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A Novel Insulinoma Tumor Suppressor Gene Locus on Chromosome 22q with Potential Prognostic Implications

Departments of General Surgery (A.W., P.L., B.C., D.K.B.) and Pathology (A.R.), Philipps University of Marburg, D-35043 Marburg, Germany

Address all correspondence and requests for reprints to: Detlef K. Bartsch, M.D., Department of General Surgery, Philipps University of Marburg, Baldingerstrasse, D-35043 Marburg, Germany. E-mail: bartsch{at}mailer.uni-marburg.de

Abstract

The molecular mechanisms contributing to the tumorigenesis of insulinomas are still poorly understood. As moderate to high rates of LOH have been found on chromosome 22q in gastrinomas, we performed a finer deletion mapping study of chromosome 22q with 8 microsatellite markers in 15 insulinomas (4 malignant and 11 benign). Fourteen of 15 (93%) insulinomas revealed LOH on chromosome 22q, whereas the shortest region of overlap implicated a deletion of approximately 700 kb at 22q12.1-q12.2 with an LOH rate of up to 57% (8 of 14). Although the expressed sequence tag marker A006E25 that is localized in the hSNF5/INI1 gene on 22q11.2 revealed LOH in 50% of informative cases (7 of 14), no alterations in this gene could be identified by single strand conformational polymorphism analysis, direct DNA sequencing, or RNA expression analysis. Remarkably, the four malignant tumors showed a common deleted region between markers D22S345 and D22S1144 compared with none of the 11 benign insulinomas.

The observed high frequency of chromosome 22q12 deletions in insulinomas is suggestive for a region compatible with harboring a tumor suppressor gene. The hSNF5/INI1 gene is most likely not the candidate gene, because no alterations could be identified. The distinct pattern of allelic loss identified in this chromosomal region appears to be an attractive candidate marker for further evaluation with regard to the discrimination between benign and malignant insulinomas.

PANCREATIC ENDOCRINE TUMORS (PETs) arise from the pancreatic islet cells and belong to the amine and precursor uptake and decarboxylation system. They are rare, accounting for only 1–5% of pancreatic tumors. Insulinomas arising from the ß-cells are the most common type, representing approximately 70% of PETs. Most insulinomas are less than 2 cm in size, and only 10% are malignant (1). However, to determine malignancy in these tumors by histopathology is occasionally very difficult, making genetic markers that could reliably indicate malignancy very valuable. About 10–15% of insulinomas are associated with the autosomal dominant inherited MEN1, a tumor syndrome that predisposes to develop PETs (2). The genetic defects that have been most frequently identified in sporadic human insulinomas include somatic MEN1 gene mutations in 17% of the cases (3). LOH including the region of the MEN1 gene was found in 50% of sporadic insulinomas (4, 5). Most studies of the K-ras oncogene and the TP53 tumor suppressor gene have failed to implicate a role for these two genes in the pathogenesis of insulinomas (6, 7). However, in one small study four of six malignant and two of eight benign insulinomas revealed K-ras mutations at codon 12 (8). No alterations of the DPC4/Smad4 tumor suppressor gene were identified in insulinomas, in contrast to a mutation rate of 55% in nonfunctioning endocrine pancreatic carcinomas (9). Although inactivation of the p16^INK4a tumor suppressor gene by either hypermethylation or homozygous deletion was identified in up to 90% of gastrinomas and nonfunctioning neuroendocrine tumors (10, 11, 12), alterations of the p16^INK4a gene occur in only a small subset of insulinomas (13). Genome-wide allelotyping of PETs revealed significant allelic losses on chromosomal arms 3p, 3q, 11p, 11q, 16p, and 22q, but very few insulinomas were included in the study (14). The allelic deletion of chromosome 22q is a common somatic alteration in a variety of human tumors, such as breast (15), colorectal (16), ovarian (17), and brain (18) cancers. There are a number of candidate tumor suppressor genes on 22q, including the neurofibromatosis type 2 gene (NF2) and the hSNF5/INI1 gene, which are reported to play significant roles in the tumorigenesis of different types of cancer (19, 20, 21). Therefore, we performed for the first time a fine deletion mapping of chromosome 22q and a mutation analysis of the hSNF5/INI1 gene in human insulinomas.

Materials and Methods

Patients and tissue samples

Fifteen fresh-frozen insulinoma specimens were obtained from the tumor bank of the Department of General Surgery, Philipps University of Marburg (Marburg, Germany). Fourteen cases were sporadic, and one patient had MEN1 endocrinopathy (Table 1). The diagnosis of insulinoma required symptomatic hypoglycemia (<40 mg/dl) with concomitant endogenous hyperinsulinemia (>0.12 nmol/liter) during a supervised fast and positive immunohistochemistry of the tumor for insulin. Four insulinomas were unequivocally malignant as determined by the presence of metastases or infiltrating growth. Clinical follow-up was obtained through the patient’s personal physician or at out-patient attendance. Survival was calculated from the time of surgical resection to either death or most recent contact. Informed consent was obtained by all patients. All investigations and all patient material in this study were assessed under a research protocol approved by the Philipps University of Marburg ethic committee. The tumor samples used for DNA isolation had a neoplastic cellularity between 85–95% after microdissection, whereas constitutional normal DNA was derived from blood lymphocytes. Genomic DNA from tissue and blood samples was isolated using the QIAamp DNA kits (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. Total RNA from tumor tissue was extracted with the TRIzol kit as recommended by the manufacturer (Life Technologies, Inc., Karlsruhe, Germany).

Immunohistochemical stainings for Ki-67, proinsulin, and chromogranin A were performed by a standard avidin-biotin-peroxidase complex technique using commercially available antibodies and following standard methods (22, 23). Staining results were interpreted by an experienced pathologist (A.R.) without knowledge of clinical parameters and LOH data. The Ki-67 results were expressed as the percentage of positive cells. The remaining stainings were graded as follows: -, negative; +, less than 10% positive cells; 2+, 10–50% positive cells; and +++, more than 50% positive cells.

LOH analysis

Fluorescence-based LOH analysis was performed on a 310 Genetic Analyzer (PE Applied Biosystems, Foster City, CA). The location and sequence of seven highly polymorphic microsatellite markers on 22q (D22S351, D22S345, D22S929, D22S1174, D22S1167, D22S1144, and D22S280) and one expressed sequence tag (EST) marker (A006E25), localized in the hSNF/INI1 gene were confirmed by the published National Center for Biotechnology Information chromosome 22q sequences and are available at http://www.ncbi.nlm.nih.gov. In addition, LOH on chromosome 3p and 11q13 were determined by microsatellite markers D3S1295, D3S1110, and D11S4946, because they might provide prognostic information (5, 24, 25). PCR amplification was performed with fluorescence-labeled oligonucleotides on a Primus 25 PCR cycler (MWG Biotech, Ebersberg, Germany) with a standard protocol. LOH was defined as a reduction in intensity of 50% or more in either of the two alleles compared with those in constitutional normal DNA by semiquantitative analysis on a 310 Genetic Analyzer (PE Applied Biosystems). Data were analyzed using Genescan 2.1.1 software (PE Applied Biosystems).

Single strand conformational polymorphism (SSCP) analysis

The oligonucleotides for SSCP analysis of exons 1–9 of the hSNF5/INI gene have previously been published (20) with the exception of exon 7 (Ex7.1 forward, 5'-GACAAGGACCACATGCAG-3'; Ex7.1 reverse, 5'-GCTCCGAGCACAGCTTCAG-3'; Ex7.2 forward, 5'-GCCTCAGCTGAACATCCATG-3'). PCR amplification and SSCP analysis were performed as described previously (26). Variant DNA bands were confirmed in several independent PCR reactions under at least two different gel conditions, then reamplified by PCR and directly sequenced by Taq cycle sequencing (ABI Prism BigDye Terminator kit, PE Applied Biosystems). Data were analyzed using Sequencing Analysis 3.0 and Sequence Navigator 3.01 software (PE Applied Biosystems).

RT-PCR of hSNF5/INI1

For two insulinomas with LOH in the hSNF5/INI1 locus enough tumor material was available for total RNA preparation. RT-PCR analysis was performed with the One Step RT-PCR kit as described by the manufacturer’s protocol (Life Technologies, Inc.). The following oligonucleotides were used for RT amplicon 1 (exons 1–5): SNF5-RT-1 forward, 5'-CGCTGAGCAAGACCTTCGGGC-3'; and SNF5-RT-5 reverse, 5'-CGATCTCCATGTCCAGCCGG-3' producing a 567-bp product; and for RT amplicon 2 (exons 4–9): SNF5-RT-4 forward, 5'-CAGCTCCCACCACTTAGATGC-3'; and SNF5-RT-9 reverse, 5'-CGATCCTCAGGATGGCGC-3', coding for a 856-bp amplicon. A 300-bp glyceraldehyde-3-phosphate dehydrogenase amplicon was amplified as a positive control for every reaction. The products were visualized on polyacrylamide mini gels, followed by ethidium bromide staining.

Results

Four of 15 insulinomas showed clear evidence of malignancy by infiltrating overgrowth and/or metastases. As in the absence of local invasiveness or metastases, there are no unequivocal clinical or histopathological methods to classify insulinomas, we evaluated some indirect indicators of the phenotype. Immunohistochemistry of Ki-67 showed a high index of expression in all 4 malignant insulinomas, but in only 2 of 9 tumors without signs of malignancy. Chromogranin A and proinsulin were expressed in moderate to high levels in all insulinomas (Table 1). LOH on chromosome 3p was detected in 3 of the malignant tumors compared with 2 of 11 insulinomas without signs of malignancy. 11q13 allelic loss was observed in 2 of 3 informative malignant insulinomas and in 2 of 9 informative tumors with a potentially benign phenotype.

The eight chromosome 22q markers used in this study are localized on chromosomal bands 22q11.2 to 22q13.1 and are distributed over a genomic region of 16 Mb according to the published sequence data of chromosome 22q (GenBank accession no. NT_001454; http://www.sanger.ac.uk/HGP/Chr22/). Fourteen of 15 insulinomas (93%) revealed LOH of chromosome 22q. LOH results of chromosome 22q are visualized in Fig. 1, and examples of LOH are presented in Fig. 2. Two of the chosen markers are associated with different candidate gene regions on chromosome 22q formerly identified to be involved in the tumorigenesis of different types of cancer. The EST marker A006E25 is localized in the tumor suppressor gene hSNF5/INI1 on 22q11.2, and D22S929 represents an intragenic microsatellite marker in the NF2 gene on 22q12.3. They revealed LOH in 50% (7 of 14) and 40% (6 of 15) of informative cases, respectively. No homozygous deletions in 22q were observed as described by other groups in some types of cancer (20). The smallest common region of LOH centered at chromosome 22q12.1-q12.2, corresponding to markers D22S1167 and D22S1144, was localized in between approximately 700 kb. Remarkably, the 4 malignant tumors showed a common deleted region of about 3.5 Mb that involved markers D22S345, D22S1174, D22S1167, and D22S144, respectively (Fig. 1). In contrast, benign insulinomas showed at most 2 deleted markers in this region. Thus, 100% (4 of 4) of the malignant insulinomas showed this specific LOH pattern compared with none of the 11 benign tumors.

View larger version (28K): [in this window] [in a new window]   Figure 1. Results of LOH analysis on chromosome 22q in insulinomas. , LOH; , retention of both alleles; NI, not informative. The black bar shows the smallest common region of deletion in 22q12.1-q12.2, whereas the boxes indicate the common specific LOH pattern in malignant insulinomas.

View larger version (16K): [in this window] [in a new window]   Figure 2. LOH analysis for the markers A006E25 and D22S1144. T, tumor tissue; N, corresponding normal tissue. Arrows indicate the deleted alleles. The intern size length standard is given as gray numbers. A, LOH analysis of the EST marker A006E25 localized in 22q11.2 in tumor and in normal DNA of patient SW151222. B, LOH analysis of the polymorphic marker D22S1144 on 22q12.2 in tumor and normal DNA of patient 168/98.

View larger version (37K): [in this window] [in a new window]   Figure 3. SSCP analysis and sequence analysis of exon 7 of the hSNF5/INI1 gene in insulinoma patient 79/97. Variant bands and nucleotides are indicated by arrows. A, SSCP analysis of exon 7 of the hSNF5/INI1 gene. 79/97, Patient’s sample with variant bands; 101/97, patient’s sample with normal band pattern; WT1 to WT3, different control samples of healthy probands. WT3 showed the same band pattern as patient 79/97, indicating a benign polymorphism. B, Sequence analysis of exon 7 of the hSNF5/INI1 gene. The wild-type DNA (WT) sequence (TCG) is altered in the DNA of patient 79/97 on nucleotide position 966 by a transition from a guanine to an adenine residue, resulting in a silent sequence alteration (c.966G > A, S309S).

View larger version (30K): [in this window] [in a new window]   Figure 4. Sample for RT-PCR analysis of hSNF5/INI1 gene expression. A, RT-PCR analysis is shown for RT amplicon 1 of the hSNF5/INI1 transcript. Lane M contains the length standard (50-bp ladder). Lanes 1 and 2 contain the RT amplicons for tumor specimens F11.T and SW151222.T, respectively. Both demonstrated LOH in the intragenic EST marker A006E25, but showed normal transcript length compared with the transcript from wild-type RNA in lane 3. Control for DNA contamination in wild-type RNA was loaded in lane 4; lane 5 contains the negative control. B, Control RT-PCR with the glyceraldehyde-3-phosphate dehydrogenase gene transcript of 300 bp length.

LOH studies are a powerful tool to assess the status of particular genes in the development of human neoplasms (27). They have also played an important role in positional cloning of novel tumor suppressor genes, such as the retinoblastoma gene (28), the DPC4/Smad4 gene (29, 30), and the MEN1 gene (31). In this study we showed that 93% (14 of 15) of insulinomas had allelic loss on the long arm of chromosome 22. The presence of confined deletions in most of the positive cases allows assignment of the smallest common region of loss to 22q12.1-q12.2 corresponding to markers D22S1167 and D22S1174. This smallest overlapping region is located telomeric to the recently cloned tumor suppressor gene hSNF5/INI1 and centromeric to the NF2 gene. The LOH rate in this region was at most 57% at marker D22S1174, which represents the highest allelic loss in insulinomas reported to date despite an approximately high LOH rate of 55% at the MEN1 locus (4, 5). Previous reports of chromosome surveys in PETs identified no to only moderate (29%) LOH on chromosome 22q (14, 25, 32). There are some possible explanations for this discrepancy. First, the earlier studies did not extensively use polymorphic microsatellite markers, which significantly enhance the sensitivity of this type of analysis. Second, only 2 markers per chromosomal arm were used in previous studies, and this limited the chances of finding LOH. Considering that insulinomas are characterized by relatively low fractional allelic loss of 0.07 (14), our finding is suggestive for the presence of one or more tumor suppressor genes at 22q12.1-22q12.2 that might be important for the tumorigenesis of insulinomas. Taking advantage of the availability of the complete human chromosome 22 sequence (33) and the information on the precise position of polymorphic markers, it is possible to estimate the locus of interest as approximately 700 kb.

It has been previously reported that the long arm of chromosome 22q contains some important tumor suppressor genes involved in human tumorigenesis, including the NF2 and hSNF5/INI1 genes. Somatic mutations in the NF2 gene on 22q12.3 have been identified in neurofibromatosis type 2- related tumors, such as sporadic meningiomas and vestibular schwannomas (19). As 40% of insulinomas revealed LOH at the NF2 locus, this gene needs to be analyzed in the future to determine its role in the development of insulinomas. The tumor suppressor gene, hSNF5/INI1 is located on 22q11.2 and involved in the regulation of gene expression (34). Numerous loss of function mutations and homozygous deletions have been identified in malignant rhabdoid tumors as well as in primitive neuroectodermal tumors of the central nervous system and medulloblastomas (20, 21, 35, 36, 37). Lung and breast cancers, Wilms’ tumor, sarcomas, and chronic myeloid leukemias did not show any hSNF5/INI1 alterations, although some of those revealed allelic loss at this gene locus (20, 21, 36, 38, 39, 40). As the deleted region in some insulinomas did include hSNF5/INI1 and because the LOH rate at this gene locus was 50%, hSNF5/INI1 was a compelling candidate for mutation analysis. However, SSCP analysis of all 9 coding exons including splice junctions could not detect any relevant mutations in the 15 insulinomas. RT-PCR analysis in 2 tumors with LOH at the hSNF5/INI locus revealed intact expression of hSNF5/INI1 gene compared with the expression in control RNAs. Thus, hSNF5/INI1 does not appear to play an important role in the tumorigenesis of insulinomas.

About 5–10% of insulinomas are malignant, even when the tumors are small. Currently there is no histopathological feature other than the presence of gross metastases or infiltrating growth that can reliably distinguish benign from malignant insulinomas. High expression of proinsulin and chromogranin A are discussed to indicate malignant tumors (5), but our data and the results of other groups (23, 41, 42) revealed no correlation between a particular expression level and malignancy in insulinomas. As previously reported (43) we also confirmed a higher Ki-67 index in malignant insulinomas (mean, 7.2%) than in potentially benign tumors (mean, 1.5%). In our study allelic loss at chromosome 22q11.2-q12.3 appeared to be associated with the malignant phenotype, as all 4 malignant insulinomas revealed a distinct LOH pattern with allelic loss of markers D22S345 to D22S1144 compared with none of the 11 benign insulinomas. However, large scale studies are warranted to clarify this observation. Previously, LOH on chromosome 3p has been also proposed as potential marker for malignancy in PETs, including insulinomas (5, 24, 25). Chung and collaborators (24), for example, found LOH on chromosome 3p in 3 of 3 malignant and in only 1 of 14 tumors with a benign phenotype. The presented data confirm the potential prognostic value of 3p allelic loss, as 3 of 4 malignant insulinomas revealed LOH of 3p compared with only 2 of 11 potentially benign tumors. A recent study by Hessman and co-workers (5) suggested that allelic loss on chromosome 11q13 is also associated with the malignant phenotype of nonfamilial PETs. We detected LOH on chromosome 11q13 in 2 of 4 malignant and 2 of 7 informative benign tumors. Thus, these results can neither confirm nor reject such an association due to the small number of informative tumors in our study. Although the genotyping of PETs appears to be a promising approach for the identification of markers that might reliably indicate malignancy, these associations await confirmation in carefully designed controlled, large scale studies. If these data can be confirmed, it might be an important goal of the future to characterize PETs, including insulinomas, not only according to histopathology but also by genotype.

In summary, the high frequency of chromosome 22q12 deletions in insulinomas indicates a locus compatible with harboring one or more tumor suppressor genes that may be associated with the development and/or progression of these tumors. The hSNF5/INI1 gene as one of the potential candidates in this region does not appear to play an important role in the molecular pathogenesis of insulinomas. Studies of a larger population of these tumors are warranted to determine whether a distinct pattern of allelic loss in this region may be a reliable molecular marker that helps to distinguish benign from malignant insulinomas.

Acknowledgments

We are grateful to all patients for their participation in the study.

Footnotes

This work was supported by the Deutsche Krebshilfe (Grant no. 10-1674-BaI).

Abbreviations: EST, Expressed sequence tag; PET, pancreatic endocrine tumor; SSCP, single strand conformational polymorphism.

Received January 19, 2001.

Accepted September 6, 2001.

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