zachary d. goodman luigi m. terracciano · 2013-12-20 · because of chronic liver disease may...

Tumours and tumour-like lesions of the liver

Zachary D. Goodman Luigi M. Terracciano

Benign hepatocellular tumours and tumour-like lesions 762

Malignant hepatocellular tumours 768

Benign biliary tumours and tumour-like lesions 788

Malignant biliary tumours 790

Benign vascular tumours 795

Malignant vascular tumours 797

Other benign tumours and tumour-like lesions 800

Other malignant tumours 803

Mass lesions in the liver represent a number of disease processes that frequently prompt patients to seek medical attention. Rapidly growing tumours produce abdominal symptoms and even slowly growing tumours, both benign and malignant, may outgrow their blood supply with sub-sequent infarction and haemorrhage into the tumour or the peritoneal cavity. Some lesions, particularly small benign tumours, may be clinically silent and are only discovered when the patient is evaluated for some other reason. Small tumours may also be detected in asymptomatic patients in screening programs because of increased risk of hepatic malignancy; for example those with chronic viral hepatitis at risk for hepatocellular carcinoma (HCC), or those with primary sclerosing cholangitis at risk for cholangiocarci-noma. This chapter deals with the principal neoplasms and other mass lesions that may occur in the liver. The classifi ca-tion (Table 15.1) follows that of the World Heath Organization Classifi cation of Tumours1 and the Armed Forces Institute of Pathology Atlas of Tumour Pathology,2 rearranged to emphasize the histogenesis of the lesions. Although the emphasis is on primary liver tumours, it is important to remember that metastases far outnumber primary tumours, and so these are always considered in differential diagnosis.

The clinical scenario is an important feature in the dif-ferential diagnosis of any mass lesion in the liver. Age, gender and predisposing factors such as underlying liver disease or exposure to drugs, chemicals, or parasites infl u-ence the likelihood of development of many tumours (Table 15.2). Consequently, one should be very circumspect about diagnosing a tumour of infancy, such as hepatoblastoma, in

an adult or a tumour such as hepatocellular adenoma that is strongly associated with oral contraceptive steroids in an elderly male. Rare cases may occur in atypical hosts, but such diagnoses should be questioned and verifi ed. Some tumours, especially HCC, have a striking geographic distri-bution, due primarily to its association with chronic hepa-titis B infection and other chronic liver diseases.

Detection and evaluation of hepatic mass lesions nearly always involves radiological imaging techniques.3,4 These have evolved rapidly over the past three decades, and the technology continues to advance. The major modalities used to identify and characterize mass lesions are variations of ultrasound (US),5,6 computed tomography (CT),7,8 and magnetic resonance imaging (MRI) (Chapter 1).9,10 When combined with various contrast media, these can provide images to characterize both the lesions and the blood supply to the lesions. The images may be suffi ciently characteristic to provide fi rm diagnosis of some tumours, especially cav-ernous haemangiomas, or strongly suggest the diagnosis of others, such as focal nodular hyperplasia. In most instances, however, a biopsy is required for defi nitive diagnosis.

In some cases, clinical information can support imaging studies to give a strong presumptive diagnosis; for example, a mass shown to enlarge on sequential imaging studies over several months in a patient with chronic viral hepatitis and cirrhosis and rising serum α-fetoprotein is almost certainly HCC. Consequently, patients at risk for developing HCC because of chronic liver disease may undergo periodic sur-veillance with imaging, most often ultrasound, and serum tumour markers, most often α-fetoprotein, in an attempt to detect tumours at a curative stage. It has been suggested that

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criteria such as two compatible imaging studies can be used in such cases for definitive diagnosis without biopsy, but this remains controversial.11,12 Clinical considerations deter-mine the need for obtaining tissue but, in general, a defini-tive diagnosis of a liver tumour requires pathological examination.

Benign hepatocellular tumours and tumour-like lesions

Hepatocellular adenoma and adenomatosis

Hepatocellular adenoma (HCA) is a benign neoplasm that arises in a normal liver composed of cells that closely resem-ble normal hepatocytes. When multiple (usually more than 10) adenomas are present, the condition is called ‘liver adenomatosis’.

Aetiology

HCA typically develops in women in the reproductive age group (15 to 45 years), nearly always associated with oral contraceptive steroid use. Although the absolute risk has always been very low, epidemiological case–control studies conducted in the 1970’s found an annual incidence of about 3 to 4 per 100 000 long-term ( 5 year) oral contraceptive users, but only 1 per million in non-users or women with less than 2 year’s exposure.13,14 The incidence appears to have decreased in recent decades with the introduction of lower-dose oral contraceptive preparations.15 The exact mechanism by which adenomas are produced is not known, but experimental evidence suggests that sex hormones are promoters rather than initiators of hepatocellular neo-plasms.16,17 This is supported by the observation that in several cases, unresectable adenomas have been observed to regress after contraceptive steroid use was stopped.18,19

Furthermore, other steroid hormones, including non-contraceptive oestrogens20 and anabolic/androgenic ste-roids,21 have been associated with HCA. Since hepatocellular adenomas nearly always occur in long-term users of oral contraceptives, any cases outside this setting are highly suspect, and may actually be a different benign lesion such

Table 15.1 Abbreviated classification of primary tumours and tumour-like lesions of the liver

Benign Malignant

Hepatocellular tumours Hepatocellular adenoma Hepatocellular carcinoma

Focal nodular hyperplasia Fibrolamellar hepatocellular carcinoma

Dysplastic nodule Combined hepatocellular-cholangiocarcinoma

Carcinosarcoma

Hepatoblastoma

Biliary tumours Von Meyenburg complex Biliary cystadenocarcinoma

Bile duct cyst Cholangiocarcinoma

Ciliated foregut cyst

Peribiliary gland hamartoma

Biliary papillomatosis

Biliary cystadenoma

Vascular tumours Haemangioma Angiosarcoma

Infantile haemangioendothelioma Epithelioid haemangioendothelioma

Other tumours Angiomyolipoma Primary lymphomas

Mesenchymal hamartoma Other sarcomas and rare tumours

Inflammatory pseudotumour

Table 15.2 Clinical features of liver tumours

Tumours of infancy and young Infantile haemangioendothelioma

children Mesenchymal hamartoma

Hepatoblastoma

Tumours of older children and Fibrolamellar hepatocellular

young adults carcinoma

Embryonal sarcoma

Tumours much more frequent Hepatocellular carcinoma

in men

Tumours much more frequent Hepatocellular adenoma

in women Biliary cystadenoma

Tumours associated with chronic Hepatocellular carcinoma

liver disease and cirrhosis

Tumours associated with Hepatocellular adenoma

chemical or drug exposure Hepatocellular carcinoma

Angiosarcoma

Tumours associated with Cholangiocarcinoma

parasitic infections and

inflammatory diseases of the

biliary tract

Tumours associated with Hepatocellular carcinoma

congenital anomalies and Cholangiocarcinoma

metabolic diseases

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as focal nodular hyperplasia (FNH) or a well-differentiated hepatocellular carcinoma. Cases of HCA have been reported rarely in men, children, and women not taking oral contra-ceptives, but at least some of these are probably misdiag-noses. Older reports of so-called hepatocellular adenomas in cirrhotic livers are undoubtedly examples of macroregen-erative nodules.

HCA has been reported in association with conditions and drug exposures other than steroid hormones, but these are so rare that they are probably coincidental, with two exceptions. One of these is in the inherited glycogen storage diseases, especially Type Ia but also Types III and IV.22,23

Patients with these disorders may develop multiple adeno-mas. The other exception is a form of autosomal dominant familial diabetes mellitus, termed ‘maturity-onset diabetes of the young, type 3′ or MODY3.24–26 Patients with this dis-order also develop multiple adenomas, and they have been found to have a germline mutation of the TCF1 gene, which codes hepatocyte factor 1a (HNF1a), a transcription factor that controls numerous liver genes. Somatic mutations with HNF1a inactivation are also found in 60% of sporadic or contraceptive steroid-associated HCA, suggesting that it may be important in pathogenesis.

Clinical features

There is very little recent literature on the subject, and it is quite possible that the decreasing incidence of contraceptive steroid-associated HCA may have altered the conventional view. Nevertheless, in two large series compiled in the 1970’s, the mean age was 30, and most patients were between 20 and 39.14,27 Patients usually come to medical attention when symptoms develop, with only 5–10% found inciden-tally; 25–35% were aware of an abdominal mass; 20–25% had chronic or mild episodic abdominal pain; and 30–40% had acute abdominal pain, due to haemorrhage into the tumour or into the peritoneal cavity. Intraperitoneal haem-orrhage, the most serious complication of HCA, regardless of aetiology, often requires emergency surgery and causes circulatory collapse and death in 20% of patients.

Surgical excision is usually advised for HCA to avoid possible rupture and haemorrhage, and because of the risk of malignant transformation. Steroid-associated tumours often regress if the patient stops taking the exogenous hormones,28 while those associated with glycogen storage disease may regress with dietary therapy.29 Unresectable tumours or multiple tumours in liver adenomatosis may be treated by liver transplantation. Malignant transformation is rare, since most hepatocellular adenomas are resected when discovered, but there are documented cases of HCC arising in unresected solitary as well as multiple adenomas.30

Pathology

HCA, by defi nition, always arises in a non-cirrhotic liver. A similar-appearing lesion in a cirrhotic liver would be con-sidered a macroregenerative or low-grade dysplastic nodule. Hepatocellular adenoma is a solitary nodule, although

occasional patients may have more than one, and those with glycogen storage disease or other forms of adenomato-sis have multiple nodules. The tumours often bulge from the surface of the liver and occasionally are pedunculated. They may measure up to 30 cm in diameter, although the majority are 5–15 cm. They are usually unencapsulated, but on cut section they are well demarcated from the surround-ing liver. The colour varies from yellow or tan to brown, and there may be green areas of bile production as well as areas of necrosis or haemorrhage (Fig. 15.1) and sometimes irreg-ular scars from previous necrosis.

Microscopically, HCA is composed of benign hepatocytes arranged in sheets and cords without acinar architecture (Figs 15.2 and 15.3). The tumour cells are usually, but not always, larger and paler than non-tumour hepatocytes in the surrounding tissue (Fig. 15.2), due to increased cyto-plasmic glycogen and/or fat. The fat may be quite abundant, simulating fatty liver (Fig. 15.4). Other features that are variably present include bile production (Fig. 15.5), occa-sionally with pseudogland formation around dilated canaliculi, cytoplasmic lipofuscin granules, Dubin–Johnson

Fig. 15.1 • Hepatocellular adenoma. A resected tumour with areas of necrosis and haemorrhage.

Fig. 15.2 • Hepatocellular adenoma. The tumour (left) is composed of sheets of hepatocytes that are larger and paler than those of the surrounding liver, which shows some compression and mild steatosis. H&E.


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pigment and rarely Mallory bodies. The nuclei of the tumour cells are typically uniform and regular, the nuclear–cytoplasmic ratio is low, and mitoses are almost never seen. Nucleoli are seldom prominent. Occasional tumours, espe-cially in patients with long exposure to contraceptive ste-

roids, may have a few pleomorphic nuclei, resembling large-cell change in non-neoplastic chronic liver disease.

HCA lacks the central scar and large arteries of focal nodular hyperplasia, but areas of fibrosis and septum for-mation may be present, especially in those associated with glycogen storage disease, presumably reflecting long dura-tion. A well developed reticulin framework is usually present in the tumour. The sinusoids, with flattened endothelial lining cells, are usually compressed, thus contributing to the sheet-like appearance. Sometimes the sinusoids are dilated, a finding which can be mistaken for peliosis hepatis. Bile ducts are not found in HCA, but ductules and progenitor cells may be present.31 The presence of dilated sinusoids and ductules (Fig. 15.6) have caused some tumours to be classi-fied as a telangiectatic variant of focal nodular hyperplasia, but molecular studies have shown these to be HCA.32 Thin-walled vascular channels and small arteries are scattered throughout the tumours, but large arteries are only seen around the periphery. Kupffer cells are present though usually inconspicuous, stellate cells are occasionally seen. Haematopoietic elements are noted in the sinusoidal lumen of some tumours, and rare cases have had non-caseating granulomas in the tumour.33 Areas of haemorrhage may be present as well as recent or old infarcts, and areas of scarring containing haemosiderin-laden macrophages from old haemorrhages.

Differential diagnosis

Among benign lesions, focal nodular hyperplasia can some-times be difficult to distinguish from HCA, especially in small biopsies. The finding of areas of scarring that contain large arteries and the presence of chronic cholestatic fea-tures in the hepatocytes of the lesion favour the diagnosis of focal nodular hyperplasia. Distinction of HCA from well-differentiated HCC can be difficult and sometimes impos-sible, but can usually be made on histological grounds. Recognition of a trabecular growth pattern and cytological features of malignancy, including high nuclear/cytoplasmic ratios and nuclear irregularities, are most helpful. When a lesion with all the features of HCA has a moderate degree

Fig. 15.3 • Hepatocellular adenoma. The tumour has no focally acinararchitecture. The cells are large and pale, due to cytoplasmic glycogen. H&E.

Fig. 15.4 • Hepatocellular adenoma. The tumour has unpaired arteries butno portal areas. Some tumour cells have cytoplasmic fat, simulating hepaticsteatosis. H&E.

Fig. 15.5 • Hepatocellular adenoma. Bile production by the tumour cellswith canalicular bile plugs (arrows). H&E.

Fig. 15.6 • Hepatocellular adenoma. There is sinusoidal dilatation as well asinflammation and focal ductular proliferation, which has led such tumours tobe misdiagnosed as telangiectatic focal nodular hyperplasia. H&E.

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of nuclear irregularity and hyperchromatism, one may wish to take the history into account. If the patient had been taking oral contraceptives or other sex steroids, then the tumour is best regarded as an atypical HCA; but if it is certain that there is no such history, it is probably a well-differentiated HCC. Histochemical and immunohisto-chemical stains add little to the differential diagnosis of HCA from HCC in the diffi cult cases. Reticulin fi bres are generally decreased in the trabeculae of HCC compared to benign lesions, but well-differentiated carcinomas may have abundant reticulin. Markers of proliferation, such as Ki67, are much more frequent in malignant than benign hepato-cellular tumours, but some clearly malignant tumours are negative for these. Markers of endothelial differentiation such as CD34 are expressed only in periportal or periseptal sinusoids in non-neoplastic liver. Although they are usually strongly expressed in HCC, HCA can sometimes have an identical staining pattern.34

Focal nodular hyperplasia

Focal nodular hyperplasia (FNH) is a tumour-like malfor-mation composed of hyperplastic nodules of hepatocytes, separated by fi brous septa which often form typical stellate scars. FNH usually occurs in a liver that is otherwise histo-logically normal, although rare examples of such lesions have also been described in cirrhotic livers. Thick-walled arteries are present in the scars and septa, and ductules (but not ducts) are typically located within the fi brous tissue and at the junction with the parenchymal component. Focal nodular hyperplasia occurs in both sexes and all ages, but they are most commonly found in adult women.2 Occasional patients present because of a palpable mass but the vast majority are discovered incidentally at surgery, usually for diseases of the gallbladder, or in imaging studies performed for evaluation of abdominal symptoms, most of which are unrelated to the FNH. Lesions discovered during laparo-scopic cholecystectomy or by imaging are often not excised, but rather undergo needle biopsy, which may pose a diag-nostic challenge. If imaging has demonstrated a central scar, and the suspicion of FNH is high, then the patient may be followed without biopsy.

Despite the fact that FNH is most common in women, contraceptive steroids are not thought to play an aetiological role, unlike HCA. Most evidence points to abnormal blood fl ow as the key component in pathogenesis, although the exact sequence is unclear. The hepatocellular component is polyclonal,35 whereas HCA is monoclonal. The blood fl ow is predominantly arterial through arteries that are abnor-mally large for the region of the liver that they perfuse,36,37

while the extracellular matrix of both the septa and sinu-soids is similar to that of cirrhotic liver.38 Furthermore, angiopoietin gene expression is up-regulated in the lesions.39

Pathology

FNH can be large, at times occupying an entire lobe of the liver, but over 85% are less than 5 cm in diameter.2

Approximately 20% of patients have more than one FNH in the liver, and there is a frequent association with hepatic haemangiomas. The gross appearance is highly characteris-tic (Figs 15.7 and 15.8). The lesions are usually well circum-scribed but non-encapsulated. They frequently bulge from one of the surfaces of the liver and often have a depressed centre, resembling a metastasis. The colour is lighter than that of the adjacent normal liver. On cut section, the lesion is subdivided into smaller nodules by fi brous septa that often run into a stellate scar that may be central or eccentric, or in large lesions there may be several large scars. Microscopically, the lesions invariably have fi brous septa, often with the large central scar (Figs 15.9–15.11). These contain numerous vessels, both arteries and to a lesser extent veins, that course through the septa and the large scars (Fig. 15.12). The large arteries often show eccentric thickening due to intimal proliferation, fi bromuscular hyperplasia and disruption of the elastic lamina. The septa are often infi ltrated by varying numbers of infl ammatory cells, and numerous ductules (Fig. 15.13), but not true bile ducts, are present at the junction of the fi brous septa with the hepatocellular component. The hepatocellular

Fig. 15.7 • Focal nodular hyperplasia. On cut section, the lesion has a typical central scar with surrounding nodules that bulge from the cut surface of this fresh specimen.

Fig. 15.8 • Focal nodular hyperplasia. This wedge resection was sectioned after fi xation, showing the umbilicated central scar, radiating fi brous septa and pale parenchymal nodules.


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may be slightly larger and paler. They may contain fat, espe-cially if the rest of the liver is fatty, and even Mallory bodies if the patient has steatohepatitis. Liver cells adjacent to the fi brous septa of the lesion often show features of chronic cholestasis, with cholate stasis (‘pseudoxanthomatous trans-formation’) and copper storage, demonstrable with special stains. These features of chronic cholestasis are present in almost all cases of FNH and can be very helpful in differ-ential diagnosis.40 The absence of true bile ducts and a con-nection to the biliary outfl ow tract in part explains the cholestatic features.

Differential diagnosis

When the entire lesion is excised, together with a rim of normal adjacent parenchyma, the diagnosis is not diffi cult. The central stellate scar, fi brous septa, ductules and hepato-cellular nodules all serve to distinguish FNH from other benign hepatocellular lesions, especially HCA. Needle biop-sies may cause diffi culty, but if the pathologist is aware that the biopsy is from a mass lesion, then the recognition of scars and septa, ductules and chronic cholestatic features in the hepatocytes all serve to distinguish FNH from other

Fig. 15.9 • Focal nodular hyperplasia. The large central scar contains blood vessels and fi brous septa radiating from the scar, separating cirrhosis-like nodules of hepatocytes. H&E.

Fig. 15.10 • Focal nodular hyperplasia. The Masson trichrome stain shows the fi brous septa radiating from the central scar. H&E.

Fig. 15.11 • Focal nodular hyperplasia. Fibrous septa become thin in the periphery of the lesions. Ductules are present at the junction of the septa and parenchyma. Masson trichrome stain.

component is arranged in liver cell plates that are generally two cells thick and supported by a well-developed reticulin framework. They are separated by sinusoidal spaces lined by inconspicuous endothelial cells. The liver cells resemble normal hepatocytes in the adjacent parenchyma, but they

Fig. 15.12 • Focal nodular hyperplasia. There is a large artery in the centre of the central scar. H&E.

Fig. 15.13 • Focal nodular hyperplasia. Ductules and cholate stasis at the junction between a septum and the parenchyma. H&E.

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benign hepatocellular lesions, especially HCA. In difficult cases, the constellation of findings should be used as diag-nostic criteria rather than any one individual histological feature. As noted earlier, HCA can sometimes have a few septa and ductules, but not all of the features of FNH. Lesions classified as ‘telangiectatic FNH’ because of the pres-ence of ductules have been shown to be HCA,32 and it seems likely that other lesions reported to be atypical variants of FNH41 may also have been misclassified. Distinction from cirrhosis, especially a biliary cirrhosis, may be more diffi-cult, but if part of the specimen shows normal acinar archi-tecture, while part appears cirrhotic, one should suspect FNH. The presence of large, abnormal arteries in the septa is also a valuable clue in distinguishing FNH from cirrhosis.

Dysplastic nodules and putative premalignant lesions

The terminology of these lesions has undergone change in the past decade and remains somewhat confusing.2,42–45

There may be further changes but, at present, dysplastic nodule (DN) has come to encompass lesions previously called ‘adenomatous hyperplasia’, ‘macroregenerative nodule’ and a variety of less popular terms. These are nodules in a cirrhotic liver that are macroscopically distinct from the surrounding cirrhotic nodules. They are usually larger than surrounding nodules and may be detected by imaging studies. They may also differ in colour or texture and may bulge from the surface of the liver. Histological examination is required to distinguish a DN from a small HCC, and they are further classified as low-grade or high-grade, based on morphological features.

Hepatocellular changes associated with the development of HCC are of two types, large-cell change and small-cell change. Large-cell change, originally called ‘liver cell dysplasia’46 refers to cellular enlargement, bizarre nuclear pleomorphism and hyperchromatism with occasional mul-tinucleation of liver cells occurring in groups or sometimes occupying whole cirrhotic nodules. Although associated with the development of HCC, hepatocytes with large-cell change have a low proliferation rate but a greater degree of apoptosis than normal hepatocytes, suggesting a derange-ments of the normal process of hepatocyte polyploidiza-tion.47 This could possibly be due to chronic inflammation-induced DNA damage, yielding a population of enlarged liver cells with characteristic nuclear changes but, while it may be a habitual feature of cirrhosis and a regular accompaniment of HCC, the preponderance of evidence sug-gests that it is not a direct precursor of malignancy.

Small-cell change is less readily recognized than large-cell change. It refers to clusters of cells, often forming small nodules within a cirrhotic nodule, with increased nuclear/cytoplasmic ratios and cytoplasmic basophilia. In contrast to large-cell change, hepatocytes with small-cell change have increased proliferative activity and seem more likely to be a direct precursor of HCC.48,49

Low-grade dysplastic nodules contain portal areas within the nodule, and are composed of liver cells that are mini-

mally abnormal, although large-cell change may be present (Fig. 15.14) The nuclear/cytoplasmic ratio is normal or slightly increased. Nuclear atypia is minimal and there are no mitoses. Steatosis may be present and there may be Mallory bodies. Iron may be increased or decreased com-pared to the surrounding cirrhotic liver.

High-grade dysplastic nodules are characterized by small-cell change and features that suggest increased cellular pro-liferation—plates more than two cells thick, pseudogland formation, cytoplasmic basophilia, high nuclear/cytoplas-mic ratio, nuclear hyperchromasia or an irregular nuclear contour. These features are often confined to one or more foci within the nodule, giving the appearance of ‘nodule-in-nodule’ formation (Fig. 15.15).

It is generally accepted that high-grade DN are precursors of HCC based on several lines of evidence, including mor-phological features intermediate between low-grade nodules and HCC, the presence of foci of HCC in otherwise high-grade dysplastic nodules (Fig. 15.16) and follow-up showing progression to malignancy in a few cases.2,43–45, 50,51 Whether low-grade DN can also progress is less clear.

Fig. 15.14 • Low-grade dysplastic nodule. The nodule is much larger thanthe surrounding cirrhotic nodules. It contains portal areas and is cytologicallynormal. H&E.

Fig. 15.15 • High-grade dysplastic nodule. There are numerous smallernodules within the large dysplastic nodule (nodule-in-nodule). H&E.

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Nodular regenerative hyperplasia

Nodular regenerative hyperplasia (NRH) is usually consid-ered a reaction to heterogeneous blood fl ow within the liver which can be of many causes (Chapter 13).52 The usual case, a cirrhosis-like micronodular transformation of the liver that mimics cirrhosis but without the scarring typical of that condition, is not likely to be confused with a neoplasm. Occasionally, however, as in cirrhosis, there may be large regenerative nodules among the small cirrhosis-like nodules, causing confusion with hepatocellular adenoma. Furthermore, some cases of liver adenomatosis have been confused with NRH, and sometimes the terms have been used synonymously. The key to recognizing cases of NRH with adenoma-like nodules is in sampling the tissue that surrounds the large nodules. If it shows features of NRH with diffuse nodularity and no intervening normal paren-chyma, then the diagnosis is NRH. If the intervening paren-chyma is normal or only compressed by the large nodules, then it is adenomatosis.2

Malignant hepatocellular tumours

Hepatoblastoma

Hepatoblastoma is the most frequent liver tumour in children, with an annual incidence of approximately 1 per million in children under 15 years of age.53 It mimics the developing fetal or embryonal liver histologically and may contain other heterologous mesenchymal or epithelial ele-ments. At least 90% occur before the age of 5 years, and the diagnosis in cases reported in older children and adults is always suspect.

Aetiology

As in many other tumours of early childhood, genetic abnormalities appear to play a very strong role. Congenital anomalies are present in approximately 5% of patients and there are strong associations with Beckwith–Wiedemann syndrome and familial adenomatous polyposis coli (APC),

along with a number of other rare syndromes. Numerous chromosomal abnormalities have been found in the tumours and sometimes germline mutations in the patients. Gains of chromosomes 2 and X are common, and loss of heterozygosity of 11p15 is especially important in Beckwith–Wiedemann syndrome-associated hepatoblastoma.54–56

Cytogenetic studies and comparative genomic hybridiza-tion have revealed that tumour cells usually harbour a limited number of chromosomal abnormalities with a mean of three changes per tumour.57 Recurrent genetic alterations involve chromosomes 2, 20, 1, 8 and X.55,58 Frequent triso-mies 2q and 20, often seen as the only abnormality, have also been observed in other embryonic tumours such as embryonal rhabdomyosarcoma. The DNA content is fre-quently 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 11.59

Abnormalities of the Wnt signalling pathway involving mutations of the b-catenin and APC genes play a central role, as in a number of other tumours.60,61 Several studies revealed that genes encoding components of the wingless/Wnt signal transduction pathway, including the APC, AXIN1 and b-catenin genes, are mutated in a large proportion of cases.62–65

These mutations lead to abnormal activation and induction of growth-promoting genes. The central molecule of this pathway is β-catenin, which carries mutations in more than half of hepatoblastoma cases. β-catenin is a multifunctional protein that affects cell adhesion and gene expression, and has a pivotal role in cell adhesion through its participation in the cadherin–catenin complex of adherens junction. Activation of insulin-like growth factor 2 (IGF-2) and altered expression of members of the IGF-axis have also been involved, but mutations or allelic deletions of p53 are infre-quent in hepatoblastoma.

Clinical features

Approximately 75% of children with hepatoblastoma are male. They usually present because of abdominal enlarge-ment noted by a parent. Weight loss, anorexia, nausea, vom-iting, abdominal pain and jaundice are seen less frequently. A right upper quadrant mass is often palpable in the abdomen on physical examination. Serum α-fetoprotein (AFP) is elevated at the time of diagnosis in up to 90% of patients. Radiological studies may be suggestive, but biopsy is usually recommended for defi nitive diagnosis.

Pathology

Hepatoblastomas usually present as a single mass that may be up to 25 cm in diameter. They are typically lobulated on cut surface and may be yellow, brown, green or variegated, depending on the differentiation of the tumour and whether a mesenchymal component is present (Figs 15.17 and 15.18).

Histologically, hepatoblastoma may be classifi ed as epi-thelial type, with four subtypes, or mixed epithelial and mesenchymal, with two subtypes.2 In the Armed Forces

Fig. 15.16 • Hepatocellular carcinoma arising in a high-grade dysplastic nodule. H&E.

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Institute of Pathology (AFIP) series,2,66,67 slightly more than half of tumours were epithelial. These were subdivided into those with a pure fetal epithelial pattern (31% of all cases) and those with fetal mixed with embryonal (19%), macro-trabecular (3%) and/or small cell undifferentiated (3%) components. The fetal epithelial pattern is composed of sheets and thin trabeculae of cells resembling fetal hepato-cytes with a small round nucleus and 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. 15.19). Haematopoietic cells are almost always present, mimicking the typical extramedullary haematopoiesis of normal fetal liver (Fig. 15.20). The embryonal pattern, when present along with fetal, consists of sheets, clusters or single small, angulated, hyperchromatic cells with a high nuclear:cyto-plasmic ratio. Embryonal cells often cluster into glandular, acinar or pseudorosette formations (Fig. 15.21). The macro-trabecular pattern refers to tumours in which a significant proportion is composed of trabeculae more than 10 cells in thickness with either fetal or embryonal type cells or even larger cells that resemble those of hepatocellular carcinoma. The small-cell undifferentiated pattern is composed of non-cohesive sheets of small cells similar to those of other ‘small blue cell’ paediatric neoplasms. In both macrotrabecular

and small-cell undifferentiated tumours, at least part of the mass must resemble more conventional epithelial hepato-blastoma to establish the diagnosis.

The mixed epithelial and mesenchymal pattern of hepa-toblastoma contains areas of fetal and embryonal epithelial cells along with primitive mesenchyme and various

Fig. 15.17 • Hepatoblastoma. The tumour occupies the entire right lobe andhas a typically lobulated appearance.

Fig. 15.18 • Hepatoblastoma. A lobectomy specimen, showing a tumourmass bulging from the surface with lobulations on cut section.

Fig. 15.19 • Hepatoblastoma, fetal epithelial type. The tumour has acharacteristic ‘light and dark’ appearance at low magnification. H&E.

Fig. 15.20 • Hepatoblastoma, fetal epithelial type with abundantextramedullary haematopoiesis. H&E.

Fig. 15.21 • Hepatoblastoma, epithelial type with embryonal epithelium(left) and fetal epithelium (right). H&E.

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mesenchymally derived tissues (Figs 15.22–15.25). In approximately three-quarters of mixed tumours, the mes-enchymal component consists of immature and mature fibrous tissue and frequently osteoid-like tissue and carti-lage. The cells in the osteoid-like areas express cytokeratin and other epithelial markers, indicating that they are meta-plastic rather than of truly mesenchymal origin. The remain-der of the mixed hepatoblastomas are considered teratoid and may contain a variety of tissues including striated muscle, bone, cartilage, stratified squamous epithelium, mucinous epithelium and melanin-containing epithelium. Tumours that are resected following chemotherapy may show extensive necrosis, while the amount of osteoid-like tissue is frequently increased.68

Staging

Several staging systems have been proposed, but the one most widely used is that of the Children’s Cancer Study Group (CCSG) and Paediatric Oncology Group (POG), which classifies the tumours postoperatively by their resect-ability.57 This classifies tumours as Stage I (complete resec-tion), Stage II (microscopic residual disease), Stage III

(macroscopic residual tumour), or Stage IV (distant metas-tases). At presentation, approximately 38% of hepatoblasto-mas are Stage I, 9% are Stage II, 24% Stage III and 29% Stage IV.67,69 With preoperative chemotherapy and liver transplan-tation, as noted above, the majority of the 53% ‘unresect-able’ (Stages III and IV) cases can be rendered ‘resectable’.

Treatment and prognosis

Surgery is the mainstay of treatment of hepatoblastoma, and complete resection is the only chance for cure. Preoperative chemotherapy to shrink the tumour has resulted in improved resectability and has improved the overall 5-year survival rate to 75% from 35% in the 1970s.57

Liver transplantation has been used in children whose tumours remain unresectable after chemotherapy. Multivariate analyses have shown that the stage of the tumour at the time of initial resection is the key prognostic factor in determining survival.2,57,67,69 The histological pattern does not independently affect survival when adjusted for age, sex and stage, except perhaps for the small-cell undifferentiated variant, which has a high mortality, even if the initial resection appears complete.67,70

Fig. 15.22 • Hepatoblastoma, mixed type. There is a fetal epithelialcomponent (right) and an embryonal component mixed with primitivemesenchyme (left). H&E.

Fig. 15.23 • Hepatoblastoma, mixed type, with osteoid. H&E.

Fig. 15.24 • Hepatoblastoma, mixed type. Partially calcified osteoid in themidst of a fetal epithelial component. H&E.

Fig. 15.25 • Hepatoblastoma, mixed type. An area with primitivemesenchyme and cartilage. H&E.

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Hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common primary hepatic malignancy of adults. It is a malignant neoplasm composed of cells that differentiate in some way in the manner of hepatocytes. HCC is a tumour that is of interest to investigators in many fi elds, especially to those interested in the pathogenesis of human cancer. There is striking geographic variation in the incidence of HCC, and its association with viral infections and exposure to chemi-cal agents and other forms of chronic liver injury have provided important clues to carcinogenetic mechanisms. Furthermore, the association of HCC with underlying chronic liver disease is so strong that development of cancer is frequently the terminal event for many patients with chronic liver disease.

Epidemiology and aetiology

One of the most striking features of HCC is the wide varia-tion in its incidence in different parts of the world (Table 15.3).71 Overall, HCC is the fi fth most common malignancy among men and the eighth most common among women worldwide, but east Asia and Subsaharan 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 approxi-mately 4.1 per 100 000.71,72 It is currently the seventh leading cause of cancer deaths in the US, but the incidence has been

rising, more than doubling between 1975 and 1998.73 In general, regions of the world with a high incidence of HCC are those that have a high prevalence of chronic hepatitis B infection, but even within these areas there is geographic variability; for example in western Africa the country of Gambia has nearly 5 times the incidence of Nigeria, suggest-ing that environmental cofactors such as afl atoxin exposure may also be important. Hepatitis C infection is associated with many cases in countries such as Japan where the preva-lence of hepatitis B is intermediate but the incidence of HCC is relatively high.

The incidence of HCC generally increases with age, although there are geographic differences.71 In Europe and the United States, the peak age of incidence is in the seventh decade, while in Qidong province in China, which has the highest incidence in the world, the peak is in the fi fth 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.74 Nevertheless, HCC can occur in younger individuals and even young children.54 Regardless of geo-graphic location, HCC occurs more frequently in men than women, with male : female ratios in various countries ranging from 2 : 1 to 5 : 1. The precise reason is not known, but it has been shown that many tumours have androgen receptors,75 raising the possibility 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.

Cirrhosis. The majority of patients who develop HCC have underlying cirrhosis and, consequently, cirrhosis is a major risk factor for HCC. Macronodular cirrhosis has always been more strongly associated with HCC than micronodular cirrhosis, and cirrhosis of virtually any cause may be complicated by the development of HCC.76 This also shows geographic variation, so that in Japan and other Asian countries, approximately 90% of patients have under-lying cirrhosis,77 whereas in some parts of Africa only 50–63% are cirrhotic, although most have precirrhotic forms of chronic viral hepatitis.78 Series from North America have reported 46–91% with cirrhosis.79 Nevertheless, every series includes some patients without cirrhosis, and some with no evidence of any underlying liver disease.

Viral hepatitis. The majority of cases of HCC in the world are due to hepatitis B virus (HBV), with the number of hepatitis C (HCV)-associated cases increasing in the Western world. HBV and HCV are the main causal agents of chronic hepatitis. Approximately 5–10% of HBV and 75% of HCV infections become chronic.71,76,80 The epidemiological asso-ciation of chronic HBV or HCV infection with HCC is well established.71,72,74,76,81–89 The two viruses are different and the precise mechanisms by which they can cause cancer are not yet well clarifi ed. Besides a direct effect of the virus on the genome, HCC could arise also as indirect result of the cycle of infl ammation–necrosis–regeneration that occurs in the setting of chronic hepatitis.90,91 On the other hand, it has been shown that in many instances the cause of HCC

Table 15.3 Relative incidence of primary liver cancer in men in different parts of the world; age adjusted rates per 100 00071

High

Eastern Asia 35.5

Central Africa 24.2

Southeast Asia 18.3

Pacifi c Islands 18.3

Medium

East Africa 14.4

West Africa 13.5

Southern Europe 9.8

Caribbean 7.6

Low

South Africa 6.2

Eastern Europe 5.8

Western Europe 5.8

Western Asia 5.6

North Africa 4.9

South America 4.8

North America 4.1

Australia/New Zealand 3.6

South Central Asia 2.8

Northern Europe 2.6

Central America 2.1


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may be accurately determined on the basis of its ‘molecular signature’.92–94

Hepatitis B. Of all risk factors, HBV infection has the strongest association with the development of HCC. The relative risk of HCC in patients serologically positive for hepatitis B surface antigen (HBsAg) is 98 times that of patients who are negative.74 Those who are also positive for e antigen (HBeAg), indicating active viral replication, have 3.6 times the risk of those who are surface antigen positive but e antigen negative, suggesting that the activity of the disease plays a role in pathogenesis.95 Even patients with occult HBV infection (surface antigen negative, with hepa-titis B DNA demonstrable in tissue by PCR) may develop HCC, although the incidence and relative risk are not known.96

HBV is the prototype member of a virus family called ‘hepadnaviridae’, which also includes the woodchuck hepa-titis virus (WHV), ground squirrel hepatitis virus (GSHV), duck hepatitis B virus (DHBV) and heron hepatitis B virus (HHBV).97,98 There are biological and genetic similarities between these viruses,80,97–100 such as a narrow host range, hepatotropism and tendency to chronicity. Among them, WHV infection is most often associated with the develop-ment of HCC.101

HBV consists of a partially double-stranded DNA genome of 3.2 kb enclosed by envelope proteins (HBsAg) (Chapter 8). The genome is packaged with a core protein (HBcAg) and a DNA polymerase. After penetration of the virus in the cell, its genome becomes a covalently closed, totally double-stranded molecule that can eventually integrate into the host genome. Protein synthesis proceeds from four open reading frames: envelope proteins from the S gene, pre-S1 and pre-S2; HBeAg and HBcAg from the C gene and pre-C gene sequence; the DNA polymerase from the P gene; and X protein from the X gene.80,99,102 DNA replication depends upon reverse transcription of a RNA intermediate in the nucleus. The virions are then built in the cytoplasm and released by the hepatocyte.103,104

The integration of HBV in sites within the host genome has been seen as a possible carcinogenetic mechanism. In WHV-induced HCC, WHV DNA integrates adjacent to cel-lular N-myc genes101 and studies with transgenic mice have shown that this could interfere with the p53 axis.105 In man the integration is, however, not specifi c and only rarely associated with activation of cellular oncogenes.106–111 In a minority of HCC cases, integration of DNA sequences into the host cell genome can activate cellular genes through a mechanism of modifi cation of its expression (i.e. cis-acting mechanism).112 Moreover, 20% of patients with HCC asso-ciated with HBV do not integrate the DNA of the virus. On the other hand, integrated HBV-DNA can generate chromo-somal instability and integration can also target the telom-erase gene.92

Several HBV genes have been found in infected tissues more frequently than others, including truncated pre-S2/S, hepatitis Bx gene (HBx) and a novel spliced transcript of HBV, named hepatitis B spliced protein (HBSP).113 The proteins expressed from these integrated genes have been shown to have intracellular effects that may account for

their association with HCC, including effects on cellular growth and apoptosis. Among these genes, HBx seems to play a pivotal role in hepatocarcinogenesis. HBx gene, indeed, harbours weak transcriptional transactivation activ-ity. The 154-amino-acid viral product ‘X’ (HBx) has been shown to be essential for HBV and WHV infection ‘in vivo’.80,99,114,115 It may be a prime candidate for mediating HBV pathological effects. With regard to oncogenesis, it can directly inactivate the tumour suppressor p53 and the nega-tive growth regulator p55sen,102,116–118 both involved in the pathway of senescence, and can transcriptionally down-regulate p21 and sui1, both of which inhibit hepatocellular growth.102 Other biological effects of HBx can be summa-rized as follows:

1. HBx transactivates several cellular promoters by protein–protein interactions, interacting with proliferation, apoptosis, and response to DNA damage.119–121

2. HBx infl uence proteasome function, interfering with the degradation of viral and cellular proteins.122

3. HBx may inhibit the DNA repair system.99,102

4. HBx infl uence the mitochondria function and interacts with calcium homeostasis.123–125

5. HBx has also been implicated in DNA repair.99,102

The real functional consequences in hepatocarcinogene-sis of these effects are, however, still unclear.92,99 One model is that HBx acts (through its effect on calcium homeostasis and consequent activation of calcium-dependent kinases) on NFkB, a transcription factor involved in the control of immune response126 that has also been associated with HCV polypeptides.

Other HBV proteins have been suggested to enhance the risk for development of HCC. Studies with transgenic mice have shown association between various envelope proteins (namely L and M) and hepatocellular carcinoma, although a real ‘cause–effect’ relationship could not be demonstrated. The risk enhancement could be indirectly mediated via envelope-induced cellular stress.127–129

Hepatitis C. Hepatitis C rivals hepatitis B in importance as an aetiological factor in HCC, and it appears to be largely responsible for the rising incidence of liver cancer in the United States, where 21% of cases are associated with HCV, and it is also believed to be responsible for the relatively high incidence of HCC in Japan and some other coun-tries.76,82,130,131 Most cases of HCC associated with hepatitis C have occurred after the development of cirrhosis, and the risk is multiplied in the presence of other factors, such as male gender, advanced age, coinfection with other viruses (HBV and/or HIV), alcohol, diabetes or hepatic steatosis. Patients infected with both HBV and HCC have a much greater risk of HCC than with either virus alone.76

HCV is a positive strand RNA virus belonging to the Hepaciviridae genus (Flaviviridae family) (Chapter 8). The genome is 9-kb-long single-stranded, linear RNA. Its repli-cation occurs in the rough endoplasmic reticulum (RER), without reverse transcriptase activity.132–134 The RNA is translated by ribosomes of RER and the resulting polyprot-ein is modifi ed and cleaved giving rise to 10 polypeptides,

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including three structural (core, E1 and E2) and multiple non-structural (NS1 to NS5).135–137 The non-structural pro-teins associate with ER membrane to form the viral replicase.138–140

The mechanism of liver damage in HCV infection is the result of immune response and direct cytopathic effect.141,142

The continuous process of necrosis and regeneration may make the cells prone to the action of various procarcino-genic substances (HCV proteins?) with genetic instability and subsequent malignant transformation. Based largely on experimental ‘in vitro’ results, an oncogenic role has been reported for three HCV proteins: core protein, NS3 and NS5a.99,141 Core protein intervenes in several cellular func-tions as apoptosis, signal transduction and transforma-tion.99,141 It modulates p21WAF1 expression, and physically interacts with p53,143–145 promoting apoptosis and cell pro-liferation. Moreover, core protein stimulates NFkB, increas-ing its levels and DNA binding activity and modifying the response to TNF-α.119 In summary, by modulating the activ-ity of transcription factors and cytokines, it could promote cellular transformation.99 Also NS3 protein represses the transcription of p21WAF1, both acting on the promoter and via p53.146–148 NS5a is part of the viral replicase complex, although no defi ned role in virus replication has been revealed.80 It has effects on cell-cycle-regulatory kinases and transcription activation machinery (p21 WAF1, p53, cyclins), blocking apoptosis in HCV infection, so contribut-ing to hepatocarcinogenesis.

In general, a possible mechanism of viral-induced hepa-tocarcinogenesis may involve endoplasmic reticulum (ER) and oxidative stress. ER stress is a homeostatic mechanism that controls cellular metabolism in response to perturba-tions in protein biosynthesis.149 Extreme or prolonged ER stress can lead to apoptosis.150 Non-cytopathic viruses, such as HCV and HBV, inducing ER stress at sublethal levels, can produce alterations in cells that may lead to transformation. On the other side, ER stress is strictly linked to intracellular redox state, so that its ultimate consequence is oxidative stress.99,151 This is linked to changes in cellular proliferation and to DNA damage; therefore several studies have impli-cated oxidative stress in the development of HCC in HCV hepatitis.152,153 Oxidative stress activates intracellular signal-ling pathways that may promote transformation, such as mitogen-activated protein kinases (MAPKs) pathway.154

MAPKs are a group of enzymes that include extracellular signal-regulated protein kinase (ERK), c-JUN N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 subfamilies.99 They respond to various stimuli in different ways; for instance ERK is stimulated by mitogens and endorses survival, while JNK/SAPK and p38 are stimulated by various stresses and promote apoptosis.155,156

Afl atoxin B1. Afl atoxins, a family of mycotoxins produced by fungi of the Aspergillus genus, are powerful carcinogens in experimental animals. Contamination of food, particu-larly grains and peanuts, by these toxins is common in the very same parts of the world where HCC is most common, namely China and southern Africa,157,158 and indeed, before the association with hepatitis B was recognized, afl atoxin B1 was thought to be a principal cause of HCC.159 Afl atoxin

is converted by cytochrome P450 to 8,9-exo-epoxide, that damages DNA. Afl atoxin B1 (AFB1) binding to guanine resi-dues of DNA can produce G to T mutations,160,161 that are not found in human HCC in areas with low AFB1 expo-sure.162,163 Such mutations in codon 249 of the p53 tumour suppressor gene are thought to play an important role in carcinogenesis in parts of the world with a high incidence of HCC.160 There is a synergistic cooperation between HBV and AFB1 in hepatocarcinogenesis, and the combination of chronic hepatitis B infection with dietary afl atoxin expo-sure more than triples the risk of developing HCC.164,165

Alcohol. There is very little evidence that alcohol is directly carcinogenic or genotoxic, but alcoholic cirrhosis is the single most frequent risk factor in the United States and many Western countries.72,166 Non-genotoxic mechanisms are most probably related to causation. Alcohol is metabo-lized to acetaldehyde by alcohol dehydrogenase and cytochrome P450 2E1 (CYP2E1), that is associated with microsomes. CYP2E1 produces reactive oxygen species that can lead to DNA damage and transformation.167 Moreover, polymorphism in alcohol dehydrogenase and CYP2E1 may infl uence the risk of hepatocarcinogenesis.166,168 In alcohol-induced cirrhosis, the reserve of S-adenosylmethionine, the methyl donor for DNA methylation, is decreased.166,169 The effect could be hypomethylation of DNA, with consequent DNA instability, that could be associated with the develop-ment of cancer. Alcohol may be synergistic with other risk factors, however, increasing the likelihood of HCC in patients with hepatitis B or C, diabetes, obesity or smoking.166,170–172

Heavy alcohol consumption is associated with a six-fold increase in risk of HCC in cirrhotic patients.172

Diabetes, obesity and fatty liver disease. Diabetes mellitus is associated with a two to three-fold increase in risk of HCC, regardless of other risk factors,173–175 while obesity is associated with a two to four-fold increased risk.175–178 Most patients with diabetes or obesity who develop HCC have cirrhosis, and since both diabetes and obesity can lead to cirrhosis through non-alcoholic steatohepatitis (Chapter 7); this presumably accounts for most of the cases. When viral hepatitis, alcohol, or other risk factors are present, the fatty liver disease may be synergistic.171,172 Approximately one-third of cases of HCC that occur in the United States have no known aetiology, and it is possible that non-alcoholic fatty liver disease (NAFLD)-related cirrhosis is a predispos-ing factor in may of these.72,175,179 This hypothesis is sup-ported not only by epidemiological data, but also by the suspected pathogenesis and molecular ‘lesions’ of NAFLD. Lipid peroxidation with production of free oxygen radicals is thought to be a central event in the pathogenesis of ste-atohepatitis.180 This oxidative stress may lead to prolifera-tion of oval cells, in man as in experimental models.181 The reactive oxygen species (ROS) can induce mutations, for instance, in the p53 gene. The progression of the neoplastic cells can to be facilitated by the numerous alterations in growth factors and cytokines (TGF or TNF), which have been shown to stimulate oval cell proliferation.182

Metabolic diseases. Cases of HCC have occurred in a number of metabolic diseases, but the strongest associa-tions are with genetic haemochromatosis (GH),


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tyrosinaemia and α1-antitrypsin defi ciency.183–185 Deve-lopment of HCC is a frequent terminal event following the development of cirrhosis in GH (approximately 20%), hereditary tyrosinaemia (37%) and α1-antitrypsin defi -ciency (15%). Prevention of cirrhosis by phlebotomy in GH and diet with NTBC (2-(2nitro-4-trifl uoromethylbenzoyl)-1,3-cyclohexanedione) therapy in tyrosinaemia can prevent HCC in all but a few cases. There is also an increased inci-dence of HCC in patients with porphyria cutanea tarda, but since that disease is frequently associated with alcohol use and chronic hepatitis C infection, the role of the porphyria is unclear. A few cases of HCC have been reported in patients with glycogen storage diseases, types I and III, in associa-tion with hepatocellular adenomas and presumably repre-senting malignant transformation of an adenoma. HCC has also rarely been reported in patients with other forms of porphyria, hypercitrullinaemia, fructosaemia, Wilson disease, Byler disease and Alagille syndrome.

Because of its relative frequency, GH is the metabolic disease that is the most important risk factor in develop-ment of HCC, and conversely HCC is recognized as one of the most important complications of GH. The risk of devel-oping HCC in GH individuals is signifi cantly higher (up to 200 times) than the risk of the general population.186–188 It is not completely clear if this is due to the iron overload in itself or to the development of cirrhosis that invariably occurs in untreated hereditary haemochromatosis (HH) patients.184 An important role of iron overload in hepatocar-cinogenesis, however, has also been demonstrated in patients with iron excess without HH as thalassaemia or the so-called ‘African overload’.189–190 Several studies (in vivo and in vitro) point to a causative role for iron excess in hepatocarcinogenesis.184 Iron can act directly on cellular proliferation, for instance by inactivating p53. It can also act indirectly, for example by infl uencing lipid peroxidation and production of reactive oxygen radicals. Iron is, in fact, a substrate for cell proliferation and could initiate the process of carcinogenesis.191,192 The effect of free iron could be due both to direct damage to chromosomes and to muta-tion, via the increased production of ROS.193,194 Interestingly, in patients with HH a particular type of mutation of p53 has been described (A220G) in the DNA binding site of the protein.195,196 At the same time, it has been hypothesized that the formations of adduct due to lipid peroxidation may lead to DNA damage in patients with GH.

Anabolic and contraceptive steroids. A number of hepato-cellular tumours have been reported in patients taking ana-bolic steroids.197 Some have been classifi ed as hepatocellular adenoma, others as HCC, based on histological features, but even those called HCC regress when the drug is dis-continued, and none has been reported to metastasize. Contraceptive steroids are clearly associated with hepatocel-lular adenoma. Several case–control series have reported an association with HCC, but others have found a protective effect of contraceptive steroid use as well as other factors that lead to high oestrogen exposure, including multiple pregnancies and early menarche, so the matter remains unresolved.198,199

Molecular pathology

During the last 25 years, molecular genetic and phenotypic cellular changes have been extensively studied in human and experimental hepatocarcinogenesis.92,101,141,165,200–206 A variety of genetic and epigenetic alterations (point muta-tions, deletions, amplifi cations, methylations), which may result in the distortion of either gene expression or the biochemical function of genes, have been detected. As in tumours of other sites, the majority of genes and proteins affected have a physiological role (control of differentiation, proliferation, apoptosis, etc.), but their dysregulation is involved in carcinogenesis and they are therefore been called proto-oncogene or tumour suppressor genes.207–209

Although a number of general pathways have been discov-ered,102,210 the type of genetic alterations and their sequence of occurrence may be very variable,211 refl ecting under this aspect the wide range of aetiological factors of HCC. Each HCC has probably its own profi le and this heterogeneity can be found also within a given tumour.212–214 Moreover, while many discrepancies have been observed in molecular genetic changes that appear during hepatocarcinogenesis triggered by various oncogenic agents in different species, similarities in specifi c phenotypic cellular changes are evident.215–220

At the present stage of knowledge, two main ‘pathways’ regarding hepatocarcinogenesis have been proposed:

1. malignant cell clones arise from hepatocytes by mutations and clonal selections (dedifferentiation hypothesis)

2. activation of hepatic progenitor cells defective in maturation lead to malignant clones (maturation arrest hypothesis)

Of course, these two ‘pathways’ are not mutually exclusive, and could simultaneously be involved during hepatocar-cinogenesis. An intriguing current hypothesis is the possible role of hepatic progenitor cells as target cells for hepatocar-cinogenesis, since these cell type are prevalent in early dys-plastic foci of human livers affected with the most prevalent carcinogenic conditions, such as viral hepatitis and alco-holic liver disease.

Molecular genetic alterations in HCC

As noted above, most HCCs develop in the setting of chronic hepatopathy as a multistep process.42,102 Its evolution may take up to 30 years.94 Foci of dysplastic hepatocytes (DH), low-grade dysplastic nodules (LG-DN) and high-grade dys-plastic nodules (HG-DN) have been identifi ed as possible preneoplastic lesions. A minority of HCC arises, however, in the absence of underlying hepatopathy or in children.71

That underlines the importance of a genetic predisposition in the development of this neoplasm.

Epigenetic alterations in preneoplastic lesions. In preneo-plastic lesions, an elevated expression of transforming-growth factor alpha (TGF-α) and insulin-like growth factor 2 (IGF-2) is observed, that is responsible for enhanced hepa-tocyte proliferation.205,206,218 This up-regulation is due to the

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zachary d. goodman luigi m. terracciano · 2013-12-20 · because of chronic liver disease may...

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