Abstract
Pituitary ademonas are benign tumours from the pituitary gland but may have an invasive and destructive growth pattern. There is little understanding of the growth and progression control of pituitary tumours. In the present study, we investigated the expression of vascular endothelial growth inhibitor (VEGI), a vascular endothelial growth and apoptosis regulator and VEGI receptor Death Receptor-3 (DR3), in clinical pituitary tumours. Pituitary tumours from 95 patients were included in the study. Fresh pituitary tumours were obtained immediately after surgery and processed for histological and molecular-based analyses. Histopathological and clinical information including tumour size, tumour invasion and endocrine status were analyzed against the gene transcript expression of VEGI, DR3 and VEGF. VEGI and VEGF family and VEGF receptors were quantitatively determined for their gene transcript expression. The expression levels of VEGI were significantly lower in pituitary tumours which invaded the sella floor, and with suprasellar extension than in non-invasive tumours (p=0.0073). VEGI levels were also negatively correlated with cavernous sinus invasion stage (p<0.0001), in that a high level of VEGI was associated with low tumour grade. Multivariate analysis indicated that VEGI is an independent factor predictive of invasion (p=0.05). It was further demonstrated that the relationship between VEGI and pituitary tumour invasion were independent of the expression of VEGF and its receptors. Low levels of VEGI transcripts were associated with the intratumoural haemorrhage (p=0.05). Out of all the pituitary tumours, 59 were non-functional. Out of the functional tumours, it was found that follicle stimulating hormone (FSH)-expressing and gonadotrophic tumours tended to have markedly low levels of VEGI transcripts, compared with non-functional tumours (p=0.0026 and p=0.003, respectively). The opposite was seen with thyroid-stimulating hormone (TSH)-secreting tumours. Levels of DR3 in tumours with sella destruction were also lower than in those without destruction. VEGI, possibly via DR3, suppresses the aggressive nature of pituitary tumours and its expression level is closely linked to the invasion and destruction of the suprasellar and sella regions. It also has implications for the endocrine nature of these tumours. VEGI thus has an important predictive and prognostic value in patients with pituitary tumours.
Pituitary adenoma is a common type of benign tumours of the central nervous system, but there are a number of biological behaviours similar to malignant tumours, including invasion of the surrounding structures such as the cavernous sinus, hypothalamus, and sphenoid sinus, which is termed invasive adenoma (1). Most invasive pituitary adenomas are characterized by rapid growth, large size, poor treatment efficacy, and a high recurrence rate. However, there are also some small tumour showing obvious invasive behaviour and apoplexy early. Although there is still little consensus about what constitutes an invasive pituitary adenoma, the utility of biomarkers is rapidly evolving. Potential markers of pituitary adenoma invasiveness that have been studied include oncogenes, such as pituitary tumor transforming gene (PTTG); proliferation-related factors, such as Ki-67, proliferating cell nuclear antigen (PCNA), and P53 (2-4); and angiogenic factors, such as vascular endothelial growth factor (VEGF) (5, 6).
Vascular endothelial growth inhibitor, VEGI, is a protein that was initially discovered from endothelial cells as a naturally occurring endothelial growth inhibitor. It was subsequently found to be the same as tumour necrosis factor superfamily member-15 (TNFSF15), a member of the TNF superfamily. The primary function of VEGI in endothelial cells appears to be an inhibitory role in cellular growth and also in cellular migration (7, 8). VEGI is known to operate via two receptors, Death Receptor-3 (DR-3) and the Decoy Recoptor-3 (DcR-3). DR-3 is a member of the TNF receptor superfamily and is also known as TNFRSF25, a protein of approximate 47 kDa in size. DR3 is known to induce apoptosis when activated. DR3 is so far the only known functional receptor for VEGI. A decoy receptor for VEGI, namely DcR3, which is a secreted soluble protein also belonging to the TNFR superfamily, also exists (9-14).
VEGI has been shown to be an angiogenesis inhibitor in vitro and in vivo. Due to its significant role in angiogenesis in cancer, its expression has been investigated in a variety of human tumours including mammary tumours, prostate cancer, bladder cancer, kidney cancer, malignant melanoma and colorectal cancer (15-22). Collectively, these studies have demonstrated that VEGI expression reduced in aggressive and late stage tumours and in most cases is associated with patient survival. Although it was natural to link VEGI and tumour progression by the inhibitory effect of VEGI in angiogenesis, hence loss or reduction of VEGI may result in active angiogenesis in solid tumours, it was recently shown that VEGI has a direct effect on epithelial and cancer cells by also exerting an inhibitory effect on the migration and growth of cancer cell. Thus, VEGI acts directly on both endothelial cells and cancer cells (23-25).
The vast majority of pituitary tumours are adenomas, with or without endocrine functions. However, despite their slow growth, there are adenomas that display an aggressive pattern, with the ability to invade the surrounding tissues, namely the sella fossa within which the pituitary gland is housed. The aggressive growth of pituitary tumours ranges from invasion to destruction of the sella tissues (1, 2, 26-28). Presently, it is not clear what biological features of the pituitary tumours are responsible for sella invasion and destruction. In the light of the biological functions of VEGI and its receptor and indeed the role of VEGI in solid human tumours, we hypothesised that VEGI may have a potential role in the biological behaviour of pituitary tumours and that a loss or low level of VEGI may lead to an aggressive phenotype. In the present study, we report on the expression pattern of VEGI and DR3 in pituitary tumours and their association with the clinical and pathological features of these tumours.
Materials and Methods
Clinical and pathological demographics of the patients. Patients' clinical history, diagnostic images and endocrine tests were routine recorded. This study included a total of 95 patients with pituitary adenomas who underwent trans-sphenoidal or craniotomy surgical resection from Jan 2012 to Dec 2012 at the Department of Neurosurgery of Beijing TianTan Hospital. Age, gender and hormonal functioning were reviewed. The diagnosis was confirmed by postoperative pathology. Immunohistochemical examination was used to determine the endocrine type. Preoperative magnetic resonance imaging (MRI) was performed to determine image characteristics: tumour size, cystic lesion, intratumour haemorrhage and invasion type. The greatest diameter of tumour obtained on the gadolinium (Gd)-enhanced T1WI MRI was measured as the tumour size. Intratumoral haemorrhage and cystic lesion were defined by preoperative MRI and were confirmed intraoperatively. The invasion type was determined based on the invasion site of the tumour and included cavernous sinus invasion, sphenoid sinus invasion, and suprasellar invasion. The criterion for suprasellar invasion was tumour growth toward the suprasellar region with invasion in the third ventricle and/or lateral ventricle. The criterion for sphenoid sinus invasion was tumour growth downward to the sphenoid sinus cavity or tumour growth into the clivas. Cavernous sinus invasion was defined as tumour extending laterally to the lateral tangent of the intra- and supracavernous internal carotid artery (ICA) or beyond that (Grade 3 or 4), with reference to the classification by Knosp et al. (29) and the method proposed by Vieira et al. (30, 31). Full details of the information is given in Table I.
Clinical and pathological information of the patients and expression levels of VEGI and DR3.
Primers used in the present study.
Collection of pituitary adenomas. Pituitary adenoma samples were freshly collected immediately after microsurgical resection at the Department of Neurosurgery of Beijing TianTan Hospital, supported by an ethics approval by the Local Research Ethics Committee with patient's consent. A total of 95 patients entered the current study. The tissues were immediately frozen and stored in liquid nitrogen until use.
Tissue processing and generation of genetic materials for genetic-based analyses. Frozen tumour tissues were frozen-sectioned at 10 μm. A few sections were used for histological evaluation and the rest were combined and then homogenised in an RNA extraction solution (Sigma-Aldrich, Dorset, UK). Total RNA was extracted according to the manufacturer's instructions and quantified using a spectrophotometer. Equal amount of total RNA was used to generate complementary DNA, cDNA, using a reverse transcription kit from Promega (Southampton, England, UK). The quality of cDNA samples was verified using Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a house keeping control.
Quantitative gene transcript analyses. This was based on the Amplifluor™ Technology in quantitative gene transcript analysis. Briefly, pairs of specific primers were designed to amplify a region of VEGI and DR3 (Table II). To one primer of the primer sets was added a Z sequence which is complementary to the Amplifluor probe (FAM tagged; Biosearch Technologies, Inc., Novato, CA, USA). Each reaction was comprised of a forward primer, a reserve Z primer at one tenth of the strength, the Amplifluor probe, cDNA from tumours and custom master mix (Life Technologies, Paisley, UK). An internal standard was included as control. Quantitative PCR analysis was conducted using the SteponePlus instrument (ABI, Paisley, Scotland, UK). GAPDH transcript which was also simultaneously quantified was used as a house keeping control.
Statistical analysis. Statistical package used was SigmaPlot (Version 11). Student t-test and ANOVA testd were used for normally distributed data and Mann-Whitney U-test and Kruskall-Walis test for non-normally distributed data.
Results
Clinical information. A total of 95 patients with histopathologically-confirmed pituitary adenomas were identified. There were 51 (53.7%) males and 44 (46.3%) females with a mean age of 45.4±12.8 years (21–72 years). A total of 59 patients had non-functioning pituitary adenomas; 36 were functioning tumours (Table I). The percentages of each histological type are listed in Table I. The average tumour maximum diameter was 3.1±1.4 cm (0.8-6.6 cm). Cystic component was noted in 46 cases (48.4%) and intratumoral haemorrhage was noted in 18 cases (18.9%). Tumour invasion into the cavernous sinus according to Knosp's grading system was: grade 0-2 in the majority of the cases (Table I). Most of the cases had only one invasion site, while a small number of cases were associated with multiple sites or multiple types of invasion.
VEGI and DR3 were expressed in pituitary tumours and linked to invasion. Transcripts of VEGI and DR3 were both detectable in pituitary tumour tissues, although the level of VEGI was generally higher than that of DR3 (Table I). VEGI levels had no association with sex or age of the patients, or the size of tumours.
However, the most striking observation was that pituitary adenomas with supersella invasion had significantly low levels of VEGI transcript compared with those without invasion (639±201 vs. 181±80, p=0.0073) (Figures 1 and 2). The same was not seen with DR3 (Figures 1 and 2).
VEGI levels reflect the cavernous sinus invasion. Based on the cavernous sinus staging it was found that Knosp stage 0-2 tumours had markedly high levels of VEGI compared with Knosp stage 3-4 tumours (620±180 vs. 69±44, p=0.004). This was not observed with DR3 (Table I).
VEGI and DR3 are inversely linked to the presence of cysts and intratumoral haemorrhage in pituitary tumours. Pituitary tumours with cystic lesions tended to have low levels of VEGI (151±111 vs. 705±225 for tumour with and without cystic lesions, p=0.032). Similarly, cystic lesions were also associated with low levels of DR3 (for tumour with and without cystic lesions, p=0.05, Figure 3).
Another noteworthy observation is that the high levels of VEGI and DR3 were also a significant factor for the intratumour haemorrhage. VEGI transcript in tumours with no signs of haemorrhage was 346±140 compared with 978±316 with haemorrhage. Similarly, DR3 levels in tumours without and with haemorrhage were 0.19±0.11 vs. 0.66±0.53 (Table I).
VEGI and the endocrine function of pituitary tumours. One special feature of some pituitary tumours is active endocrine secretion associated with the respective syndrome. It was very interesting to note that FSH- and luteinizing hormone (LH)-active tumours had markedly low levels of VEGI compared with endocrine inactive tumours (p=0.0026 and p=0.0027, respectively) (Figure 4 top). Growth hormone (GH)-, Adrenocorticotropic Hormone (ACTH)-, Thyroid-stimulating Hormone (TSH)-expressing and mixed tumours did not have marked difference in VEGI levels from non-active tumours. Interestingly, FSH-, TSH- and LH-secreting tumours had also low levels of DR3 compared with non-active tumours (Figure 4, bottom), although the difference did not reach statistical significance.
Magnetic Resonance Imaging (MRI) images of invasion in pituitary tumours with different Vascular endothelial growth inhibitor (VEGI) level. A1-3: Pituitary adenoma with high VEGI expression, characteristic image without invasion. Arrows indicate the pituitary tumours; arrowheads indicate sellar fossa unaffected by the tumours. B1-3: Pituitary adenoma with very low VEGI expression, associated with multiple site invasion. Arrows indicate pituitary tumours with suprasellar invasion. Arrowheads in B1 and B2 indicate sellar fossa invaded by tumour. The tumour grew toward the suprasellar region with invasion in the third ventricle and temporal lobe. Arrowhead in B2 indicates cavernous sinus invasion, the tumour invaded the cavernous sinus to surround the cavernous segment of the carotid artery. With sphenoid sinus invasion, the tumour grew downward to break through the floor of the sella into the sphenoid sinus. The diagnosis based on the imaging study was also confirmed in surgery.
Tumours with bone invasion had significantly low levels of VEGI (left) and DR3 (right). *p=0.0073 vs. non-invasive tumours.
Magnetic Resonance Imaging (MRI) images of intratumour haemorrhage (A1-A3) and cystic lesions (B1-B3) of pituitary tumours with very low Vascular endothelial growth inhibitor (VEGI) expression. Arrows in A1-A3 indicate a small pituitary adenoma with low VEGI expression, but with early intratumour haemorrhage. Arrows in B1-B3 indicate cystic lesions within large pituitary adenomas which had low levels of VEGI expression. B1-B3 was from a patient who had large cystic lesions and lost vision rapidly. It is thought that the multiple cystic lesions were transformed from intratumour haemorrhage.
VEGI and DR3 levels did not correlate with angiogenic factors. The levels of the VEGF family were assessed in previous studies of these patients including VEGF, VEGFR1, VEGFR2 and VEGFR3 (data not published). A Spearman rank correlation analysis did not reveal any significant correlation between VEGI, DR3 and VEGF and VEGFR.
Discussion
The present study reports, to our knowledge for the first time, that VEGI and its receptor DR3 have a distinct pattern of expression in human pituitary tumours and that levels of VEGI, and to some degree DR3, are linked to the invasion of the sellar fossa, haemorrhage and intratumoral cysts of pituitary tumours. They are also linked to the endocrine function of pituitary tumours.
One of the most interesting observations of the current study is that a low level of VEGI is associated with intratumoural haemorrhage. VEGI is better known for its anti-angiogenesis function and both in vitro and in vivo studies have shown that it acts as a powerful naturally-occurring angiogenesis inhibitor. Lack of VEGI has been shown to be negatively correlated with angiogenesis in solid tumours, due to the removal of the inhibitory mechanisms in the tissues (13, 17). It is thus argued here that in a similar pattern, lack of VEGI in pituitary tumours may trigger an increase in angiogenesis in the tumour tissues. The abundance of new immature vessels may have lead to the occurrence of bleeding. Owing to the size and lack of availability of pituitary tumours, the present study was unable to evaluate the microvessel density in pituitary tumours, but will warrants for a new study to be implemented to evaluate this aspect.
Expression of Vascular endothelial growth inhibitor (VEGI) and Death receptor-3 (DR3) in relation to the endocrine function of pituitary tumours. FSH- and LH-secreting tumours had significantly lower levels of VEGI than non-active tumours. FSH-, TSH and LH- secreting tumours had non-statistically significantly lower levels of VEGI. ACTH: Adrenocorticotropic Hormone; GH: growth hormone; LH: luteinizing hormone; PRL: prolactin; TSH: thyroid-stimulating hormone.
One initial hypothesis that VEGI may be inversely linked to the invasiveness of pituitary tumours is also confirmed by the data from the study. Indeed, pituitary tumours which have signs of invasion of the sellar floor had markedly reduced levels of VEGI. This may be part of the mechanism by which VEGI mediates the biological behaviour of pituitary tumour and tumour cells. Via regulation of angiogenesis as discussed earlier, and by a possible effect on pituitary cells, VEGI may inhibit the migration and growth of endothelial cells and possibly pituitary glandular cells. Lack of VEGI may lead to the aggressive growth of pituitary tumour cells. Human pituitary tumour cells and cell lines are not widely available. It would be interesting to conduct in vitro-based research to test this possibility (1, 2, 32-35).
The link between VEGI levels and endocrine function, a fundamental characteristic of pituitary tumours, is interesting. Functional pituitary adenoma accounted for almost half of cases of the disease. Due to endocrine dysfunction and the appearance of clinical symptoms, such tumours are relatively easy to diagnose early and treat. Hence, there is a relatively small number of functional pituitary adenomas in our group. FSH- and LH-secreting tumours had significantly low levels of VEGI than non-active tumours. FSH-, TSH- and LH-secreting tumours had a lower but non-statistically significant level of VEGI. More samples will be collected to identify the relationship between the endocrine functions and VEGI levels.
Finally, we have also shown that VEGI receptor, DR-3 is also linked to the aggressiveness of pituitary tumours, and particularly with intratumour haemorrhage. It is anticipated that this link is via the same mechanism as VEGI, in that reduced ligand and receptor, namely DR3, together lifted an inhibitory mechanism of the protein pair on angiogenesis and growth of pituitary tumours (14-16, 34-37). Nonetheless, the link between DR3 and pituitary tumours is not as strong as that of VEGI, in that for example, the association with invasion was not significant. One possibility is that other mechanisms for VEGI may also operate, namely the presence of DcR3. DcR3 is a decoy receptor for VEGI and in some ways may act as an antagonist to VEGI/DR3. Assessing DcR3 in pituitary tumours will be of significant interest.
In conclusion, the present study is, as far as we know, the first to demonstrate a link between a lack of expression of VEGI and the aggressive nature of pituitary tumours. Loss or reduced VEGI is a common feature in this tumour type and is associated with tumour invasion of the sellar fossa, intratumour haemorrhage and presence of cysts, interestingly some of the endocrine functions of pituitary tumours. It is suggested that VEGI is a suppressive molecule in pituitary tumour and may have both prognostic and therapeutic value in patients.
Acknowledgements
The Authors wish to thank Cancer Research Wales and the Albert Hung Foundation for supporting their work.
- Received June 20, 2013.
- Revision received July 17, 2013.
- Accepted July 19, 2013.
- Copyright© 2013 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
