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ene expression profile of ovarian serous papillaryarcinomas: identification of metastasis-associated genes

liana Bignotti, PhD; Renata A. Tassi, PhD; Stefano Calza, PhD; Antonella Ravaggi, PhD; Elisabetta Bandiera, PhD;lisa Rossi, PhD; Carla Donzelli, MD; Brunella Pasinetti, MD; Sergio Pecorelli, MD; Alessandro D. Santin, MD

BJECTIVE: The purpose of this study was to identify genes that areighly differentially expressed in metastatic serous papillary ovar-

an tumors (MET) when compared with primary ovarian serous car-inomas (OSPC).

TUDY DESIGN: An oligonucleotide microarray with probe sets com-lementary to �14,500 human genes was used to determine whetheratterns of gene expression may differentiate OSPC from MET in 31nap-frozen serous papillary ovarian carcinomas (ie, 14 primary OSPCnd 17 omental metastasis [MET]).

ESULTS: Hierarchic cluster analysis of gene expression in OSPCnd MET identified 156 genes that exhibited � 2-fold differencesP � .05) and that distinguished OSPC from MET. A number ofnvasion and metastasis predictive genes (including plasminogenctivator; matrix metalloproteinase; matrix structural constituentenes encoding products with collagen, heparin, and hyaluroniccid binding activity; genes encoding receptors for insulin-likerowth factors; vascular endothelial growth factor; endothelin type

oi: 10.1016/j.ajog.2006.10.874

ntegrins, and chemokines [stromal cell-derived factor 1CXCL12)]) were found among the 120 genes that were highly dif-erentially overexpressed in MET, when compared with OSPC.own-regulated genes in MET compared with OSPC included hep-in and testisin. Overexpression of CXCL12, matrix metalloprotein-se, plasminogen activator, type A and B integrins, and hepsinenes was validated by quantitative real-time polymerase chain re-ction in all samples. Finally, overexpression of CXCL12 in MET,hen compared with OSPC, was validated at the protein level by

mmunohistochemistry.

ONCLUSION: Gene expression profiling may differentiate meta-tatic ovarian cancer from primary OSPC. The identification of me-astasis-associated genes may provide a foundation for the devel-pment of new type-specific diagnostic strategies and treatment foretastatic ovarian cancer.

ey words: complementary DNA, gene expression profiling,etastasis-associated gene, microarray, ovarian cancer, ovarian

; fibroblast growth factor; thrombospondin 1 and 2; type A and B serous papillary carcinoma

ite this article as: Bignotti E, Tassi RA, Calza S, et al. Gene expression profile of ovarian serous papillary carcinomas: identification of metastasis-associatedenes. Am J Obstet Gynecol 2007;196:245.e1-245.e11.

varian carcinoma remains themost lethal gynecologic tumor;

2,220 new cancer cases and 16,210eaths were estimated in 2005 in thenited States alone.1 Ovarian serous

papillary carcinoma (OSPC) representsthe most common histologic type ofovarian carcinoma.2 Because of the lackof reliable screening tests and the insidi-ous onset of the disease, two thirds of pa-

tients have advanced metastatic diseasewhen diagnosed; although many pa-tients with disseminated tumors respondinitially to standard combinations of cy-toreductive surgery and platinum-basedtherapy, approximately 90% will experi-ence recurrence and inevitably die of thedisease.2 Understanding the molecularbasis of ovarian carcinoma metastaticspread may have the potential to refinesignificantly the management of theseserous tumors and lay the groundworkfor the development of novel and morespecific and more effective treatmentmodalities against this disease.

Comprehensive high-throughput tech-nologies for assaying gene expression(such as high-density oligonucleotide andcomplementary DNA microarrays) mayallow precise and accurate grouping of hu-man tumors and may have the potential to

rom the Division of Gynecologic Oncology (Drs Bignotti, Tassi, Ravaggi, Bandiera,asinetti, and Pecorelli), the Section of Medical Statistics and Biometry, Department ofiomedical Sciences and Biotechnology (Dr Calza), and the Department of Pathology (Drsossi and Donzelli), University of Brescia Medical School, Brescia, Italy, and the Division ofynecologic Oncology, University of Arkansas for Medical Sciences, Little Rock, AR (Drantin).

eceived Jul. 1, 2006; accepted Oct. 11, 2006.

eprints: Alessandro D. Santin, MD, UAMS Medical Center, Department of Obstetrics &ynecology, 4301 W Markham, Slot 518, Little Rock, AR 72205-7199;[email protected].

upported by grants from the Nocivelli, the Camillo Golgi and the Berlucchi Foundationsrescia, Italy, and from grants from the Istituto Superiore di Sanita’ (Programmancotecnologico), Rome, Italy, and the Centre for Innovative Diagnostics and Therapeutics

IDET), Brescia, Italy.

002-9378/$32.002007 Mosby, Inc. All rights reserved.

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dentify patients who are unlikely to be

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ured by conventional therapy. Consistentith this view, in the last few years severalroups have used microarray technologyo identify genes that are involved in ovar-an carcinogenesis.3,4 To date, however,

icroarray-based studies in ovarian can-er have focused mainly on comparison ofrimary and/or metastatic tumors withormal ovarian samples or primary tumoramples at different stages of disease.3,4 In-eed, very little information has addressedirectly the potential difference in gene ex-ression profiling between primary ovar-

an carcinoma vs metastatic ovarian dis-ase. This point is noteworthy becausepecific metastatic fingerprints have beendentified recently in several human solidumors, which include lung, breast, andolon cancer.5-7 In this study, we used anligonucleotide microarray with probeets complementary to �14,500 humanenes to determine whether patterns ofene expression may differentiate primarySPC from metastatic tumors. We reportn a number of genes that are expressedighly differentially in metastatic diseasehen compared with primary ovarian

ancer. Some of the identified genes haveeen reported previously to be up-regu-

ated in ovarian cancer, which further val-dates our criteria for the determination ofifferentially expressed genes; other genesepresent novel findings. It is our hope thatome of the metastatic tumor markers thatere identified in this work may be used

eadily for early detection of disease recur-ence and for the development of novelherapies against metastatic ovarianarcinoma.

ATIENTS AND METHODSvarian tissue samplestotal of 31 snap-frozen unpaired OSPCs

hat were collected from the primary ovar-an tumor (ie, 14 samples), and omental

etastatic ovarian disease (ie, 17 samples)

TABLE 1Clinical and pathologic characteris

CharacteristicPatients(n)

A

M

Primary OSPC 14 64...................................................................................................................

MET 17 59

ere obtained from the Division of Gyne- w

45.e2 American Journal of Obstetrics & Gynecol

ologic Oncology at the University of Bres-ia, Italy. Study approval was obtainedrom the institutional review board, and allatients signed an informed consent, ac-ording to institutional guidelines. All theatients with ovarian cancer were treatedurgically in the Department of Gyneco-ogic Oncology of the University of Bresciauring the last 3 years. They underwent aadical surgical tumor debulking and aomplete staging procedure, which wereollowed by platinum-based chemother-py. No patients received chemotherapyefore surgery. Patient clinical and patho-

ogic characteristics are showed in Table 1.Briefly, ovarian tumor tissues were iden-

ified, sharp-dissected, and snap-frozen iniquid nitrogen within 30 minutes afterurgery. The samples were embedded inCT medium, macrodissected in a cryo-

tat at –20°C, and stained with hematoxy-in and eosin to check epithelial purity.ach sample was analyzed histologically bystaff pathologist, and only tumor samples

hat contained at least 70% tumor epithe-ial cells were retained for further totalNA extraction.

otal RNA extraction and genehip hybridizationotal RNA was extracted from all 31 tu-or samples with TRIZOL reagent (Lifeechnologies, Inc, Carlsbad, CA) and fur-

her purified with RNeasy Min-elutelean-up Columns (Qiagen, Valencia,A), as described by the manufacturers.issue samples, approximately 50 mg,ere pulverized previously in a chilledortar, TRIZOL reagent was added, and

he tissue was homogenized for 20 secondsith Ultra-turrax T8 (IKA-Werke,tauffen, Germany). All tissue sampleuality was controlled strictly to verify theNA integrity before use in microarray ex-eriments. RNA quantity was evaluatedpectrophotometrically, and the quality

s of the patientsy) Grade (n)

SD 1 2 3

15 0 0 14.........................................................................................................................

14 0 2 15

as assessed with the Agilent 2100 bioana- m

ogy MARCH 2007

yzer (Agilent Technologies Inc, Palo Alto,A). Only samples with good RNA yieldnd no RNA degradation (28S:18S �1.7nd RNA integrity number �8.5) were re-ained for further experiments. Labeling ofamples and hybridization to the Af-ymetrix Human U133A oligonucleotide

icroarray chips (Santa Clara, CA) thatontained 22,283 probe sets were per-ormed according to the manufacturer’srotocols, as previously described.8

ata analysis and clusteringene expression values were computedith the MAS5 algorithm, with targetalue set to 500. To select highly differen-ially expressed genes, a nonspecific filter-ng was first applied. Genes were retained ift least 75% of the samples in the overex-ressed group had presence call and an in-erquartile range of�0.6. The comparisonetween OSPC and MET samples was per-ormed with the Student t test, with foldiscovery rate correction as previously de-cribed.8 Genes were considered of interestf the absolute value of the estimated foldhange was �2 and if the fold discoveryate was � 0.2. A hierarchic clustering withearson correlation as the distance matrixere performed to show graphically the re-

ults of the analysis. All the analyses wereerformed with the Bioconductor soft-are (Fred Hutchinson Cancer Researchenter, Seattle, WA).8

alidation of gene expression byuantitative real-time polymerasehain reactioneal-time polymerase chain reaction (RT-CR) was performed in triplicate withrimer sets and probes that were specific

or 5 selected genes that were found to beignificantly differentially expressed inSPCtissuesamples, comparedwithMET

amples. These genes were chemokineXC motif ligand 12 (CXCL12), matrix

Stage (n)

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1 1 10 2..................................................................................................................

0 0 14 3

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etalloproteinase 11 (MMP11), plasmin-

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gen activator (PLAU), integrin, beta-4ITGB4) and hepsin (HPN). All reactionsere carried out on the ABI PRISM 7000equence Detection System (Appliediosystems, Cheshire, UK) with the usef the TaqMan Universal PCR master Mixnd the following Assays on DemandApplied Biosystems): Hs00171022_m1CXCL12), Hs00171829_m1 (MMP11),s00170182_m1 (PLAU), Hs00174009_m1

ITGB4), and Hs00170096_m1 (HPN). Inrief, complementary DNA that had beenbtainedfrom50ngoftotalRNAwasampli-edina25-�LPCRreactionaccordingtotheanufacturer’s recommended protocol

nd amplification steps: denaturation for0 minutes at 95°C, followed by 40 cyclesfdenaturationat95°Cfor15seconds,andnnealing-extension at 60°C for 1 minute.he comparative threshold cycle methodas used for the calculation of amplifica-

ion fold, as specified by the manufacturer.he housekeeping gene glyceraldehyde-3-

FIGURE 1Unsupervised hierarchical clusterincarcinomas (ie, 14 primary OSPC a

endrogram that resulted from unsupervised cluene expression profiling.

hosphate dehydrogenase was used to a

ormalize the quantity of complementaryNA that was used in the PCR reactions.

pearman rank correlation was used to es-imate the degree of association between

icroarray and quantitative RT-PCR dataor each genes.

mmunohistochemistry onormalin-fixed tissueso evaluate protein expression levels for 1f the genes that was found to be up-regu-

ated in MET compared with OSPC, im-unohistochemical staining for CXCL12as performed on all samples (ie, 14 OSPC

nd 17 MET). Formalin-fixed, paraffin-mbeddedtissue that representedthespec-lar section of the tumor biopsy specimens

hat were used for microarray analysis (tu-or percentage, �70%) were cut and

tained with hematoxylin and eosin.riefly, slides were deparaffinized and re-ydrated in graded solutions of ethanol

of 31 serous papillary ovarian17 omental metastasis, MET)

analysis that differentiated MET from OSPC by

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ase was blocked by incubation with per-xidase-blocking solution (Novolink-OVOCASTRA, Menarini, Italy) for 5inutes, followed by rinsing in Tris-buff-

red saline solution. Nonspecific stainingas blocked by treatment with proteinlock that was ready to use for 5 minutes.he immunohistochemical method in-olved sequential application of primaryntibody to CXCL12 (mouse monoclonalntibody, clone 79018, dilution 1:50; R&Dystems Inc, Minneapolis, MN) for 30inutes and a postprimary block ready to

se (NOVOCASTRA, Menarini, Italy) for5 minutes. The immunoprecipitate wasisualized by treatment with 3=3-diamino-enzidine (Novolink-NOVOCASTRA)or 7 minutes and counterstained by he-

atoxylin (Dako Corporation, Carpinte-ia, CA). Cases with less than 10% mem-ranous staining in tumor cells wereonsidered negative for claudin expres-ion. The intensity of membranous immu-oreactivity for CXCL12 in tumor cellsas scored subjectively in the followinganner: (a) 1�, weak staining; (b) 2�,edium staining; and (c) 3�, intense

taining. Negative controls, in which therimary antibodies were not added, wererocessed in parallel. The statistical signif-

cance of the difference in CXCL12 stain-ng between OSPC and MET was evalu-ted with the Fisher’s exact test.

ESULTSene expression analysis andlustering of OSPC and METomprehensive gene expression profilesf 14 OSPCs and 17 snap frozen METsere generated with high-density oligonu-

leotide arrays with 22,283 probe sets,hich in total interrogated approximately4,500 genes. Using unsupervised hierar-hical cluster analysis with 6577 probe sets,ith a single exception (ie, a primary tu-or collected from a patient who experi-

nced rapid progression during platinum-ased chemotherapy), we identified theifferences in gene expression betweenSPC and MET, which readily distin-

uished the 2 groups. As shown in Figure 1,ll 17 METs were found to group togethern the rightmost columns of the dendro-ram.Similarly, in the leftmostcolumns13

gnd

ster

f 14 OSPCs were found to cluster tightly



2

TABLE 2Up-regulated genes expressed at least 2-fold higher in metastasis vs primary OSPCCode Symbol Gene name FC

204320_at COL11A1 Collagen, type XI, alpha 1 8.23................................................................................................................................................................................................................................................................................................................................................................................

37892_at COL11A1 Collagen, type XI, alpha 1 6.33................................................................................................................................................................................................................................................................................................................................................................................

217430_x_at COL1A1 Collagen, type I, alpha 1 5.67................................................................................................................................................................................................................................................................................................................................................................................

201150_s_at TIMP3 Tissue inhibitor of metalloproteinase 3 (Sorsby fundus dystrophy, pseudoinflammatory) 5.52................................................................................................................................................................................................................................................................................................................................................................................


214701_s_at FN1 Fibronectin 1 5.4................................................................................................................................................................................................................................................................................................................................................................................

210511_s_at INHBA Inhibin, beta A (activin A, activin AB alpha polypeptide) 4.94................................................................................................................................................................................................................................................................................................................................................................................

201842_s_at EFEMP1 EGF-containing fibulin-like extracellular matrix protein 1 4.86................................................................................................................................................................................................................................................................................................................................................................................

206439_at DSPG3 Dermatan sulfate proteoglycan 3 4.36................................................................................................................................................................................................................................................................................................................................................................................

221730_at COL5A2 Collagen, type V, alpha 2 4.07................................................................................................................................................................................................................................................................................................................................................................................

215446_s_at LOX Lysyl oxidase 4.03................................................................................................................................................................................................................................................................................................................................................................................


213764_s_at MFAP5 Microfibrillar associated protein 5 4.01................................................................................................................................................................................................................................................................................................................................................................................

210809_s_at POSTN Periostin, osteoblast specific factor 3.97................................................................................................................................................................................................................................................................................................................................................................................

203325_s_at COL5A1 Collagen, type V, alpha 1 3.95................................................................................................................................................................................................................................................................................................................................................................................

203083_at THBS2 Thrombospondin 2 3.91................................................................................................................................................................................................................................................................................................................................................................................

202766_s_at FBN1 Fibrillin 1 (Marfan syndrome) 3.9................................................................................................................................................................................................................................................................................................................................................................................

209955_s_at FAP Fibroblast activation protein, alpha 3.84................................................................................................................................................................................................................................................................................................................................................................................


214336_s_at COPA Coatomer protein complex, subunit alpha 3.81................................................................................................................................................................................................................................................................................................................................................................................

209621_s_at PDLIM3 PDZ and LIM domain 3 3.75................................................................................................................................................................................................................................................................................................................................................................................

211571_s_at CSPG2 Chondroitin sulfate proteoglycan 2 (versican) 3.74................................................................................................................................................................................................................................................................................................................................................................................


214587_at COL8A1 Collagen, type VIII, alpha 1 3.7................................................................................................................................................................................................................................................................................................................................................................................

213338_at RIS1 NA 3.44................................................................................................................................................................................................................................................................................................................................................................................

203876_s_at MMP11 Matrix metalloproteinase 11 (stromelysin 3) 3.37................................................................................................................................................................................................................................................................................................................................................................................

201843_s_at EFEMP1 EGF-containing fibulin-like extracellular matrix protein 1 3.28................................................................................................................................................................................................................................................................................................................................................................................

209541_at IGF1 Insulin-like growth factor 1 (somatomedin C) 3.26................................................................................................................................................................................................................................................................................................................................................................................

212464_s_at FN1 Fibronectin 1 3.23................................................................................................................................................................................................................................................................................................................................................................................

220988_s_at NA NA 3.23................................................................................................................................................................................................................................................................................................................................................................................

206658_at UPK3B Uroplakin 3B 3.2................................................................................................................................................................................................................................................................................................................................................................................

201262_s_at BGN Biglycan 3.16................................................................................................................................................................................................................................................................................................................................................................................


202311_s_at COL1A1 Collagen, type I, alpha 1 3.12................................................................................................................................................................................................................................................................................................................................................................................

209754_s_at TMPO Thymopoietin 3.11................................................................................................................................................................................................................................................................................................................................................................................



202238_s_at NNMT Nicotinamide N-methyltransferase 3.05................................................................................................................................................................................................................................................................................................................................................................................

211668_s_at PLAU Plasminogen activator, urokinase 3.02................................................................................................................................................................................................................................................................................................................................................................................

216442_x_at FN1 Fibronectin 1 3.02................................................................................................................................................................................................................................................................................................................................................................................

209754_s_at TMPO Thymopoietin 3.11................................................................................................................................................................................................................................................................................................................................................................................



202238_s_at NNMT Nicotinamide N-methyltransferase 3.05

Continued on page 245.e5.

45.e4 American Journal of Obstetrics & Gynecology MARCH 2007


TABLE 2Up-regulated genes expressed at least 2-fold higher in metastasis vs primary OSPCContinued from page 245.e4.Code Symbol Gene name FC

211668_s_at PLAU Plasminogen activator, urokinase 3.02................................................................................................................................................................................................................................................................................................................................................................................


213765_at MFAP5 Microfibrillar associated protein 5 3................................................................................................................................................................................................................................................................................................................................................................................

204989_s_at ITGB4 Integrin, beta 4 2.99................................................................................................................................................................................................................................................................................................................................................................................

212344_at SULF1 Sulfatase 1 2.98................................................................................................................................................................................................................................................................................................................................................................................

201108_s_at THBS1 Thrombospondin 1 2.94................................................................................................................................................................................................................................................................................................................................................................................


202237_at NNMT Nicotinamide N-methyltransferase 2.91................................................................................................................................................................................................................................................................................................................................................................................

217428_s_at COL10A1 Collagen, type X, alpha 1(Schmid metaphyseal chondrodysplasia) 2.9................................................................................................................................................................................................................................................................................................................................................................................

201852_x_at COL3A1 Collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant) 2.89................................................................................................................................................................................................................................................................................................................................................................................

201559_s_at CLIC4 Chloride intracellular channel 4 2.85................................................................................................................................................................................................................................................................................................................................................................................

212952_at CALR Calreticulin 2.82................................................................................................................................................................................................................................................................................................................................................................................

204298_s_at LOX Lysyl oxidase 2.8................................................................................................................................................................................................................................................................................................................................................................................

206002_at GPR64 G protein-coupled receptor 64 2.79................................................................................................................................................................................................................................................................................................................................................................................

210892_s_at GTF2I General transcription factor II, i 2.79................................................................................................................................................................................................................................................................................................................................................................................

202274_at ACTG2 Actin, gamma 2, smooth muscle, enteric 2.78................................................................................................................................................................................................................................................................................................................................................................................

205428_s_at CALB2 Calbindin 2, 29 kd (calretinin) 2.77................................................................................................................................................................................................................................................................................................................................................................................

204589_at ARK5 NA 2.75................................................................................................................................................................................................................................................................................................................................................................................

206227_at CILP Cartilage intermediate layer protein, nucleotide pyrophosphohydrolase 2.73................................................................................................................................................................................................................................................................................................................................................................................

201109_s_at THBS1 Thrombospondin 1 2.72................................................................................................................................................................................................................................................................................................................................................................................

207173_x_at CDH11 Cadherin 11, type 2, OB-cadherin (osteoblast) 2.69................................................................................................................................................................................................................................................................................................................................................................................

205907_s_at OMD Osteomodulin 2.66................................................................................................................................................................................................................................................................................................................................................................................



212354_at SULF1 Sulfatase 1 2.65................................................................................................................................................................................................................................................................................................................................................................................

200974_at ACTA2 Actin, alpha 2, smooth muscle, aorta 2.64................................................................................................................................................................................................................................................................................................................................................................................

201792_at AEBP1 AE binding protein 1 2.64................................................................................................................................................................................................................................................................................................................................................................................

205941_s_at COL10A1 Collagen, type X, alpha 1(Schmid metaphyseal chondrodysplasia) 2.63................................................................................................................................................................................................................................................................................................................................................................................

215076_s_at COL3A1 Collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant) 2.63................................................................................................................................................................................................................................................................................................................................................................................

201744_s_at LUM Lumican 2.58................................................................................................................................................................................................................................................................................................................................................................................

202310_s_at COL1A1 Collagen, type I, alpha 1 2.58................................................................................................................................................................................................................................................................................................................................................................................

221541_at CRISPLD2 Cysteine-rich secretory protein LCCL domain containing 2 2.58................................................................................................................................................................................................................................................................................................................................................................................

205991_s_at PRRX1 Paired related homeobox 1 2.56................................................................................................................................................................................................................................................................................................................................................................................


202998_s_at LOXL2 Lysyl oxidase-like 2 2.5................................................................................................................................................................................................................................................................................................................................................................................

221731_x_at CSPG2 Chondroitin sulfate proteoglycan 2 (versican) 2.5................................................................................................................................................................................................................................................................................................................................................................................

205018_s_at MBNL2 Muscleblind-like 2 (Drosophila) 2.46................................................................................................................................................................................................................................................................................................................................................................................

212667_at SPARC Secreted protein, acidic, cysteine-rich (osteonectin) 2.46................................................................................................................................................................................................................................................................................................................................................................................

209466_x_at PTN Pleiotrophin (heparin binding growth factor 8, neurite growth-promoting factor 1) 2.42................................................................................................................................................................................................................................................................................................................................................................................

203939_at NT5E 5’-Nucleotidase, ecto (CD73) 2.41................................................................................................................................................................................................................................................................................................................................................................................

202450_s_at CTSK Cathepsin K (pycnodysostosis) 2.4................................................................................................................................................................................................................................................................................................................................................................................

203868_s_at VCAM1 Vascular cell adhesion molecule 1 2.39................................................................................................................................................................................................................................................................................................................................................................................

211161_s_at COL3A1 Collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant) 2.37................................................................................................................................................................................................................................................................................................................................................................................

213139_at SNAI2 Snail homolog 2 (Drosophila) 2.37

Continued on page 245.e6.

MARCH 2007 American Journal of Obstetrics & Gynecology 245.e5

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2

ogether. After the most “absent” genesere filtered out, the significance analysisf microarrays test revealed a total of 156

TABLE 2Up-regulated genes expressed at lContinued from page 245.e5.Code Symbol

213496_at LPPR4...................................................................................................................

208879_x_at C20orf14...................................................................................................................

212105_s_at DHX9...................................................................................................................

201438_at COL6A3...................................................................................................................

213125_at OLFML2B...................................................................................................................

212353_at SULF1...................................................................................................................

207172_s_at CDH11...................................................................................................................

202388_at RGS2...................................................................................................................

201110_s_at THBS1...................................................................................................................

210139_s_at PMP22...................................................................................................................

213548_s_at H41...................................................................................................................

205547_s_at TAGLN...................................................................................................................

209365_s_at ECM1...................................................................................................................

215236_s_at PICALM...................................................................................................................

210973_s_at FGFR1...................................................................................................................

209257_s_at CSPG6...................................................................................................................

200906_s_at KIAA0992...................................................................................................................

213872_at C6orf62...................................................................................................................

203851_at IGFBP6...................................................................................................................

209651_at TGFB1I1...................................................................................................................

212154_at SDC2...................................................................................................................

204464_s_at EDNRA...................................................................................................................

213909_at LRRC15...................................................................................................................

204051_s_at SFRP4...................................................................................................................

203666_at CXCL12...................................................................................................................

209758_s_at MFAP5...................................................................................................................

200604_s_at PRKAR1A

...................................................................................................................

203570_at LOXL1...................................................................................................................

200841_s_at EPRS...................................................................................................................

201617_x_at CALD1...................................................................................................................

204114_at NID2...................................................................................................................

201506_at TGFBI...................................................................................................................

37005_at NBL1...................................................................................................................

212257_s_at SMARCA2...................................................................................................................

209465_x_at PTN...................................................................................................................

202404_s_at COL1A2...................................................................................................................

211277_x_at APP...................................................................................................................

201476_s_at RRM1...................................................................................................................

NA, not available.

robe sets that showed a � 2-fold change O

45.e6 American Journal of Obstetrics & Gynecol

nd a fold discovery rate � 0.2. Of 156enes, 120 genes were found to be up-reg-lated in METs, when compared with

st 2-fold higher in metastasis vs pri

Gene name

NA.........................................................................................................................

Chromosome 20 open reading frame 14.........................................................................................................................

DEAH (Asp-Glu-Ala-His) box polypeptide 9.........................................................................................................................

Collagen, type VI, alpha 3.........................................................................................................................

Olfactomedin-like 2B.........................................................................................................................

Sulfatase 1.........................................................................................................................

Cadherin 11, type 2, OB-cadherin (osteoblast).........................................................................................................................

Regulator of G-protein signaling 2, 24 kd.........................................................................................................................

Thrombospondin 1.........................................................................................................................

Peripheral myelin protein 22.........................................................................................................................

NA.........................................................................................................................

Transgelin.........................................................................................................................

Extracellular matrix protein 1.........................................................................................................................

Phosphatidylinositol binding clathrin assembly protein.........................................................................................................................

Fibroblast growth factor receptor 1 (fms-related tyros.........................................................................................................................

Chondroitin sulfate proteoglycan 6 (bamacan).........................................................................................................................

NA.........................................................................................................................

Chromosome 6 open reading frame 62.........................................................................................................................

Insulin-like growth factor binding protein 6.........................................................................................................................

Transforming growth factor beta 1 induced transcript.........................................................................................................................

Syndecan 2 (heparan sulfate proteoglycan 1, cell sur.........................................................................................................................

Endothelin receptor type A.........................................................................................................................

Leucine rich repeat containing 15.........................................................................................................................

Secreted frizzled-related protein 4.........................................................................................................................

Chemokine (C-X-C motif) ligand 12 (stromal cell-deriv.........................................................................................................................

Microfibrillar associated protein 5.........................................................................................................................

Protein kinase, cyclic adenosine monophosphate-dep(tissue specific extinguisher 1)

.........................................................................................................................

Lysyl oxidase-like 1.........................................................................................................................

Glutamyl-prolyl-transfer RNA synthetase.........................................................................................................................

caldesmon 1.........................................................................................................................

Nidogen 2 (osteonidogen).........................................................................................................................

Transforming growth factor, beta-induced, 68 kd.........................................................................................................................

Neuroblastoma, suppression of tumorigenicity 1.........................................................................................................................

SWI/SNF related, matrix associated, actin dependent.........................................................................................................................

Pleiotrophin (heparin binding growth factor 8, neurite.........................................................................................................................

Collagen, type I, alpha 2.........................................................................................................................

Amyloid beta (A4) precursor protein (protease nexin-I.........................................................................................................................

Ribonucleotide reductase M1 polypeptide.........................................................................................................................

SPCs. Included in this list are PLAU, a

ogy MARCH 2007

MP11, matrix structural constituentenes that express products endowed withollagen/heparin/hyaluronic acid binding

ry OSPC

FC

2.34..................................................................................................................

2.33..................................................................................................................

2.33..................................................................................................................

2.32..................................................................................................................

2.32..................................................................................................................

2.3..................................................................................................................

2.29..................................................................................................................

2.28..................................................................................................................

2.27..................................................................................................................

2.27..................................................................................................................

2.26..................................................................................................................

2.25..................................................................................................................

2.25..................................................................................................................

2.22..................................................................................................................

inase 2, Pfeiffer syndrome) 2.21..................................................................................................................

2.19..................................................................................................................

2.18..................................................................................................................

2.18..................................................................................................................

2.17..................................................................................................................

2.17..................................................................................................................

associated, fibroglycan) 2.17..................................................................................................................

2.15..................................................................................................................

2.15..................................................................................................................

2.14..................................................................................................................

actor 1) 2.12..................................................................................................................

2.12..................................................................................................................

nt, regulatory, type I, alpha 2.11

..................................................................................................................

2.1..................................................................................................................

2.09..................................................................................................................

2.09..................................................................................................................

2.08..................................................................................................................

2.07..................................................................................................................

2.06..................................................................................................................

lator of chromatin 2.05..................................................................................................................

th-promoting factor 1) 2.04..................................................................................................................

2.03..................................................................................................................

heimer disease) 2.03..................................................................................................................

2.01..................................................................................................................

ea ma

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ctivity, genes encoding receptors for insu-

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in-like growth factors, vascular endothe-ial growth factor, endothelin type A, fibro-last growth factor, thrombospondin 1nd 2, alpha 6 extracellular, type A and Bntegrins (ITGB4), and chemokinesCXCL12; Table 2). The second profile wasepresented by 36 genes that were under-xpressed in METs and overexpressed inSPCs (Table 3). This list includes HPN

nd testisin.

alidation of gene expression byuantitative RT-PCRive highly differentially expressed genesetween OSPC and MET (ie, CXCL12,MP11, PLAU, ITGB4, and HPN ) were

elected for quantitative RT-PCR analysis.comparisonof themicroarrayandquan-

itative RT-PCR data for these genes ishown in Figure 2. For all 5 genes that wereested, the quantitative RT-PCR data wereorrelated significantly to the microarrayata (P � .001; Spearman correlation co-fficient: CXCL12, 0.79; MMP11, 0.88;LAU, 0.78; ITGB4, 0.83; and HPN, 0.84).hus, quantitative RT-PCR data suggest

hatmostarrayprobesetsare likely tomea-ure accurately the levels of the intendedranscript within a complex mixture ofranscripts.

alidation of protein expressiony immunohistochemicaltainingo confirm gene expression results at therotein level, immunohistochemistry

or CXCL12 was carried out on 14 for-alin-fixed primary OSPC tumors and

ll 17 omental metastases. As depicted inable 4 and representatively shown in

he right panel of Figure 3, a strong cyto-lasmatic staining (3� scoring) forXCL12 was detected in 12 of 17 MET

amples (70%); only 5 of 14 OSPCs35%; Figure 3 left panel) showed strongositivity for CXCL12 (P � .045).

OMMENThis study represents the first communi-ation of an investigation that involvedhe genome-wide examination of differ-nces in gene expression between pri-ary vs metastatic OSPCs. We found

hat, with a single exception, hierarchic
lustering of the samples led to the un- t
mbiguous separation of OSPCs fromETs. We detected 156 genes that were

xpressed differentially between OSPCsnd METs; the average change in expres-ion level between the 2 groups was ateast 2-fold. The known function ofome of these genes may provide insightsn the molecular pathogenesis and theighly aggressive biologic behavior ofetastatic serous ovarian cancer, al-

hough others may prove to be useful di-gnostic and therapeutic markers againstetastatic disease.A large number of invasion and metas-

asis predictive genes have been identi-ed in our analysis. Among these genesre PLAU, MMP11 and 2) throm-ospondin 1 and 2, and CXCL12. Of in-erest, a considerable proportion of theene-expression signature that is associ-ted with metastasis seems to be derivedrom nonepithelial components of theumors. Examples of these include genesncoding for several type of extracellularatrix structural constituents (such as

ifferent type of collagens) the expres-ion of which is restricted to fibroblastsr actin �2 (a marker of smooth mus-le). Importantly, these gene expres-ion signatures that were associatedith metastasis likely may have beenissed had only malignant epithelial

ells been isolated before expressionrofiling.Urinary plasminogen activator (PLAU

r uPA) was found among the most up-egulated genes in metastatic ovarian tu-ors. This gene is well-known to play a

ritical role in tissue remodeling, in theegulation of tumor cell migration, in ex-racellular matrix invasion and during

etastatic tumor spread by binding to itsell-surface receptor, uPAR. Several in-estigations have reported a direct corre-ation between increased uPA levels andhe invasive potential of several humanumors that includes ovarian tumors,nd a high concentration of uPA in thescites and plasma of patients with ovar-an cancer has been shown to correlateith poor prognosis and poor response

o chemotherapy.9 Furthermore, in vitrond in vivo experimental models dem-nstrated that (1) uPA induces ovarianancer cell proliferation and migra-
ion,10,11 (2) uPA and uPAR overexpres- M
MARCH 2007 America

ion confers cancer cells with invasivend metastatic potentials12 and (3) in-ibiting uPA function with specific in-ibitors significantly decreases ovarianancer invasion and metastasis.13,14

aken together, our gene expression re-ults that show a significant up-regula-ion of uPA in metastatic ovarian cancerhen compared with primary ovarian

umors are consistent with previousndings and further support the view

hat a selective inhibition of uPA and/orts pathway may have significant thera-eutic effects against metastatic ovarianisease.Thrombospondins are multimodular

ecreted proteins that associate with thextracellular matrix and possess a varietyf biologic functions that include tissueemodeling, cell migration, adhesion,nd modulation of angiogenesis. Never-heless, the functional role of throm-ospondins in human tumor growthnd progression is still a matter of de-ate. Some authors have shown thathrombospondin 1 (TSP-1) may haventiangiogenetic effects and thereforeay function as an inhibitor of tumoretastasis in human fibrosarcomas.15

n the other hand, it has been shownhat thrombospondins may play a role inumor progression of several human tu-

ors and therefore may represent trueetastasis-associated genes.16,17 Consis-

ent with this view, in ovarian cancer,revious reports have suggested an asso-iation between thrombospondins over-xpression and aggressive tumor pheno-ypes.18 In our study, TSP-2 was found toe �5-fold up-regulated in ovarian can-er metastasis, when compared with pri-ary tumors, although TSP-1 was over-

xpressed 3-fold higher in metastaticisease when compared with primaryarcinomas. Thus, our gene expressionrofiling results seem consistent with anctive role of TSP-1 and -2 during theetastatic process of ovarian cancer.MMP11, also known as Stromelysin

, is a protease that belongs to the ma-rix metalloproteinase family that isndowed with the unique feature to de-rade the serine protease inhibitorslpha1-antitripsin and insulin-likerowth factor binding protein-1.
MP11 was shown originally to be


2

TABLE 3Up-regulated genes expressed at least 2-fold higher in primary OSPC vs metastasisCode Symbol Gene name FC

219873_at COLEC11 Collectin subfamily member 11 7.69................................................................................................................................................................................................................................................................................................................................................................................

203824_at TSPAN8 Tetraspanin 8 4.55................................................................................................................................................................................................................................................................................................................................................................................

209242_at PEG3 Paternally expressed 3 4.17................................................................................................................................................................................................................................................................................................................................................................................

218804_at TMEM16A Transmembrane protein 16A 4.17................................................................................................................................................................................................................................................................................................................................................................................

209443_at SERPINA5 Serine (or cysteine) proteinase inhibitor, clade A (alpha-1antiproteinase, antitrypsin)

4

................................................................................................................................................................................................................................................................................................................................................................................

204450_x_at APOA1 Apolipoprotein A-I 3.85................................................................................................................................................................................................................................................................................................................................................................................

204934_s_at HPN Hepsin (transmembrane protease, serine 1) 3.85................................................................................................................................................................................................................................................................................................................................................................................

202992_at C7 Complement component 7 3.7................................................................................................................................................................................................................................................................................................................................................................................

202991_at STARD3 START domain containing 3 3.03................................................................................................................................................................................................................................................................................................................................................................................

204187_at GMPR Guanosine monophosphate reductase 3.03................................................................................................................................................................................................................................................................................................................................................................................

219568_x_at SOX18 SRY (sex determining region Y)-box 18 2.94................................................................................................................................................................................................................................................................................................................................................................................

221491_x_at HLA-DRB3 Major histocompatibility complex, class II, DR beta 3 2.94................................................................................................................................................................................................................................................................................................................................................................................

202350_s_at MATN2 Matrilin 2 2.86................................................................................................................................................................................................................................................................................................................................................................................

221874_at KIAA1324 NA 2.86................................................................................................................................................................................................................................................................................................................................................................................

204591_at CHL1 Cell adhesion molecule with homology to L1CAM (closehomolog of L1)

2.78

................................................................................................................................................................................................................................................................................................................................................................................

205862_at GREB1 NA 2.56................................................................................................................................................................................................................................................................................................................................................................................

210059_s_at MAPK13 Mitogen-activated protein kinase 13 2.56................................................................................................................................................................................................................................................................................................................................................................................

214079_at DHRS2 Dehydrogenase/reductase (SDR family) member 2 2.56................................................................................................................................................................................................................................................................................................................................................................................

208498_s_at AMY1A Amylase, alpha 1A 2.5................................................................................................................................................................................................................................................................................................................................................................................

204203_at CEBPG CCAAT/enhancer binding protein (C/EBP), gamma 2.38................................................................................................................................................................................................................................................................................................................................................................................

205485_at RYR1 Ryanodine receptor 1 (skeletal) 2.38................................................................................................................................................................................................................................................................................................................................................................................

205975_s_at HOXD1 Homeo box D1 2.38................................................................................................................................................................................................................................................................................................................................................................................

204205_at APOBEC3G Apolipoprotein B messenger RNA editing enzyme, catalyticpolypeptide-like 3G

2.27

................................................................................................................................................................................................................................................................................................................................................................................

209921_at SLC7A11 Solute carrier family 7, (cationic amino acid transporter,y� system) member 11

2.27

................................................................................................................................................................................................................................................................................................................................................................................

204529_s_at TOX NA 2.22................................................................................................................................................................................................................................................................................................................................................................................

201656_at ITGA6 Integrin, alpha 6 2.13................................................................................................................................................................................................................................................................................................................................................................................

202152_x_at USF2 Upstream transcription factor 2, c-fos interacting 2.13................................................................................................................................................................................................................................................................................................................................................................................

205166_at CAPN5 Calpain 5 2.13................................................................................................................................................................................................................................................................................................................................................................................

214868_at PIWIL1 Piwi-like 1 (Drosophila) 2.13................................................................................................................................................................................................................................................................................................................................................................................

203921_at CHST2 Carbohydrate (N-acetylglucosamine-6-O) sulfotransferase 2 2.08................................................................................................................................................................................................................................................................................................................................................................................

205258_at INHBB Inhibin, beta B (activin AB beta polypeptide) 2.08................................................................................................................................................................................................................................................................................................................................................................................

218417_s_at FLJ20489 NA 2.08................................................................................................................................................................................................................................................................................................................................................................................

218454_at FLJ22662 NA 2.08................................................................................................................................................................................................................................................................................................................................................................................

220051_at PRSS21 Protease, serine, 21 (testisin) 2.08................................................................................................................................................................................................................................................................................................................................................................................

201883_s_at B4GALT1 UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase,polypeptide 1

2

................................................................................................................................................................................................................................................................................................................................................................................

210010_s_at SLC25A1 Solute carrier family 25 (mitochondrial carrier) 2................................................................................................................................................................................................................................................................................................................................................................................

NA, not available.

45.e8 American Journal of Obstetrics & Gynecology MARCH 2007

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MARCH 2007 America

verexpressed in human breast cancernd, later on, in several other humanarcinomas that were endowed withggressive biologic behavior.19-21 Ouricroarray results that show highMP11 expression in metastatic

varian disease confirm previous ob-ervations in ovarian cancer and fur-her suggest an association between

MP11 overexpression and the bio-ogic aggressiveness of metastatic ovar-an disease.21

Chemokines are a superfamily of-10 kd proteins that play an impor-ant role in tumor progression that in-uences cell proliferation, angiogenic/ngiostatic processes, cell migrationnd metastasis, and regulating im-une cell recruitment into the tumorass. We found CXCL12 (stromal cell-

erived factor-1), a member of theXC chemokine family binding to theXCR4 receptor, to be expressedighly differentially in metastatic ovar-

an cancer, when compared with pri-ary ovarian disease. Although previ-

us studies have suggested a crucialole played by the CXCL12/CXCR4xis during the dissemination of ovar-an cancer in the peritoneal cavity,22 tour knowledge, no studies have yetemonstrated a differential expressionf CXCL12 in metastatic ovarian dis-ase, when compared with primaryvarian tumors. Our data, validated byT-PCR and at the protein level by im-unohistochemistry in all 31 tumor

amples that were included in thetudy, suggest that overexpression ofXCL12 in metastatic disease may rep-

esent a further crucial step for ovarianancer dissemination in the peritonealavity. Consistent with this view, re-ent evidence supports the notion thathigh concentration of CXCL12 in as-

ociation with vascular endothelialrowth factor efficiently and synergis-ically induces in vivo angiogenesis invarian cancer.23 These data, com-ined with observations on the role ofumor cell CXCL12 in attracting tu-

or-promoting predendritic cells intohe microenvironment of human ovar-an cancer,24 suggest that the CXCR4/XCL12 axis may provide important

FIGURE 2Comparison of microarray and qRT-PCR results for 5 selected genes

uantitative RT-PCR and microarray expression analysis of CXCL12, MMP11, PLAU, ITGB4, andPN genes differentially expressed between MET and OSPC. Quantitative RT-PCR data wereorrelated highly to the microarray data (P � .001).

aracrine and autocrine signals that, in


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ddition to facilitating abdominalpreading, may inhibit host immuneesponses, thus further promoting ma-ignant progression.

In conclusion, multiple markers thatre predictive of invasion and highetastatic behavior have been identi-

FIGURE 3Immunohistochemical staining for

epresentative immunohistochemical staining for1�) cytoplasmatic staining for CXCL12. Rightisplays a strong (3�) cytoplasmatic positivity

TABLE 4CXCL12 protein expression in 14 pmetastasis, MET

Patient

CXCL12immunohistochemistryscoring

MET 1 2�...................................................................................................................

MET 2 3�...................................................................................................................

MET 3 3�...................................................................................................................

MET 4 3�...................................................................................................................

MET 5 2�...................................................................................................................

MET 6 3�...................................................................................................................

MET 7 2�...................................................................................................................

MET 8 3�...................................................................................................................

MET 9 3�...................................................................................................................

MET 10 3�...................................................................................................................

MET 11 2�...................................................................................................................

MET 12 3�...................................................................................................................

MET 13 2�...................................................................................................................

MET 14 3�...................................................................................................................

MET 15 3�...................................................................................................................

MET 16 3�...................................................................................................................

MET 17 3�

45.e10 American Journal of Obstetrics & Gyneco

ed in this study. Although our resultsay be limited by a relatively small

ample size and must be validated byurther studies, the identification ofXCL12 among the most highly dif-

erentially expressed genes in meta-tatic ovarian carcinoma suggest that

CL12

CL12. Left panel, Primary OSPC shows a weaknel, Omental ovarian cancer metastasis (MET)CXCL12.

ary OSPC and 17 omental

Patient

CXCL12immunohistochemistryscoring

OSPC 2 3�..................................................................................................................

OSPC 3 1�..................................................................................................................

OSPC 4 3�..................................................................................................................

OSPC 6 2�..................................................................................................................

OSPC 7 3�..................................................................................................................

OSPC 8 1�..................................................................................................................

OSPC 9 3�..................................................................................................................

OSPC 10 2�..................................................................................................................

OSPC 12 2�..................................................................................................................

OSPC 13 1�..................................................................................................................

OSPC 14 2�..................................................................................................................

OSPC 17 2�..................................................................................................................

OSPC 18 2�..................................................................................................................

OSPC 19 3�..................................................................................................................

..................................................................................................................

..................................................................................................................

c

logy MARCH 2007

herapeutic strategies targeting theXCL12/CCR4 axis by monoclonalntibodies or by selective CXCR4 in-ibitors25 may represent a novel, po-entially effective therapy for the treat-

ent of metastatic ovarian disease.he future design and implementationf clinical trials in this regard ulti-ately will determine the validity of

hese approaches. f

CKNOWLEDGMENTSe thank Prof Fabio Facchetti, Prof Piergio-

anni Grigolato, Dott Marcella Falchetti, Mrsnna Galletti, and Mrs Lucia Fontana for theirxcellent technical support to the project.

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cs of ovarian cancer. Am J Med Genet002;115:157-63.. Le Page C, Provencher D, Maugard CM,uellet V, Mes-Masson AM. Signature of a si-

ent killer: expression profiling in epithelial ovar-an cancer. Exp Rev Mol Diagn 2004;4:157-67.. Ramaswamy S, Ross KN, Lander ES, GolubR. A molecular signature of metastasis in pri-ary solid tumors. Nat Genet 2003;33:49-54.. Weigelt B, Glas AM, Wessels LF, WitteveenT, Peterse JL, van’t Veer LJ. Gene expressionrofiles of primary breast tumors maintained inistant metastases. Proc Natl Acad Sci U S A003;100:15901-5.. D’Arrigo A, Belluco C, Ambrosi A, et al., Met-static transcriptional pattern revealed by genexpression profiling in primary colorectal carci-oma. Int J Cancer 2005;115:256-62.. Bignotti E, Tassi RA, Calza S, et al. Differen-ial gene expression profiles between tumor bi-psies and short-term primary cultures of ovar-

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CX

CXpafor

rim

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.........

.........

.........

.........

.........

.........

.........

.........

.........

.........

.........

.........

.........

ancer cells: characterization of structural ele-

mL1(mc1Heits21aie21fiat1

1Tpmfd1fitt21TpR1mi12Sc

2Wil22Br22Csm62erp72aar


ents required for growth factor function. FEBSett 1998;438:101-5.2. Rabbani SA, Xing RH. Role of urokinase

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nhibitor is mediated through inhibition of pro-ein kinase C- and MEK/ERK/c-Jun-dependentignaling pathways. J Biol Chem 2001;76:2015-22.4. Sato S, Kopitz C, Schmalix WA, et al. High-ffinity urokinase-derived cyclic peptides inhib-

ting urokinase/urokinase receptor-interaction:ffects on tumor growth and spread. FEBS Lett002;528:212-6.5. Volpert OV, Lawler J, Bouck NP. A humanbrosarcoma inhibits systemic angiogenesisnd the growth of experimental metastases viahrombospondin-1. Proc Natl Acad Sci U S A
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