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Department of Pathology (A.P., T.R., A.S., P.U.H., P.K.), Institute of Surgical Pathology, University Hospital, CH-8091 Zürich, Switzerland; Institute of Pathology (A.P.), Technische Universitaet Muenchen, D-81675 Munich, Germany; Department of Pathology (M.A., T.H., G.K.), University of Kiel, D-24105 Kiel, Germany; Department of General, Visceral, and Pediatric Surgery (A.R., W.T.K.), University of Düsseldorf, D-40225 Düsseldorf, Germany; Department of General, Visceral, and Vascular Surgery (O.G., H.D.), University of Halle, D-06120 Halle, Germany; Departments of Molecular Neuroscience and Anatomy and Cell Biology (E.W.), University of Marburg, D-35037 Marburg, Germany; and Institute of Pathology (P.K.), Stadtspital Triemli, CH-8063 Zurich, Switzerland
Address all correspondence and requests for reprints to: Aurel Perren, M.D., Institute of Surgical Pathology, Department of Pathology, University Hospital Zürich, 8091 Zürich, Switzerland. E-mail: aurel.perren{at}usz.ch.
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Design: We hypothesized that monohormonal endocrine cell clusters observed in MEN1 patients are small neoplasms with loss of heterozygosity of the MEN1 locus. Using a technique combining fluorescence in situ hybridization of the MEN1 locus and the centromeric region of chromosome 11q with hormone immunostaining, we examined resection specimens from four MEN1 patients. We focused our investigations on the following: 1) typical microadenomas; 2) monohormonal endocrine cell clusters; 3) endocrine and exocrine structures entrapped in microadenomas; and 4) morphologically normal islets.
Results: Loss of one MEN1 allele was found in all 27 microadenomas and 19 of 20 (95%) monohormonal endocrine cell clusters. By contrast, it was absent in islets and ductal or acinar structures. Our results indicate that monohormonal endocrine cell clusters represent a minute form of microadenomas.
Conclusion: The frequent presence of single nonneoplastic insulin cells in microadenomas and the occurrence of microadenomas in islets suggest an islet origin of microadenomas. Islet hyperplasia does not seem to be an obligatory stage in pancreatic MEN1-associated tumor development.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The presence of multiple microadenomas (i.e. endocrine tumors up to 5 mm in diameter) is a hallmark of MEN1 in the pancreas (6, 7, 8). Because almost all MEN1 patients show microadenomatosis (9), pancreatic tissue of these patients represents a unique possibility to study early neoplasms and their potential precursor lesions.
Two molecular studies examined MEN1-associated pancreatic microadenomas (5, 10). Using microdissection and PCR-based microsatellite analysis, a loss of the wild-type MEN1 allele on chromosome 11q13 in all examined microadenomas was described. Additionally, Vortmeyer et al. (10) found 11q13 loss of heterozygosity (LOH) in duct-associated lesions but not islets, and their conclusion was a nonislet cell origin of pancreatic islet cell tumors.
Recently we reappraised the morphological features and hormone expression pattern of MEN1-associated pancreatic microadenomas (9). We observed endocrine cell clusters, suggesting an origin of at least some tumors from islets. In addition, we identified islet-like, glucagon-rich structures, whose neoplastic potential could not be determined. We hypothesized that these endocrine cell clusters represent small neoplasms and that MEN1 LOH could be a molecular marker for neoplastic growth.
To tackle the questions related to the site of origin of endocrine tumors in the MEN1 pancreas and the identification of early neoplastic lesions, we initiated a study using a combined fluorescence in situ hybridization (FISH) and hormone immunostaining technique. This technique enabled us to simultaneously and precisely analyze allelic deletions and hormone expression in single endocrine cells and determine the allelic loss of the MEN1 gene locus in the following: 1) classical microadenomas, 2) monohormonal endocrine cell clusters (MECCs), 3) endocrine and exocrine structures entrapped in microadenomas, and 4) in normal and enlarged islets.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The study included four MEN1 patients suffering from pancreatic endocrine tumors (two men and two women; 25, 29, 35, and 41 yr old at the time of surgery), whose specimens were collected between 1980 and 2005 in the archives of the Departments of Pathology of the Universities of Kiel (Germany) and Zürich (Switzerland). Morphological findings of microadenomatosis of three patients were reported in a previous study (9). All patients fulfilled the clinical criteria of the MEN1 syndrome, i.e. at least two MEN1-related endocrine tumors or one of these tumors together with a first-degree relative suffering from MEN1 (11) Three patients were proven to have a MEN1 germline mutation. One patient suffered from hyperinsulinemic hypoglycemia. Two patients exhibited a Zollinger-Ellison syndrome caused by duodenal gastrinomas. One patient underwent surgery for multiple pancreatic endocrine tumors without hormonal syndrome. A total of 18 paraffin blocks were selected from the surgical specimens and included in the analysis. Specimens from three patients who had a Whipple resection for pancreatic ductal adenocarcinoma (three men; mean age 53 yr; range 39–69) were used as control tissues.
Immunohistochemistry
Serial sections (3–4 µm) were cut from paraffin-embedded tissue blocks. For histological analysis, two sections were stained with hematoxylin and eosin (H&E) and periodic acid-Schiff. Serial sections were immunostained with the following antibodies: chromogranin A (mouse monoclonal, dilution 1:40 and 1:4 for immunofluorescence; BioGenex, San Ramon, CA), synaptophysin (mouse monoclonal, dilution: 1:100 and 1:10 for immunofluorescence; HyTest, Turku, Finland, and rabbit, polyclonal, dilution 1:50, Dako, Glostrup, Denmark), somatostatin (rabbit, polyclonal, dilution 1:200 and 1:50 for immunofluorescence; Dako, Hamburg, Germany; and rat monoclonal, dilution 1:100 for immunofluorescence; Acris, Hiddenhausen, Germany), insulin (guinea pig, monoclonal, dilution 1:6000 and 1:600 for immunofluorescence; Dako; and mouse monoclonal, dilution 1:40; Biogenex), glucagon (rabbit, polyclonal, dilution 1:60, Biogenex, and goat polyclonal, dilution 1:600 for immunofluorescence, Santa Cruz Biotechnology, Santa Cruz, CA), pancreatic polypeptide (rabbit, polyclonal, dilution 1:5,000, Paesel and Lorei, Hanau, Germany; and sheep polyclonal, dilution 1:60 for immunofluorescence; Binding Site, Birmingham, UK) (6, 9). In addition, antisera against gastrin, vasoactive intestinal polypeptide, ACTH, and calcitonin were used to analyze the expression of ectopic peptides, as previously described (9). After heat-induced epitope retrieval and blocking with nonimmune serum for 20 min, the primary antibody was applied for 45 min and the reaction was detected with species-specific biotinylated secondary antibodies (Dianova, Hamburg, Germany) for 45 min (12). The slides were washed three times in PBS and incubated for 30 min with the ABC reagents (Vectastatin Elite ABC kit, Boehringer, Ingelheim, Germany). The immunoreactions were visualized with 3'3-diaminobenzidine (Sigma, Deisenhofen, Germany), which resulted in brown staining. Immunostained sections were analyzed and photographed with an Axioskop 50 microscope (Zeiss, Oberkochen, Germany).
Fluorescence microscopy
The sections were covered with a mixture of the relevant primary antibodies in appropriate dilutions for 2 h at room temperature. Before antibody application, the tissue sections were incubated in 4x sodium chloride sodium citric acid (SSC) containing 5% nonfat dry milk for 10 min and 4x SSC/0.05% Tween 20 to reduce autofluorescence and block unspecific binding sites. The slides were incubated with the primary antibodies for 2 h and then washed in 4x SSC/0.05% Tween 20. Subsequently the slides were incubated with the secondary antibodies for 1 h at 37 C (Alexa 488 antimouse, 1:1000, Alexa 488 antirabbit, 1:1000, Alexa 488 antigoat 1:1000; both Molecular Probes, Eugene, OR; and Cy3 antimouse 1:1000, Cy3 antirabbit, 1:1000, Cy 3 antigoat 1:1000, both Dianova). The sections were counterstained for nuclei using A33528 dye (Hoechst, Franfurt, Germany) at a concentration of 0.025 mg/ml and extensively washed in 4x SSC/0.05% Tween 20. Then the sections were mounted using ProLong antifade mounting medium (Molecular Probes). For the double fluorescence analysis, a BX 61 fluorescence microscope (Olympus, Hamburg, Germany) was used.
Classification of endocrine lesions
In accordance with the recently published World Health Organization criteria for endocrine tumors of the pancreas we distinguished between microadenomas (
5 mm) and macrotumors (>5 mm) (11). Microadenomas were characterized by the presence of a trabecular, gyriform or solid growth pattern, and/or sclerosis. We additionally recorded lesions only recognizable by immunohistochemical stainings for peptide hormones. Such MECCs were defined by a predominant expression of one islet hormone (or complete absence of islet hormone expression).
Further structures that were analyzed included endocrine cells in the duct epithelium, ductuloinsular complexes, scattered endocrine cells within the acinar parenchyma, areas of atypical acinar nodules, normal islets, and enlarged hyperplastic islets showing an increase in glucagon cells.
LOH analysis of chromosomal regions 11q13 and centromere 11
FISH was performed on all specimens using a chromosome 11-specific centromere probe (pLC11A) in combination with an 11q13-specific probe (RP1CTD-2220I9) containing the MEN1 gene. Both FISH probes were labeled with fluorescein-12-dUTP (spectrum green) or biotin by nick translation (Boehringer, Mannheim, Germany) and precipitated in ethanol in the presence of 50x Cot1-DNA, sodium acetate, and glycogen. The DNA pellet was resuspended in the hybridization buffer (50% formamide, 10% dextran sulfate, 2x SSC) with a final concentration of 250 ng probe per 10 µl. After dehydration in an ethanol series and blocking for 30 min in 1% methanol H2O2, the slides were pretreated in HCL 0.2 M at room temperature for 20 min and in 1 M NaSCN for 30 min at 80 C. Proteinase treatment was performed by incubation in 0.4 g/liter pepsin in 10 mM Tris HCL solution for 10 min at 37 C followed by postfixation in 4% formalin for 10 min at room temperature. After sequential washings in 2x SSC and distilled water, both probes were applied to the tissue specimens, denaturated at 80 C for 5 min, and then incubated at 37C overnight in a humidified chamber. Posthybridization washes were performed at 45 C in 50% formamide/2x SSC (twice for 5 min). The hybridized fluorescein-labeled probes were detected by using a cascade of rabbit anti-fluorescein isothiocyanate (FITC) immunoglobulins (1:1000, Dako), FITC-conjugated swine antirabbit immunoglobulins (1:100, Dako), and FITC-conjugated rabbit antiswine immunoglobulins (1:1000, Dako). Detection of the biotin-labeled probes was performed using a cascade of avidin tetramethylrhodamine isothiocyanate (1:100, Vector, Burlingame, CA), biotinylated anti-avidin D (1:100) and avidin tetramethylrhodamine isothiocyanate (1:100). The slides were washed in 4x SSC/0.05% Tween 20, air dried, and then mounted in Vectashild (Vector) containing 0.5 µg/ml 4',6-diamidino-2-phenylindole-antifade (Sigma) for nuclear counterstaining. Images were recorded with Analysis software (Olympus Biosystems).
Simultaneous FISH and hormone expression analysis
The specificity and sensitivity of simultaneous FISH/immunofluorescence detection of endocrine cells was first tested on normal pancreatic parenchyma from the three control patients and then applied to the patients’ tissue specimens. The specimens were dehydrated in an ethanol series, 4x SSC Tween 20 for 10 min, and then pretreated overnight in a 4x SSC/5% nonfat dry milk solution at 37 C. Denaturation, hybridization, and posthybridization were performed as described above. The 11q13 signal was amplified with a cascade of rabbit anti-FITC immunoglobulins (1:1000), FITC-conjugated swine antirabbit immunoglobulins (1:100), and FITC-conjugated rabbit antiswine immunoglobulins (1:1000). Primary antibodies against insulin or somatostatin were applied in the second step of the detection cascade in the final dilutions described above and incubated for 45 min. The slides were washed in 4x SSC/0.05% Tween 20 and then incubated with the secondary antibodies for 1 h at 37 C (Alexa 488 antimouse, 1:1000, Alexa 488 antirabbit, 1:1000, Alexa 488 anti-guinea pig 1:1000) together with the FITC-conjugated rabbit antiswine immunoglobulins. Counterstaining and microscopic analysis were performed as described above.
Specificity controls and evaluation
The specificity controls for the FISH and the immunostainings included: 1) omission of individual steps of the detection cascade, 2) performance of the FISH/immunohistochemistry protocol without FISH probes or 3) without primary antibodies, and 4) scanning and digitalization of immunohistochemically stained sections and comparison with FISH results subsequently obtained on the same sections.
For comparative evaluation of histological, immunohistochemical and LOH data, multiple sets of six serial sections of each tissue specimen were cut and stained in the following order: H&E, synaptophysin, insulin, glucagon, somatostatin or pancreatic polypeptide, and simultaneous FISH for 11q13 (red) and C11 (green). Very stringent criteria for LOH definition could be applied: lesions with fewer than 5% of cells showing two signals were interpreted as showing LOH. For macrotumors and classical microadenomas, at least 500 cells were counted; for MECCS, hyperplastic, and normal islets, all cells were evaluated. Normal duodenal tissue specimens or connective tissue in the vicinity of the lesions served as internal controls. They exhibited nuclei with two probe signals at a frequency of more than 80%. Only scattered cells showed one signal due to cutting artifacts.
Ethics
This project was approved by the Ethics Committee of the University of Kiel (D430/2005).
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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A total of 34 pancreatic endocrine tumors were identified in the tissue specimens of the four patients. These were randomly distributed within the pancreatic parenchyma. Seven macrotumors (mean 12 mm; range 7–22 mm) and 27 microadenomas (mean 0.9 mm; range 100 µm to 4.5 mm) showed the typical histology of endocrine neoplastic growth, i.e. trabecular, gyriform, solid, or desmoplastic (Table 1
and Fig. 1). Immunohistochemically the majority of these neoplasms (28 of 34; 82%) were homogeneously positive for one hormone [glucagon, n = 20; insulin, n = 4; somatostatin, n = 2; pancreatic polypeptide (PP), n = 2] with minor populations of scattered endocrine cell expressing other islet hormones. Six tumors (18%) expressing synaptophysin and chromogrannin-A failed to express any hormone (Table 1). Expression of ectopic peptides was not detected. LOH on chromosome 11q13 was detected in 33 of 34 tumors (97%). In addition, 30 tumors (88%) revealed LOH in the centromere 11 region (Table 1 and Fig. 1). Three patients (no. 1, 3, and 4) showed different deletion/retention patterns in synchronous tumors (Table 1). Single entrapped endocrine cells expressing hormones other than that characterizing the tumor and the entrapped cells of the duct epithelium consistently retained both MEN1 alleles (Fig. 1). We did not detect any chromosome 11 gains or homozygous deletions in any tumor.
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In addition to microadenomas, we identified 20 endocrine cell aggregates with a monohormonal immunohistochemical profile similar to that of the microadenomas (mean diameter, 188 µm; range, 50–300 µm; Table 1
). Eight MECCs were intimately associated with an islet (Table 1, tumors 35–42; Figs. 2 and 4, A–F); five MECCs could not be clearly distinguished from islets without hormone immunostaining (Table 1, tumors 43–47; Figs. 3 and 4, J–L); six MECCs were composed of micronodular endocrine cell complexes embedded within sclerotic tissue (Table 1, tumors 40–47; Fig. 4, G–I), and one MECC was in close contact with duct epithelium (Table 1, tumor 54; Fig. 4, M–O). The MECCs were immunoreactive for glucagon (n = 14; 70%) or PP (n = 1; 5%). In five MECCs (25%) none of the hormones tested was expressed. LOH at the MEN1 locus was detected in all but one of these lesions (95%). In addition, 17 of them revealed LOH in the centromere 11 region (85%). Single entrapped endocrine cells expressing a hormone different from that characterizing the MECC and endocrine cells in the duct epithelium consistently retained both MEN1 alleles.
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Most islets showed a normal distribution of endocrine cells and normal size. Approximately 3% of the islets, however, were enlarged, displaying a diameter of more than 400 µm and/or were of an irregular shape. In these enlarged or irregularly shaped islets the ratio of glucagon cells was over 50% of all islet cells. Trabecular, gyriform, solid, or desmoplastic growth was absent (Fig. 5). LOH analysis of 600 islets including 15 enlarged islets consistently showed retention of the 11q13 and centromere 11 signals (Fig. 5).
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The high ratio of chromosome 11q13 LOH in MEN1-associated macrotumors, frequently encompassing both centromere 11 and the MEN1 locus, corresponds well with the LOH rates of similar tumors described in the literature (4, 5). The only macrotumor in our series lacking MEN1 LOH produced insulin and led to an insulinoma syndrome. Interestingly, Pannett and Thakker (13) also described a MEN1-associated insulinoma without LOH and demonstrated a second somatic mutation of the MEN1 gene. We therefore assume that a similar mechanism of inactivation of the MEN1 wild-type allele could also have been active in the insulinoma of the present series.
In three patients the allelic deletion/retention pattern differed among individual tumors. For example, one patient showed a loss of both the centromere 11 and the MEN1 regions in one macrotumor and seven microadenomas, a loss of the MEN1 region sparing the C11 region in one macrotumor and two microadenomas, but no allelic deletion in one macrotumor. This finding suggests that the second hits inactivating the tumor suppressor gene (MEN1) are independent events in individual MEN1-associated tumors, making them clonally different and independent neoplasms. In duodenal gastrinomas, such a polyclonal origin of synchronous tumors has been demonstrated by analysis of the X-inactivation pattern of the human androgen receptor gene (HUMARA) (4) and recently by FISH examination (14). The finding of a homogenous deletion pattern in individual tumors is in contrast to those of other chromosomal loci, which is characterized by intratumoral heterogeneity (5). The homogenous pattern is suggestive of an early occurrence of 11q13 LOH in tumorigenesis, i.e. before chromosomal instability.
In addition to microadenomas, we identified small aggregates of endocrine cells, some of them embedded in sclerotic stroma or integrated in duct epithelium (duct associated), which we suspected to be neoplastic but found to be difficult to distinguish from islet tissue. Because these lesions showed the same monohormonal staining pattern as microadenomas, we termed them MECCs with and without sclerosis or MECCs with duct association. LOH analysis clearly revealed the neoplastic nature of all but one of the MECCs and showed that the hormonal expression pattern is a valuable adjunct for recognizing these endocrine cell aggregates as neoplasms. The majority of the microadenomas and MECCs we were able to identify (37 of 46, 80%) were found to be less than 500 µm in diameter.
Vortmeyer et al. (10) described 11q LOH in microdissected atypical structures in pancreatic resection specimens of MEN1 patients, termed type A1, A2, A3, and A4. Based on the histological pictures provided, we believe that these structures represent MECCs. Whereas the A3 structure appears to correspond to a classical microadenoma, the A1 lesion corresponds to a MECC with sclerosis and the A2 structure to a MECC with duct association. Unfortunately, it was not clearly stated in this publication at what frequency these different lesions were observed or how many of them could be examined for LOH. The A4 structures, corresponding to atypical acinar nodules, retained both MEN1 alleles (10). We confirmed these data and in addition show that LOH was consistently absent in acini, normal ducts, and ducts entrapped in microadenomas. Furthermore, all insulin and somatostatin positive single endocrine cells interspersed in microadenomas and MECCs showed retention of heterozygosity. This was also true of islets, regardless of their size and number of glucagon cells.
Studies on human tissue allow only indirect conclusions regarding the dynamics of a disease. In our study, however, the various tumors detected in the pancreas of a MEN1 patient probably represent neoplasms at different stages of development (Fig. 6). We may therefore gain some insights into the question as to the cell compartment in the pancreas from which they originate. Our observations indicate that the tumors may arise from different pancreatic structures. First, the identified nonneoplastic insulin cells in glucagon-producing microadenomas could represent the remaining cells of an islet from which the tumor originated. This was indeed demonstrated in the eight MECCs developing in preexisting islets. Second, we found one MECC integrated in duct epithelium. These results suggest that MEN1-associated endocrine tumors seem to arise from at least two different endocrine cell compartments of the pancreas, i.e. most frequently from islets and rarely from ducts. To date evidence of an origin from the acinar compartment is lacking.
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Islet hyperplasia in humans with the MEN1 syndrome is characterized by islet enlargement and an increased number of glucagon cells, which, however are always accompanied by insulin cells. LOH was never found in these enlarged islets. A MECC was found only once in association with such an islet, whereas seven other MECCs originated from normal appearing islets. The presence of an increased number of enlarged islets (i.e. islet hyperplasia) may therefore be a lesion associated with the MEN1 germline mutation, but it does not appear to be necessarily involved in the development of microadenomas. Indeed, hyperplastic islets as defined here are not more frequent than microadenomas, which would be very unusual for an obligatory precursor lesion.
In summary, we showed that pancreatic microadenomas including MECCs are monoclonal. The frequent presence of single nonneoplastic insulin cells in microadenomas and the occurrence of microadenomas in intimate association with islets point to an islet origin of these microadenomas. The occurrence of enlarged islets also seems to be associated with the MEN1 germline mutation but is probably not a obligatory step for neoplastic transformation.
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Acknowledgments |
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Footnotes |
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The authors have nothing to disclose.
First Published Online December 19, 2006
1 A.P. and M.A. contributed equally to this study. 
Abbreviations: FISH, Fluorescence in situ hybridization; FITC, fluorescein isothiocyanate; H&E, hematoxylin and eosin; LOH, loss of heterozygosity; MECC, monohormonal endocrine cell cluster; MEN1, multiple endocrine neoplasia type 1; PP, pancreatic polypeptide; SSC, sodium chloride sodium citric acid.
Received September 6, 2006.
Accepted December 11, 2006.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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