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Kisspeptin and GPR54 immunoreactivity in a cohort of 518 patients defines favourable prognosis and clear… Prentice, Leah M; Klausen, Christian; Kalloger, Steve; Köbel, Martin; McKinney, Steven; Santos, Jennifer L; Kenney, Challayne; Mehl, Erika; Gilks, C B; Leung, Peter; Swenerton, Ken; Huntsman, David G; Aparicio, Samuel A Nov 15, 2007

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ralssBioMed CentBMC MedicineOpen AcceResearch articleKisspeptin and GPR54 immunoreactivity in a cohort of 518 patients defines favourable prognosis and clear cell subtype in ovarian carcinomaLeah M Prentice1,2, Christian Klausen3, Steve Kalloger1,2, Martin Köbel2,4, Steven McKinney1, Jennifer L Santos5,6, Challayne Kenney2, Erika Mehl2, C Blake Gilks2, Peter Leung3, Ken Swenerton7,8, David G Huntsman*2 and Samuel AJ Aparicio1Address: 1Molecular Oncology and Breast Cancer Program, British Columbia Cancer Research Centre and Department of Pathology, University of British Columbia, Vancouver, British Columbia, Canada, 2Genetic Pathology Evaluation Centre of the Prostate Centre and Departments of Pathology of Vancouver Coastal Health Research Institute, British Columbia Cancer Agency, and University of British Columbia, Vancouver, British Columbia, Canada, 3Department of Obstetrics and Gynaecology, University of British Columbia, Vancouver, British Columbia, Canada, 4Institute of Pathology, Charité Hospital, Berlin, Germany, 5Department of Gynaecology, Vancouver General Hospital, Vancouver, British Columbia, Canada, 6Cheryl Brown Ovarian Cancer Outcomes Unit, Department of Gynaecologic Oncology, British Columbia Cancer Agency, Vancouver, British Columbia, Canada, 7Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada and 8Department of Medical Oncology, British Columbia Cancer Agency, Vancouver, British Columbia, CanadaEmail: Leah M Prentice - Leah.Prentice@vch.ca; Christian Klausen - klausen@interchange.ubc.ca; Steve Kalloger - skalloger@mac.com; Martin Köbel - Martin.Koebel@vch.ca; Steven McKinney - smckinney@bccrc.ca; Jennifer L Santos - jsantos@bccancer.bc.ca; Challayne Kenney - Challayne.Smith@vch.ca; Erika Mehl - Erika.Mehl@vch.ca; C Blake Gilks - Blake.Gilks@vch.ca; Peter Leung - peleung@interchange.ubc.ca; Ken Swenerton - kswener@bccancer.bc.ca; David G Huntsman* - dhuntsman@bccancer.bc.ca; Samuel AJ Aparicio - saparicio@bccrc.ca* Corresponding author    AbstractBackground: Kisspeptins and their G-protein coupled receptor, GPR54 are required for GnRHrelease and have been associated with anti-metastatic tumour cell behaviour in model systems. Thelatter might suggest that their overexpression would be associated with a better prognosis incancer. However, kisspeptin/GPR54 interactions (autocrine, paracrine, and/or endocrine) couldalso impact tumour behaviour in a negative manner. Here, for the first time, we associate theimmunoreactivity of the kisspeptin/GPR54 ligand-receptor pair with favourable prognosis in a largecohort of ovarian carcinomas.Methods: Immunohistochemical analysis for kisspeptin and GPR54 was performed on a tissuemicroarray (TMA) consisting of 518 early stage ovarian carcinomas, all with linked clinical outcomedata. The TMA was scored using a staining intensity scale of 0 (negative), +1 (mild-moderate), and+2 (strong). Strong staining cases were considered either kisspeptin or GPR54 positive anddesignated as 1, while all other cases were considered negative and designated 0. All statisticalanalysis was conducted using two-sided tests and a p value equal to or less than 0.05 wasconsidered significant.Published: 15 November 2007BMC Medicine 2007, 5:33 doi:10.1186/1741-7015-5-33Received: 1 June 2007Accepted: 15 November 2007This article is available from: http://www.biomedcentral.com/1741-7015/5/33© 2007 Prentice et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 12(page number not for citation purposes)Results: Kisspeptin and GPR54 immunoreactive cases show a favourable prognosis in univariabledisease specific survival (p = 0.0023, p = 0.0092), as well as in overall survival (p = 0.0006, p =BMC Medicine 2007, 5:33 http://www.biomedcentral.com/1741-7015/5/330.0002). Furthermore, kisspeptin is an independent marker for favourable prognosis as determinedby multivariable disease specific (p = 0.0046) and overall survival analysis (p = 0.0170), while GPR54is an independent marker for overall survival only (p = 0.0303). Both kisspeptin positive and GPR54positive cases are strongly associated with the ovarian carcinoma clear cell subtype (p < 0.0001, p< 0.0001), and GPR54 is significantly associated with favourable prognosis in overall survival withinthe clear cell subtype (p = 0.0102).Conclusion: Kisspeptin and GPR54 immunoreactivity are significantly associated with favourableprognosis in both disease specific and overall survival, as well as being significantly associated withthe clear cell ovarian carcinoma subtype, thereby creating the first independent prognosticbiomarkers specific for ovarian clear cell carcinomas.BackgroundThe early diagnosis and management of ovarian cancer isa major area of unmet medical need. Central to the lack ofprogress in clinical management has been the virtualabsence of prognostic or predictive molecular markers forovarian cancer. Key to addressing these questions is theavailability of sufficiently large, clinically annotated tissuemicroarrays (TMA) that offer the prospect of defining theprognostic or predictive value of any given molecularmarker. Therefore we have constructed a large ovariancancer TMA (518 patients) with associated clinical demo-graphic and outcome information and have used this tosystematically address the value of possible biomarkers ofdisease prognosis. In the present study, we have tested theprognostic value of kisspeptin and GPR54 immunoreac-tivity in ovarian cancer. Kisspeptins (Kp-54, Kp-14, Kp-13,Kp-10) are the canonical, physiologically occurring andhigh affinity RF-amide peptide ligands that activate trans-membrane signalling via a classical (7TM1) family G-pro-tein coupled receptor, GPR54. Kisspeptins were firstdiscovered through microcell-mediated chromosometransfer experiments that defined the KiSS-1 locus as asuppressor of melanoma tumour metastasis [1,2]. Subse-quently, kisspeptins were associated as endogenous lig-ands for the GPR54 receptor. Furthermore, a physiologicalrole in the regulation of placental trophoblast invasionhas been suggested [3] and in migratory cell lines, activa-tion of GPR54 signalling abrogates migratory behaviour[1,4-6]. Specifically overexpression of KiSS-1 in an ovariancell line expressing endogenous GPR54 suppressed itsmetastatic phenotype [7].In 2003, we uncovered in human and mouse genetic stud-ies, the major physiological functions of kisspeptin-GPR54 signalling, as being gatekeepers for GnRH releasein the hypothalamus [8,9]. In the absence of functionalkisspeptin [10] and GPR54 [9,11-13] neither humans normice undergo puberty and are unable to generate pituitaryrelease of gonadotropins that drive sex-steroid release.Several subsequent physiological studies have confirmed[14-25], and are thus required as physiological regulatorsof sex-steroid release. The mechanistic relationshipbetween GPR54 regulation of the hypothalamic-pituitary-gonadal axis, and possible effects on epithelial cell migra-tion remains unclear, however several anecdotal studieson human tumours have suggested possible associationsof loss/absence of expression, with poor prognosis [26-33]. Recently, Zhang et al [34] and Hata et al [35] surveyedRNA expression of the KiSS-1 and GPR54 loci in smallcohorts (< 100 cases) of ovarian cancer and observe atrend towards favourable prognosis where KiSS-1/GPR54RNA expression is elevated. None of these studies havebeen sufficiently powered to address cell type and prog-nostic associations in major epithelial malignancies. Weshow in the present study of 518 ovarian cancer cases thatkisspeptin and GPR54 immunoreactivity are very signifi-cantly associated with a clear cell carcinoma subtype, andthat both kisspeptins and GPR54 are independent mark-ers for favourable prognosis as determined by multivaria-ble analysis.MethodsOvarian tumour samples and TMA constructionApproval for the study was obtained from the ethics com-mittee of the University of British Columbia. Mostwomen diagnosed with ovarian cancer in British Colum-bia are treated at the British Columbia Cancer agency(BCCA) and provincial treatment guidelines are followed.Outcomes are tracked via The Cheryl Brown Ovarian Can-cer Outcomes Unit as an ovarian cancer database of theBCCA. A total of 3501 patients with invasive epithelialovarian carcinoma were referred to the BCCA between1984 and the year 2000. The focus of this study was 834patients who had ovarian carcinoma with no macroscopicresidual disease after surgery. For 202 cases, the slides ofthe primary ovarian tumour were not available for reviewand these cases are excluded. A gynaecological pathologist(CBG) then did a blinded full slide review of the remain-ing 632 cases. Tumour cell type and grade (Silverberg)were assessed; all clear cell carcinomas were considered toPage 2 of 12(page number not for citation purposes)that kisspeptins act as neuroendocrine peptides thatswitch on or off the GnRH axis in humans and mammalsbe grade 3, as per World Health Organization recommen-dations. After review, 518 cases of invasive ovarian carci-BMC Medicine 2007, 5:33 http://www.biomedcentral.com/1741-7015/5/33noma were available in tissue blocks for tissue microarrayconstruction. A representative area of each tumour wasselected and a duplicate core TMA was constructed(Beecher Instruments, Silver Springs, MD, USA); thecohort is described in Table 1. Serial 4 µm sections werecut for immunohistochemical (IHC) analysis.ImmunohistochemistryKisspeptinSections from formalin-fixed and paraffin-embedded tis-sues were deparaffinized with xylene and rehydrated witha graded series of alcohols. Wet heat-induced antigenretrieval was performed in a steamer for 20 min with amodified citrate buffer (pH 6.1, Dako, Mississauga,Ontario, Canada). Following antigen retrieval, sectionswere treated with 3% hydrogen peroxide (H2O2) in phos-phate buffered saline (PBS) for 30 min to quench endog-enous peroxidase activity. All of the aforementioned stepswere followed by three washes with PBS for 5 min each.Slides were subsequently blocked for 30 min with serum-free protein block (Dako) and incubated overnight at 4°Cwith a polyclonal goat anti-(KiSS-1) antibody (C-20,Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted1:400 in serum-free protein block. Kisspeptin immunore-activity (IR) was detected with the CSA II biotin-free tyra-mide signal amplification system and 3,3'-diaminobenzidine chromogen solution (Dako). Specifi-cally, rabbit anti-goat horseradish peroxidase conjugate(HRP) was applied for 15 min followed by fluorescyl-tyra-mide amplification reagent for 15 min and anti-fluores-cein HRP for 15 min. All of the steps subsequent to theincubation with primary antibody were followed by threewashes with Tris-buffered saline containing 1% Tween(TBST) for 5 min each. Slides were counterstained withHarris hematoxylin (Sigma-Aldrich, Oakville, Ontario,Canada) and mounted in a xylene-based mountingmedium. Based on previously published data showingcell-type restriction of GPR54 and kisspeptins in differenttrophoblast layers of human placenta [3], less than 10-week old human placenta was used as a specificity control(courtesy of Vancouver Coastal Health archives), in con-junction with two blocking peptides (21 residues and 54residues; Figure 1). Omission of the primary antibody wasused as a negative control.GPR54TMA 4 µm sections were processed using a Ventana Dis-covery XT automated system (Ventana Medical Systems,Tucson, AZ, USA) as per manufacturer's protocol withproprietary reagents. After slides were baked at 60°C for 1h, they were deparaffinized on the automated system withEZ Prep solution (Ventana). Heat-induced antigenretrieval method was used in Cell Conditioning solution(CC1-Tris based EDTA buffer, pH 8.0, Ventana). The pol-yclonal rabbit GPR54 antibody was obtained from MBLInternational Corporation (Woburn, MA, USA) specificfor the N-terminal extracellular domain (cataloguenumber LS-A1929) and used with heat at a 1:25 concen-tration in Ventana antibody diluent. The Ventana Univer-sal Secondary Antibody was used for 32 min at 37°C. Thedetection system used was the Ventana DABMap kit, andslides were then counterstained with Hematoxylin andtreated with a proprietary bluing agent (Ventana). Allwashes were conducted with the Ventana Reaction Buffer.Dehydration steps and coverslip procedure were com-pleted manually as per manufacturer's recommendations.Specificity was determined by Western blot (Additionalfile 1) and by using less than 10-week old human placentaas a positive control (Figure 1) and omission of primaryantibody as a negative control.PhotomicrographsThe TMA was digitally scanned with a BLISS (Bacus Labo-ratories Inc., Slide Scanner) automated system (Bacus Lab-oratories, Lombard, IL, USA) as previously described [36].These images are available on our webslide server that ispublicly available [37].Statistical analysisSurvival time dependant recursive partitioning was usedto binarise the raw kisspeptin and GPR54 data. Univaria-ble survival analysis was performed by the generation ofKaplan-Meier curves [38] and differences between thegroups were assessed using Log-rank Statistic [39]. Multi-Table 1: Clinicopathological characteristics of the cohortParameter NHistopathological subtypeAdenocarcinoma 4Clear cell 132Endometrioid 125Mucinous 31Serous 212Squamous cell 1Transitional 6Undifferentiated 7Grade1 1062 1143 298Stage1 2142 2193 85Age (years)Mean (SD) 58 (12.8)Median 57Range (min-max) 25–89Page 3 of 12(page number not for citation purposes)variable survival analysis was performed using the CoxProportional Hazards Model [39,40]; the adenocarci-BMC Medicine 2007, 5:33 http://www.biomedcentral.com/1741-7015/5/33noma, squamous cell, transitional, and undifferentiatedovarian subtypes were excluded from multivariable anal-ysis due to insufficient sample size. Contingency tablesand the Pearson Chi-square statistic were used to test theperformed using JMP version 6.0.3 (SAS Institute, CaryNC, USA).IHC controlsFigure 1IHC controls. Less than 10-week-old human placenta used as a positive control. (A) Kisspeptin-IR shows intense cell-type specific staining in the syncytiotrophoblasts (black arrow), while the cytotrophoblast layers remain unaffected (black arrow-head). (B) GPR54-IR shows intense staining in the villous cytotrophoblasts (black arrow), the extravillous cytotrophoblasts (black arrowhead), and moderate staining on the syncytiotrophoblast membrane (grey arrow). (C) Schematic of the 1–145 amino acid (aa) KiSS-1 pro-peptide. Metastin (Kp-54) is encoded within the 68–121 aa sequence, while Kp-10 is encoded within this same region from 112–121 aa. The specific blocking peptide is encoded within the 100–120 aa sequence. (D) Varying kisspeptin-IR was found among the different blocking peptides used. Blocking the primary antibody with full-length metastin (Kp-54) and blocking peptide resulted in complete loss of immunoreactivity, while Kp-10 was unable to block any detectable staining.Page 4 of 12(page number not for citation purposes)change in the distribution of kisspeptin and GPR54expression across primary cell types [41]. All analyses wereBMC Medicine 2007, 5:33 http://www.biomedcentral.com/1741-7015/5/33ResultsKisspeptin positivity is an independent marker for favourable prognosisKisspeptin-IR was tested on human placenta less than 10weeks old as a positive control (Figure 1). There was celltype specificity demonstrated by intense staining in syncy-tiotrophoblast cells as previously determined [3,42], butnot in other cell layers of the trophoblast. Pre-absorptionwith two different blocking peptides (metastin (Kp-54,68–121 amino acids (aa)) and kisspeptin 100–120 aa),fully blocked kisspeptin-IR, whereas Kp-10 (112–121 aa)showed little or no block (Figure 1).For the 518 case ovarian tissue microarray, kisspeptin-IRwas scored as 0 for negative cases, +1 for mild staining,and +2 for intense staining (Figure 2). Of the 518 cases, 44stained at +2, 98 had +1 staining intensity, 354 cases werenegative for kisspeptin-IR, and 22 cases were uninterpret-able. The negative (0) and mildly reactive (+1) cases weregrouped for statistical analysis and assigned the designa-tion 0 and considered kisspeptin negative, while the +2cases were considered kisspeptin positive and designatedas 1. Univariable disease specific survival analysis showedthat kisspeptin-IR significantly associated with favourableprognosis (p = 0.0023), as did overall survival (p =0.0006, Figure 3). Further, multivariable survival analysisincluding; stage, grade, histological subtype, age andGPR54-IR, indicated kisspeptin-IR as an independentmarker for favourable prognosis in disease specific (p =0.0046, Table 2) and overall survival (p = 0.0170, Table3).GPR54 positivity is an independent marker for favourable prognosis in overall survivalThe GPR54 antibody and protocol were tested on lessthan 10-week old human placenta and specifically stainedboth villous and extravillous cytotrophoblasts and thesyncytiotrophoblasts as described by previous groups[3,42] (Figure 1). Although GPR54 is a 7-transmembraneprotein, there was some reactivity in the cytoplasm ofImmunoreactivity patternsFigure 2Immunoreactivity patterns. Three types of kisspeptin-IR (A-C) and GPR54-IR (D-F) observed in the ovarian TMA. (A, D) Representative samples of negative cases show complete lack of staining and are classified as 0. (B) Moderate GPR54-IR shows a patchy staining pattern with light and dark brown regions of reactivity shown throughout the tumour core, and (E) Mild kisspeptin-IR shows uniform light brown staining throughout the sample: both B and E are classified as +1 immunoreactivity. (C, F) Examples of +2 intense immunoreactivity exhibit dark brown staining in all tumour cells. Scale bar represents 100 µm. Insets in each panel show a more detailed view of the staining pattern that is demonstrated in the larger image; inset scale bar Page 5 of 12(page number not for citation purposes)represents 25 µm.BMC Medicine 2007, 5:33 http://www.biomedcentral.com/1741-7015/5/33some tumour cells (this is not entirely surprising asGPR54 is a transmembrane protein and can be recycledthrough the cytoplasm) but only membranous stainingwas taken into consideration while assessing immunore-activity. Three immunoreactivity patterns were observedwithin the TMA for GPR54. Specifically, negative or veryweak reactivity in less than 5% of cells was designated as0 (103 cases), while patchy or moderate staining in 5–75% of cells was designated +1 (282 cases), and strongstaining in greater than 75% of cells were considered +2(104 cases, Figure 2). The remaining nine cases were unin-terpretable. As with kisspeptin, the 0 and +1 GPR54 caseswere group together and considered as loss of receptorand designated 0, while the strong staining +2 cases wereconsidered positive and designated as 1. Univariable sur-vival analysis determined GPR54 as a significant markerfor favourable prognosis in disease specific (p = 0.0092)and overall survival (p = 0.0002, Figure 3). Similar tokisspeptin-IR, GPR54 maintained significance in multi-variable overall survival (p = 0.0303, Table 3). However,GPR54 was not found to be a significant independentmarker in disease specific survival (p = 0.1118, Table 2).Kisspeptin positivity correlates with GPR54 positive casesKisspeptin positive cases had a moderate correlation withGPR54 positivity as determined by Kendall's tau-b [43] (τ= 0.3837, p < 0.0001). There were 31 cases that were bothkisspeptin and GPR54 positive, 90 cases that werekisspeptin negative and GPR54 positive, 12 cases withkisspeptin positivity and had loss of GPR54, 356 casesthat had loss of both kisspeptin and GPR54, and theremaining 29 cases were uninterpretable. When kisspep-tin-IR and GPR54-IR cases are grouped together (G+ K+),patients have a more favourable outcome than those thathave loss of either one or both (G- K+, G+ K-, G- K-). Thereis a significant difference between survival for double pos-itive patients (G+ K+) as compared to double negativepatients (G- K-) in both disease specific (p = 0.0067) andoverall survival (p = 0.0002, Figure 3).Kisspeptin and GPR54 positive staining are significantly associated with clear cell carcinoma histopathological subtypeThe percentage of kisspeptin and GPR54 positive caseswithin each histopathological subtype is listed in Table 4.The proportionality of primary histopathological cell typein the entire cohort, kisspeptin positive cases, and GPR54positive cases are represented in Table 5. Testing for anassociation between ovarian carcinoma subtype andkisspeptin status, there was a highly significant positiveassociation with clear cell carcinoma, and a significantnegative association with serous carcinoma subtype (χ2, p< 0.0001). GPR54 positive cases also had a significantpositive association with clear cell carcinoma subtype anda negative association with the serous subtype (χ2, p <0.0001).When disease specific survival and overall survival wereTable 3: Multivariable overall proportional hazardsParameter Risk ratio (95% CI) p ValueStage < 0.00011 0.7258 (0.5842 to 0.8961)2 0.7149 (0.6457 to 0.9476)3 1.0000Histological grade 0.53561 0.8268 (0.5818 to 1.1642)2 1.1113 (0.8647 to 1.4148)3 1.0000Subtype 0.6763Clear cell 1.2126 (0.7934 to 1.8513)Endometrioid 0.8120 (0.5508 to 1.1914)Mucinous 1.0192 (0.5652 to 1.6841)Serous 1.0000Age < 0.0001N/AGPR54 0.0303Positive 0.5959 (0.3684 to 0.9523)Negative 1.0000Kisspeptin 0.0170Positive 0.4844 (0.2443 to 0.8841)Negative 1.0000Table 2: Multivariable disease specific proportional hazardsParameter Risk ratio (95% CI) p ValueStage < 0.00011 0.6404 (0.4901 to 0.8278)2 0.7149 (0.5647 to 0.8994)3 1.0000Histological grade 0.07201 0.6234 (0.3829 to 0.9808)2 1.3899 (1.0193 to 1.8888)3 1.0000Subtype 0.2508Clear cell 1.4519 (0.8853 to 2.3847)Endometrioid 0.6804 (0.4105 to 1.1032)Mucinous 1.0721 (0.5119 to 1.9699)Serous 1.0000Age 0.0747N/AGPR54 0.1118Positive 0.6475 (0.3738 to 1.1052)Negative 1.0000Kisspeptin 0.0046Positive 0.3508 (0.1426 to 0.7408)Negative 1.0000*RR for age is not available because it is a continuous variable.Page 6 of 12(page number not for citation purposes)analyzed within each ovarian carcinoma subtype, the log-rank test for kisspeptin-IR status failed to achieve signifi-*RR for age is not available because it is a continuous variable.BMC Medicine 2007, 5:33 http://www.biomedcentral.com/1741-7015/5/33Page 7 of 12(page number not for citation purposes)Disease specific (left) and overall (right) survival curves for kisspeptin and GPR54Figure 3Disease specific (left) and overall (right) survival curves for kisspeptin and GPR54. The top two graphs demon-strate the significant survival curves for GPR54, while the middle two graphs demonstrate kisspeptin related survival. For the bottom two graphs, the p value refers to the distance between the GPR54 positive/kisspeptin positive cases (G+ K+, solid light grey) and the GPR54 negative/kisspeptin negative cases (G- K-, solid dark grey).BMC Medicine 2007, 5:33 http://www.biomedcentral.com/1741-7015/5/33cance (due to insufficient sample size), although for theclear cell cases statistical significance was approached (p =0.1042, p = 0.0859, results not shown). Of note, none ofthe kisspeptin positive patients that were not clear cellsubtype (16 cases) died from their disease. Similarly,when assessing GPR54 positivity within each ovarian can-cer subtype, disease specific survival did not reach signifi-cance within the clear cell subtype (p = 0.0656), althoughsignificance was achieved for overall survival (p = 0.0102,Figure 4).DiscussionAlthough clear cell carcinomas comprise fewer than 5% ofovarian malignancies, they are notoriously difficult totreat due to their tendency to resist platinum based chem-otherapy [44]. To date, clinical stage has been the onlyprognostic marker for clear cell ovarian carcinoma. Herewe show for the first time, that kisspeptin and GPR54immunoreactivities mark distinctly for favourable prog-nosis, with kisspeptin being independent of pathologicsubtype, stage, grade, or age in both overall and diseasespecific survival, while GPR54 is an independent markerin overall survival. Within clear cell carcinomas, GPR54expressers have a favourable prognosis and to our knowl-edge this is the first molecular marker of prognosis specif-ically applicable to clear cell ovarian cancer. Althoughseveral studies have suggested possible relationshipsbetween GPR54 and kisspeptin expression and clinicaloutcome [26,27,30-34,45], these studies have consisted ofsmaller cohorts and while some associations have beennoted, some studies might not have been sufficientlypowered to address possible prognostic or cell type spe-cific effects with rigour. To date, the present study is thelargest systematic analysis of GPR54 and kisspeptinexpression determined by immunoreactivity for an epi-thelial malignancy. In part, this could be due to difficul-ties in obtaining sufficiently specific antisera anddetection protocols, in that short peptides and GPCRs arenotoriously difficult antibody targets. The antibodies andimmunodetection protocols used in this study were veri-fied by the use of either Western blotting and cell-typespecific expression (GPR54), or cell-type specific expres-sion and specific blocking peptides (kisspeptin). This isbased on previous work showing differential expressionof kisspeptin and GPR54 in human placental trophoblastcell types [3,45]. We note that while Muir et al [46] dem-onstrated a 75 kDa fragment as GPR54 by Western blot,our data show a fragment much closer to the predicted42.5 kDa molecular weight for GPR54. The basis of thisdifference is unknown, but could result from post-transla-tional modification arising in different tissues (brain vstumour cell lines). Although blocking peptides suggestspecificity of the antisera used, the precise spectrum ofimmunoreactivity of the kisspeptin antiserum to kisspep-tin fragments remains to be determined. As with manyantibodies, it remains possible that other proteins couldbe detected however this does not diminish their utility asmarkers of prognosis. Very recently a survey of 76 ovariancancer patients using Q-PCR detection of GPR54 andkisspeptin transcripts [35] demonstrated a negative corre-lation between KiSS-1 and GPR54 mRNA levels withresidual disease, although they showed no correlationwith histopathological subtype (possibly due to the rela-tively small number of clear cell ovarian cancers in thatcohort), however the overall correlation observed in thisstudy is in agreement with our observations.The mechanisms responsible for the association ofkisspeptin and GPR54 expression with disease behaviourin ovarian cancer requires definitive studies, however sev-eral possibilities arise. It is possible that expression ofkisspeptins and/or GPR54 result in higher endogenousGPR54 signalling in malignant cells. Although no studieshave directly addressed the degree of GPR54 signalling inepithelial malignancies in relation to clinical outcomes,the present study shows that both kisspeptin and GPR54expression are associated with a better prognosis. Further-more, patients with double positive tumours (G+ K+)have the most favourable prognosis (Figure 3). Theseobservations together with previous evidence of the effectsof GPR54 signalling on cell migration, suggest some formof autocrine or paracrine loop could exist in clear cell car-cinomas. GPR54 is exquisitely sensitive to kisspeptin lig-and stimulation [3,5] and receptor overexpression alonemight be enough to increase basal signalling throughGPR54.The interplay of mechanisms could be complicated by themajor physiological role of GPR54, which is to regulateGnRH secretion at the hypothalamic level. Kisspeptinscan cross from the peripheral circulation to act on thehypothalamus, as has been shown in numerous mamma-lian [22,24,47-49] and one human study [21]. It is possi-ble that kisspeptin overexpressing tumours could result instimulation of the hypothalamic-pituitary axis, resultingin the release of gonadotropins and other derived pep-tides with a possible paracrine/endocrine effect onTable 4: Percentage of kisspeptin and GPR54 positive cases within the histological subtypesHistological subtypeKisspeptin positive (%)GPR54 positive (%)Clear cell 21.88 66.41Endometrioid 8.13 20.33Mucinous 12.00 10.34Serous 1.49 3.37Page 8 of 12(page number not for citation purposes)tumour growth. Indeed Nash et al [50], have shown thatmelanoma cells unable to signal on exposure toBMC Medicine 2007, 5:33 http://www.biomedcentral.com/1741-7015/5/33kisspeptins, can still be suppressed from metastasis byexogenous kisspeptin, suggesting that paracrine effectscould operate in these cases. Finally, some evidence sug-gests that kisspeptins and GPR54, which are expressed inovarian epithelium and granulosa cells, might co-modu-late the activity of gonadotropins in sex steroid release[42]. If such a mechanism were operational in clear cellovarian cancers, it would imply that tumour behaviour isalso linked to co-modulatory peptides.Beyond the salient observation of prognostic significancein this study, the nature of the proteins involved suggestsa number of possible areas for intervention. First,kisspeptins, the products of the KiSS-1 gene locus, are nat-urally occurring peptides that can be detected in humanserum and other tissues [51-53]. It is possible that serumkisspeptide levels could be developed as a biomarker ofdisease activity in patients with clear cell carcinoma. How-ever, diagnostic grade antibodies would have to be devel-oped before routine immunohistochemical-basedanalysis of kisspeptin and GPR54 could be undertaken.Secondly, kisspeptins are naturally occurring peptide hor-mones that have activity in humans [21]. As such they arehighly amenable to use as therapeutic agents, either aloneor as modified peptides. We anticipate that the strongassociation of GPR54 and kisspeptin expression with out-come and clear cell type in ovarian carcinoma will stimu-late fresh approaches to what is still a lethally intractabledisease.ConclusionKisspeptin and GPR54 are significantly associated withfavourable prognosis in both disease specific and overallsurvival, as well as being significantly associated with theclear cell ovarian carcinoma subtype, thereby creating thefirst independent prognostic biomarkers specific for ovar-ian clear cell carcinomas.Table 5: Kisspeptin and GPR54 proportions within the cohortHistological subtype Whole cohort Kisspeptin positive GPR54 positiveProportion Count Proportion Count Proportion CountClear cell 0.2640 132 *0.6364 28 **0.7131 87Endometrioid 0.2500 125 0.2273 10 0.2049 25Mucinous 0.0620 31 0.0682 3 0.0246 3Serous 0.4240 212 *0.0682 3 **0.0574 7*χ2 p value < 0.0001**χ2 p value < 0.0001Kaplan-Meier disease specific (left) and overall (right) survival curves for GPR54 positivity within the clear cell subtypeFigure 4Page 9 of 12(page number not for citation purposes)Kaplan-Meier disease specific (left) and overall (right) survival curves for GPR54 positivity within the clear cell subtype.BMC Medicine 2007, 5:33 http://www.biomedcentral.com/1741-7015/5/33Competing interestsThe author(s) declare that they have no competing inter-ests.Authors' contributionsLMP was responsible for the IHC data accruement andanalysis, participated in the study design, wrote the initialmanuscript, and implemented the manuscript revisions.CK optimized the Kisspeptin IHC, acquired the data andassisted in the study design.SK was responsible for the statistical analysis, and assistedwith the editing of the manuscript. MK was responsiblefor the IHC analysis and assisted with the editing of themanuscript. SM assisted with the data analysis and editingof the manuscript.KS and JLS led the ovarian cancer cohort design, dataaccruement for the cohort, and assisted with editing of themanuscript. CK and EM were responsible for GPR54 IHCoptimization and staining. CBG assisted in the studydesign, data accruement, data analysis and editing of themanuscript. PL assisted with the study design. DGH andSAJA led the study design, writing and editing of the man-uscript.All authors have read and approved the final manuscript.Additional materialAcknowledgementsLMP is a Michael Smith Foundation for Health Research (MSFHR) Trainee jointly funded by Vancouver General Hospital and University of British Columbia Hospital Foundation and a Canadian Institute for Health gram. MK and SK are supported through the OvCaRe research team of the Vancouver General Hospital and British Columbia Cancer Agency founda-tions. DGH is supported by a Michael Smith Foundation for Health Research Scholarship. SAJA is a Canada Research Chair in molecular oncol-ogy. Molecular histopathology was conducted in part by the Genetic Pathology Evaluation Centre (GPEC), which is supported by a MSFHR unit grant and an unrestricted educational grant from sanofi-aventis. We thank Dr A Rajput for assistance in compiling data and scoring tissue microarrays, and Samuel Leung for assistance with the TMA micrographs.References1. Lee JH, Welch DR: Suppression of metastasis in human breastcarcinoma MDA-MB-435 cells after transfection with themetastasis suppressor gene, KiSS-1.  Cancer Res 1997,57:2384-2387.2. Lee JH, Miele ME, Hicks DJ, Phillips KK, Trent JM, Weissman BE,Welch DR: KiSS-1, a novel human malignant melanomametastasis-suppressor gene.  J Natl Cancer Inst 1996,88:1731-1737.3. 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J Neuroendocrinol2006, 18:349-354.15. Nazian SJ: Role of metastin in the release of gonadotropin-releasing hormone from the hypothalamus of the male rat.Additional file 1Western blot demonstrating GPR54 specificity. A total of 30 µg of pro-tein was run on 12% SDS-PAGE and transferred to a nitrocellulose mem-brane. The membrane was blocked for 1 h at room temperature with TBST 5% non-fat milk powder and incubated overnight at 4°C on a rocking incubator with 1/1000 GPR54 MBL antibody. The blot was washed four times with TBST for 5 min each and incubated with a 1/20000 secondary anti-rabbit antibody for 1 h at room temperature. The blot was then incu-bated with SuperSignal Chemiluminescent (Pierce, San Francisco, CA, USA) for 5 min and exposed to film for 20 s before developing. Loading control β-actin was detected using 1/2500 anti-(β-actin) antibody incu-bated on the same blot for 1 h at room temperature and visualized with an anti-mouse secondary antibody and enhanced chemiluminescence (ECL) for a 12 min exposure. The cell lines were kept in tissue culture and passaged as per distributors' recommendations. Protein lysate was col-lected using the standard RIPA buffer method.Click here for file[http://www.biomedcentral.com/content/supplementary/1741-7015-5-33-S1.tiff]Page 10 of 12(page number not for citation purposes)Research Canadian Graduate Scholar. CK is supported by a fellowship from the Interdisciplinary Women's reproductive Health Research Training Pro-J Androl 2006, 27:444-449.16. 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Zhonghua Fu Chan Ke Za Zhi 2005, 40:689-692.Publish with BioMed Central   and  every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime."Sir Paul Nurse, Cancer Research UKYour research papers will be:available free of charge to the entire biomedical communitypeer reviewed and published immediately upon acceptancecited in PubMed and archived on PubMed Central BMC Medicine 2007, 5:33 http://www.biomedcentral.com/1741-7015/5/33Pre-publication historyThe pre-publication history for this paper can be accessedhere:http://www.biomedcentral.com/1741-7015/5/33/prepubyours — you keep the copyrightSubmit your manuscript here:http://www.biomedcentral.com/info/publishing_adv.aspBioMedcentralPage 12 of 12(page number not for citation purposes)


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