Related Experiment Video
Updated: May 13, 2026
Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology
Published on: March 21, 2018
Increased detection rates of EGFR and KRAS mutations in NSCLC specimens with low tumour cell content by 454 deep
Evgeny A Moskalev1, Robert Stöhr, Ralf Rieker
1Department of Pathology, University Medical Center, Erlangen, Germany.
Abstract:
Detection of activating EGFR mutations in NSCLC is the prerequisite for individualised therapy with receptor tyrosine kinase inhibitors (TKI). In contrast, mutant downstream effector KRAS is associated with TKI resistance. Accordingly, EGFR mutation status is routinely examined in NSCLC specimens, but the employed methods may have a dramatic impact on the interpretation of results and, consequently, therapeutic decisions. Specimens with low tumour cell content are at particular risk for false-negative EGFR mutation reporting by sequencing with Sanger chemistry. To improve reliability of detecting clinically relevant mutant variants of EGFR and KRAS, we took full advantage of 454 deep sequencing and developed a two-step amplification protocol for the analysis of EGFR exons 18-21 and KRAS exons 2 and 3. We systematically addressed the sensitivity, reproducibility and specificity of the developed assay. Mutations could be reliably identified down to an allele frequency of 0.2-1.5 %, as opposed to 10-20 % detection limit of Sanger sequencing. High reproducibility (0-2.1 % variant frequency) and very low background level (0.4-0.8 % frequency) further complement the reliability of this assay. Notably, re-evaluation of 16 NSCLC samples with low tumour cell content ≤40 % and EGFR wild type status according to Sanger sequencing revealed clinically relevant EGFR mutations at allele frequencies of 0.9-10 % in seven cases. In summary, this novel two-step amplification protocol with 454 deep sequencing is superior to Sanger sequencing with significantly increased sensitivity, enabling reliable analysis of EGFR and KRAS in NSCLC samples independent of the tumour cell content.
Insights
This study introduces a sensitive 454 deep sequencing method for detecting EGFR and KRAS mutations in non-small cell lung cancer (NSCLC). This advanced technique improves upon Sanger sequencing, especially for samples with low tumor cell content, aiding personalized therapy decisions.
Area of Science:
- Molecular Biology
- Oncology
- Genetics
Background:
- Activating EGFR mutations in non-small cell lung cancer (NSCLC) are crucial for targeted therapy with receptor tyrosine kinase inhibitors (TKIs).
- KRAS mutations are linked to TKI resistance, making simultaneous detection of both EGFR and KRAS mutations important for treatment planning.
- Current methods like Sanger sequencing struggle with low tumor cell content, risking false-negative results for EGFR mutations.
Purpose of the Study:
- To develop a highly sensitive and reproducible method for detecting EGFR and KRAS mutations in NSCLC.
- To improve the reliability of mutation detection in samples with low tumor cell content.
- To compare the performance of the new method with conventional Sanger sequencing.
Main Methods:
- Development of a two-step amplification protocol for analyzing EGFR exons 18-21 and KRAS exons 2 and 3.
- Utilized 454 deep sequencing technology for enhanced mutation detection sensitivity.
- Systematic evaluation of assay sensitivity, reproducibility, and specificity.
Main Results:
- The novel assay reliably detected mutations down to 0.2-1.5% allele frequency, significantly lower than Sanger sequencing's 10-20% limit.
- High reproducibility (0-2.1% variant frequency) and low background noise (0.4-0.8% frequency) were achieved.
- Re-analysis of 16 NSCLC samples with low tumor content previously negative for EGFR mutations by Sanger sequencing identified clinically relevant mutations in seven cases (0.9-10% allele frequency).
Conclusions:
- The two-step amplification protocol combined with 454 deep sequencing offers superior sensitivity for EGFR and KRAS mutation detection in NSCLC.
- This method enables reliable genetic analysis regardless of tumor cell content, overcoming limitations of Sanger sequencing.
- Improved detection capabilities can lead to more accurate therapeutic decisions for NSCLC patients.
