Detecting ALK Rearrangement with RT-PCR: A Reliable Approach Compared with Next-Generation Sequencing in Patients with NSCLC

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.

A Highly Sensitive XNA-Based RT-qPCR Assay for the Identification of ALK, RET, and ROS1 Fusions in Lung Cancer

“…Kuang et al conducted an analysis of RT-qPCR and NGS for EML4-ALK detection, reporting a concordance rate of 95.16%. In approximately 5% of discordant samples, RT-qPCR further identified additional ALK fusions among those with negative NGS results [ 42 ]. Lu et al reported the incidence of EML4-ALK detected by IHC (9.51%), RT-qPCR (11.62%), and NGS (5.84%) [ 43 ], suggesting that RT-qPCR has the potential advantage in clinical scenarios for ALK fusion detection.…”

Detecting the Neuraminidase R294K Mutation in Avian Influenza A (H7N9) Virus Using Reverse Transcription Droplet Digital PCR Method

“…Meanwhile, there are still some limitations to the RT-dd PCR method developed in this study. Compared with the advantages of NGS in detecting various types of mutations (including new or unknown drug-resistant mutations) [23,24], RT-dd PCR can only detect specific known mutation sites and cannot discover new or rare mutations [25,26]. In addition, RT-dd PCR also requires larger sample sizes and higher mutation frequency to achieve more reliable results [27].…”

Targeted RNA-sequencing analysis for fusion transcripts detection in tumor diagnostics: assessment of bioinformatic tools reliability in FFPE samples

“…Indeed, not only pre-analytical and analytical conditions, but also the biology of carcinomas could contribute to increasing the percentage of false-negative, since it is well known that carcinoma, differently from STT [30], often express chimeric transcripts at low levels [1]. Other evidence is present in the literature that supports this, e.g., the comparison of RT-PCR with an NGS pipeline for the detection of EML4-ALK fusions in non-small cell lung cancer FFPE samples [38].…”