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% (×=3.1; n=17). Amplification ≥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.     

Real-time quantitative PCR of microdissected paraffin-embedded breast carcinoma: an alternative method for HER-2/neu analysis

We studied the feasibility of using real-time quantitative PCR to determine HER-2 DNA amplification and mRNA expression in microdissected formalin-fixed, paraffin-embedded breast tumors and compared this with standard immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) methods. Study cases (27 carcinomas and 3 ductal breast carcinoma in situ (DCIS) cases) showed varying Her-2 expression as determined by IHC (HercepTest). In carcinomas, there was a good correlation between HER-2 DNA amplification and strong HER-2 protein expression detected by FISH and IHC, respectively. A single DCIS case was amplified in FISH, but not in IHC. Both HER-2 gene amplification and expression could be quantified in microdissected paraffin-embedded tumors using real-time PCR, DNA and RNA being successfully detected in 146 of 150 (97%) and 141 of 150 (94%) samples, respectively. PCR analysis for HER-2 DNA amplification using the LightCycler HER2/neu DNA Quantification kit (Roche Molecular Biochemicals, Mannheim, Germany) correlated fairly well with IHC and FISH. All IHC HER-2 3+ tumors were amplified according to the kit, as was the FISH-amplified DCIS case. DNA-PCR identified five additional tumors as being amplified. Interestingly, all these scored 2+ with the HercepTest, but were negative using FISH. We believe that real-time quantitative PCR analysis of HER-2 DNA amplification following microdissection represents a useful supplementary or perhaps even an alternative technique for establishing HER-2 status in paraffin-embedded tumors.     

HER-2 DNA quantification of paraffin-embedded breast carcinomas with LightCycler real-time PCR in comparison to immunohistochemistry and chromogenic in situ hybridization

OBJECTIVES: To compare the detection of HER-2 status by real-time PCR, on paraffin-embedded breast carcinomas, in respect to immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH). DESIGN AND METHODS: Paraffin-embedded breast carcinomas collected from 85 patients diagnosed with early stage breast cancer were analyzed for HER-2 gene amplification by real-time PCR and CISH, as well as for HER-2 protein expression by IHC. RESULTS: HER-2 gene amplification was observed in 19 (22.4%) of 85 breast cancer patients by real-time PCR and in 19 (22.4%) of 85 patients by CISH. Strong (3+) HER-2 protein over-expression was observed in 13 (15.3%) out of 85 patients. Moreover, there were 4 out of 85 (4.7%) patients that had moderate (2+) HER-2 protein over-expression, while 68 out of 85 (80%) patients had no HER-2 protein over-expression by IHC. There were strong concordance rates between real-time PCR and IHC (79/85, 92.9%, p<0.0001) and real-time PCR and CISH (77/85, 90.6%, p<0.0001). The concordance rate between the three methods was 90.6% (p<0.0001). CONCLUSIONS: Our data show that the results obtained for amplification of HER-2 by real-time PCR on the LightCycler are comparable to those obtained by IHC and CISH.     

Predicting tissue HER2 status using serum HER2 levels in patients with metastatic breast cancer

BACKGROUND: Immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) are reliable ways to identify overexpression or amplification of the HER-2/neu (HER2, symbol ERBB2) gene, but each technique requires a high-quality tissue sample, which may not be available. We investigated whether serum concentrations of the HER2 extracellular domain (ECD) can be used as an alternative to tissue HER2 status in metastatic breast cancer, and we defined an optimal decision-level concentration of serum HER2 for prediction of tissue HER2 status. METHODS: In 195 patients with metastatic breast cancer, we determined HER2 expression by IHC and performed FISH analysis on tumors for which IHC staining was graded as 2+. We measured serum HER2 by immunoassay and used ROC curve analysis to determine optimal serum HER2 ECD concentrations for differentiation between positive and negative HER2 status. RESULTS: IHC results were 0/1+ for 30 (15%) of the patients, 2+ for 89 (46%), and 3+ for 76 (39%). FISH revealed HER2 amplification in 19 (21%) of the IHC 2+ tumors. Mean (SE) serum HER2 ECD was 22.2 (5.1) microg/L in the tissue HER2-negative group, significantly lower than the concentration of 363 (96) microg/L in the tissue HER2-positive group (P<0.0001). ROC curve analysis showed 95% specificity and 62% sensitivity for tissue HER2 positivity at 37 microg/L of serum HER2. CONCLUSION: To use serum HER2 concentration as an alternative to direct determination of tissue HER2 status, we suggest 37 microg/L as a cutoff for predicting positive tissue HER2 with 95% specificity. Sensitivity, however, is low.     

HER-2/neu gene copy number quantified by real-time PCR: comparison of gene amplification,heterozygosity, and immunohistochemical status in breast cancer tissue

BACKGROUND: Amplification of the oncogene HER-2/neu influences breast cancer pathogenesis, and therapy and prognosis may be affected by the degree of amplification. The extent of amplification or protein overexpression typically is analyzed by fluorescence in situ hybridization or immunohistochemistry (IHC), but quantitative PCR techniques have been described that may provide alternatives to these methods. METHODS: We developed a rapid-cycle, real-time PCR assay for quantification of HER-2/neu gene status. We compared results obtained with this assay with short tandem repeat findings by capillary electrophoresis (CE) and with protein overexpression assessments by IHC. Accuracy and linearity were tested on cell lines and with simulation experiments. We analyzed the amplification of HER-2/neu in 51 clinical tissue samples from patients with suspected breast cancer. RESULTS: The intra- and interrun CVs for HER-2/neu quantification by real-time PCR were 12% and 18%, and the CV for different simulated amplification and deletion experiments was <7%. The results for HER-2/neu gene status in cell lines matched the values reported in literature. We detected HER-2/neu amplification by real-time PCR in 11 samples, all from patients with invasive ductal carcinoma. Allelic imbalances were found by CE analyses in three samples and by protein overexpression in six samples; five of these were also detected by real-time PCR. Comparison of the quantification results with known prognostic indices yielded results similar to those reported in several other published studies. CONCLUSIONS: The assay is suitable for accurate and precise quantification of HER-2/neu copy numbers in tumor tissue samples obtained in routine clinical practice.     

乳腺癌HER-2检测方法比价及其与ER、PR、淋巴结转移的相关性分析

【目的】比较免疫组化（IHC）与荧光原位杂交（FISH ）检测乳腺癌 HER‐2的一致性;分析 HER‐2与 ER 、PR 及淋巴结转移的相关性。【方法】采用回顾性分析,统计本院2010年至2014年 IHC 法检测的1003例乳腺癌手术标本 HER‐2蛋白及 ER 、PR 的表达情况,其中434例浸润性癌行 FISH 检测 HER‐2基因扩增状态,比较 IHC 与 FISH 方法检测 HER‐2的差异;分析 IHC 与 FISH 检测乳腺癌 HER‐2与 ER 、PR 的相关性;同时分别分析 HER‐2蛋白表达情况及 HER‐2基因扩增情况与淋巴结转移的相关性。【结果】① IHC 检测1003例乳腺癌 HER‐2蛋白表达（处）阳性率为23．93％,其中行 FISH 检测的434例浸润性癌 HER‐2基因扩增率为26．27％;IHC 检测 HER‐2蛋白（处）的病例与 FISH 检测 HER‐2基因扩增符合率为85．71％;②HER‐2蛋白表达（处）和 HER‐2基因扩增均与 ER 、PR 表达呈显著负相关;③ HER‐2蛋白表达阳性（处）病例中淋巴结转移率为45．16％;HER‐2蛋白表达阴性（0／＋）病例中淋巴结转移率为46．99％,HER‐2蛋白表达可疑（触）病理中淋巴结转移率为49．45％,组间比较差异无统计学意义（ P ＝0．1398）;HER‐2基因扩增病例中淋巴结转移率为51．58％,HER‐2基因无扩增病例中淋巴结转移率为51．99％,两者差异无统计学意义（P ＝0．346）。【结论】IHC 检测 HER‐2蛋白（－）／（＋）和（处）的病例与 FISH 检测 HER‐2基因扩增有较好的一致性;HER‐2蛋白（触）的病例需要进一步行 FISH 检测确定 HER‐2基因扩增状态,以准确指导把向药物治疗;IHC 与 FISH 检测乳腺癌 HER‐2均与 ER 、PR 呈显著负相关性,与淋巴结转移无相关性。     

FISH和IHC检测乳腺癌患者HER2/neu基因影响因素的研究

目的 观察乳腺癌石蜡包埋组织荧光原位杂交(FISH)和免疫组化(IHC)检测乳腺癌患者HER2/neu基因状态,比较不同切片方法及不同结果判读人员的经验区别.方法 55例乳腺癌石蜡包埋组织分组切片和观察判读,观察1组:由技术人员选定区域切片,平行检测HER2/neu基因状态;观察2组:先切片做HE染色,由初级病理医师根据病理图像选定区域后切片,平行检测HER2/neu基因状态,并由初级病理医师分析结果;观察3组,由中、高级病理医师根据病理图像选定区域后重新切片检测,判读分析.结果 观察1组和观察2组,IHC(-)和IHC(1+)与FISH检测一致性较好,IHC(2+)和IHC(3+)与FISH检测的相符率分别为33.33%/33.33%,50.00%/100.00%;2组的一致性分别为中等和较好(K1=0.478,K2=0.659);共有10例患者入选观察3组,重新切片后,6例患者结果不同,其中3例为取材误差,3例为判读误差,3例取材误差均出现在观察1组.结论 FISH和IHC检测乳腺癌患者HER2/neu基因状态受到制片、结果判读等多种因素影响,实验室应制定标准化的操作程序,严密的质量控制和质量保证措施,才可得到准确而可靠的结果.     

HER2 testing in patients with breast cancer: poor correlation between weak positivity by immunohistochemistry and gene amplification by fluorescence in situ hybridization

OBJECTIVE: To evaluate amplification of the HER-2/neu gene by fluorescence ir situ hybridization (FISH) in tumors with weakly positive (2+) immunohistochemical staining. METHODS: A total of 1556 breast tumor biopsy specimens were referred to Mayo Medical Laboratories, Rochester, Minn, for HER2 testing between August and December 2000. Immunohistochemical (IHC) analysis was performed with use of a diagnostic test for the assessment of HER2 overexpression, the HercepTest. The IHC-stained slides were interpreted and scored on a scale ranging from 0 to 3+ according to Food and Drug Administration-approved guidelines. All specimens scored as 2+ were also routinely evaluated by FISH with use of a HER-2/neu DNA probe kit (PathVysion). Specimens were determined to be amplified if the ratio of HER-2/neu signals to chromosome 17 centromere (CEP17) signals was higher than 2.0. RESULTS: Thirty-eight percent of the specimens evaluated with the HercepTest were scored 0, 35% were 1+, 14% were 2+, and 13% were 3+. Of the 216 tumor specimens scored as 2+, 26 (12%) had a high level of HER-2/neu gene amplification, 54 (25%) demonstrated duplication of HER2, 4 (2%) deleted HER-2/neu and/or CEP17, and 123 (57%) had no apparent HER-2/neu anomaly, no apparent CEP17 anomaly, nor apparent single gain (aneusomy) of CEP17. CONCLUSION: We recommend that all specimens with a 2+ HercepTest result be evaluated by FISH for HER-2/neu gene amplification. The results of both assays should be considered before making a decision to recommend anti-HER2 therapy.     

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。     

Quantification of HER2/neu gene amplification by competitive pcr using fluorescent melting curve analysis

BACKGROUND: Molecular detection methods for HER2/neu gene amplification include fluorescence in situ hybridization (FISH) and competitive PCR. We designed a quantitative PCR system utilizing fluorescent hybridization probes and a competitor that differed from the HER2/neu sequence by a single base change. METHODS: Increasing twofold concentrations of competitor were coamplified with DNA from cell lines with various HER2/neu copy numbers at the HER2/neu locus. Competitor DNA was distinguished from the HER2/neu sequence by a fluorescent hybridization probe and melting curve analysis on a fluorescence-monitoring thermal cycler. The percentages of competitor to target peak areas on derivative fluorescence vs temperature curves were used to calculate copy number. RESULTS: Real-time monitoring of the PCR reaction showed comparable relative areas throughout the log phase and during the PCR plateau, indicating that only end-point detection is necessary. The dynamic range was over two logs (2000-250 000 competitor copies) with CVs < 20%. Three cell lines (MRC-5, T-47D, and SK-BR-3) were determined to have gene doses of 1, 3, and 11, respectively. Gene amplification was detected in 3 of 13 tumor samples and was correlated with conventional real-time PCR and FISH analysis. CONCLUSION: Use of relative peak areas allows gene copy numbers to be quantified against an internal competitive control in < 1 h.     

Prognostic markers in breast cancer: the reliability of HER2/neu status in core needle biopsy of 325 patients with primary breast cancer

INTRODUCTION: The assessment of HER2/neu overexpression in tissue provides information about one of the most relevant prognostic and predictive markers in breast cancer: overexpression of HER2/neu is associated with worse prognosis in primary breast cancer. Since core needle biopsy is increasingly used for the diagnosis of breast cancer, the purpose of this study was to assess the reliability of HER2/neu evaluation using this technique in patients with primary breast cancer. PATIENTS AND METHODS: We investigated the accuracy of immunohistochemical assessment of HER2/neu in core needle biopsies compared with surgically obtained specimens in 325 patients with primary breast cancer. In patients strongly positive for HER2/neu, additional fluorescence in situ hybridization (FISH) analysis of needle biopsies was performed. RESULTS: Using immunohistochemistry alone, accuracy of HER2/neu assessment in core biopsies in relation to surgically removed specimens was 92% and increased to 96% with additional FISH analysis (weighted Kappa coefficient: 0.86). DISCUSSION: As proven with this large series of patients, the assessment of HER2/neu status by core needle biopsy in breast cancer is accurate. Notwithstanding, in order to minimize the number of false-positive results, strongly positive core needle biopsies identified using immunohistochemistry should be confirmed by FISH analysis.     

HER-2/neu assessment in breast cancer by immunohistochemistry and fluorescence in situ hybridization: comparison of results and correlation with survival.

BACKGROUND: HER-2/neu immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) results guide breast cancer therapy; however, few studies compared the results and no published studies have correlated them with patient outcome. METHODS AND RESULTS: We compared results, cost, and turnaround time in 117 archival, invasive breast carcinomas and compared 50-month survival in 65 of these cases using commercial HER-2/neu IHC and FISH assays. Twenty-one of 112 FISH (19%) and 33 of 117 IHC cases (28%) were positive. Concordance was high overall (88%; 98 of 112 cases) and in IHC 3+ cases (88%; 14 of 16 cases) but low in IHC 2+ cases (35%; six of 17 cases). Survival correlated with IHC results in 3+ cases (P =.02) and FISH cases with signal ratio greater than 4.0 (P =.03), but not in IHC 2+ cases (P=.7). Cost and turnaround time were greater for FISH. CONCLUSION: IHC is appropriate for initial HER-2/neu assessment; however, patients with tumors scored less than 3+, particularly those interpreted as 2+, would benefit from FISH to more accurately assess HER-2/neu status and avoid inaccurate prognostication and inappropriate treatment.     

乳腺癌HER-2基因扩增和蛋白表达与ER、PR的关系及临床意义

目的探讨HER-2基因及其蛋白在乳腺癌中扩增及表达状况,并研究其与ER、PR的相关性。方法用FISH技术和IHC技术检测51例乳腺癌新鲜标本中的HER-2基因的扩增及蛋白表达情况,并检测ER、PR进行对比分析。结果51例中10例HER-2蛋白表达强阳性（＋＋＋）,6例阳性（＋＋）,2例弱阳性（＋）,33例阴性,其中基因过表达率（IHC检测＋＋／＋＋＋）为31．4％;FISH检测中基因扩增14例,37例无扩增,扩增率为27．5％。IHC（＋＋／＋＋＋）与FISH检测符合率87．5％（P〈0．05）,ER、PR表达情况与HER-2基因及蛋白扩增呈负相关,ER和PR均为阴性的HER-2基因扩增率及蛋白表达率明显高于ER和（或）PR阳性的患者（IHC50％与24．3％,P〈0．05;FISH42．9％与19．4％,P〈0．05）。结论HER-2的扩增和蛋白过度表达提示乳腺癌预后不良,HER-2与ER、PR在乳腺癌的治疗中起重要作用,IHC可以作为检测HER-2基因状态的首选方法,但对于HER-2（＋＋）需要进一步进行FISH检测。     

人类表皮生长因子受体2在乳腺癌表达与临床的相关病理特征分析

目的 评估人类表皮生长因子受体2(HER2)预测价值,并分析其与常见的组织病理学参数的相关性。方法 收集陕西省人民医院2011年～2014年之间160例接受手术治疗的乳腺癌患者组织标本,通过免疫组织化学方法(IHC)和荧光原位杂交(FISH)检测 HER2水平,通过χ²检验用来评估HER2基因扩增状况及不同临床病理特性的相关性,临床参数包括:肿瘤大小、组织学分级、雌激素受体(ER)和孕激素受体(PR)表达,年龄、绝经情况和Ki-67指数。结果 HER-2表达与组织学分级、淋巴结转移、ER水平、PR,Ki-67指数差异存在统计学意义(均P<0.05)。相对于HER-2⁺组患者,HER-2^-病变多表现为雌激素ER阴性、孕激素阴性、ER阴性、淋巴结阴性、ki-67≥20%。结论 HER-2表达与多种临床病理因素存在相关性。     

中国人群胰腺癌与胃癌HER2/neu基因表达

  目的  探讨胰腺癌与胃癌组织中HER2/neu基因表达状态及其在治疗与评估临床结局中的作用。  方法  应用免疫组织化学和多色荧光原位杂交技术, 检测北京协和医院手术切除的81例胰腺导管癌及癌旁胰腺组织和100例胃癌及癌旁胃组织标本中HER2/neu蛋白表达及基因状态的变化, 分析HER2/neu蛋白表达与基因状态间的关系及其与胰腺癌和胃癌临床病理改变间的关系。  结果  免疫组织化学显示, 81例胰腺癌组织中9例(11.1%)HER2/neu蛋白表达阳性(2+及3+), 100例胃癌组织中13例(13%)HER2/neu蛋白表达阳性(2+及3+); 荧光原位杂交结果显示, 81例胰腺癌组织中15例(18.5%)HER2/neu基因扩增, 100例胃癌组织中11例(11%)HER2/neu基因扩增。胰腺癌HER2/neu基因扩增与淋巴结转移有显著相关性(P=0.001)。胃癌组织中6例HER2/neu蛋白3+病例均显示HER2/neu基因扩增, 且胃癌组织HER2/neu蛋白表达与其基因扩增有显著相关性(P < 0.0001)。无论是癌旁胰腺组织还是癌旁胃组织均未检测到HER2/neu蛋白表达及基因扩增。  结论  胰腺癌组织HER2/neu蛋白表达与基因扩增不一致, 而胃癌组织HER2/neu蛋白表达与基因扩增有显著相关性。HER2/neu基因异常可能在胰腺癌及胃癌的发生发展中起着重要作用, 对胰腺癌及胃癌患者进行HER2/neu基因状态检测可能对其靶向治疗具有一定的指导意义。     

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.     

乳腺癌HER-2基因荧光原位杂交检测的临床应用

目的研究荧光原位杂交(FISH)用于乳腺癌HER-2检测的临床应用及HER-2基因扩增与乳腺癌临床病理的相关性。方法应用FISH技术和免疫组化(IHC)技术检测40例乳腺浸润性导管癌石蜡包埋标本,对比分析。结果IHC检测CerbB-2(3+)/(2+)/(1+或0),其FISH阳性率分别为85.7%、50%、5.9%。24例腋窝淋巴结阳性者,其FISH检测HER-2基因扩增10例(P=0.0399)。ER/PR阴性8例,其HER-2基因扩增6例(P0.05)。结论IHC检测HER-2蛋白有很高的假阳性及假阴性,FISH有效性及准确性显著高于IHC,可在临床广泛推广应用。HER-2基因扩增与腋窝淋巴结阳性相关。     

Impact of polysomy 17 on HER-2/neu immunohistochemistry in breast carcinomas without HER-2/neu gene amplification

Her-2/neu, a proto-oncogene located on chromosome 17, is an important biomarker in breast carcinoma. Immunohistochemistry (IHC) is currently the most widely used method for assessing Her-2/neu status. Some IHC-positive cases do not show Her-2/neu gene amplification by fluorescence in situ hybridization (FISH). It has been suggested that some of these IHC “false positive” results may in part be due to increased copy number of chromosome 17 resulting in increased Her-2/neu protein expression. We analyzed IHC and FISH data from 561 cases of invasive breast carcinoma to test this hypothesis. IHC and FISH for Her-2/neu were performed on formalin-fixed, paraffin-embedded sections of 561 invasive breast carcinomas. The IHC results were interpreted as 0, 1+, 2+, or 3+ according to the manufacturer’s recommended criteria. The FISH results were expressed as a ratio of Her-2/neu/chromosome 17 and were interpreted as positive (> = 2.0) or negative (<2.0) for gene amplification according to the manufacturer’s recommended scoring system. We found that in IHC 3+/FISH-negative cases (n = 15) both the average chromosome 17 copy number and the average Her-2/neu copy number were significantly higher than that in IHC (0 to 2+)/FISH-negative cases (n = 411) (2.45 vs. 1.68; P < 0.0001, and 3.19 vs. 1.95; P < 0.0001, respectively). In contrast, the IHC 2+/FISH-negative cases did not exhibit a significantly increased number of chromosome 17 compared to IHC 0 to 1+ cases. In addition, the average copy number of chromosome 17 in FISH-positive cases (n = 135) was significantly higher than that in FISH-negative cases (n = 426) (2.27 vs. 1.70; P < 0.0001), indicating a general association of increased chromosome 17 copy number with Her-2/neu gene amplification. Thus, our data suggest that IHC 3+ immunostaining without scorable gene amplification may indeed be, at least in some cases, the result of increased Her-2/neu protein expression secondary to an increased copy number of chromosome 17, associated with an increased total number of Her-2/neu gene copies per tumor cell.     

HER-2/neu在乳腺癌血清中的表达及意义

目的 分析乳腺癌HER-2／neu表达水平并探讨其临床意义。方法 用ELISA方法分析127例乳腺癌患者，30例良性疾病及40名健康体检者血清可溶性HER-2／neu水平并用免疫组化（mc）方法分析乳腺癌组织切片中癌细胞的HER-2／neu染色。结果乳腺癌患者血清中可溶性HER-2／neu水平与正常对照和良性乳腺疾病相比，有显著性差异（P〈0．05）。随TNM分期进展，乳腺癌患者可溶性HER-2／neu阳性率逐步升高。结论 乳腺癌患者血清中可溶性HER-2／neu水平呈过度表达，而且与免疫组化结果呈正相关。