Use of Anchored Multiplex PCR Enrichment for Detection of Gene Fusions in Solid Tumors by Next Generation Sequencing

Abstract

BackgroundThe known genomic complexities of human cancer are being used to combat the disease with novel targeted therapies. Next generation sequencing (NGS) technologies have been introduced to determine a human cancer profile that is then used to develop a therapeutic management strategy. To date most NGS assays assess SNVs, Indels and some CNVs in various cancer related genes. Gene fusions are another type of mutation that have recently been implicated in the diagnosis and treatment of human cancers. In this study we assessed the ability to detect gene fusions using an anchored multiplex PCR‐enriched NGS assay.MethodsRNA was extracted from 15 formalin fixed, paraffin embedded tissue samples and 10 cell lines using the AllPrep DNA/RNA FFPE kit. cDNA synthesis and library preparation were performed using 200 ng of RNA. RNA quantity and quality were evaluated using the Qubit HS RNA Assay and the Archer PreSeq RNA QC Kit, respectively. Libraries were quantified using the KAPA Library Quantification Kit and sequenced on the Illumina MiSeq System using the Archer FusionPlexTM Solid Tumor panel which detects fusions in 53 genes. Data analysis was performed using the Archer Analysis platform (v4.0.11). We examined the limit of detection (LOD) by varying the amount of total RNA input (200‐2.5 ng), precision in two sequencing runs using two different operators, and accuracy by comparison of results to results from independent FISH and NGS assays.ResultsWe established QC metrics that included: (1) tumor content, (2) RNA quantification, (3) RNA quality, (4) library quantification, and (5) post‐sequencing analysis. While NGS results could be obtained using 10 ng of input RNA, more robust results were obtained when we used higher RNA inputs. Accuracy was determined to be 100% with detection of for all known fusions in the samples used. The following fusions were present in these samples: EML4‐ALK, ETV6‐NTRK3, EWSR1‐FLI1, TPM3‐NTRK1, TNM4‐NRG1, FGFR2‐COL14A1, MBNL1‐RAF1, ETV6‐RUNX1, BRD4‐NUTM1, and TMPRSS2‐ERG. All positive (CCD6‐RET, EML4‐ALK, SLC34A2‐ROS1) and negative controls gave the expected results.ConclusionsThis study demonstrates the feasibility of using the Archer Solid Tumor FusionPlex assay for detection of gene fusions in human cancers. The assay detects fusions of any partner gene with the 53 genes in the panel and thus makes novel fusion discovery possible.