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Background Gliomas comprise the most common type of primary brain tumor, are highly invasive, and often fatal. IDH-mutated gliomas are particularly challenging to image and there is currently no clinically accepted method for identifying the extent of tumor burden in these neoplasms. This uncertainty poses a challenge to clinicians who must balance the need to treat the tumor while sparing healthy brain from iatrogenic damage. The purpose of this study was to investigate the feasibility of using resting-state blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI) to detect glioma-related asynchrony in vascular dynamics for distinguishing tumor from healthy brain. Methods Twenty-four stereotactically localized biopsies were obtained during open surgical resection from ten treatment-naïve patients with IDH-mutated gliomas who received standard-of-care preoperative imaging as well as echo-planar resting-state BOLD fMRI. Signal intensity for BOLD asynchrony and standard-of-care imaging was compared to cell counts of total cellularity (H&E), tumor density (IDH1 & Sox2), cellular proliferation (Ki67), and neuronal density (NeuN), for each corresponding sample. Results BOLD asynchrony was directly related to total cellularity (H&E, P = 4 × 10–5), tumor density (IDH1, P = 4 × 10–5; Sox2, P = 3 × 10–5), cellular proliferation (Ki67, P = .002), and inversely related to neuronal density (NeuN, P = 1 × 10–4). Conclusions Asynchrony in vascular dynamics, as measured by resting-state BOLD fMRI, correlates with tumor burden and provides a radiographic delineation of tumor boundaries in IDH-mutated gliomas.

BACKGROUND AND PURPOSE: Malignant glioma is a highly infiltrative malignancy that causes variable disruptions to the structure and function of the cerebrovasculature. While many of these structural disruptions have known correlative histopathologic alterations, the mechanisms underlying vascular dysfunction identified by resting-state blood oxygen level–dependent imaging are not yet known. The purpose of this study was to characterize the alterations that correlate with a blood oxygen level–dependent biomarker of vascular dysregulation. Materials and Methods: Thirty-two stereotactically localized biopsies were obtained from contrast-enhancing ( n = 16) and nonenhancing ( n = 16) regions during open surgical resection of malignant glioma in 17 patients. Preoperative resting-state blood oxygen level–dependent fMRI was used to evaluate the relationships between radiographic and histopathologic characteristics. Signal intensity for a blood oxygen level–dependent biomarker was compared with scores of tumor infiltration and microvascular proliferation as well as total cell and neuronal density. Results: Biopsies corresponded to a range of blood oxygen level–dependent signals, ranging from relatively normal ( z = −4.79) to markedly abnormal ( z = 8.84). Total cell density was directly related to blood oxygen level–dependent signal abnormality ( P = .013, R 2 = 0.19), while the neuronal labeling index was inversely related to blood oxygen level–dependent signal abnormality ( P = .016, R 2 = 0.21). The blood oxygen level–dependent signal abnormality was also related to tumor infiltration ( P = .014) and microvascular proliferation ( P = .045). Conclusions: The relationship between local, neoplastic characteristics and a blood oxygen level–dependent biomarker of vascular function suggests that local effects of glioma cell infiltration contribute to vascular dysregulation.

OBJECTIVEExtent of resection is an important prognostic factor in patients undergoing surgery for glioblastoma (GBM). Recent evidence suggests that intravenously administered fluorescein sodium associates with tumor tissue, facilitating safe maximal resection of GBM. In this study, the authors evaluate the safety and utility of intraoperative fluorescein guidance for the prediction of histopathological alteration both in the contrast-enhancing (CE) regions, where this relationship has been established, and into the non-CE (NCE), diffusely infiltrated margins.METHODSThirty-two patients received fluorescein sodium (3 mg/kg) intravenously prior to resection. Fluorescence was intraoperatively visualized using a Zeiss Pentero surgical microscope equipped with a YELLOW 560 filter. Stereotactically localized biopsy specimens were acquired from CE and NCE regions based on preoperative MRI in conjunction with neuronavigation. The fluorescence intensity of these specimens was subjectively classified in real time with subsequent quantitative image analysis, histopathological evaluation of localized biopsy specimens, and radiological volumetric assessment of the extent of resection.RESULTSBright fluorescence was observed in all GBMs and localized to the CE regions and portions of the NCE margins of the tumors, thus serving as a visual guide during resection. Gross-total resection (GTR) was achieved in 84% of the patients with an average resected volume of 95%, and this rate was higher among patients for whom GTR was the surgical goal (GTR achieved in 93.1% of patients, average resected volume of 99.7%). Intraoperative fluorescein staining correlated with histopathological alteration in both CE and NCE regions, with positive predictive values by subjective fluorescence evaluation greater than 96% in NCE regions.CONCLUSIONSIntraoperative administration of fluorescein provides an easily visualized marker for glioma pathology in both CE and NCE regions of GBM. These findings support the use of fluorescein as a microsurgical adjunct for guiding GBM resection to facilitate safe maximal removal.

Immune checkpoint inhibitors have been successful across several tumor types; however, their efficacy has been uncommon and unpredictable in glioblastomas (GBM), where

OBJECTIVE Somatic variants are a recognized cause of epilepsy-associated focal malformations of cortical development (MCD). We hypothesized that somatic variants may underlie a wider range of focal epilepsy, including nonlesional focal epilepsy (NLFE). Through genetic analysis of brain tissue, we evaluated the role of somatic variation in focal epilepsy with and without MCD. METHODS We identified somatic variants through high-depth exome and ultra-high-depth candidate gene sequencing of DNA from epilepsy surgery specimens and leukocytes from 18 individuals with NLFE and 38 with focal MCD. RESULTS We observed somatic variants in 5 cases in SLC35A2, a gene associated with glycosylation defects and rare X-linked epileptic encephalopathies. Nonsynonymous variants in SLC35A2 were detected in resected brain, and absent from leukocytes, in 3 of 18 individuals (17%) with NLFE, 1 female and 2 males, with variant allele frequencies (VAFs) in brain-derived DNA of 2 to 14%. Pathologic evaluation revealed focal cortical dysplasia type Ia (FCD1a) in 2 of the 3 NLFE cases. In the MCD cohort, nonsynonymous variants in SCL35A2 were detected in the brains of 2 males with intractable epilepsy, developmental delay, and magnetic resonance imaging suggesting FCD, with VAFs of 19 to 53%; Evidence for FCD was not observed in either brain tissue specimen. INTERPRETATION We report somatic variants in SLC35A2 as an explanation for a substantial fraction of NLFE, a largely unexplained condition, as well as focal MCD, previously shown to result from somatic mutation but until now only in PI3K-AKT-mTOR pathway genes. Collectively, our findings suggest a larger role than previously recognized for glycosylation defects in the intractable epilepsies. Ann Neurol 2018.

BackgroundDespite extensive molecular characterization, we lack a comprehensive understanding of lineage identity, differentiation, and proliferation in high-grade gliomas (HGGs). We sampled the cellular milieu of HGGs with massively-parallel single-cell RNA-Seq.ResultsWhile HGG cells can resemble glia or even immature neurons and form branched lineage structures, mesenchymal transformation results in unstructured populations. Glioma cells in a subset of mesenchymal tumors lose their neural lineage identity, express inflammatory genes, and co-exist with marked myeloid infiltration, reminiscent of molecular interactions between glioma and immune cells established in animal models. Additionally, we discovered a tight coupling between lineage resemblance and proliferation among malignantly transformed cells. Glioma cells that resemble oligodendrocyte progenitors, which proliferate in the brain, are often found in the cell cycle. Conversely, glioma cells that resemble astrocytes, neuroblasts, and oligodendrocytes, which are non-proliferative in the brain, are generally non-cycling in tumors.ConclusionsThese studies reveal a relationship between cellular identity and proliferation in HGG and distinct population structures that reflects the extent of neural and non-neural lineage resemblance among malignantly transformed cells.

BACKGROUND AND PURPOSE: The complex MR imaging appearance of glioblastoma is a function of underlying histopathologic heterogeneity. A better understanding of these correlations, particularly the influence of infiltrating glioma cells and vasogenic edema on T2 and diffusivity signal in nonenhancing areas, has important implications in the management of these patients. With localized biopsies, the objective of this study was to generate a model capable of predicting cellularity at each voxel within an entire tumor volume as a function of signal intensity, thus providing a means of quantifying tumor infiltration into surrounding brain tissue. MATERIALS AND METHODS: Ninety-one localized biopsies were obtained from 36 patients with glioblastoma. Signal intensities corresponding to these samples were derived from T1-postcontrast subtraction, T2-FLAIR, and ADC sequences by using an automated coregistration algorithm. Cell density was calculated for each specimen by using an automated cell-counting algorithm. Signal intensity was plotted against cell density for each MR image. RESULTS: T2-FLAIR ( r = −0.61) and ADC ( r = −0.63) sequences were inversely correlated with cell density. T1-postcontrast ( r = 0.69) subtraction was directly correlated with cell density. Combining these relationships yielded a multiparametric model with improved correlation ( r = 0.74), suggesting that each sequence offers different and complementary information. CONCLUSIONS: Using localized biopsies, we have generated a model that illustrates a quantitative and significant relationship between MR signal and cell density. Projecting this relationship over the entire tumor volume allows mapping of the intratumoral heterogeneity in both the contrast-enhancing tumor core and nonenhancing margins of glioblastoma and may be used to guide extended surgical resection, localized biopsies, and radiation field mapping.

In the version of this article originally published, the graph in Extended Data Fig. 2c was a duplication of Extended Data Fig. 2b. The correct version of Extended Data Fig. 2c is now available online.

Although immune signaling has emerged as a defining feature of the glioma microenvironment, how the underlying structure of the glioma-infiltrating T-cell population differs from that of the blood from which it originates has been difficult to measure directly in patients. High-throughput sequencing of T-cell receptor (TCR) repertoires (TCRseq) provides a population-wide statistical description of how T cells respond to disease. We have defined immunophenotypes of whole repertoires based on TCRseq of the α- and β-chains from glioma tissue, nonneoplastic brain tissue, and peripheral blood from patients. Using information theory, we partitioned the diversity of these TCR repertoires into that from the distribution of VJ cassette combinations and diversity due to VJ-independent factors, such as selection due to antigen binding. Tumor-infiltrating lymphocytes (TILs) possessed higher VJ-independent diversity than nonneoplastic tissue, stratifying patients according to tumor grade. We found that the VJ-independent components of tumor-associated repertoires diverge more from their corresponding peripheral repertoires than T-cell populations in nonneoplastic brain tissue, particularly for low-grade gliomas. Finally, we identified a “signature” set of TCRs whose use in peripheral blood is associated with patients exhibiting low TIL divergence and is depleted in patients with highly divergent TIL repertoires. This signature is detectable in peripheral blood, and therefore accessible noninvasively. We anticipate that these immunophenotypes will be foundational to monitoring and predicting response to antiglioma vaccines and immunotherapy.

Immunotherapy for glioblastoma (GBM) is the subject of numerous clinical trials due to the potential ability of the adaptive immune response to combat this diffusely infiltrating tumor. However, rational application of immunotherapy to these tumors is challenging because of the peculiar immune privilege of the brain and the molecular heterogeneity of glioma antigens. Little is known about patient-to-patient variability in the potential to generate anti-glioma immune responses of their systemic and local T cell populations. We conducted a foundational study of the population-wide characteristics of both the peripheral blood and local, tissue-infiltrating T lymphocytes (TILs) of glioma patients using TCRseq. Through reverse transcription and pan-repertoire amplification of the TCR-alpha and -beta chains we generated TCRseq libraries from matched surgical tissue and peripheral blood cells. Using peripheral blood samples from primary GBM, low-grade glioma, and non-tumor patients (N = 14), we define new immunophenotypes using statistical metrics based on information theory - specifically, the clonality associated with antigen-binding function in the TCRs of TIL, and the antigen-binding associated divergence of the TIL population from the blood. We found that diversification of this repertoire was characteristic of high-grade tumors, and correlated strongly with the activation of specific immune and inflammatory pathways. Furthermore, we found strong correlation between usage of a subset of TCR sequences in patients' peripheral blood and the divergence of their TIL population from the peripheral repertoire. We anticipate that these immunophenotypes will be key to monitoring and predicting response to anti-glioma vaccines and immunotherapy, and understanding the immunological mechanisms determining their efficacy.

BACKGROUND: The primary treatment of GBM is surgical resection of the contrast enhancing mass, while regions of non-enhancing tumor are left behind, and inevitably gives rise to recurrence. Thus, there is therapeutic significance to understanding the cellular and molecular features of the non-enhancing margins of GBM. Advances in comparative genetic and molecular analyses have uncovered patterns of gene expression and genetic alterations in GBM, and led to the recognition of clinically significant tumor subtypes. However, most previous studies have been based on analysis of tissue that was removed during surgery without documenting the radiographic location. This has precluded the systematic study of intra-tumoral heterogeneity, and its correlation with radiographic regions. We propose a method of sequential image guided biopsies, allowing for specimen localization, intra-tumoral comparative analysis, and the systematic study of infiltrated tissue beyond the borders of contrast enhancement. METHODS: In 77 patients with GBM, multiple stereotactic-guided biopsies were obtained from radiographically distinct regions prior to resection. The radiographic localization of each biopsy was mapped using the 3D intraoperative neuro-navigation system (BrainLab). Sampled regions included areas of contrast enhancement (CE) and adjacent non-enhancing FLAIR+ (NEF+). Samples from each region were divided into pieces processed for RNA-seq and histological analyses. RESULTS: There were significant differences in the cellular density and cellular composition between the CE and NEF+ samples. The CE samples had higher cellularity than NEF+ samples, and were more likely to contain the histological hallmarks of GBM, including vascular proliferation and pseudopalisading necrosis. In contrast, the NEF+ regions showed the histological features of diffusely infiltrating glioma with neoplastic glial cells intermingled with non-neoplastic and reactive cells. RNA-seq showed that the CE and NEF+ regions also had markedly different expression patterns. Comparisons between our image guided biopsies and the TCGA data set showed that the CE regions were enriched for genes associated with the mesenchymal, classical and proneural subtypes while the NEF+ region were enriched for genes of the neural subtype. Further analysis revealed that region-specific and subtype-specific differences in expression patterns were predominantly due to differences in the cellular composition. CONCLUSIONS: Cellular composition is a major determinant of the expression patterns seen at the non-enhancing margins of GBM. The different GBM subtypes show distinct expression patterns that relate the contrast enhancing centers to the non-enhancing margins of the tumors. Understanding these patterns provides a means to infer the molecular and cellular features of glioma tissue left behind after surgery. SECONDARY CATEGORY: Tumor Biology.

Glioblastomas (GBMs) diffusely infiltrate the brain, making complete removal by surgical resection impossible. The mixture of neoplastic and non-neoplastic cells that remain after surgery form the biological context for adjuvant therapeutic intervention and recurrence. We performed RNA-Seq and histological analysis on radiographically guided biopsies taken from different regions of GBM and showed that the tissue contained within the contrast-enhancing (CE) core of tumors have different cellular and molecular compositions compared to tissue from the non-enhancing (NE) margins of tumors. Comparisons with the TCGA dataset showed that the samples from CE regions resembled the Proneural, Classical, or Mesenchymal subtypes of GBM, while the samples from the NE regions predominantly resembled the Neural subtype. Computational deconvolution of the RNA-seq data revealed that contributions from non-neoplastic brain cells significantly influence the expression pattern in the NE samples. Gene ontology showed that the cell type-specific expression patterns were functionally distinct and highly enriched in genes associated with the corresponding cell phenotypes. Comparing the RNA-Seq data from the GBM samples to that of normal brain revealed that the differentially expressed genes are distributed across multiple cell types. Notably, the patterns of cell type-specific alterations varied between the different GBM subtypes; the NE regions of Proneural tumors were enriched in oligodendrocyte progenitor genes while the NE regions of Mesenchymal GBM were enriched in astrocytic and microglial genes. These subtype-specific patterns in the molecular and cellular composition of the infiltrative margins of GBM make it possible to infer key characteristics of the tissue left behind after surgery.

Meeting abstracts Immunotherapy for glioblastoma (GBM) is the subject of numerous clinical trials, given the potential for the adaptive immune response to combat this diffusely infiltrating tumor. However, rational application of immunotherapy to these tumors is challenging because of the peculiar