Detection and quantitation of melanoma cells in the circulation of patients

Abstract

A method for the quantitation of tyrosinase mRNA in the bloodstream of melanoma patients has been developed by using competitive polymerase chain reaction (PCR) with a heterologous DNA (PCR MIMIC) as an internal standard. The method was validated by demonstration of similar amplification efficiencies for both molecules and by accurate quantitation of an artificial fourfold difference in the level of tyrosinase mRNA. The ratio of amplified target to amplified standard (At/As ratio) was determined for a range of melanoma patients who had previously tested positive for tyrosinase. Sequential samples were also analysed to examine the level of tyrosinase in individual patients over time. When tyrosinase levels in melanoma cell lines were compared for a constant amount of total RNA, or for a constant number of cells, tyrosinase mRNA was found to vary more than a thousand-fold between cell lines. Because of this, the At/As ratio of patients was compared with the At/As ratios obtained when 10-fold serial dilutions of cells from a control melanoma cell line (MM200) were added to 2 ml of packed blood. An 'MM200 equivalence' value was thus calculated, giving the equivalent number of MM200 cells in the bloodstream of melanoma patients. Prolonged follow-up will be needed to determine the prognostic significance of the detection and levels of tyrosinase mRNA in the bloodstream of melanoma patients.