Marker Of Melanoma Progression Research Articles

Detection of circulating melanoma cells by specific amplification of tyrosinase complementary DNA is not a reliable tumor marker in melanoma patients: a clinical two-center study.

Recently, the reverse-transcription polymerase chain reaction (RT-PCR) of tyrosinase messenger RNA (mRNA) was reported to be a useful tool for the detection of circulating tumor cells in the peripheral blood of melanoma patients. Our aim was to evaluate critically the diagnostic value of this marker by investigating a significant number of patients in different stages of the disease in a two-center study. Different techniques of blood collection, RNA isolation, and RT-PCR were compared, and the detectability of tyrosinase mRNA was tested using nine different melanoma cell lines. The sensitivity of the method was confirmed by blood spiking experiments and the specificity by restriction enzyme analysis. Subsequently, a total of 153 blood samples from 137 individuals (30 healthy subjects, five basal cell carcinoma, and 102 melanoma patients) were investigated. The detection level of melanoma cells differed between the cell lines tested. However, we could reproducibly detect single melanoma cells by spiking whole blood samples from healthy volunteers. One of 43 patients with primary melanoma (2.3%), zero of 15 patients with regional metastasis (0%), and 12 of 44 patients with advanced disease (27.3%) were found to be RT-PCR positive. All blood samples obtained from controls and patients with basal cell carcinoma were tyrosinose mRNA negative. Our data support the recent doubts that the detection of circulating tumor cells in melanoma patients using the tyrosinase mRNA RT-PCR is not sensitive enough to be used either as a melanoma progression marker in early stages of the disease or to monitor therapy in advanced stages of the disease.

In situ expression of transforming growth factor beta is associated with melanoma progression and correlates with Ki67, HLA-DR and beta 3 integrin expression.

Transforming growth factor-beta (TGF beta), which is secreted by cultured melanoma cells constitutively, inhibits the proliferation of normal melanocytes and of most melanoma cells in vitro, but some melanoma cells from advanced stages of the disease develop resistance to TGF beta-dependent growth inhibition, without developing any change in TGF beta cell surface binding. In vitro TGF beta also downregulates the expression of HLA-DR molecules on melanoma cells, and upregulates the expression of the beta 3 integrin subunit on some cell lines. Immunohistochemical analysis of 53 melanocytic lesions (12 naevi, 30 primary melanomas and 11 metastases) revealed a trend of increasing expression of TGF beta and TGF beta receptor type III with tumour progression, and a significantly higher expression of both TGF beta (P < 0.0001) and the receptor (P < 0.05) in metastatic and thick (> 1 mm) primary melanomas compared with thin (< 1 mm) primary melanomas. The expression of TGF beta correlated with expression of a marker of proliferation, Ki67, and with HLA-DR and beta 3 integrin subunit expression. Coexpression of the four molecules was observed in all metastases and in most thick primary melanomas. These findings argue against an inhibitory effect of TGF beta on cell proliferation or HLA-DR antigen expression in melanoma, and suggest the upregulation of the beta 3 subunit. TGF beta protein appears to be a biological marker of melanoma progression in situ.

Serum levels of sICAM-1 and 5-S-cysteinyldopa as markers of melanoma progression.

The serum levels of the soluble form of intercellular adhesion molecule-1 (sICAM-1) and 5-S-cysteinyldopa (5-S-CD) were determined by double determinant immunoassay and high-performance liquid chromatography, respectively. Fifty-three melanoma patients (stage 1, 12 patients; stage II, 11; stage III, 19; and stage IV, 11; total number of samples, 116) and 31 age- and sex-matched healthy control subjects were analysed. Both the sICAM-1 and 5-S-CD levels were significantly higher in stage IV patients than those in stage I, II and III patients (sICAM-1; all P < 0.001; 5-S-CD; all P < 0.05). The serum levels of 5-S-CD were elevated only in stage IV patients. sICAM-1 levels elevated gradually with disease progression. Testing of sequential bleedings showed that both sICAM-1 and 5-S-CD levels were elevated in most of the patients whose disease had progressed. However 5-S-CD levels were not elevated in those patients whose metastases were amelanotic. There was a statistically significant correlation between sICAM-1 and 5-S-CD levels (R = 0.6 55, P < 0.001). Serum levels of 5-S-CD may be a useful parameter for monitoring the clinical course of the disease in patients whose metastases are melanotic. Analysis of both sICAM-1 and 5-S-CD levels in serum will contribute greatly to monitoring the clinical course of melanoma patients.

Tyrosinase fails as an early melanoma progression marker

Gläser, R.; Hauschild, A.; Lohmeier, Ch; Bartels, J.; Mrowietz, U.; Schlüter, C.; Christophers, E.Author Information

Serum factors as markers of melanoma progression

Serum factors as markers of melanoma progression

Evaluation of melanin-related metabolites as markers of melanoma progression

Urinary excretion of 5-S-cysteinyldopa (5-S-CD) has been used as a biochemical marker of melanoma progression. Melanomas produce not only 5-S-CD but also 5,6-dihydroxyindole-2-carboxylic acid (5,6DHI2C) as major intermediates in melanin formation. 5,6DHI2C is then metabolized to the two O-methyl derivatives, 5H6MI2C and 6H5MI2C. The aim of this study was to determine which marker in serum and urine most sensitively reflected the progression of melanoma. Serum and 24-hour urine samples were collected and assayed serially by high-performance liquid chromatography every 1 to 4 months in 28 patients with primary or recurrent melanomas, for up to 48 months. Serum concentration and urinary excretion of 5-S-CD and 6H5MI2C in patients with melanoma without metastases were close to those obtained from normal subjects. Metastases developed in 9 of the 28 patients. In seven of these nine patients, serum or urinary 5-S-CD values were elevated before or at the time of clinical detection of visceral metastases. However, serum 5-S-CD was elevated significantly earlier and reflected melanoma progression better than the physical examination and/or laboratory tests, such as scintigraphy and echography. Serum 6H5MI2C values exceeded the normal range shortly before death in three patients, and urinary 6H5MI2C did not increase at any stage in most patients, therefore these metabolites did not reflect progression of disease. Among the four markers, serum 5-S-CD appears to be the best biochemical marker for the detection of progression of melanotic melanoma, a value of more than 10 nmol/l suggesting the presence of metastasis.

Serum factors as markers of melanoma progression

Serum factors as markers of melanoma progression

Usefulness of serum 5-S-cysteinyldopa level as a biochemical marker of melanoma progression

Usefulness of serum 5-S-cysteinyldopa level as a biochemical marker of melanoma progression

Serum 5‐S‐Cysteinyldopa (5‐S‐CD) as a Marker of Melanoma Progression

We previously reported that serum 5-S-cysteinyldopa (5-S-CD) tended to elevate earlier and reflect melanoma progression better than urinary 5-S-CD. In patients without metastatic melanomas, serum concentration and urinary excretion of 5-S-CD and 6-hydroxy-5-methoxy-indole-2-carboxylic acid (6H5MI2C) were within the upper limits of normal controls. In this report, we presented more precisely the changes in these melanin-related markers and clinical courses of four melanoma patients. Serum and 24-hour urine samples were serially collected and assayed every 1 to 4 months. Three of them developed stage IV malignant melanomas and died of metastatic disease. 6H5MI2C in serum and urine did not reflect the progression of disease. Among the 4 parameters considered, 5-S-CD in serum appeared to be the best biochemical marker for melanoma progression. Serum 5-S-CD over the upper limit of 10 nmol/L was suggested as a serious sign of the progression of melanoma.

Normal values of urinary excretion and serum concentration of 5-S-cysteinyldopa and 6-hydroxy- 5-methoxyindole-2-carboxylic acid, biochemical markers of melanoma progression

The urinary excretion of the pheomelanin precursor 5-S-cysteinyldopa (5-S-CD) has been used as a tumour marker for metastatic melanoma. The eumelanin-related metabolite 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5M12C) is also excreted at high levels in some melanoma patients. In order to compare normal values, we measured urinary excretion and serum concentration of 5-S-CD and 6H5M12C in 33 Japanese normal subjects. The mean values of 5-S-CD and 6H5M12C in urine were 0.45 and 0.39 mumol/day, respectively, and those in serum 4.3 and 3.6 nmol/l. Levels of these markers in urine were much more variable than those in serum. We have adopted 1.5 mumol/day and 10 nmol/l as the upper limits for normal ranges of the urinary excretion and serum concentration, for both markers. No significant differences were found between men and women. There were no correlations among the four markers. The urinary excretion of both markers showed significant decrease in elderly subjects as compared with middle-aged subjects, while the serum concentration showed no age-dependent differences. These results suggest that the levels of 5-S-CD and 6H5M12C in serum are more reliable as tumour markers for the estimation of melanoma progression than those in urine.

Cytostatic and Cytotoxic Effects of Recombinant Tumor Necrosis Factor-α on Sensitive Human Melanoma Cells In Vitro May Result in Selection of Cells with Enhanced Markers of Malignancy

Monolayer cultures of the human melanoma cell lines StML-12, StML-11, StML-14 (third, respectively, twenty-fifth subculture), and SKMel-28 derived from specimens representing different stages of tumor progression were treated with 10-10,000 U/ml rTNF-alpha applied for 72 h. The effects of rTNF-alpha on cell proliferation, DNA synthesis, cell viability, cloning efficiency, cell division, cell morphology, and the immunophenotype were studied in triplicate experiments. The cell line StML-14(3) revealed a significantly dose-dependent reduction of growth due to both cytostatic and cytotoxic activities of rTNF-alpha as well as a decrease of CE. Increased numbers of cells in prophase were observed 24 h after addition of r-TNF-alpha. In addition, dislocation of chromosomes in the metaphase, formation of micronuclei, and dose-dependent increases of cells exhibiting micronuclei and the DNA amount per cell were detected at the end of treatment. On the other hand, only a slight sensitivity to the anti-proliferative effect of rTNF-alpha was observed with StML-14(25) and SKMel-28, whereas StML-12 and StML-11 were significantly resistant. The last four cell lines were serially subcultivated and presented common phenotypic patterns with more malignant characteristics than the cell line StML-14(3) before treatment. Overall, rTNF-alpha enhanced the malignant immunophenotype of the cell lines tested. It increased the expression of the "late" melanoma progression markers A.10.33 and A.1.43, and Ki67, and it decreased the expression of the "early" progression marker K.1.2. The expression of HLA-I, HLA-DR, and ICAM-1 was also enhanced after rTNF-alpha treatment, whereas in contrast to other cytokines, rTNF-alpha did not induce the de novo expression of HLA-DR in HLA-DR-negative melanoma cell lines. These findings indicate that rTNF-alpha induces cytostasis and decreases cell viability of certain rTNF-alpha-sensitive melanoma cells. These effects may result in selection of rTNF-alpha-non-sensitive human melanoma cell populations with higher proliferation rates and a more aggressive immunophenotype in vitro.

Antitumor Activities of Interferon Alpha, Beta, and Gamma and their Combinations on Human Melanoma Cells In Vitro: Changes of Proliferation, Melanin Synthesis, and Immunophenotype

The antitumor activities of human interferon (IFN) alpha, beta, and gamma alone or in combination were studied on four human melanoma cell lines (StML-11, StML-12, StML-14, and SKMel-28) in various concentrations (1-50,000 IU/ml IFN alpha, 0.1-1000 IU/ml IFN beta, 1-10,000 IU/ml IFN gamma) in vitro. In all experiments IFN beta exhibited the most potent antiproliferative effect of all IFN tested. After 3 d of incubation a 50% growth inhibition was achieved with 20-40 IU/ml for natural IFN beta and with 600-1200 U/ml for recombinant IFN gamma. Substantially higher doses (7,000 to more than 50,000 IU/ml) of recombinant IFN alpha 2a were required to achieve a 50% growth inhibition. A strong synergistic antiproliferative activity resulted from the combination of IFN alpha with IFN gamma and IFN beta with IFN gamma. None of the IFN tested induced terminal differentiation of melanoma cells in vitro. The formation of dendrites was inhibited, and the portion of differentiated cells in vitro was reduced after treatment with IFN in comparison to the untreated controls (untreated controls: 100%; portion of differentiated cells after treatment with IFN alpha: 58%-74%, IFN beta: 48%-96%, IFN gamma: 10%-33%). The melanin synthesis was slightly elevated after treatment with IFN alpha (untreated controls: 100%; after treatment with IFN alpha: 103%-157%, ns.) and decreased significantly after treatment with IFN beta (49%-71%, p less than 0.05) as well as with IFN gamma (80%-88%, ns.). Cell surface markers were modulated varyingly by the IFN: HLA-I antigens were enhanced by all IFN, with IFN beta emerging as the most potent inducer. Only IFN gamma, however, induced a de novo expression of HLA-DR and -DQ antigens and increased the expression of the ICAM-1 molecule and of the melanoma progression marker A.1.43. Possibly, these findings indicate a biologically more aggressive phenotype of melanoma cells.

Evaluation of urinary and serum 5-S-cysteinyldopa and 6-hydroxy-5-methoxyindole-2-carboxylic acid as markers of melanoma progression

Evaluation of urinary and serum 5-S-cysteinyldopa and 6-hydroxy-5-methoxyindole-2-carboxylic acid as markers of melanoma progression

The Stability of 5‐S‐Cysteinyldopa and 6‐Hydroxy‐5‐Methoxyindole‐2‐Carboxylic Acid in Human Urine

5-S-cysteinyldopa and 6-hydroxy-5-methoxyindole-2-carboxylic acid are important intermediate metabolites in the formation of cutaneous melanin pigment. Since they both are serious candidates as markers of melanoma progression, their stability in urine has been investigated during storage at various conditions. The results show that storage at -20 degrees C is necessary. Both compounds are nonstable at room temperature, particularly if the urine was not acidified to pH 4-5. Reference levels were obtained from analysis of urine from 31 men and 40 women. The mean (SD) excretion of 5-S-cysteinyldopa was 32 (12.5) mumol/mol creatinine (women). Corresponding figures for 6H5MI2C were 23 (10.3) and 24 (8.1) mumol/mol creatinine for men and women respectively.