The Value of Soluble CD146 in the Diagnosis of Ovarian Cancer
WANG Ya-qin, ZHANG Jun*, WANG Huan-ying, ZHANG Hao-feng
Department of Obstetrics and Gynecology, Capital Medical University Affiliated Beijing Anzhen Hospital, Beijing Institute for Heart, Lung and Blood Vessel Diseases, Beijing, 100029, China
*Corresponding Author: ZHANG Jun, E-mail: drzhangj@outlook.com
Abstract

Objective: To study the possible diagnostic value of soluble CD146 (sCD146) in patients with ovarian cancer.Methods: A total of 33 patients with benign ovarian tumor and 30 with ovarian cancer from Department of Obstetrics and Gynecology, Anzhen Hospital were respectively selected as observation group A and observation group B, while 31 healthy women from Medical Center, Anzhen Hospital were served as control group. There was no significant difference in the age among three groups. Enzyme immunoassay and chemiluminescence were used to detect the expression of sCD146 and CA125, respectively.Results: The sCD146 level was lower in observation group A and observation group B when compared with control group (P<0.01). Moreover, serum sCD146 in observation group B was further lower than that in observation group A (P<0.05). The CA125 level increased in observation group B compared with those in control group and observation group A (P<0.001). And the CA125 level in observation group A was markedly higher than that in control group, and the difference was statistically significant (P<0.001). If either single target was tested to be positive, it was advisable to choose combined detection of CA125 and sCD146, which could improve the diagnostic specificity.Conclusion: sCD146 may provide a potential marker for the diagnosis and treatment of patients with ovarian cancer.

Key words: sCD146; CA125; Ovarian cancer; Specificity; Sensitivity
Introduction

Ovarian cancer is one of the common gynecologic malignancies, and its mortality rate ranks the first in female genital malignancies [1]for the majority of early-stage cancers are asymptomatic. It is often latent until the tumor has been metastasized, that is too late to treat. Although there have been many reports on the pathology of ovarian cancer, such as the continuous stimulation of estrogen, incessant ovulation gonadotropin and androgen stimulation [2] as well as higher body mass index (BMI) [3] that have been postulated as contributing factors, and the exposure of ovaries to pelvic contaminants and carcinogens [4]. However, the exact molecular events leading to tumorigenesis and metastasis of ovarian tumor cells have not yet been well elucidated, and lacking of early diagnostic methods results in poor survival. Ovarian cancer has a poor prognosis, with 5-year survival rate being just 40% in patients [5].

An American report demonstrated that although the age-adjusted ovarian cancer mortality rate in the USA declined by 23% over the last 40 years, which paralleled a decline in incidence, it was largely caused by changes in reproductive risk factors, and there was no contribution from improvements in early detection or treatment [6]. Therefore, many studies now have focused on how to identify or prevent ovarian cancer, which basically includes genetic testing for susceptibility genes of ovarian cancer, early screening for normal population, and preventive bilateral salpingo-oophorectomy [5, 6, 7]. A report showed that the presence of circulating tumor cells (CTCs) in the blood of ovarian cancer patients was correlated with decreased overall survival [8], suggesting that serum might be a valuable source of biomarkers. Currently, serum biomarkers for early diagnosis of ovarian cancer receive more attentions [9, 10, 11].

CA125 is the most commonly used serum marker for ovarian cancer [12], but shows a high-false-positive rate for several benign diseases like endometriosis and benign tumor [13, 14]. Therefore, in recent years, many studies have focused on finding a more sensitive biomarker to detect ovarian cancer at its earliest stage, or seeking for some biomarkers or technologies for joint inspection [15, 16]. CD146 is a cell adhesion molecule localized on the endothelial junction belonging to the immunoglobulin super family, which has been originally identified as a marker for melanoma (MCAM) [17]. It is a 113-kDa integral membrane glycoprotein, whose amino acid sequence consists of a signal peptide, an extracellular fragment structure of iv immunoglobulin like domains (V-V-C2-C2-C2), a transmembrane region and a short cytoplasm tail [18]. Increasing amounts of evidences have demonstrated that CD146 is over-expressed in a variety of carcinomas, including melanoma, prostate cancer, epithelial ovarian cancer, and breast cancer [19, 20, 21, 22] as well as cervical and endometrial cancer. Therefore, further studies were carried out to explore the expression of soluble CD146 (sCD146) in the serum of patients with ovarian cancer.

Soluble form of CD146 - sCD146, was first detected in Human Umbilical Vein Endothelial Cells (HUVEC) culture medium [23]. Elevated level of serum sCD146 has been reported in vasculitis [24], acute decompensated heart failure [25], and carotid atherosclerotic plaques [26]. Targeting sCD146 with a neutralizing antibody inhibits vascularization, growth and survival of CD146-positive tumors [27]. In addition, reduced level of serum sCD146 has been reported in active inflammatory bowel disease [28]. Recently, Kaspi et al. [29] identified sCD146 as a regulator for trophoblast migration in placental vascular development during pregnancy, with a physiological down-regulation of sCD146 throughout pregnancy. However, sCD146 expression and detection in ovarian cancer has not been investigated. Therefore, the purpose of this study was to facilitate the diagnosis of ovarian cancer through detection of sCD146 expression..

Materials and Methods
Subjects and samples

Patients with benign ovarian tumors and ovarian cancer were collected from Department of Obstetrics and Gynecology, Anzhen Hospital, while healthy women from Medical Center of Anzhen Hospital. The study included 33 patients with benign ovarian tumor (mean age: 42 years) and 30 patients with ovarian cancer (mean age: 55 years) as observation groups A and B respectively. In addition, 31 healthy women (mean age: 38 years) were selected as control group.. Serum samples were collected from peripheral venous blood at least 2 mL. They were centrifuged at 2 000 rpm/min for 15 min and frozen at -80℃ until assay. Informed consent forms were signed by participants in this study. All procedures were approved by the Ethics Committee of Capital Medical University Affiliated Beijing Anzhen Hospital, Institute of Beijing Heart, Lung and Blood Vessel Diseases and Institute of Biophysics, Chinese Academy of Sciences.

Antibodies and reagents

Soluble CD146 standard product was purchased from Biocytex (Marseille, France). All mouse anti-CD146 monoclonal antibodies, AA1, AA98, and targeting distinct epitopes of CD146 were generated in the Protein and Peptide Pharmaceutical Laboratory (CAS-University of Tokyo Joint Laboratory of Structural Virology and Immunology, Institute of Biophysics, Beijing, China). Biotin-conjugated AA98 was labeled by Tianjin Sungene Biotech Co., Ltd. Horseradish peroxidase (HRP)-conjugated streptavidin was bought from Dianova (Rodeo, CA, USA).

Enzyme Immunoassay for sCD146

The level of sCD146 in serum samples was determined by enzyme-linked immunosorbent assay using anti-CD146 mAbs AA1 (capture antibody, 2.5 μ g/mL) and biotin-conjugated AA98 (detection antibody, 2 μ g/mL). HRP-conjugated streptavidin (0.5 μ g/mL) served as the detection enzyme. Standards were diluted in 1% bovine serum albumin-phosphate buffered saline (BSA-PBS) to form 80-1.25 ng/mL for standard curve determination. Serum samples were diluted in 1% BSA-PBS by 1:10 prior to measurement, and then all samples were tested in duplicate (50 μ L each well). A solution of tetramethylbenzidine (TMB) was used as a substrate for the HRP enzyme. Sample absorption at a wavelength of 450 nm was measured using a BioRad ELISA reader (Richmond, CA, USA).

Chemiluminescence for CA125

Automated chemiluminescence analysis system (Abbott AXSYM, US.) was used to detect the level of serum CA125. The assay was performed according to the manufacturer’ s instructions. All specimens were tested in duplicate.

Statistical analysis

Statistical analysis was conducted using SPSS software. The significant differences between the groups and the possible contribution to diagnosis of ovarian cancer were evaluated using the analysis of variance and LSD-test. P< 0.05 was considered to be statistically significant.

Results
Comparative analysis of sCD146 level among three groups

From the analysis of variance, the results indicated significant differences among three groups (F=13.623, P=0.000). The sCD146 level was lower in observation group A and observation group B when compared with control group (P< 0.01). Moreover, serum sCD146 in observation group B was further lower than that in observation group A (P< 0.05) (Figure 1).

Figure 1 Comparison of sCD146 and CA125 levels in Serum in Observation Group A (n=33), Observation Group B (n=30), and Control Group (n=31) Using an ELISA Sandwich System and Automated Chemiluminescence Analysis System respectively.

Comparative analysis of CA125 level among three groups

The analysis of variance showed significant differences among three groups (F=13.950, P=0.000). The CA125 level increased in observation group B compared with those in control group and observation group A (P< 0.001). And the CA125 level in observation group A was markedly higher than that in control group, and the difference was statistically significant (P< 0.001)(Figure 1).

CA125 combined with sCD146 in the diagnosis of ovarian cancer

CA125 was considered to be positive if its value was > 35 μ /mL, whereas sCD146 was considered to be positive when its value was lower than 169.69 ng/mL. The sensitivity and specificity of CA125 were consistent with the literature. The sensitivity of sCD146 was higher than that of the CA125. If either single target was tested to be positive, it was advisable to choose combined detection of CA125 and sCD146, which could improve the diagnostic specificity (Tables 1 and 2).

Table 1 Comparison of sCD146 and CA125 Levels Among Three Groups (ヌ± S)
Table 2 Specificity, Sensitivity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) of sCD146 and CA125 in Diagnosis of Ovarian Cancer (ヌ± S, %)
Discussion

Ovarian cancer is the most lethal gynecological malignancy. It is usually diagnosed at the advanced stage, with a 5-year survival rate of 15%-20%, limiting the effectiveness of debulking surgery and chemotherapy [1, 4], while the 5-year survival rate for patients with early ovarian cancer up to 90%. In 1983, Bast[30] found a relationship between CA125 and ovarian cancer, and established a reliable radioimmunoassay, and since then, CA125, as a marker of ovarian cancer diagnosis and monitoring of ovarian cancer, has been widely used [14, 15]. But the sensitivity and specificity of CA125 for the general asymptomatic population screening for early ovarian cancer are not high enough, which are improved by a variety of tumor markers joint inspection or examination to promote the early diagnosis of ovarian cancer [15, 16].

CD146 is a transmembrane glycoprotein expressed at the junction of endothelial cells. It is involved in the control of cell-cell cohesion and in angiogenesis [24]. Increasing amounts of evidences have demonstrated that CD146 is over-expressed in a variety of carcinomas, and considered to be a potential marker for tumor diagnosis, prognosis and treatment [19, 20, 21, 22]. sCD146, as the soluble form of CD146, can be detected in the plasma of healthy individuals according to the opinions of Bardin et al. [31] who proposed that the release of sCD146 in plasma could reflect the physiological mobilization of the protein, and then speculated that pathophysiological settings affecting the junction could modify sCD146 release. sCD146 boosted therapeutic effect of endothelial colony-forming cells (ECFC) in ischemic cardiovascular diseases through improving the cells’ survival capacity [32]. The mechanism of sCD146 on ECFC plays its role through a signalosome located in lipid rafts, containing angiomotin, the short isoform of CD146 (shCD146), VEGFR1, VEGFR2, and presenilin-1. sCD146 induces a sequential proteolytic cleavage of shCD146. The generated intracellular part of shCD146 is directed towards the nucleus to modulate the transcription of genes involved in cell survival (FADD, Bcl-xl) and angiogenesis (eNOS). In this study, it was found that sCD146 level decreased in observation group A, and much lower in observation group B, which showed an opposite trend to the tissue forms of CD146. In this regard, it was envisioned that the reduced sCD146 level was associated with the increased expression of tissue forms of CD146, the enhancement of CD146 adhesion capacity in ovarian tumors, consequently causing the reduced release of sCD46. This decreasing trend was consistent with the results reported by Stalin et al. [32], namely in patients with active Crohn disease, decreased levels of sCD146 were associated with the increased expression of membrane CD146 in areas of neovascularization. Some soluble forms of adhesion molecules have been described as active as competitive inhibitors of membrane-bound forms, such as the function way of sCD31 inhibiting the heterotypic adhesion mediated by CD31 expressed on the surface of transfected cells.

This study indicated that sCD146 level deceased in observation group A, and much lower in observation group B, which suggested different trends of sCD146 in different tissues; the sCD146 level decreased more significantly in observation group A when compared with that in control group, while CA125 level increased; the sensitivity of sCD146 was higher than that of CA125. The above results verified that the specificity was up to 90% in the diagnosis of ovarian cancer when CA125 combined with sCD146. These suggest that sCD146 could be a new potential marker in the diagnosis of ovarian cancer. Although sCD146 couldn’ t be used alone to diagnose ovarian cancer, it could play a significant complementary role in combination with CA125, so as to improve the sensitivity and specificity of CA125 in the diagnosis of ovarian cancer. However, for the small sample sizes, further studies are necessary to verify the exact role of sCD146 in ovarian cancer.

In conclusion, sCD146 constitutes a potential marker to evaluate the severity of endothelial functional alterations in patients with ovarian cancer, which effectively compensates for the diagnostic deficiency of CA125.

Declaration

The authors of this manuscript declare that they have no conflict of interest.

Acknowledgment

We wish to thank for the great help of Professor YAN Xi-yun, Protein and Peptide Pharmaceutical Laboratory, Chinese Academy of Sciences-University of Tokyo Joint Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences. This study was supported by Capital Medical University Joint Research Fund for General Basic Clinical Topics (12JL59).

Reference
[1] Renata R. Urban, Hao He, Raphael Alfonso, et al. Ovarian cancer outcomes: Predictors of early death. Gynecologic Oncology, 2016, 140(3): 474-480. doi: http://dx.doi.org/10.1016/j.ygyno.2015.12.021. [本文引用:2]
[2] Jeon SY, Hwang KA, Choi KC. Effect of steroid hormones, estrogen and progesterone, on epithelial mesenchymal transition in ovarian cancer development. J Steroid Biochem Mol Biol, 2016, 158: 1-8. doi: 10.1016/j.jsbmb.2016.02.005. [本文引用:1]
[3] Nagle CM, Dixon SC, Jensen A, et al. Obesity and survival among women with ovarian cancer: results from the Ovarian Cancer Association Consortium. Br J Cancer. 2015, 113(5): 817-826. doi: 10.1038/bjc.2015.245. [本文引用:1]
[4] Holschneider CH, Berek JS. Ovarian cancer: epidemiology, biology, and prognostic factors. Semin Surg Oncol, 2000, 19(1): 3-10. doi: 10.1002/1098-2388(200007/08)19:1<3::aid-ssu2>3.0.co;2-s. [本文引用:2]
[5] Jacobs IJ, Menon U, Ryan A, et al. Ovarian cancer screening and mortality in the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS): a rand omised controlled trial. Lancet, 2016, 387(10022): 945-956. doi: 10.1016/S0140-6736(15)01224-6. [本文引用:2]
[6] Victoria Sopik, Barry Rosen, Vasily Giannakeas, et al. Why have ovarian cancer mortality rates declined? Part III. Prospects for the future. Gynecol Oncol, 2015, 138(3): 757-761. doi: 10.1016/j.ygyno.2015.06.019. [本文引用:2]
[7] Russell MR, Walker MJ, Williamson AJ, et al. Protein Z: A putative novel biomarker for early detection of ovarian cancer. Int J Cancer, 2016, 138(12): 2984-2992. doi: 10.1002/ijc.30020.Epub2016Feb19. [本文引用:1]
[8] Blassl C, Kuhlmann JD, Webers A. Gene expression profiling of single circulating tumor cells in ovarian cancer-Establishment of a multi-marker gene panel. Mol Oncol, 2016, 10(7): 1030-1042. doi: http://dx.doi.org/10.1016/j.molonc.2016.04.002. [本文引用:1]
[9] Timms JF, Arslan-Low E, Kabir M, et al. Discovery of serum biomarkers of ovarian cancer using complementary proteomic profiling strategies. Proteomics Clin Appl, 2014, 8(11-12): 982-993. doi: 10.1002/prca.201400063. [本文引用:1]
[10] Park BJ, Cha MK, Kim IH. Thioredoxin 1 as a serum marker for ovarian cancer and its use in combination with CA125 for improving the sensitivity of ovarian cancer diagnoses. Biomarkers, 2014, 19(7): 604-610. doi: 10.3109/1354750X.2014.956793. [本文引用:1]
[11] Rohr I, Braicu EI, En-Nia A, et al. Y-box protein-1/p18 as novel serum marker for ovarian cancer diagnosis: A study by the Tumor Bank Ovarian Cancer (TOC). Cytokine, 2016, 85: 157-164. doi: 10.1016/j.cyto.2016.06.021. [本文引用:1]
[12] Pelissier A, Bonneau C, Chéreau E, et al. Dynamic analysis of CA125 decline during neoadjuvant chemotherapy in patients with epithelial ovarian cancer as a predictor for platinum sensitivity. Anticancer Res, 2016, 36(4): 1865-1871. [本文引用:1]
[13] Shen A, Xu S, Ma Y, et al. Diagnostic value of serum CA125, CA19-9 and CA15-3 in endometriosis: A meta-analysis. J Int Med Res, 2015, 43(5): 599-609. doi: 10.1177/0300060515583076. [本文引用:1]
[14] Sasaki A, Akita K, Ito F, et al. Difference in mesothelin-binding ability of serum CA125 between patients with endometriosis and epithelial ovarian cancer. Int J Cancer, 2015, 136(8): 1985-1990. doi: 10.1002/ijc.29185. [本文引用:2]
[15] Menon U, Talaat A, Rosenthal AN et al. Performance of ultrasound as a second line test to serum CA125 in ovarian cancer screening. BJOG, 2014, 121(Suppl 7): 35-39. doi: 10.1111/1471-0528.13211. [本文引用:3]
[16] Zhang Y, Qiao C, Li L, et al. Serum HE4 is more suitable as a biomarker than CA125 in Chinese women with benign gynecologic disorders. Afr Health Sci, 2014, 14(4): 913-918. doi: 10.4314/ahs.v14i4.20. [本文引用:2]
[17] Lehmann JM, Holzmann B, Breitbart EW, et al. Discrimination between benign and malignant cells of melanocytic lineage by two novel antigens, a glycoprotein with a molecular weight of 113, 000 and a protein with a molecular weight of 76, 000. Cancer Res, 1987, 47: 841-845. [本文引用:1]
[18] Johnson JP, Rothbächer U, Sers C, et al. The progression associated antigen MUC18: a unique member of the immunoglobulin supergene family. Melanoma Res, 1993, 3: 337-340. doi: 10.1097/00008390-199310000-00006. [本文引用:1]
[19] Wu Z, Wu Z, Li J, et al. MCAM is a novel metastasis marker and regulates spreading, apoptosis and invasion of ovarian cancer cells. Tumor Biol, 2012, 33(5): 1619-1628. doi: 10.1007/s13277-012-0417-0. [本文引用:2]
[20] Feng G, Fang F, Liu C, et al. CD146 gene expression in clear cell renal cell carcinoma: a potential marker for prediction of early recurrence after nephrectomy. Int Urol Nephrol, 2012, 44(6): 1663-1669. doi: 10.1007/s11255-012-0255-4. [本文引用:2]
[21] Li Y, Yu JM, Zhan XM, et al. Correlation of CD146 expression and clinic pathological characteristics in esophageal squamous cell carcinoma. Oncol Lett, 2014, 8: 859-863. doi: 10.3892/ol.2014.2227. [本文引用:2]
[22] Zhang H, Zhang J, Wang Z, et al. CD146 is a potential marker for the diagnosis of malignancy in cervical and endometrial cancer. Oncol Lett, 2013, 5(4): 1189-1194. doi: 10.3892/ol.2013.1147. [本文引用:2]
[23] Bardin N, Frances V, Combes V, et al. CD146: biosynthesis and production of a soluble form in human cultured endothelial cells. FEBS Letters, 1998, 421: 12-14. doi: 10.1016/s0014-5793(97)01455-5. [本文引用:1]
[24] Zhang BR, Li L, Feng LQ, et al. Elevated levels of soluble and neutrophil CD146 in active systemic vasculitis. Labmedicine, 2009, 40: 351-356. doi: 10.1309/lm92sm1llmwwseiq. [本文引用:2]
[25] Gayat E, Caillard A, Laribi S, et al. Soluble CD146, a new endothelial biomarker of acutely decompensated heart failure. Int J Cardiol, 2015, 199: 241-247. doi: 10.1016/j.ijcard.2015.07.039. [本文引用:1]
[26] Qian YN, Luo YT, Duan HX, et al. Adhesion molecule CD146 and its soluble form correlate well with carotid atherosclerosis and plaque instability. CNS Neurosci Ther, 2014, 20(5): 438-445. doi: 10.1111/cns.12234. [本文引用:1]
[27] Stalin J, Nollet M, Garigue P, et al. Targeting soluble CD146 with a neutralizing antibody inhibits vascularization, growth and survival of CD146-positive tumors. Oncogene, 2016. doi: 10.1038/onc.2016.83. [本文引用:1]
[28] Bardin N, Reumaux D, Geboes K, et al. Increased expression of CD146, a new marker of the endothelial junction in active inflammatory bowel disease. Inflamm Bowel Dis, 2006, 12(1): 16-21. doi: 10.1097/01.mib.0000194181.46930.88. [本文引用:1]
[29] Kaspi E, Guillet B, Piercecchi-Marti MD, et al. Identification of soluble CD146 as a regulator of trophoblast migration: potential role in placental vascular development. Angiogenesis, 2013, 16(2): 329-342. doi: 10.1007/s10456-012-9317-6. [本文引用:1]
[30] 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(15): 883-887. doi: 10.1056/NEJM198310133091503. [本文引用:1]
[31] Bardin N, Moal V, Anfosso F, et al. Soluble CD146, a novel endothelial marker, is increased in physiopathological settings linked to endothelial junctional alteration. Thromb Haemost, 2003, 90(5): 915-920. doi: 10.1160/TH02-11-0285. [本文引用:1]
[32] Stalin J, Harhouri K, Hubert L. Soluble CD146 boosts therapeutic effect of endothelial progenitors through proteolytic processing of short CD146 isoform. Cardiovasc Res, 2016, 111(3): 240-251. doi: 10.1093/cvr/cvw096. [本文引用:2]