Multiplexed Cytokine Profiling for Early Detection ofImmunobead-Based Ovarian Cancer

Elieser Gorelik, Douglas P. Landsittel, Adele M. Marrangoni, et al. Cancer Epidemiol Biomarkers Prev 2005;14:9 1!9 ".

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Cancer Epidemiology, Biomarkers & Prevention

Short Communication

Multiplexed Immuno ead!Based Cytokine Pro"iling "or Early #etection o" $varian Cancer
Elieser %orelik, #ouglas P( )andsittel, +dele M( Marrangoni, 3,' ' ' ,rancesmary Modugno, )yudmila -elikokhatnaya, Matthe. /( 0inans, ' 3,' 3,' 0illiam )( Big ee, 1onald B( 2er erman, and +nna E( )okshin
#epartments o" &Pathology and Immunology, *Biostatistics, and 3Epidemiology, 3#ivision o" 2ematology4$ncology, ' 5niversity o" Pitts urgh School o" Medicine and 5niversity o" Pitts urgh Cancer Institute, Pitts urgh, Pennsylvania

&,'

'

+ stract
Early detection o" ovarian cancer might improve clinical outcome( Some studies have sho.n the role o" cytokines as a ne. group o" tumor markers "or ovarian cancer( 0e hypothesi6ed that a panel comprised o" multiple cytokines, .hich individually may not sho. strong correlation .ith the disease, might provide higher diagnostic po.er( /o evaluate the diagnostic utility o" cytokine panel, .e used a novel multianalyte )a M+P pro"iling technology that allo.s simultaneous measurement o" multiple markers( Concen! trations o" *3 cytokines 7cytokines4chemokines, gro.th, and angiogenic "actors8 in com ination .ith cancer antigen!&*' 7C+!&*'8, .ere measured in sera o" 33 patients .ith early! stage ovarian cancer, 3' healthy .omen, and 39 patients .ith enign pelvic tumors( Six markers, i(e(, interleukin 7I)8!:, I)! ;, epidermal gro.th "actor 7E%,8, vascular endothelial gro.th "actor 7-E%,8, monocyte chemoattractant protein!& 7MCP!&8, and C+!&*', sho.ed signi"icant di""erences in serum concentrations et.een ovarian cancer and control groups( $ut o" this group, I)!:, I)!;, -E%,, E%,, and C+! &*', .ere used in a classi"ication tree analysis that resulted in ;3< sensitivity at ='< speci"icity( /he receiver operator characteristic curve created using the com ination o" markers produced sensitivities et.een =>< and &>>< in the area o" ;>< to =>< speci"icity, .hereas the receiver operator characteristic curve "or C+!&*' alone resulted in sensitivities o" 9>< to ;><( /he classi"ication tree analysis "or discrimination o" enign condition "rom ovarian cancer used C+!&*', granulocyte colony!stimulating "actor 7%!CS,8, I)!:, E%,, and -E%, resulting in ;:('< sensitivity and =3(>< speci"icity( /he presented data sho. that simulta! neous testing o" a panel o" serum cytokines and C+!&*' using )a M+P technology may present a promising ap! proach "or ovarian cancer detection( 7Cancer Epidemiol Biomarkers Prev *>>'?&3738@=;& A 98

Introduction
$varian cancer represents the third most "reBuent cancer o" the "emale genital tract( /he maCority o" early!stage ovarian cancers are asymptomatic, and over three!Buarters o" clinical diagnoses are made at a time .hen the disease has already esta lished regional or distant metastases( #espite aggressive cytoreductive surgery and platinum! ased chemotherapy, the '!year survival "or patients .ith clinically advanced ovarian cancer is only &'< to *><, in striking contrast to the cure rate "or stage I disease, .hich is usually D=>< 7&8( /hese statistics provide the primary rationale to improve ovarian cancer screening and early detection( #ue to the lo. prevalence o" spontaneous ovarian cancer in the general population, a screening strategy must have sensitivity o" at least ;>< in early!stage disease and near! per"ect speci"icity o" at least ==(:< 7*8( +t present, there are t.o screening tests "or ovarian cancer@ serologic screening "or tumor antigen using cancer antigen!&*' 7C+!&*'8, and imaging using transvaginal sonography 7*!:8( 2o.ever, .ith a cuto"" o" 3> to 3' units4m), serum C+!&*' has een sho.n to have a
,ax@ 3&*!:*3!99>3( E!mail@ lokshinaFupmc(edu Copyright # *>>' +merican +ssociation "or Cancer 1esearch(

1eceived :434>3? revised &&4**4>3? accepted &*4&>4>3( %rant support@ EI2 grant &1$& C+>=;:3*!>&+& and the #$# grant #+M#&9!>3!&!>:=: 7+(E( )okshin8, and EI2 grant 1$3 C+&>*;;; 7E( %orelik8( /he costs o" pu lication o" this article .ere de"rayed in part y the payment o" page charges( /his article must there"ore e here y marked advertisement in accordance .ith &; 5(S(C( Section &933 solely to indicate this "act( 1eBuests "or reprints@ +nna E( )okshin, 2illman Cancer Center, 5niversity o" Pitts urgh Cancer Institute, '&&9 Centre +venue, Pitts urgh, P+ &'*&3( Phone@ 3&*!:*3!99>:?

Cancer Epidemiol Biomarkers Prev *>>'?&3738( +pril *>>'

Cytokine Pro"iling in $varian Cancer #etection =3(=< speci"icity 7&*, &38( /here"ore, at present, proteomic pro"iling, .hereas promising, does not possess the reBuired diagnostic discrimination "or primary ovarian cancer screen! ing( +dditional approaches are necessary to provide the reBuired high level o" speci"icity and positivity "or an e""ective high!throughput screening "or ovarian cancer( #uring the last t.o decades, a large num er o" serologic tumor markers have een evaluated "or their a ility to detect early!stage epithelial ovarian cancer( Biomarkers that have a sho.n association .ith ovarian cancer include cancer antigens, di""erentiation markers, anti odies to mutated onco! genes, and cytokines 7revie.ed in re"( &38( Cytokines are a diverse group o" proteins comprised o" hematopoietic gro.th "actors, inter"erons, lymphokines, and chemokines 7&'8( Serum cytokines that possess diagnostic value in ovarian cancer include interleukin 7I)8!:, I)!;, macrophage colony!stimulating

sensitivity o" only '>< to :>< .ith the speci"icity o" D=;<, "or early!stage disease 73, 9, ;8( /ransvaginal sonography alone or com ined .ith #oppler and morphologic indices, are only sensitive and speci"ic "or esta lished tumors, and are, there"ore, not suita le "or early diagnostics o" ovarian cancer 7:, =8( 1ecently, a novel technology, sur"ace!enhanced laser desorption4ioni6ation time!o"!"light mass spectrometry has een o""ered "or early detection o" ovarian cancer 7&>8( /his technology .as reported to allo. "or discriminating serum protein pro"iles .ith &>>< sensitivity and &>>< speci"icity 7&&8( 2o.ever, in t.o other studies o" early detection o" ovarian cancer using sur"ace!enhanced laser desorption4ioni6ation time!o"!"light mass spectrometry, the results .ere less opti! mistic, demonstrating 9*(;< to ='(9< sensitivity and ;*(:< to

Cancer Epidemiol Biomarkers Prev *>>'?&3738( +pril *>>'

Cytokines are implicated in many aspects o" tumor gro.th 7revie.ed in re"( **8( /umor cells express and produce various angiogenic "actors, such as -E%,, ro last I)!:, gro.th "actor 7 ,%,8, platelet!derived gro.th asic "actor"i7P#%,8, I)! ; 7*3!*:8, and other cytokines, such as MCP!&, granulocyte CS, 7%!CS,8, M!CS,, tumor necrosis "actor!a 7/E,a8, I)!&a,

Patient group Control 7n G 3'8 Early!stage ovarian cancer 7n G

+ge, median 7range8 3: 73:!9:8 3: 733!;;8

2istologic types Papillary serous carcinoma Carcinoma, endometrioid 7n G &38 7n G &>8

"actor 7M!CS,8, MCP!&, tumor necrosis "actor receptor 7/E,18, and vascular endothelial gro.th "actor -E%, 7re"s( &:!*&8( /a le &( Patient characteristics

and I)!&h 7*3, *9!*=8( /umor!produced cytokines could ind to their receptors expressed y endothelial and hematopoietic4lymphoid cells and trigger production o" additional types o" cytokines 7**8( /his leads to the 7ascites8 as .ell as systemically 7in the lood8( Cytokine pro"iles could e cancer!speci"ic since malignant cells o" di""erent histologic types could produce di""erent patterns o" proangiogenic "actors, gro.th "actors, and chemokines( /hus, cytokine panels could serve as cancer iomarkers that can e used "or early diagnosis and assessment o" therapy response( Previous pu lications sho.ed that none o" these markers, used alone, is su""iciently diagnostic o" malignancy 7&3, 3>8( In some studies, com inations o" several markers have een evaluated "or early detection o" ovarian cancer using conventional E)IS+ assays( +nalysis o" the diagnostic po.er o" individual serologic markers in com ination .ith C+!&*' #ue to the limitations o" E)IS+ 7.hich is expensive, time! consuming, and each assay encompasses only one marker at a time8, none o" the tested marker com inations thus "ar .as su""iciently comprehensive and achieved the reBuired char! acteristics "or diagnosis o" ovarian cancer( /here"ore, "urther research is necessary to identi"y a multi iomarker panel allo.ing "or early detection o" ovarian cancer .ith reBuired high sensitivity and speci"icity( /o test this, .e have used a novel multianalyte )a M+P pro"iling technology 7)uminex Corp(, +ustin, /H8, that allo.s simultaneous measurement o" multiple iomarkers in serum or plasma o" ovarian cancer patients and control healthy .omen( In this study, a panel o" *3 serologic markers including cytokines, chemokines, gro.th and angiogenic "actors, and C+!&*' .as analy6ed in lood sera o" ovarian cancer patients 7stages I and II8, patients .ith enign pelvic disease, and control healthy .omen( $ur studies sho. that a panel o" cytokines in com ination .ith C+!&*' sho.ed increased speci"icity and sensitivity as compared .ith C+!&*' alone(
Benign tumors 7n G 398 33(' 7*;!;98

Carcinoma, mucinous 7n G 98 Carcinoma, poorly di""erentiated 7n G :8 +denocarcinoma, serous Carcinoma, clear cell 7n G 38 +deno"i roma, serous 7n G &8 Brenner tumor 7n G &8 Crystadeno"i roma, serous 7n G *8 Cyst, paratu al 7n G *8 Cyst, serous 7n G &8 Cyst, simple 7n G 38 Cystadeno"i roma, serous 7n G 38 Cystadenoma, mucinous 7n G ;8 Cystadenoma, serous 7n G =8 ,i rosis 7n G &8 $vary enign 7n G 38 Mucinous enign 7n G *8

su Cect or "rom his or her guardian( Sample collection .as done a"ter approval y the Institutional 1evie. Board and in accord .ith an assurance "iled .ith and approved y the 5(S( #epartment o" 2ealth and 2uman Services(

Patients and Methods

Patient Populations( /he serum samples "rom 33 patients diagnosed .ith early!stage 7I and II8 ovarian cancer, 39 patients .ith enign pelvic masses, and 3' healthy age! matched controls .ere tested( Serum samples "rom patients .ith early!stage 7I and II8 ovarian cancer, and .omen .ith enign pelvic disease, .ere provided y the %ynecologic $ncology %roup 7Cleveland, $28( Patients .ere enrolled y the %ynecologic $ncology %roup under their Institutional 1evie. Board protocols( In"ormation a out gynecologic diagnoses and ovarian cancer staging as .ell cancer histology and grade .as provided y the %ynecologic $ncology %roup( Eo data allo.ing identi"ication o" patients .ere provided( +ll maCor types o" epithelial ovarian cancer and a variety o" enign pelvic conditions .ere represented in these series 7/a le &8( Control serum samples "rom healthy, age!matched .omen .ere received "rom the +llegheny County Case!Control Eet.ork( 0ritten in"ormed consent .as o tained "rom each

Collection and Storage o" Blood Serum( /en microliters o" peripheral lood .as dra.n "rom su Cects using standardi6ed phle otomy procedures( 2andling and processing .as similar "or all three groups o" patients( Samples .ere o tained "rom patients diagnosed .ith ovarian cancer, prior to surgery, and e"ore administration o" anesthesia( Blood samples .ere collected .ithout anticoagulant into red top vacutainers and allo.ed to coagulate "or *> to 3> minutes at room temperature( Sera .ere separated y centri"ugation, and all specimens .ere immediately aliBuoted, "ro6en and stored in a dedicated ;>CC "ree6er( Eo more than t.o "ree6e!tha. cycles .ere allo.ed "or each sample( Multiplex +nalysis( /he )a M+P technology 7)uminex8 com ines the principle o" a sand.ich immunoassay .ith the "luorescent! ead! ased technology allo.ing individual and multiplex analysis o" up to &>> di""erent analytes in a single microtiter .ell 73>8( /he )a M+P serum assays .ere done in =:!.ell microplate "ormat according to the protocol y Biosource International 7Camarillo, C+8( + "ilter! ottom, =:! .ell microplate

7Millipore, Billerica, M+8 .as locked "or &> minutes .ith PBS4 ovine serum al umin( /o generate a standard curve, '!"old dilutions o" appropriate standards .ere prepared in serum diluent( Standards and patient sera .ere pipetted at '> +) per .ell in duplicate and mixed .ith '> +) o" the ead mixture( /he microplate .as incu ated "or & hour at room temperature on a microtiter shaker( 0ells .ere then .ashed thrice .ith .ashing u""er using a vacuum mani"old( Phycoerythrin 7PE8!conCugated secondary anti ody .as added to the appropriate .ells and the .ells .ere incu ated "or 3' minutes in the dark .ith constant shaking( 0ells .ere .ashed t.ice, assay u""er .as added to each .ell, and samples .ere analy6ed using the Bio!Plex suspension array system 7Bio!1ad )a oratories, 2ercules, C+8( +nalysis o" experimental data .as done using "ive!parametric!curve "itting( #evelopment o" )a M+P +ssays "or C+!&*'( +ssay "or C+!&*' .as developed in our la oratory according to the protocol y )uminex( +nti ody pair "or C+!&*' .as purchased "rom ,it6gerald Industries International

7Concord, M+8( #etection anti ody .ere iotinylated using the EI!)ink sul"o!E2S! iotinylation kit 7Pierce, 1ock"ord, I)8 according to the manu"acturerJs protocol( /he extent o" iotin incorporation .as determined using the 2+B+ assay 7Pierce8 and .as "ound to e *> mol o" iotin per mole o" protein "or all o" the iotinylation reactions( /he capture anti ody .as covalently coupled to individual spectrally addressed car oxylated polystyrene microspheres purchased "rom )uminex( Covalent coupling o" the capture anti odies to the microspheres .as done y "ollo.ing the procedures recom! mended y )uminex( In short, microsphere stock solutions .ere dispersed in a sonication ath 7Sonicor Instrument Corporation, CopiaBue, EK8 "or * minutes( +n aliBuot o" *(' &>: microspheres .as resuspended in microtiter tu es containing >(& mol4) sodium phosphate u""er 7p2 :(&8, to a "inal volume o" ;> +)( /his suspension .as sonicated until a homogeneous distri ution o" the microspheres .as visually o served( Solutions o" E !hydroxy!sul"osuccinimide and &!ethyl!3!73!dimethylaminopropyl8!car odiimide hydro! chloride 7Pierce8, oth at '> mg4m), .ere prepared in phosphate u""er, and &> +) o" each solution .as seBuentially added to sta ili6e the reaction and activate the microspheres( /his suspension .as incu ated "or &> minutes at room temperature and then resuspended in *'> +) o" PBS containing '> +g o" anti ody( /he mixture .as incu ated at room temperature overnight in the dark .ith continuous shaking( Microspheres .ere then incu ated .ith *'> +) o" PBS!>(>'< /.een *> "or 3 hours( +"ter aspiration, the eads .ere locked .ith & m) o" PBS!&< ovine serum al umin! >(&< sodium a6ide( /he microspheres .ere counted .ith a hemacytometer and stored at a "inal concentration o" &>: microspheres4m) in the dark at 3CC( /he coupling e""iciency o" monoclonal anti odies .as tested y staining *,>>> microspheres .ith phycoerythrin!conCugated goat anti!mouse Ig% 7B# Biosciences, San #iego, C+8( Microspheres .ere analy6ed y Bio!Plex system, and the mean "luorescence intensity o" D&',>>> .as accepted as an indicator o" su""icient coupling e""iciency( /he minimum detection levels "or C+!&*' .as L' international units4m)( Interassay varia ility, expressed as a coe""icient o" variation, .as calculated ased on the average "or &> patient samples and standards that .ere measured in "our separate assays( /he interassay varia ility .ithin the replicates presented as an average coe""icient o" variation .as in the range o" '(3< to =(&< 7data not sho.n8( Intraassay varia ility .as evaluated y testing Buadruplicates o" each standard and &> samples measured thrice( /he varia ilities o" these samples .ere et.een '(:< and =(:< 7data not sho.n8( C+!&*' assay .as "urther validated in comparison .ith standard clinical E)IS+ 7Centocor, Malvern, P+8 and has sho.n =3('< correlation( *3!plex assay "or I)!&h, I)!*, I)!3, I)!', I)!:, I)!;, I)!&>, I)! &*p3>, I)!&3, I)!&', I)!&9, I)!&;, /E,a, I,Eg, %M!CS,, E%,, -E%,, %!CS,, asic "i ro last gro.th "actor, hepatocyte gro.th "actor 72%,8, 1+E/ES, macrophage in"lammatory protein 7MIP8!&a, MIP!&h, and MCP!& purchased "rom Biosource International( Interassay varia ilities "or individual cytokines in *3!plex .ere in the range o" 3('< to =(;< and intraassay varia ilities .ere in the range o" 3(:< to &*(:< 7in"ormation provided y Biosource International8( Each assay .as validated against appropriate E)IS+ demonstrating ='< to ==< correlations 7in"ormation provided y Biosource International8( Statistical +nalysis o" #ata( #escriptive statistics and graphical displays 7i(e(, dot plots8 .ere prepared to sho. the distri ution o" each marker "or each disease state( /he 0ilcoxon rank!sum test .as used to evaluate the signi"icance o" di""er! ences in marker expression et.een each disease state( Spear! manJs 7nonparametric8 rank correlation .as also calculated to Buanti"y the relationships et.een each pair o" markers(

#iscrimination o" ovarian cancer status .as accomplished using classi"ication and regression trees 7C+1/? re"( 3&8 implemented through S!Plus statistical so"t.are 73*8( Classi"i! cation trees discriminate et.een outcome classes 7e(g(, cancer patients versus controls8 y "irst searching the range o" each potential predictor 7e(g(, a given cytokine8 and "inding the split that maximi6es the likelihood o" the given data set( 0ithin each resulting su set 7or node8, the algorithm again searches the range o" each varia le to choose the optimal split( /his process is continued until all o servations are per"ectly discriminated, or the sample si6e .ithin a given node is too small to divide "urther 7i(e(, n G ' or less8( $nly t.o o servations in the data set had missing values "or any o" the markers and .ere excluded "rom the analysis( /he "inal output o" the resulting classi"ication tree is a graphical display o" decision criteria "or each split and resulting predicted pro a ilities o" eing a case across the "inal splits 7i(e(, terminal nodes8( Several other methods 7logistic regression and neural net.orks8 .ere also implemented .ith similar, ut some.hat less optimal results 7results not sho.n8( /en!"old cross!validation 733, 338 .as implemented to assess classi"ication accuracy using independent data( Speci"ically, the data .ere randomly split into &> su sets o" eBual si6e 7or as eBual as possi le? n k G ;!= "or these data8( ,or each su set, a model .as "it to =>< o" the data outside that su set? the resulting model 7or tree8 .as then applied to &>< o" data .ithin the given su set( /he resulting estimate o" classi"ication accuracy there"ore uses separate su sets o" data "or model "itting and validation, and thus avoids resu stitution ias( /he resulting sensitivity and speci"icity are reported across a range o" decision rules 7i(e(, cut!points "or classi"ying a given predicted pro a ility as either a case or control8 to generate the receiver operator characteristic 71$C8 curve( Since cross! validation produces a potentially di""erent model "or each su set o" the data, ho.ever, the classi"ication tree produced using all o servations 7i(e(, .ithout cross!validation8 .as displayed "or purposes o" descri ing the optimal model( 0hen not other.ise stated, o servations .ith a predicted pro a ility D>(' are classi"ied as a case 7or as a enign condition "or the comparison o" enign versus controls8(

1esults
)a M+P!Based +nalysis o" Serum Concentrations o" Cytokines and C+!&*' in $varian Cancer Patients( Concentrations o" *3 di""erent serum markers elonging to di""erent "unctional groups .ere evaluated in a multiplexed assay using )a M+P technology, in serum samples o" patients "rom three clinical groups@ .omen .ith early stage 7I and II8 ovarian cancer, .omen .ith enign pelvic masses, and age! matched healthy controls 7/a le &8( Serum levels o" I)!*, I)!3, I)!', I)!&>, I)!&3, I)!&', I)!&9, I)!&;, /E,a, and I,Eg .ere undetecta le in either control or patientsJ sera( I)!&h, I)! &*p3>, MIP!&a, MIP!&h, 2%,, 1+E/ES, ,%,, and %M!CS, sho.ed measura le serum concentrations .hich did not di""er et.een the control and patient groups 7data not sho.n8( Serum concentrations o" I)!:, I)!;, C+!&*', and -E%, .ere "ound to e signi"icantly higher in ovarian cancer patients as compared .ith controls 7P L >(>' ! P L >(>>&? /a le *? ,ig( &8( )a M+P assays sho.ed relatively high serum concentrations o" E%, 7**3 , &* pg4m)8 and MCP!& 73;3 , *& pg4m)8 in the serum o" healthy .omen 7/a le *? ,ig( &8( 2o.ever, serum levels o" E%, and MCP!& .ere signi"icantly lo.er 7P L >(>' ! P L >(>>&8 in ovarian cancer patients as compared .ith controls 7/a le *? ,ig( &8( Serum o" patients .ith enign tumors had signi"icantly elevated levels o" -E%, 7P L >(>'8, %!CS, 7P L >(>&8, I)!: 7P L >(>>&8, and C+!&*' 7P L >(>&8 as compared .ith controls 7/a le *8( In addition, patients .ith enign tumors had

/a le *( )evels o" serum markers

+nalytes4patients E%, I)!: %!CS, I)!; -E%, C+!&*' MCP!& mean , SE median 7range8 mean , SE median 7range8 mean , SE median 7range8 mean , SE median 7range8 mean , SE median 7range8 mean , SE median 7range8 mean , SE median 7range8 2ealthy controls **3(; , &&(3: *3; 7*=(;!3>*(:8 ;(; , *('> > 7>!:3(&8 *&(; , ;(33 > 7>!*'9(:8 &>(* , &(:; : 7*(3!'&(38 =>(9 , &>('* :9 7&;!3>:8 &>(3 , *(*; :(> 7>!;98 33&(; , *&(33 3*:(; 7&3'('!:='(98 $varian cancer &&>(9 , &'(';MMM 93(= 7>!3=:(=8 :3(* , &*(9*MMM *3(; 7>!*;>(*8 ES 3=(* , &*(>3 > 7>!*=>(;8 *3(> , '(=;MM =(: 7*(>!&;>(:8 &'3(' , &=(='M &>: 7*;!''*8 &'3(9 , 33(>3MMM '&(> 7>!&3&*8 *&>(3 , *>('3MMM &9*(= 7&9(&!'>*(38 Benign =;(: , &*(3'MMM =3(= 7>!*9:(38 *;(> , =(3MMM 9(: 7>!*9'(38 99(3 , &3(>3MM > 7>!33=(&8 &*(3 , 3(&& 9(: 73(>!&*9(;8 *';(; , *:(>3M *&; 73;!::*8 '&(; , &3(*3MM &:(> 7>!39*8 &=:(3 , &:(>:MMM &9;(* 733(=!333(:8

E$/E@ Comparison o" ovarian cancer or enign patients .ith controls@ M, P L >(>'? MM, P L >(>&? MMM, P L >(>>&? ES, not signi"icant(

signi"icantly lo.er levels o" E%, and MCP!& as compared .ith controls 7P L >(>>& "or oth? /a le *8( 0hen cytokine levels .ere compared et.een cancer and enign groups, signi"icantly lo.er circulating concentrations o" I)!:, I)!;, and C+!&*' .ere o served in sera o" enign cases 7P L >(>' "or all? ,ig( &8( %!CS, concentration .as signi"icantly 7P L >(>'8 higher in the enign group as compared .ith the cancer group 7,ig( &8( Patients .ith enign pelvic disease did not di""er "rom patients .ith early!stage ovarian cancer .ith regard to o" concentrations o" E%,, -E%,, and MCP!& 7,ig( &8( Statistical +nalysis o" Serum Cytokines as $varian Cancer Biomarkers Comparison o" Early!Stage $varian Cancer versus 2ealthy Controls( /a le 3 illustrates classi"ication results using each individual cytokine to distinguish early!stage ovarian cancer "rom controls( 1esults sho. that the individual markers led to only moderately accurate prediction o" early!stage cancer( $nly C+!&*', E%,, and I)!: correctly classi"ied D;>< o" the test set su Cects 7/a le 38(

,igure *+ displays the classi"ication tree using C+1/ methodology "or discriminating controls "rom early!stage ovarian cancer( /he model in ,ig( *+ used all o servations in either group to "it the model 7as opposed to cross!validation, .hich is used "or su seBuent estimation o" classi"ication accuracy as explained in su seBuent paragraphs8( /he classi! "ication tree used "ive o" the eight markers, including C+!&*', E%,, -E%,, I)!:, and I)!;( /he num ers speci"ied "or each o"

the "inal groups 7i(e(, terminal nodes8 represent the pro a ility o" eing a case .ithin each su set( 1ates o" classi"ication accuracy 7in discriminating controls "rom early!stage cancer8 .ere then o tained using &>!"old cross!validation( ,igure *B displays the resulting 1$C curve( +s descri ed in Patients and Methods, the sensitivity and speci"icity depend on the cut!point 7i(e(, predicted pro a il! ity "rom the classi"ication tree8 used to classi"y each su Cect as either a case or control( 5sing the standard cut!point o" >(' 7i(e(, everyone .ith a predicted pro a ility D>(' is classi"ied as a cancer case8 gives &>>< sensitivity, ;:< speci"icity, and =3< correctly classi"ied( ,ixing the speci"icity at =&< still leads to a very high sensitivity, at =:< 7again .ith =3< correctly classi"ied8( +lternatively, a speci"icity o" ='(3< corresponds to a sensitivity o" ;3(&< 7and =>(>< correctly classi"ied8( /he total area under the 1$C curve .as near one, at >(=::( +dditionally, the 1$C curve .as created using only C+!&*' 7again ased on &>!"old cross!validation? ,ig( *C8( Comparing this curve to the com ination o" markers clearly sho.s a su stantial gain "rom using multiple markers to predict cancer status( Speci"ically, in the area o" ;>< to =>< speci"icity 7i(e(, et.een >(& and >(* on the x ! axis8, the "inal model 7using multiple markers8 produces sensitivities et.een =>< and &>><, .hereas C+!&*' only produces sensitivities in the area o" 9>< to ;><( Several models provided compara le high sensitivity and speci"icity "or early diagnosis o" ovarian cancer( /here"ore, the resulting com ination o" cytokines should not e vie.ed as a

,igure &( #istri ution o" serum levels o" markers in the three study groups( Serum levels o" cytokines and gro.th "actors in healthy controls, ovarian cancer patients at stages I and II and patients .ith enign gynecologic disease( Sera .ere collected "rom 3' patients .ith early!stage 7I and II8 ovarian cancer, 33 patients .ith enign pelvic masses, and "rom 39 age! and sex!matched healthy controls( Circulating concentrations o" cytokines and gro.th "actors .ere measured using )a M+P technology as descri ed in Patients and Methods( Measurements .ere done t.ice( 2ori6ontal lines, mean values? M, statistical signi"icance et.een controls and cancer patients or et.een patients .ith enign pelvic disease and patients .ith ovarian cancer? M, P L >(>'? MM, P L >(>&? MMM, P L >(>>&(

/a le 3( Predictive values "or single serum markers "or early!stage ovarian cancer
Cytokine C+!&*' I)!: E%, I)!; MCP -E%, %!CS, < Correctly classi"ied ;'(& ;'(& ;>(' 9=(3 9;(* 93(: 93(: Sensitivity ='(' ;3(& ;3(& ;;(: ;3(& 9=(' 9*(9 Speci"icity 93(3 ;:(> 9:(9 :=(; 9*(& :9(3 93(3

using conventional E)IS+ assays 73;, '&!'38( Increased levels o" cytokines in the lood o" cancer patients may e due to secretion y malignant or y normal stromal cells, i(e(, immune or endothelial cells( /o the est o" our kno.ledge, this study is the "irst to o serve reduced levels o" E%, in patients .ith ovarian cancer and enign pelvic disease( )o.er circulating levels o" MCP!& in ovarian cancer as compared .ith controls .ere previously reported y Penson et al( 7*>8( )o.er serum concentrations o" E%, .ere o served in patients .ith di""erentiated thyroid carcinoma 73'8 and reast cancer, ut not in patients .ith pancreatic, lung, and head and neck

uniBue su set o" markers( $ther models .ith the same num er o" cytokines 7data not sho.n8, o"ten led to very similar results( ,or instance, all o" the tested three!varia le models led to very similar classi"ication rates( /he large num er o" possi le com inations, and the computational demands o" iteratively partitioning the training and test sets, prevented an exhaustive search o" all possi le models( Comparison o" Controls and Early!Stage $varian Cancer versus Benign Conditions( /o assess the validity o" serum iomarker panel "or discrimination o" enign pelvic tumors "rom the other groups, separate classi"ication tree models .ere "it to predict 7a 8 enign conditions versus early!stage cancer, and 7 8 enign conditions versus controls( /he same &>!"old cross! validation procedure .as used to assess classi"ication accu! racy( ,or the comparison o" enign versus cancer, ;>(*< o" su Cects .ere correctly classi"ied, .ith a sensitivity o" ;3(&< and a speci"icity o" 9'(9<( /he classi"ication tree "or compar! ison o" enign versus cancer 7data not sho.n8 used "ive markers 7C+!&*', %!CS,, I)!:, E%,, and -E%,8( ,or the comparison o" enign versus controls, =>(>< o" su Cects .ere correctly classi"ied, .ith a sensitivity o" ;:('< and a speci"icity o" =3(><( /he classi"ication tree "or comparison o" enign versus controls 7data not sho.n8 used six o" the eight markers, including E%,, -E%,, %!CS,, C+!&*', I)!:, and I)!;( Correlation Bet.een Biomarkers( +nalysis o" correlations et.een individual markers using Spearman rank correlation method revealed that most o" the markers .ere relatively uncorrelated 7/a le 38( $nly MCP!& and E%, 7r G >(3'8 had a correlation D>(3? I)!: and I)!; 7r G >(338 .ere the only other markers having a correlation D>(3( /he maCority o" markers had a correlation o" >(&> or less 7in a solute value8, suggesting that marker com inations may provide complementary classi"ica! tion in"ormation(

#iscussion
0e used )a M+P technology "or analy6ing *3 cytokines and C+!&*' antigen in sera o" .omen .ith early!stage ovarian cancer in comparison .ith matched healthy controls and patients .ith enign pelvic tumors( /he sensitivity o" the )a M+P assay is compara le to E)IS+ and 1I+ 73>8( In "act, in our experiments, circulating levels o" all *' proteins in healthy .omen .ere very similar to those measured y E)IS+ or 1I+ and that reported in previously pu lished o servations 7*>, 3'!'>8( 0e have identi"ied six circulating proteins that sho.ed an association .ith ovarian cancer, i(e(, E%,, MCP!&, C+!&*', -E%,, I)!:, and I)!;( 0e o served t.o distinct patterns o" changes in the sera o" .omen .ith ovarian cancer@ the concentrations o" -E%,, I)!:, I)!;, and C+!&*' .ere higher in patients .ith ovarian cancer, .hereas decreased concen! trations o" E%, and MCP!& .ere "ound in ovarian cancer as compared .ith the healthy controls( /hese elevated levels o" -E%,, I)!:, I)!;, and C+!&*' in the circulation o" ovarian cancer patients have een previously reported "rom studies

,igure *( Classi"ication tree and 1$C discriminating early!stage ovarian cancer "rom healthy controls( +( Classi"ication tree@ rectangles, splitting nodes containing cytokine and cytokine cuto""( /he range o" data speci"ied at each split represents the su set o" data .hich is "urther su divided y ranches to the le"t( /he num ers speci"ied "or each o" the "inal groups 7i(e(, terminal nodes8 represent the pro a ility o" eing a case .ithin each su set( B( 1$C curve "or iomarker panel( 1esults "rom &>!"old cross!validation o" classi"ication tree analysis o" early! stage ovarian cancer versus healthy controls( C( 1$C curve "or C+!&*' alone( 1esults "rom &>! "old cross validation o" early!stage ovarian cancer versus healthy controls(

/a le 3( Spearman rank correlations

Marker E%, -!E%, MCP!& %!CS, C+!&*' I)!: I)!; I)!&* E%, &(>> >(&> >(3' >(>; >(*: >(>9 >(&' >(&* -!E%, MCP!& %!CS, C+!&*' I)!: &(>> >(>& >(>& >(>9 >(>* >(>; >(>3 I)!; I)!&*

&(>> >(>3 >(&: >(>: >(*& >(*3

&(>> >(>' >(&3 >(&> >(&3

&(>> >(** >(&> >(>*

&(>> >(33 &(>> >(>' >(>& &(>>

cancers or melanoma(: /here"ore, decreased circulating levels o" E%, may e speci"ic "or particular type7s8 o" cancer( $varian cancer cells express E%, receptor, and E%, is an autocrine gro.th "actor "or ovarian cells 7'3, ''8( 0e have o served the a sorption o" E%, and MCP!& y ovarian tumor cells in vitro incu ated .ith serum,: indicating that lo.er circulating levels o" these molecules in ovarian cancer patients might e due to the consumption y ovarian tumor expressing speci"ic receptors 7':, '98( +nalysis o" serum iomarkers in patients .ith enign pelvic masses revealed increased levels o" -E%, and %!CS, that could e explained y their proangiogenic e""ects and stimulation o" angiogenesis and "ormation o" a lood vessel net.ork that is essential "or supporting gro.th not only o" malignant ut also enign tumors( 2o.ever, in comparison .ith ovarian cancer serum, no signi"icant increase in I)!; concentrations in the serum o" patients .ith enign tumors .as o served( /he serum levels o" I)!: and C+!&*' .ere "ound to e elevated in patients .ith enign tumors ut not to the same extent as in patients .ith ovarian cancer( #ecreased concentrations o" E%, and MCP!& .ere "ound to e similar in the serum o" patients .ith enign masses and ovarian cancer( It is possi le that receptors "or E%, and MCP!& are also expressed y enign tumor cells( Expression o" E%,1 y normal epithelial cells has een reported 7''8( #iscrimination o" ovarian cancer "rom enign samples has presented a di""icult pro lem in past studies, in .hich a relatively high percentage o" "alse!positive classi"ication o" enign neoplasms has een o served 7&*, ';8( Statistical analysis sho.ed that although correlation o" each o" the a ove markers .ith ovarian cancer .as modest .hen evaluated alone, a com ined iomarker panel sho.ed very strong association .ith malignant disease, and can have potential utility "or early diagnosis o" ovarian cancer( Com inations o" several serum markers as measured y )a M+P technology provided a sensitivity o" ;3< at a speci"icity o" ='< in this sample set( #ue to the lo. prevalence o" spontaneous ovarian cancer in the general population, a screening strategy must have sensitivity o" at least ;>< in early!stage disease and near!per"ect speci"icity o" at least ==(:< 7*8( 0e should there"ore ideally evaluate the sensitivity o" a given model using a cuto"" that produces very high speci"icity( Sensitivities are reported here "or "ixed speci"icities as high as ='<( +lthough reporting the modelJs sensitivity at higher speci"icities 7e(g(, =;< or ==<8 .ould e pre"era le, such results could not e reached 7.ith the given model and the given data sets8 due to the small num er o" controls( 0e expect to improve on such results through the "uture collection o" larger data sets and an expanded panel o" ovarian!associated iomarkers( It should also e noted that the &>>< sensitivity and speci"icity results apply to samples o tained "rom ovarian cancer cases already clinically diag! nosed, along .ith healthy controls, and not "rom a prospective screening trial( /he results in a prospective

screening trial are likely to e lo.er than o tained .ith preoperative samples( /hese results, ho.ever, sho. a strong potential to accurately discriminate cancer status .ith only a moderate num er o" samples( ,or an estimate o" the optimal classi"ication tree, .e presented the model "it to the entire data set, .hich is su seBuently re"erred to as the overall model( It should e noted that the cross!validation procedure used here produ! ces a di""erent model "or each o" the &> training data sets( Each o" these &> classi"ication trees, ho.ever, .as either the same as, or su sets o" the overall model( Eone o" the &> models "it through the cross!validation procedure included any markers that .ere not in the overall model 7i(e(, MCP!& or %!CS,8( Seven o" the &> cross!validation models included "our o" the "ive markers in the overall model( +lthough some ias may result "rom this cross!validation procedure, as opposed to separate training and test sets, the latter approach is not "easi le unless one has very large sample si6es( 0ith the given sample si6es, separate training and test sets .ould lead to more unsta le estimates o" sensitivity and speci"icity, since each o servation can only e used "or training or prediction( ,or the given data, the &>!"old cross! validation approach represents a reasona le alternative to at least partially avoid classi"ication ias 7imposed .hen the same data are used "rom oth training and prediction8, and estimate classi"ication measures 7e(g(, sensitivity and speci! "icity8 .ith improved precision( 2o.ever, .hereas this approach su stantially reduces the pro lem o" classi"ication ias, it does not easily allo. ready calculation o" con"idence limits, .hich is a limitation o" the cross!classi"ication approach, compared .ith splitting the data into t.o independent training and test sets( /he predictive po.er o" com ined serologic markers "or early!stage ovarian cancer, as determined y )a M+P technology, is at least as good as that identi"ied in pu lished studies "or sur"ace!enhanced laser desorption4ioni6ation time!o"!"light mass spectrometry technology 7&*8( 2o.ever, )a M+P technology is less expensive and permits a high! throughput approach( ,urthermore, the "lexi ility o" )a ! M+P technology allo.s "or the addition o" ne. markers and there"ore "or the opportunity to incrementally increase the diagnostic po.er o" the com ined assay( /o the est o" our kno.ledge, the reported multiplexed cytokines4C+!&*' o""ers the highest predictive po.er, as compared .ith other pu lished assays using de"ined protein serologic markers 73, &3, 3>, 3*!3=, '=!:*8( In conclusion, .e sho. here that analysis o" multiple serum iomarkers using a novel )a M+P technology is a promising approach "or the development o" a diagnostic assay "or ovarian cancer( /he predictive po.er o" the cytokines4C+!&*' panel is still lo.er than that reBuired "or general population ovarian cancer screening as de"ined y Naco s 7*8( $ptimi6ation o" cytokine4C+!&*' panel y including additional markers .ith high association .ith ovarian cancer .ould likely increase the diagnostic po.er o" the assay( ,urther validation o" this assay in retrospective studies .ith a larger num er o" samples .ill allo. "or con"irming the clinical diagnostic utility o" )a M+P! ased assay(

+ckno.ledgments
0e thank #rs( ,rederick Moolten and Merryl Egorin "or critically reading the manuscript(

1e"erences
&( *( 2olschneider C2, Berek NS( $varian cancer@ epidemiology, iology, and prognostic "actors( Semin Surg $ncol *>>>?&=@3 A &>( Naco s I( $vervie.Oprogress in screening "or ovarian cancer( In@ Sharp ,, Berek N, Bast 1, editors( $varian cancer '( $x"ord@ Isis Medical Media? &==;(

$ur unpu lished o servations(

3( 3( '( :( 9( ;( =( &>( &&(

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2ensley M), Castiel M, 1o son ME( Screening "or ovarian cancer@ .hat .e kno., .hat .e need to kno.( $ncology 72untingt8 *>>>?&3@&:>& A 9? discussion &:>;@&:&3 A :( 5r an E, McIntosh M0, +ndersen M, Parlan BK( $varian cancer screening( 2ematol $ncol Clin Eorth +m *>>3 +ug?&9@=;= A &>>'? ix( Menon 5, Naco s I( Screening "or ovarian cancer( Best Pract 1es Clin $ stet %ynaecol *>>* +ug?&:@3:= A ;*( /ailor +, Bourne /2, Camp ell S, $kokon E, #e. /, Collins 0P( 1esults "rom an ultrasound! ased "amilial ovarian cancer screening clinic@ a &>!year o servational study( 5ltrasound $ stet %ynecol *>>3 +pr?*&@39; A ;'( Bast 1C Nr, Hu ,N, Ku K2, Barnhill S, Ihang I, Mills %B( C+ &*'@ the past and the "uture( Int N Biol Markers &==; $ct!#ec?&3@&9= A ;9( Naco s I, Bast 1C Nr( /he C+ &*' tumour!associated antigen@ a revie. o" the literature( 2um 1eprod &=;= Nan?3@& A &*( Cohen ), ,ishman #+( 5ltrasound and ovarian cancer( Cancer /reat 1es *>>*?&>9@&&= A 3*( Petricoin E,, +rdekani +M, 2itt B+, et al( 5se o" proteomic patterns in serum to identi"y ovarian cancer( )ancet *>>*?3'=@'9* A 9( Ihu 0, 0ang H, Ma K, 1ao M, %limm N, Povach NS( #etection o" cancer! speci"ic markers amid massive mass spectral data( Proc Eatl +cad Sci 5 S + *>>3 #ec =?&>>@&3::: A 9&( Po6ak P1, +mneus M0, Pusey SM, et al( Identi"ication o" iomarkers "or ovarian cancer using strong anion!exchange ProteinChips@ potential use in diagnosis and prognosis( Proc Eatl +cad Sci 5 S + *>>3 $ct &3?&>>@ &*333 A ;( -lahou +, Schorge N$, %regory B0, Coleman 1)( #iagnosis o" ovarian cancer using decision tree classi"ication o" mass spectral data( N Biomed Biotechnol *>>3?*>>3@3>; A &3( Bast 1C Nr( Status o" tumor markers in ovarian cancer screening( N Clin $ncol *>>3 May &'?*&@*>> A '( )iles 0C, -an -oorhis 0C( 1evie.@ nomenclature and iologic signi"icance o" cytokines involved in in"lammation and the host immune response( N In"ect #is &==' #ec?&9*@&'93 A ;>( Eash M+, ,errandina %, %ordinier M, )oercher +, ,reedman 1S( /he role o" cytokines in oth the normal and malignant ovary( Endocr 1elat Cancer &=== Mar?:@=3 A &>9( ,oti E, ,errandina %, Martucci 1, et al( I)!:, M!CS, and I+P cytokines in ovarian cancer@ simultaneous assessment o" serum levels( $ncology &=== $ct?'9@*&& A '( %adducci +, ,erdeghini M, Castellani C, et al( Serum levels o" tumor necrosis "actor 7/E,8, solu le receptors "or /E, 7''! and 9'!k#a s/E,r8, and solu le C#&3 7sC#&38 in epithelial ovarian cancer( %ynecol $ncol &==' +ug?';@&;3 A ;( %adducci +, ,erdeghini M, ,anucchi +, et al( Serum preoperative vascular endothelial gro.th "actor 7-E%,8 in epithelial ovarian cancer@ relationship .ith prognostic varia les and clinical outcome( +nticancer 1es &===?&=@&3>& A '( Penson 1/, Pronish P, #uan I, et al( Cytokines I)!&h, I)!*, I)!:, I)!;, MCP!&, %M!CS, and /E,a in patients .ith epithelial ovarian cancer and their relationship to treatment .ith paclitaxel( Int N %ynecol Cancer *>>>?&>@33 A 3&( Su6uki M, Po ayashi 2, $h.ada M, /erao /, Sato I( Macrophage colony! stimulating "actor as a marker "or malignant germ cell tumors o" the ovary( %ynecol $ncol &==; Nan?:;@3' A 9( Muller ), Pa.elec %( Cytokines and antitumor immunity( /echnol Cancer 1es /reat *>>3 Nun?*@&;3 A =3( Sonoda /, Po ayashi 2, Paku /, 2iraka.a /, Eakano 2( Expression o" angiogenesis "actors in monolayer culture, multicellular spheroid and in vivo transplanted tumor y human ovarian cancer cell lines( Cancer )ett *>>3 Nul &>?&=:@**= A 39( )idor KN, Hu ,N, Martine6!Ma6a $, et al( Constitutive production o" macrophage colony!stimulating "actor and interleukin!: y human ovarian sur"ace epithelial cells( Exp Cell 1es &==3 +ug?*>9@33* A =( #i Blasio +M, Carniti C, -igano P, -ignali M( Basic "i ro last gro.th "actor and ovarian cancer( N Steroid Biochem Mol Biol &=='?'3@39' A =( -ersnel M+, 2aar rink M, )angerak +0, et al( 2uman ovarian tumors o" epithelial origin express P#%, in vitro and in vivo ( Cancer %enet Cytogenet &==3?93@:> A 3( Eegus 1P, Stamp %0, 1el" M%, et al( /he detection and locali6ation o" monocyte chemoattractant protein!& 7MCP!&8 in human ovarian cancer( N Clin Invest &==' May?='@*3=& A :( Savarese /M, Mitchell P, McQuain C, et al( Coexpression o" granulocyte colony stimulating "actor and its receptor in primary ovarian carcinomas( Cancer )ett *>>& Nan &>?&:*@&>' A &'( %le6erman M, Ma6ot M, Maymon E, et al( /umor necrosis "actor!a and interleukin!: are di""erently expressed y "resh human cancerous ovarian tissue and primary cell lines( Eur Cytokine Eet. &==; Nun?=@&9& A =( Berek NS, Bast 1C Nr( $varian cancer screening( /he use o" serial complementary tumor markers to improve sensitivity and speci"icity "or early detection( Cancer &==' Eov &'?9:@*>=* A :( 0oolas 1P, Cona.ay M1, Hu ,, et al( Com inations o" multiple serum markers are superior to individual assays "or discriminating malignant "rom enign pelvic masses( %ynecol $ncol &=='?'=@&&& A :( %adducci +, ,erdeghini M, 1ispoli %, Prontera C, Bianchi 1, ,ioretti P( Comparison o" tumor!associated trypsin inhi itor 7/+/I8 .ith C+&*' as a marker "or diagnosis and monitoring o" epithelial ovarian cancer( Scand N Clin )a Invest Suppl &==&?*>9@&= A *3(

33( Inoue M, ,uCita M, Eaka6a.a +, $ga.a 2, /ani6a.a $( Sialyl!/n, sialyl! )e.is Hi, C+ &=!=, C+ &*', carcinoem ryonic antigen, and tissue polypeptide antigen in di""erentiating ovarian cancer "rom enign tumors( $ stet %ynecol &==* Mar?9=@333 A 3>( 33( Mills %B, Bast 1C Nr, Srivastava S( ,uture "or ovarian cancer screening@ novel markers "rom emerging technologies o" transcriptional pro"iling and proteomics( N Eatl Cancer Inst *>>& $ct 3?=3@&339 A =( 3'( Pan6a E, Pacilio %, Campanella ), et al( Cancer antigen &*', tissue polypeptide antigen, carcinoem ryonic antigen, and h!chain human chorionic gonadotropin as serum markers o" epithelial ovarian carcinoma( Cancer &=;; Nan &?:&@9: A ;3( 3:( Peters!Engl C, Medl M, $gris E, )eodolter S( /umor!associated trypsin inhi itor 7/+/I8 and cancer antigen &*' 7C+&*'8 in patients .ith epithelial ovarian cancer( +nticancer 1es &==' Eov A #ec?&'@*9*9 A 3>( 39( Schutter EM, #avelaar EM, van Pamp %N, -erstraeten 1+, Penemans P, -erheiCen 12( /he di""erential diagnostic potential o" a panel o" tumor markers 7C+ &*', C+ &'!3, and C+ 9*!3 antigens8 in patients .ith a pelvic mass( +m N $ stet %ynecol *>>* +ug?&;9@3;' A =*( 3;( /holander B, /au e +, )indgren +, et al( Pretreatment serum levels o" C+! &*', carcinoem ryonic antigen, tissue polypeptide antigen, and placental alkaline phosphatase, in patients .ith ovarian carcinoma, orderline tumors, or enign adnexal masses@ relevance "or di""erential diagnosis( %ynecol $ncol &==> $ct?3=@&: A *'( 3=( 2ogdall E-, 2ogdall CP, /ingulstad S, et al( Predictive values o" serum tumour markers tetranectin, $-H&, C+S+ and C+&*' in patients .ith a pelvic mass( Int N Cancer *>>> Eov *>?;=@'&= A *3( 3>( -ignali #+( Multiplexed particle! ased "lo. cytometric assays( N Immunol Methods *>>> Sep *&?*33@*33 A ''( 3&( Brieman ),N, $lshen 1+, Stone CN( Classi"ication and regression trees( Monterey@ 0ads.orth and Brooks4Cole? &=;3( 3*( -ena les 0, 1ipley B#( Modern applied statistics .ith S!Plus( Ee. Kork@ Springer!-erlag? &==9( 33( E"ron B, /i shirani 1, Storey N, /usher -( Empirical Bayes analysis o" a microarray experiment( N +m Stat +ssoc *>>&?=:@&&'& A :>( 33( /i shirani 1, E"ron B( Pre!validation and in"erence in microarrays( Stat +ppl %enet Mol Biol *>>*?&( 3'( Eedvidkova N, Eemec N, Stol a P, -avreCnova -, Bednar N( Epidermal gro.th "actor 7E%,8 in serum o" patients .ith di""erentiated carcinoma o" thyroids( Eeoplasma &==*?3=@& A *>( 3:( )ev!1an +, 2.ang #), +hmad B, Bix y 2( Immunoreactive epidermal gro.th "actor in serum, plasma, platelets, and urine in patients on chronic dialysis( Eephron &==&?'9@&:3 A :( 39( 2e"ler ), /emp"er C, 2ein6e %, et al( Monocyte chemoattractant protein!& serum levels in ovarian cancer patients( Br N Cancer &=== Eov?;&@;'' A =( 3;( Mayerho"er P, Bodner P, Bodner!+dler B, et al( Interleukin!; serum level shi"t in patients .ith ovarian carcinoma undergoing paclitaxel!containing chemotherapy( Cancer *>>&?=&@3;; A =3( 3=( Matsu ara P, $chi 2, Pitaga.a 2, Kamanaka P, Pusanagi K, Ito M( Concentrations o" serum granulocyte!colony!stimulating "actor in normal pregnancy and preeclampsia( 2ypertens Pregnancy &===?&;@=' A &>:( '>( Bux N, 2o"mann C, 0elte P( Serum %!CS, levels are not increased in patients .ith anti ody!induced neutropenia unless they are su""ering "rom in"ectious diseases( Br N 2aematol &=== Nun?&>'@:&: A 9( '&( /emp"er C, Ieisler 2, Sliut6 %, 2aeusler %, 2an6al E, Pain6 C( Serum evaluation o" interleukin : in ovarian cancer patients( %ynecol $ncol &==9 Nul?::@*9 A 3>( '*( Maccio +, )ai P, Santona MC, Pagliara ), Melis %B, Mantovani %( 2igh serum levels o" solu le I)!* receptor, cytokines, and C reactive protein correlate .ith impairment o" / cell response in patients .ith advanced epithelial ovarian cancer( %ynecol $ncol &==; Nun?:=@*3; A '*( '3( Bahner #, Plucke C, Pit6e B, et al( Inter"eron!h!& increases serum interleukin!&* p3> levels in primary progressive multiple sclerosis patients( Eeurosci )ett *>>* Nun *;?3*:@&*' A ;( '3( Baron +/, )a"ky NM, Boardman C2, et al( Serum sEr B& and epidermal gro.th "actor levels as tumor iomarkers in .omen .ith stage III or Iepithelial ovarian cancer( Cancer Epidemiol Biomarkers Prev &===?;@&*= A 39( ''( Maihle EN, Baron +/, Barrette B+, et al( E%,4Er B receptor "amily in ovarian cancer( Cancer /reat 1es *>>*?&>9@*39 A ';( ':( Pack S#, +lper $M, Strom erg P, et al( Simultaneous suppression o" epidermal gro.th "actor receptor and c!er B!* reverses aneuploidy and malignant phenotype o" a human ovarian carcinoma cell line( Cancer 1es *>>3 ,e &?:3@9;= A =3( '9( %orelik E, Ed.ards 1P, ,eng H, et al( I)!&* receptor!mediated up!regulation o" ,as) in human ovarian carcinoma cells( Int N Cancer *>>3 Eov *>?&&*@:*> A 9( ';( Sedlac6ek P, ,rydecka I, %a rys M, -an #alen +, Einarsson 1, 2arlo6inska +( Comparative analysis o" C+&*', tissue polypeptide speci"ic antigen, and solu le interleukin!* receptor a levels in sera, cyst, and ascitic "luids "rom patients .ith ovarian carcinoma( Cancer *>>* Eov &?='@&;;: A =3( '=( Pim N2, Skates SN, 5ede /, et al( $steopontin as a potential diagnostic iomarker "or ovarian cancer( N+M+ *>>* +pr 3?*;9@&:9& A =( :>( Mok SC, Chao N, Skates S, et al( Prostasin, a potential serum marker "or ovarian cancer@ identi"ication through microarray technology( N Eatl Cancer Inst *>>& $ct 3?=3@&3'; A :3( :&( 1osenthal +, Naco s I( $varian cancer screening( Semin $ncol &==;?*'@3&' A *'( :*( van 2aa"ten!#ay C, Shen K, Hu ,, et al( $-H&, macrophage!colony stimulating "actor, and C+!&*'!II as tumor markers "or epithelial ovarian carcinoma@ a critical appraisal( Cancer *>>& #ec &?=*@*;39 A 33(