The Distribution Of TERT Promoter Mutations In Gliomas And The Study Of Prognostic Stratification

Telomere is the component of chromosome ends of eukaryotic cells, with the cell division and gradually shortened. Telomerase can be activated by the telomerase reverse transcriptase (TERT) to maintain the integrity of the telomere, and provide cells with unlimited proliferation. TERT is highly expressed in glioma cells, and is closely related to the malignant degree of gliomas, whereas there is no expression of TERT in normal brain tissue. It is found that TERT promoter regions harbor two somatic mutations:C228T and C250T, and leads to TERT promoter transcriptional activity increased by 2 to 4 times. Studies found TERT promoter mutations occur frequently in the glioblastoma, and influence the prognosis. Our study aimed to clarify its distribution rule and effect on the prognosis by TERT promoter mutation detection in large sample of the Chinese population, hoping to find a prognostic molecular marker of glioma.In the first part of this study, we examined 887 glioma samples of different levels, different histopathological subtypes by whole genome DNA extraction, nested PCR amplifying particular region of TERT promoter mutation, tapping after recovery and purification of Sanger sequencing. Through comparative analysis the fragments amplified C228T and C250T mutations situation, mutation rate statistics, drawing C228T and C250T mutations in different levels, different histological subtypes of gliomas distribution of spectrum, and reveal its enrichment regularity. Results showed that:in 887 gliomas samples,274 cases were detected to contain C228T mutation, accounted for 30.89% of the total number of samples,83 cases with C250T mutation, accounted for 9.36% of the total number of samples. Only 1 case samples co-occurred with C228T and C250T mutation. In addition to the 10 cases of homozygous mutations, the rest samples were all hybrid mutation.we analyzed the distribution of TERT according to the WHO classification (â…¡,â…¢, â…£) Of gliomas. Results show that in 498 cases WHO grade II gliomas samples,197 cases were detected to contain TERT mutation, the mutation rate is 39.56%; In 139 cases WHO grade III glioma samples,56 cases were detected to contain TERT mutation, the mutation rate is 40.29%; In 250 cases of WHO level IV glioma samples,104 cases were detected with TERT mutations, mutation rate is 41.6%. The mutation rate with the increase of glioma level increased slightly. Besides, TERT promoter mutation has the highest percentage in oligodendrogliomas tumor. The lowest frequency of TERT promoter mutation (22/203,10.84%) was detected in astrocytomas, whereas the oligodendrogliomas exhibited high frequency of such alternation, accounting for 75.71%(53/70). For more detailed descriptions of the mutation in the glial cell distribution, we further analyzed the frequency of TERT mutation in primary and secondary gliomas, respectively. Results showed that 44.72%(89/199) of primary glioblastoma contains TERT promoter mutation, while only 29.41%(15/51) of the secondary glioblastoma contained this mutation. Through the above results, we found that TERT promoter mutant highly enriched in the oligodendrogliomas, mutation rate has increasing trend along with the increase of age, and C228T mutation and C250T mutations are obviously mutual exclusion. The frequency of TERT promoter mutation in Chinese population was slightly different from the foreign report, which can be considered as a prognostic molecular marker of glioma based on the Chinese people for stratification research.Currently, barriers of glioma treatment mainly based on the following aspects: invasive glioma cells limit the scope of surgical resection; drug-resistant cell clones will soon appear after chemical treatment; Tumor cells exist in a variety of carcinogenic activation of signaling pathways and the interaction of genetic mutants and so on. For these reasons, survival rate of glioma, especially for the patients with glioblastoma, has been very poor; with the media survival is generally shorter than 15 months. However, there are still about 3-5% of the patients with glioblastoma can survive for three to five years. Reports have pointed out that young female patients with glioma who carried O6-Methylguanine-DNA methyltransferase (MGMT) promoter methylation have access to a longer lifetime than other patients. Therefore, looking for characteristic diagnostic or prognostic significance of molecular markers and prognostic stratification has important guiding significance on the individualized treatment of glioma.In the second part of this study, we are aimed at TERT promoter mutant prognostic stratification in different levels, different histological of glioma, and evaluate the prognostic value of the TERT promoter mutation in Proneural, Neural, Classical, Mesenchymal molecular subtypes of TCGA classification through annotations of glioma samples by the RNA microarray data. The result showed that the survival of patients with TERT promoter mutant was obviously higher than that of TERT promoter wide types (P< 0.05), and patients harbored C250T mutation have the better clinical outcome than people with C228T mutation. In the WHO grade â…¡ gliomas, survival advantage of patients with TERT promoter mutant is more obvious. When using TCGA classification standard to note molecular subtypes of samples we found that, TERT promoter mutation in Proneural and Mesenchymal molecular subtypes revealed a significant prognostic stratification, in Classical and Neural subtype of the delamination is not obvious. Accordingly we draw the conclusion: TERT promoter mutation as a molecular marker for judging prognosis of glioma is more obvious in the TCGA molecular classification and low grade gliomas, and can be used as a stable prognostic indicator to evaluate the prognosis of patients with glioma.In recent years, the study found that the process of the occurrence and development of glioma are based on the different genetic background, one or a few characteristic of genetic changes always runs through these genetic background. And the characteristic genetic changes directly or indirectly influence the evolution and prognosis of glioma. Currently studies recognized IDH mutation is an early event of genome change in glioma, and TP53 mutations, ATRX mutations and 1P19Q LOH were run through in the process of the evolution in astrocyte tumors and oligodendrogliomas, respectively. TP53 mutation was considered as the genetic marker of process of astrocyte tumors from a low grade glioma into the secondary glioblastoma. While the EGFR amplification is the characteristic of primary glioblastoma molecular marker.In the third part of the study, we introduce the prognostic molecular biomarkers in glioma, including of IDH mutation,1P19Q LOH, TP53 mutations, PTEN mutation, EGFR amplification and MGMT promoter methylation, and further analyzed the prognostic impact of TERT mutations in glioma patients with background of these genetic alterations.The results showed that:in the environment of 1P19Q deletion and IDH1 mutation, the advantage of prognostic impact of TERT promoter was more obvious. In addition, we found that TERT promoter mutations could still be used as a good prognostic marker in the condition of absence with the PTEN mutation or EGFR amplification. PTEN mutation and EGFR amplification had inhibitory effect on the prognosis of TERT promoter mutation, and the inhibitory effect was more obvious in the TP53 mutant group.To sum up, this study used the Nest-PCR, Sanger sequencing, fully sequenced transcripts, mRNA chip data and the survival prognosis to analyze the molecular markers, including of TERT promoter mutation, TP53 mutation, IDH mutation, loss of 1P19Q and EGFR amplification in glioma, draw the TERT promoter concentration spectrum in glioma, evaluated TERT promoter mutation characteristic as a molecular marker in prognosis value. These results laid the foundation for the subsequent research in mechanism of TERT promoter mutation affecting the prognosis of glioma, and also provides a theoretical reference in looking for diagnostic and prognostic molecular markers for molecular therapy of glioma.