Detection of circulating melanoma cells in peripheral blood by a two-marker RT-PCR assay

Abstract

The aim of this study was to develop a highly sensitive two-marker assay for the detection of circulating melanoma cells in patients' blood using a reverse transcriptase-polymerase chain reaction (RT-PCR). We analysed the usefulness of two different sets of markers: tyrosinase and MUC-18 (TYR/MUC-18), and tyrosinase and MART 1 (TYR/MART 1). Total cellular RNA was isolated from 337 blood samples from 80 melanoma patients at different stages of the disease. All patients had undergone primary surgery. Assay sensitivity and specificity were confirmed using three different melanoma cell lines and two different fibroblast lines. In addition, blood from 47 healthy subjects and 10 patients with non-melanoma cancer was used as a negative control. We found that two-marker analysis is more accurate than the single tyrosinase assay. The frequency of melanoma cell detection in patients' blood was about 10% higher when the TYR/MART 1 two-marker assay was used. Using this assay we did not find any statistical correlation between the molecular markers and the UICC stage of disease or the Breslow thickness or Clark level of the primary melanoma. The frequency of melanoma cell detection with the TYR/MUC-18 two-marker assay was even higher than the TYR/MART 1 assay, but unfortunately the MUC-18 transcript was also present in about 20% of healthy subjects. Therefore we do not recommend the use of MUC-18 as a standard value marker.

Detection of Circulating Melanoma Cells by a Two-marker Polymerase Chain Reaction Assay in Relation to Therapy

“…This ratio is consistent with an earlier report (Curry et al, 1999), as well as the hypothesis that most melanoma tumors and cell lines express this gene (Schittek et al, 1999). In agreement with previous reports (Kulik et al, 2001;Taback et al, 2001), the frequency of melanoma cell detection in a patients blood was higher in our study when the two-marker assay was used. However, Melan A transcripts were still detectable in 43% of the patients following chemotherapy.…”

“…This finding is consistent with other studies (Hoon et al, 1995;Kulik et al, 2001;Taback et al, 2001). Since detection limits are roughly the same for both transcripts, the higher detection rate for Melan A may be associated with the characteristics of the tumor cell.…”

“…This information may be useful in identifying patients who may benefit from more intense therapies, and provide an opportunity for therapeutic intervention at an earlier stage. However, the assessment of single markers has limitations in sensitivity and specificity, based on the heterogeneity of the marker expression among tumors (Kulik et al, 2001). Furthermore, although specific molecular techniques have been utilized for diagnostic purposes, the After Therapy III 14 --8 57 I V 6 1 9 6 1 0 0 correlation between molecular detection of circulating tumor cells and the response to therapy has not been addressed satisfactorily.…”

Prognostic Value of Multiple Reverse Transcription-PCR Tyrosinase Testing for Circulating Neoplastic Cells in Malignant Melanoma

“…In our 5-year study, in common with other authors, we have experienced extreme variability in TYR test results in staging, prognosis, and evaluating the effects of different therapies (29,30 ). We recently provided an explanation for this variability by demonstrating that it is attributable to low concentrations of CMCs in the peripheral blood [more than 10-fold below the detection threshold of the assay (31 )].…”

“…Blood was centrifuged at 400g for 5 min, and the supernatant was decanted. The total RNA was extracted by the method of Chomczynski and Sacchi (38 ) with some modifications (30 ). RNA integrity was checked electrophoretically.…”