应用荧光原位杂交技术检测骨肉瘤患者人表皮生长因子2受体基因扩增状态

目的 应用荧光原位杂交技术检测骨肉瘤人表皮生长因子2受体(HER-2/neu)基因是否存在扩增.方法 以HER-2/neu及17号染色体为探针,应用荧光原位杂交技术分析23例骨肉瘤病例HER-2/neu基因扩增情况,以HER-2/neu基因扩增阳性的乳腺癌病例作为阳性对照.结果 FISH检测23例骨肉瘤标本,无一例出现HER-2/neu基因扩增.结论 骨肉瘤中出现HER-2/neu基因扩增的频率极低,骨肉瘤患者可能不适合应用以HER-2/neu基因为靶点的靶向性药物.     

Distribution of hsp-27 and HER-2/neu in in situ and invasive ductal breast carcinomas

Background: The overexpression of heat shock protein 27 (hsp-27) in early-stage breast cancer is associated with histopathologic features of poor prognosis and clinically with an increased probability of disease recurrence. Hsp-27 is overexpressed in 25% of invasive ductal carcinomas (IDC); however, its distribution in ductal carcinoma in situ (DCIS) and DCIS associated with IDC has not been investigated. We postulated that hsp-27 might be detected and variably expressed in DCIS and, like HER-2/neu oncoprotein expression, might be a tumor-specific marker worthy of future clinical investigation. Methods: To test these hypotheses, the distribution of hsp-27 in noncomedo and comedo DCIS, and DCIS associated with IDC, was evaluated by immunohistochemistry and compared with HER-2/neu expression within the same cancers. Results: Hsp-27 was overexpressed in 28 of 47 (60%) cases of DCIS; expression in pure DCIS was 16 of 24 (67%), and 12 of 23 (50%) in DCIS associated with IDC. Hsp-27 expression by in situ and invasive components of the same neoplasm were concordant in 22 of 23 (95%) cases tested. Comedo variants appeared to have somewhat higher hsp-27 expression than noncomedo DCIS, whether or not there was an associated IDC. These results are reminiscent of HER-2/neu oncoprotein expression in DCIS and DCIS associated with IDC observed by others. However, although 4 of 22 (18%) cancers containing DCIS + IDC expressed HER-2/neu, no relationship with hsp-27 expression in the same cancers was observed. Conclusions: We found a high incidence of hsp-27 overexpression in DCIS and in DCIS associated with IDC. This rate is twice that previously observed in IDC alone. Hsp-27 expression is independent of HER-2/neu expression.     

HER-2/neu基因在原发性肝癌中的表达及意义

目的 探讨原癌基因HER-2／neu在原发性肝癌(PHC)、肝硬化及正常肝组织中的表达状况及意义。探讨应用单克隆抗体Herceptin治疗肝癌的可能性。方法 采用免疫组织化学的方法检测肝癌、肝硬化及正常肝组织中HER-2／neu蛋白的表达。结果 HER-2／neu在(PHC)、肝硬化及正常肝组织中的表达率分别为37．5％(15／40)、10％(1／10)和无表达；其表达与PHC的类型无关，与肿瘤包膜侵犯、病理分级及肿瘤转移有关；而与肿瘤大小、AFP水平及肝硬化无关。结论 HER-2／neu基因在PHC的侵袭性生长转移和发展中可能具有一定的促进作用，可作为PHC独立的预后因子；可考虑应用Herceptin对PHC进行生物学靶向治疗。     

Expression of cyclooxygenase-2 in breast carcinogenesis and its relation to HER-2/neu and p53 protein expression in invasive ductal carcinoma

The purpose of this study was to evaluate cyclooxygenase-2 (COX-2) expression in the successive steps of breast carcinogenesis and to determine its correlation with HER-2/neu and p53 expression in invasive ductal carcinomas of the breast. Immunohistochemical staining with anti-COX-2 antibody was performed in normal breast tissue, usual hyperplasia, ductal carcinoma in situ, and invasive ductal carcinoma. Expression of COX-2 in invasive ductal carcinoma was correlated with immunohistochemical expression of HER-2/neu and p53 protein. COX-2 expression was found to be progressively elevated along the continuum from normal breast tissue to invasive ductal carcinoma (P<0.001). COX-2 expression significantly correlated with p53 and HER-2/neu protein expression (P<0.05 and P<0.001). On multivariate analysis, only TNM stage and elevated COX-2 expression correlated with survival. Our results suggest that COX-2 may be involved in the carcinogenesis of the breast and may be an independent prognostic indicator in patients with invasive ductal carcinoma. HER-2/neu and p53 are likely to be involved in the regulation of COX-2 expression in invasive ductal carcinomas of the breast.     

Prognostic values of p53 and HER-2/neu coexpression in invasive bladder cancer in Taiwan

OBJECTIVES: To explore the clinical significance of p53 and HER-2/neu coexpression by immunohistochemistry in patients with invasive bladder cancer in Taiwan. METHODS: Paraffin-embedded tumor blocks were obtained from 67 patients with invasive bladder cancer subjected to radical cystectomy, bilateral lymph node dissection, and urinary diversion with or without systemic chemotherapy. Two observers (N.H.C. and T.S.T.), blinded to clinical outcome, reviewed the immunohistochemical staining for p53 (PAb1801) and HER-2/neu (Ab-17). The results were analyzed for progression-free survival and patient survival. RESULTS: Positive staining for p53 and HER-2/neu was found in 30 (44.8%) and 39 (58.2%) patients. In contrast to HER-2/neu, p53 expression was significantly associated with tumor grade and pathologic stage (p = 0.040 and 0.004, respectively), and tended to be related to the nodal status (p = 0.080). Most importantly, coexpression of p53 and HER-2/neu significantly correlated with nodal metastases (p = 0.020). Univariate and multivariate analysis revealed p53 and nodal status as two independent prognostic factors. Additionally, patients with p53 and HER-2/neu coexpression had the shortest time to relapse and overall survival, irrespective of whether adjuvant chemotherapy was given or not (p = 0.005 and 0.030). CONCLUSIONS: In invasive bladder cancer, p53 was an important prognostic factor since its expression correlated with tumor grade and stage, even nodal status, whereas HER-2/neu did not show prognostic significance. Tumors with p53 and HER-2/neu coexpression were associated with nodal metastases, probably resulting in decreased progression-free survival. Although some basic studies provide some important supports, studies including larger patient cohorts would still be required to prove the hypothesis that p53 and HER-2/neu-coexpressing tumors have a worse prognosis and are more resistant to a cisplatin-based multidrug regimen.     

Detection of HER-2/neu gene amplification in breast cancer using a novel polymerase chain reaction/ligase detection reaction technique

BACKGROUND: Gene amplification is the primary mechanism of HER-2/neu overexpression in breast cancer and is a strong predictor of prognosis. Currently screening for HER-2/neu gene amplification in breast cancer is done by fluorescent in-situ hybridization (FISH), which is accurate but costly and labor intensive. We have evaluated a new PCR (polymerase chain reaction)-based assay for the detection of HER-2/neu gene amplification in human breast cancer. STUDY DESIGN: A total of 15 breast cancer cell lines and 14 breast cancer specimens were evaluated. HER-2/neu status of the tumors was evaluated by FISH and then assessed using a quantitative polymerase chain reaction/ligase detection reaction (PCR/LRD) technique. RESULTS: Amplification of the HER-2/neu gene was detected in seven cell lines previously reported to have amplification and no amplification was found in any of the six that had been reported not to have amplification. In the assessment of breast specimens the PCR/LDR and FISH assays were in complete agreement. All 10 tumors with amplification by FISH were also amplified by PCR/LDR. CONCLUSIONS: The PCR/LDR technique successfully detects HER-2/neu gene amplification in clinical breast cancer specimens and shows 100% concordance with FISH. This technique is an accurate and rapid alternative to FISH with the potential for automation and high throughput analysis of HER-2/neu status in breast cancer.     

DNA, RNA and immunohistochemical characterization of the HER-2/neu oncogene in transitional cell carcinoma of the bladder

HER-2/neu overexpression appears to play a role in determining the malignant potential of some human cancers. To date, no urothelial malignancies appear to have been evaluated for HER-2/neu DNA amplification, mRNA expression and protein overproduction. By Southern hybridization we detected DNA amplification and a possible structural rearrangement of the HER-2/neu oncogene in one of 12 bladder tumors. A 14 kb DNA fragment in addition to the expected 12.5 Kb fragment was found. Additionally, the HER-2/neu oncogene was amplified sixfold in the tumor compared to placental DNA. Five of 14 (36%) bladder tumors overexpressed HER-2/neu mRNA three to 38-fold compared to normal urothelium. HER-2/neu overexpression occurred in superficial and invasive tumors. Immunohistochemical analysis was performed on the one tumor with DNA amplification and the 14 tumors evaluated for mRNA expression. The tumor with DNA amplification and three of the five tumors with HER-2/neu mRNA overexpression stained positively for the p185HER-2/neu protein. These findings suggest that DNA amplification occurs infrequently in bladder cancer. Thirty-six percent of bladder cancers overexpress HER-2/neu mRNA. Immunohistochemical analysis with a p185HER-2/neu polyclonal antibody, on formalin fixed, paraffin embedded tissue, was specific for HER-2/neu overexpression but not as sensitive as Northern analysis.     

CC3/TIP30蛋白在乳腺癌中的表达及其与HER-2/neu基因的相关性研究

目的 分析CC3/TIP30蛋白在乳腺癌中的表达及临床意义,并探讨CC3/TIP30与HER-2/neu的mRNA相关性.方法 采用免疫组化EnVision二步法检测2004年1月至2005年1月手术治疗并病理证实的112例乳腺癌标本中CC3/TIP30、HER-2/neu蛋白的表达,分析其表达与临床病理特征及预后的相关性,同时应用化学合成靶向敲除CC3/TIP30 mRNA的siRNA,并转染SK-BR-3细胞株48 h后,应用real-time PCR法测定CC3/TIP30、HER-2/neu基因mRNA的表达水平.结果 免疫组化显示CC3/TIP30蛋白在乳腺癌组织中表达与TNM分期、淋巴结转移、HER-2和分子分型相关(P=0.048、0.019、0.027、0.011),而与患者年龄、肿瘤大小,以及雌激素受体和孕激素受体表达状态无关.实时定量PCR结果示CC3/TIP30 siRNA转染导致CC3/TIP30 mRNA的表达下降,同时伴随着HER-2/neu基因mRNA水平的下降,差异有统计学意义(F=56.797,F=165.101;P均=0.000).分子分型为HER-2阳性型的乳腺癌患者中CC3/TIP30蛋白表达阳性、阴性组患者5年总生存率差异有统计学意义(X2=10.732,P=0.001).结论 CC3/TIP30蛋白表达缺失与乳腺癌的发生、发展有关,提示乳腺癌患者早期发生转移复发可能,可作为HER-2阳性型乳腺癌亚组分型的指标之一.     

Immunohistochemical her-2/ Neu expression with gene amplification by fluorescence in situ hybridization for assessment in breast carcinomas

Objective: To compare gene amplification of HER-2/neu gene by fluorescence in situ hybridization (FISH) in moderate to strong immunohistochemically (IHS) positive HER-2/neu cases of invasive breast carcinomas. Study Design: Cross- sectional study. Place and Duration of study: Section of Histopathology, The Aga Khan University Hospital, Karachi, from January 2004 to December 2006. Patients and Methods: Forty one (41) diagnosed cases of invasive breast carcinomas were included in this study in which already determined immunohistochemical HER-2/neu expression was scored as either 2+ or 3+, based on the intensity of membranous staining. These cases were further evaluated for gene amplification by FISH. For gene amplification, a ratio of HER-2/CEP Z 2 was accepted as positive gene amplification. Results: Out of a total 41 cases, which were scored as 2+ and 3+ by IHC, 14 cases (34.1%; 95% confidence interval: 19%-49.3% ) showed gene amplification by FISH. Proportion of FISH positivity in IHC 2+ cases alone was found to be 25% (95% confidence interval: 10.5%-41%). In contrast, a majority of IHC 3+ cases (5 of 6) were positive by FISH studies. Conclusion: IHC is appropriate for initial HER-2/neu assessment and patients with tumors scored as 3+ may be treated alone based on this information provided strict quality control and 95% concordance with FISH assays; however, patients with tumors interpreted as 2+, would benefit from gene amplification by FISH studies for more accurate assessment to avoid inaccurate prognostication and treatment.     

Clinical evaluation of a multi-target fluorescent in situ hybridization assay for detection of bladder cancer

PURPOSE: The UroVysion fluorescence in situ hybridization assay (UroVysion Bladder Cancer Recurrence Kit, Vysis, Inc., Downers Grove, Illinois) is a multi-target assay that detects aneuploidy of chromosomes 3, 7 and 17, and loss of the 9p21 band in exfoliated cells in urine from patients with transitional cell carcinoma. We performed 2 multicenter trials. In 1 trial we compared the sensitivity of the FISH assay to the BTA Stat test (Bion Scientific, Redmond, Washington) and voided cytology in the detection of transitional cell carcinoma. In a separate study of healthy volunteers and patients with other (nontransitional cell carcinoma) conditions we determined the specificity of the FISH assay. MATERIALS AND METHODS: A total of 176 patients with transitional cell carcinoma in the previous 9 months provided voided urine before cystoscopy. Each specimen was split, preserved and shipped to a central laboratory where all 3 tests were performed. All sites were blinded to results. Sensitivity calculations were based on central pathology review of resected tissue. Specificity was determined by testing 275 volunteers who were healthy and with nontransitional cell carcinoma conditions. RESULTS: The 21 sites enrolled 176 patients with a history of transitional cell carcinoma, with 62 recurrences while undergoing surveillance. Overall sensitivities (with 95% CI) were FISH 71% (95% CI 58 to 82), BTA Stat test 50% (37 to 63) and cytology 26% (16 to 39). FISH was negative in 260 of the 275 healthy volunteers or patients with no history of transitional cell carcinoma (specificity 94.5%). CONCLUSIONS: Sensitivity of the FISH assay is superior to that of cytology and at least equivalent to the BTA Stat test in detecting recurrent transitional cell carcinoma. Its specificity approaches that of cytology. Further testing of its clinical use is warranted.     

荧光原位杂交技术在前列腺癌诊断中的临床应用

目的建立TMPRSS2/ETS(ERG、ETV1、ETV4)融合基因的荧光原位杂交(FISH)检测和评估方法,并探讨其对于前列腺癌的诊断价值。方法对50例前列腺癌样本、15例正常前列腺组织及20例良性前列腺增生样本应用三组国产FISH探针顺序检测TMPRSS2/ERG、TMPRSS2/ETV1及TMPRSS2/ETV4融合基因,建立FISH技术诊断前列腺癌的阈值,并计算敏感性、特异性、阳性预测值(+PV)和阴性预测值(-PV)。结果三组探针的联合敏感性达到90%,其中TMPRSS2-ERG检测敏感性为78%,总体特异性为100%,阳性预测值为100%,阴性预测值为87.5%。结论应用FISH技术检测TMPRSS2/ETS(ERG、ETV1、ETV4)融合基因诊断前列腺癌具有较高的敏感性和特异性,显示出良好的临床应用前景。     

Analytical validation and interobserver reproducibility of EnzMet GenePro: a second-generation bright-field metallography assay for concomitant detection of HER2 gene status and protein expression in invasive carcinoma of the breast

Fluorescence in situ hybridization (FISH) has both excellent sensitivity and specificity in detecting HER2 gene amplification in invasive breast carcinoma. FISH has not been widely implemented in clinical practice because of reagent costs and the special instrumentation and expertise required to perform and integrate the assay. Immunohistochemistry (IHC) for HER2 protein is widely used, but false-positive and false-negative results are problematic. We developed a bright-field assay to visualize HER2 gene amplification and concomitant HER2 protein expression (EnzMet GenePro). This assay detects HER2 gene amplification via deposition of metallic silver by enzyme metallographytrade mark (EnzMettrade mark, Nanoprobes, Yaphank, NY) combined with HER2 protein detection by IHC using alkaline phosphatase and fast red K substrate visualization (CB11;Ventana, Tucson, AZ). The assay was performed on 94 invasive breast carcinomas, for which FISH (PathVysiontrade mark, Vysis, Downer's Grove, IL), conventional IHC (CB11), and enzyme metallography (EnzMettrade mark) results were known. The EnzMettrade mark component of the assay was scored as either HER2 gene amplified, polysomic, or nonamplified. The IHC component was scored using the conventional FDA scale of 0 to 3+. Concordance of the EnzMet component of the assay versus FISH was assessed and showed an excellent correlation (Pearson coefficient of 0.95; P < 0.001). The combination of gene and protein detection (EnzMet GenePro) displayed a specificity of 100% and an accuracy of 92.6% (95% confidence interval 85.3-97.0), facilitated recognition of gene/protein discordances, and allowed for efficient interpretation of the slide by conventional light microscopy. The interobserver kappa for each component was excellent (IHC, kappa = 0.94; and EnzMettrade mark, kappa = 0.96). EnzMet is the first bright-field ISH assay in our experience that routinely and nonambiguously detects endogenous HER2 signals, essential for a reliable clinical HER2 assay, and in combination with HER2 protein enables improved diagnosis in borderline cases.     

The influence of polysomy 17 on HER2 gene and protein expression in adenocarcinoma of the breast: a fluorescent in situ hybridization, immunohistochemical, and isotopic mRNA in situ hybridization study

Breast carcinomas with amplification of HER2 on chromosome 17 are associated with HER2 protein overexpression, adversely affecting prognosis and predicting response to Herceptin therapy. Chromosome 17 polysomy is encountered in assessing HER2 gene status, and its impact on HER2 gene and protein expression remains unclear. This impact was investigated in breast carcinomas identified by fluorescence in situ hybridization (FISH) to have a gain of chromosome 17 (CEP17+; n = 56), using a dual probe assay, which detects HER2 gene copy number and enumerates chromosome 17 (HER2/CEP17; Vysis). Cases were immunostained for HER2 protein (CB-11, Ventana), and scored blinded to FISH. A subgroup was evaluated by isotopic in situ hybridization for HER2 mRNA expression. Controls included ten HER2 amplified and ten nonamplified tumors, eusomic for chromosome 17. Immunohistochemistry (IHC) for HER2 protein was negative (0 or 1+) in 69% (39 of 56), 2+ in 27% (15 of 56), and 3+ in 3% (2 of 56) of CEP17+ cases. The mean CEP17 copy number among the three groups was similar (3.1, 3.0, and 3.1 for IHC 0/1+, 2+, and 3+, respectively). Isotopic in situ hybridization for HER2 mRNA performed on 26 CEP17+ cases (16 IHC 0-1+, 10 IHC 2+ or 3+) showed no increased HER2 mRNA expression (normalized to beta-actin mRNA). The mRNA expression and the IHC staining of the HER2-amplified and nonamplified controls was concordant with their FISH status. These results suggest that chromosome 17 polysomy in the absence of HER2 amplification does not have a significant biologic influence on HER2 gene expression in breast carcinoma.