Expression of the Carbohydrate Tumour Marker SLeX, SLeA (CA19-9), LeY and Thomsen-Friedenreich (TF) Antigen on Normal Squamous Epithelial Tissue of the Penis and Vagina IRMI WIEST 1, SANDRA SCHULZE 1, CHRISTINA KUHN 1, CHRISTIAN SELIGER 2, ROLAND HAUSMANN 3, PETER BETZ 3, DORIS MAYR 4, KLAUS FRIESE 1 and UDO JESCHKE 1 1 I. Department of Obstetrics and Gynaecology, and 4 Institute of Pathology, LMU Munich, Munich; 2 Department of ENT and 3 Institute of Legal Medicine, University of Erlangen, Germany Abstract. Background: Sialyl Lewis x (SLeX), sialyl Lewis a (SLeA), Lewis Y (LeY) and the Thomsen-Friedenreich (TF) antigen are carbohydrate motifs that mediate the adhesion between tumour cells and the endothelium. These antigens are usually not expressed in non-malignant tissue. Overexpression of SLeX and SLeA is combined with poor prognosis and malignant relapse. In this study, we analysed the combined expression of SLeX, SLeA, LeY and TF in normal squamous epithelium tissue of the penis shaft, glans and foreskin and in addition of the vagina and vulva. Materials and Methods: Paraffin-embedded slides of vaginal tissue (8), vulva tissue (8) and penis shaft (8) and glans tissue (8) were fixed and incubated with monoclonal antibodies against SLeX (IgM), SLeA (IgM), LeY (IgM) and TF (IgM). Staining reaction was performed with ABC reagent. The intensity of immunohistochemical reaction on images of the slides was analyzed using a semiquantitative score. Results: Strong focal expression of both sialyl Lewis antigens was found in the uretra of the penis shaft and on epithelial tissue of the glans, and permanent moderate expression of SLeX and SLeA in squamous epithelial tissue of the vagina. Moderate expression of TF was observed in male squamous epithelial tissues of the glans and foreskin and faint expression of TF was found in vulval epithelial tissue. Faint expression of Le Y was observed in female vulval epithelial tissue. Conclusion: Expression of SLeX, SLeA, LeY and especially of the TF antigen in normal non malignant epithelial tissue is surprising and can be explained by the function of this tissue in human reproduction. In addition, Correspondence to: PD Dr. Udo Jeschke, Ludwig-Maximilians- University of Munich, Department of Obstetrics and Gynaecology, Maistrasse 11, D-80337 Munich, Germany. Tel: +49 89 5160 4266, Fax: +49 89 5160 4916. Key Words: Carbohydrate tumour marker, SLeX, SLeA (CA19-9), LeY, Thomsen-Friedenreich (TF) antigen. moderate TF expression seems to be restricted to epithelial tissue of the penis glans and foreskin. Carbohydrates are functionally involved in different physiological processes like in cell-cell adhesion. E-selectin carbohydrate [sialyl Lewis x (SLeX) / sialyl Lewis a (SLeA)] interactions play a major role in leukocyte homing as well as in blood borne metastasis (1-4). The homing of lymphocytes is regulated by a unique posttranslational modification of sialic acid moieties that occurs specifically in sulfated selectin ligands, whereby the sialic acid loses its N- acetyl group and is converted into a 1.5 cyclic derivative. By contrast, nonsulfated carbohydrate ligands of the selectins, such as SLeX, are mainly involved in the recruitment of leukocytes during inflammation. The increase of SLeX expression upon inflammatory stimuli is mediated by transcriptional induction of fucosyltransferase genes (2). The sialyl Lewis antigens also, play a major role as ligands on tumour cells that are involved in interactions between E-selectin on endothelial cells. This interaction is the first step of building metastases via floating tumour cells sticking to the endothelium (5-7). Malignant transformation is associated with abnormal glycosylation, resulting in synthesis and expression of altered carbohydrate determinants, including SLeA and SLeX. These determinants appear in sera of patients with cancer and are extensively utilized for serum diagnosis of cancer in Japan. SLeA and SLeX are involved in selectin-mediated adhesion of cancer cells to vascular endothelium and these determinants are thought to be closely associated with hematogenous metastasis of cancer (6). In addition, we showed in a former study that sialyl Lewis carbohydrate antigens and amniotic fluid glycoproteins containing SLeX modulate the endocrine function of trophoblasts in culture by up-regulating progesterone production (8, 9). There is a focus on Lewis a and Lewis b antigens as the only two components of the Lewis blood group system. But 0250-7005/2007 $2.00+.40 1981
Table I. Antibodies used for the study. Antigen Antibody Isotype Concentration Source Sialyl Lewis a KM231 Mouse IgG1 2 Ìg/ml Calbiochem Sialyl Lewis x KM93 Mouse IgM 2 Ìg/ml Calbiochem Lewis Y A70-C/C8 Mouse IgM 2 Ìg/ml Glycotope GmbH Thomsen-Friedenreich NemodTF1 Mouse IgM 2 Ìg/ml Glycotope GmbH Thomsen-Friedenreich NemodTF2 Mouse IgM 2 Ìg/ml Glykotope GmbH fucosylation of these antigens produces Lewis isomers which include Lewis Y (LeY). Numerous -(1, 3)-fucosyltransferase genes have been characterized that can perform this function (10). This oligosaccharide structure may play a key role in lymphocyte traffic and inflammatory responses (11, 12). The Lewis Y (LeY) antigen, which is one of the type 2 human blood group-related antigens, also is thought to behave as an onco-developmental cancer-associated antigen (13). The Thomsen-Friedenreich (TF) antigen (or, more precisely, epitope) on the other hand has long been recognized as a tumour-associated antigen (14). Primary and metastatic carcinomas are epithelial in origin and comprise the largest group of malignant tumours in humans. In most of these tumours, the TF antigen is uncovered and immunoreactive. In all other tissues the TF antigen is masked and not accessible to the immune system; they are generally precursors of normal complex carbohydrate chains. Thus, carcinomas have antigens recognized as foreign by the patients immune system. The expression of the TF antigen has pathogenic and clinical consequences and the antigen itself is a powerful histological marker in carcinoma diagnosis and frequently in prognosis (14). The TF antigen is a carbohydrate epitope related to blood group antigens and consists of galactose- 1-3-N-acetylgalactose (Gal 1-3GalNAc-). In epithelial cells, the TF epitope is carried by mucin1 (Muc1), which belongs to a family of highly glycosylated proteins present on the surface of many glandular epithelial cells. On tumour cells, Muc1 is posttranslationally modified resulting in incomplete O-glycosylation and exposure of TF epitope. TF carbohydrate epitopes occur only in limited amounts in normal adult human tissues (15). The TF antigen is, however, expressed by foetal epithelia and mesothelia (16). In addition it is also found on transferrin isolated from human amniotic fluid (17) and produced by the trophoblast (18). The presence of TF during an early foetal phase, its absence in noncarcinomatous postfoetal tissues and its association with carcinomas suggest that TF is a stage-specific oncofoetal carbohydrate antigen. Carbohydrate antigens play a primary role in the initial gamete binding that precedes the existence of the foetalmaternal interphase (19). The aims of this study were to investigate the expression of SLeX, SLeA, LeY and the TF antigen in normal squamous epithelium tissue of the penis shaft, glans and foreskin, and in addition of the vagina and vulva and to identify any gender-specific expression of one or more of these markers in the tissues described. Materials and Methods Tissue samples. Squamous epithelial tissue samples from penis glans, shaft and foreskin and normal vaginal and vulval tissue samples were obtained from the Institute of Legal Medicine, University of Erlangen. Immunohistochemistry. Immunohistochemistry on paraffin sections (2-3 Ìm) of the different tissue specimens was performed as described elsewhere (20, 21). Briefly, sections were incubated in methanol/h 2 O 2 (30 min) to inhibit endogenous peroxidase activity, washed in PBS (5 min) and treated with goat serum (20 min, 22ÆC) to reduce nonspecific background staining. Incubation with the primary antibody (Table I) was done overnight at 4ÆC. Then sections were thoroughly incubated with the biotinylated secondary anti-mouse antibody (1 h, 22ÆC) and avidin-biotinylated peroxidase (45 min, RT). Between each step, sections were washed with phosphate-buffered saline (PBS, ph 7.4). Peroxidase staining reaction was performed with diaminobenzidine/h 2 O 2 (1 mg/ml; 5 min) and stopped in tap water (10 min). Sections were counterstained in haemalaun (1 min) and then coverslipped. As negative control, the primary antibody was replaced by pre-immune horse serum. Malignant tumor tissue was used as positive control. All specimens were evaluated by a pathologist with experience in immunohistochemistry (D.M.). Immunohistochemical evaluation and statistical analysis. The intensity and distribution patterns of specific carbohydrate antigen immunohistochemical staining reaction was evaluated by two blinded, independent observers, including a gynaecological pathologist (D. M.), using a semi-quantitative score as described elsewhere (22). Briefly, the staining intensity score was graded as 0=no staining; 1=weak staining; 2=moderate staining and 3=strong staining. Digital images were obtained with a 3CCD colour camera (JVC, Victor Company of Japan, Japan) and a Leitz (Wetzlar, Germany) microscope and saved on computer. The Mann-Whitney rank-sum test was used to compare the means of the staining intensity (SPSS; Chicago, IL, USA). Significance of differences was assumed at p 0.05. 1982
Wiest et al: Carbohydrate Tumour Markers in Normal Epithelial of the Penis and Vagina Figure 1. SLeX expression in (a) vaginal tissue (magnification x10), in (b) male foreskin tissue (magnification x10), (c) penis glans tissue (magnification x10) and (d) penis shaft (magnification: x10). Results Expression of SLeX in vaginal and penis tissue. Moderate expression of SLeX in squamous epithelial tissue of normal human vagina was found (Figure 1a). There was no SLeX expression on normal vulval epithelial tissue (figure not shown). In squamous epithelial tissue of male foreskin strong but focal staining for this antigen (Figure 1b) was found. The same staining pattern could be identified in the epithelial tissue of penis glans (Figure 1c). On the epithelial tissue of the penis shaft we identified a uniform moderate expression of SLeX (Figure 1d). Expression of SLeA in vaginal, vulval and penis tissue. In comparison to the SLeX expression moderate expression of SLeA in tissue slides of normal human vagina was found (Figure 2a). There was no SLeA expression on normal vulval epithelial tissue (Figure 2b) or on male foreskin tissue (Figure 2c). We identified a consistent moderate to strong expression of SLeA on sqamous epithelial tissue of penis glans and shaft (Figure 2d, 2e). Expression of LeY in vulval sqamous epithelial tissue. In contrast to SLeX and SLeA expression, normal vaginal epithelial tissue did not express LeY (Figure 3a). Moderate expression of LeY was found on squamous epithelial tissue of the vulva (Figure 3b). Penis tissue did not express LeY (foreskin: Figure 3c, penis glans: Figure 3d). Expression of TF in penis tissue. The TF antigen was not expressed in vaginal epithelial tissue (Figure 4a). Faint expression of TF was found in vulval epithelial tissue (Figure 4b). Moderate expression was found in penis foreskin (Figure 4c) and glans tissue (Figure 4d) stained with the anti-tf antibody Nemod TF1. This antibody is capable of binding single TFs and is not cross-reactive with Core-2. Also Nemod TF2 showed faint staining of TF in vulval epithelial tissue (Figure 4e) and moderate staining of TF in penis foreskin (Figure 4f) and glans epithelial tissue (Figure 4g). This antibody detects Gal 1-3GalNAc- (TF >TF ) and is cross-reactive in ELISA with GlcNAc 1-6[Gal 1-3]GalNAc-, Core-2, cluster of 2 TFs at a distance of about 13 amino acids. 1983
Figure 2. SLe expression in (a) vaginal tissue (magnification x10), (b) the vulva (magnification x10) and in (c) foreskin tissue (magnification x10), (d) penis glans tissue (magnification x10) and (e) penis shaft (magnification x10). Discussion In this study we analysed the combined expression of SLeA, SLeX, LeY and TF in squamous epithelial tissues of the vagina and vulva and in addition penis foreskin, glans and shaft. We identified a simultaneous expression of SLeA and SLeX in vaginal tissue and in penis glans and shaft squamous epithelial tissue. Vulval tissue was negative for both antigens and penis foreskin was only positive for SLeX. Both carbohydrate epitopes are tumour markers and natural ligands for selectins (4, 23). Selectins are glycoproteins bearing a lectin domain that is specifically recognized by the tetrasaccharides SLeX or SLeA (24). Depending on individual conditions, selectins are permanently or temporarily expressed on leucocytes (Lselectin) (25), platelets (P-selectin) (3) and activated endothelial cells (E-selectin, P-selectin) (26). They are 1984
Wiest et al: Carbohydrate Tumour Markers in Normal Epithelial of the Penis and Vagina Figure 3. LeY expression in (a) vaginal tissue (magnification x10), (b) the vulva (magnification x25), (c) foreskin tissue (magnification x10) and (d) penis glans tissue (magnification x10). involved in initial steps of the attachment of circulating leucocytes but also tumour cells to activated vascular endothelial cells (27). Blocking the lectin domain by exogenous probes could offer a way to interfere with the pathological processes caused by the adhesion of circulating tumour cells from the blood stream to vessel walls (28). Efficient blocking probes should bear the minimum recognized tetrasaccharide structure SLeX or SLeA in a pattern that fits to the cellular accommodation of selectins (29). This pattern is unknown. Expression of SLeX or SLeA is not connected to an inhibitory function of the probe. Moreover, multivalent binding of the probe to the receptor pattern is necessary to reach high efficacy, because of the low affinity of a single carbohydrate lectin interaction (30). Because both antigens (SLeX or SLeA) are expressed by vaginal tissue and in penis glans and shaft squamous epithelial tissue they cannot be used as marker for gender specific tissue expression. Over-expression of both antigens in tumour cells is connected with high metastatic potential. In the tissue investigated both Lewis antigens could play a role in cell-cell adhesion procedures. LewisY (LeY) was expressed only in squamous epithelial tissue of the vulva. Vaginal tissue and the different penis tissues were completely negative for this tumour antigen. LeY belongs to the Lewis antigens that are structurally related to the ABH blood group system. Lewis and related antigens may occur as free oligosaccharides in milk and urine, in protein bound by an O-linkage via an N- acetylgalactosamine to a serine or threonine residue or as glycolipids located mainly in the outer plasma membrane (31, 32). Circulating Lewis antigens can be acquired by red cells, lymphocytes, and platelets (32). Therefore this antigen may play a key role in lymphocyte traffic, inflammatory responses but also in metastatic procedures (11). In addition, LeY showed gender specific expression in the vulva but not in male sqamous epithelial tissue. Therefore this tumour marker could be useful for the discrimination between male and female tissue for medical investigations of legal nature. The TF epitope was identified on epithelial tissue of the penis foreskin and glans. Vaginal tissue showed no staining for this antigen. The vulval tissue showed faint expression of this 1985
Figure 4. TF expression in (a) vaginal tissue (magnification x10), (b) vulva epithelial tissue (magnification x25), (c) foreskin tissue (magnification x25) and (d) penis glans tissue (magnification x25). Another anti-tf antibody (Nemod TF2) also showed faint expression of TF in (e) vulval tissue (magnification x25), moderate expression in (f) foreskin (magnification x25) and (g) penis glans epithelial tissue (magnification x25). 1986
Wiest et al: Carbohydrate Tumour Markers in Normal Epithelial of the Penis and Vagina antigen. TF epitope was first identified in adenocarcinomata and not in benign or healthy tissues. TF-antigenic specificities are thus human carcinoma-associated. All humans possess anti-tf antibodies. Patients with breast tract carcinoma showed statistically significant alteration of anti-tf titer levels when compared to patients with benign disease and to healthy controls. Breast carcinoma patients but not healthy people showed cellular immunity to TF antigen in vitro and in vivo (33). Former investigations on human fetal tissues showed that the most intense staining of TF was in epithelial and mesothelial components; the mesenchyme stained more faintly. The presence of immunoreactive TF during an early phase of fetal development, and its known absence in noncarcinomatous postfetal tissues suggests that TF, in addition to its association with CA, is a stage-specific oncofetal antigen in pretolerogenic differentiation phases (16). The TF antigen is also expressed in the placenta where especially extravillous trophoblast cells and the syncytiotrophoblast were positive for this antigen (34-38). We identified this tumour marker on normal penis foreskin and glans. This is the first known study on TF expression in normal adult epithelial tissue. Formerly it was believed that TF is not expressed in benign or healthy tissues. Although we identified TF expression on normal penis tissue, this antigen may be involved in immunomodulation of the female reproductive tract. In addition, it could be a useful marker for the identification of male tissue cells in the vagina for medical investigations of legal nature. Conclusion We analysed the combined expression of SLeX, SLeA and TF in normal squamous epithelium tissue of the penis shaft, glans and foreskin and in the vagina and vulva. Expression of SLeX, SLeA, LeY and especially of the TF antigen in normal nonmalignant epithelial tissue is surprising and can be explained by the function of this tissue involved in human reproduction. In addition, since moderate TF expression seems to be restricted to the epithelial tissue of the penis glans and foreskin, the TF epitope could be a useful candidate for the identification of male foreskin or glans cells in the vagina of women after sexual harassment events without ejaculation for medical investigations of legal nature. References 1 Uchimura K, Kadomatsu K, El-Fasakhany FM, Singer MS, Izawa M, Kannagi R, Takeda N, Rosen SD and Muramatsu T: N-acetylglucosamine 6-O sulfotransferase-1 regulates expression of L-selectin ligands and lymphocyte homing. 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