Benign and Malignant Tumors of the Liver

Clinical Features

Hepatocellular adenomas (HCAs) are rare tumors that constitute less than 2% of all liver tumors, with an incidence of 2 to 3 per 1 million per year in the Western world. Young women in the reproductive age group are most commonly affected, while occurrence in men and children is rare. The incidence is increasing, mostly because of the obesity epidemic. Tumors in older women (>50 years of age) and men are being increasingly encountered.

The tumor may come to attention incidentally on imaging, or due to signs such as abdominal pain, mass demonstrate variable echogenicity in HCAs, and is not helpful in diagnosis. HCAs typically show arterial enhancement on CT with contrast, with persistent enhancement in the delayed phase. MRI with liver-specific contrast agents can often distinguish areas of fat and telangiectasia, and it can help determine the subtype of HCA ; however, a definitive diagnosis of HCA based on imaging is often not possible.

The serum alkaline phosphatase level may be elevated in HCA, but serum α-fetoprotein (AFP) levels are typically either normal or only minimally elevated.

Pathogenesis and Risk Factors

Oral contraceptive and anabolic steroid use are well-known risk factors for HCA. HCAs may regress with cessation of oral contraceptive use.

Other risk factors include obesity, metabolic syndrome, and inherited metabolic disorders such as glycogen storage diseases I and IV, galactosemia, tyrosinemia, and familial diabetes mellitus. ^, The genetic basis is briefly discussed with each subtype in the following sections and in further detail in Chapter 45 .

Pathological Features

Adenomas may be single or multiple. They are rather arbitrarily designated as adenomatosis when >10 tumors are present. HCAs are unencapsulated tumors most often with an irregular, ill-defined border. They occur mostly in noncirrhotic livers. However, rare cases of inflammatory HCA have been described in patients with cirrhosis. The tumors are usually soft, tend to bulge on cut section, and are lighter in color than the surrounding liver parenchyma. However, the appearance can vary with the degree of necrosis or hemorrhage. Inflammatory HCAs can be particularly ill-defined. In some cases, they may impart a congestive appearance, or bogginess to the parenchyma. HCAs usually lack significant fibrosis or nodularity, but these features can be present, especially in inflammatory HCA. ^, Rarely, HCAs may have a slate gray to black color caused by a large amount of lipofuscin pigment (black adenoma).

Microscopically, HCAs are composed of a relatively uniform population of hepatocytes arranged in cell plates that are most typically one to three cells thick ( Fig. 56.1 ). The reticulin framework of the cell plates is usually largely intact ( Fig. 56.2 ). However, the sinusoids may appear compressed, resulting in a uniform and solid appearance. The tumor cells can be slightly smaller or larger than normal hepatocytes, but the nucleus-to-cytoplasm ratio is usually normal. The cytoplasm of tumor cells may be eosinophilic or clear, or it may contain fat, bile, lipofuscin pigment, or Mallory hyaline (rare). Multinucleated tumor cells or rare hepatocytes with large atypical nuclei may be present. Mitotic figures are typically absent. Unpaired arterioles (without accompanying bile ducts) are a characteristic feature of HCA. Kupffer cells may be present in adenomas, but are fewer than in normal liver. Rarely, a partial capsule surrounds the tumor, showing foci of tumor cells that merge with adjacent liver parenchyma at sites where the capsule is absent. A bile ductular reaction is typically absent, but can be seen especially in inflammatory HCA, and this feature may simulate normal portal tracts (see Fig. 56.1 ). Portal tracts are occasionally found within HCAs at the border of the tumor. Areas of infarction and hemorrhage are frequent findings, especially in larger tumors. Peliosis-like changes can be seen in HNF1A -inactivated and inflammatory subtypes.

The WHO recognizes four subtypes of HCA based largely on genetic analysis: (1) hepatocyte nuclear factor 1α (HNF1A) -inactivated HCA (H-HCA), (2) inflammatory HCA (previously termed telangiectatic HCA ), (3) β-catenin–activated HCA, and (4) unclassified HCA. These subtypes can be identified by immunohistochemistry ( Tables 56.1 and 56.2 ). ^,

H-HCAs are characterized by somatic or germline HNF1A mutations. Germline mutations can be associated with maturity onset diabetes of the young (MODY3). These tumors often show marked steatosis, lack cytological atypia, and have a low association with hepatocellular carcinoma (HCC; Fig. 56.3A ). In some of these tumors, foci with pseudoacinar architecture can be prominent. These HCAs show loss of cytoplasmic liver fatty acid binding protein (LFABP) on immunohistochemistry ( Fig. 56.3B ). Transformation to HCC can occur but is uncommon. It likely occurs in less than 5% of cases.

Inflammatory HCAs (previously termed telangiectatic HCAs ) are characterized by activation of the interleukin (IL)-6 signaling pathway, most commonly caused by mutations in the IL6ST gene. ^, The typical morphological features of this subtype are sinusoidal dilation and lymphocytic inflammation ( Fig. 56.4A ). A variable degree of ductular reaction and fibrous septa can be present in these tumors, which closely mimic focal nodular hyperplasia (FNH). Immunohistochemistry for inflammatory markers such as serum amyloid A (SAA) and C-reactive protein (CRP) show granular cytoplasmic staining and helps confirm the diagnosis ( Fig. 56.4B ; Fig. 56.5 ). CRP is more sensitive and is positive in nearly 100% of cases, while SAA is more specific with a sensitivity of 80% to 90%. Transformation to HCC can occur, usually through β-catenin activation, which is observed in 10% of cases.

β-catenin–activated tumors frequently exhibit atypical cytological features and have a high association (40% to 50%) with concurrent or subsequent HCC. Hence these tumors are best placed in a separate atypical category, rather than as a subtype of HCA. The designation of atypical hepatocellular neoplasm (AHN) and borderline tumor has been used in the WHO 2019 scheme. The term hepatocellular neoplasm of uncertain malignant potential (HUMP) has been proposed by an international group of liver pathologists, but it is not included in the WHO scheme. There is lack of consensus on the use of terminology for these tumors, and all three terms are appropriate as long as the significance is properly discussed in a comment. These tumors often occur in men (∼40%). They are characterized by activation of the Wnt signaling pathway, most commonly resulting from a CTNNB1 (β-catenin) exon 3 mutation. In a small minority of cases, mutations in other components of the Wnt signaling pathway (APC, AXIN) may be involved. Cytological atypia, prominent nucleoli, small cell change, a pseudoacinar pattern, and bile plugs may be present ( Fig. 56.6A ). ^, Nuclear staining with β-catenin occurs in a subset of cases (<50%), but it is not sensitive for the diagnosis of this subtype (especially on biopsy) as the staining may be focal or even negative despite the presence of an exon 3 β-catenin mutation ( Fig. 56.7 ). Nuclear β-catenin leads to overexpression of glutamine synthetase (GS). Diffuse GS staining (moderate to strong cytoplasmic staining in ≥50% of tumor cells) is a better marker of β-catenin activation ( Fig. 56.6B ). Diffuse GS staining can be diffuse homogeneous (>90% of tumor cells) or diffuse heterogeneous (50% to 90% of tumor cells) ( Fig. 56.8 ). ^, A diffuse homogeneous pattern is nearly always related to mutations associated with a high level of β-catenin activation, while a diffuse heterogeneous pattern is more common with mutations associated with an intermediate level of β-catenin activation (such as S45); however, the correlation of the pattern of staining and type of mutation is not perfect. For practical purposes, diffuse GS staining irrespective of whether it is diffuse homogeneous or heterogeneous is currently regarded as evidence of β-catenin activation.

The unclassified group of tumors lack typical features of the other subtypes. For instance, HCAs with a CTNNB1 (β-catenin) exon 7 or 8 mutation show weaker β-catenin activation, generally lack diffuse GS, and thus typically fall into this category based on morphology and immunohistochemistry. A sonic hedgehog–activated subtype (shHCA) has been recently described, which is associated with a high risk of bleeding. Immunohistochemistry for prostaglandin D synthase may help identify this variant, but it has not been widely tested. Argininosuccinate synthetase 1 (ASS1) has been proposed as a marker of shHCA, but it is also positive in other subtypes like inflammatory HCA. Thus its diagnostic use in this setting has not yet been well established.

Differential Diagnosis

The main differential diagnosis of HCA includes FNH and well-differentiated HCC ( Table 56.3 and Table 56.4 ). The presence of nodular architecture, a central scar, fibrous septa, a prominent ductular reaction, and large arteries within fibrous stroma favor FNH, but these findings overlap with HCA, especially the inflammatory subtype. A maplike GS pattern (FNH), SAA/CRP (inflammatory HCA), and LFABP ( HNF1A -inactivated HCA) help in confirming a diagnosis of HCA.

Lack of cytological atypia, thin cell plates, absence of prominent small cell change or pseudoacini, and an intact reticulin framework help distinguish HCC from HCA. Tumors with β-catenin activation and/or focal or mild cytoarchitectural atypia that is insufficient for a diagnosis of HCC are best categorized as atypical hepatocellular neoplasm. ^, It is important to note that LFABP loss, positive SAA, and/or CRP can be observed in HCC. Hence these stains should not be used to distinguish HCA and HCC, but they should be used to subclassify HCA after the diagnosis of HCA has been made based on a combination of morphology and a reticulin stain.

Increased sinusoidal staining with CD34 is common in HCA and is not helpful for distinction from HCC. Positivity with glypican 3 (GPC3), an oncofetal protein, favors HCC but is positive in less than 50% of well-differentiated HCCs.

In small biopsies, distinction between HCA and normal liver may also be difficult. Diffuse staining with CD34 and patchy GS staining in regions distant from the perivenous region of the liver help favor HCA. SAA and CRP should be interpreted with caution because these stains can also be positive in adjacent nonlesional liver.

Treatment

HCAs >5 cm in size are at a higher risk for hemorrhagic complications and HCC. Thus surgical excision is recommended for tumors of this size and those with high-risk features such as male gender and β-catenin activation. Because resection is recommended for HCAs >5 cm and in males (any size), it can be argued that subtyping and risk subtyping is not necessary for patient management. For smaller tumors in women, the decision to perform a resection may rest on pathological risk factors including β-catenin activation. Accurate assessment of GS and β-catenin immunohistochemistry is crucial in these cases. If the staining pattern with GS and β-catenin immunohistochemical results are unclear based on the biopsy, sequencing assays can be used to confirm mutations in the Wnt signaling pathway. For patients with multiple HCAs, the management is guided by the size and risk factors of the individual nodules. Nodules with a higher risk are resected, whereas others may be followed up by imaging.

Clinical Features

FNH is a benign, nonneoplastic lesion that is most commonly encountered in young women. The estimated incidence is 0.9% to 3%. Most are asymptomatic and are thus discovered incidentally. However, large ones can be symptomatic. FNH is difficult to characterize on ultrasonography, but it is well visualized on contrast-enhanced CT imaging with hyperattenuation in the arterial phase and isoattenuation in the delayed phase. A central scar with a hypoattenuated appearance in the arterial phase can be seen in 70% of cases. On MRI, FNH gives an isointense to hypointense signal on T1-weighted images and appears hyperintense or isointense on T2-weighted images.

FNH may increase in size with oral contraceptive use, but this is not thought to be a risk factor in its development. Multiple FNH has been described in association with hepatic hemangioma, vascular malformation, meningioma, and astrocytoma. FNH has no known malignant potential. It is not considered a precursor of fibrolamellar HCC despite its rare association with this malignant tumor.

Pathogenesis

The pathogenesis of FNH has not been well established, but it is thought to result from a hyperplastic or altered growth response to alterations in parenchymal blood flow. The presence of numerous abnormal muscular vessels in FNH and the occurrence of similar nodules ( FNH-like lesions ) in association with hemangiomas, vascular abnormalities, and adjacent to other mass lesions lend support to this theory. FNH can also develop in association with vascular disorders (heart disease, Budd-Chiari syndrome) and hereditary conditions such as hereditary hemorrhagic telangiectasia (Osler-Weber-Rendu disease). The terms FNH-like regenerative nodules and FNH-like lesions have been used in these clinical settings, but the morphology in most of these cases is similar to sporadic FNH.

Most cases that were previously designated as telangiectatic FNH were likely inflammatory HCA. However, sinusoidal dilation (telangiectasia) can be seen in FNH as well.

Pathological Features

FNH is usually a solitary lesion, but it can be multifocal in about 20% of cases. ^, The multinodular appearance can simulate cirrhosis in some cases. FNH tends to be lighter in color than the surrounding liver parenchyma. Occasionally, FNH may be pedunculated. FNH is normally well demarcated from the adjacent normal liver parenchyma, but a fibrous capsule is usually absent. FNH may vary considerably in size, ranging from 1 mm to 19 cm. FNH has a central fibrous scar in 60% to 70% of cases, which consists of fibrovascular tissue, not dense scar tissue.

Microscopically, FNH is composed of normal-appearing benign hepatocytes arranged in incomplete nodules and partially separated by fibrous tissue that extends outward from the central fibrous zone of the tumor. A ductular reaction is normally present in the fibrous septa in most cases, but FNH does not contain interlobular bile ducts. Atypia of the hepatocytes is not typically present. However, in some cases, wider cell plates (3 to 4 cells thick), focal pseudoacinar structures, and focal reticulin loss may be seen, especially at the periphery of the lesion. The hepatocytes may demonstrate increased glycogen, fat, ballooning degeneration, Mallory hyaline, bile, lipofuscin, iron, and copper or copper-associated protein. Occasional atypical hepatocytes containing larger-sized nuclei and mild hyperchromasia, either with or without conspicuous nucleoli, may be seen.

Medium- to large-sized, thick-walled, muscular vessels that often exhibit myointimal myxoid or fibromuscular hyperplastic changes are characteristic of FNH and are normally present in the central scar and fibrous septa ( Figs. 56.9 and 56.10 ). Portal tracts are absent in FNH, except at the periphery of the lesion, although a bile duct of intermediate or large caliber may be found in the central fibrous zone in rare cases. The sinusoids may be dilated, and Kupffer cells may be evident. Inflammatory cell infiltrates are relatively common and consist mainly of lymphocytes, although neutrophils and eosinophils may be present, particularly surrounding bile ductular structures. Rarely, granulomas are present as well.

The reticulin framework is largely intact in FNH; focal loss can be seen at the periphery of tumors and encircling small groups of hepatocytes in some cases. Immunohistochemistry with GS demonstrates intense staining of broad interconnected groups of hepatocytes, creating a maplike (geographical) pattern of staining ( Fig. 56.11 ). However, small areas in the periseptal region are often negative. A maplike pattern may not be clearly apparent around the central scar.

Differential Diagnosis

Nodular architecture, fibrous septa, ductular reaction, and large, thick-walled arterioles in fibrous stroma favor FNH over HCA, but there is overlap in the morphological features of FNH and inflammatory HCA (see Table 56.3 ). Immunohistochemistry with GS and SAA/CRP plays an important role in diagnosis on needle biopsy. SAA can be positive in 10% to 20% of FNH cases, while CRP typically shows periseptal staining that fades in areas away from the septa. Although focal plate thickening and focal reticulin loss can mimic HCC, these changes are confined to small areas of FNH (typically the periphery). The focality of these changes as well as a maplike GS pattern helps in its distinction from HCC.

Treatment

Surgical resection is usually not necessary for FNH unless the lesion is pedunculated, large, or symptomatic. Of course, excision of FNH is justified if the lesion cannot be differentiated from HCC or HCA on biopsy.

Macroregenerative and Low-Grade Dysplastic Nodules

In cirrhosis, benign nodules without significant atypia larger than 1 cm in size are referred to as large regenerative nodules or macroregenerative nodules. These are otherwise similar to conventional regenerative cirrhotic nodules, and they lack cytoarchitectural atypia or reticulin abnormalities. Portal tracts can be present within these nodules ( Figs. 56.14 and 56.15 ). Fibrous septa can extend into the nodule and may have a few scattered arterioles without accompanying bile ducts. Increased sinusoidal CD34 staining may be seen, but diffuse staining is typically absent. Low-grade dysplastic nodules are believed to represent a clonal proliferation of hepatocytes that lack high-grade features ( Table 56.5 ). Findings such as Mallory hyaline, bile stasis, clear cytoplasm, iron or copper deposits, a slight decrease in cell size, and focal or diffuse fatty changes may be present in these nodules. Low-grade dysplastic nodules are often indistinguishable from large regenerative nodules unless clonality analysis is performed. Low-grade dysplastic nodules lack a strong association with HCC and do not require specific treatment. Hence the distinction of large regenerative nodule and low-grade dysplastic nodules is not clinically relevant.

Regenerative nodules may occur in a noncirrhotic liver in a variety of clinical settings, such as nodular regenerative hyperplasia, vascular diseases such as Budd-Chiari syndrome, congenital heart disease and portal vein thrombosis, or as sequela of hepatocyte necrosis. These nodular proliferations can have a morphological appearance of an HCA or FNH, and the distinction is based on the overall clinical setting.

High-Grade Dysplastic Nodules

High-grade dysplastic nodules, previously referred to as borderline nodules, are preneoplastic lesions that can arise in cirrhosis of any etiology. These come to clinical attention on imaging, or they may be encountered by the pathologist in resections or explants. The reported prevalence in cirrhosis varies widely with numbers ranging from 11% to 40%.

Clinical Features

HCC is the most common primary malignant tumor in the liver. The presenting signs and symptoms include abdominal pain, weight loss, and hepatomegaly. In some cases, a palpable mass may be clinically apparent. Features of cirrhosis or underlying liver disease can be present. Rare cases can be associated with paraneoplastic syndromes such as hypoglycemia, erythrocytosis, hypercholesterolemia, hypercalcemia, precocious puberty, gynecomastia, hypertrophic pulmonary osteoarthropathy, hyperthyroidism, dysfibrinogenemias, and skin manifestations.

In the United States, approximately 85% of HCCs occur in patients with cirrhosis. The incidence is low in Europe and North America (2 to 7 cases per 100,000 persons) and high in eastern Asia and southern Africa (30 cases per 100,000 persons). The mean age of occurrence depends on the specific geographical location, being approximately 60 years of age in North America, approximately 35 years in Africa, and 40 to 60 years in Taiwan. HCC is three times more common among men than women, and it is the fifth most common malignancy in men and the eighth most common in women worldwide.

AFP, an oncofetal protein, is synthesized by the yolk sac and fetal hepatocytes, but it is very low or undetectable in healthy adults. Elevated serum AFP is observed in 70% to 80% of patients with HCC, with levels of >400 ng/mL in about 50% of cases. Large tumor size, poor differentiation, and advanced stage correlate with higher AFP levels. However, AFP may be normal in tumors <2 cm, nearly 40% of early cases, and even 10% to 15% of advanced cases. On the other hand, mild to moderate elevations (typically <400 ng/mL) are often observed in benign conditions such as tyrosinemia, steatosis, chronic hepatitis, and cirrhosis. Elevated AFP levels can also occur in nonhepatocellular tumors such as gastric/colonic adenocarcinomas, ovarian tumors, and germ cell tumors. Patients with cirrhosis are screened every 6 months by ultrasound and serum AFP level.

Imaging

HCC may be more echogenic or less echogenic than adjacent liver parenchyma. Contrast-enhanced CT or MRI has high sensitivity for the diagnosis, but lesions smaller than 2 cm may not show the characteristic enhancement in the arterial phase and washout in the venous phase. The Liver Imaging Reporting and Data System (LI-RADS) is used to classify liver lesions into five categories based on radiological features: LR-1 (definitely benign), LR-2 (probably benign), LR-3 (moderate probability of benign or malignant), LR-4 (probably malignant), and LR-5 (definitely malignant). Liver biopsy is recommended for diagnosis for mass lesions in noncirrhotic liver and in smaller (<2 cm) and radiologically indeterminate lesions in cirrhotic liver.

Risk Factors

The major etiological association is cirrhosis, which is present in more than 80% of cases. Cirrhosis related to alcohol use and viral hepatitis accounts for most of the HCC cases in the United States. There is also a high risk of HCC in the setting of cirrhosis resulting from hemochromatosis or α ₁ -antitrypsin deficiency. HCC can also develop with chronic viral hepatitis (B and C) in the absence of cirrhosis. Diabetes, obesity, and metabolic syndrome have been increasingly linked to higher risk for HCC. ^, Other risk factors include exposure to thorium dioxide (Thorotrast), aflatoxins, and estrogenic steroids.

Pathogenesis

HCC results from a multistep process that often occurs in the setting of various risk factors (e.g., chronic hepatitis), exposure to certain toxic or viral agents, and genetic alterations (see Chapter 44 for details).

Pathological Features

On gross examination, HCC may form a solitary mass or multiple scattered discrete nodules. Rarely, the entire lesion is composed of multiple nodules similar in size to typical cirrhotic nodules ( Fig. 56.16 ), and this has been referred to as cirrhosis-like or cirrhotomimetic variant. Most tumors have a circumscribed border and are well delineated from the surrounding liver, while a minority of tumors have an infiltrative appearance. A fibrous capsule can be present. Most HCCs lack abundant stroma and hence are soft, with variable necrosis. In contrast, abundant stroma is present in fibrolamellar and scirrhous variants, which tend to be firm and gray-white. HCC nodules may have a yellow hue if rich in fat, while a green color can result from bile.

Invasion of the portal or hepatic vein or their large branches can be seen, particularly in association with large tumors. Small HCCs (<2 cm in diameter) usually lack vascular invasion, necrosis, and hemorrhage. The nonneoplastic liver often shows cirrhosis.

The tumor manifests a variety of growth patterns: trabecular, pseudoacinar, and compact. The trabecular (platelike) pattern is the most common ( Figs. 56.17 and 56.18 ) and mimics the cell plate architecture of normal liver. However, the cell plates are typically three or more cells thick and are lined by endothelial cells. Occasionally, the tumor cell plates or trabeculae may be separated by fibrous tissue instead of endothelial cells ( Fig. 56.19 ). The pseudoacinar (pseudoglandular) pattern is characterized by glandlike or pseudoacinar spaces lined by tumor cells ( Fig. 56.20 ) and can lead to a misdiagnosis of adenocarcinoma when prominent. The spaces represent dilated canaliculi and may contain bile or proteinaceous material. The spaces can also develop as a result of central necrosis within trabeculae and contain protein, cellular debris, or macrophages. The pseudoacinar pattern is frequently admixed with the trabecular pattern ( Fig. 56.21 ). The compact (solid) pattern of HCC is characterized by sheets of tumor cells that lack clearly identifiable endothelial cell–lined trabeculae or cell plates ( Fig. 56.22 ). It is more common in poorly differentiated HCCs. Compressed trabeculae may be highlighted with endothelial cell markers in some cases.

The tumor cells often show small cell change (see Figs. 56.17 and 56.18 ). Large cell change is less frequent, except in high-grade tumors. Admixed foci of small and large cell change can occur. Kupffer cells are typically absent in HCC.

The tumor cells in HCC tend to be polygonal with some resemblance to normal hepatocytes, at least in well-differentiated and moderately differentiated cases. Most tumors reveal a variable degree of atypia in the form of nuclear pleomorphism and prominent nucleoli. Intranuclear vacuoles (composed of cytoplasmic invaginations) and glycogenation of nuclei are common ( Fig. 56.23 ). The cytoplasm may be more basophilic compared with normal hepatocytes, may have a granular appearance, or may be brightly eosinophilic as a result of a large number of mitochondria. Mallory hyaline, globular acidophilic bodies, or cytoplasmic inclusions (related to proteins such as albumin, fibrinogen, α ₁ -antitrypsin, or ferritin) may be seen (see Fig. 56.23 ).

Fat ( Fig. 56.24 ) or glycogen may be prominent in some HCCs, leading to a clear cell appearance. Other less frequent cytoplasmic changes include pale bodies, which are round to oval, lightly eosinophilic or clear cytoplasmic structures most frequently seen in fibrolamellar HCCs. Ground-glass cells containing hepatitis B surface antigen are found in some patients with HBV infection, and dark brown to black pigment similar to that seen in the Dubin-Johnson syndrome can be present.

In HCC, reticulin stain typically shows loss or fragmentation of the reticulin framework, although the reticulin pattern can be intact in very-well-differentiated cases (see Fig. 56.18 ). Reticulin stain is not helpful for HCC with prominent fat because reticulin loss can also be present in nonneoplastic liver with steatosis ( Fig. 56.25 ). Areas with no or less prominent steatosis should be sought in the tumor to evaluate the reticulin framework.

Histological Grading of Hepatocellular Carcinoma

The original system developed by Edmondson and Steiner in 1954 had four grades. However, the WHO 2019 scheme categorizes HCC into three grades : well-differentiated, moderately differentiated, and poorly differentiated.

• 1.

  Well-differentiated tumor cells resemble mature hepatocytes, with minimal to mild nuclear atypia. The cytoplasm ranges from abundant and eosinophilic to moderate and basophilic.

• 2.

  Moderately differentiated tumor cells resemble hepatocytes and show moderate nuclear atypia. Multinucleated cells occasionally can be present. The cytoplasm ranges from abundant and eosinophilic to moderate and basophilic.

• 3.

  Poorly differentiated tumor cells may or may not appear hepatocellular on morphology, and they show marked nuclear pleomorphism. Anaplastic giant cells can be present, and cytoplasm is scant to moderate and usually basophilic.

“Small” (Early and Progressed) Hepatocellular Carcinoma

By definition, small HCCs are tumors that measure 2 cm or less in diameter. These are divided into early HCC and progressed HCC . Early HCCs are very-well-differentiated tumors with indistinct margins and no capsule. Microscopically, these tumors often show fat, pseudoacinar architecture, few intratumoral portal zones, thin cell plates (<3 cells thick), few unpaired arteries, and sparse sinusoidal staining with CD34. These HCCs closely resemble high-grade dysplastic nodules, but they are distinguished by the hallmark feature of stromal invasion. The latter is characterized by invasion of tumor cells into adjacent septa, hepatic parenchyma, or portal tracts. Invasion into intratumoral portal tracts can also be present. Unlike regenerative nodules in cirrhosis, areas of stromal invasion lack a ductular reaction at the tumor-stroma interface, a feature that can be highlighted by CK7 staining. In contrast, small progressed HCCs have distinct outlines and often show a capsule. Microscopically, these tumors are indistinguishable from larger HCCs with clearly evident cytoarchitectural atypia, lack of portal tracts, more unpaired arterioles, and increased sinusoidal staining with CD34.

Histological Variants of Hepatocellular Carcinoma

Several histological variants of HCC are recognized in the WHO 2019 classification and together account for a third of all HCCs ( Table 56.6 ). These include scirrhous, fibrolamellar, steatohepatitic, clear cell, lymphocyte-rich, neutrophil-rich, macrotrabecular massive, chromophobe, and sarcomatoid variants. The importance of recognizing these histological variants is primarily to avoid errors in diagnosis. Apart from fibrolamellar HCCs, there are no treatment differences based on histological variants of HCC.

The scirrhous variant (4% of all HCCs) is characterized by abundant fibrous stroma composing more than 50% of the tumor (see Fig. 56.22 ) and can be mistaken for cholangiocarcinoma or metastatic adenocarcinoma. Atypical radiological features, low sensitivity of Hep Par 1 and pCEA, as well as frequent staining with CK7/CK19/MOC31, can add to the diagnostic confusion. Arginase-1 and glypican-3 are positive in most cases. The term sclerosing hepatic carcinoma was used in older literature and likely included both HCCs and cholangiocarcinomas associated with hypercalcemia; it is not a recognized variant, and this term should be avoided.

The fibrolamellar variant (1% of all HCCs) occurs mainly in young adults without cirrhosis (mean age, 26 years) and shows a higher incidence in women than in men. The most common clinical symptoms include abdominal pain or swelling, anorexia, weight loss, jaundice, and hemoperitoneum (rare). No definite risk factors have been identified in patients with this tumor type. FNH-like changes have been observed adjacent to fibrolamellar HCC in some cases, ^, but this is likely secondary to vascular changes adjacent to the tumor, and not a true precursor lesion. Except in rare cases, serum AFP levels are usually normal. ^,

Fibrolamellar HCCs are typically well-circumscribed, nodular, yellow- to brown-colored tumors with extensive fibrosis. A prominent central fibrous zone, as seen with FNH, may be identified in some cases. Larger tumors may show foci of hemorrhage and necrosis. Satellite lesions are rare. A triad of microscopic features is essential to establish an accurate diagnosis: dense bands of lamellar fibrosis ( Fig. 56.26 ), polygonal-shaped tumor cells with eosinophilic granular cytoplasm, and large vesicular nuclei with prominent nucleoli. Cytoplasmic pale bodies, which may contain fibrinogen and albumin can be present, but they are not specific because they can occur in conventional HCC as well. Other features that may be present include acinar structures, bile, mucin (rare), eosinophilic globules, multinucleated tumor cells, copper, fat, epithelioid granulomas, and peliosis hepatis.

Fibrolamellar HCCs are typically positive for hepatocellular markers by immunohistochemistry. In addition, most tumors are positive for CK7 and CD68; negative results with these markers should raise doubt regarding the diagnosis, but positive results are not diagnostic because they can also be positive in conventional HCCs. Staining for CK19 and epithelial cell adhesion molecule (EpCAM) is positive in up to 30% of cases. Neuroendocrine markers may be focally positive as well.

In classic cases of fibrolamellar HCC, it is not difficult to differentiate it from benign lesions such as FNH and adenomas. Strict reliance on the characteristic triad of microscopic features is essential to distinguish it from conventional HCCs and other variants such as scirrhous HCC.

Fibrolamellar HCC is characterized by deletion of 400 kb on chromosome 19 that leads to fusion of the promoter and first exon of DNAJB1, a heat shock protein, with nine exons of PRKACA, the catalytic domain of protein kinase. The resulting fusion transcript codes for a chimeric protein that likely plays a role in the pathogenesis of the tumor. This deletion has been reported in nearly all fibrolamellar HCCs, but not in other liver tumors including conventional HCC, hepatoblastoma, or intrahepatic cholangiocarcinoma. This fusion can be demonstrated by fluorescence in situ hybridization (FISH) and can help in the diagnosis of morphologically ambiguous cases. The same fusion was recently described in oncocytic pancreatic and biliary neoplasms. ^,

Fibrolamellar HCCs, in general, have a more favorable prognosis when compared with HCC as a whole, but the outcome is similar when compared with stage-matched conventional HCCs in patients without cirrhosis. Complete resection of the involved lobe is the preferred form of therapy. When tumor location precludes surgical resection, liver transplantation is an option.

Steatohepatitic variant (5% to 20% of all HCCs) shows features of steatohepatitis in the tumor cells in the form of steatosis, ballooning, Mallory hyaline, inflammation, and pericellular fibrosis ( Fig. 56.27 ). ^, It has been suggested that at least three of these features should be present for a diagnosis of this variant and that changes should involve at least 50% of the tumor ; however, these cutoffs are not included in the WHO classification. This variant typically occurs in the setting of metabolic risk factors for fatty liver disease, and the nonneoplastic liver may show steatohepatitis. A minority of cases can occur in other settings such as hepatitis C.

Clear cell variant (<10% of all HCCs) is characterized by cytoplasmic glycogen accumulation in the tumor cells imparting a clear appearance involving at least 80% of the tumor cells. ^{, ,} Occasional fat vacuoles can be present. This variant is thought to have a more favorable prognosis based on a limited number of cases. The main importance of this variant is its morphological resemblance to other clear cell neoplasms such as renal cell carcinoma. The distinction is usually straightforward on immunohistochemistry as clear cell HCCs are positive for hepatocellular markers and yield negative results with renal markers like PAX8.

Lymphocyte-rich variant (<1% of all HCCs) refers to HCCs with a prominent lymphoid infiltrate in which the lymphocytes outnumber the tumor cells in most fields ( Fig. 56.28 ). The prominent lymphoid infiltrate can mimic a lymphoma. A syncytial growth pattern similar to nasopharyngeal carcinoma can be seen. Epstein-Barr virus (EBV) is typically negative in the tumor cells, although rare positive cases have been described. Microsatellite instability is not observed in these tumors. These tumors have a more favorable prognosis and may have high PDL-1 expression with a potential for immunotherapy.

Neutrophil-rich variant (<1% of all HCCs) refers to HCCs with marked diffuse neutrophilic infiltrate in the tumor ( Fig. 56.29 ), usually as a result of granulocyte colony-stimulating factor production by the tumor cells. ^, The patient may have systemic symptoms like fever and leukocytosis, which along with the histological picture dominated by neutrophils, can be mistaken for an infectious process. A poorer prognosis has been ascribed to these tumors.

Macrotrabecular massive variant (5% of all HCCs) is a recently described variant characterized by thick trabeculae (≥10 cells thick per the WHO classification, ≥6 cells thick in some studies) composing more than 50% of the tumor. ^{, ,} This variant has been associated with high serum AFP, TP53 mutations, FGF19 amplification, and a poor prognosis.

Chromophobe variant (<5% of all HCCs) is a recently described and not yet fully characterized variant that shows light staining cytoplasm (chromophobe), mostly bland tumor nuclei, and focal areas of marked nuclear atypia. Scattered pseudocysts can be present. This variant has been associated with alternative lengthening of telomeres but has no known prognostic significance.

Sarcomatoid variant (<5% of all HCCs) was recognized as a variant of HCC in the prior WHO scheme, but it is not mentioned as a variant in the WHO 2019 scheme and is regarded as an example of poorly differentiated HCC. Other terms like spindle cell carcinoma and carcinosarcoma have also been used synonymously, while the latter has been employed by some for cases with heterologous elements. The sarcomatoid component may show malignant spindle, epithelioid, or pleomorphic cells; areas of conventional HCC are necessary to make the diagnosis ( Fig. 56.30 ). Heterologous differentiation along the lines of leiomyosarcoma, rhabdomyosarcoma, chondrosarcoma, and osteosarcoma can occur in classical HCC following chemotherapy or transarterial chemoembolization. Immunohistochemistry for pancytokeratin is positive in the sarcomatous area in a majority of cases, while hepatocellular markers are usually negative. The diagnosis cannot be established with certainty in the absence of conventional HCC areas. The clinical setting of cirrhosis and positive keratin may help favor sarcomatoid HCC in these cases. The prognosis is poorer compared with classical HCC. ^,

Immunohistochemical Markers in Hepatocellular Carcinoma

Hepatocellular Markers

Arginase-1 (Arg-1) is a urea cycle enzyme that is produced only in hepatocytes. Cytoplasmic staining, either with or without nuclear staining with Arg-1, has high sensitivity and specificity for HCC (both >80%). Staining is also present in 70% to 80% of poorly differentiated and scirrhous cases. ^, Arg-1 can be negative in 5% of well-differentiated cases. Most nonhepatocellular tumors are negative for Arg-1, but focal staining has been described in various adenocarcinomas in rare cases. Furthermore, hepatoid adenocarcinomas in the gastrointestinal tract, pancreas, and in other sites are typically positive for Arg-1.

Hepatocyte antibody (i.e., clone OCH1E5.2.10 or Hep Par-1 antibody) demonstrates a granular cytoplasmic pattern in most HCCs, but it may be negative in approximately half of poorly differentiated HCCs and scirrhous HCCs. Aberrant staining is present in many adenocarcinomas, including those from the stomach and lung, and less commonly cholangiocarcinomas and neuroendocrine neoplasms.

Glypican-3 (GPC-3) is an oncofetal antigen that has a high degree of sensitivity for poorly differentiated and scirrhous HCC, while more than 50% of well-differentiated HCCs as well as benign hepatocytes are typically negative ( Fig. 56.31 ). Occasional cirrhotic nodules and high-grade dysplastic nodules may also show patchy GPC-3 staining. Many nonhepatocellular tumors, such as squamous cell carcinoma, melanoma, nonseminomatous germ cell tumors, and rare cases of cholangiocarcinoma can also be GPC-3 positive.

Polyclonal antibody to carcinoembryonic antigen (pCEA) yields a canalicular pattern of staining in most HCCs. It cross-reacts with biliary glycoprotein in the bile canaliculus. Cytoplasmic and/or luminal staining is usually present in most adenocarcinomas. However, the sensitivity of pCEA is low in poorly differentiated HCC and scirrhous HCC. Occasionally, it can be difficult to distinguish a canalicular pattern of staining from a membranous or luminal staining pattern commonly seen in adenocarcinomas.

Albumin in situ hybridization (not immunohistochemistry) is a useful marker of primary hepatic origin, as it is positive in both HCC (80% to 90%) and intrahepatic cholangiocarcinomas (iCCAs; 60% to 70%), but is negative in most other carcinomas. Pancreatic acinar cell carcinomas can be strongly positive, while focal staining can occur in carcinomas from a variety of sites such as gallbladder, breast, and lung.

Other hepatocellular markers include CD10, bile salt export pump (BSEP), and villin, which show a canalicular pattern similar to pCEA in HCCs. AFP has a low sensitivity (30%) and is usually negative in well-differentiated cases.

Differential Diagnosis

HCC must be distinguished from FNH, adenoma, and high-grade dysplastic nodules at the well-differentiated end of the spectrum (see earlier sections) and from cholangiocarcinoma and nonhepatocellular tumors for moderate or poorly differentiated cases ( Tables 56.7 to 56.9 ).