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CD99 is a unique 32-kDa cell surface molecule with broad cellular expression but still poorly understood biological functions. In cancer cells, CD99 is highly expressed in virtually all Ewing's sarcoma (ES). Engagement of CD99 induces fast homotypic aggregation of ES cells and caspaseindependent apoptosis. In this study, we analysed signal transduction after CD99 engagement on ES cells. Findings obtained with selective inhibitors indicated that only actin cytoskeleton integrity was essential for cell-cell adhesion and apoptosis of ES cells. Indeed, CD99 stimulation induced actin repolymerization, further supporting the role of cytoskeleton in CD99 signaling. Gene expression profiling of ES cells after CD99 engagement showed modulation in the expression of 32 genes. Among the pool of upregulated genes reported to be involved in cell adhesion, we chose to analyse the role of zyxin, a cytoplasmic adherens junction protein found to play a role in the regulation of the actin cytoskeleton. Overexpression of zyxin after CD99 ligation was confirmed by real-time PCR and Western blot. Treatment of ES cells with zyxin antisense oligonucleotides inhibited CD99-induced cell aggregation and apoptosis, suggesting a functional role for this protein. Therefore, our findings indicate that CD99 functions occur through reorganization of cytoskeleton and identify actin and zyxin as the early signaling events driven by CD99 engagement.

DNA copy number amplifications activate oncogenes and are hallmarks of nearly all advanced tumors. Amplified genes represent attractive targets for therapy, diagnostics and prognostics. To investigate DNA amplifications in different neoplasms, we performed a bibliomics survey using 838 published chromosomal comparative genomic hybridization studies and collected amplification data at chromosome band resolution from more than 4500 cases. Amplification profiles were determined for 73 distinct neoplasms. Neoplasms were clustered according to the amplification profiles, and frequently amplified chromosomal loci (amplification hot spots) were identified using computational modeling. To investigate the site specificity and mechanisms of gene amplifications, colocalization of amplification hot spots, cancer genes, fragile sites, virus integration sites and gene size cohorts were tested in a statistical framework. Amplification-based clustering demonstrated that cancers with similar etiology, cell-oforigin or topographical location have a tendency to obtain convergent amplification profiles. The identified amplification hot spots were colocalized with the known fragile sites, cancer genes and virus integration sites, but global statistical significance could not be ascertained. Large genes were significantly overrepresented on the fragile sites and the reported amplification hot spots. These findings indicate that amplifications are selected in the cancer tissue environment according to the qualitative traits and localization of cancer genes.

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Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with regard to its clinical course. The limitations of the methods currently available for prognostic assessment in CLL do not allow accurate prediction of the risk of disease progression in individual patients. The recently developed cDNA array technique provides a unique opportunity to study gene expression in various malignancies. To identify new molecular markers for prognostication of CLL patients, we analyzed cDNA arrays by using hierarchical clustering and standard statistic t-test on 34 CLL patients. We found significant expression differences in 78 genes compared to the reference tonsillar B lymphocytes. A cluster of genes, LCP1, PARP, BLR1, DEK, NPM, MCL1, SLP76, STAM, HIVEP1, EVI2B, CD25, HTLF, HIVEP2, BCL2, MNDA, PBX3, EBI2, TCF1, CGRP, CD14, IL8, GZMK, GPR17 and CD79B, was associated (P Ͻ 0.05) with the unfavorable 11q deletion and also with the unfavorable Binet stages B and C. We present here gene expression profiling that is associated with CLL patients with the 11q23 deletion. Many of the genes in the cluster have not previously been shown to be related to the initiation or progression of CLL. These novel findings provide fundamental information for further attempts to understand the interaction of the clustered genes in the leukomogenesis of CLL in order to better design treatments aimed at specific molecular target(s). Leukemia (2001) 15, 1721-1728.

To identify new potential diagnostic markers for lung cancer, the expression profiles of 37 lung tumours were analysed using cDNA arrays. Seven samples were from small-cell lung cancer (SCLC), two from large-cell neuroendocrine tumours (LCNEC), and 28 from other non-small-cell lung cancers (mainly squamous cell cancer and adenocarcinoma). Principal component analysis and the permutation test were used to detect differences in the gene expression profiles and a set of genes was found that distinguished high-grade neuroendocrine carcinomas (SCLC and LCNEC) from other lung cancers. In addition, several genes, such as caveolin-1 (CAV1) and caveolin-2 (CAV2), were constantly deregulated in all types of tumour sample, compared with normal tissue. The expression of these two genes was investigated further at the protein level on a tissue microarray containing tumours from 161 patients and normal tissues. Immunostaining for CAV1 was negative in 48% of tumours, whereas 28% of the tumours did not express CAV2. Lack of CAV1 protein expression was not caused by methylation or mutation. In stage I adenocarcinomas, CAV2 protein expression correlated with shorter survival. In conclusion, the present study was able to identify genes that have not previously been implicated in lung cancer by the combined use of two different array techniques. Some of these genes may provide novel diagnostic markers for lung cancer.

DNA copy number amplification at the chromosomal region of 17q is frequent in gastric cancer. Recently 17q21 was identified as the critical region for the amplicon formation because this region harbors the ERBB2 oncogene and several other targets, such as TOP2A and DARPP32. In our study, we characterized the amplification (52 cases) and expression (29 cases) levels of ERBB2, TOP2A and DARPP32 in gastric cancer samples. These 3 genes were concomitantly amplified in 17% of the intestinal type of gastric adenocarcinoma. However, the expression levels were independent, showing overexpression of DARPP32 (48%), TOP2A (17%) and ERBB2 (3%) studied by quantitative real-time PCR. The most frequently overexpressed gene, DARPP32, exhibited strong protein overexpression in 45% (30/66) of the cases in immunohistochemical study of gastric cancer tumor tissue array. Additional studies are required to thoroughly understand the biological significance of these genes in gastric cancer.