Development of an APC and TP53-based duplex sequencing assay to positively predict colorectal cancer response to anti-EGFR therapy

PURPOSE: EGFR inhibitor (EGFRi) therapies have been FDA-approved for metastatic colorectal cancer (CRC). However, extended RAS/RAF testing required in the drug labels, identifies only non-responders, and only ~50% of selected patients respond to therapy, suggesting an unmet need to develop additional biomarkers. METHODS: We previously reported combined mutations in APC and TP53 as a potential positive biomarker to identify EGFRi-sensitive patients. By leveraging the TwinStrand Duplex Sequencing (DS) technology, this study developed an ultrasensitive 6-gene panel DS assay that adds a positive filter for APC(A) and TP53(P) mutations in addition to the negative KRAS(K), BRAF(B), and NRAS(N) mutation filters for EGFRi therapy. RESULTS: The 6-gene DS assay was analytically validated using reference cell lines (n = 9, individually sequenced to > 3,000x Duplex depth). The assay yielded exceptionally high assay performance on (1) accuracy, (2) sensitivity, (3) specificity and (4) precision. Application to fresh frozen (FF):FFPE paired tissues from 21 CRC patients demonstrates that the ultrasensitive DS assay can accurately detect additional "new" mutations at low allelic frequencies compared to a standard NGS method (13 of the 17 new mutations had < 10% VAF) that may ultimately be responsible for drug resistance. Furthermore, Kaplan-Meier analysis on Duplex-sequenced EGFRi FFPE samples showed that the third-line metastatic CRC patients harboring combined APC and TP53 mutations (AP/APK(N) versus others) tended to have longer TOT (6.65 versus 3.60 months, p = 0.048, n = 29). CONCLUSION: These data suggest the potential of the 6-gene Duplex Sequencing assay to improve EGFRi patient selection and therapeutic outcomes.