Analytical Validation of a Practical Molecular Assay Prognostic of Survival in Nonsquamous Non-Small Cell Lung Cancer
*Thoracic Oncology Program, Department of Surgery ‡Department of Epidemiology and Biostatistics, University of California, San Francisco, San Francisco †Pinpoint Genomics, Mountain View, CA.Diagnostic molecular pathology: the American journal of surgical pathology, part B (Impact Factor: 2.28). 04/2013; 22(2). DOI: 10.1097/PDM.0b013e318273fb61
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- and 2-fold input RNA. The median CT 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.
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ABSTRACT: Introduction Adjuvant chemotherapy improves survival for some patients with non-small-cell lung cancer (NSCLC) and is recommended by National Comprehensive Cancer Network (NCCN) guidelines for stage Ib-IIa patients with certain “high-risk” characteristics. An internationally validated, 14-gene expression assay has been shown to better stratify mortality risk in non-squamous NSCLC than either conventional staging or these high risk clinicopathologic features. Patients and Methods A blinded chart review of 52 patients with prospective molecular risk stratification by the 14-gene test compared recurrence outcomes with a mean follow up of 15.2 ±11.7 months of patients with high- or low-risk determined by either NCCN criteria or the molecular assay. Results Molecular risk assessment was discordant from NCCN criteria in 14 of 23 patients in stages Ib and IIa (61%). Recurrence was not observed among any of 31 molecular intermediate- or low-risk patients, including 10 NCCN high-risk patients, whereas 33% of recurrences occurred among NCCN low-risk patients. Recurrences in stages I or IIa were seen in 2 of 18 NCCN high-risk patients (11%, both were stage IIa and both received a high-risk molecular designation), and in 4 out 18 patients (22%) with a high-risk molecular score, including 1 stage Ia and 1 stage Ib patient. Conclusion This small cohort study suggests that a 14-gene prognostic assay more accurately stratifies risk among early stage NSCLC patients than current NCCN criteria. NCCN guidelines already advocate risk stratification within TNM stages. This molecular assay has clinical utility in better identifying high-risk patients and might improve NCCN adjuvant chemotherapy recommendations.Clinical Lung Cancer 08/2014; 15(6). DOI:10.1016/j.cllc.2014.07.004 · 3.10 Impact Factor
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