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.

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