Cytokeratins (CKs) belong to a group of proteins known as intermediate filaments that constitute the cytoskeletal structure of virtually all epithelial cells. Being intermediate between microfilaments (6nm) and microtubules (25nm), the intermediate filaments comprise five characteristic groups based on cellular origin: CKs (epithelium), glial (astrocytes), neurofilaments (nerve cells), desmin (muscle) and vimentin (mesenchymal cells). More recently, this family of cytoskeletal proteins the intermediate filaments — have been reclassified into six subtypes (Table 1) (Miettinen, 1993). Intermediate filament proteins are composed of a 310 amino acid residue central region known as the rod domain. This is flanked by end domains of varying length and sequence, known as the head and tail. It is these flanking sequences that are the most immunogenic, responsible for the different properties and functions of the intermediate filament proteins. Being exposed, these molecules are also sensitive to fixation artefact due to the formation of crosslinkages. It is also important to note that due to the 30-50% sequence homology between the amino acid sequences of intermediate filaments of different types, monoclonal antibodies may crossreact with different intermediate filament types (Battifora, 1988).
Table 1. Classification of intermediate filaments
I Acidic cytokeratin (CK9-CK20)
II Basic cytokeratin (CK1-CK8)
III Vimentin (mesenchymal cells), desmin (muscle), glial fibrillary acid protein (glial cells and astrocytes), peripherin (neuronal cells)
IV Neurofilaments protein triplet (neurons)
V Nuclear laminin proteins (nuclear lamina)
VI Nestin (CNS stem cells)
CKs are present in both benign and malignant epithelial cells, independent of cellular differentiation. However, CK immunohistochemistry utilizing subset selective antibodies has extended beyond the typing of epithelial tumors, with recent descriptions of non-epithelial cells and tumors expressing CK.
The CKs are a family of proteins coded by different genes and the expression in epithelial cells is dependent on the embryonic development and degree of cellular differentiation. Practically, the most important CKs have been classified and numbered, based on the catalog of Moll et al (numbered 1 to 20) (Moll et al, 1982). These CKs were identified by the biochemical properties in two-dimensional gel electrophoresis of tissue extracts with their identification based on their isoelectric points and molecular weight. Hence, two groups of CKs emerge: type I/A (CK 9-20) with an acidic isoelectric point and type II/B (keratins 1-8) with a basic-neutral isoelectric point. Apart from a few exceptions, CKs are numbered from the highest to the lowest molecular weight in each group (Table 2) (Miettinen, 1993).
An interesting phenomenon is the existence of the keratin intermediate filaments as pairs. With some exceptions, all other CKs form polymers with their corresponding member from each type (Table 2). Hence, it follows that all epithelial cells contain at least two CKs. For example, whilst hepatocytes harbor a single pair of CK 8 and 18, keratinocytes may contain as many as ten CKs.
Thus, these laws governing the expression of various CKs are observed in part by neoplastic cells, forming the basis for the application of antibodies to CKs within neoplastic cells (indicating epithelial differentiation) using immunohistochemical methods (Schaafsma & Ramaekers, 1994).
Table 2. Keratins 1-20 with their molecular weight and most important distribution (Modified from Miettinen, 1993)
TYPE II MW(kd) Distribution Type I MW(kd)
Epidermis-alms and soles 9 64
1 67 Epidermis, keratinizing squamous epithelia 10 56.5
2 65 11 56
3 63 Cornea 12 55
4 59 Non-keratinizing squamous epithelia (internal organs) 13 51
5 58 Basal cells squamous and glandular epithelia, 14 50 myoepithelium, mesothelium Squamous epithelia 15 50
6 56 Squamous epithelia (hyperproliferative) 16 48
7 54 Simple epithelial 17 46 Basal cells glandular epithelia, myoepithelium Simple epithelia
8 52 18 45 Simple epithelia, most glandular, some squamous 19 40 epithelia (basal) Simple epithelia intestines and stomach, 20 46
Merkel cells
Keratin pairs in the same line, MW, molecular weight, KD, kilodalton
The emergence of selective monoclonal antibodies identifying individual CKs now offers the advantage of immunohistochemical detection with morphological correlation (Heatley, 1996). Monoclonal antibodies to CKs may be divided into two categories: (i) a broad group that recognizes many members of the keratin family (see later) and (ii) a selective group that reacts with isolated CKs; in this regard, only CKs 7 and 20 will be considered in detail. Nevertheless, Table 3 provides a list of the most important CK subtypes in some epithelial neoplasms (Miettinen, 1993). In addition, popular commercially available antibodies to broad groups of CKs (Table 4) will also be detailed individually. False negativity due to masking of keratin epitopes and loss of Table 3 Summary of the most important keratin subtypes of some epithelial tumors (Modified from Miettinen, 1993) Carcinoma type Keratin composition (Moll's catalog)
45781314171819 20
Squamous cell carcinoma, skin + + +*
Squamous cell Ca of esophagus + + + +
Ductal carcinoma of breast + + +* +* + +
Malignant mesothelioma + + + + + +
Adenocarcinoma, lung + + + +
Adenocarcinoma, colon + + + +
Adenocarcinoma, pancreas + + + + +*
Hepatocellular carcinoma + + +* +**
Carcinoid tumor/small cell carcinoma + +
Merkel cell carcinoma + + + +
Renal (cell) adenocarcinoma + + + **
Transitional cell carcinoma, low gr. + + + + + + + *
Transitional cell carcinoma, high gr. + + + * + +
Thyroid carcinoma, papillary + + +
Thyroid carcinoma, follicular + + + *
Adenocarcinoma of prostate + * + + +
Adenocarcinoma of ovary + + + + + *
* Occasionally present/minor component
** Often but inconsistently present
Table 4 Specificities of selected-cytokeratin antibodies **
MW 35ß
bbH11 34ß
bbE12 AE1 AE3 *Anti *Anti Cam KL1 MNF 116
(kD) bovine callus 5.2
keratin keratin
39 ++
40+ +
45 +++
48 ++
50++ +
51 +
52++ +
52.5 ++
54 +
56 ++++
56.5 + ++
57 +
58 + ++++
60 +
64 +
65 +
65.5 +
66 +
67 +
68 +
Antibody sources: 35bH11 and 34 bE12 (Dakopatts, California, USA); antibovine keratin, anticallus keratin (Dakopatts, California, USA); AE1, AE3 (available as AE1/3 cocktail) Boehringer, Sydney, Australia; Dakopatts, California, USA); Cam 5.2 (Becton Dickinson, California, USA); KL1 (Immunotech, Marseilles, France); MNF116 (Dakopatts, California, USA). *polyclonal antisera +specificities as supplied by manufacturers antigenicity warrants antigen retrieval in most instances. Hence, the need for extensive and carefully controlled optimization of every new antibody before diagnostic application cannot be overemphasized.
References
•Battifora H 1988. Diagnostic uses of antibodies to keratins: a review and immunohistochemical comparison of seven monoclonal and three polyclonal antibodies. In: Fenoglio-Preiser CM, Wolff M, Rilke F (eds) Progress in surgical pathology, vol. VIII. Berlin: Springer-Verlag, pp 1-15.
•Heatley, MK 1996. Cytokeratins and cytokeratin staining in diagnostic histopathology (commentary). Histopathology 28: 479-483.
•Miettinen M 1993. Keratin immunohistochemistry: update of applications and pitfalls. In: Rosen PP, Fechner RE (eds) Pathology annual, part 2/vol 28. New York: Appleton and Lange pp 113-143. (eds) Pathology annual, part 2/vol 28. New York: Appleton and Lange pp 113-143.
•Moll R, Franke WW, Schiller DL, Geiger B, Krepler R 1982. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 31: 11-24.
•Schaafsma HE, Ramaekers FCS 1994. Cytokeratin subtyping in normal and neoplastic epithelium: basic principles and diagnostic applications. In: Rosen PP, Fechner RE (eds) Pathology annual, part I/vol 29. New York: Appleton and Lange pp 21-62.
Bibliografía
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
Table 1. Classification of intermediate filaments
I Acidic cytokeratin (CK9-CK20)
II Basic cytokeratin (CK1-CK8)
III Vimentin (mesenchymal cells), desmin (muscle), glial fibrillary acid protein (glial cells and astrocytes), peripherin (neuronal cells)
IV Neurofilaments protein triplet (neurons)
V Nuclear laminin proteins (nuclear lamina)
VI Nestin (CNS stem cells)
CKs are present in both benign and malignant epithelial cells, independent of cellular differentiation. However, CK immunohistochemistry utilizing subset selective antibodies has extended beyond the typing of epithelial tumors, with recent descriptions of non-epithelial cells and tumors expressing CK.
The CKs are a family of proteins coded by different genes and the expression in epithelial cells is dependent on the embryonic development and degree of cellular differentiation. Practically, the most important CKs have been classified and numbered, based on the catalog of Moll et al (numbered 1 to 20) (Moll et al, 1982). These CKs were identified by the biochemical properties in two-dimensional gel electrophoresis of tissue extracts with their identification based on their isoelectric points and molecular weight. Hence, two groups of CKs emerge: type I/A (CK 9-20) with an acidic isoelectric point and type II/B (keratins 1-8) with a basic-neutral isoelectric point. Apart from a few exceptions, CKs are numbered from the highest to the lowest molecular weight in each group (Table 2) (Miettinen, 1993).
An interesting phenomenon is the existence of the keratin intermediate filaments as pairs. With some exceptions, all other CKs form polymers with their corresponding member from each type (Table 2). Hence, it follows that all epithelial cells contain at least two CKs. For example, whilst hepatocytes harbor a single pair of CK 8 and 18, keratinocytes may contain as many as ten CKs.
Thus, these laws governing the expression of various CKs are observed in part by neoplastic cells, forming the basis for the application of antibodies to CKs within neoplastic cells (indicating epithelial differentiation) using immunohistochemical methods (Schaafsma & Ramaekers, 1994).
Table 2. Keratins 1-20 with their molecular weight and most important distribution (Modified from Miettinen, 1993)
TYPE II MW(kd) Distribution Type I MW(kd)
Epidermis-alms and soles 9 64
1 67 Epidermis, keratinizing squamous epithelia 10 56.5
2 65 11 56
3 63 Cornea 12 55
4 59 Non-keratinizing squamous epithelia (internal organs) 13 51
5 58 Basal cells squamous and glandular epithelia, 14 50 myoepithelium, mesothelium Squamous epithelia 15 50
6 56 Squamous epithelia (hyperproliferative) 16 48
7 54 Simple epithelial 17 46 Basal cells glandular epithelia, myoepithelium Simple epithelia
8 52 18 45 Simple epithelia, most glandular, some squamous 19 40 epithelia (basal) Simple epithelia intestines and stomach, 20 46
Merkel cells
Keratin pairs in the same line, MW, molecular weight, KD, kilodalton
The emergence of selective monoclonal antibodies identifying individual CKs now offers the advantage of immunohistochemical detection with morphological correlation (Heatley, 1996). Monoclonal antibodies to CKs may be divided into two categories: (i) a broad group that recognizes many members of the keratin family (see later) and (ii) a selective group that reacts with isolated CKs; in this regard, only CKs 7 and 20 will be considered in detail. Nevertheless, Table 3 provides a list of the most important CK subtypes in some epithelial neoplasms (Miettinen, 1993). In addition, popular commercially available antibodies to broad groups of CKs (Table 4) will also be detailed individually. False negativity due to masking of keratin epitopes and loss of Table 3 Summary of the most important keratin subtypes of some epithelial tumors (Modified from Miettinen, 1993) Carcinoma type Keratin composition (Moll's catalog)
45781314171819 20
Squamous cell carcinoma, skin + + +*
Squamous cell Ca of esophagus + + + +
Ductal carcinoma of breast + + +* +* + +
Malignant mesothelioma + + + + + +
Adenocarcinoma, lung + + + +
Adenocarcinoma, colon + + + +
Adenocarcinoma, pancreas + + + + +*
Hepatocellular carcinoma + + +* +**
Carcinoid tumor/small cell carcinoma + +
Merkel cell carcinoma + + + +
Renal (cell) adenocarcinoma + + + **
Transitional cell carcinoma, low gr. + + + + + + + *
Transitional cell carcinoma, high gr. + + + * + +
Thyroid carcinoma, papillary + + +
Thyroid carcinoma, follicular + + + *
Adenocarcinoma of prostate + * + + +
Adenocarcinoma of ovary + + + + + *
* Occasionally present/minor component
** Often but inconsistently present
Table 4 Specificities of selected-cytokeratin antibodies **
MW 35ß
bbH11 34ß
bbE12 AE1 AE3 *Anti *Anti Cam KL1 MNF 116
(kD) bovine callus 5.2
keratin keratin
39 ++
40+ +
45 +++
48 ++
50++ +
51 +
52++ +
52.5 ++
54 +
56 ++++
56.5 + ++
57 +
58 + ++++
60 +
64 +
65 +
65.5 +
66 +
67 +
68 +
Antibody sources: 35bH11 and 34 bE12 (Dakopatts, California, USA); antibovine keratin, anticallus keratin (Dakopatts, California, USA); AE1, AE3 (available as AE1/3 cocktail) Boehringer, Sydney, Australia; Dakopatts, California, USA); Cam 5.2 (Becton Dickinson, California, USA); KL1 (Immunotech, Marseilles, France); MNF116 (Dakopatts, California, USA). *polyclonal antisera +specificities as supplied by manufacturers antigenicity warrants antigen retrieval in most instances. Hence, the need for extensive and carefully controlled optimization of every new antibody before diagnostic application cannot be overemphasized.
References
•Battifora H 1988. Diagnostic uses of antibodies to keratins: a review and immunohistochemical comparison of seven monoclonal and three polyclonal antibodies. In: Fenoglio-Preiser CM, Wolff M, Rilke F (eds) Progress in surgical pathology, vol. VIII. Berlin: Springer-Verlag, pp 1-15.
•Heatley, MK 1996. Cytokeratins and cytokeratin staining in diagnostic histopathology (commentary). Histopathology 28: 479-483.
•Miettinen M 1993. Keratin immunohistochemistry: update of applications and pitfalls. In: Rosen PP, Fechner RE (eds) Pathology annual, part 2/vol 28. New York: Appleton and Lange pp 113-143. (eds) Pathology annual, part 2/vol 28. New York: Appleton and Lange pp 113-143.
•Moll R, Franke WW, Schiller DL, Geiger B, Krepler R 1982. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 31: 11-24.
•Schaafsma HE, Ramaekers FCS 1994. Cytokeratin subtyping in normal and neoplastic epithelium: basic principles and diagnostic applications. In: Rosen PP, Fechner RE (eds) Pathology annual, part I/vol 29. New York: Appleton and Lange pp 21-62.
Bibliografía
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
