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The Lazarus effect is a rare condition that happens when someone seemingly dead shows signs of life. The epidermal growth factor receptor (EGFR) represents a target in the fatal neoplasm glioblastoma (GBM) that through a series of negative clinical trials has prompted a vocal subset of the neuro-oncology community to declare this target dead. However, an argument can be made that the core tenets of precision oncology were overlooked in the initial clinical enthusiasm over EGFR as a therapeutic target in GBM. Namely, the wrong drugs were tested on the wrong patients at the wrong time. Furthermore, new insights into the biology of EGFR in GBM vis-à-vis other EGFR-driven neoplasms, such as non-small cell lung cancer, and development of novel GBM-specific EGFR therapeutics resurrects this target for future studies. Here, we will examine the distinct EGFR biology in GBM, how it exacerbates the challenge of treating a CNS neoplasm, how these unique challenges have influenced past and present EGFR-targeted therapeutic design and clinical trials, and what adjustments are needed to therapeutically exploit EGFR in this devastating disease.

Glioblastoma (GBM) is an immunologically “cold” tumor characterized by poor responsiveness to immunotherapy. Standard of care for GBM is surgical resection followed by chemoradiotherapy and maintenance chemotherapy. However, tumor recurrence is the norm, and recurring tumors are found frequently to have acquired molecular changes (e.g., mutations) that may influence their immunobiology. Here, we compared the immune contexture of de novo GBM and recurrent GBM (rGBM) using high-dimensional cytometry and multiplex IHC. Although myeloid and T cells were similarly abundant in de novo and rGBM, their spatial organization within tumors differed and was linked to outcomes. In rGBM, T cells were enriched and activated in perivascular regions and clustered with activated macrophages and fewer regulatory T cells. Moreover, a higher expression of phosphorylated STAT1 by T cells in these regions at recurrence was associated with a favorable prognosis. Together, our data identify differences in the immunobiology of de novo GBM and rGBM and identify perivascular T cells as potential therapeutic targets. See related Spotlight by Bayik et al., p. 787

Differentiation of true tumor progression (TP) from pseudoprogression (PsP) is a major unmet need in patients with glioblastoma. [18F]Fluciclovine is a synthetic amino acid PET radiotracer that is FDA-approved in biochemical recurrence in prostate cancer. The study aim was to assess the value of [18F]Fluciclovine PET in differentiation of histologically confirmed (“true”) TP and PsP in post-treatment of glioblastoma. METHODS: 23 patients with glioblastoma with new contrast-enhancing lesions or lesions showing increased enhancement ( > 25% increase) on standard MRI after completion of radiation underwent 60-minutes dynamic [18F]Fluciclovine PET imaging. Patients subsequently underwent resection of enhancing lesion and tumor percentage vs. treatment-related changes were quantified on histopathology. Patients were considered "true” TP if tumor represented ≥ 50% of the resected specimen, mixed TP-PsP if < 50% and > 10%, and PsP if tumor represented ≤ 10%. Summed 30- to 40-minute post-injection PET images were used to measure SUVpeak and SUVmax (g/mL units). RESULTS: 15 patients with “true” TP, 3 with mixed TP-PsP, and 5 with PsP were included. There was a positive correlation between SUVpeak by PET and tumor percentage by histology (Rho= 0.56, p= 0.006). Patients who demonstrated “true” TP had significantly higher SUVpeak compared to patients with histological PsP (4.8±1.6 vs 2.9± 1.0, p= 0.02, AUC= 0.91, n=20). SUVpeak cut-off of 3.3 provided 93% sensitivity, 80% specificity, and 90% accuracy for differentiation of “true” TP from PsP. Patients with “true” TP/mixed TP-PsP also had significantly higher SUVpeak than patients with PsP (4.6±1.5 vs 2.9± 1.0, p= 0.03, AUC= 0.88, n=23). SUVmax and partial volume-corrected SUVpeak and SUVmax exhibited similar performance. CONCLUSION: Our results indicated that [18F]Fluciclovine PET imaging can accurately differentiate “true” TP from PsP. Further studies are required to confirm these promising early results and determine optimal criteria for interpreting [18F]Fluciclovine PET to distinguish PsP from TP.

Malignant solid tumors are characterized by aberrant vascularity that inhibits T cell adhesion to vasculature and impedes T cell delivery into the tumors, contributing to glioblastoma (GBM) resistance to T cell-based immunotherapy such as adoptive chimeric antigen receptor (CAR)–T transfer. Vascular abnormality is driven by pro-angiogenic pathway activation and genetic reprogramming in tumor endothelial cells (ECs). Here, our kinome-wide functional screening of mesenchymal-like transcriptional activation in human GBM-derived ECs identifies p21-activated kinase 4 (PAK4) as a selective regulator of genetic programming and aberrant vascularization in tumor. Genetic ablation of PAK4 reprograms EC transcriptome and inhibits mesenchymal-like transformation. Interestingly, deficiency of PAK4 induces adhesion protein re-expression in tumor ECs, reduces vascular abnormalities in tumors, improves T cell infiltration into the tumors, and inhibits GBM growth in mice. Moreover, pharmacological PAK4 inhibition normalizes the tumor vascular microenvironment and sensitizes GBM to Egfrviii CAR–T cell immunotherapy. Finally, we reveal a MEF2D/ZEB1- and SLUG-mediated mechanism by which PAK4 reprograms the EC transcriptome and downregulates claudin-14 and VCAM-1 expression, enhancing vessel permeability and reducing T cell adhesion to the endothelium. Thus, targeting PAK4-mediated EC plasticity may offer exciting opportunities to recondition the vascular microenvironment and strengthen cancer immunotherapy.

BACKGROUND Tumor-associated macrophages (TAMs) are a key component of glioblastoma (GBM) tumor microenvironment. Considering the differential role of different TAM phenotypes in iron metabolism with the M1 phenotype storing intracellular iron, and M2 phenotype releasing iron in the tumor microenvironment, here we investigated non-invasive quantitative susceptibility mapping (QSM) and T2* MRI relaxometry to quantify iron as imaging biomarkers for TAMs in adult patients with GBM. METHODS In this prospective study, 21 adult patients with GBM were enrolled between 2016 to 2019. Patients underwent a 3D single echo gradient echo sequence in addition to standard anatomical sequences on a 3 Tesla MRI. QSM images were reconstructed using the morphology-enabled dipole inversion (MEDI) algorithm. In 3 subjects, ex vivo imaging of surgical specimens was performed on a Bruker 9.4 Tesla 8.9 cm vertical bore MR using 3D multi-echo GRE scans, and R2* (1/T2*) maps were generated by a pixel-wise monoexponential fitting. Each specimen was stained with hematoxylin and eosin, as well as with CD68, CD86, CD206, and L-Ferritin. RESULTS Significant positive correlation was observed between mean susceptibility for the tumor enhancing zone and the L-ferritin positivity percent (r =0.56, p=0.018) and the combination of tumor’s enhancing zone and necrotic core and the L-Ferritin positivity percent (r=0.72; p=0.001). Moreover, the mean susceptibility significantly correlated with positivity percent for CD68 (ρ=0.52, p=0.034) and CD86 (r=0.7 p=0.001), but not for CD206 (ρ=0.09; p=0.7). There was a positive correlation between mean R2* values and CD68 positive cell counts (r =0.6, p=0.016). Similarly, mean R2* values significantly correlated with CD86 (r=0.54, p=0.03) but not with CD206 (r=0.15, p=0.5). High mean susceptibility in the necrotic core was associated with inferior PFS (hazard ratio,4.5, p=0.016). CONCLUSION MR Susceptibility Imaging can quantify the iron content of GBM and provide a non-invasive method for TAM quantification and phenotyping.

Glioblastoma (GBM) has high metabolic demands, which can lead to acidification of the tumor microenvironment. We hypothesize that a machine learning model built on temporal principal component analysis (PCA) of dynamic susceptibility contrast-enhanced (DSC) perfusion MRI can be used to estimate tumor acidity in GBM, as estimated by pH-sensitive amine chemical exchange saturation transfer echo-planar imaging (CEST-EPI). We analyzed 78 MRI scans in 32 treatment naïve and post-treatment GBM patients. All patients were imaged with DSC-MRI, and pH-weighting that was quantified from CEST-EPI estimation of the magnetization transfer ratio asymmetry (MTRasym) at 3 ppm. Enhancing tumor (ET), non-enhancing core (NC), and peritumoral T2 hyperintensity (namely, edema, ED) were used to extract principal components (PCs) and to build support vector machines regression (SVR) models to predict MTRasym values using PCs. Our predicted map correlated with MTRasym values with Spearman’s r equal to 0.66, 0.47, 0.67, 0.71, in NC, ET, ED, and overall, respectively (p

Background We aimed to determine whether plasma cell-free DNA (cfDNA) concentration is associated with survival in patients with isocitrate dehydrogenase (IDH) wild-type glioblastoma (GBM). Methods Pre-operative and post-chemoradiotherapy blood samples were prospectively collected from patients with newly diagnosed IDH wild-type GBM. Patients underwent surgical resection or biopsy and received adjuvant radiotherapy with concomitant temozolomide. Cell-free DNA (cfDNA) was isolated from plasma and quantified using SYBR Green-based q polymerase chain reaction (qPCR). Results Sixty-two patients were enrolled and categorized into high vs. low cfDNA groups relative to the pre-operative median value (25.2 ng/mL, range 5.7–153.0 ng/mL). High pre-operative cfDNA concentration was associated with inferior PFS (median progression-free survival (PFS), 3.4 vs. 7.7 months; log-rank P = .004; hazard ratio [HR], 2.19; 95% CI, 1.26–3.81) and overall survival (OS) (median OS, 8.0 vs. 13.9 months; log-rank P = .01; HR, 2.43; 95% CI, 1.19–4.95). After adjusting for risk factors, including O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status, pre-operative cfDNA remained independently associated with PFS (HR, 2.70; 95% CI, 1.50–4.83; P = .001) and OS (HR, 2.65; 95% CI, 1.25–5.59; P = .01). Post-hoc analysis of change in cfDNA post-chemoradiotherapy compared to pre-surgery (n = 24) showed increasing cfDNA concentration was associated with worse PFS (median, 2.7 vs. 6.0 months; log-rank P = .003; HR, 4.92; 95% CI, 1.53–15.84) and OS (median, 3.9 vs. 19.4 months; log-rank P < .001; HR, 7.77; 95% CI, 2.17–27.76). Conclusions cfDNA concentration is a promising prognostic biomarker for patients with IDH wild-type GBM. Plasma cfDNA can be obtained noninvasively and may enable more accurate estimates of survival and effective clinical trial stratification.

Tumor heterogeneity is one of the key reasons for therapeutic failure in Glioblastoma (GBM). Our chimeric antigen receptor (CAR) T cell clinical trial (NCT02209376) against Epidermal growth factor receptor (EGFR) variant III (EGFRvIII) demonstrated successful trafficking of T cells across the blood brain barrier into GBM active tumor sites. However, CART cell infiltration was associated with a selective loss of EGFRvIII+ tumor cells and upregulation of immunosuppressive molecules. Post-CAR T treated tumor specimens showed continued presence of EGFR amplification and oncogenic EGFR extracellular domain (ECD) missense mutations. To study this further We generated two structurally different CARs by fusing the scFv of mAb806 to 4-1BB and Killer immunoglobulin like receptor (KIR) co-stimulatory domains. Both 4-1BB and KIR based EGFR806 CAR T cells specifically lysed tumor cells and secreted cytokines when co-cultured with U87-MG (expressing low levels of EGFR) and U87-MG EGFR (U87-MG transduced with EGFR wild type for amplified background) cell lines and engineered to ectopically express targeted EGFR-ECD missense mutations and EGFRvIII. Unlike EGFR-specific cetuximab based CAR, EGFR-806CART cells did not kill EGFR wild type expressing fetal brain primary astrocytes and keratinocytes in vitro. We further evaluated the in vivo efficacy of 806-CARs and EGFRvIII CART currently in clinic in NSG mice. While EGFRvIII CART cells cleared EGFRvIII+ tumors alone 806 CART cells controlled EGFRvIII, amplified EGFR and EGFRR108K-mutant bearing tumors. KIR-CAR treated tumors are GVHD free and enhanced survival compared to 4-1BB based CARs. The broad specificity of EGFR806 CART cells to amplified EGFR and its mutant variants gives us the potential to clear various forms of EGFR. The enhanced anti-tumor efficacy by KIR based CAR in vivo setting provides us with additional therapeutic options.

AI-based methods have shown great promise in a variety of biomedical research fields, including neurooncologic imaging. For example, machine learning methods have offered informative predictions of overall survival (OS) and progression-free survival (PFS), differentiation between pseudoprogression (PsP) and progressive disease (PD), and estimation of mutational status from imaging data. Despite their promise, AI, and especially the emerging deep learning (DL) methods, are challenged by several factors, including imaging heterogeneity across scanners and lack of sufficiently large and diverse training datasets, which limits their reproducibility and general acceptance. These challenges prompted the development of the ReSPOND (Radiomics Signatures for PrecisiON Diagnostics) consortium on glioblastoma, a growing effort to bring together a community of researchers sharing imaging, demographic, clinical and (currently) limited molecular data in order to address the following aims: 1) pool and harmonize data across diverse hospitals and patient populations worldwide; 2) derive robust and generalizable AI models for prediction of (initially) OS, PFS, PsP vs. PD, and recurrence; 3) test these predictive models across multiple sites. In its first phase, ReSPOND aims to pool together approximately 3,000MRI scans (from 10institutions plus TCIA), along with demographics, KPS, and (for a subset) MGMT/IDH1 status. We present initial results testing the generalization of a previously trained model of OS on 505Penn datasets to 2independent cohorts from Case Western Reserve University and University Hospitals (N=44), and Penn (N=67). The results indicate good generalization, with correlation coefficients between OS/predicted-OS between 0.25 to 0.5, depending on variable availability, which is comparable to cross-validated accuracy previously obtained from the training set itself (N=505). Additional preliminary studies evaluating prediction of future recurrence from baseline pre-operative scans in de novo patients (Penn model applied to CWR) indicated potential for guiding targeted dose escalation and supra-total resection (excellent predictions in 6/12 patients, modest in 1/12, and poor in 5/12).

Background Plasma cell-free DNA (cfDNA) concentration is lower in glioblastoma (GBM) compared to other solid tumors, which can lead to low circulating tumor DNA (ctDNA) detection. In this study, we investigated the relationship between multimodality magnetic resonance imaging (MRI) and histopathologic features with plasma cfDNA concentration and ctDNA detection in patients with treatment-naive GBM. Methods We analyzed plasma cfDNA concentration, MRI scans, and tumor histopathology from 42 adult patients with newly diagnosed GBM. Linear regression analysis was used to examine the relationship of plasma cfDNA concentration before surgery to imaging and histopathologic characteristics. In a subset of patients, imaging and histopathologic metrics were also compared between patients with and without a detected tumor somatic mutation. Results Tumor volume with elevated (>1.5 times contralateral white matter) rate transfer constant (Kep, a surrogate of blood–brain barrier [BBB] permeability) was independently associated with plasma cfDNA concentration (P = .001). Histopathologic characteristics independently associated with plasma cfDNA concentration included CD68+ macrophage density (P = .01) and size of tumor vessels (P = .01). Patients with higher (grade ≥3) perivascular CD68+ macrophage density had lower volume transfer constant (Ktrans, P = .01) compared to those with lower perivascular CD68+ macrophage density. Detection of at least 1 somatic mutation in plasma cfDNA was associated with significantly lower perivascular CD68+ macrophages (P = .01). Conclusions Metrics of BBB disruption and quantity and distribution of tumor-associated macrophages are associated with plasma cfDNA concentration and ctDNA detection in GBM patients. These findings represent an important step in understanding the factors that determine plasma cfDNA concentration and ctDNA detection.

Chimeric antigen receptor (CAR)-engineered T cells have demonstrated remarkable success in the treatment of B cell malignancies. FDA approval of these therapies represents a watershed moment in the development of therapies for cancer. Despite the successes of the last decade, many patients will unfortunately not experience durable responses to CAR therapy. Emerging research has shed light on the biology responsible for these failures, and further highlighted the hurdles to broader success. Here, we review the recent research identifying how interactions between cancer cells and engineered immune cells result in resistance to CAR therapies.

Summary: A number of mitochondrial diseases arise from single-nucleotide variant (SNV) accumulation in multiple mitochondria. Here, we present a method for identification of variants present at the single-mitochondrion level in individual mouse and human neuronal cells, allowing for extremely high-resolution study of mitochondrial mutation dynamics. We identified extensive heteroplasmy between individual mitochondrion, along with three high-confidence variants in mouse and one in human that were present in multiple mitochondria across cells. The pattern of variation revealed by single-mitochondrion data shows surprisingly pervasive levels of heteroplasmy in inbred mice. Distribution of SNV loci suggests inheritance of variants across generations, resulting in Poisson jackpot lines with large SNV load. Comparison of human and mouse variants suggests that the two species might employ distinct modes of somatic segregation. Single-mitochondrion resolution revealed mitochondria mutational dynamics that we hypothesize to affect risk probabilities for mutations reaching disease thresholds. : Morris et al. use independent sequencing of multiple individual mitochondria from mouse and human brain cells to show high pervasiveness of mutations. The mutations are heteroplasmic within single mitochondria and within and between cells. These findings suggest mechanisms by which mutations accumulate over time, resulting in mitochondrial dysfunction and disease. Keywords: single mitochondrion, single cell, human neuron, mouse neuron, single-nucleotide variation

Glioblastoma treatment options remain limited due to its aggressive and invasive nature. It is increasingly appreciated that molecular heterogeneity between tumors and within tumors likely contributes to the lack of therapeutic advances. To maintain the inherent heterogeneity of glioblastoma, we employed a novel method to rapidly culture glioblastoma organoids (GBOs) directly from neurosurgical resection. GBOs are routinely generated around two weeks following initial resection. Comprehensive histologic and sequencing analyses demonstrated similarity to primary tumors. Leveraging clinical molecular and sequencing data, selected GBOs were treated with radiation/temozolamide and targeted inhibitor therapies. The effect on proliferation was measured by the percentage of KI67+ cells and gene set enrichment (GSEA) analysis was performed to compare the pre-treated expression signature amongst responsive and non-responsive tumors. Treatment of organoids with radiation/temozolamide led to a decrease in the percentage of KI67+ cells in four of eight patient-derived organoid lines with some evidence of correlative radiographic response Gene sets associated with radiation response and TNF signaling were enriched in radiation/temozolamide sensitive GBOs. GBO response to EGFR inhibition via gefitinib treatment was specific to EGFR altered tumors, whose expression also enriched for EGF signaling pathway expression. Two GBOs had downstream NF1 mutations that responded to the MEK inhibitor trametinib. On GSEA, gene expression of NF1 mutated GBOs enriched for RAS signaling. One GBO line was found to have a PI3K mutation and responded dramatically to mTOR inhibition via everolimus. Dichotomous efficacy of MEK and mTOR inhibition was also noted by tumor-specific changes in GBO diameter following treatment. This novel culturing method of GBOs maintains intertumoral and intratumoral heterogeneity and allows for therapeutic testing within two weeks of neurosurgical resection. As clinical sequencing because increasingly prevalent, GBOs may become a valuable tool to functionally test mutation-specific treatment strategies in a patient-specific manner within a clinically relevant time frame.

BACKGROUND This study builds on the results of the University of Pennsylvania sponsored phase I study of a single peripheral infusion of chimeric antigen receptor (CAR) T cells targeting epidermal growth factor receptor variant III (EGFRvIII) in recurrent glioblastoma (GBM) (NCT02209376). A dose of 5x108 CART-EGFRvIII cells was safe, and the cells were able to expand in the host and reach the GBM tumor in the brain. In addition, there was no cross-reactivity of CART-EGFRvIII cells with wild-type EGFR normally expressed by human tissues. Some patients required tumor resection after CAR T cell infusion. In situ evaluation of the tumor microenvironment demonstrated increased and robust expression of inhibitory molecules, such as programmed death-ligand 1 (PD-L1), compared to pre–CART-EGFRvIII tumor specimens. Therefore, we hypothesized that using a combination of CART-EGFRvIII cells and a PD-1 inhibitor would improve the outcome of the treatment. METHODS This single-center study (NCT03726515) has a single-arm, open-label, phase 1 design and will enroll 7 patients with newly diagnosed, O6-methylguanine-methyltransferase (MGMT)-unmethylated, EGFRvIII+ GBM. Following maximal safe tumor resection, patients receive a short course of adjuvant radiation with a total dose of 40 Gy administered in 15 fractions. Peripheral IV infusions of 2x108 CART-EGFRvIII cells and 200mg pembrolizumab begin 2–3 weeks after completing radiation therapy. Thereafter, subjects receive CART-EGFRvIII cells + pembrolizumab in 3-week cycles for up to 3 infusions of CART-EGFRvIII cells and 4 infusions of pembrolizumab. The primary endpoint of the study is the safety and tolerability of administering multiple infusions of CART-EGFRvIII cells in combination with pembrolizumab, as measured by the occurrence of study-related adverse events. Secondary endpoints include overall survival, progression-free survival, and objective response rate. PROGRESS: At 5 June 2019, 2 patients have been enrolled and treated on study.

High grade glioma is the most common malignant primary brain tumor. Despite the best available therapy, the median survival is limited. Chimeric antigen receptor (CAR)-T cells effectively attack target positive tumor cells, demonstrating a promising possible treatment. Based on our previous clinical and pre-clinical studies, we utilized epidermal growth factor receptor variant III (EGFRvIII) and interleukin 13 receptor subunit alpha 2 (IL13Rα2) targeting single-chain variable fragments (scFvs) to generate bi-specific tandem CAR T cells and bi-specific parallel CAR T cells to decrease the potential of antigen escape and tumor recurrence. 5, 10, 15 or 20 flexible amino acids were utilized to link scFvs in tandem CARs. Ribosomal “skipping” 2A peptides were utilized to generate parallel CAR T cells. Flow-based IL13Rα2 staining on glioma stem cells (GSCs) showed 7 out 11 cases positive (64%) heterogeneous intratumoral expression. Tandem CAR T cells and parallel CAR T cells upregulated CD69 expression (P< 0.0001), generated IFNγ, interleukin 2 and TNFα (P< 0.0001), as well as exhibiting cytotoxic activity (P< 0.0001) when compared against un-transduced T cells, in co-culture with either single- or double-target positive GSCs. There was no significant difference between different lengths of linker in tandem CAR T cells. Parallel CAR T cells generated a larger cytokine response (P< 0.0001) and more effectively killed target positive cells (P< 0.05) than tandem CAR T cells. In summary, glioma associated target expression is heterogeneous in GSCs. Multivalent tandem CAR T cells and parallel CAR T cells effectively respond to target expressing tumor cells. The function of tandem CAR T cells is not relative with the length of linker. Parallel CAR T cells are a more promising strategy in generating gene modified multivalent targeting T cells.

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults and carries a dismal prognosis. Significant challenges in the care of patients with GBM include marked vascular heterogeneity and arteriovenous (AV) shunting, which results in tumor hypoxia and inadequate delivery of systemic treatments to reach tumor cells. In this study, we investigated the utility of different MR perfusion techniques to detect and quantify arteriovenous (AV) shunting and tumor hypoxia in patients with GBM. Macrovascular shunting was present in 33% of subjects, with the degree of shunting ranging from (37–60%) using arterial spin labeling perfusion. Among the dynamic susceptibility contrast-enhanced perfusion curve features, there were a strong negative correlation between hypoxia score, DSC perfusion curve recovery slope (r = −0.72, P = 0.018) and angle (r = −0.73, P = 0.015). The results of this study support the possibility of using arterial spin labeling and pattern analysis of dynamic susceptibility contrast-enhanced MR Imaging for evaluation of arteriovenous shunting and tumor hypoxia in glioblastoma.

Molecular profiling of glioblastoma has revealed complex cytogenetic, epigenetic, and molecular abnormalities that are necessary for diagnosis, prognosis, and treatment. Our neuro-oncology group has developed a data-driven, institutional consensus guideline for efficient and optimal workup of glioblastomas based on our routine performance of molecular testing. We describe our institution’s testing algorithm, assay development, and genetic findings in glioblastoma, to illustrate current practices and challenges in neuropathology related to molecular and genetic testing. We have found that coordination of test requisition, tissue handling, and incorporation of results into the final pathologic diagnosis by the neuropathologist improve patient care. Here, we present analysis of O 6 -methylguanine-DNA-methyltransferase promoter methylation and next-generation sequencing results of 189 patients, obtained utilizing our internal processes led by the neuropathology team. Our institutional pathway for neuropathologist-driven molecular testing has streamlined the management of glioblastoma samples for efficient return of results for incorporation of genomic data into the pathological diagnosis and optimal patient care.

© 2019 Elsevier B.V. Objective: Corpus callosum (CC) involvement is a poor prognostic factor in patients with glioblastoma (GBM). The purpose of this study was to determine whether diffusion tensor imaging (DTI) can quantify occult tumor infiltration in the CC and predict the overall survival in GBM patients. Methods: Forty-eight patients with pathologically proven GBM and 17 normal subjects were included in this retrospective study. Patients were divided into four groups based on CC invasion and overall survival: long survivors without CC invasion; short survivors without CC invasion; long survivors with CC invasion; short survivors with CC invasion. All patients underwent DTI at 3T MRI scanner. Fractional anisotropy (FA) and mean diffusivity (MD) values were measured from genu, mid-body, and splenium of the CC. The mean values of these parameters were compared between different groups and Kaplan Meier curves were used for prediction of overall survival. Results: Patients with short survival and CC invasion had the lowest FA values (0.64 ± 0.05) from the CC compared with other groups (p < 0.05). Receiver operator characteristic curve (ROC) analysis indicated that a FA cutoff value of 0.70 was the best predictor for overall survival with an area under the curve (AUC) of 0.77, sensitivity 1, specificity 0.59. Kaplan-Meier survival curves demonstrated that the mean survival time was significantly longer for patients with high FA (>0.70) compared with those with low FA (

EGFRvIII targeted chimeric antigen receptor T (CAR-T) cell therapy has recently been reported for treating glioblastomas (GBMs); however, physiology-based MRI parameters have not been evaluated in this setting. Ten patients underwent multiparametric MRI at baseline, 1, 2 and 3 months after CAR-T therapy. Logistic regression model derived progression probabilities (PP) using imaging parameters were used to assess treatment response. Four lesions from “early surgery” group demonstrated high PP at baseline suggestive of progression, which was confirmed histologically. Out of eight lesions from remaining six patients, three lesions with low PP at baseline remained stable. Two lesions with high PP at baseline were associated with large decreases in PP reflecting treatment response, whereas other two lesions with high PP at baseline continued to demonstrate progression. One patient didn’t have baseline data but demonstrated progression on follow-up. Our findings indicate that multiparametric MRI may be helpful in monitoring CAR-T related early therapeutic changes in GBM patients.

PURPOSE Differentiation of true tumor progression from pseudoprogression (PsP) is a major unmet need in patients with glioblastoma (GBM). [18F]Fluciclovine is a synthetic amino acid PET radiotracer that is FDA approved in the setting of biochemical recurrence in prostate cancer. The aim of this study was to assess the value of [18F]Fluciclovine PET in differentiation of true tumor progression and PsP in post-treatment of glioblastoma. METHODS 15 patients with GBM with new contrast-enhancing lesions or lesions showing increased enhancement (>25% increase) on standard MRI after completion of radiation underwent 60-minutes dynamic [18F]Fluciclovine PET imaging. Patients subsequently (within 1 week) underwent resection of the enhancing lesion and the tumor percentage vs treatment-related changes were quantified on histopathology. Patients were considered true tumor progression if tumor represented ≥ 50% of the resected specimen and considered PsP if treatment-related changes represented ≥70% of the resected specimen. Summed 30- to 40-minute post-injection PET images were used to measure SUVpeak, SUVmax, and 50% threshold SUVmean. RESULTS 10 patients with true tumor progression and 5 patients with PsP were included. Patients who demonstrated true tumor progression had significantly higher SUVpeak compared to patients with PsP (5.3±1.4 vs 3.1± 0.9, p=0.002, AUC=0.92, p DISCUSSION Our preliminary results indicated that [18F]Fluciclovine PET imaging can accurately differentiate true tumor progression from PsP. Further studies are required to confirm these promising early results and determine the optimal criteria for interpreting [18F]Fluciclovine PET to distinguish PsP from true tumor progression.

PURPOSE: Mutational status of isocitrate dehydrogenase (IDH1) is a defining feature of the World Health Organization classification scheme for high grade gliomas (HGGs). IDH-mutant HGGs confer significantly improved prognoses when compared with IDH-wildtype, which typically describe the most common malignant primary HGGs in adults, namely glioblastoma. HGGs are densely cellular, pleomorphic tumors with high mitotic activity, with glioblastoma having either microvascular proliferation, or necrosis, or both. We hypothesize that integrative analysis of multi-parametric magnetic resonance imaging (mpMRI) via multivariate machine learning (ML), will enhance subtle yet important radiographic HGG characteristics, and reveal imaging signatures determinant of IDH1 mutational status. METHODS: 86 HGG patients were retrospectively identified with available pre-operative clinically-acquired mpMRI data (T1, T1-Gd, T2, T2-FLAIR, DTI, DSC-MRI). Each HGG was delineated into sub-regions of enhancement, non-enhancement, and peritumoral edema/invasion. 342 quantitative imaging phenomic (QIP) features extracted across sub-regions from all mpMRI, comprising descriptors of size, morphology, texture, intensity, and biophysical growth modeling. Cross-validated sequential feature selection determined the most discriminative QIP features for our integrative ML predictor of IDH1 status. The predicted classifications, following a 10-fold cross-validation, were compared with the IDH1 status obtained by next generation sequencing, or immunohistochemistry. RESULTS: 61 QIP features, primarily descriptive of tumor texture, were determined as most important for an IDH1 imaging signature. Using this signature, our predictor classified IDH1 mutational status with an accuracy of 88.4% (sensitivity=66.7%, specificity=92.9%). CONCLUSION: Quantitative analysis of clinically-acquired mpMRI reveals subtle/visually-imperceptible, yet informative features, which integrated via ML yield a non-invasive in vivo IDH1 imaging signature in HGG. Knowledge of IDH1 mutational status at initial presentation can influence therapeutic decision-making, which will have a significant impact on patient care. Particularly in this precision medicine era, as mutant-IDH enzyme inhibitors and immunotherapy targeting IDH-mutant tumor cells are developed, imaging to diagnose and follow IDH-mutant tumors can be invaluable. *equal contribution

BACKGROUND: The promoter methylation status of the gene encoding for the repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) indicates increased efficacy of current standard of care therapy, which is concomitant adjuvant chemoradiotherapy with temozolomide. The MGMT promoter methylation status (MGMTpms) is typically determined as MGMT-methylated or MGMT-unmethylated by tissue-based polymerase chain reaction assays, which can be limited by inadequate specimen or assay failures. Thus, we investigate the hypothesis that integration of subtle, yet distinctive, quantitative imaging phenomic (QIP) features using machine learning may lead to non-invasive determination of MGMTpms. METHODS: We identified a retrospective cohort of 122 (46 MGMT-methylated) pathology-proven de novo glioblastoma patients with available baseline pre-operative multi-parametric magnetic resonance imaging (mpMRI) data (T1, T1-Gd, T2, T2-FLAIR, DSC, DTI). MGMTpms was obtained through MGMT methylation testing (pyrosequencing across 4 CpG sites in the MGMT promoter). Following delineation of distinct abnormal sub-regions (enhancing, non-enhancing, edematous), comprehensive and diverse QIP features were extracted using the Cancer Imaging Phenomics Toolkit (CaPTk, www.cbica.upenn.edu/captk), capturing intensity, volume, morphology, statistics, and texture of each sub-region. A support vector machine multivariately integrated these features towards a non-invasive marker of MGMTpms. RESULTS: The cross-validated accuracy of our MGMT marker in classifying the mutation status in individual patients was 84.43% (sensitivity=80.43%, specificity=86.84%, area under the curve [AUC]=0.85). Our marker revealed MGMT-methylated tumors with lower neovascularization and cell density, when compared with MGMT-unmethylated tumors, and a distinct spatial distribution pattern between MGMT-methylated and MGMT-unmethylated tumors, with the latter being more lateralized to the right hemisphere. CONCLUSION: Multivariate integrative analysis of QIP features extracted from mpMRI yields an accurate, non-invasive marker of MGMTpms in glioblastoma. The proposed non-invasive MGMT marker may contribute to (i) MGMTpms determination for patients with inadequate tissue/inoperable tumors, (ii) stratification of patients into clinical trials, (iii) patient selection for targeted therapy, and (iv) personalized treatment planning. *equal contribution

Despite many clinical efforts, the prognosis of GBM patients has not improved in the last 10 years. Epigenetic silencing of the O6-methylguanine-DNA-methyltransferase (MGMT) gene is the only predictive biomarker that is associated with temozolomide (TMZ) response. The establishment of new biomarkers, for personalized treatment is of significant interest. The epidermal growth factor receptor (EGFR) variant III (EGFRvIII) is expressed in approximately 20–30% of GBM. The aim of this study was to investigate if EGFRvIII is a predictive biomarker for TMZ response by analyzing the TMZ sensitivity of isogenic cell cultures and tumour xenografts with/without endogenous EGFRvIII expression. Using two, unique isogenic pairs of MGMT deficient GBM cells we observed that endogenous EGFRvIII expression was associated with a significant increase in TMZ sensitivity in vitro and in vivo. Knockdown of EGFRvIII restored TMZ resistance, confirming EGFRvIII dependence. We observed prolonged accumulation in S/G2-phase in EGFRvIII+ cells after TMZ treatment, indicating increased replication stress induced by TMZ and a significantly higher number of DNA double strand breaks. Because TMZ sensitivity in MGMT negative cells is predominantly determined by functional mismatch repair (MMR), we next analyzed MMR protein expression. We observed that EGFRvIII expression was associated with upregulated MMR proteins and that conversely, even a moderate decrease in MSH6 and MLH1 expression yielded a pronounced increase in TMZ resistance. In accordance with our findings, analysis of clinical data revealed that EGFRvIII positive GBM patients displaying MGMT promotor methylation have significantly better outcomes than EGFRvIII negative patients. In summary, we have shown that MGMT negative EGFRvIII expressing GBM cells and tumours are significantly more sensitive to TMZ compared to isogenic EGFRvIII negative counterparts due to upregulated MMR. Our data highlight a new mechanism of TMZ sensitivity and we conclude that EGFRvIII is a predictive biomarker for response to TMZ in MGMT methylated GBM patients.

Summary Background Rindopepimut (also known as CDX-110), a vaccine targeting the EGFR deletion mutation EGFRvIII, consists of an EGFRvIII-specific peptide conjugated to keyhole limpet haemocyanin. In the ACT IV study, we aimed to assess whether or not the addition of rindopepimut to standard chemotherapy is able to improve survival in patients with EGFRvIII-positive glioblastoma. Methods In this randomised, double-blind, phase 3 trial, we recruited patients aged 18 years and older with glioblastoma from 165 hospitals in 22 countries. Eligible patients had newly diagnosed glioblastoma confirmed to express EGFRvIII by central analysis, and had undergone maximal surgical resection and completion of standard chemoradiation without progression. Patients were stratified by European Organisation for Research and Treatment of Cancer recursive partitioning analysis class, MGMT promoter methylation, and geographical region, and randomly assigned (1:1) with a prespecified randomisation sequence (block size of four) to receive rindopepimut (500 μg admixed with 150 μg GM-CSF) or control (100 μg keyhole limpet haemocyanin) via monthly intradermal injection until progression or intolerance, concurrent with standard oral temozolomide (150–200 mg/m 2 for 5 of 28 days) for 6–12 cycles or longer. Patients, investigators, and the trial funder were masked to treatment allocation. The primary endpoint was overall survival in patients with minimal residual disease (MRD; enhancing tumour 2 post-chemoradiation by central review), analysed by modified intention to treat. This trial is registered with ClinicalTrials.gov, number NCT01480479. Findings Between April 12, 2012, and Dec 15, 2014, 745 patients were enrolled (405 with MRD, 338 with significant residual disease [SRD], and two unevaluable) and randomly assigned to rindopepimut and temozolomide (n=371) or control and temozolomide (n=374). The study was terminated for futility after a preplanned interim analysis. At final analysis, there was no significant difference in overall survival for patients with MRD: median overall survival was 20·1 months (95% CI 18·5–22·1) in the rindopepimut group versus 20·0 months (18·1–21·9) in the control group (HR 1·01, 95% CI 0·79–1·30; p=0·93). The most common grade 3–4 adverse events for all 369 treated patients in the rindopepimut group versus 372 treated patients in the control group were: thrombocytopenia (32 [9%] vs 23 [6%]), fatigue (six [2%] vs 19 [5%]), brain oedema (eight [2%] vs 11 [3%]), seizure (nine [2%] vs eight [2%]), and headache (six [2%] vs ten [3%]). Serious adverse events included seizure (18 [5%] vs 22 [6%]) and brain oedema (seven [2%] vs 12 [3%]). 16 deaths in the study were caused by adverse events (nine [4%] in the rindopepimut group and seven [3%] in the control group), of which one—a pulmonary embolism in a 64-year-old male patient after 11 months of treatment—was assessed as potentially related to rindopepimut. Interpretation Rindopepimut did not increase survival in patients with newly diagnosed glioblastoma. Combination approaches potentially including rindopepimut might be required to show efficacy of immunotherapy in glioblastoma. Funding Celldex Therapeutics, Inc.

Purpose: The epidermal growth factor receptor variant III (EGFRvIII) mutation has been considered a driver mutation and therapeutic target in glioblastoma, the most common and aggressive brain cancer. Currently, detecting EGFRvIII requires postoperative tissue analyses, which are ex vivo and unable to capture the tumor's spatial heterogeneity. Considering the increasing evidence of in vivo imaging signatures capturing molecular characteristics of cancer, this study aims to detect EGFRvIII in primary glioblastoma noninvasively, using routine clinically acquired imaging. Experimental Design: We found peritumoral infiltration and vascularization patterns being related to EGFRvIII status. We therefore constructed a quantitative within-patient peritumoral heterogeneity index (PHI/ϕ-index), by contrasting perfusion patterns of immediate and distant peritumoral edema. Application of ϕ-index in preoperative perfusion scans of independent discovery (n = 64) and validation (n = 78) cohorts, revealed the generalizability of this EGFRvIII imaging signature. Results: Analysis in both cohorts demonstrated that the obtained signature is highly accurate (89.92%), specific (92.35%), and sensitive (83.77%), with significantly distinctive ability (P = 4.0033 × 10−10, AUC = 0.8869). Findings indicated a highly infiltrative-migratory phenotype for EGFRvIII+ tumors, which displayed similar perfusion patterns throughout peritumoral edema. Contrarily, EGFRvIII− tumors displayed perfusion dynamics consistent with peritumorally confined vascularization, suggesting potential benefit from extensive peritumoral resection/radiation. Conclusions: This EGFRvIII signature is potentially suitable for clinical translation, since obtained from analysis of clinically acquired images. Use of within-patient heterogeneity measures, rather than population-based associations, renders ϕ-index potentially resistant to inter-scanner variations. Overall, our findings enable noninvasive evaluation of EGFRvIII for patient selection for targeted therapy, stratification into clinical trials, personalized treatment planning, and potentially treatment-response evaluation. Clin Cancer Res; 23(16); 4724–34. ©2017 AACR.

OBJECTIVES/SPECIFIC AIMS: In patients with recurrent glioblastoma (GBM) who undergo a second surgery following standard chemoradiotherapy, histopathologic examination of the resected tissue often reveals a combination of viable tumor and treatment-related inflammatory changes. However, it remains unclear whether the degree of viable tumor Versus “treatment effect” in these specimens impacts prognosis. We sought to determine whether the percentage of viable tumor Versus “treatment effect” in recurrent GBM surgical samples, as assessed by a trained neuropathologist and quantified on a continuous scale, is associated with overall survival. METHODS/STUDY POPULATION: We reviewed the records of 47 patients with histopathologically confirmed GBM who underwent surgical resection as the first therapeutic modality for suspected radiographic progression following standard radiation therapy and temozolomide. The percentage of viable tumor Versus “treatment effect” in each specimen was estimated by one neuropathologist who was blinded to patient outcomes. RESULTS/ANTICIPATED RESULTS: After adjusting for other known prognostic factors in a multivariate Cox proportional hazards model, there was no association between the degree of viable tumor and overall survival (HR 0.83; 95% CI, 0.20–3.4; p=0.20). DISCUSSION/SIGNIFICANCE OF IMPACT: These results suggest that, in patients who undergo resection for recurrent GBM following standard first-line chemoradiotherapy, histopathologic quantification of the degree of viable tumor Versus “treatment effect” present in the surgical specimen has limited prognostic influence and clinical utility.

We have initiated a first-in-human pilot study of intravenous delivery of autologous T cells re-directed to the EGFR variant III mutation by means of a lentiviral vector encoding a chimeric antigen receptor (CAR). Patients with recurrent or incompletely resected glioblastoma (GBM) have their tumors screened for the presence of the EGFRvIII mutation by a next-generation sequencing based assay. Eligible patients undergo leukapheresis for collection of autologous T cells, which are then manufactured to form CART-EGFRvIII T cell products. We report preliminary results on the first six patients we have treated. We targeted a challenging subset of GBM patients, with multi-focal recurrent, residual, progressive disease refractory to standard and experimental therapies, including chemotherapy, radiation, bevacizumab and prior immunotherapy. To date, we have found that infusion of CART-EGFRvIII cells is safe, without evidence of off-tumor toxicity such as cross-reactivity to wild type EGFR. No clinical or laboratory signs of systemic cytokine release syndrome have been observed. One patient developed a seizure and non-convulsive status epilepticus nine days after CART-EGFRvIII infusion. Seizures resolved with anti-epileptic medications, and the patient was treated with steroids and anti-IL6 cytokine therapy. All patients have had significant expansion of CART-EGFRvIII cells as measured by flow cytometry and quantitative PCR in peripheral blood samples, despite the use of steroids in two patients. In one of two patients who had operative resection of presumed residual tumor, pathologic evaluation demonstrated inflammation, T cell infiltration, and EGFRvIII antigen loss. In total, two of six heavily pretreated patients are clinically stable three months post-CART infusion. These findings provide preliminary evidence that CART-EGFRvIII cells are safe, without evidence of off-target toxicity and without evidence of cytokine release syndrome, and that these are active T cell products that expand in the blood.