Transbronchial Biopsy Needle Rinse Solution Used for Comprehensive Biomarker Testing in Patients with Lung Cancer Yuichi Sakairi, MD, PhD, Kenichi Sato,

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Transbronchial Biopsy Needle Rinse Solution Used for Comprehensive Biomarker Testing in Patients with Lung Cancer Yuichi Sakairi, MD, PhD, Kenichi Sato, PhD, Sakae Itoga, PhD, Fumie Saegusa, CT, Kazuyuki Matsushita, MD, PhD, Takahiro Nakajima, MD, PhD, Shigetoshi Yoshida, MD, PhD, Yuichi Takiguchi, MD, PhD, Fumio Nomura, MD, PhD, Ichiro Yoshino, MD, PhD Journal of Thoracic Oncology Volume 9, Issue 1, Pages 26-32 (January 2014) DOI: 10.1097/JTO.0000000000000024 Copyright © 2014 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 1 Sampling sequence for primary lesions and metastatic nodes. For primary lesions, TBNA with needle and TBFB was performed by using conventional bronchofiberscopy. For metastatic nodes, EBUS-TBNA was performed. Used needles were washed in saline and divided into three bottles, and these uMSs were used for bacterial culture, cytological analysis, and molecular analysis. All samples in this study were confirmed as malignant cells in each modality by rapid onsite cytological evaluation. TBNA, transbronchial needle aspiration; TBFB, transbronchial forceps biopsy; EBUS, endobronchial ultrasound-guided; uMS, ultra-microsamples. Journal of Thoracic Oncology 2014 9, 26-32DOI: (10.1097/JTO.0000000000000024) Copyright © 2014 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 2 Case enrollment. From 149 samples, 15 samples were omitted because of the pathological/cytological final evaluation. Only non–small-cell lung cancer samples were enrolled in this study. Ad, adenocarcinoma; Sq, squamous-cell carcinoma; TBNA, transbronchial needle aspiration; TBFB, transbronchial forceps biopsy; EBUS-TBNA, endobronchial ultrasound-guided transbronchial needle aspiration; uMS, ultra-microsample. Journal of Thoracic Oncology 2014 9, 26-32DOI: (10.1097/JTO.0000000000000024) Copyright © 2014 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 3 DNA and cDNA analyses for EGFR, KRAS, and BRAF mutation and ALK, RET, and ROS1 fusion genes. HRM analysis and direct sequencing of EGFR-, KRAS-, and BRAF-positive samples are shown (A–C). The blue baseline shows the HRM curve of the wild-type sample, and the red curves show the existence of each mutation (A: EGFR exon21, L858R; B: KRAS exon12, G12D; C: BRAF V600E) by HRM analysis. Loci were identified by direct sequencing by using the PCR products from HRM analysis. HRM analysis and direct sequencing of EML4-ALK–, CCDC6-RET– and SCL34A2-ROS1–positive samples are shown (D–F). D, HRM curves for EML4-ALK variant 1 (uMS) and variant 2 (histological core and uMS) and the results of direct sequencing. Each variant showed a different curve. E, HRM curve and sequencing for CCDC6-RET fusion gene–positive samples. F, HRM curve for the TPM3-ROS1 fusion sequence (artificial sequence for the positive control) and HRM curve for the detected SCL34A2-ROS1 fusion gene. The sequence of the SCL34A2-ROS1 fusion gene was identified. EGFR, epidermal growth factor receptor; ALK, anaplastic lymphoma kinase; ROS1, receptor tyrosine kinase; uMS, ultra-microsample; HRM, high-resolution melting; PCR, polymerase chain reaction; KRAS, K-rat sarcoma; BRAF, B-rapidly accelerated fibrosarcoma. Journal of Thoracic Oncology 2014 9, 26-32DOI: (10.1097/JTO.0000000000000024) Copyright © 2014 International Association for the Study of Lung Cancer Terms and Conditions