Histologic, Immunofuorescence, and Ultrastructural ... - Europe PMC

Histologic, Immunofuorescence, and Ultrastructural Study of Malignant Islet-Cell Tumors of the Pancreas Induced in Hamsters by BK Human Papovavirus

From the Institutes of Pathologic Anatomy, University of Parma and University of Ferrara, and the Institute of Microbiology, University of Ferrara, Ferrara, Italy

CESARE BORDI, MD, OLIMPIA DE VITA, MD, CORRADO FERRARI, MD, GIUSEPPE ALTAVILLA, MD, ALFREDO CORALLINI, MD, and GIUSEPPE BARBANTI-BRODANO, MD

Histologic, immunofluorescence and ultrastructural studies were performed in 17 c^ases of pancreatic carcinomas induced by the BK virus in Syrian hamsters, a unique model of experimentally induced malignant islet cell tumors. The tumors were composed of small, poorly differentiated cells mostly arranged in a trabecular structure. By immunofluorescence all four islet cell types were found in the tumors, though with different frequency. Insulin cells were present in 16 cases, glucagon cells in 11, somatostatin cells in 7, PP cells in 6. Thirteen tumors contained more than one cell type. Insulin cells were the most frequent cell type in 13 cases, and glucagon cells predominated in 1 case. Insulin-containing cells usually occupied a central position within tumor-cell aggregates,

while the other cell types were mostly located in a peripheral position, a distribution reminiscent of that seen in normal islets. Gastrin and calcitonin immunoreactivities were not observed. Immunoreactive cells were more abundant in tumors with trabecular structure. Argyrophil cells revealed by the Grimelius method often exceeded the cumulative number of immunoreactive cells in the same tumor, which suggests that there were additional cell types. Multiple cell types were also found in liver metastases. Ultrastructurally most neoplastic cells were poorly granulated. The occurrence of many damaged cells suggests hormone leakage, which may account, at least in part, for the deregulated hormone release from the tumors. (Am J Pathol 1985, 118:256-265)

EXPERIMENTALLY induced tumors of the pancreatic islets have aroused particular interest mainly in their use as a source of islet tissue for biochemical and functional investigation. The experimental procedures mostly employed for the tumor induction were X-ray exposure` and administration of streptozotocin combined with nicotinamide5-8 or with the nicotinamide isomer picolinamide.9 Moreover, induction of islet-cell tumors was also reported after treatment with streptozotocin alone,9 with several pyrrolizidine alkaloids,10°11 and with monocrotaline."2 Tumor-cell transplantation was successfully attained with either the X-ray'3 14 or the streptozotocin-nicotinamide8-induced tumors, as it was with a spontaneous islet-cell tumor of the Syrian hamster.'5 These experiments yielded permanent cell lines maintaining the morphologic and functional characteristics of the parent tumor cells.8' 3 14 With the exception of the transplantable spontaneous tumor of the Syrian hamster, which has a very low

content of insulin"6 and an uncertain cytologic characterization, 15.17.18 all the aforementioned, experimentally induced insuloinas are benign tumors composed of welldifferentiated cells without any clinical and/or pathologic evidence of malignancy. In contrast, clearly malignant, metastasizing islet-cell tumors have been recently obtained in the hamster by intravenous inoculation of BK virus (BKV), a human papovavirus. 19'20 This unique experimental model of malignant islet-cell tumors may be useful in evaluating the efficacy of chemotherapeutic or other antitumor agents, because the relative rarSupported by grants from the Italian Ministry of Public Education and the Italian National Research Council (CNR), Progetto Finalizzato "Oncologia." Accepted for publication September 13, 1984. Address reprint requests to Cesare Bordi, MD, UniversitA di Parma, Istituto di Anatomia Patologica, 43100 Parma, Italy.

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ity of the corresponding human forms severely limits adequate clinical investigation. The aim of the present work, therefore, is to characterize the morphofunctional features of these tumors by means of a parallel histologic, immunohistochemical, and ultrastructural study.

Materials and Methods Tumor Induction and Pathologic Examination Tumors were induced by intravenous inoculation of purified BKV in either normal or immunosuppressed hamsters.'9'2122 BKV (prototype Gardner strain) was concentrated and purified by sedimentation onto a cushion of a potassium bromide solution followed by two cycles of equilibrium density gradient centrifugation in cesium chloride. The purified virus preparation had a hemagglutinating titer of 105' hemagglutinating units/ml and an infectious titer of 109 4 fluorescent antibody focus-forming units/ml. Syrian golden hamsters, 18-22 days old, were inoculated intravenously with 0.2 ml purified BKV in Tris buffer (0.02 M Tris-HCl, pH 7.4). Virus neutralization was performed by mixing equal amounts of purified BKV and specific guinea pig serum (neutralizing titer, 1:2560) diluted 1:2 in Tris buffer. The virus-serum mixture was incubated for 1 hour at room temperature and then immediately injected intravenously (0.4 ml/ animal). Additional series of experiments were performed in animals immunosuppressed with either rabbit antihamster lymphocyte serum, methyl-prednisolone acetate (MPA), or total body radiation with 60Co y-rays followed by MPA. Details on the immunosuppressive procedures are described in earlier reports.2,22 Animals appearing ill or found dead on daily inspections were subjected to necroscopic examination. Surviving animals, with or without tumors, were killed and underwent necropsy 12 months after inoculation. In this series of experiments, pancreatic tumors showed a frequency of 12.337o in normal hamster and of 15.31% in immunosuppressed hamsters. They were found between 5 and 11 months after BKV inoculation. Symptoms observed in hamsters with pancreatic islet-cell tumors were somnolence, weakness, contracture of the legs, intense diarrhea, loss of weight, and palpable abdominal masses. However, three tumors were observed by chance in the absence of signs. On macroscopic examination, pancreatic tumors measured from 3-5 to 10-30 mm in diameter, and appeared as uninodular or multinodular, very soft masses with frequent hemorrhagic areas. Liver metastases were present in 5007o of animals and were often multinodular.

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Light Microscopy and Immunofluorescence Material was available from the primary pancreatic tumors in 17 animals and from liver metastases in 3 animals. Tumor tissues were fixed in 1007o buffered formalin or in Bouin's fluid and embedded in paraffin. Sections were stained with hematoxylin and eosin (H&E) and the Grimelius silver method23 as well as with the indirect immunofluorescence technique using the following antisera: 1) guinea pig anti-insulin (dilution, 1:200) (produced by Dr. P. H. Wright, Indianapolis, Indiana, and donated by Dr. L. Orci, Geneva, Switzerland); 2) rabbit anti-glucagon 02K (directed against the C-terminal region of the glucagon molecule) (1:50); 3) rabbit anti-glucagon 05Y (directed against the Nterminal region) (1:200) (both gifts of Dr. R. H. Unger, Dallas, Texas); 4) rabbit antisomatostatin (1:120) (KB18, commercially purchased from Milab, Malmoe, Sweden); 5) rabbit anti-bovine pancreatic polypeptide (PP) (1:200) (a gift from R. E. Chance, Indianapolis, Ind); 6) rabbit antigastrin (1:320) (B36, commercially purchased from Milab, Malmoe, Sweden); 7) rabbit anticalcitonin (1:200) (Dako A576, commercially purchased from Labometrics, Milan, Italy). After 2 hours' incubation at room temperature with the specific antisera, sections were subsequently exposed to fluorescein-labeled anti-guinea pig (Pasteur Institute, Paris, France) or anti-rabbit (Behring Institute, L'Aquila, Italy) y-globulin sera for 1 hour and examined in a Zeiss Photomicroscope III equipped with the fluorescence vertical illuminator, III RS. Controls for specificity of reactions were performed by incubating the consecutive, serial section with the same antiserum absorbed with an excess of the respective antigen. Antisera against islet hormones (1 to 5 of the above list) were found to react heavily with the respective cell type in normal, extratumoral islets of the hamster pancreas, with the exception of the antiglucagon antiserum 02K. This antiserum, in fact, showed only feeble reactivity for the hamster normal A cells, in sharp contrast to the intense binding of 05Y antiglucagon antiserum to the same cells. Antigastrin antiserum was effective in localizing the hormone in the duodenal mucosa of the hamster, as was anticalcitonin antiserum in cells of thyroid medullary carcinomas. In our material the antigenicity of the various hormones was equally preserved with both types of fixative employed (formalin and Bouin's fluid). Electron Microscopy Specimens for ultrastructural investigations were available from 8 pancreatic tumors. They were fixed in 2.5% glutaraldehyde in phosphate-buffered saline (PBS)

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Figure 1-Panoramic survey of a pancreatic tumor induced by inoculation of BK virus. Note the scarcity of stroma and the defined borders of the tumor. (H&E, x 100) Figure 2-The most common histologic structure of the tumors is illustrated. Trabeculas of varying thickness are associated with elongated sinusoids and dilated vascular spaces filled with blood cells. Tumor cells are poorly differentiated, with frequent mitoses. (H&E, x 195) Figure 3Tumor composed of spindle-shaped cells arranged in variously oriented bundles. This histologic pattern was associated with the lowest frequency of immunoreactive cells. (H&E, x 195) Figure 4-Tumor composed of noncohesive cells. These are pleomorphic, with multinucleated forms. Foci of necrosis are also evident. The immunofluorescence of this tumor is shown in Figure 10. (H&E, x 300)


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(0.2 M, pH 7.2) for 4 hours, postfixed in 1% OS04 in PBS for 4 hours, dehydrated, and embedded in Araldite. Ultrathin sections were stained with uranyl acetate and lead citrate and were examined in a Siemens Elmiskop I or in a Zeiss EM 109 electron microscope.

Results Light Microscopy The histologic arrangement of the tumors was characterized by irregular and anastomosing, thick trabeculas and solid cords supported by a delicate connective tissue stroma containing dilated blood vessels (Figure 1). In several tumors, the vessels expanded into large, bloodfilled cavities, giving an angiomatous appearance to neoplastic tissue. Tumor cells were poorly differentiated, being usually small, with scarce to moderate cytoplasm and hyperchromatic round or oval nuclei (Figure 2). Pronounced polymorphism, multinucleated cells, and mitoses were frequently encountered, especially in most undifferentiated areas, where necrotic and hemorrhagic foci were also common. Variations of this basic histologic pattern included arrangement of spindle-shaped cells into solid bundles (Figure 3, predominant in 2 cases) and loss of tumor-cell cohesiveness (Figure 4). The latter finding, which was associated with the highest degree of cellular atypia, was present either in discrete areas or, in 2 cases, in the whole tumor. Stromal fibrosis was rarely encountered. Although tumor borders were usually distinct and sometimes showed a thin fibrous capsule, infiltration of the capsule and/or the surrounding structures (acinar and fat tissue or, if present, duodenal wall) was seen in all 14 tumors in which the available material allowed proper evaluation. In most cases invasion of venous and/or lymphatic vessels was apparent. Peritumoral lymph nodes were rarely included in the specimens, but one of them showed metastatic tumor tissue. Metastases in the liver reproduced the same pattern of the primary tumors and were associated with neoplastic colonization of small veins in the portal tracts. Immunofluorescence The results of immunofluorescence investigations are summarized in Table 1. In all tumors but one, immunoreactive cells were found, 13 neoplasms containing more than one cell type. All the hormones elaborated in normal pancreatic islets (ie, insulin, glucagon, somatostatin, and PP) were detected in tumor tissue (Figures 5 to 8), though with different frequency and in various combinations. Insulin-containing cells occurred in 16 cases and were the only detectable cell type

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Table 1-Multiple Hormone Production by BK VirusInduced Islet-Cell Carcinomas of the Hamster as Revealed by Immunofluorescence Cell frequency (percentage of Number of tumors with the total tumor immunoreactive cells cell population) Cell type Insulin cells Glucagon cells

16/17 11/17

Somatostatin cells PP cells Gastrin cells Calcitonin cells

7/17

1-2

6/17 0/17 0/17

1 to 10 (usually 1-2)

1 to 50 (usually 20-30) 1 to 40 (usually less than 5)

in 3 tumors. When abundant, they occupied large central areas of the neoplastic cell clusters (Figure 5). The intensity of immunostaining varied from very faint to pronounced. Glucagon cells were the second most frequent cell type encountered in this study and, in a single case (Figure 9), the most abundant immunofluorescent cell type, with a frequency (40%0) more than twofold that of insulin cells in the same tumor. Glucagon cells were usually aggregated in small nests at the periphery of tumor-cell clusters (Figure 6). In keeping with the results obtained in A cells of normal hamster pancreatic islets, the antiserum directed toward the Nterminal region of the glucagon molecule stained a larger number of cells than the antiserum directed toward the C-terminal region. Somatostatin cells always appeared as single or coupled cells with a constant peripheral location in contact with fibrovascular septa (Figure 7). Their size was larger than that of other cell types. PP cells also were grouped in peripheral clusters (Figure 8). Although the possibility of multiple hormone production by single tumor cells could not be ruled out, in general, different hormones were located in different areas of the tumors. Gastrin- and calcitonincontaining cells were not found. Moreover, in all tumors at least 50'7o of cells did not react with any of the antisera tested. In general, spindle-cell tumors were the most unreactive, and those with a trabecular structure showed a relative abundance of immunofluorescent cells. Isolated hormone-containing cells were well represented also in tumors with a high degree of noncohesiveness (Figure 10). Argyrophil cells were demonstrated by the Grimelius method in virtually all tumors. Although in several cases large clusters of strongly stained cells were found (Figure 11), the intensity of the staining was most often faint and concentrated at the periphery of the cell cytoplasm or in cytoplasmic processes. The frequency of Grimelius-positive cells in tumors always exceeded that of immunofluorescent glucagon and PP cells, the cell

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Figure 5 -Immunofluorescence localization of insulin-containing cells. These are aggregated in large groups extending to the center of neoplastic cell (x 200) Figure 6 - Immunofluorescence localization of glucagon-containing cells. These are mostly distributed at the periphery of tumor cell (x 200) Figure 7-Occasional somatostatin-containing cells are depicted by immunofluorescence in a peripheral position. (x 200) Figure 8-Cluster of PP-containing cells lying in close contact with a stromal septum. (x 300)

masses. masses.

types detected by the Grimelius method in normal islets. This suggests occurrence of additional tumoral cell types besides those revealed immunohistochemically. Immunofluorescence analysis of liver metastases showed a heterogeneous pattern. In 1 case all four isletcell types were present, as they were in the primary tumor, although with contrasting variations in respective frequency. No immunofluorescent cells were found in

the other 2 cases, in which the primary tumor either contained few insulin and PP cells or was composed of cells totally unreactive. Electron Microscopy Tumor cells were characterized by marked heterogeneity in shape and size (Figure 12). Nuclear profiles were

Figure 9-Antiglucagon immunofluorescence in the single tumor showing predominance of glucagon-containing cells. (x 250) Figure 10-Numerous discrete insulin-containing cells revealed by immunofluorescence in the same tumor, composed of noncohesive cells, of Figure 4. (x 200)

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(Figure 12). Damaged cells showed large vacuoles with light floccular content, often in relationship with dilated sacs of round endoplasmic reticulum, swollen mitochondria, and fragmented plasma membranes. In addition, cellular debris, including typical secretory granules, were seen both in close association with collagen bundles and within large phagosomes of macrophages scattered among damaged tumor cells (Figure 14). Viruslike particles were never encountered. Findings suggesting ductal or acinar differentiation of tumor cells were never seen. Discussion

Figure 11-Numerous intensely stained argyrophil cells are irregularly distributed throughout the tumor tissue. Although the reaction was usually less intense than that illustrated in this figure, the frequency of argyrophil cells often exceeded that of immunoreactive cells in the same tumor, indicating additional hormone productions. (Grimelius, x 250)

irregular, with pronounced indentations. The cytoplasm showed a rather dense matrix with numerous free ribosomes and polyribosomes (Figures 13). In contrast, rough endoplasmic reticulum was only moderately represented. It consisted of short and long elements that usually did not aggregate to form the habitual stacks of parallel sacs. The Golgi complex was frequently prominent. Mitochondria were abundant and frequently elongated. Secretory granules were usually very scarce and either scattered over the entire cytoplasm or distributed along the cell plasma membrane. In addition, they were found to accumulate in cytoplasmic processes extending between adjacent cells. Because of the concomitant cell characterization by immunofluorescence, a systematic ultrastructural differentiation of secretory granules was not attempted in this study. Most of the granules were round, small (size range, 120-170 nm) and dense without features specific for any of the established islet-cell types. However, wellgranulated cells with typical beta granules (size range, 180-300 nm) were occasionally seen. Secretory granules were also found in tumor cells undergoing mitosis. A common finding in all tumors was the close association of well-preserved cells with a variable number of cells showing moderate or severe signs of damage

The present immunofluorescence investigation has demonstrated that islet-cell carcinomas induced by BKV may synthesize all four islet cell hormones. Most of these tumors may be classified as insulinomas because of the striking predominance of insulin-producing cells. As shown previously,'92' these tumors are usually associated with high circulating levels of insulin and low blood glucose. Moreover, some tumors may be regarded as glucagonomas. In agreement with a predominant glucagon-producing cell population, another tumor (not included in this study) was found to contain as much as 4270 ng/g of glucagon and only 17.2 ,U/mg of insulin.19 This tumor was associated with an overt diabetic state (blood glucose, 2.70 mg/ml). Tumors predominantly composed of glucagon-producing cells were not induced so far with other experimental procedures. On the other hand, the predominance of insulinproducing cells in the BKV-induced tumors is shared by other experimentally induced islet-cell tumors; in contrast, the paucity of somatostatin-producing cells is at variance with the relative abundance of this cell type in islet-cell tumors induced chemically7 8 24 and by radiation. 13.25 No calcitonin-containing tumor cells were found in the present investigation. However, two islet cell carcinomas in the same series of experiments, from which material was no longer available for immunofluorescence investigation, were found to contain large amounts of calcitonin.19 It is noteworthy that both these tumors were associated with osteosarcomas.19 The occurrence of multiple cell types which we observed within the same neoplasm is a well-recognized finding also in human islet-cell tumors.26 In this study we detected different cell types in liver metastases as well, confirming that they are of a neoplastic nature and not remnants of islets trapped by the tumors. Whether production of different hormones reflects a polyclonal or a monoclonal origin of tumor cells is an unsettled question. Cloning experiments with fetal human pancreatic tissue have revealed that each clone produced

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Figure 12-Ultrastructural survey of tumor cells. These usually show a low content of secretory granules (encircled areas). Damaged cells (asterisks) are often found in close association with well-preserved cells. (x 4300)

a single hormone.28 In contrast, cloning experiments with cell lines derived from the radiation-induced isletcell tumor of the rat"4 resulted in clones containing all the hormones of the parent tumor, though one of them

usually predominated.2529 These experiments suggested that acquisition of the phenotype for peptide hormone elaboration is the result of a terminal differentiation event of a totipotential stem cell.29 Although the distribution of different cell types within the tumor tissue was irregular, a tendency for cells producing hormones other than insulin to be localized at the periphery of the neoplastic structures was evident (Figures 7-9), in keeping with the distribution of the corresponding cell types within the normal islets.30 Of interest is that the preferential peripheral distribution of non-B-cells can also be reproduced in vitro when dissociated islet cells of monolayer cultures are induced to reorganize into islet-like organoids.31 The

factors responsible for the ordered distribution of the various islet-cell types are unknown but our results indicate that they are still active in the present poorly differentiated tumors. The ultrastructural appearance of tumor cells was consistent with that of the spontaneous transplantable hamster islet-cell tumor-1`832 but differed in several aspects from that of well-differentiated tumors induced either chemically7'8.33.34 or by radiation exposure. 13 In particular, these latter tumors showed an abundance of well-granulated cells with typical granule morphology and a large amount of rough endoplasmic reticulum with a lamellar disposition, features that were both lacking in BKV-induced tumors. The majority of secretory granules observed in this study corresponded to the small, nonspecific granules found to have wide distribution in human islet-cell tumors.27 35 Because these granules are known to be argyrophil2735,36 they ac-

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Figure 13-The ultrastructure of a relatively well granulated cell is shown. Besides peripherally distributed mature secretory granules, less dense, probably "maturing" granules (asterisks) are seen in close association with the Golgi stalks (Go). Rough endoplasmic reticulum (rer) is sparse and irregularly arranged. Small, dense particles scattered throughout the cytoplasmic matrix (encircled areas) are polyribosomes. (x 17,500) Figure 14-A macrophage having engulfed cellular debris with clearly recognizable secretory granules is adjacent to tumor cells. (x 7900)

counted at least in part for the Grimelius silver reaction displayed by tumor cells. Their functional significance is a matter of speculation (for a review see Bordi and Tardini35) but remains essentially unknown. For this reason electron microscopy is less effective than immunocytochemistry in the morphofunctional characterization of malignant, poorly differentiated isletcell tumors. The occurrence of necrotic or leaking cells has already been described in human37 or animal18 33 isletcell tumors, regardless of their benign or malignant nature. It has been suggested that hormone leakage from these altered cells may contribute to the autonomous, nonregulated hormone release from the tumors.37 The islet cell carcinomas induced by BKV, with their large number of damaged cells, may represent a favorable model to verify such a hypothesis. A limitation of the present series of experiments is the relatively low incidence of islet cell tumors produced by BKV inoculation. The rate of tumor induction was

in fact about one-fourth of that obtained with X-ray exposure' 4 and one-sixth to one-eighth that of streptozotocin-nicotinamide induced' 38 tumors. However, Uchida et a120 noted that the insulinoma-inducing ability varied in different BKV mutants, some mutants producing islet-cell tumors at a frequency up to 47% and 92%. These results prompted investigations on the possible role of BKV in the development of human islet cell tumors. Indeed, BKV DNA has been detected in a human insulin-secreting adenoma.39 Viral DNA was in a free state in tumor cells, and no evidence was found of viral sequences integrated into cellular DNA. BKV DNA was not detected in circulating leukocytes of the patient, which suggests that it might have been specifically associated with the tumor. By transfection of human embryonic fibroblasts with tumor DNA, a variant of BKV was rescued which transforms hamster kidney cells in vitro and is oncogenic for hamsters (unpublished observations). In view of these data and of

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the experimental evidence discussed in this paper, it is reasonable to consider the possibility of a relationship between BKV and human tumors of pancreatic islets.

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Acknowledgments The authors thank Dr. Roger H. Unger, Dallas, Texas, Dr. Ronald E. Chance, Indianapolis, Indiana, and Dr. Lelio Orci, Geneva, Switzerland for generous supply of antisera and Professor R. Starcich, Institute of Medical Pathology, University of Parma, for allowing use of his fluorescence microscope facilities. The skilled technical assistance of Ms. Elmina Ferri, Mr. Gino Brignoli, Mr. Marco Visconti, and Ms. Silvia Sartori is gratefully acknowledged.