Cooperative Role of Thrombopoietin and Vascular Endothelial Growth Factor-A in the Progression of Liver Cirrhosis to Hepatocellular Carcinoma.

Primary thrombopoietic mediator thrombopoietin (THPO) is mainly produced by the liver; it may act as a growth factor for hepatic progenitors. Principal angiogenesis inducer vascular endothelial growth factor-A (VEGF-A) is critical for the complex vascular network within the liver architecture. As a cross-regulatory loop between THPO and VEGF-A has been demonstrated in the hematopoietic system, the two growth factors were hypothesized to cooperatively contribute to the progression from liver cirrhosis (LC) to hepatocellular carcinoma (HCC). The mRNA and protein expression levels of THPO, VEGF-A, and their receptors were examined, compared, and correlated in paired cancerous and LC tissues from 26 cirrhosis-related HCC patients, using qRT-PCR and immunohistochemistry. THPO and VEGF-A were alternatively silenced by small interfering RNA (siRNA) in human liver cancer cell lines Huh7 and HepG2. THPO and VEGF-A expressions significantly increased in tumor versus LC tissues. HCC and paired LC cells expressed similar levels of THPO receptor (R), whereas vascular endothelial growth factor receptor (VEGFR) -1 and VEGFR-2 levels were higher in HCC than in corresponding LC tissue samples. A significant linear correlation emerged between THPO and VEGF-A transcripts in HCC and, at the protein level, THPO and THPOR were significantly correlated with VEGF-A in tumor tissues. Both HCC and LC expressed similar levels of gene and protein hypoxia inducible factor (HIF)-1α. Positive cross-regulation occurred with the alternative administration of siRNAs targeting THPO and those targeting VEGF-A in hypoxic liver cancer cell lines. These results suggest THPO and VEGF-A might act as interdependently regulated autocrine and/or paracrine systems for cellular growth in HCC. This might be clinically interesting, since new classes of THPOR agonistic/antagonistic drugs may provide novel therapeutic options to correct the frequent hemostatic abnormality seen in HCC patients.

Impact of Tissue Enolase 1 Protein Overexpression in Esophageal Cancer Progression.

Enolase (ENO) 1 is a key glycolytic enzyme and important player in tumorigenesis. ENO1 overexpression has been correlated with tumor progression and/or worse prognosis in several solid malignancies. However, data concerning the impact of ENO1 in cancer conflict. The study correlated local and circulating ENO1 protein levels in esophageal cancer (EC) with clinicopathological data, to assess its potential clinical value. ENO1 expression was analyzed by immunohistochemistry in paired tumor and non-tumor tissue samples from 40 EC cases and mucosal biopsies from 45 Barrett's esophagus (BE) cases, plus in plasma from these patients and 25 matched healthy controls. ENO1 was abnormally elevated in cancer-cell cytoplasm in both EC types, in esophageal squamous cell carcinoma and in adenocarcinoma (EAC), increasing significantly with tumor stage progression and the transition from BE to EAC. EAC patients exhibited significantly lower ENO1 plasma concentrations than normal subjects. Neither local nor systemic ENO1 expression levels were significantly associated with overall survival. These results indicate ENO1 as potential biomarker, delineating a population of patients with Barrett's esophagus at high risk of cancer, and as new therapeutic opportunity in EC patient management. However, further confirmation might be necessary.

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.

A Xenogenic Bone Derivative as a Potential Adjuvant for Bone Regeneration and Implant Osseointegration: An In Vitro Study.

Several clinical conditions may limit the success of bone regeneration and/or implant osseointegration. For this reason, many compounds have been tested for their ability to stimulate this biological process. Synthetic hydroxyapatite (HA), mimicking natural bone hydroxyapatite, and extra-cellular matrix proteins, such as type I collagen, are potential candidates. However, the synthetic origin of HA and the denaturing conditions required for extracting collagen from skin and derma are sources of potential drawbacks. This study examines the in vitro effects of a natural bone derivative (NBD) extracted from equine bone and containing both natural, non-synthetic bone hydroxyapatite and native, non-denatured, type I bone collagen as a possible active compound for stimulating bone regeneration and implant osseointegration. The activity of NBD was tested on bone marrow stromal cells (BMSCs), evaluating their growth/viability by the methylthiazol tetrazolium (MTT) assay and their migration potential by a scratch assay. Moreover, expression of the hyaluronic acid receptor (CD44) and the C-X-C chemokine receptor type 4 (CXCR4, CD184) on the surface of BMSCs was assessed by flow cytometry, and the release of Transforming Growth Factor (TGF)-ß, Interleukin (IL)-1α and IL-6 was quantified using an enzyme-linked immunosorbent assay (ELISA). The effect of NBD-coated implants on human osteoblasts was tested by measuring alkaline phosphatase (ALP) activity with the p-nitrophenyl phosphate (pNPP) degradation test. NBD stimulated BMSC growth/viability, migration, CD184 surface expression and the release of TGF-ß1. NBD-coated implants increased ALP activity of human osteoblasts. These results indicate that NBD may be an adjuvant to accelerate both bone regeneration and osseointegration.

Enhancement of the Immunostimulatory Functions of Ex Vivo-Generated Dendritic Cells from Early-Stage Colon Cancer Patients by Consecutive Exposure to Low Doses of Sequential-Kinetic-Activated IL-4 and IL-12. A Preliminary Study.

Dendritic cells (DCs), specialized antigen-presenting cells bridging innate and adaptive immunity, play a crucial role in determining specific immune response to tumors. Because of their potent immunoregulatory capacities, DCs have been exploited in anticancer vaccination, with limited success thus far. This pilot study compared low-dose interleukin (IL)-4 and IL-12 prepared by sequential kinetic activation (SKA) with standard doses of the same recombinant human cytokines on functional activity of ex vivo-generated monocyte-derived (Mo) DCs from colon carcinoma patients and normal subjects. MoDCs were exposed to medium alone, SKA-IL-4 (0.5 fg/ml), or SKA-IL-12 (2 fg/ml), alone or consecutively combined, in parallel with rhIL-4 (50 ng/ml) and rhIL-12 (1 ng/ml). Primary allogeneic one-way mixed lymphocyte reaction (MLR) was the end point to assess in vitro T-lymphocyte proliferation in response to MoDCs, and secreted IL-12p70 and interferon-γ in MLR supernatants measured by ELISA to assay for T-helper 1-promoting MoDC phenotype. No single agent enhanced the compromised allostimulatory activity of MoDCs from colon cancer patients, unlike healthy donors. However, MoDCs from nonmetastatic colon cancer patients, after sequential exposure to SKA-IL-4 (48 hours) and SKA-IL-12 (24 hours), displayed increased T-cell stimulatory capacity by MLR and acquired driving T-helper 1 polarization activity, although less markedly than the effects induced by recombinant human cytokines or found in normal subjects. These results point to an immunomodulatory capacity of low-dose SKA-IL-4 and SKA-IL-12 and encourage further investigation to provide clues for the rational development of new and more effective immunotherapeutic strategies against cancer.

Eosinophilic inflammation of chronic rhinosinusitis with nasal polyps is related to OX40 ligand expression.

The aims of this study were to investigate OX40 ligand expression in sinus tissue from patients with nasal polyposis compared with patients with chronic rhinosinusitis without nasal polyps (NPs), and to determine if OX40 ligand expression is related to eosinophilic sinus infiltration. Twenty patients with chronic rhinosinusitis (11 with and nine without NPs) and seven controls were enrolled in the study. The mRNA expression of OX40 ligand and thymic stromal lymphopoietin and its receptor were analyzed. The immunoreactivity score for OX40 ligand and the eosinophil count were obtained. The mRNA expression and immunoreactivity score of OX40 ligand were higher in patients with nasal polyposis than in patients without NPs, as well as healthy controls. The mRNA expression of thymic stromal lymphopoietin and its receptor was significantly higher in nasal polyposis than in the control, but not significantly higher than in chronic rhinosinusitis without NPs. A correlation between the number of OX40 ligand-positive cells and the number of eosinophils in sinus biopsies was found only in patients with nasal polyposis. In conclusion, the thymic stromal lymphopoietin/OX40 ligand axis is up-regulated in nasal polyposis and is related to the intensity of eosinophilic inflammation.

Low-doses of sequential-kinetic-activated interferon-γ enhance the ex vivo cytotoxicity of peripheral blood natural killer cells from patients with early-stage colorectal cancer. A preliminary study.

Natural killer (NK) cells are innate immune-system lymphocytes capable of killing tumor cells. They secrete cytokines, including interferon (IFN)-γ, which participate in shaping the initial inflammatory and downstream adaptive immune responses. Its potent immunoregulatory action means that IFN-γ might be beneficial in cases of tumor rejection, but its severe side-effects limit clinical applications. This pilot study compared low-dose IFN-γ prepared by sequential-kinetic-activation (SKA), with standard-dose recombinant (r) IFN-γ, in terms of ex-vivo cytotoxic activity of peripheral blood (PB)-NK cells from colorectal carcinoma (CRC) patients. This was tested against the NK-sensitive K562 cell line and the less-sensitive human CRC Caco-2 and HT-29 cell lines. Twenty primitive non-metastatic CRC patients, five metastatic CRC patients, and thirteen healthy donors were enrolled. PB lymphocytes (PBL) were exposed to medium alone, SKA-IFN-γ (0.25fg/ml) or rIFN-γ (1ng/ml). NK-cell cytolytic activity was examined via short-term (51)Cr-release. Pretreatment of PBL from non-metastatic patients with SKA-IFN-γ caused a significant increase in NK-cell cytotoxicity, compared to those from normal donors, although less markedly than pretreatment with rIFN-γ against all three cell lines. In contrast, PBL from metastatic CRC patients displayed significantly decreased NK-cell activity and responsiveness to both rIFN-γ and SKA-IFN-γ treatments. These results demonstrate in principle the immunomodulatory capacity of low-dose SKA-IFN-γ, and might open the door to the possibility of generating a novel, safe, and feasible approach to enhancing NK-cell antitumor activity in early-stage CRC patients.

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.

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.

Innate and lymphocytic response of birch-allergic patients before and after sublingual immunotherapy.

Functional imbalance in Th1/Th2 cell response toward allergens is a recognized hallmark of allergic patients and a major role of dendritic cells (DCs) in redirecting T-cell phenotypes after specific immunotherapy has been suggested. This study investigates the proliferative and cytokine responses of T cells cocultured with monocyte-derived DCs (MoDCs) after allergen stimulation in birch-allergic patients compared with controls and investigates whether sublingual immunotherapy (SLIT) could change the DC-driven immune response. T cells were stimulated with the major birch pollen allergen (nBet v1) and MoDCs from eight birch-allergic patients with seasonal allergic rhinitis and eight nonallergic controls. Proliferation and cytokine production were measured before and after one course of SLIT with birch allergoid. Significantly lower levels of proinflammatory (IL-1beta, p = 0.027; IL-6, p = 0.030; TNF-alpha, p = 0.019) and Th1 (interferon gamma, p = 0.032; IL-12, p = 0.05) cytokines were measured in supernatants of T cells and MoDCs cultures from allergic patients compared with nonallergic controls. After SLIT, significant increase in IL-12 (p = 0.039), IL-1beta (p = 0.040), IL-6 (p = 0.041), TNF-α (p = 0.048), and IL-10 (p = 0.048) and significant decrease in IL-13 (p = 0.001) were observed. MoDCs/T-cell cocultures, pulsed with the specific allergen, produced lower quantities of proinflammatory and Th1 cytokines in allergic patients compared with healthy subjects, suggesting an allergen-specific impairment of natural immunity and Th1 immune response. A single course of SLIT was able to enhance allergen-specific innate immunity and to modify lymphocyte response, promoting Th1 and T-cell regulatory activity.

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.

Unexplained chronic cough and vitamin B-12 deficiency.

BACKGROUND: Chronic cough is characterized by sensory neuropathy. Vitamin B-12 (cobalamin) deficiency (Cbl-D) causes central and peripheral nervous system damage and has been implicated in sensory neuropathy and autonomic nervous system dysfunction. OBJECTIVE: We evaluated whether Cbl-D has a role in chronic, unexplained cough. DESIGN: Laryngeal threshold (histamine concentration that provokes a 25% decrease in the midinspiratory flow), bronchial threshold (histamine concentration that provokes a 20% decrease in the forced expiratory volume in 1 s), and cough threshold (histamine concentration that causes ≥5 coughs) in response to an inhaled histamine were assessed in 42 patients with chronic, unexplained cough [27 Cbl-D patients and 15 patients without Cbl-D (Cbl-N)] before and after intramuscular injections of cobalamin for 2 mo. Laryngeal, bronchial, and cough hyperresponsiveness was diagnosed when histamine concentration thresholds were ≤8 mg/mL. Seven Clb-D and 3 Cbl-N patients underwent an oropharyngeal biopsy before treatment. RESULTS: Cbl-D patients had a higher prevalence of laryngeal hyperresponsiveness than did Cbl-N patients (92.6% compared with 66.7%; P = 0.03), a thinner oropharyngeal epithelium [133.7 µm (95% CI: 95, 172 µm) compared with 230.8 µm (95% CI: 224, 237 µm); P = 0.002], a lower number of myelinated nerve fibers [2.25/mm(2) (95% CI: 1.8, 2.7/mm(2)) compared with 3.44/mm(2) (95% CI: 3, 3.8/mm(2)); P = 0.05], and a higher immunoreactive score for nerve growth factor (NGF) [6.7 (95% CI: 6, 7.3) compared with 2.8 (95% CI: 2.5, 3.1); P = 0.02]. After cobalamin supplementation, symptoms and laryngeal, bronchial, and cough thresholds were significantly improved in Cbl-D but not in Cbl-N patients. CONCLUSIONS: This study suggests that Cbl-D may contribute to chronic cough by favoring sensory neuropathy as indicated by laryngeal hyperresponsiveness and increased NGF expression in pharyngeal biopsies of Cbl-D patients. Cbl-D should be considered among factors that sustain chronic cough, particularly when cough triggers cannot be identified.

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.

Abnormal expression of Endoglin and its receptor complex (TGF-ß1 and TGF-ß receptor II) as early angiogenic switch indicator in premalignant lesions of the colon mucosa.

The precise timing of the angiogenic switch in colorectal cancer development is still unclear. The simultaneous expression of Endoglin (CD105), transforming growth factor (TGF)-ß1 and TGF-ß receptor (R) II were quantified in surgical specimens comprising normal human colon, pre-malignant dysplastic tissue, in situ, and invasive colon cancer specimens, at mRNA and protein levels, respectively by real-time PCR and immunohistochemistry. Serum concentrations of soluble Endoglin and TGF-ß1 were evaluated. mRNA and CD105+-microvessel density (MVD) increased significantly in dysplastic colon and carcinoma versus normal tissues; values correlated respectively with dysplasia degree and Dukes' stages. TGF-ß1 expression was significantly upregulated in most severe dysplastic adenoma specimens, while TGF-ß1 transcript and protein signals were intense in carcinoma, positively-correlated with tumor progression. TGF-ß1 RII was overexpressed in adenoma and carcinoma versus normal samples, but unrelated with dysplasia or Dukes' stage. Soluble Endoglin serum levels were equivalent in adenoma and normal tissues; in carcinoma the highest levels were in invasive tumor. Circulating TGF-ß1 levels were increased in severe dysplasia and progressed with tumor progression. Correlations between adenoma dysplasia degree and TGF-ß RII and CD105+-MVD, and between tumor Dukes' staging and TGF-ß1 and CD105+-MVD, were significant. TGF-ß1 and Endoglin and TGF-ß1 serum levels, TGF-ß1 staining and CD105+-MVD were significantly and inversely associated with disease-free survival. TGF-ß1 levels were an independent and significant prognostic factor of disease-free survival. These findings suggest active angiogenesis occurs in many pre-malignant colon cases and supports more careful evaluation of different chemopreventive agents.

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.

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.

Ski/SnoN expression in the sequence metaplasia-dysplasia-adenocarcinoma of Barrett's esophagus.

Barrett's esophagus (BE) is a precancerous condition. However, the mechanisms underlying the transformation from metaplastic to dysplastic to adenocarcinomatous epithelium are still poorly understood. As loss of transforming growth factor-beta growth inhibition is considered a hallmark of several human neoplasms, we evaluated the expression of Ski and SnoN (proteins that antagonize transforming growth factor-beta signaling through physical interaction with Smad complex and by recruiting histone deacetylases), as markers of the transforming growth factor-beta signaling pathway, in BE with and without dysplasia. Biopsy samples from 37 patients (26 men, aged 60 +/- 8 years) with histologically proven BE were evaluated; 10 patients had concomitant low-grade dysplasia, 7 high-grade dysplasia (HGD), and 6 HGD associated with adenocarcinoma. Ski and SnoN expression was assessed immunohistochemically. Neither Ski nor SnoN was expressed in normal esophageal epithelium, but both were strongly expressed in BE tissue, with intense cytoplasmic positivity. Expression of these proteins decreased markedly in dysplastic areas in patients with low-grade dysplasia and was absent in those with HGD or HGD/adenocarcinoma. Ski and SnoN proteins are overexpressed in BE and may be involved in abnormal signaling elicited by transforming growth factor-beta in this epithelium, enhancing the tumorigenesis process. These observations might help to elucidate the molecular mechanisms involved in the BE tumorigenesis process.

Transforming growth factor-beta binding receptor endoglin (CD105) expression in esophageal cancer and in adjacent nontumorous esophagus as prognostic predictor of recurrence.

BACKGROUND: We hypothesized that the potent neovascularization marker endoglin (CD105), by differentially highlighting a subset of microvessels (MV) in esophageal cancer (EC), could provide better prognostic/therapeutic information than the panendothelial marker CD34, which also highlights MV. METHODS: Endoglin messenger ribonucleic acid (mRNA) expression in normal, malignant, and adjacent nontumorous esophagus tissue was quantified by real-time reverse-transcription polymerase chain reaction (RT-PCR). Sections of formalin-fixed, paraffin-embedded tissues were analyzed immunohistochemically for CD105 and CD34. MV density was counted following a standard protocol. Circulating soluble endoglin levels were determined in patient and control sera, and compared with clinical outcome. RESULTS: CD105 mRNA was upregulated by a median factor of 2.89 in ECs versus controls. In 28% of patients, CD105 mRNA was upregulated by a median factor of 2.65 in adjacent non-tumorous versus normal tissue. In tumor tissues, CD105 was stained negatively or positively only in a subset of MV. CD34 always showed positive extensive MV staining. In adjacent nontumorous esophagus, CD105 rarely showed diffuse MV staining, while CD34 stained blood-vessel endothelial cells in all non-neoplastic tissue. CD105 expression was high in residual highly dysplastic Barrett's-type mucosa associated with some adenocarcinomas. No statistically significant difference in endoglin serum levels appeared between patients and normal subjects. Correlation with clinicopathological data showed higher intra-tumor MV-CD105+ scores at more-advanced clinical stages. High-scoring MV-CD105+ patients had significantly shorter disease-free and overall survival; MV-CD34+ density was not survival related. Diffuse CD105 expression in adjacent nontumorous esophagus predicted poorer disease-free and overall survival. CONCLUSIONS: Our findings could help identify EC patients who may benefit from targeted anti-angiogenic therapies.

Loss of interstitial cells of Cajal network in severe idiopathic gastroparesis.

AIM: To report a case of severe idiopathic gastroparesis in complete absence of Kit-positive gastric interstitial cells of Cajal (ICC). METHODS: Gastric tissue from a patient with severe idiopathic gastroparesis unresponsive to medical treatment and requiring surgery was analyzed by conventional histology and immunohistochemistry. RESULTS: Gastric pacemaker cells expressing Kit receptor had completely disappeared while the local level of stem cell factor, the essential ligand for its development and maintenance, was increased. No signs of cell death were observed in the pacemaker region. CONCLUSION: These results are consistent with the hypothesis that a lack of Kit expression may lead to impaired functioning of ICC. Total gastrectomy proves to be curative.