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Pyrosequencing-based methods reveal marked inter-individual differences in oncogene mutation burden in human
S Weidlich1, K Walsh, D Crowther
1Biomedical Research Institute, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK.
Background:
The epidermal growth factor receptor-targeted monoclonal antibody cetuximab (Erbitux) was recently introduced for the treatment of metastatic colorectal cancer. Treatment response is dependent on Kirsten-Ras (K-Ras) mutation status, in which the majority of patients with tumour-specific K-Ras mutations fail to respond to treatment. Mutations in the oncogenes B-Raf and PIK3CA (phosphoinositide-3-kinase) may also influence cetuximab response, highlighting the need for a sensitive, accurate and quantitative assessment of tumour mutation burden.
Methods:
Mutations in K-Ras, B-Raf and PIK3CA were identified by both dideoxy and quantitative pyrosequencing-based methods in a cohort of unselected colorectal tumours (n=102), and pyrosequencing-based mutation calls correlated with various clinico-pathological parameters.
Results:
The use of quantitative pyrosequencing-based methods allowed us to report a 13.7% increase in mutation burden, and to identify low-frequency (<30% mutation burden) mutations not routinely detected by dideoxy sequencing. K-Ras and B-Raf mutations were mutually exclusive and independently associated with a more advanced tumour phenotype.
Conclusion:
Pyrosequencing-based methods facilitate the identification of low-frequency tumour mutations and allow more accurate assessment of tumour mutation burden. Quantitative assessment of mutation burden may permit a more detailed evaluation of the role of specific tumour mutations in the pathogenesis and progression of colorectal cancer and may improve future patient selection for targeted drug therapies.
Insights
Quantitative pyrosequencing improves detection of low-frequency mutations in colorectal cancer, aiding assessment of tumor mutation burden for targeted therapies like cetuximab.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Cetuximab (Erbitux) is a monoclonal antibody for metastatic colorectal cancer.
- Treatment response to cetuximab depends on Kirsten-Ras (K-Ras) mutation status.
- Tumor-specific K-Ras mutations often predict non-response to cetuximab.
Purpose of the Study:
- To assess tumor mutation burden in colorectal cancer.
- To evaluate the role of K-Ras, B-Raf, and PIK3CA mutations in cetuximab response.
- To compare dideoxy and pyrosequencing methods for mutation detection.
Main Methods:
- Identified mutations in K-Ras, B-Raf, and PIK3CA using dideoxy and quantitative pyrosequencing.
- Analyzed a cohort of 102 unselected colorectal tumors.
- Correlated pyrosequencing mutation calls with clinico-pathological parameters.
Main Results:
- Quantitative pyrosequencing identified a 13.7% increase in mutation burden compared to dideoxy sequencing.
- Low-frequency mutations (<30% mutation burden) were detected by pyrosequencing but not dideoxy sequencing.
- K-Ras and B-Raf mutations were mutually exclusive and linked to advanced tumor phenotypes.
Conclusions:
- Pyrosequencing enables accurate assessment of tumor mutation burden by detecting low-frequency mutations.
- Quantitative mutation burden assessment can refine understanding of colorectal cancer pathogenesis and progression.
- Improved patient selection for targeted therapies may result from quantitative mutation analysis.
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