Custom gene capture and next-generation sequencing to resolve discordant ALK status by FISH and IHC in lung adenocarcinoma

Jin Sung Jang, Xiaoke Wang, Peter T. Vedell, Ji Wen, Jinghui Zhang, David W. Ellison, Jared M. Evans, Sarah H. Johnson, Ping Yang, William R. Sukov, Andre M. Oliveir, George Vasmatzis, Zhifu D Sun, Jin Jen, Eunhee S. Yi

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Introduction: 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. Methods: 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. Results: 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. Conclusions: ALK FISH results appeared to be falsepositive 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 formalinfixed paraffin embedded samples was effective for detecting rearrangements involving ALK and other actionable oncogenes.

Original languageEnglish (US)
Pages (from-to)1891-1900
Number of pages10
JournalJournal of Thoracic Oncology
Volume11
Issue number11
DOIs
StatePublished - Nov 13 2016

Fingerprint

Fluorescence In Situ Hybridization
Genes
Gene Fusion
Clone Cells
Adenocarcinoma of lung
Transcriptome
Paraffin
Phosphotransferases
Micrococcal Nuclease
Kinesin
Proto-Oncogenes
Antibodies
Gene Rearrangement
Telomerase
Oncogenes
Formaldehyde

Keywords

  • ALK
  • Discordant
  • FISH
  • IHC
  • Lung
  • NGS

ASJC Scopus subject areas

  • Oncology
  • Pulmonary and Respiratory Medicine

Cite this

Custom gene capture and next-generation sequencing to resolve discordant ALK status by FISH and IHC in lung adenocarcinoma. / Jang, Jin Sung; Wang, Xiaoke; Vedell, Peter T.; Wen, Ji; Zhang, Jinghui; Ellison, David W.; Evans, Jared M.; Johnson, Sarah H.; Yang, Ping; Sukov, William R.; Oliveir, Andre M.; Vasmatzis, George; Sun, Zhifu D; Jen, Jin; Yi, Eunhee S.

In: Journal of Thoracic Oncology, Vol. 11, No. 11, 13.11.2016, p. 1891-1900.

Research output: Contribution to journalArticle

Jang, JS, Wang, X, Vedell, PT, Wen, J, Zhang, J, Ellison, DW, Evans, JM, Johnson, SH, Yang, P, Sukov, WR, Oliveir, AM, Vasmatzis, G, Sun, ZD, Jen, J & Yi, ES 2016, 'Custom gene capture and next-generation sequencing to resolve discordant ALK status by FISH and IHC in lung adenocarcinoma', Journal of Thoracic Oncology, vol. 11, no. 11, pp. 1891-1900. https://doi.org/10.1016/j.jtho.2016.06.001
Jang, Jin Sung ; Wang, Xiaoke ; Vedell, Peter T. ; Wen, Ji ; Zhang, Jinghui ; Ellison, David W. ; Evans, Jared M. ; Johnson, Sarah H. ; Yang, Ping ; Sukov, William R. ; Oliveir, Andre M. ; Vasmatzis, George ; Sun, Zhifu D ; Jen, Jin ; Yi, Eunhee S. / Custom gene capture and next-generation sequencing to resolve discordant ALK status by FISH and IHC in lung adenocarcinoma. In: Journal of Thoracic Oncology. 2016 ; Vol. 11, No. 11. pp. 1891-1900.
@article{9ef5dfecbc04468b911c80fdf49ec347,
title = "Custom gene capture and next-generation sequencing to resolve discordant ALK status by FISH and IHC in lung adenocarcinoma",
abstract = "Introduction: 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. Methods: 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. Results: 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. Conclusions: ALK FISH results appeared to be falsepositive 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 formalinfixed paraffin embedded samples was effective for detecting rearrangements involving ALK and other actionable oncogenes.",
keywords = "ALK, Discordant, FISH, IHC, Lung, NGS",
author = "Jang, {Jin Sung} and Xiaoke Wang and Vedell, {Peter T.} and Ji Wen and Jinghui Zhang and Ellison, {David W.} and Evans, {Jared M.} and Johnson, {Sarah H.} and Ping Yang and Sukov, {William R.} and Oliveir, {Andre M.} and George Vasmatzis and Sun, {Zhifu D} and Jin Jen and Yi, {Eunhee S.}",
year = "2016",
month = "11",
day = "13",
doi = "10.1016/j.jtho.2016.06.001",
language = "English (US)",
volume = "11",
pages = "1891--1900",
journal = "Journal of Thoracic Oncology",
issn = "1556-0864",
publisher = "International Association for the Study of Lung Cancer",
number = "11",

}

TY - JOUR

T1 - Custom gene capture and next-generation sequencing to resolve discordant ALK status by FISH and IHC in lung adenocarcinoma

AU - Jang, Jin Sung

AU - Wang, Xiaoke

AU - Vedell, Peter T.

AU - Wen, Ji

AU - Zhang, Jinghui

AU - Ellison, David W.

AU - Evans, Jared M.

AU - Johnson, Sarah H.

AU - Yang, Ping

AU - Sukov, William R.

AU - Oliveir, Andre M.

AU - Vasmatzis, George

AU - Sun, Zhifu D

AU - Jen, Jin

AU - Yi, Eunhee S.

PY - 2016/11/13

Y1 - 2016/11/13

N2 - Introduction: 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. Methods: 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. Results: 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. Conclusions: ALK FISH results appeared to be falsepositive 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 formalinfixed paraffin embedded samples was effective for detecting rearrangements involving ALK and other actionable oncogenes.

AB - Introduction: 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. Methods: 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. Results: 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. Conclusions: ALK FISH results appeared to be falsepositive 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 formalinfixed paraffin embedded samples was effective for detecting rearrangements involving ALK and other actionable oncogenes.

KW - ALK

KW - Discordant

KW - FISH

KW - IHC

KW - Lung

KW - NGS

UR - http://www.scopus.com/inward/record.url?scp=84995976636&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84995976636&partnerID=8YFLogxK

U2 - 10.1016/j.jtho.2016.06.001

DO - 10.1016/j.jtho.2016.06.001

M3 - Article

C2 - 27343444

AN - SCOPUS:84995976636

VL - 11

SP - 1891

EP - 1900

JO - Journal of Thoracic Oncology

JF - Journal of Thoracic Oncology

SN - 1556-0864

IS - 11

ER -