Rapid and highly sensitive approach for multiplexed somatic fusion detection

Abstract

Somatic gene translocations are key to making an accurate diagnosis in many cancers including many pediatric sarcomas. Currently available molecular diagnostic approaches to identifying somatic pathognomonic translocations have limitations such as minimal multiplexing, high cost, complex computational requirements, or slow turnaround times. We sought to develop a new fusion-detection assay optimized to mitigate these challenges. To accomplish this goal, we developed a highly sensitive multiplexed digital PCR-based approach that can identify the gene partners of multiple somatic fusion transcripts. This assay was validated for specificity with cell lines and synthetized DNA fragments. Assay sensitivity was optimized using a tiered amplification approach for fusion detection from low input and/or degraded RNA. The assay was then tested for the potential application of fusion detection from FFPE tissue and liquid biopsy samples. We found that this multiplexed PCR approach was able to accurately identify the presence of seven different targeted fusion transcripts with a turnaround time of 1 to 2 days. The addition of a tiered amplification step allowed the detection of targeted fusions from as little as 1 pg of RNA input. We also identified fusions from as little as two unstained slides of FFPE tumor biopsy tissue, from circulating tumor cells collected from tumor-bearing mice, and from liquid biopsy samples from patients with known fusion-positive cancers. We also demonstrated that the assay could be easily adapted for additional fusion targets. In summary, this novel assay detects multiple somatic fusion partners in biologic samples with low tumor content and low-quality RNA in less than two days. The assay is inexpensive and could be applied to surgical and liquid biopsies, particularly in places with inadequate resources for more expensive and expertise-dependent assays such as next-generation sequencing.

Clinically relevant fusion oncogenes: detection and practical implications

“…This method was recently used to measure gene fusions from as little as 1 pg of RNA with great success. 195 , 196 …”

“…This method was recently used to measure gene fusions from as little as 1 pg of RNA with great success. 195,196 To conclude, traditional and novel PCR-based tools represent a gold standard for fusion detection in cancer and are widely used in clinical practice, research, and development.…”

“…256 Zhang. 257 RT 178 Cruz-Rico et al, 180 Lyu et al, 192 Sorber et al, 194 Abbou et al, 195 5 Sorokin et al, 197 Winters et al, 225 Peng et al 240 Single-cell RNA-seq Measures clonality of tumors.…”

Experimentally Deduced Criteria for Detection of Clinically Relevant Fusion 3′ Oncogenes from FFPE Bulk RNA Sequencing Data

“…Furthermore, reverse transcription PCR (RT-PCR) is a standard method for detecting the presence of chimeric transcripts including fusion oncogenes, and several kits are commercially available to detect NTRK1-3 , ALK , RET , and ROS1 fusion RNAs in FFPE biosamples [ 8 , 12 ]. Another method—digital multiplex PCR—measures the presence of fusion RNA from as little as 1pg of RNA in miniature droplets, each encompassing a single RNA molecule, allowing a “yes–no” assessment of PCR results [ 13 ]. However, all those PCR methods are sensitive only when both fusion partners are known and included within specific panels, and not informative otherwise, e.g., for the RTK fusions with rare or new partners.…”