DPC4 Genes Research Articles

Secrete and Exude-Pancreatic Neuroendocrine Tumour

Pancreatic neuroendocrine tumour is derived from neuroendocrine cells of pancreas or hormone secreting cells within vicinity of pancreas and is categorized into indolent well differentiated neuroendocrine tumour (WDNET) and the aggressive, rapidly progressive poorly differentiated neuroendocrine carcinoma(PDNEC). Well differentiated neuroendocrine tumour (WDNET) demonstrates inactivation of MEN1, genetic mutation or loss of DAXX / ATRX, altered mTOR pathway, incrimination of PTEN, TSC2, PIK3CA genes with alteration of VHL. Poorly differentiated neuroendocrine carcinoma and mixed neuroendocrine / non neuroendocrine neoplasm delineates alterations within TP53, RB, KRAS, APC, BRAF or SMAD4 / DPC4 genes. Neuroendocrine tumour delineates an organoid architecture with solid nests, trabeculae, gyri, cords, festoons, ribbons, glandular, pseudoacinar or tubulo-acinar articulations. Miniature to intermediate tumour cells are permeated with eosinophilic, amphophilic, finely granular cytoplasm, centric, spherical to elliptical, uniform nuclei with finely stippled ‘salt and pepper’ nuclear chromatin, inconspicuous nucleoli and an abundant circumscribing vascular network. Pancreatic neuroendocrine tumour is immune reactive to CK8 / CK18, CAM 5.2, OSCAR, AE1 / AE3, synaptophysin, chromogranin A, INSM1, CD56, neuron specific enolase (NSE) or CD57. Pancreatic neuroendocrine tumour pancreas requires segregation from neoplasms such as acinar cell carcinoma, pancreatoblastoma, solid pseudo-papillary neoplasm, pancreatic ductal adenocarcinoma, paraganglioma, clear cell tumours as clear cell sarcoma, metastatic renal cell carcinoma, PEComa or solid variant of serous cystadenoma. Comprehensive surgical eradication is recommended for treating well differentiated neuroendocrine tumour. Poorly differentiated neuroendocrine tumour is appropriately treated with platinum based chemotherapy.

Molecular features of pancreaticobiliary neoplasms: Implications for diagnosis, prognostication, and therapy selection.

Molecular diagnostics has impacted the diagnosis, prediction of prognosis, and selection of targeted therapy for many tumor types. While pulmonary adenocarcinomas and melanomas are among the neoplasms most associated with molecular diagnostics and targeted therapy, malignancies of the pancreaticobiliary system have also been impacted by precision medicine. We undertook an electronic search using PubMed and Embase to review the published literature to determine what forms of molecular testing, mutations and oncogenetic pathways are associated with neoplasms of the pancreaticobiliary system. Keywords utilized were pancreas, bile duct, mutations, ERCP, FNA, KRAS, SMAD4, TP53, next-generation sequencing, serous cystadenoma, pancreatic ductal adenocarcinoma, intraductal papillary mucinous neoplasm, cystic mucinous neoplasm, solid pseudo-papillary neoplasm. A search between 1999 and 2022 yielded 6874 manuscripts. Screening of these yielded 302 more focused manuscripts of which 55 were used for the study. Ductal adenocarcinoma of the pancreas is associated with a progression of mutations beginning wit KRAS mutations and ending with a set of mutations in the TP53, SMAD4, and DPC4 genes. Similar mutations are found in neoplastic mucinous cysts. Specific mutations characterize serous cystadenomas, solid, and pseudo papillary neoplasms and adenocarcinomas of the bile ducts. Mutational analysis of cytologic specimens obtained by fine-needle aspiration, and duct brushings and washings are helpful in the diagnosis of pancreaticobiliary neoplasms and may supply prognostic information.

Negative control of TRAIL-R1 signaling by transforming growth factor β1 in pancreatic tumor cells involves Smad-dependent down regulation of TRAIL-R1

Pancreatic ductal adenocarcinoma (PDAC) is characterized by both, overexpression of transforming growth factor (TGF)β and resistance of the tumor cells to many apoptosis-inducing stimuli. The latter negatively impacts the outcome of therapeutic efforts and represents one important mechanism which tumor cells utilize to escape the immune surveillance. Since TGFβ acts as a tumor promoter in advanced tumor stages and suppression of apoptosis is a known driver of tumor progression, it is possible that TGFβ functions as a crucial determinant of tumor cell sensitivity to apoptosis in PDAC. Here, we have studied the impact of TGFβ on TNF-related apoptosis inducing ligand (TRAIL)-induced signaling in PDAC cells.In TGFβ-responsive Panc1 and Colo357 cells, TGFβ1 reduced total and plasma membrane-associated levels of TRAIL-R1 but not those of TRAIL-R2. Consistent with the known predominant role of TRAIL-R1 in TRAIL-mediated signaling in PDAC, TGFβ1 inhibited TRAIL-induced DISC formation and apoptosis as well as phosphorylation of MAPKs and IκBα. Similarly, it also reduced signaling of TRAIL-R1 following its specific activation with an agonistic antibody. In contrast, specific TRAIL-R2 signaling remained unchanged. The TGFβ1 effect on TRAIL-R1 expression was mimicked by ectopic expression of a kinase-active version of the TGFβ type I receptor ALK5 (ALK5-T204D) but not by ALK5 double mutant lacking the ability to phosphorylate Smad proteins (RImL45-T204D). Moreover, TGFβ regulation of TRAIL-R1 was absent in two PDAC cell lines lacking the Smad4 gene DPC4 and siRNA-mediated silencing of Smad4 in Smad4-positive Panc1 cells abolished the TGFβ-mediated decrease in TRAIL-R1 expression, together showing that ALK5/Smad4 signaling is crucial for TGFβ regulation of TRAIL-R1 expression. Our results suggest a novel tumor-promoting function of TGFβ1. By downregulating TRAIL-R1, TGFβ1 may not only promote tumor escape from immune surveillance but also negatively impact on TRAIL- or TRAIL-R1-based therapy regimens for treatment of PDAC.

Large-scale analysis for treatment strategy according to genetic alterations of KRAS and DPC4 (SMAD4) genes in pancreatic ductal adenocarcinoma.

361 Background: To investigate correlation of genetic alterations of pancreatic ductal adenocarcinoma (PDAC) with patients’ survival, recurrence patterns, and treatment for recurrent disease. Methods: We reviewed genetic alterations of major 4 genes (K-ras, DPC4, p53, and c-erbB-2) in 699 patients who underwent surgical resection, and correlated with clinical outcomes. Results: Median survival of all patients was 21.7 months, and 5-year survival rate was 20.4%. Alterational rates of each gene were as follows: K-ras, 48.3%; DPC4, 68.1%; p53, 40.8%; c-erbB-2, 14.0%. Mutation of K-ras and inactivation of DPC4 genes were associated with shorter patients’ survival in univariate analysis. In multivariate analysis, mutation of K-ras gene was independently correlated with patients’ survival (especially, GAT and TGT subtypes). Inactivated DPC4 gene had no independent correlation with overall survival, but it was strongly associated with distant metastasis following pancreatectomy. Survival after recurrence (SAR) was evaluated in subdivided groups according to DPC4 gene function (intact or loss)/recurrence patterns (locoregional or distant)/treatment options (local control or systemic therapy). In these 8 groups, patients in intact/locoregional/local control group showed the longest survival (24.2 months of median SAR), and intact/locoregional/systemic therapy and loss/distant/systemic therapy groups were associated with much shorter survival (9.5 and 9.2 months of median SAR, respectively). Conclusions: Assessment of genetic features of PDAC may assist in deciding targeted surveillance or treatment for primary as well as recurrent PDAC, and further studies for therapeutic strategies according to genetic analyses should be devised.

Abstract SY20-01: Role of Notch signaling in colon cancer invasion and metastasis

Abstract While there have been substantial advances in our understanding of the mechanisms of cancer metastasis, efficient remedies for prevention and treatment of metastasis are still missing. The invasion-metastasis cascade consists of local invasion, intravasation, transport, extravasation, formation of micrometastases, and colonization (1,2). This sequence is completed only infrequently, causing metastatic inefficiency, and the least efficient of these steps appears to be colonization (3). Spread of metastatic cancer cells via blood circulation is responsible for the majority of distant metastases although they may travel also through the lymph ducts to nodes (4). In colorectal cancer, metastatic tropism to the liver and lungs can be explained largely by the organization of venous circulation of the intestines through portal vein to the liver, and further to the lungs through pulmonary artery. Among endogenous colon tumor models, the widely used Apc (Adenomatous polyposis coli) mutant mice form adenomas in the small intestine, with various multiplicities (3᠄300 per animal) depending on the mutational allele, although several adenomas are also found in the colon (5). Additional mutations introduced into Apc mutant mice can modify the tumor phenotype. For example, knocking out Smad4 gene in the TGF- family signaling converts the benign intestinal adenomas to very invasive adenocarcinomas (6). Even in such a model, the adenocarcinomas are only locally invasive, and neither intravasation nor distant metastasis is observed during the short life span of these mice. Accordingly, we have screened for candidate genes whose inactivation can stimulate metastasis of mouse colon cancer cells from the rectum to the liver, the commonest site of metastasis using an orthotopic transplantation model. We found such a metastasis suppressor of CRC; Aes that turned out to be an endogenous inhibitor of Notch signaling transcription that is carried out by a complex of Notch intracellular domain (NICD), Maml and Rbpj (7, 8). Upon introduction of a conditional Aes knockout mutation into adenomatous intestinal epithelium of Apc+/∆716 polyposis mice, their tumors become malignant due to Notch signaling activation, showing marked submucosal invasion and intravasation. Consistently, transendothelial migration (TEM) is increased significantly, when CRC cells are activated for Notch signaling and placed on an endothelial cell (EC) layer in culture. When human CRC cells that were inhibited for Aes expression were transplanted to the nude mouse rectum, the number of metastatic foci increased significantly. Thus, reduced level of Aes and stimulation of Notch signaling are implicated in the invasion and intravasation of CRC cells during metastasis. The molecular mechanism of Notch inhibition by Aes is through transcriptional repression by sequestering Rbpj, NICD and Maml1 to the nuclear matrix. The cellular mode of metastasis suppression includes inhibition of transendothelial migration (TEM) of tumor cells, which blocks intravasation. Heterotypic interaction between cancer epithelium and host stromal cells activates Notch signaling and promotes TEM of metastasizing cancer cells. Because TEM in metastasis is common to many solid tumors, this mechanism is important in various types of cancers. As a model for colon cancer invasion and intravasation, compound mutant mice for the Apc and Aes genes should be useful to evaluate new therapeutics including Notch signaling inhibitors against colon cancer metastasis. Some more recent advances will be presented and discussed regarding the molecular mechanism responsible for metastatic invasion downstream of Notch signaling activation.

Carcinogenesis of Pancreatic Adenocarcinoma: Precursor Lesions

Pancreatic adenocarcinoma displays a variety of molecular changes that evolve exponentially with time and lead cancer cells not only to survive, but also to invade the surrounding tissues and metastasise to distant sites. These changes include: genetic alterations in oncogenes and cancer suppressor genes; changes in the cell cycle and pathways leading to apoptosis; and also changes in epithelial to mesenchymal transition. The most common alterations involve the epidermal growth factor receptor (EGFR) gene, the HER2 gene, and the K-ras gene. In particular, the loss of function of tumor-suppressor genes has been documented in this tumor, especially in CDKN2a, p53, DPC4 and BRCA2 genes. However, other molecular events involved in pancreatic adenocarcinoma pathogenesis contribute to its development and maintenance, specifically epigenetic events. In fact, key tumor suppressors that are well established to play a role in pancreatic adenocarcinoma may be altered through hypermethylation, and oncogenes can be upregulated secondary to permissive histone modifications. Indeed, factors involved in tumor invasiveness can be aberrantly expressed through dysregulated microRNAs. This review summarizes current knowledge of pancreatic carcinogenesis from its initiation within a normal cell until the time that it has disseminated to distant organs. In this scenario, highlighting these molecular alterations could provide new clinical tools for early diagnosis and new effective therapies for this malignancy.

Correlation of Smad4 Status With Outcomes in Patients Receiving Erlotinib Combined With Adjuvant Chemoradiation and Chemotherapy After Resection for Pancreatic Adenocarcinoma

Although adjuvant therapy may reduce the risk of local and distant recurrence, it remains unclear why some patients experience widely disseminated disease whereas others experience recurrence only locally or not at all. Identification of biomarker predictors for local and distant recurrence could guide the administration of targeted therapies in the adjuvant setting. A potential prognostic biomarker is the tumor-suppressor gene DPC4(SMAD4) (1). DPC4 encodes the Smad4 protein, which functions as a central mediator of the canonical transforming growth factor (TGF)-β signaling pathway involved in cell proliferation, differentiation, apoptosis, and migration.

Peroxisome proliferator-activated receptor gamma and regulations by the ubiquitin-proteasome system in pancreatic cancer.

Pancreatic cancer is one of the most lethal forms of human cancer. Although progress in oncology has improved outcomes in many forms of cancer, little progress has been made in pancreatic carcinoma and the prognosis of this malignancy remains grim. Several molecular abnormalities often present in pancreatic cancer have been defined and include mutations in K-ras, p53, p16, and DPC4 genes. Nuclear receptor Peroxisome Proliferator-Activated Receptor gamma (PPARγ) has a role in many carcinomas and has been found to be overexpressed in pancreatic cancer. It plays generally a tumor suppressor role antagonizing proteins promoting carcinogenesis such as NF-κB and TGFβ. Regulation of pathways involved in pancreatic carcinogenesis is effectuated by the Ubiquitin Proteasome System (UPS). This paper will examine PPARγ in pancreatic cancer, the regulation of this nuclear receptor by the UPS, and their relationship to other pathways important in pancreatic carcinogenesis.

Genetic and Epigenetic Alterations in Pancreatic Carcinogenesis

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide. Despite significant progresses in the last decades, the origin of this cancer remains unclear and no efficient therapy exists. PDAC does not arise de novo: three remarkable different types of pancreatic lesions can evolve towards pancreatic cancer. These precursor lesions include: Pancreatic intraepithelial neoplasia (PanIN) that are microscopic lesions of the pancreas, Intraductal Papillary Mucinous Neoplasms (IPMN) and Mucinous Cystic Neoplasms (MCN) that are both macroscopic lesions. However, the cellular origin of these lesions is still a matter of debate. Classically, neoplasm initiation or progression is driven by several genetic and epigenetic alterations. The aim of this review is to assemble the current information on genetic mutations and epigenetic disorders that affect genes during pancreatic carcinogenesis. We will further discuss the interest of the genetic and epigenetic alterations for the diagnosis and prognosis of PDAC. Large genetic alterations (chromosomal deletion/amplification) and single point mutations are well described for carcinogenesis inducers. Mutations classically occur within key regions of the genome. Consequences are various and include activation of mitogenic pathways or silencing of apoptotic processes. Alterations of K-RAS, P16 and DPC4 genes are frequently observed in PDAC samples and have been described to arise gradually during carcinogenesis. DNA methylation is an epigenetic process involved in imprinting and X chromosome inactivation. Alteration of DNA methylation patterns leads to deregulation of gene expression, in the absence of mutation. Both genetic and epigenetic events influence genes and non-coding RNA expression, with dramatic effects on proliferation, survival and invasion. Besides improvement in our fundamental understanding of PDAC development, highlighting the molecular alterations that occur in pancreatic carcinogenesis could provide new clinical tools for early diagnosis of PDAC and the molecular basis for the development of new effective therapies.

DPC4 Gene Status of the Primary Carcinoma Correlates With Patterns of Failure in Patients With Pancreatic Cancer

Contrary to the extensive data accumulated regarding pancreatic carcinogenesis, the clinical and molecular features characteristic of advanced stage (stage III and IV) disease are unknown. A comprehensive study of pancreatic cancers from patients who have succumbed to their disease has the potential to greatly expand our understanding of the most lethal stage of this disease and identify novel areas for intervention. Rapid autopsies were performed on 76 patients with documented pancreatic cancer. The histologic features of end stage disease were determined and correlated to the stage at initial diagnosis, patterns of failure (locally destructive v metastatic disease) and the status of the KRAS2, TP53, and DPC4 genes. At autopsy, 30% of patients died with locally destructive pancreatic cancer, and 70% died with widespread metastatic disease. These divergent patterns of failure found at autopsy (locally destructive v metastatic) were unrelated to clinical stage at initial presentation, treatment history, or histopathologic features. However, Dpc4 immunolabeling status of carcinoma tissues harvested at autopsy, a sensitive marker of DPC4 genetic status, was highly correlated with the presence of widespread metastasis but not with locally destructive tumors (P = .007). Pancreatic cancers are represented by distinct genetic subtypes with significantly different patterns of failure. Determinations of DPC4 status at initial diagnosis may be of value in stratifying patients into treatment regimens related to local control versus systemic therapy.

Expression of novel markers of pancreatic ductal adenocarcinoma in pancreatic nonductal neoplasms: additional evidence of different genetic pathways

Solid pseudopapillary tumor, pancreatoblastoma, undifferentiated carcinoma with osteoclastic-like giant cells, and acinar cell carcinomas are rare pancreatic nonductal neoplasms. Compared to the significant advances in our understanding of the pathogenesis of pancreatic ductal adenocarcinomas in the last decades, the molecular mechanisms underlying pancreatic nonductal neoplasms are poorly understood. In order to elucidate their molecular pathogenesis, we constructed tissue microarrays to study the expression of some novel pancreatic ductal adenocarcinoma-associated tumor markers in these nonductal pancreatic neoplasms. We analyzed nine markers including tumor suppressor gene (14-3-3 sigma), proliferation marker (topoisomerase II alpha), epithelial markers (prostate stem cell antigen, mesothelin and cytokeratin 19), stromal markers (fascin, hsp47 and fibronectin), and gamma-synuclein whose function is not delineated. In addition, we included tumor suppressor gene DPC4 and oncogene Beta-catenin to further confirm their expression in pancreatic nonductal tumors. Our results showed that in contrast to pancreatic ductal adenocarcinomas that show loss of Dpc4 protein in 55% of cases, loss of Dpc4 expression is absent in pancreatic nonductal neoplasms. Expression of 14-3-3 sigma is frequently seen in both pancreatic nonductal neoplasms (25–100%) and ductal adenocarcinomas (89%). Aberrant nuclear expression of beta-catenin is common in pancreatic nonductal neoplasms, specifically in solid pseudopapillary tumors (88%) and pancreatoblastomas (100%) but is rarely seen in pancreatic ductal adenocarcinomas (<5%). Expression of topoisomerase II alpha is not seen in solid pseudopapillary tumors and undifferentiated carcinomas with osteoclastic-like giant cells but is focally seen in pancreatoblastomas (50%) and acinar cell carcinomas (85%). Expression of PSCA and mesothelin was observed in pancreatic nonductal neoplasms but their expression was seen less frequently (0–50%) and weaker than that in pancreatic ductal adenocarcinomas (60–100%). CK19, a marker of pancreatic ductal adenocarcinomas, is not expressed in pancreatic nonductal neoplasms. Expression of gamma-synuclein as well as stromal markers (fascin, hsp47 and fibronectin) is frequently seen in both. Our findings indicate pancreatic nonductal neoplasms have distinctive patterns of protein expression relative to pancreatic ductal adenocarcinomas and suggest that pancreatic nonductal neoplasms have different genetic pathways from the more common pancreatic ductal adenocarcinomas.

Mucinous cystic carcinoma of the pancreas: a unique cell line and xenograft model of a preinvasive lesion

Pancreatic mucinous cystic tumors (MCT) are proliferations of mucin-producing epithelia supported by an ovarian-like stroma. They are classified into adenomas (MCA), borderline (MCB) and noninvasive or invasive carcinomas (MCC). The molecular mechanisms underlying their clinical behavior are poorly understood, partly due to the lack of cellular models. We report the establishment of MCC1, the first cell line from a pancreatic MCT, deriving from the highly dysplastic cell component of a noninvasive MCC. MCC1 has mutations in codon 12 of K-RAS (GGT>GAT), codon 58 of P16 (CGA>TGA) and codon 132 of P53 (AAG>AGG). The FHIT and DPC4 genes are unaltered. Immunohistochemistry shows abnormal expression of MUC1 and p53, loss of p16 and retention of Fhit and Dpc4 in both the cell line and the highly dysplastic cells of the primary lesion. The morphological and molecular features of MCC1 and its corresponding primary tumor are consistent with a model for non-invasive MCC, where K-RAS, P16, P53 and MUC1 alterations are pre-invasive changes associated with progression of malignancy of MCT from adenoma to carcinoma. MCC1 is sensitive to 5-fluorouracil, representing the first assessment of drug sensitivity for MCC. Finally, MCC1 is a suitable model for preclinical studies, as it grows in immunodeficient mice.

Immunohistochemical Study of DPC4 and p53 Proteins in Gallbladder and Bile Duct Cancers

Gallbladder and bile duct carcinomas belong to the family of biliary tract tumors, but they demonstrate different clinical behavior. We evaluated a series of biliary tract carcinomas to determine whether they also had genotypic differences by analysis of the tumor suppressor genes DPC4 and p53. Twenty-one gallbladder cancers, 20 intrahepatic bile duct carcinomas, and 10 extrahepatic bile duct carcinomas were retrieved from the surgical pathology files of Kaohsiung Medical University Hospital. Sections were immunostained with monoclonal antibodies to the DPC4 and P53 proteins. Statistical differences between gallbladder cancer and bile duct carcinomas were determined using chi2 analysis or the Fisher's exact test, when appropriate. Two of the 21 gallbladder cancers (9.5%), 7 of the 20 intrahepatic bile duct carcinomas (35%), and five of the 10 extrahepatic bile duct carcinomas (50%) were negatively labeled for DPC4. The differences were significant between gallbladder carcinoma and both intrahepatic bile duct carcinomas (p = 0.023) and extrahepatic bile duct carcinomas (p = 0.012). A higher frequency of P53 overexpression was found in gallbladder cancers (61.9%) than in intrahepatic bile duct carcinomas (26.3%) (p = 0.024). This study suggests that the DPC4 gene may play a limited role in gallbladder carcinoma; however, p53 gene mutation is more frequently found in gallbladder cancers. In contrast, DPC4 deletion may be more common in bile duct carcinomas, especially in those arising from the extrahepatic bile duct. These findings support the concept that gallbladder and bile duct carcinomas are different tumors with differing etiologies and tumorigenesis.

Loss of chromosome 18q and DPC4 (Smad4) mutations in appendiceal adenocarcinomas.

Appendiceal adenocarcinomas are uncommon, and the genetic alterations present in these tumors are not well characterized. We studied genetic alterations including loss of chromosome 18q (location of DCC, DPC4, and JV-18 genes), and mutations of the DPC4 (SMAD4) and beta-catenin genes in 28 appendiceal adenocarcinomas, consisting of 17 mucinous and 11 nonmucinous carcinomas. Chromosome 18q loss was present in 57% (12/21) of appendiceal carcinomas including 54% (7/13) of mucinous and 63% (5/8) of nonmucinous carcinomas. Mutation of the DPC4 gene was present in 14% (three of 22) of the carcinomas occurring in one tumor with chromosome 18q loss and in two with unassessed chromosome 18q status. beta-catenin gene mutation was present in 0% (0 of 25) of the carcinomas. Chromosome 18q loss status was not associated with any clinicopathological features. The presence of chromosome 18q loss and DPC4 mutations in appendiceal adenocarcinomas suggests involvement of DPC4 and nearby genes on chromosome 18q (DCC and/or JV-18) in the pathogenesis of appendiceal adenocarcinomas.

Alteration of somatostatin receptor subtype 2 gene expression in pancreatic tumor angiogenesis.

To explore the difference of somatostatin receptor subtype 2 (SST2R) gene expression in pancreatic cancerous tissue and its adjacent tissue, and the relationship between the change of SST2R gene expression and pancreatic tumor angiogenesis related genes. The expressions of SST2R, DPC4, p53 and ras genes in cancer tissues of 40 patients with primary pancreatic cancer, and the expression of SST2R gene in its adjacent tissue were determined by immunohistochemiscal LSAB method and EnVision(TM) method. Chi-square test was used to analyze the difference in expression of SST2R in pancreatic cancer tissue and its adjacent tissue, and the correlation of SST2R gene expression with the expression of p53, ras and DPC4 genes. Of the tissue specimens from 40 patients with primary pancreatic cancer, 35 (87.5%) cancer tissues showed a negative expression of SST2R gene, whereas 34 (85%) a positive expression of SST2R gene in its adjacent tissues. Five (12.5%) cancer tissues and its adjacent tissues simultaneously expressed SST2R. The expression of SST2R gene was markedly higher in pancreatic tissues adjacent to cancer than in pancreatic cancer tissues (P<0.05). The expression rates of p53, ras and DPC4 genes were 50%, 60% and 72.5%, respectively. There was a significant negative correlation of SST2R with p53 and ras genes (chi(1)(2)=9.33, chi(2)(2)=15.43, P<0.01), but no significant correlation with DPC4 gene (chi(2)=2.08, P>0.05). There was a significant difference of SST2R gene expression in pancreatic cancer tissues and its adjacent tissues, which might be one cause for the different therapeutic effects of somatostatin and its analogs on pancreatic cancer patients. There were abnormal expressions of SST2R, DPC4, p53 and ras genes in pancreatic carcinogenesis, and moreover, the loss or decrease of SST2R gene expression was significantly negatively correlated with the overexpression of tumor angiogenesis correlated p53 and ras genes, suggesting that SST2R gene together with p53 and ras genes may participate in pancreatic cancerous angiogenesis.

Genetic Alterations in Goblet Cell Carcinoids of the Vermiform Appendix and Comparison with Gastrointestinal Carcinoid Tumors

Goblet cell carcinoid is a relatively rare neuroendocrine tumor of the vermiform appendix with poorly understood molecular pathogenesis. We studied the clinicopathologic features and genetic alterations, including allelic loss of chromosomes 11q, 16q, and 18q; sequencing of the K-ras, β-catenin, and DPC4 (SMAD4) genes; and p53 overexpression and loss of DPC4 by immunohistochemistry; in 16 goblet cell carcinoids. We compared the allelic loss in goblet cell carcinoids to those in 18 gastrointestinal carcinoid tumors. For goblet cell carcinoids, appendiceal perforation was the most common (70%, 7/10) clinical presentation. The mean tumor size was 2.0 ± 1.5 cm (range, 0.4 to 4.5 cm). The tumor invaded to appendiceal serosa in 50% (8/16) of patients, and two patients had metastasis in lymph nodes or adjoining viscera. With mean follow-up of 24 ± 14 months (median, 23 mo), 1 of 10 patients had died of disease, and 2 others had tumor recurrence. All four patients with metastases, recurrences, and/or death from disease had serosal involvement at presentation (P = .02). Loss of heterozygosity of chromosome 11q was present in 25% of goblet cell carcinoids, 14% of ileal carcinoid tumors, and 9% of nonileal carcinoid tumors; of chromosome 16q in 38%, 29%, and 0 (P = .02); and of chromosome 18q in 56%, 86%, and 9% (P = .002), respectively. No mutations of K-ras, β-catenin, or DPC4 genes; p53 overexpression; or loss of staining for DPC4 was present in any tumors. These findings suggest that allelic loss of chromosomes 11q, 16q, and 18q in goblet cell carcinoids and ileal carcinoids may have an important role in the pathogenesis of these tumors.

DPC-4 (Smad-4) and K-ras gene mutations in biliary tract epithelium in children with anomalous pancreaticobiliary ductal union

Background/Purpose: Recent studies have found that anomalous pancreaticobiliary ductal union (APBDU) is a substantial risk factor for biliary tract cancer at a younger age. DPC-4 (Smad-4) is a new tumor suppressor gene frequently inactivated in pancreatic and bile duct adenocarcinoma. To clarify carcinogenesis in APBDU, the authors investigated possible DPC-4 and K-ras mutations in 35 pediatric patients. Methods: DNA was extracted from biliary tract epithelial cells, which were resected surgically and histologically purified using microdissection. Polymerase chain reaction (PCR) primers were designed specifically for exons 8-11 of DPC-4 (18q21.1) and exons 1-2 of the K-ras oncogene (12p12.1). DNA sequences were determined using the direct DyeDeoxy Terminator Cycle method. Results: Of 35 children, 30 had wild-type DPC-4 and K-ras genes. K-ras mutations were detected in 5 patients, 4 of whom showed epithelial hyperplasia or metaplasia. In a 12-year-old girl with adenocarcinoma arising from a choledochal cyst, K-ras and DPC-4 (homozygous deletion) mutations were identified simultaneously. Conclusions: These results suggest that carcinogenesis in the biliary tract epithelium in APBDU is accompanied by multistep genetic mutational events; K-ras gene mutation occurs early in epithelial hyperplasia or metaplasia, whereas inactivation of the DPC-4 gene accumulates late in the progression of biliary tract adenocarcinoma. J pediatr Surg 38:694-697. © 2003 Elsevier Inc. All rights reserved.

Immunohistochemical and Genetic Analysis of Non–Small Cell and Small Cell Gallbladder Carcinoma and Their Precursor Lesions

Gallbladder carcinomas can be highly lethal neoplasms. Relatively little is known about the genetic abnormalities that underlie these tumors, particularly with respect to their timing in neoplastic progression. The authors evaluated 5 noninvasive dysplasias and 33 invasive gallbladder carcinomas (6 small cell carcinomas, 27 non–small cell carcinomas, of which 16 were accompanied by an in situ carcinoma component) for expression of the protein products of the p16, p53, Dpc4, and pRB tumor suppressor genes by immunohistochemistry. Neoplasms were also evaluated for the presence of activating K-ras oncogene mutations. Seventy-five percent of non–small cell gallbladder carcinomas demonstrated loss of p16 expression, whereas 63% accumulated high levels of p53. Loss of Dpc4 and pRB expression was less frequent, seen in 19% and 4% of the neoplasms, respectively. Thirty percent of neoplasms harbored activating K-ras mutations. In contrast, 100% of the small cell carcinomas of the gallbladder demonstrated inactivation of the pRB/p16 pathway; 67% showed loss of pRB expression, and the other 33% lost p16 expression. Eighty-three percent of small cell carcinomas accumulated high levels of p53, whereas loss of Dpc4 expression and activating K-ras mutations were not found. Among 15 evaluable in situ components, 13 harbored the same alterations found in the invasive component. Inactivation of p16 and p53 occur in the majority of non–small cell gallbladder carcinomas. Dpc4 inactivation and K-ras mutations occur in a significant minority of cases. pRB loss is uncommon in non–small cell gallbladder carcinoma, but virtually all small cell carcinomas inactivate the p16/pRB pathway, usually by retinoblastoma protein loss. It is noteworthy that all of these alterations occur at the level of carcinoma in situ.

Genetic profile of 22 pancreatic carcinoma cell lines. Analysis of K-ras, p53, p16 and DPC4/Smad4.

The K-ras, p53, p16 and DPC4 genes are among those most frequently altered in pancreatic ductal carcinoma. We analyzed 22 cell lines for alterations in these genes by direct sequence analysis and methylation-specific polymerase chain reaction. These cell lines showed mutations in K-ras and p53 at frequencies of 91% and 95%, respectively. Alterations in p16INK4a were found in all cases and included nine homozygous deletions, seven mutations and promoter methylation in six cases. Eight cell lines (36%) had an alteration of DPC4, including one mutation and seven homozygous deletions. The most typical mutational profile involved K-ras, p53, and p16INK4a, concurrently aberrated in 20 cases (91%). Eight cell lines had alterations in all four genes. Inactivation of DPC4 was always accompanied by alteration of all of the other three genes. This comprehensive data regarding the cumulative genetic alterations in pancreatic carcinoma cell lines will be of great value for studies involving drug sensitivity or resistance that may be associated with inactivation of a particular gene or molecular pathway.

Allelic Loss Is Often the First Hit in the Biallelic Inactivation of the p53 and DPC4 Genes During Pancreatic Carcinogenesis

The presumed precursor lesions of pancreatic ductal adenocarcinoma were recently classified according to their increasing grade of dysplasia and were designated as pancreatic intraepithelial neoplasia (PanIN) 1 through 3. In this study, we tested whether molecular genetic alterations can be correlated with this classification and may help to further categorize the various PanIN grades. We determined the frequencies of allelic loss at chromosomal arms 9p, 17p, and 18q in 81 microdissected duct lesions of various PanIN grades, using a combination of whole genome amplification and microsatellite analysis. In addition we examined the p53 and Dpc4 protein expression patterns by immunohistochemical analysis. In PanIN-1, we did not detect allelic losses. In PanIN-2, allelic losses were found in increasing frequency, and were particularly high in those lesions with moderate-grade dysplasia (low grade, 20, 33, and 17%, loss at 9p, 17p, and 18q, respectively; moderate grade, 46, 77, and 58%). PanIN-3 and invasive carcinomas exhibited abundant losses. Abnormal p53 and Dpc4 protein expression was only rarely identified in PanIN-2 lesions, but occurred frequently in PanIN-3 lesions and invasive carcinomas. The combined genetic and protein expression data support a model in which allelic loss is the first hit in the biallelic inactivation of the p53 and DPC4 tumor suppressor genes. In addition, our data indicate that allelic loss analysis may be useful in separating PanIN-2 lesions with low-grade dysplasia from those PanIN-2 lesions with moderate-grade dysplasia, each potentially representing a distinct progression step toward invasive carcinoma.