ANTICANCER RESEARCH 25: 1761-1764 (2005)

Expression of Glycodelin in Human Breast : Immunohistochemical Analysis in Mammary In Situ, Invasive and their Lymph Node Metastases

NAIM SHABANI1*, IOANNIS MYLONAS1*, CHRISTIANE KUNERT-KEIL2, VOLKER BRIESE3, WOLFGANG JANNI1, BERND GERBER1, KLAUS FRIESE1 and UDO JESCHKE1*

1I. Department of Obstetrics and Gynaecology, Ludwig-Maximilians-University of Munich, Maistrasse 11, 80337 Munich; 2Ernst Moritz Arndt University of Greifswald, Institute of Pathophysiology, Karlsburg; 3University of Rostock, Department of Obstetrics and Gynaecology, Faculty of Medicine, Rostock, Germany

Abstract. Objective: Glycodelin is a 28 kDa glycoprotein, carcinoma) without lymph node metastases, expression of previously known as placental protein 14 (PP 14). Glycodelin glycodelin was demonstrated in 9 cases (9/10), of whom 3 cases displays immunosuppressive and contraceptive properties. It also showed a strong and 6 cases a mediate to weak expression of suppresses the cytolytic capacity of human natural killer (NK) cells glycodelin. Only 1 case was negative. In the group of invasive breast in vitro and inhibits binding of sperm cells to the zona pellucida cancer with axillary lymph node metastases, in 5 cases (4 ductal (the outer membrane of the oocyte). Glycodelin is expressed in and 1 lobular carcinoma) there was no expression of glycodelin, normal glandular of the endometrium as well as in either in the primary tumour tissue, or in the metastatic infiltration endometrial, ovarian and cervical carcinoma cells. Glycodelin is of the axillary lymph nodes (5/10). In only one case (invasive also expressed in normal and malignant glandular cells outside the ) was there a strong expression of glycodelin in reproductive tract, like hidroadenoma, parabronchial , sweat the primary tumour and a weak expression in the metastasis in glands and pancreatic . Recently, glycodelin was axillary lymph node. In 4 other cases (2 ductal, 1 lobular and 1 demonstrated to be expressed in normal and cancerous human mucinous), there was a weak staining in primary breast tissue. Materials and Methods: Paraffin-embedded slides of cells and no expression in the metastatic lymph node. Conclusion: carcinoma in situ (8 DCIS, 2 CLIS), invasive carcinomas without These results demonstrate that invasive breast carcinomas without lymph node metastases (9 invasive duct carcinomas, 1 invasive axillary lymph node metastases (better prognosis) are more likely to lobular carcinoma) and invasive carcinomas (7 invasive duct express glycodelin. In contrast, the cases of breast cancer with carcinomas, 2 invasive lobular carcinomas, 1 mucinous metastatic infiltration of the axillary lymph nodes showed no (or carcinoma) with corresponding lymph node metastases were used. weak) expression of glycodelin (worse prognosis). On the basis of Immunohistochemical staining reaction was used to detect these results, we speculate that glycodelin expression could be used glycodelin expression in the different types of carcinoma in situ, in as a prognostic marker for breast cancer. This has to be confirmed invasive carcinoma of the human breast and in metastatic in larger studies. carcinoma in axillary lymph nodes. Results: Glycodelin expression was found in all cases of carcinoma in situ (10/10). In the group Carcinoma of the breast is the most frequent malignant of invasive carcinoma of the breast (ductal and lobular tumour in women, with high incidence and mortality in most developed countries. Early detection and adequate therapy are very important (1). Among many prognostic factors, the most important are the tumour size, histological grade and *N. Shabani, I. Mylonas and U. Jeschke contributed equally to this lymph node involvement. Lymph node involvement is a work. powerful prognostic factor. Numerous studies have shown that patients with axillary lymph node metastases have an Correspondence to: PD Dr. Udo Jeschke, Ludwig-Maximilians- independently increased risk for relapse and for cancer- University of Munich, I. Department of Obstetrics and associated death, compared to patients without nodal Gynaecology, Maistrasse 11, 80337 Munich, Germany. Tel: 0049- metastasis (2). 89-5160-4266, Fax: 0049-89-5160-4916, e-mail: udo.jeschke@ med.uni-muenchen.de Glycodelin, previously known as placental protein 14 (PP14) (3), which is immunologically indistinguishable from Key Words: Glycodelin, breast cancer, metastasis. chorionic ·2-microglobulin (CAG-2) (4) and placental

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Figure 1. A. Expression of glycodelin in ductal carcinoma in situ (DCIS) with comedo necrosis, 10x. B. Expression of glycodelin in invasive ductal carcinoma without metastasis, 25x. C. Expression of glycodelin in invasive ductal carcinoma with lymph node metastasis, 10x. D. Expression of glycodelin in lymph node metastasis, 10x.

Table I. Antibodies used in the study.

Epitope Antibody Isotype Concentration Source

Glycodelin A A87-D/F4 Mouse IgG 2 Ìg/ml NEMOD AG

Glycodelin - Rabbit IgG 2 Ìg/ml Bioscience AG peptide antibody

specific ·2-microglobulin (PAMG-2) (5), is expressed in glandular epithelium of the endometrium (6) and decidua Figure 2. Staining intensity of glycodelin in breast cancer subtypes (7). Glycodelin suppresses the cytolytic capacity of NK-cells [carcinoma in situ (Ca in situ), invasive carcinoma without lymph node metastasis (Ca w/o LNM), invasive carcinoma with lymph node (natural killer cells) in vitro (8). Our own investigations (9) metastasis (Ca w LNM) and lymph node metastases (LNM)] determined suggested a relationship between serum levels of glycodelin by computerised analysis of the immunohistochemical reaction on the and threatened abortion. different tissue slides.

1762 Shabani et al: Glycodelin in Human Breast Cancer

Glycodelin was shown to have a molecular weight of Zeiss, Germany). For standardization of the measurement, in each 28,000, with two identical subunits held together by picture the optical density of white background colour was attuned noncovalent bonds and a carbohydrate content of 17.5% with to 250. For all sections, we assessed the mean optical density and the quantity of pixels which had a positive reaction for glycodelin a unique carbohydrate configuration, consistent with using the KSRun software (imaging system KS400, release 3.0; sialylated LacdiNAc structures that are very unusual for Zeiss, Vision GmbH, Germany). mammals (10). Julkunen and coworkers confirmed the findings of homology between glycodelin and ‚-lactoglobulins Statistical methods. The SPSS/PC software package, version 6.01 by deducing its complete amino acid sequence (11). Two iso- (SPSS GmbH, München, Germany), was used for collection, forms of glycodelin have been described; glycodelin A and a processing and statistical analysis of all data. All p values resulted similar glycoprotein, glycodelin S, which is found in seminal from two-sided statistical tests and p≤05 was considered to be significant. plasma, but is differently glycosylated from glycodelin A (12). There are several other prognostic and predictive factors Results for breast cancer, which are still under investigation. The present study was designed to analyse glycodelin expression in Expression of glycodelin in breast cancer tissue. We found a different types of in situ and invasive breast carcinoma, and to strong expression of glycodelin in tissue slides of carcinoma address its role in metastatic tissue of breast cancer. in situ, with 100% of the cases (10/10) being positive for this Materials and Methods antigen (Figure 1A). Invasive carcinoma tissue without axillary lymph node metastases (LNM) showed similar, but Forty formalin-fixed paraffin-embedded tissue blocks from partially reduced, expression of glycodelin compared to patients, who underwent surgery for breast tumour were obtained. carcinoma in situ (Figure 1B). We identified expression of The tissue samples were classified according to the histological this antigen in 90% of the invasive carcinomas without classification of breast cancer: intraepithelial neoplasia (8 patients axillary LNM. A reduced expression of glycodelin was with DCIS, 2 patients with CLIS), invasive carcinomas without metastasis (9 invasive ductal carcinomas, 1 invasive lobular identified in tumour cells with LNM (Figure 1C). We found carcinoma) and invasive carcinomas (7 invasive ductal carcinomas, an expression of this antigen in 50% of the cases with node- 2 invasive lobular carcinomas, 1 invasive mucinous carcinoma) with negative breast cancer. The expression of glycodelin in LNM corresponding lymph node metastases (Figure 1D) was comparable to that in the primary tumour. Computerized quantification of glycodelin expression in breast Immunohistochemistry. Immunohistochemistry was performed on cancer tissue. The computerized analysis of staining intensity paraffin sections (7 Ìm) of the different breast cancer tissue specimens, as described previously (13). Briefly, sections were (optical density) is summarized in Figure 2. We identified incubated in methanol/H2O2 (30 min) to inhibit endogenous significant differences of glycodelin staining between peroxidase activity, washed in phosphate-buffered saline (PBS) carcinoma in situ and LNM (p=0.028). (5 min) and treated with goat serum (20 min, 22ÆC) to reduce non- specific background staining. Incubation with the primary antibody Discussion (Table I) was done overnight at 4ÆC. Sections were then thoroughly incubated with the biotinylated secondary anti-mouse antibody In this study, we analysed the expression of the glycoprotein (1 h, 22ÆC) and avidin-biotinylated peroxidase (45 min, room temperature). Between each step, the sections were washed with glycodelin in forty formalin-fixed paraffin-embedded tissue PBS (pH 7.4) three times. Peroxidase staining reaction was done blocks from patients who underwent surgical removal of a with diaminobenzidine/H2O2 (1 mg/ml; 5 min) and stopped in tap breast tumour. Glycodelin is mainly synthesized in secretory water (10 min). Sections were counter stained in haemalaun (1 min) endometrial glands, by gestational decidua, in seminal and then cover-slipped. In controls, the primary antibody was vesicles (6, 7, 13, 14), in the ovary (15), as well as in replaced with pre-immune mouse serum. Positive and negative megakaryocytic/erythroid precursors of the bone marrow controls were always included. All specimens were evaluated by a (16). Furthermore, glycodelin is expressed in a variety of pathologist with experience in immunohistochemistry. The classification of the stained cells as either micrometastases or tumour tumours. Investigations of glycodelin expression in ovarian cells also required histomorphological characterisation. Unspecific cancer (15) showed that polyclonal and monoclonal reactions or staining of endothelial cells in the marginal sinus of antibodies against PP14 stained negative in serous lymph nodes were not considered to be related to tumour cells. and mucinous ovarian tumours. The same antibodies were used to investigate the glycodelin Computerized analysis of antigen expression. The level of antigen expression in breast cancer cells (17). Glycodelin expression expression was determined in a blinded fashion in one run with was found in ductal carcinomas, tubular carcinomas, identical staff, equipment and chemicals. From each section, 5 digital pictures were taken at random of different areas of the mucinous carcinomas, mixed ductal/tubular carcinomas and tumour tissue (200-fold magnification; 3CCD colour camera; lobular carcinomas. The results were confirmed by Northern Hitachi HV-C20M; Hitachi Denshi Ltd, Japan, and Axiolab, Carl blot analysis and RT-PCR. Investigations with a polyclonal

1763 ANTICANCER RESEARCH 25: 1761-1764 (2005) antibody against a synthetic glycodelin peptide sequence 8 Okamoto N, Uchida A, Takakura K, Kariya Y, Kanzaki H, (18) on endometrial, ovarian and cervical cancer showed Riittinen L, Koistinen R, Seppala M and Mori T: Suppression that glycodelin expression is elevated in ovarian and by human placental protein 14 of natural killer cell activity. Am endometrial cancer tissue. These results were confirmed at J Reprod Immunol 26: 137-142, 1991. 9 Tomczak S, Briese V, Kunkel S and Muller H: Serum placental the mRNA-level by RT-PCR. In addition, transfection of protein 14 (PP14) levels in patients with threatened abortion. glycodelin cDNA into breast cancer cells induced an Arch Gynecol Obstet 258: 165-169, 1996. alteration of the cell growth behaviour, leading to formation 10 Dell A, Morris HR, Easton RL, Panico M, Patankar M, of acinar configurations and apoptosis. The glycodelin- Oehniger S, Koistinen R, Koistinen H, Seppala M and Clark expressing cells displayed strongly up-regulated expression GF: Structural analysis of the oligosaccharides derived from of markers of organized epithelia. The rate of proliferation glycodelin, a human glycoprotein with potent immuno- was also suppressed. These results indicate that the suppressive and contraceptive activities. J Biol Chem 270: 24116-24126, 1995. expression of glycodelin is accompanied by a phenotype of 11 Julkunen M, Seppala M and Janne OA: Complete amino acid organized glandular epithelium in breast cancer cells (19). sequence of human placental protein 14: a progesterone- Our results seem to confirm these findings. In summary, regulated uterine protein homologous to beta-lactoglobulins. invasive breast carcinomas without metastasis (better Proc Natl Acad Sci USA 85: 8845-8849, 1988. prognosis) are more likely to express glycodelin. In addition, 12 Koistinen H, Koistinen R, Dell A, Morris HR, Easton RL, no (or weak) expression of glycodelin (worse prognosis) was Patankar M, Oehniger S, Clark GF and Seppala M: Glycodelin observed in positive breast cancer cases. On the basis of from seminal plasma is a differentially glycosylated form of contraceptive glycodelin A. Mol Hum Reprod 2: 759-765, 1996. these results, we suggest that glycodelin expression could be 13 Julkunen M, Wahlstrom T, Seppala M, Koistinen R, Koskimies used in the future as a prognostic marker for breast cancer. A, Stenman UH and Bohn H: Detection and localization of This has to be confirmed in larger studies. placental protein 14-like protein in human seminal plasma and in the male genital tract. Arch Androl 12: 59-67, 1984. Acknowledgements 14 Seppala M, Suikkari AM, Koistinen R, Riittinen L and Julkunen M: Endometrial protein secretion with respect to We thank C. Kuhn, I. Wiest, S. Kunze, S. Schulze and J. Adolph endometrial and ovarian function. Eur J Obstet Gynecol for their excellent technical assistance. Furthermore, we are Reprod Biol 36: 250-257, 1990. grateful to Dr. R. Jarchow (Computing Center, University of 15 Kamarainen M, Leivo I, Koistinen R, Julkunen M, Karvonen Greifswald, Germany) for his support. U, Rutanen EM and Seppala M: Normal human ovary and ovarian tumours express glycodelin, a glycoprotein with References immunosuppressive and contraceptive properties. Am J Pathol 148: 1435-1443, 1996. 16 Kamarainen M, Riittinen L, Seppala M, Palotie A and 1 Black RJ, Bray F, Ferlay J and Parkin DM: Cancer Incidence Andersson LC: Progesterone-associated endometrial protein – and Mortality in the European Union: Cancer Registry Data a constitutive marker of human erythroid precursors. Blood 84: and Estimates of National Incidence for 1990. Eur J Cancer 33: 467-473, 1994. 1075-1107, 1997. 17 Kamarainen M, Halttunen M, Koistinen R, von Boguslawsky K, 2 Haybittle JL, Blamey RW and Elston CW: A prognostic index von Smitten K, Andersson LC and Seppala M: Expression of in primary breast cancer. Br J Cancer 45: 361-366, 1982. glycodelin in human breast and breast cancer. Int J Cancer 83: 3 Bohn H, Kraus W and Winckler W: New soluble placental 738-742, 1999. tissue proteins: their isolation, characterization, localization and 18 Horowitz IR, Cho C, Song M, Flowers LC, Santanam N, quantification. Placenta Suppl 4: 67-81, 1982. Parthasarathy S and Ramachandran S: Increased glycodelin 4 Petrunin DD, Griaznova IM, Petrunina IA and Tatarinov IS: levels in gynecological malignancies. Int J Gynecol Cancer 11: Comparative immunochemical and physicochemical 173-179, 2001. characteristics of human placental chorionic alphal- and alpha2- 19 Kamarainen M, Seppala M, Virtanen I and Andersson LC: microglobulins. Bull Exp Biol Med 85: 658-661, 1978. Expression of glycodelin in MCF-7 breast cancer cells induces 5 Tatarinov YS, Kozlaeva GA, Petrunin DD and Petrunina YA: differentiation into organized acinar epithelium. Lab Invest 77: Proteins and hormones. In: The Human Placenta, Klopper A, 565-753, 1997. Genazzi A and Crosignani PG (eds) London Academic Press pp. 35-46, 1980. 6 Julkunen M, Koistinen R, Sjoberg J, Rutanen EM, Wahlstrom T and Seppala M: Secretory endometrium synthesizes placental protein 14. Endocrinology 118: 1782-1786, 1986. 7 Julkunen M, Rutanen EM, Koskimies A, Ranta T, Bohn H and Seppala M: Distribution of placental protein 14 in tissues and body fluids during pregnancy. Br J Obstet Gynaecol 92: 1145- Received August 2, 2004 1151, 1985. Accepted October 31, 2004

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