Glycoprotein Nonmetastatic Melanoma Protein B, a Potential Molecular Therapeutic Target in Patients with Glioblastoma Multiforme

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Abstract
Purpose: More brain tumor markers are required for prognosis and targeted therapy. We have identified and validated promising molecular therapeutic glioblastoma multiforme (GBM) targets: human transmembrane glycoprotein nonmetastatic melanoma protein B (GPNMBwt) and a splice variant form (GPNMBsv, a 12-amino-acid in-frame insertion in the extracellular domain). Experimental Design: We have done genetic and immunohistochemical evaluation of human GBM to determine incidence, distribution, and pattern of localization of GPNMB antigens in brain tumors as well as survival analyses. Results: Quantitative real-time PCR on 50 newly diagnosed GBM patient tumor samples indicated that 35 of 50 GBMs (70%) were positive for GPNMBwt+sv transcripts and 15 of 50 GBMs (30%) were positive for GPNMBsv transcripts. Normal brain samples expressed little or no GPNMB mRNA. We have isolated and characterized an anti-GPNMB polyclonal rabbit antiserum (2640) and two IgG2b monoclonal antibodies (mAb; G11 and U2). The binding affinity constants of the mAbs ranged from 0.27 × 108 to 9.6 × 108 M−1 measured by surface plasmon resonance with immobilized GPNMB, or 1.7 to 2.1 × 108 M−1 by Scatchard analyses with cell-expressed GPNMB. Immunohistochemical analysis detected GPNMB in a membranous and cytoplasmic pattern in 52 of 79 GBMs (66%), with focal perivascular reactivity in ∼27%. Quantitative flow cytometric analysis revealed GPNMB cell surface molecular density of 1.1 × 104 to 7.8 × 104 molecules per cell, levels sufficient for mAb targeting. Increased GPNMB mRNA levels correlated with elevated GPNMB protein expression in GBM biopsy samples. Univariate and multivariate analyses correlated expression of GPNMB with survival of 39 GBM patients using RNA expression and immunohistochemical data, establishing that patients with relatively high mRNA GPNMB transcript levels (wt+sv and wt), >3-fold over normal brain, as well as positive immunohistochemistry, have a significantly higher risk of death (hazard ratios, 3.0, 2.2, and 2.8, respectively). Conclusions: Increased mRNA and protein levels in GBM patient biopsy samples correlated with higher survival risk; as a detectable surface membrane protein in glioma cells, the data indicate that GPNMB is a potentially useful tumor-associated antigen and prognostic predictor for therapeutic approaches with malignant gliomas or any malignant tumor that expresses GPNMB.
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