基于微流体PCR和新一代测序技术的乳腺癌基因突变检测研究

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Early breast cancer detection in patients could significantly increase their survival rate and require less severe treatment. Both germline and somatic gene mutations have been indicated to be associated with breast cancer. Highly sensitive mutation detection could be used for early detection in breast cancer for high-risk identification, disease prognosis，treatment response prediction, and monitoring therapy. We will use candidate-gene approach to investigate the germline and somatic mutaiton of 10 top candidate genes in breast cancer and evaluate a gene panel for breast cancer early diagnostics by mutataion detection. A novel microfluidic Access Array system will be applied for next generation sequencing to detect gene mutation in the exons and promoter regions of the 10 candidate genes. The tumor tissue and blood DNA from over 200 breast cancer patients and normal controls will amplified on Access Array then sequenced on Illumina GAII sequencer. The association of gene mutation level of the candidate genes with breast cancer disease risk, ER/PR/Her-2 status, lymph node metastasis, treatment response will be evaluated. In addition, the gene expression level of the 10 candidate genes in the breast cancer tumor /normal tissues will be analyzed using real-time RT-PCR and correlated with their mutation level. We will develop a critical gene mutation panel based on our results and blindly test it in 100 new clinic samples. This panel would be useful tool for breast cancer early detection, high-risk identification and therapy prediction.

基因突变是乳腺癌肿瘤发生的重要发病机制之一，本研究基于国际癌症突变数据库及乳腺癌全基因组测序的最新数据结果，筛选了10个在乳腺癌中最常见的候选靶基因，应用Access Array纳米微流体PCR芯片和新一代测序技术，将在200例以上的乳腺癌患者外周血及肿瘤组织DNA样本中分析这些靶基因的胚系突变及体细胞突变情况，研究它们与中国人群乳腺癌发病风险的关联，并分析它们与患者病理类型、ER/PR/Her-2状况、腋淋巴结转移、治疗反应等的相关性。同时检测乳腺癌肿瘤组织中这10个靶基因的基因表达水平。从多层次、多角度深入研究基因突变与乳腺癌发病机制的相关性，发现与中国人群乳腺癌相关的关键靶基因突变组合，并在100例新的临床样本中进行验证检测，探索其作为乳腺癌早期诊断及病理分型的生物标记的可能性。

基因突变是乳腺癌肿瘤发生的重要发病机制之一，本研究基于国际癌症突变数据库及乳腺癌全基因组测序的最新数据结果，筛选了10个在乳腺癌中最常见的候选靶基因(BRCA1、BRCA2、TP53、PIK3CA、GATA3、MAP3K1、PTEN、AKT1、CDH1、RB1)，应用Access Array 纳米微流体PCR 芯片和新一代测序技术，在313例乳腺癌患者肿瘤组织DNA样本中分析了这些靶基因的体细胞突变及胚系突变情况，在10个基因中发现不同频率的胚系及体细胞突变。研究它们与中国人群乳腺癌发病风险的关联，并分析它们与患者病理类型、ER/PR/Her-2 状况、腋淋巴结转移等临床特征的相关性，发现了多个临床病理指标与突变存在显著相关性。在新的乳腺癌外周血样本中检测了10个基因的突变情况。同时我们检测了乳腺癌肿瘤组织中这10个靶基因的基因表达水平，发4个基因在肿瘤与正常组织中存在显著差异。从多层次、多角度深入研究基因突变与乳腺癌发病机制的相关性，研究发现了多个基因的突变与临床相关病理信息相关，这些信息为今后为乳腺癌诊断、分型和治疗策略提供参考依据。