近藤 征史

Lymphocyte profiles in mediastinal lymph nodes may reflect the immune status of patients with sarcoidosis. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is useful for the diagnosis of diseases with mediastinal lymphadenopathy including sarcoidosis. The purpose of this study was to determine lymphocyte profiles of lymph nodes in sarcoidosis by analyzing EBUS-TBNA samples. We prepared single cell suspensions from EBUS-TBNA samples of mediastinal lymph nodes from patients with sarcoidosis or lung cancer and analyzed surface markers (CD3, CD4, CD8, CD19, CD25) and FoxP3 expression in the resultant lymphocytes using flow cytometry. We studied 26 patients with sarcoidosis and 16 with lung cancer with mediastinal lymph node metastases. In sarcoidosis, the CD4/CD8 ratio was significantly more elevated in lymph nodes than in bronchoalveolar lavage fluid (P<0.001), although both were strongly correlated. The CD4/CD8 ratio was significantly higher in stage I than in stage II both in the BAL fluid and lymph nodes. When compared with lung cancer lymph node metastasis, the CD4/CD8 ratio was significantly higher in sarcoidosis, whereas the CD3/CD19 ratio was significantly higher in lung cancer. The proportion of regulatory T cells (CD4+, CD25+, FoxP3 high) did not differ between sarcoidosis and lung cancer samples. Lymphocyte profiles in mediastinal lymphadenopathy can be analyzed by flow cytometry of EBUS-TBNA samples. These findings might help elucidate the immunopathology of sarcoidosis.

Background Alectinib, a potent, highly selective, CNS-active inhibitor of anaplastic lymphoma kinase (ALK), showed promising efficacy and tolerability in the single-arm phase 1/2 AF-001JP trial in Japanese patients with ALK-positive non-small-cell lung cancer. Given those promising results, we did a phase 3 trial to directly compare the efficacy and safety of alectinib and crizotinib. Methods J-ALEX was a randomised, open-label, phase 3 trial that recruited ALK inhibitor-naive Japanese patients with ALK-positive non-small-cell lung cancer, who were chemotherapy-naive or had received one previous chemotherapy regimen, from 41 study sites in Japan. Patients were randomly assigned (1: 1) via an interactive web response system using a permuted-block method stratified by Eastern Cooperative Oncology Group performance status, treatment line, and disease stage to receive oral alectinib 300 mg twice daily or crizotinib 250 mg twice daily until progressive disease, unacceptable toxicity, death, or withdrawal. The primary endpoint was progression-free survival assessed by an independent review facility. The efficacy analysis was done in the intention-to-treat population, and safety analyses were done in all patients who received at least one dose of the study drug. The study is ongoing and patient recruitment is closed. This study is registered with the Japan Pharmaceutical Information Center (number JapicCTI-132316). Findings Between Nov 18, 2013, and Aug 4, 2015, 207 patients were recruited and assigned to the alectinib (n=103) or crizotinib (n=104) groups. At data cutoff for the second interim analysis, 24 patients in the alectinib group had discontinued treatment compared with 61 in the crizotinib group, mostly due to lack of efficacy or adverse events. At the second interim analysis (data cutoff date Dec 3, 2015), an independent data monitoring committee determined that the primary endpoint of the study had been met (hazard ratio 0.34 [99.7% CI 0.17-0.71], stratified log-rank p&lt; 0.0001) and recommended an immediate release of the data. Median progression-free survival had not yet been reached with alectinib (95% CI 20.3-not estimated) and was 10.2 months (8.2-12.0) with crizotinib. Grade 3 or 4 adverse events occurred at a greater frequency with crizotinib (54 [52%] of 104) than alectinib (27 [26%] of 103). Dose interruptions due to adverse events were also more prevalent with crizotinib (77 [74%] of 104) than with alectinib (30 [29%] of 103), and more patients receiving crizotinib (21 [20%]) than alectinib (nine [9%]) discontinued the study drug because of an adverse event. No adverse events with a fatal outcome occurred in either treatment group. Interpretation These results provide the first head-to-head comparison of alectinib and crizotinib and have the potential to change the standard of care for the first-line treatment of ALK-positive non-small-cell lung cancer. The dose of alectinib (300 mg twice daily) used in this study is lower than the approved dose in countries other than Japan; however, this limitation is being addressed in the ongoing ALEX study.

In patients with pulmonary diseases such as idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome, progressive pulmonary fibrosis is caused by dysregulated wound healing via activation of fibroblasts after lung inflammation or severe damage. Migration of fibroblasts toward the fibrotic lesions plays an important role in pulmonary fibrosis. Fibrotic tissue in the lung is much stiffer than normal lung tissue. Emerging evidence supports the hypothesis that the stiffness of the matrix is not only a consequence of fibrosis, but also can induce fibroblast activation. Nevertheless, the effects of substrate rigidity on migration of lung fibroblasts have not been fully elucidated. We evaluated the effects of substrate stiffness on the morphology, α-smooth muscle actin (α-SMA) expression, and cell migration of primary human lung fibroblasts by using polyacrylamide hydrogels with stiffnesses ranging from 1 to 50 kPa. Cell motility was assessed by platelet-derived growth factor (PDGF)-induced chemotaxis and random walk migration assays. As the stiffness of substrates increased, fibroblasts became spindle-shaped and spread. Expression of α-SMA proteins was higher on the stiffer substrates (25 kPa gel and plastic dishes) than on the soft 2 kPa gel. Both PDGF-induced chemotaxis and random walk migration of fibroblasts precultured on stiff substrates (25 kPa gel and plastic dishes) were significantly higher than those of cells precultured on 2 kPa gel. Transfection of the fibroblasts with short interfering RNA for α-SMA inhibited cell migration. These findings suggest that fibroblast activation induced by a stiff matrix is involved in mechanisms of the pathophysiology of pulmonary fibrosis.

To identify potential therapeutic targets for lung cancer, we performed semi-genome-wide shRNA screening combined with the utilization of genome-wide expression and copy number data. shRNA screening targeting 5043 genes in NCI-H460 identified 51 genes as candidates. Pathway analysis revealed that the 51 genes were enriched for the five pathways, including ribosome, proteasome, RNA polymerase, pyrimidine metabolism and spliceosome pathways. We focused on the proteasome pathway that involved six candidate genes because its activation has been demonstrated in diverse human malignancies, including lung cancer. Microarray expression and array CGH data showed that PSMA6, a proteasomal subunit of a 20S catalytic core complex, was highly expressed in lung cancer cell lines, with recurrent gene amplifications in some cases. Therefore, we further examined the roles of PSMA6 in lung cancer. Silencing of PSMA6 induced apoptosis or G2/M cell cycle arrest in cancer cell lines but not in an immortalized normal lung cell line. These results suggested that PSMA6 serves as an attractive target with a high therapeutic index for lung cancer.

© 2016 Elsevier Inc. Background Nanoparticle albumin-bound (nab) paclitaxel is a promising new therapeutic agent for all histologic types of non–small-cell lung cancer (NSCLC). We recently performed a phase 2 study of weekly nab-paclitaxel in patients with previously treated advanced NSCLC, finding promising activity and acceptable toxicity for this regimen. We have now designed a randomized phase 3 intergroup study (J-AXEL, UMIN000017487) to examine the clinical benefit and safety of nab-paclitaxel compared to docetaxel in patients with previously treated advanced NSCLC. Patients and Methods Patients are randomized to receive either docetaxel (60 mg/m2on day 1 every 3 weeks, control arm) or nab-paclitaxel (100 mg/m2on days 1, 8, and 15 every 3 weeks, experimental arm), with each drug being administered until disease progression or unacceptable toxicity. The study will evaluate the noninferiority of nab-paclitaxel relative to docetaxel for the primary end point of overall survival. Conclusion If the primary objective is achieved, this study will provide evidence for a new alternative treatment option for patients with previously treated advanced NSCLC.

Predicting the feasibility of platinum-based chemotherapy remains an important issue in elderly (over 70 years) patients with non-small cell lung cancer (NSCLC). The aim of this study was to identify the risk factors for the early serious adverse events (SAEs) (during cycles 1-2) in elderly receiving platinum-based chemotherapy, and to explore the clinical characteristics of patients who require early treatment termination without progressive disease (PD). One hundred and ninety-eight consecutive elderly NSCLC patients receiving platinum-based chemotherapy were retrospectively reviewed. The median age was 73 years (range 70-83). 161 (81 %) were males, and 190 (95 %) were PS 0-1. Fifty-one (29 %) and 39 (19 %) patients developed early non-hematological SAEs and hematological SAEs, respectively. Multivariate analysis identified low serum albumin (&lt; 3.0 g/dl) as an independent risk factor for non-hematological SAEs, while low creatinine clearance (&lt; 45 ml/min) for hematological SAEs. In all, 24 (12 %) patients needed early treatment termination without PD. The major reason for this event was the development of non-hematological SAEs (4.5 %), followed by grade 2 non-hematological adverse events (AEs) (3 %). In multivariate analysis, age over 75 years and low serum albumin were associated with this event. The median overall survival (OS) in patients with this event was only 6.0 months, while the development of early SAE was not associated with poor OS. Baseline serum albumin might be useful for predicting the feasibility of platinum-based chemotherapy, and the risk estimation of early treatment termination without PD might be beneficial for the treatment selection in elderly NSCLC patients.

Background: Several pre-clinical and clinical studies suggest a potential predictive role of epidermal growth factor receptor (EGFR) mutation in responsiveness to cytotoxic chemotherapy. The aim of this phase II study was to evaluate the efficacy and safety of pemetrexed-carboplatin combination as first-line chemotherapy in advanced non-squamous non-small cell lung cancer (NSCLC) limited to EGFR-wild-type cases. Patients and Methods: In this single-arm, multicenter clinical trial, patients received pemetrexed (500 mg/m(2)) and carboplatin (area under the curve=6) intravenously on day 1 every 3 weeks for three to six cycles. The objective response rate (ORR) was the primary end-point; secondary end-points included the disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and safety. Results: A total of 54 patients were enrolled and 53 patients received therapy. No complete response was observed and partial response was observed in 19 (35.8%) cases, resulting in an ORR of 35.8% [95% confidence interval (CI)=23.1-50.2%]. Stable disease was observed in 20 (37.7%) patients and therefore the DCR was 73.6% (95% CI=59.7-84.7%). The median PFS was 5.4 months (95% CI=4.1-6.8 months) and the median OS was 12.7 months (95% CI=9.3-16.1 months). Treatment-related grade 3 or 4 hematological toxicities were neutropenia, leukopenia, anemia and thrombocytopenia in 35.8%, 11.3%, 30.2%, and 32.1% of patients, respectively. No grade 3 febrile neutropenia was observed, and grade 3 or 4 non-hematological toxicities were mild. There was no treatment-related death. Conclusion: The pemetrexed-carboplatin combination was effective and well-tolerated in patients with EGFR-wild-type non-squamous NSCLC (UMIN-CTR number: UMIN000003393).

Rheumatoid arthritis (RA)-related pulmonary disorders specifically airway abnormalities and interstitial pneumonia (IP) are important extra-articular manifestations. The forced oscillation technique (FOT) is a useful method to assess respiratory impedance, respiratory resistance (Rrs) and reactance (Xrs), at different oscillatory frequencies during tidal breathing. The aim of this study was to characterize the respiratory mechanics of patients with RA and to relate them to parameters of the pulmonary function test and findings of chest CT images. Respiratory impedance of RA patients (n = 69) was measured as a function of frequency from 4 to 36 Hz using the FOT device and compared with that of healthy subjects (n = 10). Data were retrospectively reviewed. Patients were female-dominant (60.9 %) and 95.7 % had abnormal CT findings including airway and parenchymal abnormalities. Thirty-seven of 69 patients (53.6 %) were smokers. Rrs was significantly frequency-dependent in RA patients but not in the healthy subjects. Xrs were significantly frequency-dependent in both RA and healthy groups. Rrs was significantly higher during an expiratory phase in both RA and healthy groups. Xrs was significantly lower (more negative) during an expiratory phase than that during an inspiratory phase in RA patients but not in healthy subjects. Xrs of the RA group was significantly more negative than that of the normal control. There was no difference in impedance parameters between the airway lesion dominant (n = 27) and IP dominant groups (n = 23) in the RA group. The impedance parameters of the RA group significantly correlated with most parameters of the pulmonary function test. In pulmonary function test results, % of the predicted value for forced expiratory flow from 25 to 75 % of forced vital capacity was significantly lower and % of the predicted value for diffusing capacity of the lung for carbon monoxide was higher in the airway lesion dominant group than those in the IP dominant group. Krebs von den Lungen-6, a serum indicator of IP, was significantly higher in the IP group than that in the airway lesion dominant group. Taken together, the impedance results reflect abnormalities in pulmonary functions and structures in patients with RA.

Objective Endobronchial ultrasonography with a guide sheath (EBUS-GS) and virtual bronchoscopic navigation (VBN) improves the diagnostic yield in patients with peripheral pulmonary lesions (PPLs). Most previous reports on EBUS-GS-guided transbronchial biopsy (TBB) have included patients with benign and malignant diseases. We aimed to determine the factors that predicted a successful diagnosis by EBUS-GS-guided TBB diagnostic in patients with small peripheral lung cancer, with a focus on the high-resolution computed tomography (HRCT) findings before bronchoscopy. Methods We retrospectively reviewed the medical records of 173 consecutive patients with 175 small (&lt;= 30 mm) PPLs who were diagnosed with primary lung cancer between June 2010 and October 2013 at Nagoya University Hospital. All patients underwent EBUS-GS-guided TBB with VBN using a ZioStation computer workstation (Ziosoft, Osaka, Japan). We analyzed the patient characteristics, HRCT findings, diagnostic yield, and the diagnostic factors in small peripheral lung carcinoma. Results The EBUS probe position was within the PPL in 83 of the 175 lesions (47%) and 112 (64.0%) cases were successfully diagnosed by EBUS-GS-guided TBB. A univariate analysis revealed that the following factors were associated with a significantly higher diagnostic yield: CT bronchus sign positivity, a lesion of &gt;20 mm in diameter, a solid nodule, and a probe position that was within the lesion. The following factors were not significant: the lesion location, the number of biopsies, and the lung cancer histology. A multivariate analysis revealed that the following factors significantly affected the diagnostic yield: CT bronchus sign positivity [ odds ratio (OR) =2.479]; a probe position that was within the lesion (OR=2.542); and a solid nodule (OR=2.304). Conclusion The significant factors that were significantly associated with a successful diagnosis using EBUS-GS-guided TBB in small peripheral lung carcinoma were as follows: CT bronchus sign positivity, a solid nodule, and a probe position that was within the lesion.

Both pro- and anti-oncogenic roles of miR-221 and miR-222 microRNAs are reported in several types of human cancers. A previous study suggested their oncogenic role in invasiveness in lung cancer, albeit only one cell line (H460) was used. To further evaluate involvement of miR-221 and miR-222 in lung cancer, we investigated the effects of miR-221 and miR-222 overexpression on six lung cancer cell lines, including H460, as well as one immortalized normal human bronchial epithelial cell line, HBEC4. miR-221 and miR-222 induced epithelial-to-mesenchymal transition (EMT)-like changes in a minority of HBEC4 cells but, unexpectedly, both the microRNAs rather suppressed their invasiveness. Consistent with the prior report, miR-221 and miR-222 promoted growth in H460; however, miR-221 suppressed growth in four other cell lines with no effects in one, and miR-222 suppressed growth in three cell lines but promoted growth in two. These are the first results to show tumor-suppressive effects of miR-221 and miR-222 in lung cancer cells, and we focused on clarifying the mechanisms. Cell cycle and apoptosis analyses revealed that growth suppression by miR-221 and miR-222 occurred through intra-S-phase arrest and/or apoptosis. Finally, lung cancer cell lines transfected with miR-221 or miR-222 became more sensitive to the S-phase targeting drugs, possibly due to an increased S-phase population. In conclusion, our data are the first to show tumor-suppressive effects of miR-221 and miR-222 on lung cancer, warranting testing their potential as therapeutics for the disease.

© 15CBMS-0001. In the present study, we propose immuno-wall lab-on-a-chip companion diagnostic devices for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) such as erlotinib and gefitinib. The lysates of cytological samples including pleural effusion in lung cancer patients were successfully analyzed within 20 minutes. This is the first experiment demonstrating the detection of mutated EGFRs in the pleural effusion by microdevices. Our devices have a great potential to become the next generation companion diagnostic devices which overcome the problems of currently available methods.

Airway smooth muscle (ASM) cells within the airway walls are continually exposed to mechanical stimuli, and exhibit various functions in response to these mechanical stresses. ATP acts as an extracellular mediator in the airway. Moreover, extracellular ATP is considered to play an important role in the pathophysiology of asthma and chronic obstructive pulmonary disease. However, it is not known whether ASM cells are cellular sources of ATP secretion in the airway. We therefore investigated whether mechanical stretch induces ATP release from ASM cells. Mechanical stretch was applied to primary human ASM cells cultured on a silicone chamber coated with type I collagen using a stretching apparatus. Concentrations of ATP in cell culture supernatants measured by luciferin-luciferase bioluminescence were significantly elevated by cyclic stretch (12 and 20% strain). We further visualized the stretch-induced ATP release from the cells in real time using a luminescence imaging system, while acquiring differential interference contrast cell images with infrared optics. Immediately after a single uniaxial stretch for 1 second, strong ATP signals were produced by a certain population of cells and spread to surrounding spaces. The cyclic stretch-induced ATP release was significantly reduced by inhibitors of Ca2+-dependent vesicular exocytosis, 1,2-bis(o-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid tetraacetoxymethyl ester, monensin, N-ethylmaleimide, and bafilomycin. In contrast, the stretch-induced ATP release was not inhibited by a hemichannel blocker, carbenoxolone, or blockade of transient receptor potential vanilloid 4 by short interfering RNA transfection or ruthenium red. These findings reveal a novel property of ASM cells: mechanically induced ATP release may be a cellular source of ATP in the airway.

One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca2+ concentration ([Ca2+](i)) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10-30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca2+](i) transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca2+](i) The stretch-induced [Ca2+](i) elevation was attenuated in Ca2+-free solution. In contrast, the increase of [Ca2+](i) by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd3+, ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca2+](i) elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca2+ influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP. (C) 2014 Elsevier Inc. All rights reserved.