Spongiotic Pericytoma: A Benign Neoplasm Deriving from the ...

American Journal of Pathology, Vol. 146, No. 4, April 1995 Copyright s American Society for Investigative Pathology

Spongiotic Pericytoma: A Benign Neoplasm Deriving from the Perisinusoidal (Ito) Cells in Rat Liver

Philipp Stroebel, Frank Mayer, Heide Zerban, and Peter Bannasch From the Abteilung fur Cytopathologie, Deutsches Krebsforscbungszentrum, Heidelberg, Germany

Spongiosis hepatis has been knownfor some time to develop frequently in livers of rats and fish treated with hepatocarcinogens and was considered to derive from the perisinusoidal (Ito) ceUs (PSC). Using rat liver treated with N-nitrosomorpholine at different dose levels, we studied the celular composition and origin as weU as the proliferation kinetics of spongiosis hepatis by immunohistochemical demonstration of desmin, vimentin, and a-smooth-muscle actin, and by autoradiographic determination of [3H]thymidine incorporation, respectively. The vast majority of the ceUs forming spongiosis hepatis were positive for desmin and vimentin but negative for ao-smooth-muscle actin, confirming the celular origin of spongiosis hepatis from PSC In addition, immunohistochemical demonstration of desmin and vimentin revealed that spongiosis hepatis is an integral part of larger lesions consisting offocal PSC aggregates. These aggregates show a significantly increased incorporation of [3H1-thymidine compared with PSC in the extrafocal tissue and in the liver tissue of untreated control animals. In stop experiments, this increased labeling index was maintained many months after withdrawal ofthe carcinogen, in line with the earlier observation of a progressive behavior of spongiosis hepatis. We conclude that PSC may give rise toproliferative lesions appearing as PSC aggregates associated with more or less pronounced spongiosis hepatis. The persistence, the proliferative activity, and the slow expansive growth of these lesions suggest a benign neoplastic behavior. We therefore propose to classify these lesions as spongioticpericytoma. Malignant tumors possibly originatingfrom spongioticperi-

cytoma should consequently by classified as pertisinusoidal (Ito) ceU sarcomas. (Am J Pathol 1995, 146:903-913)

Ever since Ito and Nemoto1 described perisinusoidal cells (PSC; synonyms: Ito cells, fat-storing cells, pericytes) in the human liver, this cell type has been characterized extensively in various species.2 Two main functions have been ascribed to PSC: a role in the lipid metabolism, especially the metabolism of vitamin A,3-5 and participation in the production of extracellular components of connective tissue, such as collagen types 1, 111, IV, V, and VI; fibronectin; laminin; and tenascin.6 10 Most of the studies on the behavior of PSC under pathological conditions focused on their role in fibrogenesis, during which a pronounced proliferation of this cell type is regularly observed.11,12 Although pericytomas have been described as a rare entity in humans,13'14 the pathogenesis of this tumor type is poorly understood. In experimental models of liver carcinogenesis, however, a lesion classified as spongiosis hepatis (SH) possibly progressing to pericytomas is often observed, which has been related to PSC and may thus help to understand neoplastic transformation of this cell type. SH is a pathomorphological entity that occurs frequently in the livers of carcinogen-treated rats15-17 and fish.18-21 SH is a multilocular cyst-like lesion of variable size lacking both epithelial and endothelial lining. SH may consist of only one or two pseudocysts, but frequently progresses to large lesions, which may replace wide areas of normal parenchyma. Sometimes SH is combined with preneoplastic foci of altered hepatocytes (FAH), hepatocellular adenomas, and hepatocellular carcinomas. 15 As a rule, the pseudocysts are filled with a finely flocculent material Accepted for publication January 13, 1995. Address reprint requests to Professor Dr. Peter Bannasch, Deutsches Krebsforschungszentrum, Abteilung fOr Cytopathologie, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.

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that has been shown to contain acid mucopolysaccharides. Ultrastructural and biochemical features indicated that PSC is the constituting cell type of SH. 15,18 In the livers of rats treated with nitrosamines, SH has been considered to represent a preneoplastic lesion that may rarely progress to a malignant tumor, previously classified as pericytoma.2223 Based on observations in fish exposed to diethylnitrosamine, Couch and Courtney18'20 proposed that SH may not only represent a preneoplastic but a benign neoplastic lesion that has the potential to undergo malignant transformation. To further clarify the possible neoplastic nature and the biological behavior of SH, we used an immunohistochemical approach to demonstrate PSC by antibodies to desmin, which is regarded as a reliable marker of this cell type in the liver of the rat.24-26 In addition, proliferation of pericytes inside and outside spongiotic lesions was evaluated by determination of [3H]-thymidine incorporation.

21, 30, 40, 50, 60, 70, and 80 under light ether anesthesia. The liver of each animal was excised, sliced, and fixed in Carnoy's fluid and embedded in paraffin. Two-p thick sections were cut on a Jung microtome and stained with hematoxylin and eosin (H&E), alcian blue, and the periodic acid-Schiff (PAS) reaction for demonstration of glycogen. From each liver lobe probes were snap frozen in isopentane precooled with liquid nitrogen at -140 C and stored at -80 C. For immunostaining, 6-p thick cryostat sections were cut on a Jung cryostat (Leica, Bensheim, Germany), air dried and fixed in acetone analytical grade at -20 C for 10 minutes. In addition, parallel sections were fixed in periodic acid and stained with toluidine blue. Animals from experiments 3 to 5 were treated with different dose levels of NNM as summarized in Table 1 (for detailed description of these experiments see refs. 27 to 30). From these experiments, only animals that displayed unequivocal SH in toluidine bluestained cryostat sections were selected.

[3H]-Thymidine Labeling

Materials and Methods Animal Model Livers from five different experimental groups treated for limited time periods (stop experiments) or continuously with various doses of N-nitrosomorpholine (NNM) were studied. The NNM was a generous gift of Professor Preussmann (Abteilung fOr Umweltcarcinogene, DKFZ Heidelberg, Germany). All animals were male Sprague-Dawley rats (purchased from Zentralinstitut fOr Versuchstierzucht, Hannover, Germany) with an initial body weight of -200 g, maintained under constant conditions and fed Altromin diet (Lage, Lippe, Germany) ad libitum. In experiment 1, 50 animals were treated continuously with 1 mg NNM/kg body wt/day, administered in the drinking water until 4 weeks before sacrifice. In experiment 2, 50 rats were given 3 mg NNM/kg body wt/day in the drinking water over a time period of 7 weeks and received tap water thereafter. 50 untreated animals served as controls. Five animals each from experiments 1 and 2, and five control animals, were killed at weeks 7, 14,

Animals from experiment 5 [30] were subcutaneously implanted with an osmotic minipump (Alzet, Palo Alto, CA, Model 2 ml 1) for 48 hours. The pumps contained 2 ml saline-diluted [3H]-thymidine with an activity of 3 mCi and a pumping rate of 10 pi [3H]-thymidine/hour. Thus, each animal was given a volume of 480 pi with a total activity of 0.72 mCi [3H]-thymidine. Livers were excised and further processed as described above.

Immunohistochemistry Desmin, a-smooth-muscle actin (SMA), and vimentin were demonstrated by an indirect immunoperoxidase method on cryostat sections. For desmin staining a monoclonal mouse anti-desmin antibody (Boehringer Mannheim, Germany, cat. no. 814 377) was used ac-

cording to the following procedure: (1) Inhibition of endogenous peroxidase with hydrogen peroxide 0.5% in absolute methanol for 30 minutes at room temperature (RT), (2) incubation with 3% bovine serum albumin/tris buffered saline (BSA/TBS) for 30

Table 1. Summary of SH-Bearing Animals Used from Experimental Groups 1 to 5

treatment

Duration of experiment (weeks)

No. of SHbearing animals used

Continuous 7 weeks Continuous Continuous 7 weeks

60-71 30-71 50 37 30-41

2 4 6 2 10

Dose of NNM

Duration of

Experiment

(mg/kg day)

1 2 3 4 5

1 3 6 12 12

Spongiotic Pericytoma

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minutes to minimize nonspecific binding of antibodies, (3) incubation with the anti-desmin antibody (1:8 in 1% BSA/TBS) for 1 hour at RT and (4) incubation with a peroxidase-conjugated goat anti-mouse antibody (1:60 in 1% BSA/TBS) for 1 hour at RT. Sections were stained using amino-ethyl-carbazol as chromogen, and counterstained with Mayer's haematoxylin for 5 seconds. Slides were mounted in Kaiser's glycerol gelatin (Herck, Darmstadt, Germany). a-SMA staining was performed by means of a monoclonal mouse-anti-a-SMA as primary antibody (Sigma, Deisenhofen, Germany, cat. no. A 2547), which was diluted 1:800 in 1% BSA/TBS, then following the same procedure as described above. For the demonstration of the cytoskeletal protein vimentin we used a monoclonal mouse anti-vimentin primary antibody (Progen, Heidelberg, Germany, cat. no. 61 013) at a dilution of 1:800 in 1% BSA/TBS. Slides were stained with 3,3-diaminobenzidine for 15 minutes, rinsed in tap water, dehydrated in graded alcohols, and mounted in Eukitt (Kindler, Freiburg, Germany). For all immunohistochemical reactions, controls were performed by replacing the primary antibody by 1%

BSA/TBS.

Autoradiography To identify PSC, desmin staining was performed as described above. Sections were dipped in 10 ml autoradiography emulsion NTB-2 (Nuclear track emulsion, Eastman Kodak Co., Rochester, NY), air dried, and stored in a dark chamber for 2 weeks. The emulsion was developed for 3 minutes (EDF Photochemicals (Kodak), and diluted 1:2 in tap water). Sections were counterstained with Mayer's hematoxylin. Only desmin-positive cells with a visible nucleus were counted. Nuclei were considered positive when at least five silver grains resulting from [3H]-thymidine incorporation were observed. The labeling index (LI) was calculated by dividing the number of labeled cells by the total number of desmin-positive cells in the respective lesion. The LI is given as number of labeled cells/100 cells ± SEM. For comparison, 50 randomly chosen nuclei of desmin-positive cells in the remaining tissue were counted. Additionally, 50 desmin-positive nuclei in randomly selected fields of vision in the livers of untreated control animals were

eyepiece. Cell density was determined inside and outside of SH and related proliferative lesions by counting PSC in a total of 26 lesions, and in the same number of randomly selected areas of liver tissue outside of these lesions. In addition, cell density of PSC in control animals was evaluated.

Statistical Analysis Differences in proliferation rates were tested for significance by Fisher's exact and x2 tests. Differences in cell density were tested for significance by the Wilcoxon test.

Results

Histological Findings Histological and cytological changes induced in the liver parenchyma, the intrahepatic biliary system, and the vascular system of rats by NNM at most of the dose levels used have been described in detail previously.23' 27-30 In addition to SH, particular attention has been given in these studies to the characterization of preneoplastic FAH, hepatocellular adenomas, hepatocellular carcinomas, and angiosarcomas. In the two experimental groups (1 and 2) which were not analyzed in detail earlier, similar types of FAH, hepatocellular adenomas, hepatocellular carcinomas, and angiosarcomas were found in H&E-stained paraffin sections. The incidence of SH and liver neoplasms detected in the paraffin sections is given in Table 2. In toluidine blue-stained frozen sections, which were used for comparison with the immunohistochemical preparations, SH appeared as a lesion composed of multiple pseudocysts of very variable size. In about 27% of SH, the holes of the spongiotic formations were filled with a finely flocculent material that showed metachromasia, a phenomenon indicative of acid mucopolysaccharid deposits (Figure 1). Of 150 SH diagnosed, 114 were located within morphologically normal tissue, and 36 were associated with FAH. Sometimes hepatocytes within or in the immediate vicinity of SH showed signs of fatty degeneration and occasionally underwent single-cell necrosis.

counted.

Immunohistochemical Findings Cell Density Cell density was evaluated on the basis of the number of PSC per mm2 tissue using a net micrometer disc

Immunohistochemical demonstration of desmin revealed that SH was integrated into the parenchyma without an obvious structural disturbance. These lesions were of variable size; the largest among them

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Table 2. Incidence of SH, Hepatocellular Adenomas (HCA), Hepatocellular Carcinomas (HCC) and Angiosarcomas (AS) at Different Time Points after Continuouis Oral Administration of 1 mg (1 cont.) or limited exposure to 3 mg/kg bwlday of NNM (3 stop)

NNM (mg/kg

bw/day) 1 1 1 1 3 3 3 3 3

cont. cont. cont. cont. stop stop stop stop stop

Week

Incidence of SH

Total number of SH occurrences

30 50 60 70 40 50 60 70 80

1/5 4/5 2/5 2/5 2/5 2/5 3/5 4/5 1/5

1 5 9 8 2 3 3 23 4

Incidence of epithelial and endothelial neoplasms

HCA 1/5 HCC 5/5, AS 2/5 HCA 1/5

HCC 1/5, AS 1/5 HCC 1/5

Figure 1. Spongiosis hepatis. The content of the spongiotic cavities displays pronounced metachromasia, thus indicating acid mucopolysaccharides. Toluidine blue. (X97.5)

extended over three to four lobuli. In comparison with PSC of the remaining parenchyma, these otherwise normal-shaped cells showed a marked increase in desmin-positivity (Figure 2a). At places, PSC encircled the hepatocytes with their processes, thus building a net similar to the typical structure of SH, but without any formation of spongiotic cavities (Figure 2b). The diagnostic criteria for this type of lesion were: (1) clearly demarcated aggregation of PSC, (2) enhanced desmin-positivity compared with PSC of the surrounding tissue, and (3) no spongiotic cavities.

Most of these lesions were negative for a-SMA, thus showing a phenotype that was previously described to be specific for perisinusoidal cells.24,31 Furthermore, we diagnosed a total of 150 SH, ranging from very small lesions, which consisted of only one or two single cavities (Figure 3), to large multiloculated formations that replaced several lobuli. In nearly all SH (97%), the majority of cells composing these lesions stained strongly positive for desmin. As a rule, SH were negative for a-SMA. Only in 7% of SH, a weak immunohistochemical reaction for a-SMA was


907


a

t: 'S

L.

*,*

WM

J.

Figure 2. Aggregates of PSC with increased desmin positivitv as demonstrated by monoclonal antibody to desmin at (a) lower (x 78) and (b) higher (x 195) magnifications.

observed at places. Small SH were usually embedded in larger aggregations of PSC, which often by far exceeded the size of SH (Figure 4, a and b). In contrast, extended SH were in most cases only surrounded by a narrow rim of PSC aggregates (Figure 4c). Sometimes, no additional PSC aggregates were seen in the vicinity of SH. With respect to the focal

increase in PSC, the enhanced desmin-positivity of these cells and the outer shape of the individual lesions, and aggregates of PSC with and without SH, were very similar. Although all lesions observed were positive for vimentin, immunohistochemical reaction for this intermediate filament protein did not spot the lesions as clearly as the reaction for desmin. In ad-

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Figure 3. Small spongiosis hepatis. Focal proliferation of PSC, which at places encircle hepatocytes with their processes, thus forming possible precursors forfurther spongiotic cavities. Monoclonal antibody to desmin. (X 195)

dition, there were seven distinct spongiotic lesions with a massive increase in cellular density, indicating a particularly enhanced proliferation (Figure 5).

39%). Because these data differed considerably from those of the remaining SH, they were not included in the statistical evaluation.

Autoradiographic Findings

Cellular Density

Average Lls of focal aggregates of PSC and SH were significantly higher than those of the surrounding liver tissue as well as those of the livers of untreated control animals. The mean of the LI was 3.3 1.9 in aggregates of PSC, 3.2 ± 0.8 in SH with surrounding PSC aggregates and 3.2 1.2 in SH without PSC aggregates, with the differences between the different types of lesions not being significant (P = 0.4). The overall LI of PSC aggregates with and without SH was 3.2, whereas that of extrafocal PSC was 0.5, resulting in a highly significant difference (P < 0.0001). Lls of extrafocal PSC and PSC in control animals were not significantly different from each other (P = 0.8). A summary of these autoradiographic data is given in Table 3. Of the seven SH with high density of PSC mentioned above, only two were available for autoradiography. The light-microscopic impression of pronounced cell proliferation in these lesions was substantiated by a high [3H]-thymidine LI (73%,

Cellular densities of PSC aggregates, SH, and transitional forms between these lesions are summarized in Table 4. They were significantly increased in comparison with PSC of the surrounding tissue (P = 0.0001) and of the liver tissue of untreated control animals (P = 0.0001). The different types of lesions showed no significant difference in cellular density (P = 0.3). Cellular density of PSC in the extrafocal tissue of treated animals was significantly higher than in untreated control animals (P = 0.0001).

Discussion The immunohistochemical demonstration of desmin in SH showed that the vast majority of these lesions are built up by desmin-positive cells. As desmin is generally considered to be an appropriate marker of perisinusoidal cells in rat liver24-26 these results strongly support the conception previously inferred


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Figure 4. Different developmental stages of focal proliferations of PSC and spongiosis hepatis. Monoclonal antibody to desmin. (a and b) Small spongiosis hepatis embedded in a large aggregate of PSC, (a) x 97.5 and (b) x 78. (c) Extended spongiosis hepatis exhibiting septae formed by PSC. The immediate vicinity of the spongiotic lesion does not display any pronounced PSC aggregate. (x 122)

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Figure 5. Spongiosis bepatis with a massive increase in cellular density of PSC with strong desmin-positivity. (X 122)

Table 3. Average f-Hi-7Thymidine Labeling Indices of PSC in Focal Aggregates of PSC, SH With and Without Surrounding PSC Aggregate, and PSC in Extrafocal Tissue and Tissue of Untreated Contral Animals (Number of Labeled Cells/i00 Cells (Mean + SEAl)

[3H]-Thymidine Types of lesions and tissues

PSC aggregates SH with PSC aggregates SH without PSC aggregates Extrafocal tissue Tissue of untreated control

labeling index (mean ± SEM) 3.3 3.2 3.2 0.4 0.5

± ± ± ± ±

1.9 0.8 1.2 0.2 0.3

animals Table 4. Cellular Density of PSC in PSC aggregates, in SH With and Without Surrounding PSC Aggregate, in Extrafocal Liver Tissue and in Untreated Control Animals (Numbers Are Given as Mean + SEM) Types of lesions and tissues

PSC aggregates SH with PSC aggregates SH without PSC aggregates Extrafocal liver tissue Tissue of control animals

Cellular density (cells/mm2)

94.2 122.9 91.4 22.9 11.4

±

+ ±

±

+

10.3 17.7 11.4 1.7 2.5

from light- and electron-microscopical studies that the cells constituting SH derive from PSC, which largely maintain their phenotype in these lesions, but usually lose the feature of fat storage.15 The appear-

ance of the few SH negative for desmin could be explained by the existence of desmin-negative populations of PSC, which have been described recently.32 The most interesting new result of our investigations is the detection of focal, approximately spherical aggregates of perisinusoidal cells with and without spatial relationship to SH. When stained for desmin, PSC in these lesions showed an increased cell density and desmin-positivity. The appearance of the focal aggregates of PSC suggested a proliferative nature of these lesions. This assumption was confirmed by our autoradiographic studies. In double labeling for desmin and [3H]-thymidine, PSC of these aggregates displayed a significantly higher LI than PSC of the surrounding tissue and of untreated control animals. Although some of the PSC aggregates did not show any pseudocysts typical of SH, and were thus only detected after immunohistochemical demonstration of desmin, most of the proliferative lesions contained spongiotic areas of variable size ranging from small single cavities to extended multilocular SH. Large SH frequently replaced almost the whole parenchyma within the proliferative lesion. It is evident from these findings that SH as detected by microscopic inspection of conventionally stained tissue sections (eg, H&E, toluidine blue, PAS reaction) does not reflect the whole dimension of the underlying cellular alterations but is rather an integral part of a more complex entity that as a whole can only be dem-


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onstrated by desmin staining. Nevertheless, SH retains its diagnostic value as a reliable indicator of a focal proliferation of PSC in conventional histological sections. The focal proliferations associated with SH are clearly discernible from proliferations of PSC occurring in the course of fibrogenesis. During fibrogenesis, PSC exhibit increased proliferation rates, 11,12,33,34 transform into myofibroblast-like cells and then regularly express a-SMA,31'34 whereas most of the lesions diagnosed as PSC aggregates were a-SMA-negative. Furthermore, PSC aggregates of the SH type appear to be of a spherical shape, whereas myofibroblasts in the course of fibrosis assemble in septae-like formations connecting the central veins11 or portal tracts. In addition, after administration of NNM in concentrations of 1 mg and 3 mg/kg body wt/day used in experimental groups 1 and 2, no fibrotic changes developed, not even at later time points. The weak expression of a-SMA in small subpopulations of advanced SH remains to be clarified but might indicate some fibrogenesis in these lesions. It has been described previously under various experimental conditions, including stop experiments with NNM, that both the number and the size of SH in rats increase with time.15'20 This was largely confirmed in the experiments reported in this study. The exceptional low incidence of SH in the animals studied 80 weeks after withdrawal of a 3 weeks' treatment with NNM at a dose level of 3 mg/kg body wt/day appears to be due to chance, because all types of preneoplastic and neoplastic liver lesions were relatively rare in these animals. Degenerative changes of the hepatocytes within and in the immediate vicinity of SH have been demonstrated by electron microscopical investigations.15 Desmin decoration of the focal aggregates of PSC often shows connections between the cytoplasmic processes of neighboring cells resulting in a tight network, which in many ways may compromise the enclosed hepatocytes. One important factor may be that acid mucopolysaccharides, which are probably produced by the PSC,35 start to be accumulated in early stages of SH. Thus, the accumulation of mucopolysaccharides may contribute to the degeneration of hepatocytes. Whereas this alteration was only detected in about 27% of SH by metachromasia in frozen sections (Figure 1), it has earlier been shown by specific stains (alcian blue, colloidal iron binding) in paraffin sections that the accumulation of mucopolysaccharides is typical of all small SH.15 Once pseudocysts have emerged by cell death, the cavities could enlarge by further accumu-

lation of mucopolysaccharides and confluence of adjacent pseudocysts. In addition to proliferation of PSC, enlargement of SH seems to be the result of a gradual replacement of parenchyma by spongiotic formations within the area initially demarcated by the focal aggregates of PSC. This expansion, however, does not generally cause any visible compression of the surrounding liver parenchyma. This also holds true for the rare SH demonstrating a massive increase in density of PSC. Although SH has been shown to occur rarely in old untreated control animals, a high incidence and number of SH has hitherto only been observed after administration of hepatocarcinogens in rats15-17'23 and fish.18-21 In the rat, some results suggested a progression from SH with high cellular density to malignant tumors classified as pericytomas. From these findings it has been concluded that SH should be regarded as a preneoplastic lesion rarely progressing to pericytomas.23 However, basing their view on the appearance of SH in fish, Couch and Courtney20 extended these observations and suggested that SH might represent a benign neoplastic lesion, possibly leading to pericytoma. Our immunohistochemical and autoradiographic findings of focal proliferating aggregates of perisinusoidal cells associated with SH (which in stop experiments show a progressive biological behavior even in the absence of the carcinogenic stimulus) strongly support this notion and argue in favor of the classification of these lesions as benign neoplasia rather than preneoplasia. We propose the term "spongiotic pericytoma" for this benign neoplasm. Malignant mesenchymal tumors that may derive from these lesions should consequently be called "perisinusoidal (Ito) cell sarcoma."

Acknowledgments The authors would like to thank Erika Filsinger, Dagmar Bock, and Ulrike Beckhaus for their excellent technical assistance; and Dr. Annette Kopp-Scheider for her kind and valuable statistical advice.

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