Optimization of the tyrosinase mRNA probe to detect circulating melanocytes with reverse transcription and polymerase chain reaction

It was recently suggested that reverse transcription polymerase chain react ion (RT-PCR)-based detection of tyrosinase messenger RNA (mRNA) in peripher al blood is useful in the early detection of circulating tumor cells, since tyrosinase is thought to be a melanocyte-specific marker. However, the sen sitivity of detection of these cells in circulation is controversial, some authors reporting 0% effectiveness, others obtaining 100% efficacy. We deve loped a modification of a technique to process blood samples to detect tyro sinase mRNA, and tested the method with 50 samples from as many patients wi th histologically confirmed malignant melanoma in different stages. Whole b lood was processed by discarding the plasma and extracting RNA from density gradient-isolated peripheral blood lymphocytes. The RNA samples were teste d with a sensitive nested primer RT-PCR assay. Sensitivity was tested using RNA extracted from SK-mel-1 human melanoma cells diluted serially with per ipheral blood obtained from healthy control subjects. A lymph node from a p atient with confirmed disseminated melanoma served as the positive control. Our technique was able to detect tyrosinase mRNA in samples from the 37 pa tients with progressive metastatic melanoma The test detected tyrosinase mR NA from both the melanoma cell line and the positive lymph node. Our method to extract RNA from whole blood improves the specificity and sensitivity o f tyrosinase mRNA detection by RT-PCR. The test should be of use in determi ning the prognosis of patients with melanoma, and in deciding when to initi ate early treatment in patients with malignant melanoma.