Microarray Glycoprofiling of CA125 Improves Differential Diagnosis of

Article pubs.acs.org/jpr

Microarray Glycoprofiling of CA125 Improves Differential Diagnosis of Ovarian Cancer Kowa Chen,†,‡ Aleksandra Gentry-Maharaj,‡,§ Matthew Burnell,§ Catharina Steentoft,† Lara Marcos-Silva,†,∥ Ulla Mandel,† Ian Jacobs,§,⊥ Anne Dawnay,# Usha Menon,*,‡,§ and Ola Blixt*,†,‡ †

Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark § Gynaecological Cancer Research Centre, Women’s Cancer, University College London EGA Institute for Women’s Health, 149 Tottenham Court Road, London W1T 7DN, United Kingdom # Clinical Biochemistry, University College London Hospitals, London W1T 4 EU, United Kingdom ⊥ Faculty of Medical and Human Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom ∥ IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Portugal S Supporting Information *

ABSTRACT: The CA125 biomarker assay plays an important role in the diagnosis and management of primary invasive epithelial ovarian/tubal cancer (iEOC). However, a fundamental problem with CA125 is that it is not cancer-specific and may be elevated in benign gynecological conditions such as benign ovarian neoplasms and endometriosis. Aberrant O-glycosylation is an inherent and specific property of cancer cells and could potentially aid in differentiating cancer from these benign conditions, thereby improving specificity of the assay. We report on the development of a novel microarraybased platform for profiling specific aberrant glycoforms, such as Neu5Acα2,6GalNAc (STn) and GalNAc (Tn), present on CA125 (MUC16) and CA15-3 (MUC1). In a blinded cohort study of patients with an elevated CA125 levels (30−500 kU/L) and a pelvic mass from the UK Ovarian Cancer Population Study (UKOPS), we measured STn-CA125, ST-CA125 and STn-CA15-3. The combined glycoform profile was able to distinguish benign ovarian neoplasms from invasive epithelial ovarian/tubule cancer (iEOCs) with a specificity of 61.1% at 90% sensitivity. The findings suggest that microarray glycoprofiling could improve differential diagnosis and significantly reduce the number of patients elected for further testing. The approach warrants further investigation in other cancers. KEYWORDS: CA125, glycosylation, differential diagnosis, antibody microarray, ovarian cancer

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INTRODUCTION Aberrant O-glycosylation is an inherent and specific hallmark of cancer cells and could potentially aid in the differentiation of healthy from cancer patients.1 Assays to detect current cancer antigens such as CA125 for primary invasive epithelial ovarian/ tubal cancers (iEOC) use protein specific monoclonal antibodies (mAbs) and have moderate sensitivity and specificity as the marker is not truly cancer specific. Levels are elevated in some benign gynecological conditions such as benign neoplasms, fibroids and endometriosis as well as in inflammatory disease and physiological states such as menstruation and pregnancy. The difficulty in differential diagnosis applies especially to moderately elevated CA125 levels in the range of 30−500 kU/L as higher levels strongly correlate with malignancy. Patients with elevated CA125, as measured by the current CA125 commercial kits therefore require further investigations in the form of imaging2,3 and additional markers.4 The latter efforts including CA15−3 based on the mucin 1 (MUC1) antigen,5 have had limited success. © 2013 American Chemical Society

The only potential new protein marker so far that might add to the clinical utility of CA125 is Human Epididymis Protein 4 (HE4), which performs better in not detecting CA125 in patients with endometriosis.4,6−8 Numerous indices such as Risk of Malignancy Index9,10 and Risk of Ovarian Malignancy Algorithm11,12 are used to aid differential diagnosis and triage patients to conservative management or surgery by general gynecologists versus surgery by specialist gynecological oncologists.13,14 However, there is a continuing need to develop more reliable tests to improve the accuracy of preoperative assessment. One line of investigation has been using advanced massspectrometry-, nuclear magnetic resonance- and chromatographic glycoprofiling to better understand the CA125 structural antigenic complexity.15−18 However, characterization is complicated19 as the CA125 antigen or MUC16, is a complex high molecular Received: November 6, 2012 Published: January 29, 2013 1408

dx.doi.org/10.1021/pr3010474 | J. Proteome Res. 2013, 12, 1408−1418

Journal of Proteome Research

Article

Figure 1. Schematic representation of antibody capture microarray glycoprofiling concept. (A) Secreted tumor proteins from normal, cosmc mutated Simple Cell (sc) and tumor cells. (B) Structure of CA125 (MUC16). (C) Microarray antibody capture and detection of glycoforms with labeled mAbs and lectins.

Ovarian Cancer Population Study (UKOPS), a biobanking study.29 Results suggest that this approach has the potential to improve the differential diagnosis of iEOC from benign and borderline (low malignant potential) ovarian neoplasms.

weight heavily O-glycosylated protein (mucin) with a core protein of 22,152 amino acids4,15,20,21 (Figure 1). The typical change in O-glycosylation found in cancer is the loss of complextype branched core-2 or core-4 O-glycans and overexpression of simple core-1 O-glycans (Neu5Acα2−3Galβ1−3GalNAcα, ST and Galβ1−3GalNAcα, T) or truncated core-1 such as Neu5Acα2−6GalNAcα (STn) and GalNAcα (Tn).1,22 Switch in glycan core structures could be an important detection signal of malignancy and potentially improve the performance of the current CA125 assay. Recent reports measured the CA19-9 carbohydrate antigen (Sialyl-Lewisa) carried on CA125 in pancreatic cancer patients23 and a tumor specific CA125 glycoprofile discriminating primary iEOC from benign ovarian neoplasms using mass-spectrometry.24 However, little has been done to improve the specificity of the CA125 serum assay per se.25−28 In the present work we demonstrate that aberrant O-glycoforms of CA125 (MUC16) and MUC1 (CA15-3) are present in serum from primary iEOC patients and can be detected with O-glycan specific monoclonal antibodies and lectins (Figure 1). The antibody capture microarray platform was first evaluated with a tumor cell line model system designed to express and secrete CA125 and MUC1 with STn and Tn glycoforms. The assay was validated with sera from patients with a pelvic mass and moderately elevated CA125 levels recruited to the UK

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MATERIALS AND METHODS

Study Design and Participants

All serum samples used in this study were obtained from women prior to surgery for suspected ovarian neoplasm. They include samples from: (i) Initial pilot studywomen participating in the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS) which recruited postmenopausal women aged 50−74 from the general population.30,31 It consisted of 10 women with serum CA125 levels between 200 and 500 kU/L prior to surgery, 5 of whom were diagnosed with primary invasive EOC and 5 with benign ovarian neoplasm following surgery; and (ii) the validation studywomen recruited to the UK Ovarian Cancer Population Study (UKOPS) a biobanking study involving ten gynecological oncology departments in England, Wales and Northern Ireland.29 For the validation study, 229 women were identified with serum CA125 levels between 30 and 500 kU/L prior to surgery. One woman was excluded since she was erroneously classified as primary iEOC when she had a primary gastrointestinal cancer. A further 4 samples were excluded because of 1409



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with two thin (0.5 mm) spacers allowing even distribution of each sample within each well. The paired slides were kept in a humidified chamber at 4C and gently agitated for 36−48 h (all incubations throughout).38 Slides were washed by gentle dipping three times into PBS and PBS containing 0.05% Tween-20 (PBST), dried by slide centrifugation, and further incubated with Neuraminidase (EC.3.2.1.18, Sigma, St Louis, MO) (0.1 U/mL in PLIP) at 37 °C for 1 h. After washing, captured glycoproteins were incubated with anticarbohydrate IgM antibodies 3C9 (Galβ1−3GalNAcα, T-specific) and 1B2 (Galβ1−4GlcNAc, LacNAc, type-2 specific (hybridoma supernatants, 1:4 dilution) or biotinylated GalNAcα (Tn) specific Vicia Villosa Lectin (VVL, VVA) and protein specific mAbs X306-biotin (2 μg/mL) recognizing a different antigenic site39 and HMFG2-biotin (2 μg/mL) recognizing the same site but on a multimeric tandem repeat. For detection, slides with IgM antibodies were incubated with Cy5-conjugated goat antimouse IgM (Southern Biotech, Birmingham, AL) (1:2000 dilution in PLIP buffer) and biotinylated lectins/mAbs with fluorescently labeled StreptavidinAlexa Fluor 555 (1:5000 dilution in PLIP buffer) for 1 h at room temperature. After washings fluorescent images were visualized by using a confocal array scanner (Pro Scan Array HT Microarray, Perkin-Elmer Life and Analytical Sciences, Boston, MA) at 10 μm resolution, using fixed scanner settings for each detection channels. The ScanArray Express software v3.0 (Perkin-Elmer Life and Analytical Sciences) was used to quantify the intensity of each spot, using the adapted circle method. The local background was subtracted to compensate for possible local defects and cases and controls were applied randomly on each slide to minimize assay variations. The coefficient of correlation for both intra-assays and for interassays was higher than 0.99.

duplication or incomplete collection of microarray data. Overall, 224 women were included of whom 97 were diagnosed with primary iEOC, 35 with borderline (low malignant potential) ovarian neoplasms and 92 with benign ovarian neoplasms. Samples from the validation study were collected and processed according to the PRoBe study design32 and uncoded after laboratory analysis were completed. All serum samples were collected and processed as previously described and before any intervention and before the disease status was known.33 Disease status was subsequently identified by histopathological examination of the excised tissue. The median patient age was 64.4, 57.1, and 51.3 years in individuals with primary iEOC, borderline and benign ovarian neoplasms, respectively. The distribution of the primary iEOC subtypes was similar to the distribution seen for primary iEOC as previously reported in the literature. Ethics

All participants provided written consent for the use of samples in secondary studies. Approval for the UKCTOCS study was obtained from the Joint UCL/UCLH Committees on the Ethics of Human Research (Committee A) (Amendment 11 December 2010) and for the UKOPS study from the Joint UCL/UCLH Committees on the Ethics of Human Research (Committee A) (Reference No. 05/Q0505/58). Measurement of CA125

Circulating CA125 was measured at a central laboratory (Tumour Marker Laboratory at UCL) using an electro-chemiluminescence immunoassay on a Roche Elecsys 2010 analyzer (Roche Diagnostics, Burgess Hill, UK) employing OC125 as the detection antibody and M11 as the capture antibody. The microarray capture and detection sensitivities were comparable with the Roche Elecsys 2010 analyzer and other ELISA based assays34 with overall high reproducibility with low inter- and intraslide variations (Supporting Information Table S6). The coefficient of variation of signals detected from triplicate spots using a CA125 standard (BioSite, A97180H) averaged 3% within the same slide and 6−8% between slides (Supporting Information Table S5).

Statistical Analysis of Performance

In order to investigate if combining the information of glycoforms STn-CA125, STn-MUC1, ST-CA125 (plus age at sample) improves predictive performance, a multivariate classifier approach was evaluated. Quadratic discriminant analysis (QDA) was used to predict whether a sample was either a control (benign or borderline) or case (iEOC) and compared with the discriminatory power of each glycoform alone using (1) area under the ROC curve and (2) achieved specificity for fixed sensitivities of 50, 60, 70, 80 and 90%. In the absence of a suitable external data set for validation, the results were based on leave-one-out cross validation, where the discriminant function was determined with each sample left out in turn, and then used to classify that sample. This, though not as rigid as using an independent validation set, will reduce some of the upward bias resulting from using the same set to model and predict outcomes. ROC curves were produced for each glycoform singly and in combination, and in addition the AUCs for the best glycoform (STn-CA125) and for the combination were compared by statistically testing the null hypothesis of no difference using an algorithm suggested by DeLong.40 Secondary analyses repeated the classification approach when the distinction was (1) benign versus borderline, (2) benign and borderline versus primary iEOC, and (3) benign versus primary iEOC.

Preparation of Antibody Capture Microarrays

Schott Nexterion Slide H (Schott Nexterion Slide H, Schott AG, Mainz, Germany) were coated with antimouse-IgG(H+L) antibody (Southern Biotech, Birmingham, AL) (10 μg/mL) diluted in phosphate buffer (PB) (150 mM, pH 8.5 containing 0.005% CHAPS).35 After deactivating remaining amino-reactive N-hydroxy succinimide groups (50 mM ethanolamine in 50 mM borate buffer, pH 9.2) for 1 h, mouse monoclonal (mAb) antibodies to CA125, X52 (IgG) (Thermo Scientific, Rockford, IL) and to MUC1, HMFG2,36 were spotted (200−600 μg/mL in PB) in triplicate by BioRobotics MicroGrid II arrayer (Genomics Solution) with a 300 μm pitch using Stealth 3B Micro Spotting Pins (Telechem International ArrayIt Division). Immobilized mAbs were analyzed with antimouse-IgG for quality purposes to ensure efficient printing. After printing, slides were stored in a closed slide container at 4 °C up to 3 months until assayed.

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General Procedure for Microarray Glycoprofiling Assay

Cell line secreted antigens (wt/sc Capan-1 and wt/sc T47D), prepared as described previously,37 or human serum samples were diluted (1/5 by volume) into incubation buffer PLIP (0.5 M NaCl, 3 mM KCl, 1.5 mM KH2PO4, 6.5 mM Na2HPO4, 1% BSA, 1% Triton-X-100, pH = 7.4) and applied (5 μL) onto a BSAcoated and inactivated Slide-H MPX48 Teflon-patterned glass slide serving as a sample reservoir. The printed antibody slide was then reversely mounted on top of the Teflon slide separated

RESULTS

Glycoprofiling of CA125 and MUC1 from Tumor Cell Lines with Core-1 T-Synthase Cosmc Chaperon Deletion

Protein specific mouse monoclonal antibodies to the serum proteins CA125 (X52 mAb) and MUC1 (HMFG2 mAb) were immobilized onto a hydrogel coated glass slide by microarray contact printing (See Material and Methods). As a glycoprofiling 1410



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Figure 2. Microarray glycoprofiling of glycoengineered (T47D and Capan-1) cell line derived CA125 and MUC1. Wild type (wt); cosmc deleted SimpleCell (sc); X dilution factor. Error bars represent SEM of three data replicates. Detection with biotinylated anti-CA125 and anti-MUC1 mAbs (A) X-306 and (F) HMFG2. Detection of Tn with VVL lectin before (B;G) and after neuraminidase treatment (+Neu) (C;H). Detection of T with 3C9 after neuraminidase treatment (D;I) and detection of LacNAc with 1B2 after neuraminidase treatment (E;J).

Tn- O-glycoforms (SimpleCells, sc).41,42 First we showed (Figure 2) that CA125 from both wild type (wt) and sc Capan-1 cells were

model system, we evaluated the tumor cell lines, T47D and Capan-1, genetically engineered to only express the STn- and 1411



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Figure 3. CA125 glycoprofiling of a pilot-set of benign (n = 5, light bars) and primary iEOC sera (n = 5, dark bars). (A) CA125 levels as meassured by commercial kit and as meassured with biotinylated X306 mAb on X52 microarray captured CA125 (D). Detection of Tn-glycoforms with VVL (B) before and (E) after neruaminidase treatment (+Neu). Detection of T-glycoforms with 3C9 (C) before and (F) after neruaminidase treatment (+Neu). A positive CA125 control produced in OVCAR3 cell line was present throughout. Error bars represent SEM of three data replicates.

500 kU/L (Materials and Methods). Analysis of Tn−glycoform with VVL and T-glycoform with 3C9 mAb revealed essentially a complete lack of nonsialylated O-glycans (Figure 3B, C), which is in agreement with our previous findings that circulating CA125 in cancer appears to only carry sialylated O-glycans,45 possibly due to rapid removal of nonsialylated species by innate lectin receptors on macrophages, hepatocytes, and dendritic cells.46 We did find weak reactivity for unsialylated LacNAc (1B2 mAb) in two of five cancer patients (Supporting Information Figure S2A, upper panel). Thus, when pretreating slides with neuraminidase, we found exposed Tn- and T- (Figure 3C, D) as well as increase in terminal LacNAc (Supporting Information Figure S2A, lower panel). Importantly, the exposure of Tn and T was substantially higher in cancer sera compared to benign sera, a trend that was not found for LacNAc. The efficiency of the on-slide neuraminidase treatment was also assessed by loss of reactivity of a Neu5Acα2,6 sialic acid binding lectin, SNA (Supporting Information Figure S2B, compare upper and lower panels). We also tested and concluded with glycan microarray analysis35 that the VVL lectin does not cross-react with blood group A oligosaccharide possessing a terminal Tn-moiety (data not shown). It was previously established that MUC1 in addition to CA125 is present in primary iEOC patients47 and we could confirm this by detecting circulating MUC1 in the same sera and found essentially the same glycoprofiling trends (Supporting Information Figure S3).

captured and detected with biotinylated anti-CA125 mAb X306B whereas T47D (wt and sc) were CA125 negative. Analytical sensitivity with our method for CA125 protein detection with X306-B mAb, was determined to 15 ng/mL (3.3 U/mL) by 3SD over mean background using a commercial CA125 standard (BioSite, Copenhagen, Denmark) (Supporting Information Figure S1) which is comparable to other ELISA based systems with same or similar antibodies.34 The MUC1 antigen secreted from both sc cell lines was readily captured with HMFG2 mAb and detected with HMFG2-biotin whereas wt did show significantly lower binding suggesting restricted HMFG2 mAb specificity due to interference with extended glycans43 (Figure 2A and F). As expected, strong detection of the Tn-glycoform with Vicia Villosa Lectin (VVL) was seen in the sc cell lines compared to wt predominantly expressing the extended Core-1 and Core-2 glycoforms (Figure 2B,C and G,H). Detection of Core-1 (T-glycoform) with 3C9 mAb after neuraminidase treatment was found on both CA125 and MUC1 from Capan-1 wt cells44 but not in sc (Figure 2D and I). Detection of terminal type-2 (Galβ1−4GlcNAc, LacNAc) Core-2 extensions was mostly found in wt Capan-1 cells after neuraminidase treatment. Some LacNAc-reactivity could also be detected in sc possibly part of an N-glycan structure commonly present on both cell types (Figure 2E and J). No terminal LacNAc was detected on MUC1 in cosmc mutated sc after neuraminidase treatment. At this stage, we concluded that secreted CA125 and MUC1 glycoprotein could be captured and differentially detected correctly based on their post-translational glycosylation level using glycan specific lectins and antibodies.

Evaluation of the Diagnostic Performance of the CA125 and MUC1 Glycoprofiling Microarray Assay

Next we evaluated serum glycoprofiling of CA125 and MUC1 in a blinded set of prediagnostic clinical samples from a cohort of 224 patients with a pelvic mass and an elevated CA125 level (30− 500 kU/L) from the UK Ovarian Cancer Population Study (UKOPS) (Table 1). The range of 30−500 kU/L for elevated

Detection of STn- and ST-glycoforms on CA125 and MUC1 in Serum Distinguishes Benign from Cancer Patients

A pilot study was undertaken in 10 women (5 benign ovarian neoplasm, 5 primary iEOC) with CA125 levels between 200 and 1412



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Table 1. Description of Histological Subtypes Amongst the Women with Benign, Borderline Ovarian Neoplasm or Primary Invasive Ovarian/Tubal Cancer, Distribution of Age tand CA125 Values no. of women histological subtype

premenopausal

age

CA125 (KU/L)

postmenopausal

total

median in years (25th−75th percentile)

median (25th to 75th percentile)

23 12 3 3 7 0 2 4

31 14 7 3 26 2 3 6

51.3 (43.0−66.0)

62 (41−128)

10 16

15 20

57.1 (48.3−71.5)

66 (50−114)

42 6 13 9 4 5 2 0 1

47 9 17 10 5 5 2 1 1

64.4 (53.7−73.2)

176 (96−273)

Benign ovarian neoplasm Cystadenoma/Cystadenofibroma 8 Fibroma/Fibrothecoma 2 Mature teratoma 4 Brenner tumor 0 Endometriosis/Endometriotic cyst 19 Ovarian abscess 2 Fibroid 1 Other 2 Borderline/Low Malignant Potential ovarian neoplasm Serous 5 Mucinous 4 Primary Invasive Ovarian/Tubal cancer Serous 5 Endometrioid 3 Mucinous 4 Clear cell 1 Mixed 1 Carcinosarcoma/MMMT 0 Carcinoma NOS 0 Transitional cell 1 Fallopian Tube cancer 0

between four groups: benign ovarian neoplasms (BE) versus borderline ovarian neoplasms (BD) and primary iEOC (OC), BE and BD versus OC; BE versus BD and BE versus OC (Figure 4). ROC curves demonstrated that a combination of the three glycoforms was superior to each of them separately, though the difference in the AUCs between the combination and STn-CA125 was modest (p = 0.045, 0.062, 0.902, and 0.016 respectively) for the four separate comparisons. The combined glycoform profile including age was able to distinguish benign ovarian neoplasms from primary iEOC with a specificity of 61.1% at 90% sensitivity whereas the CA125 specificity for commercial assay was only 41.3% at the same sensitivity (Supporting Information Table S4). When restricting to the postmenopausal women, the “combined glycoforms”, including the age, outperforms CA125 but the combination of both is better (Supporting Information Table S4). Of note, the CA125 levels in the OC patients were only moderately elevated (Table 1). 35 women with benign ovarian neoplasms (7 endometriosis, 17 ovarian cystadenoma/cystadenofibroma, 5 fibroma/fibrothecoma, 1 mature teratoma, 1 Brenner tumor, 1 fibroid, 1 ovarian abscess, 1 Leydig cell hyperplasia, 1 paratubal cyst) were misclassified as cancer by the combined glycoform profile (Supporting Information Table S7). The glycoforms (either alone or combined) in the benign versus borderline comparison did not achieve any discrimination between the groups.

CA125 levels was chosen as the difficulty in differential diagnosis applies most to this range as higher levels strongly correlate with malignancy. The set consisted of 97 patients with primary iEOC cancer with median CA125 levels of 176 kU/L (IQR 96−273), 35 with borderline/low malignant potential ovarian neoplasms with median CA125 of 66 kU/L (IQR 50−114) and 92 benign ovarian neoplasms with median CA125 levels of 62 kU/L (IQR 41−128). The Stage and Grade of the primary iEOC and those with borderline/low malignant potential ovarian neoplasms is presented in Table 2. The STn-CA125 and ST-CA125 and STnTable 2. Stage and Grade Distribution in the Women with Primary Invasive OC and Borderline Ovarian Tumors no. of women Stage I II III IV Not available Grade 1 2 3 Not available

ovarian/FT cancer

borderline ovarian tumor

42 4 43 8 0

23 0 1 0 11

15 21 55 6

Glycoforms and Histological Subtypes and Stage

We also addressed whether the glycoforms varied by histological subtypes and stage of iEOC and of borderline/low malignant potential tumor group. As shown in Figure 5A-D both the STn and ST CA125 glycoforms were significantly increased in serous and endometrioid but not mucinous iEOC compared to benign ovarian neoplasms. There was no difference in S-LacNAc glycoforms between the three groups or the cancer subtypes. The STn-MUC1 glycoform showed a significant discriminative tendency for serous, mucinous and clear cell carcinoma but not for

MUC1 glycoforms were significantly increased in iEOC compared to the benign ovarian neoplasms, whereas no discriminatory power of ST-MUC1 or S-LacNAc glycoforms was observed. Using a quadratic discriminant analysis (QDA), the predictive performance of the 3 best-performing glycoforms (STn-CA125, STCA125, STn-MUC1) was tested. The ability of these glycoforms alone or in combination to discriminate samples were compared 1413



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Figure 4. ROC curves comparing (A) Benign ovarian neoplasms (BE) versus Borderline ovarian tumors (BD) and primary iEOC (OC) combined; (B) BE and BD vs OC; (C) BE vs BD; (D) BE vs OC.

study sets, this has immediate clinical value in differential diagnosis and triage of patients to surgery in specialist gynecological oncology centers versus conservative management or surgery by general gynecologists. While it is well-known that aberrant post-translational glycosylation occurs in disease, particularly cancer, with several different approaches investigated for their detection49 there have been limited efforts to further develop the CA125 diagnostic assay. In primary iEOC patients, previous reports have characterized glycosylation changes on serum immunoglobulins and changes of O-glycans in ascitic fluid using mass spectrometry and lectin chromatography methods.19,27 These early studies did not detect the STn-CA125 glycoform. However, more recently it has been identified in peritoneal fluid from ovarian cancer patients26 including detection of elevated MUC1,50 in keeping with our finding of elevated serum STn glycoforms of CA125 and MUC1 in iEOC patients. The benign conditions that were picked up by the combined glycoforms, included ovarian cystadenoma/cystadenofibroma (17 of 33), ovarian fibroma/fibrothecoma (5 of 14) and endometriosis (7 of 26), (Supporting Table S7). There is good evidence that endometriosis is a risk factor for invasive primary iEOC especially endometriod and clear cell subtypes.51 It would be interesting to speculate whether secretion of aberrant

the endometrioid subtype cancer (Figure 5F). There was no discrimination between cancer and benign with ST and S-LacNAc glycoforms which is in agreement with previous characterization of circulating MUC1 glycoforms.48 While the levels of circulating MUC1 and CA125 appear to be unrelated in the study population, it was clear that the STn-MUC1 glycoform was predominantly found in primary iEOC whereas the ST-glycoform was not. Despite six of the nine primary invasive primary iEOC not detected using the combined glycoform being stage I, there was no apparent statistical association between stage and test result (p = 0.658). No significant influence of age on each of the glycoforms was found and in the regression models, there was no evidence for effect of age on the levels of glycoforms detected when each group (benign, borderline, ovarian cancer) was considered (Supporting Information Figure S8).

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DISCUSSION

The present study demonstrates for the first time that a qualitative glycoprofiling assay to detect changes of secreted serum CA125 glycoprotein can improve the diagnostic utility of current assays. In patients with moderately elevated (30−500 kU/L) CA125 levels and a pelvic mass, a combined glycoform profile (STn-CA125, ST-CA125 and ST-MUC1) was able to detect 90% of those with primary iEOC while only detecting 40% (specificity 61.1%) of those with benign masses. If validated in independent 1414



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Figure 5. Glycoprofiling distributions classified by subtype of primary iEOC stages (n = 95), benign controls (n = 91). Protein specific detection of CA125 and MUC1 with (A) X306-B and (E) HMFG2-B mAbs. Glycoprofiling of CA125 (B−D) and MUC1 (F−H). The multiple comparisons were performed by Kruskal−Wallis tests and the summary for p value of

Microarray Glycoprofiling of CA125 Improves Differential Diagnosis of

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