br Acknowledgements br A subgroup of
A subgroup of pediatric solid neoplasms, collectively referred to as small round cell tumors (SRCTs) of childhood, demonstrates undifferentiated small round cell morphological characteristics. The SRCTs include neuroblastomas, lymphomas, and several bone and soft tissue sarcomas. These tumors often pose significant diagnostic challenges because they can be indistinguishable from each other microscopically. Although neuroblastomas and lymphomas can typically be diagnosed on the basis of anatomical location and immunohistochemistry, bone and soft tissue sarcomas, including Ewing sarcoma (ES), embryonal and alveolar rhabdomyosarcoma (aRMS), desmoplastic SRCT (DSRCT), and congenital fibrosarcoma (CFS), may occur at diverse anatomical sites, and can be difficult to diagnose using routine anatomical pathological tools alone., , Therefore, strict criteria on the basis of molecular diagnostic modalities are needed to accurately classify these tumors. Several childhood sarcomas are characterized by pathognomonic gene fusions that are associated with chromosomal translocations, including ETS fusions (predominantly and ) in ES,, , , , - and - in aRMS,, , - in DSRCT,, and in CFS. Molecular detection of tumor-specific gene fusions has become an essential ancillary approach to accurately classify childhood sarcomas and assign them to appropriate treatment regimens., , The standard of practice to identify pathognomonic gene fusions is to use RT-PCR or fluorescence hybridization (FISH) assays., Detection of fusion transcripts uses selected primer pairs and RG7388 to generate amplification products that are visualized by gel electrophoresis or a fluorescent signal when using fluorescent-labeled probes for RT-PCR., Detection depends on DNA amplification and generation of an expected end product size, rather than by providing the exact nucleotide sequence of the target. This shortcoming can be overcome by subjecting the observed RT-PCR products to Southern blot analysis or direct sequencing. However, the former requires the use of radioactive probes and the latter is typically achieved through gel isolation of RT-PCR products, followed by Sanger sequencing. Both additional steps are labor intensive and time consuming for most clinical laboratories. A popular alternative to RT-PCR for gene fusion detection is FISH, because commercial breakpoint-spanning probes designed for standard break-apart FISH or fusion assays have become widely available. An additional advantage of FISH-based methods is their broad applicability to formalin-fixed, paraffin-embedded tissue (FFPE) blocks., However, such as RT-PCR, FISH is labor intensive and time consuming, and also requires significant expertise, instrumentation, and quality assurance. Another shortcoming of both RT-PCR and FISH assays in the clinical setting is the difficulty in performing any form of multiplexing. This precludes the ability to screen for multiple fusion transcripts simultaneously when a diagnosis is uncertain. Therefore, multiple individual RT-PCRs are typically run, each under different reaction conditions, until a diagnosis is achieved. This approach extends the work flow and increases personnel time and laboratory expenditure considerably. Last, for an RT-PCR assay to detect a specific gene fusion, both translocation partners must be known for primer design. Consequently, fusion products will not be detected by conventional RT-PCR assays in cases in which the fusion site is not already characterized at the nucleotide level. To overcome the previously mentioned limitations of RT-PCR and FISH, we sought to develop a next-generation sequencing (NGS)–based method for the molecular diagnosis of fusion-associated childhood sarcomas. Herein, we present a novel multiplex assay, designated ChildSeq-RNA, which combines target-capture chemistry, with high-throughput RNA sequencing, to provide nucleotide resolution detection of select known fusion transcripts. In this proof-of-concept version, ChildSeq-RNA was designed to detect the following gene fusions: , , and in ES, , and in aRMS, in DSRCT, and in CFS. Our assay offers several advantages over RT-PCR and FISH assays: i) simultaneous detection of all known pathognomonic fusions indicated for a particular differential diagnosis in a single reaction, ii) identification of novel and/or rare fusion transcript(s) or subtypes(s) when both partner regions are captured, iii) scalability of up to 16 tumor samples assayed in a single run, and iv) capacity to report expression for the targeted genes. ChildSeq-RNA uses 120 base gene–specific DNA probes specifically designed to target the exons of known fusion partner genes, thereby selectively capturing all fusion transcripts involving those exons. All captured transcripts are sequenced using an Ion Torrent Personal Genome Machine (IonT PGM; Life Technologies, Inc., Carlsbad, CA). To facilitate the analysis of ChildSeq-RNA data, we also developed a companion bioinformatics application, which allows for the identification of fusion transcripts using a web-based portal. Herein, we report results for gene fusion detection and gene expression estimation in four ES cell lines and 33 clinical specimens of ES, aRMS, DSRCT, and CFS using the ChildSeq-RNA method.