Virchows Archiv B Cell Pathol (1991) 60:373-380
9 Springer-Verlag 1991
Morphologic correlation between liver epithelium and mesenchyme allows insight into histogenesis of focal nodular hyperplasia (FNH) of the liver Hans-Peter Fischer and Georg Lankes Center of Pathology, University of Giel3en, W-6300 Giessen, Federal Republic of Germany Received March 6 / Accepted April 24, 1991
Summary. The microanatomic organization of focal nodular hyperplasia (FNH) of the liver was analyzed to obtain information about the histogenesis of this tumor-like lesion. All of the 11 examples of FNH studied showed subdivision into multiple pseudolobules, which were characterized by fibrovascular and ductular areas radiating from perilobular septa, and an expanding periphery of normal appearing hepatocytes. Immunohistochemical analysis showed continuous transitions from normal hepatocytes in the periphery of the pseudolobules, which expressed only the keratins 8 and 18, to small hepatocytes and ductular aggregates in the center of the pseudolobules, both of which also expressed the keratins 7 and 19. Ductular metaplasia of hepatocytes was always accompanied by sinusoidal endothelial cells stained by the endothelial markers BMA 120, M 616, and by an increase in collagenous fibers especially of type III. Further development of this fibrovascular and ductular transformation lead to subdivision of the involved pseudolobules. The pseudolobules had similar mean sizes, irrespective of their site in the periphery or the center of the FNHs, showing that proliferation, fibrovascular and ductular transformation and subdivision of these micronodules are a basic histogenetic phenomenon in FNH. The findings indicate that local changes in the interrelations between liver epithelial and mesenchymal cells influence substantially the abnormal but nevertheless regulated growth of liver parenchyma which gives rise to FNH.
Key words: Liver - Focal nodular hyperplasia - Histogenesis - Immunohistochemistry
Introduction Focal nodular hyperplasia of the liver (FNH) is a distinct benign tumor-like lesion which is morphologically charOffprint requests to." H.-P. Fischer, Institute of Pathology, Universi-
ty of Bonn, Sigmund Freud Strasse 23, W-5300 Bonn, Federal Republic of Germany
acterized by liver cell nodules and bile duct aggregates, surrounded by fibrous septa radiating from a central stellate scar (Knowles and Wolff 1976; Gibson and Sobin 1978; Goodman 1987; Craig et al. 1989). The characteristic morphological appearance of FNH permits its easy differentiation from all other liver neoplasms, especially from liver cell adenomas. Because of its complex morphologic composition, including different cell types which are normally found in liver tissue, e.g. liver epithelial cells, Kupffer cells (Phillips et al. 1973) and Ito cells (Callea et al. 1982), FNH is usually considered to be a reactive process or a hamartoma rather than a true neoplasm. The exact pathogenetic principles underlying FNH are as yet unclear (Goodman 1987; Kreitner et al. 1987). Insight into the histogenesis of epithelial liver tumors has grown in recent years through immunohistochemical studies of hepatocellular carcinoma and combined hepatocholangiocarcinoma with antibodies that react specifically with certain keratin polypeptides (Fischer et al. 1987; Van Eyken et al. 1988a; Johnson et al. 1988). Since hepatocytes and bile duct epithelia contain characteristic keratin subsets (Denk etal. 1982; Moll and Franke 1986), antibodies specifically directed against a certain keratin allow differentiation between hepatocytes and bile duct epithelia, and are helpful in detecting possible transformations between these two cell types in liver neoplasms (Fischer et al. 1988), non-neoplastic liver diseases (Thung 1990), and liver embryogenesis (van Eyken etal. 1988b; Shah and Gerber 1989; Ruebner etal. 1990). In a former study, we demonstrated ductular metaplasia of hepatocytes in one case of FNH by monoclonal keratinpeptide antibodies (Fischer et al. 1987). Recently this metaplastic process was analyzed in a detailed keratin immunohistochemical study (van Eyken et al. 1989). Now we have investigated the topology of normal and transforming hepatocytes as well as of bile duct aggregates in micronodules of FNH, and their morphologic correlation with mesenchymal elements, especially sinusoidal endothelial cells, parasinusoidal cells and collagen subtypes. Since the interaction between liver epithelium and fibrovascular stroma, as me-
diated by growth factors and their corresponding membrane receptors, seems to play an important role in the growth regulation and differentiation of non-neoplastic hyperregenerative liver tissue (Roijkind and Greenwel 1988; Fausto and Mead 1989), analysis of the microanatomic interrelation of these cell types should also be of help in understanding the histogenesis and nature of FNH.
Materials and methods Resection specimensor needle biopsies from 11 patients with FNH (six females aged 25-49 years and five males aged 50-71 years) were studied. All tumors were fixed in formaldehydeand stained with hematoxylineand eosin (H & E), periodic acid-schiff(PAS), Domagk's connective tissue stain and Gomori's reticulum stain. Tissue samples from two tumors were fresh-frozenin liquid nitrogen and stored at -70 ~ C. The immunohistochemicalreactions were detectedwith the alkalinephosphatase-anti-alkalinephosphatase (standard APAAP) method (Cordell et al. 1984). The following mouse monoclonal antibodies were used (Table 1): CK-7, an antibody specificallyreacting with keratin polypeptide 7 (Toelle etal. 1985) (Boehringer GmbH, Mannheim, FRG); Troma-l, specifically reacting with keratin polypeptide 8 (Toelle et al. 1985); CK-2, specific for keratin polypeptide 18 (Debus et al. 1982) (Boehringer, Mannheim,FRG); KA4, whichreacts strongly with keratin 19 and less strongly with keratins 14, 15, 16 (Nagle et al. 1985); b170, an antibody specificallyreacting with keratin polypeptide 19 (kindly provided by Dr. M. Osborn, Max Planck Institute of BiophysicalChemistry,G6ttingen, FRG); V9, an antibody specific for vimentin (Osborn et al. 1984); BMA 120, strongly reacting with a 200 kD-glycoprotein of endothelial cells (Alles and Bosslet 1986) (BehringWerke, Marburg, FRG); M 616, an antibody directed against factor VIII-related antigen (Dako Diagnostika GmbH, Hamburg, FRG), HHF-35, directed against actin isotypesof sceletal, cardiac and smoth musclecells (Tsukada et al. 1987a, b) (ENZO Biochem.,Inc., New York, NY). For collagen typing we used purified polyclonalgoat antibodies (Southern BiotechnologyAssociates, Inc.) reacting with collagen type I, III, and IV (Southern BiotechnologyAssociates,Inc). Planimetryof the FNHs was performedon tumor cross-sections (magnificationx 10) by opto-manual, semiautomaticquantitative image analysiswith a MOP-system(Kontron AG, Munich, FRG).
Macroscopic and histopathologic findings The maximum cross-sectional area of the FNH lesions ranged from 5410.0 mm 2 to 12.5 mm 2 for those of which the dimensions were recorded. The lesions showed expansive growth but were partly encapsulated in two examples only. One pedunculated FNH was connected by a broad stalk to subcapsular liver tissue, and another FNH presented as a bicentric lesion (Fig. 1 a). Characteristically, the FNHs contained a central scar with radiating septa which surrounded hyperplastic liver cell nodules. In four examples the scar contained large vessels with eccentric wall thickening and fibromyxoid myointimal proliferation. The nodules were subdivided homogeneously into coalescing pseudolobules or micronodules without portal triads or central veins resembling micronodular cirrhosis (Fig. 1 a-c). Fibrovascular stroma arising from perilobu-
lar septa radiated from one side into the center of the pseudolobules. This intralobular fibrovascular tissue was constantly interspersed with ductular aggregates which merged imperceptibly with expansive growing hepatocytes of normal appearance in the periphery of the pseudolobules (Fig. 1 b). Sometimes the fibroductular aggregates involved large non-expansive parts of pseudolobules, so that their demarcation from interlobular septa was blurred. These ductules were surrounded by a dense lymphocytic infiltration (Fig. 2a). The ductular epithelia showed no mitotic figures but regressive cellular changes were sometimes seen, especially nuclear and cellular shrinkage which gave the impression of a degenerative process leading to dissolution of the involved part of a pseudolobule. Although the degenerating ductular aggregates preserved for some time the shape of the former pseudolobule from which they developed, they were finally replaced by new stromal tissue in which only a few residual ducts were still incorporated (Fig. 2a).
Immunohistochemical findings The distinct morphologic interrelations between hepatocytes, ductular elements and fibrovascular stroma of FNH were demonstrable more precisely by immunohistochemical analysis (Table 1). Unfixed, fresh frozen material from two FNHs was investigated using all of the keratin polypeptide antibodies, whereas the formalinfixed material of all tumors was studied only with the antibodies KA4 and b170, both reacting with keratin 19. Both antibodies showed identical reactivity in unfixed and formalin-fixed tissue. The ductular and pseudoductular aggregates of the FNH and the bile ducts of the surrounding normal liver tissue reacted with the antibodies CK-7 (directed against keratin 7), Troma 1 (directed against keratin 8), CK-2 (directed against keratin 18), KA4 and bl70 (reacting with keratin19) (Fig. 1 c, 2a, b). Remarkably, some ductular sprouts of two FNHs were additionally decorated by the vimentin antibody V9 (Fig. 2a, inset). The hepatocytes of normal liver tissue and most FNH hepatocytes were stained only by the antibodies Troma 1 and CK-2. However, the hepatocytes merging with the ductular aggregates of the pseudolobules were additionally decorated by the antibodies CK-7, KA4 and b170 (Fig. 2b) which in normal liver tissue stain only bile duct epithelia, thus giving the impression of a continuous transformation of hepatocytes into ductular elements. The capillaries and the sinusoids accompanying these transforming liver epithelial cells consisted of endothelial cells which reacted strongly with the endothelial marker BMA 120 (Fig. 2c). However, the sinusoids between normal CK-7-, KA4-, and b170-negative hepatocytes of the pseudolobules as well as the sinusoids of normal liver parenchyma surrounding the FNHs showed only very few BMA 120-positive endothelial cells. The same reactivity pattern was found in lesser intensity with the monoclonal antibody M 616 directed against factor VIII-related antigen. Pericapillary and perisinu-
Fig. I a-c. Bicentric focal nodular hyperplasia with central scars (X) and radiating septa (a, • 1,5, Gomori's reticulum stain). Transverse section of a pseudolobule with expansive growth of normal hepatocytes on the right and a fibroductular nidus on the left (b, • 32, Gomori's reticulum stain). Cross-section of a pseudolobule
with a stellate fibrovascular center interspersed with, and surrounded, by keratin 19-positive ductular aggregates stained by the antibody b170 (e, x 20, alakaline phosphatase antialkaline phosphatase stain)
Table 1. Immunohistochemical antigen expression of different cell types of focal nodular hyperplasia Primary antibody
CK-7 Troma 1 CK-2 KA4
keratin 7 keratin 8 keratin 18 keratin 19 (14, 15, 16) keratin 19 vimentin endothelial cells factor VIII related AG muscular actin
b170 V9 B 120 M 616 HHF-35
Sinusoidal cells between normal HC
Sinusoidal cells between transform. HC
Perisinusoidal mesenchymal cells
a Unfixed, fresh frozen material from 2 FNH b Some ductular sprouts of 2 FNH reacted with the vimentin antibody V9 PERIPHERY EXPANSIVE GROWTH
HEPATOCYTES KP 8, 18
CENTRAL HIDUS DIFFEREHTIATON
T~HSFO~ING HEPA~YTES KP 7, 8, 18, 19
M~AP~STIC I DUC~LES KP 7, 8, 18, 19
, I .
EH~HELIAL CELLS B ~ 120, F VIII
PERISIHUSOIDAL C E L L S ~ Vi~ntln (muscular aetl
~ R T ~ TRACT I EQUIV~EHTS
Fig. 3. Scheme of the morphologic interrelation between liver epithelium and mesenchyme of micronodules in FNH
COL~GEN TYPE III#~ ; TYPE ~ ~ ; TYPE I
soidal cells of these transitional areas were stained by the vimentin antibody V9 and by the antibody H H F - 3 5 which is directed against muscle actin isotypes. B M A 120-positive capillaries and sinusoids between transforming hepatocytes and ductular aggregates were
a--d. A small pseudolobule completely transformed into keratin 19-positive ductular aggregates stained by the antibody b170. Degenerative changes and dense lymphocyte infiltration is seen (a, • 32). Some of the ductules also express vimentin marked by the antibody V9 (a, • 80, inset). Continuous transitions from normal keratin 19-negative hepatocytes to keratin 19-positive hepatocytes and ductular aggregates stained by the antibody b170 (b, • 200). The sinusoids of these transforming parenchymal areas are lined by endothelial cells reacting with the antibody BMA 120 in contrast to the sinusoidal cells between normal hepatocytes (e, x 200). The metaplastic ductules and transforming hepatocytes are surrounded by new collageneous fibers of type III marked by a polyclonal antibody (d, • 200) (All alkaline phosphatase antialkaline phosphatase stain) Fig. 2
surrounded by broadening fibrous bands which contained type III collagen (Fig. 2d) and to a lesser extent type IV collagen, whereas only small, inconstant amounts of type I collagen were present. Summarizing the light microscopic and immunohistochemical findings, it can be stated that the characteristic pseudolobules of F N H consist of a fibroductular " n i d u s " which arises from perilobular stromal tissue. This nidus is surrounded by expansively growing normal hepatocytes. In particular, the reticulum stain and the immunohistochemical demonstration of type III collagen illustrate the direction of the expansive growth which m a y result in a secondary modification of the microarchitecture by coalescence or compression of neighboring micronodules (Fig. 3).
Morphometric analysis The geometric mean area of the pseudolobules measured in seven F N H s ranged from 3.0 m m z to 7.26 m m 2 (medi-
378 Table 2. Comparison of the geometric mean areas of pseudolobules measured separately in the periphery and the center of different FNHs
E E v
Geometric mean areas of pseudolobules periphery mm 2
1 2 3 4 5 Total
center mm 2
378 116 216 88 241
5.5 8.4 4.9 6.0 3.4
6.1 5.3 5.1 5.6 3.3
an 5.59 mm2). The mean areas of the pseudolobules showed no correlation to the maximum cross-sectional area of the FNHs (p =0.05, Spearman rank correlation) (Fig. 4). In five FNHs the pseudolobules of the interior and the peripheral portions of the lesions were measured separately9 In accordance with the morphologic aspect of the cross-sections, the pseudolobules had similar mean areas (Table 2). Despite the small number of FNHs, the morphometric analysis demonstrates a relative homogeneity of the mean size of the pseudolobules irrespective of the size of the FNHs and their localization in the lesions.
MEAN AREA OF THE PSEUDOLOBULES k
Fig. 4. A correlation between the maximum cross-sectional area F of the measured FNHs ( n = 7 ) and the geometric mean area L of their pseudolobules cannot be prooved (p=0.05; Spearman rank correlation)
Focal nodular hyperplasia of the liver is generally accepted to be a distinct and benign morphological entity with predominance among females, especially in young women of reproductive age. Various pathogenetic mechanisms have been proposed for FNH. It has been thought to be a hamartoma (Benz and Baggenstoss 1953 ; Phillips et al. 1973; Rhodes et al. 1978), a response to a trauma (Benz and Baggenstoss 1953), a result of myointimal alterations and thrombosis (Nime et al. 1979; Lough etal. 1980) or vascular malformation (Whelan et al. 1973; Stocker and Ishak 1981; Wanless et al. 1985)9 In particular, a preexisting arterial spiderlike malformation has been suggested to induce hyperplastic liver cell nodules around terminating arterial branches (Wanless et al. 1985). However an abnormous arterial vascularization alone would not explain the lack of a sufficient system of large bile ducts9 The insufficient bile drainage is held to be responsible for many of the histologic features in another pathogenetic concept of FNH (Butron Vila et al9 1984). We wondered whether, apart from an abnormous vascularization and insufficient bile drainage, the apparent and characteristic morphologic interrelations between hepatocytes, bile duct like structures and fibrovascular stroma, might allow insight into the histogenesis of FNH. Light microscopic analysis showed the FNHs to be subdivided into pseudolobules consisting of expansile growths of hepatocytes arranged around a radiating fibrovascular center, which spreads from the interlobular
septa. The centrilobular fibrovascular stroma is always accompanied by ductular aggregates that merge imperceptibly with normal hepatocytes in the periphery of the micronodules (Stocker and Ishak 1981 ; Knowles and Wolff 1976; Rhodes et al. 1978; Gold et al9 1978 ; Goodman 1987; Craig et al. 1989). According to light microscopic and electron microscopic findings (Rhodes et al. 1978), continuous transitions from normal hepatocytes to ductular aggregates have also been demonstrated immunocytochemically with different panels of antibodies reacting specifically with distinct keratinpolypeptides (Fischer et al. 1987; van Eyken et al. 1989, this study)9 In contrast to normal hepatocytes (containing only the keratins 8 and 18) transforming hepatocytes in FNHs studied here also express keratins 7 and 19, but less strongly as the adjacent ductular aggregates. Van Eyken et al. (1989) showed that keratin 7 is expressed before keratin 19 in the transforming hepatocytes of FNH. The morphological and immunocytochemical continuity of this transformation makes it evident that the ductular aggregates in FNH do not arise from proliferations of preexisting ducts but derive from ductular metaplasia of hepatocytes, and that keratin 7 is expressed before keratin 19 in this metaplastic process9 The additional surprising expression of vimentin in some ductular aggregates is analogous to the coexpression of keratin and vimentin in damaged and regenerating tubular epithelial cells of the kidney (Gr6ne et al. 1987) or in newly formed bile ducts in secondary biliary
fibrosis in rats (Milani et al. 1989). This is clearly only a transient phenomenon, since it is not seen in normal hepatocytes or in true ducts with lumina. In FNH the ductular differentiation of hepatocytes is constantly accompanied by transformation of the surrounding fibrovascular tissue. The sinusoids and capillaries between transforming liver epithelial cells always consist of endothelial cells that express the endothelial markers BMA 120 and M 616, whereas the sinusoidal cells between normal-looking hepatocytes in the periphery of the pseudolobules do not react with these antibodies. Electron microscopically, transitions between Ito cells, myofibroblasts and fibroblasts have been observed in the immediate vicinity of, or in close contact with, the ductules of FNH (Callea et al. 1982). The demonstrated coexpression of vimentin and muscle actin by these mesenchymal cells also showed their fibromyoblastic nature immunohistochemically. These sinusoidal and perisinusoidal cell types have been shown to be involved in the synthesis of collagen type I, III, and IV in vitro (DeLeeuw et al. 1984; Irving et al. 1984; Reid et al. 1988) and in vivo (Reid et al. 1988; Milani et al. 1990), and they also seem to be responsible for the interstitial fibrosis between the transforming hepatocytes of FNH (Callea et al. 1982; Butron Vila et al. 1984). These findings indicate that the morphologic organization of the pseudolobules in FNH is the result of an intimate interplay between fibrovascular stroma and the adjacent liver epithelial cells. In the context of these findings, our morphometric analysis of FNH further enlightens the histogenetic process leading to the generation of pseudolobules of FNH. Remarkably, we could not find a statistic correlation between the mean size of the pseudolobules of the measured FNHs and the size of the entire lesion. Micronodules in the central and peripheral areas of the same FNH had similar mean sizes, although one would expect that central pseudolobules which represent the oldest parts of the lesion, which would have had the chance to become larger than the younger peripheral micronodules. This relative homogeneity of the pseudolobules can only be the result of a regulated proliferation and differentiation process in FNH which leads to subdivision of the pseudolobules when they have reached a certain size, irrespective of their position in the lesion. Subdivision proceeds by splitting and spreading of a centrilobular fibrovascular nidus in combination with ductular metaplasia of adjacent hepatocytes. At a later stage of growth, the centrilobular septa will be shifted between the new pseudolobules. They form portal tract eqivalents with residual ducts in a dense matrix of fibrosis (Fig. 3). Subsequently, this hyperplastic process will give rise to the three-dimensional stromal network around a fibrovascular stellate center by which FNH is characterized. Local conditions, especially differences in the vascularization within the lesion or compression of pseudolobules by adjacent, more expansive micronodules, will secondarily modify the microarchitecture of FNH. If one followed the favored pathogenetic concept for FNH, a focal excess of arterial blood flow by a preexisting arterial malformation (Wanless et al. 1985) could
modify the interrelations between fibrovascular stroma and liver epithelium resulting in FNH. However, one has to consider the alternative that primary deviated interrelations between liver epithelium and mesenchyme might be responsible for the abnormal, but nevertheless regulated, proliferation and differentiation of liver parenchyma in FNH with abnormal vascularization, perfusion and insufficient bile drainage as a secondary consequence. Acknowledgement. This work was supported by Deutsche Forschungsgemeinschaft, 5300 Bonn 2, FRG (DFG Fi 437/1-1)
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