The immunohistochemical expression of CD24 and CD171 adhesion molecules in borderline ovarian tumors

Introduction

Ovarian tumors grow silently, in many instances attaining very large size, and their behavior usually can only be predicted postoperatively after histological evaluation. In this regard, most of them fall into one out of three categories, namely benign, borderline or malignant. This scheme is especially applicable for the most common of the ovarian tumors, which derive from the surface epithelium. Still, classic histological classification is not always adequate for proper and effective management and prognosis. Currently, molecular markers have become quite promising in tumor diagnostics and therapeutics. Among them recognition and adhesion molecules seem to play an important role in carcinogenesis and tumor progression.

CD24 is a sialoglycoprotein that is anchored to the cell surface by a glycosyl phosphatidylinositol linkage. It was originally described as a B-cell specific marker, which is expressed in the early stages of B-cell development, as well as in several solid tumors, including breast, colon, prostate, periampullary, and renal cell carcinomas, and neuroblastoma [1-4]. CD171, also known as L1CAM, is a 200-kDa transmembrane glycoprotein. It is a neuronal cell adhesion molecule involved in axon guidance and cell migration [5] with a strong implication in treatment-resistant cancers [6-8]. L1CAM belongs to the immunoglobulin superfamily of recognition molecules, and participates in heterophilic interactions with other adhesion molecules such as laminin, integrins, proteoglycans and CD24. It is also expressed by hematopoietic cells and a variety of tumor cell lines [2, 9]. We undertook the task of investigating these molecules in ovarian neoplasms, focusing on the borderline tumor category, since literature concerning the expression of these molecules in ovarian cancer is still limited [10-16].

Material and methods

Our material included 50 borderline ovarian tumors (33 serous, 16 mucinous and 1 endometrioid). In addition, we studied 29 ovarian cystadenomas (10 serous, 10 mucinous cystadenomas and 9 endometrioid cysts), and 25 ovarian invasive carcinomas (10 serous, 5 mucinous, and 10 endometrioid), which were used as controls. Three patients with borderline tumors (all of them serous type) had peritoneal implants, and 6 patients with ovarian carcinoma (4 serous and 2 endometrioid) had metastases to the omentum.



Immunohistochemical technique



Immunohistochemical detection was performed on formalin fixed, paraffin embedded tissue sections. The following monoclonal antibodies were applied: CD24 (dilution 1 : 20, clone 5G3, eBioscience, San Diego, CA, USA) and CD171 (dilution 1 : 20, clone SN3, NeoMarkers, Fremont, CA, USA). A standardized automated (Nexes, Ventana, Tucson, Arizona, USA), streptavidin-biotin immunohistochemical detection system was used (I-VIEW Paraffin DAB; Ventana). Normal tonsils were used as a positive control for CD24, and normal peripheral nerves were used as a positive control for CD171. A negative control for immunostaining was carried out by replacing the primary antibody with non-immune rabbit serum.



Immunohistochemistry scoring



Both the intensity and the extent of immunostaining were evaluated for each antibody, in a semiquantitative way. The intensity of immunostaining was graded on a scale of 0 to 3, where 0 – negative reaction, 1 – weak, 2 – moderate and 3 – strong reaction. The extent of immunoreactivity was expressed as the percentage of immunoreactive cells, and was recorded separately for each of the intensity categories (0 to 3). The sum of the products for each intensity category (intensity grade X percentage of positive cells) was the final score of each case (score range: 0 to 300). In order to interpret better our results, we regarded our cases as negative (score = 0), mildly positive (score = 1-100), moderately positive (score = 101-200), and strongly positive (score = 201-300).



Statistical analysis



IBM SPSS version 17 for Windows and Microsoft Excel version 10 for Windows were used for statistical analysis of the collected data. Student’s t-test was performed for comparison of the mean values between different categories of tumors.

Results

The mean age of the patients with benign cystadenomas was 39.31 ±15.30, the mean age of the patients with borderline tumors was 41.11 ±12.14, and the mean age of the patients with malignant tumors was 57.91 ±14.70. The results of CD24 and CD171 immunostaining of ovarian tumors are shown in Table I.



CD24 immunoreactivity



The cellular staining pattern was either apical (more commonly) or both membranous and cytoplasmic. 1) Apical staining: Normal ovarian epithelium showed no immunoreaction, benign cystadenomas had negative to weak staining intensity, while borderline tumors and invasive carcinomas generally showed much stronger immunostaining (Fig. 1). The difference between the benign tumors on the one hand, and the borderline and malignant tumors on the other hand, almost reached statistical significance (benign vs. borderline tumors p = 0.06, benign vs. malignant tumors p = 0.09, borderline vs. malignant p = 0.46). No significant differences were observed between the different histological types (serous vs. mucinous vs. endometrioid) except for the malignant category of tumors, where serous carcinomas had on average almost double the scores of mucinous carcinomas (p = 0.06). Borderline tumors without peritoneal implants did not differ significantly in their staining intensity from cases with peritoneal implants. However, low-grade carcinomas had much lower scores than high-grade carcinomas (p = 0.05), and carcinomas with peritoneal metastases had on average more than double the staining scores of cases without peritoneal metastases (p = = 0.08). 2) Membranous and cytoplasmic staining: 3/50 (6%) borderline tumors, 5/7 (71.43%) serous carcinomas, and 1/8 (12.50%) endometrioid ovarian adenocarcinomas presented CD24 cytoplasmic staining, which was accentuated in a membranous pattern along the circumference of tumor cells (Fig. 2). Two out of the five cases (40%) with serous carcinoma showing cytoplasmic CD24 staining pattern had metastases to the omentum.



CD171 immunoreactivity



The staining pattern of CD171 was always membranous (Fig. 3). The lowest immunoreactivity scores were recorded in normal ovaries and benign ovarian tumors, while the highest scores were found in malignant tumors (Table I). However, none of these differences reached statistical significance (benign vs. borderline, p = 0.10; benign vs. malignant, p = 0.09; borderline vs. malignant p = 0.49). It is noteworthy that only one case was strongly positive for CD171, a serous carcinoma presenting extensive peritoneal metastases. The tumor cells showed increased immunoreactivity especially at the invasive front of the tumor. There were no significant differences between tumors of different histological type, between borderline tumors with and without peritoneal implants, low-grade and high-grade carcinomas, and between malignant tumors with and without metastases to the omentum.

Discussion

CD24 is a ligand of P-selectin, an adhesion receptor on activated endothelial cells and platelets, and thus might contribute to the metastasizing capacities of CD24-expressing tumor cells [17, 18]. Immunohistochemical expression of CD24 has been investigated in various carcinomas, including those of the stomach, colon, gallbladder, breast, endometrium, pancreas, and the lung [1, 19, 20]. In general, malignant tumors, especially those of higher histological grade and those with metastatic disease, tend to present stronger immunoreaction, while CD24 expression has been associated with survival and prognosis. Previous studies in ovarian cancer cases have shown that overexpression of CD24 is an independent factor associated with tumor metastasis, and poor survival [10-14]. In this study the expression of CD24 was considerably higher in borderline tumors and ovarian carcinomas than normal ovaries and benign neoplasms. However, the intensity scores of this marker were very close when considering borderline tumors and carcinomas. This implies that the latter two categories may share some molecular pathways. In addition, high-grade carcinomas and carcinomas with metastases to the omentum had considerably higher CD24 scores, confirming the results of previous reports [10-14]. The polarized apical expression of CD24 was maintained in benign cystadenomas and most borderline tumors, while progression to carcinoma made prominent a non-polarized, cytoplasmic expression of CD24, an observation made by other authors as well [10-13]. This change of expression pattern may be due to overproduction, disturbance of protein distribution or degradation within the cell [11]. Accumulative data from our results and those of the literature [10-13] support the view that cytoplasmic expression of CD24 in a borderline or a malignant ovarian tumor may suggest a more aggressive phenotype.

Overexpression of CD171 has been shown to promote tumor cell growth, cell motility and migration as well as angiogenesis [7, 8]. Several reports suggest that L1CAM is also involved in apoptosis resistance [6]. Tumors of various sites express this molecule, including neoplasms of the female genital tract, colon, stomach, pancreas, skin, and the kidneys [6-8, 21-23]. Expression is often found at the invasive front of primary tumors [24], strongly supporting a role for L1CAM in metastasis. Increased expression of L1CAM has been correlated with lymph node metastases, advanced tumor stage and reduced patient survival [24, 25]. In our material CD171 expression was more prominent in borderline and malignant tumors than benign cystadenomas, but it did not relate to tumor grade or stage of the disease. Still other authors have found an association of CD171 expression with tumor stage and survival in ovarian cancer [15, 16], and so further investigation is warranted.

In conclusion, CD24 and CD171 expression in the ovaries seems to be associated with tumor malignancy, as is the case in tumors of other organs, and may indicate that a neoplasm has attained a more aggressive phenotype. If such a correlation is established, it may have therapeutic implications, and potentially lead to the development of targeted therapies with a better patient response [24, 29, 30].



The authors declare no conflict of interest.

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Address for correspondence



Dimosthenis Miliaras

14 Ethnikis Amynis Street

54621 Thessaloniki, Greece

fax: +30 2310 241 810

e-mail: dmiliara@med.auth.gr