Znt. J . Cancer: 46, 99CL997 (1990) 0 1990 Wiley-Liss, Inc.
Publication of t h e International Union Against Cancer Publication de I'Union Internationale Contre 1e Cancer
MONOCLONAL ANTIBODY AGAINST A TUMOR-ASSOCIATED SIALOGLYCOPROTEIN OF SUPERFICIAL PAPILLARY BLADDER TUMORS AND CERVICAL CONDYLOMAS Yves FRADET'.~,HCEne LARUE',Carmen PARENT-VAUGEOIS~, Alain BERGERON', Colette DUFOUR',Lucie BOUCHER'and k n a r d BERNIER~ Tentre de Recherche en Canctrologie de L'Universitt Laval, L'Hbtel-Dieu de Qukbec, Qukbec, Canada; and 2Department of Pathology, L'H6tel-Dieu de Qdbec, Qutbec, Canada. A mouse IgG, monoclonal antibody (MAb), 19A2I I,defining a tumor-associated cell-surface antigen of superficial papillary bladder tumors, was generated by immunizing with fresh bladder tumor cells mice neonatally injected with normal human urothelial cells. The reactivity of the antibody with cell lines was examined by indirect immunofluorescence staining and was restricted t o 3/14 bladder cancer lines and 3/31 cancer cell lines of non-bladder origin, including HeLa cervical cancer. No normal fibroblast, kidney cells, EBVlymphocytes, erythrocytes or leukocytes expressed the antigen. Reactivity of MAb l9A2l I was well preserved on tissue paraffin sections. lmmunoperoxidase staining of normal adult or fetal tissues showed no reactivity except for a patchy or uniform staining of umbrella cells in 6/23 adult and 1/4 fetal urothelium samples. Positive and often heterogeneous staining was observed on 24/38 papillary superficial tumors (Ta) and 415 carcinoma in situ bladder lesions but on only 4/20 infiltrating tumors. It was also observed on 5/6 cervical condylomas and one bladder condyloma, but none of 6 penile or vulvar condylomas. All other tumors tested were negative. The antigenic determinant is present on a heterogeneous group of proteins with molecular weights ranging from 90 t o 200 kDa. It is sensitive t o periodate treatment and t o neuraminidase but only partially sensitive t o proteases. MAb l9A2l I is different from other reported MAbs with similar reactivity t o superficial bladder tumors and umbrella cells of normal urothelium. When tested in competition assays, several of these MAbs, but not I9A2 II,were found to react with Lewis X blood group determinant. Our results suggest that l9A2l I may be useful for detection and stratification of bladder tumors.
Superficial papillary tumors constitute 60-70% of bladder carcinomas. They have a high rate of recurrence and an unpredictable behavior in that a small percentage of those that recur will be infiltrating tumors. Superficial tumors are not frequently detected by conventional urine cytology because of the well-differentiated phenotype of the malignant cells which makes it difficult to distinguish them from their normal counterparts. A specific and reliable method of differentiating malignant cells from normal ones would be a great asset for early diagnosis and follow-up. The search for specific tumor markers in the form of new molecules or modulated pre-existing antigens has been a long quest that has met with some success. Although strictly specific tumor markers are rare, quantitative differences in the expression of antigens between normal and malignant cells may be large enough to be exploited for diagnosis or therapy. Several antigenic systems have already been associated with bladder cancer. The loss of expression of ABH blood group antigens (Decenzo et al., 1975; Limas and Lange, 1982), or modifications in the expression of Lewis blood group antigens (Limas and Lange, 1985; Orntoft et al., 1987a, 1989; CordonCardo et al., 1988) have been associated with malignant transformation of the urothelium. A correlation has also been observed between the pattern of expression of cytokeratins and the grade of transitional-cell carcinomas (Feitz et al., 1985; Moll et al., 1988). A series of antigens of transitional-cell carcinoma have been defined by MAbs (Fradet et al., 1984,
1986; Messing et al., 1984; Young et al., 1985; Masuko et al., 1984; Ben-Aissa et al., 1985; Huland et al., 1987; Chopin et al., 1988; Baricordi et al., 1985; Summerhayes et al., 1985; Longin et al., 1989). However, they mostly show a preferential reactivity with higher-grade tumors. We have recently described a tumor-associated antigen, M344, defined by a MAb obtained after immunization of mice concomitantly with fresh bladder tumor cells and mouse serum hyperimmunized against normal human urothelial cells (Fradet et al., 1987). In the present study, a different approach was used based on the observed immunotolerance of mice to some antigens introduced in utero or at birth (Jamieson and Ahmed, 1988). Neonatal mice were immunized with normal urothelial cells and 6 weeks later immunized with cells from superficial papillary bladder tumors. We thus obtained 19A211 MAb, selectively reacting with well-differentiated bladder tumors and carcinoma in situ of the bladder as well as cervical condylomas. It characterizes a sialylated antigen not related to any blood group antigen. MATERIAL AND METHODS
Tissues and cells Fresh tissues, obtained from the Department of Surgical Pathology, were embedded in OCT compound (Tissue Tek, Miles, McLean, VA), frozen in liquid nitrogen, and stored at - 70°C. Sections were also obtained from formalin-fixed, paraffin-embedded tissues. Bladder tumor-cell suspensions for immunization and serological analysis were prepared from fresh specimens by mechanical dispersion in phosphatebuffered saline (PBS). Normal urothelial cells were obtained by scraping off the mucosa of normal bladder specimens obtained from cadaver kidney donors. The resulting cell suspensions were verified by flow cytometry with the urothelial specific T16 MAb (Fradet et al., 1984) and judged to be >95% pure. They were stored frozen in minimum essential medium containing 10% dimethyl sulfoxide and 15% fetal calf serum. Cultured human cancer cell lines were obtained from the collection of the Sloan-Kettering Institute (New York), except for the bladder cancer cell lines MGH-U3 and U4 which were kindly provided by Drs. C.W. Lin and G. Rout (Massachusetts General Hospital, Boston) (Lin et al., 1985). Short-term cultures of normal human kidney cells and fibroblasts were obtained as previously described (Fradet et al., 1987). Tests for Mycoplasma contamination were routinely performed. Production of monoclonal antibodies Attempts to decrease reactivity to normal cells were made by
'To whom correspondence and reprint requests should be sent, at the Centre de Recherche en Cancerologie de I'Universitk Laval, L'HBtel-Dieu de Quebec, Quebec, Quebec GIR 2J6, Canada.
Received: May 15, 1990 and in revised form July 23, 1990.
99 1
NEW ANTIGEN OF HUMAN BLADDER TUMORS
injecting neonatal BALB/c mice (
Publication of t h e International Union Against Cancer Publication de I'Union Internationale Contre 1e Cancer
MONOCLONAL ANTIBODY AGAINST A TUMOR-ASSOCIATED SIALOGLYCOPROTEIN OF SUPERFICIAL PAPILLARY BLADDER TUMORS AND CERVICAL CONDYLOMAS Yves FRADET'.~,HCEne LARUE',Carmen PARENT-VAUGEOIS~, Alain BERGERON', Colette DUFOUR',Lucie BOUCHER'and k n a r d BERNIER~ Tentre de Recherche en Canctrologie de L'Universitt Laval, L'Hbtel-Dieu de Qukbec, Qukbec, Canada; and 2Department of Pathology, L'H6tel-Dieu de Qdbec, Qutbec, Canada. A mouse IgG, monoclonal antibody (MAb), 19A2I I,defining a tumor-associated cell-surface antigen of superficial papillary bladder tumors, was generated by immunizing with fresh bladder tumor cells mice neonatally injected with normal human urothelial cells. The reactivity of the antibody with cell lines was examined by indirect immunofluorescence staining and was restricted t o 3/14 bladder cancer lines and 3/31 cancer cell lines of non-bladder origin, including HeLa cervical cancer. No normal fibroblast, kidney cells, EBVlymphocytes, erythrocytes or leukocytes expressed the antigen. Reactivity of MAb l9A2l I was well preserved on tissue paraffin sections. lmmunoperoxidase staining of normal adult or fetal tissues showed no reactivity except for a patchy or uniform staining of umbrella cells in 6/23 adult and 1/4 fetal urothelium samples. Positive and often heterogeneous staining was observed on 24/38 papillary superficial tumors (Ta) and 415 carcinoma in situ bladder lesions but on only 4/20 infiltrating tumors. It was also observed on 5/6 cervical condylomas and one bladder condyloma, but none of 6 penile or vulvar condylomas. All other tumors tested were negative. The antigenic determinant is present on a heterogeneous group of proteins with molecular weights ranging from 90 t o 200 kDa. It is sensitive t o periodate treatment and t o neuraminidase but only partially sensitive t o proteases. MAb l9A2l I is different from other reported MAbs with similar reactivity t o superficial bladder tumors and umbrella cells of normal urothelium. When tested in competition assays, several of these MAbs, but not I9A2 II,were found to react with Lewis X blood group determinant. Our results suggest that l9A2l I may be useful for detection and stratification of bladder tumors.
Superficial papillary tumors constitute 60-70% of bladder carcinomas. They have a high rate of recurrence and an unpredictable behavior in that a small percentage of those that recur will be infiltrating tumors. Superficial tumors are not frequently detected by conventional urine cytology because of the well-differentiated phenotype of the malignant cells which makes it difficult to distinguish them from their normal counterparts. A specific and reliable method of differentiating malignant cells from normal ones would be a great asset for early diagnosis and follow-up. The search for specific tumor markers in the form of new molecules or modulated pre-existing antigens has been a long quest that has met with some success. Although strictly specific tumor markers are rare, quantitative differences in the expression of antigens between normal and malignant cells may be large enough to be exploited for diagnosis or therapy. Several antigenic systems have already been associated with bladder cancer. The loss of expression of ABH blood group antigens (Decenzo et al., 1975; Limas and Lange, 1982), or modifications in the expression of Lewis blood group antigens (Limas and Lange, 1985; Orntoft et al., 1987a, 1989; CordonCardo et al., 1988) have been associated with malignant transformation of the urothelium. A correlation has also been observed between the pattern of expression of cytokeratins and the grade of transitional-cell carcinomas (Feitz et al., 1985; Moll et al., 1988). A series of antigens of transitional-cell carcinoma have been defined by MAbs (Fradet et al., 1984,
1986; Messing et al., 1984; Young et al., 1985; Masuko et al., 1984; Ben-Aissa et al., 1985; Huland et al., 1987; Chopin et al., 1988; Baricordi et al., 1985; Summerhayes et al., 1985; Longin et al., 1989). However, they mostly show a preferential reactivity with higher-grade tumors. We have recently described a tumor-associated antigen, M344, defined by a MAb obtained after immunization of mice concomitantly with fresh bladder tumor cells and mouse serum hyperimmunized against normal human urothelial cells (Fradet et al., 1987). In the present study, a different approach was used based on the observed immunotolerance of mice to some antigens introduced in utero or at birth (Jamieson and Ahmed, 1988). Neonatal mice were immunized with normal urothelial cells and 6 weeks later immunized with cells from superficial papillary bladder tumors. We thus obtained 19A211 MAb, selectively reacting with well-differentiated bladder tumors and carcinoma in situ of the bladder as well as cervical condylomas. It characterizes a sialylated antigen not related to any blood group antigen. MATERIAL AND METHODS
Tissues and cells Fresh tissues, obtained from the Department of Surgical Pathology, were embedded in OCT compound (Tissue Tek, Miles, McLean, VA), frozen in liquid nitrogen, and stored at - 70°C. Sections were also obtained from formalin-fixed, paraffin-embedded tissues. Bladder tumor-cell suspensions for immunization and serological analysis were prepared from fresh specimens by mechanical dispersion in phosphatebuffered saline (PBS). Normal urothelial cells were obtained by scraping off the mucosa of normal bladder specimens obtained from cadaver kidney donors. The resulting cell suspensions were verified by flow cytometry with the urothelial specific T16 MAb (Fradet et al., 1984) and judged to be >95% pure. They were stored frozen in minimum essential medium containing 10% dimethyl sulfoxide and 15% fetal calf serum. Cultured human cancer cell lines were obtained from the collection of the Sloan-Kettering Institute (New York), except for the bladder cancer cell lines MGH-U3 and U4 which were kindly provided by Drs. C.W. Lin and G. Rout (Massachusetts General Hospital, Boston) (Lin et al., 1985). Short-term cultures of normal human kidney cells and fibroblasts were obtained as previously described (Fradet et al., 1987). Tests for Mycoplasma contamination were routinely performed. Production of monoclonal antibodies Attempts to decrease reactivity to normal cells were made by
'To whom correspondence and reprint requests should be sent, at the Centre de Recherche en Cancerologie de I'Universitk Laval, L'HBtel-Dieu de Quebec, Quebec, Quebec GIR 2J6, Canada.
Received: May 15, 1990 and in revised form July 23, 1990.
99 1
NEW ANTIGEN OF HUMAN BLADDER TUMORS
injecting neonatal BALB/c mice (
