ANTICANCER RESEARCH 26: 4943-4948 (2006) CD46 Expression is Indicative of Shorter Revival-free Survival for Ovarian Cancer Patients PAWEL SUROWIAK1,2,3, VERENA MATERNA1, ADAM MACIEJCZYK3, IRINA KAPLENKO4, MAREK SPACZYNSKI4, MANFRED DIETEL1, HERMANN LAGE1 and MACIEJ ZABEL2,5 1Institute of Pathology, Charité Campus Mitte, D-10117 Berlin, Germany; 2Chair and Department of Histology and Embryology, University School of Medicine, ul. Chalubinskiego 6a, 50-356 Wroclaw; 3Lower Silesian Centre of Oncology, pl. Hirszfelda 12, 53-413 Wroclaw; 4Chair and Department of Obstetrics and Gynaecology and 5Chair and Department of Histology and Embryology, University School of Medicine, ul. Swiecickiego 6, 60-781 Poznan, Poland Abstract. Background: The membrane cofactor protein CD46 cure very rarely. Despite the introduction of novel represents a complement inhibitor, which protects autologous chemotherapy regimens, the frequency of 5 - year survival cells from complement - mediated cytotoxicity. CD46 may of patients at all clinical stages has not exceeded 40%, in the exhibit the potential to protect tumor cells from the immune last 20 years (2). Therefore, intense efforts are being made responses of the host. The present study aimed to evaluate the in numerous centres to detect new prognostic factors, which prognostic significance of CD46 expression in ovarian cancers. might prove valuable towards studies on new therapeutic Materials and Methods: The analyses were performed on 73 approaches. ovarian cancer samples. Immunohistochemical reactions were The absence of the host’s immune response to the performed on paraffin sections of tumors using monoclonal presence of tumor cells represents one of the circumstances, antibodies directed against CD46. The immunohistochemical which promotes development of the tumor. The reactions and the clinical observations results were subjected to phenomenon may be explained by the expression of statistical analysis. Results: Expression of CD46 was complement regulatory proteins (CRPs) on tumor cells (3). demonstrated in 60% of primary laparotomy cases and in 70% Presence of CRPs on the cell membrane inhibits the secondary cytoreduction cases. Kaplan-Meier analysis showed formation of C3/C5 complexes. CD46 (MCP – membrane that a significantly shorter revival-free time was linked to cases cofactor protein) is a membrane protein (4) present on the with CD46 expression at PL (p=0.01). Conclusion: Ovarian surface of multiple types of epithelial cells, on endothelium, cancers manifest CD46 expression that is linked to a less and blood cells (3). However, it is absent from erythrocytes. favourable prognosis. The protein functions as a complement inhibitor. Expression of the protein has also been demonstrated in Ovarian cancers are frequent gynaecological tumors in various types of tumors and in cell lines derived from those women. About 190,000 new cases and 114,000 deaths from neoplasms (5-7). Expression of CD46 is more pronounced ovarian cancer are estimated to occur worldwide annually. on tumor cells than in the surrounding healthy tissues (8). The highest rates are reported in Scandinavia and Eastern The augmented expression of CD46 on tumor cells has been Europe, the USA and Canada (1). Due to their localisation suggested to represent the mechanism, which protects the and consequent late diagnosis, as well as the aggressive cells from the immune reaction (7, 8). To date, no studies course of the disease, therapy of ovarian cancers results in a have been performed on the potential prognostic significance of the estimation of CD46 expression in ovarian cancers. Thus, the aim of the present study was to evaluate the prognostic value of the immunohistochemical estimation Correspondence to: PD Dr. Hermann Lage, Charité Campus Mitte, of CD46 expression, in a group of ovarian cancer samples. Institute of Pathology, Charitéplatz 1, D-10117 Berlin, Germany. Tel: +49-30-450 536 045, Fax: +49-30-450 536 900, e-mail: Materials and Methods [email protected] Patients and specimens. Immunohistochemical examination was Key Words: Ovarian carcinoma, CD46, prognosis, immuno- performed retrospectively on tissue samples taken for routine histochemistry. diagnostic purposes. Forty three patients operated in 1999-2002 0250-7005/2006 $2.00+.40 4943 ANTICANCER RESEARCH 26: 4943-4948 (2006) on account of ovarian carcinoma at the Department of Table I. Patient and tumor characteristics. Gynaecology and Obstetrics, University Medical School in Poznan, Poland, were qualified for the studies. The cases were Characteristics No. (%)3 selected based on the availability of tissue and were not stratified for known pre-operative or pathological prognostic factors. The All patients study was approved by an Institutional Review Board (IRB) and 43 (100) 1 the patients gave their informed consent, before their inclusion Age (mean 51.0) in the study. Following the primary laparotomy (PL), all the ≤50 20 (47) 50-60 16 (37) patients were subjected to chemotherapy, using platinum-based >60 7 (16) schemes (Table I). Thirty-six patients from the same group were Grade1 subjected also to the secondary cytoreduction (SCR). In 7 cases, 1 7 (16) no secondary cytoreduction was performed due to advancement 2 18 (42) of the disease. In 6 cases, no tumor cells were detected in the 3 18 (42) material originating from the SCR. The patients were monitored FIGO1 via periodic medical check-ups, serum CA-125 levels, I 1 (2) ultrasonographic and radiological examinations. During the II 1 (2) follow-up period, 22 patients (51%) had recurrent disease and III 41 (95) 13 patients (30%) died of the disease. The mean revival-free Histology1 survival time was 16.9 months (range 0 to 52 months), while the Serous 37 (86) mean overall-disease-free survival time was 24.6 months (range 6 Endometrioid 3 (7) to 52 months). Other 3 (7) Samples from studied tumors were fixed in 10% buffered Clinical response2 formalin and then embedded in paraffin. In each case, hematoxylin Complete response 16 (37) and eosin stained preparations were subjected to histopathological Stable disease 5 (12) evaluation by two pathologists. The stage of the tumors was Progressive disease 22 (51) assessed according to the International Federation of Gynaecology Chemotherapy (in total) and Obstetrics (9). Tumors were graded according to the Silverberg Cisplatin/Paclitaxel 31 (72) grading system (10). Cisplatin/Cyclophosphamide/Adriblastin 6 (14) Cisplatin/Cyclophosphamide/Paclitaxel 3 (7) Cisplatin/Cyclophosphamide/Paclitaxel/Adriblastin 2 (5) Immunohistochemistry. Formalin-fixed, paraffin-embedded tissue Carboplatin/Paclitaxel 1 (2) was freshly cut (4 Ìm). The sections were mounted on Superfrost slides (Menzel Gläser, Germany), dewaxed with xylene and 1Data are given for the first operation/diagnosis implemented. gradually rehydrated. Activity of endogenous peroxidase was 2According to RECIST (Response Evaluation Criteria in Solid blocked by 30 min incubation in 1% H2O2. The sections were Tumours) (13). boiled for 15 min in a microwave oven in Antigen Retrieval 3Differences in the sum in groups are due to rounding up. Solution (DakoCytomation, Denmark) at 250W. This was followed by immunohistochemical reactions using the monoclonal mouse antibodies (clone 169-1-E4.3) against CD46 (Ancell Corporation, MN, USA). The antibodies were diluted 1:400 in the Antibody Control reactions. The control reactions included: (A) six samples Diluent, Background Reducing (DakoCytomation, Denmark). The of healthy human ovaries (from the archive of the Chair and sections were incubated with the antibody for one hour at room Department of Histology and Embryology, University School of temperature. Subsequently, they were incubated with biotinylated Medicine, Wroclaw, Poland) and (B) three smears of human blood, antibodies (15 min, room temperature) and with the streptavidin- fixed in a 1:1 mixture of acetone and methanol at –20ÆC (procedure biotinylated peroxidase complex (15 min, room temperature) such as that in the case of paraffin sections, but omitting xylene, (LSAB+, HRP, DakoCytomation, Denmark). NovaRed (Vector series of alcohols and boiling in the Target Retrieval Solution). Laboratories, UK) was used as a chromogen, employing 10 min incubation at room temperature. All the sections were Statistics. Statistical analysis of the results was performed in two stages. counterstained using Meyer’s hematoxylin. Each reaction was In the first stage, using the Statistica 98 PL (Statsoft, Poland) software, accompanied by the negative control, in which the specific relationships were examined between the expression of studied antibody was substituted by the Primary Mouse Negative Control antigen and clinical/pathological variables of studied cases (¯2 test), (DakoCytomation, Denmark). and, in the second stage, employing the Mann-Whitney U-test, CD46 was examined in the material originating from PL and SCR. Kaplan- Scoring of immunostaining results. Intensity of immunohistochemical Meier statistics and log-rank tests were performed using SPSS reactions was estimated independently, by two pathologists. In cases software (release 10.0; SPSS Inc., Chicago, IL, USA) to estimate the of controversy, a re-evaluation was performed with the use of a significance of differences in survival times. The length of revival-free double-headed microscope. The case was diagnosed as positive [1] survival was defined as the time from the primary surgical treatment, when the expression was observed in all tumor cells or in numerous until the