bluuebunny/crossref_metadata_2025_split huggingface dataset:
P06.03.A IMMORTALIZED MACROPHAGES AS A TOOL TO STUDY PHAGOCYTOSIS AND POLARIZATION IN VITRO
Authors: RoyChoudhury R , Hossain J A , Bjerkvig R , Miletic H 
Published: September 2023

Abstract

BACKGROUND
Macrophages, an important element of the tumour-microenvironment (TME), are classified based on polarization state: tumour inhibiting ‘classically-activated macrophages’ M1 and tumour promoting ‘alternatively-activated macrophages’ M2. It is wellknown that the glioblastoma (GBM) TME is dominated by immunosuppressive M2 macrophages contributing to tumor growth by promoting angiogenesis, invasion and immunosuppression. However, macrophage culture is difficult to maintain as the cells do not proliferate and expand, making co-culture studies challenging. In this work, we successfully immortalized and expanded bone marrow derived mouse macrophages (BMDMs) that can be used as a tool to study phagocytosis and polarization in co-culture with tumor cells.

MATERIAL AND METHODS
Primary macrophages, obtained from the bone marrow of C57/BL6 mice, were transduced with two lentiviral vectors: hTERT with antibiotic hygromycin (Hy) and Simian virus 40 (SV40) with mCherry selective markers. Viral-titers were prepared by co-transfecting HEK293T with hTERT and SV40 constructs and plasmids MDL-X (Gag/Pol):Vpx. Vpx is a virion-packaged accessory protein that induces degradation of SAMHD1, a GTP- activated dNTP hydrolase that inhibits lentiviral infection of macrophages. BMDMs transduced with hTERT were subjected to gradient Hy selection after 5 days infection and SV40.mCherry positive cells were sorted by flow cytometry. To assess phagocytotic capacity, BMDMs were incubated with apoptotic bodies of a murine GBM cell-line (GL261) which was treated with HSV-TK/ganciclovir (GCV) suicide gene therapy. Phagocytosis was measured by fluorescence microscopy and flow cytometry. Further, BMDMs were externally induced with either E. coli lipopolysaccharide (LPS) and interferon gamma (IFNγ) or interleukin 4 (IL4) to activate M1 and M2 polarization. Flow cytometry was used to detect iNOS, CD40 and CD86 markers for M1 phenotype and Arginase1 and CD206 for M2.

RESULTS
hTERT.MDL-X.Vpx.Hy BMDM showed no clear signs of immortalization with limited proliferation followed by cell death. In contrast, SV40.MDL-X.Vpx.mCherry BMDM exhibited proliferative expansion from single colony, efficient freeze-thaw cycles and were used for further studies. BMDMs showed successful phagocytosis of apoptotic bodies from HSV-tk/GCV treated GL261 cells with an increasing capacity upto 18 hours of application. The phagocytic capacity was higher compared to commercial cell lines such as BV2 and Raw 264.7. Immortalized BMDMs tend to have a M2 phenotype without external induction and it is possible to activate M1 phenotype by LPS and IFNγ induction.

CONCLUSION
Here, we successfully immortalized bone-marrow derived macrophages and showed that they can be used as a tool to study macrophage polarization and phagocytic capability in a tumor immunotherapy context.
P06.07.B DEVELOPMENT OF A NOVEL CO-CULTURE MODEL OF GLIOBLASTOMA NEUROSPHERES WITH AUTOLOGOUS PERIPHERAL BLOOD MONONUCLEAR CELLS TO INVESTIGATE THE EFFICACY OF ONCOLYTIC ADENOVIRUS THERAPY
Authors: Stavrakaki E , van den Bossche W B L , Vogelezang L B , Teodosio C , van Dongen J J M , Dirven C M F , Balvers R K , Lamfers M L 
Published: September 2023

Abstract

BACKGROUND
The dismal prognosis of glioblastoma (GBM) patients, with a median survival of less than 15 months despite maximal therapy urgently warrants new therapeutic approaches. Cancer immunotherapies such as oncolytic virotherapy have shown encouraging results against GBM, but a large fraction of patients fails to respond to treatment. As the mechanisms that malignant cells employ to avoid elimination by the immune system are diverse, we hypothesized that development of an ex vivo co-culture model that would capture the complex interactions between different immune cell types, would allow us to explore the nature and degree of immune response induced by oncolytic viruses. To this end, we established a co-culture model of primary GBM neurospheres with autologous patient peripheral blood mononuclear cells (PBMCs).

MATERIAL AND METHODS
Different culture parameters such as co-culture media and cell ratios were explored to select the optimal co-culture setting. Furthermore, we developed a 20-color flow cytometry panel to evaluate the phenotype of the PBMCs; markers such as CD69, HLA-DR, TIGIT on T cells, as well as M1 and M2-related signatures on monocyte-derived macrophages were assessed. Co-culture supernatants were evaluated for the presence of pro-inflammatory cytokines and chemokines.

RESULTS
We established that viability and effector functions of PBMCs were retained in our co-culture conditions. Co-culture of GBM cells with PBMCs induced a pro-tumoral microenvironment. However, treatment with oncolytic adenovirus led to downregulation of M2-related signatures on monocyte-derived macrophages as well as upregulation of activation markers on CD8+ T cells. We also observed increase in a number of pro-inflammatory cytokines and chemokines such as IFNγ, TNFα, CCL4 as well as granzyme b. The extent of immune activation upon treatment, however, was distinct per patient, indicating that our system may offer a tool to assess patient-specific responses to oncolytic virus treatment. Importantly, we showed that patient exposure to dexamethasone prior to PBMC isolation did not preclude the IFNγ production in our system.

CONCLUSION
We established an autologous GBM/PBMCs co-culture model which reflects inter-tumoral heterogeneity in response to oncolytic virotherapy. This may allow the establishment of an ex vivo assay to assess and predict individual patient response to oncolytic virotherapy and possibly in the future also to other immunotherapies.
P06.11.B NEUROTOXICITY AND MANAGEMENT OF PRIMARY AND SECONDARY CNS LYMPHOMA AFTER ADOPTIVE IMMUNOTHERAPY WITH CD19-DIRECTED CAR T-CELLS
Authors: Karschnia P , Arrillaga-Romany I C , Eichler A , Forst D A , Gerstner E , Jordan J T , Ly I , Plotkin S R , Wang N , Martinez-Lage M , Winter S F , Tonn J , Rejeski K , von Baumgarten L , Cahill D P , Nahed B V , Shankar G M , Abramson J S , Barnes J A , El-Jawahri A , Hochberg E P , Johnson P , Soumerai J D , Takvorian R W , Chen Y , Frigault M J , Dietrich J 
Published: September 2023

Abstract

BACKGROUND
Chimeric antigen receptor (CAR) T-cells targeting CD19 have been established as a leading engineered T-cell therapy for systemic B-cell lymphomas; however, data for patients with CNS involvement are limited. Whether CAR T-cells represent a viable therapeutic avenue for such patients remains therefore unclear.

MATERIAL AND METHODS
We retrospectively searched our institutional database for patients with primary and secondary CNS lymphoma who were treated with CD19-directed CAR T-cells for active CNS disease. Clinical data, CNS-specific toxicities, imaging findings, and outcome were analyzed.

RESULTS
We identified 45 consecutive CAR T-cell transfusions for active CNS lymphoma at the Massachusetts General Hospital over a five-year period. This cohort includes 17 patients with primary CNS lymphoma (PCNSL; one patient with two CAR T-cell transfusions) and 27 patients with secondary CNS lymphoma (SCNSL). Mild neurotoxicity (grade 1-2) was observed after 19/45 transfusions (42.2%) and severe neurotoxicity (grade 3-4) after 7/45 transfusions (15.6%). A larger increase in C-reactive protein (CRP) levels and higher rates of neurotoxicity were detected in SCNSL. Early fever and baseline C-reactive protein levels predicted neurotoxicity occurrence. CNS response was seen in 31 cases (68.9%), including a complete response of CNS disease in 18 cases (40.0%) which lasted for a median of 11.4 ± 4.5 months. Dexamethasone dose at time of lymphodepletion (but not at or after CAR T-cell transfusion) was associated with an increased risk for CNS progression (HR per mg/d: 1.16, p = 0.031). If bridging therapy was warranted, the use of Ibrutinib translated into favourable CNS-progression free survival (5 versus 1 month, HR 0.28, CI 0.1-0.7; p = 0.010).

CONCLUSION
CD19-directed CAR T-cells exhibit promising anti-tumor effects and a favourable safety profile in patients with CNS lymphoma. Further evaluation on the role of bridging regimens and corticosteroids is urgently warranted.
P06.18.B OVERVIEW OF THE GLITIPNI TRIAL - LOCAL ADMINISTRATION OF IMMUNE CHECKPOINT INHIBITION AND/OR DENDRITIC CELLS FOLLOWING RESECTION OF RECURRENT GLIOBLASTOMA
Authors: Duerinck J , Tijtgat J , Dirven I , Geens W , Brock S , Michotte A , Geeraerts X , Stevens L , Thuyaerts S , Everaert H , Vanbinst A , Bruneau M , Rogiers A , Neyns B 
Published: September 2023

Abstract

BACKGROUND
Intravenous (IV) administration of the Immune checkpoint inhibitor (ICI) nivolumab (NIVO) for recurrent glioblastoma (rGB) has proven mostly ineffective, and the combination of IV ipilimumab (IPI) plus nivolumab (NIVO) is associated with a high incidence of irAE. Combinatorial immunotherapy strategies are likely to be more effective, and direct Intracerebral (iCE) administration following the resection of rGB could be a more effective and safer alternative to iv-dosing

METHODS
We previously demonstrated that intra-operative intracerebral (iCE) administration of the Immune checkpoint inhibitors (ICI) ipilimumab (IPI) and nivolumab (NIVO) after resection of recurrent glioblastoma (rGB) is safe and resulted in encouraging survival. The adaptive phase I Glitipni trial was further expanded with more cohorts (C) exploring the implantation of an Ommaya reservoir for intracavitary (iCav) administration of escalating doses of ICI, complementary iCE injection of increasing numbers of myeloid dendritic cells (myDC). We provide an overarching overview of all patients included in the program.

RESULTS
102 patients have been included in the Glitipni trial. In C1 and 2 (3 and 24 patients) iCE IPI and IPI+NIVO were followed by IV NIVO every two weeks. Next, we combined this peroperative backbone with postoperative iCav injection of NIVO 1-/5-/10 mg every two weeks through an Ommaya reservoir following stereotactic biopsy (C3, 16 patients) or resection (C4, 16 patients). No dose-related toxicity was seen for iCav NIVO. iCE injection of BDCA-1 and BDCA-3+ myDC (escalating from 1-/10- to 20.10E6) was added to the backbone of C3/4 with adjuvant 10mg iCav NIVO (resection in C5, 11 patients, biopsy in C6, 2 patients). In C7 (27+patients), patients had resection, iCE IPI+NIVO, and postoperative IV NIVO with iCav NIVO 10mg and IPI escalating 1/5/10mg. Dose limiting toxicity (aseptic neutrophilic pleocytosis) was encountered with iCav IPI (5-10mg Q2w). Median Overall Survival (mOS) was 38 weeks (C1/2), 23 weeks (C3), 42 weeks (C4), 44 weeks (C5/6) and 40 weeks (C7)

CONCLUSION
Peroperative iCer administration of IPI and NIVO is feasible and associated with an acceptable safety profile. A potential beneficial effect on overall survival was restricted to patients with resectable recurrences. Addition of myDC my improve PFS and OS. There is no indication for an additional benefit of adjuvant iCav IPI or NIVO. Further research will expand on iCer myDC administration and investigate complementary neo-adjuvant ICI administration.
P07.03.A COMPLETE LONG-LASTING RESPONSE TO MULTIMODAL THIRD LINE TREATMENT WITH NEUROSURGICAL RESECTION, CARMUSTINE WAFER IMPLANTATION AND DABRAFENIB PLUS TRAMETINIB IN A BRAFV600E MUTATED HIGH-GRADE GLIOMA
Authors: Fonte C , Castelli B , Tellini M , Guidi M , Giunti L , Di Nicola M , Buccoliero A , Caporalini C , Desideri I , Greto D , Genitori L , Sardi I 
Published: September 2023

Abstract

BACKGROUND
BRAF mutations are drivers of oncogenesis in a variety of tumors and they can be successfully targeted with small molecule inhibitors. In primary central nervous system tumors, BRAFV600E mutations are most frequently found in pleomorphic xanthoastrocytoma, ganglioglioma, pilocytic astrocytoma and epithelioid glioblastoma. Dabrafenib is a potent and selective inhibitor of BRAF-mutant kinase that was approved, as monotherapy or in combination with trametinib (mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor), for unresectable or metastatic BRAF-mutated melanoma, advanced non-small cell lung cancer and anaplastic thyroid cancer harboring BRAFV600E mutation.

MATERIAL AND METHODS
In February 2016, a 21-year-old female was admitted to our Unit for seizures. A brain magnetic resonance imaging (MRI) revealed a frontal contrast enhanced lesion. Neurosurgical resection was performed. The histological analysis diagnosed anaplastic pleomorphic xanthoastrocytoma (PXA) WHO grade III. A volumetric modulated radiotherapy course was delivered for a total dose of 59,4 Gy in 33 fractions with concomitant and adjuvant temozolomide therapy (Stupp protocol). In October 2017, a brain MRI showed relapsing disease. Six courses of chemotherapy with procarbazine, lomustine and vincristine (PCV) were administered, and then neurosurgical partial removal and carmustine wafer implantation were performed. The histological analysis confirmed BRAF V600E mutated anaplastic PXA.

RESULTS
In April 2019 the 24-year-old female patient with BRAF mutated anaplastic PXA started her third-line therapy with dabrafenib. The well-known tumor residue was less evident on the subsequent MRIs performed every three/four months. Given the literature data of the most effectiveness and the best control of the side effects, in August 2020 the patient was put on combined treatment with dabrafenib plus trametinib. Since December 2021 the residual tumor has not been longer visible. Last MRI evaluation, performed on January 26th 2023, showed no recurrence of the disease, four years after BRAF inhibitor treatment beginning.

CONCLUSION
Dabrafenib plus trametinib is a promising new therapy for patients affected by BRAFV600E-mutant glioma, with high overall response and manageable toxicity. Due to very poor prognosis, there are a few described cases of high-grade glioma (HGG) patients in third-line therapy, showing complete response and for such a long time. The emergence of optimized sequencing strategies and targeted agents, including multimodal treatment and systemic therapy with dabrafenib plus trametinib, will continue to broaden personalized therapy in HGG and to improve patient outcomes.
P07.04.A ELASTIC IMAGE FUSION OF INTRAOPERATIVE ULTRASOUND AND PREOPERATIVE MRI FOR IMPROVING NAVIGATION ACCURACY IN BRAIN TUMOR SURGERY
Authors: Onken J S 
Published: September 2023

Abstract

BACKGROUND
In brain surgery, neuronavigation based on preoperative MRI is limited by various physical and operational factors, such as surgery-induced brain shift. Using navigated intraoperative ultrasound (iUS) imaging and 3D iUS image reconstruction may help improve neuronavigation. However, the registration of MRI and US scans can lead to spatial inconsistencies between the two modalities. This study aims to evaluate the clinical integration of navigated iUS imagining and its potential to improve the accuracy of resection during brain tumor surgery by comparing the performance of rigid and elastic MRI-iUS image fusion algorithms during retrospective evaluation.

MATERIAL AND METHODS
N = 50 patients with brain tumors were prospectively enrolled in this trial. The neuronavigation system (BrainLab AG) used in the study allowed for the reconstruction and integration of 3D iUS imaging, which was acquired multiple times throughout the surgery using a tracked ultrasound probe (BK Medical). Prototypes of automatic rigid and elastic MRI-iUS image fusion algorithms (Brainlab Elements Image Fusion) were retrospectively applied to improve co-registration accuracy between the MRI and iUS data. To evaluate the image co-registration accuracy, anatomical landmarks were defined in the MRI and iUS scans and used to measure Euclidean distances between each landmark pair.

RESULTS
Improvements in the image quality over the course of the study indicated that skills in US handling are essential. Contrast behavior and tumor structure influence the visibility of the tumor in the US. Artifacts caused by hemostatic agents challenge the US image quality and the fluid-filled resection cavity requires experience in probe handling. In terms of image fusion, significant mismatches of the iUS and MRI data were reported. For scans acquired before resection, rigid fusion using a novel co-registration algorithm may enhance the registration accuracy with mean target registration errors for two representative patients of 6.5/8.9 mm and 3.7/3.4 mm before and after registration update, respectively. For iUS scans acquired after tumor resection, rigid fusion is inherently limited due to non-linear brain shifts. Therefore, elastic image fusion has been applied to provide deformable MR-iUS image co-registration.

CONCLUSION
Navigated iUS imaging can be integrated into clinical routine and the use of MR-iUS image co-registration for updating navigation is a promising tool to compensate brainshift during surgical resection. However, further studies are needed to quantify the accuracy and assess whether iUS could potentially replace the use of iMRI in resection control of selected cases.
P07.07.B INDUCTION AND TARGETING OF DIFFUSE MIDLINE GLIOMA IN A NOVEL HUMAN PONTINE ORGANOID MODEL
Authors: Bessler N , Ruiz Moreno C , Wezenaar A , Dommann N , Keramati F , Ariese H C R , Honhoff C , de Blank S , Barrera Román M , Alieva M , Wehrens E J , Alemany A , Stunnenberg H G , Rios A C 
Published: September 2023

Abstract

BACKGROUND
Diffuse Midline Glioma (DMG) is a rare and aggressive pediatric brain cancer with no chance of survival, highlighting a critical need for therapy development. Disease representative models can aid the search for effective treatments, but human preclinical models that reflect the unique developmental features and anatomical environment of DMG, are so far lacking. Latest research on DMG suggest a fetal neurodevelopmental origin with a stem cell-like cellular profile. Here, we developed a novel pontine hindbrain model to create de novo H3K27M DMG and applied it to study early tumorigenesis and immunotherapy response.

MATERIAL AND METHODS
Timely sequenced morphogens were applied to developing cerebral organoids, to create a new pontine patterned brain organoid model. Bulk sequencing and 3D imaging over a period of 16 weeks were used to determine ideal patterning conditions, confirm the correct brain regionality and reconstruct cellular developmental dynamics. Introducing H3K27M with common pontine mutations P53 and PDGFRA-D842V via electroporation resulted in de novo H3K27M DMG, which subsequently was characterized based on WHO-histopathological criteria and single cell sequencing. Exploiting this new human DMG model, we applied barcode-based genetic lineage tracing paired with single cell sequencing, to delineate tumorigenesis, and anti-GD2 chimeric antigen receptor (CAR) T cell therapy to investigate CAR T cell responses.

RESULTS
Our novel pontine hindbrain organoid model inherently gives rise to all relevant macroglia and pons-specific neurons, resembling the same developmental dynamics as seen in humans. Moreover, de novo DMG introduced in these pontine organoids strongly resembles patient cancer, including its cellular human-specific heterogeneity and invasive nature, outperforming existing gold standard PDX and cell line models. As a first model for investigating DMG, the applied barcoded lineage-tracing delineates cancerous transforming precursor states and how they contribute to the diverse lineage among known DMG cancer cell populations. Finally, administration of CAR T cells recreates treatment outcomes observed in patients and demonstrates a high level of CAR T cell heterogeneity.

CONCLUSION
We generated a bona fide DMG pontine hindbrain organoid model, the first human in vitro model for this specific region of the brain. The matching resemblance to patient tumor paired with the relevant healthy brain environment gives this model the potential for new insights into DMG early tumorigenesis and microenvironmental impact, as well as next generation therapy development.
P07.11.B A NOVEL CRISPR/CAS9 KNOCKOUT LIBRARY UNCOVERS PAN-ESSENTIAL MIRNAS IN HUMAN CANCER CELL LINES
Authors: Merk D J , Paul L , Tsiami F , Hohenthanner H , Mohseni-Kouchesfahani G , Walter B , Haeusser L A , Brown A , Persky N S , Root D E , Tabatabai G 
Published: September 2023

Abstract

BACKGROUND
MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that play an important role during cancer initiation and progression. Genome-wide functional screening using the CRISPR/Cas9 system is a powerful tool to uncover genetic dependencies for distinct tumor entities, and has revealed novel insights into pan-cancer fitness genes. Current libraries are focused on single guide RNAs (sgRNAs) targeting protein-coding genes, thereby limiting functional genomics-based investigations of miRNA function.

MATERIAL AND METHODS
We here designed a novel CRISPR/Cas9 knockout library of 8,107 distinct sgRNAs targeting a total of 1,769 human miRNAs. Using a total of 45 human cancer cell lines, representing 16 different tumor entities, we performed negative selection screens to identify miRNA fitness genes. Screen hits per cell line were scored on the basis of a combination of a supervised Bayesian gene essentiality analysis (BAGEL2) and an unsupervised gene ranking algorithm (MAGeCK). miRNA genes that scored across all cell lines, i.e. potentially pan-essential miRNAs, were identified using a combination of the Adaptive Daisy Model (ADaM) algorithm and the Fitness Percentile (FiPer) method.

RESULTS
In silico analyses revealed that our library has significantly lower off-target activity for protein-coding genes as compared to previously described miRNA-targeting sgRNA libraries, while providing a higher coverage of miRNAs present in the human genome. In total, only 13% (n=230) of all targeted miRNAs induced a fitness effect on one or more cell lines, and the majority (83%) of these genes induced a dependency in less than 50% of the tested cell lines. Pan-cancer fitness gene detection by ADaM or FiPer differed solely on the level of stringency. As such, ADaM and FiPer identified 34 core fitness or 54 common essential miRNAs, respectively, and almost all core fitness genes (97%) were identified as common essential. Preliminary network analyses revealed that protein-coding genes that are regulated by core fitness miRNAs are significantly enriched for cell cycle and p53 signaling networks, providing a first rationale for their dependency profiles in human cancer cell lines.

CONCLUSION
We here established a novel method for functional investigation of miRNAs by means of genome-wide CRISPR/Cas9 knockout screens. Our sgRNA library outperforms previously established libraries in terms of specificity and overall miRNA coverage, making it a novel tool to investigate miRNA function in a genome-wide fashion. We here use this library to define a first-of-its-kind genome-wide functional annotation of miRNAs, revealing a subset of pan-cancer fitness miRNAs that might regulate essential cellular processes such as cell cycle progression. Our data highlight the wide potential of this library in investigating miRNA function in diverse biological settings.