- Email: [email protected]
Expression of Cytokeratins in Oral Adenosquamous Carcinoma: Specific Detection of Individual Cytokeratins by Monoclonal Antibodies
Expression Cytokeratins in 2002;14:232-239. Oral Adenosquamous Carcinoma Asian J OralofMaxillofac Surg ORIGINAL RESEARCH
Expression of Cytokeratins in Oral Adenosquamous Carcinoma: Specific Detection of Individual Cytokeratins by Monoclonal Antibodies Masakatsu Fukuda,1 Akio Tanaka,1 Aya Hamao,1 Munenori Kitada,1 Fumie Fukuda,1 Seiji Suzuki,1 Yumiko Kebusa,2 Yoshiro Yamamoto,3 Hideaki Sakashita,1Kaoru Kusama2 1 Second Department of Oral and Maxillofacial Surgery, Meikai University School of Dentistry, Saitama, Japan 2 Department of Oral Pathology, Meikai University School of Dentistry, Saitama, Japan 3 First Department of Oral and Maxillofacial Surgery, Meikai University School of Dentistry, Saitama, Japan
Abstract Objective: Adenosquamous carcinoma is an exceptionally uncommon malignant tumour with the histological features of both adenocarcinoma and squamous cell carcinoma. This study investigated the histogenetic aspects of adenosquamous carcinoma with the aim of assisting the differential diagnosis. Patients and Methods: Immunohistochemical methods using monoclonal antibodies against cytokeratin 10/13, 14, 18, and 19 were used to clarify the antigenic profile of adenosquamous carcinoma. Results: The distribution of cytokeratin 10/13 was localised in the squamous components of adenosquamous carcinoma and squamous cell carcinoma, except for the basal layer. The reaction for cytokeratin 14 was distinctly positive in the basal cells. The positive reaction for cytokeratin 18 was mainly observed in the luminal columnar cells of adenosquamous carcinoma, whereas it was negative in oral squamous cell carcinoma. No reaction for cytokeratin 19 was observed in either adenosquamous carcinoma or squamous cell carcinoma. Conclusion: These findings indicate that the immunoprofile of cytokeratins might make them useful markers for discriminating between adenosquamous carcinoma and squamous cell carcinoma in the diagnosis of oral tumours. Key Words: Carcinoma, adenosquamous, Immunohistochemistry, Oral medicine
Introduction Adenosquamous carcinoma (AC) is a rare neoplasm that, as the name implies, shows both adenoid and squamous cell differentiation. There have been reports of such tumours in many sites in the body. Gerughty et al described 10 patients, of whom 5 had this carcinoma in the nasal or laryngeal areas and 5 were in the mouth.1 Ellis and Gnepp reviewed 40 patients, and found that only minor oral glands were affected.2 Correspondence: Masakatsu Fukuda, Second Department of Oral and Maxillofacial Surgery, Meikai University School of Dentistry, 1-1 Keyakidai, Sakado, Saitama 350-0283, Japan. Tel: (81 492) 85 5511, Fax: (81 492) 79 1193 E-mail: [email protected]
232
The main sites appear to be the floor of the mouth, the posterior tongue, and the faucial area. Men are affected more than women, at a ratio of 2:1, and the peak age incidence is in the sixth and seventh decades of life. Clinically, the tumour resembles oral squamous cell carcinoma (SCC). AC has intermingled areas of adenocarcinoma with squamous carcinoma but, unlike mucoepidermoid carcinoma, distinct and separate areas of both types of carcinoma are also seen. AC should be discriminated from pseudoglandular areas in SCC, as in adenoid squamous carcinoma (ASC). ASC is also uncommon, and is Asian J Oral Maxillofac Surg Vol 14, No 4, 2002
Fukuda, Tanaka, Hamao, et al
known as a variant type of SCC, in which gland-like spaces are created by acantholysis in islands of tumour cells. ASC is an entirely different entity from AC, which is composed of both squamous and glandular structures.3
had undergone radiotherapy or chemotherapy before surgery. Samples were fixed in 10% buffered formalin and embedded in paraffin wax. Tissue sections 3 µm thick were prepared for immunohistochemistry.
The expression of cytokeratins (CKs), which are epithelial cell-specific intermediate filament proteins, has been widely investigated in human neoplasms, including those of the salivary glands. The patterns of CK expression are of practical significance in cancer diagnosis as they are generally tissue-specific. Immunohistochemical observations suggest that the expression of keratins is tightly linked with epithelial differentiation, the composition and patterns of expression in normal epithelium being reflected to a large extent in their neoplastic lesions.4
Chemicals and reagents were obtained from the following companies: monoclonal antibody (mAb) DE-K13 against CK10/13 (Santa Cruz Biotechnology, Inc, Santa Cruz, USA); mAb against CK14 (Biømeda Corp, Foster City, USA); mAb DC10 against CK18 (Santa Cruz Biotechnology, Inc.); streptavidin-peroxidase (GIBCO-BRL, Life Technologies Inc., Gaithersburg, USA); biotinylated horse anti-mouse immunoglobulin (Ig) G (H+L) antibody (Vector Laboratories Inc, Burlingame, USA); and 3,3'-diaminobenzidine tetrahydrochloride (DAB, Sigma Chem Ind, St Louis, USA).
Extensive reviews have dealt with the regulation of expression, assembly, interactions with other cell structures, and possible functions of CKs.5 Keratin filaments are assembled from heterodimeric subunits composed of type I acidic (numbers 9 to 21) and type II basic (numbers 1 to 8) cytokeratins, and each is encoded by its own gene.6 Although many keratins are present in a particular epithelial cell, the acidic and basic keratins are present in equimolar amounts. In particular, CK10 and 13 are strongly expressed in a subset of SCCs, while CK18 is expressed in a majority of adenocarcinomas and basal cell carcinomas.7,8 Furthermore, CK14 shows obvious expression in the basal layer of SCC. It has also been reported that CK19 is expressed in basal keratinocytes of some stratified epithelia9 and in striated duct luminal cells of salivary glands.4 In this study, immunohistochemical methods using monoclonal antibodies against CK10/13, 14, 18, and 19 were used to clarify the antigenic profile of AC, with the aim of investigating the histogenetic aspects as well as assisting in the differential diagnosis of AC from SCC.
Patients and Methods Tissues from 4 patients with AC and 4 patients with SCC who were initially diagnosed on the basis of the World Health Organization (WHO) classification were involved in this study. None of the patients Asian J Oral Maxillofac Surg Vol 14, No 4, 2002
Deparaffinised sections were immersed in absolute methanol containing 0.3% H2O2 for 20 minutes at room temperature to block endogenous peroxidase activity. After being washed with phosphate-buffered saline (PBS, pH 7.4), each section was immersed in 0.01 M citrate buffer (pH 6.0) and heated in a microwave oven for 15 minutes as described by Shi et al.10 After being washed with PBS, the sections were incubated in 2% bovine serum albumin in PBS for 15 minutes at room temperature to block non-specific reactions. Appropriately diluted mAb DE-K13, anti-CK14 antibody, DC-10, or anti-CK19 antibody was applied to the sections for 60 minutes at room temperature. After being washed with PBS, the slides were incubated with biotinylated horse anti-mouse IgG (H+L) antibody (1:200) for 30 minutes at room temperature. Appropriately diluted streptavidinperoxidase was applied to the tissue sections for 30 minutes. The sections were immersed for 10 minutes in 0.05% 3,3'-diaminobenzidine tetrahydrochloride in 0.05 M Tris-HCl buffer (pH 7.6) containing 0.01% hydrogen peroxide (H2O2) and then counterstained with Mayer’s hematoxylin.
Results Histopathological examinations revealed that areas of squamous cell carcinoma mixed with foci of 233
Expression of Cytokeratins in Oral Adenosquamous Carcinoma
a
b
neoplastic ductal structures characteristic of adenocarcinoma were noted in all ACs. The surface epithelium displayed a range of cells from epithelial dysplasia to carcinoma. The dysplastic epithelium exhibited an abrupt transition to an invasive epithelial neoplasm with squamous and glandular components (Figures 1a and b). The squamous cell component was keratinising in all tumours. The adenocarcinomatous pattern consisted of neoplastic ducts and small glandular structures lined by single or double layers of cuboidal cells and occasional basaloid cells (Figures 1c and d). The hematoxylin and eosin-stained sections of SCC revealed a range of epithelial dysplasia and an invasive proliferation of malignant epithelial cells involving the submucosal layer (Figure 1e). The immunostaining patterns of the CKs for the major tumour cell types of AC are summarised in Table 1. Immunostaining of the CKs varied according to the cellular type and the position of the cells
c
d
e
Figure 1. Hematoxylin and eosin-stained sections. (a) Presence of dysplastic epithelium; (b) invasive epithelial neoplasm with squamous and glandular components in adenosquamous carcinoma; (c) carcinomatous nests composed of squamous nests with keratin pearl formation; (d) glandular nests with luminal structures; and (e) range of epithelial dysplasia to an invasive proliferation of malignant epithelial cells involving the submucosal layer in squamous cell carcinoma.
234
Asian J Oral Maxillofac Surg Vol 14, No 4, 2002
Fukuda, Tanaka, Hamao, et al
a
b
c
d
Figure 2. Non-keratinised stratified squamous epithelium stained for cytokeratin 10/13. All suprabasal keratinocytes are positive. (a) The basal keratinocytes are clearly negative in non-neoplastic epithelium adjacent to the tumour of adenosquamous carcinoma and squamous cell carcinoma; (b) dysplasia or invasive lesions of the squamous components of adenosquamous carcinoma; (c) squamous cell carcinoma are negative; and (d) the duct-like structure of adenosquamous carcinoma is entirely negative.
in the squamous epithelial and glandular tumour arrangements. In AC and SCC, independent of their topography, the stratified squamous epithelium cells were predominantly positive for CK10/13. However, the basal Patient number*
Cytokeratin profile Squamous component
1 2 3 4 5 6 7 8
layers were distinctly negative in non-neoplastic epithelium adjacent to the tumour (Figure 2a), whereas the immunoreactivity of CK10/13 changed to negative in dysplasia or invasive lesions of the squamous components of both AC and SCC (Figures 2b and c). The immunoreactivities for CK10/13 were entirely
Glandular component
10/13
14
18
19
10/13
14
18
19
+ + + + ± + + +
+ + ++ ++ ++ ++ + +
— — — — +† +‡ — —
— — — — — — — —
— — — —
+ ++ ++ +
+ ± ± +
— — — —
* Patients 1 to 4 had adenosquamous carcinoma and patients 5 to 8 had squamous cell carcinoma. Rare positive cells identified as Merkel cells. ‡ Invasive lesion. †
Table 1. Expression of different types of cytokeratins in adenosquamous carcinoma and squamous cell carcinoma.
Asian J Oral Maxillofac Surg Vol 14, No 4, 2002
235
Expression of Cytokeratins in Oral Adenosquamous Carcinoma
a
b
c
d
Figure 3. Orthokeratinised stratified squamous epithelium stained for cytokeratin 14. (a) Positive cells are generally restricted to the basal layer in non-neoplastic epithelium adjacent to the tumour of adenosquamous carcinoma and squamous cell carcinoma; (b) cytokeratin 14 is strongly positive in the invasive lesion of the squamous components of adenosquamous carcinoma; (c) cytokeratin 14 is strongly positive in the invasive lesion of the squamous components of squamous cell carcinoma; and (d) cytokeratin 14 is strongly positive in the invasive lesion of the duct-like structure of adenosquamous carcinoma.
negative in the duct-like structure of AC (Figure 2d). The immunoreactivity of CK14 was usually strongly positive in the basal layer of non-neoplastic epithelium adjacent to the tumour, although it was also expressed by suprabasal keratinocytes (Figure 3a). The positive reaction for CK14 was also observed in the invasive region of the squamous components of both AC and SCC (Figures 3b and c) and in the duct-like structures of AC (Figure 3d), in contrast to the pattern of immunoreactivity of CK10/13. The immunoreactivity of CK18 was usually negative, except in some cells such as Merkel cells, in the squamous epithelium. The positive reaction for CK18 corresponded with Merkel cells in the basal layer of SCC (Figure 4a). However, expression of CK18 appeared in the invasive front of only 1 case of SCC (Figure 4b). 236
Simultaneously, in the glandular-pattern areas of AC, the immunoreactivity for CK18 was heterogeneously and discretely observed (Figure 4c). No reaction for CK19 was observed in either AC or SCC.
Discussion AC is a relatively rare oral tumour that has been defined by the WHO as a malignant tumour with histological features of both adenocarcinoma and squamous cell carcinoma. The histological characteristics of AC in this study were similar to those classically described in the literature.11,12 Two major growth patterns were identified: squamous epithelial and gland-like patterns. The main cellular types were squamous, columnar, and cuboid. The squamous epithelium areas showed various stratified squamous cells. Asian J Oral Maxillofac Surg Vol 14, No 4, 2002
Fukuda, Tanaka, Hamao, et al
The expression of CKs, epithelial cell-specific intermediate proteins, has been widely investigated in human neoplasia, including neoplastic lesions of salivary glands. The patterns of expression of CKs are of practical significance in cancer diagnosis, as they are generally expressed in a tissue-specific manner.
was absent in most buccal mucosal squamous carcinomas,13 and Kannan et al regarded CK10 and 11 expression as confirmation that a lesion was not dysplastic.14 It was reported that CK10 is lost as dysplasia becomes more severe15 and is not expressed by malignant cells, even where a carcinoma shows keratinisation.
Based on immunohistochemical observations, the expression of keratins is tightly linked to epithelial differentiation, in that the composition and patterns of expression in normal epithelium are reflected to a large extent in their neoplastic lesions.4 Extensive overviews and reviews have dealt with the regulation of expression, assembly, interactions with other cell structures and possible functions of CKs.5
On the other hand, differences in the distribution of CK14 have also been reported. A high level of CK14 expression is not seen in epithelium, but suprabasal CK14 expression in keratinised oral epithelium, sometimes extending through the entire thickness of the rete ridges, has been reported. 9,16 These findings suggest that the immunoreactivity of CK14 may appear where there is dysplasia or an invasive lesion of the squamous components and duct-like structures. Suprabasal keratinocytes may express a more basaloid phenotype, for example CK14, particularly when invasion supervenes.15 However, it has been shown that, in some tumours, CK14 may be lost. This suggests that CK14 is a primitive cytokeratin and may appear wherever a tumour is proliferating such as in a dysplastic or invasive lesion.
The expression of CK10/13 may be lost where there is cell atypia. Vaidya et al reported that CK13 a
Merkel cells, derived from the neural crest and thought to act as some form of receptor, are found scattered within the basal layer of stratified epithelia, often in close apposition to nerve fibres.17,18 In this study, the positive reaction for CK18 corresponded with Merkel cells in the basal layer of SCC. Furthermore, it has been reported that CK18 is expressed in b
c
Figure 4. The stratified squamous epithelium and glandular pattern stained for cytokeratin 18. (a) Occasional Merkel cells are seen in the basal layer; (b) cytokeratin 18 is expressed in the invasive front of only 1 squamous cell carcinoma; and (c) the immunoreactivity of cytokeratin 18 is heterogeneous and discrete in adenosquamous carcinoma.
Asian J Oral Maxillofac Surg Vol 14, No 4, 2002
237
Expression of Cytokeratins in Oral Adenosquamous Carcinoma
severe epithelial dysplasia and frank carcinoma, regardless of the degree of differentiation, mainly at the invasive front.13,19-21 Ogden et al have reported that the clinically normal oral stratified epithelium adjacent to the sites of malignancy may also revert in this way.22 In our study, this inconsistency suggested that there might not be normal translation to produce the actual protein of CK18. However, Balm et al have suggested that the CK18-negativity of oral SCC was a factor that allowed it to be distinguished from similar carcinomas of laryngeal or hypopharyngeal origin.23 Therefore, we believe that CK18 is a reliable marker that would be able to distinguish AC from ASC, which has glandlike spaces created by acantholysis in islands of tumour cells. CK19 has been reported to be found in the basal layer. However, such expression has been proposed to be the result of inflammation.24, 25 Although all of these results are largely consistent with those of immunohistochemical staining in previous studies, there are various expressional characteristics of CKs in oral tumours. These results indicate that the immunoprofile of CKs in oral tumour diagnosis might be useful for discriminating between AC and SCC. In addition, it is important to recognise the pattern of expression of various CKs in relation to morphological characteristics for accurately discriminating between AC and SCC.
References 1. Gerughty RM, Hennigar GR, Brown FM. Adenosquamous carcinoma of the nasal, oral and laryngeal cavities. Cancer 1968;22:1140-1155. 2. Ellis GL, Gnepp DR. Unusual salivary gland tumors. In: Gnepp DR, editor. Pathology of the head and neck. New York: Churchill Livingstone; 1988:627-631. 3. Jones AC, Freedman PD, Kerpel SM. Oral adenoid squamous carcinoma: a report of three cases and review of the literature. J Oral Maxillofacial Surg 1993;51:676-681. 4. Li C, Okamoto Y, Ohmura H, Ogawa K, Shrestha P, Mori M. Expression of cytokeratins in Warthin’s tumor (adenolymphoma) of parotid glands: Specific detection of individual cytokeratin types by 238
monoclonal antibodies. Eur J Cancer B Oral Oncol 1996;32B:352-358. 5. van de Klundert FAJM, Raats JM, Bloemendal H. Intermediate filaments: regulation of gene expression and assembly. Eur J Biochem 1993; 214:351-366. 6. Hatzfeld M, Franke WW. Pair formation and promiscuity of cytokeratins: formation in vivo of heterotypic complexes and intermediate sized filaments by homologous and heterogeneous recombinations of purified polypeptides. J Cell Biol 1985;101:1826-1846. 7. Silen A, Wiklund B, Andersson EL, Nilsson S. A novel IRMA and ELSA for quantifying cytokeratin 8 and 18 fragments in the sera of healthy individuals and cancer patients. Scan J Clin Lab Invest 1995;55:153-161. 8. Silen A, Wiklund B, Norlen BJ, Nilsson S. Evaluation of a new tumor marker for cytokeratin 8 and 18 fragments in healthy individuals and prostate cancer patients. Prostate 1994;24: 326-332. 9. Morgan PR, Leigh IM, Purkis PE, Gardner ID, van Muijen GNP, Lane EB. Site variation in keratin expression in human oral epithelia: an immunocytochemical study of individual keratins. Epithelia 1987;1:31-43. 10. Shi SR, Key ME, Kalra KL. Antigen retrieval in formalin-fixed and paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 1991;39: 741-748. 11. Bhaskar SN, Bernier JL. Mucoepidermoid tumors of major and minor salivary gland: Clinical features, histology, variations, natural history, and results of treatment for 144 cases. Cancer 1962; 15:801-817. 12. Healey WV, Perzin KH, Smith L. Mucoepidermoid carcinoma of salivary gland origin: classification, clinical-pathologic correlation, and results of treatment. Cancer 1970;26:368-388. 13. Vaidya MM, Borges AM, Pradhan SA, Bhisey AN. Cytokeratin expression in squamous cell carcinomas of the tongue and alveolar mucosa. Eur J Cancer B Oral Oncol 1996;2B:333-336. 14. Kannan S, Balaram P, Chandran GJ, Pillai MR, Mathew B, Nalinakumari KR, Nair MK. Alterations in expression of terminal differentiation Asian J Oral Maxillofac Surg Vol 14, No 4, 2002
Fukuda, Tanaka, Hamao, et al
markers of keratinocytes during oral carcinogenesis. Pathobiol 1994;62:127-133. 15. Heyden A, Huitfeldt HS, Koppang HS, Thrane PS, Bryne M, Brandtzaeg P. Cytokeratins as epithelial differentiation markers in premalignant and malignant oral lesions. J Oral Pathol Med 1992;21:7-11. 16. Mackenzie ICM, Rittman G, Gao Z, Leigh I, Lane EB. Patterns of cytokeratin expression in human gingival epithelia. J Periodont Res 1991;26: 468-478. 17. Moll R, Moll I, Franke WW. Identification of Merkel cells in human skin by specific cytokeratin antibodies: changes in cell density and distribution in fetal and adult plantar epidermis. Differentiation 1984;28:136-154. 18. Gould VE, Moll R, Moll I, Franke WW. Neuroendocrine (Merkel) cells of the skin: hyperplasias, dysplasias, and neoplasms. Lab Invest 1985;52:443-453. 19. Su L, Morgan PR, Lane EB. Protein and mRNA expression of simple epithelial keratins in normal, dysplastic and malignant oral epithelia. Am J Pathol 1994;145:1349-1357. 20. Ogden GR, Chisholm DM, Adi M, Lane EB.
Asian J Oral Maxillofac Surg Vol 14, No 4, 2002
Cytokeratin expression in oral cancer and its relationship to tumor differentiation. J Oral Pathol Med 1993;22:82-86. 21. Schaafsma HE, van der Velden LA, Manni JJ, Peters H, Link M, Ruiter DJ, Ramaekers FCS. Increased expression of cytokeratins 8, 18 and vimentin in the invasion front of mucosal squamous cell carcinoma. J Path 1993;170:77-86. 22. Ogden GR, Lane EB, Hopwood DV, Chisholm DM. Evidence for field change in oral cancer based on cytokeratin expression. Brit J Cancer 1993;67:1324-1330. 23. Balm AJ, Hageman PC, van Doornewaard MH, Groeneveld EM, Ivanyi D. Cytokeratin 18 expression in squamous cell carcinoma of the head and neck. Eur Archs Oto Rhino Laryngol 1996;253:227-233. 24. Bosch FX, Ouhayoun JP, Bader BL, Collin C, Grund C, Lee I, Franke WW. Extensive changes in cytokeratin expression patterns in pathology affected human gingiva. Vir Arch (B) 1989;58:59-77. 25. Ouhayoun JP, Goffaux JC, Sawaf MH, Shabana AHM, Collin C, Forest N. Changes in cytokeratin expression in gingiva during inflammation. J Periodont Res 1990;25;283-292.
239
Expression of Cytokeratins in Oral Adenosquamous Carcinoma: Specific Detection of Individual Cytokeratins by Monoclonal Antibodies Masakatsu Fukuda,1 Akio Tanaka,1 Aya Hamao,1 Munenori Kitada,1 Fumie Fukuda,1 Seiji Suzuki,1 Yumiko Kebusa,2 Yoshiro Yamamoto,3 Hideaki Sakashita,1Kaoru Kusama2 1 Second Department of Oral and Maxillofacial Surgery, Meikai University School of Dentistry, Saitama, Japan 2 Department of Oral Pathology, Meikai University School of Dentistry, Saitama, Japan 3 First Department of Oral and Maxillofacial Surgery, Meikai University School of Dentistry, Saitama, Japan
Abstract Objective: Adenosquamous carcinoma is an exceptionally uncommon malignant tumour with the histological features of both adenocarcinoma and squamous cell carcinoma. This study investigated the histogenetic aspects of adenosquamous carcinoma with the aim of assisting the differential diagnosis. Patients and Methods: Immunohistochemical methods using monoclonal antibodies against cytokeratin 10/13, 14, 18, and 19 were used to clarify the antigenic profile of adenosquamous carcinoma. Results: The distribution of cytokeratin 10/13 was localised in the squamous components of adenosquamous carcinoma and squamous cell carcinoma, except for the basal layer. The reaction for cytokeratin 14 was distinctly positive in the basal cells. The positive reaction for cytokeratin 18 was mainly observed in the luminal columnar cells of adenosquamous carcinoma, whereas it was negative in oral squamous cell carcinoma. No reaction for cytokeratin 19 was observed in either adenosquamous carcinoma or squamous cell carcinoma. Conclusion: These findings indicate that the immunoprofile of cytokeratins might make them useful markers for discriminating between adenosquamous carcinoma and squamous cell carcinoma in the diagnosis of oral tumours. Key Words: Carcinoma, adenosquamous, Immunohistochemistry, Oral medicine
Introduction Adenosquamous carcinoma (AC) is a rare neoplasm that, as the name implies, shows both adenoid and squamous cell differentiation. There have been reports of such tumours in many sites in the body. Gerughty et al described 10 patients, of whom 5 had this carcinoma in the nasal or laryngeal areas and 5 were in the mouth.1 Ellis and Gnepp reviewed 40 patients, and found that only minor oral glands were affected.2 Correspondence: Masakatsu Fukuda, Second Department of Oral and Maxillofacial Surgery, Meikai University School of Dentistry, 1-1 Keyakidai, Sakado, Saitama 350-0283, Japan. Tel: (81 492) 85 5511, Fax: (81 492) 79 1193 E-mail: [email protected]
232
The main sites appear to be the floor of the mouth, the posterior tongue, and the faucial area. Men are affected more than women, at a ratio of 2:1, and the peak age incidence is in the sixth and seventh decades of life. Clinically, the tumour resembles oral squamous cell carcinoma (SCC). AC has intermingled areas of adenocarcinoma with squamous carcinoma but, unlike mucoepidermoid carcinoma, distinct and separate areas of both types of carcinoma are also seen. AC should be discriminated from pseudoglandular areas in SCC, as in adenoid squamous carcinoma (ASC). ASC is also uncommon, and is Asian J Oral Maxillofac Surg Vol 14, No 4, 2002
Fukuda, Tanaka, Hamao, et al
known as a variant type of SCC, in which gland-like spaces are created by acantholysis in islands of tumour cells. ASC is an entirely different entity from AC, which is composed of both squamous and glandular structures.3
had undergone radiotherapy or chemotherapy before surgery. Samples were fixed in 10% buffered formalin and embedded in paraffin wax. Tissue sections 3 µm thick were prepared for immunohistochemistry.
The expression of cytokeratins (CKs), which are epithelial cell-specific intermediate filament proteins, has been widely investigated in human neoplasms, including those of the salivary glands. The patterns of CK expression are of practical significance in cancer diagnosis as they are generally tissue-specific. Immunohistochemical observations suggest that the expression of keratins is tightly linked with epithelial differentiation, the composition and patterns of expression in normal epithelium being reflected to a large extent in their neoplastic lesions.4
Chemicals and reagents were obtained from the following companies: monoclonal antibody (mAb) DE-K13 against CK10/13 (Santa Cruz Biotechnology, Inc, Santa Cruz, USA); mAb against CK14 (Biømeda Corp, Foster City, USA); mAb DC10 against CK18 (Santa Cruz Biotechnology, Inc.); streptavidin-peroxidase (GIBCO-BRL, Life Technologies Inc., Gaithersburg, USA); biotinylated horse anti-mouse immunoglobulin (Ig) G (H+L) antibody (Vector Laboratories Inc, Burlingame, USA); and 3,3'-diaminobenzidine tetrahydrochloride (DAB, Sigma Chem Ind, St Louis, USA).
Extensive reviews have dealt with the regulation of expression, assembly, interactions with other cell structures, and possible functions of CKs.5 Keratin filaments are assembled from heterodimeric subunits composed of type I acidic (numbers 9 to 21) and type II basic (numbers 1 to 8) cytokeratins, and each is encoded by its own gene.6 Although many keratins are present in a particular epithelial cell, the acidic and basic keratins are present in equimolar amounts. In particular, CK10 and 13 are strongly expressed in a subset of SCCs, while CK18 is expressed in a majority of adenocarcinomas and basal cell carcinomas.7,8 Furthermore, CK14 shows obvious expression in the basal layer of SCC. It has also been reported that CK19 is expressed in basal keratinocytes of some stratified epithelia9 and in striated duct luminal cells of salivary glands.4 In this study, immunohistochemical methods using monoclonal antibodies against CK10/13, 14, 18, and 19 were used to clarify the antigenic profile of AC, with the aim of investigating the histogenetic aspects as well as assisting in the differential diagnosis of AC from SCC.
Patients and Methods Tissues from 4 patients with AC and 4 patients with SCC who were initially diagnosed on the basis of the World Health Organization (WHO) classification were involved in this study. None of the patients Asian J Oral Maxillofac Surg Vol 14, No 4, 2002
Deparaffinised sections were immersed in absolute methanol containing 0.3% H2O2 for 20 minutes at room temperature to block endogenous peroxidase activity. After being washed with phosphate-buffered saline (PBS, pH 7.4), each section was immersed in 0.01 M citrate buffer (pH 6.0) and heated in a microwave oven for 15 minutes as described by Shi et al.10 After being washed with PBS, the sections were incubated in 2% bovine serum albumin in PBS for 15 minutes at room temperature to block non-specific reactions. Appropriately diluted mAb DE-K13, anti-CK14 antibody, DC-10, or anti-CK19 antibody was applied to the sections for 60 minutes at room temperature. After being washed with PBS, the slides were incubated with biotinylated horse anti-mouse IgG (H+L) antibody (1:200) for 30 minutes at room temperature. Appropriately diluted streptavidinperoxidase was applied to the tissue sections for 30 minutes. The sections were immersed for 10 minutes in 0.05% 3,3'-diaminobenzidine tetrahydrochloride in 0.05 M Tris-HCl buffer (pH 7.6) containing 0.01% hydrogen peroxide (H2O2) and then counterstained with Mayer’s hematoxylin.
Results Histopathological examinations revealed that areas of squamous cell carcinoma mixed with foci of 233
Expression of Cytokeratins in Oral Adenosquamous Carcinoma
a
b
neoplastic ductal structures characteristic of adenocarcinoma were noted in all ACs. The surface epithelium displayed a range of cells from epithelial dysplasia to carcinoma. The dysplastic epithelium exhibited an abrupt transition to an invasive epithelial neoplasm with squamous and glandular components (Figures 1a and b). The squamous cell component was keratinising in all tumours. The adenocarcinomatous pattern consisted of neoplastic ducts and small glandular structures lined by single or double layers of cuboidal cells and occasional basaloid cells (Figures 1c and d). The hematoxylin and eosin-stained sections of SCC revealed a range of epithelial dysplasia and an invasive proliferation of malignant epithelial cells involving the submucosal layer (Figure 1e). The immunostaining patterns of the CKs for the major tumour cell types of AC are summarised in Table 1. Immunostaining of the CKs varied according to the cellular type and the position of the cells
c
d
e
Figure 1. Hematoxylin and eosin-stained sections. (a) Presence of dysplastic epithelium; (b) invasive epithelial neoplasm with squamous and glandular components in adenosquamous carcinoma; (c) carcinomatous nests composed of squamous nests with keratin pearl formation; (d) glandular nests with luminal structures; and (e) range of epithelial dysplasia to an invasive proliferation of malignant epithelial cells involving the submucosal layer in squamous cell carcinoma.
234
Asian J Oral Maxillofac Surg Vol 14, No 4, 2002
Fukuda, Tanaka, Hamao, et al
a
b
c
d
Figure 2. Non-keratinised stratified squamous epithelium stained for cytokeratin 10/13. All suprabasal keratinocytes are positive. (a) The basal keratinocytes are clearly negative in non-neoplastic epithelium adjacent to the tumour of adenosquamous carcinoma and squamous cell carcinoma; (b) dysplasia or invasive lesions of the squamous components of adenosquamous carcinoma; (c) squamous cell carcinoma are negative; and (d) the duct-like structure of adenosquamous carcinoma is entirely negative.
in the squamous epithelial and glandular tumour arrangements. In AC and SCC, independent of their topography, the stratified squamous epithelium cells were predominantly positive for CK10/13. However, the basal Patient number*
Cytokeratin profile Squamous component
1 2 3 4 5 6 7 8
layers were distinctly negative in non-neoplastic epithelium adjacent to the tumour (Figure 2a), whereas the immunoreactivity of CK10/13 changed to negative in dysplasia or invasive lesions of the squamous components of both AC and SCC (Figures 2b and c). The immunoreactivities for CK10/13 were entirely
Glandular component
10/13
14
18
19
10/13
14
18
19
+ + + + ± + + +
+ + ++ ++ ++ ++ + +
— — — — +† +‡ — —
— — — — — — — —
— — — —
+ ++ ++ +
+ ± ± +
— — — —
* Patients 1 to 4 had adenosquamous carcinoma and patients 5 to 8 had squamous cell carcinoma. Rare positive cells identified as Merkel cells. ‡ Invasive lesion. †
Table 1. Expression of different types of cytokeratins in adenosquamous carcinoma and squamous cell carcinoma.
Asian J Oral Maxillofac Surg Vol 14, No 4, 2002
235
Expression of Cytokeratins in Oral Adenosquamous Carcinoma
a
b
c
d
Figure 3. Orthokeratinised stratified squamous epithelium stained for cytokeratin 14. (a) Positive cells are generally restricted to the basal layer in non-neoplastic epithelium adjacent to the tumour of adenosquamous carcinoma and squamous cell carcinoma; (b) cytokeratin 14 is strongly positive in the invasive lesion of the squamous components of adenosquamous carcinoma; (c) cytokeratin 14 is strongly positive in the invasive lesion of the squamous components of squamous cell carcinoma; and (d) cytokeratin 14 is strongly positive in the invasive lesion of the duct-like structure of adenosquamous carcinoma.
negative in the duct-like structure of AC (Figure 2d). The immunoreactivity of CK14 was usually strongly positive in the basal layer of non-neoplastic epithelium adjacent to the tumour, although it was also expressed by suprabasal keratinocytes (Figure 3a). The positive reaction for CK14 was also observed in the invasive region of the squamous components of both AC and SCC (Figures 3b and c) and in the duct-like structures of AC (Figure 3d), in contrast to the pattern of immunoreactivity of CK10/13. The immunoreactivity of CK18 was usually negative, except in some cells such as Merkel cells, in the squamous epithelium. The positive reaction for CK18 corresponded with Merkel cells in the basal layer of SCC (Figure 4a). However, expression of CK18 appeared in the invasive front of only 1 case of SCC (Figure 4b). 236
Simultaneously, in the glandular-pattern areas of AC, the immunoreactivity for CK18 was heterogeneously and discretely observed (Figure 4c). No reaction for CK19 was observed in either AC or SCC.
Discussion AC is a relatively rare oral tumour that has been defined by the WHO as a malignant tumour with histological features of both adenocarcinoma and squamous cell carcinoma. The histological characteristics of AC in this study were similar to those classically described in the literature.11,12 Two major growth patterns were identified: squamous epithelial and gland-like patterns. The main cellular types were squamous, columnar, and cuboid. The squamous epithelium areas showed various stratified squamous cells. Asian J Oral Maxillofac Surg Vol 14, No 4, 2002
Fukuda, Tanaka, Hamao, et al
The expression of CKs, epithelial cell-specific intermediate proteins, has been widely investigated in human neoplasia, including neoplastic lesions of salivary glands. The patterns of expression of CKs are of practical significance in cancer diagnosis, as they are generally expressed in a tissue-specific manner.
was absent in most buccal mucosal squamous carcinomas,13 and Kannan et al regarded CK10 and 11 expression as confirmation that a lesion was not dysplastic.14 It was reported that CK10 is lost as dysplasia becomes more severe15 and is not expressed by malignant cells, even where a carcinoma shows keratinisation.
Based on immunohistochemical observations, the expression of keratins is tightly linked to epithelial differentiation, in that the composition and patterns of expression in normal epithelium are reflected to a large extent in their neoplastic lesions.4 Extensive overviews and reviews have dealt with the regulation of expression, assembly, interactions with other cell structures and possible functions of CKs.5
On the other hand, differences in the distribution of CK14 have also been reported. A high level of CK14 expression is not seen in epithelium, but suprabasal CK14 expression in keratinised oral epithelium, sometimes extending through the entire thickness of the rete ridges, has been reported. 9,16 These findings suggest that the immunoreactivity of CK14 may appear where there is dysplasia or an invasive lesion of the squamous components and duct-like structures. Suprabasal keratinocytes may express a more basaloid phenotype, for example CK14, particularly when invasion supervenes.15 However, it has been shown that, in some tumours, CK14 may be lost. This suggests that CK14 is a primitive cytokeratin and may appear wherever a tumour is proliferating such as in a dysplastic or invasive lesion.
The expression of CK10/13 may be lost where there is cell atypia. Vaidya et al reported that CK13 a
Merkel cells, derived from the neural crest and thought to act as some form of receptor, are found scattered within the basal layer of stratified epithelia, often in close apposition to nerve fibres.17,18 In this study, the positive reaction for CK18 corresponded with Merkel cells in the basal layer of SCC. Furthermore, it has been reported that CK18 is expressed in b
c
Figure 4. The stratified squamous epithelium and glandular pattern stained for cytokeratin 18. (a) Occasional Merkel cells are seen in the basal layer; (b) cytokeratin 18 is expressed in the invasive front of only 1 squamous cell carcinoma; and (c) the immunoreactivity of cytokeratin 18 is heterogeneous and discrete in adenosquamous carcinoma.
Asian J Oral Maxillofac Surg Vol 14, No 4, 2002
237
Expression of Cytokeratins in Oral Adenosquamous Carcinoma
severe epithelial dysplasia and frank carcinoma, regardless of the degree of differentiation, mainly at the invasive front.13,19-21 Ogden et al have reported that the clinically normal oral stratified epithelium adjacent to the sites of malignancy may also revert in this way.22 In our study, this inconsistency suggested that there might not be normal translation to produce the actual protein of CK18. However, Balm et al have suggested that the CK18-negativity of oral SCC was a factor that allowed it to be distinguished from similar carcinomas of laryngeal or hypopharyngeal origin.23 Therefore, we believe that CK18 is a reliable marker that would be able to distinguish AC from ASC, which has glandlike spaces created by acantholysis in islands of tumour cells. CK19 has been reported to be found in the basal layer. However, such expression has been proposed to be the result of inflammation.24, 25 Although all of these results are largely consistent with those of immunohistochemical staining in previous studies, there are various expressional characteristics of CKs in oral tumours. These results indicate that the immunoprofile of CKs in oral tumour diagnosis might be useful for discriminating between AC and SCC. In addition, it is important to recognise the pattern of expression of various CKs in relation to morphological characteristics for accurately discriminating between AC and SCC.
References 1. Gerughty RM, Hennigar GR, Brown FM. Adenosquamous carcinoma of the nasal, oral and laryngeal cavities. Cancer 1968;22:1140-1155. 2. Ellis GL, Gnepp DR. Unusual salivary gland tumors. In: Gnepp DR, editor. Pathology of the head and neck. New York: Churchill Livingstone; 1988:627-631. 3. Jones AC, Freedman PD, Kerpel SM. Oral adenoid squamous carcinoma: a report of three cases and review of the literature. J Oral Maxillofacial Surg 1993;51:676-681. 4. Li C, Okamoto Y, Ohmura H, Ogawa K, Shrestha P, Mori M. Expression of cytokeratins in Warthin’s tumor (adenolymphoma) of parotid glands: Specific detection of individual cytokeratin types by 238
monoclonal antibodies. Eur J Cancer B Oral Oncol 1996;32B:352-358. 5. van de Klundert FAJM, Raats JM, Bloemendal H. Intermediate filaments: regulation of gene expression and assembly. Eur J Biochem 1993; 214:351-366. 6. Hatzfeld M, Franke WW. Pair formation and promiscuity of cytokeratins: formation in vivo of heterotypic complexes and intermediate sized filaments by homologous and heterogeneous recombinations of purified polypeptides. J Cell Biol 1985;101:1826-1846. 7. Silen A, Wiklund B, Andersson EL, Nilsson S. A novel IRMA and ELSA for quantifying cytokeratin 8 and 18 fragments in the sera of healthy individuals and cancer patients. Scan J Clin Lab Invest 1995;55:153-161. 8. Silen A, Wiklund B, Norlen BJ, Nilsson S. Evaluation of a new tumor marker for cytokeratin 8 and 18 fragments in healthy individuals and prostate cancer patients. Prostate 1994;24: 326-332. 9. Morgan PR, Leigh IM, Purkis PE, Gardner ID, van Muijen GNP, Lane EB. Site variation in keratin expression in human oral epithelia: an immunocytochemical study of individual keratins. Epithelia 1987;1:31-43. 10. Shi SR, Key ME, Kalra KL. Antigen retrieval in formalin-fixed and paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 1991;39: 741-748. 11. Bhaskar SN, Bernier JL. Mucoepidermoid tumors of major and minor salivary gland: Clinical features, histology, variations, natural history, and results of treatment for 144 cases. Cancer 1962; 15:801-817. 12. Healey WV, Perzin KH, Smith L. Mucoepidermoid carcinoma of salivary gland origin: classification, clinical-pathologic correlation, and results of treatment. Cancer 1970;26:368-388. 13. Vaidya MM, Borges AM, Pradhan SA, Bhisey AN. Cytokeratin expression in squamous cell carcinomas of the tongue and alveolar mucosa. Eur J Cancer B Oral Oncol 1996;2B:333-336. 14. Kannan S, Balaram P, Chandran GJ, Pillai MR, Mathew B, Nalinakumari KR, Nair MK. Alterations in expression of terminal differentiation Asian J Oral Maxillofac Surg Vol 14, No 4, 2002
Fukuda, Tanaka, Hamao, et al
markers of keratinocytes during oral carcinogenesis. Pathobiol 1994;62:127-133. 15. Heyden A, Huitfeldt HS, Koppang HS, Thrane PS, Bryne M, Brandtzaeg P. Cytokeratins as epithelial differentiation markers in premalignant and malignant oral lesions. J Oral Pathol Med 1992;21:7-11. 16. Mackenzie ICM, Rittman G, Gao Z, Leigh I, Lane EB. Patterns of cytokeratin expression in human gingival epithelia. J Periodont Res 1991;26: 468-478. 17. Moll R, Moll I, Franke WW. Identification of Merkel cells in human skin by specific cytokeratin antibodies: changes in cell density and distribution in fetal and adult plantar epidermis. Differentiation 1984;28:136-154. 18. Gould VE, Moll R, Moll I, Franke WW. Neuroendocrine (Merkel) cells of the skin: hyperplasias, dysplasias, and neoplasms. Lab Invest 1985;52:443-453. 19. Su L, Morgan PR, Lane EB. Protein and mRNA expression of simple epithelial keratins in normal, dysplastic and malignant oral epithelia. Am J Pathol 1994;145:1349-1357. 20. Ogden GR, Chisholm DM, Adi M, Lane EB.
Asian J Oral Maxillofac Surg Vol 14, No 4, 2002
Cytokeratin expression in oral cancer and its relationship to tumor differentiation. J Oral Pathol Med 1993;22:82-86. 21. Schaafsma HE, van der Velden LA, Manni JJ, Peters H, Link M, Ruiter DJ, Ramaekers FCS. Increased expression of cytokeratins 8, 18 and vimentin in the invasion front of mucosal squamous cell carcinoma. J Path 1993;170:77-86. 22. Ogden GR, Lane EB, Hopwood DV, Chisholm DM. Evidence for field change in oral cancer based on cytokeratin expression. Brit J Cancer 1993;67:1324-1330. 23. Balm AJ, Hageman PC, van Doornewaard MH, Groeneveld EM, Ivanyi D. Cytokeratin 18 expression in squamous cell carcinoma of the head and neck. Eur Archs Oto Rhino Laryngol 1996;253:227-233. 24. Bosch FX, Ouhayoun JP, Bader BL, Collin C, Grund C, Lee I, Franke WW. Extensive changes in cytokeratin expression patterns in pathology affected human gingiva. Vir Arch (B) 1989;58:59-77. 25. Ouhayoun JP, Goffaux JC, Sawaf MH, Shabana AHM, Collin C, Forest N. Changes in cytokeratin expression in gingiva during inflammation. J Periodont Res 1990;25;283-292.
239