bluuebunny/crossref_metadata_2025 huggingface dataset:
P03.05.B PRIMARY GAMMA KNIFE RADIOSURGERY FOR PINEAL REGION TUMORS, A SYSTEMATIC REVIEW AND POOLED ANALYSIS OF AVAILABLE LITERATURE WITH HISTOLOGICAL STRATIFICATION
Authors: Gagliardi F , De Domenico P , Garbin E , Bailo M , Snider S , Mortini P
Published: September 2023

BACKGROUND

Pineal region tumors (PTs) represent extremely rare pathologies, characterized by highly heterogeneous histological patterns. Most of the available evidence for Gamma Knife radiosurgical (GKSR) treatment of PTs arises from multimodal regimens, including GKSR as an adjuvant modality following surgery or as a salvage treatment at recurrence. We aimed to gather the existing evidence on the topic and analyze single-patient level data to address the efficacy and safety of primary GKSR when only a biopsy was attempted or biochemical or neuroimaging diagnosis was available.

MATERIAL AND METHODS

This is a systematic review of literature (Pubmed, Embase, Cochrane, Science Direct) and pooled analysis of single-patient-level data. A total of 1054 original works were retrieved. After excluding duplicates and irrelevant works, we included 13 papers in our final analysis comprising 64 patients. Additional 12 patients were included from the authors’ original series.

RESULTS

A total of 76 patients reached the final analysis. Presumptive diagnoses included intrinsic pineal low-grade (46%), glial low-grade (LGG, 5%), pineoblastoma (4%), germ cell tumors (GCTs, 4%), pineal tumors of intermediate differentiation (3%), other pineal tumors (3%), glial high-grade (HGG, 2%) and meningioma (1%). In 32% of cases, the diagnosis was unclear. Only 57% of cases received histological confirmation. The mean age at treatment was 38.7y and the mean lesional volume was 4.2 ± 4 cc. All patients received GKSR with a mean marginal dose (50% isodose) of 14.7 ± 2.1 Gy. At a median 36mo follow-up, local control was achieved in 84% of cases. Ten patients showed progression within a mean time of 24.2mo. Progression was more common in HGG and GCTs (p=0.03) compared to intrinsic pineal tumors. Overall mortality was 13.2%. The median OS was not reached for all included lesions, except high-grade gliomas (8mo). The 3y-OS was 100% for LGG, 90% for the intrinsic pineal, 60% for GCTs, 50% for HHG, and 82% for undetermined tumors. The 3y-PFS was 100% for LGG, 90% for the intrinsic pineal, 32% for GCTs, and 0% for HHG. Median PFS was 5mo for HHG and 34mo for GCTs. The radionecrosis rate was 6%, and cystic degeneration was observed in 2%. Ataxia as presenting symptom strongly predicted mortality (OR 104, p=0.02), while GCTs and HHG histology well predicted PD (OR: 13, p=0.04).

CONCLUSION

These results support the efficacy and safety of primary GKSR treatment of pineal region tumors. Further studies are needed to validate these results, which highlight the importance of the initial presumptive diagnosis for choosing the best therapeutic strategy.
P03.07.B DOSIMETRIC ACCURACY OF CYBERKNIFE STEREOTACTIC RADIOSURGERY FOR BENIGN PERIOPTIC TUMOR
Authors: Yoon K , Jeong C , Park M , Goh Y , Kim S , Cho B , Kwak J , Cho Y
Published: September 2023

BACKGROUND

The Cyberknife (CK) SRS delivers a highly focused ablative radiation dose to tumors to effectively control tumors with minimized radiation toxicity to surrounding normal tissues. This study aimed to evaluate the dosimetric accuracy of Cyberknife (CK) for benign perioptic tumor using patient specific head phantom.

METHODS

Patient specific head phantoms was fabricated using a 3D-printer to be dosimetrically equivalent to actual target regions of benign perioptic tumor case treated via Cyberknife radiosurgery. Head phantom QA plan was produced using the original CK contour set (target and optic nerve). The head phantom containing the Gafchromic EBT3 film was irradiated using Cyberknife 6D skull tracking method. The dose distributions calculated by MultiPlan Treatment Planning System (version 5.6) was compared with those measured by film dose using gamma analysis method. After moved rotating (1 degree) and translating (1-5mm) the couch table prior to beam delivery, we checked the 6D skull tracking accuracy according to the beam irradiation.

RESULTS

Gamma passing rates for the 2%/1 mm and 2%/2 mm criteria was found to be 84.11 ± 2.12% and 93.61±1.39% greater than the acceptance criteria 80% and 90%. The difference between measured and calculated dose to the optic nerve in contact with the tumor was less than 3%.

CONCLUSION

Dosimetric verification with patient-specific head phantoms could be successfully implemented as the evaluation method for CK perioptic tumor radiosurgery delivery with 6D skull tracking system.
P04.08.A PATIENT-DERIVED ORGANOIDS FOR MODELING AND CHARACTERIZING PEDIATRIC BRAIN TUMORS
Authors: Lago C , Federico A , Leva G , Ballabio C , Gianesello M , Abballe L , Kool M , Miele E , Tiberi L
Published: September 2023

BACKGROUND

Central nervous system (CNS) tumors are the most common solid tumors in the pediatric population and the leading cause of death among childhood cancers. They present common features related to the development and produce symptoms based on their location, rate of tumor growth and age of the child. Tumors of embryonal origin, such as medulloblastoma or primitive neuroectodermal tumor, have a higher incidence in younger patients, whereas older ones tend to present tumors of glial origin. Most of CNS-embryonal tumors are highly malignant and consequently leading to poor prognoses. Despite the understanding of their underlying molecular landscape that has helped in clinical advances to improve life expectancies of the patients, there is still an emerging need to minimize morbidity and manage the long-term therapy effects.

MATERIAL AND METHODS

Human samples were collected according to protocols approved by the institutional review board with written consent. Patient-Derived Organoids (PDOs) and Patient-Derived Xenograft Organoids (PDXOs) were obtained using scalpels by chopping in small pieces the tumor tissue and were grown in PDOs medium. PDOs and PDXOs were analyzed through DNA methylation, immunostainings, sc-RNA sequencing and orthotopic nude mice engraftment. Therapeutic regimens were adapted according to patients’ treatment protocols depending on the tumor of origin.

RESULTS

Developing new models to better understand the complexity and heterogeneity of pediatric tumors and to test personalized treatment strategies remains a clinical challenge. We tried to answer this medical need with PDOs and PDXOs established through the direct in vitro culture of different kinds of primary tumors (ependymoma EPN, medulloblastoma MB and low-grade glial tumors) and MB patient derived xenograft (PDX)-derived tumors. We have developed a method that recapitulates mutational profiles, histological features, and cellular heterogeneity of the corresponding parental tumors. We have obtained promising results from the DNA methylation profile analysis, nowadays used for an accurate routine CNS tumors diagnosis. Through this new model, we confirmed the action of clinically used therapeutic regimens, i.e., for EPN with ZFTA-RELA fusion and G3 MB-derived PDOs. PDOs were subjected to the same treatment the patients underwent and responded similarly to the patients.

CONCLUSION

We found a suitable in vitro culture condition to generate and maintain PDOs from different kinds of pediatric brain tumors. Their reliability has been proved by different analyses, confirming they still resemble the parental tumors. Through this new model, we confirmed the action of clinically used therapeutic regimens, suggesting its potential at the translation level as a reliable tool for testing personalized medicine, drug screening and development of newer and more specific therapeutic strategies.
P06.02.B GSDME-MEDIATED PYROPTOSIS IN GLIOBLASTOMA
Authors: Solel E , Brudvik E , Afroz S , Choudhury R , Bjerkvig R , Hossain J , Miletic H
Published: September 2023

BACKGROUND

Immunotherapeutic strategies have largely failed for glioblastoma , the most frequent primary brain tumor. Beyond apoptosis and autophagy, little is known about cell death mechanisms in glioblastoma, despite their potential impact on the immune microenvironment. Pyroptosis, unlike apoptosis, is an immunogenic and lytic type of programmed cell death, where gasdermin proteins form pores into the plasma membrane, releasing cell content to the tumor microenvironment. Gasdermin E (GSDME), often inactivated in cancers by mutations/low expression, is proteolytically cleaved by caspase-3 upon apoptotic stimuli leading to a shift from apoptosis to pyroptosis. Here, we investigated the relevance of GSDME-mediated pyroptosis for glioblastoma. According to The Cancer Genome Atlas, GSDME is highly expressed in glioblastomas compared to many other cancers and no mutations are found.

MATERIAL AND METHODS

Using the drug raptinal to induce apoptosis, we assessed expression of full length and cleaved GSDME as well as cleaved caspase-3 in syngeneic mouse and human patient-derived glioblastoma stem-like cell lines by western blotting. Lytic cell death and viability were measured by lactate dehydrogenase (LDH) and WST-1 assays respectively. PI uptake assay was performed to capture pyroptotic morphology by Incucyte®️ S3. BAPTA-AM, a fast Ca2+ chelator, was used to block plasma membrane repair in human glioblastoma cell lines and analyze its effect on pyroptosis. GSDME was knocked out by CRISPR/Cas9.

RESULTS

GSDME and caspase-3 cleavage were confirmed after raptinal treatment in mouse and human glioblastoma cell lines, indicating that the molecular pyroptotic machinery is intact. A significant drop in viability with raptinal was shown indicating successful execution of cell death. A significant LDH release was detected in mouse glioblastoma cell lines, which could be blocked by GSDME knockout. In contrast, human glioblastoma cells, although executing GSDME cleavage, showed less or no LDH release. Pyroptotic morphology was detected after raptinal treatment in mouse glioblastoma cell lines, while it was delayed/inhibited in human glioblastoma cells. Application of BAPTA-AM in human glioblastoma cell lines enhanced pyroptotic morphology, indicating a role for plasma membrane repair in inhibiting pyroptosis.

CONCLUSION

The molecular machinery to execute pyroptosis is functioning in mouse and human glioblastoma cells. However, while mouse glioblastoma cells show clear execution of pyroptosis upon apoptosis induction, human glioblastoma cells indicate less efficient pyroptotic ability which partly can be reverted by blocking plasma membrane repair. While pyroptosis can induce anti-tumor immunity in experimental cancer models, future in vivo experiments must delineate if pyroptosis is a viable strategy for immunotherapy of glioblastoma.
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

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

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

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

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

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

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

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

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.
P07.12.A DIAGNOSTIC POTENTIAL OF INFRARED SPECTROSCOPY IN MENINGIOMA PATIENTS
Authors: Richter S , Galli R , Kirsch M , Eyüpoglu I , Uckermann O
Published: September 2023

BACKGROUND

Vibrational spectroscopy probes molecular vibrations in the tissue and can provide optical diagnosis of brain tumors. Here, we investigated its potential for the identification of aggressive meningioma.

MATERIAL AND METHODS

Tissue samples were obtained during routine surgery from 113 meningioma patients (WHO 1 n= 33; WHO 2 n=39; WHO 3 n=41). Infrared (IR) spectroscopic imaging was performed at three positions (170 x 170 µm, 16x16 = 256 spectra) on frozen sections in reference to HE histology. Spectra were labeled according to the clinical diagnosis of the patient. Baseline correction, area normalization and fourfold spectral binning was performed. Spectra displaying artifacts were identified by visual inspection and were excluded from further analysis. The data was split into test and training set. 42825 spectra of 57 specimens (training set) were utilized to develop an algorithm using linear discriminant analysis to identify aggressive WHO 3 meningioma.

RESULTS

The developed algorithm assigned 88.6% of the 40626 spectra of the independent test set to the correct class. Based on these

RESULTS

, the class assignment for each measurement position was calculated as mean of class assignment of the respective spectra: All WHO 1 (16/16) samples, 16/19 WHO 2 and 17/21 WHO 3 meningioma were correctly assigned at all three positions. One sample of meningioma WHO 3 was misclassified at all measurement positions. However, in case of six patients inconclusive results were obtained for the samples as the measurement positions were assigned to different classes. Comparison of IR spectral signatures revealed increased bands at 1114, 1242, 1336 and 1450 cm-1 and decreased bands at 1544 and 1651 and 1734 cm-1 in meningioma WHO 3. This suggests increased phospholipids and decreased proteins as main difference to meningioma WHO 1 and 2 and might form the basis for diagnostic exploitation of IR spectral information.

CONCLUSION

IR spectroscopy qualifies as reliable tool for the identification of aggressive meningioma. Based on the classification results of this study, translational strategies may be deduced. The data suggests, that analysis of the tumor needs to be performed at multiple sites to obtain reliable clinical information. In case of conflicting results at different measurement positions, the tissue should be evaluated as unratable. As infrared spectroscopy can be applied in situ using fiber based systems, it might allow intraoperative meningioma grading and tumor delineation in future translational settings.
P07.15.B NEUROMUSCULAR HIGH-RESOLUTION ULTRASOUND IN PERIPHERAL NEUROTOXICITY OF SYSTEMICALLY TREATED CANCER PATIENTS
Authors: Mäurer I , Hammersen J , Heiling B , Lawson McLean A , Risse C , Leistner N A , Schütze S , Schnetzke U , Schwab M , Hilgendorf I , Senft C , Hartinger S
Published: September 2023

BACKGROUND

Up to 40 % of cancer patients treated with neurotoxic cytostatic chemotherapies develop treatment-induced peripheral neuropathy. Furthermore, in patients treated with immune checkpoint inhibitors neuromuscular immune-related adverse events (irAEs) are the most common neurological complication. The differentiation between various types of peripheral neurotoxicity, neurological comorbidities and (para-)neoplastic conditions requires experienced clinical and neurophysiological diagnostics within an interdisciplinary team of experts. High-resolution ultrasound (HRUS) is a widely used diagnostic tool for peripheral neuropathies. As there are no generally approved biomarkers of treatment-related neurotoxicity so far, HRUS might extend the repertoire of diagnostic and neuromonitoring methods in patients with neuropathies induced by cytostatic agents, immunotherapy, and targeted therapy.

MATERIAL AND METHODS

We present the integration of HRUS into a standardized algorithm for suspected peripheral neurotoxicity by characterizing a case series from our neuro-oncological center. We show representative HRUS findings of seven cancer patients (diagnosed with breast cancer, cholangiocarcinoma, colorectal carcinoma, or multiple myeloma) treated with platinum derivates, immune checkpoint inhibitors, bortezomib, or lenalidomide.

RESULTS

We describe the potential of HRUS independently of a certain cancer treatment regimen and beyond that in patients with both neurological co-morbidities and concomitant neoplastic infiltration of the nervous system. In our retrospective cohort, HRUS revealed partly continuous and partly discontinuous nerve swelling as well as compression syndromes. Moreover, we detected enlargements of dorsal roots and vagus nerve in cancer patients with treatment-induced neuropathies for the first time. In addition, we identified significant discordance between clinical, electrophysiological and HRUS findings and discuss how peripheral neurotoxicity may be amenable to complementary HRUS assessment and neuromonitoring.

CONCLUSION

At our institution, neuromuscular ultrasound is an integral part of the interdisciplinary algorithm for peripheral neuropathies in systemically treated cancer patients. HRUS expands the neurological tool inventory for cancer patients and accelerates the differentiation of peripheral neurotoxicity, neurological comorbidities, and neoplastic affection of the nervous system in a non-invasive and cost-sparing manner. Our data may help to detect distinct patterns of nerve enlargement as new biomarkers of peripheral neurotoxicity. Thus, we employed our data to conduct a prospective, cross-sectional trial (PARTICIPATE), which has the potential to define the diagnostic relevance of HRUS for patients treated with agents affecting the PNS and its impact on cancer patient care.
P10.06.B APPLYING TUMOR TREATING FIELDS (TTFIELDS) FOR ENHANCEMENT OF TEMOZOLOMIDE AND LOMUSTINE EFFICACY IN GLIOBLASTOMA CELL LINES
Authors: Fishman H , Monin R , Dor-On E , Kinzel A , Haber A , Giladi M , Weinberg U , Palti Y
Published: September 2023

BACKGROUND

Tumor Treating Fields (TTFields) are electric fields that disrupt cellular processes critical for cancer cell viability and tumor progression, ultimately leading to cell death. TTFields therapy concurrent with maintenance temozolomide (TMZ) is approved for treatment of newly-diagnosed glioblastoma (GBM). Recently, benefit was demonstrated in patients with O6-methylguanine DNA methyltransferase (MGMT) promoter methylation for the combination of TMZ with lomustine (CCNU). The addition of adjuvant TTFields to this treatment regimen further improved patient outcomes, leading to a CE mark. The current study aimed to elucidate the mechanism underlying the benefit of this treatment protocol by utilizing GBM cell lines with different MGMT expression status.

MATERIAL AND METHODS

Human GBM cell lines with different MGMT promoter methylation status [LN-18 (unmethylated MGMT promoter), U-118 MG (partially methylated MGMT promoter), and U-87 MG and LN-229 (methylated MGMT promoter)] were examined for MGMT expression levels. Next, the cells were treated with TTFields (intensity of 0.83 V/cm RMS; frequency of 200 kHz) using the inovitro system. Cell count and colony formation were employed to detect the effectiveness of TTFields concomitant with TMZ and/or CCNU. Western blot allowed the detection of expression levels of DNA repair proteins in control versus TTFields-treated cells.

RESULTS

The cell lines displayed comparable responses to TTFields and CCNU, while they differed in sensitivity to TMZ according to MGMT expression status. TTFields concomitant with TMZ displayed an additive effect, irrespective of MGMT expression levels. The effect of concomitant TTFields with CCNU was additive in MGMT-expressing cell lines, whereas it trended toward synergy in MGMT-non-expressing cell lines. Expression of proteins from the Fanconi Anemia (FA)-BRCA pathway for DNA repair was downregulated following TTFields application. TTFields concurrent with both TMZ and CCNU provided higher efficacy relative to TMZ plus CCNU or TTFields apart, with additivity in MGMT-expressing cell lines and a tendency toward synergy in MGMT-non-expressing cell lines.

CONCLUSION

The results support the clinical benefit demonstrated for concomitant TTFields with TMZ plus CCNU, showing that the efficacy of TMZ and CCNU may be enhanced by TTFields, with synergism seen for the latter case in cells not expressing MGMT. The BRCAness state induced by TTFields rationalizes this, as in the absence of MGMT DNA damage induced by CCNU requires the FA-BRCA pathway for repair.
P10.14.B PROGRESSION OF IDH-MUTANT ASTROCYTOMAS TO GRADE 4 UNDER TREATMENT-RELATED SELECTION PRESSURE
Authors: Rautajoki K J , Jaatinen S , Hartewig A , Tiihonen A M , Annala M , Salonen I , Valkonen M , Simola V , Vuorinen E M , Kivinen A , Rauhala M J , Nurminen R , Maass K K , Lahtela S , Jukkola A , Yli-Harja O , Helén P , Pajtler K W , Ruusuvuori P , Haapasalo J , Zhang W , Haapasalo H , Nykter M
Published: September 2023

BACKGROUND

The progression of low-grade diffuse astrocytomas into grade 4 tumors significantly impacts patient prognosis, therefore a better understanding of this process is of paramount importance for improved patient care.

MATERIAL AND METHODS

We analyzed next-generation sequencing data from matched IDH-mutant astrocytomas before and after progression to grade 4 from up to 27 patients as well as publicly available The Cancer Genome Atlas (TCGA) data from up to 595 cases. All patients in our discovery cohort with genome-wide sequencing data received radiation, all but one underwent chemotherapy, and no patient received temozolomide before progression to grade 4.

RESULTS

One case in the discovery cohort exhibited a hypermutation signature associated with inactivation of the MSH2 and DNMT3A genes. In other patients, the number of novel chromosomal rearrangements and deletions increased in grade 4 tumors. Tumor progression was associated with upregulated cell cycle progression, migration, and DNA damage repair-related genes. Upon progression, the cell cycle checkpoint genes CDKN2A or RB1 were most commonly inactivated, especially after postoperative radiation and chemotherapy, followed by activating growth factor receptor gene alterations in cases with homozygous CDKN2A deletion. We also detected alterations in DNA repair pathway genes associated with therapy resistance and the promotion of error-prone DNA repair, thus facilitating tumor progression. The homologous recombination repair gene RAD51B was hemizygously deleted in all but one progressed tumor in our discovery cohort. In our retrospective nonrandomized survival analysis, treatment including both radio- and chemotherapy improved relapse-free and overall survival compared to other treatments in cases with primary grade 3 diagnosis. Similar benefit was not observed for cases with primary grade 2 diagnosis, and radiation as sole postoperative treatment was not significantly improving the prognosis in any of the analyses.

CONCLUSION

Our results support the previous notions that treatment-induced DNA damage can contribute to tumor progression to grade 4 and suggest that altered DNA repair has a role in treatment resistance and tumor progression.
P10.26.B EXPLORING POTENTIAL ANTITUMOR EFFECTS OF CANNABIDIOL AND TETRAHYDROCANNABINOL IN A PATIENT-DERIVED GLIOBLASTOMA MODEL
Authors: Zekanovic S , van den Bent M J , Dirven C M F , Lamfers M L M , Leenstra S
Published: September 2023

BACKGROUND

Glioblastoma (GBM) is a CNS malignancy which is in dire need of novel treatment options. Drug repurposing has become a means to circumvent major obstacles that accompany drug development for GBM. The cannabinoids cannabidiol (CBD) and tetrahydrocannabinol (THC) have garnered the interest of the scientific community for the treatment of GBM as they possess pleiotropic effects in other pathologies. In the present study we are investigating potential antitumor effects of these cannabinoids by using our patient derived GBM model which recapitulates GBM in its genetic make-up and heterogeneity.

MATERIAL AND METHODS

To assess potential antitumor effects in vitro, we performed a screen on 20 patient derived GBM cultures to obtain a sensitivity profile for both cannabinoids. These cultures ranged in background regarding primary/recurrence and IDH mutation. To investigate effects of these cannabinoids on GBM radio-and-chemosensitivity, combination studies with either lomustine (CCNU) or irradiation (RTx) were performed on recurrent GBM cultures. The zero-interaction potential method was used to quantify synergy between the cannabinoids. The enhancement factor was used to quantify potential radiosensitization or ratio-effects between the cannabinoids.

RESULTS

Screening of both cannabinoids revealed CBD to possess a higher level of cytotoxicity than THC. IC50 values of CBD ranged from 2.5 - 18.5 µM and the values of THC ranged from 7.20 - 47.60 µM. In a panel of 8 cultures, combinations of CBD with THC, CBD with CCNU and THC with CCNU was tested to gain insight in combination effects. In 1/8 cultures synergy was observed between CBD and THC. In 1/8 cultures synergy between CBD and CCNU was observed and in 4/8 cultures synergy between THC and CCNU. When combined with irradiation, no enhancement was observed in any of the 4 cultures tested. No ratio-dependent enhancement between the cannabinoids was observed when combined in a 1:1, 1:4 or 4:1 ratio of CBD:THC.

CONCLUSION

Screening of the cannabinoids CBD and THC revealed that CBD displays a stronger cytotoxic effect than THC. Combinations of either of the two with each other or CCNU revealed moderate additive effects in the majority of cultures and synergy in a small subset. We did not observe radiosensitizing effects in our patient derived GBM model. Relating in vitro found cytotoxic concentrations to clinically achievable concentrations, we found that CBD requires approximately 3 times higher plasma concentrations and THC >40 times to achieve an antitumor effect. Currently we are investigating long term treatment effects of CBD and its mechanism of action.