Marlène Guérin

Johann Guillemot

Erwan Thouënnon

Alice Pierre

Fatima-Zohra El-Yamani

Maité Montero-Hadjadje

Christophe Dubessy

Rabia Magoul

Isabelle Lihrmann

Youssef Anouar

Laurent Yon
email: laurent.yon@univ-rouen.fr

Granins and their derived peptides in normal and tumoral chromaffin tissue: Implications for the diagnosis and prognosis of pheochromocytoma

Introduction

The granin proteins are widely distributed in secretory granules of endocrine, neuroendocrine and nerve cells [START_REF] Rosa | The granin protein family: markers for neuroendocrine cells and tools for the diagnosis of neuroendocrine tumors[END_REF][2][3], and have also been described in other secreting cells like exocrine cells, cardiomyocytes or immune system cells [4][5][START_REF] Saruta | Expression and localization of chromogranin A gene and protein in human submandibular gland[END_REF]. At present, the granin family can loosely be divided into two groups, the chromogranins (Cg) and the secretogranins (Sg), distinguishable by the presence of a disulfide-bonded loop at the N-terminus of Cg but not in Sg proteins [START_REF] Taupenot | The chromogranin-secretogranin family[END_REF][START_REF] Helle | The granin family of uniquely acidic proteins of the diffuse neuroendocrine system: comparative and functional aspects[END_REF]. They constitute a family of acidic glycoproteins whose major members are chromogranin A (CgA), which was first isolated from chromaffin cells of the adrenal medulla [START_REF] Banks | The release of protein from the stimulated adrenal medulla[END_REF]; chromogranin B (CgB, previously also called secretogranin I), initially characterized in a rat pheochromocytoma cell line [START_REF] Lee | Tyrosine-O-sulfated proteins of PC12 pheochromocytoma cells and their sulfation by a tyrosylprotein sulfotransferase[END_REF]; and secretogranin II (SgII, previously also called chromogranin C), which was originally described in the anterior pituitary [START_REF] Rosa | Secretogranins I and II: two tyrosine-sulfated secretory proteins common to a variety of cells secreting peptides by the regulated pathway[END_REF]. The granin family has further expanded to include other members such as secretogranin III (SgIII or 1B1075) [START_REF] Ottiger | 1B1075: a brain-and pituitary-specific mRNA that encodes a novel chromogranin/secretogranin-like component of intracellular vesicles[END_REF], secretogranin IV (SgIV or HISL-19) [START_REF] Krisch | Monoclonal antibody HISL-19 as an immunocytochemical probe for neuroendocrine differentiation. Its application in diagnostic pathology[END_REF], secretogranin V (SgV or 7B2) [START_REF] Iguchi | Tissue distribution and molecular forms of a novel pituitary protein in the rat[END_REF], secretogranin VI (SgVI or NESP55) [START_REF] Ischia | Molecular cloning and characterization of NESP55, a novel chromogranin-like precursor of a peptide with 5-HT1B receptor antagonist activity[END_REF], secretogranin VII (SgVII or VGF) [START_REF] Helle | The granin family of uniquely acidic proteins of the diffuse neuroendocrine system: comparative and functional aspects[END_REF][START_REF] Levi | Molecular cloning of a gene sequence regulated by nerve growth factor[END_REF] and proSAAS [START_REF] Fricker | Identification and characterization of proSAAS, a granin-like neuroendocrine peptide precursor that inhibits prohormone processing[END_REF].

Granins are characterized by a high proportion of acidic amino acids (aspartic and glutamic acids) that confers the acidic nature of these proteins. This property enables them to play a role in the formation of secretory vesicles and sequestration of hormones in neuroendocrine cells [START_REF] Chanat | Milieu-induced, selective aggregation of regulated secretory proteins in the trans-Golgi network[END_REF][START_REF] Iacangelo | Chromogranin A: current status as a precursor for bioactive peptides and a granulogenic/sorting factor in the regulated secretory pathway[END_REF][START_REF] Kim | Chromogranin A, an "on/off" switch controlling dense-core secretory granule biogenesis[END_REF]. In addition, granins encompass in their sequence numerous pairs of basic amino acids which are potential cleavage sites for prohormone convertases and which may give rise to bioactive peptides through post-translational proteolytic processing. Indeed, numerous studies have shown that peptides derived from granin maturation occur in various tissues and exhibit autocrine, paracrine, or endocrine activities [START_REF] Montero-Hadjadje | Chromogranins A and B and secretogranin II: evolutionary and functional aspects[END_REF][START_REF] Zhao | New insights into granin-derived peptides: evolution and endocrine roles[END_REF]. The ubiquitous distribution of granins in endocrine and neuroendocrine tissues from which they are secreted into the bloodstream, makes granins useful markers of normal and tumoral neuroendocrine cells. In fact, multiple studies have documented the clinical value of detecting granins and their derived peptides in tissues and measuring their circulating levels [START_REF] Taupenot | The chromogranin-secretogranin family[END_REF]. In particular, measurement of CgA levels in plasma can be used to diagnose or monitor the progression of neuroendocrine tumors [START_REF] Ferrari | The biological characteristics of chromogranin A and its role as a circulating marker in neuroendocrine tumours[END_REF]. The highest accuracy has been observed in tumors characterized by an intense secretory activity, although the specificity and sensitivity remain high also in non-functioning tumors [START_REF] Kimura | Plasma chromogranin A in pheochromocytoma, primary hyperparathyroidism and pituitary adenoma in comparison with catecholamine, parathyroid hormone and pituitary hormones[END_REF].

Among neuroendocrine tumors, pheochromocytomas are rare catecholamine-producing neoplasms primarily (80-85%) arising from chromaffin cells of the adrenal medulla or extra-adrenal paraganglia (Fig. 1). Tumors from extra-adrenal location are referred to as paragangliomas. Most of pheochromocytomas occur sporadically but approximately 25% result from germline mutations in 6 genes identified to date. Mutations of the VHL tumor suppressor gene predispose to the development of von Hippel-Lindau (VHL) syndrome, those of NF1 gene to von Recklinghausen's syndrome, and those of the RET protooncogene to multiple endocrine neoplasia (MEN) type 2A and 2B [START_REF] Thouënnon | Identification of potential gene markers and insights into the pathophysiology of pheochromocytoma malignancy[END_REF]. More recently, mutations identified in genes encoding succinate dehydrogenase (SDH) subunits B, C and D have been shown to predispose to familial paraganglioma syndromes [START_REF] Burnichon | The succinate dehydrogenase genetic testing in a large prospective series of patients with paragangliomas[END_REF]. Clinical presentation of pheochromocytoma can vary greatly, with similar signs and symptoms produced by many other clinical conditions. Most but not all the clinical manifestations of pheochromocytoma are due to the indirect actions of secreted catecholamines. Hypertension, tachycardia, pallor, headache and feeling of panic or anxiety, usually dominate the clinical presentation. Metabolic effects include hyperglycemia, lactic acidosis and weight loss. Less common signs and symptoms are nausea, fever and flushing [START_REF] Lenders | Phaeochromocytoma[END_REF]. Despite the increasing availability of molecular diagnostic and prognostic markers, it remains impossible to predict the development of malignant disease, based on histological findings in a resected tumor. Only the presence of metastases of tumoral chromaffin tissue establishes a definite diagnosis of malignant pheochromocytoma [START_REF] Lenders | Phaeochromocytoma[END_REF][START_REF] Scholz | Diagnostic management of benign and malignant pheochromocytoma[END_REF]. It should be noted that the incidence of pheochromocytomas is only 2-8 cases per 1,000,000 subjects and that malignant pheochromocytomas only represent about 10% of all pheochromocytomas.

In this review, we have explored the literature concerning the occurrence of granins in normal and tumoral chromaffin cells, and the clinical usefulness of these proteins and their derived peptides for the diagnosis and the prognosis of pheochromocytomas.

Chromogranin A and derived peptides in pheochromocytoma

Chromogranin A

CgA is the major soluble protein, co-stored and co-released with catecholamines from storage vesicles of the adrenal medulla (Fig. 2). In 1983, using a radioimmunoassay (RIA) O'Connor and colleagues described the widespread distribution of CgA in polypeptide hormone-producing tissues and particularly in the medulla of the adrenal gland [START_REF] Connor | Chromogranin: widespread immunoreactivity in polypeptide hormone producing tissues and in serum[END_REF]. Thereafter, the ubiquitous distribution of CgA in bovine tissues was investigated by immunohistochemistry, showing the occurrence of a specific CgA immunostaining in adrenal medullary chromaffin cells [START_REF] O'connor | Chromogranin A: immunohistology reveals its universal occurrence in normal polypeptide hormone producing endocrine glands[END_REF]. Immunohistochemical labeling for CgA was then demonstrated in normal human adrenomedullary chromaffin cells, as well as in pheochromocytoma cells [START_REF] O'connor | Immunoreactive human chromogranin A in diverse polypeptide hormone producing human tumors and normal endocrine tissues[END_REF] (Table 1). O'Connor and Bernstein [START_REF] O'connor | Radioimmunoassay of chromogranin A in plasma as a measure of exocytotic sympathoadrenal activity in normal subjects and patients with pheochromocytoma[END_REF] were the first to report the measurement of immunoreactive CgA in human plasma. They have shown that in patients with pheochromocytoma, CgA concentration was higher than in healthy volunteers (1614 ± 408 ng/ml vs 129 ± 12 ng/ml, respectively). Accumulated data, thereafter, have confirmed high concentrations of plasma or serum CgA in patients with pheochromocytoma, and numerous assays have been developed using different markers (enzymatic or luminescent), other standard peptide (bovine CgA) or other antibodies (polyclonal, monoclonal or mixed system) [START_REF] Dillen | Enzyme-linked immunosorbent assay for chromogranin A[END_REF][START_REF] Degorce | A new human chromogranin A (CgA) immunoradiometric assay involving monoclonal antibodies raised against the unprocessed central domain (145-245)[END_REF]. Interestingly, Cleary and collaborators [START_REF] Cleary | Chromogranin A expression in phaeochromocytomas associated with von Hippel-Lindau syndrome and multiple endocrine neoplasia type 2[END_REF] showed that pheochromocytoma tissues from MEN2 patients expressed substantially more CgA than tumors from VHL patients at both the mRNA and protein levels. Moreover, they indicated that, relative to the increase in plasma catecholamines, patients with pheochromocytoma associated with MEN2 had higher plasma concentrations of CgA than those with tumors in VHL syndrome.

CgA has been proposed as an alternative diagnostic test to catecholamines for the detection of pheochromocytoma, because its measurement in plasma is not substantially altered by drugs commonly used to diagnose or treat pheochromocytoma, such as phentolamine, tyramine, clonidine, and metoprolol [START_REF] Hsiao | Chromogranin A storage and secretion: sensitivity and specificity for the diagnosis of pheochromocytoma[END_REF]. Hsiao and collaborators [START_REF] Hsiao | Chromogranin A storage and secretion: sensitivity and specificity for the diagnosis of pheochromocytoma[END_REF][START_REF] Hsiao | Chromogranin A in familial pheochromocytoma: diagnostic screening value, prediction of tumor mass, and post-resection kinetics indicating two-compartment distribution[END_REF] first examined the value of CgA determination for the diagnosis of pheochromocytoma and reported a diagnostic sensitivity of 83% and a specificity of 96% (Table 1, see the legend for the definition of the terms sensitivity and specificity). Similar results were obtained by Nobels et al. [START_REF] Nobels | Chromogranin A as serum marker for neuroendocrine neoplasia: comparison with neuron-specific enolase and the alpha-subunit of glycoprotein hormones[END_REF] while studying a relatively small number of cases (n= 9). It was found that the accuracy of plasma CgA measurement for the diagnosis of pheochromocytoma was lower than that of catecholamines or metanephrines [START_REF] Boomsma | Sensitivity and specificity of a new ELISA method for determination of chromogranin A in the diagnosis of pheochromocytoma and neuroblastoma[END_REF]. These authors concluded that CgA determination did not offer additional information with respect to the other markers currently used in diagnostic setting of pheochromocytoma. However, Stridsberg and Husebye [START_REF] Stridsberg | Chromogranin A and chromogranin B are sensitive circulating markers for phaeochromocytoma[END_REF] reported that elevated CgA was observed in 90% of cases, and that no correlation was found between plasma CgA and urine adrenaline or noradrenaline, indicating that CgA could be considered as an independent biomarker for pheochromocytoma. They proposed that the combined assessment of blood CgA and urinary catecholamines could improve the diagnosis of pheochromocytoma. Similarly, Giovanella et al. [START_REF] Giovanella | Serum chromogranin-alpha immunoradiometric assay in the diagnosis of pheochromocytoma[END_REF] reported that measurement of circulating CgA displayed a higher sensitivity (100%), specificity (96%) and accuracy (96%) than all other tests performed (i.e. 24-hour urinary adrenaline, noradrenaline, vanillylmandelic acid and metanephrines), and concluded that the CgA assay might be used as a single test for the diagnosis of pheochromocytoma (Table 1). D'Herbomez et al. [START_REF] Herbomez | Chromogranin A assay and (131)I-MIBG scintigraphy for diagnosis and follow-up of pheochromocytoma[END_REF] reported a 90.8% concordance between CgA levels and 131 I-MIBG scintigraphy data and showed that a CgA level in the reference range is highly predictive of normal scintigraphy findings. More recently, the same authors [START_REF] Herbomez | An analysis of the biochemical diagnosis of 66 pheochromocytomas[END_REF] investigated in a group of 66 pheochromocytoma patients, the performance of several markers currently used for the diagnosis of this tumor. They showed that the more sensitive variables were plasma and urinary normetanephrines and plasma CgA. Finally, other authors have reported a high sensitivity of CgA assays and confirmed that combination with other tests like urinary catecholamines or metanephrines, increases the sensitivity for the diagnosis of chromaffin cell tumors [START_REF] Giovanella | Chromogranin A immunoradiometric assay in diagnosis of pheochromocytoma: comparison with plasma metanephrines and 123I-MIBG scan[END_REF][START_REF] Grossrubatscher | The role of chromogranin A in the management of patients with phaeochromocytoma[END_REF] (Table 1).

Although CgA is a reliable sensitive marker for the diagnosis of pheochromocytoma, it has a poor diagnostic specificity [START_REF] Hsiao | Chromogranin A storage and secretion: sensitivity and specificity for the diagnosis of pheochromocytoma[END_REF][START_REF] Bravo | Evolving concepts in the pathophysiology, diagnosis, and treatment of pheochromocytoma[END_REF][START_REF] Bravo | Pheochromocytoma: state-of-the-art and future prospects[END_REF][START_REF] Algeciras-Schimnich | Plasma chromogranin A or urine fractionated metanephrines follow-up testing improves the diagnostic accuracy of plasma fractionated metanephrines for pheochromocytoma[END_REF]. This is in part due to the fact that the kidneys play a major role in the clearance of CgA from the circulation, so that even mild degrees of renal impairment can lead to a significant increase (false positive) in serum concentration of CgA. Canale and Bravo [START_REF] Canale | Diagnostic specificity of serum chromogranin-A for pheochromocytoma in patients with renal dysfunction[END_REF] indicated that in hypertensive patients with creatinine clearance less than 80 ml/min, the specificity of the CgA test assay for the diagnosis of pheochromocytoma was only 50% with a positive predictive value of 38%. However, the authors reported that combining CgA with plasma catecholamines provided a lower sensitivity, but excellent specificity, accuracy, and positive predictive value. In clinical practice, there is another major drawback of CgA measurement, i.e. elevated levels of CgA are common in patients under treatment with proton pump inhibitors, although no neuroendocrine tumor is present [START_REF] Algeciras-Schimnich | Plasma chromogranin A or urine fractionated metanephrines follow-up testing improves the diagnostic accuracy of plasma fractionated metanephrines for pheochromocytoma[END_REF][START_REF] Stridsberg | Measurements of chromogranin A, chromogranin B (secretogranin I), chromogranin C (secretogranin II) and pancreastatin in plasma and urine from patients with carcinoid tumours and endocrine pancreatic tumours[END_REF][START_REF] Sanduleanu | Serum chromogranin A as a screening test for gastric enterochromaffin-like cell hyperplasia during acid-suppressive therapy[END_REF][START_REF] Igaz | Marked chromogranin A elevation in a patient with bilateral adrenal incidentalomas, and its rapid normalization after discontinuation of proton pump inhibitor therapy[END_REF]. It was shown that patients taking proton pump inhibitors alone or in combination with antihypertensive medications had significantly higher CgA levels (mean 635 ng/ml) than subjects without any treatment (mean 174 ng/ml; P b 0.001) [START_REF] Algeciras-Schimnich | Plasma chromogranin A or urine fractionated metanephrines follow-up testing improves the diagnostic accuracy of plasma fractionated metanephrines for pheochromocytoma[END_REF]. Other reasons for non-tumor related increase of CgA are type A gastritis and heart failure [START_REF] Borch | Basal chromogranin A and gastrin concentrations in circulation correlate to endocrine cell proliferation in type-A gastritis[END_REF][START_REF] Spadaro | Serum chromogranin-A in hepatocellular carcinoma: diagnostic utility and limits[END_REF]. Currently, CgA is the only granin whose assay is used routinely by clinicians for the diagnosis of neuroendocrine tumors. However, Stridsberg and collaborators [START_REF] Stridsberg | A comparison between three commercial kits for chromogranin A measurements[END_REF] compared the usefulness of three commercial kits (CGA-RIA CT; CIS bio international, Gif-sur-Yvette cedex, France; DAKO Chromogranin A ELISA kit; DAKO A/S, Glostrup, Denmark; and CgA EuroDiagnostica, Malmö, Sweden) in different types of neuroendocrine tumors including pheochromocytomas and showed that, depending on the kit used, the sensitivity of the CgA test varied between 67 and 93%. Thus, in addition to false positive, plasma measurement of CgA may also produce falsenegative test results in the diagnosis of neuroendocrine tumors, whose rate may depend on the CgA test used.

Several authors [START_REF] Hsiao | Chromogranin A storage and secretion: sensitivity and specificity for the diagnosis of pheochromocytoma[END_REF][START_REF] Hsiao | Chromogranin A in familial pheochromocytoma: diagnostic screening value, prediction of tumor mass, and post-resection kinetics indicating two-compartment distribution[END_REF][START_REF] Giovanella | Serum chromogranin-alpha immunoradiometric assay in the diagnosis of pheochromocytoma[END_REF][START_REF] Herbomez | Chromogranin A assay and (131)I-MIBG scintigraphy for diagnosis and follow-up of pheochromocytoma[END_REF][START_REF] Giovanella | Chromogranin A immunoradiometric assay in diagnosis of pheochromocytoma: comparison with plasma metanephrines and 123I-MIBG scan[END_REF][START_REF] Bilek | Chromogranin A, a member of neuroendocrine secretory proteins as a selective marker for laboratory diagnosis of pheochromocytoma[END_REF] observed a link between circulating CgA concentration and tumor burden, suggesting that CgA could be a good predictor of tumor size and catecholamine secretion. In contrast, low-secreting or non-functioning chromaffin tumors such as head and neck paragangliomas may go undetected when assessed for CgA. The granin can also be used in the follow-up of patients after resection of tumor and as a prognostic marker. Indeed, it was shown that CgA levels fall to normal shortly after removal of the tumor [START_REF] Kimura | Plasma chromogranin A in pheochromocytoma, primary hyperparathyroidism and pituitary adenoma in comparison with catecholamine, parathyroid hormone and pituitary hormones[END_REF][START_REF] Hsiao | Chromogranin A storage and secretion: sensitivity and specificity for the diagnosis of pheochromocytoma[END_REF][START_REF] Hsiao | Chromogranin A in familial pheochromocytoma: diagnostic screening value, prediction of tumor mass, and post-resection kinetics indicating two-compartment distribution[END_REF][START_REF] Herbomez | Chromogranin A assay and (131)I-MIBG scintigraphy for diagnosis and follow-up of pheochromocytoma[END_REF][START_REF] Grossrubatscher | The role of chromogranin A in the management of patients with phaeochromocytoma[END_REF][START_REF] Bilek | Chromogranin A, a member of neuroendocrine secretory proteins as a selective marker for laboratory diagnosis of pheochromocytoma[END_REF][START_REF] Stridsberg | Measurements of chromogranin B can serve as a complement to chromogranin A[END_REF][START_REF] Cotesta | High plasma levels of human chromogranin A and adrenomedullin in patients with pheochromocytoma[END_REF]. Rao and co-workers [START_REF] Rao | Malignant pheochromocytoma. Chromaffin granule transmitters and response to treatment[END_REF] observed that CgA levels were significantly different in benign vs malignant pheochromocytomas. Indeed, plasma CgA rose progressively from control subjects (48.0 ± 3.0 ng/ml) to patients with benign (188 ± 40.5 ng/ml) or malignant tumor (2932 ± 960 ng/ml). However, these results were not confirmed in another study which reported similar CgA levels in patients with benign or malignant neoplasms [START_REF] Grossrubatscher | The role of chromogranin A in the management of patients with phaeochromocytoma[END_REF]. Nevertheless, Bilek et al. [START_REF] Bilek | Chromogranin A, a member of neuroendocrine secretory proteins as a selective marker for laboratory diagnosis of pheochromocytoma[END_REF] found a significant relationship between plasma CgA concentrations and the PASS score [START_REF] Thompson | Pheochromocytoma of the Adrenal gland Scaled Score (PASS) to separate benign from malignant neoplasms: a clinicopathologic and immunophenotypic study of 100 cases[END_REF] rating the malignancy of pheochromocytoma. Moreover, while performed on a low number of tumors, an immunohistochemical study using six CgA region-specific antibodies reported that the peptide expression pattern differed between benign and malignant pheochromocytomas. The benign neoplastic cells exhibited immunoreactivity to all region-specific antibodies, whereas those of the malignant pheochromocytomas expressed immunoreactivity to only one fragment (CgA 176-195 ) [START_REF] Portela-Gomes | Chromogranin A in human neuroendocrine tumors: an immunohistochemical study with region-specific antibodies[END_REF]. Finally, CgA may also be indicative of response to therapy in patients with malignant pheochromocytoma. It was shown that, in 5 responders to chemotherapy, there was a significant decline in CgA levels, whereas in 4 nonresponders CgA concentrations did not change, CgA varying longitudinally with tumor response and relapse [START_REF] Rao | Malignant pheochromocytoma. Chromaffin granule transmitters and response to treatment[END_REF].

WE-14

WE-14 is a 14 amino-acid long peptide derived from the proteolytic processing of CgA (human CgA 324-337 ) and was first isolated from ileal carcinoid tumor and pheochromocytoma tissues [START_REF] Curry | Isolation and primary structure of a novel chromogranin A-derived peptide, WE-14, from a human midgut carcinoid tumour[END_REF][START_REF] Conlon | Isolation of peptides arising from the specific posttranslational processing of chromogranin A and chromogranin B from human pheochromocytoma tissue[END_REF] (Fig. 2). WE-14 is widely distributed in neuroendocrine tissues including rat and human chromaffin cells [START_REF] Gleeson | Occurrence of WE-14 and chromogranin A-derived peptides in tissues of the human and bovine gastroentero-pancreatic system and in human neuroendocrine neoplasia[END_REF]. Moreover, WE-14 immunostaining was also observed in tumoral chromaffin cells [START_REF] Montero-Hadjadje | Localization and characterization of evolutionarily conserved chromogranin A-derived peptides in the rat and human pituitary and adrenal glands[END_REF]. HPLC analysis combined with RIA detection demonstrated that WE-14 was present as a single immunoreactive peak in pheochromocytoma extracts. Measurement of circulating levels of WE-14 revealed significantly higher concentrations in patients with pheochromocytoma compared to healthy volunteers, the median preoperative level of WE-14 being about 4-fold higher [START_REF] Anouar | Development of novel tools for the diagnosis and prognosis of pheochromocytoma using peptide marker immunoassay and gene expression profiling approaches[END_REF]. In vitro, using cultured cells from a benign pheochromocytoma, it was demonstrated that metoclopramide, a gastroprokinetic and antiemetic agent, induced a concentration-dependent increase in WE-14 production with an EC 50 reaching 3.84 × 10 -8 M, and E max of 78 ± 12% [START_REF] Guillemot | Metoclopramide stimulates catecholamine-and granin-derived peptide secretion from pheochromocytoma cells through activation of serotonin type 4 (5-HT4) receptors[END_REF]. Moreover, the WE-14 response to metoclopramide was inhibited by GR 113808 (10 -7 M), a 5-HT4 receptor antagonist. Cisapride, a 5-HT4 receptor agonist, and dopamine were also shown to exert stimulatory effects on WE-14 release from pheochromocytoma cells [START_REF] Guillemot | Metoclopramide stimulates catecholamine-and granin-derived peptide secretion from pheochromocytoma cells through activation of serotonin type 4 (5-HT4) receptors[END_REF]. These observations indicate that the levels of CgA-derived peptides may vary in pheochromocytomas in response to therapeutic interventions.

EL35

EL35 (human CgA 402-436 ) is a well-conserved peptide derived from the C-terminal domain of CgA (Fig. 2). Consistent with WE-14, EL35-like labeling is found in chromaffin cells of the rat and human normal adrenal glands as well as in benign pheochromocytomas [START_REF] Montero-Hadjadje | Localization and characterization of evolutionarily conserved chromogranin A-derived peptides in the rat and human pituitary and adrenal glands[END_REF]. However, HPLC characterization revealed only minute amounts of authentic EL35 in pheochromocytoma extracts while other EL35-immunoreactive forms were observed. The identity of this immunoreactive material was not determined but it should be mentioned that EL35 possesses an internal pair of basic residues which could be cleaved to give rise to shorter peptides. Therefore, the proteolytic processing of CgA to generate this peptide could be altered during tumorigenesis [START_REF] Montero-Hadjadje | Localization and characterization of evolutionarily conserved chromogranin A-derived peptides in the rat and human pituitary and adrenal glands[END_REF].

Pancreastatin

Pancreastatin (PST, human CgA 250-301 ) is a 52 amino-acid peptide located in the internal part of the CgA sequence, showing a low sequence similarity in vertebrate species [START_REF] Montero-Hadjadje | Chromogranins A and B and secretogranin II: evolutionary and functional aspects[END_REF] (Fig. 2). Using an antibody directed against synthetic porcine PST, Bishop et al. [START_REF] Bishop | The occurrence of pancreastatin in tumours of the diffuse neuroendocrine system[END_REF] reported the occurrence of PST-like immunoreactivity in a series of human neuroendocrine tumors. PST levels have been determined through RIA measurement in pheochromocytomas (from 8.0 to 69.0 pmol/g). However, no labeling could be observed by immunocytochemistry in the same study. This discrepancy was ascribed to the fact that the antiserum to PST was raised against the porcine peptide. The development of a specific assay for the measurement of circulating human PST allowed Stridsberg and Husebye [START_REF] Stridsberg | Chromogranin A and chromogranin B are sensitive circulating markers for phaeochromocytoma[END_REF] to investigate the usefulness of this peptide for the diagnosis of patients with pheochromocytoma. They found a low diagnostic sensitivity for this tumor (43%), since plasma PST levels were elevated in only nine out of twenty one patients (Table 1). This finding does not speak for the usefulness of PST as a marker for the diagnosis of pheochromocytoma.

Chromacin

Using antibodies directed against six regions of the CgA molecule including the chromacin peptide (human CgA 176-197 ) (Fig. 2), Portela-Gomes et al. [START_REF] Portela-Gomes | Chromogranin A in human neuroendocrine tumors: an immunohistochemical study with region-specific antibodies[END_REF] described a positive labeling for this CgA region in different neuroendocrine tumors studied including pheochromocytomas. Moreover, semi-quantitative analysis revealed a more intense chromacin-specific labeling in benign than in malignant pheochromocytomas, but this finding was not confirmed by other biochemical analyses.

Chromogranin B and derived peptides in pheochromocytoma

Chromogranin B

CgB is an abundant secretory protein found in a large variety of endocrine cells and neurons [START_REF] Benedum | The primary structure of human secretogranin I (chromogranin B): comparison with chromogranin A reveals homologous terminal domains and a large intervening variable region[END_REF] (Fig. 2). CgB has been described in chromaffin cells of different mammalian species [START_REF] Fischer-Colbrie | Immunological studies on the distribution of chromogranin A and B in endocrine and nervous tissues[END_REF][START_REF] Hagn | Chromogranin A, B, and C in human adrenal medulla and endocrine tissues[END_REF] and in pheochromocytoma extracts [START_REF] Lloyd | Distribution of chromogranin A and secretogranin I (chromogranin B) in neuroendocrine cells and tumors[END_REF]. In benign pheochromocytomas, CgB was consistently detected, whereas in two out of three malignant tumors, the granin protein was absent [START_REF] Weiler | Immunological studies on the occurrence and properties of chromogranin A and B and secretogranin II in endocrine tumors[END_REF]. In pheochromocytomas, while variations in the expression pattern occurred, no significant semiquantitative differences were noted between benign and malignant tumors [START_REF] Portela-Gomes | Expression of chromogranins A, B, and C (secretogranin II) in human adrenal medulla and in benign and malignant pheochromocytomas. An immunohistochemical study with region-specific antibodies[END_REF]. Nevertheless, in all malignant pheochromocytomas, the antibodies raised against the C-terminal region of CgB revealed a noticeable population of immunoreactive large spindle-shaped tumor cells, characterized by elongated processes. Interestingly, this cell type was only observed in one out of 25 benign tumors. Therefore, the authors suggested that the use of antibodies raised against epitopes of the C-terminal region of CgB could facilitate the diagnosis of malignant pheochromocytomas [START_REF] Portela-Gomes | Expression of chromogranins A, B, and C (secretogranin II) in human adrenal medulla and in benign and malignant pheochromocytomas. An immunohistochemical study with region-specific antibodies[END_REF]. Stridsberg and Husebye [START_REF] Stridsberg | Chromogranin A and chromogranin B are sensitive circulating markers for phaeochromocytoma[END_REF] developed a specific assay for the measurement of circulating CgB, in order to investigate the usefulness of this protein for the diagnosis of patients with pheochromocytoma. They found a diagnostic sensitivity of the CgB test assay of 81%, the granin level being elevated in 17 out of 21 patients with pheochromocytoma (Table 1). While the CgA levels fell to normal shortly after the tumor was removed, the CgB levels decreased towards normal over the course of several days [START_REF] Stridsberg | Chromogranin A and chromogranin B are sensitive circulating markers for phaeochromocytoma[END_REF].

GAWK

GAWK (human CgB 420-493 ) is a 74 amino-acid peptide originally isolated from human pituitary gland extracts [START_REF] Benjannet | GAWK, a novel human pituitary polypeptide: isolation, immunocytochemical localization and complete amino acid sequence[END_REF][START_REF] Benjannet | Chromogranin B (secretogranin I), a putative precursor of two novel pituitary peptides through processing at paired basic residues[END_REF] (Fig. 2). Bishop et al. [START_REF] Bishop | The distribution of GAWK-like immunoreactivity in neuroendocrine cells of the human gut, pancreas, adrenal and pituitary glands and its co-localisation with chromogranin B[END_REF] have shown that GAWK-like immunoreactivity is present in chromaffin cells of the human adrenal medulla. Using two different antibodies (directed against the GAWK 1-17 and GAWK 20-38 fragments), a consistent positive immunoreactive staining was seen in all pheochromocytomas studied (n = 6) [START_REF] Sekiya | Production of GAWK (chromogranin-B 420-493)-like immunoreactivity by endocrine tumors and its possible diagnostic value[END_REF]. In contrast, only antibodies against GAWK 20-38 yielded an immunostaining in cells of normal adrenal medulla. RIA quantification revealed that GAWK 20-38 and GAWK 1-17 concentrations were about 15-fold higher in tumoral compared to normal chromaffin cells [START_REF] Sekiya | Production of GAWK (chromogranin-B 420-493)-like immunoreactivity by endocrine tumors and its possible diagnostic value[END_REF]. The GAWK levels were at least 10 times higher in pheochromocytomas than in any other GAWK-producing tumor. While circulating GAWK 1-17 levels were systematically more elevated in pheochromocytoma patients than in healthy volunteers (121 ± 66 pmol/l vs 7.0 ± 1.1 pmol/l), this was not the case for the GAWK 20-38 peptide (114 ± 19 pmol/l vs 74 ± 5.0 pmol/l) [START_REF] Sekiya | Production of GAWK (chromogranin-B 420-493)-like immunoreactivity by endocrine tumors and its possible diagnostic value[END_REF] (Table 1).

Secretogranin II and derived peptides in pheochromocytoma

Secretogranin II

SgII was observed in human adrenomedullary tissue and pheochromocytomas [START_REF] Hagn | Chromogranin A, B, and C in human adrenal medulla and endocrine tissues[END_REF][START_REF] Weiler | Immunological studies on the occurrence and properties of chromogranin A and B and secretogranin II in endocrine tumors[END_REF][START_REF] Schober | Comparison of chromogranins A, B, and secretogranin II in human adrenal medulla and pheochromocytoma[END_REF]. In the tumors, while variations in the staining pattern with different SgII antibodies were observed, no significant semi-quantitative differences were noted between benign and malignant neoplasms [START_REF] Portela-Gomes | Expression of chromogranins A, B, and C (secretogranin II) in human adrenal medulla and in benign and malignant pheochromocytomas. An immunohistochemical study with region-specific antibodies[END_REF]. However, these authors described SgII-immunoreactive large spindle-shaped pheochromocytoma cells preferentially labeled in malignant tumors, as previously observed with CgB labeling. Guillemot et al. [START_REF] Guillemot | Expression and processing of the neuroendocrine protein secretogranin II in benign and malignant pheochromocytomas[END_REF] showed that SgII mRNA was overexpressed in pheochromocytoma compared to non-tumoral chromaffin tissue and in benign compared to malignant tumors. In addition, western blot quantification revealed that the SgII protein was significantly more abundant in benign than in malignant neoplasms. Using region-specific antisera, Stridsberg et al. [START_REF] Stridsberg | Measurements of secretogranins II, III, V and proconvertases 1/3 and 2 in plasma from patients with neuroendocrine tumours[END_REF] showed differential occurrence of SgII-derived fragments in the plasma of pheochromocytoma patients.

Secretoneurin

The SgII-derived peptide SN (human SgII 152-184 ) is a highly conserved 33 amino-acid peptide [START_REF] Wiedermann | Secretoneurin: a functional neuropeptide in health and disease[END_REF][START_REF] Fischer-Colbrie | Secretoneurin: a new player in angiogenesis and chemotaxis linking nerves, blood vessels and the immune system[END_REF] (Fig. 2) that was initially described in the rat and human adrenal medulla [START_REF] Kirchmair | Secretoneurin-a neuropeptide generated in brain, adrenal medulla and other endocrine tissues by proteolytic processing of secretogranin II (chromogranin C)[END_REF][START_REF] Schmid | Immunohistochemical detection of secretoneurin, a novel neuropeptide endoproteolytically processed from secretogranin II, in normal human endocrine and neuronal tissues[END_REF]. It was shown that SgII processing to yield SN was limited in the adrenal medulla and pituitary compared to other organs. Ischia et al. [START_REF] Ischia | Levels and molecular properties of secretoneurin-immunoreactivity in the serum and urine of control and neuroendocrine tumor patients[END_REF] determined the levels of SN in the serum of healthy subjects and patients suffering of pheochromocytoma. They first showed that the mean serum concentration of SN in 42 controls was 22.2 ± 1.1 fmol/ml (range 9.1-37.9). They observed that SN levels were age-dependent with higher concentrations in newborns followed by a continuous decline until 12 years, where adult levels were already reached. It was shown that the contribution of the adrenal gland to serum SN-immunoreactivity was limited since, in patients with unilateral or bilateral adrenalectomy, no significant reduction in serum SN was observed [START_REF] Ischia | Levels and molecular properties of secretoneurin-immunoreactivity in the serum and urine of control and neuroendocrine tumor patients[END_REF]. In patients with pheochromocytoma, serum SN levels were elevated up to 5-fold (99.9 ± 25.6 fmol/ml) with only few patients within the normal range (Table 1). After surgical resection of the tumor, SN values returned to normal (18.3 ± 2.9 fmol/ml) [START_REF] Ischia | Levels and molecular properties of secretoneurin-immunoreactivity in the serum and urine of control and neuroendocrine tumor patients[END_REF]. Similar results were obtained by Stridsberg et al. [START_REF] Stridsberg | Measurements of secretogranins II, III, V and proconvertases 1/3 and 2 in plasma from patients with neuroendocrine tumours[END_REF], although these authors found concentrations of SN 10-fold higher in healthy volunteers with a reference range of 220 fmol/ml and concentrations up to 5400 fmol/ml in patients with pheochromocytoma. These differences could probably be ascribed to the different biological tools and methods used. However, these studies agree on the significant elevation of SN in pheochromocytoma patients.

EM66

EM66 (human SgII 187-252 ) is a 66 amino-acid long peptide that was initially identify by us (Fig. 2) [START_REF] Anouar | Molecular cloning of frog secretogranin II reveals the occurrence of several highly conserved potential regulatory peptides[END_REF][START_REF] Anouar | Identification of a novel secretogranin II-derived peptide (SgII(187-252)) in adult and fetal human adrenal glands using antibodies raised against the human recombinant peptide[END_REF]. We demonstrated that EM66 was produced in chromaffin cells of the adult and fetal human adrenal gland [START_REF] Montero-Hadjadje | Biochemical characterization and immunocytochemical localization of EM66, a novel peptide derived from secretogranin II, in the rat pituitary and adrenal glands[END_REF]. Subsequently, the presence of EM66-immunoreactivity was shown in rat and bovine adrenochromaffin cells [START_REF] Montero-Hadjadje | Biochemical characterization and immunocytochemical localization of EM66, a novel peptide derived from secretogranin II, in the rat pituitary and adrenal glands[END_REF][START_REF] Guillemot | Involvement of multiple signaling pathways in PACAP-induced EM66 secretion from chromaffin cells[END_REF] (Table 1). Immunohistochemical analysis demonstrated the occurrence of authentic EM66 in both benign and malignant pheochromocytomas [START_REF] Yon | Identification of the secretogranin II-derived peptide EM66 in pheochromocytomas as a potential marker for discriminating benign versus malignant tumors[END_REF]. RIA quantification revealed that a low EM66 concentration in pheochromocytoma was associated with a malignant status of this tumor [START_REF] Guillemot | Expression and processing of the neuroendocrine protein secretogranin II in benign and malignant pheochromocytomas[END_REF][START_REF] Yon | Identification of the secretogranin II-derived peptide EM66 in pheochromocytomas as a potential marker for discriminating benign versus malignant tumors[END_REF]. It was also shown that pheochromocytes in primary culture had the ability to release high basal levels of EM66, suggesting that, in vivo, the peptide is secreted from the tumor in the general circulation [START_REF] Guillemot | Circulating EM66 is a highly sensitive marker for the diagnosis and follow-up of pheochromocytoma[END_REF]. In accordance, RIA determination revealed that plasma levels of EM66 were significantly higher in patients with pheochromocytoma compared to healthy volunteers (median 2.6, [1.9-3.7] ng/ml vs 26.9 [7.3-44] ng/ml, respectively) and returned to normal values after removal of the tumor [START_REF] Guillemot | Circulating EM66 is a highly sensitive marker for the diagnosis and follow-up of pheochromocytoma[END_REF]. In contrast, urinary EM66 levels in patients with pheochromocytoma were not significantly different from those of controls. Stridsberg et al. [START_REF] Stridsberg | Measurements of secretogranins II, III, V and proconvertases 1/3 and 2 in plasma from patients with neuroendocrine tumours[END_REF] found no change of EM66-like concentrations in patients with pheochromocytoma (see Section 4.1). This discrepancy may be ascribed to the antiserum used recognizing SgII 225-242 i.e. C-terminal part of EM66, and/or to the low number of cases studied (n = 3). As observed for the WE-14 peptide (see Section 2.2), metoclopramide, cisapride and dopamine dose-dependently stimulated EM66 release from cultured pheochromocytoma cells [START_REF] Guillemot | Metoclopramide stimulates catecholamine-and granin-derived peptide secretion from pheochromocytoma cells through activation of serotonin type 4 (5-HT4) receptors[END_REF], again indicating that the levels of granin peptides are prone to variation under therapeutic treatment, at least with drugs targeting aminergic systems.

Other granins and their derived peptides in pheochromocytoma

While less studied, secretogranins III-VII and proSAAS also belong to the granin family and are characterized by numerous pairs of basic amino acids which may serve as potential cleavage sites for processing by the co-stored prohormone converting enzymes PC1/3 and PC2 in secretory granules [START_REF] Helle | The granin family of uniquely acidic proteins of the diffuse neuroendocrine system: comparative and functional aspects[END_REF] (Fig. 2). However, their presence and their use as circulating markers for pheochromocytomas have not been extensively explored (Table 1).

SgIII (or 1B1075)

SgIII is distinct from other granins as it is not found either in the adrenal gland or in pheochromocytomas [START_REF] Stridsberg | Measurements of secretogranins II, III, V and proconvertases 1/3 and 2 in plasma from patients with neuroendocrine tumours[END_REF].

SgIV (or HISL-19)

SgIV is a protein expressed by neuronal and peptide hormone-producing cells which shares many biochemical and molecular key features with the granin family [START_REF] Krisch | Monoclonal antibody HISL-19 as an immunocytochemical probe for neuroendocrine differentiation. Its application in diagnostic pathology[END_REF]. Few studies reported the presence of SgIV in human adrenal medulla and in pheochromocytoma [START_REF] Krisch | Monoclonal antibody HISL-19 as an immunocytochemical probe for neuroendocrine differentiation. Its application in diagnostic pathology[END_REF][START_REF] Buffa | Identification of the endocrine cells detected by the monoclonal antibody HISL-19 in human tissues[END_REF][START_REF] Shimizu | Immunohistochemical, biochemical and immunoelectron microscopic analysis of antigenic proteins on neuroendocrine cell tumors using monoclonal antibody HISL-19[END_REF]. Immunoelectron microscopic analysis revealed SgIV immunoreactivity in secretory granules, which was apparently more pronounced in malignant compared with benign pheochromocytomas [START_REF] Shimizu | Immunohistochemical, biochemical and immunoelectron microscopic analysis of antigenic proteins on neuroendocrine cell tumors using monoclonal antibody HISL-19[END_REF]. No data are available concerning circulating SgIV in pheochromocytoma patients (Table 1).

SgV (or 7B2)

7B2 is an acidic protein contained in most neuroendocrine cells. The presence of 7B2 was demonstrated in chromaffin tissue of the normal adrenal gland [START_REF] Iguchi | Presence of the novel pituitary protein "7B2" in bovine chromaffin granules: possible co-release of 7B2 and catecholamine as induced by nicotine[END_REF][START_REF] Brayton | Cloning, characterization, and sequence of a porcine cDNA encoding a secreted neuronal and endocrine protein[END_REF][START_REF] Marcinkiewicz | Identification and localization of 7B2 protein in human, porcine, and rat thyroid gland and in human medullary carcinoma[END_REF][START_REF] Hacker | Multiple peptide production and presence of general neuroendocrine markers detected in 12 cases of human phaeochromocytoma and in mammalian adrenal glands[END_REF] and also in pheochromocytoma [START_REF] Hacker | Multiple peptide production and presence of general neuroendocrine markers detected in 12 cases of human phaeochromocytoma and in mammalian adrenal glands[END_REF][START_REF] Vieau | Expression of the neuroendocrine cell marker 7B2 in human ACTH secreting tumours[END_REF] (Table 1). In human, Natori and co-workers [START_REF] Natori | Plasma 7B2 (a novel pituitary protein) immunoreactivity concentrations in patients with various endocrine disorders[END_REF] determined plasma 7B2 levels in healthy volunteers and in patients with pheochromocytoma. They measured significantly higher plasma 7B2 concentrations in patients with pheochromocytoma than those in age-matched normal subjects (221± 82.8 ng/l vs 55.8 ± 1.2 ng/l).

SgVI (or NESP55)

NESP55 (neuroendocrine secretory protein of MW 55,000) is a soluble, acidic, heat-stable secretory protein [START_REF] Fischer-Colbrie | Neuroendocrine secretory protein 55: a novel marker for the constitutive secretory pathway[END_REF] (Fig. 2) which was described in the rat and bovine adrenal medulla with a preferential localization in adrenaline-synthesizing cells [START_REF] Ischia | Molecular cloning and characterization of NESP55, a novel chromogranin-like precursor of a peptide with 5-HT1B receptor antagonist activity[END_REF][START_REF] Bauer | The new chromogranin-like protein NESP55 is preferentially localized in adrenalinesynthesizing cells of the bovine and rat adrenal medulla[END_REF]. NESP55 immunoreactivity was also shown in pheochromocytomas, with the same strong labeling pattern in both benign and malignant tumors [START_REF] Jakobsen | NESP55, a novel chromogranin-like peptide, is expressed in endocrine tumours of the pancreas and adrenal medulla but not in ileal carcinoids[END_REF][START_REF] Nilsson | Importance of vesicle proteins in the diagnosis and treatment of neuroendocrine tumors[END_REF][START_REF] Srivastava | Neuroendocrine secretory protein-55 (NESP-55) expression discriminates pancreatic endocrine tumors and pheochromocytomas from gastrointestinal and pulmonary carcinoids[END_REF] (Table 1).

SgVII (or VGF) and proSAAS

SgVII/VGF and proSAAS represent the most recently characterized members of the granin family (Fig. 2). VGF was originally identified as a neurotrophin-regulated gene product in PC12 cells [START_REF] Levi | Molecular cloning of a gene sequence regulated by nerve growth factor[END_REF]. ProSAAS was identified as the precursor of several peptides that accumulate in the obese, carboxypeptidase E-deficient Cpefat/fat mouse, and was shown to inhibit prohormone convertase 1 activity [START_REF] Fricker | Identification and characterization of proSAAS, a granin-like neuroendocrine peptide precursor that inhibits prohormone processing[END_REF]. VGF and proSAAS are stored in and released from secretory vesicles and are expressed widely in nervous, endocrine, and neuroendocrine tissues [START_REF] Fricker | Identification and characterization of proSAAS, a granin-like neuroendocrine peptide precursor that inhibits prohormone processing[END_REF][START_REF] Mzhavia | Processing of proSAAS in neuroendocrine cell lines[END_REF][START_REF] Chakraborty | Quantification of VGF-and pro-SAAS-derived peptides in endocrine tissues and the brain, and their regulation by diet and cold stress[END_REF]. Variable proVGF-immunoreactive fragments were observed in human adult adrenal medulla and in pheochromocytomas [START_REF] Rindi | Peptide products of the neurotrophin-inducible gene vgf are produced in human neuroendocrine cells from early development and increase in hyperplasia and neoplasia[END_REF]. ProSAAS mRNA is primarily expressed in the brain but also occurs in endocrine organs and cells including the adrenal gland and the rat pheochromocytoma PC12 cell line [START_REF] Fricker | Identification and characterization of proSAAS, a granin-like neuroendocrine peptide precursor that inhibits prohormone processing[END_REF] (Table 1). Two proSAAS-derived peptides (SAAS and LEN) were detected in the rat adrenal medulla [START_REF] Feng | Distribution of proSAAS-derived peptides in rat neuroendocrine tissues[END_REF] (Fig. 2).

Concluding remarks

Malignant pheochromocytomas are orphan diseases for which there exist a difficulty for the management and a disparity in clinical practices. To date, the universally admitted criterion of malignancy for a pheochromocytoma is the occurrence of metastasis [110,[START_REF] Pacak | An assessment of biochemical tests for the diagnosis of pheochromocytoma[END_REF]. Therefore, numerous efforts have been undertaken to discover reliable tools for the early diagnosis and prognosis of malignant pheochromocytomas. While pathological analysis utilizing multiple histological criteria appears to hold some value for distinguishing benign from malignant disease, these evaluations are not practiced routinely as predictors of malignancy [START_REF] Thompson | Pheochromocytoma of the Adrenal gland Scaled Score (PASS) to separate benign from malignant neoplasms: a clinicopathologic and immunophenotypic study of 100 cases[END_REF][START_REF] Gao | Development and validation of pheochromocytoma of the adrenal gland scaled score for predicting malignant pheochromocytomas[END_REF][START_REF] Strong | Prognostic indicators of malignancy in adrenal pheochromocytomas: clinical, histopathologic, and cell cycle/apoptosis gene expression analysis[END_REF]. Among pheochromocytoma susceptibility genes, it is now established that SDHB mutation is associated with the highest risk of malignancy [START_REF] Brouwers | Gene expression profiling of benign and malignant pheochromocytoma[END_REF]. It has also been shown that malignant pheochromocytomas are characterized by an enhanced production of noradrenaline and that extra-adrenal (paragangliomas) tumors secrete dopamine [START_REF] Eisenhofer | Biochemical and clinical manifestations of dopamine-producing paragangliomas: utility of plasma methoxytyramine[END_REF]. A global and promising approach of gene expression profiling of benign and malignant pheochromocytomas, has allowed to identify a set of genes that could discriminate the two types of tumors [START_REF] Brouwers | Gene expression profiling of benign and malignant pheochromocytoma[END_REF][START_REF] Thouënnon | Genetic markers for the diagnosis and prognosis of pheochromocytoma[END_REF]. As indicated in the present review, except SgIII, all members of the granin family and several of their derived peptides have been shown to be present in normal chromaffin cell and in pheochromocytomas (Table 1). However, to date, CgA is the only granin currently used for the diagnosis of chromaffin cell tumors; a positive CgA immunostaining indicating the neuroendocrine nature of the neoplasm in general, without specificity for pheochromocytoma. Currently, besides CgA, other biochemical tests used for the diagnosis of pheochromocytoma are the measurement of plasma and urinary catecholamine metabolites (metanephrine and normetanephrine). While they offer a high sensitivity, these tests also have limitations. To date, a biochemical test alone is obviously not sufficient for the screening of pheochromocytoma. False-negative and false-positive results using biological tests remain a problem, leading to cost-effective and time-consuming additional imaging examinations [117,[START_REF] Sawka | The economic implications of three biochemical screening algorithms for pheochromocytoma[END_REF]. It is therefore tempting to propose that the measurement of CgA and metanephrine levels should be combined with the determination of other granins or granin-derived peptides for the diagnosis/prognosis of pheochromocytoma.

CgA levels are correlated with tumor mass since malignant pheochromocytomas which are often more bulky than benign ones display higher CgA concentrations [START_REF] Herbomez | An analysis of the biochemical diagnosis of 66 pheochromocytomas[END_REF]. Thus, CgA may serve as a prognostic marker in certain conditions but there are several restrictions such as the existence of malignant chromaffin tumors of the head and neck. Other drawbacks of CgA limit the clinical use of the protein (falsenegative or false-positive test results) and therefore, there is a need to identify other granin or granin-derived peptides for the diagnosis and prognosis of pheochromocytomas. In this respect, CgB did not seem to have a better diagnostic sensitivity than CgA but was shown to be a complementary tool for the management of neuroendocrine tumors, since its concentrations were not elevated in patients with renal failure or under treatment with proton pump inhibitors [START_REF] Stridsberg | Measurements of chromogranin B can serve as a complement to chromogranin A[END_REF]. It was also postulated by the group of Stridsberg and Portela-Gomes that the use of antibodies raised against epitopes of the C-terminal region of CgB could facilitate the diagnosis of malignant pheochromocytoma [START_REF] Portela-Gomes | Expression of chromogranins A, B, and C (secretogranin II) in human adrenal medulla and in benign and malignant pheochromocytomas. An immunohistochemical study with region-specific antibodies[END_REF]. Our data showed that SgII mRNAs were down-regulated in malignant compared to benign pheochromocytomas and that malignancy was associated with reduced levels of SgII and SgII-derived peptides, in accordance with the low concentrations of EM66 found in malignant tumors [START_REF] Guillemot | Expression and processing of the neuroendocrine protein secretogranin II in benign and malignant pheochromocytomas[END_REF][START_REF] Yon | Identification of the secretogranin II-derived peptide EM66 in pheochromocytomas as a potential marker for discriminating benign versus malignant tumors[END_REF]. Taken together, these observations strengthen the notion that granins other than CgA and their derived peptides may provide new diagnostic and prognostic markers of pheochromocytoma.

An important issue raised by A. Tischler, is to define more precisely the term "malignant" concerning pheochromocytoma/paraganglioma since the potential lethality of extensive local invasion is ignored by a definition of malignancy based only on metastasis [START_REF] Tischler | Pheochromocytoma and extra-adrenal paraganglioma[END_REF]. He suggested that pheochromocytoma and extra-adrenal paraganglioma could be classified as "non-invasive", "minimally invasive" and "extensively invasive", in order to stratify the risk of metastasis and aggressiveness for a better management of patient treatment [START_REF] Tischler | Pheochromocytoma and extra-adrenal paraganglioma[END_REF]. In this regard, the development of additional markers derived from granins and their peptides used solely or in combination, i.e. through array of different peptide-recognizing antibodies, may provide a mean to accurately discriminate different pheochromocytoma subtypes. However, a precise description of the relative affinity of each antibody raised against a granin-derived peptide towards the peptide itself and its precursor will be needed because this important information is not always available. Other challenges to the diagnosis and prognosis of pheochromocytoma will be to detect tumors that do not contain or produce excess catecholamines, metanephrines or CgA. Concerning the prognostic value of histopathology of pheochromocytoma tumors, to date there is no consensus on the reliability of the PASS score [START_REF] Thompson | Pheochromocytoma of the Adrenal gland Scaled Score (PASS) to separate benign from malignant neoplasms: a clinicopathologic and immunophenotypic study of 100 cases[END_REF]. Further studies should combine histological (PASS), immunohistochemical (Ki67) and biochemical (adrenergic/noradrenergic, granins) features to predict metastatic potential of tumors [START_REF] Strong | Prognostic indicators of malignancy in adrenal pheochromocytomas: clinical, histopathologic, and cell cycle/apoptosis gene expression analysis[END_REF][START_REF] Kimura | Histological grading of adrenal and extra-adrenal pheochromocytomas and relationship to prognosis: a clinicopathological analysis of 116 adrenal pheochromocytomas and 30 extraadrenal sympathetic paragangliomas including 38 malignant tumors[END_REF][START_REF] Bílek | Chromogranin A, a member of neuroendocrine secretory proteins as a selective marker for laboratory diagnosis of pheochromocytoma[END_REF]. However, it should be kept in mind that these different markers would carry different weights depending on the adrenal or extra-adrenal localization and the genetic background of the tumor [START_REF] Tischler | Pheochromocytoma and extra-adrenal paraganglioma[END_REF]. In addition, chromaffin tumors are rare, heterogeneous, and further work aimed at identifying new predictive factors of malignancy should be performed with large and well characterized cohorts of patients. Finally, in vitro experiments, performed on tumor cell lines, suggest that CgA may affect tissue remodeling and tumor growth [START_REF] Colombo | Chromogranin A expression in neoplastic cells affects tumor growth and morphogenesis in mouse models[END_REF]. Therefore, in addition to their potential use as predictors for the diagnosis and prognosis, granin-derived peptides could also be considered for use in therapeutic strategies for the treatment of pheochromocytoma and other neuroendocrine tumors.

Fig. 2 .

 2 Fig. 2. Schematic representation of human granins and their derived peptides. Only granins and their processing products mentioned in the present review because of their presence in normal and/or tumoral chromaffin cells, or in patients with pheochromocytoma, are indicated. The black boxes correspond to signal peptides, full arrows indicate multibasic cleavage sites and dotted arrows indicate other cleavage sites. CgA, chromogranin A; CgB, chromogranin B; SgII-VII, secretogranin II-VII; K, lysine residue; R, arginine residue. The number of amino acids contained in the signal peptides, granins and derived peptides is indicated. No data are available for the sequence of the SgIV (HISL-19) protein.

Table 1

 1 Detection of granins and derived peptides in normal and tumoral chromaffin cells, and in circulation. Indication of the test assay sensitivity for the diagnosis of pheochromocytoma.

					Distribution		Test assay sensitivity**
			Normal chromaffin cells	Tumoral chromaffin cells	Circulation
					(human pheochromocytoma)
	CgA	CgA	+	h, p, b, m, r	+	+	71%-100%*
		WE-14	+	h, r	+	+	84%(n = 25)[66]
		EL35	+	h, r	+	ND	ND
		PST	+	h, pg	+	+	43%(n = 21)[40]
		chromacin	+	b	+	ND	ND
	CgB	CgB	+	h, b, r	+	+	81%(n = 21)[40]
		GAWK	+	h, b	+	+	56%GAWK 1-17 (n = 9)[77]
							33%GAWK 20-38 (n = 12)
	SgII	SgII	+	h, p, b, d, m, r	+	+	ND
		SN	+	h, b, r	+	+	78%(n = 9)[87]
		EM66	+	h, b, r	+	+	92%(n = 12)[66]
	SgIII (1B1075)		-		-	+	N D
	SgIV (HISL-19)		+	h	ND	+	ND
	SgV (7B2)		+	h, b, r	+	+	ND
	SgVI (NESP55)		+	h,b, r	+	ND	ND
	SgVII (VGF)		+	h, m, r	+	ND	ND
	ProSAAS	proSAAS	+	h, m, r	PC12 cells	ND	ND
		SAAS	+	r	ND	ND	ND
		LEN	+	r	ND	ND	ND

A plus sign indicates the presence of the protein, granin-derived peptide and/or messenger RNA; a minus sign indicates absence or undetectable levels. b, bovine; CgA, chromogranin A; CgB, chromogranin B; d, dog; h, human; m, mouse; ND, not determined; p, primate; pg, pig; PST, pancreastatin; r, rat; SgII-VII, secretogranin II-VII; SN, secretoneurin. *The CgA sensitivity test assay for the diagnosis of pheochromocytoma differs according to the following studies: 71% (n = 45)

[START_REF] Boomsma | Sensitivity and specificity of a new ELISA method for determination of chromogranin A in the diagnosis of pheochromocytoma and neuroblastoma[END_REF]

; 83% (n = 29)

[START_REF] Hsiao | Chromogranin A storage and secretion: sensitivity and specificity for the diagnosis of pheochromocytoma[END_REF]

; 86% (n = 50)

[START_REF] Canale | Diagnostic specificity of serum chromogranin-A for pheochromocytoma in patients with renal dysfunction[END_REF]

; 89% (n = 9)

[START_REF] Nobels | Chromogranin A as serum marker for neuroendocrine neoplasia: comparison with neuron-specific enolase and the alpha-subunit of glycoprotein hormones[END_REF]

; 90% (n = 21)

[START_REF] Stridsberg | Chromogranin A and chromogranin B are sensitive circulating markers for phaeochromocytoma[END_REF]

; 91% (n = 22)

[START_REF] Grossrubatscher | The role of chromogranin A in the management of patients with phaeochromocytoma[END_REF]

; 95% (n = 44)

[START_REF] Giovanella | Chromogranin A immunoradiometric assay in diagnosis of pheochromocytoma: comparison with plasma metanephrines and 123I-MIBG scan[END_REF]

; 100% (n = 15)

[START_REF] Giovanella | Serum chromogranin-alpha immunoradiometric assay in the diagnosis of pheochromocytoma[END_REF]

. **Sensitivity: refers to the probability for a (diagnostic) test to be positive in a population of individuals with the target disorder (tumor, disease…) = true-positive / true-positive + false-negative. Specificity: refers to the probability for a (diagnostic) test to be negative in a population of individuals without the target disorder = true-negative / true-negative + false-positive. False-negative: individuals with the target disorder and negative for the test; False-positive: individuals without the target disorder and positive for the test; True-negative: individuals without the target disorder and negative for the test; True-positive: individuals with the target disorder and positive for the test.