Genomic profiles in stage I primary non small cell lung cancer using comparative genomic hybridization analysis of cDNA microarrays

To investigate the genomic aberrations that are involved in lung tumorigenesis and therefore may be developed as biomarkers for lung cancer diagnosis, we characterized the genomic copy number changes associated with individual genes in 14 tumors from patients with primary non small cell lung cancer (NSCLC). Six squamous cell carcinomas (SQCAs) and eight adenocarcinomas (ADCAs) were examined by high-resolution comparative genomic hybridization (CGH) analysis of cDNA microarray. The SQCAs and ADCAs shared common frequency distributions of recurrent genomic gains of 63 genes and losses of 72 genes. Cluster analysis using 57 genes defined the genomic differences between these two major histologic types of NSCLC. Genomic aberrations from a set of 18 genes showed distinct difference of primary ADCAs from their paired normal lung tissues. The genomic copy number of four genes was validated by fluorescence in situ hybridization of 32 primary NSCLC tumors, including those used for cDNA microarray CGH analysis; a strong correlation with cDNA microarray CGH data emerged. The identified genomic aberrations may be involved in the initiation and progression of lung tumorigenesis and, most importantly, may be developed as new biomarkers for the early detection and classification of lung cancer.     

Detection of gene amplification by genomic hybridization to cDNA microarrays

Gene amplification is one of the major mechanisms of oncogene activation in tumorigenesis. To facilitate the identification of genes mapping to amplified regions, we have used a technique based on the hybridization of total genomic DNA to cDNA microarrays. To aid detection of the weak signals generated in this complex hybridization, we have used a tyramide-based technique that allows amplification of a fluorescent signal up to 1000-fold. Dilution experiment suggests that amplifications of 5-fold and higher can be detected by this approach. The technique was validated using cancer cell lines with several known gene amplifications, such as those affecting MYC, MYCN, ERBB2, and CDK4. In addition to the detection of the known amplifications, we identified a novel amplified gene, ZNF133, in the neuroblastoma cell line NGP. Hybridization of NGP cDNA on an identical array also revealed over expression of ZNF133. Parallel analysis of genomic DNA for copy number and cDNA for expression now provides rapid approach to the identification of amplified genes and chromosomal regions in tumor cells.     

Expression analysis onto microarrays of randomly selected cDNA clones highlights HOXB13 as a marker of human prostate cancer

In a strategy aimed at identifying novel markers of human prostate cancer, we performed expression analysis using microarrays of clones randomly selected from a cDNA library prepared from the LNCaP prostate cancer cell line. Comparisons of expression profiles in primary human prostate cancer, adjacent normal prostate tissue, and a selection of other (nonprostate) normal human tissues, led to the identification of a set of clones that were judged as the best candidate markers of normal and/or malignant prostate tissue. DNA sequencing of the selected clones revealed that they included 10 genes that had previously been established as prostate markers: NKX3.1, KLK2, KLK3 (PSA), FOLH1 (PSMA), STEAP2, PSGR, PRAC, RDH11, Prostein and FASN. Following analysis of the expression patterns of all selected and sequenced genes through interrogation of SAGE databases, a further three genes from our clone set, HOXB13, SPON2 and NCAM2, emerged as additional candidate markers of human prostate cancer. Quantitative RT-PCR demonstrated the specificity of expression of HOXB13 in prostate tissue and revealed its ubiquitous expression in a series of 37 primary prostate cancers and 20 normal prostates. These results demonstrate the utility of this expression-microarray approach in hunting for new markers of individual human cancer types.     

Progressive genetic aberrations detected by comparative genomic hybridization in squamous cell cervical cancer

Genetic changes orchestrated by human papillomaviruses are the most important known factors in carcinogenesis of the uterine cervix. However, it is clear that additional genetic events are necessary for tumour progression. We have used comparative genomic hybridization to document non-random chromosomal gains and losses within a subset of 37 cervical carcinomas matched for clinical stage Ib, but with different lymph node status. There were significantly more chromosomal changes in the primary tumours when the lymph nodes were positive for metastases. The most frequent copy number alterations were loss of 3p, 11q, 6q and 10q and gain of 3q. The smallest areas of loss and gain on chromosome 3 were 3p14-22 and 3q24-26. The study identifies progressive DNA copy number changes associated with early-stage invasive cervical cancers with and without lymph node metastases, a factor of potential prognostic and therapeutic value.     

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.     

基因芯片在乳腺癌研究中的应用

基因芯片因能快速检测肿瘤组织和细胞中的基因表达情况而广泛应用于乳腺癌研究中。可以根据乳腺癌基因表达情况对乳腺癌进行分子分型、预测预后，且能够有效地检测内分泌治疗、化学治疗和分子靶向治疗的敏感性。基因芯片技术与乳腺癌临床研究结合将会提高乳腺癌临床治疗水平。     

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.     

High-resolution cDNA microarray-based comparative genomic hybridization analysis in neuroblastoma

Neuroblastoma (NB) is one of the most common pediatric solid tumors and displays a broad variety of genomic alterations. Array-based comparative genomic hybridization (A-CGH) is a novel technology enabling the high-resolution detection of DNA copy number aberrations. In this article, we outline features of this new technology and approaches of data analysis. We focus on stage specific DNA copy number variations in neuroblastoma detected by cDNA array-based comparative genomic hybridization (A-CGH). We also discuss hypothetic evolutionary models of neuroblastoma progression that can be derived from A-CGH data.     

Detection of recurrent copy number loss at Yp11.2 involving TSPY gene cluster in prostate cancer using array-based comparative genomic hybridization

Prostate cancer is the second leading cause of cancer deaths among American men. The loss of Y chromosome has been frequently observed in primary prostate cancer as well as other types of cancer. Earlier, we showed that introduction of the human Y chromosome suppresses the in vivo tumorigenicity of the prostate cancer cell line PC-3. To further characterize the Y chromosome, we have developed a high-density bacterial artificial chromosome (BAC) microarray containing 178 BAC clones from the human Y chromosome. BAC microarray was used for array comparative genomic hybridization on prostate cancer samples and cell lines. The most prominent observation on prostate cancer specimens was a deletion at Yp11.2 containing the TSPY tandem gene array. Out of 36 primary prostate tumors analyzed, 16 (44.4%) samples exhibited loss of TSPY gene copies. Notably, we observed association between the number of TSPY copies in the blood and the incidence of prostate cancer. Moreover, PC-3 hybrids with an intact Yp11.2 did not grow tumors in nude mice, whereas PC-3 hybrids with a deletion at Yp11.2 grew tumors in nude mice.     

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.     

Genome-wide array comparative genomic hybridization analysis reveals distinct amplifications in osteosarcoma

Background  

Osteosarcoma is a highly malignant bone neoplasm of children and young adults. It is characterized by extremely complex karyotypes and high frequency of chromosomal amplifications. Currently, only the histological response (degree of necrosis) to therapy represent gold standard for predicting the outcome in a patient with non-metastatic osteosarcoma at the time of definitive surgery. Patients with lower degree of necrosis have a higher risk of relapse and poor outcome even after chemotherapy and complete resection of the primary tumor. Therefore, a better understanding of the underlying molecular genetic events leading to tumor initiation and progression could result in the identification of potential diagnostic and therapeutic targets.     

Molecular profiling and genomic microarrays in prostate cancer

In the present review article a global approach regarding the usefulness of genomic microarrays in prostate cancer management, is attempted. Cancer is a multistep process of mutations in key regulatory genes and epigenetic alterations that result in loss of balanced gene expression. A complete knowledge of the interaction between the genetic variability of the neoformation (tumor profiling) and the genetic variability of the host (inherited genome profiling), will be able to determine the better strategy against the cancer and the less toxicity for the patient. Alterations in the sequence of the hormone binding domain of the androgen receptor as well as mutations in some genes, determine radioresistance and resistance or sensitivity to some chemotherapeutic drugs. New therapies using monoclonal antibodies directed against specific extracellular binding domains of some receptors are based on molecular alterations observed in tumors.     

Global gene expression profiling in Barrett's esophagus and esophageal cancer: a comparative analysis using cDNA microarrays

In order to identify and contrast global gene expression profiles defining the premalignant syndrome, Barrett's esophagus, as well as frank esophageal cancer, we utilized cDNA microarray technology in conjunction with bioinformatics tools. We hybridized microarrays, each containing 8000 cDNA clones, to RNAs extracted from 13 esophageal surgical or endoscopic biopsy specimens (seven Barrett's metaplasias and six esophageal carcinomas). Hierarchical cluster analysis was performed on these results and displayed using a color-coded graphic representation (Treeview). The esophageal samples clustered naturally into two principal groups, each possessing unique global gene expression profiles. After retrieving histologic reports for these tissues, we found that one main cluster contained all seven Barrett's samples, while the remaining principal cluster comprised the six esophageal cancers. The cancers also clustered according to histopathological subtype. Thus, squamous cell carcinomas (SCCAs) constituted one group, adenocarcinomas (ADCAs) clustered separately, and one signet-ring carcinoma was in its own cluster, distinct from the ADCA cluster. We conclude that cDNA microarrays and bioinformatics show promise in the classification of esophageal malignant and premalignant diseases, and that these methods can be applied to small biopsy samples.     

Unanswered questions in screening for prostate cancer

Prostate cancer fulfils some of the conditions required of a disease that might be managed by population screening. In a cohort of 50- to 60-year-old men, carrying out a rectal examination and prostate specific antigen (PSA) test will detect clinically suspicious areas within the prostate in approximately 5%, and approximately 10% will have a raised PSA. We are however unsure which of the prostate cancers that are known to be present in approximately 30-40% of men aged over 60 years will be detected. Eventually after such screening, around 4% of men with an otherwise normal prostate will be found to have prostate cancers. The use of rectal examination may increase the number of tumours found, but will reduce compliance. The use of free/total PSA ratios will reduce the number of unnecessary biopsies at the expense of missing some tumours. Of more concern, we remain uncertain how effective aggressive local treatment is in altering the natural history of the disease. The risk of a 50-year-old man with a 25 year life expectancy of having microscopic cancer is 42%, of having clinically evident cancer is 9.5%, and of dying of prostate cancer 2.9%. Only a small proportion of cancers known to be present become clinically evident: more men die with prostate cancer than of it. Screening will identify some men with cancer who will not benefit from treatment. It is unclear whether screening would be followed by a reduction in morbidity and mortality. Recent data suggest a screening effect has been observed in the USA with: an increase in incidence, a decrease in men with distant metastases. The small decrease in mortality recently observed (many times smaller than the increase in incidence) may be confounded by inappropriate 'attribution' of cause of death, the detection of men with better prognosis distant metastatic disease responsive to hormonal ablation and changes in social factors such as diet. Future changes may incorporate molecular markers that might aid identification of men best treated aggressively because of a risk of progression. Tests to identify genetic pre-disposition may also allow targeted screening. New treatments and early chemoprevention or dietary strategies will again shift the ground on which these arguments are being rehearsed. The most urgent evidence required concerns the effectiveness of treatment strategies.     

Genome-wide screening for prognosis-predicting genes in early-stage non-small-cell lung cancer

In early-stage non-small-cell lung cancer (NSCLC), a substantial proportion of patients can be cured by surgery. Development of distant metastases is the most frequent cause of therapeutic failure. The possibility to accurately predict a patient's risk for developing distant metastasis would help to identify patients that are candidates for further intervention such as conventional adjuvant chemotherapy or experimental drugs. Current molecular biology techniques allow the genome-wide screening for differentially expressed genes; and adequate bioinformatics approaches are developed at a rapid pace to improve prognosis prediction. Genes associated with metastasis do not necessarily play a role in disease pathogenesis but rather reflect the activation of specific signal-transduction pathways that are associated with enhanced migration and invasion capability. In our own work, we have identified several genes (e.g. thymosin beta-4, elF4A1), including a novel non-coding RNA (MALAT-1) to be expressed at significantly higher levels in stage-I and stage-II NSCLC primary tumours that subsequently metastasised. As a consequence, patients with high-level expression of these genes were shown to have significantly worse survival compared to patients with low-level expression of these genes. These data support the hypothesis that gene-expression patterns in primary tumours determine the tumours' likelihood to metastasise. In the near future, this information will be used for tailored therapy approaches for patients with early-stage NSCLC.     

Genome-wide cDNA microarray screening to correlate gene expression profile with survival in patients with advanced lung cancer

We conducted a study using cDNA microarray analysis to determine whether expression levels of genes in tumors were correlated with survival after chemotherapy. Between September 2000 and December 2001, 47 patients were registered in the study. Eighteen patients had small cell lung cancer (SCLC), and the others had non-small cell lung cancer (NSCLC). All patients except three received platinum-based chemotherapy. Transbroncheal biopsy specimens of tumors were obtained before chemotherapy. The expression levels of 1176 genes in tumor specimens were analyzed using the Atlas Human Cancer 1.2 Array. The expression levels of three genes, G1/S-specific cyclin, type II cGMP-dependent protein kinase and hepatocyte growth factor-like protein, were significantly correlated with survival (p<0.01). Ten of the 47 patients who showed an elevated expression level of one or more of the three genes had a significantly increased chance of survival (p=0.0056). In conclusion, some survival-related genes were detected in the tumor tissue of lung cancer patients using cDNA microarray analysis. A prospective study is required to confirm whether expression levels of these genes can be used for prognosis.     

Genetic alterations in lobular breast cancer by comparative genomic hybridization

Infiltrating lobular carcinoma (ILC) and infiltrating ductal carcinoma (IDC) are distinguished by their histopathological appearance. However, little is known about the differences in genetic changes between lobular cancers and ductal cancers. We used comparative genomic hybridization (CGH) and compared aberrations in 19 ILCs and 46 IDCs. The total number of aberrations was lower in ILC than in IDC. While the average number of DNA copy number losses did not reach significance between them, copy number gains were significantly lower in ILCs. Fifteen of 19 ILCs (79%) showed increased copy number of 1q, and 12 cases (63%) revealed loss of 16q. The presence of these aberrations was independent of nodal status, histologic subtypes (pleomorphic or classic ILC), or BrdUrd-labeling index. ILCs had a higher frequency of 16q loss than did ductal cancers, and a lower frequency of 8q and 20q gains. Our data suggest that the altered growth pattern and clinical presentation which characterize infiltrating lobular cancers are correlated with distinct genetic alterations. Int. J. Cancer 74:513–517, 1997. © 1997 Wiley-Liss, Inc.     

Ethical issues in genetic screening for cancer

Genetically-based diseases with a late onset, such as BRCA1-dependent breast cancer or Huntington's disease, can be predicted by the screening of relevant mutations in members of high-risk families. Genetic screening is characterized by a conflict between respect for autonomy – e.g., the right not to know – and responsibility toward future generations (the duty to know for the sake of one's descendants). Other ethical conflicts are related to uncertainty as to benefits deriving from screening for mutations, since for most conditions no clearly effective therapeutical strategy has as yet been defined. In addition to monogenic high-penetrance conditions, polygenic low-penetrance susceptibility is attracting increasing attention, in particular with respect to environmental-genetic interactions (metabolic polymorphisms). A simple approach to genetic screening would be to weigh the benefits and costs of genetic screening against those of primary prevention, and a superficial conclusion might be that genetic screening is less expensive and, overall, more practicable than restriction of toxic exposures or other known risk factors for the disease. Economic advantage notwithstanding, however, giving precedence to screening over primary prevention would be unacceptable. A serious hazard of genetic screening is the implicit limitation of research efforts aimed at primary prevention, and a serious drawback is its potential application for selection of non-susceptible employees. The principle of equity is easily violated by genetic screening of workers in view of the fact that genetically-based metabolic polymorphisms are distributed unevenly among different ethnic groups.