Prognostic value of serial blood S100B determinations in stage IIB-III melanoma patients: a corollary study to EORTC trial 18952.

S100B is a prognostic factor for melanoma as elevated levels correlate with disease progression and poor outcome. We determined its prognostic value based on updated information using serial determinations in stage IIb/III melanoma patients. 211 Patients who participated in the EORTC 18952 trial, evaluating efficacy of adjuvant intermediate doses of interferon α2b (IFN) versus observation, entered a corollary study. Over a period of 36 months, 918 serum samples were collected. The Cox time-dependent model was used to assess prognostic value of the latest (most recent) S100B determination. At first measurement, 178 patients had S100B values <0.2 µg/l and 33 ≥ 0.2 µg/l. Within the first group, 61 patients had, later on, an increased value of S100B (≥ 0.2 µg/l). An initial increased value of S100B, or during follow-up, was associated with worse distant metastasis-free survival (DMFS); hazard ratio (HR) of S100B ≥ 0.2 versus S100B < 0.2 was 5.57 (95% confidence interval (CI) 3.81-8.16), P < 0.0001, after adjustment for stage, number of lymph nodes and sex. In stage IIb patients, the HR adjusted for sex was 2.14 (95% CI 0.71, 6.42), whereas in stage III, the HR adjusted for stage, number of lymph nodes and sex was 6.76 (95% CI 4.50-10.16). Similar results were observed regarding overall survival (OS). Serial determination of S100B in stage IIb-III melanoma is a strong independent prognostic marker, even stronger compared to stage and number of positive lymph nodes. The prognostic impact of S100B ≥ 0.2 µg/l is more pronounced in stage III disease compared with stage IIb.

Melanoma progression and serum L-dopa/L-tyrosine ratio: a comparison with S100B.

The challenge to find a reliable tumour marker for the management of melanoma patients still remains. In this study, the serum L-dopa/L-tyrosine ratio was compared with serum S100B as a reference marker. A total of 89 melanoma patients were sampled and staged according to the American Joint Committee on Cancer (AJCC) classification. Of these, 19 stage III and 28 stage IV patients were evaluated for disease progression at 1.5 years and 6 months post-sampling, respectively. Serum L-dopa and L-tyrosine were measured by high performance liquid chromatography (HPLC) (normal value for ratio < 16 x 10(-5)) and S100B using the LIA-mat Sangtec 100 assay (normal value < 0.10 microg/l). Non-parametric tests (Kruskal-Wallis analysis of variance, Dunn's and Spearman) were used for the statistical analysis. The median serum L-dopa/L-tyrosine ratio was 16.0 x 10(-5) (range 2.7-545.1 x 10-5 and the median S100B level was 0.15 microg/l (range < 0.10-13.8 microg/l), with a sensitivity of 51% for the ratio and 66% for S100B. There was a 47% discordance and no correlation between the two markers (r = 0.149). The ratio was higher in stage IV than in other stages (P < 0.05), as was the S100B level (P < 0.0001). Both markers were higher in patients with evolutive disease (n = 23) than in stable patients (n = 24), with values of 20.8 x 10(-5) versus 13.1 x 10(-5) for the ratio (P < 0.05) and 0.89 microg/l versus 0.16 microg/l for S100B (P < 0.001); for the ratio, this difference was more pronounced in stage III than in stage IV patients. The overall sensitivity and specificity of the markers to predict disease progression were 78% and 67%, respectively, for the ratio, and 74% and 83%, respectively, for S100B (using an ROC cut-off of 0.38 microg/l). In conclusion, the serum L-dopa/L-tyrosine ratio correlates with melanoma progression and has predictive value, especially in stage III patients. This tumour marker, like S100B, could serve as an additional tool in the management of melanoma.

Evaluation of standard tyrosinase RT-PCR in melanoma patients by the use of the LightCycler system.

BACKGROUND: Haematogenous spread influences outcome in melanoma patients. The clinical relevance of detecting circulating melanoma cells in peripheral blood by tyrosinase mRNA RT-PCR is, however, questioned as rates of positivity considerably vary between studies. Standard tyrosinase-nested RT-PCR was here compared with a real-time PCR technique. METHODS: Forty-three blood samples from 20 stage III--IV melanoma patients were analyzed. Mononuclear cells were isolated using a Ficoll Hypaque gradient technique. Total RNA extracted by the acid guanidinum thiocyanate-phenol-chloroform method was reverse transcribed using random hexamers or specific primers. Standard tyrosinase-nested PCR was performed on Touchdown machine (Hybaid) and real-time PCR on a LightCycler instrument (Roche). RESULTS: Only two samples from stage IV patients (one from random hexamers, one from antisense primers) were found tyrosinase positive with a 100% agreement between the two PCR techniques. A 10-fold dilution of the first-round products improved the PCR kinetic and the final amount of amplified product of positive samples, but not the rate of positivity. CONCLUSIONS: Efficiency of the PCR reaction can be monitored in an online fashion by the LightCycler instrument allowing technical improvements. However, tyrosinase mRNA RT-PCR cannot be yet considered as a useful technique in the monitoring of melanoma patients.

[Current biological markers of cutaneous melanoma progression]. / Marqueurs biologiques actuels de progression du mélanome cutané.

Melanoma is the most agressive skin cancer in humans. The most important prognostic factors are the histological features of the tumor, while the clinical ones play a secondary role. Melanoma progression is characterized by the metastatic process which directly threatens the patients life. Unfortunately, routine imaging methods cannot estimate early enough this metastatic risk. Are biologic markers of cancer progression more efficient than those applied in everyday practice? Are they able to evaluate the metastatic risk and thus help the therapeutic strategy? In this review, we analysed the analytical and the clinical aspects of biologic markers of cutaneous melanoma currently available or in development. At the present time it is very difficult to distinguish one single marker of melanoma progression in the blood which correlates with the stage and the prognosis of melanoma. The most specific and sensitive enough are the melanoma associated antigens protein S-100, MIA (melanoma inhibiting activity) and the melanin precursors 5-S-cysteinyldopa and the ratio L-dopa/L-tyrosine. Tyrosinase mRNA remains the best target for the detection of circulating metastatic melanoma cells by RT-PCR. Simultaneous detection of several markers might be useful if they are carefully selected. Despite the progress in the field, more clinical studies should be performed for the development of new techniques or improvements of the existing ones for the follow-up of cutaneous melanoma.

Development of metastases in malignant melanoma is associated with an increase in the plasma L-dopa/L-tyrosine ratio.

In this prospective study we evaluated a new biochemical approach in which the plasma ratio of the melanin precursors L-dopa and L-tyrosine serves as a marker of metastatic dissemination in malignant melanoma. Control values (11.20 x 10(-5) +/- 2.92 x 10(-5)) were determined. The L-dopa/L-tyrosine ratio was evaluated in the plasma of 90 patients with malignant melanoma (stage I/II, n = 33; stage III, n = 33; stage IV, n = 24) classified according to the tumour/node/metastasis (pTNM) classification. A total of 106 samples were studied. Serial measurements were performed in eight stage III-IV patients. The L-dopa/L-tyrosine ratio was significantly elevated in melanoma patients with clinical stage III (15.23 x 10(-5) +/- 3.34 x 10(-5)) compared with stage I (10.88 x 10(-5) +/- 2.52 x 10(-5)). Stage IV patients showed a significant increase in the plasma L-dopa/L-tyrosine ratio (45.73 x 10(-5) +/- 61.75 x 10(-5)) compared with the other groups. The ratio was higher for those with two rather than one metastatic site and markedly higher for those with widespread metastases. The development of metastases was associated with an increase in plasma L-dopa, a decrease in plasma L-tyrosine and a significant increase in the plasma L-dopa/L-tyrosine ratio. These data suggest that the plasma L-dopa/L-tyrosine ratio reflects the tumour burden and correlates with the progression of malignant melanoma.

Simultaneous analysis of tyrosinase mRNA and markers of tyrosinase activity in the blood of patients with metastatic melanoma.

Determination of blood tyrosinase mRNA by RT-PCR and markers of tyrosinase activity (L-DOPA/L-tyrosine ratio) by HPLC have been proposed as biological tools for the detection of metastases in melanoma patients. We prospectively evaluated their significance and clinical value in a group of 30 stage III (n = 10) and IV (n = 20) melanoma patients and one with melanosis of Dubreuilh. L-DOPA/L-tyrosine ratio was elevated in 30% of stage III, 41% of stage IV patients (range: 7.5-261.0 x 10(5)) and in melanosis of Dubreuilh (184.8) (reference values: 6-16 X 10(5)). One stage III and four stage IV melanoma patients were positive for tyrosinase mRNA. In stage IV patients, tyrosinase mRNA positivity was associated with disease progression (P<0.01). The presence of tyrosinase mRNA in blood is more related to clinical status than level of melanin precursors, which probably reflects tumor burden.