Marlijn Waaijers

Immunoreactivity Score Using an Anti-sst2A Receptor Monoclonal Antibody Strongly Predicts the Biochemical Response to Adjuvant Treatment with Somatostatin Analogs in Acromegaly

CONTEXT Somatostatin receptor subtype 2 (sst2A) protein expression has been demonstrated to positively correlate with somatostatin analog treatment outcome in GH-secreting adenomas. Recently, a new rabbit monoclonal anti-sst2A antibody (clone UMB-1) has been validated as a reliable method to selectively detect sst2A protein levels in formalin-fixed tissues. OBJECTIVE The aim of the study was to establish whether the evaluation of sst2A protein levels, assessed with a routine reproducible immunohistochemistry protocol using UMB-1 antibody, may predict the successful adjuvant therapy with somatostatin analogs in acromegalic patients. DESIGN, SETTING, AND PATIENTS Thirty-six acromegalic patients from our referral hospital were evaluated retrospectively. Sst2A expression analysis was performed by immunohistochemistry in 25 patients and by quantitative RT-PCR in 26 patients. Sst2A immunoreactivity was evaluated using an immunoreactivity score (IRS), which takes into account both the percentage of positive cells and staining intensity. INTERVENTIONS Patients with persistent disease after surgery (n = 26) were treated with somatostatin analogs for a median duration of 6 months. MAIN OUTCOME MEASURE GH and IGF-I levels were measured before and after postoperative treatment. RESULTS Sst2A IRS showed a significant positive correlation with both GH (P = 0.039) and IGF-I (P = 0.001) suppression by octreotide. Sst2A IRS was negatively associated with IGF-I levels reached after treatment (P = 0.001), and patients that achieved IGF-I normalization showed significantly higher sst2A IRS compared to the group that was not normalized (P = 0.002). A sst2A IRS of at least 5 showed a sensitivity of 86% and a specificity of 91% in predicting IGF-I normalization during adjuvant octreotide treatment. CONCLUSION Sst2A IRS with the anti-sst2A antibody UMB-1 represents a valid tool in the clinical practice to identify acromegalic patients likely to be responders to adjuvant therapy with the currently available somatostatin analogs.

IFN-β is a highly potent inhibitor of gastroenteropancreatic neuroendocrine tumor cell growth in vitro

IFN-α controls hormone secretion and symptoms in human gastroenteropancreatic neuroendocrine tumors (GEP-NET) but it rarely induces a measurable tumor size reduction. The effect of other type I IFNs, e.g., IFN-β, has not been evaluated. We compared the antitumor effects of IFN-α and IFN-β in BON cells, a functioning human GEP-NET cell line. As determined by quantitative reverse transcription-PCR analysis and immunocytochemistry, BON cells expressed the active type I IFN receptor mRNA and protein (IFNAR-1 and IFNAR-2c subunits). After 3 and 6 days of treatment, IFN-β significantly inhibited BON cell growth in a time- and dose-dependent manner. IC50 and maximal inhibitory effect on day 6 were 8 IU/mL and 98%, respectively. In contrast, the effect of IFN-α resulted significantly in a less potent effect (IC50: 44 IU/mL, maximal inhibition: 26%). IFN-α induced only cell cycle arrest, with an accumulation of the cells in S phase. IFN-β, apart from a more potent delay in S-G2-M phase transit of the cell cycle, also induced a strong stimulation of apoptosis, evaluated by flow cytometry (Annexin V and 7-AAD) and measurement of the DNA fragmentation. Besides, only IFN-β severely suppressed chromogranin A levels in the medium from BON cells after 6 days of treatment. In conclusion, IFN-β is much more potent, compared with IFN-α, in its inhibitory effect on GEP-NET cell proliferation in vitro through the induction of apoptosis and cell cycle arrest. Further studies are required to establish whether IFN-β has comparable potent tumor growth inhibitory effects in vivo . (Cancer Res 2006; 66(1): 554-62)

Internalisation of isotope-coupled somatostatin analogues

With the technique of in vivo somatostatin (SRIF) receptor (sst) scintigraphy a variety of human sst-positive tumours can be visualised 24-48 h after intravenous injection of the radiolabelled (SRIF) analogue [111In-DTPA-D-Phe1]octreotide. This rather long residence time of radioactivity on tumours suggests that the radioligand is internalised by the tumour cells; literature data for normal pituitary and pancreatic islet cells agree with this. Internalised SRIF has been found to be associated with cytoplasmic organelles, especially the Golgi apparatus, lysosomes and secretory granules. We have found that mouse AtT20 and primary cultures of human growth-hormone-secreting pituitary adenoma cells internalised a high amount of radio-iodinated [Tyr3]octreotide. Since octreotide binds with high affinity to the sst2 and sst5 subtypes and because both sst subtypes are expressed in these cells, one or both subtypes may be involved in this process. We also found that unlabelled octreotide, SRIF-14 or SRIF-28 can increase the internalisation of the radioligand. These observations, together with other studies on the manipulation of sst expression, may help to optimise the uptake of radioligand in in vivo sst scintigraphy in humans and improve the potential effect of radiotherapy with radiolabelled (subtype-specific) SRIF analogues.

Interferon-β is a potent inhibitor of cell growth and cortisol production in vitro and sensitizes human adrenocortical carcinoma cells to mitotane

Adrenocortical carcinoma (ACC) is an aggressive tumor with very poor prognosis. Novel medical treatment opportunities are required. We investigated the effects of interferon-β (IFN-β), alone or in combination with mitotane, on cell growth and cortisol secretion in primary cultures of 13 human ACCs, three adrenal hyperplasias, three adrenal adenomas, and in two ACC cell lines. Moreover, the interrelationship between the effects of IGF2 and IFN-β was evaluated. Mitotane inhibited cell total DNA content/well (representing cell number) in 7/11 (IC50: 38±9.2 μM) and cortisol secretion in 5/5 ACC cultures (IC50: 4.5±0.1 μM). IFN-β reduced cell number in 10/11 (IC50: 83±18 IU/ml) and cortisol secretion in 5/5 ACC cultures (IC50: 7.3±1.5 IU/ml). The effect of IFN-β on cell number included the induction of apoptosis. IFN-β strongly inhibited mRNA expression of STAR, CYP11A1, CYP17A1, and CYP11B1. Mitotane and IFN-β induced an additive inhibitory effect on cell number and cortisol secretion. IGF2 (10 nM) inhibited apoptosis and increased cell number and cortisol secretion. These effects were counteracted by IFN-β treatment. Finally, IFN-β inhibited IGF2 secretion and mRNA expression. In conclusion, IFN-β is a potent inhibitor of ACC cell growth in human primary ACC cultures, partially mediated by an inhibition of the effects of IGF2, as well as its production. The increased sensitivity of ACC cells to mitotane induced by treatment with IFN-β may open the opportunity for combined treatment regimens with lower mitotane doses. The inhibition of the expression of steroidogenic enzymes by IFN-β is a novel mechanism that may explain its inhibitory effect on cortisol production.

Long‐term in‐vitro treatment of human growth hormone (GH)‐secreting pituitary adenoma cells with octreotide causes accumulation of intracellular GH and GH mRNA levels

OBJECTIVE We studied the effects of long‐term in‐vitro exposure of human GH secreting pituitary adenoma cells to octreotide on GH release, intracellular GH concentrations and GH messengerbonuclelc acid (mRNA) levels.

Dopamine D2 receptor expression in the corticotroph cells of the human normal pituitary gland

The dopamine D2 receptor is the main dopamine receptor expressed in the human normal pituitary gland. The aim of the current study was to evaluate dopamine D2 receptor expression in the corticotroph cell populations of the anterior lobe and pars intermedia, as well as posterior lobe of the human normal pituitary gland by immunohistochemistry. Human normal pituitary gland samples obtained from routine autopsies were used for the study. In all cases, histology together with immunostaining for adrenocorticotropic hormone, melanocyte-stimulating hormone, prolactin, and neurofilaments were performed and compared to the immunostaining for D2 receptor. D2 receptor was heterogeneously expressed in the majority of the cell populations of the anterior and posterior lobe as well as in the area localized between the anterior and posterior lobe, and arbitrary defined as “intermediate zone”. This zone, characterized by the presence of nerve fibers included the residual pars intermedia represented by the colloid-filled cysts lined by the remnant melanotroph cells strongly expressing D2 receptors, and clusters of corticotroph cells, belonging to the anterior lobe but localized within the cysts and adjacent to the posterior lobe, variably expressing D2 receptors. D2 dopamine receptor is expressed in the majority of the cell populations of the human normal pituitary gland, and particularly, in the different corticotroph cell populations localized in the anterior lobe and the intermediate zone of the pituitary gland.