REVIEW REVIEW Histopathologic Features of Hepatocellular Carcinoma Elizabeth M. Brunt, M.D. Paradoxically, with the recognized increase in hepatocellular carcinoma, liver biopsy is used less frequently for diagnosis unless there are atypical imaging characteristics. 1,2 Thus, unless pathologists practice in a center with high liver surgery volume, experience with this tumor may diminish. However, whether for tissue acquisition for management decisions of the atypical lesions, the incidental lesion encountered in a blind percutaneous biopsy, or for final diagnostic reporting of resected or transplanted hepatectomies, familiarity with the histopathologic lesions of hepatocellular carcinoma remains important for practicing pathologists. As noted in recent reviews, clinical management choices and decisions, and prognosis of primary liver carcinoma are fields of rapid growth. Arguments are now being made that for optimum clinical outcomes, management also necessitates the most reliable classification of tumors, including histopathologic evaluation. 3 5 This review discusses current criteria, useful immunohistochemistry stains, and differential diagnostic considerations for hepatocellular carcinoma. Hepatocellular Carcinoma Hepatocellular carcinoma (HCC), a malignant tumor of hepatocytes, may exhibit any or all of the cytologic and/or architectural characteristics of hepatocellular differentiation, along with features of malignancy. Identification of intercellular bile and/or canaliculi are diagnostic of the cellular origin of the tumor. Figures 1-6 show hematoxylin and eosin (H&E) stained images of routine HCC. Architectural Considerations. The trabecular and pseudoacinar patterns of HCC are the most easily recognized. The former recapitulates hepatic cords, but the width is 3 nuclei; in some cases, floating trabeculae may be appreciated in which there are apparent cross-sections of cords that are not attached or are floating freely. If considered threedimensionally, this could represent a cross-section from a complex growth of tumor with multiple projections. In nontumorous liver, this appearance is seen only in large vein outflow obstruction with sinusoidal dilatation, but the cords are 2 nuclei wide. Pseudoacinar formations may resemble cholestatic rosettes when small; pseudoacini may also be dilated and large, infrequent to numerous, empty or filled with eosinophilic material. More challenging to recognize as HCC is the compact (solid) pattern in which nearly confluent sheets of tumor are present. A characteristic feature of so-called progressed HCC is the presence of unpaired arteries within the tumor nodule. Finally, intravascular or intraductal tumor and stromal invasion are uncommon but diagnostic of malignancy. Stromal invasion is best recognized on large tissue sections of encapsulated HCC in a background of cirrhosis; keratin 7 6 and keratin 19 7 show progressive loss of the perinodular ductular reaction from cirrhotic nodules to dysplastic nodules to HCC. In some cases, apparent overrun of residual portal tracts may also be appreciated in stromal invasion in HCC. Cytologic Considerations. Malignant hepatocytes manifest almost all the features of benign hepatocytes; the exception is accumulation of iron granules. HCC in the setting of iron overload is iron-free. The cytoplasm may be deeply eosinophilic, basophilic, or clear. Cytoplasmic aggregates may include steatosis, fibrinogen (so-called pale bodies), glycogen (clear cells) or glycoprotein inclusions (þ/ periodic acid Schiff with diastase-positive), and Mallory-Denk bodies; intranuclear inclusions include glycogen, cytoplasmic pseudo-inclusions, or large nucleoli. Nuclear contours are often irregular, and the nuclear/cytoplasmic ratio is increased. Giant, multinucleated cells may occur in HCC. An important concept in HCC is that once tumors are no Abbreviations: DN, dysplastic nodule; FLC, fibrolamellar hepatocellular carcinoma; GPC-3, glypican-3; GS, glutamine synthetase; H&E, hematoxylin and eosin; HCC, hepatocellular carcinoma; HSP70, heat shock protein 70; LEL, lympho-epithelial-like carcinoma; pcea, polyclonal carcinoembryonic antigen. From the Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO Potential conflict of interest: Nothing to report. View this article online at wileyonlinelibrary.com VC 2012 by the American Association for the Study of Liver Diseases doi: 10.1002/cld.98 194 Clinical Liver Disease, Vol. 1, No. 6, December 2012 An Official Learning Resource of AASLD
FIGURE 1. H&E-stained HCC. There are examples of broad trabeculae (white arrows), pseudoglands or pseudoacini (orange arrows), and a bile plug (black arrow). Nuclear contours are irregular and large nucleoli are notable. H&E, magnification 20. FIGURE 3. HCC with large pseudoglands/pseudoacini, deeply eosinophilic cells, and thick fibrous bands coursing through the tumor. This could be confused for fibrolamellar HCC if care is not taken to know the context (cirrhosis, not shown) and features of the cells and stroma. H&E, magnification 20. FIGURE 2. HCC biopsy shows both thin trabeculae and pseudoacini. The lack of normal parenchymal portal tracts or terminal hepatic venules is the initial clue as to the neoplastic nature of this hepatocellular tumor. The pleomorphism and pseudoacini would not be acceptable for adenoma were this in a noncirrhotic liver. H&E, magnification 20. longer well-differentiated, pleomorphism of cytologic phenotype may occur throughout the tumor. Useful Stains. Table 1 lists commonly used stains for routine HCC. It is worth noting that no single stain is entirely sensitive or specific, 8 and the premier stain for diagnosis remains hematoxylin and eosin. Reticulin and immunohistochemistry stains can be used, with care, to confirm (or rarely, exclude) the diagnosis, as discussed below. Immunohistochemistry also plays a large role in the evaluation of FIGURE 4. Well-differentiated HCC (left) is separated from the nontumorous liver (right) by a dense capsule. Without appreciating the dense capsule that lacks ductular reaction, this well-differentiated HCC could be confused for a high-grade dysplastic nodule. There is marked nuclear crowding and a high N/ C ratio; all of this can best be appreciated in comparison to the adjacent nontumorous liver. H&E, magnification 10. phenotypic differentiation of liver carcinoma, as discussed below. Figures 7-10 show stains used commonly for HCC. Histologic Subtypes of Hepatocellular Carcinoma It is increasingly recognized that morphologic subtypes of liver carcinoma exist beyond the obvious broad distinctions of hepatocellular and cholangiocarcinoma. Commonly accepted subtypes are fibrolamellar HCC (FLC), scirrhous HCC, sarcomatoid HCC, and the so-called inflammatory 195 Clinical Liver Disease, Vol. 1, No. 6, December 2012 An Official Learning Resource of AASLD
HCC or lympho-epithelial-like carcinoma (LEL). 9 The first, FLC, is characterized by large polygonal tumor cells with prominent nuclei and dark nucleoli along with bands of lamellar fibrosis coursing through it. Tumor cells may contain pale body inclusions. FLC and LEL both are found predominantly in noncirrhotic livers. Scirrhous HCC, on the other hand, may be noted in livers with foci of otherwise typical HCC; the scirrhous component may be subcapsular. Neither the cytology of the tumor cells nor the character of the fibrous component are to be confused with FLC; in addition, it has been noted that treated HCC may become scirrhous in some areas. 9 These tumors are considered separately from FIGURE 5. Peliotic foci are found within the central portion of the well-differentiated HCC shown in Fig. 4. Peliotic formations are not diagnostic of HCC, as they may also occur in hepatocellular adenomas. H&E, magnification 20. FIGURE 6. Reticulin stain in a biopsy of HCC highlights loss of reticulin as well as demonstration of pseudoglands. Gomori s reticulin, magnification 20. TABLE 1: Stains for HCC Stain/IHC Primary Usefulness in HCC Other Useful Findings Comments Reticulin K8 and K18 K7 K20 CD34 pcea CD10 HEPAR1 GPC-3 Highlights loss of normal cord architecture; referred to as loss of reticulin Keratins of hepatocytes; likely not positive in most adenocarcinomas May label cells in up to 50% of cases of HCC Has been found in scattered cells in up to 20% of cases of HCC Labels the endothelial cells of the sinusoids of HCC but not nontumorous liver Labels canaliculi between benign and malignant hepatocytes; will not be as numerous as nontumorous liver Canalicular marker; specific but not as sensitive as pcea Labels mitochondrial enzyme (granular) in mature hepatocytes; thus, may be negative in HCC Useful when positive, not useful when negative; reported to be negative in hepatocellular adenoma Useful when positive, not useful when Very strong and diffuse labeling is highly suggestive of biphenotypic differentiation Is a marker of capillarization, loss of endothelial cell fenestration Cholangiocarcinoma is positive in cytoplasm For clear cell carcinoma, CD10 can be useful to evaluate for cytoplasmic reactivity expected in renal cell carcinoma Best used in comparison with nontumorous liver; easily misinterpreted and does not necessarily distinguish adenoma and HCC Not useful in the K7/K20 algorithm for evaluating carcinomas Caution: may be positive focally and/or diffusely in hepatocellular adenoma Nontumorous liver is strong positive control Nontumorous liver is strong positive control Very limited application in evaluation; may be positive in other cell types Caution: can be patchy ; has been reported in chronic hepatitis C and in DNs GS Zone 3 hepatocytes are normally Characteristic map-like pattern in FNH negative; may be positive in adenoma strongly positive for GS HSP70 Useful in combination with GPC-3 and GS* Caution: may be positive in DNs Annexin-2 Positive in hepatocytes and/or sinusoids Caution: may also be positive in similar patterns in DNs and hepatocellular adenoma Abbreviations: IHC, immunohistochemistry; K, keratin. *See text. 196 Clinical Liver Disease, Vol. 1, No. 6, December 2012 An Official Learning Resource of AASLD
FIGURE 7. CD34 immunohistochemistry demonstrates the neovascularization of this HCC. Nontumorous hepatic sinusoids do not react with CD34 antibodies, as the endothelial cells are fenestrated and do not rest on a basement membrane. This pattern of sinusoidal reactivity, however, is not in itself diagnostic of HCC, as it may also occur diffusely or in areas of hepatocellular adenoma. Anti-CD34 immunohistochemistry, magnification 10. FIGURE 9. Polyclonal carcinoembryonic antigen (pcea) immunostaining. pcea cross-reacts with biliary glycoprotein and is reactive in the canaliculi of benign and malignant hepatocytes. Thus, it is a very strong marker of hepatocellular differentiation, as no other cell type has canaliculi. When nontumorous liver is present in a biopsy or resection, it serves as a useful control. It is clear from this example that in HCC the canalicular structures are not uniform. AntipCEA, magnification 40. FIGURE 8. The darkly stained structures in this photomicrograph are keratin 19 (K19)-positive. On the right side, there is a cirrhotic nodule virtually surrounded by K19-positive ductular cells, whereas the tumor on the left of the photomicrograph has no K19 ductular reaction around the edges. For further description, see Ref. 7. Anti-K19 immunohistochemistry, magnification 10. FIGURE 10. GPC-3, an oncofetal protein that is not found in hepatocellular adenoma but may be present in HCC and DNs and has been reported in chronic hepatitis C. The reactivity is as shown: irregular within the tumor and within the cells. The reactivity may be pericanalicular, granular in the cytoplasm, or membranous. Anti-GPC-3, magnification 20. the sclerosing HCC that is associated with hypercalcemia. 9 Sarcomatoid HCC is diagnosed as such when the majority of tumor cells are spindled; this phenotype may also be a result of treatment. With enough sections, identifiable typical HCC is commonly found. 8,9 LEL may or may not be related to Epstein-Barr viral infection as other lympho-epithelial carcinomas are. 9 Varying quantities of mononuclear cells are present within these tumors. Many of the tumors have cholangiolar differentiation. Immunogenic stimuli are not understood. New Topics in Pathology of Hepatocellular Carcinoma Thanks to the careful work of our Japanese colleagues, there is now recognition of two types of HCC that are both <2 cm but have separate long-term outcomes. 10 The first, distinctly nodular, advanced, 10 or progressed HCC, 5 is the classical form of encapsulated HCC with unpaired arteries, usually moderately differentiated but with tumoral cytologic 197 Clinical Liver Disease, Vol. 1, No. 6, December 2012 An Official Learning Resource of AASLD
FIGURE 11. Schematic of the currently accepted concepts of progression of clonal populations in chronic liver disease to DNs, early HCC, and progressed HCC. Reprinted with permission from Archives of Pathology & Laboratory Medicine. 12 Copyright College of American Pathologists. FIGURE 12. Differential diagnosis of nonmetastatic nodules in adult liver. Abbreviations: AML, angiomyolipoma; AS, angiosarcoma; CCa, cholangiocarcinoma; EHE, epithelioid hemangioendothelioma; FNH, focal nodular hyperplasia; HCC, hepatocellular carcinoma; HGDN, high-grade dysplastic nodule; IPT, inflammatory pseudotumor; MRN, macroregenerative nodule. heterogeneity, and the long-term prognosis similar to its larger counterparts. CD34 is positive in sinusoids of the tumor. There is a small but documented risk of intrahepatic tumor metastases, and invasion into portal veins. The second form of small HCC is known as early HCC. 10,11 The lesion is vaguely nodular, does not have a capsule, and even though unpaired arteries are present, portal tracts are as well. These tumors receive both arterial and portal blood supply, and CD34 is not uniformly present. Compared with surrounding parenchyma, portal tract numbers are fewer. Microscopically, the tumors are well-differentiated with thin trabeculae, with or without pseudoacini, and blend imperceptibly into adjacent nontumorous parenchyma. Steatosis is common in early HCC; the N/C ratio is increased, lending 198 Clinical Liver Disease, Vol. 1, No. 6, December 2012 An Official Learning Resource of AASLD
References 1. Bruix J. Liver cancer: Still a long way to go. Hepatology 2011;54:1-2. 2. Bruix J, Sherman M. Management of hepatocellular carcinoma: An update. Hepatology 2011;53:1020-1022. 3. Parisi G. Should a radiological diagnosis of hepatocellular carcinoma be routinely confirmed by a biopsy? Yes. Eur J Intern Med 2012;23:34-6. 4. Schirmacher P, Bedossa P, Roskams T, Tiniakos DG, Brunt EM, Zucman-Rossi J, et al. Fighting the bushfire in HCC trials. J Hepatol 2011;55:276-277. 5. Roncalli M, Park YN, Di Tommaso L. Histopathological classification of hepatocellular carcinoma. Dig Liver Dis 2010;42(suppl 3):S228-S234. 6. Park YN, Kojiro M, Di Tommaso L, Dhillon AP, Kondo F, Nakano M, et al. Ductular reaction is helpful in defining early stromal invasion, small hepatocellular carcinomas, and dysplastic nodules. Cancer 2007;109:915-923. 7. Lennerz JK, Chapman WC, Brunt EM. Keratin 19 epithelial patterns in cirrhotic stroma parallel hepatocarcinogenesis. Am J Pathol 2011;179:1015-1029. 8. Bedossa P, Paradis V. Hepatocellular carcinoma. In: Saxena R, ed. Practical Hepatic Pathology: A Diagnostic Approach. St. Louis, MO: W.B. Saunders; 2011:489-501. 9. Theise ND, Curado MP, Franceschi S, Hytiroglou P, Kudo M, Park YN, et al. Hepatocellular carcinoma. In: Bosman FT, Carneiro F, Hruban RH, Theise ND, eds. WHO Classification of Tumours of the Digestive System. 4th ed. Lyon, France: International Agency for Research on Cancer; 2010:205-216. an overall crowded appearance to the involved area. This lesion may be a challenge to distinguish from dysplastic nodules, and there may be East-West discordance in individual cases. 10,11 The definitive demonstration of carcinoma is stromal invasion into the intratumoral portal tracts, but sufficient tumor sampling may not be present in a biopsy sample. Early HCC, as defined, carries a prognosis of slower tumor progression. 9 A schematic of the progression of dysplastic nodules to early and progressed HCC was published and discussed recently. 12 Figure 11 is a reprint of the schematic. With increased application of immunohistochemical panels and molecular studies, several investigators have shown the existence of liver carcinomas that share genetic and phenotypic expression of both hepatocellular and biliary differentiation, regardless of histologic findings. The tumors may be quite homogenous within themselves, or may be heterogenous with areas of clear-cut classical HCC or, less likely, intrahepatic cholangiocarcinoma. Many of the tumors seemingly have nearly blended histologic features in differing fields, while others show an abrupt transition between the two. In many cases, tumor nests, cords, and gland-like structures are present within sclerotic stroma. Terminology continues to be worked out by investigators in this field. The 2010 World Health Organization Classification of Tumors of the Digestive System 9 refers to the entire group as combined hepatocellular-cholangiocarcinoma with subgroups of classical type, and subtypes with stem cell features. Within the latter, there are three further subdivisions: typical, intermediate cell, and cholangiocellular types. Roncalli et al. 5 have proposed three types: (1) HCC with stem/progenitor cell immunophenotype; (2) mixed hepatobiliary carcinoma, classical type; and (3) mixed hepatobiliary carcinoma with stem/progenitor cell phenotype and immunophenotype. These tumors are under intensive investigation, as it is apparent they are neither uncommon nor restricted to cirrhotic (or noncirrhotic) livers. Differential Diagnostic Considerations of HCC The differential diagnostic considerations of nodules in the liver largely depend on the status of the background nontumorous liver. The possibilities for cirrhotic liver differ from those of noncirrhotic liver, as shown in Fig. 12. The potentially most challenging lesions for pathologists to distinguish from HCC in cirrhotic livers are high-grade dysplastic nodules (DN), 10,12 and in noncirrhotic livers, hepatocellular adenomas. 13 Proposals for the first type have been made, including detailed analysis of various routine histologic features such as cord thickness, vascular and capsular invasion and nuclear features, 14 or utilization of immunomarkers with heat shock protein 70 (HSP70), glutamine synthetase (GS), and glypican-3 (GPC-3) in various combinations. 15 One group has suggested the use of annexin-2. 16 Di Tommaso et al. 17 showed that in all DNs in cirrhotic livers studied, positivity for all three (HSP70, GS, and GPC-3) was never seen, but the combination correctly identified 43.7% of HCCs; 72.7% of DN were negative for all three markers. The value of two or three positive markers in liver biopsies has also been published. 11,17 Annexin-2 was most useful in its diffuse sinusoidal pattern (82% of HCCs versus 0 DNs) compared with the hepatocellular or zonal sinusoidal patterns. 16 References are provided for an in-depth review of adenoma and HCC. 13 n CORRESPONDENCE Elizabeth M. Brunt, M.D., Department of Pathology and Immunology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. E-mail: ebrunt@path.wustl.edu. 10. Kojiro M, Roskams T. Early hepatocellular carcinoma and dysplastic nodules. Semin Liver Dis 2005;25:133-142. 11. Roskams T, Kojiro M. Pathology of early hepatocellular carcinoma: conventional and molecular diagnosis. Semin Liver Dis 2010;30:17-25. 12. Park YN. Update on precursor and early lesions of hepatocellular carcinomas. Arch Pathol Lab Med 2011;135:704-715. 13. Bioulac-Sage P, Cubel G, Balabaud C, Zucman-Rossi J. Revisiting the pathology of resected benign hepatocellular nodules using new immunohistochemical markers. Semin Liver Dis 2011;31:91-103. 14. Quaglia A, Jutand MA, Dhillon A, Godfrey A, Togni R, Bioulac-Sage P, et al. Classification tool for the systematic histological assessment of hepatocellular carcinoma, macroregenerative nodules, and dysplastic nodules in cirrhotic liver. World J Gastroenterol 2005;11:6262-6268. 15. Di Tommaso L, Franchi G, Park YN, Fiamengo B, Destro A, Morenghi E, et al. Diagnostic value of HSP70, glypican 3, and glutamine synthetase in hepatocellular nodules in cirrhosis. Hepatology 2007;45:725-734. 16. Longerich T, Haller MT, Mogler C, Aulmann S, Lohmann V, Schirmacher P, et al. Annexin A2 as a differential diagnostic marker of hepatocellular tumors. Pathol Res Pract 2011;207:8-14. 17. Di Tommaso L, Destro A, Seok JY, Balladore E, Terracciano L, Sangiovanni A, et al. The application of markers (HSP70 GPC3 and GS) in liver biopsies is useful for detection of hepatocellular carcinoma. J Hepatol 2009;50: 746-754. 199 Clinical Liver Disease, Vol. 1, No. 6, December 2012 An Official Learning Resource of AASLD