Glioblastoma evolution and heterogeneity from a 3D whole-tumor perspective.

Treatment failure for the lethal brain tumor glioblastoma (GBM) is attributed to intratumoral heterogeneity and tumor evolution. We utilized 3D neuronavigation during surgical resection to acquire samples representing the whole tumor mapped by 3D spatial coordinates. Integrative tissue and single-cell analysis revealed sources of genomic, epigenomic, and microenvironmental intratumoral heterogeneity and their spatial patterning. By distinguishing tumor-wide molecular features from those with regional specificity, we inferred GBM evolutionary trajectories from neurodevelopmental lineage origins and initiating events such as chromothripsis to emergence of genetic subclones and spatially restricted activation of differential tumor and microenvironmental programs in the core, periphery, and contrast-enhancing regions. Our work depicts GBM evolution and heterogeneity from a 3D whole-tumor perspective, highlights potential therapeutic targets that might circumvent heterogeneity-related failures, and establishes an interactive platform enabling 360° visualization and analysis of 3D spatial patterns for user-selected genes, programs, and other features across whole GBM tumors.

Quantitative Assessment of Preanalytic Variables on Clinical Evaluation of PI3/AKT/mTOR Signaling Activity in Diffuse Glioma.

Biomarker-driven therapeutic clinical trials require the implementation of standardized, evidence-based practices for sample collection. In diffuse glioma, phosphatidylinositol 3 (PI3)-kinase/AKT/mTOR (PI3/AKT/mTOR) signaling is an attractive therapeutic target for which window-of-opportunity clinical trials could facilitate the identification of promising new agents. Yet, the relevant preanalytic variables and optimal tumor sampling methods necessary to measure pathway activity are unknown. To address this, we used a murine model for isocitrate dehydrogenase (IDH)-wildtype glioblastoma (GBM) and human tumor tissue, including IDH-wildtype GBM and IDH-mutant diffuse glioma. First, we determined the impact of delayed time-to-formalin fixation, or cold ischemia time (CIT), on the quantitative assessment of cellular expression of 6 phosphoproteins that are readouts of PI3K/AK/mTOR activity (phosphorylated-proline-rich Akt substrate of 40 kDa (p-PRAS40, T246), -mechanistic target of rapamycin (p-mTOR; S2448); -AKT (p-AKT, S473); -ribosomal protein S6 (p-RPS6, S240/244 and S235/236), and -eukaryotic initiation factor 4E-binding protein 1 (p-4EBP1, T37/46). With CITs ≥ 2 hours, typical of routine clinical handling, all had reduced or altered expression with p-RPS6 (S240/244) exhibiting relatively greater stability. A similar pattern was observed using patient tumor samples from the operating room with p-4EBP1 more sensitive to delayed fixation than p-RPS6 (S240/244). Many clinical trials utilize unstained slides for biomarker evaluation. Thus, we evaluated the impact of slide storage conditions on the detection of p-RPS6 (S240/244), p-4EBP1, and p-AKT. After 5 months, storage at -80°C was required to preserve the expression of p-4EBP1 and p-AKT, whereas p-RPS6 (240/244) expression was not stable regardless of storage temperature. Biomarker heterogeneity impacts optimal tumor sampling. Quantification of p-RPS6 (240/244) expression in multiple regionally distinct human tumor samples from 8 patients revealed significant intratumoral heterogeneity. Thus, the accurate assessment of PI3K/AKT/mTOR signaling in diffuse glioma must overcome intratumoral heterogeneity and multiple preanalytic factors, including time-to-formalin fixation, slide storage conditions, and phosphoprotein of interest.

Whole tumor analysis reveals early origin of the TERT promoter mutation and intercellular heterogeneity in TERT expression.

BACKGROUND: The TERT promoter mutation (TPM) is acquired in most IDH-wildtype glioblastomas (GBM) and IDH-mutant oligodendrogliomas (OD) enabling tumor cell immortality. Previous studies on TPM clonality show conflicting results. This study was performed to determine whether TPM is clonal on a tumor-wide scale. METHODS: We investigated TPM clonality in relation to presumed early events in 19 IDH-wildtype GBM and 10 IDH-mutant OD using 3-dimensional comprehensive tumor sampling. We performed Sanger sequencing on 264 tumor samples and deep amplicon sequencing on 187 tumor samples. We obtained tumor purity and copy number estimates from whole exome sequencing. TERT expression was assessed by RNA-seq and RNAscope. RESULTS: We detected TPM in 100% of tumor samples with quantifiable tumor purity (219 samples). Variant allele frequencies (VAF) of TPM correlate positively with chromosome 10 loss in GBM (R = 0.85), IDH1 mutation in OD (R = 0.87), and with tumor purity (R = 0.91 for GBM; R = 0.90 for OD). In comparison, oncogene amplification was tumor-wide for MDM4- and most EGFR-amplified cases but heterogeneous for MYCN and PDGFRA, and strikingly high in low-purity samples. TPM VAF was moderately correlated with TERT expression (R = 0.52 for GBM; R = 0.65 for OD). TERT expression was detected in a subset of cells, solely in TPM-positive samples, including samples equivocal for tumor. CONCLUSIONS: On a tumor-wide scale, TPM is among the earliest events in glioma evolution. Intercellular heterogeneity of TERT expression, however, suggests dynamic regulation during tumor growth. TERT expression may be a tumor cell-specific biomarker.

PRDM6 promotes medulloblastoma by repressing chromatin accessibility and altering gene expression.

SNCAIP duplication may promote Group 4 medulloblastoma via induction of PRDM6, a poorly characterized member of the PRDF1 and RIZ1 homology domain-containing (PRDM) family of transcription factors. Here, we investigated the function of PRDM6 in human hindbrain neuroepithelial stem cells and tested PRDM6 as a driver of Group 4 medulloblastoma. We report that human PRDM6 localizes predominantly to the nucleus, where it causes widespread repression of chromatin accessibility and complex alterations of gene expression patterns. Genome-wide mapping of PRDM6 binding reveals that PRDM6 binds to chromatin regions marked by histone H3 lysine 27 trimethylation that are located within, or proximal to, genes. Moreover, we show that PRDM6 expression in neuroepithelial stem cells promotes medulloblastoma. Surprisingly, medulloblastomas derived from PRDM6-expressing neuroepithelial stem cells match human Group 3, but not Group 4, medulloblastoma. We conclude that PRDM6 expression has oncogenic potential but is insufficient to drive Group 4 medulloblastoma from neuroepithelial stem cells. We propose that both PRDM6 and additional factors, such as specific cell-of-origin features, are required for Group 4 medulloblastoma. Given the lack of PRDM6 expression in normal tissues and its oncogenic potential shown here, we suggest that PRDM6 inhibition may have therapeutic value in PRDM6-expressing medulloblastomas.

Spatial genomic, biochemical and cellular mechanisms underlying meningioma heterogeneity and evolution.

Intratumor heterogeneity underlies cancer evolution and treatment resistance, but targetable mechanisms driving intratumor heterogeneity are poorly understood. Meningiomas are the most common primary intracranial tumors and are resistant to all medical therapies, and high-grade meningiomas have significant intratumor heterogeneity. Here we use spatial approaches to identify genomic, biochemical and cellular mechanisms linking intratumor heterogeneity to the molecular, temporal and spatial evolution of high-grade meningiomas. We show that divergent intratumor gene and protein expression programs distinguish high-grade meningiomas that are otherwise grouped together by current classification systems. Analyses of matched pairs of primary and recurrent meningiomas reveal spatial expansion of subclonal copy number variants associated with treatment resistance. Multiplexed sequential immunofluorescence and deconvolution of meningioma spatial transcriptomes using cell types from single-cell RNA sequencing show decreased immune infiltration, decreased MAPK signaling, increased PI3K-AKT signaling and increased cell proliferation, which are associated with meningioma recurrence. To translate these findings to preclinical models, we use CRISPR interference and lineage tracing approaches to identify combination therapies that target intratumor heterogeneity in meningioma cell co-cultures.

Deconstructing Intratumoral Heterogeneity through Multiomic and Multiscale Analysis of Serial Sections.

Tumors may contain billions of cells, including distinct malignant clones and nonmalignant cell types. Clarifying the evolutionary histories, prevalence, and defining molecular features of these cells is essential for improving clinical outcomes, since intratumoral heterogeneity provides fuel for acquired resistance to targeted therapies. Here we present a statistically motivated strategy for deconstructing intratumoral heterogeneity through multiomic and multiscale analysis of serial tumor sections (MOMA). By combining deep sampling of IDH-mutant astrocytomas with integrative analysis of single-nucleotide variants, copy-number variants, and gene expression, we reconstruct and validate the phylogenies, spatial distributions, and transcriptional profiles of distinct malignant clones. By genotyping nuclei analyzed by single-nucleus RNA-seq for truncal mutations, we further show that commonly used algorithms for identifying cancer cells from single-cell transcriptomes may be inaccurate. We also demonstrate that correlating gene expression with tumor purity in bulk samples can reveal optimal markers of malignant cells and use this approach to identify a core set of genes that are consistently expressed by astrocytoma truncal clones, including AKR1C3, whose expression is associated with poor outcomes in several types of cancer. In summary, MOMA provides a robust and flexible strategy for precisely deconstructing intratumoral heterogeneity and clarifying the core molecular properties of distinct cellular populations in solid tumors.

Noninvasive Autopsy-Validated Tumor Probability Maps Identify Glioma Invasion Beyond Contrast Enhancement.

BACKGROUND AND OBJECTIVES: This study identified a clinically significant subset of patients with glioma with tumor outside of contrast enhancement present at autopsy and subsequently developed a method for detecting nonenhancing tumor using radio-pathomic mapping. We tested the hypothesis that autopsy-based radio-pathomic tumor probability maps would be able to noninvasively identify areas of infiltrative tumor beyond traditional imaging signatures. METHODS: A total of 159 tissue samples from 65 subjects were aligned to MRI acquired nearest to death for this retrospective study. Demographic and survival characteristics for patients with and without tumor beyond the contrast-enhancing margin were computed. An ensemble algorithm was used to predict pixelwise tumor presence from pathological annotations using segmented cellularity (Cell), extracellular fluid, and cytoplasm density as input (6 train/3 test subjects). A second level of ensemble algorithms was used to predict voxelwise Cell, extracellular fluid, and cytoplasm on the full data set (43 train/22 test subjects) using 5-by-5 voxel tiles from T1, T1 + C, fluid-attenuated inversion recovery, and apparent diffusion coefficient as input. The models were then combined to generate noninvasive whole brain maps of tumor probability. RESULTS: Tumor outside of contrast was identified in 41.5% of patients, who showed worse survival outcomes (hazard ratio = 3.90, P < .001). Tumor probability maps reliably tracked nonenhancing tumor on a range of local and external unseen data, identifying tumor outside of contrast in 69% of presurgical cases that also showed reduced survival outcomes (hazard ratio = 1.67, P = .027). CONCLUSION: This study developed a multistage model for mapping gliomas using autopsy tissue samples as ground truth, which was able to identify regions of tumor beyond traditional imaging signatures.

Challenges in the discovery of tumor-specific alternative splicing-derived cell-surface antigens in glioma.

Despite advancements in cancer immunotherapy, solid tumors remain formidable challenges. In glioma, profound inter- and intra-tumoral heterogeneity of antigen landscape hampers therapeutic development. Therefore, it is critical to consider alternative sources to expand the repertoire of targetable (neo-)antigens and improve therapeutic outcomes. Accumulating evidence suggests that tumor-specific alternative splicing (AS) could be an untapped reservoir of antigens. In this study, we investigated tumor-specific AS events in glioma, focusing on those predicted to generate major histocompatibility complex (MHC)-presentation-independent, cell-surface antigens that could be targeted by antibodies and chimeric antigen receptor-T cells. We systematically analyzed bulk RNA-sequencing datasets comparing 429 tumor samples (from The Cancer Genome Atlas) and 9166 normal tissue samples (from the Genotype-Tissue Expression project), and identified 13 AS events in 7 genes predicted to be expressed in more than 10% of the patients, including PTPRZ1 and BCAN, which were corroborated by an external RNA-sequencing dataset. Subsequently, we validated our predictions and elucidated the complexity of the isoforms using full-length transcript amplicon sequencing on patient-derived glioblastoma cells. However, analyses of the RNA-sequencing datasets of spatially mapped and longitudinally collected clinical tumor samples unveiled remarkable spatiotemporal heterogeneity of the candidate AS events. Furthermore, proteomics analysis did not reveal any peptide spectra matching the putative antigens. Our investigation illustrated the diverse characteristics of the tumor-specific AS events and the challenges of antigen exploration due to their notable spatiotemporal heterogeneity and elusive nature at the protein levels. Redirecting future efforts toward intracellular, MHC-presented antigens could offer a more viable avenue.

Perivascular NOTCH3+ stem cells drive meningioma tumorigenesis and resistance to radiotherapy.

Meningiomas are the most common primary intracranial tumors. Treatments for patients with meningiomas are limited to surgery and radiotherapy, and systemic therapies remain ineffective or experimental. Resistance to radiotherapy is common in high-grade meningiomas and the cell types and signaling mechanisms that drive meningioma tumorigenesis and resistance to radiotherapy are incompletely understood. Here we report NOTCH3 drives meningioma tumorigenesis and resistance to radiotherapy and find that perivascular NOTCH3+ stem cells are conserved across meningiomas from humans, dogs, and mice. Integrating single-cell transcriptomics with lineage tracing and imaging approaches in genetically engineered mouse models and xenografts, we show NOTCH3 drives tumor initiating capacity, cell proliferation, angiogenesis, and resistance to radiotherapy to increase meningioma growth and reduce survival. To translate these findings to patients, we show that an antibody stabilizing the extracellular negative regulatory region of NOTCH3 blocks meningioma tumorigenesis and sensitizes meningiomas to radiotherapy, reducing tumor growth and improving survival.

Fc-enhanced anti-CTLA-4, anti-PD-1, doxorubicin, and ultrasound-mediated BBB opening: A novel combinatorial immunotherapy regimen for gliomas.

BACKGROUND: Glioblastoma is a highly aggressive brain cancer that is resistant to conventional immunotherapy strategies. Botensilimab, an Fc-enhanced anti-CTLA-4 antibody (FcE-aCTLA-4), has shown durable activity in "cold" and immunotherapy-refractory cancers. METHOD: We evaluated the efficacy and immune microenvironment phenotype of a mouse analogue of FcE-aCTLA-4 in treatment-refractory preclinical models of glioblastoma, both as a monotherapy and in combination with doxorubicin delivered via low-intensity pulsed ultrasound and microbubbles (LIPU/MB). Additionally, we studied 4 glioblastoma patients treated with doxorubicin, anti-PD-1 with concomitant LIPU/MB to investigate the novel effect of doxorubicin modulating FcγR expressions in tumor associated macrophages/microglia (TAMs). RESULTS: FcE-aCTLA-4 demonstrated high-affinity binding to FcγRIV, the mouse ortholog of human FcγRIIIA, which was highly expressed in TAMs in human glioblastoma, most robustly at diagnosis. Notably, FcE-aCTLA-4 mediated selective depletion of intra-tumoral regulatory T cells (Tregs) via TAM-mediated phagocytosis, while sparing peripheral Tregs. Doxorubicin, a chemotherapeutic drug with immunomodulatory functions, was found to upregulate FcγRIIIA on TAMs in glioblastoma patients who received doxorubicin and anti-PD-1 with concomitant LIPU/MB. In murine models of immunotherapy-resistant gliomas, a combinatorial regimen of FcE-aCTLA-4, anti-PD-1, and doxorubicin with LIPU/MB, achieved a 90% cure rate, that was associated robust infiltration of activated CD8+ T cells and establishment of immunological memory as evidenced by rejection upon tumor rechallenge. CONCLUSION: Our findings demonstrate that FcE-aCTLA-4 promotes robust immunomodulatory and anti-tumor effects in murine gliomas and is significantly enhanced when combined with anti-PD-1, doxorubicin, and LIPU/MB. We are currently investigating this combinatory strategy in a clinical trial (clinicaltrials.gov NCT05864534).