Mapping uncharted territory: a gene expression signature for precision glioblastoma therapeuticsSkinner, Kasey, R;Nabors, L, Burt;Miller, C, Ryan
doi: 10.1093/neuonc/noaa242pmid: 33257985
Diffusely invasive glioma, the most common and malignant primary brain tumor, comprises a diverse group of diseases. These tumors range from lower-grade astrocytomas and oligodendrogliomas (grades II and III) to the deadly glioblastoma (GBM, grade IV) when diagnosed based on the current World Health Organization (WHO) classification.1 GBM has historically been treated as a single disease on the basis of histological criteria; however, advances in molecular technology and the advent of next-generation sequencing have revealed significant heterogeneity both within and among tumors. In 1999, the NCI Director’s Challenge asked “the scientific community to harness the power of comprehensive molecular analysis technologies to make the classification of tumors vastly more informative.” In response, The Cancer Genome Atlas (TCGA) and others identified mutational drivers in GBM and classified tumors into subtypes according to gene expression. Alterations in tumor suppressors such as CDKN2A, TP53, and PTEN and oncogenes including EGFR were among those most frequently found, delineating signaling pathways commonly affected in this disease.2 Additionally, tumors harboring point mutations in the genes encoding isocitrate dehydrogenase (IDH1 or IDH2) were found to be associated with longer survival than their IDH wild-type (IDHwt) counterparts.3 Because of these newly discovered genomic alterations and their implications for the clinic, the 2016 WHO classification of central nervous system tumors was historic in that it included both molecular and histological criteria for the diagnosis of GBM.1 IDH mutational status is now routinely used to classify GBM for both disease management and clinical trial enrollment. Thus, dividing GBM into subtypes according to genomic alterations is useful for both rational treatment design and explaining variability in patient response to therapy. Additional stratification based on transcriptome signatures holds promise for further improving care. To that end, in this issue of Neuro-Oncology, Johnson et al identify a novel prognostic transcriptome signature that classifies IDHwt GBM by predicted patient survival. Despite comprising the largest group of GBM and having poor prognosis, IDHwt tumors lack biomarkers that are predictive of overall survival. Although the Phillips and TCGA gene expression subtypes correlate with biological and clinical characteristics, they have not been shown to have prognostic value in samples containing only IDHwt GBM or in clinical trial cohorts.4,5 One biomarker that has been validated for prediction of overall survival in IDHwt GBM is O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation, which is associated with both better response to standard-of-care temozolomide and radiation therapy and longer overall survival than tumors with unmethylated MGMT promoters.6 However, stratifying patients by MGMT methylation status still yields variability in response to treatment and survival, suggesting that additional biomarkers and improved classification schemes are needed. Johnson et al aimed to develop such a biomarker for IDHwt tumors by identifying a prognostic gene expression signature using a NanoString platform.7 This study assessed gene expression from 512 formalin-fixed, paraffin-embedded (FFPE) tumor samples from newly diagnosed IDHwt glioma patients enrolled in the ARTE, TAMIGA, and EORTC 26101 clinical trials (collectively the ATE trials) to identify a prognostic signature of 9 genes from different biological pathways, referred to as the ATE score. This score predicted overall patient survival in both the ATE cohort and the independent validation cohorts comprising samples from the AvaGlio trial, the GLARIUS MGMT nonmethylated GBM trial, and a retrospective UCLA cohort. Excitingly, the prognostic value of the ATE score was not diminished after adjusting for clinical variables and MGMT promoter methylation status, suggesting that this signature represents valuable, independent prognostic information. This study has generated a novel platform for stratifying IDHwt GBM based on predicted patient survival, with great potential to enhance both therapeutic decision making and clinical trial design. The use of NanoString adds to the clinical relevance of the ATE signature because it allows the assessment of the 9 signature genes using FFPE tumor samples. Indeed, utility of this biomarker technology has been validated and is routinely used in breast cancer. Further, while patients are already stratified in trials by MGMT promoter methylation status, the ATE score represents an additional prognostic metric by which IDHwt GBM can be classified. For example, in the recent Checkmate 143 trial of programmed cell death 1 checkpoint inhibition with nivolumab in recurrent GBM, patients treated with nivolumab showed no improved survival benefit over those treated with bevacizumab. MGMT promoter methylation status was not significantly different among treatment groups; however, retrospectively comparing ATE score could be useful to determine whether lack of benefit from nivolumab could be partially explained by a disproportionate number of poor ATE prognosis patients.8 It would also be interesting to see whether the ATE score maintains its prognostic value in molecular GBM (which does not meet the histologic criteria for GBM but harbors GBM-like mutations), as well as in the setting of recurrent IDHwt GBM.9 Some questions remain as to how the new ATE may be implemented clinically. Gene expression is heterogeneous within a single GBM, with all 4 TCGA subtypes sometimes found in the same tumor.10 Therefore, it is unclear how the ATE score varies within a tumor based on regional sampling. Johnson et al began to explore this question by analyzing single-cell RNA-seq data and found that the fraction of cells expressing ATE signature genes was highest in tumor cells relative to other cell types in the microenvironment. However, this approach does not address the varied morphological and regional “biomes” found in IDHwt GBM.10 Clinical implementation will also require prospective validation of the ATE score as an inclusion criterion or retrospective validation as a secondary endpoint in clinical trials. This work represents the first gene expression biomarker that can successfully classify the otherwise largely uncharted territory of IDHwt GBM and is very likely to be clinically useful in developing therapies to better treat these devastating tumors. It also serves as a reminder for the field that GBM is not one disease, but many, and that this heterogeneity must be a foremost consideration in attempting to understand and treat it. Conflict of interest statement. None. References 1. Louis DN , Perry A, Reifenberger G, et al. The 2016 World Health Organization classification of tumors of the central nervous system: a summary . Acta Neuropathol. 2016 ; 131 ( 6 ): 803 – 820 . Google Scholar Crossref Search ADS PubMed WorldCat 2. Brennan CW , Verhaak RG, McKenna A, et al. ; TCGA Research Network. The somatic genomic landscape of glioblastoma . Cell. 2013 ; 155 ( 2 ): 462 – 477 . Google Scholar Crossref Search ADS PubMed WorldCat 3. Parsons DW , Jones S, Zhang X, et al. An integrated genomic analysis of human glioblastoma multiforme . Science. 2008 ; 321 ( 5897 ): 1807 – 1812 . Google Scholar Crossref Search ADS PubMed WorldCat 4. Verhaak RG , Hoadley KA, Purdom E, et al. ; The Cancer Genome Atlas Research Network. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1 . Cancer Cell. 2010 ; 17 ( 1 ): 98 – 110 . Google Scholar Crossref Search ADS PubMed WorldCat 5. Phillips HS , Kharbanda S, Chen R, et al. Molecular subclasses of high-grade glioma predict prognosis, delineate a pattern of disease progression, and resemble stages in neurogenesis . Cancer Cell. 2006 ; 9 ( 3 ): 157 – 173 . Google Scholar Crossref Search ADS PubMed WorldCat 6. Hegi ME , Diserens AC, Gorlia T, et al. MGMT gene silencing and benefit from temozolomide in glioblastoma . N Engl J Med. 2005 ; 352 ( 10 ): 997 – 1003 . Google Scholar Crossref Search ADS PubMed WorldCat 7. Johnson RM , Phillips HS, Bais C, et al. Development of a gene expression-based prognostic signature for IDH wild-type glioblastoma . Neuro Oncol. 2020 . In press. doi: 10.1093/neuonc/noaa157. Google Scholar OpenURL Placeholder Text WorldCat 8. Reardon DA , Brandes AA, Omuro A, et al. Effect of nivolumab vs bevacizumab in patients with recurrent glioblastoma: the CheckMate 143 phase 3 randomized clinical trial . JAMA Oncol. 2020 ; 6 ( 7 ): 1003 – 1010 . Google Scholar Crossref Search ADS PubMed WorldCat 9. Brat DJ , Aldape K, Colman H, et al. cIMPACT-NOW update 3: recommended diagnostic criteria for “Diffuse astrocytic glioma, IDH-wildtype, with molecular features of glioblastoma, WHO grade IV” . Acta Neuropathol. 2018 ; 136 ( 5 ): 805 – 810 . Google Scholar Crossref Search ADS PubMed WorldCat 10. Prabhu A , Kesarwani P, Kant S, Graham SF, Chinnaiyan P. Histologically defined intratumoral sequencing uncovers evolutionary cues into conserved molecular events driving gliomagenesis . Neuro Oncol. 2017 ; 19 ( 12 ): 1599 – 1606 . Google Scholar Crossref Search ADS PubMed WorldCat © The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: [email protected] This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
Radiotherapy innovations to optimize brain metastases controlGondi,, Vinai;Mehta, Minesh, P
doi: 10.1093/neuonc/noaa244pmid: 33089325
brain metastases, hippocampal avoidance, whole-brain radiotherapy Radiotherapeutic management of brain metastases has improved considerably over the past decade with several practice-changing advances. Technologic improvements in the stereotactic non-invasive delivery of focal high-dose radiotherapy to multiple macrometastases have provided efficient approaches to providing durable long-term local control and preventing symptomatic progression. Incorporation of neuroprotective approaches of hippocampal avoidance1 and prophylactic memantine2 has led to safer delivery of whole-brain radiotherapy (WBRT) to control micrometastatic disease in the brain. The judicious and complementary utilization of these radiotherapy innovations yields the most optimal intracranial control available to brain metastasis patients irrespective of underlying histology, as illustrated in the manuscript by Westover et al.3 The authors report on a single-arm phase II trial, which treated 50 brain metastasis patients with hippocampal-avoidant (HA) WBRT to 20 Gy in 10 fractions with simultaneous integrated boost (SIB) to macrometastatic disease to 40 Gy in 10 fractions. They report intracranial control comparable to modern series of sequential WBRT plus stereotactic radiosurgery (SRS), better neurocognitive outcomes compared with historical trials of conventional WBRT, and an acceptable toxicity profile. Specifically, HA-WBRT + SIB led to one-year rates of 91% local control of boosted macrometastatic disease and 87.5% distant brain control. These are dramatic and durable intracranial control rates, not achieved with either radiosurgery alone or whole brain radiotherapy alone. Table 1 demonstrates the comparability of these outcomes with prior prospective trials of combined WBRT and SRS. Interestingly, the high distant brain control rate comparable to historical series of WBRT was achieved in spite of an approximate 40% reduction in biologically equivalent dose for the HA-WBRT component. In fact, the dose of 20 Gy in 10 fractions represents a 23% reduction in biologically equivalent dose relative to doses commonly employed for prophylactic cranial irradiation. Thus, the results of this phase II trial suggest that a lower radiotherapy dose may be sufficient to durably sterilize micrometastatic brain disease. Table 1. Summary of intracranial control following WBRT + SRS or SIB Study . Treatment . 1-Year Outcomes . . . . Local Control . Distant Brain Control . RTOG 95–084 WBRT 71% 67% WBRT + SRS 82% 73% EORTC 229525 SRS 70% 56% WBRT + SRS 87% 72% MDACC6 SRS 67% 45% WBRT + SRS 100% 73% JROSG-99–17 SRS 76% 37% WBRT + SRS 90% 58% Alliance N05748 SRS 73% 70% WBRT + SRS 90% 92% Summary of WBRT + SRS Arms 82–100% 58–92% Popp et al9 HA-WBRT 30 Gy/12 fx + SIB 51Gy/42Gy/12 fx for metastases/resection cavities 98% 69% Westover et al3 HA-WBRT 20 Gy/10 fx + SIB 40 Gy/10 fx for metastases 91% 87% Study . Treatment . 1-Year Outcomes . . . . Local Control . Distant Brain Control . RTOG 95–084 WBRT 71% 67% WBRT + SRS 82% 73% EORTC 229525 SRS 70% 56% WBRT + SRS 87% 72% MDACC6 SRS 67% 45% WBRT + SRS 100% 73% JROSG-99–17 SRS 76% 37% WBRT + SRS 90% 58% Alliance N05748 SRS 73% 70% WBRT + SRS 90% 92% Summary of WBRT + SRS Arms 82–100% 58–92% Popp et al9 HA-WBRT 30 Gy/12 fx + SIB 51Gy/42Gy/12 fx for metastases/resection cavities 98% 69% Westover et al3 HA-WBRT 20 Gy/10 fx + SIB 40 Gy/10 fx for metastases 91% 87% Open in new tab Table 1. Summary of intracranial control following WBRT + SRS or SIB Study . Treatment . 1-Year Outcomes . . . . Local Control . Distant Brain Control . RTOG 95–084 WBRT 71% 67% WBRT + SRS 82% 73% EORTC 229525 SRS 70% 56% WBRT + SRS 87% 72% MDACC6 SRS 67% 45% WBRT + SRS 100% 73% JROSG-99–17 SRS 76% 37% WBRT + SRS 90% 58% Alliance N05748 SRS 73% 70% WBRT + SRS 90% 92% Summary of WBRT + SRS Arms 82–100% 58–92% Popp et al9 HA-WBRT 30 Gy/12 fx + SIB 51Gy/42Gy/12 fx for metastases/resection cavities 98% 69% Westover et al3 HA-WBRT 20 Gy/10 fx + SIB 40 Gy/10 fx for metastases 91% 87% Study . Treatment . 1-Year Outcomes . . . . Local Control . Distant Brain Control . RTOG 95–084 WBRT 71% 67% WBRT + SRS 82% 73% EORTC 229525 SRS 70% 56% WBRT + SRS 87% 72% MDACC6 SRS 67% 45% WBRT + SRS 100% 73% JROSG-99–17 SRS 76% 37% WBRT + SRS 90% 58% Alliance N05748 SRS 73% 70% WBRT + SRS 90% 92% Summary of WBRT + SRS Arms 82–100% 58–92% Popp et al9 HA-WBRT 30 Gy/12 fx + SIB 51Gy/42Gy/12 fx for metastases/resection cavities 98% 69% Westover et al3 HA-WBRT 20 Gy/10 fx + SIB 40 Gy/10 fx for metastases 91% 87% Open in new tab Popp et al9 reported similar results in a recently published single-institution feasibility trial of HA-WBRT + SIB: HA-WBRT 30 Gy in 12 fractions, SIB 51 Gy/42 Gy in 12 fractions for macrometastases/surgical cavities. The neurocognitive function component of this approach is being evaluated further in HIPPORAD,10 a randomized phase II trial of HA-WBRT + SIB versus WBRT + SIB for patients with at least 4 brain macrometastases (but not more than 10) and at least 1 macrometastasis ≥5 mm (none within 7 mm of the hippocampus). One of the limitations of the phase II trial reported by Westover et al3 is that the historical comparator employed conventional WBRT to 30 Gy in 10 fractions as opposed to the 20 Gy in 10 fractions HA-WBRT. This confounds the historical comparison of neurocognitive results. However, the practice-changing neurocognitive and patient-reported symptom benefits of hippocampal avoidance added to WBRT plus memantine have already been established by the phase III trial NRG Oncology CC001.1 Another important consideration of the phase II trial reported by Westover et al3 is the eligibility limit of a maximum number of 8 brain metastases. In our experience, limiting the hippocampal dose in HA-WBRT with SIB techniques is highly dependent on the number, size, and location of macrometastases, as well as the technical details of treatment planning. Therefore, broad generalizability of these results cannot be assumed, and sequential HA-WBRT and SRS could be considered as an alternative. As advances in systemic therapy improve the survival of brain metastases patients, the question of when to use HA-WBRT in combination with SIB or SRS to provide optimal brain metastasis control becomes even more salient. This question will be addressed by NRG Oncology BN009, a phase III trial of salvage SRS versus salvage HA-WBRT plus SRS for first or second distant brain relapse after upfront SRS with brain metastasis velocity (BMV) of 4 or more brain metastases per year. This trial is based on prior data from a cohort of 737 brain metastasis patients reported by Farris et al11 who observed that BMV at first or second distant brain relapse after upfront SRS predicted overall survival. In a larger validation dataset of >2000 brain metastasis patients from 9 institutions, BMV remained prognostic with nearly identical median survival outcomes. Specifically, patients who had a BMV ≥4 brain metastases/year had a 7-month shortening in median survival as compared with patients with BMV <4 brain metastases/year (P < 0.0001). BMV at first distant brain relapse was also predictive of BMV at second distant brain relapse, highlighting the ability of BMV to serve as a surrogate marker for intracranial control. The prognostic value of BMV has since been validated in 2 additional published series.12,13 Further, BMV at first or second distant brain relapse after upfront SRS predicted for neurologic death following salvage SRS.11 Patients with BMV ≥4 brain metastases/year were nearly 2-fold more likely to suffer neurologic death than patients with BMV <4 brain metastases/year. A recent analysis of brain metastasis patients treated with SRS in the immunotherapy era confirmed that BMV remained prognostic for both overall survival and neurologic death, with >7-fold increased risk of neurologic death in patients with BMV ≥4 brain metastases/year (P = 0.005).14 The summation of these findings underscores the capacity of BMV following upfront SRS to distinguish a subset of patients (BMV ≥4 brain metastases/y) for whom optimizing intracranial control with combined HA-WBRT plus SRS may prevent neurologic death from being a primary contributor to survival. NRG Oncology BN009, scheduled to activate in winter 2020, will test this hypothesis. The reimbursement landscape for Medicare patients is anticipated to change to a fixed payment model for an “episode of care,” which amounts to an almost 4-month period of time during which only one predefined amount for all care would be provided. Whether such a change on the reimbursement landscape motivates providers to shift from a “treat/image/re-treat” approach as seen with SRS followed by repeat SRS, to broader utilization of HA-WBRT plus SIB or SRS remains to be seen. In summary, the phase II trial results reported by Westover et al are an important contribution to our understanding of how recent radiotherapy innovations, when combined together, provide a safe and effective approach to optimizing brain metastases control. Acknowledgment The text is the sole product of the authors and no third party had input or gave support to its writing. References 1. Brown PD , Gondi V, Pugh S, et al. ; for NRG Oncology. Hippocampal avoidance during whole-brain radiotherapy plus memantine for patients with brain metastases: Phase III trial NRG oncology CC001 . J Clin Oncol. 2020 ; 38 ( 10 ): 1019 – 1029 . Google Scholar Crossref Search ADS PubMed WorldCat 2. Brown PD , Pugh S, Laack NN, et al. ; Radiation Therapy Oncology Group (RTOG). Memantine for the prevention of cognitive dysfunction in patients receiving whole-brain radiotherapy: a randomized, double-blind, placebo-controlled trial . Neuro Oncol. 2013 ; 15 ( 10 ): 1429 – 1437 . Google Scholar Crossref Search ADS PubMed WorldCat 3. Westover KD , Mendel JT, Dan T, et al. Phase II trial of Hippocampal-Sparing Whole Brain Irradiation with Simultaneous Integrated Boost (HSIB-WBRT) for metastatic cancer . Neuro Oncol. 2020 . doi: 10.1093/neuonc/noaa092. Google Scholar OpenURL Placeholder Text WorldCat 4. Andrews DW , Scott CB, Sperduto PW, et al. Whole brain radiation therapy with or without stereotactic radiosurgery boost for patients with one to three brain metastases: phase III results of the RTOG 9508 randomised trial . Lancet. 2004 ; 363 ( 9422 ): 1665 – 1672 . Google Scholar Crossref Search ADS PubMed WorldCat 5. Kocher M , Soffietti R, Abacioglu U, et al. Adjuvant whole-brain radiotherapy versus observation after radiosurgery or surgical resection of one to three cerebral metastases: results of the EORTC 22952-26001 study . J Clin Oncol. 2011 ; 29 ( 2 ): 134 – 141 . Google Scholar Crossref Search ADS PubMed WorldCat 6. Chang EL , Wefel JS, Hess KR, et al. Neurocognition in patients with brain metastases treated with radiosurgery or radiosurgery plus whole-brain irradiation: a randomised controlled trial . Lancet Oncol. 2009 ; 10 ( 11 ): 1037 – 1044 . Google Scholar Crossref Search ADS PubMed WorldCat 7. Aoyama H , Shirato H, Tago M, et al. Stereotactic radiosurgery plus whole-brain radiation therapy vs stereotactic radiosurgery alone for treatment of brain metastases: a randomized controlled trial . JAMA. 2006 ; 295 ( 21 ): 2483 – 2491 . Google Scholar Crossref Search ADS PubMed WorldCat 8. Brown PD , Jaeckle K, Ballman KV, et al. Effect of radiosurgery alone vs radiosurgery with whole brain radiation therapy on cognitive function in patients with 1 to 3 brain metastases: a randomized clinical trial . JAMA. 2016 ; 316 ( 4 ): 401 – 409 . Google Scholar Crossref Search ADS PubMed WorldCat 9. Popp I , Rau S, Hintz M, et al. Hippocampus-avoidance whole-brain radiation therapy with a simultaneous integrated boost for multiple brain metastases . Cancer. 2020 ; 126 ( 11 ): 2694 – 2703 . Google Scholar Crossref Search ADS PubMed WorldCat 10. Grosu AL , Frings L, Bentsalo I, et al. Whole-brain irradiation with hippocampal sparing and dose escalation on metastases: neurocognitive testing and biological imaging (HIPPORAD)—a phase II prospective randomized multicenter trial (NOA-14, ARO 2015-3, DKTK-ROG) . BMC Cancer. 2020 ; 20 ( 1 ): 532 . Google Scholar Crossref Search ADS PubMed WorldCat 11. Farris M , McTyre ER, Cramer CK, et al. Brain metastasis velocity: a novel prognostic metric predictive of overall survival and freedom from whole-brain radiation therapy after distant brain failure following upfront radiosurgery alone . Int J Radiat Oncol Biol Phys. 2017 ; 98 ( 1 ): 131 – 141 . Google Scholar Crossref Search ADS PubMed WorldCat 12. Yamamoto M , Aiyama H, Koiso T, et al. Validity of a recently proposed prognostic grading index, brain metastasis velocity, for patients with brain metastasis undergoing multiple radiosurgical procedures . Int J Radiat Oncol Biol Phys. 2019 ; 103 ( 3 ): 631 – 637 . Google Scholar Crossref Search ADS PubMed WorldCat 13. Fritz C , Borsky K, Stark LS, et al. Repeated courses of radiosurgery for new brain metastases to defer whole brain radiotherapy: feasibility and outcome with validation of the new prognostic metric brain metastasis velocity . Front Oncol. 2018 ; 8 : 551 . Google Scholar Crossref Search ADS PubMed WorldCat 14. LeCompte M , Hughes R, Farris M, et al. Impact of salvage modality on neurologic death for distant brain failure after initial stereotactic radiosurgery . Int J Radiat Oncol. 2019 ; 105 : E79 - E80 . Google Scholar Crossref Search ADS WorldCat © The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: [email protected] This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
Penetrating the brain tumor space with DNA damage response inhibitorsBindra, Ranjit, S
doi: 10.1093/neuonc/noaa228pmid: 33059370
DNA repair, GBM, PARP inhibitors, pharmacodynamics, pharmacokinetics, Phase 0 DNA damage response (DDR) inhibitors have rapidly emerged in the clinic as promising anticancer agents for a wide range of tumor types (reviewed by Yap et al1). Poly(ADP)-ribose polymerase (PARP) inhibitors are the most well-known agents within this class, which are now FDA approved primarily for the treatment of selected cancers with defects in a key DNA double-strand break repair pathway, homologous recombination (HR). PARP inhibitors catalytically inhibit the function of PARP proteins, which play key roles in both base excision repair and single-strand break repair.2 A subset of PARP inhibitors, including olaparib, are thought to “trap” PARP at DNA lesions. PARP trapping activity appears to greatly enhance PARP inhibitor-induced DNA damage and cell killing, compared with drugs in this class which predominantly act via catalytic inhibition, such as veliparib.3 The interaction between PARP inhibitors and HR defects, arising most commonly from BRCA1 and BRCA2 mutations, is defined as a “synthetic lethal” interaction based on 2 seminal papers published in the early 2000s.4,5 In addition, PARP inhibitors are synergistic with certain DNA damaging agents such as topoisomerase inhibitors and monofunctional alkylating agents, such as temozolomide (TMZ),6 which suggests that these combinations can be efficacious independent of HR status. Silencing of O6-methylgaunine methyltransferase (MGMT) gene expression occurs commonly in glioblastoma multiforme (GBM), and loss of this DNA repair gene confers exquisite sensitivity to TMZ (reviewed by Gupta et al7). These findings have formed the basis for multiple clinical trials testing the safety and efficacy of PARP inhibitors and TMZ in many tumor types, including GBM (eg, NCT04166435, NCT02152982). In this issue of Neuro-Oncology, Hanna et al report on the results of a phase I trial testing the safety, tolerability and pharmacokinetics of olaparib and TMZ for recurrent GBM.8 In this study, adult patients with recurrent GBM were treated with a range of olaparib and TMZ dosing regiments to identify the recommended phase II dose (RP2D). A subset of patients received olaparib prior to surgical resection, and drug levels were measured in plasma, tumor core, and tumor margin by mass spectrometry. In parallel, a series of preclinical pharmacokinetic (PK) studies were performed to assess tissue distribution in rats and tumor-bearing mice in vivo, and the radiosensitizing effects of olaparib were measured in GBM cell lines using clonogenic survival assays in vitro. The latter was performed as a pharmacodynamic (PD) proxy, to define the levels of drug in tumor tissue which would be necessary for chemo/radiosensitization. This elegant study addresses a critical unmet need in GBM trials to rigorously evaluate drug levels in T1-postcontrast enhancing and non-enhancing areas of tumor, and to understand whether these levels are sufficient for antitumor activity. We recently convened an Adult Brain Tumor Consortium workshop to elucidate the key issues that must be addressed, in order to appropriately evaluate and further develop novel agents for brain tumors in clinical trials.9 As outlined in Fig. 1A, drug levels need to be measured and compared with plasma concentrations, preferably as the free and bound fractions. Equally important is to understand whether these intratumoral levels engage the intended target (Figure 1B) and whether the level of target engagement induces the expected or desired outcome (Figure 1C). These measurements should be performed in enhancing and non-enhancing areas of disease for glioma (when feasible) for 2 key reasons: (i) it has been argued that while drugs may penetrate enhancing areas of disease in a manner similar to that observed for gadolinium, this may not hold true for non-enhancing disease areas, in which a more intact blood–brain barrier (BBB) limits drugs permeability; and (ii) unlike enhancing disease, which typically is removed as part of a gross total resection, non-enhancing disease areas are not amenable to resection, and thus drug delivery in these areas is critical for durable tumor control (reviewed by Sarkaria et al10). Fig. 1 Open in new tabDownload slide Outline of the key features that must be addressed in brain tumor clinical trials: (A) BBB permeability; (B) target engagement; and (C) functional consequences of target engagement (created with BioRender.com). Fig. 1 Open in new tabDownload slide Outline of the key features that must be addressed in brain tumor clinical trials: (A) BBB permeability; (B) target engagement; and (C) functional consequences of target engagement (created with BioRender.com). In this study, the authors found that olaparib was detected in 71 of 71 tumor core specimens across 27 patients who received olaparib prior to a surgical resection. There was a relatively wide range of concentrations of drug found in these specimens, from 97 to 1374 nM, with a median of concentration of 496 nM. In order to assess drug penetration in non-enhancing areas of disease, tumor margin specimens were also obtained and analyzed in 9 patients, in which drug levels ranged 97–1237 nM (median, 512.3 nM). The mean ratio of brain tumor to plasma was highly variable in these studies (0.01–0.9), with a mean of 0.25. However, free versus bound olaparib levels were not measured in this study, which could provide a clearer picture of drug exposure in the tumor. Importantly, the authors also demonstrate in preclinical models that olaparib does not appear to penetrate the “undisturbed” BBB, using whole body autoradiography in rats, although the drug was detected in orthotopic (intracranial) GBM xenografts in mice. These findings highlight a rather disconcerting issue that preclinical modeling of BBB penetration animals may not always translate into humans, and thus better models are needed for more accurate predictions. Parallel in vitro studies with GBM cell lines revealed that doses of 100 and 500 nM were sufficient to induce radiosensitization in clonogenic survival assays, which suggests that the in situ concentrations in GBM samples in their clinical trial appear to be functionally relevant. The authors noted they were unable to develop a suitable PARP inhibitor PD assay, which formed the basis for using these parallel in vitro assays as a proxy. Previous studies have utilized a reduction in PARylation and an induction of key DDR markers, such as phosphorylated H2AX (γH2AX), as PD biomarkers for PARP inhibitor activity. However, these assays are highly dependent on the tumor tissue type, biopsy technique, and timing of the specimen acquisition, which can limit their widespread implementation in trials (reviewed by Wilsker et al11). Brain tumors represent an additional challenge for the development of PD assays: unlike extracranial solid tumor and liquid cancer trials, a baseline biopsy is rarely possible, such that target engagement and functional activity must be measured without an untreated control sample from the same patient. In addition, the work presented here provides insights into the potential and path forward for PARP inhibitor and DNA damaging agent combinations in GBM. PARP inhibitors, such as rucaparib, were originally developed as chemosensitizers for metastatic solid tumors in the early 2000s. However, dose-limiting toxicities (primarily myelosuppression) in the setting of modest efficacy limited their further clinical development at the time (reviewed by Curtin and Szabo2). The discovery that mutations in 2 HR genes, BRCA1 and BRCA2, confer an exquisite synthetic lethal interaction with PARP inhibitors revealed a strategy to address this apparent therapeutic index issue.4,5 This ultimately led to FDA approval of 4 PARP inhibitors as monotherapies for several types of cancers.2 As true HR defects are rarely found in GBM, PARP inhibitors are unlikely to have efficacy as monotherapies, and thus combinations with DNA damaging agents are likely needed for activity. Yet this brings us back to the same therapeutic index problems that were encountered almost 20 years ago with such combinations. This problem is further compounded by BBB penetration, with current CNS-penetrant PARP inhibitors having brain/plasma ratios ranging 0.09–0.4,12,13 such that the “effective therapeutic index” between tumor in the brain and in the bone marrow is further narrowed. Newer, potent PARP-trapping PARP inhibitors have been developed, which include olaparib, but these drugs appear to have enhanced activity against both tumor and bone marrow. Nevertheless, the current study was able to identify an RP2D of 150 mg q.d. (3 d/wk) with daily TMZ (75 mg/m2), although this represents a marked dose reduction for olaparib, which is normally given at a dose of 300 mg b.i.d. continuously (a nearly ~10-fold olaparib dose reduction). The observed progression-free survival rate at 6 months was 39%, comparing favorably with recent clinical trials, although it was not robust enough to continue developing this combination in recurrent GBM. Taken together, the study by Hanna et al is elegant and well designed, highlighting the feasibility, challenges, and overall importance of incorporating rigorous assessments of drug PK and PD into GBM trials. A key open question will be whether a favorable therapeutic index can be obtained with CNS-penetrant PARP inhibitors and chemotherapy agents such as TMZ, and a focus on MGMT-silenced tumors or other DNA repair defects may address this issue. To this end, we await the results of a recently concluded phase III trial testing veliparib with TMZ in newly diagnosed, MGMT-silenced GBM (NCT02152982).13 Fulton and colleagues also recently began testing olaparib-based combinations with TMZ and radiotherapy in newly diagnosed GBM cohorts stratified by MGMT status,14 and it will be interesting to see these data mature. Finally, other biomarkers for PARP inhibitor sensitivity in glioma, including possibly mutant isocitrate dehydrogenase 1 or 2–induced HR defects, may help to further widen the therapeutic index, which is currently being tested in clinical trials.15 Acknowledgments Reviews are primarily cited because of formatting constraints that limit the number of references, and thus we apologize to those whose work could not be referenced here. References 1. Yap TA , Plummer R, Azad NS, et al. The DNA damaging revolution: PARP inhibitors and beyond . Am Soc Clin Oncol Educ Book. 2019 ; 39 : 185 – 195 . Google Scholar Crossref Search ADS PubMed WorldCat 2. Curtin NJ , Szabo C. Poly(ADP-ribose) polymerase inhibition: past, present and future . Nat Rev Drug Discov. 2020 ; 19 ( 10 ): 711 – 736 . Google Scholar Crossref Search ADS PubMed WorldCat 3. Murai J , Huang S-YN, Das BB, et al. Trapping of PARP1 and PARP2 by clinical PARP inhibitors . Cancer Res. 2012 ; 72 : 5588 – 5599 . Google Scholar Crossref Search ADS PubMed WorldCat 4. Bryant HE , Schultz N, Thomas HD, et al. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase . Nature . 2005 ; 434 ( 7035 ): 913 – 917 . Google Scholar Crossref Search ADS PubMed WorldCat 5. Farmer H , McCabe N, Lord CJ, et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy . Nature. 2005 ; 434 ( 7035 ): 917 – 921 . Google Scholar Crossref Search ADS PubMed WorldCat 6. Murai J , Zhang Y, Morris J, et al. Rationale for poly(ADP-ribose) polymerase (PARP) inhibitors in combination therapy with camptothecins or temozolomide based on PARP trapping versus catalytic inhibition . J Pharmacol Exp Ther. 2014 ; 349 ( 3 ): 408 – 416 . Google Scholar Crossref Search ADS PubMed WorldCat 7. Gupta SK , Smith EJ, Mladek AC, et al. PARP inhibitors for sensitization of alkylation chemotherapy in glioblastoma: impact of blood-brain barrier and molecular heterogeneity . Front Oncol. 2018 ; 8 : 670 . Google Scholar Crossref Search ADS PubMed WorldCat 8. Hanna C , Kurian KM, Williams K, et al. Pharmacokinetics, safety and tolerability of olaparib and temozolomide for recurrent glioblastoma: results of the phase I OPARATIC trial . Neuro Oncol. 2020 : noaa104 . doi:10.1093/neuonc/noaa104. Epub ahead of print. Google Scholar OpenURL Placeholder Text WorldCat 9. Grossman SA , Romo CG, Rudek MA, et al. ; Adult Brain Tumor Consortium. Baseline requirements for novel agents being considered for phase II/III brain cancer efficacy trials: conclusions from the adult brain tumor consortium’s first workshop on CNS drug delivery . Neuro Oncol. 2020 : noaa142 . doi:10.1093/neuonc/noaa142. Epub ahead of print. Google Scholar OpenURL Placeholder Text WorldCat 10. Sarkaria JN , Hu LS, Parney IF, et al. Is the blood-brain barrier really disrupted in all glioblastomas? A critical assessment of existing clinical data . Neuro Oncol. 2018 ; 20 ( 2 ): 184 – 191 . Google Scholar Crossref Search ADS PubMed WorldCat 11. Wilsker DF , Barrett AM, Dull AB, et al. Evaluation of pharmacodynamic responses to cancer therapeutic agents using DNA damage markers . Clin Cancer Res. 2019 ; 25 ( 10 ): 3084 – 3095 . Google Scholar Crossref Search ADS PubMed WorldCat 12. Xiong Y , Guo Y, Liu Y, et al. Pamiparib is a potent and selective PARP inhibitor with unique potential for the treatment of brain tumor . Neoplasia. 2020 ; 22 ( 9 ): 431 – 440 . Google Scholar Crossref Search ADS PubMed WorldCat 13. Gupta SK , Smith EJ, Mladek AC, et al. PARP inhibitors for sensitization of alkylation chemotherapy in glioblastoma: impact of blood-brain barrier and molecular heterogeneity . Front Oncol. 2019 ; 8 : 670 . Google Scholar Crossref Search ADS PubMed WorldCat 14. Fulton B , Short SC, James A, et al. PARADIGM-2: Two parallel phase I studies of olaparib and radiotherapy or olaparib and radiotherapy plus temozolomide in patients with newly diagnosed glioblastoma, with treatment stratified by MGMT status . Clin Transl Radiat Oncol. 2018 ; 8 : 12 – 16 . Google Scholar Crossref Search ADS PubMed WorldCat 15. van den Bent MJ , Mellinghoff IK, Bindra RS. Gray areas in the gray matter: IDH1/2 mutations in glioma . Am Soc Clin Oncol Educ Book. 2020 ; 40 : 1 – 8 . Google Scholar PubMed OpenURL Placeholder Text WorldCat © The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: [email protected] This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
The duration of adjuvant temozolomide in patients with glioblastoma and the law of diminishing returnsGrossman, Stuart, A
doi: 10.1093/neuonc/noaa243pmid: 33074333
The law of diminishing returns is a highly regarded principle of economics and business which states that not every unit of input will lead to a proportional increase in output. Furthermore, it asserts that recurrent investments in one input, while holding all other inputs constant, yield progressively smaller output results. This principle is frequently taught using an agricultural example. While one application of fertilizer may dramatically increase crop yields, with successive applications the increase in yield falls and eventually productivity diminishes while fertilizer-related toxicities and costs rise. Thus, it is critically important to identify the “point of diminishing returns” if the ultimate goal is to continually improve outcomes while focusing on a single interventional strategy. This principle also applies to chemotherapy and survival outcomes. The pivotal study that led to the approval of temozolomide (TMZ) in patients with newly diagnosed glioblastoma randomized patients to 6 weeks of radiation or 6 weeks of radiation with concurrent TMZ followed by 6 months of adjuvant TMZ.1 The results demonstrated a significant increase in median survival (12 vs 14.5 mo) and survival at 2 years (10% vs 26%). However, the survival benefit from adding TMZ is largely restricted to patients with O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation.2 The design of this important study was simple and appropriate. Survival was compared in patients receiving no TMZ and those receiving extensive TMZ (concurrent and adjuvant). With regulatory approval, the full complement of TMZ was fixed as the standard of care for all patients with newly diagnosed glioblastoma. Fifteen years have now passed since the original publication of these results. Multiple attempts have been made to improve this regimen. Higher dose adjuvant TMZ was studied in a large, randomized, prospective study.3 This resulted in significant increases in toxicity, but no improvement in survival. In addition, several retrospective analyses have concluded that prolonging the duration of adjuvant TMZ also increases toxicities without a survival benefit.4,5 It is of particular importance that even in MGMT methylated patients, who might be expected to benefit most from TMZ, no survival benefit was observed in these studies exploring higher doses or longer durations of adjuvant TMZ. The GEINO 14-01 study group is to be commended for conducting the first prospective randomized trial designed specifically to address the risks and benefits of prolonged adjuvant TMZ.6 Patients who had no evidence of tumor progression after completing standard therapy (6 weeks of concurrent radiation and TMZ and 6 months of adjuvant TMZ) were randomized to stop TMZ or to receive an additional 6 months of adjuvant TMZ. In retrospect, there are several aspects of this study design which were not ideal. The primary endpoint of the study was progression-free survival (PFS) rather than overall survival. Unfortunately, PFS is difficult to accurately quantify, especially in the post-radiation setting and in a population of patients rich in MGMT methylation who are prone to pseudoprogression. The investigators were asking a non-inferiority question but chose to approach this using a “phase II” design. The overall sample size was too small given the many important variables that need to be addressed, such as age, performance status, MGMT methylation status, isocitrate dehydrogenase (IDH) mutational status, and extent of resection. In addition, IDH mutational status was incompletely assessed. There are reasonable explanations for these shortcomings. Accruing patients with a terminal illness to this type of study is difficult as clinicians and patients prefer promising novel therapies intended to improve survival rather than non-inferiority and dose de-escalation studies. Knowing this, the investigators chose a phase II design rather than a non-inferiority design, which would have required much higher accrual numbers. They also chose a PFS endpoint which shortens the time to reach the primary endpoint. The lack of complete IDH evaluations is also understandable given that the study was designed and initiated before the importance of IDH mutations was fully understood. In spite of these shortcomings, this prospective, randomized, multicenter study confirms what has been reported in retrospective studies posing the same question. They found no improvement in PFS or overall survival associated with longer durations of adjuvant TMZ, even in MGMT methylated patients. They also found that prolonged TMZ was associated with higher toxicity rates. Other factors to be considered with prolonged TMZ administration include: (i) higher medical costs for TMZ, anti-emetics, pneumocystis jerovecii pneumonia prophylaxis, blood counts, and complications of therapy; (ii) prolonged TMZ-induced lymphopenia which may affect outcomes with immunotherapy or infections; and (iii) extended disruption of quality time for some patients who instinctively limit travel and important social interactions while receiving chemotherapy. Given the outcomes of studies documenting that higher doses and longer durations of adjuvant TMZ result in more harm than good, it is reasonable to wonder if even the standard 6 months of adjuvant TMZ is beyond the “point of diminishing returns” described by economists (Fig. 1). There are no studies that convincingly document the value of the adjuvant TMZ. In the study by the European Organisation for Research and Treatment of Cancer that led to FDA approval of TMZ, patients received a median of 3 rather than 6 cycles of adjuvant TMZ and yet the results were positive.1 Is it possible that adjuvant TMZ adds nothing to the 6 weeks of daily TMZ with radiation? This is important to consider as all of us would quickly move to explore novel adjuvant regimens in this patient population if we knew that adjuvant TMZ added little to survival. Although dose de-escalation and non-inferiority studies are often discouraged in diseases where outcomes are poor, it is imperative that we understand which components of this 15-year-old treatment regimen are truly critical. As astutely noted by economists: More is not always better—sometimes it is just more. Fig. 1 Open in new tabDownload slide TMZ and the Law of Diminishing Returns. Data from GEINO 14-01 and several retrospective studies strongly suggests that 12 months of adjuvant TMZ adds toxicity without additional survival benefit. As a result, this should be considered beyond the “point of negative returns.” Additional information is needed to determine how much adjuvant TMZ is required to reach the “point of diminishing returns.” Fig. 1 Open in new tabDownload slide TMZ and the Law of Diminishing Returns. Data from GEINO 14-01 and several retrospective studies strongly suggests that 12 months of adjuvant TMZ adds toxicity without additional survival benefit. As a result, this should be considered beyond the “point of negative returns.” Additional information is needed to determine how much adjuvant TMZ is required to reach the “point of diminishing returns.” Acknowledgment This text is the sole product of the author. No third party had input or gave support to its writing. There are no potential conflicts of interest. References 1. Stupp R , Hegi ME, Mason WP, et al. ; European Organisation for Research and Treatment of Cancer Brain Tumour and Radiation Oncology Groups; National Cancer Institute of Canada Clinical Trials Group. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial . Lancet Oncol. 2009 ; 10 ( 5 ): 459 – 466 . Google Scholar Crossref Search ADS PubMed WorldCat 2. Hegi ME , Diserens AC, Gorlia T, et al. MGMT gene silencing and benefit from temozolomide in glioblastoma . N Engl J Med. 2005 ; 352 ( 10 ): 997 – 1003 . Google Scholar Crossref Search ADS PubMed WorldCat 3. Gilbert MR , Wang M, Aldape KD, et al. Dose-dense temozolomide for newly diagnosed glioblastoma: a randomized phase III clinical trial . J Clin Oncol. 2013 ; 31 ( 32 ): 4085 – 4091 . Google Scholar Crossref Search ADS PubMed WorldCat 4. Blumenthal DT , Gorlia T, Gilbert MR, et al. Is more better? The impact of extended adjuvant temozolomide in newly diagnosed glioblastoma: a secondary analysis of EORTC and NRG Oncology/RTOG . Neuro Oncol. 2017 ; 19 ( 8 ): 1119 – 1126 . Google Scholar Crossref Search ADS PubMed WorldCat 5. Gramatzki D , Kickingereder P, Hentschel B, et al. Limited role for extended maintenance temozolomide for newly diagnosed glioblastoma . Neurology. 2017 ; 88 ( 15 ): 1422 – 1430 . Google Scholar Crossref Search ADS PubMed WorldCat 6. Balana C , Vaz MA, Sepúlveda JM, et al. A phase II randomized, multicenter, open-label trial of continuing adjuvant temozolomide beyond six cycles in patients with glioblastoma (GEINO 14-01) . Neuro Oncol . 2020 . doi: 10.1093/neuonc/noaa107. Epub ahead of print. Google Scholar OpenURL Placeholder Text WorldCat © The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: [email protected] This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
Young children with medulloblastoma: important open questions and the high-risk dilemmaMynarek,, Martin;Rutkowski,, Stefan
doi: 10.1093/neuonc/noaa241pmid: 33091120
When choosing the postoperative therapy concept for young children with medulloblastoma, the question of whether or not to irradiate during primary treatment is key for all other therapeutic decisions. Craniospinal irradiation (CSI), a highly efficient treatment component for medulloblastoma, has detrimental effects on the developing brain and leads to significant impairment of neurocognitive functioning in surviving patients, especially in the youngest patients. Therefore, trial consortia recently investigated to avoid the use of CSI in these children by various chemotherapy strategies, (i) in combination with high-dose chemotherapy/autologous stem cell rescue (HDCT/ASCR) as described here by the Head-Start group, (ii) in combination with intraventricular methotrexate (MTX), and (iii) by conventional, systemic chemotherapy alone.1–4 All of these trials confirmed the prognostic impact of histologic/molecular features, with sonic hedgehog (SHH)–activated desmoplastic/extensively nodular medulloblastoma (DMB/MBEN) representing a favorable risk group and rather infrequent need for radiotherapy, and non-SHH classic medulloblastoma/large cell anaplastic medulloblastoma (CMB/LCAMB) carrying a high risk of relapse. Although sample sizes for these trials precluded randomized comparisons, results suggest that despite being low-risk, a substantial proportion of SHH-activated DMB/MBEN cannot be cured by conventional, systemic chemotherapy alone and therefore require treatment intensification either by HDCT/ASCT or by intraventricular MTX. Those trials that did not include such a component3,4 reported progression-free survival (PFS) rates of 55% (5-y PFS in SJYC07-low risk stratum) and 52% (2-y PFS in ACNS-1221), and compare unfavorably to the 93% 5-year PFS in patients treated with intraventricular MTX2 and 89% 5-year PFS for all DMB/MBEN patients (93% for M0 DMB/MBEN patients) treated with HDCT/ASCT in the series published in this edition.1 However, important questions remain. One is the impact of histopathological features within biologically defined subgroups of medulloblastoma: Do patients with SHH-activated CMB carry a higher risk of relapse as suggested by retrospective analysis,5 and how frequent are those? If biology is primarily driving the prognosis, to which extent do the at least in part subjective histopathological features contribute to refine the genetically defined subtype? Unfortunately, the current study cannot contribute to answer this question, because information on genetically defined subgroups is unavailable. Another question is related to the role of germline mutations, as some groups have suggested a worse prognosis for children with Gorlin syndrome and SUFU (suppressor of fused homolog) mutations.6 Other questions are: What is the optimal therapy for young children with low-risk SHH-activated medulloblastoma, and what is the role of metastatic disease in this context? “Standard-dose” chemotherapy without additional intraventricular MTX and without HDCT/ASCT do not seem to confer sufficient event-free/radiotherapy-free survival rates for all infant SHH-MB,1–4,7 but might be an option for patients with very-low-risk infant SHH-II medulloblastoma. However, the very positive outcome has only been described in one series so far,4 and was less clear in the other series with biological characterization available.2,3 Although the long-term toxicities of both intraventricular MTX and HDCT/ASCT are less detrimental than CSI, they are related to different acute and long-term side effects, and survivors after either therapy show relevant neurocoginitve impairments.1,2,8,9 Randomized comparisons of both therapeutic strategies including neurocognitive assessments are highly desirable but challenging to perform, given the low incidence of the disease and the complexity of such a trial. Another very important question is: What can be offered to young children with medulloblastoma and biological high-risk features (non-SHH-MB)? The current 21% 5-year radiotherapy-free event-free survival reported for CMB/LCAMB patients in the series presented by Dhall et al1 is similar to the 39% CSI-free survival in M0 CMB/LCAMB patients from the HIT-group2 and the approximately 10% PFS for Group 3/Group 4 MB reported in the St Jude series.4 These data suggest that despite the intensive chemotherapy regimens used in these series, a very high proportion of children will require radiotherapy to survive their disease. In light of CSI-associated late effects, this is a critical tradeoff to be made by many affected families and treating physicians. Since most conventional chemotherapeutic regimens have failed so far to achieve substantial CSI-free survival rates, new approaches are urgently needed for these patients, as well as for older children with high-risk medulloblastoma. Some novel concepts are currently in preclinical or early-phase clinical trial development. In spite of the achieved improvements, there are still significant challenges remaining. Conflict of interest statement Stefan Rutkowski has received research funding from Riemser Pharma, Germany. References 1. Dhall G , O’Neil SH, Ji L, et al. Excellent outcome of young children with nodular desmoplastic medulloblastoma treated on “Head Start” III: a multi-institutional, prospective clinical trial . Neuro Oncol. 2020 . doi: 10.1093/neuonc/noaa102. Google Scholar OpenURL Placeholder Text WorldCat 2. Mynarek M , von Hoff K, Pietsch T, et al. Nonmetastatic medulloblastoma of early childhood: results from the prospective clinical trial HIT-2000 and an extended validation cohort . J Clin Oncol. 2020 ; 0 : JCO1903057 . doi: 10.1200/JCO.19.03057. Google Scholar OpenURL Placeholder Text WorldCat 3. Lafay-Cousin L , Bouffet E, Strother D, et al. Phase II study of nonmetastatic desmoplastic medulloblastoma in children younger than 4 years of age: a report of the Children’s Oncology Group (ACNS1221) . J Clin Oncol. 2020 ; 38 ( 3 ): 223 – 231 . Google Scholar Crossref Search ADS PubMed WorldCat 4. Robinson GW , Rudneva VA, Buchhalter I, et al. Risk-adapted therapy for young children with medulloblastoma (SJYC07): therapeutic and molecular outcomes from a multicentre, phase 2 trial . Lancet Oncol. 2018 ; 19 ( 6 ): 768 – 784 . Google Scholar Crossref Search ADS PubMed WorldCat 5. Hicks D , Rafiee G, Schwalbe EC, et al. The molecular landscape and associated clinical experience in infant medulloblastoma: prognostic significance of second-generation subtypes . Neuropathol Appl Neurobiol. 2020 . doi: 10.1111/nan.12656. Google Scholar OpenURL Placeholder Text WorldCat 6. Guerrini-Rousseau L , Dufour C, Varlet P, et al. Germline SUFU mutation carriers and medulloblastoma: clinical characteristics, cancer risk and prognosis . Neuro Oncol. 2017 . doi: 10.1093/neuonc/nox228. Google Scholar OpenURL Placeholder Text WorldCat 7. Robinson GW , Gajjar A. Genomics paves the way for better infant medulloblastoma therapy . J Clin Oncol. 2020 ; 38 ( 18 ): 2010 – 2013 . Google Scholar Crossref Search ADS PubMed WorldCat 8. Ottensmeier H , Schlegel PG, Eyrich M, et al. Treatment of children under 4 years of age with medulloblastoma and ependymoma in the HIT2000/HIT-REZ 2005 trials: neuropsychological outcome 5 years after treatment . PLoS One. 2020 ; 15 ( 1 ): e0227693 . Google Scholar Crossref Search ADS PubMed WorldCat 9. Sands SA , Oberg JA, Gardner SL, Whiteley JA, Glade-Bender JL, Finlay JL. Neuropsychological functioning of children treated with intensive chemotherapy followed by myeloablative consolidation chemotherapy and autologous hematopoietic cell rescue for newly diagnosed CNS tumors: an analysis of the Head Start II survivors . Pediatr Blood Cancer. 2010 ; 54 ( 3 ): 429 – 436 . Google Scholar Crossref Search ADS PubMed WorldCat © The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: [email protected] This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
Highlights from the LiteraturePurow,, Benjamin;Wright,, Karen;Abounader,, Roger;Ghosh,, Sourav;Charest,, Alain
doi: 10.1093/neuonc/noaa246pmid: N/A
Reality Check Re: The Right Middleman for Anti-PD1 in Glioma Immunotherapy with checkpoint inhibition like anti-PD1 antibodies has led to dramatic prolongations in survival for subsets of patients with melanoma, lung cancer, and several other cancers. However, it has typically yielded disappointing results for patients with glioma, albeit with a glimmer of hope from recent trials of neoadjuvant anti-PD1 therapy.1,2 Anti-PD1 checkpoint inhibition has been thought to operate primarily through increased activation of T cells, although it also has been found to increase activation of macrophages.3 A new report from Rao et al. suggests that the latter may be more important for anti-PD1 effects against glioma.4 The authors generated gliomas in mice via the RCAS-tva model in wild-type or in a CD8alpha knockout background, yielding mice without CD8 T cells. Interestingly, the CD8-/- mice had increased CD11b+ myeloid-derived cells in the glioma microenvironment, a subset that includes microglia and macrophages. Treatment with anti-PD1 antibodies had efficacy in the wild-type setting, but notably this efficacy was retained in the mice lacking CD8 T cells. These effects were seemingly mediated by action on tissue-resident microglia and blood-borne macrophages, with two different mechanisms evident. Microglia and macrophages displayed an increase in M1 markers, consistent with a shift to a tumor-suppressive phenotype. There was also a decrease in M2 marker-bearing microglia and macrophages, and this appeared to be mediated in part by penetration of the anti-PD1 antibodies into the brain and their binding to PD1-expressing tumor-promoting microglia/macrophages, leading to destruction of these cells by antibody-mediated cellular cytotoxicity. These results provide important new understanding of mechanisms for anti-PD1 antibodies action against glioma and also suggest that the innate immune system—specifically microglia and macrophages—is sufficient to mediate anti-glioma effects. Hopefully, these key findings will be verified in other immunocompetent mouse models as well, and certain aspects could also be tested in patient samples obtained from surgery performed after neoadjuvant anti-PD1 is given. It will be critical in such studies to carefully distinguish not only microglia and macrophages with M1 versus M2 markers but monocytic MDSCs (myeloid-derived suppressor cells) as well. Certain aspects of this work, such as the substantial penetration of the anti-PD1 antibodies into high-grade glioma, may be more applicable in mouse models than in patients, and this too could be investigated. The increased understanding of mechanisms for anti-PD1 activity in high-grade glioma could lead to more rational and effective immunotherapeutic combinations for this dreaded cancer. References 1. Cloughesy TF , Mochizuki AY, Orpilla JR, et al. Neoadjuvant anti-PD-1 immunotherapy promotes a survival benefit with intratumoral and systemic immune responses in recurrent glioblastoma . Nat Med. 2019 ; 25 ( 3 ): 477 – 486 . Google Scholar Crossref Search ADS PubMed WorldCat 2. Schalper KA , Rodriguez-Ruiz ME, Diez-Valle R, et al. Neoadjuvant nivolumab modifies the tumor immune microenvironment in resectable glioblastoma . Nat Med. 2019 ; 25 ( 3 ): 470 – 476 . Google Scholar Crossref Search ADS PubMed WorldCat 3. Gordon SR , Maute RL, Dulken BW, et al. PD-1 expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity . Nature. 2017 ; 545 ( 7655 ): 495 – 499 . Google Scholar Crossref Search ADS PubMed WorldCat 4. Rao G , Latha K, Ott M, et al. Anti-PD-1 induces M1 polarization in the glioma microenvironment and exerts therapeutic efficacy in the absence of CD8 cytotoxic T cells . Clin Cancer Res. 2020 ; 26 ( 17 ): 4699 – 4712 . Google Scholar Crossref Search ADS PubMed WorldCat Single-cell RNA-seq Reveals Cellular Hierarchies and Impaired Developmental Trajectories in Pediatric Ependymoma Despite molecular advances in the characterization of ependymoma subgroups, we have yet to understand the role of grade in prognosis and identify effective, long-term therapies. Moreover, chemotherapy has long been touted as ineffective in ependymoma, but we have yet to understand why that may be the case. Additionally, posterior fossa A ependymomas tend to demonstrate inferior outcomes compared with other subgroups. To begin to address some of these questions, Gojo and colleagues applied single-cell transcriptomics, which has provided insight into both the genetic underpinnings of diffuse intrinsic pontine glioma and medulloblastoma as well as numerous other non-malignant diseases, to now investigate the gene expression level and intratumoral heterogeneity of subgroup-specific ependymoma cells by measuring the mRNA concentration of thousands of genes.1 Single-cell analysis offers the opportunity to further delineate the transcriptional and histopathological challenges of these disease groups. The identification of distinct neuronal, astrocytic and ependymal-like tumor cell types provides further subgroup insight. Prognostically favorable posterior fossa B tumors, subependymomas, and supratentorial YAP1 tumors, for example, were enriched for more differentiated ependymoma-like cell types as were ependymomas in older patients. Aggressive subtypes like that of posterior fossa A tumors in young patients, in contrast, harbored undifferentiated neural stem cell-like and early neuronal precursor-like tumor cell types. Posterior fossa neuronal precursor-like cells also expressed ABCC5, a multidrug resistance-associated protein transporter of the blood-brain barrier that participates in exporting several chemotherapeutic agents and may help to partly explain the ongoing argument that ependymomas are relatively chemo-unresponsive. The authors also showed a shift from differentiated cell types to undifferentiated cell types at recurrence. In summary, their findings suggest that the clinical course of ependymoma is predicated upon the underlying stem cell signatures as opposed to cell cycle activation and tumor grade. Therefore, improving future treatment strategies will need to focus on inhibiting pathways responsible for maintaining these undifferentiated cell states. However, whether application of single-cell sequencing will impact our treatment choices for patients in the future remains to be seen. Reference 1. Gojo J , Englinger B, Jiang L, et al. Single-cell RNA-Seq reveals cellular hierarchies and impaired developmental trajectories in pediatric ependymoma . Cancer Cell. 2020 ; 38 ( 1 ): 44 – 59.e9 . Google Scholar Crossref Search ADS PubMed WorldCat MGMT Genomic Rearrangements Contribute to Temozolomide Resistance in Gliomas Temozolomide (TMZ) is a chemotherapeutic drug that significantly extends the overall survival of glioblastoma (GBM) and high-risk low-grade glioma patients. TMZ is an orally bioavailable alkylating agent. The enzyme O6-methylguanine-DNA methyltransferase (MGMT) counteracts the effects of TMZ by repairing the main TMZ-induced toxic DNA adduct, the O6-methylguanine lesion. Epigenetic silencing of MGMT via promoter methylation inhibits MGMT synthesis and consequently increases tumor sensitivity to TMZ. MGMT promoter hypermethylation is therefore a biomarker of sensitivity to TMZ, and hypomethylation causes resistance to the drug. A recent study published in the journal Nature Communications uncovered a new MGMT-associated mechanism of resistance to TMZ.1 Specifically, the study discovered MGMT gene fusions in recurrent gliomas that lead to MGMT overexpression and resistance to TMZ. The study analyzed 252 TMZ-treated recurrent gliomas and found that 7 patients (3% of the total) had eight different rearrangements that led to MGMT gene fusions. These were: BTRC-MGMT, CAPZB-MGMT, GLRX3-MGMT, NFYC-MGMT, RPH3A-MGMT, SAR1A-MGMT, CTBP2-MGMT, and FAM175B-MGMT. MGMT fusions, MGMT hypomethylation, and DNA hypermutation in the tumors were mutually exclusive, suggesting different roles in glioma progression. Gliomas with MGMT fusions or hypomethylated MGMT promoter had significantly higher MGMT expression than MGMT-methylated and DNA-hypermutated tumors. To further study the role of MGMT fusions in gliomas, the authors used CRISPR/Cas9 technology to generate GBM cell lines and primary cells that express four of the MGMT fusions. Expression of the MGMT fusions led to overexpression of MGMT in the cells. The MGMT fusion-expressing cells were resistant to TMZ, both in vitro and when implanted to generate GBM xenografts in vivo. Interestingly, the study also found that the fusions can be detected in tumor-derived exosomes in cell culture and in mice bearing GBM xenografts with MGMT fusions. This study uncovered a new MGMT-associated mechanism of resistance to TMZ that is present in a subset of recurrent TMZ-treated glioma patients. It is not known if the MGMT fusions were already present in the newly diagnosed tumors and selected for during recurrence, or if they are acquired after exposure to TMZ. The existence of the fusions and their possible detection in exosomes collected from liquid biopsies could be used to inform therapeutic decisions about a potential reuse of TMZ in recurrent glioma patients. Reference 1. Oldrini B , Vaquero-Siguero N, Mu Q, et al. MGMT genomic rearrangements contribute to chemotherapy resistance in gliomas . Nat Commun. 2020 ; 11 ( 1 ): 3883 . Google Scholar Crossref Search ADS PubMed WorldCat Machine Learning-enabled Overall Survival Prediction in Glioblastoma Brain tumors, in particular, glioblastoma, are characterized by intrinsic biological heterogeneity. Efforts to correlate heterogeneity with overall survival (OS) outcomes have included molecular stratification schemes based on cataloging somatic mutations in the genome or unsupervised hierarchical clustering of the transcriptome. With the exception of certain molecular parameters such as IDH, MGMT or 1p14q deletion, predicting OS has remained an outstanding challenge. Glioblastoma heterogeneity is also reflected in its MRI imaging. BraTS (Brain Tumor Segmentation) challenge is a concerted effort, begun in 2012, that is aimed at segmentation of multimodal MRI scans through computational algorithms using a uniform training, validation, and testing dataset. Since 2017, patient OS predictions have been included as part of this initiative. In a recent Frontiers of Computational Neuroscience article, Baid et al. published their prediction algorithm for segmentation, extraction of radiomic features from region of interest (ROI), and prediction of OS by using a multilayer percepton (MLP) neural network.1 The algorithm was trained on data from 163 patients. This method had a 0.571 and 0.558 accuracy on the 53-patient validation and the 130-patient test datasets, respectively. A patch-based 3D U-Net architecture was used for segmentation. ROI was decomposed into two sub-bands using low-pass (L)- and high-pass (H)-filters. After repeating this step, gray-level co-occurrence matrices (GLCM) features were extracted from the 4 bands – LL, LH, HL, and HH. Combination of whole tumor, necrosis with enhancing tumor, and enhancing tumor only were used for extraction of radiomic features from the FLAIR and T1ce channels. A total of 679 features were reduced to 54 based on their correlation coefficients and used to predict 3 survival groups: <300 days (short survivors), 300–900 days (mid survivors), and >900 days (long survivors) determined by unsupervised two-step hierarchical clustering. Notably, the accuracy of OS prediction for short survivors was 0.804. A number of algorithms such as that of Sun et al.2 and others (listed in 3) achieved similar or greater accuracy with the test dataset in the BraTS challenge, heralding significant advances in neural network and deep learning that can automate quantitative analyses of pre-operative MRI scans for risk assessment and treatment strategies. References 1. Baid U , Rane SU, Talbar S, et al. Overall survival prediction in glioblastoma with radiomic features using machine learning . Front Comput Neurosci. 2020 ; 14 : 61 . Google Scholar Crossref Search ADS PubMed WorldCat 2. Sun L , Zhang S, Chen H, Luo L. Brain tumor segmentation and survival prediction using multimodal MRI scans with deep learning . Front Neurosci. 2019 ; 13 : 810 . Google Scholar Crossref Search ADS PubMed WorldCat 3. Bakas S , Reyes M, Jakab A, et al. Identifying the best machine learning algorithms for brain tumor segmentation, progression assessment, and overall survival prediction in the BRATS challenge . arXiv:1811.02629. 2018 . https://ui.adsabs.harvard.edu/abs/2018arXiv181102629B. Immune Phenotyping of Diverse Syngeneic Murine Brain Tumors Identifies Immunologically Distinct Types The advent of immunotherapies for the treatment of primary brain cancers solidifies the need for accurate models of gliomas. There are various approaches to modeling malignant gliomas in mice. Models based on the growth of patient-derived xenografts (PDXs) in immunocompromised animals are certainly tumor-cell relevant but unfortunately ignore the contribution of the immune system to glioma biology. On the other hand, genetically engineered mice (GEM) are immunocompetent models that permit a genetic control over initiating events and are suited for longitudinal studies on the effects of the immune system on glioma initiation, maintenance, and explosive growth. When GEM model strains are created and maintained on fully inbred genetic backgrounds, cells from the resulting gliomas can be allografted back into recipient mice of matching strain. Finally, cells from carcinogen-induced gliomas can also be utilized in a syngeneic fashion, although their oncogenetic drivers (often Kras mutations) are less relevant to glioma biology. All of these models have advantages and disadvantages associated with them. With regards to their preclinical use for the development of novel immunotherapy modalities, the use of GEM models and the tumor cells derived therefrom are considered the gold standard. In a study by Khalsa et al, the authors extensively described the immune composition of 4 such syngeneic mouse models that are derived from GEM and carcinogen-induced models.1 Using bulk RNA seq and CyTOF, the authors determined the different immune cell population contributing to these glioma tumors. They observed that certain lines (GL261 and 005) are immunologically active (as defined by an abundance in expression of immune-specific genes), whilst the other lines studied (Mut3 and CT2A) are immunologically inert. Further analyses showed that the immunologically inert gliomas have an immune-suppressive phenotype composed of higher levels of exhausted and classical CD8 T cells and resident macrophages and lower levels of activated microglia, eosinophils, and SiglecF+ macrophages. One very interesting observation made by the authors is that surgical resection (a key component of the standard of care for glioblastoma, GBM) of immunologically inert CT2A tumors promotes infiltration of immune cells that have an activated phenotype. Further studies to determine if there is reversion of immune cell activation status over time post-resection are warranted and should yield clinically relevant results. Finally, the authors performed parallel CyTOF evaluations of patient GBM samples and were able to demonstrate that one of the lines studied (005) matched more closely to patient material in terms of composition of immune cells. Overall, this powerful study demonstrates the variation in immune cell composition amongst 4 independent syngeneic models of malignant glioma and serves to provide invaluable information on these widely used models. Reference 1. Khalsa JK , Cheng N, Keegan J, et al. Immune phenotyping of diverse syngeneic murine brain tumors identifies immunologically distinct types . Nat Commun. 2020 ; 11 ( 1 ): 3912 . Google Scholar Crossref Search ADS PubMed WorldCat © The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: [email protected] This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
Ad-CD40L mobilizes CD4 T cells for the treatment of brainstem tumorsWongthida, Phonphimon; Schuelke, Matthew R; Driscoll, Christopher B; Kottke, Timothy; Thompson, Jill M; Tonne, Jason; Stone, Cathy; Huff, Amanda L; Wetmore, Cynthia; Davies, James A; Parker, Alan L; Evgin, Laura; Vile, Richard G
doi: 10.1093/neuonc/noaa126pmid: 32459315
Abstract Background Diffuse midline glioma, formerly DIPG (diffuse intrinsic pontine glioma), is the deadliest pediatric brainstem tumor with median survival of less than one year. Here, we investigated (i) whether direct delivery of adenovirus-expressing cluster of differentiation (CD)40 ligand (Ad-CD40L) to brainstem tumors would induce immune-mediated tumor clearance and (ii) if so, whether therapy would be associated with a manageable toxicity due to immune-mediated inflammation in the brainstem. Methods Syngeneic gliomas in the brainstems of immunocompetent mice were treated with Ad-CD40L and survival, toxicity, and immune profiles determined. A clinically translatable vector, whose replication would be tightly restricted to tumor cells, rAd-Δ24-CD40L, was tested in human patient–derived diffuse midline gliomas and immunocompetent models. Results Expression of Ad-CD40L restricted to brainstem gliomas by pre-infection induced complete rejection, associated with immune cell infiltration, of which CD4+ T cells were critical for therapy. Direct intratumoral injection of Ad-CD40L into established brainstem tumors improved survival and induced some complete cures but with some acute toxicity. RNA-sequencing analysis showed that Ad-CD40L therapy induced neuroinflammatory immune responses associated with interleukin (IL)-6, IL-1β, and tumor necrosis factor α. Therefore, to generate a vector whose replication, and transgene expression, would be tightly restricted to tumor cells, we constructed rAd-Δ24-CD40L, the backbone of which has already entered clinical trials for diffuse midline gliomas. Direct intratumoral injection of rAd-Δ24-CD40L, with systemic blockade of IL-6 and IL-1β, generated significant numbers of cures with readily manageable toxicity. Conclusions Virus-mediated delivery of CD40L has the potential to be effective in treating diffuse midline gliomas without obligatory neuroinflammation-associated toxicity. adenovirus, brainstem tumors, CD40L, immunotherapy, tertiary lymphoid structure Key Points 1. Ad-CD40L therapy improves survival through induction of immune cell infiltration. 2. CD4 T cells are important in Ad-CD40L therapy. Importance of the Study New, aggressive therapies for pediatric brainstem tumors including diffuse midline gliomas are urgently required. CD40/CD40L engagement has generated potent antitumor responses in anatomic locations where high levels of inflammation are acceptable. Our study here is the first to investigate whether such CD40L-induced inflammation in brainstem tumors can be tolerated. We demonstrate here that when CD40L expression was tightly restricted to tumor cells, Ad-CD40L significantly prolonged survival of brainstem tumor–bearing mice. Depletion of CD4 T cells abolished Ad-CD40L therapy and decreased levels of antigen-presenting cells. A replication competent adenovirus engineered to replicate only in tumor cells (rAd-Δ24-CD40L) had cytopathic effects on patient-derived diffuse midline glioma cells and improved survival of mice bearing brainstem tumors in vivo. Our results show that it is possible to develop a novel, potentially highly effective immunotherapy for diffuse midline glioma, without unacceptable toxicity, which can be translated into clinical trials. Brainstem gliomas represent about 10–15% of pediatric central nervous system tumors.1 Of these, diffuse midline glioma (formerly diffuse intrinsic pontine glioma [DIPG]) is the major cause of death, with survival rates of fewer than 9 months from diagnosis.1 Focal radiation to the pons remains the main therapy for newly diagnosed disease and the addition of chemotherapy, targeted therapies, differentiation agents, and radiation sensitizers has not significantly impacted outcomes. Moreover, surgery is not an option due to the critical location within the brain.2 Therefore, new therapeutic modalities are urgently needed. Cancer immunotherapy has dramatically improved treatment of several tumors with immune checkpoint inhibitors and chimeric antigen receptor T-cell therapies, although not appreciably so far in children with brain tumors.3 Oncolytic virotherapy utilizes engineered viruses which preferentially replicate in tumor cells, leading to cell death and stimulation of the immune system and has been clinically investigated in brain tumors.4 Immunotherapies for tumors in the brainstem pose unique potential dangers associated with inflammation in this location. However, we have shown that diverse immunotherapies can extend survival in mice bearing brainstem tumors without catastrophic neurological toxicity,5 opening the path for testing novel immunotherapies for this disease. Cluster of differentiation (CD)40 ligand (CD40L:CD154) is expressed both as a membrane-bound form on activated helper T cells and as a soluble form by platelets.6 CD40/CD40L engagement develops T helper (Th)–dependent immune responses, provides proliferation/differentiation signals to B cells, and triggers maturation of antigen-presenting cells (APCs) for induction of cytotoxic T lymphocytes.7 Therefore, agonistic antibodies against CD40, and viral vectors expressing CD40L, have been successfully tested against several malignancies, including glioblastoma,8 and are associated with priming cytotoxic CD8+ T cells, reducing regulatory T cells, and activating tumor-associated macrophages.9 In models of subcutaneous melanoma, where local inflammation was well tolerated, we demonstrated that CD40L expressed from a replication-defective adenoviral vector (Ad-CD40L) significantly enhanced survival and primed antitumor CD8+ T cells.10,11 Others have shown that Ad-CD40L activates dendritic cells, increases the intratumoral Teffector/Tregulatory cell ratio,12 and combines with checkpoint inhibitors to eradicate tumors.13 Therefore, we investigated whether Ad-CD40L could be an effective treatment for brainstem tumors, while simultaneously monitoring whether the inflammation associated with antitumor therapy could be controlled/tolerated. Using immunocompetent models to assess the efficacy/toxicity balance of adenovirus expressing CD40L, we show here that tumor-restricted expression of CD40L represents a novel, potentially highly effective immunotherapy for diffuse midline glioma with an acceptable toxicity profile. Materials and Methods Additional detailed methods are provided in the Supplementary Material. Cell Lines GL261, CT2A, and 293A cell lines were grown in DMEM (HyClone) supplemented with 10% fetal bovine serum (Life Technologies). Mixed astrocytes were cultured in DMEM (HyClone) supplemented with 10% fetal bovine serum (Life Technologies), 1% penicillin/streptomycin (Corning), and 1% sodium pyruvate (Sigma). SJPDGF1 and SF7761 diffuse midline glioma neurospheres were cultured in trichoderma selective media. Viruses Ad-GFP or Ad-CD40L serotype 5 replication-defective adenoviruses express the green fluorescent protein (GFP) or murine CD40L, respectively, under the cytomegalovirus promoter in E1.11 Two replication-competent adenoviruses—rAd-Δ24-luc and rAd-Δ24-CD40L—are expressing luciferase or murine CD40L in which 24 bp of E1A was deleted.14–16 In Vivo Studies All studies were approved by the Mayo Foundation Institutional Animal Care and Use Committee. Female C57BL/6 mice (6–8 mice/group) were challenged (2 µL) with tumor cells or virus via stereotactic implantation using established coordinates in the frontal lobe or brainstem.17 Flow Cytometry Brain-infiltrating leukocytes (BILs) were harvested in Roswell Park Memorial Institute medium from perfused brains. BILs were prepared using dounce homogenization and Percoll gradient.18 Immune cells were analyzed using FlowJo 10. Histology and Immunofluorescence Brains were fixed in 10% formalin and stained with hematoxylin and eosin by Mayo Clinic Histology Core Facility. Immunofluorescence staining was as described.19 RNA-Sequencing Analysis of Gene Expression Ten days established GL261 tumor-bearing mice were intratumorally injected (2 µL) with phosphate buffered saline (PBS) or 5 × 106 plaque-forming units (pfu) Ad-GFP or Ad-CD40L. On day 8, brains were dissected for RNA extraction (RNeasy, Qiagen). RNA samples were then subjected to RNA-seq analysis at the Genome Analysis Core, Mayo Clinic. Tumor-Specific Immunoglobulin G Detection Sera were harvested on day 8 post-virus injection and used as primary antibody. Stained were 1 × 105 GL261 cells, or mixed astrocytes (1:10 dilution) at 4°C for 30 min, and 1:100 of Alexa Fluor 488 goat anti-mouse immunoglobulin (Ig)G (heavy + light chain) antibody (Invitrogen) was added for flow analysis. Viability Assays GL261 cells were plated at 1 × 104 cells for overnight; 24 hours later, GL261, SJPDGF1, or SF7761 cells (1 × 105) were infected with viruses at different multiplicity of infections (MOIs). At indicated time points, cell viability was assessed by alamarBlue Cell Viability Reagent (ThermoFisher Scientific). For CD4+ T cell co-culture assays, GL261 cells were seeded in 96-well plates in triplicate and treated with CD4+ T cells at an effector:target ratio of 10:1. Then, 48 h later, wells were washed gently 3x in PBS to remove non-adherent cells. Surviving cells were trypsinized and resuspended in PBS and viable cells were counted (viability was assessed by alamarBlue Cell Viability Reagent [ThermoFisher Scientific]). Luciferase Assay SJPDGF1 cells (1 × 105) were infected with rAd-∆24-luc at different MOIs. At indicated time points, luciferase was assessed by Steady-Glo Luciferase Assay System (Promega). Statistics The Mantel–Cox log-rank test was used to analyze Kaplan–Meier survival curves. Student’s t-tests were used for in vitro and ex vivo analysis where appropriate. Statistical significance was determined at P < 0.05. All analyses were performed using GraphPad Prism 8 software. Results Restricting Ad-CD40L to Tumor Cells Induces Rejection Without Toxicity To test the hypothesis that ligation of the CD40/CD40L axis would promote brainstem tumor clearance with manageable toxicity, we used the GL261/C57BL/6 immunocompetent models of tumor growth in the brainstem. Although not derived from pediatric diffuse midline gliomas, we have shown that GL261 can be reproducibly grown as transplantable tumors in the pons in the anatomic location that mirrors diffuse midline glioma growth in patients.5 Infection of GL261 cells with replication-defective Ad-GFP or Ad-CD40L at various MOIs (0.1–10) did not induce direct cytotoxicity, but transgene expression was detected by 48 h post-infection (Figure 1A, B). To determine the efficacy of CD40L as a mediator of immune rejection in the brainstem, GL261 cells were pre-infected with Ad-GFP or Ad-CD40L immediately prior to implantation into the brainstem using established coordinates.17 Implantation of tumor cells pre-infected with Ad-GFP (MOI 10) did not prolong survival of mice relative to mice treated with PBS (Figure 1C). However, mice implanted with GL261 cells pre-infected with Ad-CD40L at MOI of 1 or 10 (but not 0.1) rejected tumors completely with no overt toxicity (Figure 1C). To determine whether surviving mice developed long-term immunologic memory, they were rechallenged with the same tumor cells in the frontal lobe. While naïve mice succumbed to tumor by day 40, all mice which survived the brainstem challenge rejected the frontal lobe rechallenge (Figure 1D). These results suggest that highly targeted delivery of Ad-CD40L to tumors in the brainstem might improve survival with manageable toxicity and represents a valuable model to explore further as a novel immunotherapy of diffuse midline glioma. Fig. 1 Open in new tabDownload slide Restricted expression of CD40L in tumor cells exerts antitumor response. (A) GL261 cells were infected with different MOIs of Ad-GFP or Ad-CD40L, and cell viability was measured using MTT assay. Error bars ± SD. (B) GL261 cells were infected with Ad-GFP (MOI 1) and GFP expression was detected using florescence microscopy (20x). Scale bar, 100 µm. (C) Survival curves of mice treated with GL261 cells pre-infected with viruses and implanted into the brainstem (day 0). (D) Survival curves of mice which survived in (C) and were rechallenged with parental GL261 in the frontal lobe. *P ≤ 0.05, **P ≤ 0.01. Fig. 1 Open in new tabDownload slide Restricted expression of CD40L in tumor cells exerts antitumor response. (A) GL261 cells were infected with different MOIs of Ad-GFP or Ad-CD40L, and cell viability was measured using MTT assay. Error bars ± SD. (B) GL261 cells were infected with Ad-GFP (MOI 1) and GFP expression was detected using florescence microscopy (20x). Scale bar, 100 µm. (C) Survival curves of mice treated with GL261 cells pre-infected with viruses and implanted into the brainstem (day 0). (D) Survival curves of mice which survived in (C) and were rechallenged with parental GL261 in the frontal lobe. *P ≤ 0.05, **P ≤ 0.01. Ad-CD40L Induces Recruitment of Immune Cells into the Tumor Microenvironment To characterize the mechanisms by which Ad-CD40L induced antitumor responses in the brainstem, we analyzed the induction of BILs 8 days following implantation of tumor cells pre-infected with PBS, Ad-GFP, or Ad-CD40L (MOI 1). Treatment with Ad-CD40L induced a profound infiltration of CD45+ cells (Figure 2A), largely composed of CD4+ and CD8+ T cells, CD19+ (B) cells, and natural killer cells (Figure 2B). Ad-CD40L induced CD11b+Gr1+ cell infiltration (Figure 2C) and importantly activated class II and CD86 expression in CD11c+ and CD45midCD11b+ cells (Figure 2D, E). As shown by bromodeoxyuridine labeling, Ad-CD40L significantly promoted local proliferation of activated (CD44+) CD4+ T, CD8+ T, and CD19+ (B) cells (Figure 2F). In separate studies, we have also observed significant proliferation of adoptively transferred tumor-specific T cells in the draining lymph nodes following intratumoral expression of CD40L. Fig. 2 Open in new tabDownload slide Ad-CD40L induces immune cell infiltration in brain tumors. Mice were implanted with GL261 tumors pre-infected with PBS, Ad-GFP, or Ad-CD40L (MOI 1) on day 0. Mice were given bromodeoxyuridine (BrdU) on day 7, and brains were perfused and harvested on day 8 for flow analysis. Numbers of total (A) CD45+, (B) CD4+ T, CD8+ T, CD19+, NK1.1+, (C) CD11b+Gr1+, (D) CD11c+II+, (E) CD45midCD11b+, (F) CD44+BrdU+, (G) CXCR5+, (H) CD4+PD1+ICOS+. Error bars ± SD (n = 4). Student’s t-test: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. (I) Representative images (40x) of brains day 8 post-implantation stained with DAPI, B220, and CD4. Scale bar, 50 µm. Fig. 2 Open in new tabDownload slide Ad-CD40L induces immune cell infiltration in brain tumors. Mice were implanted with GL261 tumors pre-infected with PBS, Ad-GFP, or Ad-CD40L (MOI 1) on day 0. Mice were given bromodeoxyuridine (BrdU) on day 7, and brains were perfused and harvested on day 8 for flow analysis. Numbers of total (A) CD45+, (B) CD4+ T, CD8+ T, CD19+, NK1.1+, (C) CD11b+Gr1+, (D) CD11c+II+, (E) CD45midCD11b+, (F) CD44+BrdU+, (G) CXCR5+, (H) CD4+PD1+ICOS+. Error bars ± SD (n = 4). Student’s t-test: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. (I) Representative images (40x) of brains day 8 post-implantation stained with DAPI, B220, and CD4. Scale bar, 50 µm. In addition, Ad-CD40L induced recruitment of CXCR5+CD4+ T and B cells (Figure 2G). Consistent with C-X-C chemokine receptor 5 binding to C-X-C chemokine ligand 13, a chemokine important in the generation of secondary/tertiary lymphoid structures (TLS), these CD4+ T cells expressed programmed cell death 1 and inducible co-stimulator (ICOS), which are markers of follicular CD4+ T cells (Figure 2H). Similarly, CD4+ T cells and B220+ B cells clustered around the meninges following Ad-CD40L treatment, reminiscent of TLS formation (Figure 2I). CD4+ T Cells Are Critical for Ad-CD40L Therapy To identify critical cellular types for therapy, we performed multiple antibody-mediated depletions of CD4+ T, CD8+ T, or B cells. Of these, only depletion of CD4+ T cell significantly diminished the total number of BILs (Figure 3A), under conditions which we confirmed led to depletions of CD4+ T, CD8+ T, or B cells (Figure 3B). Depletion of B cells did not alter the number of CD4+ or CD8+ T cells. However, CD4 depletion reduced the number of CD19+CD40+ and CD19+II+ cells (Figure 3C) and CD11c+CD40+, CD11c+II+, CD11b+F4/80+, or CD11b+Gr1+ cell numbers were all significantly decreased (Figure 3D, E). CD4 depletion also suppressed microglia activation as evidenced by levels of CD45midCD11b+CD86+ and CD45midCD11b+II+ cells (Figure 3F). Fig. 3 Open in new tabDownload slide CD4 depletion abrogates Ad-CD40L therapy. Immune cells were depleted 3 days before implanting pre-infected cells. Brains were harvested on day 8. Number of total (A) BILs, (B) CD45+, (C) CD19+, (D) CD11c+, (E) CD11b+, (F) CD45midCD11b+ cells. Error bars ± SD (n = 3). Student’s t-test: *P ≤ 0.05, **P ≤ 0.01. (G) Survival curves of immune cell-depleted mice implanted with pre-infected GL261 cells. **P ≤ 0.01. (H) Survival curves of rechallenge of surviving mice from (G). All treatments **P ≤ 0.01 over naïve control. (I, J) Mice with 10 day established GL261 tumors were injected with PBS or with 5 × 106 pfu of Ad-GFP or Ad-CD40L. Five days later, CD4+ T cells were purified from splenocytes by magnetic bead separation. CD4+ T cells pooled from 3 mice per treatment group were co-cultured with 104 GL261 target cells pretreated for 24 h with murine IFN-γ (20 U/mL) at an effector:target ratio of 10:1 (triplicate wells). The number of surviving GL261 cells 48 h later is shown (I) for GL261 cells co-cultured with: 1: no CD4+ T cells; 2: CD4+ T cells from naïve mice (no tumor, no treatment); 3: CD4+ T cells from tumor bearing mice treated with PBS; 4: CD4+ T cells from tumor bearing mice treated with Ad-GFP; 5: CD4+ T cells from tumor bearing mice treated with Ad-CD40L. J. Supernatants harvested from these cultures were assayed for levels of IFN-γ by enzyme-linked immunosorbent assay. Fig. 3 Open in new tabDownload slide CD4 depletion abrogates Ad-CD40L therapy. Immune cells were depleted 3 days before implanting pre-infected cells. Brains were harvested on day 8. Number of total (A) BILs, (B) CD45+, (C) CD19+, (D) CD11c+, (E) CD11b+, (F) CD45midCD11b+ cells. Error bars ± SD (n = 3). Student’s t-test: *P ≤ 0.05, **P ≤ 0.01. (G) Survival curves of immune cell-depleted mice implanted with pre-infected GL261 cells. **P ≤ 0.01. (H) Survival curves of rechallenge of surviving mice from (G). All treatments **P ≤ 0.01 over naïve control. (I, J) Mice with 10 day established GL261 tumors were injected with PBS or with 5 × 106 pfu of Ad-GFP or Ad-CD40L. Five days later, CD4+ T cells were purified from splenocytes by magnetic bead separation. CD4+ T cells pooled from 3 mice per treatment group were co-cultured with 104 GL261 target cells pretreated for 24 h with murine IFN-γ (20 U/mL) at an effector:target ratio of 10:1 (triplicate wells). The number of surviving GL261 cells 48 h later is shown (I) for GL261 cells co-cultured with: 1: no CD4+ T cells; 2: CD4+ T cells from naïve mice (no tumor, no treatment); 3: CD4+ T cells from tumor bearing mice treated with PBS; 4: CD4+ T cells from tumor bearing mice treated with Ad-GFP; 5: CD4+ T cells from tumor bearing mice treated with Ad-CD40L. J. Supernatants harvested from these cultures were assayed for levels of IFN-γ by enzyme-linked immunosorbent assay. Despite the fact that Ad-CD40L therapy recruited both CD8+ T cells and B cells, depletion of neither CD8+ T nor B cells significantly altered survival compared with the IgG control. In contrast, CD4+ T cell depletion drastically reduced Ad-CD40L therapy, showing CD4+ T cells were absolutely and obligatorily required for therapy (Figure 3G). Regardless of whether mice survived following depletion of either CD8+ T or B cells, all of these surviving mice developed long-term antitumor memory responses against a rechallenge with GL261 cells in the frontal lobe (Figure 3H). In addition to providing helper function to generate intrinsic antitumor immune responses, CD4+ T cells have also been reported to have direct cytotoxicity against tumor cells.20,21 In this respect, CD4+ T cells isolated from spleens of mice treated with Ad-CD40L significantly inhibited the proliferation of target GL261 cells in vitro compared with CD4+ T cells from untreated mice, or from mice treated with Ad-GFP (Figure 3I). This inhibition of tumor cell proliferation may be contributed by some direct cytotoxicity and/or through a directly cytostatic effect exerted by the CD4+ T cells. This anti-proliferative activity on GL261 targets was also associated with low, but detectable levels of interferon-gamma (IFN-γ) secretion by the CD4+ T cells from Ad-CD40L-treated mice. In contrast, neither inhibition of proliferation against GL261 cells nor IFN-γ secretion was observed from CD4+ T cells from mice treated with PBS or Ad-GFP (Figure 3J). CD4+ T cell mediated anti-proliferative effects against GL261 targets in vitro absolutely required pretreatment of GL261 cells with IFN-γ. Although major histocompatibility complex (MHC) class II expression was undetectable on GL261 cells growing in vitro, pretreatment for 24 h with IFN-γ induced relatively high levels (Supplementary Figure 1A). Consistent with GL261 tumors being the target of direct CD4+ T-cell mediated inhibition of proliferation following Ad-CD40L treatment, GL261 tumors excised from the brainstems of mice treated with PBS had significantly lower levels of MHC class II expression than did GL261 tumors from brainstems of mice treated with Ad-CD40L (Supplementary Figure 1B). In addition, GL261 tumors showed significant induction of IFN-γ in Ad-CD40L-treated tumors compared with PBS or Ad-GFP-treated tumors (Supplementary Figure 1C), which is consistent with induction of MHC class II presented targets on tumors for CD4+ T-cell mediated recognition. We have not tested tumors directly from patients for MHC class II expression. However, although neither of the 2 diffuse midline glioma cell lines SJPDGF1 and SF7761 express detectable MHC class II molecules in normal cell culture, pretreatment for 24 h with human IFN-γ did induce class II expression (200-fold, SJPDGF1; 1000-fold, SF7761). So, although we do not know if patient tumors in situ express class II, patient-derived lines (cultured for multiple passages in vitro) have the capacity to do so. Taken together, these data are consistent with a model in which Ad-CD40L induces a population of CD4+ T cells which exerts anti-proliferative effects on GL261 tumors growing in situ, in part through an in vivo upregulation of MHC class II on the tumor cells as a result of the pro-inflammatory tumor microenvironment induced by Ad-CD40L. Therefore, CD4+ T cells were necessary to activate antigen-presenting cells, B cells and microglia, and were critical mediators of Ad-CD40L therapy—at least in part through a highly anti-proliferative activity against tumor cells. Intratumoral Injection of Ad-CD40L Improves Survival of Glioma-Bearing Mice Next, we assessed efficacy and toxicity of Ad-CD40L in an intratumoral injection model of established brainstem tumors. Direct injection of Ad-GFP at 106 pfu did not prolong survival of mice, all of which died of tumor growth (Figure 4A). When mice were treated with 106 pfu of Ad-CD40L, significant acute toxicity was observed, with loss of up to 20% body weight (Figure 4B) and some deaths (Figure 4A) within the first 30 days post treatment. However, with supportive care (fluid treatment), about 40% of these mice recovered and survived long term (Figure 4A, B). Treatment with Ad-CD40L was dose dependent as neither 5 × 104 nor 105 pfu significantly extended survival through tumor treatment (Figure 4A), but neither did they induce high-level toxicity (Figure 4B). Significant numbers of long-term tumor cures could be induced by increasing the dose of Ad-CD40L as high as 5 × 106 or 107 pfu, compared with mice treated with Ad-GFP at 107 pfu (Figure 4C), when fluids and nutrient support were provided over a period of 2 weeks (Figure 4D). Fig. 4 Open in new tabDownload slide Intratumoral injection of Ad-CD40L results in tumor clearance with acute toxicity. (A, C) Survival curves of tumor-bearing mice treated with viruses. (B, D) Percent body weight loss of GL261-bearing mice treated with Ad-CD40L. Tumor bearing mice were intratumorally injected with viruses. On day 8, total RNA was isolated for RNA-seq analysis. (E) Top 12 canonical pathways compared between Ad-GFP and Ad-CD40L. (F) Heat map of genes expressed in Ad-GFP or Ad-CD40L over PBS (expression log ratio ≥3) in top 5 canonical pathways. Fig. 4 Open in new tabDownload slide Intratumoral injection of Ad-CD40L results in tumor clearance with acute toxicity. (A, C) Survival curves of tumor-bearing mice treated with viruses. (B, D) Percent body weight loss of GL261-bearing mice treated with Ad-CD40L. Tumor bearing mice were intratumorally injected with viruses. On day 8, total RNA was isolated for RNA-seq analysis. (E) Top 12 canonical pathways compared between Ad-GFP and Ad-CD40L. (F) Heat map of genes expressed in Ad-GFP or Ad-CD40L over PBS (expression log ratio ≥3) in top 5 canonical pathways. RNA-seq transcriptional profiling 8 days post-injection of tumor-bearing mice treated with PBS, Ad-GFP, or Ad-CD40L using Ingenuity Pathway Analysis (https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis/) showed that Ad-CD40L induced genes involved in signaling pathways of neuroinflammation, T and B cell signaling, Th1 and Th2 activation, communication between innate and adaptive immune cells, and dendritic cell maturation (Figure 4E). Among genes which were most differentially expressed in each pathway (log2FC≥ 3), IL-1β, IL-6, IFN-γ, tumor necrosis factor alpha (TNF-α), CD40, IL-12-β, and Toll-like receptor 2 were shared between each of the top 5 pathways (Figure 4F). Consistent with Figure 2, genes responsible for B- and T-cell recruitment were also strongly upregulated in Ad-CD40L treatment. Thus, Ad-CD40L activated pro-inflammatory cytokines/chemokines, which are candidates as mediators of efficacy, toxicity, or both. Consistent with the ability of Ad-CD40L to reverse the immune suppressive tumor microenvironment, treatment with Ad-CD40L induced both significant reductions in the expression of immune suppressive factors, such as transforming growth factor beta and forkhead box P3, as well as increased expression of other cytokines, including IFN-γ and IL-6, compared with PBS or Ad-GFP treatment (Supplementary Figure 1). Ad-CD40L Therapy Is Associated with Tertiary Lymphoid Structure-Like Formation Brains of tumor-bearing mice 9 days post treatment showed reproducibly smaller, or the absence of, tumors following treatment with Ad-CD40L (Figure 5A, left panel) along with consistently higher levels of CD4+ T cells (Figure 5A, middle panel). B and CD3+ T cells were present along the meninges of Ad-CD40L-treated brains, organized in a manner that resembled TLS as found in inflamed or tumoral tissue22 (Figure 5A, right panel). In this respect, levels of claudin-5, a marker of vessel formation in the brain,23 were significantly upregulated following Ad-CD40L treatment compared with PBS or Ad-GFP (Supplementary Figure 1G) The formation of these TLS and increased vascularization are compatible with a wound-healing type of response to the inflammatory effects of Ad-CD40L. Brains harvested on days 2, 3, and 8 post-infection showed significantly increased numbers of CD45+ BILs following treatment with Ad-CD40L compared with Ad-GFP, as early as 72 hours post-infection (Figure 5B). Ad-CD40L also significantly increased CD11b+CD11c+, CD11b+Gr1+, CD11c+II+, CD45midCD11b+CD40+, and CD45midCD11b+II+ cells (Supplementary Figure 2). Similar to the pre-infection model, the majority of total cells in Ad-CD40L–treated mice were CD4+ T, CD8+ T, and B cells (Figure 5C–E). Interestingly, when Ad-CD40L was intracranially injected without tumor present, total BILs or CD45+ cells were not significantly different in mice injected with Ad-GFP or Ad-CD40L (Supplementary Figure 3A, B). Notably, we detected a marginal enhancement in CD8+ T and B cells (Supplementary Figure 3D, E), whereas CD4+ T and natural killer cells were decreased in Ad-CD40L-treated tumor-free animals (Supplementary Figure 3C, F). Therefore, the specific profile of immune cells depends on the presence of tumors in the brain. Fig. 5 Open in new tabDownload slide Intratumoral injection of Ad-CD40L induces immune cell infiltration and antitumor antibody. (A) Representative images (40x) of tumor-bearing mice treated with viruses. Left panel, hematoxylin/eosin staining; middle panel (arrow), brain tissues stained with DAPI, B220, and CD4 (scale bar, 100 µm); right panel (inset), brain tissues stained with dapi, B220, and CD3 (scale bar, 50 µm). (B–F) Flow analysis of brains treated with PBS, Ad-GFP, or Ad-CD40L. Numbers of total (B) CD45+, (C) CD4+ T, (D) CD8+ T, (E) CD19+ cells. Error bars ± SD (n = 3). Student’s t-test; *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. (F) Average of median fluorescent intensity of IgG levels. Error bars ± SD (n = 3). Student’s t-test; *P ≤ 0.05, **P ≤ 0.01. (G) Mice injected with GL261 brainstem tumors on day 1 were injected with control IgG or with depleting antibodies against CD4+T, CD8+T, or natural killer cells on days 6, 8, 10, and 12. Mice (n = 6) were injected intratumorally with PBS or with 1 × 106 pfu of Ad-CD40L on day 10. Survival with time is shown. Fig. 5 Open in new tabDownload slide Intratumoral injection of Ad-CD40L induces immune cell infiltration and antitumor antibody. (A) Representative images (40x) of tumor-bearing mice treated with viruses. Left panel, hematoxylin/eosin staining; middle panel (arrow), brain tissues stained with DAPI, B220, and CD4 (scale bar, 100 µm); right panel (inset), brain tissues stained with dapi, B220, and CD3 (scale bar, 50 µm). (B–F) Flow analysis of brains treated with PBS, Ad-GFP, or Ad-CD40L. Numbers of total (B) CD45+, (C) CD4+ T, (D) CD8+ T, (E) CD19+ cells. Error bars ± SD (n = 3). Student’s t-test; *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. (F) Average of median fluorescent intensity of IgG levels. Error bars ± SD (n = 3). Student’s t-test; *P ≤ 0.05, **P ≤ 0.01. (G) Mice injected with GL261 brainstem tumors on day 1 were injected with control IgG or with depleting antibodies against CD4+T, CD8+T, or natural killer cells on days 6, 8, 10, and 12. Mice (n = 6) were injected intratumorally with PBS or with 1 × 106 pfu of Ad-CD40L on day 10. Survival with time is shown. Sera from tumor-bearing mice treated with Ad-CD40L significantly increased IgG levels specifically against GL261, but not against normal brain-derived mixed astrocyte cells24 as a negative control (Figure 5F), showing that Ad-CD40L induces both adaptive and humoral antitumor immune responses. However, the role of these tumor-specific antibodies remains unclear, since depletion of B cells had no significant effects on therapy (Figure 3G). Finally, consistent with the results of Figure 3G with the pre-transduced model, depletion of CD4+ T cells completely abolished in vivo therapy following direct delivery of Ad-CD40L virus to established tumors (Figure 5G). In this model, however, depletion of CD8+ T cells also reduced therapeutic efficacy compared with CD8+ intact mice, suggesting that effector CD8+ T cell responses may also play a role in therapy of direct virus delivery (Figure 5G). Replication-Competent Adenovirus Expressing CD40L Improves Antitumor Efficacy While Minimizing Off-Tumor Toxicity We hypothesized that off-target expression of Ad-CD40L in nontumor brain tissue led to pro-inflammatory cytokine/chemokine production and the acute toxicity of Figure 4. Therefore, for clinical translation of CD40L therapy for diffuse midline gliomas, it will be important to restrict CD40L expression to tumor cells in vivo. To achieve this goal, we exploited the tumor-specific targeting conferred upon the replication competency of adenoviruses afforded by the incorporation of a 24 bp deletion in the E1A viral gene (rAd-Δ24) responsible for retinoblastoma protein (pRB) binding.25 Since many gliomas, including diffuse midline gliomas, have defects in the p16/retinoblastoma (Rb)/E2F pathway,26,27 the 24 bp deletion in the E1A gene confers rAd-Δ24 with high levels of selectivity to glioma cells. Therefore, we constructed rAd-Δ24-CD40L in which expression of CD40L will ultimately be under the control of the highly tumor selective replication of the virus. To validate the replication and antitumor efficacy of this virus in human tumors, the patient-derived diffuse midline glioma cell line SJPDGF1 was infected with rAd-Δ24 expressing luciferase (rAd-Δ24-luc). Virus infection and transgene expression were observed within 3 days, which decreased with time due to the cytotoxicity associated with replication and oncolysis (Figure 6A). Consistent with this, rAd-Δ24-luc and rAd-Δ24-CD40L killed almost 60% of SJPDGF1 cells by day 6 (Figure 6B), results which were reproduced in a second human patient–derived diffuse midline glioma cell line, SF7761 (Figure 6B). Fig. 6 Open in new tabDownload slide rAd-Δ24-CD40L treats brainstem tumors. (A) SJPDGF1 cells were infected with rAd-Δ24-luc at different MOIs and luciferase activity measured. (B) SJPDGF1 or SF7761 cells were infected with rAd-Δ24-luc or rAd-Δ24-CD40L and cell viability assessed by MTT assay. (C) Survival curves of GL261 tumor-bearing mice injected with PBS, rAd-Δ24-luc or rAd-Δ24-CD40L ± anti-IL-6 and anti-IL-1β neutralizing antibodies. *P ≤ 0.05, **P ≤ 0.01. (D) Percent body weight loss of GL261-bearing mice treated with rAd-Δ24s. (E) Survival curves of CT2A tumor-bearing mice injected with PBS, rAd-Δ24-luc or rAd-Δ24-CD40L. **P ≤ 0.01. (F) Percent body weight loss of CT2A-bearing mice treated with rAd-Δ24s. (G) Representative images of hematoxylin/eosin staining. (H) Tumor area of GL261-bearing mice treated with rAd-Δ24s. Error bars ± SD (n = 3). Fig. 6 Open in new tabDownload slide rAd-Δ24-CD40L treats brainstem tumors. (A) SJPDGF1 cells were infected with rAd-Δ24-luc at different MOIs and luciferase activity measured. (B) SJPDGF1 or SF7761 cells were infected with rAd-Δ24-luc or rAd-Δ24-CD40L and cell viability assessed by MTT assay. (C) Survival curves of GL261 tumor-bearing mice injected with PBS, rAd-Δ24-luc or rAd-Δ24-CD40L ± anti-IL-6 and anti-IL-1β neutralizing antibodies. *P ≤ 0.05, **P ≤ 0.01. (D) Percent body weight loss of GL261-bearing mice treated with rAd-Δ24s. (E) Survival curves of CT2A tumor-bearing mice injected with PBS, rAd-Δ24-luc or rAd-Δ24-CD40L. **P ≤ 0.01. (F) Percent body weight loss of CT2A-bearing mice treated with rAd-Δ24s. (G) Representative images of hematoxylin/eosin staining. (H) Tumor area of GL261-bearing mice treated with rAd-Δ24s. Error bars ± SD (n = 3). Assessing the activity of rAd-Δ24-CD40L in patient-derived xenografts would require immunocompromised mice, thereby eliminating the immune-mediated effects of CD40L expression. Therefore, we tested the antitumor efficacy of rAd-Δ24-CD40L in 2 different murine immune competent models where either GL261 or CT2A cells were implanted into the brainstem on day 0, and rAd-Δ24 was intratumorally injected on day 10. While these murine cells only partially support adenoviral replication, they represent the effects of intact immune subsets on both antitumor efficacy and toxicity. Injection of rAd-Δ24-CD40L significantly improved survival of tumor-bearing mice over PBS or rAd-Δ24-luc in both immune-competent GL261 and CT2A scenarios, and led to at least 50% cure rates in these aggressive models (Figure 6C, E). Most importantly, rAd-Δ24-CD40L did not induce weight loss to the same degree as replication-defective Ad-CD40L (Figure 4). GL261-bearing mice treated with rAd-Δ24-CD40L lost less than 25% body weight 2 days post-infection, and recovered within 14 days (Figure 6D). Similarly, CT2A-bearing mice treated with rAd-Δ24-CD40L had weight loss less than 20% (Figure 6F). To investigate the relative toxicity of the rAd-Δ24-CD40L virus to tumor and normal brain tissue, mixed astrocyte/glial cultures from normal brains of C57BL/6 mice were infected with increasing MOI of the rAd-Δ24-CD40L virus. Despite the relatively poor ability of replication competent adenoviruses to replicate in murine cells, both CT2A and GL261 glioma cell lines showed significant cytotoxicity upon infection with rAd-Δ24-CD40L (Supplementary Figure 4), consistent with the cytotoxicity observed against human diffuse midline glioma cell lines (Figure 6B). In contrast, similar MOI of rAd-Δ24-CD40L used to infect normal astrocyte/glial cultures did not cause significant reduction in cell numbers compared with mock infection (Supplementary Figure 4). These data confirm that the Δ24 mutation confers a tight degree of targeting of virus replication upon transformed glioma cell types, as opposed to normal brain cells. Our data in Figure 4 demonstrated that both IL-6 and IL-1β were upregulated in Ad-CD40L treated tumors which were associated with high levels of toxicity. Therefore, we hypothesized that blocking these cytokines during therapy would reduce the toxicity associated with CD40L treatment while not necessarily diminishing therapy. In this respect, blockade of IL-6 and IL-1β using neutralizing antibodies—a strategy which is directly translatable to clinical studies28—allowed us to escalate the intratumoral treatment dose of rAd-Δ24-CD40L as high as 107 pfu in the GL261 model with manageable and nontoxic levels of body weight loss (Figure 6D). Treatment with neither anti-IL-6, anti-IL-1β, nor a combination of the two had any effect on therapy of GL261 tumors. Consistent with these results, histopathologic studies of the mouse brains on day 20 post-injection demonstrated that rAd-Δ24-CD40L treated mice exhibited smaller tumors than control mice (Figure 6G, H). Discussion New, aggressive therapies for diffuse midline gliomas and other pediatric brainstem tumors are urgently required. CD40/CD40L engagement has generated potent antitumor responses in multiple preclinical models in anatomic locations where high levels of inflammation can be readily tolerated.6,7 Therefore, in this study, we sought to address two major questions. First, we hypothesized that direct delivery of adenovirus expressing CD40L (Ad-CD40L) to brainstem tumors would induce immune-mediated antitumor clearance. Second, we tested whether such therapy would be associated with manageable toxicity due to immune-mediated inflammation in the brainstem. To investigate the twin components of immune-mediated efficacy and toxicity of the Ad-CD40L strategy, immune-competent preclinical models are essential. Unfortunately, we were unable to grow explanted cell lines derived from the replication competent avian-like sarcoma (RCAS) spontaneous model of diffuse midline glioma29 reproducibly in our mice. Therefore, for the current studies, we used GL261 and CT2A murine gliomas. Although these lines are not derived from diffuse midline gliomas, we have shown that they can be reproducibly grown as transplantable tumors in the pons in the anatomic location that mirrors diffuse midline glioma growth in patients.5 Therefore, although the models of therapy and delivery that we have used here are not perfect replicates of the human situation, we believe that CD40L-mediated treatment of these realistically located tumors will reflect the efficacy and, importantly, the immune-mediated toxicity associated with immunotherapy for diffuse midline gliomas. Although Ad-CD40L did not induce a direct cytopathic effect (Figure 1A), pre-infection of tumor cells before implantation into the brainstem induced complete tumor rejection and long-lasting antitumor memory without overt toxicity (Figure 1C, D). Our data show that CD4+ T cells were absolutely required for Ad-CD40L therapy, as depletion of these cells abolished APC activation and abrogated tumor rejection (Figure 3G). The data of Figure 3I, J show that, consistent with other reports of the therapeutic role of antitumor cytotoxic CD4+ T cells,20,21 this dependence upon CD4+ T cells for antitumor therapy is, at least in part, due to the generation of CD4+ T cells which exert a direct anti-proliferative effect upon target tumor cells. Central to this mechanism of action is the conversion of the tumor microenvironment from an immune quiescent phenotype to an immune inflamed phenotype which allows for upregulation of MHC class II molecules on the target tumor (Supplementary Figure 1A–C). Moreover, the cumulative data of Figures 2, 3 suggest that CD40L-activated CD4+ T cells were central in maintaining and licensing APCs to relay CD4+ T-cell help to cytotoxic T lymphocytes which have at least some role in directly delivered antitumor therapy (Figure 5G). These data are consistent with reports showing that the interaction of CD40/CD40L on APCs and activated CD4+ T cells promotes priming of potent antitumor T cells6,30,31 and helps to maintain neuroprotection through MHC class II+ APCs.32 In addition, we observed structures reminiscent of TLS around the meninges near the tumor areas in mice treated with Ad-CD40L—structures which could be a positive prognostic factor33 acting as privileged sites for local priming, activation, and proliferation of long-lasting memory Th, cytotoxic cells, memory B cells, and antibody-producing plasma cells.34–36 It may, therefore, be that these lymphoid-like aggregates found in Ad-CD40L–treated brains are local sites of antigen presentation for B- and T-cell activation.37 However, further studies are required to understand the role of these structures and the generation of the antiglioma immune responses in our model. In addition, ongoing phenotyping studies of the effector CD4+ and CD8+ T cells induced by CD40L treatment suggest that additional combination with immune checkpoint blockade may add further therapeutic potential to this approach—although additional toxicities may also be induced. The interaction of follicular CD4+ T cells and B cells promotes B cell differentiation into antibody-secreting cells.38 In this respect, sera from tumor-bearing mice treated with Ad-CD40L significantly increased IgG levels specifically against GL261, but not against normal brain-derived astrocyte cells.24 Although it is tempting to speculate that CD40L-induced antitumor antibody contributed to therapy, the depletion of B cells had no significant effects on tumor treatment and so the role of these antibodies remains to be elucidated (Figure 3G). Our data from the pre-infection model showed that tumor-restricted expression of CD40L represents an excellent candidate to induce immune-mediated rejection of tumor cells growing in the brainstem without necessarily inducing catastrophic toxicity. However, direct delivery of Ad-CD40L to established tumors did induce acute toxicity before day 30. Mice which survived that toxicity with fluid and nutrient support were cured of tumor (Figure 4A, C). We believe that this toxicity was a result of viral infection of nontumor tissue following direct injection, which did not occur with pre-infection of tumor cells with the replication-defective Ad-CD40L. Consistent with this, intracranial injection of Ad-CD40L into mice without tumors induced neurologic symptoms as early as 5 days post injection, almost certainly associated with CD40L expression in nontumor brain tissue leading to pro-inflammatory cytokines/chemokine production and acute toxicity, as supported by our RNA-seq transcriptional profile data (Figure 4). Clinically, our ultimate goal will be to restrict CD40L expression to tumor cells in vivo. Therefore, we exploited the tumor specific targeting conferred upon the replication competency of adenoviruses afforded by the incorporation of a 24-bp deletion in the E1A viral gene (rAd-Δ24) responsible for pRB binding.25 The abnormalities of p16/Rb/E2F pathway allow oncolytic adenovirus with 24-bp deletion in E1A gene to preferentially replicate in gliomas26 which have disrupted Rb function, and confers antiglioma responses.25,39 Moreover, the backbone of this virus (without CD40L) has entered clinical trials for diffuse midline glioma.39 As expected of these tumor-specific, replication competent, oncolytic viruses, both the control rAd-Δ24-luc and the rAd-Δ24-CD40L viruses were cytolytic for patient-derived diffuse midline glioma cell lines SJPDGF1 and SF7761. Therefore, although it would be possible to assess the direct oncolytic activity of rAd-Δ24-CD40L in patient-derived xenografts, these experiments would have to be performed in immune deficient mice and both viruses would likely achieve similar antitumor effects. However, tumor therapy experiments in immune deficient mice would not reflect the immune-mediated effects of CD40L expression. Therefore, to assess the combined oncolytic and immunotherapeutic effect of CD40L expressed from the virus, we tested the antitumor efficacy of rAd-Δ24-CD40L in 2 different murine immune competent models (GL261 and CT2A). While these murine cells only partially support adenoviral replication, they do reflect the effects of intact immune subsets on both antitumor efficacy and toxicity. Importantly, the rAd-Δ24-luc virus could not prolong the survival of brainstem tumor–bearing mice (Figure 6). In contrast, incorporation of CD40L into the E1A mutant Δ24 adenovirus resulted in long-term cures of 50‒70% of GL261 or CT2A brainstem tumor–bearing mice (Figure 6). Because of the very limited replication of adenoviruses in these murine cells, our results here likely underestimate the levels of CD40L transgene spread and expression (and potentially therapy) that could be achieved in a human tumor where replication is more efficient. Importantly, however, the inclusion of the transgene into this vector whose replication is restricted to tumor cells greatly ameliorated the toxicity that we had observed with the Ad-CD40L vector, and this could be entirely rescued by nutrient and fluid support. We used RNA-seq transcriptional profiling analysis (Figure 4) to improve the safety profile still further. These data showed that IL-6 and IL-1β were consistently among the most differentially expressed cytokines between all 5 pathways upregulated by CD40L treatment of brainstem tumors (Figure 4F). Since antibody neutralization of both of these cytokines is clinically useful in other scenarios,40 we hypothesized that blocking these cytokines during therapy would reduce the toxicity associated with CD40L treatment. Consistent with this, blockade of IL-6 and IL-1β allowed us to escalate the intratumoral treatment dose of rAd-Δ24-CD40L as high as 107 pfu in the GL261 model with manageable and nontoxic levels of body weight loss (Figure 6D, G, H). In conclusion, we show here that CD40L expressed from an adenoviral platform can potentially reverse the immunosuppressive environment in glioblastoma by recruiting and maintaining innate and adaptive antitumor immune responses in the brainstem. This is the first report, to our knowledge, showing that CD4+ T cells play a critical role in Ad-CD40L therapy for brainstem tumors. We also show that the tumor-restricted, replication-competent rAd-Δ24-CD40L has cytotoxicity against human patient–derived diffuse midline glioma cell lines in vitro, generates tumor cures against orthotopic brainstem gliomas in 2 different, fully immune-competent, murine models, and can be used in combination with cytokine neutralization at high intratumoral titers with readily manageable toxicity. Therefore, we believe that by building on the rAd-Δ24 backbone which is already in clinical trials for diffuse midline glioma,39 rAd-Δ24-CD40L represents a promising therapeutic for clinical evaluation as a treatment of brainstem tumors in the context of direct intratumoral injection with careful dose escalation. Funding This work was supported by the European Research Council, the Richard M. Schulze Family Foundation, Mayo Foundation, Cancer Research UK, National Institutes of Health (R01CA175386 to R.V., R01CA108961 to R.V.), the Shannon O’Hara Foundation, and Hyundai Hope on Wheels. Authorship statement Conception and design: P. Wongthida, R. Vile. Development of methodology: P. Wongthida, M. Schuelke, L. Evgin. Data acquisition: P. Wongthida, M. Schuelke, J. Thompson, T. Kottke, J. Tonne, C. Stone, J. Davies, A. Parker, C. Wetmore. Analysis and interpretation of data: P. Wongthida, M. Schuelke, L. Evgin, T. Kottke, C. Driscoll, A. Huff, R. Vile. Writing, review and/or revision of the manuscript: P. Wongthida, L. Evgin, M. Schuelke, T. Kottke, J. Thompson, C. Driscoll, A. Huff, J. Tonne, C. Stone, J. Davies, A. Parker, C. Wetmore, R. Vile. Study supervision: R. Vile Acknowledgments We thank Toni Woltman for expert editorial assistance, Dr Katayoun Ayasoufi for analysis of immune infiltrates, Christina McCarthy for mixed astrocytes, and the Genome Analysis and Bioinformatics Core Facilities for RNA-seq. Conflict of interest statement None. References 1. Guillamo JS , Doz F, Delattre JY. Brain stem gliomas . Curr Opin Neurol. 2001 ; 14 ( 6 ): 711 – 715 . Google Scholar Crossref Search ADS PubMed WorldCat 2. Cohen KJ , Jabado N, Grill J. Diffuse intrinsic pontine gliomas-current management and new biologic insights. Is there a glimmer of hope? Neuro Oncol. 2017 ; 19 ( 8 ): 1025 – 1034 . Google Scholar Crossref Search ADS PubMed WorldCat 3. Foster JB , Madsen PJ, Hegde M, et al. Immunotherapy for pediatric brain tumors: past and present . Neuro Oncol. 2019 ; 21 ( 10 ): 1226 – 1238 . Google Scholar Crossref Search ADS PubMed WorldCat 4. Donnelly OG , Errington-Mais F, Prestwich R, et al. Recent clinical experience with oncolytic viruses . Curr Pharm Biotechnol. 2012 ; 13 ( 9 ): 1834 – 1841 . Google Scholar Crossref Search ADS PubMed WorldCat 5. Schuelke MR , Wongthida P, Thompson J, et al. Diverse immunotherapies can effectively treat syngeneic brainstem tumors in the absence of overt toxicity . J Immunother Cancer. 2019 ; 7 ( 1 ): 188 . Google Scholar Crossref Search ADS PubMed WorldCat 6. Quezada SA , Jarvinen LZ, Lind EF, Noelle RJ. CD40/CD154 interactions at the interface of tolerance and immunity . Annu Rev Immunol. 2004 ; 22 : 307 – 328 . Google Scholar Crossref Search ADS PubMed WorldCat 7. Brunekreeft KL , Strohm C, Gooden MJ, et al. Targeted delivery of CD40L promotes restricted activation of antigen-presenting cells and induction of cancer cell death . Mol Cancer. 2014 ; 13 : 85 . Google Scholar Crossref Search ADS PubMed WorldCat 8. Chonan M , Saito R, Shoji T, et al. CD40/CD40L expression correlates with the survival of patients with glioblastomas and an augmentation in CD40 signaling enhances the efficacy of vaccinations against glioma models . Neuro Oncol. 2015 ; 17 ( 11 ): 1453 – 1462 . Google Scholar Crossref Search ADS PubMed WorldCat 9. Kosaka A , Ohkuri T, Okada H. Combination of an agonistic anti-CD40 monoclonal antibody and the COX-2 inhibitor celecoxib induces anti-glioma effects by promotion of type-1 immunity in myeloid cells and T-cells . Cancer Immunol Immunother. 2014 ; 63 ( 8 ): 847 – 857 . Google Scholar Crossref Search ADS PubMed WorldCat 10. Sanchez-Perez L , Kottke T, Daniels GA, et al. Killing of normal melanocytes, combined with heat shock protein 70 and CD40L expression, cures large established melanomas . J Immunol. 2006 ; 177 ( 6 ): 4168 – 4177 . Google Scholar Crossref Search ADS PubMed WorldCat 11. Galivo F , Diaz RM, Thanarajasingam U, et al. Interference of CD40L-mediated tumor immunotherapy by oncolytic vesicular stomatitis virus . Hum Gene Ther. 2010 ; 21 ( 4 ): 439 – 450 . Google Scholar Crossref Search ADS PubMed WorldCat 12. Schiza A , Wenthe J, Mangsbo S, et al. Adenovirus-mediated CD40L gene transfer increases Teffector/Tregulatory cell ratio and upregulates death receptors in metastatic melanoma patients . J Transl Med. 2017 ; 15 ( 1 ): 79 . Google Scholar Crossref Search ADS PubMed WorldCat 13. Singh M , Vianden C, Cantwell MJ, et al. Intratumoral CD40 activation and checkpoint blockade induces T cell-mediated eradication of melanoma in the brain . Nat Commun. 2017 ; 8 ( 1 ): 1447 . Google Scholar Crossref Search ADS PubMed WorldCat 14. Stanton RJ , McSharry BP, Armstrong M, Tomasec P, Wilkinson GW. Re-engineering adenovirus vector systems to enable high-throughput analyses of gene function . Biotechniques. 2008 ; 45 ( 6 ): 659 – 662, 664–668 . Google Scholar Crossref Search ADS PubMed WorldCat 15. Uusi-Kerttula H , Legut M, Davies J, et al. Incorporation of peptides targeting EGFR and FGFR1 into the adenoviral fiber Knob domain and their evaluation as targeted cancer therapies . Hum Gene Ther. 2015 ; 26 ( 5 ): 320 – 329 . Google Scholar Crossref Search ADS PubMed WorldCat 16. Uusi-Kerttula H , Davies JA, Thompson JM, et al. Ad5NULL-A20: a tropism-modified, αvβ6 integrin-selective oncolytic adenovirus for epithelial ovarian cancer therapies . Clin Cancer Res. 2018 ; 24 ( 17 ): 4215 – 4224 . Google Scholar Crossref Search ADS PubMed WorldCat 17. Caretti V , Zondervan I, Meijer DH, et al. Monitoring of tumor growth and post-irradiation recurrence in a diffuse intrinsic pontine glioma mouse model . Brain Pathol. 2011 ; 21 ( 4 ): 441 – 451 . Google Scholar Crossref Search ADS PubMed WorldCat 18. Cumba Garcia LM , Huseby Kelcher AM, Malo CS, Johnson AJ. Superior isolation of antigen-specific brain infiltrating T cells using manual homogenization technique . J Immunol Methods. 2016 ; 439 : 23 – 28 . Google Scholar Crossref Search ADS PubMed WorldCat 19. Hofman FM , Taylor CR. Immunohistochemistry . Curr Protoc Immunol. 2013 ; 103 : 21.4.1 – 21.4.26 . Google Scholar Crossref Search ADS WorldCat 20. Perez-Diez A , Joncker NT, Choi K, et al. CD4 cells can be more efficient at tumor rejection than CD8 cells . Blood. 2007 ; 109 ( 12 ): 5346 – 5354 . Google Scholar Crossref Search ADS PubMed WorldCat 21. Quezada SA , Simpson TR, Peggs KS, et al. Tumor-reactive CD4(+) T cells develop cytotoxic activity and eradicate large established melanoma after transfer into lymphopenic hosts . J Exp Med. 2010 ; 207 ( 3 ): 637 – 650 . Google Scholar Crossref Search ADS PubMed WorldCat 22. Dieu-Nosjean MC , Goc J, Giraldo NA, Sautès-Fridman C, Fridman WH. Tertiary lymphoid structures in cancer and beyond . Trends Immunol. 2014 ; 35 ( 11 ): 571 – 580 . Google Scholar Crossref Search ADS PubMed WorldCat 23. He L , Vanlandewijck M, Mäe MA, et al. Single-cell RNA sequencing of mouse brain and lung vascular and vessel-associated cell types . Sci Data. 2018 ; 5 : 180160 . Google Scholar Crossref Search ADS PubMed WorldCat 24. McCarthy KD , de Vellis J. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue . J Cell Biol. 1980 ; 85 ( 3 ): 890 – 902 . Google Scholar Crossref Search ADS PubMed WorldCat 25. Fueyo J , Gomez-Manzano C, Alemany R, et al. A mutant oncolytic adenovirus targeting the Rb pathway produces anti-glioma effect in vivo . Oncogene. 2000 ; 19 ( 1 ): 2 – 12 . Google Scholar Crossref Search ADS PubMed WorldCat 26. Fueyo J , Gomez-Manzano C, Yung WK, Kyritsis AP. The functional role of tumor suppressor genes in gliomas: clues for future therapeutic strategies . Neurology. 1998 ; 51 ( 5 ): 1250 – 1255 . Google Scholar Crossref Search ADS PubMed WorldCat 27. Diaz AK , Baker SJ. The genetic signatures of pediatric high-grade glioma: no longer a one-act play . Semin Radiat Oncol. 2014 ; 24 ( 4 ): 240 – 247 . Google Scholar Crossref Search ADS PubMed WorldCat 28. Shimabukuro-Vornhagen A , Gödel P, Subklewe M, et al. Cytokine release syndrome . J Immunother Cancer. 2018 ; 6 ( 1 ): 56 . Google Scholar Crossref Search ADS PubMed WorldCat 29. Misuraca KL , Hu G, Barton KL, Chung A, Becher OJ. A novel mouse model of diffuse intrinsic pontine glioma initiated in Pax3-expressing cells . Neoplasia. 2016 ; 18 ( 1 ): 60 – 70 . Google Scholar Crossref Search ADS PubMed WorldCat 30. Grewal IS , Flavell RA. CD40 and CD154 in cell-mediated immunity . Annu Rev Immunol. 1998 ; 16 : 111 – 135 . Google Scholar Crossref Search ADS PubMed WorldCat 31. Moran AE , Kovacsovics-Bankowski M, Weinberg AD. The TNFRs OX40, 4-1BB, and CD40 as targets for cancer immunotherapy . Curr Opin Immunol. 2013 ; 25 ( 2 ): 230 – 237 . Google Scholar Crossref Search ADS PubMed WorldCat 32. Schetters STT , Gomez-Nicola D, Garcia-Vallejo JJ, Van Kooyk Y. Neuroinflammation: microglia and T cells get ready to tango . Front Immunol. 2017 ; 8 : 1905 . Google Scholar Crossref Search ADS PubMed WorldCat 33. Sautès-Fridman C , Petitprez F, Calderaro J, Fridman WH. Tertiary lymphoid structures in the era of cancer immunotherapy . Nat Rev Cancer. 2019 ; 19 ( 6 ): 307 – 325 . Google Scholar Crossref Search ADS PubMed WorldCat 34. Germain C , Gnjatic S, Tamzalit F, et al. Presence of B cells in tertiary lymphoid structures is associated with a protective immunity in patients with lung cancer . Am J Respir Crit Care Med. 2014 ; 189 ( 7 ): 832 – 844 . Google Scholar Crossref Search ADS PubMed WorldCat 35. Kroeger DR , Milne K, Nelson BH. Tumor-infiltrating plasma cells are associated with tertiary lymphoid structures, cytolytic T-cell responses, and superior prognosis in Ovarian cancer . Clin Cancer Res. 2016 ; 22 ( 12 ): 3005 – 3015 . Google Scholar Crossref Search ADS PubMed WorldCat 36. Martinet L , Garrido I, Filleron T, et al. Human solid tumors contain high endothelial venules: association with T- and B-lymphocyte infiltration and favorable prognosis in breast cancer . Cancer Res. 2011 ; 71 ( 17 ): 5678 – 5687 . Google Scholar Crossref Search ADS PubMed WorldCat 37. Mitsdoerffer M , Peters A. Tertiary lymphoid organs in central nervous system autoimmunity . Front Immunol. 2016 ; 7 : 451 . Google Scholar Crossref Search ADS PubMed WorldCat 38. Borst J , Ahrends T, Bąbała N, Melief CJM, Kastenmüller W. CD4+ T cell help in cancer immunology and immunotherapy . Nat Rev Immunol. 2018 ; 18 ( 10 ): 635 – 647 . Google Scholar Crossref Search ADS PubMed WorldCat 39. Tejada S , Alonso M, Patiño A, Fueyo J, Gomez-Manzano C, Diez-Valle R. Phase I trial of DNX-2401 for diffuse intrinsic pontine glioma newly diagnosed in pediatric patients . Neurosurgery. 2018 ; 83 ( 5 ): 1050 – 1056 . Google Scholar Crossref Search ADS PubMed WorldCat © The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] © The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.