Multimarker quantitative real-time PCR detection of circulating melanoma cells in peripheral blood: relation to disease stage in melanoma patients.
BACKGROUND
Detection of melanoma cells in circulation may be important in assessing tumor progression. The objective of this study was to develop a specific, reliable multimarker quantitative real-time reverse transcription-PCR (qRT) assay for detecting melanoma cells in patients' blood.


METHODS
We developed qRT assays for the mRNA of four melanoma-associated markers: MART-1, GalNAc-T, PAX-3, and MAGE-A3. In optimization studies, we tested 17 melanoma cell lines and 49 peripheral blood leukocyte (PBL) samples from volunteers. We performed RNA and melanoma cell dilution studies to assess the detection limits and imprecision of the assays. We measured the mRNAs in blood specimens from 94 melanoma patients [American Joint Committee on Cancer (AJCC) stage I, n = 20; II, n = 20; III, n = 32; IV, n = 22].


RESULTS
All markers were frequently detected in melanoma cell lines, whereas none of the markers was detected in PBLs from volunteers. The qRT assay could detect 1 melanoma cell in 10(7) PBLs in the melanoma cell-dilution studies. Markers were detected in 15%, 30%, 75%, and 86% of melanoma patients with AJCC stage I, II, III, and IV disease, respectively. The number of positive markers and AJCC stage were significantly correlated (Spearman correlation coefficient = 0.58; P < 0.0001).


CONCLUSIONS
Multimarker qRT can detect circulating melanoma cells in blood. Measurement of the studied molecular markers in blood may be useful in detection of metastasis and monitoring treatment response of melanoma patients.
Koyanagi
Kazuo
K
Department of Molecular Oncology, John Wayne Cancer Institute at Saint John's Health Center, Santa Monica, CA 90404, USA.
Kuo
Christine
C
Nakagawa
Taku
T
Mori
Takuji
T
Ueno
Hideaki
H
Lorico
Arnulfo R
AR
Wang
He-Jing
HJ
Hseuh
Eddie
E
O'Day
Steven J
SJ
Hoon
Dave S B
DS
D000951:Antigens, Neoplasm / Q000097:blood / Q000235:genetics; D014408:Biomarkers, Tumor / Q000097:blood* / Q000235:genetics; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000097:blood / Q000235:genetics; D004311:Double-Blind Method; D018628:Gene Dosage; D006801:Humans; D007962:Leukocytes / Q000737:chemistry; D008198:Lymph Nodes / Q000737:chemistry; D058965:MART-1 Antigen; D008545:Melanoma / Q000175:diagnosis / Q000473:pathology*; D017350:N-Acetylgalactosaminyltransferases / Q000097:blood / Q000235:genetics; D009362:Neoplasm Metastasis; D009363:Neoplasm Proteins / Q000097:blood / Q000235:genetics; D009367:Neoplasm Staging; D009360:Neoplastic Cells, Circulating; D000072041:PAX3 Transcription Factor; D051761:Paired Box Transcription Factors; D012333:RNA, Messenger / Q000097:blood; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012878:Skin Neoplasms / Q000175:diagnosis / Q000473:pathology*; D014157:Transcription Factors / Q000097:blood / Q000235:genetics
D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D004268:DNA-Binding Proteins; C072013:MAGEA3 protein, human; D058965:MART-1 Antigen; C548667:MLANA protein, human; D009363:Neoplasm Proteins; D000072041:PAX3 Transcription Factor; C489409:PAX3 protein, human; D051761:Paired Box Transcription Factors; D012333:RNA, Messenger; D014157:Transcription Factors; D017350:N-Acetylgalactosaminyltransferases