Konstantin Stoitchkov

Post-surgery adjuvant therapy with intermediate doses of interferon alfa 2b versus observation in patients with stage IIb/III melanoma (EORTC 18952): randomised controlled trial

BACKGROUND Individuals affected by melanoma with thick primary tumours or regional node involvement have a poor outlook, with only 30-50% alive at 5 years. High-dose and low-dose interferon alfa have been assessed for the treatment of these patients, with the former having considerable toxicity and a consistent effect on disease free survival, but not on overall survival, and the latter no consistent effect on either. Our aim was, therefore, to assess the effect of two regimens of interferon of intermediate dose versus observation alone on distant metastasis-free interval (DMFI) and overall survival in such patients. METHODS We did a randomised controlled trial in 1388 patients who had had a thick primary tumour (thickness > or = 4 mm) resected (stage IIb) or regional lymph node metastases dissected (stage III) and had been assigned to 13-months (n=553) or 25 months (n=556) of treatment with subcutaneous interferon alfa 2b, or observation (n=279). Treatment comprised 4 weeks of 10 million units (MU) of interferon alfa (5 days per week) followed by either 10 MU three times a week for 1 year or 5 MU three times a week for 2 years, to a total dose of 1760 MU. Our primary endpoint was DMFI. Analyses were by intent to treat. FINDINGS After a median follow-up of 4.65 years, we had recorded 760 distant metastases and 681 deaths. At 4.5 years, the 25-month interferon group showed a 7.2% increase in rate of DMFI (hazard ratio 0.83, 97.5% CI 0.66-1.03) and a 5.4% improvement in overall survival. The 13-month interferon group showed a 3.2% increase in rate of DMFI at 4.5 years (0.93, 0.75-1.16) and no extension of overall survival. Toxicity was acceptable, with 18% (195 of 1076) of patients going off study because of toxicity or as a result of refusal of treatment because of side-effects. INTERPRETATION Interferon alfa as used in the regimens studied does not improve outcome for patients with stage IIb/III melanomas, and cannot be recommended. With respect to efficacy of the drug, duration of treatment seemed more important than dose, and should be assessed in future trials.

Genetic polymorphism of NK receptors and their ligands in melanoma patients: prevalence of inhibitory over activating signals.

Antitumor cytotoxicity of NK cells and T cells expressing NK-associated receptors is regulated by interaction between their cell surface killer immunoglobulin-like receptors (KIRs) and CD94/NKG2 heterodimers with MHC class I ligands on target cells. To test the hypothesis that KIR and/or HLA polymorphisms, and KIR/HLA combinations could contribute to the tumorigenesis, association studies were performed in 50 patients with malignant melanoma (MM) in different stages of disease and 54 controls. Our data showed that the frequency of inhibitory and activating KIR genes and KIR genotypes did not differ significantly between healthy individuals and melanoma patients. HLA haplotype distribution showed statistically significant increased frequencies of A*01-B*35-Cw*04 (0.069 vs 0.000; pc<0.05; OR=19.9), A*01-B*08-DRB1*03 (0.079 vs 0.019; pc<0.05; OR=4.5), and A*24-B*40-DRB1*11 (0.026 vs 0.000; pc<0.05; OR=7.1) in melanoma patients compared with healthy controls. Individuals homozygous for group 2 HLA-C ligands were less frequent in the patient group compared with the control cohort (12% vs 31.5%; p<0.017). In addition, we observed an increased frequency (88.0% vs 68.5%; p=0.017; OR=2.80) of KIR2DL2/2DL3 in combination with their group 1 HLA-C ligands, while the presence of these KIRs in the absence of the putative ligands was decreased (12.0% vs 31.5%; p=0.017) in the patient group. Furthermore, an increased frequency of activating KIR2DS1 in the absence of the putative HLA-CLys80 ligands was found in melanoma patients (16.0% vs 9.2%). In contrast, KIR2DS2 was absent in patients more often (38.0% vs 25.9%) when the presumptive HLA-CAsn80 ligands were present. A slightly higher incidence of KIR3DL1 in combination with the less effective Bw4Thr80 ligands was seen in patients with primary (20.8%) compared with metastatic (4.2%) disease. The data obtained in this study imply that there may not be a direct association between KIR gene content in the genome and the presence of malignant melanoma, or melanoma progression. However, some HLA haplotypes could be predisposing to MM in the Bulgarian population. Furthermore, distinct KIR/HLA ligand combinations may be relevant to the development of malignancy whereby inhibition overrides activation of NK cells and T cells expressing NK-associated receptors, which in turn might facilitate tumor escape and progression.

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 × 10−5) and S100B using the LIA-mat Sangtec 100 assay (normal value < 0.10 μg/l). Non-parametric tests (Kruskal–Wallis analysis of variance, Dunns and Spearman) were used for the statistical analysis. The median serum l-dopa/l-tyrosine ratio was 16.0 × 10−5 (range 2.7–545.1 × 10−5 and the median S100B level was 0.15 μg/l (range < 0.10–13.8 μg/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 × 10−5 versus 13.1 × 10−5 for the ratio (P < 0.05) and 0.89 μg/l versus 0.16 μg/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 μg/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.

INV 12 Surgical approach on melanoma patients: an EORTC melanoma group survey

Alessandro Testori, Javier Soteldo, François Sales, Lorenzo Borgognoni, Zbigniew Nowecki, Ferdy Lejeune, Konstantin Stoitchkov, Alexander Eggermont, Barry Powell European Institute of Oncology, Centro Regionale di Riferimento per il Melanoma, Ospedale S. M. Annunziata, Italy, Institut Jules Bordet, Brussels, Belgium, Centre Hospitalier Universitaire Vaudois, Switzerland, Erasmus University Medical center, Rotterdam, The Netherlands, St. Georges Hospital, UK