Genomic and immune landscape Of metastatic pheochromocytoma and paraganglioma.

The mechanisms triggering metastasis in pheochromocytoma/paraganglioma are unknown, hindering therapeutic options for patients with metastatic tumors (mPPGL). Herein we show by genomic profiling of a large cohort of mPPGLs that high mutational load, microsatellite instability and somatic copy-number alteration burden are associated with ATRX/TERT alterations and are suitable prognostic markers. Transcriptomic analysis defines the signaling networks involved in the acquisition of metastatic competence and establishes a gene signature related to mPPGLs, highlighting CDK1 as an additional mPPGL marker. Immunogenomics accompanied by immunohistochemistry identifies a heterogeneous ecosystem at the tumor microenvironment level, linked to the genomic subtype and tumor behavior. Specifically, we define a general immunosuppressive microenvironment in mPPGLs, the exception being PD-L1 expressing MAML3-related tumors. Our study reveals canonical markers for risk of metastasis, and suggests the usefulness of including immune parameters in clinical management for PPGL prognostication and identification of patients who might benefit from immunotherapy.

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.

First proof of association between autoimmune polyglandular syndrome and multiple endocrine neoplasia in humans.

Autoimmune Addison's disease (AAD) is a rare condition occurring either in isolation or associated with other autoimmune diseases as part of an autoimmune polyglandular syndrome (APS) type 1, 2 or 4. Multiple endocrine neoplasia (MEN) type 1, 2 or 4 is a hereditary autosomal dominant cancer syndrome. Medullary thyroid carcinoma and pheochromocytoma are neoplasms common to MEN-2a and MEN-2b. We describe a unique, complex case of a man resulted affected by both APS-2 and MEN-2a. The patient developed Hashimoto's thyroiditis, diabetes mellitus type 1 and AAD, despite testing negative for adrenal cortex autoantibodies (ACA) and steroid 21-hydroxylase autoantibodies (21-OHAb). Moreover, he had also a family history for MEN-2a and he first developed medullay thyroid cancer, then bilateral pheochromocytoma on the adrenal substrate of an AAD. On adrenal histology we found complete bilateral cortical atrophy in the presence of a lymphocytic infiltration and fibrosis, confirming an ACA and 21-OHAb-negative AAD. This datum is the first documented in a living individual and confirms that the absence of autoantibodies is not incompatible with an autoimmune disease and confirms that AAD is a cell-mediated autoimmune disease limited to the adrenal cortex and sparing medullary. In the light of a literature review concerning the association between APS and MEN, this is the first proven case to be reported in humans. Finally, our findings suggest that adrenal medullary tumor can develop even on an adrenal gland with cortical atrophy due to autoimmune adrenalitis.

Chromogranin A as potential target for immunotherapy of malignant pheochromocytoma.

Currently, no effective treatment for malignant pheochromocytoma exists. The aim of our study was to investigate the role of chromogranin A (CgA) as a specific target molecule for immunotherapy in a murine model for pheochromocytoma. Six amino acid-modified and non-modified CgA peptides were used for dendritic cell vaccination. Altogether, 50 mice received two different CgA vaccination protocols; another 20 animals served as controls. In vitro tetramer analyses revealed large increases of CgA-specific cytotoxic T cells (CTL) in CgA-treated mice. Tumors of exogenous applied pheochromocytoma cells showed an extensive infiltration by CD8+ T cells. In vitro, CTL of CgA-treated mice exhibited strong MHC I restricted lysis capacities towards pheochromocytoma cells. Importantly, these mice showed strongly diminished outgrowth of liver tumors of applied pheochromocytoma cells. Our data clearly demonstrate that CgA peptide-based immunotherapy induces a cytotoxic immune response in experimental pheochromocytoma, indicating potential for therapeutic applications in patients with malignant pheochromocytoma.

[Clinicomorphological aspects of adrenal pheochromocytoma diagnostics].

The removed pheochromocytomas of 25 patients aged from 25 to 72 years have been studied. Among them 5 tumors have had bilateral character, 8 pheochromocytomas have been diagnosed as malignant, 4 patients have had metastases. The average size of benign pheochromocytomas has been 4.0 cm, malignant - 4.5 cm. The majority of malignant tumors have had areas with diffusing growth patterns. The results of immunohistochemical study of benign and malignant pheochromocytomas have had small differences. The study of tumor ultrastructure hasn't found out their biological potential.

Chromogranin a as potential tumour antigen in pheochromocytoma.

Chromogranin A (CgA) is a dense core vesicle-associated protein and, therefore, represents a specific marker of neuroendocrine cells and tumours including pheochromocytomas. CgA has already been investigated for its immunogenic properties. Importantly, in vitro studies demonstrated the presence of naturally occurring CgA-specific cytotoxic T cells in patients with neuroendocrine cancers. Therefore, it is worthwhile to investigate the potential role of CgA as tumour antigen in pheochromocytoma. Depending on the results of these ongoing in vitro and in vivo studies, a clinical application may arise in the near future.

Overexpression of protein kinase C-delta plays a crucial role in interleukin-6-producing pheochromocytoma presenting with acute inflammatory syndrome: a case report.

Pheochromocytomas are tumors that may produce a variety of substances in addition to catecholamines. To date, among several cases of systemic inflammatory syndrome associated with interleukin-6 (IL-6) secretion, IL-6-producing pheochromocytomas, have been reported. However, the mechanism underlying IL-6 oversecretion in these cases has not yet been clarified. This report describes a patient with pheochromocytoma who exhibited pyrexia and marked inflammatory signs including C-reactive protein elevation. The inflammatory symptoms were easily controlled by the administration of loxoprofen, a nonsteroidal anti-inflammatory drug. The plasma concentration of IL-6 and 11-d-TXB(2), a stable metabolite of thromboxane A(2) (TXA(2)), were significantly elevated in parallel with an elevation of norepinephrine in the samples obtained by selective venous sampling. A left adrenalectomy was performed, and the acute inflammatory symptoms naturally diminished without loxoprofen. Cultured tumor cells obtained from the resected specimen spontaneously released IL-6, and indomethacin inhibited the IL-6 release. According to a cDNA microarray analysis, mRNA of protein kinase C-delta (PKC-delta), prostaglandin D synthase, and arachidonate release-relating enzymes were significantly overexpressed in the tumor tissue in comparison to the adjacent nontumor tissue. The constitutive phosphorylation of PKC-delta was observed in the tumor tissue. These results strongly suggest that the systemic inflammatory syndrome in IL-6-producing pheochromocytoma, at least in part, is caused by the overexpression of PKC-delta, resulting in an excess of arachidonate derivatives such as prostaglandins.

Antigenic cross-reactivity of nerve growth factors from diverse source: activity versus toxicity of NGF.

NGF used in these studies was from various sources: cobra venom (V-NGF), mouse submaxillary glands (M-NGF), honeybee venom (B-NGF), human serum (H-NGF), and cobra serum (CS-NGF). This investigation reports the antigenic cross-reactivity of NGFs from the above mentioned diverse sources. Antigenic cross-reactivity of NGFs was determined by immunological test, using anti-NGFs against V-NGF, M-NGF, B-NGF, and H-NGF. Results revealed that NGF was a conserved protein showing antigenic cross-reactivity among the NGFs from diverse source, except for NGFs from cobra and bee venoms. Anti B-NGF reacted poorly with cobra venom V-NGF and vice versa. Anti M-NGF showed higher antigenic reactivity with human serum H-NGF than with honeybee B-NGF and cobra serum CS-NGF. This research also reports that the toxicity of NGF is directly related to the biological activity of producing neurite outgrowth on PC12 cells. Cobra venom NGF produced neurites at 1-5 ng, and it was toxic at 10-20 ng on PC12 cells. After reducing the activity of NGF, it was found to be non-toxic to PC12 cells at 50 X concentration.

Interleukin-6 (IL-6) producing phaeochromocytoma: direct IL-6 suppression by non-steroidal anti-inflammatory drugs.

A 35-year-old Japanese woman presented with a phaeochromocytoma and demonstrated marked inflammatory reactions and pyrexia as a result of excessive production of interleukin-6 (IL-6) by the tumour. Serum IL-6 level was 262 ng/l (normal; < 4.0 ng/l). Fever and inflammatory markers were largely overcome by the administration of the nonsteroidal anti-inflammatory drug, naproxen, and all symptoms disappeared soon after the tumour was excised. Immunohistochemical study revealed positive staining using an antihuman IL-6 antibody and Northern analysis showed increased IL-6 mRNA levels in the tumour. Cultured tumour cells showed IL-6 protein synthesis, and nonsteroidal anti-inflammatory drugs such as naproxen and indomethacin directly inhibited IL-6 release. These results indicate that the effects of naproxen in vivo were due, at least in part, to direct suppression of IL-6 secretion from the tumour.

Improved gene transfer to neuroblastoma cells by a monoclonal antibody targeting RET, a receptor tyrosine kinase.

Receptor-mediated gene transfer is an effective strategy among nonviral vector systems. It is, however, crucial to develop various types of monoclonal antibodies satisfying both the binding specificity for cell targeting and the capacity of endocytosis required for gene transfer. In the present study, we generated a novel monoclonal antibody (NBL-1) to RET, a receptor tyrosine kinase expressed in both neuroblastoma cells and cells present in substantia nigra, a responsive locus of Parkinson's disease. NBL-1, when added to the culture medium of the neuroblastoma cells, was incorporated by endocytosis in a wortmannin-sensitive manner. Using a biotinylated NBL-1 complexed with plasmid DNAs based on electrostatic interaction through avidin-conjugated polylysines, exogenous luciferase genes were expressed in neuroblastoma cells at a more than 10-fold higher level. The expression level of the gene based on NBL-1 was comparable to that obtained by a geneporter system, an improved nonviral gene transduction method. Furthermore, the NBL-1-based gene transfer mediated the formation of more than 20-fold higher numbers of drug-resistant colonies. In contrast, RET-negative cells, which included HeLa, HT1080, Caco-2, and Colo205 cells, did not show any increased expression of an exogenous gene by NBL-1. These data suggest that the RET molecules enable selective gene transduction, and that NBL-1 may possibly be applied to gene therapy for neuroblastomas and Parkinson's disease.

Interleukin-6 secreting phaeochromocytoma associated with clinical markers of inflammation.

Phaeochromocytomas have been shown to produce not only catecholamines but other neuropeptides and hormones, with a variety of clinical manifestations. We report a 70-year-old female patient with phaeochromocytoma exhibiting sustained hypertension, low-grade fever, thrombocytosis, and elevated levels of plasma fibrinogen and C-reactive protein. Serum interleukin (IL)-6 levels were significantly elevated, whereas serum IL-1 alpha and IL-1 beta were not detectable. After surgical removal of the tumour, hypertension and low-grade fever disappeared, and the laboratory finding including serum IL-6 concentrations became normal. Immunohistochemical study of the tumour showed positive staining for IL-6. Culture of the resected tumour revealed the production of large amounts of IL-6. It is suggested that IL-6 secreted by the tumour was responsible for some of the clinical manifestations in this patient.

Malignant pheochromocytoma associated with Jaccoud's-type arthropathy, Raynaud's phenomenon, positive antinuclear antibody and rheumatoid factor.

We describe a patient with malignant pheochromocytoma who developed Jaccoud's-type arthropathy and Raynaud's phenomenon as initial manifestations of malignant pheochromocytoma. Serologic findings included positive antinuclear antibody (ANA) and rheumatoid factor (RF) was also found in this patient. To our knowledge, this is the first time Jaccoud's-type arthropathy with positive ANA and RF has been reported as rheumatic manifestations of pheochromocytoma.

Immunoreactive C-type natriuretic peptide in human adrenal glands and adrenal tumors.

C-type natriuretic peptide (CNP) in human adrenal glands and adrenal tumors was measured with a specific radioimmunoassay for CNP. Tissue immunoreactive (IR-) CNP concentrations were 0.54 +/- 0.40 pmol/g wet tissue (gwt) (mean +/- SD) in 14 pheochromocytomas, 0.69 +/- 0.19 pmol/gwt in six adrenocortical tumors, and 0.49 +/- 0.22 pmol/gwt in seven normal adrenal glands (cortex and medulla mixed). These concentrations were comparable to those found in tissues from human brains. Sephadex G-50 superfine column chromatography and reverse-phase high performance liquid chromatography revealed that IR-CNP in normal adrenal glands and pheochromocytoma consisted of at least two components: a component in low molecular weight form chromatographically identical to CNP-22 and the other, a high molecular weight form very similar to human CNP-53. This study has shown that IR-CNP is present in human adrenal glands and adrenal tumors with similar molecular forms and comparable concentrations to those in the human brain.

Polyoma-induced neoplasms of the mouse adrenal medulla. Characterization of the tumors and establishment of cell lines.

BACKGROUND: Pheochromocytomas that are usually noradrenergic arise commonly in the adult rat adrenal medulla. The widely studied PC12 cell line, that is representative of these rat adrenal tumors, is also noradrenergic. The reasons for the absence of epinephrine production by most rat pheochromocytoma cells are unknown, and there are currently no adrenergic adrenal medullary cell lines. Pheochromocytomas are rare in mice. EXPERIMENTAL DESIGN: Tumors induced by polyoma virus in the adrenal medullas of postnatal mice were studied immunocytochemically for catecholamine biosynthetic enzymes in order to determine how their profiles of catecholamine production compared with those of rat pheochromocytomas. Clonal cell lines were established from a representative tumor and were evaluated for responsiveness to agents known to affect the development and function of normal and neoplastic rat chromaffin cells. RESULTS: Although adrenal medullary cells from normal rodents produce epinephrine before birth, polyoma-induced mouse adrenal tumor cells are immature or poorly differentiated. They synthesize norepinephrine, but not epinephrine, which during normal development is produced later than norepinephrine. They also produce relatively large quantities of dihydroxyphenylalanine, suggesting an abnormality of catecholamine biosynthesis such that tyrosine hydroxylase is not rate-limiting. Secretory granules are sparse, as demonstrated by electron microscopy or by staining for chromogranin A, and catecholamine stores are low. Further, the tumor cells appear to be phenotypically unstable, as judged from heterogeneous staining for tyrosine hydroxylase even in early passage, twice-cloned cell lines. Tumor cell morphology and catecholamine profiles appear to be unaffected or minimally affected by nerve growth factor, forskolin or dexamethasone, which are known to affect normal or neoplastic rat chromaffin cells. However, tumors formed after subcutaneous injection of cell lines into mice show up to a 10-fold increase in catecholamine stores, suggesting that the cells are subject to some forms of regulation. The cloned cell lines do not produce detectable polyoma virus, but express all three viral T antigens, including a characteristic, truncated form of large T. CONCLUSIONS: The findings suggest that the process of neoplastic transformation and/or the presence of polyoma virus T antigens results in suppression of the adrenergic phenotype in mouse adrenal chromaffin cells. T antigens might therefore be useful as tools for studying mechanisms that regulate the differentiation and maturation of chromaffin cells in normal and neoplastic states. Furthermore, although polyoma virus cannot be readily used to produce adrenergic cell lines from the mouse adrenal medulla, the lines that are produced might substitute for PC12 cells in some types of studies that require a mouse model.

Malignant phaeochromocytoma and hypercalcaemia.

We describe a case of hypercalcaemia secondary to recurrent malignant phaeochromocytoma. Parathyroid-related protein (PTHrp 1-86) immunoreactivity was identified in plasma and PTHrp was identified by immunocytochemistry in tumour tissue.

HMB-45 reactivity in adrenal pheochromocytomas.

Melanoma-specific antibody (HMB-45) is highly specific for junctional nevi, malignant melanomas, and related lesions. Rarely have other benign or neoplastic tissues demonstrated positivity with the monoclonal antibody following purification. In the present study, four of 12 pheochromocytomas contained chief cells that reacted with HMB-45. We discuss the clinical and pathologic implications of this finding.

Immunohistochemical characterization of HLA-DR-antigen positive dendritic cells in phaeochromocytomas and paragangliomas as a prognostic marker.

Twelve cases of phaeochromocytoma (PCC) and four cases of paraganglioma (PGG) were studied by immunohistochemistry and immunoelectron microscopy in order to demonstrate HLA-DR (Ia)-antigen-positive dendritic cells (IaDCs). Dense infiltration of IaDCs was detected in the majority of PCCs revealing high urinary or serum catecholamine levels, but in aggressively growing PCCs, a familial PCC and all PGGs, few IaDCs were demonstrated. Interestingly, these IaDCs were negative for S-100 protein. Although S-100-protein-positive sustentacular-like cells (SCs), morphologically similar to IaDCs, were also present, these were clearly distinguished from IaDCs by our double immunostaining method. Ultrastructurally, IaDCs had smooth or slightly indented nuclei and contained a moderate amount of endoplasmic reticulum, small mitochondria and vacuoles, extending elongated cytoplasmic processes. These results suggest that determination of the quantity of IaDCs is a highly effective method of assessing the character of PCCs, in particular, their prognosis.