Ultrasensitive Detection of Lung Cancer Mutation Pattern Favoring Minimal Invasive Liquid Biopsy - An Experimental Approach

Abstract

Lung cancer is the most commonly diagnosed cancer type and the leading cause of cancer death in men and women worldwide. Early detection of lung cancer is problematic as it requires surgery to extract tissue specimens. In contrast, liquid biopsy is a minimally invasive method requiring only a blood sample to perform early diagnosis and treatment monitoring of cancer. Analyzing tumor-derived biomarkers, such as circulating tumor cells (CTCs) and/or circulating tumor DNA (ctDNA), require ultrasensitive detection methods for profiling lung cancer. The purpose of this study is to analyze the ultrasensitive detection of lung cancer mutations (ctDNAs) in patients’ plasma samples using the UltraSEEK Lung Panel. Results of this study will be integrated in the collaboration project “The Brain Metastases” between Universitätsklinikum Hamburg-Eppendorf and Agena Bioscience. Plasma samples of seventeen lung cancer patients have been collected for this study. The quality analysis of cfDNA and preliminary on-site tests, using reference material, were used to verify the performance of the UltraSEEK Lung Panel. In addition, participating in a ring trial was an appropriate opportunity to evaluate the performance of the UltraSEEK Lung Panel. Silica membrane adsorption technique and magnetic beads binding technology were the two different methods used to extract cfDNA. NanoDrop, Qubit, LabChip, and LiquID IQ Panel were used to quantify the cfDNA concentration. Finally, the MassARRAY System technology combined with the UltraSEEK Lung Panel was used to detect the mutation in plasma samples of the seventeen lung cancer patients. During verification and validation of the UltraSEEK Lung Panel, differences in cfDNA fragment lengths were observed in synthetically manufactured cfDNA versus human cfDNA. DNA fragmentation has an impact on the ability of the analysis panel to detect low mutation frequencies. The verification test, using reference materials, showed a high sensitivity (91-100%) of mutation detection for mutation with allele frequencies (AF) between 0.5% to 2.0%. The sensitivity of mutation detection decreased to 44%-70% for AF in the range of 0.1% to 0.3%. The high performance of the UltraSEEK Lung Panel was confirmed by the Reference Institute for Bioanalytics, a ring trial supervising laboratory. Finally, in the UltraSEEK Lung Panel clinical study, the assay detected mutations (on genes such as KRAS, EGFR, and PIK3CA) in 9 out of 17 lung cancer patient samples. As a conclusion, the UltraSEEK Lung Panel was shown to be able to detect mutations (ctDNA) of lung cancer patients in blood samples with AF as small as 0.1%, and could therefore be used for early detection and treatment monitoring of lung cancer using minimal invasive liquid biopsies.