Samir Tawadros

Soluble ligands for NK cell receptors promote evasion of chronic lymphocytic leukemia cells from NK cell anti-tumor activity

Natural killer (NK) cells are a major component of the anti-tumor immune response. NK cell dysfunctions have been reported in various hematologic malignancies, including chronic lymphocytic leukemia (CLL). Here we investigated the role of tumor cell-released soluble and exosomal ligands for NK cell receptors that modulate NK cell activity. Soluble CLL plasma factors suppressed NK cell cytotoxicity and down-regulated the surface receptors CD16 and CD56 on NK cells of healthy donors. The inhibition of NK cell cytotoxicity was attributed to the soluble ligand BAG6/BAT3 that engages the activating receptor NKp30 expressed on NK cells. Soluble BAG6 was detectable in the plasma of CLL patients, with the highest levels at the advanced disease stages. In contrast, NK cells were activated when BAG6 was presented on the surface of exosomes. The latter form was induced in non-CLL cells by cellular stress via an nSmase2-dependent pathway. Such cells were eliminated by lymphocytes in a xenograft tumor model in vivo. Here, exosomal BAG6 was essential for tumor cell killing because BAG6-deficient cells evaded immune detection. Taken together, the findings show that the dysregulated balance of exosomal vs soluble BAG6 expression may cause immune evasion of CLL cells.

Early Detection of Erlotinib Treatment Response in NSCLC by 3′-Deoxy-3′-[18F]-Fluoro-L-Thymidine ([18F]FLT) Positron Emission Tomography (PET)

Background Inhibition of the epidermal growth factor receptor (EGFR) has shown clinical success in patients with advanced non-small cell lung cancer (NSCLC). Somatic mutations of EGFR were found in lung adenocarcinoma that lead to exquisite dependency on EGFR signaling; thus patients with EGFR-mutant tumors are at high chance of response to EGFR inhibitors. However, imaging approaches affording early identification of tumor response in EGFR-dependent carcinomas have so far been lacking. Methodology/Principal Findings We performed a systematic comparison of 3′-Deoxy-3′-[18F]-fluoro-L-thymidine ([18F]FLT) and 2-[18F]-fluoro-2-deoxy-D-glucose ([18F]FDG) positron emission tomography (PET) for their potential to identify response to EGFR inhibitors in a model of EGFR-dependent lung cancer early after treatment initiation. While erlotinib-sensitive tumors exhibited a striking and reproducible decrease in [18F]FLT uptake after only two days of treatment, [18F]FDG PET based imaging revealed no consistent reduction in tumor glucose uptake. In sensitive tumors, a decrease in [18F]FLT PET but not [18F]FDG PET uptake correlated with cell cycle arrest and induction of apoptosis. The reduction in [18F]FLT PET signal at day 2 translated into dramatic tumor shrinkage four days later. Furthermore, the specificity of our results is confirmed by the complete lack of [18F]FLT PET response of tumors expressing the T790M erlotinib resistance mutation of EGFR. Conclusions [18F]FLT PET enables robust identification of erlotinib response in EGFR-dependent tumors at a very early stage. [18F]FLT PET imaging may represent an appropriate method for early prediction of response to EGFR TKI treatment in patients with NSCLC.

Comparison of necrosis in human ovarian tissue after conventional slow freezing or vitrification and transplantation in ovariectomized SCID mice.

This paper examines and compares necrosis in human ovarian tissue after conventional slow freezing or vitrification and ensuing xenotranplantation. Slow cryoconserved or vitrified ovarian tissue samples and fresh controls from nine patients were subcutaneously transplanted into SCID mice. The tissue samples were explanted after 6 weeks and the necrotic areas were examined by staining with Lucifer yellow SV. The size of the necrotic areas in parallel cultivated ovarian tissue samples was compared, as was necrosis in cultivated prostate tumour spheroids where the emergence of necrosis and its pathophysiological correlation have been described. Examinations showed no significant rise in the proportion of necrotic areas after slow cryoconservation/transplantation and in the controls (transplanted fresh tissue, not transplanted fresh tissue, long-term culture). The proportion of necrotic areas in the tumour spheroids was significantly higher than in the ovarian tissue. Vitrification could, after these results, be presented as an alternative to conventional slow cryoconservation.

Re-vascularisation in human ovarian tissue after conventional freezing or vitrification and xenotransplantation

OBJECTIVE In ovarian tissue grafts there is a massive loss of follicles during the ischaemic period until re-vascularisation is established. The aim of our study was to investigate the influence of different cryopreservation techniques on the ability for the re-vascularisation of ovarian tissue transplanted to SCID mice. STUDY DESIGN Ovarian fragments from five patients were cut into pieces (approximately 0.5 mm x 1.0 mm x 1.0 mm) and randomly distributed into three groups: fresh non-treated tissue (group A); tissue conventionally frozen in standard 0.5 ml insemination straws with 1.5 M ethylene glycol+0.1 M sucrose, with thawing in a 40 degrees C water bath and step-wise removal of cryoprotectants at room temperature in 0.5 M, 0.25 M and 0.15 M sucrose with gentle agitation (group B); tissue vitrified in 2.62 M dimethylsulphoxide+2.6 M acetamide+1.31 M propylene glycol+0.0075 M polyethylene glycol, with warming by direct plunging of solid specimens with ovarian pieces into 20 ml of 50% vitrification solution pre-warmed to 40 degrees C and dilution of cryoprotectants in a decreasing concentration of vitrification solution (25%, 12.5%) at room temperature (group C). We used a xenograft model in which ovarian tissue pieces of all three groups were subcutaneously transplanted in SCID mice. The animals were sacrificed on the third day after ovarian tissue transplantation and then weekly during 1 month to obtain the ovarian tissue grafts. These samples were examined by immunohistochemical staining with the endothelial cell-specific marker platelet endothelial cell adhesion molecule-1 (PECAM-1) to determine angiogenesis. Histological observation of tissue after explantation was performed and quality and quantity of follicles were assessed. RESULTS No PECAM-1 staining was observed in all treatment groups prior to grafting. After warming and in vivo culture of ovarian tissue, the beginning of angiogenesis in pieces from all treatment groups on the third day was detected by PECAM-1 staining. After 4 weeks of in vivo culture the overall area of PECAM-1-positive blood vessels significantly increased (P<0.05), independent of the type of cryopreservation (groups B and C vs. group A). It was found that transplantation technique had negative influence on the integrity of follicles independent of the type of treatment during in vivo culture. The duration of in vivo culture has a negative, but not statistically significant, influence on follicle quality in long-cultured transplants inside each treatment group (P>0.5). CONCLUSION The process of re-vascularisation of transplanted ovarian tissue is independent of the type of treatment and does not influence follicle quality.

Heat Shock Protein 90 Inhibitor BIIB021 (CNF2024) Depletes NF-κB and Sensitizes Hodgkin's Lymphoma Cells for Natural Killer Cell–Mediated Cytotoxicity

Purpose: In Hodgkins lymphoma, constitutive activation of NF-κB promotes tumor cell survival and proliferation. The molecular chaperone heat shock protein 90 (HSP90) has immune regulatory activity and supports the activation of NF-κB in Hodgkins lymphoma cells. Experimental Design: We analyzed the effect of HSP90 inhibition on viability and NF-κB activity in Hodgkins lymphoma cells and the consequences for their recognition and killing through natural killer (NK) cells. Results: The novel orally administrable HSP90 inhibitor BIIB021 (CNF2024) inhibited Hodgkins lymphoma cell viability at low nanomolar concentrations in synergy with doxorubicin and gemcitabine. Annexin V/7-aminoactinomycin D binding assay revealed that BIIB021 selectively induced cell death in Hodgkins lymphoma cells but not in lymphocytes from healthy individuals. We observed that BIIB021 inhibited the constitutive activity of NF-κB and this was independent of IκB mutations. Furthermore, we analyzed the effect of HSP90 inhibition on NK cell–mediated cytotoxicity. BIIB021 induced the expression of ligands for the activating NK cell receptor NKG2D on Hodgkins lymphoma cells resulting in an increased susceptibility to NK cell–mediated killing. In a xenograft model of Hodgkins lymphoma, HSP90 inhibition significantly delayed tumor growth. Conclusions: HSP90 inhibition has direct antitumor activity in Hodgkins lymphoma in vitro and in vivo. Moreover, HSP90 inhibition may sensitize Hodgkins lymphoma cells for NK cell–mediated killing via up-regulation of ligands engaging activating NK cell receptors. (Clin Cancer Res 2009;15(16):5108–16)

Magnetic resonance imaging in an orthotopic rat model: Blockade of epidermal growth factor receptor with EMD72000 inhibits human pancreatic carcinoma growth

The purpose of our research was to investigate the antiangiogenic effect of the epidermal growth factor receptor monoclonal antibody (anti‐EGF‐R MAB) EMD72000, in an orthotopic human pancreatic carcinoma model in rats, assessed by magnetic resonance (MR) imaging using angiogenic surrogate markers in comparison with histopathologic findings. Human pancreatic adenocarcinoma cells L3.6pl were injected orthotopically in the pancreas of 12 athymic nude rats. Through a 21‐day course, groups of 6 rats were treated intraperitoneally with either EMD72000 or with saline solution for control animals. Dynamic contrast‐enhanced MR imaging was performed before and after the treatment to assess microvascular permeability, estimated by the endothelial transfer coefficient (KPS) and fractional plasma volumes (fPV) of the pancreatic tumors. EMD72000‐treated animals showed significantly less tumor volume progression (1,080 mm3 ± 1,244; p = 0.012) and significantly lower values for microvascular permeability (KPS = 4.2 ml min−1 100 ml−1 of tissue ± 2.8; p = 0.015), fractional plasma volume (fPV = 0.018 ml ml−1 of tissue ± .015; p = 0.003) and microvessel density (MVD = 13 ± 4 (0.159 mm2); p = 0.001) than saline‐treated animals (6,544 mm3 ± 5,202; 9.5 ml min−1 100 ml−1 of tissue ± 4.3, 0.056 ml ml−1 of tissue ± 0.019 and 25 ± 5 (0.159 mm2), respectively). KPS and fPV values showed moderate positive correlation with MVD (r = 0.5, p = 0.103; r = 0.6, p = 0.065, respectively). Intraperitoneal injection of EMD72000 inhibits orthotopic human pancreatic carcinoma growth in rats. Antiangiogenic effects of anti‐EGF‐R MAB EMD72000 can be quantified and monitored noninvasively by dynamic MR imaging.

Terminal B cell differentiation is skewed by deregulated interleukin-6 secretion in β2 integrin-deficient mice

Absence of the common β chain (CD18) of β2 integrins leads to leukocyte‐adhesion deficiency type‐1 (LAD1) in humans. Mice with a CD18 null mutation suffer from recurrent bacterial infections, impaired wound healing, and skin ulcers, closely resembling human LAD1. Previous findings in CD18−/− mice demonstrated a skewed terminal B cell differentiation with plasmacytosis and elevated serum immunoglobulin G (IgG). As interleukin‐6 (IL‐6) is a potent enhancer of plasma cell formation and Ig secretion, we assessed IL‐6 serum levels of CD18−/− and wild‐type (WT) mice kept under a conventional or barrier facility or specific pathogen‐free (SPF) conditions. We detected an up to 20‐fold increase in IL‐6 in serum of CD18−/− mice compared with WT controls when kept under conventional or barrier facility conditions, respectively. Under SPF conditions, no significant differences in terms of IL‐6 serum levels were found between CD18−/− and WT mice. However, histological alterations of secondary lymphoid tissues, plasmacytosis, abnormal plasmacytoid cells (Mott cells), and hypergammaglobulinemia persisted. To further analyze the role of IL‐6 in these pathological alterations, we established a CD18−/− IL‐6−/− double‐deficient mouse mutant. In these mice, serum IgG levels were normal, and the altered plasma cell phenotype, including Mott cells, was no longer detectable. The CD18−/− IL‐6−/− double‐deficient mouse model thus demonstrated that IL‐6 is responsible for parts of the phenotype seen in the CD18−/− mouse mutants. It may be of interest to examine human leukocyte‐adhesion deficiency type‐1 patients closer and search for pathological changes possibly induced via overproduction of IL‐6.

Portal Vein Arterialization Increases Hepatocellular Apoptosis and Inhibits Liver Regeneration

BACKGROUND Portal vein arterialization is performed in particular situations to guarantee sufficient blood flow in the portal vein. In addition, some authors have postulated a proliferation-promoting influence of portal vein arterialization on the liver tissue. However, portal vein arterialization is an unphysiological procedure: It increases portal blood flow and blood pressure as well as oxygenation of the liver tissue. On the other hand, it reduces the influx of hepatotrophic factors from the portal venous blood. The aim of these experiments was to investigate apoptosis and proliferation of hepatocytes during various conditions of the portal perfusion. MATERIALS AND METHODS After 70% liver resection in Lewis rats, the following four experimental groups were formed differing in portal perfusion: (I) hyperperfused, nonarterialized; (II) flow-regulated, nonarterialized; (III) hyperperfused, arterialized; (IV) flow-regulated, arterialized. A warm ischemia of 30 min was kept in all groups. RESULTS Portal vein arterialization of 70% reduced rat livers significantly reduced liver regeneration as shown by a significant reduction in liver weight, body weight, and liver function after 6 wk, in contrast to the group with 70% liver mass reduction and portal venous inflow of the portal vein. Furthermore, we found a significantly elevated number of apoptotic hepatocytes after portal vein arterialization. These results were independent from blood flow regulation of the arterialized portal vein, which caused no improvement of the results. CONCLUSIONS Portal vein arterialization should be performed only temporarily and is clinically not recommended as a permanent option, because of the increased hepatocellular apoptosis and the very distinctive, negative long-term effects on liver weight.

Mutational analysis of the IκBα gene in activated B cell-like diffuse large B-cell lymphoma

The lymphoma cells of the activated B cell‐like (ABC‐) subtype of diffuse large B‐cell lymphoma (DLBCL) show constitutive activity of the transcription factor, nuclear factor κB (NFκB). We sought to determine whether mutations in the IκBα gene – the predominant inhibitor of NFκB – might play a role in the pathogenesis of ABC‐DLBCL. All exons of the IκBα gene were directly sequenced from 10 cases of immunohistochemically classified ABC‐DLBCL and from six non‐ABC‐DLBCL cases. Two novel polymorphisms were identified, based on their presence in tumour as well as non‐tumour DNA of the respective patients: a duplication near the transcriptional start and a single nucleotide exchange in exon 1. A somatic missense mutation was identified in exon 3, in addition to a wild‐type sequence in only one ABC‐DLBCL case. Thus, also in this case no clonal biallelic inactivating mutation was present in the IκBα gene. We conclude that mutations in the IκBα gene do not play a dominant role in the pathogenesis of ABC‐DLBCL.

Metalloproteinase inhibition augments antitumor efficacy of the anti-CD30 immunotoxin Ki-3(scFv)-ETA' against human lymphomas in vivo.

There is increasing evidence that the shedding of extracellular antigen domains impedes selective immunotherapy. One example is CD30, which is overexpressed on the surface of malignant lymphoma cells and has been identified as a promising target for antibody‐based immunotherapy. However, CD30 is cleaved from the surface of target cells and the resulting soluble ectodomain (sCD30) is protecting the cells from antibody binding. Shedding can be inhibited by hydroxamate inhibitors of metalloproteinases such as BB‐3644. We thus evaluated the influence of BB‐3644 on the efficacy of the anti‐CD30 single‐chain immunotoxin Ki‐3(scFv)‐ETA′. In vitro, the addition of BB‐3644 augmented the antitumor effect of Ki‐3(scFv)‐ETA′ against Hodgkin‐derived L540Cy cells by a factor of 2.75. Severe combined immunodeficiency (SCID) mice challenged with CD30‐positive L540Cy cells were treated with the immunotoxin. One single nontoxic dose of BB‐3644 increased the mean survival time of animals treated concomitantly with Ki‐3(scFv)‐ETA′ to 93 days as compared with 35 days in the control (p = 0.0017). When BB‐3644 was continuously delivered using subcutaneously implanted pumps, this effect was even more pronounced with no observed tumor growth in the animals within 200 days. Thus, concomitant application of metalloproteinase inhibitors might become clinically relevant in antibody‐based immunotherapy against targets known to be shed from tumor cells.