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Although signaling from phosphatidylinositol 3-kinase (PI3K) and AKT to mechanistic target of rapamycin (mTOR) is prominently dysregulated in high-grade glial brain tumors, blockade of PI3K or AKT minimally affects downstream mTOR activity in glioma. Allosteric mTOR inhibitors, such as rapamycin, incompletely block mTORC1 compared with mTOR kinase inhibitors (TORKi). Here, we compared RapaLink-1, a TORKi linked to rapamycin, with earlier-generation mTOR inhibitors. Compared with rapamycin and Rapalink-1, TORKi showed poor durability. RapaLink-1 associated with FKBP12, an abundant mTOR-interacting protein, enabling accumulation of RapaLink-1. RapaLink-1 showed better efficacy than rapamycin or TORKi, potently blocking cancer-derived, activating mutants of mTOR. Our study re-establishes mTOR as a central target in glioma and traces the failure of existing drugs to incomplete/nondurable inhibition of mTORC1.

BACKGROUND Unlike cancers affecting many solid organs, gliomas exist within the context of complex neural circuitry. It remains unknown whether glioma-neuron interactions play a role in maintaining functional circuits underlying cognition. We test the hypothesis that malignant gliomas remodel functional circuits through glioma-neuron interactions. METHODS Using language processing as a model for functional circuit dynamics, we enrolled 53 patients with dominant hemisphere IDH-wild-type glioblastoma. Task related circuit dynamics were measured using electrocorticography. Magnetoencephalography measures of functional connectivity identified intratumoral connectivity (HFC) and suppressed connectivity (LFC) regions. Primary patient samples and cultures from HFC and LFC-sites were assessed by single-cell RNA sequencing, pre/post-synaptic marker expression, cocultured with murine hippocampal neurons, and induced neuron organoids. Hippocampal tumor xenografts were created. Language/survival statistics were performed to correlate with functional connectivity measures. RESULTS Speech production evokes neuronal population spikes within the entire area of tumor-infiltrated cortex, far beyond the cortical territory normally involved in expressive language. Primary patient samples from HFC-regions are enriched for glioblastoma cells with a synaptogenic profile as characterized by pre-and post-synaptic marker expression at both tissue and cellular levels. RNA-sequencing and proteomic analyses from HFC samples revealed a neurogenic signature including thrombospondin-1 originating from glioma cells in HFC-regions and non-tumor astrocytes in LFC-regions. HFC xenografts demonstrated increased total number of synapses. Importantly, when compared with gliomas without intratumoral functional connectivity, connected gliomas have worse language task performance (r= -0.54,p=0.03) and shorter OS (medianOS-64 weeks compared with 107-weeks,p=0.04). CONCLUSION Glioma infiltrated regions generate task-relevant neural responses, with speech production evoking neuronal activity throughout tumor-involved cortex in the dominant hemisphere. An enriched population of synaptogenic glioma cells are organized within functionally connected intratumoral regions and this confers negative functional and survival outcomes. Together, these findings indicate that malignant gliomas can functionally remodel neural circuitry, thereby impairing neurological function and promoting tumor progression.

The modified Edmonston vaccine strain of measles virus (MV) has shown potent oncolytic efficacy against various tumor types and is being investigated in clinical trials. Our laboratory showed that MV effectively kills medulloblastoma tumor cells in both localized disease and when tumor cells are disseminated through cerebrospinal fluid (CSF). Although the safety of repeated intracerebral injection of modified MV in rhesus macaques has been established, the safety of administering MV into CSF has not been adequately investigated. In this study, we assessed the safety of MV-NIS (MV modified to express the human sodium iodide symporter protein) injected into the CSF of measles-immunized and measles virus-susceptible transgenic (CD46, IFNαRko) mice. Treated animals were administered a single intraventricular injection of 1 × 105 or 1 × 106 TCID50 (50% tissue culture infective dose) of MV-NIS. Detailed clinical observation was performed over a 90-day period. Clinically, we did not observe any measles-related t...

Author(s): Calixto-Hope, Lucas; Lee, Julieann; Sloan, Emily; Hofmann, Jeffrey; Van Ziffle, Jessica; Onodera, Courtney; Grenert, James; Devine, Patrick; Kline, Cassie; Banerjee, Anu; Clarke, Jennifer; Taylor, Jennie; Ann Oberheim-Bush, Nancy; Buerki, Robin; Butowski, Nicholas; Chang, Susan; McDermott, Mike; Aghi, Manish; Theodosopoulos, Philip; Hervey-Jumper, Shawn; Berger, Mitchel; Raffel, Corey; Gupta, Nalin; Kleinschmidt-DeMasters, Bette; Wood, Matthew; Grafe, Marjorie; Guo, Hua; Sun, Peter; Torkildson, Joseph; Cooney, Tabitha; Fata, Cynthia; Scharnhorst, David; Samuel, David; Bannykh, Serguei; Khatib, Ziad; Maher, Ossama; Chamyan, Gabriel; Pelaez, Liset; Brathwaite, Carole; Jin, Lee-way; Lechpammer, Mirna; Born, Donald; Vogel, Hannes; Lee, Han; Phillips, Joanna; Pekmezci, Melike; Bollen, Andrew; Tihan, Tarik; Perry, Arie; Solomon, David | Abstract: Abstract BACKGROUND Rosette-forming glioneuronal tumor (RGNT) is an uncommon CNS tumor originally described in the fourth ventricle characterized by a low-grade glial neoplasm admixed with a rosette-forming neurocytic component. METHODS We reviewed clinicopathologic features of 42 patients with RGNT. Targeted next-generation sequencing was performed, and genome-wide methylation profiling is underway. RESULTS The 20 male and 22 female patients had a mean age of 25 years (range 3–47) at time of diagnosis. Tumors were located within or adjacent to the lateral ventricle (n=16), fourth ventricle (15), third ventricle (9), and spinal cord (2). All 31 tumors assessed to date contained FGFR1 activating alterations, either in-frame gene fusion, kinase domain tandem duplication, or hotspot missense mutation in the kinase domain (p.N546 or p.K656). While 7 of these 31 tumors harbored FGFR1 alterations as the solitary pathogenic event, 24 contained additional pathogenic alterations within PI3-kinase or MAP kinase pathway genes: 5 with additional PIK3CA and NF1 mutations, 4 with PIK3CA mutation, 3 with PIK3R1 mutation (one of which also contained focal RAF1 amplification), 5 with PTPN11 mutation (one with additional PIK3R1 mutation), and 2 with NF1 deletion. The other 5 cases demonstrated anaplastic features including hypercellularity and increased mitotic activity. Among these anaplastic cases, 3 harbored inactivating ATRX mutations and two harbored CDKN2A homozygous deletion, in addition to the FGFR1 alterations plus other PI3-kinase and MAP kinase gene mutations seen in those RGNT without anaplasia. CONCLUSION Independent of ventricular location, RGNT is defined by FGFR1 activating mutations or rearrangements, which are frequently accompanied by mutations involving PIK3CA, PIK3R1, PTPN11, NF1, and KRAS. Whereas pilocytic astrocytoma and ganglioglioma are characterized by solitary activating MAP kinase pathway alterations (e.g. BRAF fusion or mutation), RGNT are genetically more complex with dual PI3K-Akt-mTOR and Ras-Raf-MAPK pathway activation. Rare anaplastic examples may show additional ATRX and/or CDKN2A inactivation.

The design and evaluation of immunotherapies in IDH-mutant lower grade gliomas (LGG) is hindered by a poor understanding of the LGG T-cell repertoire. We present data on the temporal evolution, intratumoral spatial distribution, and prognostic value of the T-cell repertoire in IDH-mutant LGGs. We performed immunogenomic profiling using T-cell receptor beta-chain sequencing of 163 glioma and peripheral blood samples from 33 immunotherapy-naive glioma patients (22 astrocytomas, 11 oligodendrogliomas). T-cell repertoire evolution was analyzed in a subset of 26 patients (69 samples) with matched primary (WHO grade II) and recurrent (WHO grade II-IV) glioma samples. T-cell repertoire diversity was defined as the number of unique T-cell clonotypes by V-gene, J-gene, and CDR3 nucleotide sequences. Malignant transformed (Grade III or IV) recurrent gliomas demonstrated increased T-cell repertoire diversity compared to their patient-matched primary tumors (p=0.0023), but grade II recurrences did not show the same increased diversity (p=0.26). This increase in T-cell repertoire diversity was greater in patients who underwent transformation in the context of TMZ-associated hypermutation compared to spontaneously transformed counterparts (p=0.035). In grade II primary astrocytomas (n=17), T-cell repertoire diversity above the median (186 unique T-cell clonotypes per sample) was associated with worse transformation-free (HR=4.2, p=0.045) and overall survival (HR=6.4, p=0.025). Next, we evaluated intratumoral immune heterogeneity in 7 patients by sampling from up to 10 distinct and maximally-separated intratumoral sites per LGG (64 samples). Eighty-two to 96% of unique clonotypes within a given tumor were present only within a single sampled site. Despite this heterogeneity, six LGG patients harbored T-cell clonotypes present tumor-wide across all sampled sites within a given tumor. Ten of 24 (42%) tumor-wide T-cell clonotypes were enriched in the glioma compared to matched peripheral blood, suggesting glioma-specificity. Taken together, T-cell receptor profiling in LGGs may have utility both as a prognostic biomarker and to identify glioma-specific T-cells.

This clinical trial evaluated whether whole exome sequencing (WES) and RNA sequencing (RNAseq) of paired normal and tumor tissues could be incorporated into a personalized treatment plan for newly diagnosed patients (

Although tumor-propagating cells can be derived from glioblastomas (GBM) of the proneural and mesenchymal subtypes, a glioma stem-like cell (GSC) of the classic subtype has not been identified. It is unclear whether mesenchymal GSCs (mGSC) and/or proneural GSCs (pGSC) alone are sufficient to generate the heterogeneity observed in GBM. We performed single-cell/single-nucleus RNA sequencing of 28 gliomas, and single-cell ATAC sequencing for 8 cases. We found that GBM GSCs reside on a single axis of variation, ranging from proneural to mesenchymal. In silico lineage tracing using both transcriptomics and genetics supports mGSCs as the progenitors of pGSCs. Dual inhibition of pGSC-enriched and mGSC-enriched growth and survival pathways provides a more complete treatment than combinations targeting one GSC phenotype alone. This study sheds light on a long-standing debate regarding lineage relationships among GSCs and presents a paradigm by which personalized combination therapies can be derived from single-cell RNA signatures, to overcome intratumor heterogeneity. Significance: Tumor-propagating cells can be derived from mesenchymal and proneural glioblastomas. However, a stem cell of the classic subtype has yet to be demonstrated. We show that classic-subtype gliomas are comprised of proneural and mesenchymal cells. This study sheds light on a long-standing debate regarding lineage relationships between glioma cell types. See related commentary by Fine, p. 1650. This article is highlighted in the In This Issue feature, p. 1631

Glioma is recognized to be a highly heterogeneous CNS malignancy, whose diverse cellular composition and cellular interactions have not been well characterized. To gain new clinical- and biological-insights into the genetically-bifurcated IDH1 mutant (mt) vs wildtype (wt) forms of glioma, we integrated multiplexed immunofluorescence single cell data for 43 protein markers across cancer hallmarks, in addition to cell spatial metrics, genomic sequencing and magnetic resonance imaging (MRI) quantitative features. Molecular and spatial heterogeneity scores for angiogenesis and cell invasion differ between IDHmt and wt gliomas irrespective of prior treatment and tumor grade; these differences also persisted in the MR imaging features of peritumoral edema and contrast enhancement volumes. Longer overall survival for IDH1mt glioma patients may reflect generalized altered cellular, molecular, spatial heterogeneity which manifest in discernable radiological manifestations.

BackgroundChromosomal instability is associated with earlier progression in isocitrate dehydrogenase (IDH)–mutated astrocytomas. Here we evaluated the prognostic significance of polysomy in gliomas tested for 1p/19q status.MethodsWe analyzed 412 histologic oligodendroglial tumors with use of 1p/19q testing at 8 institutions from 1996 to 2013; fluorescence in situ hybridization (FISH) for 1p/19q was performed. Polysomy was defined as more than two 1q and 19p signals in cells. Tumors were divided into groups on the basis of their 1p/19q status and polysomy and were compared for progression-free survival (PFS) and overall survival (OS).ResultsIn our cohort, 333 tumors (81%) had 1p/19q loss; of these, 195 (59%) had concurrent polysomy and 138 (41%) lacked polysomy, 79 (19%) had 1p/19q maintenance; of these, 30 (38%) had concurrent polysomy and 49 (62%) lacked polysomy. In agreement with prior studies, the group with 1p/19q loss had significantly better PFS and OS than did the group with 1p/19q maintenance (P < 0.0001 each). Patients with 1p/19q loss and polysomy showed significantly shorter PFS survival than patients with 1p/19q codeletion only (P < 0.0001), but longer PFS and OS than patients with 1p/19q maintenance (P < 0.01 and P < 0.0001). There was no difference in survival between tumors with >30% polysomic cells and those with ConclusionsThe presence of polysomy in oligodendroglial tumors with codeletion of 1p/19q predicts early recurrence and short survival in patients with 1p/19q codeleted tumors.

Author(s): Lee, Julieann C; Mazor, Tali; Lao, Richard; Wan, Eunice; Diallo, Alpha B; Hill, Nicholas S; Thangaraj, Naina; Wendelsdorf, Katherine; Samuel, David; Kline, Cassie N; Banerjee, Anuradha; Auguste, Kurtis; Raffel, Corey; Gupta, Nalin; Berger, Mitchel; Raleigh, David R; Shai, Anny; Phillips, Joanna J; Bollen, Andrew W; Tihan, Tarik; Perry, Arie; Costello, Joseph; Solomon, David A

High-grade neuroepithelial tumor with BCOR exon 15 internal tandem duplication (HGNET BCOR ex15 ITD) is a recently proposed tumor entity of the central nervous system (CNS) with a distinct methylation profile and characteristic genetic alteration. The complete spectrum of histologic features, accompanying genetic alterations, clinical outcomes, and optimal treatment for this new tumor entity are largely unknown. Here, we performed a comprehensive assessment of ten new cases of HGNET BCOR ex15 ITD. The tumors mostly occurred in young children and were located in the cerebral or cerebellar hemispheres. On imaging all tumors were large, well-circumscribed, heterogeneous masses with variable enhancement and reduced diffusion. They were histologically characterized by predominantly solid growth, glioma-like fibrillarity, perivascular pseudorosettes, and palisading necrosis, but absence of microvascular proliferation. They demonstrated sparse to absent GFAP expression, no synaptophysin expression, variable OLIG2 and NeuN positivity, and diffuse strong BCOR nuclear positivity. While BCOR exon 15 internal tandem duplication was the solitary pathogenic alteration identified in six cases, four cases contained additional alterations including CDKN2A/B homozygous deletion, TERT amplification or promoter hotspot mutation, and damaging mutations in TP53, BCORL1, EP300, SMARCA2, and STAG2. While the limited clinical follow-up in prior reports had indicated a uniformly dismal prognosis for this tumor entity, this cohort includes multiple long-term survivors. Our study further supports inclusion of HGNET BCOR ex15 ITD as a distinct CNS tumor entity and expands the known clinicopathologic, radiographic, and genetic features.

Pleomorphic xanthoastrocytoma (PXA) is an astrocytic neoplasm that is typically well circumscribed and can have a relatively favorable prognosis. Tumor progression to anaplastic PXA (WHO grade III), however, is associated with a more aggressive biologic behavior and worse prognosis. The factors that drive anaplastic progression are largely unknown. We performed comprehensive genomic profiling on a set of twenty-three PXAs from 19 patients, including 15 with anaplastic PXA. Four patients had tumor tissue from multiple recurrences, including two with anaplastic progression. We find that PXAs are genetically defined by the combination of CDKN2A biallelic inactivation and RAF alterations that were present in all 19 cases, most commonly as CDKN2A homozygous deletion and BRAF p.V600E mutation but also occasionally BRAF or RAF1 fusions or other rearrangements. The third most commonly altered gene in anaplastic PXA was TERT, with 47% (7/15) harboring TERT alterations, either gene amplification (n=2) or promoter hotspot mutation (n=5). In tumor pairs analyzed before and after anaplastic progression, two had increased copy number alterations and one had TERT promoter mutation at recurrence. Less commonly altered genes included TP53, BCOR, BCORL1, ARID1A, ATRX, PTEN, and BCL6. All PXA in this cohort were IDH and histone H3 wildtype, and did not contain alterations in EGFR. Genetic profiling performed on six regions from the same tumor identified intratumoral genomic heterogeneity, likely reflecting clonal evolution during tumor progression. Overall, anaplastic PXA is characterized by the combination of CDKN2A biallelic inactivation and oncogenic RAF kinase signaling as well as a relatively small number of additional genetic alterations, with the most common being TERT amplification or promoter mutation. These data define a distinct molecular profile for PXA and suggest additional genetic alterations, including TERT, may be associated with anaplastic progression. This article is protected by copyright. All rights reserved.

Glioma is recognized to be a highly heterogeneous CNS malignancy, whose diverse cellular composition and cellular interactions have not been well characterized. To gain new clinical- and biological-insights into the genetically-bifurcated IDH1 mutant (mt) vs wildtype (wt) forms of glioma, we integrated data from protein, genomic and MR imaging from 20 treatment-naïve glioma cases and 16 recurrent GBM cases. Multiplexed immunofluorescence (MxIF) was used to generate single cell data for 43 protein markers representing all cancer hallmarks, Genomic sequencing (exome and RNA (normal and tumor) and magnetic resonance imaging (MRI) quantitative features (protocols were T1-post, FLAIR and ADC) from whole tumor, peritumoral edema and enhancing core vs equivalent normal region were also collected from patients. Based on MxIF analysis, 85,767 cells (glioma cases) and 56,304 cells (GBM cases) were used to generate cell-level data for 24 biomarkers. K-means clustering was used to generate 7 distinct groups of cells with divergent biomarker profiles and deconvolution was used to assign RNA data into three classes. Spatial and molecular heterogeneity metrics were generated for the cell data. All features were compared between IDH mt and IDHwt patients and were finally combined to provide a holistic/integrated comparison. Protein expression by hallmark was generally lower in the IDHmt vs wt patients. Molecular and spatial heterogeneity scores for angiogenesis and cell invasion also differed between IDHmt and wt gliomas irrespective of prior treatment and tumor grade; these differences also persisted in the MR imaging features of peritumoral edema and contrast enhancement volumes. A coherent picture of enhanced angiogenesis in IDHwt tumors was derived from multiple platforms (genomic, proteomic and imaging) and scales from individual proteins to cell clusters and heterogeneity, as well as bulk tumor RNA and imaging features. Longer overall survival for IDH1mt glioma patients may reflect mutation-driven alterations in cellular, molecular, and spatial heterogeneity which manifest in discernable radiological manifestations.

PBTC-042 was a phase I trial of palbociclib to determine the maximum tolerated dose (MTD) and describe toxicities in children. Palbociclib is an oral, selective cyclin dependent kinase 4/6 inhibitor. METHODS: A rolling-6 design was utilized. Eligible patients were children ≥4 and ≤21 years-old with a progressive/refractory CNS tumor with intact retinoblastoma protein, measurable disease, and ability to swallow capsules. Pharmacokinetic studies were performed during the first course. Here, we report on the heavily pretreated stratum, which included patients who received >4 prior treatment regimens (either chemotherapy or biologic agent), and/or craniospinal irradiation, and/or myeloablative chemotherapy plus stem cell rescue. Palbociclib was initiated at 50 mg/m2/day for 21 consecutive days of a 28-day course. This was one dosage level below the MTD for the less heavily pretreated stratum (75 mg/m2). RESULTS: Fourteen eligible patients were enrolled (median age 12.8 years; male 79%). Eleven patients (79%) had either ependymoma or medulloblastoma. Four eligible and evaluable patients were enrolled at 50 mg/m2 with no DLTs. This prompted a dosage increase to 75 mg/m2. Ten eligible subjects were enrolled and 7 were evaluable for DLT assessment. One of 7 evaluable patients experienced a DLT (grade 3 thrombocytopenia). This established 75 mg/m2 as the MTD for more heavily pretreated patients. Mean ± SD palbociclib apparent oral clearance was 34.6 ± 18.4 L/h/m2. CONCLUSION: The MTD for palbociclib on a 3 week on/1 week off schedule in children with brain tumors is 75 mg/m2 and does not appear to be influenced by the degree of prior therapy.

We report a comprehensive proteogenomics analysis, including whole-genome sequencing, RNA sequencing, and proteomics and phosphoproteomics profiling, of 218 tumors across 7 histological types of childhood brain cancer: low-grade glioma (n = 93), ependymoma (32), high-grade glioma (25), medulloblastoma (22), ganglioglioma (18), craniopharyngioma (16), and atypical teratoid rhabdoid tumor (12). Proteomics data identify common biological themes that span histological boundaries, suggesting that treatments used for one histological type may be applied effectively to other tumors sharing similar proteomics features. Immune landscape characterization reveals diverse tumor microenvironments across and within diagnoses. Proteomics data further reveal functional effects of somatic mutations and copy number variations (CNVs) not evident in transcriptomics data. Kinase-substrate association and co-expression network analysis identify important biological mechanisms of tumorigenesis. This is the first large-scale proteogenomics analysis across traditional histological boundaries to uncover foundational pediatric brain tumor biology and inform rational treatment selection.

OBJECTIVE To estimate the 6-month Progression Free Survival (PFS6) associated with everolimus for progressive/recurrent pLGGs and to determine if activated PI3K/Akt/mTOR pathway as measured by positive phosphorylated-ribosomal protein S6 (p-RPS6) status was associated with response. METHOD: Patients 3–21 years of age with recurrent or progressive pLGG were enrolled. Everolimus was administered orally at 5 mg/m2 daily. Tissue availability for molecular analysis was mandatory. Immunohistochemistry (IHC) for p-RPS6 was performed centrally. An adaptive Simon two-stage design was employed based on p-RPS6 status. Based on results of the first stage, enrollment in the second stage was either limited to pathway activated patients or open to all subjects. RESULTS From December 2012 to July 2019 a total of 65 subjects enrolled [median age 9 years (range 3–19); 43% female]. As of December 15, 2019 median number of treatment cycle is 8 (range 1–24); 7 patients remain on treatment. Toxicity profile is similar to published reports with rash and elevated lipid profiles as most common adverse events. PFS6 for the entire cohort is 63%; PFS6 is 64% for the activated and 61% for the non-activated patients. Central imaging review (n=52) revealed 1 partial response, 1 complete response, 33 stable disease, and 17 progressive disease at the end of study treatment. Initial molecular analysis identified BRAF alterations in 35/65 patients. CONCLUSION Everolimus is well tolerated and active in a subset of pLGGs. Ongoing analyses will assess predictive biomarkers of response and will be reported at the meeting.

The Children’s Brain Tumor Network (formerly known as Children’s Brain Tumor Consortium- CBTTC) is a global organization pioneering a model of open-science medical research to improve treatment and discover cures. Started in 2011, our objective was to utilize a regulatory, agreement, and governance architecture to remove existing research barriers that slowed down the pace of research and collaboration. Our network now includes 17 institutions working together to empower research. As of December 2019, over 3,600 subjects have been enrolled resulting in collection of over 45,000 specimens. Clinical data collection is longitudinal and includes medical history, diagnosis, treatment, pathology slides and reports, radiology imaging and reports, and outcome data. The tissue is collected flash-frozen, in freezing media, and fresh for the generation of pre-clinical models including cell lines. Blood is collected from the subject, with blood or saliva collected from the parents for germline comparison. Additionally, the Children’s Brain Tumor Network- Pediatric Brain Tumor Atlas has generated 952 WGS and RNAseq, 221 proteomics, with annotated clinical data. All of this data, both generated raw and processed data, has been made available broadly to the scientific community via cloud-based platforms, including the Gabriella Miller Kids First Data Resource Portal, Cavatica, and PedCbioportal. As of January 2020, we have 45 approved biospecimen requests and 80 genomic/molecular data requests. In summary, the Children’s Brain Tumor Network’s goal is to accelerate the pace of discovery by providing resources and expanding the network of scientists working towards a cure.

Increased overall survival for patients with glioma brain tumours is associated with mutations in the metabolic regulator isocitrate dehydrogenase 1 (IDH1). Gliomas develop within a mechanically challenged microenvironment that is characterized by a dense extracellular matrix (ECM) that compromises vascular integrity to induce hypoxia and activate HIF1α. We found that glioma aggression and patient prognosis correlate with HIF1α levels and the stiffness of a tenascin C (TNC)-enriched ECM. Gain- and loss-of-function xenograft manipulations demonstrated that a mutant IDH1 restricts glioma aggression by reducing HIF1α-dependent TNC expression to decrease ECM stiffness and mechanosignalling. Recurrent IDH1-mutant patient gliomas had a stiffer TNC-enriched ECM that our studies attributed to reduced miR-203 suppression of HIF1α and TNC mediated via a tension-dependent positive feedback loop. Thus, our work suggests that elevated ECM stiffness can independently foster glioblastoma aggression and contribute to glioblastoma recurrence via bypassing the protective activity of IDH1 mutational status.

While immunotherapy may offer promising new approaches for high grade meningiomas, little is currently known of the immune landscape in meningiomas. We sought to characterize the immune microenvironment and a potentially targetable antigen mesothelin across WHO grade I-III cases of meningiomas, and how infiltrating immune populations relate to patient outcomes. Immunohistochemistry was performed on tissue microarrays constructed from 96 meningioma cases. The cohort included 16 WHO grade I, 62 WHO grade II, and 18 WHO grade III tumors. Immunohistochemistry was performed using antibodies against CD3, CD8, CD20, CD68, PD-L1, and mesothelin. Dual staining using anti-PD-L1 and anti-CD68 antibodies was performed, and automated cell detection and positive staining detection algorithms were utilized. Greater degree of PD-L1 expression was found in higher grade tumors. More specifically, higher grade tumors contained increased numbers of intratumoral CD68-, PD-L1+ cells (p = 0.022), but did not contain higher numbers of infiltrating CD68+, PD-L1+ cells (p = 0.30). Higher PD-L1+/CD68- expression was independently predictive of worse overall survival in our cohort when accounting for grade, performance status, extent of resection, and recurrence history (p = 0.014). Higher expression of PD-L1+/CD68- was also present in tumors that had undergone prior radiotherapy (p = 0.024). Approximately quarter of meningiomas overexpressed mesothelin to levels equivalent to those found in pancreatic carcinomas and malignant mesotheliomas. The association with poor survival outcomes in our study suggests that PD-L1 may play a significant biologic role in the aggressive phenotype of higher grade meningiomas. Thus, immunotherapeutic strategies such as checkpoint inhibition may have clinical utility in PD-L1 overexpressing meningiomas.

Rationale and Objectives The treatment of glioma with bevacizumab results in decreased enhancement, making it challenging to diagnose tumor recurrence. Therefore, the primary aim of this retrospective study was to determine if the underlying biological processes occurring within regions of recurrent glioblastoma in patients undergoing bevacizumab therapy influence morphologic and diffusion-weighted magnetic resonance (MR) imaging characteristics. Materials and Methods In this Health Insurance Portability and Accountability Act–compliant and institutional review board–approved study, 26 patients treated with bevacizumab for high-grade glioma underwent lesion-wide apparent diffusion coefficient analysis before therapy and at the time of clinical/radiological progression, allowing for stratification by the presence or absence of reduced diffusion. Lesions with reduced diffusion were classified into “block” or “salt & pepper” phenotypes. Eight of the 26 patients underwent image-guided tissue sampling at the time of suspected disease recurrence allowing for direct biological correlation. Clinical, histologic, and MR imaging differences between diffusion groups were assessed using a two-sample Welch t test. Results All patients had histologic evidence of recurrent disease with or without a background of treatment effect. Sixty-two percent of the cohort had developed reduced diffusion at clinical progression following bevacizumab. Image-guided tissue sampling demonstrated that treatment effect was not observed within regions of reduced diffusion. Recurrent tumor intermixed with treatment effect was more likely to be observed within the “salt & pepper” phenotype when compared to “block” phenotype. Conclusions Following bevacizumab therapy, recurrent glioblastoma can manifest as nonenhancing regions characterized by reduced diffusion. Histologically, these MR imaging characteristics correlate with aggressive biological features of disease recurrence.

OBJECT There is evidence that 5-aminolevulinic acid (ALA) facilitates greater extent of resection and improves 6-month progression-free survival in patients with high-grade gliomas. But there remains a paucity of studies that have examined whether the intensity of ALA fluorescence correlates with tumor cellularity. Therefore, a Phase II clinical trial was undertaken to examine the correlation of intensity of ALA fluorescence with the degree of tumor cellularity. METHODS A single-center, prospective, single-arm, open-label Phase II clinical trial of ALA fluorescence-guided resection of high-grade gliomas (Grade III and IV) was held over a 43-month period (August 2010 to February 2014). ALA was administered at a dose of 20 mg/kg body weight. Intraoperative biopsies from resection cavities were collected. The biopsies were graded on a 4-point scale (0 to 3) based on ALA fluorescence intensity by the surgeon and independently based on tumor cellularity by a neuropathologist. The primary outcome of interest was the correlation of ALA fluorescence intensity to tumor cellularity. The secondary outcome of interest was ALA adverse events. Sensitivities, specificities, positive predictive values (PPVs), negative predictive values (NPVs), and Spearman correlation coefficients were calculated. RESULTS A total of 211 biopsies from 59 patients were included. Mean age was 53.3 years and 59.5% were male. The majority of biopsies were glioblastoma (GBM) (79.7%). Slightly more than half (52.5%) of all tumors were recurrent. ALA intensity of 3 correlated with presence of tumor 97.4% (PPV) of the time. However, absence of ALA fluorescence (intensity 0) correlated with the absence of tumor only 37.7% (NPV) of the time. For all tumor types, GBM, Grade III gliomas, and recurrent tumors, ALA intensity 3 correlated strongly with cellularity Grade 3; Spearman correlation coefficients (r) were 0.65, 0.66, 0.65, and 0.62, respectively. The specificity and PPV of ALA intensity 3 correlating with cellularity Grade 3 ranged from 95% to 100% and 86% to 100%, respectively. In biopsies without tumor (cellularity Grade 0), 35.4% still demonstrated ALA fluorescence. Of those biopsies, 90.9% contained abnormal brain tissue, characterized by reactive astrocytes, scattered atypical cells, or inflammation, and 8.1% had normal brain. In nonfluorescent (ALA intensity 0) biopsies, 62.3% had tumor cells present. The ALA-associated complication rate among the study cohort was 3.4%. CONCLUSIONS The PPV of utilizing the most robust ALA fluorescence intensity (lava-like orange) as a predictor of tumor presence is high. However, the NPV of utilizing the absence of fluorescence as an indicator of no tumor is poor. ALA intensity is a strong predictor for degree of tumor cellularity for the most fluorescent areas but less so for lower ALA intensities. Even in the absence of tumor cells, reactive changes may lead to ALA fluorescence.

The M2 isoform of pyruvate kinase (PK) is upregulated in most cancers including glioblastoma. Although PKM2 has been reported to use dual kinase activities to regulate cell growth, it also interacts with phosphotyrosine (pY)-containing peptides independently of its kinase activity. The potential for PKM2 to use the binding of pY-containing proteins to control tumor growth has not been fully examined. We here describe a novel mechanism by which PKM2 interacts in the nucleus with the RNA binding protein HuR to regulate HuR sub-cellular localization, p27 levels, cell cycle progression and glioma cell growth. Suppression of PKM2 in U87, T98G and LN319 glioma cells resulted in increased p27 levels, defects in entry into mitosis, increased centrosome number, and decreased cell growth. These effects could be reversed by shRNA targeting p27. The increased levels of p27 in PKM2 knock-down cells were caused by a loss of the nuclear interaction between PKM2 and HuR, and a subsequent cytoplasmic re-distribution of HuR, which in turn led to increased cap-independent p27 mRNA translation. Consistent with these results, the alterations in p27 mRNA translation, cell cycle progression and cell growth caused by PKM2 suppression could be reversed in vitro and in vivo by suppression of HuR or p27 levels, or by introduction of forms of PKM2 that could bind pY, regardless of their kinase activity. These results define a novel mechanism by which PKM2 regulates glioma cell growth, and also define a novel set of potential therapeutic targets along the PKM2-HuR-p27 pathway.

Item does not contain fulltext Primitive neuroectodermal tumors of the central nervous system (CNS-PNETs) are highly aggressive, poorly differentiated embryonal tumors occurring predominantly in young children but also affecting adolescents and adults. Herein, we demonstrate that a significant proportion of institutionally diagnosed CNS-PNETs display molecular profiles indistinguishable from those of various other well-defined CNS tumor entities, facilitating diagnosis and appropriate therapy for patients with these tumors. From the remaining fraction of CNS-PNETs, we identify four new CNS tumor entities, each associated with a recurrent genetic alteration and distinct histopathological and clinical features. These new molecular entities, designated "CNS neuroblastoma with FOXR2 activation (CNS NB-FOXR2)," "CNS Ewing sarcoma family tumor with CIC alteration (CNS EFT-CIC)," "CNS high-grade neuroepithelial tumor with MN1 alteration (CNS HGNET-MN1)," and "CNS high-grade neuroepithelial tumor with BCOR alteration (CNS HGNET-BCOR)," will enable meaningful clinical trials and the development of therapeutic strategies for patients affected by poorly differentiated CNS tumors.

Infiltrating low-grade gliomas (LGGs) are a class of terminal central nervous system tumors that comprise malignant neuroglia. Histopathological diagnosis of tumor grade is performed using criteria set by the World Health Organization (WHO) and is based on factors that include nuclear atypia, proliferative capacity, tumor neovascularization, and necrosis.1 The clinical outcome for patients with LGG is variable, with some lesions following a more indolent disease course, while others recur more rapidly and often after undergoing malignant transformation (MT) to a higher grade.2 Lesions that have thus transformed to a grade III anaplastic glioma or grade IV secondary glioblastoma multiforme (GBM) are managed with additional, more aggressive treatments. To date, little is known regarding the nature of recurrent disease, and the most significant prognostic factors for patients diagnosed with LGG are the presence of somatic driver mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) oncogenes and the codeletion of the 1p and 19q chromosomal arms, which have been associated with increased survival and sensitivity to the treatment given.2–5 IDH mutations have been implicated as an initiating event in gliomagenesis and are key to reprogramming the tumor epigenome and metabolome, largely through neomorphic production and accumulation of 2-hydroxyglutarate.6 Given uncertainties regarding the effectiveness of options available for treating LGGs, IDH mutations have garnered significant attention as a targetable therapeutic pathway, and there are several novel therapies on the horizon.7–9 Until these therapeutics become available, the clinical mainstay of treatment for patients with LGG consists of surgical resection, with radiation therapy and alkylating chemotherapy being typically reserved for recurrences that were either subtotally resected or have undergone MT. MRI is an integral component of brain tumor diagnosis and monitoring. Recent advances in state-of-the-art techniques have enabled the noninvasive assessment of tumor morphology and physiology, with primary GBM having been studied most extensively. Although the ability to noninvasively detect MT would be of significant clinical interest for diagnosis and treatment planning, few studies have focused on addressing this question. Multiparametric MRI holds significant promise for comprehensively characterizing the structural and physiological properties of the tumor. We hypothesize that these advanced imaging techniques may also provide improved characterization of MT in patients with recurrent LGG. The objective of this study was to establish multiparametric MRI profiles of patients with tumors prior to image-guided surgery in order to relate metrics obtained from these methodologies to histopathological grade. Volumetric regions with abnormal imaging features were calculated to provide a robust assessment of the entire recurrent tumor lesion. Regions of viable tumor were targeted for image-guided tissue sampling in order to associate in vivo parameters with histopathological features and to strengthen our understanding of the link between glioma tumor biology and parameters from noninvasive imaging.

Somatic mutations of the H3F3A and HIST1H3B genes encoding the histone H3 variants, H3.3 and H3.1, were recently identified in high-grade gliomas arising in the thalamus, pons and spinal cord of children and young adults. However, the complete range of patients and locations in which these tumors arise, as well as the morphologic spectrum and associated genetic alterations remain undefined. Here, we describe a series of 47 diffuse midline gliomas with histone H3-K27M mutation. The 25 male and 22 female patients ranged in age from 2 to 65 years (median = 14). Tumors were centered not only in the pons, thalamus, and spinal cord, but also in the third ventricle, hypothalamus, pineal region and cerebellum. Patients with pontine tumors were younger (median = 7 years) than those with thalamic (median = 24 years) or spinal (median = 25 years) tumors. A wide morphologic spectrum was encountered including gliomas with giant cells, epithelioid and rhabdoid cells, primitive neuroectodermal tumor (PNET)-like foci, neuropil-like islands, pilomyxoid features, ependymal-like areas, sarcomatous transformation, ganglionic differentiation and pleomorphic xanthoastrocytoma (PXA)-like areas. In this series, histone H3-K27M mutation was mutually exclusive with IDH1 mutation and EGFR amplification, rarely co-occurred with BRAF-V600E mutation, and was commonly associated with p53 overexpression, ATRX loss (except in pontine gliomas), and monosomy 10.

BACKGROUND The prognostic importance of maximal resection of contrast enhancing and non-contrast enhancing disease has been established. Nonetheless, glioblastomas exist within the framework of complex neural circuitry serving cognition, movement, and behavior consequential leading to neurological impairments. The prognostic importance of neurological impairments on survival remains poorly understood. METHODS This is a retrospective, single cohort study from UCSF including 316 eligible patients diagnosed over 20 years with 9.6 years of follow-up. All patients underwent surgical resection for newly diagnosed glioblastoma for whom survival, molecular, preoperative and postoperative MRI images, and clinical data were available. All patients had chemoradiation treated IDH-wild-type glioblastoma with available preoperative and 1-month post-surgical resection neurological outcomes. We employed survival models and recursive partitioning (RPA) to investigate multivariate relationships of overall survival (OS). RESULTS Preoperative neurological impairments were present in 75.6% (n= 239) and new post resection impairments were identified in 37.3% (n=117). Univariate analysis confirmed that new postoperative cognitive impairment [HR 7.91, 95% CI 2.47-25.33] and hemiplegia [HR 3.38, 95% CI 0.83-13.67] (not hemiparesis) impact OS. Risk stratified grouping by RPA demonstrated that gross total resection of contrast enhancing tumor in patients with no new postoperative neurological impairments confers the longest OS (median OS 27.1 months 95%CI 21.5-33.7). Patients with any residual tumor volume after surgery but no new neurological deficits experience a similar survival to younger patients (under 65) with 1 or more new postoperative neurological deficits (median OS 16.6 months 95%CI 15.2-19.2). Shortest OS is identified in patients with any volume of residual tumor plus 1 or more new postoperative neurological deficits and age over 65 (median OS 11.4 months, 95%CI 9.3-13.5). CONCLUSIONS This study confirms that new postoperative neurological impairments impact overall survival in patients with chemoradiation treated IDH-wild-type glioblastoma.

INTRODUCTION Current WHO guidelines emphasize classification of diffuse gliomas by genetic alterations into three subgroups: 1) IDH-wildtype; 2) IDH-mutant, 1p/19q-codeleted; and 3) IDH-mutant, 1p/19q-non-codeleted. Non-invasive genetic characterization can benefit patients with inoperable lesions or who are administered molecularly-targeted therapy before surgery. Prior studies that use anatomical images and convolutional neural networks (CNNs) to distinguish either IDH-mutant from IDH-wildtype tumors, or 1p/19q-codeleted from non-codeleted tumors have resulted in misclassification of nonenhancing IDH-wildtype and enhancing IDH-mutant tumors. This study investigated the benefit of a priori separation of enhancing from nonenhancing lesions and the inclusion of ADC maps from diffusion MRI to genetic subgroup classification. METHODS 3D T2-weighted, T2-FLAIR, and post-contrast T1-weighted images were acquired preoperatively from 254 patients with newly-diagnosed gliomas. IDH1R132H mutations[VJ1] [CJ2], 1p19q-codeletions, ATRX alterations, and p53 mutations were assessed from the resected tissue to determine subtype stratification: IDH-wildtype (n=95), IDH-mutant, 1p/19q-codeleted (n=62), and IDH-mutant, non-codeleted (n=97). 3-channel input images were constructed for each patient using T2-FLAIR, T1-post-contrast, and either T2-weighted or ADC images. Three VGG-16 CNNs pre-trained on ImageNet were re-trained for: 1) lesions without enhancement, 2) enhancing lesions, and 3) all lesions together[VJ3]. RESULTS A network trained on only enhancing lesions predicted the IDH-wildtype subtype with the highest class accuracy (ADC 94%, T2-weighted 100%) compared to using all lesions combined (ADC 90%, T2-weighted 90%). Models trained using non-enhancing lesions and ADC yielded the highest accuracy classifying 1p/19q-codeleted/non-codeleted subgroups (87%/90% for the non-enhancing network vs 83%/81% for combined network). CONCLUSIONS Our results support a strategy that first considers whether a lesion is enhancing when predicting molecular subgroup and includes ADC if the lesion is non-enhancing. Analysis is underway to test this model framework on independent TCIA data.

Pathological evaluation of tumor tissue images stained with hematoxylin and eosin (H&E) is pivotal in diagnosis and predictive of outcome, yet only a small fraction of the rich phenotypic information on the slide is currently used for clinical care. In this study, we developed a computational approach based on deep learning to predict overall survival within distinct molecular subtypes of glioma patients and to extract prognostic biomarkers from microscopic images of tissue biopsies. Whole-slide images from 766 unique patients [IDH: 336 IDH-wildtype, 364 IDH-mutant, 1p/19q: 142 1p/19q-codeleted, 620 1p/19q-non-codeleted] were obtained from The Cancer Genome Atlas (TCGA). Sub-images that were free of artifacts and that contained viable tumor with descriptive histologic characteristics were extracted, which were used for training and testing the deep neural-network. Our unified survival deep learning framework (SDL) uses a residual CNN network integrated with a traditional survival model to predict patient risk from digitized whole-slide images. We employed statistical sampling techniques and randomized transformation of images to address challenges in learning from histology images. Univariable and multivariable Cox proportional-hazards regression models were used to evaluate the significance of predicted patient risk with and without controlling for known prognostic factors. The integrated SDL framework showed substantial prognostic power achieving a median c-index of 0.79 [95 % CI 0.77 - 0.81]. In multivariable Cox regression analysis, SDL risk was significantly associated with overall survival (hazard ratio of 1.65, 95% CI 1.49-1.83, p < 0.001) after adjusting for age, grade, IDH status, ATRX status, 1p19q codeletion and CDKN2A/2B status. Only IDH-status and age were also significant in the multivariable model. Preliminary findings highlight the emerging role of AI in precision medicine and suggest the utility for computational analysis of tumor tissue images for objective and accurate prediction of outcome for glioma patients and also for risk stratification for targeted clinical therapy.

The determinants of the tumor-associated immune response in brain tumors are poorly understood. Using tumor samples from two molecularly distinct subtypes of lower grade glioma, MAPK-driven glioma with biallelic inactivation of CDKN2A (n=30) and IDH-mutant, 1p/19q-intact astrocytoma (n=29), we demonstrate qualitative and quantitative differences in the tumor-associated immune response and we investigate the molecular mechanisms involved. Histologically the MAPK-driven gliomas were comprised of pleomorphic xanthoastrocytoma (PXA) (n=11) and anaplastic PXA (n=19). Seven patients had paired samples from two sequential surgeries. Immune cell populations and their activity were determined by quantitative multiplex immunostaining and Digital Spatial Profiling and gene expression was analyzed by Nanostring. Functional studies were performed using established cell lines and two new patient-derived lines from MAPK-driven LGGs. MAPK-driven tumors exhibited an increased number of CD8+ T cells and tumor-associated microglial/macrophage (TAMs), including CD163+ TAMs, as compared to IDH-mutant astrocytoma. In contrast, IDH-mutant tumors had increased FOXP3+ immunosuppressive T regulatory cells. Transcriptional and protein level analyses in MAPK-driven tumors suggested an active cytotoxic T cell response with robust expression of granzyme B, present on 27% of CD8+ T cells, increased MHC class I expression, and altered cytokine profiles. Interestingly, MAPK-driven tumors also had increased expression of immunosuppressive molecules, including CXCR4, PD-L1, and VEGFA. Expression differences for cell surface and secreted proteins were confirmed in patient-derived tumor lines and functional relationships between altered chemokine expression and immune cell infiltration was investigated. Our data provide novel insights into the immune contexture of MAPK driven LGGs and suggest MAPK driven gliomas with biallelic inactivation of CDKN2A may be particularly vulnerable to immunotherapeutic modulation

2506 Background: Temozolomide, a commonly used alkylating agent to treat gliomas, can induce somatic hypermutation. The prevalence and clinical implications of this phenomenon are not well characterized. Methods: We used targeted and whole exome sequencing from a cohort of 82 patients with recurrent IDH-mutant low grade gliomas undergoing re-operation to evaluate the prevalence as well as the clinical implications of hypermutation. Results: Hypermutation was identified at transformation in 57% of recurrent gliomas exposed to Temozolomide, 94% of which were transformed to higher WHO grades. All patients who developed hypermutation were exposed to Temozolomide. Hypermutation was associated with transformation to higher WHO grade (OR 12.0 95% CI 2.5 – 115.5, p = 0.002) and shorter survival after transformation (HR 2.1, 95% CI 1.1-4.0, p = 0.018) compared with non-hypermutated transformed tumors, controlling for grade, molecular subtype, age, and prior radiotherapy. Patients with transformation to glioblastoma had poor survival regardless of hypermutation (p = 0.78). Hypermutated tumors were associated with development of discontiguous disease at a significantly higher frequency (p = 0.003), including four cases with spinal dissemination. Conclusions: TMZ-induced hypermutation is associated with high grade transformation, unique patterns of dissemination and shortened survival after transformation. Next generation sequencing should be considered in this patient population. These data have important implications for the management of newly diagnosed and recurrent IDH-mutant low grade gliomas.

Recurrent high grade gliomas (rHGG) are difficult to diagnose accurately as they are often confounded with treatment-induced effects (TxE) because on conventional T1w and T2w anatomical MR imaging these two phenomena appear identical. This could result in removing a patient from an effective second-line therapy, confound the results of a clinical trial of a new therapeutic, and expose a patient to unnecessary surgical intervention. Advanced MR imaging modalities have shown promise in distinguishing between TxE and rHGG, but prior studies are unable to account for heterogeneity within the same lesion and often lack pathological confirmation. The goal of this study was to identify imaging parameters that could spatially map directly to pathology to account for heterogeneity within lesions. 1–8 tissue samples were collected upon surgical resection of suspected rHGG. A total of 484 samples were collected from 183 patients; 332 of these (163 patients) were labeled as either pathologically confirmed treatment effect or having no tumor cells (tumor score=0) within the imaging abnormality (TxE; 80/51 samples/patients) or rHGG (tumor score=2–3; 252/118 samples/patients) by a pathologist. Preoperative 3T MRI scans included T1w-Gd and T2w FLAIR; diffusion tensor imaging (b=1000s/mm2); 3D MRSI; and dynamic susceptibility-contrast perfusion MRI. Univariate models were created using generalized estimating equations to evaluate each variable’s ability to distinguish TxE from rHGG tissue samples. Normalized choline (median=1.1 TxE & 1.2 rHGG), choline-to-NAA index (CNI, median=2.4 TxE & 3.5 rHGG), and normalized cerebral blood volume (nCBV, median=1.2 TxE & 1.4 rHGG) were significantly associated with biopsy-level classification (p=0.034, 0.015, 0.017). These results are being consolidated into a multiparametric algorithm for predicting TxE or rHGG, which could be of clinical importance in managing patients with recurrent rHGG. Downstream analysis will create a spatial map of rHGG to help guide future surgical sampling.

Although the overall prognostic significance of maximal surgical resection of contrast-enhancing tumor in glioblastoma patients is well established, prior studies have not evaluated the combined importance of resection, molecular markers, patient characteristics, and chemoradiation. Incorporation of these factors may redefine the relative benefit of cytoreductive surgery and establish differing thresholds for extent of resection in varying clinical presentations. In the first study of its kind, we examine the interactive effects of volumetric extent of resection with molecular and clinical factors to develop a new roadmap for cytoreductive surgery. Based on a 20-year retrospective cohort of 850 glioblastoma patients who had initial surgery at UCSF, we employed survival models and recursive partitioning (RPA) to investigate multivariate relationships of overall survival (OS), both in the entire cohort as well as a subset diagnosed since 2005 (Stupp-era) with IDH1 mutation status available (n=470). For the entire cohort and the Stupp-era subset, the RPAs elucidate the combinatorial consequence of treatment, age, IDH1 status (in the subset), and resection of both enhancing and non-enhancing tumor. In the Stupp-era, temozolomide-treated patients that are IDH-wildtype and >65 clearly benefit from a reduction of the enhancing tumor (median OS: 10.1 vs 15.8 months). IDH-wildtype, temozolomide-treated patients under 65 benefit from reduction of both enhancing and non-enhancing tumor with a median survival similar to that of IDH-mutant, temozolomide-treated patients (combined median OS: 33.7 months). The patients faring worst are those that did not receive temozolomide that are >65 and/or have ≥0.3 cm(3) residual enhancing tumor (median OS: 4 months). These risk models outperform all published prognostic models. This is the first study to combine resection of contrast-enhancing and non-enhancing tumor in conjunction with molecular and clinical information in a large single-institution study, and paves the way for rethinking surgical strategies for individual patients with newly diagnosed glioblastoma.

70–90% of low-grade gliomas and secondary glioblastomas are characterized by mutations in isocitrate dehydrogenase (IDH1mut). Mutant IDH produces the oncometabolite 2-hydroxyglutarate (2HG), which drives tumorigenesis. Inhibitors of phosphoinositide-3-kinase (PI3K) pathway are currently under clinical investigation for IDH1mut gliomas with positive outcome results, but early response imaging biomarkers are missing. The goal of this study was to identify potential magnetic resonance (MR) detectable biomarkers of IDH1mut glioma response to the dual PI3K/mammalian target of rapamycin (mTOR) inhibitor XL765. We used two genetically-engineered IDH1mut models: a U87 glioblastoma-based model (U87IDHmut) and an immortalized normal human astrocyte-based model (NHAIDHmut), as well as a patient derived IDH1mut model (BT142). The reduction of PI3K/mTOR pathway signaling was validated with western blots of p4E-BP1 for all models. We then used MR spectroscopy (MRS) to investigate the impact of treatment on cell metabolism. Using (1)H-MRS, we observed that XL765 induced a significant ~70% and ~50% reduction in steady-state levels of 2HG and glutamate in U87IDHmut while in NHAIDHmut the drop was ~90% and ~70% for 2HG and glutamate respectively. In the case of BT142 there was a ~35% drop in glutamate levels while 2HG was MRS undetectable in both groups. The translatability of our findings was evaluated in mice bearing orthotropic U87IDHmut xerographs treated with XL765 orally twice a day. XL765-treatment of mice led to an apparent slower tumor growth, which was associated with significantly increased animal survival. Importantly, in vivo (1)H-MRS spectroscopy showed a significant reduction in 2HG levels. The results were verified by (1)H-MRS of tumor extracts. Collectively, our results indicate that treatment with XL765 is associated with response in IDH1mut models. To our knowledge, this is the first time in vivo (1)H-MRS detected reduction in 2HG in gliomas undergoing treatment with a non-IDH1mut-specific inhibitor. Thus, 2HG could potentially be used as a response biomarker.

IntroductionAlterations in the CDK4/6-RB signaling pathway are common causes of cell cycle dysregulation in many cancers, including glioblastoma. Palbociclib is an oral inhibitor of CDK4/6, which leads to phosphorylation of RB1 and cell-cycle arrest. We conducted a two-arm study evaluating efficacy and tissue pharmacokinetics/pharmacodynamics of palbociclib in patients with recurrent glioblastoma.MethodsEligibility criteria included confirmation of RB1 proficiency by IHC; ≤ 3 relapses; KPS ≥ 60; no limit on prior treatments. Arm 1 received palbociclib for 7days prior to indicated resection followed by adjuvant palbociclib. Arm 2 received palbociclib without resection. Primary objective was PFS6; secondary included toxicity, OS, and ORR. Exploratory aims included biomarker assessment and pharmacokinetic/pharmacodynamic effects in surgical patients.ResultsTotal of 22 patients were enrolled; 6 on Arm 1 and 16 on Arm 2. Trial was stopped early secondary to lack of efficacy, with 95% of evaluable patients progressing within 6months. Median PFS was 5.14 weeks (range 5days-142weeks) and median OS was 15.4weeks (range 2-274weeks). Two patients (10%) had related grade ≥ 3 AEs. In Arm 1, 5 patients had tissue concentrations of palbociclib felt to be sufficient for biological effect and paired samples available for RB1 IHC. There were no consistent changes in RB1 expression or cell proliferation in the paired tissue.ConclusionIn this trial, despite adequate tissue PK, palbociclib monotherapy was not an effective treatment for recurrent glioblastoma. However, these were heavily pretreated patients and targeting the CDK4/6 pathway may still deserve further exploration.

The mTOR signaling is dysregulated prominently in human cancers including glioblastoma, suggesting mTOR as a robust target for therapy. Inhibitors of mTOR have had limited success clinically, however, in part because their mechanism of action is cytostatic rather than cytotoxic. Here, we tested three distinct mTOR kinase inhibitors (TORKi) PP242, KU-0063794, and sapanisertib against glioblastoma cells. All agents similarly decreased proliferation of glioblastoma cells, whereas PP242 uniquely induced apoptosis. Apoptosis induced by PP242 resulted from off-target cooperative inhibition of JAK2 and protein kinase C alpha (PKCα). Induction of apoptosis was also decreased by additional on-target inhibition of mTOR, due to induction of autophagy. As EGFR inhibitors can block PKCα, EGFR inhibitors erlotinib and osimertinib were tested separately in combination with the JAK2 inhibitor AZD1480. Combination therapy induced apoptosis of glioblastoma tumors in both flank and in patient-derived orthotopic xenograft models, providing a preclinical rationale to test analogous combinations in patients. Significance: These findings identify PKCα and JAK2 as targets that drive apoptosis in glioblastoma, potentially representing a clinically translatable approach for glioblastoma.

The goal of this research was to elucidate the relationship between WHO 2016 molecular classifications of newly diagnosed, nonenhancing lower grade gliomas (LrGG), tissue sample histopathology, and magnetic resonance (MR) parameters derived from diffusion, perfusion, and 1H spectroscopic imaging from the tissue sample locations and the entire tumor. A total of 135 patients were scanned prior to initial surgery, with tumor cellularity scores obtained from 88 image-guided tissue samples. MR parameters were obtained from corresponding sample locations, and histograms of normalized MR parameters within the T2 fluid-attenuated inversion recovery lesion were analyzed in order to evaluate differences between subgroups. For tissue samples, higher tumor scores were related to increased normalized apparent diffusion coefficient (nADC), lower fractional anisotropy (nFA), lower cerebral blood volume (nCBV), higher choline (nCho), and lower N-acetylaspartate (nNAA). Within the T2 lesion, higher tumor grade was associated with higher nADC, lower nFA, and higher Cho to NAA index. Pathological analysis confirmed that diffusion and metabolic parameters increased and perfusion decreased with tumor cellularity. This information can be used to select targets for tissue sampling and to aid in making decisions about treating residual disease.

Pleomorphic xanthoastrocytoma (PXA) is a type of astrocytic glial neoplasm classified as WHO grade II and anaplastic PXA as WHO grade III. These tumors are known to commonly harbor BRAF (p.V600E, c.1799T>A) mutation leading to dysregulation of mitogen-activated protein kinase (MAPK) pathway. Using targeted next-generation sequencing analysis, two gene fusions ATG7-RAF1/CRAF and NRF1-BRAF were identified in BRAF p.V600 wild-type anaplastic PXA patients. We sought to functionally characterize above two fusions using the heterologous cell model systems, which stably expressed ATG7-RAF1 and NRF1-BRAF. Immunoblot analysis showed increased phosphorylation of PI3K/mTOR and MAPK pathways in both the cell lines expressing ATG7-RAF1 and NRF1-BRAF fusions. In protein-protein interaction analysis of ATG7-RAF1 and NRF1-BRAF by co-immunoprecipitation we found both fusions to dimerize suggesting activation of PI3K/mTOR and MAPK pathways due to dimerization. ATG7-RAF1 and NRF1-BRAF preferred homo dimerization compared to hetero dimerization to the full form CRAF and BRAF partners. Soft agar proliferation assay showed significant increase in colony formation in both the fusion expressing cell lines compared to the control (p

Adenomatoid tumors are the most common neoplasm of the epididymis, and histologically similar adenomatoid tumors also commonly arise in the uterus and fallopian tube. To investigate the molecular pathogenesis of these tumors, we performed genomic profiling on a cohort of 31 adenomatoid tumors of the male and female genital tracts. We identified that all tumors harbored somatic missense mutations in the TRAF7 gene, which encodes an E3 ubiquitin ligase belonging to the family of tumor necrosis factor receptor-associated factors (TRAFs). These mutations all clustered into one of five recurrent hotspots within the WD40 repeat domains at the C-terminus of the protein. Functional studies in vitro revealed that expression of mutant but not wild-type TRAF7 led to increased phosphorylation of nuclear factor-kappa B (NF-kB) and increased expression of L1 cell adhesion molecule (L1CAM), a marker of NF-kB pathway activation. Immunohistochemistry demonstrated robust L1CAM expression in adenomatoid tumors that was absent in normal mesothelial cells, malignant peritoneal mesotheliomas and multilocular peritoneal inclusion cysts. Together, these studies demonstrate that adenomatoid tumors of the male and female genital tract are genetically defined by TRAF7 mutation that drives aberrant NF-kB pathway activation.

Background: Magnetic resonance spectroscopy (MRS) studies have identified elevated levels of the phospholipid precursor phosphocholine (PC) and phosphoethanolamine (PE) as metabolic hallmarks of cancer. Unusually, however, PC and PE levels are reduced in mutant isocitrate dehydrogenase 1 (IDHmut) gliomas that produce the oncometabolite 2-hydroxyglutarate (2-HG) relative to wild-type IDH1 (IDHwt) gliomas. The goal of this study was to determine the molecular mechanism underlying this unusual metabolic reprogramming in IDHmut gliomas. Methods: Steady-state PC and PE were quantified using 31P-MRS. To quantify de novo PC and PE synthesis, we used 13C-MRS and measured flux to 13C-PC and 13C-PE in cells incubated with [1,2-13C]-choline and [1,2-13C]-ethanolamine. The activities of choline kinase (CK) and ethanolamine kinase (EK), the enzymes responsible for PC and PE synthesis, were quantified using 31P-MR-based assays. To interrogate the role of 2-HG, we examined IDHwt cells incubated with 2-HG and, conversely, IDHmut cells treated with the IDHmut inhibitor AGI-5198. To examine the role of hypoxia-inducible factor 1-α (HIF-1α), we silenced HIF-1α using RNA interference. To confirm our findings in vivo and in the clinic, we studied IDHwt and IDHmut orthotopic tumor xenografts and glioma patient biopsies. Results: De novo synthesis of PC and PE was reduced in IDHmut cells relative to IDHwt. Concomitantly, CK activity and EK activity were reduced in IDHmut cells. Pharmacological manipulation of 2-HG levels established that 2-HG was responsible for reduced CK activity, EK activity, PC and PE. 2-HG has previously been reported to stabilize levels of HIF-1α, a known regulator of CK activity. Silencing HIF-1α in IDHmut cells restored CK activity, EK activity, PC and PE to IDHwt levels. Our findings were recapitulated in IDHmut orthotopic tumor xenografts and, most importantly, in IDHmut patient biopsies, validating our findings in vivo and in the clinic. Conclusions: This study identifies, to our knowledge for the first time, a direct role for 2-HG in the downregulation of CK and EK activity, and thereby, PC and PE synthesis in IDHmut gliomas. These results highlight the unusual reprogramming of phospholipid metabolism in IDHmut gliomas and have implications for the identification of MRS-detectable metabolic biomarkers associated with 2-HG status. This work was supported by the following grants: NIH R01CA172845 (SMR), NIH R01CA197254 (SMR), NIH R01CA154915 (SMR), FP7/2007-2013 REA600396 (JLI), SAF2014-59118-JIN (JLI), UCSF Brain Tumor Loglio Collective (SMR, ROP, JJP), NICO (SMR), UCSF Brain Tumor Research Center SPORE Tissue Core (P50CA97257) and UCSF Brain Tumor Research Center SPORE Career Development Grant (P50CA97257 to PV). Sí

Interstitial fluid pressure (IFP) presents a barrier to drug uptake in solid tumors, including the aggressive primary brain tumor glioblastoma multiforme (GBM). It remains unclear how fluid dynamics impacts tumor progression and can be targeted therapeutically. To address this issue, a novel telemetry-based approach was developed to measure changes in IFP during progression of GBM xenografts. Antisecretory factor (AF) is an endogenous protein that displays anti-secretory effects in animals and patients. Here, endogenous induction of AF protein or exogenous administration of AF peptide reduced IFP and increased drug uptake in GBM xenografts. AF inhibited cell volume regulation of GBM cells, an effect that was phenocopied in vitro by the sodium-potassium-chloride co-transporter 1 (SLC12A2/NKCC1) inhibitor bumetanide. As a result, AF induced apoptosis and increased survival in GBM models. In vitro, the ability AF to reduce GBM cell proliferation was phenocopied by bumetanide and NKKC1 knockdown. Next, AF9s ability to sensitize GBM cells to the alkylating agent temozolomide, standard of care in GBM patients, was evaluated. Importantly, combination of AF induction and temozolomide treatment blocked regrowth in GBM xenografts. Thus, AF-mediated inhibition of cell volume regulation represents a novel strategy to increase drug uptake and improve outcome in GBM. Implications: Antisecretory factor treatment represents a novel approach to increase drug uptake and reduce tumor growth in GBM.

BACKGROUND Previous studies of solid tumors have been restricted in their ability to map how heterogeneous cell populations evolved within the tumor in three-dimensional (3D) space due to insufficient sampling, typically one sample per tumor, and a lack of knowledge of where within the tumor the sample was obtained. Knowledge of the extensivity of heterogeneity and how it is spatially distributed is crucial for assessing whether a therapeutic target is truly tumor-wide, and for exploring how mutations relate to heterogeneity in the local microenvironment. METHODS We developed a novel platform to integrate and visualize in 3D multi-omics data generated from each of 8–10 spatially mapped samples per tumor. Together, the 171 samples collected using this approach from 16 adult diffuse glioma at diagnosis and recurrence form a novel resource – the 3D Glioma Atlas. RESULTS By maximally sampling the tumor geography without excluding samples based on low cancer cell fraction (CCF), we identify a subpopulation of glioblastoma with pervasively lower CCF likely excluded by other atlases, such as the TCGA, that used stringent CCF cutoffs. Exome sequencing of 3D-mapped samples from lower-grade tumors revealed that clonal expansions are typically spatially segregated, implying minimal tumor-wide intermixing of genetically heterogenous cells. Heterogeneity is less spatially segregated for faster-growing high-grade tumors, suggesting that cell populations expand in these tumors differently. Recurrent low-grade tumors have greater intratumoral mutational heterogeneity than newly diagnosed tumors, though this did not translate into greater dissimilarity in gene expression profiles for recurrent tumors, suggesting minimal functional impact of this additional mutational diversity on gene expression. CONCLUSIONS The delineation of spatial patterns of heterogeneity that our work provides enables more informed interpretation of biopsies and greater insight into the factors shaping intratumoral variation of gene expression patterns. Ongoing work is exploring the spatial patterning of amplification events and the tumor microenvironment.

Background Precision medicine trials targeting the epidermal growth factor receptor (EGFR) in glioblastoma patients require selection for EGFR-amplified tumors. However, there is currently no gold standard in determining the amplification status of EGFR or variant III (EGFRvIII) expression. Here, we aimed to determine which technique and which cutoffs are suitable to determine EGFR amplification status. Methods We compared fluorescence in-situ hybridization (FISH) and real-time quantitative (RT-q)PCR data from patients screened for trial inclusion into the Intellance 2 clinical trial, with data from a panel-based next generation sequencing (NGS) platform (both DNA and RNA). Results By using data from >1000 samples, we show that at least 50% of EGFR amplified nuclei should be present to define EGFR gene amplification by FISH. Gene amplification (as determined by FISH) correlates with EGFR expression levels (as determined by RT-qPCR) with receiver operating characteristics analysis showing an area under the curve of up to 0.902. EGFR expression as assessed by RT-qPCR therefore may function as a surrogate marker for EGFR amplification. Our NGS data show that EGFR copy numbers can strongly vary between tumors, with levels ranging from 2 to more than 100 copies per cell. Levels exceeding 5 gene copies can be used to define EGFR-amplification by NGS; below this level, FISH detects very few (if any) EGFR amplified nuclei and none of the samples express EGFRvIII. Conclusion Our data from central laboratories and diagnostic sequencing facilities, using material from patients eligible for clinical trial inclusion, help define the optimal cutoff for various techniques to determine EGFR amplification for diagnostic purposes.

Pleomorphic xantoastrocytoma (PXA) is a typically well circumscribed astrocytic neoplasm that can progress to anaplastic PXA with an aggressive biologic behavior and worse prognosis. Histological features of anaplastic progression include increased proliferation, necrosis, microvascular proliferation, loss of pericellular reticulin, and increased infiltrative growth. Genetically anaplastic PXA is characterized by the combination of CDKN2A biallelic inactivation and oncogenic RAF kinase signaling, as observed in PXA, as well as a relatively small number of additional genetic alterations, with the most common being TERT amplification or promoter mutation (Phillips et al 2018). Common to both PXA and anaplastic PXA is the perivascular accumulation of lymphocytes. Given the potential importance of the immune response in brain tumors we asked whether the immune response in anaplastic PXA was different than that in PXA and in IDH-mutant diffuse glioma. To do this we used quantitative multiplex immunofluorescence of scanned slides and quantified the populations and phenotypes of T cells and microglia/macrophages in tumors identified from the UCSF Brain Tumor Center Biorepository and from the archives of the UCSF Neuropathology Division. All tumors were molecularly characterized by immunostaining and/or comprehensive genomic profiling. A total of 30 PXA, including 6 paired tumors before and after tumor progression, and 30 diffuse glioma were examined. Immunostaining for T cell subsets included CD3, CD8, and FOXP3 and for microglial/macrophages included Iba1, CD204, and CD163. Using quantitative multiplex immunostaining we assess immune cell subsets and their localization within the tissue highlighting the perivascular CD3+ lymphocytes. Our data suggest differences in the quantity and quality of immune cells in anaplastic PXA.

BACKGROUND: Although the contrast-enhancing (CE) lesion on T 1 -weighted MR images is widely used as a surrogate for glioblastoma (GBM), there are also non-enhancing regions of infiltrative tumor within the T 2 -weighted lesion, which elude radiologic detection. Because non-enhancing GBM (Enh−) challenges clinical patient management as latent disease, this study sought to characterize ex vivo metabolic profiles from Enh− and CE GBM (Enh+) samples, alongside histological and in vivo MR parameters, to assist in defining criteria for estimating total tumor burden. Methods: Fifty-six patients with newly diagnosed GBM received a multi-parametric pre-surgical MR examination. Targets for obtaining image-guided tissue samples were defined based on in vivo parameters that were suspicious for tumor. The actual location from where tissue samples were obtained was recorded, and half of each sample was analyzed for histopathology while the other half was scanned using HR-MAS spectroscopy. Results: The Enh+ and Enh− tumor samples demonstrated comparable mitotic activity, but also significant heterogeneity in microvascular morphology. Ex vivo spectroscopic parameters indicated similar levels of total choline and N -acetylaspartate between these contrast-based radiographic subtypes of GBM, and characteristic differences in the levels of myo-inositol, creatine/phosphocreatine, and phosphoethanolamine. Analysis of in vivo parameters at the sample locations were consistent with histological and ex vivo metabolic data. CONCLUSIONS: The similarity between ex vivo levels of choline and NAA, and between in vivo levels of choline, NAA and nADC in Enh+ and Enh− tumor, indicate that these parameters can be used in defining non-invasive metrics of total tumor burden for patients with GBM.

To investigate whether quantitative dynamic susceptibility-weighted contrast-enhanced (DSC) perfusion magnetic resonance imaging (MRI) metrics are influenced by cellular and genomic expression patterns of glioblastoma angiogenesis.Twenty-five stereotactic neurosurgical tissue samples were prospectively obtained from enhancing and non-enhancing tumour regions from 10 patients with treatment-naïve glioblastoma. Using monoclonal antibodies, histopathological features of angiogenesis were examined: total microvascular density, vascular morphology, and hypoxia. Angiogenic expression patterns of tissue samples were investigated using RNA microarrays. DSC perfusion MRI metrics were measured from the tissue sampling sites. MRI and histopathological variables were compared using Pearson's correlations. Microarray analysis was performed using false discovery rate (FDR) statistics.Thirteen enhancing and 12 non-enhancing MR image-guided tissue specimens were prospectively obtained. Enhancing tumour regions demonstrated a significant difference in DSC perfusion and histopathological metrics of angiogenesis when compared to non-enhancing regions. Four angiogenic pathways (vascular endothelial growth factor [VEGF], hypoxia inducible factor [HIF], platelet-derived growth factor [PDGF], fibroblast growth factor [FGF]; 25 individual genes) were significantly up-regulated within enhancing regions when compared to non-enhancing regions (adjusted p0.05, FDR0.05). A statistically significant correlation was observed between VEGF-A expression, microvascular density, microvascular morphology, and DSC perfusion MRI metrics (p0.05).Pro-angiogenic genomic and cellular expression patterns of treatment-naïve primary glioblastoma significantly influences morphological and physiological DSC perfusion metrics suggesting that expression levels of therapeutically relevant genetic signatures can be quantified using MRI.

Mutant isocitrate dehydrogenase 1 (IDH1) catalyzes the production of 2-hydroxyglutarate but also elicits additional metabolic changes. Levels of both glutamate and pyruvate dehydrogenase (PDH) activity have been shown to be affected in U87 glioblastoma cells or normal human astrocyte (NHA) cells expressing mutant IDH1, as compared with cells expressing wild-type IDH1. In this study, we show how these phenomena are linked through the effects of IDH1 mutation, which also reprograms pyruvate metabolism. Reduced PDH activity in U87 glioblastoma and NHA IDH1 mutant cells was associated with relative increases in PDH inhibitory phosphorylation, expression of pyruvate dehydrogenase kinase-3, and levels of hypoxia inducible factor-1α. PDH activity was monitored in these cells by hyperpolarized 13C-magnetic resonance spectroscopy (13C-MRS), which revealed a reduction in metabolism of hyperpolarized 2-13C-pyruvate to 5-13C-glutamate, relative to cells expressing wild-type IDH1. 13C-MRS also revealed a reduction in glucose flux to glutamate in IDH1 mutant cells. Notably, pharmacological activation of PDH by cell exposure to dichloroacetate (DCA) increased production of hyperpolarized 5-13C-glutamate in IDH1 mutant cells. Furthermore, DCA treatment also abrogated the clonogenic advantage conferred by IDH1 mutation. Using patient-derived mutant IDH1 neurosphere models, we showed that PDH activity was essential for cell proliferation. Taken together, our results established that the IDH1 mutation induces an MRS-detectable reprogramming of pyruvate metabolism, which is essential for cell proliferation and clonogenicity, with immediate therapeutic implications. Cancer Res; 75(15); 2999–3009. ©2015 AACR.

Integrated sequencing strategies have provided a broader understanding of the genomic landscape and molecular classifications of multiple cancer types and have identified various therapeutic opportunities across cancer subsets. Despite pivotal advances in the characterization of genomic alterations in glioblastoma, targeted agents have shown minimal efficacy in clinical trials to date, and patient survival remains poor. In this review, we highlight potential reasons why targeting single alterations has yielded limited clinical efficacy in glioblastoma, focusing on issues of tumor heterogeneity and pharmacokinetic failure. We outline strategies to address these challenges in applying precision medicine to glioblastoma and the rationale for applying targeted combination therapy approaches that match genomic alterations with compounds accessible to the central nervous system.

Lower-grade (World Health Organization Grades II and III) gliomas vary widely in clinical behavior and are classified as astrocytic, oligodendroglial, or mixed. Anaplasia depends greatly on mitotic activity, with CDKN2A loss considered as the most common mechanism for cell cycle dysregulation. We investigated whether loss of the CDKN2A gene is associated with overall survival across pathologically and genetically defined glioma subtypes. After adjustment for IDH mutation, sex, and age, CDKN2A deletion was strongly associated with poorer overall survival in astrocytomas but not in oligodendrogliomas or oligoastrocytomas. Molecular classification of astrocytomas by IDH mutation, TP53 mutation, and /or ATRX loss of expression revealed that CDKN2A loss in IDH/TP53 mutated tumors was strongly associated with worse overall survival. CDKN2A loss in IDH mutated tumors with ATRX loss was only weakly associated with worse overall survival. These findings suggest that CDKN2A testing may provide further clinical aid in lower-grade glioma substratification beyond IDH mutation and 1p19q codeletion status, particularly in IDH/TP53 mutated astrocytomas.