Survey of gene amplifications during prostate cancer progression by high-throughout fluorescence in situ hybridization on tissue microarrays

Prostate cancer development and progression is driven by the accumulation of genetic changes, the nature of which remains incompletely understood To facilitate high-throughput analysis of molecular events taking place in primary, recurrent, and metastat prostate cancer, we constructed a tissue microarray containing small 0.6-mm cylindrical samples acquired from 371 formalin-fixed blocks, including benign prostatic hyperplasia (n = 32) and primary tumors (n = 223), as well as both locally recurrent tumors (n = 54) and metastases (n = 62) from patients with hormone-refractory disease. Fluorescence in situ hybridization (FISH) was applied to the analysis of consecutive tissue microarray sections with probes for five different genes. High-level (> or =3X) amplifications were very rare (<2%) in primary prostate cancers However, in metastases from patients with hormone-refractory disease, amplification of the androgen receptor gene was seen in 22%, MYC in 11%, and Cyclin-D1 in 5% of the cases. In specimens from locally recurrent tumors, the corresponding percentages were 23, 4, and 8%. ERBB2 and NMYC amplifications were never detected at any stage of prostate cancer progression. In conclusion, FISH to tissue microarray sections enables high-throughput analysis of genetic alterations contributing to cancer development and progression. Our results implicate a role for amplification of androgen receptor in hormonal therapy failure and that of MYC in the metastatic progression of human prostate cancer.     

Candidate genes in breast cancer revealed by microarray-based comparative genomic hybridization of archived tissue

Genomic imbalances in 31 formalin-fixed and paraffin-embedded primary tumors of advanced breast cancer were analyzed by microarray-based comparative genomic hybridization (matrix-CGH). A DNA chip was designed comprising 422 mapped genomic sequences including 47 proto-oncogenes, 15 tumor suppressor genes, as well as frequently imbalanced chromosomal regions. Analysis of the data was challenging due to the impaired quality of DNA prepared from paraffin-embedded samples. Nevertheless, using a method for the statistical evaluation of the balanced state for each individual experiment, we were able to reveal imbalances with high significance, which were in good concordance with previous data collected by chromosomal CGH from the same patients. Owing to the improved resolution of matrix-CGH, genomic imbalances could be narrowed down to the level of individual bacterial artificial chromosome and P1-derived artificial chromosome clones. On average 37 gains and 13 losses per tumor cell genome were scored. Gains in more than 30% of the cases were found on 1p, 1q, 6p, 7p, 8q, 9q, 11q, 12q, 17p, 17q, 20q, and 22q, and losses on 6q, 9p, 11q, and 17p. Of the 51 chromosomal regions found amplified by matrix-CGH, only 12 had been identified by chromosomal CGH. Within these 51 amplicons, genome database information defined 112 candidate genes, 44 of which were validated by either PCR amplification of sequence tag sites or DNA sequence analysis.     

应用组织微阵列技术检测Kiss-1和KAI-1在胆囊癌中的表达

目的：探讨转移相关基因表达蛋白Kiss-1和KAI-1在人胆囊癌转移发展过程中的意义。方法：应用组织微阵列技术和免疫组织化学（En Vision）技术研究Kiss-1和KAI-1在59例胆囊癌（其中转移病例23例）、7例癌旁和6例正常胆囊组织中的表达。结果：与正常和癌旁组织相比,癌组织中Kiss-1和KAI-1的表达明显降低（P〈0．05）;与癌旁组织相比,Kiss-1、KAI-1在转移病例中的表达明显下降（P〈0．01）。两者的表达下调与胆囊癌的部分恶性生物学行为相关。Kiss1与KAI-1的阳性表达均与胆囊癌的Nevin分期呈明显负相关（P=0．000）,且胆囊癌中的Kiss-1的阳性表达与KAI．1的阳性表达呈正相关（r=0．419,P=0．001）。提示Kiss-1与KAI-1均与胆囊癌的高转移潜能相关。结论：Kiss-1或KAI-1表达可能是反映胆囊癌侵袭和转移潜力及预后的重要生物学标记。检测Kiss-1与KAI-1的表达可能对早期发现胆囊癌和评价其预后有重要临床意义。     

应用组织微阵列技术研究肺癌组织中IGF-Ⅱ的表达

目的构建高通量组织微阵列(tissue microarray)/组织芯片(tissue chip),检测其上IGF-Ⅱ的表达情况,以明确IGF-Ⅱ表达与肺癌的相关性.方法用组织阵列仪制备按不同组织学类型排布的组织芯片,然后用免疫组织化学SP法检测一张组织芯片上54例肺癌肿瘤组织和10例正常肺组织样本中的IGF-Ⅱ表达情况,并分析其表达与各临床病理学参数之间的相关性.结果IGF-Ⅱ蛋白在实验组中的阳性表达率为42.6%(23/54),对照组为阴性,两者之间具有显著性差异(P＜0.05);IGF-Ⅱ表达阳性率与大体类型、组织学分级、临床分期有关(P＜0.05),而与性别、年龄、组织学分型及淋巴结转移无关(P＞0.05).结论IGF-Ⅱ的表达与肺癌的恶性行为有关,检测此指标对预测肺癌的预后和指导治疗有一定的参考价值.组织芯片是一较高的技术平台,可以应用此技术进行大规模组织样本的检测.     

Candidate driver genes in microsatellite-unstable colorectal cancer

Defects in the mismatch repair system lead to microsatellite instability (MSI), a feature observed in ～ 15% of all colorectal cancers (CRCs). Microsatellite mutations that drive tumourigenesis, typically inactivation of tumour suppressors, are selected for and are frequently detected in MSI cancers. Here, we evaluated somatic mutations in microsatellite repeats of 790 genes chosen based on reduced expression in MSI CRC and existence of a coding mononucleotide repeat of 6-10 bp in length. All the repeats were initially sequenced in 30 primary MSI CRC samples and whenever frameshift mutations were identified in >20%, additional 70 samples were sequenced. To distinguish driver mutations from passengers, we similarly analyzed the occurrence of frameshift mutations in 121 intronic control repeats and utilized a statistical regression model to determine cut-off mutation frequencies for repeats of all types (A/T and C/G, 6-10 bp). Along with several know target genes, including TGFBR2, ACVR2, and MSH3, six novel candidate driver genes emerged that harbored significantly more mutations than identical control repeats. The mutation frequencies in 100 MSI CRC samples were 51% in G8 of GLYR1, 47% in T9 of ABCC5, 43% in G8 of WDTC1, 33% in A8 of ROCK1, 30% in T8 of OR51E2, and 28% in A8 of TCEB3. Immunohistochemical staining of GLYR1 revealed defective protein expression in tumors carrying biallelic mutations, supporting a loss of function hypothesis. This is a large scale, unbiased effort to identify genes that when mutated are likely to contribute to MSI CRC development.     

应用组织芯片检测肝细胞癌组织中8种p53相关基因的表达

背景及目的:肝细胞癌(hepatocellularcarcinoma,HCC)的发生是一个多步骤、多基因参与的过程。目前已知与HCC相关的癌基因和抑癌基因有十余种,其中p53基因家族的功能变化是HCC发生发展过程中的重要分子事件。本研究利用组织芯片高通量的优点,探讨肝细胞癌及癌旁肝组织中p53基因家族及相关基因中8种癌基因及抑癌基因的表达差异。方法:应用Instrumedics公司生产的组织芯片制作仪,取样针直径2mm,制作成3个肝细胞癌组织芯片蜡块,包括273例肝细胞癌组织、144例癌旁肝组织及10例非肝病死亡的尸检正常肝组织。应用免疫组织化学方法检测了p16INK4a、p21WAF1、p33ING1b、p53、p57KIP2、p73、mdm2和ATM8种基因在HCC及癌旁肝组织中的表达率,同时观察乙肝病毒(hapatitisBvirus,HBV)感染与这8种基因表达间的关系。结果:3个肝癌组织芯片分别含有136、143和148个位点。8种基因在HCC和癌旁肝组织中的阳性表达率分别为p16INK4a35.9%和9.0%、p21WAF141.8%和11.1%、p33ING1b43.65%和14.6%、p5346.2%和12.5%、p57KIP239.2%和16.7%、p739.5%和2.8%、mdm241.4%、9.0%和ATM6.6%和1.4%。在癌组织中上述基因的表达强于癌旁肝组织(P<0.05);不同分化程度的HCC组织中8种基因的阳性表达率差异均不显著(P>0.05);除了p16INK4a、p21WAF1、     

胃癌基因表达谱芯片差异基因的生物信息学分析

目的：筛选胃癌发生发展过程中的关键基因和信号通路,为寻找有价值的胃癌分子标志物提供依据。方法：从GEO数据库下载5个胃癌基因芯片数据集：GSE35809、GSE54129、GSE79973、GSE66229和GSE51105。合并5个数据集中的样本,去除数据集间的批次效应,对合并后的基因表达数据进行标准化,并通过主成分分析监测数据标准化情况。利用R语言中的limma包筛选胃癌组织和正常组织中表达差异的基因。利用DAVID数据库对胃癌发生发展过程中的差异基因进行功能富集分析,并通过STRING数据库和Cytoscape分析差异基因编码蛋白之间的相互作用网络并进行可视化。结果：总共筛选出1 205个差异基因,包括480个上调基因,725个下调基因。差异基因的生物学功能主要富集于细胞-细胞信号传导、炎症反应的调节、细胞粘附、细胞凋亡和离子的跨膜转运。KEGG信号通路分析显示差异基因主要富集于p53信号通路、PI3K-Akt信号通路、NF-κB信号通路。通过构建蛋白质相互作用网络筛选出了CENPE、KIF15、MELK、KIF2C、CENPF、KIF11、NUSAP1、UBE2C、TTK、AURKB、DLGAP5、TOP2A等29个Hub基因。结论：通过合并不同数据集,利用生物信息学方法筛选出胃癌发生发展过程中的关键基因和信号通路,为胃癌的诊疗提供新的候选标志物。     

Identification of cervical cancer markers by cDNA and tissue microarrays

The Pap test has effectively reduced the incidence and mortality of cervical cancer. However, because of the morphological basis of this test, sensitivity and specificity are less than ideal, a situation that complicates the clinical management of women diagnosed with low-grade cervical abnormalities. In an attempt to understand the molecular basis of cervical tumorigenesis and to discover molecular markers for accurate cervical cancer screening, we used cDNA microarrays containing >30,000 Unigene clones to examine the gene expression patterns of 34 cervical tissues from different clinically defined stages. It was found that global gene expression patterns separated normal cervical tissues and low-grade squamous intraepithelial lesions from cervical cancers and most of the high-grade squamous intraepithelial lesions (HSILs). Among the top 62 genes/(expressed sequence tags) that were overexpressed in tumors and HSIL tissues, 35 were confirmed using in situ hybridization on cervical tissue micorarrays. Many of these genes were overexpressed in high-grade dysplastic and malignant cervical epithelium or in stroma adjacent to the diseased tissues, with cellular proliferation and extracellular matrix-associated genes being the most common. In general, the extent of gene overexpression increased as the lesions progressed from low-grade squamous intraepithelial lesions to HSILs and finally to cancer. It is hoped that with additional development, some of these markers will improve the interpretation of cervical screening tests and provide useful information for patient management decisions.     

组织芯片技术检测蛋白激酶C-α蛋白在肺癌组织中的表达

背景与目的:蛋白激酶C(protein kinase C,PKC)在癌变过程中的作用使其成为肿瘤治疗的潜在重要靶点,非小细胞肺癌(non-small cell lung cancer,NSCLC)中存在PKC-α的异常表达和活性增高。本研究探讨组织芯片技术检测PKC-α蛋白在肺癌中的表达及其意义。方法:应用免疫组织化学SP方法检测肺癌组织芯片中PKC-α蛋白的表达,其中肿瘤组织182例,癌旁组织18例。182例肿瘤组织中,NSCLC为160例,SCLC为22例;Ⅰ期102例,Ⅱ～Ⅲ期80例。结果:PKC-α蛋白在182例肺癌组织中的表达率为48.3%(88/182),而18例癌旁组织的表达率为16.7%(3/18)(P<0.01);PKC-α蛋白在NSCLC组织中的表达率(53.1%)明显高于在SCLC中的表达率(13.6%),差异有显著性(P<0.01);而PKC-α的表达在腺癌及鳞癌之间无明显差异(P>0.05);PKC-α的表达与患者的性别、年龄等因素无明显相关;PKC-α的表达与NSCLC特别是腺癌的临床分期、淋巴结转移密切相关(P<0.05),而与SCLC的临床分期、淋巴结转移无明显相关;PKC-α的表达与腺癌的分化程度密切相关(P<0.01),而与鳞癌的分化程度无明显相关。结论:在NSCLC组织中存在PKC-α的高表达,PKC-α的表达在NSCLC特别是腺癌的浸润、转移和发展中具有重要作用。     

应用组织芯片技术检测肺癌组织中MRP-1/CD9的表达

目的MRP-1/CD9是穿膜四超家族成员之一,具有抑制细胞移动的功能,本研究旨在探讨其在肺癌组织中的表达情况及与病理分级、临床分期、转移与预后的关系.方法应用组织芯片技术,采用免疫组化S-P方法检测54例肺癌组织和10例同期正常肺组织中MRP-1/CD9的表达.统计学方法采用x2检验,检验水准为a=0.05.结果MRP-1/CD9在肺癌组织中阳性率为42.60%,与正常肺组织相比表达下调,统计学上具有显著性差异(P＜0.05);其蛋白表达情况与患者的年龄、性别、肿瘤肉眼类型无关,而与肿瘤的组织学类型、临床分期、分化程度、淋巴结转移有关.其中非小细胞肺癌(NSCLC)组表达显著高于小细胞肺癌(SCLC)组;高-中分化组高于低-未分化组;早期(Ⅰ Ⅱ)组高于中晚期(Ⅲ Ⅳ)组;无淋巴结转移组高于有淋巴结转移组,均具有显著性意义(P＜0.05).结论肺癌的进展可能与MRP-1/CD9的表达下调密切相关,尤其小细胞肺癌MRP-1/CD9的表达缺失更具有重要意义;MRP-1/CD9的表达降低促进了肿瘤的淋巴结转移,此指标有望成为判断肿瘤预后的新标志.     

Ki67 expression in invasive breast cancer: the use of tissue microarrays compared with whole tissue sections

Background

Although the prognostic value of Ki67 in breast cancer is well documented, using optimal cut-points for patient stratification, reproducibility of the scoring and interpretation of the results remains a matter of debate particularly when using tissue microarrays (TMAs). This study aims to assess Ki67 expression assessed on TMAs and their matched whole tissue sections (WTS). Moreover, whether the cut-off used for WTS is reproducible on TMA in BC molecular classes and the association between Ki67 expression cut-off, assessed on TMAs and WTS, and clinicopathological parameters and patient outcome were tested.

Method

A large series (n = 707) of primary invasive breast tumours were immunostained for Ki67 using both TMA and WTS and assessed as percentage staining and correlated with each other, clinicopathological parameters and patient outcome. In addition, MKI67 mRNA expression was correlated with Ki67 protein levels on WTS and TMAs in a subset of cases included in the METABRIC study.

Results

There was moderate concordance in Ki67 expression between WTS and TMA when analysed as a continuous variable (Intraclass correlation coefficient = 0.61) and low concordance when dichotomised (kappa value = 0.3). TMA showed low levels of Ki67 with mean percentage of expression of 35 and 22% on WTS and TMA, respectively. MKI67 mRNA expression was significantly correlated with protein expression determined on WTS (Spearman Correlation, r = 0.52) and to a lesser extent on TMA (r = 0.34) (p < 0.001). Regarding prediction of patient outcome, statistically significant differences were detected upon stratification of patients with tumours expressing Ki67 at 10, 15, 20, 25 or 30% in TMA. Using TMA, ≥20% Ki67 provided the best prognostic cut-off particularly in triple-negative and HER2-positive classes.

Conclusion

Ki67 expression in breast cancer can be evaluated using TMA although different cut-points are required to emulate results from WTS. A cut-off of ≥20% for Ki67 expression in BC provides the best prognostic correlations when TMAs are used.

     

Expression analysis of imbalanced genes in prostate carcinoma using tissue microarrays

To identify candidate genes relevant for prostate tumour prognosis and progression, we performed an exhaustive gene search in seven previously described genomic-profiling studies of 161 prostate tumours, and four expression profiling studies of 61 tumours. From the resulting list of candidate genes, six were selected for protein-expression analysis based on the availability of antibodies applicable to paraffinised tissue: fatty acid synthase (FASN), MYC, beta-adrenergic receptor kinase 1 (BARK1, GRK2) the catalytic subunits of protein phosphatases PP1alpha (PPP1CA) and PP2A (PPP2CB) and metastasis suppressor NM23-H1. These candidates were analysed by immunohistochemistry (IHC) on a tissue microarray containing 651 cores of primary prostate cancer samples and benign prostatic hyperplasias (BPH) from 175 patients. In univariate analysis, expression of PP1alpha (P=0.001) was found to strongly correlate with Gleason score. MYC immunostaining negatively correlated with both pT-stage and Gleason score (P<0.001 each) in univariate as well as in multivariate analysis. Furthermore, a subgroup of patients with high Gleason scores was characterised by a complete loss of BARK1 protein (P=0.023). In conclusion, our study revealed novel molecular markers of potential diagnostic and therapeutic relevance for prostate carcinoma.     

Proteomic analysis of human breast cancer tissue with laser-capture microdissection and reverse-phase protein microarrays

Despite recent advances in breast cancer therapy, women with similar types of breast cancers may respond very differently to standard treatments. The emerging field of clinical proteomics has the potential to revolutionize breast cancer therapy. The ultimate goal of clinical proteomics is to characterize information flow through protein cascades for individual patients. After the protein networks have been elucidated, drug therapies may be specially designed for each patient. The following review describes the proteomic technologies of laser-capture microdissection (LCM) and reverse-phase protein arrays (RPPAs). These technologies allow scientists to analyze relative abundances of key cellular signaling proteins from pure cell populations. Cell survival and apoptotic protein pathways are currently being monitored with LCM and RPPAs at the National Institutes of Health, in phase II clinical trials of metastatic breast and ovarian cancers. Ultimately, proteomics will become an integral component of tracking and managing individualized breast cancer therapy.     

Analysis of Metastatic-Related Gene Expression in Gastric Cancer by Low-Density cDNA Microarrays

OBJECTIVE To screen metastatic-related genes in human gastric cancer by a low-density cDNA microarray technique. METHODS A total of 18 paired gastric cancer and adjacent normal mu-cosa were examined by a low-density cDNA microarray containing 23 genes. RT-PCR was used for further verification. RESULTS The mRNA expression of MMP -7, heparanase, S100A4, hTERT, hRad17 in gastric cancers was higher than that in coupled normal mucosa (P=0.002, 0.00011, 0.000072, 0.002, 0.00016 respectively), whereas nm23H1, and CDH1 were lower (P=0.003, 0.012 respectively). The concordance was verified further by RT-PCR with a correlation coefficient of 0.774. In gastric primary lesions the mRNA expression of MMP-7, heparanase and S100A4 was higher in the serosa involved compared to non-involved (P=0.003, 0.009, 0.012 respectively), whereas nm23H1, CDH1, KAI1 were lower (P=0.001, 0.001, 0.006 respectively). With respect to the area of serosa involvement, MMP-7 and heparanase expressions were higher in an area of more than 20 cm2 compared to an area of less than 20 cm2 (P=0.001, 0.02 respectively), whereas nm23H1, CDH1 and KAI1 were lower (P=0.030, 0.041, 0.031 respectively). MMP-7 and hTERT expressions were higher in the heavier lymph node metastatic cases (no less than 7) than in the lighter lymph node metastatic cases (no more than 6, P=0.001, 0.005 respectively). CONCLUSION Expression of MMP -7, S100A4, heparanase, hTERT, KAI1, CDH1 and nm23H1 correlated closely with invasion and metastasis in gastric carcinomas. The low-density cDNA microarrays can be used to examine the expression of many genes simultaneously, parallely and quickly.     

C-myc蛋白在睾丸肿瘤中的表达及意义

目的　探讨C myc基因蛋白与睾丸肿瘤生物学行为及预后的关系。方法　采用免疫组化S P法对 38例睾丸肿瘤和 10例正常睾丸组织中C myc蛋白表达进行了定量分析。结果　 31例 (81.6 % )睾丸肿瘤显示阳性反应 ,正常睾丸组织为阴性反应。肿瘤组和正常组组织阳性细胞率分别为 2 4 .99%和 3.2 6 % ,两者相比有非常显著性差异 (P <0 .0 1)。在睾丸肿瘤中C myc蛋白的表达程度与肿瘤病理分级、临床分期无关 (P >0 .0 5 )。C myc表达在精原细胞瘤组及非精原细胞瘤组间无显著性差异 (P >0 .0 5 )。结论　C myc蛋白的异常表达可能在睾丸肿瘤的发生、发展过程中起着促进作用。     

Global gene expression analysis of gastric cancer by oligonucleotide microarrays.

To gain molecular understanding of carcinogenesis, progression, and diversity of gastric cancer, 22 primary human advanced gastric cancer tissues and 8 noncancerous gastric tissues were analyzed by high-density oligonucleotide microarray in this study. Based on expression analysis of approximately 6800 genes, a two-way clustering algorithm successfully distinguished cancer tissues from noncancerous tissues. Subsequently, genes that were differentially expressed in cancer and noncancerous tissues were identified; 162 and 129 genes were highly expressed (P < 0.05) >2.5-fold in cancer tissues and noncancerous tissues, respectively. In cancer tissues, genes related to cell cycle, growth factor, cell motility, cell adhesion, and matrix remodeling were highly expressed. In noncancerous tissues, genes related to gastrointestinal-specific function and immune response were highly expressed. Furthermore, we identified several genes associated with lymph node metastasis including Oct-2 or histological types including Liver-Intestine Cadherin. These results provide not only a new molecular basis for understanding biological properties of gastric cancer, but also useful resources for future development of therapeutic targets and diagnostic markers for gastric cancer.     

Clinical validation of candidate genes associated with prostate cancer progression in the CWR22 model system using tissue microarrays

To explore molecular mechanisms of prostate cancer progression, we applied tissue microarrays (TMAs) to analyze expression of candidate gene targets discovered by cDNA microarray analysis of the CWR22 xenograft model system. A TMA with 544 clinical specimens from different stages of disease progression was probed by mRNA in situ hybridization and protein immunohistochemistry. There was an excellent correlation (r = 0.96; n = 16) between the expression levels of the genes in the xenografts by cDNA microarray and mRNA in situ hybridization on a TMA. One of the most highly overexpressed genes in hormone-refractory CWR22R xenografts was the S100P gene. This gene, coding for a calcium signaling molecule implicated in the loss of senescence, was also significantly associated with progression in clinical tumors by TMA analysis (P < 0.001), suggesting dysregulation of this pathway in hormone-refractory and metastatic prostate cancers. Conversely, two genes that were down-regulated during tumor progression in the CWR22 model system were validated in vivo: crystallin mu (CRYM) and a LIM-domain protein LMO4 both showed significantly lower mRNA levels in hormone-refractory tumors as compared with primary tumors (P < 0.001). These results illustrate a strategy for rapid clinical validation at the mRNA and protein level of gene targets found to be differentially expressed in cDNA microarray experiments of model systems of cancer.