The Association Of Rs1063478 Polymorphism And Glioma Susceptibility And The Association Between MTOR Pathway And And Prognosis In High-grade Gliomas

Part I Association of HLA-DMA rs1063478 Polymorphism and Gene-Environment Interactions with GliomaBackgroundsGlioma is one of the most common brain malignant tumors, surgery treatment is the main treatment of glioma, radiotherapy and chemotherapy only as adjuvant therapy. As characterized by the diffuse invasion of distant brain tissue and the special nature of glioma grow area, the prognosis remains poor, with high mortality rate and high recurrence. There is no doubt that exploration about susceptibility and pathogenesis of glioma contributes to the effective prevention and finding of new targeted treatment, thus improve the prognosis of glioma patients. HLA-DMA is closely connected with human immune system. Many cancers are caused by the drawback of human immune surveillance. Owing to these reasons, HLA-DMA gene is connected with the incidence of many cancers. These results suggest that HLA-DMA gene mutation and polymorphism may be associated with the occurrence and development of glioma, however, research in this area is still very little at present. High doses of ionizing radiation is the only proven environmental risk factors of glioma, but only a few population exposure to ionizing radiation eventually develop glioma. What is the relationship between environmental factors and risk of glioma? How environmental factors induced glioma? All the mechanisms remain to be further explored. The traditional view as follows, gene-environment interactions contributed to mutations, and then promoting a multi-stage carcinogenesis. Studies have shown that gene-environment interactions may be related to glioma, but there is no research focused on HLA-DMA gene and environment interactions on glioma. In order to detect the correlation between the interactions and glioma susceptibility among Chinese Han population, we explore the interaction of HLA-DMA rs1063478 polymorphism and environmental factors on glioma, with method of case-only study.Objective1. To explore environmental factors associated with the risk of glioma.2. To detect HLA-DMA rs1063478 polymorphism genotype in case and control groups.3. To explore the relationship of HLA-DMA gene polymorphism with the glioma susceptibility.4. To investigate HLA-DMA gene polymorphism-environment interactions with the glioma susceptibility.Methods1. The subjects were interviewed face to face with standardized structured questionnaire containing basic demographic data,Chi-square test was used for analyzing the features of the participants to explore environmental exposure factors associated with the risk of glioma.2. HLA-DMA rs 1063478 polymorphism was tested by the method of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 90 glioma patients and 110 healthy controls.3. Distribution consistency of genotypes and alleles of the two groups of patients was determined by odds ratio (OR) and 95% confidence interval (CI), logistic regression analysis was used to explore the relationship of HLA-DMA gene polymorphism with the glioma susceptibility.4. To investigate HLA-DMA gene polymorphism-environment interactions with the glioma susceptibility by multivariate regression analysis..Results1. Analysis of Basic information associated with the risk of glioma 90 patients diagnosed as glioma and 110 healthy individuals were interviewed face to face with standardized structured questionnaire containing basic demographic data, smoking and drinking status, family history of glioma, the exposure to chemical toxins and ionizing radiation and etc. Chi-square test was used for analyzing the basic information, Factors, such as age, gender, histories of smoking and drinking had no significant differences among the two groups (p>0.05).10 patients with glioma family history accounted for 11.1% in cases, which had apparent higher proportion than controls (0.9%), and the difference had statistical significance (p=0.002). Compared with the controls (0.9%), there were 9 cases (10.0%) with contact history of chemical toxins, that was possessed with statistical significance (p=0.003). Owing to radiation therapy in hospital (4) and professional exposure (9), there were 13 patients with ionizing radiation in case group. The proportion of people suffered from ionizing radiation showed significant difference in the two groups (cases 14.4% vs. controls 1.8%; p=0.001). The result showed that glioma family history, chemical toxins and ionizing radiation were significantly associated with glioma risk.2. HLA-DMA rs1063478 Genotype and allele distributions in case and control groups HLA-DMA rs 1063478 polymorphism was tested by the method of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 90 glioma patients and 110 healthy controls. Three kinds of C/T polymorphism genotypes were detected, CC genotype, CT genotype, TT genotype respectively. In case groups,40 CC genotype,38 CC genotype,12 TT genotype,50 CT+TT genotype respectively, but in control groups,68 CC genotype,36 CC genotype,6 TT genotype,42 CT+TT genotype respectively. Genotype and allele distributions in case and control groups were according with HWE.3. The relationship of HLA-DMA gene rs 1063478 polymorphism with glioma susceptibility. Through univariate analysis, TT homozygous genotype frequency in case group was higher than that in control group, with 3.40 times in terms of increasing the risk of glioma. Heterozygous genotype CT had no significant association with glioma risk. CT+TT genotype had more glioma risk than CC genotype(OR= 2.204,95% CI=1.149-3.556). After adjusted with environment factors, CT+TT genotype still increased the risk of glioma(OR= 1.984,95% CI= 1.084-3.630).4. Correlation between HLA-DMA gene polymorphism-environment interactions and the glioma susceptibility There are 2 cases showed homozygous CC genotype and 7 patients showed CT+TT genotype in 9 cases of glioma patients with chemical toxins exposure. To use Chi-square test, we find that although chemical toxin was signicantly associated with glioma risk, the interactions between rs 1063478 polymorphism and chemical toxin had no statistical significance (p=0.289, OR=3.093,95% CI=0.065-15.801). There are 2 cases showed homozygous CC genotype and 11 patients showed CT+TT genotype in 13 cases of glioma patients with ionizing radiation exposure. Through Chi-square test, we find that the interactions between rs 1063478 polymorphism and the interactions between rs 1063478 polymorphism and ionizing radiation could make glioma risk rise(p=0.033, OR=5.359,95% CI=1.13-25.797).ConclusionsHLA-DMA rs 1063478 polymorphism is related to glioma susceptibility. Besides, it can make the susceptibility of glioma rise along with ionizing radiation.Part II Prognostic Significance of mTOR Pathway and Ki67 in High-grade Glioma PatientsBackgroundGlioma is the most common adult primary brain tumors. According to the WHO classification, which is based on morphological criteria, gliomas are divided into four grades, and high-grade gliomas comprise all gliomas with grade ? and grade IV.High-grade glioma (HGG) are optimally treated with maximum safe surgery, followed by radiotherapy (RT) and/or systemic chemotherapy (CT). Although the comprehensive treatment has been given,The prognosis of high-grade gliomas is still poor, its survival period is 14 weeks to 2 years, and the average survival time is 1 year reported in the literatures. It is the most important means to predict prognosis of the patients according to high grade glioma pathology classification, which has beenaccepted widely for the majority of neurosurgeon.The WHO classification reflect the malignancy of the tumor and serves as a criterion to estimate the prognosis of patients. So far, histological evaluation remains the gold standard for glioma diagnosis and for grading the tumors. Clinical experiences derived from the prospective randomized clinical trials have shown that the histomorphological criteria alone might not be sufficient to predict the clinical outcome, part because of the subjegtivity of The WHO classification. Different pathologist may be arrive at different diagnosis from the same specimens, And specimens from tumor samples may not reflect the real situation due to the heterogeneity of gliomas.Integrated the above two factors, deviation often exist when pathological diagnosis resulted from histological performance, thus affecting the evaluation of patients prognosis. Rovei et al think that HGG prognosis is often determined by their important biological marker, so it is necessary to study objective indicators based on molecular biology level for determining glioma grade and clinical prognosis. In recently years, increasingly importance has been attached to the association of mTOR pathway and growth and development about tumors. There is evidence that mTOR signaling pathway is significantly correlated with grade of tumors, and glioma pathological grade is significantly correlated with its prognosis, so we have reason to believe that the target protein expression level may be associated with HGG prognosis. Research concern it is very little currently, and it is the key elements of our study. Ki-67 is one of the proliferative markers strongly linked to cell cycle control, so Ki-67 can be used as an indicator reflecting cell proliferation and cell cycle status. Ki-67 has proven to be a immunohistochemica prognosis biomarkers of various tumors, such as breast cancer, stomach cancer, et al. Studies have shown that Ki-67 was an ideal marker to evaluate glioma cell proliferation, it maf reflect the degree of malignancy. Whether ki-67 is an independent HGG prognosis factor, this study attempts to answer this question.Up to now, few studies combined with Ki-67 and mTOR signaling pathway on the prognosis of HGG patients.This study was conducted immunohistochemical analysis of p-AKT, p-mTOR, p-p70S6K, and Ki-67 of 60 HGG cases, and then explore its prognostic sigenificance.Objective1. To detect the protein expression of p-AKT, p-mTOR, p-p70S6K, and Ki-67 in different HGG glioma patients.2. To investigate the effect of mTOR signaling pathway proteins on HGG prognosis.3. To explore the effect of Ki-67 expression on different HGG prognosis.Methods1. To record age, gender, preoperative kamofsky Pedonnance Status, PFS (progression free survival), and survival time.2. For each case, Formalin-fixed paraffin-embedded samples were used.a representative block containing adequate tumor cells tissue was selected. Sections were cut. Sample Slice received immunohistochemistry staining about p-AKT,p-mTOR,p-p70S6K and Ki-67.expression level of p-AKT, p-mTOR, p-p70S6K and Ki-67, to determining the expression level of p-AKT, p-mTOR, p-p70S6K, and the Ki-67 according to staining intensity and percentage of positive cells.3. Kaplan-Meier estimates were used to analyze 12 prognostic factors of 60 cases of high-grade gliomas, to study the effects of these factors for HGG prognosis.4. Multivariate COX regression analysis was applied for those factors with p<0.05 in single factor analysis, to further explore the correlation between the relevant factors and HGG prognosis.Results1. Immunohistochemical staining showed a moderately positive in patients with WHO grade ?, thus immunohistochemical staining showed a strong positive in patient with WHO grade ?.79.2%(19/24) of grade ? were classified AKT positive, and 75.0%(27/36) of grade IV.75.0%(18/24) of grade ? classified mTOR positive, and 72.2%(26/36) of grade ?. for p70S6K and Ki-67,70.8%(17/24) of grade ? were classified as positive,respectively,then 80.5%(29/36) of grade IVwere classified as positive.2. When Kaplan-Meier estimates were used to analyze 12 prognostic factors, we found high level expressions of p-AKT, p-mTOR, p-p70S6K, Ki-67 were significant correlation with overall survival(OS)(p<0.05).3. When Multivariate COX regression analysis was applied for those factors with p< 0.05 in single factor analysis, Both p-AKT (low, high) and Ki-67 (low, high) had no correlation with survival (p> 0.05). But p-mTOR and p-p70S6K were significantly correlated with survival.4. p-mTOR positive rate was negatively correlated with progression-free survival (r=-0.838, p<0.001), and also with overall surviva (r=-0.777, p<0.001). p-p70s6k positive rate was negatively correlated with progression-free survival (r=-0.509, p<0.001), and also with overall survival(r=-0.531, p<0.001).5. Relationship of p-mTOR,p-p70S6K expression with progression-free survival and OS was made into Kaplan-Meier survival curves, differences of PFS and OS were statistically significant in two groups patients(p<0.05).Conclusion1. High level expression of p-mTOR and p-p70S6K in mTOR signaling pathway are closely correlated with prognosis in patients with high-grade gliomas.2. No significant correlation between Ki-67 expression and high-grade glioma prognosis.