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Introduction

Fibrolamellar hepatocellular carcinoma (FL-HCC) is a distinct malignant liver cell tumor that differs from classical HCC at histological, cytological, immunohistochemical, and molecular levels. It mainly occurs in adolescents and young adults and is histologically characterized by strands of large, eosinophilic hepatocyte-like cells embedded in a fibrous stroma forming collagen-rich lamellae. FL-HCC was first described in 1956 (Edmondson 1956) and subsequently confirmed by Craig et al. (1980) and Berman et al. (1980). The typical clinicopathologic and molecular features of this tumor entity have been reviewed (Soreide et al. 1986; Vecchio 1988; Saab and Yao 1996; McLarney et al. 1999; El-Serag and Davila 2004; Torbenson 2007; Liu et al. 2009; Lim et al. 2014; Darcy et al. 2015a). After the first description, it took a rather long time period until the tumor entity was defined and accepted. Several alternative terms had been employed to denote this lesion, including hepatocellular carcinoma with laminar fibrosis, hepatocellular carcinoma with polygonal cell type and fibrous stroma, oncocytic hepatocellular carcinoma, and eosinophilic hepatocellular carcinoma with lamellar fibrosis.

Epidemiology

FL-HCC accounts for 1–9 % of HCCs overall, this impressive frequency range being caused by markedly variable prevalences in different geographic niches and differences in diagnostic criteria used (Craig et al. 1980; McLarney et al. 1999). Among 46,392 cases of HCC registered in the SEER program between 2000 and 2010, 191, tumors were FL-HCC, with an incidence of clearly less than 0.1 per 100,000 people (Eggert et al. 2013). In contrast to conventional HCC, FL-HCC is not associated with cirrhosis of the liver and other typical risk factors, although a relation to genetic syndromes has been suggested in part of the cases, e.g., Gardner syndrome (Gruner et al. 1998) and Fanconi anemia (LeBrun et al. 1991). FL-HCC is typically a tumor of adolescents and young adults, but rarely also occurs in older individuals. FL-HCC is rare in infants and small children (Cruz et al. 2008), but FL-HCC in older children has more often been encountered (Dellaportas et al. 2011). There is usually no gender predominance, but a predominance of the female gender has also been reported (Chagas et al. 2015). The tumor has been diagnosed during pregnancy (Kroll et al. 1991; Louie-Johnsun et al. 2003). In reviewing studies there is an age range of 5–69 years (mean in Western patients : 23 years), and most patients present in their second and third decades (McLarney et al. 1999). In contrast, conventional HCC is less common than FL-HCC in individuals younger than 40 years (Hernandez-Castillo et al. 2005).

The incidence of FL-HCC is relatively high in Western countries, but is rare in Oriental countries. Analysis of Japanese cases revealed an age range of 15–45 years, with a mean of 21.9 years, findings similar to those in Western countries (Kohno et al. 1988; Haratake et al. 1990; Tanaka et al. 1994; Hoshino et al. 1996; Nojiri et al. 2000; Magata et al. 2001; Yoshimi et al. 2002; Yoshinaga et al. 2002; Kanai et al. 2004; Morise et al. 2005). FL-HCC is apparently not uncommon in sub-Saharan Africa (Moore et al. 2004; Bhaijee et al. 2009) and Latin America (Arista-Nasr et al. 2002) .

Clinical Features

FL-HCC characteristically manifests as a large hepatic mass in the absence of liver cirrhosis, elevated serum AFP values, or known risk factors for HCC. In most cases, FL-HCC presents with vague and nonspecific clinical signs and symptoms, often with abdominal discomfort or pain, malaise, and weight loss (Craig et al. 1980; Saab and Yao 1996; Hemming et al. 1997; reviews: Torbenson 2007; Chun and Zimmitti 2013). Due to its mass effect, FL-HCC can cause recurrent obstructive jaundice (Albaugh et al. 1984; Soyer et al. 1991a). It can, similar to ordinary HCC, invade large bile ducts and cause obstructive jaundice, sometimes with subsequent migration of tumor fragments to the common bile duct and distal biliary obstruction (intrabiliary tumor thrombus; Eckstein et al. 1988; Kunz et al. 2002; De Gaetano et al. 2013; Arora 2015). FL-HCC reveals invasion of large liver veins less commonly than conventional HCC, but Budd-Chiari syndrome has been observed as the first manifestation of FL-HCC (Lamberts et al. 1992; Asrani and LaRusso 2012). Caval compression syndrome has been reported as a complication of FL-HCC (Kanai et al. 2004). Additional and usually less common or rare disorders sometimes associated with FL-HCC include severe anemia (Tanaka et al. 1994), nonbacterial thrombotic endocarditis (Vaideeswar et al. 1993), tumor-induced ascites in the case of peritoneal spread (Gupta et al. 1999), a clinical syndrome mimicking hepatic pyogenic abscess (Debray et al. 1994), and cold agglutinin disease (Al-Matham et al. 2011). FL-HCC has been observed in association with ulcerative colitis with primary sclerosing cholangitis (Snook et al. 1989).

FL-HCC patients can show complex patterns of metabolic disorders (Table 1).

Table 1 Metabolic disorders in fibrolamellar hepatocellular carcinoma
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The tumor shows abnormalities in the metabolism of vitamin B12 (cobalamin). An increase of serum levels of vitamin B12-binding capacity in patients with FL-HCC has first been reported in 1982 (Paradinas et al. 1982) and confirmed in later analyses (Sheppard et al. 1983; Wheeler et al. 1986; Lildballe et al. 2011). Increasing vitamin B12-binding capacity has been found in postoperative recurrence of FL-HCC (Kanai et al. 2004). However, elevation of vitamin B12 and B12-related proteins seems to be a common feature in HCCs and not only in FL-HCC (Simonsen et al. 2014). Transcriptional profiling of FL-HCC revealed an endocrine signature, in that 4 of the 16 genes most significantly overexpressed in pure FL-HCC were neuroendocrine genes, i.e., prohormone convertase 1/PCSK1, neurotensin, delta/notch-like EGF repeat containing, and calcitonin (Malouf et al. 2014). Serum neurotensin levels (especially levels of the C-terminal part) may be elevated in patients with FL-HCC, caused by secretion of this peptide by the tumor (Collier et al. 1984; Read et al. 1991). Neurotensin was originally isolated from bovine hypothalamus and is an important gastrointestinal regulatory peptide that affects pancreatic secretion, gut motility, gut mucosal growth, and the translocation of fatty acids from the intestinal lumen. The neurotensin gene is expressed in fetal human liver (but not adult liver) and in FL-HCCs (Ehrenfried et al. 1994). In the tumor itself, the neurotensin (6–13) peptide was detectable (Read et al. 1991).

FL-HCC can be associated with gynecomastia (McCloskey et al. 1988; Hany et al. 1997; Agarwal et al. 1998; Sher et al. 1998), mainly in cases of large tumor mass/metastasis. Gynecomastia may be the presenting sign of FL-HCC (McCloskey et al. 1988). Gynecomastia in this tumor is caused by increased aromatase P450 activity in the neoplasm (hyperaromatase syndrome; Hany et al. 1997; Agarwal et al. 1998; Muramori et al. 2011), which gives rise to markedly elevated serum levels of estrone and estradiol-17 beta, suppressing FSH and LH, respectively, and consequently testosterone (Agarwal et al. 1998). Northern analysis indicated the presence of P450 aromatase transcripts in total RNA from FL-HCC but not in the adjacent liver (Agarwal et al. 1998). FL-HCC can rarely produce beta-HCG and by this cause vaginal bleeding (Dahan and Kastell 2002). In part of patients, FL-HCC is associated with a recurrent non-hepatic/non-cirrhotic form of hyperammonemia (paraneoplastic hyperammonemia) complicated by encephalopathy. This complication may mimic a disorder of urea synthesis, e.g., ornithine transcarbamylase deficiency (Sulaiman and Geberhiwot 2014).

Imaging Features

Abdominal imaging shows a well-circumscribed, lobulated, heterogeneous mass that may resemble focal nodular hyperplasia (FNH) . CT images document that FL-HCCs are predominantly solitary, well-delineated, and hypervascular lesions which are predominantly hypoattenuating compared with the liver (Bedi et al. 1988; Soyer et al. 1991b; Ichikawa et al. 2000; Marrannes et al. 2005; Selaru et al. 2007; Yen and Chang 2009; Terzis et al. 2010). Almost all tumors were heterogeneous on non-enhanced CT images. In portal venous phase and relative to enhanced liver, FL-HCCs appeared isoattenuating in 48 %, hyperattenuating in 16 %, and hypoattenuating in 36 % (Ichikawa et al. 1999). A central scar resembling the stellate scar seen in FNH is present in CT images in about a third to about 70 % of the cases (the “scar sign”; Kane et al. 1987; Soyer et al. 1991b; Ichikawa et al. 1999). In one study, 82 % of the scars showed a stellate morphology on CT images (Ichikawa et al. 1999). However, central or eccentric scars are also found in conventional HCC, cholangiocarcinoma, and some hepatic metastases (review: Kim et al. 2009). Among 31 cases, CT revealed well-defined margins in 77 % and ill-defined margins in seven cases (Ichikawa et al. 1999). PET-CT scans are a valuable modality in the diagnosis of FL-HCC (Liu et al. 2011). Tumor calcifications in FL-HCC have been reported in 15–68 % of cases at CT and occur in a wide variety of patterns (Soyer et al. 1991b; Stoupis et al. 1998; Ichikawa et al. 1999). Calcifications are more frequent in FL-HCC than in FNH (Caseiro-Alves et al. 1996).

Classification

FL-HCC has been divided into two separate entities, i.e., pure FL-HCC and mixed FL-HCC, differing in clinical presentation and course (Malouf et al. 2012). Pure FL-HCC typically occurs in patients younger than 30 years of age and is often complicated by lymph node metastasis at the time of diagnosis and more frequently shows extrahepatic recurrence. So far, combined or mixed FL-HCC has been regarded as rare lesion. However, in a more recent investigation of 54 patients, the mixed form was found in 25 % of the patients. In contrast to pure FL-HCC, the mixed variant appeared to resemble HCC, occurring in patients aged >40 years, often involving the liver as primary site of disease recurrence, and imparting an increased risk of death (Malouf et al. 2012). Pure FL-HCCs have a distinct transcriptomic signature characterized by strong expression of neuroendocrine genes, including prohormone convertase 1, neurotensin, delta/notch-like EGF repeat containing, and calcitonin, suggesting a complex cellular lineage (Malouf et al. 2014).

Pathology

Macroscopy

The tumors tend to be more common in the left liver lobe (about two thirds of cases; Craig et al. 1980), but relatively often involve both lobes (Ichikawa et al. 1999). In one study, 55 % of the cases had involvement of three or more hepatic segments, and 32 % had involvement of two segments (Ichikawa et al. 1999). Multifocality, gross vascular invasion, and cirrhosis are typically absent, but the tumor often demonstrates aggressive local invasion and nodal and distant metastases (Wong et al. 1982; Friedman et al. 1985; Francis et al. 1986; Gibson et al. 1986; Brandt et al. 1988; Ruffin 1990; Ichikawa et al. 1999; McLarney et al. 1999; Smith et al. 2008). Macroscopically, the tumors are well-delineated masses with a polycyclic border and an expansive growth pattern. A tumor capsule (usually incomplete) is seen in approximately half of the neoplasms. In a study of 31 cases, tumor diameter ranged from 3 to 27 cm, with an average of 13 cm (Ichikawa et al. 1999). In 80–90 % of the cases, a solitary tumor is present. In the remaining cases, there is a mass with small peripheral satellite lesions (10–15 %), a bilobed mass (5 %), or rarely a diffusely/multifocal mass (less than 1 %). Up to 20 % of the tumors show some degree of pedunculation. On cut surfaces, the neoplasms are yellow to tan and often show a nodular or radiated structure caused by radiating fibrous band that originates from central fibrous scar-like areas (Figs. 1 and 2).

Fig. 1
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Fibrolamellar hepatocellular carcinoma. On cut surfaces, the neoplasm exhibits a lobulated texture, lobules, or nodules separated from each other by fibrous septa

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Fig. 2
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Nodular structures in fibrolamellar hepatocellular carcinoma can form intertwining ribbons, resulting in a gyriform pattern

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In case of marked bile accumulation, a greenish discoloration of the tumor is seen. The neoplasms are encapsulated in about half of the cases, the pseudocapsule being mostly incomplete. A central scar-like structure resembling that seen in focal nodular hyperplasia was detected in about three quarters of cases (Ichikawa et al. 1999). Small necroses and/or hemorrhages may be noted, but are grossly visible in only about 10 %. Rare examples of tumors have revealed massive necrosis and/or hemorrhage, resulting in a multicystic morphology mimicking a primarily cystic liver tumor, but primary multicystic variants of FL-HCC also occur (Pombo et al. 1993). The adjacent liver does not show cirrhosis.

Histopathology

The typical cell of FL-HCC is a large polygonal cells having an abundant, deeply eosinophilic, faintly granular cytoplasm and well-defined cell borders . These oxyphilic cells are, however, cytologically different from oncocytes (Altmann 1990). The nuclei are large, round to ovoid and vesicular, and usually single, and they show a peripheral condensation of chromatin and a prominent, centrally placed amphophilic nucleolus. The typical morphologies of the cells and nuclei, together with lamellar fibrosis, are the three main criteria for diagnosis (Figs. 3, 4, 5, 6, and 7).

Fig. 3
figure 3

Fibrolamellar hepatocellular carcinoma. Solid strands and ribbons of large eosinophilic tumor cells are separated by fibrous hypocellular tracks, the fibrolamellae (hematoxylin and eosin stain)

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Fig. 4
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Fibrolamellar hepatocellular carcinoma. The epithelial tumor cells are in direct contact with collagenous bands , without a visible interposed cellular stroma (hematoxylin and eosin stain)

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Fig. 5
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Fibrolamellar hepatocellular carcinoma. Focally, stroma of slightly higher cellularity can occur, sometimes associated with dissociation of tumor cells (hematoxylin and eosin stain)

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Fig. 6
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Fibrolamellar hepatocellular carcinoma. In the fibrous lamellae, collagen-rich connective tissue may undergo hyaline change (hyaline sclerosis of stroma, center of figure; hematoxylin and eosin stain)

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Fig. 7
figure 7

Fibrolamellar hepatocellular carcinoma. The typical tumor cell is large, with abundant oxyphilic and granular cytoplasm . Nuclei are spherical to ovoid, with a hyperchromatic periphery and less dense center, and with prominent nucleoli. Pseudotubular lumina can be present (hematoxylin and eosin stain)

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A part of the nuclei display intranuclear cytoplasmic pseudoinclusions (cytoplasmic invaginations). Large invaginations cause so-called empty nuclei. Mitotic figures are much less common than in ordinary HCC (Farhi et al. 1983) and may have previously suggested that the tumor is of low malignancy, what is not the case. Less than 10 % of the cells exhibit PAS-positive globular cytoplasmic inclusions (2–7 μm in diameter; Farhi et al. 1982). About half of the tumor cells contain large ovoid cytoplasmic inclusions that are much less eosinophilic or are amphiphilic, the so-called pale bodies (Fig. 8) .

Fig. 8
figure 8

Fibrolamellar hepatocellular carcinoma. Part of neoplastic cells contain large pale cytoplasmic inclusions (pale bodies, center of figure, hematoxylin and eosin stain)

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Pale bodies sometimes show a central hyaline core. Cholestasis is often seen. The tumor cells commonly show canaliculus-like lumina present between adjacent cells containing bile. Tumor cholestasis is, like in ordinary HCC, associated with deposition of copper (copper-binding protein in many cases (Lefkowitch et al. 1983). Small calcifications are sometimes seen, mostly in the central scar and within the fibrous bands. In a minority of FL-HCC, a mild macrovesicular steatosis may be found.

The solid components of medium- to large-sized polygonal cells are irregularly subdivided by connective tissue bands rich in collagen (the “fibrolamellae” that have given the tumor’s name). The amount of collagen in the bands varies markedly, ranging from sclerosed bands to bands with a loose texture of tissue rich in glycosaminoglycans and higher spindle cell cellularity. However, it has been shown that the connective tissue content in FL-HCC is overall greater than in other liver tumors (Sarosi et al. 1991). The bands stain red in the van Gieson stain and bright blue with the trichrome stain. In the silver stain, the stroma shows a fine, dense, and complex network of reticulin fibers. The fibrolamellae often end in frayed edges. The fibrous matrix is predominantly composed of collagens I, III, and V, tenascin, and (less abundantly) basement membrane proteins such as laminin. In situ hybridization studies have shown that the fibroblastoid cells embedded in the matrix express collagen III-mRNA (Nerlich et al. 1992). FL-HCC cells may develop apparently intracellular lumina or spaces containing erythrocytes (Fig. 9).

Fig. 9
figure 9

Fibrolamellar hepatocellular carcinoma. Neoplastic cells can develop apparently intracellular lumina or spaces that contain erythrocytes (hematoxylin and eosin stain)

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Rare variants of FL-HCC, or certain regions in these tumors, may display a markedly solid component (Kojima et al. 2004). Based on the distinct cellular features and the distinct epithelial-stromal composition of FL-HCC, this neoplasm has been diagnosed by cytological methods (Suen et al. 1985; Perez-Guillermo et al. 1999; Jain et al. 2002; Kunz et al. 2002; Sarode et al. 2002; Mansouri et al. 2006; Gulati and Saran 2009). FL-HCC is an angioinvasive neoplasm that tends to invade large veins. Vascular invasion is present in about 40 % of the tumors (Farhi et al. 1983; Kakar et al. 2005). It was reported that FL-HCC arose in a background of focal nodular hyperplasia (Imkie et al. 2005). In a patient with generalized AA amyloidosis, amyloid was also deposited in the stroma of an FL-HCC (Llortea et al. 1994).

Ultrastructure

The neoplastic cells are rich in organelles and contain nuclei with often prominent cytoplasmic invaginations, the latter corresponding to the clear nuclear pseudoinclusions seen in light microscopy. The cytoplasm frequently contains numerous and densely packed, swollen mitochondria which are uniform in size and shape and often contain dense inclusions, resulting in a picture resembling oncocytes (Andreola et al. 1986). The endoplasmic reticulum is prominent, sometimes with dilated profiles. Dense cytoplasmic inclusions have been noted (Farhi et al. 1982; Andreola et al. 1986; Hasegawa 1996). Fully developed Mallory-Denk bodies are absent, but filamentous material resembling that found in these bodies has been detected (Caballero et al. 1985). Between two adjacent cells, lumina with microvilli can be found, representing abortive biliary canaliculi (Sato et al. 1997). The pale bodies seen at light microscopy ultrastructurally represent intracellular lumina lined with numerous microvilli. The central hyaline cores seen in part of the pale bodies are depositions of fine granular material (An et al. 1983). In part of the tumors, ultrastructural signs of neuroendocrine differentiation have been found (Payne et al. 1986; Garcia de Davila et al. 1987; Lloreta et al. 1994). The tumor cells are separated by broad, homogeneous bands of dense collagen with characteristic periodicity. The bands of collagen are lined by spindle cells with the features of fibroblasts or myofibroblasts, and these cells are interposed between collagen and tumor cells (Farhi et al. 1982).

Immunohistochemistry of the Epithelial Component

The immunophenotype of epithelial cells in FL-HCC has been analyzed in detail (Caballero et al. 1985; Teitelbaum et al. 1985; Berman et al. 1988). The cells of FL-HCC are positive for HepPar [hepatocyte paraffin 1] (Wennerberg et al. 1993; Klein et al. 2005; Ward et al. 2010; Patonai et al. 2013), suggesting that a hepatocyte-like differentiated cell is involved. Similar to conventional HCC, FL-HCC expresses the heparan sulfate proteoglycan, glypican-3, the positivity rate ranging from 17 % (Abdul-Al et al. 2010) to 59 % (Ward et al. 2010) and 64 % of cases (Shafizadeh et al. 2008). The tumor cells display canalicular membrane expression of pCEA (Ward et al. 2010). Interestingly, FL-HCC cells show a circumferential staining for pCEA, suggesting a loss of the normal apical-basolateral polarization of hepatocytes (own observations). The tumor cells may be positive for copper and copper-binding protein (Lefkowitch et al. 1983). The cells of FL-HCC typically and strongly express cytokeratin-7 in the cytoplasm (Van Eyken et al. 1990). Positivity for cytokeratin-7 has been confirmed in later studies (Gornicka et al. 2005; Klein et al. 2005; Abdul-Al et al. 2010; Fig. 10).

Fig. 10
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Fibrolamellar hepatocellular carcinoma. Cells typically express cytokeratin-7 (CK7 immunostain)

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In one study, all cases of FL-HCC were positive for epithelial membrane antigen (EMA), and 36 % of FL-HCCs showed staining for cytokeratin-19, B72.3, and EpCAM (Ward et al. 2010). It has, however, been shown that FL-HCC is less often positive for CK19 than ordinary HCC (Abdul-Al et al. 2010). In ordinary HCC, expression of CK19 and EpCAM is associated with worse prognosis and the potential involvement of a bipotential progenitor cell (see the respective chapter). Cells of FL-HCC usually show a beta-catenin staining pattern similar to that of hepatocytes (Fig. 11).

Fig. 11
figure 11

Fibrolamellar hepatocellular carcinoma. The tumor cells show membranous reactivity for beta-catenin (beta-catenin immunostain)

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Interestingly, the cells of FL-HCC are positive for CD68, a marker of lysosomal/endosomal membranes and typically positive in macrophages. The CD68 immunostaining, which involves almost all cases of FL-HCC, presents a distinctive granular, dot-like, or stippled pattern of the cytoplasm and had a sensitivity of 96 % and a specificity of 80 % (Ross et al. 2010). A part of FL-HCCs are immunoreactive for CD99 (Vasdev and Nayak 2003). Cells of FL-HCC produce several proteins that are also typical products of normal hepatocytes, including ferritin (Caballero et al. 1985) and alpha-1 antitrypsin, the latter sometimes accumulated in cytoplasmic globular inclusions or around such inclusions. FL-HCCs more often than ordinary HCCs express claudin-2 and claudin-5 (Patonai et al. 2011). FL-HCCs demonstrate hepatocyte growth factor receptor (c-met) levels similar to normal (Schoedel et al. 2003). In contrast to common HCC, FL-HCC has not been shown to express survivin (Kannangai et al. 2005). Similar to HCC, FL-HCC can express the CCK-B/gastrin receptor Caplin et al. 1999).

Immunohistochemistry of the Matrix Component

Immunohistochemically, the abundant extracellular matrix of FL-HCCs has been shown to contain large amounts of tenascin (Scoazec et al. 1996) and fibronectin (Jagirdar et al. 1985), whereas basement membrane proteins such as laminin seem to be less expressed. As FL-HCC cells more often express TGF-beta than HCC, it was suggested that fibrosclerosis and matrix protein production may be regulated by this fibrokine (Nerlich et al. 1992; Orsatti et al. 1997). The tumor cells express a set of surface proteins that are important for cell-matrix interactions. The large epithelial cells are positive for alpha-1 integrin, E-cadherin, and the hepatocyte N-related cadherin, but lack beta-4 integrin (Scoazec et al. 1996). The tumor cells display a strong expression of CD44 (Washington et al. 1997), a widely distributed integral membrane protein and cell adhesion protein that has been implicated in cell-matrix contact, invasion, and metastatic spread. Most FL-HCCs revealed increased expression of active matrix metalloproteinase-2, in contrast to ordinary HCC (Schoedel et al. 2003). The sinusoid-like vascular channels of FL-HCC have diffusely CD34-positive endothelia (Abdul-Al et al. 2010).

Growth Features of FL-HCC

FL-HCCs typically grow with broad pushing borders. Invasion of adjacent parenchyma may be seen histologically, and portal tracts may be entrapped in the growing tumor front. FL-HCC has a very low mitotic activity, mitotic figures being normally structured in most cases. In a study of seven cases, the mitotic index was very low, i.e., 0–1 mitotic figure/10 high-power fields. The percentage of Ki-67 nuclear staining ranged from 1.0 to 29.7 %. There was no reactivity for S-phase kinase-associated protein (Skp) 2; all cases showed moderate to strong nuclear p16INK4 positivity (Dhingra et al. 2010 ).

Clear Cell Variant of Fibrolamellar Hepatocellular Carcinoma

A clear cell variant of FL-HCC has been described (Cheuk and Chan 2001). A 59-year-old female known to be a hepatitis B virus carrier was found to have a liver mass on routine checkup, visualized at CT as a solitary mass in the right liver lobe. Serum AFP was not elevated. The lesion was resected via right hepatectomy, and the patient was without of recurrence 13 months later. Grossly, the tumor was circumscribed, bosselated and formed a tumor measuring up to 50 mm in diameter. Histologically, the tumor consisted of oxyphilic cells and clear cells of about 50 % each, embedded in stroma consisting of strands of hyalinized collagen. Transitional forms between the two main cell types were seen. Pale bodies were found in part of the cells. Both the oxyphilic cells and the clear cells showed diffuse immunostaining for HepPar1, whereas antimitochondrial antibody stained the oxyphilic cells but weakened the clear cells. This variant has also been diagnosed by fine needle aspiration cytology (Kaplan and Hoda 2007.)

Glandular Variant of Fibrolamellar Hepatocellular Carcinoma

This probably rare and somewhat intriguing variant of FL-HCC is characterized by components of acinar or pseudoglandular structures, sometimes with mucin production (Goodman et al. 1985; Tanaka et al. 2005). The cells lining the gland-like spaces are structurally similar to other FL-HCC cells, but are usually smaller. Mucin is detectable within the neoplastic cells but also in the lumina of acinar profiles, and the mucinous substance is positive for mucicarmine and alkaline alcian blue (Goodman et al. 1985). The cells lining the pseudoglandular spaces immunohistochemically share features with cholangiocellular elements, i.e., positivity for biliary-type cytokeratins. This phenotype was also found in lymph node metastases of the tumor, a phenomenon which may exclude a collision tumor (Tanaka et al. 2005). This is the reason why such neoplasms were also termed, combined fibrolamellar carcinoma and cholangiocarcinoma, with biphenotypic antigen expression (Tanaka et al. 2005). This cholangiocyte-like differentiation is not associated with other histologic features of true cholangiocarcinoma; the biologic behavior of such combined tumors seems to be more in keeping with pure FL-HCC.

Combined Patterns

Combined tumors, albeit very rare, are of some theoretical interest because they may shed light on the cell of origin of FL-HCC. Exceptionally, FL-HCC occurred in combination with conventional HCC (Hasegawa 1996; Okano et al. 1998; Seitz et al. 2002), the FL-HCC component being cytokeratin-7 positive, in contrast to the HCC component (Seitz et al. 2002; Zermani et al. 2005). In the tumor described by Hasegawa (1996), a common HCC element with trabecular structure existed at the periphery of the FL-HCC. In contrast, the tumor described by Seitz et al. (2002) showed HCC at the center of a large FL-HCC. In few cases, FL-HCC transformed into common HCC in recurrent lesions of the residual liver and in a remote metastasis (Yamamoto et al. 1999; Chang et al. 2003). FL-HCC has also been found to be associated synchronously with common HCC located in another part of the liver (Okada et al. 1993).

FL-HCC has been observed 5 years after hepatocellular adenoma in a 14-year-old girl (Terracciano et al. 2004). Nodular hyperplasia associated with or surrounding FL-HCC has been observed (Saul et al. 1987; Saxena et al. 1994). In a 14-year-old girl, a 9 cm subcapsular nodule was present in the right lobe of the liver, with a distinct zonation: ca central grayish white area of FL-HCC and peripheral fleshy, tan-colored rim of nodular hyperplastic liver parenchyma resembling FNH. It was suggested that the hyperplasia was a reaction to an abnormal arterialization of the tissue, the arteries originating from the central FL-HCC (Saxena et al. 1994). It has been assumed that FL-HCC may be the malignant counterpart of FNH (Vecchio et al. 1984), but there is no convincing evidence for this.

Biology of Disease

FL-HCC reveals a metastasizing pattern different from ordinary HCC. FL-HCC tends to metastasize within the abdominal cavity. This may lead to intriguing and misleading patterns, such as metastasis to the pancreatic head (Thirabanjasak et al. 2009) or the skeletal muscle (Kutluk et al. 2001). FL-HCC can metastasize to the ovaries (Benito et al. 2012) and has been shown to produce ovarian Krukenberg tumor as first tumor manifestation (Montero et al. 2007; Bilbao et al. 2008). FL-HCC is also a primary liver tumor that typically metastasizes to locoregional lymph nodes (Stipa et al. 2006; Tsilividis et al. 2010; Gras et al. 2012, Fig. 12), lymph node positivity representing a negative prognosticator and considered to be an indication for lymph node dissection (Yamamoto et al. 1995; Hiramatsu et al. 1999; Jaeck 2003).

Fig. 12
figure 12

Lymph node metastasis of fibrolamellar hepatocellular carcinoma. The typical tumor morphology, with strongly eosinophilic cells and fibrous bands, is recapitulated in metastatic disease (hematoxylin and eosin stain)

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As ordinary HCC, FL-HCC can invade large vessels, including the inferior vena cava, and spread to the heart cavities and pulmonary arteries (Asrani and LaRusso 2012; Knudson et al. 2012).

Several studies have advocated that FL-HCC is less aggressive than conventional HCC (Berman et al. 1980; Craig et al. 1980; Lack et al. 1983; Wetzel et al. 1983; Nagorney et al. 1985; Hodgson 1987; Kaczynski et al. 1996; Pinna et al. 1997; Okuda 2002; El-Serag and Davila 2004; Meriggi and Forni 2007), and even in textbooks it is or was mentioned that FL-HCC is associated with a favorable prognosis (Everson and Trotter 2003; Sherlock and Dooley 2003; Ferrell 2004). There are single reports of long-term survival after surgery alone (Ishikawa et al. 2007), but in contrast to older suggestions, there is now evidence that the biology of FL-HCC in comparison with ordinary HCC is a complex issue, the presence or absence of differences in part being affected by artifacts of methodology (Njei 2014). For example, in patients with FL-HCC, the better outcome in some patients is due to the absence of cirrhosis in these patients, and the presentation at a non-advanced stage, rather than its distinct clinicopathologic features (Stevens et al. 1995; Zografos et al. 1997). There is also evidence that tumor stage was in fact the most significant factor for prognosis in patients treated with resection and transplantation (Ringe et al. 1992; El-Gazzaz et al. 2000). The biology of FL-HCC shows, nevertheless, striking differences in comparison with conventional HCC, mainly what regards metastatic patterns and causes of death (Epstein et al. 1999). A study of the Pediatric Oncology Group (Pediatric Intergroup Hepatoma Protocol), analyzing 46 patients, showed that children with FL-HCC did not have a favorable prognosis and do not respond any differently to current therapeutic regimens than patients with common HCC (Katzenstein et al. 2003). In a study of 20 resected cases, the 5-year survival was 45 %, the overall mortality was 60 %, and mortality of FL-HCC was higher with metastatic disease at presentation. Age, gender, and tumor size did not correlate with survival (Kakar et al. 2005). In another retrospective investigation, however, age was a prognosticator, in that patients diagnosed before 23 of age had a worse outcome than those diagnosed after age 23 (Moreno-Luna et al. 2005). In a study on 41 patients, median tumor size in 28 patients treated with resection was 9 cm, 50 % of the patients had lymph node metastases, and nodal metastases were the only negative prognostic factor. Seventeen of these patients (61 %) underwent a second resection for recurrent disease. The 5-year survival of resected patients was 76 %, but 5-year recurrence-free survival was only 18 %, illustrating that this tumor has a characteristic tendency for recurrence (Stipa et al. 2006). In a series of ten patients, FL-HCC was treated with resection followed by close surveillance and aggressive management of relapse. Relapse occurred in all ten cases at a median of 2.2 years. With a combination of re-resection, systemic chemotherapy, and radiotherapy, the overall median survival was 9.3 years (Maniaci et al. 2009). FL-HCC is different from common HCC in its recurrence pattern after liver transplantation. Late recurrence (>1000 days) is typical for FL-HCC, and the tumors tend to be associated predominantly with extrahepatic or combined extra- and intrahepatic recurrence, while well-differentiated HCCs tended to recur within the liver (Schlitt et al. 1999). In the recent SIOPEL/Childhood Liver Tumour Strategy Group study on 24 patients with FL-HCC, long-term overall survival in FL-HCC and ordinary HCC was similar (Weeda et al. 2013). In contrast, the results of a nationwide survey using SEER data show that surgically treated patients with FL-HCC had better long-term outcomes than those with conventional HCC (Mayo et al. 2014). In the setting of a systematic review and meta-analysis of 368 FL-HCC patients, it turned out that overall there was a significant increase in 5-year survival for FL-HCC versus conventional HCC, but this difference was not detectable in the subgroup of non-cirrhotic patients (Njei et al. 2014).

Differential Diagnosis

There are conventional HCCs having a central scar and a scalloped tumor margin resembling FNH and FL-HCC. In one study, such tumors predominantly occurred in non-cirrhotic livers of patients in their 60s and were associated with a good surgical outcome (Yamamoto et al. 2006). There are other primary liver carcinomas with a marked stromal component, which may cause diagnostic problems (Malouf et al. 2009). Histologically, sclerosing hepatocellular carcinoma may mimic FL-HCC, but these tumors have smaller epithelial cells, the stroma is more cellular and not band-like, and the tumors occur in middle-aged persons, show a male predominance, and are associated with the typical HCC risk factors (Haratake and Horie 1989).

Pathogenesis

Cell Lineage(s) Involved

The cell lineage of FL-HCC is still not well known, despite the fact that the tumor cells resemble hepatocytes with oxyphilic features and that the cells share with hepatocytes immunoreactivity for a hepatocyte marker, HepPar. Other findings that support a hepatocyte-like lineage in FL-HCC is the positivity for glypican-3, a canalicular staining pattern with polyclonal CEA/pCEA (see above), and the positivity for albumin mRNA by in situ hybridization (Ward et al. 2010). In contrast to normal hepatocytes, FL-HCC cells are usually positive for cytokeratin-7 and sometimes for cytokeratin-19 and for epithelial membrane antigen (EMA), normally expressed in cholangiocytes of the normal liver. These results have been interpreted to suggest that FL-HCCs show a dual differentiation, i.e., both hepatocellular and cholangiocellular (Ward et al. 2010). There is also immunohistochemical evidence for a stem cell-associated phenotype of FL-HCC. The tumor cells were positive for the stem cell markers CD133 and CD44, revealed reduced cell cycle progression, and displayed evidence of reduced differentiation (Zenali et al. 2010).

Cytogenetic Findings

FL-HCC generally reveals less ploidy and chromosomal aberrations than ordinary HCC and hence a greater genomic homogeneity (Lapis et al. 1990; Orsatti et al. 1994; Sirivatanauksorn et al. 2001; Ward and Waxman 2011). Specifically, 8p- and 17q+, which are typical for HCC, hardly occur in FL-HCC (Terraciano and Tornillo 2003). Also allele loss seems to be much less common that in conventional HCC (Ding et al. 1993). FL-HCCs with chromosomal alterations seem to behave more aggressively than tumors without cytogenetic abnormalities (Kakar et al. 2009). By the use of comparative genomic hybridization, chromosomal imbalances were identified in 6 out of 11 (55 %) of cases, and the mean number of aberrations per case was 3.9 for all cases and 7.2 in abnormal cases. The most common abnormalities were found in chromosomes 1, 7, 8 and 18, with gains in 1q and 8q and loss of 18q being the most common alterations (Kakar et al. 2009; review: Ward and Waxman 2011). As outlined in the next paragraph, 7p has a gene (AGR2) that is overexpressed in FL-HCC and affects cancer cell spread. Some of the tumors show complex chromosomal patterns, such as a nearly triploid karyotype (Hany et al. 1997). Genomic hybridization (CGH) studies demonstrated striking differences in recurrent cytogenetic aberrations between FL-HCC and common HCC (Wilkins et al. 2000). Loss of heterozygosity (LOH) on 6q at the mannose 6-phosphate/insulin-like growth factor II receptor locus has found in 33 % of FL-HCCs, in comparison with 69 % in common HCCs, suggesting that this gene may act as a tumor suppressor in HCC and FL-HCC (DeSouza et al. 1995). Previously (and in apparent contrast to conventional HCC), epigenetic instability manifesting as methylation of tumor suppressor promoters had been reported to be rare in FL-HCC (Vivekanandan and Torbenson 2008). In a later study, it was shown that FL-HCCs display frequent and distinct gene-specific hypermethylation in the absence of significant global hypomethylation. Frequent aberrant hypermethylation was found for the cyclin D2 and the RASSF1A genes as well as for the microRNA genes mir-9-1 and mir-9-2 (Tränkenschuh et al. 2010). The genomic features of FL-HCC loose homogeneity as a function of the tumor’s spread, and recurrent metastatic tumors have complex karyotypes (Lowichik et al. 1996).

Molecular Findings

FL-HCC displays a distinct genomic landscape (Darcy et al. 2015b) and shows a unique genomic profile uncovering three robust molecular classes, i.e., a proliferation class, an inflammation class, and an unannotated class (Cornella et al. 2015). Recently, a recurrent DNAJB1-PRKACA chimeric transcript, arising as a result of a ~400 kb deletion on chromosome 19, was detected in FL-HCC. The chimeric RNA was predicted to code for a homolog of the molecular chaperone DNAJ, fused in frame with PRKACA, the catalytic domain of protein kinase A. The chimeric protein is expressed in tumor tissue and retains kinase activity (Honeyman et al. 2014; Xu et al. 2015).

FL-HCCs display various other molecular abnormalities. FL-HCC overexpresses epidermal growth factor receptor (EGFR) , similar to conventional HCC (Buckley et al. 2008), probably due to polysomy 7 rather than gene amplification. Immunohistochemically, EGFR was strongly overexpressed on the cell membranes of more than 90 % of tumors tested (Buckley et al. 2006). FL-HCC exhibits overexpression of genes in the RAS, MAPK, PIK3, and xenobiotic degradation pathways (Kannangai et al. 2007) and reveals mTORC1 activation and FGFR1 overexpression (Riehle et al. 2015). FL-HCC cells express anterior gradient 2 (AGR2 ; Vivekanandan et al. 2009), a protein that is critical for normal embryonic development. AGR2 is located on chromosome 7p21.3 (Petek et al. 2000) and encodes the human homolog of a secreted protein identified in Xenopus (cement gland-specific gene), where it determines the formation of anterior structures during the generation of ectoderm. In the adult mammalian organism, AGR2 is mainly expressed in cells of the gastrointestinal tract. In the small intestine of mice, expression is found predominantly in Paneth, neuroendocrine, and goblet cells (Komiya et al. 1999; Wang et al. 2008). It has been shown that AGR2 encodes a protein disulfide isomerase (enzymes that aid protein folding and assembly), which is essential for the production of the intestinal mucin MUC2 (Park et al. 2009). A critical interacting protein of AGR2 is the ATP-binding protein, reptin (Maslon et al. 2010). AGR2 is expressed in several cancers, including pancreatic cancer, breast cancer, and lung cancer, where it affects cancer cell survival (via the p53 pathway), cancer cell motility, and cancer spread. Specifically, AGR2 promotes metastatic spread and its expression is associated with poor survival. This metastasis-promoting activity of AGR2 is repressed by ErbB3-binding protein 1 (Zhang et al. 2010). In FL-HCC, overexpression of AGR2 is associated with several polymorphisms, but no mutations (Vivekanandan et al. 2009). In contrast to ordinary HCC, where missense mutations and interstitial deletions of the beta-catenin gene and nuclear accumulation of beta-catenin are common, beta-catenin gene alterations were not yet detected in FL-HCCs (Terris et al. 1999), so that the Wnt-beta-catenin signaling pathway does not seem to be involved in the carcinogenesis of this tumor.

FL-HCC reveals alterations of mitochondrial structure and function. It is well known that ordinary HCCs show mitochondrial DNA mutations. In one molecular study, approximately 50 % of ordinary HCCs had lower levels of total mitochondrial DNA than paired nonneoplastic tissue, associated with deletions of the mitochondrial DNA control region. Despite their apparently increased numbers of mitochondria, primary FL-HCCs had lower levels of total mitochondrial DNA, while metastatic FL-HCCs had greatly increased mitochondrial DNA levels. Complete sequencing of the entire mitochondrial genome in FL-HCC identified several somatic mutations, but no consistent pattern of mutations was found (Vivekanandan et al. 2010).

HBV Infection

Ordinary HCC has an etiologic association with HBV in numerous cases. It was therefore of interest to test whether FL-HCC is also related to HBV infection, although this tumor is not associated with liver cirrhosis. The finding of integrated HBV DNA in certain instances of FL-HCC tissue may be consistent with an oncogenic role of HBV in at least a subset of the tumors (Davison et al. 1990; Dadke et al. 2002). This question has to be addressed in more detail with larger numbers of tumors.