title: An accurate and rapid detection of lymph node metastasis in non-small cell lung cancer patients based on one-step nucleic acid amplification assay.
abstract: A sublobar resection is currently recognized as an option for early small-sized non-small cell lung cancer (NSCLC), and intraoperative rapid and accurate lymph node assessment is required for a complete resection. To solve this issue, we investigated the clinical utility of one-step nucleic acid amplification (OSNA) assay, an automated rapid molecular diagnostic method and its optimal mRNA marker for detection of lymph node metastasis in lung cancer. We extracted 16 target candidate mRNA markers with high expression in lung cancer from a genetic database, and then quantified their expression levels by quantitative RT-PCR using surgically dissected lymph nodes with or without metastasis. Cytokeratin 19 (CK19), cytokeratin 7 (CK7), stratifin (SFN), and anterior gradient homolog 2 (AGR2) showed significant differences for mRNA expression between metastasis-negative and -positive lymph nodes in quantitative-RT-PCR screening. CK19 and CK7 were finally selected as potential target markers and were quantified using OSNA assay findings of 165 dissected lymph nodes obtained from 49 lung cancer patients. The OSNA assay with CK19 and CK7 were completed within 40 min and their positive predictive value, negative predictive value, and accuracy comparing to pathological diagnosis with hematoxylin-eosin staining and immunohistochemistry were shown to be 95.0%, 99.3%, and 98.8%, and 85.0%, 97.9%, and 96.4%, respectively, using a cut-off value of 250 copies/μL. Among the 165 lymph nodes tested, 1 false negative result was due to massive necrosis of cancer cells and 1 false positive was caused by the allocation bias of cancer cells in the sampling in patient with pleural dissemination. The best performance was observed when CK19 was used as a marker, while the addition of CK7 mRNA as a marker did not increase sensitivity or specificity. In conclusion, an OSNA assay using CK19 could be effective for molecular diagnosis of lymph node metastasis in lung cancer. This is the first report suggesting the potential clinical utility of OSNA assay for intraoperative rapid diagnosis of nodal status in lung cancer.
authors.affiliation: Department of General Thoracic Surgery, Osaka University Graduate School of Medicine, Suita, Japan. mi@thoracic.med.osaka-u.ac.jp
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Inoue
authors.forename: Kayo
authors.initials: K
authors.lastname: Hiyama
authors.forename: Kadzuki
authors.initials: K
authors.lastname: Nakabayashi
authors.forename: Eiichi
authors.initials: E
authors.lastname: Morii
authors.forename: Masato
authors.initials: M
authors.lastname: Minami
authors.forename: Noriyoshi
authors.initials: N
authors.lastname: Sawabata
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Shintani
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Nakagiri
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Susaki
authors.forename: Jun
authors.initials: J
authors.lastname: Maeda
authors.forename: Masahiko
authors.initials: M
authors.lastname: Higashiyama
authors.forename: Jiro
authors.initials: J
authors.lastname: Okami
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Yoshida
authors.forename: Junyi
authors.initials: J
authors.lastname: Ding
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Otomo
authors.forename: Meinoshin
authors.initials: M
authors.lastname: Okumura
mesh_terms: D000230:Adenocarcinoma / Q000175:diagnosis* / Q000378:metabolism / Q000556:secondary; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis* / Q000378:metabolism / Q000556:secondary; D005260:Female; D006801:Humans; D007432:Intraoperative Period; D053539:Keratin-19 / Q000235:genetics / Q000378:metabolism; D053552:Keratin-7 / Q000235:genetics / Q000378:metabolism; D008175:Lung Neoplasms / Q000175:diagnosis* / Q000378:metabolism / Q000473:pathology; D008198:Lymph Nodes / Q000378:metabolism / Q000473:pathology; D008207:Lymphatic Metastasis; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques; D009088:Mucoproteins; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015513:Oncogene Proteins; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
chemical_list: C502296:AGR2 protein, human; D014408:Biomarkers, Tumor; D053539:Keratin-19; D053552:Keratin-7; D009088:Mucoproteins; D015513:Oncogene Proteins; D011506:Proteins; D012333:RNA, Messenger