Abstract LB156: Early mutation-mediated detection of cancers via biomarker production

Abstract Background: Cancer remains one of the leading causes of mortality worldwide, with early detection pivotal to improving patient outcomes. Current diagnostic methods often lack the sensitivity and specificity to identify cancers at their earliest stages, especially for KRAS-driven cancers such as pancreatic cancer and non-small cell lung cancer (NSCLC). This challenge is further exacerbated for high-risk populations, where invasive biopsies, imaging, or endogenous biomarkers fail to meet clinical needs. There is a critical need for non-invasive, reliable, and continuous cancer detection methods capable of diagnosing tumors at nascent stages. To address this, we developed a mutation-mediated synthetic biomarker-based blood test specifically targeting early-stage cancers with KRAS G12 mutations. This study evaluates its performance in detecting KRAS-driven cancers using synthetic biomarkers and controls. Methods: A CRISPR-based gene editing system was designed to insert synthetic biomarker genes, such as Gaussia luciferase (GLuc), into cancer cells harboring KRAS G12 mutations. Using Gibson assembly, we constructed a donor plasmid carrying the GLuc gene alongside a plasmid encoding Cas9 VQR and sgRNA targeting the KRAS G12V locus. These plasmids were co-transfected into NCI-H727 human lung tumor cells. Single-cell sorting yielded 29 clonal lines with stable GLuc expression. Sequencing confirmed precise knock-in at the KRAS G12V target in 21 lines, with qPCR verifying exclusive insertion. GLuc secretion was measured via luciferase assays, and limits of detection (LOD) were assessed in vitro. To test biomarker detection in vivo, GLuc-expressing tumor cells were inoculated into SCID/NOD mice at varying numbers. Serum GLuc levels were measured using luciferase assays at defined time points. Results: In vitro, the LOD for GLuc-expressing cancer cells was 5,775 cells/mL. In vivo, SCID/NOD mice injected with GLuc-expressing tumor cells showed time-dependent increases in serum luminescence. Two weeks post-inoculation with 106 cells, serum luminescence in the experimental group was 8.24-fold higher than controls (8.24 ± 0.32), with a tumor lesion size of 29.86 ± 7.26 mm3. Conclusion: This mutation-mediated synthetic biomarker platform detected tumor lesions as small as millimeter-scale in vivo with high sensitivity and specificity after a single injection. Its non-invasive, scalable design offers the potential to transform early detection and monitoring of KRAS-driven cancers, addressing critical unmet needs for high-risk populations. Citation Format: Shengyue Piao, Miller Harris, Kevin McHugh. Early mutation-mediated detection of cancers via biomarker production [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB156.