Lung cancer is the leading cause of cancer-related death in America and across the world, accounting for nearly 1.6 million deaths per year. Somatic mutations in the EGFR, KRAS or TP53 gene are common in lung cancer, and EGFR mutational status is an important factor in choosing the optimal form of molecularly targeted therapy. Liquid biopsy is the detection of tumor materials in blood and other body fluids, including the detection of cell-free DNA (cfDNA) and its component tumor-derived DNA (termed circulating tumor DNA or ctDNA in the blood). ctDNA detection is less invasive than traditional tissue biopsy and usually has a faster turnaround time than tissue sequencing. While next-generation sequencing (NGS)-based ctDNA detection is very sensitive in later-stage disease, it is less sensitive for early-stage cancers. Additionally, in early lung cancer, bronchoscopy and bronchoalveolar lavage (BAL) is used for diagnosis and staging. BAL samples the lung periphery by directly washing the tumor area with a sterile saline solution which is traditionally used for detecting tumor cells. However, BAL fluid (BALF) may be a better source of cfDNA for the detection of lung cancer mutations than blood, with equal or higher diagnostic sensitivity for early-stage disease. This project aims to detect and quantify targetable EGFR mutations in the blood or BALF of NSCLC patients using ddPCR. 

The patient sample consisted of ​​2 positive control stage IV patients who had known mutation by tissue sequencing, 7 negative control stage IV patients with a KRAS G12 mutation, a known mutually exclusive mutation, 4 negative control stage IV patients lacking the mutation of interest, and 10 negative control patients determined to be cancer-free on biopsy. 

The assay used is specific at detecting positive and negative controls for the EGFR E746_A750 deletion mutation, however, the small sample size should be noted. Further, no false positive droplets were detected. Importantly, the assay detected mutant copies in the positive control sample; it did not detect any in the positive control, however, this variant was missed by a commercial ctDNA assay as well, suggesting a very low variant allele fraction. In the future, we intend to perform the same background analysis conducted on the blood plasma samples on the BALF controls. We also intend to perform this assay on plasma and BALF samples from stage II/III NSCLC patients to test hypotheses for greater sensitivities to determine assay sensitivity in the setting of earlier-stage disease.