Identification of astrocytoma associated genes including cell surface markers

Abstract

Background: Despite intense effort the treatment options for the invasive astrocytic tumors are still limited to surgery and radiation therapy, with chemotherapy showing little or no increase in survival. The generation of Serial Analysis of Gene Expression (SAGE) profiles is expected to aid in the identification of astrocytoma-associated genes and highly expressed cell surface genes as molecular therapeutic targets. SAGE tag counts can be easily added to public expression databases and quickly disseminated to research efforts worldwide.

Methods: We generated and analyzed the SAGE transcription profiles of 25 primary grade II, III and IV astrocytomas 1. These profiles were produced as part of the Cancer Genome Anatomy Project's SAGE Genie 2, and were used in an in silico search for candidate therapeutic targets by comparing astrocytoma to normal brain transcription. Real-time PCR and immunohistochemistry were used for the validation of selected candidate target genes in 2 independent sets of primary tumors.

Results: A restricted set of tumor-associated genes was identified for each grade that included genes not previously associated with astrocytomas (e.g. VCAM1, SMOC1, and thymidylate synthetase), with a high percentage of cell surface genes. Two genes with available antibodies, Aquaporin 1 and Topoisomerase 2A, showed protein expression consistent with transcript level predictions.

Conclusions: This survey of transcription in malignant and normal brain tissues reveals a small subset of human genes that are activated in malignant astrocytomas. In addition to providing insights into pathway biology, we have revealed and quantified expression for a significant portion of cell surface and extra-cellular astrocytoma genes.

Figure 1

Representative immunostaining of paraffin sections with antibodies against Topoisomerase 2A and Aquaporin 1. (A) Upper panel shows two TOP2A positive cores from GBMs; lower panel shows two negative cores on the same tissue micro-array. (B) Upper panel from left to right: cortex and white matter; lower panel from left to right: spinal cord and hypocampus. All stained with anti-TOP2A antibodies. (C – D) Paraffin section of GBM showing nuclear staining of TOP2A; D shows a higher magnification and E is the corresponding negative control with mouse IgG1 and no primary antibodies. (F – G) Paraffin section of GBM showing cytoplasmatic staining with anti-aquaporin 1 antibodies; G shows a higher magnification and H is the negative control.