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Updated: Jul 15, 2026
Ultra-Fast Amplicon-Based Next-Generation Sequencing in Non-Squamous Non-Small Cell Lung Cancer
Published on: September 8, 2023
EGFR and K-ras mutation analysis in non-small cell lung cancer: comparison of paraffin embedded versus frozen
Mariëlle I Gallegos Ruiz1, Karijn Floor, Frank Rijmen
1Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
Background:
Mutational analysis of the Epidermal Growth Factor Receptor (EGFR) and K-ras genes to select non-small cell lung cancer (NSCLC) patients for treatment with novel EGFR tyrosine kinase inhibitors is an appealing possibility currently under investigation. Although frozen tumor tissue would probably be the optimal source for analysis, the most common source of tumor material is fixed and paraffin embedded (FPE) archival specimens. Here, we evaluate how different procedures of tissue sample processing and preservation may affect the outcome of EGFR and K-ras mutation analysis. Furthermore, we compare the sensitivity of the analysis using genomic DNA (gDNA) versus RNA.
Methods:
We used PCR amplification and direct sequencing to analyze EGFR and K-ras genes in paired FPE and frozen tumor samples corresponding to 47 NSCLC patients. In frozen samples, the analysis was carried out using both gDNA and RNA extracted in parallel.
Results:
Whereas 100% of frozen samples were successfully amplified, the rate of successful PCR amplification in FPE samples was approximately 50%. We detected three previously described EGFR point mutations in 2 samples. In ten other samples, a K-ras mutation was observed. These mutations were detected in DNA extracted from frozen samples as well as in DNA obtained from FPE tissue. In addition, 10 nucleotide changes, were detected in FPE samples that were not detected in the frozen specimens. Upon re-analysis, these nucleotide changes could not be confirmed and were most likely the result of paraffin embedding and fixation procedures. All mutations found in gDNA were also detected in the corresponding RNA and, in two cases, the presence of the mutant allele was easier to identify by using RNA.
Conclusions:
Our results indicate that RNA extracted from frozen tissue is the preferred source for EGFR and K-ras mutation testing. When analyzing FPE samples, reducing the size of the amplified fragments would increase PCR success rate, and care should be taken to control for false-positive results.
Insights
Frozen tissue RNA is best for Epidermal Growth Factor Receptor (EGFR) and K-ras mutation testing in non-small cell lung cancer (NSCLC). Fixed and paraffin-embedded (FPE) samples show lower success rates and potential false positives.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Accurate mutational analysis of Epidermal Growth Factor Receptor (EGFR) and K-ras genes is crucial for selecting non-small cell lung cancer (NSCLC) patients for targeted therapies.
- While frozen tumor tissue is ideal, fixed and paraffin-embedded (FPE) archival specimens are more commonly available for molecular testing.
- Understanding the impact of tissue processing and preservation on mutation detection is essential for reliable clinical application.
Purpose of the Study:
- To evaluate how different tissue processing and preservation methods affect EGFR and K-ras mutation analysis in NSCLC.
- To compare the sensitivity of mutation detection using genomic DNA (gDNA) versus RNA.
- To determine the optimal sample type and nucleic acid source for EGFR and K-ras mutation testing.
Main Methods:
- Paired fixed and paraffin-embedded (FPE) and frozen non-small cell lung cancer (NSCLC) tumor samples from 47 patients were analyzed.
- Polymerase Chain Reaction (PCR) amplification and direct sequencing were used to examine EGFR and K-ras genes.
- Both genomic DNA (gDNA) and RNA were extracted and analyzed from frozen samples.
Main Results:
- PCR amplification success rates were 100% for frozen samples versus approximately 50% for FPE samples.
- EGFR and K-ras mutations were detected in both frozen and FPE samples, with all mutations found in gDNA also present in RNA.
- RNA analysis facilitated easier identification of mutant alleles in two cases, and FPE samples yielded unconfirmed nucleotide changes likely due to processing.
Conclusions:
- RNA extracted from frozen tumor tissue is the preferred source for EGFR and K-ras mutation testing in NSCLC.
- For FPE samples, reducing PCR amplicon size may improve success rates, and rigorous controls are needed to mitigate false-positive results.
- These findings have implications for patient selection for targeted therapies based on molecular profiling.