A Simple Method to Determine Pheomelanin Content and Structure in FFPE Human Melanoma Specimens

Human cutaneous malignant melanoma is a skin cancer that develops from melanocytes, the cells specialised in the production of eu- and pheomelanin. A growing body of evidence suggests that pheomelanin in particular is involved in melanoma development. The aim of this study was to develop a new method enabling the determination of the pheomelanin in formalin-fixed paraffin-embedded (FFPE) tissue specimens of human nodular (NM) and superficial spreading (SSM) melanomas. The pheomelanin level was evaluated in a small amount of material obtained from FFPE melanoma samples (less than 1 mg), using a multi-step procedure of paraffin removal, tissue rehydration, and homogenisation, omitting the melanin isolation step. The obtained product was studied for pheomelanin content using the Py-GC/MS/MS method operating in a multiple reaction monitoring (MRM) mode. The results of our research confirmed the presence of all the pheomelanin markers in the FFPE human melanoma specimens and showed that the tissues analysed contained different amounts of pheomelanin isomers (5-S-cysteinylDOPA and 2-S-cysteinylDOPA). The developed Py-GC/MS/MS procedure enables sensitive quantification of pheomelanin in FFPE human melanoma samples, facilitating broader studies on its role in melanoma development and progression. This method opens new avenues for investigating pheomelanin’s involvement in melanoma malignancy.