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Ultra-sensitive detection of melanoma NRAS mutant ctDNA based on programmable endonucleases
Zuoying Zhang1, Qing Ji2, Zhanfang Zhang1
1Medical School, Faculty of Medicine, Tianjin University, Tianjin, 300072, China.
Background:
Melanoma is a complex and often fatal disease, with NRAS being one of the most frequently mutated genes in this type of cancer. Liquid biopsies, specifically tests for circulating tumor DNA (ctDNA), represent a promising and less invasive approach to diagnosis. This study aims to develop an ultra-sensitive assay for detecting melanoma NRAS mutant ctDNA.
Methods:
To detect rare NRAS mutant ctDNA, we developed the NRAS PASEA assay by screening CRISPR-Cas proteins that recognize the PAM sequence 5'-TTN-3'. This method employs CRISPR-Cas proteins to continuously shear wild-type alleles during isothermal amplification, resulting in exponential amplification of mutant alleles to a detectable level by Sanger sequencing.
Results:
The developed NRAS Q61R/L/K mutation detection method can detect simulated ctDNA samples with mutant allele fractions (MAF) as low as 0.01 % with 30 mins of PASEA treatment. Notably, the NRAS Q61 K mutation was accurately identified by FnCas12a-based NRAS PASEA, even with the nucleotide at the "N" position in the PAM site "TTN." The method successfully detected ctDNA in patients with malignant melanoma. All patients (5/5) from 15 melanoma blood samples with NRAS Q61R (4/4) and NRAS Q61 K (1/1) mutations were accurately identified, with no false positives among patients with wildtype NRAS Q61.
Conclusion:
Detecting ctDNA from peripheral blood samples is highly significant for melanomas in areas where imaging evaluation is challenging. Our assay demonstrated 100 % consistency with tumor tissue NGS, providing a new analytical strategy for companion diagnosis and dynamic assessment of therapeutic efficacy and disease progression in melanoma.
Insights
A new ultra-sensitive assay detects melanoma NRAS mutant circulating tumor DNA (ctDNA) in blood. This method offers a less invasive diagnostic tool for melanoma, showing high accuracy in patient samples.
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.
