MRP3: a molecular target for human glioblastoma multiforme immunotherapy.

<p>Abstract</p> <p>Background</p> <p>Glioblastoma multiforme (GBM) is refractory to conventional therapies. To overcome the problem of heterogeneity, more brain tumor markers are required for prognosis and targeted therapy. We have identified and validated a promising molecular therapeutic target that is expressed by GBM: human multidrug-resistance protein 3 (MRP3).</p> <p>Methods</p> <p>We investigated MRP3 by genetic and immunohistochemical (IHC) analysis of human gliomas to determine the incidence, distribution, and localization of MRP3 antigens in GBM and their potential correlation with survival. To determine MRP3 mRNA transcript and protein expression levels, we performed quantitative RT-PCR, raising MRP3-specific antibodies, and IHC analysis with biopsies of newly diagnosed GBM patients. We used univariate and multivariate analyses to assess the correlation of RNA expression and IHC of MRP3 with patient survival, with and without adjustment for age, extent of resection, and KPS.</p> <p>Results</p> <p>Real-time PCR results from 67 GBM biopsies indicated that 59/67 (88%) samples highly expressed <it>MRP3 </it>mRNA transcripts, in contrast with minimal expression in normal brain samples. Rabbit polyvalent and murine monoclonal antibodies generated against an extracellular span of MRP3 protein demonstrated reactivity with defined <it>MRP3</it>-expressing cell lines and GBM patient biopsies by Western blotting and FACS analyses, the latter establishing cell surface MRP3 protein expression. IHC evaluation of 46 GBM biopsy samples with anti-MRP3 IgG revealed MRP3 in a primarily membranous and cytoplasmic pattern in 42 (91%) of the 46 samples. Relative RNA expression was a strong predictor of survival for newly diagnosed GBM patients. Hazard of death for GBM patients with high levels of <it>MRP3 </it>RNA expression was 2.71 (95% CI: 1.54-4.80) times that of patients with low/moderate levels (p = 0.002).</p> <p>Conclusions</p> <p>Human GBMs overexpress MRP3 at both mRNA and protein levels, and elevated MRP3 mRNA levels in GBM biopsy samples correlated with a higher risk of death. These data suggest that the tumor-associated antigen MRP3 has potential use for prognosis and as a target for malignant glioma immunotherapy.</p

Induction of lethal experimental allergic encephalomyelitis in nonhuman primates and guinea pigs with human glioblastoma multiforme tissue

✓ The introduction of active specific immunotherapy as an adjunct to conventional therapy of the brain-tumor patient creates the risk of the concomitant induction of experimental allergic encephalomyelitis (EAE). The lack of resolution concerning the total group of central nervous system (CNS) antigens which may be encephalitogenic, and the lack of definition of the necessary conditions for the induction of an anti-CNS myelin response complicate the design of an immunotherapeutic regimen for brain-tumor patients. We report here the ready induction of EAE in four of four guinea pigs and both of two nonhuman primates (Macaca fascicularis) with human glioblastoma multiforme (GBM) tissue injected with either complete or incomplete Freund's adjuvant (CFA, IFA). Immunization protocols utilizing encephalitogenic GBM tissue and adjuvant which did not result in EAE induction were established in both of two macaques, and the production of significant levels of antibodies specifically reactive with immunizing GBM-derived cultured cell lines in all of 12 macaques without EAE induction was demonstrated. As the lower detection limit of the sodium dodecyl sulfate-polyacrylimide gel electrophoresis (SDS-PAGE) assay for human myelin basic protein (HBP) was 0.6 µg HBP/gel, and an extract prepared from WR-GBM tumor tissue contained less than 0.6 µg of detectable HBP/25 µg of pH 3 extractable protein, and as 100 to 1000 µg of purified human basic protein (HBP) failed to induce EAE in three of three macaques, it was hypothesized that 1) GBM tissue may act as an adjuvant and markedly lower myelin basic protein (MBP) threshold doses for EAE induction, that 2) MBP encephalitogenic fragments capable of EAE induction may be present in GBM tissue but difficult to quantitate in precipitates by in vitro methods, or that 3) secondary encephalitogenic antigens unrelated to MBP may be present in GBM tissue. The threat of EAE induction and the potential difficulty of its detection in the deteriorating brain-tumor patient receiving active specific immunotherapy warrants a biological screen in immunizing CNS material in experimental animals prior to administration to patients in immunotherapy protocols.</jats:p