Liver-cell adenoma

Liver-cell adenomas are solitary or occasionally multiple tumours composed of hepatocytes. Macroscopically they are well defined but often not encapsulated. The cells of the tumour closely resemble normal hepatocytes (Fig. 11.1). Nuclei are small and regular and mitoses are almost never seen. These features are evident in fine-needle aspiration biopsies.¹ The cells are arranged in normal or thickened trabeculae interspersed with prominent arteries and thin-walled blood vessels. In adenomas, reticulin is normal or reduced, but extensive loss is in most cases confined to areas of necrosis or haemorrhage. The latter are characteristically found in adenomas in oral contraceptive users, and are responsible for pain and for the serious complication of haemoperitoneum. They probably also explain the fibrous scars which are sometimes found in the lesions. Regular septa, portal tracts and bile ducts are, however, absent; this distinguishes liver-cell adenomas both from non-neoplastic liver and from macroregenerative nodules (large regenerative nodules) in cirrhosis as well as focal nodular hyperplasia. An exception to this rule may occur in patients with multiple adenomas (adenomatosis²) where bile ducts can become entrapped within the lesions.³

Figure 11.1 Hepatocellular adenoma.

Liver cells appear normal or contain fat vacuoles. Blood vessels but no portal tracts are seen within the lesion. (Operative specimen, H&E.)

Adenomas may show small liver-cell rosettes (acini) with a central bile-filled or empty lumen, as in cholestatic liver. They may contain Dubin–Johnson-like pigment⁴ or show steatohepatitis with Mallory bodies.⁵ Non-necrotising granulomas within adenomas are also described.⁶ Genetic–histological correlations have allowed subclassification of adenomas into several types.⁷ Approximately 30–50% of adenomas show HNF-1α mutations and these typically contain fat but show no cytological atypia.^8,9 Activating β-catenin gene mutations are seen in some 10–15% of adenomas with cytological atypia and acini⁸ and these tumours are more likely to show transformation to hepatocellular carcinoma (HCC). A third type of adenoma (‘inflammatory and/or telangiectatic adenoma’) with chronic inflammatory cell infiltrates and/or sinusoidal dilatation has been linked to IL6ST gene mutations and increased interleukin-6 signalling¹⁰ and represents some 35% of adenomas.

The distinction of adenoma from HCC is occasionally difficult. Loss of reticulin, nuclear atypia and mitotic activity and the presence of many acinar structures favour carcinoma. Immunohistochemical demonstration of nuclear and/or cytoplasmic β-catenin overexpression is often helpful evidence of transition to carcinoma, but is not invariably present.¹¹

Most liver-cell adenomas arise in women of childbearing age, usually after prolonged use of oral contraceptives.¹² Use of anabolic/androgenic steroids is a risk factor for both adenoma and HCC,¹³ particularly in Fanconi’s anaemia.^14,15 Adenomatosis,^16,17 in which multiple tumours are seen throughout the liver, is much less common, is associated with HNF-1α mutations and shows female predominance.² A subgroup of these cases is familial and associated with diabetes.^2,17–19 Adenomatosis is also seen in patients taking anabolic/androgenic steroids²⁰ or in patients without risk factors.²¹ Liver-cell adenomas may also arise in patients with diabetes²² or type I glycogen storage disease,²³ and in children or young adults (see Neoplasms and nodules in children).

Macroregenerative nodules (large regenerative nodules)

The macroregenerative nodule (MRN) or large regenerative nodule is an unusually large regenerative nodule, which usually develops in cirrhotic liver. Because the MRN is one of several possible settings in which HCC may develop, it is covered in greater detail later in this chapter (see Precursors of hepatocellular carcinoma).

Focal nodular hyperplasia

Focal nodular hyperplasia (FNH) is a fairly common lesion, seen in either sex and at any age. FNH is a reactive, hyperplastic response of polyclonal²⁴ hepatocytes, fibrous stroma and bile ductules due to a putative pre-existing arterial malformation.^25–28 FNH, unlike liver-cell adenoma, does not appear to be caused by oral contraceptives. Although oral contraceptives may cause an increase in size and vascularity,²⁹ they do not appear to influence the number or size of these lesions.³⁰ Bleeding and rupture are rare, as is recurrence after resection.³¹ Features of FNH and adenoma are only very occasionally seen in the same tumour, and the occurrence of the two lesions in the same liver may be coincidental.³² There may be multiple FNHs in the same patient, and such individuals often have other lesions, including vascular anomalies (hepatic haemangioma, telangiectasis of the brain, berry aneurysm, dysplastic systemic arteries, portal-vein atresia), central nervous system neoplasms (meningioma, astrocytoma)^33,34 and hemihypertrophy.³⁵

Macroscopically, the nodules are well demarcated from the normal hepatic parenchyma. They are usually pale, and are dissected by fibrous septa into nodules, giving them an appearance very like that of cirrhosis. There may be a prominent central fibrous scar (Fig. 11.2) with closely associated smooth muscle actin immunostain-positive activated stellate cells.³⁶ Histologically, the appearance is also very like that of inactive cirrhosis. The dense fibrous septa contain large thick-walled and sometimes narrowed arteries, as well as bile-duct-like structures probably derived from metaplastic liver-cell plates³⁷ or from progenitor cells.²⁷ Cytokeratin 7 immunostain is useful for demonstration of the bile ductular structures and helps distinguish FNH from adenoma.³⁸ The presence of bile-duct cells in fine-needle aspiration cytology of FNH is helpful in distinguishing this lesion from HCC.³⁹ In radiologically guided needle biopsies, the pathologist should be made aware that a mass lesion is being sampled, since the proliferated bile-duct-like structures and reactive stroma may otherwise suggest the diagnosis of mechanical bile-duct obstruction⁴⁰ (Fig. 11.2).

Figure 11.2 Focal nodular hyperplasia (FNH).

A: Part of a central scar with abnormal arterioles has been sampled. Radiating fibrous septa show small bile-duct-like structures at their edges (arrowheads). The parenchyma is nodular. (Operative specimen, H&E.) B: Needle core biopsy from FNH has sampled a portion of the fibrous scar and lesional parenchyma. A portal tract-like region (arrow) with oedematous stroma and bile ductular structures shows changes resembling those of large bile-duct obstruction. Inset above: Ductular structures in oedematous stroma. (Needle biopsy, H&E.) Inset below: Cytokeratin 7 immunostaining pattern of bile ductular structures in FNH. (Operative specimen, specific immunoperoxidase.)

Lesions that grossly resemble FNH are also occasionally seen in Budd–Chiari syndrome.⁴¹ Microscopically, these masses show hyperplastic, regenerative nodules in combination with other features, including central scars and multiple arterial structures. Some vary histologically so as to suggest crossover lesions between large regenerative nodules, FNH and liver-cell adenoma.⁴² They appear to result from hyperarterialisation of regions of decreased hepatic venous blood flow.^42,43 FNH is also seen after liver transplantation in allografts with vascular perfusion abnormalities.^43a

Nodular regenerative hyperplasia

In nodular regenerative hyperplasia (NRH) multiple hyperplastic parenchymal nodules with thickened liver-cell plates are seen but fibrosis is absent or slight⁴⁴ (Fig. 11.3). This distinguishes the lesion from cirrhosis. In some cases perisinusoidal fibrosis is found in the compressed liver tissue between nodules. Portal tracts may be found at the centres of the nodules but this is not invariable. Diagnosis is often difficult in needle-biopsy specimens. The nodularity may be more clearly seen in reticulin preparations (Fig. 11.4). A wedge liver biopsy may be required to establish the diagnosis and to exclude an important differential: incomplete septal cirrhosis (see Ch. 10).

Figure 11.3 Nodular regenerative hyperplasia.

This abnormal, nodular growth pattern is not accompanied by fibrosis and therefore differs from cirrhosis. The parenchymal nodules (N) are often adjacent to (nodule at left) or surrounding portal tracts. The intervening liver shows flattened and compressed liver-cell plates and/or sinusoidal dilatation. (Post-mortem liver, H&E.)

Figure 11.4 Nodular regenerative hyperplasia.

Reticulin stain of a field similar to that shown in Fig. 11.3 highlights the regenerative nodules and the absence of fibrosis. (Post-mortem liver, reticulin.)

NRH is associated with a wide range of conditions, mainly rheumatic diseases, myeloproliferative disorders and chronic venous congestion.^45–47 Patients with NRH may have received therapeutic drugs, including corticosteroids, anabolic steroids, oral contraceptives, antineoplastics,⁴⁸ anticonvulsants and immunosuppressive agents.^45,49,50 NRH has also been associated with the toxic oil syndrome,⁵¹ Behçet’s disease,⁵² early histological stages of primary biliary cirrhosis,⁵³ coeliac disease with anti-cardiolipin antibodies,⁵⁴ livers containing metastatic neuroendocrine tumours⁵⁵ and non-cirrhotic livers in which HCC has developed.⁵⁶ Some patients with NRH have portal hypertension. Serum alkaline phosphatase and γ-glutamyl transpeptidase levels may be elevated.^47,53

Wanless and co-workers⁵⁷ have postulated that the basic lesion is portal venous thrombosis, leading to atrophy and compensatory hyperplasia. Arterial lesions, particularly arteriosclerosis of ageing, may also contribute to these changes.⁴⁷ Portal venous thrombosis has also been invoked in the pathogenesis of the rare partial nodular transformation, in which somewhat larger nodules are found, often localised to the perihilar region.^58,59 NRH, FNH and partial nodular transformation share the common feature of liver-cell hyperplastic growth in the form of nodules; they have accordingly been grouped under the umbrella heading of ‘nodular transformation’ by Wanless.⁶⁰ The main features of the various non-cirrhotic nodular conditions so far discussed are summarised in Table 11.2.

Table 11.2 Non-cirrhotic parenchymal nodules

Bile-duct adenoma

Bile-duct adenomas are small, grey-white, usually subcapsular nodules measuring from 1 to 20 mm in diameter,⁶¹ which may represent hamartomatous peribiliary glands rather than a neoplasm.⁶² They are more often solitary than multiple. Histologically, they are composed of small, well-formed ducts embedded in a stroma of mature fibrous tissue which may contain chronic inflammatory cells, often densely aggregated at the periphery of the lesion^61,63,64 (Fig. 11.5). Their chief importance is that they may be mistaken for metastatic carcinoma, both macroscopically and microscopically. They differ from microhamartomas (von Meyenburg complexes) in that the ducts are smaller and more numerous, are usually not dilated and do not contain bile.^61,65 Periodic acid–Schiff (PAS)-positive, diastase-resistant globules of α₁-antitrypsin within the bile-duct epithelium of multiple adenomas were described in a patient with heterozygous α₁-antitrypsin deficiency.⁶⁶ The bile-duct adenoma should also be distinguished from the rare biliary adenofibroma, a much larger tumour composed of tubulocystic bile-duct structures with apocrine metaplasia and intraluminal bile embedded in fibrous stroma, resembling fibroadenoma of the breast.⁶⁷

Figure 11.5 Bile-duct adenoma.

This subcapsular tumour consists of closely packed bile ducts set in a dense fibrous stroma. A dense collection of lymphocytes is seen at the edge of the lesion (bottom). (Operative specimen, H&E.)

Biliary cystadenoma

Biliary cystadenoma is a multilocular tumour, the cystic spaces of which contain mucoid fluid and are lined by columnar, mucin-secreting epithelium which may form papillary projections. A variant with subepithelial mesenchymal stroma containing myofibroblasts occurs in women.^68,69 Malignant change is common.⁷⁰

Haemangioma

The cavernous haemangioma is the most common benign tumour of the liver, found incidentally at autopsy or operation and occasionally seen in biopsy material.⁷¹ A few reach a large and clinically significant size. As in other sites, the lesions are composed of endothelium-lined channels supported by a fibrous stroma (Fig. 11.6). Lesional tissue sometimes extends irregularly into adjacent liver.⁷² Complications include thrombosis, sclerosis and calcification.⁷³ Spontaneous rupture is recorded but uncommon. A distinction should be made between cavernous haemangiomas and peliosis (see Ch. 12); the latter lacks the complete endothelial layer and fibrous trabeculae. Lymphangioma of the liver has been reported as part of multiorgan lymphangiomatosis or as a solitary hepatic lesion,⁷⁴ but is very rare. The endothelium-lined channels of this neoplasm are empty or contain lymph with occasional leucocytes. It should not be mistaken for mesenchymal hamartoma (see Neoplasms and nodules in children).

Figure 11.6 Haemangioma.

Blood-filled spaces are separated by fibrous septa. A thick capsule is seen at right. (Operative specimen, H&E.)

Mesenchymal and neural tumours

Connective-tissue elements, adipocytes and smooth muscle of the liver, nerve sheaths of intrahepatic nerves and other mesenchymal cells may give rise to rare tumours, including lipomas, myelolipomas, angiomyelolipomas,^75,76 schwannomas and neurofibromas,^77–79 solitary fibrous tumours⁸⁰ and chondromas.⁸¹ Angiomyolipomas resemble their more common renal counterparts, and contain blood vessels, smooth muscle (myoid cells) and fat.⁸² These components allow subcategorisation into mixed, lipomatous, myomatous and angiomatous types, in decreasing order of frequency.⁸³ Multiple tumours may be present.^84,85 Muscle cells may be partly of epithelioid type, with finely granular eosinophilic cytoplasm and pleomorphic nuclei^86–89 (Fig. 11.7). These may be mistaken for hepatocytes or malignant cells, particularly in cases where the component of fat is minimal. Megakaryocytes and other bone marrow elements are commonly present. Positive HMB-45 immunostaining of the myoid cells is a major diagnostic feature.^83,89 Pseudolipomas⁹⁰ probably represent separated nodules of peritoneal fat which become embedded in the liver capsule.

Figure 11.7 Angiomyolipoma.

The tumour shows myoid cells with ample granular cytoplasm resembling hepatocytes. Fat vacuoles, seen at right, were variably scattered through the tumour, as were small blood vessels. (Operative specimen, H&E.)

Inflammatory pseudotumour

Lesions of inflammatory pseudotumour may be solitary or multiple and usually occur in young, male patients with constitutional symptoms, fever and weight loss. They may sometimes involve structures near the porta hepatis with resultant biliary problems or portal hypertension. The microscopic hallmark of inflammatory pseudotumour is the extensive polyclonal plasma-cell infiltrates which are intermixed with lymphocytes, eosinophils, foamy histiocytes and variable degrees of stromal proliferation, including spindle cells in bundles and whorls with associated fibrosis⁹¹ (Fig. 11.8). Granulomas and partly obliterated blood vessels may be present. The lesion falls within a diagnostically controversial spectrum ranging from an inflammatory–reparative process (possibly infectious in aetiology) to a low-grade stromal malignancy termed inflammatory myofibroblastic tumour (IMT).^92,93 Some have been thought to be follicular dendritic cell tumours related to Epstein–Barr virus infection.⁹⁴ Immunostains are helpful to characterise individual cases. Among these, smooth muscle actin will highlight the extent of the myofibroblastic component and activin-like kinase 1 (ALK1) expression in the spindle cells favours a diagnosis of IMT.^92,93 Surgical resection is the treatment of choice, when possible.

Figure 11.8 Inflammatory pseudotumour.

Dense infiltrates of plasma cells, lymphocytes and histiocytes with interwoven bundles of collagen are seen. Inset: Plasma cells are unusually prominent. (Operative specimen, H&E.)

Precursors of hepatocellular carcinoma

A number of hepatocellular changes and nodular lesions have been considered pre-malignant or precursors of hepatocellular carcinoma (HCC) and these are discussed below. Despite refinements in the terminology of these lesions (Table 11.3) provided by panels of hepatic pathologists,^95–97 the precise sequence of histological and molecular changes in the presumed multistep pathogenesis of HCC in humans has not been established. The importance of recognition of these worrisome lesions is based on the need for close patient surveillance and possible surgical resection (or liver transplantation) once they are identified pathologically. The presence of one or more of these lesions should be clearly stated in the pathologist’s report.

Table 11.3 Nomenclature of putative precursors of hepatocellular carcinoma

Non-neoplastic liver tissue may show varying degrees of liver-cell dysplasia (LCD) of either large- or small-cell type (see Figs 10.8, 10.9). Large-cell LCD (large-cell change) is the type most often observed and features cell and nuclear enlargement, nuclear pleomorphism, multinucleation and multiple nucleoli and increased nuclear staining⁹⁸ (see Figs 10.8 and 11.33). Its distribution is random within lobules or cirrhotic nodules and should be distinguished from the variations in nuclear morphology seen in perivenular hepatocytes with ageing, in the presence of cholestasis or in methotrexate therapy. This type of dysplasia was first associated with hepatitis B virus infection, cirrhosis and HCC^99,100 and subsequently with a four- to five-fold increased risk of HCC in several studies.^101,102 Affected cells are usually aneuploid¹⁰³and may have attendant chromosomal abnormalities.¹⁰⁴ However, it has been considered merely an effect of cholestasis¹⁰⁵ or a derangement in normal liver-cell polyploidisation¹⁰⁶ and has not been proven to be a direct pathogenetic precursor lesion of HCC. Nevertheless, it is a strong independent risk factor for the development of HCC^107,108 and thereby identifies patients requiring more diligent surveillance.

Small-cell LCD (small-cell change) is characterised by enlarged, hyperchromatic nuclei within small hepatocytes (increased nuclear–cytoplasmic ratio) arranged in crowded clusters¹⁰⁹ (see Figs 10.9 and 11.34). These foci show high cellular proliferation rates¹¹⁰ and an overall cytological resemblance to HCC, and may originate from progenitor cells.¹⁰⁷ These features have lent support to small-cell LCD as a true precursor lesion that is subject to the later cellular events leading to the development of HCC.¹¹¹

Other cellular changes cited as indicators of pre-malignancy include intracytoplasmic Mallory bodies,¹¹² irregular areas of regeneration showing hepatocyte glycogenosis, oncocytic change (see Ch. 9) or bulging nodularity,¹¹³ iron-negative foci in siderotic macroregenerative nodules¹¹⁴ and ‘iron-free foci’ in livers of patients with hereditary haemochromatosis;¹¹⁵ the last may show large-cell LCD.¹¹⁵ Clusters of large-cell or small-cell dysplastic hepatocytes less than 1 mm in diameter have been termed dysplastic foci by an international working party.⁹⁶

The macroregenerative nodule (MRN) is an unusually large regenerative nodule measuring 0.8 cm or more in diameter which develops in cirrhosis or other chronic liver disease¹¹⁶ (Fig. 11.9). MRNs are particularly common in macronodular cirrhosis.¹¹⁷ They may be paler or more bile-stained than the surrounding liver.⁹⁶ The cirrhotic liver may harbour several MRNs, which may coexist with HCC elsewhere in the liver or may contain foci of carcinoma. Cirrhotic explant livers should be carefully examined for these lesions^95,118 and for liver-cell dysplasia.¹¹⁹

Figure 11.9 Macroregenerative nodule.

This low-magnification view demonstrates the increased size of the nodule at right compared with the cirrhotic nodules at left. (Operative specimen, H&E.)

(Illustration kindly provided by Dr Kamal Ishak, Washington, DC, USA.)

The MRN histologically shows hyperplastic liver parenchyma arranged in plates two or three cells thick, which is typical of cirrhosis. The nodule contains portal tracts and fibrous septa with bile ducts, hepatic arteries and portal-vein branches, and shows no cellular atypia or disorder in the liver-cell plate arrangement. Steatosis, haemosiderin, bile plugs and Mallory bodies may be present.^116,120 The term ‘adenomatous hyperplasia’, a former synonym of MRN, as well as subdivisions into MRN types I and II¹¹⁷ are not currently advocated for use.⁹⁶

The dysplastic nodule (borderline nodule) shows atypical architectural and/or cytological features that are not acceptable for a benign macroregenerative nodule, but which fall diagnostically short of frank HCC.^96,121 Dysplastic nodules may show varying degrees of large and small-cell LCD, increased cellularity and foci where the liver cords are less cohesive, focal loss of reticulin fibres or pseudoacini^{95,96,118,121} (Fig. 11.10).

Figure 11.10 Dysplastic nodule.

The dysplastic nodule at right shows hepatocytes arranged in pseudoacini, with a less cohesive growth pattern centrally. A cirrhotic nodule is present at lower left. The patient had an inactive cirrhosis due to tyrosinaemia. (Explant liver, H&E.)

The major diagnostic concern is to distinguish MRNs and dysplastic nodules from HCC. Certain features seen in these nodules are associated with high risk of progression to carcinoma, including an increased ratio of nuclear density, clear-cell change, small-cell dysplasia and fatty change.¹²² Increased mitotic activity, loss of reticulin fibres, formation of broad trabeculae and an infiltrative margin are helpful evidence of carcinoma.^{95,118,123,124} Studies that have demonstrated clonality and loss of heterozygosity¹²⁵ and increased cell proliferative indices¹²⁶ combined with newer techniques such as spectral karyotype (SKY)¹²⁷ and gene microarray¹²⁸ analysis are promising avenues for understanding the key biological and morphological steps in the pathogenesis of HCC.

Hepatocellular carcinoma

Hepatocellular carcinoma (HCC) ranks fifth among malignant tumours worldwide.¹⁰⁰ Epidemiological and other studies of HCC have defined geographical variations in the incidence and prevalence of this tumour, as well as a multifactorial aetiology.¹²⁹ Chronic necrosis and inflammation of the liver are important driving forces in the multistep process of hepatocarcinogenesis^130,131 in the context of underlying risk factors such as hepatitis B and C viral infections,¹⁰⁰ iron overload,¹³² aflatoxin exposure¹³³ and the presence of fatty liver disease.^134–136 At the molecular level, identification of genetic changes that control cell cycling and apoptosis,¹³⁷ as well as oncogene expression,¹³⁸ mutation of tumour suppressor genes such as p53,¹³⁹ expression of vascular and cellular growth factors^140–145 and proliferation of hepatic stem cells or their progeny^146,147 constitute a large and growing literature on this subject.

The majority of HCCs develop in cirrhotic liver.¹⁴⁸ The cause of the cirrhosis is usually known, even in many cases labelled as ‘cryptogenic’ where risk factors for non-alcoholic fatty liver disease (see Ch. 7) become apparent.¹³⁵ The non-cirrhotic setting accounts for a substantial number of cases from North America¹⁴⁹ and elsewhere¹⁵⁰ and can be seen in hepatitis B virus carriers^151,152 or in those with suspected occult hepatitis B¹⁵⁰ and in individuals infected with hepatitis C virus.¹⁵³ HCC may even develop within ectopic liver.¹⁵⁴ The cirrhosis associated with carcinoma is often macronodular in pattern, except for the micronodular cirrhosis seen in genetic haemochromatosis and chronic hepatitis C. The cirrhosis is usually inactive, although inflammation and necrosis may be seen near the tumour itself. Tumours may be multifocal.¹⁵⁵ Intrahepatic tumour spread is both portal (via portal-vein branches) and lobular.¹⁵⁶ Rarely, HCC may spontaneously regress.^100,157,158 Following transplantation, cirrhotic explant livers require careful examination for small carcinomas and precursor lesions which are clinically undetected.¹⁵⁹ Pathology reports on explants or partial resections with HCC should specify the number of lesions and their sizes, as well as the histological grade and evidence of vascular invasion¹⁶⁰ since these factors affect TNM staging and other prognostic classifications.¹⁶¹

The outstanding histological features of HCC are the resemblance of the tumour cells to normal hepatocytes, and of their arrangement to the trabeculae of normal liver (Fig. 11.11). However, the trabeculae are for the most part thicker and reticulin is often scanty or even absent (Fig. 11.12). This paucireticulin pattern is helpful in fine-needle aspiration biopsies (see Cytopathological diagnosis). In exceptional cases where there may be an increase in reticulin, other histological features and/or the clinical behaviour of the tumour must be used as diagnostic criteria of malignancy. Rarely, the trabecular pattern and even bile production is mimicked by primary tumours (‘hepatoid carcinomas’) of the stomach, ovary and other sites¹⁶² (see section on Metastatic tumour). Between the tumour trabeculae in HCC there is a network of vascular channels lined by endothelium which is positive with immunostains for CD34,^140,141 factor VIII-related antigen and Ulex europaeus lectin.¹⁶³ The endothelial lining of these channels is a particularly helpful diagnostic feature in fine-needle aspirates (see Fig. 11.36). The absence of portal tracts and a cohesive connective tissue framework in the tumour results in a characteristic fragmentation of needle biopsy specimens with separation of tumour trabeculae that is readily observed at low magnification. Although connective tissue stroma is uncommon except in fibrolamellar carcinoma (described below), focal areas of fibrosis may follow tumour necrosis. A form of HCC designated as sclerosing carcinoma¹⁶⁴ is often associated with hypercalcaemia; some of these tumours appear to be of bile duct rather than hepatocellular origin. The adenoid (acinar) variant of HCC (Fig. 11.13) should not be confused with adenocarcinoma of the biliary tree. Bile-duct carcinomas are usually scirrhous, mucin-secreting tumours, whereas the characteristic secretion of HCCs is bile, seen in a minority of tumours in spaces homologous with normal bile canaliculi. Mixed or combined tumours designated hepatocellular–cholangiocarcinoma are also well described, with special stains and immunohistochemical features representative of both hepatocellular and bile-duct epithelial derivation. Progenitor/stem cell constituents are sometimes present¹⁶⁵ as shown by cytokeratin 7 or 19 positivity (discussed below). Occasionally HCC is infiltrated by T lymphocytes and shows an improved prognosis.¹⁶⁶

Figure 11.11 Hepatocellular carcinoma.

Note the trabecular–sinusoidal structure and resemblance of the tumour cells to normal hepatocytes. (Needle biopsy, H&E.)

Figure 11.12 Hepatocellular carcinoma.

Reticulin is scanty in this example. (Needle biopsy, reticulin.)

Figure 11.13 Hepatocellular carcinoma.

Adenoid pattern. Other areas of this tumour showed a more typical trabecular structure. (Post-mortem liver, H&E.)

At a cellular level variants include giant-cell forms with multinucleated tumour cells (a bad prognostic sign¹⁶⁷) (Fig. 11.14), spindle-cell or sarcomatoid tumours^168,169 and clear-cell carcinomas. The last must be distinguished from metastatic renal adenocarcinoma.^170,171 Fine-needle aspiration yields diagnostic material in a high proportion of patients.^172–175 Histological grading of HCC from 1 to 4 is based on nuclear features, with grade 1 HCC resembling normal hepatocytes and grade 2 showing prominent nucleoli, hyperchromatism and nuclear membrane irregularities.¹⁷⁶ Grades 3 and 4 show progressively greater nuclear pleomorphism, the latter featuring anaplastic and giant tumour cells (Fig. 11.14).

Figure 11.14 Hepatocellular carcinoma: cytological grading.

Grades 1–4 are illustrated in the respective panels. Grade 1 (well differentiated) tumours have small, round nuclei similar to those of normal and cirrhotic liver. Grades 2 and upwards show progressive alterations in nuclear contour, chromatin coarseness and chromaticity. Grade 4 shows marked anaplasia with giant, multinucleated tumour cells and atypical mitotic figures. (Needle biopsies, H&E.)

When there is doubt about the hepatocellular origin of a carcinoma, further evidence can sometimes be gained from the characteristics of the tumour cells. In hepatocellular carcinoma these often contain fat and glycogen, and may also contain α₁-antitrypsin globules, even in patients without genetic α₁-antitrypsin deficiency. Mallory bodies are commonly found in the cytoplasm of the tumour cells.¹⁷⁷ Evidence of hepatocellular origin is also provided when immunohistochemical stains of paraffin sections are positive for albumin, fibrinogen, liver-cell cytokeratins (8 and 18), α₁-antitrypsin or α₁-antichymotrypsin.^178–182

There are several possible immunohistochemical strategies for confirming the diagnosis of HCC (Table 11.4). One established approach is to use the quartet of cytokeratin 7 (CK7), cytokeratin 20 (CK20), Hep Par 1 (‘hepatocyte’) and polyclonal carcinoembryonic antigen (pCEA). HCC typically is negative for both CK7 and CK20,¹⁸³ while Hep Par 1 stains normal and malignant hepatocytes (and, rarely, several extrahepatic tumours¹⁸⁴). Hep Par 1 staining may be only patchy in needle biopsies of HCC or negative with more poorly differentiated tumours, liabilities which can be surmounted using more recently recommended immunostains (described below). Polyclonal carcinoembryonic antigen (CEA) provides positive staining of the carbohydrate moiety of biliary glycoprotein on the apical surfaces or canalicular structures of the HCC cells (see Fig. 11.15) and on bile canaliculi in non-neoplastic liver tissue. CD10 shows similar results to pCEA but is far less sensitive.¹⁸⁵ α-Fetoprotein is an unreliable immunostain for HCC,¹⁸⁶ in contrast to hepatoblastoma where most cases stain positively. Interestingly, the immunostain for thyroid transcription factor-1 (TTF-1), often used in the diagnosis of lung carcinomas, showed positive cytoplasmic (not nuclear) staining in the majority of HCCs in one study,¹⁸⁷ which may be helpful in specific diagnostic settings.

Table 11.4 Immunohistochemical stains in the evaluation of hepatic tumours

^* Staining is canalicular or apical.

^** See Ref. 183.

^*** GPC-3: glypican-3; GS: glutamine synthetase; HSP70: heat shock protein 70. At least 2 of the 3 should be positive (see Ref. 188).

Figure 11.15 Immunohistochemical demonstration of bile canalicular structures in a hepatocellular carcinoma.

The branching spaces are here outlined by the use of polyclonal anti-CEA.

The trio of glypican-3 (GPC-3), glutamine synthetase (GS) and heat shock protein 70 (HSP70) immunostains is proving to be an exceptionally robust combination for the diagnosis of HCC, particularly when any two of the three are positive.¹⁸⁸ GPC-3, a cell-surface heparan sulphate proteoglycan, usually shows cytoplasmic positivity in the tumour cells, but may also be membranous or canalicular. It has particular value in staining poorly differentiated HCCs that are negative with Hep Par 1 and is also applicable to fine-needle aspiration specimens.¹⁸⁹ However, cirrhotic nodules and hepatocytes in chronic hepatitis C with high-grade necroinflammatory activity may also show strong GPC-3 positivity.^190,191 Positive GPC-3 staining may also be seen in certain germ cell tumours,¹⁹² ovarian clear-cell carcinoma¹⁹³ and in squamous cell carcinoma of the lung.¹⁹⁴ GS staining in non-neoplastic liver is restricted to the cytoplasm of perivenular hepatocytes, but HCC shows diffuse strong lesional staining.^97,188 HSP70 shows focal nucleocytoplasmic positivity in HCC.⁹⁷ This panel of three immunostains also helps distinguish dysplastic lesions from HCC.¹⁸⁸

Fibrolamellar carcinoma

This tumour usually develops in non-cirrhotic liver in older children and adults and carries a better prognosis (because of its resectability¹⁹⁵ and absence of cirrhosis^195a) than typical HCC.^196–199 The lesions are solitary or multiple and occasionally resemble focal nodular hyperplasia macroscopically in having a central fibrous scar.²⁰⁰ The unique histological features distinguish this tumour from routine hepatocellular carcinoma. Fibrous lamellae arranged in parallel separate groups of large, densely eosinophilic tumour cells^198,201 which produce transforming growth factor-β²⁰² (Fig. 11.16). The eosinophilia is due to the presence of abundant mitochondria.^197,203 Tumour cells commonly contain eosinophilic, diastase–PAS-negative globules which stain immunohistochemically for C-reactive protein, fibrinogen and α₁-antitrypsin, as well as cytoplasmic ‘pale bodies’ which are reactive for fibrinogen.¹⁹⁸ Additional features include bile production (as in other forms of hepatocellular carcinoma), copper and copper-associated protein within tumour cells,^204,205 and stainable CEA in bile canaliculi.²⁰⁶ Some fibrolamellar carcinomas have neuroendocrine features,^207,208 mucicarmine-positive pseudoglands^208a or show features of both fibrolamellar and typical hepatocellular carcinoma.²⁰⁹ Despite isolated reports such as the association of Fanconi’s anaemia with fibrolamellar carcinoma,²¹⁰ the pathogenesis of this tumour is uncertain and risk factors are not apparent.

Figure 11.16 Hepatocellular carcinoma: fibrolamellar type.

Groups of large, eosinophilic tumour cells are surrounded by fibrous septa in parallel arrays. (Needle biopsy, H&E.) Inset: Tumour cells contain ‘pale bodies’ (top centre). Several hyaline bodies are also evident in tumour cells at top, left of centre. (Explant liver, H&E.)

Bile-duct carcinoma (cholangiocarcinoma)

Carcinoma of the bile ducts can arise anywhere between the papilla of Vater and the smaller branches of the biliary tree within the liver. It is not usually associated with cirrhosis. There are two major anatomic types:²¹¹ one arising either from extrahepatic bile ducts or at the birfurcation of right and left hepatic ducts (the latter perihilar cholangiocarcinoma sometimes referred to as a Klatskin tumour²¹²), and a second type presenting as an intrahepatic mass.²¹³ Carcinoma of the hepatic ducts is an important cause of biliary obstruction, which may be missed at laparotomy unless the intrahepatic bile ducts are explored or visualised. The commonest known predisposing factors to bile-duct cancer are infestation with oriental flukes, primary sclerosing cholangitis²¹⁴ and congenital cystic lesions of the biliary tree.²¹⁵ Of these, Caroli’s disease and choledochal cysts are important precursors, but carcinoma may also arise in von Meyenburg complexes (bile-duct microhamartomas)²¹⁶ and in congenital hepatic fibrosis.²¹⁷ Development of carcinoma in bile-duct adenoma is also reported.²¹⁸ In Japan, hepatitis C virus infection has been suggested as a risk factor²¹⁹ and intrahepatic cholangiocarcinoma is a known consequence of hepatolithiasis.²²⁰ Investigations at the molecular level indicate pathogenetic roles for K-ras and p53 mutations, increased expression of receptor tyrosine kinase c-erB-2 and c-met and expression of human telomerase reverse transcriptase (hTERT).²¹¹

Microscopically, bile-duct carcinomas are mucin-secreting adenocarcinomas with a reactive, desmoplastic fibrous stroma (Fig. 11.17). A fairly uniform gland size (medium to small) is often maintained within these tumours, in comparison with the wide size variations seen in glands of metastatic pancreatic carcinoma. The cholangiolocellular carcinoma subtype shows interanastomosing duct-like structures composed of small cuboidal cells with scant cytoplasm that appear to evolve from progenitor cells in the region of the canal of Hering.²²¹ The tumour cells are cuboidal or columnar and may assume a papillary pattern. Adenosquamous, squamous, mucinous,²²² clear-cell²²³ and anaplastic histological types are less common.²²⁴ Intraneural and perineural invasion is common. The presence of free stromal mucin, small groups and isolated tumour cells in fibrous stroma and the concurrence of apparently normal epithelium and abnormal tumour cells within a duct-like structure all help to distinguish cholangiocarcinoma from metastatic tumour.²²⁵ Cholangiocarcinoma must be distinguished from the acinar type of hepatocellular carcinoma, a distinction usually made with confidence on the basis of mucin or bile secretion, respectively. In difficult cases positive staining for epithelial membrane antigen,²²⁶ tissue polypeptide antigen,²²⁷ biliary cytokeratins¹⁷⁹ (7 and 19), Lewis(x) and Lewis(y) blood group-related antigens²²⁸ and α-amylase²²⁹ helps to exclude hepatocellular carcinoma. In the uncommon tumour which shows combined hepatocellular–cholangiocarcinoma, cytokeratins 7 and 19 and epithelial membrane antigen immunostaining is positive in the cholangiocellular component.^230,231 Other rare mixed tumours show sarcomatoid²³² or fibrolamellar regions.²³³ The differential diagnosis of bile-duct cancer includes epithelioid haemangioendothelioma. Bile-duct tumours are very occasionally of neuroendocrine type, with characteristic neurosecretory granules in their cytoplasm.

Figure 11.17 Bile-duct carcinoma.

There are islands of adenocarcinoma in the connective tissue. The appearances are different from those of the hepatocellular carcinoma of adenoid pattern shown in Fig. 11.13. (Operative specimen, H&E.)

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