Glucagon Cell Adenomatosis: A Newly Recognized Disease of the ...

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Glucagon Cell Adenomatosis: A Newly Recognized Disease of the Endocrine Pancreas Tobias Henopp,* Martin Anlauf,* Anja Schmitt, Regina Schlenger, Attila Zalatnai, Anne Couvelard, Philippe Ruszniewski, Klaus-Peter Schaps, Yvonne M. H. Jonkers, Ernst-Jan M. Speel, Natalia S. Pellegata, Philipp U. Heitz, Paul Komminoth, Aurel Perren,† and Gu¨nter Klo¨ppel† Departments of Pathology (T.H., M.A., G.K.) and Forensic Medicine (R.S.), University of Kiel, 24105 Kiel, Germany; Department of Pathology (T.H.), University of Tu¨bingen, 72076 Tu¨bingen, Germany; Institute of Pathology (M.A.), 41462 Facharztzentrum Neuss, Germany; Department of Surgical Pathology (A.S., P.U.H., A.P.), University Hospital of Zu¨rich, 8091 Zurich, Switzerland; 1st Institute of Pathology and Experimental Cancer Research (A.Z.), Semmelweis University, 1085 Budapest, Hungary; Department of Pathology (A.C.), Hospital Beaujon, 92110 Clichy, France; Service of Gastroenterology (P.R.), Hospital Beaujon, 92118 Clichy, France; Praxis fu¨r Innere Medizin (K.-P.S.), 26388 Wilhelmshaven, Germany; Department of Molecular Cell Biology (Y.M.H.J., E.-J.M.S.), GROW-School for Oncology and Developmental Biology, Maastricht University Medical Center, 6200 MD Maastricht, The Netherlands; Institute of Pathology (N.S.P.), Helmholtz Zentrum Munich, 85764 Neuherberg, Germany; Department of Pathology (A.P.), Technische Universita¨t, 81675 Mu¨nchen, Germany; and Department of Pathology (P.K.), Triemli Hospital, 8063 Zu¨rich, Switzerland

Background: Glucagon-producing tumors are either solitary neoplasms of the pancreas, occasionally associated with a glucagonoma syndrome, or multiple neoplasms associated with multiple endocrine neoplasia type 1 (MEN1). We observed a previously undescribed multicentric glucagonproducing tumor disease that is not related to MEN1. Methods: Pancreatic tissue from four patients showing multiple neuroendocrine microadenomas and in two cases also macrotumors were screened for hormones using immunohistochemical and morphometric methods. MEN1, von Hippel-Lindau, and p27 germ line and somatic mutation analysis was performed. Deletion of MEN1 (11q13), von Hippel-Lindau (3p25), and the centromere 11 and 3 gene locus was determined by fluorescence in situ hybridization. DNA copy number changes were studied using array comparative genomic hybridization. Results: The pancreatic tissue from the four patients contained more than 870 microadenomas and 10 macrotumors, all of which expressed exclusively glucagon and none of which showed evidence of malignancy. In addition, many islets were unusually large and showed glucagon cell hyperplasia. There was no clinical or molecular evidence of any hereditary tumor disease, and changes in the MEN1 gene were only seen in individual tumors. Array comparative genomic hybridization of one macrotumor and 20 pooled microadenomas revealed a homogeneous diploid chromosome set. Conclusions: The findings are sufficiently distinctive to suggest a new neoplastic disease of the endocrine pancreas that we recommend calling glucagon cell adenomatosis. Clinically, this disease may be an incidental finding, or it may lead to a glucagonoma syndrome. (J Clin Endocrinol Metab 94: 213–217, 2009)

ancreatic endocrine tumors (PETs) that express glucagon, either alone or in concert with other hormones, are usually sporadic solitary neoplasms (1, 2). A fraction of these tumors produce a glucagonoma syndrome, and are large and malignant (3, 4).

P

Nonsyndromatic glucagon-producing tumors have been described in three different conditions: 1 as solitary tumors that become symptomatic because of their size and/or malignant growth; 2) as microtumors (⬍0.5 cm) found incidentally; and 3)

ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2009 by The Endocrine Society doi: 10.1210/jc.2008-1300 Received June 16, 2008. Accepted October 16, 2008. First Published Online October 28, 2008 * T.H. and M.A. share first authorship for this manuscript. † A.P. and G.K. share senior authorship for this manuscript.

Abbreviations: CGH, Comparative genomic hybridization; FISH, fluorescence in situ hybridization; MEN1, multiple endocrine neoplasia type 1; PET, pancreatic endocrine tumor; VHL, von Hippel-Lindau.

J Clin Endocrinol Metab, January 2009, 94(1):213–217

jcem.endojournals.org

213

nk 0

30/f

44/m

3

4

DM, Diabetes mellitus; f, female; m, male; LSPPT, left-sided partial pancreatectomy; nk, not known; PT, pancreatectomy.

Yes (4-fold) Glucagonoma syndrome with unknown primary No DM Negative

No None Negative

43/f 2

Acute lymphatic leukemia Weight loss, abdominal pain, deep vein thrombosis, necrolytic migratory erythema

No None Negative

25/m

Abdominal pain

Autopsy

⬎100

nk 0 nk

Autopsy

⬎50

None 2 ⬎120 Yes (25-fold)

LSPPT

8 ⬎600 No

Multiple microcystic endocrine tumors Mucinous cystic tumor Incidental finding No None Negative

Metastases

Total PT

No. of macrotumors Increased serum glucagon Initial clinical diagnosis Other endocrine diseases Family history

1

Clinical presentation Table 1 summarizes the clinicopathological features of the four patients. None of the patients had extrapancreatic tumors. Patient 1, a 25-yr-old man, presented with a 3-month history of repeated episodes of acute pancreatitis. Magnetic resonance pancreatography showed a diffuse enlargement of the pancreas containing numerous tiny lesions and a few tumors up to 2 cm in diameter. Somatostatin receptor scintigraphy revealed diffuse labeling of the whole pancreas and no evidence of extrapancreatic involvement (Fig. 1A). A total pancreatectomy and splenectomy were performed. Four years after pancreatectomy, the patient is doing well. Diabetes mellitus is well controlled, with rare hypoglycemic episodes.

Clinical presentation

Results

TABLE 1. Clinicopathological features and mutational analysis of patients with glucagon cell adenomatosis

Four patients (two men and two women; mean age 35.5 yr; range 25– 44) with no evidence of hereditary pancreatic endocrine diseases (1, 7) were studied (Table 1). Patients 1–3 were already included in a previous study (5). From each patient and three age-matched controls, all available formaldehyde-fixed, paraffin-embedded pancreatic tissue blocks were examined histologically. A neuroendocrine neoplasm was identified by its positive immunostaining for synaptophysin and chromogranin A. The number of glucagon-producing tumors was recorded, and the tumors were classified according to the World Health Organization criteria (1). Microadenomas (250 ␮m to 5 mm) and macrotumors (ⱖ5 mm) were distinguished as previously described (5, 8). In all four patients, the density, number, size, and glucagon to insulin cell ratio of the islets were analyzed by point counting and the data compared with those from controls. The size and distribution of 100 islets per case (with a diameter ⬍100, 100 –500, and ⬎500 ␮m) were recorded. To calculate a glucagon to insulin cell ratio in relation to the size of the islet, 60 size-matched islets per case were evaluated. A total of 69 microadenomas from patients 1 and 2 was analyzed for loss of the MEN1 gene, and 55 microadenomas from patients 1–3 were for loss of the von Hippel-Lindau (VHL) gene locus by double-target fluorescence in situ hybridization (FISH), as described recently (9, 10). For the germ line mutation analysis of the MEN1 and VHL genes, DNA was extracted from the nontumorous pancreatic and parapancreatic parenchyma of patients 1–3. For the analysis of somatic mutations of the MEN1 gene, DNA was extracted from 15 microadenomas and/or macroadenomas from patient 1 (with known MEN1 deletion status), and from one macroadenoma and 20 pooled microadenomas from patient 2. For the analysis of somatic mutations of the VHL gene and for array comparative genomic hybridization (CGH), DNA was extracted from 20 pooled microadenomas and from one macroadenoma from patient 1, as well as 20 pooled microadenomas from patient 2. Further conditions were as described recently (5, 11, 12). With tissue from patients 1, 2, and 4, a mutational analysis of the p27 gene was performed as recently published (13). This project was approved by the Ethics Committee of the University of Kiel (D430/2005).

Specimen

Patients and Methods

Microtumors

MEN1/VHL germ line mutations

as multiple microadenomas and macrotumors in patients with multiple endocrine neoplasia type 1 (MEN1) (5, 6). Here, we report a fourth condition of which we believe that we are dealing with a new disease of the endocrine pancreas.

None


Acute pancreatitis

Glucagon Cell Adenomatosis

Age (yr)/ sex

Henopp et al.

Patient no.

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The tumors expressed almost exclusively glucagon (Fig. 1C), and were all positive for synaptophysin and chromogranin A (Table 1 and Figs. 1D and 2). None of the patients had extrapancreatic tumors. There were normal numbers of islets. However, a considerable fraction of the islets was enlarged with diameters between 176 and 726 ␮m [mean diameter 324 (⫾384); median: 200), which significantly exceeded the mean and median diameter of islets in the control specimens (mean diameter: 117, ⫾61; median: 100). Immunohistochemical examination revealed a reverse glucagon to insulin ratio with increasing islet size compared with that of controls.

FIG. 1. A, Somatostatin receptor scintigraphy showing diffuse labeling of the entire pancreas. B, Surgical specimen from patient 1 showing the cut surface of the pancreas and spleen. C, Double-immunofluorescence analysis of tumor cells for glucagon (GLU) (green) and insulin (INS) (red) reveals almost exclusively glucagon cells and only individual scattered insulin cells. Magnification, ⫻250. D, The pancreatic parenchyma is studded with multiple small neuroendocrine tumors immunostained with synaptophysin (SYN). Magnification, ⫻10.

Patient 2, a 43-yr-old woman, had suffered from unspecific upper abdominal pain for 14 d. Imaging revealed a cystic lesion in the tail of the pancreas with a diameter of 8 cm, suggesting a mucinous cystic neoplasm. A left-sided partial pancreatectomy was performed. The postoperative outcome was without complications. The patient is alive and has not developed any new neoplasms or signs of endocrine hyperactivity but showed 25fold increased serum glucagon levels on follow-up. Patient 3, a 30-yr-old woman, died of pneumonia in the course of acute lymphatic leukemia. The changes in the pancreas were an incidental postmortem finding. Patient 4, a 44-yr-old man, presented with a 9-month history of erythematous, pruritic papules on the soles of the feet and legs that finally involved the trunk and the scalp. Cheilitis, anemia, hypoproteinemia, diabetes, weight loss (10 kg), and deep vein thrombosis in the right leg followed. Treatment with prednisolone, azathioprine, and methotrexate had no effect. After developing necrolytic migratory erythema and high serum glucagon levels, a glucagonoma syndrome was diagnosed. Imaging and octreotide scintigraphy failed to detect a tumor. The patient developed a fever and progressive cardiac decompensation. He was then treated with octreotide. After a transitional period of relief, the skin lesions and his general status worsened, and he died of septic shock. A postmortem examination was performed. Pathology The pancreas of patient 1 was enlarged and showed eight randomly distributed macrotumors (up to 2 cm) and numerous microadenomas (Fig. 1 B). The pancreas of patient 2 showed a cystic tumor (8 ⫻ 6 cm in diameter) and a small solid tumor (0.5 cm in diameter). Patients 3 and 4 both had a normal-sized pancreas. Histologically, all patients had well-differentiated microadenomas randomly distributed in the parenchyma, classified as benign PET according to World Health Organization criteria (1).

Molecular genetics Germ line mutational analysis of the MEN1, VHL, and p27 genes (9, 13–15) was negative in patients 1 and 2. Due to insufficient DNA quality, mutational analysis failed in patients 3 and 4. Array CGH of 20 pooled microadenomas and one macroadenoma from patient 1 revealed a homogeneous diploid chromosome set. FISH analysis of the MEN1 gene in 54 microadenomas from patients 1–3 showed normal findings in 53 microadenomas, but allelic deletion at the 11q13 locus in one microadenoma. Analysis of 15 additional microadenomas from patient 1 revealed another tumor with an allelic deletion at 11q13 and one in the centromere 11 region. The same microadenoma was microdissected, and mutation analysis revealed a missense mutation in exon 10 of the menin gene (T580K), indicating that both MEN1 alleles were inactivated. FISH analysis of the VHL gene in 15 microadenomas from patient 1 followed by somatic mutation analysis did not reveal any evidence of somatic mutations or allelic deletions of the VHL gene. A total of 55 further tumors from this patient analyzed by FISH alone failed to reveal any allelic deletions of chromosome 3p25 and the centromere 3 region.

FIG. 2. Synaptophysin (SYN) stained pancreatic endocrine tissue in patients 2– 4 (A–C, identical magnifications) compared with that of a control pancreas (D). pat, Patient; ref, reference.

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Discussion Multiple glucagon-producing PETs are a key finding in MEN1 patients (5, 6). Multiple PETs may also occur in VHL patients (14, 16). Very recently, p27 germ line mutations have been identified as a further cause of a MEN syndrome (13). We have described four patients with multiple glucagon-producing tumors and islet glucagon cell hyperplasia that occurred unrelated to MEN1, VHL, and p27 germ line mutations, and in one patient caused a severe glucagonoma syndrome. The patients had multiple small PETs. In two cases there were also macrotumors. Although such findings are suggestive of MEN1 (5, 6) and may occasionally also be observed in a VHL patient (14), the patients in this study had neither the clinical features nor the genetic findings characteristic of MEN1 or VHL (8, 9). Moreover, not a single tumor expressed a hormone other than glucagon. These features clearly distinguished this multicentric tumor disease from that seen in MEN1, VHL, and p27 patients. Because inactivation of the wild-type MEN1 and VHL alleles due to loss of heterozygosity is responsible for the development of early neoplasms in the MEN1 and VHL syndromes (8, 9, 15), we also analyzed such conditions in our patients and failed to detect allelic deletions or somatic mutations of the VHL gene. The loss of one copy of the MEN1 gene was present in two of 69 tumors (2.9%). These findings indicate that losses of the wildtype MEN1/VHL allele are not a decisive factor in tumor development, and an association with MEN1, VHL, or p27 can be excluded. There are also no indications in the family history of the patients of an inherited disease, though this statement has to be made with caution because of the occasional asymptomatic nature of inherited conditions. A noteworthy finding was that many of the islets showed glucagon cell hyperplasia. Some of these islets were difficult to distinguish from small glucagon cell neoplasms, and in some giant islets (⬎300 –500 ␮m), there appeared to be an imperceptible transition to glucagon cell neoplasms. This suggested that the development of the tumors was preceded by glucagon cell hyperplasia, which affected the entire glucagon cell population. The reason for the increased proliferation of the glucagon cells is not known. However, the observation that the two microadenomas that carried MEN1 gene alterations showed distinct deletion patterns indicates that these two PETs, and probably also all other PETs, were caused by independent mutations and, thus, derived from different cell clones. So far, none of the glucagon-producing PETs in the four patients showed criteria of malignancy, such as increased mitotic activity, lymphangio-invasion, or metastatic spread. The benign nature of these tumors is also supported by their homogeneous diploid chromosome set, as revealed by the array CGH analysis. However, because of the small number of cases, any statements on the biological behavior of the tumors need to be made with caution. Three of the four patients did not have any symptoms related to the increase in glucagon cells. Only the fourth patient had diabetes as a result of the severe glucagonoma syndrome that led to his death.


The occurrence of multiple pancreatic neoplasms exclusively producing glucagon, associated with glucagon cell hyperplasia of the islets and unrelated to MEN1, VHL, and the recently identified p27 MEN syndrome (13), is a novel observation. A recently published case report of a 60-yr-old patient seems to represent the same disease (17). To give it a name and emphasize the so-far benign nature of the tumors, we suggest the term “glucagon cell adenomatosis.”

Acknowledgments We thank Maike Pacena, Anja Bredtmann, Sonja Schmid, Thomas Rudolf, Sandra M. H. Claessen, Marion Bawohl, Franziska No¨tzli, and Klaus Scho¨nheinz for their excellent technical assistance. We are grateful to Kay Dege for editing the manuscript. Address all correspondence and requests for reprints to: Tobias Henopp, M.D., Department of Pathology, University of Tu¨bingen, Liebermeisterstr. 8, 72076 Tu¨bingen, Germany. E-mail: Tobias.Henopp@med. uni-tuebingen.de. This work was supported by the Hensel Stiftung, Kiel, Germany (F370011) (to M.A. and G.K.), the German Society of Pathology (to M.A.) and Novartis, Oncology, Nu¨rnberg, Germany (to M.A. and G.K.), and The Swiss National Foundation (SNF 31-108257) (to A.P. and P.K.). T.H. has a fellowship sponsored by Ipsen GmbH, Ettlingen. Disclosure Statement: A.S., R.S. A.Z., A.C., P.R., K.-P.S., Y.M.H.J., E.-J.M.S., N.S.P., and P.U.H. have nothing to disclose.

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