Temozolomide Chemotherapy Research Articles

DNAR-04. MICRORNA DEGRADATION ENHANCES GLIOBLASTOMA INTRA-TUMORAL HETEROGENEITY TO AUGMENT ACQUIRED TEMOZOLOMIDE RESISTANCE

Abstract INTRODUCTION O6 methyl-guanine methyltransferase (MGMT) restores alkylated DNA to its undamaged form and dictates clonal evolution in response to chemotherapy. As such, MGMT is a major determinant of glioblastoma resistance to the standard-of-care chemotherapy temozolomide. miR-181d is a MGMT suppressing microRNA, whose expression inversely correlate with survival expectation after temozolomide treatment. Here, we characterize the expression of miR-181d and MGMT in response to temozolomide treatment. Method: Laboratory characterization, single cell analysis, and murine modeling RESULTS Treatment of glioblastoma cells with temozolomide (TMZ) induced a feed-forward cascade resulting in an ataxia telangiectasia and Rad3 (ATR) and polyribonucleotide nucleotidyl transferase 1 (PNPT1) dependent degradation of miR-181d. Single-cell analyses revealed such miR-181d degradation: 1) increased mean level of MGMT expression in a cell population and 2) enhanced cell-to-cell variability in MGMT expression to increase intra-tumoral heterogeneity in DNA repair capacity. This increased intra-tumoral heterogeneity enhanced glioblastoma resistance to temozolomide through a mechanism distinct from lowering the mean MGMT expression of the tumor population. Suppression of this heterogeneity by exogenous miR-181d over-expression enhanced the tumoricidal activity of temozolomide against glioblastomas. CONCLUSION We identified an interface between DNA damage response and miRNA degradation that mediate intra-tumoral heterogeneity and subsequent temozolomide resistance. This resistance mechanism is distinct from modulation of the mean MGMT expression of the population.

DDDR-07. TARGETING THE CIRCADIAN CLOCK WITH SMALL MOLECULE COMPOUNDS RESULTS IN ANTI-TUMOR EFFECTS SPECIFICALLY IN GLIOBLASTOMA STEM CELLS

Abstract Glioblastoma multiforme (GBM) is the most common primary brain tumor, with an average survival time of less than one year following diagnosis. Treatment success remains challenging despite aggressive, multimodal current standard-of-care consisting of maximal surgical resection followed by concurrent temozolomide (TMZ) chemotherapy and radiation. The presence of GBM stem cells (GSCs) is thought to make treatment exceptionally difficult as they are highly invasive, resistant to chemoradiation, promote angiogenesis, support an immune suppressive environment, and can recapitulate the primary tumor following surgical resection. We have reported that GSCs, however, are dependent on core circadian clock proteins, Brain and Muscle ARNTL-Like 1 (BMAL1) and Circadian Locomoter Output Cycles Protein Kaput (CLOCK), which is not observed in differentiated GBM cells or normal neuronal stem cells. In this study, we explore the anti-GSC effects of multiple families of novel small molecule circadian clock compounds that either lower BMAL1 transcription (REV-ERB agonists) or inhibit BMAL1::CLOCK heterodimer transcriptional activity (Cryptochrome (CRY) stabilizers or Casein Kinase (CK) 1/2 inhibitors). Here we report that compared to compared to non-cancerous cells, U2OS human osteosarcoma cells, and differentiated GSCs (DGCs), GSCs display increased sensitivity to clock compounds at single agent and combinatory doses of clock compounds. Additionally, clock compounds are significantly more effective in targeting GSCs than TMZ and combinations of clock compounds with TMZ can lower TMZ IC50 value in different GBM cell lines and types. Application of the CRY stabilizer SHP1705 in GBM PDX patient derived xenograft (PDX) models resulted in decreased tumor growth rate and increased survival time compared to vehicle control. These results highlight the therapeutic potential of circadian clock compounds as single agents, in combinations and as adjuvants to existing therapies by specifically targeting the GSC population in GBM.

EXTH-12. CYTOCHROME C OXIDASE SUBUNIT 4 ISOFORM 1 MEDIATES ONCOLYTIC HERPES SIMPLEX VIRUS-1 EFFICACY IN HIGH-GRADE GLIOMA

Abstract RATIONALE Outcomes for patients with high-grade glioma (HGG) are dismal despite standard of care surgical resection, radiation, and temozolomide (TMZ) chemotherapy. RESULTS from our Phase I trial of pediatric patients with recurrent HGG suggest oncolytic herpes simplex virus-1 (oHSV) is safe with promising efficacy. However, optimization of oHSV therapy is needed to achieve more durable responses. Based on our prior work with matched pair HGG cell lines and patient-derived xenografts (PDXs) with and without acquired TMZ resistance, oHSV efficacy is enhanced in TMZ-resistant cells with no difference in oHSV entry receptor (CD111) expression. Microarray transcriptomic data of four PDXs with and without TMZ resistance revealed downregulation of interferon (IFN) responses. Cytochrome c oxidase subunit 4 isoform 1 (COX4I1) is expressed in recurrent HGG samples and cell lines. While COX4I1 positively regulates influenza virus replication, no study has investigated the role of COX4I1 in mediating oncolytic virotherapy efficacy. HYPOTHESIS We hypothesized that COX4I1 expression mediates HGG sensitivity to oHSV through suppression of IFN signaling. METHODS Using previously published models, oHSV cytotoxicity was assessed in vitro using Alamar Blue and CellTiter-Glo assays. oHSV infectivity and CD111 protein expression was assessed by flow cytometry. COX4I1 protein expression was measured by immunoblot. mRNA expression of IFN-stimulated genes was assessed by qPCR. RESULTS COX4I1 overexpression increased oHSV cytotoxicity and infectivity compared to empty vector controls. Short hairpin RNA knockdown of COX4I1 decreased oHSV cytotoxicity and infectivity in TMZ-resistant cells compared to pLKO controls. IFN-stimulated genes IFNB1, TMEM173, DDX58, IFIH1, and OAS1 were all significantly decreased in COX4I1 overexpressing cells and TMZ-resistant cells compared to parental lines. CONCLUSIONS Overexpression of COX4I1 sensitizes HGG cells to oHSV, and COX4I1 knockdown de-sensitizes TMZ-resistant HGG cells to oHSV, suggesting a novel role of COX4I1 in mediating HGG sensitivity to oHSV.

CTNI-58. UPDATED RESULTS AND MOLECULAR SUBGROUP ANALYSES FROM THE RANDOMIZED PHASE 3 MIRAGE TRIAL ON MARIZOMIB IN PATIENTS WITH NEWLY DIAGNOSED GLIOBLASTOMA

Abstract BACKGROUND The standard of care for patients with newly diagnosed glioblastoma includes maximum safe resection followed by radiotherapy (RT) with concomitant and maintenance temozolomide (TMZ) chemotherapy (TMZ/RT→TMZ). Among potential new therapeutic strategies, targeting the proteasome has been considered promising because of its implication in numerous biological processes in tumors cells. Marizomib is a novel small molecule proteasome inhibitor that crosses the blood brain barrier and showed signs of activity in recurrent glioblastoma. METHODS EORTC 1709/CCTG CE.8 was a randomized, controlled, multicenter, open label phase 3 superiority trial. Major inclusion criteria were histologically confirmed newly diagnosed glioblastoma, age of 18 years or older and a Karnofsky performance status (KPS) > 70. Patients with tumors known to harbor an isocitrate dehydrogenase mutation were not eligible. RESULTS The trial was opened at 82 institutions in Europe, Canada and the US and 749 patients (of 750 planned) were randomized in a 1:1 ratio. Following a pre-planned interim analysis and a recommendation of the independent data monitoring committee, enrolment was discontinued. No significant difference in the median OS (mOS) was observed (standard arm: 17 months, marizomib arm: 16.5 months; p = 0.64; HR = 1.04). Median PFS was 6.0 vs. 6.3 months (p = 0.67; HR = 0.97). Patients with tumors harboring an unmethylated O6-methylguanine DNA methyltransferase (MGMT) promoter had a mOS of 14.5 months in the standard therapy group and 15.1 months in the marizomib arm (p = 0.27, HR = 1.13). Among patients with an MGMT promoter-methylated tumor, median OS was 29.4 months in the marizomib arm and 25.5 months in the standard arm (p = 0.41, HR = 0.86). More CTCAE grade 3/4 treatment-emergent adverse events were noticed in the marizomib arm than in the standard arm. CONCLUSIONS The addition of marizomib to standard temozolomide-based radiochemotherapy was associated with more toxicity but did not confer a survival benefit in glioblastoma patients, independent of the MGMT promoter methylation status.

EXTH-05. ANTI-TUMOR IMMUNE RESPONSE OF ONCOMAGNETIC MONOTHERAPY IN GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) is the most lethal primary malignant brain tumor with a median survival of 15–20 months. Concurrent temozolomide (TMZ) chemotherapy and radiation (XRT) remain the current standard of care (SOC) treatment for newly diagnosed GBM. The addition of tumor treating fields (TTFs) improves median survival for newly diagnosed GBM from 16 to 20.9 months. This modest improvement underscores the urgent need to identify a new treatment modality as a safe and effective therapy. PURPOSE The noninvasive Oncomagnetic device developed in our laboratory selectively kills glioblastoma (GBM) in vitro by generating the spinning oscillating magnetic field (sOMF) which disrupts electron transport chain, leading to a marked increase in reactive oxygen species (ROS) and consequently to cancer cell death. We studied the cellular and molecular effects of sOMF treatment on GBM cell lines and investigated the immune response to Oncomagnetic monotherapy (OMT) in a syngeneic mouse model. RESULTS sOMF significantly reduces cell proliferation and cell survival in vitro . It induces DNA damage and arrests cells in G1-phase of the cell cycle. Whole-body sOMF stimulation caused a substantial retardation of tumor growth and reduction of the contrast-enhanced tumor volume in 9.4T MRI scans. OMT showed significant increase in overall survival in treated mice. We also observed upregulated immune response indicated by RNA sequencing data obtained from sOMF-treated LN229 cells as well as in treated GBM mouse tumor sections by imaging mass cytometry analysis. The tSNE analysis of various immune clusters suggests significant increase in immune response by activating CD4+, CD8+, murine-macrophages and M1-macrophages located at tumor site in the treated group. CONCLUSION The obtained data indicate significant anti-tumor response by inhibiting cancer signaling pathways and an increased immune response. Our findings suggest unique mechanism of action for sOMF stimulation and provide a strong rationale for standalone OMT for GBM.

CTNI-18. DESIGNING AND DELIVERING A RANDOMISED CONTROLLED TRIAL OF MEDICINAL CANNABINOIDS IN RECURRENT GLIOBLASTOMA - THE ARISTOCRAT TRIAL

Abstract BACKGROUND ARISTOCRAT is a randomised, double-blind, placebo-controlled phase II trial of temozolomide chemotherapy, with or without cannabinoids (Nabixomols) in patients with MGMT-methylated recurrent GBM and has recently opened to recruitment across the UK. METHODS A multi-disciplinary team of co-applicants and trial management group (TMG) members was assembled comprising clinicians, statisticians, trialists, Patient & Public Involvement (PPI), pharmacists and quality of life (QoL) researchers to design a pragmatic phase II randomised study. The protocol randomises on a 2:1 ratio between Nabiximols or matched placebo alongside standard dose temozolomide in patients at first recurrence of IDH wild-type GBM. Patients titrate to an individualised dose based on tolerance to a maximum of 12 oro-mucosal sprays per day. The primary outcome measure is overall survival time with key secondary outcomes including survival at 6, 12 and 24 months, progression-free survival time, health-related QoL and adverse events. Intermediate assessment of feasibility to confirm safety, compliance and achievability of recruitment will be undertaken after 40 patients are randomised. PPI input has been critical in ensuring sensitive and appropriate patient & public messaging and in developing patient information videos. RESULTS ARISTOCRAT was rapidly funded via a novel crowdfunding campaign organised by The Brain Tumour Charity during the COVID-19 pandemic. The trial office has since responded to >150 patient enquiries, demonstrating the high level of patient and public enthusiasm for this trial. Coverage in the press also shows a more broad level of interest in the use of cannabis-derived medicines. The study opened to recruitment on 03-Feb-2023 and the first patient was recruited on 08-Mar-2023. The study aims to recruit a total of 234 patients over an 18 month period. CONCLUSION ARISTOCRAT will provide robust clinical data on the role of cannabinoids in improving overall survival and the impact on QoL for patients with recurrent GBM.

CTNI-56. FEASIBILITY AND EFFICACY OF AI-GUIDED PERSONALIZED PRECISION RADIATION THERAPY IN NEWLY DIAGNOSED GLIOBLASTOMA: A MATCHED-CONTROL STUDY

Abstract PURPOSE The aim of this study was to evaluate the feasibility and efficacy of AI-guided personalized precision radiation therapy (PPRT) in improving outcomes for patients with newly diagnosed glioblastoma. METHODS In an open-label trial conducted at the University of Pennsylvania (NCT03477513), between August 2018 and April 2021, 17 patients with IDH-wildtype glioblastoma who underwent maximal safe resection were enrolled. They were matched with a cohort of glioblastoma patients based on age, sex, extent of resection (EOR), O6-methylguanine-DNA methyltransferase promoter (MGMTp) methylation status, and IDH status. Propensity Score Matching (PSM) was employed to assess the impact of the intervention on the likelihood of survival, using logistic regression to estimate the propensity score. Four patients were disqualified based on exclusion criteria. The PPRT group received personalized radiation dose escalation guided by AI-based predictive modeling of recurrence, along with concomitant and maintenance temozolomide chemotherapy. The control group received standard-of-care chemoradiotherapy between 2012 and 2020. In this study, we utilized a previously published and evaluated (retrospectively and prospectively) predictive AI model, which accurately estimated neoplastic cell infiltration and predicted future tumor recurrence using preoperative, multi-parametric MRIs. RESULTS Median overall survival was 24.4 months in the PPRT-temozolomide group and 17.9 months in the standard-of-care treatment group (hazard ratio, 0.34; 95% CI: 0.15-0.77; p = 0.009). PPRT was well-tolerated, with low-grade adverse events during radiation. Radiation necrosis occurred in 54% of the PPRT-temozolomide group and 36% of the standard-of-care group. CONCLUSION AI-guided PPRT in newly diagnosed glioblastoma patients demonstrated feasibility in routine clinical practice and led to a statistically significant improvement in overall survival compared to matched controls receiving standard-of-care treatment. These findings highlight the potential of personalized precision radiation therapy, with focused dose escalation, in enhancing outcomes for glioblastoma patients and emphasize the need for prospective validation in a randomized controlled clinical trial.

Targeting the non-coding genome and temozolomide signature enables CRISPR-mediated glioma oncolysis

Glioblastoma (GBM) is the most common lethal primary brain cancer in adults. Despite treatment regimens including surgical resection, radiotherapy, and temozolomide (TMZ) chemotherapy, growth of residual tumor leads to therapy resistance and death. At recurrence, a quarter to a third of all gliomas have hypermutated genomes, with mutational burdens orders of magnitude greater than in normal tissue. Here, we quantified the mutational landscape progression in a patient's primary and recurrent GBM, and we uncovered Cas9-targetable repeat elements. We show that CRISPR-mediated targeting of highly repetitive loci enables rapid elimination of GBM cells, an approach we term "genome shredding." Importantly, in the patient's recurrent GBM, we identified unique repeat sequences with TMZ mutational signature and demonstrated that their CRISPR targeting enables cancer-specific cell ablation. "Cancer shredding" leverages the non-coding genome and therapy-induced mutational signatures for targeted GBM cell depletion and provides an innovative paradigm to develop treatments for hypermutated glioma.

County-level disparities in care for patients with glioblastoma.

Racial and socioeconomic disparities in neuro-oncological care for patients with brain tumors remain underexplored. This study aimed to analyze county-level disparities in glioblastoma (GBM) care in the United States, focusing on access to surgery and the use of adjuvant temozolomide chemotherapy and radiation therapy. Using repeated cross-sectional data from the Surveillance, Epidemiology, and End Results 17 database; the Area Health Resources File; and the American Community Survey, from 2010 to 2019, the authors performed multivariate regression analyses to understand the associations between county-level racial and socioeconomic characteristics, as well as the rates of surgery performed, delays in surgery, and use of adjuvant chemotherapy and radiation therapy for newly diagnosed GBM. In total, 29,609 GBM patients from 602 different US counties over a decade were included in this study. Counties with lower rates of surgery for GBM were associated with a higher percentage of Black residents (coefficient [CE] -0.001, 95% CI -0.002 to 0; p < 0.05) and being located in the Midwest (CE -0.132, 95% CI -0.195 to -0.069; p < 0.001) or West (CE -0.127, 95% CI -0.189 to -0.065; p < 0.001) relative to the Northeast. Counties with delayed surgical treatment were more likely to lack neurosurgeons (adjusted OR [aOR] 2.52, 95% CI 1.77-3.60; p < 0.001), have a higher percentage of Black residents (aOR 1.011, 95% CI 1.00-1.02; p < 0.05), and be located in the Midwest (aOR 3.042, 95% CI 1.12-8.24; p < 0.05) or West (aOR 3.175, 95% CI 1.12-8.97 p < 0.05). Counties with high rates of adjuvant radiation therapy were less likely to have higher percentages of Black residents (aOR 0.987, 95% CI 0.980-0.995; p < 0.01) and uninsured individuals (aOR 0.962, 95% CI 0.937-0.987; p < 0.01). Counties without neurosurgeons and those with a higher percentage of Black patients have delays in surgical care and demonstrate lower overall rates of surgery and adjuvant therapy for GBM. This study underscores the need for targeted interventions and policies that address structural barriers in healthcare access, improve equitable distribution of the neurosurgery workforce, and ensure timely and comprehensive GBM care to all populations.

A novel strategy to increase the therapeutic potency of GBM chemotherapy via altering parenchymal/cerebral spinal fluid clearance rate

Patients with glioblastoma (GBM) face a poor prognosis with a median survival of less than two years. Escalating the dose of chemotherapy is often impossible due to patient comorbidities; thus, we focused on modulating brain clearance as a mechanism to enhance drug accumulation. Given the recently identified interconnectivity between brain parenchymal fluid and cerebral spinal fluid (CSF), we reasoned enhancing drug concentration in the CSF also increases drug concentration in the parenchyma where a GBM resides. To improve drug accumulation in the CSF, we impair the motility of ependymal cell cilia. We identified FDA-approved therapeutics that interact with cilia as a “side effect.” Therapeutics that inhibit airway cilia also inhibit ependymal cilia. Multiple cilia-inhibiting drugs, when administered in combination with GBM chemotherapy temozolomide (TMZ), significantly improved the overall survival of mice bearing orthotopic GBM. Combining TMZ with lidocaine results in 100% of animals surviving tumor-free to the study endpoint. This treatment results in a ~ 40-fold increase in brain TMZ levels and is well-tolerated. Mice bearing MGMT methylated, human PDX orthotopic GBM also responded with 100% of animals surviving tumor-free to the study endpoint. Finally, even mice bearing TMZ-resistant, orthotopic GBM responded to the combination treatment with 40% of animals surviving tumor-free to the study endpoint, implying this strategy can sensitize TMZ-resistant GBM. These studies offer a new concept for treating malignant brain tumors by improving the accumulation of TMZ in the CNS. In the future, this regimen may also improve the treatment of additional encephalopathies treated by brain-penetrating therapeutics. SignificanceWe exploit the interconnectivity of parenchymal and cerebral spinal fluid to enhance the amount of temozolomide that accumulates in the central nervous system to improve the survival of mice bearing brain tumors.

BCL2A1 is associated with tumor-associated macrophages and unfavorable prognosis in human gliomas.

B-cell lymphoma 2-related protein A1 (BCL2A1) is a member of the BCL-2 family. Previous studies have shown that BCL2A1 is closely related to the tumorigenesis and resistance to chemotherapy of multiple solid tumors, such as breast cancer. However, the expression pattern and potential biological function of BCL2A1 in glioma remain unknown. For the first time, we found that the expression of BCL2A1 was higher in human glioma tissues than in normal brain tissues (NBTs) in both public datasets and an in-house cohort. High BCL2A1 expression was associated with advanced WHO grade, IDH 1/2 wild type and the mesenchymal (ME) subtype, and its overexpression in glioma predicted resistance to temozolomide (TMZ) chemotherapy and unfavorable prognosis. In addition, Gene set enrichment analysis (GSEA), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that BCL2A1 was significantly correlated with the immune response and immune-related pathways, and BCL2A1 expression was positively correlated with microenvironmental parameters (immune, stromal, and ESTIMATE scores) and macrophage infiltration. Interestingly, bioinformatic prediction and immunohistochemical/immunofluorescence staining analysis revealed that BCL2A1 expression was obviously associated with the tumor-associated macrophages (TAMs) markers CD68 and CCL2. Notably, knockdown of BCL2A1 significantly inhibited cell proliferation of U87 and U251 in vitro, induced smaller tumor size and prolonged survival time of mice in vivo. Co-culture experiments of macrophages and GBM cells showed that BCL2A1 knockdown inhibited macrophage migration. Meanwhile, knockdown of BCL2A1 was associated with low expression of CD68 and CCL2 in intracranial xenograft model. This may suggest that BCL2A1 promotes the progression of glioma and influences the prognosis of patients by participating in TAMs infiltration. In conclusion, these findings suggest that BCL2A1 could serve as a promising prognostic indicator and immunotherapy target in gliomas.

Intraoperative and postoperative complications for repeat high-grade glioma resections with concurrent chemotherapy: patient series.

High-grade gliomas are aggressive primary brain tumors, the most common of which is glioblastoma multiforme. Despite advances in treatment, the prognosis for these patients remains poor. The most common chemotherapeutic agents used in the treatment of this pathology include temozolomide (TMZ), procarbazine, lomustine, and vincristine. It is unclear whether chemotherapy should be held during resection for high-grade gliomas, because the perioperative risk profile is not clearly defined. The authors report a case series of 18 surgeries to investigate the effects of concurrent TMZ and lomustine chemotherapy on surgical complications in patients undergoing repeat resection for recurrent high-grade gliomas. The authors found no postoperative infections, self-limiting postoperative complications, or excessive intraoperative blood loss and found one intraoperative complication. There may not be a need to pause TMZ and lomustine chemotherapy during recurrent resections for high-grade gliomas, and continuing these medications throughout the perioperative period may be appropriate. This case series suggests that patients receiving TMZ and lomustine chemotherapy who need a repeat resection for recurrent high-grade gliomas should consider remaining on their chemotherapy regimen because it has been shown in the literature to improve recurrence-free survival time.

Glioblastoma in a Patient with Neurofibromatosis Type 1: A Case Report and Literature Review

Neurofibromatosis type 1 (NF1) is an autosomal dominant inherited affection characterized by a higher chance of development of tumors. There have been only few cases of glioblastoma (GBM) in adult NF1 patients. In this paper, we present the case of 43 years old patient who presented initially amnesia and seizures. The patient underwent gross total surgical excision, followed by the Stupp protocol. He received concomitant chemo-radiotherapy with temozolomide then adjuvant temozolomide chemotherapy. We have also reviewed the literature and reported the cases of GBM in NF1 patients previously published.

Tumor Microenvironment Activated Photoacoustic-Fluorescence Bimodal Nanoprobe for Precise Chemo-immunotherapy and Immune Response Tracing of Glioblastoma.

Synergistic therapy strategy and prognostic monitoring of glioblastoma's immune response to treatment are crucial to optimize patient care and advance clinical outcomes. However, current systemic temozolomide (TMZ) chemotherapy and imaging methods for in vivo tracing of immune responses are inadequate. Herein, we report an all-in-one theranostic nanoprobe (PEG/αCD25-Cy7/TMZ) for precise chemotherapy and real-time immune response tracing of glioblastoma by photoacoustic-fluorescence imaging. The nanoprobe was loaded with TMZ and targeted regulatory T lymphocyte optical dye αCD25-Cy7 encapsulated by glutathione-responsive DSPE-SS-PEG2000. The results showed that the targeted efficiency of the nanoprobe to regulatory T lymphocytes is up to 92.3%. The activation of PEG/αCD25-Cy7/TMZ by glutathione enhanced the precise delivery of TMZ to the tumor microenvironment for local chemotherapy and monitored glioblastoma's boundary by photoacoustic-fluorescence imaging. Immunotherapy with indoleamine 2,3-dioxygenase inhibitors after chemotherapy could promote immunological responses and reduce regulatory T lymphocyte infiltration, which could improve the survival rate. Photoacoustic imaging has in real-time and noninvasively depicted the dynamic process of immune response on a micrometer scale, showing that the infiltration of regulatory T lymphocytes after chemotherapy was up-regulated and would down-regulate after IDO inhibitor treatment. This all-in-one theranostic strategy is a promising method for precisely delivering TMZ and long-term dynamically tracing regulatory T lymphocytes to evaluate the immune response in situ for accurate tumor chemo-immunotherapy.

Hypoxia-Inducible Transgelin-2 Confers Treatment Resistance through Activation of PI3K/Akt/GSK3β Pathway in Glioblastoma

Glioblastoma (GBM) patients with wild-type IDH experience worse survival response to the standard treatment of surgery followed by radiation therapy (RT) and temozolomide (TMZ) chemotherapy compared to their mutant IDH counterparts. This treatment has remained relatively ineffective partly due to the highly invasive phenotype of GBM leading to therapeutic resistance and tumor recurrence. Hypoxia is one of the key characteristic features of GBM which results in cancer metastasis and confers treatment resistance. Therefore, it is paramount to identify targets to help overcome hypoxia-induced treatment resistance in glioblastoma. Our lab has identified transgelin-2 (TAGLN2) to be significantly upregulated in IDH-wt GBM through multiple molecular profiling studies. This study aims to understand the mechanisms by which TAGLN2 confers treatment resistance for developing additional treatments for GBM. Additionally, active drug development efforts are also underway to target TAGLN2 for circumventing these therapeutic resistance mechanisms for effective GBM therapy. RNAi-mediated TAGLN2 knockdown (KD) approach was employed to assess the functions of TAGLN2 in GBM patient-derived xenograft (PDX) cell lines. Series of in vitro functional assays were performed to assess the role of TAGLN2 in these cell lines. Cell proliferation, invasion ± RT and/or TMZ were assessed by MTS and Trans-well invasion assays. Subsequently, WB analysis of oncogenic signaling pathways was performed following Transgelin-2 KD. Co-IP assays and Biacore/SPR analyses were performed to study the binding affinity and kinetics for the interaction of PTEN with TAGLN2. Further, cells were intracranially implanted in nude mice to assess the role of TAGLN2 on tumor growth in vivo. Conditional KD of TAGLN2 reduces cell proliferation, survival and invasive potential of GBM PDX cell lines. TAGLN2 KD also improved the sensitivity of these cells to both TMZ and radiation in vitro, as assessed by proliferation, survival, clonal expansion, and invasion. Histopathological studies of human GBM tumors and mouse xenograft tumors showed elevated expression of TAGLN2 in the peri-necrotic region of the tumors indicating that TAGLN2 protein level was upregulated by hypoxia. We also show that TAGLN2 is induced in hypoxic microenvironments with GBM PDX cell lines and its overexpression may enhance cellular resistance towards conventional therapy. Subsequently, we also show that hypoxia-induced TAGLN2 activates the PI3K/Akt oncogenic pathway through binding and inhibition of PTEN. Finally, in vivo data using an orthotopic xenograft mouse model shows reduction of tumor growth with knockdown of TAGLN2. Our in vitro and in vivo xenograft studies suggest that TAGLN2 confers treatment resistance to GBM contributing to tumor recurrence. Altogether, TAGLN2 may serve as a potential therapeutically vulnerable target in GBM specifically through its role in cell survival and invasion.

Adjuvant Chemoradiotherapy within One Year of Resection for Molecularly Defined Astrocytoma

Treatments for diffuse low-grade gliomas (LGG) are controversial. Level I evidence supports the use of adjuvant radiotherapy (RT) and PCV chemotherapy for histologic LGG, but integration of molecular biomarkers in recent WHO classification and the emergence of temozolomide chemotherapy for gliomas necessitates additional investigation of the optimal treatment and timing of postoperative interventions. We hypothesized molecularly-defined LGG (IDH-mutant astrocytoma (astro) and IDH-mutant, 1p/19q-codeleted oligodendroglioma (oligo)) may have different clinical outcomes following adjuvant RT (aRT) with chemotherapy (aRT+chemo) vs observation or chemo alone. A retrospective analysis of consecutive adult patients diagnosed with WHO Grade 2 astrocytoma or oligodendroglioma who underwent initial resection at a single institution from January 1998 to November 2017 was performed. Wilcoxon rank sum and Chi-squared tests were used to compare continuous and categorical variables, respectively. Survival analyses were performed using the Kaplan-Meier method and Cox proportional hazards models. Patients without clinical progression or death were censored at the date of last follow-up. Pre-operative and post-operative T2 FLAIR hyperintense tumor volumes were quantified using 3D Slicer to calculate extent of resection (EOR). A total of 342 patients with molecularly-defined LGG (178 astro, 164 oligo) were identified with a median follow up of 9.1 yr. 171 (50%) patients received RT during their treatment course, of which 31 (18%) were treated with aRT within 1 year of diagnosis. The median aRT dose was 54 Gy (range: 40-60 Gy). aRT was more likely for astro (58%) vs oligo (41%, p = 0.001) and for patients who had resections with lower median EOR (88% vs 95%, p = 0.014). 53 patients (15%) were treated with chemo alone, and 136 patients (40%) were treated with aRT+chemo. Temozolomide was used for 161 patients (85%). For astro, aRT+chemo was associated with longer PFS (median 14.9 yr) compared to observation (4.8 yr, p = 0.05), aRT without chemo (5.2 yr, p = 0.01), or chemo alone (4.7 yr, p = 0.02). For oligo, aRT+chemo was associated with longer PFS (median not reached) compared to aRT without chemo (1.6 yr, p = 0.03), but not when compared to observation (median not reached, p = 0.47), or chemo alone (7.9 yr, p = 0.45). Multivariate analysis showed preoperative tumor volume, EOR, and aRT+chemo (but not aRT or chemo alone) were independently associated with astro PFS compared to observation. Propensity matching based on pre-operative tumor volume, EOR, and age demonstrated longer astro PFS after aRT+chemo (14.9 yr) compared to observation or chemo alone (4.5 yr, p = 0.015), without significant difference in OS (18.2 vs. 11.5 yr, p = 0.40). Retrospective data from a single institution support the use of adjuvant radiotherapy with chemotherapy for patients with molecular astrocytomas, while the role of this approach for oligodendrogliomas is unclear in this cohort.

P03.01.A “TO BE SMART” IN FOLLOW-UP AFTER CRANIAL RADIOTHERAPY: REPORT OF TWO CASES

Abstract BACKGROUND Stroke-like Migraine Attacks after Radiation Therapy Syndrome (SMART) is a rare, delayed complication of radiation therapy and its real incidence is unknown. In this study, clinical and treatment features of two cases with clinical and radiological diagnosis of SMART are presented. METHODS Case-1: 49 years old male was diagnosed with grade-3 Oligodendroglioma; after gross total surgery treated with 60 Gy local cranial radiotherapy and temozolomide. Patient presented with acute left-sided weakness and numbness, episodic myoclonic jerking of the left arm and leg, 6 years after treatment. Contrast-enhanced MRI and MR angiography revealed no sign of a recurrent tumor but contrast uptake at right parietooccipital cortical giriform in the lobe of the right temporal lobe on post-contrast images (FLAIR and T1) and an increase in cortical blood volume are observed in perfusion MRI (Figure A-B). The clinical and radiologic diagnosis was SMART syndrome. Pulse dose steroids was planned as the primary treatment, and neurological symptoms was completely disappeared. After 3.5 years of the primary episode, patient re-applied with acute headache, homonymous hemianopsia of the left eye and left sided hemiparesia. Cranial MRI revealed the similar features and after treatment with steroids for 7 days complete radiological and clinical regression was experienced. Case-2: 60 years old female with a right occipital grade-3 oligodendroglioma treated with resection followed by local cranial radiotherapy (60 Gy) and temozolomide chemotherapy. Ten years after the treatment she presented with severe headache, expressive aphasia and homonymous hemianopsia of the left eye. Contrast-enhanced MRI and MR angiography revealed no sign of a recurrent tumor but contrast uptake at right occipital cortical giriform on post-contrast images (FLAIR and T1) and an increase in cortical blood volume are observed in perfusion MRI (Figurec. The diagnosis was SMART syndrome. After one week of steroid treatment she completely recovered. Similar complaints recurred 9 months after the first episode and completely recovered with steroids in one week. Radiological finding disappeared in 2 months. CONCLUSION SMART syndrome represents an acute-onset rare condition which may result in serious consequences if not diagnosed and treated properly. It usualy appears years after cranial radiation therapy. Clinical signs and radiological findings may resemble tumor recurrence and distinction from real tumor progression is essential. Treatment is symptomatic, most patients recover with steroids and radiological signs disappear completely, however SMART episodes may recur during the follow-up which can be treated similarly.

P17.16.B INVESTIGATION OF REGORAFENIB TUMOR SENSITIVITY AND UNDERLYING TREATMENT INDUCED MOLECULAR MECHANISMS IN GLIOBLASTOMA RECURRENT TUMORS

Abstract BACKGROUND Glioblastoma (GB) is the most aggressive brain malignant tumor with a highly unfavorable prognosis. Maximal surgery resection, temozolomide chemotherapy (TMZ), and radiotherapy (RT), currently remain the best treatment option. Recently the phase II REGOMA clinical trial, has shown that regorafenib (REGO), a multikinase inhibitor that targets angiogenic, stromal and oncogenic tyrosine kinase receptors, significantly improves survival in GB patients.The aim of this study was to investigate Regorafenib tumor sensitivity and underlying molecular mechanisms in GB recurrent tumors leveraging on an ex vivo drug response functional precision medicine approach, that we have developed recently consisting of an in vitro vital patient-derived GB 3D organoid model and NAD(P)H Fluorescence Lifetime Imaging (FLIM). MATERIAL AND METHODS In-vitro 3D GB organoids were derived from 17 patients’ resected recurrent GB tumors. For 5 of these we had the matched primary tumor, and for other 3 we collected also the tumor peripheral portion. Totally, we obtained 25 samples. After 3 days from culture, organoids were treated with TMZ and REGO and drug response, using NAD(P)H FLIM, was assessed in the following 72 hours to stratify tumors in Sensitive (S) and Non Sensitive (NS). Whole-exome was performed on 14 tissue samples, comprising 5 primary/recurrence tumor pairs and whole-transcriptome on 11 recurrent samples before and after treatment. RESULTS Stratification of tumor cases in S and NS groups was performed both for TMZ and REGO treatment. We found 32% (n=8) and 76% (n= 19) of TMZ and REGO S tumors, respectively. Interestingly there was never a TMZ S case that was NS to REGO. Heterogeneity was found in the TMZ and REGO responsiveness for the tumor core/peripheral pair portions and in the primary/recurrence tumors pairs. It is noteworthy that all primary tumors were responsive to REGO.The drug-responsive stratification performed using our approach was also well reflected at the molecular level, as shown from transcriptome and exome RESULTS , where differently expressed genes and mutated genes were found in S versus NS tumors. CONCLUSION GB recurrent tumors resulted more responsive to REGO when compared to TMZ treatment, as expected. Thanks to our functional precision medicine approach, we were able to study the molecular alterations occurring due to the treatment, discovering biomarkers of sensitivity or resistance.

P16.01.A THE CASE OF AGGRESSIVE DIFFUSE LEPTOMENINGEAL GLIONEURONAL TUMOR IN ADULT TREATED WITH CRANIOSPINAL IRRADIATION AND TEMOZOLOMIDE

Abstract BACKGROUND Diffuse leptomeningeal glioneuronal tumor (DLGNT) is a rare tumor, which was introduced as a separate entity in the 2016 World Health Organization classification of central nervous system tumors. It is most common in pediatric age, but very rare in adults. Literature demonstrates limited reports regarding treatment strategy in adult. We present a case of young female affected by DLGNT with aggressive behavior. MATERIAL AND METHODS A 28-year-old woman presented with severe headache and vomiting. Brain MRI showed diffuse leptomeningeal enhancement with hydrocephalus. At first, she was under suspicion of tuberculosis meningitis with mild increased protein level in CSF study. Her symptom rapidly improved after ventriculoperitoneal shunt and she started anti-tuberculosis medication. Despite 8-month of medication, her neurological status gradually deteriorated with paraparesis and follow-up MR images showed progression of disease. She underwent open biopsy of superficial brain lesions. RESULTS Intraoperatively, multiple nodular masses were attached inner side of dura mater and diffuse adhesion on subarachnoid space of brain surface. Histological examination revealed small round cells with oligodendroglial-like aspects. Immunohistochemistry showed positivity for Olig2, synaptophysin and S100. Chromosome 1p deletion was confirmed by FISH. Next-generation sequencing profile disclosed mutation of H3K28M, FGFR1 and ATRX. Diagnosis was consistent with DLGNT. She received craniospinal irradiation of 36 Gy in 20 fractions, then started chemotherapy with temozolomide (TMZ) at a dose 200mg/m2/day for 5 days in 4 weeks. After 4 cycles of TMZ, MR images demonstrated stable disease. CONCLUSION DLGNT is a very rare tumor in adult. Diagnosis needed high level of suspicion and histological confirmation. We present a very rare adult DLGNT patient with aggressive clinical features who showed stable state treated with craniospinal irradiation and TMZ chemotherapy.

P10.27.A THE INFLUENCE OF DEXAMETHASONE ON TOTAL DNA METHYLATION LEVEL IN GLIOBLASTOMA CELL LINES

Abstract BACKGROUND Dexamethasone (DEX) is a first-line antiedematic drug for glioblastoma (GBM) patients. There is also some evidence it also inhibits GBM cell proliferation and migration, thus impacting patients’ outcomes. However, other studies show that the use of DEX is associated with poor treatment outcomes in GBM patients. The exact mechanism of DEX action is unclear, and no definitive conclusion has yet been reached on its benefits in neurooncological treatment. Temozolomide (TMZ) is a first-line chemotherapeutic in GBM. Epigenetics offers a connection between genetic and environmental factors that influence the development of the disease. The best-characterized epigenetic mark is 5-methylcytosine (m5C) in DNA. The aim of that project is to show the effects of DEX administration on the total DNA methylation level. MATERIAL AND METHODS Using the nucleotide post-labeling method, we analyzed the total amount of m5C in DNA of GBM (T98G, U118, U138), cancer (HeLa) and normal (HaCaT) cell lines treated with DEX, and a combination of DEX and with TMZ. RESULTS We adjusted the DEX doses to the ones achieved in the central nervous system during treatment. We observed dose-dependent increase in total DNA methylation in all cell lines. However, the exposition of GBM cells to the combination of DEX and TMZ caused an adverse synergistic effect resulting in DNA demethylation in high doses of both drugs. In lower concentrations of both drugs DEX kept the increased DNA methylation and attenuated the demethylating effect of TMZ. CONCLUSION Total DNA methylation changes in glioma cell lines under DEX treatment suggest the new mechanism of that drug action and promote clinical implications for adjusting DEX and TMZ therapy in GBM patients. Our results show the potential and possible obstacles of the combined therapy of TMZ with DEX. Our experiments show that combined therapy with both drugs leads to total DNA hypomethylation in high doses of both drugs. Therefore the conclusion would be to adjust DEX administration during TMZ chemotherapy.