{"paper_id":"690a7837-867b-4daa-82e8-2002945432e6","body_text":"www.ogscience.org234\nReceived: 2013.2.14.   Revised: 2013.5.6.  Accepted: 2013.5.20.\nCorresponding author: Seung Cheol Kim\nDepartment of Obstetrics and Gynecology, Ewha Womans \nUniversity Mokdong Hospital, Ewha Womans University School of \nMedicine, 1071 Anyangcheon-ro, Y angcheon-gu, Seoul 158-710, Korea \nTel: +82-2-2650-5587  Fax: +82-2-2647-9860\nE-mail: onco@ewha.ac.kr\nArticles published in Obstet Gynecol Sci are open-access, distributed under the terms of \nthe Creative Commons Attribution Non-Commercial License (http://creativecommons.\norg/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, \nand reproduction in any medium, provided the original work is properly cited.\nCopyright © 2013 Korean Society of Obstetrics and Gynecology \nIntroduction\nOvarian cancer is the second most common gynecological \ncancer in Western countries as well as the leading cause \nof gynecological cancer-related death, with an incidence \nestimated at 8.8 per 100,000 women-years in USA [1-4]. \nIn Korea, ovarian cancer is also the second most common \ngynecological cancer, with an incidence of 8.0 per 100,000 \nwomen-years in 2010 [4,5]. Though ovarian cancer can \nbe curable in early-diagnosed cases where the disease is \nlimited to the ovary, however most patients are diagnosed \nClinical efficacy of serum human epididymis protein 4 as \na diagnostic biomarker of ovarian cancer:  A pilot study\nShin Hye Chung\n1\n, Soo Yoon Lee\n2\n, Woong Ju\n1\n, Seung Cheol Kim\n1\n1\nDepartment of Obstetrics and Gynecology, Ewha Womans University Mokdong Hospital, Ewha Womans University School of Medicine, Seoul; \n2\nDepartment of Obstetrics and Gynecology, Cheil General Hospital and Women’s Healthcare Center, Kwandong University College of Medicine, Seoul, \nKorea\nObjective\nTo compare accuracy of serum human epididymis protein 4 (HE4) levels with cancer antigen 125 (CA-125) levels as \nbiomarkers for ovarian cancer. \nMethods \nThe study population included 94 Korean women, including 32 patients with a diagnosis of ovarian cancer and 62 \npatients with a diagnosis of benign ovarian tumor. All diagnoses were confirmed by histopathological analysis. Serum \nHE4 levels were assessed using an HE4 enzyme immunoassay, which were performed according to the manufacturer’s \ninstructions. Serum CA-125 levels were determined using a Modular analytics E170 module. \nResults\nThe median serum CA-125 and HE4 levels were significantly higher in patients with ovarian cancer than those with \nother benign tumors (CA-125, 394.1 U/mL vs. 22.7 U/mL; HE4, 56.7 pM vs. 18.5 pM; P < 0.05 in both). An additional \ncomparison revealed that the patients with endometriosis had greater median serum CA-125 levels than those with \nother benign ovarian tumors (32.0 U/mL vs. 17.9 U/mL, P = 0.03). Conversely, the median serum HE4 levels were similar \namong the two benign ovarian tumor groups, with no statistically significant difference observed (19.0 pM vs. 18.2 pM, \nP = 0.49). The receiver operating characteristics curve analysis for the benign ovarian tumor and ovarian cancer patients \nshowed that HE4 showed a greater area under the curve with borderline significance when compared with CA-125 in \nboth groups (0.93 vs. 0.85).\nConclusion \nSerum HE4 levels may not only allow for the detection of ovarian cancer, but also allow for better differentiation of \ncases of ovarian cancer versus other benign ovarian tumors compared with serum CA-125.\nKeywords: Biochemical tumor markers; CA-125; HE4 protein; Koreans; Ovarian cancer\nOriginal Article\nObstet Gynecol Sci 2013;56(4):234-241\nhttp://dx.doi.org/10.5468/ogs.2013.56.4.234\npISSN 2287-8572 · eISSN 2287-8580\n\nwww.ogscience.org 235\nShin Hye Chung, et al. Serum HE4 as a marker of ovarian cancer\nwhen at more advanced stages (International Federation of \nObstetrics and Gynecology [FIGO] stage III-IV) [6].\nAmong gynecologic cancers, the incidence of cervical can -\ncer has been decreasing in Korea [5], which has been attrib-\nuted to earlier diagnoses secondary to routine pap smears. \nHowever, increasing the rate of earlier diagnosis for ovarian \ncancer has remained difficult due to the relative dearth of \nassociated symptoms and lack of specific serum biomarker.\nCurrently used as a diagnostic marker for ovarian cancer, \ncancer antigen 125 (CA-125) is elevated in roughly 80% of \npatients with ovarian cancer and 30% of patients with other \nprimary cancers with extensive intra-abdominal disease. \nAccordingly, serum CA-125 levels are not only elevated in \novarian malignancies, but also benign ovarian diseases as \nwell as any other inflammatory conditions of the perito -\nnieum, pleura and pericardium [7-10]. Moreover, as CA-125 \nlevels are elevated in less than half of cases of early stage \novarian cancer [11,12], a new biomarker for ovarian cancer \nis clearly needed.\nFirst identified in the epithelium of the distal epididymis, \nhuman epididymis protein 4 (HE4) was originally believed \nto represent a protease inhibitor for sperm maturation and \ncontribute to intrinsic immunity. HE4 is also one of 14 ho -\nmologous genes on chromosome 20q12-13.1 that encodes \nproteins with a whey acidic protein-type four disulphide core \ndomain [13-15]. Emerging data now suggests that serum \nlevels of HE4 is elevated in ovarian cancer patients, demon -\nstrating similar sensitivity and increased specificity for ovar -\nian cancer when compared with CA-125 [16]. HE4 is also \nelevated in lung adenocarcinoma, transitional cell, breast, \nrenal and pancreatic carcinomas [17].\nIn the current study, we analyzed the serum levels of HE4 \nand CA-125 among patients with ovarian cancer as well as \nother benign ovarian tumors in order to assess the possible \nrole of serum HE4 levels as an ovarian cancer biomarker.\n \nMaterials and methods\n1. Study population\nIn the current case-controlled 1:2 matching study, patients \nwere recruited from Ewha Woman’s University Mokdong \nHospital in Seoul, Korea between October 2005 and March \n2010. Informed consent was obtained in all cases prior to \nenrollment. The inclusion criteria were: no other diagnosed \ngynecologic disease except ovarian mass, the ovarian mass \nhad to be the primary diagnosis availability of complete \nclinical records, informed consent and agreement to have \nadditional testing for new markers, clinical and histological \ndiagnosis with staging and grading of ovarian cancer, ac -\ncording to the current classification and guidelines. And if \nany cases were not satisfied in criteria, they were excluded.\nDuring the period, 367 women underwent operation, 47 \nwomen received a diagnosis of cancer, and 320 women \nreceived a diagnosis of benign ovarian tumor. Based on the \ninclusion criteria, a total of 94 women were enrolled. The 32 \ncases of ovarian cancer included 16 serous, 5 clear-cell, 5 \nmucinous, 4 mixed, 2 endometrioid carcinomas. Of 32 ovar-\nian cancer patients, 6 (18.8%) had stage I disease, 4 (12.5%) \nhad stage II disease, 20 (62.5%) had stage III disease and \n2 (6.25%) had stage IV disease as per the International \nFederation of Gynecology and Obstetrics (FIGO) criteria. His-\ntopathology of 62 patients with benign ovarian tumors were \nas follows: 23 endometriomas (37.1%), 16 mature cystic \nteratomas (25.8%), 8 mucinous cystadenomas (12.9%), 8 \nserous cystadenomas (12.9%), 7 other non-specified neo -\nplasms (11.3%). All enrolled patients underwent laparos -\ncopy or laparotomy, and all diagnoses were histopathologi -\ncally confirmed by pathologic examination at Ewha Womans \nUniversity Mokdong Hospital.\n2. CA-125 and HE4 levels\nIn all cases, patient sera was obtained on the day prior to the \nlaparotomy/laparoscopy and was stored frozen at -80\no\nC until \nanalysis. \nSerum HE4 levels were measured by HE4 enzyme immuno-\nassay (Fujirebio Diagnostics Inc., Malvern, PA, USA), which \nwere performed as per the manufacturer’s instructions. Spe-\ncifically, the HE4 assay is a solid-phase immunoassay derived \nfrom the direct sandwich technique, which uses biotinylated \nanti-HE4 monoclonal antibody (MAb), streptavidin coated \nmicrostrips, and HRP labeled anti-HE4 MAb. To date, no de-\nfinitive diagnostic thresholds for HE4 have been reported in \nKorean women, however previous data from other western \ncountries identified 74.2 pM as a cut-off point, as this value \ncorresponded with the upper 95% among healthy individuals \nfrom Verona, Italy [18].\nSerum CA-125 levels were determined by Modular analyt -\nics E170 module (Roche Diagnostics, Mannheim, Germany), \nan electrochemiluminescence immunoassay derived from the \n\nwww.ogscience.org236\nVol. 56, No. 4, 2013\nsandwich principle using two monoclonal antibodies, a bioti-\nnylated monoclonal CA-125−specific antibody, and a mono-\nclonal CA-125−specific ruthenium complex-labeled antibody. \nNotably, this assay is able to measure CA-125 levels between \n0.600 to 5,000 U/mL, though the manufacturer’s suggested \ncut-off level is 35 U/mL.\n3. Statistical analysis\nAll the data were analyzed using SPSS ver. 21.0 (IBM, Ar -\nmonk, NY , USA). The median values of the serum HE4 and \nCA-125 levels were calculated separately for individuals \nwith other benign ovarian tumor and the patients with a \ndiagnosis of ovarian cancer. The relative serum tumor marker \nlevels were compared among the two groups using the Wil -\ncoxon signed-rank test because they did not follow a normal \ndistribution. In all cases, P-values <0.05 were defined as \nstatistically significant.\nReceiver operating characteristic (ROC) curves were as -\nsessed for both serum values of HE4 and CA-125. Values \nwith the best diagnostic performance as per the ROC curve \nwere identified in order to estimate the area under the curve \n(AUC). \nResults\nThe clinical characteristics and study groups demograph -\nics are presented in Table 1. There were some demographic \ndifferences between two groups. The mean age of ovarian \ncancer group is older than that of benign ovarian tumor \ngroup and menopausal patients were more larger in ovarian \ncancer group. \nThe median serum levels of CA-125 and HE4 were signifi -\ncantly higher among individuals with ovarian cancer when \ncompared with those with other benign ovarian tumors, \nwith the values for each group reaching statistical signifi -\ncance (CA-125, 394.1 U/mL vs. 22.7 U/mL; HE4, 56.7 pM \nvs. 18.5 pM; P<0.05 in both) (Table 2, Fig. 1). \nThe patients with benign ovarian tumors were further \nstratified by known endometriosis as confirmed by a patho -\nlogic diagnosis. The median serum CA-125 and HE4 levels \nwere then recalculated for both groups (Table 2) revealing \nsignificantly higher serum CA-125 levels in the ovarian \nendometrioma group when compared with the patients \nwith other benign ovarian tumors (31.95 U/mL vs. 17.9 U/\nmL, P = 0.03). Conversely, the median serum HE4 levels did \nTable 2. Comparison of the serum human epididymis-specific protein E4 (HE4) and CA-125 levels among patients with ovarian cancer \nversus other benign ovarian tumors\nPathologic diagnosis \nCA-125 (U/mL) HE4 (pM)\nMedian (range) P-value\na)\nMedian (range) P-value\na)\nCancer & benign\nOvarian cancer 394.1 (6.7−12,643) <0.001 56.68 (3.2−867) 0.018\nBenign tumor 22.7 (4.8−306.6)    18.5 (0.2−378)\nOvarian endometrioma & others\nEndometrioma 31.95 (4.9−306.6)   0.030    19 (0.2−378) 0.490\nOther benign ovarian tumors   17.9 (4.8−126.3) 18.2 (5.6−118)\na)\nP-value, calculated using the Wilcoxon signed-rank test.\nTable 1. The demographic and clinical characteristics of the study group\nCharacteristic Ovarian cancer\n(n=32)\nBenign ovarian tumor\n(n=62) P-value\nAge (yr) 49.5 (38-71) 35.5 (13-71) <0.001\nMarital status (%) 29 (90.6)  43 (69.4) 0.008\nMenopause 14 (43.8)    8 (12.9) 0.003\nGravida 3.1 2.0 0.030\nParity 1.9 1.2 0.020\nValues are presented as median (range) or number (%).\n\nwww.ogscience.org 237\nShin Hye Chung, et al. Serum HE4 as a marker of ovarian cancer\nnot vary significantly between groups (19.0 pM vs. 18.2 \npM, P = 0.49). Furthermore, serum CA-125 and HE4 values \nwere compared between patients with ovarian cancer and \nthe ovarian endometrioma subgroup, showing significantly \nelevated serum levels of both biomarkers among the ovarian \ncancer group (CA-125, P = 0.004; HE4, P = 0.001).\nThe ROC curve analysis of the diagnostic performance of \npatients with ovarian cancer revealed a higher AUC with \nborderline significance for HE4 when compared with CA-\n125 (0.93 [95% confidence interval, CI: 0.90−0.97] vs. 0.85 \n[95% CI, 0.77−0.92]) (Fig. 2, Table 3). Additionally, the AUC \nfor the combination of the two serum markers was 0.89 \n(95% CI, 0.83−0.95), but a significant difference was not \nfound when comparing HE4 and CA-125 alone.\nUsing a serum cut-off level of 76.0 pM for HE4 a sensi -\ntivity and specificity of 78.1% and 86.8% was observed. \nUsing a serum cut-off level of 37.45 U/mL for serum CA-\n125, a sensitivity and specificity of 84.4% and 67.4% was \nobserved. \n7.074.5\n2,237.7\n454\n306.6\n170.6\n96.2\n39.4\n27.4\n20.9\n18.5\n10.8\n6.7\nCA-125\nGroup\nCancer Benign\n394.1\n22.7\nP < 0.001\n 378\n256.4\n118\n70\n53.8\n36.8\n21\n19.4\n18.6\n14\n9\n0.2\nHE4\nGroup\nCancer Benign\n56.68\n18.5\nP < 0.05\nA B\nFig. 1. Comparison of  (A) the serum CA-125 and (B) human epididymis-specific protein E4 (HE4) levels between ovarian cancer and be -\nnign ovarian tumor.\nFig. 2. Comparison of the area under the curve from the receiver \noperating characteristics (ROC) curve analysis for serum CA-125 \nand human epididymis-specific protein E4 (HE4) levels.\n1.0\n0.8\n0.6\n0.4\n0.2\n0.0\nSensitivity\n1-Specificity\nROC\nHE4\nCA-125\nCA-125+HE4\nReference line\n0.0          0.2           0.4           0.6          0.8           1.0\nTable 3. Analysis of the receiver operating characteristics (ROC) curve analysis for the serum CA-125 and HE4 levels\nCharacteristic ROC-AUC\n95% Confidence interval\nStandard error P-value\nLower limit Upper limit\nHE4 0.93 0.90 0.97 0.019 <0.001\nCA-125 0.85 0.77 0.92 0.039\nCA-125 and HE4 0.89 0.95 0.030\nHE4, human epididymis-specific protein E4; ROC, receiver operating characteristics; AUC, area under the curve.\n\nwww.ogscience.org238\nVol. 56, No. 4, 2013\nDiscussion\nCA-125 is the most widely used serum biomarker in ovarian \ncancer screening, as the utility of CA-125 in determining \ntreatment response or monitoring recurrent disease status \nhas been established [19]. Previous data indicates that at a \nserum level of 35 U/mL CA-125 has a sensitivity of 73.2% \nand a specificity of 79.2%, which are comparable to other \nbiomarkers in predicting ovarian malignancy [20]. Neverthe-\nless, CA-125 is not only increased in cases of ovarian cancer \nbut also other benign conditions.\nFor these reasons, several novel ovarian cancer tumor \nmarkers have been assessed for use in screening patients \nfor ovarian cancer, including haptoglobin, osteopontin, HE4, \nmesothelin (SMRP), B7-H4, prostasin, macrophage colony \nstimulating factor, vascular endothelial growth factor, sev -\neral interleukins (IL-6, IL-8), eosinophil-derived neurotoxin, \nCOOH-osteopontin fragments, OVX1, lysophophatidic acid, \napolipoprotein A1, and transthyretin [21]. Of these, HE4 \nhas demonstrated high sensitivity and specificity (90% and \n77.6%, respectively) in identifying cases of ovarian cancer. \nIn detecting cases of stage I ovarian cancer, HE4 has the \nhighest sensitivity when compared to CA-125, SMRP , CA-\n72-4, andosteopontin [22].\nThe results presented here suggest a possible role for \nserum HE4 as a diagnostic marker for detecting ovarian can-\ncer. Serum HE4 levels were significantly higher in the ovar -\nian cancer group when compared with patients with other \nbenign ovarian tumors ( P<0.05), and showed comparable \nsensitivities in detecting ovarian cancer to CA-125. \n Moreover, HE4 demonstrated a significantly lower false \npositive rate, especially in cases of other benign ovarian \ndiseases such as endometriosis. Several previous studies \nsuggest that serum levels of HE4 are significantly higher \nin patients with both endometrial and ovarian malignan -\ncies, though not ovarian endometriomas or other types of \nendometriosis. In comparison, serum CA-125 levels were el-\nevated in patients with ovarian cancer, as well as advanced \nendometriosis with peritoneal or deep lesions, and ovarian \nendometriomas, though not in the patients with endometrial \ncancer [23]. \nAs such, we stratified the other benign ovarian tumor \ngroup for ovarian endometriomas, as this common benign \ntumor has been associated with false elevations in serum \nCA-125 level. In a comparison between groups of patients \nwith ovarian endometrioma versus other benign ovarian \ntumors, serum CA-125 but not serum HE4 levels were found \nto be significantly increased in the setting of endometrioma. \nAccordingly, serum HE4 levels had a lower false positive rate \nin the data presented here, as in other recent studies. \nWe also compared AUC for the ROC analysis for CA-125, \nHE4, and the combination of the two markers. Specifically, \nHE4 demonstrated a higher AUC than other two groups in \ndistinguishing benign and malignant pelvic masses. In a pre-\nvious study, risk of ovarian malignancy, showed excellent di-\nagnostic performance for the detection of epithelial ovarian \ncancer in post-menopausal women, but just the dual marker \ncombination of HE4 and CA-125 did not exhibit any greater \naccuracy than HE4 alone [18]. In another cohort of Sweden \nwomen, HE4 seems like a CA-125 for diagnostic marker for \novarian mass, although the AUC for the HE4 ROC curve is \nnot greater that CA-125. Nonetheless, the sensitivity for HE4 \ncombined with CA-125 was greater than two other serum \nHE4 and CA-125 group [24]. Given these findings, HE4 may \nrepresent a useful diagnostic marker for excluding ovarian \ncancer in patients with a known pelvic mass. \nIn the current study, the appropriate cut-off level that \nyielded a higher sensitivity and specificity was 76.0 pM, a \nvalue consistent with data from other studies [18]. \nHerein, we report early data indicating that serum HE4 \nlevels may represent a new marker for identifying ovarian \ncancers in Korean women. Until now, many studies for ovar-\nian mass tried to distinguish between ovarian cancer and \nbenign mass in Korean women. Serum CA-125 levels as \nwell as other methods have also been evaluated for ovarian \nmass screening in Korean women in the past. These previous \nstudies assessed pulsatility index, transvaginal sonographic \nscoring system and CA-125 preoperatively, and transvaginal \nDoppler color flow imaging may be useful clinical tools for \nthe differential diagnosis of malignant ovarian tumors [25]. \nAnother study showed that the combination of CA 15-3, \nTumor-associated glycoprotein (TAG) 72, and CA-125 may \nreach an acceptable sensitivity and excellent specificity in \ndifferentiating malignant from benign pelvic masses, par -\nticularly among patients over 50 years of age. Specifically, \nthese authors collected preoperative serum samples from 78 \npatients with pelvic masses and measured tumor-associated \nantigens CA-125, CA-15-3, and TAG 72 by immunoradio -\nmetric assay in order to evaluate the efficacy of these mark -\ners in differentiating benign and malignant pelvic masses, \n\nwww.ogscience.org 239\nShin Hye Chung, et al. Serum HE4 as a marker of ovarian cancer\nfinding that among patients over 50 years of age the three \nmarker combination was associated with a sensitivity of \n79% and a specificity of 100% [26]. Another study of 56 \nnewly-diagnosed epithelial ovarian cancer patients, showed \nthat of five serum biomarkers–leptin, prolactin, osteoponin, \ninsulin-like growth factor-II and CA-125–only the preopera -\ntive serum CA-125 level had a significant positive correla -\ntion with cancer stage (P<0.01) [27].\nWith specific regard to the association between HE4 and \novarian cancer in Korean women, the initial study recruited \n159 women with adnexal masses, including 78 patients \nwith ovarian cancer [28], as well as 224 healthy controls. In \nthis study, serum HE4 and CA-125 levels were found to be \nsignificantly elevated in the ovarian cancer patients when \ncompared with those from patients with benign disease \nor healthy controls (HE4, 80.0 pM; CA-125, 216.8 U/mL; \nP<0.0001 in both). But no definitive diagnostic threshold \nfor these tests was ever determined in Korean women. So \nwe need to perform more studies about serum HE4 level \nin Korean healthy women, then we may determinate the \nproper definitive diagnostic threshold. A larger case-control \nstudy of Korean females was done in 2011. The population \nof that study comprised 2,182 healthy women, 72 pregnant \nwomen, 66 women with ovarian cancers, and 257 women \nwith benign gynecologic disease. The authors suggested an \nHE4 cut-off level of 33.2 pmol/L for 97% upper reference \nlimits. Using this value as a cutoff point, the sensitivity and \nspecificity for diagnosing ovarian cancer as differentiated \nfrom benign gynecologic disease were 90.9% and 94.1%, \nrespectively. The cut-off HE4 level was different from that in \nour study due to the use of different machines and methods \n[29]. \nThe primary limitation of the present study is the relatively \nsmall size of sample number. As such, no statistical analysis \nwas performed according to the ovarian cancer histological \nsubtype. Furthermore, the serum HE4 and CA-125 levels \nwere also not compared among the healthy controls. A sec -\nond limitation is that age and menstruation status were not \nincluded in the analysis, both of which may have influenced \nthe serum HE4 level. In one prior study, the serum HE4 level \nincreased with increasing age, while serum CA-125 levels \nwere lower in older subjects. However, the upper limits of \nserum HE4 levels did not vary significantly in the individu -\nals without ovarian cancer regardless of menopausal status \n[29]. Given these findings, the differences in age between \nthe two groups may have influenced another demographic \nfactor, and a larger scale age−matched case−control study \nis needed to better characterize this relationship. \nIn conclusion, serum HE4 likely represents a useful tumor \nmarker for ovarian cancer in Korean women. Assessing se -\nrum HE4 levels has the potential to increase the accuracy \nof ovarian cancer screening and provide better information \nin differentiating ovarian cancer from other benign ovarian \ntumors. We did this study as a pilot study and larger, more \nextended studies are needed to confirm the accuracy of se -\nrum HE4 as a tumor marker for the early diagnosis of ovar -\nian cancer in patient with ovarian masses.\n     \nConflict of interest\nNo potential conflict of interest relevant to this article was \nreported.\nAcknowledgments\nThis study was supported by a grant of the Korean Health \nTechnology R&D Project, Ministry of Health & Welfare, Re -\npublic of Korea (A120071). \nReferences\n  1. Ozols RF. Recurrent ovarian cancer: evidence-based \ntreatment. J Clin Oncol 2002;20:1161-3.\n  2. Parkin DM, Pisani P , Ferlay J. Estimates of the worldwide \nincidence of 25 major cancers in 1990. Int J Cancer \n1999;80:827-41.\n  3. 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Correlation between preop-\nerative serum levels of five biomarkers and relationships \nbetween these biomarkers and cancer stage in epithelial \noverian cancer. J Gynecol Oncol 2009;20:169-75.\n28. Kim YM, Whang DH, Park J, Kim SH, Lee SW, Park HA, et \nal. Evaluation of the accuracy of serum human epididy -\nmis protein 4 in combination with CA125 for detecting \n\nwww.ogscience.org 241\nShin Hye Chung, et al. Serum HE4 as a marker of ovarian cancer\novarian cancer: a prospective case-control study in a Ko-\nrean population. Clin Chem Lab Med 2011;49:527-34.\n29. Park Y , Kim Y , Lee EY , Lee JH, Kim HS. Reference ranges \nfor HE4 and CA125 in a large Asian population by auto-\nmated assays and diagnostic performances for ovarian \ncancer. Int J Cancer 2012;130:1136-44.","source_license":"CC0","license_restricted":false}