Abstract
Background
Circulating tumor DNA has emerging clinical applications in several cancers; however, previous studies have shown low sensitivity in glioma. We investigated if 3 key glioma gene ...mutations IDH1, TERTp, and EGFRvIII could be reliably detected in plasma by droplet digital polymerase chain reaction (ddPCR) thereby demonstrating the potential of this technique for glioma liquid biopsy.
Methods
We analyzed 110 glioma patients from our biobank with a total of 359 plasma samples (median 4 samples per patient). DNA was isolated from plasma and analyzed for IDH1, TERTp, and EGFRvIII mutations using ddPCR.
Results
Total cfDNA was significantly associated with tumor grade, tumor volume, and both overall and progression-free survival for all gliomas as well as the grade 4 glioblastoma subgroup, but was not reliably associated with changes in tumor volume/progression during the patients’ postoperative time course. IDH1 mutation was detected with 84% overall sensitivity across all plasma samples and 77% in the preoperative samples alone; however, IDH1 mutation plasma levels were not associated with tumor progression or survival. IDH1m plasma levels were not associated with pre- or postsurgery progression or survival. The TERTp C228T mutation was detected in the plasma ctDNA in 88% but the C250T variant in only 49% of samples. The EGFRvIII mutation was detected in plasma in 5 out of 7 patients (71%) with tissue EGFRvIII mutations in tumor tissue.
Conclusions
Plasma ctDNA mutations detected with ddPCR provide excellent diagnostic sensitivity for IDH1, TERTp-C228T, and EGFRvIII mutations in glioma patients. Total cfDNA may also assist with prognostic information. Further studies are needed to validate these findings and the clinical role of ctDNA in glioma.
INTRODUCTION: Glioblastoma multiforme (GBM) cells release glutamate through expression of system x
c
−
, which exchanges intracellular glutamate for extracellular cysteine. Lack of glutamate uptake ...through low excitatory amino acid transporter 2 (EAAT2) expression permits high extracellular glutamate levels, causing damaging excitotoxicty to surrounding parenchyma and epileptogenesis at the peritumoural edge. PPARγ agonists upregulate EAAT2 expression and prevent glutamate mediated excitotoxicity in stroke models. We investigated PPARγ agonists in GBM glutamate regulation. METHOD: Glioma cell lines U87MG and U251, and Glioma stem cells (GSCs) isolated from freshly resected tumour specimens were exposed to increasing concentrations of Pioglitazone. Effects on cell viability, protein expression and extracellular glutamate levels were evaluated with an ATP assay, Western Blotting and HPLC respectively. RESULTS: EAAT2 expression was upregulated in a dose and time dependent manner in both U87MG and U251 cells. Glutamate levels were reduced with the addition of pioglitazone, where statistical significance was reached in U251 cells at a concentration of 30 µM pioglitazone (n = 3, p < 0.05). Pioglitazone significantly reduced the cell viability of U87MG and U251 cells at concentrations of ≥ 30 µM (n = 4, p < 0.05) and 100 µM (n = 6, p < 0.05) respectively. Photomicrographs revealed morphological changes in the glioma cell lines and inability of the GSCs to form neurospheres with increasing pioglitazone concentrations. CONCLUSION: This study provides preliminary evidence of PPARγ-mediated regulation of EAAT2 expression in human glioblastoma cells and suggests a novel treatment strategy for glioma associated seizures.
INTRODUCTION: Glioblastoma multiforme (GBM) cells release glutamate through expression of system xc-, which exchanges intracellular glutamate for extracellular cysteine. Lack of glutamate uptake ...through low excitatory amino acid transporter 2 (EAAT2) expression permits high extracellular glutamate levels, causing damaging excitotoxicty to surrounding parenchyma and epileptogenesis at the peritumoural edge. PPAR gamma agonists upregulate EAAT2 expression and prevent glutamate mediated excitotoxicity in stroke models. We investigated PPAR gamma agonists in GBM glutamate regulation. METHOD: Glioma cell lines U87MG and U251, and Glioma stem cells (GSCs) isolated from freshly resected tumour specimens were exposed to increasing concentrations of Pioglitazone. Effects on cell viability, protein expression and extracellular glutamate levels were evaluated with an ATP assay, Western Blotting and HPLC respectively. RESULTS: EAAT2 expression was upregulated in a dose and time dependent manner in both U87MG and U251 cells. Glutamate levels were reduced with the addition of pioglitazone, where statistical significance was reached in U251 cells at a concentration of 30 mu M pioglitazone (n = 3, p < 0.05). Pioglitazone significantly reduced the cell viability of U87MG and U251 cells at concentrations of greater than or equal to 30 mu M (n = 4, p < 0.05) and 100 mu M (n = 6, p < 0.05) respectively. Photomicrographs revealed morphological changes in the glioma cell lines and inability of the GSCs to form neurospheres with increasing pioglitazone concentrations. CONCLUSION: This study provides preliminary evidence of PPAR gamma -mediated regulation of EAAT2 expression in human glioblastoma cells and suggests a novel treatment strategy for glioma associated seizures.
PDF
