IJC International Journal of Cancer

Difference in mesothelin-binding ability of serum CA125 between patients with endometriosis and epithelial ovarian cancer Aya Sasaki1,2*, Kaoru Akita1*, Fumitake Ito2, Taisuke Mori2, Jo Kitawaki2 and Hiroshi Nakada1 1 2

Department of Molecular Biosciences, Faculty of Life Sciences, Kyoto Sangyo University, Kyoto, Japan Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, Kyoto, Japan

The glycoprotein carrying the CA125 antigen, which is encoded by the MUC16 gene in humans, is initially synthesized as a type I integral membrane mucin glycoprotein, and is highly expressed in epithelial ovarian carcinoma (EOC).1,2 The ectodomain of CA125 is often proteolytically released from the plasma membrane into the extracellular space and consequently into the circulation. Currently, CA125 is used in differential diagnosis and monitoring for EOC. The serum level of this mucin is elevated in about 80% of patients with EOC,3 but is also increased in healthy women during menstruation and in patients with several benign diseases such as

Key words: CA125, mesothelin, ovarian cancer, endometriosis Additional Supporting Information may be found in the online version of this article. Conflict of interest: Nothing to report *A.S. and K.A. contributed equally to this work Grant sponsor: JSPS KAKENHI; Grant number: 25830106; Grant sponsor: ASTEM RI/KYOTO DOI: 10.1002/ijc.29185 History: Received 8 Apr 2014; Accepted 26 Aug 2014; Online 5 Sep 2014 Correspondence to: Hiroshi Nakada, Department of Molecular Biosciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo-Motoyama, Kita-ku, Kyoto 603-8555, Japan, Tel.: 181-75-705-1888, Fax: 181-75-705-1888, E-mail: [email protected]

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endometriosis.4 It was recently found that the glycosylation of CA125 in patients with EOC is different from that in healthy subjects and patients with benign ovarian neoplasms, providing an attractive target for qualitative evaluation of this mucin.5–7 However, the biochemical tools for specifically detecting such altered glycosylation are limited. The CA125 core protein is modified with a variety of Nand O-glycans.8 These carbohydrate chains are involved in the binding of CA125 to mesothelin, siglec-9, galectin-1 and selectins.9–13 Although the latter three lectins bind not only to CA125 but also to various other glycoconjugates, mesothelin binds to CA125 with high specificity, suggesting that the expression and/or precise positioning of unique carbohydrate chain(s) on the core protein of this mucin may be responsible for its binding to mesothelin.9,10 Mesothelin is a glycosylphosphatidylinositol-linked cell surface glycoprotein that is expressed in mesothelial cells lining the body cavity.14 It has been suggested that the CA125–mesothelin interaction plays a role in promoting the attachment of EOC cells to the peritoneum.9,10 At present, which carbohydrate structure(s) on the CA125 core protein is involved in the binding to mesothelin, and whether the mesothelin-binding carbohydrate chain(s) is altered on transformation of the cells remain unclear. There is increasing evidence that human epididymal secretory protein E4 (HE4; WAP four-disulfide core domain protein 2, WFDC2) is a promising EOC biomarker to complement CA125.15–17 In our study, we developed a novel assay for determining the serum level of CA125meso,

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The epithelial ovarian carcinoma (EOC) is an aggressive malignant tumor, and is currently the leading cause of gynecologic cancer death. CA125 is the most commonly used serum marker for EOC, but shows a high-false-positive rate for several benign diseases such as endometriosis. The purpose of our study is therefore to identify a useful biochemical tool for detecting qualitative differences between CA125 from patients with endometriosis and EOC, and to facilitate differential diagnosis of these diseases. In our study, using two different CA125-binding molecules, i.e., recombinant mesothelin and an anti-CA125 monoclonal antibody, a novel sandwich ELISA for determining the serum levels of CA125 with mesothelin-binding ability (CA125meso) was developed, and tested for patients with endometriosis (n 5 59) and EOC (n 5 36). We found that both the serum CA125meso level and the ratio of the serum CA125meso to CA125 levels (CA125meso/CA125) were significantly higher in patients with EOC than in patients with endometriosis (p < 0.00005 and p < 0.000001, respectively). Furthermore, receiver operating characteristic analysis showed that the CA125meso assay was superior to the conventional antibody-based CA125 assay in discriminating endometriosis from EOC. Thus, mesothelin-binding ability may be a useful indicator for qualitatively evaluating CA125 in patients with endometriosis and EOC.

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Measurement of serum CA125 with mesothelin-binding ability

What’s new? Diagnostic tests using the CA125 antigen have a high false-positive rate for ovarian cancer (OC) when conditions such as endometriosis are present. Is there a way to distinguish between these two diseases? In this study, the authors developed a “sandwich” ELISA based on CA125 with mesothelin-binding ability (CA125meso). They found that serum CA125meso levels were significantly higher in patients with OC, as was the ratio of serum CA125meso to CA125 (CA125meso/CA125). This assay may thus enable a more accurate differential diagnosis between endometriosis and OC.

evaluated its utility in discriminating endometriosis and EOC and also discussed its performance compared with HE4.

Material and Methods Human subjects

Serum specimens from women diagnosed with uterine myomas, endometriosis, endometrial cancer, cervical cancer or EOC were used in our study. Their numbers, ages and clinicopathological characteristics are given in Supporting Information Materials and methods. Immunoassay

For measurement of serum CA125meso, the wells of Maxisorp Immunoplates (Nunc, Roskilde, Denmark) were coated with a recombinant human mesothelin (R&D Systems, Minneapolis, MN). After blocking, specimens were added to the wells, followed by incubation. Bound CA125 was detected with a biotinylated anti-CA125 monoclonal antibody (mAb) (clone X306; HyTest, Turku, Finland), HRP-conjugated streptavidin polymer (Sigma, St Louis, MO) and tetramethyl benzidine (TMB) substrate. Further details of the CA125meso assay, other serum assays and the materials used can be found in Supporting Information Materials and methods. Statistical analysis

A detailed description of the statistical analysis is available in Supporting Information Materials and methods.

Results

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Measurement of serum CA125meso levels in patients with endometriosis and EOC

To determine the serum levels of CA125meso, here we developed a sandwich ELISA, which is schematically shown in Figure 1a. In this assay, CA125meso was captured on assay plates through its interaction with immobilized recombinant mesothelin, and was detected with a biotinylated antiCA125 mAb. To examine the linearity and sensitivity of the CA125meso assay, serum from two randomly selected patients with endometriosis and EOC, and OVCAR-3 cellsproduced CA125 were tested using various concentrations of CA125 (0–100 U/ml). As shown in Figure 1b, the CA125meso signal exhibited a linear correlation with the tested CA125 concentration, and was detectable when more than 6.25 U/ml of CA125 was analyzed. Notably, the signal intensity of CA125meso was clearly different between specimens.

We next performed the CA125meso assay using serum specimens from patients with endometriosis (n 5 59) and EOC (n 5 36) (Fig. 1c). Furthermore, patients with uterine myomas (n 5 17), endometrial cancer (n 5 15) and cervical cancer (n 5 4) served as the control group (Supporting Information Fig. 1A). The serum CA125 levels in these subjects were also determined by means of the conventional antibody-based sandwich ELISA (Fig. 1d and Supporting Information Fig. 1B). In our study, all cancer patients had primary invasive tumors, patients with borderline tumors being excluded. Generally, the cutoff value for serum CA125 in EOC screening is set at 35 U/ml.3,18 When this cutoff value was used in our subjects, the positive rate for serum CA125 was 83.3% for EOC, and the false-positive rate was 79.7% for endometriosis (Supporting Information Fig. 1B). When it was set at 15 U/ml for the CA125meso assay, a similar positive rate (77.8%) was obtained in patients with EOC, whereas the false-positive rate in patients with endometriosis decreased to 47.5% (Supporting Information Fig. 1A). There was no elevation of either the CA125 or CA125meso level in most of the patients with uterine myomas, endometrial cancer and cervical cancer, indicating that the CA125meso assay could produce reliable signals even in subjects with the basal or a low serum level of CA125 (Supporting Information Figs. 1A and 1B). As shown in Figures 1c and 1d, the serum levels of CA125 and CA125meso were significantly different between patients with endometriosis and EOC. However, the statistical score in the CA125meso assay (p 5 0.00001) was more reliable than that in the antibody-based CA125 assay (p 5 0.001). Consistent with this, the receiver operating characteristic (ROC) analysis for discriminating endometriosis from EOC showed a significantly higher area under the curve (AUC) for the CA125meso levels (AUC 5 0.771) when compared with the CA125 levels (AUC 5 0.697) (Fig. 1e). The serum level of CA125meso varies depending on at least two factors: the serum level of CA125 and its ability to bind mesothelin. To quantify the mesothelin-binding ability of CA125, we calculated the ratio of the serum CA125meso to CA125 levels (CA125meso/CA125) as an index. As shown in Figure 2a, the levels of CA125meso/CA125 were significantly different between patients with endometriosis and EOC (p 5 0.0000006). Furthermore, a positive correlation between the serum CA125 and CA125meso/CA125 levels was found in patients with EOC (correlation coefficient 5 0.537) (Fig. 2b). The mean age of patients with EOC (mean age, 61 years) was higher than that of patients with endometriosis (mean

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age, 40 years). To next examine whether the mesothelinbinding ability of CA125 is related to the age distribution of the subjects in each cohort, we compared the CA125meso/ CA125 levels between premenopausal (n 5 7; mean age, 47 years) and postmenopausal (n 5 29; mean age, 65 years) patients with EOC. As shown in Figure 2c, no significant difference was found in the CA125meso/CA125 levels between these patients. The results for the control subjects also indicated that there was no relationship between the CA125meso/ CA125 levels and the age distribution (Supporting Information Fig. 1C). Furthermore, there was no clear relationship between the CA125meso/CA125 levels and the clinical stage, and the histological type of EOC (Figs. 2d and 2e). It has been reported that the level of mesothelin released from the cell surface into the serum is elevated in patients with EOC.19 In our specimens, the levels were distributed from 6.2 to 195.5 ng/ml (Fig. 3a). As described under Material and Methods, we used plates on which 250 ng of recombinant mesothelin was immobilized, and maximally 9.8 C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V

ng of endogenous mesothelin might have been included in the specimens. To determine whether the endogenous soluble mesothelin competitively inhibited the binding of CA125meso to the immobilized recombinant mesothelin in our assay, we analyzed the relationship between the serum level of endogenous mesothelin and the CA125meso/CA125 levels in patients with EOC. As shown in Figure 3b, no significant negative correlation was found between these factors (correlation coefficient 5 0.062). Furthermore, the competitive inhibition assay showed that 95% of the binding signal was retained even when samples including 35 U/ml of CA125 were assayed in the presence of 640 ng/ml of recombinant soluble mesothelin (Fig. 3c). These results indicate that the competitive inhibitory effect of endogenous mesothelin on the CA125meso assay is virtually negligible, and are also consistent with previous reports that CA125 much more preferentially binds to immobilized than soluble mesothelin.10,20 An analogous sugar structure(s) on other serum mucins may competitively inhibit the CA125–mesothelin binding in the

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Figure 1. A sandwich ELISA involving a recombinant mesothelin and anti-CA125 mAb (CA125meso assay). (a) A schematic diagram of the CA125meso assay. (b) Linearity study of the CA125meso assay. Serum specimens from patients with endometriosis (n 5 2) and EOC (n 5 2), and OVCAR-3 cells-produced CA125 were diluted to give the indicated concentrations of CA125 and then analyzed by means of the CA125meso assay. (c and d) The serum levels of CA125meso (c) and CA125 (d) were examined in patients with endometriosis (n 5 59) and EOC (n 5 36). The horizontal dotted lines represent the arbitrary cutoff values; 15 U/ml in (c) and 35 U/ml in (d). (e) ROC curves for the CA125 and CA125meso assays for discriminating endometriosis from EOC.

Measurement of serum CA125 with mesothelin-binding ability

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Figure 2. Evaluation of the mesothelin-binding ability of CA125 (CA125meso/CA125). (a) Comparison of the CA125meso/CA125 levels between patients with endometriosis and EOC. (b) Relationship between the serum CA125 and CA125meso/CA125 levels in patients with EOC. (c) Comparison of the CA125meso/CA125 levels between premenopausal and postmenopausal patients with EOC. (d) Comparison of the CA125meso/CA125 levels according to the clinical stage of EOC. (e) Comparison of the CA125meso/CA125 levels according to the histological type of EOC. In (a) and (c)–(e), the horizontal bars represent the mean values. In (a), (d) and (e), the horizontal dotted line represents the arbitrary cutoff value (1.08).

CA125meso assay. Elevated serum levels of MUC1 have been reported in patients with EOC.21 Consistent with this, CA15-3 antigen, a carbohydrate epitope expressed on MUC1, was

detected in our serum specimens (Fig. 3d). However, there was no negative correlation between the serum level of CA15-3 and the CA125meso/CA125 levels in patients with EOC (correlation C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V

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coefficient 5 0.450) (Fig. 3e), suggesting that, at least, MUC1 in specimens does not affect the CA125meso assay.

Discussion As shown in Figure 2a and Supporting Information Figure 1C, the CA125meso/CA125 levels in 50% (18 of 36) of the patients with EOC were higher than the mean 6 3 SD (1.08) of those in the patients with endometriosis. On the other hand, the percentage of endometriosis patients with elevated CA125meso/CA125 levels (>1.08) was less than 2% (1 of 59). Notably, the CA125meso/CA125 levels were elevated in 55% (11 of 20) of the patients with early-stage EOC (Fig. 2d), and in 57% (four of seven) of the patients with mucinous EOC (Fig. 2e), even though relatively low levels of serum CA125 have been reported in these populations.22–25 As the mesothelin-binding ability of CA125 is a key factor for the CA125meso assay, the difference in CA125meso/CA125 levels between patients with endometriosis and EOC implies that C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V

this assay may have an advantage over the conventional antibody-based CA125 assay for a differential diagnosis. It has been reported that HE4 is a useful EOC marker to complement CA125.15 For example, elevated serum levels of HE4 are found in over half of CA125-negative patients with EOC.16 Furthermore, HE4 exhibits a higher sensitivity (71.4%) than CA125 (64.3%) in discriminating endometriosis from EOC at 95% specificity.17 Our results revealed that CA125meso also showed a higher sensitivity (55.6%) than CA125 (38.9%) in discriminating these diseases at the same specificity. However, 47.5% of the patients with endometriosis were still positive for CA125meso (>15 U/ml) (Fig. 1c). Thus, these results indicate the usefulness of measuring CA125meso in combination with HE4. It is well known that CA125 is expressed in a number of gynecologic and nongynecologic cancers.4 To investigate the wider applicability of the CA125meso assay, further study will be needed to determine whether the mesothelin-binding ability of CA125 is elevated in cancers other than EOC.

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Figure 3. Effects of soluble mesothelin and MUC1 on the measurement of CA125meso. (a and d) The serum levels of mesothelin (a) and CA153 antigen (d) were determined in patients with endometriosis (n 5 59 or 32) and EOC (n 5 36 or 30). (b and e) Relationship between the CA125meso/CA125 levels and the endogenous serum mesothelin (b) or CA15-3 (e) level. (c) OVCAR-3 cells-produced CA125 was diluted to 35 U/ml and then analyzed by means of the CA125meso assay in the presence of the indicated concentrations of the recombinant soluble human mesothelin (0–640 ng/ml). The data represent the means 6 SE (n 5 4). In (a) and (d), the horizontal bars represent the mean values.

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Measurement of serum CA125 with mesothelin-binding ability

In summary, here we developed a novel ELISA method for determining the serum level of CA125meso. Our results suggest that

the CA125meso assay allows better discrimination of endometriosis from EOC when compared with the conventional CA125 assay.

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Yin BW, Lloyd KO. Molecular cloning of the CA125 ovarian cancer antigen: identification as a new mucin, MUC16. J Biol Chem 2001;276: 27371–5. O’Brien TJ, Beard JB, Underwood LJ, et al. The CA 125 gene: an extracellular superstructure dominated by repeat sequences. Tumour Biol 2001;22:348–66. Bast RC, Jr, Klug TL, St John E, et al. A radioimmunoassay using a monoclonal antibody to monitor the course of epithelial ovarian cancer. N Engl J Med 1983;309:883–7. Bast RC, Jr, Xu FJ, Yu YH, et al. CA 125: the past and the future. Int J Biol Markers 1998;13: 179–87. Akita K, Yoshida S, Ikehara Y, et al. Different levels of sialyl-Tn antigen expressed on MUC16 in patients with endometriosis and ovarian cancer. Int J Gynecol Cancer 2012;22: 531–8. Chen K, Gentry-Maharaj A, Burnell M, et al. Microarray glycoprofiling of CA125 improves differential diagnosis of ovarian cancer. J Proteome Res 2013;12:1408–18. Saldova R, Struwe WB, Wynne K, et al. Exploring the glycosylation of serum CA125. Int J Mol Sci 2013;14:15636–54. Kui Wong N, Easton RL, Panico M, et al. Characterization of the oligosaccharides associated with the human ovarian tumor marker CA125. J Biol Chem 2003;278:28619–34. Rump A, Morikawa Y, Tanaka M, et al. Binding of ovarian cancer antigen CA125/MUC16 to mes-

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Difference in mesothelin-binding ability of serum CA125 between patients with endometriosis and epithelial ovarian cancer.

The epithelial ovarian carcinoma (EOC) is an aggressive malignant tumor, and is currently the leading cause of gynecologic cancer death. CA125 is the ...
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