Circulating EM66 is a highly sensitive marker for the diagnosis and follow-up of pheochromocytoma

Johann Guillemot (1, 2) , Youssef Anouar (1, 2) , Maïté Montero-Hadjadje (1, 2) , Eric Grouzmann (3) , Luca Grumolato (1, 2) , Joana Roshmaninho-Salgado (4) , Val Erie Turquier (1, 2) , Céline Duparc (1, 2) , Hervé Lefebvre (1, 2) , Pierre-François Plouin (5) , Marc Klein (6) , Mihaela Muresan (6) , Billy K C Chow (7) , Hubert Vaudry (1, 2) , Laurent Yon (1, 2)

1 Neuroendocrinologie cellulaire et moléculaire
2 IFRMP 23 - Institut Fédératif de Recherches Multidisciplinaires sur les Peptides
3 CHUV - Centre Hospitalier Universitaire Vaudois = Lausanne University Hospital [Lausanne]
4 Universidade de Coimbra [Coimbra]
5 HEGP - Hôpital Européen Georges Pompidou [APHP]
6 CHU Nancy - Centre Hospitalier Universitaire de Nancy
7 HKU - The University of Hong Kong

We have previously demonstrated that measurement of tissue concentration of the novel secretogranin II-derived peptide EM66 may help to discriminate between benign and malignant pheo-chromocytomas. The aim of the present study was to characterize EM66 in plasma and urine of healthy volunteers and pheochromo-cytoma patients, in order to further evaluate the usefulness of this peptide as a circulating marker for the management of the tumors. HPLC analysis of plasma and urine samples demonstrated that the EM66-immunoreactive material coeluted with the recombinant peptide. In healthy volunteers, plasma and urinary EM66 levels were, respectively, 2.6 (1.9–3.7) ng/ml and 2.9 (1.9– 4.6) ng/ml. In patients with pheochromocytoma, plasma EM66 levels were 10-fold higher than those of healthy volunteers (26.9 (7.3– 44) ng/ml), and returned to normal values after removal of the tumor. In contrast, urinary EM66 levels were not significantly different from those of healthy volunteers (3.2 (2.2–3.9) ng/ml). Measurement of total or free plasma metanephrines and 24 hr uri-nary metanephrines in our series of patients revealed that these tests, taken separately, are less sensitive than the EM66 determination. Pheochromocytes in primary culture secreted high levels of EM66, suggesting that the chromaffin tumor was actually responsible for the increased plasma peptide concentrations in the patients. These data indicate that EM66 is secreted in the general circulation and that elevated plasma EM66 levels are correlated with the occurrence of pheochromocytoma. Thus, EM66 is a sensitive plasma marker that should be considered as a complementary tool in the management of pheochromocytoma. ' 2005 Wiley-Liss, Inc. Chromogranins (Cgs) constitute a family of acidic glycopro-teins encompassing chromogranin A (CgA), chromogranin B (CgB) and secretogranin II (SgII) that are widely distributed in neuroendocrine tissues where they are packaged in secretory granules. 1 The physiological role of these proteins is unclear but a number of potential biological functions have been postulated, including regulation of secretory granule formation and production of bioactive peptides through proteolytic processing. 2 It has been demonstrated that Cgs and Cgs-derived peptides are secreted together with hormones and neurotransmitters and can be detected in blood by radioimmuno assay (RIA) techniques. 3–5 This observation combined with their ubiquitous distribution in neuroendo-crine, endocrine and nervous tissues makes Cgs useful markers of secretion of normal and tumoral neuroendocrine cells. In particular , measurement of CgA levels in plasma can be used to diagnose or monitor the progression of neuroendocrine tumors. 6 The highest accuracy has been observed in tumors characterized by an intense secretory activity, although the specificity and sensitivity remain high also in nonfunctioning tumors. 7 However, CgA levels may also be elevated in patients with hyperplasia 3 and may therefore not be reliable for distinguishing neuroendocrine hyperplasia from adenoma or carcinoma. In addition, CgA measurement showed a low sensitivity in certain neuroendocrine tumors such as insulino-mas, pituitary adenomas and medullary thyroid carcinomas. 8,9 Thus, measurement of other Cgs or Cgs-derived peptides may be helpful for the diagnosis of different neuroendocrine tumors. It has been reported that plasma levels of GAWK and CCB, 2 CgB-derived peptides, are elevated in patients with pancreatic islet-cell tumors 10,11 or with bronchial tumors. 12 Similarly, enhanced plasma concentrations of the SgII-derived peptide secre-toneurin (SN) are associated with various endocrine tumors such as gastroenteropancreatic endocrine tumors or oat cell lung carci-nomas, and are also related to the progression of prostatic carcino-mas. 5,13 SgII is the precursor of 2 highly conserved peptides, i.e. SN and a 66-amino-acid C-terminal flanking peptide termed EM66. 14 Using specific polyclonal antibodies directed against recombinant EM66, we have previously shown the presence of EM66 in the rat pituitary and adrenal glands, 15 and in chromaffin cells of fetal and adult human adrenals. 16 We have recently demonstrated the occurrence of EM66 in human pheochromocytomas and found that low tissue concentrations of the peptide are associated with malignant differentiation of the tumor while benign pheochromocytomas contain significantly higher quantities of EM66, indicating that EM66 is a potential prognostic marker of chromaffin cell tumors. 17 Pheochromocytomas are usually benign but ~20% of these tumors are malignant, and to date the only evidence for malignancy is the occurrence of metastases, which are associated with a very poor survival rate. 18–20 The characterization of novel circulating markers is therefore crucial for the management of these tumors. As a further step in the evaluation of EM66 as a marker for the diagnosis and prognosis of pheochromocytoma, the aim of the present study was to characterize this peptide in plasma and urine, and to compare its concentrations between healthy volunteers and patients with pheochromocytoma. The ability of pheochromocytes in primary culture to release EM66 was also investigated.

Mots clés: secretogranin II; neuroendocrine tumor; chromogranins; plasma marker; EM66; pheochromo- cytoma

Domaines: Sciences du Vivant [q-bio]

Johann Guillemot, Youssef Anouar, Maïté Montero-Hadjadje, Eric Grouzmann, Luca Grumolato, et al.. Circulating EM66 is a highly sensitive marker for the diagnosis and follow-up of pheochromocytoma. International Journal of Cancer, 2006, 118 (8), pp.2003 - 2012. ⟨10.1002/ijc.21571⟩. ⟨hal-01706431⟩