Detection of N-myc gene expression in neuroblastoma tumors by in situ hybridization.

The presence of N-myc DNA amplification in human neuroblastoma tumors has been shown to be an independent prognostic factor predicting rapid progression of disease. Southern blot analysis has been used previously to detect N-myc amplification in these tumors. The authors report an analysis of N-myc gene expression by in situ hybridization in 28 human neuroblastoma tumors previously studied by Southern blot analysis. In the LA-N-5 human neuroblastoma cell line known to be amplified for N-myc, reaction conditions favoring RNA-RNA hybridization yielded an optimal signal. Using these hybridization conditions, in situ hybridization analysis of N-myc expression in 28 human neuroblastoma tissues correlated perfectly with N-myc DNA amplification in these tumors as detected by Southern blot analysis. In particular, there were no tumors in which N-myc RNA expression was found by in situ hybridization analysis in the absence of DNA amplification detectable by Southern blot, nor were there tumors that had DNA amplification in the absence of RNA expression. Heterogeneity of N-myc RNA expression was observed both among cells in any given tumor area, as well as within different areas of a single tumor. N-myc expression by in situ hybridization analysis was not observed in those tumors with more neuronally differentiated, ganglioneuroma histology. It is concluded that in situ hybridization of tissue sections is as effective as Southern blot analysis of tumor cell DNA in identifying human neuroblastoma tumors in which the N-myc gene is of prognostic significance.     

N-myc amplification was rarely detected by fluorescence in situ hybridization in retinoblastoma

In retinoblastoma, genetic alteration of N-myc amplification different from the alteration of the RB1 gene on chromosome 13q14 has been described. This study is to determine the frequency of N-myc amplification by fluorescence in situ hybridization method in retinoblastoma. This study was prospectively derived from 26 patients who were diagnosed as having unilateral retinoblastoma (highly progressive large retinoblastoma, group 5 in Reese-Ellsworth classification) and underwent enucleation. We performed locus-specific fluorescence in situ hybridization probes for N-myc gene. Our results demonstrated that in only one of 26 patients was N-myc amplification found in retinoblastoma tissue. N-myc amplification has been regarded as one characteristic of retinoblastoma cell line and an adverse prognostic factor. However, our study indicates that N-myc amplification is not frequently found in retinoblastoma.     

Detection of amplified N-myc gene in neuroblastoma by in situ hybridization using the single-step silver enhancement method

Amplification of the N-myc oncogene in 27 cases of neuroblastoma group tumor was examined by in situ hybridization using the single-step silver enhancement technique. The N-myc gene copy numbers of these neuroblastoma group tumors were previously examined by dot-blot hybridization using DNA extracted from formalin-fixed, paraffin-embedded tissues (H Tsuda et al., Lab Invest, 59:321, 1988). Silver grains were deposited just over the nuclei of tumor cells, but no or only faint deposition was observed over those of infiltrating lymphocytes, stromal fibroblasts, and endothelial cells. We judged a case to be positive for gene amplification when silver grains precipitated over the nuclei of the tumor cells to a greater extent than over nuclei of lymphocytes or endothelial cells in the same section. According to this criterion, 14 cases (12 cases of neuroblastoma and 2 cases of ganglioneuroblastoma) were positive for N-myc gene amplification of 27 cases (18 cases of neuroblastoma, 5 cases of ganglioneuroblastoma, and 4 cases of composite ganglioneuroblastoma). These results corresponded well to those of the dot-blot hybridization analysis using the same materials. Thirteen of 15 cases that carried 4 copies or more of N-myc gene were judged positive, and 11 of 12 cases that carried less than 4 copies of the N-myc gene were judged negative by in situ hybridization. In neuroblastoma group tumors with amplification of the N-myc gene, tumor cells were stained almost homogenously, except for two cases of ganglioneuroblastoma, in which less differentiated small round tumor cells were stained more intensely than differentiated ganglion-like cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

胃癌c-erbB-2基因扩增的荧光原位杂交检测

目的：研究胃癌ｃｅｒｂＢ２ 基因扩增。方法：使用荧光原位杂交技术（ＦＩＳＨ） 检测５５ 例胃癌。结果：胃癌肿瘤区ｃｅｒｂＢ２基因扩增阳性率为３６－４％ 。在胃癌移行区内阳性率为１０－９％ ,在正常粘膜区阳性率为３－６ ％ ,相互比较,三个区域之间差异有高度显著性（ Ｐ＜ ０－０１）。在肠型和弥漫型胃癌,ｃｅｒｂＢ２ 基因扩增阳性率为５１－６％ 和１６－７％ ,两者差异有显著性（ Ｐ＜０－０５）。ｃｅｒｂＢ２ 基因扩增阳性与淋巴结内胃癌转移有明显相关性（ Ｐ＜ ０－０５）。结论：ｃｅｒｂＢ２ 基因扩增与胃癌的发生,发展有明显关联,有助于判断胃癌预后。     

Detection of MYCN amplification in three neuroblastoma cell lines by non-radioactive chromosomal in situ hybridization.

A non-radioactive chromosomal in situ hybridization technique utilizing a biotin-streptavidin-polyalkaline-phosphatase complex was successfully applied to three neuroblastoma cell lines for detection of MYCN amplification. These cell lines, designated PER-106, PER-107, and PER-108, were derived from consecutive bone marrow samples taken from a patient with stage IV neuroblastoma. The cell line derived at diagnosis (PER-106) exhibited MYCN amplification in the form of variable numbers of double-minute chromosomes, small fragments, and rings of varying sizes. This observed variability of MYCN amplification may explain the reported heterogeneity of both MYCN mRNA and protein expression among individual cells of some neuroblastomas. The cell lines derived from subsequent samples (PER-107 and PER-108) contained amplified MYCN as two consistent homogeneously staining regions in every cell. These were located on the short arms of chromosomes 6 and 14. Thus, amplified MYCN was identified in each cell line and demonstrated the concurrent evolution of amplification with cytogenetic abnormalities.     

应用荧光原位杂交检测人喉癌中EGFR基因扩增

目的 检测人喉癌Ｈｅｐ－２细胞系和５个喉癌组织中表皮生长因子受体（ｅｐｉｄｅｒｍａｌ ｇｒｏｗｔｈ ｆａｃｔｏｒ ｒｅｃｅｐｔｐｒ,ＥＧＦＲ）基因扩增。方法 荧光原位杂交技术。结果 在Ｈｅｐ－２细胞和２例喉癌组织中期染色体和间期细胞核中,检测到明显的集中成簇和多个斑点分散排布的杂效信号,另３例喉癌组织间期细胞核中杂交信号未见增强或数目增加。结论 以正常二倍休 色体和间期细胞核为对照,在喉癌Ｈｅｐ－     

应用荧光原位杂交技术在组织标本中检测宫颈上皮病变的端粒酶RNA基因扩增

目的 用荧光原位杂交(FISH)技术检测端粒酶RNA基因(TERC)在宫颈上皮内瘤变(CIN)和宫颈鳞状细胞癌(SCC)组织中的扩增,探讨其用于SCC筛查及CIN诊断的可行性及实际意义.方法 选取手术切除及活检的子宫颈组织标本196例,有效样本150例,采用FISH技术在石蜡包埋组织芯片上检测TERC基因的扩增情况.结果 150例组织芯片样本中,正常宫颈鳞状上皮24例,CIN 78例(CIN Ⅰ级25例,Ⅱ级21例,Ⅲ级32例),SCC 48例.正常宫颈鳞状上皮组织中TERC基因无扩增,从CIN Ⅰ级到SCC组织中,TERC基因的扩增率依次为8.0%(2/25)、47.6%(10/21)、71.9%(23/32)和87.5%(42/48),差异有统计学意义(P＜0.05).组间两两比较:正常宫颈鳞状上皮与CINⅡ级、Ⅲ级、SCC组,组间比较差异均有统计学意义(P＜0.05);CIN Ⅰ级与CINⅡ级、Ⅲ级及SCC组组间比较,差异有统计学意义(P＜0.05);CINⅡ级组与SCC组相比,差异有统计学意义(P＜0.05).正常宫颈鳞状上皮与CIN Ⅰ级组、CINⅡ级与Ⅲ级组、CINⅢ级与SCC组,组间差异均无统计学意义(P＞0.05).高度癌前病变(CINⅡ～Ⅲ级)与低度癌前病变(CIN Ⅰ级)的TERC基因扩增结果:25例CIN Ⅰ级病变中,2例扩增,23例不扩增;53例CINⅡ～Ⅲ级病变中,33例扩增,20例不扩增.经公式计算,敏感性为62.3%、特异性为92.0%、准确度为71.8%、阳性预测值为94.3%、阴性预测值为53.5%.结论 在石蜡包埋CIN及SCC组织切片上应用FISH技术进行TERC基因检测,方法可行,结果可靠;可作为辅助CINⅡ级的诊断;TERC基因扩增可以预测CIN发展为癌的风险,具有一定的实用价值.

Abstract：

Objective To explore the feasibility and practical value of fluorescence in situ hybridization (FISH) detection of TERC gene amplification in cervical intraepithelial lesions (CIN) and squamous cell carcinoma (SCC). Methods Tissue microarray was constructed to cover 150 cases of various cervical conditions, including 24 cases of normal cervical mucosa, 78 cases of CINs ( CIN Ⅰ , 25 cases; CIN Ⅱ , 21 cases and CIN Ⅲ, 32 cases) and 48 cases of SCC. FISH was used to detect TERC gene amplification. Results TERC gene amplification was detected in 8% (2/25) CIN Ⅰ , 47.6% ( 10/21 )CIN Ⅱ, 71.9% (23/32) CIN Ⅲ and 87.5% (42/48) SCC. There were significant differences among these groups (P ＜ 0.05 ). The amplification rates of TERC gene in SCC, CIN Ⅲ and CIN Ⅱ were significantly higher than those of normal cervical epithelium and CIN Ⅰ ( P ＜ 0.05 ). Significant differences were also observed among CIN Ⅰ and CIN Ⅱ , CIN Ⅲ and SCC ( P ＜ 0.05 ), and between CIN Ⅱ and SCC(P ＜0.05). There were no significant differences between normal cervical epithelium and CIN Ⅰ , CIN Ⅱ and CIN Ⅲ, and between CIN Ⅲ and SCC (P ＞ 0.05 ). FISH detection of amplification of TERC gene in CIN Ⅰ and CIN Ⅱ -Ⅲ demonstrated the following statistics: sensitivity of 62. 3%, specificity of 92.0%,accuracy of 71.8%, positive and negative predictive values of 94.3% and 53.5%, respectively.Conclusions FISH detection is a reliable method in detecting TERC gene amplification using paraffin tissue sections. When histological evaluation becomes difficult, TERC amplification detectable by FISH may offer a diagnostic distinction of CIN Ⅰ from CIN Ⅱ. Moreover, TERC amplification may be used as a biomarker in predicting CIN progression to invasive cancer.     

Investigation of MYCN status in neuroblastoma by fluorescence in situ hybridization

Neuroblastoma, a form of neuroblastic tumor, is one of the most common neoplasms seen in early childhood. The diverse clinical behavior of this tumor is most probably explainable by the heterogeneity in the associated genetic changes. We investigated 12 neuroblastoma patients for MYCN amplification and chromosome 2 aneusomy by fluorescence in situ hybridization (FISH) and results were correlated with conventional cytogenetics. Samples from both primary tumor tissue and bone marrow metastasis were available in two cases. The copy number of MYCN oncogene paralleled that of chromosome 2 in 10 cases, whereas two cases (16.7%) showed numerous distinct signals within the nuclei of the tumor cells, consistent with MYCN amplification as double minute (dmin). The morphology of dmin in one case was of an extremely small type and might potentially be missed by conventional chromosome analysis. Discordant cytogenetics and FISH results was observed between primary tumor and metastasis disease in one case, with loss of chromosome 2 tetrasomic and pentasomic cells as well as over-representation of chromosome 2 disomic cells harboring MYCN amplification in bone marrow deposits. Our study reaffirmed the need for MYCN status to be determined in light of chromosome 2 copy number, as recommended by published guidelines. We also showed that genetic heterogeneity might occur between primary tumor and bone marrow metastasis. Finally, atypical dmin morphology when encountered would need confirmation by FISH study.     

N-myc gene product expression in neuroblastoma.

The presence and distribution of N-myc gene product were studied in 13 neuroblastomas and five ganglioneuroblastomas, using immunohistochemical techniques. Nine tumors (eight neuroblastomas and one ganglioneuroblastoma of composite type) contained neuroblastoma cells with positive nuclei for N-myc protein. Microscopic examination showed that most of the positive neuroblastoma cells seemed to be immature, with no apparent neuronal differentiation. Nine of 11 tumours with amplified N-myc gene copies exhibited tumour cells with positive immunostaining for the N-myc gene product, while none of the seven non-N-myc amplified cases contained immunoreactive tumour cells. The survival of the patients positive for N-myc protein was significantly low compared with that of the negative ones. It is concluded that immunohistochemical staining for the N-myc gene product will facilitate prediction of the prognosis of patients with neuroblastoma.     

Her2 amplification: correlation of chromogenic in situ hybridization with immunohistochemistry and fluorescence in situ hybridization.

Detecting Her2 gene amplification has become routine in predicting therapeutic responsiveness in patients with breast carcinoma. Fluorescence in situ hybridization (FISH) is a common technique for detecting Her2 amplification, yet dark field fluorescence microscopy remains problematic for many pathologists. Thus, a technique such as chromogenic in situ hybridization (CISH), in which the more familiar light microscopy can be used, is appealing. Paraffin-embedded sections from 61 breast carcinomas were tested for Her2 amplification by immunohistochemistry (IHC) and CISH. FISH was used to confirm CISH results. Excellent correlation was found between IHC and CISH except in cases considered negative (1+ on the DAKO scale) by IHC. CISH detected low-level Her2 amplification in 4 of 9 of these cases. Amplification was subsequently confirmed by FISH in all but 1 case. When compared with FISH, CISH was more sensitive than IHC for detecting low levels of Her2 gene amplification. Moreover, excellent concordance was found between FISH and CISH, supporting the conclusion that the CISH assay for Her2 gene amplification provides an accurate, effective, and practical alternative to FISH.     

Detection of subtle reciprocal translocations by fluorescence in situ hybridization

Three different subtle reciprocal translocations were detected on long, well-banded chromosomes. The same translocations were examined using fluorescence in situ hybridization (FISH) with chromosome-specific libraries and unique DNA sequences. Our findings show that FISH allows rapid and unequivocal detection and characterization of this type of chromosome rearrangement. This approach is especially useful for prenatal diagnosis when one of the parents is a balanced carrier of such small fragment translocations.     

Gold-facilitated in situ hybridization: a bright-field autometallographic alternative to fluorescence in situ hybridization for detection of Her-2/neu gene amplification

Fluorescence in situ hybridization (FISH) represents an excellent method for profiling gene amplification in situ, but correlation with tissue morphology is difficult because of dark-field visualization. Validation of a bright-field assay for assessment of HER-2/neu gene amplification was investigated. Streptavidin-Nanogold was used to generate bright-field gene copy signals using GoldEnhance gold-based autometallography, catalyzed reported deposition, and a biotin-labeled probe. One hundred cases of invasive breast carcinoma were evaluated for which FISH gene copy results, and mRNA and oncoprotein gene expression, were known. Autometallographic signals were qualitatively evaluable without the use of oil immersion microscopy. Results correlated well with indirect and direct label FISH. Autometallographic gold-based in situ hybridization represents a promising bright-field assay for the assessment of HER-2/neu gene amplification.     

Feasibility of using tissue microarrays for the assessment of HER-2 gene amplification by fluorescence in situ hybridization in breast carcinoma.

Tissue microarrays (TMAs) have been commonly used to study protein expression by immunohistochemistry (IHC). However, limited data exist on the validity of using TMAs to study gene amplification. In this study, we evaluated the feasibility of using breast carcinoma TMAs to study HER-2 gene amplification by fluorescence in situ hybridization (FISH). In addition, hormonal receptor status (ER and PR) and HER-2 protein overexpression by IHC were also studied, and results were compared with whole tissue sections. FISH for HER-2 was performed on formalin-fixed paraffin-embedded tissue from 114 invasive breast carcinomas both on whole tissue sections and on TMAs containing the same tumors. The TMA was created using 0.6-mm tissue cores with four sampled cores per tumor from the same tissue block used for whole section FISH. The PathVysion HER-2 probe kit was used for the FISH analysis. A ratio of HER-2:Chromosome17 > or =2.0 was interpreted as positive for gene amplification. The ER or PR was interpreted as positive when nuclear staining was detected in more than 10% of tumor cells. The HER-2 IHC (HercepTest; DAKO Corp, Carpinteria, CA) results were interpreted as 0, 1+, 2+, and 3+ according to standard criteria. The FISH results in the TMA and whole sections were concordant in 99 out of 101 successfully analyzed cases (99%). The FISH scores were consistent among the two to four cores in the majority of the cases. ER and PR results were concordant between whole sections and TMA cores in 97% (107/110) and 89% (97/109) cases, respectively. The overall concordance for HER-2 status by IHC between whole sections and TMA cores was 86% (94 out of 109 cases). TMAs are a reliable approach to study HER-2 gene amplification in a high throughput manner.     

生物素标记探针原位杂交法检测细胞中c-myc基因的表达

本文采用生物素标记的c-mye基因作探针，通过细胞原位分子杂交技术，对人舌癌、膀胱癌和正常细胞中c-myc基因的转录量进行了定性和半定量分析。结果表明与正常细胞相比，舌癌和膀胱癌细胞中c-myc基因异常高度表达。     

应用荧光原位杂交技术检测肺癌患者染色体数目的改变

目的应用荧光原位杂交(FISH)技术研究肺癌患者染色体数目改变及其意义。方法用人的全套染色体特异探针与肺癌患者中期染色体进行杂交检测。结果肺癌患者细胞为异倍体,染色体数目以亚二倍体居多,常见2、5、7、8、10、11、14和17号染色体增多及1、4号染色体的丢失。结论 FISH技术可检测肺癌患者染色体数目的改变,肺癌遗传学变异主要是2、5、7、8、10、11、14、17号染色体的增加,并对肺癌的发生、发展和预后有一定的提示作用。     

N-myc amplification and cell proliferation rate in human neuroblastoma

In neuroblastoma, N-myc amplification has been found to be strikingly associated with rapid tumour progression and poor prognosis. Recent studies have demonstrated that cell proliferative activity also significantly predicts the clinical outcome in patients with neuroblastoma. In order to define the correlation between N-myc amplification and cell proliferation rate, in the present investigation the two parameters were first assessed in 48 neuroblastoma tumours. N-myc amplification was evaluated in frozen specimens by Southern-blot analysis using the NB 19-21 probe and it was detected in nine patients. Cell proliferative activity was determined by measuring the AgNOR protein area in histological sections selectively stained by silver. The mean AgNOR protein area value of neuroblastomas with N-myc amplification (3·63±1·62 μm²) was not significantly different from that of neuroblastomas without N-myc amplification (2·46±1·57 μm²; P=0·30). On the other hand, both N-myc amplification and AgNOR protein expression were found to be significantly related to the clinical outcome of the disease (P<0·001 and P=0·0143, respectively; median follow-up time=47 months; range 18–106 months). In a second set of experiments, the relationship between N-myc amplification and cell proliferation rate was assessed in seven established human neuroblastoma cell lines. N-myc amplification was found to be completely independent of the population doubling time (DT), which, on the contrary, was strictly related to the quantitative expression of AgNOR protein (r=−0·947; P<0·001). Altogether, the present results indicate that N-myc amplification and cell proliferation rate are not interrelated in neuroblastoma, each representing an independent biological parameter of cancer cells associated with the clinical behaviour of the disease. © 1997 John Wiley & Sons, Ltd.     

PGD for dystrophin gene deletions using fluorescence in situ hybridization

Duchenne muscular dystrophy and Becker muscular dystrophy (DMD and BMD) are caused by mutations in the dystrophin gene (Xp21). In two-thirds of DMD/BMD cases, the mutation is a large deletion of one or several exons. We have established PGD for DMD/BMD using interphase fluorescence in situ hybridization (FISH) analysis on single nuclei from blastomeres for the detection of deletions of specific exons in the dystrophin gene. We performed PGD for two carrier females; one had a deletion of exons 45-50 (DMD), and the other had a deletion of exons 45-48 (BMD). An exon 45-specific probe was used in combination with probes for the X and Y centromeres. Using this straightforward approach, we can distinguish affected and unaffected male embryos as well as carrier female and normal female embryos. Three cycles were performed for each patient, which resulted in a pregnancy and the birth of a healthy girl. To the best of our knowledge, this approach for PGD has not been previously reported. The use of interphase FISH is an attractive alternative to sexing or PCR-based mutation detection for PGD patients with known deletions of the dystrophin gene.     

用荧光原位杂交从母血中检测胎儿细胞

目的 从母血中分离胎儿细胞并确定其来自胎儿。方法 从孕早、中期各２０名、分娩后１５名母血中富集并分离有核细胞。用Ｙ特异性探针（ＰＹ３．４）行荧光原位杂交,从中识别胎儿细胞。结果 孕早、中期孕妇各怀１５名男胎。阳性细胞比例是１：６５２８．０及１：２７３．８。与同期１０名女胎阳性细胞相比,差别有高度显著性。分娩１周内的３名,阳必民孕中期的差别没有显著性。分娩３个月后的阳性率与比,差别有高度显著性。分娩     

Oncogene amplification in medulloblastoma: analysis of a case by comparative genomic hybridization and fluorescence in situ hybridization

We describe amplification of the MYCC oncogene in a medulloblastoma with aggressive clinical behavior. The patient was a six year old boy who underwent gross total surgical excision of a cerebellar tumor. Despite chemotherapy and total neuraxis radiation, the clinical course was one of relentless progression, with extensive subarachnoid spread and death within eight months of presentation. The pathological features were consistent with the recently described, "large cell variant" of medulloblastoma. Tumor cells exhibited large vesicular nuclei, prominent nucleoli and strong immunoreactivity for synaptophysin. Polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) assay revealed no evidence of MYCN amplification or 1p deletion in the tumor. FISH analysis revealed evidence of MYCC amplification in the 20- to 30-fold range. Comparative genomic hybridization (CGH) revealed regions of gains and amplification in three locations, with gains of chromosome 7, amplification of 8q24 (corresponding to the MYCC locus) and gains of the long arm of chromosome 17 (suggestive of isochromosome 17q). While conventional karyotypic analysis was not successful in the present case, CGH provided invaluable information about gene amplification and losses/gains of chromosomes and chromosomal regions. Thus, CGH is a powerful technique applicable to frozen or paraffin-embedded material which helps to ascertain the presence of gene amplification even without prior knowledge of the gene to be tested.