Evaluation of the use of tyrosinase-specific and melanA/MART-1-specific reverse transcriptase-coupled-polymerase chain reaction to detect melanoma cells in peripheral blood samples from 299 patients with malignant melanoma

Background There is a current need for a reliable prognostic marker for mel anoma patients, particularly those with stage 2 and stage 3 disease, so tha t adjuvant therapies can be directed appropriately, Objectives To establish whether or not the use of tyrosinase-specific or me lanA/MART-1-specific reverse transcriptase-coupled-polymerase chain reactio n (RT-PCR) of peripheral blood cells detects preclinical disease progressio n in patients with malignant melanoma. Methods Two hundred and ninety-nine patients with melanoma in clinical stag es 1-4 were observed in this study, Samples were obtained sequentially from 153 of these patients at 4-week intervals over a period of up to 2 years a nd correlated with clinical evidence of disease activity. Tyrosinase and me lanA/MART-1 amplicons were analysed by agarose gel electrophoresis and Sout hern blot hybridization subsequent to a single round of amplification. Results We demonstrated a statistically significant increase in tyrosinase RT-PCR positivity with advancing stage of melanoma progression. The percent age tyrosinase positivity in 910 samples tested was: stage 1, 135 samples, 34% positive; stage 2, 196 samples, 51% positive; stage 3, 423 samples, 50% positive: and stage 4. 156 samples, 65% positive. The positivity rate for individual patients tested sequentially was higher if only one positive tes t was required to label a patient positive, at 42%, 65%, 82% and 81% for pa tients in stages 1-4, respectively. However, we did not find a clear patter n of conversion from negativity to positivity in patients who progressed du ring the study from stage 2 to stage 3 or stage 3 to stage 4, and found no clear evidence of increased positivity rates in the 6-week period following melanoma-related surgery in patients with stage 3 and 4 disease, The posit ivity rate for melanA/MART-1 was lower for both patients and samples, and n o melanA/MART-1-positive sample was negative for tyrosinase, Conclusions We conclude that the presence of circulating tyrosinase-positiv e cells as detected by this method appears to be a discontinuous rather tha n a continuous phenomenon, even in patients with stage 4 disease. For this reason the assay cannot be recommended as a method of sequentially monitori ng individual patients in a clinical setting.