Special Studies - Clinical Tree

The pathologist’s H&E (hematoxylin and eosin) is like the clinician’s H&P (history and physical)—basic examinations performed on every specimen or patient forming the cornerstone of diagnosis. A wide variety of additional special studies are available to evaluate pathologic processes, from simple histochemical stains to global gene expression patterns. Pathologists are now clinical cell biologists with the “special power” of being able to link morphology to biology.

Histochemistry

Histochemical stains are critically important for the evaluation of many pathologic lesions ( Table 5.1 ). Standard panels of stains are ordered on some types of specimens such liver and kidney biopsies (see Chapter 3 ). Almost all stains are suitable for formalin-fixed tissues.

TABLE 5.1

HISTOCHEMICAL STAINS

STAIN	COMPONENTS STAINED	POSSIBLE USES AND COMMENTS
AFOG (acid fuschin orange G) (modified Masson’s trichrome)	Nuclei—brown Connective tissue—blue Basement membrane—blue Proteins, fibrin, readsorption droplets in cells, immune complexes—red/orange/yellow RBC’s—yellow	Evaluation of renal biopsies.
Alcian blue	Acid mucins—blue (e.g., normal intestinal glands) Nuclei—red Cytoplasm—pink	Sometimes used to identify mucosubstances in mesotheliomas or intestinal metaplasia. Affected by pH. Hyaluronidase digestion can be used to identify hyaluronic acid.
Alcian blue/PAS	Intestinal metaplasia—dark purple Normal stomach—pink	Demonstrates both acid and neutral mucins.
Alcian yellow	Free mucus—yellow Bacteria—dark blue	Identification of H. pylori in gastric biopsies.
Acid fast bacilli stains (Fite-Faraco, Ziehl-Neelson, Kinyoun)	TB—red and beaded MAI—red Nocardia —pink Tissue—blue	Identification of mycobacteria. Modifications are used to demonstrate M. leprae or Nocardia . Carnoy’s fixed tissues cannot be used and B-5 is suboptimal. Slides must be examined under oil.
Alizarin Red S	Calcium—orange red, polarizes	Identifies calcium in tissues.
Bile	Bile—dark green on a yellow background	Identification of bile.
Bodian’s	Nerve fibers and neurofibrils—black Nuclei—black Tissue—blue	Neural tumors, identification of axons.
Chloroacetate esterase (Leder) (CAE)	Mature myeloid cells, mast cells—red granules Nuclei—blue	Evaluation of leukemias. Identification of mast cells. Cannot be used if fixed in Zenker’s or B978-0-323-54632-4.
Congo Red	Amyloid—orange red with apple green birefringence after polarization with optimal optics; additional colors are seen with standard optics Nuclei—blue	Detection of amyloid. Immunoperoxidase studies can be used to identify specific types. Overstaining can result in false positives.
Dieterle	Spirochetes, Legionella, other bacteria—brown to black Tissue—pale yellow or tan	Infectious lesions. Melanin, chromatin, formalin pigment, and foreign material may also stain.
Diff Quik (a modified Giemsa stain)	H. pylori —dark blue Other bacteria—blue Nuclei—dark blue Cytoplasm—pink	Evaluation of chronic gastritis.
Elastic stains (Verhoeff–van Gieson)	Elastic fibers—blue black to black Nuclei—blue to black Collagen—red Other tissue—yellow	Identification of arteries and veins, vasculitis, invasion of lung tumors into visceral pleura, abnormal elastic fibers in elastofibromas.
Fontana–Masson	Melanin, argentaffin granules, chromaffin granules, some lipofuschin—black Nuclei—red	Identification of melanin in melanomas and secretory granules in neuroendocrine tumors. Cryptococcus—some capsule negative forms produce melanin and will be positive. This stain has largely been replaced by immunohistochemistry.
Giemsa (May–Grunwald)	Bacteria (e.g., H. pylori )—blue Parasites (Leishmania , Plasmodium )—blue Mast cells—red to purple granules Nuclei—blue Cytoplasm of leukocytes—pink to blue depending on cell type and differentiation	Lymphoproliferative disorders (good nuclear and cytoplasmic detail). Identification of bacteria, rickettsias, and Toxoplasma gondii.
Gram (Brown–Hopps, Brown–Brenn)	Gram-positive bacteria—blue Gram-negative bacteria—red Nuclei—red Tissue—variable	Identification of bacteria, some cases of actinomycetes, nodcardia, coccidiomycosis, blastomycosis, cryptococcosis, aspergillosis, rhinosporidiosis, and amebiasis.
Grimelius	Argentaffin and argyrophil granules—dark brown to black Nuclei—red Background—pale yellow–brown	Evaluation of neuroendocrine tumors (largely replaced by the use of immunohistochemistry for chromogranin).
Hematoxylin and eosin	Nuclei—dark blue or purple Cytoplasm—pink to red	Standard stain for the routine evaluation of tissues.
Iron (colloidal iron)	Ferric iron (e.g., hemosiderin)—blue Nuclei—red Background—pink	Bone marrow (iron stores, myelodysplasias), liver (hemochromatosis). Chromophobe renal cell carcinomas are positive.
Jone’s Silver Methenamine	Basement membrane—dark maroon to black	Evaluation of renal biopsies.
Melanin bleach	Removes melanin from tissue, usually for IHC.	Melanin can be difficult to distinguish from IHC positivity.
Methyl green-pyronin Y	DNA (nuclei)—green to blue–green RNA—red Goblet cells—mint green Plasma cell and immunoblast cytoplasm—pink to red Mast cells—orange Background—pale pink to colorless	Plasma cell lesions (largely replaced by immunohistochemistry). Does not work well on tissues decalcified with formic acid.
Mucicarmine (Mayer)	Mucin—deep rose to red Capsule of cryptococcus —deep rose to red Nuclei—black Tissue—blue or yellow	Identification of adenocarcinomas, identification of Cryptococcus . Rarely Cryptococcus is negative for mucicarmine (capsule negative) (see Fontana–Masson).
Oil red O	Fat—red Nuclei—blue	Requires frozen sections (lipids are dissolved by most fixatives or during processing). Tissue fixed in formalin can be used if tissue is frozen.
Periodic acid Schiff (PAS)	Glycogen—red Basement membranes—red Mucins—red Colloid—red Fungi—red	Classification of tumors with glycogen (e.g., Ewing sarcoma, rhabdomyosarcoma, renal cell carcinoma), glomerular diseases (BM), identification of adenocarcinomas (mucin), fungal diseases (especially in argentophillic areas—neutrophils and debris), spironolactone bodies in adrenal adenomas treated with this drug.
Periodic acid Schiff with diastase digestion (PAS-D)	As above except glycogen has been digestedand will not be stained	Identification of glycogen in tumors. Identification of fungus in glycogen rich tissue (e.g., skin). PAS-D-resistant deposits in liver are present in alpha-1-antitrypsin deficiency.
Phosphotungistic acid Hematoxylin (Mallory PTAH)	Glial fibers—blue Nuclei—blue Neurons—salmon Myelin—blue Skeletal muscle cross striations—blue Fibrin—blue Collagen—red brown	Identification of neural lesions. Skeletal muscle differentiation (Zenker’s fixative is preferred). This stain has been replaced by IHC for muscle markers.
Reticular fibers (Gomori’s reticulin, Gordon and Sweets, Snook)	Reticulin—black Mature collagen, type 1 brown Immature collagen, types 3 and 4—black	Bone marrow (myelophthisis), liver (fibrosis, veno-occlusive disease), carcinoma versus sarcoma (reticular network) (but largely replaced by IHC).
Silver stain (Grocott methenamine–silver nitrate—GMS)	Fungi—black Pneumocystis —black Mucin—taupe to gray Tissue—green	Evaluation of infectious diseases. Bacteria will also stain black.
Steiner	Spirochetes, H. pylori , Legionella , other bacteria—dark brown to black Tissue—light yellow	Evaluation of infectious diseases.
Sulfated Alcian Blue	Myocytes—yellow Connective tissue—red-purple Amyloid—sea-foam green	Identification of amyloid in cardiac biopsies.
Toluidine blue	Mast cells—deep violet Background—blue	Mast cell diseases, chronic cystitis.
Trichrome (Gomori, Masson)	Mature collagen, type 1—dark blue Immature collagen, types 3 and 4—light blue Mucin—green or blue Nuclei—black Cytoplasm, keratin, muscle fibers—red	Liver (fibrosis).
Trichrome—modified	Viable myocardium—magenta to brick red Nonviable myocardium—dusky gray to mauve	Evaluation of myocardial biopsies.
Von Kossa calcium	Calcium—black Tissue—red	Demonstration of phosphate and carbonate radicals with calcium in tissues, identification of malakoplakia (Michaelis–Gutmann bodies).
Warthin Starry	Spirochetes—black Cat scratch bacillus and Bartonella henselae (bacillary angiomatosis)—black Other bacteria—black Tissue—pale yellow to light brown	Infectious lesions.
Wright’s	Eosinophilic granules—pink Neutrophilic granules—purple Lymphocytic cytoplasm—blue Nuclei—blue to purple	Blood smears.

Immunohistochemical Studies

The development of methods to detect antigens on tissue sections was a major advance in surgical pathology. Immunohistochemical (IHC) studies link histology to molecular biology by allowing the visualization of proteins across tissues and in subcellular locations. IHC studies are the most common special studies in pathology and are used for many purposes including the following:

Classification of tumors (e.g., carcinoma vs. lymphoma vs. melanoma vs. sarcoma)
Identification of in situ lesions versus invasive carcinomas (e.g., myoepithelial markers in breast cancers, basal cell markers in prostate)
Prognostic factors (e.g., Ki-67 [MIB1] in glioblastomas)
Predictive factors to guide specific therapy (e.g., CD117 [c-kit] for gastrointestinal stromal tumors, hormone receptors and HER2 for breast cancer, PD-L1 for many cancers)
Identification of extracellular material (e.g., ß-2 microglobulin amyloid, other types of amyloid)

Identification of infectious agents (e.g., CMV or HSV)

A good resource for proteins and corresponding IHC assays is the Human Protein Atlas ( http://proteinatlas.org ; accessed 1/26/2022).

Factors Affecting Immunogenicity

Numerous variables can affect antigenicity ( Table 5.2 ). Laboratories need to standardize protocols in order to limit day to day variation. Pathologists need to be aware of these factors in order to be alert to assay conditions that may yield erroneous IHC results. Studies on tissues or slides not prepared in the routine fashion for a laboratory, or that may have lost antigenicity, must be interpreted with caution or not at all.

TABLE 5.2

VARIABLES THAT CAN ALTER THE RESULTS OF IMMUNOHISTOCHEMICAL ASSAYS

STAGE IN PROCESS	FACTOR	COMMENT
Acquisition of specimen
	Cautery	Heat can diminish immunoreactivity of antigens. Cautery artifact can make interpretation impossible.
	Cold ischemic time	Optimally, <1 hour. Prolonged ischemia can result in tissue changes and antigen degradation.
Specimen processing
	Type of fixative	The majority of IHC assays are optimized for formalin. Other fixatives can alter results.
	Length of fixation	Formalin causes protein cross-links. If fixation is too short (e.g., <6 hours for small biopsies) or prolonged (many days to weeks), antigenicity may be impaired.
	Decalcification	The use of strong acids can reduce antigenicity. Weak acids have a lesser effect on well-fixed tissue. The length of treatment should be minimized.
	Tissue processing and paraffin embedding	Changes in the tertiary structure of proteins can occur due to ethanol and clearing agents.
Preparation of unstained slides
	Thickness of sections
	Drying temperature and time	The length of time and the temperature used to dry slides can alter antigenicity.
	Time since slide was cut	Antigenicity diminishes over time after a glass slide is prepared. Preferably, only recently cut slides are used.
IHC assay
	Type of antibody	The specific clone of antibody is often important.
	Titer of antibody	The optimal titer produces the greatest specific staining compared to background staining and may be different for each batch of antibody. Over and understaining can lead to erroneous results.
	Mouse versus rabbit antibody	Rabbit antibodies can have a higher affinity and require less antigen retrieval.
	Monoclonal antibody	Detects a specific epitope.
	Polyclonal antibody	Multiple antibody types produce broader detection of proteins but lower specificity. Cross-reactivity with other antigens can be an issue.
	Antibody expiration date	Controls must be monitored with every assay to ensure antibodies are acceptable.
	Antigen retrieval method	These methods using protease treatment or heat reverse changes due to fixation and processing to restore antigenicity.
	Blocking agents	Nonspecific antibody binding and endogenous enzyme activity must be blocked in order to minimize background staining.
	Detection system	Often required in order to amplify the signal. Ultrasensitive systems can result in atypical positivity.
	Chromogen	Brown and red chromogens are in common use and a blue chromogen is available.
	Counterstain	The counterstain should enable the tissue to be seen but not interfere with detection of the chromogen.
	Multiplex assays	Multiple antigens (generally 2–3) can be detected on a single tissue section. The antigens should be in different sites (e.g., nucleus, cytoplasm, or membrane) and/or different chromogens should be used.
Interpretation
	External controls	External positive and negative controls need to be checked for each assay.
	Internal controls	Internal controls are superior to external controls, as the cells have experienced the identical conditions as the target cells. However, they are not available in all specimens. Appropriate positive and negative results should be assessed.
	Location of reactivity	The location of the positivity should be appropriate for the antibody and lesion.
	Reporting	For some assays, results can be reported as positive or negative. Other assays have very specific criteria for reporting the results (e.g., HER2, PD-L1).

Acquisition of biopsy or excision. Removal of tissue using cautery can damage proteins as well as introduce artifacts that preclude, interpretation. Delay in placing biopsies in fixative also allows degradation of the tissue. Cutting core needle biopsies are often optimal specimens, as they are not exposed to heat and can be fixed immediately.

Type of fixative. The majority of antibodies have been optimized on formalin-fixed tissue. Formalin causes cross-linking of proteins, which alters antigens. Some other types of fixatives also alter antigens (e.g., Bouin’s diminishes estrogen immunoreactivity and keratins are not well preserved in B5). IHC cannot be assumed to be equivalent for fixatives other than formalin unless validation studies have been performed.

Length of time of formalin fixation . Protein cross-linking continues while the specimen is in formalin. Antigenicity can be altered by too brief or too lengthy fixation. For example, the American Society of Surgical Oncologists and the College of American Pathologists recommend that breast cancers be fixed between 6 and 72 hours in formalin for optimal preservation of hormone receptors and HER2. ^, To some extent, loss of antigenicity due to cross-linking can be reversed using antigen retrieval methods.

Decalcification . There are multiple methods to soften bone in order to allow sectioning. In general, decalcification using strong acids (e.g., hydrochloric acid) decreases antigenicity of some epitopes (predominantly nuclear) but may not alter others (predominantly cytoplasmic). Weaker acids including EDTA and formic acid (if used for a brief time) have fewer effects on antigenicity. However, when possible, it is preferable to perform IHC studies on tissue that has not been decalcified.

Length of time since the glass slide was cut . The immunoreactivity of the majority of antigens declines over days to weeks with potential complete loss at 1 month. ^, The loss may be due to exposure of tissue to air with oxidation of amino acids, as the immunogenicity of tissue deeper in the block can be preserved for many years. Antigen retrieval methods do not completely restore the antigenicity of old slides. Coating slides with paraffin, storing the slides in a nitrogen desiccator, and/or storing at lower temperatures can partially preserve antigenicity. However, studies should be performed on newly cut slides, if possible. Negative results on older slides should be interpreted with caution or not reported.

Tissue is often dislodged from normal glass slides during the treatments required for IHC. Thus, slides must be coated (e.g., with glue, poly-L-lysine, gelatin, albumin) or special commercial slides must be used.

Antigen retrieval procedures (e.g., proteolysis, heating [microwave, steam], special incubation fluids). To some extent these methods reverse the effects of formalin fixation. Variable effects are observed for different antibodies.

Type of antibody (polyclonal vs. monoclonal vs. mixture of different monoclonals, epitope detected, mouse vs. rabbit). Very different results can be obtained with different antibodies to the same protein or different commercial sources of the same antibody. Laboratories need to validate each antibody.

Interpretation of Immunohistochemical Assays

The first important step is to generate a differential diagnosis after examination of the H&E stained slides. IHC is then used to gain evidence for or against diagnostic possibilities. “Trolling” cases through an immunohistochemistry laboratory by ordering numerous antibody studies without a clear reason in mind is more likely to lead to misguided diagnosis due to aberrant immunoreactivity than to provide an unexpected correct diagnosis.

Panels

In general, a diagnosis should not be based solely on the results of one IHC result, but rather on a pattern of immunoreactivity. Aberrant immunoreactivity or loss of immunoreactivity is occasionally observed for all antibodies, either due to biologic variability (e.g., occasional keratin positive melanomas) or technical factors (e.g., impure antibodies, cross reaction with other antigens, failure to preserve antigenicity). Thus, IHC markers are used most effectively as panels of markers with interpretation based on an IHC profile.

Controls

Controls are essential for the appropriate interpretation of IHC studies and ensure that all steps of this complicated procedure have been performed adequately.

Positive controls consisting of tissues known to be immunoreactive should be included each time an assay is performed. External controls (performed on tissue other than the diagnostic specimen) are important to track results for each antibody and over time. Internal positive controls should always be evaluated when present, as they control not only for the assay but also for the antigenicity of the tissue under investigation. Some laboratories have used vimentin as a control to determine if a specimen retains immunogenicity, as almost all tissue should demonstrate positivity. Given the wide and nonspecific distribution of vimentin, smooth muscle alpha actin may be more useful in this context as pericytes, vascular smooth muscle, and myoepithelial cells present in most tissues are immunoreactive.

Examples of internal positive controls:

S100: Normal nerves, melanocytes, and Langerhans cells in epidermis, cartilage, some myoepithelial cells, skin adnexa
Estrogen and progesterone receptors: Normal luminal cells in ducts and lobules of the breast

CD31, ERG: Vascular endotheliumc-KIT (CD117): Mast cells

Smooth muscle alpha actin: Blood vessel walls, myoepithelial cells in the breast

Vimentin: Blood vessels, stromal cells

High MW keratin: Squamous epithelium

Low MW keratin: Glandular epithelium

CD15: Polymorphonuclear leukocytes

Negative controls usually consist of replacing the primary antibody with nonimmune animal serum diluted to the same concentration as the primary antibody. No positive reaction should be present. If multiple primary antibodies are used reactive with different target antigens, then they may serve as negative controls for each other. Although the best negative control would be to use antibody preabsorbed against the target antigen, this is rarely practical in a diagnostic laboratory.

Diagnostic slides should also be evaluated for internal negative controls. If normal cells are staining in an inappropriate way, this would indicate a serious problem with the assay such as nonspecific staining or use of the wrong antibody.

The following features must be taken into consideration when evaluating immunoperoxidase studies:

Normal Sites of Immunoreactivity

Antigens are present in specific sites ( Fig. 5.1 ). Some antigens may be present in more than one location or be extracellular.

Figure 5.1, Examples of the normal location of antigens (purple indicates the location of the immunoreactivity).

Nonspecific positivity should be suspected when immunoreactivity is present in atypical locations:

Background: Suspect nonspecific positivity if normal cells or noncellular components are positive. This can occur with suboptimal performance of the assay or suboptimal antibodies.
Edge artifact: Antibodies can pool at edges or holes in tissue. True positivity should also be present in the center of the tissue. Some membrane antigens (e.g., HER2) can show artifactual edge enhancement on discohesive cells (e.g., lobular carcinomas). However, if tissue has not been processed appropriately, cells in the center of the tissue may be poorly fixed and lack immunogenicity.
Necrosis or crushing of cells: Nonspecific positivity can be seen in disrupted cells. Although keratin is generally reliable in necrotic tumors, other markers generally should not be interpreted.
Inappropriate location (e.g., cytoplasm instead of nucleus): Occasionally ER or PR is present in the cytoplasm instead of the nucleus. This is not interpreted as a positive result. Plasma cells have large amounts of cytoplasmic immunoglobulins that can cross-react with many antibodies.

Aberrant Sites of Immunoreactivity

If an expected antigen is not in the appropriate location, the most likely possibility is that the wrong antibody was used or there is something wrong with the assay. However, in rare cases, immunoreactivity in an unusual location is of diagnostic importance ( Table 5.3 ).

TABLE 5.3

ABERRANT SITES OF IMMUNOREACTIVITY OF DIAGNOSTIC IMPORTANCE

ANTIBODY	NORMAL LOCATION	ABERRANT LOCATION	TUMORS
TTF-1	Nucleus	Cytoplasm	Hepatocellular carcinoma
Ki-67 (MIB1)	Nucleus	Cytoplasm	Trabecular hyalinizing adenoma of thyroid Sclerosing hemangioma of lung
ß-catenin	Membrane, cytoplasm	Nucleus	Solid-pseudopapillary tumor of pancreas Pancreatoblastoma Endometrioid endometrial carcinoma (20%) Desmoid-type fibromatosis (70%)
ß-catenin	Membrane, cytoplasm	Nucleus and membrane	Colon carcinomas (majority)
NPM1	Nucleus	Cytoplasm	ALK+ anaplastic lymphoma with NPM1-ALK fusion gene has ALK positivity in nucleus and cytoplasm and aberrant NPM1 in cytoplasm. In contrast, ALK+ anaplastic lymphoma with variant translocations has ALK positivity in cytoplasm (or rare membrane) and NPM1 shows normal nuclear location
E-cadherin	Membrane	Absent or cytoplasm or perinuclear	Lobular neoplasms of the breast, signet ring cell carcinomas at other sites
p120 catenin	Membrane	Cytoplasm	Lobular neoplasms of the breast, signet ring cell carcinomas at other sites; with loss of E-cadherin, p120 localizes to the cytoplasm

Manual of Surgical Pathology —Chapter 5.

Identification of Immunoreactive Cells

Immunoreactivity of tumor cells must be distinguished from immunoreactivity of normal entrapped cells (desmin (+) skeletal muscle cells infiltrated by tumor, S100 (+) Langerhans cells in tumors, smooth muscle alpha actin (+) blood vessels, etc.). Plasma cells have large amounts of cytoplasmic immunoglobulin and can react nonspecifically with many antibodies. They should not be mistaken for tumor cells in lymph nodes.

Intensity of Immunoreactivity

Some weak immunoreactivity may be present as a nonspecific finding. It is important to compare positive cells with control slides and with normally nonimmunoreactive cells to determine if the immunoreactivity is significant.

Age of the Slide

Alkaline phosphatase chromogens (red color) fade over time. DAB (a brown color) is more permanent. This is not a problem in evaluating current pathology specimens. However, if immunoperoxidase slides are reviewed after a period of time, some chromogens may fade and once positive results may appear to be negative.

Criteria for Assessment

The majority of assays are interpreted simply as “positive” or “negative.” Diffuse strong staining is a clear positive, whereas weak, focal, and/or heterogeneous staining must be interpreted with caution and in the context of the clinical case.

“Positive” should always refer to whether or not immunoreactivity is present and “negative” to the absence of immunoreactivity, as these meanings are clear and unambiguous. If these terms are used to indicate whether or not the result supports a diagnosis, these terms become conditional on the observer’s expectations. For example, if the absence of SMAD4 (DPC4) is reported as “positive” because it supports a diagnosis of pancreatic carcinoma, this would be reported as “negative” if another diagnosis was favored.

Some assays have very strict criteria for evaluation, especially those that are used to determine prognosis or the treatment of patients. These include estrogen and progesterone receptors, HER2, and PD-L1. For these assays, the specific antibody used can also be important.

The Histo-score or H-score is a semiquantitative method to evaluate IHC results based on both the intensity of immunoreactivity and the percent of cells showing positivity. In the original version, the % of cells at each intensity is multiplied by the respective intensity score and added together, resulting in a possible score from 0 to 400. For example, a cancer with 20% of the cells with weak staining and 50% of the cells with moderate staining would have a score of 20 × 1 + 50 × 2 = 120. In a simplified version, the % of cells of any staining intensity is multiplied by the average intensity of staining. In another simplified version used for Allred classification for hormone receptor results in breast cancer, the proportion score (based on the percent of staining cells) is added to an intensity score (based on the intensity of staining) which results in eight possible values.

ALLRED MODIFICATION OF H-SCORE CATEGORIES (QUICK SCORE)

PROPORTION SCORE (PS)	% POSITIVE CELLS	INTENSITY SCORE (IS)	INTENSITY OF POSITIVITY
0	0	0	None or trace
1	<1%	1	Weak
2	1–10%	2	Moderate
3	10–33%	3	Intense
4	33–66%
5	>66%

THE PS AND IS ARE ADDED TOGETHER FOR A TOTAL SCORE

TOTAL SCORE (TS) PS + IS	INTERPRETATION
0, 2	Negative
3, 4, 5, 6, 7, 8	Positive

Multiplex Assays (“Cocktails”)

In some diagnostic situations, evaluating more than one antibody on the same slide has advantages. For diagnostic purposes using chromogenic detection methods, these assays are usually limited to either two or three different antibodies. The different antigens can be identified by using chromogens of different colors or by their location in different cellular components.

Evaluation of a small number of cells: A small focus of cells may or may not be present in deeper levels of a block. It can be very difficult or impossible to identify the same group of cells on two different slides with two different antibodies. Having both antibodies on the same slide facilitates interpretation.
Expression of two antigens in the cell: In some cases it is important to assess if the same cell expresses two antigens. This can be difficult, if not impossible, using multiple levels. If the antigens are in different locations (e.g., cytoplasm and nucleus), the presence of both can be assessed using the same chromogen. If the two antigens are in the same location (e.g., both in cytoplasm), two different chromogens may give a different color when present—but this can be difficult to assess.
Evaluation of the distribution of two populations of cells: Having two markers for both populations of cells facilitates determining their relationship to each other.

The most commonly used multiplex assays are the following:

Prostate: The assay is used to determine if a small focus of atypical glands is an area of invasive carcinoma. A triple assay includes a high-molecular-weight cytokeratin, p63, and AMACR (P504S). Benign glands have basal cells positive for keratin and p63, and the glandular cells will be negative for AMACR. Prostatic intraepithelial neoplasia (PIN) has basal cells, but the glandular cells express AMACR. Cancers lack basal cells and are usually positive for AMACR. Examples include TriView Prostate (Integrated Oncology; AMACR [red cytoplasm], p63 [brown nucleus], cytokeratin 903/34ßE12 [brown cytoplasm]) and PIN4 (CellNetix; AMACR [red cytoplasm], p63 (brown nucleus), high-molecular-weight cytokeratin [brown cytoplasm]).

Breast: Assays combining a myoepithelial marker with a low-molecular-weight cytokeratin positive in luminal cells are very useful for distinguishing invasive carcinoma from sclerosing lesions. These assays are also helpful for identifying foci of microinvasion associated with DCIS.

Assays that also include a high-molecular-weight keratin can also be used to distinguish usual ductal hyperplasia from atypical ductal hyperplasia and ductal carcinoma in situ. Examples include ADH-5 (Biocare Medical; cytokeratin 5/14 [brown cytoplasm], p63 [brown nuclear], cytokeratin 7/18 [red cytoplasm]), TriView Breast (Integrated Oncology; cytokeratin 5/6 [brown cytoplasm], cytokeratin 8/18 [red cytoplasm], p63 [brown nucleus]), and Breast Triple Stain (Clarient; cytokeratin 5 [brown cytoplasm], cytokeratin 8/18 [red cytoplasm], p63 [brown nucleus]).

Assays can include both E-cadherin and p120 to aid in distinguishing lobular neoplasms from ductal neoplasms. However, because both E-cadherin and p120 are normally located on the membrane, and abnormally can be present in the cytoplasm, a multiplex assay can be more difficult to interpret than each assay alone. An example is LC/DC Breast Cocktail (Biocare; E-cadherin [brown membrane], p120 [red cytoplasm]).

Pleural fluids: It can be difficult to distinguish mesothelial cells from metastatic carcinoma and histiocytes in fluids. A cytokeratin AE1/AE3 and WT1 double stain distinguishes mesothelial cells (either benign or malignant—positive for cytokeratin and nuclear WT1) from metastatic carcinoma (generally negative for nuclear WT1—with the exception of serous carcinomas of the ovary—and positive for keratin) from histiocytes (negative for both).

Melanoma: Combinations of markers for melanoma have been used to provide broader coverage for the detection of melanocytic cells (e.g., MART-1, HMB 45, tyrosinase, and S100). Combinations of a melanocytic marker with a marker of proliferation (MIB-1 or Ki-67) have been used to assist in determining proliferation.

Common Panels for Immunohistochemical Studies

Numerous antibodies are currently in use for IHC studies used for diagnosis, prognosis, and prediction of response to treatment ( Table 5.4 ). IHC studies for diagnosis are best used as panels of multiple antibodies. The following tables include typical results for common diagnostic entities. Because of the many differences in specific antibodies, laboratory assays, and criteria for considering a pattern of immunoreactivity “positive,” results typically vary for different institutions. The percent of lesions that are expected to be positive are coded by term and by shading ( Table 5.5 ). Note that “%” refers to the number of tumors reported to be positive, not the number of cells positive within a tumor.

TABLE 5.4

ANTIBODIES FOR IMMUNOHISTOCHEMISTRY

NAME (ALTERNATE NAME)	ANTIGEN (LOCATION)	NORMAL CELLS AND TISSUES	TUMORS	USES	COMMENTS
GENERAL MARKERS
2SC ( S-(2-succino)-cysteine)	Proteins show abnormal increase in succination when fumarate is increased (Nucleus and cytoplasm—cytoplasm staining alone is not specific)	Normally absent	Fumarate hydratase (FH)-deficient tumors due to germline or sporadic mutations—FH (−) and 2SC (+)	ID of tumors associated with germline or sporadic FH mutations—some cases of papillary renal cell carcinoma and leiomyoma with bizarre nuclei.	Mutations in FH cause loss of function and an increase in fumarate. This results in increased succination of proteins such as 2SC. Germline FH mutations are associated with Hereditary Leiomyomatosis and Renal Cell Carcinoma (HLRCC) syndrome.
Actin (alpha smooth muscle actin (SMA, SM-ACT)	Smooth muscle isoform of actin (Cytoplasm or membrane)	Smooth muscle, myoepithelial cells, blood vessel walls, pericytes, some stromal cells of intestine, testis, and ovary, myofibroblasts in desmoplastic stroma. Not in striated muscle or myocardium.	Smooth muscle tumors, myofibroblastic tumors, PEComas, glomus tumors, Kaposi sarcoma, some spindle cell carcinomas (e.g., those with features of myoepithelial cells)	ID of smooth muscle differentiation (muscle or myofibroblasts) in tumors. Noninvasive lesions of breast (myoepithelial cells present if benign or DCIS) versus invasive carcinoma. Microglandular adenosis also lacks myoepithelial cells.	Good marker for myoepithelial cells of the breast but also positive in myofibroblasts and vessels in stroma. P63 is more specific, but the target (nucleus) is smaller and fewer cells may be visualized.
Actin (muscle-specific actin) (HHF35, MSA, muscle common actin, EM ACT)	Alpha and gamma smooth muscle actins, recognizes a common epitope of alpha in skeletal, cardiac, and smooth muscle (Cytoplasm)	Smooth, striated and cardiac muscle, smooth muscle of blood vessels, pericytes, myoepithelial cells, myofibroblasts	Numerous tumors including tumors of muscle, glomus tumor, PEComa, GIST, DFSP, dermatofibroma, myofibroblastic tumors, spindle cell carcinomas, salivary gland tumors, mesothelioma, others	ID of muscle differentiation in tumors.	Sensitive but not specific. Present in tumors not of muscle origin.
Adenovirus	Viral proteins resulting in enlarged nuclei with basophilic chromatin (“smudge cells”) (Nucleus of infected cell)	Not present		ID of infections due to adenovirus (typical in lining cells and in necrotic debris).
ALK protein (Anaplastic lymphoma kinase, ALK, p80, CD246)	The ALK gene (2p23) (a tyrosine kinase receptor) is translocated to part of the nucleophosmin ( NPM1 ) gene (5q35) to form the fusion protein p80 and is overexpressed (Cytoplasm, nucleus)	Nervous system, T cells	Anaplastic (CD30+) large-cell lymphomas (about 1/3 have the fusion protein t(2;5)(p23; q35)). ALK negative anaplastic lymphomas may have trisomy 2. Rare diffuse large B cell lymphomas Inflammatory myofibroblastic tumors (~50%) Epithelioid fibrous histiocytoma	ID of anaplastic large-cell lymphomas. Lung adenocarcinomas with EML4-ALK rearrangements. Inflammatory myofibroblastic tumor versus other spindle cell neoplasms.	ALK+ anaplastic lymphoma with NPM1 - ALK fusion gene has ALK positivity in nucleus and cytoplasm and aberrant NPM1 in cytoplasm. In contrast, ALK+ anaplastic lymphoma with variant translocations has ALK positivity in cytoplasm (or rare membrane) and NPM1 shows normal nuclear location.
Alpha fetoprotein ( AFP, Alpha 1-fetoprotein)	Glycoprotein present in fetal liver ( Cytoplasm, granular, membrane)	Fetal liver, regenerating liver cells (e.g., chronic hepatitis)	Hepatocellular carcinoma (HCC)(but not the fibrolamellar variant), hepatoblastomas, yolk sac tumors, embryonal carcinoma (but less commonly)	HCC (+/−) versus other cell types (however, AFP is rarely present in other carcinomas such as breast and ovary). Yolk sac tumors (+) versus other germ cell tumors (−/+).	Correlates with extracellular hyaline eosinophilic globules in yolk sac tumors.
Alpha 1-antitrypsin (AAT, alpha-1-AT)	Glycoprotein inhibiting proteolytic enzymes produced in the liver (Cytoplasm)	Histiocytes, reticulum cells, mast cells, Paneth cells, salivary gland	HCC, germ cell tumors, true histiocytic neoplasms, colon and lung carcinoma, others	Accumulates in liver cells in AAT deficiency.	Not specific for tumor type. CD68 is somewhat more specific for macrophages.
AMACR ( alpha-methylacyl-CoA racemase, P504S)	Mitochondrial and peroxisomal enzyme involved in the metabolism of branched-chain fatty acid and bile acid intermediates (Cytoplasm)	Not present in normal tissues	Colorectal carcinoma (92%), colonic adenomas (75%), prostate carcinoma (83%), nephrogenic adenoma (58%), breast cancer (44%), ovarian carcinoma, urothelial cell carcinoma, lung carcinoma, RCC, lymphoma, melanoma Prostatic intraepithelial neoplasia (64%)	Can be combined with p63 to distinguish prostate carcinoma (AMACR +, p63 absent in basal cells) from benign mimics (AMACR -, p63 present in basal cells). However, ~20% of small cancers on core may be negative for AMACR.
Androgen receptor (AR)	Mediates the function of androgens (Nucleus)	Prostate, skin, oral mucosa, breast luminal cells (especially with apocrine metaplasia)	Osteosarcoma, prostatic carcinoma, breast carcinoma (80% of ER positive carcinomas, 20% of ER negative carcinomas), salivary duct carcinoma, ovarian carcinoma, others	Identifies a subset of "triple negative" (negative for hormone receptors and HER2) breast cancers that often show apocrine features ("luminal AR").
ARG-1 ( arginase-1)	Urea cycle enzyme (Cytoplasm)	Hepatocytes	Hepatocellular neoplasms (>90%), cholangiocarcinoma (~60%)	More sensitive for hepatocellular carcinoma than Hep-Par-1 for poorly differentiated carcinomas.
ARID1A (AT rich interactive domain 1A, encodes BAF250A )	Member of SWI/SNF ATP-dependent chromatin-remodeling complexes (Nucleus)	Normal cells	Present in most tumors, lost in some		Loss of expression associated with poorer prognosis for some tumors (e.g., clear cell carcinoma of ovary).
ATRX (Alpha-thalassemia/mental retardation syndrome X-linked, ATDX)	Chromatin remodeling protein involved in DNA repair (Nucleus)	Normal cells	Present in most tumors, loss of expression in some	Loss of expression in ~1/2 of anaplastic astrocytomas, glioblastoma, IDH-mutant tumors.	Associated with a rare genetic disorder (ATRX syndrome).
B978-0-323-54632-4.3 (Tumor-associated glycoprotein 72, TAG-72, CA 72-4)	Oncofetal glycoprotein, may be a precursor of the MN blood group system, sialosyl-Tn antigen (Cytoplasm, membrane)	Not present in most benign adult epithelial cells (may be present in secretory endometrium, gastric, colonic, and duodenal mucosa, apocrine metaplasia, and fetal GI tract)	Adenocarcinomas (especially ovary, colon, breast), others	Adenocarcinoma (+ >90%) versus mesothelioma (5%) or mesothelial cells (−).	Other markers are more useful for mesothelioma versus adenocarcinoma.
BAP1 ( BRCA1-associated protein-1)	A deubiquitinating enzyme ( Nuclear—cytoplasmic positivity is nonspecific)	Present in most normal cells	Present in many tumors and lost in some (e.g., uveal melanoma, renal cell carcinoma)	Distinguish mesothelioma (loss in ~40–70%) versus benign pleural proliferations (+).
Bcl-2 (B-cell lymphoma 2)	Protein involved in inhibition of apoptosis (Membrane, cytoplasm)	Medullary lymphocytes and epithelial cells of the normal thymus, mantle and T zone small lymphocytes	Synovial sarcoma, solitary fibrous tumor, myofibroblastic tumors, schwannoma, neurofibroma, granular cell tumor, GIST, KS, melanoma Small lymphocytic lymphoma/CLL, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma (MALT), some large B cell lymphoma	Synovial sarcoma (+/-) versus mesothelioma (−). Thymic carcinomas strongly express bcl-2 compared to thymomas. Small lymphocytic lymphoma, mantle cell lymphoma, and marginal zone lymphoma (MALT) (+) versus reactive follicles (−).	The bcl-2 gene is involved in the t(14;18) found in follicular lymphomas.
Bcl-10 ( B-cell lymphoma 10)	Encodes a member of the CBM complex. The c-terminal of BCL10 shows homology with an enzyme produced by pancreatic acinar cells ( Cytoplasm)	Pancreatic acinar cells	Acinar cell carcinoma, Some B cell lymphomas		Highly sensitive and specific marker of acinar cells—more sensitive than trypsin.
BCOR ( BCL6 transcriptional corepressor)	Protein involved in transcriptional repression ( Nucleus)	Spermatogonia	Many tumors	Distinguish round cell sarcomas with BCOR alterations (+) versus similar tumors without alterations.	Upregulated due to BCOR fusions and duplications and amplifications result in overexpression.
Ber-EP4 ( Epithelial-specific antigen (ESA), Ep-CAM)	Glycoprotein ( Membrane)	All epithelial cells except superficial layers of epidermis	Most carcinomas	Adenocarcinoma (+; strong and diffuse in 60–100%) versus mesothelioma (− or focal in 26%).	Other markers are better for distinguishing adenocarcinoma versus mesothelioma.
Beta-amyloid ( 6F/3D)	Amyloid present in Alzheimer’s disease (AD) and in cerebral amyloid angiopathy ( Extracellular)	None	AD (amyloid plaques, neurofibrillary tangles, cerebral amyloid angiopathy)	Diagnosis of AD, other diseases.	Found in AD, Lewy body dementia, Down’s syndrome, hereditary cerebral amyloidosis (Dutch type).
Beta-catenin	Component of the adherens junction that binds to e-cadherin and functions in cell adhesion and anchoring the cytoskeleton; signaling molecule of the Wnt/wingless pathway ( Membrane, cytoplasm—aberrant nuclear expression can be helpful for diagnosis)	Urothelium, breast epithelium, colon, esophagus, stomach, thyroid, fibroblasts, endothelial cells	Urothelial cell carcinoma, colonic adenocarcinomas and adenomas, breast carcinoma, esophageal squamous cell carcinoma, head and neck squamous cell carcinomas, gastric carcinoma, ovarian carcinoma, thyroid carcinoma, prostate carcinoma, HCC, brain neoplasms Nuclear positivity in solitary fibrous tumor (40%), endometrial stromal sarcoma (40%), synovial sarcoma (28%)	Aberrant nuclear expression in solid-pseudopapillary tumors of the pancreas (95%) and pancreatoblastomas (78%). Aberrant nuclear expression in medulloblastomas in the WNT-activated group. Aberrant nuclear expression in desmoid fibromatosis (80% deep, 56% superficial) versus low-grade myofibroblastic sarcoma (30%), solitary fibrous tumor (22%), infantile fibrosarcoma (20%), desmoplastic fibroblastomas (6%). Absent or weak in some LCIS.
Beta-2 microglobulin	Immunoglobulin-associated protein ( Extracellular deposits of amyloid)	Plasma cells		Identification of amyloid in patients on dialysis.	Amyloid tends to accumulate around joints and in the GI tract.
BG8	Lewis blood group y antigen ( Cytoplasm)	Red blood cells, endothelial cells	Adenocarcinomas (95%), rare mesotheliomas (about 5%)		Other markers are better for this purpose.
Blood group antigens	A, B, and H antigens ( Membrane)	Epithelial cells and red blood cells, endothelial cells	Lost or abnormally expressed in many carcinomas	May be helpful to identify specimens if patients’ blood types are known but DNA testing is more definitive.	H is diminished by decalcification but not A and B antigens.
Brachyury	Transcription factor active in notocord development (Nucleus)	Notocord, spermatogonia	Chordoma (+)	ID of chordoma versus other cancers that are negative (RCC, chondrosarcoma, myoepithelial tumors, germ cell tumors).
BRAF (V600E mutation) ( B1 homolog of v-raf murine sarcoma viral oncogene, VE1 antibody)	Serine threonine protein kinase—mutation causes constitutive expression (Cytoplasm)	None	Melanoma, papillary thyroid carcinoma, colon carcinoma, some CNS tumors, Langerhans cell histiocytosis, borderline ovarian carcinoma, others	Melanoma—predicts response to treatment (vemurafenib). Colon carcinoma—separates Lynch syndrome cancers (−) from MSI cancers (40–50% +). Thyroid—ID of papillary carcinoma. Ganglioglioma, pleomorphic xanthoastrocytoma, epithelioid glioblastoma.	This mutation results in constitutive expression and increased protein levels. If IHC is equivocal, mutation can be identified by sequencing. Patients are eligible for BRAF inhibitor treatment.
CA 125 ( OC125)	Mucin-like glycoprotein, antibody to ovarian carcinoma antigen ( Luminal surface)	Epithelial cells, mesothelial cells	Adenocarcinomas of ovary, breast, lung (lepidic patter), and others (rarely colon), urothelial cell carcinoma, adenomatoid tumor of the uterus, squamous cell carcinoma, seminal vesicle carcinoma, anaplastic lymphoma	Seminal vesicle carcinoma (+)versus prostate carcinoma (−).	Used as a serum marker for monitoring ovarian cancer.
CA19-9 ( Carbohydrate antigen 19-9 )	Antigen of sialyl Lewisa-containing glycoprotein; antibody to colon carcinoma ( Cytoplasm)	Epithelial cells of breast, colon, kidney, liver, lung, pancreas, salivary gland, others	Adenocarcinomas of GI tract, pancreas, ovary, lung, and bladder, rare in mesotheliomas Chronic pancreatitis		Used as a serum marker for monitoring gastrointestinal and pancreatic carcinomas.
Cadherin-17 (CDH17, liver-intestinal cadherin, human peptide transporter-1)	Transmembrane cell adhesion molecule that binds to catenins for cell polarization, glandular differentiation, and stratification (Membrane)	Intestinal epithelial cells (small and large intestine, appendix, and rectum)	Colorectal and appendiceal adenocarcinomas	ID of metastatic intestinal adenocarcinoma (some gastroesophageal and pancreatobiliary adenocarcinomas show some positivity—carcinomas from other sites are negative).	Sensitivity and specificity is similar to CDX2 for identifying intestinal adenocarcinomas.
Calcitonin	Peptide hormone produced by C cells ( Cytoplasm and extracellular amyloid)	C-cells of the thyroid	Medullary carcinoma of the thyroid (within tumor cells and in amyloid)	ID of C-cell hyperplasia. ID of medullary thyroid carcinoma.	Used as a serum marker for medullary carcinoma.
Caldesmon ( h-caldesmon )	Actin and calmodulin binding protein in smooth muscle ( Cytoplasm)	Vascular and visceral smooth muscle cells, some myoepithelial cells of the breast	Smooth muscle tumors, PEComa, GIST	Smooth muscle tumors (+) versus myofibroblastic lesions (−) or endometrial stromal tumors (−).
Calponin (CALP)	Protein that binds to calmodulin, F-actin, and tropomyosin to regulate smooth muscle contraction ( Cytoplasm)	Vascular and visceral smooth muscle cells, myoepithelial cells of the breast, periacinar and periductal myoepithelial cells of the salivary gland, myofibroblasts	Myoepithelioma, some smooth muscle tumors, myofibroblastic lesions	Can be helpful to identify myoepithelial cells in breast lesions.
Calretinin	Intracellular calcium-binding protein of the troponin C superfamily with an EF-hand domain ( Cytoplasm, nucleus)	Subsets of neurons, pineal cells, germinal epithelium of ovary, mesothelial cells, keratinocytes, breast, sweat glands, neuroendocrine cells, thymus, adipocytes	Epithelial mesotheliomas (less + in sarcomatoid type), adenomatoid tumor, some lung squamous cell carcinomas, rare adenocarcinomas, mesenchymal tumors (e.g., synovial sarcoma), granular cell tumor, Leydig cell tumor, granulosa cell tumor, others	Epithelial mesotheliomas (> 90%) versus adenocarcinoma (<10%).	Useful positive marker for mesotheliomas.
Carcinoembryonic antigen ( CEA. CD66e)	Glycoproteins with immunoglobulin-like regions found in fetal tissues ( Cytoplasm)	Fetal tissues	Adenocarcinomas (liver, colon, pancreas, bile duct, and lung more than breast and ovary), urothelial cell carcinoma, medullary carcinoma of the thyroid Usually absent in RCC, prostate carcinoma, and papillary or follicular thyroid carcinomas	Adenocarcinoma (+) versus mesothelioma (−). HCC: polyclonal CEA has a canalicular pattern.	Different reactivity patterns occur with different antibodies and with polyclonal versus monoclonal antibodies.
CD5 ( Leu 1)	Transmembrane glycoprotein ( Membrane)	T cells and B cell subsets (mantle zone)	Thymic carcinoma, adenocarcinomas, mesothelioma (cytoplasmic) T cell leukemias and lymphomas, aberrantly expressed in low-grade B cell lymphomas (CLL or mantle cell lymphoma)	Thymic carcinoma (+/-) versus thymoma (−). Thymic carcinoma (+/−) versus metastatic squamous carcinoma (−). Classification of low-grade B cell lymphomas. Evaluation of T cell lymphomas (this marker is frequently lost).
CD10 ( CALLA ( c ommon a cute l eukemia a ntigen), J5)	Cell surface metalloendopeptidase that inactivates peptides ( Membrane)	Precursor B cells, granulocytes, rare cells in reactive follicles, myoepithelial cells of breast, bile canaliculi, fibroblasts, brush border of kidney and gut	Endometrial stromal sarcoma, RCC (clear cell and papillary types), HCC, urothelial cell carcinoma, rhabdomyosarcoma, pancreatic carcinoma, schwannoma, melanoma Precursor lymphoblastic lymphoma/leukemia, follicular lymphoma, Burkitt lymphoma, CML, angioimmunoblastic lymphoma	Myoepithelial cell marker in breast. Endometrial stromal sarcoma (+) versus leiomyosarcoma (−/+) (but caldesmon is preferred for this purpose). Evaluation of low-grade lymphomas. Evaluation of leukemias.	Not specific for nonlymphoid neoplasms.
CD15 ( LeuM1)	3-fucosyl-N-acetyllactos-amine, X-hapten—CHO moiety linked to cell membrane protein ( Membrane and cytoplasm)	Granulocytes, monocytes	Adenocarcinomas CMV infected cells Reed Sternberg cells (not LP Hodgkin disease) in a membranous and Golgi pattern, some large T cell lymphomas, MF, some leukemias	Adenocarcinomas (+) versus mesotheliomas (−). Evaluation of Hodgkin disease.
CD30 ( Ber-H2, Ki-1)	Single chain transmembrane glycoprotein homologous to the nerve growth factor superfamily ( Cytoplasm, membrane, and golgi)	Activated B and T cells, some plasma cells, immunoblasts, interdigitating cells, histiocytes, follicular center cells, decidualized endometrium, reactive mesothelial cells, most other tissues negative	Embryonal carcinoma, some vascular tumors (50% of angiosarcomas; not KS), some mesotheliomas Anaplastic large-cell (CD30+) lymphomas, mediastinal large B cell lymphoma, primary effusion lymphoma, HD (but not LP HD), some other B and T cell lymphomas, EBV transformed B cells	ID of anaplastic large-cell (CD30+) lymphomas. Evaluation of Hodgkin disease (RS cells are positive except in LP HD). ID of peripheral T cell lymphoma (large cells may be positive).
CD31 ( PECAM-1, platelet-endothelial cell adhesion molecule)	Transmembrane glycoprotein functioning in cell adhesion ( Cytoplasm, membrane)	Endothelial cells, platelets, megakaryocytes, plasma cells, histiocytes, other hematopoietic cells	Vascular tumors (> 80% of angiosarcomas), Kaposi sarcoma, histiocytic neoplasms, PEComa, very rarely other tumors	ID of endothelial differentiation in tumors. Evaluation of angiogenesis.	Sensitive and specific marker for endothelial cells.
CD34 ( HPCA-1, hematopoietic progenitor cell, class 1, QBEnd10)	Single chain transmembrane glycoprotein, leukocyte differentiation antigen ( Cytoplasm, membrane)	Hematopoietic progenitor cells (decreases with maturation), endothelial cells, fixed connective tissue cells (e.g., in skin), fibroblasts	Acute leukemia, sarcomas of vascular origin, KS, epithelioid sarcoma, GIST, DFSP, solitary fibrous tumor, neurofibroma, schwannoma, spindle cell lipoma, phyllodes tumor, fibroadenoma	ID of endothelial or fibroblastic differentiation in tumors. Evaluation of angiogenesis. Evaluation of the number of blasts in bone marrow in acute leukemia. Solitary fibrous tumor (+) versus sarcomatoid mesothelioma (−). DFSP (+) versus dermatofibroma (−). Absent in breast desmoid type fibromatosis and nodular fasciitis but positive in other stromal cell proliferations.	Not specific but can be useful in context with other features.
CD44v3 ( CD44 variant 3, H-CAM)	Transmembrane glycoprotein mediating cell adhesion ( Membrane)	Many, including myometrium	Many, including endometrial carcinomas	Possibly helpful to distinguish cellular leiomyoma (+) from endometrial stromal sarcoma (−).	Many splice variants of CD44 are present in normal and malignant cells.
CD57 ( Leu 7, HNK-1)	Lymphocyte antigen that cross reacts with a myelin-associated glycoprotein ( Membrane)	T cell subsets, NK cells, myelinized nerves, neuroendocrine cells, prostate, pancreatic islets, adrenal medulla	Nerve sheath tumor (occasional), leiomyosarcoma, synovial sarcoma, rhabdomyosarcoma, neuroblastoma, glioma, neuroendocrine carcinoma, neurofibroma, some prostate carcinomas Angioimmunoblastic lymphoma, T gamma lymphoproliferative disorder (large granular cell lymphocytic leukemia)	ID of neuroendocrine differentiation in tumors. ID of angioimmunoblastic T cell lymphoma. Evaluation of NK neoplasms.	Not very specific for solid tumors.
CD63 ( NKI/C3, (melanoma-associated antigen, ME491)	Member of the tetraspanin or transmembrane four superfamily (TM4SF) found on lysosomes ( Cytoplasm or membrane)	Melanocytes, mast cells, histiocytes, salivary gland cells, sweat gland cells, pancreatic cells, islets of Langerhans, prostatic cells, Paneth cells, peribronchial glands, pituitary	Nevi, melanoma, carcinoid, medullary carcinoma of the thyroid, some adenocarcinomas	Cellular neurothekoma (NKI/C3 + and S100 −) versus melanocytic lesions (NKI/C3 and S100 +). ID of melanocytic lesions.	May be negative in desmoplastic melanomas.
CD68 ( KP1, CD68-PG-M1, Mac-M)	Intracellular glycoprotein associated with lysosomes ( Cytoplasm, membrane)	Macrophages, monocytes, neutrophils, basophils, large lymphocytes, Kupffer cells, mast cells, osteoclasts	Neurofibroma, schwannoma, MPNST, granular cell tumor, PEComa, melanoma, atypical fibroxanthoma, RCC Some lymphomas, histiocytic sarcoma, APML, Langerhans proliferative disorders	Best general marker for macrophages, although not specific to this cell type.	The antibody PG-M1 does not react with granulocytes. Not very specific for solid tumors.
CD99 ( MIC-2, 12E7, Ewing sarcoma marker, E2 antigen, HuLy-m6, FMC 29, O13 (different epitope))	MIC2 gene product—glycoproteins (p30 and p32) involved in rosette formation with erythrocytes ( Membrane, cytoplasm) Membrane immunoreactivity is more specific than cytoplasmic	Cortical thymocytes, T lymphocytes, granulosa cells of ovary, pancreatic islet cells, Sertoli cells, some endothelial cells, urothelium, ependymal cells, squamous cells	Ewing sarcoma, chondroblastoma, mesenchymal chondrosarcoma, synovial sarcoma, solitary fibrous tumors, GIST, some alveolar rhabdomyocarcomas, desmoplastic small-cell tumors, small-cell carcinomas, granulosa cell tumors, yolk sac components of germ cell tumors, Sertoli-Leydig cell tumors, atypical fibroxanthoma, meningioma B and T cell precursor lymphoblastic lymphoma/leukemia	Thymic carcinomas (lymphocytes +) versus other carcinomas. ID of Ewing sarcoma (immunoreactivity should be clearly membranous in the majority of the cells). Evaluation of lymphoblastic lymphoma/leukemia.	O13 is the most commonly used antibody. Immunoreactivity is highly dependent upon the antigen retrieval system used.
CD117 ( c-kit, stem cell factor receptor)	Transmembrane tyrosine kinase receptor (ligand is stem cell factor)—apoptosis is inhibited when the ligand is bound ( Cytoplasm, membrane)	Mast cells, interstitial cells of Cajal (ICC—pacemaker cells of the GI tract found through-out the muscle layers and in the myenteric plexus), epidermal melanocytes, mono-nuclear bone marrow cells, Leydig cells, early spermatogenic cells, trophoblast	GIST (> 95%), seminomas (> 70%), intratubular germ cell neoplasia, mature teratomas (>70%), papillary renal cell (cytoplasmic—associated with mutations), chromophobe renal cell (membrane—not associated with mutations), some melanomas (focal), mast cell tumors, some carcinomas (including adenoid cystic carcinoma of salivary gland and breast), some brain tumors, some Ewing sarcoma, some angiosarcomas AML (> 50%), CML in myeloid blast crisis	ID of GIST (+) versus leiomyomas (−) and schwannomas (−). ID of seminomas ID of mast cells (mastocytosis)—abnormal mast cells commonly have the imatinib resistant mutation D816V. Adenoid cystic carcinoma of breast—some CD117 antibodies are positive in luminal tumor cell component.	Mast cells are an excellent internal control. CD117 (+) does not correlate with mutations and/or oncoprotein activity in tumors not known to have activating mutations and is, in general, not of clinical or therapeutic significance in this setting (e.g., to detect tumors likely to respond to therapy directed against the protein).
CD123 ( interleukin-3 receptor alpha chain)	Alpha chain of the IL-3 receptor ( Membrane)	Myeloid precursors, macrophages, dendritic cells, mast cells, basophils, megakaryocytes	Plasmacytoid dendritic cell tumors
CD141 ( Thrombomodulin, TM)	Transmembrane glycoprotein, receptor for thrombin ( Cytoplasm (epithelial cells), membrane (mesothelial cells))	Endothelium, platelets, monocytes, synovial cells, syncytiotrophoblast, mesothelial cells, dermal keratinocytes, islet cells, peripheral nerves	Mesotheliomas, urothelial cell carcinoma, Kaposi sarcoma, squamous cell carcinomas, choriocarcinomas, rarely adenocarcinomas, benign and malignant vascular tumors	Mesothelioma (+ 80%) versus adenocarcinoma (+ 10%) (but variable results have been reported in other studies).	Other markers are better for distinguishing adenocarcinoma versus mesothelioma.
CD146 ( melanoma cell adherin molecule, MELCAM, MCAM, MN-4, MUC18, A32 antigen, S-Endo-1 )	Membrane cell adhesion glycoprotein of the Ig gene superfamily ( Membrane)	Implantation site intermediate trophoblast, myofibroblasts, endothelium, pericytes, Schwann cells, ganglion cells, smooth muscle, cerebellar cortex, breast luminal and myoepithelial cells, external root sheath of hair follicle, subcapsular epithelium of thymus, follicular dendritic cells, basal cells of bronchus and parathyroid, subpopulations of activated T cells	Melanoma, angiosarcoma, Kaposi sarcoma, leiomyosarcoma, placental site trophoblastic tumor, choriocarcinoma May be focally positive in squamous cell carcinoma and small-cell carcinoma of the lung, mucoepidermoid carcinoma, breast carcinoma, some leukemias, neuroblastoma	ID of placental site trophoblastic tumors.
CD163 ( M130)	Endocytic receptor to scavenge haptoglobin and hemoglobin complexes ( Membrane, cytoplasm)	Tissue macrophages (high expression), monocytes (low expression) including Kupffer cells, Hofbauer cells but not follicular dendritic cells or plasmacytoid monocytes	Neoplasms of histiocytic differentiation Leukemias of monocytic differentiation Synovial type giant cell tumors of the vertebral column Langerhans cell histiocytosis (~60%), benign fibrous histiocytoma (~67%) Littoral cell angioma of the spleen	ID of true histiocytic derivation of tumors.	More specific for monocyte/histiocyte derivation than CD68.
CDK4 ( cylin-dependent kinase 4)	A kinase involved in cell cycle regulation ( Nucleus)	None	Liposarcoma, glioblastoma, anaplastic astrocytoma, large B cell lymphoma, osteosarcoma (parosteal and central about 33%; high grade 12%), breast carcinoma, cardiac sarcoma	Atypical lipomatous tumor/well-differentiated liposarcoma and dedifferentiated liposarcoma (>90% +) versus benign adipose tumors (<5% +).	MDM2 can also be used for this differential diagnosis. Best used together as a panel.
CDX2 (caudal-related homeobox transcription factor, CDX-88)	Homeobox nuclear transcription factor specific for the intestinal tract that regulates MUC2 expression ( Nucleus)	Small intestine, colon, and endocrine pancreas	Colon carcinoma (usually strong and diffuse), small intestine carcinoma, mucinous ovarian carcinoma, bladder adenocarcinoma, some gastric, esophageal, pancreatic, and bile duct carcinoma HCC, breast, lung, and head and neck carcinomas are usually negative.	ID of colon carcinomas and other carcinomas of the gastrointestinal tract. However, other carcinomas (e.g., mucinous ovarian carcinoma or mucinous lung lepidic pattern carcinoma) can also be positive.	Cadherin-17 is also a good marker for intestinal neoplasms.
Chromo granin A	Acidic glycoprotein in neurosecretory granules ( Cytoplasm, granular)	Islet cells of pancreas, bronchial Kulchitsky cells, parathyroid, adrenal medulla, anterior pituitary, C-cells of thyroid	Pheochromocytoma, carcinoid (not rectal), small cell carcinoma, neuroblastoma, some breast and prostatic carcinomas, Merkel cell tumor, islet cell tumor, medullary carcinoma of the thyroid, parathyroid lesions, Brenner tumor	ID of neuroendocrine differentiation in tumors. Not present in pituitary prolactinomas. Pheochromocytoma (+) versus adrenal cortical carcinoma (−). Parathyroid (+) versus thyroid (−).	Most specific marker of neuroendocrine differentiation but also present in some breast and prostate carcinomas. Also can be detected in serum. Bouin solution or B5 fixation may increase immunogenicity.
Claudin-1 ( CLDN1)	Protein component of the tight junction complex ( Membrane—not cytoplasmic)	Epithelial cells, perineurial cells, some endothelial cells (venules)	Perineurioma (30%), synovial sarcoma (epithelioid areas, lower in spindle cell areas), carcinomas Some perineurial cells may be present in neurofibromas and schwannomas	Perineurioma (+ 30%) versus DFSP (−), fibromatosis (−), low-grade fibromyxoid sarcoma (−). Meningiomas (50% +)
Claudin-4	Tight junction protein ( Membrane)	Normal epithelial cells	Carcinomas	Carcinoma (+) versus mesothelioma (−).
Collagen IV	Major constituent of basement membranes (Basement membrane)	Mesangial cells within glomeruli, basement membranes, basal lamina of capillaries	Tumors with external lamina (schwannomas, smooth muscle tumors)	Absence or loss may be associated with stromal invasion by carcinomas.
Cytomegalovirus (CMV)	Viral inclusions	None		ID of CMV infections.
D2-40 ( podoplanin, M2A)	Oncofetal Membrane O-linked sialoglycoprotein (Membrane)	Lymphatic endothelium, germ cells of testis, interstitial cells of Cajal, follicular dendritic cells, some myoepithelial cells of breast	Lymphatic tumors, some angiosarcomas, some epithelioid hemangioendotheliomas, epithelioid mesotheliomas, seminomas, ITGCN, Kaposi sarcoma, GIST, ovarian serous carcinomas, some triple negative breast carcinomas	Identification of lymphovascular invasion. ID of seminoma (+, diffuse) versus embryonal carcinoma (− or focal) Epithelioid mesothelioma (+) versus adenocarcinoma (−). ID of follicular dendritic cell sarcoma.	Myoepithelial cells of the breast can show cytoplasmic positivity—limiting usefulness in the breast for LVI.
DDIT3 ( DNA damage-inducible transcript 3)	A proapoptotic transcription factor ( Nucleus)	Not present	Myxoid liposarcoma	ID of myxoid liposarcoma (~100%).	Myxoid liposarcomas have FUS-DDIT3 fusion (~90%) or EWSRI-DDIT3 fusion (~10%). Highly sensitive and specific.
Desmin	Intermediate filament in muscle ( Cytoplasm)	All striated muscle (Z bands) and many smooth muscle cells, myofibroblasts, smooth muscle of some blood vessels	Rhabdomyosarcoma (80% +), leiomyosarcoma (50–70% +), PEComa, desmoplastic small round cell tumors (usually dot-like), some myofibroblastic tumors, endometrial stromal sarcoma	ID of muscle differentiation in tumors (leiomyosarcoma, rhabdomyosarcoma, desmoplastic small round cell tumor, others).
DOG1 ( Discovered On GIST-1; ANO1)	Calcium-activated chloride channel anocatamin ( Cytoplasm or membrane)	Interstitial cells of Cajal, salivary gland serous acini, intercalated ducts	GIST (>95%; positivity in other tumor types is < 10%) Acinic cell carcinoma, adenoid cystic carcinoma (majority), epithelial/myoepithelial carcinomas (~50%)	ID of GIST (may be + in some CD117 neg GIST). More sensitive for gastric epithelioid GIST than CD117. Pos in 79% of GIST with PDGFRA mutations, whereas CD117 (+) in 9% of this group.	2% of GIST are negative for DOG1 and CD117.
E-cadherin	Transmembrane cell adhesion molecule that binds to catenins for cell polarization, glandular differentiation, and stratification (Membrane)	Epithelial cells	Most carcinomas—may be lost in poorly differentiated carcinomas	Ductal (+) versus lobular (−) lesions of the breast. Loss in signet ring cell carcinoma of stomach and other sites.	Can be helpful to distinguish DCIS from LCIS. Can also use ß-catenin and p120. Patients with germline mutations develop gastric signet ring cell carcinomas—rarely lobular carcinoma.
EGFR (epidermal growth factor receptor, HER1)	Transmembrane protein receptor of the type 1 growth factor family with tyrosine kinase activity (Membrane positivity scored, cytoplasmic positivity is not scored)	Many types of epithelium, skin eccrine and sebaceous glands, mesenchymal cells, perineurium. The strongest membrane positivity is present in hepatocytes, bile ducts, basilar squamous cells, pancreatic ducts, breast epithelium, lung alveolar lining cells, mesothelial cells, prostate epithelium, endometrial glands and stroma	Adenocarcinomas (especially colon), squamous cell carcinomas, urothelial cell carcinoma, neural tumors, sarcomas	Expression is increased in tumors of higher grade and poorer prognosis. Colon carcinomas (80–90% positive)—response to cetuximab is not related to IHC score. Lung adenocarcinoma—specific mutations (but not IHC) predict response to tyrosine kinase inhibitors GBM—40% show gene amplification and overexpression by IHC. ISH is preferred over IHC. Tyrosine kinase domain mutations are rare.
Epithelial membrane antigen ( EMA, MUC1, HMFG, DF3, CA 15-3, CA 27.29, PEM, many others)	Episialin, glycoprotein found in human milk fat globule membranes ( Cytoplasm (more common in malignant cells), membrane (more common in benign cells))	Epithelial cells, perineurial cells, meningeal cells, plasma cells, usually negative in non-neoplastic mesothelial cells	Carcinomas, mesotheliomas (thick membrane pattern), some sarcomas (synovial sarcoma, epithelioid sarcoma, leiomyosarcoma, some osteosarcomas), adenomatoid tumor, chordoma, perineurioma, neurofibroma, meningioma, desmoplastic small round cell tumor, Sertoli cell tumor Some anaplastic large-cell lymphomas (CD30 +), plasma cell neoplasms	ID of epithelial differentiation in tumors—however, keratin is more specific for this purpose. Synovial sarcoma (typically focal positivity) versus other sarcomas. Positivity on apical surface demonstrates the “inside out” pattern of invasive micropapillary breast carcinoma; this pattern is also seen with MUC1.	There are over 50 monoclonal antibodies recognizing different glycosylation patterns in normal tissues and tumors.
Epstein–Barr virus EBV-encoded nonpolyadenylated early RNAs ( EBERS )	RNA produced by EBV ( Nucleus)	EBV infected B cells	All EBV-related tumors	Most sensitive marker for EBV.	Detected by in situ hybridization for RNA on paraffin sections.
LMP-1	Latent membrane protein ( Membrane)	EBV infected B cells	Nasopharyngeal carcinomas, RS cells (not LP HD), transplant lymphomas, AIDS-related lymphomas, endemic Burkitt lymphoma (rare in sporadic cases)	Evaluation of EBV-related neoplasms.
EBNA 2 (nuclear antigen 2)	Nuclear protein ( Nucleus)	EBV infected B cells	Transplant-related lymphomas, AIDS-related lymphomas. Not present in Burkitt lymphoma, nasopharyngeal carcinomas, or HD.	Evaluation of transplant and AIDS-related lymphomas.
ERG ( Ets-related gene)	Member of the Ets family of transcription factors ( Nucleus)	Endothelial cells (blood vessels and lymphatics), early myelocytes, mature lymphocytes, fetal mesenchymal cells	Angiosarcoma (95%; most sensitive and specific marker), prostate carcinoma ~50%), subset of AML, Ewing sarcoma (5%)	Prostate cancer with TMPRSS2-ERG translocation (~50%). Myeloid sarcomas (majority) Epithelioid sarcoma (N terminus 68%, C terminus <5%). Ewing sarcoma ( EWSR1-ERG fusion gene)(5%) EWSR1-SMAD3 -rearranged fibroblastic tumor.	Results depend on antibody detecting N terminus or C terminus. N-terminus antibodies cross react with epithelioid sarcoma more than C-terminus antibodies.
Estrogen receptor ( ER, 1D5, SP1, 6F11, H222, others)	Steroid binding protein ( Nucleus)	Breast luminal cells (not myoepithelial cells), myofibroblasts, smooth muscle, epithelial and myometrial cells of the uterus	Breast carcinomas (> 70%), myofibroblastoma of breast, gynecologic carcinomas, some skin appendage tumors, rare in other carcinomas, present in some meningiomas, smooth muscle tumors, some melanomas, some thyroid tumors, desmoid tumors, vulvovaginal stromal tumor	Prognosis and prediction of response to endocrine therapy of breast cancer. Only nuclear positivity is scored. ID of estrogen receptor positive metastatic breast cancer.	Antibodies recognize different epitopes and have varying sensitivities in formalin-fixed tissue. Antigenicity may be diminished after decalcification or exposure to heat during surgery.
Factor VIII-related antigen ( VWF, FVIII:RAg, von Willebrand’s factor)	Glycoprotein involved in coagulation, part of FVIII complex ( Cytoplasm)	Endothelial cells, megakaryocytes, platelets, and mast cells, endocardium	Vascular tumors (but often absent in angiosarcomas) Not present in Kaposi sarcoma or PEComa Megakaryocytic AML (M7)	ID of endothelial differentiation in tumors (specific but not very sensitive). Evaluation of angiogenesis. Evaluation of megakaryocytic (M7) leukemias.	May not detect smaller blood vessels (see CD 31 and 34). Present in Weibel–Palade bodies. Not a sensitive marker for vascular neoplasms.
Factor XIIIa ( Factor XIII subunit A)	Transglutaminase involved in the coagulation pathway ( Cytoplasm)	Fibroblasts, dendritic reticulum cells in reactive follicles, dermal dendrocytes, liver, placenta, platelets, mega-karyocytes, monocytes, macrophages	Fibroblastic neoplasms, dermatofibroma		Not very specific.
Fascin ( p55)	Actin binding protein thought to be involved in the formation of microfilament bundles and cell motility ( Cytoplasm )	Interdigitating reticulum cells in lymph nodes, dendritic cells of lymph node, thymus, spleen and peripheral blood, histiocytes, smooth muscle, endothelial cells, squamous mucosal cells, lining cells of splenic sinuses, neurons	Reed-Sternberg cells and their variants (but not LP HD), rare non Hodgkin lymphomas Dendritic cell tumors Some sarcomas Many carcinomas, especially those of advanced stage Glial tumors (more common in high-grade tumors) Langerhans cell histocytosis		Not very specific.
Fibronectin	Glycoproteins found in BM’s and extracellular matrix, bind to integrins ( Extracellular)	Normal stroma	Stroma of many tumors
FLI-1 ( Friend leukemia integrin-site 1)	Transcription factor (ETS family)—translocation in Ewing sarcoma can result in an EWS-FLI-1 fusion protein ( Nucleus)	Endothelial cells (hemangioblasts, angioblasts), small lymphocytes	Ewing sarcoma, vascular tumors (including Kaposi sarcoma), Merkel cell carcinoma, melanoma Can also be weakly present in lymphomas, synovial sarcoma, some carcinomas	ID of vascular tumors. ID of Ewing sarcoma—not specific but very sensitive (70%).	Reactivity can be variable with high background and may be difficult to interpret; ERG is a better vascular marker.
FOS ( c-Fos)	Proto-oncogene transcription factor homologous to the viral oncogene v-fos ( Nucleus)	Epidermis, striated muscle, endothelial cells, pericytes, chondrocytes, reactive and proliferative bone forming lesions	Osteoid osteoma, osteoblastoma, pseudomyogenic epitheliod hemangioendothelioma (with FOS rearrangements), carcinomas	Differentiation of osteoid osteoma from other bone tumors.	Osteoid osteoma and osteoblastoma have rearrangements of FOS (~87%) and FOSB (~3%); immunoreactivity diminished after 3 days of acidic decalcification.
FOSB ( FosB, fosB)	Transcription factor that is part of the same complex as FOS ( Nucleus)		Pseudomyogenic hemangioendothelioma, epithelioid hemangioma, Hodgkin disease, some non-Hodgkin lymphoma, some carcinomas	ID of pseudomyogenic hemangioendothelioma (+ ~100%) (due to SERPINE1-FOSB or ACTB-FOSB fusion) and some epithelioid hemangiomas (+ ~50%) (due to FOSB gene fusions).
FOXJ1	Member of the forkhead gene family that regulates motile cilia in epithelial cells (Nucleus)	Fallopian tube, ependymal cells, ependymal circumventricular subcommissural organ of the posterior third ventricle, Rathke’s cleft cells	Endosalpingiosis, low grade and serous neoplasms, Rathke’s cleft cysts, ependymal neoplasms, papillary tumor of pineal region
FH ( fumarate hydratase)	Enzyme that converts fumarate into malate in the Krebs cycle ( Cytoplasmic)	Normally present	Fumarate hydratase (FH)-deficient tumors due to germline or sporadic mutations—FH (−) and 2SC (+)	ID of tumors associated with germline or sporadic FH mutations—some cases of papillary renal cell carcinoma and leiomyoma with bizarre nuclei.	Mutations in FH cause loss of function and an increase in fumarate. This results in increased succination of proteins such as 2SC. Germline FH mutations are associated with Hereditary Leiomyomatosis and Renal Cell Carcinoma (HLRCC) syndrome.
Galectin-3 ( Gal-3)	Lectin with antiapoptosis function (galactoside-binding protein) ( Nucleus, cytoplasm, membrane, extracellular matrix)	Many epithelial cells, lymphocytes, mesenchymal cells, macrophages, activated endothelial cells	Many carcinomas, adenomas, lymphomas, soft tissue tumors	Thyroid carcinomas (papillary and to a lesser extent follicular) show higher expression than benign lesions. In some carcinomas, expression is diminished in higher grade lesions.
GATA3	Transcription factor (Nucleus)	Epidermis, breast luminal cells, sebaceous glands, apocrine glands, parathyroid, some T cells	Breast, urothelial, and skin carcinomas are more commonly positive than other types Paraganglioma, pheochromocytoma, choriocarcinoma and gestational trophoblastic tumor, renal cell carcinoma, adnexal tumors, neuroblastoma, others	Breast cancer (67%), triple negative breast cancer (43%), metaplastic breast cancer (54%) versus other carcinomas. Squamous cell carcinoma of skin (+) versus lung (low). Lobular breast (+) versus gastric signet ring cell carcinoma (low). Urothelial carcinoma (+) versus prostate (low). Mesothelioma (+) versus lung adenocarcinoma (low).
Glial fibrillary acidic protein ( GFAP)	Intermediate filament ( Cytoplasm)	Normal and reactive astrocytes, developing and reactive ependymal cells, choroid plexus, Schwann cells, enteric glial cells, posterior pituitary cells, chondrocytes	Tumors of astrocytes, ependymal cells, and oligodendrocytes, MPNST, myoepitheliomas (salivary glands and soft tissue), sweat gland tumors, Merkel cell carcinomas, chordomas Some high-grade gliomas are negative	ID of neural differentiation in tumors (30% of MPNST’s are +). Neuroblastomas are negative, schwannomas may be focally +. Merkel cell carcinoma (+) versus small-cell carcinoma (−) (but CK20 is a better marker for this purpose). ID of soft tissue myoepitheliomas and myoepithelial carcinomas.
GLUT-1 (glucose transporter 1)	Component of transmembrane glucose transport (Membrane)	Erythrocytes, perineurium, blood vessels, trophoblasts, renal tubules, germinal center cells	Urothelial cell carcinoma, lung carcinoma, squamous cell carcinoma, adenocarcinomas of colon, lung, bile ducts, kidney, ovary, pancreas, stomach, and endometrium, germ cell tumors		Not very specific.
Glutamine synthetase ( GS)	Enzyme that plays an essential role in the metabolism of nitrogen ( Cytoplasm)	Centrilobular (zone 3) hepatocytes (perivenular or negative patterns)	Liver neoplasms, solid pseudo-papillary tumor of pancreas	ID of liver lesions: HCC—diffuse pattern Focal nodular hyperplasia—map-like pattern (patchy). Hepatic adenoma—perivenular pattern or negative.
Glypican-3 ( GPC3)	Heparan sulfate proteoglycan important for cell growth and differentiation ( Cytoplasm and membrane)	Fetal tissue—adult tissues are negative	HCC (84%), hepatoblastoma, yolk sac tumor, hepatoid adenocarcinoma, squamous cell carcinoma of the lung (55%)	Well-differentiated HCC (+) versus hepatic adenoma (−) HCC (+) versus cholangiocarcinoma or metastatic carcinoma (−).
Gross cystic disease fluid protein-15 ( GCDFP, CDP, BR-2, BRST-2)	Protein found in breast cyst fluid ( Cytoplasm)	Apocrine sweat glands, apocrine metaplasia of the breast	Breast carcinomas (60%), sweat gland carcinomas, some salivary gland tumors, some prostate carcinomas	ID of apocrine differentiation in tumors. ID of breast metastases (however, only positive in about 60%).
H3F3A ( K27M)	H3F3A and H3F3B are genes coding for the histone H3.3 protein that is mutated in some tumors ( Nucleus)	None	Pediatric high-grade gliomas arising in the midline (brainstem, thalamus, spinal cord, or rarely cerebrum), giant cell tumor		Missense mutations occur in H3F3A in multiple tumor types. The antibody detects the mutated protein (the mutation is K27M).
H3K27me3	H3K27 is the 27th amino acid in histone H3 and can undergo trimethylation and acts as a gene transcription repressor ( Nucleus)	Normal tissue, in women the accumulated protein on the inactive X chromosome forms an eccentric intranuclear dot	Many tumors	Loss of expression due to SUZ12 or EED mutations, leading to loss of PRC2 (methylating) function in MPNST (useful for diagnosis). Loss of expression in subsets of meningiomas, radiation-associated sarcomas, melanomas, Merkel cell carcinomas, others.	This study can be helpful if the staining for H3F3A(K27M) is difficult to interpret (high background or weak).
HepPar-1 (hepatocyte paraffin-1, HP1)	Recognizes carbamoyl phosphate synthetase 1, an essential component of the urea cycle ( Cytoplasm, coarsely granular)	Hepatocytes	HCC, some cases of gastric adenocarcinoma, esophageal adenocarcinoma, others negative or only rarely positive	HCC (80–95%) versus metastatic carcinomas to the liver.
HBME-1	Antigen to microvilli on mesothelioma cells ( Membrane and cytoplasm)	Mesothelial cells, epithelial cells	Mesotheliomas (epithelial type—thick, membrane staining), adenocarcinomas, chordomas, chondrosarcomas	Positivity higher in thyroid carcinomas than in adenomas. May be absent in thyroid carcinomas with Hurthle cell features.	Not a specific marker for mesotheliomas.
Hepatitis B virus	Viral proteins ( Nucleus or cytoplasm depending on antibody)	Absent in normal tissue		ID of hepatitis B infection.
HER2 ( HER2/neu, c-erbB2, A0485, Sp3)	Growth factor receptor (tyrosine kinase) homologous to epidermal growth factor receptor ( Membrane)	Present in normal cells but at low level not detected by immunohistochemistry	Breast carcinomas (20–30%), Paget disease of nipple (> 90%), less frequently in other carcinomas (lung, ovary, uterus, GI, pancreas), some synovial sarcomas	Predictive factor in breast carcinoma, colorectal carcinoma, and esophageal carcinoma for response to HER2 targeted therapy—there are different scoring criteria for HER2 positivity for each type of carcinoma. ID of Paget disease of nipple.	Only membrane positivity is scored. Gene amplification (detected by ISH) correlates with strong complete membrane immunoreactivity. Rarer HER2 mutations have not been reported to result in increased protein.
Herpes Virus 1 and 2 ( HSV)	Viral proteins ( Nucleus)	Absent in normal tissue		ID of viral infections.	The antibodies do not distinguish well between HPV 1 and 2.
HHV8 ( Kaposi sarcoma virus, KSHV)	Latent nuclear antigen of Human Herpes Virus type 8 ( Nucleus)	Absent in normal tissue	KS (endothelial cells and some perivascular cells) Primary effusion lymphoma (PEL), AIDS-associated multicentric Castleman’s disease	Evaluation of KS and PEL.
HMB-45 ( E-MEL, melanoma-specific antigen)	Oligosaccharide side-chain of a melanosomal antigen, gp100/pmel17 ( Cytoplasm)	Fetal melanocytes and some normal adult superficial melanocytes, retinal pigment epithelium	Melanoma (epithelioid but not spindle cell or desmoplastic type), clear cell sarcoma, PEComa, tumors associated with tuberous sclerosis, melanotic schwannoma, others	ID of metastatic melanoma. Melanophages can also be positive. Melan-A may be more specific. ID of PEComa.	NKI-betab detects the same protein. Tissues fixed in B5 may have high background staining.
HMGA2 ( high-mobility group AT-hook 2)	Nonhistone protein involved in DNA structure ( Nucleus)		Pleomorphic adenoma of salivary gland, pulmonary chondroid hamartoma, lipoma, uterine leiomyoma, soft tissue chondroma, extraskeletal osteochondroma, aggressive angiomyxoma		Numerous tumors have translocations with HMGA2 and a variety of partners resulting in increased expression.
Hormones (ER and PR are listed separately)	Insulin, gastrin, glucagon, somato-statin, calcitonin, ACTH, FSH, LH, PRL, TSH, others (Cytoplasm)	Hormone producing cells	Hormone producing tumors	ID of hormone products in tumors.	May not correlate well with serum levels of the same markers.
Human chorionic gonadotropin ( hCG, B-HCG)	Beta chain of the hormone ( Cytoplasm)	Syncytiotrophoblasts	Choriocarcinoma, giant cells in seminomas, placental site tumors (weak)	ID of trophoblastic differentiation in tumors.
Human placental lactogen ( HPL, hPL)	Hormone ( Cytoplasm)	Trophoblast	Choriocarcinoma (may be weak), complete moles (strong), partial moles (weak), some lung and stomach carcinomas	ID of trophoblastic differentiation in tumors.
IDH1 ( isocitrate dehydrogenase 1, IDH1-R132H)	Metabolic enzyme in the Kreb’s cycle ( Nucleus)	Absent in normal tissue	Low-grade gliomas or high-grade gliomas arising from progression of low-grade tumors (“secondary”). Rare in primary high-grade GBM. Present in 20% of cytogenetically normal AML.	Presence confirms origin as low-grade CNS neoplasm. Favorable prognostic factor—predicts response to chemotherapy.	The antibody detects a specific point mutation found in many gliomas.
IGF-2 ( insulin-like growth factor)	Polypeptide growth factor (Cytoplasm, nucleus—normal cells, juxtanuclear Golgi in cortical carcinomas)	Most normal cells		ID of adrenocortical carcinoma—juxtanuclear Golgi pattern (90%). Adrenal cortical adenomas show cytoplasmic positivity but not the abnormal pattern.	Alterations of the IGF2/H19 locus lead to abnormally large forms of IGF-2 in carcinomas that are seen in an abnormal pattern. Careful attention to antibody dilution and interpretation is critical.
Inhibin—alpha subunit	Hormone produced by ovarian granulosa cells and prostate, inhibits FSH production ( Cytoplasm)	Ovarian granulosa cells, Sertoli cells, pregnancy luteomas, ovarian follicles, syncytiotrophoblast, adrenal cortex, hepatocytes	Granulosa cell tumors, juvenile granulosa cell tumors, Sertoli and Leydig cell tumors, ovarian stromal cells around other tumors, hydatidiform moles, choriocarcinoma, thecofibroma, adrenal cortical tumor, granular cell tumor, hemangiopericytoma	ID of sex cord stromal differentiation in ovarian tumors. Distinguishes adrenal cortical tumors (>70% +) versus HCC (<5% +) and RCC (<5% +).
INSM1 ( insulinoma-associated protein 1, IA1)	Zinc finger transcription protein ( Nucleus)	Normal neuroendocrine tissues (adrenal medulla, pineal gland, pituitary gland, islet cells, C cells of thyroid, developing axons)	Carcinoid, medullary thyroid, medulloblastoma, Merkel cell, neuroblastoma, paraganglioma, pheochromocytoma, pituitary tumors, retinoblastoma, small cell carcinoma of lung, others	ID of neuroendocrine and neuroepithelial neoplasms. Can be positive in breast and prostate cancer without neuroendocrine features.	As sensitive and specific as chromogranin, synaptophysin, and CD56.
INI1 ( Integrase interactor 1; BAF47, hSnF5, SMARCB1)	Chromatin remodeling complex ( Nucleus)	All normal cells	Loss of expression can be helpful for diagnosis: Poorly differentiated chondrosarcoma, extraskeletal myxoid chondrosarcoma (17%), epithelioid MPNST, renal medullary carcinoma, myoepithelial carcinoma Deleted or mutated in pediatric rhabdoid tumors (tumors are negative) and CNS atypical teratoid/rhabdoid tumors	Lost in 90% of epithelioid sarcomas (conventional and proximal) versus epithelioid hemangioendothelioma (+). Lost in 50% of epithelioid MPNST. ID of rhabdoid tumors and atypical teratoid/rhabdoid tumors (−) versus choroid plexus carcinoma (+).	Germline INI1 mutations result in sporadic schwannomatosis (10% of cases) or rhabdoid tumors.
ISLET1	Pancreatic islet cell-specific transcription factor ( Nucleus)	Normal islet cells of pancreas	Pancreatic neuroendocrine tumors	Pancreatic NE tumors (+) versus midgut (small bowel) NE tumors (carcinoid, neg).	PAX8 is also helpful for this differential.
Keratins	Intermediate filaments ( Cytoplasm)	Epithelial cells	Carcinomas, mesotheliomas, desmoplastic small round cell tumors (dot-like pattern), thymomas, chordomas, synovial sarcomas, epithelioid sarcoma, leiomyosarcoma, trophoblastic tumors, some other sarcomas, rarely melanomas	ID of poorly differentiated carcinomas. Cytokeratins 7 and 20 can be used to help identify the site of origin of carcinomas.	Keratins are relatively resistant to destruction. Positivity can sometimes be identified in tissue that is necrotic or cauterized.
AE1/AE3	Two monoclonal antibodies. AE1 detects keratins 10, 15, 16, and 19; AE3 detects keratins 1–8. ( Cytoplasm)	Epithelial cells, mesothelial cells	Most carcinomas. The only common carcinomas that are frequently negative are HCC (70% negative) and RCC, clear cell type (20% negative), spindle cell carcinomas of breast Noncarcinomas that are positive include epithelioid hemangioendothelioma, epithelioid sarcoma, synovial sarcoma, mesothelioma, adenomatoid tumor, germ cell tumors	ID of epithelial differentiation in tumors. HCC (−/+) versus cholangiocarcinoma and metastatic carcinomas (+).	Good broad spectrum keratin.
CAM 5.2	Keratins 8 and 18 ( Cytoplasm)	Simple and glandular epithelium	Most carcinomas including those usually negative for Ck7 and 20: HCC, prostatic carcinoma, thymic carcinoma, gastric carcinoma, renal cell carcinoma, small-cell carcinoma Carcinoid tumor, thymoma, germ cell tumors, mesothelioma, dendritic cells Synovial sarcoma, epithelioid sarcoma Many squamous cell carcinomas are negative.	ID of carcinomas that may be negative for Ck7 and Ck20. Paget disease (+) versus squamous cell carcinoma (−).	May be positive when other keratins are negative. Positivity for dendritic cells in lymph nodes and elsewhere may be confused for micrometastases.
Keratin 5/6	Keratins 5 and 6 ( Cytoplasm)	Basal cells, stratum spinosum of epidermis, mesothelial cells	Squamous cell carcinomas, urothelial cell carcinoma, epithelioid mesotheliomas, squamous metaplasia in adenocarcinomas, thymic carcinoma Less frequent in nonsquamous carcinomas.	Epithelioid mesothelioma (+) versus pulmonary adeno (−). Present in some “triple negative” breast cancers—may identify a poor prognostic group. Usual ductal hyperplasia of breast (patchy positivity) versus ADH/DCIS (−).
Keratin 7	Keratin 7 ( Cytoplasm)	Simple epithelia, respiratory epithelium, transitional epithelium, endothelial cells of small veins and lymphatics Not present in squamous epithelium	Most adenocarcinomas of glandular epithelial origin, urothelial cell carcinoma, mesothelioma, neuroendocrine neoplasms Not Merkel cell carcinoma or colon carcinoma. Rare in clear cell RCC (but present in other variants), prostate carcinoma, HCC, lung small cell carcinoma, thymoma, carcinoid Not present in squamous cell carcinomas of the skin, but may be present in squamous cell carcinomas arising from nonkeratinizing epithelium (e.g., cervical carcinoma).	The combination of Ck7 and Ck20 is used to distinguish carcinomas arising at different sites (see tables).
Keratin 14	Keratin 14 ( Cytoplasm)	Squamous cells, myoepithelial cells	Squamous cell carcinomas, thymoma, myoepithelial neoplasms, oncocytic neoplasms (Hurthle cell adenoma of the thyroid), some triple negative (“basaloid”) breast cancers	ID of keratin in spindle cell breast carcinomas and other triple negative breast cancers.
Keratin 20	Keratin 20 ( Cytoplasm)	Gastric foveolar epithelium, intestinal villi and crypt epithelium, Merkel cells, taste buds, umbrella cells of urothelium, subsets of epithelial cells. Not present in nonepithelial cells.	Colon carcinoma, Merkel cell carcinoma, urothelial cell carcinoma, adenocarcinoma of the bladder, pancreatic carcinoma, cholangiocarcinoma, mucinous ovarian carcinoma, esophageal adenocarcinoma	Merkel cell carcinomas are CK20 positive, whereas most similar tumors are negative ID of metastatic colon carcinomas (the pattern of Ck7 negative, Ck20 positive, is most frequently seen in this carcinoma, but can rarely be seen in other types).
PAN-K ( MNF-116)	Broad spectrum detection of keratins including 5, 6, 8, 17, and 18 ( Cytoplasm)	Epithelial cells including simple epithelium and squamous cells		Detection of keratin in all carcinomas, including poorly differentiated carcinomas (especially spindle cell squamous cell carcinomas). May be more sensitive than AE1/AE3 for triple negative breast carcinomas with myoepithelial (“basal”) features due to inclusion of the “basal” keratin Ck17.
34ßE12 ( 903)	High molecular weight keratins including 1, 5, 10, 14 ( Cytoplasm)	Complex epithelia, basal cells, myoepithelial cells	Urothelial cell carcinoma, cholangiocarcinoma, squamous cell carcinoma, nonmucinous lepidic pattern lung carcinoma, RCC (papillary and chromophobe types), mesothelioma, papillary thyroid carcinoma, thymic carcinoma, lymphoepithelial carcinoma	Urothelial cell carcinoma (+) versus prostate carcinoma (−) or RCC (−). Prostate carcinoma (no basal cells) versus benign lesions (with some + basal cells present). Can be combined with p63 for this use. May be more sensitive than AE1/AE3 for triple negative breast carcinomas with myoepithelial (“basal”) features due to inclusion of the “basal” keratin Ck14. (Ck14 is also available separately).
Ki-67 ( MIB-1)	Protein found during the entire cell cycle but not in G0 and early G1 (Nucleus)	Any cycling cell	Any cycling tumor	Used to grade some tumors and dysplasias. Used for prognosis for some tumors. Detects number of cycling cells in Burkitt lymphoma and large B cell lymphoma. Aberrant membrane and cytoplasmic immunoreactivity is present in trabecular hyalinizing adenoma of the thyroid and sclerosing hemangioma of the lung.	MIB-1 recognizes an epitope preserved in formalin-fixed tissue.
Laminin	Component of basement membranes ( Basement membrane)	Basement membranes of normal tissues	Nerve sheath tumors, smooth muscle tumors	Loss associated with stromal invasion by carcinomas. Present surrounding tubules of microglandular adenosis of the breast.	Multiplex studies with keratin have been used to evaluate microinvasion.
LFABP ( liver-type fatty acid binding protein, FABP1)	Involved in metabolism of long chain fatty acids in the liver ( Cytoplasm)	Hepatocytes	Hepatocellular carcinoma (~75%), Fibrolamellar hepatocellular carcinoma (~50%), hepatocellular adenoma (~75%)	Loss of expression in HNF1A -inactivated hepatic adenomas and loss or partial loss in some cases of associated fibrolamellar hepatocellular carcinoma.
Lysozyme ( Ly)	Muramidase which is a mucolytic enzyme ( Cytoplasm)	Circulating monocytes, some tissue macrophages, granulocytes, salivary gland, lacrimal gland, stomach and colon epithelial cells (in flamed or regenerative), apocrine glands, Paneth cells, some other epithelial cells	Salivary gland tumors, stomach and colon carcinomas AML with monocytic differentiation,	Marker for histiocytes but not specific. May mark activated phagocytic macrophages. Evaluation of myeloid leukemias.	Not specific for identification of solid tumors.
MAC 387 ( L1 antigen, calprotectin, calgranulin, cystic fibrosis antigen)	Three polypeptide chains released with activation or death of neutrophils ( Cytoplasm)	Neutrophils, monocytes, some tissue macrophages, eosinophils, squamous mucosa, reactive skin, synovial lining cells	Lung carcinomas (not small cell or carcinoid), squamous cell carcinomas Histiocytic neoplasms (but not Langerhans cells)	Marker for macrophages (but not as specific as CD68).	Belongs to the S100 protein family. Cells can passively take up antigen resulting in false positive results.
Mamma globin ( MGB1)	Secretory glycoprotein ( Cytoplasm)	Breast epithelium, sweat glands, endocervix, endometrium	Breast cancer (50%), endometrioid adenocarcinoma (~40%), salivary gland carcinoma (~20%), melanoma (~6%)	ID of metastatic breast cancer (+ in about 50%).	More sensitive than GCDFP-15 but less specific.
MCPyV (Merkel cell virus)	Merkel cell polyoma virus large T antigen	Not seen in normal cells	Merkel cell carcinoma	ID of Merkel cell carcinoma (+ ~60%; more common in Northern hemisphere and less common in populations with high UV exposure) versus other high-grade neuroendocrine carcinomas.	The virus is integrated into the genome of Merkel cell carcinomas.
MDM2 ( murine double minute 2 homolog)	A ubiquitin protein ligase that regulates p53 stability ( Nucleus)	Not seen in normal cells	Liposarcomas (> 90%) and MPNST (40%), solitary fibrous tumor, myxofibroma, cardiac sarcoma, intimal sarcoma (majority), osteosarcoma	Atypical lipomatous tumor/well-differentiated liposarcoma and dedifferentiated liposarcoma (>90% +) versus benign adipose tumors (<5% +). Low-grade osteosarcoma (+) versus benign lesions (−). High-grade osteosarcomas are positive for MDM2 and CDK2 (low-grade osteosarcomas are CDK2 negative).	CDK4 has a similar pattern. Best used together as a panel. MDM is amplified in liposarcomas and can be detected by FISH.
MELAN-A or MART-1 (melanoma antigen recognized by T cells, A103, M2-7C10)	Melanocyte differentiation antigen ( Cytoplasm)	Antibody MC-7C10 is positive in melanocytes of skin, uvea, and retina Antibody A103 is also positive in adrenal cortex, granulosa and theca cells of the ovary, Leydig cells	Melanomas (but < 50% of spindle cell or desmoplastic melanomas), PEComas Antibody A103 is also positive in adrenocortical tumors, Leydig cell tumor, granulosa cell tumor. Antibody M2-7C10 is not positive in adrenal cortical carcinomas.	ID of melanomas. The antibodies are not positive in melanophages and may be more specific for the detection of micrometastases in lymph nodes. Antibody A103 distinguishes adrenocortical tumors (≥50% +) versus HCC (−) and RCC (−).	More sensitive than HMB978-0-323-54632-4. Peptides are used for melanoma immunotherapy. A103 has a broader spectrum of immunoreactivity than MC-7C10.
MITF (microphthalmia transcription factor, Mit-f, D5, MiTF)	Basic-helix-loop-helix-leucine zipper transcription factor that regulates tyrosinase and other melanogenic proteins (Nucleus)	Melanocytes, pigmented cells of the retina, mast cells, osteoclasts	Melanoma, PEComa, angiomyolipoma, clear cell sarcoma	Melanoma versus other tumors—not as specific as other melanoma markers (also present in other tumors).	Mutations result in autosomal dominant Waardenburg syndrome type 2a (hereditary deafness and skin hypopigmentation).
MLH1 (human mutS homologue 2) , MSH2 (human mutL homologue 1), MSH6, PMS2	Proteins involved in mismatch repair of DNA (the first two genes account for 95% of HNPCC) ( Nucleus)	Most normal tissues May be lost in areas of chronic inflammation	Expression (or nonexpression) is not specific for tumor type MLH1 and PMS2 loss: likely sporadic MSH2 and MSH6 loss: likely familial PMS2 loss: likely familial MSH6 loss: likely familial	Absence is associated with germline mutations in Lynch syndrome patients and with gene silencing by methylation in 10–15% of sporadic colon carcinomas—correlated with characteristic clinical, pathologic, and treatment response features. IHC will not detect the 5% of patients with mutations in other genes or rare patients with mutated gene products that are immunoreactive.	Other assays for microsatellite instability utilize PCR (90% sensitive for MSI).
MTAP ( methylthioadenosine phosphorylase)	Enzyme involved in adenosine monophosphate and methionine salvage ( Cytoplasm)	Many normal cells	Present in many tumors and lost in many tumors	Distinguish mesothelioma (loss in ~66%) versus benign pleural proliferations (present).	MTAP is often co-deleted with CDKN2A (coding for p16) which occurs in ~66% of mesotheliomas.
MUC1 and MUC2 ( mucin-1, mucin-2)	Mucins ( Cytoplasm or membrane)	MUC1 is typical of pancreaticobiliary-type differentiation and MUC2 of intestinal differentiation. MUC1 is found in normal breast but not MUC2.	Many adenocarcinomas	Identification of colonic metastases (expression more common than in lung or ovary). For pancreatic/ampullary tumors, MUC2 positive tumors may have better prognosis than MUC1 tumors. Cholangiocarcinoma (MUC1 80%) versus HCC (neg) IPMN is usually MUC2+/MUC1−, most PanIN are MUC2−/MUC1+, ductal adenocarcinoma is MUC1+ except colloid type which is MUC2+. Apical positivity identifies the “inside out” morphology seen in invasive micropapillary breast carcinoma.	MUC2 is a marker of intestinal cells—similar pattern as CDX2.
MUC4 ( mucin-4)	Transmembrane glycoprotein ( Cytoplasm, membrane in some epithelial cells)	Lung, colon, vagina, breast, others	Low-grade fibromyxoid sarcoma (99%), sclerosing epithelioid fibrosarcoma (70%) Overexpressed in many carcinomas including pancreas, bile duct, breast, colon, ovary, lung, and prostate	Low-grade fibromyxoid sarcoma (+) versus synovial sarcoma (epithelioid component), monophasic synovial sarcoma (30%, usually focal), epithelioid GIST (focal) versus other sarcomas (−; perineurioma, MPNST).
MYB	Member of the MYB (myeloblastosis) transcription factor family; important for hematopoiesis ( Nucleus)	Proliferating epithelial, endothelial, and hematopoietic cells, others	Adenoid cystic carcinomas of various sites, dermal cylindroma, some salivary gland neoplasms and some carcinomas (weak to moderate, focal) Leukemias and lymphomas	Adenoid cystic carcinoma (~50–80%) versus other tumor types (positivity ~15%).	Adenoid cystic carcinoma and dermal cylindromas often have a MYB-NFIB fusion resulting in increased expression.
MYC ( c-Myc)	Similar to the myelocytomatosis viral oncogene—a transcription factor (8q24) that activates many genes ( Nucleus)	Should not be present.	Breast cancer (80%), colon cancer (70%), GYN cancer (90%), lymphomas, many others	Postradiation cutaneous angiosarcoma (+) versus atypical vascular proliferation (−). Primary angiosarcomas are negative. Burkitt lymphoma and other high-grade B-cell lymphomas with MYC rearrangements.	Only detects c-Myc. Does not detect MYCN (N-Myc) located at 2p24.
Myf-4 ( MYF4, MRF4, myogenin)	Human homologue of myogenin—muscle regulatory protein ( Nucleus)	Striated muscle	Rhabdomyosarcoma	ID of skeletal muscle differentiation in tumors.	Better than MyoD1.
MyoD1	Nuclear phosphoprotein, role in myogenic regulation ( Nucleus)	Developing muscle tissues (myoblasts), adult muscle is negative	Rhabdomyosarcoma (especially spindle cell/sclerosing type), mixed Mullerian tumors	ID of skeletal muscle differentiation in tumors.	Older antibodies often showed cytoplasmic background; new antibody only shows expected nuclear staining.
Myoglobin	Oxygen binding protein ( Cytoplasm)	Striated muscle (including cardiac muscle), not smooth muscle	Tumors of striated muscle (rhabdomyosarcoma + 50%), but often negative in poorly differentiated tumors	ID of skeletal muscle differentiation in tumors.	More specific but less sensitive than actin and desmin.
Myosin—smooth muscle myosin heavy chain ( SM-MHC; SMMS-1, M3558)	Contractile protein in smooth muscle that interacts with actin ( Cytoplasm)	Visceral and vascular smooth muscle, myoepithelial cells of the breast	Tumors with myoepithelial cells	Marker for myoepithelial cells in the breast (less likely to be positive in vascular smooth muscle cells and myofibroblasts compared to other antibodies used to identify myoepithelial cells).	Antibodies to different isoforms will detect different types of muscle fibers.
Myosin—fast myosin (MY-32, M4276)	Contractile protein in skeletal muscle that interacts with actin ( Cytoplasm)	Striated muscle—Type 2 fibers (not present in cardiac or smooth muscle)	Rhabdomyosarcoma (some; especially pleomorphic variant)	ID of skeletal muscle differentiation in tumors.
NANOG	Embryonic stem cell transcription factor ( Nucleus)	Embryonic cells, normal duct cells of pancreas	Embryonal carcinoma, seminoma, CNS germinoma, transitional cell carcinoma, glioblastoma multiforme epithelioid type, ovarian serous carcinoma, oral squamous cell carcinoma	Seminoma and embryonal carcinoma versus other germ cell tumors. May detect “stem cells” in tumors.	Stronger and more diffusely positive compared to OCT3/4. Named after Tir Na Nog, the mythologic Celtic land of eternal youth.
Napsin A	Aspartic proteinase ( Cytoplasm)	Lung in type II pneumocytes (plays a key role in the maturation of surfactant protein B), alveolar macrophages, kidney tubular cells	Lung adenocarcinoma (~80%), renal cell carcinoma (~80% papillary carcinomas, ~35% clear cell carcinomas), tall cell variant of papillary thyroid carcinoma	Metastatic breast cancer (−) versus lung adenocarcinoma (70–80% +). Lung adenocarcinoma versus mesothelioma (−).
NB84	Uncharacterized antigen on neuroblastoma ( Cytoplasm and neuropil)	Placental trophoblast, colonic epithelia, pulmonary alveolar epithelium (granular), basal squamous cells	Neuroblastoma, Ewing sarcoma, medulloblastoma, desmoplastic small round cell tumor	Neuroblastoma versus Wilms tumor (−), rhabdomyosarcoma (−), and lymphoma (−).	Highly sensitive but not specific marker of neuroblastoma.
Nestin	Intermediate filament ( Cytoplasm)	Neural stem/progenitor cells, embryonic neural cells, neuronal and glial cells Retina, striated muscle, cardiac muscle, skin, liver, pancreas, kidneys, testes, adrenals	Neurocytomas, neuroblastomas, gliomas, glioblastomas, astrocytomas, ependymomas, medulloblastomas, Schwannomas Carcinomas, GIST, others
NeuN ( NEUronal Nuclei, A60)	DNA binding neuron-specific protein expressed at terminal differentiation ( Nucleus)	Neuronal cells including cerebellum, cerebral cortex, peripheral ganglion cells Absent in glia, pineocytes, Schwann cells	Central neurocytomas May be focally positive in other CNS neoplasms	ID of neuronal differentiation.
Neurofilaments ( 70 + 200 kD, NFP)	Intermediate filaments with three subunits ( Cytoplasm)	Neuronal cells, adrenal medulla	Tumors of neuronal origin or with neuronal differentiation, neuroblastoma, medulloblastoma, retinoblastoma, Ewing sarcoma, esthesioneuroblastoma, Merkel cell carcinoma, some endocrine tumors (carcinoids, pheochromocytomas)	ID of neuronal differentiation in tumors. ID of Merkel cell carcinomas. ID of axons in benign peripheral nerve sheath tumors.
Neuron-specific enolase ( NSE—do not confuse with the enzyme nonspecific esterase)	Gamma-gamma enolase isoenzyme ( Cytoplasm)	Neuroectodermal and neuroendocrine cells, more weakly striated and smooth muscle, megakaryocytes, T cells, some platelets, neurons, pituitary cells, hepatocytes	Neuroectodermal and neuroendocrine tumors, melanomas (including desmoplastic melanomas), many breast carcinomas, germ cell tumors, alveolar soft part sarcoma	ID of neuronal or neuroendocrine differentiation in tumors.	Lacks specificity.
NPM1	Nucleophosmin ( Nucleus)		Adult AML with NPM1 mutation (NPMc+AML: ~30%)—aberrant cytoplasm positivity	ALK+ anaplastic lymphoma with NPM1-ALK fusion gene has ALK positivity in nucleus and cytoplasm and aberrant NPM1 in cytoplasm. In contrast, ALK+ anaplastic lymphoma with variant translocations has ALK positivity in cytoplasm (or rare membrane) and NPM1 shows normal nuclear location.
NR4A3 ( nuclear receptor 4A3, NOR1)	Member of the nuclear receptor family of transcription factors ( Nucleus)		Acinic cell carcinoma of salivary gland		Acinic cell carcinoma is associated with a t(4;9)(q13;q31) that upregulates NR4A3. Highly sensitive and specific.
NUT ( nuclear protein in testis, C15orf55)	Unknown function ( Nucleus)	Germ cells in testis and ovary	NUT midline carcinomas (carcinomas with a translocation with the NUT gene)		These carcinomas have BRD4-NUT or BRD3-NUT fusion genes resulting in overexpression of NUT.
OCT4 ( OCT3, OCT3/4, POU5F1)	POU-domain transcription factor (POU5F1 gene) ( Nucleus)	Embryonic stem cells and pluripotent germ cells	Seminoma, intratubular germ cell neoplasia, embryonal carcinoma Absent in yolk sac tumors, spermatocytic seminoma, choriocarcinoma, and teratoma	ID of seminoma and embryonal carcinoma. Other epithelioid and round cell neoplasms are negative.	More specific than PLAP for this purpose.
OLIG2	Member of the basic helix-loop-helix transcription factor family ( Nucleus)	Oligodendrocytes	Diffuse glioma (100%), may be positive in other CNS tumors. Sparse positivity in ependymomas. T-ALL with OLIG2 translocation.	Primary CNS tumor (+) versus metastasis (−).
p16 (MTS1, CDKN2)	Binds to and inhibits the cyclin-dependent kinases cdk4 and cdk6 ( Cytoplasm and nucleus)	Absent	Cervical squamous cell carcinomas and adenocarcinomas (both in situ and invasive), endocervical carcinoma Some basaloid squamous cell carcinomas of the tonsil in young patients that are associated with HPV16.	Evaluation of cervical lesions. Possible use predicting tonsillar site for metastatic squamous cell carcinoma of the head and neck. Endocervical carcinoma (+) versus endometrial carcinoma (− or weak).	Overexpression is due to HPV-induced cell cycle dysregulation.
p40	Detects an isoform of p63 (deltaNp63) that is specific for squamous differentiation ( Nucleus)	Cells with squamous or myoepithelial features	Tumors with squamous or myoepithelial features	Squamous cell carcinoma of the lung (+) versus adenocarcinoma and other poorly differentiated malignancies.	p63 recognizes two isoforms—deltaNp63 and Tap63—and therefore is less specific for squamous differentiation compared to p40.
p53	Tumor suppressor gene product—probably most frequently mutated gene in malignancy ( Nucleus)	A normal pattern is scattered positive cells. Diffuse positivity or the absence of expression are abnormal	Many malignant tumors—but not specific for malignancy Fallopian tube intraepithelial carcinoma, triple negative breast carcinomas (common)	Overexpression may be used as a prognostic factor.	Different antibodies recognize different wild type and mutant forms of the protein and will give different results.
p57 ( kip2, p57 ^KIP2 )	Cyclin-dependent kinase inhibitor (CDKI) acting to inhibit cell proliferation, paternally imprinted ( Nucleus)	Cytotrophoblast, intermediate trophoblast, villous stromal cells, decidual stromal cells, absent in syncytiotrophoblast	Partial hydatidiform mole and hydropic abortion	Diploid complete moles show absent or low expression in cytotrophoblast and villous stromal cells (may be present in villous intermediate trophoblast and decidual stromal cells) versus partial moles and hydropic abortions that have normal expression.	Usually only transcribed from the maternal allele which is absent in a complete mole.
p63	Protein with at least six major isotypes, including deltaNp63, member of the p53 family ( Nucleus)	Proliferating basal cells of cervix, urothelium, prostate, and myoepithelial cells of breast, basal squamous cells, squamous metaplasia	Squamous cell carcinomas, Urothelial cell carcinoma, adenomyoepithelioma and adenoid cystic carcinoma (in the myoepithelial cell-like component), nasopharyngeal carcinoma, “basal type” breast carcinomas, papillary carcinoma of the thyroid, epithelioid angiosarcoma, others	ID of myoepithelial cells in breast lesions. ID of spindle cell breast carcinomas. Dx of prostatic carcinoma by showing absence of basal cells (more sensitive when combined with 34BE12). Basaloid squamous lung cancer (+) versus small cell (−). ID of metastatic poorly differentiated squamous cell carcinomas (p40 is more specific for this purpose).	Easier to interpret than SMA in breast lesions as myofibroblasts and vessels are negative; some tumor cells can be positive. P40 is more specific for squamous differentiation as it only recognizes the deltaNp63 isoform.
p120 ( p120 catenin)	E-cadherin binding protein ( Membrane)	Normal epithelial cells and some nonepithelial cells	Many tumors	Aberrant cytoplasmic staining is seen in tumors that lack E-cadherin expression including lobular lesions of the breast and signet ring cell carcinomas.	p120 connects E-cadherin to actin cytoskeleton and localizes to cytoplasm when E-cadherin is abnormal.
Pan-Trk ( Pan tropomyosin receptor kinase, neurotrophic Trk or NTRK)	Three member family of tyrosine kinases receptors ( Cytoplasm, Trk 3 may also be in nucleus)	Testis, muscle, nerve	Tumors with NTRK fusions: Secretory carcinoma of breast, mammary analog secretory carcinoma of salivary gland, infantile fibrosarcoma, cellular (some mixed) congenital mesoblastic nephroma, uterine sarcoma with features of fibrosarcoma	ID of tumors with NTRK fusions.	Pan-Trk recognizes the c-terminal of all 3 Trk proteins. Tumors with Trk fusions are eligible for treatment with Trk inhibitors. Antibody not entirely specific.
PAX2 ( paired-box 2)	Member of paired box (PAX) family of transcription factors (Nucleus)	Renal epithelium, fallopian tube, rete testis, seminal vesical, epithelial cells of endocervix and endometrium	Renal cell carcinoma, oncocytoma, nephrogenic adenoma, Mullerian tumors, cervix, medulloblastoma, others	Hemangioblastoma (rarely positive) versus metastatic RCC (almost all positive).
PAX3 ( paired-box 3)	Member of paired box (PAX) family of transcription factors (Nucleus)	Expressed during development	Biophenotypic sinonasal sarcoma (BSS), alveolar rhabdomyosarcoma (~60%), melanoma, medulloblastoma, glioblastoma, others	ID of BSS versus other spindle cell tumors of the nasal cavity.	BSS has PAX3 gene rearrangements resulting in overexpression. Sensitive and specific.
PAX8 ( paired-box 8)	Member of paired box (PAX) family of transcription factors (Nucleus)	Renal tubules, epithelial cells of fallopian tube, normal endometrium, lymphocytes, thyroid follicles	Thyroid carcinomas, including anaplastic carcinoma Gynecologic carcinomas (especially high-grade serous carcinomas) Nephroblastoma, renal cell carcinoma	ID of anaplastic thyroid carcinoma (positive in 79%). ID of gynecologic carcinomas. Hemangioblastoma (neg) versus metastatic RCC (positive).
PD-L1 ( programmed death ligand 1, PDL1)	Checkpoint protein expressed in activated immune cells and tumor cells ( Membrane and cytoplasm)	Can be present in stroma and inflammatory cells	Can be present in stroma, inflammatory cells, and/or tumor cells	Used to select patients for treatment trials.	Eligibility for treatment with different agents requires evaluation using a specific antibody using site specific criteria.
PDGFRA ( platelet-derived growth factor receptor A)	Receptor tyrosine kinase ( Cytoplasm)		Gastrointestinal stromal tumor (GIST) (~10%), monophasic synovial sarcoma (~50%), inflammatory fibroid polyps (~50%), inflammatory myofibroblastic tumor (~50%), plexiform fibromyxomas (~25%)	ID of GIST with PDGFRA mutations (~10% of GIST).	GIST with PDGFRA D842V mutations are resistant to conventional therapy. Patients are eligible for other treatment. IHC is highly sensitive and moderately specific for detecting these tumors.
PDX1	Pancreatic and duodenal homeobox 1 ( Nucleus)	Normal gastric glands	NE tumors of pancreas (often), gastric (some), duodenal (some), midgut (rare), lung (rare) Pancreatic acinar cell carcinoma or ductal adenocarcinoma (variable)	ID of metastatic well-differentiated NE tumors of pancreas (+) versus NE tumors of midgut (rare) or lung (rare).
PHOX2B	Transcription factor for autonomic nerve differentiation ( Nucleus)	Enteroendocrine cells	Pheochromocytoma (more common in malignant tumors), pheochromocytoma, neuroblastoma, ganglioneuroblastama		Best marker for neuroblastoma.
Placental alkaline phosphatase ( PLAP)	Alkaline phosphatase secreted by trophoblast ( Cytoplasm)	Placenta (trophoblast)	Germ cell tumors (but not spermatocytic tumor or immature teratomas), intratubular germ cell neoplasia, dysgerminoma, germinoma, partial moles, some carcinomas of breast, ovary, lung, stomach, and pancreas, some rhabdomyosarcomas (especially alveolar type)	Sensitive for ID of germ cell tumors but not specific Seminoma (+) versus spermatocytic tumor (−). ID of intratubular germ cell neoplasia.
PLAG1 ( pleomorphic adenoma gene 1)	Zinc finger protein ( Nucleus)		Pleomorphic adenoma of salivary gland, lipoblastoma, skin and soft tissue adenomyoepithelioma (especially with ductal differentiation)		Detects products of P LAG1 gene rearrangements resulting in high expression.
Progesterone receptor ( PR, PgR, PgR636)	Steroid binding protein ( Nucleus)	Normal breast epithelial cells, endometrial cells, many smooth muscle cells, breast lobular stroma	Breast carcinomas, gynecologic carcinomas, some skin adnexal tumors, secretory meningiomas, endometrial stromal sarcomas, some leiomyomas, myofibroblastic tumors, rarely other tumors.	Evaluated for prognosis, staging, and treatment of breast cancer. ID of PR positive metastatic breast cancer.
Prealbumin ( Trans thyretin, TTR)	Plasma transport protein for retinol and thyroxin ( Cytoplasm)	Pancreatic islet cells, choroid plexus, retinal pigment epithelium, liver	Pancreatic islet cell tumors, carcinoid tumors, choroid plexus papillomas, choroid plexus carcinomas (may be focal or absent)	ID of choroid plexus neoplasms. Evaluation of some forms of amyloidosis.	Major subunit protein in some forms of inherited systemic amyloidosis.
Prostate specific antigen ( PSA)	Member of kallikrein family of serine protease isolated from human seminal plasma ( Cytoplasm)	Normal prostatic epithelium, urachal remnants, endometrium, transitional cells of bladder	Prostatic carcinomas, some breast carcinomas.	ID of prostatic carcinomas (may be lost in some poorly differentiated carcinomas). Seminal vesicle carcinomas are negative.	More specific than PAP. Used as a serum screening test for prostate cancer.
Prostatic acid phosphatase ( PrAP, PAP)	Isoenzyme of acid phosphatase produced by prostate ( Cytoplasm)	Normal prostatic epithelium, periurethral glands, anal glands, macrophages	Prostatic carcinomas, urothelial cell carcinoma, rectal carcinoids	ID of prostatic carcinomas (may be lost in some poorly differentiated carcinomas).
RB ( retinoblastoma gene)	RB protein is a tumor suppressor that inhibits progression through the cell cycle (Nucleus)	Normal cells	Present in many tumors	Retinoblastoma (lost) due to germline mutation in 40%. Myofibroblastoma, spindle cell/pleomorphic lipoma, and cellular angiofibroma (lost) due to deletion.	Rb can be inactivated by many different mechanisms in many tumors.
RCC ( Renal cell carcinoma marker, gp200)	Glycoprotein on surface of renal tubules, breast epithelial cells, epididymis ( Cytoplasm, membrane)	Renal tubules, breast, epididymis	Clear cell and papillary RCC, breast carcinoma, embryonal carcinoma, salivary acinar cell tumor, parathyroid adenoma, pituitary adenoma, squamous cell carcinomas, others	Clear cell and papillary RCC (+) versus chromophobe carcinoma (−/+) and oncocytoma (−).
RET ( Rearranged during transfection)	RET-proto-oncogene—Surface glycoprotein of the receptor tyrosine kinase family ( Cytoplasm)	Neurons, embryonic kidney	Papillary thyroid carcinomas (78%), follicular variant of papillary carcinoma (63%), Hurthle cell carcinoma (57%), insular carcinoma (50%), medullary carcinoma, not present in follicular carcinomas or benign lesions Neuroblastoma, pheochromocytoma	Evaluation of thyroid tumors.	Germline mutations occur in MEN2A and 2B (10q11.2), familial medullary thyroid carcinoma, and some cases of Hirschsprung’s disease.
ROS1 ( D4D6)	ROS1 gene rearrangement product ( Cytoplasm)	Reactive pneumocytes, giant cells, macrophages		Routinely tested in lung adenocarcinomas to detect ROS1 gene rearrangements in 1–2% (100% sensitive and 98% specific for the gene rearrangement). Rearrangement in 5% of inflammatory myofibroblastic tumors.	ROS1 gene rearrangements in 1–2% of lung adenocarcinomas. May predict response to tyrosine kinase inhibitor crizotinib.
S-100 protein	Calcium binding protein isolated from the CNS (member of the EF hand family) ( Nucleus and cytoplasm)	Glial and Schwann cells, melanocytes, chondrocytes, adipocytes, myoepithelial cells, Langerhans cells, macrophages, reticulum cells of lymph nodes, eccrine glands, others	Melanoma (including spindle cell and desmoplastic types), clear cell sarcoma, schwannoma, chordoma, ependymoma, astroglioma, Langerhans proliferative disorders, some carcinomas (e.g., breast, ovary endometrial, thyroid), granular cell tumor, histiocytic sarcoma, myoepithelioma	ID of melanoma (if negative, melanoma is highly unlikely). ID of Langerhans proliferative disorders. Sustentacular cells in pheochromocytomas (loss may be poor prognostic factor). ID of neural tumors. ID of cellular schwannomas (more strongly and diffusely positive than in MPNST).	Langerhans cells and macrophages in tumors may be misinterpreted as positivity in the tumor itself. S100 is very soluble and may be eluted from frozen tissues.
SALL4	Embryonic stem cell transcription factor ( Nucleus)	Spermatogonia, primary spermatocytes	Yolk sac tumors, intratubular germ cell neoplasia, embryonal carcinoma, seminoma, spermatocytic seminoma, and a subset of teratomas and choriocarcinomas	ID of germ cell tumors—however a few carcinomas can also be positive.
SARS-CoV2 spike protein	Viral protein	None		ID of tissue from patients in acute phase of infection (hyaline membranes, alveolar macrophages, pneumocytes, endothelium).	Specificity is a problem with some antibodies
SATB2 (special AT-rich sequence binding protein)	SATB2 homeobox 2, encodes a DNA binding protein involved in transcriptional regulation and chromatin remodeling ( Nucleus)	Lower GI tract, ductal epithelium of testis and epididymis, some neurons, some lymphoid cells	Colorectal tumors, tumors with osteoblastic differentiation, renal and urothelial tumors	Metastatic colon carcinoma versus metastatic carcinoma of other sites. Confirmation of osteoblastic differentiation.
SDHB ( succinate dehydrogenase iron-sulfur subunit, mitochondrial)	Mitochondrial protein that catalyzes the oxidation of succinate (Cytoplasm- granular)	Parotid gland	Warthin’s tumor, sporadic pheochromocytoma, paraganglioma, and GIST	Pheochromocytomas and paragangliomas related to SDHB mutations are negative. If a GIST is negative, a germline SDHx mutation may be present in ~12%.	SDHB is a nuclear gene at 1 p36.1-p35. Germline mutations cause familial paraganglioma/pheochromocytoma and patients may develop GIST. Mutations also occur in SDHC and SDHD.
SMAD4 (DPC4 - homozygously deleted in pancreatic carcinoma, locus 4)	Transcriptional regulator interacting with the TGFbeta signaling pathway ( Nucleus)	Normal tissues	Expressed in most carcinomas	Mucinous ovarian carcinoma (+) versus metastatic pancreatic carcinoma (neg in 55%). Loss can help identify pancreatic carcinoma (~55%) and PAN IN-3 (~30%).	Mutated in familial juvenile polyposis in 25–60% of cases.
SMARCE1 ( SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily E member 1)	Part of the chromatin modeling complex SWI/SNF ( Nucleus and cytoplasm )	Present in normal cells	Present in many tumors	Lost in clear cell meningioma in ~50%.	Germline mutations in SMARCE1 predispose to clear cell meningioma
Smoothelin	Smooth muscle-specific cytoskeletal protein ( Cytoplasm)	Fully differentiated smooth muscle cells (not present in noncontractile smooth muscle cells or myofibroblasts), weak in perivascular smooth muscle	Some smooth muscle tumors Aberrant nuclear staining can be seen in some leiomyosarcomas and GISTs	May distinguish muscularis propria (+, strong, diffuse) versus muscularis mucosae (− or weak and focal) in urinary bladder biopsies in order to determine depth of invasion.
Somatostatin receptor 2a ( SSTR2A)	Receptor for somatostatin ( Membrane, cytoplasm)	Many normal cells including arachnoid cap cells (meningothelial cells)	NE tumors, meningiomas, GIST, olfactory neuroblastoma, paraganglioma, pheochromocytoma, others	Meningioma (+) versus rhabdoid meningiomas (−) versus other tumors.
SOX2 ( SRY [sex determining region Y]-box 2)	Member of SRY-related HMG-box family of transcription factors—embryonic stem cell transcription factor ( Nucleus)	Fetal CNS	Embryonal carcinoma, breast carcinoma, pancreatic carcinoma	Embryonal carcinoma (+) versus seminoma (−).
SOX10 ( SRY [sex determining region Y]-box 10)	Member of SRY-related HMG-box family of transcription factors involved in neural crest and peripheral nervous system development ( Nucleus)	Melanocytes, Schwann cells, myoepithelial cells	Melanoma, clear cell sarcoma, nerve sheath tumors, sustentacular cells of pheochromocytoma/paraganglioma, carcinoid tumor (50%), triple negative breast carcinoma	ID of neuroectodermal neoplasms including melanoma and PNST. ID of metastatic triple negative breast carcinoma.
Spirochete	Bacterial protein (Cytoplasm)	None	None	Recognizes Treponema pallidum (syphilis), Borrelia burgdorferi (Lyme), and Brachyspira species (intestinal spirochetosis).
SS18-SSX	Member of the SWI/SNF chromatin remodeling complex ( Nucleus)		Synovial sarcoma (product of t(X;18) resulting in SS18-SSX fusion protein)	ID of synovial sarcoma.	Highly sensitive and specific for synovial sarcoma. The SSX antibody (to a conserved sequence of the C terminus) is highly sensitive but less specific.
STAT6 ( signal transducer and activator of transcription 6)	Transcription factor that moves a receptor complex to the nucleus to activate gene expression (Nucleus)	Low levels can be seen in the nucleus and cytoplasm of scars and adipose tissue	Solitary fibrous tumor (due to the pathognomonic NAB2-STAT6 fusion), subset of dedifferentiated liposarcoma (due to STAT6 amplification), Reed-Sternberg cells in Hodgkin lymphoma, some lymphomas	ID of solitary fibrous tumor.	STAT6 is a very sensitive and specific marker for SFT.
Synaptophysin	Transmembrane glycoprotein found in presynaptic vesicles ( Cytoplasm)	Neuroectodermal and neuroendocrine cells, neurons	Medulloblastoma, neuroblastoma, pheochromocytoma, paragangliomas, carcinoids, small-cell carcinoma, medullary carcinoma of the thyroid, neural neoplasms, pancreatic islet cell tumors	ID of neuroendocrine differentiation in tumors. ID of neuronal differentiation in CNS tumors.
Synuclein-1	Neuron-specific protein associated with synaptosomes ( Lewy bodies)	Brain	Present in Lewy bodies (Lewy body dementia and Parkinson’s disease)
Tau	Microtubule-associated protein ( Cytoplasm, extracellular)	Normal neuronal cell bodies and dendrites, neuropil, glial cells	Abnormal amounts in Alzheimer’s disease in neurofibrillary tangles and senile plaques	Evaluation of Alzheimer’s disease, Pick’s disease, supranuclear palsy corticobasal degeneration, others.
TLE1 ( transducin-like enhancer of split)	Transcriptional repressor essential in hematopoiesis as well as epithelial and neuronal differentiation ( Nucleus)	Basal keratinocytes, adipocytes, perineurial cells, endothelial cells, mesothelial cells	Synovial sarcoma (90%), neurofibromas (33%), schwannomas (100%), MPNST (30%)	Synovial sarcoma (+) versus other tumors.	TLE1 protein expression correlates well with t(X;18) in synovial sarcoma.
TFE3	Transcription factor ( Nucleus)	Not reported	Alveolar soft part sarcoma and MiT family translocation-associated children and young adults, epithelioid hemangioendothelioma	Other tumors and normal adult tissues are negative.	These carcinomas have translocations involving the TFE3 gene resulting in its overexpression.
Thyroglobulin	Glycoprotein produced by thyroid follicular cells ( Cytoplasm)	Thyroid follicles	Thyroid carcinomas (papillary, follicular, and Hurthle cell types, rarely present in medullary carcinomas)	ID of metastatic thyroid carcinoma.	Thyroglobulin can diffuse into metastatic tumors to the thyroid.
TTF-1 ( thyroid transcription factor 1)	Transcription factor for thyroglobulin, thyroid peroxidase, Clara cell secretory protein, and surfactant proteins ( Nucleus; aberrant cytoplasm positivity in HCC)	Thyroid, lung, and some brain tissues	Thyroid carcinomas (including medullary carcinoma; may be negative in anaplastic carcinoma), lung adenocarcinomas (75%—but lower in mucinous lepidic pattern carcinomas), small cell carcinoma of lung (> 90%), HCC (cytoplasmic), absent or focal in most other adenocarcinomas	Mesothelioma (−) versus adenocarcinoma (+/−). Lung adenocarcinoma (+/−) versus metastatic breast carcinoma (−). Small cell carcinoma of lung (+) versus metastasis from other sites. (−), but some extrapulmonary small cell carcinomas can also be (+). HCC (cytoplasmic 71%), rare in other tumor types.	The detection of cytoplasmic TTF-1 may depend on the specific antibody used and the antigen-retrieval method.
Tyrosinase	Melanogenic protein ( Cytoplasm)	Melanocytes		Melanoma versus other tumors (sensitivity similar to MART-1 and HMB-45).
Ulex ( Ulex Europaeus I lectin, UEA 1)	Lectin, fucose residues on blood group H ( Cytoplasm)	Endothelial cells	Vascular tumors, some carcinomas	Evaluation of angiogenesis.	Not very specific.
Uroplakin II ( UPK2)	Transmembrane protein that may function in permeability or stability of the membrane ( Membrane)	Apical surface of umbrella cells	Transitional cell carcinomas		More sensitive than uroplakin III; lower positivity in plasmacytoid and micropapillary subtype.
Uroplakin III ( UPK3a)	Transmembrane protein that may function in permeability or stability of the membrane ( Membrane)	Apical surface of umbrella cells	Transitional cell carcinomas
YAP1 ( yes-associated protein 1, YAP, YAP65)	Transcriptional regulator of cell proliferation ( Nucleus and cytoplasm)	Present in normal cells	Present in many tumors	Absent in poroma (~80%) and porocarcinomas (~60%) with YAP1 fusions. Absent in rare epithelioid hemangioendotheliomas with YAP1-TFE3 translocation.
Varicella zoster ( VZV)	Viral inclusions			ID of infections.
Vimentin	Intermediate filament ( Cytoplasm )	Mesenchymal cells, fibroblasts, endothelial cells, chondrocytes, histiocytes, lymphocytes, many glial cells, myoepithelial cells, smooth muscle	All mesenchymal tumors, neural tumors, melanomas, meningiomas, chordoma, Leydig cell tumor, granulosa cell tumor, Sertoli cell tumor, adrenal cortical adenoma May be co-expressed with keratin in carcinomas of endometrium, thyroid, kidney (clear cell), adrenal cortex, lung, salivary gland, ovary, and liver		Can be used as an internal control for immunogenicity. Not a specific marker for tumor type or line of differentiation.
WT1 ( Wilms’ tumor 1 protein)	Zinc finger transcription factor ( Cytoplasm, nucleus)	Sertoli cells, decidual cells of uterus, granulosa cells of ovary, blood vessels, myelocytic cells, mesothelial cells	Wilms tumors (epithelial and blastemal components), epithelial mesotheliomas (nuclei—80–90%), acute leukemia (nuclei), adenocarcinomas (cytoplasmic; especially breast (mucinous breast cancers can have nuclear positivity (64%), ovary), desmoplastic small-cell tumors (nuclear and cytoplasmic), rhabdomyosarcoma	Mesothelioma (+, nuclear) versus adenocarcinoma (adenocarcinoma usually negative for nuclear positivity except for ovarian)—use mouse monoclonal antibody.	Gene located on 11p13 and is inactivated in 5–10% of sporadic Wilms’ tumors and nearly 100% of Denys–Drash syndrome patients. ABs detect epitopes at different ends of the protein and may give different results. Not very specific.
HEMATOPATHOLOGY MARKERS
ALK protein ( Anaplastic lymphoma kinase, ALK, p80, CD246)	The ALK gene (2p23) (a tyrosine kinase receptor) is translocated to part of the nucleophosmin (NPM1) gene (5q35) to form the fusion protein p80 and is overexpressed ( Cytoplasm, nucleus)	Nervous system, T cells	Anaplastic (CD30+) large cell lymphomas (about one third have ALK-NPM1 fusions). ALK negative anaplastic lymphomas may have trisomy 2. Some inflammatory myofibroblastic tumors	ID of anaplastic large-cell lymphomas.	ALK fusion genes with different partners are seen in lymphomas, lung cancers and others. The pattern of immunoreactivity varies with the translocation present.
Alpha-1-antichymo-trypsin ( ACH)	Serine protease inhibitor ( Cytoplasm)	Histiocytes, granulocytes, others	Histiocytic tumors, many adenocarcinomas, melanomas, many sarcomas.	Marker for histiocytes but CD68 is more specific.	Not specific for tumor type.
Alpha-1-antitrypsin ( AAT, alpha-1-AT)	Glycoprotein synthesized in the liver that inhibits proteolytic enzymes (especially elastase) ( Cytoplasm)	Histiocytes, reticulum cells, mast cells, Paneth cells, salivary gland	HCC, germ cell tumors, histiocytic neoplasms, colon and lung carcinomas, others	Accumulates in liver cells in AAT deficiency.	Not specific for tumor type. CD68 is a more specific marker for macrophages.
Bcl-2	Protein involved in inhibition of apoptosis ( Membrane, cytoplasm)	Medullary lymphocytes and epithelial cells of the normal thymus, mantle and T zone small lymphocytes	CLL, mantle cell lymphoma, follicular center cell (FCC) lymphoma, marginal zone lymphoma Synovial sarcoma, other soft tissue tumors	FCC lymphomas (+) versus reactive follicles (−). Hyperplastic marginal zones of the spleen, abdominal LN’s, and ilial lymphoid tissue are (+). Malignant thymomas may have greater reactivity than other thymomas. Synovial sarcoma is more frequently positive compared to mesothelioma.	Involved in the t(14;18) found in 90% of FCC lymphomas. Not specific for ID of solid tumors.
Bcl-6	Proto-oncogene—Kruppel-type zinc finger protein with homology to transcription factors ( Nucleus)	Normal germinal center B cells	Follicular lymphomas, diffuse large B-cell lymphomas, Burkitt lymphoma, mediastinal large B cell lymphoma, LP HD Not present in B-CLL, hairy cell leukemia, mantle cell lymphoma, and marginal zone lymphomas	Evaluation of B cell lymphomas.	Involved in gene rearrangements at 3q27 in lymphomas.
Blood group antigens	A, B, and H antigens ( Membrane)	Epithelial cells, endothelial cells, erythrocytes	Abnormally expressed or lost in many carcinomas	Have been used to identify specimens—however, DNA testing is preferred.	H is diminished by decalcification but not A and B antigens.
BOB.1 ( B-cell Oct-binding protein 1)	Coactivator that interacts with Oct transcription factors in B cells ( Cytoplasm)	B cells (including plasma cells)	B cell lymphomas and leukemias Reed-Sternberg cells in LP HD, usually absent in other HD types	Evaluation of HD.	BOB.1 and Oct2 are necessary (but not sufficient) for Ig expression.
BSAP ( B-cell-specific activator protein, PAX-5)	Transcription factor encoded by the Pax-5 gene that regulates B-lineage-specific genes ( Nucleus)	B cells	All B cell neoplasms and HD	Merkel cell tumors and pulmonary small-cell carcinomas have been reported to be positive.	Not reliable in Zenker’s-fixed tissue.
CD1a ( T6)	Membrane glycoprotein ( Membrane)	Cortical thymocytes (immature T cells), Langerhans cells, dendritic cells	Langerhans proliferative disorders, lymphoblastic lymphoma	Evaluation of Langerhans proliferative disorders. Evaluation of lymphoblastic lymphoma.
CD2 ( TE, T11, rT3, Leu 5a + b, LFA-2)	Glycoprotein mediating adhesion of activated T cells and thymocytes with antigen presenting cells and target cells, functions in E rosette formation ( Membrane)	T cells, NK cells, cortical thymocytes	T cell neoplasms, may be aberrantly lost in peripheral T cell neoplasms	Pan T cell marker.
CD3 ( T3)	C3 antigen (five polypeptide chains) ( Membrane, cytoplasm)	T cells, cortical thymocytes	T cell neoplasms, may be aberrantly lost in peripheral T cell neoplasms Anaplastic large-cell lymphoma is often negative.	Best pan T cell marker.	In paraffin sections, NK cells may also be positive.
CD4 ( TH, T4, Leu 3)	Transmembrane glycoprotein, HIV receptor ( Membrane)	T helper / inducer cells, macrophages, Langerhans cells	MF, other T cell neoplasms	Evaluation of MF. Evaluation of T cell neoplasms.
CD5 ( Leu 1)	Transmembrane glycoprotein ( Membrane)	T cells and B cell subsets (mantle zone)	T cell leukemias and lymphomas, aberrantly expressed in low-grade B cell lymphomas (CLL or mantle cell lymphoma). Thymic carcinoma, adenocarcinomas, mesothelioma (cytoplasmic)	Classification of low-grade B cell lymphomas. Evaluation of T cell lymphomas (this marker is frequently lost). Thymic carcinoma (~40%) versus thymoma (< 10%) versus pulmonary squamous cell carcinoma (< 5%).
CD7 ( Leu 9)	Membrane bound glycoprotein ( Membrane)	Precursor T cells, T cell subsets, NK cells, thymocytes	T cell lymphomas and leukemias	Frequently (50%) lost in T cell lymphomas versus reactive T cells (+). Evaluation of T cell leukemias.
CD8 ( T8, Leu 2)	Two glycoprotein chains ( Membrane)	T cell subsets, NK cells, T cytotoxic/suppressor cells	T cell lymphomas and leukemias	Evaluation of MF and T cell lymphomas (this marker is frequently lost).
CD10 ( CALLA [common acute leukemia antigen], J5, neprilysin)	Cell surface metalloendopeptidase that inactivates peptides ( Membrane)	Precursor B cells, granulocytes, rare cells in reactive follicles, myoepithelial cells of breast, bile canaliculi, fibroblasts, brush border of kidney and gut	Follicular lymphomas, pre-B-ALL, Burkitt lymphoma, CML, angioimmunoblastic lymphoma RCC (clear cell and papillary), HCC, rhabdomyosarcoma, endometrial stromal sarcoma	Evaluation of follicular center cell lymphomas. Evaluation of leukemias. Myoepithelial cell marker in breast. Endometrial stromal sarcoma (+) versus leiomyosarcoma (−) (but caldesmon is preferred for this purpose).
CD11b ( Mac-1)	Cell surface receptor for the C3bi complement fragment ( Membrane)	Granulocytes, monocytes, macrophages	Myelomonocytic leukemias
CD11c	Member of the beta(2) integrin family that mediates adhesion to vascular endothelium, transendothelial migration, chemotaxis, and phagocytosis ( Membrane)	Myeloid cells, NK cells, dendritic cells, activated lymphoid cells	Hairy cell leukemia, B-cell prolymphocytic leukemia, some B-CLL, marginal zone lymphoma (MALT)
CD13 ( My 7)	Aminopeptidase-N, a type II integral membrane metalloprotease functioning in cell surface antigen presentation, receptor for coronaviruses ( Membrane, cytoplasm)	Granulocytes, macrophages, bone marrow stromal cells, osteoclasts, renal tubules, intestinal brush border, cells lining bile duct canaliculi, endothelial cells, fibroblasts, brain cells	AML, CML with blast crisis, some ALL	Classification of leukemias.	Requires frozen tissue.
CD15 ( Leu-M1)	3-fucosyl-N-acetyllactosa-mine, X-hapten—CHO moiety linked to cell membrane protein ( Membrane and granular perinuclear)	Granulocytes, monocytes	Reed-Sternberg cells (not LP HD), some large T cell lymphomas, MF, some leukemias, some epithelial cells (adenocarcinomas), CMV infected cells	Adenocarcinomas (+) versus mesotheliomas (−). Evaluation of HD.
CD16	Low affinity transmembrane Fc receptor for IgG ( Membrane)	NK cells, granulocytes, activated macrophages, subsets of T cells	Extranodal NK/T-cell lymphoma, some hepatosplenic T-cell lymphomas
CD19 ( B4)	B cell type I integral membrane glycoprotein (Membrane)	B cells, follicular dendritic cells, early myelomonocytic cells	pre-B-ALL and B cell neoplasms (but not plasma cell lesions)	Good pan B cell marker.	Fresh or frozen tissue required.
CD20 ( L26, B1, Leu16)	B cell nonglycosylated phosphoprotein functioning as a receptor during B cell activation and differentiation ( Membrane, cytoplasmic)	B cells, monocytes, not plasma cells	B cell lymphomas, Reed-Sternberg cells in LP HD, not plasmacytomas	Best pan B cell marker. Evaluation of B cell lymphomas. Evaluation of HD.	L26 is best for formalin-fixed tissue. May be preserved in necrotic tissue.
CD21 ( B2)	Type I integral membrane glycoprotein functioning as the receptor for the C3d fragment of complement C3, CR2, receptor for EBV (Membrane)	Follicular dendritic cells, mature B cells	Marginal zone (MALT) lymphomas, CLL (B cell), some T cell ALL, follicular dendritic cell tumors	ID of residual follicular structure in LP HD and other diseases. Evaluation of low-grade B cell lymphomas. ID of follicular dendritic cell sarcoma.
CD22 ( BL-CAM)	Type I integral membrane glycoprotein ( Membrane. cytoplasm)	B cells, precursor B cells	B cell neoplasms (but not plasma cell lesions)	Pan B cell marker.
CD23	Membrane glycoprotein, low affinity IgE receptor ( Membrane)	Subpopulation of peripheral B cells, follicular dendritic cells	CLL, but usually not mantle zone lymphoma, Maltomas, or follicular lymphomas	Evaluation of low-grade B cell lymphomas.
CD25 ( IL-2 receptor)	Interleukin-2 receptor ( Membrane, cytoplasm)	Subpopulation of T cells, myeloid precursors, oligodendrocytes HTLV-1 transformed T and B cells	Hairy cell leukemia, adult T cell lymphoma/leukemia, some T cell prolymphocytic leukemia, precursor lymphoblastic lymphoma, and anaplastic large-cell lymphoma	Evaluation of cutaneous T cell lymphomas for potential anti-CD25 therapy. Aberrant expression by a subset of neoplastic mast cells.
CD30 ( Ki-1, BERH2)	Single chain transmembrane glycoprotein, homologous to the nerve growth factor superfamily ( Cytoplasm, membrane and golgi)	Activated B and T cells, some plasma cells, immunoblasts, interdigitating cells, histiocytes, follicular center cells, decidualized endometrium, reactive mesothelial cells, most other tissues negative	Anaplastic (CD30+) large cell lymphomas, large B cell lymphoma, primary effusion lymphoma, mediastinal large B cell lymphoma, Reed-Sternberg cells (not LP HD), enteropathy T cell lymphoma, peripheral T cell lymphoma, EBV transformed B cells Embryonal carcinoma, vascular tumors (not KS), some mesotheliomas, rarely carcinomas are positive	Evaluation of anaplastic (CD30+) lymphomas. Evaluation of HD (Reed-Sternberg cells are positive except in LP HD). Evaluation of peripheral T cell lymphoma (large cells may be positive).
CD33 ( My 9)	Myeloid-specific receptor (sialic acid-binding immunoglobulin-like lectin or Siglec-3) ( Membrane)	Granulocytes, monocytes	AML	Evaluation of leukemias.	Gemtuzumab ozogamicin is a humanized CD33 antibody linked to an antitumor antibiotic calicheaminin for the treatment of AML.
CD34 ( HPCA-1, QBEnd10)	Single chain transmembrane glycoprotein ( Cytoplasm, membrane)	Lymphoid and myeloid hematopoietic progenitor cells, endothelial cells, some skin cells, myofibroblasts	Acute leukemia Neurofibroma, angiosarcoma, KS, epithelioid hemangioendothelioma, solitary fibrous tumor, DFSP, epithelioid sarcoma, GIST, myofibroblastic tumors	ID of endothelial or myofibroblastic differentiation in tumors. Evaluation of angiogenesis. Evaluation of the number of blasts in bone marrow in acute leukemia.	Not specific for endothelial cells.
CD35 ( CR1, C3b/C4b R)	Transmembrane protein that binds complement components C3b and C4b and mediates phagocytosis ( Membrane)	Erythrocytes, B cells, a subset of T cells, monocytes, neutrophils, eosinophils, glomerular podocytes, follicular dendritic cells	Marginal zone (MALT) lymphoma, follicular dendritic cell tumors	Detects follicular dendritic cells. ID of follicular dendritic cell sarcomas.
CD38	Type II transmembrane glycoprotein with enzymatic action for the formation and hydrolysis of cADPR ( Membrane)	Immature B and T lymphocytes, thymocytes, mitogen-activated T cells, Ig-secreting plasma cells, monocytes, NK cells, erythroid and myeloid progenitors, brain cells	Acute leukemias, plasma cell lesions Neurofibrillary tangles in Alzheimer’s disease	ID of plasma cell lesions.	Immunoreactivity may be a poor prognostic marker for patients with CLL.
CD43 ( Leu 22, L60)	Cell surface glycoprotein ( Membrane)	T cells, macrophages, granulocytes	AML (chloromas), T cell neoplasms, aberrant expression in some low-grade B cell neoplasms (e.g., mantle cell lymphoma, SLL/CLL, marginal zone lymphoma), some MALT lymphomas	Evaluation of T cell lymphomas and leukemias. Evaluation of low-grade B cell lymphomas.	Less specific than UCHL-1 for T cells.
CD45, Leukocyte common antigen ( LCA, CLA)	Five or more membrane glycoproteins ( Membrane, cytoplasm)	Lymphocytes, leukocytes, histiocytes, not plasma cells, erythrocytes, platelets	Non-Hodgkin’s lymphomas, some anaplastic (CD30+) large-cell lymphomas, Reed-Sternberg cells in LP HD (but not other types)	ID of poorly differentiated neoplasms as lymphomas. However, some anaplastic lymphomas and plasmacytomas may be negative.	Preserved in necrotic tissue. Best general marker for hematologic neoplasms. Note: CLA also refers to a different antigen, HECA-452.
CD45RA ( DPB)	Restricted form of leukocyte common antigen ( Membrane, cytoplasm)	B cells, monocytes, some T cells	B cell neoplasms, Hairy cells (not specific)	Pan B cell marker that can be used in Zenker’s-fixed tissue.	Not completely specific—other B cell markers are preferred.
CD45RO ( UCHL-1)	Isoform of CD45 (leukocyte common antigen) (Membrane, cytoplasm)	T cells (memory), granulocytes, monocytes	T cell neoplasms, histiocytic sarcoma, some B cell lymphomas (plasmacytic, HIV associated)	Good pan T cell marker (CD3 is more specific).
CD56 ( NCAM)	Neural cell adhesion molecule—cell surface glycoprotein ( Membrane)	Neurons, astrocytes, Schwann cells, NK cells, subset of activated T cells	Some T /NK cell lymphomas, plasmacytomas Neuroblastoma	Evaluation of panniculitis-like T-cell lymphoma (both CD56+ and CD56-) and T/NK lymphomas.
CD57 ( Leu 7, HNK-1)	Lymphocyte antigen that cross reacts with a myelin-associated glycoprotein ( Membrane)	T cell subsets, NK cells, myelinized nerves, neuroendocrine cells, prostate, pancreatic islets, adrenal medulla	Angioimmunoblastic T cell lymphoma Nerve sheath tumors (occasional), leiomyosarcoma, synovial sarcoma, rhabdomyosarcoma, neuroblastoma, gliomas, neuroendocrine carcinomas, neurofibromas, some prostate carcinomas	ID of T gamma lymphoproliferative disorder (large granular cell lymphocytic leukemia). ID of neuroendocrine differentiation in tumors. Evaluation of NK neoplasms.	Not very specific for solid tumors.
CD61 ( GPIIIa, platelet glycoprotein IIIa)	Glycoprotein, receptor for fibrinogen, fibronectin, von Willebrand factor, and vitronectin ( Cytoplasm)	Megakaryocytes, platelets	Megakaryocytic leukemias	ID of megakaryocytic differentiation.
CD68 ( KP1, CD68-PGM1, Mac-M)	Intracellular glycoprotein associated with lysosomes ( Cytoplasm, membrane)	Macrophages, monocytes, neutrophils, basophils, large lymphocytes, Kupffer cells, mast cells, osteoclasts	Some lymphomas, histiocytic sarcomas, APML, Langerhans proliferative disorders Neurofibroma, schwannoma, MPNST, granular cell tumors, PEComa, melanomas, atypical fibroxanthoma, RCC	Best general marker for macrophages, although not specific to this cell type.	The antibody PG-M1 does not react with granulocytes.
CD74 ( LN2)	Subunit of MHC II-associated invariant chain ( Membrane)	B cells, monocytes, histiocytes	B cell neoplasms, Hairy cell leukemia, plasma cell lesions	Pan B cell marker.
CDw75 ( LN1)	Sialylated glycoconjugate present in surface Ig-positive B cells ( Membrane, cytoplasm)	Mature B cells, T cell subsets, fetal colon, epithelial cells	Reed-Sternberg cells of LP HD (not other types), follicular lymphomas Colon carcinomas (50%), gastric carcinomas	Evaluation of HD.
CD77 ( BLA.36, PK antigen)	Globotriaosylceramide, glycolipic membrane from Burkitt lymphoma cell line (Cytoplasm, membrane)	Tonsillar B cells, dendritic reticulum cells, sinus-lining cells, macrophages, endothelial cell, epithelial cells	HD, Burkitt lymphoma, rarely other B and T cell lymphomas	Evaluation of RS cells.
CD79a ( mb-1 protein)	Heterodimer of mb-1 (CD79a) and B29 (CD79b) polypeptides, B cell antigen receptor ( Membrane)	B cells, plasma cells	Precursor B cell ALL, B cell lymphomas, plasma cell lesions, but not primary effusion lymphoma	Evaluation of B cell neoplasms (may be the only B cell marker present).
CD79b	See above ( Membrane)			Absent from CLL, Hairy cell leukemia
CD95 ( Fas)	Transmembrane glycoprotein member of the nerve growth factor receptor/tumor necrosis factor superfamily—mediates apoptosis ( Membrane)	Activated T and B cells, epithelial cells	Panniculitis-like T cell lymphoma (if CD56+)
CD99 ( MIC-2, 12E7, Ewing sarcoma marker, E2 antigen, HuLy-m6, FMC 29, O13 (different epitope))	MIC2 gene product—glycoproteins (p30 and p32) involved in rosette formation with erythrocytes ( Membrane—more specific than cytoplasm positivity)	Cortical thymocytes, T lymphocytes, granulosa cells of ovary, pancreatic islet cells, Sertoli cells, some endothelial cells, urothelium, ependymal cells, squamous cells	B and T cell precursor lymphoblastic lymphoma/leukemia Ewing sarcoma, chondroblastoma, synovial sarcoma, solitary fibrous tumors, GIST, some alveolar rhabdomyocarcomas, desmoplastic small-cell tumors, small cell carcinomas, granulosa cell tumors, yolk sac components of germ cell tumors, Sertoli-Leydig cell tumors, atypical fibroxanthoma, meningioma	Evaluation of lymphoblastic lymphoma/leukemia Thymic carcinomas (lymphocytes +) versus other carcinomas. ID of Ewing sarcoma (immunoreactivity should be clearly membranous in the majority of the cells).	O13 is the most commonly used antibody. Immunoreactivity is highly dependent upon the antigen retrieval system used.
CD103	Mucosal integrin alphaEbeta7 with specificity for e-cadherin ( Cytoplasm)	T cells	Enteropathy type T cell lymphoma, Hairy cell leukemia		Requires frozen tissue or cell suspension.
CD117 ( c-kit, stem cell factor receptor)	Transmembrane tyrosine kinase receptor (ligand is stem cell factor)—apoptosis is inhibited when the ligand is bound ( Cytoplasm, membrane)	Mast cells, interstitial cells of Cajal (ICC—pacemaker cells of the GI tract found through-out the muscle layers and in the myenteric plexus), epidermal melanocytes, mono-nuclear bone marrow cells (4%), Leydig cells, early spermatogenic cells, trophoblast, breast epithelium	GIST (> 95%), seminomas (> 70%), intratubular germ cell neoplasia, mature teratomas (> 70%), some melanomas (focal), mast cell tumors, some carcinomas, some brain tumors, some Ewing sarcoma, some angiosarcomas AML (> 50%), CML in myeloid blast crisis	ID of GIST (+) versus leiomyomas (−) and schwannomas (−). ID of seminomas. ID of mast cells (mastocytosis).	Mast cells are an excellent internal control. CD117 positivity does not correlate with mutations and/or oncoprotein activity in tumors not known to have activating mutations and is, in general, not predictive of tumors likely to respond to tyrosine kinase inhibitors in this setting.
CD123	Alpha chain of the IL-3 receptor ( Membrane)	Myeloid precursors, macrophages, dendritic cells, mast cells, basophils, megakaryocytes	Plasmacytoid dendritic cell tumors
CD138 (Syndecan-1)	Transmembrane heparin sulfate glycoprotein that interacts with extracellular matrix and growth factors ( Membrane)	Pre-B cells, immature B cells, Ig-producing plasma cells, basolateral surface of epithelial cells, vascular smooth muscle, endothelium, neural cells	Plasma cell lesions, primary effusion lymphoma, plasma cell component of other B cell lymphomas Squamous cell carcinomas, other carcinomas	ID of plasma cells and their neoplasms. Expression may be diminished or lost in poorly differentiated carcinomas.
CD163 ( M130)	Endocytic receptor to scavenge haptoglobin and hemoglobin complexes ( Membrane, cytoplasm)	Tissue macrophages (high expression), monocytes (low expression) including Kupffer cells, Hofbauer cells but not follicular dendritic cells or plasmacytoid monocytes	Neoplasms of histiocytic differentiation Leukemias of monocytic differentiation Synovial type giant cell tumors of the vertebral column Langerhans cell histiocytosis (~60%), benign fibrous histiocytoma (~67%) Littoral cell angioma of the spleen	ID of true histiocytic derivation of tumors.	More specific for monocyte/histiocyte derivation than CD68.
CD207 ( Langerin)	Langerhans cell-specific C-type lectin (Cytoplasm)	Langerhans cells of epidermis and epithelia	Langerhans cell histiocytosis	ID of Langerhans lesions.	Induces formation of Birbeck granules.
Clusterin ( Apolipoprotein J, complement lysis inhibitor, gp80, SGP-2, SP40, TRPM2, T64, ApoJ)	Multifunctional protein involved in lipid transport, complement regulation, immune regulation, cell adhesion, other functions ( Membrane, cytoplasm, nucleus)	Many tissues	Anaplastic large-cell lymphoma (Golgi pattern) Alzheimer’s disease—present in amyloid plaques and cerebrovascular deposits Many types of carcinomas
Cyclin D1 ( PRAD1, bcl-1)	Cyclin regulating cyclin-dependent kinases during G1 in the cell cycle, phosphorylates and inactivates the retinoblastoma tumor suppressor protein ( Nucleus)	Cycling cells (however, lymphocytes usually express only cyclins D2 and D3)	Mantle cell lymphoma Breast cancer (especially lobular carcinomas and other ER positive carcinomas), esophageal cancer, bladder cancer, lung cancer, HCC, colon carcinoma, pancreatic carcinoma, head and neck squamous cell carcinomas, pituitary tumors, sarcomas Parathyroid adenomas (inversion involving cyclin D1 gene and the parathormone receptor)	ID of mantle cell lymphoma.	Involved in t(11;14)(q13;q32) translocation in mantle cell lymphoma.
DBA.44 ( HCL)	B cell antigen ( Cytoplasm, membrane)	Mantle zone B cells, some immunoblasts	Hairy cell leukemia (> 95%), B cell lymphomas (30%)	Evaluation of Hairy cell leukemia.
Epithelial membrane antigen ( EMA, MUC1, HMFG, DF3, CA 15-3, CA 27.29, PEM, many others)	Episialin, glycoprotein found in human milk fat globule membranes ( Cytoplasm (more common in malignant cells), membrane (more common in benign cells))	Epithelial cells, perineurial cells, meningeal cells, plasma cells, usually negative in mesothelial cells, monocytes	Some anaplastic large-cell lymphomas (CD30+), plasma cell neoplasms, malignant histiocytosis, erythroleukemia, AML (M4 and M5), LP HD Carcinomas, mesotheliomas, some sarcomas (synovial sarcoma, epithelioid sarcoma), adenomatoid tumor, chordomas, perineurioma, neurofibroma, meningiomas, desmoplastic small round cell tumor, Sertoli cell tumor	ID of epithelial differentiation in tumors—however, keratin is more specific for this purpose. Beware of EMA in some large-cell lymphomas. Synovial sarcoma typically shows focal positivity.	There are over 50 monoclonal antibodies recognizing different glycosylation patterns in normal tissues and tumors.
Epstein–Barr virus EBV-encoded nonpolyadenylated early RNAs (EBERS )	RNA produced by EBV ( Nucleus)	EBV infected B cells	All EBV-related tumors	Most sensitive marker for EBV.	Detected by in situ hybridization for RNA on paraffin sections.
LMP-1	Latent membrane protein ( Membrane)	EBV infected B cells	Nasopharyngeal carcinomas, Reed-Sternberg cells (not LP HD), transplant lymphomas, AIDS-related lymphomas, endemic Burkitt lymphoma (rare in sporadic cases)	Evaluation of EBV-related neoplasms.
EBNA 2 (nuclear antigen 2)	Nuclear protein ( Nucleus)	EBV infected B cells	Transplant-related lymphomas, AIDS-related lymphomas. Not present in Burkitt lymphoma or nasopharyngeal carcinomas.	Evaluation of transplant and AIDS-related lymphomas.
Fascin	Actin bundling protein regulated by phosphorylation ( Cytoplasm)	Interdigitating reticulin cells from the T cell zones, dendritic cells, reticular network, histiocytes, smooth muscle, endothelium, squamous cells, splenic sinuses	Reed-Sternberg cells (but not in LP HD) High-grade breast carcinomas	ID of Reed Sternberg cells in classical HD. Fascin positivity has also been reported in anaplastic large cell lymphoma.
FMC7	Antigen on subgroups of mature B cells, epitope of CD20 ( Cytoplasm )	B cells	B cell lymphomas	Not expressed by CLL.	Pan B cell marker. Epitope of CD20 but reactivity low in cells with low cholesterol.
Glycophorin A ( GPA)	A glycosylated erythrocyte membrane protein ( Membrane)	Erythroid elements at all stages	Erythroleukemia	ID of erythroid elements (normal and neoplastic).
Granzyme B	Neutral serine proteases stored in granules in cytotoxic T cells and in NK cells involved in target cell apoptosis by exocytosis ( Cytoplasm)	Cytotoxic T cells and NK cells	Some T cell lymphomas, Reed-Sternberg cells of some cases of EBV-positive HD
Heavy immunoglobulin chains ( G, A, M, D)	Heavy chain of immunoglobulins [Cytoplasm (plasma cells), membrane (lymphocytes)]	Plasma cells (G>A>M>D)	Plasma cell tumors (monotypic expression of usually G or A), mantle zone lymphomas and WDLL/CLL may coexpress M and D, lymphoplasmocytic lymphoma (M)	ID of monoclonal populations of plasma or plasmacytoid cells.
HECA-452 ( endothelial cell antigen, cutaneous lymphocyte-associated antigen, CLA)	Cell surface glycoprotein ( Membrane)	T cells, more common in cutaneous T cells	Mycosis fungoides and other cutaneous T cell lymphomas		Note: CLA is also used to refer to CD45.
Hemoglobin ( Hb)	Hemoglobin ( Cytoplasm)	Erythroid cells	Some leukemias	Marker for erythroid cells.
HHV8	Latent nuclear antigen of Human Herpes Virus type 8 (Nucleus)	Absent in normal tissue	Primary effusion lymphoma (PEL), AIDS-associated multicentric Castleman’s disease Kaposi’s sarcoma (endothelial cells and some perivascular cells),	Evaluation of Kaposi’s sarcoma and primary effusion lymphoma.
HLA-DR	Major histocompatibility complex Class II gene ( Membrane)	B lymphocytes, macrophages, Langerhans’ cells, dendritic cells, activated T cells, some endothelial and epithelial cells	Leukemic myeloblasts		Not very specific for cell type.
Light immunoglobulin chains ( lambda [L], kappa [K])	Light chain of immunoglobulins ( Cytoplasm)	Plasma cells (normally K > L), B cells	Plasma cell tumors, B cell lymphomas	ID of monoclonal populations of plasma cells and B cells. ID of some types of amyloid.	May require frozen tissue for assessment of B lymphoid cells. Excellent Ig preservation in plasma cells in B5 or Zenker’s-fixed tissue.
Lysozyme ( Ly)	Muramidase ( Cytoplasm)	Circulating monocytes, some tissue macrophages, granulocytes, salivary gland, lacrimal gland, stomach and colon epithelial cells (inflamed or regenerative), apocrine glands, some other epithelial cells	AML with monocytic differentiation, salivary gland tumors, stomach and colon carcinomas.	Marker for histiocytes but not specific. May mark activated phagocytic macrophages. Evaluation of myeloid leukemias. Strongly positive in monocytoid leukemias.	Not specific for solid tumor identification.
Mast cell tryptase	Serine protease ( Cytoplasm)	Mast cells	Mast cell neoplasms	ID of mast cell differentiation.
Myeloperoxidase ( MPO)	Enzyme in primary granules of myeloid cells ( Cytoplasm)	Myeloid cells, monocytes	AML, chloromas	Classification of leukemias.	Can be used with tissue fixed in Zenker’s fixative.
Oct2 ( Octomer transcription factor)	Transcription factor of the POU homeo-domain family binding to the Ig gene octomer sites regulating B specific genes ( Nucleus)	B cells	B cell lymphomas and leukemias Reed-Sternberg cells in LP HD (but not other types)	Evaluation of HD.	Interacts with the transcriptional coactivator BOB.1. BOB.1 and Oct are necessary (but not sufficient) for Ig expression.
Perforin	Pore-forming protein in cytoplasmic granules of cytotoxic T-cells ( Cytoplasm)	NK cells, large granular lymphocytes, gamma/delta T cells	NK cell lymphomas, anaplastic large-cell lymphoma	Evaluation of T cell lymphomas.
PU.1	Ets transcription factor ( Nucleus)	B cells, monocytic/macrophage/histiocytic lineage	B cell and monocytic/macrophage/histiocytic lineage lymphomas	Good nuclear marker for histiocytic sarcoma.
TCR ( T cell antigen receptor, JOVI 1)	Two polypeptide chains (alpha and beta)	Peripheral T cells	Many T cell lymphomas	Evaluation of T cell lymphomas.	Alpha/beta and gamma/delta T cell receptors can be evaluated in frozen tissue.
Terminal deoxytransferase ( TdT)	Enzyme that catalyzes addition of nucleotides to single strand DNA ( Nucleus)	Immature T and B cells	Lymphoblastic lymphoma/ALL	Lymphoblastic lymphoma (+) versus Burkitt lymphoma (−).
TIA-1 ( T-cell intracellular antigen)	A cytolytic granule-associated protein expressed in some CD8+ T cells ( Cytoplasm)	T cells, mast cells, polymorphonuclear leukocytes, eosinophils	Many T cell lymphomas	Evaluation of T cell lymphomas.
traf-1 ( Tumor necrosis factor receptor-associated factor)	Membrane bound proteins that activate the nuclear factor-(kappa)B (NF-(kappa)B) transcription factor resulting in cell proliferation ( Cytoplasm)	Usually absent	Hodgkin lymphoma, primary mediastinal large B cell lymphoma	Negative in most DLBCL and ALCL.	May interact with LMP1.

NAME: The most common name used to refer to the marker. The name may refer to the antigen, a CD number, or a specific antibody raised to the antigen. In some cases more than one name is commonly used. The majority of CD numbers correspond to a specific gene product. However, some CD numbers correspond to antigens formed from post-translational modifications. For example, CD15 (LeuM1) is a carbohydrate side chain linked to a protein.

ALTERNATE NAME: This list includes abbreviations, antibody names (sometimes recognizing different epitopes), or other terms for the marker.

ANTIGEN: The antigen recognized by the antibody.

LOCATION: The normal location of the antigen. In some cases, only certain locations of the antigen are considered a positive result (e.g., nuclear immunoreactivity for estrogen receptor, membrane immunoreactivity for HER2).

NORMAL CELLS AND TISSUES: The presence of the marker in normal cells and tissues. These cells serve as important internal positive controls. Abnormal positive immunoreactivity is also an important control for the specificity of the study.

TUMORS: The tumors in which immunoreactivity is typically expected. Refer to the Tables for additional information.

USES: The most common uses for the marker. Different pathologists and institutions will often have preferences for the use of certain markers.

COMMENTS: Additional comments regarding the marker.

Abbreviations: AD , Alzheimer’s disease; AIDS , acquired immunodeficiency syndrome; ALL , acute lymphocytic leukemia; AML , acute myelogenous leukemia; APML , acute promyelogenous leukemia; BM , basement membrane; CML , chronic myelogenous leukemia; CMV , cytomegalovirus; DFSP , dermatofibrosarcoma protuberan; EBV , Epstein–Barr virus; FISH , fluorescence in situ hybridization; GIST , gastrointestinal stromal tumor; HCC , hepatocellular carcinoma; HD , Hodgkin’s disease; HNPCC , Hereditary Non-Polyposis Colorectal Cancer; ID , identification; KS , Kaposi’s sarcoma; LP HD , lymphocyte predominant Hodgkin’s disease; MF , mycosis fungoides; MPNST , malignant peripheral nerve sheath tumor; NK , natural killer; PIN , prostatic intraepithelial neoplasia; PNET , primitive neuroectodermal tumor; RCC , renal cell carcinoma; RS , Reed Sternberg.

TABLE 5.5

CRITERIA USED FOR RESULTS IN THE IMMUNOHISTOCHEMICAL TABLES


ANTIBODY RESULTS	CATEGORY	% OF TUMORS
INTERPRETATION	Positive (POS)	> 90%
Almost always positive; a negative result would be unusual	High	60–90%
Most tumors are positive	Moderate (Mod)	40–60%
May or may not be positive—usually the least useful type of marker	Low	10–40%
Most tumors are negative	Negative (neg)	<10%
Almost all tumors are negative; a positive result would be unusual	Blank

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