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Updated: Jun 5, 2026
Ultra-Fast Amplicon-Based Next-Generation Sequencing in Non-Squamous Non-Small Cell Lung Cancer
Published on: September 8, 2023
A platform for rapid detection of multiple oncogenic mutations with relevance to targeted therapy in non-small-cell
Zengliu Su1, Dora Dias-Santagata, Markeesa Duke
1Vanderbilt-Ingram Cancer Center, the Department of Medicine/Division of Hematology-Oncology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Abstract:
The identification of somatically acquired tumor mutations is increasingly important in the clinical management of cancer because the sensitivity of targeted drugs is related to the genetic makeup of individual tumors. Thus, mutational profiles of tumors can help prioritize anticancer therapy. We report herein the development and validation of two multiplexed assays designed to detect in DNA from FFPE tissue more than 40 recurrent mutations in nine genes relevant to existing and emerging targeted therapies in lung cancer. The platform involves two methods: a screen (SNaPshot) based on multiplex PCR, primer extension, and capillary electrophoresis that was designed to assess for 38 somatic mutations in eight genes (AKT1, BRAF, EGFR, KRAS, MEK1, NRAS, PIK3CA, and PTEN) and a PCR-based sizing assay that assesses for EGFR exon 19 deletions, EGFR exon 20 insertions, and HER2 exon 20 insertions. Both the SNaPshot and sizing assays can be performed rapidly, with minimal amounts of genetic material. Compared with direct sequencing, in which mutant DNA needs to compose 25% or more of the total DNA to easily detect a mutation, the SNaPshot and sizing assays can detect mutations in samples in which mutant DNA composes 1.56% to 12.5% and 1.56% to 6.25% of the total DNA, respectively. These robust, reliable, and relatively inexpensive assays should help accelerate adoption of a genotype-driven approach in the treatment of lung cancer.
Insights
We developed two multiplexed assays to detect over 40 lung cancer mutations in FFPE tissue. These rapid, sensitive, and cost-effective tests aid genotype-driven lung cancer treatment.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Somatic tumor mutations are crucial for personalized cancer therapy.
- Tumor mutational profiles guide the selection of targeted anticancer drugs.
- Accurate identification of mutations enhances treatment efficacy.
Purpose of the Study:
- To develop and validate multiplexed assays for detecting recurrent mutations in lung cancer.
- To enable genotype-driven treatment strategies through rapid mutational profiling.
- To improve the sensitivity and accessibility of targeted therapy selection.
Main Methods:
- Development of two multiplexed assays: SNaPshot (multiplex PCR, primer extension, capillary electrophoresis) and a PCR-based sizing assay.
- SNaPshot assay detects 38 somatic mutations in eight key genes (AKT1, BRAF, EGFR, KRAS, MEK1, NRAS, PIK3CA, PTEN).
- Sizing assay identifies EGFR and HER2 exon mutations (deletions and insertions).
Main Results:
- The assays detect over 40 recurrent mutations in nine genes relevant to lung cancer targeted therapies.
- High sensitivity achieved: mutations detected at 1.56%–12.5% mutant DNA levels, significantly lower than direct sequencing.
- Both assays are rapid, require minimal genetic material, and are cost-effective.
Conclusions:
- The developed SNaPshot and sizing assays are robust, reliable, and facilitate the detection of clinically relevant mutations in FFPE lung cancer tissue.
- These assays can accelerate the clinical adoption of genotype-driven approaches for lung cancer treatment.
- The assays offer a sensitive and efficient method for identifying actionable mutations to guide targeted therapy selection.
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