|Year : 2022 | Volume
| Issue : 3 | Page : 623-628
Assessment of angiogenesis using endoglin in salivary gland tumors – An immunohistochemical study
Pratyusha Prakash Gaonkar1, Sangeeta R Patankar2, Gokul Sridharan2
1 Department of Medical Affairs, Lupin Limited, Mumbai, Maharashtra, India
2 Department of Oral Pathology and Microbiology, YMT Dental College and Hospital, Navi Mumbai, Maharashtra, India
|Date of Submission||03-Jan-2021|
|Date of Decision||28-Apr-2021|
|Date of Acceptance||30-Apr-2021|
|Date of Web Publication||28-Jan-2022|
Pratyusha Prakash Gaonkar
Lupin Limited, Chunabhatti (East), Mumbai - 400 022, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: Endoglin, a co-receptor of transforming growth factor (TGF)-β1 and TGF-β2, is indispensable for endothelial cell proliferation and modulation of tumor promotion activities of TGF-β1. The assessment of neovascularization using endoglin expression has been considered a potential predictor of prognosis in various solid malignancies.
Aims and Objectives: To analyze the expression of endoglin by immunohistochemistry in both benign and malignant salivary gland tumors.
Materials and Methods: Fifteen cases of benign salivary gland tumors and seventeen cases of malignant salivary gland tumors were included in the study, and immunohistochemistry was performed using anti-CD105 antibody using standard protocol.
Results and Conclusion: The study demonstrated that there is increased endoglin expression in malignant tumors as compared to their benign counterparts which is suggestive of increased angiogenic activity in tumor areas and could be responsible for the aggressive behavior of the malignancies. The highest density of endoglin-positive blood vessels was observed in the inflammatory tumor stromal areas. Furthermore, a significant increase in endoglin expression was evident as the grade of malignant salivary gland tumor increased. The results of the study indicate that the increased expression of endoglin in high-grade malignancies contributes to their aggressive nature.
Keywords: Angiogenesis, endoglin, salivary gland tumors
|How to cite this article:|
Gaonkar PP, Patankar SR, Sridharan G. Assessment of angiogenesis using endoglin in salivary gland tumors – An immunohistochemical study. J Can Res Ther 2022;18:623-8
|How to cite this URL:|
Gaonkar PP, Patankar SR, Sridharan G. Assessment of angiogenesis using endoglin in salivary gland tumors – An immunohistochemical study. J Can Res Ther [serial online] 2022 [cited 2022 Aug 10];18:623-8. Available from: https://www.cancerjournal.net/text.asp?2022/18/3/623/336704
| > Introduction|| |
Salivary gland tumors are exceedingly rare, estimated as occurring in between 0.25 and 2.5 per 100,000 of the population, accounting for only 1% or 2% of all tumors and comprising about 3–10% of all head-and-neck neoplasms., Despite their rarity, these heterogeneous groups of tumors are clinically significant because of their histological and behavioral diversity. Because of the complexity in their biologic behavior, very little is known about the pathways implicated in tumor dissemination of salivary gland cancer. Angiogenesis, a dynamic process of formation of new blood vessels from preexisting ones, is indispensible for tumor growth and metastasis of solid tumors and hence is regarded as the hallmark of cancer. A range of angiogenic (pan-endothelial) immunohistochemical markers such as CD34, CD31, factor VIII, and vascular endothelial growth factor (VEGF) have been documented in the literature. These markers detect both tumor and parenteral vessels and hence are not specific for tumor vasculature., However, in the recent past, with research gaining momentum toward antibody-based therapeutic strategies in cancers, a new and specific angiogenic marker, endoglin/CD105, became popular. CD105 (endoglin) is a major glycoprotein of human vascular endothelium. It is a disulfide-linked homodimeric cell membrane glycoprotein of 180 kDa, vital for angiogenesis and vascular development, which is predominantly expressed in vascular endothelial cells. It is a transmembrane phosphorylated glycoprotein, a component of the receptor complex of transforming growth factor-beta. Endoglin is highly expressed in syncytiotrophoblasts and more weakly expressed in stromal cells, fibroblasts, and hematopoietic progenitor cells. In solid malignancies, endoglin is highly expressed on peri- and intratumoral endothelial cells and sometimes on the stroma of the tumor. As opposed to the pan-endothelial markers, CD105 is preferentially expressed on activated endothelial cells that participate in tumor angiogenesis but weakly so in vascular endothelium of normal tissues., Furthermore, with the ability to stain the newly formed microvessels, endoglin is the marker of choice for tumor angiogenesis. Several studies have explored endoglin expression in cancers of the endometrium, cervix, and breast, and it was found to be a sensitive and specific marker for angiogenesis. It has also been documented that there is overexpression of endoglin in head-and-neck squamous cell carcinoma. In the light of this literature, the following study was conducted with the intention of evaluating the expression of endoglin in salivary gland tumors in order to get a better understanding of the biologic behavior of the salivary gland tumors and thereby evaluate the role of endoglin as a marker of tumor progression.
| > Materials And Methods|| |
Forty-two archival cases of benign and malignant salivary gland tumors were retrieved from pathology files at a dental college and hospital. The baseline clinical data including the patient's age and gender as well as the site of the tumor were obtained from patients' registered medical documents. H- and E-stained sections from representative formalin-fixed, paraffin-embedded tissue blocks were re-evaluated to confirm the diagnosis. There were 15 cases of benign salivary gland tumors (pleomorphic adenoma, Warthin's tumor, and oncocytoma) and 17 malignant salivary gland tumors. The control group was ten sections of the normal salivary gland. The malignant salivary gland tumors comprised low-grade tumors (mucoepidermoid carcinoma, acinic cell carcinoma, and adenocarcinoma), intermediate-grade tumors (adenoid cystic carcinoma), and high-grade tumors (mucoepidermoid carcinoma, salivary duct carcinoma, adenocarcinoma, and squamous cell carcinoma of salivary gland).
Immunostaining was performed on 4-μm paraffin sections. Sections were deparaffinized with xylene and rehydrated in graded ethyl alcohol. Sections were cut from paraffin blocks and mounted on superfrost silane-coated slides (Fisherbrand size 25 mm × 75 mm × 1 mm). On dewaxed slides, antigen retrieval procedure was carried out in EZ retriever system (V.2.1, BioGenex, USA). The slides were then flooded with peroxidase block for 15 min and treated with the power block for 5 min. Primary antibody (anti-CD105 – BioGenex) was applied for 1 h. Polymer horseradish peroxidase (BioGenex) was applied for 40 min. Slides were cleaned with phosphate-buffered saline (PBS) and cleaned properly. Working solution of diaminobenzidine (DAB) (1 μl DAB buffer + 1 drop of chromogen + 5 μ H2O2) was applied as chromogen in H2O2 for 10 min. Sections were counterstained and mounted with (DePeX [Distrene 80: A commercial polystyrene, a plasticizer, e.g., dibutyl phthalate and xylene]). For positive control, sections of the tonsil for CD105 were treated in a similar manner as the test sample. Negative control samples were treated as above, except that the primary antibody was replaced by a solution of bovine serum albumin in PBS.
Quantitative evaluation of endoglin-positive cells – Briefly, in an optical microscope, hotspot areas for endoglin expression in discrete blood vessels were initially identified by scanning the entire tumor in low power (×10). Based on the hotspot areas under low power, the number of endoglin-highlighted vessels in five of these areas was then counted in high-power magnification (×40) using the binocular microscope from Motic attached to a computer with Motic Advanced Images 3.2 software (Motic, Hong Kong). The fields containing artifactual changes such as chatter and tears were omitted from the study. Any stromal positivity for endoglin, except for blood vessels, was not taken into account. Intensity of staining was not considered for assessment. The mean count of endoglin expression from the five hotspot areas was obtained.
ANOVA test was used to compare the immunoreactivity of endoglin between normal salivary gland, benign, and malignant salivary gland tumors. Further, to note the exact difference, Student's t-test was used for the analysis of pairwise comparisons. t-test was used for comparison of endoglin expression between normal salivary glands and salivary gland tumors. Similarly, ANOVA test was used for the comparison of means of endoglin-positive blood vessels within the three grades of malignant salivary gland tumors, and t-test was done for the analysis of pairwise comparisons. P <0.05 was considered statistically significant.
| > Results|| |
The results of immunohistochemical staining for the normal salivary gland and the benign and malignant salivary gland tumor samples are displayed in [Table 1], [Table 2], [Table 3] and [Figure 1], [Figure 2], [Figure 3]. Furthermore, the results of endoglin immunoreactivity within different grades of malignant salivary gland tumors are shown in [Table 4] and [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9].
|Table 1: Mean of endoglin-positive cells and ANOVA test result for quantitative analysis in three groups|
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|Table 3: Mean of endoglin-positive cells and t-test result for quantitative analysis in two groups|
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|Figure 1: Photomicrograph (×40) – Immunohistochemical staining of endoglin in normal salivary gland|
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|Figure 2: Photomicrograph (×40) – Immunohistochemical staining of endoglin in benign salivary gland tumor|
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|Figure 3: Photomicrograph (×40) – Immunohistochemical staining of endoglin in malignant salivary gland tumor|
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|Table 4: Mean of endoglin-positive cells and ANOVA test result for quantitative analysis in three grades of malignant salivary gland tumors|
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|Figure 4: Photomicrograph (×40) – Immunohistochemical staining of endoglin in low-grade malignant salivary gland tumor|
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|Figure 5: Photomicrograph (×40) – Immunohistochemical staining of endoglin in intermediate-grade malignant salivary gland tumor|
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|Figure 6: Photomicrograph (×40) – Immunohistochemical staining of endoglin in high-grade malignant salivary gland tumor|
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|Figure 7: Photomicrograph (×10) – Immunohistochemical staining of endoglin in low-grade malignant salivary gland tumor|
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|Figure 8: Photomicrograph (×10) – Immunohistochemical staining of endoglin in intermediate-grade malignant salivary gland tumor|
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|Figure 9: Photomicrograph (×10) – Immunohistochemical staining of endoglin in high-grade malignant salivary gland tumor|
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| > Discussion|| |
The stromal microenvironment plays a critical role in tumor biology, with the blood vessels found within the tumor tissues being fundamental components of the tumor microenvironment. Tumor angiogenesis is indispensible for the growth and establishment of solid tumors and is a well-accepted paradigm. Intratumoral blood vessels are known to play a crucial role in cancer growth by supplying oxygen and nutrients, excreting metabolic products and are also associated with metastasis. Thus, the quantification of various aspects of tumor vasculature might provide an indication of angiogenic activity. One often quantitated aspect of tumor vasculature is microvessel density which is quantified by immunohistochemical staining for endothelial cell markers. Studies over the last few years have demonstrated the value of using tumor microvessel density as a prognostic indicator for a wide range of cancers.
In the past, many immunohistochemical studies have assessed angiogenesis using pan-endothelial markers such as von Willebrand factor, CD31, and CD34 but were found to lack specificity to tumor vessels., Antibodies against these factors react with large and small vessels in addition to lymphatic, tumoral, and inflammatory cells and may be confused with tumor microvessels. During recent years, a lot of effort has been put into identifying new and specific markers of angiogenesis (Meng et al., 2012). CD105 is a powerful marker of neovascularization in solid malignancies and has been accepted as a more accurate marker than other pan-endothelial markers such as CD31 and CD34 in the evaluation of tumor angiogenesis. Endoglin is upregulated in endothelial cells of tumor tissues than in their normal counterpart and hence it is considered as a more selective marker for the assessment of microvessel density of tumors., It is, therefore, a proven preferred prognostic marker in various tumors such as breast, prostate, colon, and lung tumors. The role of angiogenesis in the onset and progression of salivary gland malignancies is well recognized, with an angiogenic switch having been reported during the malignant transformation of pleomorphic adenoma to carcinoma ex-pleomorphic adenoma. The purpose of the present study was to assess the endoglin expression and hence to evaluate angiogenesis in the salivary gland tumors. In this study, endoglin-positive blood vessels were scanty in normal salivary gland tissue. The endoglin expression was slightly increased in benign salivary gland tumors as compared to the normal tissue, but it increased significantly in the salivary gland malignancies. In benign salivary gland tumors, the pleomorphic adenomas with endoglin expression showed blood vessels with weak intensity. Thus, in this study, there was a significant increase of endoglin expression in malignant salivary gland tumors as compared to the benign tumors indicating increased development of blood vessels in malignancies. Increased demand for blood supply because of accelerated growth or acquired ability of tumor cells to produce angiogenic factors could explain this finding. Results similar to our study findings were reported in earlier studies by Tadbir et al. (2012) and Cardoso et al. (2009). They concluded that there is a higher rate of angiogenesis in malignant salivary gland tumor as compared to their benign counterparts suggesting their role in the malignancy and aggressive behavior of the tumors. Reports of multiple-step tumorigenesis model tumors suggest that angiogenesis was found to be activated in mid-stage lesions before the appearance of full-blown tumors suggesting that neovascularization is a prerequisite to the rapid clonal expansion associated with the formation of macroscopic tumors. Furthermore, in the macroscopic tumor, the main factor affecting the density of blood vessels is the metabolic needs of tumor cells which usually increase with tumor progression (Sharma et al., 2005). Thus, in malignant tumors with severe invasiveness and progression, metabolic needs and consequently the development of blood vessels increase (Romani et al., 2006).
In the present study, when endoglin expression in different grades of salivary gland malignancies was computed and subjected to statistical analysis, there was an increase in endoglin expression as the grade of the malignancy increased. A statistically significant result was found in the frequency of CD105 expression between the low and high grades and also between the intermediate and high grades of malignant salivary gland tumors. The increased expression of endoglin expression in the high-grade malignancies such as high-grade mucoepidermoid carcinoma, salivary duct carcinoma, high-grade adenocarcinoma, and squamous cell carcinoma of the salivary glands represents the increased angiogenesis in them. The increased vascularization contributes to the known aggressiveness of these lesions. However, studies conducted by Cardoso et al. and Costa et al. (2008) showed less frequent staining of vessels by endoglin in adenoid cystic carcinoma than mucoepidermoid carcinoma. According to Cardoso et al., the intratumoral microvessel density is not apparently correlated to the aggressiveness of these lesions, but their myoepithelial content may explain such a difference. Faur et al. (2014) investigated different types of salivary gland tumors and found low microvessel density in mucoepidermoid carcinoma and adenoid cystic carcinoma. Shi et al. (2007) mentioned that microvessel density (MVD) in MEC was related to clinical stage, histologic grade, and tumor recurrence. They stated that high-grade MECs have higher MVD compared to low-grade tumors which was in accordance with our study. In the current study, the highest density of endoglin-positive blood vessels was found in the inflammatory tumor stromal areas. This was in concordance with the study conducted by Mărgăritescu et al. wherein angiogenesis in acinic cell carcinoma was investigated by assessing CD105 microvessel density and the expression of VEGF. Furthermore, in our study, it was observed that the tumor vessels were mostly of aberrant morphology, tortuous, with or without clear lumen. The tumor cells of Warthin's tumor and oncocytoma showed a weak cytoplasmic reactivity with endoglin. The in vitro study of Zhang and Peng has suggested that adenoid cystic carcinomatous cells with higher metastatic potential could present a greater stimulus to angiogenesis. On the other hand, according to Dhanuthai K et al. (2012), although malignant salivary gland tumors showed higher MVD than their correspondent benign neoplasm, MVD itself could not be an indicator to distinguish between benign and malignant salivary gland tumors. Costa AF et al. (2008) comparing adenoid cystic carcinomas with and without distant metastases did not observe an increase in CD34-MVD nor in CD105-MVD in the group with metastases. In addition, Cardoso et al. recorded that the mean intratumoral MVD assessed by CD105 was not significantly different between the entire samples of malignant tumors that did or did not metastasize. Therefore, the authors suggested that angiogenesis is neither an absolute determinant nor required for acquisition of metastatic phenotype in these salivary gland tumors. One possible explanation for this variation in the metastatic potential of salivary gland tumors might be the differences in intrinsic properties of cancer cells themselves and/or of the tumor microenvironment. All these aspects of salivary gland tumor angiogenesis must be recognized, especially in the light of their implication in the field of antiangiogenic therapy. It is well known that CD105 promoter is predominantly active in proliferating endothelial cells, and this molecule is currently being evaluated as an ideal target for antiangiogenic therapies that aim to prevent the development of tumor neovasculature.
| > Concluding Remarks|| |
The results of the present work contribute to the knowledge on the biology of salivary gland tumors, since increased endoglin expression was observed in malignant tumors, which is suggestive of increased angiogenic activity in tumor areas. Further studies are required to understand the mechanism of CD105 upregulation and its potential role as a target of antiangiogenic therapy.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
[Table 1], [Table 2], [Table 3], [Table 4]