本文選題：非特殊型浸潤性乳腺癌　切入點(diǎn)：HER-2　出處：《山東大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：乳腺癌中HER-2基因表達(dá)狀態(tài)直接影響到患者靶向藥物Herceptin的應(yīng)用與否,目前臨床HER-2的檢測首先選用免疫組織化學(xué)(Immunohistochemistry, IHC)技術(shù)進(jìn)行初篩,尤其是對于HER-2表達(dá)為2+者,應(yīng)用熒光原位雜交(Fluorescence in situ hybridization, FISH)方法進(jìn)一步驗(yàn)證有無HER-2基因擴(kuò)增,有擴(kuò)增者方適用Herceptin。 FISH方法具有較高的精確性,但在應(yīng)用方面也存在一定的局限性,例如探針昂貴,需要暗室熒光顯微鏡下進(jìn)行觀察,熒光容易淬滅,且人工計(jì)數(shù)困難等。本研究中我們采用實(shí)時(shí)熒光定量PCR技術(shù)(Quantitive Real-time PCR,qPCR)對石蠟包埋非特殊型浸潤性乳腺癌標(biāo)本中HER-2基因mRNA的表達(dá)狀況進(jìn)行檢測,同時(shí)與IHC和FISH相應(yīng)檢測結(jié)果進(jìn)行比較,觀察qPCR技術(shù)在HER-2基因檢測中的特異性和敏感性,以期為檢測乳腺癌HER-2基因標(biāo)找到一種更加簡便、可靠、實(shí)用的方法。方法：選取山東省千佛山醫(yī)院病理科2010年1月至2014年6月經(jīng)手術(shù)切除病理明確診斷為非特殊型浸潤性乳腺癌的400例乳腺癌組織標(biāo)本,挑選合適的組織蠟塊(有相當(dāng)比例的浸潤癌組織和正常乳腺組織),采用用全自動(dòng)免疫組化儀進(jìn)行HER-2免疫組織化學(xué)染色,經(jīng)有經(jīng)驗(yàn)的病理診斷醫(yī)師按照新版ASCO/CAP和中國指南HER-2判讀標(biāo)準(zhǔn)進(jìn)行判定,選出結(jié)果分別為HER-2 (0、1+、2+、3+)的乳腺癌標(biāo)本各100例。將入選的病例組織蠟塊按厚度3-4μ m進(jìn)行切片,按照FISH操作規(guī)程進(jìn)行,暗室操作,熒光顯微鏡下觀察、采圖并計(jì)數(shù),請有經(jīng)驗(yàn)的病理診斷醫(yī)師根據(jù)新版ASCO/CAP和中國指南判讀標(biāo)準(zhǔn)進(jìn)行評估。將入選的病例組織蠟塊以8-10μm連續(xù)切片2-3片,烤片后,根據(jù)HE染色切片畫定腫瘤組織部位進(jìn)行分割,刮取腫瘤組織至EP管中,脫蠟后進(jìn)行RNA的提取并進(jìn)行qPCR擴(kuò)增檢測。將qPCR檢測結(jié)果與IHC和FISH結(jié)果進(jìn)行對比分析,同時(shí)分組比較各自與患者臨床病理特征的關(guān)系。結(jié)果：100例HER-2(0)和HER-2(1+)的標(biāo)本FISH和qPCR的檢測結(jié)果均無擴(kuò)增,符合率100%； 100例(2+)的標(biāo)本中,FISH檢測有20例擴(kuò)增,陽性率20%,qPCR檢測有25例擴(kuò)增,陽性率25%；100例HER-2(3+)的標(biāo)本中,FISH檢測有92例擴(kuò)增,符合率92%,qPCR檢測有90例擴(kuò)增,符合率為90%。對四組結(jié)果分別用Kappa檢驗(yàn)進(jìn)行一致性分析,IHC檢測HER-2(2+)的標(biāo)本,FISH和qPCR檢測結(jié)果之間k=0.73(P0.001),二者一致性強(qiáng)；IHC檢測HER-2(3+)的標(biāo)本,FISH與qPCR檢測結(jié)果k=0.634(P0.001),二者具有較好的一致性,同時(shí)FISH檢測結(jié)果92%陽性,qPCR陽性率為90%,二者與IHC檢測結(jié)果具有較強(qiáng)的一致性。臨床病理聯(lián)系分析表明：qPCR、IHC和FISH所檢測的HER-2表達(dá)結(jié)果均與組織的病理學(xué)分級、ER、PR以及Ki67的表達(dá)密切相關(guān)(P0.05),三者對乳腺癌患者的臨床指標(biāo)反應(yīng)具有較好的一致性。結(jié)論：非特殊型浸潤性乳腺癌患者中采用qPCR方法可以有效的檢測HER-2基因的狀態(tài),在HER-2 IHC檢測為0、1+和3+的乳腺癌患者中,qPCR、FISH二者具有較強(qiáng)的一致性；在IHC表達(dá)為HER-2(2+)的乳腺癌標(biāo)本,qPCR方法和FISH法對HER-2基因的檢測亦具有較好的一致性且可以互為補(bǔ)充。
[Abstract]:Objective: whether the application directly affects patients with drugs targeting Herceptin gene expression of HER-2 in breast cancer, the detection of clinical HER-2 first used immunohistochemical techniques (Immunohistochemistry, IHC) were screened, especially on the expression of HER-2 2+, using fluorescence in situ hybridization (Fluorescence in situ hybridization, FISH) there is no method to further verify the accuracy of HER-2 gene amplification, amplification method for Herceptin. FISH method is high, but the application also has some limitations, such as expensive probe, darkroom fluorescence microscope observation, fluorescence quenching and artificial easily, difficult to count. In this study, we used real time fluorescence quantitative PCR (Quantitive Real-time PCR, qPCR) on paraffin embedded non special type of HER-2 in invasive breast cancer specimens, mRNA gene expression status detection, At the same time with IHC and FISH detection results were compared to observe the sensitivity and specificity of qPCR in detection of HER-2 gene in breast cancer, in order to detect the HER-2 gene target to find a more simple, reliable and practical method. Methods: Department of pathology Shandong hospital in Qianfo Hill province from January 2010 to 2014 6 menstrual surgical pathology diagnosis of 400 cases of breast cancer tissue samples of non special type of invasive breast cancer, select appropriate paraffin blocks (a considerable proportion of the infiltration of cancer tissue and normal breast tissue), using HER-2 for immunohistochemical staining by automatic immunohistochemistry was confirmed by pathology physicians with experience and according to the new edition of ASCO/CAP China guide HER-2 interpretation standard of judgment, which were HER-2 (0,1+, 2+, 3+) of breast cancer samples from 100 patients. Patients were selected according to the thickness of wax tissue 3-4 m slices, In accordance with the operating procedures, FISH darkroom operation were observed by fluorescence microscopy, image collecting and counting, please pathology physicians have experience according to the new version of ASCO/CAP and Chinese guide interpretation standards for evaluation. The selected cases of paraffin blocks with 8-10 m serial sections of 2-3 pieces of toast, according to the HE staining of tumor mark tissue segmentation, scraping the tumor tissue to EP tube, after dewaxing to RNA extraction and qPCR amplification. The detection results of qPCR and IHC and FISH were analyzed, and compared their relationship with clinical and pathological features of patients. Results: 100 cases of HER-2 (0) and HER-2 (1+ the specimens of FISH and qPCR) detection results were not amplified, the coincidence rate was 100%; 100 cases (2+) specimens, 20 cases of amplified FISH detection, the positive rate was 20%, qPCR detected 25 cases were positive rate of 25%; 100 cases of HER-2 (3+) specimens, detection of FISH 92 Cases of amplification, the coincidence rate was 92%, qPCR detected 90 cases with the rate of 90%. amplification, the results of four groups were used Kappa test consistency analysis, detection of IHC HER-2 (2+) specimens, k=0.73 between FISH and qPCR results (P0.001), the two strong consistency detection; IHC HER-2 (3+). FISH and qPCR samples, the detection result of k=0.634 (P0.001), the two have good consistency, and FISH detection results of 92% positive, the positive rate of qPCR was 90%, two and IHC test results have strong consistency. Clinical pathological correlation analysis showed that the qPCR expression of IHC and FISH, the results of detection were HER-2 with the pathological grading, ER, PR and Ki67 expression are closely related (P0.05), three clinical indicator of breast cancer patients have good consistency. Conclusion: non special infiltrating can effectively detect HER-2 gene using qPCR method in breast cancer patients in the state of HER-2 IHC detected 0,1+ and 3+ in breast cancer patients, qPCR and FISH two have strong consistency; in IHC expression of HER-2 (2+) of breast cancer specimens, qPCR method and FISH method for the detection of HER-2 gene has good consistency and can complement each other.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R737.9

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 董海新;張仁亞;劉啟龍;趙元明;孫卓祥;郭煥;趙廣章;陳濤;;熒光原位雜交技術(shù)檢測乳腺癌Her-2/neu基因與免疫組化法檢測C-erbB2蛋白的相關(guān)性研究[J];分子診斷與治療雜志;2009年02期

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本文編號：1572579