DEVELOPMENT AND APPLICATION OF A HIGHLY SENSITIVE PNA CLAMP PCR ASSAY FOR DETECTION OF NRAS MUTATIONS IN MELANOMA USING TISSUE AND CFDNA
Abstract
Melanoma is known to be one of the common cutaneous cancers worldwide. NRAS mutations are found in 15%–20% of melanomas. Non-NRAS-mutant subset of melanoma is less aggressive and associated with higher outcomes in comparison to NRAS- mutant melanoma. New treatment methods for this subset might be developed with improved understanding of NRAS-mutant melanoma. Therefore, a highly sensitive technique for detection of NRAS mutation in melanoma is imperative. The aims of this research were to develop a highly sensitive PCR assay to detect low tumour cell percentage in formalin fixed paraffin embedded (FFPE) tissue and plasma. The assay was developed on stable cell lines containing NRAS (codon 61) mutations. Peptide nucelic acide (PNA) Clamp PCR was used for the selective amplification of DNA target sequences. Total DNA was extracted from 48 FFPE tissues and 20 blood plasma (14 matched) stage II-IV tumours and screened for NRAS mutations. PNA Clamp PCR on FFPE tissue showed 25% (n=12) NRAS mutant of which p.Q61R c.182A>G was the most frequent. Sample cases were independently tested. For NRAS Circulating cell free DNA (cfDNA) analysis only one case was mutated. This demonstrates an innovative and highly sensitive technique for the detection of the common driver mutations in melanoma using exceptionally low tumour burden samples, representing a useful tool for future research and clinical application.
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References
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