Sialyl Lewis x expression in cervical scrapes of premalignant lesions

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1 Sialyl Lewis x expression in cervical scrapes of premalignant lesions NOÉ VELÁZQUEZ-MÁRQUEZ 1, GERARDO SANTOS-LÓPEZ 1, LUCIO JIMÉNEZ-ARANDA 2, JULIO REYES-LEYVA 1 and VERÓNICA VALLEJO-RUIZ 1, * 1 Laboratorio de Biología Molecular y Virología, Centro de Investigación Biomédica de Oriente, Instituto Mexicano del Seguro Social, Km 4.5 Carretera Federal Atlixco-Metepec, Atlixco, Puebla, Mexico C.P Clínica de Displasias, Hospital General de Zona No. 1, Instituto Mexicano del Seguro Social, Tlaxcala, Tlaxcala, Mexico *Corresponding author (Fax, ; , veronica_vallejo@yahoo.com) Sialylated oligosaccharides of glycoproteins and glycolipids have been implicated in tumour progression and metastases. Altered expression of glycosidic antigens has been reported in cervical cancer. In cervix premalignant lesions, an increased expression of sialic acid has been reported. In the present study we determined the expression profiles of the glycosidic antigens Tn, sialyl Tn (stn), Lewis a (Le a ), sialyl Lewis a (sle a ), Lewis x (Le x ) and sialyl Lewis x (sle x )in cervical scrapes with cytological diagnoses of normal, low-grade squamous intraepithelial lesions (LGSIL) and highgrade squamous intraepithelial lesions (HGSIL). Cervical scrapings were collected to detect tumour antigens expressions by flow cytometry using monoclonal antibodies. Cytometry analysis of Tn, stn, Le a and Le x did not reveal differences at the expression level among groups. The number of positive cells to sle a antigen increased in the HGSIL group with respect to the normal group (p=0.0495). The number of positive cells to sle x antigen in the samples increased with respect to the grade of squamous intraepithelial lesion (SIL) (p<0.001, Mann Whitney U test). The intensity of expression of this antigen increased in the HGSIL samples with respect to normal samples (p<0.0068). sle x antigen could be a candidate to be used as biomarker for the early diagnosis of cervical cancer. [Velázquez-Márquez N, Santos-López G, Jiménez-Aranda L, Reyes-Leyva J and Vallejo-Ruiz V 2012 Sialyl Lewis x expression in cervical scrapes of premalignant lesions. J. Biosci ] DOI /s z 1. Introduction Malignant transformation is often associated with changes in glycosylation. The most frequent changes in cancer glycosylation are the expression of branched structures, highly sialylated glycans, premature termination in the glycan biosynthesis giving truncated or incomplete structures, and re-expression of fetal-type glycosidic antigens. Aberrant glycosylation is found in both membrane glycoproteins and glycolipids (Takada et al. 1993; Dall Olio 1996; Marcos et al. 2004; Yu et al. 2005). Some of these alterations may provide a selective advantage for tumour cells (Cazet et al. 2010; Lau and Dennis 2008). Overexpression of glycosidic antigens including Tn, sialyl Tn (stn), Lewis a (Le a ), sialyl Lewis a (sle a ), Lewis x (Le x ) and sialyl Lewis x (sle x ) at the surface of cancer cells has been reported (Marcos et al. 2004; Mizuguchi et al. 2007; Ratei et al. 2008). The sle x and sle a serve as ligands of the selectins family and are associated with the binding of tumour cells to endothelial cells. They have been implicated in tumour metastasis and poor prognosis of cancer patients (Ohyama et al. 1999; Ugorski and Laskowska 2002; Paganuzzi et al. 2003; Saito et al. 2003; Akamine et al. 2004; Tozawa et al. 2005). Analysis of Tn and stn in the human cervix showed that Tn antigen expression increases in association with the grade of malignant transformation, and stn has been detected in normal cervix, severe dysplasia and in invasive carcinoma with no significant differences (Carrilho et al. 2000). A previous study reported that stn is not present in normal epithelia but is present in severe dysplasia, carcinoma in situ and invasive carcinoma. Tn antigen has been detected only in invasive carcinoma; in addition, Tn antigen expression in Keywords. Cervical cancer; glycosylation; Sialyl Lewis x; squamous intraepithelial lesions; tumour antigens , * Indian Academy of Sciences 999 Published online: 30 September 2012

2 1000 Noé Velázquez-Márquez et al. premalignant lesions has been detected using lectins (Santaella-Verdejo et al. 2007; Teresawa et al. 1996). In invasive cervical carcinoma, a loss of Le x and Le a antigens expression has been observed, as well as overexpression of the Le y antigen (Moro-Rodríguez and Álvarez-Fernández 2008). However, in patients with carcinoma of the uterine cervix, an increased concentration of sialic acid in serum and in local cervical tissue has been observed (Lagana et al. 1995; Roy and Chakraborty 2005). Our group analysed the expression of sialic acid in premalignant lesions and has identified an enhanced expression of α2,3-linked sialic acid and α2,6-linked sialic acid in concordance with the grade of squamous intraepithelial lesion (SIL) (López-Morales et al. 2010). Enhanced expression of sialyltransferase genes has also been detected (López-Morales et al. 2009). Expression profile of tumour antigens in cervical scrapes with diagnosis of premalignant lesions has not been reported. Based on our previous results, we decided to analyse sialylated tumour antigens and their non-sialylated forms. The present study analysed the expression profile of Tn, stn, Le a, sle a,le x and sle x antigens in cervical scrapes with diagnosis of normal cervix, LGSIL and HGSIL by flow cytometry. Findings revealed an enhanced expression of sle x antigen related with the grade of the cervical neoplasia. 2. Materials and methods 2.1 Patients Samples were collected from patients referred to the Colposcopy Unit from the General Hospital Zone No.1 of the Mexican Institute of Social Security (IMSS) located in Tlaxcala, Mexico, between January 2007 and July The study was approved by the institutional ethics committee. The purpose and procedures of the study were explained to patients, and informed consent was obtained from all participants. Two cervical samples were obtained from each patient. The first sample was used for cytological diagnosis. Papanicolaou smears were evaluated by a pathologist according to the Bethesda diagnostic criteria (Solomon et al. 2002). The second sample was used to analyse the expression profile of tumour antigens. In addition to Pap smear, each woman was subjected to a colposcopy, and when necessary, a biopsy was obtained for pathological diagnosis. No biopsy was obtained for research purposes. 2.2 Sample collection and processing Cervical cells were collected from the endo- and exocervices with a cytobrush and a spatula, respectively, and were placed in tubes containing 3 ml of ice-cold phosphatebuffered saline (PBS). Samples were stored at 4 C and processed within 24 h of their arrival to the laboratory. Cells were centrifuged at 4000 rpm for 15 min at room temperature and the cell pellets were suspended in 1 ml of PBS and centrifuged at 2000 rpm for 5 min at room temperature. The washed cell pellets were then suspended in 1 ml of PBS. We collected a group of 86 samples for all tumour antigens analysis, and according to the results obtained we included 24 more samples for the sle x antigen (a total of 110). 2.3 Detection of carbohydrate antigens with flow cytometry To optimize the same amount of cells in each assay, 0.5 ml of suspended cells were stained with the Guava Viacount Reagent (Millipore, Billerica, MA) according to manufacturer s instructions and counted in a Guava PCA cytometer (Millipore). A minimum of cells/ml were obtained in all samples. For analysis of the expression of the carbohydrate antigens, cells ( ) were incubated on ice for 90 min with antibodies specific for the Tn antigen (Biomeda Corporation, Foster City, CA), stn (Biomeda Corporation), Le a (Biomeda Corporation), sle a (Biomeda Corporation), Le x (Chemicon, Temecula, CA) or sle x (Chemicon) to a final concentration of 2 μg/ml in agitation. Cells were washed three times with ice-cold PBS and incubated for 30 min in the dark with Goat F(ab ) 2 Mouse IgG- Phycoerythrin (USBio, Marblehead, MA) or with Goat F(ab ) 2 anti-mouse IgM-Phycoerythrin (USBio) to a final concentration of 5 μg/ml. Cells were rinsed again with ice-cold PBS and suspended in 100 μl PBS and analysed in a Guava PCA flow cytometer using CytoSoft (Millipore) software (v.2.1). At least 1000 events were acquired. Cell intensity was expressed as median fluorescence intensity ratio (arbitrary units). To normalize the assay condition, a negative control sample was used without primary antibody (secondary antibody only). To determine the percentages of positive cells, events counts were compared with the gated events. Histogram statistics included the percentage of histogram events (all data in the histogram) and the gated data as a percentage of all data in the histogram (% of positive cells). 2.4 Statistical analysis Results were analysed with SPSS software (SPSS v.12, SPSS Inc., Chicago, IL). All data were analysed using the nonparametric Mann Whitney U test to compare for differences between the percentage of positive cells and the levels of expression of the tumour antigens in the SILs and normal samples of the uterine cervix; p-value < 0.05 was considered statistically significant.

3 Sialyl Lewis x in cervical premalignant lesions Results A total of 83 samples were analysed for all the antigens, except for sle x, where 110 samples were analysed. The mean age of women enrolled in this study was 37.8 years (range: 17 to 74 years). The mean age of women with HGSIL was 41.3 years, 35.9 years for those with LGSIL and 36.4 years for normal cytology. There was no statistical difference between age and lesion grade. 3.1 Analysis of Tn, stn, Le a, sle a,le x and sle x antigens expression in cervical scrapes To determine the expression profile of the Tn, stn, Le a, sle a,le x, and sle x antigens and whether these changes concur with the grade of cervical neoplasia, cervical scrapes with different cytological diagnoses (normal, LGSIL and HGSIL) were analysed by flow cytometry. All antigens were identified in the three groups but not in all samples (table 1). The number of positive samples varied among the different antigens. In the group with a normal diagnosis, Tn was the antigen with the lowest percentage of positive samples (42%) and sle x was the antigen with the highest number of positive samples (89%) (table 1). For the sle x antigen, all samples with a diagnosis of SIL were positive. The analysis of positive and negative samples to the different antigens did not permit discrimination between the groups of patients, but when we analysed the percentage of positive cells in each sample, we found differences between some of the antigens and groups of patients. The percentage of positive cells for the Tn and stn antigens in all samples was remarkably low in all groups, and statistical analysis did not reveal significant differences (data not shown). The analysis of Le a antigen in the three groups showed a similar percentage of positive cells, but the percentage of positive cells for the sle a antigen increased in relation to the grade of neoplasia (figure 1). Statistical analysis showed significant differences between normal and HGSIL (p=0.0495). When the expression level of the Le x antigen was analysed, we observed that the percentage of % positive cells Normal LGSIL HGSIL Normal LGSIL HGSIL Le(a) sle(a) Figure 1. Flow cytometry expression analysis of Le(a) and sle(a) antigens in cervical scrapes. Percentage of positive cells in patients with diagnosis of normal, LGSIL or HGSIL. The horizontal line represents the median. Statistical significance was determined by the Mann Whitney U test. positive cells was higher with respect to the other antigens, with a higher percentage of positive cells in the HGSIL group, but statistical analysis did not show differences among groups (figure 2). The percentage of positive cells for the sle x antigen increased in relation to the grade of neoplasia. The percentage of positive cells to sle x antigen was higher in both the LGSIL and the HGSIL groups as compared with normal samples (figure 2). Mann Whitney analysis showed differences between the normal and LGSIL groups (p=0.0021), the normal and HGSIL groups (p<0.0001), and between the LSIL and HGSIL groups (p<0.0001). The intensity of expression of each antigen was also evaluated and the results showed significant differences only for sle x between the normal and HGSIL groups (p<0.0068) (table 2). The results obtained for sle x showed that the percentage of positive cells increased in relation to the grade of neoplasia and that the intensity of its expression was higher in the HGSIL group. Table 1. Diagnosis Positive samples to tumour antigens in the normal, LGSIL and HGSIL groups analysed by flow cytometry Tn stn Le a sle a Le x sle x n (%) a n (%) a n (%) a n (%) a n (%) a n (%) a Normal 11/26 (42) 13/26 (50) 20/26 (77) 12/26 (46) 23/26 (88) 31/35 (89) LGSIL 16/36 (44) 22/36 (61) 29/36 (80) 22/36 (61) 34/36 (94) 52/52 (100) HGSIL 13/21 (62) 8/21 (38) 16/21 (76) 13/21 (62) 20/21 (95) 23/23 (100) LGSIL: low-grade squamous intraepithelial lesion; HGSIL: high-grade squamous intraepithelial lesion. n: number of positive cases/total of cases. a Percentage of positive cases.

4 1002 Noé Velázquez-Márquez et al. % positive cells Normal LGSIL HGSIL Normal LGSIL HGSIL Le(x) 4. Discussion sle(x) Figure 2. Flow cytometry expression analysis of Le(x) and sle(x) antigens in cervical scrapes. Percentage of positive cells in patients with diagnosis of normal, LGSIL or HGSIL. The horizontal line represents the median. Statistical significance was determined by the Mann Whitney U test. Glycans structure changes with development of cancer. The most common changes include synthesis of highly branched and sialylated glycans, synthesis of truncated structures and re-expression of fetal antigens (Dall Olio 1996). Tn and stn antigens represent truncated forms of O-glycans and are often expressed in tumours of different types of cancers, including breast, colon, stomach and pancreas (Nanashima et al. 1999; Nakagoe et al. 2002a, b). Studies have been conducted based on immunohistochemical analyses of the uterine cervix, determining the expression of Tn and stn (Carrilho et al. 2000; Santaella- Verdejo et al. 2007; Teresawa et al. 1996). Tn and stn antigens expression have been detected in severe dysplasia and invasive carcinoma but not in normal epithelium (Teresawa et al. 1996). Carrilho et al., in 2000, detected expression of Tn and stn antigens in 10% and 80% of Table 2. Mean of the fluorescence intensity for Le(x) and sle(x) in the normal, LGSIL, and HGSIL groups analysed by flow cytometry Mean of fluorescence intensity Diagnosis Le(x) sle(x) Normal LGSIL HGSIL LGSIL: low-grade squamous intraepithelial lesion; HGSIL: highgrade squamous intraepithelial lesion. normal samples, respectively. In the severe dysplasia group, Tn and stn were detected in 30% and 90% of the samples, respectively; these antigens were weakly expressed using immunohistochemistry. The present study using cervical scrapes showed that Tn and stn antigens were present in the normal and SIL groups with no differences in the percentage of positive cells or in fluorescence intensity. The results obtained in this study and previous studies suggest that stn and Tn are not good markers for the early diagnosis of cervical cancer. Some discrepancies have been reported among different studies that could have resulted from the specificity of the antibodies employed. Analysis of tumour antigens by immunohistochemistry or in cervical scrapes by cytometry could give different results because of the type of sample. Altered expression of other antigens such as the Lewis-type has been reported for many cancers, including lung, cholangiocarcinoma, breast, colon and cervix (Moro- Rodríguez and Álvarez-Fernández 2008; Satoh et al. 1997; Juntavee et al. 2005; Kurebayashi et al. 2006; Doekhie et al. 2008). In the cervical epithelium, the analysis of some Lewis-type antigens (Le a, sle a and Le b ) revealed expression in normal tissue with a loss of expression in neoplastic epithelium (Sanders et al. 1990). Expression of Le x,le y and the sialyl-dimeric Le x has been analysed in normal and cervical carcinoma. No differences were detected in the immunoreactivity for these antigens; however, the expression pattern was very different between primary lesions and metastatic lesions in cervical carcinomas (Ogawa et al. 1994). Most of the samples analysed showed expression of Le a,le x and Le y in normal tissue. There was a tendency to decrease in the SIL groups, but the carcinoma group showed an important loss of Le a and Le x expression and overexpression of the Le y antigen (Moro-Rodríguez and Álvarez- Fernández 2008). In this study, the expression of Le a and Le x in cervical scrapes did not show differences among the analysed groups, in neither the percentage of positive cells nor the level of expression. We did not detect a tendency toward decreasing their expression; however, it is important to mention that the type of sample may influence the results obtained. When antigen expression is analysed immunohistochemically, the expression profile can be determined in all layers of the epithelium. When we analysed cervical scrapes, we detected that cells mainly in the superficial layers might provide a different result. For the sle a antigen, we detected an increase in the percentage of positive cells in the HGSIL group. The percentage of positive cells to the sle x antigen was increased in LGSIL and HGSIL with respect to normal samples. The level of expression was also increased in HGSIL. Because the results showed that both the intensity and the percentage of positive cells showed differences for the sle x antigen, this antigen could be a good target to be studied in a larger number of samples to confirm if this antigen can be used in the development of a diagnostic

5 Sialyl Lewis x in cervical premalignant lesions 1003 method and to determine sensitivity, specificity and cutoff values to discriminate between normal and LGSIL or HGSIL samples. The analysis of a larger number of samples could also help to determine which measurements must be considered for the diagnosis, whether the percentage of positive cells, the mean fluorescence intensity or both. This antigen has not been analysed previously in cervical neoplasia and seems to change in relation to the grade of neoplasia. The sle x antigen is a sialylated and fucosylated glycan and member of the Lewis blood group structures found at the N-linked or O-linked glycans on glycoproteins and glycolipids (Wilkins et al. 1996). The ST3Gal III and ST3Gal IV participate in the synthesis of this antigen (Harduin- Lepers et al. 2001). Previous studies performed by our group showed an increase of mrna levels of certain sialyltransferases (ST6Gal I, ST3Gal III and ST3Gal IV) and sialic acid in both α2,3 and α2,6 linkage in neoplastic lesions (López-Morales et al. 2009, 2010). In cervical cancer tissue, an increase has been reported in the total sialic acid value (Roy and Chakraborty 2005) as well as increased levels of mrna sialyltransferases. ST3Gal III mrna level is significantly increased in patients with lymph node metastases compared to those without lymph node metastases (Wang et al. 2002). Although changes of the expression level of sle x antigen in cervical cancer may be due to differences in the relative activities of α2,3-sialyltransferases and α1,3-fucosyltransferases, it remains to be determined whether glycosyltransferases are involved in the biosynthesis of the sle x antigen in cervical cancer. The sle x antigen serves as a ligand of the selectin family and has been associated with tumour formation and metastasis (Okuno et al. 2003; Horstkorte et al. 2004). Moreover, it has also been implicated clinically in poor prognosis of cancer patients (Paganuzzi et al. 2003; Akamine et al. 2004). Clinical studies have shown that patients with cancer whose tumours express high levels of sle x have a significantly higher risk of developing invasion and metastasis than patients with tumours that express low levels of this antigen (Hoff et al. 1989; Ono et al. 1996; Nakagoe et al. 2002a, b). Increased expression levels of sle x correlate with recurrence, poor prognosis and survival rate of cancer patients (Yu et al. 2005; Mizuguchi et al. 2007). In summary, the expression of sle x in cervical scrapes increased in relation to the grade of cervical neoplasia, showing that glycosylation changes are present during early transformation and are not exclusive of cancer. 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