Ultra-sensitive detection of melanoma NRAS mutant ctDNA based on programmable endonucleases

Area of Science:

• Oncology
• Molecular Diagnostics
• Genetics

Background:

• Melanoma is a significant cause of cancer mortality.
• NRAS mutations are common in melanoma.
• Liquid biopsies for circulating tumor DNA (ctDNA) offer less invasive diagnostic potential.

Purpose of the Study:

• To develop an ultra-sensitive assay for detecting NRAS mutant ctDNA in melanoma patients.
• To enable early and accurate diagnosis of melanoma through liquid biopsy.

Main Methods:

• Developed the NRAS PASEA assay utilizing CRISPR-Cas proteins targeting the 5'-TTN-3' PAM sequence.
• Employed continuous shearing of wild-type alleles during isothermal amplification for exponential mutant allele amplification.
• Utilized Sanger sequencing for detectable amplification of mutant ctDNA.

Main Results:

• The NRAS PASEA assay detected simulated ctDNA with mutant allele fractions as low as 0.01%.
• Accurate identification of NRAS Q61R, Q61L, and Q61K mutations, including variations at the 'N' position of the PAM site.
• Successfully detected NRAS mutant ctDNA in 100% of tested melanoma patient samples (5/5), with no false positives.

Conclusions:

• ctDNA detection in peripheral blood is crucial for melanoma management, especially in challenging imaging scenarios.
• The developed assay demonstrated 100% concordance with tumor tissue next-generation sequencing (NGS).
• This assay provides a novel strategy for companion diagnostics and monitoring therapeutic response and disease progression in melanoma.