Immunohistochemical features of the gastrointestinal tract tumors
Department of Pathology, City of Hope National Medical Center, Duarte, California, USA
Review Article
Immunohistochemical features of the gastrointestinal tract tumors
Department of Pathology, City of Hope National Medical Center, Duarte, California, USA
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Abstract
Gastrointestinal tract tumors include a wide variety of vastly different tumors and on a whole
are one of the most common malignancies in western countries. These tumors often present at late
stages as distant metastases which are then biopsied and may be difficult to differentiate without the aid
of immunohistochemical stains. With the exception of pancreatic and biliary tumors where there are no
distinct immunohistochemical patterns, most gastrointestinal tumors can be differentiated by their unique
immunohistochemical profile. As the size of biopsies decrease, the role of immunohistochemical stains will
become even more important in determining the origin and differentiation of gastrointestinal tract tumors.
Key words
Immunohistochemistry; gastrointestinal neoplasms; diagnosis
Submitted Mar 10, 2012. Accepted for publication Mar 29, 2012.
DOI: 10.3978/j.issn.2078-6891.2012.019 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Esophagus
Squamous cell carcinoma (SCC)
SCC of the esophagus has been associated with various
geographic, ethnic and lifestyle risk factors. Compared
to adenocarcinoma of the esophagus which is the more
common tumor in the United States, SCC is much more
common in Asian countries, where up to 40% have been
linked to HPV infection (1). SCC is more common in
males, particularly African American males and lifestyle
risk factors such as smoking and alcohol are believed to
increase the risk of SCC up to 90% (1,2). Patients may
present with dysphagia, odynophagia and weight loss.
Although SCC can develop in any part of the esophagus but
are more commonly found in the middle and lower third
portions of the esophagus (3,4). On gross examination the
tumor is usually circumferential with sharp margin and
are often ulcerate. Polypoid forms may also be seen (1).
Microscopically, the tumors resemble their counterparts
in the skin and show varying degrees of squamous
differentiation with extensive keratinization in the welldifferentiated
forms and lack of cohesiveness, with even
a pseudoglandular configuration in poorly-differentiated
forms. The immunohistochemical profile of SCC is similar
to that of its skin counterpart: CK7-, CD20-, CK5/6+,
CK10+ and CK14+ (Figure 1A). SCC is always positive for
p63 (Figure 1B) (5-9). Additionally, most cases of esophageal
SCC are also positive for p53, a finding not seen in normal
esophageal mucosa (8). As mentioned before, HPV has been
found to be associated with esophageal SCC, particularly in
cases reported from China (10) and Africa (11) where up to
20-40% of esophageal cases have been shown to be positive
for HPV, particularly type 11, 16 and 18. Many of these
HPV cases have been found to be positive for p16 as well,
much similar to cases of cervical SCC (12,13).
Figure 1 Immunohistochemical features of esophageal squamous cell carcinoma. A. CK14 highlights the tumor cells; B. p63 shows nuclear
positivity in the tumor cells
Intestinal metaplasia(IM)
IM (Barrett’s esophagus) is defined as the presence of
specialized intestinal epithelium in the distal esophagus
above the level of the lower esophageal sphincter (14,15), and
according to the American College of Gastroenterology
Barrett’s mucosa is defined as a change in the esophageal
epithelium of any length that can be recognized by
endoscopy and is confirmed to be intestinal metaplasia
(IM) by biopsy. Most patients with IM are adults,
although this condition may develop in children with
gastroesophageal reflux and following chemotherapy
(16,17). Gastroesophageal reflux is believed to play a role
in IM as up to 10% of patients with IM suffer from reflux.
The importance of IM lies mainly in its association with
the development of adenocarcinoma since more than 80%
of patients with adenocarcinoma have been shown to have
associated IM. Histologically, IM is quite similar to normal
small intestinal mucosa with the presence of absorptive cells,
goblet cells and Paneth cells. IM is further classified into three categories based on the degree of dysplasia: negative for
dysplasia, indefinite for dysplasia and positive for dysplasia
(low-grade and high grade). These are based on evaluation
of surface maturation in comparison to underlying glands,
architecture of the glands, cytologic features, inflammation
and the presence of erosions/ulcers (18). In additional to
its unique morphologic features, IM also shows a unique
immunohistochemical profile. Greater than 95% cases of
IM have characteristic superficial CK20 staining pattern
along with a strong superficial and deep CK7 staining (19-21). Unlike normal gastric mucosa where cells are positive
for MUC1, MUC5AC and MUC6 but negative for MUC2
the cells in IM/Barrett’s esophagus are positive for MUC2.
The intensity of MUC2 staining varies according to the
number of goblet cells, being higher in complete IM and
lower in incomplete IM. Other monocolonal antibodies
which are specific to gastric or colonic mucosa have also
been used to confirm the diagnosis of IM such as Das-
1 antibody which binds to colonic epithelial protein in
absorptive cells, and HepPar-1 which is expressed in small
intestinal mucosa but not normal gastric and colonic
mucosa. Another marker that has been found to be useful
in distinguishing the degree of dysplasia is AMACAR.
AMACAR is a marker for prostate adenocarcinoma, and
is also expressed in normal small intestinal and colorectal
mucosa. AMCAR has also been found to be expressed in
cases of IM with dysplasia, with an incidence of 20%, 40%
and 80% in cases of indefinite, low grade and high-grade
dysplasia, respectively (22) but is negative in cases of IM
without dysplasia (22,23). p53 expression can also aid in
the classification of dysplasia as up to 60% of cases of highgrade
dysplasia and carcinoma express p53 in comparison
to just 30% of cases classified as indefinite for dysplasia and
low grade dysplasia (24,25). The increase in p53 expression
is accompanied by increased Ki-67 labeling (26,27). IM
with and without dysplasia can also be separated by using
a combination of the markers described above. IM without
dysplasia is usually positive for HepPar-1 and MUC2 and
negative for AMCAR, whereas IM with dysplasia and
adenocarcinoma often express AMCAR but not HepPar-1
or MUC2 (28,29).
Esophageal adenocarcinoma is rapidly increasing in
incidence in the United States (30,31). Predisposing factors
include male gender, white race, obesity, Barrett’s esophagus,
smoking and alcohol consumption (32). Most cases of
esophageal adenocarcinoma involve the lower one third of
the esophagus and show glandular differentiation. These
tumors usually express CK7, variable CK20, AMACAR,
and weak focal CDX-2, an immunohistochemical pattern
similar to that of gastric adenocarcinoma. P16 is negative in
esophageal adenocarcinoma unlike SCC (26).
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Stomach
Gastric Epithelial Dysplasia (GED)
GED most commonly occurs in men in their fifth to
seventh decades, and is more common in westernized
countries. There is often no gross features which can be
recognized endoscopically but microscopically there may
be glandular crowding, branching, budding, cytologic
atypia, decreased apical mucin and frequent mitoses. GED
may arise in either native gastric or intestinalized gastric
epithelia, and is divided into three categories: indefinite
for dysplasia, low-grade and high-grade dysplasia (33).
Studies have show that 15% of low-grade dysplasia may
show progression to carcinoma while cases of carcinoma from high-grade dysplasia is seen in 80-85% (34,35).
Immunohistochemical stains may assist in the assessment
of dysplasia as p53 expression and Ki-67 positive dysplastic
cells also increase as dysplasia increases (36).
Gastric Intestinal Metaplasia (GIM)
GIM is similar histologically and immunohistochemically to
Barrett’s esophagus/esophageal adenocarcinoma. It is defined
as the development of goblet and/or Paneth cells within the
normal gastric mucosa. The two main types of GIM are the
complete type (type I) characterized by its resemblance to
normal small intestinal mucosa with absorptive cells, Paneth
cells and goblet cells; and the incomplete type (types II and
III) where there are columnar and goblet cells. Most cases
of gastric carcinoma arise within areas of incomplete GIM,
and show CK7 and CK20 in the superficial and deep crypt
cells (37-39). GIM is also positive for HepPar-14 and CDX-
2 (40). The complete type of GIM is negative for MUC1 and
MUC5AC but positive for MUC2 while incomplete GIM
is positive for both (22). H. pylori infection has been found
in over 80% of patients with GIM (37) which can then be
identified by using the Das-1 antibody which stains H. pylori
in gastric associated GIM (36).
Gastric adenocarcinoma (GA)
GA is the second most common cancer worldwide with the
highest rates in Asia. It is more common in males and has
been associated with risk factors such as low socioeconomic
status, cigarette smoking, nitrites, chronic gastritis and H.
pylori (41-43). The majority of gastric adenocarcinomas is
located in the pylorus and antrum (50-60%), followed by
the cardia (25%), and the body or fundus (15-25%) and may
be exophytic, flat or ulcerated. There are two classifications
of GA, the intestinal type, which has well-formed glands
lined by columnar to cuboidal epithelial cells (Figure 2),
and the diffuse type which shows single to poorly formed nests of cells growing in an infiltrate pattern (signet ring
cell carcinoma) (Figure 3A) (43,44). Intestinal type GA shows
variable expression of CK7 (Figure 2B), CK20 (Figure 2C),
CDX-2 (Figure 2D), MUC1, and MUC5AC (45-47).
Diffuse type of GA usually develops de novo, and is not
associated with H. pylori induced IM. Over 70% of cases of the
diffuse type of GA are positive for CDX-2 (Figure 3B), CK7
(Figure 3C), HepPar-1 (Figure 3D) and variable expression
of CK20 (Figure 3E), MUC2 and MUC5AC, but negative
for MUC1 and E-cadherin (Figure 3F) (48,49). Cases of
poorly differentiated adenocarcinoma with prominent
lymphoplasmacytic stroma may also be positive for EBV
(50,51).
Figure 2 Histologic and immunohistochemical features of gastric adenocarcinoma – intestinal type. A. Gastric adenocarcinoma-intestinal type; B. CK7 shows variable expression in tumor cells; C. CK20 with variable expression; D. CDX-2 diffuse nuclear positivity
Figure 3 Histologic and immunohistochemical features of gastric adenocarcinoma - diffuse type/signet ring cell carcinoma. A. Gastric adenocarcinoma- diffuse type/signet ring cell carcinoma; B. Variable CDX-2 positivity; C. CK7 positivity; D. HepPar-1 expression; E. CK20
shows variable positivity; F. E-cadherin is negative
Tumors of the upper gastrointestinal tract such as
Barrett’s esophagus, esophageal adenocarcinoma and gastric
adenocarcinoma may show similar immunohistochemical
findings, Table 1 compares each of their unique
immunohistochemical profile (52,53).
Gastrointestinal stromal tumor (GIST)
Stromal tumors comprise the majority of primary nonepithelial neoplasms in the stomach, and GIST is the
most common GI mesenchymal neoplasm. GIST may occur
anywhere within the GI tract but is most common in the
stomach (60%) (53), with prognosis varying according to
their location (54). Histologically, GISTs resemble smooth
muscle tumors with spindle or epithelioid cells. Gastric
GIST generally have a better prognosis compared to small
intestinal GIST and may have either a predominantly
spindle cell pattern or an epithelioid pattern. Features
associated with a more aggressive behavior include a high
mitotic rate (>5/per 50 hpf), large size (>5 cm), invasion,
location within the fundus or gastrointestinal junction,
coagulative necrosis, ulceration and epithelioid morphology
(55,56). The vast majority of GISTs show a diffuse
cytoplasmic staining with membranous accentuation of
CD117 (KIT) (Figure 4A). CD117 is the product of the
c-kit gene and is a type-3 tyrosine kinase receptor which
is normally expressed in the interstitial cells of Cajal, mast
cells, melanocytes, fetal endothelial cells and CD34-positive
hematopoietic stem cells. CD117 is also positive in a variety of
tumors such as mastocytoma, seminoma, pulmonary small cell
carcinoma and blastic types of myeloid sarcoma just to name a
few (57). Although CD117 positivity is present in most GIST,
it is not required for diagnosis (58), since 5-10% of gastric
GIST and 4% of small intestinal GIST may be negative for
CD117 (57). Most CD117 negative GISTs are positive for
another GIST marker-DOG-1 (Figure 4B). The diagnosis
of GIST then requires examination of the morphologic,
immunohistochemical and molecular PDGRFRA mutation
analysis. Other immunohistochemical markers which may
be positive in GIST include PDGFRA5, CD34 (80%),
SMA (20%) (55), DOG1 (79%), and CK18 (59). Antibody
cocktails for keratin such as AE1/AE3 are generally negative
in gastric GIST as they are negative for CK7, CK17, CK19
and CK20. S-100 is also only positive in <1% of gastric
GISTs (57). GFAP is negative in GIST and thus helps in
differentiating from gastrointestinal schwannoma which is
GFAP positive.
Figure 4 Immunohistochemical features of gastrointestinal stromal tumors (GIST). A. CD117 shows diffuse cytoplasmic staining with membranous accentuation; B. DOG-1 also shows diffuse positivity
Extranodal marginal zone lymphoma of mucosaassociated
lymphoid tissue (MALT)
MALT is the most common type of lymphoma to occur
in the stomach (60). Development of MALT has been
associated with Helicobacter pylori infection with induction
of remission reported by antibiotic treatment of the H.
pylori (61). The lymphoma cells are B-cells and infiltrate
the marginal zone around the preserved follicles. The cells
are small to medium in size with a monocytoid appearance.
Plasmacytic differentiation is often present in gastric
MALT lymphomas (60). Tumor cells are positive for CD20,
CD79a and Pax-5 but negative for CD5, CD10, and CD23.
Aberrant CD5 co-expression has been described while
co-expression of CD43 has been reported in one-third
of cases (62). Cytogenetic abnormalities in MALT include
t[11;18], t[1;14], t[14;18] and t[3;14] with t[11;18] being the
most common translocation in MALT lymphomas involving
the stomach (63,64).
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Small intestine
The small intestine includes the duodenum, jejunum and
ileum extending from the pylorus to ileocecal valve, yet
neoplasms in the small intestine are extremely rare. Global
incidence of small intestinal neoplasms is less than 1.0
per 100,000, and in the United States they represent only 0.4% of total cancers (65). The different types of primary
small intestinal tumors include adenocarcinomas, carcinoid
tumors, lymphomas and sarcomas (66,67).
Adenocarcinoma of the small intestine
Adenocarcinoma of the small intestine is the most common
type of primary malignancy in the small bowel, and
generally presents in older males with a higher incidence in
African Americans than Caucasians. Most cases are sporadic
but reported risk factors include sporadic adenomatous
polyps, familial adenomatous polyposis and Crohn’s disease.
Presentation may include obstruction, jaundice, GI bleeding
and abdominal pain, and often presents at an advanced
stage. The most common locations for adenocarcinoma are
the duodenum and proximal jejunum. Adenocarcinomas
may present as polypoid, infiltrative or constricting lesions,
with tumors in the duodenal and ampullary regions
generally being exophytic in nature (67). Histologically,
these tumors are similar to colorectal adenocarcinomas and
are characterized by the degree of pleomorphism, complex
glandular architecture, luminal necrosis and invasion. Small
intestinal adenocarcinomas are CK7+ in more than half of
all cases (Figure 5A), unlike normal small intestinal mucosa
which is CK7- and colorectal adenocarcinomas which are
CK7-/CK20+ (68). Adenocarcinomas of the small bowel are
also positive for CK20 (Figure 5B), CDX-2 (Figure 5C), and
villin (68).
Figure 5 Immunohistochemical features of small intestinal adenocarcinoma. A. CK7 positivity; B. CK20; C. CDX-2showing diffuse positivity
Adenocarcinoma of ampulla of Vater
Adenocarcinoma of ampulla of Vater comprise about
5-6% of cancers arising (69) in the head of the pancreas.
These tumors cause obstruction of the bile duct even at
a very small size and hence patients often present early
in the disease course with jaundice. Two major histologic
types have been described: an intestinal type, arising from
the overlying intestinal mucosa of the papilla (intestinal
type adenocarcinoma of duodenal papillary origin) and
a pancreatobiliary type, derived from the ductal epithelium
which penetrates the duodenal muscularis propria (ampullary
carcinoma of pancreatobiliary origin) (69). The intestinal type
adenocarcinoma is much more common and has a much
better prognosis (70), hence it is important to differentiate
these two entities. Fortunately, the immunophenotype
of these two types differ, with the intestinal type
adenocarcinoma of duodenal papillary origin being
positive for CK7, CK20, MUC2 and CDX-2 but negative
for MUC1, MUC5AC and CK17; whereas ampullary
carcinoma of pancreatobiliary origin is positive for MUC1,
CK7, and CK17 but negative for MUC2 (69,70).
Gastrointestinal neuroendocrine tumors
Gastrointestinal neuroendocrine tumors are tumor
derived from endocrine cells and can arise anywhere in
the gastrointestinal tract. Most neuroendocrine tumors
and carcinomas (carcinoids) in the GI tract are well
differentiated. The location of these carcinoid tumors
can be divided based on their embryologic derivation
into carcinoids of the foregut (esophagus, stomach and
duodenum), midgut (jejunum, ileum, appendix and
ascending colon) and hindgut (transverse colon, descending
colon, sigmoid and rectum) (71). Tumors from each
different region of the gastrointestinal tract may secrete
different hormones as well. Foregut and midgut carcinoid
often produce serotonin and substance P while hindgut
carcinoids may produce glucagon like peptide, pancreatic
polypeptide, and polypeptide YY (72-78). In spite of these
differences, these tumors share similar morphologic features
such as clusters/sheets/nests of neuroendocrine cells with
round to ovoid nuclei, “salt and pepper” chromatin and
moderate amounts of clear cytoplasm. All gastrointestinal
neuroendocrine tumors are positive for the generic markers
of neuroendocrine differentiation such as chromogranin A,
synaptophysin and NSE, as well as PGP 9.5, and CD56 (79).
Determining the origin of the tumor may be challenging;
however, immunohistochemical stains can be very helpful. Carcinoids from the foregut and midgut are generally
positive for chromogranin A and CD56, while those from
the hindgut are usually negative (73,80,81). Hindgut
carcinoids on the other hand often express prostatic acid
phosphatase (82). A less helpful marker is CDX-2, which
although positive for most colorectal carcinomas has an
immunoreactivity of about 40% in well differentiated
carcinoids (83-87) but has a reported 80% expression rate
in poorly differentiated carcinoids (80).
Carcinoid tumors make up about a third of the neoplasms
in the small intestine. They most often occur in the ileum
and rarely in the duodenum and can be separated by their
location: duodenal and jejunoilieal carcinoids. Duodenal
carcinoids, similar to any carcinoid in the gastrointestinal
tract can be further divided by the type of cells which
make up the tumor into gastrinomas (G-cell tumors),
somatostatinomas (D-cell tumors) and a small percentage
of the undefined type (88). Classification of neuroendocrine
tumors is based on the degree of differentiation. Most
carcinoids are well-differentiated carcinoid (50-75%),
well-differentiated neuroendocrine carcinoma and poorly
differentiated neuroendocrine carcinoma (<1-3%) (88).
Carcinoid tumors usually show a monotonous proliferation
of small bland polygonal cells with round nuclei, “salt and
pepper” chromatin and moderate amounts of cytoplasm in
either a nested (type A), trabecular (type B) or acinar (type
C) pattern. Distinction between benign and malignant
carcinoid is based on the presence or absence of metastasis
rather than just on histology.
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Esophagus
Colorectal cancer (CRC)
CRC is the third most common cancer diagnosed in the
United States and third most common cause of cancer deaths.
Risk for development of colorectal carcinoma increases
significantly after the age of 40. In addition to age, lifestyle
modifiers and genetic risk factors all play a role in CRC.
Family history and genetics plays an important role as well,
particularly in patients less than 50 years. Approximately
25% of CRC arise in patients with a family history of
disease while 5% arise in the setting of an established
familial syndrome (89). The genetic syndromes associated
with CRC can be divided into the hereditary polyposis
colon cancers (HPCC) and hereditary nonpolyposis colon
cancers (HNPCC). Categories of HPCC include: (I)
Familial adenomatous polyposis; (II) MUTYH-associated
polyposis; (III) hyperplastic polyposis syndrome; (IV) Peutz-
Jeghers syndrome; and (V) Juvenile polyposis syndrome (89).
Of the polyposis CRC familial adenomatous polyposis (FAP)
is the most common. FAP is an autosomal dominant disease
with 100% penetrance. Patients with FAP develop hundreds
to thousands of adenomatous colonic polyps starting in
the second decade of life with a 100% risk of CRC (89,90).
Another category of HPCC is the MUTYH - associated
polyposis, an autosomal recessive colon cancer syndrome
which accounts for 0.5% to 1% of all CRC (91,92). Patients
with MUTYH - associated polyposis may have zero to
thousands of polyps like FAP, with an estimated lifetime risk
of CRC around 80% (92). Hyperplastic polyposis syndrome
(HPS) is characterized by the development of numerous,
large hyperplastic and sessile serrated polyps, with a 35% to
54% prevalence of CRC development (93). Peutz-Jeghers
syndrome (PJS) is a rare autosomal dominant disease
characterized by the development of pigmented macules
on lips, mucosa, hands and feet, along with development
of hamartomatous polyps as well as cancers in the CRC,
stomach, small bowel, pancreas, breast, sex cord, uterus,
cervix and skin. Patients with PJS have a 39% lifetime
risk of CRC and 93% risk for any other malignancy (94).
Juvenile polyposis syndrome (JPS) typically presents in
childhood and has an associated 10-38% lifetime risk of
developing colon cancer (95). Lynch syndrome/HNPCC
is the most common autosomal dominant inherited colon
cancer family syndrome responsible for 10% of colon cancer
cases before the age of 50 years (96). The risk of CRC
is related to the development of innumerable adenomas.
Diagnosis of HNPCC is based on the Amsterdam criteria
taking into account the extracolonic malignancies which are
common in HNPCC involving the endometrium, stomach,
ovary, urinary collecting system, skin, pancreatic and
biliary tract (97). Patients with HNPCC have a seven fold
increased risk of CRC and present at least 20 years younger
than the general population (98).
The histopathologic types of CRC recognized by the
World Health Organization include adenocarcinoma,
mucinous adenocarcinoma, signet ring carcinoma, small
cell carcinoma, adenosquamous carcinoma, squamous cell
carcinoma and undifferentiated carcinoma.
Colorectal adenocarcinoma (CA)
CA may be polypoid, exophytic, ulcerative, or infiltrative,
and can present anywhere within the colon. Polypoid
tumors are more common in the cecum and right colon,
while ulcerative tumors are more common in the left
colon and rectum. Microscopically, CA form glands with
mucin (Figure 6A) and are classified as well, moderate
or poorly differentiated. In addition to the genetic
syndromes discussed previously, sporadic CAs have
been associated with mutations in the APC, K-ras and
p53. CA is characterized by a CK7 negative and CK20 positive (Figure 6B) immunophenotype, and thus can
be differentiated from non-ampullary small intestinal
adenocarcinomas by their lack of expression of CK7
and positivity for CK20, Table 2 summarizes several
key immunohistochemical stains which can help in
distinguishing these two entities.
Figure 6 Histologic and immunohistochemical features of colon adenocarcinoma. A: Colon adenocarcinoma; B: Diffuse CK20 positivity in tumor cells; C: Villin shows diffuse positivity; D: CDX-2 is diffusely positive; E: MUC2 positive in mucin producing cells
Appendiceal adenocarcinomas with mucinous
differentiation, as well as rectal adenocarcinomas on
the other hand may also show expression of CK7 and
thus differentiation from metastatic ovarian mucinous
tumors is required. Other markers for CA include
villin which is positive in 80% of CA (Figure 6C),
CDX-2 which is positive (Figure 6D) in almost all well
differentiated CA and adenomas but less than 10-20%
of poorly differentiated adenocarcinomas may be weakly
positive or negative (99). CDX-2 is a transcription
factor involved in the proliferation and differentiation
of intestinal epithelial cells, and the incidence of CDX-
2 expression in adenocarcinoma of the gastrointestinal
tract increases from esophagus to rectum and in cases
where it is positive all tumor cells show a strong staining
pattern. In addition to expression in CA, CDX-2 may
also be expressed in ovarian mucinous adenocarcinomas
and bladder adenocarcinomas (99-101). CA with
mucinous features have an immunophenotype similar
to conventional CA with tumor cells positive for CK20,
CDX-2, MUC2 (Figure 6E) and β-catenin (102), while
the signet ring type of CA is also positive for CDX-2,
CK20 and MUC2.
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Appendix
The appendix may develop any of the tumors described
above from the small and large intestines; however, there
are a few unique entities at this site including mucinous
neoplasms and goblet cell carcinoid tumors.
Goblet cell carcinoid of the appendix (GCC)
GCC is a distinct type of carcinoid tumor within the colorectum
which exhibits both neuroendocrine and intestinal-type
goblet cell morphology (103). Up to 50% of patients with
GCC present with disseminated disease hence it is an
important differential to consider particularly in female
patients, where an ovarian primary is often one of the
first considerations (104). GCC arise from the base of
the epithelial crypts of the appendiceal mucosa without
associated epithelial dysplasia, and show a submucosal
growth pattern with invasion of the appendiceal wall
comprised of goblet or signet ring cells in clusters or
glands separated by smooth muscle stroma (Figure 7A) (105).
The signet ring cells are positive for PAS, mucicarimine,
pancytokeratin, CDX-2 (Figure 7B), CK20, MUC2 and
CEA; as well as focally positive for chromogranin (Figure 7C)
and synaptophysin. Up to 25% of cases are negative for
neuroendocrine markers (106,107).
Figure 7 Histologic and immunohistochemical features of goblet cell carcinoid tumor of the appendix. A. Goblet cell carcinoid tumor of the appendix; B. Tumor cells positive for CDX-2; C. Focal positivity for chromogranin
Mucinous neoplasms of the appendix
Mucinous neoplasms of the appendix are the most
common type of epithelial neoplasms in the appendix.
These neoplasms present in a wide spectrum ranging
from mucinous cystadenoma, low-grade mucinous
neoplasm, and disseminated peritoneal adenomucinosis or
cystadenocarcinoma, mucinous carcinoma, and peritoneal
mucinous carcinomatosis (108). These tumors are associated
with pseudomyxoma peritonei, a clinical condition of
gelatinous ascites, commonly also seen in ovarian mucinous
neoplasms (109-111). The classification of mucinous
neoplasms within the appendix remains a controversial issue.
Broadly speaking, mucinous neoplasms of the appendix
can be divided into two major types: those that resemble
conventional colonic adenocarcinoma with potential for
destructive growth, nodal or solid organ metastasis; and
those, which are predominantly low-grade mucinous
neoplasms with potential for peritoneal dissemination (108).
Their immunophenotype is similar to that of other mucinous
tumors in the lower gastrointestinal tract being positive for
MUC-2, CK20, CDX-2 and beta-catenin, but with lower
expression of CDX-2 and beta-catenin. In addition, mucinous
adenocarcinomas of the appendix with positivity for CK7
(113), hence differentiation from upper GI and mucinous
neoplasms from other areas is necessary.
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Anal tumors
The anal canal is defined as the region located between the
junction of the colorectal-type glandular mucosa and the
junction between the squamous mucosa lined distal portion.
Despite its short length, the anal canal produces a wide
variety of tumor types. Tumors within the anal canal
include: (I) squamous cell tumors including condyloma
acuminatum, flat squamous dysplasia, invasive squamous
cell carcinoma and its variants; (II) adenocarcinoma rectal
type, anal gland adenocarcinoma, fistula-related mucinous
adenocarcinoma and intraepithelial adenocarcinoma (Paget
disease); (III) neuroendocrine neoplasms; (IV) melanoma;
(V) mesenchymal tumors and (VI) lymphoma.
Squamous cell carcinoma
Squamous cell carcinoma is the most common type of
tumor within the anal canal. The incidence of SCC of
the anal region is higher in females (114). There is also
an increase in incidence in high-risk patient population
(HIV positive patients) and an association with HPV
(115). Three distinctive subtypes are recognized based on
their distinctive histologic features: verrucous carcinoma,
squamous cell carcinoma with mucinous microcysts and
small cell (anaplastic) carcinoma (116).
Adenocarcinoma of the anal canal
Adenocarcinoma of the anal canal is much less common, accounting for about 10% of all anal cancers (117).
Similar to squamous cell carcinoma of the anal canal,
adenocarcinomas in this region have been associated
with high-risk HPV types. Other risk factors include
inflammatory conditions such as Crohn’s disease and
chronic anal fistulas (118).
Of the various types of adenocarcinomas in this region,
Paget disease is the one most likely to cause difficulties
in diagnosis. Paget disease of the anal canal may arise
from an underlying anal gland adenocarcinoma, adnexal
(eccrine gland) adenocarcinoma or an underlying visceral
malignancy, most commonly a colorectal adenocarcinoma.
The use of immunohistochemistry can help differentiate
these as those arising from anal gland adenocarcinoma
would be CK7+/CK20+/CDX-2+/GCDFP-15- (119,120),
from adnexal adenocarcinoma would be CK7+/CK20-/
CDX-2-/GCDFP-15+ and that from a colorectal
adenocarcinoma would be CK7-/CK20+/CDX-2+/
GCDFP-15+- (119-124). These tumors may also need to
be differentiated from mammary Paget disease (CK7+/
CEA+/EMA+/HER-2/neu+/MUC1+/ER+/CK20-/CDX-
2-/GCDFP-15+) (125-133) and Paget disease of the vulva
(CK7+/CEA+/EMA+/HER-2/neu-/MUC1+/ER-/CK20-/
CDX-2-/GCDFP-15-) (133-136).
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Pancreas
Although pancreatic tumors are one of the less common
tumors within the gastrointestinal tract, it is the 4th leading
cause of cancer mortality in the United States in both
men and women (137). Due to the nature of the disease,
pancreatic cancers often do not cause symptoms until the
later stages. In fact, less than 10% of pancreatic cancers are
detected at a stage where cure is possible. The overall survival
for this group of cancers is only about 5% (137). Based on the
histological features, pancreatic tumors can be divided into
three main categories: exocrine neoplasms, neuroendocrine
tumors and mixed exocrine-endocrine tumors.
Pancreatic ductal adenocarcinomas
Pancreatic ductal adenocarcinomas make up the majority
(>95%) of pancreatic tumors. Pancreatic cancer is more
common among the elderly, with a higher incidence in men
than in women and more common in blacks compared to
other races (137). Risk factors include cigarette smoking,
family history, diabetes mellitus and obesity (138).
Presentation often occurs late in the disease course as
epigastric pain, weight loss, painless jaundice, light claycolored
stools, dark urine, pruritus, and nausea. Pancreatic
ductal carcinomas often present as poorly defined masses
involving the head of the pancreas (>60%) with variable
degrees of necrosis which may lead to the formation of
cysts (139). Depending on the degree of differentiation
these tumors may show well formed glands in a haphazard
pattern (Figure 8A) or individual cells forming sheets,
single cell infiltration or poorly formed glands in poorly
differentiated adenocarcinoma. Variants of adenocarcinoma
included adenosquamous carcinoma, colloid carcinoma,
hepatoid carcinoma, medullary carcinoma, signet ring cell
carcinoma and undifferentiated carcinoma (139). Most cases
show expression of CK7 (Figure 8B), while a subset focally
express CK20 (40%) (Figure 8C), a feature which allow for
differentiation from extra-pancreatobiliary non-mucinous
adenocarcinomas. Pancreatic ductal carcinomas are also
positive for CK8, CK17 (Figure 8D), CK18, CK19, CEA,
CA19-9, Dupan-2, MUC1, MUC4 and MUC5AC (140-143).
Figure 8 Histologic and immunohistochemical features of pancreatic ductal carcinoma. A. Pancreatic ductal carcinoma; B. Diffuse CK7 positivity in tumor cells; C. CK20 positive; D. CK17 positivity in tumor cells
Pancreatic intraepithelial neoplasia (PanIN)
Pancreatic intraepithelial neoplasia (PanIN) has been
speculated to be the precursor lesion of pancreatic ductal
adenocarcinomas for over fifty years, but it is only recently
that its significance and role in pancreatic carcinoma has
been established. It is one of three major categories of
precursor lesions defined by ongoing epidemiological and
molecular studies, the other two being intraductal papillarymucinous
neoplasm (IPMN) and mucinous cystic neoplasm
(MCN) (144). PanIN is the most common and most
defined precursor lesion of pancreatic ductal carcinoma
(145). These lesions are often found at the same time as the
diagnosis for pancreatic adenocarcinoma, and share similar
genetic alterations such as K-ras mutation, inactivation
of tumor suppressor genes and both show expression of
MUC1 and MUC5AC but not MUC2 (143,146,147).
Mucin-producing cystic neoplasms of the pancreas
Mucin-producing cystic neoplasms of the pancreas
comprise of two entities: mucinous cystic neoplasm (MCN)
and intraductal papillary mucinous neoplasm (IPMN).
Mucinous cystic neoplasms occur almost exclusively in
perimenopausal women in the body or tail of pancreas.
These lesions generally do not show any communication
with the pancreatic duct system and often has a thick wall
and are multiloculated (148). Histologically, the cyst is lined
at least focally by columnar mucinous epithelium and has
an ovarian-type stroma (149). These tumors are positive for
CK7, CEA, CA19-9, pancytokeratin, MUC-2 (in goblet
cells) and EMA (150). Most MCNs also express MUC5AC
while MUC-1 is only expressed in invasive MCNs (151).
The ovarian-type stroma present in MCN may be positive
for ER, PR, inhibin and frequently CD10 (152-154).
Intraductal papillary mucinous neoplasms
Intraductal papillary mucinous neoplasms are more common
in older men and are most often located at the head of
the pancreas. These lesions comprise of an intraductal
proliferation of mucinous epithelium within the main
pancreatic and/or the branching ducts (155,156), usually in
a papillary arrangement without ovarian-type stroma. The
epithelial cells may be of intestinal type, pancreatobiliary
type or null type (similar to gastric foveolar epithelium)
or morphologically unclassifiable (150). Intestinal-type
IPMN often show a colloid-type pattern of invasion and are
frequently positive for CDX-2, and MUC2 but negative for
MUC1, while the pancreatobiliary type is more aggressive
and is negative for CDX-2, MUC2 and positive for
MUC1 (150). The null type on the other hand is generally
negative for MUC1, CDX-2 and MUC2 (157). Mucinous
carcinomas which arise from IMPN are frequently positive
for MUC1 but less often positive for MUC5AC (158).
Solid-pseudopapillary neoplasm (SPN)
SPN is an uncommon pancreatic tumor most often found
in young women (159). Patients present with nonspecific
symptoms related to the intra-abdominal mass such as
abdominal pain and early satiety. SPNs are generally large,
well circumscribed tumors which can occur anywhere
within the pancreas (160). Microscopically, they form
dense nests of uniform eosinophilic cells surrounding
delicate vasculature resembling ependymal rosettes. The
tumor cells often have nuclei with grooves and clear
vacuolated cytoplasm (159). Slide preparations from
material obtained by fine-needle aspirate biopsy show a
distinct “Chinese character-like” appearance due to the
branching capillaries are surrounded by small uniform
tumor cell and show prominent nuclear grooves and/
or inclusions in the tumor cells and background of
metachromatic myxoid material (161). The tumor cells are
positive for alpha-1-antitrypsin, vimentin, NSE, ER-β, PR, CK8/18, CD10, CD56 and synaptophysin (153,162,163).
These tumors have a mutation of the β-catenin gene
and show a diffuse cytoplasmic and nuclear positivity in
virtually all cases by immunohistochemistry (164). Because
the β-catenin complex activates transcription of cyclin D1,
nuclear cyclin D1 immunoreactivity is detected in up to
75% of SPNs (165). SPNs have also been found to show
a loss of cell-cell adhesion molecule and thus are negative
for E-cadherin (166).
Serous cystic neoplasms (SCN)
SCN are neoplasms composed of glycogen-rich, ductularlike
epithelial cells. Most SCNs are benign while others
may be precursors to invasive cancer. Correlation with the
patient’s age, gender, relationship between cysts and larger
pancreatic ducts, cysts contents (serous fluid, mucin or
necrotic debris), lining cell and nature of the stroma are
all required in evaluation. Serous cystadenomas are more
common in females and often present with nonspecific
symptoms such as pain, nausea, weight loss. These tumors
are well-circumscribed masses which on sectioning shows
innumerable small cysts with a “honeycomb” appearance
and often a central scar (167). The cells have a central round
to oval nuclei, inconspicuous nucleoli and clear cytoplasm
and are positive on periodic acid-Schiff (PAS) stain due to the
abundant intracytoplasmic glycogen. SCNs are positive for
low molecular weight keratins (CK7, CK8, CK18 and CK19),
MUC1, EMA (168), alpha inhibin, NSE, and MUC6 but
negative for MUC5A, CK17 and CEA (markers positive in
pancreatic ductal adenocarcinoma) as well as chromogranin,
synaptophysin, vimentin, PR, and β-catenin (169).
Pancreatic neuroendocrine tumors (PNETs)
Pancreatic neuroendocrine tumors (PNETs) are rare neoplasms
with an incidence of 1 per 100,000 individuals per year and
comprising just 1-2% of all pancreatic tumors (170). Pancreatic
neuroendocrine tumors can present at any age but are
most common during the 4th to 6th decades of life with no
sex predilection (170). Although most tumors are sporadic
there is an association with hereditary endocrinopathies
such as multiple endocrine neoplasia type I (MEN I), von
Hippel-Lindau syndrome, neurofibromatosis and tuberous
sclerosis. PNETs can be broadly divided into functional
and nonfunctional tumors. Functional neuroendocrine
tumors are tumors which produce a variety of clinical
syndromes due to an excess in hormones and include
insulinoma, gastrinoma, glucagonoma, VIPoma, and
somatostatinoma (171). The non-functional PNETs
may also produce hormones but generally do not have
symptoms due to the hormone production. These tumors
are classified according to the WHO classification into
well differentiated endocrine tumor, well differentiated
endocr ine carcinoma and poorly d i fferent i ated
endocrine carcinoma based on size, mitotic count, Ki-67
proliferation index, angioinvasion and metastasis. PNETs
are diffusely positive for synaptophysin consistently while
chromogranin A may show a more focal staining pattern of
variable intensity (170). They also express CD56, CD57,
PDG 9.5 and NSE (172,173), as well a CK8 and 18. In
differentiating PNETs from neuroendocrine tumors from
other primary sites, CDX-2 may also be helpful as it has
been reported to be positive in 20-30% of PNET cases
(83,84). Other markers shown to be positive in pancreatic
endocrine tumors include trypsin, chymotrypsin and lipase
(174,175).
Pancreatoblastoma
Pancreatoblastoma is the most common pancreatic
neoplasm of childhood. Most cases occur in children
less than 10 years of age (176), and there is a slight male
predominance and association with Beckwith-Weidemann
syndrome (177). These tumors are generally large, and may
arise in either the head or the tail of the pancreas as wellcircumscribed
and lobulated masses. Histologically, the
tumor has a lobular appearance with well-defined islands
of small epithelial cells separated by fibrous bands with a
geographic pattern of lighter and darker staining cells due
to the different cell types present. The tumor cells in the
darker staining areas are small with centrally placed nuclei
and prominent nucleoli with scant cytoplasm, while cells
in the lighter areas have abundant eosinophilic cytoplasm
and may be spindled in shape with a whorling pattern. The
presence of occasional squamoid nests is characteristic for
this lesion (178). The immunophenotype of the tumor cells
often shows acinar differentiation as they are PAS-positive
with diastase resistant cytoplasmic granules and positive
for trypsin, chymotrypsin and lipase. Despite the presence
of squamoid nests, pancreatoblastomas are negative for
squamous markers (negative for high molecular weight
keratins CK14, CK5/6, and CK17) and CK7 (179) but
positive for CK8, CK18, CK19, EMA and cytoplasm and
membranous β-catenin (180). Up to half of the tumors
may exhibit neuroendocrine differentiation with focal
chromogranin and synaptophysin positivity while the cells of
ductal differentiation are highlighted by their production of
mucin, CEA and CA19 positivity (181). Pancreatoblastomas
have also been found to show alterations in the β-catenin/
APC pathway in up to 80% of cases, hence its positivity by
immunohistochemistry (180).
Acinar cell carcinoma
Acinar cell carcinoma is more common in adults and
presents with non-specific gastrointestinal symptoms such
as abdominal pain, nausea and weight loss. Some patients
may have subcutaneous fat necrosis and polyarthralgia due
to increase levels of serum lipase (159). These tumors are
often large and can occur anywhere within the pancreas
but are more often found at the head of the pancreas.
Microscopically, acinar cell carcinomas show nests of
pyramidal cells arranged in solid or acinar patterns.
Tumor cells have basally oriented nuclei, single prominent
nucleoli and granular cytoplasm. Acinar cell carcinomas
are positive for pancytokeratin, CK8, CK18, zymogen,
trypsin, chymotrypsin and lipase, but negative for CK7 and
CK19 (182,183). Scattered cells positive for neuroendocrine
markers are present in one-third of cases. A few cases may
demonstrate the APC/β-catenin gene mutation (184).
Mixed exocrine-endocrine tumors
Mixed exocrine-endocrine tumors are defined as malignant
epithelial neoplasms where the ductal and endocrine cells are
intimately mixed in the primary tumor with at least one-third
to one-half of tumor cells showing positivity for endocrine
markers (185). Ductal differentiation is defined as ductular
formation and mucin production (174) and presence of
ductal markers like CEA, CK19 and CA19.9, while ductal
acinar cells can be highlighted by pancreatic enzymes like
trypsin, chymotrypsin and lipase (186,187). Endocrine
cells can be characterized by positivity for endocrine
markers chromogranin A and synaptophysin. These mixed
tumors generally behave as ductal adenocarcinomas (187).
It is important to remember that 40-80% of usual ductal
adenocarcinomas may contain endocrine cells, but the
metastases from these tumors generally lack endocrine cells
(174,187).
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Liver
Primary tumors of the liver are divided into epithelial and
non-epithelial (mesenchymal) lesions and then further into
benign and malignant categories. The majority of the mass
lesions within the liver are benign lesions such as focal
nodular hyperplasia (FNH), regenerative nodules, adenoma,
cirrhosis, and vascular lesions. Of the malignant lesions
metastatic tumors are far more common than primary
hepatocellular carcinomas.
Hepatocellular carcinoma (HCC)
HCC is the sixth most common malignancy and third most
common cause of mortality from cancer worldwide (188).
Risk factors for HCC include chronic liver disease such
as chronic hepatitis C virus, hepatitis B virus, cirrhosis,
obesity related liver disease and alcohol-related liver
disease (189). Symptoms of HCC include abdominal pain,
fullness, mass or signs and symptoms of cirrhosis, with
the most helpful indicator being elevated serum levels of
alpha fetal protein (AFP). On gross examination, HCC
presents as a single large mass which may or may not have
satellite nodules. Histologically, well-differentiated HCC is
difficult to differentiate from normal liver as the polygonal
cells resemble hepatocytes and are arranged in trabecular
pattern lined by sinusoids mimicking normal liver but have
intracytoplasmic bile (Figure 9A) (188). Differentiation
between HCC and normal/benign liver is therefore very
difficult especially on small needle-core biopsies, and
immunohistochemical stains are thus very helpful (189,190).
Two immunohistochemical stains that can differentiate HCC
from normal/benign liver are Glyican-3 (Figure 9B), a marker
which is exclusively expressed in neoplastic processes and
not normal tissue in humans (191), and CD34 (Figure 9C) a
vascular marker which highlights the increased vascularity seen
in HCC (192-196). Another marker which is only expressed
in HCC and not normal liver is AFP (Figure 9D) (197). Other
markers for HCC include CD10 (Figure 10A), polyclonal
CEA (Figure 10B) which highlights the canaliculi (198,199),
HepPar-1 (Figure 10C) which reacts with both neoplastic
and normal liver tissue. and AFP (200). HCC express only a
limited number of keratin markers, namely CK8 and CK18
and thus most metastatic carcinomas can be excluded as
they generally express a larger variety of keratin markers
such as CK5/6, CK7, CK14 or CK20 in comparison to
HCC (201).
Figure 9 Histologic and unique immunohistochemical features of hepatocellular carcinoma. A. Hepatocellular carcinoma; B. Glypican-3 shows diffuse positivity in tumor cells; C. CD34 highlights the increased vascularity within the tumor; D. AFP is aberrantly expressed
Figure 10 Immunohistochemical features of hepatocellular carcinoma. A. CD10 shows a canalicular staining pattern; B. polyclonal CEA also highlights the canaliculi; C. HepPar-1 with diffuse intracytoplasmic granular positivity
Cholangiocarcinoma (CC)
CC make up approximately 3% of all gastrointestinal
cancers worldwide (202). These tumors are more common
in elderly men and have been associated with cirrhosis,
hepatitis C, infections by Clonorchis sinesis and Opisthorchisis
viverrini, primary sclerosing cholangitis, Thorotrast
exposure, genetic hemochromatosis, alpha-1-antitrypsin
deficiency and contraceptive steroid use (202-204). CC
arises from the intrahepatic bile duct epithelial cells and
can be divided based on the location of origin, intrahepatic/
peripheral CC arise at the confluence of the right and left
hepatic ducts, while the extrahepatic CC arise between the
ampulla of Vater and the hepatic hilium (205). Depending
on their location the presenting symptoms may also differ.
Histologically, CC is similar to ductal adenocarcinoma
of the pancreas with tumor cells arranged in tubules and glands which may be cribriform or form nests, solid cords
and papillary structures (205). CC is positive for CK7,
CK17, mucin, CEA (cytoplasmic and luminal), CAM 5.2,
CK19, EMA and CK20 (30-70%) (206).
Hepatoblastomas (HB)
HB are the most common primary liver tumors in
children, with the majority occurring in children less
than 2 years of age (207). These tumors have a slight
predominance in males, low-birth weight infants and have
been associated with familial adenomatous polyposis, and
various chromosomal abnormalities as well as mutations in
the β-catenin gene (208). HB generally present as solitary
masses in the right lobe of liver and are classified based
on their histology into six main patterns: fetal pattern,
embryonal pattern, macrotrabecular pattern, small cell
undifferentiated pattern, mixed epithelial and mesenchymal
pattern and mixed pattern with teratoid features (209).
Immunohistochemically HBs are positive for HepPar-1,
AFP and EMA while those with the small cell pattern may
be positive for cytokeratin with the mesenchymal areas
being positive for vimentin (210).
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Gallbladder
Benign bile duct proliferations: Benign bile duct
proliferations such as bile duct hamartomas (also know as
von Meyenburg complexes) and bile duct adenomas are
usually small, incidental asymptomatic lesions identified at
time of autopsy. Bile duct hamartomas are believed to be
formed by failure of the embryonic ductal plates in the liver
to involute. These lesions are often subcapsular, small white
nodules that may require differentiation from metastatic
adenocarcinoma or cholangiocarcinoma (211). Bile duct
adenomas are also small subcapsular nodules consisting
of acini and tubules and may be confused for a malignant
lesion (212). Both of these benign bile duct proliferations
have an immunohistochemical profile similar to that of
pancreatic ductal adenocarcinoma and are positive for CK7,
CK17, MUC1 and MUC5AC (213). They also share an
immunophenotype with bile duct carcinoma, and are all
positive for CK7, focally positive for CK20, and CDX-2;
however, they are negative for p53 and monoclonal CEA
which is positive in bile duct carcinoma (214). Hence, it
is important to correlate with radiological and clinical
findings.
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Conclusions
Tumors of the gastrointestinal tract are varied, yet can
often prove to be diagnostically challenging. Understanding
the unique immunohistochemical profiles of each entity
will greatly assist in the diagnosis of these tumors. Table 3
provides a summary of the immunohistochemical profile of
several key gastrointestinal tumors.
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Acknowledgements
Disclosure: The authors declare no conflict of interest.
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References
Cite this article as: Wong HH, Chu P. Immunohistochemical
features of the gastrointestinal tract tumors. J Gastrointest Oncol
2012;3(3):262-284. DOI: 10.3978/j.issn.2078-6891.2012.019
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