title: Decentral gene expression analysis: analytical validation of the Endopredict genomic multianalyte breast cancer prognosis test.
abstract: BACKGROUND
  EndoPredict (EP) is a clinically validated multianalyte gene expression test to predict distant metastasis in ER-positive, HER2-negative breast cancer treated with endocrine therapy alone. The test is based on the combined analysis of 12 genes in formalin-fixed, paraffin-embedded (FFPE) tissue by reverse transcription-quantitative real-time PCR (RT-qPCR). Recently, it was shown that EP is feasible for reliable decentralized assessment of gene expression. The aim of this study was the analytical validation of the performance characteristics of the assay and its verification in a molecular-pathological routine laboratory.
  
  
  METHODS
  Gene expression values to calculate the EP score were assayed by one-step RT-qPCR using RNA from FFPE tumor tissue. Limit of blank, limit of detection, linear range, and PCR efficiency were assessed for each of the 12 PCR assays using serial samples dilutions. Different breast cancer samples were used to evaluate RNA input range, precision and inter-laboratory variability.
  
  
  RESULTS
  PCR assays were linear up to Cq values between 35.1 and 37.2. Amplification efficiencies ranged from 75% to 101%. The RNA input range without considerable change of the EP score was between 0.16 and 18.5 ng/μl. Analysis of precision (variation of day, day time, instrument, operator, reagent lots) resulted in a total noise (standard deviation) of 0.16 EP score units on a scale from 0 to 15. The major part of the total noise (SD 0.14) was caused by the replicate-to-replicate noise of the PCR assays (repeatability) and was not associated with different operating conditions (reproducibility). Performance characteristics established in the manufacturer's laboratory were verified in a routine molecular pathology laboratory. Comparison of 10 tumor samples analyzed in two different laboratories showed a Pearson coefficient of 0.995 and a mean deviation of 0.15 score units.
  
  
  CONCLUSIONS
  The EP test showed reproducible performance characteristics with good precision and negligible laboratory-to-laboratory variation. This study provides further evidence that the EP test is suitable for decentralized testing in specialized molecular pathological laboratories instead of a reference laboratory. This is a unique feature and a technical advance in comparison with existing RNA-based prognostic multigene expression tests.
authors.affiliation: Sividon Diagnostics GmbH, Nattermannallee 1, 50829, Cologne, Germany. Kronenwett@sividon.com
authors.forename: Ralf
authors.initials: R
authors.lastname: Kronenwett
authors.forename: Kerstin
authors.initials: K
authors.lastname: Bohmann
authors.forename: Judith
authors.initials: J
authors.lastname: Prinzler
authors.forename: Bruno V
authors.initials: BV
authors.lastname: Sinn
authors.forename: Franziska
authors.initials: F
authors.lastname: Haufe
authors.forename: Claudia
authors.initials: C
authors.lastname: Roth
authors.forename: Manuela
authors.initials: M
authors.lastname: Averdick
authors.forename: Tanja
authors.initials: T
authors.lastname: Ropers
authors.forename: Claudia
authors.initials: C
authors.lastname: Windbergs
authors.forename: Jan C
authors.initials: JC
authors.lastname: Brase
authors.forename: Karsten E
authors.initials: KE
authors.lastname: Weber
authors.forename: Karin
authors.initials: K
authors.lastname: Fisch
authors.forename: Berit M
authors.initials: BM
authors.lastname: Müller
authors.forename: Marcus
authors.initials: M
authors.lastname: Schmidt
authors.forename: Martin
authors.initials: M
authors.lastname: Filipits
authors.forename: Peter
authors.initials: P
authors.lastname: Dubsky
authors.forename: Christoph
authors.initials: C
authors.lastname: Petry
authors.forename: Manfred
authors.initials: M
authors.lastname: Dietel
authors.forename: Carsten
authors.initials: C
authors.lastname: Denkert
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D057089:Pathology, Molecular / Q000379:methods*; D011379:Prognosis; D012313:RNA / Q000032:analysis*; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D012313:RNA