Evaluating Pancreatic and Biliary Neoplasms with Small Biopsy-Based Next Generation Sequencing (NGS): Doing More with Less

Pancreatic cancer and cholangiocarcinoma are lethal diseases mainly diagnosed at an inoperable stage. As pancreatobiliary surgical specimens are often unavailable for further molecular testing, this review aimed to highlight the diagnostic, prognostic, and therapeutic impact of next-generation sequencing (NGS) performed on distinct small biopsies, including endoscopic ultrasound fine-needle aspirations and biopsies of pancreatic solid and cystic lesions, biliary duct brushings, and also “liquid biopsies” such as the pancreatic juice, bile, and blood. NGS could clarify indeterminate pancreatic lesions or biliary strictures, for instance by identifying TP53 or SMAD4 mutations indicating high-grade dysplasia or cancer. It could also stratify pancreatic cystic lesions, by distinguishing mucinous from non-mucinous cysts and identifying high-risk cysts that should be excised in surgically fit patients, whereas the combination of cytology, elevated cystic CEA levels and NGS could improve the overall diagnostic accuracy. When NGS is performed on the pancreatic juice, it could stratify high-risk patients under surveillance. On the plasma, it could dynamically monitor the disease course and response to therapy. Notably, the circulating tumor DNA (ctDNA) levels have been associated with staging, grading, and survival. Lastly, NGS has shown potential in identifying potentially actionable molecular alterations. In conclusion, NGS applied on small biopsies could carry significant diagnostic, prognostic, and therapeutic value.

Isolation, Characterization and Acid/Alkaline Tolerance of Lactic Acid Bacteria Present in Fermented Rye, Wheat, Oat and Barley

Objective: This study determined the lactic acid bacteria (LAB) present in fermented rye, wheat, oat and barley grains, and evaluated their survival in simulated gastric juice and pancreatic juice. Methods: Samples of rye, wheat, oat and barley grains were fermented for 72 hours at room temperature. Lactic acid bacteria (LAB) strains were isolated using MRS agar and were enumerated. Isolated LAB strains were cultured with MRS broth and the fermentation patterns of the isolated strains were characterized using API 50 CH kit (Biomerieux, France). Each isolated LAB strain was exposed to simulated gastric juice at pH of 2.0 for 80 minutes at 370C, followed by exposure to simulated pancreatic juice at pH of 8.0 for 120 minutes at 370C. Aliquots were taken at 0 minute and 80 minutes at pH of 2.0 and 0 minutes and 120 minutes at pH of 8.0 for enumeration of LAB strains. Results: The total LAB cell count ranged from 6.6 * 108 ± 11 cfu/ml in the rye sample to 9.5*109 ± 7 cfu/ml in the oat sample. 13 LAB strains were isolated from the four selected cereal grains and were characterized as six strains of Lactobacillus plantarum1, five strains of L. brevis 1 and one strain each of L. collinoides and Leuconostoc citreum. All the isolated LAB strains from the four selected cereals survived in the simulated gastric juice at pH of 2.0 (before and after incubation at 0min and 80a min) and after addition of simulated pancreatic juice at pH of 8.0 (before and after incubation at 80b min and 200 min respectively). The mean viable counts of all the strains ranged from 2.0 *108 in R3 at 80b min to 1.54 * 1010 in B4 at 80b minutes. Conclusion: LAB associated with fermentation of rye, wheat, oat and barley grains are likely to survive transport through the harsh acidic and alkaline conditions of the GIT.

Migration of aerobic bacteria from the duodenum to the pancreas with tumors: a mechanistic understanding

More aerobic bacteria are found in the pancreas with tumors than in the healthy pancreas. We provide a mechanistic understanding of the migration of intestinal bacteria from the duodenum to the pancreas with tumors. Mathematical models of migration of aerobic bacteria from the duodenum to the pancreas with tumors in the hepatopancreatic duct were developed. In addition, the behaviors of GFP E. coli under a pH gradient in a microfluidic device were analyzed. Moreover, upstream migrations of Pseudomonas fluorescens against flow were measured in a polydimethylsiloxane (PDMS) T-shaped cylinder mimicking a pancreatic duct. The simulated bacterial concentration of the pancreas with tumors was higher than that of the healthy pancreas and agreed reasonably well with the literature. Migration of aerobic bacteria in the hepatopancreatic duct is counteracted by bile and pancreatic juice flow but facilitated greatly by bacterial pH taxis from lower pH in duodenum fluid toward slightly alkaline pH in pancreatic juice, favorable for them. Migration of bacteria to the pancreas with tumors is made easier by solid tumors on the pancreatic duct, which compresses the pancreatic duct and thus reduces the fluid flow rate. On the other hand, GFP E. coli migrated under the pH gradient in a microfluidic device from acidic areas toward neutral or slightly alkaline pH, validating pH taxis. Furthermore, Pseudomonas fluorescens migrated upstream from hydrochloride solution but not from bicarbonate solution against bicarbonate flow at >20 µm/s, with an advancing velocity of approximately 60 µm/s, validating the models (244 words).

Enterococcus spp. have higher fitness for survival, in a pH-dependent manner, in pancreatic juice among duodenal bacterial flora

Objectives: Bacterial infection is involved in the progression of many gastrointestinal diseases, including cancer; however, how and which bacteria colonize in pancreatic juice and tissue have yet to be elucidated. Recently, we reported that Enterococcus faecalis exists in the pancreatic juice and tissues of patients with chronic pancreatic disease. Here, we investigated the survival of E. faecalis in duodenal juice with different pH conditions. Methods: Pancreatic juice samples from 62 patients with cancers of the duodeno-pancreato-biliary region were evaluated for the presence of E. faecalis. 16S ribosomal RNA PCR and 16S-based metagenome analyses were performed to determine the bacterial composition. The survival of E. faecalis in various pancreatic juice conditions was evaluated. Results: Of 62 samples, 27% (17/62) were positive for Enterococcus spp., among which 71% (12/17) contained E. faecalis. Enterococcus spp. showed the highest fitness for survival in alkaline pancreatic juice among various bacterial species. The microbiome of pancreatic juice from patients with pancreatic and bile duct cancer showed diversity, but Enterococcus spp. were enriched among duodenal tumors and intraductal papillary mucinous neoplasms. Conclusions: Alkalinity is important for the selective survival of E. faecalis among microbiota. E. faecalis may induce pancreatic inflammation with changes in pancreatic juice conditions.

Survival of Lactobacillus paracasei subsp. paracasei LBC 81 in Fermented Beverage from Chickpeas and Coconut in a Static In Vitro Digestion Model

The objective of this study was to evaluate in a static in vitro digestion model the survival of Lactobacillus paracasei subsp. paracasei LBC 81 in fermented chickpea and coconut beverage. The fermented beverage was stored for 1 and 8 days at 4 °C and then submitted to gastric juice, pancreatic juice, or sequential exposure to gastric and pancreatic juice. The experiment controls were (i) control 1—suspension of cells in 0.85% saline solution; (ii) control 2—cell suspension in chickpea and coconut beverage. The survival of L. paracasei was determined in log CFU/mL and expressed as a survival percentage. The survival of L. paracasei in the fermented beverage after exposure to gastric juice and sequential exposure to gastric and pancreatic juice was 99.47 + 2.05% and 93.21 + 0.43%, respectively. These values were higher than those found for controls 1 and 2. The storage condition of the fermented beverage for 1 or 8 days at 4 °C did not affect the survival after exposure to gastric juice, pancreatic juice, or sequential exposure. The results obtained in this study conclude that the fermented beverage of chickpeas and coconut is an excellent carrier for L. paracasei LBC 81, capable of enhancing survival to gastrointestinal conditions and ensuring a greater number of viable cells reaching the intestinal epithelium.

Pancreatic acinar cells utilize tyrosine to synthesize L-dihydroxyphenylalanine

The pancreatic β cells can synthesize dopamine by taking L-dihydroxyphenylalanine, but whether pancreatic acinar cells synthesize dopamine has not been confirmed. By means of immunofluorescence, the tyrosine hydroxylase -immunoreactivity and aromatic amino acid decarboxylase (AADC)- immunoreactivity were respectively observed in pancreatic acinar cells and islet β cells. Treatment with L-dihydroxyphenylalanine, not tyrosine, caused the production of dopamine in the incubation of INS-1 cells (rat islet β cell line) and primary isolated islets, which was blocked by AADC inhibitor NSD-1015. However, only L-dihydroxyphenylalanine, but not dopamine, was detected when AR42J cells (rat pancreatic acinar cell line) were treated with tyrosine, which was blocked by tyrosine hydroxylase inhibitor AMPT. Dopamine was detected in the coculture of INS-1 cells with AR42J cells after treatment with tyrosine. In an in vivo study, pancreatic juice contained high levels of L-dihydroxyphenylalanine and dopamine. Both L-dihydroxyphenylalanine and dopamine accompanied with pancreatic enzymes and insulin in the pancreatic juice were all significantly increased after intraperitoneal injection of bethanechol chloride and their increases were all blocked by atropine. Inhibiting TH with AMPT blocked bethanechol chloride-induced increases in L-dihydroxyphenylalanine and dopamine, while inhibiting AADC with NSD-1015 only blocked the dopamine increase. Bilateral subdiaphragmatic vagotomy of rats leads to significant decreases of L-dihydroxyphenylalanine and dopamine in pancreatic juice. These results suggested that pancreatic acinar cells could utilize tyrosine to synthesize L-dihydroxyphenylalanine, not dopamine. Islet β cells only used L-dihydroxyphenylalanine, not tyrosine, to synthesize dopamine. Both L-dihydroxyphenylalanine and dopamine were respectively released into the pancreatic duct, which was regulated by the vagal cholinergic pathway. The present study provides important evidences for the source of L-dihydroxyphenylalanine and dopamine in the pancreas.