Click
here to close Hello! We notice that
you are using Internet Explorer, which is not supported by Echinobase
and may cause the site to display incorrectly. We suggest using a
current version of Chrome,
FireFox,
or Safari.
Detecting ALK Rearrangement with RT-PCR: A Reliable Approach Compared with Next-Generation Sequencing in Patients with NSCLC.
Kuang Y
,
Xu P
,
Wang J
,
Zheng Y
,
Sun X
,
Li Z
,
Gan R
,
Li H
,
Guo Y
,
Yao F
,
Zhu C
,
Ke Z
,
Tang K
.
Abstract
BACKGROUND: Precise detection of anaplastic lymphoma kinase (ALK) rearrangement guides the application of ALK-targeted tyrosine kinase inhibitors (ALK-TKIs) in patients with non-small-cell lung cancer (NSCLC). Next-generation sequencing (NGS) has been widely used in clinics, but DNA-based NGS used to detect fusion genes has delivered false-negative results. However, fusion genes can be successfully detected at the transcription level and with higher sensitivity using RNA-based reverse transcription polymerase chain reaction (RT-PCR).
OBJECTIVE: This study compared the performance of RT-PCR and NGS in the detection of echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion in Chinese patients with NSCLC.
METHODS: Formalin-fixed paraffin-embedded tissues from 153 patients who were pathologically diagnosed as having NSCLC were collected from November 2017 to October 2019. Both DNA/RNA-based NGS and RNA-based RT-PCR were used to detect EML4-ALK fusion. For samples with discordant ALK status results, fluorescence in situ hybridization (FISH) or Sanger sequencing was used to further confirm the ALK status.
RESULTS: In total, 124 samples were successfully analyzed using both NGS and RT-PCR. For 118 samples, results were consistent between NGS and RT-PCR, with 25 reported as ALK fusion positive and 93 as ALK fusion negative, achieving a concordance rate of 95.16%. Among the six samples with disconcordant results, five were positive using RT-PCR but negative using NGS, and one was positive using NGS but negative using RT-PCR. Four of six cases with disconcordant results (three RT-PCR positive and one NGS positive) were successfully validated using either FISH or Sanger sequencing.
CONCLUSIONS: Compared with NGS, RT-PCR appears to be a reliable method of detecting EML4-ALK fusion in patients with NSCLC.
Fig. 1. Flowchart showing the selection of study participants. ALK anaplastic lymphoma kinase, FISH fluorescence in situ hybridization, NGS next-generation sequencing, NSCLC non-small-cell lung cancer, RT-PCR reverse transcription polymerase chain reaction
Fig. 2. Consistency of the two methods of detecting EML4-ALK fusion. a The relative level of EML4-ALK fusion (2−ΔCT value) tested using RT-PCR in DNA-NGS-positive, RNA-NGS-positive, and NGS-negative samples was compared. b The CT value of EML4-ALK in NGS-positive and NGS-negative samples (red dots indicate high CT values). *P <0.05 unless specified otherwise. ALK anaplastic lymphoma kinase, EML4 echinoderm microtubule-associated protein-like 4, CT cycle threshold, NGS next-generation sequencing, RT-PCR reverse transcription polymerase chain reaction
Fig. 3. Results of ALK status confirmed using fluorescence in situ hybridization (FISH) or Sanger sequencing. ALK status of a specimen F182120-1A tested using reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing; b specimen P01002-1B tested using RT-PCR and FISH (magnification × 60); c specimen 1911802 tested using RT-PCR and FISH (magnification × 60); d specimen 1910754-1 tested using RT-PCR and FISH (magnification × 60); e specimen 1902861 tested using RT-PCR; and f specimen 1910882 tested using FISH (magnification × 60). White arrows indicate split red-green signal indicative of EML4-ALK fusion, red arrows indicate isolated red signals indicative of EML4-ALK fusion. ALK anaplastic lymphoma kinase
Benayed,
High Yield of RNA Sequencing for Targetable Kinase Fusions in Lung Adenocarcinomas with No Mitogenic Driver Alteration Detected by DNA Sequencing and Low Tumor Mutation Burden.
2019, Pubmed
Benayed,
High Yield of RNA Sequencing for Targetable Kinase Fusions in Lung Adenocarcinomas with No Mitogenic Driver Alteration Detected by DNA Sequencing and Low Tumor Mutation Burden.
2019,
Pubmed
Davies,
DNA-Based versus RNA-Based Detection of MET Exon 14 Skipping Events in Lung Cancer.
2019,
Pubmed
Heriyanto,
The Prevalence of the EML4-ALK Fusion Gene in Cytology Specimens from Patients with Lung Adenocarcinoma.
2020,
Pubmed
Kamps,
Next-Generation Sequencing in Oncology: Genetic Diagnosis, Risk Prediction and Cancer Classification.
2017,
Pubmed
Lee,
Lower T Regulatory and Th17 Cell Populations Predicted by RT-PCR-Amplified FOXP3 and RORγt Genes Are Not Rare in Patients With Primary Immunodeficiency Diseases.
2020,
Pubmed
Letovanec,
Evaluation of NGS and RT-PCR Methods for ALK Rearrangement in European NSCLC Patients: Results from the European Thoracic Oncology Platform Lungscape Project.
2018,
Pubmed
Li,
Intergenic Breakpoints Identified by DNA Sequencing Confound Targetable Kinase Fusion Detection in NSCLC.
2020,
Pubmed
Lih,
Analytical Validation of the Next-Generation Sequencing Assay for a Nationwide Signal-Finding Clinical Trial: Molecular Analysis for Therapy Choice Clinical Trial.
2017,
Pubmed
Liu,
Frequency, clinical features and differential response to therapy of concurrent ALK/EGFR alterations in Chinese lung cancer patients.
2019,
Pubmed
Lu,
Comparison of EML4-ALK fusion gene positive rate in different detection methods and samples of non-small cell lung cancer.
2020,
Pubmed
Rogers,
Comparison of methods in the detection of ALK and ROS1 rearrangements in lung cancer.
2015,
Pubmed
Schmittgen,
Analyzing real-time PCR data by the comparative C(T) method.
2008,
Pubmed
Shaw,
Ceritinib in ALK-rearranged non-small-cell lung cancer.
2014,
Pubmed
Smits,
The estimation of tumor cell percentage for molecular testing by pathologists is not accurate.
2014,
Pubmed
Soda,
Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer.
2007,
Pubmed
,
Echinobase
Solomon,
First-line crizotinib versus chemotherapy in ALK-positive lung cancer.
2014,
Pubmed
Tao,
Distribution of EML4-ALK fusion variants and clinical outcomes in patients with resected non-small cell lung cancer.
2020,
Pubmed
,
Echinobase
Teixidó,
Concordance of IHC, FISH and RT-PCR for EML4-ALK rearrangements.
2014,
Pubmed
,
Echinobase
Thunnissen,
The challenge of NSCLC diagnosis and predictive analysis on small samples. Practical approach of a working group.
2012,
Pubmed
Wang,
EML4-ALK Fusion Detected by RT-PCR Confers Similar Response to Crizotinib as Detected by FISH in Patients with Advanced Non-Small-Cell Lung Cancer.
2015,
Pubmed
Wang,
Feasibility of cytological specimens for ALK fusion detection in patients with advanced NSCLC using the method of RT-PCR.
2016,
Pubmed
Wen,
Genomic Signature of Driver Genes Identified by Target Next-Generation Sequencing in Chinese Non-Small Cell Lung Cancer.
2019,
Pubmed
Wu,
Comparison of IHC, FISH and RT-PCR methods for detection of ALK rearrangements in 312 non-small cell lung cancer patients in Taiwan.
2013,
Pubmed
,
Echinobase
Zheng,
Genotyping of Cerebrospinal Fluid Associated With Osimertinib Response and Resistance for Leptomeningeal Metastases in EGFR-Mutated NSCLC.
2021,
Pubmed