Overexpression of MicroRNA miR-7-5p Is a Potential Biomarker in Neuroendocrine Neoplasms of the Small Intestine.

BACKGROUND/AIMS: Neuroendocrine neoplasms of the small intestine (SI-NENs) constitute 25-30% of all gastroenteropancreatic NEN. These tumors arise from enterochromaffin cells, and little is known about their microRNA (miRNA) expression. The purpose of this study was to characterize the expression of miRNAs in SI-NEN and to determine the potential of miRNAs as noninvasive blood-based biomarkers. METHODS: miRNA was purified from 15 tumor and 7 control tissue samples, converted to cDNA, and applied to a miScript miRNA PCR. The small nucleolar RNA, SNORD95, was used as an endogenous control. RESULTS: Microarray analysis revealed 7 miRNAs that showed a promising distinction between tumorous and healthy tissue. The miRNAs miR-7-5p and miR-96-5p were clearly upregulated in the tumor compared to the healthy tissue. In contrast, miRNAs miR-9-5p, miR-122-5p, miR-124-3p, miR-143-3p, and miR-144-3p showed a distinct downregulation in the tumor compared to the healthy tissue. These results were validated on a further 15 tumor samples, and the findings held true. As the miR-7-5p was significantly upregulated and revealed a low range across tumor samples, its presence was tested in the sera of 32 tumor patients and 25 healthy controls. Sera from all patients with SI-NENs had significantly higher levels of miR-7-5p than those from the 25 healthy controls (p = 0.0002), whereas there was no correlation with age, gender, or T-stage or UICC stage. CONCLUSION: The miRNA miR-7-5p may be a promising biomarker test for SI-NEN, which should be validated in a large-scale prospective study.

Loss of Chromosome 18 in Neuroendocrine Tumors of the Small Intestine: The Enigma Remains.

BACKGROUND/AIMS: Neuroendocrine tumors of the small intestine (SI-NETs) exhibit an increasing incidence and high mortality rate. Until now, no fundamental molecular event has been linked to the tumorigenesis and progression of these tumors. Only the loss of chromosome 18 (Chr18) has been shown in up to two thirds of SI-NETs, whereby the significance of this alteration is still not understood. We therefore performed the first comprehensive study to identify Chr18-related events at the genetic, epigenetic and gene/protein expression levels. METHODS: We did expression analysis of all seven putative Chr18-related tumor suppressors by quantitative real-time PCR (qRT-PCR), Western blot and immunohistochemistry. Next-generation exome sequencing and SNP array analysis were performed with five SI-NETs with (partial) loss of Chr18. Finally, we analyzed all microRNAs (miRNAs) located on Chr18 by qRT-PCR, comparing Chr18+/- and Chr18+/+ SI-NETs. RESULTS: Only DCC (deleted in colorectal cancer) revealed loss of/greatly reduced expression in 6/21 cases (29%). No relevant loss of SMAD2, SMAD4, elongin A3 and CABLES was detected. PMAIP1 and maspin were absent at the protein level. Next-generation sequencing did not reveal relevant recurrent somatic mutations on Chr18 either in an exploratory cohort of five SI-NETs, or in a validation cohort (n = 30). SNP array analysis showed no additional losses. The quantitative analysis of all 27 Chr18-related miRNAs revealed no difference in expression between Chr18+/- and Chr18+/+ SI-NETs. CONCLUSION: DCC seems to be the only Chr18-related tumor suppressor affected by the monoallelic loss of Chr18 resulting in a loss of DCC protein expression in one third of SI-NETs. No additional genetic or epigenetic alterations were present on Chr18.

Ileal neuroendocrine tumors show elevated activation of mammalian target of rapamycin complex.

BACKGROUND: Neuroendocrine tumors (NETs) of the ileum are sporadic tumors derived from submucosal gastrointestinal stem cells. They often show clinical symptoms only after hepatic metastasation when curative therapy is limited or impossible. In this study, we analyzed the expression of the candidate genes mammalian target of rapamycin (mTOR), alpha thalassemia/mental retardation syndrome X-linked (ATRX), and death domain-associated protein (DAXX) to investigate the specific oncogenetics and potential therapeutic options for ileal NETs. METHODS: In a prospective database, all patients who underwent surgical removal of a NET of the ileum between 2001 and 2011 were specified. Expression analysis was performed for mTOR, ATRX, and DAXX by immunohistochemistry of paraffin-embedded tumor samples. To evaluate the results the immunoreactive score was applied. Normal tissue and tumor tissue were analyzed for the comparison of gene expression levels using quantitative-real-time polymerase chain reaction for ATRX and mTOR genes. Results were correlated under pathologic and clinical aspects. RESULTS: A total of 69 patients were admitted to the study. Positive cytosolic expression of the potential oncogene mTOR was immunohistochemically detected in 76.2% of the human probes. A loss of nuclear ATRX expression was detected in 13.0% of the samples. A nonexpression of the DAXX-protein in cell nuclei was not found (0%). Gene transcript levels did not show a significant alteration in ileal NETs in comparison with normal tissue. CONCLUSIONS: mTOR is overexpressed in ileal NETs. Additionally, the loss of ATRX expression was registered, thus underlying a tumorigenic role in a subgroup of these tumors. To enable potential therapeutic application of mTOR inhibitors, further trials with larger study groups are needed.

Diagnostic accuracy of magnetic resonance imaging: a prospective evaluation of patients with suspected appendicitis (diamond).

PURPOSE: To show the effect of standard magnetic resonance imaging (MRI) in patients with suspected appendicitis on negative laparotomy and perforation rate. Moreover, the economic impact on hospital resources was evaluated. MATERIALS AND METHODS: In all, 52 patients (21 female; mean age 44.7 years) were prospectively included in this Institutional Review Board (IRB)-approved study. Abdominal MRI including coronal inversion recovery, axial T2-weighted, and contrast-enhanced axial T1-weighted sequences was performed. MRI results were compared to final clinical outcome determined by follow-up or histopathology. Change of treatment was evaluated according to the final clinical outcome. Economic impact was evaluated by comparing the costs of MRI to the savings due to a change in treatment after MRI. Negative laparotomy and perforation rate as well as sensitivity and specificity were derived. RESULTS: Negative laparotomy and perforation rate were 0% (0/52) and 8% (1/13). Sensitivity and specificity for detecting acute appendicitis were 85% (11/13) and 97% (38/39). In 40% of patients therapy changed due to the MRI. The overall effect on the use of hospital resources was a net saving of €2,335. CONCLUSION: Abdominal MRI in the evaluation of patients with suspected appendicitis and equivocal clinical findings is safe, reliable, and cost-effective. It should be considered an important alternative to computed tomography.

Hedgehog inhibition with the orally bioavailable Smo antagonist LDE225 represses tumor growth and prolongs survival in a transgenic mouse model of islet cell neoplasms.

BACKGROUND: This study was designed to evaluate the role of the hedgehog pathway in tumor progression of murine islet cell tumors. Blockade of aberrant hedgehog activation has recently been proposed as a therapeutic target, but effects in models of islet cell tumors with a new orally bioavailable Smoothened (Smo) antagonist LDE225 have not been examined. MATERIAL AND METHODS: To assess in vivo effects, transgenic Rip1Tag2 mice, which develop islet cell neoplasms, were treated with vehicle or LDE225 (80 mg/kg/d) from week 5 until death. The resected pancreata were evaluated macroscopically and microscopically by iummohistochemsistry. Quantitative real-time polymerase chain reaction was performed for hedgehog target genes with RNA from islet, isolated from treated and untreated Rip1Tag2 mice. RESULTS: LDE225 significantly reduced tumor volume by 95% compared with untreated control mice. Hedgehog inhibition with LDE225 significantly prolonged median survival in the used transgenic mouse model (105 vs 116 days; P = 0.02). Quantitative real-time polymerase chain reaction for downstream hedgehog target genes demonstrated significant downregulation in the islet cell tumors of Rip1Tag2 mice treated with LDE225, confirming the ability to achieve effective pharmacologic levels of LDE225 within the desired tissue site, in vivo. CONCLUSION: This is the first study to show that the orally bioavailable Smo antagonist LDE225 may provide a new option for therapy of islet cell neoplasms.

Neuroendocrine pancreatic tumors are risk factors for pancreatic fistula after pancreatic surgery.

BACKGROUND: Postoperative pancreatic fistula (PF) is still regarded as a major complication in pancreatic surgery. In the present study, we evaluated the risk of PF in a large study population of patients with neuroendocrine pancreatic tumors (NPT), pancreatic cancer (PC), and chronic pancreatitis (CP). METHODS: Patients who underwent pancreatic surgery between 1989 and 2008 at our institution were retrospectively evaluated. Patients were analyzed regarding age, gender, BMI, alcohol, smoking, preoperative diabetes, reason for operation, operative procedure, and PF. Three different grades of PF (grades A, B, C) were defined. RESULTS: 133 patients with NPT, 212 patients with PC, 52 patients with CP, and 76 patients with other reasons were evaluated. Patients with a NPT had a significant higher risk of developing a PF than patients with PC, CP, or other reasons (p = 0.0001). Enucleation of the tumor was associated with the highest rate of PF (p = 0.001). In a multivariate analysis, BMI >26, and preoperative diabetes were associated with a higher rate of PF (p = 0.042 and p = 0.02, respectively). CONCLUSION: We demonstrated that after excluding factors like different definitions of PF, or different peri- or postoperative management, patients with NPT have a significantly higher risk of postoperative PF than patients with other pancreatic diseases.

C-Reactive Protein as a New Prognostic Factor for Survival in Patients With Pancreatic Neuroendocrine Neoplasia.

CONTEXT: Patients with pancreatic neuroendocrine neoplasia (pNEN) show great variability in prognosis and treatment response. Additional prognostic markers might help in individual therapeutic decision making. OBJECTIVE: The objective of the study was to investigate the association between preoperative plasma levels of C-reactive protein (CRP) and overall survival (OS) in pNEN. DESIGN: This was a single-center, retrospective analysis of long-term prospective patient-database. SETTING: The study was conducted at a tertiary referral center. PATIENTS: All 149 patients with sporadic pNENs were eligible for retrospective analysis. MAIN OUTCOME MEASURE: Cumulative overall survival, compared between patients with elevated and normal CRP levels, was measured. RESULTS: Median OS for patients with elevated CRP levels was 1093 days (SE 1261, 95% confidence interval [CI] 0-3565), compared with 6859 days (SE 1252, 95% CI 4405-9313) for patients with normal CRP levels. Log rank test showed a significant correlation between CRP and OS (P < .001). In univariate Cox regression, patients with elevated CRP levels had a significantly higher hazard ratio for death (3.27; 95%-CI 1.74-6.16; P < .001). This finding persisted after multivariable adjustment. Furthermore, OS was associated with the presence of liver metastases (hazard ratio 3.17; 95% CI 1.88-5.35; P < .001), incomplete resection (R1/R2 status; hazard ratio 3.99; 95% CI 2.16-7.35; P < .001) and Ki-67 percentage (hazard ratio 5.05; 95% CI 2.17-11.76; P < .001). CONCLUSION: CRP is an independent prognostic marker in patients with pNEN. Pretreatment CRP measurements should be considered for incorporation into prospective studies of outcome in patients with pNENs and clinical trials of systemic therapies for these tumors.