Precursor Lesions in Patients With Multiple Endocrine Neoplasia Type 1--Associated Duodenal Gastrinomas
2005; Elsevier BV; Volume: 128; Issue: 5 Linguagem: Inglês
10.1053/j.gastro.2005.01.058
ISSN1528-0012
AutoresMartin Anlauf, Aurel Perren, Cora Lu Meyer, Sonja Schmid, Parvin Saremaslani, Marie L. Kruse, Eberhard Weihe, Paul Komminoth, Philipp U. Heitz, Günter Klöppel,
Tópico(s)Pancreatic and Hepatic Oncology Research
ResumoBackground & Aims: The identification of precursor lesions has a great impact on the understanding of tumorigenesis. Precursor lesions of endocrine tumors are known to occur in the setting of the MEN1 syndrome. The aim of this study was to test the hypothesis that MEN1-associated duodenal gastrinomas originate from diffuse preneoplastic gastrin cell changes. Precursor lesions may precede the development of duodenal gastrinomas because, in contrast to sporadic gastrinomas, these tumors are usually multiple. Methods: The distribution of endocrine cells in the nontumorous duodenal tissue was analyzed qualitatively and quantitatively for 25 patients operated on for a duodenal gastrinoma. MEN1 status was assessed clinically and by polymerase chain reaction-based mutational analysis. Results: Fourteen of 25 patients with gastrinoma had proliferative, hyperplastic lesions consisting of gastrin cells in the nontumorous duodenal mucosa, similar to the gastric enterochromaffin-like cell lesions observed in chronic atrophic gastritis. All patients with Zollinger-Ellison syndrome with proven MEN1 had such proliferative gastrin cell lesions, and all patients with Zollinger-Ellison syndrome without precursor lesions were MEN1 negative. Conclusions: Duodenal gastrinomas in MEN1, but not sporadic duodenal gastrinomas, are associated with proliferative gastrin cell changes within the nontumorous mucosa. It is likely that these lesions precede the development of MEN1-associated duodenal gastrinomas. Background & Aims: The identification of precursor lesions has a great impact on the understanding of tumorigenesis. Precursor lesions of endocrine tumors are known to occur in the setting of the MEN1 syndrome. The aim of this study was to test the hypothesis that MEN1-associated duodenal gastrinomas originate from diffuse preneoplastic gastrin cell changes. Precursor lesions may precede the development of duodenal gastrinomas because, in contrast to sporadic gastrinomas, these tumors are usually multiple. Methods: The distribution of endocrine cells in the nontumorous duodenal tissue was analyzed qualitatively and quantitatively for 25 patients operated on for a duodenal gastrinoma. MEN1 status was assessed clinically and by polymerase chain reaction-based mutational analysis. Results: Fourteen of 25 patients with gastrinoma had proliferative, hyperplastic lesions consisting of gastrin cells in the nontumorous duodenal mucosa, similar to the gastric enterochromaffin-like cell lesions observed in chronic atrophic gastritis. All patients with Zollinger-Ellison syndrome with proven MEN1 had such proliferative gastrin cell lesions, and all patients with Zollinger-Ellison syndrome without precursor lesions were MEN1 negative. Conclusions: Duodenal gastrinomas in MEN1, but not sporadic duodenal gastrinomas, are associated with proliferative gastrin cell changes within the nontumorous mucosa. It is likely that these lesions precede the development of MEN1-associated duodenal gastrinomas. The majority of duodenal neuroendocrine tumors are gastrinomas, which can lead to Zollinger-Ellison syndrome (ZES).1Ellison E.H. Wilson S.D. The Zollinger-Ellison syndrome reappraisal and evaluation of 260 registered cases.Ann Surg. 1964; 160: 512-530Crossref PubMed Scopus (288) Google Scholar, 2Zollinger R.M. The ulcerogenic (Zollinger-Ellison) syndrome gastrinoma.in: Friesen S.R. Thompson N.W. Surgical endocrinology clinical syndromes. 2nd ed. JB Lippincott, Philadelphia, PA1990: 267-278Google Scholar, 3Thom A.K. Norton J.A. 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Furthermore, it is likely that the adenomatous changes of the parathyroid in patients with the MEN1 syndrome originate from hyperplastic changes.11Heppner C. Kester M.B. Agarwal S.K. Debelenko L.V. Emmert-Buck M.R. Guru S.C. Manickam P. Olufemi S.E. Skarulis M.C. Doppman J.L. Alexander R.H. Kim Y.S. Saggar S.K. Lubensky I.A. Zhuang Z. Liotta L.A. Chandrasekharappa S.C. Collins F.S. Spiegel A.M. Burns A.L. Marx S.J. Somatic mutation of the MEN1 gene in parathyroid tumours.Nat Genet. 1997; 16: 375-378Crossref PubMed Scopus (369) Google Scholar, 12Crabtree J.S. Scacheri P.C. Ward J.M. Garrett-Beal L. Emmert-Buck M.R. Edgemon K.A. Lorang D. Libutti S.K. Chandrasekharappa S.C. Marx S.J. Spiegel A.M. Collins F.S. A mouse model of multiple endocrine neoplasia, type 1, develops multiple endocrine tumors.Proc Natl Acad Sci U S A. 2001; 98: 1118-1123Crossref PubMed Scopus (404) Google Scholar, 13Libutti S.K. Crabtree J.S. Lorang D. Burns A.L. Mazzanti C. Hewitt S.M. O'Connor S. Ward J.M. Emmert-Buck M.R. Remaley A. Miller M. Turner E. Alexander H.R. Arnold A. Marx S.J. Collins F.S. Spiegel A.M. Parathyroid gland-specific deletion of the mouse Men1 gene results in parathyroid neoplasia and hypercalcemic hyperparathyroidism.Cancer Res. 2003; 63: 8022-8028PubMed Google Scholar All such conditions, except for the enterochromaffin-like (ECL) cell hyperplasia in chronic atrophic gastritis, are associated with an inherited endocrine disorder.14Bordi C. D'Adda T. Azzoni C. Ferraro G. Pathogenesis of ECL cell tumors in humans.Yale J Biol Med. 1998; 71: 273-284PubMed Google Scholar, 15Delle Fave G. Marignani M. Corleto V.D. Angeletti S. D'Ambra G. Ferraro G. D'Adda T. Azzoni C. Jensen R.T. Annibale B. Bordi C. Progression of gastric enterochromaffin-like cells growth in Zollinger-Ellison syndrome and atrophic body gastritis patients.Dig Liver Dis. 2002; 34: 270-278Abstract Full Text PDF PubMed Scopus (18) Google Scholar, 16Peghini P.L. Annibale B. Azzoni C. Milione M. Corleto V.D. Gibril F. Venzon D.J. Delle Fave G. Bordi C. Jensen R.T. Effect of chronic hypergastrinemia on human enterochromaffin-like cells insights from patients with sporadic gastrinomas.Gastroenterology. 2002; 123: 68-85Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar In 1990, it was noticed that many duodenal gastrinomas arising in the setting of MEN1 are multiple, in contrast to sporadic gastrinomas.4Pipeleers-Marichal M. Somers G. Willems G. Foulis A. Imrie C. Bishop A.E. Polak J.M. Hacki W.H. Stamm B. Heitz P.U. et al.Gastrinomas in the duodenums of patients with multiple endocrine neoplasia type 1 and the Zollinger-Ellison syndrome.N Engl J Med. 1990; 322: 723-727Crossref PubMed Scopus (340) Google Scholar Our working hypothesis is that these multifocal gastrinomas originate from diffuse proliferative changes of the duodenal gastrin cells. The aim of this study was therefore to (1) screen the duodenum of patients with gastrinomas for preneoplastic changes in gastrin cells, (2) quantify their extent and proliferative capacity, and (3) determine whether they are associated with the MEN1 syndrome. The study included 25 patients treated for duodenal gastrinomas whose specimens were collected between 1980 and 2002 in the consultation files of the departments of pathology of the University of Kiel (Germany) and the University of Zurich (Switzerland). Three patients underwent endoscopic polypectomy, 14 patients were treated by local excision, 3 patients underwent partial duodenectomy, and 5 patients had a Whipple resection. In addition to the duodenal gastrinomas, the presence of other endocrine tumors known to occur in patients with the MEN1 syndrome was recorded. An MEN1 status was assessed if at least 2 main MEN1-related endocrine tumors (parathyroid adenoma, enteropancreatic endocrine tumors, pituitary tumors, adrenocortical tumors, foregut carcinoids) were present. Patients with multifocal or recurrent endocrine tumors were defined as clinically suspicious for MEN1.17DeLellis R.A. Heitz P.U. Lloyd R.V. Eng C. WHO classification of tumors. Pathology and genetics of tumours of endocrine organs. IARC Press, Lyon, France2004Google Scholar From each patient, representative paraffin-embedded tissue blocks containing tumor and extratumoral duodenal mucosa fixed in either 4% formaldehyde or Bouin's solution were examined. All gastrin cell tumors >300 μm and associated with a ZES were classified as gastrinomas. Tumors >2 mm were referred to as macrotumors and tumors from 300 μm to 2 mm as microtumors. The occurrence of 2 or more duodenal gastrinomas was defined as multifocality. Samples from different regions of the duodenum of 3 patients who had a Whipple resection for pancreatic ductal adenocarcinoma (3 men; mean age, 53 years; range, 39–69 years) were used as control tissue. The duodenal endocrine cell populations were examined by staining with the antibodies listed in Table 1. To assess proliferation in gastrinomas and in extratumorous gastrin cells, the expression of the nuclear proliferation antigen Ki-67 was determined (Table 1).Table 1List of Primary AntibodiesAntigenCodeSource/referenceDilutionSpeciesGastrinG 17Paesel, Frankfurt, Germany1:3000/1:200aImmunofluorescence.Rabbit, polyclonalCholecystokininCCK-8 YP030Yanaihara, Shizuoka, Japan1:40,000Rabbit, polyclonalMotilinY120Yanaihara, Shizuoka, Japan1:40,000Rabbit, polyclonalSecretinY030Yanaihara, Shizuoka, Japan1:40,000Rabbit, polyclonalXenin2815/3Anlauf et al35Anlauf M. Weihe E. Hartschuh W. Hamscher G. Feurle G.E. Localization of xenin-immunoreactive cells in the duodenal mucosa of humans and various mammals.J Histochem Cytochem. 2000; 48: 1617-1626Crossref PubMed Scopus (67) Google Scholar1:6000Rabbit, polyclonalSomatostatinA 0566Dako, Hamburg, Germany1:200Rabbit, polyclonalGastric inhibitory peptideGIP Y101Yanaihara, Shizuoka, Japan1:50,000Rabbit, polyclonalSerotonin5HT H209Dako, Hamburg, Germany1:20Mouse, monoclonalSerotonin5HT 43H37RINC/IBL, Hamburg, Germany1:60,000Rabbit, molyclonalVesicular monoamine transporter 11/10Erickson et al36Erickson J.D. Schafer M.K. Bonner T.I. Eiden L.E. Weihe E. Distinct pharmacological properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter.Proc Natl Acad Sci U S A. 1996; 93: 5166-5171Crossref PubMed Scopus (379) Google Scholar1:2000Rabbit, molyclonalα–human chorionic gonadotropin823Biogenex, San Ramon, CA1:100Mouse, monoclonalCgAE 001Linaris, Wertheim, Germany1:2/1:1aImmunofluorescence.Mouse, monoclonalSynaptophysinA0010Dako, Hamburg, Germany1:50Rabbit, polyclonalKi-67Ki-67Kreipe et al37Kreipe H. Wacker H.H. Heidebrecht H.J. Haas K. Hauberg M. Tiemann M. Parwaresch R. Determination of the growth fraction in non-Hodgkin's lymphoma by monoclonal antibody Ki-S5 directed against a formalin-resistant epitope of the Ki-67 antigen.Am J Pathol. 1993; 142: 1689-1694PubMed Google Scholar1:1/1:30aImmunofluorescence.Mouse, monoclonala Immunofluorescence. Open table in a new tab Deparaffinized sections (3–4 μm thick) were rehydrated and subjected to heat-induced epitope retrieval procedures as previously described.18Sipos B. Weber D. Ungefroren H. Kalthoff H. Zuhlsdorff A. Luther C. Torok V. Kloppel G. Vascular endothelial growth factor mediated angiogenic potential of pancreatic ductal carcinomas enhanced by hypoxia an in vitro and in vivo study.Int J Cancer. 2002; 102: 592-600Crossref PubMed Scopus (29) Google Scholar, 19Feurle G.E. Anlauf M. Hamscher G. Arnold R. Kloppel G. Weihe E. Xenin-immunoreactive cells and extractable xenin in neuroendocrine tumors of duodenal origin.Gastroenterology. 2002; 123: 1616-1626Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar Before application of the primary antibody, blocking with nonimmune serum was performed for 20 minutes. After incubation for 45 minutes, the reaction was detected with species-specific biotinylated secondary antibodies (Dianova, Hamburg, Germany) for 45 minutes, washed several times in phosphate-buffered saline, and incubated for 30 minutes with the ABC reagents (Vectastatin Elite ABC kit; Boehringer, Ingelheim, Germany). Immunoreactions were visualized with 3,3′-diaminobenzidine tetrahydrochloride (Sigma, Deisenhofen, Germany), which resulted in brown staining. The number of gastrin- and chromogranin A (CgA)-positive cells in neuroendocrine tumors was estimated semiquantitatively as an approximate percentage and scored on a scale from 1 (<20%) to 5 (80%–<100%). To examine nuclear expression of Ki-67 in gastrinomas, 20 high-power fields (40× lens) were analyzed. Immunostained sections were analyzed and photographed with an Axioskop 50 microscope (Zeiss, Oberkochen, Germany). The gastrin cell lesions were classified in a double-blind fashion by 2 observers in analogy to the system proposed for the ECL cells of the stomach.20Solcia E. Bordi C. Creutzfeldt W. Dayal Y. Dayan A.D. Falkmer S. Grimelius L. Havu N. Histopathological classification of nonantral gastric endocrine growths in man.Digestion. 1988; 41: 185-200Crossref PubMed Scopus (346) Google Scholar In most samples, at least 10 serial sections at a distance of more than 0.5 cm from the tumor were assessed. The frequency of gastrin cells in the duodenal crypts and Brunner's glands was scored using a scale from 0 to 6 (0, absent; 1, <10%; 2, 10%–<20%; 3, 20%–<40%; 4, 40%–<60%; 5, 60%–<80%; 6, 80%–100%). Gastrin cell density was analyzed in 3 patients with gastrinoma with associated nontumorous gastrin cell lesions versus 3 patients with gastrinoma without any evidence of nontumorous gastrin cell lesions and 3 control patients. For each patient, 3 tissue samples obtained from the first portion of the duodenum at least 0.5 cm from the excised tumor were examined. Cell density was determined by the hot spot method as previously described.21Oberholzer M. Heitz P.U. Kloppel G. Ehrsam R.E. Morphometry in endocrine pathology.Pathol Res Pract. 1984; 179: 220-224Crossref PubMed Scopus (7) Google Scholar, 22Weidner N. Semple J.P. Welch W.R. Folkman J. Tumor angiogenesis and metastasis-correlation in invasive breast carcinoma.N Engl J Med. 1991; 324: 1-8Crossref PubMed Scopus (5459) Google Scholar In crypts and Brunner's glands, gastrin-immunoreactive cells showing a clearly visible nucleus were counted (40× lens) and expressed as number of mucosal cells per millimeter of the muscularis mucosae and number of cells per square millimeter of Brunner's glands, as described in earlier studies.23Creutzfeldt W. Arnold R. Creutzfeldt C. Track N.S. Mucosal gastrin concentration, molecular forms of gastrin, number and ultrastructure of G-cells in patients with duodenal ulcer.Gut. 1976; 17: 745-754Crossref PubMed Scopus (89) Google Scholar, 24Müller J. Kirchner T. Müller-Hermelink H.K. Gastric endocrine cell hyperplasia and carcinoid tumors in atrophic gastritis type A.Am J Surg Pathol. 1987; 11: 909-917Crossref PubMed Scopus (68) Google Scholar The length of the muscularis mucosae and the area of Brunner's glands were determined with an occulometer (Netzmicron; Carl Zeiss, Jena, Germany). The minimum length of duodenal mucosa examined per case was 2 cm, and the minimum area of Brunner's glands was 2 cm2. Data are given as mean and SD. Confocal laser scanning microscopy was used to analyze gastrin/CgA coexpression and the proliferation index of duodenal gastrin cells. The analysis was performed in patients with gastrinoma with nontumorous gastrin cell lesions, in patients with gastrinoma without these lesions, and in control patients. Data are given as approximate percentage of gastrin/CgA-copositive and gastrin/Ki-67–copositive cells. The sections were covered with a mixture of the respective primary antibodies in appropriate dilutions and subsequently labeled with the species-specific secondary antibodies bearing the specific fluorochromes. Before antibody application, the tissue sections were incubated in 1% sodium borohydride in phosphate-buffered saline for 30 minutes to reduce autofluorescence and subsequently stained with 1% Sudan black B (Sigma, Taufkirchen, Germany) for 10 minutes. Nonspecific binding sites were blocked in blocking buffer containing 0.1% bovine serum albumin and 0.2% glycine in Tris-buffered saline (TBS) for 1 hour at room temperature. The slides were incubated with the antibodies overnight in TBS. After extensive washing in TBS, the slides were incubated with the secondary antibodies for 1 hour at 37°C (goat anti-rabbit Alexa Fluor 488 [1:1000] and goat anti-mouse Alexa Fluor 546 [1:1000]; Molecular Probes, Eugene, OR). The sections were counterstained for nuclei using Hoechst A33528 dye at a concentration of 0.025 mg/mL. After extensive washing in TBS, the sections were mounted using ProLong anti-fade mounting medium (Molecular Probes). Laser scanning analysis was performed using a Zeiss LSM510 confocal laser-scanning microscope (Carl Zeiss). All recordings were done using multitracking with 400× original magnification and pinhole diameter set at 1.0 Airy unit. All tissue specimens that were formalin fixed were subjected to mutation analysis. Three Bouin-fixed specimens, however, could not be used for this purpose. From each case, 10 sections (10 μm thick) were cut and every fifth section was stained with H&E. The other sections were used for microdissection. Precisely identified normal connective tissue without any content of epithelial cells was scraped off using a 24-gauge needle or a razor blade under a stereomicroscope (Stemi DV4; Zeiss). The paraffin material was placed in a 1.5-mL Eppendorf tube containing 500 μL of DNA extraction buffer (100 mmol/L Tris, pH 8.0, 5 mmol/L EDTA, 0.5% Nonidet P-40, 0.5% Tween 20) and heated at 95°C until the paraffin was dissolved. After centrifugation for 10 minutes at 4°C, the paraffin was removed and the remnant was digested overnight with 20 mg/mL Proteinase K (Sigma Chemical Co, St Louis, MO) at 55°C. The DNA was purified by phenol-chloroform extraction and quantified by spectrophotometry following standard procedures. In 2 patients, DNA was additionally extracted from the peripheral blood using a commercially available kit (Puregene, DNA Purification System; Gentra Systems, Minneapolis, MN) as recommended by the manufacturer. DNA was resuspended in Tris-EDTA buffer (10 mmol Tris, pH 8.0, 1 mmol EDTA) at a concentration of 0.2–1.5 μg/μL and stored at 4°C. Primers with GC clamps for amplification of exons 2–10 of the MEN1 gene were designed for denaturing gradient gel electrophoresis (DGGE) analysis. The primers are summarized in Table 2. Polymerase chain reaction (PCR) mixtures were prepared in 0.5-mL thin-walled reaction tubes (GeneAmp; Perkin Elmer, Norwalk, CT). They contained 100 ng of template DNA; 0.2 mmol/L of deoxyadenosine triphosphate, deoxythymidine triphosphate, deoxyguanosine triphosphate, and deoxycytidine triphosphate; 50 pmol of each sense and antisense primer; 1.5 mmol MgCl2; 10 mmol/L Tris-HCl (pH 8.3); 50 mmol/L KCl; and 1 U of Ampli Taq Gold DNA polymerase (Perkin Elmer) in a final volume of 50 μL. PCR was performed in a programmable thermal cycler (DNA thermal cycler 9600; Perkin Elmer) using the following conditions: initial denaturation for 7 minutes at 94°C, 35–45 cycles of each denaturation for 45 seconds at 94°C, annealing for 60 seconds at 45°C–58°C, and extension for 60 seconds at 72°C. After a final extension for 300 seconds at 72°C, heteroduplex formation was induced for DGGE analysis by initial denaturation for 10 minutes at 98°C followed by incubation at 55°C for 30 minutes and 37°C for 30 minutes.Table 2Primers, PCR Conditions, and Methods Used to Detect MutationsPrimer (code)Position sequenceLength (base pairs)ConditionMutation analysisM2 neuF/neuR5′-CCT TAG CGG ACC CTG GGA-3′3651.5 mmol/L MgCl25% SSCP, 16 h 8 W5′-ATA GAG GGC GGC GAT GAT AGA-3′M2 NNF/NR5′-TCA ACC GCG TCA TCC CTA CC-3′4051.5 mmol/L MgCl25% SSCP, 16 h 8 W5′-CAC CTG CCG AAC CTC ACA AG-3′M2.2 F/R5′-CCC AGA AGA CGC TGT TCC-3′2051.5 mmol/L MgCl25% SSCP, 16 h 8 W5′-CCA CGG GAA AGT AGG TGA GG-3′M2.3 F/R5′-CTT CGT GGA GCA TTT TCT GG-3′1881.5 mmol/L MgCl25% SSCP, 16 h 8 W5′-ACC CCC TTC TCG AGG ATA GA-3′M2c neu F/R 5′-aGC11: cg.CCC GCT TCA CCG CCC AGA T-3′5′-bGC3: cgc ccg ccg cgc ccc gcg ccc ggc ccg ccg ccc ccg ccc g.TGG AGG GTT TTG AAG AAG TGG GTC A-3′2721.5 mmol/L MgCl2, 10% DMSODGGE 20%–80% U/F, 15 h 100 VM3 F/Rgc 5′-cGC12: gcg cg.GCA CAG AGG ACC CTC TTT CAT-3′5′-aGC11: cg.CTA CAG TAT GAA GGG GAC AAG G-3′3351.5 mmol/L MgCl2, 10% DMSODGGE 20%–80% U/F, 15 h 100 VM4 F/Rgc 5′-aGC11: cg.TGG GCC ATC ATG AGA CAT AA-3′5′cGC12: gcg cg.-CCC ACA GCA AGT CAA GTC TG-3′2721.5 mmol/L MgCl2, 10% DMSODGGE 20%–80% U/F, 15 h 100 VM5 F/Rgc 5′-cGC12: gcg cg.CCT GTT CCG TGG CTC ATA AC-3′5′bGC3: cgc ccg ccg cgc ccc gcg ccc ggc ccg ccg ccc ccg ccc g.-CCT GGC CAC TTC CCT CTA-3′1481.5 mmol/L MgCl2, 10% DMSODGGE 20%–80% U/F 15 h 100 VM6 F/Rgc 5′cGC12: gcg cg.-GGT GGC AGC CTG AAT TAT GA-3′5′dGC8: cgc ccg ccg cgc ccc gcg ccc gcg ccc ggc ccg ccg ccc ccg ccc gaa ata ata aa.-TTAGGGTCTCCCTTCTGCAC-3′2131.5 mmol/L MgCl2, 10% DMSODGGE 20%–80% U/F, 15 h 100 VM7 F/Rgc 5′cGC12: gcg cg.-GGT GGG AGT GGA GAT GGA GAG G-3′5′cGC12: gcg cg.-GGA CGA GGG TGG TTG GAA ACT G-3′3981.5 mmol/L MgCl2, 10% DMSODGGE 20%–80% U/F, 15 h 100 VM7a F/Rgc 5′-dGC8: cgc ccg ccg cgc ccc gcg ccc gcg ccc ggc ccg ccg ccc ccg ccc gaa ata ata aa.ACT CCC TGG GAT CTT CCT GT-3′5′-aGC11: cg.GTG GTA GCC AAG CCA GGT ACA-3′2391.5 mmol/L MgCl2DGGE 50%–90% U/F, 15 h 60 VM7b F/Rgc 5′dGC8: cgc ccg ccg cgc ccc gcg ccc gcg ccc ggc ccg ccg ccc ccg ccc gaa ata ata aa.-TGC CTC AGC CCA AGA CCT ACT-3′5′-eGC13: cgt ccc gc.GAT GGA GGG GAA GAA AGG AC-3′2381.5 mmol/L MgCl2, 10% DMSODGGE 50%–90% U/F, 15 h, 60 VM8 NF/R 5′-AGA CCC CTT CAG ACC CTA CAG AG-3′5′-CCA TCC CTA ATC CCG TAC ATG C-3′2731.5 mmol/L MgCl25% SSCP, 16 h 8 WM8a F/Rgc 5′-dGC8: cgc ccg ccg cgc ccc gcg ccc gcg ccc ggc ccg ccg ccc ccg ccc gaa ata ata aa.CCT TCA GAC CCT ACA GAG ACC-3′5′-aGC11: cg.CTG CCT CCT TCA GCA GGT T-3′1771.5 mmol/L MgCl2, 10% DMSODGGE 20%–80% U/F, 15 h 100 VM8b F/R 5′-ACA ACT ACT GCC GGG AAG AC-3′5′-GTG GGA GGC TGG ACA CAG-3′1731.5 mmol/L MgCl2, 10% DMSO5% SSCP, 16 h 8 WM9 F/Rgc 5′-cGC12: gcg cg.CTG CTA AGG GGT GAG TAA GAG AC-3′5′-bGC3: cgc ccg ccg cgc ccc gcg ccc ggc ccg ccg ccc ccg ccc g.AAA AGT CTG ACA AGC CCG TG-3′3341.5 mmol/L MgCl2, 10% DMSODGGE 20%–80% U/F, 15 h 100 VM9a F/Rgc 5′-bGC3: cgc ccg ccg cgc ccc gcg ccc ggc ccg ccg ccc ccg ccc g.CCC TCT GCA AGG GGT GAG T-3′5′-cGC12: gcg cg.GAC TGC CCT CCT CCC ATT-3′2001.5 mmol/L MgCl2, 10% DMSODGGE 50%–90% U/F, 15 h 60 VM10a altF/Rgc 5′-bGC3: cgc ccg ccg cgc ccc gcg ccc ggc ccg ccg ccc ccg ccc g.CCT TGC TCT CAC CTT GCT CT-3′5′-cGC12: gcg cg.TCT GGA AAG TGA GCA CTG GA-3′3921.5 mmol/L MgCl2, 10% DMSODGGE 50%–90% U/F, 15 h 60 VM10b altF/Rgc 5′-bGC3: cgc ccg ccg cgc ccc gcg ccc ggc ccg ccg ccc ccg ccc g.GAG GGT CCA GTG CTC ACT TT-3′5′dGC8: cgc ccg ccg cgc ccc gcg ccc gcg ccc ggc ccg ccg ccc ccg ccc gaa ata ata aa.GGG TTC TGA GCT GGA GAA AA-3′4231.5 mmol/L MgCl2, 10% DMSODGGE 20%–80% U/F, 15 h 100 VM10 NF/NNR 5′-TTG CTC TCA CCT TGC TCT CC-3′5′-CTT GAT GGC GCT CGA GTT-3′4091.5 mmol/L MgCl2, 10% DMSO5% SSCP, 16 h 8 WM10 NNF/NR 5′-AAG ATG AAG GGC ATG AAG GA-3′5′-GCT GGA GAA AAT CGT GGG TT-3′3321.5 mmol/L MgCl2, 10% DMSO5% SSCP, 16 h 8 WSSCP, single-strand conformation polymorphism; DMSO, dimethyl sulfoxide; U/F, urea and formamide.a GC11: cg.b GC3: cgc ccg ccg cgc ccc gcg ccc ggc ccg ccg ccc ccg ccc g.c GC12: gcg cg.d GC8: cgc ccg ccg cgc ccc gcg ccc gcg ccc ggc ccg ccg ccc ccg ccc gaa ata ata aa.e GC13: cgt ccc gc. Open table in a new tab SSCP, single-strand conformation polymorphism; DMSO, dimethyl sulfoxide; U/F, urea and formamide. DGGE and single-strand conformation polymorphism analysis were performed as described by Komminoth et al.25Komminoth P. Kunz E. Hiort O. Schroder S. Matias-Guiu X. Christiansen G. Roth J. Heitz P.U. Detection of RET proto-oncogene point mutations in paraffin-embedded pheochromocytoma specimens by nonradioactive single-strand conformation polymorphism analysis and direct sequencing.Am J Pathol. 1994; 145: 922-929PubMed Google Scholar The PCR products were then visualized by silver staining.25Komminoth P. Kunz E. Hiort O. Schroder S. Matias-Guiu X. Christiansen G. Roth J. Heitz P.U. Detection of RET proto-oncogene point mutations in paraffin-embedded pheochromocytoma specimens by nonradioactive single-strand conformation polymorphism analysis and direct sequencing.Am J Pathol. 1994; 145: 922-929PubMed Google Scholar, 26Komminoth P. Kunz E.K. Matias-Guiu X. Hiort O. Christiansen G. Colomer A. Roth J. Heitz P.U. Analysis of RET protooncogene point mutations distinguishes heritable from nonheritable medullary thyroid carcinomas.Cancer. 1995; 76: 479-489Crossref PubMed Scopus (139) Google Scholar Samples with an aberrant banding pattern were purified using a QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and then cycle sequenced on an automated sequencer (ABI Prism; Applied Biosystems, Foster City, CA). Sequencing results were compared by means of BLAST (http://www.ncbi.nlm.nih.gov/Blast/) to the men1 gene GI 18860855 and protein NP 570715. The procurement of human tissue for this study was approved by the ethics committees of the University of Kiel and the University of Zurich. All patients gave written informed consent. Table 3 summarizes the most important clinicopathologic data on the 25 patients included in this study.Table 3Clinicopathologic Features of Duodenal Gastrinomas and Their Association With Proliferative Gastrin Cell Lesions and Clinical MEN1 StatusPatient no.Age (y)/sexGastrin IRCgA IRhCG IRKi 57 IR (%)TopographyMetastasisSurgeryMacrotumor (≥2 mm)Microtumor (<2 mm)Other endocrinopathiesClinical MEN1 statusMultifocal gastrinomas associated with proliferative gastrin cell lesions150/M3+2++1BuNoExcision10/6aIndicates submucosal localization of tumors within Brunner's glands and without any signs of mucosal infiltration.1aIndicates submucosal localization of tumors within Brunner's glands and without any signs of mucosal infiltration./0.8aIndicates submucosal localization of tumors within Brunner's glands and without any signs of mucosal infiltration./0.5aIndicates submucosal localization of tumors within Brunner's glands and without any signs of mucosal infiltration.HPTYes260/M4+3++2PdLN, LiverWhipple6/4/4aIndicates submucosal localization of tumors within Brunner's glands and without any signs of mucosal infiltration.1.8aIndicates submucosal localization of tumors within Brunner's glands and without any signs of mucosal infiltration./1.3aIndicates submucosal localization of tumors within Brunner's glands and without any signs of mucosal infiltration./0.5aIndicates submucosal localization of tumors within Brunner's glands and without any signs of mucosal infiltration.Pancreatic microadenomatosisSuspicious367/F5+3+−3PdLV, LiverExcision9aIndicates submucosal localization of tumors within Brunner's glands and without any signs of mucosal infiltration./3aIndicates submucosal localization of tumors within Brunner's glands and without any signs of mucosal infiltration.0.5aIndicates submucosal localization of tumors within Brunner's glands and without any signs of mucosal infiltration.Two doudenal somatostatinomas, multiple oxyntic ECLomas, parathyroid adenomaYes444/F5+4+−3Bu, PdLNWhipple9/50.8Neuroendocrine tumors lung (functionally not active), pancreatic glucagonomaYes553/M5+2++2BuLNWhipple11/3aIndicates submucosal localization of tumors within Brunner's glands and without any signs of mucosal infiltration./2aIndicates
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