Abstract
Objectives:
To assess the expression of endoglin in head and neck paragangliomas and the soluble endoglin level in serum of paraganglioma patients.
Methods:
Seven tumor samples of patients operated for cervical paraganglioma were assessed, as well as serum samples collected preoperatively, on days 4 and 28 postoperation. Serum level of endoglin in healthy controls was also determined. Tumor samples were subjected to immunofluorescent staining and examined with confocal microscope. The level of soluble endoglin in serum samples was examined using the immunoenzymatic assay (ELISA).
Results:
Endoglin was highly expressed in all tumor samples. The level of soluble endoglin was significantly higher in paraganglioma patients compared to healthy controls and correlated with the tumor size. The serum level of s-endoglin was reduced after surgical excision of the tumor and remained stable after 4 weeks in all patients with complete resection of the tumor.
Conclusion:
Endoglin is an important factor in the pathophysiology of head and neck paragangliomas and may be a potential diagnostic and prognostic marker in these types of tumors.
Introduction
Paragangliomas are rare, usually nonmalignant tumors, derived from paraganglia of the autonomic nervous system. Paragangliomas of the head and neck region account for about 3% of all paragangliomas. 1 Most commonly they develop in the carotid body, followed by jugular, tympanic, and vagal localizations. The specific feature of paragangliomas is its high level of vascularization. 2 Endoglin, also known as CD105, is a transmembrane glycoprotein that serves as an accessory receptor for transforming growth factor beta (TGF-β). It is mostly expressed on vascular endothelial cells, with a very poor expression on fibroblasts, stromal, and vascular smooth muscle cells. 3 The expression of endoglin is remarkably upregulated in actively proliferating endothelial cells. Studies have shown anti-CD105mAb stained with high intensity vascular endothelial cells in tissues undergoing active angiogenesis, in contrast to no or weak staining for CD105 in normal blood vessels. 4 Therefore, endoglin can be considered as an appropriate marker for tumor-related angiogenesis and neovascularization. In solid tumors, endoglin is expressed on endothelial cells of both peri- and intratumoral blood vessels and on tumor stromal components. 3 Its overexpression has been shown in gastric, esophageal, colorectal, and breast cancer tumor tissues. In these types of malignancies, high expression of CD105 has correlated with lower patient survival rates, presence of nodes metastases. and distant metastatic disease. 5
Studies have shown that the ectodomain of endoglin is released through proteolytic cleavage by membrane-type matrix metalloproteinase 14 (MMP-14) and exist in the circulation as a soluble form of endoglin (s-endoglin). High amounts of circulating s-endoglin were found in breast and colorectal cancer patients with metastatic disease as well as non–small cell lung cancer patients.3,6 These results suggest that serum endoglin levels may have a diagnostic as well as prognostic value in various types of tumors.
The aim of the present study was to assess the expression of CD105 in tumor tissue and s-endoglin serum level in patients operated for head and neck paraganglioma.
Materials and Methods
Study Population
A total number of 7 patients, operated for paraganglioma of the head and neck region in the Otolaryngology, Head and Neck Surgery Department of Warsaw Medical University between 2015 and 2016, was included in the study. Seven tumor samples were collected during surgery. Three blood samples were taken from each patient: 1 prior to surgery and others on postoperative days 4 and 28. Twenty-four–hour urinary collection for catecholamines and metanephrines test was performed in all patients to rule out the presence of simultaneous pheochromocytoma. The volume of each tumor was estimated by the same experienced radiologist, based on cross-sectional measurement of tumor dimension in 3 different planes on a computed tomography (CT) or magnetic resonance imaging (MRI) scan. Additionally, 7 blood samples of healthy volunteers were taken, as controls.
In all cases, informed consent was obtained from patients included in the study. The concept of the study was reviewed and approved by the local bioethics committee. The study procedures conformed to the ethical guidelines of the World Medical Association Declaration of Helsinki.
Tissue Samples Preparation and Confocal Microscopy
Tumor samples were deep frozen in liquid nitrogen directly after surgical excision and then stored at −80°C for further use.
Frozen sections were cut serially in a cryostat into 10-μm sections, fixed in 4% paraformaldehyde in PBS at room temperature for 20 minutes, and stained with a primary antibody for 1 hour. The primary antibody used was mouse anti-CD105 (Abcam, Cambridge, UK) in 1:300 concentration.
Subsequently, after 3 washes in PBS, the sections were incubated with donkey secondary antibody: Alexa Fluor 647 anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania USA) for 1 hour, washed as previously described, and counterstained with Hoechst (Sigma, St. Louis, Missouri, USA) or Dapi, according to the manufacturer’s recipe. Sections were mounted with DAKO immunofluorescent mounting medium (DAKO, Glostrup, Denmark) and viewed under a Leica confocal microscope, type TCS SP5.
Blood Sampling and Processing, S-Endoglin Determination
The s-endoglin concentrations were evaluated in serum by commercially available sandwich enzyme-linked immunosorbent assay kit Quantikine Human Endoglin/CD105 Immunoassay (R&D Systems Inc, Minneapolis, Minnesota, USA). The level of absorbance for each tested sample was measured using the Microplate Reader 550 (BIO-RAD, Hercules, California, USA). The concentration of CD105 in each sample was calculated based on corresponding standard calibration curve and expressed in ng per ml. The assay sensitivity was 0.03 ng/ml.
The statistical analysis has been performed using the t test and Wilcoxon signed-rank test.
Results
Clinical characteristics of patients included in the study are presented in Table 1.
Clinical Data of All the Patients Included in the Study.
Histologic Studies
Microscopically assessed tumors were formed of nests of polygonal cells, separated by capillaries (Figure 1). Immunohistochemical staining revealed intense endoglin expression in all tumors, with particularly marked expression on endothelial cells (Figure 2).
The expression of endoglin (CD 105) in tumor tissue. Serial sections of carotid paraganglioma were stained with Mabs to CD105 (green marker). The cell nuclei are stained with Hoehst (blue marker).
The localization of endoglin (CD 105) in tumor vasculature. An intense staining with Mabs to CD105 is visible in the endothelium of the tumor vessel.
Endoglin Serum Levels
Significantly elevated CD105 levels were seen in paraganglioma patients compared with controls (Figure 3). The mean level of s-endoglin in paraganglioma patients before surgical intervention was 8.02 ± 1.67 ng/ml (median = 8.10 ng/ml; minimum = 6.25; maximum = 11.6) compared to 4.81 ± 0.58 ng/ml (median = 4.87 ng/ml; minimum = 3.54; maximum = 5.69) in healthy volunteers (P < .001).
The serum level of s-endoglin in patients preoperatively and healthy controls was expressed in ng/ml. Each dot represents a single patient/control; mean values of s-endoglin concentration in each group are indicated as dashed lines, and the median values are shown as solid lines.
There was a positive correlation between preoperative s-endoglin level and tumor size, expressed as a tumor volume in cm3 (Figure 4).
Correlation between the s-endoglin serum level and tumor volume, expressed in cm3. Each dot represents a single patient; a trendline is indicated.
The s-endoglin level decreased on day 4 after the operation in all patients (Figure 5). The mean level of s-endoglin in paraganglioma patients 4 days after surgical intervention was 6.59 ± 1.86 ng/ml (median = 6.09 ng/ml; minimum = 5.3; maximum = 10.9) compared to 8.02 ± 1.67 ng/ml (median = 8.1 ng/ml; minimum = 6.25; maximum = 11.6) preoperatively (P ≤ .05 in Wilcoxon signed-rank test).
Changes of s-endoglin serum level in patients on day 4 after the excision of the tumor in comparison to preoperative values. Each line represents a single patient; s-endoglin concentration is expressed in ng/ml.
The level of s-engolin on day 28 after the operation was measured in 6 patients (Table 2). The s-endoglin level stabilized in 4 patients who underwent radical procedure (median = 5.8 ng/ml; minimum = 4.9; maximum = 11.25, compared to median = 6.09 ng/ml; minimum = 5.3; maximum = 10.9 on day 4). The s-endoglin level increased in 1 patient with partial excision of the tumor (8.45 ng/ml vs 5.6 ng/ml on day 4) and in 1 with multicentric tumor localization (7.7 ng/ml vs 5.5 ng/ml on day 4).
The S-Endoglin Level in ng/ml, in Patients’ Serum Before the Surgical Intervention and on Day 28 After the Surgical Intervention.
Patient with bilateral tumor.
Patient with nonradical resection of the tumor.
Patient lost from follow-up.
Discussion
Endoglin is highly expressed in proliferating endothelial cells; therefore, it is considered as a reliable marker for tumor angiogenesis. Studies have shown that CD105 antibody stains angiogenic blood vessels, whereas it reacts poorly with normal micro vessels that are usually stained with pan-endothelial markers, such as CD31, CD34, or VEGFR2.7,8 Furthermore, anti-endoglin antibody reacts specifically with endothelial cells, with a very weak staining of stromal or inflammatory cells that are also present in most kinds of neoplasms.9-11 That limits the false-negative staining rate observed while using pan-endothelial markers. El-Gohary et al 12 have demonstrated that micro vessel density (MVD) calculated using CD105 antibody but not CD31 or CD34 antibody correlated with poor prognosis in prostatic cancer patients. Similar results have been observed in colorectal cancer patients. 13
As it has been mentioned before, endoglin has been identified in several solid malignant tumors samples, such as non–small lung, prostatic, colorectal, brain, and lung cancer. As it has been reported previously by Eleno et al, 14 we have identified the CD105 expression in all head and neck paraganglioma tumor samples, which is a potential source of s-endoglin in patient’s serum.
A soluble form of endoglin is present in small amounts in serum of healthy subjects. Elevated level of s-endoglin in serum has been observed in patients with various malignant tumors, including breast, liver, colorectal, and non–small cell lung carcinoma. Studies have shown that higher serum endoglin levels correlate with the presence of metastatic disease.15,16 In prostate cancer, CD105 is a predictor of pelvic lymph node metastasis as well as recurrence after prostatectomy.17,18 All of these data taken together suggest that s-endoglin serum level may be a useful marker for classifying patients with advanced disease as well as monitoring cancer recurrence in a long-term follow-up.
In our study, s-endoglin serum level was significantly higher in head and neck paraganglioma patients than healthy controls. Moreover, s-endoglin level correlated with the tumor size. Four days after surgical excision of the tumor, the level of s-endoglin decreased in all patients when compared to preoperative values. It remained stable 4 weeks after the surgical intervention in the patients that were diagnosed with a single tumor that was completely removed. However, in the case of bilateral tumor, the level of s-endoglin increased 4 weeks after the operation and reached higher values than preoperatively. A similar increase was observed in the patient who underwent nonradical resection of the tumor. All of these data taken together support the conclusion that tumor tissue is a main source of s-endoglin in serum of paraganglioma patients. Therefore, the s-endoglin serum level might be a useful diagnostic marker in the management of head and neck paragangliomas that would help monitor disease progression and recurrence. Nevertheless, this observation requires further studies on a larger group of patients.
In summary, the results of our preliminary study have shown that endoglin is highly expressed in human head and neck paragangliomas. At the same time, the level of s-endoglin is significantly elevated in serum of paraganglioma patients compared to healthy controls and correlates with the tumor volume. The surgical excision of the tumor reduces the level of s-endoglin in a short time after the operation. However, in patients with nonradical resection or multicentric tumor, the s-endoglin serum level fluctuates and can reach the preoperative values in a distant postoperative period. The study was conducted on a small series of cases, and such factors as localization and genetics of the tumor were not taken into consideration. Nevertheless, all of these data taken together suggest that endoglin is an important factor in the pathophysiology of head and neck paragangliomas and may be a potential diagnostic and prognostic marker in these types of tumors. The exact mechanisms that regulate the expression of CD105 in paraganglioma tissue and its subsequent shedding to blood circulation require further investigation on a larger group of patients.
Footnotes
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by statutory funds of the Otolaryngology, Head and Neck Surgery Department of Medical University of Warsaw. The authors have no other source of founding to declare.