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Rare RAS Mutations in Metastatic Colorectal Cancer Detected During Routine RAS Genotyping Using Next Generation
Alexandre Harlé1,2,3, Pierre Filhine-Tresarrieu4, Marie Husson4
1Faculté de Pharmacie, Université de Lorraine, 54001, Nancy, France. a.harle@nancy.unicancer.fr.
Background:
Overall survival of metastatic colorectal cancer (mCRC) patients has been improved with the addition of targeted therapy such as anti-epithelial growth factor receptor monoclonal antibodies (anti-EGFR mAbs) to standard chemotherapy. Retrospective studies and randomized trials showed that the presence of RAS mutations was linked to the absence of clinical response to anti-EGFR mAbs. Patients harboring KRAS and NRAS mutations on exons 2, 3 or 4 have little or no benefit from anti-EGFR therapies. Polymerase chain reaction (PCR)-based assays are routinely used to assess KRAS and NRAS status, whereas deep sequencing with next generation sequencing (NGS) currently represents an alternative method.
Objective:
The objective of our study was to identify KRAS and NRAS non-hotspot mutations using NGS of mCRC tumor samples.
Method:
DNA was extracted from 188 consecutive formalin-fixed paraffin embedded samples of histologically proven colorectal cancer tumor tissue from patients with mCRC. Following amplification, DNA was sequenced by ultra-deep pyrosequencing. Non-hotspot mutations identified by NGS (frequency of mutated allele range [1.8-70.6 %]) were confirmed by Sanger direct-sequencing when possible.
Results:
NGS procedure was applicable in 94 % of the cases and detected mutations in 62 % of the samples. Nine uncommon mutational profiles were found with a frequency of mutated allele > 1 %. Silent mutations were found in 3.6 % of the samples. Mutations at or near functional domains of RAS proteins, other than defined hotspots, were found in 3.6 %. NGS proved to be accurate, sensitive and suitable for routine RAS genotyping.
Conclusion:
Clinical responses to anti-EGFR mAbs are potentially impaired in the presence of these uncommon RAS mutations.
Insights
Next-generation sequencing (NGS) identified uncommon RAS mutations in metastatic colorectal cancer (mCRC) patients. These non-hotspot mutations may affect response to anti-epithelial growth factor receptor monoclonal antibodies (anti-EGFR mAbs).
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Metastatic colorectal cancer (mCRC) treatment improved with anti-epithelial growth factor receptor monoclonal antibodies (anti-EGFR mAbs).
- RAS mutations (KRAS, NRAS) predict poor response to anti-EGFR mAbs in mCRC patients.
- Current PCR-based assays primarily detect hotspot RAS mutations.
Purpose of the Study:
- To identify KRAS and NRAS non-hotspot mutations in mCRC tumor samples using next-generation sequencing (NGS).
- To evaluate the utility of NGS for comprehensive RAS mutation profiling in mCRC.
Main Methods:
- DNA extraction from 188 mCRC tumor samples.
- Ultra-deep pyrosequencing using NGS for mutation detection.
- Confirmation of non-hotspot mutations by Sanger sequencing.
Main Results:
- NGS was successful in 94% of samples and detected mutations in 62%.
- Nine uncommon RAS mutational profiles were identified, with mutated allele frequencies >1%.
- Mutations outside defined hotspots, including near functional domains, were found in 3.6% of samples.
Conclusions:
- NGS is accurate, sensitive, and suitable for routine RAS genotyping in mCRC.
- Uncommon RAS mutations identified by NGS may impair clinical response to anti-EGFR mAbs.
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