[CANCER RESEARCH 61, 5186–5192, July 1, 2001] Genetic Evidence for Early Divergence of Small Functioning and Nonfunctioning Endocrine Pancreatic Tumors: Gain of 9Q34 Is an Early Event in Insulinomas1 Ernst J. M. Speel,2 Alexander F. Scheidweiler, Jianming Zhao, Claudia Matter, Parvin Saremaslani, Ju¨rgen Roth, Philipp U. Heitz, and Paul Komminoth3 Department of Molecular Cell Biology, University of Maastricht, Research Institute Growth and Development, 6200 MD Maastricht, The Netherlands [E. J. M. S.], Department of Pathology [A. F. S., J. Z., C. M., P. S., P. U. H., P. K.], and Division of Cell and Molecular Pathology [J. R., P. K.], University of Zurich, 8091 Zurich, Switzerland ABSTRACT fields), and/or exhibit Յ2% Ki-67 positive cells. Most insulinomas fall into this category, as do nonfunctioning (micro)adenomas that are The malignant potential among endocrine pancreatic tumors (EPTs) a rather common finding in carefully examined postmortem pancre- varies greatly and can frequently not be predicted using histopathological ases (4). EPTs that are Ͼ2 cm or show angioinvasion, higher numbers parameters. Thus, molecular markers that can predict the biological behavior of EPTs are required. In a previous comparative genomic hy- of mitoses, or percentages of Ki-67 positive cells are at risk of bridization study, we observed marked genetic differences between the malignancy. They include many of the functioning tumors other than various EPT subtypes and a correlation between losses of 3p and 6 and insulinomas. gains of 14q and Xq and metastatic disease. To search for genetic alter- Our understanding of the molecular mechanisms underlying the ations that play a role during early tumor development, we have studied tumorigenesis of EPTs is still scarce. A small percentage of EPTs 38 small (<2 cm) EPTs, including 24 insulinomas and 10 nonfunctioning occur in association with the dominantly inherited MEN 1 and, to a EPTs. Small EPTs are usually classified as clinically benign tumors in the lesser extent, VHL syndromes, the susceptibility genes of which have absence of histological signs of malignancy. Using comparative genomic been cloned and mapped to chromosome bands 11q13 and 3p25.5, hybridization, we identified chromosomal aberrations in 27 EPTs (mean, respectively (5, 6). The vast majority of EPTs, however, occur spo- 4.1). Interestingly, the number of gains differed strongly between non- radically, and only a subset (15–20%) of these tumors harbor somatic functioning and functioning EPTs (3.4 versus 1.5, respectively; and MEN 1 mutations (7). In contrast, VHL mutations appear not to be (30 ؍ as did the number of aberrations in the benign (n ,(0.0526 ؍ P In the relevant in the pathogenesis of sporadic EPTs (8), as are alterations of .(0.0022 ؍ tumors (3 versus 8.4, respectively; P (8 ؍ malignant (n insulinomas, 9q gain (common region of involvement: 9q34) was most the K-ras,N-ras, and TP53 genes (9–11). The involvement of chro- common (50%) and in nonfunctioning EPTs, gain of 4p was most common mosomal regions 9p21 (p16 gene) and 17q12-q21 (c-erbB-2 gene) in (40%). Most frequent losses in insulinomas involved 1p (20.8%), 1q, 4q, EPT tumorigenesis is controversial (10–14). 11q, Xq, and Y (all 16.7%) and in nonfunctioning EPTs, 6q. Losses of 3pq Recently, we have applied CGH to identify chromosomal alter- and 6q and gains of 17pq and 20q proved to be strongly associated with ations that are important in the pathogenesis of EPTs (14). Various malignant behavior in all of the small EPTs (P < 0.0219). Our results significant genetic changes could be identified, including losses of demonstrate marked genetic differences between small functioning and chromosomes 3, 6, 11, X, and Y and gains of chromosomes 5, 7, 9, 14, nonfunctioning EPTs, indicating that these subtypes evolve along different and 17. The total number of genetic changes per EPT appeared to be genetic pathways. In addition, our study endorses the importance of chromosomes 3 and 6q losses to discriminate EPTs with a malignant strongly correlated with both tumor size and disease stage. Moreover, behavior from benign ones. genetic differences could be detected in the various hormonal sub- types, indicating that these groups may evolve along genetically different pathways. The objective of the present study was to charac- INTRODUCTION terize the genetic alterations of EPTs with a diameter Յ2 cm, because 4 these lesions may provide information about the initiating genetic EPTs make up only 1–2% of all of the pancreatic tumors and can events in the pathogenesis of these tumors. We found marked genetic cause syndromes by uncontrolled expression of biologically active differences between small functioning and nonfunctioning EPTs, in- hormones. Such tumors are classified as functioning EPTs, whereas dicating that they evolve along different genetic pathways. In addi- EPTs that do not release any hormone or produce hormones that do tion, by correlating genetic alterations with tumor subtype and malig- not lead to clinical symptoms are called nonfunctioning tumors (1). nant potential, additional evidence was obtained showing that losses Because these clinical parameters do not provide information with of chromosomes 3 and 6q are related to malignancy in EPTs. respect to the biological behavior of EPTs, more indicative his- topathological classification schemes to predict prognosis have been established (2, 3). Accordingly, well-differentiated EPTs are classified MATERIALS AND METHODS Յ as benign if they are 2 cm in size, confined to the pancreas, Tumor Material and Patient Data. Thirty-eight EPTs with a maximum nonangioinvasive (with two or fewer mitoses per 10 high-power diameter of 2 cm (21 females, mean age 55.2 Ϯ 21.3 years and 17 males, mean age 57.1 Ϯ 15.1 years) were selected from the archives of the Departments of Received 12/22/00; accepted 5/2/01. Pathology, Universities of Zurich and Basel, Switzerland and University of The costs of publication of this article were defrayed in part by the payment of page Turin, Turin, Italy (Table 1). The samples included 15 frozen and 23 formalin- charges. This article must therefore be hereby marked advertisement in accordance with fixed, paraffin-embedded EPTs, which were all sporadic tumors and not 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by Swiss Cancer League Grant SKL-649-2-1998 and Swiss National associated with the inherited MEN 1 or VHL syndromes. They were classified Science Foundation Grant 31-53625.98. according to the most recent classification (15) and consisted of 10 nonfunc- 2 To whom requests for reprints should be addressed, at Department of Molecular Cell tioning (6 benign and 4 malignant) and 28 functioning EPTs, including 24 Biology, Research Institute Growth and Development (GROW), University of Maastricht, insulinomas (22 benign and 2 malignant), 3 glucagonomas (2 benign and 1 P.O. Box 616, 6200 MD Maastricht, The Netherlands. Phone: 31-43-3881425; Fax: 31-43-3884151; E-mail: [email protected]. malignant), and 1 malignant gastrinoma. Thirty-six patients had localized 3 Present address: Institute for Pathology, Department of Medical Service, Kantons- disease, as defined by the absence of extrapancreatic spread of the tumor, spital Baden, CH-5404 Baden, Switzerland. whereas only 2 patients had advanced disease (patients 23 and 24 in Table 1), 4 The abbreviations used are: EPT, endocrine pancreatic tumor; CGH, comparative with tumor spread into the lymph nodes (one case) or the liver (one case). genomic hybridization; CRI, common region of involvement; TSC, tuberous sclerosis; FISH, fluorescence in situ hybridization; MEN 1, multiple endocrine neoplasia type 1; DNA Extraction. Genomic DNA from frozen samples was isolated by TSG, tumor suppressor gene; VHL, von Hippel Lindau. homogenizing ϳ5mm3 of each sample prior to DNA extraction using the 5186 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2001 American Association for Cancer Research. GENETIC ALTERATIONS IN SMALL EPTS Table 1 Patient characteristics and genetic findings in small EPTs Number of changes FISH resultsb Size Nra Sex Age Diagnosis Ben/mal (cm) CGH results All Gains Losses 9c–9q34 6c–6q21 3c–4p16 Functioning EPTs 1 Female 57 Ins Ben 1.1 0 0 0 0 2 Female 73 Ins Ben 1 0 0 0 0 Disomy Disomy 3 Female 42 Ins Ben 1.5 0 0 0 0 Disomy Disomy 4 Female 59 Ins Ben 1.2 0 0 0 0 5 Male 62 Ins Ben 1.2 0 0 0 0 6 Female 26 Ins Ben 2 0 0 0 0 7 Female 22 Ins Ben 1.5 0 0 0 0 8 Male 52 Ins Ben 1.6 0 0 0 0 9 Female 72 Ins Ben 1.2 1pqϪ 202 10 Female Ins Ben 1.5 9q34ϩ 1 1 0 3–6 Trisomy 11 Female 80 Ins Ben 1 9q34ϩ 1 1 0 3–3/4–4 (37/37%) Trisomy 12 Male 40 Ins Ben 1 9q33–34ϩ 110 13 Female 75 Ins Ben 1.3 9qϩ 110 14 Female 32 Ins Ben 1.5 XpqϪ 202 15 Male 53 Ins Ben 1.2 5pqϩ, 9q33–34ϩ,Xpϩ 4 4 0 2–3 (51%) Disomy 16 Male 43 Ins Ben 1.2 9q22–34ϩ, 14q22-terϩ 220 17 Male 36 Ins Ben 1.9 16q21-terϪ, 22qϪ,Yϩ 312 18 Male 36 Ins Ben 1 11pqϪ, 13qϪ, 21qϩ 413 19 Male 37 Ins Ben 1.3 1p12–31Ϫ,4qϪ,9qϩ,YϪ 413 20 Male 72 Ins Ben 1.5 1pqϪ, 4pqϪ, 7pqϩ, 9pqϩ, 11pqϪ, 12 5 7 2–1 15qϩ,YϪ 21 Female 42 Ins Ben 1 4p15.2-qterϪ,5qϩ, 7pqϩ, 8pqϪ,9qϩ, 13 5 8 11p13–15.2Ϫ, 11q13-terϪ, 20qϩ, XpqϪ 22 Male 58 Ins Ben 2 1p12–31Ϫ, 1q23-terϪ, 2q22–33Ϫ, 10 3 6 9q31–34ϩ, 11q13-terϪ, 14qϩ, 15qϩ, 18q11.2–22Ϫ, Xq21.3-terϪ, Yp11.2– q11.2Ϫ 23 Female 26 Ins Mal 1.5 4p15.2-qterϪ, 6q12–22Ϫ,9qϩ, 13q21- 10 4 6 terϪ, 14q22-terϩ, 15qϩ, 20qϩ, Xp21-qterϪ 24 Male 56 Ins Mal 1.9 1p13–31Ϫ,1qϪ, 2q23–33Ϫ, 3p14.1- 13 6 6 4–4/4–8 (44/25%) 2–1 qterϪ,6qϪ,9qϩ, 12q23-terϩ, 17pqϩ,