Squamous cell

Squamous cell carcinomas are typically not primary to the liver. These tumours often undergo necrosis with suppuration that mimics abscesses and obscures tumour cells. Keratin-induced granulomas can also be seen.

Small/intermediate cell

Both HCC and ICC may have small-cell features. Apart from the usual list of small round cell tumours, the pathologist must be aware of inflammatory/reactive haematological processes that also have small-cell features, although haematological malignancies tend to be diffuse when they involve the liver (see haematopoietic pattern later). Small round cell tumours are relatively common in the paediatric age-group.

Clear cell

The clear cell pattern is typically caused by glycogen deposits or fatty change of various types that can result in clear to bubbly cytoplasm. The classic prototypes of clear cell carcinomas are renal cell and adrenocortical carcinomas, but both HCC and ICC may also have clear cell features.

Fat-containing cell

Hepatocellular nodular lesions with fatty change (steatosis) should be distinguished from steatosis of the background liver. It is also important to consider lipomatous lesions or other tumours that may contain fat cells (e.g. angiomyolipoma) and neoplastic/degenerative conditions that may contain cells mimicking intracytoplasmic ‘fat’ vacuoles.

Pleomorphic and giant cell

These tumours are typically poorly differentiated malignancies with high-grade pleomorphic and giant cell features (but not spindle cells). The giant cells are much larger and with multinucleation; tumour giant cells may exhibit extraordinary malignant atypia and mitotic activity, and these types are common in the more poorly differentiated HCCs. Histiocytic giant cells, as well as neoplastic giant cells of carcinomas and sarcomas (including undifferentiated/embryonal sarcoma of the liver), also could fall into this category.

Spindle cell

The spindle cell pattern consists of elongated or fusiform cells, often arranged in interlacing fascicles. Differential diagnoses range from reactive fibroblastic stroma to mesenchymal tumours. Sarcomatoid carcinomas may also have this pattern and, when primary in the liver, may be of hepatocellular or biliary origin.

Haematopoietic/inflammatory cell

These tumours range from inflammatory/reactive to proliferative lesions. Intrahepatic and perihilar inflammatory masses may form as a result of distal obstructions, and such obstructions in turn may result from a malignant stricture or intraductal papillary neoplasm. Additionally, marked inflammatory reactions (both neutrophilic and lymphocytic) can be rarely seen in HCC or in tumours with extensive necrosis. Thus the clinician should consider the possibility of a mixture of reactive and malignant components.

Other cells

Germ cell tumours, such as teratoma and yolk sac tumour, have characteristic features.

Aetiology

The pattern of presentation of hepatocellular adenoma (HCA) has changed in the recent past such that the incidence is increasing, particularly in certain clinical and aetiological settings. As a result, we now recognize at least four subtypes of HCA: hepatocyte nuclear factor 1α-inactivated (HHCA), inflammatory (IHCA), β-catenin-activated (BHCA) and unclassified, each with different aetiological, histological and molecular characteristics ( Table 13.5 ). HCA is a benign neoplasm that typically arises in noncirrhotic liver; reports of HCAs in cirrhotic liver almost always represent macroregenerative nodules or extremely well-differentiated hepatocellular carcinomas, except in rare cases when definitive subtyping of an adenoma can be proven. HCA typically still occurs in women in the reproductive age-group (15–45 years). In the more remote past, its association with oral contraceptive (OC) use was well established, but the incidence of OC-related adenomas greatly decreased in the last few decades with the introduction of lower-dose OC preparations. HCA can also increase in size during pregnancy, when oestrogen levels are high. This association supports that sex hormones play a role in tumourigenesis by promoting cell proliferation, but are not the initiators of hepatocellular neoplasms. Higher risk of HCA is also associated with noncontraceptive oestrogens and anabolic-androgenic steroids.

HCA has been reported in association with several hereditary disorders, and multiple adenomas can be seen in these patients. HCA can occur in type Ia glycogen storage diseases and less often in types III and IV. Patients with these disorders may develop multiple adenomas. Maturity-onset diabetes of the young, type 3 (MODY3), a form of autosomal dominant familial diabetes mellitus with germline mutations involving TCF1 gene, can be associated with HCAs (see subtypes later) and multiple adenomas as well. Other, less common associations are familial adenomatous polyposis, McCune–Albright syndrome, Fanconi anaemia, clomiphene use and Klinefelter syndrome.

More recently, a significant increase in HCA has been related to rising levels of obesity and metabolic syndrome. In addition, some specific forms of HCA associated with obesity now appear to be increasing in incidence, especially the inflammatory variants and the β-catenin-activated subtype. These obesity-related lesions present in a slightly older age-group in women, but also in men. Moreover, it is proposed that overweight/obesity could represent a major risk of malignant transformation of HCA, possibly via the IL-6 pathway. Chronic vascular disorders may also increase the risk for adenomas.

Clinical features

The mean age for patients with HCA was 30 years in older series, whereas the mean age has been 37–41 years in more recent series, reflecting changes in aetiology and risk factors for HCA. HCA occasionally occurs outside the reproductive age-group, but these are still uncommon. In the earlier series, patients usually came to medical attention with symptoms of abdominal pain, mass or haemorrhage. In more recent series, however, the majority of tumours were discovered incidentally on imaging, and about 20% of patients present with acute abdominal pain caused by haemorrhage into the tumour or into the peritoneal cavity, which may be life threatening.

HCAs demonstrate variable echogenicity on ultrasound (US) and cannot readily be distinguished from other lesions. On computed tomography (CT), HCAs are iso- or hypoattenuating and often heterogeneous with hyperattenuating areas corresponding to recent bleeding. On multiphasic CT, there is arterial enhancement and a persistent enhancement in the delayed phase. On magnetic resonance imaging (MRI), HCAs are typically hyper- or isointense relative to adjacent liver on T1-weighted images and slightly hyperintense on T2-weighted images. The presence of fat and telangiectasia lead to distinctive appearances on contrast-enhanced US and MRI and may be able to determine HCA subtypes with an accuracy approaching 85%.

Pathology

By definition, HCA is a benign neoplasm composed of hepatocytes and almost always arises in a noncirrhotic liver. Multiple tumours may be present, and when ≥10 are present, this process has been designated as ‘adenomatosis’ (see later). The tumours often bulge from the surface of the liver and occasionally are pedunculated. The majority of tumours are 5–15 cm but can measure up to 30 cm in diameter. The colour varies from yellow or tan to brown ( Fig. 13.1 ), and there may be green areas of bile production as well as areas of necrosis or haemorrhage. Irregular areas of fibrosis may be present. Lesions may be well circumscribed or not, and in the latter case, the adenoma can be difficult to differentiate from the surrounding liver ( Fig. 13.2 ).

Hepatocellular adenoma. The tumour is tan to brown (after formalin fixation), similar to background liver and well demarcated. Zones of fibrosis (darker brown) are present.

Hepatocellular adenomas, inflammatory type. This lesion has a red-brown appearance with irregular, poorly circumscribed borders (fresh sample). There are three larger lesions and at least two smaller lesions on the left liver sample, the latter noted as small areas of darker red-brown parenchyma. On the right sample, there are at least four small inflammatory adenomas.

Microscopically, HCA is typically composed of hepatocytes arranged in thin cell plates (typically one or two cells thick) without cytological atypia ( Fig. 13.3 ). Tumour cells are typically uniform in size and shape, but some variation can be seen, which may result from regenerative and atrophic/ischaemic changes within the tumour. The reticulin framework is intact, outlining the cell plates in a pattern similar to that seen in normal liver without loss or fragmentation ( Fig. 13.4 ). In certain HCA subtypes, however, particularly the HHCA, there may often be near to complete encircling of small groups of tumour cells by reticulin fibres (‘packeting’). The nuclei of tumour cells are typically uniform and regular; the nuclear/cytoplasmic ratio is low; and mitoses are almost never seen (see Fig. 13.3 ). Nucleoli are seldom prominent. Occasional tumours, especially in patients with long exposure to contraceptive steroids, may have a few pleomorphic nuclei resembling large-cell change in cirrhosis.

Hepatocellular adenoma. The tumour cells are uniform with minimal cytological atypia and are arranged in architecturally normal hepatic plates. Minimal fatty change and mild focal inflammatory infiltrate are also present in this case.

Hepatocellular adenoma. The cell plate reticulin framework is intact, demonstrating a pattern similar to that seen in normal liver. This example shows plates that are one to two cells thick. Mild fatty change is also present. (Reticulin stain.)

Features that are variably present include fat, glycogen production, cytoplasmic lipofuscin granules, Dubin–Johnson pigment (pigmented liver cell adenoma) and rarely, Mallory–Denk bodies. The fat may be abundant, simulating fatty liver, particularly in some HHCA and IHCA subtypes. The sinusoids can be compressed, leading to a sheet-like appearance. Sometimes the sinusoids are dilated and can be mistaken for peliosis hepatis. Thin-walled vascular channels and small arteries are scattered throughout the tumours, whereas large arteries are seen at the periphery. The arteries are unpaired (lack corresponding bile ducts), because interlobular bile ducts are not found in HCA. However, bile ductules and progenitor cells may be present, particularly in the IHCA subtype. Kupffer cells are present but usually inconspicuous; stellate cells are occasionally seen. Haematopoietic elements may be noted in the sinusoids, and rare cases with noncaseating granulomas have been described. Areas of fresh haemorrhage and haemosiderin-laden macrophages from past haemorrhages may be seen, along with recent or previous infarcts. Immunohistochemically, the endothelial cells lining the plates can stain positively to variable degrees for CD34, similar to that seen in hepatocellular carcinoma (HCC). Other markers typically used for HCC, such as glypican 3 (GPC3) and alpha fetoprotein (AFP), are negative.

Cytologically, FNA aspirates are hypercellular, comprising small cohesive groups of almost normal-looking hepatocytes with transgressing endothelium in the absence of bile duct epithelium ( Fig. 13.5 ). The neoplastic hepatocytes may display fat vacuoles, pale glycogen-laden cytoplasm or pigments. They have relatively small, uniform central round nuclei with normal nuclear/cytoplasmic ratio (≤1 : 3 by estimating comparison of nuclear and cell diameters). Differential diagnoses include other well-differentiated hepatocellular nodules. Cell blocks may not prove sufficient for immunohistochemical (IHC) subtyping. An indeterminate cytodiagnosis may be proffered in the absence of appropriate clinical and radiological information.

Hepatocellular adenoma; aspiration biopsy. Small, cohesive groups of neoplastic cells resemble normal hepatocytes, with ample granular cytoplasm, uniform central round nuclei, small nucleolus and normal nuclear/cytoplasmic ratio. (May–Grünwald Giemsa stain [MGG] stain.)

Histological subtypes

Correlation of specific genetic mutations with clinical and histological features led to the recognition of subtypes of HCA ( Table 13.5 ), which formed the basis of the 2010 classification adopted by the World Health Organization (WHO).

Hepatocyte nuclear factor 1α-inactivated (HHCA)

Biallelic inactivation of TCF1 gene that codes for hepatocyte nuclear factor 1α (HNF1α) was observed in about 40% of sporadic HCAs in early studies of adenoma subtypes. The gene defect was found to be somatic in 90% but germline in 10%. These tumours tend to occur in young women, are associated with OC use as well as MODY3 and are often characterized by prominent intralesional steatosis ( Fig. 13.6 ). Both large and small droplets can be present, but in many cases, small-droplet fat can predominate and may be overlooked as an important diagnostic finding. Minor architectural irregularities such as small acinar structures can be seen ( Fig. 13.6 ). Inflammation and cytological atypia are typically absent, but the reticulin framework pattern often has a ‘packeted’ appearance in which reticulin fibres can partially encircle small clusters of hepatocytes ( Fig. 13.7 ). HNF1α positively regulates the FABP1 gene, which codes for liver fatty acid-binding protein (LFABP). LFABP is expressed in the cytoplasm in normal hepatocytes, as demonstrated by immunohistochemistry (IHC), but is low or absent in HNF1α-inactivated adenoma (HHCA) ( Fig. 13.8 ). This is one of the subtypes that may be more frequently associated with adenomatosis, so the liver parenchyma should be examined for the presence of small fatty nodules (also LFABP negative), which represent microadenomas ( Fig. 13.9 ). The risk for malignant transformation is likely very low.

Hepatocellular adenoma, hepatocyte nuclear factor 1α-inactivated (HHCA) subtype. The tumour cells are relatively uniform in size, but most contain cytoplasmic, small or large droplets of fat. Small acinar structures are also present.

Hepatocellular adenoma, HHCA subtype. The reticulin framework is intact, but there is a prominent pericellular pattern of reticulin fibres nearly or completely encircling many of the tumour cells. Some mild variation is cell plate size is also present, imparting a slightly nodular microscopic appearance. (Reticulin stain.)

Hepatocellular adenoma, HHCA subtype. Normal hepatocytes stain positively for liver fatty acid-binding protein (LFABP) (upper left), but tumour cells in the HHCA subtype are negative. Note that the junction of the tumour and adjacent liver is irregular. (LFABP immunostain.)

Hepatocellular adenoma, HHCA subtype. The microscopic pale zone of hepatocytes with fatty change in patient with large HHCA was found to be LFABP negative (not shown) and thus represents a small microadenoma, not focal fatty change.

Challenges in HHCA diagnosis

Steatosis is not a specific finding because it can be observed in other subtypes (particularly the inflammatory type), while rare HHCAs may lack steatosis. Loss of LFABP is useful to establish the diagnosis, but the IHC staining has to be carefully titrated because staining in normal hepatocytes can be weak. Hepatocellular carcinoma (HCC) arising in HHCA is rare (<5%) and can be associated with β-catenin activation. Since LFABP loss can be observed in HCC, it should be used for histological subtyping after the diagnosis of HCA has been established based on other histological features, such as cytological and architectural findings.

Inflammatory (formerly ‘telangiectatic’) (IHCA)

The IHCA subtype accounts for 40–50% of HCAs and is most common in women, although it also can occur in men with appropriate risk factors. IHCA is often associated with obesity and metabolic syndrome; steatosis and steatohepatitis can be present in the non-neoplastic liver. These tumours are characterized by activation of the interleukin (IL)-6 signalling pathway (IL6/JAK/STAT3 pathway) resulting from somatic gain-of-function mutations in the IL6ST gene that encodes the signalling co-receptor gp130. These mutations are observed in 60% of IHCAs, leading to activation of signal transducer and activator of transcription 3 (STAT3) in the absence of ligand. Less often, somatic mutations in other genes involved in the pathway, such as Fyn-related kinase ( FRK ), STAT3 , GNAS and Janus kinase 1 ( JAK1 ), have been implicated in IHCA, all of which promote the constitutive activation of STAT3.

The histological hallmarks of IHCA are sinusoidal dilation, ductular reaction, haemorrhage and inflammation ( Figs 13.10 and 13.11 ). Arterioles, often in clusters, and surrounded by a small amount of fibrous tissue with or without ductular reaction at the interface, can mimic portal tracts, but the arterial zones lack interlobular bile ducts (‘pseudo-portal tracts’) ( Fig. 13.11 ). Ductular reaction is seen in almost half the cases. Fibrous septa may be present, and therefore, in combination with ductular reaction, the findings can mimic focal nodular hyperplasia (FNH), although a nodular architecture typical of FNH is seen in <10% of cases. However, because of the similarities to FNH, many of these cases were previously termed ‘telangiectatic FNH’. Based on imaging characteristics, clonality and protein-clustering studies, it is now recognized that most of these lesions are IHCA. The distinction cannot be made by the presence of sinusoidal dilation alone; this feature can be present in a minority of FNH. C-reactive protein (CRP) and serum amyloid-associated (SAA) protein, both acute-phase reactants, are overexpressed in the neoplastic hepatocytes, so IHC staining for these markers helps to confirm the diagnosis ( Fig. 13.12 ).

Hepatocellular adenoma, inflammatory (IHCA) subtype. Characteristic features of this subtype illustrated include sinusoidal dilation as well as a fibrous zone with ductular reaction, mild chronic inflammatory infiltrates and small arterioles.

Hepatocellular adenoma, IHCA subtype. Higher magnification of Fig. 13.10 details ductular reaction and inflammatory reaction as well as small arterioles in the fibrous zones.

Hepatocellular adenoma, IHCA subtype. The serum amyloid A (SAA) stains strongly, noted as small globules in the hepatocytes. (SSA immunostain.)

Challenges in IHCA diagnosis

Sinusoidal dilation and/or inflammation may not be seen in up to 10% of IHCAs. Both SAA and CRP typically stain these lesions in a diffuse manner, although SAA can be negative or minimal in 5–10% of cases with otherwise typical morphology, while CRP will be diffusely positive in an estimated >80% of tumour cells in essentially all these lesions. Thus, CRP has higher sensitivity, and completely negative results are rare. Since both SAA and CRP can be positive in the adjacent non-neoplastic liver, careful correlation with morphology and CD34 staining is necessary to avoid interpreting this as a positive result in the tumour, especially in limited needle biopsies. It is important to note that activation of β-catenin is seen in approximately 10% of IHCAs, resulting in diffuse staining with glutamine synthetase (GS) with or without nuclear β-catenin staining; these tumours are thought to have a higher risk of progression to HCC. A combination of gp130 alterations and β-catenin mutation is likely in these cases.

β-catenin-activated (BHCA)

The BHCA subtype occurs more often in men than do the other subtypes and often harbours atypical morphological features, such as cytological atypia, small-cell change and pseudoglandular/acinar architecture. Multifocal reticulin loss may occur ( Fig. 13.13 ), raising concerns for transition to HCC. Association with concurrent or subsequent HCC may occur in almost half the cases. These tumours are characterized by mutations in the exon 3 of CTTNB1 (β-catenin) gene, which is a critical component in the Wnt signalling pathway. In normal cells, β-catenin is located in the membranous region and can be demonstrated by IHC ( Fig. 13.14 ). Cytoplasmic β-catenin binds to a protein complex that targets it for proteasomal degradation. Deletions or point mutations in the exon 3 region interfere with the binding of β-catenin to the protein complex, which in turn interferes with the cytoplasmic degradation of the protein and leads to its nuclear translocation. Nuclear β-catenin activates T-cell factor/lymphoid enhancer factor (TCF/LEF) family of proteins, leading to expression of a variety of Wnt target genes that play an important role in cell proliferation. IHC demonstration of nuclear β-catenin can help to identify tumours with β-catenin-activation ( Fig. 13.14 ). Activated β-catenin leads to transcriptional upregulation of GLUL gene, which codes for GS, and diffuse GS staining can be demonstrated by IHC as evidence of β-catenin activation ( Fig. 13.15 ).

Hepatocellular adenoma of uncertain malignant potential, β-catenin (BHCA) subtype. The reticulin framework in this tumour shows focal loss and fragmentation, increasing the concern for hepatocellular carcinoma. (Reticulin stain.)

Hepatocellular adenoma, BHCA subtype. Focal, strong nuclear staining indicates a positive sign for this subtype. The cytoplasmic membrane staining is also seen in normal (or nonmutated) liver, so is not diagnostic. (β-Catenin immunostain.)

Hepatocellular adenoma, BHCA subtype. Strong, diffuse homogeneous staining with glutamine synthetase (GS) in this subtype strongly correlates with beta-catenin activation (BHCA on right, background liver on left).

Challenges in diagnosis of BHCA

In view of the atypical histological findings, frequent reticulin loss, high risk of HCC and rare instance of metastases in these BHCAs, it may be better to regard these tumours as extremely well-differentiated variants of HCC rather than adenomas. Cytogenetic abnormalities such as gains of 1q and 8q are typical of HCC and are often seen in adenoma-like tumours with β-catenin activation, further supporting their classification as HCC. In some cases, adenoma-like tumours with β-catenin activation do not show features that are sufficient for definite diagnosis of HCC. It has been suggested that these tumours should be characterized as ‘atypical hepatocellular neoplasm’ or ‘hepatocellular neoplasms with uncertain malignant potential’ (HUMP). In addition to tumours with borderline histological features and those with β-catenin activation, this designation has also been recommended for all adenoma-like tumours that occur in men, women >50 years, rare aberrant IHC features (e.g. GPC3 or HSP70 positivity) and those occurring in unusual clinical settings (e.g. glycogen storage disease, Fanconi anaemia). In addition, it is possible that the β-catenin activation can occur in any other HCA subtype (HHCA, IHCA or unclassified) as a ‘second hit’ in the progression of HCA to HCC, and thus BHCA may not truly represent a subtype of HCA. In initial studies, the BHCA subtype accounted for 10–15% of total HCA, but the numbers in more recent studies are less than 5%, perhaps reflecting that many tumours initially considered as BHCAs are now being classified as ‘extremely well-differentiated HCC’ if enough cytoarchitectural atypia is present.

Nuclear β-catenin staining is present in less than half of the tumours with known β-catenin mutation or deletion (see Fig. 13.14 ), whereas diffuse GS staining has greater sensitivity and better correlation with β-catenin activation (see Fig. 13.15 ). Therefore, absence of nuclear β-catenin on IHC does not exclude β-catenin activation in a tumour. When present, nuclear β-catenin staining can be focal, and any unequivocal nuclear staining irrespective of number of positive tumour cells should be regarded as a positive result. Because of possible nonspecific background cytoplasmic staining, it is recommended that cytoplasmic β-catenin staining in the absence of nuclear staining not be considered positive.

The interpretation of GS immunohistochemistry plays a critical role in the classification of HCA subtypes. GS is an enzyme involved in ammonia detoxification by combining it with glutamate to produce the amino acid glutamine. In normal liver, GS expression is limited to a narrow rim of hepatocytes around the central vein. This zonation is thought to be the result of β-catenin activation in centrizonal hepatocytes, presumably as a result of Wnt signalling from the central vein. Activation of β-catenin in neoplastic hepatocytes leads to diffuse cytoplasmic expression of GS in the tumour cells. Due to the low sensitivity of β-catenin IHC, it has become standard clinical practice in diagnostic pathology to use diffuse GS immunostaining as a marker of β-catenin activation. In this context, diffuse staining has been defined as ‘moderate to strong’ cytoplasmic staining in >50% of tumour cells. Diffuse staining can be seen in almost all the tumour cells (diffuse homogeneous staining), or it can be seen in >50% but not all tumour cells (diffuse heterogeneous staining). The interpretation of diffuse homogeneous GS staining is often straightforward, but it can be difficult to discern whether staining is seen in >50% of tumour cells when a diffuse heterogeneous pattern is encountered. In contrast to the near-perfect correlation between diffuse GS staining and β-catenin activation in HCAs reported in some studies and in the WHO classification, recent studies have shown that GS is not a perfect marker of β-catenin activation, and many cases with diffuse GS staining (perhaps 20–30%) may not have evidence of β-catenin mutation or deletion. The correlation is likely to be higher for diffuse homogeneous GS staining. The reasons for the discrepancy are unclear but may be related to activation of other components of Wnt signalling pathway, such as AXIN1 mutation or mechanisms unrelated to mutations in genes in Wnt signalling, including alterations in vascular flow. It thus seems prudent to diagnose these tumours as HCA or HUMP with diffuse GS staining and convey that an underlying β-catenin ( CTTNB1 ) mutation or deletion is likely but not certain. GS staining in other subtypes of HCA tends to be patchy and, by definition, involves <50% of hepatocytes. This patchy staining can be perivenular and is often more prominent compared to normal liver (‘expanded perivascular staining’). Peripheral accentuation can be seen. In some instances, the patchy areas of staining at the periphery can connect with each other, creating a pattern reminiscent of map-like pattern seen in FNH (‘pseudo-map-like’).

Mutations in exons 7 and 8 of CTTNB1 have recently been described in 10% of HCAs, many of which were previously categorized as unclassified. These cases do not show atypical morphological characteristics or diffuse GS staining typical of tumours with exon 3 CTTNB1 mutations. Based on initial results, it is thought that mutations in exons 7 and 8 do not confer the malignant potential associated with exon 3 CTTNB1 mutations, but may result in diffuse heterogeneous staining in a small subset of cases.

Specific clinicopathological settings

Treatment

Discontinuation of OCs, hormone-containing intrauterine devices (IUDs) and anabolic steroids may lead to regression of HCA. HCAs associated with GSD may regress with dietary therapy. Resection is recommended for tumours ≥5 cm because of the risk of rupture, haemorrhagic complications and associated HCC. Nonsurgical options such as embolization can be considered for patients who are poor surgical candidates. Since some HCAs have been reported to increase in size despite the discontinuation of OCs or anabolic steroids, and the development of HCC has been reported despite regression in size, it is recommended that patients with smaller tumours have CT or MRI at 6-month intervals for 2 years, with subsequent follow-up dictated by the growth pattern and stability of the tumour over time.

Differential diagnosis

Focal nodular hyperplasia can sometimes be difficult to distinguish from HCA. The presence of fibrous septa containing large arteries and ductular reaction favors FNH (see next section). However, these features can also be seen in IHCA, so the distinction can be challenging, especially in small biopsies. In these settings, the map-like GS staining in conjunction with histological features can confirm the diagnosis of FNH. Notes of caution, however, include that the periphery of HCAs may show a vague anastomosing pattern of staining reminiscent of the map-like pattern of FNH. In addition, SAA can be positive in 10–15% of FNH cases, typically in a focal manner, while CRP staining in the periseptal region is seen in most FNH cases without the diffuse staining typical of IHCA, with diffuse staining restricted to a small minority of FNH cases.

Distinguishing HCA from well-differentiated HCC can also be challenging and may be impossible on needle biopsies. The presence of wide cell plates, prominent pseudoglandular/acinar architecture, zones of small-cell change, cytological atypia and reticulin loss favour HCC (see Table 13.5 ). Diffuse sinusoidal staining for CD34 also favours HCC but is insufficient alone for the diagnosis. GPC3 and heat shock protein 70 (HSP70), if positive, strongly point to HCC. Similarly, the demonstration of cytogenetic abnormalities such as gains of chromosome 1 and 8 and TERT promoter mutation would indicate HCC, but these are not currently available for routine diagnostic use. If the histological changes are borderline, or if high-risk features such as male gender and β-catenin activation are present, it is prudent to designate the tumour as ‘atypical hepatocellular neoplasm’ or ‘HUMP’.

Pathology

More than 85% of FNH lesions are <5 cm in diameter, but larger lesions can occur. FNH is usually well circumscribed but nonencapsulated. The colour is lighter than that of the adjacent normal liver. On cut section, the lesion is subdivided into smaller nodules by fibrous septa that often run into a stellate scar that may be central or eccentric ( Fig. 13.16 ). Several scars may be seen in large lesions. A central scar is absent in 20% of cases. Microscopically, FNH is characterized by hyperplastic nodules that are separated by fibrous septa, often radiating from a central scar. The fibrous septa and central scar contain dystrophic thick-walled arteries and to a lesser extent, veins. The arteries are often eccentrically thickened and show intimal and fibromuscular hyperplasia with poorly formed elastic lamina ( Figs 13.17 and 13.18 ). The scar and fibrous septa typically show a ductular reaction, most prominent at the interface between the nodules and fibrous stroma, and variable lymphocytic inflammation, while interlobular bile ducts are absent. The hepatocellular component is arranged in plates one to two cells thick, with intact reticulin framework, but thicker plates may be present focally, often near the center and periphery of the lesion ( Fig. 13.19 ). Complete circling of small groups of hepatocytes by reticulin fibres (packeting) is also a common feature. Fat accumulation can be seen, especially in the patient with fatty liver disease (see Fig. 13.18 ). Steatohepatitic changes such as ballooned hepatocytes and Mallory–Denk bodies may also be present and should not be confused with steatohepatitic variant of HCC. The periseptal hepatocytes adjacent to the fibrous septa of the lesion often show chronic cholestasis, with cholate stasis (‘pseudoxanthomatous transformation’) and copper accumulation. These features of chronic cholestasis are present in many cases of FNH and can be very helpful in differential diagnosis.

Focal nodular hyperplasia, wedge resection. This lesion is well circumscribed, with a multinodular appearance. Note the centrally placed scar, with fibrous septa extending from the center toward the periphery.

Focal nodular hyperplasia. The central scar-like zone contains dystrophic, thick, irregularly shaped arteries. Fibrous septa radiate from the central scar, partially separating the nodular parenchyma. Ductular reaction is present, typically located at the interface of parenchyma and fibrous tissue. (Trichrome stain.)

Focal nodular hyperplasia. Fibrous septa with abnormal arteries and ductular reaction. Some sinusoidal dilation is present, as well a focal steatosis. A few scattered inflammatory cells are present in sinusoids as well as septa.

Focal nodular hyperplasia. The reticulin stain shows an intact framework, some of which is increased in amount of staining over that of normal liver. In addition, in this zone near the central scar, the cell plates are more irregularly shaped with rounded forms and more widened plates than seen in normal liver. (Reticulin stain.)

On IHC, FNH shows a highly characteristic geographical or map-like pattern on GS staining, with strong cytoplasmic staining of broad anastomosing groups of hepatocytes ( Fig. 13.20 ). The map-like pattern can be focal in needle biopsies and may not be well developed around the central scar or areas with fatty change. In normal liver, GS expression is limited to a narrow rim of hepatocytes around the central vein. This zonation most likely results from β-catenin activation in the centrizonal hepatocytes, which in turn may be the result of Wnt signalling from the central vein. Expansion of the β-catenin-activated centrizonal region leads to GS overexpression in the map-like pattern in FNH. β-catenin mutations are not observed. The ductular reaction is highlighted by cytokeratins 7 and 19 and neuronal cell adhesion molecule (NCAM). CD34 usually shows patchy sinusoidal staining, with diffuse staining in a small number of cases.

Focal nodular hyperplasia. Glutamine synthetase (GS) immunostain demonstrates focal broad zones of hepatocyte staining, alternating with zones of nonstaining hepatocytes, originally described as ‘map-like’.

On FNA aspiration cytology, a polymorphous population of cells is seen, comprising hyperplastic hepatocytes with normal nuclear/cytoplasmic ratio (≤1 : 3, by estimating comparison of nuclear and cell diameters), biliary elements, endothelium and Kupffer cells. Non-neoplastic hepatocytes in FNH show some mild variation in cell and nuclear size but maintain the normal nuclear/cytoplasmic ratio; they are not monotonous or pleomorphic, similar to that seen within a family of siblings, unless they are identical twins, so-called sibling variation. Anisonucleosis is also a distinct characteristic of non-neoplastic hepatocytes. Another helpful benign feature is the presence of biliary ductular clusters. They represent ductular reaction at the hepatocellular-stromal interface of the scar and appear as curved, double-stranded rows of small cells with overlapping, oval nuclei and scanty or absent cytoplasm ( Fig. 13.21 ). The rows can be seen emanating from hepatocytes. Ductular clusters should be distinguished from bile duct epithelium, which appears as flat sheets of uniform equidistant cells with discernible cytoplasm. Ductular reaction can easily be mistaken for adenocarcinoma, such as cholangiolocellular carcinoma. Endothelial and Kupffer cells are difficult to separate cytologically and are recognized as spindle/comma-shaped nuclei and nuclear streaks. Stromal fragments are rare and may mimic leiomyosarcoma. Regenerative and dysplastic nodules are the primary differential diagnoses.

Focal nodular hyperplasia; aspiration biopsy. Hyperplastic hepatocytes occur in cords up to two cells thick. The cells exhibit ample granular cytoplasm, central round nucleus, anisonucleosis, distinct nucleolus, normal nuclear/cytoplasmic ratio and binucleation. Note the double-stranded row of ductular cells with oval nuclei and absent cytoplasm overlapping the hepatocytes. (Papanicolaou stain.)

Variant forms

Telangiectatic FNH refers to a subset of cases with prominent sinusoidal dilation. Based on imaging features, clonality results and gene expression studies, most but not all ‘telangiectatic FNH’ cases are now classified as IHCA. ‘Focal nodular hyperplasia-like’ (FNH-like) lesion has been used to describe lesions morphologically similar to FNH, but that occur in the setting of liver disease (e.g. cirrhosis) and vascular disorders (e.g. idiopathic portal hypertension, hereditary haemorrhagic telangiectasia, haemangiomas, Budd–Chiari syndrome). These FNH-like lesions can be multiple and may not show the characteristic map-like pattern of GS staining seen in true FNH.

Differential diagnosis and treatment

FNH is a non-neoplastic lesion and does not require surgical resection even if large, unless the patient is symptomatic or the location of the lesion compromises liver function. Thus it is important to distinguish FNH from HCA and HCC (see Table 13.5 ). The nodular architecture, central stellate scar, fibrous septa, bile ductules and hepatocellular nodules distinguish FNH from HCA. However, these features can be seen in IHCA, and the distinction can be challenging on histological grounds alone, especially in needle biopsies. Map-like GS pattern is diagnostic of FNH and is not seen in IHCA. Staining with inflammatory markers such as SAA is typically negative or focal, while CRP staining is usually restricted to periseptal areas; diffuse staining typical of IHCA is uncommon in FNH. Sinusoidal dilation (telangiectasia) is more common in HCA but can be seen in 15–20% of FNH, and it cannot be relied on to distinguish these entities. Core needle biopsies can mimic ductopenic cirrhosis, but the clinical setting and presence of dystrophic arterioles suggest FNH.

Aetiology and molecular aspects

Congenital anomalies are present in approximately 5% of patients with HB, and reported associations with other congenital diseases include Beckwith–Wiedemann syndrome, familial adenomatous polyposis coli, Li–Fraumeni syndrome, trisomy 18, GSD types I–IV, hemihypertrophy and Simpson–Golabi–Behmel syndrome. Rare cases of congenital HB have been reported.

Cytogenetic studies and comparative genomic hybridization have revealed that tumour cells have a relatively stable genome and usually harbour a limited number of chromosomal abnormalities (mean, three changes per tumour). Recurrent genetic alterations involve chromosomes 2, 20, 1, 8 and X. Frequent trisomies 2 and 20, often seen as the only abnormality, have also been observed in other embryonic tumours such as embryonal rhabdomyosarcoma. The DNA content is frequently diploid in the fetal type and aneuploid in half the embryonal and small, anaplastic cell types. Microsatellite analysis revealed regions with allelic loss of chromosomes 1 and 11.

Several studies revealed that genes encoding components of the wingless/Wnt signal transduction pathway, including the APC , AXIN1 and CTTNB1 genes are mutated in a large proportion of cases (>80% in total). These mutations lead to abnormal activation and induction of growth-promoting genes and other survival pathways (e.g. Notch pathway) that can be differentially expressed in different histological subtypes of HB. The central molecule of this pathway is β-catenin, which carries mutations, mostly in-frame deletions, in more than half of HB cases. An additional gene reported to be affected by recurrent mutations in HB is NFE2L2 , which encodes a transcription factor involved in the antioxidant response pathway.

Other typical molecular features include aberrant reprogramming of imprinted genes such as IGF2, H19, DLK1, GTL2, PEG3, PEG10, BEX1, MEG3 and NDN, which are abundantly expressed in fetal liver. Based on recent transcriptional data, HB can be classified into two distinct groups. The C1 subclass recapitulates liver features at late stages of intrauterine life, with a mostly fetal phenotype and the expression of markers of mature hepatocytes. The C2 subclass has a predominantly embryonal phenotype, with the expression of markers of hepatic progenitor cells (e.g. K19, EpCAM).

Studies of gene expression profiling have compared HB with HCC and identified upregulation of expression of MIG6, TGFβ1, DLK1 and IGF2 in HB. These genes were differentially expressed between HB and HCC, but they did not separate HB histological subtypes.

Clinical features

Approximately 75% of children with HB are male. They usually present with abdominal swelling and less frequently with weight loss, anorexia, nausea, vomiting, abdominal pain and jaundice. Thrombocytosis and marked elevation of serum AFP is present at diagnosis in most patients. Although biopsy is necessary to confirm the diagnosis, imaging plays an important role to determine the location and extent of tumour, assess feasibility of surgery and evaluate vascular invasion and extrahepatic extension.

Pathology

Hepatoblastomas usually present as a single mass but can be multifocal and may measure up to 25 cm in diameter. The right lobe is more often involved. The non-neoplastic liver is usually normal. HB typically has a lobulated cut surface and may be yellow, brown, green or variegated, depending on the differentiation of the tumour and whether a mesenchymal component is present. In post-therapy resections, a heterogeneous appearance with areas of haemorrhage and necrosis is common. Calcification and ossification may be present. Specific attention should be paid to gross vascular invasion and surgical margins, and fresh tissue should be set aside for special studies whenever possible.

Histologically, HB may be classified as epithelial or mixed epithelial-mesenchymal, each with several recognized subtypes. The epithelial type is the most common and has several subtypes.The fetal epithelial pattern comprises one-third of the cases and is composed of sheets and thin trabeculae of polygonal cells resembling fetal hepatocytes with a small round nucleus, inconspicuous nucleolus, clear to finely granular cytoplasm with variable amounts of cytoplasmic glycogen and lipid giving a ‘light and dark’ pattern to the tumour when viewed at low-power magnification ( Fig. 13.22 ). Haematopoietic cells are almost always present, mimicking the typical extramedullary haematopoiesis of normal fetal liver. HBs that are composed exclusively of fetal pattern are referred to as pure fetal HB . By definition, the entire tumour is composed of uniform cells, and mitoses are <2 in 10 high power fields. In the crowded fetal subtype , the tumour cells are more closely packed, have higher nuclear/cytoplasmic ratios, decreased cytoplasmic glycogen, more eosinophilic cytoplasm and higher mitotic activity (≥2 in 10 high power fields) compared to the fetal pattern ( Fig. 13.23 ).

Hepatoblastoma. The tumour cells at lower magnification in fetal areas often have variable amounts of cytoplasmic glycogen and lipids that result in a ‘light and dark’ pattern of staining.

Hepatoblastoma. Fetal epithelial pattern (left) of a crowded type, merging with embryonal pattern (right).

The fetal pattern is mixed with the embryonal pattern in 20% of cases, and the crowded fetal pattern may merge into the embryonal pattern (see Fig. 13.23 ). The embryonal pattern corresponds to the sixth to eighth week of the embryonic liver and consists of sheets, clusters or single small, angulated, hyperchromatic cells with a high nuclear/cytoplasmic ratio, prominent nucleolus and frequent mitoses. Embryonal cells often cluster into glandular, acinar or pseudorosette formations. Extramedullary haematopoiesis is often more prominent than the fetal pattern. The macrotrabecular pattern is seen in about 3% of cases and is composed of trabeculae >10 cells in thickness with either fetal or embryonal type cells ( Fig. 13.24 ). However, the tumour cells may rarely be larger and closely resemble HCC. In most cases, this pattern is mixed with other patterns of HB. The small-cell pattern is seen in about 3% of cases and is composed of noncohesive sheets of small cells similar to those of other ‘small blue cell’ paediatric neoplasms. The mitotic rate is often high, but some tumours do not show significant mitoses. Rhabdoid phenotype with eccentric nucleus and eosinophilic cytoplasmic globules can be seen. This pattern has been variably called small-cell anaplastic or small-cell undifferentiated HB. A tumour should contain at least 70% small-cell component to be considered a pure small-cell HB. Smaller components of this morphology should also be recognized because of a potential for adverse outcome.

Hepatoblastoma. Macrotrabecular pattern consists of the trabeculae >10 cells thick, primarily composed of fetal-type pattern.

Ductular differentiation can be seen in the epithelial variant as in fetal HB, often at the periphery of the tumour. When ductular differentiation is prominent, the tumour is referred to as cholangioblastic HB. Ductular reaction can occur at the periphery of the tumour, especially after chemotherapy, and should not be considered as the cholangioblastic variant.

The mixed epithelial-mesenchymal pattern contains areas of fetal and/or embryonal epithelial cells along with neoplastic mesenchymal elements ( Fig. 13.25 ). The mesenchymal component consists of immature and mature fibrous tissue, osteoid-like tissue and in some cases, hyaline cartilage. The cells in the mesenchyme and osteoid-like areas share staining characteristics with the epithelial component, being positive for keratin and showing nuclear β-catenin staining, indicating that they are metaplastic epithelial cells rather than truly of mesenchymal origin.

Hepatoblastoma, mixed epithelial-mesenchymal pattern. The epithelial pattern is mostly fetal type and is admixed with both a characteristic primitive stromal mesenchyme and osteoid.

Teratoid HB (HB with heterologous elements) refers to mixed HB with heterologous elements such as neural/neuroectodermal differentiation represented by mature brain, primitive neuroepithelial components forming tubules and rosettes, as well as melanin and retinal pigment. Rhabdomyosarcomatous elements can be present. Squamous and mucinous glands may be present, but these elements can also be seen without a neural component in epithelial HB. Rarely, glandular elements with subnuclear and supranuclear vacuoles reminiscent of yolk sac tumour can be present, but these are interspersed with other areas of typical HB. These tumours are not related to teratomas, and representations from all three germ layers typical of teratoma are not seen.

After chemotherapy, HB may show extensive necrosis and haemorrhage with nodules of fibrous tissue, and there may be no residual tumour. Osteoid-like tissue is frequently present and may be the only evidence of viable tumour. Viable epithelial areas may be seen grossly as pale friable areas within fibrotic nodules, while the mixed elements appear as tiny white areas of osteoid. The amount of residual tumour is variable, and it can be difficult to differentiate fetal pattern from entrapped benign hepatocytes. The histological subtype may be difficult to identify because of secondary changes such as marked fat accumulation and bizarre nuclei. Squamous epithelium with keratinization and foreign body giant cell reaction is often seen in post-therapy specimens.

Immunohistochemistry

Both fetal and embryonal elements in HBs express pancytokeratin, and hepatocellular markers such as hepatocyte paraffin 1 (HepPar 1), polyclonal carcinoembryonic antigen (pCEA) and AFP. GPC3 is positive in >90% of cases. Nuclear β-catenin is seen in the embryonal component but is absent or rare in most fetal HBs. However, rare cases of fetal HB may show prominent β-catenin nuclear staining. GPC3 staining in the fetal areas is slightly more intense in the eosinophilic cells compared with clear cells. GS shows diffuse cytoplasmic staining in both embryonal and fetal areas. The staining pattern in the crowded fetal pattern is similar to the fetal pattern, but GPC3 and GS staining can be stronger. SALL4, a marker of germ cell tumours, can be positive in embryonal HB. The ductular elements can be highlighted by cytokeratins 7 and 19. The tumour cells in small-cell variant are negative for HepPar 1, AFP, GS and GPC3. The tumour cells express cytokeratin and vimentin; INI1 may be lost in some tumours, especially those with rhabdoid phenotype. Cytokeratin 19 can be positive in scattered tumour cells. Strong and diffuse nuclear staining with β-catenin is uniformly present.

Cytology

FNA cytology findings in epithelial HB depend on whether differentiated (fetal, embryonal and/or macrotrabecular subtypes) or undifferentiated elements are aspirated. Fetal elements resemble immature hepatocytes ( Fig. 13.26 ). Neoplastic cells are slightly smaller and have ample cytoplasm with small, central round nucleus, finely granular chromatin and normal nuclear/cytoplasmic ratio. Pleomorphism and mitoses are not seen. Embryonal subtype is characterized by cells with scant cytoplasm, angulated nucleoli, coarsely granular chromatin, high nuclear/cytoplasmic ratio and mitoses. The cells are usually arranged in acinar groupings, in a papillary pattern or in sheets. Extramedullary haematopoiesis may be discernible. Differentiated epithelial HB can resemble HCC cytologically. Cytological subtyping can be difficult without resorting to ancillary tests, including ultrastructural studies. The undifferentiated type consists of anaplastic, small round cells and is one of the differential diagnoses of small round cell tumours in childhood. In the mixed epithelial-mesenchymal type, mesenchymal tissue is rarely seen in FNA cytology.

Hepatoblastoma, fetal epithelial type, aspiration biopsy. Hypercellular aggregates of narrow arborizing cords of neoplastic cells resembling immature hepatocytes. Elongated nuclei and nuclear streaks represent transgressing endothelium. (Papanicolaou stain.)

Differential diagnosis

Calcifying nested stromal-epithelial tumour

Calcifying nested stromal-epithelial tumour of the liver is an extremely rare tumour that is composed of a spindle cell component along with epithelial, ductular and osteoid components; thus it could be confused with HB ( Fig. 13.27 ). This tumour affects children and young adults, some of whom may present with Cushing syndrome. Cytokeratin, vimentin and hormones (e.g. ACTH) are often positive, whereas GPC3 and nuclear β-catenin are negative.

Calcifying nested stromal-epithelial tumour. The tumour is composed of nests of epithelial cells, which frequently contain calcifications, embedded in a spindle cell stroma.

Courtesy of Dr. Hala Makhlouf.

Staging

The most widely used staging systems are is that of the Children’s Oncology Group (COG) and PRETEXT schemes. The COG scheme classifies the tumours postoperatively by their resectability into four groups: stage I (complete resection), stage II (microscopic residual disease), stage III (macroscopic residual tumour) and stage IV (distant metastases). At presentation, approximately 38% of HBs are stage I, 9% stage II, 24% stage III and 29% stage IV. The majority of the 53% of unresectable (stages III and IV) cases can be rendered resectable with preoperative chemotherapy. Liver transplantation (LT) is an option for unresectable disease.

Developed by the International Childhood Liver Tumor Strategy Group (SIOPEL), the PRETEXT staging system is based on segmental involvement of the liver and is used to determine tumour extension before therapy: stage I (1 section involved, 3 adjoining sections are free), stage II (1 or 2 sections involved, 2 adjoining sections are free), stage III (2 or 3 sections involved, no two adjoining sections are free) and stage IV (all 4 sections involved). The sections in the PRETEXT staging are defined by grouping of liver segments: segments II and III (left lateral section), segment IV (left medial section), segments V and VIII (right anterior section) and segments VI and VII (right posterior section). In addition, the PRETEXT system also includes assessment of tumour involvement of inferior vena cava (IVC), hepatic veins (V), portal veins (P), caudate lobe, multifocality, rupture/intraperitoneal haemorrhage, lymph node involvement, extrahepatic abdominal disease (E) and distant metastases (M).

Treatment and prognosis

Surgery is the mainstay of treatment of HB, and complete resection is the only chance for cure. All patients with COG stage I tumours with pure fetal pattern are treated with surgery alone without chemotherapy because 5-year survival is almost 100%. The other histological variants as well as higher-stage tumours receive a combination of chemotherapy and surgical resection, the order determined by tumour resectability at presentation. For nonpure fetal histology, the disease-free survival is 97–100% for COG stage I and II, 70% for stage III and 40% for stage IV disease. Preoperative chemotherapy to shrink the tumour has resulted in improved resectability and improved the overall 5-year survival rate to 75% from 35% in the 1970s. Cisplatin/doxorubicin or cisplatin/5-FU/vincristine are the usual regimens. LT is an option for tumours that remain unresectable after chemotherapy.

The stage of the tumour at initial resection is the key prognostic factor in determining survival. Small-cell and macrotrabecular subtypes have an adverse outcome. The other histological patterns do not independently affect survival when adjusted for age, gender and stage. Other unfavorable prognostic factors include involvement of multiple lobes, vascular invasion, slow decline in AFP after therapy, aneuploidy, nuclear β-catenin staining, low p27 expression and high cyclin D1 expression.

Clinical features

The clinical presentation of hepatocellular carcinoma (HCC) is related to effects of the mass or signs and symptoms of underlying chronic liver disease, including upper abdominal pain, weight loss, abdominal enlargement and hepatomegaly with or without a palpable mass. Signs of decompensated liver disease, such as jaundice, ascites, variceal haemorrhage, hepatic encephalopathy or obstructive jaundice, can be present. Rarely, patients may present with distant metastases or paraneoplastic syndromes such as hypoglycaemia, erythrocytosis, hypercholesterolaemia, hypercalcaemia, isosexual precocious puberty (in children), gynaecomastia (in the absence of cirrhosis), carcinoid syndrome, hypertrophic pulmonary osteoarthropathy, osteoporosis, hypertension, hyperthyroidism, dysfibrinogenaemias, porphyria cutanea tarda and a variety of other cutaneous changes.

High levels of serum alpha fetoprotein (AFP), an oncofetal antigen, can help in the screening, diagnosis and follow-up of HCC. The utility of AFP is limited by low sensitivity and specificity. Many other serum biomarkers, such as des-γ-carboxy prothrombin, are becoming available, but their usefulness remains to be proved. Serum AFP level and US of the liver are recommended every 6 months for screening of patients at risk of developing HCC.

CT and MRI are the most common modalities used for radiographic diagnosis of HCC. The criteria proposed by the American Association for the Study of Liver Diseases (AASLD) state that for tumours >1 cm in cirrhotic liver, biopsy is not necessary to confirm the diagnosis if classical features of HCC are seen on multiphasic contrast-enhanced CT or MRI. The classic features are enhancement of the lesion in the arterial phase and washout in the venous phase. Atypical imaging features are common in small (typically <2 cm) and well-differentiated tumours. Additional imaging (CT or MRI) is recommended for these patients, and biopsy is often required. Functional imaging of HCC using hepatocyte-specific gadolinium chelate agents is increasingly used and may increase sensitivity and specificity for HCC diagnosis compared to multiphasic MRI or CT. Of note, small tumours are increasingly being detected by surveillance in high-risk patients.

A system of standardized reporting of CT and MRI findings for HCC diagnosis in high-risk patients is called the Liver Imaging Reporting and Data System (LI-RADS). This system combines arterial enhancement with tumour size, venous washout, presence of capsule and growth compared to prior imaging to yield five diagnostic categories: 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).

Epidemiology and aetiology

One of the most striking features of HCC is the wide variation in its incidence in different parts of the world. Overall, HCC represents the third leading cause of cancer-related mortality worldwide. It is the fifth most common cancer in men and the seventh in women worldwide, but East Asia and sub-Saharan Africa have by far the greatest number of cases, and in the countries of those regions, HCC is among the leading causes of death. In areas of low incidence, such as the United States, carcinoma of the liver (primarily HCC) accounts for only 2.3% of cancer deaths in recent years, with an annual incidence of approximately 4.1 cases per 100,000 population. However, the incidence of HCC in the United States has more than doubled over the past two decades and is anticipated to continue increasing over the next 20 years because of the growing number of patients with advanced hepatitis C virus (HCV) infection and NASH. At its current pace, HCC is projected to surpass breast and colorectal cancers to become the third leading cause of cancer-related death in the United States by 2030. In general, regions of the world with a high incidence of HCC are those that have a high prevalence of chronic hepatitis B virus (HBV) infection, but even within these areas there is geographical variability, e.g. in western Africa the country of Gambia has nearly five times the incidence of Nigeria, suggesting that environmental cofactors such as aflatoxin exposure may also be important. In contrast to the rising incidence in the United States, the incidence of HCC has been declining in some high-incidence areas, such as China and Hong Kong, partly related to HBV vaccination of children and reduction of aflatoxin exposure in grains. HCV infection is associated with many cases in countries such as Japan, where the prevalence of hepatitis B is intermediate but the incidence of HCC relatively high.

The incidence of HCC generally increases with age, although geographical differences exist. In Europe and the United States the peak age-specific incidence is in the seventh decade, while in Qidong province in China where the incidence is the highest in the world, the peak is in the fifth decade. In South Africa the average age of patients with HCC is 35 years, and 40% are 30 or younger, whereas in Taiwan (another area of high incidence), the majority of patients are 40–60 years old, with a peak incidence in the eighth decade. Nevertheless, HCC can occur in younger individuals and even young children. Regardless of geographical location, HCC occurs more frequently in men than women, with male/female ratios in various countries ranging from 2 : 1–5 : 1. The precise reason is not known, but it has been shown that many tumours have androgen receptors, suggesting that androgens may promote tumour development and growth. There is also a male predominance in risk factors, such as chronic viral hepatitis, alcoholism and smoking, which undoubtedly also play a role. Other authors, however, have attributed this gender bias to female sex hormones, because HCC incidence greatly increases in postmenopausal women who do not take hormone replacement therapy. Rare HCCs arise in association with HCA (see previous section).

Molecular pathology

Molecular pathology of HCC is also discussed in Chapter 2 , so this section provides only a brief overview of molecular classifications of HCC with possible clinicopathological implications ( Table 13.7 ). As noted previously, HCC is a multistage and a heterogeneous disease linked to environmental, dietary/lifestyle factors, hereditary disorders and chronic liver disease or cirrhosis. In a small proportion of cases, HCC is observed in absence of liver disease. These facts, along with the different aetiologies responsible for liver damage (e.g. viral hepatitis, alcohol, iron overload, other causes of cirrhosis), account for the high molecular variability of HCC. During the last decade, several genomic alterations have been reported, including high-level DNA amplifications in chromosome 6p21 ( VEGFA ), observed in 6.7% of HCCs, and 11q13 ( FGF19/CNND1 ), as well as homozygous deletions in chromosome 9 ( CDNK2 ). Interestingly, improved survival of sorafenib-treated patients with VEGFA -amplified HCCs has been reported. Despite these research efforts, however, no molecular classification has yet been introduced in clinical practice.

Several studies have attempted to establish molecular classifications of HCCs based on global gene expression profiles. Such classifications seek to group HCCs based on specific dysregulation of a limited set of biological pathways, allowing personalized treatment decisions targeting the ‘driver’ molecules responsible for cancer growth in each particular case. Four main molecular classifications of HCC have been published to date, reporting two, three, five or six discrete HCC subgroups, based on gene expression profiling in tumour samples. Recently, a transcriptome meta-analysis involving several hundred HCC tumours depicted a molecular classification onto which the subclasses, gene signatures and somatic DNA alterations can be mapped. According to this large meta-analysis, HCCs can be classified into two main subgroups ( Table 13.7 ). The first group is characterized by more aggressive biological and clinical features, showing increased genetic instability, cellular proliferation, ubiquitination, activation of prosurvival signals such as E2F1 and MET pathways, impairment of tumour suppressor TP53, gene signature of KRT19, larger and less differentiated tumour, higher incidence of tumour recurrence and poorer prognosis. The remaining tumours are characterized by less aggressive features, including preserved hepatocyte function, smaller and more differentiated tumour and better prognosis. The aggressive tumours are further subdivided into two groups: S1 subclass, characterized by relatively higher activation of TGFβ pathway and hepatocyte/cholangioma-like gene signature, and S2 subclass, characterized by positivity of stemness markers, epithelial cell adhesion molecule (EpCAM), high AFP and GPC3, activation of IGF2 pathway, relative suppression of interferon target genes and hepatoblastoma-like gene signature. The subset of less aggressive HCC tumours, S3 subclass, is characterized by somatic mutations accumulated in exon 3 of CTNNB1 (β-catenin gene) accompanied by induction of specific target genes, GLUL, LGR5 and SLC1A2, but not with canonical Wnt pathway target genes. Of note, slightly different sets of canonical Wnt pathway target genes are more preferentially induced in the S1 or S2 subclass, suggesting biological context-specific induction of WNT target genes in HCC. Interestingly, a recent transcriptome analysis has developed a molecular signature, based on combined expression level of five genes: HN1, RAN, RAMP3, KRT19 and TAF9. This molecular five-gene score is associated with outcomes of patients with HCC treated by resection in different clinical settings worldwide, independent of other clinicopathological features of HCC.

Pathology

The tumour may form a single mass that replaces an entire lobe, or there may be multiple scattered discrete nodules. Numerous cirrhosis-like nodules can occur (‘cirrhosis-like HCC’) and can be missed on imaging and gross examination ( Fig. 13.36 ). Some tumours form an expanding mass well demarcated from the surrounding liver, with or without a capsule, whereas others appear to infiltrate the surrounding liver tissue at the tumour margin. Encapsulated tumours usually arise in a cirrhotic liver, with the expanding tumour causing compression atrophy of the surrounding cirrhotic nodules and incorporation of cirrhotic scars to form the fibrous capsule. Most HCCs are soft, often with areas of necrosis, with the exception of fibrolamellar and scirrhous variants, which can be grey-white and firm due to abundant stroma. HCCs can be variably tan or yellow, and green if they produce bile. Vascular invasion is common and the portal vein, hepatic veins as well as the vena cava may be involved. Invasion of major bile ducts is uncommon but may cause biliary obstruction.

Cirrhosis-like hepatocellular carcinoma. The tumour is composed of innumerable cirrhosis-like nodules that cannot be distinguished from the cirrhotic background.

Microscopically, HCC can have a highly variable appearance from tumour to tumour, even in different lesions within the same liver, as well as within the same lesion. Tumour cells in HCC resemble normal hepatocytes to a variable extent, depending on the degree of differentiation. HCC tumour cells are often smaller than normal hepatocytes (small-cell change; see earlier) ( Fig. 13.37 ), with high nuclear/cytoplasmic ratio (see cytology section later). Nucleoli and hyperchromasia can be variable, but prominent nucleoli can often be seen in less well-differentiated or pleomorphic tumour cells. Intranuclear inclusions (vacuoles) may occur ( Fig. 13.38 ). The cell membranes are often distinct ( Fig. 13.39 ), and tumour cells typically have a moderate amount of eosinophilic, finely granular cytoplasm. However, the cytoplasm can be variable on staining, from more basophilic than normal hepatocytes to highly eosinophilic. The basophilia is a feature often associated with small-cell change within HCC (see Fig. 13.37 ), whereas the eosinophilia can take the form of oncocytic change (see Fig. 13.39 ), typically caused by accumulation of abundant mitochrondria.

Hepatocellular carcinoma. Small-cell change with increased nuclear/cytoplasmic ratio. The tumour cell nuclei are small, round, and lack prominent nucleoli, and the cytoplasm is more basophilic than normal hepatocytes. A few small to medium-sized pseudoglands are also present.

Hepatocellular carcinoma, poorly differentiated. The trabeculae are very wide (macrotrabeculae). An intranuclear inclusion/vacuole is present (top center), as well as scattered necrosis/apoptosis within the tumour plates and focal inflammatory infiltrate (upper right) .

Hepatocellular carcinoma, trabecular type. The tumour cells are more eosinophilic to oncocytic, with focally distinct cytoplasmic borders. Small-cell change is present, but some larger nuclei with nucleoli are also present. Trabeculae are lined by endothelial cells and vary from thin to thick; sinusoidal space is focally prominent.

The tumour cells can grow in several architectural patterns ( Table 13.8 ). The trabecular pattern is the most common ( Fig. 13.40 ; see also Figs 13.37–13.39 ), in which the tumour cells grow in variably thick cell plates (typically three or more cells thick) separated by vascular sinusoids and lined by endothelial cells which phenotypically resemble capillary endothelium (CD34+) ( Fig. 13.41 ), rather than normal hepatic sinusoidal endothelium (CD34−). Dilation or expansion of canalicular forms at the centre of the trabeculae leads to a pseudoglandular or pseudoacinar pattern, and the prominence of this pattern can be highly variable, from scattered foci to near-complete involvement of large zones of tumour by this transformation ( Figs 13.42 and 13.43 ; see also Fig. 13.37 ). The trabeculae can compress the sinusoids, producing a compact pattern with sheets of tumour cells without obvious trabeculae formation ( Fig. 13.44 ). The peliotic pattern is characterized by tumour cells interspersed by large vascular lakes, mimicking peliosis hepatis. Bile pigment may be present in tumour cells or in dilated canaliculi in about 50% of tumours. Cytoplasmic fat is present in about two-thirds of tumours and abundant in about 10%; fat can be present as small or large droplets. It tends to be more prominent in smaller tumours (particularly notable in early HCC; see previous discussion) and often disappears in advanced cases. Large amounts of cytoplasmic fat and glycogen can cause the cytoplasm to appear white in routine sections, producing a clear cell appearance ( Figs 13.45 and 13.46 ). The term clear cell HCC has been used if the clear cytoplasm is a prominent finding. Cytoplasmic MDBs are present in approximately 20% of tumours and hyaline globules in another 20%, which may represent AAT or other proteins ( Figs 13.47 and 13.48 ). Ground-glass cytoplasmic inclusions similar to those observed in chronic hepatitis can be seen in the tumour cells. Pale eosinophilic cytoplasmic inclusions (‘pale bodies’) are most characteristic of the fibrolamellar variant but can be seen in classic HCC and represent accumulations of fibrinogen. Rare findings include bizarre multinucleated cells ( Fig. 13.49 ), osteoclast-like giant cells ( Fig. 13.50 ) and extramedullary haematopoiesis. Necrosis and inflammatory changes may also be present, particularly in more poorly differentiated tumours (see Fig. 13.38 ). When prominent, lymphocytic or neutrophilic inflammatory infiltrates in the tumour are recognized as distinct variants (see later). Connective tissue stroma is typically sparse and is a helpful diagnostic clue for HCC. Abundant fibrous stroma is present in scirrhous and fibrolamellar variants.

Hepatocellular carcinoma, trabecular type. This tumour shows variable differentiation from moderate (left) to poor (right) but still maintains a predominantly trabecular pattern.

Hepatocellular carcinoma. CD34 highlights endothelial cells along the trabeculae, in this case in a somewhat fragmented pattern, but many HCCs with well-formed trabeculae may show diffuse staining. (CD34 immunostain.)

Hepatocellular carcinoma, pseudoglandular (pseudoacinar) pattern. The pseudoglands are focally large in this example and contain abundant pink, proteinaceous material. Focal intraluminal haemorrhage is also present.

Hepatocellular carcinoma, mixed trabecular and pseudo­glandular pattern. This lesion shows moderate nuclear atypia, varying from small-cell change to large-cell change, the latter with focal prominent nucleoli. The trabeculae are lined by endothelial cells, and pseudoglands have formed within the trabeculae. The cytoplasm is eosinophilic to oncocytic.

Hepatocellular carcinoma, compact type. The tumour has a solid appearance, without obvious trabeculae or pseudoglands, but some vessels can be seen tracking through the tumour.

Hepatocellular carcinoma with diffuse fatty change. Both large- and small-droplet fat expands tumour cells to render an overall ‘clear cell’ pattern.

Hepatocellular carcinoma, clear cell pattern. Tumour cells have clear cytoplasm, in this case caused mostly by glycogen, with possibly some degree of fat as well.

Hepatocellular carcinoma. Tumour cells contain large- and small-droplet fat, as well as microvesicular fat, the latter cells with cytoplasmic inclusions consistent with Mallory–Denk bodies (Mallory hyaline). Thin fibrous bands separate clusters of tumour cells.

Hepatocellular carcinoma, trabecular type. Note cytoplasmic hyaline inclusions, some of which may be Mallory–Denk bodies. Nuclei are variably small to large, many with prominent nucleoli.

Hepatocellular carcinoma. Poorly differentiated HCC with bizarre giant cell forms, admixed with focal necrosis.

Hepatocellular carcinoma. Focal osteoclast giant cells in HCC staining faintly with bile.

The reticulin network of the hepatic plates is typically fragmented or lost in HCC ( Fig. 13.51 ). Thus, histochemical stain for reticulin fibres is one of the most useful techniques for the diagnosis of HCC. The reticulin network can be present in some cases, particularly in early HCC and in extremely well-differentiated to well-differentiated HCC, and thus does not exclude the diagnosis. However, in many of these cases, the framework in HCC has an abnormal pattern, such as increased deposition around single cells or small clusters of cells ( Fig. 13.52 ), accentuation of greatly widened trabeculae or acinar structures ( Fig. 13.53 ) or abnormal architecture admixed with loss or fragmentation of reticulin ( Fig. 13.54 ), not typical of benign hepatocellular lesions such as HCA. Of note, widened trabeculae (>3 cells) alone is not an indicator of HCC, because focal reticulin irregularities in plate width and architecture can also be seen in benign lesions, especially FNH (see Fig. 13.19 ) and at times, regenerative nodules. Also, the near-complete encircling, or ‘packeting,’ of small groups of tumour cells by the reticulin fibres is not specific for HCC and can also be seen in HCA (see Fig. 13.12 ) and FNH (see Fig. 13.19 ). Similarly, loss and fragmentation of reticulin staining are frequently seen with fat accumulation in hepatocytes, even in non-neoplastic or benign settings. Therefore the reticulin stain has limited utility in the diagnosis of HCC in the presence of prominent intralesional steatosis.

Hepatocellular carcinoma, reticulin stain. Diffuse loss and fragmentation of reticulin framework in a somewhat compact pattern of HCC. Nuclei vary from small- to large-cell change.

Hepatocellular carcinoma. Reticulin staining is activated, but in abnormal pericellular pattern in this example of a β-catenin-activated HCC.

Hepatocellular carcinoma. Reticulin is present, but surrounds zones with loss or fragmentation of reticulin, resulting in the division of the tumour into small groups of cells. (Reticulin stain.)

Hepatocellular carcinoma. Reticulin is focally fragmented and lost in centre, but is present at periphery in abnormal packeting pattern (left) and highlighting larger cell clusters (right). (Reticulin stain.)

Immunohistochemistry

A wide armamentarium of immunohistohemical stains is available to confirm hepatocellular differentiation and to distinguish HCC from other primary or metastatic neoplasms (see Table 13.8 and later section on metastatic tumours ). Since limited tissue is often available for biopsies, judicious selection of markers is imperative for diagnosis. The strengths and limitations of various markers are outlined next.

Hepatocellular markers

Arginase 1

Arg1 is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of arginine to ornithine and urea; it is found only in the liver. Arg1 shows strong cytoplasmic staining in normal as well as neoplastic hepatocytes ( Fig. 13.55 ). Nuclear staining can be seen in some cases.

Hepatocellular carcinoma. Arginase-1 immunostain shows diffuse cytoplasmic and focal nuclear staining.

Strengths.

Arg1 is highly sensitive and specific marker for hepatocellular differentiation. These results are true across the spectrum of differentiation of HCC. Variants such as scirrhous HCC are usually positive. The staining tends to be diffuse in most cases, implying that this marker will yield similarly high sensitivity in biopsies. Most nonhepatocellular tumours are negative for Arg1.

Limitations.

Rare instances of focal positive staining in adenocarcinomas of the prostate, pancreas, colon, breast and biliary tree have been reported. Hepatoid adenocarcinomas that can occur in the gastrointestinal (GI) tract, pancreas and other sites are typically positive for Arg1. Occasional cases of HCC can be negative for Arg1, and thus a panel of markers is recommended. Since Arg1 is positive in both benign and malignant hepatocellular neoplasms, it is not helpful for distinguishing HCA from HCC.

Hepatocyte paraffin 1

HepPar 1 is a monoclonal antibody that was initially obtained using tissue from a formalin-fixed failed allograft liver. It is now known that it reacts with the urea cycle enzyme carbamoyl phosphate synthetase 1 of liver mitochondria, with a granular cytoplasmic staining in normal and neoplastic hepatocytes ( Fig. 13.56 ).

Hepatocellular carcinoma. HepPar-1 immunostain shows variable intense to moderate staining in a granular cytoplasmic pattern.

Strengths.

HepPar 1 has high sensitivity and specificity (both >80%) for the diagnosis of HCC. A majority of adenocarcinomas from most sites, including biliary tree, pancreas, colorectum, breast, urinary bladder and prostate, are negative or only focally positive. Other tumours that usually enter the differential diagnosis of HCC, such as neuroendocrine neoplasms, renal cell carcinoma, adrenocortical carcinoma, malignant melanoma and epithelioid angiomyolipoma, are typically negative.

Limitations.

HepPar 1 has low sensitivity (<50%) for poorly differentiated and scirrhous HCC. The staining is patchy in 20% of HCC cases and thus can be negative in the tumour on needle biopsies. Positive staining can be seen in pulmonary adenocarcinomas and less often in adenocarcinomas from other sites, such as stomach, oesophagus and biliary tree. Hepatoid carcinomas are positive for HepPar 1, and it is not useful for distinction of benign and malignant hepatocellular lesions.

Glypican 3

GPC3 is a membrane-anchored heparin sulfate proteoglycan normally expressed in fetal liver and placenta. It is an oncofetal protein and is not expressed in normal or benign hepatocytes. GPC3 shows cytoplasmic staining in HCC, with membranous and Golgi pattern in some cases ( Fig. 13.57 ).

Hepatocellular carcinoma. Glypican-3 immunostain shows diffuse cytoplasmic positivity. Focal fatty change is also present in this HCC.

Strengths.

GPC3 has moderate to high sensitivity (65–80% in most studies) for HCC. Most cases of poorly differentiated and scirrhous HCC express GPC3. Since GPC3 is negative in normal liver, FNH and adenomas, it can be useful in distinguishing benign hepatocellular lesions from HCC.

Limitations.

GPC3 is not specific for HCC, and can be expressed in a wide variety of other tumours, including squamous cell carcinoma, melanoma, nonseminomatous germ cell tumours (e.g. yolk sac tumour, choriocarcinoma), gastric adenocarcinoma and rare cases of cholangiocarcinoma. The sensitivity is low (<50%) in well-differentiated HCC. GPC3 can occasionally be positive in cirrhotic nodules and areas of high necroinflammatory activity in hepatitic disease. High-grade dysplastic nodules can be variably positive for GPC3.

Polyclonal antibody to carcinoembryonic antigen

Polyclonal CEA cross-reacts with biliary glycoprotein I in the bile canaliculus, leading to a characteristic canalicular pattern of staining in normal and neoplastic hepatocytes. Most adenocarcinomas show cytoplasmic and/or luminal staining. Monoclonal CEA also shows cytoplasmic reactivity in adenocarcinomas, but the sensitivity is lower (~60%). HCC is nonreactive with monoclonal CEA.

Strengths.

The overall sensitivity for HCC is high (70–80%), and the canalicular pattern observed with pCEA is considered to be specific for hepatocellular differentiation ( Fig. 13.58 ).

Hepatocellular carcinoma. Polyclonal carcinoembryonic antigen (pCEA) immunostain shows a patchy canalicular pattern of pattern, including focal ‘dot-like’ staining.

Limitations.

The sensitivity of pCEA is low in poorly differentiated and scirrhous HCC. Cytoplasmic staining can be seen in some HCC cases. Occasionally, it can be difficult to distinguish canalicular from membranous or luminal staining pattern seen in adenocarcinomas.

Other hepatocellular markers

Alpha fetoprotein (AFP) is an oncofetal protein produced by the liver and yolk sac. Its expression in a tumour is specific for hepatocellular differentiation if germ cell tumours can be excluded. However, staining tends to be patchy, and sensitivity is low (30–50%) and almost always essentially absent in small HCCs. The low sensitivity and availability of better antibodies have rendered AFP as a less useful option for diagnosis. However, serum AFP levels remain helpful in the diagnosis and monitoring response to therapy for HCC.

The staining pattern with CD10, villin and bile salt excretory protein (BSEP) is similar to the canalicular pattern of pCEA and has the same drawbacks.

Thyroid transcription factor 1 (TTF-1) is expressed in nuclei of epithelial cells and tumours of the thyroid and lung. Cytoplasmic TTF-1 staining has been observed in 70% of HCCs. Since the staining often parallels the results obtained with HepPar 1, and the immunoreactivity varies with the clone and antigen retrieval technique, TTF-1 offers no benefit if HepPar 1 is part of the panel.

Histological variants of hepatocellular carcinoma

Fibrolamellar hepatocellular carcinoma

Fibrolamellar HCC, or fibrolamellar carcinoma (FLC) per WHO (2010), typically occurs in young adults, with a mean age of 26 years at diagnosis. The tumour generally occurs in the absence of chronic liver disease or cirrhosis, and serum AFP level tends to be normal or with only mild elevation in 10–15% of cases. Serum B12 binding protein is often elevated. There are no known risk factors. Since FLC is a rare tumour (1–8% of HCCs), classic HCC is more common than FLC, even in children and young adults.

FLCs are distinct from typical HCCs at the histological, immunophenotypical and molecular levels. On gross examination, FLCs are tan-white tumours that are typically larger than classic HCC ( Fig. 13.59 ). A central scar can be present, often with calcifications, and can be detected radiographically. Microscopically, FLC is characterized by a trio of histological features, as follows:

• 1.
  

  Large polygonal tumour cells with abundant eosinophilic granular cytoplasm ( Fig. 13.60 ). The granular appearance is related to abundant mitochondria, lysosomes or endosomal cytoplasmic accumulations. Eosinophilic cytoplasmic inclusions paler than the tumour cell cytoplasm and with a ground-glass appearance (pale bodies) are often present and are thought to be caused by fibrinogen accumulation. However, pale bodies are not specific for FLC and can be seen in classic HCC.

  
  

  
  

  
  

  

  
  

  
  

  Fibrolamellar hepatocellular carcinoma. The tumour is composed of groups of large, polygonal, eosinophilic cells with distinct cell borders, admixed with a dense, lamellar fibrous stroma.

  
  
  
  
• 2.
  

  Prominent, usually single, eosinophilic macronucleoli, generally associated with vesicular nuclear chromatin or margination of nuclear chromatin ( Fig. 13.61 ).

  
  

  
  

  
  

  

  
  

  
  

  Fibrolamellar hepatocellular carcinoma. Tumour cells are large and eosinophilic, with large nuclei containing a single prominent nucleolus. Lamellar fibrosis is also present, as well as focal small calcification (upper centre).

  
  
  
  
• 3.
  

  Lamellar fibrosis comprising parallel plate-like stacks of collagen (see Figs 13.60 and 13.61 ). The characteristic fibrous stroma can be patchy and may not be present in a needle biopsy or metastasis.

Fibrolamellar hepatocellular carcinoma. The tumour is hard and yellow to tan with areas of fibrosis. The cut surface stands out above the surrounding noncirrhotic liver.

Additional variable findings within the tumour include degenerative atypia characterized by enlarged nuclei with smudgy chromatin as a common finding, occasional pseudoglandular/acinar structures with mucin, granulomas and focal calcifications (see Fig. 13.61 ). A clear cell variant of FLC has been reported. It is recommended that all three criteria listed above should be met for an unequivocal diagnosis, because it is important to use strict morphological criteria for the diagnosis of FLC. Of note, in retrospect, it is likely that stringent criteria were not used in the diagnosis of rare cases that have been described in cirrhotic liver.

Similar to classic HCC, hepatocellular markers such as HepPar 1, pCEA (canalicular pattern) and Arg1 are positive in FLC. GPC3 immunoreactivity is observed in 17–64% of cases, but positive results with AFP are rare, correlating with the often unremarkable serum AFP. However, K7 is positive in almost all cases, compared to only 20–30% of classic HCCs. CD68, a macrophage marker, is positive in almost all cases as well, but it is not specific for this variant since staining is also seen in 10–25% of classic HCC. It has been argued that the diagnosis of FLC should be made with caution if K7 or CD68 is negative. In some cases, neuroendocrine markers such as nonspecific enolase and chromogranin can be positive.

As noted, focal pseudoglandular differentiation, presence of mucin and strong K7 staining are not uncommon findings and thus should not be mistaken for cholangiocarcinoma (CC), metastatic adenocarcinoma or mixed FLC-CC. However, rare cases of true mixed FLC-CC have been described that demonstrate distinct, well-characterized FLC and CC components. Some FLC cases express EpCAM, NCAM, CD133 and CD44, which are thought to be progenitor cell-associated markers, suggesting that these tumours have stem cell-like characteristics. The plate-like fibrosis typical of FLC can be seen in a minority of scirrhous HCCs, but the typical cytological features of FLC are not present.

A remarkable recent molecular finding in many cases of FLC distinguishing this lesion from classical HCC is a consistent somatic deletion leading to DNAJB1-PRKACA fusion transcripts. Whole-genome sequencing has revealed that the fusion results from a 400-kb deletion on chromosome 19. The fusion transcript encodes a chimeric protein that couples a segment of the heat shock protein, DNAJB1, with the catalytic domain of protein kinase A (PKA) and exhibits full retention of PKA activity. Although the tumourigenic mechanism for this fusion protein is not yet proved, the fusion protein appears to drive overexpression of PRKACA, a key regulatory kinase with numerous downstream signalling targets. The fusion protein can be detected in paraffin-embedded tissue on a transcriptional and a genomic level using a reverse-transcriptase polymerase chain reaction (RT-PCR) and FISH assay, respectively. Of note, further data later found that some HCCs of non-FLC type may also have this deletion, so even though this deletion may be a highly consistent finding in FLC, this molecular marker may not be 100% specific.

Based on initial studies, FLC was thought to have a favourable outcome compared to classic HCC. However, FLC is an aggressive tumour, and recent data show similar outcomes in FLC and classic HCC occurring in noncirrhotic liver. The 5-year survival in FLC is 50–60% in most series, and surgical resectability is the most important prognostic factor. Adjuvant chemotherapy is not effective, and the median survival for unresectable cases is 1 year. LT has been used for unresectable cases. Peritoneal spread and lymph node metastasis are common, the latter occurring in up to 70% of cases (versus <5% for classic HCC). Therefore, regional lymph node dissection is often done along with lobectomy.

Other histological subtypes

A variety of other histological subtypes, although not recognized as HCC variants in the WHO 2010 classification, are sufficiently distinctive to merit discussion.

Steatohepatitic hepatocellular carcinoma

In this variant the tumour shows typical morphological features of steatohepatitis in an otherwise classic HCC ( Figs 13.62 and 13.63 ). These features include steatosis, ballooning, MDBs and pericellular fibrosis. Most affected patients have steatohepatitis in the non-neoplastic liver, and risk factors associated with metabolic syndrome are often present. This variant can also occur in the absence of metabolic risk factors and steatohepatitis in non-neoplastic liver. The outcome is similar to classic HCC.

Hepatocellular carcinoma, steatohepatitic variant. Numerous ballooned hepatocytes similar to those seen in steatohepatitis are present, embedded in an irregular pattern of fibrosis and admixed with a scattered inflammatory cell infiltrate of mostly lymphocytes and neutrophils.

Hepatocellular carcinoma, steatohepatitic variant. Higher magnification of the ballooned tumour cells in Fig. 13.62 demonstrates cytoplasmic Mallory–Denk bodies.

Granulocyte colony-stimulating factor-producing hepatocellular carcinoma

This unusual variant is characterized by a striking neutrophilic infiltrate, which can obscure the tumour cells and mimic an infectious/inflammatory process. Fever and leukocytosis can accompany the tumour. Serum levels of granulocyte colony-stimulating factor (G-CSF) are high, and G-CSF has been demonstrated by IHC in the tumour cells.

Clear cell hepatocellular carcinoma

Clear cell HCC is not a distinct histological subtype (see Fig. 13.46 ) because clear cells can be seen in any subtype of HCC. Thus the importance of recognizing this variant pattern is its distinction from other clear cell tumours, such as clear cell renal cell carcinoma. The distinction is usually straightforward because hepatocellular markers (e.g. Arg1, HepPar 1) are negative in renal cell carcinoma, whereas staining for renal transcription factors PAX-2 and PAX-8 is negative in HCC.

Diffuse cirrhosis-like hepatocellular carcinoma

Diffuse cirrhosis-like HCC is also not a distinct histological subtype, but it represents a characteristic growth pattern comprising multiple small nodules admixed with regenerative nodules in cirrhotic liver (see Fig. 13.36 ), with no evidence of a single, dominant, larger HCC within the liver. The tumour nodules may mimic cirrhotic nodules on imaging and gross examination. AFP is normal or mildly elevated in most cases. Histologically, the tumour nodules show features of well- or moderately differentiated HCC and typically show a similar morphology among all the nodules throughout the liver.

Cytology

HCCs are notably heterogeneous in differentiation and histological patterns within the same lesion. Aspirating different parts of the tumour may yield more classic features, obviating the need for costly ancillary tests. On the other hand, the aspirate may not be representative. An impression of tumour architecture can also be gleaned from smears. Grading of HCC is based on nuclear features and follows the criteria outlined for histological grading.

Gross inspection of the hypercellular smears reveals trails of cohesive arborizing aggregates of malignant hepatocytes imparting a granular pattern of spread ( Fig. 13.64 ). The broad, tongue-like cords may be wrapped by peripheral endothelium ( Figs 13.65 and 13.66 ). Transgressing endothelium with basement membrane material resembles pink ‘tramlines’ on May–Grünwald Giemsa (MGG) smears ( Fig. 13.67 ). Pseudoglands are surrounded by neoplastic hepatocytes, similar to adjacent sibling cells ( Fig. 13.68 ). In the better-differentiated lesions, the hepatocytic characteristics with increased nuclear/cytoplasmic ratio (>1 : 3) are easily recognizable ( Fig. 13.69 ). Atypical bare or stripped hepatocytic nuclei may be identified in all grades of HCC ( Fig. 13.70 ). Multinucleated tumour giant cells, which may be of ‘osteoclastic’ or pleomorphic type, are encountered with increasing frequency in higher-grade lesions ( Fig. 13.71 ). Bile pigment can best be seen in MGG smears as blackish blue clumps in hepatocytic cytoplasm, ‘ropy cords/broken twigs’ within canaliculi or bile plugs within pseudoacini ( Fig. 13.72 ). Intracytoplasmic fat and glycogen vacuoles are also best observed in MGG smears. HCC cells with fatty change exhibit large cytoplasmic vacuoles with eccentric nuclei, accompanied by free fat globules in the background ( Fig. 13.73 ). HCC cells loaded with glycogen exhibit clear cell change ( Fig. 13.74 ). Intracytoplasmic inclusions (hyaline globules, MDBs, pale bodies) and intranuclear cytoplasmic inclusions may be seen ( Fig. 13.75 ). Bile duct epithelial cells, if present, are few and far apart. The background may be haemorrhagic or necrotic, especially after locoregional ablational therapies.

Hepatocellular carcinoma, aspiration biopsy. Gross inspection of the smear shows a granular pattern of spread. (Papanicolaou stain.)

Hepatocellular carcinoma, aspiration biopsy. Scanning view shows cohesive clusters with arborizing tongue-like projections of broad cords of tumour cells. (Papanicolaou stain.)

Hepatocellular carcinoma, aspiration biopsy. Broad cords of malignant hepatocytes wrapped by peripheral endothelium. (Papanicolaou stain.)

Hepatocellular carcinoma, aspiration biopsy. Transgressing endothelium with pink basement membrane material seen amid cords of malignant hepatocytes exhibiting mild fatty change. A group of non-neoplastic hepatocytes contain bile droplets in the cytoplasm. (MGG stain.)

Hepatocellular carcinoma, well differentiated, aspiration biopsy and resection. A, Well-differentiated malignant hepatocytes display pseudoglandular formation. The vague circular gaps are best appreciated by focusing up and down on the cellular aggregates. (Papanicolaou stain.) B, The histological counterpart shows trabecular-sinusoidal and pseudoglandular patterns. The polygonal tumour cells encircling the spaces are similar to the adjacent sibling cells.

Hepatocellular carcinoma, moderately differentiated, aspiration biopsy. Note classic hepatocytic features such as polygonal cells with ample granular cytoplasm, central round nucleus with well-defined nuclear membrane, distinct nucleolus, coarsely granular chromatin and increased nuclear/cytoplasmic ratio >1 : 3. (Papanicolaou stain.)

Hepatocellular carcinoma, aspiration biopsy. Bare or stripped atypical nuclei with prominent nucleolus retain hepatocytic nuclear characteristics. (Papanicolaou stain.)

Hepatocellular carcinoma, giant cell type, aspiration biopsy. Highly pleomorphic, hepatocytic tumour giant cells exhibit multinucleation, bizarre hyperchromatic nuclei and high nuclear/cytoplasmic ratio. (Papanicolaou stain.)

Hepatocellular carcinoma; aspiration biopsy. A well-defined, magenta -coloured, pale body larger than a nucleus is discernible in the cytoplasm of a tumour cell. A blackish bile plug is seen, presumably within a pseudogland. (MGG stain.)

Hepatocellular carcinoma with fatty change, aspiration biopsy. Dissociated malignant hepatocytes contain cytoplasmic fat vacuoles of varying size, pushing the nucleus to one side. Free-lying fat globules are evident in the background. (MGG stain.)

Hepatocellular carcinoma with clear cell change, aspiration biopsy. Cords of malignant hepatocytes show clear, bubbly cytoplasm surrounding central round nucleus. (Papanicolaou stain.)

Hepatocellular carcinoma arising in large regenerative nodule, aspiration biopsy. Two distinct populations of hepatocytes are present in this nodule-in-nodule lesion. The neoplastic cells exhibit pleomorphism with dense cytoplasm and increased nuclear/cytoplasmic ratio (top). The two-cell-thick cords of non-neoplastic hepatocytes are larger, with ample pale cytoplasm and lower nuclear/cytoplasmic ratio (bottom). (Papanicolaou stain.)

Cytological features of malignancy are lacking at the well-differentiated HCC (WDHCC) end of the spectrum, whereas resemblance to hepatocytes is lacking at the poorly differentiated HCC (PDHCC) end. Cohesion is the rule in HCC. However, a tendency to dissociation is noted in highly WDHCC with cords ≤2 cells thick; and in PDHCC with virtually absent reticulin. WDHCC cells tend to be conspicuous by their small size, monotony, subtle increase in nuclear/cytoplasmic ratio and nuclear crowding (see Table 13.8 ). Another pitfall is the ‘nodule-in-nodule’ lesion, where an HCC subnodule arises within a ‘parent’ nodule, whether dysplastic or regenerative ( Fig. 13.75 ). This raises the issue of adequacy of fine-needle aspiration (FNA) evaluation, given the focality of proliferative clones within such nodules. Small HCC, as with other well-differentiated hepatocellular nodules, are prone to fatty change. Lesional fatty change in the absence of steatosis is a useful parameter indicating representative sampling. Cytodifferentiation of highly WDHCC from other well-differentiated hepatocellular nodules is extremely challenging, often with indeterminate reports. On the other hand, PDHCC cells are highly pleomorphic with thin nuclear membranes, irregular nuclear contours and even absent nucleolus ( Fig. 13.76 ). Immunohistochemistry is necessary for separating PDHCC from ICC and metastatic carcinoma.

Hepatocellular carcinoma, poorly differentiated, aspiration biopsy. Discohesive, poorly differentiated tumour cells show marked pleomorphism and high-grade features with minimal resemblance to hepatocytes. (Papanicolaou stain.)

On cell block/microbiopsies, representative HCC tissue may yield invaluable architectural details, unpaired arteries, diffuse sinusoidal capillarization (CD34), canalicular formation (pCEA, CD10), abnormal/deficient reticulin and stromal invasion. Ductular reaction (K7 or K19) should be lacking within or at the invasive front of HCC. A panel of GPC3, Arg1, HepPar 1, pCEA, CD10, CD34, MOC31 and K7 is helpful in diagnosing and differentiating HCC from metastatic adenocarcinoma on FNA biopsies.

Variant histological patterns of HCC have special features in FNA. FNA cytology of the fibrolamellar variant is characterized by discohesive, large polygonal cells with abundant oncocytic granular cytoplasm, large nucleus and nucleolus, and low nuclear/cytoplasmic ratio ( Fig. 13.77 ). Intracytoplasmic hyaline globules and well-defined pale bodies are common. The other distinctive feature is lamellar fibrosis, seen as pink parallel bands of fibrous tissue within tumour fragments in MGG smears. Trabecular arrangement is not observed.

Fibrolamellar hepatocellular carcinoma, aspiration biopsy. Loosely cohesive aggregates of large, plump, neoplastic hepatocytes with oncocytic cytoplasm, large nuclei and pale bodies are accompanied by pink collagenous bands. Proliferating capillaries abound. (MGG stain.)

Other variations in histology may include steatosis, as well as a prominence of clear, small, spindle or giant cells. HCC with fatty change can be focal or diffuse (see Fig. 13.73 ). Highly WDHCC with fatty change can easily be misinterpreted as non-neoplastic hepatocytes from steatosis or focal fatty change. PDHCC with fat vacuoles can mimic lipoblasts or signet-ring cell adenocarcinoma. In HCC, clear cell type, the tumour cells exhibit pale, finely vacuolated and fragile cytoplasm surrounding a small, central round nucleus (see Fig. 13.74 ). Mimics include metastatic clear cell tumours, especially renal cell and adrenocortical carcinomas. In HCC of small-cell type, the tumour cells are small with scanty cytoplasm, round to oval nuclei with granular chromatin, high nuclear/cytoplasmic ratio and tendency to dissociation and microacinar formation ( Fig. 13.78 ). They mimic neuroendocrine tumours, but the chromatin is not of the ‘salt and pepper’ type. Closer scrutiny may reveal classic HCC features. Other small, round cell malignancies need to be excluded. Spindle cell HCC, or purely sarcomatoid HCC, is rare and often seen in conjunction with tumour giant cells. The spindle cells exhibit various degrees of pleomorphism. Ancillary tests are required to distinguish them from sarcomas and other types of sarcomatoid carcinomas. HCC can also contain, or can be composed entirely of, giant cells. The pure giant cell variant is rare and must be distinguished from giant cell sarcomas. There is an abundance of multinucleated tumour giant cells of the pleomorphic type. Discohesion, bizarre nuclei and abundant mitoses, including abnormal forms, are features ( Fig. 13.71 ).

Hepatocellular carcinoma, small cell type, aspiration biopsy. Loosely cohesive malignant cells show scanty cytoplasm, small round to oval nuclei, granular chromatin, small nucleolus and high nuclear/cytoplasmic ratio. (Papanicolaou stain.)

Lastly, in undifferentiated HCC, the tumour cells are pleomorphic, undifferentiated and larger than the small-cell category ( Fig. 13.79 ). Because the tumour cells bear no obvious resemblance to hepatocytes, it is difficult to differentiate them from other types of undifferentiated carcinomas cytologically.

Undifferentiated carcinoma, aspiration biopsy. Loosely cohesive group of nondescript pleomorphic malignant cells exhibit scanty/absent cytoplasm, oval nuclei, nuclear membrane irregularities, coarse chromatin and high nuclear/cytoplasmic ratio. (Papanicolaou stain.)

Grading and staging

A histological grading system for HCC was first described by Edmondson and Steiner in 1954. The tumours were graded on a scale of I–IV, with increasing nuclear irregularity, hyperchromasia and nuclear/cytoplasmic ratio associated with decreasing cytological differentiation for each successively higher grade, and a greater emphasis on the amount and appearance of the cytoplasm and the nuclear/cytoplasmic ratio, so that grade IV tumours had scant cytoplasm, even though the nuclei might be minimally anaplastic. This system is not widely used in practice, and the system proposed by WHO in 2010 based on a combination of cytological features and differentiation is generally employed for grading ( Table 13.9 ). A correlation between the grade and prognosis has been reported. PDHCC are associated with higher recurrence after LT.

The staging system proposed by the American Joint Committee on Cancer (AJCC) is based on a combination of tumour characteristics (size, number, vascular invasion, direct extrahepatic extension), nodal status and distant metastasis ( Table 13.10 ). The clinical utility of this system is limited because most HCCs arise in the setting of underlying liver disease, which complicates therapy and leaves the patient at risk for developing new tumours, even if the current HCC is successfully eradicated. Therefore, several other staging systems have been proposed that combine tumour characteristics with clinical and laboratory features, including the Barcelona Clinic Liver Cancer (BCLC) staging ( Table 13.11 ), Cancer of the Liver Italian Program (CLIP) score, Japan Integrated Staging (JIS) and Hong Kong Liver Cancer (HKLC) staging system. The BCLC system has been validated in many studies and incorporated into the AASLD guidelines for treatment of HCC. However, the system that allows for the best prognostic stratification continues to be debated.

Spread and metastases

The average doubling time of small, untreated tumours is estimated to be 5–6 months. From an encapsulated mass in the early stages, the tumours infiltrate the surrounding liver as they enlarge and can form satellite nodules. Microvascular invasion is common, found in approximately 50% of tumours, and intrahepatic metastases, presumably by haematogenous spread, are present in 60% of tumours <5 cm in diameter and >95% of those >5 cm. Nodules growing from intrahepatic venous involvement or those adjacent to the main tumour are likely to be intrahepatic metastasis, whereas other nodules likely represent multicentric tumours. Presence of concurrent multifocal early HCC, a well-differentiated component at the periphery or distinct histologies in tumour nodules would favour multicentricity over intrahepatic metastasis. Thrombosis of the portal vein or its branches occurs in 65–75% and that of the hepatic veins in 20–25% of advanced tumours. Tumours with large-vessel vascular invasion involving vessels with a muscular wall or >1 cm are twice as likely to recur after resection.

Invasion of large bile ducts with obstructive jaundice occurs in approximately 5% of HCC patients. Advanced tumours often invade the diaphragm and gallbladder. Breach of Glisson capsule is rare, so peritoneal dissemination is uncommon. Extrahepatic metastases are common, being found at autopsy in more than half of cases. Lymph node metastasis is uncommon in classic HCC and is observed in <5% of patients at presentation. Haematogenous spread is observed in 40–60% of cases, with lung being the most common site. Other potential sites for metastases include adrenal glands, bone, stomach, heart, pancreas, kidney, spleen and ovary.

Treatment and prognosis

Surgical resection offers the only potential for cure for HCC and works best for resectable tumours in noncirrhotic liver or in cirrhotic patients with adequate liver reserve. Liver transplantation (LT) offers the possibility of cure for cirrhotic patients with HCC. Patients selected based on Milan criteria (solitary tumour ≤5 cm, or up to three tumours and none >3 cm) have a 5-year survival exceeding 70%. It has been argued that Milan criteria are restrictive, and expanded criteria (e.g. UCSF criteria: solitary tumour ≤6.5 cm, or up to three nodules with the largest ≤4.5 cm, and total tumour diameter ≤8 cm) have been successfully used to obtain 5-year survival of >60%. If LT is not an option, US-guided percutaneous ablation can be done using alcohol or thermal techniques such as radiofrequency waves, microwaves, laser or cryoablation. Ethanol injection and radio­frequency ablation have become the most widely used methods to cure small lesions (usually <3 cm) or as a bridge measure for patients awaiting definitive therapy by resection or LT (for lesions 3–5 cm). Since HCC receives its blood supply from the hepatic artery, transarterial embolization (TAE) can lead to tumour necrosis and prolong survival and is recommended for unresectable tumours >5 cm. The embolic material may be combined with lipoidol and antineoplastic drugs such as doxorubicin (TACE). Transarterial injection of yttrium-90 microspheres has been used for unresectable tumours (radioembolization). Systemic radiation, chemotherapy and hormonal therapy have largely proved unsuccessful in HCC. However, stereotactic body radiation therapy (SBRT) can deliver large doses of radiation and can be helpful to ablate the tumour. Sorafenib, a multikinase inhibitor, has been shown to prolong survival in patients with advanced HCC. National Comprehensive Cancer Network (NCCN) guidelines state that sorafenib can be considered after ablative or arterial therapy if there is persistent tumour and liver function is adequate.

Favourable prognostic factors for HCC are age <50 years, female gender, resectable tumour, better-differentiated tumour, low mitotic index, absence of vascular invasion, encapsulated tumour and absence of cirrhosis. The overall 5-year survival in HCC is 15–20% but can be as high as 75% for tumours <5 cm. For patients with advanced disease, survival beyond 1 year is unusual.