非特殊型浸润性乳腺癌HER-2检测方法的对比研究

发布时间：2018-03-06 00:28

本文选题：非特殊型浸润性乳腺癌　切入点：HER-2　出处：《山东大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的：乳腺癌中HER-2基因表达状态直接影响到患者靶向药物Herceptin的应用与否,目前临床HER-2的检测首先选用免疫组织化学(Immunohistochemistry, IHC)技术进行初筛,尤其是对于HER-2表达为2+者,应用荧光原位杂交(Fluorescence in situ hybridization, FISH)方法进一步验证有无HER-2基因扩增,有扩增者方适用Herceptin。 FISH方法具有较高的精确性,但在应用方面也存在一定的局限性,例如探针昂贵,需要暗室荧光显微镜下进行观察,荧光容易淬灭,且人工计数困难等。本研究中我们采用实时荧光定量PCR技术(Quantitive Real-time PCR,qPCR)对石蜡包埋非特殊型浸润性乳腺癌标本中HER-2基因mRNA的表达状况进行检测,同时与IHC和FISH相应检测结果进行比较,观察qPCR技术在HER-2基因检测中的特异性和敏感性,以期为检测乳腺癌HER-2基因标找到一种更加简便、可靠、实用的方法。方法：选取山东省千佛山医院病理科2010年1月至2014年6月经手术切除病理明确诊断为非特殊型浸润性乳腺癌的400例乳腺癌组织标本,挑选合适的组织蜡块(有相当比例的浸润癌组织和正常乳腺组织),采用用全自动免疫组化仪进行HER-2免疫组织化学染色,经有经验的病理诊断医师按照新版ASCO/CAP和中国指南HER-2判读标准进行判定,选出结果分别为HER-2 (0、1+、2+、3+)的乳腺癌标本各100例。将入选的病例组织蜡块按厚度3-4μ m进行切片,按照FISH操作规程进行,暗室操作,荧光显微镜下观察、采图并计数,请有经验的病理诊断医师根据新版ASCO/CAP和中国指南判读标准进行评估。将入选的病例组织蜡块以8-10μm连续切片2-3片,烤片后,根据HE染色切片画定肿瘤组织部位进行分割,刮取肿瘤组织至EP管中,脱蜡后进行RNA的提取并进行qPCR扩增检测。将qPCR检测结果与IHC和FISH结果进行对比分析,同时分组比较各自与患者临床病理特征的关系。结果：100例HER-2(0)和HER-2(1+)的标本FISH和qPCR的检测结果均无扩增,符合率100%； 100例(2+)的标本中,FISH检测有20例扩增,阳性率20%,qPCR检测有25例扩增,阳性率25%；100例HER-2(3+)的标本中,FISH检测有92例扩增,符合率92%,qPCR检测有90例扩增,符合率为90%。对四组结果分别用Kappa检验进行一致性分析,IHC检测HER-2(2+)的标本,FISH和qPCR检测结果之间k=0.73(P0.001),二者一致性强；IHC检测HER-2(3+)的标本,FISH与qPCR检测结果k=0.634(P0.001),二者具有较好的一致性,同时FISH检测结果92%阳性,qPCR阳性率为90%,二者与IHC检测结果具有较强的一致性。临床病理联系分析表明：qPCR、IHC和FISH所检测的HER-2表达结果均与组织的病理学分级、ER、PR以及Ki67的表达密切相关(P0.05),三者对乳腺癌患者的临床指标反应具有较好的一致性。结论：非特殊型浸润性乳腺癌患者中采用qPCR方法可以有效的检测HER-2基因的状态,在HER-2 IHC检测为0、1+和3+的乳腺癌患者中,qPCR、FISH二者具有较强的一致性；在IHC表达为HER-2(2+)的乳腺癌标本,qPCR方法和FISH法对HER-2基因的检测亦具有较好的一致性且可以互为补充。
[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.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.9

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