Artigo Acesso aberto Revisado por pares

Development and Evaluation of a Pan-Sarcoma Fusion Gene Detection Assay Using the NanoString nCounter Platform

2017; Elsevier BV; Volume: 20; Issue: 1 Linguagem: Inglês

10.1016/j.jmoldx.2017.09.007

ISSN

1943-7811

Autores

Kenneth Tou En Chang, Angela Goytain, Tracy Tucker, Aly Karsan, Cheng‐Han Lee, Torsten O. Nielsen, Tony Ng,

Tópico(s)

Cardiac tumors and thrombi

Resumo

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-fixed, paraffin-embedded material. We designed a NanoString assay targeting 174 unique fusion junctions in 25 sarcoma types. The study cohort comprised 212 cases, 96 of which showed fusion gene expression by the NanoString assay, including all 20 Ewing sarcomas, 11 synovial sarcomas, and 5 myxoid liposarcomas tested. Among these 96 cases, 15 showed fusion expression not identified by standard clinical assay, including EWSR1-FLI1, EWSR1-ERG, BCOR-CCNB3, ZC3H7B-BCOR, HEY1-NCOA2, CIC-DUX4, COL1A1-PDGFB, MYH9-USP6, YAP1-TFE3, and IRF2BP2-CDX1 fusions. There were no false-positive results; however, four cases were false negative when compared with clinically available fluorescence in situ hybridization 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. 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-fixed, paraffin-embedded material. We designed a NanoString assay targeting 174 unique fusion junctions in 25 sarcoma types. The study cohort comprised 212 cases, 96 of which showed fusion gene expression by the NanoString assay, including all 20 Ewing sarcomas, 11 synovial sarcomas, and 5 myxoid liposarcomas tested. Among these 96 cases, 15 showed fusion expression not identified by standard clinical assay, including EWSR1-FLI1, EWSR1-ERG, BCOR-CCNB3, ZC3H7B-BCOR, HEY1-NCOA2, CIC-DUX4, COL1A1-PDGFB, MYH9-USP6, YAP1-TFE3, and IRF2BP2-CDX1 fusions. There were no false-positive results; however, four cases were false negative when compared with clinically available fluorescence in situ hybridization 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. Sarcomas are malignant mesenchymal tumors arising from soft tissues and bones. Diagnosis of sarcomas can be challenging for pathologists because of the large number of entities, many with overlapping histomorphologies and immunophenotypes. Nonetheless, accurate diagnosis is critical because certain diagnoses require specific treatment strategies or affect eligibility for clinical trials. Subtype diagnoses confer important prognostic information that affects immediate local management and follow-up strategies. From a genetic perspective, sarcomas can be classified into two main groups. Approximately three-quarters of sarcomas are karyotypically complex and lack recurrent genetic alterations.1Guillou L. Aurias A. Soft tissue sarcomas with complex genomic profiles.Virchows Arch. 2010; 456: 201-217Crossref PubMed Scopus (103) Google Scholar The other quarter (including most pediatric, adolescent, and young adult tumors) have simple genetic alterations that often define specific sarcoma entities.2Mertens F. Antonescu C.R. Hohenberger P. Ladanyi M. Modena P. D'Incalci M. Casali P.G. Aglietta M. Alvegard T. Translocation-related sarcomas.Semin Oncol. 2009; 36: 312-323Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar Many sarcomas in the latter category can be classified on the basis of pathognomonic chromosomal translocations. These generate fusion genes encoding chimeric transcription factors that alter the epigenome and dysregulate transcription of target genes or function as chimeric protein kinases or overexpressed growth factors.2Mertens F. Antonescu C.R. Hohenberger P. Ladanyi M. Modena P. D'Incalci M. Casali P.G. Aglietta M. Alvegard T. Translocation-related sarcomas.Semin Oncol. 2009; 36: 312-323Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar The importance of molecular genetic testing for accurate diagnosis of sarcomas was demonstrated in the Clinical Effect of Molecular Methods in Sarcoma Diagnosis (GENSARC) study, in which the histologic diagnosis of 53 of 384 sarcomas (13.8%) was amended by molecular findings, with implications for patient management and prognostication.3Italiano A. Di Mauro I. Rapp J. Pierron G. Auger N. Alberti L. Chibon F. Escande F. Voegeli A.C. Ghnassia J.P. Keslair F. Lae M. Ranchere-Vince D. Terrier P. Baffert S. Coindre J.M. Pedeutour F. Clinical effect of molecular methods in sarcoma diagnosis (GENSARC): a prospective, multicentre, observational study.Lancet Oncol. 2016; 17: 532-538Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar However, even when molecular techniques optimized for formalin-fixed, paraffin-embedded (FFPE) pathology tissue are available, diagnostic identification of such fusion genes can be challenging in the clinical setting for the following reasons. First, for some sarcoma types, such as Ewing sarcoma, multiple fusion combinations involving several partner genes are possible.4Barr F.G. Womer R.B. Molecular diagnosis of ewing family tumors: too many fusions…?.J Mol Diagn. 2007; 9: 437-440Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar Second, the specific break point in each partner gene can be variable, resulting in a variety of exon-exon fusion combinations at the transcript level. Third, current standard clinical assays lack multiplex capabilities, potentially entailing multiple costly consecutive investigations with long net turnaround times when identifying unusual gene fusions. There is, therefore, a clinical need for a single pan-sarcoma gene fusion identification assay that is both cost-effective and has a short turnaround time that meets patient care requirements. Current standard molecular diagnostic methods for identifying gene fusions in use in hospital laboratories include conventional cytogenetics, fluorescence in situ hybridization (FISH), and RT-PCR.5Demicco E.G. Sarcoma diagnosis in the age of molecular pathology.Adv Anat Pathol. 2013; 20: 264-274Crossref PubMed Scopus (22) Google Scholar Conventional cytogenetics is a well-established technique that provides a gross pan-chromosomal survey and can detect large chromosomal alterations without prior knowledge of specific alterations.6Turc-Carel C. Pedeutour F. Durieux E. 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Use of FISH to detect chromosomal translocations and deletions: analysis of chromosome rearrangement in synovial sarcoma cells from paraffin-embedded specimens.Am J Pathol. 1993; 143: 15-19PubMed Google Scholar, 8Taylor C. Patel K. Jones T. Kiely F. De Stavola B.L. Sheer D. Diagnosis of Ewing's sarcoma and peripheral neuroectodermal tumour based on the detection of t(11;22) using fluorescence in situ hybridisation.Br J Cancer. 1993; 67: 128-133Crossref PubMed Scopus (72) Google Scholar FISH is not amenable to multiplexing, and multiple iterations of individual FISH assays (each requiring additional tissue, technical time, and quality control procedures) may need to be performed to identify rare fusion genes in tumors with multiple possible partner genes. RT-PCR likewise can be performed on fresh/frozen or FFPE tissue, although it is less sensitive on the latter.9Chen Q.R. Vansant G. Oades K. Pickering M. Wei J.S. Song Y.K. Monforte J. Khan J. Diagnosis of the small round blue cell tumors using multiplex polymerase chain reaction.J Mol Diagn. 2007; 9: 80-88Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar A single RT-PCR assay can determine the presence of a translocation using primer pairs targeting each partner gene. However, the possibility of multiple partner genes and permutations of possible exonic break points in several sarcoma types make RT-PCR even less sensitive than FISH in this setting. The advent of high-throughput molecular techniques has opened up new possibilities for the diagnosis of sarcomas bearing gene fusions. One option, the NanoString nCounter analysis system, uses nucleic acid hybridization probes with specific and distinguishable fluorescent bar code tags that allow detection of multiple specific short nucleic acid sequences within a given sample.10Geiss G.K. Bumgarner R.E. Birditt B. Dahl T. Dowidar N. Dunaway D.L. Fell H.P. Ferree S. George R.D. Grogan T. James J.J. Maysuria M. 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Ang D. Ito T. Kimura Y. Drilon A. Guha U. Rusch V. Kris M.G. Zakowski M.F. Rizvi N. Khanin R. Ladanyi M. Identification of KIF5B-RET and GOPC-ROS1 fusions in lung adenocarcinomas through a comprehensive mRNA-based screen for tyrosine kinase fusions.Clin Cancer Res. 2012; 18: 6599-6608Crossref PubMed Scopus (156) Google Scholar In this study, we investigate the accuracy and feasibility of a NanoString-based assay using a custom-designed CodeSet composed of target-specific probes complementing a list of sarcoma fusions that detect all common and most uncommon gene translocations in bone and soft tissue tumors. 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