Materials and Methods br Results
Materials and Methods
Discussion For several decades, the diagnosis of translocation-associated childhood sarcomas with overlapping morphological characteristics has been facilitated by pathognomonic gene fusion detection through RT-PCR or FISH. However, these assays have several shortcomings, including their inability to identify gene fusions at nucleotide resolution and the lack of multiplexing. To overcome these issues and to take advantage of NGS technologies, we developed an NGS-based assay called ChildSeq-RNA that uses the Ion Torrent sequencing platform. In the present study, we used previously molecularly characterized childhood sarcoma cell lines and fresh frozen clinical samples to validate the ChildSeq-RNA method. In the ES cell line panel, ChildSeq-RNA accurately identified all of the EWSR1-ETS fusion transcripts, including several subtypes, some of which were concurrently being expressed. ChildSeq-RNA also successfully identified a previously unsequenced EWSR1-ERG fusion subtype in the cell line COG-E-352. Thus, unlike RT-PCR, ChildSeq-RNA can identify previously unrecognized fusion transcripts involving known partner genes without any prior modifications to the assay. By using a cohort of clinical SRCT cases with previously confirmed fusion events, ChildSeq-RNA correctly identified gene fusions in 15 of 16 ES samples, all six aRMS samples, the two DSRCT samples, and one CFS sample. The single failed ES case (which also failed in an RT-PCR experiment that we performed) was the result of compromised RNA quality, because the RNA integrity number score was barely higher than acceptable levels (RNA integrity number = 6). For clinical samples 4 and 7, we also identified relatively rare EWSR1-FLI1 fusion subtypes involving gsk3 inhibitor 8/exon 6 and exon 7/exon 8, respectively. This demonstrates the ability of the assay to capture fusions that would be missed by conventional RT-PCR methods, which target known fusion transcripts on the basis of the initial primer design. The ability of ChildSeq-RNA to detect rare (and dominant) EWSR1-FLI1 subtypes, including exon 10/exon 5 (present in <2% of ES cases according to COSMIC) and exon 10/exon 6 fusion (present in <3% of ES cases according to COSMIC), highlights another advantage of the ChildSeq-RNA assay over RT-PCR. This capability was further demonstrated in the sequencing and identification of an EWSR1-FLI1 fusion transcript with a truncated exon in clinical sample 19. This fusion transcript would not have been detected by a TaqMan-based RT-PCR assay. The fluorescent reporter probes of the TaqMan assay would be designed to bind to the expected fusion junction, which was actually truncated, thus leading to a false negative. Thus, our results show that ChildSeq-RNA has high sensitivity (96.43%) and can detect fusions with anomalous features that likely lead to false negatives in conventional assays. Also, ChildSeq-RNA did not report fusion transcripts for any samples that were known to be fusion negative, indicating that ChildSeq-RNA has high specificity (100%), with no examples of false positives. Overall, our results, despite a small validation cohort, demonstrate that ChildSeq-RNA identifies fusion transcripts with high accuracy (96.97%), high sensitivity and specificity. A salient advantage of ChildSeq-RNA over conventional methods is its ability to detect multiple genotypes (or fusion subtypes) in a single run. This has important implications for reducing the turnaround time for detection of pathognomonic gene fusions and thus streamlining appropriate clinical protocol assignment for SRCT patients. Another important feature of ChildSeq-RNA is its ability to report gene expression, consistent with the findings of a previous study that used targeted RNA-Seq for gene expression analysis. Taken together, the development of a standardized diagnostic modality for the identification of pathognomonic translocations, along with gene expression estimation as part of a routine diagnostic workup, may open new avenues for personalized therapy.