Histopathological Analysis of Tumor Microenvironment and Angiogenesis in Pheochromocytoma.

Pheochromocytomas (PHEOs) are relatively rare catecholamine-producing tumors derived from adrenal medulla. Tumor microenvironment (TME) including neoangiogenesis has been explored in many human neoplasms but not necessarily in PHEOs. Therefore, in this study, we examined tumor infiltrating lymphocytes (CD4 and CD8), tumor associated macrophages (CD68 and CD163), sustentacular cells (S100p), and angiogenic markers (CD31 and areas of intratumoral hemorrhage) in 39 cases of PHEOs in the quantitative fashion. We then compared the results with pheochromocytoma of the adrenal gland scaled score (PASS), grading system for pheochromocytoma and paraganglioma (GAPP) and the status of intra-tumoral catecholamine-synthesizing enzymes (TH, DDC, and PNMT) as well as their clinicopathological factors. Intratumoral CD8 (p = 0.0256), CD31 (p = 0.0400), and PNMT (p = 0.0498) status was significantly higher in PHEOs with PASS <4 than PASS ≧4. In addition, intratumoral CD8+ lymphocytes were also significantly more abundant in well-than moderately differentiated PHEO according to GAPP score (p = 0.0108) and inversely correlated with tumor size (p = 0.0257). Intratumoral CD68+ cells were significantly higher in PHEOs with regular or normal histological patterns than those not (p = 0.0370) and inversely correlated with tumor size (p = 0.0457). The status of CD163 was significantly positively correlated with that of CD8 positive cells (p = 0.0032). The proportion of intratumoral hemorrhage areas was significantly higher in PHEOs with PASS ≧4 (p = 0.0172). DDC immunoreactivity in tumor cells was significantly positively correlated with PASS score (p = 0.0356) and TH status was significantly higher in PHEOs harboring normal histological patterns (p = 0.0236) and cellular monotony (p = 0.0219) than those not. Results of our present study did demonstrate that abundant CD8+ and CD68+ cells could represent a histologically low-scored tumor. In particular, PHEOs with increased intratumoral hemorrhage should be considered rather malignant. In addition, abnormal catecholamine-producing status of tumor cells such as deficient PNMT and TH and increased DDC could also represent more aggressive PHEOs.

From clinic to mechanism: Proteomics-based assessment of angiogenesis in adrenal pheochromocytoma.

Adrenal pheochromocytoma (PCC) is a very rare tumor that stems from chromaffin cells, which can develop into malignant tumor. During the operation, abundant blood vessels were often observed in PCC than other adrenal tumors, which increases the difficulty and risk of the surgery. Therefore, it is important to investigate the mechanism of PCC angiogenesis. Twelve surgical specimens of PCC from Ruijin Hospital, Shanghai Jiaotong University were grouped into high and low microvessel density (MVD) group. They were also divided into rich blood supply and nonenriched blood supply group, according to computed tomography (CT) manifestation. Comparative proteomic analysis based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatics analysis revealed that 206 proteins differentially regulated in the high MVD group compared with low MVD group (p < 0.05). Besides, 61 proteins were discovered to be significantly changed when the 12 samples were grouped according to CT manifestation. By intersecting the differentially changed protein from MVD and CT grouping, 25 proteins were filtered out, with pathological function. COX4I2 was verified to be increased gradually with angiogenesis with increasing severity, and PLAT was shown to be decreased with angiogenesis in PCC, by quantitative reverse-transcription polymerase chain reaction and immunohistochemistry. The quantitative proteomics result indicated that the tumor angiogenesis in PCC is associated with hypoxia. COX4I2 and PLAT were highly correlated with blood supply in PCC which contribute to angiogenesis in PCC, which could be used as biomarkers to better indicate tumor angiogenesis, or targets to regress tumor angiogenesis as treatment.

Preoperative adrenal artery embolization followed by surgical excision of giant hypervascular adrenal masses: report of three cases.

BACKGROUND: Transcatheter arterial embolization (TAE) is an effective minimally invasive adjunct to surgery for the management and/or palliation of adrenal tumors. METHODS: In this case study, we reported three patients who underwent preoperative TAE before adrenalectomy for large hypervascular adrenal tumors. All patients underwent preoperative embolization 24 h before the operation and were then followed up at the intensive care unit surgery. RESULTS: The largest diameter of the adrenal lesions ranged between 8 and 17 cm. Hypertensive attack occurred in one patient with pheochromocytoma during embolization. No other complications associated with the procedure was noted. The adrenal tumors were totally excised in all patients. The major intraoperative findings associated with preoperative embolization were marked reduction in hypervascularity and the decrease in the size of collateral vessels. No major hemorrhage necessitating blood transfusion were noted during surgery. CONCLUSIONS: Preoperative embolization of large hypervascular adrenal masses could reduce perioperative blood loss by reducing tumor vascularity.

Emerging role of dopamine in neovascularization of pheochromocytoma and paraganglioma.

Dopamine is a catecholamine that acts both as a neurotransmitter and as a hormone, exerting its functions via dopamine (DA) receptors that are present in a broad variety of organs and cells throughout the body. In the circulation, DA is primarily stored in and transported by blood platelets. Recently, the important contribution of DA in the regulation of angiogenesis has been recognized. In vitro and in vivo studies have shown that DA inhibits angiogenesis through activation of the DA receptor type 2. Overproduction of catecholamines is the biochemical hallmark of pheochromocytoma (PCC) and paraganglioma (PGL). The increased production of DA has been shown to be an independent predictor of malignancy in these tumors. The precise relationship underlying the association between DA production and PCC and PGL behavior needs further clarification. Herein, we review the biochemical and physiologic aspects of DA with a focus on its relations with VEGF and hypoxia inducible factor related angiogenesis pathways, with special emphasis on DA producing PCC and PGL.-Osinga, T. E., Links, T. P., Dullaart, R. P. F., Pacak, K., van der Horst-Schrivers, A. N. A., Kerstens, M. N., Kema, I. P. Emerging role of dopamine in neovascularization of pheochromocytoma and paraganglioma.

Hypoxia Pathway Mutations in Pheochromocytomas and Paragangliomas.

Pheochromocytomas (PCC) and sympathetic paragangliomas (PGL) are rare neuroendocrine tumors, which derive from chromaffin cells occurring in the adrenal medulla and extra-adrenal sympathetic paraganglia. PCC and PGL are often benign, catecholamine-producing tumors, responsible for a myriad of symptoms that may be potentially hazardous to the patient. In contrast, nonsecreting parasympathetic PGL, derived from chief cells, develop mainly in the head and neck region. Although PCC/PGL are more commonly sporadic tumors, germline mutations are present in up to 40% of the patients, ranking these tumors among those with the highest degree of heritability. PCC/PGL are associated with a variety of hereditary syndromes, comprising genetic alterations in RET, NF1, VHL, and SDHx genes, the last 2 being involved in regulating the hypoxia pathway. Additional hypoxia pathway-related genes have been recently associated with PCC/PGL development, namely EGLN1 and EPAS1. Thus, dysregulation of the hypoxia pathway seems to play a major role in PCC/PGL development, in particular through the stabilization of hypoxia-inducible factors and the appearance of a pseudohypoxia signature. This article is focused on reviewing the tumorigenic mechanisms resultant from VHL, SDHx, EGLN1, and EPAS1 mutations, as well as the associated tumors, namely PCC/PGL, and extra manifestations such as polycythemia. In the light of the recent discoveries, hypoxia pathway molecules appear as key players in PCC/PGL development.

Vascular pattern analysis for the prediction of clinical behaviour in pheochromocytomas and paragangliomas.

Pheochromocytomas (PCCs) are neuroendocrine tumors arising from chromaffin cells of the adrenal medulla. Related tumors that arise from the paraganglia outside the adrenal medulla are called paragangliomas (PGLs). PCC/PGLs are usually benign, but approximately 17% of these tumors are malignant, as defined by the development of metastases. Currently, there are no generally accepted markers for identifying a primary PCC or PGL as malignant. In 2002, Favier et al. described the use of vascular architecture for the distinction between benign and malignant primary PCC/PGLs. The aim of this study was to validate the use of vascular pattern analysis as a test for malignancy in a large series of primary PCC/PGLs. Six independent observers scored a series of 184 genetically well-characterized PCCs and PGLs for the CD34 immunolabeled vascular pattern and these findings were correlated to the clinical outcome. Tumors were scored as malignant if an irregular vascular pattern was observed, including vascular arcs, parallels and networks, while tumors with a regular pattern of short straight capillaries were scored as benign. Mean sensitivity and specificity of vascular architecture, as a predictor of malignancy was 59.7% and 72.9%, respectively. There was significant agreement between the 6 observers (mean κ = 0.796). Mean sensitivity of vascular pattern analysis was higher in tumors >5 cm (63.2%) and in genotype cluster 2 tumors (100%). In conclusion, vascular pattern analysis cannot be used in a stand-alone manner as a prognostic tool for the distinction between benign and malignant PCC, but could be used as an indicator of malignancy and might be a useful tool in combination with other morphological characteristics.

Both sunitinib and sorafenib are effective treatments for pheochromocytoma in a xenograft model.

Pheochromocytomas and paragangliomas are rare neuroendocrine tumors which develop from chromaffin cells of the adrenal medulla and extra-adrenal sites, leading to excess catecholamine release and hypertension. Many of the tumors are characterized by a high vascularity, suggesting the possible implementation of anti-angiogenic therapies for patients. Here, the efficacy of the tyrosine kinase inhibitors sunitinib and sorafenib was investigated in vivo and in vitro. Oral treatment with either sunitinib or sorafenib (40mg/kg/day) for 14days induced a marked reduction in the volume and weight of PC12 pheochromocytoma cell tumor xenografts in mice. Assessment of tumoral neo-angiogenesis, assessed by morphometric analysis of the vascular network after CD31 immunolabeling, showed that both sunitinib and sorafenib reduced the microvessel area (-85% and -80%, respectively) and length (-80% and -78%, respectively) in treated compared to control tumors. In addition, the number of vessel nodes was significantly lower in treated tumors (-95% and -84%, respectively). Furthermore, cleaved caspase 3 immunolabeling revealed a marked increase in the number of apoptotic cells in tumors from treated animals. Sunitinib and sorafenib could exert a direct effect on PC12 cell viability in vitro. While sunitinib induced a rapid (4h) and pronounced (5-fold) increase in caspase-3/7-dependent apoptosis, sorafenib seems to exert its cytotoxic activity through a different mechanism. Altogether, our data demonstrate that sunitinib and sorafenib have the ability to impair pheochromocytoma development by inhibiting angiogenesis and reducing tumor cell viability. These results strongly suggest that both sunitinib and sorafenib could represent valuable therapeutic tools for pheochromocytoma.

Role of VEGF-A and its receptors in sporadic and MEN2-associated pheochromocytoma.

Pheochromocytoma (PHEO), a rare catecholamine producing tumor arising from the chromaffin cells, may occurs sporadically (76%-80%) or as part of inherited syndromes (20%-24%). Angiogenesis is a fundamental step in tumor proliferation and vascular endothelial growth factor (VEGF-A) is the most well-characterized angiogenic factor. The role of angiogenic markers in PHEO is not fully understood; investigations were therefore made to evaluate the expression of VEGF-A and its receptors in PHEO and correlate to clinical parameters. Twenty-nine samples of PHEO were evaluated for VEGF-A, VEGF receptor-1 (VEGFR-1) VEGFR-2 expression and microvessel density (MVD) by immunohistochemistry. Clinical data were reviewed in medical records. The mean age of patients was 38±14 years, and 69% were woman. VEGF-A, VEGFR-1 and VEGFR-2 staining were detected in nearly all PHEO samples. No significant correlation was observed between VEGF-A, VEGFR-1, VEGFR-2 expression or MVD and age at diagnosis, tumor size or sporadic and hereditary PHEO. However, the levels of expression of these molecules were significantly higher in malignant PHEO samples (p=0.027, p=0.003 and p=0.026, respectively).VEGF-A and its receptors were shown to be up-regulated in malignant PHEO, suggesting that these molecules might be considered as therapeutic targets for unresectable or metastatic tumors.

Interferon alpha and rapamycin inhibit the growth of pheochromocytoma PC12 line in vitro.

INTRODUCTION: Pheochromocytomas are benign or malignant neuroendocrine tumours. The unsatisfactory efficacy of the traditional therapeutic methods for patients with metastatic disease results in a continuing search for more effective and targeted agents. Due to the increased vascularisation of these tumours, inhibitors of angiogenesis could be potentially a new group of drugs in pheochromocytoma/paraganglioma therapy. MATERIAL AND METHODS: The aim of this study was to evaluate the influence of angiomodulators: VEGF (vascular endothelial growth factor) and five endogenous and exogenous antiangiogenic compounds (endostatin; IFN-alpha [interferon alpha]; rapamycin - mTOR [mammalian target of rapamycin] inhibitor; JV1-36 and SU5416 (semaxinib]) on the growth of rat pheochromocytoma PC12 cell line. RESULTS: IFN-alpha (10(5) U/mL) strongly inhibited PC12 growth in a 72 h culture, increasing apoptosis and arresting the cell cycle. Rapamycin in a wide range of concentrations (10(-5) to 10(-8) M) induced a slight inhibitory effect on PC12 viability and decreased cell proliferation at the concentration of 10(-5) M. VEGF, endostatin and JV1-36 did not influence the growth of PC12. CONCLUSIONS: The study has shown for the first time that IFN-a inhibited the growth of pheochromocytoma PC12 line and confirmed the inhibitory action of rapamycin on these cells. The results suggest that IFN-alpha and mTOR inhibitors could be potentially effective in the therapy of malignant pheochromocytoma, and encourage further study in this field.

Adrenal venous catecholamine concentrations in patients with adrenal masses other than pheochromocytoma.

The aim of this study was to retrospectively examine adrenal venous catecholamine concentrations and related indices in non-pheochromocytoma patients and to estimate from the obtained results whether measurements of adrenal venous catecholamine concentrations by adrenal venous sampling (AVS) are useful for localizing adrenal pheochromocytoma. The study population comprised 15 patients in whom AVS was performed for evaluation of adrenal non-pheochromocytoma masses (primary aldosteronism, n = 8; Cushing syndrome, n = 5; non-hyperfunctioning tumor, n = 2) without hormonal intervention and was successful in bilaterally judging adrenal vein to infra-renal inferior vena cava cortisol ratios as >3.0. Wide ranges of catecholamine concentrations were seen for both right (epinephrine, 35-175,821 pg/ml; norepinephrine, 115-32,102 pg/ml; dopamine, 9-232 pg/ml) and left (epinephrine, 16-27,251 pg/ml; norepinephrine, 155-9,267 pg/ml; dopamine, 5-234 pg/ml) adrenal veins. High- to low-side adrenal vein concentration ratios also showed wide ranges of up to 779 for epinephrine, 22.5 for norepinephrine, and 7.8 for dopamine. Adrenal venous catecholamine concentrations obtained by AVS and simple comparisons between bilateral adrenal veins might not be useful to localize adrenal pheochromocytoma, as wide variations in concentrations and high- to low-side adrenal vein concentration ratios were noted in patients with adrenal non-pheochromocytoma.

Microvascular density and mast cells in benign and malignant pheochromocytomas.

Pheochromocytomas, uncommon adrenal tumors, have an uncertain behavior. Recently, PASS criteria were proposed for differentiating between benign and malignant cases. These are not perfect, however. The aim of the study was to investigate angiogenesis and mast cell density in context of the clinical behavior and morphologic characteristics of pheochromocytomas. Mean intratumoral chymase positive cell count was 14.50 for malignant, 15.73 for benign cases; mean subcapsular chymase positive cell count was 12.50 for malignant, 11.27 for benign cases. Mean intratumoral tryptase positive cell count was 17.50 for malignant and 17.91 for benign cases; mean subcapsular tryptase positive cell count was 15.25 for malignant and 15.73 for benign cases. Mean intratumoral CD31 positive vessel count was 46.98 for malignant and 51.02 for benign cases; mean subcapsular CD31 positive vessel count was 44.86 for malignant and 39.81 for benign cases. Mean intratumoral CD105 positive vessel count was 37.84 for malignant and 35.95 for benign cases; mean subcapsular CD105 positive vessel count was 26.36 for malignant and 22.03 for benign cases. The differences between benign and malignant cases were not significant. All the vascular counts were correlated with mast cells counts. PASS index was inversely correlated with mast cell counts.

Contrast-enhanced ultrasound for imaging of adrenal masses.

PURPOSE: The number of incidentally discovered adrenal masses is growing due to the increased use of modern high-resolution imaging techniques. However, the characterization and differentiation of benign and malignant adrenal lesions is challenging. This study aimed to evaluate contrast-enhanced ultrasound for the characterization of adrenal masses. MATERIALS AND METHODS: We studied 58 patients with adrenal masses detected with computed tomography, magnetic resonance imaging, or ultrasound. 7 patients had bilateral adrenal lesions. Contrast-enhanced ultrasound was performed using high-resolution ultrasound (3.5 - 7 MHz) and intravenous injection of 2.4 ml SonoVue. The contrast enhancement pattern of all adrenal lesions was documented. RESULTS: The 18 malignant adrenal tumors were significantly larger at the time of diagnosis compared to the 40 benign lesions (p < 0.03). The majority of benign adrenal lesions (37 / 40) had a nonspecific type of contrast enhancement (24 / 40) or a peripheral to central contrast filling (13 / 40) described as the iris phenomenon. Similar findings were observed in malignant adrenal tumors: most malignant lesions also showed nonspecific (6 / 18) or peripheral to central contrast filling (9 / 18). Peripheral to central contrast filling had 50 % sensitivity (26 - 74 %) and 68 % specificity (51 - 81 %) for indicating malignancy. CONCLUSION: Contrast-enhanced ultrasound facilitates the visualization of vascularization even in small adrenal masses, but it does not help to distinguish malignant and benign lesions.

[Clinical manifestation and multiphasic spiral CT scanning features of abdominal pheochromocytoma: report of 70 cases].

OBJECTIVE: The purpose of this study was to assess the imaging characteristics of abdominal pheochromocytoma in multiphasic spiral CT scanning, and to determine whether these image characteristics can aid in differentiating pheochromocytoma from other types of tumors or not. METHODS: The image data of dynamic enhanced CT of 79 pathologically confirmed pheochromocytomas in 70 patients were retrospectively reviewed. RESULTS: Among the 70 patients, there were 41 patients with endocrine symptoms related to pheochromocytoma, 15 had a latent pheochromocytoma and the remaining 14 cases presented with a non-functioning pheochromocytoma. There were totally 79 tumors in 70 patients, with a single lesion in 63 cases, while multiple lesions in the other 7. Sixty cases had a tumor located in the adrenal gland, while 8 in retroperitoneal space, and 2 cases had both intraadrenal and ectopic lesions simultaneously. Sixty patients had a benign pheochromocytoma, the other 10 had a malignant one or relapse after operation. The average size of the tumor was 5.8 cm (range, 2 approximately 15 cm in the longest diameter). Seventy-seven pheochromocytomas had a well defined boundary except two big ones with a unclear margin, which were finally proven to be malignant. Homogeneous enhancement was found in 6 lesions with a diameter

Use of the tyrosine kinase inhibitor sunitinib in a patient with von Hippel-Lindau disease: targeting angiogenic factors in pheochromocytoma and other von Hippel-Lindau disease-related tumors.

CONTEXT: von Hippel-Lindau disease is characterized by highly vascularized tumors of multiple organs. EVIDENCE ACQUISITION: We present a patient with von Hippel-Lindau disease with multiple renal and pancreatic tumors and a malignant pheochromocytoma infiltrative of the sacrum and associated with lymph nodule metastases. The pheochromocytoma expressed high protein level of vascular endothelial growth factor and platelet-derived growth factor-beta receptor. The patient presented with a poor performance status, severe pelvic pain, weight loss, and manifestations of catecholamine excess. EVIDENCE SYNTHESIS: Treatment against malignant pheochromocytoma with surgery, chemotherapy, or participation in clinical trials was not feasible because of the patient's poor performance status, the presence of multiple tumors, and the extension of the pheochromocytoma into the bones. Patient was treated with sunitinib, a potent tyrosine kinase inhibitor of vascular endothelial growth factor, platelet-derived growth factor, RET, c-KIT, and FLT-3 receptors. Six months of treatment with sunitinib was associated with normalization of the patient's performance status and blood pressure, absence of symptoms of catecholamine excess, weight gain, disappearance of pain, shrinkage of each of the tumors (50% in the largest renal tumor, 38% in the largest islet cell tumor, 21% in the pelvic malignant pheochromocytoma), and reduction of plasma normetanephrines and chromogranin A. CONCLUSION: This study provides evidence that targeting tyrosine kinase receptors such as the vascular endothelial growth factor pathway and the platelet-derived growth factor-beta receptor may have value in the treatment of VHL-related tumors including pheochromocytoma.

The use of autologous femoral vein for the repair of the common iliac vein after resection of a pheochromocytoma.

AIM: To illustrate the use of autologous femoral vein for grafting ilio-caval vein defects following abdomino-pelvic tumour resections. METHODS: Case report and literature review. RESULTS: Durable restoration of ilio-caval patency was achieved, with minimal morbidity from graft harvesting. CONCLUSIONS: Autologous femoral vein presents a viable graft option for the immediate reconstruction of large intra-abdominal vein deficits.

Laparoscopic resection of extra-adrenal pheochromocytoma--case report and review of the literature in pediatric patients.

Surgery for pheochromocytoma differs from that of other tumors owing to the potential release of catecholamines, which may lead to severe intraoperative hemodynamic changes. The present standard of care for resection of adrenal pheochromocytoma has become a laparoscopic approach for surgical excision. Extra-adrenal pheochromocytoma is a very rare entity, especially in the pediatric age group; the utility of the laparoscopic approach is not established in this population. We present a case report of a child with hormonally active extra-adrenal pheochromocytoma originating in the organ of Zuckerkandl that was resected laparoscopically. We found the laparoscopic approach gave excellent exposure, allowing for proper identification of the tumor's origin and its relation to surrounding structures; a complete resection with excellent control of the feeding blood vessels was performed. Herein we present the details of this case and a review of the relevant literature. After our initial experience we can recommend laparoscopic exploration for similar cases of suspected extra-adrenal pheochromocytoma as an appropriate tool to identify extension of the disease and estimate resectability. Proper patient preparation and monitoring are critical for success.