Abstract 4259: Conversion of the Lung Cancer Risk Test (LCRT) to a next generation sequencing (NGS) platform
Authors: Crawford Erin L. , Blomquist Thomas M. , Willey James C. 
Published: August 2015

Abstract
Background: The Lung Cancer Risk Test (LCRT) is a promising, gene-expression-based test that was developed for use in individuals at risk for but not yet diagnosed with lung cancer (Blomquist et al. Cancer Research 2009). A blinded, prospective cohort study is underway in which normal bronchial epithelial cells (NBEC), blood and demographic information were collected from 384 subjects aged 50-90 years at enrollment (between 2010 and 2012) with at least a 20 pack-year smoking history and no lung cancer. These subjects underwent a chest CT at enrollment to confirm absence of suspicious lung lesions and follow-up is ongoing.
Purpose: A recently developed next generation sequencing (NGS) method enables reliable, quantitative measurement of gene expression using custom amplicon libraries (Blomquist et al. PLoS ONE 2013). Due to the reduced cost and sample consumption of this method we were motivated to convert the LCRT from capillary electrophoresis (CE)-based to NGS method.
Methods/Design: NBEC cDNA samples from 12 individuals for which the LCRT had been previously measured using the CE method were co-amplified with a newly created mixture of internal standards (MIS) designed for NGS analysis, using the original LCRT primers. The resultant PCR products were re-amplified with primers complementary to internal regions of each target gene to yield two 100 bp amplicons for NGS analysis. Sample-specific barcodes and platform adaptors were added by PCR and samples were pooled for sequencing. NGS was conducted using the Illumina Hiseq 2000 with the 100 cycle paired-read method. PCR amplification for each sample was conducted in triplicate. Two replicates were sequenced together in one run and the third replicate was sequenced in a separate run. Gene expression levels for each gene comprising the LCRT were compared to levels obtained using the CE method.
Results: There was a small molarity difference between the original and new MIS. A delta analysis was performed using 6/12 samples and a correction factor was applied to both the second 6 sample set and all 12 samples. Gene expression values for the LCRT as measured by CE and NGS were highly correlated. The slope and R2 values for the second sample set and all 12 samples were m = 0.95, R2 = 0.96 and m = 0.93, R2 = 0.95 respectively. Comparison of the two replicate PCR measurements detected using NGS in the same sequencing run demonstrated a high degree of reproducibility (m = 1.00, R2 = 0.99). The slope was 1.03 and R2 was 0.94 when these replicates were compared to the third analyzed on a separate sequencing run.
Conclusions: Measurement of the LCRT using NGS of custom amplicon libraries will yield reliable results that are comparable to those previously reported (Blomquist et al. 2009). Inter-amplification and inter-sequencing run variation is low and, thus, it is expected that results using NGS will be highly reproducible. Conversion of the LCRT from CE to NGS methods should lead to reduced costs and sample consumption.
Citation Format: Erin L. Crawford, Thomas M. Blomquist, James C. Willey. Conversion of the Lung Cancer Risk Test (LCRT) to a next generation sequencing (NGS) platform. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4259. doi:10.1158/1538-7445.AM2015-4259