A selective human H(4)-receptor agonist: (-)-2-cyano-1-methyl-3-[(2R,5R)-5- [1H-imidazol-4(5)-yl]tetrahydrofuran-2-y] methylguanidine

A series of 16 compounds related to chiral 4(5)-(5-aminomethyltetrahydrofuran-2-yl)imidazoles (1) have been designed, synthesized, and examined in vitro by radioligand displacement studies and functional assays for both the human H(3)- and H(4)-receptors expressed in SK-N-MC cells. Among them, the (2S,5S)-isomer 1d of amino compounds showed approximately 300-fold higher selectivity at the H(3)-receptor than the H(4)-receptor. On the other hand, (2R,5S)- and (2R,5R)-cyanoguanidines 3b and 3c, in which the amino group of the compounds 1b and 1c was substituted by the cyanoguanidino moiety, bound to the H(4)-receptor with a pEC(50) value of 6.65 and 7.11, respectively, and had >40-fold selectivities over the H(3)-receptor. As such, 3b and 3c are the first selective H(4) receptor agonists.     

Fruit and vegetable consumption and lymphoma risk in the European Prospective Investigation into Cancer and Nutrition (EPIC)

Introduction Lymphomas are a heterogeneous group of malignant diseases of cells of the immune system. The best-established risk factors are related to dys-regulation of immune function, and evidence suggests that factors such as dietary or lifestyle habits may be involved in the etiology. Material and methods In the European Prospective Investigation into Cancer and Nutrition (EPIC), 849 lymphoma cases were identified in a median follow-up period of 6.4 years. Fruit and vegetable consumption was estimated from validated dietary questionnaires. Cox proportional hazard models were used to examine the association between fruit and vegetable intake with the risk of lymphomas overall and subentities. Results There was no overall association between total fruit and vegetable consumption and risk of lymphoma [hazard ratio (HR) = 0.95, 95% confidence interval (CI) 0.78–1.15 comparing highest with lowest quartile]. However, the risk of diffuse large B-cell lymphomas (DLBCL) tended to be lower in participants with a high intake of total vegetables (HR = 0.49, 95% CI 0.23–1.02). Conclusion In this large prospective study, an inverse associations between fruit and vegetable consumption and risk of lymphomas overall could not be confirmed. Associations with lymphoma subentities such as DLBCL warrant further investigation. The work described in this article was carried out with the financial support of: Europe Against Cancer Program of the European Commission (SANCO); Deutsche Krebshilfe, Deutsches Krebsforschungszentrum, German Federal Ministry of Education and Research; Danish Cancer Society; Health Research Fund (FIS) of the Spanish Ministry of Health, Spanish Regional Governments of Andalucia, Asturia, Basque Country, Murcia and Navarra; the ISCIII Network RCESP (C03/09), Spain; Cancer Research UK; Medical Research Council, United Kingdom; Stroke Association, United Kingdom; British Heart Foundation; Department of Health, United Kingdom; Food Standards Agency, United Kingdom; Wellcome Trust, United Kingdom; Greek Ministry of Health; Greek Ministry of Education; Italian Association for Research on Cancer (AIRC); Italian National Research Council, Fondazione-Istituto Banco Napoli, Italy; Compagnia di San Paolo; Dutch Ministry of Public Health, Welfare and Sports; World Cancer Research Fund; Swedish Cancer Society; Swedish Scientific Council; Regional Government of Sk?ne, Sweden; Norwegian Cancer Society; Research Council of Norway; French League against Cancer (LNCC); National Institute for Health and Medical Research (INSERM), France; Mutuelle Générale de l’Education Nationale (MGEN), France; 3M Co, France; Gustave Roussy Institute (IGR), France; and General Councils of France.     

Direct and indirect impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on adult mouse Leydig cells: an in vitro study

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related substances are ubiquitous environmental pollutants that exert adverse effects on reproductive processes. In testis, Leydig cells which produce testosterone are under hormonal and local control exerted by cytokines including TNFα. Using mouse Leydig primary cell cultures as a model, we studied the effects of TCDD on the steroidogenic outcome of Leydig cells and the gene expression levels of Ccl5 and Cxcl4, previously shown to be target genes of TCDD in testis. We found that TCDD did not alter the steroidogenic outcome of Leydig cells but that it up-regulated Cxcl4 gene expression levels. TCDD also impacted Ccl5 gene expression when cells had been co-treated with TNFα. TCDD action probably initiated with binding to the aryl hydrocarbon receptor (AhR) present on Leydig cells. TCDD regulated the gene expression levels of AhR (transient down-regulation) and its repressor AhRR and Cyp1b1 (up-regulation). The trophic human chorionic gonadotropin (hCG) hormone did not impact AhR, its repressor AhRR or Cyp1b1 but it opposed the TCDD-enhanced AhRR mRNA levels. Conversely, TNFα stimulated AhR gene expression levels. Collectively, it is suggested that the impact of TCDD on expression of target genes in Leydig cells may operate under the complex network of hormones and cytokines.     

Identification of a novel allosteric binding site in the CXCR2 chemokine receptor

We have shown previously that different chemical classes of small-molecule antagonists of the human chemokine CXCR2 receptor interact with distinct binding sites of the receptor. Although an intracellular binding site for diarylurea CXCR2 antagonists, such as N-(2-bromophenyl)-N'-(7-cyano-1H-benzotriazol-4-yl)urea (SB265610), and thiazolopyrimidine compounds was recently mapped by mutagenesis studies, we now report on an imidazolylpyrimidine antagonist binding pocket in the transmembrane domain of CXCR2. Using different CXCR2 orthologs, chimeric proteins, site-directed mutagenesis, and in silico modeling, we have elucidated the binding mode of this antagonist. Our in silico-guided mutagenesis studies indicate that the ligand binding cavity for imidazolylpyrimidine compounds in CXCR2 is located between transmembrane (TM) helices 3 (Phe130(3.36)), 5 (Ser217(5.44), Phe220(5.47)), and 6 (Asn268(6.52), Leu271(6.55)) and suggest that these antagonists enter CXCR2 via the TM5-TM6 interface. It is noteworthy that the same interface is postulated as the ligand entry channel in the opsin receptor and is occupied by lipid molecules in the recently solved crystal structure of the CXCR4 chemokine receptor, suggesting a general ligand entrance mechanism for nonpolar ligands to G protein-coupled receptors. The identification of a novel allosteric binding cavity in the TM domain of CXCR2, in addition to the previously identified intracellular binding site, shows the diversity in ligand recognition mechanisms by this receptor and offers new opportunities for the structure-based design of small allosteric modulators of CXCR2 in the future.     

Neutrophils promote aerogenous spread of lung adenocarcinoma with bronchioloalveolar carcinoma features.

PURPOSE: Adenocarcinoma with bronchioloalveolar carcinoma (BAC) features is a subtype of non-small cell lung cancers characterized by an intense inflammatory reaction composed of macrophages and neutrophils and by a distinct natural history with intrapulmonary spread leading to death due to respiratory failure. We hypothesized that neutrophils could promote aerogenous spread of lung adenocarcinoma with BAC features. EXPERIMENTAL DESIGN: We examined the effect of neutrophils on A549 cell line detachment in vitro and we quantified desquamation of tumor cells on tumor tissue (n = 25) and on matched bronchioloalveolar lavage (n = 17) in vivo in a series of patients with adenocarcinoma with BAC features. RESULTS: Neutrophils induced A549 detachment mediated by signals through cell-to-cell contact. Detached A549 cells were still viable and able to proliferate in vitro. Neutralization studies identified several membrane-bound molecules involved in detachment (i.e., intercellular adhesion molecule-1/lymphocyte function-associated antigen-1, tumor necrosis factor alpha/tumor necrosis factor alpha receptor inhibitor, interleukin-1alpha /interleukin-1alpha receptor, and neutrophil elastase). In tumor tissue, shedding was detected in all samples, with a median shedding score of 42% (range, 4-95%). Micropapillary clusters were detected in 23 of the 25 tumor tissue samples, with a median micropapillary score of 1.40 (range, 0-2.1), and tumor cells were detected in 7 of 17 lavages. The micropapillary score was associated with a high neutrophil count in bronchioloalveolar lavage (P = 0.051). The shedding cell percentage was a significant factor in shorter survival (P = 0.034, univariate Cox analysis). CONCLUSIONS: Tumor shedding is induced by neutrophils. It is a significant factor of shorter survival and may be an important event in adenocarcinoma progression.     

Baseline results of the Depiscan study: a French randomized pilot trial of lung cancer screening comparing low dose CT scan (LDCT) and chest X-ray (CXR)

BACKGROUND: Lung cancer has the highest mortality-rate per cancer, with an overall 5-year survival <15%. Several non-randomized studies pointed out the high sensitivity of low dose computed tomography (LDCT) to detect early stage lung cancer. In France, Depiscan, a pilot RCT of LDCT versus chest X-ray (CXR), started on October 2002 to determine the feasibility of enrollment by general practitioners (GPs), investigations and diagnostic procedures by university hospital radiologists and multidisciplinary teams, data management by centralized clinical research assistants, and anticipate the future management of a large national trial. METHODS: GPs and occupational physicians (OPs) selected and enrolled 1000 subjects in 1 year. Eligible subjects were asymptomatic males or females aged 50-75 years with a current or former cigarette smoking history of >/=15 cigarettes per day for at least 20 years (former smokers having quit <15 years prior to enrollment). Based to randomization, annual LDCT or CXR screenings were planned at baseline and annually for 2 years. RESULTS: Between October 2002 and December 2004, 765 subjects were enrolled by 89 out of the 232 participating GPs and OPs. Complete clinical and imaging baseline data were available for 621 individuals out of the 765 enrolled, due to 144 noncompliant subjects who withdrew their consent. At least one nodule was detected in 152 out of 336 subjects (45.2%) in the LDCT screening, versus 21 out of 285 subjects (7.4%) in the CXR screening arm. Eight lung cancers were detected in the LDCT arm and one in the CXR arm. DISCUSSION: This pilot trial allows estimating that non-calcified nodules are 10 [6.36-17.07] times more often detected from LDCT than from CXR. However enrollment by GPs was more difficult than expected with 41% active investigators and a high rate (19%) of noncompliant patients. This experience speaks to the need for a high level of GPs formation and a large, coordinated clinical research team in such a trial. Trial registration number: 02526.     

Are we HER-ting for innovation in neoadjuvant breast cancer trial design?

Through the use of surrogate markers of efficacy, neoadjuvant studies may facilitate the implementation of new treatments into clinical practice. However, disease-free survival is the current standard outcome endpoint for registration of a novel treatment. The coupling of smaller neoadjuvant 'proof of principle' studies with larger adjuvant registration trials offers the promise of speeding up the time to market of new therapies. Clever new designs, such as the 'biological window' and 'learn on the way', can provide valuable insight regarding mechanisms of action and resistance of these novel drugs by identifying patients who are most likely to respond to a novel therapy early in the drug development process. Using the ongoing neoadjuvant trials with HER2 (human epidermal growth factor receptor 2)-directed therapy as a paradigm, this article discusses recent innovations in study design and the challenges of conducting translational research in the neoadjuvant setting.     

Frequent expression of PD‐L1 on circulating breast cancer cells

Immune checkpoint regulators such as PD‐L1 have become exciting new therapeutic targets leading to long lasting remissions in patients with advanced malignancies. However, in view of the remarkable costs and the toxicity profiles of these therapies, predictive biomarkers able to discriminate responders from non‐responders are urgently needed. In the present paper, we provide evidence that PD‐L1 is frequently expressed on metastatic cells circulating in the blood of hormone receptor‐positive, HER2‐negative breast cancer patients. We performed western blot, flow cytometry and immunocytochemical analyses to demonstrate the specificity of the PDL1 antibody used in our study and established immunoscores for PDL1 expression on single tumor cells. We then selected sixteen patients with circulating tumor cells (CTCs) using the CellSearch® system and found PD‐L1(+) CTCs in 11 patients (68.8%). The fraction of PD‐L1(+) CTCs varied from 0.2 to 100% in individual patients. This is the first report demonstrating the expression of PD‐L1 on CTCs. The established CTC/PD‐L1 assay can be used for liquid biopsy in future clinical trials for stratification and monitoring of cancer patients undergoing immune checkpoint blockade.     

The axon guidance molecule semaphorin 3F is a negative regulator of tumor progression and proliferation in ileal neuroendocrine tumors

Gastro-intestinal neuroendocrine tumors (GI-NETs) are rare neoplasms, frequently metastatic, raising difficult clinical and therapeutic challenges due to a poor knowledge of their biology. As neuroendocrine cells express both epithelial and neural cell markers, we studied the possible involvement in GI-NETs of axon guidance molecules, which have been shown to decrease tumor cell proliferation and metastatic dissemination in several tumor types. We focused on the role of Semaphorin 3F (SEMA3F) in ileal NETs, one of the most frequent subtypes of GI-NETs.SEMA3F expression was detected in normal neuroendocrine cells but was lost in most of human primary tumors and all their metastases. SEMA3F loss of expression was associated with promoter gene methylation. After increasing endogenous SEMA3F levels through stable transfection, enteroendocrine cell lines STC-1 and GluTag showed a reduced proliferation rate in vitro. In two different xenograft mouse models, SEMA3F-overexpressing cells exhibited a reduced ability to form tumors and a hampered liver dissemination potential in vivo. This resulted, at least in part, from the inhibition of mTOR and MAPK signaling pathways.This study demonstrates an anti-tumoral role of SEMA3F in ileal NETs. We thus suggest that SEMA3F and/or its cellular signaling pathway could represent a target for ileal NET therapy.     

Cell-surface antigen profiling of pediatric brain tumors: B7-H3 is consistently expressed and can be targeted via local or systemic CAR T-cell delivery

BackgroundImmunotherapy with chimeric antigen receptor (CAR) T cells is actively being explored for pediatric brain tumors in preclinical models and early phase clinical studies. At present, it is unclear which CAR target antigens are consistently expressed across different pediatric brain tumor types. In addition, the extent of HLA class I expression is unknown, which is critical for tumor recognition by conventional αβTCR T cells.MethodsWe profiled 49 low- and high-grade pediatric brain tumor patient-derived orthotopic xenografts (PDOX) by flow analysis for the expression of 5 CAR targets (B7-H3, GD2, IL-13Rα2, EphA2, and HER2), and HLA class I. In addition, we generated B7-H3-CAR T cells and evaluated their antitumor activity in vitro and in vivo.ResultsWe established an expression hierarchy for the analyzed antigens (B7-H3 = GD2 >> IL-13Rα2 > HER2 = EphA2) and demonstrated that antigen expression is heterogenous. All high-grade gliomas expressed HLA class I, but only 57.1% of other tumor subtypes had detectable expression. We then selected B7-H3 as a target for CAR T-cell therapy. B7-H3-CAR T cells recognized tumor cells in an antigen-dependent fashion. Local or systemic administration of B7-H3-CAR T cells induced tumor regression in PDOX and immunocompetent murine glioma models resulting in a significant survival advantage.ConclusionsOur study highlights the importance of studying target antigen and HLA class I expression in PDOX samples for the future design of immunotherapies. In addition, our results support active preclinical and clinical exploration of B7-H3-targeted CAR T-cell therapies for a broad spectrum of pediatric brain tumors.