High-dose interferon alfa-2b does not diminish antibody response to GM2 vaccination in patients with resected melanoma: results of the Multicenter Eastern Cooperative Oncology Group Phase II Trial E2696.

PURPOSE: High-dose interferon alfa-2b (IFNalpha2b) is the only established adjuvant therapy of resectable high-risk melanoma. GM2-KLH/QS-21 (GMK) is a chemically defined vaccine that is one of the best developed of a range of vaccine candidates for melanoma. A single-institution phase III trial conducted at Memorial Hospital served as the impetus for an intergroup adjuvant E1694/S9512/C509801 trial, which recently completed enrollment of 880 patients. To build on the apparent benefit of IFNalpha2b in resectable high-risk American Joint Committee on Cancer (AJCC) stage IIB or III melanoma, this phase II study was designed to evaluate the combination of GMK and IFNalpha2b. The E2696 trial was undertaken to evaluate the toxicity and other effects of the established adjuvant high-dose IFNalpha2b regimen in relation to immune responses to GMK and to evaluate the potential clinical and immunologic effects of the combined therapies. PATIENTS AND METHODS: This trial enrolled 107 patients with resectable high- or very high-risk melanoma (AJCC stages IIB, III, and IV). RESULTS: The results demonstrate that IFNalpha2b does not significantly inhibit immunoglobulin M or G serologic responses to the vaccine and that the combination of high-dose IFNalpha2b and GMK is well tolerated in this patient population. CONCLUSION: Cox analysis of the results of the combination with IFNalpha2b show improvement in the relapse-free survival of patients with very high-risk melanoma (including those with resectable M1 disease).

Gene therapy with autologous, interleukin 2-secreting tumor cells in patients with malignant melanoma.

We vaccinated metastatic melanoma patients with irradiated, autologous melanoma cells genetically engineered to secrete interleukin 2 (IL-2) to investigate whether an anti-tumor immune response would be induced. Melanoma cell cultures were established from surgical specimens and were engineered to secrete IL-2 by infection with recombinant retrovirus. Twelve patients were vaccinated subcutaneously one, two, or three times with approximately 10(7) irradiated, autologous, IL-2-secreting tumor cells. Treatment was well tolerated, with local reactions at 11 of 24 injection sites and minor systemic symptoms of fever and headache after 6 injections. One patient developed anti-tumor DTH after the first vaccination and showed an increased response after the second vaccination. Anti-autologous tumor CTLs could be detected prevaccination in the peripheral blood of seven patients and their activity increased after vaccination in four patients. No UICC-defined clinical responses were seen, but three patients had stable disease for 7-15 months, one of whom has not yet progressed (15+ months). Thus, patient vaccination with autologous, genetically engineered tumor cells is feasible and safe. Anti-tumor DTH and CTLs can be induced in some patients with such a vaccine.

Automatic multiparameter fluorescence imaging for determining lymphocyte phenotype and activation status in melanoma tissue sections.

A system has been developed that combines multiparameter fluorescence imaging and computer vision techniques to provide automatic phenotyping of multiple cell types in a single tissue section. This system identifies both the nuclear and cytoplasmic boundary of each cell. A routine based on the watershed algorithm has been developed to segment an image of Hoechst-stained nuclei with an accuracy of greater than 85%. Deformable splines initially positioned at the nuclear boundaries are applied to images of fluorescently labelled cell-surface antigens. The splines lock onto the peak fluorescence signal surrounding the cell, providing an estimate of the cell boundary. From measurements acquired at this boundary, each cell is classified according to antigen expression. The system has been piloted in biopsies from melanoma patients participating in a clinical trial of the antibody R24. Thin tissue sections have been stained with Hoechst and three different fluorescent antibodies to antigens that permit the typing and evaluation of activity of T-cells. Changes in the infiltrates evaluated by multiparameter imaging were consistent with results obtained by immunoperoxidase analysis. The multiparameter fluorescent technique enables simultaneous determination of multiple cell subsets and can provide the spatial relationships of each cell type within the tissue.

Analysis of therapeutic and immunologic effects of R(24) anti-GD3 monoclonal antibody in 37 patients with metastatic melanoma.

BACKGROUND: Antitumor effects of antibodies against ganglioside antigens of melanoma have been reported, but neither optimal doses nor mechanisms have been established. METHODS: This Phase IB trial of the murine immunoglobulin IgG(3) monoclonal antibody R(24) against disialoganglioside GD3 was conducted with 37 patients to define better the dose-response relation and mechanism of action of R(24) in patients with metastatic melanoma. RESULTS: Dose-limiting toxicity consisted of a pulmonary capillary leak syndrome in 3 of 5 patients in the 80 mg/M(2)/day dosage tier. Serial blood and tumor biopsy samples were obtained prior to therapy and on Days 5, 9, and 22 following R(24) infusion. Tumor biopsy-infiltrating lymphocytes were enumerated in peritumoral, endotumoral, and perivascular compartments: endotumoral CD4(+) and CD8(+) T cells and HLA-DR(+) T cells increased over time on R(24) antibody. Endotumoral CD4 lymphoid infiltrate activation (DR expression) and antibody-dependent cytotoxicity were the greatest in the one patient who achieved a complete response. CONCLUSIONS: Clinical response was associated with depression in natural killer (CD56(+) and CD56(+)DR(+)) blood cells (P = 0.03) and was associated with R(24) dosage (P = 0.01). A complete response that lasted 2 years and a partial response that lasted 2 months occurred at a dose of 1 mg/M(2)/day. The limited number of clinical responses observed in this trial hampered the correlation of antitumor and immune parameters but provided a rational foundation for the future evaluation of antiganglioside antibodies.

Molecular analysis of melanoma precursor lesions.

Atypical (dysplastic) nevi are melanocytic lesions, which are precursors of melanoma as well as markers of increased melanoma risk. Although these lesions exhibit distinct clinical and histological features, their molecular features are largely unknown. To determine whether atypical, compared to benign nevi, from patients with a clinical history of malignant melanoma reveal molecular changes, we analyzed these lesions for the expression of two growth factors (basic fibroblast growth factor and transforming growth factor alpha), their receptors (fibroblast growth factor receptor-1 and epidermal growth factor receptor), and two cell adhesion molecules (MUC18 and alpha v beta 3 integrin), all of which are expressed in primary and metastatic melanomas. The results demonstrated a statistically significant correlation (P = 0.02) between increasing degrees of histological atypia and expression of epidermal growth factor receptor in the epidermal keratinocytes of atypical melanocytic lesions. Furthermore, both atypical and benign nevi revealed considerably high levels of overall gene activity in their dermal melanocytic and epidermal keratinocytic compartments. In contrast, the epidermal-dermal junction wherein melanoma evolves showed little gene activity, suggesting that molecular events occurring adjacent to this junction may be important for melanocytic transformation.