Yan Aalto

DNA Copy Number Losses in Human Neoplasms

This review summarizes reports of recurrent DNA sequence copy number losses in human neoplasms detected by comparative genomic hybridization. Recurrent losses that affect each of the chromosome arms in 73 tumor types are tabulated from 169 reports. The tables are available online at http://www.amjpathol.org and http://www. helsinki.fi/ approximately lglvwww/CMG.html. The genes relevant to the lost regions are discussed for each of the chromosomes. The review is supplemented also by a list of known and putative tumor suppressor genes and DNA repair genes (see Table 1, online). Losses are found in all chromosome arms, but they seem to be relatively rare at 1q, 2p, 3q, 5p, 6p, 7p, 7q, 8q, 12p, and 20q. Losses and their minimal common overlapping areas that were present in a great proportion of the 73 tumor entities reported in Table 2 (see online) are (in descending order of frequency): 9p23-p24 (48%), 13q21 (47%), 6q16 (44%), 6q26-q27 (44%), 8p23 (37%), 18q22-q23 (37%), 17p12-p13 (34%), 1p36.1 (34%), 11q23 (33%), 1p22 (32%), 4q32-qter (31%), 14q22-q23 (25%), 10q23 (25%), 10q25-qter (25%),15q21 (23%), 16q22 (23%), 5q21 (23%), 3p12-p14 (22%), 22q12 (22%), Xp21 (21%), Xq21 (21%), and 10p12 (20%). The frequency of losses at chromosomes 7 and 20 was less than 10% in all tumors. The chromosomal regions in which the most frequent losses are found implicate locations of essential tumor suppressor genes and DNA repair genes that may be involved in the pathogenesis of several tumor types.

Online Access to CGH Data of DNA Sequence Copy Number Changes

Listing of thechromosomal locations of recurrent DNA copy numberchanges in 73 tumor types from 283 reports available atthe end of last year can be accessed online at http://www.helsinki.fi/;lgl_www/CMG.html.When reviewing the CGH literature, we encounteredseveral problems, mainly the following:1. Different CGH systems (software applications) hadbeen used.2. There were no consensus criteria for thresholds oflosses, gains, and amplifications. In our compilation, wechose to apply an intensity ratio of 1.5 or higher as thethreshold value for amplifications.3. The results, with some exceptions, had not beenconfirmed using other techniques.Thus, one should be careful in comparing the originalpaper and our data file.The online files include a figure constructed from thecomposite profiles of the listed recurrent DNA copy num-ber sequence changes (Figure 1). Major data updates ofthe files are scheduled for July and December 2000. Inthe meantime, occasional reports will be added to thecompilation.Sakari KnuutilaKirsi AutioYan Aalto

Gene Expression Profiling of Ameloblastoma and Human Tooth Germ by Means of a cDNA Microarray

The molecular and genetic characteristics of ameloblastoma are still poorly understood. We analyzed gene expression in fresh-frozen ameloblastomas and human fetal tooth germs, using a cDNA microarray. Thirty-four genes exhibited significant changes in expression levels in the ameloblastoma. Eleven genes were overexpressed more than three-fold, and 23 genes were underexpressed to below 0.4 of the control level. The oncogene FOS was the most overexpressed gene (from eight- to 14-fold), followed by tumor-necrosis-factor-receptor 1 (TNFRSF1A). Genes for sonic hedgehog (SHH), TNF-receptor-associated-factor 3 (TRAF3), rhoGTP-ase-activating protein 4 (ARHGAP4), deleted in colorectal carcinoma (DCC), cadherins 12 and 13 (CDH12 and 13), teratocarcinoma-derived growth-factor-1 (TDGF1), and transforming growth-factor-ß1 (TGFB1) were underexpressed in all tumors. In selected genes, a comparison between cDNA microarray and real-time RT-PCR confirmed similar relative gene expression changes. The gene expression profile identifies candidate genes that may be involved in the origination of ameloblastoma and several genes previously unidentified in relation to human tooth development.

Molecular mechanisms of CD99-induced caspase-independent cell death and cell-cell adhesion in Ewing's sarcoma cells: Actin and zyxin as key intracellular mediators

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 Ewings sarcoma (ES). Engagement of CD99 induces fast homotypic aggregation of ES cells and caspase-independent 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.

Abnormal expression of apoptosis-related genes in haematological malignancies: overexpression of MYC is poor prognostic sign in mantle cell lymphoma.

Summary. The expression of apoptosis‐related genes BCL2, BAX, BCL2L1, BCL2A1, MCL1, DAPK1 and MYC was studied by quantitative real‐time polymerase chain reaction on total RNA samples from patients with acute lymphoblastic leukaemia (ALL, n = 16), acute myeloid leukaemia (AML, n = 27), chronic myeloid leukaemia (CML, n = 12), mantle cell lymphoma (MCL, n = 19) and chronic lymphoid leukaemia (CLL, n = 32). BCL2, BAX, BCL2A1, MCL1, DAPK1 and MYC were overexpressed in all patient groups. BCL2L1 was underexpressed in CLL and CML, but not in AML, ALL and MCL. MCL1 levels were significantly higher in CD13 and CD33‐positive ALL, and in CD56‐positive AML samples. BCL2, BCL2L1, BCL2A1 and MCL1 were overexpressed and DAPK1 was underexpressed in CLL samples with a 11q23 deletion. MYC overexpression was significantly associated with shorter overall survival in MCL (P < 0·01). AML patients with a normal karyotype showed a higher frequency of BCL2A1 overexpression (P < 0·001) than those with an abnormal karyotype.

Chromosomal alterations in human pancreatic endocrine tumors

Comparative genomic hybridization (CGH) was used to investigate changes in DNA copy numbers in 25 paraffin‐embedded samples of pancreatic endocrine tumors from 23 patients. Insulin was the dominant hormone in 12, glucagon in 7, somatostatin in 1, and pancreatic polypeptide in 2 tumors. One to 15 (mean, 8.1) changes in DNA copy numbers were observed in 22 of the 25 tumors. The most recurrent aberration, found in 68% of the tumors, involved gains in chromosome 7 with a minimal overlapping region at 7q11.2. Other frequent gains included chromosomes 19 (60%) and 14 (56%). Chromosome arm 20q was amplified in 48% of the cases with the minimal overlapping region of 20q11.1–13.1. The two most frequent DNA losses were found at 11q21–22 in 32% and at 11p13–15 in 24% of the cases. The amplified chromosomal regions contain several candidate genes that may be involved in islet cell tumorigenesis. The regions with most frequent losses are likely to contain still uncharacterized tumor suppressor genes. Genes Chromosomes Cancer 29:83–87, 2000.

Distinct gene expression profiling in chronic lymphocytic leukemia with 11q23 deletion.

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 < 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).

Gene expression analysis of 1,25(OH)2D3-dependent differentiation of HL-60 cells: a cDNA array study.

Summary. The alterations in gene expression associated with 1,25(OH)2D3‐induced differentiation of HL‐60 cells were studied in order to identify potential targets for further investigation of the genetic basis of acute myeloid leukaemia. Atlas human haematology filters, including 406 genes (Clontech), were used to study gene expression in response to 1,25(OH)2D3 (concentration, 5 × 10−8 mol/l) for 24 and 72 h. Compared with untreated cells, expression differences were found in 43 genes. Downregulated genes at both time‐points were: IL2RA, CMYC, NPM, DEK, AF4, FLI1, htlf, MNDA, BCR, IKAROS, BPI and NFAT4. Upregulated genes at both time‐points were IL1B, CD14 and MCL1. CD55, CD58, IRF2, CREB1, ATF4, RAC1, TIAR, KIAA0053, BAT2, BTK, RCK, EV12B and EDN were downregulated at 24 h, while SPI1, MKK3, BTG1 and IL8 were upregulated. At 72 h the upregulated genes were IL1RA, IL2RG, CXCR4, SCYA1, SCYA3, SCYA4, SCYA5, SCYA22, ANX2, CD83 and UPAR. cDNA array results were confirmed on randomly selected genes using quantitative real‐time polymerase chain reaction for three upregulated (CXCR4, IL1B and CD14) and three downregulated (DEK, AF4 and FLI1) genes. Gene expression analysis after differentiation induction may provide a tool to study the roles of DEK, AF4 and FLI1 in cell proliferation and differentiation. To demonstrate the genes that initiate differentiation, sequential gene expression analysis has to be performed during the first 24 h of the differentiation process.

Clinical Importance of Genomic Imbalances in Synovial Sarcoma Evaluated by Comparative Genomic Hybridization

The t(X;18)(p11.2;q11.2) (SYT/SSX1 or SSX2) is represented in more than 95% of synovial sarcoma. Even if recent data has implicated that the type of fusion gene (SYT/SSX1 or SYT/SSX2) can be of prognostic importance, the cellular and molecular mechanisms underlying the clinical behavior of synovial sarcoma are still poorly understood. To approach this issue, we investigated whether secondary genetic aberrations may influence the clinical outcome of synovial sarcoma. Clinical outcome with reference to comparative genomic hybridization (CGH) findings (losses or gains of genetic material) were analyzed for a uniquely large modern material of 69 synovial sarcomas. Thirty-five of 69 specimens showed DNA sequence copy number changes. The frequency of aberrations/tumor were higher (mean 4.7) for monophasic tumors than for biphasic tumors (mean 2.1). Gains of the whole or parts, including the long arm, of chromosome 8 were significantly overrepresented in large tumors (> 5 cm), suggesting that tumors with this genetic abnormality have an increased growth rate. No difference regarding metastasis-free or overall survival was seen between patients with or without tumors containing secondary copy number changes. No specific copy number change was linked to a significantly improved or impaired metastasis-free survival.

DNA copy number changes in familial malignant mesothelioma

Malignant mesothelioma (MM) is predominantly a sporadic malignancy linked to exposure to asbestos. Clustering of MM in families suggests genetic susceptibility as a contributing factor. We performed comparative genomic hybridization (CGH) analysis on tumor samples from members of a family with MM of the pleura and a history of parental cancer. Our specific aim was to find a recurrent copy number loss indicating the chromosomal area to which a gene underlying the development of MM could be assigned according to the Knudson two-hit hypothesis. We found losses at 1p, 6q, 9p, 13q, and 14q. The copy number changes were very similar to those reported in sporadic cases. Our findings and results from sporadic cases highlight the importance of cloning the genes in the loss sites at 1p, 6q, 14q, and 22q.