Molecular detection of circulating tyrosinase mRNA: optimization in a preclinical xenograft mouse melanoma model and further evaluation in samples from advanced melanoma patients.

We designed high-affinity primers for the mRNA sequence of human tyrosinase to test the value of molecular detection of circulating melanoma cells by reverse transcription-polymerase chain reaction (RT-PCR). The optimization process included in vitro settings and in vivo studies in a xenograft mouse model. We detected tyrosinase expression with at least 40 pg and 1.5 pg of total RNA extracted from cultured SKmel human melanoma cells, using a first round of PCR amplification and nested PCR, respectively. Human tyrosinase expression was found in the blood of nude mice bearing subcutaneous SKmel tumors, and the expression bands were stronger after manipulation of the tumor mass. We also examined the fate of circulating melanoma cells in the present melanoma model. Tyrosinase expression declined in blood 6 h after a direct intravenous injection of SKmel cells. A preliminary study in human blood samples demonstrated a baseline positive tyrosinase determination in 64% (16/25) of advanced melanoma patients using the RT-PCR nested assay. Baseline tyrosinase expression was significantly associated with disease progression after 12 months, and sequential determination during follow-up of the remaining disease-free patients showed a progressive increase of negative results.