HLA-G expression in melanoma: A way for tumor cells to escape from immunosurveillance

Considering the well established role of nonclassical HLA-G class I molecules in inhibiting natural killer (NK) cell function, the consequence of abnormal HLA-G expression in malignant cells should be the escape of tumors from immunosurveillance. To examine this hypothesis, we analyzed HLA-G expression and NK sensitivity in human malignant melanoma cells. Our analysis of three melanoma cell lines and ex vivo biopsy demonstrated that (i) IGR and M74 human melanoma cell lines exhibit a high level of HLA-G transcription with differential HLA-G isoform transcription and protein expression patterns, (ii) a higher level of HLA-G transcription ex vivo is detected in a skin melanoma metastasis biopsy compared with a healthy skin fragment from the same individual, and (iii) HLA-G protein isoforms other than membrane-bound HLA-G1 protect IGR from NK lysis. It thus appears of critical importance to consider the specific role of HLA-G expression in tumors in the design of future cancer immunotherapies.

Identification of a Human VPF/VEGF 3′ Untranslated Region Mediating Hypoxia-induced mRNA Stability

Hypoxia is a prominent feature of malignant tumors that are characterized by angiogenesis and vascular hyperpermeability. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) has been shown to be up-regulated in the vicinity of necrotic tumor areas, and hypoxia potently induces VPF/VEGF expression in several tumor cell lines in vitro. Here we report that hypoxia-induced VPF/VEGF expression is mediated by increased transcription and mRNA stability in human M21 melanoma cells. RNA-binding/electrophoretic mobility shift assays identified a single 125-bp AU-rich element in the 3′ untranslated region that formed hypoxia-inducible RNA-protein complexes. Hypoxia-induced expression of chimeric luciferase reporter constructs containing this 125-bp AU-rich hypoxia stability region were significantly higher than constructs containing an adjacent 3′ untranslated region element without RNA-binding activity. Using UV-cross-linking studies, we have identified a series of hypoxia-induced proteins of 90/88 kDa, 72 kDa, 60 kDa, 56 kDa, and 46 kDa that bound to the hypoxia stability region element. The 90/88-kDa and 60-kDa species were specifically competed by excess hypoxia stability region RNA. Thus, increased VPF/VEGF mRNA stability induced by hypoxia is mediated, at least in part, by specific interactions between a defined mRNA stability sequence in the 3′ untranslated region and distinct mRNA-binding proteins in human tumor cells.

Functional Hierarchy of Simultaneously Expressed Adhesion Receptors: Integrin α2β1 but Not CD44 Mediates MV3 Melanoma Cell Migration and Matrix Reorganization within Three-dimensional Hyaluronan-containing Collagen MatricesV⃞

Haptokinetic cell migration across surfaces is mediated by adhesion receptors including β1 integrins and CD44 providing adhesion to extracellular matrix (ECM) ligands such as collagen and hyaluronan (HA), respectively. Little is known, however, about how such different receptor systems synergize for cell migration through three-dimensionally (3-D) interconnected ECM ligands. In highly motile human MV3 melanoma cells, both β1 integrins and CD44 are abundantly expressed, support migration across collagen and HA, respectively, and are deposited upon migration, whereas only β1 integrins but not CD44 redistribute to focal adhesions. In 3-D collagen lattices in the presence or absence of HA and cross-linking chondroitin sulfate, MV3 cell migration and associated functions such as polarization and matrix reorganization were blocked by anti-β1 and anti-α2 integrin mAbs, whereas mAbs blocking CD44, α3, α5, α6, or αv integrins showed no effect. With use of highly sensitive time-lapse videomicroscopy and computer-assisted cell tracking techniques, promigratory functions of CD44 were excluded. 1) Addition of HA did not increase the migratory cell population or its migration velocity, 2) blocking of the HA-binding Hermes-1 epitope did not affect migration, and 3) impaired migration after blocking or activation of β1 integrins was not restored via CD44. Because α2β1-mediated migration was neither synergized nor replaced by CD44–HA interactions, we conclude that the biophysical properties of 3-D multicomponent ECM impose more restricted molecular functions of adhesion receptors, thereby differing from haptokinetic migration across surfaces.

Contact with fibrillar collagen inhibits melanoma cell proliferation by up-regulating p27KIP1

It is known that the extracellular matrix regulates normal cell proliferation, and it is assumed that anchorage-independent malignant cells escape this regulatory function. Here we demonstrate that human M24met melanoma cells remain responsive to growth regulatory signals that result from contact with type I collagen and that the effect on proliferation depends on the physical structure of the collagen. On polymerized fibrillar collagen, M24met cells are growth arrested at the G1/S checkpoint and maintain high levels of p27KIP1 mRNA and protein. In contrast, on nonfibrillar (denatured) collagen, the cells enter the cell cycle, and p27KIP1 is down-regulated. These growth regulatory effects involve contact between type I collagen and the collagen-binding integrin α2β1, which appears restricted in the presence of fibrillar collagen. Thus melanoma cells remain sensitive to negative growth regulatory signals originating from fibrillar collagen, and the proteolytic degradation of fibrils is a mechanism allowing tumor cells to escape these restrictive signals.

Species-specific metastasis of human tumor cells in the severe combined immunodeficiency mouse engrafted with human tissue.

We have attempted to model human metastatic disease by implanting human target organs into the immunodeficient C.B-17 scid/scid (severe combined immunodeficiency; SCID) mouse, creating SCID-hu mice. Preferential metastasis to implants of human fetal lung and human fetal bone marrow occurred after i.v. injection of human small cell lung cancer (SCLC) cells into SCID-hu mice; the homologous mouse organs were spared. Clinically more aggressive variant SCLC cells metastasized more efficiently to human fetal lung implants than did cells from classic SCLC. Metastasis of variant SCLC to human fetal bone marrow was enhanced in SCID-hu mice exposed to gamma-irradiation or to interleukin 1 alpha. These data indicate that the SCID-hu mice may provide a model in which to study species- and tissue-specific steps of the human metastatic process.

Generation and maturation of dendritic cells for clinical application under serum-free conditions

Monocyte-derived dendritic cells (MoDCs) in clinical use for cancer immunotherapy are ideally generated in serum-free medium (SFM) with inclusion of a suitable maturation factor toward the end of the incubation period. Three good manfacturing practice (GMP) grade SFMs (AIM-V, X-VIVO 15, and X-VIVO 20) were compared with RPMI-1640, supplemented with 10% fetal bovine serum or 10% human serum. DCs generated for 7 days in SFM were less mature and secreted less interleukin (IL) 12p70 and IL-10 than DCs generated in 10% serum. DC yield was comparable in SFMs, and a greater proportion of cells was viable after maturation. Toll-like receptor (TLR) ligands were compared for their ability to induce cytokine secretion under serum-free conditions in the presence of interferon (IFN) gamma. With the exception of Poly I:C, TLR ligands stimulated high levels of IL-10 secretion. High levels of IL-12p70 were induced by two TLR4-mediated stimuli, lipopolysaccharide and Ribomunyl, a clinical-grade bacterial extract. When T-cell responses were compared in allogeneic mixed leukocyte reaction, DCs stimulated with Ribomunyl induced higher levels of IFN gamma than DCs stimulated with the cytokine cocktail: tumor necrosis factor-alpha, IL-1 beta, IL-6, and prostaglandin E-2. In the presence of IL-10 neutralizing antibodies, DC IL-12p70 production and T-cell IFN gamma were increased in vitro. Similarly, DCs stimulated with Ribomunyl, IFN gamma, and anti-IL-10 induced high levels of tetanus toxoid-specific T-cell proliferation and IFN gamma secretion. Thus, MoDCs generated ill SFM efficiently stimulate T-cell IFN gamma production after maturation in the presence of a clinical-grade TLR4 agonist and IL-10 neutralization.

External quality assessment of BRAF molecular analysis in melanoma

The availability of BRAF inhibitors has given metastatic melanoma patients an effective new treatment choice and molecular testing to determine the presence or absence of a BRAF codon 600 mutation is pivotal in the clinical management of these patients. This molecular test must be performed accurately and appropriately to ensure that the patient receives the most suitable treatment in a timely manner. Laboratories have introduced such testing; however, some experience low sample throughput making it critical that an external quality assurance programme is available to help promote a high standard of testing, reporting and provide an educational aspect for BRAF molecular testing. Laboratories took part in three rounds of external quality assessment (EQA) during a 12-month period giving participants a measure of the accuracy of genotyping, clinical interpretation of the result and experience in testing a range of different samples. Formalin fixed paraffin embedded tissue sections from malignant melanoma patients were distributed to participants for BRAF molecular testing. The standard of testing was generally high but distribution of a mutation other than the most common, p.(Val600Glu), highlighted concerns with detection or reporting of the presence of rarer mutations. The main issues raised in the interpretation of the results were the importance of clear unambiguous interpretation of the result tailored to the patient and the understanding that the treatment is different from that given to other stratified medicine programmes. The variability in reporting and wide range of methodologies used indicate a continuing need for EQA in this field.

BRAF mutation testing algorithm for vemurafenib treatment in melanoma: recommendations from an expert panel.

The incidence of melanoma has increased rapidly over the past 30 years, and the disease is now the sixth most common cancer among men and women in the U.K. Many patients are diagnosed with or develop metastatic disease, and survival is substantially reduced in these patients. Mutations in the BRAF gene have been identified as key drivers of melanoma cells and are found in around 50% of cutaneous melanomas. Vemurafenib (Zelboraf(®) ; Roche Molecular Systems Inc., Pleasanton, CA, U.S.A.) is the first licensed inhibitor of mutated BRAF, and offers a new first-line option for patients with unresectable or metastatic melanoma who harbour BRAF mutations. Vemurafenib was developed in conjunction with a companion diagnostic, the cobas(®) 4800 BRAF V600 Mutation Test. The purpose of this paper is to make evidence-based recommendations to facilitate the implementation of BRAF mutation testing and targeted therapy in patients with metastatic melanoma in the U.K. The recommendations are the result of a meeting of an expert panel and have been reviewed by melanoma specialists and representatives of the National Cancer Research Network Clinical Study Group on behalf of the wider melanoma community. This article is intended to be a starting point for practical advice and recommendations, which will no doubt be updated as we gain further experience in personalizing therapy for patients with melanoma.

Evaluation of Adoptive T Cell Therapy and Oncolytic Virotherapy for Treatment of Brain Tumors

Adoptive immunotherapy and oncolytic virotherapy are two promising strategies for treating primary and metastatic malignant brain tumors. We demonstrate the ability of adoptively transferred tumor-specific T cells to rapidly mediate the clearance of established brain tumors in several mouse models. Similar to the clinical situation, tumor recurrences are frequent and result from immune editing of tumors. T cells can eliminate antigen-expressing tumor cells but are not effective against antigen loss variant (ALV) cancer cells that multiply and repopulate a tumor. We show that the level of tumor antigen present affects the success of adoptive T cell therapy. When high levels of antigen are present, tumor stromal cells such as microglia and macrophages present tumor peptide on their surface. As a result, T cells directly eliminate cancer cells and cross-presenting stromal cells and indirectly eliminate ALV cells. We were able to show the first direct evidence of tumor antigen cross-presentation by CD11b+ stromal cells in the brain using soluble, high-affinity T cell receptor monomers. Strategies that target brain tumor stroma or increase antigen shedding from tumor cells leading to increased crosspresentation by stromal cells may improve the clinical success of T cell adoptive therapies. We evaluated one potential strategy to complement adoptive T cell therapy by characterizing the oncolytic effects of myxoma virus (MYXV) in a syngeneic mouse brain tumor model of metastatic melanoma. MYXV is a rabbit poxvirus with strict species tropism for European rabbits. MYXV can also infect mouse and human cancer cell lines due to signaling defects in innate antiviral mechanisms and hyperphosphorylation of Akt. MYXV kills B16.SIY melanoma cells in vitro, and intratumoral injection of virus leads to robust, selective and transient infection of the tumor. We observed that virus treatment recruits innate immune cells iii to the tumor, induces TNFα and IFNβ production in the brain, and results in limited oncolytic effects in vivo. To overcome this, we evaluated the safety and efficacy of co-administering 2C T cells, MYXV, and neutralizing antibodies against IFNβ. Mice that received the triple combination therapy survived significantly longer with no apparent side effects, but eventually relapsed. Based on these findings, methods to enhance viral replication in the tumor and limit immune clearance of the virus will be pursued. We conclude that myxoma virus should be further explored as a vector for transient delivery of therapeutic genes to a tumor to enhance T cell responses.

Bioenergetics of cancer cells in anoxia and role of the miRNAs in melanoma resistance to targeted therapies

Tumours are characterized by a metabolic rewiring that helps transformed cells to survive in harsh conditions. The endogenous inhibitor of the ATP-synthase IF1 is overexpressed in several tumours and it has been proposed to drive metabolic adaptation. In ischemic normal-cells, IF1 acts limiting the ATP consumption by the reverse activity of the ATP-synthase, activated by ΔΨm collapse. Conversely, IF1 role in cancer cells is still unclear. It has been proposed that IF1 favours cancer survival by preventing energy dissipation in low oxygen availability, a frequent condition in solid tumours. Our previous data proved that in cancer cells hypoxia does not abolish ΔΨm, avoiding the ATP-synthase reversal and IF1 activation. In this study, we investigated the bioenergetics of cancer cells in conditions mimicking anoxia to evaluate the possible role of IF1. Data obtained indicate that also in cancer cells the ΔΨm collapse induces the ATP-synthase reversal and its inhibition by IF1. Moreover, we demonstrated that upon uncoupling conditions, IF1 favours cancer cells growth preserving ATP levels and energy charge. We also showed that in these conditions IF1 favours the mitochondrial mass renewal, a mechanism we proposed driving apoptosis-resistance. Cancer adaptability is also associated with the onset of therapy resistance, the major challenge for melanoma treatment. Recent studies demonstrated that miRNAs dysregulation drive melanoma progression and drug-resistance by regulating tumour-suppressor and oncogenes. In this context, we attempted to identify and characterize miRNAs driving resistance to vemurafenib in patient-derived metastatic melanoma cells BRAFV600E-mutated. Our results highlighted that several oncogenic pathways are altered in resistant cells, indicating the complexity of both drug-resistance phenomena and miRNAs action. Profiling analysis identified a group of dysregulated miRNAs conserved in vemurafenib-resistance cells from distinct patients, suggesting that they ubiquitously drive drug-resistance. Functional studies performed with a first miRNA confirmed its pivotal role in resistance towards vemurafenib.

Half-Pringle Maneuver: A Useful Tool in Laparoscopic Liver Resection

Introduction: Laparoscopic liver resections are becoming a common procedure, and bleeding remains the major concern during parenchymal transection. Total vascular inflow occlusion can be performed, but ischemic reperfusion injuries can lead to postoperative morbidity. On the other hand, hemihepatic inflow occlusion, leading to hemiliver ischemia, decreases the amount of liver parenchyma submitted to reperfusion damage and offers the advantage of reduced blood loss. Objective: The aim of this work was to describe our experience with laparoscopic the half-Pringle maneuver for segmentar or nonanatomic liver resctions. Patients and Methods: Eight patients submitted to laparoscopic liver resection in a single tertiary center. Results: There were 5 women and 3 men with a mean age of 40.2 years (range, 26-54). Mean tumor size was 4.1 cm (range, 2.6-6.0), and mean hospital stay was 3.1 days (1-5). There were 3 liver adenomas, 2 hepatocellular carcinomas, 1 metastatic melanoma, 1 metastatic colorectal carcinoma, and 1 peripheral colangiocarcinoma. No postoperative complications or mortalities were observed. Conclusions: Results demonstrate that laparoscopic liver resection with the half-Pringle maneuver is feasible and safe and may be included in the technical armamentarium of laparoscopic liver resections for a selected group of patients.

Synthesis, Antimicrobial and Antiproliferative Activity of Novel Silver(I) Tris(pyrazolyl)methanesulfonateand 1,3,5-Triaza-7-phosphadamantane Complexes

Five new silver(I) complexes of formulas [Ag(Tpms)] (1), [Ag(Tpms)-(PPh3)] (2), [Ag(Tpms)(PCy3)] (3), [Ag(PTA)][BF4] (4), and [Ag(Tpms)(PTA)] (5) {Tpms = tris(pyrazol-1-yl)methanesulfonate, PPh3 = triphenylphosphane, PCy3 = tricyclohexylphosphane, PTA = 1,3,5-triaza-7-phosphaadamantane) have been synthesized and fully characterized by elemental analyses, H-1, C-13, and P-31 NMR, electrospray ionization mass spectrometry (ESI-MS), and IR spectroscopic techniques. The single crystal X-ray diffraction study of 3 shows the Tpms ligand acting in the N-3-facially coordinating mode, while in 2 and 5 a N2O-coordination is found, with the SO3 group bonded to silver and a pendant free pyrazolyl ring. Features of the tilting in the coordinated pyrazolyl rings in these cases suggest that this inequivalence is related with the cone angles of the phosphanes. A detailed study of antimycobacterial and antiproliferative properties of all compounds has been carried out. They were screened for their in vitro antimicrobial activities against the standard strains Enterococcus faecalis (ATCC 29922), Staphylococcus aureus (ATCC 25923), Streptococcus pneumoniae (ATCC 49619), Streptococcus pyogenes (SF37), Streptococcus sanguinis (SK36), Streptococcus mutans (UA1S9), Escherichia coli (ATCC 25922), and the fungus Candida albicans (ATCC 24443). Complexes 1-5 have been found to display effective antimicrobial activity against the series of bacteria and fungi, and some of them are potential candidates for antiseptic or disinfectant drugs. Interaction of Ag complexes with deoxyribonucleic acid (DNA) has been studied by fluorescence spectroscopic techniques, using ethidium bromide (EB) as a fluorescence probe of DNA. The decrease in the fluorescence of DNA EB system on addition of Ag complexes shows that the fluorescence quenching of DNA EB complex occurs and compound 3 is particularly active. Complexes 1-5 exhibit pronounced antiproliferative activity against human malignant melanoma (A375) with an activity often higher than that of AgNO3, which has been used as a control, following the same order of activity inhibition on DNA, i.e., 3 > 2 > 1 > 5 > AgNO3 >> 4.

Melastatin Expression in Ocular Melanocytic Proliferations

Purpose: Melastatin (MLSN-1) belongs to the transient receptor potential (TRP) superfamilly of calcium-permeable channels, and has been reported to be a melanocyte-specific gene. In human cutaneous melanoma, MLSN-1 mRNA expression displays a pattern of inverse correlation to disease free survival. We describe the patterns of MLSN-1 mRNA expression in conjunctival nevi, conjunctival melanoma, and uveal melanoma. Methods: In situ hybridization using two S35-labelled riboprobes for MLSN-1 was performed on formalin-fixed, paraffin-embedded tissues. A control probe for H4 histone was used to confirm mRNA integrity in these archival tissues. The 21 ocular melanocytic lesions studied included 5 conjunctival nevi, 6 conjunctival melanomas, and 10 enucleated eyes with uveal melanoma. The minimal requirement for interpretation of MLSN-1 mRNA loss was the presence of only background signal in a focus of at least 5 adjacent melanocytic cells. Results: Ubiquitous expression of MLSN-1 mRNA was found in conjunctival melanocytes in the non-lesional epithelium adjacent to the conjunctival melanocytic proliferations and in all 5 conjunctival nevi studied. Four different patterns of MLSN-1 mRNA expression were observed in conjunctival melanomas: one case showed complete preservation of MLSN-1 mRNA, two cases showed diffuse scattered loss of MLSN-1 mRNA, two cases showed focal clonal loss of MLSN-1 mRNA expression, and one case had no detected MLSN-1 mRNA. In uveal melanomas, MLSN-1 mRNA expression was partially preserved in two cases, lost by a clearly delimited subset of tumor cells (focal clonal loss) in four cases, and was not detectable in the entire tumor in four cases. MLSN-1 mRNA expression was also found in the normal iris, ciliary and choroidal melanocytes as well as in the retinal pigmented epithelium and in the inner nuclear layer of the retina. Conclusions: The patterns of MLSN-1 mRNA expression in the ocular melanocytic proliferations are similar to those reported in cutaneous melanocytic proliferations. In the conjunctiva, MLSN-1 mRNA expression appeared to correlate with tumor progression; all the benign conjunctival nevi had preserved expression of MLSN-1 mRNA and most of the conjunctival melanomas partial or complete loss of expression. In uveal melanoma, patterns of melastatin expression ranging from partial preservation to complete loss were found. Additional studies of a large number of ocular melanocytic proliferations may show a correlation with tumor progression and prognosis similar to that observed in cutaneous melanoma.

Modulation of cdk2, cyclin D1, p16INK4a, p21WAF and p27Kip1 expression in endothelial cells by TNF/IFN gamma.

BACKGROUND: Regional administration of high doses of tumor necrosis factor (TNF) and interferon gamma (IFN gamma) to metastatic melanoma patients causes selective disruption of the tumor vasculature. This effect is paralleled by decreased endothelial cell proliferation and suppressed integrin alpha V beta 3-mediated adhesion in vitro. Overexpression of the cyclin-dependent kinase (cdk) inhibitory protein p16INK4a was reported to interfere with integrin alpha V beta 3-dependent melanoma cell adhesion. MATERIALS AND METHODS: TNF- and IFN gamma-treated HUVEC were analyzed for cell cycle progression and for protein expression by flow cytometry and Western blotting, respectively. p16INK4a was overexpressed by transient transfection, and HUVEC adhesion was tested in short-term adhesion assays. RESULTS: TNF and IFN gamma synergistically induced a G1 arrest associated with reduced levels of cyclin D1 and cdk2, and increased expression of the cdk inhibitors p16INK4a, p21WAF and p27Kip1. p16INK4a overexpression, however, had no effect on alpha V beta 3-mediated adhesion. CONCLUSION: These results implicate the down-regulation of cyclin D1 and cdk-2, and up-regulation of p16INK4a, p21WAF and p27Kip1 in the suppression of endothelial cell proliferation induced by TNF/IFN gamma and demonstrate that increased p16INK4a levels are not sufficient to suppress alpha V beta 3-mediated endothelial cell adhesion.

SITC 26th annual meeting - summary.

ABSTRACT: The 26th annual meeting of the Society for Immunotherapy of Cancer took place in Bethesda on November 4 to 6, 2011 and was organized by Charles G. Drake (Johns Hopkins University) Dolores J. Schendel (Helmholtz Zentrum Muenchen - German Research Center for Environmental Health Institute of Molecular Immunology), Jeffrey Schlom (National Cancer Institute, National Institutes of Health), and Jedd D. Wolchok (Memorial Sloan-Kettering Cancer Center). It was an event marked by a number of extraordinary circumstances: it attracted a record attendance of 805 participants from 24 different countries. The gathering came in the wake of great as well as very sad news for the tumor immunology community. Good news included the approval of anti-CTLA-4 as a therapy for metastatic melanoma in April and the announcement in early October of the Nobel Prize in Physiology and Medicine awarded to pioneering studies in the field of immunology. Indeed, one part of the prize went to Dr. Bruce Beutler, Scripps Research Institute, La Jolla, USA and Dr. Jules Hoffman, Institute for Molecular Cell Biology, Strasbourg, France, for their discoveries in innate immunity and the other part to Dr. Ralph Steinman, The Rockfeller University, New York, for his discovery of dendritic cells. Sad news was the losses of two giants in the field. Jürg Tschopp of the University of Lausanne in March and Ralph Steinman, who passed away just three days before his Nobel Prize announcement. The loss of these two charismatic scientific leaders was particularly sad for the Annual Meeting as both J. Tschopp and R. Steinman were confirmed speakers at this meeting: the former to deliver the keynote lecture and the latter as recipient of the Richard V. Smalley prize.