mmunohistochemistry and special stains in gastrointestinal pathology practice

1. Immunohistochemistry
and special stains in
gastrointestinal pathology
practice
Cheng Liu
Masoumeh Ghayouri
Ian S Brown
Abstract
Immunohistochemistry and special stains play an increasingly impor-
tant role in gastrointestinal pathology practice. In neoplastic disorders
they are used to confirm the diagnosis, identify prognostic/predictive
features, and screen for an underlying genetic syndrome. In nonneo-
plastic disorders they can identify an infectious organism, clarify the
inflammatory infiltrate present, and confirm a tissue deposition. In
this review we discuss the most important and topical of these stains,
especially ones which require special care in interpretation. It should
be emphasized that, in cases with equivocal or unexpected staining
patterns, the results should be interpreted in the appropriate clinical,
endoscopic and morphologic context.
Keywords amyloid; cytomegalovirus; gastrointestinal pathology;
Helicobacter pylori; HER2; immunohistochemistry; mismatch repair;
mucin; PD-L1; SATB2; special stain
Introduction
Immunohistochemistry (IHC) and special stains are indispens-
able adjuncts in the reporting of gastrointestinal tract specimens,
whether these are procured by endoscopic biopsy or by surgical
resection. In neoplastic disorders they may confirm a neoplastic
process and/or provide clinically important information. The
latter includes the identification of the site of origin of the
neoplasm, prognostic parameters, prediction of response to
therapy, or potentially identify an underlying genetic syndrome.
Some stains serve more than one purpose, depending on clinical
context. In non-neoplastic disorders IHC and special stains help
to classify an inflammatory reaction pattern or identify an in-
fectious aetiology. Both IHC and special stains may also help
determine the nature of a tissue deposition.
This review will detail the ways in which IHC and special
stains are useful in daily practice. It is beyond the scope of this
review to detail every special stain, so we have chosen to
concentrate on stains which are of particular importance to the
gastrointestinal tract or require special attention in their inter-
pretation. We will also consider stains that are potentially
underutilized. Stains which are straightforward to interpret (e.g.
CD117 in gastrointestinal stromal tumour, HepPar-1 in hepato-
cellular carcinoma) or serve a similar purpose across organ
systems (e.g. haematolymphoid markers in lymphoma subtyp-
ing, chromogranin and synaptophysin in neuroendocrine tu-
mours) will not be further discussed.
There are two methods of presenting IHC and special stain
information, entity-based or stain-based. Both have their ad-
vantages and disadvantages. Since an entity-based approach can
be found in standard surgical pathology texts, a stain-based
approach will be used in this review. We will further subdivide
this into three broad categories, stains used in neoplastic pro-
cesses, stains used in non-neoplastic processes, and stains used
in tissue depositions.
Neoplastic processes
Increasingly IHC and special stains are being applied to
neoplastic processes, and virtually every gastrointestinal tract
neoplasm is subject to some type of additional stain. Table 1
summarises the stains we find the most useful, and a selected
number will be discussed in detail.
MLH1, PMS2, MSH2 and MSH6
Mismatch repair (MMR) is a mechanism of maintaining DNA
integrity during cell division, where incorrectly matched base
pairs are excised and repaired. This is performed by the MMR
proteins MLH1, PMS2, MSH2 and MSH6. Loss of MMR protein
function leads to progressive accumulation of errors throughout
the genome, which are accentuated at microsatellites. Micro-
satellites are small repetitive DNA sequences most sensitive to
replication error, and involvement of several predefined loci are
referred to as microsatellite instability (MSI). Thus, detection of
MMR deficiency can proceed via two ways, by detecting MMR
protein loss via IHC, or MSI by DNA sequencing-based methods.
Both methods have similar sensitivities and specificities.1
MMR IHC detects both inherited (Lynch syndrome-associated)
and sporadic (MLH1-methylated) MSI colorectal carcinomas.
Lynch syndrome accounts for 3e4% of all colorectal carci-
nomas.2
Although there is no universal agreement as to which
colorectal carcinomas should be screened for Lynch syndrome, a
growing number of international organisations have recom-
mended to screen all tumours, or at least tumours in patients less
than 70 years of age.3
In contrast, approximately 12% of colo-
rectal carcinomas arise secondary to methylation-related
silencing of MLH1. In both situations, microsatellite instability
is associated with overall more favourable prognosis.4
Recently
an additional benefit of MMR IHC has been the prediction of
response to immune checkpoint inhibitor therapy. Carcinomas
with MMR deficiency show improved response to PD-L1
blockade.5
With the approval of pembrolizumab for all MMR-
deficient tumours by the United States Food and Drug
Cheng Liu BMedSci MBBS FRCPA Envoi Specialist Pathologists,
Brisbane, Faculty of Medicine, University of Queensland, Brisbane
and The Conjoint Gastroenterology Laboratory, QIMR Berghofer
Medical Research Institute, Brisbane, Queensland, Australia.
Conflicts of interest: none declared.
Masoumeh Ghayouri MD Department of Pathology, H. Lee Moffitt
Cancer Center, Tampa, FL, USA. Conflicts of interest: none declared.
Ian S Brown BGEN MBBS FRCPA Envoi Specialist Pathologists,
Brisbane and Faculty of Medicine, University of Queensland,
Brisbane, Queensland, Australia. Conflicts of interest: none declared.
MINI-SYMPOSIUM: GASTROINTESTINAL/HEPATO-PANCREATO-BILIARY PATHOLOGY
DIAGNOSTIC HISTOPATHOLOGY 26:1 22 Ó 2019 Elsevier Ltd. All rights reserved.

2. Administration (FDA), it is increasingly likely that all gastroin-
testinal tract carcinomas will require routine MMR IHC in the
future.
IHC directed against the MMR proteins are widely available,
and whether the stains are performed on endoscopic biopsy
material or surgical resection material is a matter for individual
laboratories. Our preference is for endoscopic biopsy material if
available, because the stains are less likely to be affected by
fixation artefact,6
a subsequent surgical resection may not be
processed at the same laboratory, and neoadjuvant therapy may
alter MMR IHC result.7
There is good evidence for strong
concordance between the findings in biopsy material and resec-
tion tissue.8
Our preference is to stain for all four proteins
upfront, as this avoids a second round of IHC in the 15e20% of
colorectal cancer cases demonstrating PMS2 loss (over-
whelmingly due to MLH1 methylation),3
and it prevents falsely
interpreting weak MSH6 expression as normal in patients with
MSH2 germline mutation.9
The interpretation of MMR IHC is usually straightforward,
where a tumour will show uniform retained staining or loss of
staining of an MMR protein throughout, with retained staining in
internal control cells. The terminology used in reporting MMR
IHC must be precise, since a “positive” abnormal result is due to
loss of staining, or “negative” staining. Thus, a statement such as
“MLH1 IHC is negative” is ambiguous. Instead we recommend
using “retained” or “loss of” staining, followed by an interpre-
tation, such as “MLH1 shows loss of staining, which is an
abnormal result”.3
Sometimes interpretation is challenging when there is weak/
heterogeneous staining or equivocal internal control staining. For
example, in surgical resections where there may be areas of poor
fixation or tumour hypoxia, up to 10% of the tumour cells may
exhibit equivocal weak expression for MMR markers so long as
other areas of the tumour show uniform strong expression.10
In
tumours with MLH1 methylation, there may be variability in the
intensity of expression of both MLH1 and PMS2, with areas of
weak expression or loss of expression.11
The minimum cut off for
an abnormal result is not well established. Even though it is
sometimes recommended that any tumour staining which is at
least as strong as background internal control be regarded as
retained staining, we believe this scope is too narrow, and all
cases with difficult-to-interpret or clearly heterogeneous staining
should be flagged as possibly representing Lynch syndrome.12
Table 2 details the common patterns of staining identified with
MMR markers and their clinical significance, and Figure 1 illus-
trates selected scenarios.
Approximately 80% of colorectal carcinomas exhibiting
MLH1 loss by IHC arise from sessile serrated lesions (SSLs).
While SSLs are very common, SSLs with dysplasia are rare and
capable of progressing to carcinoma rapidly. Dysplasia in SSLs is
morphologically heterogeneous and can be very subtle. Howev-
er, as 75% of SSLs with dysplasia demonstrate MLH1 loss, loss of
staining is very helpful in confirming a diagnosis of dysplasia.13
In contrast, performing MMR IHC in conventional adenomas is of
limited value. Lynch syndrome-associated conventional ade-
nomas show loss of MMR protein staining in approximately 80%
of cases (more likely if large, with a villous component and/or
high-grade dysplasia).14
However, as non-syndromic conven-
tional adenomas are much more common in practice, routine
staining of all conventional adenomas is not an effective
screening strategy.
SATB2
SATB2 is a protein with preferential expression in the lower
gastrointestinal tract. Although its expression largely parallels
CDX2, SATB2 is more site-specific and is less prone to
methylation-related silencing than CDX2, making it more useful
in mucinous, signet ring and undifferentiated colorectal carci-
nomas.15
SATB2 is highly sensitive and specific for lower
gastrointestinal tract origin of a neuroendocrine tumour, partic-
ularly for well-differentiated neoplasms.16
Neuroendocrine car-
cinoma of the lower gastrointestinal tract demonstrates similar
high expression, however metastasis from a non-gastrointestinal
Commonly used IHC and special stains in the
gastrointestinal tract
Examples
Tumour subtyping within
gastrointestinal tract
Neuroendocrine differentiation:
chromogranin, synaptophysin,
CD56
Adenocarcinoma vs. squamous
cell carcinoma: CK7, CK20, p63,
p40, mucin stains (e.g. PAS-D,
mucicarmine)
Subtyping of gastric
adenocarcinoma: EBV (in situ
hybridization), E-cadherin, MMR
stains, p53
Prognostication and grading Neuroendocrine tumours: ki67
Colorectal carcinoma: MMR stains
Vascular invasion/serosal
involvement: elastic stains (e.g.
Verhoeff-van Gieson, orcein,
Movat)
Predict response to therapy Gastro-oesophageal and gastric
carcinoma: HER2
Colorectal carcinoma: MMR stains
Multiple malignancies: PD-L1
Diagnosis of dysplasia IBD-related dysplasia: SATB2,
p53, b-catenin
Typing of dysplasia/
adenocarcinoma
Pancreatic IPMN and associated
carcinoma: intestinal (MUC2,
CDX2), gastric (MUC5AC, MUC6),
pancreaticobiliary (MUC1)
Identifying an inherited tumour
predisposition syndrome
Lynch syndrome: MMR stains
Carney-Stratakis syndrome:
SDHA, SDHB
Identifying primary site in poorly
differentiated malignancies
Multiple possibilities: CK7, CK20,
TTF1, CDX2, SATB2, GATA3, PAX8,
ER, calretinin, CK5/6, S100,
SOX10, CD3, CD20
EBV, Epsten-Barr virus; MMR, mismatch repair; IBD, inflammatory bowel dis-
ease; IPMN, intraductal papillary mucinous neoplasm.
Table 1
MINI-SYMPOSIUM: GASTROINTESTINAL/HEPATO-PANCREATO-BILIARY PATHOLOGY
DIAGNOSTIC HISTOPATHOLOGY 26:1 23 Ó 2019 Elsevier Ltd. All rights reserved.

3. site needs to be excluded.16
SATB2 also allows for separation of
primary small intestinal adenocarcinoma from colorectal carci-
noma that has spread to the small intestine. While most primary
colorectal carcinomas express this protein, less than half of pri-
mary small intestinal adenocarcinomas do. Furthermore, unlike
the strong and diffuse staining pattern in CRC, the expression of
SATB2 is weaker in small intestinal adenocarcinoma.17
The diagnostic uses of SATB2 continue to expand and there
are recent reports of its potential role in diagnosing inflammatory
bowel disease (IBD)-associated dysplasia and carcinoma. Loss of
SATB2 staining is seen in 40% of cases of IBD related dysplasia,
while it is retained in reactive atypia and conventional ade-
nomas.18
This first allows for establishing a diagnosis of
dysplasia versus reactive atypia, and secondly it separates IBD-
related dysplasia from a conventional adenoma occurring in a
patient with IBD. Interestingly, SATB2 is also frequently lost in
IBD-associated carcinomas, but is retained in most sporadic
carcinomas.19
Figure 2 shows an example of IBD-related
dysplasia with loss of SATB2 staining.
ki67, p53 and b-catenin
These three stains are discussed together due to their similar
utility and interpretation. Within the tubular gastrointestinal
tract, b-catenin, p53 and ki67 have all been used to support a
diagnosis of dysplasia where morphology is equivocal.20
In
general, a dysplastic lesion should exhibit higher ki67 index,
increased p53 staining towards the luminal aspect, and aberrant
(cytoplasmic or nuclear) b-catenin expression, when compared
with a reactive process. However, these stains are variably pos-
itive in reactive processes, especially when associated with
inflammation or regeneration, and different studies have used
different scoring systems and cut off thresholds for positivity.
Also, the aberrant expression of b-catenin is restricted to lesions
with WNT pathway activation, generally intestinal-type ade-
nomas. The value of these stains is not an absolute cut off
threshold but rather in comparing with background mucosa. If
the lesion shows an abrupt difference in staining from the
background, this is supportive of a dysplastic interpretation.
b-catenin IHC has additional utility in unequivocally
dysplastic lesions. Until recently it was used with p53 to separate
IBD-related dysplasia from conventional adenoma, where IBD-
related dysplasia is usually negative for b-catenin and diffusely
positive for p53, while conventional adenoma shows the oppo-
site pattern.21
The potential of SATB2 in this setting has been
discussed above. Since the management of dysplastic lesions in
IBD is now dependent more on endoscopic appearance and
resectability rather than aetiology, there is less need to separate
IBD-related dysplasia from conventional adenoma.
Figure 1 Mismatch repair protein immunohistochemistry. There is loss of expression of MLH1, PMS2 and MSH6 in this colorectal carcinoma.
MSH2 is preserved (not shown). There is normal expression of the markers in the background lymphocytes and non-neoplastic epithelial cells. This
abnormal pattern of expression is usually the result of sporadic methylation-induced silencing of MLH1 gene expression with secondary loss of
PMS2 and MSH6.
Common MMR patterns and their significance
Pattern of expression Percentage Probability of Lynch syndrome Significance/further testing
MLH1, MSH2, MSH6 and PMS2 preserved 85% Very unlikely Normal pattern; no further testing unless there
is a strong clinical suspicious of Lynch
syndrome
MLH1 and PMS2 loss 15% Unlikely (sporadic in 80% of cases) BRAF mutation or MLH1 promoter methylation
testing to confirm sporadic nature; if both
absent, investigate for Lynch syndrome
MSH2 and MSH6 loss <1% Likely MSH2, followed by MSH6 germline testing
MSH6 loss <1% Likely MSH6, followed by MSH2 germline testing;
may occur secondary to neoadjuvant therapy
in rectal carcinoma
PMS2 loss <1% Likely PMS2, followed by MLH1 germline testing
Table 2
MINI-SYMPOSIUM: GASTROINTESTINAL/HEPATO-PANCREATO-BILIARY PATHOLOGY
DIAGNOSTIC HISTOPATHOLOGY 26:1 24 Ó 2019 Elsevier Ltd. All rights reserved.

4. MUC1, MUC2, MUC5AC and MUC6
Mucins are high-molecular weight glycoproteins produced by
epithelial cells, which have both gel-forming and signal trans-
duction roles. Broadly the pattern of mucin expression corre-
sponds to site-specific epithelial components of the
gastrointestinal tract. For instance, pancreaticobiliary epithelium
expresses MUC1, intestinal epithelium expresses MUC2, gastric
foveolar epithelium expresses MUC5AC and gastric pyloric
epithelium expresses MUC6. Mucin IHC can therefore be applied
in the subclassification of pancreatic intraductal papillary
mucinous neoplasm (IPMN)-derived carcinomas into pan-
creaticobiliary (MUC1), intestinal (MUC2) and gastric (MUC5AC
and MUC6) phenotypes, which have differing prognoses.22
It
should be emphasized, however, that the phenotype of these
lesions can usually be determined via morphologic means and
mucin stains have a supplementary role only. Table 3 summa-
rises the morphologic and IHC features of these phenotypes.
MUC1 and MUC2 staining may be useful in establishing a
pancreaticobiliary or intestinal phenotype in neoplasms occur-
ring at the duodenal ampulla. This is generally done in combi-
nation with CK20 and CDX2, and may guide further treatment of
these tumours.23
Unfortunately, the stains sometimes do not
establish a clear line of differentiation.24
Mucin stains may also
be useful in separating gastric phenotype dysplasia from intes-
tinal phenotype dysplasia of the upper gastrointestinal tract.
Gastric pattern dysplasia is associated with different clinico-
pathological features.25
However, as with the pancreatic IPMN,
the clinical utility of performing these stains routinely is yet to be
firmly established. In addition, many examples of glandular
dysplasia in the lower oesophagus display a hybrid pattern of
mucin expression consistent with both gastric and intestinal
differentiation.
PD-1 and PD-L1
The PD-1/PD-L1 pathway allows cytotoxic T cells to selectively
remove abnormal cells while leaving normal cells intact. To
avoid damage, normal cells express the transmembrane protein
PD-L1, which is detected by its receptor PD-1 on the cytotoxic T
cell. When a cytotoxic T cell encounters a PD-L1-expressing
normal cell, the PD-L1/PD-1 interaction inactivates the T cell,
downregulates the immune response, and may lead to apoptosis
of the T cell. In a similar vein, by expressing PD-L1, tumour cells
mimic normal cells and escape destruction by cytotoxic T cells.
The tumour cells are thus allowed to persist and proliferate. Anti-
PD-L1 therapy such as pembrolizumab “unmasks” tumour cells
from the immune system and reverses the inhibitory effect of PD-
L1.
IHC PD-L1 testing is most established in lung adenocarci-
noma, and it is now being applied to an increasing number of
tumours from diverse organ systems. However, accurate inter-
pretation of PD-L1 IHC is complicated by several factors.26
Multiple antibody clones are available, and staining is usually
heterogeneous. Different scoring systems are used for different
tumours, with some also including tumour-infiltrating lympho-
cytes. As expected, PD-L1 positive tumours are associated with
poor prognostic features,27
regardless of scoring system used,
and response to PD-1/PD-L1 blockade correlates with degree of
IHC PD-L1 positivity.28
In the gastrointestinal tract, PD-L1 IHC
testing is driven by availability of pembrolizumab. The United
States FDA data sheet for pembrolizumab includes oesophageal,
gastric, hepatocellular and colorectal carcinomas, but other tu-
mours are likely to become eligible in the future.
HER2
HER2 is a member of the epidermal growth factor receptor
family, where overexpression leads to activation of proliferation
pathways in the absence of an extracellular ligand. It is overex-
pressed in approximately 20% of gastro-oesophageal junction
and gastric adenocarcinomas, and treatment with trastuzumab
increases survival.29
Although different antibody clones are
available, the results are comparable between clones, and the
interpretation methodology is standardized (see Table 4). Of
note, interpretation differs from breast cancer in several respects.
Because gastro-oesophageal junction and gastric cancers are
commonly advanced (i.e. inoperable) at presentation, the only
tissue available for testing may be an endoscopic biopsy spec-
imen. To account for possible intratumoural heterogeneity in this
setting, the cut off for an equivocal or positive result is much
lower in biopsy compared with excision specimens. Complete
circumferential membranous staining is also not required.
Figure 2 High-grade dysplasia and invasive adenocarcinoma developing in a colonic inflammatory polyp in a patient with long standing ulcerative
colitis. There is strong nuclear expression of p53 in the dysplastic cells; in contrast, SATB2 expression is lost.
MINI-SYMPOSIUM: GASTROINTESTINAL/HEPATO-PANCREATO-BILIARY PATHOLOGY
DIAGNOSTIC HISTOPATHOLOGY 26:1 25 Ó 2019 Elsevier Ltd. All rights reserved.

5. IPMN phenotypes as determined via mucin stains
Phenotype Morphologic features MUC2 MUC5AC MUC6 MUC1
Pancreaticobiliary Columnar cells
Round, uniform, basally located nuclei
Abundant apical mucinous cytoplasm
e e e þ
Intestinal Columnar cells
Hyperchromatic, pencillate, stratified nuclei
Dense eosinophilic cytoplasm
þ Æ e e
Gastric foveolar Cuboidal to columnar cells with pale clear to
light eosinophilic cytoplasm
Hyperchromatic round to oval nuclei
Prominent nucleoli if high grade
e þ rare e
Gastric pyloric gland Closely packed tubules lined by cuboidal to
columnar epithelium with pale to eosinophilic
ground glass cytoplasm
Round basal nuclei
Nucleoli easily visible
e þ (surface) þ e
Hybrid Cytological features intermediate between the
above patterns, or an intimate admixture of
both
Æ Æ Æ Æ
Table 3
MINI-SYMPOSIUM:GASTROINTESTINAL/HEPATO-PANCREATO-BILIARYPATHOLOGY
DIAGNOSTICHISTOPATHOLOGY26:126Ó2019ElsevierLtd.Allrightsreserved.

6. SDHA and SDHB
The succinate dehydrogenase (SDH) complex is a mitochondria-
associated enzyme complex involved in oxidative phosphory-
lation, comprising four subunits SDHA, SDHB, SDHC and
SDHD. Mutation in the subunits result in Carney-Stratakis
syndrome (gastric gastrointestinal stromal tumours [GISTs]
and paragangliomas), and methylation-induced silencing of
SDHC results in Carney triad (gastric GISTs, paragangliomas
and pulmonary chondromas). Gastric GISTs that arise in the
context of SDH mutation have distinct pathologic features.30
They grow as multinodular sheets which dissect the muscu-
laris propria, display epithelioid cytology, and have a tendency
towards lymph node metastases. However, they have an indo-
lent course, so the prognostic algorithm used for usual GISTs
does not apply.
The interpretation of SDH IHC parallels that for MMR pro-
teins. As mutations in any subunit results in loss of SDHB, SDHB
IHC is an effective screening test.31
A normal result is granular
cytoplasmic (i.e. mitochondrial) staining in tumour cells. Loss of
staining is an abnormal result, which must be interpreted with an
appropriately staining internal control such as endothelial cells.
In the context of SDHB loss, SDHA IHC allows for identification
of an SDHA mutation, but IHC is not available for SDHC and
SDHD mutations. An example of a “positive” result (i.e. loss of
staining) is shown in Figure 3.
Elastic stains
Venous invasion is an independent poor prognostic feature in
malignant epithelial tumours of the gastrointestinal tract. How-
ever, its presence is underreported by pathologists. This is
particularly problematic in colorectal carcinoma where it is ex-
pected that venous invasion should be present in at least 30% of
resected tumours.32
Histological features suspicious for venous
invasion include the “orphan (or unaccompanied) arteriole”
sign, a well-circumscribed tumour nodule adjacent to a thick-
walled artery without an accompanying vein; and the “protrud-
ing tongue” sign, a circumscribed protrusion of tumour into the
pericolic fat. In these situations, at least, elastic tissue stains may
demonstrate a residual vein wall, an example of which is shown
in Figure 4. An elastin stain is also useful in confirming the
presence of venous invasion in cases originally deemed equiv-
ocal on haematoxylin and eosin (H&E)-stained sections. Some
have gone further and advocate routine use of elastin stains to
increase venous invasion detection. It has been shown that use of
Interpretation of HER2 staining in the gastrointestinal tract
HER2 score Biopsy staining Excision staining Interpretation
0 No reactivity or no membranous reactivity in
any cell
No reactivity or membranous reactivity in
<10% of cells
Negative
1þ !5 cells with a faint or barely perceptible
membranous reactivity irrespective of
percentage of cells positive
Faint or barely perceptible membranous
reactivity in !10% of cells; cells are reactive
only in part of their membrane
Negative
2þ !5 cells with a weak to moderate complete,
basolateral, or lateral membranous reactivity
irrespective of percentage of cells positive
Weak to moderate complete, basolateral or
lateral membranous reactivity in !10% of cells
Equivocal
3þ !5 cells with a strong complete basolateral, or
lateral membranous reactivity irrespective of
percentage of cells positive
Strong complete, basolateral or lateral
membranous reactivity in !10% of cells
Positive
Table 4
Figure 3 SDHB-deficient gastric gastrointestinal stromal tumour. Note loss of immunohistochemical SDHB expression in the tumour cells with
preserved internal control staining of endothelial cells. Retained internal control expression is required to ensure the stain has worked correctly.
(Photographs courtesy of Professor Anthony Gill, Royal North Shore Hospital, Sydney).
MINI-SYMPOSIUM: GASTROINTESTINAL/HEPATO-PANCREATO-BILIARY PATHOLOGY
DIAGNOSTIC HISTOPATHOLOGY 26:1 27 Ó 2019 Elsevier Ltd. All rights reserved.

7. routine elastin stains is associated with a doubling of the venous
invasion detection rate.33
Non-neoplastic processes
Most stains performed in nonneoplastic processes are for char-
acterizing an inflammatory infiltrate, or to identify an infectious
organism. Table 5 summarises the stains we find the most useful,
and a selected number will be discussed in detail.
Helicobacter pylori stains
The most common reason for performing a special stain in
inflamed gastrointestinal tract tissue is to look for an infective
cause, and Helicobacter pylori (H. pylori)-associated gastric
inflammation represents the best example of this. While a variety
of histochemical stains successfully highlight H. pylori (e.g.
Wright-Giemsa, toluidine blue, Warthin-Starry), none of these
have sensitivity or specificity that approaches the IHC, and this
has become the gold standard test.
While some laboratories advocate the routine use of stains to
look for these organisms, there seems little point in doing this
when the organism is readily identifiable on H&E in most cases.34
There are however situations where stains should be performed
if organisms are not identified in the H&E stained sections.35
These indications are listed in Box 1.
H. pylori IHC may also be useful when there has been
proton pump inhibitor therapy or incomplete antibiotic treat-
ment,36
both of which lead to a reduction in the number of
organisms. This is often accompanied by a loss or marked
reduction in active inflammation. The organisms may be found
only in the deep aspect of glands of the specialized mucosa,
often closely applied to the canaliculi of parietal cells (see
Figure 5).
Cytomegalovirus immunohistochemistry
Cytomegalovirus (CMV) is a herpesvirus which latently infects
approximately 80% of the world’s population. In immunocom-
petent individuals the virus is dormant, but reactivation occurs in
immune dysregulation, such as with steroid use, human immu-
nodeficiency virus infection, bone marrow suppression, and old
age.37
In the gastrointestinal tract any organ can be affected,
manifesting as cytomegaly and characteristic “owl’s eye” nuclear
inclusions. Infection preferentially affects stromal cells and
Figure 4 Orcein stain demonstrating the residual elastic lamina of a vein that has been invaded by colorectal carcinoma. The "orphan artery" sign is
apparent on the haematoxylin and eosin-stained section.
Common stains used in nonneoplastic processes
Purpose Example stains
Identifying infective organism Bacteria: IHC or Giemsa for H. pylori, IHC or
Warthin-Starry for T. pallidum
Mycobacteria: Ziehl-Neelsen, Wade-Fite
Fungi: PAS-D, Grocott
Viral: IHC for HSV, IHC CMV, p16 for HPV
Confirm a specific inflammatory pattern Collagenous gastritis/sprue/colitis: trichrome,
Verhoeff-van Gieson
Lymphocytic gastritis/duodenitis/colitis: CD3
Plasma cell-rich fibrosing process IgG4-related sclerosing disease: IgG, IgG4
Refractory coeliac disease Type 1: normal CD3 and CD8 in T cells
Type 2: aberrant CD3 and CD8 in T cells
IHC, immunohistochemistry; HSV, herpes simplex virus; CMV, cytomegalovirus; HPV, human papillomavirus.
Table 5
MINI-SYMPOSIUM: GASTROINTESTINAL/HEPATO-PANCREATO-BILIARY PATHOLOGY
DIAGNOSTIC HISTOPATHOLOGY 26:1 28 Ó 2019 Elsevier Ltd. All rights reserved.

8. endothelial cells, with epithelial cells being involved only in se-
vere cases. This is typically associated with active chronic
inflammation, and inflammation can be so pronounced as to
obscure the diagnostic cytologic findings.
CMV IHC aids in identifying the virus-infected cells in cases
with a high index of suspicion.38
It is of greatest utility when the
inclusions are equivocal and other entities enter the differential,
chiefly inflammation/regeneration-associated nucleolar
prominence, and a positive result in this case will always high-
light more infected cells than appreciable on H&E. On the other
hand, indiscriminate application of CMV IHC will detect latently
infected cells, which may lead to an erroneous assignment of
aetiology.39
Lastly, pigment-laden macrophages may simulate
CMV-infected cells on IHC, due to their size and haemosiderin
content.
CD3 and CD8
Refractory coeliac disease is defined as symptoms not
responding after 12 months on a gluten-free diet, or symptom
recurrence while on a gluten-free diet.40
Most cases prove to
represent an insufficient gluten-free diet or a slow healing
response. Rarely, the persisting or recurring disease represents
the development of a monoclonal T cell proliferation, which is
initially confined to the epithelium but is at high risk of pro-
gression to enteropathy-associated T-cell lymphoma. Sepa-
rating these two scenarios is clinically important, and CD3 and
CD8 are helpful in this context.41
Normally at least 90% of CD3
þ intraepithelial T cells co-express CD8, and this is maintained
in the first scenario (refractory coeliac disease type 1). Loss of
CD3 and CD8 co-expression in greater than 50% of the intra-
epithelial T cells is a worrisome finding and corresponds to the
second scenario (refractory coeliac disease type 2); an example
of this is shown in Figure 6. Refractory coeliac disease type 2 is
a difficult diagnosis and requires synthesis of clinical, H&E, IHC
and flow cytometric findings; no feature should be considered
diagnostic in isolation.
IgG and IgG4
IgG4-related sclerosing disease has been recognized in almost
all organ systems since its consensus diagnostic criteria were
published in 2012.42
The three cardinal features are (1) IgG4 þ
Indications for H. pylori stains
Acute gastritis and/or gastric ulceration (unless clearly reactive
gastropathy associated)
Active chronic gastritis
Focally enhanced gastritis
Moderate or severe chronic gastritis
Autoimmune gastritis (H. pylori infection may be a precursor)
Lymphocytic gastritis (organisms may be very sparse)
Marginal zone lymphoma (MALT type)
Previous H. pylori (to confirm eradication)
Positive urease test without organisms seen in H&E stained
sections
Gastric intestinal metaplasia
Gastric adenocarcinoma
Duodenal ulceration
MALT, mucosa-associated lymphoid tissue; H&E, haematoxylin
and eosin.
Box 1
Figure 5 Gastric biopsies in a patient taking a proton pump inhibitor. Mild active chronic inflammation is present in the gastric body. The gastric
antrum is normal. H. pylori immunohistochemical stain demonstrates organisms restricted to the gastric body and closely applied to the parietal
cell canaliculi.
MINI-SYMPOSIUM: GASTROINTESTINAL/HEPATO-PANCREATO-BILIARY PATHOLOGY
DIAGNOSTIC HISTOPATHOLOGY 26:1 29 Ó 2019 Elsevier Ltd. All rights reserved.

9. plasma cells comprising at least 40% of all IgG þ plasma cells;
(2) storiform fibrosis; and (3) obliterative phlebitis. At least two
out of three features are required for diagnosis. In addition to
the 40% minimum, an absolute number of IgG4 þ plasma cells
is also required (expressed per high power field), which differs
between organs. Recognition of the disorder is important as it
respond well to steroid treatment.43
In the gastrointestinal tract
the main organs involved are the pancreas and biliary tract. If
not considered, the prominent fibroinflammatory reaction may
be mistaken for chronic pancreatitis, primary sclerosing chol-
angitis, pancreatic ductal adenocarcinoma, or
cholangiocarcinoma.
Assessment of tissue deposition
Most tissue deposits are readily recognisable without IHC or
special stains. Table 6 lists cases where stains may be of use.
Amyloid stains
Amyloid represents abnormally aggregated protein which be-
comes deposited in extracellular sites as amorphous eosinophilic
material. Histochemical stains such as Congo red and crystal
violet can confirm the presence of amyloid, and pre-treatment of
sections by potassium permanganate further allows sub-
classification into either light chain (AL) or inflammation-
associated (AA) amyloid, the two most common subtypes. IHC
is available for a wider range of amyloid types, including AL, AA,
transthyretin and b2-microglobulin.44
However, the efficacy of
these stains is laboratory dependent, and nonspecific/equivocal
staining is common; precise subtyping is better performed via
mass spectrometry. Because gastrointestinal involvement by
amyloidosis presents with nonspecific symptoms such as diar-
rhoea, constipation and abdominal pain,45
subtle deposits are
easily missed. The deposits begin in the submucosal vessel walls,
which can be mistaken for collagen or fibrin, and subsequently
involve the lamina propria and submucosa proper. In our
opinion, it is more important to recognize amyloidosis and
recommend further investigation for a systemic cause, rather
than to subtype the amyloid.
Conclusions
It is sometimes challenging to keep abreast of available IHC and
special stains in gastrointestinal pathology, and we have
attempted to summarize the most topical and/or useful stains in
this review. It should be emphasized, however, that stains
should only be ordered to answer a specific clinical question, and
the results interpreted along with the H&E findings. A
REFERENCES
1 Lindor NM, Burgart LJ, Leontovich O, et al. Immunohistochemistry
versus microsatellite instability testing in phenotyping colorectal
tumors. J Clin Oncol 2002; 20: 1043e8.
2 Lynch HT, Snyder CL, Shaw TG, Heinen CD, Hitchins MP. Mile-
stones of Lynch syndrome: 1895-2015. Nat Rev Cancer 2015; 15:
181e94.
3 Yozu M, Kumarasinghe MP, Brown IS, Gill AJ, Rosty C. Austral-
asian Gastrointestinal Pathology Society (AGPS) consensus
guidelines for universal defective mismatch repair testing in
colorectal carcinoma. Pathology 2019; 51: 233e9.
4 Ward R, Meagher A, Tomlinson I, et al. Microsatellite instability
and the clinicopathological features of sporadic colorectal cancer.
Gut 2001; 48: 821e9.
5 Le DT, Durham JN, Smith KN, et al. Mismatch repair deficiency
predicts response of solid tumors to PD-1 blockade. Science
2017; 357: 409e13.
6 O’Brien O, Ryan E, Creavin B, et al. Correlation of immunohisto-
chemical mismatch repair protein status between colorectal
Figure 6 Refractory coeliac disease type 2 with loss of CD8 expression in >50% of CD3þ intraepithelial T cells.
Tissue deposition and their staining characteristics
Tissue deposition Example stains
Brown depositions Iron and pseudomelanosis: Perls
positive
Melanosis and lanthanum: Perls
negative
Eosinophilic depositions Amyloid: Congo red, crystal violet
Collagen: trichrome
Elastin: Verhoeff-van Gieson,
orcein, Movat
Medication capsule: PAS
Basophilic depositions Calcium/psammoma bodies,
Osmoprep: von Kossa positive
SIRT spheres: von Kossa negative
SIRT, selective internal radiation therapy.
Table 6
MINI-SYMPOSIUM: GASTROINTESTINAL/HEPATO-PANCREATO-BILIARY PATHOLOGY
DIAGNOSTIC HISTOPATHOLOGY 26:1 30 Ó 2019 Elsevier Ltd. All rights reserved.

10. carcinoma endoscopic biopsy and resection specimens. J Clin
Pathol 2018; 71: 631e6.
7 Bao F, Panarelli NC, Rennert H, Sherr DL, Yantiss RK. Neo-
adjuvant therapy induces loss of MSH6 expression in colorectal
carcinoma. Am J Surg Pathol 2010; 34: 1798e804.
8 Shia J, Stadler Z, Weiser MR, et al. Immunohistochemical staining
for DNA mismatch repair proteins in intestinal tract carcinoma:
how reliable are biopsy samples? Am J Surg Pathol 2011; 35:
447e54.
9 Pearlman R, Markow M, Knight D, et al. Two-stain immunohis-
tochemical screening for Lynch syndrome in colorectal cancer
may fail to detect mismatch repair deficiency. Mod Pathol 2018;
31: 1891e900.
10 Pai RK, Pai RK. A practical approach to the evaluation of
gastrointestinal tract carcinomas for Lynch syndrome. Am J Surg
Pathol 2016; 40: e17e34.
11 Joost P, Veurink N, Holck S, et al. Heterogenous mismatch-repair
status in colorectal cancer. Diagn Pathol 2014; 9: 126.
12 Sarode VR, Robinson L. Screening for Lynch syndrome by
immunohistochemistry of mismatch repair proteins: significance
of indeterminate result and correlation with mutational studies.
Arch Pathol Lab Med 2019; 143: 1225e33.
13 Liu C, Walker NI, Leggett BA, Whitehall VL, Bettington ML,
Rosty C. Sessile serrated adenomas with dysplasia: morpholog-
ical patterns and correlations with MLH1 immunohistochemistry.
Mod Pathol 2017; 30: 1728e38.
14 Walsh MD, Buchanan DD, Pearson SA, et al. Immunohisto-
chemical testing of conventional adenomas for loss of expression
of mismatch repair proteins in Lynch syndrome mutation carriers:
a case series from the Australasian site of the colon cancer family
registry. Mod Pathol 2012; 25: 722e30.
15 Ma C, Lowenthal BM, Pai RK. SATB2 is superior to CDX2 in
distinguishing signet ring cell carcinoma of the upper gastroin-
testinal tract and lower gastrointestinal tract. Am J Surg Pathol
2018; 42: 1715e22.
16 Bellizzi AM. SATB2 in neuroendocrine neoplasms: strong
expression is restricted to well-differentiated tumors of lower
gastrointestinal tract origin and is more frequent in merkel cell
carcinoma among poorly differentiated carcinomas. Histopathol-
ogy, 2019; https://doi.org/10.1111/his.13943 [Epub ahead of
print].
17 Kim CJ, Baruch-Oren T, Lin F, Fan XS, Yang XJ, Wang HL. Value
of SATB2 immunostaining in the distinction between small in-
testinal and colorectal adenocarcinomas. J Clin Pathol 2016; 69:
1046e50.
18 Ma C, Henn P, Miller C, Herbst C, Hartman DJ, Pai RK. Loss of
SATB2 expression is a biomarker of inflammatory bowel disease-
associated colorectal dysplasia and adenocarcinoma. Am J Surg
Pathol 2019; 43: 1314e22.
19 Iwaya M, Ota H, Tateishi Y, Nakajima T, Riddell R, Conner JR.
Colitis-associated colorectal adenocarcinomas are frequently
associated with non-intestinal mucin profiles and loss of SATB2
expression. Mod Pathol 2019; 32: 884e92.
20 Ma C, Pai RK. Predictive value of immunohistochemistry in pre-
malignant lesions of the gastrointestinal tract. Semin Diagn Pathol
2015; 32: 334e43.
21 Walsh SV, Loda M, Torres CM, Antonioli D, Odze RD. P53 and
beta catenin expression in chronic ulcerative colitis–associated
polypoid dysplasia and sporadic adenomas: an immunohisto-
chemical study. Am J Surg Pathol 1999; 23: 963e9.
22 Yonezawa S, Higashi M, Yamada N, et al. Mucins in human
neoplasms: clinical pathology, gene expression and diagnostic
application. Pathol Int 2011; 61: 697e716.
23 Ang DC, Shia J, Tang LH, Katabi N, Klimstra DS. The utility of
immunohistochemistry in subtyping adenocarcinoma of the
ampulla of vater. Am J Surg Pathol 2014; 38: 1371e9.
24 Reid MD, Balci S, Ohike N, et al. Ampullary carcinoma is often
of mixed or hybrid histologic type: an analysis of
reproducibility and clinical relevance of classification as pan-
creatobiliary versus intestinal in 232 cases. Mod Pathol 2016;
29: 1575e85.
25 Park DY, Srivastava A, Kim GH, et al. Adenomatous and foveolar
gastric dysplasia: distinct patterns of mucin expression and
background intestinal metaplasia. Am J Surg Pathol 2008; 32:
524e33.
26 Callea M, Pedica F, Doglioni C. Programmed death 1 (PD-1) and
its ligand (PD-L1) as a new frontier in cancer Immunotherapy and
challenges for the Pathologist: state of the art. Pathologica 2016;
108: 48e58.
27 Wu P, Wu D, Li L, Chai Y, Huang J. PD-L1 and survival in solid
tumors: a meta-analysis. PLoS One 2015; 10: e0131403.
28 Herbst RS, Soria JC, Kowanetz M, et al. Predictive correlates of
response to the anti-PD-L1 antibody MPDL3280A in cancer pa-
tients. Nature 2014; 515: 563e7.
29 Bang YJ, Van Cutsem E, Feyereislova A, et al. Trastuzumab in
combination with chemotherapy versus chemotherapy alone for
treatment of HER2-positive advanced gastric or gastro-
oesophageal junction cancer (ToGA): a phase 3, open-label,
randomised controlled trial. Lancet 2010; 376: 687e97.
30 Miettinen M, Wang ZF, Sarlomo-Rikala M, Osuch C, Rutkowski P,
Lasota J. Succinate dehydrogenase-deficient GISTs: a clinico-
pathologic, immunohistochemical, and molecular genetic study of
66 gastric GISTs with predilection to young age. Am J Surg Pathol
2011; 35: 1712e21.
31 Gill AJ. Succinate dehydrogenase (SDH)-deficient neoplasia.
Histopathology 2018; 72: 106e16.
32 Dawson H, Kirsch R, Driman DK, Messenger DE,
Assarzadegan N, Riddell RH. Optimizing the detection of venous
invasion in colorectal cancer: the ontario, Canada, experience and
beyond. Front Oncol 2014; 4: 354.
33 Kirsch R, Messenger DE, Riddell RH, et al. Venous invasion in
colorectal cancer: impact of an elastin stain on detection and
interobserver agreement among gastrointestinal and non-
gastrointestinal pathologists. Am J Surg Pathol 2013; 37:
200e10.
34 Batts KP, Ketover S, Kakar S, et al. Appropriate use of special
stains for identifying Helicobacter pylori: recommendations from
the rodger C. Haggitt gastrointestinal pathology society. Am J
Surg Pathol 2013; 37: e12e22.
35 Genta RM, Lash RH. Helicobacter pylori-negative gastritis:
seek, yet ye shall not always find. Am J Surg Pathol 2010; 34:
e25e34.
36 Panarelli NC, Ross DS, Bernheim OE, et al. Utility of ancillary
stains for Helicobacter pylori in near-normal gastric biopsies. Hum
Pathol 2015; 46: 397e403.
37 Griffiths P, Baraniak I, Reeves M. The pathogenesis of human
cytomegalovirus. J Pathol 2015; 235: 288e97.
MINI-SYMPOSIUM: GASTROINTESTINAL/HEPATO-PANCREATO-BILIARY PATHOLOGY
DIAGNOSTIC HISTOPATHOLOGY 26:1 31 Ó 2019 Elsevier Ltd. All rights reserved.

11. 38 Ambelil M, Saulino DM, Ertan A, DuPont AW, Younes M. The
significance of so-called equivocal immunohistochemical staining
for cytomegalovirus in colorectal biopsies. Arch Pathol Lab Med
2019; 143: 985e9.
39 Solomon IH, Hornick JL, Laga AC. Immunohistochemistry is rarely
justified for the diagnosis of viral infections. Am J Clin Pathol
2017; 147: 96e104.
40 Al-Toma A, Volta U, Auricchio R, et al. European Society for the
Study of Coeliac Disease (ESsCD) guideline for coeliac disease
and other gluten-related disorders. United Eur Gastroenterol J
2019; 7: 583e613.
41 Rubio-Tapia A, Murray JA. Classification and management of
refractory coeliac disease. Gut 2010; 59: 547e57.
42 Deshpande V, Zen Y, Chan JK, et al. Consensus statement on the
pathology of IgG4-related disease. Mod Pathol 2012; 25:
1181e92.
43 Kamisawa T, Okazaki K. Diagnosis and treatment of IgG4-related
disease. Curr Top Microbiol Immunol 2017; 401: 19e33.
44 Kebbel A, Rocken C. Immunohistochemical classification of am-
yloid in surgical pathology revisited. Am J Surg Pathol 2006; 30:
673e83.
45 Hokama A, Kishimoto K, Nakamoto M, et al. Endoscopic and
histopathological features of gastrointestinal amyloidosis. World J
Gastrointest Endosc 2011; 3: 157e61.
MINI-SYMPOSIUM: GASTROINTESTINAL/HEPATO-PANCREATO-BILIARY PATHOLOGY
DIAGNOSTIC HISTOPATHOLOGY 26:1 32 Ó 2019 Elsevier Ltd. All rights reserved.