CD146 is a potential marker for the diagnosis of malignancy in cervical and endometrial cancer

Cluster of differentiation 146 (CD146) is an endothelial cell adhesion molecule which is overexpressed in various types of malignant cancer, including ovarian cancer. However, whether CD146 is overexpressed in another two types of gynecological cancer, cervical cancer and endometrial cancer, remains unclear. In the present study, we showed that CD146 expression levels were higher in cells from cervical cancer and endometrial cancer compared with their corresponding normal tissues, using anti-CD146 mouse antibody AA4 (mAb AA4) and that mAb AA4 exhibited a high performance for specificity, sensitivity and positive predictive value in the detection of these two types of cancer. CD146 expression was positively and significantly correlated with the pathological subtype of cervical cancer and with the histological grade and depth of myometrial invasion in endometrial cancer. In addition, we confirmed that CD146 is present in the majority of blood vessels in cervical and endometrial cancer, suggesting that CD146 may be actively implicated in the metastasis of cervical and endometrial cancer via the vascular system. Thus, this study provides insights for further development of CD146 mAb in the detection of gynecological malignant cancer types and implies that a combined treatment strategy of anti-CD146 immunotherapy with other traditional chemo- or radiotherapy treatments may be a promising approach against cervical and endometrial cancer.

CD146 is a 113-kDa integral membrane glycoprotein, whose sequence of amino acids consists of a signal peptide, an extracellular fragment structure of five immunoglob-ulin-like domains (V-V-C2-C2-C2), a transmembrane region and a short cytoplasmic tail (2,18). The Protein and Peptide Pharmaceutical Laboratory, Chinese Academy of Sciences-University of Tokyo, Tokyo, raised an array of mouse antibodies (mAbs) against CD146, among which AA4 recognizes the epitope located at the second IgC2 domain (19). AA4 is able to specifically detect CD146 in tumor specimens from various organs, including breast tumors (14). CD146 overexpression has been revealed in ovarian cancer and may be a marker for poor prognosis in ovarian cancer patients (11). Thus, CD146 Abs may be a useful marker in the detection of gynecological malignancies. However, CD146 expression and detection in two other types of gynecological cancer, cervical and endometrial cancer, has not been investigated.
This study aimed to evaluate the distribution of CD146 in cervical and endometrial cancer; the diagnostic use of CD146 Abs in cervical and endometrial cancer was evaluated by calculating the correlation between clinical pathological parameters and the extent of immunohistochemical CD146 expression in tumor tissues.

Materials and methods
Antibody and reagents. The anti-CD146 monoclonal antibody AA4 was generated in the Protein and Peptide Pharmaceutical Laboratory (Chinese Academy of Sciences-University of Tokyo Joint Laboratory of Structural Virology and Immunology, Beijing, China). A CD31-specific antibody was purchased from Abcam (Cambridge, MA, USA). Biotin-conjugated secondary antibodies (goat anti-rabbit or -mouse) and HRP-conjugated streptavidin were purchased from Dianova (Rodeo, CA, USA). Goat anti-rabbit Alexa Fluor ® 488 and goat anti-mouse Alexa Fluor ® 555 were purchased from Invitrogen (San Diego, CA, USA). DAPI was purchased from Roche (Indianapolis, IN, USA). Normal goat serum and a 3,3'-diaminobenzidine (DAB) staining kit were purchased from ZSGB-BIO (China).
Ethics statement. Informed consent was obtained from all participants in this study. All procedures have been 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.
Clinical sampling. Malignant specimens were obtained from patients with gynecological cancer, including cervical carcinoma and endometrial carcinoma, by surgical excision, in Anzhen Hospital (Beijing, China) and the Fifth Affiliated Hospital of Zhengzhou University (Zhengzhou, Henan, China). Normal uterine cervical tissue and normal endometrium were collected by hysterectomy in non-cancer patients with multiple uterine leiomyomas or adenomyosis, respectively. None of the patients had received preoperative chemotherapy, radiotherapy or hormone therapy or suffered with malignant tumors, other than the gynecological cancer examined in this investigation.
Immunohistochemistry. This assay was conducted according to classically established methods (14). Briefly, tissue sections were fixed with formalin and embedded with paraffin. Tissue sections were then cut (5 µm) and stained with hematoxylin and eosin (H&E) solution. Following deparaffinization, tissue sections were stained with a CD146-or CD31-specific antibody, then with biotin-conjugated secondary antibodies (1:1000), followed by HRP-conjugated streptavidin (Dianova). The sections were counterstained with hematoxylin to visualize the nuclei.
Double-staining immunohistofluorescence analysis. Sections (5 µm) were fixed in 4% paraformaldehyde (pH 7.4) for 15 min at room temperature. Following the general procedure, which included permeabilization and blocking, the slides were co-incubated with antibodies for CD146 (mAb) and CD31 (rAb, rabbit Ab) overnight at 4˚C. After washing with PBS three times, the slides were incubated with a mixture of the two secondary antibodies (anti-rabbit Alexa Fluor ® 488 and antimouse Alexa Fluor ® 555) in the dark for 1 h. The nuclei were stained with DAPI. Fluorescent images were acquired using a confocal microscope (FV1000, Olympus, Tokyo, Japan).
Statistical analysis. Statistical analysis was conducted using SPSS software. The significant differences between the groups and the association between CD146 expression and different clinicopathological parameters were evaluated using the Chi-square test or Fisher's exact test. P<0.05 was considered to indicate a statistically significant difference.

Classification of cervical cancer specimens into categories.
The collected specimens were divided into normal tissue and cancerous tissue groups. The mean age of the patients with normal and cancerous uterine cervical tissue was 44.12±10.61 and 48.25±9.86 years, respectively. The cervical samples consisted of 16 normal (control) and 256 malignant specimens (Table I). Cervical cancer is a malignant neoplasm originating from cells in the cervix uteri and consists of two histological subtypes. The majority of cervical cancer types are squamous cell carcinomas, arising in the squamous epithelial cells that line the cervix. Adenocarcinomas, arising in glandular epithelial cells, are the second most common type of cervical cancer (20). Cancer rarely occurs in other types of cells in the cervix. In this study, 241 of the cancer samples had the squamous carcinoma subtype and only 15 samples had the adenocarcinoma subtype. Cervical cancer tissue was further classified into categories following the review of existing patient medical records. With regard to the histological grade, 46 cases were grade 1 (G1), with a high differentiation phenotype, 154 tumors were moderately differentiated (grade 2, G2) and 56 tumors had a poorly differentiated phenotype (grade 3, G3). According to the International Federation of Gynecology and Obstetrics (FIGO) systems, 226 patients were in the early stages (I-II) and 30 patients were in the advanced stages (III-Ⅳ) of the disease (Table II).
Classification of endometrial cancer specimens into categories. The mean age of the patients with normal and cancerous endometrial tissue were 33.12±10.56 and 54.69±9.43 years, respectively. Endometrial samples consisted of 18 normal (control) and 87 malignant specimens (Table I). The majority of endometrial cancer types are adenocarcinomas, originating from epithelial cells that line the endometrium and form the endometrial glands. There are numerous microscopic subtypes of endometrial carcinomas, including the common endometrioid type, the more aggressive papillary serous carcinoma and clear cell endometrial carcinomas (21). Eighty samples were endometrioid adenocarcinomas (EECs) and 7 samples were non-endometrioid adenocarcinomas (NEECs), which included 5 squamous endometrial carcinomas, 1 papillary serous endometrial carcinoma and 1 adeno-squamous endometrial carcinoma. FIGO grading showed that 30 samples were well-differentiated (G1) and 57 samples had moderate (G2) or no (G3) differentiation (Table III).
CD146 is highly expressed in cervical and endometrial cancer. To investigate the expression of CD146 in cervical and endometrial cancer, we conducted immunohistochemical assays with AA4 mAb on all the collected specimens, which consisted of 377 samples of normal and cancerous tissue. As summarized in Table I, CD146 was detected in a large number of cancer samples. The number of true positives (Table I) in     CD146 expression levels are positively correlated with the histological subtypes of cervical cancer. Immunohistochemical assays showed that CD146 expression did not occur in normal cervical squamous epithelium and mucosal glands. In cervical carcinomas, CD146 was detected mainly in the membranes and cytoplasm of squamous tumor and vascular endothelial cells (Fig. 1A). This was consistent with the localization data from fluorescent immunohistochemistical assays, as shown in Fig. 1B, in which the tumor cells with CD146 + expression were labeled with CD146 mAb AA4 and the vascular endothelial cells with CD146 + expression were confirmed with an endothelial marker for CD31. The overlapping yellow regions are indicative of the co-localization of CD31 and CD146 in vascular cells. These data demonstrated that CD146 was localized in tumor cells and vascular endothelial cells in cervical carcinomas. For patients who were <48 years old, 39% (52/133) of samples had CD146 + staining, whereas, for patients who were ≥48 years old, a higher number of cases were CD146 + and had a staining rate of 49% (60/123). There was no significant difference between the cervical cancer and normal cervical samples in the two parameters of clinical FIGO stage (I-II and III-IV stages) and histological grade (G1-G3). By contrast, as shown in Table II, the AA4 + reaction rate is significantly higher in squamous cervical carcinoma (112/241, 46.5%) than in cervical adenocarcinoma (0/15, 0%). This indicates that CD146 presence was a potential predictive marker (P=0.000) for discrimination of the two histological subtypes of cervical carcinoma, namely that a cervical carcinoma with higher CD146 expression levels was more likely to be the squamous carcinoma subtype than the adenocarcinoma subtype.
was detected in vascular endothelial cells and also in glands during the hyperplasia period. In endometrial cancer samples, CD146 was detected in the majority of tumor cells, in addition to vascular endothelial cells ( Fig. 2A). The CD146 localization in vascular endothelial cells was further confirmed using fluorescent immunohistochemistry. Similar to the results in Fig. 1B, the co-localization of CD31 and CD146 in the endothelial cells of tumor blood vessels was observed in endometrial cancer (Fig. 2B). Immunohistochemistry results showed that no statistically significant differences in CD146 expression existed between the two groups for endometrial cancer samples from patients <55 years old (70%) or ≥55 years old (65%). Furthermore, no significant difference in CD146 + expression was identified between cancer samples with different FIGO stages (P= 0.653). CD146 + staining occured in 67% (46/69) of cases in cancer samples with an early clinical stage (Ⅰ-Ⅱ phase), compared with 72% (13/18) in cancer samples with an advanced clinical stage (III-Ⅳ phase). For the histological subtypes of endometrial cancer, CD146 expression was detected in EEC and NEEC in 66% (53/80) and 86% (6/7) of samples, respectively, indicating that there is no significant difference in CD146 expression between the two subtypes of endometrial cancer (P=0.290).

Discussion
In this study, we demonstrated that the specificity, sensitivity and PPV of AA4 (a mAb for CD146) is suitable for use in the detection of cervical cancer and endometrial cancer. Results showed that CD146 expression levels were higher in cervical and endometrial cancer tissues compared with their corresponding normal tissues. Notably, CD146 expression was positively and significantly correlated with various subtypes of cervical cancer, as higher expression levels were detected in the squamous carcinoma subtype than in the adenocarcinoma subtype (Table II). The significant correlation which was identified between CD146 expression and the histological classification or the depth of myometrial invasion indicates that CD146 may be involved in the onset and development of endometrial cancer (Table III). This hypothesis was further strengthened by an immunohistofluorescent assay, where  the broad expression of CD146 in the cellular membrane of malignant cancer was confirmed. Furthermore, in accordance with previous studies (22,23), immunohistofluorescence data in this study showed that CD146 was present in the majority of cancer blood vessels ( Figs. 1 and 2), suggesting that CD146 may be actively implicated in the dissemination and metastasis of cervical cancer and endometrial cancer via the vascular system. Gynecological malignant cancer, including cervical cancer, endometrial carcinoma and ovarian cancer, is life-threatening to females (24). The incidence of cervical cancer is higher than endometrial and ovarian cancer (20) and the mortality rate of ovarian cancer is the highest among these three types of cancer (25). Therefore, effective screening methods and potential therapeutic targets have been pursued in this field. At present, the clinically used biomarkers for detection of gynecological malignancies principally include squamous cell carcinoma antigen (SCC), carcinoembryonic antigen (CEA) and sugar antigens CA125, CA199 and CA153 (26)(27)(28)(29).
However, the sensitivity and specificity are not satisfactory for the accuracy of predictive detection for gynecological malignancies (26). Therefore, seeking more reliable biomarkers is likely to aid the successful detection of tumors in the early stages of the disease and also for determining an effective therapeutic approach. Our findings of CD146 overexpression in cervical and endometrial cancer, plus the ability of AA4 to detect CD146 with high sensitivity and specificity, provides insight for further development of CD146 mAbs in the detection of malignant gynecological cancer. It also implies that a combined treatment strategy of anti-CD146 immunotherapy with other traditional chemo-or radiotherapy treatments may be a promising anticancer technique.