A Targeted Q-PCR-Based Method for Point Mutation Testing by Analyzing Circulating DNA for Cancer Management Care

Area of Science:

• Oncology
• Molecular Biology
• Genomics

Background:

• Circulating cell-free DNA (cfDNA) is released by tumor cells, reflecting tumor genome alterations.
• cfDNA analysis provides a noninvasive molecular test for cancer patients.
• Understanding cfDNA structure, fragmentation, and size is crucial for accurate analysis.

Purpose of the Study:

• To develop and validate a novel multiplexed method for cfDNA analysis.
• To enable simultaneous determination of multiple cfDNA parameters.
• To establish cfDNA analysis as a viable alternative to tumor tissue analysis for mutation detection.

Main Methods:

• Refined quantitative PCR (Q-PCR) method.
• Developed allele-specific-Q-PCR with blocker (Intplex) for multiplexed cfDNA analysis.
• Validated Intplex for detecting KRAS and BRAF mutations in metastatic colorectal cancer (mCRC) patients.

Main Results:

• Intplex enables simultaneous determination of cfDNA concentration, known point mutations, mutant cfDNA (ctDNA) concentration, mutant cfDNA proportion, and cfDNA fragmentation index.
• Clinical validation in mCRC patients demonstrated Intplex's ability to detect KRAS and BRAF mutations.
• Blood test using Intplex showed potential to replace tumor section analysis for specific mutation detection.

Conclusions:

• Intplex is a rapid, sensitive, cost-effective, and adaptable targeted approach for cfDNA mutation analysis.
• This method allows quantitative and dynamic mutation detection, supporting personalized cancer medicine.
• Intplex holds promise for noninvasive diagnosis, prognosis, theranostics, and monitoring of cancer patients.