Abstract 5037: Development of multiplexed Absolute Q digital PCR assays to rapidly quantify EGFR and KRAS genotype in advanced non-small cell lung cancer (NSCLC) patients’ plasma
Authors: Hanna Benjamin , Xu Shidong , Paweletz Cloud P. , Kuang Yanan 
Published: March 2024

Abstract
Introduction: There are needs to identify and serially monitor driver and resistance mutations (muts) in therapy-treated cancer patients (pts). Quantitative liquid biopsy in cell-free (cf)DNA has been widely adopted. We have developed and clinically validated droplet digital PCR (ddPCR) assays to detect common driver/resistance muts such as EGFR del19, L858R, T790M and KRAS G12X in the cfDNA of advanced NSCLC pts. We have established ddPCR assays for 30+ driver/resistance muts in multiple genes (BRAF, MET, ESR1, etc.) relevant to melanoma, breast, and colorectal cancer. Our assays use tumor/cell line DNA as positive controls and serve as benchmarks to develop newer assays such as plasma NGS. In this study, we converted existing/validated EGFR and KRAS G12X ddPCR assays to the QuantStudio Absolute Q digital PCR (dPCR) system. The selected mutations maximize actionable clinical information and are designed to study oncogenic and resistance mutations in NSCLC efficiently. dPCR’s accuracy and efficiency enables target multiplexing, providing comprehensive information from limited materials and contributing to liquid biopsy research progress.
Methods: Plasma from pts with tumors harboring EGFR and KRAS muts was collected at progression with advanced disease following consent to protocol DFCI #14-147. We designed a multiplex dPCR assay that identifies the most common and therapy-relevant KRAS muts in NSCLC, G12C/D/V, and detects other G12X using a drop-off probe. We also designed 2 EGFR kits, one identifying driver muts, the other resistance muts.
Results: To validate assay performance, we used gDNA derived from cell lines with EGFR or KRAS muts. We serially diluted mut gDNA samples in background wt gDNA (16 ~ 1000 mut copies in 1000 wt copies) and performed KRAS and EGFR dPCR assays. We detected 16 mut copies with sensitivities similar to those of ddPCR assays. We then studied 48 cfDNA samples from plasma of NSCLC pts with tumor confirmed KRAS G12X or EGFR L858R, del19, T790M muts. No samples had both EGFR and KRAS muts detected, suggesting our assays are specific. We detected muts in samples with an allele frequency median of 0.9% for KRAS mut, 11.9% for EGFR driver mut, 2.9% for EGFR T790M. The concordance rate is 91.3% between dPCR and ddPCR results. Our findings suggest these assays are as sensitive and specific as our validated ddPCR assays. Multiplexed Absolute Q dPCR saves cfDNA and cuts turnaround time (TAT) to 90 min for KRAS assay and 3hr for EGFR assays.
Conclusions: We developed rapid, sensitive and specific quantitative dPCR assays for KRAS and EGFR muts on the QuantStudio Absolute Q platform to quantify plasma genotyping in NSCLC. We will clinically pilot our validated rapid genotyping approach and quantify TAT advantage of multiplexed genotyping in NSCLC pts undergoing standard-of-care tumor genotyping.
Citation Format: Benjamin Hanna, Shidong Xu, Cloud P. Paweletz, Yanan Kuang. Development of multiplexed Absolute Q digital PCR assays to rapidly quantify EGFR and KRAS genotype in advanced non-small cell lung cancer (NSCLC) patients’ plasma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5037.