Abstract
BackgroundGene fusions represent promising targets for cancer therapy in lung cancer. Reliable detection of multiple gene fusions is therefore essential.MethodsFive commercially available parallel sequencing assays were evaluated for their ability to detect gene fusions in eight cell lines and 18 FFPE tissue samples carrying a variety of known gene fusions. Four RNA-based assays and one DNA-based assay were compared; two were hybrid capture-based, TruSight Tumor 170 Assay (Illumina) and SureSelect XT HS Custom Panel (Agilent), and three were amplicon-based, Archer FusionPlex Lung Panel (ArcherDX), QIAseq RNAscan Custom Panel (Qiagen) and Oncomine Focus Assay (Thermo Fisher Scientific).ResultsThe Illumina assay detected all tested fusions and showed the smallest number of false positive results. Both, the ArcherDX and Qiagen panels missed only one fusion event. Among the RNA-based assays, the Qiagen panel had the highest number of false positive events. The Oncomine Focus Assay (Thermo Fisher Scientific) was the least adequate assay for our purposes, seven fusions were not covered by the assay and two fusions were classified as uncertain. The DNA-based SureSelect XT HS Custom Panel (Agilent) missed three fusions and nine fusions were only called by one software version. Additionally, many false positive fusions were observed.ConclusionsIn summary, especially RNA-based parallel sequencing approaches are potent tools for reliable detection of targetable gene fusions in clinical diagnostics.
Highlights
Gene fusions represent promising targets for cancer therapy in lung cancer
Tumor cohort A collection of 18 formalin-fixed and paraffin-embedded (FFPE) patient tumor tissue samples, previously tested positive for ALK, ROS1, ret protooncogene (RET), FGFR2, NTRK1/3, MET or BRAF rearrangements by fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) or targeted RNA-based parallel sequencing (Archer FusionPlex panel (ArcherDX, Boulder, CO, USA)) during clinical diagnostics was selected
Study design In this study, five different commercially available assays were analyzed for their ability to reliably detect gene fusions in cell lines as well as diagnostic FFPE patient tumor tissue samples, covering RNA- and DNAbased, as well as hybrid capture- and amplicon-based enrichment approaches for targeted parallel sequencing (Fig. 1). 18 FFPE samples with known fusions in ALK, ROS1, RET, FGFR1/3, NTRK1/3, MET or BRAF were selected
Summary
Gene fusions represent promising targets for cancer therapy in lung cancer. Reliable detection of mul‐ tiple gene fusions is essential. Recurrent and therapeutically targetable fusion genes involving ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) [16, 17], ret protooncogene (RET) [18, 19], B-Raf proto-oncogene, serine/ threonine kinase (BRAF) [20], fibroblast growth factor receptor 1–3 (FGFR1-3) [21, 22], neurotrophic receptor tyrosine kinase 1–3 (NTRK1-3) [23], EGFR [24, 25] and MET proto-oncogene, receptor tyrosine kinase (MET) [26, 27] have been identified in NSCLC patients Tumors carrying these rearrangements represent unique molecular cohorts of NSCLC as they are eligible for targeted tyrosine kinase inhibitor therapies. Despite recent advances in our understanding of the genetic subgroups of NSCLC, there is still a relatively large subset of “pan-negative” patients, who do not carry any known oncogenic driver variations, suggesting that other driver alterations, including gene fusions or epigenetic alterations are yet to be identified
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