Targeting the PD-1/PD-L1 pathway in glioblastoma multiforme: Preclinical evidence and clinical interventions.

Glioblastoma multiforme (GBM), as one of the immunosuppressive and common intrinsic brain tumors in adults, remains an intractable malignancy to manage. Since the standard of care for treatment, which includes surgery and chemoradiation, has not provided a sustainable and durable response in affected patients, seeking novel therapeutic approaches to treat GBM seems imperative. Immunotherapy, a breakthrough for cancer treatment, has become an attractive tool for combating cancer with the potential to access the blood-brain-barrier (BBB). In this regard, programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1), as major immunological checkpoints, have drawn considerable interest due to their effectiveness in a spectrum of highly-aggressive neoplasms through negative regulation of the T-cell-mediated immune response. Nevertheless, due to the immunosuppressive microenvironment of GBM, the efficacy of these immune checkpoint inhibitors (ICIs), when used as monotherapy, has been unfavorable and lacks sufficient beneficial outcomes for GBM patients. A variety of clinical studies are attempting to evaluate the combination of ICIs (neoadjuvant/adjuvant) and existing treatment guidelines to strengthen their effectiveness; however, the exact mechanism of this signaling axis affects the consequences of immune therapy remains elusive. This review provides an overview of the PD-1/PD-L1 pathway, currently approved ICIs for clinical use, preclinical and clinical trials of PD-1/PD-L1 as monotherapy, and when used concomitantly with other GBM treatments.

Intratumoral Copper Modulates PD-L1 Expression and Influences Tumor Immune Evasion.

Therapeutic checkpoint antibodies blocking programmed death receptor 1/programmed death ligand 1 (PD-L1) signaling have radically improved clinical outcomes in cancer. However, the regulation of PD-L1 expression on tumor cells is still poorly understood. Here we show that intratumoral copper levels influence PD-L1 expression in cancer cells. Deep analysis of the The Cancer Genome Atlas database and tissue microarrays showed strong correlation between the major copper influx transporter copper transporter 1 (CTR-1) and PD-L1 expression across many cancers but not in corresponding normal tissues. Copper supplementation enhanced PD-L1 expression at mRNA and protein levels in cancer cells and RNA sequencing revealed that copper regulates key signaling pathways mediating PD-L1-driven cancer immune evasion. Conversely, copper chelators inhibited phosphorylation of STAT3 and EGFR and promoted ubiquitin-mediated degradation of PD-L1. Copper-chelating drugs also significantly increased the number of tumor-infiltrating CD8+ T and natural killer cells, slowed tumor growth, and improved mouse survival. Overall, this study reveals an important role for copper in regulating PD-L1 and suggests that anticancer immunotherapy might be enhanced by pharmacologically reducing intratumor copper levels. SIGNIFICANCE: These findings characterize the role of copper in modulating PD-L1 expression and contributing to cancer immune evasion, highlighting the potential for repurposing copper chelators as enhancers of antitumor immunity. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/19/4129/F1.large.jpg.

Antibodies Against Hypothalamus and Pituitary Gland in Childhood-Onset Brain Tumors and Pituitary Dysfunction.

Purpose: To detect the presence of antipituitary (APA) and antihypothalamus antibodies (AHA) in subjects treated for brain cancers, and to evaluate their potential association with pituitary dysfunction. Methods: We evaluated 63 patients with craniopharyngioma, glioma, and germinoma treated with surgery and/or radiotherapy and/or chemotherapy at a median age of 13 years. Forty-one had multiple pituitary hormone deficiencies (MPHD), six had a single pituitary defect. GH was the most common defect (65.1%), followed by AVP (61.9%), TSH (57.1%), ACTH (49.2%), and gonadotropin (38.1%). APA and AHA were evaluated by simple indirect immunofluorescence method indirect immunofluorescence in patients and in 50 healthy controls. Results: Circulating APA and/or AHA were found in 31 subjects (49.2%) and in none of the healthy controls. In particular, 25 subjects out of 31 were APA (80.6%), 26 were AHA (83.90%), and 20 were both APA and AHA (64.5%). Nine patients APA and/or AHA have craniopharyngioma (29%), seven (22.6%) have glioma, and 15 (48.4%) have germinoma. Patients with craniopharyngioma were positive for at least one antibody in 39.1% compared to 33.3% of patients with glioma and to 78.9% of those with germinoma with an analogous distribution for APA and AHA between the three tumors. The presence of APA or AHA and of both APA and AHA was significantly increased in patients with germinoma. The presence of APA (P = 0.001) and their titers (P = 0.001) was significantly associated with the type of tumor in the following order: germinomas, craniopharyngiomas, and gliomas; an analogous distribution was observed for the presence of AHA (P = 0.002) and their titers (P = 0.012). In addition, we found a significant association between radiotherapy and APA (P = 0.03). Conclusions: Brain tumors especially germinoma are associated with the development of hypothalamic-pituitary antibodies and pituitary defects. The correct interpretation of APA/AHA antibodies is essential to avoid a misdiagnosis of an autoimmune infundibulo-neurohypophysitis or pituitary hypophysitis in patients with germinoma.

Yokukansan, a Japanese Herbal Medicine, Suppresses Substance PInduced Production of Interleukin-6 and Interleukin-8 by Human U373 MG Glioblastoma Astrocytoma Cells.

BACKGROUND: Yokukansan is a traditional Japanese herbal medicine that has an antiallodynic effect in patients with chronic pain. However, the mechanisms by which yokukansan inhibits neuropathic pain are unclear. OBJECTIVE: This study aimed to investigate the molecular effects of yokukansan on neuroinflammation in U373 MG glioblastoma astrocytoma cells, which express a functional high-affinity neurokinin 1 receptor (substance P receptor), and produce interleukin (IL)-6 and IL-8 in response to stimulation by substance P (SP). METHODS: We assessed the effect of yokukansan on the expression of ERK1/2, P38 MAPK, nuclear factor (NF)-κB, and cyclooxygenase-2 (COX-2) in U373 cells by western blot assay. Levels of IL-6 and IL-8 in conditioned medium obtained after stimulation of cells with SP for 24 h were measured by enzyme-linked immunosorbent assay. All experiments were conducted in triplicate. Results were analyzed by one-way ANOVA, and significance was accepted at p < 0.05. RESULTS: Yokukansan suppressed SP-induced production of IL-6 and IL-8 by U373 MG cells, and downregulated SP-induced COX-2 expression. Yokukansan also inhibited phosphorylation of ERK1/2 and p38 MAPK, as well as nuclear translocation of NF-κB, induced by SP stimulation of U373 MG cells. CONCLUSION: Yokukansan exhibits anti-inflammatory activity by suppressing SP-induced production of IL-6 and IL-8 and downregulating COX-2 expression in U373 MG cells, possibly via inhibition of the activation of signaling molecules, such as ERK1/2, p38 MAPK, and NF-κB.

Role of myeloid cells in the immunosuppressive microenvironment in gliomas.

Glioma is the most common primary brain cancer, and half of patients present a diagnosis of glioblastoma (GBM), its most aggressive and lethal form. Conventional therapies, including surgery, radiotherapy, and chemotherapy, have not resulted in major ameliorations in GBM survival outcome, which remains extremely poor. Recent immunotherapy improvements for other tumors, coupled with growing knowledge of the complex interactions between malignant glioma cells and the immune system, led to an exponential increase in glioma immunotherapy research. However, immunotherapeutic strategies in GBM have not yet reached their full potential, mainly due to the limited understanding of the strong immunosuppressive microenvironment (TME) characterizing this tumor. Glioma-associated macrophages and microglia (GAMs) are key drivers of the local immunosuppression promoting tumor progression and its resistance to immunomodulating therapeutic strategies. Together with other myeloid cells, such as dendritic cells and neutrophils, GAMs actively shape glioma TME, modulate anti-tumoral immune response and support angiogenesis, tumor cell invasion and proliferation. In this review, we discuss the role of myeloid cells in the complex TME of glioma and the available clinical data on therapeutic strategies focusing on approaches that affect myeloid cells activity in GBM.

Slowdown intracranial glioma progression by optical hyperthermia therapy: study on a CT-2A mouse astrocytoma model.

Metallic nanorods are promising agents for a wide range of biomedical applications. We report an optical hyperthermia method capable of inducing slowdown tumor progression of an experimental in vivo CT-2A glioblastoma tumor. The tumor model used in this research is based on the transplantation of mouse astrocytoma CT-2A cells in the striatum of mice by intracranial stereotaxic surgery. Two weeks after cell implant, the resulting tumor is treated by irradiating intratumoral injected gold nanorods, biofunctionalized with CD133 antibody (B-GNRs), using a continuous wave laser. Nanoparticles convert the absorbed light into localized heat (reaching up to 44 °C) due to the effect of surface plasmon resonance. A significant slowdown in CT-2A tumor progression is evident, by histology and magnetic resonance imaging, at one (p = 0.03) and two weeks (p = 0.008) after irradiation treatment. A notable deceleration in tumor size (15%-75%) as compared to the control untreated groups, it is observed. Thus, laser irradiation of B-GNRs is found to be effective for the treatment of CT-2A tumor progression. Similarities between the pre-clinical CT-2A tumor model and the human astrocytoma disease, in terms of anatomy, metastatic behavior and histopathology, suggest that hyperthermic treatment by laser irradiation of B-GNRs administered into high-grade human astrocytoma might constitute a promising alternative treatment to limit the progression of this deadly disease.

A Novel mTORC1/2 Inhibitor (MTI-31) Inhibits Tumor Growth, Epithelial-Mesenchymal Transition, Metastases, and Improves Antitumor Immunity in Preclinical Models of Lung Cancer.

PURPOSE: We aimed to investigate efficacy and mechanism of MTI-31 (LXI-15029), a novel mTORC1/mTORC2 inhibitor currently in human trial (NCT03125746), in non-small cell lung cancer (NSCLC) models of multiple driver mutations and tyrosine kinase inhibitor (TKI)-resistance. EXPERIMENTAL DESIGN: Gene depletion, inhibitor treatment, immunological, flow cytometry, cellular, and animal studies were performed to determine in vitro and in vivo efficacy in NSCLC models of driver mutations and elucidate roles by mTOR complexes in regulating migration, epithelial-mesenchymal transition (EMT), metastasis, intracranial tumor growth, and immune-escape. RESULTS: MTI-31 potently inhibited cell proliferation (IC50 <1 µmol/L) and in vivo tumor growth in multiple NSCLC models of EGFR/T790M, EML4-ALK, c-Met, or KRAS (MED <10 mg/kg). In EGFR-mutant and/or EML4-ALK-driven NSCLC, MTI-31 or disruption of mTORC2 reduced cell migration, hematogenous metastasis to the lung, and abrogated morphological and functional traits of EMT. Disruption of mTORC2 inhibited EGFR/T790M-positive tumor growth in mouse brain and prolonged animal survival correlating a diminished tumor angiogenesis and recruitment of IBA1+ microglia/macrophages in tumor microenvironment. MTI-31 also suppressed programmed death ligand 1 (PD-L1) in EGFR- and ALK-driven NSCLC, mediated in part by mTORC2/AKT/GSK3ß-dependent proteasomal degradation. Depletion of mTOR protein or disruption of mTOR complexes profoundly downregulated PD-L1 and alleviated apoptosis in Jurkat T and primary human T cells in a tumor-T cell coculture system. CONCLUSIONS: Our results highlight mTOR as a multifaceted regulator of tumor growth, metastasis, and immune-escape in EGFR/ALK-mutant and TKI-resistant NSCLC cells. The newly characterized mechanisms mediated by the rapamycin-resistant mTORC2 warrant clinical investigation of mTORC1/mTORC2 inhibitors in patients with lung cancer.

Endogenous Neoantigen-Specific CD8 T Cells Identified in Two Glioblastoma Models Using a Cancer Immunogenomics Approach.

The "cancer immunogenomics" paradigm has facilitated the search for tumor-specific antigens over the last 4 years by applying comprehensive cancer genomics to tumor antigen discovery. We applied this methodology to identify tumor-specific "neoantigens" in the C57BL/6-derived GL261 and VM/Dk-derived SMA-560 tumor models. Following DNA whole-exome and RNA sequencing, high-affinity candidate neoepitopes were predicted and screened for immunogenicity by ELISPOT and tetramer analyses. GL261 and SMA-560 harbored 4,932 and 2,171 nonsynonymous exome mutations, respectively, of which less than half were expressed. To establish the immunogenicities of H-2Kb and H-2Db candidate neoantigens, we assessed the ability of the epitopes predicted in silico to be the highest affinity binders to activate tumor-infiltrating T cells harvested from GL261 and SMA-560 tumors. Using IFNγ ELISPOT, we confirmed H-2Db-restricted Imp3D81N (GL261) and Odc1Q129L (SMA-560) along with H-2Kb-restricted E2f8K272R (SMA-560) as endogenous tumor-specific neoantigens that are functionally immunogenic. Furthermore, neoantigen-specific T cells to Imp3D81N and Odc1Q129L were detected within intracranial tumors as well as cervical draining lymph nodes by tetramer analysis. By establishing the immunogenicities of predicted high-affinity neoepitopes in these models, we extend the immunogenomics-based neoantigen discovery pipeline to glioblastoma models and provide a tractable system to further study the mechanism of action of T cell-activating immunotherapeutic approaches in preclinical models of glioblastoma. Cancer Immunol Res; 4(12); 1007-15. ©2016 AACR.

Dexamethasone exerts profound immunologic interference on treatment efficacy for recurrent glioblastoma.

BACKGROUND: Patients with recurrent glioblastoma have a poor outcome. Data from the phase III registration trial comparing tumour-treating alternating electric fields (TTFields) vs chemotherapy provided a unique opportunity to study dexamethasone effects on patient outcome unencumbered by the confounding immune and myeloablative side effects of chemotherapy. METHODS: Using an unsupervised binary partitioning algorithm, we segregated both cohorts of the trial based on the dexamethasone dose that yielded the greatest statistical difference in overall survival (OS). The results were validated in a separate cohort treated in a single institution with TTFields and their T lymphocytes were correlated with OS. RESULTS: Patients who used dexamethasone doses >4.1 mg per day had a significant reduction in OS when compared with those who used ⩽4.1 mg per day, 4.8 vs 11.0 months respectively (χ(2)=34.6, P<0.0001) in the TTField-treated cohort and 6.0 vs 8.9 months respectively (χ(2)=10.0, P<0.0015) in the chemotherapy-treated cohort. In a single institution validation cohort treated with TTFields, the median OS of patients who used dexamethasone >4.1 mg per day was 3.2 months compared with those who used ⩽4.1 mg per day was 8.7 months (χ(2)=11.1, P=0.0009). There was a significant correlation between OS and T-lymphocyte counts. CONCLUSIONS: Dexamethasone exerted profound effects on both TTFields and chemotherapy efficacy resulting in lower patient OS. Therefore, global immunosuppression by dexamethasone likely interferes with immune functions that are necessary for the treatment of glioblastoma.

Glioblastoma exosomes and IGF-1R/AS-ODN are immunogenic stimuli in a translational research immunotherapy paradigm.

Glioblastomas are primary intracranial tumors for which there is no cure. Patients receiving standard of care, chemotherapy and irradiation, survive approximately 15 months prompting studies of alternative therapies including vaccination. In a pilot study, a vaccine consisting of Lucite diffusion chambers containing irradiated autologous tumor cells pre-treated with an antisense oligodeoxynucleotide (AS-ODN) directed against the insulin-like growth factor type 1 receptor was found to elicit positive clinical responses in 8/12 patients when implanted in the rectus sheath for 24 h. Our preliminary observations supported an immune response, and we have since reopened a second Phase 1 trial to assess this possibility among other exploratory objectives. The current study makes use of a murine glioma model and samples from glioblastoma patients in this second Phase 1 trial to investigate this novel therapeutic intervention more thoroughly. Implantation of the chamber-based vaccine protected mice from tumor challenge, and we posit this occurred through the release of immunostimulatory AS-ODN and antigen-bearing exosomes. Exosomes secreted by glioblastoma cultures are immunogenic, eliciting and binding antibodies present in the sera of immunized mice. Similarly, exosomes released by human glioblastoma cells bear antigens recognized by the sera of 6/12 patients with recurrent glioblastomas. These results suggest that the release of AS-ODN together with selective release of exosomes from glioblastoma cells implanted in chambers may drive the therapeutic effect seen in the pilot vaccine trial.

Coupling to a glioblastoma-directed antibody potentiates antitumor activity of curcumin.

Current therapies for glioblastoma are largely palliative, involving surgical resection followed by chemotherapy and radiation therapy, which yield serious side effects and very rarely produce complete recovery. Curcumin, a food component, blocked brain tumor formation but failed to eliminate established brain tumors in vivo, probably because of its poor bioavailability. In the glioblastoma GL261 cells, it suppressed the tumor-promoting proteins NF-κB, P-Akt1, vascular endothelial growth factor, cyclin D1 and BClXL and triggered cell death. Expression of exogenous p50 and p65 subunits of NF-κB conferred partial protection on transfected GL261 cells against curcumin insult, indicating that NF-κB played a key role in protecting glioblastoma cells. To enhance delivery, we coupled curcumin to the glioblastoma-specific CD68 antibody in a releasable form. This resulted in a 120-fold increase in its efficacy to eliminate GL261 cells. A very similar dose response was also obtained with human glioblastoma lines T98G and U87MG. GL261-implanted mice receiving intratumor infusions of the curcumin-CD68 adduct followed by tail-vein injections of solubilized curcumin displayed a fourfold to fivefold reduction in brain tumor load, survived longer, and about 10% of them lived beyond 100 days. Hematoxylin-eosin staining of brain sections revealed a small scar tissue mass in the rescued mice, indicating adduct-mediated elimination of glioblastoma tumor. The tumor cells were strongly CD68+ and some cells in the tumor periphery were strongly positive for microglial Iba1, but weakly positive for CD68. This strategy of antibody targeting of curcumin to tumor comes with the promise of yielding a highly effective therapy for glioblastoma brain tumors.

Recent developments on immunotherapy for brain cancer.

INTRODUCTION: Brain tumors are a unique class of cancers since they are anatomically shielded from normal immunosurveillance by the blood-brain barrier, lack a normal lymphatic drainage system and reside in a potently immunosuppressive environment. Of the primary brain cancers, glioblastoma multiforme (GBM) is the most common and aggressive in adults. Although treatment options include surgery, radiation and chemotherapy, the average lifespan of GBM patients remains at only 14.6 months post-diagnosis. AREAS COVERED: A review of key cellular and molecular immune system mediators in the context of brain tumors including TGF-ß, cytotoxic T cells, Tregs, CTLA-4, PD-1 and IDO is discussed. In addition, prognostic factors, currently utilized immunotherapeutic strategies, ongoing clinical trials and a discussion of new or potential immunotherapies for brain tumor patients are considered. EXPERT OPINION: Current drugs that improve the quality of life and overall survival in patients with brain tumors, especially for GBM, are poorly effective. This disease requires a reanalysis of currently accepted treatment strategies, as well as newly designed approaches. Here, we review the fundamental aspects of immunosuppression in brain tumors, new and promising immunotherapeutic drugs as well as combinatorial strategies that focus on the simultaneous inhibition of immunosuppressive hubs, both in immune and brain tumor cells, which is critical to consider for achieving future success for the treatment of this devastating disease.

Coupling to a cancer cell-specific antibody potentiates tumoricidal properties of curcumin.

In vitro studies have shown that curcumin, a polyphenol from the culinary component turmeric, has strong anticancer properties. However, there is no consensus on its therapeutic effect in human. Our earlier experiments involving implanted murine melanoma B16F10 cells in the neck or brain of syngeneic C57BL6 mice showed that tail vein injection of curcumin blocks formation of lesions and tumor in these mice. However, such treatment was ineffective in eliminating established tumors that already occupied ≤10% of brain volume. Possible reasons include low solubility and rapid metabolism of curcumin in vivo. To increase its efficacy, we have linked curcumin through a cleavable arm to an antibody (Ab) against the melanoma surface antigen Muc18. The antibody-coupled curcumin was 230-fold more effective in eliminating B16F10 cells in vitro, and in vivo, it rapidly decimated established, B16F10-evoked brain tumors, enabling the rescued mice to live normally far beyond 90 days from implantation of cancer cells. In contrast, mice treated with Muc18 Ab alone died of brain tumor within a month. In B16F10 cells, curcumin-Ab (adduct) treatment caused a dramatic inhibition of NF-kB: a transcription factor that is constitutively activated in cancer cells. Furthermore, overexpression of NF-kB in the B16F10 cells blocked adduct-evoked stimulation of caspase-3/7 activity. Thus, by suppressing NF-kB, the curcumin adduct inhibits other downstream tumor-promoting proteins, thereby eliminating the B16F10 cells. Our study submits a novel yet generally applicable strategy of converting curcumin into a potent anticancer agent and provides a mechanistic framework for its action.

Neutrophil degranulation and immunosuppression in patients with GBM: restoration of cellular immune function by targeting arginase I.

PURPOSE: The source of glioblastoma (GBM)-associated immunosuppression remains multifactorial. We sought to clarify and therapeutically target myeloid cell-derived peripheral immunosuppression in patients with GBM. EXPERIMENTAL DESIGN: Direct ex vivo T-cell function, serum Arginase I (ArgI) levels, and circulating myeloid lineage populations were compared between patients with GBM and normal donors or patients with other intracranial tumors. Immunofunctional assays were conducted using bulk and sorted cell populations to explore the potential transfer of myeloid cell-mediated immunosuppression and to identify a potential mechanism for these effects. ArgI-mediated immunosuppression was therapeutically targeted in vitro through pharmacologic inhibition or arginine supplementation. RESULTS: We identified a significantly expanded population of circulating, degranulated neutrophils associated with elevated levels of serum ArgI and decreased T-cell CD3ζ expression within peripheral blood from patients with GBM. Sorted CD11b(+) cells from patients with GBM were found to markedly suppress normal donor T-cell function in coculture, and media harvested from mitogen-stimulated GBM peripheral blood mononuclear cell (PBMC) or GBM-associated mixed lymphoid reactions showed ArgI levels that were significantly higher than controls. Critically, T-cell suppression in both settings could be completely reversed through pharmacologic ArgI inhibition or with arginine supplementation. CONCLUSIONS: These data indicate that peripheral cellular immunosuppression in patients with GBM is associated with neutrophil degranulation and elevated levels of circulating ArgI, and that T-cell function can be restored in these individuals by targeting ArgI. These data identify a novel pathway of GBM-mediated suppression of cellular immunity and offer a potential therapeutic window for improving antitumor immunity in affected patients.

Monoclonal antibody blockade of IL-2 receptor α during lymphopenia selectively depletes regulatory T cells in mice and humans.

Lymphodepletion augments adoptive cell transfer during antitumor immunotherapy, producing dramatic clinical responses in patients with malignant melanoma. We report that the lymphopenia induced by the chemotherapeutic agent temozolomide (TMZ) enhances vaccine-driven immune responses and significantly reduces malignant growth in an established model of murine tumorigenesis. Unexpectedly, despite the improved antitumor efficacy engendered by TMZ-induced lymphopenia, there was a treatment related increase in the frequency of immunosuppressive regulatory T cells (T(Regs); P = .0006). Monoclonal antibody (mAb)-mediated inhibition of the high-affinity IL-2 receptor α (IL-2Rα/CD25) during immunotherapy in normal mice depleted T(Regs) (73% reduction; P = .0154) but also abolished vaccine-induced immune responses. However, during lymphodepletion, IL-2Rα blockade decreased T(Regs) (93% reduction; P = .0001) without impairing effector T-cell responses, to augment therapeutic antitumor efficacy (66% reduction in tumor growth; P = .0024). Of clinical relevance, we also demonstrate that anti-IL-2Rα mAb administration during recovery from lymphodepletive TMZ in patients with glioblastoma reduced T(Reg) frequency (48% reduction; P = .0061) while permitting vaccine-stimulated antitumor effector cell expansion. To our knowledge, this is the first report of systemic antibody-mediated T(Reg) depletion during lymphopenia and the consequent synergistic enhancement of vaccine-driven cellular responses, as well as the first demonstration that anti-IL-2Rα mAbs function differentially in nonlymphopenic versus lymphopenic contexts.

Increase in tumor size following intratumoral injection of immunostimulatory CpG-containing oligonucleotides in a rat glioma model.

The immunosuppressive environment of malignant gliomas is likely to suppress the anti-tumor activity of infiltrating microglial cells and lymphocytes. Macrophages and microglial cells may be activated by oligonucleotides containing unmethylated CpG-motifs, although their value in cancer immunotherapy has remained controversial. Following injection of CpG-containing oligonucleotides (ODN) into normal rat brain, we observed a local inflammatory response with CD8+ T cell infiltration, upregulation of MHC 2, and ED1 expression proving the immunogenic capacity of the CpG-ODN used. This was not observed with a control ODN mutated in the immunostimulatory sequence (m-CpG). To study their effect in a syngeneic tumor model, we implanted rat 9L gliosarcoma cells into the striatum of Fisher 344 rats. After 3 days, immunostimulatory CpG-ODN, control m-CpG-ODN, or saline was injected stereotactically into the tumors (day 3 group). In another group of animals (day 0 group), CpG-ODN were mixed with 9L cells prior to implantation without further treatment on day 3. After 3 weeks, the animals were killed and the brains and spleens were removed. Rather unexpectedly, the tumors in several of the animals treated with CpG-ODN (both day 0 and day 3 group) were larger than in saline or m-CpG-ODN treated control animals. The tumor size in CpG-ODN-treated animals was more variable than in both control groups. This was associated with inflammatory responses and necrosis which was observed in most tumors following CpG treatment. This, however, did not prevent excessive growth of solid tumor masses in the CpG-treated animals similar to the control-treated animals. Dense infiltration with microglial cells resembling ramified microglia was observed within the solid tumor masses of control- and CpG-treated animals. In necrotic areas (phagocytic), activation of microglial cells was suggested by ED1 expression and a more macrophage-like morphology. Dense lymphocytic infiltrates consisting predominantly of CD8+ T cells and fewer NK cells were detected in all tumors including the control-treated animals. Expression of perforin serving as a marker for T cell or NK cell activation was detected only on isolated cells in all treatment groups. Tumors of all treatment groups revealed CD25 expression indicating T cells presumed to maintain peripheral tolerance to self-antigens. Cytotoxic T cell assays with in vitro restimulated lymphocytes ((51)chromium release assay) as well as interferon-gamma production by fresh splenocytes (Elispot assay) revealed specific responses to 9L cells but not another syngeneic cell line (MADB 106 adenocarcinoma). Surprisingly, the lysis rates with lymphocytes from CpG-ODN-treated animals were lower compared to control-treated animals. The tumor size of individual animals did not correlate with the response in both immune assays. Taken together, our data support the immunostimulatory capacity of CpG-ODN in normal brain. However, intratumoral application proved ineffective in a rat glioma model. CpG-ODN treatment may not yield beneficial effects in glioma patients.

IgE, allergy, and risk of glioma: update from the San Francisco Bay Area Adult Glioma Study in the temozolomide era.

The consistently observed inverse relationship of allergic conditions with glioma risk and our previous demonstration that immunoglobulin E (IgE) levels also were lower in glioma patients than controls suggest that atopic allergy may be related to a mechanism that inhibits or prevents glioma. We sought to extend these results with a new and larger series of patients (n = 535 with questionnaire data; 393 with IgE measures) and controls (n = 532 with questionnaire data; 470 with IgE measures). As expected, glioma cases were less likely than controls to report history of allergies [among self-reported cases, Odds ratios (OR) = 0.59, 95% confidence interval (CI): 0.41-0.85]. IgE levels also were lower in glioma cases versus controls (OR per unit log IgE = 0.89, 95% CI (0.82-0.98). However, this inverse relationship was only apparent among cases receiving temozolomide, a treatment which became part of the "standard of care" for glioblastoma patients during the study period. Among patients receiving temozolomide, IgE levels in cases whose blood samples were obtained within 30 days of diagnosis were slightly higher than controls, whereas IgE levels in cases whose blood sample was obtained >60 days after diagnosis were significantly lower than controls (OR = 0.80; 95% CI: 0.71-0.89). Thus, although our results robustly confirm the inverse association between allergy and glioma, the results for IgE are affected by temozolomide treatments which may have influenced IgE levels. These results have implications for the study of immunologic factors in glioma as well as for immunotherapy protocols for treating glioma.

Disseminated salmonellosis in a patient treated with temozolomide.

Temozolomide is a relatively new chemotherapeutic agent frequently associated with selective CD4+ T-lymphocytopenia. Patients with cell-mediated immune defects are at higher risk for acquiring infections with Salmonella species. We describe the first case of disseminated salmonellosis in a patient treated with temozolomide.

Immunonanoshells for targeted photothermal ablation in medulloblastoma and glioma: an in vitro evaluation using human cell lines.

We are developing a novel approach to specifically target malignant brain tumor cells for photothermal ablation using antibody-tagged, near infrared-absorbing gold-silica nanoshells, referred to as immunonanoshells. Once localized to tumor cells, these nanoshells are extremely efficient at absorbing near-infrared light and can generate sufficient heat to kill cancer cells upon exposure to laser light. In this study, we evaluated the efficacy of immunonanoshells in vitro against both medulloblastoma and high-grade glioma cell lines. We used an antibody against HER2 to target gold-silica nanoshells to medulloblastoma cells, since HER2 is frequently overexpressed in medulloblastoma. We show that treatment with HER2-targeted nanoshells, but not non-targeted nanoshells, followed by exposure to laser light, can induce cell death in the HER2-overexpressing medulloblastoma cell line Daoy.2, as well as the parental Daoy cell line, which expresses HER2 at a moderate level, but not in dermal fibroblasts that do not express HER2. In an analogous set of experiments, we conjugated gold-silica nanoshells to an antibody against interleukin-13 receptor-alpha 2 (IL13Ralpha2), an antigen that is frequently overexpressed in gliomas. We demonstrate that these immunonanoshells are capable of inducing cell death in two high-grade glioma cell lines that express IL13Ralpha2, U373 and U87, but not in A431 epidermoid carcinoma cells that do not express significant levels of IL13Ralpha2. We believe that the use of antibody-tagged gold-silica nanoshells to selectively target cancer cells presents a promising new strategy for the treatment of central nervous system tumors that will minimize the damage and resulting toxicity to the surrounding normal brain.