Sarcoma Subgrouping by Detection of Fusion Transcripts Using NanoString nCounter Technology

Abstract

Background

NanoString technology is an innovative barcode-based system that requires less tissue than traditional techniques and can test for multiple fusion transcripts in a single reaction. The objective of this study was to determine the utility of NanoString technology in the detection of sarcoma-specific fusion transcripts in pediatric sarcomas.

Design

Probe pairs for the most common pediatric sarcoma fusion transcripts were designed for the assay. The NanoString assay was used to test 22 specific fusion transcripts in 45 sarcoma samples that had exhibited one of these fusion genes previously by reverse transcription polymerase chain reaction (RT-PCR). A mixture of frozen (n = 18), formalin-fixed, paraffin-embedded (FFPE) tissue (n = 23), and rapid extract template (n = 4) were used for testing.

Results

Each of the 22 transcripts tested was detected in at least one of the 45 tumor samples. The results of the NanoString assay were 100% concordant with the previous RT-PCR results for the tumor samples, and the technique was successful using both FFPE and rapid extract method.

Conclusion

Multiplexed interrogation for sarcoma-specific fusion transcripts using NanoString technology is a reliable approach for molecular diagnosis of pediatric sarcomas and works well with FFPE tissues. Future work will involve validating additional sarcoma fusion transcripts as well as determining the optimal workflow for diagnostic purposes.

Keywords

pediatric sarcoma NanoString fusion detection sarcoma subgrouping multiplexing

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References

Helman

Meltzer

. Mechanisms of sarcoma development. Nat Rev Cancer 2003; 3(9): 685–694.

Mackall

Meltzer

Helman

. Focus on sarcomas. Cancer Cell 2002; 2(3): 175–178.

Burningham

Hashibe

Spector

Schiffman

. The epidemiology of sarcoma. Clin Sarcoma Res 2012; 2(1): 14.

Siegel

Miller

Jemal

. Cancer statistics. CA Cancer J Clin 2016; 66(1): 7–30.

Bovée

JVMG

Hogendoorn

PCW

. Molecular pathology of sarcomas: concepts and clinical implications. Virchows Arch 2010; 456(2): 193–199.

Fletcher C, Unni K, Mertens F. WHO Classification of Tumours. Pathology and Genetics of Tumours of Soft Tissue and Bone. Lyon, France: IARC; 2002. http://www.iarc.fr/en/publications/pdfs-online/pat-gen/bb5/index.php. Accessed July 9, 2018.

Hogendoorn

PCW

Collin

Daugaard

et al.

Changing concepts in the pathological basis of soft tissue and bone sarcoma treatment. Eur J Cancer 2004; 40(11): 1644–1654.

Sankar

Lessnick

. Promiscuous partnerships in Ewing’s sarcoma. Cancer Genet 2011; 204(7): 351–365.

Aurias

Rimbaut

Buffe

Dubousset

Mazabraud

. Chromosomal translocations in Ewing’s sarcoma. N Engl J Med 1983; 309: 496–497. http://www.nejm.org/doi/pdf/10.1056/NEJM198308253090817. Accessed July 9, 2018.

10.

Riggi

Cironi

Suva

M-L

Stamenkovic

. Sarcomas: genetics, signalling, and cellular origins. Part 1: the fellowship of TET. J Pathol 2007; 213: 4–20.

11.

Stegmaier

Poremba

Schaefer

et al.

Prognostic value of PAX-FKHR fusion status in alveolar rhabdomyosarcoma: a report from the cooperative soft tissue sarcoma study group (CWS). Pediatr Blood Cancer 2011; 57(3): 406–414.

12.

Missiaglia

Williamson

Chisholm

et al.

PAX3/FOXO1 fusion gene status is the key prognostic molecular marker in rhabdomyosarcoma and significantly improves current risk stratification. J Clin Oncol 2012; 30(14): 1670–1677.

13.

Geiss

Bumgarner

Birditt

et al.

Direct multiplexed measurement of gene expression with color-coded probe pairs. Nat Biotechnol 2008; 26(3): 317–325.

14.

Northcott

Shih

DJH

Remke

et al.

Rapid, reliable, and reproducible molecular sub-grouping of clinical medulloblastoma samples. Acta Neuropathol 2012; 123(4): 615–626.

15.

Beard

Abate-Daga

Rosati

et al.

Gene expression profiling using Nanostring digital RNA counting to identify potential target antigens for melanoma immunotherapy. Clin Cancer Res 2013; 19(18): 4941–4950.

16.

Reis

Waldron

Goswami

et al.

mRNA transcript quantification in archival samples using multiplexed, color-coded probes. BMC Biotechnol 2011; 11(1): 46.

17.

Erickson-Johnson

Chou

Evers

et al.

Nodular fasciitis: a novel model of transient neoplasia induced by MYH9-USP6 gene fusion. Lab Investig 2011; 91(10): 1427–1433.

18.

Hibbard

Kozakewich

Dal Cin

et al.

PLAG1 fusion oncogenes in lipoblastoma. Cancer Res 2000; 60(17): 4869–4872.

19.

Chen

Lee

. An inflammatory myofibroblastic tumor in liver with ALK and RANBP2 gene rearrangement: combination of distinct morphologic, immunohistochemical, and genetic features. Hum Pathol 2008; 39(12): 1854–1858.

20.

Lawrence

Perez-Atayde

Hibbard

et al.

TPM3-ALK and TPM4-ALK oncogenes in inflammatory myofibroblastic tumors. Am J Pathol 2000; 157(2): 377–384.

21.

Antonescu

Gerald

Magid

Ladanyi

. Molecular variants of the EWS-WT1 gene fusion in desmoplastic small round cell tumor. Diagn Mol Pathol 1998; 7(1): 24–28. http://www.ncbi.nlm.nih.gov/pubmed/9646031. Accessed July 9, 2018.

22.

Gerald

Rosai

Ladanyi

. Characterization of the genomic breakpoint and chimeric transcripts in the EWS-WT1 gene fusion of desmoplastic small round cell tumor. Proc Natl Acad Sci U S A 1995; 92(4): 1028–1032.

23.

Knezevich

McFadden

Tao

Lim

Sorensen

PHB

. A novel ETV6-NTRK3 gene fusion in congenital fibrosarcoma. Nat Genet 1998; 18(2): 184–187.

24.

Antonescu

Nafa

Segal

Dal Cin

Ladanyi

. EWS-CREB1: a recurrent variant fusion in clear cell sarcoma – association with gastrointestinal location and absence of melanocytic differentiation. Clin Cancer Res 2006; 12(18): 5356–5362.

25.

Dunham

Hussong

Seiff

Pfeifer

Perry

. Primary intracerebral angiomatoid fibrous histiocytoma. Am J Surg Pathol 2008; 32(3): 478–484.

26.

Waters

Panagopoulos

Allen

. Genetic characterization of angiomatoid fibrous histiocytoma identifies fusion of the FUS and ATF-1 genes induced by a chromosomal translocation involving bands 12q13 and 16p11. Cancer Genet Cytogenet 2000; 121(2): 109–116.

27.

Antonescu

Cin

Nafa

et al.

EWSRI-CREBI is the predominant gene fusion in angiomatoid fibrous histiocytoma. Genes Chromosom Cancer 2007; 46(12): 1051–1060.

28.

Crew

Clark

Fisher

et al.

Fusion of SYT to two genes, SSX1 and SSX2, encoding proteins with homology to the Kruppel-associated box in human synovial sarcoma. EMBO J 1995; 14(10): 2333–2340. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=398342&tool=pmcentrez&rendertype=abstract. Accessed July 9, 2018.

29.

Graham

Chilton-Macneill

Zielenska

Somers

. The CIC-DUX4 fusion transcript is present in a subgroup of pediatric primitive round cell sarcomas. Hum Pathol 2012; 43(2): 180–189.

30.

Puls

Niblett

Marland

et al.

BCOR-CCNB3 (Ewing-like) sarcoma: a clinicopathologic analysis of 10 cases, in comparison with conventional Ewing sarcoma. Am J Surg Pathol 2014; 38(10): 1307–1318.

31.

Peters

Kumar

Polikepahad

et al.

BCOR-CCNB3 fusions are frequent in undifferentiated sarcomas of male children. Mod Pathol 2015; 28(4): 575–586.

32.

Ida

Kobayashi

Taki

et al.

EWS-FLI-1 and EWS-ERG chimeric mRNAs in Ewing’s sarcoma and primitive neuroectodermal tumor. Int J Cancer 1995; 63(4): 500–504.

33.

Jeon

Davis

Braun

et al.

A variant Ewing’s sarcoma translocation (7;22) fuses the EWS gene to the ETS gene ETV1. Oncogene 1995; 10(6): 1229–1234. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=7700648. Accessed July 9, 2018.

34.

Gamberi

Cocchi

Benini

et al.

Molecular diagnosis in ewing family tumors: the Rizzoli experience – 222 consecutive cases in four years. J Mol Diagnostics 2011; 13(3): 313–324.

35.

Fitzgerald

Scherr

Barr

. Structural analysis of PAX7 rearrangements in alveolar rhabdomyosarcoma. Cancer Genet 2000; 117(1): 37–40.

36.

Barr

Nauta

Hollows

. Structural analysis of PAX3 genomic rearrangements in alveolar rhabdomyosarcoma. Cancer Genet 1998; 102(1): 32–39.

37.

Ryall

Arnoldo

Krishnatry

et al.

Multiplex detection of pediatric low-grade glioma signature fusion transcripts and duplications using the NanoString nCounter system. J Neuropathol Exp Neurol 2017; 76(7): 562–570.

38.

Kawai

Woodruff

Healey

et al.

SYT-SSX gene fusion as a determinant of morphology and prognosis in synovial sarcoma. N Engl J Med 1998; 338(3): 153–160.

39.

Chang

KTE

Goytain

Tucker

et al.

Development and evaluation of a pan-sarcoma fusion gene detection assay using the NanoString nCounter platform. J Mol Diagnostics 2018; 20(1): 63–77.

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