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Modulated expression of glycoprotein oligosaccharides identifies phenotypic differentiation in squamous carcinomas of the human cervix
Modulated Expression of Glycoprotein Oligosaccharides Identifies Phenotypic Differentiation in Squamous Carcinomas of the Human Cervix SOUMITRA BANERJEE, MB, BS, MRCOG, PETER ROBSON, MB, BS, MRCOG, W. PATRICK SOUI-IER, MSc, MD, FRCOG, AND CHRISTOPHER S. FOSTER, MD, PHD, MRCPATH This study has examined changes in expression of complex oligosaccharides during the development of invasive squamons carcinoma o f the h u m a n cervix to determine whether particular oligosaccharide structures that might influence the phenotypic behavior of individual h u m a n cervical cancers were expressed during neoplasia. An extensive panel of lectins capable of identifying all the core and antennary oligosaccharide structures commonly encountered in human epithelia was chosen to probe a range of 11 benign and 26 malignant cervical tissues, all of the latter being clinically stage I. Lectin histochemistry was performed both before and after tissue desialylation using the enzyme neuraminidase to identify masking of oligosaccharide determinants by sialic acid. Nomleoplastic cervical epithelial cells were found to express only type I antennary structures (Ga~31~3GalNAc-) usually modified by sialic acid linked 2 ~ 6 to terminal Ca/- or GalNAc residues. Type II ofigosaccharide structures (Ca//] I~4GIcNAo) were n o t identified in these normal tissues. No other terminal anteunary modifications were detected on normeoplastic cervical squamons epithelia. Conversely, neosynthesis of type II oligosaccharides was detected by Erythrina cristagalli (ECG) binding in 50% of the squamons
carcinomas. Five terminal antennary modifications were commonly identified in the carcinomas that were not identified in normal cervical epithelia and comprised the ofigosaccharides bound by lectins RCA, SBA, BS-I, LTA, and UEA-1. Synthesis of these ofigosaccharides restdted in expression of structures similar to those recognized as ligands for extracellular matrix-binding proteins. We suggest that expression o f such novel oligosaccharide structures may be an important promotor of local invasion and further dissemination of human cervical carcinomas through enhanced binding of malignant cells to stromal matrix proteins. This study has demonstrated that identification of expressed oligosaccharide structures is an objective m e t h o d of identifying individual tumor cell phenotypes and may form the basis of a useful functional classification of human cervical squamons carcinomas. HUM PATHOL 26:1005--1013. Copyright © 1995 byW.B. Saunders Company Key words: h u m a n cervix, squamons carcinoma, lectins, glycoprotein modulation. Abbreviations: PBS, phosphate-buffered saline; v/v, volume of solute per volume of solution.
Carcinoma o f the cervix uteri is the second commonest cancer in women worldwide. In the United Kingdom in 1987, the latest year for which figures are available, 19,566 new cases were registered, of which 3,972 were invasive, giving an incidence for this malignancy of 15.4 per 105 female population] In 1990, 1,721 deaths were reported in the United Kingdom, giving a rate of 6.7 per 105. Comparable figures compiled for the same time in the United States give an incidence of 8.2 per 105 with a death rate of 3.0 per 105.~ Invasive and metastatic disease together constitute the most c o m m o n cause of treatment failure and are solely responsible for the mortality of cervical cancer. At present, the likely behavior of an early cervical cancer cannot be identified at diagnosis because no markers have been developed with which to assess its functional or malignant potential independently. Morphological grade is a p o o r indicator of tumor behavior or patient survival within any stage. "~5 Unassisted morphology recognizes only gross similarities and differ-
ences between tumors, and does not identify individual tumor cells o f particular phenotypes, which may be of immense behavioral importance. Failure of morphologic grading is evidenced by recurrent disease following conventional conservative treatment. 6 Clinical stage remains the only useful parameter for determining therapeutic m a n a g e m e n t and predicting the likel~ prognosis of individual cervical squamous cell cancers. The behavioral phenotype of an epithelial cell is modulated by the composition and functional properties of its plasma membrane. Within this structure, glycoproteins link components of the cytoskeleton to the extracellular matrix and are important mediators of intercellular contact, cellular adhesion, and growth stimuli, s T h e oligosaccharide domains of glycoconjugates are synthesized by a complex system of glycosyltransferase enzymes arranged hierarchically within the endoplasmic reticulum and Golgi apparatus, and control the intracellular processing and trafficking of many glycoprotein molecules. 9 Ionic conditions within Golgi cisternae dictate the relative activities of glycosyltransferases competing for the same substrates. Failure to maintain ionic homeostasis results in the formation of novel oligosaccharides with altered expression of associated glycoconjugates at the cell surface. Conventionally, oligosaccharide c o m p o n e n t s of glycoconjugates containing the core sequence Galfll~3GalNAc-identified by the lectin p e a n u t agglutinin (PNA) are known as type I, whereas those containing the sequence Galfll-*
From the Department of Obstetrics and Gynecology, Royal Postgraduate Medical School, Hammersmith Hospital, London, and the Department of Pathology, University of Liverpool, Liverpool, UK. Accepted for publication January 24, 1995. Address correspondence and reprint requests to Christopher S. Foster, MD, PhD, Department of Pathology, Duncan Building, PO Box 147 Daulby St, University of Liverpool, Liverpool, L69 3BX, UI~ Copyright © 1995 by W.B. Saunders Company 0046-8177/95/2609-000355.00/0
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HUMAN PATHOLOGY Votume26, No. 9 (September 1995) 4GlcNAc- are identified by the lectin ECG and are known as type II. Because expression of particular oligosaccharide structures by t u m o r cells may influence their behavior, structural analysis of plasma m e m b r a n e oligosaccharides may assist in identifying individual behavioral phenotypes within a neoplasm. The use of lectins to distinguish differences in oligosaccharides expressed by normal and neoplastic cells was first reported by Aub et alJ ° Lectins are carbohydrate-binding proteins of n o n i m m u n e origin that bind only to highly restricted sugar structures and are exquisitely sensitive probes with which to investigate subtle alterations in cell m e m b r a n e oligosaccharides. 1] Using the lectins BPA, GS-2, PNA, SBA, and WGA, Byrne et al identified patterns of oligosaccharides consistently expressed by normal cervical squamous epithelium, but variably with Con-A, GS-1, and UEA-1. ~z Their findings suggested that cervical neoplasia is particularly associated with alterations in core a-Man sugars, and changes to terminal ol-//3-GalNAc-, oz-/fl-GlcNAc-, cr-//3-Gal-, and oz-Fuc- residues on the antennary re~ions of both Nlinked and O-linked glycoconjugates. ]" Binding o f PNA to cervical squamous epithelium has been reported to remain constant and not to vary with the phase of the menstrual cycle.14'15Recently, Hirao et al, indicated that although expression of the Tn-antigen, identified by the Vicia villosa agglutinin, was closely correlated with vascular permeation and lymph node metastasis by cervical cancer cells, 16 expression of the T antigen, identified by PNA, was of no predictive value. In the present study, we have c o m p a r e d the oligosaccharides expressed by h u m a n normal cervical epithelium and early invasive squamous carcinomas using a panel of 12 different lectins selected to identify the widest spectrum of c o m m o n oligosaccharide structures to be probed in these tissues. Differences in expression of oligosaccharide structures have been correlated with the appearances of individual t u m o r cells rather than the overall m o r p h o l o g y of a particular cancer. The findings support the hypothesis that it is possible to identify cells of different phenotypes within an individual squamous carcinoma and that the presence of cells expressing certain oligosaccharide determinants may indicate the likely behavior of those cervical cancers.
MATERIALS AND METHODS Specimens
Blocks of normal tissue were specifically selected to exclude features such as immature metaplastic squamous epithelium. Tumor Tissues Sections from two separate blocks were stained and examined from each cervical tumor. All tumors occurred in the region of the squamocolumnar junction and extended to a depth greater than 5 mm into the adjacent stroma; hence, their local invasion was more than "microinvasive, ''~7 although all were clinically stage I. None of the carcinomas extended beyond the limits of the tissues examined within a single tissue block so that the invasive fronts of the tumors could be identified in all cases examined. Before admission to this study, all tumors were routinely examined using the periodic acid-Schiff/Alcian blue technique to exclude poorly differentiated adenocarcinomas and adenosquamous carcinomas. In this study, analysis of lectin binding was restricted to only the invasive components of the tumors. In situ cervical cancer, as a strictly preinvasive entity, has been examined in a separate investigation and was not recorded.
kectin Histochemistry All lectins were obtained biotinylated from Sigma (Poole, UK) and are summarized in Table 1. Each was diluted to 10 #g/mL in sodium phosphate-buffered saline (PBS; pH 7.6) containing 1 mmol each of calcium chloride and manganese chloride. PBS was also used for washing. Formalin-fixed and paraffin wax-embedded tissue sections cut at 2 #m were dewaxed in TSF 30 (Infrakem Ltd, Wigan, UK) and brought to water through graded ethanols. Endogenous peroxidase was blocked with 0.3% (volume of solute per volume of solution Iv/v]) hydrogen peroxide (H202) in distilled water for 30 minutes at room temperature. Lectin staining was performed by the avidin-biotin-peroxidase complex method, 18using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Lectins were incubated for 60 minutes at room temperature on tissue sections that were then washed in PBS. Peroxidase activity was localized by addition of 3,3'-diaminobenzidine tetrahydrochloride at 250/zg/mL in PBS containing 0.03% (v/v) H202 for 10 minutes. Sections were counterstained in Harris' hematoxylin, dehydrated, cleared, and mounted. Lectin binding was assessed by light microscopic appearances, and the distribution of staining within individual cervical carcinoma cells was identified.
Neuraminidase Digestion Lectin histochemistry was performed after enzymatic desialylation. Serial tissue sections were digested with neuraminidase (ex Vibrio cholerae;, BDH Laboratories, Dagenham, UK) at a dilution of 1:10 in 200-mM sodium acetate buffer (pH 5.5) at room temperature for 60 minutes before staining was performed according to the protocol already described.
Histologically normal cervical tissues (n = 11) were obtained following abdominal hysterectomy for dysfunctional hemorrhage. Locally invasive cervical squamous carcinomas (n = 26) were obtained from radical abdominal hysterectomy specimens. Approval to use archival pathologic material was obtained from the Ethics Committee of the Hammersmith Hospital and Royal Postgraduate Medical School, London.
Paraffin sections of normal human kidney were used as positive controls of lectin binding. For the negative controls, each lectin was omitted and replaced by an equal volume of PBS.
Normal Tissues
RESULTS Tissue Morphology
Sections from two standard blocks were stained and examined from each specimen of normal cervical tissue. The contained tissue extended from the squamous resection margin of the ectocervix and through the squamocolumnar junction to include the isthmic region of endocervical epithelium.
Normal Controls
Normal Tissues None of the control tissues contained any dysplastic or neoplastic features. Junctional chronic inflammation was minimal.
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OLIGOSACCHARIDES IN CERVICAL CANCER (Banerjee et al) TABLE 1.
Lectin Concanavalin A Aracharis hypogaea Erythrina cristagalli Ricinus communis Dolichos biflorus Glycine max Triticum vulgans Banderiaera simplicifolia Lotus tetragonolobus Ulex europaeus 1 Lens culinaris Sambuccas nigra
Binding Specificities of Lectins Used in This Study
Source
Abbreviation
Jack Bean Peanut Coral tree Castor bean Horse gram Soya bean Wheat germ
Con-A PNA ECG RCA DBF SBA WGA BS 1 LTA UEA 1 LCA SNA
B. simplifcifolia seeds Asparagus pea Gorse Lentil Elderberry bark
T u m o r Tissues
All carcinomas were e x a m i n e d to d e t e r m i n e their p r e d o m i n a n t pattern of differentiation but were f o u n d to be morphologically h e t e r o g e n e o u s with different regions showing varying degrees of cytological differentiation f r o m basaloid to keratinizing squamous carcinoma. Because there is no evidence that the behavior of any individual cervical squamous carcinoma correlates with its overall morphological grade, this study concentrated on relating patterns of oligosaccharide expression to cells of identifiable cytological a p p e a r a n c e or to their location within the tumors. Lectin Histochemistry Con-A (Mana 1-~3[Mana 1--,6]Manp I~4GIcNAc/~ 1-+4) N o r m a l Cervical Epithelium. Before neuraminidase digestion, cervical squamous epithelium in all 11 (100%) n o r m a l specimens b o u n d Con-A and confirmed the presence of c o m p l e x high m a n n o s e oligosaccharide structures. Binding occurred to all squamous epithelial cells where it was distributed t h r o u g h o u t the cytoplasm. No differences in intensity or binding distribution were identified in the different layers of squamous epithelium. Following neuraminidase digestion, squamous epithelium in all 11 specimens continued to bind Con-A. Staining a p p e a r e d to be e n h a n c e d in all sections, with no differences in cellular distribution. Cervical Squamous Carcinomas. Before neuraminidase digestion, most malignant cells in all 26 (100%) squamous cell carcinomas b o u n d Con-A. T h e distribution of binding was h e t e r o g e n e o u s t h r o u g h o u t the tumors with no specific pattern of regional localization being apparent. Following neuraminidase digestion, staining was variably e n h a n c e d in different carcinomas, confirming at least partial sialylation of oligosaccharide determinants.
Structure Recognized a Man,, .. Galfll--*3GalNac Gall31-~4GlcNAc (NeuAc2--*6) Galfll ~4 GlcNAc a D GalNAc D GalNA c GlcNAc/(GlcNAcfll-~4GlcNAc) D Gala l-*3 FuceH--*6GlcNAc Fuca 1--'2 ( Gall31~4) GalNac FuccH~6GlcNAc~ [Ash] Neu5Acel2~6Gal/GaINAc
Dilution 1:1000 1:200 1:200 1:300 1:200 1:400 1:500 1:100 1:200 1:500 1:200 1:100
layers (Fig 1). Following neuraminidase digestion, 10 of the 11 cases (91%) showed staining with PNA. This staining was located t h r o u g h o u t the basal and parabasal layers, but was not present in the intermediate or superficial layers. Cervical Squamous Carcinomas. Before neuraminidase digestion, 19 of 26 (73%) primary cervical squamous carcinomas showed binding of PNA and, hence, expression of u n m a s k e d type I oligosaccharides. In these cases, binding of PNA was p r e d o m i n a n t l y to the morphologically better-differentiated squamous carcin o m a cells within the centers o f t u m o r islands. However, in a few regions, staining of t u m o r ceils m o r p h o logically resembling basal type cells at the t u m o r stromal interface was apparent. In the carcinomas, expression of u n m a s k e d type I oligosaccharide determinants increased with the d e p t h of penetration of the tumors f r o m the cervical surface and into stromal connective tissues. Following neuraminidase digestion, all 26 carcinomas (100%) b o u n d PNA. In addition, the heterogeneity of staining originally observed before desialylation was reduced so that most t u m o r cells were
PNA (Gal/51-*3GalNAc-) N o r m a l Cervical Epithelium. Before neuraminidase digestion, 4 of 11 cases (36%) stained with PNA, showing the presence of u n m a s k e d type I oligosaccharides. In these four cases, staining was distributed within the cytoplasm of the basal layer of squamous epithelial cells to the plasma m e m b r a n e s of cells in the lower parabasal
FIGURE I. Normal cervical squamous epithelium expressing. type I (GalI31-,3GalNAc-) oligosaccharide determinants only along the basal layers. (PNA without neuraminidase; original magnification ×250.)
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HUMAN PATHOLOGY
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FIGURI~2. Well-differentiated invasive squamous carcinoma expressing type I oligosaccharides along plasma membranes. (PNA following neuraminidase digestion; original magnification ×250.)
stained. T u m o r cells with basal type morphology exhibited only cytoplasmic staining, whereas staining was restricted to the plasma membranes of well-differentiated squamous carcinoma cells (Fig 2).
epithelium stained with RCA. After neuraminidase digesdon, staining of this tissue was abolished, and expression of the determinant was now apparent in a small focus in one other normal specimen. In both instances, binding of the lectin was restricted to a very small region and was not apparently associated with either an inflammatory infiltrate or intraepithelial dysplasia. Cervical Squamous Carcinomas. Before neuraminidase digestion, 16 of the 26 (62%) squamous carcinomas b o u n d RCA. Staining of the basal and parabasal type tumors was cytoplasmic and heterogeneous, with approximately 10% of cells in each of the tumors staining. O f those tumors that stained, the morphologically better-differentiated squamous carcinomas exhibited more staining along plasma membranes (Fig 5) than did the less-differentiated tumors in which staining was usually located to the cytoplasm. In all cases examined, increased staining was observed with increasing depth of tumor penetration into the cervical stroma. Following neuraminidase digestion, 21 of the 26 (77%) tumors now b o u n d RCA. No qualitative differences in distribution were observed when c o m p a r e d with the binding before neuraminidase digestion. Both before and after desialylation, several of the well-differentiated tumors did not stain, whereas some poorly differentiated tumors b o u n d the lectin within their cytoplasm (Fig 6).
ECG (Gal/~ 1--*4GIcNAc-)
DBF (GalNAc~ 1-*3GaINAc-)
Normal CervicalEpithelium.
None of the specimens of normal cervical squamous epithelium stained with the lectin Erythrina cristagalli either before or after neuraminidase digestion, demonstrating that cervical squamous epithelial cells do not synthesize type II oligosaccharide structures (Fig 3). Cervical Squamous Carcinomas. Before neuraminidase digestion, 17 of the 26 (65%) squamous carcinomas b o u n d ECG, thus demonstrating expression of the unmasked type II oligosaccharide structure. Within these tumors, expression was focal, heterogeneous, and present in only a few regions o f each tumor. Staining was present on plasma membranes as well as within the cytoplasm and was restricted to intermediate-sized tumor ceils resembling those o f the early stratum spinosum of normal epithelium. Staining was not present in large, weft-differentiated squamous carcinoma ceils. Following neuraminidase digestion, 13 o f the 26 (50%) primary carcinomas expressed type II oligosaccharide structures. In these tumors, the original heterogeneity was reduced so that many more carcinoma ceils were stained, particularly those resembling parabasal cells of the normal tissue. Well-differentiated squamous carcin o m a cells stained better than basal type cells (Fig 4). Nonkeratinizing basal type cells were invariably unstained. The four tumors that failed to stain following desialylation were all of poorly differentiated parabasal type in which the incidence of positive cells in the nondesialylated tumors was very low. RCA (NeuAc2~3Gal-)
Normal Cervical Epithelium. Before neuraminidase digestion, only one specimen (9%) of normal cervical
Normal CervicalEpithelium. None o f the 11 cases o f normal cervical squamous epithelium b o u n d this lectin, either before or after neuraminidase digestion. Cervical Squamous Carcinomas. N o n e of the 26 cases of cervical squamous carcinoma b o u n d this lectin, either before or after neuraminidase digestion. SBA (~/pGalNAc-)
Normal CervicalEpithelium. N o n e of the 11 cases of normal cervical squamous epithelium b o u n d this lectin before or after neuraminidase digestion. Cervical Squamous Carcinomas. Before neuraminidase digestion, only two of the 26 (8%) squamous carcinomas expressed the unmasked determinant. Following desialylation, good staining of well-differentiated squamous carcinoma cells, particularly in the deep infiltrating regions of the tumors was identified in nine (35%) of the carcinomas (Fig 7). Tumors composed of predominantly basal type carcinoma cells were not stained. WGA ([GIcNAc/~ 1-*4GIcNAc-]n)
Normal Cervical Epithelium. All 11 specimens of normal squamous epithelium were well stained both before and after neuraminidase digestion. Following desialylation, staining was variably enhanced. Normal squamous epithelium stained in the cytoplasm of the basal cells but luminal plasma membranes in the u p p e r layers. Cervical Squamous Carcinomas. Before neuraminidase digestion, all 26 squamous carcinomas (100%)
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OLIGOSACCHARIDES iN CERVICAL CANCER (Banerjee et al)
FIGURE 3. Nonneoplastic cervical squamous epithelium overlying a moderately differentiated invasive squamous carc noma (A) Before neuraminidase digestion showing no expression of type II (GaI~I-~4GIcNAc-) oligosaccharides by the normal epithelial layers but neoexpression within the carcinoma. (B) Following desialylation, the nonneoplastic epithelium remains unstained, whereas there is enhanced staining of the carcinoma. (ECG; original magnification ×75.)
were stained. Well-differentiated squamous carcinoma, stained both in the cytoplasm and around luminal plasma membranes, particularly in those regions of tumors infiltrating deeply into adjacent stroma, although not in the superficial tumors. No significant difference in the patterns of staining were identified following neuraminidase digestion (Fig 8).
BS- 1 (D Gal~l-~3-)
Normal CervicalEpithelium. None of the 11 cases of normal cervical squamous epithelium bound this lectin before or after neuraminidase digestion. Cervical Squamous Carcinomas. Before neuraminidase digestion, 23 of the 26 (88%) squamous carcinomas bound this lectin. Morphologically well-differenti-
FIGURE 4. Well-differentiated squamous carcinoma expressing type II oligosaccharides (A) before and (B) following desialylation revealing enhanced expression of these determinants along tumor cell plasma membranes. (ECG- original magnification ×300.) 1009
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FIGURE S. Moderately differentiated invasive squamous carcinoma expressing NeuAc2-~3Gal- structures along plasma membranes. (RCA without neuraminidase digestion; original magnification x400.)
ated carcinomas stained particularly in regions of keratinization (Fig 9). Following neuraminidase digestion, the former 23 cases together with one additional one case (92%) stained with this lectin. Previously observed heterogeneity of binding by BS-1 to the cancers was now reduced so that many more of the basal type squamous carcinoma cells also expressed this determinant. LTA (Fuc~ 1-~6GIcNAc-)
Normal Cervical Epithelium. All 11 cases of normal squamous ectocervical epithelium were negative, both before and after neuraminidase digestion. CervicaISquamous Carcinomas. In four of 26 (15%) squamous carcinomas occasional tumor cells in areas of
FIGURE 6. Poorly differentiated invasive squamous carcinoma expressing NeuAc2~3Gat- structures as cytoplasmic aggregates in approximately 10% of the tumor cells. (RCA following neuraminidase digestion; original magnification x400.)
FIGURE 7. Islands of well-differentiated and deeply invasive squamous carcinoma cells expressing a//3GalNAc- structures both within the cytoplasm and along plasma membranes. (SBA following neuraminidase digestion; original magnification x200.)
well-differentiated squamous carcinoma stained weakly before neuraminidase digesdon. Following desialylation, five of 26 carcinomas (19%) showed weak staining with similar pattern. UEA- 1 (Fuc~ 1-~2Gal[GaINAc]-)
Normal Cervical Epithelium. All 11 specimens of normal cervical squamous epithelium were negative both before and after neuraminidase digestion. Cervical Squamous Carcinomas. Before neuraminidase digestion, 10 of the 26 carcinomas (38%) b o u n d this lectin. Those that stained were the better-differenti-
FIGURE 8. Moderately differentiated invasive squamous carcinoma expressing (GIcNAcI31~4GIcNAc-)n determinants predominantly within the cytoplasm of tumor cells at the stromal interface. Basal type cells in the centers of the tumor islands do not express this determinant. (WGA following neuraminidase digestion; original magnification x250.)
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OLIGOSACCHARIDES IN CERVICAL CANCER (Banerjee et al)
above the basal layer. After neuraminidase digestion, ectocervical basal epithelial cells stained in all 11 cases. In addition to the plasma m e m b r a n e staining previously identified, cytoplasmic staining was now apparent. Cervical Squamous Carcinomas. Before neuraminidase digestion, 13 of 26 carcinomas (50%) were stained. In all of these, cytoplasmic staining of individual cells was observed. In n o n e of these carcinomas were the basal type cells stained. After neuraminidase digestion, 17 of 26 carcinomas (65%) now stained. The staining increased with depth of invasion, so that the most prominent staining occur in those cells invading deeply and at the interface with the cervical stroma.
DISCUSSION FIGURE 9. Invasive squamous carcinoma in which the central, well-differentiated cells express terminal D-GaI,~1~ 3- structures. The surrounding tumor cells with basal type morphology, including those at the stromal interface, do not express this determinant, in contrast to the determinants recognized by WGA and UEA-1 (Figs 8 and 10). (BS-1 without neuraminidase digestion; original magnification ×120.)
ated squamous tumors. After neuraminidase digestion, staining of 15 carcinomas (58%) occurred either a r o u n d the stromal interface at the periphery of the invading cells (Fig 10) or in well-differentiated regions of squamous carcinoma. In these tumors, intensity of staining was enhanced. LCA ( M a n ~ 1 - * 3 / 6 M a n f l l - * 4 G I c N A c f l l - * 4 [ F u c a l - * 6 ] GIcNAc-)
Normal CervicalEpithelium. Before neuraminidase digestion, 10 of 11 specimens o f normal squamous epithelium stained with LCA. Staining was restricted to basal squamous epithelial cells and occurred weakly, both within the cytoplasm and around the plasma membranes. Following desialylation, all 11 specimens were stained, particularly of the nonkeratinizing basal epithelial cells. Cervical Squamous Carcinomas. Before neuraminidase digestion, 17 of 26 carcinomas (65%) were stained. Staining occurred in those tumor cells immediately adjacent to the stroma. Here, staining was entirely cytoplasmic. E n h a n c e d staining o f tumor cells appeared to occur with increasing squamous differentiation. Following neuraminidase digestion, 24 of 26 carcinomas (92%) were stained. The pattern was that of the welldifferentiated squamous cells showing staining, whereas the basal type cells were not stained. T h e r e was an increase in intensity of staining so that an additional seven tumors were now stained by the lectin. SNA ( N e u 5 A c ~ 2 - * 6 G a l / G a l N A c - )
Nominal CervicalEpithelium. Before neuraminidase digestion, plasma membranes of ectocervical basal epithelial ceils were stained in 10 of 11 control cases. T h e r e was no staining of differentiated squamous epithelium
This study has shown that h u m a n nonneoplastic cervical squamous epithelial cells characteristically express complex oligosaccharide structures that are core fucosylated (Manal~3/6Manfll-~4GlcNAcfll~ 4[Fucal-~6] GlcNAc-) and carry type I antennary structures (Galfll~3 GalNAc-) usually modified by sialic acid linked 2 --* 6 to terminal Gal- or GalNAc-residues (Table 2). Type II oligosaccharide structures (Galfll~ 4GlcNAc-) were not synthesized by nonneoplastic cervical squamous epithelium, and no other terminal antennary modifications were identified in these tissues. In the squamous carcinomas, neosynthesis of type II oligosaccharide structures, identified by ECG binding, was recognized in 50% of the cases examined. An additional four terminal antennary modifications were identified in the carcinomas that were not detected in normal tissues and constituted the oligosaccharide structures b o u n d by SBA, BS-1, LTA, and UEA-1. Binding of RCA was observed in only two specimens of normal cervical tissues but was commonly observed in the carcinomas.
FIGURE 10. Invading edge of a moderately differentiated carcinoma in which intracytoplasmic and plasma membrane expression of the oligosaccharide structures Fuc,~l--*2Gal (GalNAc)- is restricted to the several layers of cells at the stromal interface. (UEA-1 following neuraminidase digestion; original magnification ×400.)
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HUMAN PATHOLOGY TABLE 2,
Volume 26, No. 9 (September 1995)
Binding of Lectins to Normal Cervical Epithelium and to 26 Primary Cervical Carcinomas
Tissue Structure
Con-A
PNA
ECG
RCA
DBF
SBA
WGA
BS-1
LTA
UEA-1
LCA
SNA
11
4
0
1
0
0
11
0
0
0
10
10
26
19
17
16
0
2
26
23
4
10
17
13
11
10
0
1
0
0
ll
0
0
0
11
ll
26
26
13
21
0
9
26
24
5
15
24
17
Before Neuraminidase Digestion Normal squamous epithelium (n = 11) Squamous carcinomas (n = 26) After Neuraminidase Digestion Normalsquamous epithelium (n = 11) Squamous carcinomas (n = 26)
Enhanced expression of terminal GaINAc-residues, particularly identified by SBA and WGA, supports the recent report suggesting that presence of the unmasked Tn-antigen oligosaccharide structure was closely correlated with vascular permeation and metastasis to ly~.~ph nodes by invasive h u m a n cervical carcinoma cells. " Our findings have extended the earlier observations ofByrne et a112'1~by identifying distinct tumor cell phenotypes with respect to cell surface oligosaccharides expressed by h u m a n cervical squamous carcinomas. We have identified several sugar structures to be expressed only by cells with characteristic cytological appearances. For example, carcinoma cells resembling the basal and parabasal cells of normal cervical squamous epithelium typically expressed oligosaccharides recognized by PNA, ECG, and RCA. Following neuraminidase digestion, this group also included BS-1. Conversely, binding of SBA and LCA was expressed by better differentiated squamous epithelial cells, but not by the basal and parabasal cell types. Therefore, it seems likely that expression of certain oligosaccharide Structures might be related to specific stages of cellular maturation within h u m a n cervical squamous epithelium and, hence, identify individual cellular phenotypes. In this respect, stage expression of cell surface oligosaccharide determinants by cervical squamous epithelium could be analogous to that which has long been recognized during lymphocyte maturation. 19 Novel fucosylation linked a l --* 3 to terminal antennary oligosaccharide structures was identified in 15 carcinomas following desialylation. Of the 13 carcinomas found to express type II oligosaccharides following neuraminidase digestion, six were ozl ~ 3 fucosylated. Only type II oligosaccharide structures are able to be fucosylated with this particular linkage. 2° Two additional terminal structures not found in normal cervical squamous epithelium were a/flGalNAc- and NeuAc2-~ 3Gal-, detected by SBA and RCA-1, respectively. Carcinoma cells expressing type II oligosaccharides modified by a 1~3 fucosylation might be a particularly important behavioral phenotype. The novel structure (Galfll~ 4[Fucal~3] GlcNAc-) is related to the family of oligosaccharide determinants known to bind extracellular adhesion molecules, a 2 ~ 3 sialylation of this structure yields sialylLewis× that specifically binds members of the selec-
tin receptor family.2~ The selectins comprise a family of mammalian receptors that mediate initial interactions between leukocytes and vascular endothelia, leading to lymphocyte homing, platelet binding, and neutrophil extravasation. The three selectins, LECAM-1, ELAM-1, and GMP-140, share structural features that include a calcium-dependent lectin domain that specifically binds the sialyl Lewisx carbohydrate structure.'22 Recently, the cluster differentiation antigen CD62 has also been shown to bind the sialyl Lewis× determinant. The selectin receptors are not only major adhesion molecules for peripheral blood leukocytes but also for tumor cells. Expression of ELAM-1 by endothelial cells can be selectively induced by the cytokines tumor necrosis factor and interleukin 1, both cytokines being released by many neoplasms. With respect to the present study, neosynthesis of the sialyl LewisX determinant is only possible in tumor cells following prior synthesis of the type II oligosaccharide structure because this is the required substrate for the particular a l ~ 3 fucosyltransferase, ~3'24 and type II oligosaccharides were not identified in nonneoplastic cervical squamous epithelium. Following extravasation into the general circulation, synthesis of sialyl Lewis× oligosaccharides would promote adhesion of squamous carcinoma cells to the selectins or to CD62 receptors on vascular endothelium. 25 Thus, expression of the sialyl Lewis× structure might identify one of the metastatic phenotypes of cervical squamous carcinoma cells. This study has compared the predominant oligosaccharide structures expressed by h u m a n nonneoplastic cervical squamous epithelium and early invasive squamous carcinomas. Significant and consistent differences have been identified both between the normal and neoplastic tissues, and within the cancers. Expression of certain oligosaccharide structures has been related to the different cytological types of individual invasive squamous carcinoma cells. We suggest that these differences identify phenotypic variants within h u m a n cervical cancers and may define carcinomas that will behave in a predictable manner, particularly their metastatic potential. Because type II oligosaccharides occur only in malignant cells, their expression may result in changes in binding to cell adhesion molecules and, hence, be an essential step in the metastatic malignant
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OLIGOSACCHARIDES IN CERVICAL CANCER (Banerjee et al)
process. Identification of these structures in noninvasive cervical lesions may signal an invasive potential and allow reliable differentiation of premalignant lesions from those that are harmless. Additional studies are now being performed to determine the stage of cervical neoplasia at which the identified changes in oligosaccharide structures occur and whether expression of particular oligosaccharide determinants, singly or in combination, define different behavioral phenotypes within h u m a n cervical cancer. T h e pathologic significance of this study is that it has shown an i n d e p e n d e n t m e t h o d for identifying the p h e n o t y p e s o f individual t u m o r cells within a cervical squamous c a r c i n o m a - - a n d that might be ext e n d e d to squamous carcinomas developing in o t h e r tissues. A detailed analysis o f the plasma m e m b r a n e s is now r e q u i r e d to identify those glycoproteins that are characteristic o f the different m o r p h o l o g i c a l variants of cervical squamous carcinoma, according to their oligosaccharide structures. T h e r e a f t e r , correlation between individual b a c k b o n e proteins thus identified, and the behavioral p h e n o t y p e o f individual cancers, could provide the basis for a novel " f u n c t i o n a l " classification o f h u m a n cervical squamous cancer.
Acknowledgment. We wish to t h a n k J. Ng, MD, at T h e Princess M a r g a r e t Hospital, Swindon, Wiltshire, UK, for t h e case m a t e r i a l i n c l u d e d in this study. We are grateful to Lindasy M c G r e g o r for t e c h n i c a l assistance, a n d V i v i e n n e E m o n s a n d T a r e q Ansari in t h e D e p a r t m e n t o f H i s t o p a t h o l o g y w h o provided invaluable p h o t o g r a p h i c assistance. REFERENCES 1. Office of Population Censuses and Surveys: Cancer Statistics-Registrations. London, Her Majesty's Stationary Office, 1987 2. Qualters JR, Lee NC, Smith RA, et al: Breast and cervical cancer surveillance, United States, 1973-1987. Morbidity and Mortality Weekly Report: Centers for Disease Control Surveillance Summaries. 41/SS-2:l-15, 1992 3. BeachamJB, Halvorsen T, Kolbenstvedt A: Histological classification, lymph node metastases and patient survival in stage l b cervical carcinoma. Gynecol Oncol 6:95-105, 1978 4. Goellner JR: Carcinoma of tfie celarix: Clinicopathologic correlation of 196 cases. Am J Clin Pathol 66:775-780, 1976 5. Graham J, Graham R, Hirabayaski K: Recurrent cancer of the cervix uteri. Surg Gynecol Obstet 126:799-806, 1968 6. Lanza A, Re A, d'Addato F, et al: Treatment failure in cervical
cancer: High risk factors in relapse. Eur J Gynaecol Oncol 10:326336, 1989 7. Shingleton HM, Soong SJ, Gelder MS, et al: Clinical and histopathological factors predicting recurrence and survival after pelvic exteneration for cancer of the cervix. Obstet Gynecol 73:1027-1034, 1989 8. Norris WE: Evidence for a second class of membrane glycoprotein involved in cell adhesion..1 Cell Sci 93:631-640, 1989 9. Foster CS: Processing of N-linked oligosaccbaride glycoproteins in human tumours. B r J Cancer 62:57-63, 1990 (suppl X) 10. Aub JC, Tieslau C, Lankester A: Reaction of normal and tumor cell surfaces to enzymes: Wheat germ lipase and associated mucopolysaccharides. Proc Natl Acad Sci USA 50:613-619, 1963 11. Goldstein IJ, Hughes RC, Monsigny M, et al: What should be called a lectin? Nature 285:66, 1980 12. Byrne P, Williams A, Rollason T: Studies of lectin binding to the human cervix uteri: I. Normal cervix. HistochemJ 21:311-322, 1989 13. Byrne P, Williams A, Rollason T: Studies of lectin binding to the human cervix uteri: II. Cervical intraepithelial neoplasia and invasive squamous carcinoma. Histochem J 21:323-336, 1989 14. DavinaJH, Stadhouders AM, van Haelst UJ, et al: Concanavalin A-peroxidase labeling in cervical exfoliadve cytopathology: I. Labelling of normal squamous cells and the detection of cancer. Gynaecol Oncol 22:212-223, 1985 15. Parkin DM, Laara E, Muir CS: Estimate of the worldwide frequency of 16 major cancers in 1980. I n t J Cancer 41:184-187, 1988 16. Hirao T, Sakamoto Y, Kamada M, et al: Tn antigen, a marker of potential for metastasis of uterine cervix cancer cells. Cancer 72:154-159, 1993 17. Brudenell M, Cox BS, Taylor CW: The management of dysplasia, carcinoma in situ and microcarcinoma of the cervix. J Obstet Gynecol 80:673-679, 1973 18. Hsu M, Raine L, Fanger H: The use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques: Comparison between ABC and unlabelled antibody (PAP) procedure.J Histochem Cytochem 29:577-583, 1981 19. Piguet PF, lrle C, Kollatte E, et al: Post-thymic T lymphocyte maturation during ontogenesis. J Exp Med 154:581-593, 1981 20. Pereira MEA, Kisailus EC, Greuzo F, et al: Immunochemical studies on the combining site of the blood-group H-specific lectin 1 from Ulex europaeus seeds. Arch Biochem Biophys 185:108-115, 1978 21. Takada A, Ohmori K, Takahashi N, et al: Adhesion of human cancer cells to vascular endothelium mediated by a carbohydrate antigen, sialyl Lewis A. Biochem Biophys Res Commun 179:713-719, 1991 22. Foxall C, Watson SR, Dowbenko D, et al: The three members of the selectin receptor family recognize a common carbohydrate epitope, the sialyl Lewis(x) oligosaccharide. J Cell Biol 117:895-902, 1992 23. Lowe JB, Stoolman LM, Nair RP, et al: ELAM-l-dependent cell adhesion to vascular endothelium determined by a transfected human fucosyltransferase cDNA. Cell 63:475-484, 1990 24. Koszdin KL, Bowen BR: The cloning and expression of a human alpha-l,3 fucosyltransferase capable of forming the E-selection ligand. Biochem Biophys Res Commun 187:152-157, 1992 25. Polley MJ, Phillips ML, Wayner E, et al: CD62 and endothelial cell-leukocyte adhesion molecule 1 (ELAM-1) recognize the same carbohydrate ligand, sialyl-Lewis X. Proc Nail Acad Sci USA 88:62246228, 1991
1013
carcinomas. Five terminal antennary modifications were commonly identified in the carcinomas that were not identified in normal cervical epithelia and comprised the ofigosaccharides bound by lectins RCA, SBA, BS-I, LTA, and UEA-1. Synthesis of these ofigosaccharides restdted in expression of structures similar to those recognized as ligands for extracellular matrix-binding proteins. We suggest that expression o f such novel oligosaccharide structures may be an important promotor of local invasion and further dissemination of human cervical carcinomas through enhanced binding of malignant cells to stromal matrix proteins. This study has demonstrated that identification of expressed oligosaccharide structures is an objective m e t h o d of identifying individual tumor cell phenotypes and may form the basis of a useful functional classification of human cervical squamons carcinomas. HUM PATHOL 26:1005--1013. Copyright © 1995 byW.B. Saunders Company Key words: h u m a n cervix, squamons carcinoma, lectins, glycoprotein modulation. Abbreviations: PBS, phosphate-buffered saline; v/v, volume of solute per volume of solution.
Carcinoma o f the cervix uteri is the second commonest cancer in women worldwide. In the United Kingdom in 1987, the latest year for which figures are available, 19,566 new cases were registered, of which 3,972 were invasive, giving an incidence for this malignancy of 15.4 per 105 female population] In 1990, 1,721 deaths were reported in the United Kingdom, giving a rate of 6.7 per 105. Comparable figures compiled for the same time in the United States give an incidence of 8.2 per 105 with a death rate of 3.0 per 105.~ Invasive and metastatic disease together constitute the most c o m m o n cause of treatment failure and are solely responsible for the mortality of cervical cancer. At present, the likely behavior of an early cervical cancer cannot be identified at diagnosis because no markers have been developed with which to assess its functional or malignant potential independently. Morphological grade is a p o o r indicator of tumor behavior or patient survival within any stage. "~5 Unassisted morphology recognizes only gross similarities and differ-
ences between tumors, and does not identify individual tumor cells o f particular phenotypes, which may be of immense behavioral importance. Failure of morphologic grading is evidenced by recurrent disease following conventional conservative treatment. 6 Clinical stage remains the only useful parameter for determining therapeutic m a n a g e m e n t and predicting the likel~ prognosis of individual cervical squamous cell cancers. The behavioral phenotype of an epithelial cell is modulated by the composition and functional properties of its plasma membrane. Within this structure, glycoproteins link components of the cytoskeleton to the extracellular matrix and are important mediators of intercellular contact, cellular adhesion, and growth stimuli, s T h e oligosaccharide domains of glycoconjugates are synthesized by a complex system of glycosyltransferase enzymes arranged hierarchically within the endoplasmic reticulum and Golgi apparatus, and control the intracellular processing and trafficking of many glycoprotein molecules. 9 Ionic conditions within Golgi cisternae dictate the relative activities of glycosyltransferases competing for the same substrates. Failure to maintain ionic homeostasis results in the formation of novel oligosaccharides with altered expression of associated glycoconjugates at the cell surface. Conventionally, oligosaccharide c o m p o n e n t s of glycoconjugates containing the core sequence Galfll~3GalNAc-identified by the lectin p e a n u t agglutinin (PNA) are known as type I, whereas those containing the sequence Galfll-*
From the Department of Obstetrics and Gynecology, Royal Postgraduate Medical School, Hammersmith Hospital, London, and the Department of Pathology, University of Liverpool, Liverpool, UK. Accepted for publication January 24, 1995. Address correspondence and reprint requests to Christopher S. Foster, MD, PhD, Department of Pathology, Duncan Building, PO Box 147 Daulby St, University of Liverpool, Liverpool, L69 3BX, UI~ Copyright © 1995 by W.B. Saunders Company 0046-8177/95/2609-000355.00/0
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HUMAN PATHOLOGY Votume26, No. 9 (September 1995) 4GlcNAc- are identified by the lectin ECG and are known as type II. Because expression of particular oligosaccharide structures by t u m o r cells may influence their behavior, structural analysis of plasma m e m b r a n e oligosaccharides may assist in identifying individual behavioral phenotypes within a neoplasm. The use of lectins to distinguish differences in oligosaccharides expressed by normal and neoplastic cells was first reported by Aub et alJ ° Lectins are carbohydrate-binding proteins of n o n i m m u n e origin that bind only to highly restricted sugar structures and are exquisitely sensitive probes with which to investigate subtle alterations in cell m e m b r a n e oligosaccharides. 1] Using the lectins BPA, GS-2, PNA, SBA, and WGA, Byrne et al identified patterns of oligosaccharides consistently expressed by normal cervical squamous epithelium, but variably with Con-A, GS-1, and UEA-1. ~z Their findings suggested that cervical neoplasia is particularly associated with alterations in core a-Man sugars, and changes to terminal ol-//3-GalNAc-, oz-/fl-GlcNAc-, cr-//3-Gal-, and oz-Fuc- residues on the antennary re~ions of both Nlinked and O-linked glycoconjugates. ]" Binding o f PNA to cervical squamous epithelium has been reported to remain constant and not to vary with the phase of the menstrual cycle.14'15Recently, Hirao et al, indicated that although expression of the Tn-antigen, identified by the Vicia villosa agglutinin, was closely correlated with vascular permeation and lymph node metastasis by cervical cancer cells, 16 expression of the T antigen, identified by PNA, was of no predictive value. In the present study, we have c o m p a r e d the oligosaccharides expressed by h u m a n normal cervical epithelium and early invasive squamous carcinomas using a panel of 12 different lectins selected to identify the widest spectrum of c o m m o n oligosaccharide structures to be probed in these tissues. Differences in expression of oligosaccharide structures have been correlated with the appearances of individual t u m o r cells rather than the overall m o r p h o l o g y of a particular cancer. The findings support the hypothesis that it is possible to identify cells of different phenotypes within an individual squamous carcinoma and that the presence of cells expressing certain oligosaccharide determinants may indicate the likely behavior of those cervical cancers.
MATERIALS AND METHODS Specimens
Blocks of normal tissue were specifically selected to exclude features such as immature metaplastic squamous epithelium. Tumor Tissues Sections from two separate blocks were stained and examined from each cervical tumor. All tumors occurred in the region of the squamocolumnar junction and extended to a depth greater than 5 mm into the adjacent stroma; hence, their local invasion was more than "microinvasive, ''~7 although all were clinically stage I. None of the carcinomas extended beyond the limits of the tissues examined within a single tissue block so that the invasive fronts of the tumors could be identified in all cases examined. Before admission to this study, all tumors were routinely examined using the periodic acid-Schiff/Alcian blue technique to exclude poorly differentiated adenocarcinomas and adenosquamous carcinomas. In this study, analysis of lectin binding was restricted to only the invasive components of the tumors. In situ cervical cancer, as a strictly preinvasive entity, has been examined in a separate investigation and was not recorded.
kectin Histochemistry All lectins were obtained biotinylated from Sigma (Poole, UK) and are summarized in Table 1. Each was diluted to 10 #g/mL in sodium phosphate-buffered saline (PBS; pH 7.6) containing 1 mmol each of calcium chloride and manganese chloride. PBS was also used for washing. Formalin-fixed and paraffin wax-embedded tissue sections cut at 2 #m were dewaxed in TSF 30 (Infrakem Ltd, Wigan, UK) and brought to water through graded ethanols. Endogenous peroxidase was blocked with 0.3% (volume of solute per volume of solution Iv/v]) hydrogen peroxide (H202) in distilled water for 30 minutes at room temperature. Lectin staining was performed by the avidin-biotin-peroxidase complex method, 18using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Lectins were incubated for 60 minutes at room temperature on tissue sections that were then washed in PBS. Peroxidase activity was localized by addition of 3,3'-diaminobenzidine tetrahydrochloride at 250/zg/mL in PBS containing 0.03% (v/v) H202 for 10 minutes. Sections were counterstained in Harris' hematoxylin, dehydrated, cleared, and mounted. Lectin binding was assessed by light microscopic appearances, and the distribution of staining within individual cervical carcinoma cells was identified.
Neuraminidase Digestion Lectin histochemistry was performed after enzymatic desialylation. Serial tissue sections were digested with neuraminidase (ex Vibrio cholerae;, BDH Laboratories, Dagenham, UK) at a dilution of 1:10 in 200-mM sodium acetate buffer (pH 5.5) at room temperature for 60 minutes before staining was performed according to the protocol already described.
Histologically normal cervical tissues (n = 11) were obtained following abdominal hysterectomy for dysfunctional hemorrhage. Locally invasive cervical squamous carcinomas (n = 26) were obtained from radical abdominal hysterectomy specimens. Approval to use archival pathologic material was obtained from the Ethics Committee of the Hammersmith Hospital and Royal Postgraduate Medical School, London.
Paraffin sections of normal human kidney were used as positive controls of lectin binding. For the negative controls, each lectin was omitted and replaced by an equal volume of PBS.
Normal Tissues
RESULTS Tissue Morphology
Sections from two standard blocks were stained and examined from each specimen of normal cervical tissue. The contained tissue extended from the squamous resection margin of the ectocervix and through the squamocolumnar junction to include the isthmic region of endocervical epithelium.
Normal Controls
Normal Tissues None of the control tissues contained any dysplastic or neoplastic features. Junctional chronic inflammation was minimal.
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OLIGOSACCHARIDES IN CERVICAL CANCER (Banerjee et al) TABLE 1.
Lectin Concanavalin A Aracharis hypogaea Erythrina cristagalli Ricinus communis Dolichos biflorus Glycine max Triticum vulgans Banderiaera simplicifolia Lotus tetragonolobus Ulex europaeus 1 Lens culinaris Sambuccas nigra
Binding Specificities of Lectins Used in This Study
Source
Abbreviation
Jack Bean Peanut Coral tree Castor bean Horse gram Soya bean Wheat germ
Con-A PNA ECG RCA DBF SBA WGA BS 1 LTA UEA 1 LCA SNA
B. simplifcifolia seeds Asparagus pea Gorse Lentil Elderberry bark
T u m o r Tissues
All carcinomas were e x a m i n e d to d e t e r m i n e their p r e d o m i n a n t pattern of differentiation but were f o u n d to be morphologically h e t e r o g e n e o u s with different regions showing varying degrees of cytological differentiation f r o m basaloid to keratinizing squamous carcinoma. Because there is no evidence that the behavior of any individual cervical squamous carcinoma correlates with its overall morphological grade, this study concentrated on relating patterns of oligosaccharide expression to cells of identifiable cytological a p p e a r a n c e or to their location within the tumors. Lectin Histochemistry Con-A (Mana 1-~3[Mana 1--,6]Manp I~4GIcNAc/~ 1-+4) N o r m a l Cervical Epithelium. Before neuraminidase digestion, cervical squamous epithelium in all 11 (100%) n o r m a l specimens b o u n d Con-A and confirmed the presence of c o m p l e x high m a n n o s e oligosaccharide structures. Binding occurred to all squamous epithelial cells where it was distributed t h r o u g h o u t the cytoplasm. No differences in intensity or binding distribution were identified in the different layers of squamous epithelium. Following neuraminidase digestion, squamous epithelium in all 11 specimens continued to bind Con-A. Staining a p p e a r e d to be e n h a n c e d in all sections, with no differences in cellular distribution. Cervical Squamous Carcinomas. Before neuraminidase digestion, most malignant cells in all 26 (100%) squamous cell carcinomas b o u n d Con-A. T h e distribution of binding was h e t e r o g e n e o u s t h r o u g h o u t the tumors with no specific pattern of regional localization being apparent. Following neuraminidase digestion, staining was variably e n h a n c e d in different carcinomas, confirming at least partial sialylation of oligosaccharide determinants.
Structure Recognized a Man,, .. Galfll--*3GalNac Gall31-~4GlcNAc (NeuAc2--*6) Galfll ~4 GlcNAc a D GalNAc D GalNA c GlcNAc/(GlcNAcfll-~4GlcNAc) D Gala l-*3 FuceH--*6GlcNAc Fuca 1--'2 ( Gall31~4) GalNac FuccH~6GlcNAc~ [Ash] Neu5Acel2~6Gal/GaINAc
Dilution 1:1000 1:200 1:200 1:300 1:200 1:400 1:500 1:100 1:200 1:500 1:200 1:100
layers (Fig 1). Following neuraminidase digestion, 10 of the 11 cases (91%) showed staining with PNA. This staining was located t h r o u g h o u t the basal and parabasal layers, but was not present in the intermediate or superficial layers. Cervical Squamous Carcinomas. Before neuraminidase digestion, 19 of 26 (73%) primary cervical squamous carcinomas showed binding of PNA and, hence, expression of u n m a s k e d type I oligosaccharides. In these cases, binding of PNA was p r e d o m i n a n t l y to the morphologically better-differentiated squamous carcin o m a cells within the centers o f t u m o r islands. However, in a few regions, staining of t u m o r ceils m o r p h o logically resembling basal type cells at the t u m o r stromal interface was apparent. In the carcinomas, expression of u n m a s k e d type I oligosaccharide determinants increased with the d e p t h of penetration of the tumors f r o m the cervical surface and into stromal connective tissues. Following neuraminidase digestion, all 26 carcinomas (100%) b o u n d PNA. In addition, the heterogeneity of staining originally observed before desialylation was reduced so that most t u m o r cells were
PNA (Gal/51-*3GalNAc-) N o r m a l Cervical Epithelium. Before neuraminidase digestion, 4 of 11 cases (36%) stained with PNA, showing the presence of u n m a s k e d type I oligosaccharides. In these four cases, staining was distributed within the cytoplasm of the basal layer of squamous epithelial cells to the plasma m e m b r a n e s of cells in the lower parabasal
FIGURE I. Normal cervical squamous epithelium expressing. type I (GalI31-,3GalNAc-) oligosaccharide determinants only along the basal layers. (PNA without neuraminidase; original magnification ×250.)
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FIGURI~2. Well-differentiated invasive squamous carcinoma expressing type I oligosaccharides along plasma membranes. (PNA following neuraminidase digestion; original magnification ×250.)
stained. T u m o r cells with basal type morphology exhibited only cytoplasmic staining, whereas staining was restricted to the plasma membranes of well-differentiated squamous carcinoma cells (Fig 2).
epithelium stained with RCA. After neuraminidase digesdon, staining of this tissue was abolished, and expression of the determinant was now apparent in a small focus in one other normal specimen. In both instances, binding of the lectin was restricted to a very small region and was not apparently associated with either an inflammatory infiltrate or intraepithelial dysplasia. Cervical Squamous Carcinomas. Before neuraminidase digestion, 16 of the 26 (62%) squamous carcinomas b o u n d RCA. Staining of the basal and parabasal type tumors was cytoplasmic and heterogeneous, with approximately 10% of cells in each of the tumors staining. O f those tumors that stained, the morphologically better-differentiated squamous carcinomas exhibited more staining along plasma membranes (Fig 5) than did the less-differentiated tumors in which staining was usually located to the cytoplasm. In all cases examined, increased staining was observed with increasing depth of tumor penetration into the cervical stroma. Following neuraminidase digestion, 21 of the 26 (77%) tumors now b o u n d RCA. No qualitative differences in distribution were observed when c o m p a r e d with the binding before neuraminidase digestion. Both before and after desialylation, several of the well-differentiated tumors did not stain, whereas some poorly differentiated tumors b o u n d the lectin within their cytoplasm (Fig 6).
ECG (Gal/~ 1--*4GIcNAc-)
DBF (GalNAc~ 1-*3GaINAc-)
Normal CervicalEpithelium.
None of the specimens of normal cervical squamous epithelium stained with the lectin Erythrina cristagalli either before or after neuraminidase digestion, demonstrating that cervical squamous epithelial cells do not synthesize type II oligosaccharide structures (Fig 3). Cervical Squamous Carcinomas. Before neuraminidase digestion, 17 of the 26 (65%) squamous carcinomas b o u n d ECG, thus demonstrating expression of the unmasked type II oligosaccharide structure. Within these tumors, expression was focal, heterogeneous, and present in only a few regions o f each tumor. Staining was present on plasma membranes as well as within the cytoplasm and was restricted to intermediate-sized tumor ceils resembling those o f the early stratum spinosum of normal epithelium. Staining was not present in large, weft-differentiated squamous carcinoma ceils. Following neuraminidase digestion, 13 o f the 26 (50%) primary carcinomas expressed type II oligosaccharide structures. In these tumors, the original heterogeneity was reduced so that many more carcinoma ceils were stained, particularly those resembling parabasal cells of the normal tissue. Well-differentiated squamous carcin o m a cells stained better than basal type cells (Fig 4). Nonkeratinizing basal type cells were invariably unstained. The four tumors that failed to stain following desialylation were all of poorly differentiated parabasal type in which the incidence of positive cells in the nondesialylated tumors was very low. RCA (NeuAc2~3Gal-)
Normal Cervical Epithelium. Before neuraminidase digestion, only one specimen (9%) of normal cervical
Normal CervicalEpithelium. None o f the 11 cases o f normal cervical squamous epithelium b o u n d this lectin, either before or after neuraminidase digestion. Cervical Squamous Carcinomas. N o n e of the 26 cases of cervical squamous carcinoma b o u n d this lectin, either before or after neuraminidase digestion. SBA (~/pGalNAc-)
Normal CervicalEpithelium. N o n e of the 11 cases of normal cervical squamous epithelium b o u n d this lectin before or after neuraminidase digestion. Cervical Squamous Carcinomas. Before neuraminidase digestion, only two of the 26 (8%) squamous carcinomas expressed the unmasked determinant. Following desialylation, good staining of well-differentiated squamous carcinoma cells, particularly in the deep infiltrating regions of the tumors was identified in nine (35%) of the carcinomas (Fig 7). Tumors composed of predominantly basal type carcinoma cells were not stained. WGA ([GIcNAc/~ 1-*4GIcNAc-]n)
Normal Cervical Epithelium. All 11 specimens of normal squamous epithelium were well stained both before and after neuraminidase digestion. Following desialylation, staining was variably enhanced. Normal squamous epithelium stained in the cytoplasm of the basal cells but luminal plasma membranes in the u p p e r layers. Cervical Squamous Carcinomas. Before neuraminidase digestion, all 26 squamous carcinomas (100%)
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OLIGOSACCHARIDES iN CERVICAL CANCER (Banerjee et al)
FIGURE 3. Nonneoplastic cervical squamous epithelium overlying a moderately differentiated invasive squamous carc noma (A) Before neuraminidase digestion showing no expression of type II (GaI~I-~4GIcNAc-) oligosaccharides by the normal epithelial layers but neoexpression within the carcinoma. (B) Following desialylation, the nonneoplastic epithelium remains unstained, whereas there is enhanced staining of the carcinoma. (ECG; original magnification ×75.)
were stained. Well-differentiated squamous carcinoma, stained both in the cytoplasm and around luminal plasma membranes, particularly in those regions of tumors infiltrating deeply into adjacent stroma, although not in the superficial tumors. No significant difference in the patterns of staining were identified following neuraminidase digestion (Fig 8).
BS- 1 (D Gal~l-~3-)
Normal CervicalEpithelium. None of the 11 cases of normal cervical squamous epithelium bound this lectin before or after neuraminidase digestion. Cervical Squamous Carcinomas. Before neuraminidase digestion, 23 of the 26 (88%) squamous carcinomas bound this lectin. Morphologically well-differenti-
FIGURE 4. Well-differentiated squamous carcinoma expressing type II oligosaccharides (A) before and (B) following desialylation revealing enhanced expression of these determinants along tumor cell plasma membranes. (ECG- original magnification ×300.) 1009
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FIGURE S. Moderately differentiated invasive squamous carcinoma expressing NeuAc2-~3Gal- structures along plasma membranes. (RCA without neuraminidase digestion; original magnification x400.)
ated carcinomas stained particularly in regions of keratinization (Fig 9). Following neuraminidase digestion, the former 23 cases together with one additional one case (92%) stained with this lectin. Previously observed heterogeneity of binding by BS-1 to the cancers was now reduced so that many more of the basal type squamous carcinoma cells also expressed this determinant. LTA (Fuc~ 1-~6GIcNAc-)
Normal Cervical Epithelium. All 11 cases of normal squamous ectocervical epithelium were negative, both before and after neuraminidase digestion. CervicaISquamous Carcinomas. In four of 26 (15%) squamous carcinomas occasional tumor cells in areas of
FIGURE 6. Poorly differentiated invasive squamous carcinoma expressing NeuAc2~3Gat- structures as cytoplasmic aggregates in approximately 10% of the tumor cells. (RCA following neuraminidase digestion; original magnification x400.)
FIGURE 7. Islands of well-differentiated and deeply invasive squamous carcinoma cells expressing a//3GalNAc- structures both within the cytoplasm and along plasma membranes. (SBA following neuraminidase digestion; original magnification x200.)
well-differentiated squamous carcinoma stained weakly before neuraminidase digesdon. Following desialylation, five of 26 carcinomas (19%) showed weak staining with similar pattern. UEA- 1 (Fuc~ 1-~2Gal[GaINAc]-)
Normal Cervical Epithelium. All 11 specimens of normal cervical squamous epithelium were negative both before and after neuraminidase digestion. Cervical Squamous Carcinomas. Before neuraminidase digestion, 10 of the 26 carcinomas (38%) b o u n d this lectin. Those that stained were the better-differenti-
FIGURE 8. Moderately differentiated invasive squamous carcinoma expressing (GIcNAcI31~4GIcNAc-)n determinants predominantly within the cytoplasm of tumor cells at the stromal interface. Basal type cells in the centers of the tumor islands do not express this determinant. (WGA following neuraminidase digestion; original magnification x250.)
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OLIGOSACCHARIDES IN CERVICAL CANCER (Banerjee et al)
above the basal layer. After neuraminidase digestion, ectocervical basal epithelial cells stained in all 11 cases. In addition to the plasma m e m b r a n e staining previously identified, cytoplasmic staining was now apparent. Cervical Squamous Carcinomas. Before neuraminidase digestion, 13 of 26 carcinomas (50%) were stained. In all of these, cytoplasmic staining of individual cells was observed. In n o n e of these carcinomas were the basal type cells stained. After neuraminidase digestion, 17 of 26 carcinomas (65%) now stained. The staining increased with depth of invasion, so that the most prominent staining occur in those cells invading deeply and at the interface with the cervical stroma.
DISCUSSION FIGURE 9. Invasive squamous carcinoma in which the central, well-differentiated cells express terminal D-GaI,~1~ 3- structures. The surrounding tumor cells with basal type morphology, including those at the stromal interface, do not express this determinant, in contrast to the determinants recognized by WGA and UEA-1 (Figs 8 and 10). (BS-1 without neuraminidase digestion; original magnification ×120.)
ated squamous tumors. After neuraminidase digestion, staining of 15 carcinomas (58%) occurred either a r o u n d the stromal interface at the periphery of the invading cells (Fig 10) or in well-differentiated regions of squamous carcinoma. In these tumors, intensity of staining was enhanced. LCA ( M a n ~ 1 - * 3 / 6 M a n f l l - * 4 G I c N A c f l l - * 4 [ F u c a l - * 6 ] GIcNAc-)
Normal CervicalEpithelium. Before neuraminidase digestion, 10 of 11 specimens o f normal squamous epithelium stained with LCA. Staining was restricted to basal squamous epithelial cells and occurred weakly, both within the cytoplasm and around the plasma membranes. Following desialylation, all 11 specimens were stained, particularly of the nonkeratinizing basal epithelial cells. Cervical Squamous Carcinomas. Before neuraminidase digestion, 17 of 26 carcinomas (65%) were stained. Staining occurred in those tumor cells immediately adjacent to the stroma. Here, staining was entirely cytoplasmic. E n h a n c e d staining o f tumor cells appeared to occur with increasing squamous differentiation. Following neuraminidase digestion, 24 of 26 carcinomas (92%) were stained. The pattern was that of the welldifferentiated squamous cells showing staining, whereas the basal type cells were not stained. T h e r e was an increase in intensity of staining so that an additional seven tumors were now stained by the lectin. SNA ( N e u 5 A c ~ 2 - * 6 G a l / G a l N A c - )
Nominal CervicalEpithelium. Before neuraminidase digestion, plasma membranes of ectocervical basal epithelial ceils were stained in 10 of 11 control cases. T h e r e was no staining of differentiated squamous epithelium
This study has shown that h u m a n nonneoplastic cervical squamous epithelial cells characteristically express complex oligosaccharide structures that are core fucosylated (Manal~3/6Manfll-~4GlcNAcfll~ 4[Fucal-~6] GlcNAc-) and carry type I antennary structures (Galfll~3 GalNAc-) usually modified by sialic acid linked 2 --* 6 to terminal Gal- or GalNAc-residues (Table 2). Type II oligosaccharide structures (Galfll~ 4GlcNAc-) were not synthesized by nonneoplastic cervical squamous epithelium, and no other terminal antennary modifications were identified in these tissues. In the squamous carcinomas, neosynthesis of type II oligosaccharide structures, identified by ECG binding, was recognized in 50% of the cases examined. An additional four terminal antennary modifications were identified in the carcinomas that were not detected in normal tissues and constituted the oligosaccharide structures b o u n d by SBA, BS-1, LTA, and UEA-1. Binding of RCA was observed in only two specimens of normal cervical tissues but was commonly observed in the carcinomas.
FIGURE 10. Invading edge of a moderately differentiated carcinoma in which intracytoplasmic and plasma membrane expression of the oligosaccharide structures Fuc,~l--*2Gal (GalNAc)- is restricted to the several layers of cells at the stromal interface. (UEA-1 following neuraminidase digestion; original magnification ×400.)
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HUMAN PATHOLOGY TABLE 2,
Volume 26, No. 9 (September 1995)
Binding of Lectins to Normal Cervical Epithelium and to 26 Primary Cervical Carcinomas
Tissue Structure
Con-A
PNA
ECG
RCA
DBF
SBA
WGA
BS-1
LTA
UEA-1
LCA
SNA
11
4
0
1
0
0
11
0
0
0
10
10
26
19
17
16
0
2
26
23
4
10
17
13
11
10
0
1
0
0
ll
0
0
0
11
ll
26
26
13
21
0
9
26
24
5
15
24
17
Before Neuraminidase Digestion Normal squamous epithelium (n = 11) Squamous carcinomas (n = 26) After Neuraminidase Digestion Normalsquamous epithelium (n = 11) Squamous carcinomas (n = 26)
Enhanced expression of terminal GaINAc-residues, particularly identified by SBA and WGA, supports the recent report suggesting that presence of the unmasked Tn-antigen oligosaccharide structure was closely correlated with vascular permeation and metastasis to ly~.~ph nodes by invasive h u m a n cervical carcinoma cells. " Our findings have extended the earlier observations ofByrne et a112'1~by identifying distinct tumor cell phenotypes with respect to cell surface oligosaccharides expressed by h u m a n cervical squamous carcinomas. We have identified several sugar structures to be expressed only by cells with characteristic cytological appearances. For example, carcinoma cells resembling the basal and parabasal cells of normal cervical squamous epithelium typically expressed oligosaccharides recognized by PNA, ECG, and RCA. Following neuraminidase digestion, this group also included BS-1. Conversely, binding of SBA and LCA was expressed by better differentiated squamous epithelial cells, but not by the basal and parabasal cell types. Therefore, it seems likely that expression of certain oligosaccharide Structures might be related to specific stages of cellular maturation within h u m a n cervical squamous epithelium and, hence, identify individual cellular phenotypes. In this respect, stage expression of cell surface oligosaccharide determinants by cervical squamous epithelium could be analogous to that which has long been recognized during lymphocyte maturation. 19 Novel fucosylation linked a l --* 3 to terminal antennary oligosaccharide structures was identified in 15 carcinomas following desialylation. Of the 13 carcinomas found to express type II oligosaccharides following neuraminidase digestion, six were ozl ~ 3 fucosylated. Only type II oligosaccharide structures are able to be fucosylated with this particular linkage. 2° Two additional terminal structures not found in normal cervical squamous epithelium were a/flGalNAc- and NeuAc2-~ 3Gal-, detected by SBA and RCA-1, respectively. Carcinoma cells expressing type II oligosaccharides modified by a 1~3 fucosylation might be a particularly important behavioral phenotype. The novel structure (Galfll~ 4[Fucal~3] GlcNAc-) is related to the family of oligosaccharide determinants known to bind extracellular adhesion molecules, a 2 ~ 3 sialylation of this structure yields sialylLewis× that specifically binds members of the selec-
tin receptor family.2~ The selectins comprise a family of mammalian receptors that mediate initial interactions between leukocytes and vascular endothelia, leading to lymphocyte homing, platelet binding, and neutrophil extravasation. The three selectins, LECAM-1, ELAM-1, and GMP-140, share structural features that include a calcium-dependent lectin domain that specifically binds the sialyl Lewisx carbohydrate structure.'22 Recently, the cluster differentiation antigen CD62 has also been shown to bind the sialyl Lewis× determinant. The selectin receptors are not only major adhesion molecules for peripheral blood leukocytes but also for tumor cells. Expression of ELAM-1 by endothelial cells can be selectively induced by the cytokines tumor necrosis factor and interleukin 1, both cytokines being released by many neoplasms. With respect to the present study, neosynthesis of the sialyl LewisX determinant is only possible in tumor cells following prior synthesis of the type II oligosaccharide structure because this is the required substrate for the particular a l ~ 3 fucosyltransferase, ~3'24 and type II oligosaccharides were not identified in nonneoplastic cervical squamous epithelium. Following extravasation into the general circulation, synthesis of sialyl Lewis× oligosaccharides would promote adhesion of squamous carcinoma cells to the selectins or to CD62 receptors on vascular endothelium. 25 Thus, expression of the sialyl Lewis× structure might identify one of the metastatic phenotypes of cervical squamous carcinoma cells. This study has compared the predominant oligosaccharide structures expressed by h u m a n nonneoplastic cervical squamous epithelium and early invasive squamous carcinomas. Significant and consistent differences have been identified both between the normal and neoplastic tissues, and within the cancers. Expression of certain oligosaccharide structures has been related to the different cytological types of individual invasive squamous carcinoma cells. We suggest that these differences identify phenotypic variants within h u m a n cervical cancers and may define carcinomas that will behave in a predictable manner, particularly their metastatic potential. Because type II oligosaccharides occur only in malignant cells, their expression may result in changes in binding to cell adhesion molecules and, hence, be an essential step in the metastatic malignant
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OLIGOSACCHARIDES IN CERVICAL CANCER (Banerjee et al)
process. Identification of these structures in noninvasive cervical lesions may signal an invasive potential and allow reliable differentiation of premalignant lesions from those that are harmless. Additional studies are now being performed to determine the stage of cervical neoplasia at which the identified changes in oligosaccharide structures occur and whether expression of particular oligosaccharide determinants, singly or in combination, define different behavioral phenotypes within h u m a n cervical cancer. T h e pathologic significance of this study is that it has shown an i n d e p e n d e n t m e t h o d for identifying the p h e n o t y p e s o f individual t u m o r cells within a cervical squamous c a r c i n o m a - - a n d that might be ext e n d e d to squamous carcinomas developing in o t h e r tissues. A detailed analysis o f the plasma m e m b r a n e s is now r e q u i r e d to identify those glycoproteins that are characteristic o f the different m o r p h o l o g i c a l variants of cervical squamous carcinoma, according to their oligosaccharide structures. T h e r e a f t e r , correlation between individual b a c k b o n e proteins thus identified, and the behavioral p h e n o t y p e o f individual cancers, could provide the basis for a novel " f u n c t i o n a l " classification o f h u m a n cervical squamous cancer.
Acknowledgment. We wish to t h a n k J. Ng, MD, at T h e Princess M a r g a r e t Hospital, Swindon, Wiltshire, UK, for t h e case m a t e r i a l i n c l u d e d in this study. We are grateful to Lindasy M c G r e g o r for t e c h n i c a l assistance, a n d V i v i e n n e E m o n s a n d T a r e q Ansari in t h e D e p a r t m e n t o f H i s t o p a t h o l o g y w h o provided invaluable p h o t o g r a p h i c assistance. REFERENCES 1. Office of Population Censuses and Surveys: Cancer Statistics-Registrations. London, Her Majesty's Stationary Office, 1987 2. Qualters JR, Lee NC, Smith RA, et al: Breast and cervical cancer surveillance, United States, 1973-1987. Morbidity and Mortality Weekly Report: Centers for Disease Control Surveillance Summaries. 41/SS-2:l-15, 1992 3. BeachamJB, Halvorsen T, Kolbenstvedt A: Histological classification, lymph node metastases and patient survival in stage l b cervical carcinoma. Gynecol Oncol 6:95-105, 1978 4. Goellner JR: Carcinoma of tfie celarix: Clinicopathologic correlation of 196 cases. Am J Clin Pathol 66:775-780, 1976 5. Graham J, Graham R, Hirabayaski K: Recurrent cancer of the cervix uteri. Surg Gynecol Obstet 126:799-806, 1968 6. Lanza A, Re A, d'Addato F, et al: Treatment failure in cervical
cancer: High risk factors in relapse. Eur J Gynaecol Oncol 10:326336, 1989 7. Shingleton HM, Soong SJ, Gelder MS, et al: Clinical and histopathological factors predicting recurrence and survival after pelvic exteneration for cancer of the cervix. Obstet Gynecol 73:1027-1034, 1989 8. Norris WE: Evidence for a second class of membrane glycoprotein involved in cell adhesion..1 Cell Sci 93:631-640, 1989 9. Foster CS: Processing of N-linked oligosaccbaride glycoproteins in human tumours. B r J Cancer 62:57-63, 1990 (suppl X) 10. Aub JC, Tieslau C, Lankester A: Reaction of normal and tumor cell surfaces to enzymes: Wheat germ lipase and associated mucopolysaccharides. Proc Natl Acad Sci USA 50:613-619, 1963 11. Goldstein IJ, Hughes RC, Monsigny M, et al: What should be called a lectin? Nature 285:66, 1980 12. Byrne P, Williams A, Rollason T: Studies of lectin binding to the human cervix uteri: I. Normal cervix. HistochemJ 21:311-322, 1989 13. Byrne P, Williams A, Rollason T: Studies of lectin binding to the human cervix uteri: II. Cervical intraepithelial neoplasia and invasive squamous carcinoma. Histochem J 21:323-336, 1989 14. DavinaJH, Stadhouders AM, van Haelst UJ, et al: Concanavalin A-peroxidase labeling in cervical exfoliadve cytopathology: I. Labelling of normal squamous cells and the detection of cancer. Gynaecol Oncol 22:212-223, 1985 15. Parkin DM, Laara E, Muir CS: Estimate of the worldwide frequency of 16 major cancers in 1980. I n t J Cancer 41:184-187, 1988 16. Hirao T, Sakamoto Y, Kamada M, et al: Tn antigen, a marker of potential for metastasis of uterine cervix cancer cells. Cancer 72:154-159, 1993 17. Brudenell M, Cox BS, Taylor CW: The management of dysplasia, carcinoma in situ and microcarcinoma of the cervix. J Obstet Gynecol 80:673-679, 1973 18. Hsu M, Raine L, Fanger H: The use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques: Comparison between ABC and unlabelled antibody (PAP) procedure.J Histochem Cytochem 29:577-583, 1981 19. Piguet PF, lrle C, Kollatte E, et al: Post-thymic T lymphocyte maturation during ontogenesis. J Exp Med 154:581-593, 1981 20. Pereira MEA, Kisailus EC, Greuzo F, et al: Immunochemical studies on the combining site of the blood-group H-specific lectin 1 from Ulex europaeus seeds. Arch Biochem Biophys 185:108-115, 1978 21. Takada A, Ohmori K, Takahashi N, et al: Adhesion of human cancer cells to vascular endothelium mediated by a carbohydrate antigen, sialyl Lewis A. Biochem Biophys Res Commun 179:713-719, 1991 22. Foxall C, Watson SR, Dowbenko D, et al: The three members of the selectin receptor family recognize a common carbohydrate epitope, the sialyl Lewis(x) oligosaccharide. J Cell Biol 117:895-902, 1992 23. Lowe JB, Stoolman LM, Nair RP, et al: ELAM-l-dependent cell adhesion to vascular endothelium determined by a transfected human fucosyltransferase cDNA. Cell 63:475-484, 1990 24. Koszdin KL, Bowen BR: The cloning and expression of a human alpha-l,3 fucosyltransferase capable of forming the E-selection ligand. Biochem Biophys Res Commun 187:152-157, 1992 25. Polley MJ, Phillips ML, Wayner E, et al: CD62 and endothelial cell-leukocyte adhesion molecule 1 (ELAM-1) recognize the same carbohydrate ligand, sialyl-Lewis X. Proc Nail Acad Sci USA 88:62246228, 1991
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