3.2. Well-differentiated hepatocellular carcinoma versus adenoma

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

exceeding the diameter of 1 cm [6]. Morphologically DNs are classified into high-grade DN and low-grade DN. Low-grade DN, carrying low risk of transformation to HCC, is generally characterised by monotonous cell population when compared with the surrounding cirrhotic liver, mildly increased cell density and minimal cell atypia. The nuclear/cytoplasmic ratio is mildly increased, nuclear atypia is slight, mitoses are absent and cell plates are 1–2 cells thick. The reticulin network is retained. The borders of low-grade dysplastic nodule are rounded, but the adjacent liver parenchyma is not compressed [3]. In contrast, high-grade dysplastic nodules can have many of classical HCC features. The nuclear/cytoplasmic ratio is increased. Nuclei show hyperchromasia and irregular borders and can be peripherally located. Occasional mitoses can be present. Cell plates are thicker than 2 cells. Cytoplasm switches to basophilic staining. Pseudoglandular structures start to appear. Occasional unpaired arteries have been observed. Lack of invasion is the most reliable criterion in the differential diagnosis with early HCC [3]. This trait is both important and biologically substantiated as the invasion is the hallmark of malignant tumours. However, it can be notoriously difficult to apply practically. In early HCC, invasion can be absent from biopsy due to sampling error. Regarding highgrade dysplastic nodule, entrapment of perinodular hepatocytes into fibrous tissues mimics invasion. To classify the entrapped hepatocytes correctly, immunohistochemical investigation of ductular proliferation can be helpful, as further described, because these non-neoplastic

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

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

33

intraseptal hepatocytes and ductular proliferation stem from common progenitors [3].

of this panel is estimated as 60–78% [55].

Expression of GPC3 points towards malignant hepatocellular tumour, as it was previously noted. However, GPC3 expression has been reported in 3–76% of dysplastic nodules. Glutamine synthetase is expressed in 69.8% of HCC contrasting with 13.6% in high-grade DN. Heat shock protein 70 is found in 73.5% of HCC and only exceptional dysplastic nodules [3]. To distinguish high-grade DN from early HCC, immunohistochemical panel comprising heat shock protein 70, glypican-3 and glutamine synthetase has been recommended. Expression of one marker is compatible with DN, while HCC expresses at least two markers. The sensitivity

In addition, cytokeratin (CK) 7 and/or CK19 and CD34 can be useful in the assessment of architecture and reactive changes. HCC is characterised by more diffuse expression of CD34 and loss of ductular reaction at the nodule interface. Dysplastic nodule shows only focal CD34 expression in the periphery of the nodule and more marked proliferation of CK7-positive ductules surrounding DN [55]. In the ductular reaction, CK7 and CK19 usually are coexpressed. Thus, gradual loss of CK7 and CK19 positive ductular reaction in perinodular stroma correlates with progression of cirrhotic to dysplastic nodule and further to HCC. Ductular reaction is

Different systems for complex evaluation of the biological potential of hepatocellular nodule have been proposed. Integrated evaluation of haematoxylin-eosin findings together with reticulin stain and immunohistochemistry for CD34 has been suggested. A hepatocellular nodule should be classified as HCC if at least three features from the following are present: necrosis; cellular atypia; thickness of trabeculae more than 4 cells; mitotic activity or diffuse expression of CD34 in the sinusoidal endothelium [6]. Alternatively, stromal invasion, loss of reticulin network and positivity for at least two out of three markers (HSP70, GS, GPC3)

present around ≥50% of perimeter of a DN, while it is almost lost in HCC [3].

Several molecular subtypes of hepatocellular adenomas are known [62, 64], including hepatocyte nuclear factor 1α (HNF1α) inactivated type (H-HCA); β-catenin activated type (B-HCA); inflammatory HCA (I-HCA) and the unclassifiable type (U-HCA). Not surprisingly, beta-catenin activated subtype is associated with malignant transformation [62]. Beta-catenin mutations are reported in 20% of HCCs, especially in patients with underlying hepatitis C virus infection. HCC arising from B-HCA is usually well to moderately differentiated and lacks vascular invasion or satellite nodules [3]. Mutations lead to remarkable overexpression of GLUL gene (coding for glutamine synthase), thus beta-catenin activation can be assessed by intense homogeneous cytoplasmic expression of glutamine synthase and by aberrant nuclear localisation of beta-catenin [62, 63]. H-HCA shows decreased expression of liver fatty acid-binding protein, and presence of fat in neoplastic cells can be seen histologically. I-HCA is characterised by immunohistochemical positivity for serum amyloid A and C-reactive protein. Marked inflammatory infiltrate, ductular reactions and sinusoid dilation can be present in the tissue as well. U-HCA lacks gene mutations or specific immunohistochemical findings, but is diagnosed as HCA by histology [61]. Liver adenomas express hepatocellular markers and have lower proliferation activity than HCC [63]. To discriminate between adenoma and HCC, the following parameters are of importance: (1) clinical history in order to disclose risk factors that might indicate either HCA or HCC; (2) structure of surrounding liver as presence of cirrhosis favours HCC; (3) expression of HCA subtype-specific proteins; (4) presence or absence of cell atypia and invasion; (5) hepatocyte plate thickness and (6) expression of malignancy-associated HCC markers, e.g., GPC3.

#### 3.3. Well-differentiated hepatocellular carcinoma versus focal nodular hyperplasia

Focal nodular hyperplasia (FNH) is a hyperplastic hepatocellular proliferation resulting from blood flow abnormalities. It is a pathological focus characterised by nodular architecture, hypervascular central scar associated with thick fibrous septa between hepatocyte nodules, inflammatory infiltrate, presence of ductular reaction and sinusoid dilation [55, 61–63].

To distinguish FNH from HCC, GPC3, heat shock protein 70 (HSP70) and reticulin network can be assessed. Loss of reticulin framework, immunohistochemical expression of GPC3 and/ or diffuse nuclear expression of HSP70 favours HCC. Such immunohistochemical evaluation has 100% specificity for HCC although the sensitivity is only 43–46%. Typical "map-like" pattern of GS expression is evident in FNH. It is characterised by wide central positive areas in the middle of nodules. The positive foci interconnect between themselves, while periseptal areas remain negative. This reactivity pattern contrasts with normal liver showing limited perivenular reactivity in the middle of lobules [55].

### 3.4. Well-differentiated hepatocellular carcinoma versus high-grade dysplastic cirrhotic nodule

Dysplastic cirrhotic nodules (DNs) are characteristic precursors of HCC in the setting of chronic liver disease and/or liver cirrhosis. Most but not all dysplastic nodules are small, not exceeding the diameter of 1 cm [6]. Morphologically DNs are classified into high-grade DN and low-grade DN. Low-grade DN, carrying low risk of transformation to HCC, is generally characterised by monotonous cell population when compared with the surrounding cirrhotic liver, mildly increased cell density and minimal cell atypia. The nuclear/cytoplasmic ratio is mildly increased, nuclear atypia is slight, mitoses are absent and cell plates are 1–2 cells thick. The reticulin network is retained. The borders of low-grade dysplastic nodule are rounded, but the adjacent liver parenchyma is not compressed [3]. In contrast, high-grade dysplastic nodules can have many of classical HCC features. The nuclear/cytoplasmic ratio is increased. Nuclei show hyperchromasia and irregular borders and can be peripherally located. Occasional mitoses can be present. Cell plates are thicker than 2 cells. Cytoplasm switches to basophilic staining. Pseudoglandular structures start to appear. Occasional unpaired arteries have been observed. Lack of invasion is the most reliable criterion in the differential diagnosis with early HCC [3]. This trait is both important and biologically substantiated as the invasion is the hallmark of malignant tumours. However, it can be notoriously difficult to apply practically. In early HCC, invasion can be absent from biopsy due to sampling error. Regarding highgrade dysplastic nodule, entrapment of perinodular hepatocytes into fibrous tissues mimics invasion. To classify the entrapped hepatocytes correctly, immunohistochemical investigation of ductular proliferation can be helpful, as further described, because these non-neoplastic intraseptal hepatocytes and ductular proliferation stem from common progenitors [3].

with anabolic use. HCA cells appear larger due to intracellular glycogen or fat accumulation.

Several molecular subtypes of hepatocellular adenomas are known [62, 64], including hepatocyte nuclear factor 1α (HNF1α) inactivated type (H-HCA); β-catenin activated type (B-HCA); inflammatory HCA (I-HCA) and the unclassifiable type (U-HCA). Not surprisingly, beta-catenin activated subtype is associated with malignant transformation [62]. Beta-catenin mutations are reported in 20% of HCCs, especially in patients with underlying hepatitis C virus infection. HCC arising from B-HCA is usually well to moderately differentiated and lacks vascular invasion or satellite nodules [3]. Mutations lead to remarkable overexpression of GLUL gene (coding for glutamine synthase), thus beta-catenin activation can be assessed by intense homogeneous cytoplasmic expression of glutamine synthase and by aberrant nuclear localisation of beta-catenin [62, 63]. H-HCA shows decreased expression of liver fatty acid-binding protein, and presence of fat in neoplastic cells can be seen histologically. I-HCA is characterised by immunohistochemical positivity for serum amyloid A and C-reactive protein. Marked inflammatory infiltrate, ductular reactions and sinusoid dilation can be present in the tissue as well. U-HCA lacks gene mutations or specific immunohistochemical findings, but is diagnosed as HCA by histology [61]. Liver adenomas express hepatocellular markers and have lower proliferation activity than HCC [63]. To discriminate between adenoma and HCC, the following parameters are of importance: (1) clinical history in order to disclose risk factors that might indicate either HCA or HCC; (2) structure of surrounding liver as presence of cirrhosis favours HCC; (3) expression of HCA subtype-specific proteins; (4) presence or absence of cell atypia and invasion; (5) hepatocyte plate thickness and (6)

expression of malignancy-associated HCC markers, e.g., GPC3.

perivenular reactivity in the middle of lobules [55].

nodule

3.3. Well-differentiated hepatocellular carcinoma versus focal nodular hyperplasia

Focal nodular hyperplasia (FNH) is a hyperplastic hepatocellular proliferation resulting from blood flow abnormalities. It is a pathological focus characterised by nodular architecture, hypervascular central scar associated with thick fibrous septa between hepatocyte nodules, inflammatory infiltrate, presence of ductular reaction and sinusoid dilation [55, 61–63].

To distinguish FNH from HCC, GPC3, heat shock protein 70 (HSP70) and reticulin network can be assessed. Loss of reticulin framework, immunohistochemical expression of GPC3 and/ or diffuse nuclear expression of HSP70 favours HCC. Such immunohistochemical evaluation has 100% specificity for HCC although the sensitivity is only 43–46%. Typical "map-like" pattern of GS expression is evident in FNH. It is characterised by wide central positive areas in the middle of nodules. The positive foci interconnect between themselves, while periseptal areas remain negative. This reactivity pattern contrasts with normal liver showing limited

3.4. Well-differentiated hepatocellular carcinoma versus high-grade dysplastic cirrhotic

Dysplastic cirrhotic nodules (DNs) are characteristic precursors of HCC in the setting of chronic liver disease and/or liver cirrhosis. Most but not all dysplastic nodules are small, not

Nuclear atypia is absent [3].

32 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

Expression of GPC3 points towards malignant hepatocellular tumour, as it was previously noted. However, GPC3 expression has been reported in 3–76% of dysplastic nodules. Glutamine synthetase is expressed in 69.8% of HCC contrasting with 13.6% in high-grade DN. Heat shock protein 70 is found in 73.5% of HCC and only exceptional dysplastic nodules [3]. To distinguish high-grade DN from early HCC, immunohistochemical panel comprising heat shock protein 70, glypican-3 and glutamine synthetase has been recommended. Expression of one marker is compatible with DN, while HCC expresses at least two markers. The sensitivity of this panel is estimated as 60–78% [55].

In addition, cytokeratin (CK) 7 and/or CK19 and CD34 can be useful in the assessment of architecture and reactive changes. HCC is characterised by more diffuse expression of CD34 and loss of ductular reaction at the nodule interface. Dysplastic nodule shows only focal CD34 expression in the periphery of the nodule and more marked proliferation of CK7-positive ductules surrounding DN [55]. In the ductular reaction, CK7 and CK19 usually are coexpressed. Thus, gradual loss of CK7 and CK19 positive ductular reaction in perinodular stroma correlates with progression of cirrhotic to dysplastic nodule and further to HCC. Ductular reaction is present around ≥50% of perimeter of a DN, while it is almost lost in HCC [3].

Different systems for complex evaluation of the biological potential of hepatocellular nodule have been proposed. Integrated evaluation of haematoxylin-eosin findings together with reticulin stain and immunohistochemistry for CD34 has been suggested. A hepatocellular nodule should be classified as HCC if at least three features from the following are present: necrosis; cellular atypia; thickness of trabeculae more than 4 cells; mitotic activity or diffuse expression of CD34 in the sinusoidal endothelium [6]. Alternatively, stromal invasion, loss of reticulin network and positivity for at least two out of three markers (HSP70, GS, GPC3) are considered the strongest parameters discriminating HCC from high-grade dysplastic nodule [3].

#### 3.5. Hepatocellular carcinoma versus metastasis

If high-grade malignant tumour is found in the liver, the differential diagnosis includes metastatic malignancy versus HCC and cholangiocarcinoma. Any malignant tumour can ultimately spread to the liver via bloodstream, lymphogeneous dissemination or transperitoneal spread. In some biopsy series, metastatic lung, colorectal, pancreatic and breast carcinomas have been the most common secondary liver tumours [3]. However, frequency of different metastatic malignant tumours in liver biopsies depends on many factors, including the biological potential of the tumour and its incidence in the population as well as institutional approach to liver biopsy in different oncological patients. This, in turn, may depend on the patient's general status, presence of contraindications for biopsy or significant oncological treatment and the availability of effective treatment.

In order to distinguish HCC from metastatic tumours, it is advisable to combine at least two hepatocellular markers and at least two antigens that are more frequently seen in adenocarcinomas. Among hepatocellular markers, Arg1 should be combined with either HepPar1 or GPC3. Most of adenocarcinomas express cytokeratin (CK) 19, MOC-31 and CK7 [55]. The spectrum of immunohistochemical panel should be planned in accordance with tissue availability within the biopsy. The suggested minimal panel includes ARG1 and CK19 [55], while maximal investigation might include several HCC markers accounting for different grades of HCC, several adenocarcinoma markers and antigens that are characteristic for certain tissues (neuroendocrine or melanocytic differentiation) or epithelia of specific organs, e.g., breast, large bowel, lung, thyroid, kidney and others. Panels of immunohistochemical markers can disclose the location of primary tumour giving rise to metastasis. Thus, CK20 and CXD2 are typical for metastatic colorectal carcinoma; CDX2 and CK7 for gastric carcinoma; TTF-1 and napsin A for lung adenocarcinoma and oestrogen receptor, mammaglobin, GATA3 or GCDFP-15 for breast cancer [65]. The expression frequencies of different tissue- and organ-specific antigens in metastases and corresponding primary tumours are further outlined in Table 4.


When differentiating between HCC and metastasis, the peculiar immunophenotype of fibrolamellar HCC must be recognised promptly. Fibrolamellar HCC expresses hepatocellular proteins, such as HepPar1, GPC3 or pCEA; biliary (CK7), progenitor and stem cell (CK19, CD44) antigens and macrophage markers (CD68). The granular or dot-like expression of CD68 in a tumour

Table 4. Frequency of immunohistochemical expression of selected tissue- or organ-specific markers [66–80].

CDX2, caudal type homeobox 2; SATB2, special adenosine-thymidine-rich-binding protein 2; CK, cytokeratin; TTF-1, thyroid transcription factor 1; HMB-45, melanosome protein human melanoma black 45; MART-1, melanoma antigen recognized by T cells 1; PAX-8, paired box gene 8; GATA3, guanosine-adenosine -thymidine -adenosine nucleotide

Antigen Tumour Frequency, % References MART-1 Metastatic melanoma 63–82 [70, 72, 73] Tyrosinase Melanoma 71 [72] Tyrosinase Metastatic melanoma 63 [72] PAX-8 Ovarian cancer 80 [69] PAX-8 Endometrial cancer 100 [69] PAX-8 Renal cancer 83–93.3 [69, 74] PAX-8 Metastatic renal cancer 93.9 [74] Napsin A Renal cancer 50 [69] Gross cystic disease fluid protein-15 Breast carcinoma 23.9–60 [75, 76] Gross cystic disease fluid protein-15 Primary triple negative breast carcinoma 10–14 [75, 77] Gross cystic disease fluid protein-15 Primary non-triple negative breast carcinoma 69 [77] Gross cystic disease fluid protein-15 Metastatic triple negative breast carcinoma 21 [75] Mammaglobin Breast carcinoma 46.6–80 [75, 76] Mammaglobin Primary triple negative breast carcinoma 17–25 [75, 77] Mammaglobin Primary non-triple negative breast carcinoma 61 [77] Mammaglobin Metastatic triple negative breast carcinoma 41 [75] GATA3 Invasive breast cancer 82.5–94 [76, 78] GATA3 Primary triple negative breast carcinoma 20.2–87 [77–80] GATA3 Metastatic triple negative breast carcinoma 44 [79] GATA3 Luminal A breast carcinoma 99.5 [80] GATA3 Luminal B breast carcinoma 97.7 [80] GATA3 HER2-positive breast carcinoma 59.6–68.5 [76, 80]

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

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

35

The molecular classification of hepatocellular carcinoma is still developing. Thus, different approaches have been proposed. Although the present tools of molecular analysis assure the

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

4. Molecular analysis

sequences binding protein 3.


CDX2, caudal type homeobox 2; SATB2, special adenosine-thymidine-rich-binding protein 2; CK, cytokeratin; TTF-1, thyroid transcription factor 1; HMB-45, melanosome protein human melanoma black 45; MART-1, melanoma antigen recognized by T cells 1; PAX-8, paired box gene 8; GATA3, guanosine-adenosine -thymidine -adenosine nucleotide sequences binding protein 3.

Table 4. Frequency of immunohistochemical expression of selected tissue- or organ-specific markers [66–80].

When differentiating between HCC and metastasis, the peculiar immunophenotype of fibrolamellar HCC must be recognised promptly. Fibrolamellar HCC expresses hepatocellular proteins, such as HepPar1, GPC3 or pCEA; biliary (CK7), progenitor and stem cell (CK19, CD44) antigens and macrophage markers (CD68). The granular or dot-like expression of CD68 in a tumour featuring appropriate morphology is helpful in diagnosing fibrolamellar HCC [6].
