应用Ion Torrent测序技术平台对乳腺癌组织中PIK3CA基因突变区域的研究

发布时间：2018-03-23 11:07

本文选题：乳腺癌　切入点：PIK3CA　出处：《吉林大学》2017年硕士论文

【摘要】：研究目的乳腺癌是我国女性最常见的恶性肿瘤,发病率居于我国女性恶性肿瘤的首位,并且目前仍呈现逐年上升的趋势。PIK3CA基因突变是乳腺癌中最常见的基因突变之一,该以基因扩增或突变等形式激活,并通过PI3K/Akt信号通路,过度激活丝氨酸/苏氨酸蛋白激酶AKT,将促进细胞无限制地生长和繁殖,并会抑制细胞凋亡,与肿瘤的发生发展密切相关。本研究通过对281例浸润性乳腺癌以及30例乳腺良性肿瘤冰冻组织应用Ion Torrent测序平台技术检测PIK3CA基因突变情况,通过对临床和病理资料的搜集和整理,旨在研究PIK3CA基因突变与乳腺癌临床病例特征之间的关系。以期PIK3CA基因突变检测对指导临床个体化治疗有所贡献。材料与方法1.标本及资料收集:所有研究对象均获知情同意。281例原发性浸润性乳腺癌以及30例乳腺良性肿瘤的冰冻组织,取自吉林大学中日联谊医院乳腺外科2011年3月至2015年3月收治的手术患者。所取肿瘤组织均在切除后15分钟之内编号记录并放入液氮保存,并在术后石蜡切片病理证实后放入-80℃冰箱保存。所有研究对象均有完整的临床及病理资料以及免疫组化结果。2.实验方法:281例乳腺癌肿瘤组织标本提取DNA后,经过浓度及纯度初步测定合格后,根据Life Technology公司相应的标准操作流程对DNA样本进行文库构建、模板制备、模板富集之后在Ion Proton System上进行测序。并对Ion Torrent测序数据进行质控和分析。通过TUTE对突变位点进行注释。3.统计学分析:研究数据应用SPSS23.0软件进行统计学分析,组间差异性分布分析采用多个独立样本非参数检验,组间率的比较采用卡方检验。以P0.05为差异有统计学意义,P0.05为差异无统计学意义。实验结果1.在281例乳腺癌患者中共108例(突变率38.4%)患者的癌组织中检测到PIK3CA基因致病突变,其中95.6%聚集在9号、20号外显子。2.PIK3CA基因的致病突变在患者肿瘤大小、淋巴结状态、组织学分级、PR状态、HER2表达之间无明显差异(P0.05)。PIK3CA基因突变情况与ER状态存在显著相关性(χ2=6.501,p=0.011)。3.PIK3CA基因的9号、20号外显子突变在患者肿瘤大小、淋巴结状态、组织学分级、PR状态、HER2表达之间无明显差异(P0.05)。但在ER状态不同的患者中存在显著差异(χ2=7.387,p=0.007)。实验结论1、本研究入选281例浸润性乳腺癌患者肿瘤组织进行PIK3CA基因突变检测,PIK3CA基因致病突变率38.4%,出现9号、20号外显子突变例数达到全部PIK3CA基因致病突变的95.6%。20号外显子突变率明显高于9号外显子。2、PIK3CA基因突变与乳腺癌患者肿瘤大小、淋巴结病理状态、组织学分级、PR+/PR-,HER2是否表达无明显相关。但该基因突变与ER+/ER-状态存在显著相关性。3、PIK3CA基因的9号、20号外显子突变在患者肿瘤大小、淋巴结状态、组织学分级、PR状态、HER2表达之间无明显差异。但在ER状态不同的患者中存在显著差异。4、PI3K/Akt信号通路与乳腺癌发生及进展密切相关,实时监测PIK3CA基因的突变状态,并进一步明确PI3K/Akt信号通路激活状态,及时调整治疗方案,寻找精确而有效的疗效决策因子或将成为整个乳腺癌个体化治疗的重要课题之一。5、Ion Torrent测序技术具有操作简单,测序速度快、测序精度高、测序成本低等优势,尤其适用于全面筛查具有高度特异性的肿瘤基因,以更好的指导患者的治疗,提高治疗效果,适合在临床大规模应用。
[Abstract]:Objective breast cancer is the most common malignant tumors of women in our country, the incidence rate in China's first female malignancy, and still showed a trend of increasing.PIK3CA gene mutation is the most common mutation in breast cancer, to activate the gene amplification and mutation and other forms, and through the PI3K/Akt signaling pathway, excessive the activation of serine / threonine protein kinase AKT, will promote cell unrestricted growth and reproduction, and inhibition of apoptosis is closely related with the occurrence and development of tumors. This study of 281 cases of invasive breast cancer and 30 cases of benign breast tumor frozen tissue sequencing platform technology using Ion Torrent to detect the mutation of PIK3CA gene, through the to collect and collate the clinical and pathological data, to study the relationship between PIK3CA gene mutation and clinical characteristics of breast cancer. In order to detect PIK3CA gene mutation Clinical individual treatment contribute. Materials and methods 1. samples and data collection: all subjects had given informed consent.281 cases of primary invasive breast cancer and 30 cases of benign breast tumor patients with frozen tissue, from March 2011 to March 2015 were breast surgery of Jilin University in China Japan union hospital. The tumor tissues were save in resection within 15 minutes after the record number and placed in liquid nitrogen, and postoperative paraffin pathology in -80 C refrigerator. All of the subjects had complete clinical and pathological data and immunohistochemical results of.2. methods: 281 cases of breast cancer tumor tissue specimens after DNA extraction, after the preliminary determination of purity and concentration after passing, according to Life Technology's corresponding standard operating procedures for construction of DNA Library of sample preparation, template, template concentration after Ion Proton Sy Stem for sequencing. And quality control and analysis of Ion Torrent sequencing data. Through the TUTE mutation analysis of.3. statistical software data annotation: a study of application of SPSS23.0 for statistical analysis, the differences between groups of distribution analysis using multiple independent samples nonparametric test, rate comparison between groups using chi square test with P0.05. The difference was statistically significant, P0.05 was no significant difference. Results 1. in 281 cases of breast cancer patients in 108 cases (mutation rate 38.4%) mutation detection of PIK3CA gene of pathogenic in patients with cancer, 95.6% of them gathered in No. 9, exon 20 of.2.PIK3CA gene mutation in patients with tumor size, lymph node status, histological grade, PR status, no significant difference between the expression of HER2 (P0.05).PIK3CA gene mutation and ER status showed significant correlation (2=6.501, p=0.011).3.PIK3CA gene of No. 9, No. 20 The mutation of exon size in patients with tumor, lymph node status, histological grade, PR status, no significant difference between the expression of HER2 (P0.05). But there were significant differences in the different status of the ER patients (2=7.387, p=0.007). Conclusions: 1. This study selected 281 cases of invasive breast cancer tissue gland cancer detection of mutations in the PIK3CA gene, PIK3CA gene mutation rate was 38.4%, 9, 20 exon mutation cases reach all the causative mutation in the PIK3CA gene 95.6%.20 mutation in exon 9 was significantly higher than that of exon.2, PIK3CA gene mutation and tumor size in patients with breast cancer, lymph node pathology, histology PR+/PR-, grading, HER2 expression had no significant correlation. But the significant correlation between.3 gene mutation and ER+/ER-, PIK3CA gene 9, exon 20 mutation in patients with tumor size, lymph node status, histological grade, PR status, HER2 No significant difference between the expression of.4. But there were significant differences in the different status of the ER patients, PI3K/Akt signaling pathway and breast cancer incidence and progression is closely related to PIK3CA gene mutation status real-time monitoring, and further clarify the activation of the PI3K/Akt signaling pathway, adjust the treatment plan in a timely manner, to find effective and accurate decision-making factor effect or will become one of the important topics in the.5 of breast cancer treatment, Ion Torrent sequencing technology has the advantages of simple operation, fast speed and high precision sequencing, sequencing, sequencing of the advantages of low cost, especially suitable for the comprehensive screening of tumor gene has high specificity, in order to better guide the treatment of patients, improve the therapeutic effect, suitable for clinical scale application.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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