Evaluation of the quantitative analytical methods real-time PCR for HER-2 gene quantification and ELISA of serum HER-2 protein and comparison with fluorescence in situ hybridization and immunohistochemistry for determining HER-2 status in breast cancer patients

BACKGROUND: HER-2 status is generally determined by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH). Both methods are only semiquantitative, require a tumor sample, and can be difficult to reproduce. We compared these methods with 2 quantitative approaches, one measuring HER-2 gene copy number in tissue by real-time quantitative PCR (qPCR), and the other measuring shed HER-2 protein in serum by ELISA in patients with metastatic disease. METHODS: We analyzed 52 cases of metastatic breast cancer for which both serum collected at the diagnosis of metastasis and stored primary breast tumor specimens were available. The within- and between-run imprecision of real-time qPCR and ELISA were evaluated according to Clinical and Laboratory Standards Institute (formerly known as NCCLS) recommendations. Concordance among the 4 methods was assessed by calculating the kappa statistic and its 95% confidence interval (95% CI). RESULTS: The CVs for within- and between-run imprecision were both <10% with qPCR and ELISA. There was good agreement of results between qPCR and IHC (kappa = 0.81; 95% CI, 0.64-0.99), qPCR and FISH (kappa = 0.77; 95% CI, 0.58-0.96), ELISA and IHC (kappa = 0.65; 95% CI, 0.41-0.89); and ELISA and FISH (kappa = 0.69; 95% CI, 0.46-0.92). CONCLUSIONS: Measurements of HER-2 gene expression by qPCR and of serum HER-2 protein by ELISA are highly reproducible approaches for determining HER-2 status in metastatic breast cancer. In addition, ELISA eliminates the need for biopsy.     

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.     

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。     

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.     

荧光原位杂交法与免疫组织化学法检测乳腺癌HER-2基因扩增及蛋白表达的对比研究

目的比较桂林市荧光原位杂交法（FISH）和免疫组织化学法（IHC）检测乳腺癌组织中人表皮生长因子受体（HER-2）基因扩增及其蛋白表达情况的一致性．探讨FISH与IHC检测乳腺癌HER-2基因状态的临床意义。方法应用IHC和FISH对50例乳腺癌患者HER-2蛋白表达、基因扩增情况及17号染色体倍体性进行检测,分析IHC与FISH检测HER-2基因状态的差异以及HER-2蛋白状态与17号染色体倍体性的相关性。结果FISH与IHC结果总的符合率为82．0％．两者之间存在较好的一致性fkappa=0．6401,FISH检测IHC结果为3＋、2＋和0／1＋者的符合率分别为92．9％、70．0％和80．8％;IHC3＋及2＋者出现17号染色体多体性的几率比IHC0／1＋者高（P〈0．05）。结论FISH可以准确和稳定地检测乳腺癌组织中HER-2的基因状态及17号染色体倍体性：FISH检测IHC3＋者符合率较高,FISH与IHC的差异主要在于IHC2＋和IHC0／＋组,IHC仅作为检测HER-2基因状态的初筛方法．而IHC结果为2＋或0／1＋者的HER-2基因状态必须应用FISH进一步确定。     

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。     

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.     

胃癌组织HER-2基因扩增和蛋白表达检测及临床意义

目的 探讨免疫组化（immunohistochemistry,IHC）法检测胃癌HER-2蛋白表达和荧光原位杂交（fluorescence in situ hybridization,FISH）法检测HER-2基因扩增的临床意义.方法 收集256例胃癌患者手术切除及胃镜活检的胃癌组织标本,分别采用IHC法及FISH法检测胃癌组织HER-2蛋白表达及基因扩增状态,并对结果进行统计学分析.结果 256例胃癌组织标本中,有26例HER-2蛋白表达阳性,阳性率为10.16％;46例HER-2基因有扩增,阳性率17.97％.26例HER-2蛋白表达（3＋）的标本中,24例有HER-2基因扩增,2例无扩增;95例HER-2蛋白表达（2＋）的标本中,20例有基因扩增,75例无扩增;90例HER-2蛋白表达（1＋）的标本中,2例有基因扩增,88例无扩增;45例HER-2蛋白表达（0）的标本中,HER-2基因均无扩增.IHC法和FISH法检测HER-2表达阳性率差异无统计学意义（P＞0.05）.结论 IHC法检测胃癌HER-2蛋白表达可作为临床治疗的初筛,HER-2蛋白表达（2＋）的患者应常规行FISH法,以确定HER-2基因扩增状态.FISH法检测结果具有较高的稳定性和可靠性,联合IHC法可更准确和客观评价胃癌预后,并指导临床选用靶向药物治疗.     

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。     

The clinical significance between activation of nuclear factor kappa B transcription factor and overexpression of HER-2/neu oncoprotein in Taiwanese patients with breast cancer

BACKGROUND: This study investigated the role of nuclear factor-kappa B (NF-kappaB) activity in human breast cancer with overexpression of HER-2/neu oncoprotein, as well as its role on expression of different histological grades of cancer cells taken from Taiwanese breast cancer patients. MATERIALS AND METHODS: Specimens were collected from 82 female breast cancer patients. The HER-2/neu oncoprotein was measured by immunohistochemistry. NF-kappaB activity expression was assessed by the electrophoretic mobility shift assay, and confirmed by the supershift technique using anti-P65 antibody in both breast cancer tissue and the adjacent normal tissue. The histological grades were measured by Modified Bloom-Richardson Grading Scheme. RESULTS: Of the 82 cancer specimens, 81 (98.7%) showed higher or equal expressions of NF-kappaB activity when compared to the adjacent normal tissue. Fifty-five cases (67.1%) had higher levels of NF-kappaB activity in the cancerous tissue than in the adjacent normal tissue (p<0.005). With regard to tumor size, steroid receptors, stages, histological types, and node status, there were no statistically significant differences in NF-kappaB activity between cancerous tissues and adjacent normal tissues. However, significantly higher expressions of NF-kappaB activity were seen in those cases with positive HER2/neu oncoprotein, poorly differentiated histological grades, high nuclear pleomorphisms, and high mitotic counts (p<0.05). Positive HER-2/neu overexpression of oncoprotein had higher NF-kappaB activity (86%) than negative overexpression (60%) (p<0.05). It has been shown that the NF-kappaB activity increases in the HER-2/neu oncoprotein overexpression in human breast cancer. CONCLUSION: Overexpression of HER-2/neu gene could induce NF-kappaB activity in human breast cancer cells, as has been confirmed in other research on cell lines.     

应用FISH和IHC技术检测中国乳腺癌患者HER-2基因状态及蛋白表达的前瞻性多中心研究

目的 对比研究乳腺癌患者IHC检测c-erbB2蛋白表达和FISH检测HER-2/neu基因扩增情况,并探讨HER-2基因状态与各临床病理特征的相关性.方法 本研究为全国73家中心参与的前瞻性研究.收集2007年10月至2009年9月乳腺癌患者标本3 249份,应用IHC和FISH两种方法分别检测3 249份乳腺癌患者手术的石蜡标本c-erbB2蛋白表达和HER-2基因扩增情况,并分析HER-2基因状态与患者各临床病理特征的关系.结果 全组患者IHC检测c-erbB2蛋白表达阳性率为46.9%(1477/3149),FISH检测HER-2基因扩增率为42.6%(1342/3149),其中IHC评分为3+和0分时,与FISH检测的一致性较高,分别为94.1%(892/948)和89.9%(660/734),而IHC 1+和2+组与FISH的一致性较低,分别为71.0%(514/725)和55.9%(415/742).同时,HER-2基因扩增与激素状态中雌激素与孕激素均阴性(r=0.45,P<0.01)、组织分级Ⅲ级(r=0.51,P<0.01)、淋巴结转移数目多于4枚(r=0.35,P<0.01)、临床分期Ⅲ/Ⅳ期(r=0.33,P<0.01)、肿瘤直径>2 cm(r=0.38,P<0.01)、绝经后(r=0.24,P<0.01)有相关性,与年龄(r=0.36,P=0.068)、CA125(r=0.11,P=0.722)、CA153(r=0.23,P=0.45)和CEA(r=0.22,P=0.074)表达情况、淋巴结转移(r=0.15,P=0.18)、肿瘤个数(r=0.21,P=0.056)及脉管瘤栓(r=0.12,P=0.133)无相关性.结论 FISH和IHC两种方法检测HER-2表达状态具有较高的一致性.结合实际情况包括费用仪器等限制,在IHC作为初筛的基础上,仍然推荐FISH作为检测HER-2基因扩增的标准方法.FISH技术检测乳腺癌患者HER-2基因表达状态可为临床指导用药和预后评价提供更可靠的依据.     

Potential clinical utility of serum HER-2/neu oncoprotein concentrations in patients with breast cancer

BACKGROUND: The HER-2/neu oncogene and its p185 receptor protein are indicators of a more aggressive form of breast cancer. HER-2/neu status guides Herceptin therapy, specifically directed to the extracellular domain (ECD) of the HER-2/neu oncoprotein. The HER-2/neu ECD is shed from cancer cells into the circulation and is measurable by immunoassay. METHODS: We performed a systematic review of the peer-reviewed literature on circulating ECD with respect to prevalence, prognosis, prediction of response to therapy, and monitoring of breast cancer. RESULTS: The prevalence of increased ECD in patients with primary breast cancer varied between 0% and 38% (mean, 18.5%), whereas in metastatic disease the range was from 23% to 80% (mean, 43%). Some women with HER-2/neu-negative tumors by tissue testing develop increased ECD concentrations in metastatic disease. Increased ECD has been correlated with indicators of poor prognosis, e.g., overall survival and disease-free survival. Increased ECD predicts a poor response to hormone therapy and some chemotherapy regimens but can predict improved response to combinations of Herceptin and chemotherapy. Many studies support the value of monitoring ECD during breast cancer progression because serial increases precede the appearance of metastases and longitudinal ECD changes parallel the clinical course of disease. CONCLUSIONS: The monitoring of circulating HER-2/neu ECD provides a tool for assessing prognosis, for predicting response to therapy, and for earlier detection of disease progression and timely intervention with appropriate therapy.     

Relationship of serum HER-2/neu and serum CA 15-3 in patients with metastatic breast cancer

BACKGROUND: Serum HER-2/neu antigen concentrations have been reported to correlate with increased tumor volume in patients with breast cancer. We measured serum CA 15-3, a surrogate marker of disease burden, and correlated serum CA 15-3 with serum HER-2/neu and analyzed the association of both markers with clinical outcomes. METHODS: Pretreatment serum samples from 566 patients were retrospectively analyzed from 2 phase III clinical trials of estrogen receptor-positive (ER(+)), ER(-)/progesterone receptor-positive, or ER status unknown metastatic breast cancer patients randomized in two similar studies to receive second-line hormone therapy with either megestrol acetate or an aromatase inhibitor (fadrozole). The extracellular domain of the HER-2/neu (c-erbB-2) oncogene and serum CA 15-3 were measured by ELISA on the Bayer Immuno 1. RESULTS: Serum HER-2/neu protein was increased in 168 patients (30%), and CA 15-3 was increased in 337 (60%) patients. Serum CA 15-3 and HER-2/neu were weakly correlated (r = 0.39; P <0.0001). The clinical benefit (complete responses plus partial responses plus stable disease) of endocrine therapy was significantly lower in patients with increased serum HER-2/neu. When adjusted for serum HER-2/neu, serum CA 15-3 was not predictive of response rates. The median time to progression was shorter in patients with increased serum HER-2/neu (89 days) compared with patients with normal serum HER-2/neu (176 days). Survival was significantly shorter in patients with increased serum HER-2/neu (513 vs 869 days; P <0.0001) or increased serum CA 15-3 (689 vs 939 days; P <0.0001). This observation was confirmed by multivariate analysis. CONCLUSIONS: Serum HER-2/neu is a significant independent predictive and prognostic factor in hormone receptor-positive metastatic breast cancer, even when adjusted for tumor burden as measured by CA 15-3. The combination of increased serum HER-2/neu and increased serum CA 15-3 predicts a worse prognosis than does increased CA 15-3 alone.     

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.     

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.     

Bortezomib (PS-341, Velcade) increases the efficacy of trastuzumab (Herceptin) in HER-2-positive breast cancer cells in a synergistic manner

BACKGROUND: Preclinical and clinical studies have shown that the proteasome inhibitor bortezomib (PS341, Velcade) is highly effective when combined with chemotherapeutic agents. The value of trastuzumab (Herceptin) in HER-2-positive (3+ score by immunohistochemistry or fluorescence in situ hybridization positive) breast cancer is also known; however, the response rate is <40% for metastatic breast cancer. These two pharmacologic agents prevent nuclear factor-kappaB (NF-kappaB) activation and induce nuclear accumulation of the cyclin-dependent kinase inhibitor p27(kip1), suggesting that combining bortezomib with trastuzumab could increase trastuzumab efficacy. METHODS: Drug cytotoxicity, both individually and together, and drug effects on p27 localization and NF-kappaB activation were investigated on four breast cancer cell lines: SKBR-3 (HER-2+++), MDA-MB-453 (HER-2++), HER-2-transfected MCF-7 (HER-2+++), and MCF-7 (HER-2-). RESULTS: Bortezomib induced apoptosis in HER-2-positive and HER-2-negative breast cancer cells in a dose- and time-dependent manner. Together, these drugs induced apoptosis of HER-2++/+++ cells at low concentrations, which had no effect when used alone, indicating there was a synergistic effect. Sequential treatment (trastuzumab then bortezomib) induced either necrosis or apoptosis, depending on the trastuzumab preincubation time. Susceptibility to bortezomib alone and the drug combination correlated with NF-kappaB activity and p27 localization. CONCLUSIONS: The addition of bortezomib to trastuzumab increases the effect of trastuzumab in HER-2+++/++ cell lines in a synergistic way. This effect likely results from the ability of these two drugs to target the NF-kappaB and p27 pathways. The potential clinical application of this drug combination is under current evaluation by our group in a phase 1 clinical trial.     

ER、PR及HER-2受体在乳腺癌原发灶及复发灶间表达差异及意义

目的:乳腺癌内分泌治疗及免疫靶向药物赫赛汀生物治疗的前提是相应的雌激素受体、孕激素受体的阳性表达及HER-2癌基因蛋白的过表达。目前对复发转移的乳腺癌病例受体表达情况的判断主要依据原发灶,而忽略了原发灶和复发转移灶之间可能存在的差异。本研究着重研究ER、PR及HER-2癌基因蛋白在乳腺癌原发灶及复发转移灶之间的表达差异,探讨其临床意义。方法:采用免疫组织化学检测45例乳腺癌患者原发灶及复发灶中ER、PR及HER2表达,分析研究ER、PR及HER2的变化率。结果:原发灶中ER、PR的阳性率分别为62.2%,60%(28/45,27/45),明显高于复发灶(37.8%17/45,31.1%14/45)(P<0.01)。复发灶中HER-2阳性率为62.2%(28/45),明显高于原发灶(26.7%12/45)(P<0.01)。结论:ER、PR及HER-2癌基因蛋白在乳腺癌原发灶和复发灶之间的表达存在显著性差异,所以在对复发乳腺癌进行临床治疗时,应考虑上述3种指标在复发灶中的确切状况。     

Serum HER-2/neu in the management of breast cancer patients

The clinical role of HER-2/neu, a 185 kD epithelial transmembranous protein, has evolved after the approval of the anti-HER-2/neu targeted monoclonal antibody trastuzumab (Herceptin) for the therapy of metastatic breast cancer. The extracellular domain of HER-2/neu undergoes proteolytic cleavage from the full-length protein by metalloproteases, and is shed into the blood as a circulating antigen. While HER-2/neu gene amplification and/or protein overexpression are detected in approximately 25% of primary breast cancers, serum HER-2/neu levels are elevated beyond the upper limit of normal in 50 to 60% of stage IV breast cancer patients. HER-2/neu in serum can be detected by enzyme immunoassays (manual and automated versions). It has been shown to have prognostic and predictive information in breast cancer patients. Monitoring for recurrence by serum HER-2/neu reaches a high sensitivity for HER-2/neu positive tumors. Longitudinal follow-up of patients during any kind of systemic therapy allows for monitoring of the therapeutic success. When utilized in these applications, serum HER-2/neu testing is complementary to HER-2/neu tissue results and to the determination of classical tumor markers such as CA 15-3, CA 27.29 and CEA, which are not targeted by specific forms of systemic therapy.     

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.