Detection of melanoma cells in the blood of melanoma patients by melanoma-inhibitory activity (MIA) reverse transcription-PCR

Abstract

The detection of tumor-specific mRNA transcripts in the blood of patients by reverse transcription (RT)-PCR has been used as a very sensitive technique for determining systemically disseminated tumor cells. On the basis of previous expression studies, we aimed to trace melanoma cells in the blood of melanoma patients by RT-PCR of melanoma-inhibitory activity (MIA) mRNA. To detect sensitively MIA transcripts in total RNA isolated from peripheral blood mononuclear cells (PBMCs), we established a sensitive PCR-ELISA system. With this assay, we detected one melanoma cell in 2 ml of blood by a single round of 32 PCR cycles. A total of 295 PBMC samples isolated from 166 patients with melanocytic tumors were tested with the MIA RT-PCR-ELISA: (a) 58 patients (99 samples) with malignant melanomas in stage I; (b) 49 patients (65 samples) with malignant melanomas in stage II; and (c) 47 patients (116 samples) with metastasized melanomas (stages III and IV), with an additional 12 patients (15 samples) with benign melanocytic nevi. Forty-four (26.8%) of 164 samples isolated from patients with melanomas in stages I and II were positive for MIA mRNA; in stages III/IV, 33 (28.4%) of 116 samples of patients, irrespective of clinically evident disease, were positive. Eleven (84.6%) of 13 PBMC samples from patients with metastasized melanoma and clinically evident disease without treatment were MIA mRNA-positive in contrast to only 19 (25.7%) of 74 samples isolated from patients in stage IV with metastasis during chemotherapy. Furthermore, none of the 16 PBMC samples of patients in stage IV without clinically detectable metastases at that time point during chemotherapy was MIA mRNA-positive. Interestingly, of the 44 positive samples (26.8%) isolated from patients with melanomas in stages I and II, 20 were still positive when retested after complete excision of the tumor. Our results reveal that amplification of MIA mRNA from the PBMCs of patients with malignant melanomas by PCR-ELISA provides a useful means to detect tumor cells in the systemic blood circulation. A correlation between positive blood samples and tumor burden in stages III and IV was detected, and, in addition, a significant effect of chemotherapy with respect to the reduction of the number of systemically spread tumor cells was observed. However, MIA amplification seems to be of little value as a surrogate marker for clinical staging or the detection of metastatic disease.