IDH Mutations Affect Cell Proliferation Of Low-grade Glioma

Glioma is the most common malignant brain tumors with high malignancy and poor prognosis. Isocitrate dehydrogenase(IDH) mutations is the most common genetic alteration in lower-grade(Grade II-III) glioma and secondary glioblastoma, and gliomas with mutated IDH1 and IDH2 have improved prognosis compared with gliomas with wild-type IDH. IDHs play critical roles in the citric acid cycle and etc. In vivo, IDH catalyse the oxidative decarboxylation of isocitrate to α-ketoglutarate(α-KG) with reduction of NADP+ to NADPH. IDH mutations could alter the metabolism of glucose, fatty acids and amino acids. However, the detail roles of mutant IDH in glioma remain unclear.In this study, we analyzed the clinical data and glioma tissues of glioma to determine the IDH mutations in glioma. And then, based on The Cancer Genome Atlas(TCGA) database of clinical data, histological type, tumor molecular markers changes in lower-grade glioma, we analyzed the relationship between IDH mutations and prognosis of glioma. The relationship between IDH mutations and cell proliferation or microvascular density(MVD) were analyzed with immunohistochemistry. In the end, we analyzed glutamine metabolism in IDH mutant tumor cells, and found a change of glutamine metabolic pathway which possibly affected cell growth. This study not only provided a reliable data for clinical diagnosis and prognosis of glioma, also got some primary information about mechanism of IDH mutation on glutamine metabolism of glial cells.1, Isocitrate dehydrogenase mutations mainly occurs in gradeⅡ, grade Ⅲ glioma and in patients with secondary glioblastoma.Use of clinical data of glioma, we found that IDH1/2 were not mutated in WHO grade I gliomas(0/21), and IDH mutations were detected in 50.3% WHO gradeⅡgliomas(70/139), 52.8% WHO grade Ⅲ gliomas(47/89), 46.7% WHO grade IV secondary glioblastomas(7/15), but 4.1%(5/121) WHO grade IV primary glioblastoma. Therefore, we conclude that IDH mutations are mainly in WHO gradeⅡ-Ⅲ glioma and secondary glioblastoma.In order to clarify the relationship between IDH mutations and prognosis in glioma, we analyzed the data from the TCGA database, the results indicated that IDH mutations and 1p/19 q co-deletion were mainly happening in oligodendrocytes gliomas,accompanying with other genetic alterations, such as CIC, FUBP1, NOTCH1 mutations. However, IDH mutations without 1p/19 q co-deletion were mainly found in astrocytoma, which usually companied with TP53 and ATRX mutations. In wild-type IDH patients mainly were astrocytomas, usually companying with NF1, EGRF amplification, and CDKN2A/B deletion. Moreover, we found that IDH mutations with 1p/19 q co-deletion with best prognosis, followed by IDH mutations without 1p/19 q co-deletion, but IDH wild-type glioma patients with poor prognosis. These results indicated IDH mutation was a good marker to determine the prognosis. Furthermore, we found that magneticresonance spectrum(MRS) still have some difficulties on IDH mutations analysis.2, IDH mutations could increase the microvessel density(MVD) in WHO grade II-III gliomas, but not the Ki-67 labeling index(Ki-67LI).Using immunohistochemistry, we found that the expression levels of IDH1(R132H) were various in IDH1 mutant glioma, which showed a positive correlation with the Ki-67 LI and MVD in Grade II-III glioma.Furthermore,we classified the immunohistochemical results by tumor grade, but Ki-67 LI were similar in same grade gloma with or without IDH mutations. we found that IDH muations could increase the MVD in both Grade II and III glioma. These results revealed that IDH mutations couldincrease the lower-grade glioma angiogenesis, but didn’t affect the proliferation.3,IDH mutations increased the glutamine addiction of glioma cells.Using established IDH1+/R132 H HCT116 cells by homologous recombination, we proved that the HCT116 cells with IDH1(R132H) were sensitive to the deficiency of glutamine and glutamate. To confirm these results,we overexpressed IDH1(WT) and IDH1(R132H) in U-87 MG and U-118 MG glioma cells, and the results indicated that high-level glutamine(2m M) didn’t significantly affect the growth of glioma cell lines with overexpressed IDH1(WT), whereas IDH1(R132H) overexpression could slightly inhibit the growth of glioma cell lines. However, low-level glutamine(0.2 m M) significantly inhibited(reduced about 80%-90%) the growth of glioma cell lines with overexpressed IDH1(R132H). These results indicated that IDH1(R132H) overexpression could cause the glioma cells more sensitiv to the shortage of glutamine.Recently, it was reportd that the growth of gliom cells was depended on the glutamine generated by the normal glia in glioma. To further verify the effect of IDH1 mutation in the growthe of glioma, we examined the effect of 2-hydroxy-glutaric acid(D-2HG) on the activity of glutamine synthetase(GS). The results showed that glutamine synthetase activity could be suppressed by D-2HG directly, and D-2HG could inhibit the generation of glutamine in glial cells. These results suggested that D-2HG coud inhibited the activity of glutamine synthetase, and affect the generation of glutamine.In this study, depended on clinical data and IDH mutants glioma, we found that IDH mutations was commonly detected in gradeⅡ-Ⅲ gliomas and secondary glioblastomas, and patients with IDH mutations have better prognosis. IDH mutactions could increase MVD in glioma, but not Ki-67 LI. Moreover, we found that D-2HG, generated by mutant IDH, could inhibit the activity of GS and decrease the generation of glutamine in glial cells, which might inhibit the growth of IDH mutatant glioma. Thesse results could help us to understand the clinical phenomenon of better prognosis in patients with IDH mutations.