B. Ueberberg

Expression of ghrelin and its receptor in human tissues.

Ghrelin is a peptide thought to be involved in the regulation of appetite. Furthermore, significant effects on the release of growth hormone (GH) and ACTH were demonstrated. Contributing to the physiological relevance of this hormone, we investigated the expression of ghrelin and its receptor (GHS-R) in several normal human tissues. RNA samples (BD Biosciences) underwent one-step TaqMan Real-Time RT-PCR. Immunohistochemistry was performed on paraffin-embedded tissues using specific primary antibodies against ghrelin and its receptor. Relevant ghrelin mRNA levels were detected in all human tissues with the highest levels in stomach, pituitary, and small intestine. By immunohistochemistry, ghrelin peptide expression was detectable in reproductive and endocrine organs (ovary, anterior pituitary, adrenal gland), and organs of the gastrointestinal tract (stomach, pancreas). GHS-R1a mRNA expression was demonstrated in 10 of 24 human organs analyzed with the highest levels in pituitary, adrenal gland, and spinal cord. Expression of the receptor peptide was detected by immunohistochemistry in endocrine and reproductive organs (anterior pituitary, thyroid, pancreas, testis), parts of the CNS (cerebrum, cerebellum), and in single cells of bone marrow. Expression of both ghrelin and its receptor in endocrine and reproductive organs may indicate new endocrine or paracrine mechanisms of regulation in these tissues.

Differential expression of ghrelin and its receptor (GHS-R1a) in various adrenal tumors and normal adrenal gland.

Ghrelin is a newly characterized, widely distributed peptide thought to be involved in the regulation of appetite. Significant effects on the release of growth hormone (GH) and ACTH have been demonstrated. This study compares the expression of ghrelin and its receptor (GHS-R) in various adrenal tumors and normal adrenal gland. Normal adrenal tissue was obtained after autopsy. Tissue was obtained from 13 pheochromocytomas (PHEOs), 15 cortisol-secreting adenomas (CPAs), 12 aldosterone-secreting adenomas (APAs), and 16 nonfunctional adenomas (NFAs) following laparoscopic surgery. Expression of ghrelin and GHS-R1a was investigated on RNA levels by using real-time reverse transcription polymerase chain reaction (RT-PCR) and on protein levels by using immunohistochemistry. In the seven normal adrenal glands analyzed, ghrelin mRNA levels were 12-fold lower than in stomach. Ghrelin protein expression was confirmed by immunohistochemistry. In all adrenal tumors, relevant levels of ghrelin mRNA were observed, with significantly lower expression in PHEOs and APAs than in normal adrenal gland. Ghrelin protein was detected in 0% of PHEOs, 55% of APAs, 87% of CPAs, and 54% of NFAs. GHS-R1a mRNA expression was detectable in normal adrenal gland, but the receptor protein was absent. In adrenal tumors, detectable levels of receptor mRNA were found in 38% of PHEOs, 13% of CPAs, and 25% of NFAs. GHS-R1a protein was absent in the majority of adrenal tumors. Expression of ghrelin in normal adrenal gland and adrenal tumors may indicate some unknown physiological function. The pathophysiological relevance of ghrelin expression in adrenal tumors remains to be investigated.

Differential Expression of Somatostatin Receptor Subtype 1–5 Proteins in Numerous Human Normal Tissues

5 Human somatostatin receptor subtypes (sst1-5) mediate the antisecretory and antiproliferative effects of somatostatin. We examined somatostatin receptor protein expression in 28 human normal tissues. Immunostaining was performed with specific polyclonal antibodies for sst1-5. Staining pattern and distribution of ssts were evaluated. Anterior pituitary was positively stained for all 5 ssts. Pancreatic islets exhibited a positive staining for sst1-3 and sst5. Adrenal cortex expressed all 5 receptor subtypes, while the medulla was positive for sst3 and sst5 only. The thyroid expressed sst5 only, limited to single interfollicular cells. All 5 ssts were detected in the ovary, limited to luteinized granulosa cells of the corpus luteum. In the testis, sst2A was detected in the basal parts of the tubules, while sst5 was positively stained in the luminal parts. Sst1 was found in Leydig cells only. Stomach was positively stained for all 5 ssts. Investigation of the kidney revealed differential expression, with sst2A being found in the glomerules. The tubules expressed all 5 ssts. In the bone marrow cells of the granulocytopoiesis expressed sst2A only. The cerebellum expressed sst5 in a certain cell type, representing presumably Purkinje cells, while sst2A was stained in intercellular fibers. The expression of somatostatin receptor subtypes in a variety of human normal tissues may indicate a physiological role in these organs. Somatostatin analogues may offer new diagnostic and therapeutic implications for tumours related to these tissues. However, treatment of defined tumours with somatostatin analogues may also alter other normal tissues.

Association of Somatostatin Receptor 2 Immunohistochemical Expression with [111In]-DTPA Octreotide Scintigraphy and [68Ga]-DOTATOC PET/CT in Neuroendocrine Tumors

In the absence of preoperative somatostatin receptor ( SST) scans, knowledge of immunohistochemical SST2 tumor expression may help predicting the success of somatostatin analogue-based follow-up studies and treatment of neuroendocrine tumors (NET). We studied the association between SST immunostaining and tracer uptake in [(111)In]-DTPA octreotide (DTPAOC) scintigraphy and [(68)Ga]-DOTA-D-Phe(1)-Tyr(3)-octreotide (DOTATOC) positron emission tomography (PET)/computed tomography (CT). Retrospective analy-sis of 36 NET patients was carried out. In 40 tumors, immunohistochemical SST2, SST3, and SST5 expressions were analyzed using a pathological scoring, applying monoclonal ( SST2) or polyclonal antibodies (SST3, SST5). In 14 lesions, [(111)In]-DTPAOC uptake was assessed by a semiquantitative score. In 26 tumors, [(68)Ga]-DOTATOC PET/CT was quantified using an uptake score and maximal standard uptake value (SUV(max)). Combined and separate qualitative analysis of SST scans revealed significant associations between increased tracer uptake and immunohistochemical SST2 detection (combined: rho=0.56, p=0.0002, [(111)In]-DTPAOC: rho=0.63, p=0.0152, and [(68)Ga]-DOTATOC: rho=0.52, p=0.0065, respectively). In contrast, SST3 and SST5 immunostaining was not associated with tracer uptake (all p>0.14). The semiquantitative immunohistochemical score for SST2 was associated with the [(68)Ga]-DOTATOC uptake score and SUV (max) values (rho=0.67, p=0.0002 and rho=0.63, p=0.0010, respectively), but not with the [(111)In]-DTPAOC uptake score (rho=0.24, p=0.4). In patients without preoperative SST scans, knowledge of immunohistochemical SST2 expression may help estimating the value of SST imaging in the clinical follow-up, in particular in those lesions with positive SST2 immunostaining. Negativity for SST2, however, does not rule out tracer uptake in some patients, with heterogeneous SST2 expression within the tumor as a potential explanation.

Expression of the proliferation marker Ki‐67 associates with tumour staging and clinical outcome in differentiated thyroid carcinomas

Although the prognosis of differentiated thyroid carcinoma (DTC) is excellent, with 10‐year survival rates of about 90%, about one‐third of patients experiences recurrent disease. We aimed to identify novel histological prognostic factors to optimize treatment and follow‐up of patients at risks.

Transcriptional regulation of the human somatostatin receptor subtypes (sst 1–5) in adrenal cortex and medulla cells

Background: Somatostatin (SRIF) is a widely distributed peptide with inhibitory effects on cell proliferation in normal and tumor tissues. In RNA obtained from normal adrenal gland, we demonstrated significant expression of all five ssts using RT/PCR. Pheochromocytomas, Conn adenomas, Cushings adenomas and nonfunctional adenomas showed specific sst expression profiles for each tumor typ. To understand the regulation of sst expression in the adrenal gland, we investigated the transcriptional activity of the sst 1–5 promoters. Material and Methods: Transcription start sites for the various sst genes were previously identified. Promoter regions (-2725sst1, -1100sst2, -2536sst3, -984sst4, and -1740sst5) obtained by PCR on human genomic DNA were cloned upstream of the luciferase gene into a luciferase expression vector. After transient transfection, the promoter activity was determined using a luciferase reporter gene assay. We investigated the promoter activity in mouse adrenocortical Y1 cells and rat adrenal medulla PC12 cells. GH4 rat pituitary cells and Cos-7 monkey kidney cells were used as positive and negative controls, respectively. Results: GH4 demonstrated significant transcriptional promoter activity of all five ssts, whereas no significant promoter activity was found in Cos-7 cells. In Y1 cells, we demonstrated significant transcriptional activity of the sst 1–5 promoters. In contrast, the tested promoter regions showed no activity in the PC12 cells. Elements transducing the transcriptional activity in Y1 cells were localized to -368 to -233, -325 to -252, -2536 to -1896, -266 to -209, -101 to -1, for sst1, sst2, sst3, sst4, and sst5, respectively. Conclusions: Sst promoter regions identified in the pituitary may also regulate sst 1–5 expression in the adrenal cortex. In contrast, alternative promoter regions may regulate sst transcription in the adrenal medulla.