T-Antigen

A Prognostic Indicator of High Recurrence Index in Transitional Carcinoma of the Bladder

EDUARDO BLASCO, PHD,’*t JULIO TORRADO, MD,$ LUIS BELLOSO. MD,’ FERNANDO AROCENA, P H D , ~ ARlEL GUTIERREZ-HOYOS, PHD,$ AND EMlLlO CUADRADO, PHD’

Forty biopsies from 36 patients with bladder tumors were tested for T-antigen (TAg) expression on tumor cells on sections untreated or treated with neuraminidase; a 37.5% of tumors showed abnormal expression of TAg either as an aberrant expression, or absence of this antigen after removing sialic acid. These changes were not well correlated with histologic signs of anaplasia or infiltration, nor with other biologic properties of tumor cells such as the expression of blood group antigens (ABH). However, a practical utility of TAg in the study of bladder tumors, is suggested by the analysis of those biopsies with low-grade low-stage tumors, on which the abnormal expression of TAg was more discriminatory than the ABH changes in defining those patients suffering tumors with a particular aggressiveness. Circulating antibody titer was also investigated in 20 patients but all of them displayed titers in the normal range, with independence of the results observed in their corresponding bladder biopsies.

Cancer 61 :1091-1095, 1988.

ANY STUDIES SUGGEST the utility of blood group M related tissue antigens as differentiation markers which exhibit both qualitative and quantitative changes when normal cells undergo neoplasia. Since David- son’s pioneer work, numerous studies have confirmed the utility of blood group antigens associated with other clinical and laboratory blood group antigens associated with other clinical and laboratory findings, as prognostic indicators in the management of patients with bladder tumors.”’ Along with these changes in ABH antigens, other alterations may occur in the biosynthesis of related molecules such as T-antigen (TAg)”-” or Lewis deter- m i n a n t ~ ’ ~ - ’ ~ in malignant cells, which could also be helpful in diagnosis and prognosis of transitional carci- noma of the bladder (TCC).

A study was made of the expression of TAg in 40 biopsies from 36 patients with bladder tumors, and titers of anti-T agglutinin in their sera.

From the Servicios de *Inmunologia, SAnatomia Patol6gica. and 5Urolea Hospital Ntra Sra de Aranzazu, San Sebastiin, Unidad Do- cente de Medicina de San Sebastiin, San Sebastihn, Universidad del Pais Vaxo, Pais Vasco, Spain.

Supported by a grant from Depto Economia Diputacibn Foral de Guipuzcoa.

t E. Blasco is a recipient of a fellowship from F.I.S. Seguridad Social. The authors thank Mrs. C. Berasarte and Mrs. P. Prieto for excellent

A d d m for reprints: Eduardo Cuadrado, PhD, Secci6n de Inmuno-

Accepted for publication September 13, 1987.

technical assistance.

l e a , Hospital Ntra Sra de Aranzazu, San Sebastihn, Spain.

Material and Methods

Biopsy sections from patients with transitional carci- noma of the bladder were taken from paraffin embed- ded specimens stored in the Pathology Unit of Ntra Sra de Aranzazu Hospital since I98 1. Histologically normal urothelial sections from each particular patient were also included. Pathological diagnosis was established (J.T.): tumors were staged according to the classification described by Jewett and Strong.‘’ The grading was done using the Armed Forces Institute of Pathology grading system.I6 The codified sections were investigated with- out previous knowledge of clinical course or pathologic records of corresponding patients who were seen at our Hospital between 1979 and 1987. The cellular expres- sion of TAg was tested in 40 biopsies from 36 patients with bladder tumors, by immunofluorescence on sec- tions treated with or without neuraminidase, using ei- ther the monoclonal antibody A 584 (DAKO) or FITC Peanut Agglutinin (PNA) (Sigma, lot N738 1) as specific reagents for testing this antigen. Dewaxed sections were incubated for 1 hour at room temperature with appro- priated dilutions of these reagents, either directly, or after treatment with 0. I U of neuraminidase (Sigma, lot N2876) as described by Lehman et After washing with PBS, sections treated with the monoclonal were incubated with a fluoresceinated rabbit anti-mouse im- munoglobulin (Ig) (F232 DAKO) for 45 minutes and washed again. Sections were mounted with oxidized p- Phenylenediamine in glycerol.” which results in nuclear

1091

1092 CANCER March 15 1988 Vol. 61

TABLE I . Histologic Features of Bladder Tumors*

Stage

Grade A B C Total

I 18 (45) 0 0 18 (45) I I 7 (17.5) 4 (10) 0 11 (27.5)

111 2 ( 5 ) 7 (17.5) I (2 .5) 10 (25) 1v 0 I(2.5) 0 1 (2.5)

Total 27 (67.5) 12 (30) l ( 2 . 5 ) 40 ( 100)

* Biopsy sections were classified according their histologic stage and grade as is described in methods. Results are expressed as absolute number and percent (%).

staining with better visualization of the cellular mor- phology. Antibody titers against TAg were analyzed in patient's sera using neuraminidase treated human 0 erythrocytes (N'RBC) in a hemagglutination test, as de- scribed by Springer el al." The presence of blood group antigens ABH was investigated by a immunoperoxidase technique using monoclonal antibodies and Ulex Euro- peaus 1 lectin as described by Cuadrado ef a1.l0

Results

The pathologic grade and stage of tumors are pre- sented in Table 1. Sixty-six percent were classified as noninvasive tumors, and 66% of these showed low-grade anaplasia. Twenty-three biopsies were taken from pa- tients of type A, 14 of 0, l of B and 2 of AB blood groups. In all cases, the results for TAg were similar when tested by reaction with PNA or with the mono- clonal A 584.

The expression of TAg in normal urothelium was reg- ularly observed as a strong staining of membrane and cytoplasm, independent of patient's blood group when the sections were desialylated by treating with neur- aminidase (T cryptic) (Fig. 1). By contrast, in tumors, abnormal staining patterns were revealed in as many as 37.5% either as an aberrant expression in sections un- treated with neuraminidase (Fig. 2) (27.5%), or totally lacking TAg even after removing the sialic acid (10%) (Fig. 1); Table 2. These changes were not correlated with

histologic signs of anaplasia or infiltration (Table 3); however a prognostic significance of changes in T anti- gen on tumor cells is suggested by the results observed in eight patients which exhibited high recurrence index, requiring multiple biopsies along their clinical course. As can be appreciated on Table 4,75% of them showed abnormal patterns of TAg expression despite the low- grade and the absence of infiltration along their follow- up period.

The results achieved for TAg were compared with those observed for ABH antigens on the same biopsies: once again, we did not find any correlation; biopsies which displayed normal reactivity for TAg showed total or partial lack of ABH antigens, conversely, biopsies with abnormal T reactivity displayed normal expression of ABH.

Finally we studied the serum levels of anti-T aggluti- nin in 20 patients. All showed normal titers (range, 1/16 to 1/18), as compared with control values, even in those which displayed aberrant expression of TAg in the tumor cells.

Discussion

Blood group tissue antigens and related molecules have evoked a considerable interest in recent years be- cause of several clinicopathological observations in human cancer: ( I ) total or partial deletion of ABH anti- gens when normal cells undergo neo~lasia,'-'~ (2) ab- normal expression of certain specificities limited to the tumor cells in the adult or to certain differentiation stages during the on tog en^,'^-*' (3) increase in the serum level of some of these antigens or their antibodies, as sialylated Lewis or TAB, reported in gastrointestinal tumors and other types of carcinoma^,'*^^^-^^ (4) aber- rant expression of incompatible antigens such as A-like molecules in tumors from patients of blood group 025-26 or substance P in a patient of pp phenotype.27

Transitional carcinoma of the bladder is probably the tumor in which the abnormalities in expression of blood group tissue antigens have proved most useful as prog- nostic markers in tumor pathology. There is substantial

TABLE 2. Expression of TAg in Tumors From Patients of Different ABO Blood Groups*

A B 0 AB Total

Reaction T Tcr T Tcr T Tcr T Tcr T Tcr

+ 0 12 0 I 0 10 0 0 0 23 (57.5) +/- 5 8 0 0 6 3 0 2 I I (27.5) 13 (32.5) - 18 3 1 0 8 I 2 0 29 (72.5) 4 (10)

* The results are presented in each group. as number of cases show- ing the following staining reactions: (+): diffuse positive staining. (t/-): focal positive staining. (-): negative reaction. Tcr: cryptic TAg

exposed by neuraminidase treatment of tissues section. Percent values are enclosed in brackets.

No. 6 T-ANTIGEN Blasco el al. 1093

FIC sion tion Note cent theli

G. 1. Absence of TAg expres- in tumor cells in a biopsy sec- treated with neuraminidase. : the strong reaction in adja- morphologically normal uro-

um. (X16).

evidence that loss of the ABH antigen system in the urinary bladder may be an early event in malignant transformation.28 The results show good correlation with the clinical course of patients as judged by the re- currence index or subsequent aggressiveness of

Although there is some controversy about this issue, we recommend its incorporation to clinical routine work in the management of patients with blad- der tumors."

Springer established the link between an abnormal carcinoma associated antigen and a human blood group MN substance29 and also found T specificity in tissue extracts from human breast, colon, and gastric carci- n ~ m a . ~ ' - ~ ~ In addition, the level of circulating anti-T was found depressed in certain forms of ~ a n c e r ' ~ - ~ ~ and delayed hypersensitivity reactions in response to skin tests were also reported in patients with breast carci- n ~ r n a . ~ ' These findings, indicated that a specific im-

FIG. 2. Aberrant expression of TAg in tumor cells on a bladder biopsy section without previous treatment with neuraminidase. ( X W .

1094 CANCER March 15 1988 Vol. 61

TABLE 3. Distribution of TAg Abnormalities in Tumors

Grade Stage

of Different Histologic Stage and Grades

I I1 I11 IV A B C

Aberrant expression of TAB 4 7 3 7 1

Focal loss ofTcr Ag 8 3 2 9 4 TotallackofTcrAg 2 I 1 3 1

mune response to unmasked TAg and anti-T antibodies might be useful in diagnosis prognosis and may be use- ful in the therapy of some types of carcinoma. Previous studies have indicated profound changes in the expres- sion of TAg on bladder tumors,''-'* but their clinico- pathologic significance is conflicting: Coon and asso- ciates,' I described two different changes in the expres- sion of TAg on tumor cells: aberrant expression, or lack of this antigen even after treatment with neuraminidase, which were correlated with malignant histologic fea- tures. By contrast, Lehman ef al l2 were unable to find any correlation between the expression or deletion of TAg and subsequent muscle wall invasion in the Stage 0 as described by Coon. They also reported four different staining patterns for TAg in Stage B tumors: membrane, granular intracytoplasmic, intracytoplasmic globular and glycocalyx which interpret cytostructural relocali- zation of TAg in malignant cells. Our results only offer new insight on the controversial issue of practical utility of TAg in bladder tumors in the identification of pa- tients with low-grade noninvasive tumors with a partic- ular aggressiveness along their clinical course: 75% of the biopsies from patients with these clinicopathological characteristics showed abnormal patterns of reactivity for TAg, versus a 37.5% of altered expression in all the tumors (Tables 2 and 4). When the results were com- pared with the observed in low-grade, low-staged tumors with low recurrence indexes (Table 4), the abnormalities

TABLE 4. Abnormal Expression of ABH and TAg (expressed in %) Observed in Tumors of Low Stage and Grade (A-I) Classified According the Aggressiveness Observed in Their Follow-Up*

A BH T Tumor

aggressiveness Type of deletion No. Type of deletion No.

High RI1. Focal 25 Aberrant expression 25 (N = 8) Total 50 Focal deletion Tcr 37.5

Total deletion Tcr 12.5 Low RI Focal 30 Focal deletion Tcr 20

( N = 10) Total 20

*All cases were followed for at least 3 years presenting multiple recurrences along the period of study without any evidence of changes in their histologic features.

t RI: recurrence index.

observed for TAg, were more discriminative than those observed for ABH. Seventy-five percent of the first group displayed alterations versus 20% in the other group.

All staining patterns described by Lehman were found in the tumors without clear cut correlation between pathologic features and ABH expressiveness. As previ- ously suggested," the aberrant expression of the TAg most likely represents an incomplete synthesis of the MN glycoproteins. Absence of the terminal sialic acid and the presence of some staining patterns apparently exclusive of tumor cells such as localized in the Golgi region, seems to indicate a cytostructural relocalization of this antigen in malignant urothelial cells.

The analysis of agglutinin titer of anti-T in the sera from some of these patients did not offer any correlation with the abnormal expression of the TAg in the corre- sponding biopsies; this finding disagrees with the results obtained in our laboratory in patients with gastrointesti- nal tumors,33 and those reported by Springer ef al. in mammary and digestive carcinoma^.^^

The identification of those patients that do not show any clear signs of histologic malignancy and still suffer multiple recurrences constitutes a profound problem in clinical urology. Our results point out significant changes in TAg expression in this particular type tumor, which associated with the alterations in the ABO system joined to other criteria as DNA content estimated by flow ~ y t o m e t r y , ~ ~ may be helpful in the prognosis and therapy of patients with tumors with a particular focal aggressiveness.

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