Immunohistology of the Gastrointestinal Tract

This chapter divides the discussion of immunohistochemistry (IHC) of the luminal gastrointestinal (GI) tract into three sections: (1) epithelial pathology, (2) neuroendocrine lesions, and (3) spindle cell lesions. We have attempted to compile the innumerable IHC studies that have been applied to these organs into a cogent, useful, and relevant text.

Biology of Antigens and Antibodies: General and Tissue Specific

This section includes a brief discussion of antigens/epitopes often used in GI pathology. Table 14.1 shows representative assay conditions for these antibodies. Note that the conditions listed in this table are meant to be guides. All new antibodies must be analytically validated by the laboratory performing the test.

Table 14.1

Assay Conditions for Representative Antibodies

Antibody Clone Dilution Retrieval
β-Catenin 17C2 1 : 200 pH 6.1
KIT (CD117) 104D2 1 : 200 pH 9.0
CDX2 CDX2-88 1 : 50 pH 6.1
Chromogranin A DAK-A3 1 : 800 pH 9.0
Cytokeratin 7 OV-TL 12/30 1 : 400 Proteinase K
Cytokeratin 20 Ks20.8 1 : 25 Proteinase K
COX-2 SP-21 1 : 100 pH 8.5
MLH1 CM220C 1 : 10 Proprietary solution
MOC-31 MOC-31 1 : 25 Proteinase K
MSH2 FE11 1 : 25 Proprietary solution
MSH6 70834 1 : 100 pH 9.0
MUC1 Ma95 1 : 100 pH 8.5
MUC2 Ccp58 1 : 25 pH 8.5
MUC5AC CLH2 1 : 100 pH 8.5
MUC6 CLH5 1 : 25 pH 8.5
p53 DO-7 1 : 25 pH 9.0
p63 4A4 1 : 50 pH 9.0
PMS2 A16-4 1 : 10 pH 9.0
Synaptophysin Sv38 1 : 100 pH 6.1
Villin CWWB1 1 : 100 Citrate, pH 6.0

CD , Cluster designation; COX-2 , cyclooxygenase 2.

General Cytokeratins

For the purposes of diagnostic IHC, carcinomas that stain with low- and high-molecular-weight cytokeratins (HMWCKs) include adenocarcinomas of the esophagus and stomach. Those that stain predominantly with low-molecular-weight CK (LMWCK) antibodies including colorectal adenocarcinomas, neuroendocrine tumors (NETs), and high-grade neuroendocrine carcinomas (NECs). Carcinomas that stain with predominantly HMWCK antibodies include squamous cell carcinomas (SCCs) of the esophagus and anus.


β-Catenin is an 88-kDa member of the catenin family of proteins, which are important constituents of the cytoskeleton; β-catenin is important in gene expression and is a component of the Wnt signaling cascade. In certain conditions, when the normal degradation of β-catenin is disrupted, this protein accumulates in the cytoplasm and abnormally translocates to the nucleus, where it can disrupt normal gene expression.

Cytokeratin 7

Cytokeratin 7 (CK7) is an intermediate filament protein expressed predominantly by ductal epithelial cells of the pancreatobiliary tract, renal collecting ducts, and proximal GI tract. Expression of CK7 is limited to subtypes of adenocarcinomas and SCCs that arise within nonkeratinized mucosa.

Cytokeratin 20

In the small intestine, CK20 stains only the highly differentiated small bowel villous enterocytes (CK18 stains the more immature, basilar, proliferative zone cells). In the colon, CK20 stains only the surface epithelial cell layer, and CK20 staining is more extensive and stronger in small bowel neoplasms than in colonic carcinomas.


CDX2 is a homeobox gene that is an integral component of intestinal cell proliferation and differentiation. It appears to function as a tumor suppressor gene in colorectal and some pancreatobiliary and gastric adenocarcinomas.

Chromogranin A

This protein is an acidic soluble glycoprotein found within neurosecretory granules. Chromogranin A undergoes posttranslational modification, which varies between GI sites and their associated tumors. Chromogranin is more specific but less sensitive than synaptophysin. Most nonneoplastic neuroendocrine lesions and low-grade NETs diffusely and strongly stain with chromogranin, and this is proportional to the number of intracytoplasmic neurosecretory granules. However, some carcinoids stain weakly with chromogranin, which may reflect differences in the type of amines contained within the tumor cell cytoplasm.

Cyclooxygenase 2

Cyclooxygenase 2 (COX-2) is the rate-limiting enzyme in the production of various prostaglandins from arachidonic acid. It is expressed in a variety of neoplasms, including colorectal, gastric, and pancreatic carcinomas.

KIT (CD117)

The antibody against this protein stains the transmembrane and cytoplasmic KIT protein, which is a type 3 tyrosine kinase receptor. This antibody is useful for identifying GI stromal tumors (GISTs).

DNA Mismatch Repair Proteins MLH1, MSH2, MSH6, PMS2

These antibodies are directed at protein components of the mismatch repair (MMR) complex. These proteins function as heterodimers; MLH1 associates with PMS2, and MSH2 associates with MSH6. As such, these pairs of proteins often show pairwise loss of expression. Decreased or absent staining is indicative of quantitative protein deficiencies or mutated protein.


MOC-31 is one of myriad monoclonal antibodies against the epithelial adhesion molecule, Ep-CAM. MOC-31 is expressed in a host of benign epithelia and is expressed in many carcinomas, including those derived from the colon, stomach, breast, pancreas, ovary, and bile ducts.

Mucin Core Polypeptides

Mucin core polypeptides (MUCs) are the backbone molecules of GI tract mucins and are responsible for the mucus-gel layer, which covers the mucosa. MUC1 is normally expressed by enterocytes and intestinal goblet cells, MUC2 is normally secreted by intestinal goblet cells, MUC5AC is expressed by gastric foveolar mucus cells and neoplastic goblet cells, and MUC6 is secreted by gastric antral and fundic gland cells.

p53 Protein

Normal p53 protein has an extremely short half-life and is found in small quantities inside cells. As such, scattered, faint p53 positivity can be detected in normal cells by using IHC. Mutations in the TP53 gene often lead to increased nuclear accumulation of an abnormal protein, but in some cases, a truncating mutation can lead to complete loss of the p53 protein (“null” phenotype). Hence, aberrant p53 IHC staining can either be stronger and more diffuse than the normal pattern (“overexpressed”) or it can be completely negative.

p63 and p40 Proteins

The TP63 gene is a member of the TP53 gene family; it encodes for several different mRNA isoforms, formed by alternative splicing, that contain (TA isoform) or lack (ΔN isoform) the transactivation domain. The relative concentrations of these protein variants affect the expression and functionality of wild-type p63 and p53 proteins. The p63 protein is expressed in a nuclear pattern in various myoepithelia and is present in the basal layer of squamous epithelium. The p63 antibody recognizes the TAp63 isoform, whereas the p40 antibody recognizes the ΔNp63 isoform. p40 has been shown to be equivalent to p63 in sensitivity, but it is markedly superior to p63 in specificity for the diagnosis of pulmonary SCC.


This protein is a membrane glycoprotein found in calcium channels of cells. Its expression is independent of chromogranin A.


Villin is a brush border, microfilament-associated, actin-binding protein related to rootlet formation. Staining in colorectal adenocarcinomas is diffusely cytoplasmic with brush border accentuation.

Diagnostic Immunohistochemistry: Epithelial Lesions of the Gastrointestinal Tract


Barrett Esophagus

Barrett esophagus (BE) is defined as endoscopically apparent abnormal mucosa extending proximally into the esophagus from the gastroesophageal junction. In the United States, biopsy confirmation of intestinal metaplasia is required, and the most current recommendations require the endoscopically abnormal segment to exceed 1 cm in length. Many studies have demonstrated reactivity of various markers, including markers of intestinal differentiation, such as MUC2 and CDX2, in nongoblet cell columnar epithelium in patients with BE, but no studies have shown these markers to be predictors of the development of goblet cell containing BE. Because goblet cells are often readily identified on the routine hematoxylin and eosin (H&E) stain, the use of IHC to aid the diagnosis of BE is currently not recommended.

Dysplasia in Barrett Esophagus

Numerous molecular alterations associated with the development of neoplasia within BE have been described. Many of these genetic alterations were used as the foundation for IHC to assist in the morphologic diagnosis and grading of dysplasia. Of these altered proteins, p53 is the only antibody with potential utility in the diagnosis of dysplasia. Weak and focal p53 immunoreactivity in BE correlates with a normal (wild-type) TP53 gene status, whereas either strong, diffuse nuclear positivity or complete absence (“null pattern”) of staining correlates with TP53 gene mutations ( Fig. 14.1 ). In theory, aberrant p53 staining (either increased or lost) as detected by IHC should be a surrogate marker for a TP53 gene mutation and could be used in the diagnosis of dysplasia. However, this is not the case in practice, because correlation between staining and gene abnormalities is not precise, and there is substantial overlap of the patterns of p53 reactivity in epithelia negative for dysplasia, those that show reactive changes, and epithelia with low-grade dysplasia (LGD). Although a recent study indicates that p53 can be used as an ancillary marker of dysplasia in difficult cases, it cannot be used to separate LGD from high-grade dysplasia (HGD). The utility of other markers of dysplasia, such as α-methylacyl-CoA racemase (AMACR) and cyclin D1, has been reported in the literature, but more studies are needed before they can be applied clinically. Morphology currently remains the gold standard for the diagnosis of BE-associated dysplasia, and ancillary stains are not recommended for diagnosing dysplasia in BE.

Key Diagnostic Points: Barrett Esophagus

  • There are currently no immunohistochemical markers that can reliably aid in the diagnosis of BE or BE-associated dysplasia.

  • Although p53 has been shown to be a marker of dysplasia in some reports, additional studies are needed before p53 staining can be used routinely to aid in the diagnosis of BE dysplasia.


  • Complete negative staining for p53 (with proper controls) is an abnormal pattern of expression and indicates the presence of a TP53 mutation.


(A) High-grade dysplasia in Barrett esophagus. (B) Strong and diffuse nuclear staining with p53 is typically seen. However, overlap can be substantial in the patterns of p53 reactivity in epithelial cells that are reactive and in those that show low-grade dysplasia.

Esophageal Adenocarcinoma

Esophageal adenocarcinomas typically express CKs AE1/AE3, CAM5.2, CK19, and CK7; a minority of cases express CK20. CDX2 expression is variable, and although many tumors may show focal positivity, a significant minority are completely negative, and only a few are uniformly positive. Villin expression is more uniform and is present in approximately 75% of tumors. Most studies have found that esophageal adenocarcinomas are immunophenotypically identical to adenocarcinomas of the gastric cardia (see below). Some but not all studies suggest that esophageal and gastric cardia adenocarcinomas have different CK7/CK20 staining patterns based on statistical comparison of a large number of cases. However, the overlap in staining patterns between the two groups in these studies is substantial, resulting in a limited use of CK7/CK20 staining to differentiate esophageal adenocarcinomas from proximal gastric adenocarcinomas.

Key Diagnostic Points: Esophageal Adenocarcinoma

  • Esophageal adenocarcinomas are generally positive for CK7, with negative-to-variable expression of CK20 and CDX2.


  • Esophageal adenocarcinoma is immunophenotypically similar to proximal gastric adenocarcinoma. There is currently no reliable immunohistochemical panel to distinguish these two entities.

Esophageal Squamous Cell Carcinoma

SCCs generally stain strongly with medium-molecular-weight and HMWCKs; expression of low-molecular-weight (LMW) keratins is typically weak ( Fig. 14.2 ). Accordingly, most SCCs stain diffusely and strongly with CK antibodies CAM5.2, AE1/AE3, 34βE12, CK5/6, CK14, and CK19. Cytokeratin 34βE12 usually produces stronger and more diffuse staining than CK5/6 ( Fig. 14.3 ).


Cytokeratin reactivity in esophageal squamous cell carcinoma. CAM , Cell adhesion molecule; CK , cytokeratin.


(A) Esophageal squamous cell carcinoma stains diffusely and strongly with cytokeratin (CK) 34βE12. Inset: Central keratin whorl is clearly outlined against the strongly staining tumor cells that surround it. (B) In contrast, CK5/6 staining is predominantly confined to the central keratinized cell whirls. Inset: Rare isolated tumor cells also stain strongly.

The intensity of CK19 expression increases with higher tumor grade. Approximately 70% of low-grade SCCs stain with CK19 in less than half of the neoplastic cells, whereas almost all high-grade neoplasms are diffusely and strongly reactive. Squamous carcinoma in situ is also positive for CK19, whereas benign squamous mucosa is negative ( Fig. 14.4 ). Other diagnostically useful antibodies that are typically strongly positive are p63 and p40, thrombomodulin, epithelial membrane antigen (EMA), and selected monoclonal carcinoembryonic antigens (CEAs).


Cytokeratin 19 strongly stains the squamous cell carcinoma in situ on the right, whereas the benign epithelium on the left is nonreactive.

Nonreactive antibodies include CK7, CK20, 35βH11, BerEP4, thyroid transcription factor-1 (TTF-1), and Wilms tumor 1 (WT1). Although CK7 is included in this group, approximately 15% to 30% of SCCs have occasional and scattered clusters of cells that are CK7+. However, if a positive result is defined as expression in more than 50% of tumor cells, esophageal SCCs are considered CK7−/CK20−.

Primary pulmonary SCC can occasionally be distinguished from esophageal SCC with TTF-1. Although both neoplasms are usually nonreactive, occasional pulmonary SCCs may show extensive and strong nuclear TTF-1 staining, whereas esophageal SCCs are consistently negative for TTF-1.

It is occasionally important to distinguish between poorly differentiated SCC and poorly differentiated adenocarcinoma; p63, p40, and CKs 7, 20, and 5/6 are useful in this context. SCCs, including poorly differentiated nonkeratinizing neoplasms, are CK7− and CK20− and positive with CK5/6, p63, and p40, whereas adenocarcinomas of the esophagus, stomach, and lung are typically positive with CK7 and CK20 but negative with CK5/6 and p63.

Distinction between SCC and thymic carcinoma is also occasionally required. CD5 can be diffusely and strongly positive in primary thymic carcinoma and may be nonreactive in esophageal SCCs. Importantly, selective CD5 reactivity of thymic carcinomas is highly dependent on the pH of the antigen retrieval solution and the antibody clone. Some CD5 antibodies diffusely and strongly stain both thymic carcinomas and esophageal squamous carcinomas.

Mesothelioma can occasionally be morphologically and clinically similar to poorly differentiated, nonkeratinizing, primary esophageal SCC. Both neoplasms are immunoreactive with calretinin and CK5/6, leaving WT1 and p63 as differentiating positive diagnostic markers for mesothelioma.

Key Diagnostic Points: Esophageal Squamous Cell Carcinomas

  • Squamous cell carcinomas (SCCs) stain strongly and diffusely with CAM5.2, AE1/3, CK5/6, p63, and p40.

  • CK7, CK20, and CEA are either negative or focally positive in poorly differentiated SCCs, whereas these antibodies are strongly positive in poorly differentiated adenocarcinomas.

  • CD5 testing, if performed correctly, can be used to distinguish thymic carcinoma from esophageal SCC.


  • A significant minority of SCCs can be focally positive for CK7.

Squamous Cell Carcinoma Variants

Basaloid Squamous Cell Carcinoma

Basaloid squamous cell carcinoma (BSCC) is a morphologic and genetic variant of poorly differentiated SCC. Mixed basaloid/classic SCC or neoplasms of mixed BSCC/adenocarcinoma may be seen. Bcl-2 has been reported to stain BSCC, but it is nonreactive in poorly differentiated, conventional SCC. CK5/6, CK monoclonal-OSCAR, CK13, CK14, CK19, AE1/3, and p63 typically are diffusely and strongly reactive in BSCC, whereas CKs CAM5.2 and 35βH11 are often negative or weakly immunoreactive. Typically, the central cells within each nest are strongly positive for CK5/6 and p63. Moreover, the pseudopalisading, single cell layer at the periphery of carcinoma nests typically shows myoepithelial differentiation, including reactivity with CK19, S100 protein, and smooth muscle actin (SMA). Similar to some high-grade breast carcinomas, IHC features of myoepithelial cell differentiation can be diffuse.

Adenoid Cystic Carcinoma

Most reported esophageal adenoid cystic carcinomas are BSCCs; true adenoid cystic carcinomas of the esophagus are extremely rare. Esophageal salivary gland–type adenoid cystic carcinomas stain diffusely and strongly with CAM5.2 and AE1/AE3. In addition, 34βE12 and CEA stain the ductal-type cells, whereas S100, actin, and vimentin stain the basaloid-type cells.

BSCCs with a solid growth pattern, those with a cribriform growth pattern that mimic adenoid cystic carcinoma, salivary gland-type adenoid cystic carcinomas, and high-grade NECs are often morphologically similar and may be difficult to separate, especially in small biopsy fragments. IHC is useful in this context ( Fig. 14.5 ). CK5/6, CK7, 34βE12, CK19, p63, CEA, chromogranin, and synaptophysin are useful for distinguishing among these three entities. CK7 is often the single positive marker in high-grade NEC. Care should be given to avoid misinterpreting the nonspecific synaptophysin staining of necrotic debris found in BSCCs as true cytoplasmic granular immunoreactivity.

Key Diagnostic Points: Squamous Cell Carcinomas Variants

  • Central areas or nests of BSCC are CK5/6 and p63 positive, whereas the peripheral rim of palisading cells may be CK19 positive.

  • Most adenoid cystic–like carcinomas are basaloid squamous cell carcinomas (BSCCs). True adenoid cystic carcinomas of the esophagus are extremely rare.

  • High-grade (small cell) neuroendocrine carcinoma is diffusely synaptophysin and CK7 positive.


  • Nonspecific synaptophysin staining can be present in BSCCs and should not be interpreted as true cytoplasmic granular positivity.


Differential staining patterns of basaloid-patterned esophageal carcinomas. ACC , Adenoid cystic carcinoma; BSCC , basaloid squamous cell carcinoma; CK , cytokeratin; HG NEC , high-grade neuroendocrine carcinoma.

Esophageal Carcinomas With Spindle Cell or Mesenchymal Differentiation

Esophageal carcinomas can rarely show spindle cell or mesenchymal differentiation; however, the spindle cell component is often associated with recognizable epithelial differentiation. Not surprisingly, this mesenchymal differentiation shows decreased cytokeratin expression. The cytokeratin clones OSCAR and CK5/6 produce the strongest and most diffuse immunoreactivity. CK OSCAR is useful for distinguishing neoplastic spindle cells from reactive myofibroblasts; CAM5.2, 35βH11, and AE1/AE3 may give variable results. Among these three antibodies, AE1/AE3 produces the strongest and most diffuse staining. Neoplastic spindle cells can stain with actin antibodies; however, desmin is usually negative, providing the IHC distinction from leiomyosarcoma. True spindle cell rhabdomyosarcomatous differentiation can also occur, in which the cells stain with pan–muscle actin, desmin, and other markers of rhabdomyoblastic differentiation.


Nonneoplastic Conditions

Lymphocytic Gastritis

Lymphocytic gastritis is usually a manifestation of celiac disease and, occasionally, of Helicobacter pylori infection. In patients with celiac disease, the density of surface intraepithelial lymphocytes (IELs) is usually lower than that seen in the duodenum. Gastric IELs are T lymphocytes that stain with CD45RO, CD3, CD7, CD8, and T cell–restricted intracellular antigen 1 (TIA-1).

Helicobacter pylori

Proton pump inhibitor and H. pylori eradication medications decrease the density of H. pylori organisms and alter their shape from spiral to coccoid. Coccoid-shaped H. pylori can be difficult to distinguish from small mucin globules or extracellular debris on modified Giemsa or other histochemical stains. IHC is a more reliable and sensitive method for detecting H. pylori , especially when the organisms are few in number, or when they are coccoid in shape ( Fig. 14.6 ). Helicobacter IHC is not indicated in the context of a histologically normal biopsy, unless that patient had a previous diagnosis of H. pylori gastritis. In addition, some H. pylori antibodies cross-react with H. heilmannii , which can occasionally be useful when the morphology of these organisms does not unequivocally allow for their identification on routine stains.


Gastric antral mucosa with numerous Helicobacter pylori organisms. Many of the microorganisms are insinuated between the cell membranes of adjacent columnar mucus cells. The inset images across the top of the figure demonstrate the broad range of bacterial shapes that include coccoid, spiral, bacilliform, and barbell varieties.

Atrophic Gastritis, Autoimmune Type

IHC can be a useful adjunct in the diagnosis of autoimmune gastritis (AIG), most cases of which show hyperplasia of the enterochromaffin-like (ECL) cell compartment secondary to hypergastrinemia. This phenomenon can be highlighted using synaptophysin and/or chromogranin IHC. Normal mucosa shows occasional synaptophysin- and/or chromogranin-positive cells within the epithelial compartment. In AIG, intraepithelial linear arrays and/or extraepithelial nodules of synaptophysin- or chromogranin-positive ECL cells may be seen ( Fig. 14.7 ).


Autoimmune gastritis.

(A) This biopsy from the gastric body lacks oxyntic glands; instead, the mucosa has the appearance of antral mucosa. However, this is not true antral mucosa; no G cells are present on the gastrin immunostain (not shown). (B) Synaptophysin highlights enterochromaffin-like (ECL) cell hyperplasia within this atrophic body mucosa. Both forms of ECL cell hyperplasia, linear (more than five contiguous positive cells) and nodular (extraepithelial, small, round clusters), are present in this image. When interpreting immunostains in cases of possible autoimmune gastritis, it is important to ignore areas of intestinal metaplasia, because metaplastic G cells or enterochromaffin cells can often be present within the intestinal-type epithelium, and they can stain with gastrin (C, arrows ) and synaptophysin (D, arrows; true ECL cell hyperplasia is also present in the lower right of the figure).

In addition to the detection of ECL hyperplasia, IHC for gastrin can be helpful in separating atrophic “antralized” oxyntic mucosa, with complete loss of both parietal and chief cells, from true antral mucosa. In fully developed AIG, these two types of mucosa can be difficult to separate on routine histology. Gastrin-staining cells are present in the antrum and are absent in atrophic oxyntic mucosa. Of note, when assessing gastric biopsies for the presence of body-predominant atrophic gastritis, three stains—synaptophysin, chromogranin, and gastrin—must be assessed in areas free of intestinal metaplasia, because the intestinal metaplasia contains its own neuroendocrine cells that may be gastrin positive (see Fig. 14.7 ).

Fundic Gland Polyps

Fundic gland polyps occur as sporadic lesions and may be associated with long-term proton-pump inhibitor therapy. In addition, fundic gland polyps may arise in association with familial adenomatous polyposis (FAP) and Zollinger-Ellison syndromes. Morphologic distinctions between the sporadic and syndromic polyps can be subtle. CK7 has been reported to stain sporadic polyps and Zollinger-Ellison syndrome–associated polyps, whereas β-catenin expression has been described in sporadic polyps but not in FAP-associated polyps. IHC is not routinely used to evaluate fundic gland polyps.

Key Diagnostic Points Stomach: Nonneoplastic Conditions

  • T-cell markers can be used to highlight intraepithelial lymphocytes in suspected cases of lymphocytic gastritis associated with celiac disease or Helicobacter pylori infection.

  • Immunohistochemistry (IHC) staining for H. pylori is useful when treatment-associated changes reduce the number of organisms or alter their normal appearance.

  • In cases of suspected autoimmune gastritis (AIG), gastrin IHC can be used to distinguish antral from atrophic body or fundic (oxyntic) mucosa; synaptophysin and chromogranin IHC are useful in the detection of enterochromaffin-like cell hyperplasia, a characteristic feature of AIG.


  • Synaptophysin, chromogranin, and/or gastrin positive neuroendocrine cells can be present in foci of intestinal metaplasia, so these foci should be not be assessed when interpreting these stains.

Gastric Adenocarcinoma

Gastric cancer can be classified by anatomic location, histologic features, and, more recently, molecular mechanisms. Anatomically, gastric cancers can be divided into two clinically and epidemiologically distinct subtypes: proximal (cardia, gastroesophageal) and distal (noncardia, true gastric) adenocarcinomas. Histologically, gastric adenocarcinomas have generally been classified into intestinal types and diffuse (poorly cohesive/signet-ring cell type). Although it is important for pathologists to characterize gastric adenocarcinomas into one of these two main morphologic subtypes, the IHC staining pattern of these two subtypes is similar. As a result, both types are discussed together in this section. Gastric adenocarcinomas stain with various CKs ( Fig. 14.8 ). The reactivity of several other antibodies is listed in Table 14.2 , some of which will be discussed in more detail.


Cytokeratin (CK) expression in gastric adenocarcinoma. CAM , Cell adhesion molecule.

Table 14.2

Expression of Various Antibodies (Excluding Cytokeratins) in Gastric Adenocarcinomas

Antibody Reactivity Comments
AMACR (p504s) + AMACR more commonly expressed in intestinal-type and high-grade dysplasia
B72.3 +
BerEP4 +
CA-125 S Single cells or small foci evident
CA 19-9 +
CD99 S Staining confined to intestinal-type neoplasms
CDX2 S Heterogeneous and variable staining seen in 20%–90% of cases
CEA + Monoclonal antibody more discriminatory
GCDFP-15 Rare (<1%) signet-ring cells can be positive
HepPar1 S High-grade and signet-ring cells can be focally positive
MUC2 S Up to 50% of cases can be positive
MUC5AC + 38%–70% of cases can be positive
p63 R Patchy staining seen in high-grade and squamoid carcinomas
S100 S About 20% of neoplasms can have focal positivity
Villin S
Vimentin R Positive staining seen in spindle cell (sarcomatoid) carcinomas

Reactivity: −, negative; R , rarely positive; S , sometimes positive; +, positive.

AMACR , α-Methylacyl-Co-A racemase; CEA , carcinoembryonic antigen; EMA , epithelial membrane antigen; GCDFP-15 , gross cystic disease fluid protein 15; TTF-1 , thyroid transcription factor 1.


Gastric adenocarcinomas are diffusely and strongly positive for AE1/AE3 and 35βH11. Cytokeratin CAM5.2 produces diffuse strong staining in approximately two thirds of these neoplasms and produces weak to moderate, patchy staining in the other third. CKs 18 and 19 are diffusely and strongly positive.

Cytokeratin 7

CK7 expression is an important marker of committed gastric epithelial cells and gastric adenocarcinoma. Approximately 50% of gastric adenocarcinomas are strongly positive in a diffuse or patchy distribution, 30% have rare clusters of strongly reactive cells, and 20% are weakly positive or are negative ( Fig. 14.9 ).


(A) Gastric adenocarcinoma, intestinal (glandular) type. Cytokeratin 7 (CK7) is diffusely and strongly reactive. (B) Almost all of the gastric signet-ring cell adenocarcinoma cells strongly stain with CK7. (C) The glandular region of this gastric adenocarcinoma (right) is strongly CK7 positive, whereas the adjacent signet-ring cells are negative to weakly reactive. Patchy or variegated CK7 staining is characteristic of gastric adenocarcinoma.

Cytokeratin 20

Approximately 40% of gastric adenocarcinomas are strongly positive for CK20 in a patchy or diffuse distribution, 20% are weakly positive in a patchy distribution, and 40% are negative ( Fig. 14.10 ).

FIGURE 14.10

(A) Gastric signet-ring cells are relatively inconspicuous within the lamina propria. (B) Cytokeratin 20 reveals the numerous neoplastic signet-ring cells, which were not easily seen on hematoxylin and eosin stain.

Cytokeratins 7 and 20 Coordinate Staining

Gastric adenocarcinoma is extremely heterogeneous in its CK7/20 coordinate staining patterns. The search for a predominant pattern has been complicated by using different cutoff points at which staining is considered positive. The percentage of positively staining cells in the literature ranges from 1% to 25%. Given these findings, no predominant pattern of coordinate CK7/20 staining is apparent; a significant minority of gastric adenocarcinomas stain with each of the four possible CK7/20 patterns. Approximately 35% of gastric adenocarcinomas are CK7+ and CK20+, 25% are CK7−/CK20+, 25% are CK7+/CK20−, and 15% are CK7−/CK20−. These percentages vary by as much as 30% depending on the cutoff point used in the study.

CDX2 and Villin

Several studies examined CDX2 expression in gastric adenocarcinomas. CDX2 appears to be variably expressed (percent positivity ranges from 20% to 90%), but even when present, its expression tends to be heterogeneous in diffuse-type cancers as compared with strong and diffuse staining in gland-forming adenocarcinomas. Villin also has variable expression and may be slightly more reliable than CDX2.


Overall, the various mucin stains may not be as helpful as the pathologist would desire. The gastric mucin MUC5AC is positive in only 38% to 70% of cases. MUC6, another gastric mucin, is only positive in 30% to 40% of cases, and MUC2 is positive in up to 50% of cases; MUC4 has inconsistent results in the literature, staining from 57% to 100% of cases, and MUC1 stains only a minority of cases.

Estrogen and Progesterone Receptors

The issue of estrogen receptor (ER) positivity in gastric adenocarcinomas has been debated for many years. Faint ER staining of gastric adenocarcinomas was initially interpreted to be a false-positive reaction. Nuclear staining was later deemed a true positive with the lack of staining considered to be a false-negative result. The IHC detection of low-level ER expression is dependent on the antibody clone and IHC procedure, but in most studies, gastric adenocarcinomas are negative for ER. Well-differentiated adenocarcinomas are reactive more often than are poorly differentiated and undifferentiated neoplasms. Gastric adenocarcinomas are also generally negative for progesterone receptor (PR), but a few studies have reported infrequent staining. Although typically negative, focal or diffuse weak staining with ER or PR does not exclude a gastric primary.

Key Diagnostic Points: Gastric Adenocarcinoma

  • Many cytokeratins stain gastric adenocarcinoma diffusely and strongly.

  • Gastric adenocarcinoma is immunophenotypically similar to esophageal adenocarcinoma, and no reliable immunohistochemical panel is currently available to distinguish these two entities.

  • The CK7/20 coordinate staining pattern is not useful in distinguishing gastric adenocarcinoma from other adenocarcinomas.


  • Weak estrogen receptor or progesterone receptor staining in a metastatic adenocarcinoma does not rule out a primary gastric carcinoma.

Gastric Adenocarcinoma Variant: Lymphoepithelial-Like Carcinoma

Gastric lymphoepithelial-like carcinomas are undifferentiated (medullary-type) carcinomas with a lymphocyte-rich stroma. These tumors are often associated with either Epstein-Barr virus (EBV, Fig. 14.11 ; discussed in the section “ Beyond Immunohistochemistry ”) or a high level of microsatellite instability (MSI-H). The MSI designation characterizes a group of carcinomas that develop as a result of deficiencies of the DNA MMR complex. Microsatellite-unstable adenocarcinomas can be syndromic, such as with hereditary nonpolyposis colorectal cancer (HNPCC), or they can be sporadic. Antibodies against MLH1, MSH2, MSH6, and PMS2, proteins of the DNA MMR complex, can detect MSI by their lack of staining in tumor cell nuclei (for a more complete discussion, see “ Colorectal Adenocarcinoma with Microsatellite Instability ” later). Syndromic patients can show a loss of any of these antibodies, most commonly MSH2. Almost all MSI-H gastric adenocarcinomas are sporadic and show loss of MLH1 protein. Authors who have classified gastric adenocarcinomas according to cell type have found that tumors with a foveolar phenotype are often microsatellite unstable, whereas carcinomas with an intestinal phenotype are usually microsatellite stable. Intestinal-type carcinomas harbor deletions of tumor suppressor genes that can be demonstrated by staining with p53.

FIGURE 14.11

Lymphoepithelioma-like gastric carcinoma.

(A) The large neoplastic cells have prominent nucleoli and blend in with the background inflammatory cells. (B) An AE1/AE3 cytokeratin stain highlights the carcinoma cells. (C) In this neoplasm, MLH1 is lost in the tumor cell nuclei as compared with the intact (positive) staining of lymphocyte and stromal cell nuclei. (D) In this different example, the tumor cell nuclei are positive for Epstein-Barr virus mRNA (in situ hybridization), and the inflammatory and stromal cells are negative.

Rare Gastric Adenocarcinoma Variants

Spindle Cell Differentiation (Sarcomatoid Carcinoma).

Similar to the colon and esophagus, spindle cell differentiation has been described in gastric carcinomas. These neoplasms usually stain with vimentin and EMA and stain variably with cytokeratin. See “ Esophagus ,” in the earlier section “ Epithelial Lesions of the Gastrointestinal Tract ,” for additional discussion.

Yolk-Sac, Hepatoid, and Choriocarcinomatous Differentiation.

So-called yolk sac, clear cell, and hepatoid differentiation are frequent in gastric carcinomas. Although areas of yolk sac and/or hepatoid differentiation in an otherwise typical adenocarcinoma are common, pure tumors are rare. The morphology of these areas histologically resembles yolk sac or hepatocellular carcinoma. Single cells or small clusters of cells strongly stain with α-fetoprotein (AFP) in areas with yolk sac differentiation. In addition, tumors with hepatoid differentiation may stain with HepPar1 in its typical, granular staining pattern. Focal immunoreactivity with HepPar1 and/or AFP can also be seen in otherwise typical intestinal-type and signet-ring cell adenocarcinomas. Thus AFP or HepPar1 staining alone is not sufficient for the diagnosis of yolk sac or hepatoid differentiation. In addition, yolk sac or hepatoid differentiation has no prognostic significance.

Choriocarcinoma-like differentiation may also be present in otherwise usual-type adenocarcinomas. These foci usually stain with β–human chorionic gonadotropin (β-hCG) and placental alkaline phosphatase (PLAP). β-hCG staining of usual-type intestinal or signet-ring adenocarcinomas is common: approximately 33% stain strongly with polyclonal β-hCG, and 60% are immunoreactive with the monoclonal antibody.

Gastric Adenocarcinoma With Neuroendocrine Differentiation.

Typical gastric adenocarcinomas of either the intestinal or signet-ring cell type may stain with chromogranin and synaptophysin without histologic evidence of neuroendocrine differentiation. Staining with either antibody can be extensive in this setting and can be increased by using more sensitive methodologies. Staining with chromogranin or synaptophysin is so common that it could be considered within the normal immunophenotype of gastric adenocarcinoma. Gastric tumors without morphologic features of neuroendocrine differentiation that stain with synaptophysin or chromogranin should not be considered NECs but rather adenocarcinomas that express neuroendocrine markers.

Key Diagnostic Points: Gastric Adenocarcinoma Variants

  • Lymphoepithelial-like gastric carcinomas have a unique morphologic pattern, and most show either loss of MLH1 or expression of Epstein-Barr virus by in situ hybridization.

  • Other morphologic variants of gastric carcinoma have been described that include spindle cell, yolk sac, hepatoid, and choriocarcinoma variants. These variants have a corresponding immunohistochemical staining pattern that helps to confirm the morphologic impression.


  • Some antibodies, such as α-fetoprotein, HepPar1, and β-hCG, that stain morphologic variants can also stain the cells of morphologically typical adenocarcinomas. Immunostaining alone should not be used as evidence of variant differentiation without morphologic correlation.

  • Chromogranin and synaptophysin can also be positive in typical adenocarcinomas. Thus staining with neuroendocrine markers is not sufficient evidence for the diagnosis of neuroendocrine carcinoma.

Key Diagnostic Panels: Gastric Adenocarcinoma

Metastatic Breast Carcinoma Versus Primary Gastric Signet-Ring Cell Carcinoma.

Antibodies: ER, MUC1, GCDFP-15, mammaglobin, GATA3, monoclonal CEA, CDX2/villin, CK20, and HepPar1 ( Fig. 14.12 ).

FIGURE 14.12

Metastatic breast carcinoma versus gastric signet-ring cell carcinoma. CK , Cytokeratin; ER , estrogen receptor; GATA3 , trans-acting T-cell–specific transcription factor; GCDFP , gross cystic disease fluid protein; Mammo , mammoglobin; mCEA , monoclonal carcinoembryonic antigen.

Gastric signet-ring cell carcinoma and metastatic lobular breast carcinoma can be morphologically similar, and IHC can be useful in this differential diagnosis. Trans-acting T-cell–specific transcription factor (GATA3) is positive in 94% of breast carcinomas, including 100% of lobular carcinoma, whereas gross cystic disease fluid protein 15 (GCDFP-15) and mammaglobin are positive in approximately 50% and 75% of breast carcinomas, respectively ; these markers are predominantly negative (≤5%) in gastric carcinoma. Although several authors have reported ER positivity in gastric carcinoma, almost all recent studies have reported uniformly negative staining (see earlier discussion under “ Gastric Adenocarcinoma ”). The vast majority of lobular/signet-ring cell carcinomas of breast show positive ER staining in most of the cells. Expression of markers of intestinal differentiation, such as villin and CDX2, is supportive of a gastric primary, whereas breast ductal carcinomas are villin and CDX2−. Gastric signet-ring cell carcinomas are often CK20+, compared with 2% of gastric carcinomas. Monoclonal CEA (mCEA) is diffusely positive in gastric adenocarcinoma and is negative in breast carcinoma. Other CEA antibodies share epitopes and can be positive in both neoplasms.

Pancreaticobiliary Versus Gastric Adenocarcinoma.

Antibodies: CK17, CA-125.

Substantial immunophenotypic overlap exists between pancreatobiliary and gastric adenocarcinomas. CK17 is expressed in as many as 88% of pancreatobiliary cancers but is only positive in 28% of gastric cancers. CA-125 is often present in less than 10% of cells in gastric carcinoma, and a tumor with more than 50% of cells staining would support a pancreatobiliary adenocarcinoma. CKs 7 and 20 do not play a role in the differential diagnosis, because the staining patterns are similar in both tumor.

Lung Versus Gastric Adenocarcinoma.

Antibodies: CK7, CK20, TTF-1, Napsin A, Surfactant-A, CDX2 ( Fig. 14.13 ).

FIGURE 14.13

Lung adenocarcinoma versus gastric signet-ring cell carcinoma. CK , Cytokeratin; TTF-1 , thyroid transcription factor 1.

Primary pulmonary signet-ring cell carcinomas can be morphologically identical to gastric signet-ring cell adenocarcinoma. CKs 7 and 20 are only useful in differentiating between these entities when a neoplasm expresses the CK7−/CK20+ pattern seen in approximately 40% of gastric adenocarcinomas compared with 0% of primary lung adenocarcinomas with signet-ring differentiation. TTF-1, surfactant-A, and napsin A are positive in the majority of pulmonary adenocarcinomas and are negative in gastric adenocarcinomas. Approximately 60% of gastric adenocarcinomas are CDX2+, whereas lung adenocarcinomas are CDX2−.

Small Intestine

Celiac Disease

Celiac disease is one of the diseases that show increased numbers of IELs as a key histologic feature. In celiac disease, most IELs are mature T cells that include both alpha/beta and delta/gamma types. The dominant IEL T-cell immunophenotype in celiac disease is CD3+ and CD8+. In general, staining for CD3 is not indicated for routine evaluation of duodenal biopsies for celiac disease. If CD3 IHC is used to quantify IELs, caution should be used to ensure that only IELs are counted as opposed to lymphocytes in the subepithelial lamina propria. In addition, cutoffs for normal numbers of IELs using CD3 IHC have not been well established; it should not be assumed that the current cutoff of 25–30 IELs per 100 enterocytes as analyzed on H&E sections is the same when using CD3 IHC. IHC for CD3 and CD8 is useful for risk stratification in patients with refractory celiac disease (for further discussion, see “ Theranostic Applications ” below).

Neonatal Diarrheal Illness

Life-threatening diarrhea in the neonatal period is a situation that uncommonly confronts the pediatric gastroenterologist and pediatric pathologist. Although there are many causes of life-threatening diarrhea in newborns, there are three major entities that can be diagnosed on biopsy in which IHC is a useful adjunct. Microvillus inclusion disease most often results in subtotal villous atrophy without crypt hyperplasia; significantly increased inflammation is not identified. CD10 and villin IHC shows abnormal brush border morphology with intracytoplasmic signal that corresponds to the microvillus inclusions seen on electron microscopy ( Fig. 14.14A and B ). Similar to microvillus inclusion disease, tufting enteropathy does not show increased lamina propria or epithelial inflammation; the histologic hallmark of this disease is piling-up of abnormally shaped epithelial cells that assume a “teardrop” configuration. Unfortunately, the classic histologic findings can be very subtle and/or exceedingly patchy in their distribution. Recently, complete loss of membranous MOC-31 expression by IHC has been noted in cases of tufting enteropathy (see Fig. 14.14C and D ). The last diarrheal illness of neonates in which IHC has utility is enteroendocrine dysgenesis. Small intestinal biopsies from patients with this extremely rare disease are histologically normal. However, IHC for synaptophysin and/or chromogranin show markedly reduced or absent enteroendocrine cells.

FIGURE 14.14

Microvillous inclusion disease shows abnormal internalized brush border signal and punctate cytoplasmic staining on both CD10 (A) and villin (B) immunohistochemistry (IHC). Tufting enteropathy shows rare, characteristic “drop-like” enterocytes (C). IHC for MOC-31 shows complete loss of membranous staining within epithelial cells (D).

Adenocarcinoma of the Small Intestine

Small intestinal adenocarcinomas stain diffusely and strongly with CK18 and CK19. In contrast to normal small intestinal epithelium, nonampullary small intestinal adenocarcinomas tend to develop CK7 expression and lose CK20 expression. One study showed that all 24 (100%) of their small intestinal tumors were positive for CK7 and of these, two thirds coexpressed both CK7 and CK20, and the remaining were CK20−. In fact, nonampullary small intestinal carcinomas tend to display more of a gastropancreatic immunophenotype (CK7, MUC1, MUC5AC and/or MUC6) rather than intestinal or colorectal carcinoma phenotype (CK20, MUC2, and/or CDX2). More specifically, nonampullary small intestinal carcinomas express CK7 (in 35% to 100% of cases), MUC1 (50%), MUC5AC (30% to 50%), MUC 6 (about 30%), CK20 (about 45%), MUC2 (36% to 57%), CDX2 (44% to 60%), and villin (60%) ( Fig. 14.15 ). In contrast, very few colon cancers are positive for MUC5AC (6%) and MUC6 (4%), and only 10% are positive for CK7. AMACR is rarely expressed in small intestinal adenocarcinomas, but it stains 62% of colorectal adenocarcinomas. Similar to colon cancers, a minority of small bowel adenocarcinomas arise via the MSI pathway, but immunohistochemically, the vast majority of cases show loss of nuclear staining of the MMR protein MLH1 and preserved (intact) staining with MSH2. Adenocarcinomas that arise in the ampulla of Vater are discussed in Chapter 15 .

Key Diagnostic Points: Small Intestinal Adenocarcinomas

  • Nonampullary small intestinal adenocarcinomas tend to exhibit gastropancreatic markers such as CK7, MUC1, MUC5AC, and MUC6, and may lack staining for intestinal markers such as CK20, MUC, and CDX2.

  • CK7 and AMACR can help differentiate small intestinal adenocarcinomas (CK7+/AMACR−) from colorectal adenocarcinomas (CK7−/AMACR+).


  • Unlike colorectal adenocarcinomas, nonampullary small intestinal adenocarcinomas tend to express CK7, whereas the majority (66%) coexpress CK20, and one third of all tumors can be negative for CK20.

FIGURE 14.15

(A) Small intestinal adenocarcinoma (hematoxylin and eosin stain). This example strongly expresses cytokeratins 7 (B) and 20 (C) in addition to CDX2 (D).

Appendix, Colon, and Rectum


Some appendiceal epithelial neoplasms are similar to their colonic counterparts, whereas others are unique to the appendix. Classic, colonic-type hyperplastic polyps (HPs) and adenomas do exist, but they are rare. Lesions with serrated, mucinous, or villous features with or without definite epithelial dysplasia are more common. The terms “appendiceal mucinous adenoma” and “low-grade appendiceal mucinous neoplasm” encompass sessile villous, usually nonserrated lesions that tend to circumferentially involve the appendiceal lumen and are often associated with a cystically dilated appendix due to intraluminal mucin accumulation that may spread into the peritoneal cavity and cause the clinically recognized entity, pseudomyxoma peritonei. These low-grade appendiceal mucinous neoplasms, as well as low-grade mucinous adenocarcinomas, have some different IHC staining characteristics compared with colorectal mucinous adenocarcinomas ( Fig. 14.16 ). In addition to expressing CK20, approximately one third of mucinous lesions of the appendix coexpress CK7, with approximately 25% to 75% of cells staining; CDX2 is also strongly reactive in the cell nuclei ( Figs. 14.17–14.19 ). The proportion of CK7+ cells in this group of lesions is substantially greater than the pattern of rare cells and the occasional cluster of positive cells that is typical of colorectal adenocarcinomas. More than 80% of appendiceal mucinous adenocarcinomas are MUC5A+, which is similar to the proportion stained in gastric, pancreatic, and ovarian primary mucinous tumors (see Fig. 14.16 ). Similar to colorectal mucinous adenocarcinomas, appendiceal mucinous tumors diffusely and strongly stain with CKs 8, 13, 18, 19, and 20 and with MUC2, CDX2, and DPC4 (SMAD4). Nonmucinous, intestinal-type adenocarcinomas of the appendix are rare and are immunophenotypically similar to colonic adenocarcinomas.

FIGURE 14.16

Immunophenotypes of mucinous adenocarcinoma. CK , Cytokeratin.

FIGURE 14.17

Appendiceal mucinous neoplasm; low-grade, noninvasive. At higher magnification, the neoplastic cells are morphologically similar to those of so-called ovarian-primary mucinous borderline neoplasms.

FIGURE 14.18

Most appendiceal-primary mucinous neoplasms are cytokeratin 20 (CK20) positive (left) and are either negative or focally reactive with CK7 (right).

FIGURE 14.19

CDX2 is typically strongly reactive in the cell nuclei of appendiceal mucinous neoplasms.

The IHC distinction of primary and metastatic mucinous adenocarcinomas can be problematic, especially when they involve the ovary. There can be overlap of the CK7/CK20 IHC profile of appendiceal and ovarian tumors. An antibody panel of CK7, CK20, CDX2, MUC2, and MUC5A yields the most informative results (see Fig. 14.16 ). Lack of CK7 and diffuse, strong CK20 staining is supportive of a colorectal adenocarcinoma, whereas diffuse CK7 staining and nonreactive CK20 is supportive of a primary ovarian neoplasm. Even in cases in which focal or patchy staining of CK7 or CK20 is noted, the pattern of staining can be helpful. Ovarian tumors tend to show diffuse CK7 and patchy CK20 staining, whereas colorectal and appendiceal tumors tend to show patchy CK7 and diffuse CK20 staining. Strong and diffuse nuclear CDX2 staining is supportive of a primary colorectal or appendiceal mucinous adenocarcinoma; CDX2 reactivity in ovarian and pancreatic mucinous neoplasms is characteristically less intense and extensive.

Key Diagnostic Points: Mucinous Appendiceal Neoplasms

  • An antibody panel of CK7, CK20, CDX2, and MUC2 can aid in the distinction between primary and metastatic mucinous adenocarcinomas.

Hirschsprung Disease

The diagnosis of Hirschsprung disease relies on the histopathologic assessment of suction rectal biopsies. Acetylcholinesterase histochemistry can be used to highlight increased nerve fibers, but this method requires special tissue handling. Routine IHC can also aid in the diagnosis. Neuron-specific enolase (NSE), cathepsin D, Bcl-2, and calretinin, which normally stain ganglion cells, can be used to identify ganglion cells or the lack thereof. S100 protein and NSE can be used to highlight hypertrophic nerve fibers within an aganglionic segment. In addition to staining ganglion cells, calretinin stains normal nerve fibers. The presence of calretinin-positive nerve fibers in the muscularis mucosae and superficial submucosa correlates strongly with the presence of ganglion cells and may be superior to acetylcholinesterase histochemistry.

Colonic Polyps

There was little need to determine protein expression in colorectal polyps until the sessile serrated adenoma (SSA) was described as a new entity. Although SSAs have morphologic features similar to those of HPs, SSAs arise via distinct genetic pathways, frequently harbor BRAF V600E mutations, and give rise to the majority of sporadic microsatellite-unstable tumors. Several studies have shown that annexin A10 is a marker for the serrated pathway of colorectal carcinoma, and positive annexin A10 IHC expression has been shown to be 73% sensitive and 95% specific for the diagnosis of SSAs versus HPs. Although the VE1 antibody, which immunohistochemically detects the BRAF V600E mutation, has theoretical utility in the detection of SSAs, too few studies have been performed to date to recommend the use of this antibody in routine clinical practice.

Dysplasia in Inflammatory Bowel Disease

Patients with long-standing inflammatory bowel disease (IBD) are at an increased risk for developing dysplasia and colorectal carcinoma. Surveillance colonoscopy with mucosal biopsies is currently the best and most widely used method to detect dysplasia and cancer in patients with IBD. However, for the pathologist, the assessment of dysplasia, especially in inflamed mucosa, can be challenging. IHC for p53 and Ki-67 can help confirm a histologic impression of dysplasia. Both p53 and Ki-67 tend to be overexpressed in areas of dysplasia compared with reactive and nonneoplastic epithelium. Similar to the assessment of dysplasia in BE, there can be overlap of the patterns of p53 and Ki-67 expression in reactive or inflamed epithelium. Surface involvement above the basal third of the crypts by these antibodies and strong p53 positivity support the presence of dysplasia.

Colorectal Adenocarcinoma

Colorectal adenocarcinomas arise through different genetic pathways and should no longer be considered one disease. The majority of colorectal cancers arise via the chromosomal instability pathway with dysfunction of the adenomatous polyposis coli (APC)/β-catenin/Wnt signaling pathway. However, a subset of colorectal cancers arises via the MSI pathway, resulting from either a germline mutation (Lynch syndrome) or epigenetic gene silencing secondary to hypermethylation. Because all older studies on colorectal cancer viewed this cancer as one disease, the majority of this section describes the immunophenotype of colon cancer in general. But evidence is now emerging that the tumors that arise through these two main pathways have different immunophenotypic features. These differences are highlighted at the end of this section.

Colorectal cancer cells contain mostly LMW CKs, predominantly CKs 8, 18, 19, and 20 ( Fig. 14.20 ). They also stain with a broad molecular-weight spectrum of antibodies that includes AE1/AE3 and CAM5.2. A subset of typical colorectal adenocarcinomas may show focal expression of synaptophysin and/or chromogranin. Expression of these neuroendocrine markers should not be interpreted as sufficient evidence for a diagnosis of primary colorectal NEC. This latter diagnosis requires both the appropriate histologic appearance of either small cell or large cell NEC and diffuse expression of the neuroendocrine markers synaptophysin or chromogranin (colorectal NECs are further discussed below).

FIGURE 14.20

Cytokeratin (CK) expression in colorectal adenocarcinoma. CAM , Cell adhesion molecule.

Specific Antibodies in Colorectal Adenocarcinoma

Cytokeratins 7 and 20.

Most adenocarcinomas (80% to 100%) are diffusely and strongly positive for CK20, but some tumors will show only focal positivity or none at all ( Fig. 14.21 ). Decreased CK20 staining occurs in microsatellite unstable adenocarcinomas. In general, colorectal adenocarcinomas infrequently express CK7 (~13% of cases); this frequency is independent of site (primary vs. metastatic) and mucinous subtype. On the basis of these staining patterns, colorectal adenocarcinoma is the major neoplasm that can be diffusely and strongly CK20+ and completely CK7−.

FIGURE 14.21

The most common pattern of cytokeratin 20 staining in colorectal adenocarcinomas.


CDX2 is a marker of intestinal differentiation and stains the nuclei of about 90% of colorectal adenocarcinomas, but the range of tumor positivity in the literature is variable, from 72% to 100%. Similar to CK7, fewer poorly differentiated and mucinous colorectal adenocarcinomas are positive for CDX2, which may be related to MSI. In addition, CDX2 is not specific for colorectal adenocarcinomas. Staining can be seen in adenocarcinomas of pancreatobiliary, gastric, small bowel, lung, ovarian (mucinous and endometrioid), and bladder origin, especially if they show intestinal differentiation.


Villin stains the brush border of the intestines and is thus commonly positive in colorectal adenocarcinoma. The staining pattern is diffusely cytoplasmic with brush border accentuation and is seen in approximately 92% of cases ( Fig. 14.22 ); however, villin expression is not as specific as CDX2. It also stains other intestinal-type tumors, such as lung and bladder adenocarcinoma.

FIGURE 14.22

High and low magnifications of a mucinous colorectal adenocarcinoma stained with villin shows diffuse, homogeneous cytoplasmic immunoreactivity with a luminal, membranous, microvillous brush border accentuation. Although noncolonic adenocarcinomas can stain with villin, the microvillous brush border pattern is characteristic of colorectal adenocarcinoma.

Other Antibodies.

Several other antibodies are typically positive in colorectal adenocarcinoma, such as MOC-31, mCEA, and COX-2, and these are shown in Table 14.3 . CA19-9 is positive in colorectal adenocarcinoma. Other antibodies, such as CK5/6, MUC5AC, and CA125, are typically negative (see Table 14.3 ).

Table 14.3

Expression of Various Antibodies (Excluding Cytokeratins) in Colorectal Adenocarcinomas

Antibody Reactivity Comments
BerEP4 +
CA125 R Positive tumors usually show focal staining
CA 19-9 S
Calretinin R
CDX2 +
COX-2 +
ER and PR
mCEA +
MOC-31 +
MUC2 +
S100 R Positive tumors usually show focal staining
Synaptophysin and chromogranin R Focal positivity can be seen in colonic adenocarcinomas without neuroendocrine features
Villin +

Reactivity: −, negative; R , rarely positive; S , sometimes positive; +, positive.

COX-2 , Cyclooxygenase 2; ER , estrogen receptor; GCDFP-15 , gross cystic disease fluid protein 15; mCEA , monoclonal carcinoembryonic antigen; PR , progesterone receptor; TTF-1 , thyroid transcription factor 1.

Colorectal Adenocarcinoma With Microsatellite Instability

Approximately 15% to 20% of colorectal adenocarcinomas arise from deficiencies in the MMR complex function, resulting in a high level of MSI-H. Four main proteins, MLH1, MSH2, MSH6, and PMS2, comprise the DNA MMR complex. Carcinomas that arise via the MSI pathway tend to have characteristic pathologic features that include a right-sided location, patient age younger than 50 years, tumor-infiltrating lymphocytes, a lack of “dirty necrosis,” presence of peritumoral lymphoid aggregates (“Crohn-like reaction”), mucinous differentiation, medullary features, and/or a neoplasm that is well differentiated.

In nonhereditary, sporadic MSI-H adenocarcinomas, hypermethylation of the MLH1 MMR promoter gene leads to deficiencies in MLH1 protein expression, resulting in loss of nuclear protein expression in the tumor cells. In hereditary adenocarcinomas (Lynch syndrome), germline mutations most commonly involve the MSH2 gene but can also involve the MLH1, MSH6, and PMS2 genes, resulting in loss of nuclear staining of the particular protein ( Figs. 14.23 and 14.24 ).

FIGURE 14.23

High and low magnification of an invasive cecal adenocarcinoma from a 46-year-old patient with hereditary nonpolyposis colon cancer syndrome (Lynch syndrome). Neoplastic cell nuclei are completely devoid of MLH2 immunoreactivity, whereas cell nuclei of the surrounding stromal cells stain strongly. Only the complete absence of nuclear staining should be interpreted as a marker of a mismatch repair enzyme defect.

FIGURE 14.24

Characteristic intact nuclear staining with MLH1 in a patient with a microsatellite-stable cancer. This pattern of staining would be present for all four mismatch repair protein antibodies: MLH1, MSH2, MSH6, and PMS2.

Antibodies to MLH1, MSH2, MSH6, and PMS2 are used to screen for Lynch syndrome and sporadic MSI, because detection of sporadic MSI-H tumors carries therapeutic and prognostic implications (see later discussion). When interpreting these stains, it is important to note that MLH1 exists as a heterodimer with PMS2, and MSH2 heterodimerizes with MSH6. Thus, if there is a defect in MLH1, PMS2 loss will be detected as well. However, if PMS2 protein expression is compromised, MLH1 will remain intact. The MSH2/MSH6 pair shows a similar pattern of reactivity: an MSH6 gene defect causes MSH6 loss only, and MSH2 compromise causes loss of both MSH2 and MSH6. It is for this reason that some authors have proposed restricting the four-antibody panel to a two-antibody panel consisting of only PMS2 and MSH6. Complete loss of staining in tumor nuclei is required to report a complete loss of protein expression. Low or patchy levels of expression in tumor nuclei can be seen, depending on variable protein expression and levels of tissue fixation; reduction, loss, or nucleolar expression of MSH6 has been noted in tumors that have been exposed to chemotherapy and/or radiation. Hence, if a two-antibody panel is used, any abnormal or equivocal staining should be confirmed by adding the respective paired antibody and/or by confirming the MSI-H status by polymerase chain reaction (PCR).

Another screening method for Lynch Syndrome is the use of molecular testing to detect MSI ; this is discussed further in the section “ Beyond Immunohistochemistry .” Such screening methods can identify patients who should have additional genetic testing and counseling.

Compared with typical glandular adenocarcinomas, signet-ring cell and mucinous adenocarcinomas are more often MSI-H and will have absent nuclear immunoreactivity with one of the MMR proteins. MSI-H adenocarcinomas can show loss of expression of CDX2 and CK20. CK20 can be negative in as many as 32% of MSI-H colon cancers, whereas CDX2 has been reported to be negative in 22% of MSI-H tumors. Interestingly, reduced or absent CDX2 expression increases dramatically, to more than 85% of cases, when medullary-type MSI-H tumors are analyzed. As mentioned earlier, CK7 can be expressed in a minority of colon cancers, and this includes both MSI-H and microsatellite-stable tumors. To avoid potential erroneous diagnoses, especially when assessing small biopsy specimens and metastases of unknown origin, it is important to be aware that some cases of colorectal cancer can aberrantly express CK20, CDX2, and/or CK7. Assessment of MSI can be helpful in such challenging cases.

Adenocarcinoma Variants and Subtypes

Signet-Ring Cell Adenocarcinoma.

In general, colonic signet-ring cell adenocarcinomas show similar CK7, CK20, and CDX2 staining patterns compared with glandular colon adenocarcinomas. However, a higher percentage of signet-ring cell carcinomas are positive for MUC2 (100%) and MUC5AC (89%), and a lower percentage are positive for E-cadherin (56%). As noted earlier, microsatellite-unstable cancers with signet-ring cell morphology may show atypical expression of CK7, CK20, and CDX2.

Clear Cell Adenocarcinoma.

Most clear cell adenocarcinomas are immunophenotypically identical to usual-type colorectal adenocarcinomas. Rare cases of primary colonic, clear cell carcinoma stain with AFP.

Undifferentiated Neoplasms and Carcinomas With Rhabdoid Differentiation.

These neoplasms are immunohistochemically similar to poorly differentiated carcinomas with spindle cell differentiation. The two most common GI locations for rhabdoid carcinomas are the colon and stomach. This variant most often occurs as a component within typical adenocarcinoma.

Key Diagnostic Points: Colorectal Carcinomas

  • Colorectal adenocarcinomas are typically positive for CK20, CDX2, and villin and are negative for CK7.

  • Stains for the mismatch repair proteins MLH1, MSH2, MSH6, and PMS2 can be used to screen for neoplasms with a high level of MSI-H.


  • MSI-H colonic adenocarcinomas can show decreased or absent CDX2 and/or CK20 staining.

  • Colorectal adenocarcinomas without cytologic features of NEC can focally express neuroendocrine markers; focal expression of synaptophysin and/or chromogranin in an otherwise conventional colorectal adenocarcinoma is meaningless.

Key Diagnostic Panels: Colorectal Adenocarcinoma

Colon (Microsatellite-Stable) Versus Lung Adenocarcinoma.

Antibodies: CK7, CK20, CDX2, Napsin A, and TTF-1 ( Fig. 14.25 ).

FIGURE 14.25

Colorectal adenocarcinoma versus lung adenocarcinoma. CK , Cytokeratin; TTF-1 , thyroid transcription factor 1.

Diffuse CK20 staining is strongly supportive of a colorectal adenocarcinoma, whereas diffuse, strong CK7− staining is strongly supportive of a lung adenocarcinoma. Focal staining with either antibody should be considered noncontributory, because it can be seen in either lung or colorectal adenocarcinomas. TTF-1, as well as surfactant-A, stains almost all low-grade nonmucinous pulmonary adenocarcinomas, compared with 0% of colon adenocarcinomas. In addition, napsin A has been shown to be very useful in this differential diagnosis, because it is expressed in only 2% of colorectal carcinomas compared with approximately 87% of pulmonary adenocarcinomas in one study. Colorectal adenocarcinomas are CDX2+, whereas nonmucinous pulmonary adenocarcinomas are CDX2−. Primary pulmonary mucinous bronchioloalveolar and goblet cell adenocarcinomas are immunophenotypically different from usual-type pulmonary adenocarcinoma. This subset of adenocarcinomas can stain with CDX2 and CK20, simulating metastatic colorectal adenocarcinoma. Importantly, the intensity and extent of CDX2 staining in pulmonary mucinous adenocarcinomas are moderate and focal, unlike the diffuse, strong immunoreactivity seen in colorectal adenocarcinomas.

Colorectal Adenocarcinoma Versus Müllerian Endometrioid Adenocarcinoma.

Antibodies: CK7, CK20, CEA, CDX2, ER, and PAX8 ( Fig. 14.26 ).

FIGURE 14.26

Colorectal adenocarcinoma versus müllerian endometrioid adenocarcinoma. CA , Carbohydrate antigen; CEA , carcinoembryonic antigen; CK , cytokeratin; ER , estrogen receptor.

CK7 is a useful initial antibody, because it is positive in more than 95% of endometrioid adenocarcinomas and is minimally reactive in colorectal adenocarcinomas. Conversely, CK20 is nonreactive in endometrioid adenocarcinomas and positive in microsatellite stable colorectal adenocarcinomas. An antibody panel that also includes mCEA, CDX2, CA-125, and ER can aid in the distinction between these two lesions. PAX8 is often used to distinguish endometrial from colorectal carcinoma, because it is positive in up to 95% of müllerian endometrioid adenocarcinomas and is uniformly negative in colorectal adenocarcinomas.

Colorectal Adenocarcinoma Versus Urothelial Carcinoma.

Antibodies: CK7, CDX2, p63, thrombomodulin, and GATA3 ( Fig. 14.27 ).

FIGURE 14.27

Colorectal adenocarcinoma versus urothelial carcinoma. CK , Cytokeratin.

In this differential diagnosis, CDX2 is currently the only antibody that definitively allows for the positive identification of colorectal adenocarcinoma, because it has been consistently negative in transitional cell carcinomas. Diffuse, strong CK7 and thrombomodulin staining are characteristic of many transitional cell carcinomas, and either is useful for supporting a transitional cell carcinoma diagnosis if positive. Many poorly differentiated transitional cell carcinomas undergo squamous differentiation and stain with CK5, CK5/6, 34βE12, and p63. Extensive staining with p63 in particular is supportive of a urothelial adenocarcinoma. The lack of staining should be interpreted as a noncontributory finding rather than supportive of a primary colonic neoplasm. GATA3 is a specific marker for urothelial carcinoma and is helpful in distinguishing urothelial carcinoma from colon cancer; GATA3 was shown to be positive in 91% of urothelial carcinomas and only 1% of colorectal adenocarcinomas. However, GATA3 was shown to be positive in only 7% of conventional (intestinal type) primary adenocarcinomas of the bladder and 41% of signet-ring cell adenocarcinomas of the bladder, a limitation in distinguishing bladder adenocarcinomas from colorectal adenocarcinomas.

Colorectal Adenocarcinoma Versus Prostate Adenocarcinoma.

Antibodies: CDX2, CA 19-9, CEA, CK20, PSA, and NKX3.1 ( Fig. 14.28 ).

FIGURE 14.28

Colorectal adenocarcinoma versus prostatic adenocarcinoma. CA , Carbohydrate antigen; CEA , carcinoembryonic antigen; CK , cytokeratin; PSA , prostate-specific antigen.

CDX2 and CA 19-9 stain colorectal adenocarcinomas and are nonreactive in prostate adenocarcinomas. CK20 and CEA support a diagnosis of colorectal adenocarcinoma only when they are diffusely and strongly reactive; both can focally stain high-grade prostatic adenocarcinomas. Prostate-specific antigen (PSA) is positive in prostatic adenocarcinoma and nonreactive in colorectal adenocarcinoma. Similar to other antibodies, the lack of staining with PSA should not be used to support the diagnosis of nonprostatic adenocarcinoma; many high-grade prostatic adenocarcinomas are PSA negative. NKX3.1, a prostatic tumor suppressor gene, is a highly sensitive (98.6%) and specific (99.7%) IHC marker of metastatic prostatic adenocarcinoma (of note, NKX3.1 was negative in all 11 colorectal adenocarcinomas studied) and can be helpful in distinguishing metastatic prostate cancer from colorectal adenocarcinoma, especially if the tumor is poorly differentiated.


Squamous Cell Carcinoma

The incidence of anal SCC is increasing in the United States, and these tumors share some similarities with their uterine cervical counterparts, including association with high-risk human papillomavirus (HPV) infection. Similar to other sites such as the head, neck, lung, and uterine cervix, CK5, CK5/6, p63, and p40 are expressed in anal SCCs ( Fig. 14.29A–C ). CK7 is usually negative unless the carcinoma is a BSCC with an adenoid cystic pattern. Emerging evidence indicates two different histopathologic types of squamous cell in situ lesions/invasive carcinomas of the anus. A bowenoid morphology of anal intraepithelial neoplasia (AIN) or invasive cancer is associated with positive p16 staining and lack of p53 staining (see Fig. 14.29D and E ), whereas a differentiated morphology that maintains squamous maturation is associated with positive p53 staining and lack of p16 staining.

FIGURE 14.29

(A) Anal squamous cell carcinoma (SCC) invading colonic mucosa (hematoxylin and eosin stain). As with SCCs at other sites, anal tumors are also strongly positive for cytokeratin 5/6 (B) and p63 (C). This example shows strong staining for p16 (D) and lack of overexpression of p53 (E).

IHC for p16 is increasingly used to aid in the diagnosis of high-grade intraepithelial lesions associated with HPV. Full-thickness “blocklike” p16 expression in the context of suspicious cytologic features can be helpful in adjudicating lesions that can be difficult to categorize as low- or high-grade squamous intraepithelial lesions. In addition, p16 can be useful when assessing resection margins that may be involved by cautery artifact.

Anal Gland Adenocarcinoma

Anal gland adenocarcinoma is typically composed of small glands with scant mucin production. It invades the anorectal wall, has no identifiable intraluminal component, and has no association with a fistula. Anal gland adenocarcinoma is diffusely positive for CK7 and negative for CK20, CDX2, p63, and CK5/6.

Anal Paget Disease

The intraepidermal adenocarcinoma cells of anal Paget disease stain diffusely with AE1/AE3, CAM5.2, and CK7. CK7 is useful because it diffusely stains the neoplastic cells, whereas the surrounding normal squamous epithelium is nonreactive. GCDFP-15 is also expressed in cases of primary anal Paget disease. When associated with an underlying rectal or urothelial adenocarcinoma, the pagetoid cells tend to coexpress CK20 and lack GCDFP-15 expression. MUC5AC and MUC1 stain most cases of anal Paget disease, whereas MUC2 is positive in cases with associated colorectal adenocarcinoma. CEA and Ber-EP4 are also expressed in the tumor cells.

Key Diagnostic Points: Anal Malignancies

  • Anal squamous cell carcinomas express CK5, CK5/6, p63, and p40.

  • Immunohistochemistry for p16 can be useful in distinguishing low- from high-grade intraepithelial lesions associated with human papillomavirus infection.

  • Anal gland adenocarcinomas are CK7 positive and are negative for CK20, CDX2, p63, and CK5/6.

  • Paget cells in primary anal disease are CK7 and GCDFP-15 positive and CK20 negative, whereas Paget cells associated with an underlying malignancy tend to be GCDFP-15 negative and positive for CK7, CK20, and MUC2.

Diagnostic Immunohistochemistry: Neuroendocrine Lesions of the Gastrointestinal Tract

NETs arise from different types of neuroendocrine cells that are present throughout the GI tract. At least 15 distinct neuroendocrine cell types have been described. The biology and behavior of GI NETs is quite variable and is associated, in part, with the cell and site of origin. Because of these regional differences, GI NETs can be divided into foregut (stomach, duodenum, upper jejunum, and pancreas), midgut (lower jejunum, ileum, appendix, and cecum), and hindgut (colon and rectum) tumors. Because small, benign-appearing NETs can metastasize, all NETs of the GI tract should be considered potentially malignant. The older term for these tumors is carcinoid tumor, but this term is no longer commonly used and has been replaced by NET . In this chapter, the term neuroendocrine tumor represents a well-differentiated lesion; all poorly differentiated, high-grade lesions are termed NECs.

The general immunophenotype of GI NETs is similar to NETs elsewhere in the body. They are positive for LMWCKs (e.g., CAM5.2) and often, but not always, HMWCKs (e.g., AE1/AE3). CK20 is positive in as many as 25% of GI NETs, and CK7 is positive in only 11% of cases. The most commonly used antibodies to detect neuroendocrine differentiation are synaptophysin and chromogranin A. Other useful, positively staining antibodies include CD56 (neural cell-adhesion molecule [N-CAM]), CD57 (Leu-7), and NSE. NESP-55, a member of the chromogranin family, is a promising marker of pancreatic neuroendocrine tumors (PanNETs), but it is typically negative in GI NETs.

It is now standard practice to use both mitotic rate and ki-67 proliferation index to grade GI NETs. The World Health Organization (WHO) 2010 grading scheme has been clarified and modified to account for tumors with a ki-67 proliferation index between 2% and 3%. Currently, gastropancreatic NETs are graded as follows : Grade 1 NETs have less than 2 mitotic figures per 10 high power fields and/or a ki-67 proliferation index of less than 3%, grade 2 tumors have between 2 and 20 mitotic figures per 10 high power field and/or a ki-67 proliferation index of between 3 and 20%, and grade 3 tumors have greater than 20 mitotic figures per 10 high power fields and/or a ki-67 proliferation index of over 20%. Multiple studies comparing various methods of determining ki-67 proliferation index have noted that a formal manual count of 500 or more cells results in a higher accuracy compared with visual estimation of the proliferation index.

Neuroendocrine Tumors of the Esophagus

Most esophageal neuroendocrine lesions are high-grade, poorly differentiated NECs that are often the large cell rather than the small cell type and may have a component of adenocarcinoma. NETs are exceedingly rare and typically occur as small polypoid lesions in association with BE and may be found incidentally. Primary high-grade small cell NECs are also unusual and occur mainly as a component of an SCC.

Immunophenotypically, NETs stain diffusely and strongly with synaptophysin and chromogranin, whereas high-grade, large cell carcinomas may only be positive for synaptophysin. Small cell carcinomas that arise in association with SCC may express CEA, and as many as half of esophageal small cell carcinomas may be immunoreactive with TTF-1.

Dec 24, 2019 | Posted by in BIOCHEMISTRY | Comments Off on Immunohistology of the Gastrointestinal Tract
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