Development of multiplex digital PCR based droplex NSCLC panel test for detecting major mutations in patients with non-small cell lung cancer (NSCLC).

46 Background: Recently, in the treatment of non-small cell lung cancer(NSCLC) patient, immunotherapies and targeted therapies aimed at various genes have been developed. Next-Generation Sequencing(NGS) technology is currently known as the only method for detecting genetic alterations in many different genes simultaneously. However, this technology requires a substantial amount of DNA or RNA input from patients to obtain high-quality results in a clinical setting. We have developed a Droplex NSCLC Panel Test Kit based on multiplex digital PCR, which is known as ideal molecular diagnostic technique due to its simple workflow, minimal sample input, high sensitivity and specificity. In addition, it has a one-day TAT (Turn around time) and low costs compared NGS, resulting in a lower financial burden for patients. Recently, the development of non-invasive liquid biopsy diagnostics as an alternative to tissue biopsy is emerging important issue. The kit, applying a digital PCR platform, enables multiple mutation detection using plasma samples of blood. The digital PCR-based Droplex NSCLC Panel Test Kit is designed to detect over 170 key mutations in 11 genes, namely EGFR, ALK, ROS1, BRAF, MET, RET, KRAS, HER2, NTRK1, NTRK2, and NTRK3, using DNA and RNA extracted from NSCLC FFPET and plasma samples. Methods: The Droplex NSCLC Panel Test Kit consists of four Oligo Mixtures (OMs) for detecting mutations of 4 genes on DNA and two OMs for detecting 7 gene rearrangements on RNA. After extracting DNA and RNA from a single sample, cDNA synthesis from RNA is prioritized, followed by simultaneous execution of droplet generation, PCR processes, and data analysis. The kit test was based on QX600 droplet digital PCR system of Bio-rad with 6 fluorescent channels. To validate the analytical performance of the Droplex NSCLC Panel Test Kit for each type of sample, DNA and RNA samples were extracted from FFPET sections and plasma isolated from NSCLC patients. Each type of samples, contrived samples were manufactured and tested for analytical performance. Results: The results showed that DNA/RNA multiple mutations can be detected simultaneously on a 6-fluorescent channel system. The kit was highly sensitive for low-input sample in dilutions studies ranging from 5 to 20ng DNA or RNA for FFPET samples and plasma sample. The Digital PCR NSCLC panel test detected an analytical specificity of 100% and detection limit of 0.25% or higher for all genes included in the kit. Conclusions: We demonstrated that the performance of the Droplex NSCLC Panel Test is reliable and effective for the detection of above 170 clinically relevant mutations of 11 genes in FFPET and plasma samples of patients with NSCLC. Additionally, it will be necessary to evaluate the utility of the kit through further clinical studies with NSCLC patients, as well as expanding its compatibility to various digital PCR platforms.