title: Custom Gene Capture and Next-Generation Sequencing to Resolve Discordant ALK Status by FISH and IHC in Lung Adenocarcinoma.
abstract: We performed a genomic study in lung adenocarcinoma cases with discordant anaplastic lymphoma receptor tyrosine kinase gene (ALK) status by fluorescent in situ hybridization (FISH) and immunohistochemical (IHC) analysis.
  
  
  
  DNA from formalin-fixed paraffin-embedded tissues of 16 discordant (four FISH-positive/IHC-negative and 12 FISH-negative/IHC-positive) cases by Vysis ALK Break Apart FISH and ALK IHC testing (ALK1 clone) were subjected to whole gene capture and next-generation sequencing (NGS) of nine genes, including ALK, echinoderm microtubule associated protein like 4 gene (EML4), kinesin family member 5B gene (KIF5B), staphylococcal nuclease and tudor domain containing 1 gene (SND1), BRAF, ret proto-oncogene (RET), ezrin gene (EZR), ROS1, and telomerase reverse transcriptase (TERT). All discordant cases (except one FISH-negative/IHC-positive case without sufficient tissue) were analyzed by IHC with D5F3 antibody. In one case with fresh frozen tissue, whole transcriptome sequencing was also performed. Twenty-six concordant (16 FISH-positive/IHC-positive and 10 FISH-negative/IHC-negative) cases were included as controls.
  
  
  
  In four ALK FISH-positive/IHC-negative cases, no EML4-ALK fusion gene was observed by NGS, but in one case using fresh frozen tissue, we identified EML4-baculoviral AIP repeat containing 6 gene (BIRC6) and AP2 associated kinase 1 gene (AAK1)-ALK fusion genes. Whole transcriptome sequencing revealed a highly expressed EML4-BIRC6 fusion transcript and a minimally expressed AAK1 transcript. Among the 12 FISH-negative/IHC-positive cases, no evidence of ALK gene rearrangement was detected by NGS. Eleven of 12 FISH-negative/IHC-positive cases detected by ALK1 clone were concordant by repeat ALK IHC with D5F3 antibody (i.e., FISH-negative/IHC-negative by D5F3 clone). Among the 16 ALK FISH-positive/IHC-positive positive controls, whole gene capture identified ALK gene fusion in 15 cases, including in one case with Huntington interacting protein 1 gene (HIP1)-ALK. No ALK fusion gene was observed in any of the 10 FISH-negative/IHC-negative cases. Other fusion genes involving ROS1, EZR, BRAF, and SND1 were also found.
  
  
  
  ALK FISH results appeared to be false-positive in three of four FISH-positive/IHC-negative cases, whereas no false-negative ALK FISH case was identified among 12 ALK FISH-negative/IHC-positive cases by ALK1 clone, which was in keeping with the concordant FISH-negative/IHC-negative status by D5F3 clone. Our targeted whole gene capture approach using formalin-fixed paraffin embedded samples was effective for detecting rearrangements involving ALK and other actionable oncogenes.
authors.affiliation: Genome Analysis Core, Medical Genome Facility, Mayo Clinic, Rochester, Minnesota; Center for Individualized Medicine, Mayo Clinic, Rochester, Minnesota.
authors.forename: Jin Sung
authors.initials: JS
authors.lastname: Jang
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Xiaoke
authors.initials: X
authors.lastname: Wang
authors.affiliation: Division of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, Minnesota.
authors.forename: Peter T
authors.initials: PT
authors.lastname: Vedell
authors.affiliation: Department of Pathology, St. Jude Children's Research Hospital, Memphis, Tennessee.
authors.forename: Ji
authors.initials: J
authors.lastname: Wen
authors.affiliation: Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, Tennessee.
authors.forename: Jinghui
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Pathology, St. Jude Children's Research Hospital, Memphis, Tennessee.
authors.forename: David W
authors.initials: DW
authors.lastname: Ellison
authors.affiliation: Division of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, Minnesota.
authors.forename: Jared M
authors.initials: JM
authors.lastname: Evans
authors.affiliation: Center for Individualized Medicine, Mayo Clinic, Rochester, Minnesota.
authors.forename: Sarah H
authors.initials: SH
authors.lastname: Johnson
authors.affiliation: Division of Epidemiology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Ping
authors.initials: P
authors.lastname: Yang
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota.
authors.forename: William R
authors.initials: WR
authors.lastname: Sukov
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Andre M
authors.initials: AM
authors.lastname: Oliveira
authors.affiliation: Center for Individualized Medicine, Mayo Clinic, Rochester, Minnesota.
authors.forename: George
authors.initials: G
authors.lastname: Vasmatzis
authors.affiliation: Division of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, Minnesota.
authors.forename: Zhifu
authors.initials: Z
authors.lastname: Sun
authors.affiliation: Genome Analysis Core, Medical Genome Facility, Mayo Clinic, Rochester, Minnesota; Center for Individualized Medicine, Mayo Clinic, Rochester, Minnesota; Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Jin
authors.initials: J
authors.lastname: Jen
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota. Electronic address: Yi.Joanne@Mayo.edu.
authors.forename: Eunhee S
authors.initials: ES
authors.lastname: Yi
mesh_terms: D000230:Adenocarcinoma / Q000201:enzymology* / Q000235:genetics* / Q000473:pathology; D000077192:Adenocarcinoma of Lung; D000077548:Anaplastic Lymphoma Kinase; D023281:Genomics / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008175:Lung Neoplasms / Q000201:enzymology* / Q000235:genetics* / Q000473:pathology; D000090063:Proto-Oncogene Mas; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism
chemical_list: C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D020794:Receptor Protein-Tyrosine Kinases
keywords: ALK; Discordant; FISH; IHC; Lung; NGS