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Development and Evaluation of a Pan-Sarcoma Fusion Gene Detection Assay Using the Nanostring Ncounter Platform

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Development and Evaluation of a Pan-Sarcoma Fusion Gene Detection Assay Using the Nanostring Ncounter Platform The Journal of Molecular Diagnostics, Vol. 20, No. 1, January 2018 jmd.amjpathol.org Development and Evaluation of a Pan-Sarcoma Fusion Gene Detection Assay Using the NanoString nCounter Platform Kenneth T.E. Chang,*y Angela Goytain,z Tracy Tucker,x Aly Karsan,zx Cheng-Han Lee,zx{ Torsten O. Nielsen,zx{ and Tony L. Ngzx{ From the Department of Pathology and Laboratory Medicine,* KK Women’s and Children’s Hospital, Singapore, Singapore; the Duke-NUS Medical School,y Singapore, Singapore; the Department of Pathology,z University of British Columbia, Vancouver, British Columbia, Canada; the Department of Pathology,x British Columbia Cancer Agency, Vancouver, British Columbia, Canada; and the Department of Pathology,{ Vancouver General Hospital, Vancouver, British Columbia, Canada Accepted for publication September 22, 2017. The NanoString nCounter assay is a high-throughput hybridization technique using target-specific probes that can be customized to test for numerous fusion transcripts in a single assay using RNA from formalin- Address correspondence to fi fi Tony L. Ng, M.D., Ph.D., xed, paraf n-embedded material. We designed a NanoString assay targeting 174 unique fusion junctions Department of Pathology and in 25 sarcoma types. The study cohort comprised 212 cases, 96 of which showed fusion gene expression Laboratory Medicine, Vancou- by the NanoString assay, including all 20 Ewing sarcomas, 11 synovial sarcomas, and 5 myxoid lip- ver General Hospital, 855 W osarcomas tested. Among these 96 cases, 15 showed fusion expression not identified by standard clinical 12th Ave, Vancouver, BC V5Z assay, including EWSR1-FLI1, EWSR1-ERG, BCOR-CCNB3, ZC3H7B-BCOR, HEY1-NCOA2, CIC-DUX4, COL1A1- 1L3, Canada. E-mail: tony. PDGFB, MYH9-USP6, YAP1-TFE3,andIRF2BP2-CDX1 fusions. There were no false-positive results; howev- [email protected]. er, four cases were false negative when compared with clinically available fluorescence in situ hybridi- zation or RT-PCR testing. When batched as six cases, the per-sample reagent cost was less than conventional techniques, such as fluorescence in situ hybridization, with technologist hands-on time of 1.2 hours per case and assay time of 36 hours. In summary, the NanoString nCounter Sarcoma Fusion CodeSet reliably and cost-effectively identifies fusion genes in sarcomas using formalin-fixed, paraffin- embedded material, including many fusions missed by standard clinical assays, and can serve as a first- line clinical diagnostic test for sarcoma fusion gene identification, replacing multiple individual clinical assays. (J Mol Diagn 2018, 20: 63e77; https://doi.org/10.1016/j.jmoldx.2017.09.007) Sarcomas are malignant mesenchymal tumors arising from sarcomas in the latter category can be classified on the basis soft tissues and bones. Diagnosis of sarcomas can be chal- of pathognomonic chromosomal translocations. These lenging for pathologists because of the large number of en- generate fusion genes encoding chimeric transcription factors tities, many with overlapping histomorphologies and that alter the epigenome and dysregulate transcription of immunophenotypes. Nonetheless, accurate diagnosis is crit- target genes or function as chimeric protein kinases or ical because certain diagnoses require specifictreatment overexpressed growth factors.2 The importance of molecular strategies or affect eligibility for clinical trials. Subtype genetic testing for accurate diagnosis of sarcomas was diagnoses confer important prognostic information that affects immediate local management and follow-up strate- Supported by the Canadian Cancer Society grant 701582 (T.O.N.). gies. From a genetic perspective, sarcomas can be classified K.T.E.C. was a visiting fellow in the Terry Fox Foundation Strategic Health into two main groups. Approximately three-quarters of sar- Research Training Program in Cancer Research at the Canadian Institutes of comas are karyotypically complex and lack recurrent genetic Health Research (TGT-53912). The work in Singapore was funded by the VIVA-KKH Pediatric Brain and Solid Tumor Program grant alterations.1 The other quarter (including most pediatric, KRDUK16V0100 (K.T.E.C.). adolescent, and young adult tumors) have simple genetic Disclosures: T.O.N. has a licensing agreement with NanoString Tech- 2 alterations that often define specific sarcoma entities. Many nologies for the Prosigna assay, not part of the current study. Copyright ª 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0). https://doi.org/10.1016/j.jmoldx.2017.09.007 Chang et al demonstrated in the Clinical Effect of Molecular Methods in sequences within a given sample.10 These probes are cus- Sarcoma Diagnosis (GENSARC) study, in which the histo- tomizable and are assembled into pooled sets, termed logic diagnosis of 53 of 384 sarcomas (13.8%) was amended CodeSets. This technique requires neither nucleic acid by molecular findings, with implications for patient man- library preparation nor determination of sequence data, uses agement and prognostication.3 However, even when no nucleic acid amplification steps, is tolerant of poorer- molecular techniques optimized for formalin-fixed, paraffin- quality nucleic acid, and is, therefore, well suited to embedded (FFPE) pathology tissue are available, diagnostic routine diagnostic pathology settings in which FFPE mate- identification of such fusion genes can be challenging in the rial is all that is usually available. A recent publication has clinical setting for the following reasons. First, for some described use of the NanoString technology to detect sarcoma types, such as Ewing sarcoma, multiple fusion specific gene fusions in nonesmall-cell lung cancer, in combinations involving several partner genes are possible.4 which this platform performed well on diagnostic tumor Second, the specific break point in each partner gene can specimens, demonstrably improving clinical sensitivity and be variable, resulting in a variety of exon-exon fusion com- specificity compared with standard techniques.11 binations at the transcript level. Third, current standard In this study, we investigate the accuracy and feasibility clinical assays lack multiplex capabilities, potentially entail- of a NanoString-based assay using a custom-designed ing multiple costly consecutive investigations with long net CodeSet composed of target-specific probes complement- turnaround times when identifying unusual gene fusions. ing a list of sarcoma fusions that detect all common and There is, therefore, a clinical need for a single pan-sarcoma most uncommon gene translocations in bone and soft tissue gene fusion identification assay that is both cost-effective tumors. Our specific aims are as follows: first, to demon- and has a short turnaround time that meets patient care strate that the NanoString assay is at least as sensitive and requirements. specific as FISH/RT-PCR testing for identification of fusion Current standard molecular diagnostic methods for iden- oncogenes on clinical specimens; second, to determine per- tifying gene fusions in use in hospital laboratories include sample cost and turnaround time; and third, to assess the conventional cytogenetics, fluorescence in situ hybridization capability of the assay to improve diagnosis by detecting (FISH), and RT-PCR.5 Conventional cytogenetics is a well- fusion genes in cases in which no fusion gene was identified established technique that provides a gross pan- through conventional molecular techniques. chromosomal survey and can detect large chromosomal al- fi 6 terations without prior knowledge of speci c alterations. Materials and Methods However, cytogenetic analysis requires fresh tissue and cell culture techniques, is labor intensive, and can be technically Probe Design challenging, with a fairly lengthy turnaround time and an inability to detect chromosomal changes beyond the resolu- An in-depth literature search was performed for gene tion of 10 to 15 megabases. For these reasons, it is rapidly translocations involved in sarcomas. The sequence across becoming obsolete in the face of technological advances. the fusion break was collected from relevant case reports FISH can be applied to fresh/frozen or FFPE tissue. Fusion when reported.12e94 When the exact sequence was not translocation probes and break-apart probes are available, reported, the junction sequence was deduced from reported which identify gene fusions or rearrangements by means of exon/exon break points. The RNA sequences across the differentially labeled probes flanking the gene break points of junction site of the gene translocations were submitted to interest.7,8 FISH is not amenable to multiplexing, and multi- the NanoString Bioinformatics group (NanoString Tech- ple iterations of individual FISH assays (each requiring nologies, Seattle, WA) for the design of a NanoString additional tissue, technical time, and quality control proced- Elementsebased assay CodeSet. The CodeSet is composed ures) may need to be performed to identify rare fusion genes of target-specific probes, each with three oligonucleotide in tumors with multiple possible partner genes. RT-PCR probes per fusion site: a capture probe, a reporter probe, likewise can be performed on fresh/frozen or FFPE tissue, and a protector probe (Figure 1). Each reporter and capture although it is less sensitive on the latter.9 A single RT-PCR probe is approximately 50 nucleotides long, and each
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