Autologous Adjuvant Linked Fibroblasta Induce Anti-Glioma Immunity: Implications for Development of a Glima Vaccine

Objectives: Adjuvant-linked vaccines have been shown to induce anti-tumor immunity in patients with a variety of solid tumors. In this study we describe an in vitro model of active immunotherapy using autologous fibroblasts as immunogen. Correlative results from glioma patients immunized with autologous fibroblasts are also described. Methods: Peripheral blood lymphocytes (PBLs) from normal subjects were immunized in vitro against autologous skin fibroblasts coupled to the adjuvant muramyl dipeptide. The lymphocytes developed cell-mediated cytotoxicity that was measured with a short-term chromium release assay. Results of in vitro experiments were compared to data derived from glioma patients immunized with subcutaneous injection of an autologous adjuvant-linked fibroblast vaccine. Glioma target cells and fibroblast immunogens were derived from early passage primary tissue culture. Results: A comparison of autologous vs. homologous immunogen indicated that major histocompatibility complex matching was required at the sensitization stage of immunity (17.2 ± 3.4% specific lysis vs. 0.4 ± 3.1%, P < 0.01). Pre-treatment of fibroblast immunogen cells with interferon gamma (IFN-) was found to significantly increase immunity (42.2 ± 10.0%, P < 0.01), as did IFN- pre-treatment of tumor target cells (35.8 ± 9.0%, P < 0.01). The positive effect of IFN- was diminished by treatment of cells with IFN-. These in vitro results correlated well with in vivo data derived from glioma patients immunized with an autologous adjuvant-linked fibroblast vaccine. PBLs from patients developed direct cell-mediated cytotoxicity against autologous tumor cells. Lysis of tumor targets after in vivo immunization increased over a three-week interval (from 1.2 ± 3.0% to 21.0 ± 3.4%, P < 0.01) while lysis of a non-MHC matched control cell line remained essentially unchanged. Conclusions: Specific lysis of glioma targets in vitro was achieved after in vivo sensitization with autologous adjuvant-linked fibroblasts. Collectively, the data indicate that biochemically modified autologous cells can stimulate anti-glioma immunity in humans. The degree of specific immunity seen in our patients compares favorably with other published series using glioma cells as an antigenic source. Accordingly, fibroblasts may represent a practical alternative to glioma cells for vaccine construction.     

Human autologous dendritic cell-glioma fusions: feasibility and capacity to stimulate T cells with proliferative and cytolytic activity

Summary Gliomas are the most common primary neoplasm of the central nervous system. The failure of conventional treatment modalities to improve outcome over the last two decades has led to interest in alternative treatment modalities. Dendritic cell (DC)-based immunotherapy has utilized DC pulsed with tumor lysate or peptide to induce an antitumor immune response mediated largely by CD8 T cells. While this has been effective in preclinical studies, clinical efficacy remains unproven. Recently, hybrid cells produced by fusions of tumor and autologous DC have demonstrated remarkable efficacy for stimulating an anti-tumor immune response in both preclinical and clinical studies of extra-cranial neoplasms. The advantage of generating such hybrid cells is that the entire cellular material of the tumor is processed and presented in both endogenous and exogenous pathways. This leads to activation of both MHC class I restricted CD8 cells as well as MHC class II restricted CD4 T cells. Here, we examinedin vitro T cell stimulatory capacity of autologous human DC-glioma fusion in comparison to DC loaded with apoptotic glioma. DC fused with autologous tumor or loaded with apoptotic tumor cells (DC/apo) were first used to stimulate autologous non-adherent peripheral blood mononuclear cells (PBMC),in vitro. The PBMC were then examined for phenotype (CD3, CD4, CD8) and intracellular IFN-γ using flow cytometry. Lymphocyte proliferation and cytolytic responses were also assessed. Lymphocytes stimulatedin vitro with fusion or DC/apo cells showed significantly enhanced cytotoxicity and proliferation against autologous tumor cells compared with PBMC stimulated with tumor cells or DC alone. Both strategies had similar efficacy. Tumor-cytolytic responses were enhanced by the addition of CD40 ligand (CD40L), and partially blocked by anti-MHC class I antibody. Flow cytometric analysis detected CD3⁺CD8⁺ T cells, which also stained positive for intracellular IFN-γ. The study suggests that DC/glioma fusion and DC/apo have comparable efficacy for stimulation of CTL with cytolytic and proliferative activity against human malignant gliomas. These findings may have implications for future studies of DC-based immunotherapy in malignant gliomas.     

Human Autologous Dendritic Cell–Glioma Fusions: Feasibility and Capacity to Stimulate T cells with Proliferative and Cytolytic Activity

Gliomas are the most common primary neoplasm of the central nervous system. The failure of conventional treatment modalities to improve outcome over the last two decades has led to interest in alternative treatment modalities. Dendritic cell (DC)-based immunotherapy has utilized DC pulsed with tumor lysate or peptide to induce an anti-tumor immune response mediated largely by CD8 T cells. While this has been effective in preclinical studies, clinical efficacy remains unproven. Recently, hybrid cells produced by fusions of tumor and autologous DC have demonstrated remarkable efficacy for stimulating an anti-tumor immune response in both preclinical and clinical studies of extra-cranial neoplasms. The advantage of generating such hybrid cells is that the entire cellular material of the tumor is processed and presented in both endogenous and exogenous pathways. This leads to activation of both MHC class I restricted CD8 cells as well as MHC class II restricted CD4 T cells.Here, we examined in vitro T cell stimulatory capacity of autologous human DC–glioma fusion in comparison to DC loaded with apoptotic glioma. DC fused with autologous tumor or loaded with apoptotic tumor cells (DC/apo) were first used to stimulate autologous non-adherent peripheral blood mononuclear cells (PBMC), in vitro. The PBMC were then examined for phenotype (CD3, CD4, CD8) and intracellular IFN- using flow cytometry. Lymphocyte proliferation and cytolytic responses were also assessed. Lymphocytes stimulated in vitro with fusion or DC/apo cells showed significantly enhanced cytotoxicity and proliferation against autologous tumor cells compared with PBMC stimulated with tumor cells or DC alone. Both strategies had similar efficacy. Tumor-cytolytic responses were enhanced by the addition of CD40 ligand (CD40L), and partially blocked by anti-MHC class I antibody. Flow cytometric analysis detected CD3⁺CD8⁺ T cells, which also stained positive for intracellular IFN-. The study suggests that DC/glioma fusion and DC/apo have comparable efficacy for stimulation of CTL with cytolytic and proliferative activity against human malignant gliomas. These findings may have implications for future studies of DC-based immunotherapy in malignant gliomas.     

Tumor lysate and IL-18 loaded dendritic cells elicits Th1 response,tumor-specific CD8+ cytotoxic T cells in patients with malignant glioma

In this study, we demonstrate that tumor lysate-loaded dendritic cells can elicit a specific CD8+ cytotoxic T lymphocyte response against autologous tumor cells in patients with malignant glioma. CTL from three of five patients expressed strong cytolytic activity against autologous glioma cells, did not lyse autologous lymphoblasts and were variably cytotoxic against the LAK-sensitive cell line Daudi. Also, DCs pulsed normal brain lysate failed to induce cytolytic activity against autologous glioma cells, suggesting the lack of autoimmune response. Two of five patients CD8+ T cells expressed a modest cytotoxicity against autologous glioma cells. CD8+ T cells isolated during these ineffective primings secreted large amounts of IL-10, less amounts of IFN-γ as detected by ELISA, Type 2 bias in the CD8+ T cell response accounts for the lack of cytotoxic effector function from these patients. Cytotoxicity against autologous glioma cells could be significantly inhibited by anti-HLA class I antibody. These data demonstrate that tumor lysate-loaded DC can be an effective tool in inducing glioma-specific CD8+ CTL able to kill autologous glioma cells in vitro. However, high levels of tumor specific tolerance in some patients may account for a significant barrier to therapeutic vaccination. Moreover, cytotoxic responses were augmented by transfecting DC with the gene for IL-18. For all five patients, CD8+T cells treated with IL18 transfected DC produced Th1 response. These results may have important implications for the treatment of malignant glioma patients with immunotherapy. DCs loaded with total tumor lysate and IL-18 may represent a method for inducing Th1 immunoresponses against the entire repertoire of glioma antigens.     

A phase-I clinical study of autologous tumor cells plus interleukin-2-gene-transfected allogeneic fibroblasts as a vaccine in patients with cancer

Tumor cells transfected to express immunostimulatory cytokines, or admixed with similarly modified bystander cells, are able to induce immune responses against unmodified tumor cells in animal models. For treatment of human patients, a vaccine composed of autologous tumor cells and IL-2-secreting allogeneic fibroblasts was developed. Autologous tumor cells were isolated from biopsy specimens. A clone (KMST6.14) of an immortalized human fibroblast line that stably secreted 5290 IU IL-2 per 10⁶ cells and per 24 hr was obtained by cationic lipofection with an expression construct for human IL-2 and Neo^r. Fifteen patients with refractory malignant tumors received 3–4 injections of irradiated KMST6.14 and autologous tumor cells in a phase-I clinical trial. Increasing transient inflammatory responses without systemic toxicity developed at vaccination sites and after injections with irradiated tumor cells only (p < 0.05). These sites contained a dense infiltrate of CD3⁺ T cells with numbers of CD4⁺ helper cells exceeding those of CD8⁺ cytotoxic T cells (CTL). CD8⁺ T-cell lines isolated from vaccination sites of 2 malignant melanoma patients but not of renal-cell carcinoma patients exhibited a dominant lytic activity against autologous tumor cells in vitro. CD8⁺ T-cell clones established from the vaccination site of 1 of 2 renal-cell carcinoma patients preferentially lysed autologous and partially matched allogeneic renal-cell carcinoma cells. In conclusion, a vaccine composed of IL-2 gene-transfected allogeneic fibroblasts and autologous tumor cells is able to enhance specific anti-tumor T-cell responses in vivo without major side-effects. Malignant melanoma and renal-cell carcinoma appear to be promising entities for testing of similar approaches in future therapeutic trials. Int. J. Cancer, 70:269–277, 1997. © 1997 Wiley-Liss, Inc.     

Diverse effect of cytokine treatment of tumor cells on specific versus non-specific cytotoxicity

(First submitted 15 Nov 1991;accepted 11 December 1991) The effect of IFN-γ and TNF-α treatment of an ovarian carcinoma line on the sensitivity to lysis by specific CTL clones and non-specific Tumor Associated Lymphocytes (TAL), isolated from the ascites fluid, was analyzed. Thein vitro established TAL line displayed a non-specific lytic activity against the autologous tumor as well as against several allogeneic tumor lines. Pretreatment with IFN-γ alone, or in combination with TNF-α, rendered the carcinoma line less susceptible to lysis by the autologous TAL line. Conversely, susceptibility to lysis by tumor specific T cell clones, isolated from the TAL line, was increased as a result of cytokine pretreatment. Several TCR-α/β⁺, CD8⁺ T-cell clones showing a more specific pattern of lysis against the autologous tumor were isolated. Lysis of the autologous tumor by these clones involved the TCR-α/β via a MHC-class I restricted mechanism dependent on the adhesion molecules ICAM-1 and LFA-3, as inferred from antibody blocking studies. The enhanced sensitivity to specific CTL clones seen after cytokine treatment may be related to theenhanced expression of ICAM-1 molecules on the ovarian carcinoma. These results have implications for cytokine based immunotherapy, where IFN-γ may enhance the effects of tumor associated specific CTL while decreasing that of non-specific effector cells.     

Robust ability of IFN-gamma to upregulate class II MHC antigen expression in tumor bearing rat brains

Summary T cells are attractive for delivering therapy to brain tumor, especially disseminated micro-tumor. However, to trigger effector function, tumor antigen must be re-presented to T cells, via major histocompatibility complex (MHC) proteins, at the tumor site. In normal brain, MHC⁺ antigen-presenting cells (APC) are rare, but abundant after gamma interferon (IFN-γ) injection. Here we studied tumor-bearing brains. IFN-γ (or buffer) was injected stereotactically into brains with established tumors from a panel of immunologically varied glioma cell lines, some expressing b-galactosidase as a micro-tumor marker. Four days later, cryostat sections were stained for tumor and MHC proteins. In phosphate-buffered saline-injected controls, class II MHC⁺ potential APC (microglia, macrophages) were seen only at (some) tumor sites. In rats that received IFN-γ, class II⁺ potential APC were widespread, including all actual and potential micro-tumor sites and all tumor-free areas. In the same slides, neither class I nor class II MHC antigen was detected in neural cells or most tumor cells. This MHC pattern favors indirect re-presentation of tumor antigen, by tumor-adjacent APC. The robust response to IFN-γ might also be exploited in other ways: activated microglia and macrophages can attack tumor directly, and class II⁺ APC may help mark micro-tumor sites.     

T SUBSETS AND ANTITUMOR ACTIVITY OF LYMPHOCYTES INFILTRATING HUMAN PRIMARY BRAIN GLIOMAS

Glioma-infiltrating lymphocytes (GIL) were isolated from 9 surgical biopsy specimens of primary brain gliomas using mechanical and enzymatic digestion and discontinuous density gradent centrifugation. During cultured in the presence of interleukin-2 (IL-2) for a period of four weeks, GIL were expanded 48. 4-fold on the average, even up to 118-fold. GIL activated by IL- 2 had specific cytolytic activity against autologous glioma cells. Analysis of T subsets of GIL freshly isolated showed that CD3 cells were 71.0±11.9%, CD4 cells 34.2±6.1% and CD3 cells 37.0±7.6%. Ability of activated GIL to produce γ-Interferon (γ-IFN) was significantly higher than that of freshly isolated GIL and autologous peripheral blood lymphocytes (PBL). The results suggest that GIL have many advantages for an adoptive immunotherapy of patients with brain gllomas and be a new type of antitumor immune effector.     

Lysis of cancer cells by autologous T cells in breast cancer pleural effusates treated with anti-EpCAM BiTE antibody MT110

In the present study, the efficacy of a new drug, i.e. the bispecific single-chain antibody MT110 targeting the epithelial antigen EpCAM and the T-cell antigen CD3 was tested ex vivo in malignant pleural effusions (MPEs). EpCAM⁺ epithelial cells were found in 78% of the MPEs (n = 18). Ex vivo treatment of seven MPEs resulted in a dose-dependent specific lysis of 37 ± 27% (±SD) EpCAM⁺ cells with 10 ng/ml (P = 0.03) and 57 ± 29.5% EpCAM⁺ cells with 1,000 ng/ml MT110 (P = 0.016) after 72 h. As a prerequisite for redirected lysis, stimulation of the autologous CD4⁺ and CD8⁺ cells in MPE by 1,000 ng/ml MT110 resulted in 21 ± 17% CD4⁺/CD25⁺ and 29.4 ± 22% CD8⁺/CD25⁺ cells (P = 0.016, respectively) after 72 h. This was confirmed by a 22-fold release of TNF-α and 230-fold release of IFN-γ (1,000 ng/ml, 48 h, P = 0.03, respectively). Thus, relapsed breast cancer patients resistant to standard treatment might benefit from targeted therapy using MT110.     

Experession of Bax in Lung Cancer Cell Apoptosis Induced by Peroxisome Proliferator－activated Receptor－γ Anoists

Objective To discuss the relationship between Bax expression level and lung cancer cell apoptosis induced by peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists. Methods RT-PCR and Western blot analysis were used to detect PPAR-γ expression in the lung cancer cells, and TUNEL was used to detect apoptosis induced by PPAR-γ agonists, while in situ hybridization and immunohistochemistry were used to monitor the changes of Bax mRNA and protein expression levels after apoptosis induced. Results PPAR-γ expression was detectable in two kinds of lung cancer cells (including Non-small cell lung cancer and small cell lung cancer), and PPAR-γ agonists could inhibit lung cancer growth through inducing apoptosis. The apoptostic rates in control group, 15d-PGJ2 group and ciglitazone group were (1.86±0.49)%, (25.8±2.9)%, and (17.3±1.9)% (P<0.01) respectively; Bax mRNA expression rates in the three groups were (8.75±1.36)%, (66.2±12.86)%, and (29.5±6.5)% (P<0.01) respectively; Bax protein expression rates in the three groups were (9.2±1.45)%, (63±10.4)%, and (34.5±6.0)% (P<0.05) respectively. Conclusion PPAR-γ is predicted to be a new target in treating lung cancer in the future, and Bax is most likely to work in treating lung cancer apoptosis induced by PPAR-γ agonists as a factor to induced apoptosis. This proiect was supported by a grant from Hubei Provincial Natural Sciences foundation (No. 98J102).     

Expression and clinical significance of REGγ in gastric cancer tissue and variously differentiated gastric cancer cell lines

OBJECTIVE To evaluate the REGγ expression in gastric cancer tissue and gastric cancer cell lines of various differentiation levels and its clinical significance. METHODS Immunohistochemistry was used to detect the expression of REGγ protein in 70 specimens of gastric cancer and 30 specimens of normal gastric mucosa. The relationship between the expression of REGγ protein and the biological behaviors of gastric cancer was analyzed. RT-PCR and Western blot were used to detect the mRNA level and the protein expression of REGγ in normal gastric cell line GES-1, well differentiated gastric cancer cell line MKN-28, moderately differentiated gastric cancer cell line SGC-7901 and poorly differentiated gastric cancer cell line BGC-823. RESULTS The expression rate of REGγ protein in gastric cancer tissue （52/70, 74.29%） was significantly higher than that in normal gastric tissue （12/30, 40%） （P 〈 0.01）. The expression rate of REGγ was correlated with tumor size （P 〈 0.01）, lymph node metastasis （P 〈 0.05）, differentiation degree （P 〈 0.01）, infiltration depth （P 〈 0.01） and distant metastasis （P 〈 0.05）. RT-PCR analysis showed that the expression of REGγ mRNA was 0.459 ± 0.079 in the normal gastric mucosa cell ling 0.588 ±0.118 in the well differentiated gastric cancer cell line, 0.715±0.066 in the moderately differentiated gastric cancer cell line, and 0.873 ± 0.099 in the poorly differentiated gastric cancer cell line, showing a negative correlation between REGγ mRNA expression and differentiation level （P 〈 0.05）. Western blot analysis showed that the expression of REGγ protein was 0.712±0.065 in the normal gastric mucosa cell line, 1.176±0.185 in the well differentiated gastric cancer cell line, 1.533 ± 0.127 in the moderately differentiated gastric cancer cell line, and 2.061± 0.398 in the poorly differentiated gastric cancer cell line, showing a negative correlation between REGγ protein expression and differentiation level （P 〈 0.05）. CONCLUSION REGγ is expressed in gastric cancer tissue and normal gastric tissue. In gastric cancer tissues, REGγ expression is positively correlated with the tumor size, lymph node metastasis, differentiation degree, infiltration depth and distant metastasis. Detecting the expression of REGγ mRNA and protein is helpful for early diagnosis and predicting prognosis of gastric cancer.     

Anticonvulsant-induced Suppression of IFN-γ Production by Lymphocytes Obtained from Cervical Lymph Nodes in Glioma-bearing Mice

It is well known that phenytoin can cause impairment of cellular immunity. The authors investigated the potential role of other anticonvulsant drugs in the development of antitumor immunity in murine malignant glioma models.The survival rate was determined in murine glioma models using syngeneic 203 glioma cells following treatment with four anticonvuisants, which are most commonly administered to glioma patients, i.e., phenytoin, phenobarbital, valproate and zonisamide. In a second set of experiments, we further examined the effect of these drugs on interferon- (IFN-) secretion by lymphocytes prepared from cervical lymph nodes (CLN) in the same models. The IFN- production of CLN lymphocytes as measured by ELISA method was markedly impaired in the early stage of tumor-bearing mice treated with phenytoin or zonisamide, and the median survival time (MST) of controls and of mice treated with either phenytoin or zonisamide was 13, 10 and 11 days, respectively, which was not a statistically significant difference. Phenobarbital and valproate did not affect either IFN- production or their survival rate. In addition, immunohistochemistry showed a reduction in tumor-infiltrating lymphocytes containing CD4 and CD8 antigens in the mice treated with phenytoin and zonisamide.Two anticonvulsants, phenytoin and zonisamide, showed a significant inhibitory effect on IFN- production by CLN lymphocytes in murine glioma models, although there was no statistically significant difference in MST between controls and the anticonvulsant-treated mice. These drugs might have some detrimental influence on the prognosis of brain tumor patients when combined with the latent immune dysfunction accompanying the tumor-bearing state.     

Innate Anti-breast Cancer Immunity of Apoptosis-resistant Human <Emphasis Type="Italic">γδ</Emphasis>-T cells

Summary We previously identified a CD2-initiated signaling pathway which inhibits activation-induced cell death in mitogen-stimulated human γδ-T cells permitting the large-scale expansion of these cells. Here we report the innate anti-tumor activity of expanded human γδ-T cells against human breast cancer cells. Apoptosis-resistant human γδ-T cells which were expanded in vitro from cultured human peripheral blood mononuclear cells displayed lytic activity against breast cancer cell lines MDA-MB-231, MCF-7 and T-47D, but failed to kill normal human skin fibroblasts and normal human liver cells. Monoclonal antibodies (mAb) directed against the γδ-T cell receptor (TCR) or mAb directed against either the Vγ9 or the Vδ2 TCR chains were able to block γδ-T cell-mediated lysis of MDA-MB-231 cells. In addition, mAb against intercellular adhesion molecules-1 (ICAM-1/CD54) or CD18 (β subunit of ICAM-1 counter–receptor) also blocked γδ-T cell-mediated killing of MDA-MB-231 cells. Ex vivo expanded human γδ-T cells are thus able to innately recognize and kill human breast cancer cells in a γδ-TCR-dependent manner; ICAM-1 and CD18 also appear to be involved in the interactions between sensitive breast cancer cells and cytolytic γδ-T cells. As apoptosis-resistant human γδ-T cells can now readily be expanded to large numbers (clinical scale), these findings must be considered in the context of developing adoptive immunotherapy strategies to exploit γδ-T cell innate immune responses for the primary or adjuvant treatment of breast cancer.     

Identification of colon-tumor-associated antigens by T-cell lines derived from tumor-infiltrating lymphocytes and peripheral-blood lymphocytes from patients immunized with an autologous tumor-cell/bacillus calmette-Guérin vaccine

Tumor immunity developing as a response to an autologous colon-tumor/bacillus Calmette-Guerin (BCG) vaccine appears to be associated with induction of CD4 helper T cells, implied by the observation that vaccine efficacy is associated with major histocompatibility complex class-ll molecule expression on the vaccine tumor cells. Therefore, in an attempt to identify colon-tumor-associated antigens responsible for conferring immunity, we examined and compared the proliferative responses of peripheral-blood lymphocytes (PBL) from patients immunized with the autologous tumor/BCG vaccine to T-cell lines cloned expanded from colon-tumor-infiltrating lymphocytes to 5 antigens isolated on the basis of their reactivity by colon-tumor-reactive human monoclonal antibodies. Enzymatically dissociated colon tumors provided a source for establishment of cloned T-cell lines, tumor cell lines propagated in vitro or in vivo as nude-mouse xenografts and EBV-transformed B-cell lines used as antigen-presenting cells. Of 104 different T-cell lines tested, only 3 proliferated in response to CTAA 28A32-46K, and I to the CTAA28A32-32K antigen. In contrast, PBL from 64% of patients immunized with the autologous colon-tumor/BCG vaccine responded to the CTAA 28A32-32K antigen. This antigen is related to a family of calcium- and phospholipid-binding placental proteins termed annexins. Since proliferative responses developed to this antigen after vaccination in 64% of individuals, this antigen may be an important common colon-tumor-associated rejection antigen.     

Relationship between immunological parameters and survival of patients with liver metastases from breast cancer given immuno-chemotherapy

Summary We treated 33 patients with liver metastases from breast cancer by immuno-chemotherapy including adoptive cell transfer between 1987 and 1992. In this study, we examined the change of immunological parameters in the peripheral blood lymphocytes and interleukin-2 (IL-2)-cultured lymphocytes, in primary vs. metastatic breast cancer patients and before vs. after treatment. Moreover, we examined their correlation with therapeutic response and survival after treatment. The immunological parameters used werein vitro natural killer cell activity (% lysis of K562),in vitro autologous tumor-killing activity (% lysis against autologous freshly isolated tumor cells), and proliferation of lymphocytes stimulated with IL-2 and autologous sonicated tumor extract antigen in mixed culture (IL-2-enhanced MLTR). When compared with primary breast cancer patients, patients with liver metastases showed a significant decrease in % lysis of K562 and autologous tumor cells. After treatment, the stimulation index in IL-2-enhanced MLTR increased significantly from the pretreatment level and correlated with survival after treatment. Moreover, non-specific immunological parameters (performance status, lymphocyte count, and transferred cell count and proliferation rate of cultured lymphocytes) were significantly associated with response and prognosis.     

Hepatocyte growth factor in cerebrospinal fluid is associated with mortality and recurrence of glioblastoma,and could be of prognostic value

Malignant gliomas—glioblastoma multiforme and anaplastic astrocytoma—are among the most fatal forms of cancer in humans. It has been suggested that hepatocyte growth factor (HGF) is a reliable predictor of glioma malignancy; amounts of HGF are directly related to cellular proliferation, angiogenesis, low apoptotic rate, and poor prognosis (WHO III and IV). We measured the HGF content of cerebrospinal fluid (CSF) from patients with malignant glioma glioblastoma multiforme (WHO IV; n = 14), anaplastic astrocytoma (WHO III; n = 4), and meningioma (WHO I; n = 9), and from control subjects (n = 25), and found a high concentration of HGF in patients with malignant glioma. However, CSF concentrations from glioblastoma multiforme and anaplastic astrocytoma patients were not statistically significantly different (893 ± 157 vs. 728 ± 61, respectively; P > 0.01). A negative correlation between HGF and survival was found at five years of follow-up (R = −0.922, R ² = 0.850, P < 0.001). Also, the HGF concentration in CSF was a reliable means of explaining the highly variable survival of patients with malignant glioma. CSF concentrations of HGF higher than 500 pg/ml were associated with increased mortality whereas values higher than 850 pg/ml were associated with a brief tumor-free period after surgery (9 ± 0.6 vs. 6 ± 0.6 months, respectively, P < 0.001). Our findings support the idea that measurement of HGF in CSF could be a useful tool for monitoring the biological activity of malignant glioma. The findings will ultimately need to be confirmed in a much larger study.     

Serum macrophage‐derived chemokine/CCL22 levels are associated with glioma risk,CD4 T cell lymphopenia and survival time

Defects in antigen presenting cell function have been implicated in glioma immunosuppression. We measured peripheral CCL22, a dendritic cell/macrophage derived T cell trafficking chemokine, in sera from 1,208 glioma cases and 976 controls to assess whether it might provide a biomarker of glioma risk, survival and immune dysfunction. Cluster models were used to examine the relationship between CCL22 and glioma risk. Patient survival was assessed using Cox regression models. We also examined the relationship between CCL22 levels and CD4 cell counts, as well as allergy history and IgE levels. CCL22 levels were significantly lower among glioma cases compared with controls (Mean ± SEM: 1.23 ± 0.03 ng/mL in cases vs. 1.60 ± 0.03 ng/mL in controls, p < 0.0001) and this difference remained significant even after controlling for other covariates in the cluster models (highest quartile versus lowest Odds Ratio = 0.21, p < 0.0001). CD4 cell counts were positively correlated with CCL22 in glioma cases (Spearman r² = 0.51, p < 0.01) and were significantly lower in cases compared with controls. Higher CCL22 levels were associated with longer survival in all cases combined and in GBM cases (hazard ratio_allcases = 0.81; 95% CI: 0.72–0.91, p = 0.0003). CCL22 levels were not associated with IgE level or self‐reported allergies. Circulating CCL22 levels are related to both glioma risk and survival duration independent of age, histology, grade and IDH mutation status. CCL22 should be considered a marker of immune status with potential prognostic value.     

Lentivirus-mediated CD/TK fusion gene transfection neural stem cell therapy for C6 glioblastoma

A suicide gene can convert nontoxic prodrugs into toxic products to kill tumor cells. In this study, our aim was to transfect lentivirus-mediated CD/TK fusion gene into Wistar rat’s neural stem cells (NSC) and then implant the NSC into a C6 glioma model to observe a C6 glioma growth inhibition effect. Primary NSC and stable transfection CD/TK fusion gene cell lines were established. To observe the tumor size and rat survival period in different groups, C6 glioma cell apoptosis and cell viability rate were applied to analyze the tumor inhibition effect of the neural stem cells’ transfected CD/TK fusion gene. C6 cell viability showed that CDglyTK-NSC + GCV/5-Fc (group 1) was lower than CDglyTK-NSC (group 2), NSC + GCV/5-Fc (group 3), and control (group 4) from day 2 (p?<?0.05), and the apoptosis rate was higher in group 1 compared with that of other groups (50.6 %, p?<?0.05) either in vitro or in vivo (35.47 %, p?<?0.05); both cell viability and apoptosis had no significance in the other three groups. In vivo, tumor size in group 1 was 7.76?±?1.37 mm³, which is smaller than the others (group2 27.28?±?4.11 mm³, group3 27.94?±?2.08 and 28.61?±?2.97 mm³; p?<?0.05). The other groups’ tumor size was not significant (p?>?0.05). Survival time of rats treated with CDglyTK-NSC + GCV/5-Fc (group 1) was significantly longer than that of the other groups (p?<?0.05; group 1 48.86?±?1.97, group 2 28.67?±?3.75, group 3 31.5?±?1.27, group 4 29.3?±?1.33). We also showed that the transfected C6 cells had a migratory capacity toward gliomas in vivo. Transfected CD/TK fusion gene neural stem cells combined with propyl–guanosine and 5-flucytosine double prodrug significantly inhibit the development of glioma.