Telomerase and N-cadherin differential importance in adrenocortical cancers and adenomas+
Sofia S. Pereira1,2,3; Valdemar Máximo1,2,4; Ricardo Coelho1,2; Rui Batista1,2; Paula Soares1,2,4; Susana G. Guerreiro1,2; Manuel Sobrinho-Simões1,2,4,5; Mariana P. Monteiro3; Duarte Pignatelli1,2,
Instituto de Investigação e Inovação em Saúde (I3S) da Universidade do Porto, R. Alfredo Allen, 4200-135 Porto, Portugal.
2 Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Rua Júlio Amaral de Carvalho,45; 4200-135 Porto, Portugal.
3 Department of Anatomy and UMIB (Unit for Multidisciplinary Research in Biomedicine) of ICBAS, University of Porto, R. de Jorge Viterbo Ferreira n.º 228, 4050-313 Porto, Portugal.
4 *Department of Pathology and Oncology, Medical Faculty, University of Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto, Portugal
5 Department of Pathology, Hospital S.João , Alameda Prof. Hernâni Monteiro, Porto, Portugal “Department of Endocrinology, Hospital S.João, Alameda Prof. Hernâni Monteiro, Porto, Portugal.
*Corresponding Author and person to whom reprint requests should be addressed:
Duarte Pignatelli, MD, PhD I3S - Cancer Signaling and Metabolism group R. Alfredo Allen, 4200-135 Porto, Portugal.
Phone: +351 912880313
Email: dpignatelli@ipatimup.pt
Key Words: Adrenocortical tumors, Adrenal gland, TERT promoter mutations, Telomerase expression, N-cadherin expression
Running head: Telomerase and N-cadherin in ACT
Grants
Contract grant sponsor: Portuguese Foundation for Science and Technology (FCT); Contract grant number: SFRH/BD/89308/2012
Contract grant sponsor: FEDER through the Operational Programme for Competitiveness Factors (COMPETE) and National Funds through the FCT; Contract grant number: PEst-C/SAU/LA0003/2013 Contract grant sponsor: Portuguese Foundation for Science and Technology; Contract grant number: UID/Multi/00215/2013
Contract grant sponsor: Programa Operacional Regional do Norte (ON.2 - O Novo Norte) under the Quadro de Referência Estratégico Nacional (QREN) and the Fundo Europeu de Desenvolvimento Regional (FEDER); Contract grant name: “Microenvironment, metabolism and cancer”
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/jcb.25811]
Received 17 October 2016; Revised 22 November 2016; Accepted 23 November 2016 Journal of Cellular Biochemistry This article is protected by copyright. All rights reserved DOI 10.1002/jcb.25811
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ABSTRACT
Adrenocortical carcinomas (ACC) are most frequently highly aggressive tumors. We assessed the telomerase reverse transcriptase (TERT) and N-cadherin role in the biology of ACC and their potential utility as molecular biomarkers, in different types of tumoral adrenocortical tissue. A total of 48 adrenal cortex samples (39 tumoral and 9 normal adrenal glands) were studied. TERT promoter mutations were searched by PCR and Sanger sequencing in two hotspots positions (-124 and -146). Also, telomerase and N-cadherin expression were evaluated by immunohistochemistry.
TERT promoter mutations were not detected in any of the samples either malignant or benign. Telomerase nuclear expression was present in 26.6% of ACC and in 45.5% of non-functioning adenomas. It was absent in benign Cushing’s lesions and in normal adrenal glands. Contrarily, N- cadherin was always expressed in the cellular membranes of benign adenomas or normal adrenals but no expression was detected in the majority of ACC. Nuclear telomerase and membrane N- cadherin expression were positively correlated in ACCs.
We conclude that in ACC the loss of N-cadherin is a frequent phenomenon while the existence of TERT promoter mutations is not and nuclear telomerase expression is present in only a minority of cases. Since the loss of N-cadherin expression was identified in both high and low proliferative ACC, this marker should be considered important for diagnostic application. Our study also suggests the existence of a TERT non-canonical function in cell adhesion. This article is protected by copyright. All rights reserved
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INTRODUCTION
Adrenal cortex tumors (ACT) can be either malignant (adrenocortical carcinomas -ACC) or benign (adrenocortical adenomas - ACA) including secretory and non-secretory forms. The latter are among the most common tumors in humans with a prevalence generally reported as being superior to 4% in the general population [Davenport et al., 2011]. Contrarily, adrenal cortex carcinomas (ACC) are rare, but highly aggressive having an extremely poor prognosis, mainly due to the advanced stages at which they are usually diagnosed [Lafemina and Brennan, 2012; Zheng et al., 2016]. Understanding of the adrenocortical tumors’ biology and identification of progression factors will contribute to a more correct and comprehensive tumor categorization and is certainly one of the most challenging areas in adrenal pathology [Lau and Weiss, 2009].
Telomeres play an essential role regulating genomic stability by allowing the cell to distinguish between chromosome ends and double-strand DNA breaks [Dewar and Lydall, 2012]. To maintain the telomeres, cells use a specialized enzyme complex called telomerase that is able to add TTAGGG repeats to the ends of chromosomes. This complex is formed by two core subunits: the catalytic telomerase reverse transcriptase (encoded by TERT) and the telomerase RNA component (TERC) [Doksani et al., 2013; Martinez and Blasco, 2011]. Telomerase and other molecules with key roles in the regulation of telomere length and end-protection, frequently have altered expression or are affected by somatic mutations in cancers conferring these malignant cells the ability to bypass senescence while promoting genomic instability.
Many cancers display increased telomerase activity leading to sustained telomere maintenance [Cong et al., 2002; Kyo et al., 2008; Vinagre et al., 2013].Germline mutation as well as somatically acquired mutations in the promoter of TERT increase the expression of TERT and have been reported to constitute a cancer-predisposition condition [Akincilar et al., 2016; Vinagre et al., 2013; Vinagre et al., 2014]. Nevertheless, there are few studies that have analyzed their contribution to the development of adrenocortical tumors [Liu et al., 2014; Papathomas et al., 2014; Zheng et al., 2016].
Telomerase activation has been related to cellular immortalization and cancer, having been described in 90% of human cancers [Cong et al., 2002; Kyo and Inoue, 2002; Kyo et al., 2008]. However, the mechanisms leading to telomerase reactivation or re-expression and its role in carcinogenesis are not yet completely understood [Donate and Blasco, 2011; Kyo et al., 2008]. Mutations in the promoter of the telomerase catalytic reverse transcriptase subunit (TERT) located in two hotspots at -124 and -146 bp upstream the ATG start site, were found to be the most important mechanism responsible for reactivation or re-expression of telomerase in cancer cells [Akincilar et al., 2016; Vinagre et al., 2013; Vinagre et al., 2014]. This ATG start site is responsible for generating a consensus binding site for transcription factors of the E26 transformation-specific (ETS) family within the TERT promoter region that stimulate the TERT promoter activity and, consequently, TERT transcription and synthesis [Horn et al., 2013; Huang et al., 2013; Patton and Harrington, 2013]. These TERT promoter mutations have already been documented in several cancers, namely of the central nervous system, the bladder, thyroid (follicular cell-derived tumors) and in melanomas [Vinagre et al., 2014]. In the case of the adrenal cortex, however the Cancer Genomic Atlas (TCGA), a multinational project that analyzed the genomes of different human cancers only identified 4 cases of TERT promoter mutations at -124 bp upstream the ATG start site, in 91 Adrenal Cortex Cancers [Zheng et al., 2016].
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Besides to unregulated growth, cancer cells are also characterized by invasiveness. Part of the aggressiveness of cancers is highly dependent on the loss of cell to cell adherence and hence on changes in the function of cell adhesion molecules (CAM) that regulate the connection between the neoplastic cells, as well as, between cells and extracellular matrix [Cavallaro and Christofori, 2004; Wheelock et al., 2008]. CAM have also been implicated in the control of cell proliferation and hence in neoplasia formation.
Cadherins are among such molecules and their expression in several malignant tumors has been demonstrated to be reduced or at least inactivated [Cavallaro and Christofori, 2004; Hirohashi, 1998; Wheelock et al., 2008]. There are 3 main cadherin molecules E-, P- and N-cadherin, each one associated with different tissues and tumors. Changes in their level of expression have been associated with increased tumor aggressiveness [Angst et al., 2001; Halbleib and Nelson, 2006].
Cell adhesion molecules are now considered to play a significant role in the reduction of connections of cancer cells and especially metastatic cancer cells: reduced expression of E-cadherin on invasive neoplastic cells has been demonstrated in cancers of the stomach, liver and breast [Furukawa et al., 1994; Wheelock et al., 2008]. On the other hand, markedly elevated levels of soluble cadherins, like E-cadherin have been demonstrated in patient with metastatic cancer [Furukawa et al., 1994; Inge et al., 2011]. Other tumors, instead of losing the expression of a certain cadherin, switch the cadherin subtype [Wheelock et al., 2008]. This switch has been observed in various metastatic tumors such as breast and prostate cancers, suggesting that it apparently confers progression advantage to such tumors [Araki et al., 2011; Mariotti et al., 2007; Wheelock et al., 2008]. The adrenal cortex, despite being an epithelial tissue, is normally characterized by an absence of the E-cadherin and the presence of N-cadherin [Khorram-Manesh et al., 2002; Pereira et al., 2013; Tsuchiya et al., 2006]. Similarly to E-cadherin, N-Cadherin expression has been described to be altered in some types of tumors namely in the adrenal cortex [Khorram-Manesh et al., 2002; Velazquez-Fernandez et al., 2005].
The aim of our study was to evaluate cadherin expression in conjunction to telomerase promoter mutation and telomerase nuclear expression in adrenocortical tumors (both benign and malignant, secretory and non-secretory), as well as in normal adrenal tissue in order to try to identify a pattern of molecular markers that may be useful in the differential diagnosis of adrenocortical tumors and also possible targets for therapeutical drugs’ development.
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MATERIALS AND METHODS
Case Selection
The study was approved by the Ethics Committee of the Centro Hospitalar São João - Porto, Portugal. The participants provided their written informed consent to accept that a tumor sample is stored in the tumor bank of the Department of Pathologic Anatomy - Centro Hospitalar São João, Porto, to posteriorly be used in research.
Samples from adrenal tumors were obtained from 39 patients, with adrenocortical carcinoma (ACC) (n=15) and adrenocortical adenomas (ACA) (n=24), including non-functioning adenomas (n=11) and cortisol secreting lesions presenting as Cushing syndrome (CUSH) (n=13). Nine specimens of normal adrenal glands obtained in nephrectomy procedures for the treatment of kidney tumors (N- AG) (n= 9) were also used.
DNA extraction
DNA was extracted from 10 um sections of paraffin-embedded tissues after careful micro- dissection. The extraction was performed using the Ultraprep Tissue DNA Kit (AHN Biotechnologie, Nordhausen, Germany) following the manufacturer’s instructions.
PCR and Sanger sequencing for TERT
Screening of TERT promoter mutations was performed in two hotspots located at -124bp and - 146bp upstream from the ATG start site previously identified by PCR followed by Sanger sequencing. TERT promoter mutation analysis was performed with the pair of primers FwTERT: CAGCGCTGCCTGAAACTC and RwTERT: GTCCTGCCCCTTCACCTT. Amplification of genomic DNA was performed by PCR using the commercial kit Qiagen Multiplex PCR (Qiagen, Hilden, Germany) following the manufacturer instructions. Sequencing reaction was performed with the ABI Prism BigDye Terminator Kit (Perkin-Elmer, Foster City, California) and the fragments were run in an ABI prism 3100 Genetic Analyser (Perkin-Elmer).
Telomerase, Ki-67 and Cadherins Immunohistochemistry (IHC)
IHC was performed in 3um formalin-fixed paraffin embedded tissue sections mounted on adhesive microscope slides. Sections were deparaffinized, rehydrated in graded alcohols and underwent antigen retrieval performed by microwave treatment in 0.01 M-citrate buffer at pH 6.0. Then, the samples were incubated overnight at 4° C with the primary antibody for hTERT (polyclonal, rabbit, 1:500, Rockland Immunochemicals Inc., Gilbertsville, PA), Ki-67 (polyclonal, rabbit, 1:500, Cell Marque), N-cadherin (1:900, ab18203, Abcam, Cambridge, UK), E-cadherin (1:200, EP700Y, Cell Marque, Rocklin, CA, USA) or P-cadherin (1:200, HPA001767, Atlas Antibodies, Stockholm,Sweden). The detection of the immune reaction was performed using the streptavidin- biotin immunoperoxidase method (Thermo Scientific/Lab Vision,Fremont, USA). DAB (3,3’- Diaminobenzidine) was used as chromogen and hematoxylin as nuclear counterstaining. A previously tested liver cancer case was used as positive control for hTERT, tonsil for Ki-67, normal
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liver for N-cadherin, lung adenocarcinoma for E-cadherin, and normal human tonsil for P-cadherin, while the omission of primary antibody incubation was used as negative control.
Ki-67 expression was evaluated using the ImageJ software and the percentage of the stained area was obtained, as described before [Pereira et al., 2013]. Cytoplasmic and nuclear telomerase expression and membrane cadherin expression were recorded in all tissue samples and evaluated independently by two observers. An IHC score for telomerase was established, 0 for no staining; 1 for staining present in 20%-50% of nuclei; and 2 for staining present in 50%-100% of nuclei. In the statistical comparisons only TERT nuclear expression was considered. For E-, N- and P-cadherin, the membrane expression was considered 0 if there was no expression or 1 if the tissues presented membrane expression.
Statistical analysis
The IHC scores for telomerase nuclear expression and for N-cadherin membrane expression were compared among the different groups, through the X2 test. To compare the percentage of the stained area for Ki-67 between the analyzed groups, the one-way ANOVA test with the post-hoc Dunn’s was used. The correlations were performed through the Spearman Test. Statistical analysis was carried out using the SPSS software (version 20.00) for Windows and a value of p<0.05 was considered statistically significant.
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RESULTS
TERT promoter mutations
TERT promoter mutations in the hotspots located at -124bp and -146bp upstream from the ATG start site were not observed in any of the studied cases, including in ACC.
Telomerase Immunohistochemistry
Contrarily to the absence of TERT promoter mutations, telomerase expression, as assessed by immunohistochemistry, was observed both in the cytoplasm and in some cases in the nucleus. Significant differences between the groups were present (p<0.05).
Cytoplasmic expression - Telomerase cytoplasmic expression was present in all types of adrenocortical tumors studied (Figure 1). It was also observed in the cytoplasm of normal adrenal glands. Cytoplasmic expression did not differ, in statistical terms, between the different types of tumor. The only difference that was observed was that tissue areas with higher lipid droplet’s content apparently displayed lower telomerase staining. This was especially noticeable in the normal adrenal tissue where the Fasciculata layer apparently had lower cytosol staining (Figure 1).
Nuclear expression - The IHC score for nuclear expression was significantly different between the studied groups (p<0.05). The majority (73,4%) of the ACC were negative for telomerase nuclear staining (Figure 1A). The staining was positive in 26.6% of ACC being that 13.3% of those presented less than 50% of nuclei stained and 13.3% presented more than 50% of the nuclei stained (Figure 1B) (Table 1).
Non-functioning adenomas presented positive staining in 45.5% of the cases. Of these, 27.3% presented less than 50% of nuclei stained and 18.2% presented more than 50% of the nuclei stained (Figure 1C and 1D) (Table 1).
On the contrary, none of the cortisol secreting adenomas presented nuclear staining for telomerase (Figure 1E) (Table 1).
Finally, none of the analyzed normal adrenal glands presented telomerase nuclear staining (Figure 1F) (Table 1).
Cadherins expression
E-, N- and P-cadherin expression in the membrane was assessed by immunohistochemistry in all tissue samples. The E- and P-cadherin were not expressed in any of the studied cases (data not
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shown). On the contrary generalized immunostaining for N-cadherin was clearly present. However, the IHC score for N-cadherin membrane expression was significantly different between the studied groups (p<0.001). The majority of the adrenocortical carcinomas (67%) did not present N-cadherin at the membrane, while the adrenocortical adenomas, and normal adrenal glands always presented N-cadherin membrane expression (Table 2 and Figure 2).
Ki-67 expression
The proliferation levels were evaluated through the Ki-67 immunohistochemistry analysis. The percentage of stained area for Ki-67 was significantly increased in the adrenocortical carcinomas compared with the other groups (p<0.01) (Figure 3). A negative significant correlation between the N-cadherin and Ki-67 expression was verified (R2= - 0.6102; p<0.001). Contrarily, in the ACC group, no correlation was found between N-cadherin and Ki-67 (p>0.05).
Correlation of the telomerase and N-cadherin membrane expression
The majority of the ACC without N-cadherin expression in the membrane did not present nuclear telomerase expression (87.5%), while 75% of the carcinomas with N-cadherin presented nuclear telomerase expression (p<0.001) (Figure 4).
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DISCUSSION
Adrenocortical carcinomas are generally highly aggressive tumors and the understanding of its molecular pathogenesis is still limited. In consequence, the differential diagnosis between adenomas and carcinomas is sometimes difficult and the progress towards newer therapeutic tools still very limited [Lafemina and Brennan, 2012; Pereira et al., 2013; Ragazzon et al., 2010].
In the present study we investigated the frequency of TERT promoter hotspot mutations in adrenocortical tumors, while at the same time we analysed the molecular distribution of telomerase, cadherins and Ki-67 in the same tumoral cells, to understand the roles played by these molecules in the biology of these tumors and the possible use of these proteins as therapeutic targets or at least as biomarkers for the differential diagnosis between benign and malignant adrenal tumors.
Tumors that have high frequency of TERT promoter mutations originate mainly from tissues with low rates of self-renewal such as glioblastomas, melanomas and thyroid carcinomas [Killela et al., 2013; Vinagre et al., 2013]. In contrast, the adrenal cortex is an exceptionally dynamic endocrine organ with a high rate of self-renewal [Pihlajoki et al., 2015]. Mutations in the promoter region of the TERT gene are among the most common somatic genetic lesions in human cancers, but knowledge about their frequency in adrenal cortex tumors has been limited because of the heterogeneity of these tumors. In our study, none of the cases presented TERT promoter mutations, which agrees with the presence of those mutations in no more than 12% of adrenocortical carcinomas formerly reported by other authors [Liu et al., 2014; Papathomas et al., 2014] and, more recently, in 4% of the cases of the multinational project of TCGA [Zheng et al., 2016]. All of the mutations found in these studies were at-124 bp upstream the ATG start site [Liu et al., 2014; Papathomas et al., 2014; Zheng et al., 2016]. The low prevalence of TERT promotor mutations might imply that other mechanisms could be active leading to the maintenance of the telomeric function by alternative pathways. TERF2 is a gene that is related to one of such mechanisms. TERT and TERF2, were amplified respectively in 15% and 7% of the ACC analyzed in the TCGA study [Zheng et al., 2016]. Since our study had an absolute number of ACC cases that was smaller than for instance that of TCGA, these results may not be considered significantly different. Taking together all of the studies that have addressed the presence of TERT promotor mutations in ACC cancer we have to conclude that the presence these mutations in these carcinomas (12 positive cases in 178 samples of ACC) has to be considered infrequent.
On the other hand, our study is the first to evaluate the immunohistochemical expression of telomerase in adrenocortical tumors. And in fact telomerase expression occurred in 26.6% of the ACC. An interesting possibility is that these may be carcinomas in initial phases of development when the prolonging of cellular viability may be crucial to the occurrence of cellular modifications that lead to more aggressive forms of ACC. Taking into account that the majority of ACC did not have increased telomerase expression (as well as TERT promoter mutations), telomerase over- expression does not seem to be crucial for the malignant adrenocortical tumors.
A completely different situation was observed concerning the expression of N-Cadherin during tumorigenesis. Changes in cadherin expression were observed in various types of tumors and have been associated with increased tumor aggressiveness because cells lose their interaction with neighbor cells and intercellular matrix and can more easily invade the neighboring organs or migrate [Cavallaro and Christofori, 2004; Mariotti et al., 2007; Wheelock et al., 2008].
In the normal adrenal glands and in adrenal cortex adenomas the cadherin that is normally expressed is N-cadherin and our study confirmed that. There was, however, a significant loss of N-
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cadherin expression in malignant tumors. Loss of N-cadherin membrane expression was found in the majority of the adrenocortical carcinomas suggesting that this phenomenon is involved in the aggressiveness of these cases, possibly by being responsible for a reduction of cell adhesion thus facilitating the process of cellular migration and invasion that could lead to metastization.
In a previous study, we showed that the ß-catenin abnormal expression was observed in both ACC and ACA being increased in the nonfunctioning ACTs [Pereira et al., 2013]. Since the ß-catenin abnormal expression is related with the ACT functionality and not with their malignancy [Pereira et al., 2013], no association could be demonstrated between the expression of abnormal ß-catenin and N-cadherin.
The evidence of the existence of TERT non-canonical functions such as roles in apoptosis, DNA damage response, inflammation and gene expression regulation has been reported [Li and Tergaonkar, 2014; Liu et al., 2016; Perrault et al., 2005].
According to our results a significant relationship between telomerase nuclear expression and N- cadherin membrane expression does exist, since the majority of the carcinomas with telomerase expression, presented intact N-cadherin in the membrane. Curiously, in a study using a hTERT- transfected prostate tumor cell line the authors observed a concomitant overexpression of N- Cadherin and suggested that telomere elongation might affect the cadherin expression [Hirashima et al., 2013]. More recently, Liu et al generated two cell lines with TERT overexpression expression and observed that TERT expression significantly increased the cell adhesion [Liu et al., 2016]. Our study, together with these studies seems to support another non-canonical function of TERT: a TERT role in cell adhesion.
Besides that, our results support the hypothesis that different tumors use distinctive molecular approaches in order to reach advantage that may promote tumor progression, depending on the endogenous proliferative rate of the tissues where those tumors originate. Since the adrenal cortex has a high rate of self-renewal [Pihlajoki et al., 2015] and the ACC are characterized by having an increased rate of proliferation, as we confirmed through the Ki-67 immunohistochemistry, these tumors do not need an increased telomerase expression to maintain the survival of cells. In this case, what seems to be more important is space to expand and the loss of cell adhesion that allows the cells to invade and metastasize is the most important.
As a general rule we postulate that there is telomerase re-expression in carcinomas with slow proliferative capacity in order to prolong their cell’s lifespan allowing them to accumulate somatic cancerigenic mutations. These carcinomas usually maintain the cadherins expression at their membranes at least in the first phases of their transformation, while in carcinomas without increased telomerase expression the cells have an elevated proliferation rate [Pereira et al., 2013] and tend rather to loose cadherin adhesion at their membranes to facilitate the tumor expansion.
Finally, no correlation was observed between N-cadherin and Ki-67 in the ACC group, meaning that the loss of N-cadherin expression is observed in both high and low grade ACC, reinforcing the idea that this marker may in the future have a great importance for diagnostic application.
In conclusion, our study shows that TERT promoter mutations and nuclear telomerase expression are not very frequent in adrenocortical carcinomas and are not likely to be useful molecular markers for differential diagnosis or treatment target. In contrast, the loss of the N-cadherin membrane expression is frequent in adrenocortical malignant tumors and may represent a useful marker for diagnosis and/or treatment. TERT may have a non-canonical function in the cell adhesion.
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ACKNOWLEDGEMENTS
The authors have no Conflict of Interest disclosure.
This study was supported by the Portuguese Foundation for Science and Technology (FCT) through a PhD grant to Sofia S. Pereira (SFRH/BD/89308/2012). Further funding was obtained from the project “Microenvironment, metabolism and cancer” that was partially supported by Programa Operacional Regional do Norte (ON.2 - O Novo Norte) under the Quadro de Referência Estratégico Nacional (QREN) and the Fundo Europeu de Desenvolvimento Regional (FEDER). IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT. This work is funded by FEDER funds through the Operational Programme for Competitiveness Factors - COMPETE and National Funds through the FCT, under the projects “PEst-C/SAU/LA0003/2013”. Unit for Multidisciplinary Research in Biomedicine is funded by grants from the Foundation for Science and Technology (UID/Multi/00215/2013).
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LEGENDS OF THE FIGURES
Figure 1- Immunohistochemistry staining of telomerase reverse transcriptase (Scale = 20 um). A- Adrenocortical carcinoma negative for nuclear staining; B- Adrenocortical carcinoma with nuclear positive staining; C- Non-functioning adrenocortical adenoma without nuclear staining; D- Non- functioning Adrenocortical adenoma with nuclear positive staining; E- Adrenocortical adenoma with Cushing Syndrome without nuclear staining; F- Normal adrenal gland without nuclear staining. Arrow heads are showing cells of glomerulosa layer that present low levels of lipid droplets and the arrows are pointing cells of fasciculata layer that present high levels of lipid droplets.
Figure 2- Immunohistochemistry staining of N-cadherin (Scale = 20 um). A- Adrenocortical carcinoma; B- Non-functioning adrenocortical adenoma; C- Adrenocortical adenoma with Cushing Syndrome and D- Normal adrenal gland
Figure 3 - Immunohistochemistry staining of Ki-67 (Scale = 50 um). A- Adrenocortical carcinoma; B- Adrenocortical adenoma with Cushing Syndrome; C- Non-functioning adrenocortical adenoma; D- Normal adrenal gland, and E- Graphic representation of the percentage of the Ki-67 in the studied groups (ANOVA: *** p<0.001).
Figure 4 - Relation between the N-cadherin and telomerase expression in the adrenocortical carcinomas (ACC).
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Accepted Article
| Groups | n | Zone | Score | ||
|---|---|---|---|---|---|
| 0 | 1 | 2 | |||
| ACC | 15 | 11 (73.4%) | 2 (13.3%) | 2 (13.3%) | |
| ACA - Non- functioning | 11 | 6 (54.5%) | 3 (27.3%) | 2 (18.2%) | |
| ACA - Cushing Syndrome | 13 | 13 (100.0%) | 0 (0.0%) | 0 (0.0%) | |
| Normal-AG | 9 | Z Glomerulosa | 9 (100.0%) | 0 (0.0%) | 0 (0.0%) |
| 9 | Z Fasciculata | 9 (100.0%) | 0 (0.0%) | 0 (0.0%) | |
| 9 | Z Reticularis | 9 (100.0%) | 0 (0.0%) | 0 (0.0%) | |
Scoring explanation: 0-No staining; 1- Staining present in 20%-50% of nuclei; 2- Staining present in 50%-100% of nuclei
| Groups | Score n 0 1 | ||
|---|---|---|---|
| ACC | 15 | 10 (66.7%) | 5 (33.3%) |
| ACA - Non-functioning | 11 | 0 (0.0%) | 11 (100.0%) |
| ACA - Cushing Syndrome | 13 | 0 (0.0%) | 13 (100.0%) |
| Normal-AG | 9 | 0 (0.0%) | 9 (100.0%) |
Scoring explanation: 0- No N-cadherin membrane staining; 1- N-cadherin staining in the membrane
Summary table
| N-AG | ACA-Non functioning | CUSH | ACC | |
|---|---|---|---|---|
| TERT promoter mutation | - | - | - | - |
| Nuclear expression of telomerase | - | + ☒ | - | + ☒ |
| Membrane expression of N-cadherin | ++ ☒ | ++ ☒ | ++ | - |
| Nuclear expression of Ki-67 mean of the % of stained area + S.E.M. (range) | 0.05±0.012 (0.00-0.17) | 0.08+0.028 (0.00-0.4) | 0.13 ±0.021 (0.01-0.22) | 2.15±0.653 (0.08-7.43) |
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A
8
B
20 um
20 um
C
D
20 um
20 um
E
F
20 um
20 pm
Accepted Article
A
B
20 um
20 um
C
D
1
20 um
20 um
Accepted Article
A
B
E
I
3.0-
D
2.0
1.0-
0.0
ACC
Non-functioning ACA
ACA with Cushing Syndrome
N-AG
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% of ACC samples
100
Telomerase expression in 50%-100% of nuclei
50
Telomerase expression in 20%-50% of nuclei
No nuclear telomerase expression
0
No membrane Membrane Staining Staining N-cadherin expression