IDH Wild-type Tumors Research Articles

Dysfunctional dendritic cells limit antigen-specific T cell response in glioma.

Dendritic cells (DC), the most potent professional antigen presenting cells capable of effective cross-presentation, have been demonstrated to license T helper cells to induce antitumor immunity in solid tumors. Specific DC subtypes are recruited to the injured brain by microglial chemokines, locally adapting to distinct transcriptional profiles. In isocitrate dehydrogenase (IDH) type 1 mutant gliomas, monocyte-derived macrophages have recently been shown to display an attenuated intratumoral antigen presentation capacity as consequence of the local accumulation of the oncometabolite R-2-hydroxyglutarate. The functionality and the contribution of DC to the IDH-mutant tumor microenvironment (TME) remains unclear. Frequencies and intratumoral phenotypes of human DC in IDH-wildtype (IDHwt) and -mutant high-grade gliomas are comparatively assessed by transcriptomic and proteomic profiling. DC functionality is investigated in experimental murine glioblastomas expressing the model antigen ovalbumin. Single-cell sequencing-based pseudotime analyses and spectral flow cytometric analyses are used to profile DC states longitudinally. DC are present in primary and recurrent high-grade gliomas and interact with other immune cell types within the TME. In murine glioblastomas, we find an IDH-status-associated major histocompatibility class I-restricted cross-presentation of tumor antigens by DC specifically in the tumor but not in meninges or secondary lymphoid organs of tumor-bearing animals. In single-cell sequencing-based pseudotime and longitudinal spectral flow cytometric analyses, we demonstrate an IDH-status-dependent differential, exclusively microenvironmental education of DC. Glioma-associated DCs are relevantly abundant in human IDHwt and mutant tumors. Glioma IDH mutations result in specifically educated, dysfunctional DCs via paracrine reprogramming of infiltrating monocytes, providing the basis for combinatorial immunotherapy concepts against IDH mutant gliomas.

Conventional MRI features can predict the molecular subtype of adult grade 2–3 intracranial diffuse gliomas

PurposeMolecular biomarkers are important for classifying intracranial gliomas, prompting research into correlating imaging with genotype (“radiogenomics”). A limitation of the existing radiogenomics literature is the paucity of studies specifically characterizing grade 2–3 gliomas into the three key molecular subtypes. Our study investigated the accuracy of multiple different conventional MRI features for genotype prediction.MethodsGrade 2–3 gliomas diagnosed between 2007 and 2013 were identified. Two neuroradiologists independently assessed nine conventional MRI features. Features with better inter-observer agreement (κ ≥ 0.6) proceeded to consensus assessment. MRI features were correlated with genotype, classified as IDH-mutant and 1p/19q-codeleted (IDHmut/1p19qcodel), IDH-mutant and 1p/19q-intact (IDHmut/1p19qint), or IDH-wildtype (IDHwt). For IDHwt tumors, additional molecular markers of glioblastoma were noted.ResultsOne hundred nineteen patients were included. T2-FLAIR mismatch (stratified as > 50%, 25–50%, or < 25%) was the most predictive feature across genotypes (p < 0.001). All 30 tumors with > 50% mismatch were IDHmut/1p19qint, and all seven with 25–50% mismatch. Well-defined margins correlated with IDHmut/1p19qint status on univariate analysis (p < 0.001), but this related to correlation with T2-FLAIR mismatch; there was no longer an association when considering only tumors with < 25% mismatch (p = 0.386). Enhancement (p = 0.001), necrosis (p = 0.002), and hemorrhage (p = 0.027) correlated with IDHwt status (especially “molecular glioblastoma”). Calcification correlated with IDHmut/1p19qcodel status (p = 0.003). A simple, step-wise algorithm incorporating these features, when present, correctly predicted genotype with a positive predictive value 91.8%.ConclusionT2-FLAIR mismatch strongly predicts IDHmut/1p19qint even with a lower threshold of ≥ 25% mismatch and outweighs other features. Secondary features include enhancement, necrosis and hemorrhage (predicting IDHwt, especially “molecular glioblastoma”), and calcification (predicting IDHmut/1p19qcodel).

Method for the Intraoperative Detection of IDH Mutation in Gliomas with Differential Mobility Spectrometry.

Isocitrate dehydrogenase (IDH) mutation status is an important factor for surgical decision-making: patients with IDH-mutated tumors are more likely to have a good long-term prognosis, and thus favor aggressive resection with more survival benefit to gain. Patients with IDH wild-type tumors have generally poorer prognosis and, therefore, conservative resection to avoid neurological deficit is favored. Current histopathological analysis with frozen sections is unable to identify IDH mutation status intraoperatively, and more advanced methods are therefore needed. We examined a novel method suitable for intraoperative IDH mutation identification that is based on the differential mobility spectrometry (DMS) analysis of the tumor. We prospectively obtained tumor samples from 22 patients, including 11 IDH-mutated and 11 IDH wild-type tumors. The tumors were cut in 88 smaller specimens that were analyzed with DMS. With a linear discriminant analysis (LDA) algorithm, the DMS was able to classify tumor samples with 86% classification accuracy, 86% sensitivity, and 85% specificity. Our results show that DMS is able to differentiate IDH-mutated and IDH wild-type tumors with good accuracy in a setting suitable for intraoperative use, which makes it a promising novel solution for neurosurgical practice.

Deciphering of Adult Glioma Vulnerabilities through Expression Pattern Analysis of GABA, Glutamate and Calcium Neurotransmitter Genes.

Adult infiltrating gliomas are highly aggressive tumors of the central nervous system with a dismal prognosis despite intensive multimodal therapy (chemotherapy and/or radiotherapy). In this study, we studied the expression, methylation and interacting miRNA profiles of GABA-, glutamate- and calcium-related genes in 661 adult infiltrating gliomas available through the TCGA database. Neurotransmitter-based unsupervised clustering identified three established glioma molecular subgroups that parallel major World Health Organization glioma subclasses (IDH-wildtype astrocytomas, IDH-mutant astrocytomas, IDH-mutant oligodendroglioma). In addition, this analysis also defined a novel, neurotransmitter-related glioma subgroup (NT-1), mostly comprised of IDH-mutated gliomas and characterized by the overexpression of neurotransmitter-related genes. Lower expression of neurotransmission-related genes was correlated with increased aggressivity in hypomethylated IDH-wildtype tumors. There were also significant differences in the composition of the tumor inflammatory microenvironment between neurotransmission-based tumor categories, with lower estimated pools of M2-phenotype macrophages in NT-1 gliomas. This multi-omics analysis of the neurotransmission expression landscape of TCGA gliomas—which highlights the existence of neurotransmission-based glioma categories with different expression, epigenetic and inflammatory profiles—supports the existence of operational neurotransmitter signaling pathways in adult gliomas. These findings could shed new light on potential vulnerabilities to exploit in future glioma-targeting drug therapies.

Correlation of Intraoperative 5-ALA-Induced Fluorescence Intensity and Preoperative 11C-Methionine PET Uptake in Glioma Surgery.

Simple SummaryIn malignant brain tumor surgery, precise identification of the tumor is essential. 5-Aminolevulinic acid (5-ALA) labels tumor cells with red fluorescence to facilitate tumor resection. On the other hand, the nuclear medicine imaging technique, positron emission tomography with 11C-methionine (MET-PET), can delineate tumors precisely but is not widely available. This study aimed to determine the correlation between intraoperative 5-ALA-induced fluorescence and preoperative MET-PET signals of gliomas. We quantitatively measured the fluorescence intensity from tumor samples and calculated the MET-PET uptake by the tumor. Our study showed that strong tumor fluorescence correlated with high MET-PET uptake and cellular proliferation. Our findings might be valuable to rapidly provide information on tumor biology at the time of surgery in circumstances where MET-PET is inaccessible.Background: 5-Aminolevulinic acid (5-ALA) is widely employed to assist fluorescence-guided surgery for malignant brain tumors. Positron emission tomography with 11C-methionine (MET-PET) represents the activity of brain tumors with precise boundaries but is not readily available. We hypothesized that quantitative 5-ALA-induced fluorescence intensity might correlate with MET-PET uptake in gliomas. Methods: Adult patients with supratentorial astrocytic gliomas who underwent preoperative MET-PET and surgical tumor resection using 5-ALA were enrolled in this prospective study. The regional tumor uptake of MET-PET was expressed as the ratio of standardized uptake volume max to that of the normal contralateral frontal lobe. A spectrometric fluorescence detection system measured tumor specimens’ ex vivo fluorescence intensity at 635 nm. Ki-67 index and IDH mutation status were assessed by histopathological analysis. Use of an antiepileptic drug (AED) and contrast enhancement pattern on MRI were also investigated. Results: Thirty-two patients, mostly with Glioblastoma IDH wild type (46.9%) and anaplastic astrocytoma IDH mutant (21.9%), were analyzed. When the fluorescence intensity was ranked into four groups, the strongest fluorescence group exhibited the highest mean MET-PET uptake and Ki-67 index values. When rearranged into fluorescence Visible or Non-visible groups, the Visible group had significantly higher MET-PET uptake and Ki-67 index compared to the Non-visible group. Contrast enhancement on MRI and IDH wild type tumors were more frequent among the Visible group. AED use did not correlate with 5-ALA-induced fluorescence intensity. Conclusions: In astrocytic glioma surgery, visible 5-ALA-induced fluorescence correlated with high MET-PET uptake, along with a high Ki-67 index.

In vivo efficacy of decitabine as a natural killer cell-mediated immunotherapy against isocitrate dehydrogenase mutant gliomas.

Isocitrate dehydrogenase (IDH) mutations are found in more than 80% of low-grade gliomas and in the majority of secondary glioblastomas. IDH mutation (IDHmut) leads to aberrant production of an oncogenic metabolite that promotes epigenetic dysregulation by inducing hypermethylation to suppress transcription of various tumor suppressor genes. Hypermethylation in IDHmut gliomas leads to transcriptional repression of NKG2D ligands, especially UL16-binding protein (ULBP)-1 and ULBP-3, and subsequent evasion of natural killer (NK) cell-mediated lysis. The demethylating agent 5-aza-2'deoxycytodine (decitabine [DAC]) is a DNA methyltransferase 1 inhibitor that prevents hypermethylation and is capable of restoring NKG2D ligand expression in IDHmut gliomas to resensitize them to NK cells. Given its capacity for sustained epigenetic reprogramming, the authors hypothesized that DCA would be an effective immunotherapeutic agent in treating IDHmut gliomas in an NK cell-dependent manner by upregulating epigenetically repressed activating NKG2D ligands in IDHmut tumors. In this study, the authors sought to use a glioma stem cell, preclinical animal model to determine the efficacy of DAC in IDHmut and IDH wild-type (IDHwt) tumors, and to characterize whether the activity of DAC in gliomas is dependent on NK cell function. Xenograft models of IDHwt and IDHmut gliomas were established in athymic-nude mice. When tumors were grossly visible and palpable, mice were treated with either DCA or dimethylsulfoxide intraperitoneally every 7 days. Tumor sizes were measured every 2 to 3 days. After the animals were euthanized, xenografts were harvested and analyzed for the following: tumor expression of NKG2D ligands, tumor susceptibility to human and murine NK cells, immunohistochemistry for NK infiltration, and tumor-infiltrating lymphocyte characterization. DAC significantly inhibited the growth of IDHmut xenografts in the athymic nude mice. This effect was abrogated with NK cell depletion. Ex vivo analysis of tumor cells from harvested xenografts confirmed that DAC increased NKG2D ligand ULBP-1 and ULBP-3 expressions, and enhanced susceptibility to lysis of both human and murine IDHmut glial cells with corresponding NK cells. Immunohistochemical analysis of the xenografts indicated that DCA-treated IDHmut gliomas had a greater level of NK infiltration into the tumor compared with the negative control. Finally, DCA radically altered the tumor-infiltrating lymphocyte landscape of IDHmut glioma xenografts by increasing NK cells, dendritic cells, and M1 macrophages, while decreasing suppressive monocyte infiltration. DCA displayed novel immunotherapeutic functions in IDHmut gliomas. This effect was critically dependent on NK cells. Additionally, DCA significantly altered the tumor immune landscape in IDHmut gliomas from suppressive to proinflammatory.

The diagnostic value of contrast enhancement on MRI in diffuse and anaplastic gliomas.

We evaluated differentiations in gadolinium contrast enhancement (CE) between low-grade WHO °II and high-grade WHO °III gliomas in conventional MRI, which have been repeatedly questioned. Ninety-nine patients, who underwent first resection of WHO°II and °III gliomas, were retrospectively retrieved from a prospective database. The quantitative metric volume of Gd-CE in T1-weighted pre-operative MRI was measured using volumetric segmentation. The OR to detect CE in anaplastic gliomas was seven times higher than that in diffuse gliomas (CI95% 2.8-17.2,p<0.0001). No CE was seen in 50% (8/16) of focal anaplastic and in 28% (10/36) of entirely anaplastic gliomas. CE was present in 21% (10/47) of diffuse gliomas. Anaplasia correlated with a larger CE volume (r=0.49, p<0.0001) and provided additional 4 cm3of CE volume compared to entirely diffuse tumors. The OR to have CE was 3.6 times for IDH1 wild-type tumors (CI95% 1.3-10.2, p=0.05) and 4.8 for tumors with ATRX expression (CI95% 1.3-17.2,p=0.05). In all sub-groups, at least a quarter of cases showed no CE at all and there were cases with present CE. CE is associated with higher odds of unfavorable prognostic features like anaplasia, wild-type IDH1 and retained ATRX. There was no CE in one-fourth of anaplastic gliomas and half of gliomas with focal anaplasia.

Rapid early progression (REP) of glioblastoma is an independent negative prognostic factor: Results from a systematic review and meta-analysis.

BackgroundIn patients with newly diagnosed glioblastoma, rapid early progression (REP) refers to tumor regrowth between surgery and postoperative chemoradiotherapy. This systematic review and meta-analysis appraised previously published data on REP to better characterize and understand it.MethodsSystematic searches of MEDLINE, EMBASE and the Cochrane database from inception to October 21, 2021. Studies describing the incidence of REP—tumor growth between the postoperative MRI scan and pre-radiotherapy MRI scan in newly diagnosed glioblastoma were included. The primary outcome was REP incidence.ResultsFrom 1590 search results, 9 studies were included with 716 patients. The median age was 56.9 years (IQR 54.0–58.8 y). There was a male predominance with a median male-to-female ratio of 1.4 (IQR 1.1–1.5). The median number of days between MRI scans was 34 days (IQR 18–45 days). The mean incidence rate of REP was 45.9% (range 19.3%–72.0%) and significantly lower in studies employing functional imaging to define REP (P < .001). REP/non-REP groups were comparable with respect to age (P = .99), gender (P = .33) and time between scans (P = .81). REP was associated with shortened overall survival (HR 1.78, 95% CI 1.30–2.43, P < .001), shortened progression-free survival (HR 1.78, 95% CI 1.30–2.43, P < .001), subtotal resection (OR 6.96, 95% CI 4.51–10.73, P < .001) and IDH wild-type versus mutant tumors (OR 0.20, 95% CI 0.02–0.38, P = .03). MGMT promoter methylation was not associated with REP (OR 1.29, 95% CI 0.72–2.28, P = .39).ConclusionsREP occurs in almost half of patients with newly diagnosed glioblastoma and has a strongly negative prognostic effect. Future studies should investigate its biology and effective treatment strategies.

Noninvasive differentiation of molecular subtypes of adult nonenhancing glioma using MRI perfusion and diffusion parameters.

BackgroundNonenhancing glioma typically have a favorable outcome, but approximately 19–44% have a highly aggressive course due to a glioblastoma genetic profile. The aim of this retrospective study is to use physiological MRI parameters of both perfusion and diffusion to distinguish the molecular profiles of glioma without enhancement at presentation.MethodsNinety-nine patients with nonenhancing glioma were included, in whom molecular status (including 1p/19q codeletion status and IDH mutation) and preoperative MRI (T2w/FLAIR, dynamic susceptibility-weighted, and diffusion-weighted imaging) were available. Tumors were segmented semiautomatically using ITK-SNAP to derive whole tumor histograms of relative Cerebral Blood Volume (rCBV) and Apparent Diffusion Coefficient (ADC). Tumors were divided into three clinically relevant molecular profiles: IDH mutation (IDHmt) with (n = 40) or without (n = 41) 1p/19q codeletion, and (n = 18) IDH-wildtype (IDHwt). ANOVA, Kruskal-Wallis, and Chi-Square analyses were performed using SPSS.ResultsrCBV (mean, median, 75th and 85th percentile) and ADC (mean, median, 15th and 25th percentile) showed significant differences across molecular profiles (P < .01). Posthoc analyses revealed that IDHwt and IDHmt 1p/19q codeleted tumors showed significantly higher rCBV compared to IDHmt 1p/19q intact tumors: mean rCBV (mean, SD) 1.46 (0.59) and 1.35 (0.39) versus 1.08 (0.31), P < .05. Also, IDHwt tumors showed significantly lower ADC compared to IDHmt 1p/19q codeleted and IDHmt 1p/19q intact tumors: mean ADC (mean, SD) 1.13 (0.23) versus 1.27 (0.15) and 1.45 (0.20), P < .001).ConclusionsA combination of low ADC and high rCBV, reflecting high cellularity and high perfusion respectively, separates IDHwt from in particular IDHmt 1p/19q intact glioma.

Tumor Mutation Burden, Expressed Neoantigens and the Immune Microenvironment in Diffuse Gliomas.

Simple SummaryTumor mutation burden (TMB) has shown promise as a biomarker for immune checkpoint blockade therapy in some cancers, but not consistently in gliomas. The goal of our study was to systematically investigate the association between TMB, expressed neoantigens, and the tumor immune microenvironment in IDH-mutant and IDH-wildtype gliomas, which are two types of biologically distinct gliomas. We demonstrated that TMB positively correlated with expressed neoantigens, but inversely correlated with immune score in IDH-wildtype tumors but showed no correlation in IDH-mutant tumors. The antigen processing and presenting (APP) score may have potential as a clinical biomarker to predict immune therapy response in gliomas. Lastly, 19% of patients had pathogenic or likely pathogenic germline mutations, primarily in DNA damage repair genes.Background: A consistent correlation between tumor mutation burden (TMB) and tumor immune microenvironment has not been observed in gliomas as in other cancers. Methods: Driver germline and somatic mutations, TMB, neoantigen, and immune cell signatures were analyzed using whole exome sequencing (WES) and transcriptome sequencing of tumor and WES of matched germline DNA in a cohort of 66 glioma samples (44 IDH-mutant and 22 IDH-wildtype). Results: Fourteen samples revealed a hypermutator phenotype (HMP). Eight pathogenic (P) or likely pathogenic (LP) germline variants were detected in 9 (19%) patients. Six of these 8 genes were DNA damage repair genes. P/LP germline variants were found in 22% of IDH-mutant gliomas and 12.5% of IDH-wildtype gliomas (p = 0.7). TMB was correlated with expressed neoantigen but showed an inverse correlation with immune score (R = −0.46, p = 0.03) in IDH-wildtype tumors and no correlation in IDH-mutant tumors. The Antigen Processing and Presentation (APP) score correlated with immune score and was surprisingly higher in NHMP versus HMP samples in IDH-wildtype gliomas, but higher in HMP versus NHMP in IDH-mutant gliomas. Conclusion: TMB was inversely correlated with immune score in IDH-wildtype gliomas and showed no correlation in IDH-mutant tumors. APP was correlated with immune score and may be further investigated as a biomarker for response to immunotherapy in gliomas. Studies of germline variants in a larger glioma cohort are warranted.

Clinical, Morphological, and Molecular Study of Diffuse WHO Grade II and III Astrocytomas: A Retrospective Analysis from a Single Tertiary Care Institute

Abstract Introduction Astrocytomas are the most common gliomas, classified on the basis of grade and IDH mutation status according to the World Health Organization (WHO) 2016 update. IDH mutations are seen in 70 to 80% of diffuse grade II and III astrocytomas and are associated with better outcome. They serve as predictive biomarker in IDH-targeted therapies such as small-molecule inhibitors or vaccines. Objective The aim of this study was to analyze the clinical, morphological, immunohistochemical, and molecular genetic characteristics of diffuse astrocytoma (DA: grades II and III). The IDH mutant and wild-type tumors are compared and contrasted with survival analysis on follow-up. Materials and Methods This was a retrospective study conducted on surgically resected tumor specimens. The hematoxylin and eosin-stained slides were examined for histologic features. Immunohistochemistry (IHC) was performed using IDH1R132H, ATRX, p53, and Ki67. All cases of negative immunohistochemical expression of IDH1R132H were subjected to IDH1 mutation analysis by Sanger sequencing. Overall survival was estimated by the Kaplan-Meier method using the log-rank (Mantel–Cox) test. Results The study included 51 cases of DA in the age of 17 to 66 years, mean ± standard deviation was 35.5 ± 9.7 years, and male:female ratio was 2:1.The IDH1R132H cytoplasmic immunopositivity was seen in 36 cases (70.5%), of which 63.6% were of grade II and 72.5% were of grade III. ATRX showed loss of expression in 50 cases (98%), and p53 showed diffuse strong immunohistochemical expression in all the cases of IDH mutant tumors. The difference in the age at presentation for IDH mutant (32.5 years) and wild type tumors (38 years) was statistically significant. Median survival was 55.3 months and 22.2 months in of IDH mutant and wild type cases, respectively. Conclusion IHC and sequencing for IDH mutations is helpful in making an integrated diagnosis and classifying definite molecular subgroups of astrocytic tumors. Mutations in IDH core-elate with survival. IDH mutant tumors showed longer survival duration and are good prognostic indicators.

CBIO-08. ENDOGENOUS DNA DOUBLE STRAND BREAKS ACTIVATE HETEROGENOUS DNA DAMAGE SIGNALING IN IDH1/2 MUTANT GLIOMAS

Abstract Isocitrate dehydrogenase 1/2 (IDH1/2) mutations are common in astrocytic glioma and are frequently coupled with TP53 and ATRX mutations. Collectively, these alterations cause genomic instability leading to high basal DNA double strand breaks (DSBs). Understanding how IDH/TP53/ATRX mutant cells process endogenous DSBs may help exploit inhibitors of DNA damage response (DDR) for the treatment of patients with IDH mutant gliomas. Through systematic effort to uncover the mechanisms involved in repair of endogenous DSBs in IDH1/2 mutant GBMs, we have discovered that high basal phosphorylated DNA-PK (p-DNA-PK) was characteristic of an IDH1/TP53/ATRX mutant GBM164 patient derived xenograft (PDX) but not in another IDH1 mutant GBM196 PDX. Immunofluorescence (IF) studies in patient specimen from which GBM164 was derived showed that p-DNA-PK co-localized with g-H2AX, 53BP1 or H4K20me2 (but not p-RPA) the known surrogates of DSBs. In contrast, p-DNA-PK was absent in the patient specimen from which GBM196 was derived, which otherwise had equally intense g-H2AX immunostaining colocalized with p-RPA. An independent IF study involving 11 IDH1 wild-type (WT) and 11 IDH1 mutant GBM patient samples, the p-DNA-PK was observed in 3 (27%) of 11 IDH1 mutant samples while IDH1 WT tumors were negative for p-DNA-PK. A telomere specific fluorescence in situ hybridization (Tel-FISH) confirmed elevated alternative lengthening of telomere (ALT) activity in GBM196 (but not in GBM164) indicative of HR proficiency. Consistently, HR related genes, including BRCA1 and MRE11A, were found upregulated in ALT-positive GBM196 as compared to those in GBM164. Interestingly, ALT+ GBM196 cells were highly vulnerable to inhibitors of ATM and ATR pathways. In conclusion, IDH1/TP53/ATRX mutant gliomas can be subdivided into HR-mediated ALT-positive group, which repairs the endogenous DSBs by HR (e.g. GBM196) and an ALT-negative/p-DNA-PK group, which repairs DSBs by c-NHEJ (e.g. GBM164) and this subdivision can be developed as a prescient biomarker of sensitivity to DDR inhibitors.

NIMG-25. IMPROVING THE NONINVASIVE CLASSIFICATION OF GLIOMA GENETIC SUBTYPE WITH DEEP LEARNING AND DIFFUSION-WEIGHTED IMAGING

Abstract In contrast to the WHO 2016 guidelines that use genetic alterations to further stratify patients within a designated grade, new recommendations suggest that IDH mutation status, followed by 1p19q-codeletion, should be used before grade when differentiating gliomas. Although most gliomas will be resected and their tissue evaluated with genetic profiling, non-invasive characterization of genetic subgroup can benefit patients where surgery is not otherwise advised or a fast turn-around is required for clinical trial eligibility. Prior studies have demonstrated the utility of using anatomical images and deep learning to distinguish either IDH-mutant from IDH-wildtype tumors or 1p19q-codeleted from non-codeleted lesions separately, but not combined or using the most recent recommendations for stratification. The goal of this study was to evaluate the effects of training strategy and incorporation of Apparent Diffusion Coefficient (ADC) maps from diffusion-weighted imaging on predicting new genetic subgroups with deep learning. Using 414 patients with newly-diagnosed glioma (split 285/50/49 training/validation/test) and optimized training hyperparameters, we found that a 3-class approach with T1-post-contrast, T2-FLAIR, and ADC maps as inputs achieved the best performance for molecular subgroup classification, with overall accuracies of 86.0%[CI:0.839,1.0], 80.0%[CI:0.720,1.0], and 85.7%[CI:0.771,1.0] on training, validation, and test sets, respectively, and final test class accuracies of 95.2%(IDH-wildtype), 88.9%(IDH-mutated,1p19qintact), and 60%(IDHmutated,1p19q-codeleted). Creating an RGB-color image from 3 MRI images and applying transfer learning with a residual network architecture pretrained on ImageNet resulted in an 8% averaged increase in overall accuracy. Although classifying both IDH and 1p19q mutations together was overall advantageous compared with a tiered structure that first classified IDH mutational status, the 2-tiered approach better generalized to an independent multi-site dataset when only anatomical images were used. Including biologically relevant ADC images improved model generalization to our test set regardless of modeling approach, highlighting the utility of incorporating diffusion-weighted imaging in future multi-site analyses of molecular subgroup.

NIMG-53. RATIO OF T1-WEIGHTED TO T2-WEIGHTED SIGNAL INTENSITY AND IDH MUTATION IN GLIOMA

Abstract INTRODUCTION Identifying IDH mutation status before treatment is essential for Lower-grade glioma (LrGG) treatment. We have previously revealed that IDH mutated LrGG consists of tumor tissues with significantly longer T1 and T2 relaxation time and is a useful radiological feature to identify IDH mutation status. The ratio of T1-weighted to T2-weighted signal intensity (rT1/T2) is a way to retrieve semi-quantitative relaxation time information of the tissue bypassing the need to perform relaxometry. This investigation aimed to elucidate the correlation between rT1/T2 and T1-, T2-relaxation time (-relax) in glioma tissue and to explore the possibility of rT1/T2 as a radiological surrogate marker to identify IDH mutation status in LrGG. MATERIALS AND METHODS We analyzed 8 LrGGs (IDHwt:4, IDHmt:2, IDHmt&amp;1p19q-CODEL:2) in which relaxometry was performed. rT1/T2 maps were reconstructed as described in previous literature. Regions-of-interest were designed based on T2WI and FLAIR. The correlations between rT1/T2 and T1- and T2-relax were analyzed. Furthermore, We also investigated the correlation of IDH mutation status and rT1/T2. RESULTS 106,488 voxels were analyzed. The correlation between rT1/T2 and T1- and T2-relax were rT1/T2=1.6e-0.0003T1-relax and rT1/T2=1.2e-0.002T2-relax (R=0.77 and 0.70). rT1/T2 of IDH-wildtype tumor was significantly higher than that of IDH-mutant tumor (1.0 vs. 0.75, p&amp;lt; 0.0001). Voxel-wise analysis of rT1/T2 map was able to discriminate IDH-wildtype tumor from the mutant tumor with an AUC of 0.82. CONCLUSIONS rT1/T2, which can be calculated from MRI acquired during routine clinical practice, is a promising radiological surrogate marker to identify IDH mutation status in LrGG.

IMMU-14. REVEALING THE MANY MYELOID STATES IN HUMAN BRAIN TUMORS AND WAYS TO PERTURB THEM

Abstract Recent breakthroughs in immunotherapy have revolutionized treatment for many types of cancer, but unfortunately trials of these therapies have failed to provide meaningful life-prolonging benefit for brain tumor patients, potentially due to abundant immunosuppressive myeloid cells in the tumor. Our ultimate goal is to reprogram immunosuppressive tumor associated myeloid cells to an antitumor state to enable effective immunotherapy. Towards this goal, we have deeply characterized the immune microenvironment of more than 50 primary high and low grade gliomas using high-throughput single-cell RNA-sequencing to reveal recurrent myeloid cell states and immunosuppressive programs across IDH1 wild-type and mutant tumors. We have also established a brain tumor organoid model from primary patient tissue that maintains all of the tumor microenvironment, including myeloid and other immune cells. We utilize the this model to functionally test data-driven reprogramming strategies and understand how they impact the states of tumor and immune cells in the ex vivo human tumor microenvironment.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Application of Cluster Analysis of Time Evolution for Magnetic Resonance Imaging -Derived Oxygen Extraction Fraction Mapping: A Promising Strategy for the Genetic Profile Prediction and Grading of Glioma.

Background: The intratumoral heterogeneity of oxygen metabolism and angiogenesis are core hallmarks of glioma, unveiling that genetic aberrations associated with magnetic resonance imaging (MRI) phenotypes may aid in the diagnosis and treatment of glioma.Objective: To explore the predictability of MRI-based oxygen extraction fraction (OEF) mapping using cluster analysis of time evolution (CAT) for genetic profiling and glioma grading.Methods: Ninety-one patients with histopathologically confirmed glioma were examined with CAT for quantitative susceptibility mapping and quantitative blood oxygen level–dependent magnitude-based OEF mapping and dynamic contrast-enhanced (DCE) MRI. Imaging biomarkers, including oxygen metabolism (OEF) and angiogenesis [volume transfer constant, cerebral blood volume (CBV), and cerebral blood flow], were investigated to predict IDH mutation, O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation status, receptor tyrosine kinase (RTK) subgroup, and differentiation of glioblastoma (GBM) vs. lower-grade glioma (LGG). The corresponding DNA sequencing was also obtained. Results were compared with DCE-MRI using receiver operating characteristic (ROC) analysis.Results: IDH1-mutated LGGs exhibited significantly lower OEF and hypoperfusion than IDH wild-type tumors (all p < 0.01). OEF and perfusion metrics showed a tendency toward higher values in MGMT unmethylated GBM, but only OEF retained significance (p = 0.01). Relative prevalence of RTK alterations was associated with increased OEF (p = 0.003) and perfusion values (p < 0.05). ROC analysis suggested OEF achieved best performance for IDH mutation detection [area under the curve (AUC) = 0.828]. None of the investigated parameters enabled prediction of MGMT status except OEF with a moderate AUC of 0.784. Predictive value for RTK subgroup was acceptable by using OEF (AUC = 0.764) and CBV (AUC = 0.754). OEF and perfusion metrics demonstrated excellent performance in glioma grading. Moreover, mutational landscape revealed hypoxia or angiogenesis-relevant gene signatures were associated with specific imaging phenotypes.Conclusion: CAT for MRI-based OEF mapping is a promising technology for oxygen measurement and along with perfusion MRI can predict genetic profiles and tumor grade in a non-invasive and clinically relevant manner.Clinical Impact: Physiological imaging provides an in vivo portrait of genetic alterations in glioma and offers a potential strategy for non-invasively selecting patients for individualized therapies.

Identification of Gender-Specific Molecular Differences in Glioblastoma (GBM) and Low-Grade Glioma (LGG) by the Analysis of Large Transcriptomic and Epigenomic Datasets.

Differences in the incidence and outcome of glioma between males and females are well known, being more striking for glioblastoma (GB) than low-grade glioma (LGG). The extensive and well-annotated data in publicly available databases enable us to analyze the molecular basis of these differences at a global level. Here, we have analyzed The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) databases to identify molecular indicators for these gender-based differences by different methods. Based on the nature of data available/accessible, the transcriptomic profile was studied in TCGA by using DeSeq2 and in CGGA by T-test, after correction based. Only IDH1 wild-type tumors were studied in CGGA. Using weighted gene co-expression network analysis (WGCNA), network analysis was done, followed by the assessment of modular differential connectivity. Differentially affected signaling pathways were identified. The gender-based effects of differentially expressed genes on survival were determined. DNA methylation was studied as an indicator of gender-based epigenetic differences. The results clearly showed gender-based differences in both GB and LGG, whatever method or database was used. While there were differences in the results obtained between databases and methods used, some major signaling pathways such as Wnt signaling and pathways involved in immune processes and the adaptive immune response were common to different assessments. There was also a differential gender-based influence of several genes on survival. Also, the autosomal genes NOX, FRG1BP, and AL354714.2 and X-linked genes such as PUDP, KDM6A, DDX3X, and SYAP1 had differential DNA methylation and expression profile in male and female GB, while for LGG, these included autosomal genes such as CNIH3 and ANKRD11 and X-linked genes such as KDM6A, MAOB, and EIF2S3. Some, such as FGF13 and DDX3X, have earlier been shown to have a role in tumor behavior, though their dimorphic effects in males and females have not been identified. Our study thus identifies several crucial differences between male and female glioma, which could be validated further. It also highlights that molecular studies without consideration of gender can obscure critical elements of biology and emphasizes the importance of parallel but separate analyses of male and female glioma.

CLRM-13. INTRAOPERATIVE MICRODIALYSIS: GLIOMA INTELLIGENCE FROM BEHIND ENEMY LINES

INTRODUCTIONGliomas present a formidable challenge for translational progress. Heterogeneity within and between tumors may demand empirically individualized and adaptive paradigms requiring rapid mechanistic feedback. We asked if tumor-associated metabolic biomarkers from glioma extracellular fluid could impart mechanistic “intelligence” reflecting intra- and inter-tumoral heterogeneity.METHODSFive live human gliomas (2 oligos; 2 IDH-WT GBMs; 1 IDH-mutant GBM), were evaluated in situ with high molecular weight (100kDA) intraoperative microdialysis using 3 disparately placed catheters. Isotonic 3% dextran perfusate was collected in 20 min (40mL) aliquots. CSF samples (n=21) were additionally evaluated from these and other patients with diverse brain tumors. The IDH-mutant glioma-associated oncometabolite D2-hydroxyglutarate (D2-HG) was quantified with targeted Liquid Chromotography-Mass Spectrometry (LC-MS). Over 200 metabolites were further evaluated via untargeted LC-MS using the Metabolon platform. Correlation, clustering, ROC and enrichment analyses were employed to identify correlations within and between patient samples.RESULTSCSF samples from patients with IDH-mutant gliomas contained over twenty-fold higher levels of D2-HG (median 4.1 mM, range 1.6-13.2, n=7) compared to those from IDH-wild type tumors (median 0.19 mM; range 0.89-0.35, n=14). Microdialysate from IDH-mutant gliomas contained 10-953mM D2-HG, 9-63x higher than paired CSF samples. Interestingly, IDH status failed to predict the global metabolic signature of microdialysate. Microdialysate samples clustered into 2 major metabolic phenotype clusters with IDH-WT and IDH-mutant gliomas in each cluster. A superimposed metabolic signature distinguishing enhancing from non-enhancing tumor, was conserved in both patient clusters. Amino acid and carnitine metabolism predominated in microdialysate signatures. TCA cycle and Warburg-associated metabolites were differentially enriched in CSF samples after prior therapy independent of tumor burden.CONCLUSIONSIntra-operative micro-dialysis may complement currently available “intelligence” regarding the phenotype, burden, and metabolism of live human gliomas and is feasible within standard-of-care surgical procedures. Future work will evaluate utility for pharmacodynamic feedback following novel early phase candidate therapies.

Raman Spectroscopy and Machine Learning for IDH Genotyping of Unprocessed Glioma Biopsies.

Simple SummaryIsocitrate dehydrogenase (IDH) mutation is one of the most important prognostic markers in glioma tumors. Raman spectroscopy (RS) is an optical technique with great potential in intraoperative molecular diagnosis and surgical guidance. We analyzed RS’s ability to detect the IDH mutation onto unprocessed glioma biopsies. A total of 2073 Raman spectra were extracted from 38 tumor specimens. From the 103 Raman shifts screened, we identified 52 shifts (related to lipids, collagen, DNA and cholesterol/phospholipids) with the highest performance in the distinction of the two groups. We described 18 shifts never used before for IDH detection with RS in fresh or frozen samples. We were able to distinguish between IDH-mutated and IDH-wild-type tumors with an accuracy and precision of 87%. RS showed optimal accuracy and precision in discriminating IDH-mutated glioma from IDH-wild-type tumors ex-vivo onto fresh surgical specimens.Isocitrate dehydrogenase (IDH) mutational status is pivotal in the management of gliomas. Patients with IDH-mutated (IDH-MUT) tumors have a better prognosis and benefit more from extended surgical resection than IDH wild-type (IDH-WT). Raman spectroscopy (RS) is a minimally invasive optical technique with great potential for intraoperative diagnosis. We evaluated the RS’s ability to characterize the IDH mutational status onto unprocessed glioma biopsies. We extracted 2073 Raman spectra from thirty-eight unprocessed samples. The classification performance was assessed using the eXtreme Gradient Boosted trees (XGB) and Support Vector Machine with Radial Basis Function kernel (RBF-SVM). Measured Raman spectra displayed differences between IDH-MUT and IDH-WT tumor tissue. From the 103 Raman shifts screened as input features, the cross-validation loop identified 52 shifts with the highest performance in the distinction of the two groups. Raman analysis showed differences in spectral features of lipids, collagen, DNA and cholesterol/phospholipids. We were able to distinguish between IDH-MUT and IDH-WT tumors with an accuracy and precision of 87%. RS is a valuable and accurate tool for characterizing the mutational status of IDH mutation in unprocessed glioma samples. This study improves RS knowledge for future personalized surgical strategy or in situ target therapies for glioma tumors.