Frequent overexpression of cyclin D1 in sporadic pancreatic endocrine tumours.

Pancreatic endocrine tumours (PETs) occur sporadically or are inherited as part of the multiple endocrine neoplasia type-1 syndrome. Little is known about the molecular events leading to these tumours. Cyclin D1, a key regulator of the G1/S transition of the cell cycle, is overexpressed in a variety of human cancers as well as certain endocrine tumours. We hypothesized that similar to other endocrine tumours, cyclin D1 is overexpressed in human sporadic PETs. Cyclin D1 protein overexpression was found in 20 of 31 PETs (65%) when compared with normal pancreatic tIssue. Furthermore, Northern blot analysis suggests that cyclin D1 up-regulation occurs at the post-transcriptional level in some PETs. Because the key cell growth signalling pathways p42/p44/ERK (extracellular signal-regulated kinase), p38/MAPK (mitogen-activated protein kinase), and Akt/PKB (protein kinase B) can regulate cyclin D1 protein expression in other cell types, pancreatic endocrine tumours were analysed with phospho-specific antibodies against the active forms of these proteins to elucidate a tIssue-specific regulatory mechanism of cyclin D1 in PETs. We found frequent activation of the p38/MAPK and Akt pathways, but down-regulation of the ERK pathway, in cyclin D1 overexpressing PETs. This study demonstrates that cyclin D1 overexpression is associated with human sporadic PET tumorigenesis, and suggests that this up-regulation may occur at the post-transcriptional level. These findings will direct future studies of PETs towards cell cycle dysregulation and the identification of key growth factor pathways involved in the formation of these tumours.

Molecular and genetic mechanisms of tumorigenesis in multiple endocrine neoplasia type-1.

Multiple endocrine neoplasia type 1 (MEN1) is a rare but informative syndrome for endocrine tumorigenesis. Since its isolation, several groups have begun to determine the role of menin, the protein product of MEN1, in sporadic endocrine tumors as well as tumors of the MEN1 syndrome. Mutations of menin have been reported in more than 400 families and tumors, most of which are truncating mutations, thus supporting the function of menin as a tumor suppressor. The exact function of menin is unknown, but overexpression of menin inhibits proliferation of Ras-transformed NIH3T3 cells. Since menin interacts with proteins from both the TGF beta and AP-1 signaling pathways, perhaps its tumor suppressor function is related to these key cell growth pathways. In this review we will discuss the various clinical manifestations of MEN1 syndrome, potential mechanisms of MEN1 tumorigenesis, and mutations associated with MEN and sporadic endocrine tumors.

Anomalous overexpression of p27(Kip1) in sporadic pancreatic endocrine tumors.

BACKGROUND: Little is known about the cellular defects and molecular mechanisms leading to pancreatic endocrine tumors (PETs). p27(Kip1) is a universal cyclin-dependent kinase inhibitor (CDKI), which acts as a tumor suppressor and a negative regulator of cell cycle. From previous reports, quiescent cells show high levels of p27(Kip1) expression while neoplastic and proliferating cells show no detectable p27(Kip1) expression. We hypothesize that in malignant sporadic PETs, p27(Kip1) expression would be decreased compared with benign PETs and normal pancreatic tissue. METHODS: Western analysis was performed on 28 PETs (7 malignant, 21 benign), 2 nonendocrine cell lines, and 5 endocrine cell lines. Signal intensities were quantitated using densitometry and standardized to normal pancreas. RESULTS: Unexpectedly, increased p27(Kip1) expression as compared with control was seen in both benign and malignant tumors, as well as in all four pancreatic islet tumor cell lines, but not fibroblast or pituitary cell lines, evaluated. There was no difference in p27(Kip1) level between benign and malignant tumors. CONCLUSION: This represents the first report of anomalous p27(Kip1) overexpression in sporadic PETs, and is part of a growing literature describing the paradoxical overexpression of p27(Kip1) in human tumors that includes other endocrine tumors. These studies suggest a unique molecular pathway leading to endocrine tumorigenesis.

Reduced length of stay by implementation of a clinical pathway for bariatric surgery in an academic health care center.

Bariatric surgery is being performed in increasing numbers in an era when reimbursements are being reduced. Academic health centers bear the responsibility for training surgeons to perform these operations yet must keep costs to a minimum and retain high quality. The UCLA Bariatric Surgery Program developed a clinical pathway for the pre- and postoperative management for gastric bypass patients to achieve these goals. Medical records for 182 consecutive gastric bypass patients were retrospectively reviewed before implementation of the pathway (Group I) during the fiscal year of 1998/1999. Data on average length of stay, average intensive care unit length of stay, average standard variable cost, percentage readmission rate, and percentage return to the operating room were collected. This information was compared with the data collected prospectively from 182 patients after implementation of the pathway in July of 1999 (Group II) during the fiscal year of 1999/2000. Hospital cost per admission was reduced by 40 per cent in Group II compared with Group I (P < 0.02). The average length of stay was reduced from 4.05 days in Group I to 3.17 days in Group II (P < 0.033). Overall readmission rate was decreased from 4.2 per cent in Group I to 3.2 per cent in Group II (P < 0.05). There were no differences in morbidities between both groups. The pathway reduced costs by reducing the hospital length of stay, intensive care unit utilization, and readmission rates. Quality was maintained as evidenced by a similar pattern of postoperative morbidities yet readmission rates were reduced. Our results indicate that implementation of a clinical pathway for bariatric surgery reduces cost and improves quality of care in an academic institution.

Deletion of chromosome 1 predicts prognosis in pancreatic endocrine tumors.

Endocrine tumors, such as parathyroid adenomas and pheochromocytomas, frequently have deletions of chromosome 1, suggesting that inactivation of a tumor suppressor gene from chromosome 1 is important in their tumorigenesis. We hypothesized that deletion of chromosome 1 may contribute to pancreatic endocrine tumor formation. Twenty-nine sporadic and MEN1 pancreatic endocrine tumors were studied for loss of heterozygosity (LOH) with 12 chromosome 1 microsatellite markers. LOH on chromosome 1 was identified in 10 of 29 (34%) tumors studied. Allele loss occurred more frequently in tumors with hepatic metastases (7 of 8) than tumors without metastases (3 of 21) (P = 0.004). Tumors in patients with lymph node involvement and patients with multiple endocrine neoplasia type 1 did not demonstrate LOH for chromosome 1 markers. These data suggest that loss of chromosome 1 is associated specifically with the development of hepatic metastases in patients with sporadic pancreatic endocrine tumors.

Mutation of the MENIN gene in sporadic pancreatic endocrine tumors.

Pancreatic endocrine tumors occur both sporadically and as part of the multiple endocrine neoplasia type 1 (MEN1) syndrome. MEN1 is an autosomal dominant disease characterized by parathyroid hyperplasia, pancreatic endocrine tumors, and pituitary adenomas. The MEN1 gene called MENIN maps to chromosome 11q13 and is thought to function as a tumor suppressor gene. We previously demonstrated loss of heterozygosity (LOH) at 11q13 in approximately 40% of sporadic pancreatic endocrine tumors and hypothesize that MENIN is involved in the development of these tumors. Thirty-one sporadic pancreatic endocrine tumors were analyzed for mutation of MENIN by nonradioactive single-stranded conformation polymorphism. Twelve mutations were detected in 31 sporadic pancreatic endocrine tumors (34%). Twelve of these 31 tumors previously demonstrated loss of heterozygosity at 11q13. Of the tumors with LOH, seven contained mutations of the MENIN gene (58%). The majority of the MENIN mutations occurred within exon 2. Two independent mutations in MENIN were detected in a gastrinoma that also revealed LOH, leading to the possibility of another tumor suppressor gene locus at 11q13. Mutations were present in both benign and malignant pancreatic endocrine tumors, suggesting that a MENIN gene mutation is a frequent and early event in the tumorigenesis. The high incidence of truncating mutations in tumors with LOH at 11q13 support the hypothesis that MENIN is a tumor suppressor gene.

Genomic organization and cloning of the human homologue of murine Sipa-1.

Murine Sipa-1 (signal-induced proliferation associated protein) is a mitogen induced GTPase activating protein (GAP). While mapping candidate genes for multiple endocrine neoplasia type 1 (MEN1) at 11q13, we cloned the human homologue of Sipa-1. Herein, we report the complete cDNA sequence, expression, and genomic organization of SIPA-1. SIPA-1 consists of 16 exons with highly conserved exon-intron boundaries. The predicted SIPA-1 protein is highly homologous to the mouse protein, particularly in the region of the GAP-related domain at the amino terminus and the leucine zipper at the carboxy terminus. It is widely expressed, including in fetal tissues, but is most highly expressed in lymphoid organs. During the course of cloning SIPA-1, the MEN1 gene was identified, thus excluding human SIPA-1 as a candidate for this disease.

Deletion mapping of endocrine tumors localizes a second tumor suppressor gene on chromosome band 11q13.

Multiple endocrine neoplasia type 1 syndrome (MEN1, MIM 131100), an autosomal dominant disease, is characterized by parathyroid hyperplasia, pancreatic endocrine tumors, and pituitary adenomas. These tumors also occur sporadically. Both the familial (MEN1) and the sporadic tumors reveal loss of heterozygosity (LOH) for chromosome band 11q13 sequences. Based on prior linkage and LOH analyses, the MEN1 gene was localized between PYGM and D11S460. Recently, the MEN1 gene (menin) has been cloned from sequences 30-kb distal to PYGM. We performed deletion mapping on 25 endocrine tumors (5 MEN1 and 20 sporadic) by using 21 polymorphic markers on chromosome band 11q13. Of these, two (137C7A, 137C7B) were derived from PYGM-containing BAC (bacterial artificial chromosome-137C7) sequences, one from INT2-containing cosmid sequences and the marker D11S4748, a (CA)20 repeat marker that was developed by us. The LOH analysis shows that the markers close to the MEN1 (menin) gene were not deleted in three of the tumors. These tumors, however, showed LOH for distal markers. Thus, the data suggest the existence of a second tumor suppressor gene on chromosome band 11q13.

Tracking the MEN1 gene.

Multiple endocrine neoplasia type 1 (MEN1) is a hereditary autosomal dominant disease characterized by parathyroid hyperplasia, pancreatic endocrine tumors, and pituitary adenomas. Sporadic forms of these tumors are more common than their inherited counterparts and share common genetic abnormalities. We have been studying the molecular genetics of sporadic pancreatic endocrine tumors to identify the tumor suppressor gene responsible for MEN1 by positional cloning. This review introduces the reader to the fundamentals of these molecular genetic techniques and outlines the general strategy used to isolate this gene.

Pancreatic endocrine tumors with loss of heterozygosity at the multiple endocrine neoplasia type I locus.

BACKGROUND: Loss of heterozygosity (LOH) at chromosome 11q13 has been demonstrated in multiple endocrine neoplasia type I (MEN I) and sporadic parathyroid tumors, pituitary adenomas, and a few types of pancreatic endocrine tumors. Gastrinomas are the most common pancreatic endocrine tumor in MEN I. We hypothesized that all pancreatic endocrine tumors have LOH at 11q13, resulting in inactivation of the previously described tumor suppressor gene in this region. METHODS: We analyzed a sporadic gastrinoma, a MEN I-associated gastrinoma, and a nonfunctional pancreatic endocrine tumor from a patient with Von Hippel-Lindau (VHL) disease for LOH at seven loci at 11q13: D11S149, PYGM, D11S427, D11S546, SEA, D11S97, and D11S146. RESULTS AND CONCLUSIONS: We found LOH at 11q13 in all three tumors. The MEN I-associated gastrinoma we analyzed is the first tumor of this type to have LOH. This is also the first report of LOH at 11q13 in a pancreatic endocrine tumor from a patient with VHL. These findings suggest that the etiology of pancreatic endocrine tumor formation involves a common genetic pathway for sporadic, MEN I, and VHL tumors.

Novel p53 mutation in a malignant tumor secreting vasoactive intestinal peptide.

OBJECTIVE: To assess involvement of p53 mutations in development of pancreatic endocrine tumors. DESIGN: Survey of sporadic pancreatic endocrine tumors. SETTING: Hospital referral centers, mainly in the Los Angeles, Calif, area. PATIENTS: We obtained fresh surgical specimens from 25 patients (convenience sample) with no family history of endocrine tumors and no evidence of multiple endocrine neoplasia 1 or von Hippel-Lindau disease. Preoperative tests included serum peptide analysis. MAIN OUTCOME MEASURES: DNA was prepared from tumor specimens. We screened exons 5 through 8 of the p53 gene using single-strand conformation polymorphism analysis, followed by DNA sequencing when a variant was detected. RESULTS: A three-base deletion mutation (codon 239) was found in one malignant tumor secreting vasoactive intestinal peptide. CONCLUSIONS: p53 appears to have a limited role in development of pancreatic endocrine tumors. However, evidence from one of our patients suggests it may be involved in tumor progression in uncommon cases.

A 1.5-megabase physical map encompassing the multiple endocrine neoplasia type-1 (MEN1) locus on chromosome 11q13.

Linkage analysis and loss of heterozygosity studies have shown that the gene responsible for the multiple endocrine neoplasia type-1 (MEN1) syndrome localizes to a small interval between D11S427 and D11S460 on chromosome 11q13. As an initial step to clone this tumor suppressor gene, our group is the first to map the MEN1 region physically using yeast artificial chromosome, bacterial artificial chromosome (BAC), and cosmid contigs. The 1.5-Mb high-resolution, contiguous map extends from PYGM to 300 kb telomeric of D11S460. Of this, the 1.2-Mb interval between PYGM and D11S460 is isolated in cosmids and BACs and will be useful for the development of genomic sequences and transcription maps of this important region. Nine new sequence-tagged sites (STS) are also characterized from this region. The physical map and the STSs will be valuable tools for the cloning of the MEN1 gene.

Putative tumor-suppressor gene on chromosome 11 is important in sporadic endocrine tumor formation.

Endocrine tumors arising sporadically or as a manifestation of the multiple endocrine neoplasia type I syndrome (MEN I) have been shown to have mutations on chromosome 11. These mutations can be detected at the molecular level by loss of heterozygosity (LOH) for DNA markers from chromosome 11. This study represents one of the largest collections of sporadic endocrine tumors in which LOH was systematically assessed on chromosome 11 for the loci flanking the proposed MEN I region. DNA was isolated from 39 endocrine tumors and probed with 7 DNA probes spanning the region of chromosome 11q13 from the loci PYGM to INT-2. Eleven tumors demonstrated LOH at any two loci in this region. The remaining 28 tumors showed no LOH or were noninformative at these loci. Thus, nearly 30% of these tumors showed LOH in the region (from PYGM to INT-2) that is thought to contain the MEN I gene(s). Previous studies of sporadic endocrine tumors have suggested that these tumors may arise via the same mechanism as tumors of the MEN I syndrome. Namely, these sporadic tumors are thought to result from mutations leading to genetic loss on the long arm of chromosome 11, thereby inactivating a possible tumor-suppressor gene (or genes). These findings strongly support the hypothesis that sporadic pancreatic endocrine tumors share a similar etiology of tumorigenesis with tumors of the MEN I syndrome, which principally involves deletion of a tumor-suppressor element (or elements).

Molecular biology and therapy of disease.

Molecular biology will have a profound impact upon the treatment of disease. Molecular techniques provide protein products for treatment of more diseases each year. The understanding of pathophysiology at the molecular level allows for improved drug design. Antisense technology can selectively control gene expression. Gene therapy is potentially the most important aspect of molecular biology. Physical and viral transduction mechanisms are being developed toward this end. Gene replacement, creation of antisense oligonucleotides, and prodrug strategies are being developed. Currently, gene replacement and prodrug therapy are feasible in at least a few cases, but further study will yield additional applications.

A review of gene transfer techniques.

The ability to transfer genetic material from one cell to another provides a novel approach for the investigation and potential treatment of a variety of diseases. Several gene transfer techniques have been developed. Of these, the use of retroviruses as vectors has allowed highly efficient and stable introduction of foreign genes into target mammalian cells, thus making gene therapy possible. The current prospects are that some unusual lethal diseases can be corrected and that various forms of cancer will be treated in this manner.

Human Genome Project.

The Human Genome Project is an international effort to clone and sequence the entire human genome. This audacious undertaking, estimated to cost 200 million dollars per year and require 15 years to complete, promises to be one of the most revolutionary and captivating scientific endeavors ever conceived by mankind. By knowing the sequence of the estimated 3 billion base pairs of the haploid human genome and its more than 30,000 genes, many questions will be answered. Moreover, our ability to intervene at the genetic level will be possible. This review outlines the scientific goals and methods of this project and discusses some of its ethical, legal, and social ramifications.

Biologic behavior of sporadic gastrinoma located to the right and left of the superior mesenteric artery.

Among a series of 107 closely followed patients with gastrinoma, 60 patients with sporadic type tumors were identified and evaluated. There were 44 patients (73%) with tumors to the right of the superior mesenteric artery (SMA). Of these, 16 (36%) had extrapancreatic tumors, 28 (64%) had tumor within lymph nodes, and 9 (20%) had multiple tumors. In this group of patients, there were 19 (43%) cures, and only 9 (20%) patients had hepatic metastases. In contrast, in 16 patients (27%) with tumors to the left of the SMA, there were no extrapancreatic tumors, only 3 patients (19%) had tumor within lymph nodes, and 7 (44%) had multiple tumors. In this group, there was only one cure (6%), and nine (56%) patients had hepatic metastases. These findings suggest two distinct populations of sporadic gastrinoma, one to the right (gastrinoma triangle) and the other to the left (outside triangle) of the SMA, which appear to have different biologic behaviors. These differences may reflect divergent etiologies for these two groups of tumors.

Loss of heterozygosity on chromosome 11 in sporadic gastrinomas.

Gastrinomas are pancreatic endocrine neoplasms that arise either sporadically or are inherited as part of the multiple endocrine neoplasia type I syndrome (MENI). Loss of heterozygosity (LOH) in the region flanking the MENI gene at chromosome 11q13 has been documented in a few sporadic and familial pancreatic endocrine tumors, but not previously in sporadic gastrinomas. It has therefore been suggested that gastrinomas develop by a mechanism different from other tumors associated with the MENI syndrome. We report LOH on chromosome 11 in 5 of 11 sporadic gastrinomas. Four of these tumors have LOH for markers flanking the MENI region. Molecular evaluation of segments of chromosomes 3, 13, and 17 known to contain cloned or putative tumor suppressor genes fail to show LOH except at one locus in one tumor. These data suggest that a tumor suppressor DNA segment exists at 11q13 that may be involved in the development of sporadic gastrinomas.

The dichotomous distribution of gastrinomas.

Our experience with 47 sporadic gastrinomas suggests that no less than 85% of these tumors are located to the right of the superior mesenteric artery. This finding is unexpected because approximately 75% of insulinomas and glucagonomas are located to the left of the superior mesenteric artery. All of our extrapancreatic gastrinomas have been located to the right. These observations prompted us to determine if other extrapancreatic gastrinomas were also predominantly located to the right side. We searched the world's literature and found 10 cases of ovarian gastrinomas and one case of a renal gastrinoma. Nine of these remote extrapancreatic gastrinomas were located on the right side. This distribution of remote extrapancreatic gastrinomas is similar to our experience with peripancreatic gastrinomas. This unexpected right-sided preponderance of both remote and peripancreatic gastrinomas suggests a common origin for both.