|Year : 2014 | Volume
| Issue : 1 | Page : 10-15
Immunohistochemical localization of β-catenin in medulloblastoma
Rehab M Samaka1, Moshira M Abd El-Wahed1, Mona A Kandil1, Eman Abdelzaher2, Mohamed I Shaaban1, Rania Abd Allah1
1 Pathology Department, Faculty of Medicine, Menoufiya University, Shebin El Kom, Egypt
2 Pathology Department, Faculty of Medicine, Alexandria University, Alexandria, Egypt
|Date of Submission||18-Aug-2013|
|Date of Acceptance||22-Sep-2013|
|Date of Web Publication||20-May-2014|
Rehab M Samaka
Pathology Department, Faculty of Medicine, Menoufiya University, Shebin El Kom 32817
Source of Support: None, Conflict of Interest: None
The aim of this study was to explore the relationship between immunohistochemical expression of β-catenin and the relevant clinicopathological features of medulloblastoma in Egyptian patients.
Medulloblastoma is a small round blue cell malignancy of the cerebellum and is a major cause of morbidity and mortality in pediatric oncology. Identification of the signaling pathways involved in the pathogenesis of medulloblastoma represents a key challenge for medulloblastoma management. The wingless (WNT)-signaling pathway has been reported to be responsible for 15% of sporadic medulloblastoma. β-Catenin represents the downstream effector of the WNT pathway. Activation of the WNT-signaling pathway results in stabilization of β-catenin and its translocation from the cytoplasm to the nucleus where it regulates the related genes.
Patients and methods
This retrospective study was conducted on 49 tissue specimens of medulloblastoma patients for evaluation of immunohistochemical expression of β-catenin.
None of the studied medulloblastoma patients showed nuclear localization of β-catenin. However, 29 patients exhibited cytoplasmic staining and 20 patients were absolutely negative. No statistically significant difference was found when comparing patients with cytoplasmic β-catenin expression and negative patients with the studied parameters. In addition, the H-score value of patients with cytoplasmic β-catenin did not show significant relationship with the studied clinicopathologic parameters.
In view of the absence of nuclear localization of β-catenin, we could conclude that β-catenin does not play a role in the pathogenesis in all evaluated medulloblastoma patients. However, a large-scale study is recommended to elicit the exact role of the WNT-signaling pathway through dysregulation of components other than β-catenin.
Keywords: β-catenin, immunohistochemical, medulloblastoma, WNT signaling
|How to cite this article:|
Samaka RM, Abd El-Wahed MM, Kandil MA, Abdelzaher E, Shaaban MI, Abd Allah R. Immunohistochemical localization of β-catenin in medulloblastoma. Menoufia Med J 2014;27:10-5
|How to cite this URL:|
Samaka RM, Abd El-Wahed MM, Kandil MA, Abdelzaher E, Shaaban MI, Abd Allah R. Immunohistochemical localization of β-catenin in medulloblastoma. Menoufia Med J [serial online] 2014 [cited 2020 Feb 16];27:10-5. Available from: http://www.mmj.eg.net/text.asp?2014/27/1/10/132295
| Introduction|| |
Medulloblastoma is a malignant embryonic tumor of the cerebellum that accounts for 20% of pediatric brain tumors . In Egypt, according to El-Bolkainy et al. , it accounts for ∼25% of pediatric intracranial tumors and 13.15% of central nervous system tumors according to the last registry of the Egyptian National Cancer Institute .
In an era of genomic profiling, risk stratification for medulloblastoma disappointingly remains strictly clinical, based solely on age, resection extent, Chang metastasis staging, and more recently anaplasia [,4]. Multiple histopathologic, cytogenetic, and molecular factors have been found to influence the prognosis, but, to date, none have been incorporated into the risk-stratification system .
The occurrence of medulloblastoma in type 2 Turcot's syndrome, in which patients have multiple colonic polyps and germline mutations in the adenomatous polyposis coli (APC) tumor suppressor gene, has suggested that abnormalities of the wingless (WNT) pathway could be involved in the development of sporadic medulloblastomas ,. Subsequently, mutations in the components of the WNT pathway, including APC and β-catenin-encoding gene (CTNNB1), have been consistently demonstrated in ∼15% of sporadic medulloblastomas and are predicted to cause aberrant pathway activation ,. Recently, it has been described that the WNT-signaling pathway is a marker of favorable patients' outcome in medulloblastoma .
Involvement of the WNT pathway in human medulloblastoma has been studied by several approaches, including mutation analysis of the CTNNB1 gene (encoding β-catenin protein) and of APC, detection of genetic alteration by comparative genomic hybridization , real-time reverse transcriptase PCR , and immunohistochemistry ,.
To the best of our knowledge, this is the first study that explores the role of WNT pathway in the pathogenesis of medulloblastoma in Egyptian patients by detecting the immunohistochemical (IHC) expression of β-catenin and its relationship to the relevant clinicopathological features.
| Patients and methods|| |
This retrospective study included 49 medulloblastoma specimens of Egyptian patients obtained from the Pathology Department, Faculty of Medicine, Menoufiya and Alexandria Universities during the period from March 2002 to February 2012. They were randomly selected pretreatment patients based on the availability of paraffin-embedded blocks for serial cutting and histopathological evaluation. Demographic and clinical features for those patients were collected from the patients' medical records. In terms of age, according to Curran et al. , the patients were divided into three groups depending on the age at the time of diagnosis: below 3 years (infant), 3-17 years (children), and at least 18 years (adults). The tumor sites were divided into two categories: midline (cerebellar vermis) and hemispheric (cerebellar hemispheres) .
The hematoxylin and eosin-stained sections were evaluated: histological type according to the 2007 WHO classification of medulloblastoma ; extra-axial extension together with the presence of connective tissue desmoplasia; and necrosis. Apoptosis was subjectively reported as focal, diffuse, or extensive . Burger and Scheithauer  defined the anaplastic/large-cell-type medulloblastoma variant by the presence of anaplasia and/or large cell type in more than 50% of the tumor area. Thus, we considered the tumor to have anaplastic features (but not anaplastic/large cell type) when cytologic anaplasia involved less than 50% of the tumor cells. Anaplasia was defined by four parameters:
- Increased nuclear size,
- Numerous mitoses,
- Sheets or nodules of large cells with round nuclei and prominent nucleoli, and
- Angular, crowded, pleomorphic nuclei in large cells that is sometimes wrapped around one another .
IHC staining was performed on formalin-fixed, paraffin-embedded tissues that were sectioned 4 mm thick and placed on positively charged slides. A purified rabbit monoclonal antibody (Cat# RM-2101-S0), raised against β-catenin antigen (Thermo Fisher Scientific Anatomical Pathology 46360 Fremont Blvd. Fremont, CA94538, USA), was used. The optimal dilution was 1 : 100. IHC staining was performed using the Universal Dako cytomation labeled streptavidin-biotin-2 system, horseradish peroxidase (LSAB-2 System, HRP Kit, Catalogue No. k0679 (Dako Denmark A/S, Produktionsvej 42, DK-2600 Glostrup, Denmark)). All slides were deparaffinized using xylene and then redehydrated in decreasing concentrations of ethanol. Antigen retrieval using microwave heating (20 min; 10 mmol/citrate buffer, pH 6.0) after inhibition of the endogenous peroxidase activity (hydrogen peroxidase for 15 min) was performed. The primary antibody was applied to the slides. The slides were incubated overnight with the primary antibody at room temperature in humidity chamber and were washed using PBS then incubated with the secondary antibody for 15 min followed by PBS wash. Finally, the detection of bound antibody was accomplished using a modified labeled avidin-biotin reagent for 20 min and then PBS wash. A 0.1% solution of diaminobenzidine was used for 5 min as a chromogen. The slides were counterstained with Mayer's hematoxylin for 5-10 min.
Interpretation of immunohistochemically stained slides
Assessment of results was determined using a semiquantitative visual approach. Unintentional bias was prevented by coding patients' slides. The entire slide was examined for immunostaining evaluation. Scoring was carried out using an Olympus CH2 light microscope (Tokyo, Japan) with a wide angle (field size of 0.274 mm 2 and field diameter of 0.59 mm 2 ). β-Catenin staining could be detected in both the cytoplasm and the nucleus of medulloblastoma tumor cells . Thereafter, the H-score system was applied to evaluate the studied patients according to Bilalovic et al. , where both the intensity and percentage of positivity were considered using the following formula:
H score = 3 (strong intensity) × % + 2 (moderate intensity) × % + 1 (mild intensity) × %.
Data were collected, tabulated, and statistically analyzed using the Statistical Package for the Social Science program for Windows (version 16; SPSS Inc., Chicago, Illinois, USA). To test whether these variables differed according to the clinicopathological parameters and biological markers, the Fisher exact test, the χ2 -test, the Mann-Whitney U-test, and the Kruskal-Wallis test were used. All P values were two-sided; P values of 0.05 or less were considered statistically significant and P values of 0.01 or less were considered highly significant .
| Results|| |
The clinical characteristics and histopathologic features of the studied 49 medulloblastoma patients were demonstrated in [Table 1]. According to the WHO classification, three types of medulloblastoma were included in this study: 36 patients (73.5%) had classic histology, 11 patients (22.4%) had nodular/desmoplastic histology, and two patients (4.1%) had large cell/anaplastic variant [Figure 1]. The majority of medulloblastoma patients included in this study showed positive extra-axial extension (40 patients, 81.6%), whereas only nine patients (18.4%) were negative for this extension. Slightly more than one-half of the patients - 25 patients (51%) - exhibited connective tissue (CT) desmoplasia, whereas 24 patients (49%) were negative for CT desmoplasia. The studied tissue specimens were categorized as follows: 26 patients (53.1%) exhibited focal apoptosis, 15 patients (30.6%) showed diffuse apoptosis, and eight patients (16.3%) belonged to the extensive apoptosis group. In the present study, tumor cell necrosis was seen in 25 medulloblastoma patients (51%). Assessment of anaplasia in 47 medulloblastoma patients (after exclusion of the two patients with large cell/anaplastic variant) revealed that only 10 patients (21.3%) showed anaplasia in less than 50% of the tumor cells, whereas the remaining 37 patients (78.7%) were devoid of anaplasia.
|Table 1: Clinical and histopathological characteristics of the studied medulloblastoma patients|
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β-Catenin immunoreactivity results in medulloblastoma patients
None of the studied 49 medulloblastoma patients showed nuclear positivity for β-catenin. Twenty-nine patients (59.2%) with nucleonegative β-catenin expression showed evidence of cytoplasmic staining pattern. The remaining 20 (40.8%) medulloblastoma patients experienced neither nuclear staining, nor cytoplasmic brown staining [Figure 2]. The H-score values of patients with cytoplasmic β-catenin expression ranged between 10 and 160, with a mean ± SD of 59.14 ± 49.54 and a median of 40 [Table 2].
Relationship of β-catenin immunohistochemical expression with demographic, clinical, and histopathological features of the studied medulloblastoma patients
There was no significant difference between the nucleonegative medulloblastoma specimens, either those of pure cytoplasmic staining for β-catenin or specimens that were devoid of any β-catenin immunoreactivity regarding the clinical and histopathological parameters. Moreover, there was no statistically significant relationship between the H score of cytoplasmic expression for β-catenin and the clinical or histopathological parameters in medulloblastoma [Table 3].
|Table 3: Relationship between the pattern of b -catenin expression among nucleonegative medulloblastoma and the H score for these patients with clinical and histopathological parameters|
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| Discussion|| |
Medulloblastoma is the most common malignant brain tumor in children . Several types of genetic changes have been identified in this tumor. These include mutations in the Hedgehog-signaling pathway, c-Myc and N-Myc amplification, loss of portions of chromosome 17p (usually associated with isochromosome 17q formation), and mutations in the WNT-signaling pathway .
The identification of medulloblastoma molecular subgroup signature relied mainly on the advanced genomic technologies that are relatively expensive and may be unavailable in most of the Egyptian laboratories. Thus, we tried to obtain better insight into the molecular biology of medulloblastoma using an easy reliable method that can be applied on formalin-fixed paraffin-embedded tissues used routinely for pathologic evaluation.
The WNT pathway is activated in ∼15% of sporadic medulloblastomas ,,. The hallmark of its activation is nuclear localization of downstream activator β-catenin ,. In our series, none of the 49 medulloblastoma specimens expressed nuclear β-catenin immunoreactivity. Kratz et al.  injected mutant β-catenin into transgenic mice, and then IHC detection of β-catenin distribution was carried out. Data from their study revealed nuclear β-catenin expression in numerous neurons of the hippocampus, cerebral cortex, and cerebellum. In contrast, in nontransgenic mice injected with wild-type β-catenin, the protein was abundant in cytoplasm but not in the nuclei of these structures, suggesting that weak activation of the WNT signaling may occur with overexpression of wild-type β-catenin protein. We can conclude that the absence of nuclear β-catenin staining in our medulloblastoma patients suggests the absence of β-catenin mutation in the studied patients.
The critical downstream effector of the canonical WNT pathway is β-catenin . Cytoplasmic β-catenin is regulated by a multimeric protein complex that contains the APC, glycogen synthase kinase 3 β (GSK-3β), and AXIN proteins. In the 'pathway-inactive' state, GSK-3β phosphorylates β-catenin, signaling it for degradation through the ubiquitin-proteasome system. Pathway activation destabilizes the protein complex, upregulating the levels of β-catenin and enhancing its translocation to the nucleus. Here, it acts as a coactivator of Tcf/Lef transcription factors, which regulate the genes involved in cell cycle progression, apoptosis, and differentiation. Thus, nuclear accumulation of β-catenin is a marker for physiologic or abnormal activation of the WNT pathway .
The absence of β-catenin mutation in our series does not mean the disappearance of WNT signature in the studied patients, as only a small part of the WNT pathway medulloblastomas is found to be carrying mutations in the CTNNB1 gene captured by identification of the nuclear activity of β-catenin. Mutations in the APC and AXIN1/2 are rare; thus, there are still undiscovered components also in the WNT pathway ,,.
When trying to compare the expression patterns of different β-catenin (cytoplasmic vs. negative patients) in nucleonegative medulloblastoma patients together with the H score of cytoplasmic β-catenin-stained patients regarding the studied clinicopathologic parameters, we did not find any significant difference. In concordance with our results, other investigators found negative nuclear staining of β-catenin in medulloblastoma . In addition, earlier study reported that there was no correlation between cytoplasmic β-catenin staining and the proliferative activity in medulloblastoma. We should remember that nuclear β-catenin expression is related to enhanced cellular proliferation in many tumors such as medulloblastoma , colonic , lung , and hepatocellular carcinomas . Thus, these findings may simply reflect the absence of nuclear accumulation rather than the absence of relationship with proliferative activity and with other parameters in the studied medulloblastoma specimens.
| Conclusion|| |
We describe a simple available IHC technique that allows identification of medulloblastomas in which the WNT-signaling pathway is aberrantly activated. The absence of β-catenin nuclear staining in the studied medulloblastoma patients implies that other signaling pathways might take the upper hand in the pathogenesis of our patients. However, this cannot exclude the role of the WNT-signaling pathway through dysregulation of components other than β-catenin. Therefore, a large-scale study is recommended to elicit the exact role of the WNT-signaling pathway and of other proposed pathways in medulloblastoma in Egyptian patients.
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]