Seminars in Surgical Oncology 8:293-299 (1992)

Cell Surface Differentiation Antigens of Normal Urothelium and Bladder Tumors GUlDO DALBAGNI, MD, VICTOR E. REUTER, MD, JOEL SHEINFELD, MD, YVES FRADET, MD, WILLIAM R. FAIR, MD, AND CARLOS CORDON-CARDO, MD, PhD From the Memorial Sloan-Ketterhg Cancer Center, New York (C.D., V.R., j.S., W.R.F.,C.C.-C.) and L a d University, Quebec, Canada (Y.F.1 -

.~

Bladder cancer ranks as the third most common malignancy among men and tenth among women. Superficial transitional cell carcinomas (stage Ta, Tis, and TI) account for approximately 7040% of these tumors, while the remaining 20-30% are invasive (T2, T3, and T4). Approximately 70% of superficial tumors will have one or more recurrences, with 25% of these expressing a higher histologic grade and 10-1 5% subsequently developing invasive and/ or metastatic disease. The detection and prediction of tumor recurrence and/or tumor progression is crucially important if timely and appropriate therapy is to be instituted. Conventional histopathologic evaluation usually provides definitive diagnosis upon which therapeutic planning is based. However, at present there are no more reliable morphologic indicator to identify which individuals will have recurrent disease or who will progress to invasive and/or metastatic cancer. Recent advances in tumor biology have identified markers that are good candidates for clinical applications in early tumor detection, as well as for the stratification of patients with like-appearing morphological lesions with different biological and clinical behavior. The ultimate goal is to develop predictive assays that would segregate patients with high probability of failures versus patients who would be cured by localized modes of therapy. 0 1992 WiIey-Liss, Inc. KEY WORDS:bladder cancer, immunopathology, monoclonal antibodies, differentiation antigens, tumor-associated antigens

INTRODUCTION The recent introduction of immunohistochemical methods provide a very powerful tool for the identification of tissue and cellular constituents, utilizing the high specificity and sensitivity of antibodies as probes of tissue antigens. With this technique, constituents of the cell that reflect cell type, differentiation, functional change, or proliferative status are potentially identifiable by immunohistological reactions. Highly specific antibodies to cellular antigens have been produced, offering promise of fundamental insight into tumor biology in addition to potential practical applications. We believe that the immunocharacterization of bladder cancer can provide early and objective means of subclassifying urothelial tu0 1992 Wiley-Liss, Inc.

mors, aid in predicting behavior, and selecting and monitoring therapy. It is becoming increasingly evident that differentiation antigens and tumor-associated antigens play a major role in the development of human bladder tumors. Because they reflect the origin and function of the tumor, monoclonal antibodies to these antigens are of potential value to the pathologist and urologist to characterize and subclassify undifferentiated or like-appearing tumors of different clinical behavior. While it is possible that a single highly specific antibody will recognize a particular tumor, most bladder Address reprint requests to Dr. Carlos Cordon-Cardo, Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021.

294 Dalbagni et al.

cancer probably will require a battery of several anti- their use in early tumor detection and prognosis. The first mouse monoclonal antibody to react with an epibodies for their subclassification. Ultimately, we anticipate a new subclassification of tope carried by bladder tumor cells, was reported by human bladder tumors based on their immunopheno- Starling et al. in 1982. This group of researchers protypic characterization. The value of this subclassifica- duced an antibody by immunizing mice against the tion will be determined by correlation with clinical and prostate cancer cell line DU 145 [4]. They demonstrated conventional pathologic features, flowcytometry a peculiar pattern of crossreactivity of this antibody parameters, rate of tumor progression and metastases, with three out of nine bladder cancer cell lines, as well as and response to therapy and prognosis. It is conceiv- with a viral-transformed human lung cell line. The strategy followed at our institution in order to able that the most highly specific antibodies may also generate and analyze MoAb to cell surface antigens be of value for clinical imaging and/or for immunoled Fradet et al. to the production and characterizatherapy. tion of the first panel of antibodies by immunizing PRODUCTION AND CHARACTERIZATION OF mice with human bladder cancer cell lines and a bladMONOCLONAL ANTIBODIES TO der papilloma [5,6]. These antibodies were grouped DIFFERENTIATION ANTIGENS OF NORMAL into three categories based on their respective characUROTHELIUM AND BLADDER TUMORS teristics. Antibody Om5, recognizing a category 1 anThe production of monoclonal antibodies to spe- tigen, was expressed in superficial bladder tumours cific antigens was made possible by the experiments of and 60% of normal urothelial samples tested. AntiKohler and Milstein [ 11. Monoclonal antibodies bodies T43, T23, and T138, detecting category 3 anti(MoAb) are generated by immunizing mice or rats gens, were mainly expressed in invasive tumors. Fiwith an appropriate source of immunogenic determi- nally, antibodies detecting category 3 antigens, such as nants. After sacrificing these animals and excising T16, T87, and 5143, were found in all cells of urotheir spleen, spleen cells are fused with immortalized thelial origin. Those antigens were also expressed on a myeloma cells deficient in hypoxanthine guanine variety of neoplasms with the sole exception of Om5, phosphoribosyl transferase (HGPRTase). Parent and which was restricted to tumors of urothelial lineage. It hybrid cells are grown in an aminopterin rich medium should also be emphasized that antibody T13, which (HAT), conferring upon the hybridized cells a survival detects a 25 kD cell surface glycoprotein, was reported advantage. Non-fused spleen cells are unable to grow to have a very restricted pattern of distribution in in culture and myeloma cells deficient in HGPRTase normal tissues (mainly endothelial cells of small vesdie in the presence of aminopterin. sels and capillaries), while being present in most of the Highly restricted tumor-associated and tumor-spe- invasive and metastatic bladder tumors analyzed [6]. cific antigens are difficult to detect, since they repre- Two other important findings were observed during sent the minority of the total antigenic structure. An the process of characterization of these antigens. Cell alternative method has been used to generate anti- lines obtained from similar tumor types showed difbodies with great specificity to bladder tumor cells ferent phenotypes, and no correlation was shown be[2,3]. Antibodies were produced by simultaneous pas- tween antigen expression in vivo vs. in vitro with most sive immunization against dominant antigens of nor- of these antigenic systems. mal urothelium and active immunization with intact Another panel of mouse MoAbs was generated with grade I papillary bladder tumor cells. Using this im- the objective of detecting bladder antigens by Masuko munomodulation strategy and well-characterized nor- et al. in 1983. They reported six antibodies against mal tissues and tumor samples, several new antigenic human bladder cancer obtained by the immunization systems were identified that present restricted specifi- of mice with the human bladder tumor cell line KU-1 cities for low-grade superficial tumors of the urinary [7]. Antibodies HBA4, HBE3, and HBG9 recognized sialoglycoproteins and reacted with 60 to 80% of the bladder [2,3]. Table I and Figure 1 summarize some of the re- bladder tumors tested. These antibodies did not react ported monoclonal antibodies (MoAb), their im- with normal urothelium. However, HBH8 recognized munoglobulin subclass, and the antigen detected a glycolipid present in normal urothelium and 80%)of (whenever data available to us), as well as the patterns bladder neoplasms. Similarly, HBF2 identified a of immunoreactivity on normal urothelium and blad- glycolipid which was expressed on 20% of the bladder der tumors. tumors tested, as well as on some normal urothelium. Since the introduction of the hybridoma technology Finally, HBlO antigen was characterized as being a in tumor immunology, a plethora of MoAb have been glycolipid present in 60% of bladder tumours but not generated in an attempt to define biological markers for on the normal urothelium.

Cell Surface Markers 295 TABLE I. Monoclonal Antibodies to Urinary Bladder Tumors Antibody

Urothelium

Om 5 (IgGI)

+/-

TI 6 (IgG2b) T87 (IgGl) T43 (IgGI)

+ + -

T138 (IgM)

-

T23 (IgM)

-

TI 10

-

3143 (IgG1) HBA4, HBE3 HBG9 (IgM) HBEIO (IgM) HBF2 (IgM) HBH8 (IgM) 9A7 2E 1 2A6 G4, E7 (IgM)b 4-72-2 (IgGl) 3-48-2148-1 13-50-3 (IgGl)/(IgGl)/(IgGl) 8-30-3177-112-94-2 (IgG2b) I (IgG2a) I (IgG3) 3-71-1 194-3 (IgG3)/(IgG3) GF 26.7.3 (IgG2b) P7 A 5-4 (IgGI) SK 4H-12 (IgG2a) 3G2-C6 (IgG1)

+ (Bas) -

+/+l-

(Umbf)’

+ (BasJnt) + (Int,Sup) - (SUP)’

+/-

Negative Negative -

C3 (IgG1) Mano 414 (IgG1)

+(Bas)

+ )’

486 P3 / 12 (IgM)b M344 (IgG1)

- (Umb

BL 2-10D1 (IgM)

- (Umb

+y

10D1 (IgM) 7C12 (IgM) 6D1 (IgGI) 3C6 (IgM) 12F6 (IgGI) AN 43 (IgGl) BB369 (IgGI) 19A 211 (IgG1)

- (Umb

+ )’

-

-(Umb+)” - (Umb

+ )’

-

+

+

~

(Umb +

Bladder __tumora Superficial tumors 88% Invasive tumors 15% Virtually all tumors Virtually all tumors Invasive and metastatic tumors 90% Invasive and metastatic tumors 75% Invasive and metastatic tumors 25% Fibrillar staining in the extracellular matrix of all malignant tissues Virtually all tumors 60% of bladder tumors 80% of bladder tumors 60% of bladder tumors 20% of bladder tumors 80% of bladder tumors Virtually all tumors Virtually all tumors Virtually all tumors High grade, Cis and low grade tumors Virtually all tumors with different patterns Virtually all tumors with different patterns Virtually all tumors 60% of bladder tumors 80% of bladder tumors 85% of bladder tumors High grade tumors and a subset of low grade

85% of bladder tumors w lymph node mets 90% of bladder tumors 68% of superficial tumors with 15% of invasive tumors 70% of bladder tumors (All grade) Grade I BT Grade I and I1 BT Grade I BT Grade 111 BT including Cis Grade 1-11 BT 55% of BT 55% of BT 80% Cis, 63% superficial, 20% invasive

Antigen Not known

Reference [5,61

gp 48, 42 Kd gp 60 kd gp 85 kd gp 25 kd

[5,61

Not known

[5+61

gp 240 kd

[5~1

gp 140, 120, 30 kd sialogp 78, 40, 130 kd sialogp 78, 40, 130 kd Glycolipid Glycolipid Glycolipid Not known Not known Not known gp 200 kd Not known

1111

300 kd glycoprotein

[ I 11

250 kd

[ I 11

Not known 92 kd, 23 kd, 17 kd polypeptide gp 100 kd 90 kd

[121 [ 131 ~ 3 1 ~ 4 1

600 kd gp28kd gp 200 kd 300 kd-62 kd Lipid Not known Not known Not known Not known Not known Mucin? Mucin? Protein 90 kd to 200 kd

aThe number represents percentages reported. bRecognizes the LeX Blood Group Antigen [21,25]. WMB, umbrella cells; BAS, basal cells; INT, intermediate cells; SUP, superficial; BT, bladder tumor; gp, glycoprotein. Identical epitopes shed by the cells.

~ 7 1

296 Dalbagni et al.

cia1 but not in invasive tumors; however, the pattern observed for group 2 was lost early on during the A neoplastic process [ 1 I]. I Two groups reported novel antigenic systems in B 1985. Baricordi et al. generated a MoAb that reacted I with bladder and colon carcinomas. The determinant C was also found on cells of the neutrophilic lineage [12]. I This antibody, designated GF 26.7.3, was reported to I stain 60% of bladder tumors, independently of grade D or stage. On the other hand, Ben-Aissa et al. obtained I two MoAb, P7A5.4 and SK4H-12, which reacted with E 88Y0of the bladder tumors tested, but not with normal I I urothelium [ 131. Interestingly, contrary to the staining Fig. 1. Schematic illustration of patterns of immunoreactivities of pattern on normal urothelial tissues, SK4H-12 was mouse monoclonal antibodies to differentiation antigens of normal positive on normal urothelial cells in culture, suggesturothelium and bladder cancer. Antibodies have been grouped in five categories (A-E) according to their distribution in normal ing either a growth-related phenotypic change or a urothelium, superficial, and invasive tumors. A, Om5 (5.6);B,TI6 transformation process of cultured cells. (5,6),T87 (5,6),5143 (5,6),4-72-2(I I), 3-48-2( 1 I), 48-1 (1 I ) , 3-50-3 Young et al. tested 31 bladder tumours with the ( I I ) , 8-30-3( l l ) , 77-1 (Il),3-71-1( I I ) , 94-3( I I ) , 2-94-2(11). GF 26.7.3(12),Mano4/4(15).AN43(19), BB369(19);C,9A7(8).2EI antibody 3G2-C6, generated in their laboratory by (8), 2A6 (8), 486 P3112 (15)*,BL2-10DI (17)*;D, M 344 (2). immunizing mice against the MGH-U I bladder tumor 19A211 (22)*: E,T43 (5.6).TI38 (5.6). T23 (5,6),3G2-C6(14).*. cell line. It had an enhanced affinity for high-grade umbrella cells. lesions, as well as for cell lines derived from poorly differentiated TCC. Nevertheless, staining of frozen A new series of monoclonal antibodies were re- sections was reported to be quite unreliable; thus fresh ported by Messing et al. They established MoAb 9A7, cell suspensions were needed for prospective studies, 2E1, and 2A6 by immunizing mice with the 647V cell curtailing significantly its use in a clinical setting [14]. line, derived from a human bladder transitional cell Arndt et al. reported two antibodies in 1987 [15]. carcinoma (TCC) [8]. These antibodies reacted with all Mano 4 / 4 reacted with the basal layers of urothelium, the TCC cell lines and tumors included in that study; while 486 P3 / 12 recognized an epitope carried on umhowever, they did not react with normal urothelium. brella cells and granulocytes. More recently, antibody An innovative approach for the improvement of 486 P3 / 12 was proven to recognize the Lewis X blood conventional cytology was proposed by Chopin et al., group antigen [lo]. Mano 414 was found on tumor using immunocytology in bladder washes. They re- cells in 80% of patients with bladder cancers; in addiported the production and characterization of anti- tion, this antibody also reacted with nonurothelial lebodies G4 and E7, reacting with umbrella cells, a sub- sions. The antibody 5143, previously reported by Fraset of low grade transitional cell carcinomas, as well as det et al. [6], and Mano 4/4 were similarly distributed high-grade bladder tumors. The diagnostic value of in the renal glomerulus, specific cells types along the phenotypes observed were similar to the results of urinary tract (i.e., basal layers of urothelium), and conventional Papanicolaou stainings [9]. More re- bladder tumors. cently, Fradet showed that these antibodies recognize Takahashi et al. tested 71 bladder tumors with the antigenic determinants carried on the LeX blood so-called No10 MoAb antibody, generated as one of group related molecule [ 101. many reagents by immunizing mice with grade 2 bladAnother strategy was that employed by Summer- der cancer cells. It was found to be restricted to bladhayes and coinvestigators, who generated MoAb by der tumors and did not react with normal urothelium immunizing mice with membrane extracts from or any other tissue [16]. human metastatic bladder neoplasms. They categoA novel approach was adopted by Fradet et al. in rized these antibodies in three groups, based on their order to circumvent the antigenic competition present reactivity to normal urothelial cell subpopulations. when the antigen of interest represents a minority of Group 1 (Ab 4-72-2) stained the basal and intermedi- the total sample. Mice were simultaneously immuate cell layers, group 2 (Abs 3-48-2, 48-1,3-50-3) nized with superficial bladder tumors and a polyclonal stained intermediate and superficial cell layers, and serum against normal urothelium. This strategy was group 3 (Aba 8-30-3, 77.1, 2-94-2, 3-71-1, 94-3) o ~ d y successful in genzrdting a highly rcstricted antibody reacted with superficial cells. Antibodies of groups 1 (Ab M344) which showed 68% specificity for the suand 3 retained their staining characteristics in superfi- perficial bladder tumours, while being reactive with NORMAL UROTHELIUM

SUPERFICIAL

"vAs~VE

I

I

I

Cell Surface Markers 297

only 15% of the invasive lesions. The pattern of staining was observed to be in the cytosolic fraction of the cells [2]. Based on the fact that the molecule detected is a high molecular weight glycoprotein, and that may shed into the urine, this highly restricted antigen may be a target for future studies, mainly those oriented to screen patients at risk to develop bladder tumors. The BL2-1ODI MoAb was generated by Longin et al. They immunized mice with bladder carcinoma cell suspension and RT4 cells. Its affinity decreased with high-grade TCC and is comparable to the OM5 MoAb in that respect [6,7]. These reagents differ in two important characteristics: antibody Om5 reacts with 60% of normal urothelial specimens, while BL2lODl stains only occasional umbrella cells. Furthermore, Om5 Ab is more restricted to superficial TCC, staining only 88% of the low-grade papillary superficial lesions, as opposed to 20% of the invasive neoplasms screened for its reactivity [17]. Hijazi et al. reported the results of bivariate flowcytometry with five new MoAb (10D1, 7C12, 6D1, 3C6, and 12F6), showing that 3C6 recognized an antigen mainly expressed by aneuploid cells. Furthermore, lODl and 6D1 labeled low-grade lesions and 7C12 and 12F6 stained low- and medium-grade neoplasms; however, 3C6 was immunoreactive with high-grade tumours [ 181. Liebert et al. identified two antibodies reacting with a shed antigen (AN43 and BB369 MoAb). The respective epitopes were found to be identical and spatially related, being present in normal urothelium and bladder tumors [ 191. Bretton and collaborators, in 1989, performed bivariate flowcytometry (FCM) seeking an improvement in the sensitivity of the technique. Essentially, they attempted to exclude from their analyses inflammatory cells present in the bladder washes. These inflammogenic elements usually affect the accuracy of flowcytometry. This method involves sorting urothelial cells by the use of MoAb T16 and propidium iodide staining. Thus, the final result of ploidy reading is a direct measurement of epithelial cells (non-tumoral and tumoral), after exclusion of other cellular elements [20]. Recently, Fradet and coinvestigators performed multiparameter FCM by simultaneously measuring DNA content and the expression of a series of MoAbs to cell surface antigens (T16, Om5, T43, and T138 specificities) [2 11. They evaluated 68 patients affected with bladder tumors and showed that T138 expression was adding new information to ploidy. With a mean follow-up of 2 years, there were no cancer-related deaths in the T138 negative and near diploid group died of their disease. Furthermore, the aneuploid

group could also be subdivided into two classes: only 20% of the T138 negative patients died of their disease, while 63% of the TI38 positive patients succumb to bladder cancer. Fradet and collaborators more recently reported the generation and characterization of 19A211 MoAb. This antibody was obtained by using another interesting strategy also aimed at reducing the crossreactivity of the produced reagent to normal urothelial cells. Neonatal mice were injected with samples of normal urothelium prior to their inoculation with bladder tumor cells [22]. The epitope detected by 19A211 MoAb was found to be expressed by 63% of the superficial lesions tested versus only 20% of the invasive tumors analyzed. This antigenic determinant was also present in a bladder and several cervical condylomata, leading to the hypothesis that it might recognize a noneucaryotic antigen. We have further characterized the patterns of expression of MoAb M344 and 19A211 in human normal tissues and tumors [23]. This study was conducted using immuno-histochemical staining of frozen and deparaffinized sections. The antigens are stable and well preserved on deparaffinized tissue sections. None of the 14 normal urothelium and 22 cases of cystitis analyzed had detectable levels of M344 or 19A211 antigens. Neoexpression of these antigens was observed in approximately 80% of low-grade papillary superficial tumors and carcinoma in situ lesions. However, in muscle infiltrating transitional cell carcinomas, antigen expression decreased to 18% of the tumor analyzed and was found in none of five metastatic bladder cancers studied [23]. We postulated that beyond their possible biologic significance, these antibodies may provide clinically useful probes for early detection and stratification of urinary bladder tumors.

POTENTIAL APPLICATIONS OF MONOCLONAL ANTIBODIES TO CELL SURFACE ANTIGENS OF BLADDER TUMORS The classification of bladder tumors based on histopathologic criteria has not been sufficient to segregate neoplasms into defined entities with similar biologic behavior. Recent advances in the hybridoma technology provided a number of monoclonal antibodies that will help address important issues in early detection, recurrence, and prognosis. The strategy is based on the recognition of tumor cell surface antigens that are qualitatively and quantitatively different from the tissue of origin. Urinary cytology has been an invaluable tool in early detection and patient monitoring. However, cytologic diagnosis of low-grade lesions is particularly difficult due to the small number of tumor cells shed

298 Dalbagni et al.

and their close resemblance to normal urothelial cells. Flow cytometry has been an important addition to the pathologist armamentarium improving the results of conventional urinary cytology. However, the presence of intercurrent infections or lithiasis curtailed its accuracy. Monoclonal antibodies recognizing antigens restricted to bladder tumor cells may become an important tool increasing the sensitivity of urinary cytology and flow cytometry. Immunocytologic examination of bladder washes allows the pathologist to detect lowgrade lesions in the midst of normal urothelial cells. Lin et al. [24] demonstrated that cytology could be improved by immunofluorescent staining of bladder wash specimens. The sensitivity was enhanced from a mere 37.5% to 87Y0including 18 of 19 grade 1 lesions. Moreover bivariate flow cytometry was performed with the panurothelial T16 MoAb, which bind exclusively to epithelial cells excluding the inflammatory cells. At initial presentation, 75% of bladder tumors are superficial and 70% of those successfully treated recur. Identification of that subset of patients that will present with recurrent lesions is critical for therapeutic choices. Currently there are no available reagents that recognize cell surface antigens, if any, differentially expressed by urothelial cells with a propensity for neoplastic transformation. Another application of MoAb in diagnosis is related to prognosis. Bladder tumors show a marked heterogeneity in their biologic behavior. Fifty percent of patients with invasive lesions die of metastatic disease 18 months after receiving treatment for presumed localized disease. This is the area where MoAb against differentiation antigens will have the greatest impact. Limited studies have addressed this important issue, T138 expression was found to be a more important prognostic variable than grade or ploidy [lo]. Multiparameter flow cytometry with MoAb against differentiation antigens added new information to ploidy, by showing that within the aneuploid population, T138 negative fared better than the T138 patients. Last but not least, MoAb will have an impact in tumor localization by the technique of radioimmunoscitigraphy. The potential applications of MoAb to cell surface antigens are not limited to diagnosis and prognosis but also to tumor therapy. Monoclonal antibodies alone or coupled to toxins will receive a great deal of attention in the near future. CONCLUSIONS Bladder tumors include a wide variety of like-appearing morphological lesions with different biologi-

cal and clinical behavior. Recent advances in tumor biology have identified markers that are good candidates for clinical applications in early tumor detections, as well as means for the stratification of patients with similar prognosis. The ultimate goal is to develop predictive assays that would segregate patients with high probability of failures versus patients who would be cured by localized modes of therapy. Monoclonal antibodies are particularly well suited for their use as prognostic and diagnostic tools based on their high specificity and sensitivity. A new subclassification of bladder tumors may be anticipated based on their immunohistogenesis. The value of this subchsification will be determined by correlation with clinical and conventional pathologic features, tumor progression, and the response to therapy. ACKNOWLEDGMENTS This work was supported in part by NCT grants number CA-47538 (C.C.-C.) and CA-47526 (Y.F.). REFERENCES 1. Kohler G , Milstein C : Continuous cultures of fused cells se-

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Cell Surface Markers 299

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monoclonal antibodies of an antigen shed by human bladder cancer cells. Cancer Res 49:6720-6726, 1989. Bretton PR, Myc A, Cordon-Cardo C, et al.: Initial evaluation of a new epithelial antigen (T16) for bivariate flow cytometry of bladder irrigation specimens. Cytometry 10:339-366, 1989. Fradet Y, Tardif M, Bourget L, et al.: Clinical cancer progression in urinary bladder tumors: Evaluation by multiparameter flow cytometry with monoclonal antibodies. Cancer Res 50: 432437, 1990. Fradet Y , LaRue H, Parent-Vaugeois C, et al.: Monoclonal antibody against a tumor-associated sialoglycoprotein of superficial papillary bladder tumors and uterine cervix condylomas. 46:990-997, 1990. Cordon-Cardo C, Wartinger DD, Melamed MR, et al.: Immunopathological analysis of human urinary bladder cancer: Characterization of two new antigens associated with lowgrade superficial bladder tumors. Am J Pathol 140:375-385, 1992. Lin CW, Young DA, Kirley SD, et al.: Detection of tumor cells in bladder washings by a monoclonal antibody to human bladder tumor associated antigen. J Urol 140:672-677, 1988.