乳腺癌中EGFR蛋白表达和基因扩增的检测结果比较

目的：比较免疫组织化学技术检测乳腺癌中EGFR蛋白表达和荧光原位杂交检测EGFR基因扩增的结果的符合率,为EGFR靶向治疗病例的选择提供依据。方法：随机选取2005年1月到2011年12月冷水江市人民医院和湖南省肿瘤医院病理科的147例乳腺癌档案病例,采用免疫组织化学技术检测乳腺癌组织中EGFR蛋白表达,荧光原位杂交检测EGFR的基因扩增,比较两种方法阳性结果的符合率。结果：免疫组化染色结果显示EGFR在原发性和转移性乳腺癌中的阳性表达率分别为85%（105/123）和79%1（9/24）,两组比较无显著差异（P〉0.05）。FISH检测结果显示原发性和转移性乳腺癌中分别有12%（15/123）和8%（2/24）存在EGFR基因扩增,两组比较结果无显著差异（P〉0.05）。所有存在EGFR基因扩增的原发性和转移性乳腺癌的EGFR免疫组织化学结果均为阳性。在原发性和转移性乳腺癌中,免疫组化阳性和基因扩增程度间呈显著正相关（P〈0.05）,但免疫组化结果预测基因扩增的特异性较低。结论：免疫组织化学检测EGFR只能作为EGFR靶向治疗病例选择的初步筛选,进一步进行荧光原位杂交检测EGFR基因扩增是必须的。     

FISH和IHC检测HER2基因及蛋白在乳腺癌诊治中的应用

目的对比研究免疫组织化学(IHC)与荧光原位杂交(FISH)方法检测乳腺癌中C-erbB-2蛋白表达和HER2基因扩增,评估两种方法的实际应用价值。方法 IHC和FISH法分别检测58例乳腺癌组织中C-erbB-2蛋白表达和HER2基因扩增状况,并进行一致性分析。结果IHC检测蛋白阳性2+和3+共22例,占总病例的37.9%,58例乳腺癌中FISH检测出HER2基因扩增17例,占29.3%。IHC检测C-erbB-2蛋白3+的12例中11例HER2基因扩增阳性,1例无扩增;C-erbB-2蛋白2+者10例,其中5例可见HER2基因扩增;C-erbB-2蛋白1+者11例HER2基因扩增仅1例。C-erbB-2蛋白阴性病例共25例均未检测到基因扩增。结果显示IHC检测C-erbB-2蛋白与FISH检测HER2基因扩增状态有较高的一致性(k=0.681,P0.01),C-erbB-2蛋白阴性和3+标本与HER2基因扩增阳性率比较差异无统计学意义(P0.05);而C-erbB-2蛋白1+和2+病例与其HER2基因扩增差异具有统计学意义(P0.05)。结果 IHC检测C-erbB-2蛋白强阳性(3+)和阴性(0)与HER2基因扩增有较高的一致性,可作为临床是否应用Herceptin治疗的依据,而C-erbB-2蛋白2+和1+病例必须进一步行HER2基因扩增检测。     

荧光原位杂交检测乳腺癌HER-2基因的应用价值

目的:探讨荧光原位杂交(FISH)在检测乳腺癌组织HER-2基因扩增的应用价值.方法:收集50例手术切除的乳腺癌标本.经过处理和切片,FISH法检测细胞HER-2基因扩增情况,IHC法检测细胞膜上HER-2蛋白表达情况.kappa检验法对两种检测结果的一致性进行统计分析.结果:FISH法与IHC法检测结果总符合率为90%(K=0.75,P<0.05).在本组胶东半岛女性乳腺癌人群中HER-2基因扩增率为28%.结论:FISH法检测乳腺癌HER-2基因敏感性及稳定性较高,可作为最终确诊HER-2基因状态的方法.     

HPv 病毒El 蛋白多克隆抗体制备及 在宫颈上皮病变检测的运用

目的：研制人乳头瘤病毒(HPV)E1蛋白抗体,并运用于组织病理学免疫组织化学检测。方法：通过基因重组、原核表达并纯化阳HPV E1蛋白片段;经免疫兔制备多克隆抗体,并经免疫印迹及基因转染鉴定特异性;免疫组织化学法检测13例宫颈病变中E1蛋白,经原位杂交验证。结果：将PCR扩增的E1蛋白保守功能域(第362～456aa)基因片断重组于原核表达载体pEK- 318的6×His标签下游,经诱导表达和亲合层析,纯化出14kD His-E1蛋白。制备的多克隆血清经免疫印迹法检测显示不仅可与His—El蛋白结合,也与HeLa细胞内源性及基因转染表达的天然E1蛋白反应。运用免疫血清检测7例宫颈尖锐湿疣和6例宫颈上皮内瘤病(CIN)Ⅲ级病例显示：7例宫颈尖锐湿疣6例为阳性染色,阳性反应主要分布于病灶中挖空细胞的胞核。同时6例CINⅢ级病变全部阳性,着色主要为非典型增生鳞状上皮细胞核。为了确定上述病例中E1蛋白免疫组化检测的准确性,运用原位杂交法进行了验证。结果显示：7例宫颈尖锐湿疣均为HPV/11型杂交阳性,6例CINⅢ级病变全部为HPV16型杂交阳性,显色部位与免疫组化基本一致。结论：本研究结果显示研制的HPV E1蛋白抗体可以用于病理组织学HPV病毒检测。     

N-myc下游调节基因1在乳腺癌中表达的临床病理意义

目的:探讨N-myc下游调节基因1(N-myc downstream-regulated gene 1,NDRG1)在乳腺癌中表达。方法:收集乳腺癌病例及相应的临床资料包括随访资料,应用免疫组织化学技术检测良性病变(BBD)47例,无淋巴结转移乳腺癌(NMBC)83例,有淋巴结转移乳腺癌(MBC)107例及配对淋巴结转移灶(PLNM)107例中NDRG1的表达,分析NDRG1表达与乳腺癌临床病理指标间(患者年龄、肿块大小、临床分期、组织学类型和分级、淋巴结转移、雌孕激素受体和c-erb B2水平、绝经史)及生存状态的关系。结果:通过免疫组化技术检测乳腺癌中NDRG1的表达,结果显示阳性表达率分别为BBD(95.7%,45/47),NMBC(96.4%,80/83),MBC(98.1%,105/107),PMLN(90.7%,97/107),MBC组织中NDRG1阳性表达率显著高于PMLN中阳性表达率(P=0.021)。NDRG1与组织学分级相关(P=0.041),即分化越差的癌表达NDRG1越强。NDRG1的表达状态与乳腺癌患者的生存预后无显著性相关(P=0.196)。结论:NDRG1表达与乳腺癌淋巴结转移和分化有一定关系。     

Nucleobindin-2 在乳腺癌中表达的临床病理意义

目的:探讨NUCB2在乳腺癌中的表达及临床病理意义。方法:收集乳腺癌病例及相应的临床资料包括随访资料应用免疫组织化学技术检测良性病变,有/无淋巴结转移的乳腺癌及配对淋巴结转移灶中NUCB2的表达,分析NUCB2表达与乳腺癌临床病理指标和生存状态间的关系。结果:免疫组化结果:显示NUCB2在BBD、MBC、NMBC、PMLN的阳性表达率分别为90.0%(45/50),82.1%(87/106)、48.2%(40/83)、47.2%(50/106)。其中,NUCB2在BBD的阳性表达率显著高于NMBC、PMLN,而与MBC无显著性差异;在MBC中的阳性表达率显著高于NMBC;在MBC中的阳性表达率显著高于PMLN。临床病理分析结果:显示乳腺癌原发灶中NUCB2表达与淋巴结转移(p=0.000)、临床分期(p=0.001)、雌激素阳性表达(p=0.020)具有显著性相关。而与年龄、肿块大小、组织学分级、组织学类型、孕激素受体、HER2表达、绝经情况无显著相关。Kaplan-Meier分析结果:显示NUCB2阳性表达病例的总生存率比阴性病例更短(P=0.004)。结论:NUCB2在乳腺癌中表达与淋巴结转移、临床分期、雌激素受体表达和生存状态相关,可能在乳腺癌恶性演进和预后中具有重要意义。     

乳腺癌组织表皮生长因子受体、细胞角蛋白5/6、上皮钙黏蛋白表达与预后的关系研究

摘要 目的：探讨乳腺癌组织表皮生长因子受体(EGFR)、细胞角蛋白5/6(CK5/6)、上皮钙黏蛋白(E-Cad)表达与预后的关系。方法：选取我院(2015年1月～2017年12月)收治的100例接受乳腺外科手术治疗的乳腺癌患者,免疫组织化学检测乳腺癌组织和癌旁组织中EGFR、CK5/6、E-Cad表达。比较乳腺癌组织与癌旁组织中EGFR、CK5/6、E-Cad的阳性表达率,分析乳腺癌组织中EGFR、CK5/6、E-Cad表达与临床病理特征的关系,Kaplan-Meier曲线分析EGFR、CK5/6、E-Cad不同表达患者的无病生存期(DFS)和总生存期(OS)曲线,多因素Cox回归分析乳腺癌预后的影响因素。结果：乳腺癌组织中EGFR、CK5/6的阳性表达率明显高于癌旁组织,E-Cad的阳性表达率明显低于癌旁组织(P＜0.05)。乳腺癌组织中EGFR、E-Cad阳性表达率与分化程度、TNM分期相关(P＜0.05),与年龄、肿瘤直径、病理类型无关(P＞0.05);CK5/6阳性表达率与年龄、肿瘤直径、分化程度、TNM分期无关(P＞0.05),与病理类型相关(P＜0.05)。Kaplan-Meier生存曲线显示,EGFR、CK5/6阳性表达患者3年DFS和OS明显低于阴性表达患者,E-Cad阳性表达患者3年DFS和OS明显高于阴性表达患者(P＜0.05)。多因素Cox回归分析显示,TNM分期Ⅲ期(HR=5.756,95%CI：1.535～21.591)、EGFR阳性(HR=8.090,95%CI：0.954～68.616)、CK5/6阳性(HR=4.507,95%CI：0.466～43.593)为乳腺癌预后独立危险因素,E-Cad阳性(HR=0.221,95%CI：0.048～1.020)为乳腺癌预后独立保护因素(P＜0.05)。结论：乳腺癌组织中EGFR、CK5/6表达明显升高,E-Cad表达明显降低,三者为乳腺癌患者预后独立影响因素。     

CISH技术在乳腺癌Her-2基因检测中的应用

原癌基因Her-2是人类表皮生长因子受体家族的第二个成员,该家族中的受体均位于细胞膜上,在许多组织中都能发现。乳腺癌居女性癌症发病率之首,研究显示Her-2在20％-30％的乳腺癌中过度表达,与乳腺癌的侵袭转移及预后密切相关。目前随着靶向药物曲妥珠单抗（赫赛汀）的发展,乳腺癌复发率及死亡率大大降低,曲妥珠单抗（赫赛汀）靶向治疗的关键是Her-2基因是否扩增,常用的Her-2基因检测方法是免疫组化（IHC）法和荧光原位杂交（FISH）法,但其结果之间一致性较差。     

双探针显色原位杂交技术在乳腺癌HER2基因检测中的应用

目的:通过双探针显色原位杂交技术检测乳腺癌HER2基因的状态,评估双探针显色原位杂交技术的临床应用价值。方法:选取存档蜡块82例,分别用免疫组化及双探针显色原位杂交两种方法对Her-2基因及蛋白进行检测,并采用四格表卡方检验、配对卡方检验及Kappa检验作相关统计学分析。结果:两种方法检测结果不存在显著性差异(P0.05);就两种方法检测结果的一致性分析而言,两种方法所得出的检测结果存在较好的一致性(P0.05)。结论:双探针显色原位杂交技术检测乳腺癌HER2基因扩增结果与免疫组化检测HER2蛋白过表达结果高度相似,可作为检测HER2基因状态的有效方法。     

Relationship between EGFR overexpression and gene amplification status in central nervous system gliomas

OBJECTIVE: To correlate epidermal growth factor receptor (EGFR) protein overexpression, as assessed by immunohistochemistry, with EGFR gene amplification determined by fluorescence in situ hybridization in a series of gliomas. STUDY DESIGN: Forty-seven central nervous system gliomas, including 34 cases of glioblastoma multiforme, 3 oligodendrogliomas, 4 juvenile pilocytic astrocytomas and 5 low grade astrocytomas, were obtained from the files of the University of Utah Pathology Department. In each case a representative paraffin block was selected, and EGFR protein expression was quantified using immunohistochemistry. EGFR gene amplification status was determined by fluorescence in situ hybridization. RESULTS: EGFR protein overexpression was detected in 9 cases of glioblastoma multiforme. EGFR gene amplification was present in 7 of these cases. Both nonamplified glioblastomas demonstrated only 2+ overexpression of EGFR protein. None of the low grade, pilocytic or anaplastic astrocytomas demonstrated either EGFR protein overexpression or gene amplification. CONCLUSION: EGFR protein overexpression is closely associated with gene amplification. Seventy-eight percent of cases showing protein overexpression demonstrated gene amplification. All cases of central nervous system neoplasms showing protein overexpression but lacking gene amplification were associated with only 2+ protein overexpression. All central nervous system neoplasms demonstrating gene amplification and/or overexpression were high grade neoplasms.     

Variation of ER status between primary and metastatic breast cancer and relationship to p53 expression*.

OBJECTIVE: Estrogen-dependent growth of breast cancer can be blocked by anti-estrogens. Estrogen receptor (ER) presence in breast cancer implies responsiveness to endocrine therapy. However, for those patients who ultimately develop resistance to endocrine therapy, the mechanisms remain unclear. The present study attempted to compare the expression status of ER mRNA in a series of primary breast tumors with matched metastases and explored the relation between ER and mutant p53 expression. METHODS: In situ hybridization using a digoxigenin-labeled estrogen receptor cDNA probe was employed to determine the expression of ER mRNA in 52 cases of primary tumors and their matched axillary lymph node metastases. Immunohistochemical staining using a monoclonal antibody against ER was also performed. RESULTS: ER expression was observed in 53.8% (28/52) of primary tumors and 48% (25/52) of metastases, while 57.7% (30/52) of primary tumors and 53.8% (28/52) of metastases showed ER mRNA positivity. There were variations in ER status between in situ hybridization and immunohistochemistry measurements and between primary tumors and metastases. Mutant p53 expression was inversely associated with ER-negative, high-grade tumors. CONCLUSIONS: In situ hybridization may be a more specific and sensitive method for determination of ER status than immunohistochemistry. It is possible that the biologic properties of ER change, and these changes may influence tumor response to endocrine therapy. In view of the ER variation, it was suggested that the ER status of metastatic tumors in addition to primary tumors should be taken into consideration in order to better determine the benefit of clinical endocrine therapy.     

Comprehensive profiling of 8p11-12 amplification in breast cancer

In human carcinomas, especially breast cancer, chromosome arm 8p is frequently involved in complex chromosomal rearrangements that combine amplification at 8p11-12, break in the 8p12-21 region, and loss of 8p21-ter. Several studies have identified putative oncogenes in the 8p11-12 amplicon. However, discrepancies and the lack of knowledge on the structure of this amplification lead us to think that the actual identity of the oncogenes is not definitively established. We present here a comprehensive study combining genomic, expression, and chromosome break analyses of the 8p11-12 region in breast cell lines and primary breast tumors. We show the existence of four amplicons at 8p11-12 using array comparative genomic hybridization. Gene expression analysis of 123 samples using DNA microarrays identified 14 genes significantly overexpressed in relation to amplification. Using fluorescence in situ hybridization analysis on tissue microarrays, we show the existence of a cluster of breakpoints spanning a region just telomeric to and associated with the amplification. Finally, we show that 8p11-12 amplification has a pejorative effect on survival in breast cancer.     

Direct detection of herceptin/trastuzumab binding on breast tissue sections.

The protooncogene product HER-2/neu is the target of the humanized monoclonal antibody trastuzumab (Herceptin). Several tests are used clinically to identify patients with HER-2/neu overexpression based on evaluation by pathologists of gene amplification by fluorescence in situ hybridization or protein expression using immunohistochemistry (IHC). A simple technique has been developed for staining formalin-fixed, paraffin-embedded breast cancer tissue using unmodified Herceptin/trastuzumab as the primary antibody. Results were compared with staining with the commercial kit, HercepTest, as well as with polyclonal anti-HER-2/neu antibodies and with biotinylated trastuzumab. These procedures were tested using four breast cancer microarrays. There were 854 cores that were stained with all four antibodies, representing 325 cases. A standard 4-point scoring system (0-3) was used. A total of 156 cases (48%) were scored as 0 by all the methods used and 31 (9.5%) were positive (3+) by all methods. Of interest, three cases scored negative using polyclonal anti-HER-2/neu antibodies but were positive using unmodified trastuzumab. To clarify this discrepancy, whole sections of tumors were examined with both antibodies using double labeling. There were some tumors that demonstrated a mosaic pattern of staining with neighboring cells or groups of cells stained exclusively with one antibody or the other. These results demonstrate that unmodified humanized or human therapeutic antibodies could be used for preclinical testing or in a clinical laboratory setting for IHC-based selection of patients for treatment, and results of such selection could be different from those obtained using polyclonal antibody-based IHC procedure.     

Novel bright field molecular morphology methods for detection of <Emphasis Type="Italic">HER2</Emphasis> gene amplification

Profiling the amplification and over-expression of the HER2 gene is a key component for defining the prognosis and management of invasive breast carcinoma. Clinical laboratory testing for HER2 gene amplification and over expression has been complicated by an unacceptably high rate of false positive immunohistochemistry (IHC) results, poor reproducibility for the '2+' category of IHC scoring, and reluctant acceptance of alternative testing by fluorescence in situ hybridization (FISH) by the diagnostic pathology community. Novel chromogenic in situ hybridization (CISH) assays have been developed that utilize bright field microscopy and a conventional light microscope for interpretation, but the analytical sensitivity of first generation CISH systems has been problematic. Novel second generation in situ hybridization detection methods based upon polymerized lg detection chemistry, autometallography or enzyme metallography, have been developed that routinely detect endogenous HER2 signals in normal cells (on slide hybridization control) and HER2 signals in both non-amplified and amplified patterns of HER2 genomic signatures. By combining the strength of polymerized peroxidase-labeled antibodies and metallography for gene amplification, with the detection of expression of HER2 encoded protein by IHC on the same slide, both HER2 gene amplification and protein over-expression can be simultaneously evaluated on a cell-by-cell basis in each microscopic field of carcinoma.     

P-gp、MMP-2、C-erbB-2在乳腺癌原发灶和转移淋巴结中的表达对比研究

目的：探讨乳腺癌侵袭转移和多药耐药之间的关系,为治疗方案的个体化提供依据。方法：采用免疫组化方法检测46例乳腺浸润性导管癌患者乳腺原发灶及相应腋淋巴结转移灶中P-gp、MMP-2、c-erbB-2的表达,结合临床表现、病理学指标,分析其相关性。结果：46例原发灶P-gp阳性表达35例（76.1%）,MMP-2阳性表达25例（54.3%）,c-erbB-2高表达18例（39.1%）;相应腋淋巴结转移灶P-gp阳性表达28例（60.9%）,MMP-2阳性表达16例（34.8%）,c-erbB-2高表达16例（34.8%）;P-gp、MMP-2蛋白表达水平与肿块大小、淋巴结转移数目均呈正相关（P〈0.05）,c-erbB-2蛋白表达水平与腋窝淋巴结转移数量呈正相关,与ER、PR表达呈负相关,P-gp阳性表达与MMP-2和c-erbB-2的表达呈正相关（P〈0.05）。结论：肿瘤原发灶与转移灶存在异质性,P-gp、MMP-2、c-erbB-2的表达与乳腺癌的多药耐药和侵袭转移有关,检测上述基因在原发灶与转移灶的表达,为乳腺癌选择个体化的化疗、内分泌治疗及分子靶向治疗提供了分子生物学依据。     

Diagnostic utility of α-methylacyl CoA racemase (P504S) & HMWCK in morphologically difficult prostate cancer

The epidermal growth factor receptor (EGFR) family, consisting of four tyrosine kinase receptors, c-erbB1-4, seems to be influential in gliomagenesis. The aim of this study was to investigate EGFR gene amplification and expression of c-erbB1-4 receptor proteins in human anaplastic astrocytomas. Formalin-fixed and paraffin-embedded sections from 31 cases were investigated by standard immunohistochemical procedures for expression of c-erbB1-4 receptor proteins using commercial antibodies. EGFR gene amplification was studied by fluorescence in situ hybridization using paraffin-embedded tissues. Two monoclonal antibodies, NCL-EGFR-384 and NCL-EGFR, were used for EGFR detection and they displayed positive immunoreactivity in 97% and 71%, respectively. For c-erbB2 detection three monoclonal antibodies, CB11, 3B5, and 5A2, were applied and they displayed positive immunoreactivity in 45%, 100%, and 52%, respectively. Positive immunostaining for c-erbB3 and c-erbB4 was encountered in 97% and 74%, respectively. The EGFR gene was amplified in 9 out of 31 tumors (29%). After adjusting for age, Karnofsky performance status, and extent of surgical resection, Cox multiple regression analysis with overall survival as the dependent variable revealed that c-erbB2 overexpression detected by the monoclonal antibody clone CB11 was a statistically significant poor prognostic factor (P = 0.004). This study shows the convenience and feasibility of immunohistochemistry when determining the expression of receptor proteins in tissue sections of human astrocytomas. The synchronous overexpression of c-erbB1-4 proteins in anaplastic astrocytomas supports their role in the pathogenesis of these tumors. Further, c-erbB2 overexpression seems to predict aggressive behaviour.     

Detection of Long Non-Coding RNA in Archival Tissue: Correlation with Polycomb Protein Expression in Primary and Metastatic Breast Carcinoma

A major function of long non-coding RNAs (lncRNAs) is regulating gene expression through changes in chromatin state. Experimental evidence suggests that in cancer, they can influence Polycomb Repressive Complexes (PRC) to retarget to an occupancy pattern resembling that of the embryonic state. We have previously demonstrated that the expression level of lncRNA in the HOX locus, including HOTAIR, is a predictor of breast cancer metastasis. In this current project, RNA in situ hybridization of probes to three different lncRNAs (HOTAIR, ncHoxA1, and ncHoxD4), as well a immunohistochemical staining of EZH2, is undertaken in formalin-fixed paraffin-embedded breast cancer tissues in a high throughput tissue microarray format to correlate expression with clinicopathologic features. Though overall EZH2 and HOTAIR expression levels were highly correlated, the subset of cases with strong HOTAIR expression correlated with ER and PR positivity, while the subset of cases with strong EZH2 expression correlated with an increased proliferation rate, ER and PR negativity, HER2 underexpression, and triple negativity. Co-expression of HOTAIR and EZH2 trended with a worse outcome. In matched primary and metastatic cancers, both HOTAIR and EZH2 had increased expression in the metastatic carcinomas. This is the first study to show that RNA in situ hybridization of formalin fixed paraffin-embedded clinical material can be used to measure levels of long non-coding RNAs. This approach offers a method to make observations on lncRNAs that may influence the cancer epigenome in a tissue-based technique.