Oligodendrogliomas with LOH 1p/19q: Identifying Genes associated with Tumourigenesis Therapeutic Sensitivity

Abstract

Loss of heterozygosity (LOH) of the short arm of chromosome 1 (1p) and the long arm of chromosome 19 (19q) is an early event in oligodendroglioma that occurs in up to 80% of patients and is associated with therapeutic sensitivity and longer overall survival. The purpose of this study was to confirm the reported association in patients at this centre, then identify and characterise genes that were differentially expressed and may function as a tumour suppressor or contribute to therapeutic sensitivity in oligodendroglioma. A clinical review of oligodendroglioma patients treated at Royal North Shore and North Shore Private Hospitals between 1990 and 2009 confirmed the association between LOH 1p/19q and longer overall survival in WHO grade III oligodendroglioma patients. Younger age and lower tumour grade were additionally confirmed as positive prognostic factors. Exon microarrays were used to identify changes in gene expression between oligodendrogliomas with and without LOH 1p/19q. Seventeen oligodendroglioma specimens (5 WHO grade II with LOH 1p/19q, 2 WHO grade II without LOH, 5 WHO grade III with LOH 1p/19q, 5 WHO grade III without LOH) were examined by microarrays. Analysis in Partek identified 408 genes that were differentially expressed by LOH status. Six candidate genes (CHI3L1, IGF2, IQGAP1, MIG-6, PDPN and PLAG1), which all showed significantly lower expression in oligodendrogliomas with LOH 1p/19q, were selected for further study. Microarray analysis of DNA methylation in seven oligodendrogliomas with LOH 1p/19q (4 WHO grade II, 3 WHO grade III) identified DNA methylation of the PADI2 and ALX3 genes on chromosome 1p. Bisulfite sequencing, restriction analysis and qPCR confirmed PADI2 as a candidate tumour suppressor gene, methylated in oligodendrogliomas with LOH 1p/19q. qPCR in forty-­six oligodendroglioma specimens confirmed under-expression of each of the seven candidate genes in oligodendrogliomas with LOH 1p/19q. Survival analysis identified high Podoplanin (PDPN) transcript expression as a negative prognostic factor. Immunohistochemistry conducted on thirty grade III oligodendrogliomas demonstrated that protein staining scores for CHI3L1 and IQGAP1 were significantly higher in tumours without LOH 1p/19q, but found that IGF2, MIG-6 or PDPN were not suitable as markers for LOH 1p/19q or survival. Functional analysis of MIG‐6 in H423 glioblastoma cells demonstrated the ability of MIG-6 to inhibit cell invasion, validating its role as a potential tumour suppressor in glioma. As no oligodendroglioma cell line with LOH 1p/19q was available, forty-six low grade tumour specimens were cultured to form primary cell lines. While nine cell lines retained an IDH1 mutation typical of oligodendroglioma, no cell line maintained LOH 1p/19q. This work has confirmed LOH 1p/19q as a positive prognostic marker for oligodendroglioma and identified seven differentially expressed genes that, through their loss following LOH 1p/19q, could contribute to the development of oligodendroglioma or confer therapeutic sensitivity to the patient.