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.
fibrolamellar hepatocellular carcinoma
hematoxylin and eosin
heat shock protein 70
polyclonal carcinoembryonic antigen
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.
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 three-dimensionally, 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 76 and keratin 197 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.
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 (+/− 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 longer well-differentiated, pleomorphism of cytologic phenotype may occur throughout the tumor.
Table 1 lists commonly used stains for routine HCC. It is worth noting that no single stain is entirely sensitive or specific, 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 phenotypic differentiation of liver carcinoma, as discussed below. Figures 7-10 show stains used commonly for HCC.
|Stain/IHC||Primary Usefulness in HCC||Other Useful Findings||Comments|
|Reticulin||Highlights loss of normal cord architecture; referred to as “loss of reticulin”||Best used in comparison with nontumorous liver; easily misinterpreted and does not necessarily distinguish adenoma and HCC|
|K8 and K18||Keratins of hepatocytes; likely not positive in most adenocarcinomas|
|K7||May label cells in up to 50% of cases of HCC||Very strong and diffuse labeling is highly suggestive of biphenotypic differentiation|
|K20||Has been found in scattered cells in up to 20% of cases of HCC||Not useful in the K7/K20 algorithm for evaluating carcinomas|
|CD34||Labels the endothelial cells of the sinusoids of HCC but not nontumorous liver||Is a marker of “capillarization,” loss of endothelial cell fenestration||Caution: may be positive focally and/or diffusely in hepatocellular adenoma|
|pCEA||Labels canaliculi between benign and malignant hepatocytes; will not be as numerous as nontumorous liver||Cholangiocarcinoma is positive in cytoplasm||Nontumorous liver is strong positive control|
|CD10||Canalicular marker; specific but not as sensitive as pCEA||For clear cell carcinoma, CD10 can be useful to evaluate for cytoplasmic reactivity expected in renal cell carcinoma||Nontumorous liver is strong positive control|
|HEPAR1||Labels mitochondrial enzyme (granular) in mature hepatocytes; thus, may be negative in HCC||Very limited application in evaluation; may be positive in other cell types|
|GPC-3||Useful when positive, not useful when negative; reported to be negative in hepatocellular adenoma||Caution: can be “patchy”; has been reported in chronic hepatitis C and in DNs|
|GS||Useful when positive, not useful when negative; may be positive in adenoma||Zone 3 hepatocytes are normally strongly positive for GS||Characteristic “map-like” pattern in FNH|
|HSP70||Useful in combination with GPC-3 and GSa||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|
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 HCC or lympho-epithelial-like carcinoma (LEL). 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. These tumors are considered separately from the sclerosing HCC that is associated with hypercalcemia. 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. 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. The first, distinctly nodular, advanced, or progressed HCC, is the classical form of encapsulated HCC with unpaired arteries, usually moderately differentiated but with tumoral cytologic 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 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. A schematic of the progression of dysplastic nodules to early and progressed HCC was published and discussed recently. 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 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. 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. 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, or utilization of immunomarkers with heat shock protein 70 (HSP70), glutamine synthetase (GS), and glypican-3 (GPC-3) in various combinations. One group has suggested the use of annexin-2. Di Tommaso et al. 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. References are provided for an in-depth review of adenoma and HCC.