(P 050) MULTIPLEX DETECTION OF TRANSLOCATION ASSOCIATED SARCOMAS WITH COMBINED FUSION JUNCTION SPANNING PROBES AND EXON IMBALANCE ANALYSIS

Objective: Background: Efficient molecular diagnostic confirmation is important for accurate characterization of translocation associated sarcomas. Building on our previously developed NanoString nCounter based diagnostic assays using fusion spanning probes (Chang KTE et al, J. Mol. Diagn. 2018), by adding assessment for aberrant 3’/5’ exon imbalance expression we have developed a single multiplexed assay to detect the great majority of fusion oncogene sarcomas. The NanoString nCounter platform is a high-throughput hybridization technique using target-specific probes that digitally quantifies the expression of multiple genes in a single assay utilizing RNA extracted from formalin-fixed paraffin-embedded (FFPE) material, requiring no enzymatic steps or amplification. The technique is more cost effective and requires less time to perform than FISH or next generation sequencing-based alternatives.

Objective: To develop a multiplex NanoString-based assay that detects translocation-based sarcomas with a combination of fusion junction spanning probes and exon imbalance gene expression analysis.

Methods: Oligonucleotide probes were designed targeting 113 specific fusions across 16 sarcoma diagnoses, 12 exon imbalance genes involved in fusions covering 12 other sarcoma diagnoses and a 5 gene cassette consistently over expressed in CIC-rearranged sarcoma (Table 1). For common exon-exon fusions, a fluorescently labelled reporter and biotinylated capture probe pair was designed per fusion spanning probe site (Figure 1A). For genes with higher variability, up to four 5’ exons and four 3’ exons sites were targeted to assay for exon expression imbalances (Figure 1B). One site was selected for each CIC associated gene (CCND2, CRH, ETV1, ETV4, ETV5). RNA was extracted from 2-6x 10 m FFPE scrolls of core biopsy or excision samples. The pool of barcoded gene-specific oligonucleotide probes (“codeset”) was hybridized with 50-150ng tumor RNA overnight. After automated removal of unbound probes and RNA in a NanoString prep station, the remaining probe/RNA complexes were bound to a streptavidin coated cartridge and counted on a NanoString Digital analyzer at maximum sensitivity. RNA sample quality was determined by based on expression of four housekeeping genes. Fusion spanning probe positivity was set at 5x the background detectible counts for each specific fusion probe set. A minimum positive exon imbalance ratio and expression ratio was set for each gene as the average background ratio plus 5x the standard deviation.

Results: Three non-fusion mesenchymal tumor samples and 69 confirmed translocation-associated sarcoma samples were assessed with the assay (Table 2). Seven samples failed to produce an analyzable result due to poor RNA quality. The single CIC associated tumor sample showed an average over expression of the 5 gene cassette (CCDN2, CRH, ETV1, ETV4, ETV5) at 181x the mean expression of the other samples analyzed. A specific fusion was detected for 42 samples with the fusion spanning probe part of the assay. The appropriate exon imbalance was detected for all 17 samples expected to express an exon imbalance including 5 congenital fibrosarcoma cases where a fusion was also detected with fusion spanning probes. The specific fusion or expected exon imbalance was not detected for seven samples. No fusion or exon imbalance was detected for the three fusion negative tumor samples. The assay takes 36 hours to run from RNA extraction to result and costs less than USD$400 per case including reagents and technologist time.

Conclusion: The combination of both fusion spanning fusion probes and exon imbalance analysis provides an effective and efficient method for molecular diagnostic confirmation of multiple translocation-associated sarcoma entities. The assay performed with 100% specificity and 88.7% sensitivity, at a low cost and with a quick turn-around time.

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