Clinical and Epidemiologic Background (See Chapter 80)

Hepatocellular carcinoma (HCC) is the most common primary malignant tumor in the adult liver. It ranks among the most frequent neoplasms—approximately 6% of all new cancer diagnosed worldwide with an estimated incidence of about 500,000 to a million new cases per year. It is a deadly malignancy and the third most frequent cause of cancer death among men, causing 600,000 deaths worldwide per year (McGlynn & London, 2005; Sherman, 2005; Parkin et al, 2005; Bruix et al, 2004).

One of the striking characteristics of HCC is the marked geographic variation in its frequency, which is mainly related to geographic distribution of chronic liver disease risk factors. East Asia and sub-Saharan Africa have a very high incidence, whereas Italy, Spain, and Latin America are at intermediate risk. A relatively low but increasing incidence is found in Western Europe, the United States, Canada, and Scandinavia. In these countries, the incidence rate is 50 times lower than in Southeast Asia (Bosch et al, 2004; Kahn et al, 2002; Seeff & Hoofnagle, 2006).

The association between cirrhosis and HCC has been well established. Indeed, 60% to 90% of HCC arises in cirrhotic livers. Conversely, approximately 1% to 3% of patients with cirrhosis will develop HCC annually (Colombo et al, 1991; Johnson & Williams, 1987; Zaman et al, 1985). Most of the epidemiologic variation in HCC prevalence is thought to reflect differences in etiologic risk factors for liver cirrhosis that encompasses a large group of environmental factors (El-Serag et al, 2008). The major known risk factors for HCC are hepatitis viruses (chronic hepatitis B and C; see Chapter 64), toxins (alcohol, aflatoxins), metabolic diseases (metabolic syndrome, α₁ antitrypsin deficiency, Wilson disease), hereditary hemochromatosis, and immune-related diseases (primary biliary cirrhosis, autoimmune hepatitis; see Chapter 70A). Given that the burden of chronic liver diseases is expected to rise along with increasing rates of alcoholism, hepatitis B and C infection, and fatty liver disease, it is expected that the incidence of HCC will also increase in the near future (Nordenstedt et al, 2010; El-Serag, 2008).

HCC is primarily a disease of older men, and its incidence generally increases with age. In Western Europe and the United States, most patients with HCC are between 50 and 75 years of age, although there are geographic differences. It occurs more frequently in men than women, with a male/female ratio ranging from 2 : 1 to 9 : 1, although the reason for this is not clear (El-Serag et al, 2008). Routine liver function tests are variably abnormal, often reflecting the underlying cirrhosis without a consistent pattern of alterations. Serum α-fetoprotein (AFP) is elevated in most symptomatic tumors, but small cancers are associated with lower or even normal levels. Thus serum AFP is not a reliable diagnostic test for HCC screening in patients with cirrhosis (Bruix & Sherman, 2005).

It is admitted that HCC usually occurs after a mean delay of 10 years following the onset of liver cirrhosis. This observation is highly consistent with the multistep process of liver cirrhosis that implies progressive malignant transformation of preneoplastic lesions, such as macroregenerative and dysplastic cirrhotic nodules. Because of the dismal prognosis of the disease, increasing emphasis has been placed on detecting early, asymptomatic HCC that is potentially curable.

Ultrasound (US) is a sensitive technique for identifying nodules larger than 1.5 cm and is thus often used for detection, whereas magnetic resonance imaging (MRI) is a more accurate procedure for liver mass characterization (Bolondi et al, 2005; see Chapters 13 and 17). Reliable and highly specific imaging criteria now allow confident diagnosis of HCC in cirrhotic patients so that liver biopsy can be avoided when hyperarterialization associated with early washout is present in dynamic imaging (Bruix & Sherman, 2005; Forner et al, 2008). Screening programs have thus been proposed to follow cirrhotic patients by repeat US. These programs have been successful in identifying small and asymptomatic HCCs in high-incidence areas such as Japan, China, and Alaska but have been less effective in regions where the tumor incidence is low (Sangiovanni et al, 2004).

Despite some progress in managing HCC, liver cancer still carries a dismal prognosis, which is partly explained by its late diagnosis and the clinical context of the patient, which may compromise potential therapeutic strategies. Prognosis is also related to the liver tumor itself, which is prone to early angioinvasion.

HCC is a peculiar tumor for several reasons. Indeed, its morphologic patterns are various, beyond the usual classification based on growth pattern and tumor differentiation. Furthermore, molecular pathogenesis of HCC is complex, involving different molecular pathways that may reflect both etiologic factors and underlying liver disease (Thorgeirsson & Grisham, 2002; Villanueva et al, 2007).

Gross Pathology

HCC can adopt a wide range of gross configurations, and several macroscopic classifications of varying complexity have been proposed, but their clinical relevance has not yet been proven. Tumor size ranges from less than 1 cm to more than 30 cm in diameter. At the time of diagnosis, mean size of HCC arising in a cirrhotic liver is usually less than those occurring in nonfibrotic liver. HCC tumors less than 2 cm are recognized as small or early HCC (International Working Party [IWP], 1995). Recently, small HCC have been subdivided into vaguely nodular and well-circumscribed HCC, two patterns with differences in prognosis; the vaguely nodular form has a better prognosis (International Consensus Group [ICG], 2009; Hytiroglou et al, 2007). Although the diagnosis of large HCC relies mainly on imaging modalities, biopsy is often requested for the diagnosis of these small nodular lesions. Upon gross examination, HCC may display a nodular, infiltrative, or diffuse macroscopic pattern (Okuda et al, 1984).

Nodular (Expanding) Hepatocellular Carcinoma

The nodular (expanding) pattern is the most common type of HCC, and it is typically seen in association with cirrhosis. This group of tumors is characterized by a sharp demarcation between the tumor mass and the compressed and partly atrophic surrounding parenchyma (Fig. 78.1). The presence of distorted hepatic vessels, including arteries, that form curved structures on the surface of the tumor mass or are visible on the cut surface support the concept of an expanding growth pattern. The nodule may be solitary or multiple across the liver, when these develop as a complication of cirrhosis. When nodules are multiple, small (<1 cm in diameter), and adjacent to the main tumor nodule, they are known as satellite nodules (Fig. 78.2).

FIGURE 78.1 Hepatocellular carcinoma developed on liver cirrhosis with an expanding growth pattern. The tumor is sharply demarcated from the surrounding parenchyma and limited by a fibrous capsule.

FIGURE 78.2 Hepatocellular carcinoma. The tumor is massively necrotic with minute satellite nodules in the vicinity (arrows).

In cases of multiple HCCs, nodules may represent either multifocal independent tumors or intrahepatic metastasis. Such a distinction is quite impossible based on pathologic study alone but could be addressed with surrogate molecular analysis (Paradis et al, 1998; Sakamoto et al, 1989). On cut section, nodular HCC is well delineated from nontumoral liver and circumscribed totally or partially by a fibrous capsule (Okuda et al, 1977). Capsule formation is considered to begin at a tumor diameter of at least 10 mm or greater, because no distinct capsule has been noted in lesions less than 10 mm (Nakanuma et al, 1986). The prognostic significance of capsule formation has not yet been definitively settled. HCC typically forms soft masses that vary in color from gray, light brown, or yellow-green, often punctuated by foci of hemorrhage or necrosis when the tumor reaches a significant volume (Fig. 78.3).

FIGURE 78.3 Hepatocellular carcinoma. The tumor forms soft masses with foci of hemorrhage or necrosis.

Infiltrative (Massive) Hepatocellular Carcinoma

The infiltrative (massive) pattern is usually characterized by a large, solitary mass that occupies a substantial portion of the liver. The lesion is poorly circumscribed with ill-defined, invasive borders (Fig. 78.4). This pattern has been associated more commonly with noncirrhotic livers (Smalley et al, 1988; Grando-Lemaire et al, 1999). On cut section, the tumor extends into and distorts the adjacent nontumor tissue, interdigitating with surrounding parenchyma. Vessels are incorporated into, not displaced by, the tumor mass so that tortuous vessels around the tumor are not so obvious (Okuda et al, 1982).

FIGURE 78.4 Hepatocellular carcinoma with an infiltrative growth pattern. The tumor is poorly circumscribed and extends into the surrounding parenchyma. No rim of compressed tissue or capsule is visible.

Diffuse Hepatocellular Carcinoma

The diffuse pattern is the least common type of HCC and represents a widespread infiltration by numerous small nodules that virtually replace the entire liver. In this pattern, tumor consists of several unconnected small tumors of roughly similar size. Multicentric origin or intrahepatic spread after portal vein invasion have been discussed as pathogenic mechanisms (Fig. 78.5; Nakashima & Kojiro, 1986).

FIGURE 78.5 Hepatocellular carcinoma with a diffuse pattern of growth. The liver is totally replaced by numerous small, round tumor nodules.

Limits of Gross Classification

This gross classification has limits, because categorization of a tumor within one single growth pattern may be difficult; for example, expanding HCC may show areas of infiltrative pattern. Furthermore, the reproducibility of this categorization among different pathologists has not been shown. Finally, besides the diffuse pattern, which is associated with a dismal prognosis, the differentiation between expanding and infiltrative patterns does not appear as a prognostic indicator in multivariate analysis.

In nodular, advanced, and infiltrative HCC, invasion of large veins is common even at gross examination. The portal vein is more often involved than hepatic veins, inferior vena cava, or right atrium (Fig. 78.6). Portal vein invasion may be associated with thrombosis, and vascular invasion should be determined with care at the initial gross examination (macrovascular invasion). In some instances, intravascular tumor plugs in the close periphery of a large tumor may be difficult to distinguish from satellite tumor nodules. Invasion of large bile ducts may produce biliary obstruction, and hemobilia might be occasionally found.

FIGURE 78.6 Invasion of a dilated large portal vein (large arrow) and a small vein (small arrow) by a tumoral thrombus in a cirrhotic liver close to a hepatocellular carcinoma.

Histopathology

Histopathologic evaluation is no more systematic, because dynamic imaging has high diagnostic accuracy for tumors larger than 3 cm. When imaging is not diagnostic, US-guided biopsy must be performed. This is often the case for HCC smaller than 2 cm, when biomarkers have low predictive values, and hyperarterialization may be incomplete or absent. For nodules smaller than 1 cm, biopsy is generally not recommended because of its limited performance. These diagnostic criteria have been endorsed by most international liver disease associations (Bruix & Sherman, 2005; Bruix et al, 2001). Nevertheless, despite the major advances in radiologic procedures, the definitive diagnosis continues to be based primarily on accurate examination and interpretation of histologic material for any small or atypical nodule.

Histopathologically, the diagnosis of HCC is based on the distinction between tumor cells and normal hepatocytes (Anthony, 1973). Therefore, the microscopic evaluation entails assessment of cytologic characteristics of tumor cells and evaluation of their architectural pattern.

Tumor cells may be seen in varying degrees of hepatocellular differentiation within a single tumor. Nuclei are usually basophilic and often irregular, with prominent nucleoli and a high nuclear/cytoplasmic ratio. In well-differentiated HCC, the tumor cells may closely resemble normal hepatocytes, with a polygonal shape, distinct cell membranes, and eosinophilic granular cytoplasm (Fig. 78.7). Bile canaliculi can often be seen by light microscopy or can be demonstrated by immunostaining. When dilated, they might contain bile pigment, a characteristic feature of hepatocellular differentiation. Accumulation of glycogen or fat in tumor cells may produce a clear-cell appearance (Fig. 78.8). Cytoplasmic fat droplets might be more common in small HCCs. Mallory-Denk bodies, hyaline globules, and eosinophilic or so-called ground-glass cytoplasmic inclusions may also be observed.

FIGURE 78.7 Well-differentiated hepatocellular carcinoma. Tumor cells are organized in large trabeculae and resemble normal hepatocytes with a polygonal shape, distinct cell membranes, and clear cytoplasm with a high nuclear/cytoplasmic ratio.

FIGURE 78.8 Hepatocellular carcinoma with a clear-cell pattern. Malignant tumor cells of various sizes are enlarged with a clear cytoplasm.

As the tumor evolves to a poorly differentiated phenotype with cell-to-cell heterogeneity, bizarre nuclei or giant tumoral cells may appear. Mitosis and apoptotic bodies can be observed (Fig. 78.9). Different degrees of cellular differentiation are usually present within a single large tumor, although small HCC tend to be more homogeneous.

FIGURE 78.9 Poorly differentiated hepatocellular carcinoma. The tumor is highly cellular with tumoral cells organized in large trabeculae with bizarre nuclei, mitosis, and apoptotic bodies.

Growth Patterns

The arrangement of the cells contributes to the variability in the microscopic appearance. On this basis, several types of tumor have been categorized, but it has not yet been fully established whether these variations reflect differences in behavior and influence prognosis (Qin & Tang, 2002). The three main architectural patterns of growth of HCC are trabecular; acinar, or pseudoglandular; and scirrhous.

Trabecular Growth

Trabecular growth occurs when tumoral hepatocytes are arranged in plates in varying thicknesses from 2 to more than 20 cells (see Fig. 78.7). This feature resumes the normal trabecular arrangement of liver plates. Neoplastic cells are organized along simplified sinusoids lined by flat endothelial cells with few or no Kupffer cells. Compared with normal liver plates, the reticulin framework is commonly sparse or absent (Fig. 78.10). A compact or solid pattern occurs when the trabeculae are closely aligned, and the sinusoids become compressed and unapparent.

FIGURE 78.10 Reticulin staining in hepatocellular carcinoma. The lower part of the figure displays fragmentation and rarefaction of the reticular framework.

Acinar (Pseudoglandular) Growth

The acinar, or pseudoglandular, pattern results either from glandlike dilation of the canaliculi between tumor cells, in which the lumens can contain bile, or central degeneration of trabeculae, in which the lumens contain mainly degenerative products with fibrin (Fig. 78.11). As with the trabecular pattern of growth, stroma is typically sparse. The lack of a desmoplastic stroma reaction is a helpful diagnostic clue, when other glandular malignant epithelial neoplasms, especially cholangiocarcinomas, are discussed. On occasion, large vascular lakes resembling peliosis can develop within the pseudoglandular formations.

FIGURE 78.11 Hepatocellular carcinoma with a pseudoglandular pattern. Hepatocytes display glandlike organization around dilated canaliculi between tumor cells.

Histologic Variants

Fibrolamellar Hepatocellular Carcinoma

This subset, which differs from other types of HCC in clinical features and prognosis, is the only variant with clinical significance. It was first delineated as a distinct entity in 1980 (Craig et al, 1980). In a population-based study, fibrolamellar HCC constituted 0.85% of all cases of primary liver cancer and 13.4% of all cases in patients younger than age 40 (El-Serag & Davila, 2004). The clinical presentation is similar to other HCCs, except that it occurs in young people with equal gender ratio and has no association with chronic liver disease, cirrhosis, or any other known predisposing risk factors.

On gross examination, fibrolamellar HCC is a firm, mostly well-defined but unencapsulated single nodule that can range from 5 cm to more than 20 cm (Saab & Yao, 1996). On cut section, the tumor is gray to brown with scalloped borders and a solid consistency (Fig. 78.12). Prominent fibrous septa subdivide the mass and may connect with a central zone of scarring. Such features may be confused with focal nodular hyperplasia. In addition, several reports in the literature describe fibrolamellar HCC spatially associated with focal nodular hyperplasia, although filiation between these two lesions has never been convincingly demonstrated (Saul et al, 1987). Calcifications may also be observed.

FIGURE 78.12 Fibrolamellar hepatocellular carcinoma. Large lobulated tumor is well delineated from normal surrounding liver.

The distinctive histologic features are fibrous stroma and large, eosinophilic tumor cells (Fig. 78.13; Berman & McNeill, 1988). The stroma comprises dense, fibrous bands of varying thickness organized around nests, cords, and sheets of neoplastic cells (Nerlich et al, 1992). The tumor cells are usually larger than normal hepatocytes and display abundant, granular, and deeply eosinophilic cytoplasm with prominent nucleoli. Bile pigment is common, and fat or glycogen accumulation is sometimes seen. Most fibrolamellar carcinomas are histologically low grade, mitoses are usually sparse, and nuclear pleomorphism and multinucleation are infrequent. Cytoplasmic inclusions of various types are common, including ground-glass pale bodies (Fig. 78.14), eosinophilic cytoplasmic globules of variable periodic acid-Schiff positivity, and rarely Mallory-Denk bodies. In contrast to most HCC, fibrolamellar HCC seems to abundantly express type 7 cytokeratin (CK) and, in some cells, biliary-type CK19 (Van Eyken et al, 1990).

FIGURE 78.13 Fibrolamellar hepatocellular carcinoma. The tumor is composed of large eosinophilic tumor cells organized in cords between dense, acellular fibrous bands.

FIGURE 78.14 Fibrolamellar hepatocellular carcinoma. A, Tumoral cells organized in trabeculae along fibrous septa. B, High magnification showing large eosinophilic tumor cells with intracytoplasmic ground-glass pale bodies. C, Intracytoplasmic pale bodies. D, Intracytoplasmic eosinophilic globules of various size.

Fibrolamellar HCC tends to be slow growing and frequently resectable with a better prognosis than other types of carcinoma. However, when adjustment is made for tumor stage, all series do not demonstrate this better prognosis. Successfully resected patients have good chance of long-term survival despite extrahepatic recurrences.

Other Morphologic Patterns of Hepatocellular Carcinoma

It is well known that HCCs that arise in hemochromatosis fail to show accumulation of stainable iron, and iron-free foci in siderotic macroregenerative nodules in human cirrhotic livers have been suggested to be a marker for incipient neoplastic lesions (Hirota et al, 1982; Deugnier et al, 1993). Some HCCs accumulate copper, which can be detected using histochemical methods. Accumulation of large amounts of copper and copper-binding protein (orcein-positive) has been found chiefly in the fibrolamellar variant of HCC (Lefkowitch et al, 1983).

There are some unusual instances in which HCC produces substances that occur in some rare metabolic disorders (e.g., black HCC in a patient without Dubin-Johnson syndrome; Roth et al, 1982). A granulomatous sarcoid-like reaction may be encountered within HCC, the granulomas being characterized by epithelioid cells, Langerhans-type giant cells, and varying numbers of lymphocytes (Nakashima & Kojiro, 1986). Massive lymphoid stroma may also circumscribe otherwise typical HCC.

Hepatocellular Carcinoma in Noncirrhotic Liver

HCC in noncirrhotic liver encompasses several entities. It may develop during the evolution of a chronic fibrosing liver disease at the stage of incomplete cirrhosis (septal fibrosis). This is especially common in the context of chronic hepatitis B (Lam et al, 2004). Because of the possible reversibility of cirrhosis, it is not known whether HCC in incomplete cirrhosis develops during an ongoing fibrogenesis or along with the reversion of cirrhosis. In this context, HCC does not show any specific morphologic criteria. HCC may also develop in strictly normal liver or in liver with mild changes, such as steatosis. This group includes the fibrolamellar variant of HCC, that arising from malignant transformation of liver cell adenoma (see below), and a group that develops in the context of other chronic liver disease, including nonalcoholic fatty liver disease (Paradis et al, 2009; Marrero et al, 2002).

Grading and Other Pathologic Prognostic Factors

Grading of HCC relies on the Edmondson and Steiner system, which subdivides HCC into four grades, from I to IV, on the basis of histologic and cytologic resemblance to normal liver (Edmonson & Steiner, 1954). This grading has been shown to correlate with the DNA content and cellular proliferation indices of the tumor (Grigioni et al, 1989). Grade I is the well-differentiated type, in which hepatocyte-like cells are arranged in thin trabeculae. Small HCC tends to be grade I, although these are often not uniform in their differentiation. Grade II HCC is composed of larger tumor cells with abnormal nuclei; glandular structures may also be present. In grade IV, neoplastic cells are much less differentiated, with hyperchromatic nuclei and loss of trabecular pattern. In fact, most HCC seen initially is grade II or III (Kenmochi et al, 1987). Therefore, and as for other carcinomas, the general tendency is to summarize the grading using a three-tiered system comprising well-, moderately, and poorly differentiated HCC.

Nonetheless, tumor grade is a weak independent predictor of the clinical course and conveys little prognostic information (Lai et al, 1979; Chuong et al, 1982). Furthermore, grading of HCC is often heterogeneous alongside the tumor so that liver biopsy might have limited performance for grading HCC. Consequently, the histologic grading of HCC is useful, but rather as a descriptive tool; its practical value is limited (Pawlik et al, 2007).

Besides grading, other histologic features of HCC, such as architectural pattern, have little or no independent prognostic value. However, several staging systems have been proposed for HCC (Llovet et al, 1999; Marrero et al, 2005). The main prognostic factors are related to general health status; tumor stage, specifically the number and size of nodules, presence of vascular invasion, and extrahepatic spread; liver function, defined by the Child-Turcotte-Pugh classification system and by serum bilirubin and serum albumin levels; and portal hypertension. Etiology has not been identified as an independent prognostic factor.

Size is a major prognostic factor: small or minute carcinomas have a better prognosis, although with larger cancers, the tumor size does not directly correlate with outcome. Presence of satellite nodules around the main tumor has been also recognized as a prognostic factor in several studies. Improved survival has been associated with tumors that are encapsulated or fail to invade surrounding hepatic parenchyma (Ohnishi et al, 1987; Arii et al, 2000; Sutton et al, 1988).

Macroscopic and microscopic vascular invasion are important histoprognostic criteria that deserve special mention. Vascular invasion is a known predictor of recurrence and survival, directly associated with histologic differentiation, degree, and size of the main nodule (Nathan et al, 2009; Pawlik et al, 2005). Characteristically, the prevalence of microscopic vascular invasion increases with tumor size, with up to 60% to 90% in nodules larger than 5 cm. Other histologic findings are not consistently correlated with outcome, although occasional series have reported a better prognosis with clear-cell HCC and tumors of low histologic grade.

Using microarray technology, recent studies have shown that a subset of adult HCC displays phenotypic traits of hepatoblasts. These tumors retent stem cells markers and express CK7 and CK19. Interestingly, worse survival was demonstrated for this subgroup (Lee et al, 2004). Although the significance of CK7 and CK19 expression in HCC is still unclear, it might represent a relevant prognostic factor, although a larger study with multidimensional analysis should be performed.

A major influence on clinical status of the patient is the presence or absence of cirrhosis, which thus becomes a leading indicator for survival. Therefore, simultaneous biopsy of nontumoral liver is of major importance and should be systematically performed for consideration in the therapeutic decision and to help the pathologist in the diagnosis of very well-differentiated HCC.

Molecular Genetics

From experimental hepatic carcinogenesis and epidemiologic studies, it appears that liver carcinogenesis follows a multistep process (see Chapter 8C). Although a few cases of HCC arise in normal liver, the vast majority of them develop from the stepwise pathway: normal liver progresses to fibrosis then to cirrhosis and finally to HCC. Therefore, cirrhosis is recognized as a precancerous lesion (IWP, 1995).

As a result of careful follow-up of cirrhotic patients and detailed pathologic analysis of cirrhotic explanted livers, relevant insights have been gained into the early pathogenesis of HCC. Data are less clear when associated molecular events are concerned; however, a consensus supports the idea that HCC results from cumulative genetic and epigenetic events (Thorgeirsson & Grisham, 2002). Although recurrent gene abnormalities have been reported in fully developed HCC, the early molecular events are far less unknown.

Several studies using different approaches have looked for early molecular abnormalities in regular cirrhosis. Several molecular markers have been evaluated in macronodules, and none of the oncogenes or tumor suppressor genes involved in advanced HCC have been repeatedly found altered in the precancerous lesions. By contrast, proliferation markers, neoangiogenesis, telomerase expression, allelic losses, and clonality have been studied with more consistent results. Interestingly, these studies convincingly demonstrate, using clonal analysis, that among cirrhotic micronodules that looked similar on light microscope, some are already monoclonal (neoplastic), and others are polyclonal (regenerative; Paradis et al, 1998, 2000). Telomerase, an enzyme that allows unrestricted cell proliferation and that is specifically expressed in cancer, can be detected in some but not all of these clonal micronodules without any remarkable histopathologic features (Oh et al, 2003). In the same orientation, and using cell-cycle markers, studies have shown that cirrhosis associated with HCC, or that will give rise to HCC, already expresses increased cell-cycle markers. Therefore even in regular cirrhosis with a bland histopathologic appearance, foci of neoplastic clonal proliferation of liver cells with a limitless capacity of proliferation clearly are already present.

Most genomic studies address advanced HCC, and several efforts have been made to provide molecular classification of HCC based on gene expression (Villanueva et al, 2007; Chuma et al, 2003). Global gene-expression profiling may be the most appropriate technology to unravel the pathogenesis of HCC and explore its heterogeneous origin (Lee et al, 2004; Wurmbach et al, 2007; Llovet et al, 2006; Hoshida et al, 2008). In fact, application of gene-expression profiling of HCC has identified subgroups of patients according to etiologic factors, different stages of the disease, recurrence, and survival (Villanueva et al, 2008; Ladeiro et al, 2008; Boyault et al, 2007). The diversity of these tumors is also found at the molecular level, and a large number of genetic and epigenetic alterations have been described, some of them specific of risk factors. Recently, several comprehensive studies have shown that a molecular classification of HCC can be drawn, linking the transcriptomic pattern of gene expression (Boyault et al, 2007), micro-RNA profiling (Ladeiro et al, 2008), promoter methylations, chromosome gains, and deletions (Xue et al, 2008) to mutation of oncogenes and tumor suppressor genes (de la Coste et al, 1998). Major classes of tumors emerging from these comprehensive analyses are also related to important carcinogenesis pathways, such as activation of ß-catenin (de la Coste et al, 1998), AKT/mTOR (Villanueva et al, 2008), or inactivation of TP53 and RB1. Whether this molecular subclassification may provide a clue to personalized, targeted treatment or to prognosis is under active investigation (Villanueva et al, 2008). Interestingly, using genome-wide expression profiling of formalin-fixed, paraffin-embedded tissues, a recent study demonstrated that a reproducible gene-expression signature correlating with survival was only described in liver tissue adjacent to the tumor in patients with HCC (Hoshida et al, 2008).

Macroregenerative Nodules

A macroregenerative nodule (MRN) is a tumor-like hepatocellular mass that can arise in the setting of cirrhosis. These lesions have increasingly been detected, the result of both improved radiographic imaging techniques and more widespread screening of cirrhotic patients. Most MRN are large, discrete nodules that range from 1 to 3 cm in diameter. There is no minimum size threshold for definition; MRNs have to be appreciated according to the size of background cirrhotic nodules, which may be twofold to threefold larger than cirrhotic nodules (Theise et al, 1992; Terada et al, 1993). Macronodules are often well demarcated and surrounded by condensed connective tissue. MRNs are common, and they have been found after careful inspection in about 10% of cirrhotic livers at autopsy or at transplantation (Theise et al, 1992; Furuya et al, 1988; Mion et al, 1996). Histologically, most MRNs are indistinguishable from the usual parenchymal nodules seen in cirrhosis: normal-appearing hepatocytes are arrayed in plates one or two cells thick, limited by a regular sinusoid lining and bounded by typical fibrous septa containing blood vessels, bile ductules, and varying degrees of inflammatory infiltration (Fig. 78.15).

FIGURE 78.15 Low magnification of a benign regenerative macronodule in a cirrhotic liver. The macronodule is well-demarcated from surrounding liver. Fibrous septa and portal tracts are still present within the macronodule.

Dysplastic Nodules

Dysplastic nodules (DNs) are sizable lesions arising in cirrhosis, and they differ from the surrounding liver parenchyma with regard to size, color, texture, and degree of bulging of the cut surface. Based on microscopic features, DNs are further subdivided into low grade (LGDN) and high grade (HGDN), the latter being closer to HCC in the spectrum of hepatocarcinogenesis (IWP, 1995; ICG, 2009). Briefly, LGDNs display features suggestive of a clonal cell population but lack architectural atypia, whereas HGDNs show cytologic and architectural atypia but insufficient for a diagnosis of malignancy.

The premalignant nature of DNs is supported by different clues, including the common association with HCC in resected and explanted end-stage cirrhotic livers (Theise et al, 1992; Furuya et al, 1988; Libbrecht et al, 2005; Sakamoto et al, 1991); the presence of hepatocellular cyto-architectural abnormalities, featuring a lesion on the way to HCC (Ferrell et al, 1992; Ganne-Carrie et al, 1996; Takayama et al, 1990); the morphologic evidence of neoangiogenesis under the form of unpaired arteries supporting the abnormally ongoing vascular supply (Roncalli et al, 1999; Park et al, 2000; El-Assal et al, 1998); the detection of both genetic and epigenetic changes, greater than those in the surroundings but less frequent and consistent than in HCC; and a natural history showing an increased risk for malignant transformation compared with control cirrhotic nodules (Maggioni et al, 2000; Kobayashi et al, 2006; Terasaki et al, 1998; Seki et al, 2000).

Among the various cytologic alterations that characterize DNs are enlarged, crowded, or irregular nuclei with patent nucleoli and an increased nuclear/cytoplasmic ratio. Atypical architectural findings involve expansile proliferative zones, sometimes located within an MRN (nodule-in-nodule formations); focal loss of associated reticulin framework; and foci of abnormal structural patterns that includes irregular thickening of hepatic plates (IWP, 1995; Fig. 78.16). According to Eastern pathologists, stromal invasion, defined as the presence of tumor cells invading into the portal tracts or fibrous septa, is the most relevant feature distinguishing HGDN from early HCC (International Consensus Group for Hepatocellular Neoplasia, 2009). The degree and extent of these features vary greatly among patients, thus forming a histologic continuum that stretches between ordinary MRNs and obvious hepatocellular carcinoma.

FIGURE 78.16 A macronodule with a nodule-in-nodule pattern of growth. An expansile proliferative basophilic zone is located within a macronodule.

Dysplastic nodules must be differentiated from dysplastic foci, microscopic changes incidentally recognized in cirrhotic tissue. According to histopathologic criteria, dysplastic foci are split into two groups: those with large-cell changes or those with small-cell changes. Although large liver cell change, previously called large liver cell dysplasia (Fig. 78.17) consists of abnormal but nonneoplastic hepatocytes that are predictors of HCC development; small liver cell dysplasia is composed of neoplastic cells that are direct precursors of HCC (Lee et al, 1997; Borzio et al, 1995).

FIGURE 78.17 Large liver cell changes. Cluster of abnormal large liver cells with dystrophic nuclei.

Small Hepatocellular Carcinoma

A “small” liver cancer is currently defined as HCC with a maximum diameter of less than 2 cm (Nakashima & Kojiro, 1986). However, the lack of a widely accepted definition has led to confusion, and HCCs smaller than 3 to 5 cm have also been included in this group by others. Small HCCs are usually clinically silent and are often discovered incidentally on explanted liver or at autopsy (Mion et al, 1996). Small HCCs are most often a well-differentiated lesion, sometimes still containing portal triads, growing without substantial destruction of the preexisting hepatic framework and seldom showing angioinvasion (Sakamoto et al, 1991).

Small HCCs have been subdivided into two different entities: those with indistinct margins, called vaguely nodular HCC, and those with distinct margins, called distinctively nodular HCC (ICG, 2009). Whereas vaguely nodular HCC is the earliest, most indolent, and least angioinvasive type of HCC, distinctively nodular HCC is thought to have already acquired an ability to invade the vessels and to metastasize (Hytiroglou, 2007; Kojiro & Roskams, 2005; Theise et al, 2002). Therefore small HCC with distinct margins seems to be a progressed cancer in spite of its small size. On the liver biopsy, the difference between these two entities cannot be appreciated, because it requires the notion of gross features, which are not recognizable in small fragments. Therefore the term well-differentiated HCC may be better used in the biopsy report.

Natural History of Premalignant Lesions and Diagnostic Challenges

Few prospective studies have been conducted in large series of histologically proven nonneoplastic nodules detected by US during surveillance programs in cirrhosis. Taken together, these studies have shown that only a minority of macroregenerative or dysplastic nodules became malignant, those transforming are mostly in the group of HGDN. Furthermore, 40% to 60% stabilized, and a few definitely disappeared during follow-up (Borzio et al, 2003; Kondo et al, 1990). Most of these nodules are 1 to 2 cm, so they seldom display a diagnostic pattern at imaging. Therefore histologic assessment is needed at baseline, and it is worth repeating when sampling is not adequate.

Whether HCC regularly develops from DNs in a low grade–high grade–small and early–small HCC sequence in individual patients is still unclear. This hypothesis is supported by the feature of so-called nodules in nodules, which have been mainly reported in the Eastern literature (Kojiro, 2004; Roncalli et al, 2004, 2007). However, HCC has also been found to originate from outside dysplastic nodules during the surveillance of cirrhotic patients, supporting the notion that HCC can also develop “de novo,” by skipping the gradual transition of the sequence (Borzio et al, 2003).

From a clinical point of view, correct classification of hepatocellular nodules, as well as the exact number of truly malignant nodules, is crucial in order to plan the most appropriate therapy. However, the diagnostic approach to these small nodular lesions is a challenge. The prevalence of HCC is closely dependent on the size of the lesion. Indeed, almost half of those lesions smaller than 1 cm are nonmalignant, whereas the large majority of lesions exceeding 2 cm are HCC, so that in the group of lesions greater than 2 cm, a diagnosis of nonmalignancy should suggest the suspicion of a diagnostic error. It has been well established that, in cirrhosis, the progression from nonmalignant nodules to dysplastic to early and advanced HCC is characterized by the progressive loss of the portal blood with a “de novo” arterial vascularization (Hayashi et al, 2002). However, in the group of lesions sized 1 to 2 cm, about 20% of small HCC is hypovascular, so that histology is required in most of these cases, either at baseline or during follow-up, to confirm the nature of the lesions (Forner et al, 2008).

In clinical practice, liver biopsy is today mostly performed to classify small hepatocellular nodules (Bruix & Sherman, 2005). Diagnosis has to be made on tiny and often fragmented material, and clinicians need a conclusive report as to whether lesions are benign or malignant. Nevertheless, the diagnosis is not always straightforward, and other techniques may be required in addition to standard staining. Fortunately, immunocytochemical tools useful to distinguish malignant from nonmalignant nodules within the group of well-differentiated hepatocellular lesions have been developed recently, such as glypican-3 antibody (Di Tommaso et al, 2007).

Focal Nodular Hyperplasia

FNH is ten times more common than HCA and is the second most common benign liver process after hemangioma. It is predominantly diagnosed in women 30 to 50 years of age. Most FNH is found incidentally, but some is revealed by clinical symptoms or biologic alterations such as pain, liver mass, or increased γ-glutamyltransferase (GGT) levels. However, complications such as rupture or bleeding are exceptional, and no evidence of malignant transformation has been reported. In contrast to HCA, the diagnosis of FNH is strongly suggested by imaging techniques, so that histopathologic examination is required for diagnosis in a minority of cases. When performed, fine needle biopsy of FNH can be difficult, because fibrous septa and thick abnormal arteries, the hallmarks of FNH, are usually missing (Fabre et al, 2002; Makhlouf et al, 2005).

On gross examination, FNH appears usually as a solitary, discrete, rounded mass, pale to beige, and well-delineated from background normal liver but without a defined fibrous capsule (Fig. 78.21). On cut surface, FNH displays a variegated and partly nodular organization, and frequently, but not always, a central stellate scar with radiating fibrous cords are also visualized at imaging (Wanless et al, 1985; Nguyen et al, 1999).

FIGURE 78.21 Focal nodular hyperplasia. A well-circumscribed nodular lesion with a central stellate scar and radiating fibrous cords.

The typical histopathologic features of classical FNH include fibrous septa, which contain large dystrophic arteries with a peripheral ductular reaction, and an absence of interlobular bile ducts (Fig. 78.22). Hepatocytes are usually unremarkable, arranged in normal or mildly thickened plates without cytologic atypia, lined by a well-preserved sinusoidal framework. Steatosis or cholestatic degeneration with Mallory-Denk bodies may be seen.

FIGURE 78.22 Focal nodular hyperplasia. Fibrous septa contain often large, dystrophic arteries with thick walls and thin, fibrous septa.

FNH has been divided into classic and nonclassic forms (Nguyen et al, 1999). In forms of FNH other than the classic one, the diagnosis may prove difficult, because some of the distinguishing pathologic features may be unapparent. Such forms consist of FNH without a central scar, a mixed hyperplastic and adenomatous form, FNH with massive steatosis, and a form with atypia of the large-cell type (Nguyen et al, 1999). Strong evidence has been found, based on clonal features and the angiopoietin gene-expression profile, that the previously so-called telangiectatic variant of FNH may, in fact, belong to the HCA group (see below; Paradis et al, 2004).

Immunohistochemistry may provide interesting information. Immunostaining with glutamine synthetase that shows focal positive hepatocellular areas, usually centered by hepatic veins and described as a maplike pattern, is highly consistent with the diagnosis of FNH and suggests a focal maintenance of lobular organization (Bioulac-Sage et al, 2009a). In addition, the perisinusoidal spaces in FNH exhibit an aberrant extracellular matrix associated with abnormal fibrillin-1 expression (Lepreux et al, 2004). Further study showed that the phenotype of endothelial cells lining the vascular channels in FNH differs from those in the remaining liver, associated with a downregulation of angiotensin I–converting enzyme/CD143 (Grantzdorffer et al, 2004).

FNH and FNH-like nodules are now well known to develop in the context of hepatic venous outflow obstruction, including Budd-Chiari syndrome (Maetani et al, 2002; Rangheard et al, 2002), and in other circulatory disorders of the liver such as portal vein thrombosis, portal vein agenesis, hereditary telangiectasia, and even in cirrhosis; this supports the concept of a vascular trigger in the pathogenesis of FNH (Kondo, 2001; Kondo et al, 2004; Bureau et al, 2004). Evidence supports that FNH is a hyperplastic reaction resulting from an arterial malformation related to increased arterial blood flow (Wanless et al, 1985).

Similar to HCA, FNH has been observed in association with the use of oral contraceptives, although the relationship remains controversial (Scott et al, 1984). FNH may occur together with HCA (Grange et al, 1987), and it has been found to be associated with a variety of nonhepatic tumors and tumorlike conditions, such as HCA, hemangioma of the liver, and several other tumors (e.g., in siblings with glioblastoma; Handra-Luca et al, 2006). The significance of these associations is unclear, but it suggests that the pathogenesis of FNH is probably heterogeneous.

Hepatocellular Adenomas

HCA is a rare, benign liver cell neoplasm strongly associated with oral contraceptive (OC) use and androgen steroid therapy (Coombes et al, 1978; Nime et al, 1979). Its estimated incidence is 0.1 per year per 100,000 in non-OC users and reaches 3 to 4 per 100,000 in long-tem OC users. HCA can also occur spontaneously or in association with underlying metabolic diseases, including type 1 glycogen storage disease, iron overload related to β-thalassemia, and diabetes mellitus. Therefore, HCA represents a heterogeneous group of tumors in which histopathologic features may vary according to the etiologic background (Bioulac-Sage et al, 2007b).

HCA is usually solitary, sometimes pedunculated, with size ranging from a few millimeters to 30 cm. Large subcapsular vessels are commonly observed with large superficial tumors (Fig. 78.23). On analysis of cut sections, the tumor is soft, white to brown, and well delineated with little or no fibrous capsule (Fig. 78.24). Heterogeneous areas of necrosis or hemorrhage may be observed, especially in larger tumors. Histologically, HCA consists of a proliferation of benign hepatocytes of normal size or slightly enlarged with a normal nuclear/cytoplasmic ratio. Hepatocytes are arranged in a trabecular pattern without any residual portal tracts; a pseudoglandular growth pattern is possible but rare. Small, thin, and unpaired vessels without other portal tract elements—including connective tissue, bile ducts, or ductular reaction—are usually found throughout the tumor (Fig. 78.25). The cytoplasm of hepatocytes may be normal, glycogen-rich, or fatty, and cellular atypia can be impressive, especially in patients who have taken steroids for many years. In that context, differential diagnosis with HCC may be difficult. Vascular changes are frequent and include sinusoidal dilation, peliosis, infarcts, and hemorrhage. These changes may leave edematous or fibrotic regions, often with hemosiderin-laden macrophages.