Multimaterial decomposition in dual-energy CT for characterization of clots from acute ischemic stroke patients.

BACKGROUND: Nowadays, there is no method to quantitatively characterize the material composition of acute ischemic stroke thrombi prior to intervention, but dual-energy CT (DE-CT) offers imaging-based multimaterial decomposition. We retrospectively investigated the material composition of thrombi ex vivo using DE-CT with histological analysis as a reference. METHODS: Clots of 70 patients with acute ischemic stroke were extracted by mechanical thrombectomy and scanned ex vivo in formalin-filled tubes with DE-CT. Multimaterial decomposition in the three components, i.e., red blood cells (RBC), white blood cells (WBC), and fibrin/platelets (F/P), was performed and compared to histology (hematoxylin/eosin staining) as reference. Attenuation and effective Z values were assessed, and histological composition was compared to stroke etiology according to the Trial of ORG 10172 in Acute Stroke Treatment (TOAST) criteria. RESULTS: Histological and imaging analysis showed the following correlation coefficients for RBC (r = 0.527, p < 0.001), WBC (r = 0.305, p = 0.020), and F/P (r = 0.525, p < 0.001). RBC-rich thrombi presented higher clot attenuation in Hounsfield units than F/P-rich thrombi (51 HU versus 42 HU, p < 0.01). In histological analysis, cardioembolic clots showed less RBC (40% versus 56%, p = 0.053) and more F/P (53% versus 36%, p = 0.024), similar to cryptogenic clots containing less RBC (34% versus 56%, p = 0.006) and more F/P (58% versus 36%, p = 0.003) than non-cardioembolic strokes. No difference was assessed for the mean WBC portions in all TOAST groups. CONCLUSIONS: DE-CT has the potential to quantitatively characterize the material composition of ischemic stroke thrombi. RELEVANCE STATEMENT: Using DE-CT, the composition of ischemic stroke thrombi can be determined. Knowledge of histological composition prior to intervention offers the opportunity to define personalized treatment strategies for each patient to accomplish faster recanalization and better clinical outcomes. KEY POINTS: • Acute ischemic stroke clots present different recanalization success according to histological composition. • Currently, no method can determine clot composition prior to intervention. • DE-CT allows quantitative material decomposition of thrombi ex vivo in red blood cells, white blood cells, and fibrin/platelets. • Histological clot composition differs between stroke etiology. • Insights into the histological composition in situ offer personalized treatment strategies.

Validating a minipig model of reversible cerebral demyelination using human diagnostic modalities and electron microscopy.

BACKGROUND: Inflammatory demyelinating diseases of the central nervous system, such as multiple sclerosis, are significant sources of morbidity in young adults despite therapeutic advances. Current murine models of remyelination have limited applicability due to the low white matter content of their brains, which restricts the spatial resolution of diagnostic imaging. Large animal models might be more suitable but pose significant technological, ethical and logistical challenges. METHODS: We induced targeted cerebral demyelinating lesions by serially repeated injections of lysophosphatidylcholine in the minipig brain. Lesions were amenable to follow-up using the same clinical imaging modalities (3T magnetic resonance imaging, 11C-PIB positron emission tomography) and standard histopathology protocols as for human diagnostics (myelin, glia and neuronal cell markers), as well as electron microscopy (EM), to compare against biopsy data from two patients. FINDINGS: We demonstrate controlled, clinically unapparent, reversible and multimodally trackable brain white matter demyelination in a large animal model. De-/remyelination dynamics were slower than reported for rodent models and paralleled by a degree of secondary axonal pathology. Regression modelling of ultrastructural parameters (g-ratio, axon thickness) predicted EM features of cerebral de- and remyelination in human data. INTERPRETATION: We validated our minipig model of demyelinating brain diseases by employing human diagnostic tools and comparing it with biopsy data from patients with cerebral demyelination. FUNDING: This work was supported by the DFG under Germany's Excellence Strategy within the framework of the Munich Cluster for Systems Neurology (EXC 2145 SyNergy, ID 390857198) and TRR 274/1 2020, 408885537 (projects B03 and Z01).

Fluorescein-stained confocal laser endomicroscopy versus conventional frozen section for intraoperative histopathological assessment of intracranial tumors.

BACKGROUND: The aim of this clinical trial was to compare Fluorescein-stained intraoperative confocal laser endomicroscopy (CLE) of intracranial lesions and evaluation by a neuropathologist with routine intraoperative frozen section (FS) assessment by Neuropathology. METHODS: In this phase II non-inferiority, prospective, multicenter, non-randomized, off-label clinical trial (Eudra-CT: 2019-004512-58), patients above the age of 18 years with any intracranial lesion scheduled for elective resection were included. The diagnostic accuracies of both CLE and FS referenced with the final histopathological diagnosis were statistically compared in a non-inferiority analysis, representing the primary endpoint. Secondary endpoints included the safety of the technique and time expedited for CLE and FS. RESULTS: 210 patients were included by 3 participating sites between November 2020 and June 2022. Most common entities were high grade gliomas (37.9%), metastases (24.1%), and meningiomas (22.7%), A total of 6 serious adverse events in 4 (2%) patients were recorded. For the primary endpoint, the diagnostic accuracy for CLE was inferior with .87 versus .91 for FS, resulting in a difference of .04 (95% confidence interval -.10; .02; p=.367). The median time expedited until intraoperative diagnosis was 3 minutes for CLE and 27 minutes for FS, with a mean difference of 27.5 minutes (standard deviation 14.5; p<.001). CONCLUSION: CLE allowed for a safe and time-effective intraoperative histological diagnosis with a diagnostic accuracy of 87% across all intracranial entities included. The technique achieved histological assessments in real-time with a tenfold reduction of processing time compared to FS, which may invariably impact surgical strategy on the fly.

Intraoperative in vivo confocal laser endomicroscopy imaging at glioma margins: can we detect tumor infiltration?

OBJECTIVE: Confocal laser endomicroscopy (CLE) is a US Food and Drug Administration-cleared intraoperative real-time fluorescence-based cellular resolution imaging technology that has been shown to image brain tumor histoarchitecture rapidly in vivo during neuro-oncological surgical procedures. An important goal for successful intraoperative implementation is in vivo use at the margins of infiltrating gliomas. However, CLE use at glioma margins has not been well studied. METHODS: Matching in vivo CLE images and tissue biopsies acquired at glioma margin regions of interest (ROIs) were collected from 2 institutions. All images were reviewed by 4 neuropathologists experienced in CLE. A scoring system based on the pathological features was implemented to score CLE and H&E images from each ROI on a scale from 0 to 5. Based on the H&E scores, all ROIs were divided into a low tumor probability (LTP) group (scores 0-2) and a high tumor probability (HTP) group (scores 3-5). The concordance between CLE and H&E scores regarding tumor probability was determined. The intraclass correlation coefficient (ICC) and diagnostic performance were calculated. RESULTS: Fifty-six glioma margin ROIs were included for analysis. Interrater reliability of the scoring system was excellent when used for H&E images (ICC [95% CI] 0.91 [0.86-0.94]) and moderate when used for CLE images (ICC [95% CI] 0.69 [0.40-0.83]). The ICCs (95% CIs) of the LTP group (0.68 [0.40-0.83]) and HTP group (0.68 [0.39-0.83]) did not differ significantly. The concordance between CLE and H&E scores was 61.6%. The sensitivity and specificity values of the scoring system were 79% and 37%. The positive predictive value (PPV) and negative predictive value were 65% and 53%, respectively. Concordance, sensitivity, and PPV were greater in the HTP group than in the LTP group. Specificity was higher in the newly diagnosed group than in the recurrent group. CONCLUSIONS: CLE may detect tumor infiltration at glioma margins. However, it is not currently dependable, especially in scenarios where low probability of tumor infiltration is expected. The proposed scoring system has excellent intrinsic interrater reliability, but its interrater reliability is only moderate when used with CLE images. These results suggest that this technology requires further exploration as a method for consistent actionable intraoperative guidance with high dependability across the range of tumor margin scenarios. Specific-binding and/or tumor-specific fluorophores, a CLE image atlas, and a consensus guideline for image interpretation may help with the translational utility of CLE.

A region-resolved proteomic map of the human brain enabled by high-throughput proteomics.

Substantial efforts are underway to deepen our understanding of human brain morphology, structure, and function using high-resolution imaging as well as high-content molecular profiling technologies. The current work adds to these approaches by providing a comprehensive and quantitative protein expression map of 13 anatomically distinct brain regions covering more than 11,000 proteins. This was enabled by the optimization, characterization, and implementation of a high-sensitivity and high-throughput microflow liquid chromatography timsTOF tandem mass spectrometry system (LC-MS/MS) capable of analyzing more than 2,000 consecutive samples prepared from formalin-fixed paraffin embedded (FFPE) material. Analysis of this proteomic resource highlighted brain region-enriched protein expression patterns and functional protein classes, protein localization differences between brain regions and individual markers for specific areas. To facilitate access to and ease further mining of the data by the scientific community, all data can be explored online in a purpose-built R Shiny app (https://brain-region-atlas.proteomics.ls.tum.de).

Enhanced Sensitivity to ALDH1A3-Dependent Ferroptosis in TMZ-Resistant Glioblastoma Cells.

Temozolomide (TMZ) is standard treatment for glioblastoma (GBM); nonetheless, resistance and tumor recurrence are still major problems. In addition to its association with recurrent GBM and TMZ resistance, ALDH1A3 has a role in autophagy-dependent ferroptosis activation. In this study, we treated TMZ-resistant LN229 human GBM cells with the ferroptosis inducer RSL3. Remarkably, TMZ-resistant LN229 clones were also resistant to ferroptosis induction, although lipid peroxidation was induced by RSL3. By using Western blotting, we were able to determine that ALDH1A3 was down-regulated in TMZ-resistant LN229 cells. Most intriguingly, the cell viability results showed that only those clones that up-regulated ALDH1A3 after TMZ withdrawal became re-sensitized to ferroptosis induction. The recovery of ALDH1A3 expression appeared to be regulated by EGFR-dependent PI3K pathway activation since Akt was activated only in ALDH1A3 high clones. Blocking the EGFR signaling pathway with the EGFR inhibitor AG1498 decreased the expression of ALDH1A3. These findings shed light on the potential application of RSL3 in the treatment of glioblastoma relapse.

ALDH1A1 and ALDH1A3 paralogues of aldehyde dehydrogenase 1 control myogenic differentiation of skeletal muscle satellite cells by retinoic acid-dependent and -independent mechanisms.

ALDH1A1 and ALDH1A3 paralogues of aldehyde dehydrogenase 1 (ALDH1) control myogenic differentiation of skeletal muscle satellite cells (SC) by formation of retinoic acid (RA) and subsequent cell cycle adjustments. The respective relevance of each paralogue for myogenic differentiation and the mechanistic interaction of each paralogue within RA-dependent and RA-independent pathways remain elusive.We analysed the impact of ALDH1A1 and ALDH1A3 activity on myogenesis of murine C2C12 myoblasts. Both paralogues are pivotal factors in myogenic differentiation, since CRISPR/Cas9-edited single paralogue knock-out impaired serum withdrawal-induced myogenic differentiation, while successive recombinant re-expression of ALDH1A1 or ALDH1A3, respectively, in the corresponding ALDH1 paralogue single knock-out cell lines, recovered the differentiation potential. Loss of differentiation in single knock-out cell lines was restored by treatment with RA-analogue TTNPB, while RA-receptor antagonization by AGN 193109 inhibited differentiation of wildtype cell lines, supporting the idea that RA-dependent pathway is pivotal for myogenic differentiation which is accomplished by both paralogues.However, overexpression of ALDH1-paralogues or disulfiram-mediated inhibition of ALDH1 enzymatic activity not only increased ALDH1A1 and ALDH1A3 protein levels but also induced subsequent differentiation of C2C12 myoblasts independently from serum withdrawal, indicating that ALDH1-dependent myogenic differentiation relies on different cellular conditions. Remarkably, ALDH1-paralogue knock-out impaired the autophagic flux, namely autophagosome cargo protein p62 formation and LC3B-I to LC3B-II conversion, demonstrating that ALDH1-paralogues interact with autophagy in myogenesis. Together, ALDH1 paralogues play a crucial role in myogenesis by orchestration of complex RA-dependent and RA-independent pathways.

Multiscale quantification of morphological heterogeneity with creation of a predictor of longer survival in glioblastoma.

Intratumor heterogeneity is a main cause of the dismal prognosis of glioblastoma (GBM). Yet, there remains a lack of a uniform assessment of the degree of heterogeneity. With a multiscale approach, we addressed the hypothesis that intratumor heterogeneity exists on different levels comprising traditional regional analyses, but also innovative methods including computer-assisted analysis of tumor morphology combined with epigenomic data. With this aim, 157 biopsies of 37 patients with therapy-naive IDH-wildtype GBM were analyzed regarding the intratumor variance of protein expression of glial marker GFAP, microglia marker Iba1 and proliferation marker Mib1. Hematoxylin and eosin stained slides were evaluated for tumor vascularization. For the estimation of pixel intensity and nuclear profiling, automated analysis was used. Additionally, DNA methylation profiling was conducted separately for the single biopsies. Scoring systems were established to integrate several parameters into one score for the four examined modalities of heterogeneity (regional, cellular, pixel-level and epigenomic). As a result, we could show that heterogeneity was detected in all four modalities. Furthermore, for the regional, cellular and epigenomic level, we confirmed the results of earlier studies stating that a higher degree of heterogeneity is associated with poorer overall survival. To integrate all modalities into one score, we designed a predictor of longer survival, which showed a highly significant separation regarding the OS. In conclusion, multiscale intratumor heterogeneity exists in glioblastoma and its degree has an impact on overall survival. In future studies, the implementation of a broadly feasible heterogeneity index should be considered.

Design and Transition of an Emergency E-Learning Pathology Course for Medical Students-Evaluation of a Novel Course Concept.

BACKGROUND: Around the world, the emergency brought about by the COVID-19 pandemic forced medical schools to create numerous e-learning supplements to provide instruction during this crisis. The question now is to determine a way in which to capitalize on this momentum of digitization and harness the medical e-learning content created for the future. We have analyzed the transition of a pathology course to an emergency remote education online course and, in the second step, applied a flipped classroom approach including research skills training. METHODS: In the summer semester of 2020, the pathology course at the Technical University of Munich was completely converted to an asynchronous online course. Its content was adapted in winter 2021 and incorporated into a flipped classroom concept in which research skills were taught at the same time. RESULTS: Screencasts and lecture recordings were the most popular asynchronous teaching formats. Students reported developing a higher interest in pathology and research through group work. The amount of content was very challenging for some students. CONCLUSION: Flipped classroom formats are a viable option when using pre-existing content. We recommend checking such content for technical and didactic quality and optimizing it if necessary. Content on research skills can be combined very well with clinical teaching content.

ALDH1-Mediated Autophagy Sensitizes Glioblastoma Cells to Ferroptosis.

The fatal clinical course of human glioblastoma (GBM) despite aggressive adjuvant therapies is due to high rates of recurrent tumor growth driven by tumor cells with stem-cell characteristics (glioma stem cells, GSCs). The aldehyde dehydrogenase 1 (ALDH1) family of enzymes has been shown to be a biomarker for GSCs, and ALDH1 seems to be involved in the biological processes causing therapy resistance. Ferroptosis is a recently discovered cell death mechanism, that depends on iron overload and lipid peroxidation, and it could, therefore, be a potential therapeutic target in various cancer types. Since both ALDH1 and ferroptosis interact with lipid peroxidation (LPO), we aimed to investigate a possible connection between ALDH1 and ferroptosis. Here, we show that RSL3-induced LPO and ferroptotic cell death revealed RSL3-sensitive and -resistant malignant glioma cell lines. Most interestingly, RSL3 sensitivity correlates with ALDH1a3 expression; only high ALDH1a3-expressing cells seem to be sensitive to ferroptosis induction. In accordance, inhibition of ALDH1a3 enzymatic activity by chemical inhibition or genetic knockout protects tumor cells from RSL3-induced ferroptotic cell death. Both RSL-3-dependent binding of ALDH1a3 to LC3B and autophagic downregulation of ferritin could be completely blocked by ALDH inhibition. Therefore, ALDH1a3 seems to be involved in ferroptosis through the essential release of iron by ferritinophagy. Our results also indicate that ferroptosis induction might be a particularly interesting clinical approach for targeting the highly aggressive cell population of GSC.

TDP-43 condensates and lipid droplets regulate the reactivity of microglia and regeneration after traumatic brain injury.

Decreasing the activation of pathology-activated microglia is crucial to prevent chronic inflammation and tissue scarring. In this study, we used a stab wound injury model in zebrafish and identified an injury-induced microglial state characterized by the accumulation of lipid droplets and TAR DNA-binding protein of 43 kDa (TDP-43)+ condensates. Granulin-mediated clearance of both lipid droplets and TDP-43+ condensates was necessary and sufficient to promote the return of microglia back to the basal state and achieve scarless regeneration. Moreover, in postmortem cortical brain tissues from patients with traumatic brain injury, the extent of microglial activation correlated with the accumulation of lipid droplets and TDP-43+ condensates. Together, our results reveal a mechanism required for restoring microglia to a nonactivated state after injury, which has potential for new therapeutic applications in humans.

Five material tissue decomposition by dual energy computed tomography.

The separation of mixtures of substances into their individual components plays an important role in many areas of science. In medical imaging, one method is the established analysis using dual-energy computed tomography. However, when analyzing mixtures consisting of more than three individual basis materials, a physical limit is reached that no longer allows this standard analysis. In addition, the X-ray attenuation coefficients of chemically complicated basis materials may not be known and also cannot be determined by other or previous analyses. To address these issues, we developed a novel theoretical approach and algorithm and tested it on samples prepared in the laboratory as well as on ex-vivo medical samples. This method allowed both five-material decomposition and determination or optimization of the X-ray attenuation coefficients of the sample base materials via optimizations of objective functions. After implementation, this new multimodal method was successfully tested on self-mixed samples consisting of the aqueous base solutions iomeprol, eosin Y disodiumsalt, sodium chloride, and pure water. As a first proof of concept of this technique for detailed material decomposition in medicine we analyzed exact percentage composition of ex vivo clots from patients with acute ischemic stroke, using histological analysis as a reference standard.

Progressive multifocal leukoencephalopathy associated with chemotherapy induced lymphocytopenia in solid tumors - case report of an underestimated complication.

Background: JC virus reactivation causing progressive multifocal leukoencephalopathy (PML) occurs preferentially in human immunodeficiency virus (HIV) positive individuals or patients suffering from hematologic neoplasms due to impaired viral control. Reactivation in patients suffering from solid malignancies is rarely described in published literature. Case Presentation: Here we describe a case of PML in a male patient suffering from esophageal cancer who underwent neoadjuvant radiochemotherapy and surgical resection in curative intent resulting in complete tumor remission. The radiochemotherapy regimen contained carboplatin and paclitaxel (CROSS protocol). Since therapy onset, the patient presented with persistent and progredient leukopenia and lymphopenia in absence of otherwise known risk factors for PML. Symptom onset, which comprised aphasia, word finding disorder, and paresis, was apparent 7 months after therapy initiation. There was no relief in symptoms despite standard of care PML directed supportive therapy. The patient died two months after therapy onset. Conclusion: PML is a very rare event in solid tumors without obvious states of immununosuppression and thus harbors the risk of unawareness. The reported patient suffered from lymphopenia, associated with systemic therapy, but was an otherwise immunocompetent individual. In case of neurologic impairment in patients suffering from leukopenia, PML must be considered - even in the absence of hematologic neoplasia or HIV infection.

Glioblastoma Relapses Show Increased Markers of Vulnerability to Ferroptosis.

Background: Despite the availability of various therapy options and being a widely focused research area, the prognosis of glioblastoma (GBM) still remains very poor due to therapy resistance, genetic heterogeneity and a diffuse infiltration pattern. The recently described non-apoptotic form of cell death ferroptosis may, however, offer novel opportunities for targeted therapies. Hence, the aim of this study was to investigate the potential role of ferroptosis in GBM, including the impact of treatment on the expression of the two ferroptosis-associated players glutathione-peroxidase 4 (GPX4) and acyl-CoA-synthetase long-chain family number 4 (ACSL4). Furthermore, the change in expression of the recently identified ferroptosis suppressor protein 1 (FSP1) and aldehyde dehydrogenase (ALDH) 1A3 was investigated. Methods: Immunohistochemistry was performed on sample pairs of primary and relapse GBM of 24 patients who had received standard adjuvant treatment with radiochemotherapy. To identify cell types generally prone to undergo ferroptosis, co-stainings of ferroptosis susceptibility genes in combination with cell-type specific markers including glial fibrillary acidic protein (GFAP) for tumor cells and astrocytes, as well as the ionized calcium-binding adapter molecule 1 (Iba1) for microglial cells were performed, supplemented by double stains combining GPX4 and ACSL4. Results: While the expression of GPX4 decreased significantly during tumor relapse, ACSL4 showed a significant increase. These results were confirmed by analyses of data sets of the Cancer Genome Atlas. These profound changes indicate an increased susceptibility of relapsed tumors towards oxidative stress and associated ferroptosis, a cell death modality characterized by unrestrained lipid peroxidation. Moreover, ALDH1A3 and FSP1 expression also increased in the relapses with significant results for ALDH1A3, whereas for FSP1, statistical significance was not reached. Results obtained from double staining imply that ferroptosis occurs more likely in GBM tumor cells than in microglial cells. Conclusion: Our study implies that ferroptosis takes place in GBM tumor cells. Moreover, we show that recurrent tumors have a higher vulnerability to ferroptosis. These results affirm that utilizing ferroptosis processes might be a possible novel therapy option, especially in the situation of recurrent GBM.

Quantifying Heterogeneity in Tumors: Proposing a New Method Utilizing Convolutional Neuronal Networks.

Heterogeneity is a hallmark of glioblastoma (GBM), the most common malignant brain tumor, and a key reason for the poor survival rate of patients. However, establishing a clinically applicable, cost-efficient tool to measure and quantify heterogeneity is challenging. We present a novel method in an ongoing study utilizing two convolutional neuronal networks (CNN). After digitizing tumor samples, the first CNN delimitates GBM from normal tissue, the second quantifies heterogeneity within the tumor. Since neuronal networks can detect and interpret underlying and hidden information within images and have the ability to incorporate different information sets (i.e. clinical data and mutational status), this approach might venture towards a next level of integrated diagnosis. It may be applicable to other tumors as well and lead to a more precision-based medicine.

Socio-Organizational Impact of Confocal Laser Endomicroscopy in Neurosurgery and Neuropathology: Results from a Process Analysis and Expert Survey.

During brain tumor resection surgery, it is essential to determine the tumor borders as the extent of resection is important for post-operative patient survival. The current process of removing a tissue sample for frozen section analysis has several shortcomings that might be overcome by confocal laser endomicroscopy (CLE). CLE is a promising new technology enabling the digital in vivo visualization of tissue structures in near real-time. Research on the socio-organizational impact of introducing this new methodology to routine care in neurosurgery and neuropathology is scarce. We analyzed a potential clinical workflow employing CLE by comparing it to the current process. Additionally, a small expert survey was conducted to collect data on the opinion of clinical staff working with CLE. While CLE can contribute to a workload reduction for neuropathologists and enable a shorter process and a more efficient use of resources, the effort for neurosurgeons and surgery assistants might increase. Experts agree that CLE offers huge potential for better diagnosis and therapy but also see challenges, especially due to the current state of experimental use, including a risk for misinterpretations and the need for special training. Future studies will show whether CLE can become part of routine care.

Tick-Borne Encephalitis Virus (TBEV) Infection in Two Horses.

A final diagnosis in a horse with clinical signs of encephalopathy can be challenging despite the use of extensive diagnostics. Clinical signs are often not pathognomonic and need to be interpreted in combination with (specific) laboratory results and epidemiological data of the geographical region of the origin of the case(s). Here we describe the diagnostic pathway of tick-borne encephalitis virus infection in two horses using established molecular diagnostic methods and a novel in situ hybridization technique to differentiate between regionally important/emerging diseases for central Europe: (i) hepatoencephalopathy, (ii) Borna disease virus, and (iii) West Nile virus infections.

Whole-genome analysis of SARS-CoV-2 samples indicate no tissue specific genetic adaptation of the virus in COVID-19 patients' upper and lower respiratory tract.

Sample panels of SARS-CoV-2 cases were retrospectively whole-genome sequenced. In three individuals, samples of upper and lower respiratory tract resulted in identical sequences suggesting virus stability including the spike protein cleavage site. In a fourth case, low-level intra-host genomic evolution and a unique 5-nucleotide deletion was observed.

Visualizing cellularity and angiogenesis in newly-diagnosed glioblastoma with diffusion and perfusion MRI and FET-PET imaging.

PURPOSE: Combining imaging modalities has become an essential tool for assessment of tumor biology in glioblastoma (GBM) patients. Aim of this study is to understand how tumor cellularity and neovascularization are reflected in O-(2-[18F]fluoroethyl)-L-tyrosine positron emission tomography ([18F] FET PET) and magnetic resonance imaging (MRI) parameters, including cerebral blood volume (CBV), fractional anisotropy (FA) and mean diffusivity (MD). METHODS: In this prospective cohort, 162 targeted biopsies of 43 patients with therapy-naïve, isocitrate dehydrogenase (IDH) wildtype GBM were obtained after defining areas of interest based on imaging parameters [18F] FET PET, CBV, FA and MD. Histopathological analysis of cellularity and neovascularization was conducted and results correlated to imaging data. RESULTS: ANOVA analysis showed a significant increase of CBV in areas with high neovascularization. For diffusion metrics, and in particular FA, a trend for inverse association with neovascularization was found. [18F] FET PET showed a significant positive correlation to cellularity, while CBV also showed a trend towards correlation with cellularity, not reaching significant levels. In contrast, MD and FA were negatively associated with cellularity. CONCLUSION: Our study confirms that amino acid PET and MR imaging parameters are indicative of histological tumor properties in glioblastoma and highlights the ability of multimodal imaging to assess tumor biology non-invasively.