Abstract 4351: Multiplex detection of G12/G13 KRAS mutations with an electrochemiluminescent hybridization assay
Authors: Szabolcs Annamaria , Break Timothy J. , Shin Isaac H. , Harkins Seth B. , Wohlstadter Jacob N. 
Published: April 2023

Abstract
Purpose: Oncogenic Kirsten rat sarcoma virus (KRAS) mutations are the most prevalent cancer mutations in all human tumors. Also, genotyping the KRAS gene has become increasingly important with the emergence of new evidence highlighting differences in downstream signaling pathways, tumor microenvironment composition, treatment responses and prognoses linked to specific single nucleotide polymorphisms (SNPs) in the G12 or G13 codons. FDA approval of G12C-specific anti-KRAS drugs highlights the importance of the development of reliable SNP assays to interrogate the mutation status in a tumor sample. Currently available genotyping assays are based on NGS, PCR, or ddPCR. These methods can be time-consuming, costly, suffer from amplification bias, or require follow-up testing or bioinformatics skills to analyze. Building on Meso Scale Discovery’s (MSD) existing technology, we aimed to develop a method for the identification of eight KRAS SNPs located on the G12-G13 codons in a single reaction.
Methods: Ten-spot, 96-well N-PLEX plates were used for the study. SNP-specific upstream probes carrying unique 5′ leader sequences complementary to spot-specific captures on the N-PLEX plates were designed for eight KRAS genotypes (WT, G12R, G12C, G12S, G12A, G12D, G12V, G13D). Locus-specific downstream probes were 5’ phosphorylated for ligation and 3’ biotinylated for detection. Synthetic DNA targets were used as assay calibrators, and commercially available FFPE reference samples were tested for validation. DNA (10 ng) was PCR amplified with primers flanking the KRAS mutation sites. A multiplexed mixture of upstream and downstream probes was hybridized to the amplicons, and 30 cycles of ligation was performed using DNA ligase in a thermal cycler. Samples were subsequently hybridized to spot-specific capture oligonucleotides on the surface of the assay plates and detected with SULFO-TAG labeled streptavidin. Electrochemiluminescence readout was collected on an MSD imager.
Results: Optimization of probe concentrations and ligation temperature was performed to maximize signal-to-background ratios and assay specificity. Spike recovery experiments using artificial target DNA showed successful identification of KRAS mutant genotypes at spike levels as low as 0.1% over wild-type background. Validation experiments with gDNA extracted from FFPE reference samples confirmed that the multiplex SNP assay can accurately differentiate eight KRAS genotypes in samples with &gt;0.4% tumor burden.
Conclusion: These data highlight a novel approach to simultaneously identify eight KRAS genotypes from 10 ng of DNA input in a single reaction within 4-5 hours. The assay is capable of identifying double mutants and can provide a semiquantitative assessment of tumor burden without the need for follow-up testing.
Citation Format: Annamaria Szabolcs, Timothy J. Break, Isaac H. Shin, Seth B. Harkins, Jacob N. Wohlstadter. Multiplex detection of G12/G13 KRAS mutations with an electrochemiluminescent hybridization assay. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4351.