Jorge Marcondes

Quantitative analysis of somatostatin receptor subtypes (1-5) gene expression levels in somatotropinomas and correlation to in vivo hormonal and tumor volume responses to treatment with octreotide LAR

OBJECTIVE To determine whether the somatostatin receptor subtype (SSTR) expression profile correlates with hormonal and tumor volume responses to postsurgical octreotide long acting repeatable (OCT LAR) treatment. DESIGN AND METHODS Quantitative real-time RT-PCR was used to evaluate the absolute mRNA copy numbers for all five SSTR subtypes in 22 somatotropinomas. Response to OCT LAR was studied by hormone levels (GH and IGF-I) and tumor volume (sella turcica magnetic resonance imaging). RESULTS SSTR5 was present at the highest level followed by SSTR2, SSTR3, SSTR1, and SSTR4 (2327 (1046-5555), 2098 (194-23 954), 97 (0-460), 14 (0-29 480), and 0 (0-652) copies respectively). Positive correlations were found between SSTR2 levels and the percentage decrease of GH and IGF-I after 3 (r=0.49, P<0.027 and r=0.49, P<0.029 respectively) and 6 (r=0.59, P<0.006 and r=0.58, P<0.008 respectively) months of OCT LAR. A negative correlation was found between SSTR5 mRNA levels and the percentage decrease of GH after 3 months of OCT LAR (r=-0.52, P=0.016, n=21). A higher SSTR2/SSTR5 ratio was observed among patients who obtained hormonal control with OCT LAR, when compared with those uncontrolled (2.4 (0.7-10) vs 0.3 (0.1-7.7), P=0.001). A ROC curve analysis showed a SSTR2/SSTR5 ratio of 1.3 as the best predictor of disease control, with a sensitivity of 88% and a specificity of 92% - area under curve, 0.9. A positive correlation was also found between SSTR2 mRNA levels and the percentage decrease in tumor volume after 6 months of OCT LAR (r=0.79, P=0.002, n=12). CONCLUSIONS Somatostatin receptor subtype 2 mRNA expression levels in somatotropinomas correlate positively with in vivo hormonal and tumor volume responses to OCT LAR.

Expression Analysis of Dopamine Receptor Subtypes in Normal Human Pituitaries, Nonfunctioning Pituitary Adenomas and Somatotropinomas, and the Association between Dopamine and Somatostatin Receptors with Clinical Response to Octreotide-LAR in Acromegaly

CONTEXT Dopamine receptor (DR) and somatostatin receptor subtype expression in pituitary adenomas may predict the response to postsurgical therapies. OBJECTIVES Our objectives were to assess and compare the mRNA levels of DR1-5 and somatostatin receptors 1-5 in normal pituitaries (NPs), nonfunctioning pituitary adenomas (NFPAs), and somatotropinomas. In addition, we determined whether the level of DR expression correlates with the in vivo response to octreotide-LAR in acromegalic patients. DESIGN AND PATIENTS Eight NPs, 30 NFPAs, and 39 somatotropinomas were analyzed for receptor mRNA levels by real-time RT-PCR. The DR2 short variant was estimated as the DR2 long/DR2 total (DR2T). The relationship between DR expression and the postsurgical response to octreotide-LAR was assessed in 19 of the acromegalic patients. RESULTS DR3 was not detected. The relationship between expression levels of DR subtypes in NPs and somatotropinomas was DR2T>>>DR4>>DR5>DR1, whereas in NFPAs, DR2T>>>DR4>>DR1>DR5. The DR2 short variant was the predominant DR2 variant in the majority of samples. In acromegalics treated with octreotide-LAR, DR1 was negatively correlated with percent GH reduction (3 months: r = -0.67, P = 0.002; and 6 months: r = -0.58, P = 0.009), and DR5 was positively correlated with percent IGF-I reduction (3 months: r = 0.55, P = 0.01; and 6 months: r = 0.47, P = 0.04). CONCLUSIONS DR2 is the predominant DR subtype in NPs, NFPAs, and somatotropinomas. The fact that DR1, DR4, and DR5 are also expressed in many adenomas tested suggests that these receptors might also play a role in the therapeutic impact of postsurgical medical therapies in patients with NFPA and acromegaly. This was supported by the finding that the in vivo response to octreotide-LAR was negatively associated with DR1 and positively associated with DR5.

Low Aryl Hydrocarbon Receptor-Interacting Protein Expression Is a Better Marker of Invasiveness in Somatotropinomas than Ki-67 and p53

Background: Some pituitary adenomas exhibit fast growth and invade surrounding structures. To date, there is no robust marker to predict invasiveness. Aim: To evaluate Ki-67, p53 and aryl hydrocarbon receptor-interacting protein (AIP) expression and compare these between invasive and noninvasive somatotropinomas and nonfunctioning pituitary adenomas (NFPAs). Methods: Protein expression was determined by immunohistochemistry. Tumors were classified according to percentage of immunolabeled nuclei for Ki-67 and p53. AIP immunopositivity was graded according to a score encompassing pattern and intensity. Invasiveness was defined according to radiological and surgical criteria. Results: Thirty-eight sporadic somatotropinomas were studied. Median Ki-67 labeling index in invasive and noninvasive tumors was 1.6 (range 0–20.6) and 0.26 (0–2.2), respectively (p = 0.01). With a 2.3% cut-off point obtained by ROC curve analysis, invasive adenomas were distinguished with 100% specificity, 39% sensitivity, and 63% accuracy. Low AIP expression was also correlated with tumor invasiveness (p = 0.001), with sensitivity, specificity and accuracy of 78, 80, and 79%, respectively. Expression of p53 was not different among tumors. Twenty-nine NFPAs were studied, with no significant difference between Ki-67, p53 and AIP expression in invasive and noninvasive tumors. High AIP expression was more frequent in NFPAs, with Ki-67 >3% (p = 0.051), especially when only gonadotrope cell adenomas (n = 25) were considered (p = 0.012). Conclusions: These data suggest, for the first time, that AIP is a better marker of invasiveness in somatotropinomas than Ki-67 and p53. In addition, low AIP expression is observed in invasive somatotropinomas, in contrast with high AIP expression in NFPAs (mainly gonadotrope cell tumors) with high proliferative indices.

Ki-67 is a predictor of acromegaly control with octreotide LAR independent of SSTR2 status and relates to cytokeratin pattern.

INTRODUCTION Only one study has evaluated Ki-67 as a predictor of the response to somatostatin analog therapy in acromegaly; however, other predictors like somatostatin receptor type 2 (SSTR2) and cytokeratin pattern expressions were not considered. OBJECTIVE To evaluate whether Ki-67 is a predictor of octreotide LAR (OCT-LAR) response in somatotropinomas independent of SSTR2 and cytokeratin expression patterns. METHODS Protein expression was analyzed by immunohistochemistry. The percentage of cell nuclei that were immunolabeled for Ki-67 and the percentage of cells with positive SSTR2 staining were calculated. SSTR2 expression was considered high when ≥25%, and a cutoff of 2.3% was designated for Ki-67. Tumors were classified as densely or sparsely granulated according to the cytokeratin pattern. RESULTS Thirty-one somatotropinomas were studied. Fourteen patients (45.2%) were controlled with OCT-LAR therapy. The median Ki-67 labeling index (LI) was higher in patients not controlled with OCT-LAR than in those controlled (1.63 and 0.15 respectively, P=0.002). Higher SSTR2 expression and densely granulated tumors were correlated with control as well (P=0.04 and 0.038 respectively). There was no difference in Ki-67 levels between patients with high and low SSTR2 expression (P=0.651). After multivariate analysis, both Ki-67 and SSTR2 remained statistically significant as predictors of OCT-LAR response (P=0.017 and 0.012 respectively). The Ki-67 LI was higher in sparsely than in densely granulated tumors (P=0.047). CONCLUSIONS Ki-67 is a predictor of response to OCT-LAR in acromegaly, independent of SSTR2 expression and relates to cytokeratin patterns.

Tenascin-C in the extracellular matrix promotes the selection of highly proliferative and tubulogenesis-defective endothelial cells ☆ ☆☆

The extracellular matrix (ECM) contains important cues for tissue homeostasis and morphogenesis. The matricellular protein tenascin-C (TN-C) is overexpressed in remodeling tissues and cancer. In the present work, we studied the effect of different ECM-which exhibited a significant diversity in their TN-C content-in endothelial survival, proliferation and tubulogenic differentiation: autologous (endothelial) ECM devoid of TN-C, but bearing large amounts of FN; fibroblast ECM, bearing both high TN-C and FN contents; and finally, glioma-derived matrices, usually poor in FN, but very rich in TN-C. HUVECs initially adhered to the immobilized matrix produced by U373 MG glioma cells, but significantly detached and died by anoikis (50 to 80%) after 24h, as compared with cells incubated with endothelial and fibroblast matrices. Surviving endothelial cells (20 to 50%) became up to 6-fold more proliferative and formed 74-97% less tube-like structures in vitro than cells grown on non-tumoral matrices. An antibody against the EGF-like repeats of tenascin-C (TN-C) partially rescued cells from the tubulogenic defect, indicating that this molecule is responsible for the selection of highly proliferative and tubulogenic defective endothelial cells. Interestingly, by using defined substrata, in conditions that mimic glioma and normal cell ECM composition, we observed that fibronectin (FN) modulates the TN-C-induced selection of endothelial cells. Our data show that TN-C is able to modulate endothelial branching morphogenesis in vitro and, since it is prevalent in matrices of injured and tumor tissues, also suggest a role for this protein in vascular morphogenesis, in these physiological contexts.

Expression of retinoblastoma protein in human growth hormone-secreting pituitary adenomas

The retinoblastoma gene (RB1) is a tumor-suppressor gene in chromosomal region 13q14.2. Its role in the pathogenesis of pituitary tumors has not been fully clarified. Some studies have shown that losses in this chromosomal region are related to aggressive tumor behavior, although the retinoblastoma protein (pRB) is still expressed. Conversely, lack of expression of pRB was observed in one fourth of GH-secreting pituitary adenomas (GH-tumors). In order to further study the expression of pRB in GH-tumors, we evaluated this protein in 49 tumors from patients with acromegaly (20 noninvasive, 25 invasive, and 4 with no information) and 8 normal pituitaries using immunohistochemistry (IHC). Nuclear staining for pRB ranged from 0 to 90% (median 40%) in the tumors and from 40 to 80% (median 58%) in normal pituitaries. In 10 tumors (20% of total) the adenomatous cells were negative (5 cases) or had very low labeling (5 cases) for pRB. Sixty three percent (31/49) of the tumors showed staining in 10–80% of the cells and in 16% (8/49) of the cases >80% of the adenomatous cells were positive for pRB. The expression of pRB was not different in invasive and noninvasive tumors. In conclusion, pRB is underexpressed in a subgroup of GH-tumors, and this may represent an early event in the pathogenesis of this tumor subtype.

Sellar and suprasellar mixed germ cell tumor mimicking a pituitary adenoma

Germ cell tumors (GCT) are a heterogeneous group of lesions whose origin is not well established. Several cases of primary intrasellar germinomas have been reported, however non-germinomatous GCT have rarely been described. We report the case of a young adult male patient with a mixed GCT that presented with a sellar tumor with suprasellar extension. The patient seeked medical attention because of seizures and magnetic resonance imaging evidenced a tumor of the sellar region. Hyperprolactinemia was also present and dopamine agonist therapy was started. As there was a rapid tumor growth and the patient had concomitant central diabetes insipidus and elevated testosterone levels, a GCT was suspected and confirmed by elevated serum concentration of β-human chorionic gonadotrophin. Patient underwent surgical resection of the tumor and histopathological examination confirmed the diagnosis of a mixed GCT. Chemotherapy was initiated, followed by conventional radiotherapy. In conclusion, although pituitary adenomas respond for the vast majority of sellar tumors, concomitant symptoms such as central diabetes insipidus and rapid tumor growth should raise the suspicion of a diverse diagnosis. The present report intend not only to show a rare case of sellar and suprasellar mixed GCT but also to remind clinicians that if laboratory findings do not fit into patient’s diagnosis (such as high testosterone levels in our patient), then the diagnosis should be reviewed.

Tumor deletion mapping of chromosomal region 13q14 in 43 growth hormone secreting pituitary adenomas

Previous studies have reported allelic loss in chromosomal region 13q14 in pituitary tumors. However, the role of RB1 in this region has not been clarified. We performed a tumor deletion map of chromosomal region 13q14 with pituitary adenomas and matched blood samples of 43 patients with acromegaly. Twenty-one patients had non-invasive tumors, 19 had invasive tumors, and in 3 this information was not available. Results showed loss of heterozygosity in at least one microsatelite marker of region 13q14 in 12% (5 of 43) of the somatotropinomas. Retention of marker D13S1325, telomeric to RB1, suggests that the putative tumor suppressor gene is located centromeric to this region, which includes RB1 locus. The participation of RB1 was excluded in four of the five cases because retinoblastoma protein was shown to be positive in these tumors in our previous study. Allelic loss occurred in similar frequency in invasive and noninvasive adenomas. In summary, we confirmed the participation of chromosomal region 13q14 in a subset of GH-secreting adenomas with no regard to tumor grade. RB1 was not implicated, suggesting the participation of another tumor suppressor gene in this region during the first steps of somatotropinoma development.

Reversal of severe SPECT asymmetry after venous extra-intracranial high flow bypass in a patient submitted to therapeutic internal carotid occlusion: case report

Therapeutic occlusion of the internal carotid artery is the main option for the treatment of the symptomatic intracavernous internal carotid artery aneurysms, but the issue of the best way of doing the balloon test occlusion (BTO) regarding prediction of future ischemic events remains debatable. Single photon emission computerized tomography (SPECT) has been offered as one of the best option of monitoring regional cerebral blood flow (rCBF) during the BTO, where severe asymmetry is predictive of delayed ischemia. We describe a case of important SPECT asymmetry during BTO clinically negative and its complete reversal with carotid occlusion after extra-intracranial bypass with high flow safenous vein bypass between the cervical carotid artery and the middle cerebral artery.

Free-floating adult human brain-derived slice cultures as a model to study the neuronal impact of Alzheimer's disease-associated Aβ oligomers

BACKGROUND Slice cultures have been prepared from several organs. With respect to the brain, advantages of slice cultures over dissociated cell cultures include maintenance of the cytoarchitecture and neuronal connectivity. Slice cultures from adult human brain have been reported and constitute a promising method to study neurological diseases. Despite this potential, few studies have characterized in detail cell survival and function along time in short-term, free-floating cultures. NEW METHOD We used tissue from adult human brain cortex from patients undergoing temporal lobectomy to prepare 200 μm-thick slices. Along the period in culture, we evaluated neuronal survival, histological modifications, and neurotransmitter release. The toxicity of Alzheimers-associated Aβ oligomers (AβOs) to cultured slices was also analyzed. RESULTS Neurons in human brain slices remain viable and neurochemically active for at least four days in vitro, which allowed detection of binding of AβOs. We further found that slices exposed to AβOs presented elevated levels of hyperphosphorylated Tau, a hallmark of Alzheimers disease. COMPARISON WITH EXISTING METHOD(S) Although slice cultures from adult human brain have been previously prepared, this is the first report to analyze cell viability and neuronal activity in short-term free-floating cultures as a function of days in vitro. CONCLUSIONS Once surgical tissue is available, the current protocol is easy to perform and produces functional slices from adult human brain. These slice cultures may represent a preferred model for translational studies of neurodegenerative disorders when long term culturing in not required, as in investigations on AβO neurotoxicity.