Glioblastoma multiforme (GBM) is the most common and most aggressive primary brain tumor in humans. Systemic immunity against gene therapy vectors has been shown to hamper therapeutic efficacy; however, helper-dependent high-capacity adenovirus (HC-Ad) vectors elicit sustained transgene expression, even in the presence of systemic anti-adenoviral immunity. We engineered HC-Ads encoding the conditional cytotoxic herpes simplex type 1 thymidine kinase (TK) and the immunostimulatory cytokine fms-like tyrosine kinase ligand 3 (Flt3L). Flt3L expression is under the control of the regulatable Tet-ON system. In anticipation of a phase I clinical trial for GBM, we assessed the therapeutic efficacy, biodistribution, and clinical and neurotoxicity with escalating doses of HC-Ad-TetOn-Flt3L + HC-Ad-TK in rats. Intratumoral administration of these therapeutic HC-Ads in rats bearing large intracranial GBMs led to long-term survival in ~70% of the animals and development of antiglioma immunological memory without signs of neuropathology or systemic toxicity. Systemic anti-adenoviral immunity did not affect therapeutic efficacy. These data support the idea that it would be useful to develop HC-Ad vectors further as a therapeutic gene-delivery platform to implement GBM phase I clinical trials.

Glioblastoma multiforme (GBM) is a deadly primary brain tumor. Conditional cytotoxic/immune-stimulatory gene therapy (Ad-TK and Ad-Flt3L) elicits tumor regression and immunological memory in rodent GBM models. Since the majority of patients enrolled in clinical trials would exhibit adenovirus immunity, which could curtail transgene expression and therapeutic efficacy, we used high-capacity adenovirus vectors (HCAds) as a gene delivery platform. Herein, we describe for the first time a novel bicistronic HC-Ad driving constitutive expression of herpes simplex virus type 1 thymidine kinase (HSV1-TK) and inducible Tet-mediated expression of Flt3L within a single-vector platform. We achieved anti-GBM therapeutic efficacy with no overt toxicities using this bicistronic HC-Ad even in the presence of systemic Ad immunity. The bicistronic HC-Ad-TK/TetOn-Flt3L was delivered into intracranial gliomas in rats. Survival, vector biodistribution, neuropathology, systemic toxicity, and neurobehavioral deficits were assessed for up to 1 year posttreatment. Therapeutic efficacy was also assessed in animals preimmunized against Ads. We demonstrate therapeutic efficacy, with vector genomes being restricted to the brain injection site and an absence of overt toxicities. Importantly, antiadenoviral immunity did not inhibit therapeutic efficacy. These data represent the first report of a bicistronic vector platform driving the expression of two therapeutic transgenes, i.e., constitutive HSV1-TK and inducible Flt3L genes. Further, our data demonstrate no promoter interference and optimum gene delivery and expression from within this single-vector platform. Analysis of the efficacy, safety, and toxicity of this bicistronic HC-Ad vector in an animal model of GBM strongly supports further preclinical testing and downstream process development of HC-Ad-TK/TetOn-Flt3L for a future phase I clinical trial for GBM.Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, affecting ϳ18,000 new patients every year; its prognosis remains poor despite standard treatment with surgery, radiotherapy, and chemotherapy (temozolomide) (36-38, 44; J. C. Buckner, presented at the ASCO Annual Meeting, 2009). Complete resection is mostly impossible due to the highly infiltrative nature of this disease. Residual GBM cells remaining within the nonneoplastic brain parenchyma eventually lead to tumor recurrence that is resistant to conventional chemotherapy and radiotherapy, ultimately leading to the patient's death (44). Several dendritic cell vaccination strategies aiming to stimulate the patient's immune system to seek out and destroy residual brain tumor cells are currently under preclinical and clinical development and constitute a promising adjuvant treatment for GBM (18,24,42,43,45).We have developed a novel immunotherapeutic approach for GBM using first-generation adenoviral vectors (Ads) to deliver a combination of therapeutic transgenes into the tumor mass (2,7,9,12,14,17,48) which is slated to begin phase I clinical testing this...

Although ~50% of acute myeloid leukemia (AML) patients have a normal diploid karyotype by conventional cytogenetics at diagnosis, this patient subset has a variable disease course and outcome. Aberrant overexpression of the p53 protein is usually associated with TP53 alterations and a complex karyotype, but the prevalence and impact of p53 overexpression in AML with diploid cytogenetics is unknown. We examined 100 newly diagnosed AML patients to evaluate the impact of p53 expression status quantified in bone marrow core biopsy samples using immunohistochemistry and computer-assisted image analysis. A total of 24 patients had p53 overexpression defined as 3+ staining intensity in ≥5% of cells; this finding correlated with lower platelet counts (P = .002), absence of CD34 expression in blasts (P = .009), higher bone marrow blast counts (P = .04), and a higher frequency of FLT3 internal tandem duplication (P = .007). Overexpression of p53 independently predicted for shorter leukemia-free survival in patients who underwent allogeneic stem cell transplantation by univariate (P = .021) and multivariate analyses (P = .004). There was no correlation between MDM2 and p53 protein expression in this cohort. We conclude that p53 expression evaluated by immunohistochemistry in bone marrow biopsy specimens at the time of AML diagnosis may indicate distinct clinical characteristics in patients with normal diploid cytogenetics and is a potentially valuable tool that can enhance risk-stratification.

Primary bronchopulmonary mucoepidermoid carcinoma (BPMEC) is a rare tumor. The fusion protein MECT1-MAML2 has been implicated as a causative genetic event in salivary and BPMECs. Several studies have shown the impact of MECT1-MAML2 on the diagnosis and prognosis of salivary gland mucoepidermoid carcinoma; however, few studies have been published regarding MECT1-MAML2 in the context of primary BPMEC. We describe the clinicopathologic, genetic, and outcome data of 16 patients with BPMEC. Clinicopathologic features were recorded from the electronic medical records. All tumors were reviewed by two expert pulmonary pathologists and graded according to previously established criteria. The presence of MECT1-MAML2 was evaluated with reverse transcription polymerase chain reaction using RNA extracted from formalin-fixed paraffin-embedded tumor tissue. Patients included 9 women and 7 men with a median age of 50 years (range, 7 to 82 years). Tumors exhibited low (n = 14, 88%), and high (n = 2, 12%) grade histologic features. Eight of nine tested tumors (89%) were positive for MECT1-MAML2. The median follow-up time was 40.8 months (range, 1.8-120). Median overall survival for patients with high-grade tumors was 12 months, which was significantly (p = 0.002) shorter than that for patients with low-grade tumors (survival undefined). We also provide a comprehensive review of literature of cases of primary bronchopulmonary mucoepidermoid carcinoma and summarize our findings in this context. MECT1-MAML2 fusion transcript is a driver genetic event in the pathogenesis of primary BPMEC. Histologic grade continues to play a pivotal role in the survival of patients with primary bronchopulmonary mucoepidermoid carcinoma.

Functional dendritic cells (DC) are professional antigen-presenting cells (APC) and can be generated in vitro from healthy as well as from leukaemic cells from acute myeloid leukemia (AML) patients giving rise to APC of leukaemic originpresenting leukaemic antigens. We describe the generation and characterization of DC from different mononuclear cell (MNC) fractions from 50 AML patients under different serum-free culture conditions, determine the optimal culture conditions and compare the results with that from 23 healthy donors. In parallel cultures, we compared DC harvests after 7-or 14-day culture, with total or adherent MNC or T-cell depleted MNC or peripheral blood (PB) or bone marrow-MNC (BM-MNC), thawn or fresh MNC, in Xvivo or CellGro serumfree media, AE10% autologous plasma or AEFL. In detail, we could show that AML-DC harvests were higher after 10-14 days culture (healthy DC: 7 days); total or adherent PB or BM-MNC fractions yield comparable DC counts, however, from magnetic cell sorting (MACS)-depleted MNC fractions or thawn MNC lower DC counts can be generated. Whereas the addition of FL increases the DC harvest, the addition of autologous plasma in many cases has inhibitory influence on DC maturation. CellGro and Xvivo media yield comparable DC counts. Optimal harvest of vital and mature DC from AML samples was obtained with a granulocyte/macrophage-colony stimulating factor, interleukin-4, FL and tumour necrosis factor-a-containing serum-free Xvivo medium after 10-14 days of culture (36/26% DC; 38/64% vital DC; 46/51% mature DC were generated from AML/healthy MNC samples). Surface marker profiles (e.g. costimulatory antigen expressing) of DC obtained from AML samples were comparable with that of healthy DC. The leukaemic derivation of AML-DC was demonstrated by the persistence of the clonal cytogenetic aberration in the DC or by coexpression of leukaemic antigens on DC. Autologous T-cell activation of leukaemia-derived DC was demonstrated in cases with AML. Autologous T cells proliferate and upregulate DC-contact-relevant antigens. We demonstrate that the generation of leukaemia-derived DC is feasable in AML under serum-free culture conditions giving rise to DC with comparable characteristics as healthy DC and offering an anti-leukaemia-directed immunotherapeutical vaccination strategy in AML.