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      Targeted next-generation sequencing of DNA regions proximal to a conserved GXGXXG signaling motif enables systematic discovery of tyrosine kinase fusions in cancer

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          Abstract

          Tyrosine kinase (TK) fusions are attractive drug targets in cancers. However, rapid identification of these lesions has been hampered by experimental limitations. Our in silico analysis of known cancer-derived TK fusions revealed that most breakpoints occur within a defined region upstream of a conserved GXGXXG kinase motif. We therefore designed a novel DNA-based targeted sequencing approach to screen systematically for fusions within the 90 human TKs; it should detect 92% of known TK fusions. We deliberately paired ‘in-solution’ DNA capture with 454 sequencing to minimize starting material requirements, take advantage of long sequence reads, and facilitate mapping of fusions. To validate this platform, we analyzed genomic DNA from thyroid cancer cells (TPC-1) and leukemia cells (KG-1) with fusions known only at the mRNA level. We readily identified for the first time the genomic fusion sequences of CCDC6-RET in TPC-1 cells and FGFR1OP2-FGFR1 in KG-1 cells. These data demonstrate the feasibility of this approach to identify TK fusions across multiple human cancers in a high-throughput, unbiased manner. This method is distinct from other similar efforts, because it focuses specifically on targets with therapeutic potential, uses only 1.5 µg of DNA, and circumvents the need for complex computational sequence analysis.

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          Most cited references31

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          The protein kinase complement of the human genome.

          G. Manning (2002)
          We have catalogued the protein kinase complement of the human genome (the "kinome") using public and proprietary genomic, complementary DNA, and expressed sequence tag (EST) sequences. This provides a starting point for comprehensive analysis of protein phosphorylation in normal and disease states, as well as a detailed view of the current state of human genome analysis through a focus on one large gene family. We identify 518 putative protein kinase genes, of which 71 have not previously been reported or described as kinases, and we extend or correct the protein sequences of 56 more kinases. New genes include members of well-studied families as well as previously unidentified families, some of which are conserved in model organisms. Classification and comparison with model organism kinomes identified orthologous groups and highlighted expansions specific to human and other lineages. We also identified 106 protein kinase pseudogenes. Chromosomal mapping revealed several small clusters of kinase genes and revealed that 244 kinases map to disease loci or cancer amplicons.
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            Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer.

            Despite the success of tyrosine kinase-based cancer therapeutics, for most solid tumors the tyrosine kinases that drive disease remain unknown, limiting our ability to identify drug targets and predict response. Here we present the first large-scale survey of tyrosine kinase activity in lung cancer. Using a phosphoproteomic approach, we characterize tyrosine kinase signaling across 41 non-small cell lung cancer (NSCLC) cell lines and over 150 NSCLC tumors. Profiles of phosphotyrosine signaling are generated and analyzed to identify known oncogenic kinases such as EGFR and c-Met as well as novel ALK and ROS fusion proteins. Other activated tyrosine kinases such as PDGFRalpha and DDR1 not previously implicated in the genesis of NSCLC are also identified. By focusing on activated cell circuitry, the approach outlined here provides insight into cancer biology not available at the chromosomal and transcriptional levels and can be applied broadly across all human cancers.
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              Letter: A new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining.

              J D Rowley (1973)
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                Author and article information

                Journal
                Nucleic Acids Res
                nar
                nar
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                November 2010
                November 2010
                29 June 2010
                29 June 2010
                : 38
                : 20
                : 6985-6996
                Affiliations
                1Weill Graduate School of Medical Sciences, Cornell University, New York, NY 10021, USA, 2Max Planck Institute for Neurological Research with Klaus Joachim Zülch Laboratories of the Max Planck Society and the Medical Faculty, University of Cologne, Cologne, Germany, 3Department of Biomedical Informatics, Vanderbilt University School of Medicine, Nashville, TN 37232, 4Computational Biology Center, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, 5Department of Medicine, Vanderbilt-Ingram Cancer Center, Nashville, TN 37232, 6Genomics Core Laboratory, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA, 7Department I of Internal Medicine and Center of Integrated Oncology Köln – Bonn, University of Cologne and 8Chemical Genomics Center of the Max Planck Society, Dortmund, Germany
                Author notes
                *To whom correspondence should be addressed. Tel: +1 615 936 3831; Fax: +1 615 343 7602; Email: william.pao@ 123456vanderbilt.edu
                Article
                gkq579
                10.1093/nar/gkq579
                2978357
                20587502
                71b59dbd-10b6-448d-a90b-32761679412d
                © The Author(s) 2010. Published by Oxford University Press.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 8 March 2010
                : 18 May 2010
                : 9 June 2010
                Categories
                Genomics

                Genetics
                Genetics

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