title: Analytical validation of the PAM50-based Prosigna Breast Cancer Prognostic Gene Signature Assay and nCounter Analysis System using formalin-fixed paraffin-embedded breast tumor specimens.
abstract: BACKGROUND
  NanoString's Prosigna™ Breast Cancer Prognostic Gene Signature Assay is based on the PAM50 gene expression signature. The test outputs a risk of recurrence (ROR) score, risk category, and intrinsic subtype (Luminal A/B, HER2-enriched, Basal-like). The studies described here were designed to validate the analytical performance of the test on the nCounter Analysis System across multiple laboratories.
  
  
  METHODS
  Analytical precision was measured by testing five breast tumor RNA samples across 3 sites. Reproducibility was measured by testing replicate tissue sections from 43 FFPE breast tumor blocks across 3 sites following independent pathology review at each site. The RNA input range was validated by comparing assay results at the extremes of the specified range to the nominal RNA input level. Interference was evaluated by including non-tumor tissue into the test.
  
  
  RESULTS
  The measured standard deviation (SD) was less than 1 ROR unit within the analytical precision study and the measured total SD was 2.9 ROR units within the reproducibility study. The ROR scores for RNA inputs at the extremes of the range were the same as those at the nominal input level. Assay results were stable in the presence of moderate amounts of surrounding non-tumor tissue (<70% by area).
  
  
  CONCLUSIONS
  The analytical performance of NanoString's Prosigna assay has been validated using FFPE breast tumor specimens across multiple clinical testing laboratories.
authors.affiliation: British Columbia Cancer Agency, 3427 - 600 W 10TH Avenue, V5Z 4E6 Vancouver, BC, Canada. torsten@mail.ubc.ca.
authors.forename: Torsten
authors.initials: T
authors.lastname: Nielsen
authors.forename: Brett
authors.initials: B
authors.lastname: Wallden
authors.forename: Carl
authors.initials: C
authors.lastname: Schaper
authors.forename: Sean
authors.initials: S
authors.lastname: Ferree
authors.forename: Shuzhen
authors.initials: S
authors.lastname: Liu
authors.forename: Dongxia
authors.initials: D
authors.lastname: Gao
authors.forename: Garrett
authors.initials: G
authors.lastname: Barry
authors.forename: Naeem
authors.initials: N
authors.lastname: Dowidar
authors.forename: Malini
authors.initials: M
authors.lastname: Maysuria
authors.forename: James
authors.initials: J
authors.lastname: Storhoff
mesh_terms: D014408:Biomarkers, Tumor / Q000378:metabolism; D001943:Breast Neoplasms / Q000175:diagnosis* / Q000235:genetics* / Q000473:pathology; D005260:Female; D005557:Formaldehyde / Q000737:chemistry*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D016612:Paraffin Embedding / Q000379:methods*; D057089:Pathology, Molecular / Q000379:methods*; D011379:Prognosis; D012313:RNA / Q000032:analysis*; D015203:Reproducibility of Results; D054928:Validation Studies as Topic
chemical_list: D014408:Biomarkers, Tumor; D005557:Formaldehyde; D012313:RNA