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 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 (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- 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 time of diagnosis of a recurrent tumor or death. We could cell clumps. The case was regarded negative [0] when no reaction not perform a multivariate analysis, because with an univariate analysis was noted or the reaction was present only in isolated (less than no significant relationships between studied clinicopathological 10%) tumor cells. parameters (age, histology, grade) and overall survival and revival-free

4944 Surowiak et al: CD46 Expression and prognosis in Ovarian Cancer

Figure 1. Immunohistochemical localisation of CD46 expression in ovarian carcinoma cells. Strong cytoplasmic reaction (hematoxylin, x 400).

survival time of studied patients (p>0.05) were found. Since 95% of (70%). The ¯2 test has not shown any significant the studied patients were in FIGO III stage of advancement, we did relationships between CD46 expression at PL, or SCR, or not investigate the relationships between the stage and the survival histologic type, or grade of studied tumors (Table II). data. P-values <0.05 were considered significant. The Mann-Whitney U-test demonstrated no significant differences in the proportion of cases manifesting Results expression of CD46 at PL or SCR (p=0.39).

CD46 immunostaining in control preparations and in ovarian CD46 expression and patient survival. At the first stage of carcinomas. In sections originating from six healthy ovaries, statistical analysis of relationships between CD46 expression a membrane reaction in the vascular endothelium and trace and survival of the patients, the ¯2 test was used. The of reaction in the ovarian surface epithelium were found. relationship between expression of CD46 on the one hand Connective tissue and corpora albicans were negative for and response to chemotherapy on the other was examined. CD46. In the sections studied, we did not find . No significant relationship was found (Table II). Smears of human blood demonstrated no CD46 expression Using analysis of Kaplan-Meier, overall survival time and in erythrocytes. In the cases of ovarian cancer studied, a revival-free time were compared among CD46-negative and cytoplasmic and membranous reaction of a variable intensity CD46-positive patients. The analysis demonstrated that in individual cases was noted in cancer cells (Figure 1). In cases with CD46 expression at PL exhibited a significantly the PL group, a positive reaction was detected in 26 cases shorter revival-free survival time (p=0.01) (Figure 2) than (60%), while in the SCR group, 21 cases were positive the cases, which were negative for CD46 expression.

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Table II. Association between CD46 expression and various clinicopathological factors.

No. of patients (%)

PL SCR

Characteristics CD46 positive CD46 negative P-value Chi2-test CD46 positive CD46 negative P-value Chi2-test

Histological type1 0.98 - Serous 23 (53.5) 14 (32.6) - - Endometrioid 3 (6.9) 0 (0.0) - - Other 0 (0.0) 3 (6.9) - - Grade1 0.05 - 1 5 (11.7) 2 (4.8) - - 2 9 (20.9) 9 (20.9) - - 3 12 (27.9) 6 (14.0) - - Clinical response 1.0 0.26 Complete response 4 (9.3) 12 (27.9) 8 (26.7) 2 (6.7) Stable disease 3 (6.9) 2 (4.8) 7 (23.3) 6 (20) Progressive disease 19 (44.2) 3 (6.9) 4 (13.3) 1 (3.3)

PL: primary laparotomy; SCR: secondary cytoreduction. 1The relation between CD46 expression at SCR on one hand and histologic type and grade on the other was not examined.

Figure 2. Kaplan-Meier curves for survival and expression of CD46 in group of 43 ovarian cancer patients: (A) no significant differences in overall survival time between patients who were CD46 positive and negative at PL; (B) patients with CD46-negative tumors at PL had an increased revival-free time compared to those who were CD46-positive; (C) no significant differences in overall survival time between patients who were CD46-positive or -negative at SCR; (D) no significant differences in revival-free time between patients who were CD46-positive or -negative at SCR. PL: first-look laparotomy; SCR: secondary cytoreduction.

4946 Surowiak et al: CD46 Expression and prognosis in Ovarian Cancer

Discussion In the cases demonstrating expression of CD46, i.e. in "complement-resistant" tumors, relapses develop more In this study, we describe the expression of CD46 protein, frequently, despite surgical treatment and chemotherapy as detected by immunohistochemistry, in malignant than in cases which are "complement-sensitive". The epithelial ovarian tumors in sections from primary phenomenon should be examined in detail, as it can provide laparotomies, secondary cytoreductions, healthy ovaries a novel target for therapy. In the in vitro study we quoted and erythrocytes. We confirm the findings of Oglesby et al., above (12), an increase was noted in the sensitivity of according to which, CD46 protein is expressed in superface ovarian cancer cells to complement on incubation of the epithelium and vascular endothelium (11), but not those cells with antibodies directed against another representative of Fishelon et al. in erythrocytes (3). We found that CD46 of CRPs, CD59. Expression of CD46, which is in part protein is expressed in a subset of invasive ovarian membrane bound, might be of therapeutic value if targeted carcinomas in sections from PL and SCR, but there was no with therapeutic antibodies. significant difference in the percentage of CD46-positive In summary, we found that CD46 is commonly expressed cases between them. The expression of CD46 has not been in ovarian cancer and that immunohistochemically described in ovarian cancers before, and only the determined CD46 predicts shorter revival-free survival of expression of another CRP member, CD59, has been ovarian cancer patients. detected in in vitro studies on ovarian cancer cells (12). Bjorge et al. (14) have shown that soluble forms of the C1 Acknowledgements inhibitors, CD59 and CD46, and the alternative pathway inhibitors, and FHL-1, were present in the ascitis This study was supported by a grant from the Committee of fluid of ovarian carcinoma patients at concentrations Scientific Investigations, Poland (No. 3 PO5E 064 22); and by the higher than these in serum samples. In our study, we have "Berliner Krebsgesellschaft e.V.", Germany. demonstrated CD46 expression at cytoplasmic and membranous localisations in 60% of sections at PL and in References 70% of sections at SCR. We can only speculate whether this cytoplasmic expression of CD46 reflects an 1 Stewart BW and Kleihues P (eds): World Cancer Report. Lyon, IARC Press, 2003. overproduction of the protein or a disturbance of protein 2 Holschneider CH and Berek JS: Ovarian cancer: epidemiology, distribution or degradation within the cell. biology, and prognostic factors. Semin Surgical Oncol 19: 3-10, CD46 is present on the surface of multiple types of 2000. epithelial cells, on endothelium and blood cells [3]. 3 Fishelon Z, Donin N, Zell S, Schultz S and Kirschfink M: Expression of the proteins has also been demonstrated in Obstacles to cancer immunotherapy: expression of membrane various types of tumors, such as gastric, renal and breast complement regulatory proteins (mCRPs) in tumours. Mol carcinomas (5-7). As it is so widely expressed, CD46 Immunol 40: 109-123, 2003. expression cannot be used as a specific marker for ovarian 4 Barilla-LaBarca M, Liszewski MK, Lambris JD, Hourcade D and Atkinson JP: Role of membrane cofactor protein (CD46) carcinomas. in regulation of C4b and deposits on cells. J Immunol 168: Our statistical analysis has demonstrated no relationship 6298-6304, 2002. between CD46 expression on the one hand and histology, 5 Niehans GA, Cherwity DL, Stalez NA, Knapp DJ and grade or relapse on the other. The phenomenon suggests Dalmasso AP: Human carcinomas variably express the that expression of CD46 is neither linked to tumor type, complemented inhibitory proteins CD46 (membrane cofactor nor differentiation and thus, neither to dynamic protein), CD55 (decay-accaelerating factor), and CD59 development of the tumor. In addition, CD46 has shown no (protectin). Am J Pathol 149: 129-142, 1996. 6 Simpson KL, Jones A, Norman S and Holmes CH: Expression relationship to response of the tumor to chemotherapy. On of the complement regulatory proteins decay accelerating factor the other hand, expression of CD46 in PL material has (DAF, CD55), membrane cofactor protein (MCP, CD46) and been shown to predispose to earlier relapse of the CD59 in the normal uterine cervix and in premalignant cervical neoplastic disease. This phenomenon suggests that CD46 disease. Am J Pathol 151: 1455-1467, 1997. expression plays a role in the spread of the tumor. To our 7 Blok VT, Daha MR, Tijsma OM, Weissglas MG, van den knowledge, this study demonstrates, for the first time, the Broeck LJ and Gorter A: A possible role of CD46 for the significant prognostic value of CD46 expression in human protection in vivo of human renal tumor cells from ovarian cancer. complement-mediated damage. Lab Invest 80: 335-344, 2000. 8 Rushmere NK, Knowlden JM, Gee JMW, Harper ME, CD46 as a complement inhibitor protects the tumor cells Robertson JF, Morgan BP and Nicholson RI: Analysis of the from activity of the host’s complement. The relationship we level of mRNA expression of the membrane regulators of have observed suggests the significance of complement in complement, CD59, CD55 and CD46, in breast cancer. Int J the protection of the host from development of the tumor. Cancer 108: 930-936, 2004.

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9 UICC: TNM Classification of malignant tumors. Sobin LH, AT, Christian MC and Gwyther SG: New guidelines to evaluate Wittekind Ch (eds). New York, Wiley-Liss, 1997. the response to treatment in solid tumours. European 10 Shimizu Y, Kamoi S, Amada S and Silverberg SG: Toward the Organisation for Research and Treatment of Cancer, National development of a universal grading system of ovarian epithelial Cancer Institute of the United States, National Cancer Institute carcinoma: Testing of a proposed system in a series of 461 of Canada. J Natl Cancer Inst 92: 205-216, 2000. patients with uniform treatment and follow-up. Cancer 82: 893- 14 Bjorge L, Hakulinen J, Vintermyr OK, Jarva H, Jensen TS, 901, 1998. Iversen OE and Meri S: Ascitic in ovarian 11 Oglesby TJ, Longwith JE, and Heuttner PC: Human cancer. Br J Cancer 92: 895-905, 2005. complement regulator expression by the normal female reproductive tract. Anat Rec 246: 78-86, 1996. 12 Bjorge L, Hakulinen J, Wahlstrom T, Matre R and Meri S: Complement-regulatory proteins in ovarian malignances. Int J Cancer 79: 14-25, 1997. Received January 12, 2006 13 Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan Revised August 4, 2006 RS, Rubinstein L, Verweij J, Van Glabbeke M, van Oosterom Accepted September 4, 2006

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