Predictive Markers for Postsurgical Medical Management of Acromegaly: A Systematic Review and Consensus Treatment Guideline

Objective: To clarify the selection of medical therapy following transsphenoidal surgery in patients with acromegaly, based on growth hormone (GH)/insulin-like growth factor 1 (IGF-1) response and glucometabolic control.Methods: We carried out a systematic literature review on three of the best studied and most practical predictive markers of the response to somatostatin analogues (SSAs): somatostatin receptor (SSTR) expression, tumor morphologic classification, and T2-weighted magnetic resonance imaging (MRI) signal intensity. Additional analyses focused on glucose metabolism in treated patients.Results: The literature survey confirmed significant associations of all three factors with SSA responsiveness. SSTR expression appears necessary for the SSA response; however, it is not sufficient, as approximately half of SSTR2-positive tumors failed to respond clinically to first-generation SSAs. MRI findings (T2-hypo-intensity) and a densely granulated phenotype also correlate with SSA efficacy, and are advantageous as predictive markers relative to SSTR expression alone. Glucometabolic control declines with SSA monotherapy, whereas GH receptor antagonist (GHRA) monotherapy may restore normoglycemia.Conclusion: We propose a decision tree to guide selection among SSAs, dopamine agonists (DAs), and GHRA for medical treatment of acromegaly in the postsurgical setting. This decision tree employs three validated predictive markers and other clinical considerations, to determine whether SSAs are appropriate first-line medical therapy in the postsurgical setting. DA treatment is favored in patients with modest IGF-1 elevation. GHRA treatment should be considered for patients with T2-hyperintense tumors with a sparsely granulated phenotype and/or low SSTR2 staining, and may also be favored for individuals with diabetes. Prospective analyses are required to test the utility of this therapeutic paradigm.Abbreviations: DA = dopamine agonist; DG = densely granulated; GH = growth hormone; GHRA = growth hormone receptor antagonist; HbA1c = glycated hemoglobin; IGF-1 = insulin-like growth factor-1; MRI = magnetic resonance imaging; SG = sparsely granulated; SSA = somatostatin analogue; SSTR = somatostatin receptor     

ZAC1 and SSTR2 Are Downregulated in Non-Functioning Pituitary Adenomas but Not in somatotropinomas

Introduction

There are few data regarding ZAC1 expression in clinically non-functioning pituitary adenomas (NFPA). Because somatotropinomas and NFPA behave differently with respect to tumor shrinkage during somatostatin analogs (SA) therapy, we sought to compare the ZAC1 and somatostatin receptor (sstr) types 1, 2, 3 and 5 mRNA expression in these two pituitary adenoma subtypes and in normal human pituitaries.

Methods

ZAC1 and SSTR mRNA expression levels were evaluated using real-time RT-PCR (TaqMan) in 20 NFPA and compared with the expression levels in 23 somatotropinomas and five normal pituitaries. The NFPA invasiveness was evaluated using magnetic resonance imaging with Hardy’s modified criteria. Ki-67 and p53 were evaluated using immunohistochemistry.

Results

A total of 20 patients with NFPA [6 males, median age 56 years (range: 30-78)], 23 with acromegaly [12 males, median age 43 years (range: 24–57)] and five normal pituitaries [4 males, median age 48 years (range: 36–54)] were included. Four of the patients (20%) had Hardy’s grade 2 tumors; all of the others had Hardy’s grade 3 tumors. The Ki-67 median expression was 2.35 (range: 0.2–9.23), and only four of the tumors (20%) were positive for p53. The ZAC1 mRNA expression was significantly lower in NFPA than in somatotropinomas and in normal pituitaries (p<0.001 for both), as well as the SSTR2 (p=0.001 and 0.01, respectively). The SSTR3 expression was higher in the NFPA than in the somatotropinomas and in the normal pituitaries (p=0.03 and 0.02, respectively). No correlation was found between the ZAC1 mRNA expression and the tumor invasiveness, Ki-67 and p53.

Conclusion

ZAC1 and SSTR2 are underexpressed and SSTR3 is overexpressed in NFPA compared to those in somatotropinomas and in normal pituitaries, which might explain the lack of tumor shrinkage that is observed in response to commercially available SA therapy in patients with NFPA.     

Coexpression of human somatostatin receptor-2 (SSTR2) and SSTR3 modulates antiproliferative signaling and apoptosis

Background

Somatostatin (SST) via five G_i coupled receptors namely SSTR1-5 is known to inhibit cell proliferation by cytostatic and cytotoxic mechanisms. Heterodimerization plays a crucial role in modulating the signal transduction pathways of SSTR subtypes. In the present study, we investigated human SSTR2/SSTR3 heterodimerization, internalization, MAPK signaling, cell proliferation and apoptosis in HEK-293 cells in response to SST and specific agonists for SSTR2 and SSTR3.

Results

Although in basal conditions, SSTR2 and SSTR3 colocalize at the plasma membrane and exhibit heterodimerization, the cell surface distribution of both receptors decreased upon agonist activation and was accompanied by a parallel increase in intracellular colocalization. Receptors activation by SST and specific agonists significantly decreased cAMP levels in cotransfected cells in comparison to control. Agonist-mediated modulation of pERK1/2 was time and concentration-dependent, and pronounced in serum-deprived conditions. pERK1/2 was inhibited in response to SST; conversely receptor-specific agonist treatment caused inhibition at lower concentration and activation at higher concentration. Strikingly, ERK1/2 phosphorylation was sustained upon prolonged treatment with SST but not with receptor-specific agonists. On the other hand, SST and receptor-specific agonists modulated p38 phosphorylation time-dependently. The receptor activation in cotransfected cells exhibits G_i-dependent inhibition of cell proliferation attributed to increased PARP-1 expression and TUNEL staining, whereas induction of p21 and p27^Kip1 suggests a cytostatic effect.

Conclusion

Our study provides new insights in SSTR2/SSTR3 mediated signaling which might help in better understanding of the molecular interactions involving SSTRs in tumor biology.     

Cytoplasmic expression of SSTR2 and 5 by immunohistochemistry and by RT/PCR is not associated with the pharmacological response to octreotide

ObjectiveTo evaluate expression of somatostatin receptor subtypes 2 and 5 (SSTR 2 and 5) by RT/PCR and immunohistochemistry (IHC) in GH-secreting adenomas, seeking correlations with response to octreotide.MethodsSSTR2 and 5 expression was tested by IHC (n = 37), RT/PCR (n = 36) or both (n = 13) in GH-secreting adenomas from 60 patients with acromegaly who had undergone pituitary surgery; 36 had been treated preoperatively with octreotide LAR for 3–6 months, and were categorized as responders (achievement of GH <2.5 ng/mL and a normal age-adjusted IGF-1), partial responders (GH and IGF-1 reduction >50% and >30%, respectively) or non-responders. IHC was performed on a tissue microarray using specific antibodies directed to the carboxyl terminus of SSTR2 and 5.ResultsSSTR5 was the predominantly expressed receptor subtype by both IHC and RT/PCR in all tumors tested, regardless of whether they came from octreotide-naïve, octreotide-responsive, or octreotide-resistant patients. Immunostaining was concentrated in the cytoplasm. Neither SSTR2 nor SSTR5 expression correlated with baseline or post-octreotide GH or IGF-1 levels or tumor volume by either method. The agreement rate between RT/PCR and IHC was 77% in all 13 adenomas in which both methods were used.ConclusionExpression of these receptors does not guarantee an adequate response to somatostatin analogs; other functional aspects of this interaction, such as receptor homo- and heterodimerization, and the resulting signaling cascade, probably play a role in determining whether a patient will respond or not to these agents.     

Identification and characterization of subtype selective somatostatin receptor agonists.

High affinity, subtype selective non-peptide agonists of somatostatin receptor subtypes 1-5 were identified in combinatorial libraries constructed based on molecular modeling of known peptide agonists. Simultaneous traditional chemical synthesis yielded an additional series of somatostatin subtype-2 receptor (SSTR2) selective agonists. These compounds have been used to further define the physiological functions of the individual somatostatin receptor subtypes. In vitro experiments demonstrated the role of the SSTR2 in inhibition of glucagon release from mouse pancreatic alpha-cells and the somatostatin subtype-5 receptor (SSTR5) as a mediator of insulin secretion from pancreatic beta-cells. Both SSTR2 and SSTR5 regulated growth hormone release from the rat anterior pituitary gland. In vivo studies performed with SSTR2 receptor selective compounds demonstrated effective inhibition of pulsatile growth hormone release in rats. The SSTR2 selective compounds also lowered plasma glucose levels in normal and diabetic animal models. The availability of high affinity, subtype selective non-peptide agonists for each of the somatostatin receptors provides a direct approach to defining their physiological function both peripherally and in the central nervous system.     

A new D₂ dopamine receptor agonist allosterically modulates A(2A) adenosine receptor signalling by interacting with the A(2A)/D₂ receptor heteromer

The structural and functional interaction between D₂ dopamine receptor (DR) and A_2A adenosine receptor (AR) has suggested these two receptors as a pharmacological target in pathologies associated with dopamine dysfunction, such as Parkinson's disease. In transfected cell lines it has been demonstrated the activation of D₂DR induces a significant negative regulation of A_2AAR-mediated responses, whereas few data are at now available about the regulation of A_2AAR by D₂DR agonists at receptor recognition site. In this work we confirmed that in A_2AAR/D₂DR co-transfected cells, these receptors exist as homo- and hetero-dimers. The classical D₂DR agonists were able to negatively modulate both A_2AAR affinity and functionality. These effects occurred even if any significant changes in A_2AAR/D₂DR energy transfer interaction could be detected in BRET experiments.Since the development of new molecules able to target A_2A/D₂ dimers may represent an attractive tool for innovative pharmacological therapy, we also identified a new small molecule, 3-(3,4-dimethylphenyl)-1-(2-piperidin-1-yl)ethyl)piperidine (compound 1), full agonist of D₂DR and modulator of A_2A-D₂ receptor dimer. This compound was able to negatively modulate A_2AAR binding properties and functional responsiveness in a manner comparable to classical D₂R agonists. In contrast to classical agonists, compound 1 led to conformational changes in the quaternary structure in D₂DR homomers and heteromers and induced A_2AAR/D₂DR co-internalization. These results suggest that compound 1 exerts a high control of the function of heteromers and could represent a starting point for the development of new drugs targeting A_2AAR/D₂ DR heteromers.     

Immunohistochemical localization and functional characterization of somatostatin receptor subtypes in a corticotropin releasing hormone-secreting adrenal phaeochromocytoma: review of the literature and report of a case

Somastostatin receptors are frequently expressed in phaeochromocytoma but data on somatostatin receptor subtyping are scanty and the functional response to the somatostatin analogue octretide is still debated.We report an unusual case of pheochromocytoma, causing ectopic Cushing’s syndrome due to CRH production by the tumour cells, in a 50-yr-old woman. Abdominal computed tomography revealed an inhomogeneous, 9-cm mass in the right adrenal gland, and [¹¹¹In-DTPA⁰] octreotide scintigraphy showed an abnormal uptake of the radiotracer in the right perirenal region, corresponding to the adrenal mass. The patient underwent laparoscopic surgery and formalin-fixed and paraffin-embedded samples were studied. The tumour was extensively characterized by immunohistochemistry and somatostatin receptor (SSTRs) subtypes expression was analyzed. Histological and immunohistochemical examination of the surgical specimens displayed a typical pheochromocytoma, which was found to be immunoreative to S-100, chromogranin A and neurofilaments. Immunostaining for SSTR subtypes showed a positive reaction for SSTR₁, SSTR_2A, SSTR_2B, antisera on tumour cells. The intense and diffuse immunostaining for corticotropin releasing hormone (CRH) antiserum indicated that Cushing’s disease was dependent on CRH overproduction by the pheochromocytoma, in which no immunostaining for adrenocorticotropic hormone was found. Our report confirms the heterogeneity of the pattern of SSTR expression in pheochromocytomas, and provide further evidence for functional SSTR subtype SSTR_2a in a subgroup of pheochromocytomas, suggesting that these tumours may represent potential target for octreotide treatment.Key words: phaeochromocytoma, neuroendocrine tumours, somatostatin receptors, octreotide, corticotropin releasing hormone.Phaeochromocytomas are tumours derived from the chromaffin cells of the sympathoadrenal system, generally associated with cathecolamine overproduction. They represent a rare condition, occurring in less than 0.2% of patients with hypertension. The diagnosis of sporadic phaeochromocytoma is based on clinical history and features characterized by the triad episodic headache, sweating, and tachycardia, but an increasing number of these tumours are diagnosed in patients without classical symptoms (Pacak et al., 2001). Ectopic Cushing’s syndrome is one of the possible, albeit unusual, expression of pheochromocytoma. Up to date, there are few reports of pheochromocytomas producing adrenocorticotropic hormone (ACTH) and/or ACTH precursors (O’Brien T et al., 1992; Chen et al., 1995; White et al., 2000), and even more limited is the number of cases in which pheochromocytoma secrete corticotropin releasing hormone (CRH) (Eng et al., 1999; Bayraktar et al., 2006).Similar to other neuroendocrine tumours, pheochromocytomas often express somatostatin receptors (SSTR) (De Herder and Hofland, 2004), but data on the specific SSTRs subtypes expressed within the tumours are thus far sparse and conflicting and the real therapeutic effectiveness of somatostatin analogue in these tumours is still debated (Reubi et al., 1992; Kubota et al., 1994; Epelbaum et al., 1995; Hofland et al., 1999; Mundschenk et al., 2003; Unger et al., 2004; Ueberberg et al., 2005; Unger et al., 2007).     

Feeding responses to central administration of several somatostatin analogs in chicks

Somatostatin is well known as an inhibitor of growth hormone release from the anterior pituitary. Its effects are exerted via 5 subtypes of receptors, which are named SSTR1 through 5. We recently reported that intracerebroventricular (ICV) injection of somatostatin stimulates feeding behavior in chicks. However, the specific receptors which mediate this orexigenic effect have not been identified in chicks. Thus, the purpose of the present study was to identify the receptor subtypes involved in somatostatin-induced feeding using 5 somatostatin analogs. Chicks that received vapreotide and octreotide (less than 3 nmol), which are agonist of SSTR2 and SSTR5, increased their food intake. Additionally, chicks ICV injected with BIM23056 or L-817,818 (SSTR3 and SSTR5 agonists, respectively) also had increased food intake. However, ICV injection of the SSTR4 agonist L-803,087 did not cause an orexigenic effect, suggesting that SSTR4 might not be important in somatostatin-induced feeding behavior. In summary, results from this study may be interpreted as SSTR2, SSTR3 and SSTR5 are related to somatostatin-associated feeding behavior in chicks.     

Synthesis,radiolabeling, and preclinical evaluation of a new octreotide analog for somatostatin receptor‐positive tumor scintigraphy

Radiolabeled somatostatin analogs have become powerful tools in the diagnosis and staging of neuroendocrine tumors, which express somatostatin receptors. The aim of this study was to evaluate a new somatostatin analog, 6‐hydrazinopyridine‐3‐carboxylic acid‐Ser³‐octreotate (HYNIC‐SATE) radiolabeled with ^99mTc, using ethylenediamine‐N,N′‐diacetic acid and tricine as coligands, to be used as a radiopharmaceutical for the in vivo imaging of somatostatin receptor subtype 2 (SSTR2)‐positive tumor. Synthesis of the peptide was carried out on a solid phase using a standard Fmoc strategy. Peptide conjugate affinities for SSTR2 were determined by receptor binding affinity on rat brain cortex and C6 cell membranes. Internalization rate of ^99mTc‐HYNIC‐SATE was studied in SSTR2‐expressing C6 cells that were used for intracranial tumor studies in rat brain. A reproducible in vivo C6 glioma model was developed in Sprague–Dawley rat and confirmed by histopathology and immunohistochemical analysis. Biodistribution and imaging properties of this new radiopeptide were also studied in C6 tumor‐bearing rats. Radiolabeling was performed at high specific activities, with a radiochemical purity of >96%. Peptide conjugate showed high affinity binding for SSTR2 (HYNIC‐SATE IC₅₀ = 1.60 ± 0.05 n m ) and specific internalization into rat C6 cells. After administration of ^99mTc‐HYNIC‐SATE in C6 glioma‐bearing rats, a receptor specific uptake of radioactivity was observed in SSTR‐positive organs and in the implanted intracranial tumor and rapid excretion from nontarget tissues via kidneys. ^99mTc‐HYNIC‐SATE is a new receptor‐specific radiopeptide for targeting SSTR2‐positive brain tumor and might be of great promise in the scintigraphy of SSTR2‐positive tumors. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

DNA typing of HLA-DR β chain genes can discriminate between undetected alleles and real homozygotes

The polymorphism of HLA-DR antigens has been studied by Southern blot hybridization under conditions specific for the detection of the DR chain genes. Haplotype-specific patterns were defined with DNA from DR1, 2, 3, 4, 7, w8, w11, w12, and W13 homozygous typing cells, with restriction enzymes Eco RI, Bgl I, and Pvu II. Certain serological specificities, such as DR2, DR3, and DR7, can be encoded by distinct allelic forms of DR chain genes. The procedure of DNA typing was applied to family analysis of individuals expressing only a single DR specificity upon serological typing. Three cases are described here: (1) in family GR, phenotypic DR 7 homozygotes correspond to genomic heterozygotes, and a novel DR7 allele is described: (2) in family RU, the genes corresponding to a serologically undetected (blank) DR allele were identified by restriction fragment length polymorphism (RFLP); this novel DR haplotype has an RFLP pattern similar to those of the DRw52 family, even though this specificity was not expressed on the DR-blank lymphocytes; (3) in family RG, there is no blank allele, but a homozygote RFLP situation at the DR subregion.     

Oligonucleotide genotyping shows that alleles at the HLA-DR β III locus of the DRw52 supertypic group segregate independently of known DR or Dw specificities

Using locus- and allele-specific oligonucleotide probes, we have studied the polymorphism of the HLA-DR III locus within the haplotypes of the DRw52 supertypic group. DNA from a number of homozygous typing cells typed for both Dw and DR was used. The DR III polymorphisms, DRw52a and DRw52b, do not segregate with Dw typing, or with DR typing, indicating that the determinants responsible for Dw-defined T -cell response and for DR haplotypic recognition are not encoded by the DR III locus. Hence, we can conclude that these DR specificities are encoded by the other functional DR locus, DR I, while the DR III locus encodes only the supertypic product.     

Osteogenic Sarcoma of the Sella After Radiation Treatment of a Pituitary Adenoma

ObjectiveTo report an uncommon case of osteogenic sarcoma of the sella turcica after radiation treatment of a pituitary adenoma.MethodsWe present the clinical history, physical findings, laboratory data, imaging studies, and pathologic findings in a patient found to have osteogenic sarcoma of the sella after radiation therapy for a nonfunctioning pituitary adenoma.ResultsSix years after transsphenoidal resection and postoperative fractionated radiation therapy for a nonfunctioning pituitary adenoma that extended to the cavernous sinus, a 45-year-old man presented with a sinus infection, diplopia, and ophthalmoplegia of the right eye. A computed tomographic scan of the head showed a mass in the sella with involvement of the optic chiasm and right cavernous sinus. Transsphenoidal resection and debulking of the tumor revealed an osteogenic sarcoma. The patient was discharged from the hospital with residual diplopia and ophthalmoplegia. He was treated with levothyroxine, testosterone, and hydrocortisone. Six weeks later, the patient was readmitted after he was found unresponsive, and computed tomographic scans disclosed a massive cerebrovascular accident. He died a few days later.ConclusionOsteogenic sarcoma is a rare, late complication of radiation treatment of pituitary adenoma. Although radiotherapy remains an effective adjunctive treatment in patients with pituitary adenomas, particularly those with residual or recurrent tumor, potential complications must be acknowledged. (Endocr Pract. 2004;10: 335-338)     

Somatostatin receptor-2 negatively regulates β-adrenergic receptor mediated Ca dependent signaling pathways in H9c2 cells

In the present study, we report that somatostatin receptor 2 (SSTR2) plays a crucial role in modulation of β₁AR and β₂AR mediated signaling pathways that are associated with increased intracellular Ca^2 + and cardiac complications. In H9c2 cells, SSTR2 colocalizes with β₁AR or β₂AR in receptor specific manner. SSTR2 selective agonist inhibits isoproterenol and formoterol stimulated cAMP formation and PKA phosphorylation in concentration dependent manner. In the presence of SSTR2 agonist, the expression of PKCα and PKCβ was comparable to the basal condition, however SSTR2 agonist inhibits isoproterenol or formoterol induced PKCα and PKCβ expression, respectively. Furthermore, the activation of SSTR2 not only inhibits calcineurin expression and its activity, but also blocks NFAT dephosphorylation and its nuclear translocation. SSTR2 selective agonist abrogates isoproterenol mediated increase in cell size and protein content (an index of hypertrophy). Taken together, the results described here provide direct evidence in support of cardiac protective role of SSTR2 via modulation of Ca^2 + associated signaling pathways attributed to cardiac hypertrophy.     

Immunological Detection of Isoforms of the Somatostatin Receptor Subtype, SSTR2

Abstract: Somatostatin (SRIF) induces its diverse physiological actions through interactions with different receptor subtypes. Multiple SRIF receptor subtypes have recently been cloned. To analyze the physical properties of receptor subtype SSTR2, two different peptide-directed antibodies were generated against SSTR2. Antibody “2e3,” directed against the peptide SSCTINWPGESGAWYT (residues 191–206), corresponding to a region in the predicted third extracellular domain of mouse SSTR2, and antibody “2i4,” directed against the peptide SGTEDGERSDS (residues 333–343) from the predicted cytoplasmic tail of mouse SSTR2, were developed. In Chinese hamster ovary (CHO) cells stably expressing the mouse SSTR2 gene (CHOB), the antibody 2e3 recognized specifically a protein of 93-kDa protein by immunoblotting. No specific immunoreactivity was detected by 2e3 in nontransfected CHO cells or CHO cells stably expressing vector alone or human SSTR1 or mouse SSTR3 genes. The antibody 2i4 specifically immunoprecipitated SSTR2 solubilized from CHOB cells that could be labeled with the SSTR2-specific ligand ¹²⁵I-MK-678. Furthermore, both 2e3 and 2i4 specifically immunoprecipitated 93-kDa [³⁵S]methionine-labeled proteins from CHOB cells, indicating that they recognize the same proteins. In contrast to studies in CHOB cells, immunoblotting studies showed that 2e3 detected specifically a single 148-kDa protein from different regions of the rat brain that have previously been shown to express high levels of SSTR2 mRNA and SRIF receptors with high affinity for ¹²⁵I-MK-678. In contrast, no immunoreactivity was detected in rat kidney, liver, or lung, which do not express SSTR2. No 93-kDa protein was detected specifically in the rat brain. The 148-kDa protein detected by 2e3 is an SRIF receptor because 2e3 and 2i4 specifically immunoprecipitated solubilized rat brain SRIF receptors that could be reversibly labeled with ¹²⁵I-MK-678. As in rat brain, 2e3 interacted specifically with a single 148-kDa protein in rat pituitary, in the rat pancreatic cell line AR42J, and in the HEK 293 cell line derived from human kidney, all of which express SSTR2 mRNA and SRIF receptors with high affinity for ¹²⁵I-MK-678. These findings indicate that rat brain and pituitary, as well as a pancreatic and a kidney cell line, express primarily a form of SSTR2 different from CHOB cells. The multiple forms of SSTR2 may result from differential post-translational processing of SSTR2 because 2e3 immunoprecipitated 41-kDa in vitro translation products generated from mRNA extracted from CHOB and AR42J cells. This 41-kDa protein has the predicted size of unprocessed SSTR2. These results demonstrate that 2e3 and 2i4 antibodies interact specifically with SSTR2. Detection of two different size proteins by the SSTR2 peptide-directed antibodies suggests the existence of multiple forms of SSTR2.     

Serological recognition of HLA-DR allodeterminant corresponding to DNA sequence involved in gene conversion

HLA class 11 molecules were isolated from mouse L cells transfected with a DR gene and an allele, 52a, of locus DR III from an HLA-homozygous cell line, AVL, of the DR3 haplotype. The isolated molecules were found to possess a new allospecificity, named TR81. This specificity behaved allelic to the previously described DR III locus. The TR81 specificity was also present on the DR I gene product of the DR3 haplotype. The nucleotide sequence of the gene encoding TR81 differs from TR81-negative DR genes of the DRw52 family in only two codons, both located in the regions known to be involved in a gene conversion event. Consequently, the following conclusions can be formulated. (a) TR81 is a bi-locus specificity and allelic to TR22 only in its DR III locus localization. (b) The TR81 specificity is the phenotypic counterpart of the gene conversion event which led to the generation of the DR I gene of the DR3 haplotype. (c) One or both individual amino acid substitutions in the first domain of the DR chain are responsible for the TR81 allospecificity. (d) Since TR81 is expressed on the DR I chain of the DR3 haplotype, it is possible that TR81 and DR3 represent the same serological specificity.     

Loss of Somatostatin Receptor Subtype 2 in Prostate Cancer Is Linked to an Aggressive Cancer Phenotype,High Tumor Cell Proliferation and Predicts Early Metastatic and Biochemical Relapse

Somatostatin receptor subtype 2 (SSTR2) is the most frequently expressed SSTR subtype in normal human tissues. SSTR2 expression is differentially regulated in various tumor types and therapeutic somatostatin analogs binding to SSTR2 are in clinical use. In prostate cancers highly contradictory results in terms of SSTR2 expression and its consequences have been published over the past years. The aim of this study was to clarify prevalence and clinical significance of SSTR2 expression in prostate cancer. Therefore, quantitative immunohistochemistry (IHC) using a tissue microarray containing samples from 3,261 prostate cancer patients with extensive clinical and molecular cancer characteristics and oncological follow-up data was performed. IHC data was compared to publicly available Gene Expression Omnibus datasets of human prostate cancer gene expression arrays. While membranous SSTR2 staining was always seen in normal prostate epithelium, SSTR2 staining was absent in more than half (56.1%) of 2,195 interpretable prostate cancer samples. About 13% of all analyzed prostate cancers showed moderate to strong cytoplasmic and membranous SSTR2 staining. Staining intensities were inversely correlated with high Gleason grade, advanced pT category, high tumor cell proliferation (p<0.0001 each), high pre-operative PSA levels, (p = 0.0011) and positive surgical margins (p = 0.006). In silico analysis confirmed lower SSTR2 gene expression in prostate cancers vs. normal adjacent tissue (p = 0.0424), prostate cancer metastases vs. primary cancers (p = 0.0011) and recurrent vs. non-recurrent prostate cancers (p = 0.0438). PSA-free survival gradually declined with SSTR2 staining intensity (p<0.0001). SSTR2-negative cancers were more likely to develop metastases over time (p<0.05). In conclusion, most prostate cancers are indeed SSTR2-negative and loss of SSTR2 strongly predicts an unfavorable tumor phenotype and poor prognosis. Therefore, SSTR2 expression seems an important factor in the pathogenesis of prostate cancer and re-introduction of the receptor in SSTR2-negative prostate cancers may feature a promising target for novel gene therapy approaches.     

DNA typing of HLA-DR antigens in systemic lupus erythematosus

HLA-DR typing is technically difficult in systemic lupus erythematosus (SLE), where patients have low numbers of peripheral B cells, often of poor viability and weak in antigenic expression. In this series, one third of SLE patients could not be HLA-DR typed by serological techniques, highlighting the potential for systematic bias in DR antigen assignment in studies of HLA and SLE. This potential bias was examined by comparing serological DR results with DNA-DR typing, achieved by examining Taq I fragments of DR, DQ, and DQ which permitted unequivocal DR phenotyping of all patients, including the 35% for whom conventional DR typing was technically impossible. This showed the success of serological DR antigen assignment was indeed nonrandom, with HLA-DR2 and -DR3 significantly more readily identifiable than DR5 and DRw13. Our study suggests that technical problems may well have contributed to conflicting reports regarding the association of DR2 or DR3 with SLE. Here, DNA-DR phenotyping showed HLA-DR3 is significantly (P<.05) associated with an increased risk for SLE (54% in 46 patients, 36% in 134 controls) and HLA-DR2 is not.     

Cell growth inhibition and functioning of human somatostatin receptor type 2 are modulated by receptor heterodimerization

Somatostatin (SST) analogs have been successfully used in the medical treatment of acromegaly, caused by GH hypersecreting pituitary adenomas. Patients on SST analogs rarely develop tachyphylaxis despite years of continuous administration. It has been recently proposed that a functional association between SST receptor (SSTR) subtypes 2 and 5 exists to account for this behavior; however, a physical interaction has yet to be identified. Using both coimmunoprecipitation and photobleaching fluorescence resonance energy transfer microscopy techniques, we determined that SSTR2 and SSTR5 heterodimerize. Surprisingly, selective activation of SSTR2 and not SSTR5, or their costimulation, modulates the association. The SSTR2-selective agonist L-779,976 is more efficacious at inhibiting adenylate cyclase, activating ERK1/2, and inducing the cyclin-dependent kinase inhibitor p27(Kip1) in cells expressing both SSTR2 and SSTR5 compared with SSTR2 alone. Furthermore, cell growth inhibition by L-779,976 treatment was markedly extended in coexpressing cells. Trafficking of SSTR2 is also affected upon heterodimerization, an attribute corresponding to modifications in beta-arrestin association kinetics. Activation of SSTR2 results in the recruitment and stable association of beta-arrestin, followed by receptor internalization and intracellular receptor pooling. In contrast, heterodimerization increases the recycling rate of internalized SSTR2 by destabilizing its interaction with beta-arrestin. Given that SST analogs show preferential binding to SSTR2, these data provide a mechanism for their effectiveness in controlling pituitary tumors and the absence of tolerance seen in patients undergoing long-term administration.     

Effects of somatostatin and its analogues on tyrosine kinase activity in rodent tumors

The effects of native somatostatin-14 (SS-14) and of its two analogues, octreotide and CH-275, on the activity of tyrosine kinases (TK) in two rodent tumors (rat pituitary tumor and murine colonic cancer) were studied in vitro. The activity of TK was measured in tissue homogenates using gamma[(32)P]ATP as the donor of the phosphoryl group and poly(Glu(80), Tyr(20)) as a substrate. It was found that native SS-14 inhibited TK activity in both investigated tumors. Octreotide, which acts preferentially via somatostatin receptor subtype 2 (SSTR2), was very effective in inhibiting TK activity in the rat pituitary tumor, but it is a rather weak inhibitor of TK activity in murine colonic cancer. CH-275, a selective ligand of the SSTR1 subtype of SS receptors, suppressed TK activity in the pituitary tumor but was ineffective in the colonic cancer. It is hypothesized that the effect of neuropeptide somatostatin (SS-14) on murine colonic cancer is exerted via the subtype of receptor which does not interact with CH-275 and has no or low affinity for octreotide (SSTR 4, 3 or 5?).