title: Analytical validation of a practical molecular assay prognostic of survival in nonsquamous non-small cell lung cancer.
abstract: A molecular assay prognostic of survival in resected nonsquamous non-small cell lung cancer designed to meet the need for improved risk stratification in early-stage disease has recently been described. This assay measures the expression levels of 14 genes using RNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues. The assay underwent blinded clinical validation in 2 large international cohorts involving approximately 1500 patients; the analytical precision and reproducibility of this assay, however, have not yet been reported. For each of the 14 TaqMan quantitative polymerase chain reaction (PCR) primer and probe sets used in the molecular prognostic assay, the linear range, PCR efficiency, limits of blank, limits of quantitation, and quantitative bias were determined using serial dilutions of pooled RNA extracted from FFPE samples. The reproducibility of the entire molecular assay was determined by performing repeat testing of FFPE samples over multiple days. The linear range of individual quantitative TaqMan PCR primer and probe sets was between 2(10)- and 2(15)-fold input RNA. The median C(T) of the quantitative PCR primer and probe sets at 10 ng of input RNA was 24.3; the median efficiency was 91.2%. The median quantitative bias across all quantitative PCR primer and probe sets was 0.75% (range, 0.32% to 1.32%). In repeat testing, the mean SD of the risk score (scaled from 1 to 100) was 2.18, with a mean coefficient of variation of 0.08. The molecular prognostic assay presented in this study demonstrates high precision and reproducibility, validating its clinical utility as a reliable prognostic tool that can contribute to the management of patients with early-stage disease.
authors.affiliation: Thoracic Oncology Program, Department of Surgery, University of California, San Francisco, San Francisco, CA, USA.
authors.forename: Johannes R
authors.initials: JR
authors.lastname: Kratz
authors.forename: Patrick T
authors.initials: PT
authors.lastname: Tham
authors.forename: Michael S
authors.initials: MS
authors.lastname: Mulvihill
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Ziaei
authors.forename: Mahashweta Roshni
authors.initials: MR
authors.lastname: Ray
authors.forename: Jerry W
authors.initials: JW
authors.lastname: Hurst
authors.forename: Mark R
authors.initials: MR
authors.lastname: Segal
authors.forename: David M
authors.initials: DM
authors.lastname: Berryman
authors.forename: Wenjiang
authors.initials: W
authors.lastname: Chu
authors.forename: Biao
authors.initials: B
authors.lastname: He
authors.forename: David M
authors.initials: DM
authors.lastname: Jablons
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Mann
mesh_terms: D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis* / Q000235:genetics; D017931:DNA Primers; D005557:Formaldehyde / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D016014:Linear Models; D020411:Oligonucleotide Array Sequence Analysis; D016612:Paraffin Embedding / Q000379:methods; D016133:Polymerase Chain Reaction; D011379:Prognosis; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D015203:Reproducibility of Results
chemical_list: D017931:DNA Primers; D012334:RNA, Neoplasm; D005557:Formaldehyde