Liquid biopsy for improving diagnosis and monitoring of CNS lymphomas: a RANO review.

BACKGROUND: The utility of liquid biopsies is well documented in several extracranial and intracranial (brain/leptomeningeal metastases, gliomas) tumors. METHODS: The RANO (Response Assessment in Neuro-Oncology) group has set up a multidisciplinary Task Force to critically review the role of blood and CSF-liquid biopsy in central nervous system lymphomas, with a main focus on primary central nervous system lymphomas (PCNSL). RESULTS: Several clinical applications are suggested: diagnosis of PCNSL in critical settings (elderly or frail patients, deep locations, steroids responsiveness), definition of minimal residual disease, early indication of tumor response or relapse following treatments and prediction of outcome. CONCLUSIONS: Thus far, no clinically validated circulating biomarkers for managing both primary and secondary CNS lymphomas exist. There is need of standardization of biofluid collection, choice of analytes and type of technique to perform the molecular analysis. The various assays should be evaluated through well organized central testing within clinical trials.

The Traumatic Inoculation Process Affects TSPO Radioligand Uptake in Experimental Orthotopic Glioblastoma.

BACKGROUND: The translocator protein (TSPO) has been proven to have great potential as a target for the positron emission tomography (PET) imaging of glioblastoma. However, there is an ongoing debate about the potential various sources of the TSPO PET signal. This work investigates the impact of the inoculation-driven immune response on the PET signal in experimental orthotopic glioblastoma. METHODS: Serial [18F]GE-180 and O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET) PET scans were performed at day 7/8 and day 14/15 after the inoculation of GL261 mouse glioblastoma cells (n = 24) or saline (sham, n = 6) into the right striatum of immunocompetent C57BL/6 mice. An additional n = 25 sham mice underwent [18F]GE-180 PET and/or autoradiography (ARG) at days 7, 14, 21, 28, 35, 50 and 90 in order to monitor potential reactive processes that were solely related to the inoculation procedure. In vivo imaging results were directly compared to tissue-based analyses including ARG and immunohistochemistry. RESULTS: We found that the inoculation process represents an immunogenic event, which significantly contributes to TSPO radioligand uptake. [18F]GE-180 uptake in GL261-bearing mice surpassed [18F]FET uptake both in the extent and the intensity, e.g., mean target-to-background ratio (TBRmean) in PET at day 7/8: 1.22 for [18F]GE-180 vs. 1.04 for [18F]FET, p < 0.001. Sham mice showed increased [18F]GE-180 uptake at the inoculation channel, which, however, continuously decreased over time (e.g., TBRmean in PET: 1.20 at day 7 vs. 1.09 at day 35, p = 0.04). At the inoculation channel, the percentage of TSPO/IBA1 co-staining decreased, whereas TSPO/GFAP (glial fibrillary acidic protein) co-staining increased over time (p < 0.001). CONCLUSION: We identify the inoculation-driven immune response to be a relevant contributor to the PET signal and add a new aspect to consider for planning PET imaging studies in orthotopic glioblastoma models.

[New treatment strategies for primary lymphoma of the central nervous system]. / Neue Therapiestrategien beim primären Lymphom des Zentralnervensystems.

Primary central nervous system lymphomas (PCNSL) are rare highly aggressive diffuse large B cell non-Hodgkin lymphomas confined to the brain, meninges, the spinal cord and the eyes. Although the implementation of high-dose methotrexate-based chemotherapy has significantly improved the prognosis of PCNSL during the last decades, about one third of patients show refractory disease and about half of the patients eventually relapse after having achieved complete response. This highlights the need for novel treatment strategies. The most promising progress has been made in the field of molecular targeted therapy that interferes with the oncogenic signaling pathways of PCNSL. These include inhibitors of Bruton tyrosine kinase and inhibitors of the PI3K/mTOR signaling pathway. In addition, the thalidomide analogues lenalidomide and pomalidomide, which belong to the class of immunomodulators, show efficacy in the treatment of PCNSL. As immune evasion appears to play a relevant pathogenetic role in PCNSL, immunotherapies in the treatment of PCNSL are the subject of intensive research. Promising initial clinical data are available for both immune checkpoint inhibitors and cellular immunotherapy with chimeric antigen receptor (CAR) T cells. Before the widespread clinical application of these novel therapies, the efficacy needs to be confirmed in larger prospective studies. Despite high response rates, targeted therapies and immunotherapy often fail to achieve lasting tumor control. Therefore, novel approaches are currently being investigated in combination protocols.

Functional connectivity MRI provides an imaging correlate for chimeric antigen receptor T-cell-associated neurotoxicity.

Background: Treatment of hematological malignancies with chimeric antigen receptor modified T cells (CART) is highly efficient, but often limited by an immune effector cell-associated neurotoxicity syndrome (ICANS). As conventional MRI is often unremarkable during ICANS, we aimed to examine whether resting-state functional MRI (rsfMRI) is suitable to depict and quantify brain network alterations underlying ICANS in the individual patient. Methods: The dysconnectivity index (DCI) based on rsfMRI was longitudinally assessed in systemic lymphoma patients and 1 melanoma patient during ICANS and before or after clinical resolution of ICANS. Results: Seven lymphoma patients and 1 melanoma patient (19-77 years; 2 female) were included. DCI was significantly increased during ICANS with normalization after recovery (P = .0039). Higher ICANS grades were significantly correlated with increased DCI scores (r = 0.7807; P = .0222). DCI increase was most prominent in the inferior frontal gyrus and the frontal operculum (ie, Broca's area) and in the posterior parts of the superior temporal gyrus and the temporoparietal junction (ie, Wernicke's area) of the language-dominant hemisphere, thus reflecting the major clinical symptoms of nonfluent dysphasia and dyspraxia. Conclusions: RsfMRI-based DCI might be suitable to directly quantify the severity of ICANS in individual patients undergoing CAR T-transfusion. Besides ICANS, DCI seems a promising diagnostic tool to quantify functional brain network alterations during encephalopathies of different etiologies, in general.

A framework for standardised tissue sampling and processing during resection of diffuse intracranial glioma: joint recommendations from four RANO groups.

Surgical resection represents the standard of care for people with newly diagnosed diffuse gliomas, and the neuropathological and molecular profile of the resected tissue guides clinical management and forms the basis for research. The Response Assessment in Neuro-Oncology (RANO) consortium is an international, multidisciplinary effort that aims to standardise research practice in neuro-oncology. These recommendations represent a multidisciplinary consensus from the four RANO groups: RANO resect, RANO recurrent glioblastoma, RANO radiotherapy, and RANO/PET for a standardised workflow to achieve a representative tumour evaluation in a disease characterised by intratumoural heterogeneity, including recommendations on which tumour regions should be surgically sampled, how to define those regions on the basis of preoperative imaging, and the optimal sample volume. Practical recommendations for tissue sampling are given for people with low-grade and high-grade gliomas, as well as for people with newly diagnosed and recurrent disease. Sampling of liquid biopsies is also addressed. A standardised workflow for subsequent handling of the resected tissue is proposed to avoid information loss due to decreasing tissue quality or insufficient clinical information. The recommendations offer a framework for prospective biobanking studies.

Deciphering sources of PET signals in the tumor microenvironment of glioblastoma at cellular resolution.

Various cellular sources hamper interpretation of positron emission tomography (PET) biomarkers in the tumor microenvironment (TME). We developed an approach of immunomagnetic cell sorting after in vivo radiotracer injection (scRadiotracing) with three-dimensional (3D) histology to dissect the cellular allocation of PET signals in the TME. In mice with implanted glioblastoma, translocator protein (TSPO) radiotracer uptake per tumor cell was higher compared to tumor-associated microglia/macrophages (TAMs), validated by protein levels. Translation of in vitro scRadiotracing to patients with glioma immediately after tumor resection confirmed higher single-cell TSPO tracer uptake of tumor cells compared to immune cells. Across species, cellular radiotracer uptake explained the heterogeneity of individual TSPO-PET signals. In consideration of cellular tracer uptake and cell type abundance, tumor cells were the main contributor to TSPO enrichment in glioblastoma; however, proteomics identified potential PET targets highly specific for TAMs. Combining cellular tracer uptake measures with 3D histology facilitates precise allocation of PET signals and serves to validate emerging novel TAM-specific radioligands.

Translocator protein (18kDA) (TSPO) marks mesenchymal glioblastoma cell populations characterized by elevated numbers of tumor-associated macrophages.

TSPO is a promising novel tracer target for positron-emission tomography (PET) imaging of brain tumors. However, due to the heterogeneity of cell populations that contribute to the TSPO-PET signal, imaging interpretation may be challenging. We therefore evaluated TSPO enrichment/expression in connection with its underlying histopathological and molecular features in gliomas. We analyzed TSPO expression and its regulatory mechanisms in large in silico datasets and by performing direct bisulfite sequencing of the TSPO promotor. In glioblastoma tissue samples of our TSPO-PET imaging study cohort, we dissected the association of TSPO tracer enrichment and protein labeling with the expression of cell lineage markers by immunohistochemistry and fluorescence multiplex stains. Furthermore, we identified relevant TSPO-associated signaling pathways by RNA sequencing.We found that TSPO expression is associated with prognostically unfavorable glioma phenotypes and that TSPO promotor hypermethylation is linked to IDH mutation. Careful histological analysis revealed that TSPO immunohistochemistry correlates with the TSPO-PET signal and that TSPO is expressed by diverse cell populations. While tumor core areas are the major contributor to the overall TSPO signal, TSPO signals in the tumor rim are mainly driven by CD68-positive microglia/macrophages. Molecularly, high TSPO expression marks prognostically unfavorable glioblastoma cell subpopulations characterized by an enrichment of mesenchymal gene sets and higher amounts of tumor-associated macrophages.In conclusion, our study improves the understanding of TSPO as an imaging marker in gliomas by unveiling IDH-dependent differences in TSPO expression/regulation, regional heterogeneity of the TSPO PET signal and functional implications of TSPO in terms of tumor immune cell interactions.

Prognostic Value of TSPO PET Before Radiotherapy in Newly Diagnosed IDH-Wild-Type Glioblastoma.

The 18-kDa translocator protein (TSPO) is gaining recognition as a relevant target in glioblastoma imaging. However, data on the potential prognostic value of TSPO PET imaging in glioblastoma are lacking. Therefore, we investigated the association of TSPO PET imaging results with survival outcome in a homogeneous cohort of glioblastoma patients. Methods: Patients were included who had newly diagnosed, histologically confirmed isocitrate dehydrogenase (IDH)-wild-type glioblastoma with available TSPO PET before either normofractionated radiotherapy combined with temozolomide or hypofractionated radiotherapy. SUVmax on TSPO PET, TSPO binding affinity status, tumor volumes on MRI, and further clinical data, such as O 6-alkylguanine DNA methyltransferase (MGMT) and telomerase reverse transcriptase (TERT) gene promoter mutation status, were correlated with patient survival. Results: Forty-five patients (median age, 63.3 y) were included. Median SUVmax was 2.2 (range, 1.0-4.7). A TSPO PET signal was associated with survival: High uptake intensity (SUVmax > 2.2) was related to significantly shorter overall survival (OS; 8.3 vs. 17.8 mo, P = 0.037). Besides SUVmax, prognostic factors for OS were age (P = 0.046), MGMT promoter methylation status (P = 0.032), and T2-weighted MRI volume (P = 0.031). In the multivariate survival analysis, SUVmax in TSPO PET remained an independent prognostic factor for OS (P = 0.023), with a hazard ratio of 2.212 (95% CI, 1.115-4.386) for death in cases with a high TSPO PET signal (SUVmax > 2.2). Conclusion: A high TSPO PET signal before radiotherapy is associated with significantly shorter survival in patients with newly diagnosed IDH-wild-type glioblastoma. TSPO PET seems to add prognostic insights beyond established clinical parameters and might serve as an informative tool as clinicians make survival predictions for patients with glioblastoma.

Neurotoxicity and management of primary and secondary central nervous system lymphoma after adoptive immunotherapy with CD19-directed chimeric antigen receptor T-cells.

BACKGROUND: Chimeric antigen receptor (CAR) T-cells targeting CD19 have been established as a leading engineered T-cell therapy for B-cell lymphomas; however, data for patients with central nervous system (CNS) involvement are limited. METHODS: We retrospectively report on CNS-specific toxicities, management, and CNS response of 45 consecutive CAR T-cell transfusions for patients with active CNS lymphoma at the Massachusetts General Hospital over a 5-year period. RESULTS: Our cohort includes 17 patients with primary CNS lymphoma (PCNSL; 1 patient with 2 CAR T-cell transfusions) and 27 patients with secondary CNS lymphoma (SCNSL). Mild ICANS (grade 1-2) was observed after 19/45 transfusions (42.2%) and severe immune effector cell-associated neurotoxicity syndrome (ICANS) (grade 3-4) after 7/45 transfusions (15.6%). A larger increase in C-reactive protein (CRP) levels and higher rates of ICANS were detected in SCNSL. Early fever and baseline C-reactive protein levels were associated with ICANS 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 ±â€…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 (hazard ratios [HR] per mg/d: 1.16, P = .031). If bridging therapy was warranted, the use of ibrutinib translated into favorable CNS-progression-free survival (5 vs. 1 month, HR 0.28, CI 0.1-0.7; P = .010). CONCLUSIONS: CAR T-cells exhibit promising antitumor effects and a favorable safety profile in CNS lymphoma. Further evaluation of the role of bridging regimens and corticosteroids is warranted.

Integrated genetic analyses of immunodeficiency-associated Epstein-Barr virus- (EBV) positive primary CNS lymphomas.

Immunodeficiency-associated primary CNS lymphoma (PCNSL) represents a distinct clinicopathological entity, which is typically Epstein-Barr virus-positive (EBV+) and carries an inferior prognosis. Genetic alterations that characterize EBV-related CNS lymphomagenesis remain unclear precluding molecular classification and targeted therapies. In this study, a comprehensive genetic analysis of 22 EBV+ PCNSL, therefore, integrated clinical and pathological information with exome and RNA sequencing (RNASeq) data. EBV+ PCNSL with germline controls carried a median of 55 protein-coding single nucleotide variants (SNVs; range 24-217) and 2 insertions/deletions (range 0-22). Genetic landscape was largely shaped by aberrant somatic hypermutation with a median of 41.01% (range 31.79-53.49%) of SNVs mapping to its target motifs. Tumors lacked established SNVs (MYD88, CD79B, PIM1) and copy number variants (CDKN2A, HLA loss) driving EBV- PCNSL. Instead, EBV+ PCNSL were characterized by SOCS1 mutations (26%), predicted to disinhibit JAK/STAT signaling, and mutually exclusive gain-of-function NOTCH pathway SNVs (26%). Copy number gains were enriched on 11q23.3, a locus directly targeted for chromosomal aberrations by EBV, that includes SIK3 known to protect from cytotoxic T-cell responses. Losses covered 5q31.2 (STING), critical for sensing viral DNA, and 17q11 (NF1). Unsupervised clustering of RNASeq data revealed two distinct transcriptional groups, that shared strong expression of CD70 and IL1R2, previously linked to tolerogenic tumor microenvironments. Correspondingly, deconvolution of bulk RNASeq data revealed elevated M2-macrophage, T-regulatory cell, mast cell and monocyte fractions in EBV+ PCNSL. In addition to novel insights into the pathobiology of EBV+ PCNSL, the data provide the rationale for the exploration of targeted therapies including JAK-, NOTCH- and CD70-directed approaches.

Significance of molecular diagnostics for therapeutic decision-making in recurrent glioma.

Background: Targeted therapies have substantially improved survival in cancer patients with malignancies outside the brain. Whether in-depth analysis for molecular alterations may also offer therapeutic avenues in primary brain tumors remains unclear. We herein present our institutional experience for glioma patients discussed in our interdisciplinary molecular tumor board (MTB) implemented at the Comprehensive Cancer Center Munich (LMU). Methods: We retrospectively searched the database of the MTB for all recurrent glioma patients after previous therapy. Recommendations were based on next-generation sequencing results of individual patient's tumor tissue. Clinical and molecular information, previous therapy regimens, and outcome parameters were collected. Results: Overall, 73 consecutive recurrent glioma patients were identified. In the median, advanced molecular testing was initiated with the third tumor recurrence. The median turnaround time between initiation of molecular profiling and MTB case discussion was 48 ± 75 days (range: 32-536 days). Targetable mutations were found for 50 recurrent glioma patients (68.5%). IDH1 mutation (27/73; 37%), epidermal growth factor receptor amplification (19/73; 26%), and NF1 mutation (8/73; 11%) were the most detected alterations and a molecular-based treatment recommendation could be made for all of them. Therapeutic recommendations were implemented in 12 cases (24%) and one-third of these heavily pretreated patients experienced clinical benefit with at least disease stabilization. Conclusions: In-depth molecular analysis of tumor tissue may guide targeted therapy also in brain tumor patients and considerable antitumor effects might be observed in selected cases. However, future studies to corroborate our results are needed.

Unbiased chemokine receptor screening reveals similar efficacy of lymph node- and tumor-targeted T cell immunotherapy.

Localization is a crucial prerequisite for immune cell function and solid tumors evade immune control by modulating immune cell infiltration into the tumor stroma. Immunosuppressive cells like regulatory T cells are attracted, while cytotoxic CD8+ T cells are excluded. Engineering CD8+ T cells with chemokine receptors is a potent strategy to turn this mechanism of directed immune cell recruitment against the tumor. Here, we utilized fluorescent tagging to track the migratory behavior of tumor-specific T cells engineered with a library of all murine chemokine receptors in vivo. We then asked whether chemokine receptor-mediated redirection of antigen-specific T cells into tumors or tumor-draining lymph nodes showed superior anti-tumoral activity. We found that both targeting approaches showed higher therapeutic efficacy than control T cells. However, multiple receptors conveying the same homing pattern did not augment infiltration. Instead, in the MC38 colon carcinoma model, anti-tumoral efficacy as well as lymph node vs. tumor-homing patterns were mostly driven by CCR4 and CCR6, respectively. Overall, our data, based on fluorescent receptor tagging, identify the tumor-draining lymph node and the tumor itself as viable targets for chemokine receptor-mediated enhancement of adoptive T cell therapy.

Synthetic Cell-Based Immunotherapies for Neurologic Diseases.

The therapeutic success and widespread approval of genetically engineered T cells for a variety of hematologic malignancies spurred the development of synthetic cell-based immunotherapies for CNS lymphoma, primary brain tumors, and a growing spectrum of nononcologic disease conditions of the nervous system. Chimeric antigen receptor effector T cells bear the potential to deplete target cells with higher efficacy, better tissue penetration, and greater depth than antibody-based cell depletion therapies. In multiple sclerosis and other autoimmune disorders, engineered T-cell therapies are being designed and currently tested in clinical trials for their safety and efficacy to eliminate pathogenic B-lineage cells. Chimeric autoantibody receptor T cells expressing a disease-relevant autoantigen as cell surface domains are designed to selectively deplete autoreactive B cells. Alternative to cell depletion, synthetic antigen-specific regulatory T cells can be engineered to locally restrain inflammation, support immune tolerance, or efficiently deliver neuroprotective factors in brain diseases in which current therapeutic options are very limited. In this article, we illustrate prospects and bottlenecks for the clinical development and implementation of engineered cellular immunotherapies in neurologic diseases.

Shunt dependency in supratentorial intraventricular tumors depends on the extent of tumor resection.

BACKGROUND: Supratentorial intraventricular tumors (SIVTs) are rare lesions of various entities characteristically presenting with hydrocephalus and often posing a surgical challenge due to their deep-seated localization. We aimed to elaborate on shunt dependency after tumor resection, clinical characteristics, and perioperative morbidity. METHODS: We retrospectively searched the institutional database for patients with supratentorial intraventricular tumors treated at the Department of Neurosurgery of the Ludwig-Maximilians-University in Munich, Germany, between 2014 and 2022. RESULTS: We identified 59 patients with over 20 different SIVT entities, most often subependymoma (8/59 patients, 14%). Mean age at diagnosis was 41 ± 3 years. Hydrocephalus and visual symptoms were observed in 37/59 (63%) and 10/59 (17%) patients, respectively. Microsurgical tumor resection was provided in 46/59 patients (78%) with complete resection in 33/46 patients (72%). Persistent postoperative neurological deficits were encountered in 3/46 patients (7%) and generally mild in nature. Complete tumor resection was associated with less permanent shunting in comparison to incomplete tumor resection, irrespective of tumor histology (6% versus 31%, p = 0.025). Stereotactic biopsy was utilized in 13/59 patients (22%), including 5 patients who received synchronous internal shunt implantation for symptomatic hydrocephalus. Median overall survival was not reached and did not differ between patients with or without open resection. CONCLUSIONS: SIVT patients display a high risk of developing hydrocephalus and visual symptoms. Complete resection of SIVTs can often be achieved, preventing the need for long-term shunting. Stereotactic biopsy along with internal shunting represents an effective approach to establish diagnosis and ameliorate symptoms if resection cannot be safely performed. Due to the rather benign histology, the outcome appears excellent when adjuvant therapy is provided.

Single-cell transcriptomic atlas-guided development of CAR-T cells for the treatment of acute myeloid leukemia.

Chimeric antigen receptor T cells (CAR-T cells) have emerged as a powerful treatment option for individuals with B cell malignancies but have yet to achieve success in treating acute myeloid leukemia (AML) due to a lack of safe targets. Here we leveraged an atlas of publicly available RNA-sequencing data of over 500,000 single cells from 15 individuals with AML and tissue from 9 healthy individuals for prediction of target antigens that are expressed on malignant cells but lacking on healthy cells, including T cells. Aided by this high-resolution, single-cell expression approach, we computationally identify colony-stimulating factor 1 receptor and cluster of differentiation 86 as targets for CAR-T cell therapy in AML. Functional validation of these established CAR-T cells shows robust in vitro and in vivo efficacy in cell line- and human-derived AML models with minimal off-target toxicity toward relevant healthy human tissues. This provides a strong rationale for further clinical development.

Neurosurgical Interventions for Cerebral Metastases of Solid Tumors.

BACKGROUND: Metastases are the most common malignant tumors affecting the central nervous system and occur in 20-40 percent of patients with solid systemic tumors. The aim of this review is to discuss the role of neurosurgical procedures in a modern, multidisciplinary treatment approach. METHODS: An expert panel of neurosurgeons, neurologists, and radio-oncologists conducted a selective literature review on neu - rosurgical interventions for the diagnosis and treatment of cerebral metastases. Original articles, meta-analyses, and systematic reviews were included. RESULTS: There is a lack of prospective randomized studies. Based on retrospective case series, international guidelines recommend the harvesting (if required, stereotactically guided) of tissue for histological and molecular diagnosis in cases of unknown or possibly competing underlying systemic malignant diseases, in cases of suspected tumor recurrence, and with regard to the evaluation of targeted therapies taking into account molecular heterogeneity of primary and secondary tumors. Surgical resection is particularly valuable for the treatment of up to three space-occupying cerebral metastases, especially to achieve clinical stabilization to allow further non-surgical treatment. For cystic metastasis, a combination of stereotactic puncture and radiotherapy may be useful. Meningeal carcinomatosis can be treated with intrathecal medication via an intraventricular catheter system. Ventriculo-peritoneal shunts represents an effective treatment option for patients with tumor-associated hydrocephalus. CONCLUSION: Neurosurgical procedures are of central importance in the multimodal treatment of cerebral metastases. The indications for neurosurgical interventions will be refined in the light of more effective radiation techniques and systemic treatments with new targeted therapeutic approaches and immunotherapies on the horizon.

In vivo dynamics and anti-tumor effects of EpCAM-directed CAR T-cells against brain metastases from lung cancer.

Lung cancer patients are at risk for brain metastases and often succumb to their intracranial disease. Chimeric Antigen Receptor (CAR) T-cells emerged as a powerful cell-based immunotherapy for hematological malignancies; however, it remains unclear whether CAR T-cells represent a viable therapy for brain metastases. Here, we established a syngeneic orthotopic cerebral metastasis model in mice by combining a chronic cranial window with repetitive intracerebral two-photon laser scanning-microscopy. This approach enabled in vivo-characterization of fluorescent CAR T-cells and tumor cells on a single-cell level over weeks. Intraparenchymal injection of Lewis lung carcinoma cells (expressing the tumor cell-antigen EpCAM) was performed, and EpCAM-directed CAR T-cells were injected either intravenously or into the adjacent brain parenchyma. In mice receiving EpCAM-directed CAR T-cells intravenously, we neither observed substantial CAR T-cell accumulation within the tumor nor relevant anti-tumor effects. Local CAR T-cell injection, however, resulted in intratumoral CAR T-cell accumulation compared to controls treated with T-cells lacking a CAR. This finding was accompanied by reduced tumorous growth as determined per in vivo-microscopy and immunofluorescence of excised brains and also translated into prolonged survival. However, the intratumoral number of EpCAM-directed CAR T-cells decreased during the observation period, pointing toward insufficient persistence. No CNS-specific or systemic toxicities of EpCAM-directed CAR T-cells were observed in our fully immunocompetent model. Collectively, our findings indicate that locally (but not intravenously) injected CAR T-cells may safely induce relevant anti-tumor effects in brain metastases from lung cancer. Strategies improving the intratumoral CAR T-cell persistence may further boost the therapeutic success.