title: Validation of a prognostic multi-gene signature in high-risk neuroblastoma using the high throughput digital NanoString nCounter™ system.
abstract: Microarray-based molecular signatures have not been widely integrated into neuroblastoma diagnostic classification systems due to the complexities of the assay and requirement for high-quality RNA. New digital technologies that accurately quantify gene expression using RNA isolated from formalin-fixed paraffin embedded (FFPE) tissues are now available. In this study, we describe the first use of a high-throughput digital system to assay the expression of genes in an "ultra-high risk" microarray classifier in FFPE high-risk neuroblastoma tumors. Customized probes corresponding to the 42 genes in a published multi-gene neuroblastoma signature were hybridized to RNA isolated from 107 FFPE high-risk neuroblastoma samples using the NanoString nCounter™ Analysis System. For classification of each patient, the Pearson's correlation coefficient was calculated between the standardized nCounter™ data and the molecular signature from the microarray data. We demonstrate that the nCounter™ 42-gene panel sub-stratified the high-risk cohort into two subsets with statistically significantly different overall survival (p = 0.0027) and event-free survival (p = 0.028). In contrast, none of the established prognostic risk markers (age, stage, tumor histology, MYCN status, and ploidy) were significantly associated with survival. We conclude that the nCounter™ System can reproducibly quantify expression levels of signature genes in FFPE tumor samples. Validation of this microarray signature in our high-risk patient cohort using a completely different technology emphasizes the prognostic relevance of this classifier. Prospective studies testing the prognostic value of molecular signatures in high-risk neuroblastoma patients using FFPE tumor samples and the nCounter™ System are warranted.
authors.affiliation: Department of Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, TN, USA.
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Stricker
authors.affiliation: Department of Pediatrics, Comer Children's Hospital, University of Chicago, Chicago, IL, USA.
authors.forename: Andres
authors.initials: A
authors.lastname: Morales La Madrid
authors.affiliation: Department of Pediatrics, Comer Children's Hospital, University of Chicago, Chicago, IL, USA.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Chlenski
authors.affiliation: Department of Pediatrics, Comer Children's Hospital, University of Chicago, Chicago, IL, USA.
authors.forename: Lisa
authors.initials: L
authors.lastname: Guerrero
authors.affiliation: Department of Pediatrics, Comer Children's Hospital, University of Chicago, Chicago, IL, USA.
authors.forename: Helen R
authors.initials: HR
authors.lastname: Salwen
authors.affiliation: Department of Pediatrics, Ann and Robert H. Lurie Children's Hospital of Chicago, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
authors.forename: Yasmin
authors.initials: Y
authors.lastname: Gosiengfiao
authors.affiliation: Department of Pathology, Ann and Robert H. Lurie Children's Hospital of Chicago, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Perlman
authors.affiliation: Department of Hematology/Oncology, St. Jude Children's Research Hospital, Memphis, TN, USA.
authors.forename: Wayne
authors.initials: W
authors.lastname: Furman
authors.affiliation: Department of Pathology, St. Jude Children's Research Hospital, Memphis, TN, USA.
authors.forename: Armita
authors.initials: A
authors.lastname: Bahrami
authors.affiliation: Department of Pediatrics, Texas Children's Cancer Center, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Jason M
authors.initials: JM
authors.lastname: Shohet
authors.affiliation: Department of Pediatrics, Texas Children's Cancer Center, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Peter E
authors.initials: PE
authors.lastname: Zage
authors.affiliation: Department of Pathology, Texas Children's Cancer Center, Baylor College of Medicine, Houston, TX, USA.
authors.forename: M John
authors.initials: MJ
authors.lastname: Hicks
authors.affiliation: Department of Pathology and Laboratory Medicine, Children's Hospital Los Angeles, Los Angeles, CA, USA.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Shimada
authors.affiliation: Department of Pathology and Laboratory Medicine, Children's Hospital Los Angeles, Los Angeles, CA, USA.
authors.forename: Rie
authors.initials: R
authors.lastname: Suganuma
authors.affiliation: Department of Pediatrics, Seattle Children's Hospital, University of Washington, Seattle, WA, USA; Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
authors.forename: Julie R
authors.initials: JR
authors.lastname: Park
authors.affiliation: Department of Pediatrics, Seattle Children's Hospital, University of Washington, Seattle, WA, USA; Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
authors.forename: Sara
authors.initials: S
authors.lastname: So
authors.affiliation: Children's Oncology Group Statistics and Data Center, Boston, MA, USA; Boston Children's Hospital/Dana-Farber Cancer Institute, Boston, MA, USA.
authors.forename: Wendy B
authors.initials: WB
authors.lastname: London
authors.affiliation: Department of Pathology, Comer Children's Hospital, University of Chicago, Chicago, IL, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Pytel
authors.affiliation: NanoString Technologies, Seattle, WA, USA.
authors.forename: Kirsteen H
authors.initials: KH
authors.lastname: Maclean
authors.affiliation: Department of Pediatrics, Comer Children's Hospital, University of Chicago, Chicago, IL, USA. Electronic address: scohn@peds.bsd.uchicago.edu.
authors.forename: Susan L
authors.initials: SL
authors.lastname: Cohn
mesh_terms: D016000:Cluster Analysis; D015331:Cohort Studies; D020869:Gene Expression Profiling / Q000295:instrumentation; D006801:Humans; D007223:Infant; D009447:Neuroblastoma / Q000175:diagnosis / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation*; D011379:Prognosis
keywords: Gene signature; High-risk neuroblastoma; Molecular classifier; NanoString; nCounter