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PLoS One. 2019 Sep 12;14(9):e0222233. doi: 10.1371/journal.pone.0222233. eCollection 2019.

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.

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Department of Thoracic Oncology, Osaka International Cancer Institute, Osaka, Osaka, Japan.
Laboratory of Medical Genomics, Nara Institute of Science and Technology, Ikoma, Nara, Japan.



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.


We 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.


We 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.

Conflict of interest statement

I have read the journal's policy and the authors of this manuscript have the following competing interests: Laboratory of Medical Genomics in Nara Institute of Science and Technology is an endowed chair for KiK and YK provided by Gene Metrics LLC. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

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