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Purpose <p>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.</p> Methods <p>We previously reported combined mutations in <i>APC</i> and <i>TP53</i> 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 <i>APC(A)</i> and <i>TP53(P)</i> mutations in addition to the negative <i>KRAS(K), BRAF(B),</i> and <i>NRAS(N)</i> mutation filters for EGFRi therapy.</p> Results <p>The 6-gene DS assay was analytically validated using reference cell lines (<i>n</i> = 9, individually sequenced to &gt; 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 &lt; 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 <i>APC</i> and <i>TP53</i> mutations (AP/APK(N) versus others) tended to have longer TOT (6.65 versus 3.60 months, <i>p</i> = 0.048, <i>n</i> = 29).</p> Conclusion <p>These data suggest the potential of the 6-gene Duplex Sequencing assay to improve EGFRi patient selection and therapeutic outcomes.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年1月13日

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  • Mingli Yang,
  • Michael J. Schell,
  • Jesse J. Salk,
  • Jake Higgins,
  • Michael Hipp,
  • Lance Pflieger,
  • Warren Jack Pledger,
  • Timothy J. Yeatman

摘要

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.