Frequent loss of heterozygosity at chromosome 3p14 2 3p21 in human pancreatic islet cell tumours*

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1 Clinical Endocrinology (1999) 51, Frequent loss of heterozygosity at chromosome 3p14 2 3p21 in human pancreatic islet cell tumours* Marina N. Nikiforova, Yuri E. Nikiforov, Paul Biddinger, Douglas R. Gnepp, Luis A. Grosembacher**, Bernardo L. Wajchenberg, James A. Fagin and Robert M. Cohen Division of Endocrinology/Metabolism, Department of Medicine and Department of Pathology and Lab Medicine, University of Cincinnati College of Medicine, Cincinnati, Cincinnati Veterans Affairs Medical Center, Cincinnati, OH USA, Department of Pathology, Rhode Island Hospital, Providence, **Division of Endocrinology and Nuclear Medicine, Hospital Italiano, Buenos Aires, Argentina and Endocrinology Section, Hospital das Clinicas, Sao Paulo, Brazil (Received 12 November 1998; returned for revision 20 January 1999; finally revised 22 February 1999; accepted 1 April 1999) Summary OBJECTIVE Pancreatic islet bcell tumours occur either sporadically or as part of inherited neoplastic syndromes, most commonly multiple endocrine neoplasia (MEN) type 1. Recently, a transgenic mouse model has been established in which the expression of the SV40 large T antigen was targeted to bcells by the rat insulin promoter, leading to the development of multiple pancreatic bcell tumours. In the advanced stages of tumour evolution, these tumours exhibited a high prevalence of loss of heterozygosity (LOH) on mouse chromosomes 9 and 16, at regions syntenic with regions 3q, 3p21, 6q12, 15q24 and 22q of the human genome. DESIGN Loss of heterozygosity in human islet cell tumours was analysed in a PCR based approach at regions of the human genome syntenic with the mouse loci linked to pancreatic bcell tumours as well as the MEN1 gene on chromosome 11q13. These included 35 microsatellite markers in the human chromosomal regions 3q, 3p21, 6q12, 11q13, 15q24 and 22q. PATIENTS 21 patients diagnosed with insulinoma * See commentary page 19. Correspondence: Dr Robert M Cohen, Division of Endocrinology/ Metabolism, PO Box , University of Cincinnati Medical Center, Cincinnati, OH , USA. Fax: þ ; Robert.Cohen@uc.edu 1999 Blackwell Science Ltd were analysed. Histologically, 16 tumours were benign, while 5 were malignant insulinomas. RESULTS Thirteen of 21 (62%) tumours were found to have loss of genetic material on chromosome 3. The shortest region of overlap implicated a deletion at 3p14 2 3p21 region, corresponding to the marker D3S1295. We did not detect a substantial frequency of LOH in the other syntenic regions, except for the region of MEN 1 gene on 11q13 found to be deleted in 6 (29%) cases, including 3 of 4 tumours from MEN 1 families. Deletions of 3p14 2 3p21 were observed in 8 of 15 (53%) benign tumours, and in 5 of 6 (83%) malignant neoplasms. CONCLUSIONS These results indicate the high frequency of 3p14 2 3p21 deletions in human pancreatic bcell neoplasms. These finding suggest the presence of a tumour suppressor gene in this region, that may be important in the microevolution of these tumours towards malignancy. Pancreatic islet cell neoplasms are usually small tumours often reaching clinical attention as a consequence of inappropriate hormone secretion, e.g. insulin, gastrin, glucagon, somatostatin or pancreatic polypeptide, and less commonly because of a mass effect. While some occur in well described familial syndromes, e.g. multiple endocrine neoplasia (MEN) type 1, the vast proportion occur sporadically. Limited information is available on the molecular mechanisms underlying the development of these endocrine neoplasms. Loss of heterozygosity (LOH) in the region of the MEN 1 gene on chromosome 11q13 has been reported in 50% of 12 sporadic insulinomas, with 17% of tumours also exhibiting MEN1 gene mutations in the second allele (Zhuang et al., 1997). In a recently published observation of 43 pancreatic islet cell tumours 33% were found to have allelic loss at the region of 3p25, a location centromeric to the VHL gene (Chung et al., 1997). Other chromosomal regions (i.e. 1p, 5q, 7q, 9q, 13q, 16q) were found to be deleted less frequently, although the number of tumours studied was low (Patel et al., 1990; Radford et al., 1990; Ding et al., 1992; Bale, 1994). A transgenic mouse model has been established by Hanahan in which the expression of SV40 large T antigen, under the direction of a rat insulin promoter, led to the development of multiple pancreatic bcell tumours (Hanahan, 1985). In the advanced stages of tumour evolution, these tumours exhibited a high prevalence of LOH on mouse chromosomes 9 and 16, 27

2 28 M. N. Nikiforova et al. which are homologous to regions 3q, 3p21, 6q12, 15q24 and 22q of the human genome. This suggests that these loci may harbor important tumour suppressor genes for mouse bcells (Dietrich et al., 1994; Parangi et al., 1995). In this study, we examined the human regions syntenic to the aforementioned mouse loci, and also the 11q13 locus of the MEN1 gene for LOH in 21 human pancreatic bcell neoplasms. Initial screening supported a high prevalence of LOH only on chromosome 3p and that region was therefore examined in great detail. The narrowest common segment of deletion was at 3p14 2 3p21, consistent with the presence of an important tumour suppressor gene for human bcell tumours in this region. This region is distinct from the common region of LOH on 3p25 found in various types of islet cell neoplasms by Chung et al. (1997). We did not detect a signficant frequency of LOH in the other syntenic regions. Materials and methods Patients and tumours Tumors from 21 patients with pancreatic bcell neoplasms treated at the University of Cincinnati Medical Center (8 cases), Rhode Island Hospital, Providence, RI (7 cases), Hospital Italiano, Buenos Aires, Argentina (4 cases), and Hospital das Clinicas, Sao Paulo, Brazil (2 cases), were collected for this study. Patient age ranged from 26 to 64 years; there were 13 females and 8 males in this group. Seventeen tumours were sporadic, while 4 patients were diagnosed with MEN1 syndrome. Histological evaluation showed characteristic features of neuroendocrine neoplasms: all tumours revealed positive immunostaining for chromogranin A (Boehringer Mannheim, Indianapolis, IN), and/or synaptophysin (Zymed, San Diego, CA). In addition (cell origin was confirmed by immunoreactivity for insulin (Biogenex, San Ramon, CA). Fifteen tumours were benign, while 6 tumours were diagnosed as malignant based on invasive growth and presence of lymph node and/or liver metastases. All six malignant islet cell tumours were from patients with no history of MEN 1. Paraffin blocks from each case were obtained. The corresponding histological slides stained with haematoxylin and eosin were reviewed microscopically, and blocks containing either tumour tissue or normal pancreatic or lymphatic tissue were chosen for further analysis. In those cases where tumour cells were admixed with normal pancreatic tissue, the surrounding normal tissue was microdissected away from tumour. DNA extraction DNA was extracted from paraffin-embedded tissue as previously reported (Nikiforov et al., 1996). Briefly, 20 mm tissue sections were deparaffinized with xylene, hydrated through graded ethanol solutions and vacuum desiccated. Samples were then incubated in 50 mm Tris, ph 8 0, 1 mm ethylene diaminotetraacetic acid (EDTA), 0 5% sodium dodecyl sulphate (SDS), and 1 mg/ml proteinase K overnight at 55 C. The mixture was then phenol-chloroform extracted. Ammonium acetate (final concentration 0 3 M) was added to the aqueous layer, which was then ethanol precipitated. DNA was pelleted, air-dried and resuspended in water. Microsatellite loci and PCR primers Oligonucleotide primers were obtained from Research Genetics (Huntsville, AL) for the amplification of microsatellite repeats D3S1621, D3S1606, D3S1297, D3S1597, D3S1478, D3S1241, D3S1578, D3S1582, D3S1295, D3S1234, D3S1300, D3S1481, D3S1312, D3S1600, D3S1552, D3S1271, D3S1616, D3S1302, D3S1215, D3S1269, D3S1278, D3S1558, D3S1593, D3S1557, D3S1216, D3S1311, D15S519, D15S87, D6S250, D6S261, D22S343, TOPIP2, D11S913, PYGM, D11S2072. The forward primer was labelled with [ 32 P]dATP using T4 polynucleotide kinase (Promega Co., Madison, WI). Both normal and tumour DNA were subjected to 35 cycles of PCR in a Programmable Thermal Cycler (MJ Research, Inc., Watertown, MA) with the following conditions: 94 C for 45 s, C for 45 s, and 72 C for 1 minute. PCR was performed with 200 ng of genomic DNA as a template, 10 pmol of cold forward primer and 10 pmol of labelled forward primer, 20 pmol of reverse primer, 20 nmol dntps, and 1 25 U of Taq DNA polymerase (Promega Co, Madison, WI) in the PCR buffer (Promega Co, Madison, WI) in a final volume of 20 ml. Detection of allele loss 5 ml of PCR products were mixed with 5 ml of formamide loading dye and were analysed by electrophoresis through an 8% denaturing polyacrylamide gel with urea in 1X TBE (0 089 M Tris Borate ph 8 3, M EDTA) buffer. The intensity of signals in polyacrylamide gels was analysed and quantified using a Phosphoimager (Molecular Dynamics, Sunnyvale, CA) and Image Quant software. Loss of heterozygosity in informative cases was scored when at least a 50% reduction was observed in the allelic ratio between the tumour and normal DNAs from the same patient (Cryns et al., 1995). Results Two microsatellite markers were randomly selected for each of the human chromosomal regions syntenic to mouse loci deleted

3 Loss of heterozygosity in islet cell tumours 29 Fig. 1 Screening for LOH in regions syntenic to mouse loci found to be deleted in animal model of pancreatic bcell tumours. (a) Low prevalence of LOH at 6q: one tumour (#5) out of 7 informative cases shows allelic loss at D6S261. (b) High prevalence of LOH at 3p: 4 tumours (#1, #12, #15, #13) out of 7 informative cases show loss at marker D3S1295. DNA extracted from normal tissue labelled N and that from paired tumour DNA labelled T. in the transgenic model of islet cell tumours (Dietrich et al., 1994; Parangi et al., 1995), and three markers were chosen in close proximity to the recently cloned MEN1 gene at 11q13 (Chandrasekharappa et al., 1997). This initial screening showed low prevalence of LOH on chromosomes 6q12 (Fig. 1), 15q24, and 22q (Table 1). Six of 21 (29%) cases were positive for allelic loss in the MEN1 gene region of 11q13, including three of four tumours from patients with MEN1 syndrome. By Table 1 Results of screening for LOH in human pancreatic bcell tumours Chromosomal Microsatellite Number of tumours with allelic regions markers loss/number informative (%) 3q D3S1215 4/11(36%) D3S1271 3/8 (38%) 3p D3S1606 6/14(43%) D3S1295 8/14(57%) 15q D15S519 0/12 D15S87 0/12 6q D6S250 0/12 D6S261 1/12 (8%) 22q TOPIP2 0/12 D22S343 0/9 11q13 PYGM 5/18(28%) D11S913 4/15(27%) (MEN1 gene) D11S2072 4/13(31%) contrast, there was a high prevalence of LOH on chromosome 3p (Table 1). In order to further explore allelic loss on chromosome 3, 26 polymorphic microsatellite markers on this chromosome were analysed (Table 2). As a result of this screening, 13 of 21 (62%) tumours showed loss of genetic material. In 4 of these cases, the whole chromosome 3 was most likely deleted (cases P5, P7, P13 and P15). In the rest of the positive cases, confined deletions of different regions on 3p were found. The smallest common region of allelic loss was 3p21 1-p14 2 (Fig. 2), corresponding to the marker D3S1295, the only marker that revealed LOH in all 13 positive cases. Interestingly, LOH at 3p14 2 3p21 was present in 8 of 15 (53%) benign tumours, and in 5 of 6 (83%) malignant neoplasms, including two cases where deletion of the same region was detected in both liver metastasis and primary tumour. Of six cases with LOH on 11q13, three sporadic islet cell tumours demonstrated LOH of both 11q13 and 3p, while three tumours from MEN 1 families had deletions of 11q13, but no loss of genetic material on 3p. One sporadic tumour with LOH on 11q13 and 3p was malignant, while other islet cell tumours with or without simultaneous loss of 3p were benign. Discussion LOH studies are a powerful tool to assess the status of particular genes in the development and progression of human neoplasms (Stanbridge, 1990). They have also played an important role

4 30 M. N. Nikiforova et al. Table 2 Summary of allelotyping data for chromosome 3 markers in 21 human pancreatic bcell tumours Microsatellite Chromosomal markers location Tumours D3S1297 3p26 N ni ni ni N LOH N N N LOH ni ni N N N N N N ni N D3S1597 3p24 LOH N LOH LOH ni ni ni N N ni LOH LOH D3S1478 3p21 LOH N ni LOH ni ni N N N N ni LOH N N N N LOH LOH N N N D3S1241 3p21 LOH ni LOH N ni N N ni ni D3S1578 3p21-p14 2 LOH N LOH N LOH N N N N LOH LOH N N ni ni ni N ni D3S1582 3p21 1-p14 2 ni N LOH N N N N ni LOH N ni N LOH LOH ni N D3S1606 3p21 1-p14 2 ni N LOH LOH N ni N N ni LOH N ni N LOH LOH LOH N N D3S1621 3p21 1-p14 2 LOH ni LOH N N N N N ni N LOH ni LOH ni ni N N D3S1295 3p21 1-p14 2 LOH N ni ni ni N N ni LOH LOH LOH ni N ni N LOH LOH LOH LOH N D3S1234 3p14 2 LOH ni ni N LOH N N LOH N N N ni LOH N D3S1300 3p14 2 N N N N ni N N LOH N N N N N N LOH N N D3S1481 3p14 2 ni N ni ni ni LOH ni ni N LOH LOH ni N N N N N N N N N D3S1312 3p14 2 N N ni N ni N N LOH D3S1600 3p14 1 N N ni N N N N N N N N N N N N N N D3S1552 3p11 N ni ni N N ni ni N N N N N N N N N N D3S1271 3q12 N ni ni LOH N ni N N ni N LOH LOH D3S1616 3q13 N N N N N N N N D3S1302 3q13 11 N ni ni N N N ni LOH D3S1215 3q13 12 N N N LOH N LOH N N N ni LOH LOH D3S1269 3q13 13 N N ni ni N N N ni D3S1278 3q13 2 N ni LOH ni LOH N ni ni N LOH ni D3S1558 3q13 3 N N ni LOH ni ni N N N N LOH LOH D3S1593 3q22 N N N N N N N N D3S1557 3q22 N ni N N N ni N ni D3S1216 3q21 25 N ni LOH ni ni N N ni ni LOH LOH N N N N ni ni N N ni D3S1311 3q29 ni N ni LOH N LOH N ni ni ni LOH LOH LOH, informative with allelic loss; ni, uninformative; N, informative with retained alleles.

5 Loss of heterozygosity in islet cell tumours 31 Fig. 2 Representative examples of LOH in human pancreatic bcell tumours at 3p14 2 3p21. in positional cloning of novel tumour suppressor genes, such as the genes for retinoblastoma (Dryja et al., 1986; Friend et al., 1986), Wilms tumour (Call et al., 1990; Gesssler et al., 1990; Rose et al., 1990), and recently, MEN type 1 (Chandrasekharappa et al., 1997). In the present study, we utilized a large number of microsatellite markers to screen human pancreatic bcell tumours for LOH in selected chromosomal regions, and showed that 13 of 21 (62%) neoplasms had chromosomal loss for microsatellite markers on the short arm of chromosome 3. The presence of confined deletions in most of the positive cases allows assignment of the smallest overlapping region of LOH to 3p14 2 3p21. Taking advantage of information available on the precise position and size of polymorphic markers in this chromosomal area (Van den Berg et al., 1996), it is possible to estimate the locus of interest as approximately 2 cm. It has been previously reported that the short arm of chromosome 3 contains several important tumour suppressor genes involved in human carcinogenesis, including the VHL and FHIT genes (Latif et al., 1996; Negrini et al., 1996; Ohta et al., 1996; Shridhar et al., 1996; Sozzi et al., 1996). The FHIT gene, mapped to 3p14 2, is a candidate tumour suppressor gene found to be altered in many human carcinomas including colon (Ohta et al., 1996), pancreatic (Shridhar et al., 1996), breast (Negrini et al., 1996), small cell lung (SCLC) and nonsmall cell lung (NSCLC) (Sozzi et al., 1996). In the original reports, a number of microsatellite markers internal to or flanking the FHIT gene were identified, including D3S1312 (centromeric), D3S1234 (telomeric), and D3S1300 and D3S1481 (internal) (Sozzi et al., 1996). All four of these markers were utilized in our study. Of nine cases with partial deletion of 3p, five tumours demonstrated LOH in at least one of these markers, while in four cases none of the FHIT markers showed LOH. These data suggest that the FHIT gene is unlikely to be a target of genetic loss in the present group of tumours. Recently, Chung et al. (1997) studied 43 pancreatic islet cell tumours, including 17 insulinomas, for LOH at 3p21 3p26, with the primary aim to analyse the status of the VHL gene. This gene was found to be unaffected in that study, while 33% of islet cell tumours had LOH at the region of 3p25. Our findings similarly indicate that the VHL gene does not play a pathogenic role in the development of pancreatic bcell tumours, since loss of genomic material at the VHL locus was found in only 2 of 9 tumours with confined 3p deletion. However, in the present study the most commonly deleted region, located at 3p14 2 3p21, is centromeric to the locus reported by Chung et al. (1997). A number of publications suggest the presence of tumour suppressor gene/s other than VHL or FHIT on chromosome 3p. Thus, three distinct regions on chromosome 3p have been reported to be frequently deleted in oral dysplastic lesions and squamous cell carcinomas, suggesting a role for at least three tumour suppressor genes on the short arm of chromosome 3 in oral carcinogenesis (Prime et al., 1997). In addition, deletions in 3p21-p23 have been found in small cell lung cancer, a poorly differentiated neuroendocrine neoplasm (Mooibroek et al., 1987; Naylor et al., 1987; Johnson et al., 1988; Hibi et al., 1992; Killary et al., 1992). Homozygous deletions at 3p21 were also found in a number of small cell lung cancer cell lines (Daly et al., 1993; Yamakawa et al., 1993), strongly suggesting that this region may contain a novel tumour suppressor gene different from VHL or FHIT. However, no specific candidate has yet been identified. Our data also suggest that the 3p14 2 3p21 region may contain an unknown tumour suppressor gene important in pancreatic bcell tumorigenesis. The higher frequency of LOH observed in this region in malignant (cell tumours as compared to benign ones suggests that this putative tumour suppressor gene may correlate with more malignant disease. This may have clinical prognostic implications. Further studies will be required to narrow the region of chromosomal

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