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Published on: September 25, 2018
Detection of KRAS mutations in colorectal cancer with Fast COLD-PCR
Pietro Carotenuto1, Cristin Roma, Salvatore Cozzolino
1Pharmacogenomic Laboratory, CROM - Centro Ricerche Oncologiche di Mercogliano, Avellino, Italy.
Abstract:
Patients with metastatic colorectal carcinoma (mCRC) carrying activating mutations of the KRAS gene do not benefit from treatment with anti-epidermal growth factor receptor (EGFR) monoclonal antibodies. Therefore, KRAS mutation testing of mCRC patients is mandatory in the clinical setting for the choice of the most appropriate therapy. Co-amplification-at-lower denaturation-temperature PCR (COLD-PCR) is a novel modification of the conventional PCR method that selectively amplifies minority alleles from a mixture of wild-type and mutant sequences irrespective of the mutation type or position within the sequence. In this study, we compared the sensitivity of a COLD-PCR method with conventional PCR/sequencing and the real-time PCR-based Therascreen kit to detect KRAS mutations. By using dilutions of KRAS mutant DNA in wild-type DNA from colon cancer cell lines with known KRAS status, we found that Fast COLD-PCR is more sensitive than the conventional PCR method, showing a sensitivity of 2.5% in detecting G>A and G>T mutations. The detection of G>C transversions was not improved by either Fast COLD-PCR or Full COLD-PCR. We next analyzed by COLD-PCR, conventional PCR and Therascreen 52 formalin-fixed paraffin-embedded samples from mCRC patients. Among 36 samples with >30% tumor cells, 8 samples were negative by conventional PCR, Therascreen and Fast COLD-PCR; 20 mutations identified by conventional PCR were confirmed by Therascreen and Fast COLD-PCR; 8 cases undetermined by conventional PCR were all confirmed to carry KRAS G>A or G>T mutations by using either Therascreen or Fast COLD-PCR. Conventional PCR was able to detect only 2 KRAS mutations among 16 samples with <30% tumor cells (12.5%), whereas Therascreen and Fast COLD-PCR identified 6 mutants (37.5%). These data suggest that Fast COLD-PCR has a higher clinical sensitivity as compared with conventional PCR in detecting G>C to A>T changes in the KRAS gene, which represent >90% of the mutations of this oncogene in CRC.
Insights
Fast COLD-PCR enhances KRAS mutation detection sensitivity in metastatic colorectal carcinoma (mCRC) patients. This improved sensitivity is crucial for selecting effective anti-epidermal growth factor receptor (EGFR) therapies.
Area of Science:
- Molecular Oncology
- Genetics
- Cancer Diagnostics
Background:
- Activating KRAS mutations in metastatic colorectal carcinoma (mCRC) predict non-response to anti-EGFR therapies.
- Accurate KRAS mutation testing is essential for guiding mCRC treatment decisions.
- Conventional PCR methods may lack sensitivity for detecting low-allele-frequency mutations.
Purpose of the Study:
- To compare the diagnostic sensitivity of Co-amplification-at-lower denaturation-temperature PCR (COLD-PCR) against conventional PCR/sequencing and Therascreen for KRAS mutation detection in mCRC.
- To evaluate the clinical utility of COLD-PCR in formalin-fixed paraffin-embedded (FFPE) mCRC patient samples.
Main Methods:
- Sensitivity assessment using serial dilutions of KRAS mutant DNA in wild-type DNA.
- Analysis of 52 FFPE mCRC patient samples using conventional PCR, Therascreen, and COLD-PCR (Fast and Full variants).
- Comparison of mutation detection rates based on tumor cell percentage.
Main Results:
- Fast COLD-PCR demonstrated higher sensitivity (2.5%) than conventional PCR for detecting KRAS G>A and G>T mutations.
- COLD-PCR identified significantly more KRAS mutations in samples with low tumor cell content (<30%) compared to conventional PCR.
- COLD-PCR confirmed mutations missed by conventional PCR in FFPE samples, particularly G>A and G>T variants.
Conclusions:
- Fast COLD-PCR offers superior clinical sensitivity for detecting clinically relevant KRAS mutations (G>A, G>T) in mCRC patients compared to conventional PCR.
- COLD-PCR improves the detection of KRAS mutations, especially in samples with limited tumor cellularity, aiding in personalized therapy selection.
- The enhanced sensitivity of COLD-PCR has significant implications for optimizing treatment strategies for mCRC patients with KRAS-mutated tumors.
