Carurjogenesis vol.12 no.3 pp.441-447, 1991

Counrelattioim between morplhology aumdl blood groep-related aeMgeini expression! m paumcreaMc tamors induiced nm Syrian toamstieirs

Tsutomu Tomioka, Hideki Fujii, Hiroshi Egami, Yoshiyuki Takiyama and Parviz M.Pour1 The Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, NE 68198-6905, USA

pattern of induced benign and malignant pancreatic lesions with the expression of BGRA for better understanding of the biology of these lesions. Materials and methods

Expression of blood group-related antigens (BGRAs) A, B, H, Leb, Lex and Le* was examined immunohistochemicaUy in neoplastic and hyperplastic lesions induced by Nnitrosotais(2-oxopropyl)amine in Syrian hamsters to determine a relationship between histologkal and biological patterns of the lesion and expression of BGRAs. With the exception of papillary ductal cell hyperplasia, A-antigen discriminated between the benign and malignant lesions far better than the other antigens, which showed heterogeneity in the expression of benign and malignant tumors. However, production of Aantigen decreased with the degree of tumor dedifferentiatJon and was absent in anaplastic regions of tumors. Marked differences were found in the expression of A- and B-antigen in tubular complexes, one of the earliest induced changes, in relation to the type of cells populating these lesions. Overall, B-antigen seemed to be associated more with benign cells than malignant cells. On the other hand, Le*-antigen was expressed primarily in the invasive portion of some cancers and seemed to reflect active proliferative processes.

Tissues The tissues were derived from experiments conducted during the last 5 years. In all of these experiments, hamsters were treated s.c. once or weekly for 4—6 weeks with the potent pancreatic carcinogen, A'-nitrosobis(2-oxopropyl)amine, and were killed 46 weeks later. In these tissues no metastatic lesions were observed. Pancreatic tissues were fixed in Bouin's solution for 6 h, washed in 70% ethanol, dehydrated and embedded in paraffin. Initially, many specimens were screened and those representing certain histologically defined lesions (10) were selected. This type of tissue selection resulted in different numbers of each particular lesion. The paraffin blocks of these specimens were cut in serial sections and processed for immunohistochemistry as described below. From some lesions only one or a few sections could be obtained.

Introduction Pancreatic tumors have been fairly well studied histopathologically. However, because of the silent clinical course and its late and fatal manifestation, the natural history of the neoplasms is not well understood. Although anecdotal observations indicate that malignant lesions develop de novo from ductal epithelium (1,2), others postulate a hyperplasia-neoplasia sequence (3). Pancreatic tumors induced in Syrian hamsters represent an experimental counterpart of human diseases morphologically and immunologically (4,5). As in humans, the induced cancers express certain antigens, such as blood group-related substances, TAG-72, 17-1A and CA125 (6). However, contrary to humans, none of these antigens are detectable in the normal hamster pancreatic cells, except for 17-1A and, thus, their expression seems to present a marker for malignancy. Among these antigens, expression of blood group-related antigen (BGRA*) was most consistent. They appeared at early stages of carcinogenesis (3) and were demonstrable in almost all cancerous tissues of various morphologic patterns (7). Expression of these antigens also in cultured pancreatic cancer cells (8) indicates that the production of these antigens is an inherent function of malignant cells. With the use of immunohistochemistry, malignant cells could be readily distinguished from the normal cells by their reactivity with the respective monoclonal antibodies. In the present study, we compared the histomorphological •Abbreviations: BGRA, blood group-related antigen; MoAb, monoclonal antibody. © Oxford University Press

Monoclonal antibodies (MoAbs) Commercially available MoAbs against human ABO blood group antigens, produced by the hybridoma technique (DAKO Corp., Santa Barbara, CA) and Lewis-related MoAbs (Le*, Leb, Lex and Le*), kindly provided by Dr Z.Steplewski, Wistar Institute, Philadelphia, PA, were used (9). Immunoperoxidase procedure Immunoperoxidase staining was performed using a Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA). The concentration of each MoAb was 40 ftg/ml. For negative control slides, the following procedures were used: (i) sections were processed without the primary antibodies; (ii) the same concentration of P3x63Ag8 myeloma cell culture supernatant (IgM), kindly provided by Dr R.S.Metzger, Durham University, NC, and of MoAb MOPC-21 of Ig G, type (Litton Bionetics, Charleston, SQ was used instead of the primary antibodies. Method of histopathology Diagnostic criteria for induced pancreatic lesions were as described (10). Histological typing, based on the most predominant pattern of the tumor, was performed on H&E-stained slides and was compared with the pattern of the antigen expression in each lesion on immunostained serial sections. Reactivity with each of the antibodies was determined on an arbitrary scoring system. In each tumor, the number of positively stained cells was estimated as follows: 0% ( - ) , up to 5% (+); between 5 and 30% (+ +); between 30 and 70% ( + + +); and between 70 and 100% (+ + + +). The pattern of cellular staining was categorized as glycocalyx (luminal), diffuse (granular) cytoplasmic and Golgi pattern.

Results Expression of antigens in the normal hamster pancreases Control slides showed no staining with MoAbs UPC-10, MOPC-21 and culture supernatant of P3X63Ag8. There was no reactivity of any of the MoAbs in the normal pancreas. Types of induced lesions and expression of antigens (Tables I and II) Collectively, 68 hyperplastic and 105 neoplastic ductal/ductular lesions (7 benign and 103 malignant) were evaluated. Hyperplastic lesions (Table I) All papillary hyperplastic ductal epithelia were reactive with MoAbs A and B (in diffuse and glycocalyx pattern), fewer with anti-H (diffuse and glycocalyx), anti-Leb and anti-Le^ (mostly in diffuse granular patterns). Cytologically, no differences were found between the immunostained and unstained cells and the 441

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'To whom reprint requests should be sent

T.Tomloka et aJ.

reactivity was generally of mosaic pattern. No reactivity was seen with anti-Lex. In pseudo-ductular (tubular) complexes, different reactivity of the MoAbs was observed depending on the type of cells populating the tubules. The flat-cell type showed a greater reac-

tivity with anti-B than with anti-A. There were no reactivities with the remaining MoAbs. Also, the number of stained cells were less with anti-A and anti-H than with anti-B. Cytologically, there were no differences between the stained and unstained cells in the given lesion.

Table I. Immunoreactivity of the monoclonal antibodies against human blood group-related antigens with hyperplastic pancreatic lesions induced in Synan hamsters Lesions

A Papillary hyperplasia

(b) Cuboidal cell type

(c) Cylindrical cell type

(d) Eosinophilic cell type

(e) Clear cell type

Antigens Leb


2 8 1 4 0

7 6 2 0 0

15 0 0 0 0

7 5 1 2 0

5 2 2 2 0 3 8 7 9 0 0 0 1 2 3 2 1 1 0 0 1 0 0 0 0

11 1 1 0 0 11 5 5 3 0 4 1 1 0 0 4 0 0 0 0 — —

15 0 0 0 0 27 0 0 0 0 6 0 0 0 0 4 0 0 0 0 -

15 0 0 0 0

15 0 0 0 0 25 0 2 0 0 5 1 0 0 0 4 0 0 0 0 _






+ ++ +++ ++++

0 0 3 2 10

0 1 0 6 8

+ ++ +++ ++++ + ++ +++ ++++ + ++ +++ ++++ + ++ +++ ++++ + ++ +++ ++++

14 1 0 0 0 23 2 1 1 0 0 1 1 1 3 4 0 0 0 0 1 0 0 0 0 26768

No. of positive cases/ total no. examined


23 1 3 0 0 0 0 0 0 0 4 0 0 0 0 — —



Table U. Immunoreactivity of the monoclonal antibodies against human blood group-related antigens with the benign and malignant pancreatic tumors induced in Syrian hamsters Lesions


Antigens A




Le 1

1 0 0 1 0

1 0 1 0 0

2 0 0 0 0





Adenomas A Mucinous cystadenoma

B Ductular-insular adenoma

No. of positive cases/ total no. examined


0 0 1 1 0

1 0 0 2 0



1 0 1 0 0 1/3

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B Tubular complexes (a) Flat cell type


Pancreatic cancer antigenidty

Table II. (continued) Lesions








0 0 4 2 7 0 1 3 8 20 0 0 0 0 1 0 0 3 2 1 0 0 0 2 5

1 1 2 8 1 0 1 6 11 9 0 0 1 0 0 0 1 3 2 0 0 0 1 3 3

0 2 3 4 0 1 5 6 7 0 0 0 1 0 0 1 1 1 1 0 1 2 4 0 0

2 1 5 0 0 2 4 3 4 0 0 0 1 0 0 2 2 1 0 0 1 1 2 2 0

4 0 0 0 0 5 1 0 0 0

4 0 0 0 0 6 0 0 0 0

B Papillary carcinoma

0 0 0 0 10

0 0 0 5 5

1 2 2 5 0

4 2 1 1 0

4 1 2 0 0

6 0 1 0 0

C Papillary cystic carcinoma

0 0 0 0 18

0 1 4 5 9

1 2 8 5 0

6 1 5 1 1

6 7 4 0 0

11 1 2 0 0

D Adeno-squamous carcinoma

0 0 1 0 0

E Giant cell carcinoma

0 0 3 0 0

1 0 1 0 0

0 0 1 0 0

F Ductular-insular carcinoma

0 0 0 2 1

0 0 0 3 0

0 1 2 0 0

Undifferentiated tumors

0 3 1 0 0

0 1 0 0 0



A Tubular adenocarcinoma (a) Cuboidal type

(b) Cylindrical type

(c) Micro-glandular type

(e) Mucinous adenocarcinoma

No. of positive cases/ total no. examined




2 0 1 0 0





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(d) Desmoplastic type

T.Tomioka et al.


Fig. 2. A tubular adenocarcinoma (left) and hyperplasia of tubular complexes (cuboidal type, right) in two consecutive sections stained with anti-A (a) or anti-B (b) antibodies. Tubular adenocarcinoma cells and the surrounding connective tissue show a strong reactivity with anti-A antibody. There is little reactivity of anti-A antibody with tubular complexes and no reactivity with acinar cells (a). With anti-B antibody, a strong reactivity is seen with tubular complexes but a weak reactivity with adenocarcinoma cells (b). ABC method, X65.

The same staining pattern was found in cuboidal-cell type of tubular complexes (Figures 1 and 2), except that more cells were reactive with MoAbs A, B and H than in the flat cell type and the staining of a few cells was also seen with MoAbs Le* and Le*. All cylindrical types of tubular complexes were positively stained (in glycocalyx pattern) with anti-A and anti-B with a score of mostly + + + + for both MoAbs. Only a few or none of these lesions were reactive with the remaining antibodies. In tubular complexes of eosinophilic-cell type, only B-antigen was expressed in some, whereas none of the clear cell type showed reactivity with any of the MoAbs. Neoplastic lesions (Table II) All induced adenomas and carcinomas were stained with MoAbs A, B and H in various proportions (Figures 2—4). In most of the lesions the staining score of anti-A was mostly + + + +, whereas with anti-B and anti-H comparatively fewer cells were stained. There were also differences in the cellular localization of the reactive product. Diffuse and glycocalyx patterns were


seen with anti-A and anti-B, and diffuse cytoplasmic pattern with anti-H (Figure 3). The staining pattern (diffuse or granular patterns) and scoring of the MoAbs against Lewis antigens varied considerably between tumor types (Table II). Anti-Lex was reactive with the invasive portion of carcinomas, the central portion being almost unstained (Figure 5). No such predilection areas for the staining was seen for other MoAbs. Co-expression of A, B and H antigens seemed to occur in many well-differentiated carcinomas but not in poorly differentiated or anaplastic types. The reactivity of anti-A was found to be useful in detecting small malignant lesions (Ca in situ or microcarcinomas) (Figure 6). In these microscopic lesions, all the cells of the foci were strongly stained with this MoAb (Figure 2). Discussion Studies on the natural history of pancreatic cancer have been hampered because of its silent clinical course. Although there

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Fig. 1. Two consecutive sections of a cuboidal cell type of tubular complexes processed with anti-A antibody (a) or anti-B antibody (b). There are only a few cells reactive with anti-A antibody, whereas many cells are stained with anti-B antibody. X65.

Pancreatic cancer antlgenkity

Fig. 3. Reactivity of MoAb-A (a), MoAb-B (b) and MoAb-H (c) in three consecutive sections of a papillary carcinoma. MoAb-A and MoAb-B show both diffuse and glycocalyx patterns, whereas the reactivity of MoAb-H is of a diffuse cytoplasmic pattern. ABC method, X65.




Fig. 4. An adenocarcinoma showing differentiated (glandular) and poorly differentiated areas. No reactivities are seen in the poorly differentiated areas with anti-A (a) or anti-B antibodies (b), whereas a strong reactivity is seen in the glandular portion of the tumor with anti-A and a weak reactivity with anti-B antibody. ABC method, x65.

is general agreement about the biological behavior of some tumors, their histogenesis and sequential development are not well understood. The malignant potential of most tumors is judged

by the clinical course rather than cytomorphological characteristics of the lesions. For example, mucinous cystadenoma, despite its benign appearance, has been considered 445

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T.Tomioka et al.


Fig. 6. An early cancer showing a strong reactivity with anti-A antibody primarily of glycocalyx pattern. ABC method, X65.

a potentially malignant tumor based on its recurrence and metastases (11 — 13). On the other hand, solid cystic tumors, despite their cellular heterogeneity and cytology, have little tendency for metastases, and there are no histologic criteria to distinguish the potentially malignant variety from the more conservative one. Difficulties in assessing the malignant nature of such lesions include the problem of thorough sampling and, in particular, the lack of markers for malignancy. An ideal marker precludes its absence in the benign lesions and in normal tissues but shows its presence in malignant lesions. Although such markers have not been identified for human pancreatic cancer, they seem to exist for the cancers induced in hamsters. In this model, the induced rumors are morphologically, biologically and immunologically similar to the human disease (1,2,6,7). Hamster's pancreatic cancer cells share the expression of some antigens with humans. These include BGRAs, CA 125, 17-1A, TAG-72 and DU-PAN-2 (6). However, contrary to humans, the normal hamster pancreas does not express these antigens and in the fetal hamster pancreas only Leb, Le^ and Lex were found (7). However, all of these antigens are expressed early during pancreatic carcinogenesis and their concentrations 446

The expression of A-antigen in all cylindrical cell types of complexes correlates with histological evidence of their malignant potential. The presence of A-antigen in all adenocarcinomas and in mucinous cystadenomas (a lesion with malignant potential also in humans) underlines the association between malignancy and A-antigen expression. The eosinophilic or clear cell type of tubular complexes, the assumed precursors of eosinophilic (oncocytic) and clear cell carcinomas (which were not seen in the present study due to their infrequent occurrence) respectively (5,10), lacked A-antigen. This finding can be explained by three possibilities: (i) not all induced cancers express BGRAs, (ii) the observed lesions are not the precursors of cancers and (iii) they represent the 'borderline' stages of cancers. A larger number of cases are required to examine these possibilities. Although A-antigen seems to discriminate between the benign and malignant lesions far better than the other antigens, the extent of A-antigen expression decreased or was absent in poorly differentiated and anaplastic areas of cancers. This finding suggests that A-antigen is a product of differentiated tumor cells and that the process of dedifferentiation is associated with loss of A-antigen. Noteworthy is the pattern of Le* expression. This antigen was lacking in benign lesions and in the bulk of carcinomas, but was present in the invasive (most peripheral) portion of some cancers. This antigen appears during the fetal development of the pancreas and disappears in the adult pancreas. Therefore, as in humans (15-17), it presents an oncodevelopmental antigen in the hamster. Its presence at the invasive parts of some cancers may be a reflection of active cell proliferation mimicking embryonic development. However, its lack in some other invasive cancers indicates that this antigen cannot be considered a marker for

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Fig. 5. A cystic papillary carcinoma showing reactivity of cancer cells with MoAb-Lex in the invasive (peripheral) portion of the cancer. The cells in the central bulk of the tumor did not show any reactivities with this antibody. ABC method, x26.

increase with the advancing stage of tumor development (3). Therefore, these antigens could distinguish between benign and potentially malignant cells. The results of this study show that the BGRAs are expressed in various incidences and degrees in hyperplastic lesions and in benign and malignant tumors. Among these antigens, A-antigen seems to discriminate better between the benign and malignant lesions than the other antigens, papillary ductal cell hyperplasia being an exception. Whether or not the expression of A-antigen by papillary hyperplasia reflects the malignant potential of these lesions is not yet clear. A-antigen was lacking or was found only occasionally in tubular complexes of flat and cuboidal cell types. Expression of A-antigen in a few cells of some flat cell type of complexes correlates with occasional malignant changes seen in these lesions (5). Accordingly, the chance of the malignant changes in these lesions is < 1% (0.7%). Moreover, if the anti-A reactivity of the cells in these lesions represents the initiation of malignancy, we can assume that the process is multifocal, because stained cells were found in different regions of a given lesion. The cuboidal cell type of tubular complexes, which all assumed to have a greater potential for malignancy than the flat cell types (2), were mostly devoid of A-antigen. This finding indicates that not all cuboidal cell types of complexes have malignant potential, the incidence of which appears to be

Correlation between morphology and blood group-related antigen expression in pancreatic tumors induced in Syrian hamsters.

Expression of blood group-related antigens (BGRAs) A, B, H, Le(b), Le(x) and Le(y) was examined immunohistochemically in neoplastic and hyperplastic l...
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