—  AMERICAN SOCIETY FOR CLINICAL PATHOLOGY   —

The Morphologic Distinctions Between Hepatocellular Carcinoma
and Metastatic Carcinomas in Fine Needle Aspiration Cytology


Kim R. Geisinger
Wake Forest University
Winston-Salem, NC


The liver is one of the major targets for deep organ fine needle aspiration biopsies. The majority of these are performed to diagnose malignant neoplasms involving the liver. Hepatocellular carcinoma (hepatoma) accounts for only 2% to 3% of all cancers involving the liver in the United States. Still, hepatoma is the most frequent primary malignancy of this organ. Furthermore, hepatoma is much more frequent in some parts of the world such as subSaharan Africa and Southeast Asia. Ninety-five percent of all hepatic malignancies represent metastases. Common primary sites include adenocarcinomas of the stomach, pancreas, and large bowel. These organs, as well as others, have direct portal drainage and thus, the liver is a common site for metastatic disease.

Both hepatoma and metastatic carcinomas are associated with a poor prognosis. Yet, the distinction between the two has clinical importance from both therapeutic and prognostic viewpoints. The prognosis is extremely dismal in this country for individuals with a hepatoma, whereas a variety of therapeutic interventions have improved the long term outlook for individuals with metastatic carcinoma, especially those with a solitary metastasis in the right lobe.

As a general introduction, the aspiration cytology of hepatoma correlates to an extent with the histologic grade of this neoplasm. [1] Moderately differentiated hepatomas are usually easy to diagnose in aspiration smears. On the other hand, it may be very difficult at times to distinguish benign hepatocytes from a well differentiated hepatoma. [1, 2] At the other end of the spectrum are poorly differentiated hepatocellular carcinomas. Here the problem is distinguishing it from a metastasis.

Another aspect of the distinction between hepatoma and metastases is related to morphologic variants of this primary malignancy. The latter include the small cell type, the clear cell type, and pleomorphic hepatocellular carcinoma. [1, 2, 3, 4]

Well to moderately differentiated hepatocellular carcinomas are typically composed of tumor cells with polygonal contours and sharply defined cellular borders. [1, 2] In most cells, the cytoplasm is opaque and granular in quality and scant to moderate in volume. A proportion of these malignant cells may manifest cytoplasmic inclusions, including fat vacuoles and lipofuscin. Characteristically, the tumor cells possess one or more centrally positioned nuclei which may appear relatively perfectly round. The tumor cells are said to be more hyperchromatic and to possess more prominent nucleoli than benign hepatocytes. A consistent attribute of hepatoma is an increase in the nuclear-to-cytoplasmic ratios of the vast majority of the malignant cells (compared to benign hepatocytes). Another important morphologic feature is the association of endothelial cells with the malignant hepatocytes. Trabeculae can be defined as aggregates of neoplastic hepatocytes which are at least partially surrounded by endothelial cells. In addition, sinusoidal capillaries are frequently seen separating masses of hepatoma cells. Interestingly, the endothelial cells in hepatoma may be phenotypically (and possibly genetically) different from those in benign liver. [5] The smear background frequently contains large hepatocytic nuclei, at times with distinct nucleoli, and without any visible attached cytoplasm.

In many instances, the distinction between hepatoma and a metastasis is straightforward on the basis of routine microscopy. In an attempt to enhance our ability to make this distinction, Bottles et al evaluated a series of hepatomas and metastases in aspiration biopsies by logistic regression analysis. [6] Three features emerged as significant for hepatoma: polygonally shaped tumor cells with centrally positioned nuclei, tumor cells separated by sinusoidal capillaries, and cytoplasmic bile; the last feature was 100% specific but relatively insensitive. This series included metastatic carcinomas, lymphomas, sarcomas, and melanomas.

More recently, Salamào evaluated 52 hepatomas and 56 metastatic adenocarcinomas for 11 cytomorphologic features; this data was also evaluated by regression analysis. [7] Two features emerged as significant for hepatoma: polygonally shaped tumor cells with centrally positioned nuclei and sinusoidal capillaries separating the neoplastic hepatocytes. In addition, one feature was significant for metastatic adenocarcinoma, namely, the presence of lumen formation.

The Salamào study also investigated the variability in identification of specific cytomorphologic features by the individual five pathologists. [7] For example, the overall prevalence for the presence of malignant cells with polygonal contours and round, central nuclei was 68% of the specimens. However, the range was from 56% to 74% among the five individuals. Similarly, the overall prevalence for the presence of sinusoidal capillaries was 42%, with a range of 26% to 51%.

Although quite uncommon, primary adenocarcinoma of the liver (cholangiocarcinoma) needs to be distinguished from both hepatoma and metastatic adenocarcinoma. [1] The former is generally easy in that cholangiocarcinomas are usually well differentiated histologically, frequently showing well formed glands with central lumens. The difficulty arises in distinguishing cholangiocarcinoma from metastatic adenocarcinoma in smears. We are unaware of any single cytomorphologic attribute which allows one to make this distinction clearly. Furthermore, immunocytochemistry is generally not of value (except for distinguishing it from colonic carcinomas which are usually negative for cytokeratin 7). In contrast to colonic carcinomas, the smear background in cholangiocarcinomas is usually clean in that it lacks a tumor diathesis. In addition, smear cellularity may be relatively low, compared to many metastases.

One of the most common morphological variants of hepatocellular carcinoma is the fibrolamellar form. This type of carcinoma should not be difficult to distinguish from conventional hepatocellular carcinoma. The clinical history and the aspiration cytomorphology is quite suggestive.

Fortunately, many of the other morphologic variants of hepatocellular carcinoma are quite uncommon. One of these is the small cell variant, which I believe represents a high grade hepatocellular carcinoma. I have now personally examined a small subset of these neoplasms. In the vast majority, intercellular cohesion is greatly reduced. Accordingly, most of the specimen consists of individually dispersed neoplastic cells. The latter have solitary round nuclei and rather high nuclear-to-cytoplasmic ratios. Thus, it presents as a small cell malignancy. Most of the neoplastic cells will not demonstrate, by routine light microscopy, differentiation as hepatocellular carcinoma. In these cells, the nuclei are often peripherally located in the scanty cytoplasm. I have now witnessed a couple of examples in which cohesion is much better preserved. In any case, one prudent step is to examine the entire specimen carefully for a small proportion of tumor cells which demonstrate more distinct hepatocellular differentiation. This usually manifests as isolated malignant cells with greater volumes of granular, dense cytoplasm and centrally positioned nuclei.

The differential diagnosis of small cell hepatocytic carcinoma may fall into two different categories, based largely on patient age. In adults, one needs to consider primarily metastatic neoplasms, including small cell carcinoma of bronchogenic origin, islet cell neoplasms, lymphoreticular tumors, and other relatively infrequent neoplasms.

For small cell carcinoma of the lung, the clinical history is important. Morphologic attributes which may assist include neoplastic cells which possess exceedingly high nuclear-to-cytoplasmic ratios, nuclear molding, a lack of nucleoli, and a distinctly granular (salt and pepper) chromatin pattern. In addition, immunocytochemistry may be exceedingly useful. In this setting, the finding of a positive reaction for TTF-1 can be extremely useful.

The differential diagnosis also includes pancreatic endocrine neoplasms (islet cell tumors). This may be more difficult, based purely on morphology, than small cell carcinoma of the lung. This is because the neoplastic cells possess greater volumes of cytoplasm and often have an eccentric nucleus which is perfectly round. A more distinctive plasmacytoid appearance of the tumor cells may suggest a well differentiated neuroendocrine carcinoma. Endothelial cell associations, in addition, would not be expected with these pancreatic primaries. Of course, immunocytochemistry pointing to neuroendocrine differentiation would be most appreciated.

A neoplasm which closely resembles pancreatic endocrine neoplasms based purely on cytomorphology is the primary granulosa cell tumor of the ovary. One distinctive feature is the finding of well developed longitudinal nuclear grooves, which are typically more evident on the Papanicolaou stained material. In addition, Call-Exner bodies may be observed in aspiration smears. Once again, immunocytochemistry could prove most useful. One also needs to consider the possibility of nonHodgkin's lymphomas and leukemias involving the liver. These may present as individually dispersed cells with high nuclear-to-cytoplasmic ratios and small but distinct nucleoli, causing confusion with the small cell variant of hepatoma. However, true intercellular cohesion will be lacking, nuclear contours may be more irregular (better seen on the Papanicolaou stained specimens), and lymphoglandular bodies will be present in the smear background. In our experience, most of these represent large cell lymphomas of the B-cell type. A significant proportion of these are first discovered as a mass within the liver.

In the pediatric population, lymphoreticular malignancies also are important in hepatic aspirates. The same cytomorphologic attributes should allow one to distinguish these neoplasms from hepatoma. In addition, immunophenotyping by immunocytochemistry or flow cytometry can be conclusive.

One also needs to consider metastatic neuroblastoma. Several distinct morphologic features may be helpful in distinguishing this from the small cell variant of hepatoma. In neuroblastoma, the solitary malignant nuclei typically have distinctly granular, hyperchromatic chromatin. Nucleoli may or may not be evident. These cells possess exceedingly high nuclear-to-cytoplasmic ratios. Other helpful clues include identifying larger ganglion-like neoplastic cells. The latter possess polygonal contours, moderate to abundant (and at times distinctly granular) cytoplasm, and one or more peripherally located nuclei. The latter typically have a round contour, vesicular chromatin, and prominent nucleoli. Finally, the smear background may contain neuropil. This is better appreciated with the Diff-Quik stain, where it presents as brilliantly eosinophilic, fibrillary material. Finally, one must consider the rare hepatoblastoma. On a cell for cell basis, this may be difficult to distinguish from the small cell variant of hepatoma. Fortunately, this differential diagnosis is exceedingly uncommon. In hepatoblastoma, the neoplastic cells, overall, are smaller than with hepatoma. In addition, in general, cohesion is better preserved with hepatoblastoma.

Another morphologic variant of hepatoma is the pleomorphic type. This characteristically consists of absolutely bizarre-appearing tumor giant cells with one or more nuclei intermingled with (usually more numerous) smaller neoplastic hepatocytes. The malignant giant cells may or may not manifest some degree of hepatocellular differentiation.

The differential diagnosis includes the pleomorphic or sarcomatoid forms of carcinoma, as may arise in organs such as the lung, thyroid, pancreas, and kidney. With these metastases, intercellular cohesion is almost completely lacking, there is no evidence of hepatocytic differentiation, and neutrophils are a frequent and prominent component of the smears. The latter may include cytophagocytosis of the segmented leukocytes by the malignant tumor cells. One would also have to consider the possibility of a metastasis of a pleomorphic soft tissue sarcoma. [8] In the latter situation, obviously, the clinical history is paramount.

One other morphologic, thankfully rare, variant of hepatocytic carcinoma is the clear cell type. These typically resemble, for the most part, the conventional hepatocytic carcinoma except that the cytoplasm is not dense and granular. Rather, it appears optically clear. Other cancers with moderate to abundant clear cell cytoplasm obviously fall into this differential diagnosis. In this setting, the architectural arrangement of the cells (including endothelial associations), immunocytochemistry, and a lack of a primary neoplasm elsewhere are crucial.

In a significant proportion of cases, the distinction between metastatic adenocarcinoma and hepatoma can be made based purely on convention light microscopy with direct smears. At times, the architectural arrangement seen in cell blocks is also useful. Perhaps more importantly, cell blocks may be used for immunocytochemistry. Immunocytochemistry is the most widespread ancillary diagnostic procedure in this setting. A number of antibodies have been utilized in the distinction of metastatic carcinoma and hepatoma. One of the most widely utilized is polyclonal carcinoembryonic antigen (CEA). Polyclonal CEA characteristically produces a canalicular decoration with moderate levels of sensitivity and high specificity. More recently, immunostaining with CD10 resulted in results that parallel those of polyclonal CEA. Various cytokeratins may be useful in this distinction. Most hepatomas express low molecular weight cytokeratin but are nonreactive with the high molecular weight forms. If a tumor is positive for cytokeratin 20 and negative for cytokeratin 7, this is strong support for a colorectal or appendiceal primary. A number of individuals have touted the use of alpha fetoprotein (AFP) due to its high level of diagnosistic specificity. However, in my experience as well as that of many others, it possesses excessively low diagnostic sensitivity. Markers of endothelium such as CD34 or CD31 may prove useful in highlighting endothelial cells wrapping about neoplastic hepatocytes. Perhaps most useful is the antibody HepPar-1. HepPar-1 is a monoclonal antibody apparently directed against an antigen specific for the mitochondria in hepatocytes. HepPar-1 staining thus is quite specific and also possesses a high level of diagnostic sensitivity.

The use of immunocytochemistry in conjunction with the clinical picture, laboratory values, and the aspiration cytomorphology generally allows one to reliably and readily make the distinction between primary hepatocellular carcinoma and metastatic carcinomas.

References

  1. Geisinger KR, et al. Liver. In Modern Cytopathology, Churchill Livingston, 2004, New York, pp. 505-542.

  2. Wee A, Sampatanukul P. Fine Needle Aspiration Cytology of the Liver. Diagnostic Algorithms. A Southeast Asian Perspective. Year Book Publishers, 2004, Bangkok, Thailand.

  3. Singh HK, et al. Fine-needle aspiration morphology of clear-cell hepatocellular carcinoma. Diagn Cytopathol 1997;17:206.

  4. Hughes JH, et al. The role of fine needle aspiration cytology in the evaluation of clear cell tumors. Cancer Cytopathol 1999;87:380-389.

  5. Chen X et al. Novel endothelial cell markers in hepatocellular carcinoma. Mod Pathol 2004;17:1198.

  6. Bottles K et al. A step-wise logistic regression analysis of hepatocellular carcinoma. An aspiration biopsy study. Cancer 1988;62:558.

  7. Salamào DR et al. Reproducibility of proposed cytologic criteria to discriminate hepatocellular carcinoma from metastatic adenocarcinoma in fine needle aspiration. Act Cytol 2001;45:857.

  8. Geisinger KR, Abdul-Karim FW. Fine Needle Aspiration Biopsies. In: Weiss SW, Goldblum JR. Enzinger and Weiss's Soft Tissue Tumors, 4th ed. Mosby, 2001, St. Louis, pp. 147-158.

  9. Minervini MI, et al. Utilization of hepatocyte-specific antibody in the immunocytochemical evaluation of liver tumors. Modern Pathol 1997;10:686.

  10. Lampe LW, Folpe AL. The diagnostic value of hepatocyte paraffin antibody 1 in differentiating hepatocellular neoplasms from nonhepatic tumors: a review. Adv Anat Pathol 2003;10:39.