January 2006, Vol. 13, No.

1 Cancer Control 13
Introduction
The liver is one of the most common sites for metasta-
tic disease, accounting for 25% of all metastases to solid
organs.
1
In the United States and Europe, secondary
liver neoplasms are far more common than primary
hepatic neoplasms. In the adult oncology patient, most
are metastatic carcinomas, of which adenocarcinomas
are the predominant subtype, followed by squamous
cell carcinomas and neuroendocrine carcinomas.
Pathology of Liver Metastases
Barbara A. Centeno, MD
Background: The liver is the most frequent site of metastatic disease, and metastatic disease to the liver is far
more common than primary liver carcinoma in the United States. Pathologic evaluation of biopsy samples is
key to establishing a correct diagnosis for patient management. Morphologic and immunoperoxidase studies,
which are the standard for pathologic practice, accurately classify most tumors. Subclassification of carcinoma
of unknown primary remains problematic.
Methods: The author reviewed the literature for articles pertaining to liver biopsy, diagnosis of specific tumor
types, utility of immunohistochemical markers, and microarray and proteomic analysis.
Results: Sampling of liver lesions is best accomplished by combining fine-needle aspiration and needle core
biopsy. Many malignancies have distinct morphologic and immunohistochemical patterns and can be correctly
subclassified. Adenocarcinoma of unknown primary remains enigmatic since current immunohistochemical
markers for this differential diagnosis lack specificity. Microarray analysis and proteomic analysis of tumors
can provide distinct gene or protein expression profiles, respectively, for tumor classification. These technologies
can be used with fine-needle aspiration and needle core biopsy samples.
Conclusions: Most metastatic malignancies in the liver may be correctly diagnosed using standard morphology
and immunohistochemical techniques. However, subtyping of some carcinomas and identification of site of
unknown primary remains problematic. New technologies may help to further refine our diagnostic capabilities.
The science of pathologic diagnosis
of metastatic malignancies in the
liver has progressed.
Annie Toja. Nice, France, The Old Port. Acrylic.
From Pathology Services at the H. Lee Moffitt Cancer Center &
Research Institute, Tampa, Florida.
Submitted June 29, 2005; accepted November 30, 2005.
Address correspondence to Barbara A. Centeno, MD, Pathology Ser-
vices, 12902 Magnolia Drive, MCC Room 2071 H, Tampa FL 33612.
E-mail: [email protected]
No significant relationship exists between the author and the com-
panies/organizations who products or services may be referenced
in this article.
Abbreviations used in this paper: FNA = fine-needle aspiration,
NCB = needle core biopsy.
January 2006, Vol. 13, No. 1 14 Cancer Control
Other tumor types that metastasize to the liver include
melanomas, lymphomas, and rarely sarcomas.
Radiologically, metastatic disease presents as multi-
ple liver lesions, but a solitary liver lesion in the adult
oncology patient in the United States is also most like-
ly to be a metastasis. A tissue diagnosis needs to be
established before initiating diagnosis, and this is usual-
ly accomplished by image-guided sampling using fine-
needle aspiration (FNA) or needle core biopsy (NCB)
techniques of the liver lesion. Adequate sampling of
the lesion is key to obtaining a diagnosis. Key issues for
the pathologist evaluating the biopsy sample are deter-
mining the tumor type, distinguishing metastatic carci-
noma from primary hepatocellular carcinoma or pri-
mary cholangiocarcinoma, and determining the site of
primary origin. The pathologist uses morphology to
establish a differential diagnosis and then uses semi-
quantitative immunohistochemical studies to refine the
diagnosis, but these techniques have limitations.
Microarray analysis and proteomic analysis have
been used to establish tumor classifiers. Early studies
have demonstrated the feasibility of using these on FNA
and NCB samples. These technologies will improve our
ability to subclassify tumors of unknown primary.
The topic of liver metastases is broad. This discus-
sion focuses on the most significant tumor types in
adult oncology patients, with an emphasis on differen-
tial diagnosis of carcinoma of unknown primary.
Sampling Technique and Preparation
In order to establish a treatment regimen, tissue diag-
nosis of liver masses is required. FNA and NCB using
either transabdominal ultrasound or computed tomog-
raphy scanning guidance are the most frequently used
modalities and provide diagnostic specimens in over
90% of cases.
2
Endoscopic ultrasound-guided FNA
(EUS-FNA) is being used more frequently to aspirate
lesions in the left lobe of the liver. The decision to use
FNA or NCB depends on the size and location of the
lesion, the suspected diagnosis, and the risk of compli-
cations. The experience of a radiologist performing the
biopsy is also an important factor. The availability of
cytologists to evaluate the FNA for adequacy may also
play a role in selection of sampling technique.
A misconception among some clinicians is that
NCB is better than FNA because it procures more tissue.
However, studies in the literature and personal experi-
ence indicate that both are complementary, and an ade-
quate FNA with a well-prepared cellblock can provide
sufficient tissue for immunohistochemical studies.
In one recent study of 141 patients with abdominal
lesions sampled with both FNA and NCB, FNA proved
more sensitive than NCB at diagnosing malignancy
(86.1% vs 80.6%, respectively).
3
In other studies evalu-
ating FNA and NCB of abdominal organs, similar results
have been shown by other authors.
4-9
All of these stud-
ies showed the sensitivity of FNA to be 2% to 24%
greater than that of NCB. The combination of FNA and
NCB increased the overall sensitivity. A few studies have
provided results contradicting these findings.
10-12
How-
ever, none of these studies used on-site immediate assess-
ment of FNA samples by a cytologist, which has been
shown to maximize diagnostic yield and accuracy.
13-15
A well-prepared cellblock derived from an FNA
sample produces a microhistology specimen that can
provide sufficient material to evaluate for architectural
features and to perform immunohistochemical stud-
ies.
2,16-18
This provides an alternative to NCB when an
NCB cannot conveniently be performed. If only an NCB
will be obtained, then cytologic evaluation of touch
preparations of the cores can provide similar rapid
assessments of specimen adequacy.
19
Touch imprints of
core biopsies may be used to minimize the number of
biopsy procedures needed
20
by ensuring that the NCB
contains diagnostic material.
In summary, selection of guidance and biopsy tech-
nique depends on the location and site of the lesion,
the potential for complications using either technique,
and the experience and expertise of the radiologist.
FNA and NCB are complementary. On-site cytologic
assessment of FNA or NCB using touch imprints
improves the adequacy of both.
Establishing the Tumor Type
Correct clinical history is crucial to the pathologic
interpretation of a biopsy from a liver mass suspected
of being a metastasis. The most accurate interpretation
is rendered when the history of previous cancer and
other pertinent findings, such as radiological findings
and serum tumor marker levels, are provided for corre-
lation with the pathologic findings.
The first step when evaluating a liver biopsy is to
establish the general tumor type, ie, whether the neo-
plasm is a carcinoma, sarcoma, lymphoma, or melanoma.
Morphology alone is often diagnostic, but frequently
ancillary studies are needed for definitive diagnosis when
the tumor is poorly differentiated. A panel incorporating
at least cytokeratins, S100, and leukocyte-common anti-
gen (LCA) will assist in subcategorizing the neoplasms.
Carcinomas express cytokeratins, most lymphomas
(except anaplastic large-cell lymphoma) express LCA,
and S100 is the most sensitive marker for the diagnosis of
melanoma. Additional antibodies can be added once the
differential diagnosis has been narrowed down.
Carcinomas
Carcinomas are the most frequent source of metastases
to the liver. Lung, colon, pancreas, breast, and stomach
January 2006, Vol. 13, No. 1 Cancer Control 15
are the most frequent sources, accounting for 24.8%,
15.7%, 10.9 %, 10.1% and 6.1%, respectively, of all
patients with metastatic disease in one autopsy series.
21
Ovarian, endometrial, prostate, and urothelial carcino-
mas are less frequent sources of metastases, each
accounting for 4% or less.
21
The appearance of carcinomas depends on their
differentiation and includes adenocarcinomas, squa-
mous cell carcinoma, urothelial carcinoma, neuroen-
docrine carcinomas, mixed types of carcinomas such as
adenosquamous carcinoma, or specific types such as
adrenal cortical carcinoma, renal cell carcinoma, and
hepatocellular carcinoma.
Squamous Cell Carcinoma
Squamous cell carcinoma is an uncommon metastasis
to the liver. Possible primary sites include lungs, esoph-
agus, head and neck, genital primaries, or anorectal
primaries. FNA smears show polygonal cells occurring
singly and in groups with hyperchromatic, irregular
nuclei. The cytoplasm is dense and nonvacuolated, in
contrast to that of adenocarcinoma (Fig 1). Keratinized
cells will have orangeophilic cytoplasm on Papanico-
laou-stained smears. Histopathology specimens will
show cells with dense, polygonal, eosinophilic cyto-
plasm with intercellular desmoplastic junctions (Fig 2).
The presence of keratinization confirms the diagnosis,
but it is not always evident. The morphology of squa-
mous cell carcinoma is not specific to site of origin.
History is key in identifying the primary site since
immunohistochemical studies are not helpful.
Urothelial Carcinoma
The cells on cytology smears are arranged in discrete
and small syncytial cell clusters. The nuclei are central
to eccentric, and the cytoplasm is variable. A key fea-
ture is the presence of cercariform cells (Fig 3). These
are cells with nucleated globular bodies and unipolar
nontapering cytoplasmic process.
22
The pattern on
histomorphology is varied. The cells are typically ar-
ranged in sheets and have dense, amphophilic cyto-
plasm. The nuclei are typically elongated and may show
grooves (Fig 4).
Fig 1. Squamous cell carcinoma, FNA. This sheet of cells has dense
polygonal cytoplasm with junctions. The nuclei show an increased nuclear
to cytoplasmic ratio and atypia (Papanicolaou, 63).
Fig 2. Squamous cell carcinoma, core biopsy. The cells have abundant,
dense, polygonal eosinophilic cytoplasm. Intercellular junctions between
the cells are evident (hematoxylin-eosin, 40).
Fig 3. Urothelial carcinoma, FNA. The cells are dispersed as single cells.
Many of the cells have a bulbous head and a tail, characteristic of cercari-
form cells (Papanicolaou, 60).
Fig 4. Urothelial carcinoma, core biopsy. The cells are arranged in cohe-
sive nests and have abundant eosinophilic cytoplasm. The nuclei show
nuclear grooves, a feature or urothelial carcinoma (hematoxylin-eosin, 40).
January 2006, Vol. 13, No. 1 16 Cancer Control
Neuroendocrine Carcinomas
Neuroendocrine carcinomas vary in the degree of dif-
ferentiation. Low-grade tumors such as carcinoids have
a monomorphic appearance with minimal mitotic
activity and no necrosis. Smears are uniformly cellular
and composed of a monomorphic population of tumor
cells. A tumor with a plasmacytoid appearance on
cytology samples is virtually pathognomonic (Fig 5).
Histopathology samples will show similar features. The
chromatin shows a characteristic salt and pepper
appearance on both smears and core biopsy samples
(Fig 6). Carcinoids may arise anywhere in the gastroin-
testinal tract. Pancreatic endocrine tumors have identi-
cal morphologic features. High-grade tumors, such as
metastatic small-cell carcinoma from the lung, will
show nuclear molding, necrosis, and abundant mitotic
activity. The chromatin is diffusely and finely stippled
(Figs 7 and 8).
Immunohistochemical studies are useful for the
identification of a neoplasm as showing neuroen-
docrine differentiation. The standard panel is synapto-
physin, chromogranin, and neural cell adhesion mole-
cules (NCAM [CD56]). Immunohistochemical studies
are less helpful for the identification of neuroendocrine
carcinoma of unknown primary.
23
Adenocarcinomas
Adenocarcinomas are the most significant since they
are the most frequent type of carcinoma to metasta-
size to the liver. Lung, colon, pancreas, breast, and
stomach are the most frequent, representing 24.8%,
15.7%, 10.9%, 10.%, and 6.1% of cases, respectively, in
one autopsy series.
21
Ovary, endometrial, prostate,
cholangiocarcinoma, and thyroid are less frequent,
each accounting for less than 4%.
21
Adenocarcinomas
are also the most frequent type of carcinoma pre-
senting as unknown primary in the liver in the adult
oncology patient.
24,25
Adenocarcinomas are neoplasms derived from
glandular tissues. The most frequent appearance of ade-
nocarcinomas is columnar cells forming acinar struc-
tures, which recapitulate the gland formation within
Fig 5. Carcinoid tumor, FNA. The cells have abundant amphophilic cyto-
plasm and the nuclei are eccentrically placed, imparting a plasmacytoid
appearance. Some cells are binucleated. The cells exhibit the characteris-
tic salt and pepper chromatin pattern (Papanicolaou 40).
Fig 6. Carcinoid tumor, core biopsy. The cells are arranged in solid
nests with focal areas of palisading or acinar structure formation. The
nuclei are round and uniform with a salt and pepper chromatin (hema-
toxylin-eosin, 40).
Fig 7. Small cell carcinoma, FNA. In contrast to the carcinoid tumor,
these cells exhibit greater variation in nuclear contour. The cells have
scant amphophilic cytoplasm. Nuclear molding, crush artifact and necro-
sis are evident. The salt and pepper chromatin pattern typical of neuroen-
docrine neoplasms is retained (Papanicolaou, 63).
Fig 8. Small cell carcinoma, FNA, cell block. The cells have scant,
eosinophilic cytoplasm. The nuclei are elongated and fusiform. Apoptosis
is noted (hematoxylin-eosin, 40).
January 2006, Vol. 13, No. 1 Cancer Control 17
the normal organ (Fig 9). The typical adenocarcinoma
shows focal mucin production, within either the cyto-
plasm or lumen, which can be demonstrated with a
histochemical stain for mucin, such as mucicarmine.
Morphologic subtypes include the mucinous car-
cinoma or colloid-type carcinoma and signet ring cell
carcinoma. While the morphologic pattern of most
adenocarcinomas is not specific for site of origin, some
primary sites have characteristic features that lead to
their recognition on FNA or NCB specimens. These
include colorectal carcinoma, breast carcinoma, and
pancreatobiliary carcinoma.
A key feature of colorectal carcinoma is a dirty,
necrotic background on cytology smears (Fig 10). The
cells are columnar in appearance. Histopathology will
show an adenocarcinoma with abundant central necro-
sis in the glands (Fig 11). Low-grade ductal adenocar-
cinomas appear as a monomorphic population on
cytology smears. The groups are flat and angulated.
The monomorphic appearance is evident on
histopathology specimens (Fig 12). Lobular carcinoma
forms a dyshesive cell population composed of small
cells with eccentric nuclei. Histology specimens will
show cells infiltrating in single file pattern. A charac-
teristic feature of both types of mammary carcinomas,
associated most often with lobular carcinoma, is cells
with a targetoid cytoplasmic lumen (Fig 13).
Pancreatobiliary carcinomas do not exhibit a spe-
cific pattern on cytology smears, although an adeno-
carcinoma with abundant cytoplasmic mucin or clear
nuclei may be suggestive. They are typically associated
with abundant sclerotic stroma (Fig 14), and therefore
this possibility may be suggested on histopathology
specimens. However, primary intrahepatic cholangio-
carcinoma is also associated with sclerotic stroma, so
the distinction of primary cholangiocarcinoma from
metastatic pancreatobiliary carcinoma cannot be made
without the history.
The morphologic patterns of other types of adeno-
carcinomas such as those originating in the lungs,
endometrium, esophagus, or intestinal type of gastric
carcinoma do not have any specific features.
Fig 9. Adenocarcinoma, NOS, core biopsy. The cells are arranged in an
acinar pattern, characteristic of adenocarcinoma from any site. Pale,
bluish mucin is evident the cytoplasm of some cells and lumen (hema-
toxylin-eosin, 40).
Fig 10. Colonic adenocarcinoma, FNA. The smear shows columnar cells
arranged in a palisaded pattern. The background shows abundant necro-
sis (Papanicolaou, 40).
Fig 11. Colonic adenocarcinoma, core biopsy. The glands show abun-
dant central necrosis, a characteristic feature of colonic adenocarcinoma
(hematoxylin-eosin, 40).
Fig 12. Mammary carcinoma, FNA. The cells are dispersed and show
eccentric nuclei. A number of cells have intracytoplasmic mucin vacuoles
(Diff-Quik, 60).
January 2006, Vol. 13, No. 1 18 Cancer Control
Mucinous Carcinoma
Mucinous carcinoma is a morphologic subtype of ade-
nocarcinoma, defined as a carcinoma that contains
more than 50% extracellular mucin. This subtype is not
specific to any organ. Mucinous carcinomas are most
frequently identified in the colon, but they also occur in
the breast, ovaries, and pancreas and may arise any-
where in the gastrointestinal tract. Mucinous bron-
choalveolar carcinoma has similar features. A mucinous
carcinoma in the liver is most likely to be of colorectal
origin, but other primary sites need to be considered.
Aspirates show malignant glandular cells floating in
pools of mucin (Fig 15). The two histologic patterns
are tumor cells floating in pools of mucin or pools of
mucin partially lined by tumor cells (Fig 16). The tumor
cells are mucin-producing columnar cells or cells that
contain a single large vacuole.
Signet Ring Cell Carcinoma
Signet ring cell carcinoma shows single cells with a
cytoplasmic mucin vacuole that displaces the nucleus.
The nucleus is sharply angulated at the tips (Fig 17).
Gastric carcinoma is most commonly associated with
this morphology, but as for mucinous carcinomas, it
may arise in any organ in the gastrointestinal tract. The
differential diagnosis includes metastatic lobular breast
carcinoma. An immunohistochemical panel that
Fig 13. Mammary carcinoma, core biopsy. The cells are arranged in
rounded nests (hematoxylin-eosin, 40).
Fig 14. Metastatic pancreatic carcinoma. The carcinoma is surrounded
by a desmoplastic stroma (hematoxylin-eosin, 20).
Fig 15. Mucinous carcinoma, FNA. The cluster of malignant cells floats
in a background of viscous mucin (Papanicolaou, 40).
Fig 16. Mucinous carcinoma, biopsy. The carcinoma is composed pre-
dominantly of extra cellular mucin. The malignant glands cling to the stro-
ma and surround pools of mucin (hematoxylin-eosin, 20).
Fig 17. Signet ring cell carcinoma, core biopsy. Signet ring cells with
eccentric, elongated nuclei displaced by intracytoplasmic mucin vacuoles
(hematoxylin-eosin, 40).
January 2006, Vol. 13, No. 1 Cancer Control 19
includes CK7, CK20, estrogen receptors,
26
and
GCDFP15 (BRST2)
27,28
can help with this differential
diagnosis. Lobular carcinoma will express CK7, ER, and
GCDFP15 and usually does not express CK20. Proges-
terone receptor is not as specific as estrogen receptor
since its expression was identified in other carcinomas,
including gastric/esophageal.
29
Adenosquamous Carcinoma
Adenosquamous carcinoma is composed of a mixture
of squamous carcinoma and adenocarcinoma, as the
name implies. One of the components must comprise
at least 30% of the tumor. This type of carcinoma may
arise anywhere in the gastrointestinal tract, including
the pancreas and biliary system, and also the lungs.
Since primary cholangiocarcinoma may show a similar
morphology, it will not be possible to determine whether
it is primary or metastatic without clinical history.
Renal Cell Carcinoma
Renal cell carcinoma infrequently metastasizes to the
liver, but it accounts for 3% of all metastases in one
autopsy series.
21
The classic appearance is that of a car-
cinoma composed of clear cells arranged in nests with
intervening stroma and blood vessels (Fig 18). Variants
include papillary renal cell carcinoma and chromo-
phobe renal cell carcinoma. The cytoplasm of FNA will
show polygonal cells arranged singly and in clusters.
The nuclei are round with prominent nucleoli, and the
cytoplasm is clear or granular. Large groups or sheets
of cells are arranged along transgressing endothelium
(Fig 19), a pattern that mimics hepatocellular carcino-
ma on aspirates.
30
Papillary renal cell carcinoma will
not demonstrate prominent nucleoli or clear cyto-
plasm. The cytoplasm in chromophobe renal cell car-
cinoma is balloon-like and excessive. The pattern of
clear cell renal cell carcinoma may mimic that of hepa-
tocellular carcinoma, particularly the clear cell variant
of hepatocellular carcinoma.
Adrenal Cortical Carcinoma
Adrenal cortical carcinoma is a rare neoplasm. The liver
is one of its most common sites of metastasis.
31
It mer-
its mention because its morphologic pattern overlaps
Fig 18. Renal cell carcinoma, biopsy. Clear cells surround by vascular
stroma (hematoxylin-eosin, 40).
Fig 19. Renal cell carcinoma, FNA. This fragment has a central, trans-
gressing vasculature, which mimics the pattern seen in hepatocellular car-
cinoma. Stripped nuclei are seen in the background (Diff-Quik, 20).
Fig 20. Adrenal cortical carcinoma, biopsy. The cells clusters show
endothelial wrapping, similar to that shown by hepatocellular carcinoma
(hematoxylin-eosin, 20).
Fig 21. Hepatocellular carcinoma, biopsy. Classic pattern of hepatocel-
lular carcinoma showing widened trabeculae and intracytoplasmic inclu-
sions (hematoxylin-eosin, 20).
cally show a population of pleomorphic cells with
intranuclear inclusions and prominent nucleoli (Fig 23).
Immunohistochemistry is specific and sensitive for the
diagnosis of melanoma. Melanomas are S100-positive,
HMB45 and MelanA-positive. A new antigen cocktail
consisting of HMB45, tyrosinase, and MART-1 is sensi-
tive for the diagnosis of melanoma.
33
However, S100
remains the most sensitive for detection of melanoma,
although it lacks specificity.
34
Lymphomas
Lymphomas are dyshesive neoplasms on FNA smears.
The background of the smears shows lymphoglandular
bodies (Fig 24) indicating that the neoplasm is of lym-
phoid origin, but they are not specific for benignancy
or malignancy. The appearance depends on the specif-
ic type. Large-cell lymphomas, the most frequent type
of lymphoma to secondarily involve the liver, are usual-
ly easy to recognize as malignant because they are com-
posed of large lymphocytes (greater than 3 times the
size of a normal lymphocyte) with nuclear membrane
irregularities (Fig 25). Follicular lymphomas, mucosa-
with that of hepatocellular carcinoma on FNA and
NCB.
32
The cells of adrenal cortical carcinoma are
polygonal in shape, like those of hepatocellular carcino-
ma. Furthermore, on core biopsy samples, it may show
endothelial wrapping, similar to that produced by hepa-
tocellular carcinoma
30
(Figs 20 and 21). Smears show
polygonal cells arranged singly and in clusters without
transgressing endothelium. The nuclei are hyperchro-
matic and variable in size. Prominent nucleoli are not a
feature as they are for hepatocellular carcinoma.
Melanomas
Melanomas are known as the great mimickers in pathol-
ogy. Generally, melanoma must be included in the dif-
ferential diagnosis of almost any neoplasm since its
appearance can be so varied and also since patients
may present with liver metastases many years after the
diagnosis of the primary tumor. However, it is a rela-
tively infrequent source of metastases in the liver,
accounting for approximately 2.2%.
21
A single cell pop-
ulation with cytoplasmic melanin pigment is diagnostic
on FNA smears (Fig 22), but nonpigmented or amelan-
otic melanomas are difficult to diagnose. Biopsies typi-
20 Cancer Control January 2006, Vol. 13, No. 1
Fig 22. Melanoma, FNA. The smear shows a dispersed cell population.
The nuclei vary in size and shape. Some of the cells show black melanin
pigment in the cytoplasm (Diff-Quik, 40).
Fig 23. Melanoma, core biopsy. The malignant cells have atypical nuclei
with prominent nucleoli, a feature considered typical of melanoma.
Melanin pigment is visible (hematoxylin-eosin, 40).
Fig 24. Large cell lymphoma, FNA. The malignant cells are dispersed.
The nuclei are three times the size of normal lymphocytes. The back-
ground shows lymphoglandular bodies, which are characteristic for lym-
phoid processes (Diff-Quik, 63).
Fig 25. Large cell lymphoma, biopsy. The specimen shows a popula-
tion of cells with scant cytoplasm, irregular nuclear membranes and promi-
nent nucleoli (hematoxylin-eosin, 40).
and neuroendocrine tumors. Histopathology samples
demonstrate a spindle cell neoplasm with a storiform
pattern, prominent vascularity, and occasionally
skenoid fibers (Fig 27). The tumors may demonstrate
variable amounts of myxoid stroma. Paranuclear vac-
uoles are a frequent feature (Fig 28). Immunohisto-
chemical analysis shows tumor cell expression for C-kit,
CD34, and vimentin. The tumors are typically negative
for actin or desmin.
38
C-kit expression is diagnostic for
these tumors.
Leiomyosarcoma is the most common sarcoma to
metastasize to the liver. It shows greater pleomorphism
and less vascularity compared to gastrointestinal stromal
tumor (GIST) (Fig 29). The characteristic immuno-
phenotype is desmin and smooth muscle actin expres-
sion and no C-kit expression.
38
Diagnostic Dilemmas
Problems facing the pathologist when evaluating a
biopsy include distinguishing hepatocellular carcinoma
from other carcinomas with similar features (renal cell
associated lymphoma tissue (MALT) lymphomas, and
small lymphocytic lymphomas are difficult to recognize
on morphology alone but can be suspected if there is a
previous history of lymphoma. Flow cytometry and
gene rearrangement studies can be performed on sam-
ples obtained from FNA. Typically, if lymphoma is sus-
pected, an aspirate directed for flow cytometry is
requested. NCB can be used for histomorphological
grading of follicular lymphomas and immunohisto-
chemical studies. The accuracy of subclassifying lym-
phomas using cytomorphological and flow cytometric
immunophenotyping has been demonstrated.
35-37
Sarcomas
Sarcomas are usually characterized by a spindle cell
appearance. Gastrointestinal stromal tumors and
leiomyosarcomas are the most frequent sarcomas to
metastasize to the liver.
Aspirates of gastrointestinal stromal tumors dem-
onstrate relatively monomorphic and uniform spindle
cells in loose aggregates and singly. The cells may be
associated with a myxoid stroma (Fig 26). Occasional-
ly they show epithelioid features, in which case the
differential diagnosis includes melanoma, carcinomas,
Cancer Control 21 January 2006, Vol. 13, No. 1
Fig 26. Gastrointestinal stromal tumor, FNA. This field demonstrates
spindle cells embedded in myxoid stroma (Diff-Quik, 20).
Fig 27. Gastrointestinal stromal tumor, core biopsy. The plump spindle
cells tumor vascularity is prominent. Skenoid fibers are noticeable at this
power (hematoxylin-eosin, 20).
Fig 28. Gastrointestinal stromal tumor, core biopsy. The cells are spin-
dle in shape with minimal nuclear atypia. Perinuclear halos are evident
(hematoxylin-eosin, 40).
Fig 29. Leiomyosarcoma, core biopsy. The cells show a greater degree
of nuclear pleomorphism and are surrounded by a myxoid stroma. The
tumor is less vascular than the GIST (hematoxylin-eosin, 40).
January 2006, Vol. 13, No. 1 22 Cancer Control
and adrenal cortical carcinoma) and distinguishing
hepatocellular carcinoma from adenocarcinoma. A spe-
cial problem in the oncology patient is the workup of
carcinoma of unknown primary.
Primary Hepatocellular Carcinoma vs
Metastases
The morphologic features of renal cell carcinoma and
adrenal cortical carcinoma overlap with those of hepa-
tocellular carcinoma, on both cytology smears and
histopathology samples. Immunohistochemical analy-
sis differentiates among these three carcinomas. Adren-
al cortical carcinomas are weakly positive for cytoker-
atin and express vimentin and synaptophysin. MART-1,
also known as melanA, and usually expressed by
melanoma, is useful for diagnosing adrenal cortical car-
cinoma.
39,40
Adrenal cortical carcinoma also expresses
inhibin, which is slightly more sensitive but less specif-
ic.
41
Calretinin has also been shown to be expressed by
adrenal cortical cells and neoplasms.
42-44
Clear cell car-
cinoma of the kidney, the most typical type of renal
neoplasm, expresses cytokeratin, vimentin, epithelial
membrane antigen (EMA), renal cell carcinoma anti-
body, and CD10.
45-47
Exceptions are the papillary vari-
ants and chromophobe cell variants, which do not
express vimentin. Alpha-methyl CoA racemace
(AMACR) is usually expressed by papillary renal cell
carcinomas,
48
and chromophobe renal cell carcinomas
demonstrate colloidal iron not demonstrated by hepa-
tocellular carcinoma or adrenal cortical carcinoma.
Hepatocellular carcinoma can usually be identified by
expression of low-molecular-weight cytokeratin, Hep-
Par, and a canalicular pattern rather than a cytoplasmic
pattern of carcinoembryonic antigen (CEA). The
canalicular pattern occurs because the CEA will stain
the bile duct canaliculi in hepatocellular carcinoma but
not the cytoplasm. CD10 will stain hepatocellular car-
cinoma in a canalicular pattern but not in the cyto-
plasm, as it does for renal cell carcinoma.
49
Other unusual metastases that may mimic hepato-
cellular carcinoma include hepatoid yolk sac tumor,
and oxyphilic follicular carcinoma of the thyroid. Hepa-
toid yolk sac tumors are rare, and follicular carcinoma
of the thyroid rarely gives rise to liver metastases. The
immunohistochemical approach to this differential diag-
nosis is summarized in Table 1.
Adenocarcinoma vs Hepatocellular Carcinoma
Typically, the distinction of adenocarcinoma from hepa-
tocellular carcinoma is clear-cut on morphologic
grounds. Adenocarcinoma is characterized by the for-
mation of gland-like or tubular structures. The lumens
or the individual cells contain mucin. Hepatocellular
carcinoma occasionally forms acinar structures resem-
bling adenocarcinoma (Fig 30) or is poorly differentiat-
ed, in which case it cannot be distinguished from ade-
nocarcinoma. In these situations, adjunctive studies are
needed. The presence of bile pigment is pathogno-
monic for hepatocellular differentiation; therefore, if
Antigen Tumor Type
Hepatocellular Carcinoma Renal Cell Carcinoma Adrenal Cortical Carcinoma Melanoma
HMW CK +
LMW CK + + /few cells +
Vimentin +/ + + +
EMA + +
CD 10 Canalicular +
AFP +
HepPar +
MART-1 + +
S100 +
HMB45 +
Synaptophysin +
Calretinin +
HMW CK = high-molecular-weight cytokeratin
LMW CK = low-molecular-weight cytokeratin
Table 1. Immunohistochemical Panel for the Differential Diagnosis of Hepatocellular Carcinoma,
Renal Cell Carcinoma, Adrenal Cortical Carcinoma, and Melanoma
Fig 30. Hepatocellular carcinoma, biopsy. Acinar pattern demonstrated
in hepatocellular carcinoma (hematoxylin-eosin, 40).
January 2006, Vol. 13, No. 1 Cancer Control 23
the pathologist recognizes bile, the diagnosis of hepa-
tocellular carcinoma can be made with certainty. A
Halls stain for bile can help to recognize the bile pig-
ment. Adenocarcinoma typically secrets mucin, so the
identification of mucin secretion by the cells using a
histochemical stain for mucin, such as mucicarmine,
can establish the carcinoma as an adenocarcinoma.
Immunohistochemistry is helpful when morphology
and identification of secretory substances fail. Table 2
lists an immunohistochemical panel to facilitate this
distinction. Adenocarcinomas express both high- and
low-molecular-weight cytokeratin, whereas the cytoker-
atin expression of hepatocellular carcinoma is usually
limited to low-molecular-weight cytokeratin.
50
More
specifically, hepatocytes and hepatocellular carcinoma do
not express cytokeratins 1, 5, 10, 11, and 19. Immuno-
histochemical evaluation for CK19 is particularly useful
since adenocarcinomas but not hepatocellular carcino-
mas express this antigen.
51
As described in the previous
section, hepatocellular carcinoma has a specific expres-
sion pattern for CEA and CD10 in which they are
expressed only in the bile canaliculi.
49
Adenocarcinomas
show a cytoplasmic expression pattern for CEA. MOC31
is reported to be sensitive for the diagnosis of adenocar-
cinoma.
52,53
The HepPar antigen is also helpful, but it is
expressed occasionally by other tumor types such as
adrenal cortical carcinoma, yolk sac tumor, ovarian carci-
noma, colonic carcinoma, lung carcinoma, and endocervi-
cal carcinoma.
54
AFP, when expressed, is also specific for
hepatocellular carcinoma but is infrequently expressed.
55
Generally, a panel combining a number of these antigens
is most useful for this differential diagnosis.
55-57
Primary Site of Origin of Carcinoma
This problem is probably of the greatest significance to
the oncologist treating the adult cancer patient and is
frequently encountered at our institute. While most
patients presenting with metastatic adenocarcinoma
have a history of a primary elsewhere, some patients do
not have a known primary. In these cases, the onus falls
on the pathologist to navigate the treating physicians to
the most likely primary site.
As previously stated, adenocarcinomas are the most
frequent type of carcinoma presenting as unknown pri-
mary, accounting for approximately 80%.
24
Squamous
cell carcinomas account for another 15%, and metastases
of other tumor types account for the remaining 5%.
24
The
immunohistochemical workup essentially focuses on
adenocarcinomas since the immunohistochemical panels
are most applicable to this subtype of carcinoma.
Morphologic clues on the FNA or NCB may help to
distinguish some adenocarcinomas from others, as
already presented. In a patient with a solitary liver
mass, the key will be distinguishing primary cholangio-
carcinoma from metastatic adenocarcinoma. Morphol-
ogy may provide some clues since the association with
a densely sclerotic stroma characterizes cholangiocar-
cinoma, and an origin or close interconnection with
adjacent canals of Herring may be seen. Except for
some of the morphologic types already mentioned,
morphology is otherwise not useful for identification of
carcinoma of unknown primary.
Immunohistochemistry
The limitations of morphology have fueled the search
for markers of differentiation. Since the first major
publication reporting cytokeratin phenotype of CK7
and CK20 as discriminatory among different tumor
types and sites,
58
it has become the cornerstone of the
panel to evaluate tumors of unknown origin. There are
four possible expression patterns: CK7+/CK20+, CK7+/
CK20, CK7/CK20+ and CK7/CK20. One difficulty
with interpreting the CK7/CK20 phenotype is that the
criteria used to define a tumor as positive for antigen
expression have varied significantly among authors.
58-60
Some authors have required only 1% of cells while oth-
ers have required at least 50%. Adding to the difficulties
in interpreting the findings is that the antibodies used
are different and that there are differences in antigen
retrieval techniques and performance of the immuno-
histochemical studies. The CK7/CK20 phenotype is
also influenced by the degree of differentiation and the
morphologic subtype. All of these caveats mean that
the pathologic interpretation of CK7/CK20 immuno-
histochemical studies remains subjective, but it remains
most effective when based on an algorithmic and prob-
abilistic approach. However, despite these limitations,
the CK7/CK20 expression pattern is effective at nar-
rowing down possibilities.
When applying an algorithmic approach to identi-
fying the origin of an adenocarcinoma, the first step
involves distinguishing the primary from a metastasis.
In the case of cholangiocarcinoma, the diagnosis
remains one of exclusion in most cases because its phe-
notype overlaps with that of many other carcinomas.
Antigen Tumor Type
Hepatocellular Carcinoma Adenocarcinoma
LMW CK + +
HMW CK /rarely + +
CEA Canalicular +
HepPar +
B72.3 +
AFP +
MOC31 +
CK19 +
Mucicarmine +
Bile +
HMW CK = high-molecular-weight cytokeratin
LMW CK = low-molecular-weight cytokeratin
Table 2. Differential Diagnosis of
Hepatocellular Carcinoma From Adenocarcinoma
January 2006, Vol. 13, No. 1 24 Cancer Control
Cholangiocarcinomas are usually CK7+ and variably
express CK20.
59
Their morphologic expression pattern
overlaps with the pattern of many other primary sites.
The most relevant phenotype to the discussion
of metastatic adenocarcinoma to the liver is the CK7/
CK20 + phenotype because it is highly characteristic of
colorectal primary.
61
The predictive probability of this
immunophenotype is 78%.
62
Carcinomas coexpressing CK7/CK20 include
urothelial carcinoma, metastatic pancreatobiliary carci-
noma, and mucinous ovarian carcinoma.
58,63
Recent
publications have shown that mucinous colon carcino-
ma and mucinous bronchoalveolar carcinoma also
express the CK7+/CK20+ phenotype.
64-67
Morphology
can sometimes exclude metastatic urothelial carcinoma
since this is not a gland-forming neoplasm; however, its
features may overlap with those of poorly differentiated
adenocarcinoma. The CK7/CK20 phenotype is usual-
ly typical of prostate, with a probability of 76%.
62
Other
tumors rarely show this phenotype. The CK7+/CK20
subtype is the least specific.
63
Lung and breast are two
tumors that exclusively have this phenotype, with a
probability of 84% and 88%, respectively. However,
other tumors can express this phenotype, particularly if
CK20 expression is weak or focal. Of note, gastric and
esophageal adenocarcinoma have the most variable
CK7/CK20 expression pattern.
58,61
Therefore, these
need to be included in the differential diagnosis of any
phenotype. Table 3 summarizes the most frequent
CK7/CK20 expression patterns for carcinomas of dif-
ferent primary sites.
Other studies can help to further refine the differ-
ential diagnosis. Cytokeratin 17 is associated with
ampullary and pancreatobiliary carcinomas more often
than gastric or esophageal carcinomas.
68,69
Additional
markers help to further refine the identification such as
TTF1 (nonmucinous pulmonary adenocarcinomas),
64,70-
74
estrogen receptor protein staining (breast, ovarian,
endometrial),
29,75
GCDFP15 (breast),
27,76-78
WT1 (ovari-
an serous tumors),
79
PSA and PAP (prostate),
80
throm-
bomodulin,
81
and uroplakin (urothelial carcinoma).
80,82
CA125, when used as part of a panel, is useful for
identifying ovarian carcinomas.
83,84
CDX2 is used as a
marker of intestinal differentiation and can help to dif-
ferentiate colorectal, intestinal, or gastric neoplasms
from pancreatobiliary, biliary, ovarian, or pulmonary
adenocarcinomas.
65,85-87
An older antibody panels consisting of MOC31, ker-
atins, vimentin, B72.3, CA125, Ca19-9, placental alkaline
phosphatase, S100 protein, estrogen receptor protein,
PSA, thyroglobulin, GCDFP15, and CEA obtained a
sensitivity of 67%
88
for the diagnosis of carcinoma of
unknown primary. Another study evaluating GCDFP15,
breast cancer antigen 225 (BCA225), B72.3, CA15-3,
CEA, CA19-9, CA125, and estrogen receptor showed a
sensitivity of 67% for the determination of site of ori-
gin.
89
While individual studies have evaluated the sen-
sitivity and specificity of sets of markers for specific dif-
ferential diagnoses, the sensitivity of a panel including
CK7 and CK20 with some of the above listed antibod-
ies has not been determined across a broad number of
cancers in the liver.
Future Directions
Cancer therapy is primarily directed by tumor origin,
making correct pathologic diagnosis imperative for
proper patient management. As discussed, the number
of specific markers available and subjectivity of inter-
pretation are factors that limit standard pathologic
practice using morphology and immunohistochem-
istry. Tumor classification, using high throughput tech-
nologies such as microarray and proteomic screening,
promise to improve cancer diagnosis and management.
A number of publications have demonstrated the
feasibility of using gene expression profiles derived
from microarray analysis as an approach to diagno-
sis.
90-92
Proteomic analysis has the potential to yield
similar data sets.
93
In order for these technologies to be clinically rel-
evant, they must be applicable with FNA and NCB.
Recent studies have shown the feasibility of using
FNA or NCB specimens for microarray analysis.
94-99
In
our own experience, FNA of resection specimens
obtained over 1 g of total RNA for microarray analy-
sis.
100
The samples were successfully classified using
a tumor classifier derived from resection specimens.
Studies evaluating the use of cytology or NCB materi-
al for proteomic analysis are limited.
101
CK7+/CK20+ CK7+/CK20 CK7/CK20+ CK7/CK20
Urothelial carcinoma Breast Colorectal Prostate
Pancreas Lung Renal cell carcinoma
Biliary tract Esophagus/stomach Hepatocellular carcinoma
Cholangiocarcinoma Pancreas Adrenal cortical carcinoma
Esophagus/stomach Biliary
Mucinous carcinoma Cholangiocarcinoma
(ovarian, colon, mucinous bronchoalveolar) Ovary (nonmucinous)
Endometrium
Table 3. Cytokeratin Coexpression Patterns
January 2006, Vol. 13, No. 1 Cancer Control 25
The problem for any of these technologies is the
lack of standardization in specimen collection and
preparation. However, these technologies promise to
supplement and improve our current standard of prac-
tice as adjunctive techniques.
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