Pure Pancreatic Juice Samples Research Articles

Next-Generation Sequencing Revealed TP53 Mutations to Be Malignant Marker for Intraductal Papillary Mucinous Neoplasms That Could Be Detected Using Pancreatic Juice.

The aims of this study were to identify the genetic mutations associated with malignant intraductal papillary mucinous neoplasms (IPMNs) and evaluate the possibility of detecting mutations in pure pancreatic juice by next-generation sequencing. Resected tissues were collected from 50 patients with IPMN, and pure pancreatic juice samples were collected from 19 patients who had a resection. The extracted DNA was amplified by multiplex polymerase chain reaction targeting 52 cancer-related genes, including KRAS, GNAS, RNF43, and TP53; the mutations were then detected by next-generation sequencing and then analyzed for correlations with the clinicopathological characteristics. In the resected tissues, the most frequently detected mutations were in KRAS, GNAS, TP53, and RNF43, in 88%, 76%, 36%, and 30% of cases, respectively. Univariate and multivariate analyses revealed that only TP53 mutations were associated with malignant IPMNs (P = 0.023). In the pure pancreatic juice, TP53 mutations were detected in 5 of 10 resected samples with malignant IPMN and in 4 of 5 pancreatic juice samples with mutation in resected samples. From 52 cancer-related gene analysis, only TP53 mutation was associated with malignant IPMNs. TP53 mutation could also be detected in pure pancreatic juice, potentially making it a useful tool to diagnose malignant IPMNs preoperatively.

Comparative Proteomic Analysis of Whole-Gut Lavage Fluid and Pancreatic Juice Reveals a Less Invasive Method of Sampling Pancreatic Secretions.

OBJECTIVES:There are currently no reliable, non-invasive screening tests for pancreatic ductal adenocarcinoma. The fluid secreted from the pancreatic ductal system (“pancreatic juice”) has been well-studied as a potential source of cancer biomarkers. However, it is invasive to collect. We recently observed that the proteomic profile of intestinal effluent from the bowel in response to administration of an oral bowel preparation solution (also known as whole-gut lavage fluid, WGLF) contains large amounts of pancreas-derived proteins. We therefore hypothesized that the proteomic profile is similar to that of pancreatic juice. In this study, we compared the proteomic profiles of 77 patients undergoing routine colonoscopy with the profiles of 19 samples of pure pancreatic juice collected during surgery.METHODS:WGLF was collected from patients undergoing routine colonoscopy, and pancreatic juice was collected from patients undergoing pancreatic surgery. Protein was isolated from both samples using an optimized method and analyzed by LC-MS/MS. Identified proteins were compared between samples and groups to determine similarity of the two fluids. We then compared our results with literature reports of pancreatic juice-based studies to determine similarity.RESULTS:We found 104 proteins in our pancreatic juice samples, of which 90% were also found in our WGLF samples. The majority (67%) of the total proteins found in the WGLF were common to pancreatic juice, with intestine-specific proteins making up a smaller proportion.CONCLUSIONS:WGLF and pancreatic juice appear to have similar proteomic profiles. This supports the notion that WGLF is a non-invasive, surrogate bio-fluid for pancreatic juice. Further studies are required to further elucidate its role in the diagnosis of pancreatic cancer.

NPTX2 Hypermethylation in Pure Pancreatic Juice Predicts Pancreatic Neoplasms

The neuronal pentraxin II (NPTX2) gene is methylated in over 90% primary pancreatic cancer tissues but rarely in normal pancreatic ductal epithelia. Here, the authors investigated the utility of methylated NPTX2 as a diagnostic marker for pancreatic cancer in pure pancreatic juice samples of patients with benign and malignant pancreatic diseases, including pancreatic cancer, intraductal papillary mucinous neoplasm or chronic pancreatitis using methylation-specific polymerase chain reaction (MSP) and quantitative MSP. MSP assays revealed that the incidence of aberrant NPTX methylation in pure pancreatic juice samples was 64.5% (20 of 31) in patients with pancreatic cancer, 70.0% (7 of 10) in patients with malignant intraductal papillary mucinous neoplasm, 33.3% (2 of 6) in patients with benign intraductal papillary mucinous neoplasm and 21.7% (5 of 23) in patients with chronic pancreatitis. NPTX2 hypermethylation in patients with chronic pancreatitis was significantly lower than that of pancreatic cancer (P<0.01) or patients with intraductal papillary mucinous neoplasm (P<0.05). At a cutoff value of 1.39 for quantitative MSP, the incidence of aberrant NPTX2 methylation was 61.3% (19 of 31) in patients with pancreatic cancer, 50.0% (5 of 10) in patients with malignant intraductal papillary mucinous neoplasm, 0% in patients with benign intraductal papillary mucinous neoplasm and 8.7% (2 of 23) in patients with chronic pancreatitis. There was a significant difference in NPTX2 methylation between pancreatic cancer and chronic pancreatitis (P<0.01). Our findings indicate that detection of aberrant methylation of NPTX2 in pure pancreatic juice samples could be useful as a molecular marker to discriminate between patients with malignant and benign disease of the pancreas.

Combined detection of p16 aberrant methylation and K-ras gene mutation in pancreatic juice for diagnosis of pancreatic cancer

Objective To investigate the diagnostic significance of detecting p16 aberrant meth-ylation and K-ras gene mutation in pancreatic juice for pancreatic cancer. Methods Aberrant p16 methylation was detected using the methylation-specific PCR (MSP) in pure pancreatic juice(PPJ) col-lected from the pancreatic duct through a nasopancreatic tube put under endoscopic retrograde cholan-giopancreatography (ERCP). K-ras gene 12 codon mutation was detected using the m PCR-RFLP in the pure PPJ. Results The DNA was obtained from PPJ through a nasopancreatic tube. Aberrant p16 methylation and K-ras gene mutation were detected in the same samples of PPJ. Sensitivity, specifici-ty,positive predictive values, negative predictive values and accuracy of HE staining for pancreatic cancer were 40%, 100%, 100%,45.4% and 60.0%, respectively. Of the 20 cases of pancreatic canc-er,K-ras gene mutation was detected in 14 (70%) and the p16 gene was shown to be methylated in 7(35%). Of the 8 cases of chronic pancreatitis,K-ras gene mutation was detected in 2 (25%). Of the 2cases of mucinous cystoadenoma of pancreas,K-ras gene mutation was detected in 1 (50%). Aberrant methylation of p16 was not detected in pancreatic juice samples from patients with chronic pancreatitis and mucinous cystoadenoma of pancreas. Sensitivity, specificity, positive predictive values, negative predictive values and accuracy of detection K-ras gene mutation for pancreatic cancer were 70%, 70%,82.4%,53.8% and 70.0%, respectively. The sensitivity, specificity, positive predictive values, nega-tive predictive values and accuracy of detection of K-ras gene mutation and p16 methylation for pancre-atic cancer were 70%, 100%, 100%,62.5% and 80%, respectively. Conclusion Pancreatic juice can be used for molecular analysis of pancreatic juice obtained using nasopanereatic drainage during ERCP.Detecting aberrant methylation of p16 and K-ras gene mutation from pancreatic juice may be a new tool to diagnose pancreatic cancer. Key words: Pancreatic cancer; ERCP; Pancreatic neoplasms; Methylation; K-ras gene

Expression of Mesothelin mRNA in Pure Pancreatic Juice From Patients With Pancreatic Carcinoma, Intraductal Papillary Mucinous Neoplasm of the Pancreas, and Chronic Pancreatitis

In the gene expression analysis of pancreatic carcinoma (PCa) using serial analysis of gene expression (SAGE) according to Ryu et al, the tag for the mesothelin mRNA transcript was present in 7 of 8 SAGE libraries derived from PCa but not in the 2 SAGE libraries derived from normal pancreatic duct epithelial cells. Mesothelin mRNA expression was confirmed with in situ hybridization in all 4 resected primary PCa tumors and with RT-PCR in 18 of 20 PCa cell lines, whereas mesothelin protein expression was confirmed with immunohistochemistry in all 60 resected primary PCa tissues by Argani et al. We evaluated mesothelin mRNA expression in pure pancreatic juice (PPJ) obtained from patients with PCa, chronic pancreatitis (CP), and intraductal papillary mucinous neoplasm (IPMN) of the pancreas. We evaluated mesothelin mRNA expression in the PPJ obtained from 21 patients with PCa, 22 with CP, and 11 with IPMN with reverse transcriptase PCR (RT-PCR). The PCR products were analyzed with agarose gel electrophoresis. DNase I treatment before RT-PCR and direct sequencing of the RT-PCR bands were performed for the analysis of the RT-PCR bands. Two products, of 308 and 226 bp, were obtained with RT-PCR, and the 308-bp RT-PCR product was confirmed as being that derived from the genomic DNA by direct DNA sequencing. Mesothelin mRNA expression was discovered using RT-PCR in 11 (52%) of 21 patients with PCa, 5 (45%) of 11 with IPMN, and 3 (14%) of 22 with CP. Fisher's exact test revealed significant differences between PCa and CP for mesothelin mRNA (P < 0.01). Moreover, the RT-PCR product (226 bp) of mesothelin mRNA in the PPJ samples with PCa was generally stronger than that in the PPJ samples with IPMN. Expression of mesothelin mRNA in PPJ was not strictly specific to PCa and was apt to be stronger in PCa than in IPMN. Quantitative detection of mesothelin mRNA in PPJ may have potential diagnostic implications for pancreatic tumors.

Increased transforming growth factor beta in pure pancreatic juice in pancreatitis.

Cytokines play an important role in the regulation of inflammation and fibrosis in the development of chronic pancreatitis. In this study, transforming growth factor beta (TGFbeta), interleukin (IL)-6, IL-10, and tumor necrosis factor alpha (TNFalpha) were measured in pure pancreatic juice obtained through pancreatic duct cannulation. Twenty patients with chronic pancreatitis were compared with six patients thought to be free of pancreatic disease who were undergoing endoscopic retrograde cholangiopancreatography for biliary tract disorders. TGFbeta was detected in 17 of 20 patients with chronic pancreatitis tested (85%), compared with only one patient in the control group (17%). There was no measurable amount of IL-10, IL-6, or TNFalpha in any of the pure pancreatic juice samples from any of the patients. These data indicate that TGFbeta may play an active role in the advancement of pancreatitis by causing local inflammation and inducing fibroblasts to secrete collagen. This finding may be relevant in the future for identifying patients whose conditions may advance to chronic pancreatitis, and blocking the effects of TGFbeta could have therapeutic effects.

Quantitative determination of K-ras mutations in pancreatic juice for diagnosis of pancreatic cancer using hybridization protection assay.

K-ras mutations at codon 12 (KRM) have been detected in approximately 80% of samples of pure pancreatic juice (PPJ) from patients with pancreatic cancer (PCa) and are a promising potential tumor marker. However, the frequent presence of KRM was reported in PPJ from noncancerous patients as determined by a highly sensitive method, raising questions as to the cancer specificity of this marker. Therefore we evaluated whether the hybridization protection assay (HPA), which can quantitatively determine KRM in PPJ, is useful for the diagnosis of PCa, differentiating from chronic pancreatitis (CP). PPJ was collected endoscopically from 29 patients with PCa, 26 patients with CP, and the 11 cases as the control group. Polymerase chain reaction (PCR) and HPA using an acridinium ester-labeled DNA probe for KRM were performed with DNA extracted from these samples. The results were compared with those obtained by PCR-restriction fragment length polymorphism (RFLP). The mean + 2 SD of chemiluminescence in the control group was 11,020 RLUs. When 11,020 RLUs was taken as the cut-off value, KRM was detected by PCR-HPA in 19 (66%) of 29 of PCa and one (4%) of 26 of CP cases. Analysis of PPJ by PCR-RFLP demonstrated KRM in 22 (79%) of 28 of PCa and five (19%) of 26 of CP cases. However, four of five patients with CP who were KRM-positive by PCR-RFLP were defined as negative by PCR-HPA, suggesting that PCR-HPA is superior to PCR-RFLP for the discrimination between PCa and CP. These findings indicate that quantitative analysis of KRM in PPJ using the PCR-HPA method is a promising approach for the diagnosis of PCa, differentiating from CP with a suitable cut-off value, as in the case with the use of conventional serum tumor marker.

Antimicrobial activity of human pancreatic juice and its interaction with antibiotics

Pancreatic juice (PJ) should be a factor of variability in the antimicrobial activity of antibiotics eliminated by the pancreas during pancreatic infections. We studied its effects on the activity of antimicrobial drugs with different mechanisms of action. Samples of pure PJ were collected from 16 patients with stabilized external pancreatic fistulas. The antimicrobial activity of the juice at different concentrations (from 1.25 to 100%) alone and in combination with mezlocillin, imipenem, ceftriaxone, gentamicin, ofloxacin, and ciprofloxacin was studied by a microbiological method (continuous turbidimetric recording of bacterial growth). The human PJ showed dose-dependent antimicrobial activity that increased directly with the concentration. The activity of the antibiotics at bactericidal concentrations were not modified by the PJ, while the combination with subinhibitory concentrations produced the following variable and different effects: (i) additivity with mezlocillin, ceftriaxone, gentamicin, and ciprofloxacin and autonomy (no interaction) with imipenem and ofloxacin against Providencia rettgeri and (ii) additivity with ceftriaxone, ofloxacin, gentamicin, imipenem, and mezlocillin and autonomy with ciprofloxacin against Escherichia coli. In the presence of PJ, fluoroquinolones showed constant positive effects, while beta-lactams showed more variable antimicrobial activity. Antibiotic concentrations and PJ pharmacodynamics are the main factors determining the final effect of the interaction in vitro. These results may be useful in choosing antibiotics for the treatment of pancreatic infections when they are supplemented with the pharmacokinetic data for each drug.

Influence of pancreatic ducts on saturation of juice with calcium carbonate in dogs.

In several species, bicarbonate and calcium concentrations of pancreatic juice are known to vary during the different phases of pancreatic secretion. The effects of these variations on the saturation of juice with calcium carbonate, a critical factor for the formation of pancreatic stones, are not known. In this work, we studied the saturation degree of pancreatic juice with calcium carbonate in six unanesthetized beagle dogs equipped with Thomas cannulae during basal secretion and after bolus injections of cerulein (30 ng/kg) or secretin (0.25 units/kg). In the different samples of pure pancreatic juice, pH, PCO2, bicarbonate, and proteins were measured by standard methods. Total calcium (CaT) and ionized calcium (Ca2+) were determined using calcium-specific electrodes. Saturation with calcium carbonate was calculated by reference to the solubility product of calcite at 37 degrees C. Almost all the samples were found to be supersaturated with calcium carbonate but large variations of the saturation index were observed. In basal samples, obtained during periods of low secretion rate, the mean saturation index (3.35 +/- 3.01) was significantly lower than under secretion (12.10 +/- 5.14) or cerulein (18.01 +/- 8.42). This low basal saturation index, in spite of a high Ca2+ content, was explained by a low bicarbonate concentration (37.6 +/- 18.9 mmol/liter) and a high PCO2 (13.4 +/- 7.5 kPa). In contrast, in juice obtained after hormonal stimulation, PCO2 (4.8 +/- 1.6 kPa) was similar to plasma PCO2 (5.5 +/- 1.2 kPa).(ABSTRACT TRUNCATED AT 250 WORDS)

Penetration of pefloxacin into human allograft pancreatic juice

The penetration of pefloxacin (400 mg) into pancreatic juice, following a single oral dose, was investigated in five patients subjected to pancreatic transplantation as treatment for insulin-dependent diabetes mellitus. The pancreatic juice was temporarily diverted to the exterior by means of a pancreatic duct catheter. Studies on pure human pancreatic juice were thus made possible. Simultaneous blood and pure pancreatic juice samples were collected at regular intervals for up to 24 h after drug intake. The antibiotic concentrations were determined by bioassay. The average concentration of pefloxacin in pancreatic juice was 100% of that in serum. The mean peak concentration in pancreatic juice was 4.6 +/- 0.9 mg/l (+/- S.E.) and occurred at 2.5 h after drug intake. The decrease in concentration with time was parallel to the serum concentration curve. The concentrations of pefloxacin in pancreatic juice exceeded the MICs of many bacteria associated with pancreatic infections for several hours after administration of a single oral dose.

Pancreatic Juice Protein Associated with Chronic Pancreatitis and Adenocarcinoma of the Pancreas

125I-protein-radioiodinated pure pancreatic juice samples from patients with adenocarcinoma of the pancreas, chronic pancreatitis, or intact pancreas were analysed by high-resolution SDS-polyacrylamide gel electrophoresis and subsequent autoradiography. Experiments resulted in the detection of a 180K protein, probably a glycoprotein, in the pure pancreatic juice from pancreatic carcinoma (93%) and chronic pancreatitis (73%) patients, which was completely absent from pancreatic juice from intact pancreas. Sephadex G-200-isolated 180K protein was found to be different from carcinoembryonic antigen (CEA) when traced by a commercial CEA radioimmunoassay, but it seemed identical in pancreatic juice samples from patients with pancreatic carcinoma and chronic pancreatitis, at least with regard to isoelectric point. In brief, the present results suggest that 180K protein identification in pancreatic juice permits adenocarcinoma of the pancreas and chronic pancreatitis to be differentiated from normal conditions but that distinction between pancreatic carcinoma and chronic pancreatitis is unlikely.

Ribonuclease C and pancreatic secretory proteins in the peripheral circulation before and after pancreatectomy for pancreatic cancer

Increases in the activities of human circulatory ribonucleases have been associated with malignancies and other pathological conditions. To elucidate the relationship between circulatory ribonuclease and pancreatic malignancy, we have determined the activities in serum of ribonucleases selective for polycytidylic acid (ribonuclease C) and of α-amylase before and after surgery for pancreatic cancer and for several malignant and nonmalignant disorders unrelated to the pancreas. Prior to pancreatectomy, the activity of ribonuclease C in the serum of three pancreatic cancer patients was within the range of values obtained for healthy subjects. Unexpectedly, ribonuclease C began to increase within 2 hr after surgery, and amounts of ribonuclease C more than threefold higher than preoperative levels were found for at least 3 weeks after pancreatectomy. Variations in the serum concentrations of urea nitrogen and creatinine followed time courses which differed from those of ribonuclease C; thus the increases in the activity of ribonuclease C could not be attributed to renal impairment. The concentrations of α-amylase and trypsinogen in the blood of two of the pancreatic cancer patients were considerably higher than those of healthy subjects and declined rapidly to subnormal levels after pancreatectomy. Only a small amount of α-amylase and no trypsinogen were found in the blood of the third patient both before and after surgery, and samples of pure pancreatic juice obtained preoperatively after sequential intravenous administration of secretin and cholecystokinin-pancreozymin were negative for α-amylase and three proteolytic zymogens; nevertheless, the presurgical level of ribonuclease C in his serum was within the normal range. For all of the patients treated for nonpancreatic disorders, the concentrations of ribonuclease C and α-amylase in serum were not significantly altered by surgery. The rate and ultimate extent of the decrease in circulatory α-amylase following pancreatectomy indicate that ≈-80% of the α-amylase in blood is pancreatic in origin and that the half-life of pancreatic α-amylase in the peripheral circulation ≈-15 hr. From the highly significant increases in circulatory ribonuclease C which followed pancreatectomy for pancreatic cancer, we conclude that the exocrine pancreas is neither the sole nor the principal source of ribonuclease C in the peripheral circulation.

Decreased lipase activity in pure pancreatic juice and duodenal content from mutant mice with some alterations resembling cystic fibrosis

Several reports have pointed out the autosomal recessive mutation cri (cribriform degeneration) of the mouse as a possible animal model for cystic fibrosis (CF). The present work constitutes the first study of the exocrine pancreatic function in this mutation. Duodenal content and pure pancreatic juice (PPJ) samples were obtained from mutant and control mice and the lipase activity was measured. Trypsin activity in feces was also determined. The lipase activity was significantly decreased in duodenal content as well as in PPJ samples (p < 0.05 in both cases) in the cri / cri mutants, compared to their phenotypically normal siblings. The same enzymatic activity was also decreased in the normal (+/?) DBA/2J- cri mice, compared to the BALB/c mice strain. The presence of trypsin activity in stools, allowed us to rule out total exocrine pancreatic insufficiency (EPI) in cri / cri mice. The results are consistent with a partial EPI in this mutation and lend support to the concept of an animal model for CF.

Profiles of pure pancreatic secretions obtained by direct pancreatic duct cannulation in normal healthy human subjects

The development of endoscopic retrograde cannulation of the pancreatic duct allows collection of pure pancreatic juice uncontaminated by bile and duodenal secretions. We have analyzed such pure pancreatic juice to establish secretory profiles of some normal components. Fifteen healthy human volunteers were studied. Secretin (1 clinical unit per kg) was injected intravenously at time 0, and 16 min later cholecystokinin-pancreozymin (CCK-PZ) (1 Ivy dog unit per kg) was injected intravenously. Pure pancreatic juice was collected for 32 min in timed minute samples. Secretin produced an almost immediate and sustained flow of juice (3.15 ± 0.24 ml per min) with initial high concentrations of protein and enzyme. After CCK-PZ, flow increased significantly to 3.40 ± 0.29 ml per min (P < 0.02) and enzyme concentration rose some 10-fold above that found during secretin stimulation. The maximum of the CCK-PZ effect occurred 2 to 3 min after injection and its half-life was 73 to 90 sec for total protein and enzymes but only 64 sec for trypsin inhibitor. Repeat studies in 3 subjects indicate marked reproducibility for these various parameters. Trypsinogen and proelastase appeared to be secreted in a parallel fashion but nonparallel secretion for amylase, chymotrypsin, and trypsin inhibitor was observed. Amylase secretion was proportionately less and trypsin inhibitor secretion proportionately more stimulated by CCK-PZ than was chymotrypsinogen. Collections of single minute samples of pure pancreatic juice reveal a more accurate profile of secretory events than does collection of pooled samples for 10 to 20 min as hitherto performed by other workers.

Plasminogen in pancreatic disease and in pancreatic secretion.

Summary1. Serum plasminogen and amylase levels were measured in 12 dogs in whom pancreatic necrosis and/or fat necrosis was produced. An increase of plasminogen along with the expected increase in amylase was noted under the conditions described. 2. Samples of pure pancreatic juice obtained from 4 dogs were tested for the presence of plasminogen. No proteolytic activity due to the activating effect of streptokinase was present by the method employed.