Proteomic Profiling Identifies Afamin as a Potential Biomarker for Ovarian Cancer DavidJackson, 1,4 RachelA.Craven, 1 RichardC.Hutson, 2 InaGraze, 5,7 PaulLueth, 5 RobertP.Tonge, 4 JoanneL.Hartley, 4 JaniceA.Nickson, 4 SteveJ.Rayner, 4 ColinJohnston, 1 BenjaminDieplinger, 5,8 MichaelHubalek, 6 NafisaWilkinson, 3 TimothyJ.Perren, 1 SeanKehoe, 9 GeoffreyD.Hall, 1 GuenterDaxenbichler, 6 HansDieplinger, 5,7 PeterJ.Selby, 1 andRosamondeE.Banks 1 Abstract Purpose: Todiscoverandvalidateserumglycoproteinbiomarkersinovariancancerusingproteo- mic-basedapproaches. Experimental Design: Serumsamplesfroma‘‘discoveryset’’of20patientswithovariancancer or benign ovarian cysts or healthy volunteers were compared by fluorescence two-dimensional differentialin-gelelectrophoresisandparallellectin-basedtwo-dimensionalprofiling.Validationof acandidatebiomarkerwascarriedoutwithWesternblottingandimmunoassay( n =424). Results: Twenty-six proteins that changed significantly were identified by mass spectrometric sequencing.Oneofthese,confirmedbyWesternblotting,wasafamin,avitaminEbindingprotein, with two isoforms decreasing in patients with ovarian cancer.Validation using cross-sectional samplesfrom303individuals(healthycontrolsandpatientswithbenign,borderline,ormalignant ovarian conditions and other cancers) assayed by ELISA showed significantly decreased total afamin concentrations in patients with ovarian cancer compared withhealthy controls (P = 0.002)andpatientswithbenigndisease(P =0.046).However,thereceiveroperatingcharacter- istic areas for total afamin for the comparison of ovarian cancer with healthy controls or benign controls were only 0.67 and 0.60, respectively, with comparable figures for CA-125 being 0.92 and 0.88 although corresponding figures for a subgroup of samples analyzed by isoelectric focusing for afamin isoform 2 were 0.85 and 0.79. Analysis of a further121samples collected prospectively from 9 patients pretreatment through to relapse indicated complementarity of afaminwithCA-125,includingtwocasesinwhomCA-125wasnoninformative. Conclusions: Afamin shows potential complementarity with CA-125 inlongitudinal monitoring ofpatientswithovariancancer,justifyingprospectivelarger-scaleinvestigation.Changesinspe- cificisoformsmayprovidefurtherinformation. The most widely used marker for ovarian cancer is CA-125 (1). However, its poor sensitivity in detecting stage I disease and lack of specificity preclude its sole use in screening and limit its value in differential diagnosis of pelvic masses, particularly in premenopausal women. Although recommen- dations about the optimal interpretation or definition of changes in CA-125 levels vary, there is growing consensus that the main utility of CA-125 lies in the assessment of response to therapy and in the longitudinal monitoring and detection of disease recurrence, providing a median lead time of 3 to 4 months over clinical or radiological assessment. However, CA-125 alone is not informative in 10% to 20% of patients with advanced disease and further complementary markers are needed. Several potential candidate markers are the subject of current evaluation, including OVX1, macrophage colony-stimulating factor, inhibin, kallikreins, tissue polypeptide-specific antigen, and lysophosphatidic acid (1, 2). The concept of using multiple markers to overcome heterogeneity and enhance sensitivity and specificity is also being explored using both specific proteins and pattern recognition analysis of mass spectrometric profiles (3–6). With technological improvements allowing more sensitive, reproducible, and higher-throughput profiling and identification of proteins and their posttranslational modifica- tions, proteomics-based approaches are increasingly being used (7, 8) with several studies illustrating the potential for ovarian cancer (6, 9–12). Imaging, Diagnosis, Prognosis Authors’ Affiliations: 1 Cancer Research UKClinical Centre, 2 Departmentof ObstetricsandGynaecology,and 3 DepartmentofPathology,StJames’sUniversity Hospital,Leeds,UnitedKingdom; 4 DiscoveryEnablingCapabilitiesandSciences, AstraZeneca,AlderleyPark,UnitedKingdom; 5 DivisionofGeneticEpidemiology, Departmentof Medical Genetics, Molecularand Clinical Pharmacology, and 6 DepartmentofGynecologyandObstetrics,InnsbruckMedicalUniversity; 7 Vitateq BiotechnologyGmbH,Innsbruck,Austria; 8 DepartmentofLaboratoryMedicine, KonventhospitalBarmherzigeBrueder,Linz,Austria;and 9 NuffieldDepartmentof ObstetricsandGynaecology,JohnRadcliffeHospital,Oxford,UnitedKingdom Received4/3/07;revised8/17/07;accepted10/8/07. Grant support: Biotechnology and Biological Sciences Research Council Co- operativeAwards in Science and Engineering, Cancer Research UK, AstraZeneca, theAustrianIndustrialResearchFund,andKompetenzzentrumMedizinTirol. Thecostsofpublicationofthisarticleweredefrayedinpartbythepaymentofpage charges.This articlemust thereforebeherebymarked advertisement inaccordance with18U.S.C.Section1734solelytoindicatethisfact. Requests for reprints: Rosamonde Banks, Cancer Research UKClinical Centre, St. James’s University Hospital, Beckett Street, Leeds LS97TF, United Kingdom. Phone:44-113-2064927;Fax:44-113-2429886;E-mail:R.Banks@leeds.ac.uk. F 2007AmericanAssociationforCancerResearch. doi:10.1158/1078-0432.CCR-07-0747 www.aacrjournals.org Clin Cancer Res 2007;13(24) December 15, 2007 7370 Research. on April 13, 2017. © 2007 American Association for Cancer clincancerres.aacrjournals.org Downloaded from