Ebook Atlas of non-gynecologic cytology: Part 2

6

Pancreaticobiliary Tract Cytology
Judy Pang and Andrew Sciallis

Introduction
The major indications for cytologic evaluation of the pancreaticobiliary tract are a pancreatic mass and/or a bile duct
stricture. Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) is the primary modality for obtaining tissue
diagnosis; it has largely replaced percutaneous FNA because
it allows for real-time visualization of the needle, provides
better visualization of small lesions than CT guidance, and
enables the sampling of regional lymph nodes and assessment of invasion of local structures, thus providing simultaneous diagnosis and staging [1–3]. The sensitivity of
EUS-FNA for solid masses has been reported to range from
54 to 95%, with specificity approaching 100% [4]. The role
of EUS-FNA is less clear for cystic lesions, as the sensitivity
is generally lower and more variable in detecting a neoplastic
mucinous cyst than a solid neoplasm, ranging from 23 to
100% [5]. Furthermore, the sensitivity of detecting a malignancy in a neoplastic mucinous cyst is reported to be 29%,
with 100% specificity [6, 7].
Endoscopic
retrograde
cholangiopancreatography
(ERCP) with bile duct brushings for cytology is an additional minimally invasive modality to obtain material for tissue diagnosis, which can be helpful in the assessment of
pancreatic neoplasms, particularly ductal adenocarcinomas.
The sensitivity of brushings is reported to be lower than that
of EUS-FNA, 44–72% [8–11], but the specificity approaches
100%, similar to EUS-FNA.
The Papanicolaou Society of Cytopathology has proposed a terminology scheme for the reporting of pancreaticobiliary cytology, utilizing a six-tiered system, as shown
on Table 6.1 [12].

Sampling of pancreatic head masses is performed using a

transduodenal approach, whereas a transgastric approach is
used for the body and tail masses. It is important for the
pathologist to be aware of the approach so that contaminating normal duodenal mucosa (Fig. 6.1) and gastric mucosa
(Fig. 6.2) is not misinterpreted as lesional tissue.
Table 6.1  Papanicolaou society of cytopathology system for reporting
pancreaticobiliary cytology
I. Nondiagnostic
II. Negative (for malignancy)
III. Atypical
IV. Neoplastic
 
• Benign
   – Serous cystadenoma
   – Neuroendocrine microadenoma
   – Lymphangioma
 
• Others
    –  Well-differentiated neuroendocrine tumor
   – Solid pseudopapillary tumor
    – Intraductal papillary mucinous neoplasm, all grades of
dysplasia
    –  Mucinous cystic neoplasm, all grades of dysplasia
V. Suspicious (for malignancy)
VI. Positive or malignant
 •  Pancreatic ductal adenocarcinoma
 
• Cholangiocarcinoma
 •  Acinar cell carcinoma
 • Poorly differentiated (small-cell and large-cell)
neuroendocrine carcinoma

 
• Pancreatoblastoma
 
• Lymphoma
 
• Metastatic malignancy
Adapted from Pitman [12]

J. Pang, M.D. (*) · A. Sciallis, M.D.
Department of Pathology, The University of Michigan,
Ann Arbor, MI, USA
e-mail: ;
© Springer International Publishing AG, part of Springer Nature 2018
X. Jing et al. (eds.), Atlas of Non-Gynecologic Cytology, Atlas of Anatomic Pathology,
/>
157


158

a

J. Pang and A. Sciallis

b

Fig. 6.1  Duodenal epithelium is often seen in aspirates of masses from the pancreatic head or proximal body. Typically seen are flat sheets of
epithelial cells with uniform small nuclei studded with occasional goblet cells. (a) Diff-Quik stain; (b) Papanicolaou stain

Fig. 6.2  Gastric epithelium is often seen in aspirates of masses from

the pancreatic distal body or tail. It typically appears as flat sheets of
mucinous epithelial cells with uniform small nuclei. In this image, adjacent to the gastric epithelium is a small cluster of disordered malignant
cells (Papanicolaou stain)

Fig. 6.3  Benign pancreatic ductal epithelial cells have uniform small
round nuclei and are arranged in cohesive, evenly spaced honeycomb
sheets (Diff-Quik stain)

Normal Pancreas
Figures 6.3, 6.4, and 6.5 show examples of normal pancreatic elements.

Fig. 6.4  Benign acinar cells are reminiscent of “grapelike” clusters
when associated with fibrovascular tissue (Papanicolaou stain)


6  Pancreaticobiliary Tract Cytology

159

a

Fig. 6.5  Benign acinar cells are polygonal with abundant granular
cytoplasm (Papanicolaou stain)

b

Solid Pancreatic Masses
EUS-FNA of solid pancreatic masses is not always necessary when a solid mass detected on imaging is considered
to be resectable, as a benign cytology does not entirely
exclude a malignancy. It is most useful in patients who have

unresectable disease or are poor surgical candidates, in
whom tissue diagnosis is necessary prior to the initiation of
chemotherapy or radiation [13]. It is also helpful when it is
not clear from clinical and radiologic findings whether a
mass is attributable to a benign process such as pancreatitis
(Figs. 6.7, 6.8, and 6.9), when the patient has a prior history
of another malignancy, or when a lymphoma is suspected.
In these scenarios, surgical resection may not be indicated.

Fig. 6.6  Stromal fragments in chronic pancreatitis. (a) Diff-Quik
stain; (b) Papanicolaou stain

Pancreatitis
Figures 6.6 and 6.7 show features of pancreatitis.

Pancreatic Ductal Adenocarcinoma
Pancreatic ductal adenocarcinoma can be identified from a
number of characteristics of its cytomorphology, as illustrated in Figs. 6.8, 6.9, 6.10, 6.11, 6.12, and 6.13:
• Disordered, crowded epithelial sheets (“drunken
honeycomb”)
• Single malignant cells
• Irregular nuclear contours (grooves, convolutions)
• Irregular chromatin (clearing and clumping)
• Nuclear enlargement
• Prominent nucleoli
• Nuclear pleomorphism (4:1 nuclear diameter size difference within a single cluster/sheet)
• Prominent mucinous vacuolization

Fig. 6.7  Pancreatitis. Cohesive cluster of slightly crowded ductal cells
with slightly enlarged round to oval nuclei, smooth nuclear membranes,

and small nucleoli. There is little variation in nuclear diameter within
the same sheet (<4:1 difference). There is also a background of neutrophils (Papanicolaou stain)


160

J. Pang and A. Sciallis

a

Fig. 6.8  Crowded clusters of epithelial cells and loss of honeycomb
arrangement in pancreatic ductal adenocarcinoma (Diff-Quik stain)

b

c

Fig. 6.9  Single malignant cells with enlarged nuclei and prominent
nucleoli in pancreatic ductal adenocarcinoma (Diff-Quik stain)

Fig. 6.10  Loosely cohesive clusters of ductal adenocarcinoma with
nuclear enlargement, admixed with benign acinar cells (Diff-Quik stain)

Fig. 6.11  Prominent mucinous vacuolization resulting in low nuclear
to cytoplasmic (N:C) ratio, is commonly encountered in adenocarcinoma. Irregular nuclear contours, nuclear enlargement, and 4:1 nuclear
diameter size difference can also be appreciated in these images. (a)
Diff-Quik stain; (b) Papanicolaou stain; (c) Cell block section with
hematoxylin and eosin (H&E) stain

6  Pancreaticobiliary Tract Cytology

161

Pancreatic Neuroendocrine Tumor
Pancreatic neuroendocrine tumors (PanNETs) can be identified by several cytomorphologic features:
•
•
•
•

Highly cellular aspirate
Single cells often with bare nuclei
Pseudorosettes and small clusters
Uniform, round or oval, eccentrically placed nuclei
(plasmacytoid)
• Moderate to abundant cytoplasm
• Salt-and-pepper chromatin and distinct nucleoli

Fig. 6.12  Mitotic figures, although not diagnostic of malignancy, are
more frequently seen in adenocarcinoma and would rarely be seen in
benign processes such as pancreatitis. In this cluster of malignant cells,
other features of adenocarcinoma are seen, including nuclear enlargement, irregular nuclear contours, and 4:1 nuclear diameter size difference (Diff-Quik stain)

These tumors are histologically separated into well-­
differentiated (low-grade and intermediate-grade) tumors
(Figs. 6.14, 6.15, 6.16, 6.17, and 6.18) and poorly differentiated
(high-grade) neuroendocrine carcinomas (Figs. 6.19 and 6.20).

a

Fig. 6.14  Highly cellular aspirate with loose clusters in a low-grade
pancreatic neuroendocrine tumor (PanNET) (Diff-Quik stain)

b

Fig. 6.13 (a and b) Nuclear clearing and irregular nuclear contours
and grooves are often seen in adenocarcinomas. Prominent mucinous
vacuolization and 4:1 nuclear diameter size difference are also seen in
these images (Papanicolaou stain)

Fig. 6.15  Pseudorosettes and small clusters in a low-grade PanNET
(Diff-Quik stain)


162

J. Pang and A. Sciallis

a

Fig. 6.16  Aspirates of low-grade PanNETs often consist of predominantly dyscohesive cells with eccentrically placed nuclei (Diff-Quik stain)

b

Fig. 6.19 (a and b) High-grade PanNET with nuclear molding and
scant cytoplasm, similar in cytomorphology to small-cell carcinoma
(Diff-­Quik stain)
Fig. 6.17  Salt-and-pepper chromatin and distinct nucleoli in low-­
grade pancreatic neuroendocrine tumors are better visualized with

Papanicolaou stain

Fig. 6.20  High-grade PanNET with nuclear molding and scant cytoplasm (Papanicolaou stain)
Fig. 6.18  Low-grade PanNETs can appear very bland on ThinPrep.
On closer inspection, the cells have eccentrically placed nuclei and salt-­
and-­pepper chromatin


6  Pancreaticobiliary Tract Cytology

Immunostains are utilized to confirm the diagnosis, as
acinar cell carcinoma (ACC) and solid pseudopapillary
tumors (SPT) can have similar cytomorphology, as discussed
below. PanNETs are typically positive for neuroendocrine
markers such as synaptophysin (Fig. 6.21) and chromogranin
(Fig.  6.22). A panel of immunostains is recommended, as
both ACC and SPT can be positive for neuroendocrine markers (Table 6.2) [14–16].
The proliferative rate is used to grade PanNETs because
it provides prognostic information that may influence clinical management. Staining for Ki-67 has been found to be
useful in FNA specimens in this regard [17]. The Ki-67
index is less than 3% for low-grade tumors, 3–20% for
intermediate-­grade tumors, and greater than 20% for highgrade tumors [18]. Caution should be noted, as a highergrade focus may not have been sampled in FNA
specimens.

163
Table 6.2  Immunohistochemical profiles of pancreatic neuroendocrine tumor (PanNET), acinar cell carcinoma (ACC), and solid pseudopapillary tumor (SPT)
Marker
Pancytokeratin
E-cadherin
Synaptophysin

Chromogranin
CD56
Trypsin
Beta-catenin

PanNET
+
+
+
+
+
−
−

ACC
+
+/sometimes −
−/focal
−/focal
−/focal
+
−

SPT
−/focal
−
+
−/focal
+
−

+ (nuclear)

Fig. 6.23  Aspirates of acinar cell carcinoma (ACC) are typically
highly cellular, consisting of numerous isolated cells, loose aggregates,
and naked nuclei (Diff-Quik stain)

Acinar Cell Carcinoma
Fig. 6.21  PanNETs stain positive for synaptophysin

Several cytomorphologic features will point to acinar cell
carcinoma (ACC):
•
•
•
•

Highly cellular aspirate
Single cells, loose aggregates, naked nuclei
Prominent nucleoli
Granular cytoplasm

The typical “grapelike cluster” arrangement of benign
acinar cells is usually absent. Figures 6.23 and 6.24 illustrate
the appearance of aspirates from these tumors.

Solid Pseudopapillary Tumor
Solid pseudopapillary tumor (SPT) is also recognized by its
characteristic cytomorphologic features:

Fig. 6.22  PanNETs stain positive for chromogranin

• Highly cellular aspirate
• Vascular stalks lined by neoplastic cells


164

a

J. Pang and A. Sciallis

•
•
•
•

Round to oval or bean-shaped nuclei
Nuclear grooves
Hyaline globules
Delicate cytoplasm with indistinct cell borders

Figures 6.25, 6.26, 6.27, 6.28, 6.29, 6.30, 6.31, and 6.32
illustrate the identification of SPTs. As listed in Table  6.2,
immunohistochemical staining is important in the differential diagnosis.

b

Fig. 6.25  Vascular stalks lined by neoplastic cells is a helpful feature
in the diagnosis of solid pseudopapillary tumor (SPT) (Diff-Quik stain)

c

Fig. 6.24  The nuclei of ACC are round to oval, with smooth nuclear
contours. Delicate, granular cytoplasm is also seen. In contrast to normal, benign acinar cells, prominent nucleoli are usually encountered. (a
and b) Diff-Quik stain; (c) Papanicolaou stain

Fig. 6.26  Vascular stalks lined by neoplastic cells in SPT (Papanicolaou
stain)


6  Pancreaticobiliary Tract Cytology

Fig. 6.27  There is frequently a predominance of dyscohesive single
cells with round to oval, sometimes bean-shaped nuclei that are eccentrically placed. As such, low-grade PanNETs and ACC are considered
in the differential diagnosis (Diff-Quik stain)

Fig. 6.28  Hyaline globules can sometimes be seen in SPTs and can be
a helpful diagnostic clue (Diff-Quik stain)

Fig. 6.29  Cell block sections often demonstrate the vascular stalks
lined by neoplastic cells (H&E stain)

165

Fig. 6.30  Nuclear staining for beta-catenin is the key diagnostic
marker of SPTs

Fig. 6.31  Nuclear staining for beta-catenin in the neoplastic cells with
adjacent benign glandular epithelium showing only membranous staining without nuclear staining is useful as an internal negative control

Fig. 6.32  A negative E-cadherin immunostain is helpful in differentiating SPT from PanNET, which is positive for E-cadherin. This image
shows negative staining in the neoplastic cells, with positive staining in
the benign glandular epithelium as an internal positive control


166

J. Pang and A. Sciallis

Pancreatic Cysts
Cystic lesions in the pancreas are being identified with increasing frequency because of increased utilization of imaging studies to evaluate abdominal complaints. As such, many of these
are incidental findings. A CT scan is often the initial test by
which a cystic lesion is detected, and certain features can be
suggestive of a particular diagnosis [19]. The finding of a solitary, septated cystic lesion in the tail of the pancreas in a woman
is highly suggestive of mucinous cystic neoplasm (MCN).
Grapelike clusters of involved side branch ducts are the most
common finding in an intraductal papillary mucinous neoplasm (IPMN). Septa and excrescent nodules along the dilated
main pancreatic duct can be seen in main duct IPMN. EUS has
been reported to be the ideal tool for the evaluation of cystic
lesions [19]. In addition to enabling the performance of FNA,
EUS images can suggest a diagnosis. EUS of pseudocysts may
show a thick wall with floating debris. EUS of serous cystadenomas typically demonstrates a multiloculated, microcystic
lesion with little free fluid in the locules. MCNs are often unilocular and contain highly viscous, clear fluid that may be difficult to aspirate. The appearance of IPMN on EUS ranges
from simple, unilocular lesions to complex cystic masses.
Classifying pancreatic cysts as non-mucinous (i.e., pseudocyst, serous cystadenoma) versus mucinous can be challenging on cytology [5, 13]. Differentiating neoplastic mucin
from contaminating mucin from the gastrointestinal tract
with absolute certainty is difficult on cytology preparations,
as is differentiating normal gastrointestinal and pancreatic
epithelium from the epithelial lining of mucinous cysts with
minimal atypia [5, 13]. Cyst fluid CEA level is the most

accurate test for the diagnosis of a neoplastic mucinous cyst,
with a diagnostic accuracy of 79% when levels are greater
than 192 ng/mL [5, 20]. However, CEA levels do not predict
the presence or absence of malignant transformation in neoplastic mucinous cysts [20].

Fig. 6.33  Aspirates of pseudocysts are paucicellular, consisting of
mixed inflammatory cells and histiocytes. Yellow, hematoidin-like pigment can sometimes be seen

a

b

Pseudocyst
Aspirates of pseudocysts are paucicellular, consisting of
mixed inflammatory cells and histiocytes. Yellow,
hematoidin-­like pigment can sometimes be seen (Fig. 6.33).
Other than gastrointestinal contamination, no extracellular
mucin or epithelial cells are present.

Serous Cystadenoma
Aspirates of serous cystadenoma are sparse in cellularity and
consist of small, bland-appearing cuboidal cells in flat sheets
and loose clusters (Fig. 6.34). These cuboidal cells are rich in
glycogen (Figs. 6.35 and 6.36).

Fig. 6.34 (a and b) Aspirates of serous cystadenoma are sparse in cellularity and consist of small, bland-appearing cuboidal cells in flat
sheets and loose clusters, as seen in these images from a ThinPrep
preparation (Papanicolaou stain)

6  Pancreaticobiliary Tract Cytology

167

a

Fig. 6.35  The cuboidal cells of serous cystadenomas are glycogen-­
rich, as highlighted with a PAS stain

b

Fig. 6.37  Thick, colloid-like extracellular mucin is seen in aspirations
of neoplastic mucinous cysts. (a) Diff-Quik stain; (b) Papanicolaou
stain
Fig. 6.36  Predigestion with diastase abolishes the glycogen in the
cuboidal cells of a serous cystadenoma

Neoplastic Mucinous Cyst
Cytology cannot differentiate between mucinous cystic neoplasm (MCN) and intraductal papillary mucinous neoplasm
(IPMN), nor can it reliably differentiate high-grade dysplasia
from invasive adenocarcinoma (Figs.  6.37, 6.38, 6.39, and
6.40). Features of malignancy should be identified using the
same criteria as for ductal adenocarcinomas, however, as clinical management would be affected (Fig. 6.41). The management of neoplastic mucinous cysts is multifactorial and
includes assessment of the patient’s surgical risk, symptoms,
and the likelihood of a benign diagnosis on cytology. Findings
on imaging, including size, presence of mural nodule, and
dilatation of the main pancreatic duct, are also considered.
When the clinical and imaging findings do not show clear
indications for surgery, an abnormal cytology could result in

Fig. 6.38  The neoplastic mucinous epithelium (right) demonstrates
disordered nuclei and apical mucin in contrast to normal intestinal epithelium (left) (Papanicolaou stain)


168

J. Pang and A. Sciallis

a

a

b

b

Fig. 6.39 (a and b) Although the nuclei of neoplastic mucinous cysts
are disordered, enlarged, and have irregular nuclear contours, significant nuclear pleomorphism (4:1 nuclear diameter size difference)
should be absent in mucinous cysts with low or moderate dysplasia
(Papanicolaou stain)

c

Fig. 6.40 (a–c) Fibrovascular cores and papillary-like projections can
sometimes be seen in aspirates of neoplastic mucinous cysts, suggestive
of an intraductal papillary mucinous neoplasm. However, this is not
absolute, and correlation with imaging to determine communication
with or without the pancreatic duct is necessary (Diff-Quik stain)

6  Pancreaticobiliary Tract Cytology

a

169

b

Fig. 6.41 (a and b) Malignant features in this neoplastic mucinous cyst including mitotic figures and a 4:1 nuclear diameter size difference are
easily identified in these images (Papanicolaou stain)

Fig. 6.42  ThinPrep preparation of a bile duct brushing with both
benign ductal epithelium (left) and adenocarcinoma (right). Nuclear
enlargement and membrane irregularity, prominent nucleoli, and 4:1

nuclear diameter size difference are evident in the cluster of malignant
cells, in contrast to the flat honeycomb sheet consisting of uniform,
small nuclei of benign ductal epithelium (Papanicolaou stain)

triaging the patient to surgery. In the international consensus
guidelines for the management of branch-duct IPMN, a cytology interpretation of suspicious or positive for malignancy
would result in the patient being triaged to surgery [21].

patients with primary sclerosing cholangitis (PSC), where
reactive changes can mimic carcinoma. As such, adjunct
fluorescence in situ hybridization (FISH) testing using the
UroVysion™ probe set has been advocated to improve the
sensitivity of detecting malignancies, compared with routine
cytology alone [22]. The sensitivity of positive FISH testing
ranges from 34 to 53% in the literature, compared with

8–38% for positive routine cytology in head-to-head comparisons [23–30]. The specificity of FISH is slightly lower
than that of routine cytology, but it remains high (from 89 to
100%) [23–30]. These probes are directed to chromosome 3
(CEP3), chromosome 7 (CEP7), chromosome 17 (CEP17),
and the 9p21 locus. The most common FISH abnormalities

Bile Duct Brushings
The same criteria used in the diagnosis of pancreatic ductal
adenocarcinoma in EUS-FNA are used to evaluate for malignant cells in a bile duct brushing (Fig. 6.42). Given the limitations of cytology, indeterminate diagnoses such as
“atypical” or “suspicious” are not infrequent, especially in


170

J. Pang and A. Sciallis

Table 6.3  UroVysion™ fluorescence in situ hybridization (FISH) interpretation for pancreaticobiliary brushing specimens
FISH cell
type
Disomy
Trisomy
Tetrasomy

Definition
2 copies of each probe
3 copies of a single probe
(usually CEP7)
4 copies of each probe

Polysomy

>2 copies of ≥2 probesa

Cutoff
value
NA
10 cells

Test
interpretation
Negative
Equivocal

10 cells

Equivocal

5 cells

Positive

Clinical significance
Expected signal pattern in benign epithelium
Increased risk of malignancy but not diagnostic of adenocarcinoma
Increased risk of malignancy but not diagnostic of adenocarcinoma;
may represent replicating cells
High specificity for malignancy

Excluding cells with exactly four copies of each probe, which are classified as tetrasomy

a

are polysomy, trisomy, and tetrasomy. A cell with polysomy
(more than two copies) of multiple probes is defined as
“polysomy,” but if each probe displays exactly four copies,
the cell is defined as “tetrasomy.” A cell with three copies of
a single probe is defined as “trisomy.” The cutoff for considering a case positive for these abnormalities is at least five
cells with polysomy, ten or more cells with trisomy, and ten
or more cells with tetrasomy. Once the threshold for polysomy is reached, a positive interpretation is rendered. If
thresholds for polysomy and an additional abnormality, such
as trisomy, are met, the case is classified as polysomy, as this
abnormality is very specific for malignancy. Polysomy is
interpreted as a positive FISH result, whereas trisomy and
tetrasomy are considered equivocal results. Table 6.3 summarizes the FISH interpretation of pancreaticobiliary brushings [22].

References
1.Al-Haddad M, Eloubeidi MA.  Interventional EUS for the diagnosis and treatment of locally advanced pancreatic cancer. JOP.
2010;11:1–7.
2.Erickson RA, Garza AA.  Impact of endoscopic ultrasound on
the management and outcome of pancreatic carcinoma. Am J
Gastroenterol. 2000;95:2248–54.
3.Volmar KE, Vollmer RT, Routbort MJ, Creager AJ. Pancreatic and
bile duct brushing cytology in 1000 cases: review of findings and
comparison of preparation methods. Cancer. 2006;108:231–8.
4.Hewitt MJ, McPhail MJ, Possamai L, Dhar A, Vlavianos P,
Monahan KJ.  EUS-guided FNA for diagnosis of solid pancreatic neoplasms: a meta-analysis. Gastrointest Endosc. 2012;
75:319–31.
5.Thosani N, Thosani S, Qiao W, Fleming JB, Bhutani MS, Guha
S. Role of EUS-FNA-based cytology in the diagnosis of mucinous
pancreatic cystic lesions: a systematic review and meta-analysis.

Dig Dis Sci. 2010;55:2756–66.
6.Pitman MB, Genevay M, Yaeger K, Chebib I, Turner BG,
Mino-­Kenudson M, Brugge WR.  High-grade atypical epithelial
cells in pancreatic mucinous cysts are a more accurate predictor of malignancy than “positive” cytology. Cancer Cytopathol.
2010;118:434–40.
7.Pitman MB, Lewandrowski K, Shen J, Sahani D, Brugge W,
Fernandez-del Castillo C. Pancreatic cysts: preoperative diagnosis
and clinical management. Cancer Cytopathol. 2010;118:1–13.
8.Layfield LJ, Wax TD, Lee JG, Cotton PB. Accuracy and morphologic aspects of pancreatic and biliary duct brushings. Acta Cytol.
1995;39:11–8.

9.McGuire DE, Venu RP, Brown RD, Etzhorn KP, Glaws WR, Abu-­
Hammour A. Brush cytology for pancreatic carcinoma: an analysis
of factors influencing results. Gastrointest Endosc. 1996;44:300–4.
10.

Ryan ME.  Cytologic brushings of ductal lesions during
ERCP. Gastrointest Endosc. 1991;37:139–42.
11.Jing X, Wamsteker EJ, Li H, Pu RT.  Combining fine needle

aspiration with brushing cytology has improved yields in diagnosing pancreatic ductal adenocarcinoma. Diagn Cytopathol.
2009;37(8):574.
12.Pitman MB.  Overview of diagnostic terminology and reporting.
In: Pitman MB, Layfield LJ, editors. The Papanicolaou Society of
Cytopathology system for reporting pancreaticobiliary cytology.
Cham: Springer; 2015. p. 1–3.
13.Pang JC, Minter RM, Kwon RS, Simeone DM, Roh MH.  The
role of cytology in the preoperative assessment and management
of patients with pancreaticobiliary tract neoplasms. J Gastrointest
Surg. 2013;17:501–10.

14. Pitman MB. Pancreas and biliary tree. In: Cibas ES, Ducatman BS,
editors. Cytology: diagnostic principles and clinical correlates. 4th
ed. Philadelphia: Elsevier Saunders; 2014. p. 399–422.
15.Ohara Y, Oda T, Hashimoto S, Akashi Y, Miyamoto R, Enomoto T,
et  al. Pancreatic neuroendocrine tumor and solid-pseudopapillary
neoplasm: key immunohistochemical profiles for differential diagnosis. World J Gastroenterol. 2016;22:8596–604.
16.Tajima S, Waki M, Azuma M, Koda K, Ohata A.  E-cadherin-­
negative acinar cell carcinoma of the pancreas: report of a case
showing a solid pseudopapillary growth pattern. Med Mol Morphol.
2016;49:177–81.
17.Farrell JM, Pang JC, Kim GE, Tabatabai ZL. Pancreatic neuroendocrine tumors: accurate grading with Ki-67 index on fine-needle
aspiration specimens using the WHO 2010/ENETS criteria. Cancer
Cytopathol. 2014;122:770–8.
18.Klimstra DS, Modlin IR, Coppola D, Lloyd RV, Suster S.  The
pathologic classification of neuroendocrine tumors: a review of
nomenclature, grading, and staging systems. Pancreas. 2010;39:
707–12.
19.Brugge WR. The use of EUS to diagnose cystic neoplasms of the
pancreas. Gastrointest Endosc. 2009;69(2 Suppl):S203–9.
20.Petrone MC, Arcidiacono PG. Role of endosocopic ultrasound in
the diagnosis of cystic tumours of the pancreas. Dig Liver Dis.
2008;40:847–53.
21.Tanaka M.  International consensus on the management of intraductal papillary mucinous neoplasm of the pancreas. Ann Transl
Med. 2015;3:286.
22. Fritcher EGB, Kipp BR, Halling KC, Clayton AC. FISHing for pancreatobiliary tract malignancy in endoscopic brushings enhances the
sensitivity of routine cytology. Cytopathology. 2014;25:288–301.
23. Bangarulingam SY, Bjornsson E, Enders F, Barr Fritcher EG, Gores
G, Halling KC, Lindor KD. Long-term outcomes of positive fluorescence in situ hybridization tests in primary sclerosing cholangitis. Hepatology. 2010;51:174–80.
24.Fritcher EG, Kipp BR, Halling KC, Oberg TN, Bryant SC, Tarrell
RF, et  al. A multivariable model using advanced cytologic meth-

6  Pancreaticobiliary Tract Cytology
ods for the evaluation of indeterminate pancreatobiliary strictures.
Gastroenterology. 2009;136:2180–6.
25.Gonda TA, Glick MP, Sethi A, Poneros JM, Palmas W, Iqbal S,
et al. Polysomy and p16 deletion by fluorescence in situ hybridization in the diagnosis of indeterminate biliary strictures. Gastrointest
Endosc. 2012;75:74–9.
26.Kipp BR, Stadheim LM, Halling SA, Pochron NL, Harmsen S,
Nagorney DM, et al. A comparison of routine cytology and fluorescence in situ hybridization for the detection of malignant bile duct
strictures. Am J Gastroenterol. 2004;99:1675–81.
27.Moreno Luna LE, Kipp B, Halling KC, Sebo TJ, Kremers WK,
Roberts LR, et  al. Advanced cytologic techniques for the detection of malignant pancreatobiliary strictures. Gastroenterology.
2006;131:1064–72.

171
28.Smoczynski M, Jablonska A, Matyskiel A, Lakomy J, Dubowik
M, Marek I, et  al. Routine brush cytology and fluorescence in
situ hybridization for assessment of pancreatobiliary strictures.
Gastrointest Endosc. 2012;75:65–73.
29.

Charatcharoenwitthaya P, Enders FB, Halling KC, Lindor
KD. Utility of serum tumor markers, imaging, and biliary cytology
for detecting cholangiocarcinoma in primary sclerosing cholangitis. Hepatology. 2008;48:1106–17.
30.Levy MJ, Baron TH, Clayton AC, Enders FB, Gostout CJ, Halling
KC, et  al. Prospective evaluation of advanced molecular markers
and imaging techniques in patients with indeterminate bile duct
strictures. Am J Gastroenterol. 2008;103:1263–73.

7

Liver Cytology
Derek B. Allison, David Borzik, and Qing Kay Li

Introduction
Advanced imaging techniques can detect many benign and
malignant liver lesions, including simple liver cysts, focal
nodular hyperplasia, hepatic adenoma, hepatic hemangioma,
hepatocellular carcinoma, cholangiocarcinoma, and the
majority of metastatic carcinomas [1–5]. Despite these
increasingly precise techniques, it is still critical to obtain
lesional tissue for morphological diagnosis and molecular
characterization of lesions that require medical or surgical
management [1, 6, 7]. Furthermore, small lesions less than
1 cm in size may not be well characterized by a radiological
image study [8–10]. Currently, percutaneous fine needle aspiration (FNA) and needle core biopsy are the most commonly
used procedures for the morphological evaluation of liver
lesions. These procedures are usually performed under ultrasound guidance or CT guidance in an outpatient setting.
Studies have shown that these procedures have a high diagnostic accuracy, with minimal risks for patients. The sensitivity of these procedures for diagnosing malignant tumors has
been reported to be 90% (range, 67–100%), with 100% specificity [7, 10, 11]. Percutaneous FNA biopsy has been reported
to have a 100% positive predictive value for liver malignancy,
59.1% negative predictive value, and 92.4% overall accuracy
[7, 10, 11]. The main challenge of diagnosing liver masses is
to differentiate a primary from a metastatic tumor [12–14].
Several factors may affect the diagnostic sensitivity and accu-

racy, such as the operator’s skill and experience, the size and
location of the lesion, the quality of cytological smears, and

the cytopathologist’s expertise [7, 15–18].
Endoscopic ultrasound-guided FNA (EUS-FNA) is
the latest diagnostic and staging tool, with a sensitivity
of 82–94% and a specificity of 90–100% [2, 4, 5, 10, 11].
This procedure is safe and accurate but highly operator-­
dependent. EUS-FNA can access the left lobe of the
liver, hilum, proximal right lobe, gallbladder, extrahepatic biliary system, and perihilar lymph nodes. It is
especially useful for small and deep-seated left lobe
lesions, which cannot be easily accessed by percutaneous FNA [2, 4, 5, 10, 11].
During FNA and/or biopsy procedures, several types of
cytological specimens can be prepared, including direct
smears, cell block preparations, and core biopsies. Direct
smears play a critical role in the on-site assessment of lesions.
Unlike core biopsy and cell block preparations, direct smear
preparations do not require formalin fixation and can be performed during rapid on-site evaluation for specimen adequacy [7, 16, 17]. Core needle biopsy and cell block
preparations are still the preferred techniques for obtaining
tumor samples, particularly in patients whose lesion requires
ancillary studies or molecular characterization.
This chapter summarizes the key findings for the accurate
morphological diagnosis of benign, malignant, and metastatic lesions of the liver.

D. B. Allison, M.D. · D. Borzik, M.D.
Department of Pathology, The Johns Hopkins Medical Institutions,
Baltimore, MD, USA
e-mail: ;
Q. K. Li, M.D., Ph.D., F.C.A.P. (*)
Department of Pathology and Oncology, The Johns Hopkins
Medical Institutions, Baltimore, MD, USA
e-mail:

© Springer International Publishing AG, part of Springer Nature 2018
X. Jing et al. (eds.), Atlas of Non-Gynecologic Cytology, Atlas of Anatomic Pathology,
/>
173


174

D. B. Allison et al.

 enign Bile Duct Epithelium, Normal Liver
B
Cells, and Benign Lesions
The cytological features of benign bile duct epithelial cells,
benign liver cells, and the most commonly seen benign liver
lesions are summarized in Table 7.1.

Table 7.1  Summary of main cytological features of benign conditions of the liver
Conditions
Benign liver
cells

Bile duct
epithelium (bile
ductules)

Kupffer cells

Histiocytes

Focal nodular
hyperplasia
Hepatic
adenoma
Bile duct
hamartoma
Hemangiomas

Extramedullary
hematopoiesis
Mallory body

Bile pigment

Lipofuscin

Hemosiderin

Main findings
Sheets, trabeculae or tissue fragments, and/or
dispersed individual cells. Centrally located
round to oval-shaped nuclei, small nucleoli,
granular chromatin, and intranuclear
pseudoinclusions. Binucleation. Dense
cytoplasm with bile and lipofuscin pigments
Small clusters or sheets of cuboidal cells.
Epithelial appearance. Ovoid nuclei with
granular chromatin, inconspicuous nucleoli,
scant cytoplasm. Nuclear disarray may be
present

Intermediate-sized cells. Elongated nuclei,
vacuolated cytoplasm, and cytoplasmic
hemosiderin pigment
Loosely formed two-dimensional clusters or
dispersed individual cells, with coffee beanshaped nuclei, fine chromatin, inconspicuous
nucleoli, and foamy cytoplasm
Benign-appearing hepatocytes. No nuclear
atypia. Presence of benign bile duct epithelial
cells
Benign-appearing hepatocytes. Absence of bile
duct epithelial cells
Benign ductal epithelium. Benign-­appearing
hepatocytes. Scattered stromal cells
Scant tissue fragments with closely packed,
thin-walled capillaries. Benign-appearing
endothelial cells. Blood in the background

Variable amount of megakaryocytes, nucleated
red blood cells, and various stages of white
blood cell maturation
Cytoplasmic eosinophilic inclusion with
characteristic “twisted-rope” appearance. Red
color by Papanicolaou stain and blue color by
Diff-Quik stain
Cytoplasmic pigment. Dark-green color by
Papanicolaou and Diff-Quik stains
Cytoplasmic pigment. Golden-brown color by
Papanicolaou stain; green-brown color by
Diff-Quik stain
Cytoplasmic pigment. Yellow-brown color by

Papanicolaou stain; blue-brown color with
Diff-Quik stain

N:C ratio nuclear-cytoplasmic ratio

Key features of differential diagnosis
Polygonal cells with centrally located nuclei and prominent small
nucleoli and dense cytoplasm. Smooth nuclear membrane. Normal N:C
ratio
Differential diagnosis: hepatic adenoma, focal nodular hyperplasia,
well-differentiated hepatocellular carcinoma, melanoma, and metastatic
carcinomas
Cells are smaller than hepatocytes. Ovoid, darkly stained, overlapping
nuclei. Scant cytoplasm. Normal N:C ratio
Differential diagnosis: cholangiocarcinoma, metastatic adenocarcinomas

Resemble macrophages
Differential diagnosis: endothelial cells, cholangiocarcinoma, metastatic
adenocarcinoma
No nuclear atypia. Abundant cytoplasm with vacuoles and pigment.
Normal N:C ratio
Differential diagnosis: cholangiocarcinoma, hepatocellular carcinoma
No nuclear atypia. Normal N:C ratio. Benign bile duct epithelial cells
Differential diagnosis: hepatic adenoma, regenerating nodule in cirrhosis
Variable cellularity on slides. No nuclear atypia. Normal N:C ratio
Differential diagnosis: well-differentiated hepatocellular carcinoma
Scant specimen, no malignant features
Differential diagnosis: cholangiocarcinoma, metastatic adenocarcinoma
Bloody specimen. Thin-walled capillaries. Benign-appearing endothelial
cells. Hemosiderin-laden macrophages

Differential diagnosis:
spindle cell lesions such as granulomatous hepatitis, leiomyosarcoma,
melanoma, spindle cell carcinoma
Multinucleated megakaryocytes
Differential diagnosis:
Reed-Sternberg cells seen in Hodgkin lymphoma
Commonly seen in alcohol-related liver disease and hepatocellular
carcinoma
Differential diagnosis: eosinophilic body in urothelial cell carcinoma
Seen in both benign hepatocytes and hepatocellular carcinoma
Differential diagnosis: melanin pigments, lipofuscin, and hemosiderin
pigments
Normal findings due to cellular aging or degenerative changes
Differential diagnosis: melanin pigments, bile pigments, and
hemosiderin pigments
Seen in normal liver and abnormal iron metabolism


7  Liver Cytology

175

Benign Bile Duct Epithelial Cells

Benign Liver Cells

Benign bile duct epithelium appears as small clusters and/or
sheets of cohesive, uniform cells. Of note, the size of benign
bile duct epithelial cells is smaller than that of normal hepatocytes. These cells have round to ovoid nuclei, dark and
granular chromatin, inconspicuous or occasionally small

nucleoli, and scant cytoplasm. Focal nuclear overlap and
nuclear disarray are not uncommon findings and may mimic
an adenocarcinoma, but in adenocarcinoma (including primary and metastatic lesions), tumor cells will have a markedly increased nuclear-cytoplasmic ratio (N:C ratio), coarse
or hyperchromatic chromatin, prominent nucleoli, and, in
many cases, vacuolated cytoplasm. Therefore, making particular note of the nuclear features as well as imaging and
clinical characteristics will be very useful for the accurate
assessment of a cluster of epithelial cells (Fig. 7.1).

Benign liver cells (Fig. 7.2) may be present in several patterns, including scattered clusters, flat sheets, or dispersed
individual cells. The specimen also may reveal trabecular
arrangements and may contain many tissue fragments. Most
commonly, benign hepatocytes are found as sheets or as dispersed, individual, large, polygonal cells with centrally
placed round to oval nuclei, granular chromatin, small and
prominent nucleoli, and, most importantly, a normal N:C
ratio. Binucleation is often encountered and should not be
interpreted as cytologic atypia. Bile and lipofuscin pigments
are often identified in the cytoplasm. Bile stains dark green
by Papanicolaou and Diff-Quik methods, whereas lipofuscin
stains golden brown by Papanicolaou stain and green-brown
by Diff-Quik. Benign liver cells may be seen in cirrhosis,
hepatic adenomas, focal nodular hyperplasia, nodular regenerative hyperplasia, and other conditions. During the cytological evaluation of slides, it is important to know the
location of the lesion, the type of procedure performed, and
how the sample was obtained (such as the path of the needle)
because benign hepatocytes may be the predominant cell
type on the slide and can obscure rare lesional cells in certain
circumstances.

Fig. 7.1  Benign bile duct epithelial cells (Papanicolaou, 40×)

Fig. 7.2  Benign hepatocytes (Diff-Quik, 20×)

176

D. B. Allison et al.

Bile Duct Hamartoma

Cirrhosis

The bile duct hamartoma (Fig.  7.3), also known as von
Meyenburg complex, Meyenburg complex, or biliary hamartoma, is a benign tumor-like developmental malformation of
the liver [19–21]. The incidence of this lesion is estimated to
be between 1% and 3%, based on autopsy series [19–21]. It
is usually an incidental finding, but it may be associated with
autosomal dominant polycystic kidney disease and Caroli’s
disease. The bile duct hamartoma may be singular (located
throughout the liver parenchyma) or multifocal (located
mainly in the subcapsular area). These lesions are usually
smaller than 1 cm. FNA reveals bland-appearing columnar
cells haphazardly arranged in tubules, sheets, and two-­
dimensional clusters. The cells can be admixed with scattered stromal cells, which constitute the dense, fibrous
stroma that surrounds the bile ducts in the liver, but this
material is not always easily aspirated. Surrounding benign-­
appearing hepatocytes, which are more readily aspirated,
may comprise a majority of the cellularity.
The main differential diagnosis for a bile duct hamartoma
includes cholangiocarcinoma and metastatic adenocarcinoma. In cholangiocarcinomas and adenocarcinomas, tumor
cells form three-dimensional clusters with large, hyperchromatic nuclei, coarse chromatin, irregular nuclear membranes,
prominent nucleoli, and vacuolated cytoplasm. Appropriate

immunohistochemistry (IHC), based on the patient’s history
of malignancy, will be useful when a metastasis is suspected.
In combination with cytology, clinical imaging will be most
helpful in differentiating between a bile duct hamartoma and
cholangiocarcinoma [19–21].

Although not routinely assessed on FNA, cirrhotic livers
may be sampled because of the presence of an ill-defined
cirrhotic nodule, which is indeterminate for a mass on an
image study. On FNA, the specimen can be variably cellular
with hepatocytes, bile duct epithelium, and stromal cells/
fragments (Fig. 7.4). Hepatocytes may appear both normal
or mildly atypical—showing some degree of anisocytosis,
binucleation, slightly increased N:C ratios, prominent nucleoli, or intranuclear inclusions.
Liver cirrhosis is a well-known risk factor for the development of hepatocellular carcinoma (HCC), which is the
most important entity in the differential diagnosis. In fact,
the presence of abundant bile duct epithelium is a helpful
feature to distinguish cirrhosis from hepatic adenomas and
well-differentiated HCC. Hepatic adenomas do not contain
intralesional bile duct epithelium. In HCC, tumor cells show
a wide range of cytomorphological atypia. In well-­
differentiated forms, tumor cells resemble normal liver cells
and may form trabeculae, cords, or nests. Typically, however,
they have an increased N:C ratio, large round nuclei, naked
nuclei, and bile pigment in the cytoplasm. In poorly differentiated forms, malignant cells are markedly polygonal and
dyscohesive, with pleomorphic nuclei and giant tumor cells.
The most notable feature of HCC, particularly in well-­
differentiated tumors, is the identification of sinusoidal capillaries surrounding the markedly thickened trabeculae of
neoplastic cells.

Fig. 7.3  Bile duct hamartoma (Diff-Quik, 20×)

Fig. 7.4  Cirrhosis (Papanicolaou, 10×)


7  Liver Cytology

177

Fatty Liver Changes

Extramedullary Hematopoiesis

Fatty change, known as hepatic steatosis, can be the result of
a number of diverse physiologic changes and can occur in
the background of benign and malignant lesions. In fact, it
has been reported that steatosis can be identified in up to
40% of HCCs [22, 23]. It is also interesting to note that the
prevalence of fatty change decreases as HCC tumor size
increases [22, 23]. Steatosis is best appreciated by a Diff-­
Quik stain as intracytoplasmic vacuoles or as dispersed bubbles in the background of the slide (Fig.  7.5). In a
well-differentiated HCC, the finding of tumor cells with
prominent fatty change should not be confused with a benign
lesion or a histiocytic process.

Extramedullary hematopoiesis (EMH) is always an abnormal finding in adults and can be caused by bone marrow failure disorders such as myelofibrosis [20]. In such conditions,
EMH commonly involves the liver and spleen. FNA cytology reveals a variable amount of megakaryocytes, nucleated
red blood cells, and various stages of white blood cell maturation. The most easily identifiable feature of EMH is the
presence of megakaryocytes [24]. Sometimes, however,
megakaryocytes can be confused with Reed-Sternberg cells,

which are seen in Hodgkin lymphoma. Megakaryocytes are
much larger than Reed-Sternberg cells and typically have
three to five lobes (Fig.  7.6). In addition, other features of
Hodgkin lymphoma should be absent in the specimen.
Finally, the mixed cell types and lack of clonal expansion
should favor a benign process over a neoplastic process.

Fig. 7.5  Fatty liver changes (hepatic steatosis) (Diff-Quik, 20×)

Fig. 7.6 Extramedullary hematopoiesis with two megakaryocytes
present (Diff-Quik, 20×)


178

D. B. Allison et al.

Focal Nodular Hyperplasia

Hepatic Adenoma

Focal nodular hyperplasia (FNH) is typically an incidental
finding most commonly found in women of reproductive age
[20, 25, 26]. Recent studies have reported that the lesion
involves both females and males equally [25, 26]. On imaging, FNH can form a tumor-like mass that may warrant tissue
sampling, though most suspected FNH lesions can be monitored with serial imaging and do not require tissue sampling
[20, 25, 26]. When sampling is required because of indeterminate radiographic features, FNA specimens are often
bloody and contain clusters or dispersed individual, benign-­
appearing liver cells with either no atypia or, at most, mild
nuclear atypia. Cells have abundant granular cytoplasm and

normal N:C ratios. Bile duct epithelial cells, often forming
long tubular structures, are also identified in most cases
because bile ducts may proliferate at the periphery of the
lesions (Fig. 7.7). Like cirrhotic nodules, FNH nodules are
surrounded by bands of fibrosis, which may or may not be
present as stromal cells or fragments of fibrous tissue on
aspirates.
The main differential diagnosis includes hepatic adenomas and well-differentiated HCCs. In hepatic adenomas,
the bile duct epithelial cells should be absent. In HCC
(particularly well-differentiated carcinomas), tumor cells
may recapitulate the normal architecture of hepatocytes,
forming trabeculae, cords, and nests. Cytologically, however, HCC cells have increased N:C ratios, large round
nuclei, prominent nucleoli, and naked nuclei. The most
notable feature of HCC is the identification of sinusoidal
capillaries surrounding markedly thickened trabeculae of
neoplastic cells, which will be absent in both FNH and
hepatic adenomas.

Hepatic adenoma, also referred to as a liver cell adenoma, is an
uncommon benign tumor most often occurring in women of
child-bearing age who have a prolonged history of using oral
contraceptives [27–29]. In the general population, this lesion is
rare and has been associated with androgen therapy and underlying metabolic diseases such as diabetes mellitus and glycogen
storage disease. Hepatic adenomas can become large, outgrow
their vascular supply, and develop intratumoral hemorrhage or
undergo spontaneous rupture into the peritoneal cavity [27–29].
Therefore, accurate diagnosis is important because of the potential life-threatening risk of hemorrhage. Cytologically, FNA
specimens are hypercellular and contain clusters or dispersed
individual, benign-­
appearing, monotonous hepatocytes with

little to no nuclear atypia and normal N:C ratios (Fig. 7.8). No
bile duct epithelial cells are present within the lesion, but a few
bile duct epithelial cells may be present from aspiration of surrounding normal liver parenchyma.
It is important to distinguish hepatic adenomas from other
benign liver tumors such as hemangiomas (spindle cell lesion,
mixed with benign liver cells) and focal nodular hyperplasia
(presence of bile duct epithelial cells), because malignant transformation to HCC may occur in up to 10% of patients [27–29].
Unlike HCC, hepatic adenomas should have preserved reticulin
scaffolding and lack significant nuclear atypia. Finally, several
molecular subtypes have been identified in hepatic adenomas,
such as inactivating mutations in hepatocyte nuclear factor 1A,
activating mutations in β-catenin, activation of inflammatory
signaling pathways, and several other genetic alterations [29]. It
has been reported that activating mutations of the β-catenin gene
are associated with a higher risk of malignant transformation
and the development of tumor bleeding [29].

Fig. 7.7  Focal nodular hyperplasia with benign hepatocytes and bile
duct epithelium (Diff-Quik, 10×)

Fig. 7.8  Hepatic adenoma with an absence of bile duct epithelium
(Papanicolaou, 20×)


7  Liver Cytology

179

Hemangioma

Hydatid Cyst

Hepatic hemangiomas are the most common mesenchymal
tumors of the liver; they are reported to be present in up to
20% of the general population, based on several autopsy and
imaging studies [20, 30, 31]. Many of these tumors are present at the time of birth [30, 31]. The causes of this lesion are
not clear, but it has been postulated that sex hormone imbalances may play a role, as it has been found that the size of
tumors increases markedly during pregnancy [30, 31]. Most
tumors are less than 4 cm in size, are typically solitary and
subcapsular in location, and are discovered incidentally on
imaging performed for unrelated reasons. Although both
ultrasound and CT can be used for the detection of tumors,
magnetic resonance imaging (MRI) is the best imaging technique for the characterization of the tumor, particularly with
the advanced MRI techniques and usage of hepatocyte-­
specific contrast agents. On MRI, tumors show a heterogeneous appearance. Extensive scarring can mimic malignant
neoplasms of the liver such as cholangiocarcinoma, requiring tissue sampling for accurate classification [30, 31].
These tumors comprise dilated vascular spaces lined by
flat, bland-appearing endothelial cells without atypia, with
various amounts of intervening fibrous septa. On FNA, these
characteristics are often displayed as scant tissue fragments
with embedded and closely packed, thin-walled capillaries in
a background of abundant blood. The endothelial cells of
these capillaries are notably bland. The background of slides
may reveal benign liver cells, stromal cells, and hemosiderin-­
laden macrophages (Fig. 7.9).
The main differential diagnosis includes spindle cell
lesions such as granulomatous hepatitis, leiomyosarcoma,
melanoma, spindle cell carcinoma, and others. GLUT-1 is an
immunohistochemical marker that is highly specific for
hemangioma and can be used to differentiate hemangioma

from vascular malformations [32].

Hydatid cysts (echinococcal cysts) are caused by infection of the larval stage of the cestode (or tapeworm) of the
Echinococcus species. The transmission is caused by
accidental swallowing of eggs found in feces of dogs.
Larvae develop over the course of years to form fluidfilled cysts in various organs, particularly the liver.
Clinically, these present as a large, cystic lesion; though
rare in the United States, this is the most common cause
of liver cysts worldwide [33, 34]. Cysts can grow to considerable size and contain a large amount of fluid containing infectious scolices. On imaging, cysts are typically
solitary; about a quarter are lined by a wall of calcification. If suspected clinically, cysts should not be aspirated,
out of concern for cyst fluid leakage and subsequent anaphylactic shock. The cysts have an acellular wall made
from both host tissue (pericyst) and larval tissue (endocyst). FNA specimens contain fragments of the laminated
cystic wall, scolices, hooklets, and fluid (Fig.  7.10).
Morphologically, the hooklets have the distinctive appearance of shark teeth and are acid-fast positive. The differential diagnosis includes other cysts of parasitic origin,
and clinical and microbiological correlation is often
required for the accurate diagnosis.

Fig. 7.10  Hydatid cyst of the liver with fibrous cystic wall (hematoxylin and eosin [H&E], 20×)

Fig. 7.9 Hemangioma with large, intact vascular structures
(­Diff-­Quik, 20×)


180

D. B. Allison et al.

Primary Malignant Tumors
The most common primary malignant tumors of the liver are
hepatocellular carcinoma (HCC), accounting for 80–85% of

liver tumors [35, 36]. The incidence of HCC has more than
doubled over the past 30 years in the United States [35, 36],
largely owing to the increased incidence of hepatitis B and
hepatitis C infections (particularly in the so-called “baby
boomer” population), alcohol abuse, and obesity and type II
diabetes [35, 36]. It is well known that the development of
HCC is also associated with many other insults that damage
the liver, such as alcohol- and non-alcohol-related cirrhosis,
aflatoxin B1 exposure, and parasite infections [37, 38]. HCC
is most commonly seen in the sixth and seventh decades of
life and is two to three times more common in men than in
women [35, 36]. Recent molecular studies have also found
that mutations of TP53 and β-catenin (CTNNB1) are frequently associated with HCC [39].
Clinically, tumors may present as a single mass or multiple masses throughout the liver [37, 38]. Because of advances
in imaging technology, 80% of HCC cases can be diagnosed
in liver nodules larger than 2 cm. Therefore, hepatic nodules

that are sampled by FNA are more likely to be smaller than
2 cm, lacking diagnostic imaging features of HCC [40]. The
cytological diagnosis of a well-differentiated HCC, as well
as the grading of the tumor, can be challenging, particularly
in cirrhotic patients [15, 41, 42]. Other primary tumors,
including cholangiocarcinoma, hepatoblastoma, neuroendocrine tumors, and many others, are much less common and
are most difficult to distinguish from metastatic carcinomas
[5, 43].
Importantly, metastases are much more common than primary hepatic malignancies in non-cirrhotic livers [5, 43, 44].
Most metastases can be diagnosed on the basis of cytomorphology, based on the similarity of the sample with features
of the primary malignancy, but it is usually necessary to confirm the diagnosis by IHC study. Furthermore, in difficult
cases, IHC markers may be used to aid in the differentiating
a primary liver tumor from a metastasis. A panel of markers

is usually performed, including liver markers of arginase 1,
glypican-3, hepatocyte paraffin 1 (Hep Par 1), CD10, and
other IHC markers of various organ origins [45–48].
The cytological features of major types of malignant
tumors are summarized in Table 7.2.

Table 7.2  Summary of main cytological features of malignant tumors
Conditions
Hepatocellular
carcinoma
(well-differentiated)

Main findings
Wide trabeculae, cords, nests, sheets or dispersed
tumor cells. Large round nuclei, granular
chromatin, prominent nucleoli. Cytoplasmic bile
pigment. Increased N:C ratio

Hepatocellular
carcinoma (poorly
differentiated)

Dyscohesive individual tumor cells. Presence of
giant tumor cells. Markedly nuclear
pleomorphism. Numerous naked nuclei on slides

Hepatocellular
carcinoma
(fibrolamellar variant)

Polygonal tumor cells with large nuclei and
abundant oxyphilic cytoplasm. Dense lamellar
material on slides

Cholangiocarcinoma

Clusters, crowded sheets, disorganized
honeycomb groups, and dispersed individual
cuboidal cells. Large nuclei, prominent nuclei,
scant cytoplasm. High N:C ratio
In fetal cell variant, tumor cells resemble normal
liver cells. In the anaplastic variant, tumor cells
reveal a pleomorphic appearance with high
mitotic activity and abundant tumor cell necrosis.
In the embryonal and small-cell undifferentiated
variant, the tumor reveals a primitive and
undifferentiated appearance with hyperchromatic
nuclei and scant cytoplasm
Three-dimensional clusters, papillary and acinar
arrangements of columnar cells. Hyperchromatic
nuclei with prominent nucleoli, coarse chromatin,
“lacy” cytoplasm, and cytoplasmic vacuolization
(cytoplasmic mucin)
Dyscohesive clusters, loosely formed twodimensional cellular sheets, and scattered
individual polymorphic cells. Hyperchromatic
nuclei. Smudgy chromatin. With or without
cytokeratin formation

Hepatoblastoma

Metastatic
adenocarcinoma

Metastatic squamous
cell carcinoma

Key features of differential diagnosis
Resemble normal hepatocytes, but loss of the reticulin
scaffolding. Numerous naked nuclei on slides. Endothelial cell
surrounding thickened cords of tumor cells
Differential diagnosis: regenerating nodule of cirrhosis, liver
cell adenoma, focal nodular hyperplasia
Numerous pleomorphic individual tumor cells with
hyperchromatic nuclei, irregular nuclear shape, large prominent
nucleoli
Differential diagnosis: metastatic carcinoma,
cholangiocarcinoma
Large polygonal tumor cells separated by dense lamellar
material
Differential diagnosis:
other types of hepatocellular carcinoma
No cytoplasmic bile pigment, no naked nuclei on slides.
Similar to other type of adenocarcinoma
Differential diagnosis: hepatocellular carcinoma, metastatic
adenocarcinoma
Heterogeneous group of neoplasms with wide variation in their
morphology
Differential diagnosis: hepatocellular carcinoma, small-cell
carcinoma, metastatic carcinomas

Malignant columnar cells. Irregular nuclear membrane, coarse
chromatin, prominent nucleoli, and cytoplasmic vacuole
(mucin production). High N:C ratio
Differential diagnosis: cholangiocarcinoma, hepatocellular
carcinoma
Polygonal, rounded, elongated, or tadpole-shaped cells. Large,
dark nuclei, smudgy chromatin, and dense cytoplasm
(cytokeratin formation) or without cytokeratin formation
Differential diagnosis: poorly differentiated hepatocellular
carcinoma


7  Liver Cytology

181

Table 7.2 (continued)
Conditions
Metastatic small-cell
carcinoma

Carcinoid

Metastatic melanoma

Lymphoma (non-­
Hodgkin lymphoma)

Sarcomas

Main findings
Tight clusters of small, hyperchromatic cells (2–3
times the size of mature lymphocytes). Nuclear
molding and crowding, nuclear stripes (broken
nuclear material), inconspicuous nucleoli, scant
cytoplasm
Loosely cohesive clusters and scattered individual
cells. Rosette-like arrangement. Relatively
uniform tumor cells, with fine (salt-and-­pepper)
chromatin and moderate cytoplasm. No mitosis or
necrosis
Scattered individual large cells with prominent
nucleoli. Cytoplasmic melanin pigment.
Binucleation with “mirror” arrangement.
Pseudointranuclear inclusions
Dispersed individual atypical lymphoid cells with
coarse chromatin and irregular nuclear membrane,
prominent nucleoli. High N:C ratio and scant
cytoplasm. Increased mitotic activity and the
presence of background lymphoglandular bodies
Four major cytomorphologic categories
depending on predominant tumor type:
 •  Epithelioid and clear cell morphology
 •  Spindle cell morphology
 • Biphasic spindle and epithelioid
morphology
 •  Small round cell morphology

Key features of differential diagnosis
Fine chromatin (salt-and-pepper appearance), paranuclear blue

bodies, mitosis, necrosis, and apoptotic bodies
Differential diagnosis: lymphoma, basaloid squamous cell
carcinoma, poorly differentiated adenocarcinoma
Monomorphic appearance of tumor cells, with fine chromatin,
inconspicuous nucleoli. Branching capillaries in the
background. No mitosis or necrosis
Differential diagnosis: atypical carcinoid, small-cell carcinoma
Large malignant cells with prominent nucleoli, dense
cytoplasm
Differential diagnosis: poorly differentiated carcinoma,
Hodgkin lymphoma, hepatocellular carcinoma
Tumor cells range from small to large in size, depending on the
type of lymphoma. Monomorphic population of lymphocytes
in SLL/CLL. Polymorphic population in other types
Differential diagnosis: reactive lymphocytes, small-cell
carcinoma, poorly differentiated carcinoma
Heterogeneous group of mesenchymal neoplasms with wide
variation in their morphology, genetics, immunoprofile, and
clinical behavior
Differential diagnosis:

CLL chronic lymphocytic leukemia, N:C ratio nuclear-cytoplasmic ratio, SLL small B-cell lymphocytic lymphoma

Hepatocellular Carcinoma
In HCC, tumor cells show a wide range of cytomorphological variability [15, 41, 42]. Generally, FNA of HCC
reveals a hypercellular specimen composed of dispersed,
single cells and tissue fragments with various architectural and cytological features indicative of the differentiation (grade) of the tumor. Further, there should be a
notable absence of bile ducts in lesional tissue; but bile
ducts located adjacent to the carcinoma in the patient may
be aspirated, smeared, and displaced next to tumor cells

on the slide. In a well-­differentiated HCC, tumor cells
resemble relatively normal liver cells and form wide trabeculae, cords, and nests with only a mildly to moderately
increased N:C ratio. Large round nuclei, prominent nucleoli, naked nuclei, and bile pigment in the cytoplasm are
typical findings. In poorly differentiated forms, malignant
cells are polygonal and dyscohesive, with pleomorphic
nuclei, naked nuclei, and giant tumor cells. The most
notable feature of HCC, particularly in w
­ ell-­differentiated
tumors, is the presence of sinusoidal capillaries surrounding markedly thickened trabeculae of neoplastic cells—a
phenomenon referred to as “endothelial wrapping.” This
feature, not seen in benign liver cells and/or cholangiocarcinoma, is essentially pathognomonic. Another common
feature is the presence of small vessels traversing tissue
fragments.

In difficult cases, IHC markers may be used to aid in
the differential diagnosis. A panel of markers is usually
performed, including arginase 1, glypican-3, Hep Par 1,
CD10, and CAM5.2 for proving hepatocyte tissue origin
[45–47]. For the differential diagnosis of cholangiocarcinoma, the panel should include polyclonal or monoclonal
carcinoembryonic antigen (CEA), CK7, and maspin [47].
Cholangiocarcinoma is positive for CEA, CK7, and
maspin. Polyclonal CEA and CD10 demonstrate a canalicular staining pattern in HCC but a cytoplasmic pattern
in
cholangiocarcinoma.
The
demonstration
of
α-fetoprotein (AFP) positivity points toward a malignant
tumor of hepatocellular origin, but it is only positive in
30% of HCC cases [46]. It is also positive in nonseminomatous germ cell tumors and extrahepatic AFP-producing

carcinomas [46]. Hep Par 1 antibody (clone OCH1E5.2.10),
developed in 1993 by Wennerberg et al., stains normal and
neoplastic hepatocytes [48]. Several studies have reported
that this antibody is a sensitive marker for HCC (80–90%)
[46–48]. However, recent studies have found that it also
frequently stains gastric carcinomas (30–47%) [47].
Several other tumors can also stain positively for Hep Par
1, including yolk sac tumors and carcinomas of the adrenal cortex, lung, colon, and ovary. It also stains cholangiocarcinoma in 50% of cases, and unfortunately, poorly
differentiated HCCs are more likely to be negative for
Hep Par 1 [47, 48].


182

 ell-Differentiated Hepatocellular Carcinoma
W
Tumor cells of well-differentiated HCC resemble normal
liver cells and form wide trabeculae, cords, and nests with an
increased N:C ratio. Tumor cells have large, round nuclei
with prominent nucleoli. The cytoplasm may contain prominent bile pigments and/or lipofuscin pigments, which are
golden and brown in color with Papanicolaou staining—a
feature that is very helpful in distinguishing HCC from a
metastatic carcinoma. Naked nuclei are also commonly present on the slide. The most notable feature in HCC, particularly in well-differentiated tumors, is the loss of the reticulin
scaffolding pattern and the presence of endothelial wrapping, as described above (Fig. 7.11). This feature is best seen
on core biopsy specimens but can also be visualized on
smears. Unfortunately, the mild nuclear atypia seen in well-­
differentiated HCCs can be confused with many entities,
including nodular regenerative hyperplasia in cirrhosis. As a
result, clinical and radiographic correlation, such as the presence of “rapidly enlarged liver mass” is incredibly helpful in
the diagnosis.

D. B. Allison et al.

 oorly Differentiated Hepatocellular Carcinoma
P
In a poorly differentiated tumor, numerous pleomorphic
individual tumor cells with hyperchromatic nuclei, irregular
nuclear shape, prominent nucleoli, and dense granular cytoplasm are often appreciated. Tumor cells may be arranged in
cords, clusters, or dispersed as individual cells. Numerous
naked nuclei with large, prominent cherry-red nucleoli and
intranuclear pseudoinclusions are also seen on smears
(Fig. 7.12). Dense cytoplasm with cytoplasmic bile/lipofuscin pigments are less likely to be present in a poorly differentiated case, but when they are present, they are particularly
helpful in distinguishing HCC from a metastatic
carcinoma.

Fig. 7.12  Poorly differentiated hepatocellular carcinoma (Diff-Quik,
20×)

Fig. 7.11  Well-differentiated hepatocellular carcinoma with endothelial wrapping