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

• Published in: PloS one 2019-09, Vol.14 (9), p.e0222233-e0222233
• Main Authors: Kunimasa, Kei, Kato, Kikuya, Imamura, Fumio, Kukita, Yoji
• Format: Article
• Language: English
• Subjects: Adapters; Alignment; ALK protein; Alleles; Analysis; Anaplastic lymphoma kinase (ALK) inhibitor; Anaplastic Lymphoma Kinase - genetics; Biology and Life Sciences; Biopsy; Breakpoints; Cancer genetics; Carcinoma, Non-Small-Cell Lung - genetics; Care and treatment; Cell fusion; Cell-Free Nucleic Acids - analysis; Cell-Free Nucleic Acids - genetics; Crizotinib; Data analysis; Deoxyribonucleic acid; DNA; DNA Primers; DNA sequencing; Dosage and administration; Fusion protein; Gene amplification; Gene fusion; Gene Rearrangement - genetics; Gene sequencing; Genes; Genetic aspects; Genomes; Genomics; Health aspects; High-Throughput Nucleotide Sequencing - methods; Humans; Hybridization; In Situ Hybridization, Fluorescence - methods; Information management; Japan; Kinases; Laboratories; Ligands; Liquid Biopsy - methods; Lung cancer; Lung diseases; Lymphoma; Lymphomas; Mapping; Medicine and Health Sciences; Methods; Mutation; Non-Hodgkin's lymphomas; Non-small cell lung cancer; Non-small cell lung carcinoma; Oncogene Proteins, Fusion - genetics; Oncology; Patient outcomes; Pipelines; Plasma; Polymerase chain reaction; Polymerase Chain Reaction - methods; Primers; Protein Kinase Inhibitors - metabolism; Protein-tyrosine kinase; Protein-Tyrosine Kinases - antagonists & inhibitors; Quantitation; Receptor Protein-Tyrosine Kinases - genetics; Research and Analysis Methods; Sensitivity and Specificity; Small cell lung cancer; Tiling; Tumors; Tyrosine
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0222233

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.