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Updated: Jun 16, 2026
A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells
Published on: July 3, 2013
Two multiplex assays that simultaneously identify 22 possible mutation sites in the KRAS, BRAF, NRAS and PIK3CA genes
Irene Lurkin1, Robert Stoehr, Carolyn D Hurst
1Department of Pathology, Josephine Nefkens Institute, Erasmus MC, Rotterdam, The Netherlands.
Abstract:
Recently a number of randomized trials have shown that patients with advanced colorectal cancer do not benefit from therapies targeting the epidermal growth factor receptor when their tumors harbor mutations in the KRAS, BRAF and PIK3CA genes. We developed two multiplex assays that simultaneously screen 22 nucleotides in the KRAS, NRAS, BRAF and PIK3CA genes for mutations. The assays were validated on 294 tumor DNA samples from patients with advanced colorectal cancer. In these samples 119 KRAS codon 12 and 13 mutations had been identified by sequence analysis, 126 tumors were wild-type for KRAS and the analysis failed in 49 of the 294 samples due to poor DNA quality. The two mutation assays detected 130 KRAS mutations, among which were 3 codon 61 mutations, and in addition 32 PIK3CA, 13 BRAF and 6 NRAS mutations. In 19 tumors a KRAS mutation was found together with a mutation in the PIK3CA gene. One tumor was mutant for both PIK3CA and BRAF. In summary, the mutations assays identified 161 tumors with a mutation, 120 were wild-type and the analysis failed in 13. The material cost of the 2 mutation assays was calculated to be 8-fold lower than the cost of sequencing required to obtain the same data. In addition, the mutation assays are less labor intensive. We conclude that the performance of the two multiplex mutation assays was superior to direct sequencing. In addition, these assays are cheaper and easier to interpret. The assays may also be of use for selection of patients with other tumor types.
Insights
Multiplex mutation assays efficiently screen for KRAS, BRAF, PIK3CA, and NRAS mutations in colorectal cancer. These cost-effective assays offer superior performance and easier interpretation compared to traditional sequencing methods.
Area of Science:
- Oncology
- Molecular Diagnostics
- Genetics
Background:
- Targeted therapies for advanced colorectal cancer (CRC) efficacy is limited by specific gene mutations.
- KRAS, BRAF, and PIK3CA mutations predict poor response to epidermal growth factor receptor (EGFR) inhibitors in CRC patients.
Purpose of the Study:
- To develop and validate multiplex assays for simultaneous detection of mutations in KRAS, NRAS, BRAF, and PIK3CA genes.
- To compare the performance, cost, and labor intensity of these novel assays against direct sequencing.
Main Methods:
- Development of two multiplex assays for simultaneous screening of 22 nucleotides in KRAS, NRAS, BRAF, and PIK3CA genes.
- Validation of the assays on 294 tumor DNA samples from advanced colorectal cancer patients.
- Comparison of mutation detection rates, cost-effectiveness, and labor requirements with direct sequencing.
Main Results:
- The multiplex assays detected 161 tumors with mutations (including KRAS, PIK3CA, BRAF, and NRAS) and identified co-mutations.
- Assay performance was superior to direct sequencing, with higher detection rates and fewer failures.
- Material costs were 8-fold lower, and assays were less labor-intensive than sequencing.
Conclusions:
- The developed multiplex mutation assays are highly effective, cost-efficient, and easier to interpret than direct sequencing for CRC.
- These assays can aid in selecting patients for targeted therapies and may be applicable to other tumor types.
