Anna Novarino

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-γ. 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-β, 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.

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.

G-CSF administration following peripheral blood progenitor cell (PBPC) autograft in lymphoid malignancies: evidence for clinical benefits and reduction of treatment costs

Clinical value and costs of G-CSF administration following autograft with mobilized peripheral blood progenitor cells (PBPC) were evaluated in two sequential groups of 20 patients each, treated for lymphoid neoplasms in the period February 1993 to January 1996. One group was given G-CSF (Filgrastim) (5 μg/kg/day), starting on day +1 until ANC was >500/μl, the other received no G-CSF. All patients were conditioned with mitoxantrone 60 mg/m2 + L-PAM 180 mg/m2 and received large numbers of PBPC (median of 12 and 13 × 106 CD34+/kg, respectively). The median time to ANC >500/μl was 10 days in the G-CSF group vs 14 days in controls (P < 0.0001). g-csf was associated with a slightly faster platelet recovery (11 vs 13 days to plts >20 000/μl, P = 0.09). Median duration of fever (2.5 vs 5 days, P = 0.028), nonprophylactic antibiotics (8 vs 11 days, P = 0.019), and post-transplant hospitalization (13 vs 16 days, P = 0.0028) were also significantly reduced. The average cost per treatment in the G-CSF group amounted to about US

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

18 241 as compared to US

PCR-Detectable Nonneoplastic Bcl-2/IgH Rearrangements Are Common in Normal Subjects and Cancer Patients at Diagnosis but Rare in Subjects Treated With Chemotherapy

21 868 in the control group, implying a cost reduction of approximately 16%. Thus, G-CSF reduced morbidity with cost containment, supporting its use even if autograft is performed with large quantities of PBPC.

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

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with a very poor 5‐year survival rate. α‐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 α‐enolase. The present work was intended to assess the ability of α‐enolase to induce antigen‐specific T cell responses. We show that α‐enolase‐pulsed dendritic cells (DC) specifically stimulate healthy autologous T cells to proliferate, secrete IFN‐γ and lyse PDAC cells but not normal cells. In vivo, α‐enolase‐specific T cells inhibited the growth of PDAC cells in immunodeficient mice. In 8 out of 12 PDAC patients with circulating IgG to α‐enolase, the existence of α‐enolase‐specific T cells was also demonstrated. Taken as a whole, these results indicate that α‐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.

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

PURPOSE To assess whether nonneoplastic Bcl-2/IgH rearrangements act as a confounding factor in the setting of minimal residual disease analysis by evaluating their incidence in a panel of lymphoma-free subjects, including cancer-free donors and chemotherapy-naive and chemotherapy-treated cancer patients. PATIENTS AND METHODS A total of 501 nonlymphoma subjects have been assessed: 258 cancer-free patients and 243 patients with malignancies other than lymphoma, 112 of whom were chemotherapy-naive. Patients were primarily assessed by nested polymerase chain reaction (PCR), followed by real-time quantitative PCR if they scored positive. In addition, six initially PCR-positive cancer-free donors were prospectively reassessed by qualitative and quantitative PCR after 30 and 60 days. RESULTS The overall incidence of Bcl-2/IgH positivity was 9.6%, with a median number of 11 rearrangements per 1,000,000 diploid genomes (range, 0 to 2,845 rearrangements), as assessed by real-time PCR. The incidence was similar in healthy subjects and cancer patients at diagnosis (12% and 12.5%; P = not significant). In contrast, the incidence of this translocation was only 2.3% in chemotherapy-treated patients (P <.001). In addition, three initially PCR-positive cancer-free donors showed persistence of their rearrangements when assessed after 30 and 60 days. CONCLUSION The low incidence of nonneoplastic Bcl-2/IgH rearrangements following chemotherapy provides further evidence of the prognostic role of persistent PCR-positivity in the posttreatment molecular follow-up of follicular lymphoma patients.

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

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; β .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.

MACOP-B treatment for advanced stage diffuse large cell lymphoma: A multicenter Italian study

BackgroundPancreatic 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 resultsThe 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.ConclusionsAutoantibodies against Ezrin are induced early in PDAC and their combination with other serological markers may provide a predictive and diagnostic signature.

IL-18 Paradox in Pancreatic Carcinoma: Elevated Serum Levels of Free IL-18 are Correlated With Poor Survival

Recently we observed that pancreatic carcinoma cell lines constitutively express Interleukin-18 (IL-18). Bioactive IL-18 induces Interferon (IFN)-g 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 posttranslational 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-g 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.