Detection of occult melanoma cells in blood with a multiple-marker polymerase chain reaction assay.
OBJECTIVE
The objective of the study was to develop a sensitive multimarker polymerase chain reaction (PCR) assay to detect circulating melanoma cells in patient blood. The rationale was that malignant melanoma is heterogeneous in regards to antigen expression.


METHODS
A PCR assay that uses four melanoma-associated gene markers (tyrosinase, p97, MUC18, and MAGE-3) was developed. Sensitivity and specificity of the PCR assay for individual markers were assessed using 10 melanoma cell lines and peripheral-blood lymphocytes (PBL) from 39 normal volunteers as controls. The assay's sensitivity and specificity were improved using nested primers and Southern blot analysis. Patients (N = 119) with American Joint Committee on Cancer (AJCC) stages I to IV disease were evaluated for circulating melanoma cells using the four gene markers under optimal conditions.


RESULTS
All melanoma-associated gene markers were expressed in at least 80% of the melanoma lines, whereas 37 of 39 normal PBL tested negative for all markers; the remaining two PBL were positive for MUC18. Using four markers in the PCR assay was significantly better than using tyrosinase alone. There was a significant correlation between the number of positive PCR markers, AJCC stage of disease, and progression of disease. In all AJCC stages, there were more PCR-positive patients with disease than without disease.


CONCLUSIONS
A multimarker PCR assay is more reliable and sensitive than a single-marker assay for detection of melanoma cells in blood of patients. This assay can provide important insight into tumor progression kinetics without major surgical or conventional radiologic diagnostic procedures.
Hoon
D S
DS
Division of Molecular and Cellular Immunology, John Wayne Cancer Institute, Santa Monica, CA 90404, USA.
Wang
Y
Y
Dale
P S
PS
Conrad
A J
AJ
Schmid
P
P
Garrison
D
D
Kuo
C
C
Foshag
L J
LJ
Nizze
A J
AJ
Morton
D L
DL
D000951:Antigens, Neoplasm / Q000235:genetics; D001483:Base Sequence; D014408:Biomarkers, Tumor / Q000097:blood*; D015139:Blotting, Southern; D016009:Chi-Square Distribution; D005819:Genetic Markers; D006801:Humans; D008545:Melanoma / Q000175:diagnosis* / Q000235:genetics / Q000276:immunology / Q000473:pathology; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000235:genetics; D009360:Neoplastic Cells, Circulating; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D005819:Genetic Markers; D014442:Monophenol Monooxygenase