Detection of tyrosinase mRNA 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 pathway the expression of which is only found in melanin-producing cells. Because melanocytes are not normally found in the peripheral blood, we hypothesize that melanoma cells circulating in the peripheral blood could be detected by amplifying the tyrosinase mRNA using the reverse transcription-PCR (RT-PCR). The purpose of this study was to determine the sensitivity of a RT-PCR-based assay for tyrosinase mRNA from peripheral blood and evaluate correlations with tumor status in melanoma patients. RNA was isolated from the peripheral blood or tissue culture cells, and cDNA was prepared. DNA was amplified using RT-PCR with nested primers for tyrosinase and beta(2)-microglobulin. Serial dilution experiments using cells from the SK-MEL-28 cell line were performed in culture media and in whole blood. Twelve patients with melanoma, 10 healthy controls, and 15 patients with nonmelanoma malignancies were tested for tyrosinase expression in peripheral blood. The sensitivity of this assay was determined to be as low as 1 melanoma cell in 5 ml of whole blood. No tyrosinase was found in healthy subjects or other cancer control patients. Tyrosinase mRNA was detected in the blood of five melanoma patients (one stage II, two stage III, and two stage IV). Three of these tyrosinase-positive patients had biopsy-proven evidence of melanoma, whereas the other two had no clinical evidence of malignant disease after surgical resection. The remaining seven melanoma patients had no evidence of disease and tested negative for tyrosinase mRNA. This study suggests that a RT-PCR-based assay for the detection of tyrosinase mRNA in peripheral blood is feasible. Moreover, the presence of tyrosinase mRNA in the blood seems to correlate with the stage of melanoma. Further study and follow-up are needed to clarify the role of tyrosinase mRNA as a tumor marker for malignant melanoma.