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Purpose: To determine whether uveal melanoma, the most common primary intraocular malignancy in adults, requires Notch activity for growth and metastasis.Experimental Design: Expression of Notch pathway members was characterized in primary tumor samples and in cell lines, along with the effects of Notch inhibition or activation on tumor growth and invasion.Results: Notch receptors, ligands, and targets were expressed in all five cell lines examined and in 30 primary uveal melanoma samples. Interestingly, the three lines with high levels of baseline pathway activity (OCM1, OCM3, and OCM8) had their growth reduced by pharmacologic Notch blockade using the γ-secretase inhibitor (GSI) MRK003. In contrast, two uveal melanoma lines (Mel285 and Mel290) with very low expression of Notch targets were insensitive to the GSI. Constitutively active forms of Notch1 and Notch2 promoted growth of uveal melanoma cultures and were able to rescue the inhibitory effects of GSI. MRK003 treatment also inhibited anchorage-independent clonogenic growth and cell invasion and reduced phosphorylation levels of STAT3 and extracellular signal-regulated kinase (Erk)1/2. Suppression of canonical Notch activity using short hairpin RNA targeting Notch2 or CBF1 was also able to reduce tumor growth and invasion. Finally, intraocular xenograft growth was significantly decreased by GSI treatment.Conclusion: Our findings suggest that Notch plays an important role in inducing proliferation and invasion in uveal melanoma and that inhibiting this pathway may be effective in preventing tumor growth and metastasis. Clin Cancer Res; 18(3); 654–65. ©2012 AACR.

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Purpose: Multiple developmental pathways including Notch, Hedgehog, and Wnt are active in malignant brain tumors such as medulloblastoma and glioblastoma (GBM). This raises the possibility that tumors might compensate for therapy directed against one pathway by upregulating a different one. We investigated whether brain tumors show resistance to therapies against Notch, and whether targeting multiple pathways simultaneously would kill brain tumor cells more effectively than monotherapy.Experimental Design: We used GBM neurosphere lines to investigate the effects of a gamma-secretase inhibitor (MRK-003) on tumor growth, and chromatin immunoprecipitation to study the regulation of other genes by Notch targets. We also evaluated the effect of combined therapy with a Hedgehog inhibitor (cyclopamine) in GBM and medulloblastoma lines, and in primary human GBM cultures.Results: GBM cells are at least partially resistant to long-term MRK-003 treatment, despite ongoing Notch pathway suppression, and show concomitant upregulation of Wnt and Hedgehog activity. The Notch target Hes1, a repressive transcription factor, bound the Gli1 first intron, and may inhibit its expression. Similar results were observed in a melanoma-derived cell line. Targeting Notch and Hedgehog simultaneously induced apoptosis, decreased cell growth, and inhibited colony-forming ability more dramatically than monotherapy. Low-passage neurospheres isolated from freshly resected human GBMs were also highly susceptible to coinhibition of the two pathways, indicating that targeting multiple developmental pathways can be more effective than monotherapy at eliminating GBM-derived cells.Conclusions: Notch may directly suppress Hedgehog via Hes1 mediated inhibition of Gli1 transcription, and targeting both pathways simultaneously may be more effective at eliminating GBMs cells. Clin Cancer Res; 16(24); 6060–70. ©2010 AACR.

Supplementary Figures S1-S3; Supplementary Tables S1-S2.

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Supplementary Figure 1 from Notch3 Activation Promotes Invasive Glioma Formation in a Tissue Site-Specific Manner

Supplementary Figure Legend from Notch3 Activation Promotes Invasive Glioma Formation in a Tissue Site-Specific Manner

Activating RAS mutations are found in a subset of fusion-negative rhabdomyosarcoma (RMS), and therapeutic strategies to directly target RAS in these tumors have been investigated, without clinical success to date. A potential strategy to inhibit oncogenic RAS activity is the disruption of RAS prenylation, an obligate step for RAS membrane localization and effector pathway signaling, through inhibition of farnesyltransferase (FTase). Of the major RAS family members, HRAS is uniquely dependent on FTase for prenylation, whereas NRAS and KRAS can utilize geranylgeranyl transferase as a bypass prenylation mechanism. Tumors driven by oncogenic HRAS may therefore be uniquely sensitive to FTase inhibition. To investigate the mutation-specific effects of FTase inhibition in RMS we utilized tipifarnib, a potent and selective FTase inhibitor, in in vitro and in vivo models of RMS genomically characterized for RAS mutation status. Tipifarnib reduced HRAS processing, and plasma membrane localization leading to decreased GTP-bound HRAS and decreased signaling through RAS effector pathways. In HRAS-mutant cell lines, tipifarnib reduced two-dimensional and three-dimensional cell growth, and in vivo treatment with tipifarnib resulted in tumor growth inhibition exclusively in HRAS-mutant RMS xenografts. Our data suggest that small molecule inhibition of FTase is active in HRAS-driven RMS and may represent an effective therapeutic strategy for a genomically-defined subset of patients with RMS.

Detection of BRAF V600E mutation in glioblastomas (GBMs) is important because of potential therapeutic implications. Still, the relative paucity of these mutations makes molecular detection in all GBMs controversial. In the present study, we analyzed clinical, radiographic and pathologic features of 12 BRAF V600E-mutant GBMs and 12 matched controls from 2 institutions. We found that a majority of BRAF V600E-mutant GBMs displayed a combination of well-circumscribed lesions, large cystic components with thin walls and solid cortical component on MRI, but with some overlap with matched BRAF wildtype controls (p = 0.069). BRAF V600E-mutant GBMs were also apt to gross total resection (83% vs 17%, p = 0.016) and morphologically displayed epithelioid features (83% vs 0%, p

AbstractTargeted therapy has gained mainstream attention with notable successes against specific genetic mutations in many cancers. One particular mutation, the BRAF V600E mutation, is present in a small subset of gliomas in adults. Although clinical experience and trial data of RAF-targeted therapy in adults with glioma are lacking at this time, the poor prognosis of adult high-grade glioma has led neuro-oncology practitioners to consider the use of targeted therapy in these patients. In this manuscript, we describe the use of RAF and MEK inhibitors in adults with recurrent glioma. We discuss the utility of these agents, describe their toxicities, and give examples of management strategies. Given the significant toxicities of RAF and MEK inhibitors, along with the long potential duration of treatment, neuro-oncology providers should counsel patients carefully before initiating therapy and monitor them closely while undergoing treatment with RAF-targeted therapy.

Outcomes for patients with high grade gliomas have changed little over the past thirty years. This realization prompted renewed efforts to increase flexibility in the design and conduct of clinical brain tumor trials.This manuscript reviews the development of clinical trial methods, challenges and considerations of flexible clinical trial designs, approaches to improve identification and testing of active agents for high grade gliomas, and evaluation of their delivery to the central nervous system.Flexibility can be introduced in clinical trials in several ways. Flexible designs tout smaller sample sizes, adaptive modifications, fewer control arms, and inclusion of multiple arms in one study. Unfortunately, modifications in study designs cannot address two challenges that are largely responsible for the lack of progress in treating high grade gliomas: 1) the identification of active pharmaceutical agents and 2) the delivery of these agents to brain tumor tissue in therapeutic concentrations. To improve the outcomes of patients with high grade gliomas efforts must be focused on the pre-clinical screening of drugs for activity, the ability of these agents to achieve therapeutic concentrations in non-enhancing tumors, and a willingness to introduce novel compounds in minimally pre-treated patient populations.

Purpose H3 K27 mutations, most commonly in H3F3A, are common in diffuse midline glioma. The exact frequency of these mutations in adults with gliomas in the midline location is unknown. This study was conducted to define the incidence of H3 K27M mutations in this location and to compare clinicopathological features with those of patients who do not harbor this mutation. Methods Consecutive glioma cases from 2007 to 2017 were screened for gliomas in the midline location. Immunohistochemistry was performed on all available tissue for mutations of H3 K27M, IDH1, and ARTX. Results Of 850 gliomas screened, 163 cases had midline glioma on MRI. Sufficient FFPE tissue was available for 123 cases (75%). H3 K27M mutation was identified in 18 of 123 cases (15%). All except one H3 K27M-mutant tumors were WHO grade III or IV on histology, while non-mutant tumors encompassed all four grades. The most common midline locations for H3 K27M-mutated tumors were midbrain (2/3; 67%), pons (4/11; 36%), and cerebellum (6/24; 25%). As compared to H3 K27M-wildtype tumors, there were no differences in age at diagnosis, sex, tumor grade, contrast enhancement on MRI, extent of resection, or treatment received. In this cohort, median survival was longer for patients with H3 K27M-mutated tumors (n = 18; 17.6 months) compared with high-grade wildtype tumors (n = 74; 7.7 months, p = 0.03). Conclusions H3 K27M mutations are common in midline gliomas in adults and can present in all midline locations. Survival comparison between H3 K27M-mutant and wildtype midline gliomas suggests that survival may be similar or possibly improved if the mutation is present.

Background: Glioblastoma remains incurable, but detailed molecular characterization has revealed different genomic subtypes have differential survival and sensitivity to therapies. Correlating clinical with molecular data has proven challenging, however, limiting the field’s insight into the effect of specific mutations. BRAF V600E, a targetable mutation with important therapeutic implications, is not well studied in adults with high-grade gliomas. With this study we sought to establish a consolidated clinical-molecular database in order to ascertain the range of primary brain tumors with BRAF V600E mutations that occur in adults, and the clinical and histopathological features of these tumors. Methods: We created a secure, web-based database using the REDCap tool. We analyzed data from 29 adults with primary brain tumors containing the BRAF V600E mutation, recording gender, age at diagnosis, tumor pathology, molecular tumor information, dates of surgeries, time to progression, treatments received, response to treatment, and survival. Results: The patient cohort’s median age at diagnosis was 33 ± 3.2 years. These patients were followed for a median of 4.2 ± 1.5 years. Tumor grades ranged from WHO I to IV, with 59% (n=17) of tumors classified as high grade by WHO criteria (III or IV). Of the patients with high-grade gliomas, 60% (n=6) received chemotherapy and radiation within 4 months of diagnosis. Three patients were treated at progression: 1 received standard therapy, the other 2 received an experimental targeted therapy of dabrafenib and trametinib (BRAF/MEK inhibitors). Overall, the high-grade glioma cohort received a median of one treatment (range 0–4) with a median follow up of 1.2 ± 0.9 years. Conclusion: Understanding the natural history of different genomic subtypes of glioblastoma is critical for the appropriate application of novel therapeutics. Here we demonstrate that a clinical-pathological database consisting of clinical and molecular data from patients with an uncommon mutation in brain tumors (BRAF V600E) is useful for understanding the prevalence, distribution, and natural history of this disease. This novel database tool also creates a systematic manner in which to track the clinical outcome in cohorts of patients with specific mutations, which is of broad interest for patients with glioblastoma and other potentially targetable mutations.

Medulloblastoma, a common pediatric malignant central nervous system tumour, represent a small proportion of brain tumours in adults. Previously it has been shown that in adults, Sonic Hedgehog (SHH)-activated tumours predominate, with Wingless-type (WNT) and Group 4 being less common, but molecular risk stratification remains a challenge. We performed an integrated analysis consisting of genome-wide methylation profiling, copy number profiling, somatic nucleotide variants and correlation of clinical variables across a cohort of 191 adult medulloblastoma cases identified through the Medulloblastoma Advanced Genomics International Consortium. We identified 30 WNT, 112 SHH, 6 Group 3, and 41 Group 4 tumours. Patients with SHH tumours were significantly older at diagnosis compared to other subgroups (p < 0.0001). Five-year progression-free survival (PFS) for WNT, SHH, Group 3, and Group 4 tumours was 64.4 (48.0-86.5), 61.9% (51.6-74.2), 80.0% (95% CI 51.6-100.0), and 44.9% (95% CI 28.6-70.7), respectively (p = 0.06). None of the clinical variables (age, sex, metastatic status, extent of resection, chemotherapy, radiotherapy) were associated with subgroup-specific PFS. Survival among patients with SHH tumours was significantly worse for cases with chromosome 3p loss (HR 2.9, 95% CI 1.1-7.6; p = 0.02), chromosome 10q loss (HR 4.6, 95% CI 2.3-9.4; p < 0.0001), chromosome 17p loss (HR 2.3, 95% CI 1.1-4.8; p = 0.02), and PTCH1 mutations (HR 2.6, 95% CI 1.1-6.2; p = 0.04). The prognostic significance of 3p loss and 10q loss persisted in multivariable regression models. For Group 4 tumours, chromosome 8 loss was strongly associated with improved survival, which was validated in a non-overlapping cohort (combined cohort HR 0.2, 95% CI 0.1-0.7; p = 0.007). Unlike in pediatric medulloblastoma, whole chromosome 11 loss in Group 4 and chromosome 14q loss in SHH was not associated with improved survival, where MYCN, GLI2 and MYC amplification were rare. In sum, we report unique subgroup-specific cytogenetic features of adult medulloblastoma, which are distinct from those in younger patients, and correlate with survival disparities. Our findings suggest that clinical trials that incorporate new strategies tailored to high-risk adult medulloblastoma patients are urgently needed.

Background RAS effector signaling pathways such as PI3K/mTOR and ERK are frequently dysregulated in glioblastoma. While small molecule targeted therapies against these pathways have appeared promising in preclinical studies, they have been disappointing in clinical trials due to toxicity and de novo and adaptive resistance. To identify predictors of glioblastoma sensitivity to dual pathway inhibition with mTORC1/2 and MEK inhibitors, we tested these agents, alone and in combination, in a cohort of genomically characterized glioblastoma cell lines. Methods Seven genomically characterized, patient-derived glioblastoma neurosphere cell lines were evaluated for their sensitivity to the dual mTORC1/2 kinase inhibitor sapanisertib (MLN0128, TAK-228) alone or in combination with the MEK1/2 inhibitor trametinib (GSK1120212), using assessment of proliferation and evaluation of the downstream signaling consequences of these inhibitors. Results Sapanisertib inhibited cell growth in neurosphere lines, but induced apoptosis only in a subset of lines, and did not completely inhibit downstream mTOR signaling via ribosomal protein S6 (RPS6). Growth sensitivity to MEK inhibitor monotherapy was observed in a subset of lines defined by loss of NF1, was predicted by an ERK-dependent expression signature, and was associated with effective phospho-RPS6 inhibition. In these lines, combined MEK/mTOR treatment further inhibited growth and induced apoptosis. Combined MEK and mTOR inhibition also led to modest antiproliferative effects in lines with intact NF1 and insensitivity to MEK inhibitor monotherapy. Conclusions These data demonstrate that combined MEK/mTOR inhibition is synergistic in glioblastoma cell lines and may be more potent in NF1-deficient glioblastoma.

ObjectiveTo examine the feasibility, safety, systemic biological activity, and cerebral activity of a ketogenic dietary intervention in patients with glioma.MethodsTwenty-five patients with biopsy-confirmed World Health Organization grade 2 to 4 astrocytoma with stable disease after adjuvant chemotherapy were enrolled in an 8-week Glioma Atkins-Based Diet (GLAD). GLAD consisted of 2 fasting days (calories ResultsTwenty-one patients (84%) completed the study. Eighty percent of patients reached ≥40 mg/dL urine acetoacetate during the study. Forty-eight percent of patients were adherent by food record. The diet was well tolerated, with two grade 3 adverse events (neutropenia, seizure). Measures of systemic activity, including hemoglobin A1c, insulin, and fat body mass, decreased significantly, while lean body mass increased. Magnetic resonance spectroscopy demonstrated increased ketone concentrations (β-hydroxybutyrate [bHB] and acetone) in both lesional and contralateral brain compared to baseline. Average ketonuria correlated with cerebral ketones in lesional (tumor) and contralateral brain (bHBRs= 0.52,p= 0.05). Subgroup analysis of isocitrate dehydrogenase–mutant glioma showed no differences in cerebral metabolites after controlling for ketonuria.ConclusionThe GLAD dietary intervention, while demanding, produced meaningful ketonuria and significant systemic and cerebral metabolic changes in participants. Ketonuria in participants correlated with cerebral ketone concentration and appears to be a better indicator of systemic activity than patient-reported food records.Trial Registration InformationClinicalTrials.gov Identifier:NCT02286167.

INTRODUCTION BRAF mutations in glioma have been recognized as a significant driver of disease in pediatric low-grade glioma, but the implications of BRAF alterations on disease trajectory and response to treatment are unknown in adult glioma. Here, we characterize a multi-institutional cohort of adults with BRAF-altered glioma. METHODS We identified patients aged ≥ 18 years with glioma containing BRAF alteration on sequencing in multi-institutional cohorts (Dana-Farber/Brigham Cancer Center, Johns Hopkins Hospital, GENIE, TCGA). BRAF mutations were grouped into three previously-defined classes: I (RAS-independent/dimerization-independent), II (RAS-independent/dimerization-dependent), III (RAS-dependent/dimerization-dependent). RESULTS We identified 198 adults with BRAF-altered glioma (median age 42 years, range 18-85 years), including 17 WHO grade I, 33 grade II, 26 grade III, 114 grade IV, and 8 others. Glioblastoma (n=115) predominated, followed by grade II-III astrocytoma (n=31), pleomorphic xanthoastrocytoma (n=18), pilocytic astrocytoma (n=12), oligodendroglioma (n=7), or other glioma (n=15). Class I mutations (BRAFV600E) comprised 49% (n=97), class II mutations 4.5% (n=9) and class III mutations 4.5% (n=9). Rearrangements occurred in 9.6% (n=19), of which canonical fusions comprised 4% (n=8), occurring primarily in pilocytic astrocytomas; copy number gains affected 13% (n=26), and other alterations 19% (n=38). All pleomorphic xanthoastrocytomas and 43.5% of glioblastoma harbored BRAFV600E. Mutation of NF1, an inhibitor of RAS/ERK signaling, was significantly associated with class II/III BRAF mutations (64.3%) and not observed in BRAFV600E-altered glioma (0%, n=62; p< 0.0001). BRAFV600E was the sole alteration in a subset of glioma with class I mutations, while others harbored multiple genomic alterations. Among 45 patients with glioblastoma and detailed clinicopathologic data, 7 received RAF-targeted therapy, with variable clinical response. Overall survival was 26 months with BRAFV600E, 21 months with class II/III mutation, and 33 months with other BRAF-alteration. CONCLUSIONS This large cohort of BRAF-altered adult glioma demonstrates a broad range of alterations, with implications for treatment sensitivity and patient survival.

BRAF V600 mutations are being identified in patients with primary brain tumors more often as molecular testing becomes widely available. Targeted treatment with BRAF inhibitors has been attempted in individual cases with some responses, whereas others showed no response or developed resistance. Preclinical work suggests that gliomas could be more responsive to the concurrent use of BRAF and MEK inhibition for MAP kinase pathway suppression. This report presents 2 cases of malignant brain tumors with BRAF V600E mutations that were resistant to radiation and temozolomide, and reports on their response to targeted treatment with the BRAF and MEK inhibitors dabrafenib and trametinib. One patient with an anaplastic pleomorphic xanthoastrocytoma experienced a partial response for 14 months, demonstrated by progressive tumor shrinkage and clinical improvement; however, this was followed by clinical and radiographic progression. The patient with glioblastoma continued to have stable disease after 16 months of treatment. These cases are encouraging in a disease that urgently needs new treatments. Further work is necessary to understand response rates, duration, and survival in primary brain tumors.

BackgroundThe benefit of immune checkpoint inhibitors (ICIs) in patients with leptomeningeal metastases (LMM) is unknown.MethodsWe undertook a phase II trial of pembrolizumab in patients with LMM from solid tumors. Eligible patients had radiologic/cytologic LMM and Eastern Cooperative Oncology Group performance status 0–1. Pembrolizumab was administered intravenously at 200 mg q3W until disease progression/unacceptable toxicity. The primary endpoint was central nervous system (CNS) response after four cycles, defined radiologically/cytologically/clinically. Serial cerebrospinal fluid (CSF) was assessed for tumor-derived DNA (t-DNA) aneuploidy and cytokines.ResultsThirteen of a planned 16 patients were treated between April 2017 and December 2019. The study closed early for poor accrual. Median age was 57 years (range: 22–79). Sixty-two percent of patients had tumors not traditionally ICI-responsive (hormone-receptor (HR)-positive breast carcinoma=39%; high-grade glioma=23%), while 38% had ICI-responsive tumors (non-small cell lung cancer (NSCLC)=23%, head and neck carcinoma=8%, cutaneous squamous carcinoma (CSC)=8%). CNS response was observed in 38% of patients at 12 weeks (95% CI 13.9% to 68.4%) by pre-defined criteria and LM-RANO, and 2 achieved durable complete responses (CSC=1, overall survival (OS) 3+ years; NSCLC=1, OS 9 months). Median CNS progression-free survival and OS was 2.9 months (95% CI 1.3 to NR) and 4.9 months (95% CI 3.7 to NR), respectively. Grade 3+ treatment-related adverse events occurred in 15% of patients. Sensitivity for LMM detection by t-DNA and cytopathology was 84.6% (95% CI 54.6% to 98.1%) and 53.9% (95% CI 25.1% to 80.8%), respectively. Pre-therapy and on-therapy CSF cytokine analysis demonstrated complete responders clustered together.ConclusionsPembrolizumab conferred a 38% CNS response rate in patients with LMM, a tolerable safety profile, and deep responses in selected patients with ICI-responsive tumors. CSF t-DNA may be sensitive for LMM detection, and immunologic subsets of CNS response warrant further study.Trial registration numberNCT03091478

BRAF alterations are recognized as a significant driver of disease in pediatric low-grade glioma (pLGG) but the implications of BRAF alterations on the natural history and response to treatment are unclear in adult glioma. We characterized the molecular and clinical features of a multi-institutional cohort of adults with BRAF-mutated gliomas. We identified patients with glioma containing BRAF alterations on sequencing in multi-institutional cohorts (Dana-Farber/Brigham Cancer Center, Johns Hopkins Hospital, GENIE, TCGA). BRAF alterations were grouped into previously defined classes: I (V600E; RAS-independent/dimerization-independent), II (RAS-independent/dimerization-dependent), III (RAS-dependent/dimerization-dependent) in addition to BRAF copy number gains, fusions, and other. We interrogated 289 BRAF-altered gliomas (199 patients >=18yrs, 90 patients

BACKGROUND AND PURPOSE: Ketogenic diets are being explored as a possible treatment for several neurological diseases, but the physiologic impact on the brain is unknown. The objective of this study was to evaluate the feasibility of 3T MR spectroscopy to monitor brain ketone levels in patients with high-grade gliomas who were on a ketogenic diet (a modified Atkins diet) for 8 weeks. MATERIALS AND METHODS: Paired pre- and post-ketogenic diet MR spectroscopy data from both the lesion and contralateral hemisphere were analyzed using LCModel software in 10 patients. RESULTS: At baseline, the ketone bodies acetone and β-hydroxybutyrate were nearly undetectable, but by week 8, they increased in the lesion for both acetone (0.06 ± 0.03 ≥ 0.27 ± 0.06 IU, P = .005) and β-hydroxybutyrate (0.07 ± 0.07 ≥ 0.79 ± 0.32 IU, P = .046). In the contralateral brain, acetone was also significantly increased (0.041 ± 0.01 ≥ 0.16 ± 0.04 IU, P = .004), but not β-hydroxybutyrate. Acetone was detected in 9/10 patients at week 8, and β-hydroxybutyrate, in 5/10. Acetone concentrations in the contralateral brain correlated strongly with higher urine ketones (r = 0.87, P = .001) and lower fasting glucose (r = –0.67, P = .03). Acetoacetate was largely undetectable. Small-but-statistically significant decreases in NAA were also observed in the contralateral hemisphere at 8 weeks. CONCLUSIONS: This study suggests that 3T MR spectroscopy is feasible for detecting small cerebral metabolic changes associated with a ketogenic diet, provided that appropriate methodology is used.

BACKGROUND Ketogenic diet therapies (KDTs) may be beneficial by exploiting glioma metabolic vulnerabilities. The GLioma modified Atkins-based Diet study (GLAD; NCT02286167) evaluated systemic and cerebral (MR spectroscopy) biomarkers to determine the feasibility and biological effects of a KDT in glioma patients. While we observed metabolic changes in tumor and normal brain after KDT using single-voxel MRS (SV-MRS), optimal voxel placement was not always achieved. AIMS: We performed an exploratory analysis comparing cerebral metabolite changes using multi-slice MRSI (MS-MRSI) versus SV-MRS acquisition. METHODS We evaluated four patients from the GLAD study (mean age 39years; 2 female, 3 AA IDH-mutant, 1 GBM IDH-wildtype) who underwent MRS at baseline and following eight weeks of KDT. SV-MRS (sLASER, TR/TE 2.2s/34ms) was acquired from a 2x2x2cm voxel placed in the residual tumor and the contralateral homologous brain. MS-MRSI was acquired with a multi-slice spin echo sequence (TR/TE 3.6/144ms, 4 slices, nominal resolution 13x7x7mm, SENSE factor 3) and maps of total choline (tCho), total N-acetyl-aspartate (tNAA), and lactate (Lac) were reconstructed and normalized relative to creatine. Metabolite levels were measured on the MS-MRSI maps using a region of interest placed in the same areas studied with the SV-MRS. RESULTS Lesional tCho and tNAA levels showed strong correlation between SV-MRS and MS-MRSI both at baseline (Pearson’s r=0.92 and 0.97, respectively) and after 8 weeks of KDT (r=0.96 and 0.84, respectively). tCho and tNAA correlated less robustly between SV-MRS and MS-MRSI in the contralesional region (r=0.56–0.96). Lesional Lac was significantly lower after KDT (1.01±0.48 versus 0.59±0.24, paired t-test p=0.02). CONCLUSIONS While SV and MS-MRSI provided generally concordant lesional results, MS-MRSI offers added potential to map regional variations not captured by SV-MRS and thus may better define the control regions. MS-MRSI detected a decrease in tumoral lactate levels following study intervention, suggesting KDT-related changes in tumoral energy metabolism.

BACKGROUND There has been increasing interest in exploring ketogenic diet therapies (KDT) in patients with glioma given the poor prognosis. The purpose of this single-arm, open label phase 2 study was to rigorously examine the feasibility, safety, systemic biological activity, and cerebral activity of a KDT in patients with glioma. METHODS 25 patients with biopsy-confirmed WHO Grade 2–4 astrocytoma with stable disease following adjuvant chemotherapy were enrolled in an 8-week GLioma Atkins-based Diet (GLAD). GLAD consisted of 2 fasting days (calories RESULTS 21 patients completed the study. 80% of patients reached ≥40 mg/dL urine acetoacetate during the study. 48% of patients were adherent by food record. The diet was well-tolerated with two grade 3 adverse events (neutropenia, seizure). Measures of systemic activity including hemoglobin A1c, insulin, and fat body mass decreased significantly, while lean body mass increased. MR spectroscopy demonstrated increased ketone concentrations (β-hydroxybutyrate (bHB) and acetone (Ace)) in both lesional and contralateral brain, compared to baseline. Higher total choline (tCho) and glutamine (Gln) levels were observed in lesional as compared to contralateral brain at baseline, and both decreased following intervention. Average ketonuria correlated with cerebral ketones in lesional (tumor) and contralateral brain (bHB Rs0.52, p=0.05). There were no differences in cerebral metabolites in IDH-mutant glioma after controlling for ketonuria. CONCLUSIONS The GLAD dietary intervention, while demanding, produced meaningful ketonuria, and significant systemic and cerebral metabolic changes in participants. Participant ketonuria correlated with cerebral ketone concentration and appears to be a better indicator of systemic activity than patient-reported food records.

Neurosarcoidosis is a rare but important cause of stroke as it is treatable. Cases reported thus far have primarily been in young people who are relatively healthy. Here we report the case of a 73-year-old woman presenting with recurrent strokes and high-grade intracranial stenosis caused by probable neurosarcoidosis. This is unique as neurosarcoidosis is not usually considered as an etiology for recurrent strokes in our patient’s age-group. We review and categorize published cases of neurosarcoidosis causing stroke and describe a classification scheme for certainty of diagnosis. Given the implications of this diagnosis for secondary stroke prevention, we recommend that neurosarcoidosis be considered in the differential for patients with few vascular risk factors, recurrent strokes refractory to medical treatment, or possible vasculitis even in the elderly patients.

BACKGROUND While BRAF-targeted therapy can be effective in a subset of patients with glioma, resistance to treatment can emerge over time. The description and validation of mechanisms of resistance in BRAF-mutant glioma are not previously described. METHODS Pre- and post- BRAF inhibitor (BRAFi) or BRAFi/MEK inhibitor (MEKi) treated patient samples were obtained under IRB-approved protocols at University of Colorado Denver, UCSF, and Johns Hopkins. Targeted DNA sequencing or whole exome sequencing (WES), and RNA-seq were conducted on paired samples. Functional validation of putative resistance mechanisms was performed using established glioma cell lines with BRAF V600E mutation (DBTRG-5MG, AM38, B76). RESULTS Analysis of 15 tissue sample pairs identified point mutations in 15 genes (including CBL, NF1, PTEN, and MAP2K1) and expression changes in RAF1 leading to putative mechanisms of resistance. We performed functional validation of loss of NF1 and CBL as resistance mechanisms and demonstrated growth inhibition and cell death in response to BRAFi with siRNA/sgRNA-mediated knockdown of each gene. Knockdown of CBL resulted in increased EGFR expression and phosphorylation, a possible mechanism for maintaining ERK signaling within the cell. Combination therapy with a MEKi or EGFR inhibitor was able to overcome resistance to BRAFi, in NF1 knockdown and CBL knockdown, respectively. Restoration of wild-type PTEN in B76 cells (PTEN-/-) restored sensitivity to BRAFi. Ingenuity pathway analysis suggested that a switch from BRAF to CRAF dependence mediated resistance in some tumors. Indeed, inhibition of CRAF expression using siRNA in a patient-derived glioma cell line re-sensitized cells to BRAFi. CONCLUSIONS Analysis of pre-/post-treatment BRAF mutant glioma sample pairs, primarily from pediatric patients, identified a variety of putative resistance mechanisms, some of which have been validated in vitro. Resistance mechanisms to BRAFi in glioma are varied and may be susceptible to different combinations of targeted therapy, highlighting the importance of a personalized approach.

e14506 Background: Leptomeningeal metastasis (LMM) from solid tumors are rare and often refractory to standard therapies. Pembrolizumab (anti-PD-1) has efficacy in patients (pts) with brain metastases from ICI-responsive tumors, however pts with LMM are often excluded from ICI trials. We hypothesized that: 1. Pembrolizumab will lead to CNS response in LMM, and 2. Genomic/immunologic analyses on CSF could identify CNS biomarkers of response. Methods: We conducted an investigator-initiated open-label phase 2 trial of Pembrolizumab in pts with LMM from any solid tumor (NCT03091478). Eligible pts had: LMM on MRI ( > 3mm lesion) or CSF cytology, ECOG PS ≤1, and were PD-naïve. Prior RT to LMM was allowed > 3 months before study start or to non-target areas. Pembrolizumab was administered IV 200mg q3W until disease progression/unacceptable toxicity. The primary endpoint was CNS response after 12 weeks, defined as radiologic (RECIST 1.1)/cytologic/clinical response to therapy. Serial CSF samples were assessed by chromosomal copy number changes using a PCR-based approach (RealSeqS) to detect tumor-derived DNA (CSF-tDNA), and 16-color flow cytometry. Results: Thirteen of a planned 18 pts were treated 04/2017-12/2019, the study was closed early for low accrual. Median age was 58 years (22-79), 53% were female. Pts had breast carcinoma (38%, n = 5), NSCLC (23%, n = 3), high-grade glioma (23%, n = 3), HNSCC (8%, n = 1) and cutaneous squamous cell carcinoma (8%, n = 1). Median no. of prior therapies was 4 (0-8), 76% of pts had prior RT to LMM. CNS response was seen in 38% of pts (5/13: 2 = complete response (NSCLC; Squamous skin); 3 = stable disease (NSCLC, Glioma, HER2+BC) by RECIST 1.1. Treatment-related adverse events were seen in 31% (4/13) of pts, 15% (2/13) G3+ (fatigue = 1, infection = 1). CSF cytology was negative at ICI start in 6 cases, but positive by CSF-tDNA in 5/6 cases. In addition, CSF-tDNA levels can correlate with disease status. CSF flow cytometry demonstrated increase in CD45R0+ activated memory T-cells, and a shift from immature to antibody-secreting B-cells in both pts with CRs in LMM. Conclusions: Pembrolizumab was well-tolerated and demonstrated anti-tumor activity in pts with LMM in ICI-responsive tumors. Correlative analyses identified CSF-tDNA can potentially be used from diagnosis to longitudinally monitor LMM; and that T and B cell populations may be enriched in the CSF of pts whose LMM regressed. These findings support inclusion of pts with LMM in ICI studies and interrogation of CSF biomarkers. Clinical trial information: NCT03091478.

Temozolomide has been available to oncologists for over 30 years. During this time, it has become an integral part of standard therapy in patients with high-grade gliomas. Given its ability to traverse the blood-brain barrier, temozolomide has also been evaluated in other cancers that involve the central nervous system (CNS). We review its role in the management of patients with primary brain tumors, brain metastases, leptomeningeal carcinomatosis, and other selected CNS cancers. There is strong evidence that temozolomide is effective in patients with high-grade astrocytomas and oligodendrogliomas. Modest evidence supports its activity in primary CNS lymphomas and aggressive pituitary adenomas. Temozolomide, however, has minimal efficacy in a wide variety of systemic cancers. Given that concentrations of temozolomide in the CNS are only 20% of those in the blood, it is not surprising that it is generally inactive in patients with CNS metastases from solid tumors.

BACKGROUND: H3F3A mutations define the entity of Diffuse Midline Glioma, which was added to the WHO 2016 classification. There have been several reports describing the clinical, prognostic, and histopathological implications of this mutation. It is unclear, however, what proportion of adults with gliomas occurring in the midline have an H3 K27M mutation. We set out to define this in a single-institution, retrospective cohort study. METHODS: From 850 consecutive gliomas in adults we identified 163 cases with radiographically-determined midline gliomas (brainstem, thalamus, basal ganglia, corpus callosum, spinal cord, or cerebellum). Clinical cases were reviewed in accordance with IRB guidelines. FFPE tissue was obtained from 120 cases and stained for H3 K27M. RESULTS: A H3 K27M mutation was identified in 18 of 120 cases (15%). As compared to non-H3 K27M mutated tumor, average age was 45.1 ± 12.8 versus 53.1 ± 16.7 years (p=0.2). 56% were female (p=0.3). 83% had contrast enhancement on MRI (p = 0.79). All H3 K27M mutant tumors were WHO grade III or IV on histology, while non-mutant tumors encompassed all four grades (p = 0.08). The most common locations to have H3 K27M-mutated tumors were midbrain (2/2; 100%), pons (4/10; 40%), cerebellum (6/22; 27.3%), spinal cord (2/13; 15.4%), and thalamus (3/30; 10%). Median survival was 16 ± 6.0 months as compared to 8.1 ± 3.6 months in non-mutated midline high grade gliomas (p = 0.15). CONCLUSIONS: H3K27M mutated tumors are common in gliomas located along the midline and this molecular subtype should be considered in adults of all ages and grades with midline tumors, regardless of tumor location or contrast enhancement. Survival was not significantly different from non-H3 K27M mutated tumors, though a larger dataset will be necessary for confirmation.

Objective: To determine the biological and signaling effects of dual-pathway inhibition with mTORC1/2 and MEK inhibitors in glioblastoma neurosphere cell lines. Background: The PI3K/mTOR signaling pathway is frequently dysregulated in glioblastoma (GBM), making it the focus of active scientific and drug development efforts in brain tumors. Unfortunately, small molecule targeted therapies against EGFR and its downstream effector pathways, while promising in the pre-clinical setting, have been disappointing in glioma clinical trials. The mTOR pathway target ribosomal protein S6 (RPS6) can also be activated by the ERK pathway target p90-RSK. We therefore tested the hypothesis that small molecule inhibition of both ERK and mTOR signaling pathways would result in enhanced anti-tumor activity. Methods: Seven glioblastoma neurosphere lines were tested for sensitivity to the ATP-competitive mTORC1/2 inhibitor sapanisertib (MLN0128) alone or in combination with the allosteric MEK1/2 inhibitor trametinib (GSK1120212) using cell growth assays and immunoblots as indicated. Results: The dual mTORC1/2 inhibitor sapanisertib inhibits mTOR signaling and cell growth in all GBM neurosphere lines, but only partially inhibits phosphorylation of the downstream target ribosomal protein S6 (phospho-RPS6) in these models. The MEK inhibitor, trametinib, inhibited ERK phosphorylation in all neurosphere lines, though phosphorylation of downstream targets and cell growth were inhibited to varying degrees. Inhibition of RPS6 activation was dose-dependent and correlated with cell line sensitivity when treated with trametinib but not with sapanisertib. In combination, sapanisertib plus trametinib showed more profound inhibition of phospho-RPS6, as well as enhanced growth inhibition and induction of apoptosis in sensitive neurosphere lines. Conclusions: The combination of sapanisertib and trametinib may have significant antitumor activity in glioblastoma neurospheres by enhanced inhibition of S6, which serves as the convergence point between these effector pathways. Citation Format: Karisa C Schreck, Amy Allen, Jiawan Wang, Christine A Pratilas. Sapanisertib in combination with trametinib in glioblastoma models enhances target inhibition and antitumor activity [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B074. doi:10.1158/1535-7163.TARG-19-B074

A 41-year-old woman with active IV and subcutaneous heroin use complicated by recurrent methicillin-resistant Staphylococcus aureus endocarditis presented to the emergency department with 2 days of progressive dyspnea. Two days prior to presentation, she had 2 episodes of vomiting and one of diarrhea, but no headache, fever, chills, weakness, numbness, or confusion. While in the emergency department, she developed hypoxic respiratory failure requiring intubation and was admitted to the medical intensive care unit. The authors thank Dr. Thomas Lloyd, Johns Hopkins University, for assistance with electrodiagnostic studies; Jenie Morris, CDC; and David Blythe and David Schnavel, Maryland Health Department.

INTRODUCTION Therapeutic successes in neuro-oncology have lagged dramatically behind exciting laboratory discoveries, and successful translation of promising laboratory findings into clinical practice is rare. We hypothesize that one important reason for this discordance is the use of very different paradigms for designing laboratory and clinical trials, and that utilizing clinically relevant procedures could improve laboratory study impact. METHODS We identified all pre-clinical neuro-oncology therapeutic trials published in four high-impact journals between 11/2018 and 4/2019, and assigned a level of evidence (LOE) to each study using the American Academy of Neurology evidence classification system. We also extracted details about statistical techniques, funding, and institutional setting. RESULTS Of the 26 articles identified, 85% had a LOE of IV (highest-I, lowest-IV) and 15% were class III. Factors contributing to the low LOE included lack of randomization (65%) or allocation concealment (100%), absence of masked assessment (96%), and no comparison of treatment and control groups with respect to prognostically relevant characteristics (92%). Effect size and confidence interval reporting, power calculation, correction for multiple hypothesis testing, and multivariate analysis were absent in 92%, 92%, 100%, 88%, and 92% of studies respectively. The “reverse” analysis (starting with successful human trials and analyzing existing pre-clinical support studies) showed significantly more high quality laboratory studies supporting “successful” compared to “unsuccessful” trials (p=0.04). CONCLUSIONS When rigorous human clinical trials criteria are applied to therapeutic laboratory studies, laboratory study LOE is uniformly poor. For laboratory research intended to inform clinical trial design and improve clinical outcomes, this situation dooms most basic science research to fail in the clinical arena. The steps required to solve this problem are challenging but addressable, and costs associated with the solution are dwarfed by the expense of early phase human trials which are destine to failure.

INTRODUCTION Exploiting metabolic vulnerabilities via ketosis is a promising approach for gliomas. The modified Atkins diet is a ketogenic diet therapy efficacious in adults with refractory epilepsy. We evaluated the feasibility, pharmacokinetics/pharmacodynamics(PK/PD), and cerebral activity of this dietary intervention intended to induce ketosis. METHODS 25 patients with biopsy-confirmed WHO Grade 2–4 astrocytoma with stable disease after adjuvant chemotherapy were enrolled in an 8-week GLioma Atkins-based Diet (GLAD). GLAD consisted of 2 ‘intermittent fasting’ days(IF; calories < 20% of recommended daily allowance) interleaved between 5 modified-Atkins diet days(MAD; carbohydrates < 20 gm/day) each week. The primary outcome was dietary compliance. Secondary outcomes were PK assessed by urine ketones post-FAST and post-MAD, PD assessed by serum insulin and IGF-1, and cerebral activity measured by MR spectroscopy at baseline and week 8. RESULTS Grade 2(n=2;8%), Grade 3(n=11;44%) and GBMs(n=12;48%) were enrolled. While only 48% of participants satisfied pre-defined compliance criteria, overall compliance with MAD(80%) was higher than IF(72%). Weight loss was observed (-4.8 + 2.2kg,p< 0.0001) consisting primarily of decreased fat mass (-2.5 + 3.1%,p< 0.0001), with increased lean body mass (2.4 + 3.2%,p< 0.0001), stable nutritional status (phase angle, -0.26 + 0.94%,p=0.22), and normalization of BMI. Urine acetoacetate significantly increased with 55% achieving moderate ketosis at week 8 (p=0.0005). Serum insulin and IGF-1 significantly decreased indicating systemic dietary response and were associated with higher ketones post-IF, but not post-MAD. MRS demonstrated cerebral activity with increased ketones in lesional (0.06±0.03- >0.27±0.06i.u,p=0.005) and contralateral brain at week 8 (0.041±0.01- >0.16±0.04i.u.,p=0.004). Higher cerebral acetone correlated with higher urine ketones (r >0.75,p< 0.02) and lower fasting glucose (r >-0.74,p< 0.05). CONCLUSIONS The GLAD diet was challenging to maintain but demonstrated quantifiable biologic effects systemically and intratumorally. MAD was more feasible than IF, but changes in PD markers correlated most strongly with IF. The role of ketogenic diet therapy for preventing glioma growth remains uncertain.

BRAF mutations have been identified as targetable, oncogenic mutations in many cancers. Given the paucity of treatments for primary brain tumors and the poor prognosis associated with high-grade gliomas, BRAF mutations in glioma are of considerable interest. In this review, we present the spectrum of BRAF mutations and fusion alterations present in each class of primary brain tumor based on publicly available databases and publications. We also summarize clinical experience with RAF and MEK inhibitors in patients with primary brain tumors and describe ongoing clinical trials of RAF inhibitors in glioma. Sensitivity to RAF and MEK inhibitors varies among BRAF mutations and between tumor types as only class I BRAF V600 mutations are sensitive to clinically available RAF inhibitors. While class II and III BRAF mutations are found in primary brain tumors, further research is necessary to determine their sensitivity to third-generation RAF inhibitors and/or MEK inhibitors. We recommend that the neuro-oncologist consider using these drugs primarily in the setting of a clinical trial for patients with BRAF-altered glioma in order to advance our knowledge of their efficacy in this patient population.

A 70-year-old woman with a history of insulin-dependent type 2 diabetes mellitus (IDDM), breast cancer treated with lumpectomy in 2012, hyperlipidemia, hypertension, coronary artery disease, and congestive heart failure with pacemaker presented with acute-onset difficulty using her right arm and leg. She was in her usual state of health until 3 weeks before admission, when she tripped over her grandson's toy on the floor, falling on her right side. Prior to falling, neither she nor her family had noted any weakness or difficulty with walking. Because her symptoms failed to improve over 3 weeks, she eventually presented to an emergency department. The authors thank Dr. Argye Hillis for her work conceptualizing this case.

Purpose: To determine whether uveal melanoma, the most common primary intraocular malignancy in adults, requires Notch activity for growth and metastasis. Experimental Design: Expression of Notch pathway members was characterized in primary tumor samples and in cell lines, along with the effects of Notch inhibition or activation on tumor growth and invasion. Results: Notch receptors, ligands, and targets were expressed in all five cell lines examined and in 30 primary uveal melanoma samples. Interestingly, the three lines with high levels of baseline pathway activity (OCM1, OCM3, and OCM8) had their growth reduced by pharmacologic Notch blockade using the γ-secretase inhibitor (GSI) MRK003. In contrast, two uveal melanoma lines (Mel285 and Mel290) with very low expression of Notch targets were insensitive to the GSI. Constitutively active forms of Notch1 and Notch2 promoted growth of uveal melanoma cultures and were able to rescue the inhibitory effects of GSI. MRK003 treatment also inhibited anchorage-independent clonogenic growth and cell invasion and reduced phosphorylation levels of STAT3 and extracellular signal-regulated kinase (Erk)1/2. Suppression of canonical Notch activity using short hairpin RNA targeting Notch2 or CBF1 was also able to reduce tumor growth and invasion. Finally, intraocular xenograft growth was significantly decreased by GSI treatment. Conclusion: Our findings suggest that Notch plays an important role in inducing proliferation and invasion in uveal melanoma and that inhibiting this pathway may be effective in preventing tumor growth and metastasis. Clin Cancer Res; 18(3); 654–65. ©2012 AACR.

Although Notch signaling has been widely implicated in neoplastic growth, direct evidence for in vivo initiation of neoplasia by the pathway in murine models has been limited to tumors of lymphoid, breast, and choroid plexus cells. To examine tumorigenic potential in the eye and brain, we injected retroviruses encoding activated forms of Notch1, Notch2, or Notch3 into embryonic mice. Interestingly, the majority of animals infected with active Notch3 developed proliferative lesions comprised of pigmented ocular choroid cells, retinal and optic nerve glia, and lens epithelium. Notch3-induced lesions in the choroid, retina, and optic nerve were capable of invading adjacent tissues, suggesting that they were malignant tumors. Although Notch3 activation induced choroidal tumors in up to 67% of eyes, Notch1 or Notch2 activation never resulted in such tumors. Active forms of Notch1 and Notch2 did generate a few small proliferative glial nodules in the retina and optic nerve, whereas Notch3 was 10-fold more efficient at generating growths, many of which were large invasive gliomas. Expression of active Notch1/Notch3 chimeric receptors implicated the RBPjk-association molecule and transactivation domains of Notch3 in generating choroidal and glial tumors, respectively. In contrast to our findings in the optic nerve and retina, introduction of active Notch receptors, including Notch3, into the brain never caused glial tumors. Our results highlight the differential ability of Notch receptor paralogs to initiate malignant tumor formation, and suggest that glial precursors of the optic nerve, but not the brain, are susceptible to transformation by Notch3. Cancer Res; 71(3); 1115–25. ©2011 AACR.

BACKGROUND: BRAF V600E-mutations are rare but noteworthy in primary brain tumors given their potential as a targetable mutation and the lack of efficacious therapies for glioblastoma. BRAF V600E mutations may serve as a prognostic marker in pediatric and low-grade gliomas, and are associated with improved survival in young adults with glioblastoma; however, its prognostic significance in adults >35 years is uncertain given the very small number of patients evaluated to date. METHODS: Patients aged >18 with WHO III-IV glioma and a BRAF V600E mutation were identified from the National Institutes of Health, the Johns Hopkins Hospital, and a previous publication (PMID:27503138). Paired control cases were identified at each institution based on age, sex, degree of resection, performance status at diagnosis/first encounter, MGMT and IDH status, and first-line treatment. Log-rank test was used to compare survival curves. RESULTS: The present cohort consisted of 23 patients (6 from each institution and 11 from a published cohort) with median age of 39 (range 20–70 years), 78% female, and 87% with a glioblastoma diagnosis. No tumors had an IDH mutation. 39% of patients were aged >50 years. All but one were treated with radiation and temozolomide at diagnosis (exception went into hospice and died shortly thereafter). The median overall survival was 33.4 ± 8.4 months in all patients. For 13 patients aged 35 or older, median survival was 34.5 ± 12.1 months compared to 18.0 ± 3.0 months in case-matched controls (p=0.03). Two patients were treated with dabrafenib and trametinib; one is still on therapy (26 months), the other progressed after 8 months. CONCLUSIONS: Adults aged >35 with BRAF V600E mutation may have improved survival compared with matched controls, similar to results in young adults. BRAF V600E mutations occur in patients with glioblastoma aged >50 years and testing in this population should be considered as well.

Summary Brain structure and size requires precise division of neural stem cells (NSCs), which self-renew and generate intermediate neural progenitors (INPs) and neurons. The factors that regulate NSCs remain poorly understood, as do mechanistic explanations of how aberrant NSC division causes reduced brain size as seen in microcephaly. Here we demonstrate that Magoh, a component of the exon junction complex (EJC) that binds RNA, controls mouse cerebral cortical size by regulating NSC division. Magoh haploinsufficiency causes microcephaly due to INP depletion and neuronal apoptosis. Defective mitosis underlies these phenotypes as depletion of EJC components disrupts mitotic spindle orientation and integrity, chromosome number, and genomic stability. In utero rescue experiments revealed that a key function of Magoh is to control levels of the microcephaly-associated protein, LIS1, during neurogenesis. This study uncovers new requirements for the EJC in brain development, NSC maintenance, and mitosis, thus implicating this complex in the pathogenesis of microcephaly.