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

MARIANO R GABRI; VALERIA VAZQUEZ; SANTIAGO GIRÓN; MÓNICA A. CASTRO; GABRIELA CINAT; ROBERTO E. GOMEZ; FEDERICO M. SANTORO; DANIEL E. GOMEZ; DANIEL F. ALONSO

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE

D.A. Spandidos,

Lugar: Grecia; Año: 2008 vol. 21 p. 555 - 559

1107-3756

We have designed high-affinity primers to the mRNA sequence of human tyrosinase to test the value of molecular detection of circulating melanoma cells by reverse transcription-PCR (RT-PCR). The optimization process included in vitro settings and in vivo studies in a xenograft mouse model. We were able to detect 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 expression bands were stronger after manipulation of the tumor mass. We have also examined the fate of circulating melanoma cells in the present melanoma model. Tyrosinase expression declined in blood 6 hours 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.