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Updated: Jan 14, 2026
Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection
Published on: June 8, 2020
Development of an APC and TP53-based duplex sequencing assay to positively predict colorectal cancer response to
Mingli Yang1, Michael J Schell2, Jesse J Salk3
1Department of Surgery, University of South Florida, Tampa, FL, 32602, USA. mingliyang@usf.edu.
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.
Insights
A new 6-gene Duplex Sequencing assay improves selection for epidermal growth factor receptor inhibitor (EGFRi) therapy in metastatic colorectal cancer (CRC) by identifying patients with combined APC and TP53 mutations. This approach enhances patient selection and may improve treatment outcomes.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- Epidermal growth factor receptor inhibitor (EGFRi) therapies are FDA-approved for metastatic colorectal cancer (CRC).
- Current RAS/RAF testing identifies only non-responders, with only ~50% of selected patients responding, indicating a need for better biomarkers.
- Combined mutations in APC and TP53 were previously identified as potential positive biomarkers for EGFRi sensitivity.
Purpose of the Study:
- To develop an ultrasensitive 6-gene panel Duplex Sequencing (DS) assay for improved EGFRi therapy patient selection.
- To incorporate positive filters for APC and TP53 mutations alongside negative filters for KRAS, BRAF, and NRAS mutations.
- To evaluate the assay's performance and its potential to predict therapeutic outcomes in metastatic CRC patients.
Main Methods:
- Developed a 6-gene panel DS assay utilizing TwinStrand Duplex Sequencing technology.
- Analytically validated the assay using reference cell lines with high Duplex sequencing depth (>3,000x).
- Applied the assay to fresh frozen/FFPE paired tissues from 21 CRC patients and performed Kaplan-Meier analysis on 29 third-line metastatic CRC patients.
Main Results:
- The 6-gene DS assay demonstrated high accuracy, sensitivity, specificity, and precision during analytical validation.
- The assay detected low allelic frequency mutations (<10% VAF) missed by standard NGS, potentially indicating drug resistance mechanisms.
- Patients with combined APC and TP53 mutations showed a trend towards longer time on therapy (TOT) (6.65 vs. 3.60 months, p=0.048).
Conclusions:
- The 6-gene Duplex Sequencing assay shows potential for improving patient selection for EGFRi therapy in CRC.
- This ultrasensitive assay can identify novel mutations and may help predict patient response and therapeutic outcomes.
- Further validation could lead to enhanced clinical utility for guiding EGFRi treatment decisions.
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