3. Pathology

Needle biopsies followed by morphologic and immunohistochemical examination can be invaluable for the characterisation of liver masses. However, nowadays clear-cut radiologic diagnostic criteria have been established for the non-invasive diagnostics of HCC; therefore, the advantages and indications of the biopsy should be considered against the risks and contraindications. Liver biopsy is recommended only in selected patients, thoughtfully evaluating the diagnostic yield [6].

Currently, three general groups of indications (see Table 2) for liver biopsy are known: to establish the diagnosis, to assess the prognosis and/or to assist in the management of patient

Diagnosis:


Prognosis and management:


Table 2. Indications for liver biopsy [49].

<sup>•</sup> Identification and staging of parenchymal and cholestatic liver diseases (alcohol-induced liver disease; non-alcoholic steatohepatitis; primary biliary cirrhosis; primary sclerosing cholangitis; Wilson's disease, haemochromatosis)

with known liver disease [49]. Percutaneous liver core biopsy is most frequently performed to evaluate the presence and activity of inflammation and extent of fibrosis/stage of frequent liver diseases, mostly chronic viral hepatitis, alcohol-induced liver disease and NAFLD. Regarding focal liver lesions, biopsy can yield the diagnosis. Molecular analyses of tissue may help determine the most appropriate individual treatment strategy for the patient with HCC [50] but are still under development for HCC. At present, biopsy from a nodule in cirrhotic liver is indicated if the findings of radiological imaging are controversial [6].

Although biopsy is often essential, sometimes it may be difficult to undertake because of associated risks (see Table 3). Percutaneous, ultrasound-guided liver biopsy (the Menghini method) has become the worldwide standard [51]. However, it is appropriate only in cooperative patients. Thus, if the patient refuses from the procedure, it is absolutely contraindicated. Although precise blood clotting parameters are unsettled, coagulopathies should be mentioned as a serious contraindication [49]. In this case, mini-laparoscopy or transjugular liver biopsy might be considered [51]. Among relative contraindications, ascites should be pointed out, as it may prevent adequate sampling of tissue, as well as increase the risk of bleeding [49]. Biopsies of malignant liver lesions also carry a low risk of tumour seeding.

Significant complications due to liver biopsy arise in about 1% of cases, with less than 0.1% mortality [51]. The main complications are post-interventional haemorrhage and bile leakage; others, like injuries to gall bladder, lung, kidney, as well as bacteraemia are rare [49, 51].

The initial assessment of liver tissue starts with the overall evaluation of parenchymal architecture. Haematoxylin and eosin represents the generally accepted standard stain in liver pathology [6]. Helpful additional visualisation methods in liver pathology include Masson's trichrome to assess fibrosis, Gordon and Sweets reticulin to evaluate lobular architecture and hepatocyte plate thickness, Perl's iron to detect hemosiderin deposits and periodic acid-Schiff (PAS) stain to identify glycogen, mucus or chitin of certain liver parasites.

Microscopically, cells of classical HCC resemble normal hepatocytes. The similarity to normal liver is most notable in well to moderately differentiated tumours. In such cases, the loss of the normal liver cell plates and plate thickness change from 1 to 2 cell nuclei to 3 or more nuclei

• History of unexplained bleeding


Relative contraindications

• Ascites

enhancement in the tumour, exhibit satellite lesions and do not uptake liver-specific contrast

Positron emission tomography (PET) is a non-invasive radiologic visualisation that demonstrates metabolic activity in normal or pathological tissue. It is usually performed in combination with CT to ensure both anatomical imaging and metabolic evaluation. 18-fluorodeoxyglucose (FDG) is one of the radiopharmaceuticals used in PET/CT. It discloses areas of high glucose uptake as many tumours including HCC are characterised by aerobic glycolysis: the Warburg

The significance of FDG PET/CT in HCC evaluation is not unequivocal. The distinction between small, well-differentiated HCC versus regenerative or dysplastic nodules can be difficult. The positive aspect of PET/CT is the ability to detect extrahepatic metastases of HCC. Considering that PET/CT provides whole-body examination, it is recommended before liver transplantation [45, 46]. Hypothetically, prognostic role of PET/CT in HCC has been discussed as well as the ability to predict response to treatment [46]. Other radiopharmaceuticals are also under discussion, including lipid radiotracer on choline base, like 11C-choline or 18Ffluorocholine [47]. 68Ga-labelled prostate-specific membrane antigen, that is used to diagnose

Needle biopsies followed by morphologic and immunohistochemical examination can be invaluable for the characterisation of liver masses. However, nowadays clear-cut radiologic diagnostic criteria have been established for the non-invasive diagnostics of HCC; therefore, the advantages and indications of the biopsy should be considered against the risks and contraindications. Liver biopsy is recommended only in selected patients, thoughtfully evalu-

Currently, three general groups of indications (see Table 2) for liver biopsy are known: to establish the diagnosis, to assess the prognosis and/or to assist in the management of patient

• Identification and staging of parenchymal and cholestatic liver diseases (alcohol-induced liver disease; non-alcoholic steatohepatitis; primary biliary cirrhosis; primary sclerosing cholangitis; Wilson's disease, haemochromatosis)

• Identification and determination of the nature of focal/diffuse intrahepatic abnormalities on imaging studies

• Pre-treatment evaluation and staging of chronic hepatitis, e.g. chronic viral hepatitis B and C

• Evaluation of post-transplant patient with abnormal liver tests (rejection versus infection)

prostate cancer, is present in other tumours, including HCC [48].

agents [43].

24 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

effect [44].

3. Pathology

Diagnosis:

ating the diagnostic yield [6].

Prognosis and management:

• Evaluation of persistent abnormal liver biochemical tests • Evaluation of the type and extent of drug-induced liver injury

• Diagnosis of multisystem infiltrative disorders

• Evaluation of pre-transplant living-related donor

• Evaluation of treatment efficacy for liver diseases

Table 2. Indications for liver biopsy [49].


Table 3. Contraindications of liver biopsy [49].

Absolute contraindications

<sup>•</sup> Uncooperative patient

<sup>•</sup> Tendency to bleed (prothrombin time more than 3–4 seconds over control; platelet count <50,000 mm3 ; prolonged bleeding time (≥10 minutes))

across a single neoplastic cord is a feature of malignancy. In healthy liver, narrow cords of hepatocytes are running in parallel, but even well-differentiated HCC shows a disorganised pattern secondary to the increased thickness of the hepatocyte cords (usually more than 3 cells thick), that can be highlighted by reticulin staining. The invasive growth of HCC disrupts and destroys the liver plate architecture, leading to decreased amount of reticulin and disorganised pattern of it. However, the loss of reticulin is not complete. HCC is characterised by the absence of normal portal tracts and/or naked or unaccompanied arteries in accordance with the radiologic hypervascularity and high contrast in the arterial phase of contrast-enhanced CT [6]. Invasion in connective tissues is diagnostic. However, except scirrhous and fibrolamellar HCC, stroma is usually scant in HCC. Loss of perinodular ductular proliferation is a manifestation of invasive growth [6]. Vascular invasion is diagnostic if evident.

Fibrolamellar HCC is a rare subtype accounting for less than 1% of HCC. Typically, fibrolamellar carcinoma is diagnosed in young adults lacking liver cirrhosis or other known predisposing factors [3]. The mean age of diagnosis is 26 years [53]. Association with germline pathogenic variants of TP53 gene has been reported suggesting that some cases of fibrolamellar HCC might represent Li-Fraumeni syndrome. Interestingly, in the case described by Andrade et al., a germline mutation of TP53 was identified not only in proband affected by

Diagnostic Algorithm of Hepatocellular Carcinoma: Classics and Innovations in Radiology and Pathology

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27

The presence of fibrous septae and central scar with possible calcification leads to architectural similarity with focal nodular hyperplasia [3, 6]. Histologically, the neoplastic cells are arranged in trabeculae and sheets, separated by collagen fibres that undergo hyalinisation and show the typical lamellar pattern [3]. Fibrolamellar HCC is defined by triad of histologic features: (1) large, polygonal neoplastic hepatocytes with wide eosinophilic granular cytoplasm. Ground glass pale bodies and PAS-positive cytoplasmic globules can be present [3, 53] but are neither sufficient nor necessary for diagnosis. (2) Prominent single eosinophilic macronucleoli should be present, and frequently are seen on the background of vesicular chromatin structure [3, 6].

The immunophenotype of fibrolamellar HCC is also unusual, showing expression of hepatocellular markers in combination with biliary, progenitor and stem cell features as well as macrophage markers (CD68). The granular or dot-like expression of CD68 in association with

Prognosis of fibrolamellar HCC is poor. The 5-year survival is similar to conventional HCC arising in non-cirrhotic liver [53]; however, it is better than for classical HCC arising in cirrhotic

Sarcomatoid HCC can occur either primarily or within classical HCC [3]. This subtype, comprising 1.8–3.9% of HCC, is partially or fully composed of malignant spindle-shaped cells, occasionally showing heterologous (rhabdoid, osteoid or chondroid) differentiation [53]. If there is no adjacent area of classical HCC, it is difficult to distinguish sarcomatous HCC from true sarcomas, including primary or metastatic tumours, e.g., metastatic gastrointestinal stromal tumour, leiomyosarcoma or fibrosarcoma. Haematoxylin-eosin stain alone can be insufficient, necessitating immunohistochemistry [3]. Considering the high grade and remarkable anaplasia of sarcomatoid HCC, hepatocellular markers should be supplemented with pancytokeratin and specific markers for sarcoma, including CD117, DOG, actin, desmin, S-100, CD34 and CD31. Hepatocellular antigens are frequently negative, and even pancytokeratin is expressed only in 23–63% cases of sarcomatoid HCC [53]; therefore, complex assessment of morphology is man-

Scirrhous HCC is a rare type, accounting for 0.2% to 4.6% of HCC. It can develop beneath liver capsule leading to pedunculated gross view [3, 53]. Microscopically, scirrhous HCC is characterised by diffuse fibrosis surrounding thin trabeculae of neoplastic cells. Such fibrosis can occur either after various regimens of oncologic treatment (chemotherapy, transarterial chemoembolization, irradiation) or in untreated patients [3]. However, HCC exhibiting post-treatment fibrosis should not be classified as scirrhous [53]. The marked desmoplasia and morphology of the tumour cells, displaying clusters, strands and tubules, can lead to misdiagnosis as cholangiocarcinoma or

(3) Lamellar fibrosis, usually present in at least half of the tumour tissue [53].

appropriate morphology is helpful in diagnosing fibrolamellar HCC [6].

datory along with clinical history and IHC for sarcoma.

liver [3].

fibrolamellar HCC but also in her asymptomatic mother [54].

Cytologically, HCC shows both signs of hepatocellular differentiation that serves as the clue to hepatocellular origin of the tumour and atypia indicating malignant behaviour. Regarding tumour differentiation, bile production is a reliable indicator of hepatocellular origin. Bile can be found in the cytoplasm of neoplastic cells or in lumina of acinar complexes. Similarly to benign counterparts, steatosis, Mallory bodies and hyaline globules can develop in cytoplasm of tumour cells. HCC cells can have intranuclear inclusions and/or optically clear cytoplasm. Giant cells are occasionally present. Iron accumulation in cells of hepatocellular carcinoma is not seen, even in the setting of hereditary hemochromatosis. In hepatocytes, nuclear pleomorphism can be a feature of regenerative changes; therefore, mitotic activity is more suspicious of malignancy, and the presence of atypical mitoses definitively confirms the presence of a malignant tumour. However, in well-differentiated HCC, abnormal mitoses are rare and are not mandatory for diagnosis [6].

The histologic patterns of HCC include trabecular (the most common pattern), acinar (pseudoglandular), solid and scirrhous patterns. Trabecular HCC resembles normal liver architecture. In acinar or pseudoglandular HCC, the neoplastic cells are arranged in gland-like tubules containing bile or fibrin. Solid HCC is characterised by compact, sheet-like arrangement of neoplastic cells. Scirrhous HCC exhibit marked desmoplasia; it will be described in detail later. HCC is characterised by significant inter- and intratumoural heterogeneity, manifesting as variability of grade and growth patterns [3]. Grade progression can be present even in a single patient and, in fact, reflects the biology of HCC. Hepatocellular carcinoma frequently develops in foci with equivocal biological potential, e.g., dysplastic cirrhotic nodule. Such early HCC typically is well differentiated. Over the disease course, well-differentiated HCC progresses to advanced dedifferentiated tumour. The heterogeneity can lead to diagnostic problems and failures in biopsy due to sampling errors. For instance, if a small suspicious nodule was evident by radiological imaging and a biopsy was obtained, the differential diagnosis between dysplastic nodule and HCC will frequently imply the necessity to distinguish between premalignant process and well-differentiated tumour, usually lacking marked cell atypia or clear-cut invasion. In addition, both processes can be adjacent in the tissues.

HCC has several histologic variants, including fibrolamellar, sarcomatoid, scirrhous, steatohepatitic and clear cell HCC, presenting with peculiar morphological features. Some cases display lymphoepithelioma-like morphology. In addition, correlations between histological and molecular subtypes have been reported [52].

Fibrolamellar HCC is a rare subtype accounting for less than 1% of HCC. Typically, fibrolamellar carcinoma is diagnosed in young adults lacking liver cirrhosis or other known predisposing factors [3]. The mean age of diagnosis is 26 years [53]. Association with germline pathogenic variants of TP53 gene has been reported suggesting that some cases of fibrolamellar HCC might represent Li-Fraumeni syndrome. Interestingly, in the case described by Andrade et al., a germline mutation of TP53 was identified not only in proband affected by fibrolamellar HCC but also in her asymptomatic mother [54].

across a single neoplastic cord is a feature of malignancy. In healthy liver, narrow cords of hepatocytes are running in parallel, but even well-differentiated HCC shows a disorganised pattern secondary to the increased thickness of the hepatocyte cords (usually more than 3 cells thick), that can be highlighted by reticulin staining. The invasive growth of HCC disrupts and destroys the liver plate architecture, leading to decreased amount of reticulin and disorganised pattern of it. However, the loss of reticulin is not complete. HCC is characterised by the absence of normal portal tracts and/or naked or unaccompanied arteries in accordance with the radiologic hypervascularity and high contrast in the arterial phase of contrast-enhanced CT [6]. Invasion in connective tissues is diagnostic. However, except scirrhous and fibrolamellar HCC, stroma is usually scant in HCC. Loss of perinodular ductular proliferation is a manifes-

Cytologically, HCC shows both signs of hepatocellular differentiation that serves as the clue to hepatocellular origin of the tumour and atypia indicating malignant behaviour. Regarding tumour differentiation, bile production is a reliable indicator of hepatocellular origin. Bile can be found in the cytoplasm of neoplastic cells or in lumina of acinar complexes. Similarly to benign counterparts, steatosis, Mallory bodies and hyaline globules can develop in cytoplasm of tumour cells. HCC cells can have intranuclear inclusions and/or optically clear cytoplasm. Giant cells are occasionally present. Iron accumulation in cells of hepatocellular carcinoma is not seen, even in the setting of hereditary hemochromatosis. In hepatocytes, nuclear pleomorphism can be a feature of regenerative changes; therefore, mitotic activity is more suspicious of malignancy, and the presence of atypical mitoses definitively confirms the presence of a malignant tumour. However, in well-differentiated HCC, abnormal mitoses are rare and are

The histologic patterns of HCC include trabecular (the most common pattern), acinar (pseudoglandular), solid and scirrhous patterns. Trabecular HCC resembles normal liver architecture. In acinar or pseudoglandular HCC, the neoplastic cells are arranged in gland-like tubules containing bile or fibrin. Solid HCC is characterised by compact, sheet-like arrangement of neoplastic cells. Scirrhous HCC exhibit marked desmoplasia; it will be described in detail later. HCC is characterised by significant inter- and intratumoural heterogeneity, manifesting as variability of grade and growth patterns [3]. Grade progression can be present even in a single patient and, in fact, reflects the biology of HCC. Hepatocellular carcinoma frequently develops in foci with equivocal biological potential, e.g., dysplastic cirrhotic nodule. Such early HCC typically is well differentiated. Over the disease course, well-differentiated HCC progresses to advanced dedifferentiated tumour. The heterogeneity can lead to diagnostic problems and failures in biopsy due to sampling errors. For instance, if a small suspicious nodule was evident by radiological imaging and a biopsy was obtained, the differential diagnosis between dysplastic nodule and HCC will frequently imply the necessity to distinguish between premalignant process and well-differentiated tumour, usually lacking marked cell atypia or clear-cut invasion.

HCC has several histologic variants, including fibrolamellar, sarcomatoid, scirrhous, steatohepatitic and clear cell HCC, presenting with peculiar morphological features. Some cases display lymphoepithelioma-like morphology. In addition, correlations between histological and

tation of invasive growth [6]. Vascular invasion is diagnostic if evident.

26 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

not mandatory for diagnosis [6].

In addition, both processes can be adjacent in the tissues.

molecular subtypes have been reported [52].

The presence of fibrous septae and central scar with possible calcification leads to architectural similarity with focal nodular hyperplasia [3, 6]. Histologically, the neoplastic cells are arranged in trabeculae and sheets, separated by collagen fibres that undergo hyalinisation and show the typical lamellar pattern [3]. Fibrolamellar HCC is defined by triad of histologic features: (1) large, polygonal neoplastic hepatocytes with wide eosinophilic granular cytoplasm. Ground glass pale bodies and PAS-positive cytoplasmic globules can be present [3, 53] but are neither sufficient nor necessary for diagnosis. (2) Prominent single eosinophilic macronucleoli should be present, and frequently are seen on the background of vesicular chromatin structure [3, 6]. (3) Lamellar fibrosis, usually present in at least half of the tumour tissue [53].

The immunophenotype of fibrolamellar HCC is also unusual, showing expression of hepatocellular markers in combination with biliary, progenitor and stem cell features as well as macrophage markers (CD68). The granular or dot-like expression of CD68 in association with appropriate morphology is helpful in diagnosing fibrolamellar HCC [6].

Prognosis of fibrolamellar HCC is poor. The 5-year survival is similar to conventional HCC arising in non-cirrhotic liver [53]; however, it is better than for classical HCC arising in cirrhotic liver [3].

Sarcomatoid HCC can occur either primarily or within classical HCC [3]. This subtype, comprising 1.8–3.9% of HCC, is partially or fully composed of malignant spindle-shaped cells, occasionally showing heterologous (rhabdoid, osteoid or chondroid) differentiation [53]. If there is no adjacent area of classical HCC, it is difficult to distinguish sarcomatous HCC from true sarcomas, including primary or metastatic tumours, e.g., metastatic gastrointestinal stromal tumour, leiomyosarcoma or fibrosarcoma. Haematoxylin-eosin stain alone can be insufficient, necessitating immunohistochemistry [3]. Considering the high grade and remarkable anaplasia of sarcomatoid HCC, hepatocellular markers should be supplemented with pancytokeratin and specific markers for sarcoma, including CD117, DOG, actin, desmin, S-100, CD34 and CD31. Hepatocellular antigens are frequently negative, and even pancytokeratin is expressed only in 23–63% cases of sarcomatoid HCC [53]; therefore, complex assessment of morphology is mandatory along with clinical history and IHC for sarcoma.

Scirrhous HCC is a rare type, accounting for 0.2% to 4.6% of HCC. It can develop beneath liver capsule leading to pedunculated gross view [3, 53]. Microscopically, scirrhous HCC is characterised by diffuse fibrosis surrounding thin trabeculae of neoplastic cells. Such fibrosis can occur either after various regimens of oncologic treatment (chemotherapy, transarterial chemoembolization, irradiation) or in untreated patients [3]. However, HCC exhibiting post-treatment fibrosis should not be classified as scirrhous [53]. The marked desmoplasia and morphology of the tumour cells, displaying clusters, strands and tubules, can lead to misdiagnosis as cholangiocarcinoma or metastasis both in biopsy and in preoperative imaging. While conventional HCC is characterised by CT enhancement in the arterial phase and washout in the venous phase, scirrhous HCC can present with peripheral ring-like enhancement in the arterial phase and delayed central enhancement in the venous phase [53]. In addition, expression of cytokeratin 19 is frequent [52]. Haemorrhage or necrosis is usually absent. Marked CD8-positive lymphocytic infiltrate can be present [3, 53]. Regarding molecular profile, scirrhous HCC is associated with mutations in TSC1/ TSC2 genes, lack of CTNNB1 mutations, presence of epithelial to mesenchymal transformation and stem cell profile [52].

The differential diagnosis of HCC varies also depending on the underlying liver pathology. In cirrhotic liver, primary tumours such as HCC and cholangiocarcinoma are much more common than secondary tumours [3]. In contrast, in non-cirrhotic liver, HCC accounts only for about 2% of tumours and metastatic lesions predominate over primary liver neoplasms. Metastasis can mimic HCC, especially in case of clear cell renal cancer, clear cell adenocarcinoma of the female genital organs, hepatoid gastric carcinoma, adrenal carcinoma and melanoma. Metastatic gastrointestinal neuroendocrine tumours can be challenging to differentiate from HCC, especially if trabecular architecture is present [3]. In the evaluation of HCC diagnosis, arginase-1, hepatocyte paraffin-1 antigen, glypican-3, carcinoembryonic antigen by polyclonal primary antibody, CD10, glutamine synthetase and CD34 are frequently assessed. Alfa-fetoprotein is partially replaced by new markers showing higher expression frequency and less background. However, it is still helpful in some cases. Clathrin and bile salt export

Diagnostic Algorithm of Hepatocellular Carcinoma: Classics and Innovations in Radiology and Pathology

http://dx.doi.org/10.5772/intechopen.76136

29

Arginase-1 (Arg1) is occasionally considered the most sensitive and specific marker of hepatocellular differentiation [55], characterised by sensitivity and specificity of approximately 90% [55]. Arginase-1 represents manganese metalloenzyme involved in the urea cycle [56]. It catalyses the hydrolysis of arginine to ornithine and urea. Arg1 is expressed in normal human liver [6] and hepatocellular tumours, including HCC. Arg1 shows better sensitivity and specificity diagnosing HCC, compared to HepPar1 and glypican 3 [55], although other researchers prefer HepPar1 (see further) to identify hepatocellular differentiation [3]. Regarding the types of HCC that might cause diagnostic difficulties—high-grade HCC and scirrhous HCC—Arg1 is characterised by sensitivity of 85 and 85%, exceeding the sensitivity of HepPar1 (64 and 26%, respectively). Arg1 displays diffuse nuclear and cytoplasmic expression pattern in HCC [6, 55]. Most other tumours are negative for Arg1, but focal or weak expression can occur in colorectal, pancreatic, breast and prostatic carcinomas, cholangiocarcinoma or hepatoid tumours [55].

Hepatocyte paraffin-1 (HepPar1) antigen is another marker of hepatocellular differentiation. Some authors prefer HepPar1 as the best marker to confirm the hepatocellular origin of a tumour [3]. HepPar1 is a carbamoyl phosphate synthetase 1: another enzyme involved in urea synthesis. In contrast to Arg1, it is expressed not only in the liver but also in non-neoplastic small intestinal mucosa and Barrett's oesophagus [56]. HepPar1 has diffuse granular cytoplasmic staining pattern. The sensitivity and specificity in HCC reaches 80%. HepPar1 is expressed in almost all welldifferentiated HCCs. However, only less than 50% of high-grade cases express HepPar1 [3]. Most of metastatic and/or non-hepatocellular tumours, including adenocarcinomas, neuroendocrine tumours, renal cell carcinoma, adrenocortical carcinoma, melanoma and angiomyolipoma, are negative for HepPar1. However, focal reactivity is occasionally observed. Strong expression can be present in cholangiocarcinomas and metastatic oesophageal, gastric and pulmonary adenocarcinomas [55]. Positive reaction has also been reported in non-ampullary small intestinal adenocarcinomas (60%) and ampullary adenocarcinomas with intestinal (73%) differentiation while expression in ampullary adenocarcinomas exhibiting pancreatobiliary (14%) morphology

Glypican-3 (GPC3) is a member of the glypican family of heparan sulphate proteoglycans. It is bound to the external surface of plasma membrane through a glycosyl-phosphatidyl-inositol

pump protein represent promising novel targets.

or colonic (9%) adenocarcinomas is rare [56].

Lymphoepithelioma-like carcinoma is characterised by the presence of rich lymphocytic infiltrate surrounding pleomorphic, small, polygonal neoplastic cells that might show syncytial growth [1].

Steatohepatitic HCC is remarkable for similarity to steatohepatitis that can even lead to missed diagnosis in well-differentiated cases [53]. This subtype HCC is characterised by the presence of fat vacuoles in more than 5% of the tumour. The neoplastic cells also show Mallory bodies and ballooning degeneration. The stroma features pericellular and trabecular fibrosis as well as inflammatory infiltrate, consisting of neutrophils, plasma cells and lymphocytes [3]. Infiltrative borders are characteristic. Within the tumour, fibrosis can be prominent [53]. The patients can have underlying steatohepatitis due to metabolic syndrome/NASH [3] or alcoholinduced liver disease [53]. However, this phenotype of carcinoma is also seen in patients without steatohepatitic changes in the non-neoplastic liver tissue [3]. Molecularly, IL6/JAK/ STAT molecular pathway is frequently activated along with immunohistochemical C-reactive protein expression. In contrast, mutations in CTNNB1 gene or activation of Wnt/Beta-catenin pathway are not evident. Regarding immunophenotype, low expression of glutamine synthetase has been observed [52].

Clear cell HCC features optically clear cytoplasm due to the presence of glycogen and fat vesicles in the neoplastic cells. The architecture is mostly trabecular [3].

The differential diagnosis of HCC includes benign pathological processes, for instance, dysplastic nodule in a cirrhotic liver while hepatic adenoma, focal nodular hyperplasia and bile duct adenoma should be considered in non-cirrhotic liver. Parasitic infestations, e.g., echinococcosis and infrequent benign tumours, e.g., angiomyolipoma occasionally need to be ruled out. The malignant tumours that enter the spectrum of differential diagnoses of hepatocellular carcinoma include metastases of extrahepatic tumours as well as cholangiocarcinoma, hepatoblastoma and non-epithelial liver tumours.

### 3.1. Immunohistochemistry and differential diagnosis

Benign and malignant liver tumours may share morphologic similarities; thus, immunohistochemical assessment is crucial to set the correct diagnosis. The two challenging tasks are (1) to distinguish low-grade/early HCC from benign lesions like liver adenoma, focal nodular hyperplasia or dysplastic nodule and (2) to differentiate high-grade HCC from metastatic tumours in the liver.

The differential diagnosis of HCC varies also depending on the underlying liver pathology. In cirrhotic liver, primary tumours such as HCC and cholangiocarcinoma are much more common than secondary tumours [3]. In contrast, in non-cirrhotic liver, HCC accounts only for about 2% of tumours and metastatic lesions predominate over primary liver neoplasms. Metastasis can mimic HCC, especially in case of clear cell renal cancer, clear cell adenocarcinoma of the female genital organs, hepatoid gastric carcinoma, adrenal carcinoma and melanoma. Metastatic gastrointestinal neuroendocrine tumours can be challenging to differentiate from HCC, especially if trabecular architecture is present [3]. In the evaluation of HCC diagnosis, arginase-1, hepatocyte paraffin-1 antigen, glypican-3, carcinoembryonic antigen by polyclonal primary antibody, CD10, glutamine synthetase and CD34 are frequently assessed. Alfa-fetoprotein is partially replaced by new markers showing higher expression frequency and less background. However, it is still helpful in some cases. Clathrin and bile salt export pump protein represent promising novel targets.

metastasis both in biopsy and in preoperative imaging. While conventional HCC is characterised by CT enhancement in the arterial phase and washout in the venous phase, scirrhous HCC can present with peripheral ring-like enhancement in the arterial phase and delayed central enhancement in the venous phase [53]. In addition, expression of cytokeratin 19 is frequent [52]. Haemorrhage or necrosis is usually absent. Marked CD8-positive lymphocytic infiltrate can be present [3, 53]. Regarding molecular profile, scirrhous HCC is associated with mutations in TSC1/ TSC2 genes, lack of CTNNB1 mutations, presence of epithelial to mesenchymal transformation

Lymphoepithelioma-like carcinoma is characterised by the presence of rich lymphocytic infiltrate surrounding pleomorphic, small, polygonal neoplastic cells that might show syncy-

Steatohepatitic HCC is remarkable for similarity to steatohepatitis that can even lead to missed diagnosis in well-differentiated cases [53]. This subtype HCC is characterised by the presence of fat vacuoles in more than 5% of the tumour. The neoplastic cells also show Mallory bodies and ballooning degeneration. The stroma features pericellular and trabecular fibrosis as well as inflammatory infiltrate, consisting of neutrophils, plasma cells and lymphocytes [3]. Infiltrative borders are characteristic. Within the tumour, fibrosis can be prominent [53]. The patients can have underlying steatohepatitis due to metabolic syndrome/NASH [3] or alcoholinduced liver disease [53]. However, this phenotype of carcinoma is also seen in patients without steatohepatitic changes in the non-neoplastic liver tissue [3]. Molecularly, IL6/JAK/ STAT molecular pathway is frequently activated along with immunohistochemical C-reactive protein expression. In contrast, mutations in CTNNB1 gene or activation of Wnt/Beta-catenin pathway are not evident. Regarding immunophenotype, low expression of glutamine synthe-

Clear cell HCC features optically clear cytoplasm due to the presence of glycogen and fat

The differential diagnosis of HCC includes benign pathological processes, for instance, dysplastic nodule in a cirrhotic liver while hepatic adenoma, focal nodular hyperplasia and bile duct adenoma should be considered in non-cirrhotic liver. Parasitic infestations, e.g., echinococcosis and infrequent benign tumours, e.g., angiomyolipoma occasionally need to be ruled out. The malignant tumours that enter the spectrum of differential diagnoses of hepatocellular carcinoma include metastases of extrahepatic tumours as well as cholangiocarcinoma, hepatoblastoma and

Benign and malignant liver tumours may share morphologic similarities; thus, immunohistochemical assessment is crucial to set the correct diagnosis. The two challenging tasks are (1) to distinguish low-grade/early HCC from benign lesions like liver adenoma, focal nodular hyperplasia or dysplastic nodule and (2) to differentiate high-grade HCC from metastatic tumours in

vesicles in the neoplastic cells. The architecture is mostly trabecular [3].

3.1. Immunohistochemistry and differential diagnosis

and stem cell profile [52].

28 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

tase has been observed [52].

non-epithelial liver tumours.

the liver.

tial growth [1].

Arginase-1 (Arg1) is occasionally considered the most sensitive and specific marker of hepatocellular differentiation [55], characterised by sensitivity and specificity of approximately 90% [55]. Arginase-1 represents manganese metalloenzyme involved in the urea cycle [56]. It catalyses the hydrolysis of arginine to ornithine and urea. Arg1 is expressed in normal human liver [6] and hepatocellular tumours, including HCC. Arg1 shows better sensitivity and specificity diagnosing HCC, compared to HepPar1 and glypican 3 [55], although other researchers prefer HepPar1 (see further) to identify hepatocellular differentiation [3]. Regarding the types of HCC that might cause diagnostic difficulties—high-grade HCC and scirrhous HCC—Arg1 is characterised by sensitivity of 85 and 85%, exceeding the sensitivity of HepPar1 (64 and 26%, respectively). Arg1 displays diffuse nuclear and cytoplasmic expression pattern in HCC [6, 55]. Most other tumours are negative for Arg1, but focal or weak expression can occur in colorectal, pancreatic, breast and prostatic carcinomas, cholangiocarcinoma or hepatoid tumours [55].

Hepatocyte paraffin-1 (HepPar1) antigen is another marker of hepatocellular differentiation. Some authors prefer HepPar1 as the best marker to confirm the hepatocellular origin of a tumour [3]. HepPar1 is a carbamoyl phosphate synthetase 1: another enzyme involved in urea synthesis. In contrast to Arg1, it is expressed not only in the liver but also in non-neoplastic small intestinal mucosa and Barrett's oesophagus [56]. HepPar1 has diffuse granular cytoplasmic staining pattern. The sensitivity and specificity in HCC reaches 80%. HepPar1 is expressed in almost all welldifferentiated HCCs. However, only less than 50% of high-grade cases express HepPar1 [3]. Most of metastatic and/or non-hepatocellular tumours, including adenocarcinomas, neuroendocrine tumours, renal cell carcinoma, adrenocortical carcinoma, melanoma and angiomyolipoma, are negative for HepPar1. However, focal reactivity is occasionally observed. Strong expression can be present in cholangiocarcinomas and metastatic oesophageal, gastric and pulmonary adenocarcinomas [55]. Positive reaction has also been reported in non-ampullary small intestinal adenocarcinomas (60%) and ampullary adenocarcinomas with intestinal (73%) differentiation while expression in ampullary adenocarcinomas exhibiting pancreatobiliary (14%) morphology or colonic (9%) adenocarcinomas is rare [56].

Glypican-3 (GPC3) is a member of the glypican family of heparan sulphate proteoglycans. It is bound to the external surface of plasma membrane through a glycosyl-phosphatidyl-inositol

anchor. Glypicans regulate signalling via Wnt, Hedgehog, fibroblast growth factor and bone morphogenetic protein pathways. Thus, glypicans are involved in the control of cell proliferation. In HCC, GPC3 promotes cancer growth by stimulating Wnt signalling. The GPC3 molecule can be released to extracellular environment after it has been cleaved off by lipase [57]. Hence, the functional activity of GPC3 explains its role as possible serum marker or treatment target for HCC. GPC3 is normally found in foetal liver and placenta but is absent from healthy adult liver and benign hepatocellular lesions including focal nodular hyperplasia and liver adenoma [55]. Thus, expression of GPC3 in liver biopsy is highly suggestive of HCC. The staining pattern is (1) granular or diffuse cytoplasmic, possibly with membranous enhancement; (2) membranous or (3) Golgi complex-related [6, 55]. Heterogeneity can lead to focal lack of expression; therefore, negative result in biopsy does not exclude HCC. The sensitivity of GPC3 ranges from 56 to 62% in low grade (G1) HCC to 80–83% in intermediate grade (G2) HCC, 85–89% in high grade (G3) HCC and 79% in scirrhous HCC [55]. GPC3 is expressed in many extrahepatic tumours that can spread to the liver, including metastatic adenocarcinoma, squamous cell carcinoma, non-seminomatous germ cell tumours (choriocarcinoma, yolk sac tumour) and malignant melanoma (5%). Cholangiocarcinoma can be positive (5%) as well [6, 55]. The strong advantages of GPC3 include the absence of it from non-malignant liver as well as high sensitivity in high-grade HCC. Lack of specificity is the greatest pitfall [55].

found only in a thin central perivenular (zone 3) area. In contrast, extensive diffuse cytoplas-

Diagnostic Algorithm of Hepatocellular Carcinoma: Classics and Innovations in Radiology and Pathology

http://dx.doi.org/10.5772/intechopen.76136

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CD34 has multiple diagnostic roles. Within its wide expression spectrum, endothelial cells are also positive. Sinusoidal expression of CD34 is increased in both benign and malignant hepatocellular lesions, contrasting with limited expression in periportal sinusoids within normal liver [55] or in parenchymal capillaries close to fibrous septa within cirrhotic tissues [6]. In HCC, the endothelial expression of CD34 increases, until capillarisation of the sinusoids becomes complete. The capillarisation develops due to higher oxygen tension in HCC. Although incomplete CD34 expression does not exclude HCC, diffuse positive reaction is strongly suggestive of HCC. However, limited sampling in biopsy can lead to pitfalls as foci of complete CD34 expression are seen in adenomas and in periphery of cirrhotic nodules. If such foci are predominantly sampled

Clathrin is one of the novel markers appearing in the differential diagnostics between malignant and non-malignant hepatocellular nodules. Clathrin is a protein that forms airscrew-like triskelion consisting of three light chains and three heavy chains. When these molecules assemble between themselves, clathrin-coated vesicles arise and participate in endocytosis and exocytosis. Thus, clathrin participates in cell communication and signalling, in the transport of nutrients, receptors and other macromolecules. During mitosis, clathrin stabilises mitotic spindle. The heavy chain of clathrin is significantly upregulated in HCC. In the initial reports, striking contrast in the immunohistochemical staining was found between tumour and surrounding tissues suggesting high affinity and low background. The expression pattern was cytoplasmic and membranous. Expression of the heavy chain of clathrin was tested for the distinction between HCC and benign nodules. The sensitivity and specificity of the heavy chain of clathrin was 41.2 and 77.2%, and the sensitivity increased to 61.1% in combination

Bile salt export pump protein is a transport molecule that is present in bile canaliculi. By immunohistochemistry, bile salt export pump protein was expressed in 89.6% HCC, mostly (76.7%) in canalicular pattern. In comparison with cholangiocarcinomas and metastatic tumours, expression of bile salt export pump protein had 90% sensitivity and 100% specificity for HCC. The performance of bile salt export pump protein was comparable to arginase-1 showing both sensitivity and specificity of 94% and slightly better than HepPar1 characterised

Hepatocellular adenoma (HCA) is defined as benign monoclonal proliferation of well-differentiated hepatocytes. The most common risk factor for HCA is exposure to high oestrogen levels in oral contraceptives, thus the disease has strong female predominance (9:1). Adenomas are typically small, solitary lesions in non-cirrhotic liver. Occasionally, multiple tumours are observed [61]. In HCA, the neoplastic hepatocytes are arranged in cords and sheets, typically two layers thick [3, 62]. The portal triads and interlobular bile ducts are absent from adenoma tissue [63]. Pseudoglandular architecture can be observed, especially in adenomas associated

within the biopsy, false overestimation of CD34 reactivity is possible [6].

mic expression is present in 70% of HCC [6].

with glypican-3 [59].

by sensitivity 90% and specificity 97% [60].

3.2. Well-differentiated hepatocellular carcinoma versus adenoma

Carcinoembryonic antigen (CEA) family represents a class of different glycoproteins belonging to immunoglobulin superfamily. Within CEA family, adhesion molecules and pregnancy-specific glycoproteins are distinguished. The functions of CEA family include cell adhesion, as well as cell interaction in pregnancy, immune reactions and angiogenesis [58]. By immunohistochemistry, CEA is found in foetal and adult epithelial cells [6]. In liver pathology, CEA assessment by polyclonal antibody (pCEA) is strongly advised. In HCC, distinct specific canalicular or so called chicken-wire fence pattern can be observed. Metastatic adenocarcinomas show diffuse membranous, luminal and/or cytoplasmic positivity [55]. In higher grade HCC, the specific canalicular pattern is progressively lost and replaced by unspecific membranous expression [6].

CD10 is a zinc-dependent metalloproteinase, located in cell surface membranes. It exhibits neutral endopeptidase activity: cleavage of peptides at the amino side of hydrophobic residues. CD10 inactivates several hormones, as glucagon, oxytocin and bradykinin. In HCC, CD10 shows canalicular expression similarly to pCEA. However, the sensitivity of CD10 for HCC is lower, around 50% [55].

Alpha-fetoprotein (AFP), the protein encoded by AFP gene on 4q25, is the major plasma protein in developing foetus. It is produced by liver and yolk sac and might represent the foetal analogue of albumin. AFP can bind metal ions, fats and bilirubin. In adults, AFP is found in HCC and germ cell tumours but normal liver tissue does not express AFP [3]. Although the sensitivity of AFP for HCC is only 30–50% and high background can frequently limit the interpretation [55], truly positive cases in our experience were easy to recognise. In contrast to HepPar1 and pCEA, AFP positivity increases with dedifferentiation of HCC [3].

Glutamine synthetase (GS) is an enzyme that catalyses the condensation reaction between glutamate and ammonia resulting in glutamine. GS is regulated by beta-catenin molecular pathway. In normal liver tissue, immunohistochemical expression of glutamine synthetase is found only in a thin central perivenular (zone 3) area. In contrast, extensive diffuse cytoplasmic expression is present in 70% of HCC [6].

anchor. Glypicans regulate signalling via Wnt, Hedgehog, fibroblast growth factor and bone morphogenetic protein pathways. Thus, glypicans are involved in the control of cell proliferation. In HCC, GPC3 promotes cancer growth by stimulating Wnt signalling. The GPC3 molecule can be released to extracellular environment after it has been cleaved off by lipase [57]. Hence, the functional activity of GPC3 explains its role as possible serum marker or treatment target for HCC. GPC3 is normally found in foetal liver and placenta but is absent from healthy adult liver and benign hepatocellular lesions including focal nodular hyperplasia and liver adenoma [55]. Thus, expression of GPC3 in liver biopsy is highly suggestive of HCC. The staining pattern is (1) granular or diffuse cytoplasmic, possibly with membranous enhancement; (2) membranous or (3) Golgi complex-related [6, 55]. Heterogeneity can lead to focal lack of expression; therefore, negative result in biopsy does not exclude HCC. The sensitivity of GPC3 ranges from 56 to 62% in low grade (G1) HCC to 80–83% in intermediate grade (G2) HCC, 85–89% in high grade (G3) HCC and 79% in scirrhous HCC [55]. GPC3 is expressed in many extrahepatic tumours that can spread to the liver, including metastatic adenocarcinoma, squamous cell carcinoma, non-seminomatous germ cell tumours (choriocarcinoma, yolk sac tumour) and malignant melanoma (5%). Cholangiocarcinoma can be positive (5%) as well [6, 55]. The strong advantages of GPC3 include the absence of it from non-malignant liver as well as high sensitivity in high-grade HCC. Lack of specificity is the greatest pitfall [55].

30 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

Carcinoembryonic antigen (CEA) family represents a class of different glycoproteins belonging to immunoglobulin superfamily. Within CEA family, adhesion molecules and pregnancy-specific glycoproteins are distinguished. The functions of CEA family include cell adhesion, as well as cell interaction in pregnancy, immune reactions and angiogenesis [58]. By immunohistochemistry, CEA is found in foetal and adult epithelial cells [6]. In liver pathology, CEA assessment by polyclonal antibody (pCEA) is strongly advised. In HCC, distinct specific canalicular or so called chicken-wire fence pattern can be observed. Metastatic adenocarcinomas show diffuse membranous, luminal and/or cytoplasmic positivity [55]. In higher grade HCC, the specific canalicular

CD10 is a zinc-dependent metalloproteinase, located in cell surface membranes. It exhibits neutral endopeptidase activity: cleavage of peptides at the amino side of hydrophobic residues. CD10 inactivates several hormones, as glucagon, oxytocin and bradykinin. In HCC, CD10 shows canalicular expression similarly to pCEA. However, the sensitivity of CD10 for

Alpha-fetoprotein (AFP), the protein encoded by AFP gene on 4q25, is the major plasma protein in developing foetus. It is produced by liver and yolk sac and might represent the foetal analogue of albumin. AFP can bind metal ions, fats and bilirubin. In adults, AFP is found in HCC and germ cell tumours but normal liver tissue does not express AFP [3]. Although the sensitivity of AFP for HCC is only 30–50% and high background can frequently limit the interpretation [55], truly positive cases in our experience were easy to recognise. In contrast to

Glutamine synthetase (GS) is an enzyme that catalyses the condensation reaction between glutamate and ammonia resulting in glutamine. GS is regulated by beta-catenin molecular pathway. In normal liver tissue, immunohistochemical expression of glutamine synthetase is

pattern is progressively lost and replaced by unspecific membranous expression [6].

HepPar1 and pCEA, AFP positivity increases with dedifferentiation of HCC [3].

HCC is lower, around 50% [55].

CD34 has multiple diagnostic roles. Within its wide expression spectrum, endothelial cells are also positive. Sinusoidal expression of CD34 is increased in both benign and malignant hepatocellular lesions, contrasting with limited expression in periportal sinusoids within normal liver [55] or in parenchymal capillaries close to fibrous septa within cirrhotic tissues [6]. In HCC, the endothelial expression of CD34 increases, until capillarisation of the sinusoids becomes complete. The capillarisation develops due to higher oxygen tension in HCC. Although incomplete CD34 expression does not exclude HCC, diffuse positive reaction is strongly suggestive of HCC. However, limited sampling in biopsy can lead to pitfalls as foci of complete CD34 expression are seen in adenomas and in periphery of cirrhotic nodules. If such foci are predominantly sampled within the biopsy, false overestimation of CD34 reactivity is possible [6].

Clathrin is one of the novel markers appearing in the differential diagnostics between malignant and non-malignant hepatocellular nodules. Clathrin is a protein that forms airscrew-like triskelion consisting of three light chains and three heavy chains. When these molecules assemble between themselves, clathrin-coated vesicles arise and participate in endocytosis and exocytosis. Thus, clathrin participates in cell communication and signalling, in the transport of nutrients, receptors and other macromolecules. During mitosis, clathrin stabilises mitotic spindle. The heavy chain of clathrin is significantly upregulated in HCC. In the initial reports, striking contrast in the immunohistochemical staining was found between tumour and surrounding tissues suggesting high affinity and low background. The expression pattern was cytoplasmic and membranous. Expression of the heavy chain of clathrin was tested for the distinction between HCC and benign nodules. The sensitivity and specificity of the heavy chain of clathrin was 41.2 and 77.2%, and the sensitivity increased to 61.1% in combination with glypican-3 [59].

Bile salt export pump protein is a transport molecule that is present in bile canaliculi. By immunohistochemistry, bile salt export pump protein was expressed in 89.6% HCC, mostly (76.7%) in canalicular pattern. In comparison with cholangiocarcinomas and metastatic tumours, expression of bile salt export pump protein had 90% sensitivity and 100% specificity for HCC. The performance of bile salt export pump protein was comparable to arginase-1 showing both sensitivity and specificity of 94% and slightly better than HepPar1 characterised by sensitivity 90% and specificity 97% [60].
