SRF AND KI-67 EXPRESSION IN ACQUIRED CHOLESTEATOMA, EXTERNAL AUDITORY CANAL SKIN AND THORACIC SKIN
Introduction
Cholesteatoma is defined by a keratinizing squamous epithelium in the middle ear cavities. Although cho-lesteatoma is a benign disease, it can invade neighboring tissues and often recur even if surgical resection is considered to be complete. Cholesteatoma is traditionally classified as either acquired, essentially due to a chronic otitis process, or congenital. The origin of acquired cholesteatoma (AC) remains under discussion. Among the various advanced theories, the most probable one considers the migration of epithelial cells from the external auditory canal as the origin of the pathology. This migration can either start from the margins of a tympanic perforation, or from the retraction of the tympanic membrane.1,2 This hypothesis is based on two observations: Firstly, the epithelium of the external auditory canal skin (EACS) presents a histological structure similar to that of cholesteatoma. In addition, the keratinocytes of the EACS exhibit particular kinetic properties that allow them, in the absence of underlying pathological conditions, to move laterally towards the pinna (lateral keratinization).3 The particularly aggressive behavior of AC can be explained – at least partially – by disorders in growth regulation of keratinocytes. They exhibit in particular a high rate of proliferation.2
Serum response factor (SRF) is a transcription factor involved in the regulation of numerous genes mostly implicated in proliferation and migration of several cell types, in the control of cell cycle, in apoptosis and in cytoskeleton regulation. It has recently been involved in the pathogenesis of hyperproliferative skin disease.4 AC is precisely characterized by hyperproliferative keratinocytes.
The aim of this study was to investigate the potential role of SRF in the pathogenesis of AC. For this, its expression was first studied in AC, EACS without middle ear associated disease and in thoracic skin. The interest of comparing these two types of epidermis is to study an epidermis with a classical keratinization (thoracic skin) in comparison with an epidermis with a lateral keratinization (thoracic skin). Also, we studied SRF expression in EACS of patients suffering from AC and in EACS of patients suffering from non-cholesteatomatous chronic otitis media (NC-COM).
Materials and methods
Histopathologic and clinical data
This study was approved by the ethical committee of the Erasmus University Hospital (ref P2010/068). For the first part of this study, 30 AC (16 male, 14 female, average age 47.2 years) were obtained in adult patients immediately after ear surgery. All AC were subjected to the standard diagnosis routine in our department of Pathology. Specimens of normal EACS and thoracic skin (n = 9) obtained during autopsy served as controls. All autopsies were performed within a 24 hours postmortem delay to ensure a good preservation and staining of tissue antigens. For the second part of this study, samples of EACS were collected during middle-ear surgery. Ten specimens of EACS of patients with AC and ten specimens of EACS of patients with NC-COM were obtained.
All specimens were immediately fixed in 4% formaldehyde and embedded in paraffin. Sections were cut at a thickness of five µm and processed for hematoxylin-eosin staining using routine protocols.
Immunohistochemistry
Immunohistochemistry was performed on five-µm-thick sections mounted on silane-coated glass slides. First, the dewaxed tissue sections were subjected to microwave pretreatment (2 x 5 min, 850 W) in a citrate buffer (pH = 6). The sections were then incubated with a solution of hydrogen peroxide to block the endogenous peroxidase activity, and rinsed in a phosphate-buffered saline. Samples were then exposed at room temperature to solutions of the specific primary antibody against SRF (G-20, polyclonal, Santacruz) and Ki-67 (MIB-1, monoclonal, Dako), and then, after rinsing, to the corresponding biotinylated secondary antibody. After another rinsing, the samples were incubated with the avidin-biotin-peroxidase complex (Avidin/Biotin blocking kit, Vector, CA). Finally, incubation with the chromogenic substrates containing diaminobenzine and hydrogen peroxide revealed the antibodies. A counterstaining with hematoxylin completed the preparation.
Evaluation
For both antibodies, we calculated the ratio between the number of labeled nuclei and the total number of nuclei within the basal (first cellular layer) and suprabasal layer of the epithelium. The entire slide was analyzed using binocular microscopy at a 400 x magnification. All the statistical analyses were carried out using Statistica (Statsoft, Tulsa, OK).
Results
Evaluation of SRF and Ki-67 in AC, EACS without middle ear associated pathology and thoracic skin (Fig. 1)
Fig. 1. Ki-67 epithelial expression (% of marked nuclei) in AC, EACS and Thoracic skin.
Ki-67 exhibits as expected a higher rate of labeled nuclei in AC compared to EACS (p < 0,01, Mann-Whitney U test). However, this difference does not appear if we compare AC and thoracic skin (p > 0,05).
SRF exhibits a nuclear and cytoplasmic staining in all epithelia. SRF is less expressed in EACS in comparison to AC and thoracic skin (p < 0.01). Moreover, the expression of SRF between the epithelial layers exhibits some particular feature, with a preferentially suprabasal staining in AC (p < 0.001, Wilcoxon signed-rank test) and a larger basal expression in thoracic skin (p < 0.01).
Evaluation of SRF in EACS of patients with AC and in EACS of patients with NC-COM (Fig. 2)
Fig. 2. SRF epithelial expression (% of marked nuclei) in AC, EACS and Thoracic skin.
EACS of patients with AC exhibits a preferentially suprabasal staining compared to EACS of patients with NC-COM (p < 0.01, Mann-Whitney U test). Unlike the suprabasal layer, there is no difference in the SRF expression pattern of the basal layer.
Conclusion
Besides the interest of studying the potential role of SRF in the pathogenesis of cholesteatoma, this study has the benefit of being the first comparative study of the EACS in two different middle ear pathological conditions (in AC and in NC-COM). The data reported in this study suggest that the changes of keratinocytes proliferative activity in AC could be linked to the shift of the SRF expression from the basal to the suprabasal layer. Because SRF is an important regulator of cell migration and actin cytoskeleton,5 an assumption would be that potential change of expression observed within epidermal layers can alter the normal movements of keratinocytes. Moreover, the fact that this expression pattern is found in EACS of patients with AC but not in EACS of patients with NC-COM constitutes an argument in favor of the participation of the EACS in the development of AC.
Fig. 3. SRF epithelial expression in EACS of patients with AC an NC-COM.
References
1.Broekaert D. The problem of middle ear cholesteatoma: etiology, genesis and pathobiology. A review. Acta Otorhinolaryngol Belg 45(4):355–367, 1991
2.Olszewska E, Wagner M, Bernal-Sprekelsen M, Ebmeyer J, Dazert S, Hildmann H, et al. Etiopathogenesis of cholesteatoma. Eur Arch Otorhinolaryngol 261(1):6–24, 2004
3.Vennix PP, Kuijpers W, Peters TA, Tonnaer EL, Ramaekers FC. Epidermal Differentiation in the Human External Auditory Meatus. Laryngoscope 106(4):470–475, 1996
4.Koegel H, et al. Loss of serum response factor in keratinocytes results in hyperproliferative skin disease in mice. J Clin Ivest 119:899–910, 2009
5.Verdoni AM, Ikeda S, Ikeda A. Serum response factor is essential for the proper development of skin epithelium. Mamm Genome 21:64–76, 2010
Address for correspondence: Dr. Marc Vander Ghinst, mvdghins@ulb.ac.be
Cholesteatoma and Ear Surgery – An Update, pp. 207–210
Edited by Haruo Takahashi
2013 © Kugler Publications, Amsterdam, The Netherlands