The tobacco carcinogen NNK is stereoselectively reduced by human pancreatic microsomes and cytosols.

BACKGROUND/AIMS: Cigarette smoking increases the risk of cancer of the pancreas. The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the only known environmental compound that induces pancreatic cancer in laboratory animals. Concentrations of NNK are significantly higher in the pancreatic juice of smokers than in that of nonsmokers. The chiral NNK metabolite, (R,S)-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is itself a potent pancreatic carcinogen in rats. The carcinogenicity of NNAL is related to its stereochemistry; (S)-NNAL is a more potent lung tumorigen in the A/J mouse than is (R)-NNAL. In this study, we determined the potential of the human pancreas to convert NNK into NNAL. MATERIALS AND METHODS: Human pancreatic microsomes and cytosols were incubated with [5-(3)H]NNK, and the metabolic products were determined by high-performance liquid chromatography (HPLC). RESULTS: (S)-NNAL was the predominant isomer formed in all cytosolic incubations. In ten microsomal samples, NNAL was formed at an average rate of 3.8 +/- 1.6 pmol/mg/min; (R)-NNAL was the predominant isomer in this group. The average rate of NNAL formation in 18 other microsomal samples was significantly lower, 0.13 +/- 0.12 pmol/mg/min (p < 0.001); (S)-NNAL was the predominant isomer formed in this group. CONCLUSION: In human pancreatic tissues, there is intraindividual variability regarding the capacity for, and stereoselectivity of, carbonyl reduction of NNK.

The clinical efficacy of adjuvant systemic chemotherapy with gemcitabine and NSC-631570 in advanced pancreatic cancer.

BACKGROUND/AIMS: Recently we have shown that NSC-631570 (Ukrain) is a safe and effective drug in the treatment of unresectable pancreatic cancer. The aim of this study was to determine the effectiveness of the combined treatment with Gemcitabine and NSC-631570 in the adjuvant treatment of resected advanced pancreatic cancer. METHODOLOGY: 30 patients received adjuvant chemotherapy following surgical resection for pancreatic cancer. Chemotherapy consisted of Gemcitabine according to the Burris-protocol with weekly infusions of 1000 mg/sqm. Immediately following Gemcitabine infusion 20mg of NSC-631570 were administered intravenously over 15 minutes. RESULTS: WHO grade II toxicities were observed in 53%, no WHO grade III or IV toxicities occurred. In 80% of the patients recurrence of the disease was observed. The relapse-free survival time was 21.7 months. The actuarial survival rates were 86.7% after one year, 76.6% after two years, 46.7% after three years and 23.3% after five years. The median survival time according to Kaplan-Meier regression analysis was 33.8 months. CONCLUSIONS: Adjuvant chemotherapy in advanced stages of pancreatic cancer using the combination of Gemcitabine and NSC-631570 is a safe treatment and seems to lead to a prolonged survival. Although further investigation is needed to confirm these results, the combined treatment of Gemcitabine and NSC-631570 is a promising therapy for the adjuvant treatment of resectable advanced pancreatic cancer.

Detection of disseminated tumor cells in the lymph nodes of colorectal cancer patients using a real-time polymerase chain reaction assay.

INTRODUCTION: Postoperative treatment for colorectal cancer depends on tumor stage as defined by the International Union Against Cancer (UICC). Adjuvant chemotherapy is not recommended in patients without lymph node involvement (UICC stages I and II). As many as 20-30% of these patients, however, will develop recurrence. AIMS AND OBJECTIVES: We conducted this study to determine the presence of disseminated tumor cells in the lymph nodes by quantitative real-time polymerase chain reaction (QRT-PCR) for cytokeratin 20 (CK20) in an attempt to provide supplementary information compared to histopathological findings. MATERIALS AND METHODS: Using a standard QRT-PCR assay, we examined primary tumors and 391 lymph nodes from 31 patients with completely resected colorectal cancer. RESULTS: Of the 31 primary tumors, 29 were positive for CK20 by QRT-PCR. DISCUSSION: An examination of the lymph nodes from the 29 patients with CK20-positive primary tumors revealed that 35 (92.1% sensitivity) of the 38 histopathologically positive lymph nodes and 54 (16.7%) of the 324 histopathologically negative lymph nodes were positive by molecular analysis. CK20 expression was detected in 10 (100%) of 10 patients with a histopathologically positive lymph node status (pN1). In 9 (47.4%) of 19 patients with negative histopathological results (pN0), we detected a CK20 mRNA signal in at least one lymph node. Whereas eight patients with histopathologically negative lymph nodes could be upstaged on the basis of the molecular findings, no patient would be downstaged. CONCLUSION: Our results suggest that QRT-PCR for CK20 is a useful tool for the quantitative detection of micrometastases in the regional lymph nodes. We introduce a standardized procedure that integrates a molecular diagnostic technique in the clinical staging.

Systemic immune dysfunction in pancreatic cancer patients.

BACKGROUND AND AIMS: We investigated the immune status in 32 pancreatic cancer patients (PC) in comparison with healthy controls (HC). MATERIALS AND METHODS: Using flow cytometry, peripheral blood lymphocytes (PBL) were characterized by the expression of surface markers for T helper cells (CD4), T suppressor cells (CD8), B cells (CD19) and NK cells (CD56). The blastogenic response of PBL was analyzed after stimulation with concavalin A (ConA), phytohemagglutinin (PHA), pokeweed mitogen (PWM) and anti-CD3 antibodies. The serum levels of TNF-alpha, IL-1beta, IL-2, IL-10, IL-12, IL-18, IL-1RA, sIL-2R and TGF-beta were determined by ELISA. RESULTS: No differences in the distribution of peripheral immunocytes in PC were found, whereas the blastogenic response of peripheral blood lymphocytes (PBL) after stimulation with PHA or anti-CD3 antibodies was significantly decreased in PC. In PC, we found reduced serum levels of IL-2 and significantly elevated levels of TNF-alpha, TGF-beta1, IL-10, IL-2R, IL-1beta and IL-1RA. CONCLUSION: These data provide evidence for a systemic immune dysfunction in pancreatic cancer patients characterized by a shift towards a T helper cell type 2 cytokine profile, a significant elevation of substances related to T cell suppression and a reduced blastogenic response to PHA and anti-CD3 antibodies of PBL.

Pancreatic carcinoma cells induce fibrosis by stimulating proliferation and matrix synthesis of stellate cells.

BACKGROUND & AIMS: Tumor desmoplasia is one of the representative histopathologic findings in ductal pancreatic adenocarcinoma. The aims of this study were to examine the cellular and molecular mechanisms of fibrogenesis associated with pancreatic adenocarcinomas. METHODS: Immunostainings were performed with human pancreatic adenocarcinomas (n = 27) and tumors induced in nude mice (n = 36) by subcutaneously injecting MiaPaCa2, Panc1, and SW850 with and without pancreatic stellate cells. Matrix-producing cells were isolated from pancreatic adenocarcinomas and compared with pancreatic stellate cells isolated from tissue of chronic pancreatitis. Paracrine stimulation of pancreatic stellate cells by carcinoma cells was studied regarding matrix synthesis (collagen and c-fibronectin on protein and messenger RNA level) and cell proliferation (bromodeoxyuridine incorporation). RESULTS: High numbers of desmin and alpha-smooth muscle actin-positive cells were detected in 26 of 27 pancreatic adenocarcinomas. Intense fibronectin and collagen stainings were associated with these cells. By using cytofilament stainings, gene expression profiling, and morphological examinations, the matrix-producing cells obtained by the outgrowth method from pancreatic adenocarcinomas were identified as pancreatic stellate cells. Supernatants of MiaPaCa2, Panc1, and SW850 cells stimulated proliferation and collagen type I and c-fibronectin synthesis of cultured pancreatic stellate cells. Preincubation of the carcinoma cell supernatants with neutralizing antibodies against fibroblast growth factor 2, transforming growth factor beta 1, and platelet-derived growth factor significantly reduced the stimulatory effects. Subcutaneous injection of carcinoma cells and pancreatic stellate cells induced fast-growing subcutaneous fibrotic tumors in nude mice. Morphometric analysis of carcinoma cells (cytokeratin stainings) showed a high density of carcinoma cells in these tumors. CONCLUSIONS: Pancreatic stellate cells strongly support tumor growth in the nude mouse model. The increased deposition of connective tissue in pancreatic carcinoma is the result of a paracrine stimulation of pancreatic stellate cells by carcinoma cells.

Down-regulation of BRCA1 in chronic pancreatitis and sporadic pancreatic adenocarcinoma.

PURPOSE: BRCA1 and BRCA2 are considered to be breast cancer susceptibility genes that may also contribute to pancreatic cancer development because family studies revealed mutation carriers to have an increased risk of developing pancreatic cancer. However, as demonstrated for breast and ovarian cancer, inactivation of BRCA in sporadic diseases is based on alteration in gene expression or functional alteration. EXPERIMENTAL DESIGN: To study a potential correlation of BRCA1 and BRCA2 to chronic pancreatitis and development of sporadic pancreatic adenocarcinoma, we have analyzed the expression of these genes by quantitative PCR and performed immunohistochemical analyses in normal pancreatic tissues, chronic pancreatitis, and pancreatic cancer specimens. RESULTS: BRCA1 expression was down-regulated in chronic alcoholic pancreatitis, in particular on the RNA level. Furthermore, our data indicate suppressed BRCA1 expression in pancreatic cancer on both the RNA and protein levels. Quantitative analysis of BRCA1 protein expression demonstrated regular staining in 50% of tumor specimens tested and reduced staining in 50% of tumor specimens tested. Correlation with the clinical outcome revealed a significantly better 1-year overall survival for patients with BRCA1-regular as compared with BRCA1-reduced or BRCA1-absent tumors. In contrast, no substantial differences in BRCA2 expression were found in chronic pancreatitis and pancreatic cancer samples. CONCLUSIONS: Our data demonstrate alteration of BRCA1 expression in chronic pancreatitis and sporadic pancreatic adenocarcinoma. We, for the first time, provide evidence for a role of BRCA1 in pancreatic carcinogenesis of noninherited tumors and for clinical outcome.

Overexpression of Caspase-1 in adenocarcinoma of pancreas and chronic pancreatitis.

AIM: To identify the expression of Caspase-1(interleukin-1beta converting enzyme) and its role in adenoma of the pancreas and chronic pancreatitis. METHODS: The expression of Caspase-1 was assessed in 42 pancreatic cancer tissue samples, 38 chronic pancreatitis specimens, and 9 normal pancreatic tissues by immunohistochemistry and Western blot analysis. RESULTS: Overexpression of Caspase-1 was observed in both disorders, but there were differences in the expression patterns in distinct morphologic compartments. Pancreatic cancer tissues showed a clear cytoplasmatic overexpression of Caspase-1 in tumor cells of 71% of the tumors, whereas normal pancreatic tissues showed only occasional immunoreactivity. In chronic pancreatitis, overexpression of Caspase-1 was found in atrophic acinar cells (89%), hyperplastic ducts (87%), and dedifferentiating acinar cells (84%). Although in atrophic cells a clear nuclear expression was found, hyperplastic ducts and dedifferentiating acinar cells showed clear cytoplasmic expression. Western blot analysis revealed a marked expression of the 45 kDa precursor of Caspase-1 in pancreatic cancer and chronic pancreatitis (80% and 86%, respectively). Clear bands at 30 kDa, which suggested the p10-p20 heterodimer of active Caspase-1, were found in 60% of the cancer tissue and 14% of the pancreatitis tissue specimens, but not in normal pancreatic tissues. CONCLUSION: Overexpression of Caspase-1 is a frequent event in pancreatic disorders and its differential expression patterns may reflect two functions of the protease. One is its participation in the apoptotic pathway in atrophic acinar cells and tumor-surrounding pancreatitis tissue, the other is its possible role in proliferative processes in pancreatic cancer cells and hyperplastic duct cells and dedifferentiating acinar cells in chronic pancreatitis.

Cyclooxygenase-2 is overexpressed in chronic pancreatitis.

INTRODUCTION: Cyclooxygenase enzymes catalyze a critical step in the conversion of arachidonic acid to prostaglandins, which are important mediators of acute and chronic inflammation. The constitutively expressed cyclooxygenase-1 (COX-1) appears to regulate many normal physiologic functions in several cell types, whereas the inducible cyclooxygenase-2 (COX-2) enzyme mediates the inflammatory response. AIMS AND METHODOLOGY: We investigated the expression of COX-2 in tissues of 35 patients with chronic pancreatitis, 6 patients with pancreatic cancer, and 5 control patients by immunohistochemical analysis and correlations to clinicopathologic features. RESULTS: We found an overexpression of COX-2 in the atrophic acinar cells (80% of patients), hyperplastic ductal cells (86% of patients), and islets cells (97% of patients) but not in normal pancreatic tissues. The COX-2 overexpression in the tissue of patients with chronic pancreatitis was significantly correlated with the frequency of acute attacks of pancreatitis. Tissue from patients who had more than five acute attacks of pancreatitis (n = 10) exhibited COX-2 immunoreactivity of a significantly higher score in atrophic acinar cells (p = 0.004). No correlation could be found with other examined clinical features such as duration of the disease, diabetes, alcohol consumption, smoking, or pain. CONCLUSION: Our results support the hypothesis that COX-2 may be involved in inflammatory responses in chronic pancreatitis and in the progression of this chronic inflammatory disease.

Identification of tobacco-derived compounds in human pancreatic juice.

Cancer of the pancreas is the fourth leading cause of cancer mortality in the USA with an estimated 28 900 deaths in 2001. Several factors have been implicated in the etiology of this disease. However, at present, only cigarette smoking has been positively associated with pancreatic cancer. It is our working hypothesis that tobacco-derived compounds can be delivered to the pancreas where, upon metabolic activation, they can initiate carcinogenesis. Our current investigation was conducted to determine whether cotinine and tobacco-specific nitrosamines (TSNA) are present in human pancreatic juice. Smoking status was assessed by the determination of levels of urinary cotinine and was further supported by quantifying nicotine in hair. The TSNA were extracted from the pancreatic juice of 18 smokers and 9 nonsmokers by supercritical carbon dioxide that contained 10% methanol. The extracts were analyzed for TSNA, namely, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN), by gas chromatography with mass spectrometric detection using a selected ion monitoring technique (GC-SIM-MS). Twenty-three extracts of human pancreatic juice were also analyzed for the presence of the NNK metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by GC-SIM-MS and by gas chromatography interfaced wit a thermal energy analyzer (GC-TEA; TEA, a nitrosamine-specific detector). Cotinine was detected in all analyzed samples of pancreatic juice from smokers (129 +/- 150 ng/mL juice; mean +/- standard deviation) and was present in only two of the nine samples of pancreatic juice from nonsmokers. Its levels in these two samples were 7 and 9 ng/mL juice. NNK was detected in 15 of 18 samples (83%) from smokers at levels from 1.37 to 604 ng/mL pancreatic juice. In nine samples of pancreatic juice from nonsmokers, NNK ranged from not detected (in three samples) to 96.8 ng/mL juice. In pancreatic juice from smokers the mean level of NNK (88.7 +/- 161 ng/mL juice) was significantly higher (p < 0.04) than in that from nonsmokers (12.4 +/- 31.7 ng/mL juice). In addition to NNK, NNN was found in two samples of pancreatic juice of smokers at levels of 68.1 and 242 ng/mL juice; NNN was not detected in any other sample. NNAL was present in 8 of 14 pancreatic juice samples (57%) from smokers and in three of nine samples (33%) from nonsmokers. This research presents preliminary data that supports the hypothesis that pancreatic tissue is exposed to TSNA and that they may be important contributors to pancreatic carcinogenesis in humans.

NSC-631570 (Ukrain) in the palliative treatment of pancreatic cancer. Results of a phase II trial.

BACKGROUND: NSC-631570 (Ukrain) is a semisynthetic compound of thiophosphoric acid and the alkaloid chelidonine from the plant Chelidonium majus. It has been used in complementary herbal medicine for more than 20 years for the treatment of benign and malignant tumors. PATIENTS/METHODS: Between August 1999 and June 2001, 90 patients with histologically proven unresectable pancreatic cancer were randomized in a monocentric, controlled, randomized study. Patients in arm A received 1000 mg gemcitabine/m2, those in arm B received 20 mg NSC-631570, and those in arm C received 1000 mg gemcitabine/m2 followed by 20 mg NSC-631570 weekly. End point of the study was overall survival. RESULTS: In all three arms therapy was well tolerated and toxicity was moderate. At the first re-evaluation in arm A 32%, in arm B 75%, and in arm C 82% showed no change or partial remission according to WHO criteria (arm A versus arm B: P<0.01, arm A versus arm C: P<0.001). Median survival according to Kaplan-Meier analysis was in arm A 5.2 months, in arm B 7.9 months, and in arm C 10.4 months (arm A versus arm B: P<0.01, arm A versus arm C: P<0.01). Actuarial survival rates after 6 months were 26%, 65% and 74% in arms A B and C, respectively (arm A versus arm B: P<0.05, arm A versus arm C P<0.01). CONCLUSION: We could show that in unresectable advanced pancreatic cancer, NSC-631570 alone and in combination with gemcitabine nearly doubled the median survival times in patients suffering from advanced pancreatic cancer.