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Detection of EML4-ALK in lung adenocarcinoma using pleural effusion with FISH, IHC, and RT-PCR methods.

ABSTRACT: Anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) gene rearrangements occur in approximately 5% of non-small-cell lung cancers (NSCLC), leading to the overexpression of anaplastic lymphoma kinase and predicting a response to the targeted inhibitor, crizotinib. Malignant pleural effusion occurs in most patients with advanced lung cancer, especially adenocarcinoma, and tissue samples are not always available from these patients. We attempted to clarify the feasibility of detecting the EML4-ALK fusion gene in pleural effusion cells using different methods. We obtained 66 samples of pleural effusion from NSCLC patients. The pleural effusion fluid was centrifuged, and the cellular components obtained were formalin fixed and paraffin embedded. The EML4-ALK fusion gene status was determined with fluorescent in situ hybridization (FISH), reverse transcription-polymerase chain reaction (RT-PCR), and immunohistochemistry (IHC). EML4-ALK was detected in three of 66 patient samples (4.5%) with RT-PCR. When the RT-PCR data were used as the standard, one false positive and one false negative samples were identified with IHC; and one false negative sample was identified with FISH. These results suggest that a block of pleural effusion cells can be used to detect the EML4-ALK fusion gene. IHC had good sensitivity, but low specificity. FISH had low sensitivity, but high specificity. RT-PCR is a good candidate method for detecting EML4-ALK in blocks of pleural effusion cells from lung cancer patients.

SUBMITTER: Liu L

PROVIDER: S-EPMC4364974 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

Project description:BackgroundPleural effusion (PE) caused by lung cancer is prevalent, and it is difficult to differentiate it from PE caused by tuberculosis. Exosome-based liquid biopsy offers a non-invasive technique to diagnose benign and malignant PE. Exosomal miRNAs are potential diagnostic markers and play an essential role in signal transduction and biological processes in tumor development. We hypothesized that exosomal miRNA expression profiles in PE would contribute to identifying its diagnostic markers and elucidating the molecular basis of PE formation in lung cancer.MethodsThe exosomes from PE caused by lung adenocarcinoma (LUAD) and pulmonary tuberculosis were isolated and verified by transmission electron microscopy. The exosomal miRNA profiles were identified using deep sequencing and validated with quantitative real-time PCR (qRT-PCR). We performed bioinformatic analysis for differentially expressed miRNAs to explore how exosomal miRNAs regulate pleural effusion.ResultsWe identified 99 upregulated and 91 downregulated miRNAs in malignant pleural effusion (MPE) compared to tuberculous pleural effusion (TPE). Seven differentially expressed miRNAs (DEmiRNAs) were validated by qRT-PCR, out of which 5 (71.4%) were confirmed through sequencing. Gene Ontology (GO) analysis revealed that most exosomal miRNAs target genes were involved in regulating cellular processes and nitrogen compound metabolism. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, the exosomal miRNAs target genes were mainly involved in Fc gamma R-mediated phagocytosis, Rap1 signaling pathway, and breast cancer. The hub genes, including ITGAM, FOXO1, MAPK14, YWHAB, GRIN1, and PRF1, were screened through plug-in cytoHubba. The PFR1 was identified as a critical gene in MPE formation using single-cell sequencing analysis. Additionally, we hypothesized that tumor cells affected natural killer cells and promoted the generation of PE in LUAD via the exosomal hsa-miR-3120-5p-PRF1 axis.ConclusionsWe identified exosomal miRNA profiles in LUAD-MPE and TPE, which may help in the differential diagnosis of MPE and TPE. Bioinformatic analysis revealed that these miRNAs might affect PE generation through tumor immune response in LUAD. Our results provided a new theoretical basis for understanding the function of exosomal miRNAs in LUAD-MPE.

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Detection of EML4-ALK in lung adenocarcinoma using pleural effusion with FISH, IHC, and RT-PCR methods.

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