Journal Articles

'Omics analyses

Genome, transcriptome, proteome, etc. studies that help to understand and improve cancer immunotherapy

A genome-wide survey of mutations in the Jurkat cell line

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BACKGROUND: The Jurkat cell line has an extensive history as a model of T cell signaling. But at the turn of the 21st century, some expression irregularities were observed, raising doubts about how closely the cell line paralleled normal human T cells. While numerous expression deficiencies have been described in Jurkat, genetic explanations have only been provided for a handful of defects. RESULTS: Here, we report a comprehensive catolog of genomic variation in the Jurkat cell line based on whole-genome sequencing. With this list of all detectable, non-reference sequences, we prioritize potentially damaging mutations by mining public databases for functional effects. We confirm documented mutations in Jurkat and propose links from detrimental gene variants to observed expression abnormalities in the cell line. CONCLUSIONS: The Jurkat cell line harbors many mutations that are associated with cancer and contribute to Jurkat's unique characteristics. Genes with damaging mutations in the Jurkat cell line are involved in T-cell receptor signaling (PTEN, INPP5D, CTLA4, and SYK), maintenance of genome stability (TP53, BAX, and MSH2), and O-linked glycosylation (C1GALT1C1). This work ties together decades of molecular experiments and serves as a resource that will streamline both the interpretation of past research and the design of future Jurkat studies.

Author Info: (1) Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California, 92037, USA. lhgioia@scripps.edu. (2) Next Generation Sequencing Core, The Scripps Research Institute, La

Author Info: (1) Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California, 92037, USA. lhgioia@scripps.edu. (2) Next Generation Sequencing Core, The Scripps Research Institute, La Jolla, California, 92037, USA. (3) Next Generation Sequencing Core, The Scripps Research Institute, La Jolla, California, 92037, USA. (4) Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California, 92037, USA. (5) Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California, 92037, USA.

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Whole exome and transcriptome analyses integrated with microenvironmental immune signatures of lung squamous cell carcinoma

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The immune microenvironment in lung squamous cell carcinoma (LUSC) is not well understood, with interactions between the host immune system and the tumor, as well as the molecular pathogenesis of LUSC, awaiting better characterization. To date, no molecularly targeted agents have been developed for LUSC treatment. Identification of predictive and prognostic biomarkers for LUSC could help optimize therapy decisions. We sequenced whole exomes and RNA from 101 tumors and matched noncancer control Korean samples. We used the information to predict subtype-specific interactions within the LUSC microenvironment and to connect genomic alterations with immune signatures. Hierarchical clustering based on gene expression and mutational profiling revealed subtypes that were either immune defective or immune competent. We analyzed infiltrating stromal and immune cells to further characterize the tumor microenvironment. Elevated expression of macrophage 2 signature genes in the immune competent subtype confirmed that tumor-associated macrophages (TAMs) linked inflammation and mutation-driven cancer. A negative correlation was evident between the immune score and the amount of somatic copy-number variation (SCNV) of immune genes (r = -0.58). The SCNVs showed a potential detrimental effect on immunity in the immune-deficient subtype. Knowledge of the genomic alterations in the tumor microenvironment could be used to guide design of immunotherapy options that are appropriate for patients with certain cancer subtypes.

Author Info: (1) Gong Wu Genomic Medicine Institute, Seoul National University Bundang Hospital jeongsun@snu.ac.kr. (2) Department of Biomedical Sciences, Seoul National University College of Medicine. (3) Department

Author Info: (1) Gong Wu Genomic Medicine Institute, Seoul National University Bundang Hospital jeongsun@snu.ac.kr. (2) Department of Biomedical Sciences, Seoul National University College of Medicine. (3) Department of Biomedical Sciences, Seoul National University College of Medicine. (4) GMI, SNU. (5) Cancer Research Institute, Seoul National University College of Medicine. (6) Cancer Research Institute, Seoul National University College of Medicine. (7) Cancer Research Institute, Seoul National University College of Medicine. (8) Department of Thoracic and Cardiovascular Surgery, Seoul National University Hospital. (9) Thoracic and Cardiovascular Surgery, None. (10) Thoracic and Cardiovascular Surgery, Seoul National University Hospital. (11) Department of thoracic and cardiovascular surgery, Seoul National University Hospital. (12) Macrogen, Macrogen Inc. (13) Macrogen Inc. (14) Department of Thoracic and Cardiovascular Surgery, Seoul National University Hospital.

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Why do proteases mess up with antigen presentation by re-shuffling antigen sequences

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The sequence of a large number of MHC-presented epitopes is not present as such in the original antigen because it has been re-shuffled by the proteasome or other proteases. Why do proteases throw a spanner in the works of our model of antigen tagging and immune recognition? We describe in this review what we know about the immunological relevance of post-translationally spliced epitopes and why proteases seem to have a second (dark) personality, which is keen to create new peptide bonds.

Author Info: (1) Max-Planck-Institute for Biophysical Chemistry, 37077 Gottingen, Germany. (2) Department of Chemical Immunology, Leiden University Medical Center, NL-2333 ZA Leiden, The Netherlands. (3) Centre for

Author Info: (1) Max-Planck-Institute for Biophysical Chemistry, 37077 Gottingen, Germany. (2) Department of Chemical Immunology, Leiden University Medical Center, NL-2333 ZA Leiden, The Netherlands. (3) Centre for Inflammation Biology and Cancer Immunology (CIBCI) & Peter Gorer Department of Immunobiology, King's College London, SE1 1UL London, United Kingdom. Electronic address: michele.mishto@kcl.ac.uk.

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Endocytosis regulation by autophagy proteins in MHC restricted antigen presentation

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The macroautophagy machinery supports membrane remodeling and fusion events that lead to the engulfment of cytoplasmic constituents in autophagosomes and their degradation in lysosomes. The capacity of this machinery to regulate membrane adaptors and influence vesicle fusion with lysosomes seems to be used not only for autophagosomes, but also for endosomes. We summarize recent evidence that two aspects of endocytosis are regulated by parts of the macroautophagy machinery. These are recruitment of adaptors for the internalization of surface receptors and the fusion of phagosomes with lysosomes. Antigen processing for MHC presentation is affected by these alternative functions of the macroautophagy machinery. Primarily extracellular antigen presentation by MHC class II molecules after phagocytosis benefits from this regulation of phagosome maturation. Furthermore, MHC class I molecules are more efficiently internalized in the presence of the core macroautophagy machinery. The identification of these alternative functions of macroautophagy proteins not only complicates the interpretation of their deficiencies in biological processes, but could also be harnessed for the regulation of antigen presentation to T cells.

Author Info: (1) Neuroinflammation, Institute of Experimental Immunology, University of Zurich, Switzerland. (2) Viral Immunobiology, Institute of Experimental Immunology, University of Zurich, Switzerland. (3) Viral Immunobiology, Institute

Author Info: (1) Neuroinflammation, Institute of Experimental Immunology, University of Zurich, Switzerland. (2) Viral Immunobiology, Institute of Experimental Immunology, University of Zurich, Switzerland. (3) Viral Immunobiology, Institute of Experimental Immunology, University of Zurich, Switzerland. (4) Department of Pathology and Immunology, School of Medicine, University of Geneva, Switzerland. (5) Neuroinflammation, Institute of Experimental Immunology, University of Zurich, Switzerland. (6) Viral Immunobiology, Institute of Experimental Immunology, University of Zurich, Switzerland. Electronic address: christian.muenz@uzh.ch.

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MHC class I presented antigens from malignancies: A perspective on analytical characterization & immunogenicity

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The field of cancer immunotherapy has expanded rapidly in the past few years, with many new approaches entering the clinic for T cell mediated killing of tumors. Several of these clinical approaches involve the exploitation of a CD8+T cell response against MHC I presented tumor antigens. Here, we describe the types of tumor antigens which are considered as targets in the design of T cell based therapeutic approaches, the rationale for targeting MHC I antigens and the analytical tools commonly employed for the discovery of MHC I presented peptides. The advantages and disadvantages of each approach are discussed and a perspective on the future directions of the MHC I peptide exploration field and biotherapeutic strategies is given. SIGNIFICANCE: This work is the first review time an article has been written to show summarize all the various types of tumor antigens, and the analytical tools employed to discover and characterize them.

Author Info: (1) Clinical Biomarker, FivePrime Therapeutics, 111 Oyster Point Boulevard, South San Francisco, CA 94080, United States. (2) Department of Microchemistry, Proteomics & Lipidomics, Genentech Inc

Author Info: (1) Clinical Biomarker, FivePrime Therapeutics, 111 Oyster Point Boulevard, South San Francisco, CA 94080, United States. (2) Department of Microchemistry, Proteomics & Lipidomics, Genentech Inc, 1 DNA Way, South San Francisco, CA 94080, United States. Electronic address: jlill@gene.com.

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A flexible MHC class I multimer loading system for large-scale detection of antigen-specific T cells

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Adaptive immunity is initiated by T cell recognition of specific antigens presented by major histocompatibility complexes (MHCs). MHC multimer technology has been developed for the detection, isolation, and characterization of T cells in infection, autoimmunity, and cancer. Here, we present a simple, fast, flexible, and efficient method to generate many different MHC class I (MHC I) multimers in parallel using temperature-mediated peptide exchange. We designed conditional peptides for HLA-A*02:01 and H-2K(b) that form stable peptide-MHC I complexes at low temperatures, but dissociate when exposed to a defined elevated temperature. The resulting conditional MHC I complexes, either alone or prepared as ready-to-use multimers, can swiftly be loaded with peptides of choice without additional handling and within a short time frame. We demonstrate the ease and flexibility of this approach by monitoring the antiviral immune constitution in an allogeneic stem cell transplant recipient and by analyzing CD8(+) T cell responses to viral epitopes in mice infected with lymphocytic choriomeningitis virus or cytomegalovirus.

Author Info: (1) Oncode Institute and Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, Netherlands. Department of Cell Biology II, Netherlands Cancer Institute, Amsterdam

Author Info: (1) Oncode Institute and Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, Netherlands. Department of Cell Biology II, Netherlands Cancer Institute, Amsterdam, Netherlands. (2) Core Research Lab, the Second Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, China m.garstka@xjtu.edu.cn. Department of Cell Biology II, Netherlands Cancer Institute, Amsterdam, Netherlands. (3) Department of Hematology, Leiden University Medical Center, Leiden, Netherlands. (4) Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, Netherlands. (5) Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, Netherlands. (6) Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, Netherlands. (7) Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, Netherlands. (8) Department of Hematology, Leiden University Medical Center, Leiden, Netherlands. (9) Oncode Institute and Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, Netherlands j.j.c.neefjes@lumc.nl. Department of Cell Biology II, Netherlands Cancer Institute, Amsterdam, Netherlands. (10) Oncode Institute and Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, Netherlands h.ovaa@lumc.nl. Department of Cell Biology II, Netherlands Cancer Institute, Amsterdam, Netherlands.

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Molecular signatures in hepatocellular carcinoma: A step toward rationally designed cancer therapy

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Molecular characterization of hepatocellular carcinoma (HCC) has greatly improved our understanding of disease pathogenesis. Mutational analysis, RNA and microRNA expression profiling, and epigenetic characterization have revealed common aberrations in oncogenes and tumor suppressors that correlate with disease biology and serve as a guide for the rational design of targeted therapies. These approaches have also led to the discovery of novel targets, including mutations in isocitrate dehydrogenase and chromatin remodeling enzymes. With the advent of immunotherapy, RNA expression profiling of the tumor microenvironment has identified a subset of HCC with high lymphocyte infiltration that may benefit from checkpoint inhibitor therapy. Molecular signatures thus capture the biology of a tumor, providing a supplement to current staging schema, which are based on tumor size and number, for more accurate prognostication of recurrence risk and survival. Molecular signatures may also be used to guide interventional therapy by defining those most suitable for transplantation or locoregional therapy rather than surgical resection. Finally, a multiomics approach involves the aggregation and analysis of multiple signatures for a more comprehensive characterization of pathogenic mechanisms. This broader approach attempts to address issues with signaling pathway cross-talk and redundancy, which have greatly limited the potential value of targeted therapies to date. Cancer 2018. (c) 2018 American Cancer Society.

Author Info: (1) Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts. (2) Division of Surgical Oncology, Massachusetts General Hospital, Boston, Massachusetts. (3) Division of Surgical Oncology, Massachusetts

Author Info: (1) Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts. (2) Division of Surgical Oncology, Massachusetts General Hospital, Boston, Massachusetts. (3) Division of Surgical Oncology, Massachusetts General Hospital, Boston, Massachusetts.

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Tobacco Smoking-Associated Alterations in the Immune Microenvironment of Squamous Cell Carcinomas

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Background: Tobacco smoking creates DNA damage, inducing mutations and potentially altering the tumor immune microenvironment. These types of genetic and immune microenvironment alterations are critical factors known to affect tumor response to immunotherapy. Here we analyze the association between the mutational signature of tobacco smoking, tumor mutational load, and metrics of immune activity in squamous cell carcinomas arising in the head and neck and lung. Methods: Using RNA and DNA sequencing data from The Cancer Genome Atlas head and neck (HNSC; n = 287) and lung (LUSC; n = 130) squamous cell carcinoma data sets and two independent gene expression data sets (HNSC, n = 136; LUSC, n = 75), we examined associations between the mutational smoking signature, mutation count, immune cell infiltration, cytolytic activity, and interferon-gamma signaling. Results: An increasing mutational smoking signature was associated with statistically significantly increased overall mutational load in both HNSC (rho = .33, P = 1.01 x 10-7) and LUSC (rho = .49, P = 2.80 x 10-9). In HNSC, a higher mutational smoking signature was associated with lower levels of immune infiltration (rho = -.37, P = 1.29 x 10-10), cytolytic activity (rho = -.28, P = 4.07 x 10-6), and interferon-gamma pathway signaling (rho = .39, P = 3.20 x 10-11). In LUSC, these associations were reversed (rho = .19, P = .03; rho = .20, P = .02; and rho = .18, P = .047, respectively). Differentially expressed genes between smoking-high and smoking-low tumors revealed broad tobacco-induced immunosuppression in HNSC, in contrast to a tumor-inflamed microenvironment in smokers with LUSC. Conclusions: In squamous cell carcinomas, the genetic smoking signature is associated with higher mutational load, but variable effects on tumor immunity can occur, depending on anatomic site. In HNSC, smoking is predominantly immunosuppressive; in LUSC, more pro-inflammatory. Both tumor mutation load and immune microenvironment affect clinical response to immunotherapy. Thus, the mutational smoking signature is likely to have relevance for immunotherapeutic investigation in smoking-associated cancers.

Author Info: (1) Immunogenomics and Precision Oncology Platform, Memorial Sloan Kettering Cancer Center, New York, NY. Human Oncology and Pathogenesis Program, Memorial, Memorial Sloan Kettering Cancer Center

Author Info: (1) Immunogenomics and Precision Oncology Platform, Memorial Sloan Kettering Cancer Center, New York, NY. Human Oncology and Pathogenesis Program, Memorial, Memorial Sloan Kettering Cancer Center, New York, NY. (2) Immunogenomics and Precision Oncology Platform, Memorial Sloan Kettering Cancer Center, New York, NY. (3) Immunogenomics and Precision Oncology Platform, Memorial Sloan Kettering Cancer Center, New York, NY. Human Oncology and Pathogenesis Program, Memorial, Memorial Sloan Kettering Cancer Center, New York, NY. (4) Immunogenomics and Precision Oncology Platform, Memorial Sloan Kettering Cancer Center, New York, NY. Human Oncology and Pathogenesis Program, Memorial, Memorial Sloan Kettering Cancer Center, New York, NY. (5) Immunogenomics and Precision Oncology Platform, Memorial Sloan Kettering Cancer Center, New York, NY. Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY. (6) Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY. (7) Immunogenomics and Precision Oncology Platform, Memorial Sloan Kettering Cancer Center, New York, NY. Human Oncology and Pathogenesis Program, Memorial, Memorial Sloan Kettering Cancer Center, New York, NY. Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY. (8) Immunogenomics and Precision Oncology Platform, Memorial Sloan Kettering Cancer Center, New York, NY. Human Oncology and Pathogenesis Program, Memorial, Memorial Sloan Kettering Cancer Center, New York, NY. Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY.

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Mapping the tumour HLA ligandome by mass spectrometry

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The entirety of HLA-presented peptides is referred to as the HLA ligandome of a cell or tissue, in tumours often termed immunopeptidome. Mapping the tumour immunopeptidome by mass spectrometry (MS) comprehensively views the pathophysiologically relevant antigenic signature of human malignancies. MS is an unbiased approach stringently filtering the candidates to be tested as opposed to epitope prediction algorithms. In the setting of peptide-specific immunotherapies, MS-based strategies significantly diminish the risk of lacking clinical benefit, as they yield highly enriched amounts of truly presented peptides. Early immunopeptidomic efforts were severely limited by technical sensitivity and manual spectra interpretation. The technological progress with development of orbitrap mass analysers and enhanced chromatographic performance led to vast improvements in mass accuracy, sensitivity, resolution and speed. Concomitantly, bioinformatic tools were developed to process MS data, integrate sequencing results, and to deconvolute multi-allelic datasets. This enabled the immense advancement of tumour immunopeptidomics. Studying the HLA-presented peptide repertoire bears high potential for both answering basic scientific questions and translational application. Mapping the tumour HLA ligandome has started to significantly contribute to target identification for the design of peptide-specific cancer immunotherapies in clinical trials and compassionate need treatments. In contrast to prediction algorithms, rare HLA allotypes and HLA class II can be adequately addressed when choosing MS-guided target identification platforms. Herein, we review the identification of tumour HLA ligands focusing on sources, methods, bioinformatic data analysis, translational application and provide an outlook on future developments. This article is protected by copyright. All rights reserved.

Author Info: (1) University of Tubingen, Interfaculty Institute for Cell Biology, Department of Immunology, Auf der Morgenstelle 15, 72076, Tubingen, Germany. DKFZ Partner Site Tubingen, German Cancer

Author Info: (1) University of Tubingen, Interfaculty Institute for Cell Biology, Department of Immunology, Auf der Morgenstelle 15, 72076, Tubingen, Germany. DKFZ Partner Site Tubingen, German Cancer Consortium (DKTK), Auf der Morgenstelle 15, 72076, Tubingen, Germany. (2) University of Tubingen, Interfaculty Institute for Cell Biology, Department of Immunology, Auf der Morgenstelle 15, 72076, Tubingen, Germany. (3) University of Tubingen, Interfaculty Institute for Cell Biology, Department of Immunology, Auf der Morgenstelle 15, 72076, Tubingen, Germany. DKFZ Partner Site Tubingen, German Cancer Consortium (DKTK), Auf der Morgenstelle 15, 72076, Tubingen, Germany.

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Prognostic Impact of Tumor Mutation Burden in Patients with Completely Resected Non-Small Cell Lung Cancer: Brief Report

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INTRODUCTION: Tumor mutation burden (TMB), is thought to be associated with the amount of neoantigen in the tumor and to have an important role in predicting the effect of immune checkpoint inhibitors. However, the relevance of TMB to prognosis is not yet fully understood. In this study, we investigated the clinical significance of TMB in patients with non-small cell lung cancer (NSCLC) and examined the relationship between TMB and prognosis. METHODS: We calculated TMB within individual tumors by whole exome sequencing analysis using next-generation sequencing. We included 90 patients with NSCLC who underwent surgery in the Hospital of Fukushima Medical University from 2013 to 2016. No patients received chemotherapy or immunotherapy before surgery. We assessed the correlation between TMB and prognosis. RESULTS: TMB >62 was associated with worse overall survival (OS) of patients with NSCLC (hazard ratio [HR] = 6.633, P = 0.0003). Multivariate analysis showed poor prognosis with high TMB (HR = 12.31, P = 0.019). In patients with stage I NSCLC, higher TMB was associated with worse prognosis for both OS (HR = 7.582, P = 0.0018) and disease-free survival (HR = 6.07, P = 0.0072). CONCLUSION: High TMB in NSCLC is a poor prognostic factor. If high TMB is a predictor of the efficacy of immune checkpoint inhibitors, postoperative adjuvant therapy with immune checkpoint inhibitors may contribute to improvement of recurrence and OS.

Author Info: (1) Department of Chest Surgery, Fukushima Medical University, Fukushima, Japan. (2) Department of Chest Surgery, Fukushima Medical University, Fukushima, Japan. (3) Department of Chest Surgery

Author Info: (1) Department of Chest Surgery, Fukushima Medical University, Fukushima, Japan. (2) Department of Chest Surgery, Fukushima Medical University, Fukushima, Japan. (3) Department of Chest Surgery, Fukushima Medical University, Fukushima, Japan. (4) Department of Chest Surgery, Fukushima Medical University, Fukushima, Japan. (5) Department of Chest Surgery, Fukushima Medical University, Fukushima, Japan. (6) Department of Chest Surgery, Fukushima Medical University, Fukushima, Japan. (7) Department of Chest Surgery, Fukushima Medical University, Fukushima, Japan. (8) Department of Chest Surgery, Fukushima Medical University, Fukushima, Japan. (9) Department of Chest Surgery, Fukushima Medical University, Fukushima, Japan. (10) Department of Chest Surgery, Fukushima Medical University, Fukushima, Japan. (11) Department of Chest Surgery, Fukushima Medical University, Fukushima, Japan. (12) Department of Chest Surgery, Fukushima Medical University, Fukushima, Japan. (13) Department of Chest Surgery, Fukushima Medical University, Fukushima, Japan. (14) Translational Research Center, Fukushima Medical University, Fukushima, Japan. (15) Translational Research Center, Fukushima Medical University, Fukushima, Japan. (16) Translational Research Center, Fukushima Medical University, Fukushima, Japan. (17) Translational Research Center, Fukushima Medical University, Fukushima, Japan. (18) Department of Chest Surgery, Fukushima Medical University, Fukushima, Japan. Electronic address: hiro@fmu.ac.jp.

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