated with qRT-RCR using a mixture of eight histopathologically positive and eight negative LNs. The LNs were obtained from SCC and AC. (B) Second selection results. Expression of mRNA markers in histopathologically positive and negative LNs. The selected mRNA markers [KRT19, KRT15, KRT17, KRT4, SERPINB5, SERPINB3, PI3, ANXA8, and FXYD3] were evaluated by qRT-PCR using 16 histopathologically positive (● and ▲) and 16 negative (○ and △) LNs. The LNs were obtained from SCCs (▲ and △) and ACs (● and ○). LN, lymph node; KRT, cytokeratin; SERPINB, serpin peptidase inhibitor, clade B member; PI, peptidase inhibitor; ANXA8, annexin A8; FXYD, FXYD domain-containing ion transport regulator; HPV, human papillomavirus; SCC, squamous cell carcinoma; AC, adenocarcinoma. Gynecologic Oncology 2013 130, 530-536DOI: (10.1016/j.ygyno.2013.06.027) Copyright © 2013 The Authors Terms and Conditions

Fig. 3 (A) KRT19 mRNA expression in the OSNA assay carried out under the protocol shown in Fig. 1A. (Column A) Histopathologically negative LNs removed from pN0 patients. (Column B) Histopathologically negative LNs removed from pN1 patients. (Column C) Histopathologically positive LNs. Dotted lines correspond to a cutoff value of 250copies/μL. Two of the 22 histopathologically positive LNs (from 2 patients) showed fewer than 250copies/μL (37 and 100copies/μL) of KRT19 mRNA. These 2 LNs were diagnosed by histopathological examination as isolated tumor cells. (B) Receiver operating characteristic curves depicting the sensitivities and specificities of serial cutoff values (25–5000copies/μL). KRT, cytokeratin; OSNA, one-step nucleic acid amplification; LN, lymph node; AC, adenocarcinoma. Gynecologic Oncology 2013 130, 530-536DOI: (10.1016/j.ygyno.2013.06.027) Copyright © 2013 The Authors Terms and Conditions