Evaluation of lysophosphatidic acid in vaginal ﬂ uid as a biomarker for ovarian cancer: A pilot study

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Evaluation of lysophosphatidic acid in vaginal ﬂ uid as a biomarker for ovarian cancer: A pilot study

Evelyn Minis, Kevin Holcomb, Giovanni Sisti, Dimitrios Nasioudis, Tomi T. Kanninen, Aikaterini Athanasiou, Melissa K. Frey, Eloise Chapman-Davis, Thomas A. Caputo, Steven S. Witkin*

DepartmentofObstetricsandGynecology,WeillCornellMedicine,NewYork,NewYork,UnitedStates

ARTICLE INFO Articlehistory:

Received6January2019

Receivedinrevisedform11March2019 Accepted13March2019

Availableonline18March2019 Keywords:

Adnexalmass CA125

Endometrioidovariancancer Lysophosphatidicacid

ABSTRACT

Thereremainsaneedtodifferentiatebetweenwomenwithabenignoramalignantadnexalmasspriorto surgery.Aspartofanongoingevaluationofvaginalﬂuidcompoundsaspotentialtumorbiomarkerswe evaluated whether vaginal lysophosphatidic acid (LPA) predicted the subsequent diagnosis of a malignant adnexalmass.Inthis prospectivepilotstudyvaginalﬂuidwasobtained from100post- menopausalwomenreferredforevaluationofasuspiciousadnexalmassandtestedforLPAbyELISA.

ClinicaldataandserumCA125resultswereobtainedonlyaftercompletionofalllaboratorytesting.

Twentyeightof thewomenweresubsequentlydiagnosedwithanovarianmalignancy,four hada borderlinetumorand68hadabenigndiagnosis.Amongwomenwithamalignantovarianmass,11 (39.3%)hadanendometrioidadenocarcinoma+/ Clearcelltumorcomponents,6(21.4%)hadahigh grade serous carcinoma, 3 (10.7%)had amucinous tumor, 2 each (7.1%) had a malignant mixed mesodermal ora granulosa tumorand 1 each (3.6%)had aClear celltumor, amixed cell tumor, leimyosarcomaormetastaticadrenaltumor.ComparedtothemedianvaginalLPAlevelinwomenwith benignlesions(1.5m^M),^LPA^wassigniﬁcantlyelevatedonlyinwomenwithendometrioidovariancancer (7.9m^M)^(p⁼^0.0137).^Of^the⁶endometrioidtumorsinwhichvaluesforbothplasmaCA125andvaginal LPAwereavailable5werepositiveonlyforLPAwhileonewasonlyCA125positive.DetectionofLPAin vaginalsecretionsmaybeofvalueforthenoninvasivediagnosisofendometrioidovarianmalignanciesin post-menopausalwomen.

Introduction

Determination of whether a suspicious adnexal mass is an ovariancanceriscurrentlymadeonlyfollowinginvasivesurgery andpathologicalanalysisofexcisedtissue.Sinceonlyaminorityof thesewomenwillinfacthaveamalignancy[1],thediscoveryofa biomarkertononinvasivelydeterminewhetherornotanadnexal mass is malignant could greatly reduce the incidence of unnecessarysurgery.

Currently,serumCA125isthemostcommonlyusedbiomarker todetectovariancancer.However,about25%ofwomenwithstage 1ovariancancerwillhaveanormalCA125andmultiplebenign conditionsmaycauseCA125elevations [2]. To date,CA125has been shown to be most useful in predicting the response to

treatment and recurrence rather than detection of early stage malignancy[3].

Recentﬁndingsindicatethatsomeovariancancersoriginateas premalignantintraepitheliallesionsinthedistalfallopiantubeand that transformed cells subsequentlymigrate totheovary [4,5].

Womenwhohaveundergoneatuballigationhaveadecreasedrisk ofdevelopingovariancancer,especiallyendometrioidandserous types [6]. Thus, in women with ovarian cancer compounds associatedwithdevelopmentofthismalignancymaybepresent intheuppergenitaltract,migratetothelowergenitaltractand accumulateinthevagina.

Ascarcityofstudieshaveevaluatedvaginalﬂuidasapotential sourceofcancer-associatedbiomarkers.Aninvestigationin1997 determinedthatCA125aswellascarcinoembryonicantigen(CEA) and squamous cellcarcinoma (SCC)antigenweredetectable in vaginal ﬂuid and that the levels of all three compounds were highest in 20 women with benign gynecological diseases as comparedto10withsquamouscellcarcinoma,5withendometrial cancer,3withvulvacarcinoma,8healthypre-menopausaland7

* Correspondingauthor.

E-mailaddress:[email protected](S.S.Witkin).

http://dx.doi.org/10.1016/j.eurox.2019.100012

EuropeanJournalofObstetrics&GynecologyandReproductiveBiology:X2(2019)100012

ContentslistsavailableatScienceDirect

European Journal of Obstetrics & Gynecology and Reproductive Biology: X

j o u r n al h o m e p a g e : w w w . el s e v i e r . c o m / l o c a t e / e u r o x

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healthypost-menopausalwomen[7].Alaterstudyreportedthat thelevelofCA125inthevaginaandcervixwashighestinwomen with endometrial hyperplasia and endometrial cancer [8]. A comparativeevaluationofthreetumorsbiomarkers–CA125,CEA andCA19-9– invaginalwashsamplesfrom30womenin each group witheither advanced primary ovarian canceror benign ovariancystsrevealedhighlyelevatedlevelsofall3compoundsin themalignant group[9]. Inthemostrecentstudywomenwith precancerousendometrialintraepithelialneoplasiaorendometrial adenocarcinomahadhigherandsimilarvaginalconcentrationsof CA125than didwomenwith non-endometrioma ovariancysts, endometrialhyperplasiaordysfunctionaluterinebleeding[10].

Lysophosphatidic acid (LPA, 1-acyl-2hydroxy-sn-glycero-3- phosphate) is a phospholipid produced by malignantly trans- formedovarianepithelialcells.Ithasbeenshowntostimulatecell proliferationandinvasion,increaseexpressionoffactorsinvolved inangiogenesis andlevelsareelevatedinseraand ascitesfluid fromwomenwithovariancancer[11–14].LPAhasalsobeenshown to inhibit autophagy in a prostate tumor cell line [15]. We previously reported that vaginal fluid from women with a malignantadnexalmassinhibitedautophagyinperipheralblood mononuclearcellstoagreaterextentthandidvaginalfluidfrom womenwithabenignmass[16].

As a component of our ongoing investigation of possible predictivebiomarkersofamalignantadnexalmassinvaginalﬂuid, andbasedonpriorassociationsbetweencirculatingLPAlevelsand ovariancancer,weevaluatedwhethertheLPAconcentrationin vaginalﬂuidwouldbeelevatedinwomensubsequentlydiagnosed withamalignantadnexalmass.

Materialandmethods

Subjects The subjects in this prospective study were 100 womenreferredtotheDivisionofGynecologicOncologyatWeill CornellMedicineforevaluationofasuspiciousadnexalmass.All subjects were referred for evaluation due to a pelvic mass of presumed gynecologic origin noted on pelvic sonography or magneticresonanceimaging.Inclusioncriteriawerepost-meno- pausalstatus,theabilitytocollectvaginalsamplesthatcouldbe senttothelaboratoryforprocessingwithina3htimeperiodand ability to provide written informed consent. Exclusion criteria were women with prior hysterectomy, history of malignancy, presenceofvaginalbleeding,signsorsymptomsofinfectionorno plan for undergoing surgical evaluation of theirmass at Weill Cornell. Subsequent to vaginal sample collection all women underwent surgical exploration by laparoscopy or laparotomy andsurgicalresectionoftheadnexalmass,asdescribedpreviously [16].Brieﬂy,theadnexalmasseswereresectedandintraoperative pathologyconsultationwasutilized.Whenaninvasivemalignancy waspresent, furthersurgicalstagingprocedures includingtotal hysterectomy,bilateral salpingo-oopherectomy,pelvicand para- arorticlymphnodesamplingandoomentectomywereperformed when clinically indicated. The ﬁnal pathological diagnosis was determined by an attending gynecologic pathologist utilizing techniquesandtissuesamplesstandardatourhospital.Thestudy wasapprovedbytheInstitutionalReviewBoardofWeillCornell Medicineandallsubjectssignedinformedwrittenconsent.

SamplesVaginalﬂuidwasobtainedduringaspeculum-based examination by scraping a cotton swab against the posterior vaginalwallandshakingthecontentsintoatubecontainingoneml sterilephosphate-bufferedsaline.Thetubewascentrifugedand thesupernatantstoredinaliquotsat 80Cuntilassayed.Samples withvisiblebloodcontaminationwerediscarded.

AssaysThawedaliquotsofvaginalsecretionswereassayedfor LPAbyacommercialELISAkit(EchelonBiosciences,SaltLakeCity, Utah).LPAwasdetectedwithamonoclonalantibodythatdidnot

exhibitreactivitywithLPAanalogs.Valueswereconvertedto

m

^M

byreferencetoastandardcurvegeneratedinparalleltoeachassay.

Thelowerlimitofsensitivitywas0.064

m

^M.^Intra-andinter-assay variability was <10%. Peripheral blood serum was assayed for CA125 in the Weill Cornell clinical laboratory by a standard chemiluminescentimmunoassay.Thelaboratorystaffwasblinded toallclinicaldataincludingCA125resultsthatwereobtainedby chartreviewonlyaftercompletionofthelabanalyses.

StatisticsDifferencesinLPAandCA125levelsbetweenwomen with a benign or malignant mass were analyzed by the non- parametric Mann-Whitneytest since values werenot normally distributed.AserumCA125concentrationof35U/mlistypically usedasthecutoffformalignancy[3],andweutilizedthisvaluein our study. An LPA concentration >7.25

m

^M ^was operationally designatedasthecutofffordetectionofamalignantlesion,based ontheobservationthat>90%ofwomenwithabenigndiagnosis had a vaginal LPA level below this value.Differences between positivevaluesforCA125andLPAforeachtypeofmalignancywere assessed byFisher’sexact test. Atwo sidedp value <0.05 was consideredsigniﬁcant.GRAPHPADINSTAT(GraphPadSoftware, SanDiego,CA)wasutilizedfortheanalysis.

Results

CharacteristicsofthestudypopulationaredetailedinTable1.A malignantadnexalmasswasdetected in28women,fourhada borderlinetumorwhile 68had a benign lesion. Womenin the threegroupswereofasimilarmeanage,hadacomparablemean ageatmenarcheand menopauseand wereofsimilargravidity, parityandbodymassindex.Themajorityofwomenineachgroup were White. Analysis of these multiplevariables negated their possibleconfoundinginﬂuenceonvaginalLPAlevels.Therewasno difference in the use of hormone replacement therapy (HRT) betweengroups.ThemedianCA125levelwas52.2U/mlinwomen withamalignantmass,14.6U/mlinthosewithaborderlinetumor and 11.0 U/ml in those whose mass was benign (p<0.0001 malignantvs.theothertwogroups).

Amongwomenwithamalignantovarianmass,11(39.3%)had endometrioid ovarian cancer with or without Clear cell tumor components,six(21.4%)hadhighgradeserouscancer,three(10.7%) had a mucinoustumor,twoeach (7.1%)had a malignantmixed mesodermaloragranulosatumorandoneoffourwomen(3.6%)had aClearcelltumor,amixedcelltumor,leimyosarcomaormetastatic adrenaltumor.Themajorityofwomenwithabenigndiagnosishad eitheracystadenoma(44.1%)oranothertypeofcyst(33.8%).

TheconcentrationofLPAinvaginalﬂuidfromwomenwitheachof thedifferentovarianmalignanciesaswellastheindividualbenign conditionsisshowninTable2.Individualvaluesforwomenwitha malignantorbenignmassarepresentedinFig.1.Themedianvaginal LPAconcentrationwashighestinthesixwomenwithserousovarian

Table1

Characteristicsofpost-menopausalwomenevaluatedforanadnexalmass.

Characteristic Benign Borderline Malignant

N=68 N=4 N=28

Meanageinyears(SD) 63.2(9.4) 63.0(11.7) 66.2(10.2)

UsingHRT 12(18.2%) 0 9(26.5%)

Meanageatmenarche(SD) 12.5(1.2) 13.0(0) 13.3(1.9) Meanageatmenopause(SD) 49.1(5.8) 47.5(3.3) 50.7(4.1) Mediangravida(range) 2(0–10) 3(0–4) 2(0–9) Medianparity(range) 2(0–9) 2(0–3) 2(0–5) Meanbodymassindex(SD]^b 26.4(5.8) 27.1(5.2) 25.3(5.0)

PercentWhiterace 70.7% 75.0% 85.2%

MedianCA125(range) 11.0(2.8–75) 14.6(9.3–117) 52.2(5.7–7778)^a HRT,hormonereplacementtherapy.

a p<0.0001vs.benignandborderline.

b kg/m².

2 E.Minisetal./EuropeanJournalofObstetrics&GynecologyandReproductiveBiology:X2(2019)100012

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cancer(9.5

m

^M),^one^woman^with^a^granulosa^tumor^(9.1

m

^M)^and

elevenwithanendometrioidmalignancy(7.9

m

^M).^However,^most

likelyduetothelownumberofsubjectsevaluatedonlyin women with anendometrioidovarianmalignancywastheLPAlevelsigniﬁcantly elevated(p=0.0137)comparedtothemedianlevelinwomenwith benigndiagnoses(1.5

m

^M).^None^of^the^{women with}^a^malignant^mass

and 6womenwith a benign mass had an undetectable LPAlevelintheir

vaginalsample.Onlyfourwomenwithbenignconditions–twowitha serous cystadenoma and one each witha mucinous cystadenoma and a mesothelialcyst-hadavaginalLPAconcentration>7.25

m

^M.^HRT

administrationhadnoeffectonthevaginalLPAlevel(datanotshown).

ValuesforserumCA125wereavailablefor23ofthe28women withamalignantadnexalmass.TherelativeabilityofvaginalLPA and serum CA125 to detect the various adnexal mass-related malignanciesisshowninTable3.Ofsixwomenwithendometrioid ovariancancerﬁve(83.3%)werepositiveonlyforvaginalLPAwhile one(16.6%)wasonlyCA125positive.Nonewerepositiveforboth LPAandCA125.Ofsixwomenwithaserouscarcinomaﬁvewere CA125positive(threeonlyCA125positiveandtwoCA125andLPA positive)andthreewereLPApositive(oneLPAonlyandtwoLPA andCA125positive).ThemedianCA125levelwashigherinwomen with a serous carcinoma (71 U/ml) than in those with endometrioid cancer(20 U/ml). Among theothermalignancies onlyone,awomanwithagranulosatumor,wasLPApositiveand CA125negative.

Discussion

LPA was detected in vaginal fluid from all women with a malignantadnexalmassaswellasin91%ofthosewhosemasswas benign. The vaginal LPA concentration only in women with endometrioid ovarian cancerwas significantly higher than the level present invaginal fluidfromwomenwithvarious benign diagnoses. Furthermore, LPA predicted the detection of an endometrioid ovarian cancer in 83.3% of women with this diagnosiswhileCA125waspositiveinonly16.6%ofthesewomen.

In contrast,thevaginal LPAassaywas inferiortoserum CA125 determinationfordetectionofserousovariancarcinoma.Serum CA125andvaginalLPAwerepositivein83%and50%ofwomen withthisdiagnosis,respectively.Amongwomenwithamalignant adnexal mass the serum concentrationof CA125was lower in those with an endometrioid ovariantumor than in those with serousovariancancer.ThisfurthersuggeststhatCA125issuperior fordetectionofserouscarcinomathanforendmetrioidcovarian cancer.Thetentativeconclusionfromtheseobservationsis that determinationofthevaginalLPAconcentrationmightbeamore sensitivemeasurementthanserumCA125tononinvasivelydetect the presence of endometrioid ovarian cancer, but not serous carcinoma,inwomenwithasuspiciousadnexalmass.

Endometrioidovariancancerisclassifiedasatype1lowgrade malignancy.Ithasbeenestimatedtorepresentabout10%ofovarian cancers and is often associated with prior endometriosis.Prognosisis usually favorable,especiallyifconfinedtotheovary[17,18]. Two studieshavereportedthatvaginallevelsofCA125werehigherin womenwith endometrial adenocarcinoma than inwomen with benignovariancysts,endometrialhyperplasiaoruterinebleeding [7,8].Inaddition,theincidenceofendometriodovariancanceris loweredinwomenwhohaveundergoneatuballigation[6].These observationssuggesttheinvolvementofgenitaltractcomponentsin developmentof thismalignancy.ThefindingthatvaginalLPAis higher in women with this diagnosis is consistent with this suggestion.ElevatedlevelsofLPAinthevaginamightbeduetoits transduction from the systemic circulation and/or by its local productionwithinthegenitaltractandsubsequentmigrationand collectioninthevagina.TheassociationofLPAwithonlyonetypeof ovarian malignancy, endometrioid cancer, argues against transductionbeingamajorcontributortovaginalLPAlevels.

Thesource(s)ofLPAinvaginalﬂuidremaintobedetermined.

PlateletactivationhasbeenshowntobeasourceofLPArelease intothecirculation[19].Inaddition,transformedepithelialcells from women withgynecologic malignancies produce LPA [10].

Thus,bothapro—inﬂammatoryimmuneresponsethatstimulates plateletactivationandlocalproductionbytransformedcellsmay Table2

Associationbetweenlysophosphatidicacidconcentrationinvaginalsecretionsand differentialpathologicaldiagnosisinwomenwithabenignormalignantadnexal mass.

Diagnosis No.women LPA(m^M)

Malignant

Endometroid+/-Clearcell 11 7.9(1.9–24.3)^a

Serous 6 9.5(1.0–16.1)^b

Mucinous 3 2.6(1.2–15.2)

MMMT 2 1.1(0.9–1.4)

Granulosa 2 9.1(4.2–14.1)

Other 2 4.4(1.6–7.3)

Clearcell 1 1.6

Mixed 1 0.2

Borderline

Serous 2

Mucinous 2

Benign

Cystadenoma 30 1.2(0.4–6.6)

Othercysts 23 2.7(1.2–21.8)

Teratoma 6 1.1(0.5–2.3)

Fibroma 6 2.0(0.5–4.4)

Endometriosis 3 0.3(0–7.9)

Totalbenign 68 1.5(0.5–4.3)

Valuesaremedian(25%–75%)mM;Endometrioid+/ Clear cell,endometrioid tumor with and without clear cell components; MMMT, malignant mixed mesodermtumor(carcinosarcoma);Other,metastaticadrenal,leiomyosarcoma.

ap=0.0137vs.benign;^bp=0.1267vs.benign;^cp=0.1208vs.benign.

Fig.1.LPAinvaginalﬂuidfromwomenwithabenignormalignantadnexalmass.

Vaginalﬂuidwascollectedfromwomenbeingscreenedforanadnexalmassand testedforLPAbyELISA.Thetypeofmalignancywassubsequentlydeterminedbya gynecologicpathologist.

MMMT,malignantmixedmesodermtumor(carcinosarcoma).

E.Minisetal./EuropeanJournalofObstetrics&GynecologyandReproductiveBiology:X2(2019)100012 3

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likelycontributetotheelevationinvaginalLPAinwomenwith endometrioidovariancancer.

Limitationsofthestudymustbeacknowledged.Duetothelow number of subjects analyzed the study must be designated as hypothesistestingandissubjecttoconﬁrmation.Inaddition,studies on pre-menopausalwomen withan adnexal mass,as well as pre-and post-menopausalwomenwithoutanadnexalmass,alsoremainto beconducted. The use of more sophisticated measurements of vaginalLPA,forexamplebymassspectrometry-basedmethods,are neededtoprovideadditionalinformationonfattyacidchainlength andsaturation.Advantagesofthestudyarethatitwasprospective, allprocedures anddiagnoseswere froma singleinstitution and samplesforCA125andforLPAwereobtainedatthesametime.

In conclusion, detection of an association between vaginal LPA levels andanovarianmalignancyinthispilotstudysuggeststhatanalysisof vaginal components as biomarkers for this cancer is biologically plausible and deserves further investigation. The LPA assay may especiallybeofvalueforthepre-surgicaldetectionofendometrioid ovariancancerinpost-menopausalwomenwithasuspiciousadnexal mass.Furtheranalysisonalargersamplesizeandamongdifferent populationsisneededtoverifythetentativeconclusionsandassessthe potentialclinicalutilityofvaginalLPAdetermination.

Conﬂictofinterest

Theauthorshavenoconﬂictsofinteresttodeclare.

Acknowledgement

SupportedbyagrantfromTheMacyFoundation.

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Table3

ComparisonofwomenwithdifferentovarianmalignanciespositiveforvaginallysophosphatidicacidandCA125.

Diagnosis No.tested No.(%)positive

CA125only LPAonly Both Neither

Endometrioid+/ Clearcell 6 1(16.6) 5(83.3) 0 0

Serous 6 3(50.0) 1(16.7) 2(33.3) 0

Mucinous 3 1(33.3) 0 1(33.3) 1(33.3)

MMMT 2 2(100) 0 0 0

Granulosa 2 0 1(50.0) 0 1(50.0)

Other 2 0 0 1(50.0) 1(50.0)

Clearcell 1 0 0 0 1(100)

Mixed 1 1(100%) 0 0 0

Endometrioid+/ Clearcell,endometrioidtumorwithandwithoutclearcellcomponents;MMMT,malignantmixedmesodermtumor(carcinosarcoma);Other,metastatic adrenal,leiomyosarcoma.

4 E.Minisetal./EuropeanJournalofObstetrics&GynecologyandReproductiveBiology:X2(2019)100012