Abstract 2525: Low frequency TP53 mutations in airway epithelial cells serve as lung cancer risk biomarker
Authors: Craig Daniel J. , Crawford Erin L. , Massion Pierre P. , Morrison Thomas , Willey James C. 
Published: July 2021

Abstract
Background: Lung cancer is the leading cause of cancer-related death in men and women in the United States. Based on evidence from controlled trials in the United States and Europe that low dose CT (LDCT) screening significantly reduces lung cancer mortality, the United States Preventative Services Tasks Force (USPSTF) recommends LDCT screening for individuals with high demographic risk based on age and smoking history. However, a large fraction of lung cancers occur in individuals who do not meet LDCT screening threshold criteria. Thus, there is a need for biomarkers that supplement demographic factors to more accurately detect those at highest lung cancer risk and include additional individuals who will benefit from LDCT screening. An initial study from this lab demonstrated that TP53 mutations with 0.05-1.0% variant allele fraction (VAF) were significantly more prevalent (p&lt;0.005) in grossly normal airway epithelial cell (AEC) specimens from lung cancer cases compared to non-cancer controls matched for smoking and age. Here, we present an expanded follow-up study aimed at testing this biomarker according to PRospective-specimen-collection, retrospective-Blinded-Evaluation (PRoBE) design.
Methods: AEC specimens were prospectively collected through the National Cancer Institute Early Detection Research Network (EDRN) program from 60 subjects at high demographic risk for lung cancer by bronchoscopic brush biopsy at Vanderbilt University Medical Center. Genomic (g)DNA was extracted from each AEC specimen and stored at -80 degrees Celsius. Subjects matched for age and smoking history who subsequently did or did not develop lung cancer were identified and gDNA specimens, blinded with respect to cancer status, were shipped to the University of Toledo. gDNA specimens were quantified and 50,000 gDNA copies were included in each NGS library preparation. Synthetic DNA internal standards (IS) were prepared for multiple lung cancer driver mutations within the TP53 gene and mixed with each AEC gDNA specimen prior to competitive multiplex PCR NGS library preparation. By controlling for technical error, this approach enables reliable detection of mutations with VAF as low as 5 x 10-4 (0.05%) (Craig et al, BMC Cancer, 2019).
Results: Following library preparation and sequencing on Illumina Novaseq, there was sufficient library complexity to detect TP53 VAF &lt;0.05% in &gt;90% of AEC specimens. The data are currently in pipeline analysis. After analysis is complete, VCF files will be sent to Vanderbilt for unblinding and validation of the biomarker.
Conclusions: Based on preliminary data, we expect that sufficient AEC gDNA is available from most subjects enrolled in the EDRN program for TP53 low VAF biomarker analysis, and the previously reported method (Craig et al, BMC Cancer, 2019) is reliable and feasible. The current study should provide additional information regarding validity of the biomarker.
Citation Format: Daniel J. Craig, Erin L. Crawford, Pierre P. Massion, Thomas Morrison, James C. Willey. Low frequency TP53 mutations in airway epithelial cells serve as lung cancer risk biomarker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2525.