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Updated: Nov 2, 2025
Author Spotlight: Advancements in Molecular Biomarker Testing for Non-Squamous Non-Small Cell Lung Cancer
Published on: September 8, 2023
Detecting ALK Rearrangement with RT-PCR: A Reliable Approach Compared with Next-Generation Sequencing in Patients
Yukun Kuang1,2, Peihang Xu1,2, Jiyu Wang1,2
1Division of Pulmonary and Critical Care Medicine, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.
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.
Insights
Reverse transcription polymerase chain reaction (RT-PCR) is a reliable method for detecting echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion in non-small-cell lung cancer (NSCLC). RT-PCR showed higher sensitivity than next-generation sequencing (NGS) in detecting ALK fusion in NSCLC patients.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Accurate detection of anaplastic lymphoma kinase (ALK) rearrangement is crucial for guiding ALK-targeted tyrosine kinase inhibitor (ALK-TKI) therapy in non-small-cell lung cancer (NSCLC).
- While next-generation sequencing (NGS) is widely used, DNA-based NGS can yield false-negative results for fusion gene detection.
- RNA-based reverse transcription polymerase chain reaction (RT-PCR) offers higher sensitivity for detecting fusion genes at the transcription level.
Purpose of the Study:
- To compare the diagnostic performance of RT-PCR and NGS for detecting the echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion gene in Chinese NSCLC patients.
- To evaluate the reliability of RT-PCR versus NGS in identifying ALK fusions relevant for targeted therapy.
Main Methods:
- Analysis of formalin-fixed paraffin-embedded tissues from 153 NSCLC patients.
- Detection of EML4-ALK fusion using both DNA/RNA-based NGS and RNA-based RT-PCR.
- Confirmation of discordant results using fluorescence in situ hybridization (FISH) or Sanger sequencing.
Main Results:
- A total of 124 samples were successfully analyzed by both methods, with a 95.16% concordance rate (118/124).
- RT-PCR identified five cases as ALK fusion-positive that were negative by NGS.
- NGS identified one case as ALK fusion-positive that was negative by RT-PCR; four discordant cases were validated by FISH or Sanger sequencing.
Conclusions:
- RNA-based RT-PCR demonstrates superior reliability and sensitivity compared to NGS for detecting EML4-ALK fusion in NSCLC.
- RT-PCR is a valuable and accurate method for identifying patients who may benefit from ALK-TKI treatment.
