色素原位杂交法和免疫组织化学法检测乳腺癌HER-2基因扩增及蛋白表达

目的:对照色素原位杂交法(chromogenic in situ hybridization,CISH)和免疫组织化学法(immuno-histochemistry,IHC)检测乳腺癌组织中人表皮生长因子受体(human epidermal growth factor receptor-2,HER-2)基因扩增及其蛋白表达的状况.方法:采用CISH技术检测145例乳腺癌组织中HER-2的基因扩增情况,其中14例标本经过荧光原位杂交(fluorescence in si-tu hybridization,FISH)检测,随后分别用FISH和IHC方法检测的HER-2基因扩增及蛋白表达状况与之进行回顾性对照,并按照病理分级、淋巴结状态、绝经与否和雌激素受体(estrogen receptor,ER)/孕激素受体(progesterone receptor,PR)表达情况进行分层,分析HER-2表达与乳腺癌各高危因素之间的关系.结果:CISH检测发现,HER-2无扩增71例(50.0%),低扩增11例(7.6%),高扩增63例(43.4%).14例FISH与CISH检测结果比较,符合率为100.0%.145例IHC与CISH检测结果的符合率为84.8%(P<0.05).在IHC检测积分为0/ 以及 的病例中,HER-2基因扩增与蛋白表达情况基本一致,其符合率均在90%以上;而在IHC检测积分为 的标本中,HER-2基因扩增率仅为61.1%.CISH及IHC检测均显示ER/PR表达情况与HER-2阳性呈负相关,ER和PR均为阴性患者的HER-2基因扩增率和蛋白表达率明显高于ER/PR阳性的患者(CISH:68.3%vs 38.8%,P<0.01;IHC:71.7%vs 48.2%,P<0.01).HER-2状态与乳腺癌病理分级、腋淋巴结转移以及绝经与否无关(P>0.05).结论:CISH技术检测HER-2操作简便,准确性高,可以代替FISH技术,对IHC评分为 的病例应进一步确认HER-2状态.HER-2除了与ER/PR表达相关外,与其他乳腺癌高危因素无关,可作为独立指标进行检测.     

色素原位杂交法分析乳腺癌ＨＥＲ-２蛋白过表达者的基因状态

目的　采用色素原位杂交（ＣＩＳＨ）检测乳腺癌ＨＥＲ-２蛋白过表达者的ＨＥＲ-２基因状态,探讨ＨＥＲ-２蛋白表达与基因扩增的一致性。方法　采用Ｚｙｍｅｄ公司ＳｐｏＴ ＬｉｇｈｔＨＥＲ-２ＣＩＳＴ^ＴＭ试剂盒,按照美国临床肿瘤学会／美国病理医师学会（ＡＳＣＯ／ＣＡＰ）联合工作组推荐的结果判读标准,经免疫组织化学（ＩＨＣ）方法确认２３６例ＨＥＲ-２蛋白表达阳性,其中ＩＨＣ（＋＋）１４８例,ＩＨＣ（＋＋＋）８８例,对上述乳腺癌石蜡标本的ＨＥＲ-２基因行ＣＩＳＨ检测。结果　乳腺癌ＨＥＲ-２蛋白高表达者总基因扩增率为７０.３４％,其中ＨＥＲ-２表达ＩＨＣ（＋＋）者基因扩增率为５９.４６％（８８／１４８）,ＩＨＣ（＋＋＋）者基因扩增率为８８.６４％（７８／８８）,两者基因扩增状态差异有统计学意义（χ２ ＝２６.３５,Ｐ＜０.０１）。ＣＩＳＨ检测的ＨＥＲ-２基因扩增情况与雌激素受体（ＥＲ）、孕激素受体（ＰＲ）表达呈明显负相关（Ｐ＜０.０１）。结论　ＣＩＳＨ是一项简便经济的检测ＨＥＲ-２基因扩增技术,ＩＨＣ（＋＋＋）与ＣＩＳＨ阳性的一致性较高,但ＩＨＣ（＋＋）与ＣＩＳＨ阳性的符合率略低,有必要进一步行ＣＩＳＨ或荧光分子探针原位杂交（ＦＩＳＨ）检测明确基因扩增状态。     

乳腺癌HER2基因扩增的临床病理意义

目的 检测乳腺癌组织中HER2基因扩增状态,评价其临床病理意义。方法 应用FISH、IHC方法分析55例乳腺癌HER2基因扩增/蛋白表达状态与临床病理特征的关系,比对IHC法与FISH检测的一致性程度。结果 55例乳腺癌FISH检测有32例（58.2%）HER2基因扩增。IHC法HER2（+++）22例中21例（95.5%）HER2基因扩增;HER2（++）12例中10例（83.3%）HER2基因扩增;HER2（+/－）21例中1例（4.7%）HER2基因扩增。39例浸润性导管癌中30例（76.9%）有HER2基因扩增,12例浸润性小叶癌中仅1例（8.3%）HER2基因扩增。HER2基因扩增在浸润性导管癌的组织学分级间差异有统计学意义（P<0.001）,组织学Ⅲ级的浸润性导管癌较Ⅰ、Ⅱ级有较高的HER2基因扩增率。HER2基因扩增与ER、PR阴性状态及腋淋巴结转移有显著相关性（P<0.01）,与患者是否绝经无相关性（P>0.05）。结论 浸润性小叶癌,ER、PR阳性以及组织学Ⅰ级的浸润性导管癌常少有HER2基因扩增;对于组织学Ⅲ的浸润性导管癌,同时ER、PR阴性者尽管IHC检测结果为阴性,仍需做FISH检测以明确是否有HER2基因扩增。     

荧光原位杂交法检测HER2基因状态过程中两种切片预处理方法的对比

目的观察两种方法消化石蜡切片对荧光原位杂交（FISH）法检测HER2结果判读的影响。方法分别采用美国PanPath公司生产的FISH检测试剂常规消化和美国Invitrogen公司生产的显色原位杂交（CISH）试剂中的预处理液改良消化乳腺癌石蜡切片。结果常规法使细胞间质完全消化，细胞或重叠或孤立，细胞核中红绿信号清晰，在荧光显微镜下容易计数细胞；改良法细胞间质尚在，细胞核中红绿信号清晰，但不易计数单个无重叠细胞。结论对于HER2基因不扩增或高度扩增的病例，采用改良法消化石蜡组织切片，简单快速，又不影响结果判读；对于HER2基因状态为交界性（4～6个信号点）或多倍体的病例，其石蜡切片应采用常规消化，这有利于FISH法在临床上的推广应用。     

两种荧光原位杂交试剂盒检测乳腺癌HER2基因状态的比较

目的:应用两种荧光原位杂交(FISH)试剂盒检测乳腺癌组织HER2基因状态,旨在比较两者在信号的强度和染色结果上的差异.方法: 采用美国PathVysis和北京金普嘉公司生产的FISH HER2检测试剂盒检测10例免疫组化(IHC)HER2蛋白2+以上HER2的基因状态.结果: PathVysis公司试剂盒在荧光显微镜下观察:细胞核红绿信号清晰,可以采集比较好的图片,容易进行细胞计数;北京金普嘉公司试剂盒在荧光显微镜下观察:细胞核红色信号清晰,绿色信号不明显.结论: PathVysis公司试剂盒HER2和17号染色体信号强,稳定性好,重复性强;北京金普嘉公司试剂盒,简单快速,成本低,对HER2基因不扩增或高度扩增的病例不影响结果判读;当HER2基因与17号染色体比值为临界值时(1.8-2.2)或17号染色体为多倍体的病例,应采用PathVysis公司试剂盒.     

两种荧光原位杂交试剂盒检测乳腺癌HER2基因状态的比较

目的：应用两种荧光原位杂交（FISH）试剂盒检测乳腺癌组织HER2基因状态,旨在比较两者在信号的强度和染色结果上的差异。方法：采用美国PathVysis和北京金普嘉公司生产的FISHHER2检测试剂盒检测10例免疫组化（IHC）HER2蛋白2＋以上HER2的基因状态。结果：PathVysis公司试剂盒在荧光显微镜下观察：细胞核红绿信号清晰,可以采集比较好的图片,容易进行细胞计数;北京金普嘉公司试剂盒在荧光显微镜下观察：细胞核红色信号清晰,绿色信号不明显。结论：PathVysis公司试剂盒HER2和17号染色体信号强,稳定性好,重复性强;北京金普嘉公司试剂盒,简单快速,成本低,对HER2基因不扩增或高度扩增的病例不影响结果判读;当HER2基因与17号染色体比值为临界值时（1.8-2.2）或17号染色体为多倍体的病例,应采用PathVysis公司试剂盒。     

增殖抑制基因在乳腺癌组织中的表达及其意义

目的：探讨新的增殖抑制基因（hypevplasia suppressor gene，HSG）在乳腺癌组织中的表达及其意义。方法：应用免疫组织化学和原位杂交法检测55例乳腺癌标本和18例癌旁正常乳腺组织标本中HSG的表达。应用Image Proplus数码图像分析系统测定组织切片的积分吸光度（D）值，分析其与临床病理参数的相关性。结果：HSGmRNA及其蛋白的阳性染色主要集中于细胞质，呈棕黄色颗粒。55例乳腺癌标本中HSG mRNA及其蛋白的阳性表达率分别为40．00％（22／55）和41．82％（23／55），而癌旁正常乳腺组织的表达率分别为83．33％（15／18）和88．89％（16／18），2组比较，差异有统计学意义（P〈0．05）。在乳腺癌组织中HSGmRNA及其蛋白的低表达与组织学分级、淋巴结转移和雌激素受体（estrogen receptor，ER）有关（P〈0．05），与肿瘤大小和年龄等无关（P〉0．05）。结论：HSG在不同乳腺组织中的表达量不同，提示HSG可能与乳腺癌的发生、发展有密切关系。     

荧光原位杂交技术检测乳腺癌HER-2基因扩增实验方法的比较

目的:探讨荧光原位杂交(FISH)技术检测乳腺癌HER-2基因的最佳实验方法.方法:应用FISH技术检测55例乳腺癌组织标本的HER-2基因,分别采用水浴法和直接消化法,甲酰胺法和共变性法,检测HER-2基因表达成功率,对比实验方法的最佳结果.结果:两种消化法中,水浴法和直接法杂交成功率分别为86.21%(25/29)和88.46%(23/26),差异无统计学意义(P>0.05),杂信号比分别为17.24%(5/29)和46.15%(12/26),差异有统计学意义(P<0.05).两种变性法中甲酰胺法和共变性法杂交成功率分别为73.08%(19/26)和93.10%(27/29),差异有统计学意义(P<0.05).结论:FISH检测乳腺癌HER-2基因,以水浴法和共变性法杂交成功率最高.     

荧光原位杂交与免疫组化法检测乳腺癌HER-2表达的临床意义

[目的]探讨乳腺癌中荧光原位杂交(FISH)检测HER-2基因的扩增和免疫组织化学(IHC)检测HER-2蛋白表达在临床诊断及分子靶向治疗中的应用。[方法]采用FISH与IHC检测50例乳腺癌组织中HER-2基因的扩增与HER-2蛋白表达。[结果]50例浸润性乳腺癌中,FISH检测HER-2基因扩增阳性15例(30%),IHC检测HER-2蛋白表达(-～+)40例,HER-2蛋白表达(++)的2例,HER-2蛋白表达(+++)的8例。[结论]FISH检测HER-2基因的扩增与IHC检测HER-2蛋白表达(++～+++)有较高的一致性。对于IHC检测HER-2(-～++)表达时,必须进一步FISH检测。     

Prognostic and predictive value of c-erbB-2 (HER-2/neu) gene amplification in human Breast Cancer

Amplification of the c-erbB-2 (HER-2/neu) proto-oncogene is detected in 10-30% of human breast cancers and has been shown to be accompanied by the overexpression of its protein in the cancer cell membrane. c-erbB-2 gene amplification is one of the first genetic alterations to be used clinically as a prognostic indicator, a predictive factor of response to doxorubicin (adriamycin) chemotherapy, and a test of patient eligibility for therapy with trastuzumab, a humanized anti-c-erbB-2 antibody. There are two types of tests to detect c-erbB-2 amplification/overexpression: immunohistochemistry and fluorescence in situ hybridization (FISH). Accurate identification of cases with c-erbB-2 amplification/overexpression requires an optimized combination of immunohistochemical and FISH tests.     

乳腺癌HER-2过度表达的临床意义及其靶点治疗

HER-2和抗HER-2单克隆抗体Herceptin为乳腺癌研究的热点之一;HER-2被认为是一个新的判断乳腺癌预后的独立标志物和治疗靶点,可判定预后和治疗效果.Herceptin目前已被证实在乳腺癌的治疗中有着巨大的前景.     

Evaluation of HER-2/neu Overexpression in Gastric Carcinoma using a Tissue Microarray

Background: Amplification and overexpression of human epidermal growth factor receptor 2 (HER2 /neu) oncogene has considerable prognostic value in breast and gastric cancers. This study aimed to evaluatethe frequency, overexpression pattern, clinical significance, and concordance between the results for proteinexpression and gene amplification of HER-2/neu in gastric and gastro-esophageal junction carcinomas.Materials and Methods: In this study, 101 gastric tissue samples which were included in tissue microarray wereimmunohistochemically examined for overexpression of HER2/neu. Chromogenic in situ hybridization (CISH)was used for HER-2/neu amplification. The correlation of HER2/neu amplification with clinicopathologicalparameters was also assessed. In addition, concordance between CISH and IHC was detected. Results: This studydemonstrated a significant difference in the overexpression of HER2/neu in gastric tumors. The overexpression ofHER2/neu was significantly higher in intestinal type, poorly differentiated grade, large size (5 cm≤) and positivenodal involvement tumors (p-value=0.041, 0.015, 0.038 and 0.071, respectively). Also, amplification of HER2/neuaccording to CISH test, had a significant positive correlation with tumor size and tumor type (p-value=0.018 and0.058, respectively).Concordance between CISH and IHC was 76.9% in 101 evaluable samples. Conclusions:IHC/CISH differences were attributed to basolateral membranous immunoreactivity of glandular cells resultingin incomplete membranous reactivity and/or a higher rate of tumor heterogeneity in gastric cancers comparedto breast cancers. Therefore, this can be a potential marker for targeted therapy of malignant gastric tumors.     

Negative HER2/neu Amplification Using Immunohistochemistry and Chromogenic in Situ Hybridization Techniques in Skin Melanoma Cases

Background: This study was performed to evaluate the amplification of HER-2/neu in patients with melanoma.Materials and Methods: Amplification of HER-2/neu was evaluated in a group of patients with melanoma, referredto two referral centers in Tehran, using immunohistochemistry (IHC) and chromogenic in situ hybridization(CISH) techniques. Results: Forty patients with mean age 57.9±19.5 years were enrolled in this study. The mostfrequent type of melanoma was acral, while lower limbs were the most frequent sites. The amplification of HER2/neu was negative in 97.5% of patients with IHC and in 100% of patients with CISH technique. Only one case(2.5%) shows weak positive staining (+2) in IHC method. Fifty five percent of melanoma was ulcerative, and themost common stages of tumors were stages 4b and 3b. More than 47% of cases were in Clark level III, while themean of Breslow thickness was 3.56±2.87 mm. The stage of the case that showed weakly positive staining (2+) inIHC was 4b. Conclusions: The amplification of HER2/neu biomarker was negative in patients with melanoma,using both CISH and IHC techniques.     

Her-2/neu as a Predictive Marker of Response to Breast Cancer Therapy

Amplification of the HER-2/neu (c-erbB-2) gene resulting in overexpression of the p185HER-2 growth factor receptor occurs in ~25% of early stage breast cancers. HER- 2/neu has been established as an important independent prognostic factor in early stage breast cancer in large cohorts of patients and in cohorts with very long (30 year) follow-up duration. New data are emerging to suggest that HER-2/neu may be useful not only as a prognostic factor but also as a predictive marker for projecting response to chemotherapeutics, antiestrogens, and therapeutic anti-HER- 2/neu monoclonal antibodies. In this review we highlight recent data on HER-2/neu as a predictive marker of response to breast cancer therapy and discuss the clinical implications of this information. The difficulty in comparing results from different data sets due to the wide variety of reagents and technologies used to detect HER-2/neu amplification/overexpression in clinical specimens is also discussed. Finally, we report results from experimental models of HER-2/neu overexpression which have been used in an effort to understand the relationship between HER- 2/neu and response to chemotherapeutics and antiestrogens in breast cancer.     

荧光原位杂交检测乳腺癌HER2（++）扩增状态及其与临床病理的相关性

目的 使用荧光原位杂交（fluorescence in situ hybridization, FISH）检测HER2基因扩增情况,并探讨影响HER2基因扩增的因素及其与临床病理特征的关系。方法 收集新疆医科大学附属肿瘤医院2013年l月至2015年12月间IHC检测HER2（++）的乳腺癌病例325份,均采用IHC和FISH两种方法分别检测所有患者的石蜡标本HER2表达和扩增情况,并分析HER2扩增状态与患者各临床病理特征的关系。结果 全组患者经IHC检测HER2表达均为（++）,FISH检测HER2扩增率为12.9%（42/325）,对12项临床和病理指标进行单因素分析显示：HER2扩增状态与激素状态、肿瘤直径、P53显著相关（P<0.05）,而与ki67表达、组织学分级、肿瘤个数等因素均无关（P>0.05）。结论 雌孕激素表达均阴性、肿瘤直径>2 cm、P53表达阳性是预测FISH检测IHC HER2（++）扩增的独立因素。     

Kinetics of serum HER-2/neu changes in patients with HER-2-positive primary breast cancer after initiation of primary chemotherapy

BACKGROUND: The purpose of the study was to determine the utility of quantitation of the extracellular domain (ECD) of the HER-2/neu receptor in the serum for predicting response to treatment in patients with primary breast cancer receiving neoadjuvant therapy. METHODS: HER-2/neu ECD was measured in sera obtained from 39 patients with HER-2-amplified stage II-III primary breast cancer undergoing neoadjuvant chemotherapy. Patients were randomly assigned to either 4 cycles of paclitaxel followed by 4 cycles of fluorouracil, epirubicin, and cyclophosphamide (FEC) (n = 10) or to the same chemotherapy with simultaneous weekly trastuzumab for 24 weeks (n = 29). Changes in HER-2 ECD were monitored with the Bayer HER-2/neu assay over 6 months and correlated with pathological response to treatment. RESULTS: Before initiation of chemotherapy, 28.2% of patients had elevated concentration of the HER-2 ECD (>15 ng/mL). The median baseline serum HER-2 ECD concentration was 13.6 ng/mL (mean +/- SD, 20.3 +/- 35.5 ng/mL). A decrease in the median HER-2 ECD levels from baseline to Week 3 and from baseline to Week 6 of chemotherapy was seen regardless of treatment regimen. No significant difference in baseline HER-2 ECD levels was observed between the groups who achieved pathological complete response (pCR) and the group with residual disease (P = .41). However, a 9% drop from Week 3 to Week 6 after initial chemotherapy was predictive of pCR (P = .04). CONCLUSION: A decrease in serum HER-2 ECD levels early during treatment was associated with pathological response in patients receiving primary chemotherapy, particularly trastuzumab-based regimens. Serum HER-2 ECD levels may serve to monitor neoadjuvant therapy in HER-2-positive primary breast cancer.     

HER-2/neu Status: A Neglected Marker of Prognostication and Management of Breast Cancer Patients in India

Background: Categorizing breast tumors based on the ER, PR and HER/Neu 2 receptor status is necessary in order to predict outcome and assist in management of breast cancer. Herfe we assessed this question in South Indian patients. Materials and Methods: A total of 619 formalin fixed paraffin embedded breast tumor tissues were collected from pathology archives after receipt of ethical clearance. With the help of primary and secondary conjugated antibodies, expression status of ER, PR and HER2/neu was determined. All the experimental data were assessed for correlations with histopathological features of tumors and clinical presentation of the subjects. Results: In the present study, the ages ranged from 20-87 years with a mean of 50.0±12.q years, and majority of the tumors (84%) were of infiltrating duct cell carcinoma type. Assessment of ER, PR and Her-2/neu expression showed that 46% were triple negative. Interestingly, an inverse relation between ER, PR and HER-2/neu was apparent in 41.2% (p<0.0001) of the tumors, of which 24.5% (p<0.0001) were ER and PR co-negative but HER-2 positive. Conclusions: ER and PR positive tumors are less common (i.e<30%) compared to HER-2/neu positive tumors (i.e>50%) in Indian breast cancer patients, underlining the need for effective diagnostic screening and specific therapeutic managements in order to improve the survival rate of patients in low resource countries such as India.