[A rapid and effective melanin-bleaching method for molecular detection of melanoma].

Abstract

Objective: To establish a rapid and effective melanin-bleaching method to standardize and improve the molecular pathological analyses of melanoma. Methods: Fifteen cases of melanoma with melanin content exceeding 50% collected at the Precision Pathology Diagnosis Center, Weifang People's Hospital, Weifang, China between 2023 and 2024 were included in the study. These tissue samples were subject to melanin-bleaching treatments using H₂O₂, potassium permanganate (KMnO₄), Tris-HCl or PBS method. The bleaching effects in each group were evaluated using hematoxylin and eosin (HE) staining. Thereafter, the total amount, purity, and integrity of DNA extracted from bleached tissues were analyzed using UV spectrophotometry and Qsep1 Bio-fragment analyzer. Finally, in order to compare the efficiency of DNA amplification, C-KIT and PDGFRA were examined using Sanger sequencing, and BRAF was detected using real-time quantitative PCR. Results: Among the 15 cases, there were seven males and eight females whose median age was 68 (range, 63 to 71) years. Bleached tissue in Tris-HCl group had the highest HE score, which was significantly higher than those in the groups of PBS, H₂O₂, KMnO₄, and control (F=113.3, P<0.05). The total DNA amount in the control and Tris-HCl groups was significantly higher than those in the groups of PBS, H₂O₂, and KMnO₄, respectively (F=275, P<0.05). Meanwhile, the mean A₂₆₀/A₂₈₀ values of DNA obtained from bleached tissue in Tris-HCl and KMnO₄ were better than that of control, and the mean A₂₆₀/A₂₃₀ values of Tris-HCl and KMnO₄ were higher than that of control, PBS, and H₂O₂. Meanwhile, the proportion of >5 000 bp,>3 000 bp, and >1 000 bp DNA fragments in Tris-HCl was all significantly higher than those in other groups (F=147.9, F=127.9 and F=61.9, respectively, P<0.05). C-KIT and PDGFRA genes were both successfully amplified based on DNA obtained from bleached tissue in Tris-HCl group, and the sequencing peaks were clear and free of noticeable noises. Real-time quantitative PCR results showed that Tris-HCl method had lower cycle threshold values than other methods in detecting BRAF gene (F=30.36, P<0.05). Conclusions: Tris-HCl method is a fast and effective melanin-bleaching method for analyzing melanoma, which could remove melanin effectively, confirm high quality of DNA and good integrity of DNA, and improve the amplification efficiency of sequencing for melanoma related genes. This new method may help pathologists with reliable molecular typing and identifying therapeutic targets for melanoma. It may greatly improve clinical management of melanoma patients.