Abstract 4343: Development of an APC and TP53-based duplex sequencing assay to positively predict colorectal cancer response to EGFR inhibitors

Abstract Two well-characterized EGFR inhibitor (EGFRi) therapies (cetuximab, panitumumab) have been FDA approved for metastatic colorectal cancer (CRC). Unfortunately, the extended RAS/RAF testing required in the drug labels, identifies only non-responders, and only ~50% of treated patients will respond to therapy. Thus, there is an unmet need to develop additional biomarkers to identify EGFRi sensitive patients. Using an innovative hybrid approach fusing gene expression and DNA sequencing, we recently reported that combined mutations in APC and TP53 were strongly correlated with a validated gene expression signature measuring cetuximab sensitivity in CRC human tumors (Yang et al. Cancer Epidemiol Biomarkers Prev. 2019), suggesting a potential positive biomarker role of APC + TP53 mutations. This finding prompted us to develop a DNA test designed to positively select EGFRi-responsive patients as augmentation to currently used negative predictors for CRC patients. By leveraging the TwinStrand Duplex Sequencing (DS) technology, we recently developed an ultrasensitive custom 6-gene panel (APC, TP53, KRAS, BRAF, NRAS, and HRAS) CLIA-certified assay for FFPE CRC tissues. The assay has been analytically validated using reference cell lines (n=10) which were individually sequenced to >3,000x Duplex depth. The 6-gene DS assay yielded exceptionally high assay performance: (1) accuracy (passed, r2 = 0.98 for Duplex VAF vs. target VAF for Positive Control mutations); (2) sensitivity (passed, all mutations >1/5,000 were detected in all Positive Control replicates; 1 mutation at 1/5,000 was detected in 2/3 replicates), (3) specificity (passed, 2 mutations detected in the Negative Control at 1 count in 1 replicate; VAF <1/10,000), and (4) precision (passed, <2-fold variation among Positive Control replicates for VAF >1/500). Importantly, the validation analysis on fresh frozen (FF):FFPE paired tissues from 21 CRC patients shows: (1) The 6-gene DS assay performed on FFPE samples faithfully reproduces assay results on FF samples; (2) The ultrasensitive DS assay can accurately detect additional “new” mutations at low allelic frequencies compared to a standard NGS method. TwinStrand DS identified 17 mutations not identified by standard NGS; 13 of these new mutations had < 10% VAF. Furthermore, preliminary Kaplan-Meier analysis of the third-line EGFRi data shows that while the statistical significance was not achieved due to the small simple size (n=28), patients harboring combined APC and TP53 mutations(AP) (vs non-AP patients) tended to have longer progression free survival (6.65 vs 3.95 months, P=0.064) and overall survival (17.80 vs 10.65 months, P=0.14). Additional data acquisition and analysis is ongoing. Thus, there is an opportunity to change clinical practice and standards of care to ultimately improve CRC outcomes if this new, robust, sensitive test is clinically-validated. Citation Format: Mingli Yang, Michael J. Schell, Lance Pflieger, Jesse Salk, Timothy J. Yeatman. Development of an APC and TP53-based duplex sequencing assay to positively predict colorectal cancer response to EGFR inhibitors. [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 4343.