Cytokeratins (CKs) are intermediate filaments. Together with microfilaments and microtubules, IFs form cytoskeleton of all eukaryotic cells. They are polypeptide filaments radiating from the nucleus and extending to the plasma membrane. Based on their composition, they are divided into five types (From Class I through Class V) (1) (2).
|Class I||Acidic cytokeratins||Cytoplasm|
|Class II||Neutral-basic cytokeratins||Cytoplasm|
Glial fibrillary acidic protein
|Class IV||Neurofilament protein
Table 1: Intermediate filaments
The CK family is a highly complex multigene family of polypeptides, the molecular weight of which ranges from 40 to 68 kDa. Until now, 20 distinct CKs have been identified (excluding the so-called trichocytic keratins present in hair and nail-forming epithelia only). The classification and numbering (CK1-CK20) is based on the catalogue of Moll et al. (Table 2) (1). Twelve of the CKs (CK9-CK20) belong to the acidic type A (class I) and eight (CK1-CK8) to the neutral-basic type B (class II) subfamily (Table 2) . In the filaments, the CK polypeptides are always paired so that a particular type A CK forms a complex with a particular type B CK, creating heterodimers. The type B CK is generally 7-9 kDa larger than the corresponding CK of type A. From 2 to 10 different CKs are found in the individual cell (3). Using a broad spectrum CK antibody (pan-cytokeratin antibody) practically all epithelia as well as other cell expressing CK (see below) can be stained CK filaments may also be coexpressed with other IF types. (3)
CKs are generally held to belong to the most fundamental markers of epithelial differentiation. The composition of CKs reflects both cell type and differentiation status. Most CK types have been identified in several kinds of epithelium, whereas e.g. the paired CK3 and CK12 have been detected in cornea only. CKs are also found in a small number of non-epithelial cell types (Table 1: Intermediate filaments). In most CK-containing cells, the filaments are arranged in a mesh of loose bundles. However, in stratified squamous epithelia, the CK filaments are arranged in dense bundles, tonofibrils, attached to the desmosomes.
CKs in simple epithelia
All simple (one-layered, polar) epithelial cells contain the paired CK8 (52.5 kDa) and CK18 (45 kDa), representing the primary (constitutive) CKs of simple epithelia. These low molecular weight (LMW) CKs are the only CKs found in e.g. liver cells, pancreatic acinar cells, most endocrine cells and cells of the proximal renal tubules.
Epithelium of the gastrointestinal tract furthermore contains the secondary (accessory) CK19 (40 kDa), and in a number of other epithelia e.g. biliary and pancreatic ducts, lung alveoli, endometrium and renal collecting ducts, CK7 (54 kDa) is also present (Fig. 2). The thyroid gland follicular cells express CK7, but normally not CK19.
CK20 (46 kDa) shows the most restricted expression: Among simple epithelia it is almost entirely confined to the epithelial cells of the intestinal tract (Fig. 3) and gastric foveolae, as well as the endocrine Merkel cells of the skin. (4)
CKs in complex epithelia
The so-called complex epithelia reveal both the CKs found in simple epithelia (see above) and some of those belonging to the stratified epithelia (see below). The complex CK pattern is particularly found in epithelia with a prominent basal or myoepithelial cell layer such as the supraalveolar airways, breast, salivary and sweat glands and prostate. The basal and myoepithelial cells generally express the stratified-epithelial CK5 (58 kDa) (Fig. 4), CK14 (50 kDa) and CK17 (46 kDa), whereas the luminal/secretory epithelial cells mainly express the simple-epithelial CK8, CK18, CK19 and CK7. Trophoblast cells of placenta express the CKs of simple epithelia and focally some of the stratified-epithelial group.
It is quite understandable that the epithelial cells that form a robust supporting layer or myoid quality need higher molecular weight to perform that functionality and hence these cells show complex keratins, popularly known as high molecular weight cytokeratins (HMWCK). It is obvious that the secretory unit would be lined on the inside by an epithelium that usually contains simple CKs (Fig. 5).
CKs in mesothelium
Like the complex epithelia, mesothelial cells express, both, the simple-epithelial CK8, CK18, CK19 and CK7and the stratified-epithelial CK5, CK14 and (inconstantly) CK17.
CKs in transitional epithelium
The urothelium shows a complex but unique CK pattern: In addition to CK8, CK18, CK19 and CK7 present in all cell layers, the superficial umbrella cells (and occasionally also some intermediate cells) are distinguished by specific CK20 expression (Fig. 3) . Moreover, there are some stratified-epithelial CKs (Table 2), in particularCK5 and CK13 (54 kDa), in the basal/intermediate cell compartment.
CKs in stratified epithelia
The basal layer of the stratified squamous epithelia mainly expresses CK5 and CK14 (representing the primary – constitutive – high molecular weight CKs of stratified epithelia), and, except for skin, also CK19. In the suprabasal layers, specific differentiation-related CK pairs appear. While intermediate and superficial cells of non-cornified mucosal stratified squamous epithelia express the pair CK4 (59 kDa) and CK13, the cornified epidermis is characterized by the pair CK1 (68 kDa) and CK10 (56.5 kDa). In some areas of the skin (particularly in hard-worn areas), CK2e (e = epidermal; 65.5 kDa) and CK9 (64 kDa; in palmoplantar skin) are found as well. In the palate, CK2p is identified. The paired CK6 (56 kDa) and CK16 (48 kDa) are detected in normal non-cornifying stratified squamous epithelia and in diseased (i. e. hyperproliferative) epidermis. The reticular epithelium of thymus expresses several CKs of stratified epithelia, but also certain simple-epithelial CKs.
CKs in non-epithelial cells
In a few mesenchymal cells such as certain smooth muscle cells (particularly vascular wall and myometrium), myofibroblasts, arachnoidal cells, fibroblastic reticulum cells of lymph nodes and rarely endothelial cells, as well as cells of the umbilical cord and foetal myocardium, simple-epithelial CKs – mainly CK8 and CK18 – may be detected in addition to the original mesenchymal intermediate filaments (vimentin, desmin) (Table 1: Intermediate filaments). Some endothelial cells exhibit immunoreactivity for CK7.
Coexpression of CKs and other IFs in epithelial cells
Some normal cells show coexpression of CKs with one or more other IF types:
- CK and vimentin are coexpressed in most mesoderm derived epithelia (kidney, female genital tract and mesothelium) and in thyroid gland epithelium.
- CK and glial fibrillary acidic protein are coexpressed in some myoepithelial cells (e.g. salivary and sweat glands and breast).
- CK and neurofilament are coexpressed in some neuroendocrine tumours, but rarely in normal neuroendocrine cells.
- CK and desmin are coexpressed in some smooth muscle cells.
- Focal expression of CK7 and CK18 can be seen in endothelial cells along with vimentin. (5)
CKs in non-neoplastic epithelial lesions
Upon various kinds of damage, epithelial cells may change their pattern of CK expression. Some of the changes are similar to those found in neoplastic cells (see below). The changes may indicate an immature CK pattern of regenerative cells or a metaplastic process (e.g. neoexpression of CK7 in hepatocytes of diseased liver (Fig. 2). , which sometimes is not morphologically evident. (6)
Carcinomas generally express CK patterns, which at least in part represent the pattern of the putative cells of origin (Table 2). The consequent profile, which reflects both epithelial type and differentiation status, may therefore be useful in tumour diagnosis. Thus, adenocarcinomas generally express the simple-epithelial-type CK8, CK18, CK19 and, frequently, CK7 (Fig. 6). Typically, colorectal adenocarcinoma maintains the specific intestinal pattern (including CK20, not CK7). Also transitional cell carcinoma and Merkel cell carcinoma retain many features of the respective normal cells. In squamous cell carcinoma, the stratified-epithelial CK5, CK6, CK14, CK16 and CK17 are usually predominant (Fig. 7)
In table 3 , the coordinate CK7/CK20 expression in a number of neoplasms is indicated (Fig. 7). (2) (7) However, some types of carcinoma may deviate from the patterns of their normal counterpart. Certain carcinomas may lose the ability to express one or more CKs found in the putative tissue of origin, e.g. squamous cell carcinoma sometimes being devoid of CK13. Other types of carcinoma may switch on certain CKs, e.g. CK19 in thyroid carcinoma, or regain CKs which the putative cell of origin lost in fetal life, e.g. CK19in hepatocellular carcinoma or the simple-epithelial CKs CK8, CK18, CK7 and CK19 in squamous cell carcinoma (in addition to its retained – and still predominant – stratified-epithelial CKs such as CK5). Thus, an increased complexity of CK patterns is a feature of some carcinomas. Similar changes may be seen even in dysplastic lesions. It is noteworthy that in adenocarcinomas derived from complex epithelia such as breast, the CKs especially belonging to the basal and my epithelial cells are generally not expressed, but they may gain these CKs upon dedifferentiation or occurrence of a myoepithelial phenotype. After radio and/or chemotherapy of carcinomas, alterations of the CK pattern may be more pronounced.
Non-epithelial tumours are usually CK negative. However, in a number of mesenchymal tumours, CK positivity – usually faint or focal, and limited to CK8, CK18 and CK19 – has been demonstrated. This accounts for e.g. leiomyomatous tumours, rhabdomyosarcoma, malignant melanoma, schwannoma, Ewing’s sarcoma, desmoplastic small round cell tumour and a few types of malignant lymphoma, particularly anaplastic large-cell lymphoma. Epithelioid sarcoma, chordoma and adamantinoma express strong CK positivity corresponding to that of simple epithelia (CK8, CK18 and CK19). Synovial sarcoma and malignant mesothelioma usually express simple-epithelial CKs but also – particularly in areas of epithelial differentiation – certain stratified-epithelial (“high molecular weight”) CKs. (Fig. 8).
CKs are indispensable markers for immunohistochemical classification of poorly differentiated or phenotypically “deviant” tumours.
Almost all mesotheliomas and carcinomas except squamous cell carcinomas, contain simple keratin 8 and 18. Whereas it is the only type of keratin found in liver neoplasm.
CK-Pan antibodies, i.e., CK antibodies with broad specificity, i.e., CK-Pan antibodies like AE1/AE3 and KL1 are useful in distinguishing carcinomas from the majority of non-epithelial malignant tumours, and should be included in the “primary antibody panel”. They are extremely useful in identifying an undifferentiated tumor as of epithelial derivation. The detection of keratin, and therefore carcinomatous differentiation, is possible in tumours with extensive necrosis. Judkin’s and colleagues studied a small number of tumors with necrotic areas-including carcinomas, melanomas and sarcomas-with a panel of antibodies and found that 78% of carcinomas stained with at least one keratin antibody in necrotic area with 100% specificity. (8)
CK-Pan antibodies are particularly helpful for the demonstration of micrometastases or disseminated carcinoma cells, e.g. in lymph nodes and bone marrow, in the diagnosis of small cell carcinoma, lymphoepithelial and nasopharyngeal carcinoma and infiltrations of epithelial tumours with a dispersed growth pattern, such as diffuse stomach carcinoma and infiltrating lobular breast carcinoma.
Among germ cell neoplasms, seminoma/dysgerminoma is usually CK-negative (or only sparsely positive), while all other types are CK-positive.
Among primary intracranial neoplasms, CK is found in choroid plexus papilloma/carcinoma and in some meningiomas (notably those of the secretory type), while gliomas and primitive neuroectodermal tumours are usually CK-negative.
Predominant expression of stratified-epithelial (“high molecular weight”) CKs, such as CK5 and (to a lesser extent) CK13, in a carcinoma indicates a squamous differentiation and is helpful for the recognition of poorly differentiated, non-keratinizing squamous cell carcinoma. While CK8, CK18 and CK19 may be expressed both in adenocarcinomas and – to variable extent – in squamous cell carcinoma, the presence of CK5 in the absence (or low expression) of CK7 strongly argues for a squamous cell carcinoma. One should be aware of mucoepidermoid carcinoma that expresses both, CK5 and CK7 in different areas of tumor (Fig. 9).
In an adenomatous malignant tumour, expression of the stratified-epithelial CK5 (and CK14) may be suggestive of pancreatic-biliary adenocarcinoma, ovarian serous or endometrioid carcinoma, adenosquamous carcinoma, breast stem cell carcinoma or myoepithelial carcinoma or, in serosal biopsies, malignant mesothelioma. Adenocarcinomas of lung and breast rarely show CK5 expression, whereas gastrointestinal adenocarcinomas essentially lack these CKs.
Antibodies against stratified-epithelial CKs, such as CK5 or HMWCK (34ßE12) (Fig. 11), identify normal basal/myoepithelial cells and thus may be helpful to recognize invasiveness and malignancy in prostatic and breast lesions, particularly when combined with p63.
For transitional cell (urothelial) carcinoma, the coexpression of CK13 and CK20 (as well as CK7; see below) is a helpful diagnostic feature. This combination of CKs is rarely observed in other carcinoma types, thus allowing distinction of transitional cell carcinoma from e.g. prostatic adenocarcinoma and non-keratinizing squamous cell carcinoma in cases of unknown primary tumour site. De novo expression of CK14 in transitional cell carcinoma is indicative of squamous differentiation and a more unfavourable prognosis. In papillary urothelial neoplasms, a normal (luminal) CK20 expression pattern is indicative of tumour non-recurrence and may be used to make a differential diagnosis between invasive and non-invasive urothelial neoplasia.
Expression of CK7 without concomitant CK20 is typical for adenocarcinomas of the lung, breast, endometrium and thyroid gland, as well as non-mucinous ovarian adenocarcinoma and malignant mesothelioma.
Marked expression of CK20 without concomitant CK7 is typical for colorectal adenocarcinoma (in 80% of the cases, more than half of the cells are stained for CK20) (Fig. 11) as well as for Merkel cell carcinoma. (4)
Expression of both CK7 and CK20 is typical for transitional cell carcinoma, mucinous ovarian carcinoma and gastric, biliary tract and pancreatic adenocarcinomas (even though CK20 is not infrequently focal or missing). However, also high grade colorectal adenocarcinoma occasionally shows this pattern.
Hepatocellular carcinoma, adenocarcinoma of prostate, adrenal cortical carcinoma and renal cell carcinoma as well as small cell carcinoma of the lung and squamous cell carcinoma typically express neither CK7 nor CK20. However, exceptions from this rule are not unusual; particularly in hepatocellular carcinoma, CK7 and even focal CK20 may be found occasionally. Adrenal cortical carcinoma, small cell lung carcinoma and renal cell carcinoma usually express sparse amounts of CK8 and CK18, and a sensitive protocol is needed to detect CKs.
A paranuclear dot-like CK staining (due to formation of IF whorls, the so-called fibrous bodies) is particularly seen in neuroendocrine tumours like Merkel cell carcinoma, carcinoid tumours and pituitary adenoma as well as in ovarian granulosa cell tumour. In Merkel cell carcinoma, these structures contain CK20, a marker allowing their distinction from metastatic small cell carcinoma of the lung in which CK20 is usually absent.
To demonstrate CKs, the laboratory should stock at least one CK-pan antibody, one LMW
In the following, selected commonly used antibodies are described with respect to reaction pattern and epitope pretreatment. Heat induced epitope retrieval (HIER) in an alkaline buffer is strongly recommended, if not otherwise specified.
Clone AE1/AE3 (AE1 directed to acidic types 9, 10, 13, 14, 15, 16 and 19; AE3 directed to neutral-basic types CK1-8), clone MNF116 (directed to CK5, 6, 8, 17, 19) and clone KL1 (directed to CK2, 5, 6, 8, 10, 18, 19) are broad spectrum cytokeratin antibodies useful for screening and visualisation of epithelial structures. In optimized protocols (HIER, not proteolysis!), they give almost the same staining pattern. However, there are subtle differences:
Due to a relatively lower affinity to high molecular weight (HMW) CKs, KL1 may give a weaker staining in urothelial and squamous cell carcinomas. Likewise, due to a relatively lower affinity to low molecular weight (LMW) CKs, AE1/AE3 may give a weaker staining in adenocarcinomas.
In sections pre-treated with proteolytic enzymes, AE1/AE3 may give poor staining of HMW CKs, and the staining of CK8 is lost. In long time fixed brain tissue, cross reaction to glial fibrillary acidic protein is seen with proteolytic pretreatment.
MNF116 only works satisfactory after proteolytic pretreatment, which should be optimized for the individual tissue and fixation time! MNF116 gives no advantage over KL1 and AE1/AE3.
Combined subtype antibodies
Clone 34BE12 (directed to CK1, 5, 10, 14 and an unknown CK subtype) is useful for detection of HMW CKs. Due to a reactivity to the unknown CK subtype, 34BE12 also reacts with a some simple epithelia (breast, colon, biliary tract, kidney) but usually not prostate. I personally believe that HMWCK is a good stain for staining basal cells in evaluation of prostatic adenocarcinoma while CK5 is good for evaluation of breast carcinoma.
Clone D5/16 B4, is directed against CK5 and CK6, but can for practical purposes be applied as a CK5 antibody.
Clone CAM 5.2 (directed to CK8 and CK7 – previously assumed to react with CK8, 18 and 19) only works satisfactory after proteolytic pretreatment, which should be optimized for the individual tissue and fixation time! HIER causes inferior sensitivity and non-specific cross-reaction. Cam 5.2 gives no advantage over single subtype antibodies to CK8 and CK18.
Single subtype antibodies
Antibodies against most CK subtypes are commercially available. A few examples and comments are given.
CK8/18: Clone C51 against CK8 and clone DC10 against CK18 are equally useful for the detection of primary CKs of simple epithelia.
CK7: Clone OV-TL 12/30
CK19: Clones A53-B/A2.26 (concentrated or RTU), b170 and BA17 have shown the best performances, while clone RCK108 seems to be less sensitive.
CK20:Clone Ks20.8 may give false positive staining reaction in e.g., biliary tract and mesothelial cells after proteolytic pre-treatment.
CK4: In contrast to clone 6B10, clone 215B8 reacts with suprabasal and keratinizing cells in epidermis, possibly due to cross reaction with a denatured CK.
CK10: Clone DE-K10
CK13: Clones DE-K13 and KS-1A3. The former also reacts with CK10 in cryostat sections and unfixed cytological material. In formalin-fixed paraffin sections cross-reaction to lymphocytes can be seen when the antibody is used too concentrated.
CK14: Clone LL002 generally reacts weaker than antibodies against CK5 and CK5/6.
CK17: Clone E3
Recommended control tissue:
CK-Pan:We use tissue control multiblock made up of appendix, pancreas, liver and tonsil. As a minimum, both the primary CKs of squamous epithelia (CK5 and/or CK14) and of simple epithelia (CK8 and/or CK18) must be detected. The non-keratinized squamous epithelium should give a strong staining of all layers. In the liver, the majority of liver cells should reveal at least a moderate membranous staining.
LMW CKs (incl. CK8 and/or CK18):
Liver is appropriate for control. The majority of liver cells should reveal at least a moderate membranous staining.
HMW CKs (incl. CK5 and/or CK14):
Tonsillary epithelium is appropriate for control. The squamous epithelium should give a strong staining of all layers.
CK7: Liver and pancreas are appropriate for control. In the liver, the bile duct cells must be strongly positive, while the liver cells are negative. In the pacreas, the intercalated ducts must be strongly positive, while the acini are negative.
CK20: Appendix is appropriate for control. The majority of enterocytes in the crypts should be strongly positive at least including the upper 50% of the crypts.
CK19: Appendix is appropriate for control. A strong, distinct cytoplasmic reaction of virtually all the surface epithelial cells and at least a weak to moderate reaction of the epithelial cells in the basal part of the crypts should be seen.
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