Acta Neuropathol (2007) 113:585–590 DOI 10.1007/s00401-006-0184-3
O RI G I NAL PAPE R
Nuclear -catenin accumulation associates with epithelial morphogenesis in craniopharyngiomas Rolf Buslei · Annett Hölsken · Bernd Hofmann · Jürgen Kreutzer · Florian Siebzehnrubl · Volkmar Hans · Falk Oppel · Michael Buchfelder · Rudolf Fahlbusch · Ingmar Blümcke
Received: 2 October 2006 / Revised: 10 November 2006 / Accepted: 1 December 2006 / Published online: 13 January 2007 © Springer-Verlag 2007
Abstract Activation of the Wnt/wingless signalling cascade is a key mechanism in developmental morphogenesis, whereas aberrant nuclear accumulation of catenin in adult tissues seems to be associated with neoplastic transformation and tumour progression. Adamantinomatous craniopharyngiomas carry activating mutations in exon 3 of the -catenin gene, which results in a distinct pattern of nuclear -catenin accumulation in up to 95% of respective tumour specimens. To better characterise the impact of nuclear -catenin aggregation in these neoplasms, we systematically examined epithelial diVerentiation and cell cycle-associated molecules in accumulating compared to nonaccumulating tumour cell clusters using a cohort of 65 This work is supported by the Johannes and Frieda Marohn Foundation (to RB and BH).
adamantinomatous craniopharyngiomas. Monoclonal antibodies directed against cytokeratins 5/6 (CK5/6) were utilised to diVerentiate squamous from simple epithelium, the latter being identiWed by immunoreactivity for cytokeratins 8 and 18 (CK8/CK18). Intriguingly, nuclear -catenin accumulation in whorl-like tumour cell clusters was always associated with a distinct CK8 and CK18 immunoreactivity, whereas surrounding non-accumulating tumour cells showed exclusively squamous diVerentiation indicated by CK5/ 6 expression. In addition, a low proliferation activity combined with an increased expression of p21WAF1/CIP1, a key control protein of the cell cycle, was observed in -catenin accumulating cells. Our data support an impact of nuclear -catenin on diVerent cytoarchitectural and epithelial diVerentiation patterns in adamantinomatous craniopharyngiomas.
R. Buslei (&) · A. Hölsken · F. Siebzehnrubl · I. Blümcke Department of Neuropathology, Friedrich-Alexander University Erlangen-Nuremberg, Krankenhausstrasse 8-10, 91054 Erlangen, Germany e-mail:
[email protected]
Keywords Wnt-signalling · Nuclear accumulation · Cytokeratins · Morphogenesis · Craniopharyngioma · p21WAF1/CIP1
B. Hofmann · J. Kreutzer · M. Buchfelder Department of Neurosurgery, Friedrich-Alexander University of Erlangen-Nuremberg, Erlangen, Germany
Introduction
V. Hans Department of Neuropathology, Evangelic Hospital Bielefeld, Bielefeld, Germany F. Oppel Department of Neurosurgery, Evangelic Hospital Bielefeld, Bielefeld, Germany R. Fahlbusch International Neuroscience Institute, Hannover, Germany
Craniopharyngiomas (CP) are benign epithelial tumours arising from the sellar region. They constitute approximately 3% of all intracranial neoplasms and are the most common non-neuroepithelial intracerebral neoplasms in children, accounting for 5–10% of intracranial tumours in this age group [14]. The origin of CP is not Wrmly established. They are considered to arise from ectopic remnants of the pharyngeal epithelium following the embryological evagination of Rathke’s pouch or to derive from a misplaced enamel
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organ [7, 26]. CP represent, however, a heterogenous group consisting of adamantinomatous and papillary variants. Both subtypes are diVerent not only histopathologically but also with respect to the clinical manifestation and outcome. Epithelial morphogenesis of adamantinomatous CP is characterized by broad strands of multistratiWed squamous epithelium with peripheral palisading of nuclei resting on a basement membrane. In addition, a mixture of cystic and solid areas with keratin nodules (“wet keratin”) can be microscopically identiWed. Islands Wlled with cells containing translucent nuclei (“ghost cells”) and regressive changes like Wbrosis, calciWcations, old haemorrhages, lympocytic inWltrations and cholesterol deposits are further hallmarks. In contrast, papillary CP exclusively consist of solid sheets of squamous epithelium with a papillary growth pattern, missing cystic appearance as well as regressive elements. Nuclear palisading, wet keratin, ghost cells and calciWcations are absent. They occur mostly in elderly patients and are localized exclusively in the suprasellar region. Although CP are histologically benign and do not metastasize, higher risk of recurrence requires extensive surgical treatment strategies in adamantinomatous variants [6]. We and others were able to demonstrate -catenin mutations and aberrant nuclear -catenin accumulation in the vast majority of adamantinomatous CP [4, 8, 9, 11, 27]. The pathogenic impact of altered Wnt-signalling in adamantinomatous CP remains, however, to be speciWed. Under physiological conditions, binding of Wnt to a membrane receptor inactivates the cytoplasmic GSK3ß complex including APC and -catenin. As a consequence, proteosomal degradation is inhibited and ß-catenin molecules translocate into the nucleus, where they interact with members of the TCF (T cell factor) family of transcription factors. Enhanced expression of target genes plays a fundamental role in proliferation as well as pattern formation, morphogenesis and the evolution of polarity [12, 30]. In mammary secretory cells, experimental deletion of the -catenin N-terminus including GSK3 phosphorylation sites has been identiWed as an important machinery driving transdiVerentiation into epidermis and squamous metaplasia [19]. A similar mechanism may play a role in the morphogenesis of adamantinomatous craniopharyngiomas (AC) as well. CP express a broad spectrum of cytokeratins including CK5/6, CK7, CK8, CK14, CK17, CK18, CK19 and keratin M-903. However, variable intensities have been attributed to tumour subtypes, i.e. adamantinomatous CP versus papillary CP, or cellular compartments (superWcial cell layer, mid-zone of stellate cells, and outer basal layer) [1, 15, 29, 31]. In order to further clarify the consequence of an
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altered Wnt-signalling pathway with corresponding nuclear -catenin aggregation on the aVected tumour cell clusters, we immunohistochemically investigated cytokeratin expression patterns as well as cell-cycle associated molecules like Ki67 and p21WAF1/CIP1 in a cohort of 65 adamantinomatous CP.
Materials and methods Surgical specimens from 65 patients with AC were retrieved from the archives of the Departments of Neuropathology at the University of Erlangen-Nuremberg and the Evangelic Hospital in Bielefeld. There were 35 males and 30 females. The age ranged between 3 and 67 years in the group of males (mean age 32.1 years) and between 2 and 76 years in females (mean age 31.2 years). Each specimen was classiWed according to World Health Organization guidelines using haematoxylin and eosin [14]. Immunohistochemistry An immunohistochemical examination of all surgical specimens was performed using the following panel of antibodies and respective dilutions: -catenin (1:200; clone 14; BD Biosciences, Heidelberg, Germany); CK5/6 (1:200; clone D5/16B4; Zymed Laboratories, Hannover, Germany), CK8 (1:200; clone C-51, BioGenex, San Ramon, USA); CK18 (1:800; clone CY-90; Sigma, Deisenhofen, Germany); p21 (1:30; clone M7202; DAKO, Hamburg, Germany); Ki67 (1:100; clone Mib-1; DAKO). Surgical samples were Wxed overnight in 4% formaldehyde and routinely processed into liquid paraYn. Sections were cut at 5 m or in serial sections at 1 m with a microtome (Microm, Heidelberg), and mounted on positively charged slides (Superfrost + Menzel). The slides were air dried in an incubator at 37°C overnight. Immunohistochemical staining for CK5/6, -catenin and Ki67 was performed using a Ventana semiautomated staining machine (Nexus; Ventana, Illkirch, France) and the Ventana DAB staining system following the manufacturer’s recommendations. For CK8 and CK18 staining, streptavidin-coupled alkaline phosphatase (DAKO) was used and developed by Fast Red (Sigma) as substrate. For p21 staining procedure, a biotinylated secondary antibody and peroxidase-labelled streptavidin was used and 3⬘-3⬘-diaminobenzidine (Sigma) served as chromogen. Microwave or Pronase pre-treatment was performed when recommended. All slides were counterstained with haematoxylin. Grading was based on the revised WHO
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classiWcation system of tumours of the nervous system [14]. Evaluation of immunohistochemical staining Immunohistological staining patterns of the Ki67 proliferation rate, p21 as well as CK5/6, CK8 and CK18 expression was semi-quantitatively estimated using 1 m thin serial sections. Particular emphasis was paid to whorls with distinct nuclear -catenin accumulation. The grading scale included the following: ¡ = absent, + = less than 10% of -catenin accumulating cells were immunopositive, ++ = up to 50% immunoreactive, +++ = more than 50% of -catenin accumulating cells were immunoreactive. Considering the distinct segregation of cytokeratin patterns with -catenin accumulation (see Results and Figs. 1, 2), we did not perform stereological quantiWcation.
Results All tissue samples from the patients included in this study showed histological hallmarks of AC (n = 65),
Fig. 1 Immunohistochemical Wndings in adamantinomatous craniopharyngioma. a H&E staining of an adamantinomatous craniopharyngioma, showing a whorl-like cell cluster of epithelial cells, and peripheral palisading of nuclei Xanked by areas containing regressive changes (scale bar 20 m, applies also to b, c and d). b Cells in the centre of this whorl revealed strong nuclear and cytoplasmic expression of -catenin. In the palisading cell layer,
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according to the following characteristics: keratin nodules (wet keratin), regressive changes (Wbrosis, calciWcation, old haemorrhages and cholesterol clefts) and/or admixture of cystic and solid portions, peripheral palisading of nuclei (Figs. 1a, 2a), whorl-like structures (Fig. 1a) and ghost cells. Immunohistochemically, 62 of 65 tumours (95%) showed a distinct cytoplasmic and nuclear immunoreactivity for -catenin. However, neoplastic cells with nuclear accumulation of -catenin were not equally distributed throughout the tumour. They formed well-demarcated whorl-like structures of densely packed cells (Figs. 1b, 2a). -catenin staining was restricted to the membranous and cytoplasmic compartment in three neoplasms, but only small surgical specimens were available for immunohistochemical analysis in these cases. Recurrent tumour growth occurred in Wve patients with subtotal and one patient with total excision. All of these patients belong to the cohort of AC with -catenin accumulation. Immunohistochemical staining was performed in serial 1 m thin sections using antibodies against CK5/ 6, CK8 and CK18 to examine epithelial diVerentiation of tumour cells with aberrant nuclear -catenin expression compared to non-accumulating cells. All CPs
-catenin immunoreactivity was conWned to the cellular membrane. c Serial sections indicate that -catenin accumulating cells colocalize with CK8 expression. The palisading cells and the epithelial cells surrounding the cluster are negative. d In contrast, squamous epithelial diVerentiation, indicated by expression of CK5/6, is absent in cells showing aberrant -catenin expression
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Fig. 2 Simple epithelial diVerentiation in nuclear -catenin expressing tumour cell clusters. a Neoplastic whorls with distinct nuclear -catenin expression. The palisading cell layer as well as the vast majority of compact epithelial tumour cells reveals membranous staining only. b Serial sections conWrm lack of CK5/6 expression in nuclear -catenin accumulating cell clusters. However, these cells not only express CK8, as shown in Fig. 1c, but also CK18, another marker for simple epithelia (scale bar in (b) 50 m, apply also to a, c and d) (c). d There is a distinct expression of p21WAF1/CIP1 in the latter cell population
showed expression of high molecular weight cytokeratins 5/6. Mainly the internal cords and strands of stellate epithelial cells as well as the superWcial cell layer were positive, whereas the expression in the outer palisaded cell layer was variable. Intriguingly, nuclear -catenin expressing cell clusters of AC were always negative for CK5/6 (Figs. 1d, 2b). In contrast, CK5/6 negative -catenin accumulating cell clusters showed always an intense expression of CK8 (Fig. 1c). The same was observed for CK18, another marker for simple epithelium known to colocalize with CK8 (Fig. 2c). To address the potential association between nuclear -catenin aggregation, epithelial diVerentiation and cell cycle control, we immunohistochemically examined the expression pattern of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 and the proliferation-associated nuclear antigen Ki-67 (Mib-1) using serial sections. The proliferation activity was always low (
