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Published on: September 25, 2018
Suppression Probe Enrichment for Highly Sensitive and Multiplexed Detection of RAS Mutations in Colorectal Cancer
Xinglei Su1,2,3, Yunpei Si1,4, Wenle Huang4
1The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, National Center for Translational Medicine, Shanghai 200030, China.
Abstract:
Although RAS mutations are concentrated in specific hotspots, their clinical detection remains reliant on next-generation sequencing (NGS), which is hindered by high costs, long turnaround times, and substantial sample requirements. Herein, we report suppression probe enrichment (SPE), a thermodynamically driven low-frequency-mutation enrichment integrated with multiplex TaqMan probes for ultrasensitive detection of RAS mutations. The SPE assay covers 286 mutation types across the three major RAS genes, which is 86.8% of the clinically relevant mutations in the Cancer Genome Atlas database. It achieves a 20-fold sensitivity improvement over conventional methods, with a limit of detection as low as 0.05% variant allele frequency. Compared to NGS, SPE reduces detection time by approximately 7-fold, lowers costs by 3 orders of magnitude, and requires only 1 mL of plasma. We applied SPE to 57 tissue and 83 blood CRC samples, achieving 93.8% positive concordance and 100% true negative rate relative to NGS. For 30 paired tissue-plasma samples, the overall concordance was 93.33% with a positive concordance of 86.67%. These findings highlight SPE as a cost-effective, rapid, and highly sensitive approach for clinical RAS genotyping in CRC and for guiding targeted therapy.
Insights
A new ultrasensitive method called suppression probe enrichment (SPE) enables rapid and cost-effective detection of RAS mutations in cancer. This approach significantly improves upon next-generation sequencing (NGS) for guiding targeted therapies.
Area of Science:
- Molecular Biology
- Genetics
- Oncology
Background:
- RAS mutations are crucial biomarkers in cancer, particularly colorectal cancer (CRC).
- Current detection methods like next-generation sequencing (NGS) face limitations including high cost, long turnaround times, and significant sample volume requirements.
- Ultrasensitive detection of low-frequency RAS mutations is critical for effective clinical management and targeted therapy selection.
Purpose of the Study:
- To develop and validate a novel, ultrasensitive assay for detecting RAS mutations.
- To compare the performance of the new assay against conventional methods, specifically NGS.
- To establish a cost-effective and rapid method for clinical RAS genotyping in CRC.
Main Methods:
- Development of suppression probe enrichment (SPE), a method integrating thermodynamically driven enrichment with multiplex TaqMan probes.
- The SPE assay was designed to cover 286 mutation types across KRAS, HRAS, and NRAS genes.
- Validation using 57 tissue and 83 blood CRC samples, with comparison to NGS, and analysis of 30 paired tissue-plasma samples.
Main Results:
- The SPE assay demonstrated a 20-fold sensitivity improvement, with a limit of detection as low as 0.05% variant allele frequency.
- SPE reduced detection time by approximately 7-fold and costs by 3 orders of magnitude compared to NGS, requiring only 1 mL of plasma.
- High concordance rates were achieved: 93.8% positive concordance and 100% true negative rate against NGS in CRC samples. Paired sample analysis showed 93.33% overall and 86.67% positive concordance.
Conclusions:
- Suppression probe enrichment (SPE) offers a highly sensitive, rapid, and cost-effective alternative for RAS mutation detection.
- SPE is suitable for clinical RAS genotyping in colorectal cancer, aiding in the guidance of targeted therapies.
- This technology has the potential to significantly improve patient management and treatment strategies in oncology.
