Genetic disorders and insulinoma/glucagonoma.

Insulinoma and glucagonoma are two rare functioning neoplasms of the neuroendocrine cells of the pancreas, respectively, characterized by an uncontrolled over-secretion of insulin or glucagon, responsible for the development of the hypoglycemic syndrome and the glucagonoma syndrome. They prevalently arise as sporadic tumors; only about 10% of cases develop in the context of rare inherited tumor syndromes, such as multiple endocrine neoplasia type 1 (MEN1), neurofibromatosis type 1 (NF1), and tuberous sclerosis complex (TSC), being the result of an autosomal-dominant germline heterozygous loss-of-function mutation in a tumor-suppressor gene. Here, we reviewed the main epidemiological and clinical aspects of insulinoma and glucagonoma in the context of genetic syndromes.

A novel likely pathogenetic variant p.(Cys235Arg) of the MEN1 gene in multiple endocrine neoplasia type 1 with multifocal glucagonomas.

PURPOSE: Multiple endocrine neoplasia type 1 (MEN1) is a hereditary endocrine syndrome caused by pathogenic variants in MEN1 tumor suppressor gene. Diagnosis is commonly based on clinical criteria and confirmed by genetic testing. The objective of the present study was to report on a MEN1 case characterized by multiple pancreatic glucagonomas, with particular concern on the possible predisposing genetic defects. METHODS: While conducting an extensive review of the most recent scientific evidence on the unusual glucagonoma familial forms, we analyzed the MEN1 gene in a 35-year-old female with MEN1, as well as her son and daughter, using Sanger and next-generation sequencing (NGS) approaches. We additionally explored the functional and structural consequences of the identified variant using in silico analyses. RESULTS: NGS did not show any known pathogenic variant in the tested regions. However, a new non-conservative variant in exon 4 of MEN1 gene was found in heterozygosity in the patient and in her daughter, resulting in an amino acid substitution from hydrophobic cysteine to hydrophilic arginine at c.703T > C, p.(Cys235Arg). This variant is absent from populations databases and was never reported in full papers: its characteristics, together with the high specificity of the patient's clinical phenotype, pointed toward a possible causative role. CONCLUSION: Our findings confirm the need for careful genetic analysis of patients with MEN1 and establish a likely pathogenic role for the new p.(Cys235Arg) variant, at least in the rare subset of MEN1 associated with glucagonomas.

Glucagonoma With Necrolytic Migratory Erythema: Metabolic Profile and Detection of Biallelic Inactivation of DAXX Gene.

Context: Necrolytic migratory erythema (NME) occurs in approximately 70% of patients with glucagonoma syndrome. Excessive stimulation of metabolic pathways by hyperglucagonemia, which leads to hypoaminoacidemia, contributes to NME pathogenesis. However, the molecular pathogenesis of glucagonoma and relationships between metabolic abnormalities and clinical symptoms remain unclear. Patient: A 53-year-old woman was referred to our hospital with a generalized rash and weight loss. NME was diagnosed by histopathological examination of skin biopsy tissue. Laboratory tests revealed diabetes, hyperglucagonemia, marked insulin resistance, severe hypoaminoacidemia, ketosis, and anemia. Enhanced computed tomography scans detected a 29-mm pancreatic hypervascular tumor, which was eventually diagnosed as glucagonoma. Preoperative treatment with octreotide long-acting release reduced the glucagon level, improved the amino acid profile, and produced NME remission. Surgical tumor excision normalized the metabolic status and led to remission of symptoms, including NME. Interventions: Whole-exome sequencing (WES) and subsequent targeted capture sequencing, followed by Sanger sequencing and pyrosequencing, identified biallelic alteration of death-domain associated protein (DAXX) with a combination of loss of heterozygosity and frameshift mutations (c.553_554del:p.R185fs and c.1884dupC:p.C629fs) in the glucagonoma. Consistently, immunohistochemistry confirmed near-absence of DAXX staining in the tumor cells. Tumor expression of glucagon and somatostatin receptor subtype 2 and 3 messenger RNA was markedly upregulated. Conclusions: This is a report of glucagonoma with biallelic inactivation of DAXX determined by WES. The tumor manifested as glucagonoma syndrome with generalized NME. This case showed the relationship between hypoaminoacidemia and NME status. Further investigations are required to elucidate the underlying mechanisms of NME onset and glucagonoma tumorigenesis.

Foxa2, a novel protein partner of the tumour suppressor menin, is deregulated in mouse and human MEN1 glucagonomas.

Foxa2, known as one of the pioneer factors, plays a crucial role in islet development and endocrine functions. Its expression and biological functions are regulated by various factors, including, in particular, insulin and glucagon. However, its expression and biological role in adult pancreatic α-cells remain elusive. In the current study, we showed that Foxa2 was overexpressed in islets from α-cell-specific Men1 mutant mice, at both the transcriptional level and the protein level. More importantly, immunostaining analyses showed its prominent nuclear accumulation, specifically in α-cells, at a very early stage after Men1 disruption. Similar nuclear FOXA2 expression was also detected in a substantial proportion (12/19) of human multiple endocrine neoplasia type 1 (MEN1) glucagonomas. Interestingly, our data revealed an interaction between Foxa2 and menin encoded by the Men1 gene. Furthermore, using several approaches, we demonstrated the relevance of this interaction in the regulation of two tested Foxa2 target genes, including the autoregulation of the Foxa2 promoter by Foxa2 itself. The current study establishes menin, a novel protein partner of Foxa2, as a regulator of Foxa2, the biological functions of which extend beyond the pancreatic endocrine cells. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Characterization of pancreatic glucagon-producing tumors and pituitary gland tumors in transgenic mice overexpressing MYCN in hGFAP-positive cells.

Amplification or overexpression of MYCN is involved in development and maintenance of multiple malignancies. A subset of these tumors originates from neural precursors, including the most aggressive forms of the childhood tumors, neuroblastoma and medulloblastoma. In order to model the spectrum of MYCN-driven neoplasms in mice, we transgenically overexpressed MYCN under the control of the human GFAP-promoter that, among other targets, drives expression in neural progenitor cells. However, LSL-MYCN;hGFAP-Cre double transgenic mice did neither develop neural crest tumors nor tumors of the central nervous system, but presented with neuroendocrine tumors of the pancreas and, less frequently, the pituitary gland. Pituitary tumors expressed chromogranin A and closely resembled human pituitary adenomas. Pancreatic tumors strongly produced and secreted glucagon, suggesting that they derived from glucagon- and GFAP-positive islet cells. Interestingly, 3 out of 9 human pancreatic neuroendocrine tumors expressed MYCN, supporting the similarity of the mouse tumors to the human system. Serial transplantations of mouse tumor cells into immunocompromised mice confirmed their fully transformed phenotype. MYCN-directed treatment by AuroraA- or Brd4-inhibitors resulted in significantly decreased cell proliferation in vitro and reduced tumor growth in vivo. In summary, we provide a novel mouse model for neuroendocrine tumors of the pancreas and pituitary gland that is dependent on MYCN expression and that may help to evaluate MYCN-directed therapies.

Progranulin Stimulates Proliferation of Mouse Pancreatic Islet Cells and Is Overexpressed in the Endocrine Pancreatic Tissue of an MEN1 Mouse Model.

OBJECTIVES: Progranulin (PGRN) promotes cell growth and cell cycle progression in several cell types and contributes to tumorigenesis in diverse cancers. We have recently reported PGRN expression in islets and tumors developed in an MEN1 transgenic mouse. Here we sought to investigate PGRN expression and regulation after exposure to hypoxia as well as its effects on pancreatic islet cells and neuroendocrine tumors (NETs) in MEN1(+/−) mice. METHODS: Gene and protein expression were analyzed by quantitative polymerase chain reaction, immunohistochemistry, and Western blot. We also investigated PGRN expression in samples from patients carrying pancreatic NETs associated or not with the multiple endocrine neoplasia 1 syndrome, using enzyme-linked immunosorbent assay and immunohistochemistry analysis. RESULTS: Progranulin is upregulated in tumors and islets of the MEN1 mouse as well as in the serum of patients with pancreatic NETs associated with glucagonoma syndrome. In normal mice islets and pancreatic tumors, PGRN expression was strongly potentiated by hypoxia. Progranulin promotes cell proliferation in islet cells and ßTC-6 cells, a process paralleled by activation of the mitogen-activated protein kinase signaling cascade. CONCLUSIONS: Our findings identify PGRN as an effective inducer of pancreatic islet cell proliferation and a possible important factor for pancreatic endocrine tumor development.

A Novel Missense Mutation of the MEN1 Gene in a Patient with Multiple Endocrine Neoplasia Type 1 with Glucagonoma and Obesity.

A 35-year-old obese diabetic man presented with recurrent primary hyperparathyroidism during a three-year outpatient follow-up. He was clinically diagnosed with multiple endocrine neoplasia type 1 (MEN1) due to the presence of a pituitary adenoma and multiple glucagonomas. The glucagonomas may have affected his glycemic control. However, he did not demonstrate weight loss, suggesting that the patient's obesity could have obscured the early diagnosis of a glucagonoma. Genetic testing revealed a novel missense mutation at codon 561 in exon 10, resulting in an amino acid substitution from methionine to arginine (M561R) in the MEN1 gene. This mutation appeared to be responsible for the MEN1 pathogenicity.

Conditional deletion of Men1 in the pancreatic ß-cell leads to glucagon-expressing tumor development.

The tumor suppressor menin is recognized as a key regulator of ß-cell proliferation. To induce tumorigenesis within the pancreatic ß-cells, floxed alleles of Men1 were selectively ablated using Cre-recombinase driven by the insulin promoter. Despite the ß-cell specificity of the RipCre, glucagon-expressing tumors as well as insulinomas developed in old mutant mice. These glucagon-expressing tumor cells were menin deficient and expressed the mature α-cell-specific transcription factors Brain-specific homeobox POU domain protein 4 (Brn4) and v-maf musculoaponeurotic fibrosarcoma oncogene family, protein B (MafB). Moreover, the inactivation of ß-cell-specific transcription factors was observed in mutant ß-cells. Our work shows that Men1 ablation in the pancreatic ß-cells leads to the inactivation of specific transcription factors, resulting in glucagon-expressing tumor development, which sheds light on the mechanisms of islet tumorigenesis.

Alpha cell-specific Men1 ablation triggers the transdifferentiation of glucagon-expressing cells and insulinoma development.

BACKGROUND & AIMS: The tumor suppressor menin is recognized as a key regulator of pancreatic islet development, proliferation, and beta-cell function, whereas its role in alpha cells remains poorly understood. The purpose of the current study was to address this issue in relation to islet tumor histogenesis. METHODS: We generated alpha cell-specific Men1 mutant mice with Cre/loxP technology and carried out analyses of pancreatic lesions developed in the mutant mice during aging. RESULTS: We showed that, despite the alpha-cell specificity of the GluCre transgene, both glucagonomas and a large amount of insulinomas developed in mutant mice older than 6 months, accompanied by mixed islet tumors. Interestingly, the cells sharing characteristics of both alpha and beta cells were identified shortly after the appearance of menin-deficient alpha cells but well before the tumor onset. Using a genetic cell lineage tracing analysis, we demonstrated that insulinoma cells were directly derived from transdifferentiating glucagon-expressing cells. Furthermore, our data indicated that the expression of Pdx1, MafA, Pax4, and Ngn3 did not seem to be required for the initiation of this transdifferentiation. CONCLUSIONS: Our work shows cell transdifferentiation as a novel mechanism involved in islet tumor development and provides evidence showing that menin regulates the plasticity of differentiated pancreatic alpha cells in vivo, shedding new light on the mechanisms of islet tumorigenesis.

Glucagonoma diagnosed by arterial stimulation and venous sampling (ASVS).

To identify the location of pancreatic endocrine tumors, arterial stimulation and venous sampling (ASVS) is known to be useful for insulinoma and gastrinoma, but its usefulness for glucagonoma has not been verified to date. Here we report a case of glucagonoma that was diagnosed by ASVS with calcium loading, in which an approximately 6-fold increase of glucagon was observed in the splenic artery territory. MEN1 gene analysis verified the presence of a mutation and the glucagonoma was confirmed after operation. In conclusion, ASVS could be useful for the diagnosis of glucagonoma.

Co-existence of glucagonoma with recurrent insulinoma in a patient with multiple endocrine neoplasia-type 1 (MEN-1).

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by tumors of the parathyroid glands, the anterior pituitary, and the endocrine pancreas. Our patient was a 58-year-old man who manifested typical features of MEN-1 including primary hyperparathyroidism, lung carcinoid, and lipomas and insulinoma. He was admitted to our hospital because of recurrent hypoglycemia and a growth of pancreatic tumors. The first operation for insulinoma was performed when he was 20 years old. We found a germline mutation of the MEN1 gene (E45G, exon 2) in this patient. According to these examinations and his clinical course, the patient was diagnosed as having a recurrence of insulinoma. He subsequently underwent surgery for the pancreatic tumors. The majority of these tumor cells were immunohistochemically positive for insulin and negative for glucagon. A few nodules showed immunohistochemical staining positivity for glucagon but they were negative for insulin. Although it is uncommon for patients with MEN1 to exhibit insulinoma and glucagonoma, this case suggests the need for careful analysis of pancreatic tumors in patients with MEN1.

Mutational analyses of WNT7A and HDAC11 as candidate tumour suppressor genes in sporadic malignant pancreatic endocrine tumours.

OBJECTIVE: We and others have reported loss of heterozygosity (LOH) on chromosome 3p25 in sporadic malignant pancreatic endocrine tumours (PETs). A common region of deletion on chromosome 3p25 contains numerous genes, including VHL and PPARgamma, that have been excluded previously as candidate tumour suppressor genes by DNA sequencing analysis. We have analysed whether WNT7A or HDAC11 was biallelically inactivated in a group of well-characterized PETs. PATIENTS AND DESIGN: Ten PETs from eight patients were selected from a previous study, where LOH on chromosome 3p25 was found in 11 out of 22 sporadic PETs. These tumours were examined for inactivating mutations of WNT7A and HDAC11 by direct sequencing of all exons and intron-exon boundaries. Inactivation of WNT7A expression by aberrant CpG island methylation and WNT7A protein expression were evaluated by methylation-specific polymerase chain reaction (PCR) and immunohistochemistry, respectively. HDAC11 protein expression was also examined. RESULTS: No point mutations, deletion or insertions were detected in either WNT7A or HDAC11 in any of the PETs. Two polymorphisms were identified in the third exon of the WNT7A gene. CpG methylation of the WNT7A gene was not detected and the WNT7A and HDAC11 proteins were normally expressed. CONCLUSION: The absence of tumour-specific somatic events in WNT7A and HDAC11 suggests that these genes are unlikely to have a classical tumour suppressor gene role in sporadic malignant PETs. The putative 3p25 tumour suppressor remains to be identified.

[Hereditary tumours of the endocrine pancreas]. / Az endokrin pancreas öröklódó daganatai.

The pathogenesis, diagnosis and therapy of tumours originating from the endocrine pancreas represent one of the most exciting challenges of contemporary medicine. Some of these tumours appear as part of four hereditary syndromes (multiple endocrine neoplasia type 1 (MEN1), von Hippel-Lindau disease (VHL), neurofibromatosis type 1 and tuberous sclerosis) that are all inherited as autosomal dominant traits and result from mutations of tumour suppressor genes. Considering its clinical relevance, MEN1 appears to be the most important among these four syndromes. Tumours of the endocrine pancreas develop in 30-80% of patients carrying mutations of the MEN1 gene. Gastrinomas are the most frequent functioning tumours in MEN1 patients, followed by insulinomas, whereas other tumors e.g. glucagonoma, VIP-oma, GRF-oma and somatostatinoma occur very rarely. Tumours of the endocrine pancreas are infrequent in patients suffering from VHL, neurofibromatosis and tuberous sclerosis. In this review article, the authors present a synopsis of tumours of the endocrine pancreas related to these hereditary syndromes underlining the clinical characteristics, diagnostical and therapeutical possibilities.

p62 protein is expressed in pancreatic beta cells.

p62 is a cellular protein that plays an adapter role in signal transduction pathways involved in such diverse biological functions as proliferation, differentiation, reaction to oxidative stress and immune response. Furthermore, p62 has recently been detected as a component of intracytoplasmic protein aggregates (inclusion bodies), which are hallmarks of a variety of chronic degenerative disorders, such as Parkinson's disease and Alzheimer's disease, but also of steatohepatitis. Here we report that p62 and insulin are co-expressed in a diffuse fashion in beta cells in normal human pancreas as well as in primary chronic pancreatitis and in normal pancreas from mouse and swine. In contrast, p62 protein is absent from, or only focally and very weakly expressed in, insulinomas, glucagonomas or non-functioning pancreatic neuroendocrine tumours or carcinomas that express insulin or other pancreatic as well as extrapancreatic hormones. Although the biological function of p62 in beta cells is unknown, the co-expression of p62 and insulin in non-neoplastic beta cells suggests that, in the beta cell, p62 may play a role in specific insulin-related signalling. Since p62 may also be involved in pro-apototic signal transduction, the loss of p62 expression in neuroendocrine neoplasms of the pancreas may render the tumour cells less sensitive to pro-apototic signals. Further research is necessary to elucidate the role of p62 in beta cell-specific signal transduction.

[Multiple endocrine neoplasia syndroms. Type 1]. / Dauginiu endokrininiu naviku sindromai. Pirmas tipas.

Multiple endocrine neoplasia (MEN) type 1 syndrome or Wermer syndrome is a classical malignant neoplasia syndrome, inherited in the autosomal dominant pattern, when hyperplastic and/or neoplastic injury develops synchronously or metachronously in the cells of the parathyroid gland, pancreas islets, hypophysis, and rarer in other neuroendocrine organs. The syndrome develops when germinative MEN 1--neoplasia suppression gene inactivation mutations occurs. More than 95 percent of patients have this MEN 1 gene mutation, when the penetration of mutation is almost 100 percent. An early stage of this syndrome is hyperfunction in organs, with the syndromes of hormone excess, later benign and/or malign neoplasia develops, this mostly determines the prognosis for the patient. The risk of this syndrome developing should be estimated for all the patients diagnosed with endocrine organ hyperplasia, which determines hyperfunction, or endocrine organs neoplasia. For patients with multiple endocrine neoplasia type 1 syndrome, endocrine neoplasia develops earlier than in sporadic cases; multifocality is typical for them. Multifocality of neoplasia, typical combinations of injuries and anamnesis of the family helps to diagnose the syndrome. Diagnosis is confirmed through genetical analysis, which is also important in determining the inheritors of mutations, potential patients. After genetically diagnosing multiple endocrine neoplasia type 1 syndrome, it is not enough to analyze and look after patients with malignant neoplasia, or to make early diagnosis on pre-neoplasic disease and neoplasia, or to apply means of prevention and start well-timed treatment, but also to diagnose this syndrome for the patient's relatives, and to determine their risk of getting cancer. This opens new possibilities in early diagnostics and prevention of malignant neoplasia. The main purpose of this literature review is to introduce medical-practitioners to the newest theories of type 1 multiple endocrine neoplasia syndrome pathogenesis, clinical peculiarities, methods of diagnostics and treatment.

Putative tumor suppressor loci at 6q22 and 6q23-q24 are involved in the malignant progression of sporadic endocrine pancreatic tumors.

Our previous comparative genomic hybridization study on sporadic endocrine pancreatic tumors (EPTs) revealed frequent losses on chromosomes 11q, 3p, and 6q. The aim of this study was to evaluate the importance of 6q losses in the oncogenesis of sporadic EPTs and to narrow down the smallest regions of allelic deletion. A multimodal approach combining polymerase chain reaction-based allelotyping, double-target fluorescence in situ hybridization, and comparative genomic hybridization was used in a collection of 109 sporadic EPTs from 93 patients. Nine polymorphic microsatellite markers (6q13 to 6q25-q27) were investigated, demonstrating a loss of heterozygosity (LOH) in 62.2% of the patients. A LOH was significantly more common in tumors >2 cm in diameter than below this threshold as well as in malignant than in benign tumors. We were able to narrow down the smallest regions of allelic deletion at 6q22.1 (D6S262) and 6q23-q24 (D6S310-UTRN) with LOH-frequencies of 50.0% and 41.2 to 56.3%, respectively. Several promising tumor suppressor candidates are located in these regions. Additional fluorescence in situ hybridization analysis on 46 EPTs using three locus-specific probes (6q21, 6q22, and 6q27) as well as a centromere 6-specific probe revealed complete loss of chromosome 6 especially in metastatic disease. We conclude that the two hot spots found on 6q may harbor putative tumor suppressor genes involved not only in the oncogenesis but maybe also in the malignant and metastatic progression of sporadic EPTs.

Mutation and expression analyses reveal differential subcellular compartmentalization of PTEN in endocrine pancreatic tumors compared to normal islet cells.

The pathogenesis of sporadic endocrine pancreatic tumors (EPTs) is still primarily unknown. Comparative genomic hybridization studies revealed loss of 10q in a significant number (nine of 31) of EPTs. The tumor suppressor gene PTEN lies on 10q23, and so, is a candidate to play some role in EPT pathogenesis. Germline PTEN mutations are found in Cowden and Bannayan-Riley-Ruvalcaba syndromes, whereas somatic mutations and deletions are found in a variety of sporadic cancers. The mutation and expression status of PTEN in EPTs has not yet been examined. Mutation analysis of the entire coding region of PTEN including splice sites was performed in 33 tumors, revealing one tumor with somatic L182F (exon 6). Loss of heterozygosity of the 10q23 region was detected in eight of 15 informative malignant (53%) and in none of seven benign EPTs. PTEN expression was assessed in 24 available EPTs by immunohistochemistry using a monoclonal anti-PTEN antibody. Of these 24, 23 tumors showed strong immunoreactivity for PTEN. Only the EPTs with PTEN mutation lacked PTEN protein expression. Although normal islet cells always exhibited predominantly nuclear PTEN immunostaining, 19 of 23 EPTs had a predominantly cytoplasmic PTEN expression pattern. Exocrine pancreatic tissue was PTEN-negative throughout. PTEN mutation is a rare event in malignant EPTs and PTEN protein is expressed in most (23 of 24) EPTs. Thus, intragenic mutation or another means of physical loss of PTEN is rarely involved in the pathogenesis of EPTs. Instead, either an impaired transport system of PTEN to the nucleus or some other means of differential compartmentalization could account for impaired PTEN function. Loss of heterozygosity of the 10q23 region is a frequent event in malignant EPTs and might suggest several hypotheses: a different tumor suppressor gene in the vicinity of PTEN might be principally involved in EPT formation; alternatively, 10q loss, including PTEN, seems to be associated with malignant transformation, but the first step toward neoplasia might involve altered subcellular localization of PTEN.

Chromosomal alterations in human pancreatic endocrine tumors.

Comparative genomic hybridization (CGH) was used to investigate changes in DNA copy numbers in 25 paraffin-embedded samples of pancreatic endocrine tumors from 23 patients. Insulin was the dominant hormone in 12, glucagon in 7, somatostatin in 1, and pancreatic polypeptide in 2 tumors. One to 15 (mean, 8.1) changes in DNA copy numbers were observed in 22 of the 25 tumors. The most recurrent aberration, found in 68% of the tumors, involved gains in chromosome 7 with a minimal overlapping region at 7q11.2. Other frequent gains included chromosomes 19 (60%) and 14 (56%). Chromosome arm 20q was amplified in 48% of the cases with the minimal overlapping region of 20q11.1-13.1. The two most frequent DNA losses were found at 11q21-22 in 32% and at 11p13-15 in 24% of the cases. The amplified chromosomal regions contain several candidate genes that may be involved in islet cell tumorigenesis. The regions with most frequent losses are likely to contain still uncharacterized tumor suppressor genes. Wiley-Liss, Inc.