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Objective: Pheochromocytomas (PCCs) are neuroendocrine tumors that occur in the adrenal medulla, whereas paragangliomas (PGLs) arise from paraganglia in the head, neck, thorax, or abdomen. In a variety of tumors, cancer cells with stem cell-like properties seem to form the basis of tumor initiation because of their ability to self-renew and proliferate. Specifically targeting this small cell population may lay the foundation for more effective therapeutic approaches. In the present study, we intended to identify stem cells in PCCs/PGLs., Design: We examined the immunohistochemical expression of 11 stem cell markers (SOX2, LIN28, NGFR, THY1, PREF1, SOX17, NESTIN, CD117, OCT3/4, NANOG, and CD133) on tissue microarrays containing 208 PCCs/PGLs with different genetic backgrounds from five European centers., Results: SOX2, LIN28, NGFR, and THY1 were expressed in more than 10% of tumors, and PREF1, SOX17, NESTIN, and CD117 were expressed in <10% of the samples. OCT3/4, NANOG, and CD133 were not detectable at all. Double staining for chromogranin A/SOX2 and S100/SOX2 demonstrated SOX2 immunopositivity in both tumor and adjacent sustentacular cells. The expression of SOX2, SOX17, NGFR, LIN28, PREF1, and THY1 was significantly associated with mutations in one of the succinate dehydrogenase (SDH) genes. In addition, NGFR expression was significantly correlated with metastatic disease., Conclusion: Immunohistochemical expression of stem cell markers was found in a subset of PCCs/PGLs. Further studies are required to validate whether some stem cell-associated markers, such as SOX2, could serve as targets for therapeutic approaches and whether NGFR expression could be utilized as a predictor of malignancy., (© 2015 European Society of Endocrinology.)

Background: Both the European Neuroendocrine Tumor Society (ENETS) and the International Union for Cancer Control/American Joint Cancer Committee/World Health Organization (UICC/AJCC/WHO) have proposed TNM staging systems for pancreatic neuroendocrine neoplasms. This study aims to identify the most accurate and useful TNM system for pancreatic neuroendocrine neoplasms., Methods: The study included 1072 patients who had undergone previous surgery for their cancer and for which at least 2 years of follow-up from 1990 to 2007 was available. Data on 28 variables were collected, and the performance of the two TNM staging systems was compared by Cox regression analysis and multivariable analyses. All statistical tests were two-sided., Results: Differences in distribution of sex and age were observed for the ENETS TNM staging system. At Cox regression analysis, only the ENETS TNM staging system perfectly allocated patients into four statistically significantly different and equally populated risk groups (with stage I as the reference; stage II hazard ratio [HR] of death = 16.23, 95% confidence interval [CI] = 2.14 to 123, P = .007; stage III HR of death = 51.81, 95% CI = 7.11 to 377, P < .001; and stage IV HR of death = 160, 95% CI = 22.30 to 1143, P < .001). However, the UICC/AJCC/WHO 2010 TNM staging system compressed the disease into three differently populated classes, with most patients in stage I, and with the patients being equally distributed into stages II-III (statistically similar) and IV (with stage I as the reference; stage II HR of death = 9.57, 95% CI = 4.62 to 19.88, P < .001; stage III HR of death = 9.32, 95% CI = 3.69 to 23.53, P = .94; and stage IV HR of death = 30.84, 95% CI = 15.62 to 60.87, P < .001). Multivariable modeling indicated curative surgery, TNM staging, and grading were effective predictors of death, and grading was the second most effective independent predictor of survival in the absence of staging information. Though both TNM staging systems were independent predictors of survival, the UICC/AJCC/WHO 2010 TNM stages showed very large 95% confidence intervals for each stage, indicating an inaccurate predictive ability., Conclusion: Our data suggest the ENETS TNM staging system is superior to the UICC/AJCC/WHO 2010 TNM staging system and supports its use in clinical practice.

Objective: Aberrant adrenal expression of various hormone receptors has been identified in ACTH-independent macronodular adrenal hyperplasia (AIMAH) causing cortisol hypersecretion regulated by hormones other than ACTH. We aimed to determine aberrant expression of multiple hormone receptors in vivo and in vitro in adrenal tissue of a patient with AIMAH., Design: The design of the study includes clinical case description, and biochemical and immunohistochemical analysis to demonstrate aberrant expression of multiple hormone receptors in AIMAH., Methods: The subject of the study is a male diagnosed with Cushing's syndrome because of AIMAH. Directly after laparoscopic removal of the adrenals, adrenal tissue was incubated with and without test substances (ACTH, forskolin, arginine vasopressin (AVP), desmopressin, epinephrine, norepinephrine, purified human chorionic gonadotropin (hCG), metoclopramide and the combinations of AVP with ACTH, epinephrine and metoclopramide). LH/hCG-receptor (hCG-R) immunohistochemistry and RT-PCR analyses were performed to demonstrate aberrant expression of LH/hCG-R and V(1-3)-AVPR., Results: AIMAH was characterized by in vivo cortisol responsiveness to AVP and in vitro cortisol responses to AVP, hCG, epinephrine, and norepinephrine suggesting aberrant adrenal expression of the receptors for AVP (the V(1-3)-AVPRs), catecholamines (the beta-AR), and LH (the LH/hCG-R). Incubation with combinations of AVP and ACTH and of AVP with epinephrine induced a stronger cortisol response compared with incubation with the individual agents. Moreover, we demonstrated adrenal V(1-3)-AVPR and LH/hCG-R expression., Conclusions: AIMAH tissue may simultaneously express multiple aberrant hormone receptors, and individual ligands may potentiate each other regarding cell proliferation and cortisol production.

Context: A 56-yr-old woman presented with acromegaly, a pulmonary mass, and elevated levels of GHRH, GH, and IGF-I. Histological examination revealed a bronchial carcinoid with positive staining for GHRH. Somatostatin analogs (SAs) can play an important role in the treatment of neuroendocrine tumors, dependent on the somatostatin receptor subtype (sst) expression pattern. The sst pattern in bronchial carcinoids and effects of SAs have not been extensively investigated, particularly not for the recently developed universal SA SOM230 (Pasireotide) that has high affinity for sst1, 2, 3, and 5., Objective: Our objective was to investigate the in vivo response of a GHRH-producing bronchial carcinoid to octreotide (OCT), its sst-expression profile, and in vitro responses to different SAs, including SOM230., Methods: In vivo, 50 microg OCT was administered, and plasma GH and GHRH responses were determined. In vitro, the expression of ssts was analyzed by quantitative PCR. Furthermore, the effects of SOM230 and OCT on GHRH secretion were evaluated in primary cell cultures of the carcinoid tissue., Results: In vivo, OCT administration fully suppressed GH and GHRH levels. In vitro, sst1 mRNA was most abundant, followed by sst2 and sst5. Both SOM230 and OCT inhibited GHRH production dose dependently (SOM230 100 nm vs. control, P = 0.01; OCT 110 nm vs. control, P = 0.05)., Conclusions: In this case of a GHRH-producing bronchial carcinoid, we demonstrated that SOM230 was a potent inhibitor of GHRH production in vitro and was at least equally potent compared with OCT. Therefore, SOM230 may be a potential therapeutic agent to control GHRH secretion in ectopic acromegaly.

Objective: Phaeochromocytomas (PCCs) are widely known for their clinical unpredictability. This study intends to define predictive plasma markers for their variable postoperative behaviour. Furthermore, the diagnostic accuracy of these plasma tests was determined., Design and Methods: A retrospective correlative study was performed in a series of 83 operated and four autopsied patients in order to correlate preoperative catecholamine (CAT) levels of 103 PCCs with their clinical behaviour. In a subset of cases, chromogranin-A (Chr-A) and enzymes/precursors of the CAT biosynthesis were studied for their predictive value., Results: Basal CAT levels were elevated in 81/87 instances (sensitivity: 93%). Four of six cases with normal measurements showed only medullary hyperplasia. Larger PCCs, particularly those showing necrosis, capsular and vascular invasion, secreted higher CAT levels. Bilateral, hereditary tumours were less productive than their unilateral counterparts. Extra-adrenal PCCs secreted significantly lower levels of epinephrine (EPI) than intra-adrenal tumours. Fourteen patients developed metastases. According to Kaplan-Meier estimations, patients with higher levels of dopamine, norepinephrine (NE) and aromatic l-amino acid decarboxylase as well as lower ratios of EPI/EPI+NE, had significantly shorter metastases-free intervals. Existence of preoperative hypertension, left ventricular hypertrophy and measured blood pressures showed significant positive relationships with CAT levels, but not with Chr-A., Conclusions: These data showed that plasma CAT measurement is a sensitive method in the diagnostic work-up of PCCs. Those tumours producing normal levels are commonly small and asymptomatic. Furthermore, certain secretion patterns are indicative of the presence of metastases as well as the size and site of sporadic and syndrome-related PCCs.

Selecting the appropriate approach for resection and follow-up of pheochromocytomas (PCCs) is highly dependent upon reliable localization and exclusion of multifocal, bilateral, or metastatic disease. Metaiodobenzylguanidine (MIBG) scintigraphy was developed for functional localization of catecholamine-secreting tissues. Somatostatin receptor imaging (SRI) has a high sensitivity for localizing head and neck paragangliomas, but studies of intraabdominal PCCs are rare. In this study we review our experience of [(123)I]MIBG and SRI, performed since 1983 and 1989, respectively, in the work-up of primary and recurrent PCCs. Scintigraphic results were correlated with catecholamine secretion, size and site, malignancy, associated tumor syndromes, and morphological features. [(123)I]MIBG scans were performed in a total of 75 patients, in 70 cases before resection of primary PCCs and in 5 cases because of recurrent disease. Ninety-one PCCs were resected. The overall detection rates were 83.3% and 89.8% for PCCs larger than 1.0 cm. Multifocal disease was detected in 4 patients with [(123)I]MIBG. [(123)I]MIBG uptake correlated with greater size of PCC (r = 0.33; P = 0.008) and greater concentration of plasma epinephrine (r = 0.32; P = 0.006). [(123)I]MIBG-negative PCCs (n = 14) had significantly (P = 0.01) smaller diameters than [(123I)]MIBG-positive tumors. Furthermore, [(123)I]MIBG uptake was significantly higher in unilateral (P = 0.02), benign (P = 0.02), sporadic (P = 0.02), intraadrenal (P = 0.02), and capsular invasive (P = 0.03) PCCs than in bilateral, malignant, MEN2A/2B-related, extraadrenal, and noninvasive PCCs, respectively. The detection rate of SRI was only 25% (8 of 32) for primary benign PCCs. In 14 patients metastases occurred, which were effectively visualized with [(123)I]MIBG in 8 of 14 cases. SRI was able to detect metastases in 7 of 8 cases, including 3 [(123)I]MIBG-negative metastatic cases. In addition, [(123)I]MIBG and SRI detected 2 recurrences. In conclusion, [(123)I]MIBG uptake is correlated with the size, epinephrine production, and site of PCCs. Its role in bilateral and MEN2A/2B-related PCCs seems limited. In cases of recurrent elevation of catecholamines, localization of metastases and/or recurrence should be attempted with [(123)I]MIBG scintigraphy. In suspicious metastatic PCCs, SRI might be considered to supplement [(123)I]MIBG scintigraphy.

Although in situ hybridization has been used to examine the distribution of messenger RNA for somatostatin receptor subtypes (sst) in human tumors, the cellular localization of sst1 and sst2A receptors has not been reported. In this study, we describe the cellular localization of human sst1 and sst2A receptor proteins in both cryostat- and paraffin-embedded sections of 25 human tumor tissues using two recently developed polyclonal antibodies. Six somatostatin (SS) receptor (SSR) positive tumors (two gastrinomas, three carcinoids, one pheochromocytoma) and one SSR negative tumor (renal cell carcinoma), selected by positive and negative SSR autoradiography, respectively, were studied by both immunohistochemistry and Western blot analysis. The six SSR positive tumors expressed sst2A, while 4 of 5 expressed sst1 as well. The SSR negative tumor did not express either sst1 or sst2A. Western blot analysis of wheat germ agglutinin purified membrane proteins confirmed the presence of the sst1 and sst2A glycosylated receptors. The paraffin-embedded sections gave best information with respect to the subcellular localization. Sst1 immunoreactivity was observed both on the membrane and in the cytoplasm, while sst2A showed predominantly membrane-associated immunoreactivity. This subcellular distribution of sst1 or sst2A receptors was confirmed in paraffin-embedded sections of 8 additional intestinal carcinoids, 5 gastrinomas and 5 pheochromocytomas. Sst1 receptors were detected in 7 out of 8 carcinoids, in all gastrinomas, and in 4 out of 5 pheochromocytomas, while 6 out of 8 carcinoids, all gastrinomas, and 3 out of 5 pheochromocytomas expressed sst2A receptors. In conclusion, sst1 and sst2A receptors show a differential subcellular localization in human SSR positive tumors. The use of SSR subtype selective antibodies to detect the subcellular distribution of SSR subtypes in individual tumor cells is an important step forward to understand more about the pathophysiological role of the different SSR subtypes in human tumors.