Cancer genetics · 2025
Ultra-sensitive detection of melanoma NRAS mutant ctDNA based on programmable endonucleases.
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Zhang Zuoying, Ji Qing, Zhang Zhanfang, Lyu Bao, Li Pei, Zhang Liyi, Chen Kaifei, Fang Meiyu, Song Jinzhao
Аннотация
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.
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.