title: Exon scanning by reverse transcriptase-polymerase chain reaction for detection of known and novel EML4-ALK fusion variants in non-small cell lung cancer.
abstract: Chromosomal inversions within chromosome 2p, resulting in fusions between the echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) genes, are a recent focus of treatment options for non-small cell lung cancer. Thirteen EML4-ALK fusion variants have been identified, affecting eight EML4 exons. We have developed an exon scanning approach using multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify known and potential variants involving the first 22 EML4 exons. A total of 55 formalin-fixed, paraffin-embedded lung cancer tumors were screened, of which 5 (9%) were positive for EML4-ALK fusions. Four positive cases harbored known fusion variants: variant 3a, 3b, or both in three cases and variant 1 in one case. The fifth positive specimen harbored two novel variants, designated 8a and 8b, involving exon 17 of EML4. Fluorescence in situ hybridization confirmed the presence of EML4-ALK fusions in three of the four RT-PCR-positive specimens with sufficient tissue for examination, and also confirmed absence of fusions in all 19 RT-PCR-negative specimens tested. Immunohistochemistry analysis confirmed ALK protein expression in the sample containing the novel 8a and 8b variants. This RT-PCR-based exon scanning approach avoids the limitations of screening only for previously identified EML4-ALK fusions and provides a simple molecular assay for fusion detection in a clinical diagnostics setting.
authors.affiliation: Department of Hematology and Oncology, Quest Diagnostics Nichols Institute, San Juan Capistrano, CA, USA. heather.r.sanders@QuestDiagnostics.com
authors.forename: Heather R
authors.initials: HR
authors.lastname: Sanders
authors.forename: Hai-Rong
authors.initials: HR
authors.lastname: Li
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Bruey
authors.forename: Jay A
authors.initials: JA
authors.lastname: Scheerle
authors.forename: Aurelia M
authors.initials: AM
authors.lastname: Meloni-Ehrig
authors.forename: JoAnn C
authors.initials: JC
authors.lastname: Kelly
authors.forename: Constance
authors.initials: C
authors.lastname: Novick
authors.forename: Maher
authors.initials: M
authors.lastname: Albitar
mesh_terms: D000595:Amino Acid Sequence; D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis* / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000378:metabolism; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism; D005091:Exons; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D017404:In Situ Hybridization, Fluorescence; D008175:Lung Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D008969:Molecular Sequence Data; D015514:Oncogene Proteins, Fusion / Q000737:chemistry / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; C522504:EML4-ALK fusion protein, human; D015514:Oncogene Proteins, Fusion; D003851:Deoxyribonucleases