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
Autoři:
Kei Kunimasa aff001; Kikuya Kato aff002; Fumio Imamura aff001; Yoji Kukita aff002
Působiště autorů:
Department of Thoracic Oncology, Osaka International Cancer Institute, Osaka, Osaka, Japan
aff001; Laboratory of Medical Genomics, Nara Institute of Science and Technology, Ikoma, Nara, Japan
aff002
Vyšlo v časopise:
PLoS ONE 14(9)
Kategorie:
Research Article
doi:
https://doi.org/10.1371/journal.pone.0222233
Souhrn
Background
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.
Results
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.
Conclusions
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.
Klíčová slova:
Biology and life sciences – Computational biology – Introns – Genetics – Genomics – Genome complexity – Genome analysis – Transcriptome analysis – Cell biology – Cell physiology – Cell fusion – Anatomy – Body fluids – Blood – Physiology – Molecular biology – Molecular biology techniques – Artificial gene amplification and extension – Polymerase chain reaction – Research and analysis methods – Database and informatics methods – Bioinformatics – Sequence analysis – Sequence alignment – Sequencing techniques – DNA sequencing – Next-generation sequencing – Medicine and health sciences – Oncology – Cancers and neoplasms – Lung and intrathoracic tumors – Non-small cell lung cancer
Zdroje
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Článek vyšel v časopise
PLOS One
2019 Číslo 9
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