Discovery of Anthranilic Acid Derivatives as Difluoromethylornithine Adjunct Agents That Inhibit Far Upstream Element Binding Protein 1 (FUBP1) Function.

Polyamine biosynthesis is regulated by ornithine decarboxylase (ODC), which is transcriptionally activated by c-Myc. A large library was screened to find molecules that potentiate the ODC inhibitor, difluoromethylornithine (DFMO). Anthranilic acid derivatives were identified as DFMO adjunct agents. Further studies identified the far upstream binding protein 1 (FUBP1) as the target of lead compound 9. FUBP1 is a single-stranded DNA/RNA binding protein and a master controller of specific genes including c-Myc and p21. We showed that 9 does not inhibit 3H-spermidine uptake yet works synergistically with DFMO to limit cell growth in the presence of exogenous spermidine. Compound 9 was also shown to inhibit the KH4 FUBP1-FUSE interaction in a gel shift assay, bind to FUBP1 in a ChIP assay, reduce both c-Myc mRNA and protein expression, increase p21 mRNA and protein expression, and deplete intracellular polyamines. This promising hit opens the door to new FUBP1 inhibitors with increased potency.

Pancreatic Ductal Adenocarcinoma (PDAC) circulating tumor cells influence myeloid cell differentiation to support their survival and immunoresistance in portal vein circulation.

The portal venous circulation provides a conduit for pancreatic ductal adenocarcinoma (PDAC) tumor cells to the liver parenchyma sinusoids, a frequent site of metastasis. Turbulent flow in the portal circulation promotes retention of PDAC shed circulating tumor cells (CTC) and myeloid-derived immunosuppressor cells (MDSC). Excessive colony stimulating factor-1 receptor (CSF1R) signaling can induce myeloid differentiation to MDSC and transformation of MDSC to myeloid-derived fibroblasts (M-FB). Interactions between PDAC CTC and M-FB in the portal blood promotes the formation of immunoresistant clusters that enhance CTC proliferation, migration, and survival. Analysis of portal and peripheral blood samples collected intraoperatively from 30 PDAC patients undergoing pancreatico-duodenectomy showed that PDAC patient plasma contained high levels of macrophage colony stimulating factor (M-CSF/CSF1), granulocyte-macrophage colony stimulating factor (GM-CSF/CSF2), interleukin-8 (IL-8), and interleukin-34 (IL-34) compared to healthy control levels. Moreover, the level of M-CSF in portal blood was significantly higher than that detected in the peripheral blood of PDAC patients. PDAC CTC aseptically isolated by fluorescence activated cell sorting (FACS) out of freshly collected patient portal blood mononuclear cells (PortalBMC) had elevated RNA expression of IL34 (IL-34 gene) and CSF1 (M-CSF/CSF1 gene) which both signal through CSF1R. PDAC CTC also had high levels of RNA expression for CXCL8, the gene encoding chemokine interleukin-8 (IL-8) which can attract myeloid cells through their CXCR2 receptors. FACS-isolated portal PDAC CTC and M-FB co-cultured ex vivo had increased CTC proliferation, motility, and cluster formation compared to CTC cultured alone. CSF1R and CXCR2 cell surface expression were found on PDAC portal blood CTC and M-FB, suggesting that both cell types may respond to M-CSF, IL-34, and IL-8-mediated signaling. Portal PDAC CTC displayed enhanced RNA expression of CSF1 and IL34, while CTC+M-FB+ clusters formed in vivo had increased RNA expression of CSF2 and IL34. Portal M-FB were found to have high CSF1R RNA expression. CTC isolated from ex vivo 7-day cultures of PDAC patient portal blood mononuclear cells (PortalBMC) expressed elevated CSF1, IL34, and IL8 RNA, and CSF1 expression was elevated in M-FB. Treatment with rabbit anti-CSF1R antibodies decreased CTC proliferation. Treatment of PortalBMC cultures with humanized anti-CSF1R, humanized anti-IL-8, or anti-IL-34 antibodies disrupted CTC cluster formation and increased CTC apoptosis. U937 myeloid precursor cell line cultures treated with conditioned media from PortalBMC ex vivo cultures without treatment or treated with anti-IL-8 and/or anti-CSF1R did not prevent myeloid differentiation in the myeloid precursor cell line U937 to macrophage, dendritic cell, MDSC, and M-FB phenotypes; whereas, U937 cultures treated with conditioned media from PortalBMC ex vivo cultures exposed to anti-IL-34 were significantly inhibited in their myeloid differentiation to all but the M-FB phenotype. PDAC patient T cells that were found phenotypically anergic (CD3+CD25+CTLA4+PD1L1+) in PortalBMC could be re-activated (CD3+CD25+CTLA4-PD1L1-), and displayed increased interferon gamma (IFNγ) production when PortalBMC ex vivo cultures were treated with anti-CSF1R, anti-IL-8, and anti-IL-34 antibodies alone or in combination. These findings suggest that PDAC CTC have the potential to influence myeloid differentiation and/or antigen presenting cell activation in the PDAC portal blood microenvironment, and that disruption of CTC/M-FB interactions may be potential targets for reversing the immunosuppression supporting CTC survival in the portal blood.

Validation of the Orlando Protocol for endoscopic management of pancreatic fluid collections in the era of lumen-apposing metal stents.

OBJECTIVES: Although lumen-apposing metal stents (LAMS) are being increasingly used in lieu of plastic stents, the clinical approach to endoscopic management of pancreatic fluid collections (PFCs) is poorly standardized. We compared outcomes of approaches over two time intervals, initially using plastic stents and later integrating LAMS. METHODS: This was a retrospective, observational, before-after study of prospectively collected data on consecutive patients with symptomatic PFCs managed over two time periods. In the initial period (January 2010-January 2015) endoscopic treatment was undertaken with plastic stents and in the later period (February 2015-August 2020) by integration of LAMS with selective use of plastic stents. The treatment strategy in both periods were tailored to size, extent, type of PFC and stepwise response to intervention. The main outcome was treatment success, defined as resolution of PFC and presenting symptoms at 6-month follow-up. RESULTS: A total of 160 patients were treated with plastic stents and 227 patients were treated using an integrated LAMS approach. Treatment success was significantly higher for the integrated approach compared to using only plastic stents (95.6 vs. 89.4%; P = 0.018), which was confirmed to be predictive of treatment success on multivariable logistic regression analysis (odds ratio 2.7, 95% confidence interval 1.1-6.4; P = 0.028). CONCLUSIONS: A structured approach integrating LAMS with selective use of plastic stents improved treatment success in patients with PFCs compared to an approach using only plastic stents.

Comparing Needles and Methods of Endoscopic Ultrasound-Guided Fine-Needle Biopsy to Optimize Specimen Quality and Diagnostic Accuracy for Patients With Pancreatic Masses in a Randomized Trial.

BACKGROUND & AIMS: Given the lack of procedure standardization, findings vary from analyses of pancreatic tissues collected by endoscopic ultrasound-guided fine-needle biopsy. It is not clear which needle and technique yield the best specimen for analysis. We compared the specimen quality and accuracy of diagnoses made from samples collected by fine-needle biopsy needles using different collection techniques. METHODS: Patients found to have pancreatic masses during imaging (n = 129) were assigned randomly to groups from whom pancreatic tissue samples were collected by reverse-bevel, Menghini-tip, franseen, or fork-tip needles. A second randomization determined the technical sequence of biopsies in each patient (suction, no suction, and stylet retraction). Two independent pathologists, blinded to the type of needle and sampling technique, analyzed all the samples. Final diagnoses of malignancy were made based on surgical resection, death from cancer progression, or findings from radiology or clinical follow-up evaluations (reference standard). The primary objective was to compare the cellularity of the samples collected, defined as the proportion of core tissue in the biopsy sample. Secondary objectives were to compare the accuracy of diagnoses made from biopsy samples and identify factors associated with high cellularity. RESULTS: One-hundred and nine patients had a final diagnosis of malignancy (84.5%) and 20 patients had benign disease (15.5%). Samples collected by fork-tip or franseen needles had significantly higher cellularity than samples collected by reverse-bevels or Menghini-tip needles (P < .001). Neoplasias were identified with greater than 90% accuracy using samples collected by fork-tip or franseen needles (P < .001 compared with other needles). On multivariable regression analysis, use of franseen needles (odds ratio [OR], 4.42; 95% CI, 2.58-7.58; P < .001) or fork-tip needles (OR, 3.86; 95% CI, 2.24-6.64; P < .001), stylet retraction (OR, 2.13; 95% CI, 1.21-3.72; P = .008), no suction (OR, 2.74; 95% CI, 1.57-4.80; P < .001), and pancreatic mass larger than 3 cm (OR, 1.92; 95% CI, 1.21-3.05; P = .005) were associated with high cellularity of the sample. CONCLUSIONS: In patients with suspected pancreatic cancer, samples with the highest degree of cellularity in a single biopsy, resulting in a diagnostic accuracy of 90% of higher, were collected by fine-needle biopsy using the franseen or fork-tip needle. Clinicaltrials.gov no: NCT04085055.

Importance of Disconnected Pancreatic Duct Syndrome in Recurrence of Pancreatic Fluid Collections Initially Drained Using Lumen-Apposing Metal Stents.

BACKGROUND & AIMS: Indwelling plastic endoprosthesis in patients with disconnected pancreatic duct syndrome (DPDS) reduces the risk of pancreatic fluid collection (PFC) recurrence. Although lumen-apposing metal stents (LAMS) are used with increasing frequency for PFC drainage, they require timely removal and little is known about their effects in patients with DPDS and recurrence of PFC. METHODS: We performed a prospective study of patients who underwent endoscopic ultrasound-guided drainage of PFC using LAMS and were found to have DPDS. After resolution of PFC, LAMS were replaced with double-pigtail plastic stents. The primary outcome was to compare PFC recurrence between patients with DPDS who did vs did not receive replacements with plastic stents after removal of the LAMS. RESULTS: Of 188 PFC patients treated with LAMS, 94 had DPDS, 71 had intact pancreatic ducts, and duct patency was unknown in 23. In patients with DPDS, replacement of LAMS with plastic stents was successful in 70 patients (74.5%) and technically unsuccessful in 24 patients (25.5%). At a median follow up of 183 days (interquartile range, 179-188 days), although none of the patients with an intact duct had a recurrence of PFC, 7 of the 94 patients with DPDS had recurrence of PFC (7.4%) (P = .020). PFC recurred in 1 of 70 patients with DPDS in whom replacement of LAMS with plastic stent was successful (1.4%) and in 6 of 24 patients with unsuccessful stent replacement (25.0%) (P = .001). CONCLUSIONS: In treatment of PFCs with LAMS in patients with DPDS, it is important to replace the LAMS with indwelling plastic stents to minimize PFC recurrence and reduce morbidity. Clinicaltrials.gov no: NCT02422095.

Superiority of endoscopic interventions over minimally invasive surgery for infected necrotizing pancreatitis: meta-analysis of randomized trials.

BACKGROUND AND AIM: Infected necrotizing pancreatitis is a highly morbid disease managed by minimally invasive surgical (MIS) or endoscopy-based interventions. This meta-analysis compared the clinical outcomes of patients treated using either approach. METHODS: MEDLINE and EMBASE databases were searched to identify all randomized trials that compared MIS and endoscopy-based interventions for treatment of infected necrotizing pancreatitis. Main outcome measure was to compare rates of complications or death during 6-month follow-up. RESULTS: Three studies involving 184 patients met inclusion criteria. While there was no significant difference in mortality (14.5% vs. 16.1%, risk ratio [RR] = 1.02, P = 0.963), new onset multiple organ failure (5.2% vs. 19.7%, RR = 0.34, P = 0.045), enterocutaneous fistula/perforation (3.6% vs. 17.9%, RR = 0.34, P = 0.034) and pancreatic fistula (4.2% vs. 38.2%, RR = 0.13, P < 0.001) were significantly lower for endoscopic interventions compared to MIS. There was no significant difference in intraabdominal bleeding, endocrine or exocrine pancreatic insufficiency between cohorts. Length of hospital stay was significantly shorter for endoscopy (standardized mean difference, -0.41, P = 0.010). CONCLUSIONS: An endoscopy-based treatment approach, as compared to minimally invasive surgery, significantly reduces complications in patients with infected necrotizing pancreatitis.

An Endoscopic Transluminal Approach, Compared With Minimally Invasive Surgery, Reduces Complications and Costs for Patients With Necrotizing Pancreatitis.

BACKGROUND & AIMS: Infected necrotizing pancreatitis is a highly morbid disease with poor outcomes. Intervention strategies have progressed from open necrosectomy to minimally invasive approaches. We compared outcomes of minimally invasive surgery vs endoscopic approaches for patients with infected necrotizing pancreatitis. METHODS: We performed a single-center, randomized trial of 66 patients with confirmed or suspected infected necrotizing pancreatitis who required intervention from May 12, 2014, through March 24, 2017. Patients were randomly assigned to groups that received minimally invasive surgery (laparoscopic or video-assisted retroperitoneal debridement, depending on location of collection, n = 32) or an endoscopic step-up approach (transluminal drainage with or without necrosectomy, n = 34). The primary endpoint was a composite of major complications (new-onset multiple organ failure, new-onset systemic dysfunction, enteral or pancreatic-cutaneous fistula, bleeding and perforation of a visceral organ) or death during 6 months of follow-up. RESULTS: The primary endpoint occurred in 11.8% of patients who received the endoscopic procedure and 40.6% of patients who received the minimally invasive surgery (risk ratio 0.29; 95% confidence interval 0.11-0.80; P = .007). Although there was no significant difference in mortality (endoscopy 8.8% vs surgery 6.3%; P = .999), none of the patients assigned to the endoscopic approach developed enteral or pancreatic-cutaneous fistulae compared with 28.1% of the patients who underwent surgery (P = .001). The mean number of major complications per patient was significantly higher in the surgery group (0.69 ± 1.03) compared with the endoscopy group (0.15 ± 0.44) (P = .007). The physical health scores for quality of life at 3 months was better with the endoscopic approach (P = .039) and mean total cost was lower ($75,830) compared with $117,492 for surgery (P = .039). CONCLUSIONS: In a randomized trial of 66 patients, an endoscopic transluminal approach for infected necrotizing pancreatitis, compared with minimally invasive surgery, significantly reduced major complications, lowered costs, and increased quality of life. Clinicaltrials.gov no: NCT02084537.

T1R2 receptor-mediated glucose sensing in the upper intestine potentiates glucose absorption through activation of local regulatory pathways.

OBJECTIVE: Beyond the taste buds, sweet taste receptors (STRs; T1R2/T1R3) are also expressed on enteroendocrine cells, where they regulate gut peptide secretion but their regulatory function within the intestine is largely unknown. METHODS: Using T1R2-knock out (KO) mice we evaluated the role of STRs in the regulation of glucose absorption in vivo and in intact intestinal preparations ex vivo. RESULTS: STR signaling enhances the rate of intestinal glucose absorption specifically in response to the ingestion of a glucose-rich meal. These effects were mediated specifically by the regulation of GLUT2 transporter trafficking to the apical membrane of enterocytes. GLUT2 translocation and glucose transport was dependent and specific to glucagon-like peptide 2 (GLP-2) secretion and subsequent intestinal neuronal activation. Finally, high-sucrose feeding in wild-type mice induced rapid downregulation of STRs in the gut, leading to reduced glucose absorption. CONCLUSIONS: Our studies demonstrate that STRs have evolved to modulate glucose absorption via the regulation of its transport and to prevent the development of exacerbated hyperglycemia due to the ingestion of high levels of sugars.

Portal Venous Blood Circulation Supports Immunosuppressive Environment and Pancreatic Cancer Circulating Tumor Cell Activation.

OBJECTIVES: Aggressive spread and liver metastases are predominant features of pancreatic ductal adenocarcinoma (PDAC). This study investigates activation of PDAC circulating tumor cells (CTC) and immunosuppression in the portal venous system. METHODS: Portal venous and peripheral blood were collected during pancreaticoduodenectomy from patients with PDAC (n = 21) or other non-PDAC pancreatic conditions (n = 20). Circulating tumor cells were isolated by fluorescence-activated cell sorting and characterized for messenger RNA (mRNA) expression and acetylated chromatin encoding K-RAS exon 12 mutation (K-RASmut). Myeloid-derived suppressor cells (MDSC) were identified using flow cytometry. RESULTS: Pancreatic ductal adenocarcinoma K-RASmut mRNA expression in portal venous blood CTC was significantly elevated compared with preoperative and postoperative peripheral blood (P = 0.0123 and P = 0.0246, respectively). There was no significant variation in total CTC numbers between portal and peripheral blood.Portal venous M-MDSC were elevated compared with peripheral blood in PDAC patients (P = 0.0065). M-MDSC increases correlated with K-RASmut mRNA-expressing CTC present in PDAC portal blood (P < 0.0001). CONCLUSIONS: Association of MDSC with active CTC in portal venous blood may support immunosuppression within the portal venous circulation to promote PDAC CTC survival.

Endoscopic ultrasound evaluation in the surgical treatment of duodenal and peri-ampullary adenomas.

AIM: To investigate endoscopic ultrasound (EUS) for predicting depth of mucosal invasion and to analyze outcomes following endoscopic and transduodenal resection. METHODS: Records of 111 patients seen at our institution from November 1999 to July 2011 with the post-operative pathological diagnosis of benign ampullary and duodenal adenomas were reviewed. Records of patients who underwent preoperative EUS for diagnostic purposes were identified. The accuracy of EUS in predicting the absence of muscular invasion was assessed by comparing EUS reports to the final surgical pathological results. In addition, the incidence of the post-operative complications over a period of 30 d and the subsequent long-term outcome (recurrence) over a period of 30 mo associated with endoscopic and transduodenal surgical resection was recorded, compared and analyzed. RESULTS: Among 111 patients with benign ampullary and duodenal adenomas, 47 underwent preoperative EUS for 29 peri-ampullary lesions and 18 duodenal lesions. In addition, computed tomography was performed in 18 patients, endoscopic retrograde cholangio-pancreatography in 10 patients and esophagogastroduodenoscopy in 22 patients. There were 43 patients with sporadic adenomas and 4 patients with familial adenomatous polyposis (FAP)/other polyposis syndromes. In 38 (81%, P < 0.05) patients, EUS reliably identified absence of submucosal and muscularis invasion. In 4 cases, EUS underestimated submucosal invasion that was proven by pathology. In the other 5 patients, EUS predicted muscularis invasion which could not be demonstrated in the resected specimen. EUS predicted tumor muscularis invasion with a specificity of 88% and negative predictive value of 90% (P < 0.05). Types of resection performed included endoscopic resection in 22 cases, partial duodenectomy in 9 cases, transduodenal ampullectomy with sphincteroplasty in 10 cases and pancreaticoduodenectomy in 6 cases. The main post-operative final pathological results included villous adenoma (n = 5), adenoma (n = 8), tubulovillous adenoma (n = 10), tubular adenoma (n = 20) and hyperplastic polyp (n = 2). Among the 47 patients who underwent resection, 8 (17%, 5 of which corresponded to surgical resection) developed post-procedural complications which included retroperitoneal hematoma, intra-abdominal abscess, wound infection, delayed gastric emptying and prolonged ileus. After median follow-up of 20 mo there were 6 local recurrences (13%, median follow-up = 20 mo) 4 of which were in patients with FAP. CONCLUSION: EUS accurately predicts the depth of mucosal invasion in suspected benign ampullary and duodenal adenomas. These patients can safely undergo endoscopic or local resection.

ErbB3 expression promotes tumorigenesis in pancreatic adenocarcinoma.

Historically, ErbB3 has been overlooked within the ErbB receptor family due to its perceived lack of tyrosine kinase activity. We have previously demonstrated that in pancreatic cancer ErbB3 is the preferred dimerization partner of EGFR, ErbB3 protein expression level directly correlates with the anti-proliferative effect of erlotinib (an EGFR-specific tyrosine kinase inhibitor), and transient knockdown of ErbB3 expression results in acquired resistance to EGFR-targeted therapy. In this study, we develop a stable isogenic model of ErbB3 expression in an attempt to decipher ErbB3's true contribution to pancreatic cancer tumorigenesis and to examine how this receptor affects cellular sensitivity to EGFR-targeted therapy. Analysis of the EGFR-ErbB3 heterodimer demonstrates that ligand-induced PI3K-AKT signaling is limited to ErbB3-expressing cells and that this signaling cascade can be partially abrogated by inhibiting EGFR function with erlotinib. Using our model of exogenous ErbB3 expression we showed a direct relationship between ErbB3 protein levels and increased pancreatic cancer cell proliferation in vitro. In vivo, ErbB3(+)PANC-1 xenografts had a significantly larger tumor volume than PANC-1 control xenografts (ErbB3-PANC-1) and displayed increased sensitivity to EGFR-targeted therapy. In pancreatic cancer, ErbB3 appears to be critically involved in EGFR signaling as evidenced by its profound effect on cellular proliferation and its ability to influence response to EGFR-targeted therapy.