Glioma-expressed antigen 2 (GLEA2): a novel protein that can elicit immune responses in glioblastoma patients and some controls

Abstract

Glioma constitutes the most frequent brain tumour in man with glioblastoma as the most prevalent and malignant type. The average survival time of less than 16 months underlines the need for improvements in diagnosis and therapy. Here, we report the identification of a novel antigen termed glioma-expressed antigen 2 (GLEA2) causing a frequent immune response in glioma patients. Screening of 450 000 clones from a glioblastoma lambda zap expression library with autologous patient serum revealed a group of five serum-positive clones sharing a high sequence homology. Further sequence analysis showed a sequence homology to a hepatocellular carcinoma associated antigen 58 (HCA58). We localized the novel HCA homologous gene termed glioma-expressed antigen 2 (GLEA2) on chromosome 20 by somatic cell hybrid panel mapping. Using allogenic sera from 39 glioblastoma patients, we found an immune response against GLEA2 in 17 patients (43%). In addition, screening with allogenic sera from other glioma patients revealed GLEA2 directed antibodies in two out of five pilocytic astrocytomas and in one out of two astrocytomas. Unrelated tumour sera revealed no immune response and sera from healthy persons showed an immune response in two out of 14 cases (14%). Northern blot hybridization and RT-PCR showed ubiquitous GLEA2 gene expression in glioma and normal tissues. The novel HCA homologous gene, GLEA2, appears to induce a frequent immune response in glioma. In the light of the lack of useful glioma markers, it appears reasonable to consider GLEA2 as a potential future diagnostic marker.

Fig. 1

(a) Sequence comparison between GLEA2 and the hepatocellular carcinoma associated antigen 58 (HCA58). The single nucleotide insertion is indicated. (b) Predicted partial cDNA sequence of the GLEA2 gene combining sequences GLEA2, RP5-1121G-12 and DKFZp434F0272. The resulting open reading frame is indicated.

Fig. 2

Predicted amino acid sequence of GLEA2 protein. Basic amino acids are shown in bold letters.

Fig. 3

Chromosomal mapping of GLEA2 gene on human chromosome 20 using somatic hybrid DNA. 100 ng of human genomic DNA, mouse DNA, hamster DNA and DNA from the somatic cell hybrids were amplified by PCR for 26 cycles at 58°C. The expected PCR product of 100 basepairs was detected in human genomic DNA, DNA from hybrid NA10478 containing human chromosome 20 and DNA from polychromosomal hybrids NA09927 and NA09931 both of which containing chromosome 20.

Fig. 4

Results of immunoscreening using autologous patient serum (a) and heterologous sera from patients with glioma of different grades (b1–3).Phage recombinants are plated at a density of 8000 phages per agar plate (Ø14·5 cm) and hybridized with preabsorbed patient serum. A secondary antibody detected complexes of antigens with antibodies in the serum. Positive clones were isolated and subjected to a second round of screening. The immune response against enriched GLEA2 clones is demonstrated in the lower left part of each figure. The upper right part of each figure shows the hybridization of patient serum against the total library. GBM: Glioblastoma multiforme; AA: astrocytomas; PA: pilocytic astrocytomas.

Fig. 5

GLEA2 expression in RNA from normal tissues and gliomas. (a) Human multiple tissue Northern blot for GLEA2 (upper panel) and GAPDH (lower panel). (b) Northern hybridization using a GLEA2 specific probe (upper panel) and a GAPDH probe (lower panel). (c) RT-PCR using GLEA2 gene specific primers (upper panel) and GAPDH specific primers (lower panel).