Interlaboratory evaluation of a new reverse transcriptase polymerase chainreaction-based enzyme-linked immunosorbent assay for the detection of circulating melanoma cells: A multicenter study of the dermatologic cooperativeoncology group

Purpose: Reverse transcription-polymerase chain reaction (RT-PCR)-based def ection of tyrosinase mRNA is the most frequently used laboratory method for the detection of circulating tumor cells in melanoma patients. However, pr eviously published results showed considerable variability in the PCR posit ivity rates. Materials and Methods: We designed a collaborative study to assess the sens itivity, specificity, and clinical relevance of a new standardized RT-PCR-b ased enzyme-linked immunosorbent assay (ELISA) for the detection of circula ting melanoma cells. Blood samples of healthy donors mixed with cells of a melanoma cell line were prepared in a blinded fashion, and aliquots were se nt to seven participating laboratories experienced in RT-PCR. Results: The results demonstrate a high sensitivity(1 melanoma cell/mL bloo d) and specificity (no false-negatives and 7.4% [2 of 28] false-positives) of the assay and a satisfactory rate of interlaboratory reproducibility. Th e analysis of aliquots of blinded samples derived from 60 melanoma patients identified tyrosinase mRNA in 17 of 60 (28.3%): three (20%) of 15 stage I patients, two (13.3%) of 15 stage Il patients, five (35.7%) of 14 stage III patients, and seven (43.8%) of 16 stage IV patients. The interlaboratory r eproducibility of positive samples, however, was extremely low and indicate s the presence of low amounts of targe, mRNA. Conclusion: Reverse transcriptase-PCR ELISA has a high sensitivity and spec ificity for the detection of tyrosinase mRNA in peripheral blood cells. The low interlaboratory reproducibility for the detection of tumor cells in bl ood samples of melanoma patients, however, raises the question of relevance of this assay for clinical use. (C) 2001 by American Society of Clinical O ncology.