胰腺实性假乳头状瘤全基因组外显子测序及临床病理研究

发布时间：2018-05-03 09:19

本文选题：胰腺 + 实性假乳头状肿瘤　；参考：《北京协和医学院》2016年博士论文

【摘要】：胰腺实性假乳头状瘤(pancreatic solid pseudopapillary neoplasm, SPN)是一种少见的具有恶性潜能的胰腺肿瘤。胰腺SPN发病率低,仅占胰腺肿瘤的1%-2%,多见于年轻女性。肿瘤起病隐匿,大部分患者无临床症状,有症状的患者表现为腹部不适,恶心,呕吐等,位于胰头的SPN偶见黄疸。由于对此肿瘤的认识不断加深和影像学技术的改进,胰腺SPN的检出率在逐渐增加。胰腺SPN临床少见,具有独特的临床表现和生物学行为,然而目前相关基础及临床研究较少。基因突变检测目前已广泛应用于肿瘤的相关研究,对于揭示肿瘤的发生发展过程起到了重要作用。新一代测序技术(next-generation sequencing technology, NGS)以高通量测序为特点,包括全基因组测序和外显子测序。外显子测序是指利用目标序列捕获技术将基因组的全部外显子区域DNA捕获后并通过NGS技术进行测序的方法。其测序过程称为“循环芯片测序法”,是对布满DNA样品的芯片重复进行基于DNA的聚合酶反应(模板变性、引物退火杂交及延伸)以及荧光序列读取。外显子测序针对性强,覆盖度深,数据准确性高,具有简便、经济、高效等优点,已经被广泛应用于肿瘤疾病的研究中。本研究对胰腺SPN新鲜组织样本提取DNA,构建DNA文库并进行高通量外显子测序,初步筛选出突变基因共94个。进一步对突变基因进行生物信息学分析和Sanger测序验证,证实CTNNB1是胰腺SPN的显著突变基因。另对12个胰腺SPN肿瘤组织进行扩大样本PCR验证,结果表明CTNNB1的突变率为93.3%(14／15)。针对CTNNB1编码的β-catenin蛋白共进行了91例石蜡组织切片的免疫组织化学染色,共获得有效染色87例,结果显示共有72例组织切片为阳性,阳性率为82.8%(72／87)。相关性分析表明β-catenin免疫组化阳性表达程度与肿瘤直径有相关性(P=0.029)。此外,本研究回顾性分析2000至2010年之间所有接受手术并具有完整临床病理资料的病例。共入组100例病人,其中84例为女性,16例为男性,中位年龄31(13-68)岁。肿瘤平均直径6.5(1.5-18)cm。共有24例患者诊断为恶性胰腺SPN。49例患者接受了淋巴结清扫,术后病理提示仅有4例患者有淋巴结转移。单因素分析表明,术前CT/MRI显示胰周淋巴结肿大是恶性胰腺SPN的危险因素(P=0.025)。远期随访结果显示,共有两名患者术后出现了肝转移,并接受了肝转移瘤切除术。两名患者分别随访24和32个月,未出现肿瘤复发。综上所述,本论文对胰腺SPN的分子机制和临床病理特征进行了一系列研究,表明CTNNB1基因突变在胰腺SPN发生发展过程中起到重要作用,说明β-catenin表达与胰腺SPN肿瘤增殖密切相关,有希望成为胰腺SPN诊断和治疗的分子靶点。此外,本研究还证明了术前影像学胰周淋巴结肿大与恶性胰腺SPN相关。长期随访结果提示,对恶性胰腺SPN患者应进行密切随访和定期复查。
[Abstract]:Pancreatic solid pseudopapillary neoplasm (SPNs) is a rare pancreatic tumor with malignant potential. The incidence of pancreatic SPN was low, accounting for only 1-2 percent of pancreatic tumors, and was more common in young women. Most of the patients had no clinical symptoms. The symptomatic patients showed abdominal discomfort, nausea, vomiting, etc. The SPN located in the head of pancreas occasionally showed jaundice. The detection rate of pancreatic SPN is gradually increasing due to the deepening understanding of the tumor and the improvement of imaging techniques. Pancreatic SPN is rare and has unique clinical manifestations and biological behaviors. However, there are few basic and clinical studies. Gene mutation detection has been widely used in tumor related research, which plays an important role in revealing the process of tumor occurrence and development. The next-generation sequencing technology is characterized by high throughput sequencing, including whole genome sequencing and exon sequencing. Exon sequencing is a method which uses target sequence capture technology to capture all exon regions of genomic DNA and sequenced by NGS technology. The sequencing process is called "cyclic chip sequencing", which repeats the microarray covered with DNA samples by DNA based polymerase reaction (template denaturation, primer annealing hybridization and extension) and fluorescence sequence reading. Exon sequencing has been widely used in the study of tumor diseases due to its strong pertinence, deep coverage, high accuracy, simple, economical and efficient. In this study, DNA library was constructed from fresh tissue samples of pancreatic SPN, and high throughput exon sequencing was carried out. A total of 94 mutant genes were screened. Further bioinformatics analysis and Sanger sequencing confirmed that CTNNB1 was a significant mutant gene of pancreatic SPN. In addition, 12 pancreatic SPN tumor tissues were tested by PCR. The results showed that the mutation rate of CTNNB1 was 93. 3% and 14 / 15%. A total of 91 cases of paraffin sections were stained by immunohistochemistry for 尾 -catenin protein encoded by CTNNB1. 87 cases were stained effectively. The results showed that 72 cases were positive, and the positive rate was 82.8%. Correlation analysis showed that the expression of 尾 -catenin was correlated with the diameter of tumor. In addition, this study retrospectively analyzed all cases with complete clinicopathologic data between 2000 and 2010. A total of 100 patients were enrolled, of whom 84 were female and 16 were male, with a median age of 31 13-68 years. The mean diameter of tumor was 6.5 ~ 1.5-18 cm. A total of 24 patients were diagnosed as malignant pancreatic SPN.49 patients underwent lymph node dissection, and only 4 patients had lymph node metastasis after operation. Univariate analysis showed that preoperative CT/MRI showed peripancreatic lymphadenopathy as a risk factor for malignant pancreatic SPN. Long-term follow-up showed that two patients had postoperative liver metastases and underwent resection of liver metastases. The two patients were followed up for 24 and 32 months, respectively, without recurrence. In conclusion, a series of studies on the molecular mechanism and clinicopathological characteristics of pancreatic SPN have been carried out in this paper, which indicate that CTNNB1 gene mutation plays an important role in the pathogenesis and development of pancreatic SPN, suggesting that 尾 -catenin expression is closely related to the proliferation of pancreatic SPN tumors. It is promising to be a molecular target for the diagnosis and treatment of pancreatic SPN. In addition, this study also demonstrated that preoperative imaging peripancreatic lymphadenopathy was associated with malignant pancreatic SPN. Long-term follow-up results suggest that patients with malignant pancreatic SPN should be closely followed-up and regularly rechecked.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.9

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