Thymic stromal lymphopoietin in human pancreatic ductal adenocarcinoma: expression and prognostic significance.

Thymic stromal lymphopoietin (TSLP) has emerged as an important, but contradictory, player conditioning tumor growth. In certain contexts, by driving T helper (h) 2 responses via tumor-associated OX40 Ligand (OX40L)+ dendritic cells (DCs), TSLP may play a pro-tumorigenic role. The study elucidates the importance of TSPL in pancreatic ductal adenocarcinoma (PDAC), by analyzing: i) TSLP levels in PDAC cell-line supernatants and plasma from patients with locally-advanced/metastatic PDAC, pre- and post-treatment with different chemotherapeutic protocols, in comparison with healthy donors; ii) TSLP and OX40L expression in PDAC and normal pancreatic tissues, by immunohistochemistry; iii) OX40L expression on ex vivo-generated normal DCs in the presence of tumor-derived TSLP, by flow cytometry; iv) clinical relevance in terms of diagnostic and prognostic value and influence on treatment modality and response. Some PDAC cell lines, such as BxPC-3, expressed both TSLP mRNA and protein. Normal DCs, generated ex vivo in the presence of TSLP-rich-cell supernatants, displayed increased expression of OX40L, reduced by the addition of a neutralizing anti-TSLP polyclonal antibody. OX40L+ cells were detected in pancreatic tumor inflammatory infiltrates. Abnormally elevated TSLP levels were detected in situ in tumor cells and, systemically, in locally-advanced/metastatic PDAC patients. Of the chemotherapeutic protocols applied, gemcitabine plus oxaliplatin (GEMOX) significantly increased circulating TSLP levels. Elevated plasma TSLP concentration was associated with shorter overall survival and increased risk of poor outcome. Plasma TSLP measurement successfully discriminated PDAC patients from healthy controls. These data show that TSLP secreted by pancreatic cancer cells may directly impact PDAC biology and patient outcome.

Chimeric rat/human HER2 efficiently circumvents HER2 tolerance in cancer patients.

PURPOSE: Despite the great success of HER2 vaccine strategies in animal models, effective clinical results have not yet been obtained. We studied the feasibility of using DNA coding for chimeric rat/human HER2 as a tool to break the unresponsiveness of T cells from patients with HER2-overexpressing tumors (HER2-CP). EXPERIMENTAL DESIGN: Dendritic cells (DCs) generated from patients with HER2-overexpressing breast (n = 28) and pancreatic (n = 16) cancer were transfected with DNA plasmids that express human HER2 or heterologous rat sequences in separate plasmids or as chimeric constructs encoding rat/human HER2 fusion proteins and used to activate autologous T cells. Activation was evaluated by IFN-γ ELISPOT assay, perforin expression, and ability to halt HER2+ tumor growth in vivo. RESULTS: Specific sustained proliferation and IFN-γ production by CD4 and CD8 T cells from HER2-CP was observed after stimulation with autologous DCs transfected with chimeric rat/human HER2 plasmids. Instead, T cells from healthy donors (n = 22) could be easily stimulated with autologous DCs transfected with any human, rat, or chimeric rat/human HER2 plasmid. Chimeric HER2-transfected DCs from HER2-CP were also able to induce a sustained T-cell response that significantly hindered the in vivo growth of HER2(+) tumors. The efficacy of chimeric plasmids in overcoming tumor-induced T-cell dysfunction relies on their ability to circumvent suppressor effects exerted by regulatory T cells (Treg) and/or interleukin (IL)-10 and TGF-ß1. CONCLUSIONS: These results provide the proof of concept that chimeric rat/human HER2 plasmids can be used as effective vaccines for any HER2-CP with the advantage of being not limited to specific MHC. Clin Cancer Res; 20(11); 2910-21. ©2014 AACR.

Pancreatic-carcinoma-cell-derived pro-angiogenic factors can induce endothelial-cell differentiation of a subset of circulating CD34+ progenitors.

BACKGROUND: CD34+ progenitor cells comprise both hematopoietic and endothelial progenitor cells. Recent studies suggest that circulating endothelial progenitor cells are recruited into the angiogenic vascular system of several cancers, including pancreatic carcinoma, and that they correlate with clinical progress. However, whether endothelial progenitor cell mobilization occurs in response to cytokine release by tumor cells is still unclear. METHODS: The chemotactic- and/or differentiating-activities of the poorly-differentiated pancreatic carcinoma cell line PT45, and of the immortal H6c7 cell line, a line of near-normal pancreatic duct epithelial cells, on endothelial progenitor cells were investigated in vitro using circulating CD34+ as model. RESULTS: The study showed that Vascular Endothelial Growth Factor produced by PT45 cells and, at lesser extent, by H6c7 cells, predominantly chemoattract peripheral blood CD34+ expressing the type 2 relative receptor. Addition of PT45-conditioned medium to CD34+ cells, cultured under conditions supporting myeloid cell development, diverted the differentiation of a subset of these progenitor cells into cells expressing endothelial cell markers, such as CD146, CD105, VE-cadherin and von Willebrand Factor-related antigen. Moreover, these endothelial-like cells formed capillary networks in vitro, chiefly through the release of Angiopoietin-1 by PT45 cells. CONCLUSIONS: The results demonstrate that pancreatic-carcinoma cells potentially attract circulating endothelial progenitor cells to the tumor site, by releasing high levels of pro-angiogenic factors such as Vascular Endothelial Growth Factor and Angiopoietin-1, and may direct the differentiation of these cell subsets of the CD34+ cell population into endothelial cells; the latter cells may become a component of the newly-formed vessels, contributing to angiogenesis-mediated tumor growth and metastasis.

Autoantibodies to Ezrin are an early sign of pancreatic cancer in humans and in genetically engineered mouse models.

BACKGROUND: Pancreatic Ductal Adenocarcinoma (PDAC) is a highly aggressive malignancy with only a 5% 5-year survival rate. Reliable biomarkers for early detection are still lacking. The goals of this study were (a) to identify early humoral responses in genetically engineered mice (GEM) spontaneously developing PDAC; and (b) to test their diagnostic/predictive value in newly diagnosed PDAC patients and in prediagnostic sera. METHODS AND RESULTS: The serum reactivity of GEM from inception to invasive cancer, and in resectable or advanced human PDAC was tested by two-dimensional electrophoresis Western blot against proteins from murine and human PDAC cell lines, respectively. A common mouse-to-human autoantibody signature, directed against six antigens identified by MALDI-TOF mass spectrometry, was determined. Of the six antigens, Ezrin displayed the highest frequency of autoantibodies in GEM with early disease and in PDAC patients with resectable disease. The diagnostic value of Ezrin-autoantibodies to discriminate PDAC from controls was further shown by ELISA and ROC analyses (P < 0.0001). This observation was confirmed in prediagnostic sera from the EPIC prospective study in patients who eventually developed PDAC (with a mean time lag of 61.2 months between blood drawing and PDAC diagnosis). A combination of Ezrin-autoantibodies with CA19.9 serum levels and phosphorylated α-Enolase autoantibodies showed an overall diagnostic accuracy of 0.96 ± 0.02. CONCLUSIONS: Autoantibodies against Ezrin are induced early in PDAC and their combination with other serological markers may provide a predictive and diagnostic signature.

Maintenance sunitinib or observation in metastatic pancreatic adenocarcinoma: a phase II randomised trial.

BACKGROUND: New strategies to prolong disease control warrant investigation in patients with metastatic pancreatic adenocarcinoma. This open-label, randomised, multi-centre phase II trial explored the role of maintenance sunitinib after first-line chemotherapy in this setting. METHODS: Patients with pathologic diagnosis of metastatic pancreatic adenocarcinoma, performance status >50%, no progression after 6 months of chemotherapy were centrally randomised by an independent contract research organisation, which was also responsible for data collection and monitoring, to observation (arm A) or sunitinib at 37.5mg daily until progression or a maximum of 6 months (arm B). The primary outcome measure was the probability of being progression-free at 6 months (PFS-6) from randomisation. Assuming P0 = 10%; P1 = 30%, α .10; ß .10, the target accrual was 26 patients per arm. RESULTS: 28 per arm were randomised. One arm B patient had kidney cancer and was excluded. Sunitinib was given for a median of 91 days (7-186). Main grade 3-4 toxicity was thrombocytopenia, neutropenia and hand-foot syndrome (12%), diarrhoea 8%. In arm A versus B, PFS-6 was 3.6% (95% confidence interval (CI): 0-10.6%) and 22.2% (95% CI: 6.2-38.2%; P<0.01); 2 y overall survival was 7.1% (95% CI: 0-16.8%) and 22.9% (95% CI: 5.8-40.0%; P = 0.11), stable disease 21.4% and 51.9% (P = 0.02). CONCLUSION: This is the first randomised trial on maintenance therapy in metastatic pancreatic adenocarcinoma. The primary end-point was fulfilled and 2 y overall survival was remarkably high, suggesting that maintenance sunitinib is promising and should be further explored in this patient population.

FOLFOX-4 regimen or single-agent gemcitabine as first-line chemotherapy in advanced biliary tract cancer.

OBJECTIVES: We conducted a retrospective cohort study to compare 2 different chemotherapy regimens for advanced biliary tract cancer (BTC). METHODS: Records of patients consecutively treated in our institution for advanced BTC from 2001 to 2006 were retrieved. Chemotherapy treatment with FOLFOX-4 regimen was routinely offered as first option; gemcitabine (GEM) as single agent was proposed as an alternative option to patients who refused central venous catheter implantation. Toxicity, overall response rate, progression-free survival (PFS), and overall survival (OS) obtained with the 2 treatments were evaluated. RESULTS: Twenty-two patients were treated with FOLFOX-4, whereas 18 patients received GEM. In the FOLFOX-4 group, the overall response rate was 13.6% (95% confidence interval [CI], 4.7-33.3), with 1 complete response and 2 partial responses, and 54.5% (95% CI, 34.7-73.1) of disease control rate (complete response+partial response+stable disease). Median OS was 14.1 months (95% CI, 9.1-18.8) and median PFS 5.44 months (95% CI, 3.2-6.3). In the GEM group, we observed no objective response, whereas 27.7% (95% CI, 12.5-50.9) obtained disease control. Median OS was 8.3 months (95% CI, 4.7-12.9) and median PFS 3.9 months (95% CI, 2.2-5.4). Toxicity, mainly hematological, was acceptable for both treatments. On a multivariable Cox model including a propensity score, only the performance status and chemotherapy regimen were confirmed as strong predictors of OS, with an hazard ratio of 0.49 (95% CI, 0.24-0.99) in favor of FOLFOX-4. CONCLUSIONS: The combination chemotherapy with oxaliplatin and 5-fluorouracil is well tolerated and seems to provide prolonged survival than GEM alone in advanced BTC treatment, but further randomized trials are warranted.

Potential plasticity of T regulatory cells in pancreatic carcinoma in relation to disease progression and outcome.

CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) are understood to maintain peripheral tolerance to self-antigens and inhibit antitumor immune responses. However, compelling evidence suggests that, Tregs provide no anti-inflammatory protection in the tumor microenvironment, but rather contribute to a T helper 17 (Th17)-driven pro-carcinogenic process. Using three-color flow cytometry, we evaluated the percentage of circulating CD4(+)CD25(+)FoxP3(+) Tregs in the peripheral blood of pancreatic carcinoma patients prior to and after chemotherapy [gemcitabine (GEM) alone, or GEM+oxaliplatin (GEMOX) or bevacizumab+capecitabine+radiotherapy (BEV+CAPE+RT)]. Correlations were sought between Treg counts and plasma levels of cytokines relevant to controlling the Treg/Th17 balance, i.e., interleukin (IL)-23, IL-17A, IL-6 and transforming growth factor ß 1 (TGF-ß1), as measured by ELISA and the clinical features of pancreatic cancer. Treg, IL-6 and TGF-ß1 levels were higher in locally advanced and metastatic pancreatic carcinoma patients compared to controls. No parameter was correlated with disease stage except IL-6. IL-17A and TGF-ß1 were significantly associated with increased risk of poor prognosis. IL-17A was positively correlated with IL-23. Treg and IL-6 levels decreased following GEM monochemotherapy, IL-17A levels decreased after GEMOX, and IL-6 levels were reduced subsequent to BEV+CAPE+RT treatment. IL-23, IL-17A and TGF-ß1 levels were significantly lower in patients responding to chemotherapy (partial remission/stable disease) than in nonresponders to chemotherapy (progressive disease). These results suggest an impact of the Treg/Th17-balance in pancreatic carcinoma, highlighting the significance of TGF-ß1 and IL-17A as potential prognostic and predictive indicators. Immunological changes induced by mono and/or combined chemotherapy indicate specific windows of opportunity for introducing integrative interventions on a new target in pancreatic cancer, i.e. IL-17A, possibly improving survival in this highly lethal disease.

Comparative evaluation of cancer stem cell markers in normal pancreas and pancreatic ductal adenocarcinoma.

Chemoresistance and self-renewal of cancer stem cells (CSC), found in many tumors including pancreatic ductal adenocarcinoma (PDAC), are believed to underlie tumor mass regrowth. The distribution of cells carrying the putative stem-cell markers CD133, Nestin, Notch1-4, Jagged1 and 2, ABCG2 and aldehyde dehydrogenase (ALDH1) was assessed immunohistochemically using PDAC and normal pancreas tissue microarrays. The immunoreactivity was semi-quantitatively graded against the normal pancreas and was correlated with the differentiation grade and disease stage. No statistical significant differences were found between normal pancreas and PDAC in the expression of Nestin, Notch1, 3 and 4, ABCG2 or ALDH1. Notch2 and Jagged1 and 2 expression were increased in PDAC. CD133-positive cells were above-normal in PDAC, but the difference was not statistically significant. Nestin, Notch1-4, Jagged1, ABCG2 and ALDH1 immunostaining scores were not correlated with tumor grade or disease stage. CD133 and Notch2 expression was significantly inversely correlated with tumor grade, but not disease stage. Notch3 immunostaining positively correlated with tumor stage, but not with differentiation grade. Jagged2 protein expression correlated inversely with disease stage, but not with tumor grade. From the clinical standpoint, improved delineation of the tumor CSC signature, putatively responsible for tumor initiation and recurrence after initial response to chemotherapy, may offer novel therapeutic targets for this highly lethal cancer.

Circulating autoantibodies to phosphorylated α-enolase are a hallmark of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis and no diagnostic markers have, as of yet, been defined. In PDAC patients, α-enolase (ENOA) is up-regulated and elicits the production of autoantibodies. Here, we analyzed the autoantibody response to post-translational modifications of ENOA in PDAC patients. ENOA isolated from PDAC tissues and cell lines was characterized by two-dimensional electrophoresis (2-DE) Western blot (WB), revealing the expression of six different isoforms (named ENOA1,2,3,4,5,6) whereas only 4 isoforms (ENOA3,4,5,6) were detectable in normal tissues. As assessed by 2-DE WB, 62% of PDAC patients produced autoantibodies to the two more acidic isoforms (ENOA1,2) as opposed to only 4% of controls. Mass spectrometry showed that ENOA1,2 isoforms were phosphorylated on serine 419. ROC analysis demonstrated that autoantibodies to ENOA1,2 usefully complement the diagnostic performance of serum CA19.9 levels, achieving approximately 95% diagnostic accuracy in both advanced and resectable PDAC. Moreover, the presence of autoantibodies against ENOA1,2 correlated with a significantly better clinical outcome in advanced patients treated with standard chemotherapy. In conclusion, our results demonstrate that ENOA phosphorylation is associated with PDAC and induces specific autoantibody production in PDAC patients that may have diagnostic value.

Pilot study to relate clinical outcome in pancreatic carcinoma and angiogenic plasma factors/circulating mature/progenitor endothelial cells: Preliminary results.

Circulating endothelial cells (CEC) and bone marrow-derived endothelial progenitors (ECP) play important roles in tumor growth and have been proposed as non-invasive markers of angiogenesis. However, CEC and ECP levels have not been investigated in pancreatic carcinoma patients. Using four-color flow cytometry procedures, we evaluated the count of resting (rCEC) and activated (aCEC) endothelial cells and ECP in the peripheral blood of pancreatic carcinoma patients before and after chemotherapy, consisting of gemcitabine (GEM) alone or in combination with oxaliplatin (OX), or with 5-fluorouracil (5-FU). We also correlated CEC and ECP levels with plasma levels of relevant angiogenic factors, such as vascular endothelial growth factor (VEGF)-A, VEGF-D, angiopoietin (Angio)-1, and chemokine C-X-C motif ligand (CXCL)12, measured by ELISA, and with clinical features of pancreatic cancer. The aCEC, rCEC, ECP, and VEGF-A plasma levels were significantly higher in locally-advanced and metastatic patients than controls. Both ECP and VEGF-A levels correlated positively with disease stage and inversely with patient's overall survival. Measurements after the treatment course showed that VEGF-A plasma concentrations and ECP counts had decreased significantly. In particular, VEGF-A and rCEC were significantly down after treatment with GEM alone or in combination with OX. No significant differences in terms of circulating angiogenic factor or endothelial cell subtype levels were found between responders (patients entering partial remission or with stable disease) and non-responders (patients with progressive disease). The study provides insights into angiogenesis mechanisms in pancreatic carcinoma, for which anti-angiogenic targeting of VEGF-A and ECP could be of interest.

IL-18 paradox in pancreatic carcinoma: elevated serum levels of free IL-18 are correlated with poor survival.

The role of the proinflammatory interleukin (IL)-18 in cancer progression remains controversial; we thus examined the hypothesis that impaired antitumor immune response in pancreatic carcinoma patients is related to elevated levels of its natural inhibitor IL-18 binding protein (BP) and/or to alteration in the IL-18 receptor complex expression and function. IL-18 and IL-18 binding protein isoform a (BPa) was assessed in pancreatic carcinoma patients at various disease stages, and after surgery/chemotherapy; free bioactive IL-18 concentrations were calculated. IL-18 receptor complex expression in lymphocyte subsets was analyzed and signaling function was assessed versus healthy donors. Carcinoma cells exhibited below normal IL-18BPa expression and above normal IL-18 expression. Circulating IL-18BPa and IL-18 were above controls. Unexpectedly, free unbound IL-18 serum levels were correlated with disease severity and poor survival. IL-18BPa levels were unchanged by surgery but free IL-18 levels were elevated. Gemcitabine with 5-fluorouracil or oxaliplatin, but not alone, increased IL-18 and free IL-18 levels statistically significantly, without affecting IL-18BPa. Spontaneous/induced IL-18 receptor alpha and receptor beta expression in peripheral blood lymphocyte subsets from patients with advanced disease were near-normal, although CD4+ and CD8+ cells were fewer in percentage, and fully functional in inducing interferon-gamma. IL-18 is proposed as novel adjuvant cancer therapy, but free IL-18 levels are increased in the blood of pancreatic carcinoma patients, despite elevated IL-18BP levels, and are associated with poor survival; this highlights recent experimental insights into the prometastatic and proangiogenic effects of IL-18, and suggests that careful preclinical studies are needed to determine the proper application of IL-18 in cancer therapy.

An integrated humoral and cellular response is elicited in pancreatic cancer by alpha-enolase, a novel pancreatic ductal adenocarcinoma-associated antigen.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with a very poor 5-year survival rate. alpha-Enolase is a glycolytic enzyme that also acts as a surface plasminogen receptor. We find that it is overexpressed in PDAC and present on the cell surface of PDAC cell lines. The clinical correlation of its expression with tumor status has been reported for lung and hepatocellular carcinoma. We have previously demonstrated that sera from PDAC patients contain IgG autoantibodies to alpha-enolase. The present work was intended to assess the ability of alpha-enolase to induce antigen-specific T cell responses. We show that alpha-enolase-pulsed dendritic cells (DC) specifically stimulate healthy autologous T cells to proliferate, secrete IFN-gamma and lyse PDAC cells but not normal cells. In vivo, alpha-enolase-specific T cells inhibited the growth of PDAC cells in immunodeficient mice. In 8 out of 12 PDAC patients with circulating IgG to alpha-enolase, the existence of alpha-enolase-specific T cells was also demonstrated. Taken as a whole, these results indicate that alpha-enolase elicits a PDAC-specific, integrated humoral and cellular response. It is thus a promising and clinically relevant molecular target candidate for immunotherapeutic approaches as new adjuvants to conventional treatments in pancreatic cancer.

Impact of surgery and chemotherapy on cellular immunity in pancreatic carcinoma patients in view of an integration of standard cancer treatment with immunotherapy.

As surgery and chemotherapy may act as adjuvants providing antitumor immunity benefits, we ran phenotypical and functional immunomonitoring in patients with resectable pancreatic adenocarcinoma and advanced metastatic disease receiving combined treatment (cisplatin, gemcitabine, 5-FU). Blood was taken before/one month after resection; before/during chemotherapy. Controls were age- and gender-matched. Circulating lymphocyte, myeloid and plasmacytoid dendritic cell (MDC and PDC) subsets were examined by flow cytometry; functional activity by mixed lymphocyte reaction (MLR) for DC allostimulation, through 4-h 51Cr-release assay for Natural Killer (NK) and lymphokine-activated-killer (LAK) cell cytotoxicity; ELISA for spontaneous/activated cytokine release by PBMC and T cells. Significant differences occurred in several parameters between pretreatment patient and control values: fewer CD8+ cells and increased apoptosis-prone CD3+/CD95+ lymphocytes, higher frequency of MDC, reduced allostimulatory activity by ex vivo-generated DC, depressed LAK activity, elevated IL-10 and IL-12p40 production; impaired IL-12p70 and IFN-gamma production by stimulated PBMC and T cells. Only IL-12p70 level was correlated with survival. One month after radical, but not palliative surgery, the percentage of T-lymphocytes coexpressing CD3/CD95 decreased significantly, the stimulatory capacity of DC increased, and LPS-induced IL-12p70 release by PBMC rose concomitantly with the anti-CD3 stimulated-IFN-gamma production by T cells. In patients with locally advanced or metastatic disease, one and/or two combined drug cycles increased percentage of CD4+ cells and LAK cell cytotoxicity and decreased PDC frequency and spontaneous/LPS-stimulated IL-10 by PBMC. Results suggest immunological changes induced by surgical resection/combined chemotherapy indicate specific precisely-timed windows of opportunity for introducing immunotherapy in pancreatic cancer, possibly improving survival in this highly lethal disease.

Oxaliplatin, 5-fluorouracil, and leucovorin as second-line treatment for advanced pancreatic cancer.

OBJECTIVE: A phase II study was performed to assess the activity of oxaliplatin plus 5-fluorouracil (5-FU) modulated by leucovorin, as second-line treatment in locally advanced or metastatic pancreas adenocarcinoma pretreated with gemcitabine-containing schedule. METHODS: Patients received weekly intravenous infusions of oxaliplatin 40 mg/m, 5-FU 500 mg/m, and leucovorin 250 mg/m (3 weeks on, 1 week off). RESULTS: Twenty-three patients affected with metastatic (16) or locally advanced (7) pancreas adenocarcinoma were involved in this study. A total of 148 weeks of chemotherapy was delivered (median 2 courses each patient). Among 17 assessable patients, no objective response was registered and 4 patients had stable disease, whereas 13 had tumor progression. Median duration of stable disease was 14 weeks. Median time to progression of disease (TTP) was 11.6 weeks [95% confidence interval (CI), 7.6-5.6]. Kaplan-Meier estimated median overall survival (OS) was 17.1 week (95% CI, 4.0-30.1) and 3 months survival rate was 69.6%. Seven patients experienced grade 3 to 4 toxicity. The regimen was associated with 36% clinical benefit. CONCLUSIONS: The median TTP and median OS in this population with poor prognosis suggests some activity, however, only further investigations will be able to establish the clinical value of this combination.

Autoantibody signature in human ductal pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by rapid progression, invasiveness, and resistance to treatment. It is the fourth leading cause of cancer death with a 2% 5-year survival rate. Biomarkers for its early detection are lacking. This study was designed to use a proteomics-based approach as a means of identifying antigens that elicit a humoral response in PDAC patients. Antibodies against PDAC-associated antigens are useful for early cancer diagnosis and therapy. Proteins from PDAC cell lines were separated by 2-DE, and the serum IgG reactivity of 70 PDAC patients, 40 healthy subjects (HS), 30 non-PDAC tumor patients, and 15 chronic pancreatitis (CP) patients was tested by Western blot analysis. Spots specifically recognized by PDAC sera and revealed by mass spectrometry corresponded to metabolic enzymes or cytoskeletal proteins. Most were up-regulated in PDAC tissues. Thus, it seems that metabolic enzymes and cytoskeletal proteins are specific targets of the humoral response during PDAC. The results of further studies of these serological-defined antigens could be of diagnostic and therapeutic significance in PDAC.

Antagonistic interactions between gemcitabine and 5-fluorouracil in the human pancreatic carcinoma cell line Capan-2.

Although the recently-developed Gemcitabine (GEM) has renewed interest in clinical research in pancreatic carcinoma, it offers modest improvement of tumor-related symptoms and marginal survival advantage, even when combined with other currently-available chemotherapeutic agents such as 5-Fluorouracil (5-FU). We hypothesized that this disappointing result could be due to an interaction between the two drugs affecting cytotoxic activity. We measured in-vitro growth inhibition, cell cycle distribution, gene and protein expression of apoptosis regulators bcl-2, bcl-x and survivin, NFkappaB and telomerase activities of human pancreatic carcinoma cell line Capan-2 following exposure to GEM and 5-FU singly or combined, by MTT assay and median effect analysis, flow cytometry, real-time RT-PCR, Western blotting, electrophoretic mobility shift assay (EMSA) and telomeric repeat amplification protocol (TRAP) assay, respectively. We found cell growth to be inhibited by both drugs, decreasing the percentage of cells in S and G2/M phases and inducing apoptosis, dependent on the levels of bcl-2, bcl-xL and survivin expression in the case of 5-FU, but not for GEM. Moreover, while telomerase activity was reduced equally by both drugs, 5-FU but not GEM effectively downregulated NFkappaB binding activity. Intriguingly, a substantial antagonistic effect was noticed when GEM was combined with 5-FU in the concentration range tested, with the exception of the TRAP assay. These indications of an antagonistic interaction between GEM and 5-FU in some pancreatic cancer context urge further investigation of both genetic and non-genetic differences to identify the variables most relevant for optimal selection and dosing of treatment for the individual patient.

Cooperative induction of a tolerogenic dendritic cell phenotype by cytokines secreted by pancreatic carcinoma cells.

Ag presentation by dendritic cells (DC) is essential to effective antitumor T cell responses in cancer patients. Depending on their origin, maturation state, and the ambient cytokine milieu, DC can differentiate into distinct subpopulations, which preferentially either induce Th1 cell activation (CD11c+,CD123- myeloid DC (MDC)) or immunosuppressive T cell development (CD11c-,CD123+ plasmacytoid DC (PDC)). The present study was undertaken to characterize the effects of pancreatic carcinoma cell-derived cytokines on immature monocyte-derived DC (iMo-DC) in vitro and in vivo. Medium conditioned by human pancreatic carcinoma cells inhibited iMo-DC proliferation, expression of costimulatory molecules (CD80 and CD40) and of HLA-DR, and functional activity as assessed by MLR and IL-12p70 production. iMo-DC generated from pancreatic carcinoma patients in advanced stages of the disease similarly showed decreased levels of HLA-DR expression and reduced ability to stimulate MLR in response to CD40L and IFN-gamma. Moreover, in tumor-patient peripheral blood, the ratio of MDC to PDC cells was lower than in healthy controls due to reduced numbers of MDC CD11c+ cells. Importantly, rather than a single cytokine, a combination of tumor-derived cytokines was responsible for these effects; these were primarily TGF-beta, IL-10, and IL-6, but not vascular endothelial growth factor. In summary, we have identified an array of pancreatic carcinoma-derived cytokines that cooperatively affect iMo-DC activation in a manner consistent with ineffective antitumor immune responses.

Nonmyeloablative allogeneic blood stem cell transplantation in patients with metastatic solid tumors.

BACKGROUND: The aim of the study was to evaluate the feasibility and efficacy of a non-myeloablative regimen to achieve complete donor chimerism after stem cell transplantation (SCT) in patients with metastatic solid tumors. PATIENTS AND METHODS: Seven patients with renal cell carcinoma (RCC), 3 with colorectal carcinoma and 1 with soft tissue sarcoma received an allogeneic SCT after fludarabine (90 mg/m2) and TBI 200 cGy. RESULTS: At day 30, median donor chimerism was 94%. Regression of tumor metastases has been observed in 1 patient with RCC. Overall, 8 patients (73%) died from progressive disease (median progression-free survival 3.7 months) and 1 (9%) from treatment-related complications; 2 patients were alive 152 and 862 days after transplantation. CONCLUSIONS: Our preliminary results suggest that non-myeloablative SCT for metastatic solid tumors is feasible, although may lead to durable responses in a minority of patients. Careful patient selection seems to be mandatory in this transplant setting.

Circulating vascular endothelial growth factor and interferon-gamma-inducible protein-10 levels in pancreatic cancer during chemotherapy.

BACKGROUND: Chemotherapeutic anticancer properties are thought to derive from apoptosis pathway activation and/or cell division arrest, but animal models have also evidenced anti-angiogenic activity in some agents. PATIENTS AND METHODS: The impact of gemcitabine, irinotecan and oxaliplatin + 5-FU upon the serum markers vascular endothelial growth factor (VEGF) (pro-angiogenic) and IFN-gamma-inducible protein (IP)-10 (anti-angiogenic) was evaluated by ELISA in locally advanced and/or metastatic cancer versus clinical efficacy and survival. RESULTS: Patients had higher serum levels of both markers versus controls. No objective response to therapy was observed and no significant difference in either marker occurred during the first month of chemotherapy; analysis by survival showed slight transient VEGF decrease in longer survivors on day 14 and slight increase on day 28 in shorter survivors, who had baseline median IP-10 levels above longer survivors, diverging on day 14 (decrease and increase, respectively). Both groups were below baseline at day 28. Changes in IP-10 were not significant. CONCLUSION: These preliminary results provide a rationale for exploring whether continuous or frequent administration of some anti-neoplastic agents may elicit a global anti-angiogenic activity, and whether different administration schedules of the same drug could have a synergistic or an antagonistic effect, which obviously would need to be taken into account in determining combinations with new agents targeting angiogenesis.

Human pancreatic carcinoma cells secrete bioactive interleukin-18 after treatment with 5-fluorouracil: implications for anti-tumor immune response.

Recently we observed that pancreatic carcinoma cell lines constitutively express Interleukin-18 (IL-18). Bioactive IL-18 induces Interferon (IFN)-gamma production, Fas Ligand (FasL) expression, and inhibits angiogenesis, raising the issue of anti-tumor effects of a tumor-derived cytokine and motivating a more detailed analysis of IL-18 production in pancreatic carcinoma cells. This analysis included the study of effects of chemotherapeutic drugs (5-fluorouracil [5-FU], gemcitabine, cisplatin) commonly used in the treatment of pancreatic cancer patients on IL-18 production and processing. IL-18 expression and post-translational processing were determined using RT-PCR, immunoblot and ELISA in pancreatic carcinoma cell lines and in tumor tissue and serum samples from pancreatic carcinoma patients in the presence and absence of chemotherapeutic drugs. We describe expression of IL-18 in pancreatic carcinoma cells and tissues associated with significantly elevated IL-18 levels in patients sera. Specifically, Capan-2 pancreatic tumor cells produced and secreted precursor IL-18 with no apparent biological activity. However, the chemotherapeutic agent 5-FU, by inducing Caspase-1 and Caspase-3 activation, induced secretion of proteolytically processed mature and degraded IL-18 species, respectively, in Capan-2 cells. Conditioned medium from 5-FU-treated but not control Capan-2 cells induced IFN-gamma production by activated T cells in an IL-18-dependent manner. Furthermore, adjuvant polychemotherapy including 5-FU significantly increased serum levels of mature, bioactive IL-18 in pancreatic carcinoma patients. Treatment of pancreatic cancer cells with 5-FU induced Caspase-dependent processing of pro-IL18 leading to the secretion of biologically active IL-18. These findings delineate a novel mechanism by which chemotherapeutic agents may modulate local anti-tumor cell-mediated immune responses.