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


CONCLUSIONS
Tyrosinase mRNA detection may be therefore employed to monitor the melanoma patients over time in function of response to therapy.
Santonocito
Concetta
C
Laboratory of Molecular Biology, Department of Biochemistry and Clinical Biochemistry - Catholic University, Rome, Italy.
Concolino
Paola
P
Lavieri
Maria Michela
MM
Ameglio
Franco
F
Gentileschi
Stefano
S
Capizzi
Rodolfo
R
Rocchetti
Sandro
S
Amerio
Pierluigi
P
Castagnola
Massimo
M
Zuppi
Cecilia
C
Capoluongo
Ettore
E
D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D005260:Female; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D008297:Male; D008545:Melanoma / Q000097:blood / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D014442:Monophenol Monooxygenase / Q000235:genetics*; D009367:Neoplasm Staging; D009414:Nerve Growth Factors / Q000235:genetics / Q000378:metabolism*; D010751:Phosphopyruvate Hydratase / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000097:blood* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity
D014408:Biomarkers, Tumor; D009414:Nerve Growth Factors; D012333:RNA, Messenger; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; C579160:S100B protein, human; D007770:L-Lactate Dehydrogenase; D014442:Monophenol Monooxygenase; D010751:Phosphopyruvate Hydratase