Normal Pancreata Research Articles

Comprehensive analysis of matrix metalloproteinase and tissue inhibitor expression in pancreatic cancer: increased expression of matrix metalloproteinase-7 predicts poor survival.

To enable the design of improved inhibitors of matrix metalloproteinases (MMPs) for the treatment of pancreatic cancer, the expression profiles of a range of MMPs and tissue inhibitors of MMPs (TIMPs) were determined. Nine MMPs (MMPs 1-3, 7-9, 11, 12, and 14) and three TIMPs (TIMPs 1-3) were examined in up to 75 pancreatic ductal adenocarcinomas and 10 normal pancreata by immunohistochemistry. Eighteen additional pancreatic ductal adenocarcinomas and an additional eight normal pancreata were also analyzed by real-time reverse transcription-PCR and additionally for MMP-15. There was increased expression by immunohistochemistry for MMPs 7, 8, 9, and 11 and TIMP-3 in pancreatic cancer compared with normal pancreas (P < 0.0001, 0.04, 0.0009, 0.005, and 0.0001, respectively). Real-time reverse transcription-PCR showed a significant increase in mRNA levels for MMP-11 in tumor tissue compared with normal pancreatic tissue (P = 0.0005) and also significantly reduced levels of MMP-15 (P = 0.0026). Univariate analysis revealed that survival was reduced by lymph node involvement (P = 0.0007) and increased expression of MMP-7 (P = 0.005) and (for the first time) MMP-11 (P = 0.02) but not reduced by tumor grade, tumor diameter, positive resection margins, adjuvant treatment, or expression of the remaining MMPs and TIMPs. On multivariate analysis, only MMP-7 predicted shortened survival (P < 0.05); however, increased MMP-11 expression was strongly associated with lymph node involvement (P = 0.0073). We propose that the principle specificity for effective inhibitors of MMPs in pancreatic cancer should be for MMP-7 with secondary specificity against MMP-11. Moreover, these studies indicate that MMP-7 expression is a powerful independent prognostic indicator and potentially of considerable clinical value.

Expression of serum response factor (SRF) in normal and regenerating pancreas

Background: SRF is a transcription factor that controls mesodermal differentiation. To date, there has been no report demonstrating the role of SRF in endodermally derived tissue. Genetic ablation of SRF results in embryonic lethality precluding analysis of SRF in pancreatic development. We sought to determine the expression of SRF in the normal adult rodent pancreas and during pancreatic regeneration (PR). Methods: Pancreata from adult rodents were harvested and fixed in 4% paraformaldehyde. Sections were stained for SRF expression using a polyclonal antibody. 90% pancreatectomies were performed and regenerating pancreatic remnants harvested at 0, 24, 72 hrs and 7 days. Tissues were processed and stained as above. Sham laparotomies served as controls. Results: Nuclear staining for SRF was observed in normal pancreatic ductal, acinar, and islet cells. Staining was stronger in islet cells compared to ductal and acinar cells. Double staining for insulin demonstrated islet SRF staining was predominately within the beta cells. During PR, SRF expression was increased with maximal expression at 36 hours (Fig 1B). SRF expression continued to be increased compared to baseline and sham at 7 days. In contrast to normal pancreata, SRF expression in the regenerating pancreas was found predominately in the acinar cytoplasm (AC). Conclusion: For the first time, SRF expression is observed in all three cellular components of the pancreas. During PR, SRF expression is increased and redistributed to the acinar cytoplasm. The data suggests SRF may be an important regulator of pancreatic growth, replication, and differentiation.

Clinicopathological Correlates of Pancreatic Intraepithelial Neoplasia: A Comparative Analysis of 82 Cases With and 152 Cases Without Pancreatic Ductal Adenocarcinoma

Pancreatic intraepithelial neoplasia is often associated with pancreatic ductal adenocarcinoma and is presumed to be its precursor. It has been difficult to determine the frequency of these lesions because until recently, there was no consensus regarding the terminology and criteria for their grading. Here we compare the frequency and clinical correlates of pancreatic intraepithelial neoplasia in pancreata involved by ductal adenocarcinoma and in benign ones, using the criteria put forward recently. We evaluated pancreatectomy specimens from 82 patients with ductal adenocarcinoma and 152 patients who underwent pancreatectomy for reasons other than primary malignancy (trauma, pancreatitis, and metastatic tumor to pancreas) for the presence, grade, and number of foci of pancreatic intraepithelial neoplasia. Cases were graded by the highest grade of pancreatic intraepithelial neoplasia focus identified. An average of 5.3 sections of pancreas was available for evaluation (range, 1–28 sections). Overall, the frequency of pancreatic intraepithelial neoplasia lesions in ductal adenocarcinoma patients, including Grade 1A (mucinous duct lesions), was 82%, which was significantly higher than the one in benign pancreata −54%, P < .001. There was a progressive increase from normal pancreata to pancreatitis and to ductal adenocarcinoma in the frequency of overall pancreatic intraepithelial neoplasia lesions (16%, 60%, and 82%, respectively) and Grade 3 pancreatic intraepithelial neoplasia (0%, 4%, and 40%, respectively). In most instances, in any given case of higher-grade pancreatic intraepithelial neoplasia lesion, there were also several foci of lower grade lesions. The frequency of higher-grade pancreatic intraepithelial neoplasia lesions (2 and 3) in pancreata resected for ductal adenocarcinoma was 59%, significantly higher than in those without primary carcinoma (17%). This progressive increase in frequency of pancreatic intraepithelial neoplasia from incidental pancreatectomies (presumed to have a nonpathologic pancreas) to pancreatitis (considered a risk factor for carcinoma) and to ductal adenocarcinoma constitutes an indirect support for the precancerous role attributed to pancreatic intraepithelial neoplasia lesions. The relatively high absolute occurrence of pancreatic intraepithelial neoplasia Grade 1A (mucinous duct lesions) in benign conditions (43%) suggests that this group represents a combination of neoplastic and non-neoplastic lesions.

Molecular alterations in pancreatic carcinoma: expression profiling shows that dysregulated expression of S100 genes is highly prevalent.

In order to expand our understanding of the molecular changes underlying the complex pathology of pancreatic malignancy, global gene expression profiling of pancreatic adenocarcinoma compared with normal pancreatic tissue was performed. Human cDNA arrays comprising 9932 elements were interrogated with fluorescence-labelled normal and adenocarcinoma samples (nine tumours, three normal pancreata, and three cell lines). The data were analysed for differential gene expression, which was confirmed by serial analysis of gene expression (SAGE), digital differential display (DDD) analysis, and immunohistochemistry for selected cases. The array data were filtered to produce lists of a total of 75 genes significantly up-regulated or down-regulated in pancreatic adenocarcinoma. Two of those showing the highest differential were members of the S100 family of Ca-binding proteins, namely S100P and S100A6, and therefore the S100 genes were studied in more detail. By immunohistochemical analysis of custom-built, pancreas-specific tissue arrays and commercially available, normal/cancer tissue arrays that included a wide variety of different tumour types, differential expression of S100P protein was found to be almost exclusive to pancreatic cancer. S100P could therefore represent a useful biomarker for pancreatic adenocarcinomas.

Increased arylhydrocarbon receptor expression offers a potential therapeutic target for pancreatic cancer.

The arylhydrocarbon receptor (AhR) was initially identified as a member of the adaptive metabolic and toxic response pathway to polycyclic aromatic hydrocarbons and to halogenated dibenzo-p-dioxins and dibenzofurans. In the present study, we sought to determine the functional significance of the AhR pathway in pancreatic carcinogenesis. AhR expression was analysed by Northern blotting. The exact site of AhR expression was analysed by in situ hybridization and immunohistochemistry. The effects of TCDD and four selective AhR agonists on pancreatic cancer cell lines were investigated by growth assays, apoptosis assays, and induction of the cyclin-dependent kinase inhibitor p21. There was strong AhR mRNA expression in 14 out of 15 pancreatic cancer samples, weak expression in chronic pancreatitis tissues, and faint expression in all normal pancreata. In pancreatic cancer tissues, AhR mRNA and protein expression were localized in the cytoplasm of pancreatic cancer cells. TCDD and the four AhR agonists inhibited pancreatic cancer cell growth in a dose-dependent manner, and decreased anchorage-independent cell growth. DAPI staining did not reveal nuclear fragmentation and CYP1A1 and was not induced by TCDD and AhR agonists. In contrast, TCDD and AhR agonists induced the expression of the cyclin-dependent kinase inhibitor p21. In conclusion, the relatively non-toxic AhR agonists caused growth inhibition in pancreatic cancer cells with high AhR expression levels via cell cycle arrest. In addition, almost all human pancreatic cancer tissues expressed this receptor at high levels, suggesting that these or related compounds may play a role in the therapy of pancreatic cancer in the future.

Aberrant Methylation of the 5' CpG Island of TSLC1 Is Common in Pancreatic Ductal Adenocarcinoma and Is First Manifest in High-Grade PanINs

The recently identified tumor-suppressor gene TSLC1 on chromosome 11q23.2 is frequently inactivated in human non-small cell lung adenocarcinoma by DNA methylation-associated silencing. The aim of this study was to determine if TSLC1 is inactivated in adenocarcinoma of the pancreas. We analyzed 17 pancreatic cancer cell lines, 91 primary pancreatic adenocarcinoma, 46 pancreatic intraepithelial (PanIN) precursor lesions and 15 microscopically normal pancreata for methylation of the 5’ CpG island of the TSLC1 gene through methylation-specific PCR. We observed 5’ CpG methylation of TSLC1 in 4 of 17 cell lines (24%). In each cell line the aberrant methylation was associated with loss of TSLC1 expression by RT-PCR that was reversible after treatment with the DNA methyltransferase inhibitor 5-aza-2’- deoxycytidine. Furthermore, we observed that TSLC1 was methylated in 25 of 91 primary pancreatic adenocarcinomas (27%), and in 2 of 7 high-grade PanIN-3 lesions (29%), but not in low-grade PanIN (0 of 9 PanIN-2 and 0 of 30 PanIN-1) lesions or in normal pancreata (n=15). We conclude that epigenetic silencing of TSLC1 expression through 5’ CpG island associated methylation is common in pancreatic adenocarcinoma and is a late event in pancreatic neoplastic development.

Aberrant CpG island methylation in cancer cell lines arises in the primary cancers from which they were derived

A higher prevalence of epigenetic inactivation of tumor suppressor genes has been reported in cancer cell line populations compared to primary cancer populations. Cancer-related genes are commonly methylated in cancer cell lines but it is not known the extent to which tumor suppressor genes may be artificially methylated in vitro. We therefore examined 10 pancreatic cancer cell lines and corresponding primary tumors for aberrant DNA methylation of promoter CpG islands of eight genes and seven CpG islands. Using methylation-specific PCR (MSP), methylation was not detected at any of the 15 CpG islands in 15 normal pancreata or in an immortalized normal pancreatic duct epithelial (HPDE) cell line. Of 150 loci examined, 49 loci were methylated in both primary carcinomas and their corresponding cell lines, 95 loci were not methylated in either cell lines or their corresponding primary carcinomas. There were four loci methylated only in cell lines while another two loci were methylated only in primary carcinomas. Overall, the methylation status of primary carcinomas and their cell lines were concordant in 96% of cases (144 of 150) (J statistic; J=0.92, P<0.0001). We conclude that most of the DNA methylation of tumor suppressor genes observed in cancer cell lines is present in the primary carcinomas from which they were derived.

Alteration of the Langerhans islets in pancreatic cancer patients.

An abnormal glucose metabolism occurs in up to 80% of pancreatic cancer patients shortly or a few months before the first clinical admission. Reasons for this abnormality are obscure. We investigated immunohistochemically the pattern of islets in 14 pancreatic cancer specimens and used 14 chronic pancreatitis samples and 10 normal pancreata as controls. To study the topographical relationship of these islets to the cancer, islets in four different arbitrary zones within and around the cancer were evaluated. Ten out of 14 cancer specimens showed a significant loss of beta cells (p < 0.005) and eight of them also showed a significant increase of alpha cells (p < 0.005), all of them from hyperglycemic patients. Most affected islets were found within zone 1 (intratumoral) and zone 2 (peritumoral), to a lesser extent in zone 3 (acini close to tumor) and none in zone 4 (acini remote from tumor). No comparable changes were found in chronic pancreatitis patients. The incidence of 72% with alteration of islets in our material correlates with the frequency of abnormal glucose levels in human pancreatic cancer patients. Our findings support the notion that islet cell abnormalities is likely caused by substances released from cancer cells.

Changing distribution of islets in the developing human pancreas: a computer-assisted three-dimensional reconstruction study.

Tissue specimens of nine normal human pancreata from fetuses, neonates, and adults were subjected to serial sectioning and computer-assisted three-dimensional (3-D) reconstruction in an effort to study the growth of Langerhans' islets based on their distribution in the lobule and their relationship with the ducts and arterioles. Also, islet volumes were obtained by 3-D morphometry. In 24-week-old fetuses, the islets were shown to be in direct contact with the ducts, whereas in the neonates, they gradually became separated from the ducts, finally dispersing throughout the lobule in the adults. This transition seemed to allow islet hormones to have physiological effects on the exocrine function of the acinar tissue, making the pancreas achieve the endocrine-exocrine correlation. However, half of the islets remained next to the ducts even in the adult pancreata. With regard to the relationship between the islets and arterioles, "arteriolar" islets having an afferent arteriole accounted for approximately 20% in number, but their volume cumulated to approximately 70% of the total, comprising a greater part of the pancreatic endocrine gland.

Mutational activation of K-ras in nonneoplastic exocrine pancreatic lesions in relation to cigarette smoking status.

Cigarette smoking is among the few unequivocal risk factors for the development of pancreatic ductal adenocarcinoma (PDAC). Activating mutations in codon 12 of the K-ras protooncogene is a frequent and early molecular event in the pathogenesis of PDAC and a variety of nonmalignant ductal pancreatic lesions. The molecular epidemiologic relation between heavy cigarette smoking and mutational activation of K-ras in PDAC has been examined to a limited extent. The authors have examined the mutational status of K-ras in nonneoplastic pancreata in relation to cigarette smoking status. Archival formalin fixed paraffin embedded specimens of nonneoplastic pancreata (n = 39) were obtained from the American Cancer Society and evaluated histopathologically. Specimens from age- and gender-matched individuals were stratified into three groups: 1) those who never smoked cigarettes (n = 16), 2) those who smoked 1-2 packs/day for more than 20 years (n = 10 cases), and 3) those who smoked more than 2 packs/day for 20 or more years (n = 13). Cases were preselected from 77 specimens based on the quality, suitability, and cellularity of the archival tissues for analyses. Furthermore, none of the patients died of primary PDAC or had evidence of pancreatic metastases from an extrapancreatic primary tumor. Tissue sections were microdissected and deparaffinized, and genomic DNA was purified by standard proteinase K-phenol-chloroform extraction techniques. Genomic DNA was analyzed for mutations in codon 12 of the K-ras protooncogene by two mutant-allele-enriched polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assays and by multiplex PCR-based ligase chain reaction (LCR) analyses. Analyses of multiple microdissected pancreata specimens from 39 cases revealed wild-type K-ras codon 12 sequences in both nonsmoking individuals and those who smoked 1-2 packs/day for 20 or more years. K-ras codon 12 mutations were confirmed by PCR-RFLP and PCR-LCR assays in 5 of 13 pancreata cases (39%) obtained from individuals who smoked more than 2 packs of cigarettes/day for 20 years or more (P < 0.005). The K-ras mutation spectra revealed two G-->T transversions, one G-->C transversion and two G-->A transitions. There was no clear relation between the incidence or spectra of mutations and pancreatic histopathology, as overtly normal pancreata as well as pancreata with squamous metaplasia, periductal fibrosis, and ductal atypia revealed reproducible K-ras alterations. Similarly, among those 34 cases in which a wild-type K-ras sequence was revealed by both approaches, a similar histopathologic profile was evident. Mutational activation of codon 12 of the K-ras protooncogene was confirmed reproducibly by mutant allele-enriched PCR-RFLP and multiplex PCR-LCR analyses in 39% (5 of 13) of archival nonneoplastic pancreata from age- and gender-matched individuals who smoked more than 2 packs of cigarettes/day for 20 or more years. The presence of a mutated or wild-type or K-ras was independent of the histopathologic profile of the 39 cases examined. The data provide further suggestive molecular epidemiologic evidence of an association between a major and unequivocal risk factor for PDAC (heavy cigarette smoking) and mutations in a molecular target (K-ras), the activation of which is an important and early event both in the pathogenesis of PDAC and in the development of a variety of nonneoplastic ductal pancreatic lesions.

Bak expression and cell death occur in peritumorous tissue but not in pancreatic cancer cells

Bak is a pro-apoptotic member of the Bcl-2 family whose genes are involved in regulation of programmed cell death. Using in situ hybridization, immunohistochemistry, and Northern blot analysis, we studied the expression of Bak in specimens from 12 normal pancreata and 26 primary pancreatic cancers, and correlated the findings with the clinical and histopathologic data of the patients. By comparison with normal pancreas, Northern blot analysis demonstrated a 2.5-fold increase of Bak messenger RNA expression in the tumor samples ( P <0.001). Elevated levels were found in 15 of the 26 pancreatic cancer tissue specimens. In these samples Bak expression was increased 4.3 fold ( P <0.001). No association was detected between Bak expression and tumor stage. In situ hybridization and immunohistochemistry revealed that the tumor cells themselves and the stroma cells expressed only low levels of Bak. In contrast, in regions adjacent to the tumor, which showed chronic inflammation, there was always high expression in the acinar and inflammatory cells, explaining the increased Bak levels found in the tumor samples by means of Northern blot analysis. In the normal pancreas the expression of Bak was generally moderate in the acinar cells and low in the ductal and islet cells. In situ analysis using the terminal deoxynucleotidyl transferase method further showed that there was extensive cell death in the peritumorous areas with chronic inflammation. Taken together, these results suggest that in pancreatic cancer Bak expression and programmed cell death are present in cells that are localized in regions of chronic inflammation surrounding the pancreatic cancer cells but not in the tumor cells themselves, a situation that may facilitate tumor growth and spread.

Stenting does not decompress the pancreatic duct as effectively as surgery in experimental chronic pancreatitis

Background: In humans with chronic pancreatitis (CP), pancreatic interstitial pressure (IP) is elevated and pancreatic blood flow (PBF) is reduced. The efficacy of surgical decompression (SD) of the pancreatic duct (ie, pancreaticojejunostomy) is believed to be due to its ability to decrease IP and pancreatic vascular resistance (R p), which increases PBF. Pancreatic duct stenting (STE) also probably reduces IP and R p, which may explain its efficacy. The purpose of this study was to compare the efficacy of SD with STE. Methods: CP in cats was created by narrowing the main pancreatic duct. Six weeks later, CP and normal pancreata were isolated and perfused ex vivo under basal conditions and after secretin stimulation. In normal and CP glands, IP and perfusion pressure were measured and R p (U) was calculated. In two additional groups, the pancreatic duct was decompressed, either by stenting or by complete transection of the duct with a longitudinal capsulotomy. Results: In CP glands, IP and R p were increased and secretory output was markedly reduced compared with the normal (0.65 ± 0.30 mm Hg and 0.46 ± 0.04 U vs 3.90 ± 0.80 mm Hg and 1.68 ± 0.05 U; P < .05). Secretin administration (2 units) increased IP and R p in CP glands (6.60 ± 1.10 mm Hg and 2.87 ± 0.07 U; P < .05), but these values did not change in normal glands (0.81 ± 0.20 and 0.53 ± 0.03 U; NS). STE and SD decreased IP and R p in CP glands ( 2.20 ± 0.20 to 1.0 ± 0.40 mm Hg and 1.20 ± 0.015 to 0.90 ± 0.01 U, respectively; P < .05). Both methods prevented an increase of IP and R p after secretin administration. IP and R p decreased to a greater degree following SD, compared with STE ( P < .05). Conclusions: Both STE and SD decreased IP and R p in this experimental model of CP. However, SD was significantly more effective than STE. (Surgery 1998;124:561-7.)

The plasminogen activator/plasmin system is up-regulated after acute necrotizing pancreatitis in human beings

Background: Proteolysis and formation of new extracellular matrix components are important mechanisms in tissue remodeling and repair. In this study we analyzed the expression and distribution of the urokinase plasminogen activator (uPA), its membrane receptor (urokinase plasminogen activator receptor [uPAR]), and its inhibitor (plasminogen activator inhibitor-1 [PAI-1]) in acute necrotizing pancreatitis in human beings. In addition, we studied the concomitant expression of transforming growth factor–beta-1 (TGF-β 1), which is activated by uPA from its precursor and is a potent regulator and stimulator of formation of extracellular matrix. Methods: With immunohistochemistry and Northern blot analysis, the expression and cellular distribution of uPA, uPAR, PAI-1, and TGF-β 1 were determined in 12 normal pancreata obtained from organ donors and 12 pancreatic tissues obtained from patients undergoing operation because of complications of acute necrotizing pancreatitis. Results: Northern blot analysis showed enhanced expression of uPA, uPAR, and PAI-1 in eight of 12, seven of 12, and nine of 12 necrotizing pancreatitis samples, respectively, compared with normal control samples. In addition, increased TGF-β 1 mRNA expression was present in eight of 12 necrotizing pancreatitis samples. In contrast, amylase mRNA expression was markedly decreased in the samples of acute necrotizing pancreatitis. Immunohistochemistry revealed elevated uPA, uPAR, and PAI-1 immunoreactivity in the remaining acinar and ductal cells adjacent to the necrotic tissue areas. In contrast, acinar and ductal cells that were located farther from pancreatic necrosis exhibited less uPA and uPAR immunoreactivity. A similar staining pattern in samples of necrotizing pancreatitis was found for TGF-β 1. Conclusions: Up-regulation of uPA and uPAR, which activate proteolysis, might create a milieu that enhances lysis and removal of pancreatic necrosis. The increase in TGF-β 1 might result from the enhanced catalytic conversion of its precursors by uPA, which subsequently might stimulate formation of extracellular matrix, formation of granulation tissue, and fibrosis. (Surgery 1998;124:79-86.)

Up-regulation of cytochrome P450 1A2, 2C9, and 2E1 in chronic pancreatitis.

The oxidative metabolism of xenobiotics is effected mainly by cytochrome P450 enzymes (CYP), which are expressed as a family of genetically related enzymes primarily in hepatocytes. The pancreas is among the extrahepatic tissues expressing CYP, and it has been suggested that intermediates generated by them might be of pathogenetic significance for diseases of the pancreas such as chronic pancreatitis. We studied 10 surgical resection specimens by immunohistochemistry with polyclonal antibodies against recombinant human CYP 1A1, 1A2, 2C9, 2E1, and 3A and used tissues from 11 normal pancreata as controls. In addition, we assayed microsomal preparations for their capacity to metabolize verapamil. In normal pancreata weak to moderate expression of all enzymes was demonstrated immunohistochemically in up to 50% of duct epithelia, acinar cells, and islet cells. In contrast, in chronic pancreatitis an up-regulation was observed, with immunohistochemical positivity in some cases in up to 100% of duct epithelia and acinar cells. The oxidative capacity of microsomal preparations from chronic pancreatitis was higher than that of preparations obtained from control tissues; compared to liver microsomes, however, it was low. The up-regulation of CYP may have pathogenetic significance for chronic pancreatitis. Yet considering the pancreas' capacity for conjugation reactions, conceivably low levels of reactive intermediates could effectively undergo inactivation.

Determination of pancreatic ductal carcinoma histogenesis by analysis of mucous quality and K-ras mutation.

The authors sought to elucidate the histogenesis of pancreatic ductal carcinoma by correlating K-ras mutation with mucus type in normal epithelium, mucous cell hyperplasia (MCH), and carcinoma. Seventy-four solid-type carcinomas (SCs), 23 ductectatic-type carcinomas (DCs), and specimens of 24 normal pancreata were studied. By histochemical staining, normal duct epithelia, areas of MCH, and carcinomas were classified as having sulfo-type or sialo-type mucus. Foci from normal, DC, SC, sulfo-type, or sialo-type specimens were assessed for K-ras mutation at codon 12 by nested polymerase chain reaction and restriction fragment length polymorphism. Of the SCs, 9 were sulfo-type and 65 were sialo-type; all DC specimens were sialo-type, and all normal epithelia were sulfo-type. All foci of sulfo-type, nonneoplastic epithelia were negative for K-ras mutation. In contrast, 124 of 313 sialo-type MCH foci (40%) had a K-ras mutation. Of 74 SCs, only 3 of 9 sulfo-type tumors (33%) were positive for the mutation. Sixty of 65 sialo-type SCs (92%) had a K-ras mutation, whereas 15 of 23 sialo-type DCs (65%) had a mutation. K-ras mutant carcinomas (including both SCs and DCs) were associated with K-ras mutant MCH in 109 of 198 MCHs (55%), whereas carcinomas without a K-ras mutation had mutations in 6 of 68 MCHs (9%). MCH in normal pancreata revealed K-ras mutations in 9 of 51 foci (18%). In addition, in K-ras mutant carcinomas, frequency of K-ras mutation in MCH increased from 27% (11 of 41 foci) of nonpapillary MCHs to 62% (98 of 157 foci) of papillary MCHs; but in K-ras wild-type carcinoma, the mutation rate in MCH was unchanged from 12% (3 of 26 foci) to 7% (3 of 42 foci) in nonpapillary and papillary foci, respectively. These results suggest a strong relationship between the risk of pancreatic carcinoma and the presence of combinations of K-ras gene mutation, papillary growth, and expression of sialomucin in foci of MCH.

The lobular architecture of the normal human pancreas: a computer-assisted three-dimensional reconstruction study.

Tissue specimens of the normal pancreata from three adult patients were subjected to serial sectioning and computer-assisted three-dimensional reconstruction of the lobules, blood vessels, pancreatic ducts, and islets, to establish the anatomical relation among these structures. Most lobules, although more or less imperfectly demarcated by septa, were found to receive a single duct (lobular duct), justifying their definition as "glandular lobules." However, there were also lobules receiving not only a lobular duct of their own but a small accessory duct from a neighboring lobule. On the other hand, these lobules were shown not to correspond to the territories of arterial supply, leaving no ground for defining them as "vascular lobules."

Molecular pathology of primary and metastatic ductal pancreatic lesions

The molecular pathology underlying the development and progression of ductal pancreatic adenocarcinoma is poorly understood relative to that of other major cancers in industrialized societies. The frequency, nature, and distribution of p53 abnormalities, their temporal relationship to the metastatic and clinicopathologic phenotypes of sporadic and familial pancreatic cancer, and their consequent effects on the genetics and expression of critical wild-type p53-regulated genes (mdm-2 and p21/WAF-1) warrant examination in pancreatic adenocarcinoma. This molecular and immunochemical study of the p53, mdm-2, and p21/ WAF-1 genes and gene products examined the largest series of nonneoplastic, neoplastic, and metastatic ductal pancreatic lesions reported to date in relation to clinicopathologic profile. Histologically confirmed specimens of primary (n = 136) and metastatic (n = 23) sporadic and familial ductal pancreatic adenocarcinoma lesions were subjected to immunochemical analyses of p53 expression in which a panel of 3 antibodies was utilized. A panel of nonneoplastic but histologically abnormal pancreatic lesions (n = 77) from individuals with varied histories of cigarette smoking were subjected to similar immunohistochemical examinations. In addition, 3 specimens from patients with chronic pancreatitis, 2 specimens of normal fetal pancreata, and 16 specimens of normal adult pancreata were examined as control tissues. Suitable frozen and archival microdissected tumor lesions were evaluated for mutations in exons 4-9 of the p53 gene by single strand conformation polymorphism (SSCP) and dideoxy sequencing analyses in which two distinct sets of outer and nested intron-based amplification primers were used for each exon. A subset of 25 tumor specimens and 18 tumor-derived cell lines for which the p53 mutation status was known were examined for amplification and/or overexpression of the mdm-2 gene; amplification was determined by Southern hybridization and overexpression by immunohistochemical and Western blot analyses. Similarly, mutations in the coding region of p21/WAF-1 gene were examined by SSCP and DNA sequence analyses, and steady-state expression of the p21/WAF-1 protein was assessed by Western blot analysis in these subsets of tumors and tumor-derived cell lines. Positive ductal nuclear p53 immunostaining was demonstrated in 56% of primary tumors and 54% of metastatic lesions. The frequency did not differ significantly between sporadic and familial lesions, and immunostaining was not observed in ductal, acinar, or islet cell elements of normal pancreata or histologically abnormal benign pancreatic lesions from cigarette smokers. A total of 70% of tumor samples revealed reproducible SSCP abnormalities for p53; 42% of these were found in exons 7 and 8. DNA sequence analysis of cases with greater than 35% epithelial cellularity (n = 25) revealed 17 missense mutations, 12 of which were transitions. Seventy-five percent of these transitions were of G:C-->A:T type. A total of 22% of the p53 mutations identified were microdeletions, along with one insertional mutation at exon 8. None of the normal pancreata from sporadic or familial lesions revealed germ-line p53 alterations. Moreover, the frequency and spectra of p53 alterations exhibited no clear, statistically significant association with tumor grade, TNM stage, or patients' cigarette-smoking histories. The mdm-2 gene was neither amplified nor overexpressed immunochemically in a subset of ductal adenocarcinomas, and there was no clear relationship between the p53 mutation status and the status of the mdm-2 gene or protein. Similarly, SSCP and DNA sequence analysis of the p21/WAF-1 gene revealed only 2 genetic abnormalities in a series of 25 primary tumors and 15 tumor-derived cell lines; 1 of the cell lines also revealed the absence of immunoreactive p21/WAF-1 protein...

Expression of receptors for gut peptides in pancreata of BOP-treated and control hamsters.

The growth of pancreatic cancers may be influenced by certain gut peptides. However, the alteration of gut peptide receptors in the progress of pancreatic carcinogenesis is largely unknown. With storage phosphor autoradiography, this study visualized and characterized receptors for cholecystokinin (CCK), somatostatin (SST), bombesin (BBS), secretin and vasoactive intestinal peptide (VIP) in pancreata of control hamsters (n = 7) and pancreatic preneoplastic lesions (n = 10) or adenocarcinomas (n = 10) of N-nitrosobis(2-oxopropyl)amine (BOP)-treated hamsters. The specific CCK-A and secretin receptors expressed in normal pancreata were markedly reduced in pancreatic preneoplastic lesions and absent in adenocarcinomas. In the development of pancreatic tumours, the subgroup of SST receptors did not change, but both the affinity and binding capacity declined. In comparison with the binding of VIP to normal pancreata, specific VIP binding was significantly lower in preneoplastic lesions and almost absent in pancreatic adenocarcinomas. No specific binding for BBS was detected in normal pancreas or (pre)neoplastic lesions of hamster pancreas. The reduction or absence of receptors for CCK, secretin, SST and VIP in hamster pancreas with the progress of carcinogenesis suggests that in BOP-treated hamsters, pancreatic adenocarcinomas have, to a large extent, lost the hormone-dependent characteristics of the original tissue.

Amount and distribution of collagen in pancreatic tissue of different species in the perspective of islet isolation procedures

Because collagen is the major target in the enzymatic dissociation of the pancreas for islet isolation, we determined the amount of collagen and its distribution in a comparative study comprising normal pancreata of rat, dog, man, young pig, and adult pig. Collagen content was determined using a colorimetric method and its distribution was assessed in tissue sections stained with Sirius red. The collagen content is relatively low in the rat and adult pig pancreas, and the amount of collagen is relatively low in the septa of the rat and dog pancreas. Not the amount of collagen in the septa but collagen in the rest of the pancreas, mainly located between the acini, seems to determine the dissociation of the pancreatic tissue. This can be exemplified by the higher islet yields obtained from the adult vs. the young pig pancreas; the latter contains a higher total amount of collagen but a similar, relatively high, amount of collagen in the septa. A high amount of collagen surrounding the islets seems to be of secondary importance in islet isolations, because yields of the same magnitude are obtained from the canine and human pancreas containing a relatively low vs. high amount of collagen around the islets but a similar total collagen content. The rat pancreas contains both a low total amount of collagen and a high amount of collagen around the islets; therefore, the general experience that islet isolation procedures are effective in rats can be readily understood.

IDDM patients' sera recognize a novel 30-kD pancreatic autoantigen related to chymotrypsinogen.

We have examined, by western immunoblot analysis, the sera of 16 insulin-dependent diabetes mellitus patients (IDDM) for the presence of autoantibodies against proteins extracted from islet-cell enriched preparations of normal human pancreata. A novel putative autoantigen recognized by late stage IDDM patients sera was identified, and its amino acid sequence was partially determined. Islets of Langerhans were partially purified by a modified collagenase digestion procedure, and subsequent protein extracts were fractionated by one-dimensional or two-dimensional polyacrylamide gel electrophoresis (1-D or 2-D SDS-PAGE). Immunoblot analysis revealed a 30-kD species which was recognized by 4 of 16 IDDM patients sera, but none of 16 normal sera. The 30-kD protein, appeared as a single band on 1-D SDS-PAGE, but was resolved on 2-D gel electrophoresis as several distinct protein species with different isoelectric points (pI's), ranging from 7 to 9. The amino terminal sequence of one such species was partially determined by microsequencing, and the second through the fourteenth amino acids were found to be identical to the corresponding sequence in human chymotrypsinogen. The fifteenth through the eighteenth amino acids were different from the known chymotrypsinogen sequence. This region corresponds with the site that is cleaved to activate chymotrypsinogen. Based on the size and sequence homology, this antigen appears to be related to chymotrypsinogen. We conclude that this 30-kD species may be an autoantigen in some late stage IDDM patients.