**3. Malignant epithelial primary liver tumours**

Three primary liver tumours are of utmost importance. Hepatocellular carcinoma is the most frequent primary epithelial liver tumour with grave prognosis. Cholangiocarcinoma ranks second by the incidence except for endemic regions. Hepatoblastoma is notable for the occurrence in the infancy.

high power magnification must be carried out. There are many secondary phenomena rais‐ ing the similarity between HCC and liver tissue: presence of macrovesicular or microvesicu‐ lar fat, Mallory hyaline and bile. The capillaries can be dilated [27]. Among the histochemical staining methods, absent reticulin staining [44] is characteristic. PAS stain can reveal glycogen and intracytoplasmic globules; the latter structure remains positive after di‐ astase digestion [27,44]. With some experience, morphology is helpful to distinguish finely granular glycogen or rounded globules in HCC from mucus droplets in metastatic adeno‐

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121

**Figure 1.** Hepatocellular carcinoma displaying marked cytologic atypia. Note the presence of atypical mitosis. Haematox‐

Fibrolamellar hepatocellular carcinoma (FLHC) has distinctive aetiology, epidemiology and course. The general HCC risk factors are not associated with this subtype [31]. FLHC is rare, constituting only 1-4% of HCC [27]. It is less common in high-risk areas than in North America and Europe. The patients are young adults or even children [10]. FLHC is diag‐ nosed at the mean age of 25 years in contrast to mean age of 52 years in typical HCC patient group [27]. Controversial data are reported about the sex predilection: some but not all au‐ thors have noted that females are mostly affected [10,31]. Clinically, symptoms attributable to liver enlargement, parenchymal damage (elevated liver enzyme level) or tumour-related intoxication (weight loss or fever) can be present. Cirrhosis is absent. The tumour can be multifocal, and metastases can affect lungs and regional lymph nodes [31]. The histological structure is remarkable for the lamellar fibrosis. The stroma is composed of thick, parallel strands of hyalinised collagen [27]. The cells are large, polygonal, with wide eosinophilic cy‐ toplasm. The vesicular nuclei possess large nucleoli. Cytoplasmic pale bodies are more fre‐ quent (up to 50% of cases) and more abundant than in other types [10,27]. The pale bodies

carcinoma or cholangiocarcinoma.

ylin-eosin (HE), original magnification (OM) 100x.

#### **3.1. Hepatocellular carcinoma**

Hepatocellular carcinoma is defined as malignant tumour developing from hepatocytes and/or showing hepatocellular differentiation. It is the most common primary malignant tu‐ mour of the liver constituting 80-85% of primary epithelial liver malignancies [29,43]. Con‐ sidering the epidemiology, the worldwide burden of hepatocellular carcinoma can reach 1 million of new cases per year. The incidence shows major geographic differences. HCC is the 2nd most common cancer in Asia, and the 4th – in Africa [10]. The annual age-standar‐ dised incidence is the highest in East Asia, including China and Japan. Low-risk areas com‐ prise Europe, esp. northern and western parts; North and South America, Australia and New Zealand [10].The age-adjusted incidence rates in Mozambique are as high as 112.9 and 30.8/100 000 in males and females, respectively. In China these values reach 34.4 and 11.6. In contrast, the age-adjusted incidence rates in British males and females are 1.6 and 0.8, re‐ spectively [31]. The HCC risk factors include liver cirrhosis independently of cause, chronic hepatitis B or C, ethanol consumption and non-alcoholic liver steatosis as well as mycotox‐ ins. The aflatoxin B1 or other mycotoxins produced by *Aspergillus* fungi could be responsible for part of HCC in areas where grains, rice and peanuts are stored in hot and humid condi‐ tions [31]. Most of HCC cases develop from dysplastic cirrhotic nodule [29], thus the differ‐ ential diagnostics between dysplastic nodule and cancer represent evaluation of one point in a complex road of pathogenesis. Clinically, most of the patients approach doctor due to symptoms attributable to mass lesion in the liver (abdominal pain, sensation of fullness), tu‐ mour-related intoxication (weight loss, weakness, lack of appetite) and loss of liver func‐ tions (jaundice). Alternatively, the symptoms can be related to pre-existing cirrhosis and the tumour could be identified during routine control of cirrhotic patient or during workup for unspecific or unrelated symptoms [31]. Radiologically, the number and size of tumour masses can be evaluated. Ultrasonography can be used for screening. Typical findings re‐ garding vascularity include hypervascularity and thrombosis of portal vein, frequently due to invasion. If it is necessary to confirm invasion into portal vein, biopsy can be obtained from it [31].

By microscopy, the typical patterns include trabecular, acinar and ductular structure. The neoplastic cells in low-grade cases resemble liver cells by possessing wide eosinophilic cyto‐ plasm and distinct cell borders. Nuclear atypia is present and nucleo: cytoplasmic ratio is increased, although to different degree. Mitoses can be present; atypical mitoses can be ob‐ served (Figure 1). The architecture shows unequivocal deviations from normal structure such as thick trabeculae with more than 2 cell layers (in contrast to adenoma), solid areas, duct-like or gland-like structures. However, careful evaluation of the architecture under high power magnification must be carried out. There are many secondary phenomena rais‐ ing the similarity between HCC and liver tissue: presence of macrovesicular or microvesicu‐ lar fat, Mallory hyaline and bile. The capillaries can be dilated [27]. Among the histochemical staining methods, absent reticulin staining [44] is characteristic. PAS stain can reveal glycogen and intracytoplasmic globules; the latter structure remains positive after di‐ astase digestion [27,44]. With some experience, morphology is helpful to distinguish finely granular glycogen or rounded globules in HCC from mucus droplets in metastatic adeno‐ carcinoma or cholangiocarcinoma.

**3. Malignant epithelial primary liver tumours**

occurrence in the infancy.

from it [31].

**3.1. Hepatocellular carcinoma**

120 Liver Biopsy - Indications, Procedures, Results

Three primary liver tumours are of utmost importance. Hepatocellular carcinoma is the most frequent primary epithelial liver tumour with grave prognosis. Cholangiocarcinoma ranks second by the incidence except for endemic regions. Hepatoblastoma is notable for the

Hepatocellular carcinoma is defined as malignant tumour developing from hepatocytes and/or showing hepatocellular differentiation. It is the most common primary malignant tu‐ mour of the liver constituting 80-85% of primary epithelial liver malignancies [29,43]. Con‐ sidering the epidemiology, the worldwide burden of hepatocellular carcinoma can reach 1 million of new cases per year. The incidence shows major geographic differences. HCC is the 2nd most common cancer in Asia, and the 4th – in Africa [10]. The annual age-standar‐ dised incidence is the highest in East Asia, including China and Japan. Low-risk areas com‐ prise Europe, esp. northern and western parts; North and South America, Australia and New Zealand [10].The age-adjusted incidence rates in Mozambique are as high as 112.9 and 30.8/100 000 in males and females, respectively. In China these values reach 34.4 and 11.6. In contrast, the age-adjusted incidence rates in British males and females are 1.6 and 0.8, re‐ spectively [31]. The HCC risk factors include liver cirrhosis independently of cause, chronic hepatitis B or C, ethanol consumption and non-alcoholic liver steatosis as well as mycotox‐ ins. The aflatoxin B1 or other mycotoxins produced by *Aspergillus* fungi could be responsible for part of HCC in areas where grains, rice and peanuts are stored in hot and humid condi‐ tions [31]. Most of HCC cases develop from dysplastic cirrhotic nodule [29], thus the differ‐ ential diagnostics between dysplastic nodule and cancer represent evaluation of one point in a complex road of pathogenesis. Clinically, most of the patients approach doctor due to symptoms attributable to mass lesion in the liver (abdominal pain, sensation of fullness), tu‐ mour-related intoxication (weight loss, weakness, lack of appetite) and loss of liver func‐ tions (jaundice). Alternatively, the symptoms can be related to pre-existing cirrhosis and the tumour could be identified during routine control of cirrhotic patient or during workup for unspecific or unrelated symptoms [31]. Radiologically, the number and size of tumour masses can be evaluated. Ultrasonography can be used for screening. Typical findings re‐ garding vascularity include hypervascularity and thrombosis of portal vein, frequently due to invasion. If it is necessary to confirm invasion into portal vein, biopsy can be obtained

By microscopy, the typical patterns include trabecular, acinar and ductular structure. The neoplastic cells in low-grade cases resemble liver cells by possessing wide eosinophilic cyto‐ plasm and distinct cell borders. Nuclear atypia is present and nucleo: cytoplasmic ratio is increased, although to different degree. Mitoses can be present; atypical mitoses can be ob‐ served (Figure 1). The architecture shows unequivocal deviations from normal structure such as thick trabeculae with more than 2 cell layers (in contrast to adenoma), solid areas, duct-like or gland-like structures. However, careful evaluation of the architecture under

**Figure 1.** Hepatocellular carcinoma displaying marked cytologic atypia. Note the presence of atypical mitosis. Haematox‐ ylin-eosin (HE), original magnification (OM) 100x.

Fibrolamellar hepatocellular carcinoma (FLHC) has distinctive aetiology, epidemiology and course. The general HCC risk factors are not associated with this subtype [31]. FLHC is rare, constituting only 1-4% of HCC [27]. It is less common in high-risk areas than in North America and Europe. The patients are young adults or even children [10]. FLHC is diag‐ nosed at the mean age of 25 years in contrast to mean age of 52 years in typical HCC patient group [27]. Controversial data are reported about the sex predilection: some but not all au‐ thors have noted that females are mostly affected [10,31]. Clinically, symptoms attributable to liver enlargement, parenchymal damage (elevated liver enzyme level) or tumour-related intoxication (weight loss or fever) can be present. Cirrhosis is absent. The tumour can be multifocal, and metastases can affect lungs and regional lymph nodes [31]. The histological structure is remarkable for the lamellar fibrosis. The stroma is composed of thick, parallel strands of hyalinised collagen [27]. The cells are large, polygonal, with wide eosinophilic cy‐ toplasm. The vesicular nuclei possess large nucleoli. Cytoplasmic pale bodies are more fre‐ quent (up to 50% of cases) and more abundant than in other types [10,27]. The pale bodies are rounded and very lightly eosinophilic thus staining paler than the surrounding cyto‐ plasm. These structures represent cystically dilated endoplasmic reticulum. Pale bodies can be positive for fibrinogen by IHC. The immunophenotype is remarkable for diffuse expres‐ sion of CK7. The hepatocellular differentiation can be confirmed by Hep Par 1; alpha-feto‐ protein is present in approximately 20% of cases. The FLHC prognosis is better than in the general group. The mean survival is 32 months in contrast to 5.9 months in trabecular HCC [27]. However, it is found that the beneficial prognosis of FLHC is different from cancer in cirrhotic liver but not from HCC in the absence of liver cirrhosis [10].

marker in HCC diagnostics exceeds 80% and has reached 90% in several studies [6,67]. Un‐ fortunately, sensitivity is lower in high-grade HCC. The expression in non-hepatocellular tu‐ mours including colorectal, pancreatic, breast, urothelial, prostate cancer, neuroendocrine tumours, renal cell carcinoma, melanoma and angiomyolipoma is either negative or focal. However, few gastric, colorectal and lung adenocarcinomas can be positive [6,67]. In the bi‐

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123

Arginase-1 is an enzyme involved in the urea cycle as well. It is found in benign hepatocytes and hepatocellular neoplasms. The antibody has received high sensitivity estimates of 96%

Alpha-fetoprotein is an oncofetal protein produced by the liver and yolk sac endoderm. The antigen is remarkable for expression in malignant hepatocellular tumours (Figure 2) in con‐ trast to benign liver tissue, and for the high specifity. However, sensitivity is low (30-50%) and heterogeneity adds further problems in biopsy evaluation [6]. Nevertheless, positive ex‐

**Figure 2.** Heterogeneous intense cytoplasmic expression of alpha-fetoprotein in hepatocellular carcinoma. Immuno‐

Polyclonal antibodies against carcinoembryonic antigen (CEA) yield positive reaction more than in 70% of HCC cases, while monoclonal anti-CEA only rarely stains HCC. Reactivi‐ ty with polyclonal CEA antibodies mostly is observed in canaliculi; this pattern can be observed in benign or malignant liver tissues and is attributable to cross-reaction with biliary glycoprotein on the canalicular surface [67]. The canalicular pattern is specific for HCC and can be used to exclude cholangiocarcinoma and metastatic adenocarcinoma. It is not useful in the differential diagnosis between HCC and benign hepatocellular mass

opsy material, heterogeneity in the HCC can cause diagnostic problems [6].

and favourable performance characteristics [68,69].

pression is valuable.

peroxidase (IP), anti-alpha-fetoprotein, OM 100x.

IHC has an important role in the diagnostics of HCC. Frequently tested antigens include glypican-3, Hep Par 1, alpha-fetoprotein, CD10, carcinoembryonic antigen CEA, TTF-1, argi‐ nase-1, evaluation of cytokeratins and endothelial network as well as MOC-31 and markers of extra-hepatic tumours.

Glypican-3 is a cell surface protein [1] that is involved in the control of cell proliferation and survival. Glypican-3-knockout mice exhibit alterations in Wnt signalling [45]. Glypican-3 al‐ so interacts with Hedgehog signalling pathway [46]. In the practical surgical pathology, the value of glypican-3 is associated with the cancer diagnostics as it is expressed in 70-75% HCC but not in benign liver tissue [48-49] or cholangiocellular carcinoma [1]. Hepatoblasto‐ ma can be positive as well. However, glypican-3 can be expressed in metastatic melanoma [50], ovarian clear-cell carcinoma [51], choriocarcinoma, yolk sac tumour [52-53] as well as in blastomas including neuroblastoma and Wilms' tumour [54]. In addition, 10% of gastric can‐ cer cases are positive for glypican-3 [55]. In melanoma, 80% of tumours contain detectable level of glypican-3 protein and mRNA [1]. Regarding ovarian cancer, the rate of glypican ex‐ pression could be as high as 18% of all ovarian cancer cases and 60% of clear cell carcinoma cases [51]. However, negative reports regarding clear cell carcinoma of ovary are published as well [53]. Glypican-3 is silenced in breast cancer, lung adenocarcinoma and mesothelioma [56-58]. Another problem has been highlighted by Abdul-Al *et al*., who have described fre‐ quent granular cytoplasmic expression of glypican-3 in chronic active hepatitis C [59]. Re‐ generative changes were suggested as the explanation. Authors emphasized that membranous staining was not observed in hepatitis [59]. Glypican-3 has prognostic signifi‐ cance in HCC as it is associated with poor prognosis [60] and shorter recurrence-free period after liver transplantation [49]. The applications of glypican-3 could extend beyond liver bi‐ opsy – and return to it. It could possible to use glypican-3 plasma levels for diagnostics and monitoring of HCC [61-63]. Immunotherapy could be guided towards glypican-3; the present research is exploring both antibody and cell-based immunological mechanisms [64-65]. Cancer vaccine could be generated against this molecule [1]. Glypican-3 is among genes that are distinctly expressed in liver cancer stem cells; it is suggested that glypican could be promising candidate for gene therapy without inducing damage to normal liver stem cells [66].

Hep Par 1 is positive in normal liver, liver adenomas and HCC. The antibody was devel‐ oped in 1993 using an immunogen from failed liver allograft. The target antigen has been identified as carbamoyl phosphate synthetase. This enzyme catalyses the rate-limiting step in the urea cycle and is located in the mitochondria [67]. The specifity and sensitivity of this marker in HCC diagnostics exceeds 80% and has reached 90% in several studies [6,67]. Un‐ fortunately, sensitivity is lower in high-grade HCC. The expression in non-hepatocellular tu‐ mours including colorectal, pancreatic, breast, urothelial, prostate cancer, neuroendocrine tumours, renal cell carcinoma, melanoma and angiomyolipoma is either negative or focal. However, few gastric, colorectal and lung adenocarcinomas can be positive [6,67]. In the bi‐ opsy material, heterogeneity in the HCC can cause diagnostic problems [6].

are rounded and very lightly eosinophilic thus staining paler than the surrounding cyto‐ plasm. These structures represent cystically dilated endoplasmic reticulum. Pale bodies can be positive for fibrinogen by IHC. The immunophenotype is remarkable for diffuse expres‐ sion of CK7. The hepatocellular differentiation can be confirmed by Hep Par 1; alpha-feto‐ protein is present in approximately 20% of cases. The FLHC prognosis is better than in the general group. The mean survival is 32 months in contrast to 5.9 months in trabecular HCC [27]. However, it is found that the beneficial prognosis of FLHC is different from cancer in

IHC has an important role in the diagnostics of HCC. Frequently tested antigens include glypican-3, Hep Par 1, alpha-fetoprotein, CD10, carcinoembryonic antigen CEA, TTF-1, argi‐ nase-1, evaluation of cytokeratins and endothelial network as well as MOC-31 and markers

Glypican-3 is a cell surface protein [1] that is involved in the control of cell proliferation and survival. Glypican-3-knockout mice exhibit alterations in Wnt signalling [45]. Glypican-3 al‐ so interacts with Hedgehog signalling pathway [46]. In the practical surgical pathology, the value of glypican-3 is associated with the cancer diagnostics as it is expressed in 70-75% HCC but not in benign liver tissue [48-49] or cholangiocellular carcinoma [1]. Hepatoblasto‐ ma can be positive as well. However, glypican-3 can be expressed in metastatic melanoma [50], ovarian clear-cell carcinoma [51], choriocarcinoma, yolk sac tumour [52-53] as well as in blastomas including neuroblastoma and Wilms' tumour [54]. In addition, 10% of gastric can‐ cer cases are positive for glypican-3 [55]. In melanoma, 80% of tumours contain detectable level of glypican-3 protein and mRNA [1]. Regarding ovarian cancer, the rate of glypican ex‐ pression could be as high as 18% of all ovarian cancer cases and 60% of clear cell carcinoma cases [51]. However, negative reports regarding clear cell carcinoma of ovary are published as well [53]. Glypican-3 is silenced in breast cancer, lung adenocarcinoma and mesothelioma [56-58]. Another problem has been highlighted by Abdul-Al *et al*., who have described fre‐ quent granular cytoplasmic expression of glypican-3 in chronic active hepatitis C [59]. Re‐ generative changes were suggested as the explanation. Authors emphasized that membranous staining was not observed in hepatitis [59]. Glypican-3 has prognostic signifi‐ cance in HCC as it is associated with poor prognosis [60] and shorter recurrence-free period after liver transplantation [49]. The applications of glypican-3 could extend beyond liver bi‐ opsy – and return to it. It could possible to use glypican-3 plasma levels for diagnostics and monitoring of HCC [61-63]. Immunotherapy could be guided towards glypican-3; the present research is exploring both antibody and cell-based immunological mechanisms [64-65]. Cancer vaccine could be generated against this molecule [1]. Glypican-3 is among genes that are distinctly expressed in liver cancer stem cells; it is suggested that glypican could be promising candidate for gene therapy without inducing damage to normal liver

Hep Par 1 is positive in normal liver, liver adenomas and HCC. The antibody was devel‐ oped in 1993 using an immunogen from failed liver allograft. The target antigen has been identified as carbamoyl phosphate synthetase. This enzyme catalyses the rate-limiting step in the urea cycle and is located in the mitochondria [67]. The specifity and sensitivity of this

cirrhotic liver but not from HCC in the absence of liver cirrhosis [10].

of extra-hepatic tumours.

122 Liver Biopsy - Indications, Procedures, Results

stem cells [66].

Arginase-1 is an enzyme involved in the urea cycle as well. It is found in benign hepatocytes and hepatocellular neoplasms. The antibody has received high sensitivity estimates of 96% and favourable performance characteristics [68,69].

Alpha-fetoprotein is an oncofetal protein produced by the liver and yolk sac endoderm. The antigen is remarkable for expression in malignant hepatocellular tumours (Figure 2) in con‐ trast to benign liver tissue, and for the high specifity. However, sensitivity is low (30-50%) and heterogeneity adds further problems in biopsy evaluation [6]. Nevertheless, positive ex‐ pression is valuable.

**Figure 2.** Heterogeneous intense cytoplasmic expression of alpha-fetoprotein in hepatocellular carcinoma. Immuno‐ peroxidase (IP), anti-alpha-fetoprotein, OM 100x.

Polyclonal antibodies against carcinoembryonic antigen (CEA) yield positive reaction more than in 70% of HCC cases, while monoclonal anti-CEA only rarely stains HCC. Reactivi‐ ty with polyclonal CEA antibodies mostly is observed in canaliculi; this pattern can be observed in benign or malignant liver tissues and is attributable to cross-reaction with biliary glycoprotein on the canalicular surface [67]. The canalicular pattern is specific for HCC and can be used to exclude cholangiocarcinoma and metastatic adenocarcinoma. It is not useful in the differential diagnosis between HCC and benign hepatocellular mass lesions. Although good general sensitivity has been reported, it is higher in well or mod‐ erately differentiated HCC that present less problems regarding the differential diagno‐ sis with cholangiocellular carcinoma or metastasis [67]. Cytoplasmic stain is not observed in healthy liver or benign neoplasms; it is characteristic of malignancy but seen mostly in cholangiocellular carcinoma and metastatic neoplasms. The rate of cytokeratin fraction expression is 15% for CK7, 20% for CK20 and 10% for CK19. Diffuse strong expression of endothelial markers CD31 and CD34 is not characteristic for normal liver tissue in contrast to HCC [27]. The visualisation of endothelial layer is valuable also in estimat‐ ing the thickness of trabeculae. However, pattern of diffuse, strong endothelial marker expression has low sensitivity of 20-40%. The patchy expression is also difficult to evalu‐ ate in liver biopsies. The visualisation of endothelium thus is not recommended for the distinction between adenoma and carcinoma [6].

Molecular subtyping is emerging for HCC. The subtypes are distinguished by high prolifer‐ ation and chromosomal instability; by activation of Wnt signalling pathway and by interfer‐

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The requests for clinically relevant classification have resulted in the separation of HCC into early and progressed entities. The early HCC is recognized as small (not exceeding the diam‐ eter of 2 cm), well differentiated and lacking vascular invasion. The invasion into portal tracts can be present and is highlighted by lack of proliferating ductules. Macrovesicular steatosis is present in 40% of early HCC but appears mostly in Eastern cohorts. It can be attributable to incomplete neoarterialisation – the process of portal tract replacement by unpaired arteries outside the portal tracts. In early HCC, there is still comparatively large venous flow. The tumours in general may be radiologically hypovascular. The early HCC is more likely to become the biopsy target due to equivocal findings at imaging. Progressed HCC includes HCC of higher grade (moderate or poor differentiation degree, G2 or G3), possessing vascular invasion, larger size or stem/progenitor cell immunophenotype and mixed hepatobiliary differentiation. The stem cell immunophenotype can be detected by IHC for CK19, EpCAM, CD133, and mixed hepatobiliary immunophenotype – by expression of CK7 and CK19 [7]. The 5-year survival is 89% in the early HCC group in contrast to 48% in the progressed group. The intrahepatic metastatic spread must be distinguished from multifocal carcinoma that is prog‐ nostically better disease. The multifocal disease is characterised by "nodule in nodule" struc‐

The differential diagnosis includes benign hepatic lesions, metastatic malignancies and chol‐ angiocarcinoma. IHC is of major importance. Markers, that are expressed both in benign and malignant liver cells (CEA by polyclonal antibody, CD10, Hep Par 1, TTF-1 and (occa‐ sionally) cytokeratins [27]) identify the hepatocellular origin of tumour but cannot be used to prove the malignant biological potential of suspicious biopsied tissue. If these are found in high-grade tumour, diagnosis of HCC is preferable in contrast to metastasis. The expres‐ sion of alpha-fetoprotein and glypican-3 is typical for malignant tumour of hepatocellular origin [27]. These findings are important in differential diagnosis with non-hepatocellular and/or metastatic tumour in line with other markers specific for particular histogenesis. Re‐ garding the differential diagnosis of HCC and dysplastic cirrhotic nodule, a panel of immu‐ nohistochemical stains is recommended employing glypican-3, glutamine synthetase and heat-shock protein 70 [48,78-80]. In biopsy, the panel has lower sensitivity although good specifity: accuracy 60.8% for 3 markers and 78.4% for 2 markers with 100% specifity. The findings were acceptable even in the group of low-grade HCC: the accuracy still was 57%

HCC (except fibrolamellar type) mostly is associated either with cirrhosis or chronic active hepatitis with fibrosis that has not reached the degree of cirrhosis. To facilitate the differen‐ tial diagnosis between HCC and liver adenoma or FNH it is wise to take separate biopsies‐

The future pathways for molecular diagnostics of HCC include mRNA analysis of *GPC3*, *survivin* and *LYVE1* genes [78]. Glypican-3, encoded by *GPC3*, and survivin is up-regulated

on signalling due to tumour-infiltrating cells [70-77].

ture or by presence of at least one G1 nodule [7].

for 3 markers and 72.9% for 2 markers with 100% specifity [23].

from the lesion and from distant liver tissues if possible.

The transcription factor TTF-1 is expressed as intense granular cytoplasmic staining in nor‐ mal liver parenchyma [16] and hepatocellular tumours (Figure 3). The reaction is ensured by cross-reactivity with hepatocyte mitochondrial antigen and is seen with the clone 8G7G3/1 [69]. The reported sensitivity is 60-70%. However, it parallels the expression of Hep Par 1 decreasing the practical value [6]. Its expression can be retained even in metastatic HCC [16].

**Figure 3.** Granular cytoplasmic expression of TTF-1 in hepatocellular carcinoma. IP, Anti-TTF-1, OM 400x.

MOC-31 is an epithelial cell surface glycoprotein of unknown function. Evaluating liver bi‐ opsies, it is valuable as non-hepatocellular marker. MOC-31 is negative in HCC but positive in most metastatic adenocarcinomas and cholangiocellular cancer [67]. However, mesothe‐ lioma is MOC-31 negative as well; calretinin should be used in the panel to exclude this pos‐ sibility [17].

Molecular subtyping is emerging for HCC. The subtypes are distinguished by high prolifer‐ ation and chromosomal instability; by activation of Wnt signalling pathway and by interfer‐ on signalling due to tumour-infiltrating cells [70-77].

lesions. Although good general sensitivity has been reported, it is higher in well or mod‐ erately differentiated HCC that present less problems regarding the differential diagno‐ sis with cholangiocellular carcinoma or metastasis [67]. Cytoplasmic stain is not observed in healthy liver or benign neoplasms; it is characteristic of malignancy but seen mostly in cholangiocellular carcinoma and metastatic neoplasms. The rate of cytokeratin fraction expression is 15% for CK7, 20% for CK20 and 10% for CK19. Diffuse strong expression of endothelial markers CD31 and CD34 is not characteristic for normal liver tissue in contrast to HCC [27]. The visualisation of endothelial layer is valuable also in estimat‐ ing the thickness of trabeculae. However, pattern of diffuse, strong endothelial marker expression has low sensitivity of 20-40%. The patchy expression is also difficult to evalu‐ ate in liver biopsies. The visualisation of endothelium thus is not recommended for the

The transcription factor TTF-1 is expressed as intense granular cytoplasmic staining in nor‐ mal liver parenchyma [16] and hepatocellular tumours (Figure 3). The reaction is ensured by cross-reactivity with hepatocyte mitochondrial antigen and is seen with the clone 8G7G3/1 [69]. The reported sensitivity is 60-70%. However, it parallels the expression of Hep Par 1 decreasing the practical value [6]. Its expression can be retained even in metastatic HCC [16].

**Figure 3.** Granular cytoplasmic expression of TTF-1 in hepatocellular carcinoma. IP, Anti-TTF-1, OM 400x.

sibility [17].

MOC-31 is an epithelial cell surface glycoprotein of unknown function. Evaluating liver bi‐ opsies, it is valuable as non-hepatocellular marker. MOC-31 is negative in HCC but positive in most metastatic adenocarcinomas and cholangiocellular cancer [67]. However, mesothe‐ lioma is MOC-31 negative as well; calretinin should be used in the panel to exclude this pos‐

distinction between adenoma and carcinoma [6].

124 Liver Biopsy - Indications, Procedures, Results

The requests for clinically relevant classification have resulted in the separation of HCC into early and progressed entities. The early HCC is recognized as small (not exceeding the diam‐ eter of 2 cm), well differentiated and lacking vascular invasion. The invasion into portal tracts can be present and is highlighted by lack of proliferating ductules. Macrovesicular steatosis is present in 40% of early HCC but appears mostly in Eastern cohorts. It can be attributable to incomplete neoarterialisation – the process of portal tract replacement by unpaired arteries outside the portal tracts. In early HCC, there is still comparatively large venous flow. The tumours in general may be radiologically hypovascular. The early HCC is more likely to become the biopsy target due to equivocal findings at imaging. Progressed HCC includes HCC of higher grade (moderate or poor differentiation degree, G2 or G3), possessing vascular invasion, larger size or stem/progenitor cell immunophenotype and mixed hepatobiliary differentiation. The stem cell immunophenotype can be detected by IHC for CK19, EpCAM, CD133, and mixed hepatobiliary immunophenotype – by expression of CK7 and CK19 [7]. The 5-year survival is 89% in the early HCC group in contrast to 48% in the progressed group. The intrahepatic metastatic spread must be distinguished from multifocal carcinoma that is prog‐ nostically better disease. The multifocal disease is characterised by "nodule in nodule" struc‐ ture or by presence of at least one G1 nodule [7].

The differential diagnosis includes benign hepatic lesions, metastatic malignancies and chol‐ angiocarcinoma. IHC is of major importance. Markers, that are expressed both in benign and malignant liver cells (CEA by polyclonal antibody, CD10, Hep Par 1, TTF-1 and (occa‐ sionally) cytokeratins [27]) identify the hepatocellular origin of tumour but cannot be used to prove the malignant biological potential of suspicious biopsied tissue. If these are found in high-grade tumour, diagnosis of HCC is preferable in contrast to metastasis. The expres‐ sion of alpha-fetoprotein and glypican-3 is typical for malignant tumour of hepatocellular origin [27]. These findings are important in differential diagnosis with non-hepatocellular and/or metastatic tumour in line with other markers specific for particular histogenesis. Re‐ garding the differential diagnosis of HCC and dysplastic cirrhotic nodule, a panel of immu‐ nohistochemical stains is recommended employing glypican-3, glutamine synthetase and heat-shock protein 70 [48,78-80]. In biopsy, the panel has lower sensitivity although good specifity: accuracy 60.8% for 3 markers and 78.4% for 2 markers with 100% specifity. The findings were acceptable even in the group of low-grade HCC: the accuracy still was 57% for 3 markers and 72.9% for 2 markers with 100% specifity [23].

HCC (except fibrolamellar type) mostly is associated either with cirrhosis or chronic active hepatitis with fibrosis that has not reached the degree of cirrhosis. To facilitate the differen‐ tial diagnosis between HCC and liver adenoma or FNH it is wise to take separate biopsies‐ from the lesion and from distant liver tissues if possible.

The future pathways for molecular diagnostics of HCC include mRNA analysis of *GPC3*, *survivin* and *LYVE1* genes [78]. Glypican-3, encoded by *GPC3*, and survivin is up-regulated in parenchymal HCC cells while LYVE1 protein is down regulated in endothelial cells in case of malignancy. MYC pathway studies could also bring new information [29].

**3.2. Hepatoblastoma**

Hepatoblastoma is defined as a primary malignant blastomatous liver tumour showing complex differentiation towards fetal and embryonal hepatocytes as well as mature tissues including osteoid, connective tissue and striated muscle. Epidemiologically, hepatoblastoma is a rare malignant liver tumour of childhood with the incidence of 1 case / 1 million [8,10]. In children, hepatoblastoma is the most common primary liver tumour. Characteristically, the tumour develops within first five years of life: 4% of hepatoblastomas are present at birth, 69% have developed by 2 years of age and 90% - by 5 years of age. Only 3% of patients are older than 15 years [100]. The risk of hepatoblastoma is increased in *APC*-mutation-car‐ rying children from familial adenomatous polyposis (FAP) kindreds. Clinically, enlarging abdomen can be the first sign. The other possible manifestations include weight loss, ano‐ rexia, nausea, vomiting and abdominal pain. Jaundice is rarely observed [100]. Paraneoplas‐ tic syndromes can occur. Among those, anaemia and thrombocytosis are frequent. Precocious puberty due to production of chorionic gonadotropin is rare. Grossly, the tu‐ mours mostly occur as single lesions [10] measuring 5-22 cm [100]. Pseudocapsule can de‐ velop due to compression of surrounding liver tissue. Microscopically, hepatoblastoma can display any of different histological patterns, or combination of these patterns. The fetal epi‐ thelial differentiation is characterised by thin trabeculae of small cuboidal cells. The nuclei are small and round with fine chromatin and small nucleolus. The cytoplasm can be either clear or finely granular resulting in "light and dark" pattern under low magnification. Foci of extramedullary haemopoesis can be present. The combined fetal and embryonal pattern is characterised by presence of small tumour cells in solid or acinar groups. The small cells have scant cytoplasm, higher nucleo: cytoplasmic ratio and coarse chromatin. Hepatoblasto‐ ma is called macrotrabecular if the cells compose 6-12 cell layers in the trabeculae in most of the tumour. Larger cells are present in the macrotrabeculae in addition to fetal and embry‐ onal type. In teenagers, macrotrabecular hepatoblastoma must be differentiated from hepa‐ tocellular carcinoma. Small cell undifferentiated hepatoblastoma morphologically resembles small cell cancer displaying solid small blue cell pattern with focal necrosis. Mixed epithelial and mesenchymal hepatoblastomas contain mesenchymal components including fibrous tis‐ sue, osteoid, cartilage, striated muscle, bone or melanin [100]. Mixed epithelial and mesen‐ chymal hepatoblastoma with teratoid features is recognised by the presence of endodermal, neuroectodermal and complex mesenchymal tissues. The neuroectodermal component can comprise melanin, glial and neuronal cells [10].After treatment, connective tissue, necrosis and signs of haemorrhage develop in association with residual neoplastic tissue, and squa‐ mous islands become more common. Immunohistochemically, expression of alpha-fetopro‐ tein, beta-catenin and cell cycle markers is associated with the histological pattern. The fetal subtype is characterised by low proliferation that parallels the scant mitotic activity; alphafetoprotein can be present and the expression of beta-catenin is retained in the membranous localisation. The combined fetal and embryonal subtype is characterised by shift of beta-cat‐ enin expression towards the nuclei in higher grade embryonal component. An interesting circular pattern can be observed. In the rounded cell groups, the middle is occupied by pro‐ genitor-type pale, small cells displaying low proliferative activity and nuclear expression of beta-catenin. The progenitor-type cells are surrounded by intensively proliferating embry‐

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In addition, molecular studies can predict the HCC prognosis. Down-regulation of p57 accelerates the growth and invasion of HCC cells [18]. The reduced p57 expression corre‐ lates with larger tumour size, higher TNM stage, presence of extrahepatic metastases and decreased survival. In cell lines, the down-regulation of p57 increases the expression of cyclin D1 and CDK2, enhancing the cellular proliferation. The matrix metalloproteinase-1 (MMP-1) and protease activated receptor-1 (PAR-1) are expressed in HCC but not in normal liver. The up-regulation of MMP-1/PAR-1 axis has prognostic value [20] and potentially could be used in the identification of malignancy. Co-expression of stem cell transcription factors Oct4 and Nanog indicates aggressive tumour behaviour and predicts recurrence after HCC resection [22]. FOXJ1 is over-expressed in HCC. It is associated with histological grade, poor prognosis and with tumour cell proliferation [19]. Hedgehog signalling pathway mediates invasion and metastasis of HCC via ERK pathway. Up-regulation of cell proliferation is associated with down-regulation of p27 and p21 and up-regulation of cyclin D1 [81]. Osteo‐ pontin plays role in the proliferation of HCC through interaction with the cell surface recep‐ tor CD44 [82] and is considered the key mediator for vasculogenic mimicry [83]. Baxinteracting factor is over-expressed in HCC and correlates with shortened survival [84]. NY-ESO-1 protein is a potential marker for early recurrence after surgical treatment [85]. Hepatocyte nuclear factor 4 suppresses the HCC development [86]. Sulfatase 2 protects HCC cells against apoptosis [87]. Interleukins as IL-17 and IL-6 have tumour-promoting role [88]. Interaction with matrix metalloproteinases 2 and 9 is likely [89]. Up-regulation of sirtuins has been identified [90]. Typing of immune cells in biopsy is mostly done for research purposes [91]. If any of those parameters will show prognostic and predictive value, the relevant IHC analysis should be included in the protocol of liver biopsy evaluation. The technological future developments include virtual microscopy. Fractal analysis [92] and quantitative IHC can be applied [93].

Methylation studies have been carried out in HCC [94]. The expression of microRNAs is un‐ dergoing active analysis in HCC [95-96]. MicroRNAs are non-coding, short RNA molecules that can bind to messenger RNA and to prevent their translation into protein, providing ad‐ ditional regulation of gene expression. MicroRNAs act as large-scale molecular switch due to ability simultaneously down-regulate many genes. MicroRNA-181 down-regulates the differentiation and maturation of hepatocytes [96]. Suppression of microRNA-181 expres‐ sion leads to reduced motility and invasion of HCC stem cells [25]. MicroRNA-182 could promote metastasis [97]. MicroRNA-183 inhibits apoptosis [98]. MicroRNA expression can be subjected to regulation with IL-6 [25]. Reduced expression of microRNA-26 in HCC is as‐ sociated with poor prognosis. However, better response of interferon alpha postoperative adjuvant therapy can be expected [95]. MicroRNA-21 induces resistance to the anti-tumour effect of interferon and fluorouracil combination therapy [99]. Circulating microRNAs are valuable in tumour diagnosis and monitoring the treatment [24].

#### **3.2. Hepatoblastoma**

in parenchymal HCC cells while LYVE1 protein is down regulated in endothelial cells in

In addition, molecular studies can predict the HCC prognosis. Down-regulation of p57 accelerates the growth and invasion of HCC cells [18]. The reduced p57 expression corre‐ lates with larger tumour size, higher TNM stage, presence of extrahepatic metastases and decreased survival. In cell lines, the down-regulation of p57 increases the expression of cyclin D1 and CDK2, enhancing the cellular proliferation. The matrix metalloproteinase-1 (MMP-1) and protease activated receptor-1 (PAR-1) are expressed in HCC but not in normal liver. The up-regulation of MMP-1/PAR-1 axis has prognostic value [20] and potentially could be used in the identification of malignancy. Co-expression of stem cell transcription factors Oct4 and Nanog indicates aggressive tumour behaviour and predicts recurrence after HCC resection [22]. FOXJ1 is over-expressed in HCC. It is associated with histological grade, poor prognosis and with tumour cell proliferation [19]. Hedgehog signalling pathway mediates invasion and metastasis of HCC via ERK pathway. Up-regulation of cell proliferation is associated with down-regulation of p27 and p21 and up-regulation of cyclin D1 [81]. Osteo‐ pontin plays role in the proliferation of HCC through interaction with the cell surface recep‐ tor CD44 [82] and is considered the key mediator for vasculogenic mimicry [83]. Baxinteracting factor is over-expressed in HCC and correlates with shortened survival [84]. NY-ESO-1 protein is a potential marker for early recurrence after surgical treatment [85]. Hepatocyte nuclear factor 4 suppresses the HCC development [86]. Sulfatase 2 protects HCC cells against apoptosis [87]. Interleukins as IL-17 and IL-6 have tumour-promoting role [88]. Interaction with matrix metalloproteinases 2 and 9 is likely [89]. Up-regulation of sirtuins has been identified [90]. Typing of immune cells in biopsy is mostly done for research purposes [91]. If any of those parameters will show prognostic and predictive value, the relevant IHC analysis should be included in the protocol of liver biopsy evaluation. The technological future developments include virtual microscopy. Fractal analysis [92] and

Methylation studies have been carried out in HCC [94]. The expression of microRNAs is un‐ dergoing active analysis in HCC [95-96]. MicroRNAs are non-coding, short RNA molecules that can bind to messenger RNA and to prevent their translation into protein, providing ad‐ ditional regulation of gene expression. MicroRNAs act as large-scale molecular switch due to ability simultaneously down-regulate many genes. MicroRNA-181 down-regulates the differentiation and maturation of hepatocytes [96]. Suppression of microRNA-181 expres‐ sion leads to reduced motility and invasion of HCC stem cells [25]. MicroRNA-182 could promote metastasis [97]. MicroRNA-183 inhibits apoptosis [98]. MicroRNA expression can be subjected to regulation with IL-6 [25]. Reduced expression of microRNA-26 in HCC is as‐ sociated with poor prognosis. However, better response of interferon alpha postoperative adjuvant therapy can be expected [95]. MicroRNA-21 induces resistance to the anti-tumour effect of interferon and fluorouracil combination therapy [99]. Circulating microRNAs are

valuable in tumour diagnosis and monitoring the treatment [24].

case of malignancy. MYC pathway studies could also bring new information [29].

quantitative IHC can be applied [93].

126 Liver Biopsy - Indications, Procedures, Results

Hepatoblastoma is defined as a primary malignant blastomatous liver tumour showing complex differentiation towards fetal and embryonal hepatocytes as well as mature tissues including osteoid, connective tissue and striated muscle. Epidemiologically, hepatoblastoma is a rare malignant liver tumour of childhood with the incidence of 1 case / 1 million [8,10]. In children, hepatoblastoma is the most common primary liver tumour. Characteristically, the tumour develops within first five years of life: 4% of hepatoblastomas are present at birth, 69% have developed by 2 years of age and 90% - by 5 years of age. Only 3% of patients are older than 15 years [100]. The risk of hepatoblastoma is increased in *APC*-mutation-car‐ rying children from familial adenomatous polyposis (FAP) kindreds. Clinically, enlarging abdomen can be the first sign. The other possible manifestations include weight loss, ano‐ rexia, nausea, vomiting and abdominal pain. Jaundice is rarely observed [100]. Paraneoplas‐ tic syndromes can occur. Among those, anaemia and thrombocytosis are frequent. Precocious puberty due to production of chorionic gonadotropin is rare. Grossly, the tu‐ mours mostly occur as single lesions [10] measuring 5-22 cm [100]. Pseudocapsule can de‐ velop due to compression of surrounding liver tissue. Microscopically, hepatoblastoma can display any of different histological patterns, or combination of these patterns. The fetal epi‐ thelial differentiation is characterised by thin trabeculae of small cuboidal cells. The nuclei are small and round with fine chromatin and small nucleolus. The cytoplasm can be either clear or finely granular resulting in "light and dark" pattern under low magnification. Foci of extramedullary haemopoesis can be present. The combined fetal and embryonal pattern is characterised by presence of small tumour cells in solid or acinar groups. The small cells have scant cytoplasm, higher nucleo: cytoplasmic ratio and coarse chromatin. Hepatoblasto‐ ma is called macrotrabecular if the cells compose 6-12 cell layers in the trabeculae in most of the tumour. Larger cells are present in the macrotrabeculae in addition to fetal and embry‐ onal type. In teenagers, macrotrabecular hepatoblastoma must be differentiated from hepa‐ tocellular carcinoma. Small cell undifferentiated hepatoblastoma morphologically resembles small cell cancer displaying solid small blue cell pattern with focal necrosis. Mixed epithelial and mesenchymal hepatoblastomas contain mesenchymal components including fibrous tis‐ sue, osteoid, cartilage, striated muscle, bone or melanin [100]. Mixed epithelial and mesen‐ chymal hepatoblastoma with teratoid features is recognised by the presence of endodermal, neuroectodermal and complex mesenchymal tissues. The neuroectodermal component can comprise melanin, glial and neuronal cells [10].After treatment, connective tissue, necrosis and signs of haemorrhage develop in association with residual neoplastic tissue, and squa‐ mous islands become more common. Immunohistochemically, expression of alpha-fetopro‐ tein, beta-catenin and cell cycle markers is associated with the histological pattern. The fetal subtype is characterised by low proliferation that parallels the scant mitotic activity; alphafetoprotein can be present and the expression of beta-catenin is retained in the membranous localisation. The combined fetal and embryonal subtype is characterised by shift of beta-cat‐ enin expression towards the nuclei in higher grade embryonal component. An interesting circular pattern can be observed. In the rounded cell groups, the middle is occupied by pro‐ genitor-type pale, small cells displaying low proliferative activity and nuclear expression of beta-catenin. The progenitor-type cells are surrounded by intensively proliferating embry‐ onal type cells characterised by mixed nuclear and cytoplasmic expression of beta-catenin. The outermost layer of these concentric structures is composed by fetal type cells with low proliferative activity and retained membranous expression of beta-catenin. The small cell subtype lacks alpha-fetoprotein but has high proliferative activity, usually reaching 80%; cy‐ tokeratins are expressed as well. Even in the mixed epithelial and mesenchymal hepatoblas‐ toma, cytokeratins and alpha-fetoprotein can be expressed even in the ostecyte-like and osteoblast-like cells embedded in or associated with the osteoid, correspondingly [10]. In the study of Purcell *et al*., cyclin D1 and Ki-67 were two markers (out of 5, including also betacatenin, E-cadherin and alpha-fetoprotein) that were shown to have prognostic value re‐ garding survival [8].

suggested that morphology cannot reliably distinguish cholangiocarcinoma from metastatic pancreatic or colorectal cancer [31]. In case of pancreatic adenocarcinoma, the marked cellu‐ lar atypia disproportionally to better preserved architecture can be a clue. Colorectal adeno‐ carcinoma in typical cases is characterised by columnar morphology and diffuse intense expression of CK20, CDX2 and CEA and lack of CK7. Other authors have drawn attention to the impossibility to distinguish cholangiocarcinoma from metastatic gastric cancer and can‐ cer of gall bladder; metastatic pancreatic cancer also remains a problem [6]. The morphologi‐ cal differential diagnosis includes benign proliferation of bile ducts, hepatocellular carcinoma and metastatic adenocarcinoma [27]. In order to discriminate between biliary ad‐ enoma and cholangiocarcinoma, invasion (including single invasive cells and perineural in‐ vasion) and cellular atypia should be sought for. Radiologic findings are helpful as bile duct adenoma usually is smaller than 1 cm, but cholangiocarcinomas are large. The differential diagnosis with hepatocellular carcinoma can rely both on morphology and immunopheno‐ type. Immunohistochemically, markers of biliary differentiation CK7 and CK19 are positive in cholangiocellular carcinoma. Hep Par 1 can be used to exclude hepatocellular differentia‐ tion [6,29]. Proteomic analysis of differentially expressed proteins in peripheral cholangio‐

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Cavernous haemangioma, epithelioid haemangiendothelioma and angiosarcoma are endo‐ thelial tumours representing the whole spectrum of biological potential. Haemangioma is entirely benign although can cause complications due to large size; epithelioid haeman‐ gioendothelioma is notable for the peculiar structure leading to marked difficulties in the bi‐ opsy diagnostics, and angiosarcoma is a frank malignancy with grave prognosis. In addition, angiomyolipoma will be discussed as well although it should be noted that this tu‐

Haemangioma is defined as benign endothelial tumour [102]. Due to bleeding risk, it is only rarely seen in liver biopsy; in addition, the possibilities of radiological diagnostics are good and the prognosis only rarely necessitates active treatment. However, epidemiologically the lesion is the most common benign tumour of the liver with incidence 0.4% [27]. Clinically, haemangioma usually are asymptomatic due to small size and slow expansive growth. Oc‐ casionally, a giant haemangioma (10-30 cm) can cause pain due to mass effect. Thrombosis and bleeding can be dangerous complications. In neonates, blood shunting can lead to heart failure. Grossly, haemangiomas are mostly solitary (90%), of small or moderate size (less than 5 cm) and subcapsular. Microscopic structure is similar to cavernous haemangioma elsewhere in the body. Cavernous, lake-like blood spaces can be seen, separated by hypocel‐ lular fibrous septa (Figure 4). Thrombosis can be present. The immunophenotype reflects

mour has complex structure including rich vascularity as one component.

carcinoma is under research [101].

**4. Vascular tumours**

**4.1. Cavernous haemangioma**

#### **3.3. Cholangiocarcinoma**

Cholangiocarcinoma (CC) is defined as malignant epithelial liver tumour with biliary histo‐ genesis or biliary differentiation. Epidemiologically, CC is a rare tumour with male predilec‐ tion. It composes 15% of primary liver cancer [100] but the relative incidence range of cholangiocarcinoma is wide, from 5% in males and 12% in females in Osaka, Japan, to 90% in males and 94% of primary liver cancer cases in females in Thailand. The age-standardized incidence per 100 000 males ranges from 84.6 in Thailand to 2.8 in Osaka, Japan; 1.0 in France or 0.9 in Italy. The known risk factors include association with ulcerative colitis and primary sclerosing cholangitis [27]. The rate of cholangiocarcinoma in primary sclerosing cholangitis patients is estimated as 10-20%. The presence of parasites, especially *Clonorchis sinensis* and *Opisthorchis viverrini*, also increases the risk of cholangiocarcinoma. The high-in‐ cidence area in Laos and North and Northeast Thailand corresponds to the endemic area of *Opisthorchis viverrini*. Korea has high rate of cholangiocellular cancer due to endemic spread of *Clonorchis sinensis.* Clinically, the patients can present with painless jaundice [31], general malaise, mild abdominal pain and weight loss [100]. Grossly, several types exist. Peripheral tumours arise from portal bile ducts. Hilar lesions arise in large ducts. The diffuse intraduc‐ tal papillomatosis involves ducts as widespread carcinoma *in situ* lacking dominant mass but leading to severe obstruction of bile flow. Histologically, cholangiocarcinoma has adeno‐ carcinomatous structure characterised by tubular complexes and moderate amount of des‐ moplastic stroma. The architectural variants include high-grade tumour lacking the characteristic architecture, signet-ring cell tumour with presence of signet-ring cells, muci‐ nous type with extensive secretion of extracellular mucin, adenosquamous type with focal squamous differentiation and spindle cell type with pseudosarcomatoid structure, presence of malignant spindle cells and signs of epithelial differentiation. The tumour has no func‐ tional connection with bile excretory system although morphological connection in the form of invasion or cancer in situ can exist. CC arises from ductal epithelium and not from hepa‐ tocytes. Due to these two reasons, presence of bile in the lumina of malignant glands is not characteristic but eosinophilic or mucinous secretion can be present. Mucin stains as PAS or mucicarmine can be positive [44]. The immunophenotype is derived from the immunophe‐ notype of bile duct epithelium, with expression of following cytokeratins: CK19 (100%), CK7 (80-100%), CK20 (20%). Diffuse cytoplasmic expression of CEA is found by polyclonal anti‐ body in almost all cases and is frequent by monoclonal antibody as well [27]. However, it is suggested that morphology cannot reliably distinguish cholangiocarcinoma from metastatic pancreatic or colorectal cancer [31]. In case of pancreatic adenocarcinoma, the marked cellu‐ lar atypia disproportionally to better preserved architecture can be a clue. Colorectal adeno‐ carcinoma in typical cases is characterised by columnar morphology and diffuse intense expression of CK20, CDX2 and CEA and lack of CK7. Other authors have drawn attention to the impossibility to distinguish cholangiocarcinoma from metastatic gastric cancer and can‐ cer of gall bladder; metastatic pancreatic cancer also remains a problem [6]. The morphologi‐ cal differential diagnosis includes benign proliferation of bile ducts, hepatocellular carcinoma and metastatic adenocarcinoma [27]. In order to discriminate between biliary ad‐ enoma and cholangiocarcinoma, invasion (including single invasive cells and perineural in‐ vasion) and cellular atypia should be sought for. Radiologic findings are helpful as bile duct adenoma usually is smaller than 1 cm, but cholangiocarcinomas are large. The differential diagnosis with hepatocellular carcinoma can rely both on morphology and immunopheno‐ type. Immunohistochemically, markers of biliary differentiation CK7 and CK19 are positive in cholangiocellular carcinoma. Hep Par 1 can be used to exclude hepatocellular differentia‐ tion [6,29]. Proteomic analysis of differentially expressed proteins in peripheral cholangio‐ carcinoma is under research [101].
