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ICANCERRESEARCH 57. 2238—2243. June 1. 19971 Allelic Deletions on Chromosome 11q13 in Multiple Endocrine Neoplasia Type 1associated and Sporadic Gastrinomas and Pancreatic Endocrine Tumors Larisa V. Debelenko, Zhengping Zhuang, Michael R. Emmert-Buck, Settara C. Chandrasekharappa, Pachiappan Manickam, Siradanahalli C. Guru, Stephen J. Marx, Monica C. Skarulis, Allen M. Spiegel, Francis S. Collins, Robert T. Jensen, Lance A. Liotta, and Irma A. Lubensky' Laboratory of Pathology, National Cancer institute IL V. D., 1 1, M. R. E-B., L A. L, i. A. LI, National Centerfor Human Genome Research [S. C. C., P. M., S. C. G., F. S. C.], and Branches of Metabolic Diseases (S. J. M., A. M. S.], Diabetes [M. C. S.), and Digestive Diseases [R. T. ii, National Institute of Diabetes and Digestive and Kidney Diseases, N/H. Bethesda, Maryland 20892 ABSTRACT Endocrine tumors (ETs) of pancreas and duodenum occur sporadically and as a part of multiple endocrine neoplasia type 1 (MEN1). The MENJ tumor suppressor gene has been localized to chromosome 11q13 by link age analysis but has not yet isolated. Previous alleic deletion studies in enteropancreatic ETs suggested MENJ gene involvement in tumorigenesis of familial pancreatic ETs (nongastrinomas) and sporadic gastrinomas. However, only a few MEN1-associated duodenal gastrinomas and spo radic pancreatic nongastrinomas have been investigated. We used tissue microdissection to analyze 95 archival pancreatic and duodenal ETs and metastases from 50 patients for loss of heterozygosity (LOH) on 11q13 with 10 polymorphic markers spanning the area of the putative MENI gene. Chromosome 11q13 LOH was detected In 23 of 27 (85%) MEN!associated pancreatic ETs (nongastrinomas), 14 of 34 (41%) MEN!-asso ciated gastrinomas, 3 of 16 (19%) sporadic insulinomas, and 8 of 18(44%) sporadic gastrinomas. Analysis of LOH on !!q!3 showed different dele don patterns in ETs from different MEN! patients and in multiple tumors from Individual MEN! patients. The present results suggest that the MENJ gene plays a role in all four tumor types The lower rate of 1!q!3 LOH in MEN!-associated and sporadic gastrinomas and sporadic instill nomas as compared to MEN! nongastrinomas may reflect alternative genetic pathways for the development of these tumors or mechanisms of the MENJ gene inactivation that do not involve large deletions. The isolation of the MENJ gene is necessary to further define its role in pathogenesis of pancreatic and duodenal ETs. INTRODUCTION MEN] is a tumor suppressor gene (9—11).MEN1 patients are hypoth esized to inherit a mutation in one copy of the gene, and susceptible cells in the target organs are transformed through the inactivation of the wild-type copy of the gene, potentially occurring via point muta tions, deletions, or gene methylation (6, 7, 10, 11). Sporadic parathy roid and enteropancreatic ETs have also been described to exhibit somatic LOH of chromosome 11 loci, including the MEN] region, suggesting the role of the MEN] gene in the pathogenesis of such tumors(12—19). Allelic deletions on chromosome 11q13 have been reported in 63—100%of MEN1-associated parathyroid tumors and in 25—35%of sporadic parathyroid tumors (1 1—13,20). However, previous studies on 11q13 LOH in enteropancreatic ETs have been limited to a small number of cases in each series (6, 13—19,21—24).Four MEN Iassociated gastrinomas have been reported in the literature to date, and three tumors demonstrated retention of heterozygosity on 11q13 (17, 19, 24), whereas one gastrmnoma showed a small deletion at marker PYGM (13). Thus, the role of the MEN] gene in enteropancreatic endocrine tumorigenesis remains controversial. We used tissue microdissection to analyze 95 archival duodenal and pancreatic ETs and metastases for LOH on 11q13. The goal was to investigate the frequency of allelic loss at the MEN] gene locus in tumongenesis of MEN1-associated and sporadic enteropancreatic ETs. In addition, X-chromosome inactivation analysis of six synchro nous primary duodenal microgastrinomas in one FMEN1 female patient was performed to investigate clonality of MEN 1-associated gastrinomas, and the results were correlated with the LOH data. ETs2 of pancreas and duodenum may occur sporadically (I) or in association with inherited syndromes such as MEN1 (2). Sporadic duodenal and pancreatic ETs are usually solitary, whereas MEN 1- associated neoplasms are characteristically multiple in the involved organ (3, 4). Insulinomas and nonfunctional ETs (nongastrmnomas) occur exclusively in the pancreas, whereas the most common site for both sporadic and familial gastrinomas is the duodenum (1—5).Insu linomas usually follow a benign clinical course, whereas gastrinomas have high malignant potential, with regional lymph node or liver metastases developing in up to 90% of the cases. The putative MENJ tumor suppressor gene has been linked to chromosome 1 1q13 (6, 7). FMENI is an autosomal dominant syn drome in which the affected individuals develop multiple tumors in the parathyroid glands (90—97%), pancreas (30—82%), duodenum (25—60%),and anterior pituitary (35—60%;Refs. 2, 3, and 8). Loss of the wild-type allele at the MEN] locus in tumors arising in affected individuals is commonly observed, supporting the conclusion that PATIENTSAND METHODS Patient Population. Fifty patientswho underwentexploratorylaparotomy for pancreatic and duodenal ETs at the NIH were included in the study. Ninety-five formalin-fixed, paraffin-embedded primary pancreatic and duode nal ETs and metastases were obtained from the file of the Laboratory of Pathology, National Cancer Institute, NIH. Clinical and family histories were reviewed in each case. Sixteen patients (9 males and 7 females; mean age, 45; age range, 23—74 years) were diagnosed with MENI, and 34 patients (18 males and 16 females; mean age, 42; age range, 15—67years) had sporadic ET. Fourteen of 16 MEN! patients were categorized as having FMEN1 because in addition to two typical endocrine neoplasms, they had at least two first-degree relatives with MEN1-related endocrinopathies. The diagnosis of gastrinoma (Zollinger-Ellison nongastrinomas, had sporadic Received 1I/l 1/96; accepted 4/4/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I To whom requests for reprints should be addressed, at Laboratory of Pathology, National Cancer Institute, NIH, Building 10, Room 2N212. 9000 Rockville Pike, Bethesda, MD 20892. Phone: (301) 496-0549; Fax: (301)480-9488. 2 The abbreviations used are: ET, endocrine tumor; MENI, multiple endocrine nan plasia type I ; FMENI. familial MENI ; LOH, loss of heterozygosity. syndrome), insulinoma (hyperinsulinemic hypoglycemia), or nonfunctional tumor was made on clinical grounds and confirmed by pathological examination of the tumor. Seven MEN1 patients had pancreatic I 1 MEN1 insulinomas patients had gastrinomas, and gastrinomas, respectively and 16 and 18 patients (Tables 1—4).Among the 16 MEN! patients, 10 had gastrinomas only, 4 had pancreatic nongastri nomas only, and 2 (patients 3 and 4) had both gastrinomas and nongastrinomas available for the study. Nine MEN1 patients had multiple tumors evaluated (Tables 1 and 2). Microdissection. Tumor and normal cells were selected from routine 5-@tm-thickH&E-stained histological slides and microdissected under direct light microscope visualization as described previously (Fig. 1; Refs. 20 and 2238 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1997 American Association for Cancer Research. GENETIC ALTERATIONS IN ENDOCRINE TUMORS MEN!pancreaticendocrinetumors(nongastrinomas)3a4 Table 1LOHon!1q13in27 NFNFNFNFNFNFNFNFNFNFNFNFNFNFNFNF123456123478591011121314151617181920216 @4}@1516 In12 InInIn13 NFNFInc14 InNFb@4f@ . I I D11S1256― DllS956 DllS48O I D11S599 I I • 0 I 0 I I I 0 I 0 . I I 0 I I I I I I I 0 I PYGM I I I 0 I I I I I I I I I I I I I I I I — I I I I I I I I I • I I — D11S4908 0 PPP1CA I I I 0 I 0 D11S534 INT-2 I I • 0 I I • I — a Patientno. b Tumor type and tumor no. NF, nonfunctional; In, insulinoma; I, LOH; 0, retention of heterozygosity; —,not informative; blank, not done. C Liver metastasis. d Chromosome I 1q13 markers are listed in order from centromeric (top) to telomeric (bottom). 25). Normal duodenal epithelium, exocrine pancreas, or lymph node tissue was used as a control. DNA Extraction. Procuredcells were resuspendedin 30 @l of solution containing Tris-HCI (pH 8.0), 0.1 MEDTA (pH 8.0), 1% Tween 20, and 0.1 mg/mI proteinase K and incubated overnight at 37°C.Following thermal inactivation ofproteinase K (95°C for 5 mm), 1—1.5 pi ofthe DNA extract was used for PCR analysis. PCR Markers. Ten polymorphicDNA markerswere used in this study: D11S1256 (26), D11S956, D11S480 (27), D11S599 (28), D11S457 (D11S457,PYGM, and PPPJCA). Labeled amplified DNA was mixed with an equal volume of fonnamide loadingdye (95% formamide,20 mxiEDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol). The samples were denatured for 5 mm at 95°Cand resolved on a 6% polyacrylamide gel. Autoradiography was performed with Kodak X-Omat film (Eastman Kodak, Rochester, NY). The case was considered to be informative for a polymorphic marker on Ilql3 if normal tissue DNA showed two alleles (heterozygosity). Complete or near complete (90% decreased intensity) absence of an allele in tumor samples was interpreted as LOH (Fig. 2). Each experiment was repeated two or three (29), PYGM (CA)(GA) (27), D1]S4908 (20), PPP]CA (30), D]1S534 (31), and times, INT2 Combined tumor and family study in a FMEN1 patient 8 with multiple gastrmnomaswas performed with the informative marker D11S956 using nor (27). Labeling [a-32P]dCTP. of PCR product was achieved by incorporating PCR was conducted in a total volume of 10 .d that contained 1—1.5 ,.tl of DNA extract, 200 @M each dNTP, 0.1—0.5 @M each primer, 0.1 and the data were reproducible. mal and tumor DNA from the patient and her brother (Fig. 3). X-Chromosome Inactivation Analysis. Six synchronous primary duode The reactions were performedin a Perkin-ElmerCorp. thermal cycler as nal microgastrinomas from a female FMEN1 patient 2 (Tables 2 and 5) were follows: denaturation at 94°Cfor 5 rain, followed by 35 cycles of annealing for studied for X-chromosome inactivation to assess clonality (Fig. 4). Extracted 45 5,extension at 72°Cfor 1 rain,anddenaturationat 94°Cfor 45 s. Annealing tumor and normal DNA (obtained from the same microdissection procedure as temperatures for each set of primers were 62°C(D1]S599), 60°C(D11S599,), for LOH) was digested with HpaII (Life Technologies, Inc., Gaithersburg, 58°C(D]]S]256, D1]S599, D]]S480, D]]S4@YJ8,and 11(12), and 56°C MD) and amplified by PCR with primers to human androgen receptor unit Taq DNA polymerase, and standard PCR buffer (Perkin-Elmer Corp.). Table 2LOHon1 gastrinomasla 1q13 in 34 MEN! 4 j;;; G G G G G G G G 0 In lnIn p d 5 d 6 li d 7 d d 8 d 9 d d Ii In In • I • 1011 In d 0 0 d d G G G G G GGGGGGGGGGGGGG000GGG 2d 3d 4d 5d 6d 7d 8ln 9In 10ln11In12d 13d 14DllSl256c 123 1516l7l8l920212223242526272829303l323334d 0 D11S956 D11S48O 0 — 0 0 D11S599 0 0 0 PYGM D11S4908 0 I I 0 0 0 0 0 • 0 • 0 0 • 0 0 S 0 0 • • 0 0 0 0 0 0 —— 0 — • • 0 • 0 0 0 — S • I 0— • 0 0 • 00 00 I0 00 0 0 • 0 0 • 0 • 0 00 00 00 00 00 00 00 S0 0. 0 0 0 • 0 I • • 0 0 PPPICA 0 0000 INT-20 00 —— I • 0 • — 0 • • • 0 0 0 0 0 a Patientno. b Tumor site, tumor type, and tumor no. ln, lymph node; d, duodenum; p. pancreas;li, liver, G, gastrinoma; @, LOH; 0, retention of heterozygosity; —.not informative; blank, not done. C Chromosome 1 1q13 markers are listed in order from centromeric (top) to telomeric (bottom). insulinomas35a363738394041424344454647484950InInInInInInInInInInInInInInInIn78910111213141516171819202122D11S1256―000D11S9560•0000•0—S0000—0D11S480—.——0————00 Table 3 LOH on 1iqi3 in 16 sporadic pancreatic a Patient no., tumor type, and tumor no. In, insulinoma; •,LOH; 0, retention of heterozygosity; —, not informative; blank, not done. b Chromosome 11q13 markers are listed in order from centromeric (top) to telomeric (bottom). 2239 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1997 American Association for Cancer Research. GENETIC ALTERATIONS IN ENDOCRINE TUMORS Table 4 LOH on !!q!3 in 18 sporadic gastrinomas Patient no., tumor site, tumor type, and tumor no. 17 ln 18 In 19 In 20 ln 21 d 22 in 23 d 24 d 25 In 26 li 27 d 28 d 29 in 30 ln 31 d 32 p 33 li 34 li G G G G G G 0 G G G G G G G G G G G 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 0 0 0 — I I 0 — 0 0 0 I 0 — 0 0 — — o — — — — — 0 I — 0 — — I D1IS599 — — I — 0 — — — I — — 0 I — — DIIS457 PYGM PPPICA — 0 0 0 0 0 — — — 0 0 0 0 o • — — I I — — — 0 — — — — 0 0 — 0 — 0 0 — I — I I I — I 0 0 — — — 0 0 D1IS534 — — — — — — — 0 I 0 0 — — — I — — — INT-2 0 0 — 0 0 I 0 0 — 0 0 0 I 0 I 0 0 a I 1q13 DllS956a DIIS48O Chromosome markers are listed in order from centromeric (top) to telomeric (bottom). In, lymph node; d, duodenum; ii, liver; p. pancreas; — G, gastrinoma; 0 I, LOH; 0, retention of heterozygosity; —,not informative; blank, not done. creatic MEN1-associated tumors included 6 insulinomas and 21 din ically nonfunctional tumors (Table 1). Histological evaluation of tumors revealed characteristic neuroendocrine features (Fig. 1) and positive staining for chromogranin A (Boehringer Mannheim, Indian apolis, IN) and/or synaptophysin (Zymed, San Diego, CA) by immu nohistochemistry. The clinical diagnoses of gastrmnoma and insuli A NT NT D11S1256@ G13 G28 NT NT D11S956 D11S480 @ (HUMARA) G36 G37 NT NT intensity tissue ui@ G39 G40 NT NT N T (Fig. 4B). Dl 1S457 NT NT N T N T N T IPIT2 @- 1n7 Tumor Characteristics.Tissue microdissectionyieldedreliable G47 • . DNA procurement from 95 tumors in 50 patients. Four groups of tumors included: 27 pancreatic MEN1-associated ETs (nongastrmno mas; sizes, 0.8—4.0 cm), 34 MEN1-associated gastrinomas (sizes, 0.5—8.0 cm), 16 sporadic insulinomas (sizes, 0.6—4.5 cm), and 18 NF4 NF1O D11S534 G46 RESULTS @ NT • following conditions published previously (32). The gastrinoma in normal NT PPPICA was considered to be monoclonal if PCR amplification from HpaII-digested tumor DNA generated a single band as compared to two bands of equal @ NT D11S4908 D11S599 Fig. I. Multiple duodenal microgastrinomas (G8 and 09) in MEN1 patient 2 (H&E; X20). A, before microdissection; B, after microdissection of tumor G9. NT PYGM G41 @fl; 1n8 Fig. 2. Representativeresultsof llqI3 LOWretentionof heterozygositywith 10 polymorphicmarkersspanningthe area of the MEN! gene in MEN1-associatedand sporadic ETs. G, gastrinoma; In, insulinoma; NF, nonfunctional tumor; N, normal tissue; sporadic gastrinomas (sizes, 0.4—8.0 cm; Tables 1—4).The 27 pan- T,tumor.Arrows,allelicdeletions.Numbers,tumornumbersin Tables1—4. 2240 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1997 American Association for Cancer Research. @ @ @+ @@1Mp4@@ GENETIC ALTERATIONS IN ENDOCRINE TUMORS D11S956 @ I èF@8 different LOll/retention patterns. For example, 4 of 14 gastrinomas in patient 2 showed 11q13 LOH, whereas 8 tumors demonstrated reten tion of heterozygosity with the markers tested (Table 2). Likewise, patients 4 and 8 showed allelic deletions in two of three and three of four tumors, respectively. Fig. 4A illustrates LOH results with marker I 1 N G@G27G@PT B a b@ D11S480 parathyroid tumor(Lane P7) from patient 8 (Pt 8)and normal peripheral blood DNA from the patient'saffectedbrother(LaneB) wasamplifiedby PCRwithpolymorphicmarker D11S956 and analyzed by denaturing gel electrophoresis. a, disease allele shared by patient 8 and her affected brother; b, wild type allele. Arrow, all three of the patient's gastrinomas (Lanes G26_28) and the parathyroid tumor (Lane P7) demonstrate a loss of the wild-type allele b. Table 5 Combined Iiq!3 WH and X-chronwsome inactivation results in six (L)a G, gastrinoma; LOH no yes no no yes no U, only upper allele monoclonal (U) monoclonal (L) monoclonal (L) polyclonal (U,L) polyclonal (U.L) monoclonal detected in tumor as compared to normal tissue; L, only lower allele detected in tumor as compared to normal tissue; U. L, both upper and lower alleles detected in tumor. immunoreactivity MENI-associated 2. X-ChromosomeInactivationin SynchronousMEN!-associated three duodenal gastrinomas (Lanes G4, G6, and G9) showed a single clone (lower allele present), whereas gastrinoma G3 revealed presence of the upper allele, indicative of a single but different clone. Two tumors (Lanes G7 and G8) showed the presence of multiple clones (both alleles present). Two alleles detected by X-chromosome inactivation analysis in tumor G7 (with retention of heterozygosity by LOH study) may represent a confluence of two separate clones, whereas one of the two alleles in tumor G8 (allelic deletion by LOH analysis) may represent a contamination with normal stromal cells (Fig. 1; Refs. 20 and 25). 11q13 LOH in Sporadic ETs. Allelic deletions,spanningthe with glucagon, insulin, or gastrin (19%) nongastrinomas and gastrinomas. insulinomas showed deletions in the area of the MEN] gene. in tumor A cells. Twenty-one insulinomas and 21 nonfunctional ETs were located in the pancreas, and 1 ET (InS) represented insulinoma metastasis to the liver (Tables 1 and 3). Among 52 gastrinomas, 26 were located in the duodenum, 2 were in the pancreas, 19 were in the peripancreatic lymph nodes, and 5 were liver metastases (Tables 2 and 4). The tumor was considered to be malignant if regional lymph node or distant metastases were documented. All 52 gastrinomas and 2 insulinomas (1n3 and InS) were classified as malignant, and 21 nonfunctional tumors and 19 insulinomas were benign. The study included 19 gastrinomas (G2—17and G30—32)and 6 nongastrinomas (1n2—5and NF7—8)from 5 MEN1 patients (patients 2, 3, 9, 12, and 13) analyzed in part in a previous report (20). 11q13 LOll In MEN1-associated ETs. Allelic deletions, span ning the MEN] gene region on chromosome llql3, were detected in both in patient MEN] gene region on chromosome 11q13, were detected in both sporadic insulinomas and gastrinomas. The LOH results in 16 spo radic insu!inomas are summarized in Table 3 and Fig. 2. Three of 16 noma were confirmed by positive immunostain for gastrmn (DAKO Corp., Carpinteria, CA) and insulin (BioGenex, San Ramon, CA), respectively. Most nonfunctional ETs stained positively with pancre atic polypeptide immunostain (DAKO Corp.) and showed variable scattered microgastrmnomas Gastrinomas. DNA from the six synchronous microgastrinomas in a female FMEN1 patient 2 procured during the same microdissection procedure was analyzed for clonality using X-chromosome inactiva tion assay (32). Separate tumors dissected from the patient's duode num revealed different clonality patterns (Fig. 4B and Table 5). For example, synchronous duodenal gastrinomas in female patient 2 with MEN! G4 06 G7 G8 09 duodenal four gastrmnomas (Lanes G3, G6, G7, and G9) showed retention of heterozygosity. All synchronous tumors from each patient with LOH showed loss of the same allele (Fig. 4A). Fig. 3. Combined pedigree and tumor deletion data in FMENI patient 8. Extracted DNA from normal lymph node (Lane N), three gastrinomas (Lanes G26_28), and one Tumorno. ClonalityG3° in six synchronous @4— Two ofthe six tumors (Lanes G4* and G8*) showed allelic loss, and The N N G3G4G5G6G7G8G9 B N N G3 G4 G5 G6 G7 G8 G9 LOH results in 27 MEN1-associated pancreatic nongastrinomas from seven patients are summarized in Table 1, and representative examples are shown in Fig. 2. For each tumor studied, at least two polymorphic markers were informative. Twenty-three of 27 (85%) nongastrmnomas in six patients showed deletions in the area of the MEN] gene. LOH was demonstrated in 4 of 6 insulinomas and in 19 of 21 nonfunctional pancreatic ET. Fourteen of 34 (41%) gastrmnomas in 6 MEN! patients studied with 8 polymorphic markers showed deletions in the area of the MEN] gene (Table 2 and Fig. 2). LOH was detected in 9 of 20 duodenal gastrinomas, 1 of 1 pancreatic gastrinoma, 3 of 11 lymph nodes, and 1 of 2 liver metastases, regardless of size of the tumor. Multiple synchronous tumors in two MEN! patients with pancre atic nongastrmnomas (Table 1, patients 3 and 13) and in three MEN1 patients with gastrmnomas (Tables 2 and 5, patients 2, 4, and 8) showed Fig. 4. Combined I1q13 LOH and X-chromosome inactivation analysis in six syn chronous duodenal microgastrinomas in female patient 2 with FMENI . N. normal duo denal epithelium; G, gastrinoma. Nunthers, tumor numbers in Table 2. Arrowheads, alleles. A, LOH results with marker Dl 1S480. Compared to normal tissue N, the lower allele is lost in tumors G4° and G8° and retained in tumors G3, G6—7,and G9. Synchronous tumors show loss of the same allele. B. X-chromosome inactivation results. Tumor and normal DNA digested with HpaII was amplified by PCR with primers to human androgen receptor (HUMARA). Three duodenal gastrinomas (G4. Gd. and G9) show a single clone (lower), whereas G3 reveals presence of the upper allele, indicative of a single but different clone. Two tumors (G7 and G8) show the presence of several clones (both alleles present). 2241 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1997 American Association for Cancer Research. GENETIC ALTERATIONS IN ENDOCRINE TUMORS gastrinomas)AuthorTable 6 Literature data on !!q!3 LOll in MEN! and sporadic enteropancreatic ETs (nongastrinomas and NongastrinomaGastrinomaLarsson (Ref.)MENISporadicYear 2/2'@Bystrom ci al. (6)1988 1/1Radfordet al.(21)1990 2/2Teh et aL (22)1990 1/1Patel ci aL (14)1990 1/3Bale et aL ( I5)1990 0/20/6Sawicki et al. ( I7)199 1Ding et al. (18)19924/1 1/2Weber el al. (16)1992 Nongastrinoma Gastrinoma 1/1 1 3/4 0/1 2/2Eubanks et al. (23)1994 2/58/22Beckers el al. ( 19)1994 0/1Iwasakiel al. (24)1994 0/1 1/1Total1(13)1995 312/39Debelenko 1/12 1/4 et al. 23/27 14/34 3/16 (85%) (41%) (19%) the number1997 All tumors in this group were located in the pancreas and were clinically benign, and no correlation between the size of the tumor and 11q13 LOH was noted. LOH results in 18 sporadic gastrinomas are summarized in Table 4 and Fig. 2. Eight of 18 (44%) ETs in sporadic gastrinoma group showed deletions in the area of the MEN] gene. All tumors were malignant. LOH was detected in three of six duodenal and five of eight lymph node gastrinomas. Retention of heterozygosity on mul tiple markers was seen in one pancreatic gastrinoma and three gastri noma liver metastases. No relationship between the size or site of gastrmnoma and the presence of deletions was observed. Combined Tumor and Family Study in a FMEN1 Patient with Gastrinomas. To assess whether duodenal gastrinomas share the same developmental mechanism with parathyroid tumors in MEN1 patients, DNA from normal lymph node tissue (Lane N), three duo denal gastrinomas (Lanes G2628), and one parathyroid tumor (Lane PT) in patient 8 and normal peripheral blood DNA (Lane B) from the affected brother was amplified with marker D]]S956 (Fig. 3). Patient 8 was heterozygous for D]]S956, and her normal DNA (Lane N) contained two alleles, a and b. Her brother was homozygous for allele a at D]]S956 (Lane B). Patient 8 and her affected brother sharedallele a, and allele a was retained in all three of the patient's gastrinomas (Lanes G26_28) and parathyroid tumor (Lane PT), whereas allele b (derived from the unaffected parent) was lost in all four tumors. The results are consistent with a tumor suppressor gene function of the gene. DISCUSSION The MEN] gene has recently been localized between markers D]]S]883 (44%) of tumors studied showing I 1q13 LOH; denominator, the total number of tumors studied in each series.8/18 a Numerator, MEN] 5/1 and D]]S449 on chromosome 1 1q13 by recombination studies (33, 34). Tumor allelic deletion mapping data placed the gene between markers PYGM and D]]S97 (13, 20, 21 , 23). The present study represents the largest series of MEN1-associated and sporadic enteropancreatic ETs analyzed with 10 markers in the area of the putative MEN] gene to date (Table 6). On the basis of the minimal region of overlapping deletions on I 1q13 in MEN1-associated tumors, the MEN] gene boundaries are placed between markers D]]S480 (patient 5, 021) and INT2 (patient 2, G8 and G1 1, and patient 7, G23—25; Table 2). The data in the present study are consistent with previously reported boundaries in MEN1 tumors. The LOH analysis of sporadic gastrinomas suggests that the MEN] gene may be distal to PYGM (patient 21, G39, Table 4). The present results suggest the MEN] gene is involved in tumori genesis of MEN 1-associated pancreatic nongastrinomas and gastrino mas, sporadic gastrinomas, and some sporadic insulinomas. All four groups of tumors showed large deletions in chromosome 11q13 re gion. Interestingly, varying LOH rates were observed among the four groups of ETs. The lower incidence of 11q13 LOH in MEN 1-asso ciated gastrinomas and metastases (41%), sporadic gastrmnomas (44%), and sporadic insulinomas (19%) compared to a high LOH rate in MEN1-associated pancreatic nongastrmnomas (85%) may indicate that gastrinomas and some sporadic insulinomas could arise due to inactivation of the wild-type allele via point mutations or small deletions rather than via a loss of large segment of chromosome 11q13. Identification of the MEN] gene will allow testing of this possibility. Alternatively, because a 19% (3 of 16) LOH rate detected in benign sporadic insulinomas in the present study is low and similar to a 17% (1 of 6) rate reported in this tumor type to date (15, 17, 19), it is conceivable that the MEN] gene is inactivated in only a minority of sporadic insulinomas. Because gastrinomas are the only common MEN1-associated and sporadic enteropancreatic ETs with malignant potential, in addition to initial inactivation of the MEN] gene, further genetic alterations are probably necessary for their development and progression. Therefore, gastrinomas may offer a unique opportunity to study a chain of genetic events necessary for development of malig nant neoplasm in a setting of familial syndrome. The role of other tumor suppressor genes in enteropancreatic en docrine tumorigenesis presently remains uncertain. Analysis of spo radic pancreatic ETs for VHL gene (3p25.5) mutations by Chung et a!. (35) revealed no evidence of the role of the gene in their development. Furthermore, the screening of 27 MEN!-associated and sporadic pancreatic ETs (insulinomas and gastrinomas) with polymorphic markers in the areas of known tumor suppressor genes on chromo somes ip, 8p, and 3p in our laboratory did not demonstrate significant rate of LOH (data not shown). Further studies are necessary to provide a better understanding of enteropancreatic endocrine tumorigenesis. In this study, multiple synchronous nongastrinomas and gastrmno mas were analyzed in nine MEN! patients. In five of the patients, varying deletion patterns were documented in enteropancreatic ETs arising simultaneously regardless of tumor size, type, and anatomical location. Correlation of the LOH and X-chromosome inactivation data in six duodenal microgastrinomas from a FMEN1 female patient 2 demonstrates that each individual gastrinoma arises as an independent event (Fig. 4 and Table 5). Moreover, each clonal tumor may show either loss or retention of heterozygosity with a given marker tested (Fig. 4A). The data supports the hypothesis that the mechanism of the MEN] gene inactivation in gastrinomas may involve small deletions or mutations. These results stress the importance of analysis of mul 2242 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1997 American Association for Cancer Research. GENETIC ALTERATIONS IN ENDOCRINE TUMORS tiple tumors from each target organ in the study of pathogenesis of hereditarycancer syndromesand in allelic deletion mappingof the suppressor genes such as MEN] (20, 36). In view of the lower rate of large deletions, MEN1-associated gastrinomas may represent a useful resource for deletion mapping of the MEN] gene if the wild-type allele is inactivated by small deletions. Furthermore, because the rate and pattern of al!eic deletions on 1!q!3 is similar in sporadic gastri nomas and MEN1-associated gastrinomas, the LOH analysis in the former group may prove helpful to further define a minimal overlap ping deletion interval in mapping of the MEN] gene. 18. Sawicki, M. P., Wan, Y-J. Y., Jonson, C. L., Berenson, J., Gatti, R., and Passaro, E. Loss of heterozygosity on chromosome I I in sporadic gastrinomas. Hum. Genet., 89: 445—449, 1992. 19. Eubanks, P. J., Sawicki, M. P., Samara, G. J., Gatti, R., Nakamura, Y., Tsao, D., Jonson. C., Hurvitz, M., Wan, Y-J. Y., and Passaro, E. Putative tumor-suppressor Gene on chromosome I I is important in sporadic endocrine tumor formation. Am. J. Surg., 167: 180—185,1994. 20. Lubensky, I. A., Debelenko, L. V., Zhuang, Z., Emmett-Buck, M. R., Dong, Q., Chandrasekharappa, S., Guru, S., Manickam, P., Olufemi, E-S., Marx, S. J., Spiegel. A., Collins, F. S., and Lions. L. A. Allelic deletions on chromosome I 1q13 in multiple tumors from individual MENI patients. Cancer Res., 56: 5272—5278.1996. 21. BystrOm, C., Larsson, C., Blomberg, C., Sandelin, K., Falkmer, U., Skogseid, B., Oberg, K., Wermer, S., and Nordenskjöld,M. Localization of the MEN! gene to a small region within 11q13 by deletion mapping in tumors. Proc. NatI. Acad. Sci. USA, 87: 1968—1972,1990. 22. Redford, D. M., Ashley, S. 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Cancer Res., 5!: parathyroid tumors from MENI patients. Biochem. Biophys. Rca. Commun., 227: 1154—1157. 1991. 736—742. 1996. 2243 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1997 American Association for Cancer Research. Allelic Deletions on Chromosome 11q13 in Multiple Endocrine Neoplasia Type 1-associated and Sporadic Gastrinomas and Pancreatic Endocrine Tumors Larisa V. Debelenko, Zhengping Zhuang, Michael R. Emmert-Buck, et al. Cancer Res 1997;57:2238-2243. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/57/11/2238 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. 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