Analytical reliability of cell-free DNA from fine-needle aspiration rinses for BRAF and NRAS testing in metastatic melanoma

Introduction: Molecular profiling is essential for the management of metastatic melanoma, particularly for identifying patients eligible for targeted therapies against BRAF and NRAS alterations. Although cell blocks (CBs) represent the conventional source for molecular testing in cytology, specimen limitations frequently hinder analysis. Materials and methods: We retrospectively evaluated metastatic melanoma cases undergoing fine-needle aspiration at our institution from January 2018 to November 2025. Cell-free DNA (cfDNA) extracted from needle rinses collected in nuclease-free water was compared with DNA derived from CBs for BRAF p.V600E and NRAS p.Q61X mutational analysis using RT-qPCR. Results: Thirty-four cases were included. All cfDNA samples were evaluable, whereas only 26.5% of CBs yielded sufficient material for molecular testing. cfDNA analysis detected BRAF p.V600E in 61.8% and NRAS p.Q61X in 11.8% of cases, with 26.4% wild type. Among CBs, BRAF mutations were identified in 55.6%. Only one discordant case was observed, with BRAF p.V600E detected exclusively in cfDNA. Conclusions: cfDNA from fine-needle aspiration rinses provides a highly reliable and efficient substrate for molecular testing in metastatic melanoma, outperforming CB-based analysis in terms of sample adequacy. This approach preserves cellular material for ancillary studies and may reduce false-negative results. Prospective studies using broader next generation sequencing panels are warranted to confirm analytical robustness and clinical utility.