Detection of mutated BRAFV600E variant in circulating DNA of stage III-IV melanoma patients

BRAFV600E is the most represented somatic point mutation in cutaneous melanoma, thus providing a unique molecular marker for this disease. The development of efficient methods for its detection in free circulating DNA of patients may lead to the improvement of diagnostic and prognostic tools. With this aim, we evaluated whether BRAFV600E represents a detectable marker in the plasma/serum from melanoma patients in a pilot study. Circulating cell-free DNA was extracted from the serum or plasma of 15 healthy donors and 41 melanoma patients at different clinical stages and obtained either presurgery or after surgery during follow-up. Quantitative analysis showed higher levels of circulating free DNA in patients compared to controls, with the highest levels detected in samples obtained presurgery and at stage IV. Four different PCR methods were compared for their capacity to amplify a few copies of BRAFV600E in wild-type DNA. BRAFV600E was detectable in circulating DNA of 12 patients and in none of the controls; only 1 PCR method reproducibly amplified BRAFV600E. Positive samples were obtained from 8/13 patients at stage IV and from 4/24 patients at stage III, but not in 4 patients at stage I-II; half of the positives were obtained presurgery and half at follow-up. Correspondence between circulating DNA and related tumors were examined for 20 patients, and a correlation was found for stage IV patients. In conclusion, this method can be utilized for monitoring the disease in stage IV melanoma patients but it appears unsatisfactory for the early detection of melanoma.     

Benefit of sentinel lymphadenectomy for patients with nonulcerated cutaneous melanomas in the Breslow range between 0.76 and 1 mm: a follow-up study of 148 patients

Sentinel lymphadenectomy (SLNE) is now internationally accepted for the primary treatment of melanomas thicker than 1 mm. But it is still controversial whether also patients with nonulcerated melanomas in the Breslow range between 0.76 and 1 mm should be included. At the authors' department, 87 of such patients (Group A) underwent SLNE in combination with wide local excision (WLE) of their primary melanomas in the years 1995 to 2001. SLN micrometastases were found in 10 of these patients (11.5%). Radical completion lymph node dissections (CLND) were added in 4 of the 10 patients without revealing any further nodal metastases. All the 87 Group A patients remained free from recurrent disease at a median follow-up time of 74 months. The control Group B from the same department encompassed 61 consecutive stage Ia patients with melanomas in the identical Breslow range, who had undergone only WLE of their primaries without SLNE in the years 1987 to 1993 (median follow-up time 115 months). Five of these 61 patients (8.2%) developed melanoma metastases within 12 to 68 (median 19) months of follow-up, 3 of them initially in regional lymph nodes. Four of the 5 individuals died because of the final distant dissemination of the melanoma. Kaplan-Meier comparisons between Groups A and B with log-rank testing showed a significantly worse outcome of Group B with respect to recurrence-free survival (p = 0.01), regional nodal progression (p = 0.041), distant metastasis (p = 0.023) and melanoma-related mortality (p = 0.03). The overall survival was not significantly different, because expiries not related to melanoma predominated in both groups. Our data suggest that SLNE seems to nearly completely eliminate the risk of melanoma recurrences in patients with melanomas between 0.76 and 1 mm thick.     

Simultaneous blockade of VEGFR-1 and VEGFR-2 activation is necessary to efficiently inhibit experimental melanoma growth and metastasis formation

Metastasis continues to be the major cause of morbidity and mortality in malignant melanoma. In our study, we explored whether inhibition of VEGFR-1 or VEGFR-2 signaling conveys distinct suppressive effects on B16 melanoma subcutaneous growth and metastasis formation. The inhibition of VEGFR-1 or -2 alone had no significant influence on both melanoma growth and metastasis formation. In contrast, simultaneous blockade of VEGFR-1 and -2 signaling strongly suppressed progression in both B16 tumor models. There was no expression of VEGFR-1 or -2 detectable on the B16 cells used, excluding the melanoma cells as direct therapeutic targets. Analyzing the contribution of progenitor-like cells during melanoma metastasis formation, we observed an enhanced proliferation and mobilization of VEGFR-1+ myeloid and VEGFR-2+ endothelial cells with progenitor potential by the induction of melanoma lung metastasis, which was not influenced by interference with VEGFR signaling. These results indicate that the antimetastatic effects exerted by combined inhibition of VEGFR-1 and -2 signaling were mediated via targeting cell populations other than progenitors only. Sole inhibition of VEGFR-1 signaling led to a strong reduction of the CD45-positive inflammatory infiltrate in the tumor tissue. However, the formation of lung metastasis was not affected, indicating that inhibition of the inflammatory response was not sufficient to efficiently block B16 melanoma metastasis development. Taken together, our data suggest that in the utilized B16 tumor models the blockade of both the inflammatory and the VEGFR-2-dependent angiogenic response are necessary to effectively inhibit solid tumor growth and formation of lung metastasis by B16 melanoma cells.     

Cutaneous phenotype and MC1R variants as modifying factors for the development of melanoma in CDKN2A G101W mutation carriers from 4 countries

The G101W founder mutation is the most common CDKN2A mutation in Italy, Spain, and France. As the background of modifying genes, environmental exposures, and sun behavior vary across countries, studying G101W carriers from distinct countries offers a unique opportunity to evaluate possible modifying factors in melanoma development. We evaluated 76 G101W cases and 59 carrier controls from France, Italy, Spain, and the United States. Hair color and dysplastic nevi distributions differed significantly in cases and controls across the 4 study groups. Cases also varied significantly for eye color, freckling, and nevi. The distribution of MC1R variants in cases differed significantly across study groups because 12% of Italian melanoma patients had > or =2 MC1R variants vs. >50% for the other case groups. Several MC1R covariates showed significant associations with melanoma risk in all groups combined and in the American, French, and Spanish samples; no significant findings were observed in the Italian sample. In multiple-case families, the number and type of MC1R variants varied significantly between multiple-primary-melanoma and single-primary-melanoma patients from the 4 groups; there was also a significant decrease in median age at melanoma diagnosis as the number or type of MC1R variants increased. The variation in the effects of the cutaneous phenotypic and MC1R factors across the study sample suggests that these factors differentially contribute to development of melanoma even on a common genetic background of a germline CDKN2A mutation. Differences in melanoma risk across geographic regions justify the need for individual studies in each country before counseling should be considered.     

Identification of cytotoxic T-lymphocyte epitope(s) and its agonist epitope(s) of a novel target for vaccine therapy (PAGE4)

PAGE4 is an X chromosome-linked cancer testis antigen and is a potential new tumor-associated antigen that is overexpressed in prostate and uterine cancers. The purpose of this study was to identify a human CTL epitope and a corresponding agonist epitope of PAGE4 to determine if PAGE4 is a potential target for vaccine-mediated immunotherapy against PAGE4-expressing tumors. A class I PAGE4 epitope was identified with a high level of binding to HLA-A2. PAGE4 peptide-pulsed dendritic cells were then used to generate human PAGE4-specific T-cell lines. Further studies demonstrated the generation of an enhancer agonist epitope. Compared with the native peptide, the agonist (i) bound to HLA-A2 molecules at lower peptide concentrations, (ii) demonstrated a higher stability of the peptide HLA-A2 complex, (iii) induced higher levels of production of IFN-gamma, Granzyme B, TNF-alpha, IL-2 and lymphotactin by PAGE4-specific T-cell lines and (iv) T-cell lines generated against the agonist peptide were more efficient to lyse HLA-A2 human tumor cells expressing native PAGE4. The studies reported here are the first to describe a PAGE4 CTL epitope and its agonist epitope, and thus identify PAGE4 as a potentially useful target for vaccine-mediated therapy of prostate cancer.