Identification of Necrosis-Associated Genes in Glioblastoma by cDNA Microarray Analysis

Purpose: In the field of cancer research, there has been a paucity of interest in necrosis, whereas studies focusing on apoptosis abound. In neuro-oncology, this is particularly surprising because of the importance of necrosis as a hallmark of glioblastoma (GBM), the most malignant and most common primary brain tumor, and the fact that the degree of necrosis has been shown to be inversely related to patient survival. It is therefore of considerable interest and importance to identify genes and gene products related to necrosis formation. Experimental Design: We used a nylon cDNA microarray to analyze mRNA expression of 588 universal cellular genes in 15 surgically resected human GBM samples with varying degrees of necrosis. Gene expression was correlated with the degree of necrosis using rank correlation coefficients. The expression of identified genes was compared with their expression in tissue samples from 5 anaplastic astrocytomas (AAs). Immunostaining was used to determine whether genes showing the most positive correlation with necrosis were increasingly expressed in tumor tissues, as grade of necrosis increased. Results: The hybridization results indicated that 26 genes showed significant correlation with the amount of necrosis. All 26 genes had functions associated with either Ras, Akt, tumor necrosis factor α, nuclear factor κB, apoptosis, procoagulation, or hypoxia. Nine genes were positively correlated with necrosis grade, and 17 genes were negatively correlated with necrosis grade. There were significant differences in the median expression levels of 3 of the 26 genes between grade III necrosis GBM and anaplastic astrocytoma (AA) samples; all but 1 of the genes had elevated expression when comparing necrosis grade III with AA samples. Two factors, the ephrin type A receptor 1 and the prostaglandin E₂ receptor EP4 subtype, not previously considered in this context, were highlighted because of their particularly high (positive) correlation coefficients; immunostaining showed the products of these two genes to be localized in perinecrotic and necrotic regions and to be overexpressed in grade III GBMs, but not AAs. These two molecules also showed significant correlation with survival of GBM patients (P = 0.0034) in a combined model. Conclusions: The application of cDNA expression microarray analysis has identified specific genes and patterns of gene expression that may help elucidate the molecular basis of necrogenesis in GBM. Additional studies will be required to further investigate and confirm these findings.