Dataset Information

Comparison of IHC, FISH and RT-PCR methods for detection of ALK rearrangements in 312 non-small cell lung cancer patients in Taiwan.

ABSTRACT:

Background

Recently Echinoderm microtubule-associated protein-like 4- anaplastic lymphoma kinase (EML4-ALK) fusion gene has become an important biomarker for ALK tyrosine kinase inhibitor (crizotinib) treatment in NSCLC. However, the best detection method and the significance of EML4-ALK variant types remain uncertain.

Methods

Reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescence in Situ hybridization (FISH) and Immunohistochemical (IHC) stain were performed on tumor tissues of 312 NSCLC patients for detection of ALK rearrangements. Mutation analyses for EGFR and KRAS genes were also performed.

Results

Thirteen of the 312 patients (4.17%) had ALK rearrangements detected by RT-PCR. If RT-PCR data was used as the gold standard, FISH tests had a low sensitivity (58.33%), but very good specificity (99.32%). IHC stain had better sensitivity (91.67%) than FISH, but lower specificity (79.52%), when the cut off was IHC2+. All of the 8 patients with high abundance of EML4-ALK positive cells in tumor tissues (assessed by the signal intensities of the RT-PCR product), were also have high expression of ALK protein (IHC3+), and positive for FISH, except one failed in FISH. Variants 3a+3b (4/5, 80%) of EML4-ALK fusion gene were more common to have high abundance of EML4-ALK positive cells in tumor tissues than variant 1 (1/3, 33.3%). Meta-analysis of the published data of 2273 NSCLC patients revealed that variant 3 (23/44, 52.3%) was the most common type in Chinese population, while variant 1 (28/37, 75.7%) was most common in Caucasian.

Conclusions

Among the three detection methods, RT-PCR could detect not only the presence of EML4-ALK fusion gene and their variant types, but also the abundance of EML4-ALK positive cells in NSCLC tumor tissues. The latter two factors might affect the treatment response to anti-ALK inhibitor. Including RT-PCR as a diagnostic test for ALK inhibitor treatment in the prospective clinical trials is recommended.

SUBMITTER: Wu YC

PROVIDER: S-EPMC3737393 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Publications

Comparison of IHC, FISH and RT-PCR methods for detection of ALK rearrangements in 312 non-small cell lung cancer patients in Taiwan.

Wu Yi-Cheng YC Chang Il-Chi IC Wang Chi-Liang CL Chen Tai-Di TD Chen Ya-Ting YT Liu Hui-Ping HP Chu Yen Y Chiu Yu-Ting YT Wu Tzu-Hua TH Chou Li-Hui LH Chen Yi-Rong YR Huang Shiu-Feng SF

PloS one 20130807 8

<h4>Background</h4>Recently Echinoderm microtubule-associated protein-like 4- anaplastic lymphoma kinase (EML4-ALK) fusion gene has become an important biomarker for ALK tyrosine kinase inhibitor (crizotinib) treatment in NSCLC. However, the best detection method and the significance of EML4-ALK variant types remain uncertain.<h4>Methods</h4>Reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescence in Situ hybridization (FISH) and Immunohistochemical (IHC) stain were performed on tu ...[more]

PMID: 23951022

Similar Datasets

Project description:IntroductionWe 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.MethodsDNA 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.ResultsIn 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.ConclusionsALK 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.

Dataset Information

Comparison of IHC, FISH and RT-PCR methods for detection of ALK rearrangements in 312 non-small cell lung cancer patients in Taiwan.

Background

Methods

Results

Conclusions

Publications

Comparison of IHC, FISH and RT-PCR methods for detection of ALK rearrangements in 312 non-small cell lung cancer patients in Taiwan.

Similar Datasets

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