The aim of this study was to determine the tissue expressions of vascular endothelial growth factor (VEGF) and endocan in adrenal cortical tumors and the factors associated with them. The study included 6 subjects with adrenocortical adenoma (ACA), 7 subjects with adrenocortical carcinoma (ACC), and 13 control subjects with a normal adrenal cortex. The status of VEGF and endocan expression was determined by the proportions of cells staining on a scale ranging from negative (not staining at all) to strongly positive. VEGF expression was detected in 1 (16.7%) of 6 subjects in the ACA group and in 6 (85.7%) of 7 subjects in the ACC group. VEGF expression was not detected in any of the subjects in the control group. Endocan expression was detected in 6 (100%) of 6 subjects in the ACA group and in 7 (100%) of 7 subjects in the ACC group, while it was detected in only 4 (30.7%) of 13 subjects in the control group. VEGF was expressed with a high frequency in subjects with ACC and with a low frequency in subjects with ACA, but it was not expressed in subjects with normal adrenal cortex tissue. Although endocan was expressed with a higher frequency in subjects with ACC and ACA, it was also expressed in subjects with normal adrenal cortex tissue. The percentage of cells expressed endocan in subjects with ACC was also significantly higher than in subjects with both ACA and normal adrenal cortex.

Adrenal cortical tumors are divided into various molecular, pathological, and clinical tumors, which require different diagnostic and therapeutic approaches and have different development. Generally, adrenocortical lesions consist of adrenocortical hyperplasias, adrenocortical adenomas (ACAs), and adrenocortical carcinomas (ACCs). The Weiss scoring system is used to evaluate the malignancy potential of adrenal cortical tumors. Adrenal cortical tumors with a score of 2 or less in this scoring system are considered benign, and those with 3 or more are considered malignant [1].

ACA is a mostly nonfunctional benign tumor of the adrenal gland and is usually detected incidentally in different imaging methods. Although ACA is seen in all age groups, its frequency increases with age, and it is detected in 3–7% of people over 50 years of age [2]. The exact pathogenesis of ACA is not known, but it is suggested to occur as a result of mutations in some genes. ACC is a rare, highly malignant tumor with a generally poor prognosis, and its incidence is approximately 0.7–2 cases per million people per year [3]. The majority of cases occur between the ages of 30 and 50 years. Although its etiology ACC is not clear, some cases show an association between mutations in tumor suppressor genes, which are an important factor for adrenal tumorigenesis, and hereditary cancer syndromes [4].

Vascular endothelial growth factor (VEGF) is among the most important known regulators of angiogenesis, which leads to the spread of cancer cells through the bloodstream. The VEGF family of growth factors consists of several structurally related molecules, including VEGF-A, VEGF-B, VEGF-C, and VEGF-D. Major molecules involved in tumor angiogenesis are VEGF-A (usually referred to as VEGF) and VEGF receptor 2 [5]. Some studies have shown that VEGF expression is increased in cancer types such as lung, gastrointestinal and breast cancer, and VEGF expression has been found to be associated with the prognosis of cancers in some studies [5, 6].

Endocan is a proteoglycan secreted from vascular endothelium and plays a role in angiogenesis. Some previous studies have suggested that endocan plays a role in healing, inflammation, and tumorigenesis [7]. Previous studies have shown increased endocan expression in neoplasms of organs known to have high vascularity, such as the liver, brain, pituitary gland, colon and lungs [8]. Endocan expression is extensively upregulated through proangiogenic molecules such as VEGF-A and VEGF-C, which are known to be critical mediators of lymphangiogenesis, angiogenesis, and cancer progression [9]. There are limited studies investigating the expression of angiogenic factors in adrenal cortical tumors.

The aim of this study was to determine the tissue expressions of the angiogenic factors VEGF and endocan in adrenal cortical tumors and to determine the factors associated with them.

The study included subjects who underwent surgery for adrenal cortical tumor between 2006 and 2021. This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the local ethics committee (19.03.2021/No: 2021/3147), and the subjects’ demographic, laboratory, and pathological data were obtained from patient files.

Tissue samples from all subjects with adrenal cortical tumors were evaluated for Weiss scoring. The criteria of the Weiss scoring system are a high nuclear grade (Fuhrman grade 3 or 4), >5 mitoses per 50 high-power fields, atypical mitotic figures, <25% of tumor cells being clear cells, diffuse architecture (>33% of tumor), necrosis, venous invasion (smooth muscle in walls), sinusoidal invasion (no smooth muscle in walls), and capsular invasion. Adrenal cortical tumors with a score of 2 or less were considered ACA, and those with 3 or more were considered ACC. Subjects were excluded from the study if they had a biochemical diagnosis of primary hyperaldosteronism, adrenocortical hyperplasia, or metastasis according to histopathology result.

Sections with thickness of 4–5 microns were taken from paraffin blocks obtained from whole adrenal cortex tissue of subjects diagnosed with adrenal cortical tumors and control subjects with adrenal cortex tissue in normal morphology. Normal adrenal cortex tissue was obtained from individuals with normal adrenal cortex tissue adjacent to the tumor or from individuals who underwent nephrectomy and had normal adrenal cortex tissue. Two positively charged slides were obtained from all subjects. All sections obtained were stained for immunohistochemical analysis in a closed-system automatic immunohistochemical staining device. One slide was used for VEGF (ab51745, Abcam, Cambridge, MA, United States), and one slide was used for endocan (ab103590, Abcam, Cambridge, MA, United States). After staining, the sections were evaluated under a light microscope by a pathologist who was blinded to the subjects’ Weiss scores and medical data.

The status of VEGF and endocan expression was determined by the proportions of cells staining on a scale ranging from negative (not staining at all) to strongly positive. In the evaluation of VEGF expression, the degree of VEGF positivity was determined according to the percentage of stained tumor cells (negative = 0%; weakly positive = 1–5%; moderately positive = 6–25%, strongly positive = >25%). In the evaluation of endocan expression, we grouped the proportion of stained tumor cells into ten percentiles (0%, 10%, 20%, ... 70%, 80%, 90%). This grouping was performed to more precisely grade the subjects’ degree of endocan immunoreactivity [10].

The statistical analyses of the data were carried out with SPSS v20 statistical software package. The normality of the data was analyzed with the Shapiro-Wilk test. Continuous variables were expressed as mean ± standard deviation for normally distributed data, and as median (minimum-maximum) for non-normally distributed data. Categorical variables were expressed as numbers and percentages. In comparing two independent groups, the independent samples t test was used for normally distributed data and the Mann-Whitney U test was used for non-normally distributed data. Differences between three or more independent groups were evaluated with analysis of variance (ANOVA) test or Kruskal-Wallis H test.

Categorical variables were compared using the chi-squared test, Fisher’s exact test, and Yates’s correction. Correlations between numerical variables were determined by Spearman’s correlation analysis. Linear multiple regression analysis was performed to detect independent indicators of endocan and VEGF expression levels. p value <0.05 was considered to be significant.

The study evaluated 6 subjects with ACA, 7 subjects with ACC, and 13 control subjects with normal adrenal cortex tissue. Table 1 shows the demographic, radiological, and pathological features of the ACA, ACC, and control groups. ACA was mostly located in the right adrenal cortex (57.1%), and the mean ACA tumor size was 49 ± 10.5 mm. The median Ki-67 index in the ACA group was 1% (range: 0–5%), and the median total Weiss score was 0 (range: 0–2).

ACC was mostly located in the right adrenal cortex (71.4%). The mean ACC tumor size was 103 ± 35.4 mm. In this group, the median Ki-67 index was 20% (range: 15–70%), and the median total Weiss score was 6 (range: 6–7). According to the TNM staging system, 2 of the ACC cases were stage I, 3 cases were stage II, and 2 cases were stage III.

Among the subjects with ACC, the median 24-hour urinary metanephrine and normetanephrine levels were 92 (26–207) μg/day and 327 (147–528) μg/day, which are within the normal ranges (52 to 341 μg/day and 88 to 444 μg/day, respectively). One of the patients with ACC had laboratory findings consistent with ACTH-independent Cushing’s syndrome: a morning basal cortisol level of 40 μg/dL (normal range: 5 to 25 μg/dL), a midnight serum cortisol level of 38.39 μg/dL (normal range: <7.5 μg/dL), a 24-hour urine free cortisol level of 1,464 μg/24-hour (normal range: 11 to 53 μg/24-hours), and a basal morning ACTH level of 1.3 pg/mL (normal range: 10 to 60 pg/mL). The other 6 ACC patients had a median morning basal ACTH level, median morning basal cortisol level, and mean 24-hour urinary free cortisol level of 39.6 (5.2–74) pg/mL, 15.5 (9.8–19.3) μg/dL, and 52.45 ± 36.2 μg/24-hour, respectively.

The mean DHEAS levels of two patients with ACC were high with an average of 1,530.5 ± 1,320 μg/dL (normal range: 71.6–375.4 μg/dL). The mean DHEAS levels of all other ACC patients were normal with an average of 89.45 ± 36.1 μg/dL. All subjects with ACA and the control subjects were hormonally nonfunctional.

VEGF expression was detected in 1 (16.7%) of 6 subjects in the ACA group and in 6 (85.7%) of 7 subjects in the ACC group, but it was not detected in any of the subjects in the control group (Fig. 1). Weak VEGF expression was detected in 1 patient in the ACA group. Of the 6 subjects with VEGF expression in the ACC group, 4 had moderate expression and 2 had weak expression. The ACC group had a significantly higher number of subjects with VEGF expression and percentage of cells expressing VEGF than the ACA and control groups (p = 0.008, p = 0.004; p < 0.001, p < 0.001, respectively). In the ACC group, no difference in VEGF expression was found between stage I, stage II, and stage III cases.

Fig. 1

VEGF expression rates of ACA, ACC, and control groups

Endocan expression was detected in 6 (100%) of 6 subjects in the ACA group and in 7 (100%) of 7 subjects in the ACC group, while endocan expression was detected in only 4 (30.7%) of 13 subjects in the control group (Fig. 2). The median endocan expression level was 30% (10–50%) in the ACA group, 70% (60–90%) in the ACC group, and 0% (0–20%) in the control group. The ACC group had a similar number of subjects with endocan expression to that in the ACA group, but the ACC group’s percentage of cells that expressed endocan in subjects was significantly higher than in the ACA group (p = 0.53, p = 0.002, respectively).

Fig. 2

Endocan expression rates of ACA, ACC, and control groups

The ACA group had a significantly higher number of subjects with endocan expression and percentage of cells that expressed endocan in subjects than the control group (p = 0.043, p = 0.002, respectively). Compared to the control group, the ACC group had a significantly higher number of subjects with endocan expression and percentage of cells that expressed endocan in subjects (p = 0.003, p < 0.001, respectively). In the ACC group, no difference was found between stage I, stage II, and stage III cases in terms of endocan expression. Figs. 3–12 illustrate the VEGF and endocan expression in normal adrenal tissue, adrenocortical adenoma, and adrenocortical carcinoma.

Fig. 3

Normal adrenal tissue (hematoxylin and eosin (H&E))

Fig. 4

Adrenocortical adenoma (H&E)

Fig. 5

Adrenocortical carcinoma (H&E)

Fig. 6

Endocan negative in normal adrenal tissue (immunohistochemistry (IHC))

Fig. 7

Endocan at 20% cells and 1(+) staining intensity in normal adrenal tissue (IHC)

Fig. 8

Endocan at 80% cells and 2(+) staining intensity in adrenal adenoma (IHC)

Fig. 9

Endocan at 60% cells and 3(+) staining intensity in adrenocortical carcinoma (IHC)

Fig. 10

Vascular endothelial growth factor (VEGF) negative in normal adrenal tissue (IHC)

Fig. 11

VEGF negative in adrenal adenoma (IHC)

Fig. 12

VEGF at 2(+) staining intensity in adrenocortical carcinoma (IHC)

Correlation and linear regression analyzes of the ACC group revealed a relationship between VEGF expression and tumor size, but the expression of both VEGF and endocan had no relationship with age, total Weiss score, and Ki-67 index (Table 2).

The frequency of VEGF expression was quite high in ACC and low in ACA, but it was not expressed in normal adrenal cortex tissue. The frequency of endocan expression in both ACC and ACA subjects was significantly higher than in control subjects with normal adrenal cortex tissue. In addition, the percentage of cells that expressed endocan was higher in ACC subjects than in both ACA subjects and control subjects.

The symptoms of ACC are caused by an excess of autonomous adrenal hormone or an abdominal mass. Clinically, excess autonomic adrenal hormone is detected in 50–60% of subjects with ACC. Most of these subjects have Cushing’s syndrome alone or in combination with virilizing syndrome. Excesses of pure androgen, estrogen, or mineralocorticoid are detected at lower rates [11].

The formation of new vessels from pre-existing vessels is defined as angiogenesis. Induction of angiogenesis is considered a prerequisite for tumor growth and metastasis [12]. Angiogenesis is necessary to ensure the delivery of nutrients and oxygen to the tumor tissue. The process of angiogenesis is regulated by hypoxia, which triggers the expression of various growth factors or their receptors [13]. VEGF plays a key role in the regulation of angiogenesis. Other molecules shown to play a role in angiogenesis other than VEGF include transforming growth factor-β, fibroblast growth factor, tumor necrosis factor-α, soluble intercellular adhesion molecule-1, angiopoietins, and interleukin-8.

Endocrine glands are typically highly vascular organs, and their blood supply is essential for normal functioning and tight control of hormonal feedback loops. As with other endocrine glands, both the normal adrenal gland and the tumors arising from it are highly vascular structures [14]. To our knowledge, there are very few studies investigating angiogenesis-related markers in adrenal cortical tumors. One study investigated the cytosolic concentrations of angiogenesis markers in adrenal cortical tumors and found higher concentrations of VEGF-A in ACC than in ACA and transitional tumors. It was suggested that VEGF-A overexpression is associated with malignant phenotype [15].

In a study on subjects with benign or malignant adrenal tumors, serum levels of VEGF were higher in subjects with malignant tumors (ACC) than in subjects with benign tumors. The authors also reported that the serum levels of VEGF decreased after tumor resection in subjects with malignant tumors and increased significantly in subjects with relapse [16]. In another study, VEGF expression was higher in subjects with ACC than both subjects with ACA and subjects with a normal adrenal cortex [17].

In another study, VEGF expression was detected in 17 of 24 subjects with ACC, while VEGF expression was detected in only 5 of 20 subjects with ACA [18]. VEGF expression was detected in the vast majority of subjects with ACC (85%), while VEGF expression was not detected in any of the subjects with ACA and subjects with normal adrenal cortex. This finding suggests that VEGF expression may be associated with the development of ACC.

It has been suggested in some studies that endocan plays a role in tumor angiogenesis. A study on subjects with pituitary adenoma (both functional and non-functional) revealed a positive correlation between endocan and CD34 expression. The study showed that endocan expression increased significantly with the Knosp grade, which is an indicator of tumor invasion into adjacent normal pituitary tissues, adjacent cavernous sinuses, and carotid arteries. The authors also suggested that endocan expression could significantly predict tumor invasion and angiogenesis in pituitary adenomas [19].

A study on patients with renal cell carcinoma (RCC) suggested that endocan is overexpressed in these patients, and is a potential parameter that can be used to monitor RCC and tumor response to anti-angiogenic therapeutics [20]. A study on patients with non-small cell lung cancer (NSCLC) found that endocan was overexpressed in NSCLC tumor tissues compared to healthy lungs. Furthermore, the authors suggested that endocan overexpression represents a response of the tumor endothelium to proangiogenic growth factor stimulation [21].

To our knowledge, this study is the first to investigate endocan expression in adrenal cortical tumors. Endocan expression was detected in all subjects with ACC and ACA, but it was detected in only some of the subjects with normal adrenal cortex. However, the endocan expression level was significantly higher in subjects with ACC than in both subjects with ACA and those with a normal adrenal cortex. According to our results, although endocan expression is not specific to ACC, endocan overexpression may be associated with the development of ACC.

Studies investigating drugs targeting the VEGF pathway in ACC have shown that these therapies are a largely ineffective treatment strategy [22-24]. Some experimental studies have been conducted to develop drugs targeting endocan. One study found that deletion of endocan exon 2 disrupted the synthesis of the glycan chain, which is known to play a role in the protumoral effect of endocan. The authors also suggested that the polypeptide sequence encoded by exon 2 could be a target for cancer treatments [25].

It has been shown that endocan knockdown with small interfering RNAs (siRNAs) decreases cell survival and inhibits the migration and invasion of tumor cells in hepatocellular carcinoma [26]. It also inhibits tumor cell proliferation in gastric cancer [27] as well as proliferation and migration in head and neck cancers [28]. It has been suggested that endocan knockdown by short hairpin RNAs (shRNAs) inhibits tumor growth, invasion, and metastasis induced by nerve growth factor receptor in oral squamous cell carcinoma [29]. Another study examined a treatment containing microRNA-9-3p (miR-9-3p)-containing exosomes derived from bone marrow-derived mesenchymal stem cells, and the results showed that it downregulated endocan, prevented cancer progression, and prevented metastasis in a xenograft model of bladder cancer in mice [30].

Considering the results of our study, modulating both the VEGF pathway and other anti-angiogenic pathways targeting endocan is likely to be an effective therapy for ACC. However, the limitations of this study are the small sample size, especially the number of ACC cases, and the fact that we did not evaluate serum VEGF and endocan levels.

In conclusion, VEGF was expressed with high frequency in subjects with ACC and with low frequency in ACA cases, but it was not expressed in subjects with normal adrenal cortex tissue. Although endocan was expressed with higher frequency in subjects with ACC and ACA, it was also expressed in subjects with normal adrenal cortex tissue. The percentage of cells that expressed endocan in subjects with ACC was also significantly higher than in both subjects with ACA and those with a normal adrenal cortex. VEGF expression and endocan overexpression may be associated with the development of ACC, but studies with larger sample sizes are needed.

Melia Karaköse contributed to the concept of the study design, data acquisition, drafting, and critical revision of manuscript. Mustafa Can contributed to the statistical analysis, data acquisition, and drafting of the manuscript. Muhammet Kocabaş contributed to the statistical analysis, data acquisition, and drafting of the manuscript. Hacı Hasan Esen contributed to the study design and data acquisition. Mustafa Kulaksızoğlu contributed to the critical revision of the manuscript and data postprocessing. Feridun Karakurt contributed to the study design, statistical analysis, critical revision of the manuscript, and data postprocessing. All authors read and approved the final version of the manuscript.

The authors declare that there is no conflict of interest. This work was funded by Necmettin Erbakan University Scientific Research Projects.

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