Analytical validation of the Percepta Nasal Swab classifier; an RNA next-generation sequencing assay for the assessment of lung cancer risk in pulmonary nodules.

Abstract

Background: A novel molecular diagnostic test, Percepta Nasal Swab (PNS), was developed as a noninvasive lung cancer biomarker to aid in risk assessment for indeterminate pulmonary nodules in individuals who smoke or have previously smoked. Prior research has shown that exposure of the airway epithelium to cigarette smoke results in epithelial gene expression alterations throughout the respiratory tree that reflect the risk of lung cancer in a pulmonary nodule. The PNS classifier leverages this concept using whole transcriptome sequencing (RNASeq) of cells collected from the nasal epithelium and provides "high", "intermediate" and "low risk" classification calls to help guide clinical management decisions. The clinical validity of the PNS test was established on an independent validation set and demonstrated favorable sensitivity and specificity. This study aims to evaluate the analytical validity of the PNS test performance in our CLIA (Clinical Laboratory Improvement Amendments) laboratory.

Methods: The reproducibility between RNASeq runs within a laboratory and the accuracy between laboratories were estimated and compared against the performance-based acceptance criterion. The impacts from varying RNA input amount, genomic DNA and blood RNA interference were evaluated to demonstrate the analytical sensitivity and specificity of the PNS test results to known conditions that may occur in routine laboratory processing.

Results: Based on modeling the impact on clinical sensitivity/specificity, PNS test classifier scores can allow up to 0.776 score units of added noise/variability before any performance metrics drop below the pre-specified requirements. This allowable variability is six-fold higher than the observed variability estimated between runs and between laboratories under routine testing conditions, which are each less than 2% of the 98th percentile score range. In addition, PNS test results are shown to be robust against RNA input variation from 50 ng to 15 ng, up to 30% of genomic DNA by nucleic mass interference, and up to 14% of blood RNA interference.

Conclusions: This study provided sufficient evidence for the accuracy, reproducibility, sensitivity, and specificity of the PNS molecular test and supported its utilization in clinical testing.