RNA sequencing (RNA-Seq) assay has been widely used for transcript level gene quantification and fusion detection for research use in fresh frozen samples. Clinical use of RNA is complicated by the common use of formalin-fixed paraffin-embedded (FFPE) tissue storage, which can cause low yield and RNA degradation. We evaluate the feasibility, quality, and analytical performance of RNA-Seq on clinical FFPE tumor samples for gene fusion detection.
Total RNA was extracted from FFPE tumor samples and/or adjacent normal samples. Ribosomal RNA depletion, cDNA synthesis, and library preparation were used to prepare next-generation sequencing (NGS) libraries that were sequenced on Illumina HiSeq X instrument. Sequencing data were analyzed and annotated with an in-house developed pipeline. A set of experimental and data quality control parameters were set up.
The assay identified all the positive fusions from RNA reference material with 15 NTRK fusions spiked-in and ALK, RET, NTRK1 and FGFR3 fusions from 5 positive cell lines. The assay Limit of Detection was tested by diluting RNA from ALK fusion positive cell H2228C to fusion-negative cell line. Gene fusions were generally detectable down to 10% dilutions for all fusion types and as little as 5% for some fusion types. RNA-Seq assay detected 10 of 12 gene fusions detected by DNA based NGS assay, for a sensitivity of 83%. No false-positive gene fusions were identified in 28 tumor specimens that were negative for fusions, for a specificity of 100%. The assay also identified 6 novel fusions in 3 tumor specimens, which had been confirmed by RT-PCR and Sanger sequencing. Good intra-assay and inter assay reproducibility was observed with complete concordance for the presence or absence of gene fusions in 3 samples and 6 replicates. We observed 81% success rate on whole transcriptome RNA-Seq process for more than 100 FFPE samples.
RNA-Seq assay can help identify gene fusions in patients with cancer, which is a good supplement for DNA based NGS assay, especially for novel fusion detection or fusion breakpoints which are hard to design probes for DNA samples. These patients may in turn benefit from approved and investigational fusion related targeted therapies.
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Legal entity responsible for the study
H. Dong: Full / Part-time employment: 3DMed Inc. C. Wang: Full / Part-time employment: 3DMed Inc. Q. Xu: Full / Part-time employment: 3DMed Inc. Y. Guo: Full / Part-time employment: 3DMed Inc. Y. Chen: Full / Part-time employment: 3DMed Inc. B. Li: Full / Part-time employment: 3DMed Inc. S. Liu: Full / Part-time employment: 3DMed Inc. C. Chen: Full / Part-time employment: 3DMed Inc. L. Xiong: Full / Part-time employment: 3DMed Inc. F. Li: Full / Part-time employment: 3DMed Inc. All other authors have declared no conflicts of interest.