{"paper_id":"c4477b55-199d-4b3e-a8d4-b7389be7b5c0","body_text":"Braicu et al. BMC Cancer          (2022) 22:831  \nhttps://doi.org/10.1186/s12885-022-09887-5\nRESEARCH\nHE4 as a serum biomarker for the diagnosis \nof pelvic masses: a prospective, multicenter \nstudy in 965 patients\nElena Ioana Braicu1,2*†, Catherine Linn Krause1†, Uwe Torsten3, Herbert Mecke4, Rolf Richter1, \nLars Hellmeyer5, Malgorzata Lanowska6, Bodo Müller7, Elisa Koch1, Janine Boenneß‑Zaloum5, Kerstin Ames3, \nRadoslav Chekerov1, Kati Hasenbein8, Mathias Zimmermann9, Mandy Mangler6, Frank Chen4, \nRudolf Tauber10 and Jalid Sehouli1 \nAbstract \nBackground: To evaluate the diagnostic value of adding human epididymis protein 4 (HE4), cancer antigen 125 \n(CA125) and risk of malignancy algorithm (ROMA) to ultrasound for detecting ovarian cancer in patients with a pelvic \nmass.\nMethods: This was a prospective, observational, multicenter study. Patients aged > 18 years who were scheduled \nto undergo surgery for a suspicious pelvic mass had CA125 and HE4 levels measured prior to surgery, in addition \nto a routine transvaginal ultrasound scan. The diagnostic performance of CA125, HE4 and ROMA for distinguishing \nbetween benign and malignant adnexal masses was assessed using receiver operating characteristic (ROC) analysis \nand the corresponding area under the curve (AUC).\nResults: Of 965 evaluable patients, 804 were diagnosed with benign tumors and 161 were diagnosed with ovarian \ncancer. In late‑stage ovarian cancer, CA125, HE4 and ROMA all had an excellent diagnostic performance (AUC > 0.92), \nwhereas in stage I and II, diagnostic performance of all three biomarkers was less adequate (AUC < 0.77). In the dif‑\nferential diagnosis of ovarian cancer and endometriosis, ROMA and HE4 performed better than CA125 with 99 and \n98.1% versus 75.0% sensitivity, respectively, at 75.4% specificity.\nConclusions: ROMA and HE4 could be valuable biomarkers to help with the diagnosis of ovarian cancer in premeno‑\npausal patients in order to differentiate from endometriosis, whereas CA125 may be more adequate for postmeno‑\npausal patients.\nHighlights \n• Serum biomarkers can help to distinguish benign from malignant pelvic masses\n© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which \npermits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the \noriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or \nother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line \nto the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory \nregulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this \nlicence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco \nmmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.\nOpen Access\n†Elena Ioana Braicu and Catherine Linn Krause contributed equally to this \nwork.\n*Correspondence:  elena.braicu@charite.de\n2 Department of Obstetrics and Gynecology, Stanford University, California, \nUSA\nFull list of author information is available at the end of the article\n\nPage 2 of 11Braicu et al. BMC Cancer          (2022) 22:831 \nIntroduction\nOvarian cancer is a leading cause of mortality from \ngynecological malignancies, with an estimated 313,959 \nnew cases and 207,252 deaths worldwide in 2020 [1 ]. \nThe number of cases diagnosed each year is rising with \nincreasing life expectancy [2 ]. Survival rates for ovarian \ncancer have improved in recent decades, but the over -\nall 5-year survival rate for all disease stages is still only \naround 30% [3 ]. The 5-year survival rate for stage I dis -\nease is far higher at 92%; however, only 15% of cases are \ndiagnosed at this stage and there is no proven method \nfor early detection [4 ].\nOvarian cancer is widely known as a ‘silent killer’ \ndue to the lack of specific symptoms. Although most \npatients experience symptoms in the early stages of \nthe disease, these are often non-specific (for example \nfatigue, bloating and constipation) and are associated \nwith a number of common benign gastrointestinal, gen -\nitourinary and gynecological conditions, making early \ndiagnosis challenging [3 , 5]. Consequently, around 60% \nof patients have metastatic disease at the time of diag -\nnosis [5 ]. The probability of a pelvic mass being benign \nor malignant is the key factor for steering patients to \nthe correct institution and clinician; it has been shown \nthat patients with ovarian cancer treated by a gyneco -\nlogical oncologist are more likely to receive optimal \nsurgery compared with patients treated by gynecolo -\ngists or general surgeons [6 , 7]. It is therefore crucial \nthat patients are referred to the appropriate specialist \nas early as possible to give the best chance of long-term \nsurvival.\nPreoperative diagnostics are still one of the major chal -\nlenges in the clinical routine, as all single methods have \nseveral limitations [8]. The use of serum biomarkers can \nhelp to distinguish benign from malignant masses and \nthus facilitate referral of patients to the most appropri -\nate clinician. Several biomarkers have been evaluated \nfor their potential to discriminate between benign and \nmalignant pelvic masses, including CA125, which is a \nhigh molecular weight transmembrane mucin that is \noverexpressed in 80% of epithelial ovarian cancers [9], \nand HE4, which is a protein secreted by epithelial cells \nthat shows increased expression in the majority of ovar -\nian cancers [10]. In addition to its potential in first diag -\nnosis, there are ongoing studies examining the role of \nHE4 in detecting recurrence [11] and even predicting \noptimal cytoreduction in patients with primary ovarian \ncancer [12, 13].\nOngoing studies suggest that early clearance of serum \nHE4 during chemotherapy correlates with response to \nchemotherapy and thus prognosis [14].\nCA125 and HE4 measurements have also been com -\nbined with menopausal status to develop the risk of \nmalignancy algorithm (ROMA), which has exhibited 94% \nsensitivity for ovarian cancer at 75% specificity [15]. In \npivotal trials, magnetic resonance imaging was the pre -\nferred method despite the fact that in routine clinical \npractice ultrasound is generally the method most fre -\nquently used by gynecologists. Additionally, ultrasound \nis performed by an expert and has a better sensitivity \nand specificity than all biomarkers or algorithms [16]. \nHowever, ultrasound is very subjective and its accuracy \ndepends heavily on the experience of the ultrasound \nexaminer. Furthermore, while ultrasound may detect \nsome cases of ovarian cancer at an early stage, it lacks \nadequate specificity and sensitivity in this setting [3].\nThe aim of this prospective study was to evaluate the \ndiagnostic value of adding HE4, CA125 and ROMA to \ntransvaginal ultrasound (TVUS) for detecting ovarian \ncancer in patients presenting with a pelvic mass. The dif -\nferential diagnosis of endometriosis and early ovarian \ncancer is particularly challenging, as ultrasound features \ncan be difficult to interpret and CA125 is usually elevated \nin both conditions [17]. Therefore, a sub-analysis was also \nperformed to evaluate the ability of HE4 compared with \nCA125 to discriminate between endometriosis and ovar -\nian cancer.\nPatients with Borderline Tumors (BOT) were also \nincluded in the study as the differential diagnosis between \nepithelial ovarian cancer and BOT is difficult to be made \nvia ultrasound [18]. Although BOT patients have a bet -\nter outcome than ovarian cancer patients, comprehensive \nsurgical staging is needed [19]. Therefore it is important \nto identify these patients prior to surgery and to not treat \nBOT as a benign disease.\nMethods\nStudy design\nThis was a prospective, observational, multicenter study \nconducted at seven centers in Berlin, Germany. All cent -\ners are high volume centers which have a high expertise \nin the diagnosis of gynecological masses. CA125 and HE4 \n• We evaluated the diagnostic value of adding HE4, CA125 and ROMA to ultrasound for detecting ovarian cancer\n• In stage III and IV ovarian cancer all three biomarkers showed excellent performance\n• ROMA and HE4 performed better than CA125 in the differential diagnosis of ovarian cancer and endometriosis\nKeywords: Ovarian cancer, Endometriosis, HE4, CA125, Biomarkers\n\nPage 3 of 11\nBraicu et al. BMC Cancer          (2022) 22:831 \n \nlevels were measured prior to surgery in patients with a \npelvic mass to investigate the diagnostic value of HE4, \nCA125 and ROMA for detecting ovarian cancer.\nThe study was conducted in accordance with the prin -\nciples of the Declaration of Helsinki and Good Clinical \nPractice, and institutional review board approval was \ngranted by the local ethics committee of Charité Univer -\nsitätsmedizin Berlin (no. EA2/049/13). All patients pro -\nvided written informed consent for the use of their blood \nsamples for research purposes. Consent was given prior \nto ultrasound and to blood drawing.\nPatients\nEligible patients were females aged ≥18 years who were \nscheduled to undergo surgery for a suspicious adnexal \nmass and who had an available blood sample taken no \nmore than 30 days before surgery. Patients were usually \nreferred by their gynaecologists due to a pelvic mass for \na second opinion and treatment decision. A pelvic mass \nwas defined as suspicious when according to an investi -\ngator surgical management was indicated. Indication for \nsurgery was physician’s choice and might include differ -\nential diagnosis, and exclusion of malignancy, or symp -\ntom control. All included patients received a standard \nTVUS scan. Patients were excluded if they had a previ -\nous diagnosis of ovarian cancer, had undergone bilateral \noophorectomy or were known to be pregnant. Clinical \ndata relating to family history, symptoms, menopausal \nstatus and TVUS findings were documented prior to sur -\ngery and histological results were collected after surgery. \nPatients who met the inclusion criteria but who subse -\nquently did not undergo surgery were withdrawn from \nthe study.\nSample collection\nBlood samples were collected from all enrolled patients \nprior to surgery, shipped to Charité Labor Berlin cen -\ntral lab (Berlin, Germany) within 24 hours and stored at \n− 20 °C. Samples were analyzed for CA125 and HE4 con -\ncentration via electrochemiluminescence immunoassay \n(ECLIA) using a cobas ® 8000 analyzer and Roche Elec -\nsys® CA125 and HE4 assays (Roche Diagnostics GmbH, \nGermany) according to the manufacturer’s instructions.\nStudy endpoints\nThe primary study goal was the comparison of the diag -\nnostic performance of HE4, CA125 and ROMA in com -\nbination with TVUS in detecting ovarian cancer in \npatients with a pelvic mass.\nSecondary study endpoints included evaluation of the \ndiagnostic performance of HE4, CA125 and ROMA in \ncombination with TVUS in detecting stage I–II and stage \nIII–IV ovarian cancer in patients with a pelvic mass. A \nsub-analysis was also performed to evaluate the sensi -\ntivity and specificity of HE4 compared with CA125 as a \nbiomarker to discriminate between endometriosis and \novarian cancer.\nStatistical analysis\nComparison of patients with benign and malignant \nadnexal masses was performed using the chi-square test \nfor categorical variables and the Mann-Whitney U test \nfor continuous variables. The diagnostic performance \nof CA125, HE4 and ROMA for distinguishing between \nbenign and malignant adnexal masses was assessed using \nreceiver operating characteristic (ROC) analysis and the \ncorresponding area under the curve (AUC) with 95% \nconfidence intervals (95% CI). All analyses were per -\nformed with IBM SPSS Statistics Version 25.\nResults\nPatient demographics\nIn total, 1438 patients were enrolled in the study between \nJuly 2013 and December 2015. Of these, 965 patients pro-\nvided laboratory and histology data. Of the 965 evalu -\nable patients, 804 were diagnosed with benign tumors \nand 161 were diagnosed with ovarian cancer (including \n43 patients with borderline tumors). Patient demograph -\nics and disease characteristics are summarised in Table 1. \nPatients who were diagnosed with ovarian cancer were \ngenerally older at diagnosis and had higher CA125 \nand HE4 measurements, compared with patients with \nbenign tumors. The majority of patients with ovarian \ncancer had Fédération Internationale de Gynécologie et \nd’Obstétrique (FIGO) stage IIIC disease (32.3%), and the \nmost common benign tumor was cystadenoma (22.3%).\nDiagnostic performance of CA125, HE4 and ROMA \nin detecting ovarian cancer\nIn premenopausal patients HE4, CA125 and ROMA \nshowed a comparable performance (Fig.  1A). The AUC \nfor HE4 was 0.80 (95% CI 0.74–0.87) versus 0.80 (95% CI \n0.74–0.86) for CA125 versus (AUC 0.81 [95% CI 0.74–\n0.87]) for ROMA. In postmenopausal patients, CA125 \nand ROMA performed slightly better than HE4 (Fig.  1B), \nwith AUCs of 0.89 (95% CI 0.84–0.93) for CA125 and \n0.86 (95% CI 0.84–0.93) for ROMA, compared with 0.82 \n(95% CI 0.77–0.88) for HE4.\nDiagnostic performance of CA125, HE4 and ROMA \nin detecting early‑stage ovarian cancer\nIn premenopausal patients, performance of all biomark -\ners was nearly equivalent. AUCs were 0.73 (95% CI \n0.65–0.81) for CA125, 0.74 (95% CI 0.65–0.82) for HE4 \nand 0.74 (95% CI 0.66–0.83) for ROMA (Fig.  2A). In \npostmenopausal patients, CA125 and ROMA performed \n\nPage 4 of 11Braicu et al. BMC Cancer          (2022) 22:831 \nslightly better than HE4 in the detection of stage I and \nstage II disease (Fig.  2B). AUCs were 0.77 (95% CI 0.68–\n0.86) for CA125 and 0.74 (95% CI 0.64–0.83) for ROMA, \ncompared with 0.62 (95% CI 0.50–0.74) for HE4.\nDiagnostic performance of CA125, HE4 and ROMA \nin detecting late‑stage ovarian cancer\nIn premenopausal patients, performance of all biomark -\ners was nearly equivalent (Fig.  3A). AUCs were 0.94 (95% \nCI 0.87–1.00) for CA125 and 0.93 (95% CI 0.88–0.98) for \nHE4 and 0.94 for ROMA [95% CI 0.90–0.98]). Similarly, \nin postmenopausal patients, ROMA, CA125 and HE4 \nperformed nearly equivalent in detecting late-stage ovar -\nian cancer (AUCs 0.96 [95% CI 0.93–0.99], 0.94 [95% CI \n0.91–0.98] and 0.92 [95% CI 0.88–0.97], respectively) \n(Fig. 3B).\nDiagnostic performance of HE4 compared with CA125 \nto distinguish between ovarian cancer and endometriosis\nIn the sub-analysis to evaluate the sensitivity and speci -\nficity of HE4 compared with CA125 as a biomarker to \ndiscriminate between endometriosis and ovarian cancer, \nTable 1 Patient demographics and disease characteristics\nFIGO Fédération Internationale de Gynécologie et d’Obstétrique, HE4 Human epididymis protein 4\na Patients aged < 18 years were not included\nAll (N = 965) Benign tumors (N = 804) Ovarian cancer and \nborderline tumors \n(N = 161)\nMedian age at diagnosis, years \n(range)\n48 (18–86) (n =  853a) 45 (18–86) (n =  704a) 59 (27–84) (n =  149a)\nMenopausal status, n (%)\n Premenopausal 549 (56.9) 501 (62.3) 48 (29.8)\n Postmenopausal 362 (37.5) 251 (31.2) 111 (68.9)\n Unknown 54 (5.6) 52 (6.5) 2 (1.2)\nMedian biomarker measurements, U/mL (range)\n CA125 18.4 (2–11,616) 16.1 (2–6522) 159.2 (6.1–11,616)\n HE4 54.52 (13.19–5039) 51.61 (13.19–5039) 149.50 (28.28–4676)\nFIGO stage, n (%)\n IA 44 (4.6) – 44 (27.3)\n IB 3 (0.3) – 3 (1.9)\n IC 12 (1.2) – 12 (7.5)\n IIA 3 (0.3) – 3 (1.9)\n IIB 2 (0.2) – 2 (1.2)\n IIC 4 (0.4) – 4 (2.5)\n IIIA 4 (0.4) – 4 (2.5)\n IIIB 7 (0.7) – 7 (4.3)\n IIIC 52 (5.4) – 52 (32.3)\n IV 30 (3.1) – 30 (18.6)\nBenign tumor type, n (%)\n Cystadenoma 179 (18.5) 179 (22.3) –\n Functional cyst 165 (17.1) 165 (20.5) –\n Endometriosis 109 (11.3) 109 (13.6) –\n Cystadenofibroma 47 (4.9) 47 (5.8) –\n Dermoid cyst 44 (4.6) 44 (5.5) –\n Inclusion cyst 44 (4.6) 44 (5.5) –\n Teratoma 34 (3.5) 34 (4.2) –\n Endometrioid tumor 20 (2.1) 20 (2.5) –\n Tubo‑ovarian abscess 17 (1.8) 17 (2.1) –\n Fibroma 15 (1.6) 15 (1.9) –\n Benign Brenner tumor 5 (0.5) 5 (0.6) –\n Other 124 (12.8) 125 (15.2) –\n\nPage 5 of 11\nBraicu et al. BMC Cancer          (2022) 22:831 \n \n114 patients were diagnosed with epithelial ovarian can -\ncer and 104 patients were diagnosed with endometriosis. \nIn these patients, HE4 performed better than CA125 in \nthe differential diagnosis of ovarian cancer and endo -\nmetriosis (Fig.  4). AUCs were 0.91 (95% CI 0.87–0.95) \nfor HE4 and 0.81 (95% CI 0.75–0.87) for CA125. ROMA \noutperformed HE4 and CA125 with an AUC of 0.95 (95% \nCI 0.92–0.98). At a predefined specificity of 75.4%, the \nrespective sensitivities for CA125, HE4 and ROMA were \n75.0, 98.1 and 99.0%.\nDiagnostic performance of CA125, HE4 and ROMA \nto distinguish between early ovarian cancer \nand endometriosis\nIn the sub-analysis to evaluate the sensitivity and specificity \nof CA125 and HE4 compared with ROMA to discriminate \nbetween endometriosis and early ovarian cancer, 30 patients \nFig. 1 Diagnostic performance of CA125, HE4 and ROMA for detection of ovarian cancer in premenopausal (A) and postmenopausal (B) patients\n\nPage 6 of 11Braicu et al. BMC Cancer          (2022) 22:831 \nwere diagnosed with early epithelial ovarian cancer FIGO \nI-II and 104 patients were diagnosed with endometriosis. \nThe best sensitivity and specificity in identifying early stage \novarian cancer from endometriosis patients was shown by \nthe ROMA algorithm (Fig. 5). The results showed an AUC \nof 0.647 (p   = 0.015, 95%CI: 0.52–0.77), 0.804 (p   < 0.001, \n95%CI: 0.703–0.903) and 0.865 (p   < 0.001, 95%CI: 0.788–\n0.942) for CA125, HE4 and ROMA, respectively.\nDiscussion\nSerum biomarkers can help to distinguish benign tumors \nfrom malignant pelvic masses, ensuring that patients are \nquickly directed to the most appropriate clinician [3]. \nPatients who present with features suggestive of a benign \npelvic mass can be managed successfully and cost effi -\nciently by a gynecologist or a general surgeon, whereas \npatients with features suggestive of malignancy should be \nFig. 2 Diagnostic performance of CA125, HE4 and ROMA for detection of Stage I and Stage II ovarian cancer in premenopausal (A) and \npostmenopausal (B) patients\n\nPage 7 of 11\nBraicu et al. BMC Cancer          (2022) 22:831 \n \nreferred to a specially-trained and experienced gyneco -\nlogical oncologist for evaluation, as management of ovar -\nian cancer by a specialist significantly improves patient \noutcomes [3].\nIt is well established that serum CA125 levels can be \nelevated in patients with ovarian cancer, but this bio -\nmarker has a low sensitivity in the early stages of dis -\nease and raised CA125 levels have also been observed in \nother physiological or pathological conditions, including \nmenstruation, pregnancy, endometriosis and inflamma -\ntory diseases of the peritoneum [17]. HE4 has also been \nshown to be a potential diagnostic biomarker for ovar -\nian cancer. It is overexpressed in ovarian cancer [20] and \nwhen compared with multiple biomarkers (including \nCA125), HE4 demonstrated the highest sensitivity for \ndistinguishing ovarian cancer from benign disease (72.9% \nat 95% specificity) [15]. CA125 and HE4 have also been \nshown to have a potential role in predicting recurrence \nFig. 3 Diagnostic performance of CA125, HE4 and ROMA for detection of Stage III and Stage IV ovarian cancer in premenopausal (A) and \npostmenopausal (B) patients\n\nPage 8 of 11Braicu et al. BMC Cancer          (2022) 22:831 \nFig. 4 Diagnostic performance of CA125, HE4 and ROMA for the differential diagnosis of ovarian cancer and endometriosis\nFig. 5 Diagnostic performance of CA125, HE4 and ROMA for the differential diagnosis of early stage ovarian cancer and endometriosis\n\nPage 9 of 11\nBraicu et al. BMC Cancer          (2022) 22:831 \n \nafter treatment, survival after a recurrence, and surgical \noutcome [21–23] and CA125 has demonstrated diagnos -\ntic value in borderline tumors of the ovary [24].\nTo our knowledge, we report the largest prospec -\ntive study which combined biomarker data with vaginal \nultrasound. We demonstrated that in late-stage ovarian \ncancer, all of the evaluated markers (CA125, HE4 and \nROMA) had an excellent diagnostic value independent \nof menopausal status (all AUCs > 0.92). This data is in \nline with other meta- analyses which report AUCs rang -\ning from 0.78 to 0.90 for CA125, 0.89 to 0.93 for HE4 and \n0.84 to 0.96 for ROMA [25].\nHowever, in patients with early-stage cancer, the \ndiagnostic value was less accurate (all AUCs < 0.75). In \npremenopausal patients with stage I or II disease, no \nsignificant difference was observed between the three \nparameters. In postmenopausal patients, HE4 alone was \noutperformed by CA125 and ROMA (AUC 0.62 versus \n0.77 and 0.74, respectively).\nThis reflects the dilemma that early-stage ovarian can -\ncer is extremely difficult to diagnose by imaging or tumor \nmarkers and that a screening or early detection program \nis not at hand. Additionally, the cost-benefit relationship \nfavors the application of vaginal ultrasound.\nIn postmenopausal patients, CA125 and ROMA con -\nsistently outperformed HE4 alone, indicating that ROMA \nand CA125 may be the biomarkers of choice for detect -\ning ovarian cancer in this subgroup of patients. Our find -\nings suggest that in young patients with unclear imaging \nresults, HE4 and ROMA could be used as biomarkers to \nprovide additional information on the likelihood of ovar -\nian cancer being present.\nPrevious reports have shown HE4 to be more reliable \nthan CA125 in diagnosing ovarian cancer [26– 29] and in \na study of multiple biomarkers, HE4 was the best single \nmarker for stage I disease [30]. In line with our findings, a \nrecent multicenter Italian study evaluating biomarker diag-\nnostic performance in 387 patients reported sensitivities of \n69.6% for HE4 versus 65.2% for CA125 in premenopausal \npatients, and 78% for HE4 versus 88% for CA125 in post -\nmenopausal patients (all at 98% specificity) [26], further \nsuggesting that HE4 may be most informative when used \nin younger patients. Combining CA125 with HE4 has been \nreported to produce higher sensitivity (76.4% versus 72.9% \nfor HE4 alone), suggesting that a combination of the two \nbiomarkers may provide a more accurate prediction for \nmalignancy than either biomarker alone [30]. Indeed, high \nAUCs have been reported with the combination of HE4 \nand CA125, varying from 0.91 to 0.96 [30, 31]. However, a \nrecent study looking at combined use of these biomarkers \nspecifically in postmenopausal women reported no added \nvalue when HE4 was added to CA125, again suggesting \nthat HE4 is best used in premenopausal patients [31].\nSeveral algorithms have been established which com -\nbine age, menopausal status, imaging data and serum \nbiomarker measurements to estimate the risk of a mass \nbeing malignant. These include: the risk of malignancy \nindex (RMI), which combines ultrasound, menopau -\nsal status and serum CA125 levels; OVA1, which com -\nbines data from imaging, menopausal status and CA125 \nlevels with four additional biomarkers (apolipoprotein \nA1, transthyretin, transferrin and β2-macroglobulin); \nand ROMA, which combines menopausal status with \nCA125 and HE4 levels [32–34]. In this study, we used \nROMA, which has a reported sensitivity of 92% in post -\nmenopausal women and 77% in premenopausal women \n(both at 75% specificity) [34]. Some studies have con -\nfirmed the predictive value of ROMA in the detection of \novarian cancer [28, 35, 36], while others have reported \nthat ROMA performs no better than either CA125 or \nHE4 alone [37, 38]. In the current study, we observed \nthat the combination of HE4 levels, CA125 levels and \nmenopausal status in the ROMA score outperformed \neither biomarker alone in premenopausal patients in \nterms of overall, early-stage and late-stage ovarian can -\ncer. A recent study reported that the predictive power \nof ROMA is not significantly better than that of HE4 in \npremenopausal women (AUC 0.731 versus 0.732, respec -\ntively) or than that of CA125 in postmenopausal women \n(AUC 0.871 versus 0.888) [39].\nIn our sub-analysis of patients diagnosed with epi -\nthelial ovarian cancer or endometriosis, HE4 showed a \nhigher sensitivity than CA125 in distinguishing between \nthe two conditions (98.1% versus 75.0%, respectively, at \n75.4% specificity). This reflects evidence from previous \nstudies showing HE4 to be a better biomarker in this set -\nting, with similar sensitivity to CA125 (82–87% for HE4 \nversus 82–90% for CA125) but higher specificity (100% \nfor HE4 versus 49–70% for CA125) [40, 41]. The better \nperformance of HE4 is due to the fact that CA125 levels \nare often elevated in both ovarian cancer and endome -\ntriosis, whereas HE4 levels remain stable in endometri -\nosis [42]. In our study, ROMA outperformed both HE4 \nand CA125 in this setting, with 99.0% sensitivity at 75.4% \nspecificity. This was comparable in all ovarian cancer \nstages and early ovarian cancer FIGO I-II compared to \nendometriosis.\nMore research is needed into the use of HE4 for the \ndetection of ovarian cancer. Notably, cut-off values \nneed to be validated, with separate defined values for \npremenopausal and postmenopausal patients. Studies \nto date have not provided a universal HE4 reference \nrange for healthy women from different populations, \nand values used by different laboratories vary [43]. It is \nalso noteworthy that unlike CA125, HE4 levels may be \nincreased by smoking and decreased by the use of oral \n\nPage 10 of 11Braicu et al. BMC Cancer          (2022) 22:831 \ncontraception [44, 45]. Therefore, these lifestyle factors \nshould be taken into account when interpreting HE4 \nmeasurements.\nIn the present study we did not systematically analyse \nthe dynamics of the biomarkers; therefore, future tri -\nals should include preoperative analyses of CA125 and \nHE4 to evaluate whether this additional information \ncan increase the ability of these biomarkers to discrimi -\nnate between benign and malignant pelvic masses.\nIn conclusion, the results presented here add to exist -\ning evidence that ROMA and HE4 could be valuable \nbiomarkers to assist with the diagnosis of ovarian can -\ncer in premenopausal patients. The use of HE4 meas -\nurements and ROMA calculation in this setting may \nhelp to facilitate referral of patients to the appropriate \nspecialist to give the best chance of optimal treatment \nand long-term survival. In postmenopausal patients, \nCA125 may be the most accurate marker.\nAbbreviations\nHE4: Human epididymis protein 4; CA125: Cancer antigen 125; ROMA: Risk \nof malignancy algorithm; AUC : Area under the curve; TVUS: Transvaginal \nultrasound; ECLIA: Electrochemiluminescence immunoassay; ROC: Receiver \noperating characteristic; FIGO: Fédération Internationale de Gynécologie et \nd’Obstétrique; BOT: Borderline Tumor.\nAcknowledgments\nMedical writing support was provided by Angela Corstorphine and Sian‑Marie \nLucas of Kstorfin Medical Communications Ltd. and funded by Charité Univer‑\nsitätsmedizin Berlin, Department of Gynecology, Berlin, Germany.\nElena Ioana Braicu was a fellow of the clinical scientist programme of the Ber‑\nliner Institute of Health (BIH) and Charité at the time of the study. She is also a \nFeodor Lynen fellow of the Humboldt Foundation.\nAuthors’ contributions\nE.I.B. and J.S. designed the research, provided patient samples and intellectual \ninput; C.L.K. helped writing and editing the paper; R.R. analyzed data; M.Z. and \nR.T. performed HE4/CA125 analysis and helped reviewing the manuscript, U.T., \nH.M., M.L., B.M., E.K., J.B.‑Z., K.A., R.C., K.H., M.M., F.C. provided patient samples \nand helped reviewing the manuscript. All authors approved the final version \nto be submitted.\nFunding\nOpen Access funding enabled and organized by Projekt DEAL. This investiga‑\ntor‑initiated study was funded by Roche Diagnostics International Ltd.\nAvailability of data and materials\nThe data that support the findings of this study are available within TOC \nBiobank but restrictions apply to the availability of these data, which were \nused under license for the current study, and so are not publicly available. \nData are however available from the authors upon reasonable request.\nDeclarations\nEthics approval and consent to participate\nThe study was conducted in accordance with the principles of the Declaration \nof Helsinki and Good Clinical Practice, and institutional review board approval \nwas granted by the local ethics committee of Charité Universitätsmedizin \nBerlin (no. EA2/049/13). A signed consent to participate to the study was \nprovided by the patient.\nConsent for publication\nNot applicable.\nCompeting interests\nNone.\nAuthor details\n1 Department of Gynecology with Center of Oncological Surgery, Charité – \nUniversitätsmedizin Berlin, Freie Universität Berlin, Humboldt‑Universität zu \nBerlin, and Berlin Institute of Health, Campus Virchow Klinikum, Augusten‑\nburger Platz 1, 13353 Berlin, Germany. 2 Department of Obstetrics and Gyne‑\ncology, Stanford University, California, USA. 3 Department for Gynecology, \nVivantes Klinikum Neukölln, Berlin, Germany. 4 Department for Obstetrics \nand Gynecology, AVK Vivantes, Berlin, Germany. 5 Department for Obstetrics \nand Gynecolgy, Vivantes Klinikum Friedrichshain, Berlin, Germany. 6 Depart‑\nment of Gynecology, Charité – Universitätsmedizin Berlin, Freie Universität \nBerlin, Humboldt‑Universität zu Berlin, and Berlin Institute of Health, Campus \nCharité Mitte, Berlin, Germany. 7 Department for Gynecology, Vivantes Klinikum \nKaulsdorf, Berlin, Germany. 8 Department for Gynecology, Vivantes Klinikum \nHumboldthain, Berlin, Germany. 9 Central Institute of Laboratory Medicine, \nDRK Kliniken Berlin, Berlin, Germany. 10 Charité – Universitätsmedizin Berlin, \nFreie Universität Berlin, Humboldt‑Universität zu Berlin, and Berlin Institute \nof Health, Institute of Laboratory Medicine, Clinical Chemistry and Pathobio‑\nchemistry and Labor Berlin Charité, Berlin, Germany. \nReceived: 15 November 2021   Accepted: 6 July 2022\nReferences\n 1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. \nGlobal Cancer statistics 2020: GLOBOCAN estimates of incidence and \nmortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. \n2021;71(3):209–49.\n 2. Webb PM, Jordan SJ. Epidemiology of epithelial ovarian cancer. Best Pract \nRes Clin Obstet Gynaecol. 2017;41:3–14.\n 3. Yang WL, Lu Z, Bast RC Jr. The role of biomarkers in the management of \nepithelial ovarian cancer. Expert Rev Mol Diagn. 2017;17(6):577–91.\n 4. Reid BM, Permuth JB, Sellers TA. Epidemiology of ovarian cancer: a review. \nCancer Biol Med. 2017;14(1):9–32.\n 5. Doubeni CA, Doubeni AR, Myers AE. Diagnosis and management of ovar‑\nian cancer. Am Fam Physician. 2016;93(11):937–44.\n 6. Earle CC, Schrag D, Neville BA, Yabroff KR, Topor M, Fahey A, et al. Effect of \nsurgeon specialty on processes of care and outcomes for ovarian cancer \npatients. J Natl Cancer Inst. 2006;98(3):172–80.\n 7. du Bois A, Rochon J, Pfisterer J, Hoskins WJ. Variations in institutional \ninfrastructure, physician specialization and experience, and outcome in \novarian cancer: a systematic review. Gynecol Oncol. 2009;112(2):422–36.\n 8. Kawakami E, Tabata J, Yanaihara N, Ishikawa T, Koseki K, Iida Y, et al. Appli‑\ncation of artificial intelligence for preoperative diagnostic and prognostic \nprediction in epithelial ovarian Cancer based on blood biomarkers. Clin \nCancer Res. 2019;25(10):3006–15.\n 9. Bast RC Jr, Klug TL, St John E, Jenison E, Niloff JM, Lazarus H, et al. A \nradioimmunoassay using a monoclonal antibody to monitor the course \nof epithelial ovarian cancer. N Engl J Med. 1983;309(15):883–7.\n 10. Welsh JB, Zarrinkar PP , Sapinoso LM, Kern SG, Behling CA, Monk BJ, et al. \nAnalysis of gene expression profiles in normal and neoplastic ovarian tis‑\nsue samples identifies candidate molecular markers of epithelial ovarian \ncancer. Proc Natl Acad Sci U S A. 2001;98(3):1176–81.\n 11. Capriglione S, Luvero D, Plotti F, Terranova C, Montera R, Scaletta G, et al. \nOvarian cancer recurrence and early detection: may HE4 play a key role \nin this open challenge? A systematic review of literature. Med Oncol. \n2017;34(9) Available from: https:// pubmed. ncbi. nlm. nih. gov/ 28825 178/. \nCited 2022 Apr 27.\n 12. Feng LY, Liao S, bin, Li L. Preoperative serum levels of HE4 and CA125 \npredict primary optimal cytoreduction in advanced epithelial ovarian \ncancer: a preliminary model study. J Ovarian Res. 2020;13(1) Available \nfrom: https:// pubmed. ncbi. nlm. nih. gov/ 32050 995/. Cited 2022 Apr 27.\n 13. Angioli R, Plotti F, Capriglione S, Aloisi A, Montera R, Luvero D, et al. Can \nthe preoperative HE4 level predict optimal cytoreduction in patients with \nadvanced ovarian carcinoma? Gynecol Oncol. 2013;128(3):579–83 Avail‑\nable from: https:// pubmed. ncbi. nlm. nih. gov/ 23220 563/. Cited 2022 Apr \n27.\n\nPage 11 of 11\nBraicu et al. BMC Cancer          (2022) 22:831 \n \n 14. Rong Y, Li L. Early clearance of serum HE4 and CA125 in predicting plati‑\nnum sensitivity and prognosis in epithelial ovarian cancer. J Ovarian Res. \n2021;14(1) Available from: https:// pubmed. ncbi. nlm. nih. gov/ 33397 458/. \nCited 2022 Apr 27.\n 15. Moore RG, Miller MC, Disilvestro P , Landrum LM, Gajewski W, Ball JJ, et al. \nEvaluation of the diagnostic accuracy of the risk of ovarian malignancy \nalgorithm in women with a pelvic mass. Obstet Gynecol. 2011;118(2 Pt \n1):280–8.\n 16. Froyman W, Timmerman D. Methods of assessing ovarian masses: inter‑\nnational ovarian tumor analysis approach. Obstet Gynecol Clin N Am. \n2019;46(4):625–41.\n 17. Dochez V, Caillon H, Vaucel E, Dimet J, Winer N, Ducarme G. Biomarkers \nand algorithms for diagnosis of ovarian cancer: CA125, HE4, RMI and \nROMA, a review. J Ovarian Res. 2019;12(1):28.\n 18. Timmerman D, Valentin L, Bourne TH, Collins WP , Verrelst H, Vergote \nI. Terms, definitions and measurements to describe the sonographic \nfeatures of adnexal tumors: a consensus opinion from the interna‑\ntional ovarian tumor analysis (IOTA) group. Ultrasound Obstet Gynecol. \n2000;16(5):500–5.\n 19. Coumbos A, Sehouli J, Chekerov R, Schaedel D, Oskay‑Oezcelik G, Lichte‑\nnegger W, et al. Clinical management of borderline tumours of the ovary: \nresults of a multicentre survey of 323 clinics in Germany. Br J Cancer. \n2009;100(11):1731–8.\n 20. Drapkin R, von Horsten HH, Lin Y, Mok SC, Crum CP , Welch WR, et al. \nHuman epididymis protein 4 (HE4) is a secreted glycoprotein that is \noverexpressed by serous and endometrioid ovarian carcinomas. Cancer \nRes. 2005;65(6):2162–9.\n 21. Nassir M, Guan J, Luketina H, Siepmann T, Rohr I, Richter R, et al. The role \nof HE4 for prediction of recurrence in epithelial ovarian cancer patients‑\nresults from the OVCAD study. Tumour Biol. 2016;37(3):3009–16.\n 22. Braicu EI, Chekerov R, Richter R, Pop C, Nassir M, Loefgren H, et al. HE4 \nexpression in plasma correlates with surgical outcome and overall \nsurvival in patients with first ovarian cancer relapse. Ann Surg Oncol. \n2014;21(3):955–62.\n 23. Parashkevova A, Sehouli J, Richter R, Dimitrova D, Braicu EI, Muallem \nMZ. Preoperative CA‑125 value as a predictive factor for postoperative \noutcome in first relapse of platinum‑sensitive serous ovarian Cancer. \nAnticancer Res. 2018;38(8):4865–70.\n 24. Braicu EI, Van Gorp T, Nassir M, Richter R, Chekerov R, Gasimli K, et al. \nPreoperative HE4 and ROMA values do not improve the CA125 diagnos‑\ntic value for borderline tumors of the ovary (BOT) ‑ a study of the TOC \nconsortium. J Ovarian Res. 2014;7:49.\n 25. Dochez V, Caillon H, Vaucel E, Dimet J, Winer N, Ducarme G. Biomarkers \nand algorithms for diagnosis of ovarian cancer: CA125, HE4, RMI and \nROMA, a review. J Ovarian Res. 2019;12(1) Available from: /pmc/articles/\nPMC6436208/. Cited 2022 Apr 27.\n 26. Romagnolo C, Leon AE, Fabricio ASC, Taborelli M, Polesel J, Del Pup L, \net al. HE4, CA125 and risk of ovarian malignancy algorithm (ROMA) as \ndiagnostic tools for ovarian cancer in patients with a pelvic mass: an Ital‑\nian multicenter study. Gynecol Oncol. 2016;141(2):303–11.\n 27. Wilailak S, Chan KK, Chen CA, Nam JH, Ochiai K, Aw TC, et al. Distinguish‑\ning benign from malignant pelvic mass utilizing an algorithm with \nHE4, menopausal status, and ultrasound findings. J Gynecol Oncol. \n2015;26(1):46–53.\n 28. Molina R, Escudero JM, Auge JM, Filella X, Foj L, Torne A, et al. HE4 a \nnovel tumour marker for ovarian cancer: comparison with CA 125 and \nROMA algorithm in patients with gynaecological diseases. Tumour Biol. \n2011;32(6):1087–95.\n 29. Ruggeri G, Bandiera E, Zanotti L, Belloli S, Ravaggi A, Romani C, et al. \nHE4 and epithelial ovarian cancer: comparison and clinical evaluation \nof two immunoassays and a combination algorithm. Clin Chim Acta. \n2011;412(15–16):1447–53.\n 30. Moore RG, Brown AK, Miller MC, Skates S, Allard WJ, Verch T, et al. The use \nof multiple novel tumor biomarkers for the detection of ovarian carci‑\nnoma in patients with a pelvic mass. Gynecol Oncol. 2008;108(2):402–8.\n 31. Chen X, Zhou H, Chen R, He J, Wang Y, Huang L, et al. Development of \na multimarker assay for differential diagnosis of benign and malignant \npelvic masses. Clin Chim Acta. 2015;440:57–63.\n 32. Jacobs I, Oram D, Fairbanks J, Turner J, Frost C, Grudzinskas JG. A risk of \nmalignancy index incorporating CA 125, ultrasound and menopausal \nstatus for the accurate preoperative diagnosis of ovarian cancer. Br J \nObstet Gynaecol. 1990;97(10):922–9.\n 33. van den Akker PA, Aalders AL, Snijders MP , Kluivers KB, Samlal RA, Volle‑\nbergh JH, et al. Evaluation of the risk of malignancy index in daily clinical \nmanagement of adnexal masses. Gynecol Oncol. 2010;116(3):384–8.\n 34. Moore RG, McMeekin DS, Brown AK, DiSilvestro P , Miller MC, Allard WJ, \net al. A novel multiple marker bioassay utilizing HE4 and CA125 for the \nprediction of ovarian cancer in patients with a pelvic mass. Gynecol \nOncol. 2009;112(1):40–6.\n 35. Bandiera E, Romani C, Specchia C, Zanotti L, Galli C, Ruggeri G, et al. \nSerum human epididymis protein 4 and risk for ovarian malignancy algo‑\nrithm as new diagnostic and prognostic tools for epithelial ovarian cancer \nmanagement. Cancer Epidemiol Biomark Prev. 2011;20(12):2496–506.\n 36. Kim YM, Whang DH, Park J, Kim SH, Lee SW, Park HA, et al. Evaluation of \nthe accuracy of serum human epididymis protein 4 in combination with \nCA125 for detecting ovarian cancer: a prospective case‑control study in a \nKorean population. Clin Chem Lab Med. 2011;49(3):527–34.\n 37. Montagnana M, Danese E, Ruzzenente O, Bresciani V, Nuzzo T, Gelati M, \net al. The ROMA (risk of ovarian malignancy algorithm) for estimating the \nrisk of epithelial ovarian cancer in women presenting with pelvic mass: is \nit really useful? Clin Chem Lab Med. 2011;49(3):521–5.\n 38. Van Gorp T, Cadron I, Despierre E, Daemen A, Leunen K, Amant F, et al. \nHE4 and CA125 as a diagnostic test in ovarian cancer: prospective \nvalidation of the risk of ovarian malignancy algorithm. Br J Cancer. \n2011;104(5):863–70.\n 39. Han KH, Park NH, Kim JJ, Kim S, Kim HS, Lee M, et al. The power of the \nrisk of ovarian malignancy algorithm considering menopausal status: a \ncomparison with CA 125 and HE4. J Gynecol Oncol. 2019;30(6):e83.\n 40. Nikolova T, Zivadinovic R, Evtimovska N, Klisarovska V, Stanojevic M, Geor‑\ngievska J, et al. Diagnostic performance of human epididymis protein \n4 compared to a combination of biophysical and biochemical markers \nto differentiate ovarian endometriosis from epithelial ovarian cancer in \npremenopausal women. J Obstet Gynaecol Res. 2017;43(12):1870–9.\n 41. Anastasi E, Granato T, Falzarano R, Storelli P , Ticino A, Frati L, et al. The use \nof HE4, CA125 and CA72‑4 biomarkers for differential diagnosis between \novarian endometrioma and epithelial ovarian cancer. J Ovarian Res. \n2013;6(1):44.\n 42. Huhtinen K, Suvitie P , Hiissa J, Junnila J, Huvila J, Kujari H, et al. Serum HE4 \nconcentration differentiates malignant ovarian tumours from ovarian \nendometriotic cysts. Br J Cancer. 2009;100(8):1315–9.\n 43. Gasiorowska E, Kluz T, Lipski D, Warchol W, Tykarski A, Nowak‑Markwitz E. \nHuman epididymis protein 4 (HE4) reference limits in polish population \nof healthy women, pregnant women, and women with benign ovarian \ntumors. Dis Markers. 2019;2019:3890906.\n 44. Fortner RT, Vitonis AF, Schock H, Husing A, Johnson T, Fichorova RN, et al. \nCorrelates of circulating ovarian cancer early detection markers and their \ncontribution to discrimination of early detection models: results from the \nEPIC cohort. J Ovarian Res. 2017;10(1):20.\n 45. Ferraro S, Schiumarini D, Panteghini M. Human epididymis protein 4: fac‑\ntors of variation. Clin Chim Acta. 2015;438:171–7.\nPublisher’s Note\nSpringer Nature remains neutral with regard to jurisdictional claims in pub‑\nlished maps and institutional affiliations.","source_license":"CC0","license_restricted":false}