Detection of tyrosinase and tyrosinase-related protein 1 sequences from peripheral blood of melanoma patients using reverse transcription-polymerase chain reaction.
BACKGROUND
malignant melanoma has one of the highest rates of metastasis. Unlike other solid cancers, no sensitive tumor markers or laboratory tests that can provide information of the risk of metastasis and predict the prognosis have yet been established.


OBJECTIVE
the study was done to establish a RT-PCR sensitive and specific enough to detect melanocyte-specific transcripts from peripheral blood cells.


METHODS
peripheral white blood cells were collected from 30 healthy donors and 43 melanoma patients. Melanocyte-specific tyrosinase (TYR) and tyrosinase-related protein 1 (TYRP1) were selected as targets of RT-PCR. The sensitivity of detection using SK-mel-23 melanoma cells and rates of false-positiveness using non-melanoma blood were compared between single-step PCR and nested PCR. Analysis of melanoma blood samples was carried out by the single-step PCR.


RESULTS
the nested RT-PCR amplified the TYR and TYRP1 sequences from 14 and 2 of the 30 healthy bloods, respectively. However, the single-step RT-PCR did not amplify TYR or TYRP1 sequences from any of the healthy controls. Our single-step RT-PCR detected 1.7 and 0.8 SK-mel-23 melanoma cells per ml blood for TYR and TYRP1, respectively. Overall, TYR mRNA was detected in 15 of the 43 melanoma patients (34.9%), and TYRP1 mRNA in 16 of the 43 (37.2%). The specificities of detection of TYR and TYRP1 were 83.3 and 88.9%, respectively.


CONCLUSIONS
our single-step RT-PCR for TYR and TYRP1 mRNAs is more specific than the previous nested RT-PCR for TYR and applicable to detect circulating melanoma cells.
Jin
Hai-Ying
HY
Department of Dermatology, Sapporo Medical University School of Medicine, South 1, West 16, Chuo-ku, Sapporo 060-8543, Japan.
Yamashita
Toshiharu
T
Minamitsuji
Yasushi
Y
Omori
Fusayuki
F
Jimbow
Kowichi
K
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D008297:Male; D008544:Melanocytes / Q000201:enzymology; D008545:Melanoma / Q000097:blood* / Q000175:diagnosis / Q000201:enzymology; D008562:Membrane Glycoproteins; D008875:Middle Aged; D014442:Monophenol Monooxygenase / Q000097:blood*; D010088:Oxidoreductases; D011506:Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012878:Skin Neoplasms / Q000097:blood* / Q000175:diagnosis / Q000201:enzymology
D008562:Membrane Glycoproteins; D011506:Proteins; D012333:RNA, Messenger; D010088:Oxidoreductases; C489799:TYRP1 protein, human; C064056:tyrosinase-related protein-1; D014442:Monophenol Monooxygenase