【摘要】：目的： 子宮內(nèi)膜癌(Endometrial carcinoma, EC)是最常見的婦科惡性腫瘤,在世界范圍內(nèi)子宮內(nèi)膜癌的發(fā)病率和死亡率均有上升趨勢,且發(fā)病年齡呈現(xiàn)年輕化。由于目前尚缺乏篩查早期子宮內(nèi)膜癌的有效方法,子宮內(nèi)膜癌仍然嚴(yán)重威脅著全世界婦女的身體健康。因此有必要通過各種研究方法和技術(shù)手段,探索子宮內(nèi)膜癌的發(fā)病原因和發(fā)病機(jī)制,能夠幫助我們在疾病的早期階段發(fā)現(xiàn)和診斷子宮內(nèi)膜癌,并能夠進(jìn)行及時的干預(yù)以達(dá)到治療的目的。Ras相關(guān)區(qū)域家族1A (RASSFIA)基因作為抑癌基因,其啟動子異常甲基化造成的基因表達(dá)降低或缺失,提示RASSF1A基因與多種腫瘤的發(fā)生發(fā)展關(guān)系密切。因此,研究RASSF1A基因啟動子區(qū)域甲基化和RASSF1A蛋白的表達(dá)與子宮內(nèi)膜癌發(fā)生發(fā)展的關(guān)系,對子宮內(nèi)膜腺癌的分子病理診斷和個體化治療研究具有重要意義。 方法： 我們收集2002年1月至2009年12月期間在武漢大學(xué)中南醫(yī)院診斷的子宮內(nèi)膜癌的患者63例,患者年齡35～71歲。所有患者首次治療均為經(jīng)手術(shù)治療；術(shù)前均未進(jìn)行放療和／或化療；術(shù)后經(jīng)病理診斷為原發(fā)性子宮內(nèi)膜癌,排除轉(zhuǎn)移癌。子宮內(nèi)膜腺癌根據(jù)2009年國際婦產(chǎn)科聯(lián)盟(FIGO)制定的手術(shù)病理分期標(biāo)準(zhǔn)進(jìn)行分期,Ⅰ期28例,Ⅱ期23例,Ⅲ期8例,Ⅳ期4例；根據(jù)1998年FIGO制定的子宮內(nèi)膜樣癌組織學(xué)分級法分類,高分化G1級21例,中分化G2級29例,低分化G3級13例。子宮內(nèi)膜腺癌肌層浸潤≤1/2者21例,肌層浸潤1/2者42例,有淋巴轉(zhuǎn)移者11例,無淋巴轉(zhuǎn)移者52例;于此同時,選擇相同時期進(jìn)行子宮內(nèi)膜診刮術(shù)患者的正常增生期子宮內(nèi)膜20例作為陰性對照組,患者術(shù)前均無異常子宮出血,6個月內(nèi)未使用激素治療。根據(jù)國際婦科病理協(xié)會(ISGP)1988年提出的子宮內(nèi)膜癌腫瘤組織學(xué)新分類,收集其他類型子宮內(nèi)膜組織石蠟包埋樣本包括簡單性增生24例、復(fù)雜性增生25例、復(fù)雜性增生伴異性增生(癌前病變)36例。 我們提取石蠟切片組織中DNA,使用重亞硫酸鹽修飾DNA序列,再利用特異性甲基化PCR方法檢測不同子宮內(nèi)膜組織RASSF1A基因啟動子區(qū)的甲基化狀態(tài)。另外,我們還使用量子點(diǎn)標(biāo)記的鏈霉親和素系統(tǒng)檢測不同子宮內(nèi)膜組織RASSF1A基因的表達(dá)情況。 為了檢測子宮內(nèi)膜腺癌患者外周血中的RASSF1A的甲基化狀態(tài),我們建立了無創(chuàng)性的突變特異性熒光檢測體系,選擇表皮生長因子受體EGFR19號外顯子的缺失突變作為目標(biāo)基因,我們利用探針擴(kuò)增阻滯突變系統(tǒng)(amplification refractorymutation system, ARMS)的相關(guān)原理,設(shè)計EGFR19外顯子的缺失突變特異性引物,結(jié)合Taqman探針,構(gòu)建EGFR19外顯子缺失突變特異性熒光檢測體系�？梢詭椭袛嘧訉m內(nèi)膜癌的EGFR突變的情況。 結(jié)果： 研究結(jié)果顯示正常增殖期子宮內(nèi)膜RASSF1A甲基化陽性率為20.0%,簡單性增生子宮內(nèi)膜陽性率為12.5%,復(fù)雜性增生子宮內(nèi)膜陽性率為24.0%,復(fù)雜性增生伴異型增生(癌前病變)陽性率為77.8%,子宮內(nèi)膜樣腺癌的RASSF1A甲基化陽性率為76.2%。其中,子宮內(nèi)膜樣腺癌組織的RASSF1A甲基化陽性率高于正常增殖期子宮內(nèi)膜,差異存在統(tǒng)計學(xué)意義,P值為0.00；同時,復(fù)雜性增生伴異型增生(癌前病變)組的RASSF1A甲基化陽性率高于復(fù)雜性增生子宮內(nèi)膜,差異有統(tǒng)計學(xué)意義。而正常增殖期子宮內(nèi)膜,簡單性增生子宮內(nèi)膜和復(fù)雜性增生子宮內(nèi)膜的RASSF1A甲基化陽性率差異無統(tǒng)計學(xué)意義,P值分別為0.498和0.299；復(fù)雜性增生伴異型增生(癌前病變)和子宮內(nèi)膜樣腺癌組織RASSF1A甲基化陽性率差異也無統(tǒng)計學(xué)意義,P值為0.486。不同病理分期和組織學(xué)分級的比較結(jié)果顯示子宮內(nèi)膜樣腺癌Ⅰ期RASSF1A甲基化陽性率為64.3%,Ⅱ期為82.6%,Ⅲ期+Ⅳ期為91.7%；其中,子宮內(nèi)膜樣腺癌Ⅰ期RASSF1A甲基化陽性率和Ⅱ期比較無統(tǒng)計學(xué)差異,子宮內(nèi)膜樣腺癌Ⅱ期和Ⅲ期+Ⅳ期比較無統(tǒng)計學(xué)差異,而子宮內(nèi)膜樣腺癌Ⅰ期和Ⅲ期+Ⅳ期比較,差異有統(tǒng)計學(xué)意義。G1級RASSF1A甲基化陽性率為61.9%,G2級為82.7%,G3級為84.6%；三組之間相互比較,差異無統(tǒng)計學(xué)意義。子宮肌層浸潤≤1/2的子宮內(nèi)膜樣腺癌患者RASSF1A甲基化陽性率為57.1%,與此同時,子宮肌層浸潤1/2的子宮內(nèi)膜樣腺癌患者RASSF1A甲基化陽性率為85.7%,兩者差異具有統(tǒng)計學(xué)意義,P值為0.012；已經(jīng)發(fā)生淋巴結(jié)轉(zhuǎn)移的子宮內(nèi)膜樣腺癌患者RASSF1A甲基化陽性率為81.8%,而尚未發(fā)生淋巴結(jié)轉(zhuǎn)移的子宮內(nèi)膜樣腺癌患者RASSF1A甲基化陽性率為75.0%,兩者之間沒有統(tǒng)計學(xué)差異,P值為0.813。 檢測不同子宮內(nèi)膜組織RASSF1A基因的表達(dá)情況,結(jié)果顯示正常增殖期子宮內(nèi)膜RASSF1A表達(dá)陽性率為80.0%,簡單性增生子宮內(nèi)膜陽性率為16.7%,復(fù)雜性增生子宮內(nèi)膜陽性率為12.0%,復(fù)雜性增生伴異型增生(癌前病變)陽性率為8.3%,子宮內(nèi)膜樣腺癌的RASSF1A表達(dá)陽性率為11.1%。子宮內(nèi)膜樣腺癌組織的RASSF1A表達(dá)陽性率低于正常增殖期子宮內(nèi)膜,差異存在統(tǒng)計學(xué)意義；同時,簡單性增生子宮內(nèi)膜的RASSF1A表達(dá)陽性率也低于正常增殖期子宮內(nèi)膜,差異存在統(tǒng)計學(xué)意義；而簡單性增生子宮內(nèi)膜和復(fù)雜性增生子宮內(nèi)膜,復(fù)雜性增生子宮內(nèi)膜和復(fù)雜性增生伴異型增生(癌前病變),復(fù)雜性增生伴異型增生(癌前病變)和子宮內(nèi)膜樣腺癌組織的RASSF1A表達(dá)陽性率差異無統(tǒng)計學(xué)意義,P值分別為0.641、0.636和0.659。不同病理分期和組織學(xué)分級的比較結(jié)果顯示子宮內(nèi)膜樣腺癌Ⅰ期+Ⅱ期RASSF1A表達(dá)陽性率為11.7%,Ⅲ期+Ⅳ期為8.3%；兩者差異無統(tǒng)計學(xué)意義,P值為0.734；G1級RASSF1A表達(dá)陽性率為23.8%,G2級為6.9%,G3級為0(未檢出),其中高分化G1級與低分化G3級比較,RASSF1A表達(dá)差異具有統(tǒng)計學(xué)意義。子宮肌層浸潤≤1/2的子宮內(nèi)膜樣腺癌患者RASSF1A表達(dá)陽性率為23.8%,與此同時,子宮肌層浸潤1/2的子宮內(nèi)膜樣腺癌患者RASSF1A表達(dá)陽性率為4.76%,兩者差異具有統(tǒng)計學(xué)意義,P值為0.023；已經(jīng)發(fā)生淋巴結(jié)轉(zhuǎn)移的子宮內(nèi)膜樣腺癌患者RASSF1A表達(dá)陽性率為9.1%,而尚未發(fā)生淋巴結(jié)轉(zhuǎn)移的子宮內(nèi)膜樣腺癌患者RASSF1A表達(dá)陽性率為11.5%,兩者之間沒有統(tǒng)計學(xué)差異,P值為0.668。 另外,外周血表皮生長因子受體EGFR19號外顯子缺失突變特異性熒光檢測體系的構(gòu)建將能為子宮內(nèi)膜癌的無創(chuàng)檢測和病因分子機(jī)制的研究提供有力工具,為子宮內(nèi)膜癌的分子病理診斷和分子靶向治療提供適合臨床篩選的有效方法。通過設(shè)計EGFR19外顯子突變特異性引物結(jié)合Taqman探針,并模擬了不同條件,對這個體系的檢測能力進(jìn)行了測試。結(jié)果顯示在沒有野生信號和其他物質(zhì)干擾的情況下,這個熒光檢測體系可以檢測到1x1O1copies/μl的缺失突變；而在有一定程度的野生型質(zhì)粒作為背景的情況下,這個檢測體系也體現(xiàn)出了較好的檢測能力,結(jié)果顯示這個熒光檢測體系可以檢測到突變率為0.1%的缺失突變；我們還模擬了人血漿樣本進(jìn)行檢測,結(jié)果顯示在模擬人血漿樣本中這個熒光檢測體系可以檢出5x101copies/μL的缺失突變。證明該體系具有較好的檢出EGFR19外顯子缺失突變的能力,進(jìn)一步需要在子宮內(nèi)膜癌的患者樣本中進(jìn)行驗(yàn)證。 結(jié)論： 1. RASSF1A基因啟動子區(qū)域的甲基化率在正常增殖期子宮內(nèi)膜,簡單性增生子宮內(nèi)膜,復(fù)雜性增生子宮內(nèi)膜中較低,而在復(fù)雜性增生伴異型增生(癌前病變)和子宮內(nèi)膜樣腺癌組織中顯著升高,說明RASSF1A基因啟動子區(qū)域的甲基化可能參與了子宮內(nèi)膜樣腺癌的發(fā)生發(fā)展進(jìn)程,是子宮內(nèi)膜樣腺癌惡性化進(jìn)展的重要影響因素。RASSF1A甲基化陽性率和子宮內(nèi)膜樣腺癌的病理學(xué)分期,子宮肌層浸潤程度有顯著相關(guān)性,當(dāng)子宮內(nèi)膜樣腺癌進(jìn)展到Ⅲ期和Ⅳ期,RASSF1A甲基化陽性率呈上升趨勢；子宮肌層浸潤1/2的子宮內(nèi)膜樣腺癌患者具有更高的甲基化陽性率；提示RASSF1A甲基化陽性率對子宮內(nèi)膜樣腺癌的監(jiān)測和預(yù)后具有一定意義。 RASSF1A基因啟動子區(qū)域的甲基化有可能成為子宮內(nèi)膜樣腺癌的早期診斷分子標(biāo)志物。 2.量子點(diǎn)探針可以很好地檢測子宮內(nèi)膜組織中的RASSF1A蛋白表達(dá)。RASSF1A的表達(dá)陽性率在正常增殖期子宮內(nèi)膜中最高,而簡單性增生子宮內(nèi)膜,復(fù)雜性增生子宮內(nèi)膜,復(fù)雜性增生伴異型增生(癌前病變),子宮內(nèi)膜樣腺癌組織的RASSF1A表達(dá)陽性率呈現(xiàn)逐漸降低的趨勢。其中,復(fù)雜性增生伴異型增生(癌前病變),子宮內(nèi)膜樣腺癌組織的RASSF1A表達(dá)陽性率較低,和這兩組相應(yīng)的RASSF1A的甲基化陽性率較高的結(jié)果相一致,提示啟動子區(qū)的甲基化可能是RASSF1A表達(dá)降低的機(jī)制之一,表達(dá)產(chǎn)物減少或缺失與基因甲基化密切相關(guān)。RASSF1A表達(dá)陽性率和子宮內(nèi)膜樣腺癌的組織學(xué)分級和子宮肌層浸潤程度有一定相關(guān)性。 3. RASSF1A蛋白表達(dá)減少或缺失對子宮內(nèi)膜癌的發(fā)生、發(fā)展起了重要作用。可以結(jié)合RASSF1A啟動子區(qū)域甲基化作為子宮內(nèi)膜癌的分子標(biāo)志物,從而為子宮內(nèi)膜癌的早期診斷及生物靶向治療提供理論依據(jù)。 4.外周血EGFR19外顯子缺失突變檢測體系能夠較好地檢測EGFR19外顯子的缺失突變；在有野生型質(zhì)粒作為干擾的情況下,EGFR19外顯子缺失突變檢測體系能達(dá)到0.1%的檢測靈敏度；在模擬人血漿樣本中,該體系可以檢出5×101copies/μL的缺失突變。進(jìn)一步需要在子宮內(nèi)膜癌的患者樣本中進(jìn)行驗(yàn)證。
[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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R737.33

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