Abstract
Background: Abnormal catenin expression has been related to mammary carcinogenesis in both human and canine species and they are considered tumor- and invasion-suppressor molecules; however, in feline mammary tissues they have been scarcely studied. Materials and Methods: The immunohistochemical expression of α-, β- and p120-catenin was studied in a series of normal feline mammary glands, hyperplastic/dysplastic lesions and benign and malignant mammary tumors. Their relationship with clinicopathological parameters and with E- and P-cadherin expression was assessed. Results: Normal tissues, hyperplastic/dysplastic lesions and benign tumors expressed α-, β- and p120-catenin in the membrane of more than 75% of the luminal epithelial cells, while in malignant tumors, there was a reduction in their membranous expression and a p120-catenin cytoplasmic expression in 40%. Reduced α-catenin expression was related to tumor features with prognostic value, namely tumor size (p=0.0203) and necrosis (p=0.0205). The expression of α-, β- and p120-catenin were individually related to each other and collectively associated with E-cadherin expression. Conclusion: The results demonstrate a relationship between feline mammary carcinogenesis and decreased expression of catenins, suggesting that they may represent a valuable tool in the diagnosis of feline mammary neoplasms.
Tumor progression depends on the ability of tumor cells to overcome cell–cell adhesion and to invade, metastasize, and colonize distant sites (1). The cadherin–catenin complex, localized in the adherens junctions, is involved in cell-cell adhesion and the stability of this complex is required to maintain the integrity of epithelial tissues (2, 3). There exists evidence that the abnormal expression or function of the cadherin–catenin complex molecules results in decreased intercellular adhesion, tumor cell migration, invasion and metastatic dissemination in human breast cancer (3-6).
The classical E- and P-cadherin are transmembrane molecules responsible for calcium-dependent cell–cell adhesion that have three distinct domains: i) an extracellular domain that contains five cadherin repeats and forms homophilic bonds with cadherins on adjacent cells; ii) a single-pass transmembrane domain; and iii) a highly conserved cytoplasmic domain that forms complexes with a group of proteins collectively known as catenins (α-, β-, γ- and p120-catenin) (1-3, 7). Both β- and γ-catenin interact directly with the cytoplasmic carboxy-terminal catenin-binding domain of the cadherins in a mutually exclusive manner. α-Catenin is recruited to the complex through its interaction with β- or γ-catenin, linking the cadherin complex to the actin cytoskeleton, while p120-catenin connects directly to the juxtamembrane domain of the cytoplasmic tail of the cadherin molecules (2, 8).
The three catenins have distinct functions: α-catenin in addition to linking to the actin network, plays an important role in the regulation of distinct signaling pathways involved in cell proliferation, apoptosis, growth, migration and invasion, hence being considered a tumor suppressor molecule (9, 10); β-catenin is involved in cell–cell adhesion and cell signaling, as a member of the WNT/wingless signal transduction pathway (3), and in the regulation of the transcription of several proliferation and differentiation genes (11); p120-catenin stabilizes the cadherin–catenin complex and modulates cadherin intracellular trafficking, stability, adhesive capacity and cell motility (3, 12).
Although the expression of cadherins has been reported in canine and feline mammary tumors, to the best of our knowledge only β-catenin expression was studied in these species (13-19) and the characterization of the whole cadherin–catenin system in feline mammary carcinogenesis has not yet been described. In this context, we assessed the immunoexpression of α-, β- and p120-catenin in a series of normal feline mammary glands, hyperplastic/dysplastic lesions and benign and malignant mammary tumors, as well as their relation with the expression of classical P- and E-cadherin and with clinicopathological parameters with recognized prognostic value.
Materials and Methods
Tissue samples. Samples from 75 queens with naturally-occurring mammary lesions, surgically excised with curative intent, and nine normal mammary glands (obtained from queens that were humanely euthanized for reasons unrelated to neoplastic disease) were included in this study. In each case, an informed consent was granted by the owners. All specimens were fixed in 10% neutral buffered formalin and processed routinely. Consecutive histological sections (2 μm) were cut from each paraffin block. One was stained with haematoxylin and eosin (HE) for histological examination, and the others were used for immunohistochemistry (IHC). When available, local and regional lymph nodes were also processed and examined for the presence of metastases as described in Figueira et al. (20).
Histological classification of the tumors was performed independently by three observers (ACF, PDP and FG) based on the criteria of the World Health Organization (WHO) for the histological classification of mammary tumors of domestic animals (21).
Carcinomas were graded in accordance with the Nottingham grading system for human breast carcinomas, based on the assessment of three morphological features: tubule formation, nuclear pleomorphism, and mitotic counts, and tumors were classified as grade I (well-differentiated), grade II (moderately differentiated) and grade III (poorly differentiated) (22). Variables with known prognostic value, such as the mode of growth (infiltrative or expansive), tumor diameter (<2 cm, 2-3 cm, >3 cm), presence of necrosis, skin ulceration, lymph node metastases, and intravascular neoplastic emboli (23, 24) were also recorded.
α-, β- and p120-Catenin expression by immunohistochemistry. Immunohistochemistry was performed using a polymer-based system (Novolink Max Polymer Detection System, Product No: RE7280-K; Leica Biosystems, Newcastle, UK), according to the manufacturer's instructions. Sections were de-waxed in xylene and rehydrated through graded alcohols. Sections for β-catenin evaluation were treated with extran for 10 min in a microwave oven for antigen retrieval, while sections for α-catenin and p120-catenin evaluation were treated with 10 mM citrate buffer, pH 6.0, for 3 min in a pressure cooker. Endogenous peroxidase activity was blocked by treating the sections with 3% hydrogen peroxide in methanol for 10 minutes and rinsing in Tris-buffered saline (TBS, pH 7.6, 0.5 M). Sections were incubated overnight at 4°C in a humid chamber with specific mouse monoclonal antibodies against human α-catenin (clone αCAT-7A4; Zymed/Invitrogen, Camarillo, CA, USA), β-catenin (clone CAT-5H10; Zymed/Invitrogen), and p120-catenin (clone 98/pp120; BD Transduction Laboratories, Lexington, KY, USA). The antibodies were diluted 1:100, 1:300 and 1:1,000, respectively in TBS with 5% bovine serum albumin. Labelling was performed using 3,3’-diaminobenzidine at room temperature and sections were then counterstained with Mayer's haematoxylin, dehydrated and mounted. For negative controls, the primary antibody was replaced by TBS. Sections of human normal mammary gland were used as positive controls.
The immunoexpression evaluation of α-, β- and p120-catenin was assessed semiquantitatively according to the percentage of immunoreactive luminal epithelial cells with a membranous pattern, and graded as 0: <25%; 1: 25-50%; 2: 51-75% and 3: >75%, adapted from Penafiel-Verdu et al. (18). For statistical analysis, whenever >75% of luminal epithelial cells had membranous staining, the sample was considered to have preserved catenin expression, while all other cases were considered as having reduced expression. The staining pattern of primary tumors, lymph node metastases and intravascular neoplastic emboli was evaluated according to this described method.
The staining and evaluation methods for E- and P-cadherin expression were performed as described by Figueira et al. (20). To evaluate the combined expression of E-cadherin and catenins, samples were grouped according to the pattern of E-cadherin expression, as preserved or reduced and to the pattern of catenin expression as preserved expression of all catenins or reduced expression of at least one catenin.
Statistical methods. Data were organized in contingency tables and the likelihood ratio Chi-square test of associations was used to determine the significance of the relationship between the expression of the catenins and the tumors' clinicopathological parameters, as well as the cadherin expression. Whenever biologically consistent, 2×2 tables of contingency were built and Fisher's exact test was performed. All statistical analysis was performed using SAS/STAT, 1989 (SAS Institute Inc., Cary, NC, USA) (25) and, in all instances, p≤0.05 was considered to be statistically significant.
Results
Nine normal mammary gland samples, 13 hyperplastic/dysplastic lesions (7 fibrocystic disease cases and 6 of fibroadenomatous changes), 10 benign tumors (7 simple adenomas and 3 fibroadenomas) and 60 malignant tumors (32 tubulopapillary carcinomas, 16 solid carcinomas, 4 cribriform carcinomas, 6 mucinous carcinomas and 2 carcinosarcomas) were analyzed. Seven (11.67%) malignant tumors were grade I, 25 (41.67%) grade II and 28 (46.67%) grade III. In 21 (36.21%) carcinomas, neoplastic intravascular emboli were observed and in 18 (51.43%) out of the 35 cases where lymph nodes were available, metastases were identified. The P- and E-cadherin expression in this series has been previously analyzed and described by Figueira et al. (20).
Expression of α-catenin. In all normal mammary tissues, hyperplastic/dysplastic lesions, and benign tumors, α-catenin was expressed in the membrane of more than 75% of luminal epithelial cells (Figure 1a). The protein expression was reduced in 23 (38.33%) carcinomas (Figure 1b), a significant difference when compared with benign tumors (p=0.0245). Statistically significant relationships were also found between reduced α-catenin expression and larger tumor size (p=0.0203) and the presence of necrosis (p=0.0205) (Table I). Almost two-thirds (64.7%) of tumors larger than 3 cm had decreased α-catenin expression, while 72.1% of those of 3 cm or less had preserved the expression. The majority of non-necrotic carcinomas (91.7%) preserved α-catenin expression, while almost half of those with necrosis had reduced expression. There were no significant relationships between carcinoma α-catenin expression and histological type, grade or mode of growth, ulceration, neoplastic intravascular emboli or lymph node metastases (Table I).
Sixteen (88.89%) out of the 18 cases with neoplastic emboli (in three cases it was not possible to obtain representative sections for immunohistochemistry), exhibited preserved α-catenin expression (Figure 1c), while half of the 18 lymph node metastases also had preserved α-catenin expression (Figure 1d). The embolic and nodal metastatic patterns were unrelated to the expression of primary tumors.
There was a significant direct relationship between the expression of α-catenin and E-cadherin (p=0.0336), with preserved α-catenin expression in 75% of the carcinomas with preserved E-cadherin, and reduction in half of the tumors with reduced E-cadherin. No relationship was observed with the expression of P-cadherin (Table II).
Expression of β-catenin. In normal mammary glands, hyperplastic/dysplastic lesions, and benign tumors, β-catenin was expressed in the membrane of more than 75% of luminal epithelial cells (Figure 1e), while 38 (46.67%) carcinomas had reduced β-catenin expression (Figure 1f), a significant difference when compared with benign tumors (p=0.0045). In addition, nuclear β-catenin expression was observed in three carcinomas.
There were no statistical significant relationship between the expression of β-catenin and tumor histological type or grade, mode of growth, size, necrosis, or ulceration, nor with the presence of intravascular neoplastic emboli or lymph node metastases (Table I). The majority of cases with evidence of neoplastic emboli (n=16/18; 88.89%) (in three cases it was not possible to obtain representative sections for immunohistochemistry) preserved β-catenin expression by intravascular neoplastic cells (Figure 1g), while eight (44.44%) out of the 18 lymph node metastases had preserved β-catenin expression (Figure 1h). No significant relationships were established between the β-catenin expression pattern in the intravascular emboli and lymph node metastases and their primary tumors. There was no significant relationship between the expression of β-catenin, P- and E-cadherin in carcinomas (Table II).
Expression of p120-catenin. In normal mammary tissues, hyperplastic/dysplastic lesions, and benign tumors, p120-catenin was expressed in the membrane of more than 75% of the luminal epithelial cells (Figure 1i). In 17 (28.33%) carcinomas, there was a reduction in p120-catenin expression (Figure 1j), although this was not related to any of the evaluated clinicopathological parameters (Table I). Nuclear p120-catenin expression was observed in three carcinomas and one case of fibrocystic disease (Figure 2a). Although diffuse cytoplasmic p120-catenin expression was also observed in 40% of carcinomas (Figure 2b), it was not significantly associated with clinicopathological parameters or cadherin expression.
From the 20 cases with evidence of neoplastic emboli (in one case it was not possible to obtain representative sections for immunohistochemistry), the majority (n=18; 90%) exhibited preserved p120-catenin expression by intravascular neoplastic cells (Figure 1k), while 12 (66.67%) out of the 18 lymph node metastases preserved p120-catenin expression (Figure 1l).
There was a significant relationship between the protein expression in the primary tumors, their intravascular emboli (p=0.0316) and lymph node metastases (p=0.0217), all with preserved p120-catenin expression also exhibiting the same pattern in intravascular neoplastic cells (n=16; 100%), as well as in the majority of the lymph node metastases (n=11; 84.6%).
There was a significant relationship between the expression of p120-catenin and E-cadherin (p<0.001), with 90% of the malignant tumors with preserved E-cadherin also preserving p120-catenin. No relationship was demonstrated with the expression of P-cadherin (Table II).
The expressions of α-, β- and p120-catenin in malignant tumors were individually related to each other. Moreover, most carcinomas (n=21; 60%) with reduced expression of at least one catenin also had reduced expression of E-cadherin, and the majority of the tumors with preserved expression of all catenins (n=18; 72%) also preserved E-cadherin expression (p=0.0191). Considering the four elements of the complex (E-cadherin, and α-, β-, and p120-catenin), most benign tumors (n=9; 90%) preserved expression of all molecules, while 70% of the carcinomas had a reduction in at least one of the molecules that constitute the cadherin–catenin complex.
α- β- and p120-catenin expression by immunohistochemistry (IHC) in feline mammary tissue. a: Normal mammary gland with strong expression of α-catenin immunoreactivity in the membrane of luminal epithelial cells, ×400. b: Reduced membranous α-catenin expression in neoplastic cells in a grade II tubulopapillary carcinoma, ×400. c: Neoplastic intravascular embolus showing strong α-catenin expression, ×400. d: Lymph node metastases showing α-catenin expression in neoplastic cells, ×400. e: Normal mammary gland with strong expression of β-catenin immunoreactivity in the membrane of luminal epithelial cells, ×400. f: Reduced membranous β-catenin expression in neoplastic cells in a grade II tubulopapillary carcinoma, ×400. g: Neoplastic intravascular embolus showing strong β-catenin expression, ×400. h: Lymph node metastases showing β-catenin expression in neoplastic cells, ×400. i: Normal mammary gland with strong expression of p120-catenin immunoreactivity in the membrane of luminal epithelial cells, ×400. j: Reduced membranous p120-catenin expression in the membrane of epithelial cells in a grade III solid carcinoma, ×400. k: Neoplastic intravascular embolus showing strong p120-catenin expression, ×400. l: Lymph node metastases showing p120-catenin expression in neoplastic cells, ×400. Bar=50 μm.
a: Nuclear p120-catenin expression in a grade II tubulopapillary carcinoma as shown by immunohistochemistry, ×400. b: Diffuse cytoplasmic p120-catenin expression (*) in a grade III solid carcinoma as shown by immunohistochemistry, ×400. Bar=50 μm.
Discussion
Numerous studies have reported the expression of the cadherin–catenin complex molecules and their association with breast cancer (4-6). Although E-cadherin is considered a tumor and metastasis suppressor (26), it is questionable that the single loss of E-cadherin explains the neoplastic or metastatic phenotype, as the loss of adhesiveness does not necessarily induce cells to become motile or invasive (8). Since E-cadherin function is regulated by catenins (26), combined studies of cadherins and catenins may add information to the characterization of this phenotype. Such studies are scarce for feline mammary tumors (18, 19) and, to the best of our knowledge, this is the first time that the major catenins and cadherins have been simultaneously evaluated in normal and neoplastic feline mammary tissue.
The present investigation documented consistent membranous expression of α-, β-, and p120-catenin in luminal epithelial cells of the normal feline mammary gland, reflecting the importance of catenins in the maintenance of the structural and functional integrity of the normal mammary tissue in this species, mimicking similar roles in human breast (4, 27, 28).
Hyperplastic/dysplastic lesions and benign tumors maintained a similar pattern, while nearly 60% of carcinomas exhibited reduced expression of the three catenins, suggesting that, as previously shown for human (4, 5, 29, 30) and canine (13-15, 17) species, their loss contributes to the development of a malignant phenotype in queens. Our data support the widely accepted concept that the underexpression of cell adhesion molecules and loss of intercellular cohesion represent crucial events in the process of the typical architectural disorganization of mammary cancer (1, 3). α-Catenin is able to inhibit tumor formation and progression by maintaining the integrity of the cadherin–catenin complex and by regulating several signalling pathways involved in tumorigenesis such as WNT/β-catenin, Hippo/Yes-associated protein (Hippo-YAP), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), and Hedhehog pathways (10, 31). Abnormal α-catenin expression (such as reduced membrane expression and cytoplasmic or nuclear location) has been described in breast cancer (4, 27, 32, 33). Furthermore, several reports documented an association between down-regulation or loss of α-catenin in breast cancer and high tumor grade (10), metastasis (4, 10, 34) and poor survival (10), which raises the possibility that it may be considered a prognostic marker.
Our results also demonstrated a significant association between α-catenin expression and two parameters with known prognostic value, namely tumor size and necrosis. In larger tumors, a hostile metabolic microenvironment emerges, characterized by ischaemia, nutrients and energy deprivation, as a result of the discrepancies between cancer cell multiplication and the slower development of the supporting vascular network. It is widely accepted that changes in the tumor microenvironment influence several steps of carcinogenesis and neoplastic spread, acting as a potent modulator in the progression of the disease (35). Recently, Plumb et al. demonstrated that a stressful microenvironment contributes to the under-expression of α-catenin, being able to confer a selective survival and growth advantage to the neoplastic cells under ischaemic conditions (36). Furthermore, a borderline association between reduced α-catenin expression and larger tumor size in breast cancer was described by Bukholm et al. (37). Under such stressful conditions, the loss of α-catenin results in increased proliferation, decreased apoptosis and increased growth-factor signalling, thus promoting faster tumor growth that can account for larger tumor volumes and ischaemic necrosis at diagnosis (31, 36).
In our series, a significant reduction in β-catenin expression was observed in carcinomas compared to benign tumors. A similar loss of membrane-bound β-catenin, as well as its cytoplasmic or nuclear expression, have been described in human (4, 11, 34), canine (15-17) and feline (18, 19) mammary tumors. However, the prognostic value of β-catenin in human (4, 9, 34, 38) and canine (13-15) mammary tumors has not been clarified, with different studies revealing contradictory results. Similarly, this subject is not unequivocal in cats: while Penafiel-Verdu et al. associated preserved β-catenin expression with low-grade non-metastatic feline mammary carcinomas and its underexpression with metastatic carcinomas (18), Zappulli et al. failed to demonstrate an association between reduced β-catenin expression and clinicopathological features with known prognostic value (19).
Association of α-catenin, β-catenin and p120-catenin expression in malignant tumors with clinicopathological parameters.
Another interesting finding in this study relates to the subcellular p120-catenin location. While in normal mammary gland, hyperplastic and benign lesions p120-catenin was exclusively membranous, in 40% of carcinomas it was also identified in the cytoplasm. Furthermore, nearly 96% (23/24) of mammary tumors with cytoplasmatic p120-catenin expression were moderate- or high-grade carcinomas. Although significant correlation between sub-cellular p120-catenin location and the histological grade of carcinomas was not established, possibly due to the small number of tumors with this aberrant expression pattern, our data suggest that it may be relevant for the development and progression of feline mammary cancer. Previous reports on humans hypothesized that p120-catenin may play a dual role in breast carcinogenesis, acting as a tumor/metastasis suppressor as an element of the cell membrane cadherin–catenin complex, and as an oncogene/metastasis promoter when translocated to the cytoplasm (8, 26). Cytoplasmic p120-cadherin interacts with members of the Rho family GTPases (8, 12, 39) that regulate cell shape, adhesion, migration and polarity, thus promoting the invasive behaviour of neoplasms (8, 40). Its cytoplasmic translocation may also be responsible for structural changes in adherens junctions and disruption of actin filament organization, leading to significant modifications of cellular morphology and differentiation, hence justifying the higher histological grade of the lesions exhibiting this feature (39).
Association between expression of catenins and P- and E-cadherin in feline malignant mammary tumors.
Expressions of α- and β-catenin in neoplastic intravascular emboli and lymph node metastases were unrelated to their expression in corresponding primary tumors. In contrast, the expression pattern of p120-catenin in neoplastic emboli and lymph node metastases was very similar to that of the corresponding primary carcinomas. This finding corroborates the observations by Johnson et al. in breast cancer, where p120-catenin expression was maintained in distant metastases (40). Our data suggest that the staining pattern of p120-catenin is preserved during tumor progression, contradicting the previous concept that p120-catenin is lost during the metastatic process (5, 26).
In the course of metastasis, neoplastic cells must break-away from the primary tumor, move into the surrounding stroma, invade the lymphatic or vascular circulation, and re-establish growth at a secondary site. In this process, the loss or reduction of intercellular adhesion molecules has been postulated to facilitate detachment from the primary mass. However, during transit through the circulation, cohesive cell migration seems to provide a survival advantage compared to that of single dissociated cells (41). In fact, in approximately 90% of carcinomas with emboli, neoplastic intravascular cells preserve the expression of α-, β- and p120-catenin, irrespective of the expression in the primary tumor. This feature raises the possibility that the expression of the catenins in embolic cells may represent a strategy ensuring an effective spread of neoplastic cells. Cancer cells may migrate and invade both individually or as cohesive groups (42). During group cell migration, the maintenance of epithelial characteristics, namely a preserved cell–cell adhesion system, provides the group with strong mechanical, structural and functional properties; this allows the group to operate as a unit, increasing the individual chances of survival and metastasis (42-44). Our findings emphasize this concept, suggesting that catenins may contribute to intercellular physical and functional cohesion within emboli. Once at metastatic sites, the suitability of the microenvironment is an important factor in determining whether colonization occurs or not. In the lymph node metastases, we observed that the expression was preserved in 50%, 44.4% and 66.7% for α-, β- and p120-catenin, respectively. In order to overcome vascular extravasation, survive, proliferate and invade in a harsh microenvironment, metastatic cells undergo modifications that may include modifications of catenin expression that are advantageous for metastasized cells, such as the demonstrated association between underexpression of α-catenin and increased proliferation, decreased apoptosis and survival in hostile microenvironments (36).
In 70% of the carcinomas included in this series, underexpression of at least one of the members of the E-cadherin–catenin complex was observed; supporting the concept that dysfunction of the cadherin–catenin adhesion system is intimately related to malignancy in feline mammary carcinogenesis. Moreover, we found that carcinomas with underexpression of E-cadherin frequently lose α- and p120-catenin expression. Concurrent loss of β-catenin was also observed, but this feature was not statistically significant.
Considering that α-, β- and p120-catenin work together in the cadherin–catenin complex, it was not surprising that their expressions were individually related in our series of carcinomas, as has previously been described in breast cancer (4, 27).
Efforts to unveil the prognostic value of catenins in mammary carcinomas have not been conclusive in humans (29, 30, 38, 45), dogs (13-15) or cats (18, 19). While some studies associated a down-regulation of catenins with metastatic disease and poor prognosis (4, 5, 34, 45), others were unable to relate the expression of the catenins with traditional prognostic indicators (lymph node status, tumor size, steroid receptor expression, age and overall survival) (9, 27, 28).
Conclusion
This study describes membranous expression of α-, β- and p120-catenin in the luminal epithelium of normal feline mammary glands and demonstrates an association between decreased expression of these adhesion molecules and carcinogenesis in this species. In fact, underexpression of catenins was observed only in carcinomas, suggesting that catenins may represent a valuable tool assessing in the diagnosis of mammary neoplasm in queens.
Furthermore, data from this investigation point to the importance of the E-cadherin–catenin complex in mammary carcinogenesis in feline species. However, the prognostic value of these cell adhesion molecules seems to be limited in cats. Further prospective studies, with larger series of mammary carcinomas and of different carcinoma grades and types, as well as functional molecular studies, are warranted to evaluate the importance of the cadherin–catenin complex in feline mammary carcinogenesis, as well as its prognostic relevance.
Acknowledgements
The Authors would like to acknowledge the veterinarians of the Municipal Kennel of Coimbra, Filomena Ramalho and Mariana Portugal for providing samples. We thank the technical staff of the Laboratory of Veterinary Pathology of Institute of Biomedical Sciences Abel Salazar of the University of Porto for the precious technical support. This work was partially supported by Portuguese Foundation for Science and Technology FCT (PTDC/CVT/117610/2010) financed under the Programa Operacional Temático de Fatores de Competitividade (COMPETE) e do Quadro de Referência de Estratégia Nacional QREN. FCT supports ACF (Ph.D. grant SFRH/BD/69493/2010) and CG (Postdoctoral grant SFRH/ BPD/ 96510/ 2013). The Institute of Molecular Pathology and Immunology of the University of Porto is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education and is partially supported by the Portuguese Foundation for Science and Technology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the article.
Footnotes
Competing Interests
The Authors declare that they have no competing interests with regard to this study.
- Received March 6, 2015.
- Revision received March 17, 2015.
- Accepted March 19, 2015.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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