Introduction
Identification of the molecular pathways altered in benign and malignant hepatocellular tumors has significantly increased our understanding of hepatocellular tumorigenesis. The immunohistochemical markers derived from these molecular alterations have been useful in clinical pathology practice, contributing to accurate diagnosis and classification of these tumors in resection and biopsy specimens.
Benign Hepatocellular Tumors
Hepatocellular adenoma (HCA) and focal nodular hyperplasia (FNH) are benign hepatocellular tumors. HCA is a hepatocytic neoplasm, whereas FNH is a hyperplastic hepatocytic response caused by localized hepatic vascular abnormalities.
FNH and HCA occur most frequently in women of child-bearing age; these tumors are much less common in men, children, and postmenopausal women. Oral contraceptives are recognized as the main risk factor for HCA. These agents are probably not etiologically implicated in FNH. Most cases of HCA occurring in men are associated with the administration of anabolic steroids for Fanconi anemia, acquired aplastic anemia, or bodybuilding. HCAs also occur in the setting of metabolic disorders such as glycogenosis type 1 and 3, galactosemia, tyrosinemia, and familial diabetes mellitus.
The epidemiology of HCA has changed in recent years because low-dose contraceptive pills cause fewer cases of HCA than the pills used in the past. However, obesity and the metabolic syndrome are more frequently encountered in women and men with HCA, particularly the inflammatory subtype. FNH lesions, especially when multiple, may be associated with hepatic vascular disorders and tumors of the liver and other organs. FNH and HCA usually develop in livers that are otherwise histologically normal or sometimes steatotic.
Focal Nodular Hyperplasia
Molecular Changes
The data regarding molecular aberrations in FNH are limited. Analysis of clonality using the human androgen receptor assay demonstrated the polyclonal nature of the hepatocytes in 60% to 100% of cases. Studies of chromosomal gains and losses using comparative genomic hybridization or allelotyping detected alterations in 14% to 50% of cases. However, genetic analysis failed to identify somatic gene mutations in CTNNB1 (which encodes β-catenin), TP53 (tumor protein 53), APC (adenomatous polyposis coli protein), and HNF1A (hepatocyte nuclear factor 1α).
The mRNA expression levels of two angiopoietin genes, ANGPT1 and ANGPT2, involved in vascular maturation are altered in FNH, and the consistently increased ANGPT1/ANGPT2 ratio supports the role of a vascular trigger for this lesion. Studies have identified overexpression of genes encoding proteins of the extracellular matrix and cell–matrix adhesion, consistent with the finding of fibrosis in most cases of classic FNH. A twofold overexpression of the TGFB1 gene was detected, and other key genes involved in the fibrogenesis pathway, such as PDGFA and PDGFRB , were overexpressed in FNH. The finding of myofibroblasts expressing smooth muscle actin at the periphery and in the fibrotic areas of FNH is consistent with activation of the transforming growth factor-β (TGF-β) pathway in these cells. The NTS gene, which encodes neurotensin, was overexpressed in FNH, resulting in an increased ratio of NTS to HAL, a periportal area gene that encodes histidine ammonia lyase. This finding can discriminate FNH from other benign tumors.
Alterations in the expression of physiologically zonated genes of the hepatic lobule have been detected in FNH. Thirteen genes of the periportal areas were found to be downregulated, whereas six genes of the perivenous areas were upregulated, indicating altered zonation in FNH. One of the perivenous area genes, GLUL , which encodes glutamate-ammonia ligase, also known as glutamine synthetase (GS), is upregulated by β-catenin. GLUL mRNA overexpression correlates with a slight but significant CTNNB1 mRNA overexpression in FNH compared with normal liver parenchyma, suggesting that upregulation of the β-catenin signaling pathway is restricted to areas surrounding the veins of FNH nodules. Upregulation causes overexpression of the active form of β-catenin in the absence of CTNNB1 -activating mutations.
These molecular data indicate that the almost completely arterial inflow in FNH activates the β-catenin pathway, which can contribute to tumor formation by promoting hepatocyte proliferation and regeneration. However, the mechanism of β-catenin activation in FNH remains unresolved, because no alteration of the main known regulators of the WNT signaling pathway have been identified, including a lack of AXIN1 and APC inactivating mutations. Additional functional analyses of the interplay between the TGF-β and β-catenin pathways in hepatocytes will be required to understand the pathogenesis of FNH.
FNH-like nodules are lesions resembling FNH macroscopically and on imaging, but they occur in cirrhotic livers or in the setting of vascular hepatic disorders such as the Budd-Chiari syndrome. The gene expression profile of FNH-like nodules in cirrhosis is significantly different from that of classic FNH. The nodules do not show β-catenin activation, and the β-catenin–induced perivenous genes are significantly downregulated compared with those of nontumorous liver and classic FNH. The periportally zonated genes are not differentially expressed compared with nontumorous liver as determined by quantitative reverse transcription–polymerase chain reaction (RT-PCR), and the NTS/HAL expression ratio is not increased, which is observed in classic FNH.
Molecular Diagnostics
As a consequence of the deregulation of the β-catenin pathway, overexpression of GS is heterogeneously distributed in the hepatocytes of the FNH nodules. This can be demonstrated with immunohistochemical techniques as characteristic staining with relatively large areas anastomosed in maplike patterns ( Fig. 44.1 , A and B ). The GS-positive areas are often centered on veins and are usually located at a distance from the fibrous bands. This specific pattern of GS staining in FNH contrasts with that of normal liver, in which staining is restricted to a few rows of hepatocytes surrounding the terminal hepatic venules. Due to the absence of β-catenin (CTNNB1) mutations, immunohistochemistry using a β-catenin antibody does not show abnormal cytoplasmic or nuclear β-catenin staining, even in hepatocytes overexpressing GS (see Fig. 44.1, B ). The characteristic GS immunostaining and the lack of aberrant β-catenin expression provide a useful tool for the diagnosis of some FNH lesions that lack typical histologic features in surgical specimens or biopsies.
As expected from their molecular changes, FNH-like nodules show little or no parenchymal GS immunostaining. When present, GS staining is usually restricted to some persistent venous structures inside the nodules. This staining pattern is similar to that of cirrhotic nodules and quite different from that of the large, anastomosing, GS-positive areas of classic FNH.
Hepatocellular Adenoma
HCAs are a heterogenous group of monoclonal tumors in which several recurrent mutations have been identified, leading to a classification based on good genotypic-phenotypic correlations. Molecular changes and diagnostics are described for each HCA group.
Hepatocellular Adenoma with Inactivating Mutations of the HNF1A Gene
Molecular Changes
The HNF1A gene (formerly hepatic transcription factor 1 [ TCF1 ]) is located on chromosome 12q24.2 and encodes the hepatocyte nuclear factor 1α protein (HNF1A), a 681–amino acid homeodomain transcription factor that is involved in hepatocyte differentiation. HNF1A controls the expression of liver-specific genes, such as those for β-fibrinogen (FGB) , α 1 -antitrypsin (SERPINA1) , and albumin (ALB) . HNF1A is a human tumor suppressor gene involved in liver tumorigenesis.
Biallelic inactivating HNF1A mutations have been detected in 35% to 40% of HCAs. In most cases, both mutations are of somatic origin, but in less than 10% of cases, one mutation is germline and the other somatic. Sporadic HNF1A-inactivated HCAs (H-HCAs) with somatic mutations occur almost exclusively in women and usually are associated with oral contraception. Patients with heterozygous germline HNF1A mutations are younger than those with somatic mutations and frequently have a family history of liver adenomatosis or diabetes (usually maturity-onset diabetes of the young type 3 [MODY3]), which is a consequence of heterozygous germline HNF1A mutations. The spectrum of HNF1A somatic mutations differs significantly from the germline changes in MODY3.
Transcriptomic analysis showed that several metabolic pathways are altered in sporadic H-HCA, including neoglycogenesis repression, and glycolysis and fatty acid synthesis activation. The induction of glycolysis and lipogenesis in H-HCA is linked to HNF1A inactivation and is independent of the activation of sterol regulatory element–binding protein 1 (SREBP1) and MLX interacting protein-like (MLXIPL, formerly called carbohydrate response element–binding protein [ChREBP]), leading to the characteristic steatotic phenotype of these tumors. Genes for fatty acid binding protein 1 (FABP1) and UDP glucuronosyltransferase 2-B7 (UGT2B7) are positively regulated by HNF1A and are downregulated in H-HCA.
Heterozygous, germline, inactivating mutations of CYP1B1 have been detected in 15% of women with H-HCA, suggesting that deregulation of the enzyme, which is responsible for the formation of genotoxic metabolites of estrogens, may confer a predisposition to sporadic H-HCA in women. CYP1B1 mutation modifies the penetrance of the liver adenomatosis phenotype in patients with HNF1A germline mutations.
Molecular Diagnostics
Downregulation of the FABP1 gene leads to a lack of liver-type fatty acid binding protein 1 (FABP1) expression in H-HCA, contrasting with normal expression in the surrounding nontumorous liver as demonstrated by immunohistochemistry. In addition to steatosis, which is common in H-HCA cases, the absence of liver-type FABP1 expression is an excellent marker for the diagnosis of this subtype of adenoma in resection specimens and biopsy samples ( Fig. 44.2 ), irrespective of the degree of steatosis or size of the lesion. This feature also allows the diagnosis of HNF1A -inactivated adenomatosis to be made when multiple HCAs of various sizes are found throughout the liver. They are often associated with myriad steatotic microadenomas, which can be correctly identified by the lack of FABP1 staining.
Hepatocellular Adenoma with Activating Mutations of the CTNNB1 Gene
Molecular Changes
Approximately 10% to 15% of HCAs demonstrate activating mutations of the CTNNB1 gene encoding β-catenin, which is deranged in many malignant tumors, including hepatocellular carcinoma (HCC). At baseline, β-catenin is associated with a negative regulator complex in the cytoplasm that includes APC, AXIN1, and GSK3B (glycogen synthase kinase 3β) proteins. This complex phosphorylates β-catenin, leading to its cytoplasmic degradation by the proteasome machinery.
CTNNB1 mutations occur mainly in exon 3 and consist of a large in-frame deletion that excludes the amino acids normally phosphorylated by GSK3B, leading to an absence of β-catenin phosphorylation and its permanent activation. Unphosphorylated β-catenin is stabilized in the cytoplasm and translocates into the nucleus, promoting the transcription of a large number of genes involved in proliferation, metabolism, and hepatocyte functions. Uncommonly, mutations occur in exon 7, and they have been reported in HCC. In most cases, activating β-catenin mutations lead to upregulation of two main β-catenin target genes: GPR49 and GLUL . The latter encodes GS, which can be demonstrated by immunohistochemistry.
Molecular Diagnostics
As expected from the molecular changes previously described, GS is overexpressed in β-catenin–activated hepatocellular adenomas (B-HCAs) (see Fig. 44.1, C and D ). The staining pattern is usually strong and diffuse (see Fig. 44.1, C ), and it is associated with aberrant nuclear and cytoplasmic expression of β-catenin, which is often distributed in a random and heterogenous pattern (see Fig. 44.1, D inset ). Good concordance has been demonstrated between these immunohistochemical findings (i.e., GS positivity and aberrant nuclear β-catenin staining) and the β-catenin mutations found by molecular methods. Overexpression of GS is usually easier to interpret than nuclear expression of β-catenin by immunohistochemistry, particularly in small biopsy samples. However, strong or faint GS staining can be heterogeneous (see Fig. 44.1, D ), probably depending on the effects of different β-catenin mutations. Despite the variations of GS staining in B-HCA, the pattern is usually distinct from the characteristic maplike pattern of FNH (see Fig. 44.1, A and B ).
Identification of B-HCA is important, because this group of HCAs is more frequently associated with the development of unequivocal HCC than other HCA subtypes. B-HCA is overrepresented among male patients, and specific risk factors are often found in both sexes, such as male hormone administration, glycogenosis, and familial adenomatous polyposis coli. Because B-HCA often displays cytologic abnormalities and a pseudoglandular pattern of growth, it can be difficult to distinguish it from well-differentiated HCC (see Chapter 55 ). In clinical practice, B-HCA is frequently diagnosed as “borderline lesion HCA/HCC.” When in doubt, assessment of β-catenin mutations in frozen tumor material is recommended. Activating β-catenin mutations are also detected in 10% of inflammatory hepatocellular adenomas (B/I-HCAs).
Inflammatory Hepatocellular Adenoma
Molecular Changes
Inflammatory hepatocellular adenomas (I-HCAs) account for more than 50% of HCAs. This group of HCAs is characterized by overexpression of molecules of the acute-phase inflammatory response, including serum amyloid A2 (SAA2) and C-reactive protein (CRP), at the mRNA and protein levels. Approximately 60% of I-HCAs have gain-of-function mutations (i.e., small, in-frame, somatic deletions) in the interleukin 6 (IL6) signal transducer gene (IL6ST) , causing permanent activation of the signal transducer and activator of transcription pathway (IL6/JAK/STAT) independent of the ligand. The IL6ST gene encodes the signaling coreceptor glycoprotein 130 (GP130), and mutant GP130 constitutively activates STAT3 signaling in the absence of IL6 binding.
In I-HCA subsets lacking GP130 mutations, derangements of two other molecular pathways have been identified. Twelve percent of I-HCAs harbor somatic mutations of the signal transducer and activator of transcription 3 gene (STAT3) . Most mutations result in amino acid substitutions in the SRC homology 2 (SH2) domain that directs STAT3 dimerization. In contrast to wild-type STAT3, I-HCA STAT3 mutants constitutively activate the IL6 signaling pathway independent of ligand.
An additional 5% of I-HCAs have somatic activating mutations of the G protein α-subunit gene (GNAS) , which are exclusive of the GP130 and STAT3 activating mutations in the IL6ST and STAT3 genes. Mutated GNAS is found in various tumors and is known to cause the McCune-Albright syndrome. Whereas IL6ST mutations directly activate the IL6/JAK/STAT pathway, GNAS mutations indirectly activate this pathway through various kinase pathways.
Approximately 10% of I-HCAs harbor mutations of the CTNNB1 (β-catenin) gene, signifying a risk of malignant transformation. On the other hand, some HCCs demonstrate GP130-activating mutations of the IL6ST gene. These tumors arise in normal liver and accumulate β-catenin–activating mutations. It is reasonable to hypothesize that these HCCs result from malignant transformation of HCAs. These data underline the role of inflammation in hepatic tumorigenesis and suggest that activation of the IL6/JAK/STAT pathway alone promotes benign tumorigenesis, whereas participation of β-catenin activation may lead to malignant transformation.
Molecular Diagnostics
Positive immunohistochemical staining for inflammation-associated proteins (i.e., SAA and CRP) in tumor cells with a sharp demarcation from the surrounding nontumorous liver is characteristic of I-HCAs ( Fig. 44.3 ). SAA and CRP staining is restricted to hepatocytes, without any staining of the sinusoidal lining and inflammatory cells (see Fig. 44.3, E ). I-HCAs with β-catenin activation also express GS and aberrant β-catenin, as described earlier for B-HCAs.
Unclassified Hepatocellular Adenoma
Unclassified hepatocellular adenomas (U-HCAs) represent less than 10% of HCA cases. U-HCAs do not have HNF1A or CTNNB1 gene mutations, and they do not express inflammatory proteins encoded by mutated IL6ST , STAT3 , or GNAS genes. This group of tumors lacks the immunophenotypic features described for the other HCA groups.
Use of Molecular Diagnostics
Molecular studies of FNH and HCA have significantly clarified the pathogenesis of these tumors and have provided immunohistochemical markers (i.e., FABP1, GS, β-catenin, SAA, and CRP) for routine diagnostic use. The markers are used to distinguish FNH from HCA and to classify HCAs ( Table 44.1 ). An important contribution of these scientific advances was the reclassification of telangiectatic FNH as I-HCA.
| Diagnosis | Altered Gene | Usual Characteristics of Immunohistochemical Markers | |||
|---|---|---|---|---|---|
| GS | β-Catenin | SAA/CRP | FABP1 | ||
| Normal liver | Positive in a few rows of hepatocytes around centrilobular veins | − | − | + | |
| FNH | CTNNB1 activation without mutations | +, maplike pattern | − | − | + |
| B-HCA | CTNNB1 activation with mutations | +, strong and diffuse or heterogenous | +, nuclei ± cytoplasm (usually focal) | − | + |
| H-HCA | HNF1A biallelic inactivating mutations (90% somatic and 10% germline) | − (or a few positive hepatocytes around veins) | − | − | − |
| I-HCA | IL6ST (gp130) activating mutations (60%) STAT3 activating mutations (12%) GNAS activating mutations (5%) | − (or a few positive hepatocytes around veins) | − | + | + |
| B/I-HCA (10% of I-HCAs) | Same as B-HCA and I-HCA | + | + | + | + |
| U-HCA | Unknown | − | − | − | + |
Standard microscopic features (see Chapter 55 ) often allow accurate differentiation, but because of the significant clinical and prognostic differences among the HCA groups, immunophenotyping is considered mandatory to ascertain the HCA subtype in each case. Immunophenotyping may become an important tool for HCA management in the near future. When feasible, tumor tissue should be frozen for molecular characterization of difficult cases, particularly to detect B-HCA, which has a higher risk of HCC transformation.
Hepatocellular Carcinoma and Its Precursors
HCC usually arises in a background of chronic liver disease. In autopsy series, 80% of HCCs are found in livers with cirrhosis; most of the remaining cases arise in chronically diseased rather than normal livers. On a worldwide scale, infection with the hepatitis B virus (HBV) and the hepatitis C virus (HCV) are the most common predisposing factors, with at least 350 million and 170 million infected people, respectively. Approximately 85% of HCC cases are related to one of these viruses.
The carcinogenic effects of HBV are illustrated by the fact that HCCs often develop in HBV-infected patients without cirrhosis, whereas HCCs in HCV-infected patients typically occur after cirrhosis is established. Among cirrhotic patients, the highest incidence of HCC is found for those with HCV infection (5-year cumulative incidence of 30% in Japan and 17% in the West), followed by those with hereditary hemochromatosis (5-year cumulative incidence of 21%), and those with HBV infection (5-year cumulative incidence of 15% in regions of high endemicity and 10% in the West). Other causes of cirrhosis, such as alcohol-induced liver disease, autoimmune hepatitis, biliary diseases, and Wilson disease, are associated with lower incidences of HCC. HCC may occasionally arise in normal liver, and some of these cases may be derived from HCA. The risk factors for HCC are listed in Box 44.1 .
Cirrhosis
Chronic hepatitis B
Chronic hepatitis C
Alcohol-induced liver disease
Nonalcoholic steatohepatitis
Diabetes, obesity, metabolic syndrome
Hereditary hemochromatosis
α 1 -Antitrypsin deficiency
Hereditary tyrosinemia
Other (rare) metabolic disorders
Aflatoxin B1 exposure (associated with hepatitis B virus infection)
Tobacco smoking
Hepatocellular adenoma, mostly β-catenin–activated hepatocellular adenoma
Molecular Changes in Hepatocarcinogenesis
Many factors favor initiation of hepatocarcinogenesis in chronic liver diseases :
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Increased hepatic cell proliferation due to hepatocyte loss
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Oxidative stress resulting from chronic inflammation (e.g., chronic viral hepatitis, steatohepatitis) or accumulation of noxious substances (e.g., hemochromatosis)
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Deregulation of genes due to HBV DNA insertion into the host genome
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Overexpression of growth factors (e.g., transforming growth factor-α [TGF-α] and insulin-like growth factor 2 [IGF2]) due to inflammatory cytokines and viral transactivation
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Derangements of DNA methyltransferases, causing global DNA hypomethylation and promoter hypermethylation, resulting in silencing of tumor suppressor genes
HBV DNA insertion causes genomic instability and deregulates genes involved in cell signaling and replication, such as human telomerase reverse transcriptase (TERT) , platelet-derived growth factor receptor (PDGFR), and calcium signaling–related genes. The HBV X protein acts as an oncoprotein, transactivating various genes involved in signal transduction pathways and inhibiting expression of the tumor suppressor gene TP53 . However, a specific TP53 mutation in codon 249 is characteristic of aflatoxin B1 exposure.
Due to vascular changes causing parenchymal loss in cirrhotic livers, the rate of hepatocyte loss and replication is often significantly enhanced compared with the precirrhotic stages of chronic liver diseases. Simultaneously, hepatocytes become senescent, and hepatic progenitor cells are activated to repopulate the dwindling parenchymal regions. In the course of chronic liver diseases, clonal cell populations of increasing size are established, some of which may have accumulating genetic and epigenetic changes favoring neoplastic transformation ( Fig. 44.4 ). Clonal cell populations with significant molecular alterations may be identified on microscopic examination as precancerous lesions. The cell of origin of these lesions may be the hepatocyte or the hepatic progenitor cell. Molecular analysis of precancerous lesions and HCCs reveals a sharp increase in structural DNA changes compared with the surrounding hepatic parenchyma.
