Quantitative detection of ALK fusion breakpoints in plasma cell-free DNA from patients with non-small cell lung cancer using PCR-based target sequencing with a tiling primer set and two-step mapping/alignment.

Kei Kunimasa,Yoji Kukita,Fumio Imamura,Kikuya Kato,Satoshi S Nishizuka

doi:10.1371/journal.pone.0222233

Kei Kunimasa, Yoji Kukita + Show 3 more

Open Access

https://doi.org/10.1371/journal.pone.0222233

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Journal: PloS one	Publication Date: Sep 12, 2019
Citations: 9	License type: CC BY 4.0

Affiliation: Osaka International Cancer Institute

Abstract

BackgroundTyrosine kinase inhibitors targeted to anaplastic lymphoma kinase (ALK) have been demonstrated to be effective for lung cancer patients with an ALK fusion gene. Application of liquid biopsy, i.e., detection and quantitation of the fusion product in plasma cell-free DNA (cfDNA), could improve clinical practice. To detect ALK fusions, because fusion breakpoints occur somewhere in intron 19 of the ALK gene, sequencing of the entire intron is required to locate breakpoints.ResultsWe constructed a target sequencing system using an adapter and a set of primers that cover the entire ALK intron 19. This system can amplify fragments, including breakpoints, regardless of fusion partners. The data analysis pipeline firstly detected fusions by alignment to selected target sequences, and then quantitated the fusion alleles aligning to the identified breakpoint sequences. Performance was validated using 20 cfDNA samples from ALK-positive non-small cell lung cancer patients and samples from 10 healthy volunteers. Sensitivity and specificity were 50 and 100%, respectively.ConclusionsWe demonstrated that PCR-based target sequencing using a tiling primer set and two-step mapping/alignment quantitatively detected ALK fusions in cfDNA from lung cancer patients. The system offers an alternative to existing approaches based on hybridization capture.

Highlights

Tyrosine kinase inhibitors targeted to anaplastic lymphoma kinase (ALK) have been demonstrated to be effective for lung cancer patients with an Anaplastic lymphoma kinase (ALK) fusion gene
The data analysis pipeline firstly detected fusions by alignment to selected target sequences, and quantitated the fusion alleles aligning to the identified breakpoint sequences
We demonstrated that PCR-based target sequencing using a tiling primer set and two-step mapping/alignment quantitatively detected ALK fusions in cell-free DNA (cfDNA) from lung cancer patients

Summary

Introduction

Anaplastic lymphoma kinase (ALK) gene fusions are found in 3–7% of non-small cell lung cancer (NSCLC) patients [1, 2]. Tyrosine kinase inhibitors (TKIs), such as crizotinib and other generation ALK inhibitors, have demonstrated efficacy for ALK fusion-positive patients in many clinical studies [1]. Reverse transcription PCR (RT-PCR) of extracted RNA is a simple method to detect known fusion types, such as echinoderm microtubule associated protein-like 4 (EML4)-ALK variants [5, 6]. Tyrosine kinase inhibitors targeted to anaplastic lymphoma kinase (ALK) have been demonstrated to be effective for lung cancer patients with an ALK fusion gene. Application of liquid biopsy, i.e., detection and quantitation of the fusion product in plasma cell-free DNA (cfDNA), could improve clinical practice. To detect ALK fusions, because fusion breakpoints occur somewhere in intron 19 of the ALK gene, sequencing of the entire intron is required to locate breakpoints

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Results

Conclusion