AGR2 is a SMAD4-suppressible gene that modulates MUC1 levels and promotes the initiation and progression of pancreatic intraepithelial neoplasia.

The mechanisms controlling expression of the putative oncogene Anterior gradient 2 (AGR2) in pancreatic ductal adenocarcinoma (PDAC) are not well understood. We now show that AGR2 is a transforming growth factor-ß (TGF-ß)-responsive gene in human pancreatic cancer cells, whose downregulation is SMAD4 dependent. We also provide evidence supporting a role for AGR2 as an ER-chaperone for the cancer-associated mucin, MUC1. AGR2 is both sufficient and required for MUC1 expression in pancreatic cancer cells. Furthermore, AGR2 is coexpressed with MUC1 in mouse pancreatic intraepithelial neoplasia (mPanIN)-like lesions and in the cancer cells of four distinct genetically engineered mouse models of PDAC. We also show that Pdx1-Cre/LSL-Kras(G12D)/Smad4(lox/lox) mice heterozygous for Agr2 exhibit a delay in mPanIN initiation and progression to PDAC. It is proposed that loss of Smad4 may convert TGF-ß from a tumor suppressor to a tumor promoter by causing the upregulation of AGR2, which then leads to increased MUC1 expression, at which point both AGR2 and MUC1 facilitate mPanIN initiation and progression to PDAC.

Deoxyribonuclease IIalpha is required during the phagocytic phase of apoptosis and its loss causes perinatal lethality.

Deoxyribonuclease IIalpha (DNase IIalpha) is one of many endonucleases implicated in DNA digestion during apoptosis. We produced mice with targeted disruption of DNase IIalpha and defined its role in apoptosis. Mice deleted for DNase IIalpha die at birth with many tissues exhibiting large DNA-containing bodies that result from engulfed but undigested cell corpses. These DNA-containing bodies are pronounced in the liver where fetal definitive erythropoiesis occurs and extruded nuclei are degraded. They are found between the digits, where apoptosis occurs, and in many other regions of the embryo. Defects in the diaphragm appear to cause death of the mice due to asphyxiation. The DNA in these bodies contains 3'-hydroxyl ends and therefore stain positive in the TUNEL assay. In addition, numerous unengulfed TUNEL-positive cells are observed throughout the embryo. Apoptotic cells are normally cleared rapidly from a tissue; hence the persistence of the DNA-containing bodies and TUNEL-positive cells identifies sites where apoptosis occurs during development. These results demonstrate that DNase IIalpha is not required for the generation of the characteristic DNA fragmentation that occurs during apoptosis but is required for degrading DNA of dying cells and this function is necessary for proper fetal development.

Pancreatic intraepithelial neoplasia: a new nomenclature and classification system for pancreatic duct lesions.

Proliferative epithelial lesions in the smaller caliber pancreatic ducts and ductules have been the subject of numerous morphologic, clinical, and genetic studies; however, a standard nomenclature and diagnostic criteria for classifying these lesion have not been established. To evaluate the uniformity of existing systems for grading duct lesions in the pancreas, 35 microscopic slides with 35 representative duct lesions were sent to eight expert pathologists from the United States, Canada, and Europe. Kappa values for interobserver agreement could not be calculated initially because more than 70 different diagnostic terms were used by the eight pathologists. In several cases, the diagnoses rendered for a single duct lesion ranged from "hyperplasia," to "metaplasia," to "dysplasia," to "carcinoma in situ." This review therefore demonstrated the need for a standard nomenclature and classification system. Subsequently, during a working group meeting, the pathologists agreed to adopt a single standard system. The terminology pancreatic intraepithelial neoplasia (or PanIN) was selected, and diagnostic criteria for each grade of PanIN were established (http://pathology.jhu.edu/pancreas_panin). This new system was then evaluated by having the eight pathologists rereview the original 35 cases. Only seven different diagnoses were rendered, and kappa values of 0.43, 0.14, and 0.42 were obtained for PanINs 1, 2, and 3 respectively. Cases assigned other diagnoses (e.g., squamous metaplasia) collectively had a kappa value of 0.41. These results show both the potential of the classification system, and also the difficulty of classifying these lesions even with a consistent nomenclature. However, even when there is lack of consensus, having a restricted set of descriptions and terms allows a better understanding of the reasons for disagreement. It is suggested that we adopt and apply this system uniformly, with continued study of its reliability and use, and possibly further refinement. The acceptance of a standard classification system will facilitate the study of pancreatic duct lesions, and will lead ultimately to a better understanding of their biologic importance.

Racial differences in pancreatic cancer: comparison of survival and histologic types of pancreatic carcinoma in Asians, blacks, and whites in the United States.

SEER data for histologically confirmed carcinomas of the pancreas for 1973-1995 from Hawaii, San Francisco, and Seattle (n = 10,621) were analyzed to compare the survival and types of carcinomas in various racial groups. These geographic sites were selected because each included a sizable number of Asian patients. The median survival after diagnosis in unadjusted data was longer in Asian patients than in whites. After adjustment for age at diagnosis and year of diagnosis, only the survival advantage of Asian women over whites and blacks persisted as a statistically significant difference. Racial differences were no longer statistically significant when further adjustments were made for stage, grade, and morphology. The proportion of papillary carcinomas or mucinous cystadenocarcinomas was higher in Asians than in whites and blacks (p = 0.02), and patients with these neoplasms had a longer median survival than did patients with ductal adenocarcinoma (12 vs. 3.3 months). The fraction of Asian patients with lower stages and grades of carcinomas also was higher than among white and black patients. Longer survival of Asian compared with white and black patients with pancreatic carcinoma is at least partly explained by their higher proportion of less aggressive carcinomas at the time of diagnosis.

Vascular structure and microcirculation of experimental pancreatic carcinoma in rats.

OBJECTIVE: To study angiogenesis and microcirculation in experimental pancreatic carcinoma. DESIGN: Open experimental study. SETTING: 2 University hospitals, Germany and USA. ANIMALS: 16 male Lewis rats. INTERVENTIONS: Induction of a duct-like pancreatic cancer in the pancreas and peritoneum by interposition of fragments of tumour between 2 inert transparent polymethylmethacrylate plates. MAIN OUTCOME MEASURES: Microcirculation in the tumour and interaction between leucocytes, tumour, and endothelium investigated by intravital microscopy. RESULTS: The density of vessels in the carcinoma was significantly less than in normal pancreatic tissue (p = 0.0004). The vasculature of the tumour was characterised by a lack of differentiation in architecture of vessels, formation of sinusoidal and lacunar vessels and sudden changes in diameter of vessels. Red blood cell velocity differed among tumour vessels, but mean values were similar to those of normal exocrine pancreas. The mixed lymphocyte culture test indicated that the cell line DSL6A was immunogenic. However, high-affinity leucocyte-endothelium-interaction was significantly reduced in the tumour's microcirculation after both orthotopic and heterotopic implantation (p = 0.002). Rates of apoptosis were suppressed in heterotopic tumours compared with orthotopic ones. Tumour growth was faster in heterotopic tumours. CONCLUSIONS: Experimental duct-like pancreatic carcinoma can be differentiated from normal pancreas by: chaotic arrangement of vessels with loss of differentiation of architecture and heterogeneous distribution; the formation of sinusoidal or lacunar vessels; lower vascular density with similar erythrocyte velocity; increase in mean diameter of vessels; reduced leucocyte-endothelium interaction despite confirmed immunogeneity independent of wall shear rates. The site of implantation influences apoptosis and growth rates.

Role of the basic helix-loop-helix transcription factor p48 in the differentiation phenotype of exocrine pancreas cancer cells.

The majority of human pancreatic adenocarcinomas display a ductal phenotype; experimental studies indicate that tumors with this phenotype can arise from both acinar and ductal cells. In normal pancreas acinar cells, the pancreas transcription factor 1 transcriptional complex is required for gene expression. Pancreas transcription factor 1 is a heterooligomer of pancreas-specific (p48) and ubiquitous (p75/E2A and p64/HEB) basic helix-loop-helix proteins. We have examined the role of p48 in the phenotype of azaserine-induced rat DSL6 tumors and cancers of the human exocrine pancreas. Serially transplanted acinar DSL6 tumors express p48 whereas DSL6-derived cell lines, and the tumors induced by them, display a ductal phenotype and lack p48. In human pancreas cancer cell lines and tissues, p48 is present in acinar tumors but not in ductal tumors. Transfection of ductal pancreas cancers with p48 cDNA did not activate the expression of amylase nor a reporter gene under the control of the rat elastase promoter. In some cell lines, p48 was detected in the nucleus whereas in others it was cytoplasmic, as in one human acinar tumor. Together with prior work, our findings indicate that p48 is associated with the acinar phenotype of exocrine pancreas cancers and it is necessary, but not sufficient, for the expression of the acinar phenotype.

Pancreatic tumors with cystic dilatation of the ducts: intraductal papillary mucinous neoplasms and intraductal oncocytic papillary neoplasms.

Intraductal papillary mucinous neoplasms (IPMNs) and intraductal oncocytic papillary neoplasms (IOPNs) are the 2 types of intraductal neoplasms of the pancreas that may appear cystic because of dilatation of the ducts. Both are characterized by intraductal proliferation of mucinous cells usually arranged in papillary patterns. This proliferation is often associated with intraluminal mucin accumulation, which produces cystic dilatation of the ducts, mimicking mucinous cystic neoplasms. Endoscopic and radiologic studies and careful macroscopic examination are crucial for the correct diagnosis of IPMNs and IOPNs by showing the origin within the native ducts. Microscopically, these tumors display a spectrum of cytoarchitectural atypia that ranges from adenoma to borderline and to carcinoma-in-situ. Although they are defined as "intraductal tumors," IPMNs and IOPNs are associated with invasive carcinoma in about a third of the cases. It, therefore, appears that, like mucinous cystic neoplasms or pancreatic intraepithelial neoplasia involving the smaller ducts associated with ordinary ductal adenocarcinomas, these tumors are precursors of invasive carcinoma. Invasive carcinomas associated with IPMNs are of either tubular or colloid (mucinous noncystic) types, whereas those associated with IOPNs may be oncocytic. Even in the presence of invasive carcinoma, these tumors may follow a more protracted clinical course than ordinary ductal adenocarcinoma. On the other hand, rare examples of IPMNs after an aggressive clinical course despite the lack of any identifiable invasive carcinoma are on record. Therefore, IPMNs and IOPNs should be examined carefully and sampled extensively, first, to confirm that the main pathology is an intraductal process and, more importantly, to rule out the presence of an invasive carcinoma.

Simultaneous mucinous cystadenoma of ovary and mucinous cystadenocarcinoma of pancreas.

Mucinous cystic tumors were discovered synchronously in the tail of the pancreas and in the right ovary of an adult female. Both tumors were amenable to surgical resection. The pancreatic tumor was a noninvasive mucinous cystadenocarcinoma and the ovarian tumor was a mucinous cystadenoma. We feel these tumors represent two primaries, an uncommon occurrence, and not a single primary tumor with metastasis.

Growing colorectal tumors: minimizing microbial and stromal competition and assessing in vitro selection pressures.

Development of primary colorectal cancer cell lines ishampered by contamination from regional microbes, overgrowthof stromal cells, and purported genetic drift from selectionpressures in vitro. We initiated 32 primaryadenocarcinomas, 3 recurrences and 6 distant metastases incell culture. Twelve cell lines from eleven tumors weregenerated (26.8%) overall. Nine of 32 primary tumorsyielded 10 cell lines, 5 were lost to contamination, 13 wereoverwhelmed by stromal cells, and 5 demonstrated no growth.Addition of isobutyl methyl xanthine (IBMX) to culturelimited fibroblastoid growth. There was no associationbetween tumor location (p = 0.535, mid-P), degree ofdifferentiation (p = 0.850, mid-P) or clinicopathologic stage(p = 0.400, mid-P), and the ability of cells to becomeestablished in culture. The majority of cell lines hadsimilar nuclear DNA content and expression of cell-surfaceantigens compared with their parent tumors. Microbialcontamination and stromal cell overgrowth present thegreatest obstacle to capturing a representative bank ofcolon tumors in vitro.

Molecular pathology of invasive carcinoma.

Abnormalities of several oncogenes and tumor suppressor genes have been identified in carcinomas of the pancreas during the last decade, and multiple genetic changes have been demonstrated in individual carcinomas. The variety of genetic changes suggests that multiple etiologic factors contribute to carcinogenesis in the pancreas. Several of these changes are characteristically found in specific types of tumors, suggesting that different causes and molecular mechanisms are involved. One example is the loss of heterozygosity at the von Hippel-Lindau (VHL) gene locus in both wild type and hereditary serous cystadenomas, and another is the virtual absence of K-ras mutation and p53 abnormalities in acinar cell carcinomas, whereas both are frequently found in ductal adenocarcinomas. Multiple lines of evidence place K-ras mutation very early and loss of p53 and p16 as late events during ductal cell carcinogenesis. The timing and order of other genetic changes such as loss of the DPC4 tumor suppressor function is less certain.

Mechanistic analysis and chemoprevention of pancreatic carcinogenesis.

Thirteen presentations given at a workshop held in November 1997, under the sponsorship of The Princess Takamatsu Cancer Research Fund in Nara, Japan, are summarized. Topics include studies of initiation, promotion, and chemoprevention in animal models of pancreatic carcinogenesis, molecular and histopathologic studies of human carcinomas, and epidemiologic studies. A recurring focus was to explore ways that knowledge of molecular mechanisms of carcinogenesis and molecular abnormalities in carcinomas can guide approaches for prevention and treatment of pancreatic cancer.

K-ras mutation in focal proliferative lesions of human pancreas.

The K-ras gene is mutated in > or =75% of human pancreatic adenocarcinomas and in a number of hyperplastic ductal lesions from noncarcinoma patients. In this study, the incidence of K-ras mutation was determined in a spectrum of focal proliferative pancreatic lesions to evaluate their preneoplastic significance. PCR-based mutation-enriched RFLP analysis was used to identify mutations in codon 12. Immunostaining for Ki67 and p53 was also performed. Forty-seven % of intraductal nonpapillary hyperplasias (8 of 17) contained codon 12 mutations, as did 55% of adenomatoid hyperplasias (6 of 11). This rate increased to 61% in papillary hyperplasias (27 of 44) and to 78% when there was severe dysplasia (7 of 9). The fraction of cells staining for the Ki67 proliferation marker showed a general correlation with the rate of K-ras mutation. Nuclear staining for p53 protein was seen only in two ductal lesions with severe dysplasia. No mutations were found in normal acinar tissue (n = 38), squamous metaplasia (n = 13), ductal complexes (n = 8), or focal acinar cell dysplasia (n = 5). There seemed to be a general correlation of proliferative potential with the presence of K-ras mutation in ductal lesions. However, because of the high prevalence of lesions with K-ras mutations, we conclude that this mutation alone cannot be taken as proof of significant risk for progression to carcinoma. Efforts to use the presence of K-ras mutations in DNA harvested from pancreatic juice or duodenal aspirates as an approach for diagnosis of occult pancreatic carcinoma seem vulnerable to a high false-positive rate.

Azaserine-induced rat pancreas tumor model with transplantable cultured cells.

The purpose of this study was to describe the inoculation technique and patterns of growth as well as to characterize typical histological features of Lewis rat subcutaneous and intrapancreatic tumors, induced by inoculation of cultured pancreatic cancer cells (DSL-6A/C1). Subcutaneous inoculation of cultured cells produced a solid tumor that was a locally invasive, well- to moderately differentiated ductal adenocarcinoma. Tumor take was 100% in animals 5 weeks of age; tumor growth was consistent and predictable and a tumor volume of approximately 1 cm3 was reached in 8 weeks. After intrapancreatic transplantation the tumors showed the same histological features as subcutaneous tumors. During inoculation carcinoma cells easily spread around the injected area, and after 2 weeks both pancreatic tumors and superficially infiltrating carcinomas were found in the liver and spleen and around the peritoneum. Tumor take was 60% and tumor growth was somewhat indefinite and unpredictable in the pancreas. However, by reducing the injected carcinoma cell volume and solving the technical problems, 100% tumor take was achieved. The tumor volume reached 2 mm3 during 2 weeks and larger tumors showed a tendency for invasion. According to our results, subcutaneous as well as intrapancreatic tumor induction with cultured cells offers a model for pancreatic cancer studies.

Evaluation of p53 mutation in pancreatic acinar cell carcinomas of humans and transgenic mice.

Mutation of the p53 tumor suppressor gene is found in a large number of exocrine pancreatic tumors. The majority of these tumors is of the ductal cell phenotype. We examined 12 human acinar cell carcinomas and 42 transgenic mouse carcinomas (including 36 acinar cell tumors, four islet cell tumors, and two liver metastases of primary acinar cell tumors) for evidence of p53 mutation. Immunohistochemistry was used to identify p53 protein in tumor sections. To evaluate p53 exons 5-8, heteroduplex analysis was used on formalin-fixed, paraffin-embedded human tumor DNA, and single-strand conformation polymorphism analysis was used on frozen mouse tumor DNA. No molecular evidence of p53 mutation was found in any of the tumor DNAs and immunohistochemical data were regarded as negative. This study provides evidence that acinar cell carcinogenesis in both humans and transgenic mice is independent of p53 mutation.

Observations on the etiology and pathogenesis of intraductal papillary-mucinous neoplasms of the pancreas.

During the last two decades, intraductal papillary-mucinous neoplasms have been recognised as a group that should be distinguished from ductal adenocarcinomas. The literature for these tumors is confusing with the use of many different terms such as mucinous duct ectasia and, sometimes, there is a failure to distinguish them from mucinous cystic tumors. The recognition and apparently increasing incidence of these neoplasms is perhaps entirely attributable to improved diagnostic and imaging methods such as endoscopic retrograde pancreatography and computerized tomography. Experimental, molecular, and epidemiologic data suggest that the etiology of IPMN overlaps that of solid ductal adenocarcinomas.

Mechanisms for the pancreatic oncogenic effects of the peroxisome proliferator Wyeth-14,643.

Several peroxisome proliferators have been shown to produce pancreatic acinar cell hyperplasia/adenocarcinomas in 2-year bioassays with rats: ammonium perfluorooctanoate (C8), clofibrate, methylclofenapate, HCFC-123, and Wyeth-14,643 (WY). We have used in vitro (C8, WY) and in vivo (WY) approaches to examine several possible mechanisms of pancreatic tumorigenesis by peroxisome proliferating compounds. These mechanisms include cholecystokinin receptor agonism (CCK(A)), trypsin inhibition, alterations in gut fat content, cholestasis, and altered bile flow/composition. All of these mechanisms enhance pancreatic growth either by binding to the CCK(A) receptor or by increasing plasma CCK levels. In vitro experiments using a receptor competition binding assay demonstrated that WY and C8 do not bind directly to the CCK(A) receptor. In a continuous spectrophotometric assay, WY and C8 also failed to inhibit trypsin, a common mechanism for increasing plasma CCK levels. These in vitro results suggested that WY was not acting via the two most common mechanisms for modulation of pancreas growth. Two types of in vivo experiments were conducted. The subchronic study (2-month duration) was designed primarily to detect early changes in pancreatic growth such as those mediated by compounds that inhibit trypsin or act as CCK(A) receptor agonists. The chronic study (6 months) was designed primarily to evaluate whether the pancreatic lesions were secondary to hepatic changes such as cholestasis and/or altered bile flow/composition. In the in vivo experiments, male Crl:CDBR rats were fed diets containing 0 or 100 ppm WY. In the subchronic study WY-treated rats had a twofold increase in mean relative liver weights, an eightfold increase in hepatic peroxisomal proliferation, and a fourfold increase in hepatocyte cell proliferation after 1 week which remained elevated throughout the 2 months of treatment. In contrast, no pancreatic weight effects, increases in plasma CCK, or acinar cell proliferation was seen through 2 months in the WY group when compared to the control group. Fecal fat concentrations were also measured at 2 months and demonstrated no difference between control and WY-treated animals. The absence of any early pancreas changes in the subchronic study is consistent with the in vitro data which demonstrated that WY is not a CCK(A) agonist or a trypsin inhibitor. The chronic study demonstrated increases in pancreatic weights at 3 months (6% above control) and 6 months (17% above control), as well as increased CCK plasma levels in the WY-treated group. Liver effects in the chronic study paralleled those of the subchronic time points. Clinical pathology endpoints including increased serum concentrations of bile acids, alkaline phosphatase, and bilirubin were indicative of cholestasis in the chronic WY-treated group. The cholestasis may be responsible for the downward trend in total bile acid output, both of which may contribute to the modest increases in plasma CCK levels. These results indicate that chronic exposure to WY causes liver alterations such as cholestasis, which may increase plasma concentrations of CCK. Hence, WY may induce pancreatic acinar cell adenomas/adenocarcinomas via a mild but sustained increase in CCK levels secondary to hepatic cholestasis.

Small intraductal papillary-mucinous adenomas of the pancreas.

Intraductal papillary-mucinous tumors in the pancreas have recently been described as a distinct entity, but the classification of these neoplasms remains under debate. Of issue is the biologic potential of these lesions and the predictability of clinical behavior on the basis of histologic appearance. We report two cases with intraductal papillary tumors formed by highly differentiated mucin-containing columnar epithelium found incidentally at autopsy. Both were classified as adenomas on the basis of histologic features. Neither patient experienced symptoms from their lesion during life and both died of other causes. Both lesions were studied by the polymerase chain reaction and were found to harbor codon 12 mutations in the c-K-ras gene. The concept that these neoplasms represent an early stage in an adenoma-carcinoma sequence is discussed.

The case for parallel classification of biliary tract and pancreatic neoplasms.

Most primary epithelial neoplasms found in the intrahepatic and extrahepatic biliary tracts and the pancreas have similar histologic appearances. The degree of similarity of tumor types is greater than is commonly appreciated and extends to rare neoplasms. It has been suggested that parallel classifications could be used for tumors arising in these sites. However, significant differences in terms used to denote histologically identical neoplasms are found in the current World Health Organization (WHO) classification of biliary tract tumors and the new WHO classification of exocrine pancreatic neoplasms. Mutations of oncogenes and abnormalities of tumor suppressor genes found in ductal adenocarcinomas from the two sites are also similar, although they have somewhat different frequencies. These histologic and molecular similarities provide strong support for the use of parallel descriptive diagnostic terminology for neoplasms arising in the pancreas and the biliary tract. The revised WHO classification of exocrine pancreatic neoplasms accommodates most types of biliary tract neoplasms and provides a new basis for standardization of nomenclature.