Faculty Bibliography

Introduction: Increasing evidence suggests that the ratio of number of nodes harboring metastatic cancer to the total number of lymph node examined (lymph node ratio, LNR) affects survival after pancreatic resection for adenocarcinoma. We reviewed impact of lymph node status and LNR in our population of patients undergoing pancreatic resection for adenocarcinomMa.e thods: From our institutional pancreatic adenocarcinoma database, we identified 273 patients who underwent pancreatectomy during the period 1990-2011. of those, 51 had no nodes harvested in the specimen (No LN) and 86 had negative nodes (N0). Among those with positive nodes LNR wa<=s 0.1 in 27, <= 0.2 in 30, <= 0.3 in 21, <= 0.4 in 18 and > 0.4 in 40. Overall median survival was the study end point. Results: The 7 groups were similar in terms of gender, age, ECOG, primary procedure, and status of resection margins (see Table 1). T stage was higher in patients with elevated LNR (p=0.02). Survival was lower for patients with positive nodes (p < 0.01). This difference remained significant when excluding from analysis patients without harvested nodes (p = 0.005). Patient with LNR <= 0.1 had survival similar to N0 patients (20.1 vs. 20.0, p = 0.09). We observed a trend toward a worse survival in patients with higher LNR, which did not reach statistical significanCcoen. clusions: In our experience patients with LNR < 0.1 appeared to have survival similar to those with negative nodes. However LNR did not improve survival prognostication across patients with positive nodes. (Table Presented)

We have recently shown that Toll-like receptor (TLR) signaling exacerbates pancreatic fibro-inflammation and promotes carcinogenesis in mice. Paradoxically, inhibition of the TLR-MYD88 signaling pathway is pro-tumorigenic owing to the dendritic cell-mediated TH2-polarization of CD4+ T cells. TLR signaling appears to be central in pancreatic cancer-associated inflammation.

Pancreatic ductal adenocarcinoma is an aggressive cancer that interacts with stromal cells to produce a highly inflammatory tumor microenvironment that promotes tumor growth and invasiveness. The precise interplay between tumor and stroma remains poorly understood. TLRs mediate interactions between environmental stimuli and innate immunity and trigger proinflammatory signaling cascades. Our finding that TLR7 expression is upregulated in both epithelial and stromal compartments in human and murine pancreatic cancer led us to postulate that carcinogenesis is dependent on TLR7 signaling. In a mouse model of pancreatic cancer, TLR7 ligation vigorously accelerated tumor progression and induced loss of expression of PTEN, p16, and cyclin D1 and upregulation of p21, p27, p53, c-Myc, SHPTP1, TGF-beta, PPARgamma, and cyclin B1. Furthermore, TLR7 ligation induced STAT3 activation and interfaced with Notch as well as canonical NF-kappaB and MAP kinase pathways, but downregulated expression of Notch target genes. Moreover, blockade of TLR7 protected against carcinogenesis. Since pancreatic tumorigenesis requires stromal expansion, we proposed that TLR7 ligation modulates pancreatic cancer by driving stromal inflammation. Accordingly, we found that mice lacking TLR7 exclusively within their inflammatory cells were protected from neoplasia. These data suggest that targeting TLR7 holds promise for treatment of human pancreatic cancer.

BACKGROUND & AIMS: Immune cells of the liver must be able to recognize and react to pathogens yet remain tolerant to food molecules and other nonpathogens. Dendritic cells (DCs) are believed to contribute to hepatic tolerance. Lipids have been implicated in dysfunction of DCs in cancer. Therefore, we investigated whether high lipid content in liver DCs affects induction of tolerance. METHODS: Mouse and human hepatic nonparenchymal cells were isolated by mechanical and enzymatic digestion. DCs were purified by fluorescence-activated cell sorting or with immunomagnetic beads. DC lipid content was assessed by flow cytometry, immune fluorescence, and electron microscopy and by measuring intracellular component lipids. DC activation was determined from surface phenotype and cytokine profile. DC function was assessed in T-cell, natural killer (NK) cell, and NKT cell coculture assays as well as in vivo. RESULTS: We observed 2 distinct populations of hepatic DCs in mice and humans based on their lipid content and expression of markers associated with adipogenesis and lipid metabolism. This lipid-based dichotomy in DCs was unique to the liver and specific to DCs compared with other hepatic immune cells. However, rather than mediate tolerance, the liver DC population with high concentrations of lipid was immunogenic in multiple models; they activated T cells, NK cells, and NKT cells. Conversely, liver DCs with low levels of lipid induced regulatory T cells, anergy to cancer, and oral tolerance. The immunogenicity of lipid-rich liver DCs required their secretion of tumor necrosis factor alpha and was directly related to their high lipid content; blocking DC synthesis of fatty acids or inhibiting adipogenesis (by reducing endoplasmic reticular stress) reduced DC immunogenicity. CONCLUSIONS: Human and mouse hepatic DCs are composed of distinct populations that contain different concentrations of lipid, which regulates immunogenic versus tolerogenic responses in the liver.

Background: A pancreaticoduodenectomy (PD) offers the only chance of a cure for pancreatic cancer and can be performed with low mortality and morbidity. However, little is known about outcomes of a PD in octogenarians. Methods: Differences in two groups of patients (Group Y, <80 and Group O, >/=80 year-old) who underwent a PD for pancreatic adenocarcinoma were analysed. Study end-points were length of post-operative stay, overall morbidity, 30-day mortality and overall survival. Results: There were 175 patients in Group Y (mean age 64 years) and 25 patients in Group O (mean age 83 years). Octogenarians had worse Eastern Cooperative Oncology Group (ECOG) Performance Status (PS >/=1: 90% vs. 51%) and American Society of Anesthesiology (ASA) score (>2: 71% vs. 47%). The two groups were similar in underlying co-morbidities, operative time, rates of portal vein resection, intra-operative complications, blood loss, pathological stage and status of resection margins. Octogenarians had a longer post-operative stay (20 vs. 14 days) and higher overall morbidity (68% vs. 44%). There was a single death in each group. At a median follow-up of 13 months median survival appeared similar in the two groups (17 vs. 13 months). Conclusions: As 30-day mortality and survival are similar to those observed in younger patients, a PD can be offered to carefully selected octogenarians.

INTRODUCTION: Non-alcoholic steatohepatitis (NASH) is the most common cause of chronic liver dysfunction in theUnited States and can lead to cirrhosis, liver failure, and the need for liver transplantation. Dendritic cells (DC) are antigen presenting cells with an emerging role in hepatic inflammation. However, the role of DC in the progression of NASH is unknown. METHODS: NASH was induced in C57BL/6 mice by feeding a methionine-choline deficient diet. NASH progression was assessed by changes in liver inflammation, steatosis, and fibrosis. DC recruitment and activation was assessed by flow cytometry. DC depletion using diphtheria toxin (4ng/g/day) was achieved in C57BL/6 mice made chimeric with CD11c. DTR bone marrow. RESULTS: DC expand in NASH 4-5 fold (Table) and upregulate their expression of MHCII, CD40, ICAM-1, B7-1, and B7-2. DC also accumulate intracellular lipid in NASH (Table) and are primary in the clearance of necrotic hepatocytes, thereby limiting byproducts of sterile inflammation. Conversely, DC depletion in NASH (NASH-DC) markedly exacerbated intrahepatic inflammation, evidenced by increased liver cytokines, markedly increased CD45+ inflammatory infiltrate (Table), and activation of Kupffer cells (KC) and neutrophils with concomitant decrease in regulatory T cells. In NASH-DC, KC, inflammatory monocytes, and neutrophils underwent lower rates of apoptosis and produced increased TNF-alpha, IL-6 and pro IL-1beta. Further, end-organ fibrosis was significantly higher in NASH-DC mice (Table). (Table presented) CONCLUSIONS: DC become activated in NASH and contribute to steatosis by accumulating lipids. However, DC protect against intrahepatic fibro-inflammation by clearance of necrotic debris, thus limiting KC, monocyte, and neutrophil activation. Targeting DC may hold promise for NASH treatment

INTRODUCTION: Dendritic cells (DC) are professional antigen presenting cells (APC) that hold significant promise for the development of cancer vaccines. Endoplasmic reticular (ER)-stress can enhance the immunogenicity of APC. Since blockade of fatty acid synthesis increases ER-stress, we postulated that inhibition of fatty acid synthesis in DC would enhance their immune-stimulatory properties. METHODS: HumanmoDCwere generated fromPMBCby culture in GM-CSF/IL-4. Murine DC were generated from bone marrow using GM-CSF. TOFA-an acetyl-CoA carboxylase inhibitor-was used to block fatty acid synthesis. RESULTS: Blockade of fatty acid synthesis decreased DC development from precursors and mitigated DC proliferation in mice and humans. The mechanism was related to downregulation of Cyclin B-1-a pro-proliferative gene-and upregulation of Caspase 3 and PARP-apoptosis-related genes. Blockade of fatty acid synthesis enhanced DC activation and capacity for stimulation of immune effecter cells. TOFA-DC expressed elevated activated MAP-K, NF-kB, and PI3K/Akt intermediates and produced higher cytokines (Table). Peptide-pulsed TOFA-DC induced more vigorous proliferation of antigen-restricted CD4+ and CD8+ T cells, induced T cell IFN-g and TNF-a production, and enhanced CTL in vivo (Table). TOFA-DC induced higher NK expression of CD25 and production of IFN-g (Table). Accordingly, we found increased capacity for Notch signaling in TOFA-DC. TOFA-treated DC exhibited high ER-stress including increased expression of GRP-78, eIF2-a, p-eIF2-a and XBP-1, and blockade of ER-stress by a chaperone protein mitigated their enhanced immune-stimulation. (Table presented) CONCLUSIONS: Blockade of fatty acid synthesis markedly increases DC immunogenicity and capacity to induce CTL by augmenting ER stress. Our findings have significant implications for the design of immunotherapy vaccines

INTRODUCTION: Chronic pancreatitis (CP) and pancreatic cancer (PDAC) are characterized by an intense inflammatory infiltration. Regulation of this process is incompletely understood. We found that TLR7 expression is increased in CP and PDAC. We postulated that CP and PDAC depend on TLR7 signaling. METHODS: CP was induced in C57BL/6 mice using 7X daily caerulein injections (50mug/kg) for three weeks. KrasG12D mice which harbor a Kras mutation in pancreatic progenitor cells were used to model PDAC. TLR7 ligand (ssRNA40; 100mug/kg) was administered thrice-weekly. Blockade of TLR7 was accomplished with olignonucleotide inhibitors of TLR7 (IRS661). RESULTS: In CP and PDAC we observed substantially-increased TLR7-expression in both inflammatory and parenchymal cells, and significantly elevated levels of TLR7 ligands (Table). Ligation of TLR7 exacerbated CP in WT mice and accelerated tumor progression in KrasG12D mice (Table). TLR7 ligation altered epithelial cell expression of numerous oncogenic targets in PDAC including loss of PTEN, p16, and Cyclin D1 and up-regulation of p21, p27, p53, c-myc, SHPTP1, and TGF-beta. Further, TLR7 blockade protected against CP and pancreatic carcinogenesis, with marked reduction of inflammatory infiltration and complete arrest of tumor progression. TLR7 activation affected intra-pancreatic inflammation by interfacing with Notch pathway intermediates via STAT3 activation, and by inducing canonical NF-kB and MAP kinase signaling pathways- with corresponding blockade mitigating the pro-inflammatory and neoplastic effects of TLR7 activation. (Table presented) CONCLUSIONS: TLR7 ligation drives pancreatic inflammation and neoplasia via novel signaling mechanisms. Conversely, TLR7 blockade is powerfully protective. Targeting TLR7 holds promise for the treatment of CP and PDAC

The transition of chronic pancreatic fibroinflammatory disease to neoplasia is a primary example of the paradigm linking inflammation to carcinogenesis. However, the cellular and molecular mediators bridging these entities are not well understood. Because TLR4 ligation can exacerbate pancreatic inflammation, we postulated that TLR4 activation drives pancreatic carcinogenesis. In this study, we show that lipopolysaccharide accelerates pancreatic tumorigenesis, whereas TLR4 inhibition is protective. Furthermore, blockade of the MyD88-independent TRIF pathway is protective against pancreatic cancer, whereas blockade of the MyD88-dependent pathway surprisingly exacerbates pancreatic inflammation and malignant progression. The protumorigenic and fibroinflammatory effects of MyD88 inhibition are mediated by dendritic cells (DCs), which induce pancreatic antigen-restricted Th2-deviated CD4(+) T cells and promote the transition from pancreatitis to carcinoma. Our data implicate a primary role for DCs in pancreatic carcinogenesis and illustrate divergent pathways in which blockade of TLR4 signaling via TRIF is protective against pancreatic cancer and, conversely, MyD88 inhibition exacerbates pancreatic inflammation and neoplastic transformation by augmenting the DC-Th2 axis.

Stromal responses elicited by early stage neoplastic lesions can promote tumor growth. However, the molecular mechanisms that underlie the early recruitment of stromal cells to sites of neoplasia remain poorly understood. Here, we demonstrate an oncogenic Kras(G12D)-dependent upregulation of GM-CSF in mouse pancreatic ductal epithelial cells (PDECs). An enhanced GM-CSF production is also observed in human PanIN lesions. Kras(G12D)-dependent production of GM-CSF in vivo is required for the recruitment of Gr1(+)CD11b(+) myeloid cells. The suppression of GM-CSF production inhibits the in vivo growth of Kras(G12D)-PDECs, and, consistent with the role of GM-CSF in Gr1(+)CD11b(+) mobilization, this effect is mediated by CD8(+) T cells. These results identify a pathway that links oncogenic activation to the evasion of antitumor immunity.