荧光原位杂交和基因组半定量法检测乳腺癌组织中c—erbB-2基因扩增

目的利用荧光原位杂交（FISH）与基因组半定量法检测乳腺正常良性和恶性组织中原癌基因c-erbB-2的扩增情况。方法收集50例临床切除的乳腺肿瘤组织，抽提收集样品基因组DNA并扩增c—erbB-2的特异序列构建转化子，以c-erbB-2的特异序列为探针进行间期核FISH检测乳腺肿瘤组织中c-erbB-2基因的扩增，并采用基因组半定量法比较不同组别乳腺组织中c-erbB-2基因的扩增。结果FISH检测发现正常乳腺组织中未出现c-erbB-2扩增，而良性与恶性组织中有不同程度的扩增，基因组半定量分析显示恶性肿瘤组织中c-erbB-2扩增程度高于良性与正常组织。结论c-erbB-2扩增是乳腺癌变的可靠的分子指标。可用荧光原位杂交与基因组半定量法对乳腺癌进行分子生物学诊断，且前者的敏感性与稳定性优于后者。     

双色荧光原位杂交技术检测乳腺癌石蜡切片人类表皮生长因子受体2基因扩增

目的:应用荧光原位杂交技术检测乳腺癌组织人表皮生长因子受体2(human epidermal growth factor receptor 2,Her-2)基因扩增情况.并对操作方法进行优化.方法:收集2008年4月至2008年10月期间我院乳甲科进行手术治疗的乳腺癌患者的癌组织病理切片,采用荧光原位杂交(FISH)技术检测Her-2基因扩增情况,并与免疫组化结果相比较,分析两者的相关性,同时优化操作方法.结果:收集的40例乳腺癌石蜡切片中,免疫组化检测Her-2蛋白表达(+++)有6例,(++)有22例,(+)有7例,(-)有5例.其中Her-2蛋白表达(+++)的标本FISH检测结果均为阳性,基因扩增与蛋白表达的符合率为100%;Her-2蛋白表达(++)的标本FISH结果有6例阳性,16例阴性,基因扩增与蛋白表达的符合率为27.3%.Her-2蛋白表达(+)及(-)的标本FISH结果均为阴性.Her-2蛋白表达(++)及以上的标本基因扩增与蛋白表达的符合率达到42.9%(r=0.584,P＜0.01).结论:FISH检测乳腺癌Her-2基因扩增的结果与IHC检测的蛋白表达结果符合率较好,可作为乳腺癌Her-2基因检测的一项新技术;在具体操作时,应注意严格控制切片厚度、消化时间及变性杂交温度等因素.     

乳腺组织中钠/碘转运体基因及蛋白的表达与乳腺疾病发生的关系

目的:研究正常和良/恶性乳腺肿瘤组织中钠-碘转运体(sodium-iodidesymporter,NIS)基因和蛋白的表达情况,探讨NIS与乳腺疾病之间的关系。方法:乳腺组织取自4例乳腺纤维瘤患者,3例乳腺小叶增生患者,8例乳腺癌患者,5例癌旁正常乳腺组织,运用半定量反转录聚合酶链反应(RT-PCR)和免疫组织化学技术检测NIS的基因与蛋白表达情况。结果:癌旁正常乳腺组织和良性乳腺肿瘤组织未检出NIS基因和蛋白的表达,而乳腺癌组织NIS基因均见NIS基因表达,且NIS蛋白主要表达在胞浆中。结论:正常乳腺组织中未检测到NIS基因,而乳腺癌组织NIS基因表达明显增加,而NIS蛋白翻译和翻译后水平的缺陷可能是乳腺癌组织摄碘率降低的原因之一。     

乳腺组织中钠／碘转运体基因及蛋白的表达与乳腺疾病发生的关系

目的：研究正常和良／恶性乳腺肿瘤组织中钠-碘转运体(sodium-iodide symporter，NIS)基因和蛋白的表达情况，探讨NIS与乳腺疾病之间的关系。方法：乳腺组织取自4例乳腺纤维瘤患者，3例乳腺小叶增生患者，8例乳腺癌患者，5例癌旁正常乳腺组织，运用半定量反转录聚合酶链反应(RT-PCR)和免疫组织化学技术检测NIS的基因与蛋白表达情况。结果：癌旁正常乳腺组织和良性乳腺肿瘤组织未检出NIS基因和蛋白的表达，而乳腺癌组织NIS基因均见NIS基因表达，且NIS蛋白主要表达在胞浆中。结论：正常乳腺组织中未检测到NIS基因．而乳腺癌组织NIS基因表达明显增加，而NIS蛋白翻译和翻译后水平的缺陷可能是乳腺癌组织摄碘率降低的原因之一.     

荧光原位杂交技术检测乳腺癌Her-2/neu扩增

目的：探讨荧光原位杂交技术（FISH）检测乳腺癌Her-2/neu的扩增情况,比较FISH法与免疫组织化学（I HC）法检测结果的一致性。方法：收集乳腺癌患者手术切除的肿瘤标本50例。经石蜡包埋,组织切片后,应用FISH法检测Her-2/neu扩增情况,I HC法检测Her-2/neu蛋白表达情况,并对两种检测方法的结果进行统计学分析。结果：50例标本中FISH阳性17例,FISH阴性33例,FISH阳性率34%（17/50）。50例标本中I HC（～）14例,I HC（0～＋）36例,I HC蛋白表达率28%（14/50）。FISH法与I HC法检测结果的总符合率为82%（41/50）（K=0.581）。结论：FISH法具有较高的稳定性与可靠性,临床开展FISH法检测Her-2/neu基因扩增情况作为I HC法的补充,可以更加准确和客观地对乳腺癌患者的治疗和预后进行评价。     

Livin在乳腺癌中的表达及与c-erbB-2和激素受体的关系

目的 探讨Livin基因在乳腺癌中的表达及与c-erbB-2基因和激素受体之间的表达关系.方法 采用免疫组化S-P法及原位杂交技术检测Livin mRNA和其蛋白在正常乳腺组织、乳腺增生性病变、乳腺非浸润性癌和乳腺浸润性癌中表达及ER/PR和c-erbB-2的表达.结果 Livin蛋白和mRNA在正常乳腺组织、乳腺增生性病变、乳腺非浸润性癌和乳腺浸润性癌的表达阳性率之间差异显著(P<0.01),Livin蛋白与mRNA表达之间差异不显著(P>0.05).Livin表达与临床分期、淋巴结转移和患者年龄有关(P<0.05),与肿瘤大小和组织学分级无关;Livin蛋白与c-erbB-2基因在乳腺癌中的表达无相关性(P>0.05).Livin表达阳性的乳腺癌ER标记指数(32.25±34.90)%明显低于Livin表达阴性的乳腺癌(59.29±34.04)%(P<0.01),而PR的标记指数在两者之间差异不显著(P>0.05).结论 Livin基因及蛋白在乳腺癌中表达上调,提示在乳腺癌的发生、发展中起重要作用,有望成为一种有效、敏感的肿瘤标记物,可作为判断乳腺癌预后的新分子标记物和乳腺癌诱导凋亡治疗的新靶点.     

RT-PCR一步法检测HMGA2在乳腺组织中的表达及意义

目的探讨HMGA2在乳腺组织中的表达及其临床意义。方法应用RT-PCR一步法检测92例女性乳腺疾病中HMGA2基因的表达情况。结果 HMGA2在乳腺良性疾病无表达,而在乳腺癌中高度表达。结论 HM-GA2是乳腺癌的一种特异肿瘤标记物,基因表达与乳腺癌的分期和肿瘤大小密切相关,其高度表达反映乳腺癌的恶性进程。     

乳腺浸润性癌组织人表皮生长因子受体-2基因扩增状态与蛋白表达的比较及其与临床病理特征的相关性

目的探讨乳腺癌组织中人表皮生长因子受体-2(HER2)蛋白表达和基因扩增的一致性及临床意义,并分析HER2基因扩增与临床病理特征的相关性。方法采用全自动免疫组化(IHC)及荧光原位杂交(FISH)的方法,分别检测101例乳腺浸润性癌患者的蛋白表达及基因扩增状态。分析2种不同检测方法结果的差异,并揭示其临床意义。结果 101例浸润性乳腺癌中, IHC显示HER2蛋白1例为(-), 6例为(+), 81例为(++), 13例为(+++)。FISH显示HER2基因无扩增为75例, HER2基因有不同程度的扩增为26例。HER2蛋白(-)的1例及(+)的6例HER2基因均无扩增, HER2蛋白(++)的81例中HER2基因扩增的有14例, HER2蛋白(+++)的13例中HER2基因扩增的有12例。101例中FISH检测共发现9例17号染色体多体。FISH和IHC 2种检测方法的差异有统计学意义(P0.05)。FISH结果与患者的年龄相关(P0.05),与肿瘤组织学分级、肿瘤最大径、Ki67表达及患者腋窝淋巴结转移情况均无相关性(P0.05)。结论 IHC与FISH具有较高的一致性, IHC染色强度和基因扩增状态成正相关, IHC可作为临床使用赫赛汀的初筛方法,应联合应用FISH检测来确定临床用药及判断患者预后情况。     

乳腺癌中c-erbB-2与HIF-1基因表达相关性研究

目的探讨c-erbB-2与HIF 1基因过表达在乳腺癌进程中的内在相关性,寻找检测癌浸润转移的联合分子指标。方法乳腺癌组织芯片中200例样品经原位杂交检测c-erbB-2和HIF-1 mRNA表达,87例样本经荧光原位杂交检测c-erbB-2基因扩增。结果①185例乳腺癌样本中c-erbB-2和HIF 1基因有不同程度的共表达,浸润型的非特殊导管癌I级中c-erbB-2与HIF-1的表达2级以上的样本比率都超过70%。非特殊导管癌Ⅰ、Ⅱ级中c-erbB-2表达的样本比率高于HIF-1。c-erbB-2和HIF 1基因在非特殊导管癌Ⅰ、Ⅱ级中两者表达存在显著差异(P=0.003,P=0.036),小叶癌中两者表达无显著差异(P=0.607)。②浸润型的非特殊性导管癌Ⅰ级中c-erbB-2基因扩增与表达存在显著差异(P=0.046),非特殊性导管癌Ⅱ级与小叶癌中差异不显著(P=0.496m,P=0.878)。结论在乳腺癌浸润转移程度高时,c-erbB-2与HIF-1 mRNA的过表达一致,c-erbB-2扩增与其过表达一致,两者可成为乳腺癌浸润转移的联合分子指标。     

实时荧光定量RT-PCR检测乳腺癌VEGF mRNA的表达

目的：建立实时荧光定量逆转录一多聚酶链反应（real—timequantitative,RT—PCR）检测乳腺癌患者肿瘤组织VEGFmRNA表达的方法,并验证其与免疫组织化学LsAB方法检测VEGF蛋白表达的相关性。方法：提取组织总RNA,逆转录成mRNA,采用实时荧光定量RT—PCR检测44例乳腺浸润性导管癌、25例癌旁正常小叶组织、13例乳腺良性疾病组织中VEGFmRNA表达;采用免疫组织化学LSAB法和彩色图像病理分析系统半定量检测其VEGF蛋白表达。结果：乳腺癌组织中VEGFmRNA表达水平显著高于癌旁和良性病组织（｜P〈0．001）,而后两者之间无明显差异（P〉0．05）;VEGF蛋白表达在癌组织,癌旁和良性病组中的分布特点与VEGFmRNA完全一致。结论;本研究所建立的用于检测人乳腺组织VEGFmRNA表达的实时荧光定量RT—PCR方法,是一种快速有效、灵敏度高、特异性好的定量检测方法,与免疫组化LSAB方法检测的VEGF蛋白表达具有良好相关性,在乳腺癌的早期诊断和良恶性鉴别诊断中具有重要的参考价值。     

C-erbB2 oncoprotein and its soluble ectodomain: a new potential tumor marker for prognosis early detection and monitoring patients undergoing Herceptin treatment

c-erbB-2 oncoprotein (or p185) coded by c-erbB-2 oncogene can be extracted with detergent from cell membrane of breast tissue and breast tumor cell line. The ectodomain of p185 can be cleaved proteolytically from the transmembrane receptor and released into solution. In blood circulation, only the ectodomain can be found, whereas only p185 exists in the extracts of tissue and cell line. p185 can also be quantified in the fine-needle aspirate biopsies and the concentration of p185 from the biopsies of malignant breast tissue is much higher than that of the normal and benign tissue. Measuring the ectodomain of the c-erbB-2 oncoprotein not only is useful to identify breast cancer patient who will benefit from Herceptin treatment but also can be used to monitor patients during the treatment. Overexpression of p185 and the elevation of circulating ectodomain is usually associated with poor prognosis. Overexpression of p185 in breast cancer patients with positive estrogen receptor identifies a subgroup of patients who will not respond to the endocrine therapy. Activation of p185 appears to be an early event in tumorigenesis for some cancers. It is possible that the ectodomain could be used as an early tumor marker detecting benign disease.     

The c-erbB-2 oncogene amplification by competitive PCR in aneuploid cell clones flow sorted from breast cancer samples.

The amplification of c-erbB-2 oncogene has been reported to have clinical relevance as a prognostic index in breast cancer. However, controversies still remain about its interpretation, mainly due to the inaccuracy of methods used for this purpose and to the unpredictable variability of the ratio between cancer and normal cells. Accurate quantitative assay, combined with strategies for selection or enrichment of tumor cell populations, could shed a new light on the relationships between molecular alterations and their clinical relevance. In this study, amplification of c-erbB-2 was measured by competitive PCR in 21 aneuploid breast cancers using a multiple DNA competitor both in whole homogenized cancer cells and in aneuploid enriched clones obtained after flow cytometry cell sorting. Most breast cancers (10/12) carrying c-erbB-2 oncogene amplification showed a significant increase in copy number in sorted aneuploid clones, and 2/9 apparently not amplified in basal samples were found to be amplified after being sorted for the aneuploid population. A general concordance between amplification and c-erbB-2 overexpression was found. The mean degree of amplification in sorted aneuploid clones is increased in breast cancers with the highest levels of c-erbB-2 protein overexpression. These data indicate that in breast cancers the amplification of c-erbB-2 oncogene is mainly associated with aneuploid cells.     

MCM4与c-erbB-2蛋白在乳腺浸润性导管癌组织中的表达及其临床意义

目的检测MCM4、C—erbB-2蛋白在乳腺浸润性导管癌（IDC）组织中表达的改变及其在癌发生、发展中的作用。方法采用免疫组化SP法检测80例IDC、20例乳腺不典型增生和30例乳腺腺病等非癌病变旁正常乳腺组织中MCM4、c-erbB-2蛋白的表达情况。结果80例IDC、20例不典型增生组织中MCM4蛋白的阳性率分别为86．25％、70％,与正常对照组比较差异极显著（P〈0．01）。IDC组织中c—erbB-2蛋白的阳性率为65％,与不典型增生组及正常对照组比较差异显著（P〈0．05）。乳腺癌组中MCM4蛋白的阳性表达与组织学分级、TNM分期、淋巴结转移及肿瘤大小有关（P〈0．05）,与年龄无关。c-erbB-2蛋白的阳性表达与淋巴结转移有关（P〈0．05）,而与组织学分级、TNM分期、肿瘤大小及年龄无关。MCM4蛋白的阳性表达与c—erbB-2的表达呈正相关（r=O．240,P〈0．05）。结论MCM4蛋白阳性表达可作为乳腺癌发生、发展的生物学指标,联合c-erbB-2检测有助于乳腺癌预后的判断及I临床治疗的指导。     

Ⅰ型生长因子受体家族在非小细胞肺癌中的表达及其预后价值的研究

目的 探讨Ⅰ型生长因子受体家族在非小细胞肺癌（NSCLC）组织中异常表达的临床意义及预后价值。方法 用免疫组化法研究了58例NSCLC组织中c-erbB-1、c-erbB-2、c-erbFk3、c—erbB-4蛋白的表达。结果 NSCLC组织中,C-erbB-1、C-erbD-2、c—erbB-3蛋白表达高于正常组织,过表达率分别为62．07％、60．34％和41．38％。C-erbB-4表达率为63．85％,显著低于正常组织（P=0．03）。c—erbB-1蛋白过表达与PCNA指数呈正相关（r=0．248,P〈0．05）;c-erbB-2蛋白过表达与肿瘤的分化程度呈负相关（r=0．29,P〈0．05）,c-erbB-2和C-erbB-3蛋白的过表达,及二者的共过表达,与TNM分期有密切关系（P〈0．01）;C-erbB-4蛋白表迭趋向于高分化肿瘤（P〈0．01）。C-erbB-2蛋白过表达及其与c—erbB-3蛋白共过表达显示预后不良（P〈0．01）。结论 Ⅰ型生长因子受体家族在NSCLC的发生、发展中起着重要的作用,并有助于判断预后。     

HER-2 protein concentrations in breast cancer cells increase before immunohistochemical and fluorescence in situ hybridization analysis turn positive.

BACKGROUND: The level of HER-2/neu in breast cancer cells is normally measured by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH). It determines whether patients should be treated with trastuzumab (Herceptin). In this study, HER-2 protein in breast cancer tissue was measured using a quantitative method. METHODS: Tissue samples of malignant and adjacent benign breast tissue were collected from 118 consecutive women admitted for surgical treatment of breast cancer. The HER-2 protein concentration was determined by 2 HER-2 assays: ELISA and the Bayer ADVIA Centaur assay. Paraffin-embedded tissue sections of the corresponding tumors were analyzed by IHC and FISH. RESULTS: Increased HER-2 concentrations in cancer tissue were found compared to autologous reference tissue (p<0.0001, Wilcoxon test) and normal breast tissue (p<0.0001, Mann-Whitney test). Good concordance rates were observed between the methods: 95.8% for IHC and FISH; 86.4% for IHC and ELISA; and 87.3% for FISH and ELISA. The HER-2 positivity rate was determined to 26.3% by ELISA, 12.7% by IHC and 16.9% by FISH. No correlation was found with tumor grade, axillary node status or serum HER-2 levels. CONCLUSIONS: Detection of HER-2 overexpression by measuring HER-2 in tissue extracts by ELISA seems to be more sensitive than IHC and FISH. This suggests that some patients deprived of Herceptin treatment may benefit from this treatment and may also explain the conversion phenomenon from HER-2-negative to HER-2-positive observed in relapse and metastatic disease.     

Detection of urokinase plasminogen activator receptor and c-erbB-2 in sera of patients with breast and ovarian carcinoma

The role of urokinase plasminogen activator receptor (uPAR) and c-erbB-2 in breast and ovarian cancer was investigated. Eighty patients of breast and ovarian cancer and benign lesions, as well as twenty normal controls were evaluated for the expression of c-erbB-2 by Western blotting and uPAR levels by ELISA. The c-erbB-2 and uPAR showed a significant increase in both types of cancer investigated compared to normal control and benign lesions. The frequency of c-erbB-2 was significantly higher in breast cancer lesions (p < 0.01). Levels of CA15.3 in breast cancer and CA125 in ovarian cancer were significantly higher in cases expressing c-erbB-2 (p < 0.01) than in negative c-erbB-2 cases. The uPAR showed a significant positive correlation with advanced stages of breast cancer (r = 0.7971) and ovarian cancer (r = 0.83662), while significant correlations were found for CA15.3 in breast cancer (r = 0.64967) and CA125 in ovarian cancer (r = 0.83996). Taken together, our data suggest that the c-erbB-2 and uPAR in the sera of ovarian and breast cancer act as valuable markers for the evaluation of the patients preoperatively.     

乳腺癌腋淋巴结微小转移与C-erbB-2，nm23基因表达关系的相关性研究

目的：探讨乳腺癌腋淋巴结微小转移与C-erbB-2,nm23基因表达的关系。方法：采用免疫组织化学S—P法对78例淋巴结常规HE染色无癌转移的乳腺癌淋巴结1269枚进行上皮细胞角蛋白（CK19）表达和癌组织进行C-erbB-2,nm23基因表达的检测。结果：淋巴结CK19阳性率为4．25％,其病理阳性率为28．2％,C-erbB-2阳性率为43．6%,nm23阳性率为80．8％,CK19阳性组的C-erbB-2阳性率明显高于CK-19阴性组（P〈0．05）。CK-19阳性组的nm23阳性率明显低于cK-19阴性组（P〈0．05）。CK-19阳性率与患者月经状况、病理类型、临床分期、激素受体状况无关（P〈0．05）。结论：乳腺癌腋淋巴结CK19表达与癌组织C-erbB-2基因表达呈正相关,与癌组织nm23基因表达呈负相关,与患者月经状况、临床分期、病理类型、激素受体状况无关。     

C-erbB-2表达、SPF和组蛋白酶D与乳腺癌预后

目的探讨乳腺癌病人的c-erbB-2表达,SPF与预后的关系,以及cath-D和临床病理特征关系.方法通过免疫组化法,检测128例乳腺癌病人的c-erbB-2和cath-D的表达.结果c-erbB-2表达比不表达病人预后差(P=0.01).cath-D和c-erbB-2表达与肿块大小、淋巴结转移显著相关(P＜0.0001).结论c-erbB-2阳性表达和cath-D阳性表达者,恶性程度高,易转移,预后差.     

Quantitative PCR--new diagnostic tool for quantifying specific mRNA and DNA molecules: HER2/neu DNA quantification with LightCycler real-time PCR in comparison with immunohistochemistry and fluorescence in situ hybridization

Previously, polymerase chain reaction (PCR) technology has been hampered by its inability to generate quantitative results, a drawback inherent to the high degree of amplification taking place in the reaction. Recently, PCR techniques have been described with the potential of quantifying the amount of mRNA or DNA in biological samples. In this study quantitative PCR was used to investigate the role of the EGF (epidermal growth factor) system in cancer both for measurements of mRNA concentrations and for measurements of the number of copies of specific genes. It is shown that the mRNA expression of a subset of ligands from the EGF system is increased in bladder cancer. Furthermore, measurement of the mRNA concentration gives important information such as the expression of these ligands correlated to the survival of the patients. In addition to the alterations at the mRNA level, changes also can occur at the DNA level in the EGF system. Thus, it has been demonstrated that the number of genes coding for the human epidermal growth factor receptor 2 (HER2) is increased in a number of breast tumors. It is now possible to treat breast cancer patients with a humanized antibody reacting with HER2, and the treatment is considered to be justified if the tumor displays an increased amount of HER2. For this reason there is a need for techniques suitable for HER2 measurements. A LightCycler real-time PCR method used for HER2/neu DNA quantification was evaluated and the results compared with those obtained by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Tumor biopsies were collected from 112 patients diagnosed with early breast cancer from January 1990 to March 1994. The samples were analyzed for HER2 DNA amplification by real-time PCR on LightCycler and by FISH and for HER2 protein expression by IHC. Inter-assay variation for HER2 measured by LightCycler was 10% (x =3.1; n=17). Amplification > or = 2 was observed in 19% of the patients. Concordance rates between real-time PCR and the other methods were 91% (IHC) and 92% (FISH). The correlation between real-time PCR and FISH was highly significant (p < 0.001). The "LightCycler-HER2/neu DNA quantification kit" produces results with a high level of reproducibility and its ease of use allows rapid screening for amplification of HER2. In this paper useful information is given on how real-time PCR compares with FISH and IHC. The data show that results obtained for amplification of HER2 by real-time PCR on the LightCycler instrument are comparable to results obtained by IHC and FISH.