title: Sensitive and specific detection of EML4-ALK rearrangements in non-small cell lung cancer (NSCLC) specimens by multiplex amplicon RNA massive parallel sequencing.
abstract: OBJECTIVE
  Recurrent gene fusions of anaplastic lymphoma receptor tyrosine kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) have been recently identified in ∼5% of non-small cell lung cancers (NSCLCs) and are targets for selective tyrosine kinase inhibitors. While fluorescent in situ hybridization (FISH) is the current gold standard for detection of EML4-ALK rearrangements, several limitations exist including high costs, time-consuming evaluation and somewhat equivocal interpretation of results. In contrast, targeted massive parallel sequencing has been introduced as a powerful method for simultaneous and sensitive detection of multiple somatic mutations even in limited biopsies, and is currently evolving as the method of choice for molecular diagnostic work-up of NSCLCs.
  
  
  METHODS
  We developed a novel approach for indirect detection of EML4-ALK rearrangements based on 454 massive parallel sequencing after reverse transcription and subsequent multiplex amplification (multiplex ALK RNA-seq) which takes advantage of unbalanced expression of the 5' and 3' ALK mRNA regions. Two lung cancer cell lines and a selected series of 32 NSCLC samples including 11 cases with EML4-ALK rearrangement were analyzed with this novel approach in comparison to ALK FISH, ALK qRT-PCR and EML4-ALK RT-PCR.
  
  
  RESULTS
  The H2228 cell line with known EML4-ALK rearrangement showed 171 and 729 reads for 5' and 3' ALK regions, respectively, demonstrating a clearly unbalanced expression pattern. In contrast, the H1299 cell line with ALK wildtype status displayed no reads for both ALK regions. Considering a threshold of 100 reads for 3' ALK region as indirect indicator of EML4-ALK rearrangement, there was 100% concordance between the novel multiplex ALK RNA-seq approach and ALK FISH among all 32 NSCLC samples.
  
  
  CONCLUSIONS
  Multiplex ALK RNA-seq is a sensitive and specific method for indirect detection of EML4-ALK rearrangements, and can be easily implemented in panel based molecular diagnostic work-up of NSCLCs by massive parallel sequencing.
authors.affiliation: Institute of Pathology, University Hospital Erlangen, Erlangen, Germany.
authors.forename: Evgeny A
authors.initials: EA
authors.lastname: Moskalev
authors.affiliation: Institute of Pathology, University Hospital Erlangen, Erlangen, Germany.
authors.forename: Judith
authors.initials: J
authors.lastname: Frohnauer
authors.affiliation: Institute of Pathology, University Hospital Cologne, Cologne, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Merkelbach-Bruse
authors.affiliation: Institute of Pathology, University Hospital Cologne, Cologne, Germany.
authors.forename: Hans-Ulrich
authors.initials: HU
authors.lastname: Schildhaus
authors.affiliation: Institute of Pathology, St. Vincentius Hospital, Karlsruhe, Germany.
authors.forename: Arno
authors.initials: A
authors.lastname: Dimmler
authors.affiliation: Institute of Pathology, Speyer, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Schubert
authors.affiliation: Institute of Pathology, SRH-Waldklinikum, Gera, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Boltze
authors.affiliation: Practice for Pathology, Ingolstadt, Germany.
authors.forename: Helmut
authors.initials: H
authors.lastname: König
authors.affiliation: Department of Gastroenterology, Pneumology and Endocrinology, University Hospital Erlangen, Erlangen, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Fuchs
authors.affiliation: Department of Thoracic Surgery, University Hospital Erlangen, Erlangen, Germany.
authors.forename: Horia
authors.initials: H
authors.lastname: Sirbu
authors.affiliation: Institute of Pathology, University Hospital Erlangen, Erlangen, Germany.
authors.forename: Ralf J
authors.initials: RJ
authors.lastname: Rieker
authors.affiliation: Institute of Pathology, University Hospital Erlangen, Erlangen, Germany.
authors.forename: Abbas
authors.initials: A
authors.lastname: Agaimy
authors.affiliation: Institute of Pathology, University Hospital Erlangen, Erlangen, Germany.
authors.forename: Arndt
authors.initials: A
authors.lastname: Hartmann
authors.affiliation: Institute of Pathology, University Hospital Erlangen, Erlangen, Germany. Electronic address: florian.haller@uk-erlangen.de.
authors.forename: Florian
authors.initials: F
authors.lastname: Haller
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics*; D005260:Female; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D015514:Oncogene Proteins, Fusion / Q000032:analysis* / Q000235:genetics*; D012680:Sensitivity and Specificity; D017423:Sequence Analysis, RNA / Q000379:methods
chemical_list: C522504:EML4-ALK fusion protein, human; D015514:Oncogene Proteins, Fusion
keywords: 454 massive parallel sequencing; ALK rearrangement; EML4-ALK; Molecular diagnostic; NSCLC