Genetic Differences in Endocrine Pancreatic Tumor Subtypes Detected by Comparative Genomic Hybridization
1999; Elsevier BV; Volume: 155; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)65495-8
ISSN1525-2191
AutoresErnst‐Jan M. Speel, Jan Richter, H. Moch, Carole Egenter, Parvin Saremaslani, Katrin Rütimann, Jianming Zhao, André Barghorn, J x FC rgen Roth, Philipp U. Heitz, Paul Komminoth,
Tópico(s)Genomic variations and chromosomal abnormalities
ResumoThe molecular pathogenesis as well as histogenesis of endocrine pancreatic tumors (EPTs) is not well understood, and the clinical behavior of EPTs is difficult to predict using current morphological criteria. Thus, more accurate markers of risk and better understanding of tumor initiation and progression are needed to allow a precise classification of EPTs. We have studied 44 benign and malignant EPTs by comparative genomic hybridization to correlate the overall number of genetic alterations with clinical and histopathological parameters and to identify chromosomal regions which might harbor genes involved in EPT pathogenesis and progression. Aberrations were found in 36 EPTs, and chromosomal losses (mean, 5.3) were slightly more frequent than gains (mean, 4.6). The most frequent losses involved Y (45% of male EPTs), 6q (39%), 11q (36%), 3p, 3q, 11p (each 30%), 6p (27%), and 10q and Xq (each 25%), whereas most common gains included 7q (43%), 17q (41%), 5q and 14q (each 32%), 7p, 9q, 17p, 20q (each 27%), and 12q and Xp (each 25%). A correlation was found between the total number of genetic changes per tumor and both tumor size and disease stage. In particular, losses of 3p and 6 and gains of 14q and Xq were found to be associated with metastatic disease. Furthermore, characteristic patterns of genetic changes were found in the various EPT subtypes, eg, 6q loss in malignant insulinomas, indicating that these groups might evolve along genetically different pathways. The highlighted genetic aberrations, including the newly found involvement of 6q losses and sex chromosome alterations, should stimulate the further analysis of these chromosomal regions, which may lead to the discovery of novel genes important in the tumorigenesis and evolution of EPTs. The molecular pathogenesis as well as histogenesis of endocrine pancreatic tumors (EPTs) is not well understood, and the clinical behavior of EPTs is difficult to predict using current morphological criteria. Thus, more accurate markers of risk and better understanding of tumor initiation and progression are needed to allow a precise classification of EPTs. We have studied 44 benign and malignant EPTs by comparative genomic hybridization to correlate the overall number of genetic alterations with clinical and histopathological parameters and to identify chromosomal regions which might harbor genes involved in EPT pathogenesis and progression. Aberrations were found in 36 EPTs, and chromosomal losses (mean, 5.3) were slightly more frequent than gains (mean, 4.6). The most frequent losses involved Y (45% of male EPTs), 6q (39%), 11q (36%), 3p, 3q, 11p (each 30%), 6p (27%), and 10q and Xq (each 25%), whereas most common gains included 7q (43%), 17q (41%), 5q and 14q (each 32%), 7p, 9q, 17p, 20q (each 27%), and 12q and Xp (each 25%). A correlation was found between the total number of genetic changes per tumor and both tumor size and disease stage. In particular, losses of 3p and 6 and gains of 14q and Xq were found to be associated with metastatic disease. Furthermore, characteristic patterns of genetic changes were found in the various EPT subtypes, eg, 6q loss in malignant insulinomas, indicating that these groups might evolve along genetically different pathways. The highlighted genetic aberrations, including the newly found involvement of 6q losses and sex chromosome alterations, should stimulate the further analysis of these chromosomal regions, which may lead to the discovery of novel genes important in the tumorigenesis and evolution of EPTs. Endocrine pancreatic tumors (EPTs. are neoplasms with a prevalence of approximately 1/100,000. Sixty to 85% of EPTs belong to the group of functioning tumors, in that they are producing hormones such as insulin, gastrin, glucagon, or vasoactive intestinal polypeptide (VIP), which may lead to clinically distinct syndromes. The remaining tumors are nonfunctioning, in that they express no hormones that lead to a clinical syndrome.1Klöppel G In 't Veld PA Komminoth P Heitz PU The endocrine pancreas.in: Kovacs K Asa SL Functional Endocrine Pathology. Blackwell Scientific Publications, Oxford1998: 415-487Google Scholar Because the histopathological characteristics of EPTs do not provide useful information with respect to prognosis, more distinctive markers that can predict the clinical course of EPTs are urgently required. However, the molecular mechanisms underlying the tumorigenesis of EPTs are poorly understood. A small percentage of EPTs is associated with inherited syndromes such as the multiple endocrine neoplasia type 1 (MEN1) and von Hippel-Lindau (VHL. syndrome.2Chandrasekharappa SC Guru SC Manickam P Olufemi S-E Collins FS Emmert-Buck MR Debelenko LV Zhuang Z Lubensky IA Liotta LA Crabtree JS Wang Y Roe BA Weisemann J Boguski MS Agarwal SK Kester MB Kim YS Heppner C Dong Q Spiegel AM Burns AL Marx S Positional cloning of the gene for multiple endocrine neoplasia-type 1.Science. 1997; 276: 404-407Crossref PubMed Scopus (1734) Google Scholar, 3Latif F Tory K Gnarra J Yao M Duh FM Orcutt ML Stackhouse T Kuzmin I Modi W Geil L Identification of the von Hippel-Lindau disease tumor suppressor gene.Science. 1993; 260: 1317-1320Crossref PubMed Scopus (2538) Google Scholar The vast majority of EPTs, however, occur sporadically, and only a subset harbor somatic MEN1 mutations.4Zhuang Z Vortmeyer AO Pack S Huang S Pham TA Wang C Park W-S Agarwal SK Debelenko LV Kester M Guru SC Manickam P Olufemi S-E Yu F Heppner C Crabtree JS Skarulis MC Venzon DJ Emmert-Buck MR Spiegel AM Chandrasekharappa SC Collins FS Burns AL Marx SJ Jensen RT Liotta LA Lubensky IA Somatic mutations of the MEN 1 tumor suppressor gene in sporadic gastrinomas and insulinomas.Cancer Res. 1997; 57: 4682-4686PubMed Google Scholar, 5Görtz B Roth J Krähenmann A De Krijger RR Muletta-Feurer S Rütimann K Saremaslani P Speel EJM Heitz PU Komminoth P Mutations and allelic deletions of the MEN 1 gene are associated with a subset of sporadic endocrine pancreatic and neuroendocrine tumors and not restricted to foregut neoplasms.Am J Pathol. 1999; 154: 429-436Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar VHL mutations and alterations of the K-ras, N-ras, and TP53 genes appear not to be relevant in the pathogenesis of EPTs.6Chung DC Smith AP Louis DN Graeme-Cook F Warshaw AL Arnold A A novel pancreatic endocrine tumor suppressor gene locus on chromosome 3p with clinical prognostic implications.J Clin Invest. 1997; 100: 404-410Crossref PubMed Scopus (122) Google Scholar, 7Yashiro T Fulton N Hara H Yasuda K Montag A Yashiro N Straus F Ito K Aiyoshi Y Kaplan EL Comparison of mutations of ras oncogene in human pancreatic exocrine and endocrine tumors.Surgery. 1993; 114: 758-764PubMed Google Scholar, 8Evers BM Rady PL Sandoval K Arany I Tyring SK Sanchez RL Nealon WH Townsend CM Thompson JC Gastrinomas demonstrate amplification of the HER-2/neu proto-oncogene.Ann Surg. 1994; 219: 596-604Crossref PubMed Scopus (59) Google Scholar, 9Beghelli S Pelosi G Zamboni G Falconi M Iacono C Bordi C Scarpa A Pancreatic endocrine tumours: evidence for a tumour suppressor pathogenesis and for a tumour suppressor gene on chromosome 17p.J Pathol. 1998; 186: 41-50Crossref PubMed Scopus (67) Google Scholar Furthermore, contradictory data exist with respect to the involvement of other putative chromosomal regions in EPT tumorigenesis, such as 9p21 (p16 gene) and 17q12-q21 (c-erbB-2 gene).8Evers BM Rady PL Sandoval K Arany I Tyring SK Sanchez RL Nealon WH Townsend CM Thompson JC Gastrinomas demonstrate amplification of the HER-2/neu proto-oncogene.Ann Surg. 1994; 219: 596-604Crossref PubMed Scopus (59) Google Scholar, 9Beghelli S Pelosi G Zamboni G Falconi M Iacono C Bordi C Scarpa A Pancreatic endocrine tumours: evidence for a tumour suppressor pathogenesis and for a tumour suppressor gene on chromosome 17p.J Pathol. 1998; 186: 41-50Crossref PubMed Scopus (67) Google Scholar, 10Chung DC Brown SB Graeme-Cook F Tillotson LG Warshaw AL Jensen RT Arnold A Localization of putative tumor suppressor loci by genome-wide allelotyping in human pancreatic endocrine tumors.Cancer Res. 1998; 58: 3706-3711PubMed Google Scholar, 11Muscarella P Melvin WS Fisher WE Foor J Ellison EC Herman JG Schirmer WJ Hitchcock CL DeYoung BR Weghorst CM Genetic alterations in gastrinomas and nonfunctioning pancreatic neuroendocrine tumors: an analysis of p16/MTS1 tumor suppressor gene inactivation.Cancer Res. 1998; 58: 237-240PubMed Google Scholar To identify chromosomal alterations which may be important for EPT initiation and progression, we have applied comparative genomic hybridization (CGH), which allows the screening of tumor samples for DNA sequence losses (>10 Mb) and gains along all chromosome arms without need of culturing tumor cells in vitro for chromosome karyotyping.12Kallioniemi A Kallioniemi O-P Sudar D Rutovitz D Gray JW Waldman F Pinkel D Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors.Science. 1992; 258: 818-821Crossref PubMed Scopus (2837) Google Scholar Forty-four sporadic human EPTs were examined for genetic changes in relation to clinical disease stage, tumor size, and hormonal subtype. A subset of these CGH data was confirmed by interphase cytogenetics and molecular allelotyping. Our results show marked genetic differences with respect to these parameters and pinpoint several new loci that are candidates for harboring genes with a role in EPT pathogenesis. EPTs and 5 metastases of 44 patients (22 male, mean age 53.0 ± 16.0 years, and 22 female, mean age 51.3 ± 15.8 years) were selected from the files of the Departments of Pathology of the Universities of Zürich and Basel, Switzerland. The samples included 28 frozen and 16 formalin-fixed, paraffin-embedded EPTs, which were all sporadic and not associated with the inherited MEN1 or VHL syndromes. The tumors were classified according to the most recent classification13Klöppel G Classification of neuroendocrine tumors.Verh Dtsch Ges Pathol. 1997; 81: 111-117PubMed Google Scholar and consisted of 9 nonfunctioning (8 malignant, 1 benign) and 35 functioning EPTs, including 17 insulinomas (11 benign, 6 malignant), 7 gastrinomas, 7 VIPomas, and 4 glucagonomas (all malignant). Eighteen of the patients had localized disease, defined by the absence of extrapancreatic spread of the tumor, whereas 22 patients had advanced disease, with tumor spread into the surrounding soft tissue, lymph nodes, or liver. In four patients no data were available concerning the disease stage. Genomic DNA from frozen tumors was isolated using the D-5000 Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN). DNA from paraffin-embedded tumor samples was extracted as previously described using proteinase K digestion and phenol/chloroform extraction.5Görtz B Roth J Krähenmann A De Krijger RR Muletta-Feurer S Rütimann K Saremaslani P Speel EJM Heitz PU Komminoth P Mutations and allelic deletions of the MEN 1 gene are associated with a subset of sporadic endocrine pancreatic and neuroendocrine tumors and not restricted to foregut neoplasms.Am J Pathol. 1999; 154: 429-436Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 14Görtz B Roth J Speel EJM Krähenmann A De Krijger RR Matias-Guiu X Muletta-Feurer S Rütimann K Saremaslani P Heitz PU Komminoth P MEN 1 gene mutation analysis of sporadic adrenocortical lesions.Int J Cancer. 1999; 80: 373-379Crossref PubMed Scopus (94) Google Scholar, 15Richter J Jiang F Görög J-P Sartorius G Egenter C Gasser TC Moch H Mihatsch MJ Sauter G Marked genetic differences between stage pTa and stage pT1 papillary bladder cancer detected by comparative genomic hybridization.Cancer Res. 1997; 57: 2860-2864PubMed Google Scholar Only tumors with >70% tumor cell content were included in this study. DNA from normal pancreatic tissue was also isolated for microsatellite loss of heterozygosity (LOH. analysis. CGH was performed as described.15Richter J Jiang F Görög J-P Sartorius G Egenter C Gasser TC Moch H Mihatsch MJ Sauter G Marked genetic differences between stage pTa and stage pT1 papillary bladder cancer detected by comparative genomic hybridization.Cancer Res. 1997; 57: 2860-2864PubMed Google Scholar Briefly, 1 μg tumor DNA was labeled with Spectrum Green-dUTPs (Vysis, Downers Grove, IL) by nick translation (BioNick kit, Life Technologies, Basel, Switzerland). Spectrum Red-labeled normal reference DNA (Vysis. was used for cohybridization. The hybridization mixture consisted of 200 ng Spectrum Green-labeled tumor DNA, 200 ng Spectrum Red-labeled normal reference DNA, and 10 to 20 μg of human Cot-1 DNA (Life Technologies) dissolved in 10 μl of hybridization buffer (50. formamide, 2× SSC, pH 7.0). Hybridization was carried out for 3 days at 37°C to normal human metaphase spreads (Vysis). Slides were washed at 45°C three times for 10 minutes in 50% formamide/2× SSC followed by two times for 5 minutes in 2× SSC. The chromosomes were counterstained with 4,6-diamidino-2-phenylindole for identification. Digital images were collected from six to seven metaphases using a Photometrics cooled CCD camera (Microimager 1400; Xillix Technologies, Vancouver, BC) attached to a Zeiss Axioskop microscope and a Sun workstation. The software program QUIPS (Vysis) was used to calculate average green-to-red ratio profiles for each chromosome. At least four observations per autosome and two observations per sex chromosome were included in each analysis. In 22 of the 44 tumor cases two CGH analyses per tumor were carried out to confirm the reproducibility of the detected chromosomal abnormalities by CGH. In 8 cases, where no genomic imbalances could be detected by CGH, additional DNA was isolated from another part of the same tumor, leading to essentially the same CGH results. Positive, negative, and sex-mismatched controls were applied as previously described.15Richter J Jiang F Görög J-P Sartorius G Egenter C Gasser TC Moch H Mihatsch MJ Sauter G Marked genetic differences between stage pTa and stage pT1 papillary bladder cancer detected by comparative genomic hybridization.Cancer Res. 1997; 57: 2860-2864PubMed Google Scholar Gains and losses of DNA sequences were defined as chromosomal regions where both the mean green-to-red fluorescence ratio and its SD were above 1.20 and below 0.80, respectively. Overrepresentations were considered amplifications when the fluorescence ratio values in a subregion of a chromosomal arm exceeded 1.5. In negative control hybridizations, the mean green-to-red ratio occasionally exceeded the fixed 1.2 cutoff level at the following chromosomal regions: 1p32-pter, 16p, 19, and 22. Gains of these known G-C-rich regions were, therefore, excluded from all analyses. Contingency table analysis was used to analyze the relationship between genomic alterations and disease stage, tumor size, and hormonal subtype. Student's t-test and analysis of variance were applied to compare the number of genomic alterations between the different EPT hormonal subtypes. Ten EPTs, five of which showed losses of chromosome 3 DNA by CGH, were analyzed for allelic deletion of chromosome 3p loci using polymorphic microsatellite markers D3S1110 (3p25.1-p25.3), D3S1029 (3p21.2-p21.3) and D3S1076 (3p21.1) (Research Genetics, Huntsville, AL). Touch preparations of these EPTs were subjected to fluorescence in situ hybridization (FISH) using a chromosome 3 centromere probe (p α3.5) in combination with a P1 probe mapping to the 3p25 region (kindly provided by Dr. J. Gray, Resource of Molecular Cytogenetics, University of California, San Francisco, CA). Both methods were performed according to recently described protocols5Görtz B Roth J Krähenmann A De Krijger RR Muletta-Feurer S Rütimann K Saremaslani P Speel EJM Heitz PU Komminoth P Mutations and allelic deletions of the MEN 1 gene are associated with a subset of sporadic endocrine pancreatic and neuroendocrine tumors and not restricted to foregut neoplasms.Am J Pathol. 1999; 154: 429-436Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 14Görtz B Roth J Speel EJM Krähenmann A De Krijger RR Matias-Guiu X Muletta-Feurer S Rütimann K Saremaslani P Heitz PU Komminoth P MEN 1 gene mutation analysis of sporadic adrenocortical lesions.Int J Cancer. 1999; 80: 373-379Crossref PubMed Scopus (94) Google Scholar and the results were compared with each other and with the CGH data. In addition, paraffin-embedded tissue sections (5 μm) of eight EPTs of male patients showing gains of chromosome X (4/8), gain of chromosome Y (1/8), and loss of chromosome Y (5/8) by CGH, were used for independent in situ hybridization analysis with chromosome X (pBAMX5) and Y (DYZ3. centromere probes, as previously described.16Hopman AHN Van Hooren E Van de Kaa CA Vooijs GP Ramaekers FCS Detection of numerical chromosome aberrations using in situ hybridization in paraffin sections of routinely processed bladder cancers.Mod Pathol. 1991; 4: 503-513PubMed Google Scholar Figure 1A summarizes all DNA sequence copy number changes detected by CGH in the 44 EPTs, and representative CGH data are shown in Figure 2A. Genetic aberrations were found in 36 of 44 EPTs (82%), and the overall number of chromosome arm aberrations per tumor ranged from 0 to 36 (mean, 9.9). Chromosomal losses (range, 0–17; mean, 5.3) were slightly more frequent than gains (range 0–19. mean, 4.6), and no evident amplifications could be detected in the EPTs (Table 1).Figure 2A: Representative CGH results in EPTs. Individual examples of fluorescent ratio profiles (right) and digital images (left) of chromosomes with recurrent gains and losses. The red vertical bar on the left side of a chromosome ideogram (middle. indicates the region of loss and the green vertical bar on the right side of an ideogram indicates the region of gain. B: Example of microsatellite and FISH LOH analysis of a nonfunctioning EPT shows allelic loss for 2 of 3 markers (D3S1029 and D3S1076, red arrowheads; D3S1110 is not informative) on chromosome arm 3p (left) and monosomy for both the centromere 3 (red spots) and 3p25-specific probe (green spots) in DAPI-stained tumor nuclei (right). C: Example of in situ hybridization analysis on paraffin sections of a male VIPoma sample, showing the expected one copy of the X centromere per nucleus in the major cell population (left) and a loss of the chromosome Y centromere sequence in the tumor cells, whereas the stroma cells in between (arrowheads. are still positive for this DNA sequence (right).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1Genomic Changes per Case in Sporadic EPTsAberrations per tumorTumornAll changesP value*Analysis of variance.GainsP valueLossesP valueAll EPTs449.9 ± 9.94.6 ± 4.95.3 ± 5.3EPTs− metastases226.9 ± 9.70.03823.4 ± 4.7NS3.4 ± 5.20.0182EPTs + metastases1813.2 ± 8.75.9 ± 4.47.3 ± 4.6EPTs 2 cm2213 ± 10.66.2 ± 5.26.9 ± 5.7Nonfunctioning EPTs921.3 ± 13.30.000110.3 ± 6.60.000111 ± 6.90.0001Functioning EPTs357 ± 6.33.2 ± 3.13.8 ± 3.6Insulinoma benign112.5 ± 3.90.00011.4 ± 2.00.00011.1 ± 2.40.0001Insulinoma malignant615.8 ± 4.07.2 ± 2.48.7 ± 2.0Insulinoma all177.2 ± 7.63.4 ± 3.53.8 ± 4.4Gastrinoma75.4 ± 4.30.0238†P values for glucagonoma versusgastrinoma.2.1 ± 1.90.0122†P values for glucagonoma versusgastrinoma.3.3 ± 2.9NS†P values for glucagonoma versusgastrinoma.VIPoma74.9 ± 3.60.0097‡P values for glucagonoma versusVIPoma.2 ± 2.50.0263†P values for glucagonoma versusgastrinoma.2.9 ± 1.70.0141‡P values for glucagonoma versusVIPoma.Glucagonoma412.8 ± 4.36 ± 2.26.8 ± 2.6NS, not significant.* Analysis of variance.† P values for glucagonoma versusgastrinoma.‡ P values for glucagonoma versusVIPoma. Open table in a new tab NS, not significant. Chromosomal regions that were most often lost (>20% of cases. included 3pq, 6pq, 8q, 10q, and 11pq, with the highest frequency of losses on chromosome 6q (39%) and 11q (36%; Figure 1A and Table 2A). In addition, there were frequent losses of Xp in 20% and of Xq in 25% of patients. Chromosome X losses were more frequent in female than in male patients, ie, Xp loss was seen in seven (32%) and Xq loss in ten (45%) female patients, whereas Xp loss was detected in only two (9%) and Xq loss in one (5%) male patient. Chromosome Y loss was detected in 10 of 22 (45%) males. Increased DNA sequence copy number (>20% of cases) most often involved chromosomes 5pq, 7pq, 9q, 12q, 14q, 17pq, and 20q with the highest frequency of gains on chromosomes 7q (43%) and 17q (41%) (Figure 1A and Table 2A). Gains of chromosomes Xp (25%) and Xq (23%. were more frequently observed in male than in female patients, ie, Xp gain was seen in ten (45%) and Xq gain in seven (32%) male patients, whereas gain of Xp was detected only in two (9%) and Xq gain in three (14%) female patients.Table 2Most Frequent (>20%) Genomic Changes in EPTsA LocusAll EPTs n = 44EPT − meta n = 22EPT + meta n = 18P value†Contingency table analysis.EPT < 2 cm n = 12EPT > 2 cm n = 22P valueB EPT/NF n = 9EPT/F n = 35P valueIns ben n = 11Ins mal n = 6P valueLosses 3p13 (30)*The first number in each pair is the number of tumor samples showing the loss or gain; the second number (in parentheses) is the percentage showing the loss or gain.2 (9)10 (56)0.00141 (8)9 (41)0.04646 (67)7 (20)0.00620 (0)2 (33)0.0415 3q13 (30)4 (18)8 (44)NS0 (0)10 (45)0.00545 (56)8 (23)NS0 (0)3 (50)0.0098 6p12 (27)3 (14)8 (44)0.02991 (8)8 (36)NS5 (56)7 (20)0.03270 (0)3 (50)0.0098 6q17 (39)5 (23)11 (61)0.01372 (17)12 (55)0.0326 (67)11 (31)NS0 (0)6 (100)0.0001 8q10 (23)5 (23)4 (22)NS1 (8)7 (32)NS6 (67)4 (11)0.00041 (9)1 (17)NS 10q11 (25)3 (14)6 (33)NS0 (0)9 (41)0.00987 (78)4 (11)0.00010 (0)2 (33)0.0415 11p13 (30)5 (23)7 (39)NS1 (8)9 (41)0.04647 (78)6 (17)0.00041 (9)2 (33)NS 11q16 (36)6 (27)8 (44)NS1 (8)11 (50)0.01517 (78)9 (26)0.00381 (9)2 (33)NS Xq11 (25)5 (23)6 (33)NS4 (33)4 (18)NS2 (22)9 (26)NS2 (18)2 (33)NS Y10 (23)2 (9)6 (33)NS1 (8)6 (27)NS2 (22)8 (23)NS0 (0)4 (67)0.002Gains 5p10 (23)6 (27)3 (17)NS1 (8)7 (32)NS6 (67)4 (11)0.00042 (18)1 (17)NS 5q14 (32)7 (32)6 (33)NS2 (17)9 (41)NS6 (67)8 (23)0.01183 (27)3 (50)NS 7p12 (27)7 (32)4 (22)NS2 (17)6 (27)NS5 (56)7 (20)0.03271 (9)2 (33)NS 7q19 (43)8 (36)10 (56)NS3 (25)11 (50)NS7 (78)12 (34)NS1 (9)4 (67)0.0128 9q12 (27)3 (14)7 (39)NS3 (25)6 (27)NS3 (33)9 (26)NS2 (18)4 (67)0.0456 12q11 (25)4 (18)6 (33)NS0 (0)8 (36)0.01696 (67)5 (14)0.00120 (0)4 (67)0.002 14q13 (32)3 (14)10 (56)0.00491 (8)10 (45)0.0277 (78)7 (20)0.00090 (0)4 (67)0.002 17p12 (27)4 (18)6 (33)NS1 (8)9 (41)0.04644 (44)8 (23)NS0 (0)3 (50)0.0098 17q18 (41)7 (32)9 (50)NS1 (8)13 (59)0.00417 (78)11 (31)0.01170 (0)4 (67)0.002 20q12 (27)4 (18)7 (39)NS2 (17)8 (36)NS5 (56)7 (20)0.03271 (9)2 (33)NS Xp11 (25)3 (14)7 (39)NS1 (8)8 (36)NS4 (44)7 (20)NS1 (9)4 (67)0.0128 Xq10 (23)1 (5)8 (44)0.00260 (0)7 (32)0.02835 (56)5 (14)0.00840 (0)3 (50)0.01EPT, endocrine pancreatic tumor; meta, metastasis; NF, nonfunctioning; F, functioning; Ins, insulinoma; ben, benign; mal, malignant; NS, not significant.* The first number in each pair is the number of tumor samples showing the loss or gain; the second number (in parentheses) is the percentage showing the loss or gain.† Contingency table analysis. Open table in a new tab EPT, endocrine pancreatic tumor; meta, metastasis; NF, nonfunctioning; F, functioning; Ins, insulinoma; ben, benign; mal, malignant; NS, not significant. EPTs from patients with localized disease showed significantly less genomic changes and chromosomal losses than EPTs from patients with advanced disease (Table 1). In particular, losses of 3p and 6pq as well as gains of 14q and Xq proved to be associated with an advanced stage of disease (P ≤ 0.0299; Table 2). In addition, we found clear differences in chromosome aberrations in relation to tumor size. EPTs with a diameter 2 cm (P ≤ 0.0137; Table 1). Except for losses of Xq, all genomic changes found in >20% of EPTs were more frequently found in the larger tumors, 11 of which were significantly more frequent. Marked differences in chromosomal aberrations were detected in the different EPT subtypes (Table 1, Table 2). In general, the nonfunctioning EPTs exhibited more aberrations per tumor (mean, 21.3) than the functioning ones (mean, 7; P = 0.0001). This difference did not reach statistical significance comparing glucagonomas (mean, 12.8) with the nonfunctioning EPTs (Table 1). Strikingly, significant differences in CGH results were observed between benign and malignant insulinomas (mean, 2.5 vs. 15.8, respectively; P = 0.0001; Table 1). In addition, every hormonal subtype of the functioning EPTs (insulinoma, gastinoma, VIPoma, glucagonoma) showed a tendency towards a different mean number of CGH aberrations per tumor when compared with each other, although these differences only reached statistical significance when comparing glucagonomas with gastrinomas and VIPomas (P ≤ 0.0238; Table 1). Figure 1B depicts the chromosomal imbalances in the 9 investigated nonfunctioning EPTs. Gains and losses of many chromosomes were detected, mostly involving entire chromosomes or chromosomal arms. Table 2B demonstrates that 14 of 22 chromosomal loci proved to be more involved in these tumors than in the functioning ones (P < 0.0454) with losses of 10q and 11pq and gains of 7q, 14q, and 17q occurring in 7 out of 9 tumors. In contrast, the functioning EPTs seem to harbor fewer and more regional genomic aberrations (Figure 1C) with losses of 6q (common region of involvement [CRI]: 6q21–22), 11q (CRI: 11q13–22), Xq (in females: 9/18 = 50%; CRI: Xq22–23) and Y (in males: 8/17 = 47%) as well as gains of 7q (CRI: 7q11.2-q32), 9q (CRI: 9q34), 17q (CRI: 17q11.2–21. and Xp (in males: 7/17 = 41%; CRI: Xp11.3–11.4) in >25% of tumors. Interestingly, the most frequently occurring aberrations were different in the functioning EPT subtypes, ie, benign insulinomas 5q+ (3/11); malignant insulinomas 6q− (6/6), Xp+ and Y− (4/4 males) and Xq− (2/2 females); gastrinomas 3p− and 9q+ (3/7); VIPomas 11q−, Xq−, and Y− (each 3/7); and glucagonomas 7q+ (4/4; Table 2B). To validate the obtained CGH results, ten EPTs were additionally examined by microsatellite and FISH analysis for chromosome 3p alterations. Both techniques confirmed allelic loss of the entire chromosome 3 in three EPTs, including a monosomy for the centromere in the tumor nuclei (Figure 2B). In the five tumors without chromosome 3 imbalances, also no LOH was observed. FISH analysis demonstrated in one of these five EPTs, however, a tetrasomy for both the centromere and 3p25 locus in the major population of cells, and this tumor turned out to be aneuploid. In the two EPTs with only regional 3q losses detected by CGH, no losses of the centromere and 3p markers were found by microsatellite and FISH analysis, except for the microsatellite marker at 3p25, which showed LOH in both cases. This locus has been postulated to harbor a novel EPT tumor suppressor gene (TSG).6Chung DC Smith AP Louis DN Graeme-Cook F Warshaw AL Arnold A A novel pancreatic endocrine tumor suppressor gene locus on chromosome 3p with clinical prognostic implications.J Clin Invest. 1997; 100: 404-410Crossref PubMed Scopus (122) Google Scholar, 17Barghorn A Rütimann K Saremaslani P Muletta S Speel EJM Roth J Heitz PU Komminoth P Loss of heterozygosity von 3p25 in sporadischen neuroendokrinen Pankreastumoren: ein Malignitätsmarker?.Schweiz Med Wochenschr. 1998; 128: 1870Google Scholar Thus, microsatellite and FISH analysis confirmed the CGH data and, moreover, detected tetrasomy for chromosome 3 in one EPT and a microsatellite LOH of 3p25 in two tumors. This LOH was not detected with CGH, probably due to the small size of the chromosomal region lost (<10 Mb). In addition, in situ hybridization with centromere X- and Y-specific probes was applied to paraffin sections. It confirmed the CGH data of sex chromosome imbalances in eight EPTs of male patients. Four cases with gains of chromosome X in the CGH displayed a disomy for the centromere X in 50 to 70% of tumor nuclei, whereas the other tumors with no imbalances in the CGH displayed one copy of the centromere in the majority of nuclei by in situ hybridization. In five EPTs where CGH revealed chromosome Y losses, no signals for centromere Y could be detected by in situ hybridization, whereas the stromal cells showed the expected number of one copy per nucleus in most cells (Figure 2C). The three additional tumors harboring a gain of Y in one EPT and no imbalance of Y in two EPTs also exhibited the expected number of centromere copies (two and one, respectively) in the in situ hybridization. We have performed a comprehensive genome-wide survey of DNA sequence copy number changes in human sporadic EPTs with the goals of correlating the overall number of genetic alterations with clinical and histopathological parameters and identifying new locations of TSGs and oncogenes potentially involved in tumor pathogenesis. The powerful technique of CGH permitted the analysis of DNA extracted from frozen as well as formalin-fixed, paraffin-embedded tumor material. A wide spectrum of genetic aberrations was detected in the EPTs, and a correlation was found between the number of genomic changes per tumor and both disease stage and tumor size. Moreover, marked genetic differences were observed between the various EPT subtypes. The most commonly encountered genetic aberrations were losses on chromosomes 3pq, 6pq, 10q, 11pq, Xq, and Y, and gains on chromosomes 5q, 7pq, 9q, 12q, 17pq, 20q, and Xp. Hence, these chromosomal regions are likely to be important for tumor
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