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Osteosarcoma is the most frequent and aggressive bone tumor in children and adolescents, with a long-term survival rate of 30%. Interleukin-12 (IL-12) is a potent cytokine that bridges innate and adaptive immunity, triggers antiangiogenic responses, and achieves potent antitumor effects. In this work, we evaluated the antisarcoma effect of a high-capacity adenoviral vector encoding mouse IL-12. This vector harbored a mifepristone-inducible system for controlled expression of IL-12 (High-Capacity adenoviral vector enconding the EF1α promoter [HCA-EFZP]-IL-12). We found that local administration of the vector resulted in a reduction in the tumor burden, extended overall survival, and tumor eradication. Moreover, long-term survivors exhibited immunological memory when rechallenged with the same tumor cells. Treatment with HCA-EFZP-IL-12 also resulted in a significant decrease in lung metastasis. Immunohistochemical analyses showed profound remodeling of the osteosarcoma microenvironment with decreases in angiogenesis and macrophage and myeloid cell numbers. In summary, our data underscore the potential therapeutic value of IL-12 in the context of a drug-inducible system that allows controlled expression of this cytokine, which can trigger a potent antitumor immune response in primary and metastatic pediatric osteosarcoma., Graphical Abstract, Osteosarcoma is the most aggressive bone tumor in children and adolescents. In this work, the authors show the potential therapeutic value of IL-12 in the context of a drug-inducible system that allows controlled expression of this cytokine, which led to a potent antitumor immune response in primary and metastatic pediatric osteosarcoma model.

Simple Summary Clinical management of sarcomas is complex because they are rare and heterogeneous tumors. Management requires a coordinated multidisciplinary approach, especially in children. Genomic characterization of this complex group of tumors contributes to the identification of prognostic biomarkers and to the continued expansion of therapeutic options. In this article, we present the positive experience of two Spanish hospitals in the use of genomic analysis in the overall clinical management of sarcomas in children and young adults. We describe on a case-by-case basis how genomic analysis has contributed to both diagnosis and treatment. Abstract Genomic techniques enable diagnosis and management of children and young adults with sarcomas by identifying high-risk patients and those who may benefit from targeted therapy or participation in clinical trials. Objective: to analyze the performance of an NGS gene panel for the clinical management of pediatric sarcoma patients. We studied 53 pediatric and young adult patients diagnosed with sarcoma, from two Spanish centers. Genomic data were obtained using the Oncomine Childhood Cancer Research Assay, and categorized according to their diagnostic, predictive, or prognostic value. In 44 (83%) of the 53 patients, at least one genetic alteration was identified. In 80% of these patients, the diagnosis was obtained (n = 11) or changed (n = 9), and thus genomic data affected therapy. The most frequent initial misdiagnosis was Ewing’s sarcoma, instead of myxoid liposarcoma (FUS-DDDIT3), rhabdoid soft tissue tumor (SMARCB1), or angiomatoid fibrous histiocytoma (EWSR1-CREB1). In our series, two patients had a genetic alteration with an FDA-approved targeted therapy, and 30% had at least one potentially actionable alteration. NGS-based genomic studies are useful and feasible in diagnosis and clinical management of pediatric sarcomas. Genomic characterization of these rare and heterogeneous tumors also helps in the search for prognostic biomarkers and therapeutic opportunities.

BackgroundGlioblastoma (GBM) is a devastating primary brain tumor with a highly immunosuppressive tumor microenvironment, and treatment with oncolytic viruses (OVs) has emerged as a promising strategy for these tumors. Our group constructed a new OV named Delta-24-ACT, which was based on the Delta-24-RGD platform armed with 4-1BB ligand (4-1BBL). In this study, we evaluated the antitumor effect of Delta-24-ACT alone or in combination with an immune checkpoint inhibitor (ICI) in preclinical models of glioma.MethodsThe in vitro effect of Delta-24-ACT was characterized through analyses of its infectivity, replication and cytotoxicity by flow cytometry, immunofluorescence (IF) and MTS assays, respectively. The antitumor effect and therapeutic mechanism were evaluated in vivo using several immunocompetent murine glioma models. The tumor microenvironment was studied by flow cytometry, immunohistochemistry and IF.ResultsDelta-24-ACT was able to infect and exert a cytotoxic effect on murine and human glioma cell lines. Moreover, Delta-24-ACT expressed functional 4-1BBL that was able to costimulate T lymphocytes in vitro and in vivo. Delta-24-ACT elicited a more potent antitumor effect in GBM murine models than Delta-24-RGD, as demonstrated by significant increases in median survival and the percentage of long-term survivors. Furthermore, Delta-24-ACT modulated the tumor microenvironment, which led to lymphocyte infiltration and alteration of their immune phenotype, as characterized by increases in the expression of Programmed Death 1 (PD-1) on T cells and Programmed Death-ligand 1 (PD-L1) on different myeloid cell populations. Because Delta-24-ACT did not induce an immune memory response in long-term survivors, as indicated by rechallenge experiments, we combined Delta-24-ACT with an anti-PD-L1 antibody. In GL261 tumor-bearing mice, this combination showed superior efficacy compared with either monotherapy. Specifically, this combination not only increased the median survival but also generated immune memory, which allowed long-term survival and thus tumor rejection on rechallenge.ConclusionsIn summary, our data demonstrated the efficacy of Delta-24-ACT combined with a PD-L1 inhibitor in murine glioma models. Moreover, the data underscore the potential to combine local immunovirotherapy with ICIs as an effective therapy for poorly infiltrated tumors.

Last advances in the treatment of pediatric tumors has led to an increase of survival rates of children affected by primitive neuroectodermal tumors, however, still a significant amount of the patients do not overcome the disease. In addition, the survivors might suffer from severe side effects caused by the current standard treatments. Oncolytic virotherapy has emerged in the last years as a promising alternative for the treatment of solid tumors. In this work, we study the anti-tumor effect mediated by the oncolytic adenovirus VCN-01 in CNS-PNET models. VCN-01 is able to infect and replicate in PNET cell cultures, leading to a cytotoxicity and immunogenic cell death. In vivo, VCN-01 increased significantly the median survival of mice and led to long-term survivors in two orthotopic models of PNETs. In summary, these results underscore the therapeutic effect of VCN-01 for rare pediatric cancers such as PNETs, and warrants further exploration on the use of this virus to treat them.

Pediatric high-grade glioma (pHGG) and diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors in desperate need of a curative treatment. Oncolytic virotherapy is emerging as a solid therapeutic approach. Delta-24-RGD is a replication competent adenovirus engineered to replicate in tumor cells with an aberrant RB pathway. This virus has proven to be safe and effective in adult gliomas. Here we report that the administration of Delta-24-RGD is safe in mice and results in a significant increase in survival in immunodeficient and immunocompetent models of pHGG and DIPGs. Our results show that the Delta-24-RGD antiglioma effect is mediated by the oncolytic effect and the immune response elicited against the tumor. Altogether, our data highlight the potential of this virus as treatment for patients with these tumors. Of clinical significance, these data have led to the start of a phase I/II clinical trial at our institution for newly diagnosed DIPG (NCT03178032)., The oncolytic virus Delta-24-RGD is in clinical trial for adult glioma. Here, the authors show that this virus elicits an immune response in mouse models of pediatric high-grade glioma and diffuse pontine intrinsic glioma, resulting in improved survival.

Purpose: Atypical teratoid/rhabdoid tumors (AT/RT) and central nervous system primitive neuroectodermal tumors (CNS-PNET) are pediatric brain tumors with poor survival and life-long negative side effects. Here, the aim was to characterize the efficacy and safety of the oncolytic adenovirus, Delta-24-RGD, which selectively replicates in and kills tumor cells. Experimental Design: Delta-24-RGD determinants for infection and replication were evaluated in patient expression datasets. Viral replication and cytotoxicity were assessed in vitro in a battery of CNS-PNET and AT/RT cell lines. In vivo, efficacy was determined in different orthotopic mouse models, including early and established tumor models, a disseminated AT/RT lesion model, and immunocompetent humanized mouse models (hCD34+-NSG-SGM3). Results: Delta-24-RGD infected and replicated efficiently in all the cell lines tested. In addition, the virus induced dose-dependent cytotoxicity [IC50 value below 1 plaque-forming unit (PFU)/cell] and the release of immunogenic markers. In vivo, a single intratumoral Delta-24-RGD injection (107 or 108 PFU) significantly increased survival and led to long-term survival in AT/RT and PNET models. Delta-24-RGD hindered the dissemination of AT/RTs and increased survival, leading to 70% of long-term survivors. Of relevance, viral administration to established tumor masses (30 days after engraftment) showed therapeutic benefit. In humanized immunocompetent models, Delta-24-RGD significantly extended the survival of mice bearing AT/RTs or PNETs (ranging from 11 to 27 days) and did not display any toxicity associated with inflammation. Immunophenotyping of Delta-24-RGD–treated tumors revealed increased CD8+ T-cell infiltration. Conclusions: Delta-24-RGD is a feasible therapeutic option for AT/RTs and CNS-PNETs. This work constitutes the basis for potential translation to the clinical setting.

Rothmund–Thomson syndrome (RTS) is characterized by a rash that begins in the first few months of life and eventually develops into poikiloderma. Associated symptoms are alterations in the teeth, sparse hair, thin eyebrows, lack of eyelashes, low stature, bone abnormalities, hematological illnesses, gastrointestinal disease, malnutrition, cataracts, and predisposition to cancer, principally to bone tumors and skin cancer. Diagnostic certitude is provided by a genetic study involving detection of pathogenic variants of the RECQL4 gene. We hereby present a familiar case of RTS in two siblings from a Portuguese family, both diagnosed with osteosarcoma. Genomic analysis (203 genes) of both tumors as well as germline analysis of the RECQL4 gene, thus confirming the syndrome in the family, have been performed. The relevance of clinical recognition of the hallmarks of the disease and thus early diagnosis with early intervention is highlighted.

Immune enhancement of virotherapy by reshaping the tumor immune landscape may improve its success rates. IDO, an IFNγ inducible tryptophan catabolizing enzyme, is upregulated in glioblastoma, correlating with poor prognoses. IDO-mediated tryptophan depletion in the tumor-microenvironment decreases proliferation and induces apoptosis of surrounding effector T-cells. Kynurenine, a metabolite of tryptophan, induces T-cell differentiation into immunosuppressive Tregs. Excess kynurenine elicits AhR-mediated lymphocyte dysfunction and immunosuppression. The immune stimulating effect of oncolytic-virus, Delta-24-RGDOX, triggers IFNγ production contributing to a positive IDO-Kynurenine-AhR feedback loop. We hypothesized that combining Delta-24-RGDOX with IDO inhibitors will synergize to effectively treat glioblastoma. We characterized IDO and AhR in Delta-24-RGDOX infected cancers using immunofluorescence, qRT-PCR, and flow cytometry and found increased expression of both proteins in vitro and in vivo. We also observed induction of AhR in CD4+ and CD8+ T-cells by Delta-24-RGDOX in vivo. Delta-24-RGDOX also increased activity of AhR in cancer cells as indicated by an AhR responsive elements transcription assay. We used a murine glioblastoma model to test the efficacy of combining Delta-24-RGDOX with IDO inhibitor, 1MT/indoximod; the combination produced 30% more long-term survivors compared Delta-24-RGDOX alone (P=0.03), which we showed, through lymphocytic depletion studies, was dependent on CD4+ T-cell activation. We observed 100% survival in the re-challenged long-term glioblastoma survivors, indicating the establishment of immune memory by the combination. Functional studies showed significant increases in anti-tumor activity of splenocytes from combination-treated mice compared to Delta-24-RGDOX-treated mice (P=0.009). Flow cytometry studies revealed that combination-treated mice yielded the highest levels of chronically activated T-cells and lowest levels of Tregs and myeloid derived suppressor cells compared to Delta-24-RGDOX single treatment (P≤0.05). This microenvironment remodeling correlated with complete tumor elimination. Altogether, Delta-24-RGDOX activates the IDO-Kyn-AhR cascade in gliomas, identifying new targets, which when inhibited have the potential to enhance the anti-glioma effect of oncolytic-viruses by reversing tumor immunosuppression.

Atypical teratoid/rhabdoid tumors (AT/RTs) and primitive neuroectodermal tumors of the central nervous system (CNS-PNET) are rare pediatric brain tumors affecting mainly infants and young children. Current therapies for these diseases are inefficient and often cause severe side effects; thus, new therapeutic strategies are urgently needed. Here, we evaluate the anti-tumor effect of the Delta-24-RGD oncolytic adenovirus in preclinical models of CNS-PNET and AT/RT. In vitro, the virus infects and efficiently replicates in different cell culture models of AT/RT and CNS-PNET, inducing a dose-dependent cytotoxic effect (IC50 values below 1 PFU/cell) and the release of immunogenic cell death stimuli. Survival studies in mice bearing orthotopic AT/RTs (supra and infratentorial) or CNS-PNETs demonstrate the therapeutic benefit of a single Delta-24-RGD intratumoral administration (107 or 108 PFU). In virus-treated mice, median overall survival increased between 50–300% (depending on the model), obtaining up to 60% of long-term survivors. Intra-ventricular injection of Delta-24-RGD hinders the development of secondary AT/RT spinal lesions. Moreover, response to the treatment was observed even delaying viral administration up to 30 days after tumor engraftment; thus, demonstrating therapeutic benefit even in animals challenged with large well-established tumor masses. The effect of the oncolytic virus was also assessed in an immunocompetent environment using hCD34+ humanized NSG-SGM3 mice. In this model, Delta-24-RGD significantly extended overall survival (from 23 to 24 days in AT/RT model and from 28 to 55 days in CNS-PNET) with no signs of toxicity associated with inflammation. Immunophenotyping of Delta-24-RGD treated tumors by flow cytometry, RNAseq and multiplexed histology revealed that Delta-24-RGD contributes to generate a pro-inflammatory status, allowing the infiltration of immune cells and the trigger of an antitumor immune response. In conclusion, these results demonstrate that Delta-24-RGD could be a feasible therapeutic choice for AT/RT and CNS-PNET, paving the way for upcoming clinical trials.

Despite the advances in our understanding of pediatric diffuse midline gliomas (DMG) they remain the leading cause of pediatric death caused by cancer. Our group has demonstrated that the administration of Delta-24-RGD in DIPG models is safe and therapeutically efficacious. An on-going clinical trial with this virus has proven to be safe for these patients. To further improve the anti-tumoral response of the virus, we armed Delta-24-RGD with 4-1BBL (Delta-24-ACT). 41BB is a costimulatory receptor that promotes the survival and expansion of activated T cells, and the generation and maintenance of memory CD8+ T cells. Here, we showed that in vitro Delta-24-ACT can infect and express 4-1BBL in murine and human DIPG cell lines. Importantly, 4-1BBL expression in DIPG cell lines was able to activate lymphocytes and increase their IFN-gamma production. In addition, Delta-24-ACT triggered immunogenic cell death in DIPG cell lines, as shown by the release of DAMPs such as ATP, HMGB1, Hsp90a and calreticulin translocation. Delta-24-ACT administration in orthotopic DIPG models was well tolerated and safe. We confirmed the expression of the ligand within the tumor. Moreover, using flow cytometry and multispectral immunohistochemistry we observed profound changes in the tumor microenvironment with an increase in the T-cell populations and a remodeling of the myeloid component before and after treatment. Functional studies showed no differences in lymphocytes isolated from mice treated splenocytes but uncovered TILS that showed a significantly increased in the production of IFN-gamma. Delta-24-ACT treatment of mice bearing orthotopic DIPG murine tumors resulted in a significant increase in the median survival and led to free of disease long-term survivors. In summary, Delta-24-ACT is a virus that builds in our clinical experience with Delta-24-RGD in DIPG patients going a step further to boost the antitumor effect of viral therapy while maintaining a safe profile.

Despite being a rare disease, cancer is the first cause of mortality due to disease during the paediatric age in the developed countries. The current, great increase in new treatments, such as immunotherapy, constitutes a new clinical and regulatory paradigm. Cellular immunotherapy is one of these types of immunotherapy. In particular, the advanced therapy drugs with chimeric antigen receptors in the T-lymphocytes (CAR-T), and particularly the CAR-T19 cells, has opened up a new scenario in the approach to haematology tumours like acute lymphoblastic leukaemia and the B-Cell lymphomas. The approval of tisagenlecleucel and axicabtagene ciloleucel by the regulatory authorities has led to the setting up the National Plan for Advanced Therapies-CAR-T drugs in Spain. There is evidence of, not only the advantage of identifying the centres most suitable for their administration, but also the need for these to undergo a profound change in order that their healthcare activity is extended, in some cases, to the ability for the in-house manufacture of these types of therapies. The hospitals specialised in paediatric haematology-oncology thus have the challenge of progressing towards a healthcare model that integrates cellular immunotherapy, having the appropriate capacity to manage all aspects relative to their use, manufacture, and administration of these new treatments. Resumen: A pesar de ser una enfermedad rara, el cáncer es la primera causa de mortalidad por enfermedad durante la edad pediátrica en los países desarrollados. En este momento, la irrupción de nuevos tratamientos como la inmunoterapia constituye un nuevo paradigma clínico y regulatorio. Uno de estos tipos de inmunoterapia es la inmunoterapia celular. En particular, los medicamentos de terapia avanzada con receptores antigénicos quiméricos en los linfocitos T (CAR-T), y en concreto las células CAR-T19, han supuesto un nuevo escenario en el abordaje de los tumores hematológicos, como la leucemia aguda linfoblástica y los linfomas de células tipo B. La aprobación por las autoridades regulatorias de tisagenlecleucel y axicabtagene ciloleucel, ha impulsado la puesta en marcha del Plan Nacional de Terapias Avanzadas-Medicamentos CAR-T en España, evidenciándose no sólo la conveniencia de identificar los centros más adecuados para su administración, sino la necesidad de que éstos sufran una profunda transformación para que su actividad asistencial se extienda en algunos casos a la capacidad de fabricación propia de este tipo de terapias. Los hospitales especializados en hemato-oncología pediátrica tienen por tanto el reto de evolucionar hacia un modelo asistencial que integre la inmunoterapia celular, dotándose de capacidad propia para gestionar todos los aspectos relativos al uso, fabricación y administración de estos nuevos tratamientos.

Background Glioblastoma (GBM) is the most common malignant primary brain tumor in adults. Circulating biomarkers may assist in the processes of differential diagnosis and response assessment. GBM cells release extracellular vesicles containing a subset of proteins and nucleic acids. We previously demonstrated that exosomes isolated from the serum of GBM patients had an increased expression of RNU6-1 compared to healthy subjects. In this exploratory study, we investigated the role of this small noncoding RNA as a diagnostic biomarker for GBM versus other brain lesions with some potential radiological similarities. Methods We analyzed the expression of RNU6-1 in circulating exosomes of GBM patients (n = 18), healthy controls (n = 30), and patients with subacute stroke (n = 30), acute/subacute hemorrhage (n = 30), acute demyelinating lesions (n = 18), brain metastases (n = 21), and primary central nervous system lymphoma (PCNSL; n = 12) using digital droplet PCR. Results Expression of RNU6-1 was significantly higher in GBM patients than in healthy controls (P = .002). RNU6-1 levels were also significantly higher in exosomes from GBM patients than from patients with non-neoplastic lesions (stroke [P = .05], hemorrhage [P = .01], demyelinating lesions [P = .019]) and PCNSL (P = .004). In contrast, no significant differences were found between patients with GBM and brain metastases (P = .573). Receiver operator characteristic curve analyses supported the role of this biomarker in differentiating GBM from subacute stroke, acute/subacute hemorrhage, acute demyelinating lesions, and PCNSL (P < .05), but again not from brain metastases (P = .575). Conclusions Our data suggest that the expression of RNU6-1 in circulating exosomes could be useful for the differentiation of GBM from non-neoplastic brain lesions and PCNSL, but not from brain metastases.

BackgroundDiffuse Midline Gliomas (DMG) are aggressive pediatric brain tumors that arise in the brainstem of children between 5–10 years old. DMGs are the leading cause of pediatric death caused by a brain tumor, with a median survival of only 9 months.1 2 We have previously shown that the administration of the oncolytic adenovirus Delta-24-RGD is safe and lead to an increase in long-term survivors in murine models.3 4 In order to further increase the antitumor effect of Delta-24-RGD by boosting the immune response, we have constructed a new adenovirus, Delta-24-ACT, which incorporates the 4-1BBL (CD137L) into its backbone. 4-1BB is a costimulatory receptor that promotes the survival and expansion of activated T cells and NK cells and the generation and maintenance of memory CD8+ T cells, among other functions.5 6MethodsMurine and human DMG cell lines were used. 4-1BBL expression was assessed in infected cells by flow cytometry and immunofluorescence. Viral protein expression was measured by western blot, viral replication was analyzed using a method based on hexon detection and the oncolytic effect by MTS assay. For in vivo experiments, cells were injected in the pons of mice using a screw-guided system.7 A single administration of the adenovirus was injected intratumorally using the same procedure. The tumor immune populations were analyzed by flow cytometry.ResultsWe first confirmed by flow cytometry that DMG cells infected with Delta-24-ACT expressed 4-1BBL in their membrane in a dose-dependent manner. Afterwards, we analyzed the oncolytic effect of Delta-24-ACT in vitro. Delta-24-ACT was able to express viral early and late proteins in murine and human DMG cell lines and to replicate efficiently in human cells. In addition, the virus caused cell death in a dose-dependent manner. In vivo, Delta-24-ACT administration demonstrated to be safe and to produce a significant survival benefit in murine DMG models, obtaining 30–50% of long-term survivors depending on the model. More importantly, Delta-24-ACT generated immune memory, as long-term survivors were disease-free after cell rechallenge. On the other hand, we analyzed immune infiltration 7 or 10 days after the viral administration into the tumor and observed a significant increase of tumor infiltration in treated mice, which showed an activated state.ConclusionsDelta-24-ACT administration into DMG murine tumor models significantly increases the recruitment and activation of immune cells, which leads to long term survivors and immunological memory.ReferencesCooney T, Lane A, Bartels U, Bouffet E, Goldman S, Leary S, Foreman NK, Packer RJ, Broniscer A, Minturn JE, Shih C, Chintagumpala M, Hassall T, Gottardo NG, Dholaria H, Hoffman L, Chaney B, Baugh J, Doughman R, Leach JL, Jones BV, Fouladi M, Warren KE, Monje M. Contemporary survival endpoints: an International diffuse Intrinsic pontine glioma registry study. Neuro Oncol 2017;19(9):1279–1280.Grasso CS, Tang Y, Truffaux N, Berlow NE, Liu L, Debily MA, Quist MJ, Davis LE, Huang EC, Woo PJ, Ponnuswami A, Chen S, Johung TB, Sun W, Kogiso M, Du Y, Qi L, Huang Y, Hütt-Cabezas M, Warren KE, Le Dret L, Meltzer PS, Mao H, Quezado M, van Vuurden DG, Abraham J, Fouladi M, Svalina MN, Wang N, Hawkins C, Nazarian J, Alonso MM, Raabe EH, Hulleman E, Spellman PT, Li XN, Keller C, Pal R, Grill J, Monje M. Functionally defined therapeutic targets in diffuse intrinsic pontine glioma. Nat Med 2015;21(6):555–9.Martínez-Vélez N, Garcia-Moure M, Marigil M, González-Huarriz M, Puigdelloses M, Gallego Pérez-Larraya J, Zalacaín M, Marrodán L, Varela-Guruceaga M, Laspidea V, Aristu JJ, Ramos LI, Tejada-Solís S, Díez-Valle R, Jones C, Mackay A, Martínez-Climent JA, García-Barchino MJ, Raabe E, Monje M, Becher OJ, Junier MP, El-Habr EA, Chneiweiss H, Aldave G, Jiang H, Fueyo J, Patiño-García A, Gomez-Manzano C, Alonso MM. The oncolytic virus Delta-24-RGD elicits an antitumor effect in pediatric glioma and DIPG mouse models. Nat Commun 2019;10(1):2235.Garcia-Moure M, Gonzalez-Huarriz M, Labiano S, Guruceaga E, Bandres E, Zalacain M, Marrodan L, de Andrea C, Villalba M, Martinez-Velez N, Laspidea V, Puigdelloses M, Gallego Perez-Larraya J, Iñigo-Marco I, Stripecke R, Chan JA, Raabe EH, Kool M, Gomez-Manzano C, Fueyo J, Patiño-García A, Alonso MM. Delta-24-RGD, an oncolytic adenovirus, increases survival and promotes proinflamatory immune landscape remodeling in models of AT/RT and CNS-PNET. Clin Cancer Res 2021;27(6):1807–1820.Chester C, Sanmamed MF, Wang J, Melero I. Immunotherapy targeting 4-1BB: mechanistic rationale, clinical results, and future strategies. Blood 2018;131(1):49–57.Yonezawa A, Dutt S, Chester C, Kim J, Kohrt HE. Boosting cancer immunotherapy with anti-CD137 antibody therapy. Clin Cancer Res 2015;21(14):3113–20.Marigil M, Martinez-Velez N, Domínguez PD, Idoate MA, Xipell E, Patiño-García A, Gonzalez-Huarriz M, García-Moure M, Junier MP, Chneiweiss H, El-Habr E, Diez-Valle R, Tejada-Solís S, Alonso MM. Development of a DIPG orthotopic model in mice using an implantable guide-screw system. PLoS One 2017;12(1):e0170501.

BackgroundDiffuse Midline Gliomas (DMGs), encompassing Diffuse Intrinsic Pontine Gliomas (DIPGs), are the most aggressive pediatric brain tumors. Their meager survival has not changed despite the combination of radiotherapy with targeted therapies emphasizing the urgent need for effective treatments. Recent research suggested that the DIPG tumor microenvironment is neither highly immunosuppressive nor inflammatory.1 These analyses showed the lack of infiltrating lymphocytes and the abundance of CD11b+ cells. TIM-3 is a member of the T-cell immunoglobulin and mucin domain protein family expressed on multiple immune cell types, including T cells, Treg, NK cells, monocytes, dendritic cells, and microglia, where it potently regulates not only adaptive immunity but also innate immunity.2–3 Therefore, TIM-3 inhibitors could challenge several components in the tumor microenvironment, thereby providing potentially effective treatment for DMGs.MethodsNP53 and XFM murine DIPG cell lines were used for animal experiments in immunocompetent orthotopic models. The tumors were processed by mechanical and enzymatic digestion and immune populations were analyzed by a flow cytometry panel. Antibodies against NK cells (NK1.1), CD4 (GK1.5), CD8 (CD8β) were used for animal depletion experiments alone or in combination.ResultsIn silico assessment of TIM-3 expression in DIPG datasets showed a robust expression of this gene. Moreover, single-cell sequencing analyses of DIPG biopsies uncover its expression in the myeloid compartment (especially in microglia). In vivo efficacy studies showed that treatment with anti-TIM-3 antibody significantly increased the overall survival in two DIPG immunocompetent orthotopic animal models (doubling the median), lead to long-term survivors free of disease (50%) and showed immune memory. Analyses of CD45+ populations in the tumor microenvironment showed a significant increase in microglia, granulocytes, NK and CD8+ cells corresponding with a NK and T-cell activate phenotypes in treated-mice. In addition, we have a substantial decrease in the Treg population, which causes an increase in the CD8/Treg ratio. CD4 and CD8 T-cell depletion led to a significant but not total loss of treatment efficacy. NK cells depletion also reduced the effectiveness of this therapy, albeit to a lesser extent than CD4-CD8 depletion. We are currently investigating the role of microglia in the outcome of the treatment.ConclusionsOur data uncovered TIM-3 as a potential target for the treatment of DIPG tumors. Inhibition of this molecule led to a potent antitumor effect mediated by a profound tumor microenvironment remodelling.ReferencesLieberman NAP, DeGolier K, Kovar HM, et al. Characterization of the immune microenvironment of diffuse intrinsic pontine glioma: implications for development of immunotherapy. Neuro Oncol 2019;21(1):83–94. doi:10.1093/neuonc/noy145.Acharya N, Sabatos-Peyton C, Anderson AC. Tim-3 finds its place in the cancer immunotherapy landscape. J Immunother Cancer 2020;8(1):e000911. doi:10.1136/jitc-2020-000911.Wolf Y, Anderson AC, Kuchroo VK. TIM3 comes of age as an inhibitory receptor. Nat Rev Immunol 2020;20(3):173–185. doi:10.1038/s41577-019-0224-6

Metastatic breast cancer is the second leading cause of cancer related death among women. Although, conventional therapies such as surgery, adjuvant and neoadjuvant therapies have shown promising outcomes in preclinical and clinical settings, long-term survival rates remain unacceptably low for patients with recurrent disease or disseminated metastases. Breast primary and metastasic niches are characterized by an immunosuppressive microenvironment. Hence, novel therapeutic regimens that modulate this challenging phenotype are desperately needed. Our group has developed and tested in preclinical models and in clinical trials for patients with recurrent glioblastoma the anti-tumor effect of oncolytic adenovirus Delta-24-RGD, also named DNX-2401, resulting with a 20% of long-term survivors, and the awaking of a antitumoral immunity after the administration of the pathogen. This paradigm shift suggests that viroimmunotherapy can be used for the treatment of metastasic cancer. Thus, here we hypothesize that viroimmunotherapy is capable of exerting abscopal effects to eradicate primary breast tumors and their metastatic niches. To test the hypothesis, 4T1 tumor bearing mice were subjected to the local administration of Delta-24-RGD and its armed derivatives, expressing expressing immune co-stimulatory ligands OX40L (Delta-24-RGDOX or DNX-2440) and GITRL (Delta-24-GREAT). We found that, the tumors treated with oncolytic adenoviruses exhibited increase of both CD8+ T cell infiltration and CD8+/CD4+ ratio. Additionally, T-cell activity measured by IFN-γ secretion of splenocytes when in co-culture with breast cancer cells was found to be increased in mice that underwent viroimmunotherapy (P Citation Format: Sagar Shashikant Sohoni, Teresa Nguyen, Dong Ho Shin, Xuejun Fan, Jiang Hong, Sumit Gupta, Ashley Ossimetha, Yanhua Yi, Chandra Bartholomeusz, Marta Alonso, Juan Fueyo, Candelaria Gomez-Manzano. Targeting the immunosuppressive microenvironment of metastatic breast cancer with viroimmunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1786.

Gastric cancer is the third leading cause of cancer-related mortality worldwide, with a 5-year survival rate of 30% in the US despite standard treatment. Black, Asian, and non-white Hispanic populations have a 40-50% higher risk of gastric cancer than white people. The poor prognosis and limited therapies associated with gastric cancer has led us to investigate the use of these oncolytic adenoviruses as an alternative treatment. Viroimmunotherapy is showing promising results in preclinical and clinical studies. Our group developed an oncolytic adenovirus, Delta-24-RGD, also named DNX-2401, which has been tested in clinical trials for recurrent glioblastoma, with complete responses and durable survival in 20% of patients, which was associated with an anti-tumor immune response. To further enhance the immune effect of Delta-24-RGD, we developed the next generation virus armed with T cell co-stimulatory agonist, OX40L, termed Delta-24-RGDOX, clinically known as DNX-2440. To explore the sensitivity of gastric cancer to oncolytic adenoviruses, we performed viral infection and replication experiments in a panel of human gastric cancer cells (AGS, MKN-45, SNU-1) and a murine gastric cancer cell line, M12. Cell lines were infected in a dose-dependent experiment using an Ad-GFP-RGD vector, showing that >80% of the culture was infected using a multiplicity of infection of 25 for AGS, 50 for MKN-45 and SNU-1, and 100 for M12. Viral replication in gastric cancer was assessed by hexon staining of 293HEK cells incubated with cell lysates obtained from infected gastric cancer cells, illustrating that infection with either virus resulted in the production of new virions. Consistent with these data, we detected in Delta-24-RGD- and Delta-24-RGDOX-infected gastric cancer cells expression of E1A and hexon, confirming viral infection and replication, respectively. Of interest, 74% to 81% Delta-24-RGDOX-infected gastric cancer cells expressed in their surface OX40L 48 hours post-infection as assessed by flow cytometry. Efficacy of the proposed virotherapy in gastric cancer was visualized by using cell titer blue viability assay, which showed anti-cancer efficacy of the proposed therapy. Results on the efficacy and immune effects of armed-oncolytic adenovirus expressing OX40L in a M12-C57BL/6 gastric cancer immunocompetent mouse model are in progress. Current results suggest that armed oncolytic adenoviruses with positive immunomodulators can effectively infect, replicate, and lyse gastric cancer cells. Ultimately, we aim to translate these oncolytic adenoviruses to the clinic to improve the survival of patients with gastric cancer. Citation Format: Ashley Ossimetha, Teresa Nguyen, Natalie Melendez, Xuejun Fan, Marta Alonso, Juan Fueyo, Filipa Godoy-Vitorino, Candelaria Gomez-Manzano. Armed oncolytic adenovirus expressing the positive immune checkpoint OX40L for gastric cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1748.

Diffuse Intrinsic Pontine Glioma (DIPG) is the leading cause of pediatric death by brain tumors with a median survival of only 9 months, in spite of the current standard of therapy which includes radiation. The purpose of our study is to explore the use of oncolytic adenoviruses as a new therapeutic modality. Our group is leading a phase I clinical trial in naive DIPGs treated with the oncolytic adenovirus Delta-24-RGD, also named DNX-2401 (NCT03178032), which results are still pending. Of interest, clinical trials using Delta-24-RGD in adult gliomas resulted in encouraging results with long-term survivors and the presence of a strong anti-tumor immunity. In this study, we aim to test the effect of armed Delta-24-RGD with immune costimulatory ligands such as GITRL (Delta-24-GREAT) and OX-40L (Delta-24-RGDOX or DNX-2440) for the treatment of DIPGs. We used a panel of murine DIPG cell lines (NP53 and XFMA) to test the effect of these armed-oncolytic adenoviruses. Infectivity assay using an Ad-RGD-GFP vector and flow cytometry showed the expression of the ectopic GFP in more than 80% of the NP53 cells (50 MOIs) and XFMA cells (30 MOIs). Consistent results were obtained using flow cytometry and western blot to detect the expression of the ectopic ligands GITRL and OX-40L in these cultures. These cells were semipermissive for viral replication as assessed by the detection of the viral structural protein hexon in western blot analysis. Preliminary in vivo data using immunocompetent mice models where DIPG cells have been implanted into the pons showed a dose-dependent effect and the generation of immune memory. Our results suggest that oncolytic adenoviruses expressing positive immunocheckpoints might prove be a new effective modality for treating DIPG and hence, increase survival in children suffering this dismal disease. Citation Format: Sumit Gupta, Virginia Laspidea, Juan Fueyo, Oren Becher, Teresa Nguyen, Dong Ho Shin, Hong Jiang, Sagar Sohoni, Xuejun Fan, Yanhua Yi, Joy Gumin, Frederick Lang, Candelaria Gomez-Manzano, Marta M. Alonso. Armed oncolytic virus for treatment of pediatric diffuse intrinsic pontine glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3053.

Pediatric High Grade Gliomas (pHGGs), including Diffuse Midline Gliomas (DMGs), are aggressive pediatric tumors with a poor overall survival. In the last years, ONC201 has emerged as a promising agent in the field of pediatric brain tumors. Another interesting approach is virotherapy; Delta-24-RGD, which is an oncolytic virus, has demonstrated safety and effectiveness in different preclinical models and in clinical trials. Therefore, in this work we set to evaluate whether the combination of ONC201 with Delta-24-RGD could result in an increased therapeutic benefit in pHGGs and DMGs. Given that ONC201 targets mitochondrial metabolism in a preclinical setting, we assessed potential negative interactions of the combination therapy. While ONC201 treatment resulted in decreased viral protein load (E1A and fiber), there was no significant negative impact on the viral replication (measured by hexon staining). ONC201 did not disrupt the activation of mTORC1 pathway by the adenovirus. Furthermore, Delta-24-RGD did not affect the decrease in basal oxygen consumption rate induced by ONC201. Our results suggested that ONC201 and Delta-24-RGD are not antagonistic. Evaluation of the in vitro cytotoxicity in different human pHGG (CHLA-03-AA and SF188) and DMG (TP-54 and SU-DIPG-IV) cell lines showed that the combination treatment was significantly better that either agent alone. In vivo, a single local injection of Delta-24-RGD followed by a weekly ONC201 of mice bearing CHLA-03-AA cell line orthotopically significantly increased the median overall survival (PBS: 48 days; ONC201: 54.5 days; Delta-24-RGD: 62 days; ONC201+Delta-24-RGD: 95 days (P=0.0008)) of these mice leading to 20% long-term survivors, free of disease. Currently, we are evaluating the effect of the combination in immunosuppressed and immunocompetent models of DMGs. In summary, our data indicate ONC201 in combination with Delta-24-RGD could be a potential therapeutic choice for patients affected by pHGGs and DMGs.

Diffuse Intrinsic Pontine Gliomas (DIPG) are aggressive pediatric brain tumors that arise in the pons of children, being the leading cause of pediatric death caused by cancer. We have previously demonstrated that Delta-24-RGD administration is safe and efficacious in DIPG preclinical models, indicating that it could be a good candidate as therapeutic approach for DIPG. However, our data underscore that there is still room to improve the anti-DIPG effect obtained with Delta-24-RGD. For that purpose, we have constructed three new virus by engineering Delta-24-RGD with different T cell activators: 4-1BBL (Delta-24-ACT), OX40-L (Delta-24-RGDOX) and GITRL (Delta-24-GREAT), to further increase the immune response generated by the viral effect. In vitro, the three virus were able to infect murine and human DIPG cell lines, produce oncolytic effect in a dose-dependent manner and express the corresponding functional ligand (4-1BBL, OX40L or GITRL) in the membrane of infected cells (almost 100% of cells expressing them at 10 MOIs). As expected, viral replication was optimal in human cell lines but semipermissive in murine cells. In vivo, the intratumoral administration of armed oncolytic viruses was safe and significantly increased survival of mice bearing orthotopic DIPG murine tumors, leading to long-term survivors. In addition, we analyzed the effect of the virus in the tumor microenvironment by flow cytometry and immunohistochemistry, which indicated that there was a significant increase of immune infiltration in brains of treated mice. Moreover, the immune infiltrated showed a functional active phenotype. Although deeper characterization is needed, these data show that the incorporation of a positive immune modulator into Delta-24-RGD could improve the oncolytic effect of the virus by boosting the immune response, while maintaining a safe profile in immunocompetent models offering a feasible option treatment for DIPG.

Background A Phase 1, single center study is ongoing to evaluate the conditionally replicative oncolytic adenovirus, DNX-2401 (tasadenoturev), followed by radiotherapy (RT) in pediatric patients with newly diagnosed diffuse intrinsic pontine glioma (DIPG). Methods Patients 1–18 years with newly diagnosed DIPG with no prior treatment, Lansky/Karnofsky performance score ≥ 70, and adequate organ function were enrolled. A tumor biopsy was performed followed by a single intratumoral injection of 1e10-5e10 virus particles (vp) DNX-2401. Conventional radiotherapy was initiated within 1 month of DNX-2401 administration. Results Enrolled subjects (n=12) had a median age of 9 (range 3–18) and performance scores of 90–100 (n=4; 33%) or 70–80 (n=8; 67%). As part of a dose escalation design, subjects were treated with 1e10 vp (n=4) or 5e10 vp DNX-2401 (n=8), which was then followed by standard RT in 11 of 12 subjects (92%). No dose-limiting toxicities were observed and the treatment regimen was well-tolerated. Adverse events (AEs) have been primarily mild to moderate and consistent with underlying disease. The most commonly reported AEs (≥ 5 subjects), regardless of study drug relationship, include headache, asthenia, vomiting, anemia, leukocytosis, and fever. Two SAEs have been reported including grade 3 lymphopenia and grade 3 abdominal pain. Tumor reductions have been observed and efficacy evaluations are ongoing. As of 09Dec2020, 12-month survival (OS-12) was 71% and 4 of 12 patients had survived > 20 months. Four subjects continue to be followed for survival. Correlative analysis of tumor biopsy and peripheral samples is ongoing. Conclusions DNX-2401 followed by RT can be safely administered to pediatric subjects with newly diagnosed DIPG; clinical activity and preliminary survival are encouraging.

Osteosarcoma is the most common bone cancer among those with non-hematological origin and affects mainly pediatric patients. In the last 50 years, refinements in surgical procedures, as well as the introduction of aggressive neoadjuvant and adjuvant chemotherapeutic cocktails, have increased to nearly 70% the survival rate of these patients. Despite the initial therapeutic progress the fight against osteosarcoma has not substantially improved during the last three decades, and almost 30% of the patients do not respond or recur after the standard treatment. For this group there is an urgent need to implement new therapeutic approaches. Oncolytic adenoviruses are conditionally replicative viruses engineered to selectively replicate in and kill tumor cells, while remaining quiescent in healthy cells. In the last years there have been multiple preclinical and clinical studies using these viruses as therapeutic agents in the treatment of a broad range of cancers, including osteosarcoma. In this review, we summarize some of the most relevant published literature about the use of oncolytic adenoviruses to treat human osteosarcoma tumors in subcutaneous, orthotopic and metastatic mouse models. In conclusion, up to date the preclinical studies with oncolytic adenoviruses have demonstrated that are safe and efficacious against local and metastatic osteosarcoma. Knowledge arising from phase I/II clinical trials with oncolytic adenoviruses in other tumors have shown the potential of viruses to awake the patient´s own immune system generating a response against the tumor. Generating osteosarcoma immune-competent adenoviruses friendly models will allow to better understand this potential. Future clinical trials with oncolytic adenoviruses for osteosarcoma tumors are warranted.

Background There are no effective treatments for diffuse intrinsic pontine gliomas (DIPGs); these tumors cannot be surgical resected, and diagnosis is based on magnetic resonance imaging. As a result, tumor tissues for molecular studies and pathologic diagnosis are infrequent. New clinical trials are investigating novel medications and therapeutic techniques in an effort to improve treatment of patients with DIPGs. Objective To determine the safety, tolerability, and toxicity of an oncolytic adenovirus, DNX-2401, injected into the cerebellar peduncle in pediatric subjects with DIPG and to collect tumor samples of this type of tumor. Methods Phase I, single-center, uncontrolled trial. A tumor biopsy will be performed through the cerebellar peduncle, and DNX-2401 will be injected immediately after the biopsy. Standard therapy consisting of radiotherapy and chemotherapy will follow in 2 to 6 wk. Expected outcomes Improvement of overall survival and quality of life in patients with DIPG and collection of tumor specimens to study the molecular profiling of these tumors. Discussion The aims of this trial are to contribute to the sample collection of DIPG and to offer treatment during the tumor tissue biopsy using the virus. If this virus works as expected, it could kill the tumor cells with no damage to healthy tissue, functioning as a targeted therapy. It is important to note that edema has not been observed with this virus in all trials performed to date. The information obtained through this and other similar studies may be useful for developing or improving new therapies in the battle against DIPG.

Atypical teratoid/rhabdoid tumors (AT/RTs) are rare pediatric brain tumors affecting mainly infants and young children. However, AT/RTs encompass almost 10% of death caused by pediatric brain tumors, and the 2-year overall survival for these children remains below 20%. For this reason, AT/RT ranks among the deadliest pediatric brain tumors. Therefore, it is clear we need to find out new therapeutic options for these children. Delta-24-RGD oncolytic adenovirus has already demonstrated its efficacy in Phase I/II clinical trials in adult patients affected by high grade gliomas with no evidence of severe side effects. Of interest for pediatric brain tumors, the safety of Delta-24-RGD is has been demonstrated in an ongoing Phase I clinical trial for the treatment of DIPGs (NCT03178032). For these reasons, we propose to evaluate the anti-tumor effect of Delta-24-RGD in preclinical models of AT/RT. In vitro, the virus was able to infect and replicate in three different cell culture models of AT/RT, inducing a dose-dependent cytotoxic effect that results in IC50 values below 1 PFU/cell. In vivo, intratumor administration of the virus in mice bearing orthotopic localized AT/RT (supratentorial and infratentorial) extended significantly the survival the animals, leading to up to 20% of long-term survivors. We have also generated models of disseminated disease through intraventricular injection of the tumor cells, thus mimicking the lesions found in patients. AT/RT cell lines were transduced with a luc-expressing lentivirus in order to facilitate the follow up of these tumors. In disseminated AT/RT models, light emission reveals reduction of tumor growth in Delta-24-RGD treated animals in comparison to those mock treated, thus obtaining an increased overall survival. In conclusion, these results demonstrate that Delta-24-RGD could be a feasible therapeutic choice for patients affected by AT/RT.

Immune checkpoint blockade has revolutionized cancer therapy; however the therapeutic benefit is limited to only a subset of patients with immunogenic (“hot”) tumors and is compromised by immune-related adverse events. We have reported the efficacy of oncolytic adenovirus Delta-24-RGDOX (DNX-2440) in syngeneic glioma mouse models. We hypothesized that localized treatment with the virus is effective against disseminated melanomas, including intracranial melanomas. We tested the hypothesis in the subcutaneous (s.c.)/s.c. and s.c./intracranial (i.c.) melanoma models derived from luciferase-expressing B16-Red-FLuc cells in C57BL/6 mice. First, through monitoring tumor growth with bioluminescence imaging, we found that, in both s.c./s.c. and s.c./i.c. models, three injections of Delta-24-RGDOX significantly inhibited the growth of both the virus-injected s.c. tumor and untreated distant s.c. or i.c. tumor, thereby prolonging survival. Next, through cell profiling with flow cytometry, we observed that the virus increased the presence of T cells and effector T cell frequency in the virus-injected tumor and mediated the same changes in T cells from peripheral blood, tumor-draining lymph nodes (TDLNs), spleens, and brain hemispheres with untreated tumor. Moreover, Delta-24-RGDOX decreased the frequency of exhausted T cells and regulatory T cells in the virus-injected and untreated i.c. tumors. Consequently, the virus promoted recruitment and/or in situ expansion of antigen-specific T cells in tumors expressing the target antigen. Therefore, we concluded that local intratumoral injection of Delta-24-RGDOX resulted in systemic immune activity against the disseminated tumors. Furthermore, we speculate that given the immunogenicity, cancer-selectivity and intratumoral administration of the virus, Delta-24-RGDOX is expected to have an improved safety profile when compared to immune checkpoint blockade treatment strategies. This is the first report demonstrating that local administration of oncolytic adenovirus results in eradication of intracranial tumors, suggesting Delta-24-RGDOX could be used to manage brain metastases of melanoma.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumor, being the leading cause of pediatric death caused by cancer. We previously showed that administration of the oncolytic virus Delta-24-RGD to DIPG murine models was safe and led to an increase in the median survival of these animals. However, not all the animals responded, underscoring the need to improve this therapy. In order to increase the antitumoral effect of the virus, we have engineered Delta-24-RGD with the costimulatory ligand 4-1BBL (Delta24-ACT). 4-1BB is a costimulatory receptor that promotes the survival and expansion of activated T cells, and the generation and maintenance of memory CD8+ T cells. In this project, we evaluated the oncolytic effect of Delta24-ACT and the antitumor immune response in DIPG murine models. In vitro, Delta24-ACT was able to infect and induce cell death in a dose-dependent manner in murine DIPG cell lines. In addition, Delta24-ACT was able to replicate in these tumor cells and to express viral proteins. Moreover, infected cells expressed 41BBL in their membranes. Delta24-ACT could induce immunogenic cell death due to an increased secretion of ATP and calreticulin translocation to the membrane of infected cells (in no-infected cells it located in the ER), DAMPs that can trigger the immune response activation. In vivo, Delta24-ACT demonstrated to be safe in all the tested doses and was able to induce a significant increase in the median survival of the treated animals. Moreover, long-term survivors display immunological memory. Delta24-ACT treatment led to antitumoral effect in DIPG murine cell lines in vitro. Of significance, we have demonstrated that in vivo administration of Delta24-ACT is safe and results in an enhanced antitumor effect. Future in vivo studies will explore the underlying immune mechanism of the virus.

Background Viroimmunotherapy is evolving as a strong alternative for the standard treatment of malignant gliomas. Promising results from a recent clinical trial testing the anticancer effect of Delta-24-RGD in patients with glioblastoma suggested the induction of antitumoral immunity after viral administration. To further enhance the anti-glioma immune effect, we have armed Delta-24-RGD with the costimulatory ligand GITRL (Delta-24-GREAT [Glucocorticoid Receptor Enhanced Activity of T cells]). Methods We tested the infectivity and replication of Delta-24-GREAT, and the expression of ectopic GITRL in human and murine glioma cell lines. In vivo experiments involved the intracranial implantation of glioma cells into an immunocompetent model to study the anticancer effect, and rechallenging experiments to study long-term protection. Phenotypic and functional characterization of lymphocyte populations were performed by FACS and ELISA for Th1 cytokines expression, respectively. Results Our results showed that Delta-24-GREAT infects and induces the expression of GITRL. Delta-24-GREAT prolonged the survival of glioma-bearing immunocompetent mice and resulted in both anti-viral and anti-glioma immune responses, including increased frequency of central memory CD8+ T cells. Rechallenging the surviving mice with a second implantation of glioma cells did not lead to tumor growth; however, the surviving mice developed lethal tumors when B16/F10 melanoma cells were implanted intracranially, strongly indicating that the immune response was specific for glioma antigens. Conclusions GITRL-armed Delta-24-RGD treatment results in an antigen-restricted antitumor memory, an enhanced anti-glioma effect, and the generation of central immune memory. Our results strongly indicate that this strategy represents a vertical advance in virotherapy designed to treat patients with malignant brain tumors.

BACKGROUND Despite our increased understanding of the genetic make-up and new therapies for pediatric high grade glioma (pHGG) and Diffuse Intrinsic Pontine Glioma (DIPG) the outcome remains grim. Delta-24-RGD (DNX-2401 in the clinic) has been tested for adult glioblastoma presenting a safe profile and promising efficacy. Recently our group has showed that the virus is safe and effective in preclinical models of pHGG and DIPG. Moreover, we showed that the virus is able to trigger an antitumor immune response. These outstanding preclinical results allowed us to propel a phase I clinical trial for newly diagnosed DIPGs (NCT03178032) where the patients received an intratumoral viral injection followed by standard radiotherapy. MATERIALS AND METHODS A phase I clinical trial with DNX-2401 for patients with newly diagnosed DIPG to assess the MTD is taking place in our hospital (N=12). Tumor biopsy is performed through the cerebellar peduncle, followed by virus injection. The virus is injected using a cannula, MEMS cannula (Alcyone Lifesciences) that prevents the reflux. Virus will be injected starting with 1010 pv. The trial is uncontrolled, unicentric with a 3 + 3 design. The objective of this trial is to determine the safety, tolerability, and toxicity of DNX-2401 in subjects with DIPG and to collect tumor samples of this type of tumor. Secondary endpoints are overall survival at 12 months (OS12), percentage of responses and induced immune response against tumor. The follow up includes close monitoring of neurological status, blood tests and brain MRI. If this trial shows evidence of safety and efficacy will propel a multicenter clinical trial. RESULTS All the clinical data from the trial available until September 2019 will be presented during the congress, to date 8 patients have been treated within the trial. Three patients were treated with the D1=1x1010vp and because the lack of toxicity we escalated to the D2= 5x1010vp. The procedure was well tolerated and safe. Patients were home 3–4 days after the injection. All the patients displayed a reduced tumor volume after combined treatment. We performed molecular studies in 6 out of the 8 patients. We are currently assessing the immune responses to the virus. CONCLUSIONS Information acquired within this clinical study would aid to understand the response of DIPGs to viral therapies and therefore to better tailor this strategy to improve the survival and the quality of life of pediatric brain tumors.

Purpose: Intratumoral injection of oncolytic adenovirus Delta-24-RGDOX induces efficacious antiglioma immunity in syngeneic glioma mouse models. We hypothesized that localized treatment with the virus is effective against disseminated melanomas. Experimental Design: We tested the therapeutic effect of injecting Delta-24-RGDOX into primary subcutaneous (s.c.) B16-Red-FLuc tumors in s.c./s.c. and s.c./intracranial (i.c.) melanoma models in C57BL/6 mice. Tumor growth and in vivo luciferase-expressing ovalbumin-specific (OT-I/Luc) T cells were monitored with bioluminescence imaging. Cells were profiled for surface markers with flow cytometry. Results: In both s.c./s.c. and s.c./i.c. models, 3 injections of Delta-24-RGDOX significantly inhibited the growth of both the virus-injected s.c. tumor and untreated distant s.c. and i.c. tumors, thereby prolonging survival. The surviving mice were protected from rechallenging with the same tumor cells. The virus treatment increased the presence of T cells and the frequency of effector T cells in the virus-injected tumor and mediated the same changes in T cells from peripheral blood, spleen, and brain hemispheres with untreated tumor. Moreover, Delta-24-RGDOX decreased the numbers of exhausted T cells and regulatory T cells in the virus-injected and untreated tumors. Consequently, the virus promoted the in situ expansion of tumor-specific T cells and their migration to tumors expressing the target antigen. Conclusions: Localized intratumoral injection of Delta-24-RGDOX induces an in situ antovaccination of the treated melanoma, the effect of which changes the immune landscape of the treated mice, resulting in systemic immunity against disseminated s.c. and i.c. tumors.

Atypical teratoid/rhabdoid tumors (ATRTs) and primitive neuroectodermal tumors (PNETs) are two rare primary embryonal brain tumors, which are among the most frequent solid brain tumors in infants under one year old. The outcome for these children remains dismal, especially in ATRTs which have a 2-year overall survival below 20%. Therefore, we need to find out new efficient and safe therapeutic options for these children. Delta-24-RGD (DNX-2401 in the clinic) is an oncolytic adenovirus that has already been tested in Phase I/II clinical trials in patients affected by recurrent glioblastoma (NCT00805376; NCT01956734), demonstrating anti-tumor effect without any evidence of severe side effect associated with viral administration. Of interest for pediatric brain tumors, an ongoing Phase I trial using Delta-24-RGD for the treatment of diffuse intrinsic pontine gliomas (NCT03178032) has also demonstrated a safe profile in these children. For these reasons, we propose to evaluate the anti-tumor effect of Delta-24-RGD in preclinical models of ATRT and PNET. The virus was able to infect and replicate in tumor cell cultures (three models of ATRT and four of PNET), inducing a potent cytotoxic effect. In addition, this cytotoxicity resulted in the released the immunogenic cell death markers Hsp70, Hsp90, HMGB1 and ATP, as well as an increased calreticulin exposure in the outer cell surface. Although not conclusive, the presence of these markers suggest the triggering of an anti-tumor immune response. Intratumoral administration of the virus extended significantly the overall survival of mice bearing ATRT or PNET xenografts, leading to up to 40% of long-term survivors. We are currently setting up an intraventricular model of disseminated disease, since most of patients with ATRT actually die due to the presence of metastases. In conclusion, these results demonstrate that Delta-24-RGD could be a feasible therapeutic option for children with PNETs and AT/RTs.

Pediatric high grade gliomas (pHGG), including diffuse intrinsic pontine gliomas (DIPGs), are aggressive tumors with a dismal outcome. Radiotherapy (RT) is part of the standard of care of these tumors; however, radiotherapy only leads to a transient clinical improvement. Delta-24-RGD is a genetically engineered tumor-selective adenovirus that has shown safety and clinical efficacy in adults with recurrent gliomas. In this work, we evaluated the feasibility, safety and therapeutic efficacy of Delta-24-RGD in combination with radiotherapy in pHGGs and DIPGs models. Our results showed that the combination of Delta-24-RGD with radiotherapy was feasible and resulted in a synergistic anti-glioma effect in vitro and in vivo in pHGG and DIPG models. Interestingly, Delta-24-RGD treatment led to the downregulation of relevant DNA damage repair proteins, further sensitizing tumors cells to the effect of radiotherapy. Additionally, Delta-24-RGD/radiotherapy treatment significantly increased the trafficking of immune cells (CD3, CD4+ and CD8+) to the tumor niche compared with single treatments. In summary, administration of the Delta-24-RGD/radiotherapy combination to pHGG and DIPG models is safe and significantly increases the overall survival of mice bearing these tumors. Our data offer a rationale for the combination Delta-24-RGD/radiotherapy as a therapeutic option for children with these tumors. Significance Delta-24-RGD/radiotherapy administration is safe and significantly increases the survival of treated mice. These positive data underscore the urge to translate this approach to the clinical treatment of children with pHGG and DIPGs. Electronic supplementary material The online version of this article (10.1186/s40478-019-0714-6) contains supplementary material, which is available to authorized users.

Cancer virotherapy is a paradigm-shifting treatment modality based on virus-mediated oncolysis and subsequent antitumor immune responses. Clinical trials of currently available virotherapies showed that robust antitumor immunity characterizes the remarkable and long-term responses observed in a subset of patients. These data suggest that future therapies should incorporate strategies to maximize the immunotherapeutic potential of oncolytic viruses. In this review, we highlight the recent evidence that the antiviral immunity of the patients may limit the immunotherapeutic potential of oncolytic viruses and summarize the most relevant approaches to strategically redirect the immune response away from the viruses and toward tumors to heighten the clinical impact of viro-immunotherapy platforms.

Current therapies for atypical teratoid/rhabdoid tumors (AT/RTs) are suboptimal, resulting in a 2-year OS below 20% and the development of severe side effects. Therefore, we need to explore alternative therapeutic approaches for this disease. Since the virus Delta24-RGD has already demonstrated its efficacy and safety as a therapeutic agent for brain tumors, including pediatric patients, here we propose to evaluate the anti-tumor effect of Delta24-RGD in AT/RT. In vitro, Delta24-RGD infects and replicates in AT/RT cultures followed by oncolysis, obtaining IC50 values below 1 PFU/cell. In vivo, a single local injection of Delta-24-RGD in three infratentorial AT/RT models (BT-12, CHLA-06 and CHLA-266) extended significantly the median OS (50 to 78 days BT-12; 21 to 31 days CHLA-06; 64 to 110 days CHLA-266). Delta-24-RGD also increased the survival of mice bearing supratentorial CHLA-266 tumors (from 93 to 132 days). Next, we evaluated the efficacy of Delta24-RGD in a model mimicking metastatic disease through intraventricular injection of BT-12-luciferase cells. Administration of Delta24-RGD inhibited tumor growth and development of metastases, leading to an increased OS and nearly 70% of long-term survivors. The interaction between Delta24-RGD and the immune system was evaluated in humanized mice models bearing CHLA-06. In this model, Delta24-RGD treatment extended OS (from 23 to 34 days) and we characterized the anti-tumor immune landscape in control and Delta24-RGD treated mice by transcriptional and functional analyses. These results underscore the potential of Delta24-RGD as a promising therapeutic choice for patients affected by AT/RT.

The objective of this trial is to determine the safety, tolerability, and toxicity of DNX-2401 in newly diagnosed DIPG patients (NCT03178032) followed by radiotherapy. Secondary endpoints are overall survival at 12 months, percentage of responses and induced immune response against tumor. Tumor biopsy was performed through the cerebellar peduncle, followed by intratumoral injection of DNX-2401 (N=12). Three patients were treated with 1x1010vp and given the lack of toxicity we escalated to 5x1010vp. The procedure was well tolerated and reduced tumor volume was demonstrated in all patients after combined treatment (virus + radiotherapy). We performed molecular studies (RNAseq and the Oncomine Childhood Research Panel from Thermo Fisher). The immune cell composition of the biopsies pre-virus injection was assessed using multiplexed quantitative immunofluorescence. T cells were hardly detectable in these tumors while macrophages were abundant. Using a multiplexed TCR-sequencing mRNA-based assay to analyze 18 available paired pre- and post-treatment samples from the trial, we detected increased clonal T cell diversity following treatment with the virus. We also measured pre and post treatment neutralizing antibodies and their relationship with survival. Finally, we performed functional studies using 2 cell lines isolated from patients included in this trial to assess the response to the virus (infectivity, viability, T-cell recognition). In summary, the virus has shown safety and efficacy in some patients. The information obtained in this clinical study would aid understanding the response of DIPG patients to viral therapies and, therefore, to better tailor this strategy to improve the survival of these patients.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumour, being the leading cause of paediatric death caused by cancer. Despite all the advances made regarding effective therapies, the survival is dismal. Our lab has engineered the oncolytic virus Delta-24-ACT armed with the costimulatory ligand 41BBL in order to increase the antitumoral effect of the adenovirus. 41BB is a costimulatory receptor which promotes the expansion of activated T cells and the generation and maintenance of CD8 T memory cells. Therefore, we propose the use of Delta-24-ACT as a therapeutic approach for DIPG tumours. We observed that Delta-24-ACT is able to infect and replicate in NP53 and PDGFB-driven, two DIPG murine cell lines. Furthermore, 41BBL is expressed in the membranes of the infected cells and results with immunogenic cell death as shown by the different DAMPs. Injection of Delta-24-ACT in DIPG model was safe, showed no sign of toxicity and led to a significantly increase in the median overall survival, generating anti-glioma memory in long-term survivors. Mechanistic experiments, showed an increase of T cell infiltration (mainly CD8), decrease of proliferating cells and a reduction of the number of vessels in FFPE brain samples in the treated mice. We are currently performing nanostring analyses to assess the changes in the transcriptional immune phenotype of treated versus control mice. In summary, our data suggest that Delta-24-ACT is safe and induces a potent antitumor immune response in DIPG models mainly based in the activation of CD8 lymphocytes recruited by the viral activity.

Pediatric high-grade gliomas (pHGG) are heterogeneous brain tumors for which new specific diagnostic/prognostic biomarkers are needed. In this study, we aimed to identify new pHGG subgroup specific biomarkers by exploiting exosomes, known vehicles of oncogenic signals. We used plasma from 23 patients (including 6 controls) and conditioned medium from 12 patient-derived cell-lines, representing all locational and molecular subgroups. Upon exosome isolation, total RNA was extracted and miRNAs were assessed using a PCR Panel. Analysis of plasma miRNome showed that tumor exosomal samples were largely clustered together, independently from their locational and/or molecular subgroup. We identified 20 significantly upregulated and 25 downregulated miRNAs compared to controls. Interestingly, 27 miRNAs were expressed only in tumors. Furthermore, the unsupervised clustering showed a clear separation based on locational (hemispheric vs pontine) and mutational (WT vs H3.3G34R or H3.3G34R vs H3K27M) subgroup comparisons, with the identification of distinct miRNomes underlying the key role of location and mutations in defining the pHGG exosomal miRNA profile. This was further confirmed analyzing the miRNOme from cell-line derived exosomes. Moreover, we identified a pool of significantly differentially regulated miRNAs in diagnose vs relapse and biopsy vs autopsy cell-lines. Most importantly, when comparing hemispheric vs pontine and H3.3G34R vs H3.3K27M, we identified respectively four and three miRNas equally dysregulated and in common between plasma and cell-lines. Those were strongly associated mainly to transcriptional regulation and targeting TTC9, linked to cancer invasion and metastasis. Based on this, we suggest exosomal miRNAs as a powerful new pHGG diagnostic/prognostic tool.

Purpose: Osteosarcoma is the most common malignant bone tumor in children and adolescents. Despite aggressive chemotherapy, more than 30% of patients do not respond and develop bone or lung metastasis. Oncolytic adenoviruses engineered to specifically destroy cancer cells are a feasible option for osteosarcoma treatment. VCN-01 is a replication-competent adenovirus specifically engineered to replicate in tumors with a defective RB pathway, presents an enhanced infectivity through a modified fiber and an improved distribution through the expression of a soluble hyaluronidase. The aim of this study is to elucidate whether the use of VCN-01 would be an effective therapeutic strategy for pediatric osteosarcoma. Experimental Design: We used osteosarcoma cell lines established from patients with metastatic disease (531MII, 678R, 588M, and 595M) and a commercial cell line (143B). MTT assays were carried out to evaluate the cytotoxicity of VCN-01. Hexon assays were used to evaluate the replication of the virus. Western blot analysis was performed to assess the expression levels of viral proteins and autophagic markers. The antitumor effect of VCN-01 was evaluated in orthotopic and metastatic osteosarcoma murine animal models. Results: This study found that VCN-01, a new generation genetically modified oncolytic adenovirus, administered locally or systemically, had a potent antisarcoma effect in vitro and in vivo in mouse models of intratibial and lung metastatic osteosarcoma. Moreover, VCN-01 administration showed a safe toxicity profile. Conclusions: These results uncover VCN-01 as a promising strategy for osteosarcoma, setting the bases to propel a phase I/II trial for kids with this disease. Clin Cancer Res; 22(9); 2217–25. ©2015 AACR.

Glioblastoma recurrence after treatment with the anti-vascular endothelial growth factor (VEGF) agent bevacizumab is characterized by a highly infiltrative and malignant behavior that renders surgical excision and chemotherapy ineffective. Our group has previously reported that Tie2-expressing monocytes (TEMs) are aberrantly present at the tumor/normal brain interface after anti-VEGF therapies and their significant role in the invasive outgrowth of these tumors. Here, we aimed to further understand the mechanisms leading to this pro-invasive tumor microenvironment. Examination of a U87MG xenogeneic glioma model and a GL261 murine syngeneic model showed increased tumor expression of angiopoietin 2 (Ang2), a natural ligand of Tie2, after anti-angiogenesis therapies targeting VEGF or VEGF receptor (VEGFR), as assessed by immunohistochemical analysis, immunofluorescence analysis, and enzyme-linked immunosorbent assays of tumor lysates. Migration and gelatinolytic assays showed that Ang2 acts as both a chemoattractant of TEMs and an enhancing signal for their tumor-remodeling properties. Accordingly, in vivo transduction of Ang2 into intracranial gliomas increased recruitment of TEMs into the tumor. To reduce invasive tumor outgrowth after anti-angiogenesis therapy, we targeted the Ang-Tie2 axis using a Tie2 decoy receptor. Using syngeneic models, we observed that overexpression of soluble Tie2 within the tumor prevented the recruitment of TEMs to the tumor and the development of invasion after anti-angiogenesis treatment. Taken together, these data indicate an active role for the Ang2-Tie2 pathway in invasive glioma recurrence after anti-angiogenesis treatment and provide a rationale for testing the combined targeting of VEGF and Ang-Tie2 pathways in patients with glioblastoma.

Pediatric diffuse midline gliomas-H3-K27M-mutant are aggressive brain tumours that arise during childhood. Despite new advances in genomic knowledge and the significant number of clinical trial testing new targeted therapies, patient outcome is still insufficient. Cancer immunotherapy is opening new therapeutic options representing a hope for this orphan disease. Aptamers are single-stranded nucleic acid ligands design to achieve a remarkable affinity and specificity to their targets, comparable to antibodies. TIM-3, is a potential immune checkpoint target, typically involved in T-cell exhaustion. Recent studies showed that TIM-3 is also expressed in tumour and glial cells and it plays an important role in brain tumour responses mediated by myeloid cells. In this work, we examined the anti-tumour effect of an aptamer against TIM-3 alone or in combination with radiotherapy. Of importance, we tested TIM-3 aptamer in a murine glioma and DIPG model, where we not observed any toxicity. TIM-3 administration increased overall survival but was unable to control the disease. Of importance, TIM-3 combination with radiotherapy improved the overall survival of treated mice when compared with single treatments leading to 50% of long-term survivors. TIM-3 aptamer administration increase T-infiltration in the tumour site compared to non-treated or library control. Mechanistic studies performed on day 16 showed an increase in CD8 effector cells, a decrease in T-regulators Foxp3+ cells and an increase in IFN-gamma expression suggesting the triggering of an antitumor-immune response. Rechallenge experiments demonstrated immune memory in the long-term responders that led to reject tumour re-implantation, confirming that TIM-3 aptamer treatment in combination with RT elicits specific antitumor immunity in mouse glioma models. These results suggest that immuno-therapies approaches in combination with radiotherapy would be worth exploring in the treatment of deadly DMG-H3K27-Mutant tumours.

Despite our increased understanding of Diffuse Intrinsic Pontine Glioma (DIPG) the outcome remains dismal. Recently we showed that the virus Delta-24-RGD (DNX-2401 in the clinic) was effective in preclinical models of DIPG and had the ability to trigger an antitumor immune response. These data allowed us to propel a phase I clinical trial for newly diagnosed DIPGs (NCT03178032) where the patients received an intratumoral viral injection followed by standard radiotherapy. The main objective is to determine the safety, tolerability, and toxicity of DNX-2401. Secondary endpoints are overall survival at 12 months, percentage of responses and induced immune response against tumor. Tumor biopsy was performed through the cerebellar peduncle, followed by intratumoral injection of DNX-2401 (N=12). Three patients were treated with the D1=1x1010vp and because the lack of toxicity we escalated to the D2= 5x1010vp. The procedure was well tolerated and safe. All patients displayed a reduced tumor volume after combined treatment. We performed molecular studies in 9 out of the 12 patients. The immune cell composition of the biopsies was assessed using multiplexed quantitative immunofluorescence. T cells were hardly noticeable in these tumors while macrophages were abundant. We detected increased clonal T cell diversity following treatment with virus in peripheral blood lymphocytes when compared paired pre- and post-treatment samples from the trial. In addition, we measure pre and post treatment neutralizing antibodies and its relationship with survival. Finally, we performed functional studies using 2 cell lines isolated from patients included in this trial to assess the response to the virus (infectivity, viability, T-cell recognition). Overall, the administration of DNX2401 was safe, feasible and therapeutically beneficial in a subgroup of patients. This trial constitutes a proof of principle that aids to understand the response of DIPGs to viral therapies allowing to set the bases to improve this strategy for DIPG.

Mature B-cell non-Hodgkin lymphoma (B-NHL) constitutes a group of heterogeneous malignant lymphoproliferative diseases ranging from indolent to highly aggressive forms. Although the survival after chemo-immunotherapy treatment of mature B-NHL has increased over the last years, many patients relapse or remain refractory due to drug resistance, presenting an unfavorable prognosis. Hence, there is an urgent need to identify new prognostic markers and therapeutic targets. Podocalyxin (PODXL), a sialomucin overexpressed in a variety of tumor cell types and associated with their aggressiveness, has been implicated in multiple aspects of cancer progression, although its participation in hematological malignancies remains unexplored. New evidence points to a role for PODXL in mature B-NHL cell proliferation, survival, migration, drug resistance, and metabolic reprogramming, as well as enhanced levels of PODXL in mature B-NHL. Here, we review the current knowledge on the contribution of PODXL to tumorigenesis, highlighting and discussing its role in mature B-NHL progression.

Background Osteosarcoma is the most common malignant bone tumor in children and young adults that produces aberrant osteoid. The aim of this study was to assess the utility of 2-deoxy-2-[18F-] fluoro-D-glucose ([18F] FDG) and sodium [18F] Fluoride (Na [18F] F) PET scans in orthotopic murine models of osteosarcoma to describe the metabolic pattern of the tumors, to detect and diagnose tumors and to evaluate the efficacy of a new treatment based in oncolytic adenoviruses. Methods Orthotopic osteosarcoma murine models were created by the injection of 143B and 531MII cell lines. [18F]FDG and Na [18F] F PET scans were performed 30 days (143B) and 90 days (531MII) post-injection. The antitumor effect of two doses (107 and 108 pfu) of the oncolytic adenovirus VCN-01 was evaluated in 531 MII model by [18F] FDG PET studies. [18F] FDG uptake was quantified by SUVmax and Total Lesion Glycolysis (TLG) indexes. For Na [18F] F, the ratio tumor SUVmax/hip SUVmax was calculated. PET findings were confirmed by histopathological techniques. Results The metabolic pattern of tumors was different between both orthotopic models. All tumors showed [18F] FDG uptake, with a sensitivity and specificity of 100%. The [18F] FDG uptake was significantly higher for the 143B model (p

The identification of circulating biomarkers by non-invasive methods would be helpful for glioma detection and response assessment. Strong evidences have shown that GBM cells release microvesicles containing proteins and RNA. We have previously demonstrated that exosomes isolated from the serum of GBM patients had an increased expression of RNU6-1 compared to control samples; therefore it could serve as a non-invasive diagnostic biomarker for GBM. In this study, we set to investigate the role of RNU6-1 as a differential biomarker of GBM versus other brain diseases with similar radiological features. RNU6-1 expression was analysed by digital droplet PCR (ddPCR) in circulating exosomes from serum samples of GBM patients (n=18), healthy controls (n=28), and patients with different brain lesions: subacute stroke (n=30), acute-subacute haemorrhage (n=29), acute demyelinating lesions (n=19), brain metastases (n=21) and Primary CNS Lymphomas (PCNSL) (n=12). We observed that the expression of RNU6-1 was significantly higher in GBM patients (412 ± 550.48 copies/20µL) than in healthy controls (150 ± 224.35 copies/20µL; p=0.039) validating our preceding results. Furthermore, RNU6-1 levels were increased in exosomes from GBM patients than in exosomes from patients with non-neoplastic lesions (stroke [223 ± 709.8 copies/20µL; p=0,067], haemorrhage [127 ± 198.7 copies/20µL; p=0.010], demyelinating lesions [111.5 ± 250.35 copies/20µL; p=0.019]) and PCNSL [18.15 ± 245.7 copies/20µL; p=0.004]). Contrary, RNU6-1 levels were similar between brain metastases and GBM patients [325 ± 632 copies/20µL; p=0.573]. In addition, assessing RNU6-1 as a predictive marker of GBM by ROC curves analysis, we demonstrated that RNU6-1 was a robust diagnostic biomarker of GBM compared to subacute stroke [AUC=0.659; p=0.004], acute/subacute haemorrhage [AUC=0.724; p=0.006], acute demyelinating lesions [AUC=0.728; p=0.011] and PCNSL [AUC=0.814; p

In recent years, we have seen an important progress in our comprehension of the molecular basis of pediatric brain tumors (PBTs). However, they still represent the main cause of death by disease in children. Due to the poor prognosis of some types of PBTs and the long-term adverse effects associated with the traditional treatments, oncolytic viruses (OVs) have emerged as an interesting therapeutic option since they displayed safety and high tolerability in pre-clinical and clinical levels. In this review, we summarize the OVs evaluated in different types of PBTs, mostly in pre-clinical studies, and we discuss the possible future direction of research in this field. In this sense, one important aspect of OVs antitumoral effect is the stimulation of an immune response against the tumor which is necessary for a complete response in preclinical immunocompetent models and in the clinic. The role of the immune system in the response of OVs needs to be evaluated in PBTs and represents an experimental challenge due to the limited immunocompetent models of these diseases available for pre-clinical research.

Diffuse intrinsic pontine gliomas (DIPGs) are aggressive glial brain tumors that primarily affect children, for which there is no curative treatment. Median overall survival is only one year. Currently, the scientific focus is on expanding the knowledge base of the molecular biology of DIPG, and identifying effective therapies. Oncolytic adenovirus DNX-2401 is a replication-competent, genetically modified virus capable of infecting and killing glioma cells, and stimulating an anti-tumor immune response. Clinical trials evaluating intratumoral DNX-2401 in adults with recurrent glioblastoma have demonstrated that the virus has a favorable safety profile and can prolong survival. Subsequently, these results have encouraged the transition of this biologically active therapy from adults into the pediatric population. To this aim, we have designed a clinical Phase I trial for newly diagnosed pediatric DIPG to investigate the feasibility, safety, and preliminary efficacy of delivering DNX-2401 into tumors within the pons following biopsy. This case report presents a pediatric patient enrolled in this ongoing Phase I trial for children and adolescents with newly diagnosed DIPG. The case involves an 8-year-old female patient with radiologically diagnosed DIPG who underwent stereotactic tumor biopsy immediately followed by intratumoral DNX-2401 in the same biopsy track. Because there were no safety concerns or new neurological deficits, the patient was discharged 3 days after the procedures. To our knowledge, this is the first report of intratumoral DNX-2401 for a patient with DIPG in a clinical trial. We plan to demonstrate that intratumoral delivery of an oncolytic virus following tumor biopsy for pediatric patients with DIPG is a novel and feasible approach and that DNX-2401 represents an innovative treatment for the disease.

Gliomas are the most common and aggressive brain tumors and despite advances in their therapeutic management they remain fatal. Radically, different therapeutic approaches are needed to treat this aggressive disease. Oncolytic viruses that intrinsically kill cancer cells or that have been genetically engineered to specifically destroy this population are becoming a viable therapeutic options for gliomas. A number of preclinical studies have demonstrated the efficacy of oncolytic viruses as a therapeutic strategy for gliomas. Those studies have led to a number of clinical trials using oncolytic virus for glioma treatment. In this chapter, we review the different viruses (adenovirus, herpes virus, parvovirus, poliovirus, reovirus, newcastle disease virus, retrovirus, and measles viruses) that have been evaluated preclinically and clinically for the treatment of glioma.

Purpose DNX-2401 (Delta-24-RGD; tasadenoturev) is a tumor-selective, replication-competent oncolytic adenovirus. Preclinical studies demonstrated antiglioma efficacy, but the effects and mechanisms of action have not been evaluated in patients. Methods A phase I, dose-escalation, biologic-end-point clinical trial of DNX-2401 was conducted in 37 patients with recurrent malignant glioma. Patients received a single intratumoral injection of DNX-2401 into biopsy-confirmed recurrent tumor to evaluate safety and response across eight dose levels (group A). To investigate the mechanism of action, a second group of patients (group B) underwent intratumoral injection through a permanently implanted catheter, followed 14 days later by en bloc resection to acquire post-treatment specimens. Results In group A (n = 25), 20% of patients survived > 3 years from treatment, and three patients had a ≥ 95% reduction in the enhancing tumor (12%), with all three of these dramatic responses resulting in > 3 years of progression-free survival from the time of treatment. Analyses of post-treatment surgical specimens (group B, n = 12) showed that DNX-2401 replicates and spreads within the tumor, documenting direct virus-induced oncolysis in patients. In addition to radiographic signs of inflammation, histopathologic examination of immune markers in post-treatment specimens showed tumor infiltration by CD8+ and T-bet+ cells, and transmembrane immunoglobulin mucin-3 downregulation after treatment. Analyses of patient-derived cell lines for damage-associated molecular patterns revealed induction of immunogenic cell death in tumor cells after DNX-2401 administration. Conclusion Treatment with DNX-2401 resulted in dramatic responses with long-term survival in recurrent high-grade gliomas that are probably due to direct oncolytic effects of the virus followed by elicitation of an immune-mediated antiglioma response.

Delta-24-RGD (DNX-2401 in the clinic) has been tested for adult glioblastoma presenting a safe profile and promising efficacy. Our group has showed that the virus is safe and effective in preclinical models of pHGG and DIPG. Moreover, we showed that the virus is able to trigger an antitumor immune response. These results allowed us to propel a phase I clinical trial for newly diagnosed DIPGs (NCT03178032) where the patients received an intratumoral injection of DNX-2401 (N=12). Tumor biopsy is performed through the cerebellar peduncle, followed by virus injection using a cannula that prevents the reflux. The trial is uncontrolled, unicentric with a 3 + 3 design. The objective of this trial is to determine the safety, tolerability, and toxicity of DNX-2401 in subjects with DIPG. Secondary endpoints are overall survival at 12 months (OS12), percentage of responses and induced immune response against tumor. To date 9 patients have been treated within the trial. Three patients were treated with the D1=1x1010vp and because the lack of toxicity we escalated to the D2= 5x1010vp. The procedure was well tolerated and safe. Patients were home 3–4 days after the injection. All the patients displayed a reduced tumor volume after combined treatment. We performed molecular studies in 8 out of the 9 patients (RNAseq and a thermofisher pediatric panel). Subsequently we evaluated the immune cell composition in the tumor using multiplexed quantitative immunofluorescence on the biopsies pre-virus injection. T cells were hardly noticeable in these tumors while macrophages were abundant. Using a multiplexed TCR-sequencing mRNA-based assay to analyze 18 available paired pre- and post-treatment samples from the trial, we detected increased clonal T cell diversity following treatment with virus. In addition, we are assessing the existence of pre and post treatment neutralizing antibodies and its relationship with survival. Finally, we have performed functional studies using 2 cell lines isolated from patients included in this trials and confronting them with T-cells isolated from peripheral blood of the same patients before and after the treatment with the virus. Information acquired within this clinical study would aid to understand the response of DIPGs to viral therapies and therefore to better tailor this strategy to improve the survival of pediatric brain tumors.

BACKGROUND Glioblastoma (GBM) and Alzheimer’s disease (AD) are frequent among the elderly: about 50% of all GBM patients are 65 years or older, and 5–10% of people above this age threshold suffer from AD. MATERIAL AND METHODS We describe the case of an elderly patient with newly diagnosed GBM who developed a rapidly progressive severe dementia immediately following concomitant radio-chemotherapy. A purposed retrospective analysis of the surgical sample revealed Alzheimer pathology. RESULTS A 69-year old woman with no relevant past-medical history was diagnosed with a right temporo-parietal GBM on May 2017, with wild-type IDH1R132H gene and methylated MGMTP. After complete resection she received radiotherapy (60 Gy in 30 fractions) and concomitant chemotherapy with Temozolomide 75 mg/m2/day. During the last weeks of concomitant radio-chemotherapy and further on the patient developed a progressive neurological worsening with severe cognitive dysfunction, gait impairment and sphincter incontinence, becoming severely disabled and requiring continuous assistance. Brain MRI exams showed no signs of tumor recurrence, with focal white matter changes consisting on T2/FLAIR hyperintensities within the radiation field, enlarged sulci and ventriculomegaly. Underlying infectious and metabolic disorders, non-convulsive epileptic status and normal-pressure hydrocephalus were ruled out. Facing this situation the patient relatives were purposely asked for previous cognitive symptoms, and described very mild complaints of short-term memory loss during the previous year. Aiming at investigating the possibility of an underlying AD, focused examination of the surgical sample revealed the presence of frequent neuritic plaques, a neuropathological hallmark of AD. The patient developed a severe dementia with akinetic mutism by September 2017. Without evidence of tumor recurrence despite lack of adjuvant chemotherapy, she died of aspiration pneumonia on January 2019. CONCLUSION A unique retrospective study including elderly GBM patients treated only with surgery revealed that Alzheimer disease pathology was present in 42% of them. This case illustrates the need for actively addressing the previous cognitive status of elderly patients with GBM. Histopathological assessment of Alzheimer pathology might also be considered in this specific population. In positive cases, particularly in those with methylated MGMTP, radiotherapy should be avoided and treatment alone with Temozolomide might be considered.