Elsevier

Lung Cancer

Volume 78, Issue 3, December 2012, Pages 212-218
Lung Cancer

An accurate and rapid detection of lymph node metastasis in non-small cell lung cancer patients based on one-step nucleic acid amplification assay

https://doi.org/10.1016/j.lungcan.2012.08.018Get rights and content

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.

Introduction

Lung cancer is characterized by early metastasis and lymphatic spread with poor survival, and is the leading cause of malignancy related deaths in many parts of the world. The American Cancer Society estimates that 226,160 individuals in the United States will be diagnosed with lung cancer and 160,340 will die in 2012. Surgical intervention is mainly indicated for stage I and II non-small cell lung cancer (NSCLC) without mediastinal lymph node metastasis, while adjuvant chemotherapy is the commonly used evidence-based treatment for p-stage II and IIIA [1]. Thus, accurate diagnosis of lymph node metastasis is vitally important for deciding proper treatment strategies.

Recently, sublobar resection, such as a segmentectomy or wedge resection has been recognized as an option for early small-sized NSCLC, though a lobectomy remains the standard procedure in affected patients [2], [3], [4], [5]. In cases treated by sublobar resection, intraoperative lymph node exploration is necessary to avoid incomplete resection, as a proportion of small-sized NSCLC may be locally advanced diseases [6], [7], [8]. The intraoperative evaluation of lymph node is conventionally histologically diagnosed by using frozen sections, while only maximum cut-surface is usually applied for the rapid diagnosis. A rapid and accurate nodal evaluation procedure using whole nodal specimen is expected in patients with NSCLC, for whom a sublobar resection is intended.

Molecular diagnosis of nodal metastasis has been attempted for various types of cancer, with detection of nodal involvement by RT-PCR reported to be more accurate as compared with conventional histological diagnosis in lung, esophageal, gastric, and colorectal carcinomas [9], [10], [11], [12], [13], [14], [15], [16], [17], [18]. As for lung cancer, quantitative RT-PCR has been applied to lymph node metastasis detection by combining several mRNA markers [13], [15]. However, an RT-PCR assay requires 2–3 h to obtain results and is not applicable for intraoperative diagnosis. In the present study, we evaluated the utility of one-step nucleic acid amplification (OSNA) assay and its optimal mRNA marker for lung cancer, which was developed as a rapid mRNA detection system and previously reported to detect lymph node metastasis in breast, gastric and colorectal cancer cases [19], [20], [21], [22]. This is the first report suggesting the potential clinical utility of OSNA for lung cancer management.

Section snippets

Patients and surgical specimens

A total of 165 lymph nodes from 49 NSCLC patients who underwent surgery between 2007 and 2011 at Osaka University Hospital and Osaka Medical Center for Cancer and Vascular Diseases were enrolled in the present study. Following approval by an institutional review board, written informed consent was obtained from each patient. Patient characteristics are shown in Table 1. Resected lymph nodes were divided into 2 halves for routine histopathology and further study, with the latter specimen being

Screening of candidate mRNA markers from microarray database

Candidate mRNA markers for the OSNA assay were selected by comparison with those expressed in adenocarcinomas, squamous cell carcinomas, and lymph nodes. The expression profiles by histological type are shown in Fig. 2. After marker screening using microarray expression data, 16 mRNA markers, including CK19, CK7, AGR2, SFN, MUC1, FXYD3, CEACAM6, TACSTD1, CK17, CK15, SCGB1A1, SFTPB, SFTPA2, SFTPC, CK6A, and CK6B, with more than 100-fold higher mRNA expression in adenocarcinoma or squamous cell

Discussion

The present results indicate the possible clinical utility of OSNA assay findings obtained using CK19 mRNA to detect lymph node metastasis in NSCLC. To the best of our knowledge, this is the first study to suggest the availability of OSNA for nodal diagnosis in lung cancer, though several reports have confirmed the usefulness of such an assay for lymph node diagnosis in cases of breast, gastric, and colorectal carcinomas [19], [20], [21], [22], with CK19 used as a target molecule for mRNA

Conflict of interest statement

Ms. Hiyama K, Mr. Nakabayashi K, Dr. Yoshida Y, Dr. Ding J, and Dr. Otomo Y are employed by Sysmex Corporation. Department of General Thoracic Surgery, Osaka University Graduate School of Medicine and Department of General Thoracic Surgery, Osaka Medical Center for Cancer and Cardiovascular Diseases received research support of $6000/year during study period. All authors contributing to this work have no other conflict of interest to declare.

Acknowledgment

This study was supported by grant-in-aid for scientific research from Japan Society for the Promotion of Science (no. 21591812).

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