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Published on: September 25, 2018
Application of COLD-PCR for improved detection of KRAS mutations in clinical samples
Zhuang Zuo1, Su S Chen, Pranil K Chandra
1Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77054, USA.
Abstract:
KRAS mutations have been detected in approximately 30% of all human tumors, and have been shown to predict response to some targeted therapies. The most common KRAS mutation-detection strategy consists of conventional PCR and direct sequencing. This approach has a 10-20% detection sensitivity depending on whether pyrosequencing or Sanger sequencing is used. To improve detection sensitivity, we compared our conventional method with the recently described co-amplification-at-lower denaturation-temperature PCR (COLD-PCR) method, which selectively amplifies minority alleles. In COLD-PCR, the critical denaturation temperature is lowered to 80 degrees C (vs 94 degrees C in conventional PCR). The sensitivity of COLD-PCR was determined by assessing serial dilutions. Fifty clinical samples were used, including 20 fresh bone-marrow aspirate specimens and the formalin-fixed paraffin-embedded (FFPE) tissue of 30 solid tumors. Implementation of COLD-PCR was straightforward and required no additional cost for reagents or instruments. The method was specific and reproducible. COLD-PCR successfully detected mutations in all samples that were positive by conventional PCR, and enhanced the mutant-to-wild-type ratio by >4.74-fold, increasing the mutation detection sensitivity to 1.5%. The enhancement of mutation detection by COLD-PCR inversely correlated with the tumor-cell percentage in a sample. In conclusion, we validated the utility and superior sensitivity of COLD-PCR for detecting KRAS mutations in a variety of hematopoietic and solid tumors using either fresh or fixed, paraffin-embedded tissue.
Insights
Co-amplification-at-lower denaturation-temperature PCR (COLD-PCR) significantly improves KRAS mutation detection sensitivity in tumors. This method enhances mutant detection to 1.5%, offering a more sensitive approach for clinical diagnostics.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- KRAS mutations occur in ~30% of human tumors and predict response to targeted therapies.
- Conventional PCR and sequencing methods have limited KRAS mutation detection sensitivity (10-20%).
Purpose of the Study:
- To compare the sensitivity of conventional PCR with co-amplification-at-lower denaturation-temperature PCR (COLD-PCR) for KRAS mutation detection.
- To evaluate COLD-PCR's utility in various tumor types and sample preservation methods.
Main Methods:
- COLD-PCR was implemented by lowering the denaturation temperature to 80°C to selectively amplify minority alleles.
- Sensitivity was assessed using serial dilutions.
- Fifty clinical samples (20 bone marrow, 30 FFPE solid tumors) were analyzed.
Main Results:
- COLD-PCR demonstrated superior sensitivity, increasing mutation detection to 1.5%.
- The method enhanced the mutant-to-wild-type ratio by >4.74-fold.
- COLD-PCR successfully detected all mutations found by conventional PCR and was specific and reproducible.
Conclusions:
- COLD-PCR is a sensitive, specific, and reproducible method for detecting KRAS mutations.
- The technique is easily implemented without additional costs.
- COLD-PCR enhances KRAS mutation detection in both fresh and FFPE tissues from various cancers.
