The adoption of third-generation tyrosine kinase inhibitors (TKIs) to treat non-small cell lung cancer (NSCLC) for EGFR positive patient cases has led to the emergence of acquired resistance pathways. Approximately 40% of EGFRT790M-positive NSCLC cases display an acquired mutation of amino acid 797 (C797S) that mediates this resistance, which can develop after a median of 10 months following treatment. The effectiveness of treatment for patients with EGFRC797S may depend on whether the mutation is present in cis or trans with EGFRT790M, the type of sensitizing mutation (EGFRdel19 or EGFRL858R), and previous treatment(s). Patients with EGFRdel19 or EGFRL858R in conjunction with EGFRT790M and EGFRC797S remain resistant to all current targeted therapies. Several fourth-generation inhibitors are in development but have yet to be validated in clinical trials. We have developed a blood-based test that can detect two of the most common C797S nucleotide mutations (T>A and G>C) in cis or trans with EGFRT790M using the droplet digital PCR (ddPCR) technology and assays. Analytic sensitivity and specificity were assessed using synthetic DNA designed to mimic the EGFRC797S variants and their locus detected in the assay. Normal healthy donors as well as reference ddPCR positive and negative NSCLC donor samples were assessed for clinical specificity and sensitivity. Finally, the precision of the assay was evaluated with both clinical and analytical samples. Specifically, we evaluated the assay at high, medium, and low mutation frequencies over three consecutive days, including repeat runs on one day, and with multiple operators. This assay is capable of accurately and precisely detecting multiple EGFRC797S variants and their locus from blood specimens in the clinical laboratory. Consistent with other ddPCR blood-based EGFR variant assays we have developed, the limit of detection was 0.02%for C797S variants detected in both cis and trans with the T790M mutation. Once validated, the EGFR C797S assay described here may be of utility in diagnosing patients who have developed resistance to osimertinib.
Lung cancer is the leading cause of cancer-related deaths in the United States with Non-Small Cell Lung Cancer (NSCLC) being the most commonly diagnosed subtype. However, up to 30% of advanced NSCLC patients are not eligible for tissue biopsy. As a result, liquid biopsies are becoming increasingly utilized in clinical testing as they are non-invasive and have an overall decreased risk to patients. This approach also addresses other challenges associated with tissue-based profiling, including tumor heterogeneity and low yield of quality nucleic acid for the identification of actionable targets of treatment. In this study, cell-free total nucleic acid (cfTNA) was isolated from donor patient plasma samples collected and shipped at ambient temperatures in blood collection tubes to the Biodesix CAP/CLIA laboratory in Boulder, CO. cfTNA was profiled with a targeted cancer NGS (next generation sequencing) panel, using the Ion Torrent GeneStudio S5 Plus System. Variant analyses were conducted using a threshold of ≥0.3% percent variant allele frequency to assess concordance in the patient donor specimens. A high level of concordance (R2=0.99) was observed between inter-laboratory/inter-instrument NGS runs using 6 clinical samples. In the 0.1% single nucleotide variant (SNV) positive control sample which encompasses 23 hotspots, there was slightly lower concordance (R2=0.90) due to low variant allele frequency (0.06-0.3%). Validation studies are in progress and include RNA fusions and additional variants present in blood from patients diagnosed with NSCLC.