**4. Vascular tumours**

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‐

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

garding survival [8].

**3.3. Cholangiocarcinoma**

128 Liver Biopsy - Indications, Procedures, Results

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‐ mour has complex structure including rich vascularity as one component.

#### **4.1. Cavernous haemangioma**

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 the endothelial origin. In the rare situation, when biopsy is obtained from cavernous hae‐ mangioma, the differential diagnosis can include hepatic tumours with rich vascularity as adenoma and cholangiocellular carcinoma. These are diagnosed by the presence and cyto‐ logical properties of liver cells. Other vascular tumours could be considered, including in‐ fantile haemangioendothelioma, angiomyolipoma, epithelioid haemangioendothelioma and angiosarcoma. The infantile haemangioendothelioma can be recognized by capillary struc‐ ture and occurrence in infants [27]. Angiomyolipoma shows combination of fat, smooth muscle and blood vessels with radiating immature smooth muscle cells. The higher cellular‐ ity and presence of fat are features incompatible with cavernous haemangioma. Epithelioid haemangioendothelioma is discussed separately; the occurrence of vascular lakes usually is not observed. Angiosarcoma can have cavernous architecture but the hallmark of it is the cellular atypia.

dence of angiomyolipoma is increased. These patients may develop multiple angiomyolipo‐ mas in liver as well as kidney angiomyolipoma. Awareness of this condition is necessary to escape over-diagnosis of metastatic malignant tumour. Clinically, the tumour can be asymp‐ tomatic. However, large tumours can cause pain; rupture and bleeding is also possible. By radiologic studies, the tumour is hypervascular again. Grossly, angiomyolipoma usually is solitary (except in tuberous sclerosis), measuring 0.8-36 cm. The microscopic picture (Figure 5) is straightforward if all three components are present in liver biopsy and have typical structure. The smooth muscle cells can have epithelioid appearance leading to morphologi‐ cal similarity to liver parenchymal cells; the rich vascularity could lead to diagnostic confu‐ sion with hepatocellular tumour already earlier. The epithelioid cells sometimes can cause suspicion for malignancy due to large nuclei and nucleoli. However, the nucleo: cytoplasmic

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**Figure 5.** The microscopic structure of angiomyolipoma. Note the peculiar, thick-walled blood vessels, immature

In difficult cases, IHC is helpful. The smooth muscle cells express actin (Figure 6) and fat cells – S-100 protein. HMB-45 expression can be observed in perivascular epithelioid cells (Figure 7). The differential diagnosis can include hepatocellular neoplasms or spindle cell sarcomas. Actin expression and complex histological structure helps to exclude hepatocellu‐ lar origin of the tumour. Complex structure, combined immunophenotype and low prolifer‐

smooth muscle proliferation with high cellularity as well as the presence of fat. HE, OM 100x.

ratio remains low due to increased cell size.

ation help to exclude sarcoma [27].

**Figure 4.** Cavernous haemangioma in liver tissue. Note the large, cavernous spaces filled with red blood cells. HE, OM 50x.

#### **4.2. Angiomyolipoma**

Angiomyolipoma is defined as benign mesenchymal tumour with complex structure includ‐ ing immature smooth muscle, blood vessels and fat. Epithelioid cells and perivascular HMB-45-positive cells can be present. Research of the tumour histogenesis has resulted in the concept of PEComa, a tumour of perivascular epithelioid cells, showing myomatous, lip‐ omatous and melaninogenic differentiation. Epidemiologically, liver angiomyolipoma is rare. It has been diagnosed in wide age range (10-86 years). In tuberous sclerosis, the inci‐ dence of angiomyolipoma is increased. These patients may develop multiple angiomyolipo‐ mas in liver as well as kidney angiomyolipoma. Awareness of this condition is necessary to escape over-diagnosis of metastatic malignant tumour. Clinically, the tumour can be asymp‐ tomatic. However, large tumours can cause pain; rupture and bleeding is also possible. By radiologic studies, the tumour is hypervascular again. Grossly, angiomyolipoma usually is solitary (except in tuberous sclerosis), measuring 0.8-36 cm. The microscopic picture (Figure 5) is straightforward if all three components are present in liver biopsy and have typical structure. The smooth muscle cells can have epithelioid appearance leading to morphologi‐ cal similarity to liver parenchymal cells; the rich vascularity could lead to diagnostic confu‐ sion with hepatocellular tumour already earlier. The epithelioid cells sometimes can cause suspicion for malignancy due to large nuclei and nucleoli. However, the nucleo: cytoplasmic ratio remains low due to increased cell size.

the endothelial origin. In the rare situation, when biopsy is obtained from cavernous hae‐ mangioma, the differential diagnosis can include hepatic tumours with rich vascularity as adenoma and cholangiocellular carcinoma. These are diagnosed by the presence and cyto‐ logical properties of liver cells. Other vascular tumours could be considered, including in‐ fantile haemangioendothelioma, angiomyolipoma, epithelioid haemangioendothelioma and angiosarcoma. The infantile haemangioendothelioma can be recognized by capillary struc‐ ture and occurrence in infants [27]. Angiomyolipoma shows combination of fat, smooth muscle and blood vessels with radiating immature smooth muscle cells. The higher cellular‐ ity and presence of fat are features incompatible with cavernous haemangioma. Epithelioid haemangioendothelioma is discussed separately; the occurrence of vascular lakes usually is not observed. Angiosarcoma can have cavernous architecture but the hallmark of it is the

**Figure 4.** Cavernous haemangioma in liver tissue. Note the large, cavernous spaces filled with red blood cells. HE,

Angiomyolipoma is defined as benign mesenchymal tumour with complex structure includ‐ ing immature smooth muscle, blood vessels and fat. Epithelioid cells and perivascular HMB-45-positive cells can be present. Research of the tumour histogenesis has resulted in the concept of PEComa, a tumour of perivascular epithelioid cells, showing myomatous, lip‐ omatous and melaninogenic differentiation. Epidemiologically, liver angiomyolipoma is rare. It has been diagnosed in wide age range (10-86 years). In tuberous sclerosis, the inci‐

cellular atypia.

130 Liver Biopsy - Indications, Procedures, Results

OM 50x.

**4.2. Angiomyolipoma**

**Figure 5.** The microscopic structure of angiomyolipoma. Note the peculiar, thick-walled blood vessels, immature smooth muscle proliferation with high cellularity as well as the presence of fat. HE, OM 100x.

In difficult cases, IHC is helpful. The smooth muscle cells express actin (Figure 6) and fat cells – S-100 protein. HMB-45 expression can be observed in perivascular epithelioid cells (Figure 7). The differential diagnosis can include hepatocellular neoplasms or spindle cell sarcomas. Actin expression and complex histological structure helps to exclude hepatocellu‐ lar origin of the tumour. Complex structure, combined immunophenotype and low prolifer‐ ation help to exclude sarcoma [27].

**4.3. Epithelioid haemangioendothelioma**

is possible in our experience.

 

eration activity by Ki-67 (Figures 9-11).

Epithelioid haemangioendothelioma (EHE) is defined as intermediate-grade malignancy de‐ rived from endothelial cells. The mean age of patients is 47 years, ranging 12-86 years. The clinical picture can include symptoms related to enlarging mass in liver (abdominal pain, hepatomegaly) and tumour-related intoxication (fatigue, malaise, anorexia). Radiologically, the tumour can be found by computed tomography. EHE can be radiologically avascular [10,27,103]. This finding is probably related to fibrosis and scarcity of functioning blood ves‐ sels despite the endothelial origin of the tumour. Grossly, multiple tumours can involve liv‐ er or liver and lungs. In the lungs, epithelioid haemangioendothelioma is known also as intravascular bronchioloalveolar tumour. Despite the multifocality (Figure 8), slow progress

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Biopsy material is usually sufficient to diagnose the tumour. However, in our experience, immunohistochemical investigation is crucial in order to find out the presence of tumour cells on the background of stromal fibrosis and reactive inflammation, to detect the endothe‐ lial origin and to evaluate the low biological potential as reflected by low to moderate prolif‐

**Figure 8.** Multiple foci of epithelioid haemangioendothelioma in liver biopsy. The tumour is highlighted by immuno‐

histochemical visualisation of vimentin regarding its mesenchymal nature. IP, anti-vimentin, OM 50x.

**Figure 6.** Actin-positive smooth muscle component in angiomyolipoma. IP, anti-actin, OM 100x.

**Figure 7.** Expression of melanosome protein HMB-45 in angiomyolipoma. IP, anti-HMB-45, OM 400x.

### **4.3. Epithelioid haemangioendothelioma**

 

**Figure 6.** Actin-positive smooth muscle component in angiomyolipoma. IP, anti-actin, OM 100x.

132 Liver Biopsy - Indications, Procedures, Results

**Figure 7.** Expression of melanosome protein HMB-45 in angiomyolipoma. IP, anti-HMB-45, OM 400x.

Epithelioid haemangioendothelioma (EHE) is defined as intermediate-grade malignancy de‐ rived from endothelial cells. The mean age of patients is 47 years, ranging 12-86 years. The clinical picture can include symptoms related to enlarging mass in liver (abdominal pain, hepatomegaly) and tumour-related intoxication (fatigue, malaise, anorexia). Radiologically, the tumour can be found by computed tomography. EHE can be radiologically avascular [10,27,103]. This finding is probably related to fibrosis and scarcity of functioning blood ves‐ sels despite the endothelial origin of the tumour. Grossly, multiple tumours can involve liv‐ er or liver and lungs. In the lungs, epithelioid haemangioendothelioma is known also as intravascular bronchioloalveolar tumour. Despite the multifocality (Figure 8), slow progress is possible in our experience.

Biopsy material is usually sufficient to diagnose the tumour. However, in our experience, immunohistochemical investigation is crucial in order to find out the presence of tumour cells on the background of stromal fibrosis and reactive inflammation, to detect the endothe‐ lial origin and to evaluate the low biological potential as reflected by low to moderate prolif‐ eration activity by Ki-67 (Figures 9-11).

**Figure 8.** Multiple foci of epithelioid haemangioendothelioma in liver biopsy. The tumour is highlighted by immuno‐ histochemical visualisation of vimentin regarding its mesenchymal nature. IP, anti-vimentin, OM 50x.

**Figure 9.** Epithelioid haemangioendothelioma presenting as a fibrotic focus in liver biopsy. HE, OM 100x.

**Figure 11.** Expression of CD34 in epithelioid haemangioendothelioma. Note also the positive reaction in the lining of

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The tumour is growing within sinusoids and venules compressing the adjacent parenchyma. As was mentioned, the expression of endothelial markers is typical. Focal expression of cy‐ tokeratin and/or actin is possible [103] and should not cause confusion if panel of immunos‐ tains is performed. Stromal fibrosis follows than and can become marked so that neoplastic cells are obscured (Figure 9). Two cell types are described: epithelioid and dendritic. The morphological differential diagnosis includes non-neoplastic fibrosis and/or inflammation and granulation tissue, angiosarcoma and metastatic cancers with marked stromal fibrosis. The non-neoplastic conditions can be ruled out by tumour architecture as revealed by im‐ munohistochemistry. Epithelial tumours can be excluded by the predominance of endothe‐ lial markers by IHC. Among the vascular malignancies, the diagnosis of epithelioid haemangioendothelioma is preferred for lesions with low grade atypia, absence of frankly malignant spindle cells, low proliferation, limited destruction of surrounding liver tissue

Angiosarcoma is defined as malignant tumour of endothelial cells. Epidemiologically, it is characterised by rare occurrence in the liver constituting 2% of primary hepatic malignan‐ cies [11]. Elderly (50-60-year-old) males represent the largest group of affected patients [27]. The described risk factors include history of thorotrast use for arteriography, exposure to vi‐ nyl chloride in the plastics industry where it has been used for polymerisation, arsenic com‐ pounds (used as insecticides, possibly present in wine and used in the treatment of psoriasis), copper compounds, pesticides and other chemical carcinogens. In all cases, long

a venule. IP,anti-CD34, OM 400x.

and absence of necrosis.

**4.4. Angiosarcoma**

**Figure 10.** Loss of liver parenchyma due to infiltration of epithelioid haemangioendothelioma. IP, anti-cytokeratins AE1/AE3, OM 200x.

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**Figure 11.** Expression of CD34 in epithelioid haemangioendothelioma. Note also the positive reaction in the lining of a venule. IP,anti-CD34, OM 400x.

The tumour is growing within sinusoids and venules compressing the adjacent parenchyma. As was mentioned, the expression of endothelial markers is typical. Focal expression of cy‐ tokeratin and/or actin is possible [103] and should not cause confusion if panel of immunos‐ tains is performed. Stromal fibrosis follows than and can become marked so that neoplastic cells are obscured (Figure 9). Two cell types are described: epithelioid and dendritic. The morphological differential diagnosis includes non-neoplastic fibrosis and/or inflammation and granulation tissue, angiosarcoma and metastatic cancers with marked stromal fibrosis. The non-neoplastic conditions can be ruled out by tumour architecture as revealed by im‐ munohistochemistry. Epithelial tumours can be excluded by the predominance of endothe‐ lial markers by IHC. Among the vascular malignancies, the diagnosis of epithelioid haemangioendothelioma is preferred for lesions with low grade atypia, absence of frankly malignant spindle cells, low proliferation, limited destruction of surrounding liver tissue and absence of necrosis.

#### **4.4. Angiosarcoma**

**Figure 9.** Epithelioid haemangioendothelioma presenting as a fibrotic focus in liver biopsy. HE, OM 100x.

**Figure 10.** Loss of liver parenchyma due to infiltration of epithelioid haemangioendothelioma. IP, anti-cytokeratins

AE1/AE3, OM 200x.

134 Liver Biopsy - Indications, Procedures, Results

Angiosarcoma is defined as malignant tumour of endothelial cells. Epidemiologically, it is characterised by rare occurrence in the liver constituting 2% of primary hepatic malignan‐ cies [11]. Elderly (50-60-year-old) males represent the largest group of affected patients [27]. The described risk factors include history of thorotrast use for arteriography, exposure to vi‐ nyl chloride in the plastics industry where it has been used for polymerisation, arsenic com‐ pounds (used as insecticides, possibly present in wine and used in the treatment of psoriasis), copper compounds, pesticides and other chemical carcinogens. In all cases, long latent period (6-35 years) embarrass the data collection. The clinical picture can show signs and symptoms of liver damage (hepatomegaly, local pain, jaundice), disorders of blood cell function (anaemia, thrombocytopenia, disseminated intravascular coagulation), and tu‐ mour-related intoxication manifesting as weight loss. Ascites, bleeding into abdominal cavi‐ ty and liver failure is possible [27]. Grossly, multiple masses with signs of haemorrhage are present. Morphologically, the cellular atypia as well as vascular differentiation can be ob‐ served in variable extent. High-grade tumours exhibit solid growth with few vascular spaces. Immunohistochemically, endothelial markers CD31 and CD34 are expressed. How‐ ever, the immunophenotype can be not straightforward. In our experience, it is important to use several endothelial markers. At first, the reactivity can be uneven [27]. Even more, CD34 is technologically beneficial antibody characterised with high affinity. However, during the evaluation it is necessary to consider CD34 expression in non-endothelial tumours including gastrointestinal stromal tumour and solitary fibrous tumour, among others.

for liver biopsies. In the files of single university hospital, metastatic tumours constituted 45% of tumours or tumour-like liver lesions. Adenocarcinoma was the most frequent histo‐ logical type of metastases (65.5%) comprising metastases of colorectal (48.2%), pancreatic (13.5%), breast (13%), gastric (6.2%), lung (4.5%) and oesophageal cancer (3.7%). Neuroen‐ docrine carcinomas were seen frequently (16%). Lymphoma constituted 0.4% of all tu‐ mours [2]. Metastases in cirrhotic liver were rare [2]. In another study, including 130 cases of metastatic liver disease, gastrointestinal tract was found to be the most common pri‐ mary location (45.3%) of cancer metastasizing to liver followed by neuroendocrine tu‐ mours (10.7%) [104]. In children, neuroblastoma, nephroblastoma and rhabdomyosarcoma

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The spread to liver occurs in 5-10% of patients with Hodgkin's lymphoma and 15-40% of non-Hodgkin's lymphoma cases at the time of diagnosis. Leukemias can involve the liver as well. Grossly, large cell lymphoma can form masses similarly to carcinoma. In case of Hodg‐ kin's disease, the size of nodules is variable. Leukemic infiltrate can be present without visi‐ ble mass lesion. Myeloid leukemias preferentially infiltrate sinusoids, lymphoid – portal tracts, but hairy cell leukemia can involve both portal tracts and sinusoids forming small

Malignant melanoma (Figures 12-14) is one of the greatest challenges in diagnostic surgical pathology [105] due to amelanotic, clear cell, sarcomatoid, small cell, haemangiopericytoid, signet-ring cell, myxoid, metaplastic and rhabdoid forms. The diagnosis largely depends on IHC. Evaluating the intermediate filaments, melanoma expresses vimentin. Despite the re‐ ported concerns of cytokeratin expression in melanoma, this is rare event (3%) in formalinfixed tissues. Similarly, the expression of glial fibrillar acidic protein and actin is observed in 1% of melanomas [105]. Interspersed normal cells should be excluded from evaluation of cy‐ tokeratin and actin reactivity. Melanoma is characterised by nuclear and cytoplasmic expres‐ sion of S-100 protein in 97.4-98%. S-100 protein can be observed in carcinomas, histiocytic neoplasms and malignant peripheral nerve sheath tumour, therefore melanoma-specific an‐ tibodies, e.g., HMB-45 and MART-1/Melan-A must be included in the panel. Melanoma can express bcl-2, CD10, CD68, CD56, CD57, CD99, CD117 antigens leading to diagnostic confu‐ sion with lymphoma, renal cell cancer, hepatocellular cancer, GIST, seminoma and other ne‐ oplasms. Expression of Melan-A is found also in metastatic adrenocortical carcinoma (50-60%) that can be recognised by inhibin expression in around 70% of cases [6]. S-100,

are the most frequent source of metastases [103].

blood containing cavities, surrounded by neoplastic cells [103].

HMB-45, Melan-A and inhibin are absent from HCC [6].

### **5. Metastatic liver tumours**

In Western countries, metastatic tumours represent the most common malignant liver le‐ sion with the rate 94-98% among all malignant liver tumours [27]. Almost all malignant tumours, including carcinomas, sarcomas, melanomas and haematological malignancies, can secondary involve the liver by haematogenous, lymphogeneous or transperitoneal spread. Theoretically, metastatic tumour retains the morphological characteristics of the primary site. However, the balance between anaplasia and differentiation can shift towards anapla‐ sia in such degree that signs of differentiation towards specific tissue or cell type are hard‐ ly recognisable. Some tumours like squamous cell cancer and melanoma lack specifity regarding the organ of origin. Even adenocarcinomas retain few specific features. There‐ fore the differential diagnostics between primary and secondary liver tumours represents a complicated practical task. Clinical data can be absent if metastatic liver lesion presents as cancer of unknown origin. The diagnosis can be reached by logical analysis of morpholo‐ gy, IHC and molecular data. If the establishment of exact histogenesis is unsuccessful, the biopsy investigation should be directed towards the analysis of treatment possibilities. Path‐ ologist should comment in detail morphological and immunophenotype data that could either prove or disregard any particular type of treatment.

In case of liver metastasis, the primary tumour most frequently is located in colon, pancreas, stomach, breast, oesophagus, genitourinary organs [100, 103]. Lung cancer can metastasize to liver as well [6]. Neuroendocrine tumours, even small, can give rise to hepatic metastases. The clinical course in this case can be prolonged and occasionally characterised by carcinoid syndrome including flushing, diarrhoea and palpitations.

The spectrum of metastatic tumours in liver biopsies depend on the frequency of differ‐ ent tumours, the biological properties of different neoplasms predicting the possibility of metastatic spread to liver as well as by the medical paradigm considering the indications for liver biopsies. In the files of single university hospital, metastatic tumours constituted 45% of tumours or tumour-like liver lesions. Adenocarcinoma was the most frequent histo‐ logical type of metastases (65.5%) comprising metastases of colorectal (48.2%), pancreatic (13.5%), breast (13%), gastric (6.2%), lung (4.5%) and oesophageal cancer (3.7%). Neuroen‐ docrine carcinomas were seen frequently (16%). Lymphoma constituted 0.4% of all tu‐ mours [2]. Metastases in cirrhotic liver were rare [2]. In another study, including 130 cases of metastatic liver disease, gastrointestinal tract was found to be the most common pri‐ mary location (45.3%) of cancer metastasizing to liver followed by neuroendocrine tu‐ mours (10.7%) [104]. In children, neuroblastoma, nephroblastoma and rhabdomyosarcoma are the most frequent source of metastases [103].

latent period (6-35 years) embarrass the data collection. The clinical picture can show signs and symptoms of liver damage (hepatomegaly, local pain, jaundice), disorders of blood cell function (anaemia, thrombocytopenia, disseminated intravascular coagulation), and tu‐ mour-related intoxication manifesting as weight loss. Ascites, bleeding into abdominal cavi‐ ty and liver failure is possible [27]. Grossly, multiple masses with signs of haemorrhage are present. Morphologically, the cellular atypia as well as vascular differentiation can be ob‐ served in variable extent. High-grade tumours exhibit solid growth with few vascular spaces. Immunohistochemically, endothelial markers CD31 and CD34 are expressed. How‐ ever, the immunophenotype can be not straightforward. In our experience, it is important to use several endothelial markers. At first, the reactivity can be uneven [27]. Even more, CD34 is technologically beneficial antibody characterised with high affinity. However, during the evaluation it is necessary to consider CD34 expression in non-endothelial tumours including

In Western countries, metastatic tumours represent the most common malignant liver le‐ sion with the rate 94-98% among all malignant liver tumours [27]. Almost all malignant tumours, including carcinomas, sarcomas, melanomas and haematological malignancies, can secondary involve the liver by haematogenous, lymphogeneous or transperitoneal spread. Theoretically, metastatic tumour retains the morphological characteristics of the primary site. However, the balance between anaplasia and differentiation can shift towards anapla‐ sia in such degree that signs of differentiation towards specific tissue or cell type are hard‐ ly recognisable. Some tumours like squamous cell cancer and melanoma lack specifity regarding the organ of origin. Even adenocarcinomas retain few specific features. There‐ fore the differential diagnostics between primary and secondary liver tumours represents a complicated practical task. Clinical data can be absent if metastatic liver lesion presents as cancer of unknown origin. The diagnosis can be reached by logical analysis of morpholo‐ gy, IHC and molecular data. If the establishment of exact histogenesis is unsuccessful, the biopsy investigation should be directed towards the analysis of treatment possibilities. Path‐ ologist should comment in detail morphological and immunophenotype data that could

In case of liver metastasis, the primary tumour most frequently is located in colon, pancreas, stomach, breast, oesophagus, genitourinary organs [100, 103]. Lung cancer can metastasize to liver as well [6]. Neuroendocrine tumours, even small, can give rise to hepatic metastases. The clinical course in this case can be prolonged and occasionally characterised by carcinoid

The spectrum of metastatic tumours in liver biopsies depend on the frequency of differ‐ ent tumours, the biological properties of different neoplasms predicting the possibility of metastatic spread to liver as well as by the medical paradigm considering the indications

gastrointestinal stromal tumour and solitary fibrous tumour, among others.

either prove or disregard any particular type of treatment.

syndrome including flushing, diarrhoea and palpitations.

**5. Metastatic liver tumours**

136 Liver Biopsy - Indications, Procedures, Results

The spread to liver occurs in 5-10% of patients with Hodgkin's lymphoma and 15-40% of non-Hodgkin's lymphoma cases at the time of diagnosis. Leukemias can involve the liver as well. Grossly, large cell lymphoma can form masses similarly to carcinoma. In case of Hodg‐ kin's disease, the size of nodules is variable. Leukemic infiltrate can be present without visi‐ ble mass lesion. Myeloid leukemias preferentially infiltrate sinusoids, lymphoid – portal tracts, but hairy cell leukemia can involve both portal tracts and sinusoids forming small blood containing cavities, surrounded by neoplastic cells [103].

Malignant melanoma (Figures 12-14) is one of the greatest challenges in diagnostic surgical pathology [105] due to amelanotic, clear cell, sarcomatoid, small cell, haemangiopericytoid, signet-ring cell, myxoid, metaplastic and rhabdoid forms. The diagnosis largely depends on IHC. Evaluating the intermediate filaments, melanoma expresses vimentin. Despite the re‐ ported concerns of cytokeratin expression in melanoma, this is rare event (3%) in formalinfixed tissues. Similarly, the expression of glial fibrillar acidic protein and actin is observed in 1% of melanomas [105]. Interspersed normal cells should be excluded from evaluation of cy‐ tokeratin and actin reactivity. Melanoma is characterised by nuclear and cytoplasmic expres‐ sion of S-100 protein in 97.4-98%. S-100 protein can be observed in carcinomas, histiocytic neoplasms and malignant peripheral nerve sheath tumour, therefore melanoma-specific an‐ tibodies, e.g., HMB-45 and MART-1/Melan-A must be included in the panel. Melanoma can express bcl-2, CD10, CD68, CD56, CD57, CD99, CD117 antigens leading to diagnostic confu‐ sion with lymphoma, renal cell cancer, hepatocellular cancer, GIST, seminoma and other ne‐ oplasms. Expression of Melan-A is found also in metastatic adrenocortical carcinoma (50-60%) that can be recognised by inhibin expression in around 70% of cases [6]. S-100, HMB-45, Melan-A and inhibin are absent from HCC [6].

**Figure 12.** Diffuse sinusoidal spread of undifferentiated malignant tumour. By immunohistochemistry, metastatic melanoma was revealed (see also Figure 13). HE, OM 400 x.

**Figure 14.** Lack of cytokeratins AE1/AE3 in metastatic melanoma. Note the unusual sinusoidal spread. IP, anti-AE1/

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Metastatic breast cancer expresses CK7 but not CK20. However, this immunophenotype is shared by many adenocarcinomas. To identify the tumour as metastasis from breast primary tumour, gross cystic disease fluid protein fraction-15 (GCDFP-15) and/or mammaglobin can be detected. The specifity of GCDFP-15 is estimated as 99%, and the sensitivity ranges from 50 to 74%. Breast cancers of luminal molecular type express oestrogen (ER) and progester‐ one receptors (PR). Naturally, the expression of female steroid hormone receptors is shared by ovarian and endometrial cancer. Nowadays the detection of ER and PR is routine in breast cancer diagnostics but less experience is obtained with expression of hormone recep‐ tors in extra-genital carcinomas. The scientific studies report expression of ER in carcinoma of lung, stomach and thyroid [105]. The cross-reactivity can be associated by certain anti‐ body clones. Also, HER-2 positive and triple negative molecular types of breast cancer are more prone to develop visceral metastases. Thus, negative ER/PR expression cannot exclude metastatic breast cancer, and positive findings should be interpreted with caution recognis‐ ing the possibility of metastatic ovarian or endometrial cancer and cross-reactivity or true expression of hormone receptors in extra-genital tumour. ER/PR expression in lung or thy‐ roid tumour can be controlled by TTF-1 protein expression and/or evaluation for neuroen‐

AE3, OM 400x.

docrine markers and calcitonin.

**Figure 13.** Intense perinuclear expression of melanosome protein HMB-45 in metastatic melanoma. IP, anti-HMB-45, OM 400x.

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**Figure 14.** Lack of cytokeratins AE1/AE3 in metastatic melanoma. Note the unusual sinusoidal spread. IP, anti-AE1/ AE3, OM 400x.

**Figure 12.** Diffuse sinusoidal spread of undifferentiated malignant tumour. By immunohistochemistry, metastatic

**Figure 13.** Intense perinuclear expression of melanosome protein HMB-45 in metastatic melanoma. IP, anti-HMB-45,

melanoma was revealed (see also Figure 13). HE, OM 400 x.

138 Liver Biopsy - Indications, Procedures, Results

OM 400x.

Metastatic breast cancer expresses CK7 but not CK20. However, this immunophenotype is shared by many adenocarcinomas. To identify the tumour as metastasis from breast primary tumour, gross cystic disease fluid protein fraction-15 (GCDFP-15) and/or mammaglobin can be detected. The specifity of GCDFP-15 is estimated as 99%, and the sensitivity ranges from 50 to 74%. Breast cancers of luminal molecular type express oestrogen (ER) and progester‐ one receptors (PR). Naturally, the expression of female steroid hormone receptors is shared by ovarian and endometrial cancer. Nowadays the detection of ER and PR is routine in breast cancer diagnostics but less experience is obtained with expression of hormone recep‐ tors in extra-genital carcinomas. The scientific studies report expression of ER in carcinoma of lung, stomach and thyroid [105]. The cross-reactivity can be associated by certain anti‐ body clones. Also, HER-2 positive and triple negative molecular types of breast cancer are more prone to develop visceral metastases. Thus, negative ER/PR expression cannot exclude metastatic breast cancer, and positive findings should be interpreted with caution recognis‐ ing the possibility of metastatic ovarian or endometrial cancer and cross-reactivity or true expression of hormone receptors in extra-genital tumour. ER/PR expression in lung or thy‐ roid tumour can be controlled by TTF-1 protein expression and/or evaluation for neuroen‐ docrine markers and calcitonin.

**Figure 16.** Small cell cancer. Note the "salt-and-pepper" chromatin and high mitotic activity. HE, OM 400x.

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**Figure 17.** Granular cytoplasmic and perinuclear expression of chromogranin A in small cell cancer. IP, anti-chromog‐

ranin A, OM 400x.

**Figure 15.** Surfactant A in pulmonary adenocarcinoma. IP, anti-surfactant apoprotein A, OM 400x.

Adenocarcinoma, squamous cell cancer, small cell cancer and carcinoid are the most fre‐ quent lung neoplasms. Lung adenocarcinoma is characterised by expression of CK7 (100%) and TTF-1 (60-75%). Expression of CK20 is rare. Cytokeratins 5/6 and 34betaE12 can be present but are not dominant in comparison with CK7. Vimentin can be found in lung adenocarcinomas. Nuclear expression of TTF-1 and/or cytoplasmic expression of surfac‐ tant apoprotein A (Figure 15) is an evidence of pulmonary origin. Small cell cancer express‐ es neuroendocrine markers and pan-cytokeratin. The expression of chromogranin A and CK AE1/AE3 can be limited to perinuclear dot reactivity. Simultaneous detection of leukocyte common antigen can be suggested to perform differential diagnosis with haematological neoplasm. Nuclear expression of TTF-1 protein is frequently present (Figures 16-18). The high proliferation fraction by Ki-67 is characteristic albeit unspecific. The immunopheno‐ type of squamous cell cancer is unspecific and characterised by cytoplasmic expression of CK5/6 and CK 34betaE12 in association with strong nuclear reactivity with p63 protein. CK7 can be present but is not dominant. TTF-1 protein is absent. Carcinoid is characterised by neuroendocrine differentiation and low proliferative activity. The TTF-1 expression is not frequent [17,105-107].

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**Figure 16.** Small cell cancer. Note the "salt-and-pepper" chromatin and high mitotic activity. HE, OM 400x.

**Figure 15.** Surfactant A in pulmonary adenocarcinoma. IP, anti-surfactant apoprotein A, OM 400x.

frequent [17,105-107].

140 Liver Biopsy - Indications, Procedures, Results

Adenocarcinoma, squamous cell cancer, small cell cancer and carcinoid are the most fre‐ quent lung neoplasms. Lung adenocarcinoma is characterised by expression of CK7 (100%) and TTF-1 (60-75%). Expression of CK20 is rare. Cytokeratins 5/6 and 34betaE12 can be present but are not dominant in comparison with CK7. Vimentin can be found in lung adenocarcinomas. Nuclear expression of TTF-1 and/or cytoplasmic expression of surfac‐ tant apoprotein A (Figure 15) is an evidence of pulmonary origin. Small cell cancer express‐ es neuroendocrine markers and pan-cytokeratin. The expression of chromogranin A and CK AE1/AE3 can be limited to perinuclear dot reactivity. Simultaneous detection of leukocyte common antigen can be suggested to perform differential diagnosis with haematological neoplasm. Nuclear expression of TTF-1 protein is frequently present (Figures 16-18). The high proliferation fraction by Ki-67 is characteristic albeit unspecific. The immunopheno‐ type of squamous cell cancer is unspecific and characterised by cytoplasmic expression of CK5/6 and CK 34betaE12 in association with strong nuclear reactivity with p63 protein. CK7 can be present but is not dominant. TTF-1 protein is absent. Carcinoid is characterised by neuroendocrine differentiation and low proliferative activity. The TTF-1 expression is not

**Figure 17.** Granular cytoplasmic and perinuclear expression of chromogranin A in small cell cancer. IP, anti-chromog‐ ranin A, OM 400x.

 

Metastatic colorectal carcinoma can be recognised by diffuse intensive cytoplasmic expres‐ sion of CK20 and nuclear expression of CDX2 [108]. Carcinoid of the midgut and hindgut

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Neuroendocrine tumours are characterised by strong cytoplasmic expression of chromogra‐ nin A and synaptophysin and negativity for Hep Par 1 [6]. CD56 is considered to be the most sensitive neuroendocrine marker. In our experience, it shows reliable performance in small or compressed biopsies making it especially valuable tool for the evaluation of scant

Renal cell carcinoma is characterised by negativity for Hep Par 1 and CEA expression (by polyclonal anti-CEA antibody). Unfortunately, the rate of RCC expression decreases from 50-80% in primary clear cell renal carcinoma and 60-90% in papillary renal cell cancer to 20% in metastatic renal cell carcinoma. CD10 can be present both in HCC and renal cell carcinoma. Although the pattern of expression is different this can be difficult to evaluate, especially in core biopsy. PAX-2 is advised as marker of metastatic renal cell carcinoma with the expression rate 70-80%. Expression of vimentin is more characteristic in clear cell re‐ nal carcinoma (60-70%) than in hepatocellular carcinoma; chromophobe renal cell carcino‐

As tumour heterogeneity remains a source of problems [69] and the immunophenotype can be inherently complex and subjected to cross reactivity, we recommend wide IHC panels in‐ cluding several markers for HCC and cholangiocarcinoma as well as markers for metastatic

tissue material. Occasional CD56 expression in HCC is described [6].

tumour, including the organospecific antigens (see Tables 1-2).

Small cell cancer CK AE1/AE3 + ChrA+ CD56 +TTF-1 + Squamous cancer CK34betaE12+ CK7-/+ CK20 – p63+

Breast cancer CK 7 + CK20 – MG +/– ER +/– PR +/–

mammaglobin; ER, oestrogen receptor; PR, progesterone receptor

Antigen Valuable positive expression Notes

Colorectal cancer CK20+ CK7- CDX2+ TTF-1-

Malignant melanoma Vim + CK AE1 / AE3 – S-100 + HMB-45 + MART-1 / Melan A + Lung adenocarcinoma CK7+ CK20- CK34betaE12-/+ TTF-1+ Surfactant apoprotein A +

NET CK AE1/AE3 + ChrA+ TTF-1 +/ - (lung) or CDX2 + (midgut, hindgut)

**Table 1.** The immunophenotype of selected malignant tumours. Abbreviations in the Table: Vim, vimentin; CK, cytokeratin; TTF-1, thyroid transcription factor 1; ChrA, chromogranin A; NET, neuroendocrine tumour; MG,

Glypican-3 Hepatocellular carcinoma Occasional positivity in non-hepatocellular tumour Arginase-1 Hepatocellular carcinoma Sensitivity for hepatocellular carcinoma 96%

Normal liver tissue positive

Metastatic tumours rarely positive [69]

Tumour Immunophenotype

also are positive for CDX2 [109].

ma also is negative [6].

**Figure 18.** Nuclear TTF-1 expression in small cell cancer. IP, anti-chromogranin A, OM 400x.

Mesothelioma is characterised by expression of CK7, CK5/6, vimentin and calretinin (Figure 19). HBME-1 can be expressed as well but lacks specifity.

**Figure 19.** Nuclear and cytoplasmic expression of calretinin in epithelioid mesothelioma.IP, anti-calretinin, OM 400x.

Metastatic colorectal carcinoma can be recognised by diffuse intensive cytoplasmic expres‐ sion of CK20 and nuclear expression of CDX2 [108]. Carcinoid of the midgut and hindgut also are positive for CDX2 [109].

Neuroendocrine tumours are characterised by strong cytoplasmic expression of chromogra‐ nin A and synaptophysin and negativity for Hep Par 1 [6]. CD56 is considered to be the most sensitive neuroendocrine marker. In our experience, it shows reliable performance in small or compressed biopsies making it especially valuable tool for the evaluation of scant tissue material. Occasional CD56 expression in HCC is described [6].

Renal cell carcinoma is characterised by negativity for Hep Par 1 and CEA expression (by polyclonal anti-CEA antibody). Unfortunately, the rate of RCC expression decreases from 50-80% in primary clear cell renal carcinoma and 60-90% in papillary renal cell cancer to 20% in metastatic renal cell carcinoma. CD10 can be present both in HCC and renal cell carcinoma. Although the pattern of expression is different this can be difficult to evaluate, especially in core biopsy. PAX-2 is advised as marker of metastatic renal cell carcinoma with the expression rate 70-80%. Expression of vimentin is more characteristic in clear cell re‐ nal carcinoma (60-70%) than in hepatocellular carcinoma; chromophobe renal cell carcino‐ ma also is negative [6].

As tumour heterogeneity remains a source of problems [69] and the immunophenotype can be inherently complex and subjected to cross reactivity, we recommend wide IHC panels in‐ cluding several markers for HCC and cholangiocarcinoma as well as markers for metastatic tumour, including the organospecific antigens (see Tables 1-2).

**Figure 18.** Nuclear TTF-1 expression in small cell cancer. IP, anti-chromogranin A, OM 400x.

19). HBME-1 can be expressed as well but lacks specifity.

Mesothelioma is characterised by expression of CK7, CK5/6, vimentin and calretinin (Figure

**Figure 19.** Nuclear and cytoplasmic expression of calretinin in epithelioid mesothelioma.IP, anti-calretinin, OM 400x.

142 Liver Biopsy - Indications, Procedures, Results

 


**Table 1.** The immunophenotype of selected malignant tumours. Abbreviations in the Table: Vim, vimentin; CK, cytokeratin; TTF-1, thyroid transcription factor 1; ChrA, chromogranin A; NET, neuroendocrine tumour; MG, mammaglobin; ER, oestrogen receptor; PR, progesterone receptor



CD56 NET, cholangiocarcinoma Other tumours can be positive

GCDFP-15 Breast cancer Sensitivity for breast cancer 50-60%

Non-gynaecologic cancers can be occasionally positive,

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Ovarian (17%), endometrial (40-70%) and endocervical

Not associated with mammaglobin thus simultaneous

few breast carcinomas and rectal NETs have been observed

Sensitivity for thyroid cancer 79-100% and for renal cancer

Breast cancer is negative. Positivity is useful to discriminate

breast cancer from ovarian or endometrial cancer

including lung cancer (4-15-67%)

PLAP is co-expressed in germ cell tumours

(30%) carcinoma can also be positive [69]

evaluation can be recommended [69]

Sensitivity for breast cancer 40-85%

CD34 is co-expressed in GISTs

High heterogeneity

NETs can be positive

PSA Prostatic cancer Negative in 5% high-grade prostate cancers. Reactivity in

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[69]

**Table 2.** Panel of antibodies for liver biopsy evaluation. Abbreviations in the Table: AFP, alpha-fetoprotein; CK, cytokeratin; NET, neuroendocrine tumour; TTF-1, thyroid transcription factor 1; GIST, gastrointestinal stromal tumour; PLAP, placental alkaline phosphatase; GCDFP-15, gross cystic disease fluid protein-15; PSA, prostate specific antigen

The cystic biliary tumours are defined by cystic structure and development of / differentia‐ tion towards intrahepatic bile duct epithelium. The group includes malignant biliary cysta‐ denocarcinoma and benign biliary cystadenoma. Epidemiologically, cystic biliary tumours represent rare entities, with incidence of biliary cystadenocarcinoma approximately 1/10 million (corresponding to 0.01/100 000) and of biliary cystadenoma 1/100 000 - 5/100 000 [110]. Biliary cystadenocarcinoma is diagnosed mostly at the age 50-60 years [100]. Biliary cystadenoma is diagnosed in younger patients: mean age 40.6 (range 30-51) vs. 51.3 (range 41-63) years in biliary cystadenocarcinoma group [14]. In other studies even larger age dif‐ ference (17 years) is found between patients affected by benign and malignant cystic biliary tumours, respectively [111-112]. Cystic biliary tumours are more common in women: 80-100% of biliary adenoma and 63-71.4% of biliary cystadenocarcinoma are described in fe‐

71-98%

Breast, ovarian or endometrial cancer,

Mammaglobin Breast cancer High heterogeneity

Female genital tract carcinomas

Renal cell cancer

Urothelial carcinoma

endometrial stromal sarcoma

Seminoma

Oestrogen and progesterone receptors

CD117 GIST

Pax8 Thyroid cancer

P63 Squamous cell cancer

**6. Cystic biliary tumours**

synaptophysin


**Table 2.** Panel of antibodies for liver biopsy evaluation. Abbreviations in the Table: AFP, alpha-fetoprotein; CK, cytokeratin; NET, neuroendocrine tumour; TTF-1, thyroid transcription factor 1; GIST, gastrointestinal stromal tumour; PLAP, placental alkaline phosphatase; GCDFP-15, gross cystic disease fluid protein-15; PSA, prostate specific antigen

## **6. Cystic biliary tumours**

Hep Par 1 Hepatocellular carcinoma Sensitivity for hepatocellular carcinoma around 50% [69]

AFP Hepatocellular carcinoma Sensitivity for hepatocellular carcinoma around 15% [69]

liver

CK20 Metastatic colorectal cancer Useful in conjuction with CK7 for initial grouping of cancers

CDX2 Metastatic colorectal cancer and NETs Heterogeneous focal expression in gastric and pancreatic carcinomas

reported

apoprotein A

positive [69]

Hepatocellular carcinoma Expression in benign liver parenchyma is present

CD10 Renal cell carcinoma

144 Liver Biopsy - Indications, Procedures, Results

CK7 Cholangiocellular carcinoma Metastatic cancers

CK17 Cholangiocellular carcinoma Metastatic cancers

CK19 Cholangiocellular carcinoma Metastatic cancers

Calretinin Mesothelioma

Surfactant apoprotein A

TTF-1, nuclear expression

TTF-1, cytoplasmic expression:

Chromogranin A

NET

synaptophysin

and

Hepatocellular carcinoma

Adrenal cortical carcinoma

genital tract

useful, if positive

anaplastic carcinoma [69]

Sex cord-stromal tumours of the

Lung adenocarcinoma. In our experience possess high affinity and is

Metastatic pulmonary adenocarcinoma (75% of nonmucinous type and 10% of mucinous type), small cell cancer (pulmonary, 50-90%; non-pulmonary, 44-80%) or thyroid cancer including papillary, follicular and medullary but not

Gastric carcinoma can be positive

Negative in adrenal carcinoma

cancer, hepatocellular carcinoma)

showing adenocarcinomatous structure

Mucinous ovarian cancers can be positive Morules in endometrioid carcinoma are positive

Squamous carcinoma frequently positive

Reactivity in thyroid cancer (43% in small group) has been

 Regarding pulmonary adenocarcinoma, less subjected to heterogeneity-related evaluation problems than surfactant

Endometrial (17%) or breast (2.4%) cancer occasionally

Gastric or prostatic cancer can show cytoplasmic positivity

Heterogeneous expression has been observed [69]

Positivity does not exclude hepatocellular carcinoma Valuable for primary evaluation of malignant tumour within

Positivity does not exclude hepatocellular carcinoma

Positive tumours as pancreatic cancer (58%), squamous carcinoma (75%), urothelial carcinoma (75%) and cholangiocellular carcinoma (35%) can be distinguished from negative ones (gastric, colorectal, prostate, breast

> The cystic biliary tumours are defined by cystic structure and development of / differentia‐ tion towards intrahepatic bile duct epithelium. The group includes malignant biliary cysta‐ denocarcinoma and benign biliary cystadenoma. Epidemiologically, cystic biliary tumours represent rare entities, with incidence of biliary cystadenocarcinoma approximately 1/10 million (corresponding to 0.01/100 000) and of biliary cystadenoma 1/100 000 - 5/100 000 [110]. Biliary cystadenocarcinoma is diagnosed mostly at the age 50-60 years [100]. Biliary cystadenoma is diagnosed in younger patients: mean age 40.6 (range 30-51) vs. 51.3 (range 41-63) years in biliary cystadenocarcinoma group [14]. In other studies even larger age dif‐ ference (17 years) is found between patients affected by benign and malignant cystic biliary tumours, respectively [111-112]. Cystic biliary tumours are more common in women: 80-100% of biliary adenoma and 63-71.4% of biliary cystadenocarcinoma are described in fe‐

male [14]. The clinical picture reflects the presence of mass lesion and is dominated by ab‐ dominal pain [113]. The other manifestations and complications include jaundice, cholangitis, tumour rupture [114], haemorrhage [115], compression of the portal or caval veins with possible subsequent ascites [113], hemobilia [12] and mucobilia [116]. Notably, the tumour can progress slowly [117] with the clinical history of biliary cystadenocarcinoma as long as 10-15 years [112,118]. The long course is is in accordance with the low grade of malignancy and gradual development of tumour through stages of increased epithelial pro‐ liferation, dysplasia, *in situ* cancer and, finally, invasive cancer. Thus, long anamnesis of cystic hepatic mass does not exclude the possibility of malignant tumour and the need for careful follow-up if the cyst is not removed by operation. Although biopsy can be consid‐ ered in cases with unclear differential diagnosis, it is not the first choice because of the fol‐ lowing considerations. First, simple liver cyst is the main differential diagnosis of cystic biliary tumours. Although biliary cystadenocarcinoma is rare, liver cysts have high preva‐ lence being present in 2.5% of the population [119] and cannot be distinguished from cystic biliary tumours on the basis of CA19-9 and CEA levels [14,114]. However, core biopsy is un‐ likely to yield sufficient tissue in case of simple cyst or cystadenoma; it also is not suitable for the diagnostics of focal malignancy and rarely can lead to peritoneal carcinomatosis [13]. Therefore radiological diagnostics, especially computed tomography, is essential [117]. Grossly, biliary cystadenocarcinoma is multicystic. Internal mural nodules are irregularly distributed in the walls. The tumour most frequently is located within the liver (83%). Extrahepatic bile ducts (13%) or the gall bladder (0.02%) has been affected by this tumour as well [14]. The size of cystic biliary tumours (1.5-30 cm) is not helpful in the differential diagnos‐ tics between simple hepatic cyst and cystic biliary tumours; it also has no correlation with malignant biological potential [120]. The metastatic spread of biliary cystadenocarcinoma can affect the liver, regional lymph nodes in the hepatoduodenal ligament, lungs, pleura or peritoneum [100]. Histologically, biliary cystadenocarcinoma is characterised by clear-cut signs of malignancy: cellular atypia, particularly nuclear polymorphism, mitotic activity and invasion into surrounding stroma. The tumour architecture is cystic and papillary. The be‐ nign counterpart of biliary cystadenocarcinoma, the biliary cystadenoma lacks the malig‐ nant features [100] and is composed by either mucinous or serous benign epithelium. Most of cystic biliary tumours possess characteristic mesenchymal, ovarian–type stroma. Hypo‐ thetically, these tumours arise from bile ducts proximal to the hilum of the liver and share the cystic structure and presence of peculiar ovarian-type mesenchymal stroma with muci‐ nous cystic tumours of the pancreas and retroperitoneum, leading to the hypothesis that ec‐ topic ovarian stroma during embryogenesis can become incorporated along the biliary tree, in the pancreas and retroperitoneal space and cause the proliferation of the adjacent epitheli‐ um by production of the hormones and growth factors [121]. Origin from intrahepatic peri‐ biliary glands [122] or from ectopic rests of primitive foregut sequestered in the liver [114] has been hypothesised. Development from pluripotential stem cells is suggested on the ba‐ sis of the presence of albumin messenger RNA and biliary type cytokeratins in the tumour cells [123]. Biliary cystadenocarcinoma without mesenchymal stroma more frequently arises in males and carries poorer prognosis in comparison with the tumour possessing mesenchy‐ mal stroma [122]. By immunohistochemistry, increasing proliferative activity by Ki-67 ex‐

pression as well as increasing p53 protein expression from adenoma to carcinoma was shown in biliary cystadenocarcinoma without ovarian-type stroma [124]. Expression of cyto‐ keratin (CK) 7 and absence of CK20, CEA, alpha-fetoprotein, calretinin, CD31 and chromog‐ ranin is described [125]. However, presence of CK20, although typical for colorectal cancer, is described in cholangiocarcinoma, especially non-peripheral [126]. It might be expected in

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There is evidence showing that at least some cases of biliary cystadenocarcinoma originate from pre-existing biliary cystadenoma. These data include the age difference between biliary cystadenocarcinoma and biliary adenoma patients [14] as well as morphologic findings of

Radiologically, presence of internal septations allows excluding a simple cyst. Vascularity of septa is characteristic for cystic biliary tumours [14] and is considered by some specialists to be more reliable in distinguishing biliary cystadenoma from cyst than the simple presence of septations [117]. Biliary cystadenoma is characterised by smooth and thin internal septa, but presence of enhanced mural nodules in the outer wall or septa is the most important sign of malignancy. Calcification is not frequent but has been found specific for malignancy by some [14] but not all [119] authors as far as cystic biliary tumours are concerned. Size, num‐ ber of septations or location of the neoplasm does not help to differentiate between benign or malignant cystic biliary tumours [14]. Some authors have postulated that preoperative differentiation between biliary adenoma and cystadenocarcinoma by radiologic imaging is not possible therefore liver resection should be performed for all cystic biliary tumours [120]. This assumption is based on the experience that internal papillae with arterial en‐ hancement may be present in both tumours so that computed tomography and magnetic

The clinical differential diagnosis of cystic liver lesions, entering the differential diagnosis of biliary cystadenocarcinoma, include developmental, neoplastic, inflammatory and traumatic lesions as simple bile duct cyst, polycystic liver disease, biliary hamartoma, cystically degen‐ erated cases of other primary or metastatic liver tumours, abscesses, hydatid cyst, extrap‐

In conclusion, wide variety of neoplastic processes can affect the liver. Most of non-cystic tumours can be reliably diagnosed in liver biopsy. Several demographic and clinical data should be submitted along with the liver biopsy. Patient's age and presence or absence of clinical symptoms must be known. If there is history of contraceptive use it should be report‐ ed. Radiological data have high relevance: the size, localisation in respect to liver capsule and number of focal liver lesions should be known to the pathologist. The vascularity should be described. Knowing these data, pathologist should evaluate the haematoxylin-eosin stained

specimen. Wide panel of immunohistochemical stains can be recommended than.

biliary cystadenocarcinoma with growing awareness about this entity.

malignant transformation in a lesion with focally innocuous structure [127].

resonance imaging yield overlapping data.

**7. Conclusions**

ancreatic pseudocyst, hematoma and biloma [119,128].

pression as well as increasing p53 protein expression from adenoma to carcinoma was shown in biliary cystadenocarcinoma without ovarian-type stroma [124]. Expression of cyto‐ keratin (CK) 7 and absence of CK20, CEA, alpha-fetoprotein, calretinin, CD31 and chromog‐ ranin is described [125]. However, presence of CK20, although typical for colorectal cancer, is described in cholangiocarcinoma, especially non-peripheral [126]. It might be expected in biliary cystadenocarcinoma with growing awareness about this entity.

There is evidence showing that at least some cases of biliary cystadenocarcinoma originate from pre-existing biliary cystadenoma. These data include the age difference between biliary cystadenocarcinoma and biliary adenoma patients [14] as well as morphologic findings of malignant transformation in a lesion with focally innocuous structure [127].

Radiologically, presence of internal septations allows excluding a simple cyst. Vascularity of septa is characteristic for cystic biliary tumours [14] and is considered by some specialists to be more reliable in distinguishing biliary cystadenoma from cyst than the simple presence of septations [117]. Biliary cystadenoma is characterised by smooth and thin internal septa, but presence of enhanced mural nodules in the outer wall or septa is the most important sign of malignancy. Calcification is not frequent but has been found specific for malignancy by some [14] but not all [119] authors as far as cystic biliary tumours are concerned. Size, num‐ ber of septations or location of the neoplasm does not help to differentiate between benign or malignant cystic biliary tumours [14]. Some authors have postulated that preoperative differentiation between biliary adenoma and cystadenocarcinoma by radiologic imaging is not possible therefore liver resection should be performed for all cystic biliary tumours [120]. This assumption is based on the experience that internal papillae with arterial en‐ hancement may be present in both tumours so that computed tomography and magnetic resonance imaging yield overlapping data.

The clinical differential diagnosis of cystic liver lesions, entering the differential diagnosis of biliary cystadenocarcinoma, include developmental, neoplastic, inflammatory and traumatic lesions as simple bile duct cyst, polycystic liver disease, biliary hamartoma, cystically degen‐ erated cases of other primary or metastatic liver tumours, abscesses, hydatid cyst, extrap‐ ancreatic pseudocyst, hematoma and biloma [119,128].
