Background Treatment of pancreatic cancer with pharmacological ascorbate (ascorbic acid, vitamin C) decreases tumor progression in pre-clinical models. A phase I clinical trial was performed to establish safety and tolerability of pharmacological ascorbate combined with gemcitabine in patients with biopsy-proven stage IV pancreatic adenocarcinoma. Design Nine subjects received twice weekly intravenous ascorbate (15–125 g) employing Simon’s Accelerated Titration design to achieve a targeted post-infusion plasma level of ≥ 350 mg/dL (≥20 mM). Subjects received concurrent gemcitabine. Disease burden, weight, performance status, hematologic and metabolic labs, time to progression and overall survival were monitored. Results Mean plasma ascorbate trough levels were significantly higher than baseline (1.46 ± 0.02 vs. 0.78 ± 0.09 mg/dL, i.e. 83 vs. 44μM, p < 0.001). Adverse events attributable to the drug combination were rare and included diarrhea (n = 4) and dry mouth (n = 6). Dose-limiting criteria were not met for this study. Mean survival of subjects completing at least 2 cycles (8 weeks) of therapy was 13 ± 2 months. Conclusions Data suggest pharmacologic ascorbate administered concurrently with gemcitabine is well-tolerated. Initial data from this small sampling suggest some efficacy. Further studies powered to determine efficacy should be conducted.

A multiphase computed tomography scan of the chest, abdomen, and pelvis should be performed. Baseline performance status and comorbidity profile should be evaluated. The goals of care, patient preferences, psychological status, support systems, and symptoms should guide decisions for treatments. A palliative care referral should occur at first visit. Initial systemic chemotherapy (6 months) with a combination regimen is recommended for most patients (for some patients radiation therapy may be offered up front) with Eastern Cooperative Oncology Group performance status 0 or 1 and a favorable comorbidity profile. There is no clear evidence to support one regimen over another. The gemcitabine-based combinations and treatments recommended in the metastatic setting (eg, fluorouracil, leucovorin, irinotecan, and oxaliplatin and gemcitabine plus nanoparticle albumin-bound paclitaxel) have not been evaluated in randomized controlled trials involving locally advanced, unresectable pancreatic cancer. If there is local disease progression after induction chemotherapy, without metastasis, then radiation therapy or stereotactic body radiotherapy may be offered also with an Eastern Cooperative Oncology Group performance status ≤ 2 and an adequate comorbidity profile. If there is stable disease after 6 months of induction chemotherapy but unacceptable toxicities, radiation therapy may be offered as an alternative. Patients with disease progression should be offered treatment per the ASCO Metastatic Pancreatic Cancer Treatment Guideline. Follow-up visits every 3 to 4 months are recommended. Additional information is available at www.asco.org/guidelines/LAPC and www.asco.org/guidelines/MetPC and www.asco.org/guidelineswiki.

The pleiotropic effects of the Kit receptor system are mediated by Kit‐Ligand (KL) induced receptor autophosphorylation and its association with and activation of distinct second messengers, including phosphatidylinositol 3′‐kinase (PI3‐kinase), p21ras and mitogen‐activated protein kinase (MAPK). To define the role of PI3‐kinase, p21ras and MAPK in Kit‐mediated cell proliferation, survival and adhesion in bone marrow‐derived mast cells (BMMC), mutant Kit receptors were expressed in Wsh/Wsh BMMC lacking endogenous c‐kit expression. The introduction of both murine Kit(S) and KitL (isoform containing a four amino acid insert) into Wsh/Wsh BMMC restored KL‐induced proliferation, survival and adhesion to fibronectin, as well as activation of PI3‐kinase, p21ras and MAPK, and induced expression of c‐fos, junB, c‐myc and c‐myb mRNA. Substitution of tyrosine 719 in the kinase insert with phenylalanine (Y719F) abolished PI3‐kinase activation, diminished c‐fos and junB induction, and impaired KL‐induced adhesion of BMMC to fibronectin. In addition, the Y719F mutation had partial effects on p21ras activation, cell proliferation and survival, while MAP kinase activation was not affected. On the other hand, Y821F substitution impaired proliferation and survival without affecting PI3‐kinase, p21ras and MAPK activation, and induction of c‐myc, c‐myb, c‐fos and c‐jun mRNA, while KL‐induced cell adhesion to fibronectin remained intact. In agreement with a role for PI3‐kinase in Kit‐mediated cell adhesion, wortmannin blocked Kit‐mediated cell adhesion at concentrations known to specifically inhibit PI3‐kinase. We conclude, that association of Kit with p85PI3‐K, and thus with PI3‐kinase activity, is necessary for a full mitogenic as well as adhesive response in mast cells. In contrast, tyrosine 821 is essential for Kit‐mediated mitogenesis and survival, but not cell adhesion.

SUMMARYGenetic analysis of pancreatic development has provided new insights into the mechanisms underlying the formation of exocrine pancreatic neoplasia. Zebrafish sweetbread (swd) mutants develop hypoplastic acini and dysmorphic ducts in the exocrine pancreas, with impeded progression of cell division cycle and of epithelial growth. Positional cloning and allelic complementation have revealed that the swd mutations affect the transient receptor potential melastatin-subfamily member 7 (trpm7) gene, which encodes a divalent cation-permeable channel with kinase activity. Supplementary Mg2+ partially rescued the exocrine pancreatic defects of the trpm7 mutants by improving cell-cycle progression and growth and repressing the suppressor of cytokine signaling 3a (socs3a) gene. The role of Socs3a in Trpm7-mediated signaling is supported by the findings that socs3a mRNA level is elevated in the trpm7 mutants, and antisense inhibition of socs3a expression improved their exocrine pancreatic growth. TRPM7 is generally overexpressed in human pancreatic adenocarcinoma. TRPM7-deficient cells are impaired in proliferation and arrested in the G0-G1 phases of the cell division cycle. Supplementary Mg2+ rescued the proliferative defect of the TRPM7-deficient cells. Results of this study indicate that Trpm7 regulates exocrine pancreatic development via the Mg2+-sensitive Socs3a pathway, and suggest that aberrant TRPM7-mediated signaling contributes to pancreatic carcinogenesis.

Although many of the genes that regulate development of the endocrine pancreas have been identified, comparatively little is known about how the exocrine pancreas forms. Previous studies have shown that exocrine pancreas development may be modeled in zebrafish. However, the timing and mechanism of acinar and ductal differentiation and morphogenesis have not been described. Here, we characterize zebrafish exocrine pancreas development in wild type and mutant larvae using histological, immunohistochemical and ultrastructural analyses. These data allow us to identify two stages of zebrafish exocrine development. During the first stage, the exocrine anlage forms from rostral endodermal cells. During the second stage, proto-differentiated progenitor cells undergo terminal differentiation followed by acinar gland and duct morphogenesis. Immunohistochemical analyses support a model in which the intrapancreatic ductal system develops from progenitors that join to form a contiguous network rather than by branching morphogenesis of the pancreatic epithelium, as described for mammals. Contemporaneous appearance of acinar glands and ducts in developing larvae and their disruption in pancreatic mutants suggest that common molecular pathways may regulate gland and duct morphogenesis and differentiation of their constituent cells. By contrast, analyses of mind bomb mutants and jagged morpholino-injected larvae suggest that Notch signaling principally regulates ductal differentiation of bipotential exocrine progenitors.

The roles of transient receptor potential (TRP) cation channels in pancreatic tumorigenesis are essentially unknown. Here, we focus on the TRP melastatin-subfamily (TRPM) members. Expression of the thermally regulated transmembrane Ca2+-permeable channel TRPM8 is consistently up-regulated in human pancreatic adenocarcinoma cell lines and tissues. TRPM8-deficient pancreatic cancer cells have reduced ability of proliferation and cell cycle progression with elevated levels of cyclin-dependent kinase inhibitors. These results indicate that TRPM8 is aberrantly over-expressed in pancreatic adenocarcinoma and required for cellular proliferation, and they support further investigation of the potential of TRPM8 as a clinical biomarker and therapeutic target in pancreatic adenocarcinoma.

Mammalian studies have implicated important roles for the basic helix-loop-helix transcription factor Ptf1a-p48 in the development of both exocrine and endocrine pancreas. We have cloned the Ptf1a-p48 ortholog in Danio rerio. Early zebrafish ptf1a expression is observed in developing hindbrain and in endodermal pancreatic precursors. Analysis of ptf1a and insulin expression reveals a population of exocrine precursors that, throughout early development, are temporally and spatially segregated from endocrine elements. Morpholino-mediated knockdown of ptf1a confirms early divergence of these endocrine and exocrine lineages. Ptf1a morphants lack differentiated exocrine pancreas, but maintain normal differentiation and organization of the principal islet. In addition to the exocrine phenotype, ptf1a knockdown also reduces the prevalence of a small population of anterior endocrine cells normally found outside the principal islet. Together, these findings suggest the presence of distinct ptf1a-dependent and ptf1a-independent precursor populations in developing zebrafish pancreas.

SummaryThe receptor tyrosine kinase Kit and its cognate ligand KL/steel factor are encoded at the white spotting (W) and Steel (Sl) loci of the mouse, respectively. Mutations at both the W and the Sl loci affect hematopoiesis including the stem ceil hierarchy, erythropoiesis, and mast cells, as well as gametogenesis and mdanogenesis. In addition, mutant mice display an increased sensitivity to lethal doses of irradiation. The role of KL/c-kit in ceil proliferation and survival under conditions of growth factor-deprivation and ~/-irradiation was studied by using bone marrow-derived mast cells (BMMC) as a modal. Whereas apoptosis induced by growth factor deprivation in BMMC is a stochastic process and follows zero order kinetics, 3~-irradiation-induced apoptosis is an inductive process and follows higher order kinetics. In agreement with these results, 3,-irradiation-induced apoptosis in BMMC was shown to be dependent on p53 whereas apoptosis induced by deprivation is partly dependent on p53, implying that there are other mechanisms mediating apoptosis in KL-deprived BMMC. In the presence and in the absence of serum, KL stimulated proliferation by promoting cell cycle progression. The presence of KL was required only daring the early part of the G1 phase for entry into the S phase. At concentrations lower than those required for proliferation, KL suppressed apoptosis induced by both growth factor-deprivation and 3,-irradiation, and internudeosomal DNA fragmentation characteristic of apoptosis. The ability of KL to suppress apoptosis was independent of the phase of the ceil cycle in which the ceils were irradiated and suppression of apoptosis was a prerequisite for subsequent cell cycle progression. Moreover, addition of KL to 3,-irradiated and growth factor-deprived cells could be ddayed for up to I h after irradiation or removal of growth factors when cells became irreversibly committed to apoptosis. KL and IL-3 induce suppression of apoptosis in mast calls by different mechanisms based on the observations of induction of kl-2 gene expression by IL-3 but not by KL. It is proposed that the increased sensitivity of W and SI mutant mice to lethal irradiation results from paucity of the apoptosis suppressing and proliferative effects of KL.