Title: A Multiplexed Amplicon Approach for Detecting Gene Fusions by Next-Generation Sequencing
Abstract: Chromosomal rearrangements that result in oncogenic gene fusions are clinically important drivers of many cancer types. Rapid and sensitive methods are therefore needed to detect a broad range of gene fusions in clinical specimens that are often of limited quantity and quality. We describe a next-generation sequencing approach that uses a multiplex PCR-based amplicon panel to interrogate fusion transcripts that involve 19 driver genes and 94 partners implicated in solid tumors. The panel also includes control assays that evaluate the 3′/5′ expression ratios of 12 oncogenic kinases, which might be used to infer gene fusion events when the partner is unknown or not included on the panel. There was good concordance between the solid tumor fusion gene panel and other methods, including fluorescence in situ hybridization, real-time PCR, Sanger sequencing, and other next-generation sequencing panels, because 40 specimens known to harbor gene fusions were correctly identified. No specific fusion reads were observed in 59 fusion-negative specimens. The 3′/5′ expression ratio was informative for fusions that involved ALK, RET, and NTRK1 but not for BRAF or ROS1 fusions. However, among 37 ALK or RET fusion-negative specimens, four exhibited elevated 3′/5′ expression ratios, indicating that fusions predicted solely by 3′/5′ read ratios require confirmatory testing. Chromosomal rearrangements that result in oncogenic gene fusions are clinically important drivers of many cancer types. Rapid and sensitive methods are therefore needed to detect a broad range of gene fusions in clinical specimens that are often of limited quantity and quality. We describe a next-generation sequencing approach that uses a multiplex PCR-based amplicon panel to interrogate fusion transcripts that involve 19 driver genes and 94 partners implicated in solid tumors. The panel also includes control assays that evaluate the 3′/5′ expression ratios of 12 oncogenic kinases, which might be used to infer gene fusion events when the partner is unknown or not included on the panel. There was good concordance between the solid tumor fusion gene panel and other methods, including fluorescence in situ hybridization, real-time PCR, Sanger sequencing, and other next-generation sequencing panels, because 40 specimens known to harbor gene fusions were correctly identified. No specific fusion reads were observed in 59 fusion-negative specimens. The 3′/5′ expression ratio was informative for fusions that involved ALK, RET, and NTRK1 but not for BRAF or ROS1 fusions. However, among 37 ALK or RET fusion-negative specimens, four exhibited elevated 3′/5′ expression ratios, indicating that fusions predicted solely by 3′/5′ read ratios require confirmatory testing. Chromosomal rearrangements that generate fusion genes that involve oncogenic drivers have been implicated in many cancer types. These events lead to constitutive activation of the respective fusion proteins, which in turn trigger downstream signaling pathways. The detection of gene fusions in clinical specimens may serve to clarify otherwise ambiguous diagnoses and to identify patients most likely to respond to specific molecularly targeted therapies. Given the increasing number of clinically relevant gene fusions, it is imperative that detection methods move beyond single-gene tests. The paradigm of therapy targeted to an oncogenic gene fusion was first established by the successful treatment of chronic myelogenous leukemia (CML) with the kinase inhibitor imatinib, which targets the product of the BCR-ABL fusion present in approximately 95% of CML cases.1Druker B.J. Talpaz M. Resta D.J. Peng B. Buchdunger E. Ford J.M. Lydon N.B. Kantarjian H. Capdeville R. Ohno-Jones S. Sawyers C.L. 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Identification of KIF5B-RET and GOPC-ROS1 fusions in lung adenocarcinomas through a comprehensive mRNA-based screen for tyrosine kinase fusions.Clin Cancer Res. 2012; 18: 6599-6608Crossref PubMed Scopus (156) Google Scholar Control assays were included to evaluate the 3′/5′ expression ratios of 12 of the kinases, allowing us to assess kinase domain overexpression as a means of identifying the presence of fusions to unknown partner genes.45Suehara Y. Arcila M. Wang L. Hasanovic A. Ang D. Ito T. Kimura Y. Drilon A. Guha U. Rusch V. Kris M.G. Zakowski M.F. Rizvi N. Khanin R. Ladanyi M. Identification of KIF5B-RET and GOPC-ROS1 fusions in lung adenocarcinomas through a comprehensive mRNA-based screen for tyrosine kinase fusions.Clin Cancer Res. 2012; 18: 6599-6608Crossref PubMed Scopus (156) Google Scholar The multiplexed assay panel requires only 20 ng of input RNA and uses small amplicons (mean, 136 bp) to accommodate formalin-fixed, paraffin-embedded (FFPE) specimens. Data analysis entails a straightforward tabulation of reads mapped to assay amplicons. A total of 105 specimens of known gene fusion status were evaluated, and the panel revealed good concordance with FISH, real-time PCR, Sanger sequencing, and next-generation sequencing. Blocks of FFPE tumor or normal tissue, unstained sections of FFPE tissue, and frozen tissue specimens were obtained from the pathology archives of Oregon Health & Science University and the University of Pittsburgh Medical Center. For FFPE specimens, tumor-rich areas (30% to 90%) were dissected from unstained sections by comparison with an hematoxylin and eosin–stained slide. Nucleic acids were extracted manually using a Nucleospin kit (Macherey-Nagel, Bethlehem, PA) or on a QIAcube instrument using the DNeasy Blood/Tissue kit (Qiagen, Valencia, CA). Nucleic acids were extracted from homogenized frozen specimens and fine-needle aspirates manually using TRIzol reagent (Thermo Fisher Scientific, Austin, TX) or using a MagnaPure Compact instrument with the MagnaPure Compact Nucleic Acid Isolation kit (Roche, Basel, Switzerland). DNA was digested with DNase (ArcticZymes, Tromso, Norway), and purified RNA was quantified with a Qubit fluorometer (Life Technologies, Grand Island, NY). A multiplex amplicon panel composed of 200 assays divided into two pools was created using a custom AmpliSeq design pipeline (Thermo Fisher Scientific). The panel screens for 169 known gene fusions that involved 19 target genes and 94 fusion partners (Table 1). The panel also includes control amplicons representing five housekeeping gene transcripts, as well as amplicons that detect exons in the 5′ and 3′ regions of 12 of the target kinases. The latter are used to evaluate whether relative overexpression of the 3′ kinase domain is indicative of a gene fusion.Table 1Overview of Gene Fusion PanelTarget gene fusions AKT3MAGI3 ALKATIC, C2orf44, CARS, CLTC, EML4, FN1, KIF5B, KLC1, MSN, NPM1, PPFIBP1, PTPN3, SEC31A, SQSTM1, STRN, TFG, TPM3, TPM4, TRAF1, VCL BRAFAGK, AGTRAP, AKAP9, CLCN6, FAM131B, FCHSD1, GNAI1, KCTD7, KIAA1549, MAD1L1, MKRN1, NUDCD3, PLIN3, RNF130, SLC45A3, SOX6, TRIM24, ZKSCAN5 EGFREGFR variant III, CAND1, PSPH, SEPT14, SLC12A9 ERBB4EZR ERGTMPRSS2 FGFR1BAG4, CPSF6, ERLIN2, PLAG1, TACC1, ZNF703 FGFR2AFF3, AHCYL1, BICC1, CASP7, CCDC6, CIT, KIAA1967, OFD1, SLC45A3 FGFR3BAIAP2L1, TACC3 METMIR548F1, TPR NTRK1BCAN, CD74, MIR548F1, MPRIP, NFASC, TFG, TPM3, TPR NTRK2NACC2, QKI NTRK3ETV6 NRG1CD74, SLC3A2 PDGFRAKDR, SCAF11 PDGFRBNIN RAF1DAZL, ESRP1, MSS51, SRGAP3 RETAFAP1, CCDC6, ERC1, HOOK3, KIAA1468, KIF5B, NCOA4, PARG, PCM1, PRKAR1A, TRIM27, TRIM33 ROS1CCDC6, CD74, CEP85L, EZR, GOPC, KDELR2, LRIG3, SDC4, SLC34A2, TFG, TPM33′/5′ Expression Ratio Assessed ALKBRAFFGFR1FGFR2FGFR3NTRK1 NTRK2NTRK3PDGFRARAF1RETROS1Expression Control Genes HMBSITGB7LMNAMYCTBP Open table in a new tab Amplicon sequencing libraries were prepared with 20 ng of RNA (10 ng per amplicon pool), using the Ion Total RNA-Seq kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, after reverse transcription and amplification with the two multiplexed fusion primer pools, the pools were combined for each sample. Primers were subsequently digested, followed by adapter ligation and emulsion PCR. Sequencing was performed on an Ion Torrent personal genome machine with eight samples per 318 chip. For detection of gene fusions and gene expression, the raw data in FASTQ format were aligned to a custom reference genome using TMAP (from TorrentSuite version 3.4.1; https://github.com/iontorrent/TMAP; last accessed June 26, 2014) after adapter sequences were removed by Cutadapt software version 1.2.1.46Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads.EMBnet J. 2011; 17: 10-12Crossref Google Scholar The custom reference genome was assembled to contain sequences of the designed fusion transcripts, normal transcripts of the genes involved in the fusions, gene regions for differential expression analysis, and the entire hg19. FastQC software version 0.10.1 (http://www.bioinfor