The identification of tumor-suppressor genes in solid tumors by classical cancer genetics methods is difficult and slow. We combined nonsense-mediated RNA decay microarrays 1 and array-based comparative genomic hybridization 2,3 for the genome-wide identification of genes with biallelic inactivation involving nonsense mutations and loss of the wild-type allele. This approach enabled us to identify previously unknown mutations in the receptor tyrosine kinase gene EPHB2. The DU 145 prostate cancer cell line, originating from a brain metastasis, carries a truncating mutation of EPHB2 and a deletion of the remaining allele. Additional frameshift, splice site, missense and nonsense mutations are present in clinical prostate cancer samples. Transfection of DU 145 cells, which lack functional EphB2, with wild-type EPHB2 suppresses clonogenic growth. Taken together with studies indicating that EphB2 may have an essential role in cell migration and maintenance of normal tissue architecture, our findings suggest that mutational inactivation of EPHB2 may be important in the progression and metastasis of prostate cancer.Inactivation of tumor-suppressor genes (TSGs) in cancer is often a two-step process 4 involving mutation of the target gene and loss of the wild-type allele. Mapping of chromosomal deletions and losses of heterozygosity in cancer cells has been widely applied to guide the identification of TSGs. On its own, however, this approach is slow, labor-intensive and complicated by genomic instability, which often leads to numerous candidate regions for further study. In an alternative approach, the nonsense-mediated decay (NMD) mechanism, which normally targets transcripts with nonsense mutations for rapid degradation 5,6 , is blocked to cause the differential stabilization of genes that contain truncating mutations. This approach, coupled with microarrays to measure transcript levels after NMD inhibition, has been proposed for the genome-wide identification of mutated genes in cell lines 1 .Here we combined results from NMD microarray experiments highlighting putative nonsense mutations with high-resolution data on deleted genomic regions in cancer cell lines obtained with arraybased comparative genomic hybridization (CGH) 2,3 . We applied this integrated approach, which focuses on biallelic gene inactivation events, to the identification of candidate TSGs in prostate cancer.We pretreated the DU 145, PC-3 and LNCaP prostate cancer cell lines with emetine (which inhibits the NMD pathway) and then exposed them to actinomycin D to block new mRNA synthesis and to distinguish post-transcriptional shifts in mRNA stability, which indicate the presence of a nonsense mutation. We used cDNA microarrays to measure changes in transcript levels in cells treated with emetine versus untreated cells. We also carried out corresponding analyses with nonmalignant control cells to distinguish drug-induced gene expression changes from mutation-induced transcript stabilization events. We used known nonsense mutations, including the C39X...

NOTE.-Phosphomannomutase and protein were measured as described elsewhere (Van Schaftingen and Jaeken 1995; Jaeken et al. 1997a). Phosphomannose isomerase was assayed at 30ЊC in a reaction mixture (1 ml) containing 50 mM Hepes, pH 7.1, 5 mM MgCl 2 , 25 mM KCl, 1 mM dithiothreitol, 0.6 mM NAD ϩ , 0.5 mM mannose 6phosphate, 2.5 U/ml glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides, and 10 mg/ml phosphoglucose isomerase with 10 ml of an extract containing 5-20 mg protein/ml. Control and PMM deficient measures are mean values ‫ע‬ SD. Where two data are given, the values were obtained on two different subcultures.

Purpose: To comprehensively evaluate ephrin receptor B2 (EphB2) expression in normal and neoplastic tissues. EphB2 is a tyrosine kinase recently implicated in the deregulation of cell-to-cell communication in many tumors. Experimental Design: EphB2 protein expression was analyzed by immunohistochemistry on tissue microarrays that included 76 different normal tissues, >4,000 samples from 138 different cancer types, and 1,476 samples of colon cancer with clinical follow-up data. Results:We found most prominent EphB2 expression in the intestinal epithelium (colonic crypts) with cancer of the colorectum displaying the highest EphB2 positivity of all tumors. Positivity was found in 100% of 118 colon adenomas but in 33.3% of 45 colon carcinomas. EphB2 expression was also observed in 75 tumor categories, including serous carcinoma of the endometrium (34.8%), adenocarcinoma of the esophagus (33.3%), intestinal adenocarcinoma of the stomach (30.2%), and adenocarcinoma of the small intestine (70%). The occasional finding of strong EphB2 positivity in tumors without EphB2 positivity in the corresponding normal cells [adenocarcinoma of the lung (4%) and pancreas (2.2%)] suggests that deregulation of EphB2 signaling may involve up-regulation of the protein expression. In colon carcinoma, loss of EphB2 expression was associated with advanced stage (P < 0.0001) and was an indicator of poor overall survival (P = 0.0098). Conclusions: Our results provide an overview on the EphB2 protein expression in normal and neoplastic tissues. Deregulated EphB2 expression may play a role in several cancer types with loss of EphB2 expression serving as an indicator of the possible pathogenetic role of EphB2 signaling in the maintenance of tissue architecture of colon epithelium.

In the Finnish breast and ovarian cancer families six BRCA1 and five BRCA2 mutations have been found recurrently. Some of these recurrent mutations have also been seen elsewhere in the world, while others are exclusively of Finnish origin. A haplotype analysis of 26 Finnish families carrying a BRCA1 mutation and 20 families with a BRCA2 mutation indicated that the carriers of each recurrent mutation have common ancestors. The common ancestors were estimated to trace back to 7-36 generations (150-800 years). The time estimates and the geographical clustering of these founder mutations in Finland are in concordance with the population history of this country. Analysis of the cancer phenotypes showed differential ovarian cancer expression in families carrying mutations in the 5' and 3' ends of the BRCA1 gene, and earlier age of ovarian cancer onset in families with BRCA1 mutations compared with families with BRCA2 mutations. The identification of prominent and regional BRCA1 and BRCA2 founder mutations in Finland will have significant impact on diagnostics in Finnish breast and ovarian cancer families. An isolated population with known history and multiple local founder effects in multigenic disease may offer distinct advantages also for mapping novel predisposing genes.