Introduction
Hepatocellular ballooning is a common but poorly understood alteration observed with a variety of acute and chronic conditions, such as alcoholic hepatitis, nonalcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH), viral hepatitis, autoimmune hepatitis, chronic cholestasis, and toxic liver injury.1–5 Ballooned hepatocytes (BH) are characterized by a rarefied edematous cytoplasm and a distinct diagnostic feature of NAFLD and NASH,5 which are chronic liver diseases associated with the metabolic syndrome and the risk of progression to fibrosis, cirrhosis and hepatocellular carcinoma (HCC),6,7 including clear-cell and steatohepatitic HCC.8,9 NAFLD-associated HCC may appear against a noncirrhotic background.10–13 Host metabolic status is also a major determinant of progression to HCC in cases of chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infections.14,15
Most authors have interpreted hepatocellular ballooning as a sign of degeneration, increased fluid in the cytosol16 causing lytic,17 apoptotic18 or oncotic cell death.19 Accumulation of fat droplets may be a feature5 with phospholipid-rich shells containing oxidized phosphatidylcholine and alterations of so-called “PAT” proteins regulating insulin-sensitive droplet lipase activity.20,21 In NASH, lipotoxic effects have been suggested to elicit ballooning and apoptosis of hepatocytes.16 BH of NAFLD patients were found to be strongly immunoreactive for sonic hedgehog, the level of expression correlating with the severity of liver damage, including hepatocellular ballooning.22 However, caspase 9, a pivotal enzyme for mitochondrial pathway of apoptosis, is diminished in BH.23 Thus, BH may not be damaged cells on the path to cellular demise, but rather functional in nature. Many BH contain Mallory-Denk bodies (MDB),2–5 which can be induced experimentally in rodents by carcinogens and have been discussed as possible indicators of preneoplasia.24 Whereas cytokeratins (CKs) 8/18 are predominant components of MDB, the remaining cytoplasm of BH shows a loss of CK8/184 as previously observed in preneoplastic clear glycogenotic hepatocytes.25
The striking resemblance of hepatocellular ballooning to acquired glycogenotic/steatotic changes characterizing preneoplastic and highly differentiated neoplastic clear and acidophilic (eosinophilic) cell populations discovered in experimental chemical,26 hepadnaviral27,28 and hormonal29,30 hepatocarcinogenesis have remained largely unconsidered, despite corresponding changes being well documented in humans.8,30–37 This also holds true for the hypertrophy of the smooth endoplasmic reticulum (SER) in eosinophilic ground glass hepatocytes (GGH).8,30–33 The complexity of BH feature recognition due to a lack of appropriate biomarkers has been emphasized in recent considerations on the application of artificial intelligence-based imaging for diagnosis of NAFLD.38,39
We previously proposed that human BH may be particularly enlarged glycogenotic clear, acidophilic hepatocytes corresponding to those of preneoplastic cell populations in animal models.8 We have tested that hypothesis using cytomorphological and cytochemical approaches. We realize that the cirrhotic livers investigated did not include cases of NAFLD and NASH, but the phenotypic similarity of hepatocellular ballooning in various chronic liver diseases, particularly the consistent decrease in CK8/18, the frequent occurrence of MDB, and recent findings in patients suffering from NAFLD justify the inclusion of these diseases as detailed in the discussion.
Methods
Liver specimens
Liver tissues were provided by the tissue bank of the Nationales Centrum für Tumorerkrankungen (NCT, Heidelberg, Germany; Project No. 2233) in accordance with regulations and with approval of the ethics committee of the University of Heidelberg. Samples were obtained from 96 explanted livers of patients with liver cirrhosis due to various chronic liver diseases, including alcohol abuse, HBV or HCV infection, autoimmune hepatitis, primary biliary and/or sclerosing hepatitis, Morbus Byler or Wilson disease, and congenital liver fibrosis, as summarized in Table 1.
Table 1Underlying diseases in the 96 cirrhotic liver cases
| Liver cirrhosis | n/N (%) |
|---|
| Alcoholic | 35/96 (37) |
| Posthepatitic: HBV | 15/96 (16) |
| Posthepatitic: HCV | 27/96 (28) |
| Posthepatitic: autoimmune | 3/96 (3) |
| Biliary: PBC, PSC | 7/96 (7) |
| Cryptogenic | 7/96 (7) |
| Other | 4/96 (4) |
Tissue processing
Liver sampling was conducted within 45 minutes after explantation. Specimens were routinely fixed in Carnoy’s solution and embedded in paraffin.33 For Oil-red-O staining, enzyme histochemistry, and immunohistochemistry, slices of approximately 1.5 × 1.5 × 0.5 cm were immediately frozen in −120°C isopentane and stored at −80°C. Serial 2–3 µm serial sections were stained with hematoxylin and eosin (H&E), and used for the demonstration of glycogen by the periodic acid Schiff reaction (PAS) or histochemical staining with various antibodies. To avoid any elution of glycogen during the preparation of the paraffin sections for the PAS reaction, 70% ethanol was used instead of the conventional water bath. Serial 10 µm cryostat sections of representative cases were used for staining with H&E, Oil-red-O, the PAS-reaction, and the enzyme- or immunohistochemical assay of G6Pase by the lead method40 and G6PDH by the nitro blue tetrazolium method.41 Serial cryostat sections were also incubated with a battery of primary antibodies listed in Supplementary Table 1. They were aldolase A, CK18, fatty acid synthase (FASN), 3-hydroxy-3-methylglutaryl-CoA-reductase (HMGCoAR), glukokinase (IGLK), insulin receptor, insulin receptor substrate 1 (IRS1), v-akt murine thymoma viral oncogene homolog (Pan-Akt), extracellular related kinase (PanERK), pyruvate kinase 2 (PKM2), phosphorylated mammalian target of rapamycin (p-mTOR), Ras, raf1, and stearoyl-CoA desaturase (SCD1). Endogenous peroxidase was quenched with 1% hydrogen peroxide and positive reactivity was identified using an Ultravision LP detection system, alkaline phosphatase polymer, and fast red chromogen or horseradish peroxidase polymer and diaminobenzidine as chromogen substrates (Thermo Fisher Scientific, Waltham, MA, USA). Formalin-fixed, paraffin-embedded serial sections (5 µm thick) were immunostained for antigens with an automated system (Leica Biosystems, Wetzlar, Germany). Signal intensity in ballooned hepatocytes was estimated semiquantitatively by comparison with corresponding surrounding unaltered liver tissue. For negative controls, primary antibodies were omitted.
For western blotting, primary antibodies against p-Akt (S473), pan-Akt, PKM2, H-Ras, Erk1/2, IRS1, p-mTOR, and beta-actin were used (details are given in Supplementary Table 2). For quantitative real-time polymerase chain reaction (qRT-PCR), primers for human ERK1, ERK2, IRS1, KRAS, HRAS, FASN, PKM2, AKT1, AKT2 and ribonucleic acid ribosomal 18S (RNR-18) genes were used as the housekeeping control. Details are given in Supplementary File 1.
Electron microscopy
Cryostat sections (50 µm thick) were fixed in 2.5% glutaraldehyde and cut into 2 mm2 pieces with a razor blade and embedded in Glycid Ether 100. Sectioning with a Leica ultratome to 500 and 750 nm thick semithin sections was followed by staining as described by Richardson et al.42 Ultrathin 70–90 nm sections were contrasted with uranyl acetate and lead citrate and examined with a Libra 120 electron microscope (Carl Zeiss, Jena, Germany).
Quantification of BH, GGH and MDB
Fractions (%) of BH and GGH, were evaluated in H&E sections from 37 representative cases with BH, and from 15 representative cases with GGH. The average volume fraction was semiquantitatively estimated from the fraction of all H&E-stained hepatocytes at ×100 magnification, using a NIKON DS-2MV digital camera and NIKON NIS Elements Imaging Software Package 4.0. Percentage (%) was calculated as the number of BH /total numbers of hepatocytes ×100 and number of GGH/total number of hepatocytes ×100. MDB occurrence was reported as 1 for present and 0 for not present.
Discussion
In agreement with previous studies, considerably enlarged (ballooned) hepatocytes (BH) with clear, vacuolated or eosinophilic cytoplasm, pronounced loss of CK8/18, and frequently harboring CK8/18-containing MDB, were found to be common in cirrhotic livers of patients with various chronic liver diseases. After appropriate preservation during preparation, we were able to demonstrate that the typical appearance of BH was mainly due to particularly pronounced excessive storage of glycogen (glycogenosis), often associated with steatosis and ground glass features. According to biochemical studies, the capacity of hepatocytes to store glycogen is limited, and glucose that cannot be stored as glycogen is converted to fat by de novo lipogenesis.43,44 In principle, the phenotype of BH corresponds to that of usually less enlarged glycogenotic/steatotic hepatocytes described as frequent components of preneoplastic focal and premalignant nodular liver lesions developing in various chronic liver diseases.32
The morphological, enzyme- and immunohistochemical characteristics of BH all point to similarities with focal preneoplastic liver lesions in animal models of chemical, hormonal, and viral hepatocarcinogenesis, characterized by consistent activation of insulin dependent glycogenotic45–48 and lipogenic pathways,48,49 and an early elevation of glucose-6-phosphate (G6P), a central metabolite channeling glucose into the different pathways of carbohydrate metabolism.45 In line with these findings, cultured human hepatocytes and hepatoma cells lacking CK8/18 have an elevated glucose uptake, G6P formation and glycogen production compared with their counterparts containing CK8/18. These effects are further increased by activation of the insulin signaling cascade through the mTOR-dependent pathway, along with an augmentation in cell proliferation.50
The combination of glycogenosis and SER-hypertrophy in BH corresponds to observations in preneoplastic or highly differentiated neoplastic cell populations in animal models,30,26–28 human inborn glycogenosis type 1 (GSD I)51 and focal and nodular lesions in chronic human liver diseases.24 Evidence for a metabolically active G6P pool in the lumen of liver microsomal vesicles and a reversible G6P transporter in liver microsomal membranes has been provided.52,53 In GSD I, microsomal vesicles contain relatively high intravesicular G6P levels,52 suggesting that accumulation of G6P and SER-hypertrophy are closely related. The pronounced accumulation of glycogen, with and without SER-proliferation, in BH pushes the remaining organelles to peripheral and paranuclear parts of the cytoplasm, leading to a dislocation and relative reduction of the RER and mitochondria, possibly contributing to the miscommunication which has been proposed as an early and causal trigger of hepatic insulin resistance and steatosis.54 High frequency and long persistence of ballooning hepatocytes has actually been shown to be associated with glucose intolerance in patients with severe obesity.55
Acquired hepatocellular glycogenosis in animal models of hepatocarcinogenesis and chronic human liver diseases finds a counterpart in different types of GSD, such as GSD I due to G6Pase deficiency and GSD VI triggered by a genetic defect of glycogen phosphorylase, both conveying a high risk of hepatocellular adenomas and carcinomas.8,30,51 GSDI has been modeled in mice by targeted deletion of the G6Pase gene eliciting glycogenosis, steatosis, and eventually hepatocellular neoplasms.56
For acquired murine glycogenosis, it has been shown that the early decrease of G6Pase and glycogen phosphorylase activities is maintained during progression to hepatocellular neoplasms. Ever increasing activities of G6PDH and glycolytic enzymes indicate a fundamental metabolic shift toward the pentose phosphate pathway and the Warburg-type glycolysis, providing energy and precursors for nucleic acid synthesis and cell proliferation.45 Cytomorphological correlates of this metabolic reprogramming are gradual reduction of the glycogen initially stored in excess, frequent transient steatosis, and increase in ribosomes (basophilia) typical for the advanced neoplastic phenotype.30 In mice exposed to diethylnitrosamine or subjected to a liver-specific knockout of G6Pase, the accumulated glycogen has been shown to undergo liquid-liquid phase separation and formation of glycogen-liquid droplets, which via inhibition of Hippo signaling stimulates cell proliferation and promotes hepatocarcinogenesis.57 Elimination of glycogen accumulation abrogated tumor development, whereas increasing glycogen storage accelerated tumorigenesis.
In BH, glycogenosis is associated with high expression of glycolytic enzymes, corresponding to the glycogen shunt, which couples glycogen synthesis and breakdown pathways to the Warburg effect.58 During the progression from preneoplastic glycogenotic to glycogen-poor neoplastic cell populations, this process is linked to an isoenzyme shift from the pyruvate kinase characteristic of normal hepatocytes (PK-M1) to PK-M2,45,59 which is critical for regulating the glycogen shunt flux.58 A regulatory mechanism of the glycogen shunt implicated in metabolic reprogramming has also been observed in Myc1 knockout mice and hepatic cell lines, showing progressive accumulation of glycogen and a redistribution of glucose from glycogen to other metabolic pathways, including the pentose phosphate pathway and glycolysis.60 In addition to pyruvate kinase, hexokinase undergoes an isoenzyme shift during neoplastic progression. The low-affinity hexokinase (glucokinase/HK IV), is replaced by the high affinity hexokinase (HKII) during neoplastic transformation of rat hepatocytes in vivo and in vitro.45 Evidence for heightened HKII in human hepatocellular dysplasia and HCC also exists.61
In most animal models, glycogenotic/steatotic or purely steatotic hepatocytes appear only at later time points during progression from the initial hepatocellular glycogenosis to poorly differentiated HCC.28,30 Similar observations were made in type 2 diabetes and GSD I, which are both at high risk of neoplastic development.8,44 Thus, dysfunction of glucose metabolism may progress to NAFLD and eventually lead to hepatocellular neoplasms, suggesting that the combination of glycogenosis and steatosis, or steatosis alone, in BH are secondary rather than primary metabolic changes occurring predominantly during progression. Applying the PAS-reaction to paraffin sections of routinely processed liver biopsies from more than 2,000 children and adult patients, Allende et al.62 found that a focal or diffuse glycogenosis of the liver parenchyma is common in NAFLD. They also observed BH with a particularly pronounced excessive storage of glycogen, but in contrast to our observations the large majority of BH did not contain visible glycogen. Statistical evaluation revealed that glycogenosis was independently associated with a high grade of ballooning, and the presence of MDB and decreased steatosis and fibrosis. Hence, the authors concluded that glycogenosis may have a protective effect on disease progression. We would infer from the findings in different animal models, human type 2 diabetes, and GSDI8,44 that glycogenosis without steatosis and fibrosis indicates an earlier stage. The discrepancy in our observations and inference may be due to methodological differences in tissue preparation. Whereas Allende and colleagues used routinely processed liver biopsies collected from different laboratories, our specimens were not only fixed in Carnoýs solution preserving the glycogen, but were also cut and further processed without water contact to avoid elution.
Observations in both human and animal models agree that hepatocarcinogenesis may proceed with or without liver fibrosis and cirrhosis, depending on the severity of accompanying unspecific necroinflammatory changes elicited by the respective oncogenic agents.31 The fact that HCC in chronic liver diseases, including NAFLD and NASH,9–12 frequently appear without a background of fibrosis or cirrhosis implies that it is the metabolic dysfunction in altered hepatocytes, rather than the facultative necroinflammatory changes resulting in fibrosis and cirrhosis, which are essential for neoplastic development. The demonstration of pronounced glycogenotic and steatotic metabolic aberrations in BH comparable to those emerging early during hepatocarcinogenesis under a variety of conditions strongly suggests a preneoplastic or early neoplastic nature. Pronounced glycogenosis may serve as an appropriate biomarker for BH, particularly solving the dilemma recently emphasized by Li et al.39 to distinguish BH from hepatocellular edema.