RASSF1A检测在子宫内膜腺癌诊断中的应用研究
发布时间：2018-08-19 20:45
【摘要】：目的： 子宫内膜癌(Endometrial carcinoma, EC)是最常见的妇科恶性肿瘤,在世界范围内子宫内膜癌的发病率和死亡率均有上升趋势,且发病年龄呈现年轻化。由于目前尚缺乏筛查早期子宫内膜癌的有效方法,子宫内膜癌仍然严重威胁着全世界妇女的身体健康。因此有必要通过各种研究方法和技术手段,探索子宫内膜癌的发病原因和发病机制,能够帮助我们在疾病的早期阶段发现和诊断子宫内膜癌,并能够进行及时的干预以达到治疗的目的。Ras相关区域家族1A (RASSFIA)基因作为抑癌基因,其启动子异常甲基化造成的基因表达降低或缺失,提示RASSF1A基因与多种肿瘤的发生发展关系密切。因此,研究RASSF1A基因启动子区域甲基化和RASSF1A蛋白的表达与子宫内膜癌发生发展的关系,对子宫内膜腺癌的分子病理诊断和个体化治疗研究具有重要意义。 方法： 我们收集2002年1月至2009年12月期间在武汉大学中南医院诊断的子宫内膜癌的患者63例,患者年龄35～71岁。所有患者首次治疗均为经手术治疗；术前均未进行放疗和／或化疗；术后经病理诊断为原发性子宫内膜癌,排除转移癌。子宫内膜腺癌根据2009年国际妇产科联盟(FIGO)制定的手术病理分期标准进行分期,Ⅰ期28例,Ⅱ期23例,Ⅲ期8例,Ⅳ期4例；根据1998年FIGO制定的子宫内膜样癌组织学分级法分类,高分化G1级21例,中分化G2级29例,低分化G3级13例。子宫内膜腺癌肌层浸润≤1/2者21例,肌层浸润1/2者42例,有淋巴转移者11例,无淋巴转移者52例;于此同时,选择相同时期进行子宫内膜诊刮术患者的正常增生期子宫内膜20例作为阴性对照组,患者术前均无异常子宫出血,6个月内未使用激素治疗。根据国际妇科病理协会(ISGP)1988年提出的子宫内膜癌肿瘤组织学新分类,收集其他类型子宫内膜组织石蜡包埋样本包括简单性增生24例、复杂性增生25例、复杂性增生伴异性增生(癌前病变)36例。 我们提取石蜡切片组织中DNA,使用重亚硫酸盐修饰DNA序列,再利用特异性甲基化PCR方法检测不同子宫内膜组织RASSF1A基因启动子区的甲基化状态。另外,我们还使用量子点标记的链霉亲和素系统检测不同子宫内膜组织RASSF1A基因的表达情况。 为了检测子宫内膜腺癌患者外周血中的RASSF1A的甲基化状态,我们建立了无创性的突变特异性荧光检测体系,选择表皮生长因子受体EGFR19号外显子的缺失突变作为目标基因,我们利用探针扩增阻滞突变系统(amplification refractorymutation system, ARMS)的相关原理,设计EGFR19外显子的缺失突变特异性引物,结合Taqman探针,构建EGFR19外显子缺失突变特异性荧光检测体系。可以帮助判断子宫内膜癌的EGFR突变的情况。 结果： 研究结果显示正常增殖期子宫内膜RASSF1A甲基化阳性率为20.0%,简单性增生子宫内膜阳性率为12.5%,复杂性增生子宫内膜阳性率为24.0%,复杂性增生伴异型增生(癌前病变)阳性率为77.8%,子宫内膜样腺癌的RASSF1A甲基化阳性率为76.2%。其中,子宫内膜样腺癌组织的RASSF1A甲基化阳性率高于正常增殖期子宫内膜,差异存在统计学意义,P值为0.00；同时,复杂性增生伴异型增生(癌前病变)组的RASSF1A甲基化阳性率高于复杂性增生子宫内膜,差异有统计学意义。而正常增殖期子宫内膜,简单性增生子宫内膜和复杂性增生子宫内膜的RASSF1A甲基化阳性率差异无统计学意义,P值分别为0.498和0.299；复杂性增生伴异型增生(癌前病变)和子宫内膜样腺癌组织RASSF1A甲基化阳性率差异也无统计学意义,P值为0.486。不同病理分期和组织学分级的比较结果显示子宫内膜样腺癌Ⅰ期RASSF1A甲基化阳性率为64.3%,Ⅱ期为82.6%,Ⅲ期+Ⅳ期为91.7%；其中,子宫内膜样腺癌Ⅰ期RASSF1A甲基化阳性率和Ⅱ期比较无统计学差异,子宫内膜样腺癌Ⅱ期和Ⅲ期+Ⅳ期比较无统计学差异,而子宫内膜样腺癌Ⅰ期和Ⅲ期+Ⅳ期比较,差异有统计学意义。G1级RASSF1A甲基化阳性率为61.9%,G2级为82.7%,G3级为84.6%；三组之间相互比较,差异无统计学意义。子宫肌层浸润≤1/2的子宫内膜样腺癌患者RASSF1A甲基化阳性率为57.1%,与此同时,子宫肌层浸润1/2的子宫内膜样腺癌患者RASSF1A甲基化阳性率为85.7%,两者差异具有统计学意义,P值为0.012；已经发生淋巴结转移的子宫内膜样腺癌患者RASSF1A甲基化阳性率为81.8%,而尚未发生淋巴结转移的子宫内膜样腺癌患者RASSF1A甲基化阳性率为75.0%,两者之间没有统计学差异,P值为0.813。 检测不同子宫内膜组织RASSF1A基因的表达情况,结果显示正常增殖期子宫内膜RASSF1A表达阳性率为80.0%,简单性增生子宫内膜阳性率为16.7%,复杂性增生子宫内膜阳性率为12.0%,复杂性增生伴异型增生(癌前病变)阳性率为8.3%,子宫内膜样腺癌的RASSF1A表达阳性率为11.1%。子宫内膜样腺癌组织的RASSF1A表达阳性率低于正常增殖期子宫内膜,差异存在统计学意义；同时,简单性增生子宫内膜的RASSF1A表达阳性率也低于正常增殖期子宫内膜,差异存在统计学意义；而简单性增生子宫内膜和复杂性增生子宫内膜,复杂性增生子宫内膜和复杂性增生伴异型增生(癌前病变),复杂性增生伴异型增生(癌前病变)和子宫内膜样腺癌组织的RASSF1A表达阳性率差异无统计学意义,P值分别为0.641、0.636和0.659。不同病理分期和组织学分级的比较结果显示子宫内膜样腺癌Ⅰ期+Ⅱ期RASSF1A表达阳性率为11.7%,Ⅲ期+Ⅳ期为8.3%；两者差异无统计学意义,P值为0.734；G1级RASSF1A表达阳性率为23.8%,G2级为6.9%,G3级为0(未检出),其中高分化G1级与低分化G3级比较,RASSF1A表达差异具有统计学意义。子宫肌层浸润≤1/2的子宫内膜样腺癌患者RASSF1A表达阳性率为23.8%,与此同时,子宫肌层浸润1/2的子宫内膜样腺癌患者RASSF1A表达阳性率为4.76%,两者差异具有统计学意义,P值为0.023；已经发生淋巴结转移的子宫内膜样腺癌患者RASSF1A表达阳性率为9.1%,而尚未发生淋巴结转移的子宫内膜样腺癌患者RASSF1A表达阳性率为11.5%,两者之间没有统计学差异,P值为0.668。 另外,外周血表皮生长因子受体EGFR19号外显子缺失突变特异性荧光检测体系的构建将能为子宫内膜癌的无创检测和病因分子机制的研究提供有力工具,为子宫内膜癌的分子病理诊断和分子靶向治疗提供适合临床筛选的有效方法。通过设计EGFR19外显子突变特异性引物结合Taqman探针,并模拟了不同条件,对这个体系的检测能力进行了测试。结果显示在没有野生信号和其他物质干扰的情况下,这个荧光检测体系可以检测到1x1O1copies/μl的缺失突变；而在有一定程度的野生型质粒作为背景的情况下,这个检测体系也体现出了较好的检测能力,结果显示这个荧光检测体系可以检测到突变率为0.1%的缺失突变；我们还模拟了人血浆样本进行检测,结果显示在模拟人血浆样本中这个荧光检测体系可以检出5x101copies/μL的缺失突变。证明该体系具有较好的检出EGFR19外显子缺失突变的能力,进一步需要在子宫内膜癌的患者样本中进行验证。 结论： 1. RASSF1A基因启动子区域的甲基化率在正常增殖期子宫内膜,简单性增生子宫内膜,复杂性增生子宫内膜中较低,而在复杂性增生伴异型增生(癌前病变)和子宫内膜样腺癌组织中显著升高,说明RASSF1A基因启动子区域的甲基化可能参与了子宫内膜样腺癌的发生发展进程,是子宫内膜样腺癌恶性化进展的重要影响因素。RASSF1A甲基化阳性率和子宫内膜样腺癌的病理学分期,子宫肌层浸润程度有显著相关性,当子宫内膜样腺癌进展到Ⅲ期和Ⅳ期,RASSF1A甲基化阳性率呈上升趋势；子宫肌层浸润1/2的子宫内膜样腺癌患者具有更高的甲基化阳性率；提示RASSF1A甲基化阳性率对子宫内膜样腺癌的监测和预后具有一定意义。 RASSF1A基因启动子区域的甲基化有可能成为子宫内膜样腺癌的早期诊断分子标志物。 2.量子点探针可以很好地检测子宫内膜组织中的RASSF1A蛋白表达。RASSF1A的表达阳性率在正常增殖期子宫内膜中最高,而简单性增生子宫内膜,复杂性增生子宫内膜,复杂性增生伴异型增生(癌前病变),子宫内膜样腺癌组织的RASSF1A表达阳性率呈现逐渐降低的趋势。其中,复杂性增生伴异型增生(癌前病变),子宫内膜样腺癌组织的RASSF1A表达阳性率较低,和这两组相应的RASSF1A的甲基化阳性率较高的结果相一致,提示启动子区的甲基化可能是RASSF1A表达降低的机制之一,表达产物减少或缺失与基因甲基化密切相关。RASSF1A表达阳性率和子宫内膜样腺癌的组织学分级和子宫肌层浸润程度有一定相关性。 3. RASSF1A蛋白表达减少或缺失对子宫内膜癌的发生、发展起了重要作用。可以结合RASSF1A启动子区域甲基化作为子宫内膜癌的分子标志物,从而为子宫内膜癌的早期诊断及生物靶向治疗提供理论依据。 4.外周血EGFR19外显子缺失突变检测体系能够较好地检测EGFR19外显子的缺失突变；在有野生型质粒作为干扰的情况下,EGFR19外显子缺失突变检测体系能达到0.1%的检测灵敏度；在模拟人血浆样本中,该体系可以检出5×101copies/μL的缺失突变。进一步需要在子宫内膜癌的患者样本中进行验证。
[Abstract]:Objective:
Endometrial carcinoma (EC) is one of the most common gynecological malignancies. The incidence and mortality of EC are on the rise worldwide, and the age of onset is younger. Therefore, it is necessary to explore the pathogenesis and pathogenesis of endometrial cancer through various research methods and techniques, which can help us to detect and diagnose endometrial cancer in the early stage of the disease, and can make timely intervention to achieve the purpose of treatment. Ras-related region family 1A (RASSFIA) gene as an inhibitor Abnormal methylation of promoters in oncogenes may result in decreased or deleted gene expression, suggesting that RASSF1A gene is closely related to the genesis and development of various tumors. The study of body therapy is of great significance.
Method:
From January 2002 to December 2009, 63 patients with endometrial carcinoma, aged 35-71 years, were diagnosed in Zhongnan Hospital of Wuhan University. All patients were treated surgically for the first time. No radiotherapy and / or chemotherapy were performed before operation. Primary endometrial carcinoma was diagnosed by pathology and metastatic carcinoma was excluded after operation. Membranous adenocarcinoma was staged according to the surgical pathological staging criteria established by the International Federation of Gynecology and Obstetrics (FIGO) in 2009. There were 28 cases of stage I, 23 cases of stage II, 8 cases of stage III and 4 cases of stage IV. According to the histological classification of endometrioid carcinoma established by FIGO in 1998, 21 cases of well-differentiated grade G1, 29 cases of moderately differentiated grade G2 and 13 cases of poorly differentiated grade G3. At the same time, 20 normal proliferative endometrium patients undergoing endometrial curettage at the same time were selected as negative control group. All patients had no abnormal uterine bleeding before operation and no hormone treatment within 6 months. The new histological classification of endometrial carcinoma proposed by ISGP in 1988 included 24 cases of simple hyperplasia, 25 cases of complex hyperplasia and 36 cases of complex hyperplasia with heterotopic hyperplasia (precancerous lesions).
We extracted DNA from paraffin sections, modified the DNA sequence with bisulfite, and detected the methylation status of RASSF1A gene promoter region in different endometrial tissues by specific methylation PCR. In addition, we detected the expression of RASSF1A gene in different endometrial tissues by quantum dot-labeled streptavidin system. Situation.
In order to detect the methylation status of RASSF1A in peripheral blood of endometrial adenocarcinoma patients, a noninvasive mutagenesis-specific fluorescence detection system was established. The deletion mutation of EGFR19 exon was selected as the target gene, and the amplification refractory mutagenesis system was used. The specific primers of EGFR19 exon deletion mutation were designed based on the principle of tem, ARMS, and the fluorescence detection system of EGFR19 exon deletion mutation was constructed with Taqman probe.
Result:
The results showed that the positive rate of RASSF1A methylation was 20.0% in normal proliferative endometrium, 12.5% in simple hyperplasia, 24.0% in complex hyperplasia, 77.8% in complex hyperplasia with dysplasia (precancerous lesions), and 76.2% in endometrioid adenocarcinoma. The positive rate of RASSF1A methylation in endometrioid adenocarcinoma was higher than that in normal proliferative endometrium, and the difference was statistically significant (P value was 0.00). Meanwhile, the positive rate of RASSF1A methylation in complex hyperplasia with dysplasia (precancerous lesion) group was higher than that in complex hyperplasia endometrium, and the difference was statistically significant. The positive rates of RASSF1A methylation in simple hyperplasia endometrium and complex hyperplasia endometrium were not significantly different, P values were 0.498 and 0.299, respectively. The positive rates of RASSF1A methylation in complex hyperplasia with dysplasia (precancerous lesion) and endometrioid adenocarcinoma were not significantly different, P values were 0.486. The positive rate of RASSF1A methylation was 64.3% in stage I, 82.6% in stage II and 91.7% in stage III and IV of endometrioid adenocarcinoma. The positive rate of methylation of RASSF1A in G1, G2 and G3 was 61.9%, 82.7% and 84.6% respectively. There was no significant difference between the three groups. The positive rate of methylation of RASSF1A was 85.7% in endometrioid adenocarcinoma with myometrial invasion of 1/2, P value was 0.012. The positive rate of methylation of RASSF1A was 81.8% in endometrioid adenocarcinoma with lymph node metastasis, but not in endometrioid adenocarcinoma with lymph node metastasis. The positive rate was 75%, and there was no significant difference between the two groups, the P value was 0.813.
The expression of RASSF1A gene in different endometrial tissues was detected. The results showed that the positive rate of RASSF1A expression in normal proliferative endometrium was 80.0%, that in simple hyperplasia endometrium was 16.7%, that in complex hyperplasia endometrium was 12.0%, that in complex hyperplasia with dysplasia (precancerous lesion) was 8.3%, and that in endometrioid gland was 8.3%. The positive rate of RASSF1A expression in endometrioid adenocarcinoma was significantly lower than that in normal proliferative endometrium, and the positive rate of RASSF1A expression in simple proliferative endometrium was also lower than that in normal proliferative endometrium. The positive rates of RASSF1A expression in endometrium and complex hyperplasia endometrium, complex hyperplasia endometrium and complex hyperplasia with atypical hyperplasia (precancerous lesion), complex hyperplasia with atypical hyperplasia (precancerous lesion) and endometrioid adenocarcinoma were not significantly different, P values were 0.641, 0.636 and 0.659, respectively. The results of histological grading showed that the positive rate of RASSF1A expression was 11.7% in stage I+II and 8.3% in stage III+IV of endometrioid adenocarcinoma; there was no significant difference between them, P value was 0.734; the positive rate of RASSF1A expression in G1 grade was 23.8%, G2 grade was 6.9%, and G3 grade was 0 (not detected). The expression of RASSF1A was poor in well differentiated G1 grade and poorly differentiated G3 grade. The positive rate of RASSF1A expression in endometrioid adenocarcinoma with myometrial invasion less than 1/2 was 23.8%. At the same time, the positive rate of RASSF1A expression in endometrioid adenocarcinoma with myometrial invasion less than 1/2 was 4.76%. The difference was statistically significant, P value was 0.023. The positive rate of RASSF1A expression was 9.1% in adenocarcinoma patients and 11.5% in endometrioid adenocarcinoma patients without lymph node metastasis. There was no significant difference between them, P value was 0.668.
In addition, the construction of a specific fluorescence detection system for EGFR19 deletion mutation in peripheral blood epidermal growth factor receptor exon will provide a powerful tool for noninvasive detection of endometrial carcinoma and the study of molecular mechanism of etiology, and provide an effective method for molecular pathological diagnosis and molecular targeted therapy of endometrial carcinoma. The specific primers of EGFR19 exon mutation combined with Taqman probe were designed, and the detection ability of this system was tested under different conditions. The results showed that the deletion mutation of 1x1O1copies/mul could be detected by this fluorescence detection system without interference of wild signal and other substances. In the case of wild-type plasmids as background, this detection system also showed good detection ability, the results showed that the fluorescence detection system can detect mutation rate of 0.1%; we also simulated human plasma samples for detection, the results showed that the fluorescence detection system in simulated human plasma samples can be detected. A deletion mutation of 5x101 copies/mu L was identified, which proved that the system had a good ability to detect the deletion mutation of EGFR19 exon, and further need to be validated in patients with endometrial carcinoma.
Conclusion:
1. The methylation rate of RASSF1A promoter region was lower in normal proliferative endometrium, simple proliferative endometrium and complex proliferative endometrium, but significantly higher in complex hyperplasia with dysplasia (precancerous lesion) and endometrioid adenocarcinoma, suggesting that the methylation of RASSF1A promoter region may be involved. RASSF1A methylation positive rate and pathological stage of endometrioid adenocarcinoma were significantly correlated with the degree of myometrial invasion. RASSF1A methylation positive rate increased when endometrioid adenocarcinoma progressed to stage III and stage IV. The positive rate of RASSF1A methylation was higher in 1/2 of endometrioid adenocarcinoma patients with myometrial invasion.
Methylation of the promoter region of RASSF1A gene may be a molecular marker for early diagnosis of endometrioid adenocarcinoma.
2. Quantum dot probe can be used to detect the expression of RASSF1A protein in endometrial tissue. The expression of RASSF1A is the highest in normal proliferative endometrium. The expression of RASSF1A is positive in simple hyperplastic endometrium, complex hyperplastic endometrium, complex hyperplasia with dysplasia (precancerous lesion) and endometrioid adenocarcinoma. The positive rate of RASSF1A expression in endometrioid adenocarcinoma was lower than that in complex hyperplasia (precancerous lesion) and endometrioid adenocarcinoma, which was consistent with the high positive rate of RASSF1A methylation in these two groups, suggesting that methylation of promoter region may be one of the mechanisms of RASSF1A expression reduction. The decrease or deletion of RASSF1A was closely related to gene methylation. The positive rate of RASSF1A expression was correlated with histological grade of endometrioid adenocarcinoma and degree of myometrial invasion.
3. Reduced or deleted expression of RASSF1A protein plays an important role in the development of endometrial carcinoma. It can be combined with methylation of RASSF1A promoter region as a molecular marker of endometrial carcinoma, thus providing a theoretical basis for early diagnosis and biological targeted therapy of endometrial carcinoma.
4. EGFR19 exon deletion mutation detection system in peripheral blood can detect EGFR19 exon deletion mutation better; in the presence of wild-type plasmids as interference, EGFR19 exon deletion mutation detection system can achieve 0.1% detection sensitivity; in simulated human plasma samples, the system can detect 5 *101 copies/mu L deletion. Mutations need further validation in patients with endometrial cancer.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.33
[Abstract]:Objective:
Endometrial carcinoma (EC) is one of the most common gynecological malignancies. The incidence and mortality of EC are on the rise worldwide, and the age of onset is younger. Therefore, it is necessary to explore the pathogenesis and pathogenesis of endometrial cancer through various research methods and techniques, which can help us to detect and diagnose endometrial cancer in the early stage of the disease, and can make timely intervention to achieve the purpose of treatment. Ras-related region family 1A (RASSFIA) gene as an inhibitor Abnormal methylation of promoters in oncogenes may result in decreased or deleted gene expression, suggesting that RASSF1A gene is closely related to the genesis and development of various tumors. The study of body therapy is of great significance.
Method:
From January 2002 to December 2009, 63 patients with endometrial carcinoma, aged 35-71 years, were diagnosed in Zhongnan Hospital of Wuhan University. All patients were treated surgically for the first time. No radiotherapy and / or chemotherapy were performed before operation. Primary endometrial carcinoma was diagnosed by pathology and metastatic carcinoma was excluded after operation. Membranous adenocarcinoma was staged according to the surgical pathological staging criteria established by the International Federation of Gynecology and Obstetrics (FIGO) in 2009. There were 28 cases of stage I, 23 cases of stage II, 8 cases of stage III and 4 cases of stage IV. According to the histological classification of endometrioid carcinoma established by FIGO in 1998, 21 cases of well-differentiated grade G1, 29 cases of moderately differentiated grade G2 and 13 cases of poorly differentiated grade G3. At the same time, 20 normal proliferative endometrium patients undergoing endometrial curettage at the same time were selected as negative control group. All patients had no abnormal uterine bleeding before operation and no hormone treatment within 6 months. The new histological classification of endometrial carcinoma proposed by ISGP in 1988 included 24 cases of simple hyperplasia, 25 cases of complex hyperplasia and 36 cases of complex hyperplasia with heterotopic hyperplasia (precancerous lesions).
We extracted DNA from paraffin sections, modified the DNA sequence with bisulfite, and detected the methylation status of RASSF1A gene promoter region in different endometrial tissues by specific methylation PCR. In addition, we detected the expression of RASSF1A gene in different endometrial tissues by quantum dot-labeled streptavidin system. Situation.
In order to detect the methylation status of RASSF1A in peripheral blood of endometrial adenocarcinoma patients, a noninvasive mutagenesis-specific fluorescence detection system was established. The deletion mutation of EGFR19 exon was selected as the target gene, and the amplification refractory mutagenesis system was used. The specific primers of EGFR19 exon deletion mutation were designed based on the principle of tem, ARMS, and the fluorescence detection system of EGFR19 exon deletion mutation was constructed with Taqman probe.
Result:
The results showed that the positive rate of RASSF1A methylation was 20.0% in normal proliferative endometrium, 12.5% in simple hyperplasia, 24.0% in complex hyperplasia, 77.8% in complex hyperplasia with dysplasia (precancerous lesions), and 76.2% in endometrioid adenocarcinoma. The positive rate of RASSF1A methylation in endometrioid adenocarcinoma was higher than that in normal proliferative endometrium, and the difference was statistically significant (P value was 0.00). Meanwhile, the positive rate of RASSF1A methylation in complex hyperplasia with dysplasia (precancerous lesion) group was higher than that in complex hyperplasia endometrium, and the difference was statistically significant. The positive rates of RASSF1A methylation in simple hyperplasia endometrium and complex hyperplasia endometrium were not significantly different, P values were 0.498 and 0.299, respectively. The positive rates of RASSF1A methylation in complex hyperplasia with dysplasia (precancerous lesion) and endometrioid adenocarcinoma were not significantly different, P values were 0.486. The positive rate of RASSF1A methylation was 64.3% in stage I, 82.6% in stage II and 91.7% in stage III and IV of endometrioid adenocarcinoma. The positive rate of methylation of RASSF1A in G1, G2 and G3 was 61.9%, 82.7% and 84.6% respectively. There was no significant difference between the three groups. The positive rate of methylation of RASSF1A was 85.7% in endometrioid adenocarcinoma with myometrial invasion of 1/2, P value was 0.012. The positive rate of methylation of RASSF1A was 81.8% in endometrioid adenocarcinoma with lymph node metastasis, but not in endometrioid adenocarcinoma with lymph node metastasis. The positive rate was 75%, and there was no significant difference between the two groups, the P value was 0.813.
The expression of RASSF1A gene in different endometrial tissues was detected. The results showed that the positive rate of RASSF1A expression in normal proliferative endometrium was 80.0%, that in simple hyperplasia endometrium was 16.7%, that in complex hyperplasia endometrium was 12.0%, that in complex hyperplasia with dysplasia (precancerous lesion) was 8.3%, and that in endometrioid gland was 8.3%. The positive rate of RASSF1A expression in endometrioid adenocarcinoma was significantly lower than that in normal proliferative endometrium, and the positive rate of RASSF1A expression in simple proliferative endometrium was also lower than that in normal proliferative endometrium. The positive rates of RASSF1A expression in endometrium and complex hyperplasia endometrium, complex hyperplasia endometrium and complex hyperplasia with atypical hyperplasia (precancerous lesion), complex hyperplasia with atypical hyperplasia (precancerous lesion) and endometrioid adenocarcinoma were not significantly different, P values were 0.641, 0.636 and 0.659, respectively. The results of histological grading showed that the positive rate of RASSF1A expression was 11.7% in stage I+II and 8.3% in stage III+IV of endometrioid adenocarcinoma; there was no significant difference between them, P value was 0.734; the positive rate of RASSF1A expression in G1 grade was 23.8%, G2 grade was 6.9%, and G3 grade was 0 (not detected). The expression of RASSF1A was poor in well differentiated G1 grade and poorly differentiated G3 grade. The positive rate of RASSF1A expression in endometrioid adenocarcinoma with myometrial invasion less than 1/2 was 23.8%. At the same time, the positive rate of RASSF1A expression in endometrioid adenocarcinoma with myometrial invasion less than 1/2 was 4.76%. The difference was statistically significant, P value was 0.023. The positive rate of RASSF1A expression was 9.1% in adenocarcinoma patients and 11.5% in endometrioid adenocarcinoma patients without lymph node metastasis. There was no significant difference between them, P value was 0.668.
In addition, the construction of a specific fluorescence detection system for EGFR19 deletion mutation in peripheral blood epidermal growth factor receptor exon will provide a powerful tool for noninvasive detection of endometrial carcinoma and the study of molecular mechanism of etiology, and provide an effective method for molecular pathological diagnosis and molecular targeted therapy of endometrial carcinoma. The specific primers of EGFR19 exon mutation combined with Taqman probe were designed, and the detection ability of this system was tested under different conditions. The results showed that the deletion mutation of 1x1O1copies/mul could be detected by this fluorescence detection system without interference of wild signal and other substances. In the case of wild-type plasmids as background, this detection system also showed good detection ability, the results showed that the fluorescence detection system can detect mutation rate of 0.1%; we also simulated human plasma samples for detection, the results showed that the fluorescence detection system in simulated human plasma samples can be detected. A deletion mutation of 5x101 copies/mu L was identified, which proved that the system had a good ability to detect the deletion mutation of EGFR19 exon, and further need to be validated in patients with endometrial carcinoma.
Conclusion:
1. The methylation rate of RASSF1A promoter region was lower in normal proliferative endometrium, simple proliferative endometrium and complex proliferative endometrium, but significantly higher in complex hyperplasia with dysplasia (precancerous lesion) and endometrioid adenocarcinoma, suggesting that the methylation of RASSF1A promoter region may be involved. RASSF1A methylation positive rate and pathological stage of endometrioid adenocarcinoma were significantly correlated with the degree of myometrial invasion. RASSF1A methylation positive rate increased when endometrioid adenocarcinoma progressed to stage III and stage IV. The positive rate of RASSF1A methylation was higher in 1/2 of endometrioid adenocarcinoma patients with myometrial invasion.
Methylation of the promoter region of RASSF1A gene may be a molecular marker for early diagnosis of endometrioid adenocarcinoma.
2. Quantum dot probe can be used to detect the expression of RASSF1A protein in endometrial tissue. The expression of RASSF1A is the highest in normal proliferative endometrium. The expression of RASSF1A is positive in simple hyperplastic endometrium, complex hyperplastic endometrium, complex hyperplasia with dysplasia (precancerous lesion) and endometrioid adenocarcinoma. The positive rate of RASSF1A expression in endometrioid adenocarcinoma was lower than that in complex hyperplasia (precancerous lesion) and endometrioid adenocarcinoma, which was consistent with the high positive rate of RASSF1A methylation in these two groups, suggesting that methylation of promoter region may be one of the mechanisms of RASSF1A expression reduction. The decrease or deletion of RASSF1A was closely related to gene methylation. The positive rate of RASSF1A expression was correlated with histological grade of endometrioid adenocarcinoma and degree of myometrial invasion.
3. Reduced or deleted expression of RASSF1A protein plays an important role in the development of endometrial carcinoma. It can be combined with methylation of RASSF1A promoter region as a molecular marker of endometrial carcinoma, thus providing a theoretical basis for early diagnosis and biological targeted therapy of endometrial carcinoma.
4. EGFR19 exon deletion mutation detection system in peripheral blood can detect EGFR19 exon deletion mutation better; in the presence of wild-type plasmids as interference, EGFR19 exon deletion mutation detection system can achieve 0.1% detection sensitivity; in simulated human plasma samples, the system can detect 5 *101 copies/mu L deletion. Mutations need further validation in patients with endometrial cancer.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.33
【参考文献】
相关期刊论文 前10条
1 陈永平,许学岚,陈晨,傅春燕;C-erbB-2和EGFR在子宫内膜癌中的表达及其意义[J];湖南医学;2001年03期
2 林R，
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