This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Schraml, P. Articles by Moch, H. Search for Related Content PubMed PubMed Citation Articles by Schraml, P. Articles by Moch, H.

(American Journal of Pathology. 2003;163:985-992.)
© 2003 American Society for Investigative Pathology

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

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

From the Institute of Pathology,^* University of Basel, Basel, Switzerland; the Institute of Pathology, Mannheim, Germany; and Vysis Incorporated, Downers Grove, Illinois

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
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.

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.³ 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 oncogenes^4,5 as well as for quantitative mapping of amplicon structures.^6,7

Previous mCGH studies showed that chromosomal copy number gains are common in ovarian cancer. High-level amplifications were mainly observed in late-stage tumors,⁸ 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,^9-11 KRAS at 12p12.1,^12,13 CCND2 at 12p13,¹⁴ HER2 at 17q21,^12,15 and AIB1 at 20q12-13.¹⁶ 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.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Tumor Samples and Cell Lines

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.¹⁷ The histological subtype was defined according to the World Health Organization.¹⁸ 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).

mCGH

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.¹⁹ 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, 2x standard saline citrate, pH 7.0). Hybridization, image acquisition, image analysis, and control experiments were exactly as described.¹⁹ Thresholds used for definition of DNA sequence copy number gains and losses were >1.2 and <0.8, respectively. Overrepresentations were considered amplifications when the fluorescence ratio values exceeded 1.5 in a subregion of a chromosome arm according to previous recommendations.²⁰

aCGH

DNA for aCGH was prepared using the Gentra Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN). DNA quality was verified on a 1% agarose gel and DNA concentration was determined spectrophotometrically. aCGH was performed using the AmpliOnc I microarray kit (Vysis). Briefly, tumor and cell line DNA were labeled by nick translation with Alexa Fluor 488-5-dUTP (Molecular Probes, Leiden, The Netherlands) and the normal reference DNA was labeled with Alexa Fluor 594-5-dUTP (Molecular Probes). Labeled test-DNA and normal reference DNA (0.5 µg each) were mixed in 30 µl of hybridization buffer (50% formamide, 10% dextran sulfate, 2x standard saline citrate, 0.1 mg Cot-1 DNA, pH 7.0) and hybridized to AmpliOnc I microarray (Vysis). The chip contained 59 different DNA sequences deriving from P1, PAC, or BAC. Each clone was represented by three target spots. Arrays were hybridized for 16 hours, washed three times for 10 minutes in 50% formamide, 2x standard saline citrate at 40°C, followed by four times 5 minutes in 1x standard saline citrate at room temperature, and counterstained with DAPI IV mounting solution (Vysis). Imaging and data analysis of the arrays was implemented with the GenoSensor Reader System (Vysis). The software automatically captured three images of each chip, specific for the blue, the green, and the red color planes. The test/reference ratio was defined as the ratio of the sum of test intensity pixel values to the sum of reference intensity pixel values (mass ratio) after pixel intensity analysis within each spot and local background subtraction. The values were further normalized by dividing test/reference mass ratios by the test/reference ratio obtained from modal DNA copy numbers.

Validation of aCGH

Variations of the green and red ratios on the arrayed oncogenes were defined by three comparative hybridizations using normal male DNA as test and reference DNA (Vysis). The green/red fluorescence ratios for each spotted genomic clone ranged from 0.92 to 1.12 indicating consistent and reproducible hybridization for all 59 DNA targets on the AmpliOnc I microarray. Normal male DNA as reference was used as an additional control to confirm the 1:1 ratio of chromosome X copies in tumors with chromosome X loss and the 1:2 ratio of chromosome X copies in tumors without chromosome X loss. As expected, 11 ovarian tumors with complete Xq loss showed averaged fluorescence mean ratios of the AR gene (AR 3' and AR 5') ranging from 1.00 to 1.19 confirming loss of one AR copy in these tumors. The green/red ratio of AR ranged from 1.26 to 1.65 in four tumors with partial Xq deletions without affecting the AR locus Xq11-q12, in 13 tumors that retained two chromosome X copies, and in 1 tumor with increased copy number in the telomeric region of Xq. Based on these findings aCGH fluorescence ratio thresholds used for definition of DNA sequence copy number gains was >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.^4,5

TMA

The ovarian carcinoma TMA was constructed as previously described.²¹ 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.

View larger version (92K): [in this window] [in a new window]   Figure 1. A: The ovarian carcinoma TMA (overview) and two array elements (x40-fold, H&E staining) with a serous (B) and a mucinous (C) adenocarcinoma.

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.

Immunohistochemistry

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.

Statistics

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.

	Results

Top Abstract Materials and Methods Results Discussion References

 
mCGH

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%).

aCGH

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 .

View larger version (62K): [in this window] [in a new window]   Figure 2. Image of an AmpliOnc I microarray after hybridization of 59 genomic loci. Oncogenes CCND1, FGF4/FGF3, and EMS1 located on 11q13 are amplified.

View larger version (39K): [in this window] [in a new window]   Figure 3. aCGH results of 26 primary ovarian carcinomas and three ovarian carcinoma cell lines.

View larger version (36K): [in this window] [in a new window]   Figure 4. Comparison of aCGH and mCGH data obtained from tumor 4. aCGH (left): Amplified genes are represented in dark green, genes with gained copy numbers in green, and deleted genes in red columns. mCGH (right): Green and red bars indicate deleted or gained chromosomal regions (mean ratio + SD obtained from at least four chromosomes). Asterisk: Gene loss or gain not corresponding with mCGH profile.

FISH

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) .

View larger version (88K): [in this window] [in a new window]   Figure 5. A: PAK1 amplification: tumor cells with clusters of red PAK1 signals and two green centromere 11 signals. B: CCND1 gain with four to six red CCND1 signals and two to four centromere 11 signals (green) in an ovarian carcinoma on the TMA. C: Cytoplasmic PAK1 protein expression in an ovarian carcinoma. D: Nuclear CCND1 protein expression in an ovarian carcinoma.

Strong cytoplasmic PAK1 protein expression was observed in 172 of 199 ovarian cancers. PAK1 protein was most frequent in grade 3 tumors and was present in all undifferentiated ovarian carcinomas, but this trend was not significant. Nuclear CCND1 expression was seen in 65 of 260 (25%) tumors (Table 1) . Examples of positive cases are shown in Figure 5, C and D . The association between PAK1 overexpression and gene gain/amplification could be analyzed in 72 ovarian cancers with interpretable immunohistochemistry and FISH results on consecutive sections. This analysis revealed no significant association between PAK1 copy number increase and overexpression.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
aCGH revealed frequent gene copy number gains of PIK3CA at 3q26.3, PAK1 at 11q13.5-q14, KRAS2 at 12p12.1, and STK15 at 20q13 in ovarian carcinomas. Gene amplifications were detected for MET on 7q; MYC on 8q; CCND1, FGF4/FGF3, EMS1, GARP, and PAK1 on 11q; ERBB2 on 17q; and AIB1, PTPN1, ZNF217 on 20q. These results correlated well with the mCGH showing frequent overrepresentations on chromosome 3q, 8q, 11q, 12p, 17q, and 20q. Previous mCGH studies of ovarian tumors have reported similar findings.⁸ Our mCGH data further suggested that gains and low-level amplifications of different chromosomal regions are a common phenomenon in the pathogenesis of ovarian cancer, but that specific oncogenes are the targets of such large chromosomal copy number gains. Examples of oncogenes located in large amplicons, which were preferentially gained include PIK3CA at 3q26.3, PAK1 at 11q13.5-14, JUNB at 19p13.2, AIB1 and STK15 at 20q12-13. The dominating role of AIB1 within the 20q12-13 amplicon has recently been shown in ovarian cancer.²² One might therefore speculate that the remaining oncogenes in these amplicons, eg, TERC on 3q26.3; CCND1, FGF4/FGF3, EMS1, and GARP on 11q13; INSR on 19p13; MYBL2, PTPN1, and ZNF217 on 20q13 are of minor biological relevance for ovarian cancer.

On the basis of our aCGH data, PAK1 seems to be an important genetic target of the 11q13.5-14 amplicon. Whereas CCND1 on 11q13.3 is a well-described oncogene in this region, which has been implicated in many human cancers, little is known about PAK1 aberrations in ovarian cancer. The striking difference of PAK1 and CCND1 gain frequencies was decisive to further examine and validate PAK1 and CCND1 gene copy number alterations and protein expression on an ovarian carcinoma TMA.

11q13.5-q14 amplifications involving the PAK1 locus have been recently reported in bladder²³ and breast cancer.²⁴ 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.²⁵ PAK1 promotes hyperplasia in mammary epithelium by phosphorylating and transactivating estrogen receptor-.²⁶ It is also involved in angiogenesis by controlling vascular endothelial growth factor expression, vascular endothelial growth factor secretion, and function.²⁷

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.^28-31 Tumor growth suppression by transfecting murine fibrosarcoma cells with 11q23.1-containing YACs indicates a tumor suppressor gene locus in close vicinity to MLL.³¹ 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.^14,32 Although CCND1 amplification is uncommon in ovarian cancer, nuclear CCND1 overexpression has been reported in 15 to 30% of ovarian tumors^33,34 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.

	Acknowledgements

 
We thank Rita Epper, Susanne Griesshaber, Yvonne Knecht, Martina Mirlacher, Hedvika Novotny, and Sandra Schneider for their excellent technical support.

	Footnotes

 
Address reprint requests to Peter Schraml, Ph.D., Institute of Pathology, University Hospital, Schönbeinstrasse 40, 4031 Basel, Switzerland. E-mail: peter.schraml{at}unibas.ch

Supported by the Krebsforschung Schweiz grant KFS 1090-09-2000.

Accepted for publication May 30, 2003.

	References

Top Abstract Materials and Methods Results Discussion References

 

1. Boring CC, Squires TS, Tong T, Montgomery S: Cancer statistics, 1994. CA Cancer J Clin 1994, 44:7-26[Medline]
2. Shridhar 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 RC, Jr, James CD, Couch FJ, Hartmann LC, Lillie J, Smith DI: Genetic analysis of early- versus late-stage ovarian tumors. Cancer Res 2001, 61:5895-5904[Abstract/Free Full Text]
3. Isola 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, 84:1109-1114[Abstract/Free Full Text]
4. Daigo 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, 158:1623-1631[Abstract/Free Full Text]
5. Hui 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, 81:717-723[Medline]
6. Albertson 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, 25:144-146[Medline]
7. Pinkel 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, 20:207-211[Medline]
8. Knuutila S, Autio K, Aalto Y: Online access to CGH data of DNA sequence copy number changes. Am J Pathol 2000, 157:689[Free Full Text]
9. Abeysinghe 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, 114:136-143[Medline]
10. Wang 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, 66:140-148[Medline]
11. Diebold 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, 429:221-227[Medline]
12. Bian M, Fan Q, Huang S, Ma J, Lang J: Amplifications of proto-oncogenes in ovarian carcinoma. Chin Med J 1995, 108:844-848[Medline]
13. Yang-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, 48:678-681[Medline]
14. Courjal 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, 69:247-253[Medline]
15. Seki 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, 17:103-106[Medline]
16. Anzick 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, 277:965-968[Abstract/Free Full Text]
17. Sobin LH, Wittekind C: TNM Classification of Malignant Tumours. 2002 Wiley-Liss New York
18. Scully RE, Sobin LH: Histological Typing of Ovarian Tumours. 1999 Springer Berlin
19. Jiang 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, 153:1467-1473[Abstract/Free Full Text]
20. Kallioniemi A, Kallioniemi OP, Piper J, Tanner M, Stokke T, Chen L, Smith HS, Pinkel D, Gray JW, Waldman FM: Detection and mapping of amplified DNA sequences in breast cancer by comparative genomic hybridization. Proc Natl Acad Sci USA 1994, 91:2156-2160[Abstract/Free Full Text]
21. Kononen 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, 4:844-847[Medline]
22. Tanner MM, Grenman S, Koul A, Johannsson O, Meltzer P, Pejovic T, Borg A, Isola JJ: Frequent amplification of chromosomal region 20q12–q13 in ovarian cancer. Clin Cancer Res 2000, 6:1833-1839[Abstract/Free Full Text]
23. Simon R, Nocito A, Hubscher T, Bucher C, Torhorst J, Schraml P, Bubendorf L, Mihatsch MM, Moch H, Wilber K, Schotzau A, Kononen J, Sauter G: Patterns of her-2/neu amplification and overexpression in primary and metastatic breast cancer. J Natl Cancer Inst 2001, 93:1141-1146[Abstract/Free Full Text]
24. Bekri S, Adelaide J, Merscher S, Grosgeorge J, Caroli-Bosc F, Perucca-Lostanlen D, Kelley PM, Pebusque MJ, Theillet C, Birnbaum D, Gaudray P: Detailed map of a region commonly amplified at 11q13–>q14 in human breast carcinoma. Cytogenet Cell Genet 1997, 79:125-131[Medline]
25. Vadlamudi 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, 275:36238-36244[Abstract/Free Full Text]
26. Wang 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, 21:5437-5447[Medline]
27. Bagheri-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, 275:39451-39457[Abstract/Free Full Text]
28. Evans MF, Koreth J, Bakkenist CJ, Herrington CS, McGee JO: Allelic deletion at 11q23.3-q25 is an early event in cervical neoplasia. Oncogene 1998, 16:2557-2564[Medline]
29. Jin 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, 22:312-320[Medline]
30. Launonen 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, 71:299-304[Medline]
31. Koreth J, Bakkenist CJ, Larin Z, Hunt NC, James MR, McGee JO: 11q23.1 and 11q25-qter YACs suppress tumour growth in vivo. Oncogene 1999, 18:1157-1164[Medline]
32. Schraml 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, 5:1966-1975[Abstract/Free Full Text]
33. Kusume 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, 5:4152-4157[Abstract/Free Full Text]
34. Dhar 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, 81:1174-1181[Medline]

This article has been cited by other articles:

				I. Ayala, M. Baldassarre, G. Giacchetti, G. Caldieri, S. Tete, A. Luini, and R. Buccione Multiple regulatory inputs converge on cortactin to control invadopodia biogenesis and extracellular matrix degradation J. Cell Sci., February 1, 2008; 121(3): 369 - 378. [Abstract] [Full Text] [PDF]

				H. Aoki, T. Yokoyama, K. Fujiwara, A. M. Tari, R. Sawaya, D. Suki, K. R. Hess, K. D. Aldape, S. Kondo, R. Kumar, et al. Phosphorylated Pak1 Level in the Cytoplasm Correlates with Shorter Survival Time in Patients with Glioblastoma Clin. Cancer Res., November 15, 2007; 13(22): 6603 - 6609. [Abstract] [Full Text] [PDF]

				U. E. E. Rennefahrt, S. W. Deacon, S. A. Parker, K. Devarajan, A. Beeser, J. Chernoff, S. Knapp, B. E. Turk, and J. R. Peterson Specificity Profiling of Pak Kinases Allows Identification of Novel Phosphorylation Sites J. Biol. Chem., May 25, 2007; 282(21): 15667 - 15678. [Abstract] [Full Text] [PDF]

				S. K. Rayala, P. R. Molli, and R. Kumar Nuclear p21-Activated Kinase 1 in Breast Cancer Packs Off Tamoxifen Sensitivity. Cancer Res., June 15, 2006; 66(12): 5985 - 5988. [Abstract] [Full Text] [PDF]

				C. Holm, S. Rayala, K. Jirstrom, O. Stal, R. Kumar, and G. Landberg Association between Pak1 expression and subcellular localization and tamoxifen resistance in breast cancer patients. J Natl Cancer Inst, May 17, 2006; 98(10): 671 - 680. [Abstract] [Full Text] [PDF]

				Z. Varga, J.-P. Theurillat, V. Filonenko, B. Sasse, B. Odermatt, A. A. Jungbluth, Y.-T. Chen, L. J. Old, A. Knuth, D. Jager, et al. Preferential Nuclear and Cytoplasmic NY-BR-1 Protein Expression in Primary Breast Cancer and Lymph Node Metastases. Clin. Cancer Res., May 1, 2006; 12(9): 2745 - 2751. [Abstract] [Full Text] [PDF]

				J. E. Korkola, J. Houldsworth, R. S.V. Chadalavada, A. B. Olshen, D. Dobrzynski, V. E. Reuter, G. J. Bosl, and R.S.K. Chaganti Down-Regulation of Stem Cell Genes, Including Those in a 200-kb Gene Cluster at 12p13.31, Is Associated with In vivo Differentiation of Human Male Germ Cell Tumors Cancer Res., January 15, 2006; 66(2): 820 - 827. [Abstract] [Full Text] [PDF]

				T. Okada, M. Lopez-Lago, and F. G. Giancotti Merlin/NF-2 mediates contact inhibition of growth by suppressing recruitment of Rac to the plasma membrane J. Cell Biol., October 24, 2005; 171(2): 361 - 371. [Abstract] [Full Text] [PDF]

				C. Rodriguez, L. Hughes-Davies, H. Valles, B. Orsetti, M. Cuny, L. Ursule, T. Kouzarides, and C. Theillet Amplification of the BRCA2 Pathway Gene EMSY in Sporadic Breast Cancer Is Related to Negative Outcome Clin. Cancer Res., September 1, 2004; 10(17): 5785 - 5791. [Abstract] [Full Text] [PDF]

				L Tornillo, H Moch, P-A Diener, A Lugli, and G Singer CDX-2 immunostaining in primary and secondary ovarian carcinomas J. Clin. Pathol., June 1, 2004; 57(6): 641 - 643. [Abstract] [Full Text] [PDF]

				H. Yoshida, W. Cheng, J. Hung, D. Montell, E. Geisbrecht, D. Rosen, J. Liu, and H. Naora Lessons from border cell migration in the Drosophila ovary: A role for myosin VI in dissemination of human ovarian cancer PNAS, May 25, 2004; 101(21): 8144 - 8149. [Abstract] [Full Text] [PDF]

				J. H. Carter, L. E. Douglass, J. A. Deddens, B. M. Colligan, T. R. Bhatt, J. O. Pemberton, S. Konicek, J. Hom, M. Marshall, and J. R. Graff Pak-1 Expression Increases with Progression of Colorectal Carcinomas to Metastasis Clin. Cancer Res., May 15, 2004; 10(10): 3448 - 3456. [Abstract] [Full Text] [PDF]

				L. G. Shaffer and B. A. Bejjani A cytogeneticist's perspective on genomic microarrays Hum. Reprod. Update, May 1, 2004; 10(3): 221 - 226. [Abstract] [Full Text] [PDF]

				S. Balasenthil, A. A. Sahin, C. J. Barnes, R.-A. Wang, R. G. Pestell, R. K. Vadlamudi, and R. Kumar p21-activated Kinase-1 Signaling Mediates Cyclin D1 Expression in Mammary Epithelial and Cancer Cells J. Biol. Chem., January 9, 2004; 279(2): 1422 - 1428. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Schraml, P. Articles by Moch, H. Search for Related Content PubMed PubMed Citation Articles by Schraml, P. Articles by Moch, H.