Combining gene expression signatures and autoantibody profiles in human meningioma.

Gene expression profiling has emerged as powerful technique for studying the mechanisms of tumor genesis and development. Seroreactivity profiling of tumor antigens is a more recent technique that further contributes to the understanding of tumors and that offers itself for noninvasive tumor diagnosis. We performed expression profiling of 55,000 transcripts and expressed-sequence-tags for 24 meningiomas and related these data to autoantibody profiles of more than 50 antigens immunogenic in the autologous patients. The expression values of antigens in WHO grade I meningioma were significantly higher if the patients' sera reacted with these antigens as confirmed by a two-tailed Wilcoxon-Mann-Whitney test. Specifically, KIAA1344 that was previously identified as frequent antigen marker in meningioma, showed increased expression if antigens against KIAA1344 were detected in autologous patients. Our study is the first to combine genome-wide expression signatures and comprehensive seroreactivity patterns toward a more complete view on tumor immunology, especially concerning the overall role of the level of gene expression on the immunogenicity of meningioma antigens.

Altered expression of beta-catenin/E-cadherin in meningiomas.

AIMS: Meningiomas are generally slow-growing benign tumours representing approximately 20% of all primary intracranial tumours. The hallmark of tumorigenesis of meningiomas is the loss of chromosome 22, including loss of heterozygosity of the neurofibromatosis type 2 (NF2) gene. The NF2 encoded protein merlin appears to function as a tumour suppressor gene by controlling cadherin-mediated cell-cell adhesion. The E-cadherin cell adhesion system includes beta-catenin that indirectly connects cadherin to actin filaments. The aim of this study was to analyse the expression and the subcellular location of E-cadherin and beta-catenin in human meningiomas, including meningiomas of different histomorphological subtypes and different World Health Organization (WHO) grades. METHODS AND RESULTS: Immunohistochemical analysis revealed lack of E-cadherin expression at the cell membrane in 34% of meningiomas independent of their WHO grade. Loss of membranous beta-catenin occurred in 79% of meningiomas. An intense perinuclear granular immunoreactivity of beta-catenin without nuclear location was detected in the majority of meningiomas. Both immunofluorescence and Western blot analysis of fractionated meningioma cells located beta-catenin mostly on the Golgi apparatus and ER/Golgi intermediate compartment (ERGIC). Cytogenetic analysis of meningiomas showed no correlation between NF2 loss and the loss of the proper location of beta-catenin. CONCLUSIONS: The lack of membranous beta-catenin and/or membranous E-cadherin in meningiomas may indicate an altered interaction between meningioma cells independent of loss of NF2 and independent of the tumour grade.

The MGEA6 multigene family has an active locus on 14q and at least nine pseudogenes on different chromosomes.

The meningioma expressed antigen-6 (MGEA6) was originally identified as an immunogenic antigen in meningioma patients. Somatic hybrid panel mapping and fluorescence in situ hybridization revealed MGEA6-related sequences on different human chromosomes. Here we carry out database analysis to investigate the complexity of the MGEA6-related sequences and demonstrate the existence of a multigene family. We localized the active gene (spanning over 83 kb) to chromosome 14q and elucidated its exon/intron structure. We identified and characterized 9 processed pseudogenes on 9 different chromosomes including chromosomes 2, 3, 6, 7, 9, 10, 12, 13, and 18. We performed phylogenetic analysis and concluded that the MGEA6 pseudogenes may result from more than one retrotransposition event; we calculated divergence times of the pseudogenes to be between 21.5 and 28.9 million years ago.

Identification of a nuclear variant of MGEA5, a cytoplasmic hyaluronidase and a beta-N-acetylglucosaminidase.

MGEA5 was originally identified to be a novel human hyaluronidase, which is immunogenic in meningioma patients. Recently an N-acetylglucosaminidase was reported with identical sequence. Here, we define the origin of a splice variant by determining the genomic organization of the mgea5 gene. We find the splice variant missing a putative acetyltransferase domain of MGEA5. As for evolutionary analysis, we show that the MGEA5 is highly conserved in higher eukaryotes. As for expression analysis, we find both mRNA variants ubiquitously expressed in various human tissues and throughout mouse development. We generated polyclonal antibodies against MGEA5s/5 and identified proteins of 75 and 130 kDa, indicating posttranslational modifications of the larger protein. Cell fractionation revealed the cytoplasmic/cytoskeletal localization of the 130-kDa protein and the nuclear localization of the 75-kDa protein. We propose a model in which MGEA5 functions both as a hyaluronidase and an N-acetylglucosaminidase.

Novel tankyrase-related gene detected with meningioma-specific sera.

In many meningiomas, alterations of chromosome 22 can be found, and the NF2 (neurofibromatosis type 2) gene, in particular, is of great interest as a putative gene involved in meningioma. Because the NF2 gene is not mutated in all meningiomas, additional genes may be involved. Instead of looking for alterations directly at the DNA level, we used the immune response of meningioma patients to identify immunogenic antigens that may be associated with the disease. We screened a fetal brain cDNA expression library with sera pools from different patients bearing meningioma classified according to the three WHO grades, using the serological identification of antigens by recombinant expression cloning immunological screening method. Here, we report the finding of a new tankyrase-related protein. We found 16 overlapping clones with homologies to tankyrase when we screened the library with the common-type meningioma sera pool and 2 such clones when we screened the library with the atypical meningioma sera. The anaplastic meningioma sera did not identify any tankyrase-related clones. We tested some of the newly identified clones with 13 single sera, 6 of which (37.5%) reacted positively with the tankyrase-related clones. In addition, we screened the tankyrase-related clone with six sera pools from individuals without obvious disease. Although 1 of 24 (4.2%) normal sera reacted with the tankyrase-related clone, we found a striking difference in the frequency of reactivity to this clone by sera from patients bearing tumors corresponding to the three WHO meningioma grades; common-type sera was the most frequently reactive. Northern blot analysis demonstrates expression of the novel tankyrase gene in two common-type meningiomas from patients with immune response.

Probing the human natural autoantibody repertoire using an immunoscreening approach.

While an increasing number of studies report the presence of antibodies capable of recognizing self-antigens, the function of these natural autoantibodies remains elusive. A variety of concepts has been advanced ranging from evolutionarily tolerated but non-functional natural autoantibodies to autoantibodies effecting various biological functions. Known IgM, IgG, and IgA natural autoantibodies are directed against various antigens, including nuclear and cell surface proteins. To explore further autoantibodies and their autoantigens, we employed an immunological screening method called SEREX recently used to characterize tumour-expressed antigens eliciting an immune response in patients [1]. Sera from 12 individuals were used to screen a cDNA expression library prepared from a cytogenetically normal meningioma to identify antigens reactive with normal human sera from individuals without obvious disease. Nineteen reactive normal antigen clones were identified representing 15 different antigens, including nine genes with known functions, five genes with unknown functions, and one gene with a novel sequence not present in the databases. Of the 12 individual normal sera tested, 75% were reactive to one or more of the 15 different antigens with two highly reactive sera demonstrating reactivity with 33% of the antigens. When screening the same meningioma expression library with serum from the patient, eight antigens were identified that were totally different from those identified using sera from normal individuals. This SEREX immunological screening method presents a new option for probing the natural autoantibody repertoire and identifying normal antigens whose functions may provide additional insights into how natural autoantibodies effect various biological functions.

Five novel immunogenic antigens in meningioma: cloning, expression analysis, and chromosomal mapping.

Tumorigenesis of meningioma has been associated with chromosome 22, most notably the NF2 gene, but additional genes have been implicated in meningioma development. Here, we report the identification of five novel immunogenic antigens expressed in meningioma. An expression library was generated from a meningioma that retained both copies of chromosome 22. Screening with autologous patient serum identified seven cDNA clones that were indicated by antigen-antibody complexes. The clones were sequenced, and sequence comparison revealed that the seven clones represent five different genes, providing evidence that meningiomas express a spectrum of immunoreactive antigens, which were termed meningioma expressed antigens (MGEAs). One gene was identical with the connective tissue growth factor, one gene was in part homologous to an Alzheimer disease-associated gene, and a third gene was in part identical to Homo sapiens molybdenum cofactor biosynthesis proteins A and C mRNA. One gene was partially homologous to previously reported cDNA sequences of unknown function, and the fifth gene showed no significant homologies to sequences deposited in databases. Using somatic hybrid mapping, three genes were localized on chromosome 6, and two genes were localized on chromosomes 3 and 17, respectively. To distinguish the MGEAs from the so-called natural autoantigenes, we also screened the library with 12 sera from individuals without obvious disease. The clones identified by reactivity with normal sera were completely different from the clones identified by screening the same meningioma expression library with serum from the patient bearing the tumor. These data suggest that the newly identified MGEA genes may be useful for diagnosis and possibly therapy of meningioma.

Novel immunogenic antigen homologous to hyaluronidase in meningioma.

By screening a meningioma expression library with autologous serum we identified four cDNA clones representing a novel gene with striking homology to Caenorhabditis elegans hyaluronidase as indicated by BLASTP analysis. In humans hyaluronidase has been implicated in cancer development and three human genes are known to encode proteins with hyaluronidase activity. None of the human genes, however, showed any homology at the nucleotide or amino acid sequence level to the newly isolated antigen we termed meningioma expressed antigen 5 (MGEA5). Somatic cell hybrid mapping and fluorescence in situ hybridization mapped the gene for MGEA5 to chromosomal band 10q24.1-q24.3. Reverse transcription (RT)-PCR and northern blot hybridization revealed expression of the gene encoding MGEA5 in several meningioma and additional human tissues. Expression analysis also indicated an alternative splicing event giving rise to a shorter and altered transcript termed MGEA5s. The expression of MGEA5 and MGEA5s as fusion proteins revealed an approximate molecular weight of 92 and 54 kDa, respectively. Using heterologous sera we found antibodies against MGEA5s in five out of 23 meningioma patients, whereas no immune reaction was detected in 12 control sera from healthy individuals. Confirmation of hyaluronidase activity was independently achieved by turbidometric analysis and a gel matrix assay. A model for involvement of the novel hyaluronidase gene in meningioma development is proposed.