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Aberrant membranous expression of β-catenin predicts poor prognosis in patients with craniopharyngioma
Annals of Diagnostic Pathology 19 (2015) 403–408
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Annals of Diagnostic Pathology
Aberrant membranous expression of β-catenin predicts poor prognosis in patients with craniopharyngioma☆ Zongping Li, MD a,⁎, Jianguo Xu, MD b, Siqing Huang, MD b, Chao You, MD b a b
Department of Neurosurgery, Mianyang Central Hospital, Mianyang, Sichuan Province, China Department of Neurosurgery, Huaxi Hospital of Sichuan University, Chengdu, Sichuan, China
a r t i c l e
i n f o
a b s t r a c t
Keywords: Craniopharyngioma CTNNB1 gene β-Catenin Prognosis
The objective of this study is to investigate β-catenin expression in craniopharyngioma patients and determine its significance in predicting the prognosis of this disease. Fifty craniopharyngioma patients were enrolled in this study. Expression of β-catenin in tumor specimens collected from these patients was examined through immunostaining. In addition, mutation of exon 3 in the β-catenin gene, CTNNB1, was analyzed using polymerase chain reaction, denaturing high-pressure liquid chromatography, and DNA sequencing. Based on these results, we explored the association between membranous β-catenin expression, clinical and pathologic characteristics, and prognoses in these patients. Of all craniopharyngioma specimens, 31 (62.0%) had preserved membranous β-catenin expression, whereas the remaining 19 specimens (38.0%) displayed aberrant expression. Statistical analysis showed a significant correlation between aberrant membranous β-catenin expression and CTNNB1 exon 3 mutation, as well as between aberrant membranous β-catenin expression and the histopathologic type of craniopharyngioma and type of resection in our patient population. Furthermore, aberrant membranous β-catenin expression was found to be associated with poor patient survival. Results of Kaplan-Meier survival analysis and Cox regression analysis further confirmed this finding. In conclusion, our study demonstrated that aberrant membranous β-catenin expression was significantly correlated with poor survival in patients with craniopharyngioma. This raises the possibility for use of aberrant membranous β-catenin expression as an independent risk factor in predicting the prognosis of this disease. © 2015 Elsevier Inc. All rights reserved.
1. Introduction
of craniopharyngioma after surgical treatment is common and significantly contributes to mortality rates associated with the disease [6]. Recent studies have found that multiple factors, including tumor volume, histopathologic type, dimensions of cysts, lesion position, degree of residual tumor after resection, tumor mass effects, and region of invasion, correlate with the risk of recurrence [7]. However, the underlying molecular mechanism that leads to recurrence remains elusive. In addition, although some factors (eg, degree of residual tumor after resection) have been identified as predictors of recurrence and prognosis [8,9], there is still an imperative need to develop novel molecular signatures that can predict responses to surgical treatment and posttreatment outcomes of craniopharyngioma with reliable clinical significance. In recent years, dysregulation of the Wnt/β-catenin signaling pathway was demonstrated to be intimately involved in the pathogenesis of craniopharyngioma [2]. Gaston-Massuet et al [10] found that, in Hesx1Cre;Ctnnb1+/lox(ex3) mice, a degradation-resistant form of β-catenin was expressed in early Rathke pouch progenitors, inducing tumors that closely resemble human adamantinomatous craniopharyngiomas. Other previous clinical studies have also reported that most craniopharyngioma patients harbor mutations in the β-catenin gene CTNNB1 and aberrations in β-catenin expression [11,12]. In addition, researchers have documented that aberrant membranous β-catenin expression is closely correlated with poor prognoses in several human cancers, including colorectal
Craniopharyngioma is a rare type of solid or mixed solid cystic epithelial tumor of the sellar and suprasellar regions, which accounts for 2% to 4% of intracranial tumors and can be found in all age groups [1]. Although nearly always histologically benign, this tumor can often grow invasively and significantly affect surrounding critical structures, including the hypothalamus, pituitary gland, and optic chiasm, thus leading to permanent neurologic and cognitive deficits or even death [2,3]. According to Erfurth et al [3], the standardized overall mortality rate of craniopharyngioma varies from 2.9% to 9.3%, which is much higher than that of other pituitary tumors. Therefore, these tumors are widely considered to be low-grade malignancies and require intensive treatment and management [4]. Currently, surgery is the mainstay of treatment of craniopharyngioma and can be indicated in almost all cases [5]. Nevertheless, the recurrence
☆ Source of funding: This study was supported by China Postdoctoral Science Foundation (no. 20080441210). ⁎ Corresponding author at: Department of Neurosurgery, Mianyang Central Hospital, Mianyang, Sichuan Province, China, No. 12 Jingzhong Road, Mianyang, Sichuan Province, 621000 China. Tel.: +86 139 9017 7896. E-mail address: [email protected] (Z. Li). http://dx.doi.org/10.1016/j.anndiagpath.2015.10.002 1092-9134/© 2015 Elsevier Inc. All rights reserved.
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cancer, ovarian cancer, and hepatocellular carcinoma [13-15]. Based on these findings, it has been hypothesized that aberrant membranous β-catenin expression may be a potential indicator for predicting the prognosis of craniopharyngioma patients. However, there is still a lack of comprehensive clinical evidence confirming this hypothesis. In the current study, we examined the expression levels of β-catenin in tumor tissues from 50 craniopharyngioma patients and explored the association between membranous β-catenin expression, clinical and pathologic characteristics, and prognosis in these patients. Our results indicate that aberrant membranous β-catenin expression may be regarded as an independent risk factor for predicting the prognosis of craniopharyngioma. 2. Materials and methods 2.1. Acquisition of tissue specimens A total of 50 tumor tissue specimens were obtained from patients with newly diagnosed craniopharyngioma who received surgical treatment at the Department of Neurosurgery of the corresponding author's institution between January 2001 and January 2006. All specimens were collected in the operating room directly after tumor resection. The collected specimens were then immediately frozen in liquid nitrogen and stored at −70°C until use. The study protocol and acquisition of tissue specimens were approved by the Medical Research Ethics Committee of the corresponding author's institution. Before specimen collection, all patients were fully informed and provided their written informed consent. At enrollment, strict inclusion and exclusion criteria were followed to ensure the quality of this study. The inclusion criteria were as follows: (1) having newly diagnosed craniopharyngioma; (2) the surgically obtained tissue specimen was adequate for experimental use; (3) having complete clinical and pathological data including age, sex, histopathologic type, existence of calcification, tumor volume, existence of complication, type of resection, and surgical approach; (4) having complete follow-up data for the assessment of tumor recurrence and progression after surgery. The exclusion criteria were as follows: (1) having a history of other cranial tumors, (2) having other lifethreatening diseases, and (3) being unable or unwilling to provide informed consent. All patients received follow-ups every 2 months in the first year after surgery and then every 3 months thereafter. Local recurrence was defined as tumor recurrence at the site of previous resection, which was diagnosed by computed tomography and/or magnetic resonance imaging. Overall survival (OS) was defined as the time duration from surgery to death. 2.2. Immunohistochemical staining of β-catenin All craniopharyngioma tissue specimens were fixed in 10% formaldehyde and paraffin embedded according to routine protocols. Then, serial 4-μm sections were prepared and subjected to immunohistochemical (IHC) staining using a specific antibody against β-catenin (dilution 1: 400; Cell Signaling Technology, Beverly, MA) and an EliVisio Plus IHC kit (Fuzhou Maxim Biotechnology, Inc, Fuzhou, China) in accordance with manufacturers' instructions. For each patient, 10 high-power fields (HPFs) of 5 immunostained sections were randomly selected for quantitative analysis of β-catenin expression. Briefly, in each HPF, the total number of tumor cells as well as the number of cells that were positively stained for cell membrane and cytoplasma and/or nuclei was counted. The counting was performed by 2 independent pathologists who were blinded to the histopathologic features and patient data of the specimens, and the averages were recorded. Afterwards, the percentages of membranous and cytoplasmic and/or nuclear expression of β-catenin were determined by dividing the numbers of cells with positively stained membranes
and cytoplasm and/or nuclei by the total number of tumor cells. Then, the median value across all HPFs was calculated for each patient. Based on previous literature [13], if the percentage of membranous β-catenin expression was more than or equal to 70% and the percentage of cytoplasmic and/or nuclear β-catenin expression was less than 10%, expression of β-catenin was judged as “preserved.” Otherwise, expression of β-catenin was judged as “aberrant.” The detailed definition of both β-catenin expression statuses is listed in Table 1. 2.3. Mutation analysis of CTNNB1 exon 3 Genomic DNA was extracted from frozen craniopharyngioma tissue specimens using the regular phenol/chloroform method. Then, the extracted DNA was subjected to polymerase chain reaction (PCR) with the following primers for mutational analysis of CTNNB1 exon 3: forward, 5′-TTTGATGGAGTTGGACATGG-3′; reverse, 5′-TCAAAACTGCAT TCTGACTTTCA-3′. The conditions of PCR were as follows: 50 ng of genomic DNA was mixed with 0.5-μL primers (both forward and reverse), 10-μL deoxynucleotide triphosphate mix (2 mmol/L), 10-μL reaction buffer (10×) with MgCl2, and 1.5 U Taq DNA polymerase (Invitrogen, Carlsbad, CA) in a total volume of 25 μL. The reaction was processed through 35 cycles of 94°C for 30 seconds, 60°C for 1 minute, and 72°C for 2 minutes. The resultant PCR products were analyzed by denaturing high-performance liquid chromatography (DHPLC) using a DHPLC WAVE 3500 system (Transgenomic, Omaha, NE). For PCR products that generated DNA heteroduplexes, DNA sequencing was performed to identify the nucleotide mutation. 2.4. Statistical analysis All statistical analysis was performed using SPSS 12.0 software (SPSS, Inc, Chicago, IL). Categorical data are expressed as counts and percentages. The χ2 or Fisher exact test was used to evaluate relationships between 2 variables. Survival analysis was performed using the Kaplan-Meier method, and statistical significance was determined by the log-rank test. Patients who remained alive at the end of the follow-up period were censored for survival analysis. A Cox regression model was used for univariate and multivariate analyses. In multivariate analysis, models were adjusted for variables including age, sex, histopathologic type, calcification, tumor size, tumor volume, complication, type of resection, and surgical approach. P b .05 was considered sufficient for statistical significance (2 tailed). 3. Results 3.1. Demographic, clinical, and pathologic characteristics of patients The demographic, clinical, and pathologic characteristics of patients enrolled in the present study are presented in Table 2. All patients had a craniopharyngioma in the sellar/suprasellar regions. The mean age of patients was 29.1 years (range, 5-71 years), and the median age was 28.5 years. All patients completed a follow-up of at least 3.0 months.
Table 1 Definition of status of β-catenin expression Percentage of tumor cells with membranous β-catenin expressiona,b
Percentage of tumor cells with cytoplasmic overexpression and/or nuclear accumulation of β-catenina,b
Status of β-catenin expression
≥70% ≥70% b70% b70%
b10% ≥10% b10% ≥10%
Preserved Aberrant Aberrant Aberrant
a For each patient, 10 HPFs of 5 immunostained sections were randomly selected for quantitative analysis of β-catenin expression. b For each patient, the median value across all HPFs was calculated and compared with the thresholds listed above.
Z. Li et al. / Annals of Diagnostic Pathology 19 (2015) 403–408 Table 2 Demographic and clinical and pathologic characteristics of patients enrolled in the current study Variables Age (y) b35 ≥35 Sex Male Female Histopathologic type Adamantinomatous Squamous papillary Calcification Yes No Tumor volume (cm3) b25 ≥25 Complication Yes No Type of resection Total resection Subtotal or partial resection Surgical approach Subfrontal approach Pterional or transsphenoidal approach Tumor recurrence during follow-up Yes No Vital status at end of follow-up Dead Alive
No. of cases (%) 28 (56.0%) 22 (44.0%) 32 (64.0%) 18 (36.0%) 33 (66.0%) 17 (34.0%) 11 (22.0%) 39 (78.0%) 26 (52.0%) 24 (48.0%) 22 (44.0%) 28 (56.0%) 26 (52.0%) 24 (48.0%) 35 (70.0%) 15 (30.0%) 9 (18.0%) 41 (82.0%) 10 (20.0%) 40 (80.0%)
The duration of follow-up ranged from 3.0 to 92.0 months with a median of 42.5 months (final follow-up in January 2009). During the followup period, 10 patients died, and their causes of death were all related to craniopharyngioma.
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3.2. Expression of β-catenin in craniopharyngioma tissues We performed IHC staining to examine β-catenin expression in craniopharyngioma tissues, and the raw data for each patient are summarized in Supplementary Table 1. Of the 50 craniopharyngioma tissue samples collected for this study, 31 (62.0%) showed preserved membranous β-catenin expression (Fig. 1A), whereas the remaining 19 specimens (38.0%) showed aberrant expression, 8 of which had reduced membranous β-catenin expression (Fig. 1B) and 11 had cytoplasmic and/or nuclear β-catenin accumulation (Fig. 1C and D). 3.3. CTNNB1 exon 3 mutations in craniopharyngioma tissues Denaturing high-performance liquid chromatography and DNA sequencing were applied to detect CTNNB1 exon 3 mutations in craniopharyngioma tissues. Of all craniopharyngioma specimens, 43 (86.0%) had no sequence changes in exon 3 of the CTNNB1 gene (Fig. 2A), whereas 7 (14.0%) showed a synonymous point mutation at codon 46 of exon 3 [CTG (Leu) → TTG (Leu)] (Fig. 2B). 3.4. Correlation between aberrant β-catenin expression and clinical and pathologic characteristics of craniopharyngioma patients Correlation between aberrant membranous β-catenin expression and clinical and pathologic characteristics of craniopharyngioma patients is summarized in Table 3. The results indicate that most patients with CTNNB1 exon 3 mutation (6/7) had aberrant expression of β-catenin, thereby demonstrating that there was a significant correlation between aberrant β-catenin expression and CTNNB1 exon 3 mutation (P = .009). In addition, a large percentage of patients with either adamantinomatous craniopharyngioma (16/33) or subtotal or partial resection (14/24) showed aberrant β-catenin expression, thereby suggesting that aberrant β-catenin expression was also significantly correlated with the histopathologic type of craniopharyngioma and the type
Fig. 1. Various β-catenin expression patterns in craniopharyngioma tissue specimens by IHC. Representative specimen with preserved β-catenin expression (the percentage of membranous β-catenin expression was N70%, and the percentage of cytoplasmic and nuclear β-catenin expression was b10%) (A). Representative specimens with aberrant β-catenin expression (B-D). Representative specimen with reduced membranous β-catenin expression (the percentage of membranous β-catenin expression was b70%) (B). Representative specimens with cytoplasmic and/or nuclear accumulation of β-catenin (C and D). β-Catenin signals (brown) can be detected in the nucleus (C) or cytoplasm (D). Original magnification ×400.
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Fig. 2. DNA sequencing chromatograms of PCR products of exon 3 in the CTNNB1 gene. The wild-type sequence from codons 44 to 47 of exon 3 in CTNNB1 gene (A). The CTNNB1 gene with a synonymous point mutation at codon 46 [CTG (Leu) → TTG (Leu)] (B).
of resection (P = .033 for histopathologic type of craniopharyngioma and P = .004 for type of resection, respectively). However, there was no significant association between aberrant β-catenin expression and other variables including age, sex, calcification, tumor volume, complications, and surgical approach (P N .05). 3.5. Correlation between aberrant β-catenin expression and prognosis in craniopharyngioma patients During the follow-up period, 9 patients (18.0%) had locally recurrent craniopharyngioma, and aberrant membranous β-catenin expression was observed in 7 (77.8%) of those patients. By the end of the followup period, 10 patients died of craniopharyngioma-related causes, 7 (70.0%) of which had aberrant β-catenin expression. Collectively, these findings indicate that aberrant membranous β-catenin expression may be a marker for poor prognosis in craniopharyngioma patients. To further validate this finding, we performed Kaplan-Meier survival
analysis and Cox regression analysis to assess the association between aberrant β-catenin expression and prognosis in craniopharyngioma patients. As shown in Fig. 3, aberrant β-catenin expression displayed prognostic significance for OS of all cases. The median value of OS of the patients with preserved β-catenin expression was 89.0 months (95% confidence interval [CI], 78.8-99.2 months). For patients with aberrant β-catenin expression, however, the median OS was 37.0 months (95% CI, 8.0-66.0 moths), which was significantly shorter than that of the patients with preserved β-catenin expression (log-rank test, P = .0004). Results of Cox regression analysis of OS in craniopharyngioma patients are summarized in Table 4. Univariate analysis demonstrated that aberrant membranous β-catenin expression was a potential prognostic factor for the survival of craniopharyngioma patients (relative hazard ratio [HR] was 8.69; P = .003). Multivariate analysis with 2 different models further confirmed univariate analysis findings, thereby indicating that aberrant β-catenin expression was an independent risk factor for poor prognosis in craniopharyngioma patients (relative HR, 14.55, and P = .002 for the first model; relative HR, 11.21, and P = .043 for the second model).
Table 3 Correlation between β-catenin expression and clinical and pathologic variables Variables
No. of cases CTNNB1 exon 3 mutation Yes No Age (y) b35 ≥35 Sex Male Female Histopathologic type Adamantinomatous Squamous papillary Calcification Yes No Tumor volume (cm3) b25 ≥25 Complication Yes No Type of resection Total resection Subtotal or partial resection Surgical approach Subfrontal approach Pterional or transsphenoidal approach Tumor recurrence during follow-up Yes No Vital status at end of follow-up Dead Alive
β-Catenin expression Preserved
Aberrant
31
19
1 30
6 13
16 15
12 7
18 13
14 5
17 14
16 3
5 26
6 13
19 12
7 12
12 19
10 9
21 10
5 14
22 9
13 6
2 29
7 12
3 28
7 12
P
.009
.425
4. Discussion In this study, we examined membranous expression of β-catenin in craniopharyngioma specimens from 50 patients and investigated the association between aberrant β-catenin expression and clinical and pathologic characteristics, as well as the relation to the prognoses of these patients. We found that there was a significant correlation
.264
.033
.293
.093
.336
.004
.849
.018
.030
Fig. 3. Correlation of aberrant membranous β-catenin expression with OS in craniopharyngioma patients. Individuals with aberrant membranous β-catenin expression had a significantly increased risk of death when compared with those having preserved β-catenin expression. Survival analysis was performed using the Kaplan-Meier method, and statistical significance was determined by the log-rank test. Patients living at the end of the follow-up period were censored for survival analysis.
Z. Li et al. / Annals of Diagnostic Pathology 19 (2015) 403–408
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Table 4 Cox regression analysis of survival in patients with craniopharyngioma β-Catenin
Expression a b
HR (95% CI)
Preserved Aberrant
Univariate analysis
P
Multivariate analysis, model 1a
P
Multivariate analysis, model 2b
P
1.00 8.69 (2.13-35.48)
– .003
1.00 14.55 (2.66-79.68)
– .002
1.00 11.21 (1.08-116.51)
– .043
Adjusted for age, sex, and histopathologic type. Adjusted for all covariates including age, sex, histopathologic type, calcification, tumor size, tumor volume, complication, type of resection, and surgical approach.
between aberrant membranous β-catenin expression and CTNNB1 exon 3 mutation. In addition aberrant β-catenin expression was found to be associated with poor patient survival. Results of the Kaplan-Meier survival analysis and Cox regression analysis further confirmed this finding. Taken together, these findings suggest that aberrant β-catenin expression is an independent risk factor for a poor prognosis in patients with craniopharyngioma. In past decades, numerous studies have pointed out the critical role played by β-catenin in tumorigenesis and progression of craniopharyngioma [2]. As an essential component required for cadherin-dependent cell adhesion, cell membrane–localized β-catenin forms a complex with E-cadherin, α-catenin, and actin filaments, which acts on tumor cells as an “invasion suppressor” [16]. When the level of cell membrane–localized β-catenin is down-regulated in tumor cells, the cadherin/catenin cell adhesion system can be disrupted, thus contributing to enhanced cell migration and proliferation and, subsequently, invasion and metastasis [17]. On the other hand, disruption of the cadherin/catenin-based cell-cell adherens junction can release βcatenin from the adherens junction pool into cytoplasma [18]. Cytoplasmic β-catenin participates in the Wnt signaling cascade and is available for transport and accumulation in the nucleus in an aberrant manner [19]. Nuclear β-catenin forms complexes with T-cell factor and lymphoid enhancer binding factor family members, consequently inducing activation of the transcription of multiple target genes that are important for tumor invasion and progression [20]. Previous studies have demonstrated that cytoplasm/nuclear β-catenin accumulation is an exclusively characteristic morphology of craniopharyngioma, especially for adamantinomatous type [21,22]. In this study, aberrant cytoplasmic and/or nuclear accumulation of β-catenin was found in a large portion of patients who underwent subtotal or partial resection (14/24). This was also the case in most patients who experienced relapse of the disease. These findings provide further evidence that decreased levels of β-catenin at the cell membrane as well as aberrant accumulation in the cytoplasm and/or nucleus may be among the leading causes of tumor recurrence and relapse in craniopharyngioma patients. Recently, a number of studies have well documented that genetic mutations of β-catenin gene CTNNB1 can trigger increased levels of β-catenin in the cytoplasm or nucleus in craniopharyngioma tissue [11,12,23]. Most CTNNB1 mutations are localized to exon 3, which may affect the functional domains of β-catenin that are involved in interactions with its regulators, the adenomatous polyposis coli tumor suppressor protein and glycogen synthetase kinase 3-β (GSK3-β). Under normal physiological conditions, when the Wnt signaling cascade is inactive, cytoplasmic βcatenin is phosphorylated by a complex consisting of adenomatous polyposis coli tumor suppressor protein and GSK3-β as well as axin and casein kinase 1 and is then degraded via ubiquitination [16]. Nevertheless, mutations in CTNNB1 exon 3 have been found to cause amino acid substitution at GSK3-β phosphorylation sites or flank residues on these sites in the β-catenin protein, thus inhibiting the phosphorylation process and leading to cytoplasmic accumulation of β-catenin [2]. In this study, 7 patients showed a synonymous point mutation at codon 46 in CTNNB1 exon 3 [CTG (Leu) → TTG (Leu)], of which 6 had aberrant β-catenin expression. According to recent research, synonymous point mutations in the triplet code can influence protein translation efficiency and protein folding and function [24,25]. Therefore, we speculate that the synonymous point mutation at codon 46 in CTNNB1 exon 3 might
affect the folding pattern of the β-catenin protein, which blocks the exposure of phosphorylation sites to GSK3-β and subsequently inhibits the degradation of β-catenin and induces its aberrant accumulation in cytoplasma. However, further study is required to fully validate this speculation. Furthermore, Cani et al [26] found that craniopharyngioma cells without CTNNB1 mutations could also overexpress β-catenin, implying that an alternative mechanism may exist for the cytoplasmic and nuclear accumulation of β-catenin. This might be a reasonable explanation for the fact that a majority of patients without CTNNB1 exon 3 mutation also had aberrant β-catenin expression. However, the details of this underlying mechanism remain unknown. A number of clinical studies have revealed the significance of aberrant β-catenin expression in predicting the prognoses of various malignant tumors [27,28]. In our study, the results of our Kaplan-Meier survival analysis and Cox regression analysis demonstrate that aberrant β-catenin expression is an independent risk factor for a poor prognosis in patients with craniopharyngioma. As we discussed earlier, this may be attributed to tumor invasion and recurrence closely associated with a loss of membranous β-catenin expression and increased cytoplasmic/nuclear β-catenin expression. Nevertheless, it should be noted that other molecules, such as epidermal growth factor receptor, nestin, glial fibrillary acid protein, and microtubule-associated protein 2, may also be involved in the invasion process of craniopharyngioma [2]. The interactions between these molecules and the β-catenin/Wnt signaling pathway require further investigation. In summary, this study demonstrated that aberrant β-catenin expression was significantly correlated with poor survival rates in patients with craniopharyngioma. This raises the possibility for the use of aberrant β-catenin expression as an independent risk factor in predicting the prognoses of this disease. However, the small sample size and retrospective nature of this study indicate that these findings require further investigation and validation using a prospective method of study with a larger patient population. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.anndiagpath.2015.10.002.
Conflict of interests The authors declare that they have no conflict of interests regarding the current study.
Acknowledgments All authors would like to thank Ms Rebekah Burdyshaw, Cleveland State University, for her critical proofreading of the manuscript.
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Contents lists available at ScienceDirect
Annals of Diagnostic Pathology
Aberrant membranous expression of β-catenin predicts poor prognosis in patients with craniopharyngioma☆ Zongping Li, MD a,⁎, Jianguo Xu, MD b, Siqing Huang, MD b, Chao You, MD b a b
Department of Neurosurgery, Mianyang Central Hospital, Mianyang, Sichuan Province, China Department of Neurosurgery, Huaxi Hospital of Sichuan University, Chengdu, Sichuan, China
a r t i c l e
i n f o
a b s t r a c t
Keywords: Craniopharyngioma CTNNB1 gene β-Catenin Prognosis
The objective of this study is to investigate β-catenin expression in craniopharyngioma patients and determine its significance in predicting the prognosis of this disease. Fifty craniopharyngioma patients were enrolled in this study. Expression of β-catenin in tumor specimens collected from these patients was examined through immunostaining. In addition, mutation of exon 3 in the β-catenin gene, CTNNB1, was analyzed using polymerase chain reaction, denaturing high-pressure liquid chromatography, and DNA sequencing. Based on these results, we explored the association between membranous β-catenin expression, clinical and pathologic characteristics, and prognoses in these patients. Of all craniopharyngioma specimens, 31 (62.0%) had preserved membranous β-catenin expression, whereas the remaining 19 specimens (38.0%) displayed aberrant expression. Statistical analysis showed a significant correlation between aberrant membranous β-catenin expression and CTNNB1 exon 3 mutation, as well as between aberrant membranous β-catenin expression and the histopathologic type of craniopharyngioma and type of resection in our patient population. Furthermore, aberrant membranous β-catenin expression was found to be associated with poor patient survival. Results of Kaplan-Meier survival analysis and Cox regression analysis further confirmed this finding. In conclusion, our study demonstrated that aberrant membranous β-catenin expression was significantly correlated with poor survival in patients with craniopharyngioma. This raises the possibility for use of aberrant membranous β-catenin expression as an independent risk factor in predicting the prognosis of this disease. © 2015 Elsevier Inc. All rights reserved.
1. Introduction
of craniopharyngioma after surgical treatment is common and significantly contributes to mortality rates associated with the disease [6]. Recent studies have found that multiple factors, including tumor volume, histopathologic type, dimensions of cysts, lesion position, degree of residual tumor after resection, tumor mass effects, and region of invasion, correlate with the risk of recurrence [7]. However, the underlying molecular mechanism that leads to recurrence remains elusive. In addition, although some factors (eg, degree of residual tumor after resection) have been identified as predictors of recurrence and prognosis [8,9], there is still an imperative need to develop novel molecular signatures that can predict responses to surgical treatment and posttreatment outcomes of craniopharyngioma with reliable clinical significance. In recent years, dysregulation of the Wnt/β-catenin signaling pathway was demonstrated to be intimately involved in the pathogenesis of craniopharyngioma [2]. Gaston-Massuet et al [10] found that, in Hesx1Cre;Ctnnb1+/lox(ex3) mice, a degradation-resistant form of β-catenin was expressed in early Rathke pouch progenitors, inducing tumors that closely resemble human adamantinomatous craniopharyngiomas. Other previous clinical studies have also reported that most craniopharyngioma patients harbor mutations in the β-catenin gene CTNNB1 and aberrations in β-catenin expression [11,12]. In addition, researchers have documented that aberrant membranous β-catenin expression is closely correlated with poor prognoses in several human cancers, including colorectal
Craniopharyngioma is a rare type of solid or mixed solid cystic epithelial tumor of the sellar and suprasellar regions, which accounts for 2% to 4% of intracranial tumors and can be found in all age groups [1]. Although nearly always histologically benign, this tumor can often grow invasively and significantly affect surrounding critical structures, including the hypothalamus, pituitary gland, and optic chiasm, thus leading to permanent neurologic and cognitive deficits or even death [2,3]. According to Erfurth et al [3], the standardized overall mortality rate of craniopharyngioma varies from 2.9% to 9.3%, which is much higher than that of other pituitary tumors. Therefore, these tumors are widely considered to be low-grade malignancies and require intensive treatment and management [4]. Currently, surgery is the mainstay of treatment of craniopharyngioma and can be indicated in almost all cases [5]. Nevertheless, the recurrence
☆ Source of funding: This study was supported by China Postdoctoral Science Foundation (no. 20080441210). ⁎ Corresponding author at: Department of Neurosurgery, Mianyang Central Hospital, Mianyang, Sichuan Province, China, No. 12 Jingzhong Road, Mianyang, Sichuan Province, 621000 China. Tel.: +86 139 9017 7896. E-mail address: [email protected] (Z. Li). http://dx.doi.org/10.1016/j.anndiagpath.2015.10.002 1092-9134/© 2015 Elsevier Inc. All rights reserved.
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cancer, ovarian cancer, and hepatocellular carcinoma [13-15]. Based on these findings, it has been hypothesized that aberrant membranous β-catenin expression may be a potential indicator for predicting the prognosis of craniopharyngioma patients. However, there is still a lack of comprehensive clinical evidence confirming this hypothesis. In the current study, we examined the expression levels of β-catenin in tumor tissues from 50 craniopharyngioma patients and explored the association between membranous β-catenin expression, clinical and pathologic characteristics, and prognosis in these patients. Our results indicate that aberrant membranous β-catenin expression may be regarded as an independent risk factor for predicting the prognosis of craniopharyngioma. 2. Materials and methods 2.1. Acquisition of tissue specimens A total of 50 tumor tissue specimens were obtained from patients with newly diagnosed craniopharyngioma who received surgical treatment at the Department of Neurosurgery of the corresponding author's institution between January 2001 and January 2006. All specimens were collected in the operating room directly after tumor resection. The collected specimens were then immediately frozen in liquid nitrogen and stored at −70°C until use. The study protocol and acquisition of tissue specimens were approved by the Medical Research Ethics Committee of the corresponding author's institution. Before specimen collection, all patients were fully informed and provided their written informed consent. At enrollment, strict inclusion and exclusion criteria were followed to ensure the quality of this study. The inclusion criteria were as follows: (1) having newly diagnosed craniopharyngioma; (2) the surgically obtained tissue specimen was adequate for experimental use; (3) having complete clinical and pathological data including age, sex, histopathologic type, existence of calcification, tumor volume, existence of complication, type of resection, and surgical approach; (4) having complete follow-up data for the assessment of tumor recurrence and progression after surgery. The exclusion criteria were as follows: (1) having a history of other cranial tumors, (2) having other lifethreatening diseases, and (3) being unable or unwilling to provide informed consent. All patients received follow-ups every 2 months in the first year after surgery and then every 3 months thereafter. Local recurrence was defined as tumor recurrence at the site of previous resection, which was diagnosed by computed tomography and/or magnetic resonance imaging. Overall survival (OS) was defined as the time duration from surgery to death. 2.2. Immunohistochemical staining of β-catenin All craniopharyngioma tissue specimens were fixed in 10% formaldehyde and paraffin embedded according to routine protocols. Then, serial 4-μm sections were prepared and subjected to immunohistochemical (IHC) staining using a specific antibody against β-catenin (dilution 1: 400; Cell Signaling Technology, Beverly, MA) and an EliVisio Plus IHC kit (Fuzhou Maxim Biotechnology, Inc, Fuzhou, China) in accordance with manufacturers' instructions. For each patient, 10 high-power fields (HPFs) of 5 immunostained sections were randomly selected for quantitative analysis of β-catenin expression. Briefly, in each HPF, the total number of tumor cells as well as the number of cells that were positively stained for cell membrane and cytoplasma and/or nuclei was counted. The counting was performed by 2 independent pathologists who were blinded to the histopathologic features and patient data of the specimens, and the averages were recorded. Afterwards, the percentages of membranous and cytoplasmic and/or nuclear expression of β-catenin were determined by dividing the numbers of cells with positively stained membranes
and cytoplasm and/or nuclei by the total number of tumor cells. Then, the median value across all HPFs was calculated for each patient. Based on previous literature [13], if the percentage of membranous β-catenin expression was more than or equal to 70% and the percentage of cytoplasmic and/or nuclear β-catenin expression was less than 10%, expression of β-catenin was judged as “preserved.” Otherwise, expression of β-catenin was judged as “aberrant.” The detailed definition of both β-catenin expression statuses is listed in Table 1. 2.3. Mutation analysis of CTNNB1 exon 3 Genomic DNA was extracted from frozen craniopharyngioma tissue specimens using the regular phenol/chloroform method. Then, the extracted DNA was subjected to polymerase chain reaction (PCR) with the following primers for mutational analysis of CTNNB1 exon 3: forward, 5′-TTTGATGGAGTTGGACATGG-3′; reverse, 5′-TCAAAACTGCAT TCTGACTTTCA-3′. The conditions of PCR were as follows: 50 ng of genomic DNA was mixed with 0.5-μL primers (both forward and reverse), 10-μL deoxynucleotide triphosphate mix (2 mmol/L), 10-μL reaction buffer (10×) with MgCl2, and 1.5 U Taq DNA polymerase (Invitrogen, Carlsbad, CA) in a total volume of 25 μL. The reaction was processed through 35 cycles of 94°C for 30 seconds, 60°C for 1 minute, and 72°C for 2 minutes. The resultant PCR products were analyzed by denaturing high-performance liquid chromatography (DHPLC) using a DHPLC WAVE 3500 system (Transgenomic, Omaha, NE). For PCR products that generated DNA heteroduplexes, DNA sequencing was performed to identify the nucleotide mutation. 2.4. Statistical analysis All statistical analysis was performed using SPSS 12.0 software (SPSS, Inc, Chicago, IL). Categorical data are expressed as counts and percentages. The χ2 or Fisher exact test was used to evaluate relationships between 2 variables. Survival analysis was performed using the Kaplan-Meier method, and statistical significance was determined by the log-rank test. Patients who remained alive at the end of the follow-up period were censored for survival analysis. A Cox regression model was used for univariate and multivariate analyses. In multivariate analysis, models were adjusted for variables including age, sex, histopathologic type, calcification, tumor size, tumor volume, complication, type of resection, and surgical approach. P b .05 was considered sufficient for statistical significance (2 tailed). 3. Results 3.1. Demographic, clinical, and pathologic characteristics of patients The demographic, clinical, and pathologic characteristics of patients enrolled in the present study are presented in Table 2. All patients had a craniopharyngioma in the sellar/suprasellar regions. The mean age of patients was 29.1 years (range, 5-71 years), and the median age was 28.5 years. All patients completed a follow-up of at least 3.0 months.
Table 1 Definition of status of β-catenin expression Percentage of tumor cells with membranous β-catenin expressiona,b
Percentage of tumor cells with cytoplasmic overexpression and/or nuclear accumulation of β-catenina,b
Status of β-catenin expression
≥70% ≥70% b70% b70%
b10% ≥10% b10% ≥10%
Preserved Aberrant Aberrant Aberrant
a For each patient, 10 HPFs of 5 immunostained sections were randomly selected for quantitative analysis of β-catenin expression. b For each patient, the median value across all HPFs was calculated and compared with the thresholds listed above.
Z. Li et al. / Annals of Diagnostic Pathology 19 (2015) 403–408 Table 2 Demographic and clinical and pathologic characteristics of patients enrolled in the current study Variables Age (y) b35 ≥35 Sex Male Female Histopathologic type Adamantinomatous Squamous papillary Calcification Yes No Tumor volume (cm3) b25 ≥25 Complication Yes No Type of resection Total resection Subtotal or partial resection Surgical approach Subfrontal approach Pterional or transsphenoidal approach Tumor recurrence during follow-up Yes No Vital status at end of follow-up Dead Alive
No. of cases (%) 28 (56.0%) 22 (44.0%) 32 (64.0%) 18 (36.0%) 33 (66.0%) 17 (34.0%) 11 (22.0%) 39 (78.0%) 26 (52.0%) 24 (48.0%) 22 (44.0%) 28 (56.0%) 26 (52.0%) 24 (48.0%) 35 (70.0%) 15 (30.0%) 9 (18.0%) 41 (82.0%) 10 (20.0%) 40 (80.0%)
The duration of follow-up ranged from 3.0 to 92.0 months with a median of 42.5 months (final follow-up in January 2009). During the followup period, 10 patients died, and their causes of death were all related to craniopharyngioma.
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3.2. Expression of β-catenin in craniopharyngioma tissues We performed IHC staining to examine β-catenin expression in craniopharyngioma tissues, and the raw data for each patient are summarized in Supplementary Table 1. Of the 50 craniopharyngioma tissue samples collected for this study, 31 (62.0%) showed preserved membranous β-catenin expression (Fig. 1A), whereas the remaining 19 specimens (38.0%) showed aberrant expression, 8 of which had reduced membranous β-catenin expression (Fig. 1B) and 11 had cytoplasmic and/or nuclear β-catenin accumulation (Fig. 1C and D). 3.3. CTNNB1 exon 3 mutations in craniopharyngioma tissues Denaturing high-performance liquid chromatography and DNA sequencing were applied to detect CTNNB1 exon 3 mutations in craniopharyngioma tissues. Of all craniopharyngioma specimens, 43 (86.0%) had no sequence changes in exon 3 of the CTNNB1 gene (Fig. 2A), whereas 7 (14.0%) showed a synonymous point mutation at codon 46 of exon 3 [CTG (Leu) → TTG (Leu)] (Fig. 2B). 3.4. Correlation between aberrant β-catenin expression and clinical and pathologic characteristics of craniopharyngioma patients Correlation between aberrant membranous β-catenin expression and clinical and pathologic characteristics of craniopharyngioma patients is summarized in Table 3. The results indicate that most patients with CTNNB1 exon 3 mutation (6/7) had aberrant expression of β-catenin, thereby demonstrating that there was a significant correlation between aberrant β-catenin expression and CTNNB1 exon 3 mutation (P = .009). In addition, a large percentage of patients with either adamantinomatous craniopharyngioma (16/33) or subtotal or partial resection (14/24) showed aberrant β-catenin expression, thereby suggesting that aberrant β-catenin expression was also significantly correlated with the histopathologic type of craniopharyngioma and the type
Fig. 1. Various β-catenin expression patterns in craniopharyngioma tissue specimens by IHC. Representative specimen with preserved β-catenin expression (the percentage of membranous β-catenin expression was N70%, and the percentage of cytoplasmic and nuclear β-catenin expression was b10%) (A). Representative specimens with aberrant β-catenin expression (B-D). Representative specimen with reduced membranous β-catenin expression (the percentage of membranous β-catenin expression was b70%) (B). Representative specimens with cytoplasmic and/or nuclear accumulation of β-catenin (C and D). β-Catenin signals (brown) can be detected in the nucleus (C) or cytoplasm (D). Original magnification ×400.
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Fig. 2. DNA sequencing chromatograms of PCR products of exon 3 in the CTNNB1 gene. The wild-type sequence from codons 44 to 47 of exon 3 in CTNNB1 gene (A). The CTNNB1 gene with a synonymous point mutation at codon 46 [CTG (Leu) → TTG (Leu)] (B).
of resection (P = .033 for histopathologic type of craniopharyngioma and P = .004 for type of resection, respectively). However, there was no significant association between aberrant β-catenin expression and other variables including age, sex, calcification, tumor volume, complications, and surgical approach (P N .05). 3.5. Correlation between aberrant β-catenin expression and prognosis in craniopharyngioma patients During the follow-up period, 9 patients (18.0%) had locally recurrent craniopharyngioma, and aberrant membranous β-catenin expression was observed in 7 (77.8%) of those patients. By the end of the followup period, 10 patients died of craniopharyngioma-related causes, 7 (70.0%) of which had aberrant β-catenin expression. Collectively, these findings indicate that aberrant membranous β-catenin expression may be a marker for poor prognosis in craniopharyngioma patients. To further validate this finding, we performed Kaplan-Meier survival
analysis and Cox regression analysis to assess the association between aberrant β-catenin expression and prognosis in craniopharyngioma patients. As shown in Fig. 3, aberrant β-catenin expression displayed prognostic significance for OS of all cases. The median value of OS of the patients with preserved β-catenin expression was 89.0 months (95% confidence interval [CI], 78.8-99.2 months). For patients with aberrant β-catenin expression, however, the median OS was 37.0 months (95% CI, 8.0-66.0 moths), which was significantly shorter than that of the patients with preserved β-catenin expression (log-rank test, P = .0004). Results of Cox regression analysis of OS in craniopharyngioma patients are summarized in Table 4. Univariate analysis demonstrated that aberrant membranous β-catenin expression was a potential prognostic factor for the survival of craniopharyngioma patients (relative hazard ratio [HR] was 8.69; P = .003). Multivariate analysis with 2 different models further confirmed univariate analysis findings, thereby indicating that aberrant β-catenin expression was an independent risk factor for poor prognosis in craniopharyngioma patients (relative HR, 14.55, and P = .002 for the first model; relative HR, 11.21, and P = .043 for the second model).
Table 3 Correlation between β-catenin expression and clinical and pathologic variables Variables
No. of cases CTNNB1 exon 3 mutation Yes No Age (y) b35 ≥35 Sex Male Female Histopathologic type Adamantinomatous Squamous papillary Calcification Yes No Tumor volume (cm3) b25 ≥25 Complication Yes No Type of resection Total resection Subtotal or partial resection Surgical approach Subfrontal approach Pterional or transsphenoidal approach Tumor recurrence during follow-up Yes No Vital status at end of follow-up Dead Alive
β-Catenin expression Preserved
Aberrant
31
19
1 30
6 13
16 15
12 7
18 13
14 5
17 14
16 3
5 26
6 13
19 12
7 12
12 19
10 9
21 10
5 14
22 9
13 6
2 29
7 12
3 28
7 12
P
.009
.425
4. Discussion In this study, we examined membranous expression of β-catenin in craniopharyngioma specimens from 50 patients and investigated the association between aberrant β-catenin expression and clinical and pathologic characteristics, as well as the relation to the prognoses of these patients. We found that there was a significant correlation
.264
.033
.293
.093
.336
.004
.849
.018
.030
Fig. 3. Correlation of aberrant membranous β-catenin expression with OS in craniopharyngioma patients. Individuals with aberrant membranous β-catenin expression had a significantly increased risk of death when compared with those having preserved β-catenin expression. Survival analysis was performed using the Kaplan-Meier method, and statistical significance was determined by the log-rank test. Patients living at the end of the follow-up period were censored for survival analysis.
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Table 4 Cox regression analysis of survival in patients with craniopharyngioma β-Catenin
Expression a b
HR (95% CI)
Preserved Aberrant
Univariate analysis
P
Multivariate analysis, model 1a
P
Multivariate analysis, model 2b
P
1.00 8.69 (2.13-35.48)
– .003
1.00 14.55 (2.66-79.68)
– .002
1.00 11.21 (1.08-116.51)
– .043
Adjusted for age, sex, and histopathologic type. Adjusted for all covariates including age, sex, histopathologic type, calcification, tumor size, tumor volume, complication, type of resection, and surgical approach.
between aberrant membranous β-catenin expression and CTNNB1 exon 3 mutation. In addition aberrant β-catenin expression was found to be associated with poor patient survival. Results of the Kaplan-Meier survival analysis and Cox regression analysis further confirmed this finding. Taken together, these findings suggest that aberrant β-catenin expression is an independent risk factor for a poor prognosis in patients with craniopharyngioma. In past decades, numerous studies have pointed out the critical role played by β-catenin in tumorigenesis and progression of craniopharyngioma [2]. As an essential component required for cadherin-dependent cell adhesion, cell membrane–localized β-catenin forms a complex with E-cadherin, α-catenin, and actin filaments, which acts on tumor cells as an “invasion suppressor” [16]. When the level of cell membrane–localized β-catenin is down-regulated in tumor cells, the cadherin/catenin cell adhesion system can be disrupted, thus contributing to enhanced cell migration and proliferation and, subsequently, invasion and metastasis [17]. On the other hand, disruption of the cadherin/catenin-based cell-cell adherens junction can release βcatenin from the adherens junction pool into cytoplasma [18]. Cytoplasmic β-catenin participates in the Wnt signaling cascade and is available for transport and accumulation in the nucleus in an aberrant manner [19]. Nuclear β-catenin forms complexes with T-cell factor and lymphoid enhancer binding factor family members, consequently inducing activation of the transcription of multiple target genes that are important for tumor invasion and progression [20]. Previous studies have demonstrated that cytoplasm/nuclear β-catenin accumulation is an exclusively characteristic morphology of craniopharyngioma, especially for adamantinomatous type [21,22]. In this study, aberrant cytoplasmic and/or nuclear accumulation of β-catenin was found in a large portion of patients who underwent subtotal or partial resection (14/24). This was also the case in most patients who experienced relapse of the disease. These findings provide further evidence that decreased levels of β-catenin at the cell membrane as well as aberrant accumulation in the cytoplasm and/or nucleus may be among the leading causes of tumor recurrence and relapse in craniopharyngioma patients. Recently, a number of studies have well documented that genetic mutations of β-catenin gene CTNNB1 can trigger increased levels of β-catenin in the cytoplasm or nucleus in craniopharyngioma tissue [11,12,23]. Most CTNNB1 mutations are localized to exon 3, which may affect the functional domains of β-catenin that are involved in interactions with its regulators, the adenomatous polyposis coli tumor suppressor protein and glycogen synthetase kinase 3-β (GSK3-β). Under normal physiological conditions, when the Wnt signaling cascade is inactive, cytoplasmic βcatenin is phosphorylated by a complex consisting of adenomatous polyposis coli tumor suppressor protein and GSK3-β as well as axin and casein kinase 1 and is then degraded via ubiquitination [16]. Nevertheless, mutations in CTNNB1 exon 3 have been found to cause amino acid substitution at GSK3-β phosphorylation sites or flank residues on these sites in the β-catenin protein, thus inhibiting the phosphorylation process and leading to cytoplasmic accumulation of β-catenin [2]. In this study, 7 patients showed a synonymous point mutation at codon 46 in CTNNB1 exon 3 [CTG (Leu) → TTG (Leu)], of which 6 had aberrant β-catenin expression. According to recent research, synonymous point mutations in the triplet code can influence protein translation efficiency and protein folding and function [24,25]. Therefore, we speculate that the synonymous point mutation at codon 46 in CTNNB1 exon 3 might
affect the folding pattern of the β-catenin protein, which blocks the exposure of phosphorylation sites to GSK3-β and subsequently inhibits the degradation of β-catenin and induces its aberrant accumulation in cytoplasma. However, further study is required to fully validate this speculation. Furthermore, Cani et al [26] found that craniopharyngioma cells without CTNNB1 mutations could also overexpress β-catenin, implying that an alternative mechanism may exist for the cytoplasmic and nuclear accumulation of β-catenin. This might be a reasonable explanation for the fact that a majority of patients without CTNNB1 exon 3 mutation also had aberrant β-catenin expression. However, the details of this underlying mechanism remain unknown. A number of clinical studies have revealed the significance of aberrant β-catenin expression in predicting the prognoses of various malignant tumors [27,28]. In our study, the results of our Kaplan-Meier survival analysis and Cox regression analysis demonstrate that aberrant β-catenin expression is an independent risk factor for a poor prognosis in patients with craniopharyngioma. As we discussed earlier, this may be attributed to tumor invasion and recurrence closely associated with a loss of membranous β-catenin expression and increased cytoplasmic/nuclear β-catenin expression. Nevertheless, it should be noted that other molecules, such as epidermal growth factor receptor, nestin, glial fibrillary acid protein, and microtubule-associated protein 2, may also be involved in the invasion process of craniopharyngioma [2]. The interactions between these molecules and the β-catenin/Wnt signaling pathway require further investigation. In summary, this study demonstrated that aberrant β-catenin expression was significantly correlated with poor survival rates in patients with craniopharyngioma. This raises the possibility for the use of aberrant β-catenin expression as an independent risk factor in predicting the prognoses of this disease. However, the small sample size and retrospective nature of this study indicate that these findings require further investigation and validation using a prospective method of study with a larger patient population. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.anndiagpath.2015.10.002.
Conflict of interests The authors declare that they have no conflict of interests regarding the current study.
Acknowledgments All authors would like to thank Ms Rebekah Burdyshaw, Cleveland State University, for her critical proofreading of the manuscript.
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