Chapter 3 Hepatocellular Cancer: Pathologic Considerations Gregory Y. Lauwers Keywords Histology · Prognostic factors · Precursor lesions While the incidence of HCCs has been rising worldwide, there has been a steady stream of novel information related to the histologic characteristics of HCCs, includ- ing their pattern of spread, the risk factors for recurrence, and long-term prognosis. More particularly, a focus of great interest has been the diagnosis of early HCC. Understanding by histopathologists, surgeons, hepatologists, and oncologists of the nuances of the diagnosis of early HCC, as well as the importance of detailed patho- logic analysis of surgical specimens, is crucial to developing appropriate therapeutic algorithms based on precise prognostic stratification. Macroscopic Features of Hepatocellular Carcinoma Variations in the morphology of HCC are related to the size of the tumor and whether the surrounding liver is cirrhotic. Western series have emphasized that between 42 and 51% of HCCs arise in non- cirrhotic livers [1, 2]. However, some of the “noncirrhotic” cases may be better characterized as associated with limited fibrosis. Differences in the multiplicity of tumors, incidence of encapsulation, and rate of venous invasion have been reported in this group of tumors. Also, HCCs in noncirrhotic livers may grow faster and in general are larger than those in cirrhotic livers [3, 4]. In cirrhotic livers, small HCCs may be well demarcated and surrounded by a fibrous capsule, whereas advanced tumors are expansive multinodular masses, frequently accompanied by intrahepatic metastases [5]. In noncirrhotic livers, HCCs usually present as single large tumors that may infiltrate both lobes [2, 3]. G.Y. Lauwers (B) Department of Pathology, Massachusetts General Hospital, Boston, MA, USA 35 K.M. McMasters, J N. Vauthey (eds.), Hepatocellular Carcinoma, DOI 10.1007/978-1-60327-522-4_3, C Springer Science+Business Media, LLC 2011 36 G.Y. Lauwers The risk of intrahepatic and extrahepatic spread is related to the size of the tumor [6–8]. HCCs less than 5 cm in size are less likely to develop intrahepatic metastasis, portal vein tumor thrombosis, or hematogenous metastasis [6, 8, 9]. Conversely, the incidence of portal vein thrombosis rises f rom 40 to 75% when HCCs grow larger than 5 cm, and the rate of intrahepatic metastasis rises dramatically (60% vs. 96%) [6, 7]. Most HCCs are soft neoplasms, often displaying hemorrhage and necrosis. Their color ranges from tan-gray to green, the difference reflecting the degree of bile pro- duction [10]. Peritumoral capsule is found in 46% of HCCs measuring l ess than 2 cm and 84% of tumors between 2 and 5 cm in size. A capsule is present in only 45% of HCCs measuring more than 5 cm in diameter [11]. Peritumoral capsule is associated with improved survival, lower rate of intrahepatic recurrence, and lower incidence of venous invasion [1, 12]. Macroscopic Classification of HCCs The different patterns of growth are associated with various risks of spread, both intrahepatic and extrahepatic [6]. Eggel’s classification, published in 1901, remains widelyused[13]. HCCs are divided into nodular, massive, and diffuse types. The nodular type consists of well-circumscribed tumor nodules. Massive HCCs are circumscribed, huge tumor masses occupying most or all of a hepatic lobe. This type is commonly observed in patients without cirrhosis. The diffuse type is rare and characterized by innumerable indistinct small nodules studding the entire liver. Subsequently, the Liver Cancer Study Group of Japan has proposed a modification, with the nodular category being divided into three subtypes: single nodular, single nodular type with perinodular tumor growth, and the confluent multinodular subtype [10] (Fig. 3.1) HCC Is a Multicentric Disease Multicentricity is noted in 16–74% of HCCs resected in cirrhotic liver [6, 11, 14– 17]. In contrast, multifocality is reported to be only 12% in noncirrhotic liver [3]. Tumor multiplicity can be explained by either the metachronous development of tumors (i.e., multicentric carcinogenesis) or intrahepatic metastases via the por- tal system [18, 19]. Tumor nodules are considered metastatic if (a) they show a portal vein tumor thrombus or grow contiguously with a thrombus, (b) multiple small satellite nodules surround a larger main tumor, or (c) a single lesion is adja- cent to the main tumor but is significantly smaller in size and presents the same histology [18]. Intravascular and Biliary Growth Malignant thrombosis of the portal vein system plays a role in the development of intrahepatic metastases. Most patients develop recurrence within 1 year and die 3 Hepatocellular Cancer: Pathologic Considerations 37 Fig. 3.1 Macroscopic appearance of hepatocellular carcinoma (HCC). Example of large single nodular lesion involving most of a lobe. Note that the surrounding liver was not cirrhotic within 2 years after surgery [9, 20]. In some cases with thrombosis of the hepatic veins, the malignant thrombus may extend into the inferior vena cava and the right atrium [21]. Tumor extension into the hepatic duct or common bile duct or both is also rare. Patients may develop obstructive jaundice or hemobilia, at times leading to a misconstrued preoperative diagnosis of cholangiocarcinoma or choledocholithiasis [22, 23]. Microscopic Features of Hepatocellular Carcinoma Neoplastic hepatocytes exhibit various degrees of hepatocellular differentiation. They usually are polygonal with abundant eosinophilic and granular cytoplasm surrounded by distinct cell membranes. Characteristically, the nucleus is round and vesicular with a distinct nucleolus. Various intracytoplasmic inclusions can be observed. Glycogen, fat, bile, fibrinogen (pale bodies), Mallory bodies (accumula- tion of keratin and p62 stress protein) and intracellular hyaline bodies (accumula- tions of p62 stress protein), α-fetoprotein (AFP), giant lysosomes, or α 1 -antitrypsin have been reported [24, 25]. A trabecular arrangement mimicking normal hepatic cords is the basic architec- tural growth pattern of HCCs. The histologic appearance is variable, however. 38 G.Y. Lauwers Histologic Patterns of HCC The World Health Organization classification recognizes five major histologic subtypes [24]. Except for the fibrolamellar pattern, their significance is more of diagnostic value than indicative of prognosis [24]. The four other subtypes, fre- quently found simultaneously, are trabecular, pseudoglandular (acinar), compact, and scirrhous. The trabecular and acinar patterns are commonly observed in well t o moderately differentiated HCCs. The trabeculae can vary from a few cells thick (microtrabecu- lar pattern) to more than a dozen cells (macrotrabecular pattern) and are separated by sinusoid-like spaces lined by flat endothelial cells (Fig 3.2). In the acinar (pseudog- landular) variant, the cells are arranged in a rosette-like fashion with a central bile canaliculus (Fig 3.3). In the solid type, the sinusoids are compressed and obscured by the broad and compact trabeculae. Finally, the scirrhous pattern is characterized by abundant fibrous stroma separating cords of tumor cells. This pattern can be seen after radiation, chemotherapy, or infarction. Various degrees of the scirrhous pattern are found without any previous treatment in approximately 4% of cases [10, 24]. Histologic Grading of Hepatocellular Carcinomas The Edmondson grading scheme is based on the degree of differentiation of the neo- plastic cells [26]. Tumors with well-differentiated neoplastic hepatocytes arranged in thin trabeculae correspond to grade I (Fig 3.4). In grade II, the larger and more atypical neoplastic cells are sometimes organized in an acinar pattern. Architectural and cytologic anaplasia are prominent in grade III, but the neoplastic cells are readily identified as hepatocytic in origin. When composed of markedly anaplas- tic neoplastic cells not readily identified as hepatocytic origin, the tumor is grade IV (Fig. 3.5) Fig. 3.2 Macrotrabecular growth pattern of hepatocellular carcinoma composed of wide anastomosing cords wrapped by endothelial cells 3 Hepatocellular Cancer: Pathologic Considerations 39 Fig. 3.3 Pseudoglandular (acinar) pattern of hepatocellular carcinoma. The neoplastic hepatocytes are moderately atypical (grade II), and bile plugs are identified in the lumen Fig. 3.4 Grade I (well-differentiated) hepatocellular carcinoma. The well-differentiated neoplastic hepatocytes show minimal cytologic and architectural atypia. Note the scattered acinar structures An alternate four-tier histologic grading scheme is advocated by the Liver Cancer Study Group of Japan [10]. In this classification, well-differentiated HCCs that com- monly measure less than 2 cm in diameter demonstrate an increased cellular density. The small neoplastic cells are organized in irregular microtrabeculae, and focal aci- nar formation can be seen. Frequent fatty macrovesicular changes can be seen as well. Cellular and nuclear atypia are distinctly absent. Moderately differentiated HCCs are composed of neoplastic hepatocytes displaying abundant eosinophilic cytoplasm with round nuclei and distinct nucleoli. Notably, the nucleus to cytoplasm ratio is equal to that of the normal hepatocytes. These hepatocytes are organized in either trabeculae or pseudoglands. Poorly differentiated HCCs usually grow in a solid sheet-like pattern. The hep- atocytes show an increased nucleus to cytoplasm ratio. Cellular pleomorphism is noticeable, with mononucleated or multinucleated giant cells, or both. 40 G.Y. Lauwers Fig. 3.5 Spindle, sarcomatoid, high-grade variant of HCC. Spindle neoplastic cells mimicking a sarcoma are intermixed with bizarre multinucleated cells Undifferentiated HCCs are composed of tumor cells with little cytoplasm and short spindle-shaped or round nuclei. They grow in a solid or medullary pattern. The prognostic value of histologic grading is debated. Some authors report better prognosis for low-grade HCC, whereas others contest a correlation between poor prognosis and high histologic grade [1, 27]. Edmondson–Steiner grading has not been found to be a predictor of intrahepatic recurrence by some, whereas a high histologic grade has been estimated by others to be a strong predictor of portal vein invasion [12, 28]. It has also been claimed that the odds of having a high-grade HCC are twice as high for cirrhotic patients compared to those without cirrhosis [1]. Cytologic Subtypes of HCC The clear cell variant results from excessive intracytoplasmic deposits of glycogen [24]. Clear cells may be composed of only a limited portion of an otherwise typical HCC or t he entire tumor. A reportedly favorable prognosis has not been confirmed [29, 30]. Pathologists should distinguish clear cell HCC from metastatic renal cell and adrenocortical carcinomas. Pleomorphic HCCs display marked variations in shape and size of the neoplas- tic hepatocytes. Seemingly benign giant cells (osteoclast-type) or highly anaplastic bizarre cells can be observed [24]. Spindle (or sarcomatoid) tumor cells with features resembling fibrosarcoma, leiomyosarcoma, and malignant fibrous histiocytoma can be seen [24] (Fig. 3.5). Arterial chemotherapy has been implicated in the genesis of 3 Hepatocellular Cancer: Pathologic Considerations 41 this phenotype [21]. Distinction from a sarcoma is largely dependent on the identi- fication of foci morphologically typical for HCC. The differential diagnosis can be challenging, since epithelial markers (i.e., cytokeratin) are recognized in only 62% of cases [31]. Vascular Invasion is an important prognostic indicator, in part because intra- hepatic metastases occur through portal vein invasion. In its absence, the patients experience a longer overall and disease-free survival [17, 27, 32, 33]. Risk factors for portal vein invasion include tumor diameter greater than 3 cm, high histologic grade, tumor multiplicity, and high mitotic activity (>4 mitoses per 10 HPF) [28, 34] (Fig. 3.6) Fig. 3.6 Malignant tumor emboli in a small portal vein. Vascular invasion is a risk factor for intrahepatic metastasis and multicentricity Histologic Variants of Hepatocellular Carcinoma Fibrolamellar Carcinoma Fibrolamellar HCC is a rare variant of HCC (less than 5% of all cases). These tumors occur at a young age and are not associated with common risk factors such as chronic hepatitis and cirrhosis [35–38] and thus are frequently amenable to surgical resection. The extended survival compared to that of usual HCCs is likely related to the absence of cirrhosis rather than distinct biologic characteristics [38–41]. Overall 5-year survival is estimated at between 35 and 76% for patients undergoing hepatic resection [38–41]. However, a recent series pointed out that with frequent vascu- lar invasion (36%) and lymph node metastases (50%), late recurrences are common and the 5-year recurrence-free survival was only 18% [40]. Fibrolamellar carcino- mas are firm, sharply demarcated, and usually single tumors. They range in size from 7 to 20 cm [36, 41]. The surrounding parenchyma is frequently unremarkable, with cirrhosis reported in less than 5% of cases [36]. The characteristic histologic 42 G.Y. Lauwers features include large polygonal and deeply eosinophilic tumor cells embedded in hyalinized connective tissue commonly arranged in a lamellar fashion. The cells display single round vesicular nuclei with prominent nucleoli. They may also con- tain α 1 -antitrypsin, seen as proteinaceous cytoplasmic inclusions, and fibrinogen containing pale bodies, presenting as pale ground-glass cytoplasmic inclusions [24, 36, 41]. Combined Hepatocellular Carcinoma and Cholangiocarcinoma Combined HCC and cholangiocarcinomas (combined HCC–CC) contain unequiv- ocal elements of both HCC and CC [24]. Two types are recognized: HCC- predominant (the most frequent) and CC-predominant variants [42] (Fig. 3.7). These tumors show variable combinations of characteristic features of HCC, i.e., bile production, intercellular bile canaliculi, or a trabecular growth pattern, as well as elements of cholangiocarcinoma, such as glandular structures lined by biliary type epithelium; intracellular mucin production; or immunoreactivity for MUC-1, CK 7, and CK19 [42–45]. AFP levels are usually low, whereas an increase in serum carcinoembryonic antigen and carbohydrate antigen 19-9 can be detected [42, 46]. Cirrhosis is associated with most cases of the HCC-predominant type (55% of cases) and only occasionally with the CC-predominant type (13% of cases) [42]. These combined neoplasms may be more common in the setting of genetic hemochromato- sis. Reflecting the common embryologic origin of hepatocytes and cholangiocytes, two mechanisms of histogenesis have been hypothesized: (a) t he CC component could differentiate from an initial pure HCC or (b) an intermediate “stem cell” cell could give rise to both HCC and CC components [42, 43, 47]. Support for the latter hypothesis includes the presence of hepatic progenitor cells as well as the detection Fig. 3.7 Combined hepato- cellular/cholangiocarcinoma. Note the large anastomosing trabeculae of HCC surrounded by anastomosing malignant ductular structures 3 Hepatocellular Cancer: Pathologic Considerations 43 of hepatocellular (albumin RNA) and biliary markers (keratin profile) in combined HCC [47, 48]. Precursor Lesions A multistep carcinogenesis sequence of low- and high-grade dysplastic nodules and well-differentiated HCC is largely accepted as the morphologic process preceding the development of HCCs in cirrhotic livers. Low-grade dysplastic nodules are distinct from surrounding cirrhotic nodules. Their size usually varies between 0.5 and 1.5 cm, although large examples have been reported. They are not encapsulated [49], but condensation of peripheral fibrous tissue is noted. These nodules are distinguishable from cirrhotic nodules by the presence of dysplastic, architectural, and cytologic features, i.e., mild increase in cellular density of monotonous hepatocytes, usually with no cellular atypia. Architecturally, dysplasia refers to the presence of minimally thick cell plates, but acinar formation or macrotrabeculae are absent [50]. Rare unpaired arteries can be seen. Large cell changes (formerly referred to as large cell dysplasia) can be seen. These consist of cellular enlargement with nuclear pleomorphism and frequent multinucleation [51]. Differences between simple macroregenerative and dysplastic nodules can be challenging, especially on needle biopsies. Subtle nuclear atypia with densely packed, smaller-than-normal hepatocytes with increased cel- lular density, sometimes twice normal as in the surrounding tissue, are helpful hints. Thickening of the nuclear membrane, higher nucleus to cytoplasm ratios, and rare mitoses are also seen [52–56]. Architectural atypia range from irregular trabecular patterns to minimally thickened trabeculae or pseudoglandular forma- tion. High-grade dysplastic nodules can be either vaguely nodular or distinctly nodular lesions. Architectural and cytologic atypia are present but insufficient to merit a diagnosis of well-differentiated HCC. They commonly display increased cell density, cytoplasmic eosinophilia, and irregular thin trabeculae. Another notable feature is the increased number of unpaired muscularized arteries. However, differ- entiating these lesions is difficult, and significant overlap with early HCCs is seen (Fig. 3.8a,b). Early HCCs may develop within dysplastic nodules, initially preserving a seemingly normal cytologic and architectural pattern. These HCCs, by definition, measure less than 2 cm, and most have only a vaguely nodular morphology [10, 57]. They are extremely well-differentiated, with little cellular and structural atypia [10, 58, 59]. Subtle diagnostic changes include increased cell density (more than twice that of surrounding tissue) and increased nucleus to cytoplasm ratio. Cytoplasmic eosinophilia, fatty or clear cell changes, or both, as well as iron-free foci, can also be noted [10, 58–60]. An irregular thin trabecular pattern, acinar patterns, or both can be seen [10, 58, 59](Fig3.9a,b). Another notable feature is the increased number of unpaired muscularized arteries [60]. Vascular invasion is uncommon, 44 G.Y. Lauwers a b Fig. 3.8 (a) Vaguely nodular lesion characteristic of dysplastic nodules and early well- differentiated HCC. (b) The nodule is composed of hepatocytes with minimal cytologic atypia with slight increased cellular density and anastomosing one-cell-thick trabeculae with rare acinar structures a b Fig. 3.9 (a) Example of well-differentiated HCC. The neoplastic hepatocytic proliferation shows cytoarchitectural atypia, highlighted in 9b. (b) Reticulin stain demonstrating focal loss and disarray of reticulin fibers but “stromal invasion” of intratumoral portal spaces can be observed as the tumors enlarge [57, 59]. As they grow in size, fatty changes become uncommon and cel- lular dedifferentiation appears. The less differentiated component usually arises as a central subnodule expanding in a nodule-in-nodule fashion. It proliferates expansively, whereas the peripheral well-differentiated rim is compressed and even- tually replaced [42, 58, 59, 61]. Approximately 40% of HCCs measuring between 1 and 3 cm consist of more than two patterns of varying differentiation [42] (Fig. 3.10). . of hepatocellular carcinoma composed of wide anastomosing cords wrapped by endothelial cells 3 Hepatocellular Cancer: Pathologic Considerations 39 Fig. 3.3 Pseudoglandular (acinar) pattern of hepatocellular. inclusions, and fibrinogen containing pale bodies, presenting as pale ground-glass cytoplasmic inclusions [24, 36, 41]. Combined Hepatocellular Carcinoma and Cholangiocarcinoma Combined HCC and cholangiocarcinomas. 23]. Microscopic Features of Hepatocellular Carcinoma Neoplastic hepatocytes exhibit various degrees of hepatocellular differentiation. They usually are polygonal with abundant eosinophilic and granular cytoplasm surrounded