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Early detection and efficient treatment modality of early-stage peripheral lung cancer is essential. Current nonsurgical treatments for peripheral lung cancer show critical limitations associated with various complications, requiring alternative minimally invasive therapeutics. Porphysome nanoparticle-enabled fluorescence-guided transbronchial photothermal therapy (PTT) of peripheral lung cancer was developed and demonstrated in preclinical animal models. Systemically administered porphysomes accumulated in lung tumors with significantly enhanced disease-to-normal tissue contrast, as confirmed in three subtypes of orthotopic human lung cancer xenografts (A549, H460, and H520) in mice and in an orthotopic VX2 tumor in rabbits. An in-house prototype fluorescence bronchoscope demonstrated the capability of porphysomes for in vivo imaging of lung tumors in the mucosal/submucosal layers, providing real-time fluorescence guidance for transbronchial PTT. Porphysomes also enhanced the efficacy of transbronchial PTT significantly and resulted in selective and efficient tumor tissue ablation in the rabbit model. A clinically used cylindrical diffuser fiber successfully achieved tumor-specific thermal ablation, showing promising evidence for the clinical translation of this novel platform to impact upon nonsurgical treatment of early-stage peripheral lung cancer. Cancer Res; 76(19); 5870-80. ©2016 AACR., Competing Interests: no conflicts to disclose., (©2016 American Association for Cancer Research.)

Objective: The aim of this study was to analyze the spectral features of the radiofrequency of lymph nodes during endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) and to determine its diagnostic value for detecting metastatic nodes in patients with lung cancer., Methods: Ultrasound spectrums of lymph nodes during EBUS-TBNA were retrospectively analyzed. A linear regression of frequency spectrum and the ultrasonic spectral parameters midband-fit, slope, and intercept were calculated. Mean values for these parameters within lymph nodes were computed. The cutoff values for each parameter for distinguishing metastatic vs benign lymph nodes were first determined within the training set; these cutoff values were then applied to the testing set for validation., Results: Overall, 362 lymph nodes (112 metastatic, 250 benign) were analyzed as the training set, and 284 lymph nodes (74 metastatic, 210 benign) were evaluated as the testing set. In the training set, all of the parameters showed a significant difference between metastatic and benign lymph nodes (P < .001). The metastatic nodes tended to show low midband-fit, high slope, and low intercept. When midband-fit and intercept were combined, the diagnostic accuracy was maximized in the training set. In the testing set, the combination of intercept and slope produced the highest diagnostic accuracy, with the following outcomes: sensitivity, 79.7%; specificity, 84.3%; diagnostic accuracy, 83.1%; positive predictive value, 64.1%; and negative predictive value, 92.2%., Conclusions: Metastatic lymph nodes possess unique ultrasonic spectrum features, and spectrum analysis can be used as a novel diagnostic tool for differentiating between benign and malignant nodes in patients with lung cancer., (Copyright © 2016 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.)

Background: Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) and anaplastic lymphoma kinase (ALK) inhibitors have dramatically changed the strategy of medical treatment of lung cancer. Patients should be screened for the presence of the EGFR mutation or echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion gene prior to chemotherapy to predict their clinical response. The succinate dehydrogenase inhibition (SDI) test and collagen gel droplet embedded culture drug sensitivity test (CD-DST) are established in vitro drug sensitivity tests, which may predict the sensitivity of patients to cytotoxic anticancer drugs. We applied in vitro drug sensitivity tests for cyclopedic prediction of clinical responses to different molecular targeting drugs., Methods: The growth inhibitory effects of erlotinib and crizotinib were confirmed for lung cancer cell lines using SDI and CD-DST. The sensitivity of 35 cases of surgically resected lung cancer to erlotinib was examined using SDI or CD-DST, and compared with EGFR mutation status., Results: HCC827 (Exon19: E746-A750 del) and H3122 (EML4-ALK) cells were inhibited by lower concentrations of erlotinib and crizotinib, respectively than A549, H460, and H1975 (L858R+T790M) cells were. The viability of the surgically resected lung cancer was 60.0 ± 9.8 and 86.8 ± 13.9% in EGFR-mutants vs. wild types in the SDI (p = 0.0003). The cell viability was 33.5 ± 21.2 and 79.0 ± 18.6% in EGFR mutants vs. wild-type cases (p = 0.026) in CD-DST., Conclusions: In vitro drug sensitivity evaluated by either SDI or CD-DST correlated with EGFR gene status. Therefore, SDI and CD-DST may be useful predictors of potential clinical responses to the molecular anticancer drugs, cyclopedically.

Objectives: Localization of small, non-visible and non-palpable subcentimetre nodules can be challenging during video-assisted thoracoscopic surgery (VATS). Intraoperative ultrasonography is an option for localization of such lesions, yet this technology has not been fully adapted to thoracic surgery. The objective of this study was to assess a newly developed thoracoscopic ultrasound for localization and biopsy of subcentimetre pulmonary nodules in animal models., Methods: A prototype convex probe ultrasound thoracoscope (XLTF-UC180, Olympus Medical Systems Corp.) was used in this study. Multiple 5% agar pseudo-tumours were created in porcine lungs (n = 10) and assessed for localization with different frequencies (5.0-12.0 MHz) in deflated lungs. The evaluated pseudo-tumours were divided into two groups based on the distinctness of the tumour margin on the ultrasound images and compared in terms of the size and depth of the tumours. The visualization of real tumours and the biopsy capability were assessed using rabbit VX2 lung tumour models (n = 7)., Results: The thoracoscopic ultrasound clearly visualized normal lung structures within a 1.5-cm depth including small vessels and bronchioles less than 5 mm in diameter in the completely deflated lung. Twenty-eight of 30 agar pseudo-tumours (93.3%) were successfully detected in deflated lungs (average size: 8.5 ± 2.1 mm; average depth: 7.4 ± 7.5 mm and depth range: 0-24.8 mm). Two tumours were not detected due to residual air surrounding the tumour. Higher frequency (12 MHz) tended to show more distinct margins of the targets. Indistinct tumours were located significantly deeper in the lung than the distinct tumours (14.11vs 2.42 mm), regardless of them being in a similar size range. VX2 tumours were identified as heterogeneous isoechoic lesions and adequate tissue sampling for diagnosis was achieved using a dedicated needle., Conclusions: The newly developed convex probe ultrasound thoracoscope was capable of localizing subcentimetre nodules in the porcine deflated lung as well as of obtaining sufficient sampling from lung tumours in the rabbit model, which may enable single-port VATS lung nodule biopsy in a human clinical setting. However, the depth of the tumours significantly influenced the quality of ultrasound images. Complete collapse of the lung and use of high frequency may facilitate achieving distinct visualization of the targets., (© The Author 2015. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Intraoperative imaging technologies including computed tomography and fluorescence optical imaging are becoming routine tools in the cancer surgery operating room. They constitute an enabling platform for high performance surgical resections that assure local control while minimizing morbidity. New contrast agents that can increase the sensitivity and visualization power of existing intraoperative imaging techniques will further enhance their clinical benefit. We report here the development, detection and visualization of a dual-modality computed tomography and near-infrared fluorescence nano liposomal agent (CF800) in multiple preclinical animal models of cancer. We describe the successful application of this agent for combined preoperative computed tomography based three-dimensional surgical planning and intraoperative target mapping (>200 Hounsfield Units enhancement), as well as near-infrared fluorescence guided resection (>5-fold tumor-to-background ratio). These results strongly support the clinical advancement of this agent for image-guided surgery with potential to improve lesion localization, margin delineation and metastatic lymph node detection., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objectives: There has been limited utility for laboratory tumor models to predict clinical performance of cancer drugs. Clinical drug trials usually recruit patients that have advanced disease, therefore preclinical tumor models that closely reflect this characteristic will be more reliable to test candidate drugs. We evaluated the use of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) to sample metastatic lymph nodes in patients to establish patient-derived tumorxenograft (PDX) models of advanced lung cancer., Materials and Methods: Cell suspensions from TBNA aspirates were implanted into the subcutaneous tissue of NSG (NOD scid) gamma) mice. The success rate of PDX establishment was associated with tumor histopathology and the cellularity and cytopathological diagnosis of the primary EBUS-TBNA samples., Results: From December 2011 to June 2012, 19 patients were enrolled in this study. Successful engraftment was achieved in 8/19 cases (42.1%). The duration between inoculation and tumor formation averaged 62.4 days (13-144 days). The engrafted tumors included 3 adenocarcinomas (3/12: 25%), 2 squamous cell carcinomas (2/3: 67%), 1 large cell carcinoma (1/1: 100%), and 2 small cell carcinomas (2/3: 67%)., Conclusion: EBUS-TBNA samples can be used for establishment of tumor xenograft model in immunodeficient mice., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: The use of near-infrared (NIR) fluorescence imaging with indocyanine green (ICG) for sentinel lymph node (SN) mapping has been investigated in lung cancer; however, this has not been fully adapted for minimally invasive surgery (MIS). The aim of our study was to develop a minimally invasive SN mapping integrating pre-operative electro-magnetic navigational bronchoscopy (ENB)-guided transbronchial ICG injection and intraoperative NIR thoracoscopic imaging., Methods: A NIR thoracoscope was used to visualize ICG fluorescence. ICG solutions in a 96-well plate and ex vivo porcine lungs were examined to optimize ICG concentrations and injection volumes. Transbronchial ICG injection (n=4) was assessed in comparison to a traditional transpleural approach (n=3), where after thoracotomy an ICG solution (100 μL at 100 μg/mL) was injected into the porcine right upper lobe for SN identification. For further translation into clinical use, transbronchial ICG injection prior to thoracotomy followed by NIR thoracoscopic imaging was validated (n=3). ENB was used for accurate targeting in two pigs with a pseudo-tumor., Results: The ICG fluorescence at 10 μg/mL was the brightest among various concentrations, unchanged by the distance between the thoracoscope and ICG solutions. Injected ICG of no more than 500μ L showed a localized fluorescence area. All 7 pigs showed a bright paratracheal lymph node within 15 minutes post-injection, with persistent fluorescence for 60 minutes. The antecedent transbronchial ICG injection succeeded in SN identification in all 3 cases at the first thoracoscopic inspection within 20 minutes post-injection. The ENB system allowed accurate ICG injection surrounding the pseudo-tumors., Conclusions: ENB-guided ICG injection followed by NIR thoracoscopy was technically feasible for SN mapping in the porcine lung. This promising platform may be translated into human clinical trials and is suited for MIS.

The present study was designed to evaluate the efficacy of photothermal ablation therapy for lung cancer by low-power near-infrared laser and topical injection of indocyanine green (ICG). In vitro study 1: an 808 nm laser with 250 mW was irradiated for 10 minutes using different dilutions of ICG and the temporal thermal effect was monitored. ICG (1 mL of 0.5 g/L) was heated to a temperature of >30°C from the base temperature by laser irradiation. In vitro study 2: the cytotoxic effect of hyperthermia on human lung cancer cells was examined in different temperature and time settings. Cell viability was quantified by both an MTS assay and reculturing. Fatal conditions evaluated by reculturing were as follows: thermal treatment at 55°C for 5 minutes, 53°C for 10 minutes, and 51°C for 15 minutes. The MTS assay study suggested that thermal treatment at 59°C for 5 minutes and 57°C for 20 minutes showed a severe cytotoxic effect. In vivo study: nude mouse subcutaneous NCI-H460 human lung cancer xenograft models were used for the study. Saline or 0.5 g/L of ICG was injected topically into the tumor (n=3/group). Tumors were irradiated with a laser at 500 mW for 10 minutes. Although the tumor diameter reached 1 cm within 24 days after treatment in all 3 mice using saline/laser, tumor sizes were gradually reduced in all 3 mice using the ICG/laser. In 2 of the 3 mice using ICG/laser, tumors had disappeared macroscopically. The efficacy of the photothermal ablation therapy by low-power near-infrared laser and a topical injection of ICG was clarified using a mouse subcutaneous a lung cancer xenograft model.

Background: Video-assisted thoracoscopic wedge resection of multiple small, non-visible, and nonpalpable pulmonary nodules is a clinical challenge. We propose an ultra-minimally invasive technique for localization of pulmonary nodules using the electromagnetic navigation bronchoscope (ENB)-guided transbronchial indocyanine green (ICG) injection and intraoperative fluorescence detection with a near-infrared (NIR) fluorescence thoracoscope., Methods: Fluorescence properties of ICG topically injected into the lung parenchyma were determined using a resected porcine lung. The combination of ENB-guided ICG injection and NIR fluorescence detection was tested using a live porcine model. An electromagnetic sensor integrated flexible bronchoscope was geometrically registered to the three-dimensional chest computed tomographic image data by way of a real-time electromagnetic tracking system. The ICG mixed with iopamidol was injected into the pulmonary nodules by ENB guidance; ICG fluorescence was visualized by a near-infrared (NIR) thoracoscope., Results: The ICG existing under 24-mm depth of inflated lung was detectable by the NIR fluorescence thoracoscope. The size of the fluorescence spot made by 0.1 mL of ICG was 10.4 ± 2.2 mm. An ICG or iopamidol spot remained at the injected point of the lung for more than 6 hours in vivo. The ICG fluorescence spot injected into the pulmonary nodule with ENB guidance was identified at the pulmonary nodule with the NIR thoracoscope., Conclusions: The ENB-guided transbronchial ICG injection and intraoperative NIR thoracoscopic detection is a feasible method to localize multiple pulmonary nodules., (Copyright © 2015 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) allows for accurate minimally invasive mediastinal lymph node staging of lung cancer. The current convex probe EBUS (CP-EBUS) has limitations in the access to certain N1 lymph nodes (lobar and segmental) because of its size. The aim of this study was to assess the new thin CP-EBUS (TCP-EBUS) and an aspiration needle for sampling of N1 lymph nodes in a porcine model., Methods: The prototype TCP-EBUS (BF-Y0046, Olympus Medical Systems Corp.) with a thinner tip (5.9 mm) and larger bending angle (170 degrees upward) was used. Accessibility, operability, and TBNA capability of the TCP-EBUS were assessed and compared with the current CP-EBUS using porcine lungs. The endoscopic visibility range and the maximum reach were evaluated at the left upper lobe bronchus, tracheobronchus, and right lower lobe bronchus. The prototype aspiration needle (Olympus Medical Systems Corp.) was used for EBUS-TBNA., Results: In all of the evaluated bronchi (n=9), the TCP-EBUS had a greater reach (14.7 mm in the endoscopic visibility range, 16.0 mm in the maximum reach) than the current CP-EBUS. The TCP-EBUS was able to visualize 1 to 3 distal bifurcations farther compared with the current CP-EBUS. Adequate lymph node sampling from lobar and segmental lymph nodes was possible using the aspiration needle., Conclusions: The TCP-EBUS has improved accessibility to peripheral bronchi with excellent operability and is capable of sampling lobar and segmental lymph nodes using the dedicated aspiration needle.

Purpose: The aim of this work was to establish a novel orthotopic human non-small cell lung cancer (NSCLC) murine xenograft model by a nonsurgical, transbronchial approach., Description: Male athymic nude mice and human NSCLC cell lines, including A549, H460, and H520 were used. Under direct visualization of the vocal cords, a 23-gauge blunt-tip slightly curved metal catheter was introduced into the trachea to the bronchus, and 2.5×10(5) tumor cells mixed with Matrigel (BD Biosciences, Mississauga, Ontario, Canada) were administered into the lung. Mice were monitored using weekly microcomputed tomography scans for tumor formation., Evaluation: When the tumor size reached more than 4 mm in diameter, the animals were euthanized, and the tumor tissue was evaluated histopathologically. Of 37 mice studied, 34 were confirmed to have tumor formation: 29 developed solitary tumors and 5 had multifocal lesions. There was no evidence of extrapleural dissemination or effusion., Conclusions: Transbronchial delivery of tumor cells enabled the establishment of a novel orthotopic human NSCLC murine xenograft model. This clinically relevant preclinical model bearing a solitary nodule is of value for a variety of in vivo research studies., (Copyright © 2014 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: The rabbit VX2 lung cancer model is a large animal model useful for preclinical lung cancer imaging and interventional studies. However, previously reported models had issues in terms of invasiveness of tumor inoculation, control of tumor aggressiveness and incidence of complications., Purpose: We aimed to develop a minimally invasive rabbit VX2 lung cancer model suitable for imaging and transbronchial interventional studies., Methods: New Zealand white rabbits and VX2 tumors were used in the study. An ultra-thin bronchoscope was inserted through a miniature laryngeal mask airway into the bronchus. Different numbers of VX2 tumor cells were selectively inoculated into the lung parenchyma or subcarinal mediastinum to create a uniform tumor with low incidence of complications. The model was characterized by CT, FDG-PET, and endobronchial ultrasound (EBUS). Liposomal dual-modality contrast agent was used to evaluate liposome drug delivery system in this model., Results: Both peripheral and mediastinal lung tumor models were created. The tumor making success rate was 75.8% (25/33) in the peripheral lung tumor model and 60% (3/5) in the mediastinal tumor model. The group of 1.0×10(6) of VX2 tumor cells inoculation showed a linear growth curve with less incidence of complications. Radial probe EBUS visualized the internal structure of the tumor and the size measurement correlated well with CT measurements (r(2) = 0.98). Over 7 days of continuous enhancement of the lung tumor by liposomal contrast in the lung tumor was confirmed both CT and fluorescence imaging., Conclusion: Our minimally invasive bronchoscopic rabbit VX2 lung cancer model is an ideal platform for lung cancer imaging and preclinical bronchoscopic interventional studies.

Purpose: The aim of this study was to evaluate the feasibility of messenger RNA (mRNA) and microRNA (miRNA) expression analysis by microarray using samples obtained by endobronchial ultrasonography-guided transbronchial needle aspiration (EBUS-TBNA)., Description: We isolated total RNA from 24 archived clinical EBUS-TBNA samples. The purified RNA that met the quality threshold was used for two different kinds of microarray analyses: whole transcript-based (WT) array for mRNA expression and miRNA expression array., Evaluation: The RNA from 17 of the 24 samples (71%; 12 adenocarcinoma, 3 squamous cell carcinoma, 2 poorly differentiated carcinoma) met the quality threshold. After performing a one-way analysis of variance, we found genes with significant differences in histologic subtype (p<0.01) for each of the WT and miRNA expression data sets. The samples clustered discretely according to their histologic subtypes by hierarchical clustering. After adjusting for multiple testing using the Benjamini-Hochberg false discovery rate correction, there remained 16 WT genes and 4 miRNAs with a false discovery rate of 5% or less., Conclusions: Samples of EBUS-TBNA can be used for comprehensive WT and miRNA expression analysis using microarray technology., (Copyright © 2012 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Introduction: We attempted to assess the correlation between the Doppler mode image patterns during endobronchial ultrasound-guided (EBUS) transbronchial needle aspiration and the expression of angiogenesis-related molecules within lymph nodes in patients with non-small-cell lung cancer., Methods: Thirty-eight archived EBUS- transbronchial needle aspiration samples of lymph nodes (27 metastatic and 11 nonmetastatic) in patients with non-small-cell lung cancer with Doppler mode ultrasound image were analyzed. The Doppler mode image of the vasculature of the targeted lymph node was categorized into the following groups: normal blood flow, low blood flow (LBF), and high blood flow (HBF). Vascular index ratio (vascular area/lymph node area) of each metastatic lymph node was calculated. Total RNA and protein was extracted and analyzed for expression of HIF-1α, VEGF-A, and VEGF-C by quantitative RT-PCR and enzyme-linked immunosorbent assay., Results: Within the 27 metastatic lymph nodes, eight were categorized into the LBF group and 19 into the HBF group. Vascular index ratio was significantly higher in HBF than LBF (p = 0.0003). mRNA expression of HIF-1α and VEGF-A was significantly higher in metastatic lymph nodes than in benign lymph nodes (p < 0.0001). Compared with LBF and HBF, HIF-1α mRNA expression was significantly higher in LBF (p = 0.01) and VEGF-C mRNA expression was significantly higher in HBF (p = 0.0315). There was no significant difference in protein expression by enzyme-linked immunosorbent assay analysis., Conclusions: The vascularity of metastatic lymph nodes observed by EBUS correlates with the mRNA expression of HIF-1α and VEGF-C (not VEGF-A). This correlation is a clinical utility that needs to be evaluated further.

Background:   Lung cancers with mutations in the epidermal growth factor receptor (EGFR) gene respond well to treatment with EGFR inhibitors. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is considered a useful modality to obtain samples from the mediastinal and hilar lymph nodes. However, the EGFR gene status of EBUS-TBNA samples may not always match that of primary tumors., Methods:   In 14 node-positive patients diagnosed by EBUS-TBNA, EGFR mutation analysis results were compared between EBUS-TBNA samples and surgically removed primary tumors. EGFR mutation was screened with peptide nucleic acid-locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamp followed by direct sequence analysis. For one controversial case, gene mutation analyses were performed for the multiple micro-fractions of a metastatic lymph node, which exhibited the heterogeneous immunohistochemical features., Results:   EBUS-TBNA diagnosed one case of exon 21 point mutations, one case of exon 19 deletion, and 12 cases of wild-type EGFR. Results were consistent with those of surgically removed primary tumors in 13 of 14 cases. One case of wild-type EGFR diagnosed by EBUS-TBNA exhibited exon 21 point mutation in the surgically removed primary tumor. The metastatic lymph node targeted by EBUS-TBNA mostly consisted of cancer cells with wild-type EGFR; however, a minor component positive for thyroid transcription factor-1 (TTF-1) and surfactant-associated protein A (PE-10) exhibited EGFR mutation., Conclusion:   The combination of EBUS-TBNA and PNA-LNA clamp is useful for EGFR mutation analysis. However, EGFR mutation status in EBUS-TBNA samples may not be consistent with that of the primary tumor when the tumor contains few EGFR mutations., (© 2012 Tianjin Lung Cancer Institute and Blackwell Publishing Asia Pty. Ltd.)

Background: : Samples obtained by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) have been shown to be useful for molecular analysis., Methods: : The purpose of this study was to evaluate the feasibility of simultaneous isolation of DNA, RNA, and protein using EBUS-TBNA samples. We extracted DNA, RNA, and protein from 59 archived samples obtained by EBUS-TBNA. All samples were mixed with DNA, RNA, and protein-protective solution immediately after taking the biopsy and stored in -80°C for at least 1 year (range, 12 to 30 mo). We used QIAzol Lysis Reagent for the sequential isolation of total RNA, DNA, and protein. The concentration of RNA and DNA was measured and the quality of RNA was evaluated. The concentration of protein was measured using the Bradford protein assay., Results: : Total RNA was successfully isolated in all 59 samples. DNA was isolated in 58 of 59 (98.3%) samples and protein was isolated in 57 of 59 (96.6%) samples. On average, 7.18 μg of total RNA, 7.79 μg of DNA, and 3.96 mg of protein were isolated. RNA integrity number (RIN) was measured in 32 samples and the average RIN number was 6.2 (range, 2.7 to 7.3). Twenty of 32 total RNA samples (62.5%) showed a RIN of >6., Conclusions: : DNA, RNA, and protein can simultaneously be isolated from archived samples obtained by EBUS-TBNA. This method facilitates direct comparisons of alterations in the genome, transcriptome, and proteome within metastatic lymph nodes through a minimally invasive approach.

Background: : Diagnosis of lung cancer is achieved by fine-needle aspiration (FNA) techniques such as computed tomography-guided FNA and transbronchial needle aspiration. The purpose of this study was to establish an optimal storing method for molecular testing in FNA samples., Methods: : We performed FNA using a 21-gauge needle in surgically resected lung cancer samples. The aspirates were stored according to the following protocol: in group 1, the aspirate was snap frozen with liquid nitrogen and in group 2, the aspirate was mixed with RNA later. After sample collection from both groups, these samples were stored at -80°C for 6 months. RNA was extracted from each sample using a commercially available RNA extraction kit, and the quality and quantity of RNA were measured. Quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed for human actin β (hACTB) and keratin 19 (KRT19)., Results: : FNA was performed from 7 lung cancers. RNA was extracted from all samples. The median total amount of extracted RNA was 33.9 μg for group 1 and 35.8 μg for group 2. The mean RNA integrity number was 3.5 for group 1 and 6.3 for group 2. RT-PCR for hACTB and KRT19 could be successfully performed in all samples; however, the relative gene expression value showed intrasample variation., Conclusions: : Samples obtained from computed tomography-guided FNA or transbronchial needle aspiration may be used for genetic profiling of lung cancer. RNA can be extracted from FNA samples and can be used for RT-PCR. RNA quality may affect mRNA expression analysis; therefore, an optimal FNA sample storing protocol is essential.

A human lung adenocarcinoma cell line, designated KU-T1, was established from a Japanese man in Kochi Medical School. Conventional banding and multicolor fluorescence in situ hybridization (M-FISH) analyses of KU-T1 cells revealed a hyperdiploid chromosomal constitution and complex karyotypes. Comparative genomic hybridization showed several chromosomal copy number changes, and five regions that were highly amplified. Two of the five highly amplified regions, 1q and 3q, were identified from distributions of DNA sequences on a metaphase cell by FISH using chromosome microdissection-generated probes hybridized to 1q32 approximately q34 and 3q26 approximately q28, respectively. The 3q probe depicted a homogeneously staining region (hsr) in a derivative chromosome 3 of KU-T1. An hsr probe was regenerated by chromosome microdissection and was hybridized back to KU-T1 and normal metaphases. This hybridization experiment confirmed the probe derived from an hsr and indicated original locations of DNA sequences of hsr on normal chromosome 3. Intense hybridized signals shown at three loci (3p12, 3q26.3, and 3q28) suggests that oncogenes may be involved in the hsr formation. The present study provides a comprehensive analysis of the chromosomal abnormalities, including hsr formation and related oncogenes, in the KU-T1 cell line.

In following up a patient with non-Hodgkin's lymphoma, we encountered a case of pulmonary pleomorphic carcinoma with mediastinal direct invasion. A 65-year-old man with hemoptysis was found to have an abnormal shadow in the right upper lung field. A 6.4 x 4.8-cm tumor adjacent to the upper mediastinum occupied the right anterior segment of the upper lobe (S3) and invaded the superior vena cava (SVC). The serum level of neuron-specific enolase was elevated to 11.9 ng/ml. A specimen from a transbronchial lung biopsy of the right B3b bronchus revealed giant tumor cells. A right upper lobectomy with SVC reconstruction was performed. The resected tumor was diagnosed as a pulmonary pleomorphic carcinoma with a large component of giant and spindle cells, and it is considered to be a rare histologic type.

A 69-year-old woman was admitted with dyspnea on effort and left lung atelectasis on chest X-ray. Fiberoptic bronchoscopy revealed a complete obstruction of the left main bronchus due to a polypoid lesion. This lesion was diagnosed to be a schwannoma arising from the left lower bronchus. Bronchoscopic treatments were performed with electrosurgical snaring and the intratumoral injection of 99.5% ethanol. These treatments were performed once per week for 4 weeks, then were followed with a one-time application of semiconductor laser cautery. These treatments opened the airway and restored the left lung expansion. However, a residual tumor remained at the bifurcation of the left basal bronchus and B6. A cautious follow-up was conducted because schwannoma is a potentially benign tumor. A follow-up bronchoscopic examination at 21 months revealed a regrowth of the residual tumor. A complete resection using a left S6 sleeve segmentectomy was thus performed. The pathologic diagnosis of the tumor was benign schwannoma. There were no complications and no evidence of disease recurrence has been observed after the surgery.

Background: Biopsies by mediastinoscopy remain the most reliable preoperative staging method for N2 lung cancer. Because it is neither practical nor economical to recommend mediastinoscopy for all candidates for surgery, we developed indicational criteria for video-assisted mediastinoscopy (VAM) and carried out a prospective study to validate its usefulness., Methods: Patients with resectable primary lung cancer were chosen for VAM when at least one of three clinical indicators was present: (1) computed tomographic evidence of mediastinal adenopathy, (2) elevated levels of serologic tumor markers, and (3) diameters of primary cancers (> 2 to 3 cm). Patients without positive nodes (group 2) underwent thoracotomy, and patients with positive nodes (group 3) received induction therapy. When none of these criteria were met (group 1), thoracotomy with R2b lymph node dissection was performed without VAM., Results: One hundred twenty-one men and 82 women (total, 203) were eligible for the study. The mean age of the patients was 64.4 years (range, 39 to 75 years) with primary lung cancer. The patients were comprised of 135 adenocarcinomas, 46 squamous cell cancers, and 22 other carcinomas. There were 78 patients in group 1, 87 in group 2, and 38 in group 3. The stages of group 2 patients were more advanced (chi2 = 63.2668; p < 0.001) than those of group 1. As the incidence of positive indicators for VAM increased, the ratios of N2 patients increased from 2.5% (all negative) to 90.4% (triple positive: p < 0.001). The correlation of our criteria with the pathology findings revealed a diagnostic sensitivity of 95.8% and a negative predictive value of 97.4%. Using three indicators for N2 prediction, we selected 96% (46 of 48) pN2, N3 patients and avoided 37% (76 of 203) unnecessary VAMs., Conclusions: We established and validated currently useful criteria for VAMs in the management of primary lung cancer.

p27Kip1, one of the cyclin-dependent kinase (CDK) inhibitors (CDKIs), blocks progression from G1 to S phase by binding cyclin D1-CDK4 and/or cyclin E-CDK2 and inhibiting their activities. Reflecting the function of p27 as a CDKI in vitro, a reduced expression of protein p27 has recently been reported to be associated with tumor aggressiveness in some types of human cancers. In the present study, we examined the relationships between immunohistochemically detected expression of p27, cyclin D1, cyclin E proteins and clinicopathological findings in 77 patients with esophageal squamous cell carcinoma (SCC). Using specific monoclonal antibodies to p27, cyclin DI and cyclin E proteins, positive immunostaining in the nuclei was observed in 32.5% (25/77), 27.3% (21177) and 29.6% (21/71) of patients, respectively. There were no statistically significant relationships among the expressions of these 3 proteins. Using the Kaplan-Meier's method, p27 and cyclin D1 expressions were found to be independently associated with poor prognosis. When all parameters were combined into a multivariate regression analysis using the Cox model, the expressions of p27 and cyclin D1 retained a predictive value for survival. In contrast to former reports supporting a tumor-suppressive function of p27, our results suggest that altered expression of p27 and cyclin D1 may be associated with the progression of human esophageal SCC, in which cyclin E may well not play any central role.