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Background and purposeRecent advances in molecular techniques have characterized distinct subtypes of diffuse intrinsic pontine gliomas. Our aim was the identification of MR imaging correlates of these subtypes.Materials and methodsInitial MRIs from subjects with diffuse intrinsic pontine gliomas recruited for a prospective clinical trial before treatment were analyzed. Retrospective imaging analyses included FLAIR/T2 tumor volume, tumor volume enhancing, the presence of cyst and/or necrosis, median, mean, mode, skewness, kurtosis of ADC tumor volume based on FLAIR, and enhancement at baseline. Molecular subgroups based on EGFR and MGMT mutations were established. Histone mutations were also determined (H3F3A, HIST1H3B, HIST1H3C). Univariate Cox proportional hazards regression was used to test the association of imaging predictors with overall and progression-free survival. Wilcoxon rank sum, Kruskal-Wallis, and Fisher exact tests were used to compare imaging measures among groups.ResultsFifty patients had biopsy and MR imaging. The median age at trial registration was 6 years (range, 3.3-17.5 years); 52% were female. On the basis of immunohistochemical results, 48 patients were assigned to 1 of 4 subgroups: 28 in MGMT-/epidermal growth factor receptor (EGFR)-, 14 in MGMT-/EGFR+, 3 in MGMT+/EGFR-, and 3 in MGMT+/EGFR+. Twenty-three patients had histone mutations in H3F3A, 8 in HIST1H3B, and 3 in HIST1H3C. Enhancing tumor volume was near-significantly different across molecular subgroups (P = .04), after accounting for the false discovery rate. Tumor volume enhancing, median, mode, skewness, and kurtosis ADC T2-FLAIR/T2 were significantly different (P ≤ .048) between patients with H3F3A and HIST1H3B/C mutations.ConclusionsMR imaging features including enhancement and ADC histogram parameters are correlated with molecular subgroups and mutations in children with diffuse intrinsic pontine gliomas.

© 2015 Macmillan Publishers Limited. All rights reserved.The role of intermediate methylation states in DNA is unclear. Here, to comprehensively identify regions of intermediate methylation and their quantitative relationship with gene activity, we apply i

Transposable element (TE)-derived sequences comprise half of the human genome and DNA methylome and are presumed to be densely methylated and inactive. Examination of genome-wide DNA methylation status within 928 TE subfamilies in human embryonic and adult

Histopathology image evaluation is indispensable for cancer diagnoses and subtype classification. Standard artificial intelligence methods for histopathology image analyses have focused on optimizing specialized models for each diagnostic task 1,2 . Although such methods have achieved some success, they often have limited generalizability to images generated by different digitization protocols or samples collected from different populations 3 . Here, to address this challenge, we devised the Clinical Histopathology Imaging Evaluation Foundation (CHIEF) model, a general-purpose weakly supervised machine learning framework to extract pathology imaging features for systematic cancer evaluation. CHIEF leverages two complementary pretraining methods to extract diverse pathology representations: unsupervised pretraining for tile-level feature identification and weakly supervised pretraining for whole-slide pattern recognition. We developed CHIEF using 60,530 whole-slide images spanning 19 anatomical sites. Through pretraining on 44 terabytes of high-resolution pathology imaging datasets, CHIEF extracted microscopic representations useful for cancer cell detection, tumour origin identification, molecular profile characterization and prognostic prediction. We successfully validated CHIEF using 19,491 whole-slide images from 32 independent slide sets collected from 24 hospitals and cohorts internationally. Overall, CHIEF outperformed the state-of-the-art deep learning methods by up to 36.1%, showing its ability to address domain shifts observed in samples from diverse populations and processed by different slide preparation methods. CHIEF provides a generalizable foundation for efficient digital pathology evaluation for patients with cancer., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Postoperative recurrence risk for pediatric low-grade gliomas (pLGGs) is challenging to predict by conventional clinical, radiographic, and genomic factors. We investigated if deep learning of MRI tumor features could improve postoperative pLGG risk stratification., Methods: We used pre-trained deep learning (DL) tool designed for pLGG segmentation to extract pLGG imaging features from preoperative T2-weighted MRI from patients who underwent surgery (DL-MRI features). Patients were pooled from two institutions: Dana Farber/Boston Children's Hospital (DF/BCH) and the Children's Brain Tumor Network (CBTN). We trained three DL logistic hazard models to predict postoperative event-free survival (EFS) probabilities with 1) clinical features, 2) DL-MRI features, and 3) multimodal (clinical and DL-MRI features). We evaluated the models with a time-dependent Concordance Index (Ctd) and risk group stratification with Kaplan Meier plots and log-rank tests. We developed an automated pipeline integrating pLGG segmentation and EFS prediction with the best model., Results: Of the 396 patients analyzed (median follow-up: 85 months, range: 1.5-329 months), 214 (54%) underwent gross total resection and 110 (28%) recurred. The multimodal model improved EFS prediction compared to the DL-MRI and clinical models (Ctd: 0.85 (95% CI: 0.81-0.93), 0.79 (95% CI: 0.70-0.88), and 0.72 (95% CI: 0.57-0.77), respectively). The multimodal model improved risk-group stratification (3-year EFS for predicted high-risk: 31% versus low-risk: 92%, p<0.0001)., Conclusions: DL extracts imaging features that can inform postoperative recurrence prediction for pLGG. Multimodal DL improves postoperative risk stratification for pLGG and may guide postoperative decision-making. Larger, multicenter training data may be needed to improve model generalizability., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Diffuse hemispheric gliomas, H3G34R/V-mutant (DHG-H3G34), are lethal brain tumors lacking targeted therapies. They originate from interneuronal precursors; however, leveraging this origin for therapeutic insights remains unexplored. Here, we delineate a cellular hierarchy along the interneuron lineage development continuum, revealing that DHG-H3G34 mirror spatial patterns of progenitor streams surrounding interneuron nests, as seen during human brain development. Integrating these findings with genome-wide CRISPR-Cas9 screens identifies genes upregulated in interneuron lineage progenitors as major dependencies. Among these, CDK6 emerges as a targetable vulnerability: DHG-H3G34 tumor cells show enhanced sensitivity to CDK4/6 inhibitors and a CDK6-specific degrader, promoting a shift toward more mature interneuron-like states, reducing tumor growth, and prolonging xenograft survival. Notably, a patient with progressive DHG-H3G34 treated with a CDK4/6 inhibitor achieved 17 months of stable disease. This study underscores interneuronal progenitor-like states, organized in characteristic niches, as a distinct vulnerability in DHG-H3G34, highlighting CDK6 as a promising clinically actionable target., Competing Interests: Declaration of interests M.G.F. is a consultant for Twentyeight-Seven Therapeutics and Blueprint Medicines. M.M. is an SAB member for Cygnal Therapeutics. K.L.L. is founder and equity holder of Travera Inc. and receives consulting fees from BMS, Integragen, and Rarecyte, and research support from Lilly, BMS, and Amgen. D.H. has acted as an advisor for Novartis in relation to ribociclib., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: With the significant shift in the classification, risk stratification, and standards of care for gliomas, we sought to understand how the overall survival of patients with these tumors is impacted by molecular features, clinical metrics, and treatment received., Methods: We assembled a cohort of patients with a histopathologically diagnosed glioma from The Cancer Genome Atlas, Project Genomics Evidence Neoplasia Information Exchange, and Dana-Farber Cancer Institute/Brigham and Women's Hospital. This incorporated retrospective clinical, histological, and molecular data alongside prospective assessment of patient survival., Results: 4,400 gliomas were identified: 2,195 glioblastoma, 1,198 IDH1/2-mutant astrocytoma, 531 oligodendroglioma, 271 other IDH1/2-wildtype glioma, and 205 pediatric-type glioma. Molecular classification updated 27.2% of gliomas from their original histopathologic diagnosis. Examining the distribution of molecular alterations across glioma subtypes revealed mutually exclusive alterations within tumorigenic pathways. Non-TCGA patients had significantly improved overall survival compared to TCGA patients, with 26.7%, 55.6%, and 127.8% longer survival for glioblastoma, IDH1/2-mutant astrocytoma, and oligodendroglioma respectively (all p<0.01). Several prognostic features were characterized, including NF1 alteration and 21q loss in glioblastoma, and EGFR amplification and 22q loss in IDH1/2-mutant astrocytoma. Leveraging the size of this cohort, nomograms were generated to assess the probability of overall survival based on patient age, the molecular features of a tumor, and the treatment received., Conclusions: By applying modern molecular criteria, we characterize the genomic diversity across glioma subtypes, identify clinically applicable prognostic features, and provide a contemporary update on patient survival to serve as a reference for ongoing investigations., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

We investigated the effectiveness of navtemadlin (KRT-232) in treating recurrent glioblastoma. A surgical window-of-opportunity trial ( NCT03107780 ) was conducted on 21 patients to determine achievable drug concentrations within tumor tissue and examine mechanisms of response and resistance. Both 120 mg and 240 mg daily dosing achieved a pharmacodynamic impact. Sequencing of three recurrent tumors revealed an absence of TP53 -inactivating mutations, indicating alternative mechanisms of resistance. In patient-derived GBM models, the lower range of clinically achieved navtemadlin concentrations induced partial tumor cell death as monotherapy. However, combining navtemadlin with temozolomide increased apoptotic rates while sparing normal bone marrow cells in vitro, which in return underwent reversible growth arrest. These results indicate that clinically achievable doses of navtemadlin generate significant pharmacodynamic effects and suggest that combined treatment with standard-of-care DNA damaging chemotherapy is a route to durable survival benefits., Statement of Significance: Tissue sampling during this clinical trial allowed us to assess mechanisms of response and resistance associated with navtemadlin treatment in recurrent GBM. We report that clinically achievable doses of navtemadlin induce pharmacodynamic effects in tumor tissue, and suggest combinations with standard-of-care chemotherapy for durable clinical benefit.

Background: The frequency and significance of IDH mutations in glioma across age groups is incompletely understood. We performed a multi-center retrospective age-stratified comparison of patients with IDH-mutant gliomas to identify age-specific differences in clinico-genomic features, treatments, and outcomes., Methods: Clinical, histologic, and sequencing data from patients with IDH-mutant, grade 2-4 gliomas, were collected from collaborating institutions between 2013-2019. Patients were categorized as pediatric (<19y), YA (19-39y) or older adult (≥40y). Clinical presentation, treatment, histologic, and molecular features were compared across age categories using Fisher's exact test or analysis-of-variance. Cox proportional-hazards regression was used to determine association of age and other covariates with overall (OS) and progression-free survival (PFS)., Results: We identified a cohort of 379 patients (204 YA) with IDH-mutant glioma with clinical data. There were 155 (41%) oligodendrogliomas and 224 (59%) astrocytomas. YA showed significantly shorter PFS and shorter median time-to-malignant transformation (MT) compared to pediatric and adult groups, but no significant OS difference. Adjusting for pathology type, extent of resection, and upfront therapy in multivariable analysis, the YA group was independently prognostic of shorter PFS than pediatric and adult groups. Among astrocytomas, CDK4/6 copy number amplifications were associated with both shorter PFS and shorter OS. Among oligodendrogliomas, PIK3CA and CDKN2A/2B alterations were associated with shorter OS., Conclusions: IDH-mutant glioma YA patients had significantly shorter PFS and time to MT but did not differ in OS compared to pediatric and adult groups. Treatment approach varied significantly by patient age and warrant further study as addressable age-associated outcome drivers., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

To inform clinical trial design and real-world precision pediatric oncology practice, we classified diagnoses, assessed the landscape of mutations, and identified genomic variants matching trials in a large unselected institutional cohort of solid tumors patients sequenced at Dana-Farber / Boston Children's Cancer and Blood Disorders Center. Tumors were sequenced with OncoPanel, a targeted next-generation DNA sequencing panel. Diagnoses were classified according to the International Classification of Diseases for Oncology (ICD-O-3.2). Over 6.5 years, 888 pediatric cancer patients with 95 distinct diagnoses had successful tumor sequencing. Overall, 33% (n = 289/888) of patients had at least 1 variant matching a precision oncology trial protocol, and 14% (41/289) were treated with molecularly targeted therapy. This study highlights opportunities to use genomic data from hospital-based sequencing performed either for research or clinical care to inform ongoing and future precision oncology clinical trials. Furthermore, the study results emphasize the importance of data sharing to define the genomic landscape and targeted treatment opportunities for the large group of rare pediatric cancers we encounter in clinical practice., (© 2024. The Author(s).)

Background: Velcrins are molecular glues that kill cells by inducing the formation of a protein complex between the RNase SLFN12 and the phosphodiesterase PDE3A. Formation of the complex activates SLFN12, which cleaves tRNA Leu (TAA) and induces apoptosis. Velcrins such as the clinical investigational compound, BAY 2666605, were found to have activity across multiple solid tumor cell lines from the cancer cell line encyclopedia, including glioblastoma cell lines. We therefore aim to characterize velcrins as novel therapeutic agents in glioblastoma., Materials and Methods: PDE3A and SLFN12 expression levels were measured in glioblastoma cell lines, the Cancer Genome Atlas (TCGA) tumor samples, and tumor neurospheres. Velcrin-treated cells were assayed for viability, induction of apoptosis, cell cycle phases, and global changes in translation. Transcriptional profiling of the cells was obtained. Xenograft-harboring mice treated with velcrins were also monitored for survival., Results: We identified several velcrin-sensitive glioblastoma cell lines and 4 velcrin-sensitive glioblastoma patient-derived models. We determined that BAY 2666605 crosses the blood-brain barrier and elicits full tumor regression in an orthotopic xenograft model of GB1 cells. We also determined that the velcrins BAY 2666605 and BRD3800 induce tumor regression in subcutaneous glioblastoma PDX models., Conclusions: Velcrins have antitumor activity in preclinical models of glioblastoma, warranting further investigation as potential therapeutic agents., Competing Interests: P.Y.W.: Research Support: Agios, Astra Zeneca/Medimmune, Beigene, Celgene, Eli Lily, Genentech/Roche, Kazia, MediciNova, Merck, Novartis, Nuvation Bio, Oncoceutics, Vascular Biogenics, VBI Vaccines. Advisory Board: Agios, Astra Zeneca, Bayer, Boston Pharmaceuticals, CNS Pharmaceuticals, Elevate Bio Immunomic Therapeutics, Imvax, Karyopharm, Merck, Novartis, Nuvation Bio, Vascular Biogenics, VBI Vaccines, Voyager, QED, Celularity, Sapience. J.G.D.: consults for Microsoft Research, Abata Therapeutics, Servier, Maze Therapeutics, BioNTech, Sangamo, and Pfizer; consults for and has equity in Tango Therapeutics; serves as a paid scientific advisor to the Laboratory for Genomics Research, funded in part by GSK; receives funding support from the Functional Genomics Consortium: Abbvie, Bristol Myers Squibb, Janssen, Merck, and Vir Biotechnology. M.M. is the scientific advisory board chair of Isabl; consults for Bayer, DelveBio, and Interline; is an inventor of patents licensed to LabCorp and Bayer; and receives research funding from Bayer and Janssen. M.M. and H.G. receive an inventor’s share of license revenue as part of their employment for certain patent filings, including US-2016-0016913 and US-2018-0235961, which relate to aspects of the work described in this manuscript. The co-owners of these patent filings are The Broad Institute, Inc., Dana-Farber Cancer Institute, Inc., and Bayer Pharma A.G., A.D.C, and H.G. also receive research funding from Bayer. A.D.C consults for KaryoVerse. K.L.L.: Research Support: BMS; Consulting: BMS, Travera, Integragen Equity: Travera., (© The Author(s) 2024. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

A subset of patients with IDH-mutant glioma respond to inhibitors of mutant IDH (IDHi), yet the molecular underpinnings of such responses are not understood. Here, we profiled by single-cell or single-nucleus RNA-sequencing three IDH-mutant oligodendrogliomas from patients who derived clinical benefit from IDHi. Importantly, the tissues were sampled on-drug, four weeks from treatment initiation. We further integrate our findings with analysis of single-cell and bulk transcriptomes from independent cohorts and experimental models. We find that IDHi treatment induces a robust differentiation toward the astrocytic lineage, accompanied by a depletion of stem-like cells and a reduction of cell proliferation. Furthermore, mutations in NOTCH1 are associated with decreased astrocytic differentiation and may limit the response to IDHi. Our study highlights the differentiating potential of IDHi on the cellular hierarchies that drive oligodendrogliomas and suggests a genetic modifier that may improve patient stratification., Competing Interests: Declaration of interests M.L.S. is equity holders, scientific co-founder and advisory board member of Immunitas Therapeutics. I.T. is advisory board member of Immunitas Therapeutics. D.P.C. has consulted for Lilly, Incephalo, Boston Pharmaceuticals, Servier, Boston Scientific and Pyramid Biosciences (equity interest), and has received honoraria and travel reimbursement from Merck for invited lectures. J.J.M. received consulting fees from Servier. The authors declare that such activities have no relationship to the present study. M.T. reports consulting or advisory role for Servier, Novocure, Resilience, Agios Pharmaceutical, Integragen, and Taiho Oncology, honoraria for Ono, and research funding from Sanofi. P.Y.W. reports research support from Astra Zeneca, Black Diamond, Bristol Meyers Squibb, Chimerix, Eli Lily, Erasca, Global Coalition For Adaptive Research, Kazia, MediciNova, Merck, Novartis, Quadriga, Servier, VBI Vaccines and consulting or advisory role for Anheart, Astra Zeneca, Black Diamond, Celularity, Chimerix, Day One Bio, Genenta, Glaxo Smith Kline, Kintara, Merck, Mundipharma, Novartis, Novocure, Prelude Therapeutics, Sagimet, Sapience, Servier, Symbio, Tango, Telix, VBI Vaccines. K.L.L is equity holder, consultant, and co-founder of Travera, is a consultant for BMS, Blaze Biosciences and Integragen, and has grant research funding through DFCI from BMS and Lilly. L.N.G.C. has received research support from Merck & Co, and consulting fees from BMJ Best Practice and Oakstone Publishing., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Purpose To develop and externally test a scan-to-prediction deep learning pipeline for noninvasive, MRI-based BRAF mutational status classification for pediatric low-grade glioma. Materials and Methods This retrospective study included two pediatric low-grade glioma datasets with linked genomic and diagnostic T2-weighted MRI data of patients: Dana-Farber/Boston Children's Hospital (development dataset, n = 214 [113 (52.8%) male; 104 (48.6%) BRAF wild type, 60 (28.0%) BRAF fusion, and 50 (23.4%) BRAF V600E]) and the Children's Brain Tumor Network (external testing, n = 112 [55 (49.1%) male; 35 (31.2%) BRAF wild type, 60 (53.6%) BRAF fusion, and 17 (15.2%) BRAF V600E]). A deep learning pipeline was developed to classify BRAF mutational status ( BRAF wild type vs BRAF fusion vs BRAF V600E) via a two-stage process: (a) three-dimensional tumor segmentation and extraction of axial tumor images and (b) section-wise, deep learning-based classification of mutational status. Knowledge-transfer and self-supervised approaches were investigated to prevent model overfitting, with a primary end point of the area under the receiver operating characteristic curve (AUC). To enhance model interpretability, a novel metric, center of mass distance, was developed to quantify the model attention around the tumor. Results A combination of transfer learning from a pretrained medical imaging-specific network and self-supervised label cross-training (TransferX) coupled with consensus logic yielded the highest classification performance with an AUC of 0.82 (95% CI: 0.72, 0.91), 0.87 (95% CI: 0.61, 0.97), and 0.85 (95% CI: 0.66, 0.95) for BRAF wild type, BRAF fusion, and BRAF V600E, respectively, on internal testing. On external testing, the pipeline yielded an AUC of 0.72 (95% CI: 0.64, 0.86), 0.78 (95% CI: 0.61, 0.89), and 0.72 (95% CI: 0.64, 0.88) for BRAF wild type, BRAF fusion, and BRAF V600E, respectively. Conclusion Transfer learning and self-supervised cross-training improved classification performance and generalizability for noninvasive pediatric low-grade glioma mutational status prediction in a limited data scenario. Keywords: Pediatrics, MRI, CNS, Brain/Brain Stem, Oncology, Feature Detection, Diagnosis, Supervised Learning, Transfer Learning, Convolutional Neural Network (CNN) Supplemental material is available for this article. © RSNA, 2024.

Wilms tumor (WT) is the most common renal malignancy of childhood. Despite improvements in the overall survival, relapse occurs in ~15% of patients with favorable histology WT (FHWT). Half of these patients will succumb to their disease. Identifying novel targeted therapies remains challenging in part due to the lack of faithful preclinical in vitro models. Here we establish twelve patient-derived WT cell lines and demonstrate that these models faithfully recapitulate WT biology using genomic and transcriptomic techniques. We then perform loss-of-function screens to identify the nuclear export gene, XPO1, as a vulnerability. We find that the FDA approved XPO1 inhibitor, KPT-330, suppresses TRIP13 expression, which is required for survival. We further identify synergy between KPT-330 and doxorubicin, a chemotherapy used in high-risk FHWT. Taken together, we identify XPO1 inhibition with KPT-330 as a potential therapeutic option to treat FHWTs and in combination with doxorubicin, leads to durable remissions in vivo., (© 2024. The Author(s).)

Purpose: Adverse clinical events cause significant morbidity in patients with GBM (GBM). We examined whether genomic alterations were associated with AE (AE) in patients with GBM., Experimental Design: We identified adults with histologically confirmed IDH-wild-type GBM with targeted next-generation sequencing (OncoPanel) at Dana Farber Cancer Institute from 2013 to 2019. Seizure at presentation, lymphopenia, thromboembolic events, pseudoprogression, and early progression (within 6 months of diagnosis) were identified as AE. The biologic function of genetic variants was categorized as loss-of-function (LoF), no change in function, or gain-of-function (GoF) using a somatic tumor mutation knowledge base (OncoKB) and consensus protein function predictions. Associations between functional genomic alterations and AE were examined using univariate logistic regressions and multivariable regressions adjusted for additional clinical predictors., Results: Our study included 470 patients diagnosed with GBM who met the study criteria. We focused on 105 genes that had sequencing data available for ≥ 90% of the patients and were altered in ≥10% of the cohort. Following false-discovery rate (FDR) correction and multivariable adjustment, the TP53, RB1, IGF1R, and DIS3 LoF alterations were associated with lower odds of seizures, while EGFR, SMARCA4, GNA11, BRD4, and TCF3 GoF and SETD2 LoF alterations were associated with higher odds of seizures. For all other AE of interest, no significant associations were found with genomic alterations following FDR correction., Conclusions: Genomic biomarkers based on functional variant analysis of a routine clinical panel may help identify AE in GBM, particularly seizures. Identifying these risk factors could improve the management of patients through better supportive care and consideration of prophylactic therapies., (©2024 American Association for Cancer Research.)

Background: Antibody-drug conjugates (ADCs) enhance the specificity of cytotoxic drugs by directing them to cells expressing target antigens. Multiple ADCs are FDA-approved for solid and hematologic malignancies, including those expressing HER2, TROP2, and NECTIN4. Recently, an ADC targeting HER2 (Trastuzumab-Deruxtecan) increased survival and reduced growth of brain metastases in treatment-refractory metastatic breast cancer, even in tumors with low HER2 expression. Thus, low-level expression of ADC targets may be sufficient for treatment responsiveness. However, ADC target expression is poorly characterized in many central nervous system (CNS) tumors., Methods: We analyzed publicly available RNA-sequencing and proteomic data from the children's brain tumor network (N = 188 tumors) and gene-expression-omnibus RNA-expression datasets (N = 356) to evaluate expression of 14 potential ADC targets that are FDA-approved or under investigation in solid cancers. We also used immunohistochemistry to measure the levels of HER2, HER3, NECTIN4, TROP2, CLDN6, CLDN18.2, and CD276/B7-H3 protein in glioblastoma, oligodendroglioma, meningioma, ependymoma, pilocytic astrocytoma, medulloblastoma, atypical teratoid/rhabdoid tumor (AT/RT), adamantinomatous craniopharyngioma (ACP), papillary craniopharyngioma (PCP), and primary CNS lymphoma (N = 575)., Results: Pan-CNS analysis showed subtype-specific expression of ADC target proteins. Most tumors expressed HER3, B7-H3, and NECTIN4. Ependymomas strongly expressed HER2, while meningiomas showed weak-moderate HER2 expression. ACP and PCP strongly expressed B7-H3, with TROP2 expression in whorled ACP epithelium. AT/RT strongly expressed CLDN6. Glioblastoma showed little subtype-specific marker expression, suggesting a need for further target development., Conclusions: CNS tumors exhibit subtype-specific expression of ADC targets including several FDA-approved for other indications. Clinical trials of ADCs in CNS tumors may therefore be warranted., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Distinct genetic alterations determine glioma aggressiveness, however, the diversity of somatic mutations contributing to peritumoral hyperexcitability and seizures over the course of the disease is uncertain. This study aimed to identify tumor somatic mutation profiles associated with clinically significant hyperexcitability., Methods: A single center cohort of adults with WHO grades 1-4 glioma and targeted exome sequencing (n = 1716) was analyzed and cross-referenced with a validated EEG database to identify the subset of individuals who underwent continuous EEG monitoring (n = 206). Hyperexcitability was defined by the presence of lateralized periodic discharges and/or electrographic seizures. Cross-validated discriminant analysis models trained exclusively on recurrent somatic mutations were used to identify variants associated with hyperexcitability., Results: The distribution of WHO grades and tumor mutational burdens were similar between patients with and without hyperexcitability. Discriminant analysis models classified the presence or absence of EEG hyperexcitability with an overall accuracy of 70.9%, regardless of IDH1 R132H inclusion. Predictive variants included nonsense mutations in ATRX and TP53, indel mutations in RBBP8 and CREBBP, and nonsynonymous missense mutations with predicted damaging consequences in EGFR, KRAS, PIK3CA, TP53, and USP28. This profile improved estimates of hyperexcitability in a multivariate analysis controlling for age, sex, tumor location, integrated pathologic diagnosis, recurrence status, and preoperative epilepsy. Predicted somatic mutation variants were over-represented in patients with hyperexcitability compared to individuals without hyperexcitability and those who did not undergo continuous EEG., Conclusion: These findings implicate diverse glioma somatic mutations in cancer genes associated with peritumoral hyperexcitability. Tumor genetic profiling may facilitate glioma-related epilepsy prognostication and management., (Published by Oxford University Press on behalf of the Society for Neuro-Oncology 2023.)

A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames (ORFs). To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a stepwise approach using multiple CRISPR-Cas9 screens to elucidate non-canonical ORFs and putative microproteins implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream ORFs (uORFs) exhibited selective functionality independent of main coding sequences. A microprotein encoded by one of these ORFs, ASNSD1-uORF or ASDURF, was upregulated, associated with MYC-family oncogenes, and promoted medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future studies seeking to define new cancer targets., Competing Interests: Declaration of interests K.L.L. reports the following interests: equity in Travera; research funds from Bristol Myers Squibb, SEngine Precision Medicine, Multiple Myeloma Research Foundation, and Eli Lilly and Company; and being a consultant or on the scientific advisory board for Bristol Myers Squibb, Travera, and IntegraGen. P.B. receives grant funding from Novartis Institute of Biomedical Research, and has received grant funding from Deerfield Therapeutics, both for unrelated projects. P.B. has also served on a paid advisory board for qed Therapeutics, unrelated to this work. D.E.R. receives research funding from members of the Functional Genomics Consortium (AbbVie, BMS, Jannsen, Merck, and Vir) and is a director of Addgene, Inc., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: The Individualized Screening Trial of Innovative Glioblastoma Therapy (INSIGhT) is a phase II platform trial that uses response adaptive randomization and genomic profiling to efficiently identify novel therapies for phase III testing. Three initial experimental arms (abemaciclib [a cyclin-dependent kinase [CDK]4/6 inhibitor], neratinib [an epidermal growth factor receptor [EGFR]/human epidermal growth factor receptor 2 inhibitor], and CC-115 [a deoxyribonucleic acid-dependent protein kinase/mammalian target of rapamycin inhibitor]) were simultaneously evaluated against a common control arm. We report the results for each arm and examine the feasibility and conduct of the adaptive platform design., Patients and Methods: Patients with newly diagnosed O 6 -methylguanine-DNA methyltransferase-unmethylated glioblastoma were eligible if they had tumor genotyping to identify prespecified biomarker subpopulations of dominant glioblastoma signaling pathways (EGFR, phosphatidylinositol 3-kinase, and CDK). Initial random assignment was 1:1:1:1 between control (radiation therapy and temozolomide) and the experimental arms. Subsequent Bayesian adaptive randomization was incorporated on the basis of biomarker-specific progression-free survival (PFS) data. The primary end point was overall survival (OS), and one-sided P values are reported. The trial is registered with ClinicalTrials.gov (identifier: NCT02977780)., Results: Two hundred thirty-seven patients were treated (71 control; 73 abemaciclib; 81 neratinib; 12 CC-115) in years 2017-2021. Abemaciclib and neratinib were well tolerated, but CC-115 was associated with ≥ grade 3 treatment-related toxicity in 58% of patients. PFS was significantly longer with abemaciclib (hazard ratio [HR], 0.72; 95% CI, 0.49 to 1.06; one-sided P = .046) and neratinib (HR, 0.72; 95% CI, 0.50 to 1.02; one-sided P = .033) relative to the control arm but there was no PFS benefit with CC-115 (one-sided P = .523). None of the experimental therapies demonstrated a significant OS benefit ( P > .05)., Conclusion: The INSIGhT design enabled efficient simultaneous testing of three experimental agents using a shared control arm and adaptive randomization. Two investigational arms had superior PFS compared with the control arm, but none demonstrated an OS benefit. The INSIGhT design may promote improved and more efficient therapeutic discovery in glioblastoma. New arms have been added to the trial.

Background: Insulin feedback is a critical mechanism responsible for the poor clinical efficacy of phosphatidylinositol 3-kinase (PI3K) inhibition in cancer, and hyperglycemia is an independent factor associated with poor prognosis in glioblastoma (GBM). We investigated combination anti-hyperglycemic therapy in a mouse model of GBM and evaluated the association of glycemic control in clinical trial data from patients with GBM., Methods: The effect of the anti-hyperglycemic regimens, metformin and the ketogenic diet, was evaluated in combination with PI3K inhibition in patient-derived GBM cells and in an orthotopic GBM mouse model. Insulin feedback and the immune microenvironment were retrospectively evaluated in blood and tumor tissue from a Phase 2 clinical trial of buparlisib in patients with recurrent GBM., Results: We found that PI3K inhibition induces hyperglycemia and hyperinsulinemia in mice and that combining metformin with PI3K inhibition improves the treatment efficacy in an orthotopic GBM xenograft model. Through examination of clinical trial data, we found that hyperglycemia was an independent factor associated with poor progression-free survival in patients with GBM. We also found that PI3K inhibition increased insulin receptor activation and T-cell and microglia abundance in tumor tissue from these patients., Conclusion: Reducing insulin feedback improves the efficacy of PI3K inhibition in GBM in mice, and hyperglycemia worsens progression-free survival in patients with GBM treated with PI3K inhibition. These findings indicate that hyperglycemia is a critical resistance mechanism associated with PI3K inhibition in GBM and that anti-hyperglycemic therapy may enhance PI3K inhibitor efficacy in GBM patients., (Published by Oxford University Press on behalf of the Society for Neuro-Oncology 2023.)

Glioblastoma (GBM) remains the most malignant primary brain tumor, with a median survival rarely exceeding 2 years. Tumor heterogeneity and an immunosuppressive microenvironment are key factors contributing to the poor response rates of current therapeutic approaches. GBM-associated macrophages (GAMs) often exhibit immunosuppressive features that promote tumor progression. However, their dynamic interactions with GBM tumor cells remain poorly understood. Here, we used patient-derived GBM stem cell cultures and combined single-cell RNA sequencing of GAM-GBM co-cultures and real-time in vivo monitoring of GAM-GBM interactions in orthotopic zebrafish xenograft models to provide insight into the cellular, molecular, and spatial heterogeneity. Our analyses revealed substantial heterogeneity across GBM patients in GBM-induced GAM polarization and the ability to attract and activate GAMs-features that correlated with patient survival. Differential gene expression analysis, immunohistochemistry on original tumor samples, and knock-out experiments in zebrafish subsequently identified LGALS1 as a primary regulator of immunosuppression. Overall, our work highlights that GAM-GBM interactions can be studied in a clinically relevant way using co-cultures and avatar models, while offering new opportunities to identify promising immune-modulating targets., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Although circulating tumor DNA (ctDNA) assays are increasingly used to inform clinical decisions in cancer care, they have limited ability to identify the transcriptional programs that govern cancer phenotypes and their dynamic changes during the course of disease. To address these limitations, we developed a method for comprehensive epigenomic profiling of cancer from 1 ml of patient plasma. Using an immunoprecipitation-based approach targeting histone modifications and DNA methylation, we measured 1,268 epigenomic profiles in plasma from 433 individuals with one of 15 cancers. Our assay provided a robust proxy for transcriptional activity, allowing us to infer the expression levels of diagnostic markers and drug targets, measure the activity of therapeutically targetable transcription factors and detect epigenetic mechanisms of resistance. This proof-of-concept study in advanced cancers shows how plasma epigenomic profiling has the potential to unlock clinically actionable information that is currently accessible only via direct tissue sampling., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The lack of reliable predictive biomarkers to guide effective therapy is a major obstacle to the advancement of therapy for high-grade gliomas, particularly glioblastoma (GBM), one of the few cancers whose prognosis has not improved over the past several decades. With this pilot clinical trial (number NCT04135807), we provide first-in-human evidence that drug-releasing intratumoral microdevices (IMDs) can be safely and effectively used to obtain patient-specific, high-throughput molecular and histopathological drug response profiling. These data can complement other strategies to inform the selection of drugs based on their observed antitumor effect in situ. IMDs are integrated into surgical practice during tumor resection and remain in situ only for the duration of the otherwise standard operation (2 to 3 hours). None of the six enrolled patients experienced adverse events related to the IMD, and the exposed tissue was usable for downstream analysis for 11 out of 12 retrieved specimens. Analysis of the specimens provided preliminary evidence of the robustness of the readout, compatibility with a wide array of techniques for molecular tissue interrogation, and promising similarities with the available observed clinical-radiological responses to temozolomide. From an investigational aspect, the amount of information obtained with IMDs allows characterization of tissue effects of any drugs of interest, within the physiological context of the intact tumor, and without affecting the standard surgical workflow.

Background: Timely and accurate intraoperative cryosection evaluations remain the gold standard for guiding surgical treatments for gliomas. However, the tissue-freezing process often generates artifacts that make histologic interpretation difficult. In addition, the 2021 WHO Classification of Tumors of the Central Nervous System incorporates molecular profiles in the diagnostic categories, so standard visual evaluation of cryosections alone cannot completely inform diagnoses based on the new classification system., Methods: To address these challenges, we develop the context-aware Cryosection Histopathology Assessment and Review Machine (CHARM) using samples from 1,524 glioma patients from three different patient populations to systematically analyze cryosection slides., Findings: Our CHARM models successfully identified malignant cells (AUROC = 0.98 ± 0.01 in the independent validation cohort), distinguished isocitrate dehydrogenase (IDH)-mutant tumors from wild type (AUROC = 0.79-0.82), classified three major types of molecularly defined gliomas (AUROC = 0.88-0.93), and identified the most prevalent subtypes of IDH-mutant tumors (AUROC = 0.89-0.97). CHARM further predicts clinically important genetic alterations in low-grade glioma, including ATRX, TP53, and CIC mutations, CDKN2A/B homozygous deletion, and 1p/19q codeletion via cryosection images., Conclusions: Our approaches accommodate the evolving diagnostic criteria informed by molecular studies, provide real-time clinical decision support, and will democratize accurate cryosection diagnoses., Funding: Supported in part by the National Institute of General Medical Sciences grant R35GM142879, the Google Research Scholar Award, the Blavatnik Center for Computational Biomedicine Award, the Partners' Innovation Discovery Grant, and the Schlager Family Award for Early Stage Digital Health Innovations., Competing Interests: Declaration of interests K.-H.Y. is an inventor of US Patent 10,832,406, which was assigned to Harvard University. K.-H.Y. was a consultant of Curatio.DL., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Glioblastoma (GBM) patients are treated with radiation therapy, chemotherapy, and corticosteroids, which can cause myelosuppression. To understand the relative prognostic utility of blood-based biomarkers in GBM and its implications for clinical trial design, we examined the incidence, predictors, and prognostic value of lymphopenia, neutrophil-to-lymphocyte ratio (NLR), and platelet count during chemoradiation (CRT) and recurrence., Methods: This cohort study included 764 newly diagnosed glioblastoma patients treated from 2005 to 2019 with blood counts prior to surgery, within 6 weeks of CRT, and at first recurrence available for automatic extraction from the medical record. Logistic regression was used to evaluate exposures and Kaplan-Meier was used to evaluate outcomes., Results: Among the cohort, median age was 60.3 years; 87% had Karnofsky performance status ≥ 70, 37.5% had gross total resection, and 90% received temozolomide (TMZ). During CRT, 37.8% (248/656) of patients developed grade 3 or higher lymphopenia. On multivariable analysis (MVA), high NLR during CRT remained an independent predictor for inferior survival (Adjusted Hazard Ratio [AHR] = 1.57, 95% CI = 1.14-2.15) and shorter progression-free survival (AHR = 1.42, 95% CI = 1.05-1.90). Steroid use was associated with lymphopenia (OR = 2.66,1.20-6.00) and high NLR (OR = 3.54,2.08-6.11). Female sex was associated with lymphopenia (OR = 2.33,1.03-5.33). At first recurrence, 28% of patients exhibited grade 3 or higher lymphopenia. High NLR at recurrence was associated with worse subsequent survival on MVA (AHR = 1.69, 95% CI = 1.25-2.27)., Conclusions: High NLR is associated with worse outcomes in newly diagnosed and recurrent glioblastoma. Appropriate eligibility criteria and accounting and reporting of blood-based biomarkers are important in the design and interpretation of newly diagnosed and recurrent glioblastoma trials., Competing Interests: LN reports: Consulting for Ono, Brave Bio, Genmab. Advisory board for Ono, Kite/Gilead. Royalty from Wolters Kluwer (UpToDate). Clinical trial support from Merck, Astra Zeneca, Kazia, Ono, Bristol Myers Squibb. All other authors do not report relevant disclosures. RR reports research support from Project Data Sphere., (© The Author(s) 2023. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Background: Glioblastoma is one of the most lethal forms of cancer, with 5-year survival rates of only 6%. Glioblastoma-targeted therapeutics have been challenging to develop due to significant inter- and intra-tumoral heterogeneity. Telomerase reverse transcriptase gene (TERT) promoter mutations are the most common known clonal oncogenic mutations in glioblastoma. Telomerase is therefore considered to be a promising therapeutic target against this tumor. However, an important limitation of this strategy is that cell death does not occur immediately after telomerase ablation, but rather after several cell divisions required to reach critically short telomeres. We, therefore, hypothesize that telomerase inhibition would only be effective in glioblastomas with low tumor burden., Methods: We used CRISPR interference to knock down TERT expression in TERT promoter-mutant glioblastoma cell lines and patient-derived models. We then measured viability using serial proliferation assays. We also assessed for features of telomere crisis by measuring telomere length and chromatin bridge formation. Finally, we used a doxycycline-inducible CRISPR interference system to knock down TERT expression in vivo early and late in tumor development., Results: Upon TERT inactivation, glioblastoma cells lose their proliferative ability over time and exhibit telomere shortening and chromatin bridge formation. In vivo, survival is only prolonged when TERT knockdown is induced shortly after tumor implantation, but not when the tumor burden is high., Conclusions: Our results support the idea that telomerase inhibition would be most effective at treating glioblastomas with low tumor burden, for example in the adjuvant setting after surgical debulking and chemoradiation., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Precision medicine is critically dependent on better methods for diagnosing and staging disease and predicting drug response. Histopathology using hematoxylin and eosin (H&E)-stained tissue (not genomics) remains the primary diagnostic method in cancer. Recently developed highly multiplexed tissue imaging methods promise to enhance research studies and clinical practice with precise, spatially resolved single-cell data. Here, we describe the 'Orion' platform for collecting H&E and high-plex immunofluorescence images from the same cells in a whole-slide format suitable for diagnosis. Using a retrospective cohort of 74 colorectal cancer resections, we show that immunofluorescence and H&E images provide human experts and machine learning algorithms with complementary information that can be used to generate interpretable, multiplexed image-based models predictive of progression-free survival. Combining models of immune infiltration and tumor-intrinsic features achieves a 10- to 20-fold discrimination between rapid and slow (or no) progression, demonstrating the ability of multimodal tissue imaging to generate high-performance biomarkers., (© 2023. The Author(s).)

Oncogenic mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 occur in a wide range of cancers, including acute myeloid leukemia (AML) and glioma. Mutant IDH enzymes convert 2-oxoglutarate (2OG) to (R)-2-hydroxyglutarate [(R)-2HG], an oncometabolite that is hypothesized to promote cellular transformation by dysregulating 2OG-dependent enzymes. The only (R)-2HG target that has been convincingly shown to contribute to transformation by mutant IDH is the myeloid tumor suppressor TET2. However, there is ample evidence to suggest that (R)-2HG has other functionally relevant targets in IDH-mutant cancers. Here, we show that (R)-2HG inhibits KDM5 histone lysine demethylases and that this inhibition contributes to cellular transformation in IDH-mutant AML and IDH-mutant glioma. These studies provide the first evidence of a functional link between dysregulation of histone lysine methylation and transformation in IDH-mutant cancers., Significance: Mutant IDH is known to induce histone hypermethylation. However, it is not known if this hypermethylation is functionally significant or is a bystander effect of (R)-2HG accumulation in IDH-mutant cells. Here, we provide evidence that KDM5 inhibition by (R)-2HG contributes to mutant IDH-mediated transformation in AML and glioma. This article is highlighted in the In This Issue feature, p. 1275., (©2023 American Association for Cancer Research.)

Malignant pleural mesothelioma (MPM) has relatively ineffective first/second-line therapy for advanced disease and only 18% five-year survival for early disease. Drug-induced mitochondrial priming measured by dynamic BH3 profiling identifies efficacious drugs in multiple disease settings. We use high throughput dynamic BH3 profiling (HTDBP) to identify drug combinations that prime primary MPM cells derived from patient tumors, which also prime patient derived xenograft (PDX) models. A navitoclax (BCL-xL/BCL-2/BCL-w antagonist) and AZD8055 (mTORC1/2 inhibitor) combination demonstrates efficacy in vivo in an MPM PDX model, validating HTDBP as an approach to identify efficacious drug combinations. Mechanistic investigation reveals AZD8055 treatment decreases MCL-1 protein levels, increases BIM protein levels, and increases MPM mitochondrial dependence on BCL-xL, which is exploited by navitoclax. Navitoclax treatment increases dependency on MCL-1 and increases BIM protein levels. These findings demonstrate that HTDBP can be used as a functional precision medicine tool to rationally construct combination drug regimens in MPM and other cancers., (© 2023. The Author(s).)

Background: Functional profiling of freshly isolated glioblastoma (GBM) cells is being evaluated as a next-generation method for precision oncology. While promising, its success largely depends on the method to evaluate treatment activity which requires sufficient resolution and specificity., Methods: Here, we describe the 'precision oncology by single-cell profiling using ex vivo readouts of functionality' (PROSPERO) assay to evaluate the intrinsic susceptibility of high-grade brain tumor cells to respond to therapy. Different from other assays, PROSPERO extends beyond life/death screening by rapidly evaluating acute molecular drug responses at single-cell resolution., Results: The PROSPERO assay was developed by correlating short-term single-cell molecular signatures using mass cytometry by time-of-flight (CyTOF) to long-term cytotoxicity readouts in representative patient-derived glioblastoma cell cultures (n = 14) that were exposed to radiotherapy and the small-molecule p53/MDM2 inhibitor AMG232. The predictive model was subsequently projected to evaluate drug activity in freshly resected GBM samples from patients (n = 34). Here, PROSPERO revealed an overall limited capacity of tumor cells to respond to therapy, as reflected by the inability to induce key molecular markers upon ex vivo treatment exposure, while retaining proliferative capacity, insights that were validated in patient-derived xenograft (PDX) models. This approach also allowed the investigation of cellular plasticity, which in PDCLs highlighted therapy-induced proneural-to-mesenchymal (PMT) transitions, while in patients' samples this was more heterogeneous., Conclusion: PROSPERO provides a precise way to evaluate therapy efficacy by measuring molecular drug responses using specific biomarker changes in freshly resected brain tumor samples, in addition to providing key functional insights in cellular behavior, which may ultimately complement standard, clinical biomarker evaluations., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

In malignant cancer, excessive amounts of mutant p53 often lead to its aggregation, a feature that was recently identified as druggable. Here, we describe that induction of a heat shock-related stress response mediated by Foldlin, a small-molecule tool compound, reduces the protein levels of misfolded/aggregated mutant p53, while contact mutants or wild-type p53 remain largely unaffected. Foldlin also prevented the formation of stress-induced p53 nuclear inclusion bodies. Despite our inability to identify a specific molecular target, Foldlin also reduced protein levels of aggregating SOD1 variants. Finally, by screening a library of 778 FDA-approved compounds for their ability to reduce misfolded mutant p53, we identified the proteasome inhibitor Bortezomib with similar cellular effects as Foldlin. Overall, the induction of a cellular heat shock response seems to be an effective strategy to deal with pathological protein aggregation. It remains to be seen however, how this strategy can be translated to a clinical setting.