DETECTION OF TYROSINASE MESSENGER-RNA FROM THE BLOOD OF MELANOMA PATIENTS

Surgical therapy for localized melanoma is highly successful, However, if melanoma spreads beyond its primary site, the results of treatment are poor, Therefore, early detection of circulating melanoma cells in the blood may be important, Currently, circulating melanoma cells are undetectable, Tyrosinase is an enzyme in the melanin synthetic pathwa y the expression of which is only found in melanin-producing cells, Be cause melanocytes are not normally found in the peripheral blood, we h ypothesize that melanoma cells circulating in the peripheral blood cou ld be detected by amplifying the tyrosinase mRNA using the reverse tra nscription-PCR (RT-PCR), The purpose of this study was to determine th e sensitivity of a RT-PCR-based assay for tyrosinase mRNA from periphe ral blood and evaluate correlations with tumor status in melanoma pati ents. RNA was isolated from the peripheral blood or tissue culture cel ls, and cDNA was prepared, DNA was amplified using RT-PCR with nested primers for tyrosinase and beta(2)-microglobulin. Serial dilution expe riments using cells from the SK-MEL-28 cell line were performed in cul ture media and in whole blood. Twelve patients with melanoma, 10 healt hy controls, and 15 patients with nonmelanoma malignancies were tested for tyrosinase expression in peripheral blood. The sensitivity of thi s assay was determined to be as low as 1 melanoma cell in 5 mi of whol e blood, No tyrosinase was found in healthy subjects or other cancer c ontrol patients, Tyrosinase mRNA was detected in the blood of five mel anoma patients (one stage II, two stage III, and two stage IV), Three of these tyrosinase-positive patients had biopsy-proven evidence of me lanoma, whereas the other two had no clinical evidence of malignant di sease after surgical resection, The remaining seven melanoma patients had no evidence of disease and tested negative for tyrosinase mRNA. Th is study suggests that a RT-PCR-based assay for the detection of tyros inase mRNA in peripheral blood is feasible, Moreover, the presence of tyrosinase mRNA in the blood seems to correlate with the stage of mela noma, Further study and follow-up are needed to clarify the role of ty rosinase mRNA as a tumor marker for malignant melanoma.