Reverse transcription (RT) of the tyrosinase mRNA and specific cDNA amplification by nested polymerase chain reactin (PCR) have been reported to facilitate the early detection of circulating tumor cells in melanoma patients. The significance and practical value of this procedure for the diagnosis of tumor dissemination in melanoma patients are unclear. In the current study we analyzed peripheral blood samples of 65 melanoma patients of different clinical stages for the presence of tyrosinase mRNA by RT-PCR using nested oligonucleotide primers specific for tyrosinase cDNA. Furthermore, blood samples were evaluated for tumor cell growth by cell culture assays in vitro. No tyrosinase mRNA was detectable in blood samples of 26 patients with primary melanoma and 16 patients with regional lymph node metastases. In five of 13 patients with visceral metastases we found at least one blood sample positive for tyrosinase mRNA during a 2-to 4-mo interval. Analyses of different blood samples of patients with visceral metastases taken in a 2-h interval, furthermore, indicate that tumor cells only transiently persist in the peripheral blood. We obtained in vitro proliferating melanoma cells from two blood samples derived from different patients with visceral melanoma metastases. This demonstrates that viable melanoma cells indeed circulate in the peripheral blood with retained proliferative capacity in vitro. The analysis of blood samples by RT-PCR for tyrosinase mRNA, however, is not suitable for the early detection of tumor progression in melanoma patients.

Malignant transformation of melanocytes is frequently associated with abnormalities in antigen processing and in human leukocyte antigen class I antigen expression. Here, we evaluated a human leukocyte antigen class I antigen-independent approach to target cytotoxic T lymphocytes to melanoma cells by grafting cytotoxic T lymphocytes with a chimeric receptor that consists of both a domain binding to high molecular weight-melanoma associated antigen and a cellular activation domain. The binding domain is a single-chain antibody fragment (scFv) derived from the monoclonal anti-high molecular weight-melanoma associated antigen antibody 763.74 by phage display techniques. The cellular activation domain is the signaling unit of the FcepsilonRI receptor gamma chain. Both domains constitute the chimeric receptor scFv763.74-gammaR. Cytotoxic MD45 T cells grafted with the scFv763.74-gammaR receptor bind specifically to high molecular weight-melanoma associated antigen-positive melanoma cells and lyse melanoma cells in a human leukocyte antigen class I independent fashion. Pre-incubation of receptor grafted T cells with immobilized anti-idiotypic (id) monoclonal antibody MK2-23 binding to the scFv domain of the receptor enhanced the lysis of melanoma cells indicating that the specific cytolytic activity of receptor grafted T cells can be increased by costimulation with cross-linked anti-idiotypic monoclonal antibodies that recognize the antigen binding domain of the chimeric receptor.