Establishment of robust controls for the normalization of miRNA expression in neuroendocrine tumors of the ileum and pancreas.

There is need to determine tissue-specific robust controls for normalization of microRNA expression to avoid false results and misinterpretation. The aim of this study was to evaluate the expression of different small RNAs in neuroendocrine tumors (NETs) and their suitability as normalizers in miRNA real-time PCR experiments. We investigated the expression of the nine small RNAs miR-93, miR-191, SNORD48, SNORD61, SNORD68, SNORD72, SNORD95, SNORD96a, and RNU6-2 in formalin-fixed, paraffin-embedded tissue samples of 25 ileal NETs by real-time PCR determining the most stable controls for expression normalization using four different algorithms. This analysis was expended to ten pancreatic NETs. Finally, five small RNAs were further tested as normalizers for miRNA-133a expression, which is known to be downregulated in metastases of ileal NETs, in ten matched pairs of ileal NETs and their metastases. Ranking of the expression results revealed the following order of stability from high to low: SNORD61 < SNORD95 < SNORD72 < SNORD96a < SNORD68 < miR-191 < miR-93 < RNU6-2 < SNORD48 for ileal NETs and SNORD95 < miR-93 < SNORD96a < SNORD61 < SNORD68 < SNORD72 < RNU6-2 < miR-191 < SNORD48 for pancreatic NETs. The determination of SNORD61 and SNORD95 for ileal NETs and SNORD95 and miR-93 for pancreatic NETs as good normalizers presents a useful tool for experiments involving the analysis of miRNA expression.

Multiple osteolytic lesions of intraosseous adenoid cystic carcinoma in the mandible mimicking apical periodontitis.

AIM: Adenoid cystic carcinoma (ACC) is a relatively rare epithelial tumour of the salivary glands in the maxillofacial region. About 40-60% of the patients develop distant metastases, which have been documented most commonly in the lung but also in brain, bone, liver, thyroid, spleen and pancreatic gland. SUMMARY: A 55-year-old women with intraosseous ACC in the mandible mimicking apical periodontitis following curative resection and radiotherapy is presented. Three years later, multiple lung metastases were observed followed by chemotherapy. Five years after curative resection, the patient presented simultaneously with new expansive soft tissue in the pancreas and mammary gland as well as in the kidney found to be metastatic ACC. No case has been reported to date on the manifestation of distant metastases of intraosseous ACC in the breast and the kidney as described by these observations. Metastatic mammary gland ACC stained positive for epithelial growth factor receptor (EGFR) but was negative for HER-2/neu and Cyclooxygenase-2 (COX-2) expression.

Global histone modification pattern predicts poor prognosis in organic hyperinsulinism.

Here we tested whether global histone modifications predict survival in organic hyperinsulinism and whether global histone modification pattern can be used to distinguish benign from malignant primary insulinoma. A tissue microarray (TMA) was built, using samples from 63 patients with organic hyperinsulinism. The TMA was classified according to the WHO classification of 2004 [WHO 1A: benign insulinoma (wdPET); WHO 1B: unknown behavior (wdPETub); WHO 2/3: malignant insulinoma (wdPEC/pdPEC)]. The TMA consisted of tissue cores from islands of Langerhans, primary insulinomas, lymph node metastases, and hepatic metastases. Immunohistochemistry was performed on consecutive TMA slides with antibodies against H3K9Ac, H3K18Ac, H4K12Ac, H3K4diMe, and H4R3diMe. The Remmele immunoreactive scoring system was used to classify the staining. The IHC staining results were correlated to the WHO-classification of 2004 as well as to clinical follow-up data (mean: 107 months; range: 1-312 months). A nuclear staining pattern was observed for all antibodies directed against histone H3 and H4 acetylation/methylation sites. We observed significant differences in the distribution of the medians across all investigated tissue types (H3K9Ac, p=0.004; H3K18Ac, p=0.001; H4K12Ac, p=0.006; H4R3diMe, p=0.002) except for H3K4diMe (p=0.183). Correlation of the histone modification with the WHO-classification and clinical follow-up data, showed in the dichotomized groups ["low" (score 0-3), "moderate" (4-7) vs. "high" (≥8)] that patients with lower H3K18Ac levels ("low + moderate") had a significantly decreased relapse-free survival vs. patients with high H3K18Ac levels (p=0.038). The WHO classification and age were also of significant prognostic impact upon univariate analysis. A backwards Cox proportional hazards model revealed the independent prognostic effekt of H3K18Ac levels. Our data revealed low K18 acetylation levels of histone H3 as independent prognostic factor in organic hyperinsulinism. This result warrants validation with independent data sets of organic hyperinsulinism, but is in line with several previous studies in different cancer entities. The broad applicability of this potential biomarker might lead to standardized diagnostic tests in near future and may help to manage insulinoma patients more effectively.

[Precursor lesions of pancreatobiliary cancer]. / Vorläuferläsionen pankreatobiliärer Karzinome.

Precursor lesions of pancreatobiliary cancer can be divided into cystic and flat lesions. Mucinous cystic neoplasm and intraductal papillary mucinous neoplasm (IPMN) comprise the cystic precursors in the pancreas, while intraductal papillary neoplasm (IPN) represents their counterpart in the bile duct system. There is an adenoma-carcinoma sequence in the cystic precursors arising from four different types of epithelia: pancreatobiliary, oncocytic, intestinal and gastric. These subtypes of IPMN/IPN are morphologically and immunohistochemically well characterised and show clinical and prognostic relevance: the gastric subtype is associated with the best prognosis, followed by the oncocytic and intestinal subtypes, while the pancreatobiliary subtype is characterized by adverse clinical behaviour. Pancreatic intraepithelial neoplasia (PanIN) and biliary intraepithelial neoplasia (BilIN) represent the flat precursors. PanIN are morphologically and biologically well defined. PanIN with lobulocentric atrophy has recently been described as a putative precursor of pancreatic cancer. Despite well defined morphological features in BilIN, the molecular alterations seen during early tumor progression in the biliary tract are poorly understood.

Allelic deletion of the MEN1 gene in duodenal gastrin and somatostatin cell neoplasms and their precursor lesions.

BACKGROUND: Patients with a multiple endocrine neoplasia type 1 (MEN1)-associated Zollinger-Ellison syndrome (ZES) show multifocal duodenal gastrinomas and precursor lesions. AIMS: To test these lesions for loss of heterozygosity (LOH) of the MEN1 gene locus on chromosome 11q13, and to investigate whether the MEN1-related endocrine cell changes also involved somatostatin cells. MATERIAL AND METHODS: Tissue specimens from six patients with MEN1 and ZES were analysed by immunohistochemistry and immunofluorescence. LOH analysis was performed by fluorescence in situ hybridisation (FISH), using probes containing the MEN1 gene locus and the centromere 11 (C11) region. For simultaneous analysis of hormones and allelic deletions, a combined FISH/immunofluorescence protocol was established. RESULTS: 28 of a total of 33 duodenal neuroendocrine tumours (NETs) were gastrin-producing tumours; 13/28 (46.4%) revealed LOH on 11q13 and/or C11. Five of the NETs were somatostatin-expressing tumours, two revealing LOH. Allelic loss was detected in tumours as small as 300 microm (gastrin) and 400 microm (somatostatin) in diameter. The gastrin-producing tumours showed different deletion/retention patterns. Hyperplastic somatostatin cell lesions, similar to those of the gastrin cells, were present in all patients. The hyperplastic lesions of both cell lines consistently retained both 11q13 alleles. CONCLUSIONS: Allelic deletion of the MEN1 gene may reflect a pivotal event in the development of multifocal gastrin and somatostatin cell neoplasms in the duodenum of patients with MEN1. The observation of distinct deletion patterns in small synchronous tumours supports the concept that each gastrin-producing tumour in an individual MEN1 patient arises from an independent cell clone.

Hyperinsulinemic hypoglycemia due to adult nesidioblastosis in insulin-dependent diabetes.

In neonates, persistent hyperinsulinemic hypoglycemia (PHH) is associated with nesidioblastosis. In adults, PHH is usually caused by solitary benign insulinomas. We report on an adult patient who suffered from insulin-dependent diabetes mellitus, and subsequently developed PHH caused by diffuse nesidioblastosis. Mutations of the MEN1 and Mody (2/3) genes were ruled out. Preoperative diagnostic procedures, the histopathological criteria and the surgical treatment options of adult nesidioblastosis are discussed. So far only one similar case of adult nesidioblastosis subsequent to diabetes mellitus II has been reported in the literature. In case of conversion of diabetes into hyperinsulinemic hypoglycemia syndrome, nesidioblastosis in addition to insulinoma should be considered.