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Combined Array Comparative Genomic Hybridization and Tissue Microarray Analysis Suggest PAK1 at 11q13.5-q14 as a Critical Oncogene Target in Ovarian Carcinoma

2003; Elsevier BV; Volume: 163; Issue: 3 Linguagem: Inglês

10.1016/s0002-9440(10)63458-x

1525-2191

Peter Schraml, Georg Schwerdtfeger, Felix Burkhalter, Anna Raggi, Dietmar Schmidt, Teresa Ruffalo, Walter King, Kim Wilber, Michael J. Mihatsch, Holger Moch,

Cancer Genomics and Diagnostics

Amplification of chromosomal regions leads to an increase of DNA copy numbers and expression of oncogenes in many human tumors. The identification of tumor-specific oncogene targets has potential diagnostic and therapeutic implications. To identify distinct spectra of oncogenic alterations in ovarian carcinoma, metaphase comparative genomic hybridization (mCGH), array CGH (aCGH), and ovarian tumor tissue microarrays were used in this study. Twenty-six primary ovarian carcinomas and three ovarian carcinoma cell lines were analyzed by mCGH. Frequent chromosomal overrepresentation was observed on 2q (31%), 3q (38%), 5p (38%), 8q (52%), 11q (21%), 12p (21%), 17q (21%), and 20q (52%). The role of oncogenes residing in gained chromosomal loci was determined by aCGH with 59 genetic loci commonly amplified in human tumors. DNA copy number gains were most frequently observed for PIK3CA on 3q (66%), PAK1 on 11q (59%), KRAS2 on 12p (55%), and STK15 on 20q (55%). The 11q13-q14 amplicon, represented by six oncogenes (CCND1, FGF4, FGF3, EMS1, GARP, and PAK1) revealed preferential gene copy number gains of PAK1, which is located at 11q13.5-q14. Amplification and protein expression status of both PAK1 and CCND1 were further examined by fluorescence in situ hybridization and immunohistochemistry using a tissue microarray consisting of 268 primary ovarian tumors. PAK1 copy number gains were observed in 30% of the ovarian carcinomas and PAK1 protein was expressed in 85% of the tumors. PAK1 gains were associated with high grade (P < 0.05). In contrast, CCND1 gene alterations and protein expression were less frequent (10.6% and 25%, respectively), suggesting that the critical oncogene target of amplicon 11q13–14 lies distal to CCND1. This study demonstrates that aCGH facilitates further characterization of oncogene candidates residing in amplicons defined by mCGH. Amplification of chromosomal regions leads to an increase of DNA copy numbers and expression of oncogenes in many human tumors. The identification of tumor-specific oncogene targets has potential diagnostic and therapeutic implications. To identify distinct spectra of oncogenic alterations in ovarian carcinoma, metaphase comparative genomic hybridization (mCGH), array CGH (aCGH), and ovarian tumor tissue microarrays were used in this study. Twenty-six primary ovarian carcinomas and three ovarian carcinoma cell lines were analyzed by mCGH. Frequent chromosomal overrepresentation was observed on 2q (31%), 3q (38%), 5p (38%), 8q (52%), 11q (21%), 12p (21%), 17q (21%), and 20q (52%). The role of oncogenes residing in gained chromosomal loci was determined by aCGH with 59 genetic loci commonly amplified in human tumors. DNA copy number gains were most frequently observed for PIK3CA on 3q (66%), PAK1 on 11q (59%), KRAS2 on 12p (55%), and STK15 on 20q (55%). The 11q13-q14 amplicon, represented by six oncogenes (CCND1, FGF4, FGF3, EMS1, GARP, and PAK1) revealed preferential gene copy number gains of PAK1, which is located at 11q13.5-q14. Amplification and protein expression status of both PAK1 and CCND1 were further examined by fluorescence in situ hybridization and immunohistochemistry using a tissue microarray consisting of 268 primary ovarian tumors. PAK1 copy number gains were observed in 30% of the ovarian carcinomas and PAK1 protein was expressed in 85% of the tumors. PAK1 gains were associated with high grade (P < 0.05). In contrast, CCND1 gene alterations and protein expression were less frequent (10.6% and 25%, respectively), suggesting that the critical oncogene target of amplicon 11q13–14 lies distal to CCND1. This study demonstrates that aCGH facilitates further characterization of oncogene candidates residing in amplicons defined by mCGH. Ovarian carcinoma is the second most common gynecological tumor type in industrial countries. Asymptomatic clinical features and the lack of suitable screening tools account for its high mortality rate.1Boring CC Squires TS Tong T Montgomery S Cancer statistics, 1994.CA Cancer J Clin. 1994; 44: 7-26Crossref PubMed Scopus (1486) Google Scholar Recent studies characterizing genetic aberrations in ovarian cancer have implicated a number of oncogenes and tumor suppressor genes in ovarian tumor development.2Shridhar V Lee J Pandita A Iturria S Avula R Staub J Morrissey M Calhoun E Sen A Kalli K Keeney G Roche P Cliby W Lu K Schmandt R Mills GB Bast Jr, RC James CD Couch FJ Hartmann LC Lillie J Smith DI Genetic analysis of early- versus late-stage ovarian tumors.Cancer Res. 2001; 61: 5895-5904PubMed Google Scholar The details of the genetic changes in ovarian tumorigenesis, however, are not well understood. One of the major challenges in cancer research aims at generating molecular profiles of tumors to establish correlations between genetic changes and clinical parameters by screening technologies. Metaphase comparative genomic hybridization (mCGH) allows the detection of chromosomal aberrations across the entire genome with a maximal resolution of >10 Mb.3Isola J Visakorpi T Holli K Kallioniemi OP Association of overexpression of tumor suppressor protein p53 with rapid cell proliferation and poor prognosis in node-negative breast cancer patients.J Natl Cancer Inst. 1992; 84: 1109-1114Crossref PubMed Scopus (363) Google Scholar The application of new DNA-microarray technologies and the structural information being available on all human genes permit a more detailed and simultaneous quantification of gene transcripts or genomic DNA copy numbers of many DNA sequences. Arrays with selected clones of genomic DNA sequences are used for array CGH (aCGH). These microchips may be applied for comprehensive, tumor type-specific amplicon profiling of many oncogenes4Daigo Y Chin SF Gorringe KL Bobrow LG Ponder BA Pharoah PD Caldas C Degenerate oligonucleotide primed-polymerase chain reaction-based array comparative genomic hybridization for extensive amplicon profiling of breast cancers: a new approach for the molecular analysis of paraffin-embedded cancer tissue.Am J Pathol. 2001; 158: 1623-1631Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 5Hui AB Lo KW Yin XL Poon WS Ng HK Detection of multiple gene amplifications in glioblastoma multiforme using array-based comparative genomic hybridization.Lab Invest. 2001; 81: 717-723Crossref PubMed Scopus (178) Google Scholar as well as for quantitative mapping of amplicon structures.6Albertson DG Ylstra B Segraves R Collins C Dairkee SH Kowbel D Kuo WL Gray JW Pinkel D Quantitative mapping of amplicon structure by array CGH identifies CYP24 as a candidate oncogene.Nat Genet. 2000; 25: 144-146Crossref PubMed Scopus (527) Google Scholar, 7Pinkel D Segraves R Sudar D Clark S Poole I Kowbel D Collins C Kuo WL Chen C Zhai Y Dairkee SH Ljung BM Gray JW Albertson DG High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays.Nat Genet. 1998; 20: 207-211Crossref PubMed Scopus (1789) Google Scholar Previous mCGH studies showed that chromosomal copy number gains are common in ovarian cancer. High-level amplifications were mainly observed in late-stage tumors,8Knuutila S Autio K Aalto Y Online access to CGH data of DNA sequence copy number changes.Am J Pathol. 2000; 157: 689Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar suggesting an important role of oncogenes for ovarian tumor progression. A limited number of known oncogenes residing in some of these chromosomal regions were found amplified such as MYC at 8q24,9Abeysinghe HR Cedrone E Tyan T Xu J Wang N Amplification of C-MYC as the origin of the homogeneous staining region in ovarian carcinoma detected by micro-FISH.Cancer Genet Cytogenet. 1999; 114: 136-143Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 10Wang ZR Liu W Smith ST Parrish RS Young SR c-myc and chromosome 8 centromere studies of ovarian cancer by interphase FISH.Exp Mol Pathol. 1999; 66: 140-148Crossref PubMed Scopus (42) Google Scholar, 11Diebold J Suchy B Baretton GB Blasenbreu S Meier W Schmidt M Rabes H Lohrs U DNA ploidy and MYC DNA amplification in ovarian carcinomas. Correlation with p53 and bcl-2 expression, proliferative activity and prognosis.Virchows Arch. 1996; 429: 221-227PubMed Google Scholar KRAS at 12p12.1,12Bian M Fan Q Huang S Ma J Lang J Amplifications of proto-oncogenes in ovarian carcinoma.Chin Med J. 1995; 108: 844-848PubMed Google Scholar, 13Yang-Feng TL Li SB Leung WY Carcangiu ML Schwartz PE Trisomy 12 and K-ras-2 amplification in human ovarian tumors.Int J Cancer. 1991; 48: 678-681Crossref PubMed Scopus (34) Google Scholar CCND2 at 12p13,14Courjal F Louason G Speiser P Katsaros D Zeillinger R Theillet C Cyclin gene amplification and overexpression in breast and ovarian cancers: evidence for the selection of cyclin D1 in breast and cyclin E in ovarian tumors.Int J Cancer. 1996; 69: 247-253Crossref PubMed Scopus (184) Google Scholar HER2 at 17q21,12Bian M Fan Q Huang S Ma J Lang J Amplifications of proto-oncogenes in ovarian carcinoma.Chin Med J. 1995; 108: 844-848PubMed Google Scholar, 15Seki A Yoshinouchi M Seki N Kodama J Miyagi Y Kudo T Detection of c-erbB-2 and FGF-3 (INT-2) gene amplification in epithelial ovarian cancer.Int J Oncol. 2000; 17: 103-106PubMed Google Scholar and AIB1 at 20q12-13.16Anzick SL Kononen J Walker RL Azorsa DO Tanner MM Guan XY Sauter G Kallioniemi OP Trent JM Meltzer PS AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer.Science. 1997; 277: 965-968Crossref PubMed Scopus (1442) Google Scholar However, the target genes of most amplifications are still unknown. The aims of this study were to screen ovarian tumors for potential oncogene candidates by mCGH and aCGH. Using a tissue microarray (TMA) containing 268 ovarian tumors, we specifically examined amplification and protein expression of two oncogenes and their clinicopathological impact for ovarian cancer. Frozen tumor specimens were obtained from the archives of the Institute of Pathology, Mannheim, Germany. All ovarian tumor samples were histologically reviewed. Twenty-six ovarian tumor specimens with a minimum of 75% tumor cells in the sample and without necrosis were selected for the study on the basis of hematoxylin and eosin-stained tissue sections. Tumors were staged according to the International Federation of Gynecology and Obstetrics criteria for ovarian cancer.17Sobin LH Wittekind C TNM Classification of Malignant Tumours. Wiley-Liss, New York2002Google Scholar The histological subtype was defined according to the World Health Organization.18Scully RE Sobin LH Histological Typing of Ovarian Tumours. Springer, Berlin1999Crossref Google Scholar There were 14 serous, 3 clear cell, 3 endometrioid, 3 mucinous carcinomas, 1 mullerian-mixed tumor, and 2 undifferentiated ovarian tumors. One patient had stage I, 1 stage II, 16 stage III, and 3 patients stage IV disease. There were 8 grade 1, 13 grade 2, and 5 grade 3 tumors. The International Federation of Gynecology and Obstetrics stage of five tumors could not be defined retrospectively because of missing clinical information. Cell lines CRL-1978, CRL-10303, and HTB-161 (American Type Culture Collection, Manassas, VA) were cultured with OPTI-MEM (Invitrogen AG, Basel, Switzerland) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin (BioConcept, Allschwil, Switzerland). Tissue preparation and extraction of high-molecular weight DNA from the primary tumors and the cancer cell lines for mCGH analysis was as previously described.19Jiang F Richter J Schraml P Bubendorf L Gasser T Sauter G Mihatsch MJ Moch H Chromosomal imbalances in papillary renal cell carcinoma: genetic differences between different subtypes.Am J Pathol. 1998; 153: 1467-1473Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar One μg of tumor DNA was nick translated by using a commercial kit (BioNick kit; Life Technologies, Gaithersburg, MD) and Spectrum Green-dUTPs (Vysis Inc., Downers Grove, IL) for direct labeling of tumor DNA. Spectrum Red-labeled normal reference DNA (Vysis) was used for co-hybridization. The hybridization mixture consisted of 200 ng of Spectrum Green-labeled tumor DNA, 200 ng of Spectrum Red-labeled normal reference DNA, and 20 μg of Cot-1 DNA (Invitrogen AG) dissolved in 10 μl of hybridization buffer (50% formamide, 10% dextran sulfate, 2× standard saline citrate, pH 7.0). Hybridization, image acquisition, image analysis, and control experiments were exactly as described.19Jiang F Richter J Schraml P Bubendorf L Gasser T Sauter G Mihatsch MJ Moch H Chromosomal imbalances in papillary renal cell carcinoma: genetic differences between different subtypes.Am J Pathol. 1998; 153: 1467-1473Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar Thresholds used for definition of DNA sequence copy number gains and losses were >1.2 and 1.20. Ratios >2.0 were regarded as amplifications according to the recommendations of the standardized protocols (Vysis). A loss of DNA sequences was presumed at chromosomal regions in which the tumor-to-normal ratio was <0.80. Similar values were used in two previous studies.4Daigo Y Chin SF Gorringe KL Bobrow LG Ponder BA Pharoah PD Caldas C Degenerate oligonucleotide primed-polymerase chain reaction-based array comparative genomic hybridization for extensive amplicon profiling of breast cancers: a new approach for the molecular analysis of paraffin-embedded cancer tissue.Am J Pathol. 2001; 158: 1623-1631Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 5Hui AB Lo KW Yin XL Poon WS Ng HK Detection of multiple gene amplifications in glioblastoma multiforme using array-based comparative genomic hybridization.Lab Invest. 2001; 81: 717-723Crossref PubMed Scopus (178) Google Scholar The ovarian carcinoma TMA was constructed as previously described.21Kononen J Bubendorf L Kallioniemi A Barlund M Schraml P Leighton S Torhorst J Mihatsch MJ Sauter G Kallioniemi OP Tissue microarrays for high-throughput molecular profiling of tumor specimens.Nat Med. 1998; 4: 844-847Crossref PubMed Scopus (3575) Google Scholar It contained 120 serous, 68 endometrioid, 40 mucinous, 24 clear cell, and 16 undifferentiated ovarian tumors (Figure 1). All tumors were reviewed by one pathologist (HM). There were 86 grade 1, 87 grade 2, and 89 grade 3 tumors. Fifty-two tumors were stage pT1, 28 pT2, 117 pT3, and 11 were pT4. Histological tumor grading and TNM staging was done as described in Tumor Samples and Cell Lines. Tumor-specific survival data were obtained by reviewing the hospital records, by direct communication with the attending physicians, and from the Cancer Registry of Basel. The TMA sections were treated according to the Paraffin Pretreatment Reagent Kit protocol (Vysis) before hybridization. FISH was performed with Spectrum Orange-labeled CCND1 and PAK1 probes. Spectrum Green-labeled chromosome 11 centromeric probe CEP11 was used as a reference (Vysis). Hybridization and posthybridization washes were according to the LSI procedure (Vysis). Slides were counterstained with 125 ng/ml of 4′, 6-diamino-2-phenylindole in anti-fade solution. FISH signals were scored with a Zeiss fluorescence microscope equipped with double-band pass filters for simultaneous visualization of Spectrum Green and Spectrum Orange signals (Vysis). Gain was assumed in tumors in which there were more CCND1 or PAK1 signals than centromere 11 signals in at least 50% of the cells. Amplification was defined as presence of more than 10 gene signals, or tight clusters of at least five gene signals, or more than three times as many CCND1 or PAK1 than centromere 11 signals in ≥5% of tumor cells. Standard indirect immunoperoxidase procedures were used for immunohistochemistry (ABC-Elite; Vector Laboratories, Burlingame, CA). A monoclonal antibody to human CCND1 (clone P2D11F11, microwave oven, pronase III; Ventana, Tucson, AZ) and a polyclonal antibody to PAK1 (1:50 diluted, microwave oven; Cell Signaling Technology, Beverly, MA) were used for detection of CCND1 and PAK1 expression. Diaminobenzidine was used as a chromogen. The primary antibody was omitted for negative controls. Tumors were considered positive if cytoplasmic (PAK1) or nuclear (CCND1) expression was found. Contingency table analysis was used to analyze the association between CCND1 or PAK1 alterations and tumor grade, stage, and allelic loss. Survival analyses were performed using the Kaplan-Meier method. Statistical differences between groups were determined with log-rank test. A Cox proportional hazard analysis was used to test for independent prognostic information. mCGH showed chromosomal aberrations in all 29 ovarian tumors. DNA sequence copy number gains (mean 4.2 ± 3.6/tumor) were slightly more frequent than deletions (3.8 ± 3.8/tumor). The most common overrepresentations were seen at 8q (52%), 20q (52%), 5p (38%), 3q (38%), 2q (31%), 11q (21%), and 12p (21%). Chromosomal deletions were most prevalent at Xq (52%); Xp (45%); 4q (41%); 5q, 9p, and 18q (35%); and 17p (31%). A detailed oncogene profiling was obtained from all primary tumors and cell lines. An image of a hybridized microarray is shown in Figure 2. Fluorescent ratios of green-labeled tumor and normal red-labeled DNA ranged from 1.21 to 5.54 for overrepresentations and from 0.79 to 0.53 for deletions. Highest frequency of allelic gains was seen for PIK3CA (66%), PAK1, STK15, KRAS2 (55% each), CCND2 (41%), EGFR (38%), and MYC, AIB1, JUNB (35% each). Amplifications were observed on AIB1 and PTPN1 in one tumor; AIB1 and ZNF217 in one tumor; MYC in one tumor and CRL-10303; ERBB2 and MET in one tumor each; CCND1, FGF4/FGF3, and EMS1 in CRL-1987; GARP and PAK1 in HTB-161. The result of aCGH analysis is shown in Figure 3.Figure 3.aCGH results of 26 primary ovarian carcinomas and three ovarian carcinoma cell lines.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Comparing mCGH with aCGH data revealed high concordance between increased copy numbers of chromosomal regions and gains/amplifications of oncogenes located in these amplicons. Overall, we were able to compare 134 of 198 overrepresentations detected by mCGH in 29 ovarian tumors with data obtained by aCGH. Eighty of 134 (60%) gains identified by mCGH were confirmed by aCGH. Interestingly, chromosomal deletions observed by mCGH correlated well with corresponding DNA sequence copy number losses seen by aCGH. Gained and lost copies of oncogenes and the corresponding mCGH profiles yielded from one ovarian tumor are shown in Figure 4. Whereas most ovarian carcinomas showed gains of large chromosomal areas, regionary high-level amplifications were rare. Oncogenes located within one amplicon were further analyzed to determine the presence of co-gains/co-amplifications. Overrepresentations of at least two adjacent genes were seen at 3q26.3, 11q13-q14, 12p12-p13, and 20q12-q13. There were six oncogenes at 11q13-q14. PAK1 showed preferential copy number gains emphasizing the differing relevance of neighboring oncogenes for ovarian cancer (Figure 3). Gains and amplifications of oncogenes occurred in all stages and subtypes of ovarian cancer. There were no associations of specific oncogenes with tumor grade, tumor stage, and histological subtypes. High-quality hybridization signals for centromeric and gene-specific probes were obtained in 77 (PAK1) and 160 (CCND1) ovarian carcinomas, respectively. Twenty-three ovarian carcinomas (30%) showed PAK1 gains, but only seven (10.6%) of the tumors had increased copy number of CCND1. PAK1 was found amplified in one serous ovarian adenocarcinoma. None of the tumors revealed a CCND1 amplification. Examples of tumors with PAK1 amplification and CCND1 gain are shown in Figure 5, A and B. PAK1 gains were associated with the differentiation grade (P < 0.05). Most undifferentiated ovarian carcinomas (83%) displayed PAK1 gain (Table 1).Table 1PAK1/CCND1 Gain and Expression and Tumor PhenotypePAK1 gainCCND1 gainPAK1 protein expressionCCND1 protein expressionn%P valuen%P valuen%P valuen%P valueStage pT1102538540825122 pT2122026820752722 pT341526010898911229 pT440n.s.30n.s.11100n.s.1127n.s.Grade G1161960767768329 G2251267466838520 G33444<0.026514n.s.6692n.s.8726n.s.Histologic type Serous38327111849011532 Mucinous61725431714013 Endometroid192141759836721 Clear cell81414712712322 Undifferentiated683 q14 in human breast carcinoma.Cytogenet Cell Genet. 1997; 79: 125-131Crossref PubMed Scopus (132) Google Scholar Our FISH analysis demonstrated a high percentage of tumors with PAK1 gains including one tumor with a high-level amplification. This result was consistent with the data obtained from the aCGH. The high number of PAK1 protein expression in ovarian carcinoma further supports the theory that PAK1 is an important oncogene for ovarian cancer. A number of functional properties being characteristic for an oncogene have recently been reported for PAK1 in breast cancer. PAK1 is a member of a family of serine/threonine kinases and regulates anchorage-independent growth, invasiveness, and abnormal organization of mitotic spindles of human epithelial breast cancer cells.25Vadlamudi RK Adam L Wang RA Mandal M Nguyen D Sahin A Chernoff J Hung MC Kumar R Regulatable expression of p21-activated kinase-1 promotes anchorage-independent growth and abnormal organization of mitotic spindles in human epithelial breast cancer cells.J Biol Chem. 2000; 275: 36238-36244Crossref PubMed Scopus (225) Google Scholar PAK1 promotes hyperplasia in mammary epithelium by phosphorylating and transactivating estrogen receptor-α.26Wang RA Mazumdar A Vadlamudi RK Kumar R P21-activated kinase-1 phosphorylates and transactivates estrogen receptor-alpha and promotes hyperplasia in mammary epithelium.EMBO J. 2002; 21: 5437-5447Crossref PubMed Scopus (157) Google Scholar It is also involved in angiogenesis by controlling vascular endothelial growth factor expression, vascular endothelial growth factor secretion, and function.27Bagheri-Yarmand R Vadlamudi RK Wang RA Mendelsohn J Kumar R Vascular endothelial growth factor up-regulation via p21-activated kinase-1 signaling regulates heregulin-beta1-mediated angiogenesis.J Biol Chem. 2000; 275: 39451-39457Crossref PubMed Scopus (117) Google Scholar The TMA approach used in this study allowed us to evaluate the associations between PAK1 alterations and clinicopathological parameters because the number of tumors was large enough to perform statistical analyses. Importantly, PAK1 expression and copy number gains were seen in all undifferentiated ovarian carcinomas and in many high-grade tumors suggesting that PAK1 activation is responsible for the progression of ovarian cancer. Interestingly, copy number increases of PAK1 at 11q13.5-q14 were often associated with MLL loss at 11q23. The combination of 11q gain and 11q loss is suggestive of isochromosome formation and raises the question of whether the loss of MLL or the gain of PAK1 is the more important alteration. Allelic loss at 11q23 was previously described in cervical neoplasia, breast, ovarian, and head and neck carcinomas.28Evans MF Koreth J Bakkenist CJ Herrington CS McGee JO Allelic deletion at 11q23.3-q25 is an early event in cervical neoplasia.Oncogene. 1998; 16: 2557-2564Crossref PubMed Scopus (29) Google Scholar, 29Jin Y Hoglund M Jin C Martins C Wennerberg J Akervall J Mandahl N Mitelman F Mertens F FISH characterization of head and neck carcinomas reveals that amplification of band 11q13 is associated with deletion of distal 11q.Genes Chromosom Cancer. 1998; 22: 312-320Crossref PubMed Scopus (60) Google Scholar, 30Launonen V Stenback F Puistola U Bloigu R Huusko P Kytola S Kauppila A Winqvist R Chromosome 11q22.3-q25 LOH in ovarian cancer: association with a more aggressive disease course and involved subregions.Gynecol Oncol. 1998; 71: 299-304Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 31Koreth J Bakkenist CJ Larin Z Hunt NC James MR McGee JO 11q23.1 and 11q25-qter YACs suppress tumour growth in vivo.Oncogene. 1999; 18: 1157-1164Crossref PubMed Scopus (15) Google Scholar Tumor growth suppression by transfecting murine fibrosarcoma cells with 11q23.1-containing YACs indicates a tumor suppressor gene locus in close vicinity to MLL.31Koreth J Bakkenist CJ Larin Z Hunt NC James MR McGee JO 11q23.1 and 11q25-qter YACs suppress tumour growth in vivo.Oncogene. 1999; 18: 1157-1164Crossref PubMed Scopus (15) Google Scholar Because some PAK1 gains were not associated with MLL losses, increased copy numbers of PAK1 may be more significant for ovarian cancer than MLL loss. On the basis of its known function, it is highly unlikely that MLL is the target gene of the 11q deletion. Our aCGH and FISH results for CCND1 on 11q13 are consistent with recent studies showing that CCND1 amplification is rare in ovarian cancer.14Courjal F Louason G Speiser P Katsaros D Zeillinger R Theillet C Cyclin gene amplification and overexpression in breast and ovarian cancers: evidence for the selection of cyclin D1 in breast and cyclin E in ovarian tumors.Int J Cancer. 1996; 69: 247-253Crossref PubMed Scopus (184) Google Scholar, 32Schraml P Kononen J Bubendorf L Moch H Bissig H Nocito A Mihatsch M Kallioniemi O Sauter G Tissue microarrays for gene amplification surveys in many different tumor types.Clin Cancer Res. 1999; 5: 1966-1975PubMed Google Scholar Although CCND1 amplification is uncommon in ovarian cancer, nuclear CCND1 overexpression has been reported in 15 to 30% of ovarian tumors33Kusume T Tsuda H Kawabata M Inoue T Umesaki N Suzuki T Yamamoto K The p16-cyclin D1/CDK4-pRb pathway and clinical outcome in epithelial ovarian cancer.Clin Cancer Res. 1999; 5: 4152-4157PubMed Google Scholar, 34Dhar KK Branigan K Parkes J Howells RE Hand P Musgrove C Strange RC Fryer AA Redman CW Hoban PR Expression and subcellular localization of cyclin D1 protein in epithelial ovarian tumour cells.Br J Cancer. 1999; 81: 1174-1181Crossref PubMed Scopus (60) Google Scholar indicating that other molecular mechanisms are responsible for gene up-regulation. Our results support this finding because 14 of the 65 ovarian tumors with strong CCND1 protein expression had elevated gene copy numbers. Interestingly a co-gain of CCND1 and PAK1 was observed in 13 of 17 tumors. We hypothesize that CCND1 gain seems to play a minor role in ovarian cancer. In summary, the aCGH technique permits to precisely characterize the amplification status of specific oncogenes and to perform detailed regional amplicon mapping. This study demonstrates that combined application of tumor-related gene chips and TMAs facilitates the identification of new genetic targets in human tumors and will lead to a better understanding of the key events in the pathogenesis of cancer. We thank Rita Epper, Susanne Griesshaber, Yvonne Knecht, Martina Mirlacher, Hedvika Novotny, and Sandra Schneider for their excellent technical support.