Quantitative RT-PCR assessment of melanoma cells in peripheral blood during immunotherapy for metastatic melanoma

Circulating malignant cells in peripheral blood are thought to be precursors and surrogate markers of distant metastases and hence markers of a poor clinical outcome. In this study, we used the detection of MART-1 and tyrosinase (TYR) mRNA with a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay to identify circulating melanoma cells. Blood samples were obtained from 35 patients with metastatic melanoma before, during and after treatment with interleukin-2, interferon-alpha and cisplatin. In addition, MART-1 and TYR protein was identified by immunohistochemistry in consecutive biopsies from 15 of the patients. Analysis of three daily blood samples for 3 days demonstrated that four out of 11 patients examined were negative for both markers on all occasions, and two patients were positive for both markers on all occasions but one. The remaining five patients showed sporadic low positive results for one or the other of the two markers. By comparing the immunohistochemistry results from consecutive biopsies with the RT-PCR results, we demonstrated that patients with MART-1 and TYR protein in their tumour cells had circulating MART-1 and TYR mRNA in 77% and 54% of the cases, respectively. During treatment, the majority of patients who were positive for MART-1 and TYR mRNA converted to being negative. However, these conversions did not significantly correlate with objective response. The presence of TYR mRNA in one of the first two samples showed a trend towards being an independent prognostic factor for poor survival.