Maternal pheochromocytoma and childbirth in Sweden 1973-2015: a population-based study on short and long-term outcome.

PURPOSE: Data guiding management of pheochromocytoma and paraganglioma (PPGL) in pregnant women is limited, and long-term effects on the child are unknown. The aim of this retrospective registry-based case-cohort study was to assess how maternal PPGL and treatment impacts maternal and fetal outcome, including long-term outcome for the child. The main outcomes were maternal and fetal mortality and morbidity at delivery and relative healthcare consumption in children born by mothers with PPGL during pregnancy. METHODS: The National Birth Register identified 4,390,869 pregnancies between 1973-2015. Data was crosslinked with three Swedish national registers to identify women diagnosed with pheochromocytoma or paraganglioma within one year before or after childbirth. Hospital records were reviewed and register data was collected for five age-matched controls for each child until age 18. RESULTS: 21 women and 23 children were identified (incidence 4.8/1.000.000 births/year), all women with adrenal pheochromocytomas (Pc). The majority (71%) were diagnosed post-partum. Nine women (43%) were hypertensive during pregnancy. Preterm delivery was more common in Pc patients compared to controls (30% vs 6%, p < 0.001). There was no maternal or fetal mortality. Timing of tumor removal did not affect gestational weight or APGAR scores. There was no observed difference in hospital admissions between children affected by maternal Pc and controls. CONCLUSION: Pc was commonly diagnosed after delivery and raised the risk of pre-term delivery, suggesting a need for an increased awareness of this diagnosis. However, reassuringly, there was no fetal or maternal mortality or any observed long-term impact on the children.

Cytoplasmic HIF-2α as tissue biomarker to identify metastatic sympathetic paraganglioma.

Pheochromocytomas (PCCs) and paragangliomas (PGLs) are rare neuroendocrine tumors. PGLs can further be divided into sympathetic (sPGLs) and head-and-neck (HN-PGLs). There are virtually no treatment options, and no cure, for metastatic PCCs and PGLs (PPGLs). Here, we composed a tissue microarray (TMA) consisting of 149 PPGLs, reflecting clinical features, presenting as a useful resource. Mutations in the pseudohypoxic marker HIF-2α correlate to an aggressive tumor phenotype. We show that HIF-2α localized to the cytoplasm in PPGLs. This subcompartmentalized protein expression differed between tumor subtypes, and strongly correlated to proliferation. Half of all sPGLs were metastatic at time of diagnosis. Cytoplasmic HIF-2α was strongly expressed in metastatic sPGLs and predicted poor outcome in this subgroup. We propose that higher cytoplasmic HIF-2α expression could serve as a useful clinical marker to differentiate paragangliomas from pheochromocytomas, and may help predict outcome in sPGL patients.

Exposure to nonanoic acid alters small intestinal neuroendocrine tumor phenotype.

BACKGROUND: Small intestinal neuroendocrine tumors (SI-NET) are highly differentiated and genetically stable malignant tumors, yet they often present with advanced metastatic spread at the time of diagnosis. In contrast to many other types of malignant tumors, primary SI-NET are often asymptomatic and typically smaller in size compared to adjacent lymph node metastases. This study explores the hypothesis that stimulating the chemosensing olfactory receptor 51E1 (OR51E1) decreases SI-NET proliferation suggesting a mechanism that explains a difference in proliferative rate based on tumor location. METHODS: Clinical data was used to address difference in tumor size depending on location. A SI-NET tissue microarray was used to evaluate expression of OR51E1 and olfactory marker protein (OMP). Primary cultured tumor cells from 5 patients were utilized to determine the effect of OR51E1 agonist nonanoic acid on metabolic activity. The SI-NET cell line GOT1 was used to determine effects of nonanoic acid on the transcriptome as well as long-term effects of nonanoic acid exposure with regards to cell proliferation, serotonin secretion, alterations of the cell-cycle and morphology. RESULTS: Tumor size differed significantly based on location. OR51E1 and OMP were generally expressed in SI-NET. Primary SI-NET cells responded to nonanoic acid with a dose dependent altered metabolic activity and this was replicated in the GOT1 cell line but not in the MCF10A control cell line. Nonanoic acid treatment in GOT1 cells upregulated transcripts related to neuroendocrine differentiation and hormone secretion. Long-term nonanoic acid treatment of GOT1 cells decreased proliferation, induced senescence, and altered cell morphology. CONCLUSION: Our results raise the possibility that exposure of intraluminal metabolites could represent a mechanism determining aspects of the SI-NET tumor phenotype. However, we could not causally link the observed effects of nonanoic acid exposure to the OR51E1 receptor.

Independent somatic evolution underlies clustered neuroendocrine tumors in the human small intestine.

Small intestine neuroendocrine tumor (SI-NET), the most common cancer of the small bowel, often displays a curious multifocal phenotype with several tumors clustered together in a limited intestinal segment. SI-NET also shows an unusual absence of driver mutations explaining tumor initiation and metastatic spread. The evolutionary trajectories that underlie multifocal SI-NET lesions could provide insight into the underlying tumor biology, but this question remains unresolved. Here, we determine the complete genome sequences of 61 tumors and metastases from 11 patients with multifocal SI-NET, allowing for elucidation of phylogenetic relationships between tumors within single patients. Intra-individual comparisons revealed a lack of shared somatic single-nucleotide variants among the sampled intestinal lesions, supporting an independent clonal origin. Furthermore, in three of the patients, two independent tumors had metastasized. We conclude that primary multifocal SI-NETs generally arise from clonally independent cells, suggesting a contribution from a cancer-priming local factor.

Evaluation of SSTR2 Expression in SI-NETs and Relation to Overall Survival after PRRT.

(1) Purpose: Small intestinal neuroendocrine tumors (SI-NETs) often present with distant metastases at diagnosis. Peptide receptor radionuclide therapy (PRRT) with radiolabeled somatostatin analogues is a systemic treatment that increases overall survival (OS) in SI-NET patients with stage IV disease. However, the treatment response after PRRT, which targets somatostatin receptor 2 (SSTR2), is variable and predictive factors have not been established. This exploratory study aims to evaluate if SSTR2 expression in SI-NETs could be used to predict OS after PRRT treatment. (2) Methods: Using a previously constructed Tissue Micro Array (TMA) we identified tissue samples from 42 patients that had received PRRT treatment during 2006-2017 at Sahlgrenska University hospital. Immunohistochemical expression of SSTR2, Ki-67 and neuroendocrine markers synaptophysin and Chromogranin A (CgA) were assessed. A retrospective estimation of 177Lu-DOTATATE uptake in 33 patients was performed. Data regarding OS and non-surgical treatment after PRRT were collected. Another subgroup of 34 patients with paired samples from 3 tumor sites (primary tumor, lymph node and liver metastases) was identified in the TMA. The SSTR2 expression was assessed in corresponding tissue samples (n = 102). (3) Results: The patients were grouped into Low SSTR2 or High SSTR2 groups based upon on levels of SSTR2 expression. There was no significant difference in 177Lu-DOTATATE uptake between the groups. The patients in the Low SSTR2 group had significantly longer OS after PRRT than the patients in the High SSTR2 group (p = 0.049). PRRT treated patients with low SSTR2 expression received less additional treatment compared with patients with high SSTR2 expression. SSTR2 expression did not vary between tumor sites but correlated within patients. (4) Conclusion: The results from the present study suggest that retrospective evaluation of SSTR2 expression in resected tumors cannot be used to predict OS after PRRT.

SMAD4 haploinsufficiency in small intestinal neuroendocrine tumors.

BACKGROUND: Patients with small intestinal neuroendocrine tumors (SINETs) frequently present with lymph node and liver metastases at the time of diagnosis, but the molecular changes that lead to the progression of these tumors are largely unknown. Sequencing studies have only identified recurrent point mutations at low frequencies with CDKN1B being the most common harboring heterozygous mutations in less than 10% of all tumors. Although SINETs are genetically stable tumors with a low frequency of point mutations and indels, they often harbor recurrent hemizygous copy number alterations (CNAs) yet the functional implications of these CNA are unclear. METHODS: Utilizing comparative genomic hybridization (CGH) arrays we analyzed the CNA profile of 131 SINETs from 117 patients. Two tumor suppressor genes and corresponding proteins i.e. SMAD4, and CDKN1B, were further characterized using a tissue microarray (TMA) with 846 SINETs. Immunohistochemistry (IHC) was used to quantify protein expression in TMA samples and this was correlated with chromosome number evaluated with fluorescent in-situ hybridization (FISH). Intestinal tissue from a Smad4+/- mouse model was used to detect entero-endocrine cell hyperplasia with IHC. RESULTS: Analyzing the CGH arrays we found loss of chromosome 18q and SMAD4 in 71% of SINETs and that focal loss of chromosome 12 affecting the CDKN1B was present in 9.4% of SINETs. No homozygous loss of chromosome 18 was detected. Hemizygous loss of SMAD4, but not CDKN1B, significantly correlated with reduced protein levels but hemizygous loss of SMAD4 did not induce entero-endocrine cell hyperplasia in the Smad4+/- mouse model. In addition, patients with low SMAD4 protein expression in primary tumors more often presented with metastatic disease. CONCLUSIONS: Hemizygous loss of chromosome 18q and the SMAD4 gene is the most common genetic event in SINETs and our results suggests that this could influence SMAD4 protein expression and spread of metastases. Although SMAD4 haploinsufficiency alone did not induce tumor initiation, loss of chromosome 18 could represent an evolutionary advantage in SINETs explaining the high prevalence of this aberration. Functional consequences of reduced SMAD4 protein levels could hypothetically be a potential mechanism as to why loss of chromosome 18 appears to be clonally selected in SINETs.

Suppression of enteroendocrine cell glucagon-like peptide (GLP)-1 release by fat-induced small intestinal ketogenesis: a mechanism targeted by Roux-en-Y gastric bypass surgery but not by preoperative very-low-calorie diet.

OBJECTIVE: Food intake normally stimulates release of satiety and insulin-stimulating intestinal hormones, such as glucagon-like peptide (GLP)-1. This response is blunted in obese insulin resistant subjects, but is rapidly restored following Roux-en-Y gastric bypass (RYGB) surgery. We hypothesised this to be a result of the metabolic changes taking place in the small intestinal mucosa following the anatomical rearrangement after RYGB surgery, and aimed at identifying such mechanisms. DESIGN: Jejunal mucosa biopsies from patients undergoing RYGB surgery were retrieved before and after very-low calorie diet, at time of surgery and 6 months postoperatively. Samples were analysed by global protein expression analysis and Western blotting. Biological functionality of these findings was explored in mice and enteroendocrine cells (EECs) primary mouse jejunal cell cultures. RESULTS: The most prominent change found after RYGB was decreased jejunal expression of the rate-limiting ketogenic enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHMGCS), corroborated by decreased ketone body levels. In mice, prolonged high-fat feeding induced the expression of mHMGCS and functional ketogenesis in jejunum. The effect of ketone bodies on gut peptide secretion in EECs showed a ∼40% inhibition of GLP-1 release compared with baseline. CONCLUSION: Intestinal ketogenesis is induced by high-fat diet and inhibited by RYGB surgery. In cell culture, ketone bodies inhibited GLP-1 release from EECs. Thus, we suggest that this may be a mechanism by which RYGB can remove the inhibitory effect of ketone bodies on EECs, thereby restituting the responsiveness of EECs resulting in increased meal-stimulated levels of GLP-1 after surgery.

177Lu-octreotate therapy for neuroendocrine tumours is enhanced by Hsp90 inhibition.

177Lu-octreotate is an FDA-approved radionuclide therapy for patients with gastroenteropancreatic neuroendocrine tumours (NETs) expressing somatostatin receptors. The 177Lu-octreotate therapy has shown promising results in clinical trials by prolonging progression-free survival, but complete responses are still uncommon. The aim of this study was to improve the 177Lu-octreotate therapy by means of combination therapy. To identify radiosensitising inhibitors, two cell lines, GOT1 and P-STS, derived from small intestinal neuroendocrine tumours (SINETs), were screened with 1,224 inhibitors alone or in combination with external radiation. The screening revealed that inhibitors of Hsp90 can potentiate the tumour cell-killing effect of radiation in a synergistic fashion (GOT1; false discovery rate <3.2×10-11). The potential for Hsp90 inhibitor ganetespib to enhance the anti-tumour effect of 177Lu-octreotate in an in vivo setting was studied in the somatostatin receptor-expressing GOT1 xenograft model. The combination led to a larger decrease in tumour volume relative to monotherapies and the tumour-reducing effect was shown to be synergistic. Using patient-derived tumour cells from eight metastatic SINETs, we could show that ganetespib enhanced the effect of 177Lu-octreotate therapy for all investigated patient tumours. Levels of Hsp90 protein expression were evaluated in 767 SINETs from 379 patients. We found that Hsp90 expression was upregulated in tumour cells relative to tumour stroma in the vast majority of SINETs. We conclude that Hsp90 inhibitors enhance the tumour-killing effect of 177Lu-octreotate therapy synergistically in SINET tumour models and suggest that this potentially promising combination should be further evaluated.

Expression of tight-junction proteins in human proximal small intestinal mucosa before and after Roux-en-Y gastric bypass surgery.

BACKGROUND: Increased permeability and uptake of proinflammatory bacterial endotoxins from gut microbiota has been suggested as a mechanism for obesity-associated chronic inflammation that causes obesity-associated insulin resistance. We hypothesized that intestinal barrier function may be restored after Roux-en-Y gastric bypass (RYGB) surgery and thereby contribute to decreased inflammation. The objective of this study was to investigate levels of the permeability-regulating tight-junction proteins in human small intestinal mucosa before and after RYGB surgery. METHODS: Paired intraindividual jejunal mucosa samples were retrieved at the time of surgery and 6 to 8 months after surgery. Mucosal cell surface area was calculated by histomorphometry. Mucosal samples were analyzed by proteomics to find patterns of protein regulations. Based on these findings further analyses were performed by Western blotting. Ussing chambers were used to analyze permeability in the retrieved mucosal samples. RESULTS: Mucosal surface area was significantly decreased after surgery. Global protein expression analysis showed a significant increase in the cytokeratin-8 (Ck8), which was confirmed by Western blotting. Further analyses showed a significant increase in claudin-3 and -4 expression after surgery, whereas occludin and zonula occludens-1 levels were decreased. Expressions of claudin-1, -2, -5 and vinculin were unchanged. Ussing chamber experiments revealed a linear correlation between the epithelial electrical resistance and claudin-3 protein expression. CONCLUSION: Several alterations were found in the rerouted small intestine after surgery, indicating a decreased jejunal mucosal surface area and decreased paracellular permeability. These changes could contribute to decreased uptake of luminal microbiota-derived inflammatory mediators such as endotoxins after RYGB.

Erythrocyte sodium-lithium countertransport activity is inversely correlated to adiponectin, retinol binding protein 4 and body height.

BACKGROUND: We have previously described that the sodium/lithium countertransport (SLC) in the erythrocyte cell membrane is closely linked to obesity and insulin resistance. Adiponectin and retinol-binding protein 4 (RBP-4) are believed to affect obesity and insulin resistance. In the present study, we aimed to further characterize the relationship between SLC, inflammatory markers, adiponectin and RBP-4. METHODS: We included 93 clinically healthy 58-year-old men selected to display variations in insulin sensitivity. High sensitivity C-reactive protein (hs-CRP), TNF-alpha, soluble TNF-alpha-receptors (sTNFR) 1 and 2, IL-6 and RBP-4 were measured using antibody-based techniques. Adiponectin was determined by a radioimmunoassay kit. The lithium concentration in the special flux medium was measured by atomic absorption spectrophotometry. RESULTS: In univariate analyses, SLC correlated negatively with RBP-4 (r(s) = -0.256, p = -0.017) and to adiponectin (r(s) = -0.316, p = 0.003) and positively with TNF-alpha (r(s) = 0.346, p = 0.001) and hs-CRP (r(s) = 0.288, p = 0.005). There were no statistically significant correlations with sTNFR 1 or 2 or IL-6. SLC was negatively associated to body height (r(s) = -0.256, p = 0.013). CONCLUSIONS: We are the first to report that SLC correlates negatively with adiponectin and RBP-4. This finding is intriguing, as adiponectin is anti-inflammatory and anti-diabetic whereas RBP-4 supposedly decreases insulin sensitivity. We also observed a negative association between SLC activity and body height indicating that SLC activity is not primarily influenced by fat mass. The positive association of SLC with markers of inflammatory activity such as TNF-alpha and hs-CRP is in line with the proposed link between inflammation and insulin resistance.

Myxoid liposarcoma FUS-DDIT3 fusion oncogene induces C/EBP beta-mediated interleukin 6 expression.

The myxoid/round cell liposarcoma oncogene FUS-DDIT3 is the result of a translocation derived gene fusion between the splicing factor FUS and DDIT3. In order to investigate the downstream targets of DDIT3, and the transforming effects of the FUS-DDIT3 fusion protein, we have introduced DDIT3-GFP and FUS-DDIT3-GFP constructs into a human fibrosarcoma cell line. The gene expression profiles of stable transfectants were compared to the original fibrosarcoma cell line by microarray analysis. We here report that the NFkappaB and C/EBP beta controlled gene IL6 is upregulated in DDIT3- and FUS-DDIT3-expressing fibrosarcoma cell lines and in myxoid liposarcoma cell lines. Strong expression of the tumor associated multifunctional cytokine interleukin 6 was confirmed both at mRNA and protein level. Knockdown experiments using siRNA against CEBPB transcripts showed that the effect of FUS-DDIT3 on IL6 expression is C/EBP beta dependent. Chromatin immunoprecipitation revealed direct interaction between the IL6 promoter and the C/EBP beta protein. In addition, the effect of DDIT3 and FUS-DDIT3 on the expression of other acute phase genes was examined using real-time PCR. We demonstrate for the first time that DDIT3 and FUS-DDIT3 show opposite transcriptional regulation of IL8 and suggest that FUS-DDIT3 may affect the synergistic activation of promoters regulated by C/EBP beta and NFkappaB.