1p/19q (1p and/or 19q) deletions are prognostic factors in oligodendroglial tumors (OT) and predict better survival after both chemotherapy and radiotherapy. While studying 1p/19q status as a potential variable within multivariate prognosis models for OT, we have frequently encountered unknown 1p/19q status within our glioma sample database due to lack of paired blood samples for loss of heterozygosity (LOH) assay and/or failure to perform fluorescence in situ hybridization (FISH). We realized that a 1p and 19q deletion assay that could be reliably performed solely on tumor DNA samples would allow us to fill in these molecular biology data "holes". We built recombinant DNA with fragments of the selected "marker" genes in 1p (E2F2, NOTCH2), and 19q (PLAUR) and "reference" genes (ERC2, SPOCK1, and SPAG16 ) and used it as quantification standard in real-time PCR to gain absolute ratios of marker/reference gene copy numbers in tumor DNA samples, thus called comparative quantitative PCR (CQ-PCR). Using CQ-PCR, we identified 1p and/ or 19q deletions in majority of pure low-grade oligodenroglioma (OG) tumors (17/21, 81%), a large portion of anaplastic oligodendroglioma (AO) tumors (6/15, 47%), but rarely found in mixed oligoastrcytomas (OA) tumors (1/8, 13%). These data are consistent with results of LOH and FISH assays generally reported for these tumor types. In addition, 15 out 18 samples showed concordant results between FISH and CQ-PCR. We conclude that CQ-PCR is a potential means to gain 1p/19q deletion information, which prognostic and predictive values of CQ-PCR-derived 1p/19q status will be determined in a future study.

Abstract Purpose There are conflicting reports regarding the function of EFEMP1 in different cancer types. In this study, we sought to evaluate the role of EFEMP1 in malignant glioma biology. Experimental Design Real-time qRT-PCR was used to quantify EFEMP1 expression in 95 glioblastoma multiforme (GBM). Human high-grade glioma cell lines and primary cultures were engineered to express ectopic EFEMP1, a small hairpin RNA of EFEMP1, or treated with exogenous recombinant EFEMP1 protein. Following treatment, growth was assayed both in vitro and in vivo (subcutaneous (s.c.) and intracranial (i.c.) xenograft model systems). Results Cox regression revealed that EFEMP1 is a favorable prognostic marker for patients with GBM. Over-expression of EFEMP1 eliminated tumor development and suppressed angiogenesis, cell proliferation, and VEGFA expression, while the converse was true with knock-down of endogenous EFEMP1 expression. The EFEMP1 suppression of tumor onset time was nearly restored by ectopic VEGFA expression; however, overall tumor growth rate remained suppressed. This suggested that inhibition of angiogenesis was only partly responsible for EFEMP1's impact on glioma development. In glioma cells that were treated by exogenous EFEMP1 protein or over-expressed endogenous EFEMP1, the EGFR level was reduced and AKT signaling activity attenuated. Mixing of EFEMP1 protein with cells prior to s.c. and i.c. implantations or injection of the protein around the established s.c. xenografts, both significantly suppressed tumorigenicity. Conclusions Overall, our data reveals that EEFEMP1 suppresses glioma growth in vivo, both by modulating the tumor extracellular microenvironment and by altering critical intracellular oncogenic signaling pathways.

De-regulated EFEMP1 gene expression in solid tumors has been widely reported with conflicting roles. We dissected EFEMP1 to identify domains responsible for its cell context-dependent dual functions, with the goal being to construct an EFEMP1-derived tumor-suppressor protein (ETSP) that lacked tumor-promoting function. Exon/intron boundaries of EFEMP1 were used as boundaries of functional modules in constructing EFEMP1 variants, with removal of various module(s), and/or mutating an amino acid residue to convert a weak integrin binding-site into a strong one. A series of in vitro assays on cancerous features, and subcutaneous and intracranial xenograft-formation assays, were carried out for effects from overexpression of wild-type and variant forms of EFEMP1 in two glioma subpopulations characterized as tumor mass-forming cells (TMCs) or stem-like tumor initiating cells (STICs), where EFEMP1 showed cellcontext- dependent dual functions. One of the EFEMP1 variants was identified as the sought-after ETSP, which had a stronger tumor-suppression function in TMCs by targeting EGFR and angiogenesis, and a new tumor-suppression function in STICs by targeting NOTCH signaling and MMP2-mediated cell invasion. Therefore, ETSP may form the basis for further important research to develop a novel cancer therapy to treat many types of cancer by its tumor suppressor effect in the extracellular matrix compartment.

Aneuploidy with chromosome instability is a cancer hallmark. We studied chromosome 7 (Chr7) copy number variation (CNV) in gliomas and in primary cultures derived from them. We found tumor heterogeneity with cells having Chr7-CNV commonly occurs in gliomas, with a higher percentage of cells in high-grade gliomas carrying more than 2 copies of Chr7, as compared to low-grade gliomas. Interestingly, all Chr7-aneuploid cell types in the parental culture of established glioma cell lines reappeared in single-cell-derived subcultures. We then characterized the biology of three syngeneic glioma cultures dominated by different Chr7-aneuploid cell types. We found phenotypic divergence for cells following Chr7 mis-segregation, which benefited overall tumor growth in vitro and in vivo. Mathematical modeling suggested the involvement of chromosome instability and interactions among cell subpopulations in restoring the optimal equilibrium of tumor cell types. Both our experimental data and mathematical modeling demonstrated that the complexity of tumor heterogeneity could be enhanced by the existence of chromosomes with structural abnormality, in addition to their mis-segregations. Overall, our findings show, for the first time, the involvement of chromosome instability in maintaining tumor heterogeneity, which underlies the enhanced growth, persistence and treatment resistance of cancers.