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Supplementary Table from Natural Coevolution of Tumor and Immunoenvironment in Glioblastoma

Supplementary Data from Natural Coevolution of Tumor and Immunoenvironment in Glioblastoma

Supplementary Figure from Natural Coevolution of Tumor and Immunoenvironment in Glioblastoma

Isocitrate dehydrogenase (IDH) wild-type glioblastoma (GBM) has a dismal prognosis. A better understanding of tumor evolution holds the key to developing more effective treatment. Here we study GBM's natural evolutionary trajectory by using rare multifocal samples. We sequenced 61,062 single cells from eight multifocal IDH wild-type primary GBMs and defined a natural evolution signature (NES) of the tumor. We show that the NES significantly associates with the activation of transcription factors that regulate brain development, including MYBL2 and FOSL2. Hypoxia is involved in inducing NES transition potentially via activation of the HIF1A–FOSL2 axis. High-NES tumor cells could recruit and polarize bone marrow–derived macrophages through activation of the FOSL2–ANXA1–FPR1/3 axis. These polarized macrophages can efficiently suppress T-cell activity and accelerate NES transition in tumor cells. Moreover, the polarized macrophages could upregulate CCL2 to induce tumor cell migration.Significance:GBM progression could be induced by hypoxia via the HIF1A–FOSL2 axis. Tumor-derived ANXA1 is associated with recruitment and polarization of bone marrow–derived macrophages to suppress the immunoenvironment. The polarized macrophages promote tumor cell NES transition and migration.This article is highlighted in the In This Issue feature, p. 2711

Background The tumor microenvironment (TME) plays an important role in cancer progression. We investigated TME-specific gene signatures and established a risk score to predict the outcome of medulloblastoma (MB) patients. Methods We evaluated TME parameters of 240 MB patients at Beijing Tiantan Hospital Capital Medical University with the ESTIMATE algorithm. Co-expression network analysis of differentially expressed and weighted genes (WGCNA) was used to identify intersecting genes. Using least absolute shrinkage and selection operator regression and backward stepwise regression we obtained a TME-associated risk score (TMErisk) based on eight prognostic gene signatures (CEBPB, OLFML2B, GGTA1, GZMA, TCIM, OLFML3, NAT1, and CD1C), verified in a GEO dataset (GSE85217). Results The correlation between TMErisk and TME, immune checkpoint, mRNAsi, and tumor mutation burden (TMB) was analyzed. MB patients’ response to immunotherapy was evaluated using immune-phenoscore (IPS) and drug sensitivity. A high TMErisk score indicated a worse overall survival. TMErisk scores were negatively correlated with immune cells, immune checkpoints, and human leukocyte antigens. TMErisk scores correlated significantly negatively with TMB and IPS for specific molecular subtypes. Tumor mRNAsi was associated with TME-risk. Conclusions A prognostic model based on TME-specific gene signatures may be used as a biomarker for evaluating prognosis and predicting response to immunotherapy in MB patients.

Background: In previous studies, patients with intracranial germ cell tumour (iGCT) with pure choriocarcinoma or mixed germ cell tumours with choriocarcinoma element showed similar dismal prognoses, with median overall survival (OS) of 22 months and 1-year survival rate of approximately 60%. However, these conclusions need to be updated because radiotherapy, which is the milestone for this disease, was not applied in a number of patients. Methods: Clinical data of patients with iGCTs with histologically confirmed choriocarcinoma element or beta-human chorionic gonadotropin (β-HCG) > 500 IU/L were collected from the archives of our institution and retrospectively studied. Results: A total of 76 patients were eligible for this study. In terms of the initial treatment, 11 patients underwent surgery, four patients received radiotherapy, and 61 patients received chemotherapy. Except for two early deaths, all patients received radiotherapy (craniospinal irradiation [CSI], n=23; non-CSI, n=51). The median follow-up duration for the entire series was 63 months (range, 6–188 months). The 5-year event-free survival (EFS) and OS rates were 81.5% and 84.1%, respectively. Among patients who did not have early death or progressive disease after induction chemotherapy, multivariate analysis revealed that chemotherapy cycles (>4 vs. ≤4) (hazard ratio [HR] for EFS 0.144, p=0.020; HR for OS 0.111, p=0.028) and β-HCG levels (>3000 IU/L vs. ≤3000 IU/L) (HR for EFS 4.342, p=0.059; HR for OS 6.614, p=0.033) were independent factors for survival. Radiation volume (non-CSI vs. CSI) was not proven to be a prognostic factor for either EFS or OS (HR for EFS 1.902, p=0.59; HR for OS 2.425, p=0.49). Conclusions: Patients with iGCTs with choriocarcinoma element or β-HCG >500 IU/L showed improved survival with radiotherapy-based treatments. Additional chemotherapy cycles could result in additional survival benefits. Patients with β-HCG level >3000 IU/L had poorer prognosis.

BACKGROUND Basal ganglia germ cell tumors (BGGCTs) are rare intracranial germ cell tumors (iGCTs) that often presents with cognitive impairment. OBJECTIVE To assess structural brain plasticity in the presence of unilateral basal ganglia germ cell tumors (BGGCTs), and the correlation between gray matter volume (GMV) changes and cognitive tests. MATERIALS AND METHODS We applied voxel-based morphometry (VBM) to structural magnetic resonance imaging (MRI) scans to compare a sample of 41 patients with BGGCTs in the left (n = 22) or right (n = 19) and a sample of 16 patients as control group using a two-sample t-test, correcting for family-wise-errors. A battery of cognitive tests was administered to all BGGCTs patients prior to MRI. We used Pearson correlation analysis to assess the correlation between cognitive test scores and GMV changes. RESULTS In patients with left BGGCTs, whole-brain VBM analysis revealed a large cluster of voxels reflecting an increase in GMV in the left parahippocampal region (k = 529 voxels, T = 4.18, p

Glioma of the brain is a kind of tumor originating from neuroglial cells. It is the most common primary intracranial tumor, accounting for ~ 30% of all central nervous system tumors and 80% of malignant brain tumors. Glioma is characterized by high disability and recurrence rates. The disease seriously threatens the life of patients, affects their quality of life, and brings a heavy economic and psychological burden to patients, families, and society.With the progression of molecular genetic testing technology and the completion of various clinical trials, the classification scheme for glioma is increasingly well established. Diagnosis and treatment regimens, including traditional and new regimens, are becoming increasingly specialized and standardized. The purpose is to develop a clinical diagnosis and treatment guideline for glioma in the Chinese population suitable for Chinese doctors and the general population based on domestic and international glioma research progress. Thus, domestic practitioners in the field can obtain current information and provide better service to patients with glioma, promoting the development of domestic clinical medicine and basic research on glioma.

BACKGROUND In the previous studies, intracranial germ cell tumor (iGCTs) patients with pure choriocarcinoma or mixed germ cell tumors containing choriocarcinoma components showed similar dismal prognoses with median overall survival of 22 months and a one-year survival rate around 60%. However, those conclusions need update since radiotherapy, a milestone for this disease, was not applied to many patients in the published reports. Patients and METHODS Clinical data of iGCTs patients with histologically confirmed choriocarcinoma component and/or β-HCG > 500IU/L were collected from the archive of our institute and studied retrospectively. RESULTS Totally, 76 patients were eligible for the study. 11 patients took surgery as initial treatment, followed by chemoradiotherapy in terms of treatments. Sixty-one patients initiated their treatments with chemotherapy. Among them, twelve patients received second-look surgery due to residue disease. Subsequently, all received radiotherapy except for two early deaths. Four patients received radiotherapy initially, followed by chemotherapy. The median follow-up time was 63 months. The 5-year EFS and OS were 81.5% and 84.1%, respectively. Among patients who did not suffer from early death or progressed disease after induction chemotherapy (n = 68), multivariate analysis found chemotherapy cycles ( > 4 vs ≤ 4) (Hazard ratio [HR] for EFS 0.156, p = 0.021; HR for OS 0.110, p = 0.032), β-HCG level ( > 3000IU/L vs ≤ 3000IU/L) (HR for EFS 4.512, p = 0.052; HR for OS 6.604, p=0.028), and intertumoral hemorrhage (Yes vs No) (HR for EFS 23.177, p = 0.006; HR for OS 12.173, p = 0.081) were independent factors for survival. While radiation field (CSI vs non-CSI) was not proved to be a prognostic factor, either for EFS or OS. CONCLUSIONS iGCTs patients with choriocarcinoma component or β-HCG > 500IU/L showed improved survival with radiotherapy-based treatments. Patients with higher β-HCG levels and hemorrhage had a poorer prognosis. More chemotherapy cycles could bring additional survival benefits.

OBJECTIVES Given the increasing importance of patient’s perspective in adverse events reporting, Patient-Reported Outcomes version of the Common Terminology Criteria for Adverse Events (PRO-CTCAE™) has been proposed as new PRO measures in oncology; however, its implementation has not yet been evaluated in glioma patients, and standardized selection process of priority symptom terms has not been applied. METHODS A total of 16 experts (13 neurosurgeons, 2 radiotherapists, 1 psychiatrist) from 14 medical centers in China were invited to participate in a consensus-seeking 2-round online Delphi survey. Participants rated the level of their agreement with each symptom term likely to occur during adult diffuse gliomas treatment on a 5-point Likert scale. Terms not reaching consensus over the first round were modified in the second rounds. Consensus was defined as content validity index (CVI) > 0.78，coefficient of variation (CV)< 0.35 and average Likert score > 3.00. RESULTS Experts reached a consensus on 21 PRO-CTCAE™ symptom terms in the first Delphi round, and 10 terms in the second round. Likert scale scoring among all experts showed significant consistency (Round 1: Kendall’s W= 0.415，p＜0.001; Round 2: Kendall’s W = 0.351，P＜0.001). The 31 terms (38.75%) screened out of all 78 terms covered neurological symptoms(n=7), psychiatric symptoms(n=6), dermatological symptoms(n=6), gastrointestinal symptoms(n=5) and other common postoperative systemic symptoms of glioma patients. CONCLUSIONS We identified a customized PRO-CTCAE™ questionnaire containing a core set of priority symptom terms for the adult diffuse glioma through a 2-round Delphi survey. Next, a prospective, multi-center, real-word study would be performed to assess the validity, reliability and responsiveness of the customized PRO-CTCAE™ for adult diffuse glioma patients in Chinese population (VERONICA).

Background:Radiotherapy (RT) is one of the cornerstone of the glioblastoma (GBM) treatment paradigm. RT can significantly improve the prognosis of GBM; however, the resistance of tumor cells to radiation results in poor survival. The mechanism of radioresistance has not been fully elucidated.Methods：The differentially expressed genes were screened based on RNA sequencing dataset of 226 primary glioblastomas and 134 recurrent glioblastomas that have undergone radiotherapy, combined with 15 pairs of primary and recurrent GBM obtained from the Chinese Glioma Genome Atlas. Real-time quantitative PCR was performed in three irradiated GBM cell lines（U87, LN229 and U251）to verify the result. The selected gene was investigated to the relationship between mRNA levels and clinical characteristics in the CGGA and TCGA dataset. Kaplan-Meier survival analysis and Cox regression analysis were used for survival analysis. Gene ontology (GO) and KEGG pathway analysis was used for bioinformatics analysis.Results:TMEM59L expression was significantly elevated in recurrent GBM who have received radiotherapy. The result was validated in three irradiated GBM cells. The high-expression of TMEM59L was enriched in IDH mutant and MGMT methylated gliomas and associated with a better prognosis. Gene ontology and KEGG pathway analysis demonstrated that TMEM59L was closely associated with cell proliferation, apoptosis, drug response, and DNA damage. Conclusion：TMEM59L expression was increased after irradiation and resulted in longer survival. We speculated that the high expression of TMEM59L might affect radiotherapy sensitivity by contributing to reactive oxygen species and DNA damage repair.

Isocitrate dehydrogenase (IDH) wild-type glioblastoma (GBM) has a dismal prognosis. A better understanding of tumor evolution holds the key to developing more effective treatment. Here we study GBM's natural evolutionary trajectory by using rare multifocal samples. We sequenced 61,062 single cells from eight multifocal IDH wild-type primary GBMs and defined a natural evolution signature (NES) of the tumor. We show that the NES significantly associates with the activation of transcription factors that regulate brain development, including MYBL2 and FOSL2. Hypoxia is involved in inducing NES transition potentially via activation of the HIF1A–FOSL2 axis. High-NES tumor cells could recruit and polarize bone marrow–derived macrophages through activation of the FOSL2–ANXA1–FPR1/3 axis. These polarized macrophages can efficiently suppress T-cell activity and accelerate NES transition in tumor cells. Moreover, the polarized macrophages could upregulate CCL2 to induce tumor cell migration. Significance: GBM progression could be induced by hypoxia via the HIF1A–FOSL2 axis. Tumor-derived ANXA1 is associated with recruitment and polarization of bone marrow–derived macrophages to suppress the immunoenvironment. The polarized macrophages promote tumor cell NES transition and migration. This article is highlighted in the In This Issue feature, p. 2711

Intracranial germ cell tumors (iGCTs) are classified into two pathological subtypes (germinomas [GEs] and nongerminomatous germ cell tumors [NGGCTs]), with distinct treatment strategy and prognosis. Accurate preoperative determination of iGCT subtypes is essential to guide clinical decision-making and prognosis assessment.To investigate the diagnostic value of diffusion-weighted imaging (DWI), susceptibility weighted imaging (SWI), and dynamic susceptibility-contrast perfusion-weighted imaging (DSC-PWI) combined with conventional magnetic resonance imaging (cMRI) in finding subtypes of iGCTs.Retrospective.A total of 40 patients (45% male and 55% female) with iGCTs.A 3 T;lt;T1WI, T2WI, T1WI + C, DWI, SWI, DSC-PWIgt;.The parameters of DWI and DSC-PWI were calculated based on extracted parameters of multiparametric MRIs. The characteristics of SWI and cMRI were also compared in GEs and NGGCTs.The diagnostic efficacy of the minimum apparent diffusion coefficient (ADCmin), time-to-peak (TTP), relative mean transit time (rMTT), relative cerebral blood flow (rCBF), relative cerebral blood volume (rCBV) maps, and cMRI features in iGCT classification was evaluated by receiver operating characteristic curve (ROC) analyses. We calculated the sensitivity, specificity, AUC, and Youden index of the hybrid MR evaluation methods. A prospective cohort (five GEs and five NGGCTs) was designed as a simulation set to test the model. The significance threshold was set at P lt; 0.01.The ADCmin (1039.100 ± 453.830 vs. 1400.050 ± 394.650), rCBF values (20.650 ± 6.260 vs. 51.170 ± 6.570), and TTP values (24.450 ± 3.160 vs. 28.950 ± 5.120) were significantly lower in GEs than in NGGCTs. The combination of ADCmin, DSC-PWI, and cMRI showed the heights AUC (AUC = 0.962). The iGCT multiparametric framework showed the AUC was 0.958 in the simulation set.The iCGT multiparametric framework might be an effective diagnostic approach of iGCT subtype. The application of cMRI (T1WI, T2WI, and Gd-T1WI) with advanced imaging modalities (DWI, SWI, and PWI) had the best performance for classifying iGCT subtypes.3 TECHNICAL EFFICACY: Stage 2.

Whereas evidences of single-cell studies have suggested the cellular diversity of medulloblastoma, the most common malignant brain tumor in childhood, the understanding of spatial status for specific cell populations and gene modules remains rudimentary. Herein, we comprehensively analyze single-nucleus RNA expression, chromatin accessibility and spatial transcriptomic profiling from 52 human medulloblastomas spanning four molecular subgroups, complemented with the bulk whole genome and RNA sequencing data across 300 samples. We comprehensively interrogated the composition of medulloblastoma microenvironment and spatially described the developmental trajectory from progenitor-like to differentiated malignant cells. Furthermore, distinct heterogeneity within molecular subgroups leads to the identification of twelve subtypes of medulloblastoma, which unfortunately lacks the cellular biology. By deciphering the copy number variation and transcriptional programs at single-cell resolution, we in-depth characterized medulloblastoma and mapped the activity of gene expression and transcription factor binding according to individual subtype. The WNT-β, SHH-γ, Group 3-β and Group 4-γ were observed in early differentiation stage. Harmony alignment revealed subtype-dependent modules recapitulated the neurodevelopmental gene enrichment, strongly correlating to clinical outcomes. Collectively, our findings provide spatiotemporally resolved insights into transcriptome and epigenome of twelve-subtype medulloblastoma allowing further refinement regarding clinical risk stratification strategies.

Intra-cranial germ cell tumors (iGCTs) are classified into two pathological subtypes Germinomas (GEs) and nongerminomatous germ cell tumors (NGGCTs). Accurate preoperative determination of iGCT subtypes is essential to guide clinical decision making and prognosis assessment. This study aims to develop a deep-learning algorithm to automatically segment iGCTs and classify subtypes using preoperative T2-weighted (T2W) images. Brain MR imaging and corresponding pathologic information were retrospectively obtained from 594 subjects including 269 GEs and 325 NGGCTs from Beijing Tiantan Hospital between January 1, 2008, and October 31, 2020, and subdivided into training (n=416) and test (n=178) sets for iGCTs segmentation, and classification using 3D nnU-Net based on T2W images. An internal validation cohort (including 56 GEs and 17 NGGCTs) and two external validation cohorts (Center 1 including 13 GEs and 12 NGGCTs；Center 2 including 17 GEs and 12 NGGCTs) were prospectively designed as an independent sets for testing the iGCT-net. Dice scores were computed to assess tumor segmentation. Accuracy and area under the curve (AUC) were calculated to assess the GEs and NGGCTs classification. Additionally, sensitivity analysis of different tumor location (suprasellar, pineal and basal ganglia regions) was performed to access the classification accuracy. The iGCT-net for tumor segmentation achieved a mean dice score of 0.80 in all cohorts. For tumor classification, the iGCT-net achieved the mean accuracy of 90.96% of all cohorts, which was superior to that using demographics and morphologic features (mean = 67.50%, p< 0.01). For different tumor location (suprasellar region, pineal region, and basal ganglia region), the iGCT-net showed an mean accuracy of 87.94%, 89.38% and 65.74% in test set, internal validation set and external validation cohorts. A fully automatic deep learning algorithm to segment iGCT and discriminate GEs and NGGCTs with high accuracy based on only T2W images from a large dataset is successfully developed.

e14048 Background: To explore the clinical characteristics and treatment outcomes of patients with intracranial germ cell tumors (iGCTs). Methods: Clinical data of 1448 patients who were diagnosed with iGCTs based on histology and/or tumor marker elevation between 1990 and 2019 at Beijing Tiantan Hospital were studied retrospectively. Results: The median age was 12 years, and 74.3% of patients were male. In total, 32.6% of patients were of sellar origin, 32.3% were of pineal origin, 20.4% were of basal ganglia (BG) origin, and 14.7% were others. In nonmetastatic germinoma patients, multivariate analysis found that both whole-brain radiotherapy (p < 0.001) and craniospinal irradiation (p = 0.040) could significantly reduce the risk of relapse compared with focal radiotherapy. In nonmetastatic NGGCT patients, the disease control rate was similar among the above three radiation volumes. Among 87 relapsed patients, those with germinoma (n = 35) showed a significantly higher out-field relapse rate than patients with NGGCTs (54.3% vs. 32.7%, p = 0.003). In patients with NGGCTs (n = 52), the in-field (36.5%) and out-field (32.7%) relapse rates were similar. In terms of relapse sites, relapse at the parenchyma (36.4%) was documented only in patients with BG region involvement. In patients with sellar or pineal origin, > 80% of relapses were documented in ventricles and/or the spine. Conclusions: iGCT patients in our series showed unique characteristics. Considering both efficacy and potential adverse effects, the optimal radiation strategy in nonmetastatic germinoma patients should be minimum coverage of at-risk areas. In patients with nonmetastatic NGGCTs, the optimal radiation strategy needs further investigation.

2069 Background: Medulloblastoma is the most common malignant childhood tumor type with distinct molecular subgroups. While advances in the comprehensive treatment have been made, the mortality in the high-risk group is still very high, driven by an incomplete understanding of cellular diversity. Methods: We use single-nucleus RNA expression, chromatin accessibility and spatial transcriptomic profiling to generate an integrative multi-omic map in 40 human medulloblastomas spanning all molecular subgroups and human postnatal cerebella, which is supplemented by the bulk whole genome and RNA sequences across 300 cases. Results: This approach provides spatially resolved insights into the medulloblastoma and cerebellum transcriptome and epigenome with identification of distinct cell-type in the tumor microenvironment. Medulloblastoma exhibited three tumor subpopulations including the quiescent, the differentiated, and a stem-like (proliferating) population unique to cancer, which localized to an immunosuppressive-vascular niche. We identified and validated mechanisms of stem-like to differentiated process among the malignant cells that drive tumor progression. Integration of single-cell and spatial data mapped ligand-receptor networks to specific cell types, revealing stem-like malignant cells as a hub for intercellular communication. Multiple features of potential immunosuppression and angiogenesis were observed, including Treg cells and endothelial cells co-localization in compartmentalized tumor stroma. Conclusions: Our study provides an integrative molecular landscape of human medulloblastoma and represents a reference to advance mechanistic and therapeutic studies of pediatric neuro-oncological disease.

To summarize randomized controlled trials (RCTs) evidence for the effectiveness of available treatment strategies for elderly patients with glioblastoma (GBM) (defined as 60 years old and above) and to identify research gaps, we conducted this evidence map. Finally, 22 RCTs (with 3052 participants) were included. For newly diagnosed elderly patients with GBM (16 RCTs), 75% was identified about the effectiveness of chemotherapy, radiotherapy and chemo-radiotherapy, while there was relatively less evidence evaluating targeted therapy (12.5%), immunotherapy (18.75%) and tumor treating fields (TTFs) therapy (6.25%). Less evidence was identified in elderly patients with recurrent GBM (6 RCTs), including 2 RCTs for immunotherapy and 4 RCTs for targeted therapy. Current RCTs revealed some beneficial treatment strategies. However, more are needed to optimize regimens of RT and chemo-radiotherapy, explore potential new therapies such as targeted therapy, immunotherapy and combination therapies, and identify biomarkers to guide appropriate patient and treatment selection.