Frequent high-level expression of the immunotherapeutic target Ep-CAM in colon, stomach, prostate and lung cancers.

Epithelial cell adhesion molecule (Ep-CAM; CD326) is used as a target by many immunotherapeutic approaches, but little data are available about Ep-CAM expression in major human malignancies with respect to level, frequency, tumour stage, grade, histologic tumour type and impact on survival. We analysed by immunohistochemical staining tissue microarrays with 4046 primary human carcinoma samples from colon, stomach, prostate and lung cancers for both frequency and intensity of Ep-CAM expression under highly standardised conditions. A total of 3360 samples were analysable. High-level Ep-CAM expression was observed in 97.7% (n=1186) of colon, 90.7% of gastric (n=473), and 87.2% of prostate cancers (n=414), and in 63.9% of lung cancers (n=1287). No detectable Ep-CAM staining was found with only 0.4% of colon, 2.5% of gastric, 1.9% of prostate cancers, and 13.5% of lung cancers. The only significant correlation of Ep-CAM expression with tumour grading was observed in colon cancer where high-level Ep-CAM expression on grade 3 tumours was down to 92.1% (P<0.0001). Adenosquamous and squamous carcinomas of the lung had a lower percentage of high-level Ep-CAM expression compared to adenocarcinomas with 35.4 and 53.6%, respectively, and with 45.5 and 17.3% of tumours being Ep-CAM negative. With the exception of moderately differentiated colon carcinoma, where patients not expressing Ep-CAM on their tumours showed an inferior survival (P=0.0014), correlation of Ep-CAM expression with survival did not reach statistical significance for any of the other cancer indications and subgroups. In conclusion, the data strongly support the notion that Ep-CAM is a prime target for immunotherapies in major human malignancies. This is because the most common human cancers show (i) a low frequency of Ep-CAM-negative tumours, (ii) a high frequency of Ep-CAM expression on cells of a given tumour, and (iii) for most cancers, an insignificant influence of tumour staging, grading and histology on Ep-CAM expression.

[Analysis of gene expression profiles among 3 epithelial ovarian tumor subtypes using cDNA and tissue microarrays].

BACKGROUND & OBJECTIVE: Ovarian cancer is the leading cause of death among the gynecological malignancy mainly due to lacking of effective early diagnostic methods. To identify novel candidate genes and further explore their clinical significance of epithelial ovarian tumors, we developed a new method in our laboratory by combining cDNA microarray with RNA in situ hybridization in frozen tissue microarray. METHODS: cDNA microarrays were used to seek differentially expressed genes among 3 subtypes of ovarian tumors (serous borderline ovarian tumors, serous ovarian cancers, and endometrioid ovarian carcinomas). RNA in situ hybridization in frozen tissue microarray was used to further confirm the findings from cDNA microarrays. RESULTS: In the study of cDNA microarray, 40 genes and ESTs showed significant differential expression between low and high-malignancy, as well as serous and endometrioid carcinomas. EPHB6, PTPRF, GFER, ERG25, PLRP1, FLJ22060, and WISP2 were further validated by RNA in situ hybridization in tissue microarray. CONCLUSIONS: cDNA microarray combined with RNA in situ hybridization in frozen tissue microarray is an ideal choice for identifying novel oncogenes. EPHB6, PTPRF, GFER, ERG25, PLRP1, FLJ22060, and WISP2 might become the new candidate oncogenes for epithelial ovarian cancer.

Tissue microarrays for rapid linking of molecular changes to clinical endpoints.

Advances in genomics and proteomics are dramatically increasing the need to evaluate large numbers of molecular targets for their diagnostic, predictive or prognostic value in clinical oncology. Conventional molecular pathology techniques are often tedious, time-consuming, and require a lot of tissue, thereby limiting both the number of tissues and the number of targets that can be evaluated. Here, we demonstrate the power of our recently described tissue microarray (TMA) technology in analyzing prognostic markers in a series of 553 breast carcinomas. Four independent TMAs were constructed by acquiring 0.6 mm biopsies from one central and from three peripheral regions of each of the formalin-fixed paraffin embedded tumors. Immunostaining of TMA sections and conventional "large" sections were performed for two well- established prognostic markers, estrogen receptor (ER) and progesterone receptor (PR), as well as for p53, another frequently examined protein for which the data on prognostic utility in breast cancer are less unequivocal. Compared with conventional large section analysis, a single sample from each tumor identified about 95% of the information for ER, 75 to 81% for PR, and 70 to 74% for p53. However, all 12 TMA analyses (three antibodies on four different arrays) yielded as significant or more significant associations with tumor-specific survival than large section analyses (p < 0.0015 for each of the 12 comparisons). A single sample from each tumor was sufficient to identify associations between molecular alterations and clinical outcome. It is concluded that, contrary to expectations, tissue heterogeneity did not negatively influence the predictive power of the TMA results. TMA technology will be of substantial value in rapidly translating genomic and proteomics information to clinical applications.

Amplification and overexpression of PRUNE in human sarcomas and breast carcinomas-a possible mechanism for altering the nm23-H1 activity.

PRUNE, the human homologue of the Drosophila gene, is located in 1q21.3, a region highly amplified in human sarcomas, malignant tumours of mesenchymal origin. Prune protein interacts with the metastasis suppressor nm23-H1, but shows impaired affinity towards the nm23-H1 S120G mutant associated with advanced neuroblastoma. Based on these observations, we previously suggested that prune may act as a negative regulator of nm23-H1 activity. We found amplification of PRUNE in aggressive sarcoma subtypes, such as leiomyosarcomas and malignant fibrous histiocytomas (MFH) as well as in the less malignant liposarcomas. PRUNE amplification was generally accompanied by high mRNA and moderate to high protein levels. The sarcoma samples expressed nm23-H1 mostly at low or moderate levels, whereas mRNA and protein levels were moderate to high in breast carcinomas. For the more aggressive sarcoma subtypes, 9/13 patients with PRUNE amplification developed metastases. A similar situation was observed in all breast carcinomas with amplification of PRUNE. Infection of NIH3T3 cells with a PRUNE recombinant retrovirus increased cell proliferation. Possibly, amplification and overexpression of PRUNE has the same effect in the tumours. We suggest that amplification and overexpression of PRUNE could be a mechanism for inhibition of nm23-H1 activity that affect the development or progression of these tumours.

Tissue microarrays (TMAs) for high-throughput molecular pathology research.

A rapidly increasing number of genes are being suspected to play a role in cancer biology. To evaluate the clinical significance of newly detected potential cancer genes, it is usually required to examine a high number of well-characterized primary tumors. Using traditional methods of molecular pathology, this is a time consuming endeavor rapidly exhausting precious tissue resources. To allow for a high throughput tissue analysis we have developed a "tissue chip" approach (Kononen et al., Nat. Med. 1998;4:844-7). Using this tissue microarray (TMA) technology, samples from up to 1,000 different tumors are arrayed in one recipient paraffin block, sections of which can be used for all kind of in situ analyses. Section from TMA blocks can then be utilized for the simultaneous analysis of up to 1,000 different tumors on the DNA, RNA or protein level. TMAs allow a high throughput molecular analysis of thousands of tumors within a few hours. All currently available data have suggested that minute arrayed tissue specimens are highly representative of their donor tissues. There are multiple different types of TMAs that can be utilized in cancer research including multi tumor arrays (containing different tumor types), tumor progression arrays (tumors of different stages) and prognostic arrays (tumors with clinical endpoints). The combination of multiple different TMAs allows a very quick but comprehensive characterization of biomarkers of interest. We anticipate that the use of TMAs will greatly accelerate the transition of basic research findings to clinical applications.

CGH, cDNA and tissue microarray analyses implicate FGFR2 amplification in a small subset of breast tumors.

Multiple regions of the genome are often amplified during breast cancer development and progression, as evidenced in a number of published studies by comparative genomic hybridization (CGH). However, only relatively few target genes for such amplifications have been identified. Here, we indicate how small-scale commercially available cDNA and CGH microarray formats combined with the tissue microarray technology enable rapid identification of putative amplification target genes as well as analysis of their clinical significance. According to CGH, the SUM-52 breast cancer cell line harbors several high-level DNA amplification sites, including the 10q26 chromosomal region where the fibroblast growth factor receptor 2 (FGFR2) gene has been localized. High level amplification of FGFR2 in SUM-52 was identified using CGH analysis on a microarray of BAC clones. A cDNA microarray survey of 588 genes showed >40-fold overexpression of FGFR2. Finally, a tissue microarray based FISH analysis of 750 uncultured primary breast cancers demonstrated in vivo amplification of the FGFR2 gene in about 1% of the tumors. In conclusion, three consecutive microarray (CGH, cDNA and tissue) experiments revealed high-level amplification and overexpression of the FGFR2 in a breast cancer cell line, but only a low frequency of involvement in primary breast tumors. Applied to a genomic scale with larger arrays, this strategy should facilitate identification of the most important target genes for cytogenetic rearrangements, such as DNA amplification sites detected by conventional CGH. Figures on http://www.esacp.org/acp/2001/22-4/heiskanen.htm

Patterns of her-2/neu amplification and overexpression in primary and metastatic breast cancer.

BACKGROUND: Only 25% of patients with HER-2/neu-positive metastatic breast tumors respond favorably to trastuzamab (Herceptin) treatment. We hypothesized that a high failure rate of patients on trastuzamab could result if some of the metastases were HER-2 negative and these metastases ultimately determine the course of the disease. METHODS: We used tissue microarrays (TMAs) containing four samples each from 196 lymph node-negative primary tumors, 196 lymph node-positive primary tumors, and three different lymph node metastases from each lymph node-positive tumor to estimate HER-2 gene amplification by fluorescence in situ hybridization (FISH) and Her-2 protein overexpression by immunohistochemistry (IHC). RESULTS: FISH and IHC analyses gave the same result with respect to HER-2 status for 93.7% of the tissues contained in the TMAs. Tissue samples were, therefore, considered to be HER-2 positive if they were positive for either HER-2 DNA amplification or Her-2 protein expression and HER-2 negative if both FISH and IHC gave a negative result. The HER-2 status of lymph node-positive primary tumors was maintained in the majority of their metastases. For HER-2-positive primary tumors, 77% (95% confidence interval [CI] = 59% to 90%) had entirely HER-2-positive metastases, 6.5% (95% CI = 8% to 21%) had entirely HER-2-negative metastases, and 16.3% (95% CI = 5% to 34%) had a mixture of HER-2-positive and HER-2-negative metastases. For HER-2-negative primary tumors, 95% (95% CI = 88% to 98%) had metastases that were entirely negative for HER-2. CONCLUSIONS: Our data suggest that differences in HER-2 expression between primary tumors and their lymph node metastases cannot explain the high fraction of nonresponders to trastuzamab therapy.

Microarrays of bladder cancer tissue are highly representative of proliferation index and histological grade.

The number of genes suggested to play a role in cancer biology is rapidly increasing. To be able to test a large number of molecular parameters in sufficiently large series of primary tumours, a tissue microarray (TMA) approach has been developed where samples from up to 1000 tumours can be simultaneously analysed on one glass slide. Because of the small size of the individual arrayed tissue samples (diameter 0.6 mm), the question arises of whether these specimens are representative of their donor tumours. To investigate how representative are the results obtained on TMAs, a set of 2317 bladder tumours that had been previously analysed for histological grade and Ki67 labelling index (LI) was used to construct four replica TMAs from different areas of each tumour. Clinical follow-up information was available from 1092 patients. The histological grade and the Ki67 LI were determined for every arrayed tumour sample (4x2317 analyses each). Despite discrepancies in individual cases, the grade and Ki67 information obtained on minute arrayed samples were highly similar to the data obtained on large sections (p<0.0001). Most importantly, every individual association between grade or Ki67 LI and tumour stage or prognosis (recurrence, progression, tumour-specific survival) that was observed in large section analysis could be fully reproduced on all four replica TMAs. These results show that intra-tumour heterogeneity does not significantly affect the ability to detect clinico-pathological correlations on TMAs, probably because of the large number of tumours that can be included in TMA studies. TMAs are a powerful tool for rapid identification of the biological or clinical significance of molecular alterations in bladder cancer and other tumour types.

Comprehensive copy number and gene expression profiling of the 17q23 amplicon in human breast cancer.

The biological significance of DNA amplification in cancer is thought to be due to the selection of increased expression of a single or few important genes. However, systematic surveys of the copy number and expression of all genes within an amplified region of the genome have not been performed. Here we have used a combination of molecular, genomic, and microarray technologies to identify target genes for 17q23, a common region of amplification in breast cancers with poor prognosis. Construction of a 4-Mb genomic contig made it possible to define two common regions of amplification in breast cancer cell lines. Analysis of 184 primary breast tumors by fluorescence in situ hybridization on tissue microarrays validated these results with the highest amplification frequency (12.5%) observed for the distal region. Based on GeneMap'99 information, 17 known genes and 26 expressed sequence tags were localized to the contig. Analysis of genomic sequence identified 77 additional transcripts. A comprehensive analysis of expression levels of these transcripts in six breast cancer cell lines was carried out by using complementary DNA microarrays. The expression patterns varied from one cell line to another, and several overexpressed genes were identified. Of these, RPS6KB1, MUL, APPBP2, and TRAP240 as well as one uncharacterized expressed sequence tag were located in the two common amplified regions. In summary, comprehensive analysis of the 17q23 amplicon revealed a limited number of highly expressed genes that may contribute to the more aggressive clinical course observed in breast cancer patients with 17q23-amplified tumors.

ANX7, a candidate tumor suppressor gene for prostate cancer.

The ANX7 gene is located on human chromosome 10q21, a site long hypothesized to harbor a tumor suppressor gene(s) (TSG) associated with prostate and other cancers. To test whether ANX7 might be a candidate TSG, we examined the ANX7-dependent suppression of human tumor cell growth, stage-specific ANX7 expression in 301 prostate specimens on a prostate tissue microarray, and loss of heterozygosity (LOH) of microsatellite markers at or near the ANX7 locus. Here we report that human tumor cell proliferation and colony formation are markedly reduced when the wild-type ANX7 gene is transfected into two prostate tumor cell lines, LNCaP and DU145. Consistently, analysis of ANX7 protein expression in human prostate tumor microarrays reveals a significantly higher rate of loss of ANX7 expression in metastatic and local recurrences of hormone refractory prostate cancer as compared with primary tumors (P = 0.0001). Using four microsatellite markers at or near the ANX7 locus, and laser capture microdissected tumor cells, 35% of the 20 primary prostate tumors show LOH. The microsatellite marker closest to the ANX7 locus showed the highest rate of LOH, including one homozygous deletion. We conclude that the ANX7 gene exhibits many biological and genetic properties expected of a TSG and may play a role in prostate cancer progression.

Tissue microarray technology for high-throughput molecular profiling of cancer.

Tissue microarray (TMA) technology allows rapid visualization of molecular targets in thousands of tissue specimens at a time, either at the DNA, RNA or protein level. The technique facilitates rapid translation of molecular discoveries to clinical applications. By revealing the cellular localization, prevalence and clinical significance of candidate genes, TMAs are ideally suitable for genomics-based diagnostic and drug target discovery. TMAs have a number of advantages compared with conventional techniques. The speed of molecular analyses is increased by more than 100-fold, precious tissues are not destroyed and a very large number of molecular targets can be analyzed from consecutive TMA sections. The ability to study archival tissue specimens is an important advantage as such specimens are usually not applicable in other high-throughput genomic and proteomic surveys. Construction and analysis of TMAs can be automated, increasing the throughput even further. Most of the applications of the TMA technology have come from the field of cancer research. Examples include analysis of the frequency of molecular alterations in large tumor materials, exploration of tumor progression, identification of predictive or prognostic factors and validation of newly discovered genes as diagnostic and therapeutic targets.

Gene-expression profiles in hereditary breast cancer.

BACKGROUND: Many cases of hereditary breast cancer are due to mutations in either the BRCA1 or the BRCA2 gene. The histopathological changes in these cancers are often characteristic of the mutant gene. We hypothesized that the genes expressed by these two types of tumors are also distinctive, perhaps allowing us to identify cases of hereditary breast cancer on the basis of gene-expression profiles. METHODS: RNA from samples of primary tumor from seven carriers of the BRCA1 mutation, seven carriers of the BRCA2 mutation, and seven patients with sporadic cases of breast cancer was compared with a microarray of 6512 complementary DNA clones of 5361 genes. Statistical analyses were used to identify a set of genes that could distinguish the BRCA1 genotype from the BRCA2 genotype. RESULTS: Permutation analysis of multivariate classification functions established that the gene-expression profiles of tumors with BRCA1 mutations, tumors with BRCA2 mutations, and sporadic tumors differed significantly from each other. An analysis of variance between the levels of gene expression and the genotype of the samples identified 176 genes that were differentially expressed in tumors with BRCA1 mutations and tumors with BRCA2 mutations. Given the known properties of some of the genes in this panel, our findings indicate that there are functional differences between breast tumors with BRCA1 mutations and those with BRCA2 mutations. CONCLUSIONS: Significantly different groups of genes are expressed by breast cancers with BRCA1 mutations and breast cancers with BRCA2 mutations. Our results suggest that a heritable mutation influences the gene-expression profile of the cancer.

CDKNA2A mutation analysis, protein expression, and deletion mapping of chromosome 9p in conventional clear-cell renal carcinomas: evidence for a second tumor suppressor gene proximal to CDKN2A.

Inactivation of tumor suppressor genes on chromosome 9p is considered a critical event in renal cell carcinoma pathogenesis. Alterations of CDKN2A on 9p21 have been reported in renal cancer cell lines, but their relevance for primary renal carcinomas is unclear. Loss of heterozygosity (LOH) was analyzed by using four polymorphic microsatellites at D9S970 (9p12-9p13), D9S171 (9p13), D9S1748 (9p21), and D9S156 (9p21) in 113 primary conventional clear-cell renal cell carcinomas (CRCCs). Allelic deletion was detected in 21 of 88 informative CRCCs (24%) with the highest rate of LOH being observed at D9S171 on 9p13 (20%). Chromosome 9p LOH was associated with short tumor-specific survival in stage pT3 RCC (P = 0.01). Fluorescence in situ hybridization analysis of 54 CRCCs revealed no homozygous CDKN2A deletions indicating that this mechanism of CDKN2A inactivation is rare in CRCC. Sequencing of 113 CRCCs showed that 13 tumors (12%) had a 24-bp deletion abrogating codons 4 through 11 of CDKN2A. Immunohistochemical CDKN2A expression was absent in normal renal tissue and was only detected in six of 382 CRCCs (1.5%) on a renal tumor microarray. These data suggest that CDKN2A alterations are present in a small subset of CRCCs and a second, yet unknown tumor suppressor gene proximal to the CDKN2A locus, may play a role in CRCC development.

Identification of differentially expressed genes in human gliomas by DNA microarray and tissue chip techniques.

New genomic large-scale screening techniques have made the task of establishing an accurate molecular fingerprint of cancer cells feasible. Here, we have used a two-phase strategy for identification of molecular alterations in gliomas. First, cDNA microarrays (Clontech Laboratories, Inc., Research Genetics) were used to pinpoint differentially expressed genes between normal brain and diffuse astrocytomas (grades II-IV), and between a primary tumor and a later tumor reoccurrence in the same patient. More than 200 gene expression alterations were detected from glioblastomas, whereas relatively few changes were seen in grade II and grade III tumors. The most distinct progression-related expression change was the up-regulation of the insulin-like growth factor binding protein 2 (IGFBP2) gene. Second, a high-density tissue microarray of 418 brain tumors was constructed and used for clinical validation of gene expression changes. Strong expression of IGFBP2 was associated with progression and poor patient survival in diffuse astrocytomas (P < 0.0001). Third, comparisons of the data between (a) multiple spots retrieved from one predefined tumor region (IGFBP2 and vimentin immunohistochemistry, 20 tumors) or between (b) standard slides and arrayed tissues (p53 immunohistochemistry, 42 tumors) revealed very little variation. In conclusion, the combined use of DNA microarrays and tissue microarrays offers a powerful strategy for rapid identification and thorough characterization of differentially expressed genes in gliomas.

Chromosome imbalances in familial gliomas detected by comparative genomic hybridization.

Familial occurrence of gliomas, in the absence of well-defined hereditary multisystem disorders, is reported occasionally. We describe 17 families that have been afflicted with two or more gliomas but do not raise suspicion of other inheritable syndromes. The families were identified among 369 consecutive glioma patients operated at the Tampere University Hospital during 1983-1994. We applied comparative genomic hybridization (CGH) analysis on 21 gliomas occurring in these 17 families. The most frequent genetic alterations, detected in over 20% of the tumors, were losses of 6q, 10, 4q, 9p and gains of 7, 19, 20q, 1p. We compared the chromosomal alterations detected in the familial gliomas to those reported previously on 209 sporadic gliomas in nine different CGH studies. In this comparison, the familial gliomas more often showed losses of chromosome arms 4q and 6q and gains of 1p and 22q. The most frequent losses (9/21 tumors) in the familial gliomas resided on chromosome arm 6q (P = 0.005, Fisher's exact test; with Bonferroni correction, P = 0.04). The loss of 6q was also the most common intrafamilial aberration, present in four separate gliomas belonging to two families. The minimal common area of loss on this chromosome resided at 6q14-16. In conclusion, we have found several characteristic aberrations by CGH in the familial gliomas and we present new chromosomal regions possibly involved in the familial predisposition to gliomas.

Loss of NKX3.1 expression in human prostate cancers correlates with tumor progression.

NKX3.1 is a prostate-specific homeobox gene located on chromosome 8p21. In the mouse, Nkx3.1 has growth-suppressive and differentiating effects on prostatic epithelium. Mutations of the coding region of NKX3.1 were not found in human prostate cancer, failing to support the notion that NKX3.1 was a tumor suppressor gene. To study the expression o NKX3.1 protein in human tissues and prostate cancer, we derived a rabbit antiserum against purified recombinant NKX3.1. Among normal human tissues, NKX3.1 expression was seen in testis, in rare pulmonary mucous glands, and in isolated regions of transitional epithelium of the ureter. NKX3.1 was uniformly expressed in nuclei of normal prostate epithelial cells in 61 histological sections from radical prostatectomy specimens. We analyzed 507 samples of neoplastic prostate epithelium, most of which were contained on a tissue microarray that contained samples from different stages of prostatic neoplasia. We observed complete loss of NKX3.1 expression in 5% of benign prostatic hyperplasias, 20% of high-grade prostatic intraepithelial neoplasias, 6% of T1a/b samples, 22% of T3/4 samples, 34% of hormone-refractory prostate cancers, and 78% of metastases. Our data show that NKX3.1 expression is highly, but not exclusively, specific for the prostate. Loss of NKX3.1 expression is strongly associated with hormone-refractory disease and advanced tumor stage in prostate cancer (P < 0.0001).

Multiple genes at 17q23 undergo amplification and overexpression in breast cancer.

Studies by comparative genomic hybridization imply that amplification of the chromosomal region 17q22-q24 is common in breast cancer. Here, amplification and expression levels of six known genes located at 17q23 were examined in breast cancer cell lines. Four of them (RAD51C, S6K, PAT1, and TBX2) were found to be highly amplified and overexpressed. To investigate the involvement of these genes in vivo, fluorescence in situ hybridization analysis of a tissue microarray containing 372 primary breast cancers was used. S6K, PAT1, and TBX2 were coamplified in about 10% of tumors, whereas RADS1C amplification was seen in only 3% of tumors. Expression analysis in 12 primary tumors showed that RAD51C and S6K were consistently expressed in all cases in which they were amplified and also in some tumors without amplification. These data suggest that 17q23 amplification results in simultaneous up-regulation of several genes, whose increased biological activity may jointly contribute to the more aggressive clinical course observed in patients with 17q23-amplified tumors.

High-throughput tissue microarray analysis of cyclin E gene amplification and overexpression in urinary bladder cancer.

Studies by comparative genomic hybridization revealed that the 19q13 chromosomal region is frequently amplified in bladder cancer. The cyclin E gene (CCNE), coding for a regulatory subunit of cyclin-dependent kinase 2, has been mapped to 19q13. To investigate the role of cyclin E alterations in bladder cancer, a tissue microarray of 2,317 specimens from 1,842 bladder cancer patients was constructed and analyzed for CCNE amplification by fluorescence in situ hybridization and for cyclin-E protein overexpression by immunohistochemistry. Fluorescence in situ hybridization analysis showed amplification in only 30 of the 1,561 evaluable tumors (1.9%). Amplification was significantly associated with stage and grade (P: < 0.0005 each). Immunohistochemically detectable cyclin E expression was strong in 233 (12.4%), weak in 354 (18.9%), and negative in 1, 286 of the 1,873 interpretable tumors. The majority (62.1%) of CCNE-amplified tumors were strongly immunohistochemistry-positive (P: < 0.0001). The frequency of protein expression increased from stage pTa (22.2%) to pT1 (45.5%; P: < 0.0001) but then decreased for stage pT2-4 (29.4%; P: < 0.0001 for pT1 versus pT2-4). Low cyclin E expression was associated with poor overall survival in all patients (P: < 0.0001), but had no prognostic impact independent of stage. It is concluded that cyclin E overexpression is characteristic to a subset of bladder carcinomas, especially at the stage of early invasion. This analysis of the prognostic impact of CCNE gene amplification and protein expression in >1,500 arrayed bladder cancers was accomplished in a period of 2 weeks, illustrating how the tissue microarray technology remarkably facilitates the evaluation of the clinical relevance of molecular alterations in cancer.

Detecting activation of ribosomal protein S6 kinase by complementary DNA and tissue microarray analysis.

BACKGROUND: Studies by comparative genomic hybridization (CGH) have shown that chromosomal region 17q23 is amplified in up to 20% of primary breast cancers. We used microarray analyses to measure the expression levels of genes in this region and to explore their prognostic importance. METHODS: A microarray that contained 4209 complementary DNA (cDNA) clones was used to identify genes that are overexpressed in the MCF-7 breast cancer cell line as compared with normal mammary tissue. Fluorescence in situ hybridization was used to analyze the copy number of one overexpressed gene, ribosomal protein S6 kinase (S6K), and to localize it to the 17q23 region. Northern and western blot analyses were used to measure S6K gene and protein expression, and an enzymatic assay was used to measure S6K activity. Tumor tissue microarray analysis was used to study amplification of S6K and the HER-2 oncogene, another 17q-linked gene, and the relationship between amplification and prognosis was analyzed. The Kaplan-Meier method was used for data analysis, and the log-rank test was used for statistical analysis. All P values are two-sided. RESULTS: S6K was amplified and highly overexpressed in MCF-7 cells relative to normal mammary epithelium, and protein expression and enzyme activity were increased. S6K was amplified in 59 (8.8%) of 668 primary breast tumors, and a statistically significant association between amplification and poor prognosis (P =.0021) was observed. Amplification of both S6K and HER-2 implied particularly poor survival (P =.0001). CONCLUSIONS: The combination of CGH information with cDNA and tissue microarray analyses can be used to identify amplified and overexpressed genes and to evaluate the clinical implications of such genes and genomic rearrangements. S6K is likely to be one of the genes at 17q23 that is amplified during oncogenesis and may adversely affect the prognosis of patients with this amplification.

Novel findings in gene expression detected in human osteosarcoma by cDNA microarray.

cDNA microarray analysis was used to screen for gene expression alterations in human osteosarcoma cell lines. The analysis using three cell lines revealed changes in the expression of several genes in comparison with normal human osteoblasts. Among the 5,184 sequences that were analyzed, 35 showed aberrant expression in all the cell lines. Eight of these showed overexpression and 27 underexpression compared to their expression levels in osteoblasts. The most highly up-regulated genes included heat shock protein 90beta and polyadenylate-binding protein-like 1. Commonly down-regulated genes included fibronectin 1 and thrombospondin 1. RT-PCR was used to verify these changes in the cell lines and in three primary osteosarcoma samples. This study shows that (1) gene expression pattern in osteosarcoma cell lines differs considerably from normal osteoblasts, (2) osteosarcoma cell lines can be used as a model system to detect novel gene expression alterations present in primary tumors, (3) the overexpression of heat shock protein 90beta and polyadenylate-binding protein-like 1, and (4) the down-regulation of fibronectin 1 and thrombospondin 1 may play a role in the development and/or progression of osteosarcoma. This study indicates that microarray-based expression surveys may be used to establish the molecular fingerprint of osteosarcoma, however, larger cDNA chips and more tumor specimens are required to define the clinically relevant gene expression patterns.