Genetic and epigenetic alterations of familial pancreatic cancers.

BACKGROUND: Little is known about the genetic and epigenetic changes that contribute to familial pancreatic cancers. The aim of this study was to compare the prevalence of common genetic and epigenetic alterations in sporadic and familial pancreatic ductal adenocarcinomas. METHODS: DNA was isolated from the microdissected cancers of 39 patients with familial and 36 patients with sporadic pancreatic adenocarcinoma. KRAS2 mutations were detected by BstN1 digestion and/or cycle sequencing. TP53 and SMAD4 status were determined by immunohistochemistry on tissue microarrays of 23 archival familial pancreatic adenocarcinomas and in selected cases by cycle sequencing to identify TP53 gene mutations. Methylation-specific PCR analysis of seven genes (FoxE1, NPTX2, CLDN5, P16, TFPI-2, SPARC, ppENK) was done on a subset of fresh-frozen familial pancreatic adenocarcinomas. RESULTS: KRAS2 mutations were identified in 31 of 39 (80%) of the familial versus 28 of 36 (78%) of the sporadic pancreatic cancers. Positive immunolabeling for p53 was observed in 57% of the familial pancreatic cancers and loss of SMAD4 labeling was observed in 61% of the familial pancreatic cancers, rates similar to those observed in sporadic pancreatic cancers. The mean prevalence of aberrant methylation in the familial pancreatic cancers was 68.4%, which was not significantly different from that observed in sporadic pancreatic cancers. CONCLUSION: The prevalence of mutant KRAS2, inactivation of TP53 and SMAD4, and aberrant DNA methylation of a seven-gene panel is similar in familial pancreatic adenocarcinomas as in sporadic pancreatic adenocarcinomas. These findings support the use of markers of sporadic pancreatic adenocarcinomas to detect familial pancreatic adenocarcinomas.

Synchronous and metachronous extrapancreatic malignant neoplasms in patients with intraductal papillary-mucinous neoplasm of the pancreas.

BACKGROUND/AIMS: Patients with intraductal papillary-mucinous neoplasm (IPMN) of the pancreas are likely to have a better prognosis than those with conventional pancreatic ductal adenocarcinoma. Recently there have been some reports on extrapancreatic malignant neoplasms (EPM) occurring in patients with IPMN. The purpose of this study was to discover the characteristic features of IPMN with EPM compared with IPMN without EPM. METHODS: 61 patients with IPMN who underwent surgery at Tohoku University Hospital between 1988 and 2006 were retrospectively analyzed. RESULTS: The 61 patients with IPMN in this study comprised 25 with intraductal papillary-mucinous adenomas (IPMA) and 36 with intraductal papillary-mucinous carcinomas (IPMC) including 6 with invasive carcinomas. Synchronous and metachronous EPM were observed in 15 out of the 61 patients (24.6%). Three of these patients, including 2 with IPMA and 1 with invasive carcinoma associated with IPMC, died of the EPM. None of the features, including sex, age, smoking, family history, macroscopic types (main duct type or branch duct type), histological types (gastric, intestinal, pancreatobiliary or oncocytic), and aberrant expression of molecules including CDKN2A, TP53, SMAD4 and DUSP6, except for the histological diagnoses were associated with the occurrence of EPM, i.e., the EPM occurred more often in patients with IPMA (10 out of 25) than in those with IPMC (5 out of 36) in our series (p = 0.0199 by the chi(2) test, p = 0.0330 by Fisher's exact probability test, p = 0.0422 by Yates' correction). CONCLUSION: Patients with IPMA were more likely to have EPM than those with IPMC. Patients with IPMA are usually expected to have a fair prognosis but EPM could be fatal in some of them, so it must be noted during follow-up.

Sensitive and quantitative detection of KRAS2 gene mutations in pancreatic duct juice differentiates patients with pancreatic cancer from chronic pancreatitis, potential for early detection.

KRAS2 gene mutations are found in 75-90% of infiltrating pancreatic ductal adenocarcinomas but can also be present with other nonneoplastic pancreatic diseases. We recently developed a novel sensitive assay for point mutation detection, called "LigAmp", which can detect one mutant molecule in the presence of 10,000 wild-type molecules and can quantify mutant DNA over a wide dynamic range. We analyzed KRAS2 mutations in surgically-collected pancreatic duct juice samples from patients with pancreatic adenocarcinoma (n = 27) and chronic pancreatitis(n = 9). DNA sequencing demonstrated that 17 of the 27 pancreatic cancers harbored KRAS2 mutations at codon 12, including G12D (GGT-->GAT), G12V (GTT), and G12R (CGT). We determined the relative amounts of each KRAS2 mutant by simultaneously quantifying wild-type and mutant KRAS2 DNA. For all pancreatic adenocarcinoma patients, the dominant KRAS2 mutation detected in the pancreatic juice corresponded to that found in the primary cancer. Mutation levels were substantially higher in patients with pancreatic cancer (0.05 to 82% of total KRAS2 molecules) compared to those with chronic pancreatitis (0 to 0.7%). Among patients with mutant KRAS2 positive cancers, all but one (94%) had mutant KRAS2 DNA concentrations of more than 0.5% in their pancreatic juice samples, whereas only 1 of 9(11%) pancreatic juice samples from patients with chronic pancreatitis had more than 0.5% mutant KRAS2 DNA, corresponding to a sensitivity of 94% and a specificity of 89%. LigAmp quantification of mutant KRAS2 in pancreatic juice differentiates pancreatic adenocarcinoma from chronic pancreatitis, and may be a useful early detection tool for pancreatic cancer.

Characteristic clinicopathological features of the types of intraductal papillary-mucinous neoplasms of the pancreas.

OBJECTIVES: Intraductal papillary-mucinous neoplasm (IPMN) of the pancreas encompasses a spectrum of neoplasms with both morphological and immunohistochemical variations of mucin glycoproteins. Recently, a consensus nomenclature and criteria were histologically defined for classifying these variants of IPMNs into gastric, intestinal, pancreatobiliary, and oncocytic types. The purpose of this study was to determine associations between the histological types and clinicopathological features in patients with IPMN. METHODS: Sixty-one patients with IPMN operated upon at Tohoku University Hospital between 1988 and 2006 were retrospectively analyzed. RESULTS: Our series included 27 gastric-, 29 intestinal-, 4 pancreatobiliary-, and 1 oncocytic-type IPMNs. Statistically, the types of IPMN were significantly associated with the histological diagnoses, macroscopic types, and survival of the patients. Characteristically, the gastric-type IPMNs were likely to be diagnosed as benign, to be confined to branch ducts, and to have fair prognoses. On the other hand, the intestinal-type IPMNs were likely to be diagnosed as malignant, occupy the main duct, and have poor prognoses. Because of the small number of pancreatobiliary-type IPMNs and only 1 case of oncocytic-type IPMN, we were unable to determine any of their clinicopathological characteristics in our series. CONCLUSIONS: Evaluation of the histological types of IPMN may help to predict the clinical course of patients with IPMN and to design improved clinical management for these patients.

Genome-wide allelotypes of familial pancreatic adenocarcinomas and familial and sporadic intraductal papillary mucinous neoplasms.

PURPOSE: Most familial cancer susceptibility genes are tumor suppressor genes that are biallelically inactivated in familial neoplasms through somatic deletion of the wild-type allele. Identifying the genomic losses that occur in pancreatic neoplasms, particularly those that occur in familial and precursor neoplasms, may help localize the genes responsible for pancreatic cancer susceptibility. EXPERIMENTAL DESIGN: Normal and neoplastic tissue DNA was isolated from fresh-frozen surgically resected tissues from 20 patients with primary familial pancreatic adenocarcinoma (defined as having at least one first-degree relative with pancreatic cancer), 31 with sporadic intraductal papillary mucinous neoplasms (IPMN), and 7 with familial IPMNs using laser capture microdissection. Microdissected DNA was whole genome amplified using multiple strand displacement. Genome-wide allelotypes were determined using 391 microsatellite markers. The accuracy of microdissection and fidelity of the whole genome amplification were determined by comparing the genotypes of microdissected primary pancreatic cancers to the genotypes of xenografts derived from these cancers and by comparing the results of amplified to nonamplified specimens. RESULTS: The concordance of genotypes between LCM whole genome amplified primary pancreatic cancers and their corresponding pancreatic cancer xenograft DNAs was 98%. Among the 20 primary familial pancreatic adenocarcinomas, we found a high prevalence of loss of heterozygosity (LOH) with an average fractional allelic loss (FAL) of 49.9% of an aggregate of 2,378 informative markers. The level of FAL in the IPMNs (10%) was significantly lower than in the pancreatic adenocarcinomas. The most common locus of LOH in the IPMNs was at 19p (LOH at 24% of markers). The regions of frequent allelic loss observed in the familial pancreatic cancers were similar to those found in sporadic pancreatic cancers. CONCLUSIONS: The allelic loss patterns of familial and sporadic pancreatic cancers and IPMNs provide clues as to the genomic locations of tumor suppressor genes inactivated in these neoplasms.

Intraductal papillary-mucinous adenocarcinoma in the remnant pancreas after pancreatoduodenectomy for cancer of Vater's papilla associated with intraductal papillary-mucinous adenoma.

A 72-year-old woman, who had undergone pylorus-preserving pancreatoduodenectomy 3 years before for cancer of Vater's papilla associated with a branch-type intraductal papillary-mucinous adenoma (IPMA), developed dilatation of the main duct and a nodular lesion in the remnant pancreas. Total pancreatectomy was performed, which revealed that the lesion was intraductal papillary-mucinous adenocarcinoma (IPMC) with minimal invasion, suggesting the metachronous multicentric occurrence of this intraductal papillary-mucinous neoplasm (IPMN). Because there were no malignant cells at the pancreaticojejunostomy, and because the histological appearance of the main-duct IPMC was different from that of the IPMA in the primary specimen, the main-duct IPMC was thought to be of different origin from the IPMA. These findings suggest that careful surveillance of the gastrointestinal tract and careful follow up are necessary for IPMN, because an IPMN could be associated with other gastrointestinal tract malignancies.

Detecting low-abundance p16 and p53 mutations in pancreatic juice using a novel assay: heteroduplex analysis of limiting dilution PCRs.

We have developed a simple, robust, highly-sensitive assay for identifying gene mutations in clinical samples. We applied this assay to detect p53 and p16 mutations in pancreatic juice obtained from patients undergoing evaluation and treatment of pancreatic disease. The assay strategy involves PCR amplifying DNA at limiting dilution (LD-PCR) followed by screening PCR products for mutations using temperature gradient capillary electrophoresis (TGCE). Compared to conventional TGCE, TGCE after LD-PCR significantly increased the number of detectable mutations in pancreatic duct juice. Using LD-PCR, mutations in p53 and/or p16 were found in the pancreatic juice of 12 of 20 individuals with pancreatic cancer compared to only 1 of 8 patients with chronic pancreatitis, 0 of 8 individuals without evidence of pancreatic disease (p<0.02). We conclude that limiting dilution PCR is an effective strategy for improving the detection of mutations in clinical samples and when applied to pancreatic juice to detect mutations of p53 and/or p16, it can help distinguish patients with pancreatic cancer from those without evidence of pancreatic neoplasia.

Multifocal neoplastic precursor lesions associated with lobular atrophy of the pancreas in patients having a strong family history of pancreatic cancer.

We screened 116 patients with a strong family history of pancreatic cancer using a combination of endoscopic ultrasound and computed tomography. Ten of these patients underwent surgical resection at our institution, providing an opportunity to define the morphology of pancreatic precursor lesions in patients with a strong family history of pancreatic cancer. Eight of the 10 pancreata were available and these were entirely submitted for histologic examination. The number of pancreatic intraepithelial neoplasia (PanIN) lesions and intraductal papillary mucinous neoplasms (IPMNs) were compared with age-matched controls. Parenchymal changes were defined. Selected precursor neoplasms from 6 pancreata were microdissected and analyzed for KRAS gene mutations. PanINs were significantly more common in the 8 cases (mean of 10.7% of the duct profiles, range 1.0% to 27.3%) than in the controls (mean 1.9%, range 0% to 9.2%, P<0.01). Different KRAS gene mutations were identified in separately microdissected precursor lesions in 2 of 6 cases. IPMNs were identified in 4 of the 8 cases, including 2 pancreata each having 2 distinct IPMNs. Both the IPMNs and the PanINs, even the low-grade PanIN-1 lesions, were associated with lobular parenchymal atrophy. Some individuals with a strong family history of pancreatic cancer develop multifocal, noninvasive epithelial precursor lesions of the pancreas. PanINs and IPMNs produce obstructive lobular atrophy, and this atrophy is likely the source of the chronic pancreatitis-like changes seen in these patients. The multifocal nature of familial pancreatic neoplasia suggests that surveillance of these patients is warranted after partial pancreatectomy.

DNA methylation alterations in the pancreatic juice of patients with suspected pancreatic disease.

Molecular markers of pancreatic neoplasia could aid in the evaluation of visible pancreatic lesions and indicate neoplasia invisible to imaging. We evaluated methylation-specific PCR (MSP) assays that detect aberrantly methylated DNA for their use as markers of pancreatic neoplasia. Methylation analysis was done on pancreatic juice collected endoscopically or surgically from 155 individuals with suspected pancreatic disease: 56 patients had pancreatic ductal adenocarcinoma, 17 had intraductal papillary mucinous neoplasms, 26 had symptomatic chronic pancreatitis, 12 controls lacked evidence of pancreatic disease, and 44 were asymptomatic individuals at increased risk of developing familial pancreatic cancer undergoing screening for pancreatic neoplasia. Pancreatic juice DNA was analyzed for promoter methylation using conventional MSP assays for 17 genes. For six genes, pancreatic juice methylation was quantified using real-time quantitative MSP (QMSP; Cyclin D2, FOXE1, NPTX2, ppENK, p16, and TFPI2). Quantifying pancreatic juice methylation using QMSP with a cutoff of >1% methylated DNA could better predict pancreatic cancer than detecting methylation using conventional MSP. In the endoscopic group, 9 of 11 patients with pancreatic cancer, but none of 64 individuals without neoplasia had > or =1% methylation for two or more of the best five QMSP assays (82% sensitivity and 100% specificity; P < 0.0001). The prevalence of pancreatic juice methylation in patients with chronic pancreatitis was less than in patients with pancreatic cancer but higher than in controls and similar to high-risk individuals. The detection and quantification of aberrantly methylated DNA in pancreatic juice is a promising approach to the diagnosis of pancreatic cancer.

Pancreaticobiliary cancers with deficient methylenetetrahydrofolate reductase genotypes.

BACKGROUND & AIMS: Methyl group deficiency might promote carcinogenesis by inducing DNA breaks and DNA hypomethylation. We hypothesized that deficient methylenetetrahydrofolate reductase (MTHFR) genotypes could promote pancreatic cancer development. METHODS: First, we performed a case-control study of germline MTHFR polymorphisms (C677T, A1298C) in 303 patients with pancreatic cancer and 305 matched control subjects. Pancreatic neoplasms frequently lose an MTHFR allele during tumorigenesis; we hypothesized that such loss could promote carcinogenesis. We therefore evaluated the cancer MTHFR genotypes of 82 patients with pancreaticobiliary cancers and correlated them to genome-wide measures of chromosomal deletion by using 386 microsatellite markers. Finally, MTHFR genotypes were correlated with global DNA methylation in 68 cancer cell lines. RESULTS: Germline MTHFR polymorphisms were not associated with an increased likelihood of having pancreatic cancer. Fractional allelic loss (a measure of chromosomal loss) trended higher in cancers with 677T genotypes than in cancers with other genotypes (P = .055). Among cancers with loss of an MTHFR allele, cancers with 677T MTHFR alleles had more deletions at folate-sensitive fragile sites (36.9%) and at tumor suppressor gene loci (68.5%) than 677C cancers (28.7% and 47.8%, P = .079 and .014, respectively). LINE1 methylation was lower in cancers with less functional 677T/TT genotypes (24.4%) than in those with 677CT (26.0%) and CC/C genotypes (32.5%) (P = .014). CONCLUSION: Cancers with defective MTHFR genotypes have more DNA hypomethylation and more chromosomal losses. Deficient MTHFR function due to loss of an MTHFR allele by an evolving neoplasm might, by promoting chromosomal losses, accelerate cancer development.

Epigenetic down-regulation of CDKN1C/p57KIP2 in pancreatic ductal neoplasms identified by gene expression profiling.

PURPOSE: Intraductal papillary mucinous neoplasm (IPMN) of the pancreas is an increasingly identified precursor to infiltrating ductal adenocarcinoma. Although our knowledge of the clinical and pathologic features of IPMNs is increasing, the molecular mechanisms underlying these neoplasms remain poorly understood. EXPERIMENTAL DESIGNS: To provide further insight into the molecular pathobiology of IPMNs, global expression profiling was done to determine genes that are inactivated/down-regulated in IPMNs using oligonucleotide microarrays (Affymetrix). RESULTS: In total, 300 unique transcripts (217 known genes) were identified as highly underexpressed in 12 IPMNs (<10-fold lower and P < 0.05) compared with five normal pancreatic ductal epithelium samples obtained by laser capture microdissection. The differential expression of a selection of genes was confirmed using reverse-transcription PCR. One of the genes underexpressed at both the transcriptional and protein level in a significant proportion of IPMNs was the cyclin-dependent kinase inhibitor, CDKN1C/p57KIP2. CDKN1C expression was also decreased in many pancreatic cancer cell lines and was restored following treatment with a DNA methylation inhibitor (5-aza-2'-deoxycitidine) or, more potently, with a histone deacetylase inhibitor (trichostatin A). Partial methylation of the CDKN1C promoter CpG island was found in most, but not all, pancreatic cancer cell lines with reduced CDKN1C expression, and was also detectable in IPMNs. Furthermore, a subset of pancreatic cancers showed complete hypomethylation of LIT1, an imprinting control region important for the regulation of CDKN1C expression. Complete hypomethylation in these cancers was the result of deletion of the methylated LIT1 allele at 11p15.5 rather than loss of imprinting. CONCLUSIONS: These findings suggest that CDKN1C is commonly down-regulated in pancreatic ductal neoplasms through a combination of promoter hypermethylation, histone deacetylation, and loss of the maternal allele expressing CDKN1C.

Distinct progression pathways involving the dysfunction of DUSP6/MKP-3 in pancreatic intraepithelial neoplasia and intraductal papillary-mucinous neoplasms of the pancreas.

DUSP6/MKP-3 is identified as a candidate tumor suppressor gene for pancreatic cancer. The aim of this study was to elucidate the roles of DUSP6 in the pancreatic carcinogenesis through the pancreatic intraepithelial neoplasia and/or intraductal papillary-mucinous neoplasms, both of which are considered to be precursor lesions of invasive carcinoma of the pancreas, by comparing with involvements of other major tumor suppressive pathways. Expressions of DUSP6, CDKN2A, TP53, and SMAD4 were investigated by immunohistochemistry in a total of 206 lesions of dysplastic ductal precursors and carcinomas retrieved from 52 pancreata with invasive ductal carcinomas and 51 of those with intraductal papillary-mucinous neoplasms. The intensity of staining was evaluated in lesions at different atypical grades and statistically compared among them. Mutations of KRAS2 were analyzed by methods of the allele-specific oligonucleotide hybridization and nucleotide sequencing. In pancreata with invasive ductal carcinomas, expressions of DUSP6 were abrogated exclusively in the invasive carcinoma cells in contrast to its fairly preserved expressions in pancreatic intraepithelial neoplasia. In pancreata with intraductal papillary-mucinous neoplasms, abrogated expressions of DUSP6 were observed in a relatively small fraction of intraductal adenoma/borderlines and intraductal carcinomas. Most of the intraductal adenoma/borderline lesions with abrogation of DUSP6 harbored mutations of KRAS2. None of the molecules was associated with each other in any grade of lesions. Morphological variations of papillae of the intraductal papillary-mucinous neoplasms were evaluated and analyzed for their associations with abrogations of the molecules, which resulted in finding of no significant associations. Our results suggest that the abrogation of DUSP6 is associated exclusively with progression from pancreatic intraepithelial neoplasia to the invasive ductal carcinoma while it is potentially associated with initiation of intraductal papillary-mucinous neoplasms with mutated KRAS2, which is independent of other major tumor suppressive pathways in both types of neoplasms.

Chromosome 12, frequently deleted in human pancreatic cancer, may encode a tumor-suppressor gene that suppresses angiogenesis.

Several lines of evidence have suggested that the long arm of chromosome 12 may carry a tumor-suppressor gene(s) that plays a role in pancreatic ductal carcinogenesis. We have previously found a significant association between loss of heterozygosity of the 12q arm and a poor prognosis in pancreatic cancer patients. In this study, we introduced a normal copy of chromosome 12 into some pancreatic ductal carcinoma cells. Both anchorage-dependent and -independent proliferations as well as invasiveness were similar throughout the hybrid clones when compared with their corresponding parental cells. In sharp contrast, significant suppression of tumorigenesis was observed after inoculation of the hybrid clones into nude mice. Measurements made up to 1 month later showed that there was a significant delay in the growth of tumors into which the introduced normal copy of chromosome 12 had been restored. More significantly, using our dorsal skin chamber and an intravital microscopy system experiment in SCID mice, we demonstrated and visualized directly that implantation of the hybrids failed to promote the angiogenic phenotype encountered in the parental cells. Gene expression profiling using the complementary DNA microarray system identified a set of 24 genes differentially expressed between the hybrids and parental cells. An additional set of 18 genes was also identified that were differentially expressed between the hybrid clone that lost its growth-suppression activity and one that retained such activity. Another set of 25 genes mapped on 12q was detected that showed high expression levels in the hybrid clones retaining growth-suppressive activity. In summary, this study provides the first functional evidence of the existence of an additional tumor-suppressor gene(s) on chromosome 12, whose absence is responsible for the pathogenesis in pancreatic ductal carcinogenesis.

Functional analysis of chromosome 18 in pancreatic cancer: strong evidence for new tumour suppressor genes.

BACKGROUND: In a previous work, we demonstrated that loss of heterozygosity of 18q is a frequent event significantly associated with poor prognosis in pancreatic cancer. We hypothesized that restoration of heterozygosity of chromosome 18 in pancreatic cancer cells would reduce their tumorigenicity. This study was intended to provide functional evidence for the existence of new tumour suppressor gene(s) located on chromosome 18. METHOD: Restoration of heterozygosity was achieved by introducing a normal copy of chromosome 18 into pancreatic ductal carcinoma using a microcell-mediated chromosome transfer technique. The tumorigenicity and metastatic ability of both the parental cells and resulting hybrids were assessed in vitro and in vivo. RESULTS: In vitro growth of hybrid clones was significantly delayed compared to parental cells. This was paralleled by a significantly lower rate of promoting invasive carcinoma in nude mice and a longer latency with hybrid cells compared with parental tumour cells. Hybrid clones showed significant suppression in the number of surface lung metastases when compared with parental cells. CONCLUSION: These data represent strong functional evidence that chromosome 18q encodes strong tumour and metastasis suppressor activity that is able to switch human pancreatic cancer cells to a dormant phenotype.

The role of chromosome 18 abnormalities in the progression of pancreatic adenocarcinoma.

To date, the events that mediate tumor progression in pancreatic cancer are still poorly understood. Cytogenetic, allelotype, and somatic cell hybrid studies in human pancreatic adenocarcinoma have suggested that chromosome 18 may carry tumor suppressor genes (TSGs), including SMAD4. We previously identified that LOH of 18q at the SMAD4 locus, along with LOHs on 17p and 12q, positively associated with poor prognoses of pancreatic cancer patients. However, restoration of the SMAD4 gene did not suppress in vitro proliferation of pancreatic cancer cells that harbored homozygous deletion of this gene. An intraductal papillary mucinous neoplasm (IPMN ) is thought to be one of the premalignant lesions of the pancreas that progresses to carcinoma. Although there were frequent LOH (7/14, 50%) at the SMAD4 locus in IPMN samples, SMAD4 protein was observed immunohistochemically in tumor cells, and no mutations of the SMAD4 gene were observed, suggesting that it is the existence of a TSG in 18q, other than SMAD4, that suppresses cell growth. To functionally assess the activity of chromosome 18 in pancreatic cancer, we transferred a normal copy of the chromosome into pancreatic ductal carcinoma cells with and without completely inactivated SMAD4. In this study, in vitro growth of the hybrid cells was significantly suppressed compared with the parental cells, regardless of the initial SMAD4 status. To estimate the metastatic ability of the hybrids, we used a lung colonization model. At the end of the experiment, there was significant suppression of the number of surface metastases developing in mice injected with hybrids in comparison with those injected with parental cells. To identify and characterize genes that are involved in the progression of pancreatic cancer, we used micro-array expression analysis employing a 20k oligo-array system. It was revealed that there was increased expression of 4 genes relating to apoptosis in the 18 chromosome hybrids cells compared with the parental cells. We are now analyzing the function of these genes.

Restoration of SMAD4 by gene therapy reverses the invasive phenotype in pancreatic adenocarcinoma cells.

SMAD4 is a critical cofactor in signal transduction pathways activated in response to transforming growth factor-beta (TGF-beta)-related ligands, regulating cell growth and differentiation. The roles played by SMAD4 inactivation in tumours highlighted it as a tumour-suppressor gene. However, restoration of the TGF-beta antiproliferative pathway following SMAD4 gene transfer in null-tumour cell lines is controversial. Herein, we report the inhibitory effects of SMAD4 on pancreatic tumour invasion and angiogenesis. Adenoviral transfer of this gene in a panel of SMAD4 homozygous-deleted human pancreatic tumour cell lines restored SMAD4 protein expression and function. Although it did not affect proliferation significantly in vitro, SMAD4 inhibited in vivo tumour growth in immunodeficient mice. In this xenograft setting, differential suppression of tumour growth in vivo was mediated, at least in part, through downregulation of vascular endothelial growth factor and expression of gelatinases. We documented the reduced invasion and angiogenesis histologically and by intravital microscopy, and gained mechanistic insight at the messenger and protein level. Finally, we found a negative reciprocal regulation between SMAD4 and ETS-1. ETS-1 is considered a marker for tumour invasion. Upon SMAD4 deletion, we detected high expression levels of ETS-1 in pancreatic tumour cells, suggesting the shift of the pancreatic tumour toward an invasive phenotype.

Gene therapy for pancreatic cancer based on genetic characterization of the disease.

In order to develop an effective therapeutic intervention for patients with pancreatic cancer, we analyzed the association of loss of heterozygosity (LOH) with the clinicopathological features of the disease. Based on these results, we are developing a new gene therapy that targets the genetic character of pancreatic cancer, using mutant adenoviruses that are selectively replication-competent in tumor cells. LOH of 30% or more was observed on chromosome arms 17p (47%), 9p (45%), 18q (43%), 12q (34%), and 6q (30%). LOH of 12q, 17p, and 18q showed significant association with poor prognosis. These data strongly suggest that mutations of the putative tumor suppressor genes, TP53 and SMAD4, play significant roles in the disease progression. Based on this rationale, we are developing a new gene therapy that targets tumors without normal TP53 function. The E1B-55kDa-deleted adenovirus can selectively replicate in TP53-deficient human tumor cells, but not in cells with functional TP53. We evaluated the therapeutic effect of this E1B-55kDa-deleted mutant adenovirus on pancreatic cancer without normal TP53 function. The growth of a human pancreatic tumor in a severe combined immunodeficiency (SCID) mouse model was markedly inhibited by consecutive injections of the E1B-55kDa-deleted adenovirus. Furthermore, the replication-competent adenovirus is not only a strong weapon itself but it is also a useful carrier of genes that possess antitumor activities, as a virus vector specific to tumors without normal TP53 function.