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Comparison between three molecular methods for detection of blood melanoma tyrosinase mRNA. Correlation with melanoma stages and S100B, LDH, NSE biochemical markers.

Santonocito C, Concolino P, Lavieri MM, Ameglio F, Gentileschi S, Capizzi R, Rocchetti S, Amerio P, Castagnola M, Zuppi C, Capoluongo E

Laboratory of Molecular Biology, Department of Biochemistry and Clinical Biochemistry - Catholic University, Rome, Italy.

BACKGROUND: The molecular monitoring of circulating tumor cells by reverse transcriptase-PCR (RT-PCR) for patients with melanoma, is still under debate. It may be affected by: a) pre-analytical variability, b) frequency of melanoma-associated gene transcripts and c) the reliability of the methods employed. Few commercial methods are available for the detection of tyrosinase mRNA in blood. OBJECTIVE: Comparison between two RT-PCR-nested methods with a third one based on real-time methodology, for detection and quantitation of tyrosinase transcripts, respectively. METHODS: Sixty-two melanoma patients with different AJCC stages and 20 healthy subjects were enrolled. All blood samples were extracted in duplicate with two different methods. Two nested-PCR methods (one commercial and one in house) plus a real time commercial kit were employed. RESULTS: The two nested PCR methods employed were overimposable, specific and sensitive, at least in the stage III, where there was a concordance between sentinel lymph nodes detection and blood tyrosinase positivity. The different extraction methods did not affect the quality of results, while the commercial real-time kit cannot be used. CONCLUSION: Tyrosinase mRNA detection may be therefore employed to monitor the melanoma patients over time in function of response to therapy.

Published 8 November 2005 in Clin Chim Acta, 362(1): 85-93.
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