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.

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.