Glioma Longitudinal Analysis Research Articles

BIOM-49. PATIENT-CENTRIC INTEGRATED GRAPH DATABASE REVEALS CRITICAL BIOMARKERS IN THE RECURRENCE OF IDH WILD-TYPE GLIOMA

Abstract BACKGROUND IDH wild-type glioblastoma (GBM) represents a formidable therapeutic challenge due to its consistent recurrence and progression despite standard treatments. Understanding the molecular and cellular mechanisms driving GBM recurrence through data integration is crucial. This study employs a novel graph database strategy to uncover insights into these tumors and identify functional biomarkers through graph algorithms-based pathway enrichment analysis. METHOD We developed a stand-alone graph database using Neo4j for primary-recurrent paired GBMs, integrating clinical and gene expression data from the Glioma Longitudinal AnalySiS (GLASS) Consortium and internal MD Anderson cohort. Based on log2 fold-change, we used hierarchical trees for differentially expressed genes to decompose the expression profile. Nodes in the database represent patients with considerable absolute log2 values determined by hierarchical trees, while shared patients define the edges. We applied graph algorithms such as degree centrality and community detection to identify potential biomarkers and supplemented these analyses with pathway identification to refine the biomarker list. Further, we divided the data set into 80% training and 20% testing sets. We evaluated the predictive performance of these biomarkers using machine learning models, including logistic regression, random forest, SVM, and neural networks. RESULTS DEG analysis revealed 33 down-regulated and 418 up-regulated genes in recurrent tumors. The top enriched pathways included neuroactive ligand-receptor interaction and GPCR ligand binding. Our database approach implicated 35 out of 451 genes as significant drivers of recurrence in IDH wild-type GBM. The prediction performance of these biomarkers was comparable to features selected through methods such as mRMR and logistic lasso and target identification methods like WGCNA. CONCLUSION Our innovative systems biology approach reveals that ligand-receptor interactions are crucial in driving recurrence in IDH wild-type GBM. This method also yielded promising mechanistic targets for recurrence, prompting focused and therapeutically relevant functional analyses. Moreover, this methodology is not limited to our study but can be applied in other multi-omics settings, offering a robust tool for biomarker discovery and validation.

EPID-22. MUTATIONAL SIGNATURE ASSOCIATED WITH HALOALKANE EXPOSURE ENRICHED IN FIREFIGHTERS IN THE ADULT GLIOMA STUDY

Abstract Using data from The Cancer Genome Atlas (TCGA) and Glioma Longitudinal Analysis (GLASS), we identified specific environmentally related mutational signatures in glioma including a signature associated with haloalkane exposure (SBS42) from the Catalogue of Somatic Mutational Signatures (COSMIC). Haloalkanes are widely used commercially including in flame retardants and fire extinguishants and are an intriguing finding given an observed increased glioma risk in firefighters. To validate our findings, we use new study participants with data on occupation (not available in either TCGA/GLASS) from the University of California, San Francisco Adult Glioma Study (AGS) to compare mutational signatures in tumors from 17 persons with glioma and documented occupational exposure to firefighting with those of 18 matched glioma patients without history of firefighting. Using whole exome sequencing, we identified environmental exposure signatures in tumor tissue from firefighters as well as in non-firefighters who had other relevant occupational exposure (e.g. auto/truck mechanic work). In these data, glioma is largely associated with aging and mutational signatures relating to endogenous mutational processes that correlate with age, such as spontaneous or enzymatic deamination of 5-methylcytosine. However, some gliomas have detectable signatures associated with exogenous mutational processes, such as SBS42 haloalkanes. Thus, we confirm detection of haloalkane signatures in persons likely highly exposed, i.e. long-term firefighters. Identifying exogenous mutational processes in cancers are important as they may inform public health intervention strategy to reduce mutagenesis and prevent cancer inception. Identifying occupational correlates with SBS42, associated with occupational exposure to haloalkanes, will pinpoint occupational hazards that may be avoidable. This is especially important for glioma and other cancers where exogenous mutagenesis is not well established.

DNAR-16. TARGETING APOBEC CYTIDINE DEAMINASES TO ENHANCE RADIOSENSITIVITY IN GLIOMA

Abstract Glioma is the most common and aggressive primary adult brain tumor. Radiation therapy (RT) is a critical part of glioma treatment, but radioresistance severely limits its therapeutic efficacy. To better understand how radioresistance develops, we analyzed 212 paired primary and recurrent gliomas from the Glioma Longitudinal Analysis Consortium. We found a significant increase in mutational signatures associated with the activity of the Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) family of cytidine deaminases in RT-treated recurrent gliomas (p<0.001, paired Wilcoxon signed-rank test). Further analysis of post-treatment mutations showed that RT increased APOBEC mutational signatures independent of other factors (p<0.001, multivariable log-linear regression), suggesting that APOBEC proteins become activated by radiation. However, their role in promoting tumor evolution and treatment resistance is currently unknown. Further analyses of extended signature contexts and expression data nominated APOBEC3B (A3B) and APOBEC3G (A3G) as candidates for creating these mutational signatures. To investigate their roles in RT response, we knocked out (KO) A3B and A3G individually in patient-derived glioma cell lines before RT. Compared to Control cells, irradiated A3B-KO and A3G-KO cells acquired significantly fewer APOBEC mutational signatures by whole genome sequencing (p<0.05, Kruskal–Wallis test) and exhibited enhanced radiosensitivity in vitro. Interestingly, the KO cells displayed a significant decrease in mutational signatures associated with Non-Homologous End-Joining (NHEJ), one of the main pathways for repairing the RT-induced DNA damage (p<0.05, Kruskal–Wallis test), suggesting that APOBEC deletion impaired DNA repair. This was further supported by persistent gH2AX DNA damage foci 48h post-RT and reduced autophosphorylation of DNA-dependent protein kinase (DNA-PK), a vital component of the NHEJ pathway, particularly in A3G KO cells. Overall, our results identify APOBEC proteins as potential targets for radiosensitizing gliomas and highlight the clinical utility of using mutational signatures to predict and monitor response to treatment.

Clinical and molecular features of patients with IDH1 wild-type primary glioblastoma presenting unexpected short-term survival after gross total resection.

This study investigated the factors influencing short-term survivors (STS) after gross total resection (GTR) in patients with IDH1 wild-type primary glioblastoma. We analyzed five independent cohorts who underwent GTR, including 83 patients from Kitasato University (K-cohort), and four validation cohorts of 148 patients from co-investigators (V-cohort), 66 patients from the Kansai Molecular Diagnosis Network for the Central Nervous System tumors, 109 patients from the Cancer Genome Atlas, and 40 patients from the Glioma Longitudinal AnalySiS. The study defined STS as those who had an overall survival ≤ 12months after GTR with subsequent radiation therapy, and concurrent and adjuvant temozolomide (TMZ). The study included 446 patients with glioblastoma. All cohorts experienced unexpected STS after GTR, with a range of 15.0-23.9% of the cases. Molecular profiling revealed no significant difference in major genetic alterations between the STS and non-STS groups, including MGMT, TERT, EGFR, PTEN, and CDKN2A. Clinically, the STS group had a higher incidence of non-local recurrence early in their treatment course, with 60.0% of non-local recurrence in the K-cohort and 43.5% in the V-cohort. The study revealed that unexpected STS after GTR in patients with glioblastoma is not uncommon and such tumors tend to present early non-local recurrence. Interestingly, we did not find any significant genetic alterations in the STS group, indicating that such major alterations are characteristics of GB rather than being reliable predictors for recurrence patterns or development of unexpected STS.

Abstract LB246: Deep learning framework for patient recurrence and genetic marker prediction in diffuse glioma

Abstract Investigation of important molecular features of glioma patients may reveal driving mechanisms of patient recurrence and treatment failures in glioma. Applying various deep learning techniques enables the integration and systematic comparison of multi-omics features, allowing robust marker prediction and patient stratification. We extracted mutation and copy number variation data from the Glioma Longitudinal Analysis (GLASS) Consortium, consisting of 628 primary or recurrent samples. To achieve the patient recurrence classification task, we designed a transformer-based model that learns the genomic profiles and predicts patient recurrence status. Our model outperforms traditional machine learning models, including support vector machine (SVM), logistic regression, and decision trees, for up to 34% and 40% improvement of AUROC and AUPRC, respectively. Including mutational features alone can achieve the best prediction performance, while copy number variations are less informative in predicting patient recurrence status. Our approach is an early attempt that employs deep learning for dissecting the contribution of diverse genomic profiles on glioma recurrence and might provide insights into developing novel therapies for treating glioma. Citation Format: Ko-Hong Lin, Kai Zhang, Dung-Fang Lee, Xiaoqian Jiang. Deep learning framework for patient recurrence and genetic marker prediction in diffuse glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB246.

PATH-37. PROGNOSTIC IMPACT OF TUMOR MUTATIONAL BURDEN AT BASELINE IN IDH-WILDTYPE GLIOBLASTOMA

Abstract Tumor mutational burden (TMB) has emerged as a potential biomarker for survival and response to immune checkpoint therapy. However, limited data are available regarding the prognostic impact of TMB at baseline in glioblastoma (GBM). This study aimed to evaluate the somatic mutation status and survival outcomes of isocitrate dehydrogenase (IDH) wildtype GBM using a dataset of GBM samples from cBioPortal and single tertiary hospital. A total of 840 samples were extracted from cBioPortal, including 410 samples from Memorial Sloan Kettering Cancer Center (MSKCC), 234 samples from The Cancer Genome Atlas (TCGA), 106 samples from Glioma Longitudinal Analysis Consortium (GLASS), and 90 samples from Clinical Proteomic Tumor Analysis Consortium (CPTAC). Additionally, we enrolled 341 GBM patients with targeted sequencing data (OncoPanel AMC) from Asan Medical Center. The TMBHigh group was defined as the top 10% value of TMB within each cohort. Among the total of 1,081 IDH-wt GBM samples, 104 samples were classified as TMBHigh. No significance difference in overall survival (OS) was observed between the TMBHigh group and the non-TMBHigh group across the 5 cohorts. The mutations of DNA mismatch repair genes (MLH1, MSH2, MSH6, PMS2, and POLE) was significantly higher in the TMBHigh group compared with the non-TMBHigh group in 3 of 5 cohorts (pASAN=1.02x10-4, pMSKCC=1.5x10-2, and pCPTAC=1.26x10-2). However, when we calculated the indel to total mutation ratio (I-index) to dedect microsatellite Instability, only the MSKCC cohort showed a higher I-index in the TMBHigh group compared to the non-TMBHigh group (16.7% vs 13.7%, p=2.67x10-3). In summary, there is no significant association between TMB at baseline and poor survival outcomes in IDH-wildtype GBM. Further research is needed to explore the potential implications of TMB as a prognostic marker and its relevance for personalized treatment strategies in GBM patients.

Genomic characterization of IDH-mutant astrocytoma progression to grade 4 in the treatment setting

As the progression of low-grade diffuse astrocytomas into grade 4 tumors significantly impacts patient prognosis, a better understanding of this process is of paramount importance for improved patient care. In this project, we analyzed matched IDH-mutant astrocytomas before and after progression to grade 4 from six patients (discovery cohort) with genome-wide sequencing, 21 additional patients with targeted sequencing, and 33 patients from Glioma Longitudinal AnalySiS cohort for validation. The Cancer Genome Atlas data from 595 diffuse gliomas provided supportive information. All patients in our discovery cohort received radiation, all but one underwent chemotherapy, and no patient received temozolomide (TMZ) before progression to grade 4 disease. One case in the discovery cohort exhibited a hypermutation signature associated with the inactivation of the MSH2 and DNMT3A genes. In other patients, the number of chromosomal rearrangements and deletions increased in grade 4 tumors. The cell cycle checkpoint gene CDKN2A, or less frequently RB1, was most commonly inactivated after receiving both chemo- and radiotherapy when compared to other treatment groups. Concomitant activating PDGFRA/MET alterations were detected in tumors that acquired a homozygous CDKN2A deletion. NRG3 gene was significantly downregulated and recurrently altered in progressed tumors. Its decreased expression was associated with poorer overall survival in both univariate and multivariate analysis. We also detected progression-related alterations in RAD51B and other DNA repair pathway genes associated with the promotion of error-prone DNA repair, potentially facilitating tumor progression. In our retrospective analysis of patient treatment and survival timelines (n = 75), the combination of postoperative radiation and chemotherapy (mainly TMZ) outperformed radiation, especially in the grade 3 tumor cohort, in which it was typically given after primary surgery. Our results provide further insight into the contribution of treatment and genetic alterations in cell cycle, growth factor signaling, and DNA repair-related genes to tumor evolution and progression.

LONGITUDINAL WGS AND WES OF GLIOBLASTOMA REVEAL POTENTIAL PATHWAY TARGETS

Abstract AIMS Treating glioblastoma (GBM) patients remains challenging as the current regimens result in the recurrence of a treatment-resistant and fatal tumour. We aimed to identify candidate pathways that, when mutated, confer treatment resistance. Targeting these pathways may improve GBM treatment effcacy. METHOD We performed WGS and WES on 46 matched primary and recurrent GBMs (the Discovery cohort) and used data from additional 188 samples from the Glioma Longitudinal AnalySiS consortium (GLASS: Validation cohort). First, we identified variants that were shared between primary and recurrent GBMs. We then highlighted those most likely to be involved in treatment resistance, i.e. with: 1) low allele fractions at primary and high in recurrent, 2) high allele fractions in both primary and recurrent. Finally, we performed enrichment analysis using these variants to identify pathways that may drive clonal expansion from primary to recurrent. RESULTS The analysis of variants that increased from primary to recurrent revealed three significant disrupted path- ways in the Discovery cohort; “Activation of BH3 Only Proteins” (Reactome), “P53 pathway” (BIOCARTA), and “Intrinsic Pathway for Apoptosis” (REACTOME). Six distinct genes caused the aberration of these pathways. The same pathways were significantly disrupted in the recurrent variants group of the Validation cohort but were solely caused by TP53 alterations. In the Discovery cohort, “Activation of BH3 Only Proteins” was altered in 24% of the patients. CONCLUSIONS Investigating how pathway mutations alter in GBM through treatment has led to identifying potential candidate mechanisms to be further studied in the laboratory.

Decoding the prognostic significance of integrator complex subunit 9 (INTS9) in glioma: links to TP53 mutations, E2F signaling, and inflammatory microenvironments

IntroductionGliomas, a type of brain neoplasm, are prevalent and often fatal. Molecular diagnostics have improved understanding, but treatment options are limited. This study investigates the role of INTS9 in processing small nuclear RNA (snRNA), which is crucial to generating mature messenger RNA (mRNA). We aim to employ advanced bioinformatics analyses with large-scale databases and conduct functional experiments to elucidate its potential role in glioma therapeutics.Materials and methodsWe collected genomic, proteomic, and Whole-Exon-Sequencing data from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) for bioinformatic analyses. Then, we validated INTS9 protein expression through immunohistochemistry and assessed its correlation with P53 and KI67 protein expression. Gene Set Enrichment Analysis (GSEA) was performed to identify altered signaling pathways, and functional experiments were conducted on three cell lines treated with siINTS9. Then, we also investigate the impacts of tumor heterogeneity on INTS9 expression by integrating single-cell sequencing, 12-cell state prediction, and CIBERSORT analyses. Finally, we also observed longitudinal changes in INTS9 using the Glioma Longitudinal Analysis (GLASS) dataset.ResultsOur findings showed increased INTS9 levels in tumor tissue compared to non-neoplastic components, correlating with high tumor grading and proliferation index. TP53 mutation was the most notable factor associated with upregulated INTS9, along with other potential contributors, such as combined chromosome 7 gain/10 loss, TERT promoter mutation, and increased Tumor Mutational Burden (TMB). In GSEA analyses, we also linked INTS9 with enhanced cell proliferation and inflammation signaling. Downregulating INTS9 impacted cellular proliferation and cell cycle regulation during the function validation. In the context of the 12 cell states, INTS9 correlated with tumor-stem and tumor-proliferative-stem cells. CIBERSORT analyses revealed increased INTS9 associated with increased macrophage M0 and M2 but depletion of monocytes. Longitudinally, we also noticed that the INTS9 expression declined during recurrence in IDH wildtype.ConclusionThis study assessed the role of INTS9 protein in glioma development and its potential as a therapeutic target. Results indicated elevated INTS9 levels were linked to increased proliferation capacity, higher tumor grading, and poorer prognosis, potentially resulting from TP53 mutations. This research highlights the potential of INTS9 as a promising target for glioma treatment.

Genomic and Transcriptomic Remodeling by Neoadjuvant Chemoradiotherapy (nCRT) and the Indicative Role of Acquired INDEL Percentage for nCRT Efficacy in Esophageal Squamous Cell Carcinoma

The effect of genomic factors on the response of patients with esophageal squamous cell carcinoma (ESCC) to neoadjuvant chemoradiotherapy (nCRT), as well as how nCRT influences the genome and transcriptome of ESCC, remain largely unknown. In total, 137 samples from 57 patients with ESCC undergoing nCRT were collected and subjected to whole-exome sequencing and RNA sequencing analysis. Genetic and clinicopathologic factors were compared between the patients achieving pathologic complete response and patients not achieving pathologic complete response. Genomic and transcriptomic profiles before and after nCRT were analyzed. Codeficiency of the DNA damage repair and HIPPO pathways synergistically sensitized ESCC to nCRT. nCRT induced small INDELs and focal chromosomal loss concurrently. Acquired INDEL% exhibited a decreasing trend with the increase of tumor regression grade (P=.06, Jonckheere's test). Multivariable Cox analysis indicated that higher acquired INDEL% was associated with better survival (adjusted hazard ratio [aHR], 0.93; 95% CI, 0.86-1.01; P=.067 for recurrence-free survival [RFS]; aHR, 0.86; 95% CI, 0.76-0.98; P=.028 for overall survival [OS], with 1% of acquired INDEL% as unit). The prognostic value of acquired INDEL% was confirmed by the Glioma Longitudinal AnalySiS data set (aHR, 0.95; 95% CI, 0.902-0.997; P=.037 for RFS; aHR, 0.96; 95% CI, 0.917-1.004; P=.076 for OS). Additionally, clonal expansion degree was negatively associated with patient survival (aHR, 5.87; 95% CI, 1.10-31.39; P=.038 for RFS; aHR, 9.09; 95% CI, 1.10-75.36; P=.041 for OS, with low clonal expression group as reference) and also negatively correlated with acquired INDEL% (Spearman ρ=-0.45; P=.02). The expression profile was changed after nCRT. The DNA replication gene set was downregulated, while the cell adhesion gene set was upregulated after nCRT. Acquired INDEL% was negatively correlated with the enrichment of the DNA replication gene set (Spearman ρ=-0.56; P=.003) but was positively correlated with the enrichment of the cell adhesion gene set (Spearman ρ=0.40; P=.05) in posttreatment samples. nCRT remodels the genome and transcriptome of ESCC. Acquired INDEL% is a potential biomarker to indicate the effectiveness of nCRT and radiation sensitivity.

Ancient ubiquitous protein 1 (AUP1) is a prognostic biomarker connected with TP53 mutation and the inflamed microenvironments in glioma

IntroductionGlioblastoma (GBM) is the most common and lethal brain tumor. The current treatment is surgical removal combined with radiotherapy and chemotherapy, Temozolomide (TMZ). However, tumors tend to develop TMZ resistance which leads to therapeutic failure. Ancient ubiquitous protein 1 (AUP1) is a protein associated with lipid metabolism, which is widely expressed on the surface of ER and Lipid droplets, involved in the degradation of misfolded proteins through autophagy. It has recently been described as a prognostic marker in renal tumors. Here, we aim to use sophisticated bioinformatics and experimental validation to characterize the AUP1's role in glioma.Material and methodsWe collected the mRNA, proteomics, and Whole-Exon-Sequencing from The Cancer Genome Atlas (TCGA) for bioinformatics analyses. The analyses included the expression difference, Kaplan–Meier-survival, COX-survival, and correlation to the clinical factors (tumor mutation burden, microsatellite instability, and driven mutant genes). Next, we validated the AUP1 protein expression using immunohistochemical staining on the 78 clinical cases and correlated them with P53 and KI67. Then, we applied GSEA analyses to identify the altered signalings and set functional experiments (including Western Blot, qPCR, BrdU, migration, cell-cycle, and RNAseq) on cell lines when supplemented with small interfering RNA targeting the AUP1 gene (siAUP1) for further validation. We integrated the single-cell sequencing and CIBERSORT analyses at the Chinese Glioma Genome Atlas (CGGA) and Glioma Longitudinal AnalySiS (GLASS) dataset to rationale the role of AUP1 in glioma.ResultsFirstly, the AUP1 is a prognostic marker, increased in the tumor component, and correlated with tumor grade in both transcriptomes and protein levels. Secondly, we found higher AUP1 associated with TP53 status, Tumor mutation burden, and increased proliferation. In the function validation, downregulated AUP1 expression merely impacted the U87MG cells' proliferation instead of altering the lipophagy activity. From the single-cell sequencing and CIBERSORT analyses at CGGA and GLASS data, we understood the AUP1 expression was affected by the tumor proliferation, stromal, and inflammation compositions, particularly the myeloid and T cells. In the longitudinal data, the AUP1 significantly dropped in the recurrent IDH wildtype astrocytoma, which might result from increased AUP1-cold components, including oligodendrocytes, endothelial cells, and pericytes.ConclusionAccording to the literature, AUP1 regulates lipophagy by stabilizing the ubiquitination of lipid droplets. However, we found no direct link between AUP1 suppression and altered autophagy activity in the functional validation. Instead, we noticed AUP1 expression associated with tumor proliferation and inflammatory status, contributed by myeloid cells and T cells. In addition, the TP53 mutations seem to play an important role here and initiate inflamed microenvironments. At the same time, EGFR amplification and Chromosome 7 gain combined 10 loss are associated with increased tumor growth related to AUP1 levels. This study taught us that AUP1 is a poorer predictive biomarker associated with tumor proliferation and could report inflamed status, potentially impacting the clinical application.

Genetic evolution and cellular interactions within the tumour microenvironment determine glioblastoma progression

Glioblastoma (GBM) is the most aggressive form of primary brain cancer, with 5-year survival rates of less than 5%. Clinical management of GBM has not changed in the last 15 years, and current treatment approaches combine surgical resection, followed by radiotherapy and chemotherapy. Tragically, tumour recurrence is inevitable. Still, very little is known about how tumours evolve in response to therapy and become treatment resistant. In 2019, The Glioma Longitudinal AnalySiS (GLASS) consortium curated extensive, publicly accessible genomic profiling data captured from matched primary and recurrent tumours across 222 patients, along with comprehensive clinical annotations. Recently, this longitudinal genomic data resource was expanded by integrating matching transcriptomic and genomic data from 304 adult patients with isocitrate dehydrogenase (IDH)-wild-type and IDH-mutant glioma captured at two or more time points (Varn et al., 2022). This has enabled new insights into the dynamic changes in transcriptional programs, cellular compositions and microenvironment interactions within these brain tumours. In this commentary, we will focus on recurrent high-grade IDHwt and the implications of these findings for targeting tumour-microenvironment interactions that may pave new pathways for developing therapies for this type of brain tumour.

Identification, validation and biological characterisation of novel glioblastoma tumour microenvironment subtypes: implications for precision immunotherapy

New precision medicine therapies are urgently required for glioblastoma (GBM). However, to date, efforts to subtype patients based on molecular profiles have failed to direct treatment strategies. We hypothesised that interrogation of the GBM tumour microenvironment (TME) and identification of novel TME-specific subtypes could inform new precision immunotherapy treatment strategies. A refined and validated microenvironment cell population (MCP) counter method was applied to >800 GBM patient tumours (GBM-MCP-counter). Specifically, partition around medoids (PAM) clustering of GBM-MCP-counter scores in the GLIOTRAIN discovery cohort identified three novel patient clusters, uniquely characterised by TME composition, functional orientation markers and immune checkpoint proteins. Validation was carried out in three independent GBM-RNA-seq datasets. Neoantigen, mutational and gene ontology analysis identified mutations and uniquely altered pathways across subtypes. The longitudinal Glioma Longitudinal AnalySiS (GLASS) cohort and three immunotherapy clinical trial cohorts [treatment with neoadjuvant/adjuvant anti-programmed cell death protein 1 (PD-1) or PSVRIPO] were further interrogated to assess subtype alterations between primary and recurrent tumours and to assess the utility of TME classifiers as immunotherapy biomarkers. TMEHigh tumours (30%) displayed elevated lymphocyte, myeloid cell immune checkpoint, programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 transcripts. TMEHigh/mesenchymal+ patients featured tertiary lymphoid structures. TMEMed (46%) tumours were enriched for endothelial cell gene expression profiles and displayed heterogeneous immune populations. TMELow (24%) tumours were manifest as an 'immune-desert' group. TME subtype transitions upon recurrence were identified in the longitudinal GLASS cohort. Assessment of GBM immunotherapy trial datasets revealed that TMEHigh patients receiving neoadjuvant anti-PD-1 had significantly increased overall survival (P= 0.04). Moreover, TMEHigh patients treated with adjuvant anti-PD-1 or oncolytic virus (PVSRIPO) showed a trend towards improved survival. We have established a novel TME-based classification system for application in intracranial malignancies. TME subtypes represent canonical 'termini a quo' (starting points) to support an improved precision immunotherapy treatment approach.

BIOM-26. GENOME-WIDE SOMATIC COPY NUMBER ALTERATION SIGNATURES HAVE PROGNOSTIC IMPLICATIONS FOR ADULT ASTROCYTIC GLIOMA

Abstract BACKGROUND The molecular landscape of adult diffuse glioma has been extensively characterized by gene expression and DNA methylation profiling, but less attention has been paid to somatic copy number alteration (SCNA) data. This study aimed to give a rigorous, survival-focused analysis of glioma genome-wide SCNA data that builds on our previous work. METHODS Detailed survival analyses were conducted on the substructure of UMAP projections of all TCGA glioma (Nf1092), exclusively astrocytic glioma (Nf914), and exclusively IDH-wildtype glioma (Nf528). Results were validated with data from the Glioma Longitudinal Analysis Consortium (GLASS) (Nf224). Clinical factors such as age and MGMT methylation were tested in multivariate survival analyses. RESULTS A UMAP projection of TCGA glioma SCNA data generated three distinct clusters composed of entirely oligodendroglioma, predominantly IDH-mutant astrocytoma (C-IDHmut-astro), and predominantly IDH-wildtype glioma (C-IDHwt), respectively. For astrocytic tumors, cluster assignment was independently prognostic of IDH status (p< 0.001): TCGA IDH-mutant astrocytomas that clustered in C-IDHwt had poorer outcomes than their counterparts (p< 0.04) and IDH-wildtype tumors that clustered in C-IDHmut-astro fared better than those that did not (p< 0.01). The distribution of GLASS astrocytic tumors, which is skewed for better survival, supported our results in IDH-wildtype glioblastoma (p< 0.001, Fisher’s). Among four distinct subclusters of TCGA IDH-wildtype glioblastomas, the largest was significantly or marginally significantly negatively prognostic compared to each other cluster (p=0.048, p=0.059, p=0.027) and their combination (p=0.002). In the GLASS dataset, inclusion in the largest subcluster was also prognostic (p=0.013) and similar trends were observed between individual clusters (p=0.21, p=0.036, p=0.083). Furthermore, membership to the largest cluster was independently prognostic of MGMT methylation status and several published IDH-wildtype glioblastoma subtypes in the TCGA. CONCLUSIONS Unsupervised learning of genome-wide SCNA has prognostic implications for astrocytic glioma. SCNA cluster membership is independently prognostic of MGMT methylation status.

TMIC-52. RELATIONSHIP BETWEEN MACROPHAGE AND RADIOSENSITIVITY IN HUMAN PRIMARY AND RECURRENT GLIOBLASTOMA: IN SILICO ANALYSIS WITH PUBLICLY AVAILABLE DATASETS

Abstract The glioblastoma microenvironment predominantly contains tumor-associated macrophages that support tumor growth and invasion. We investigated the relationship between tumor radiosensitivity and infiltrating M1/M2 macrophage profiles in public datasets of primary and recurrent glioblastoma. We estimated the radiosensitivity index (RSI) score based on gene expression rankings. Macrophages were profiled using the deconvolution algorithm CIBERSORTx. Samples from The Cancer Genome Atlas (TCGA), Chinese Glioma Genome Atlas (CGGA), the Ivy Glioblastoma Atlas Project dataset, a single-cell RNA sequencing dataset (GSE84465), Glioma Longitudinal Analysis Consortium (GLASS), and an immunotherapy trial dataset (GSE121810) were included. RSI-high radioresistant tumors were associated with worse overall survival in TCGA and CGGA than RSI-low tumors. M1/M2 macrophage ratios and RSI scores were inversely associated, indicating that radioresistant glioblastoma tumor microenvironments contain more M2 than M1 macrophages. In the single-cell RNA sequencing dataset, the mean RSI of neoplastic cells was positively correlated with high M2 macrophages proportions. A favorable response to programmed cell death protein 1 (PD-1) therapy was observed in recurrent glioblastomas with high M1/M2 macrophage ratios and low RSI scores. In patients with recurrent glioblastoma, fewer M2 macrophages and low RSI scores were associated with improved overall survival. High M2 macrophage proportions may be involved in radioresistant glioblastoma.

JS08.7.A Integrative analysis of longitudinal genomic and non-genetic mechanisms of radiotherapy-resistance in glioma

Abstract Background Radiotherapy (RT) is a mainstay in the treatment of glioma patients, but RT-resistance is common. We have recently identified RT-associated genomic small deletion signatures, marking tumors that have developed resistance. Here, we used longitudinal molecular profiles to explore non-genetic mechanisms of RT-resistance. Material and Methods We have analyzed the multi-omic Glioma Longitudinal Analysis (GLASS) dataset, integrating DNA and RNA sequencing datasets from primary and post-treatment gliomas. Results We classified tumors as harboring temozolomide-associated hypermutation based on mutational burden increase (HM), and RT-associated signature based on small deletion burden increase (RTscars). By deconvoluting RNA profiles into cell state fractions, we observed an increase of a proliferating stem-like (PSL) cell state in 50% of patients with a RTscars+ signature which was significantly higher than RTscars-/HM- patients (P=8e-04, Fisher’s exact). PSL cell state increase associated with worse overall survival outcomes (P=1.5e-03, log-rank). We observed a significant correlation between the expression change of E2F cell cycle regulator genes (R=0.91, P=6.7e-04, Pearson) and EZH2 (R=0.81, P=8.3e-03) with the increase in the PSL cell state, nominating the E2F/EZH2-pathway as regulator of the PSL cell state. Furthermore, the RTscars signature was associated with an increase in frameshift neoantigens and significantly higher neoantigen burden at recurrence (P=2.4e-02, Kruskal-Wallis). This was accompanied by a significantly higher post-treatment T-cell fraction in RTscars+ tumors (P=8.2e-04, Wilcox), suggesting an increased T-cell infiltration in these patients. Conclusion Our analyses revealed a longitudinal increase in the proliferating stem-like cell state associated with RT-resistance and nominates the cell cycle pathway as an actionable target. The RT-associated deletion signature (RTscars) correlated with increased frameshift-neoantigens and T-cell fractions at recurrence, suggesting a potential benefit of a combinatorial immune-targeted therapy in this specific patient population.

Highlights from Recent Cancer Literature

Breaking Insights| August 03 2022 Highlights from Recent Cancer Literature Author & Article Information Online ISSN: 1538-7445 Print ISSN: 0008-5472 ©2022 American Association for Cancer Research2022American Association for Cancer Research Cancer Res (2022) 82 (15): 2659–2660. https://doi.org/10.1158/0008-5472.CAN-82-15-BI Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Cite Icon Cite Search Site Article Versions Icon Versions Version of Record August 3 2022 Citation Highlights from Recent Cancer Literature. Cancer Res 1 August 2022; 82 (15): 2659–2660. https://doi.org/10.1158/0008-5472.CAN-82-15-BI Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest Search Advanced Search As part of the Glioma Longitudinal Analysis (GLASS) Consortium aimed at understanding glioma evolution and therapeutic vulnerabilities, Varn and colleagues analyzed over 300 glioma patients wild-type or mutant for isocitrate dehydrogenase (IDH-wt and IDH-mut, respectively), with RNA and DNA sequencing data available at multiple time points. Longitudinally, the authors discovered that the IDH-wt gliomas switched from the classical subtype to the mesenchymal subtype, whereas IDH-mut gliomas remained proneural. Moreover, IDH-wt gliomas showed increases in oligodendrocytes, neural signaling pathways, and invasion. Whereas IDH-wt glioma typically present with CDKN2A deletions, IDH-mut tumors tended to acquire deletions in CDKN2A or amplifications of CCND2 at recurrence. Myeloid cells were also implicated in driving the mesenchymal subtype of glioma at recurrence, potentially through myeloid-tumor interactions such as those mediated by oncostatin M and its receptor. Expert Commentary: Diffuse gliomas undergo several changes in cell state and molecular... You do not currently have access to this content.

Comprehensive analysis of intratumoural heterogeneity of somatic copy number alterations in diffuse glioma reveals clonality-dependent prognostic patterns.

Intratumoural heterogeneity (ITH) has been implicated in tumour growth and progression as well as therapy resistance. However, the extent of ITH of somatic copy number alterations (ITH-SCNAs) as a result of tumour evolution and its influence on clinical outcomes in diffuse glioma (DG) remain poorly understood. We used an integrated computational method to infer clonal and subclonal SCNAs in 760 untreated primary DGs from The Cancer Genome Atlas. ITH-SCNAs at the genome-wide, peak (region with recurrent SCNAs) and gene level were calculated. We used the Kaplan-Meier estimators and Cox proportional hazards models to examine the associations of ITH-SCNA with patient outcomes. An independent cohort of 243 patients with paired initial and recurrent tumours from the Glioma Longitudinal Analysis Consortium was used for validation. DGs showed widespread ITH-SCNA, with a median of 25.5% of SCNAs identified as subclonal. We found that clonal SCNA burden had stronger prognostic power than total SCNAs in IDH-mutant astrocytoma. Coamplifications of receptor tyrosine kinases (RTKs) tended to be subclonal, and subclonal RTK amplification was significantly associated with high tumour proliferative potential and unfavourable clinical outcomes in IDH-wild-type glioblastoma. In addition, we found that the prognostic values of the peak-level SCNAs were related to their mutated clonal architecture, from which three clonality-dependent prognostic patterns of SCNAs were proposed, including clonal-dominant, subclonal-dominant and clonality-independent schemas. The systematic analysis of ITH-SCNAs in large cohorts of DGs highlighted the importance of considering the clonality of SCNA in discovery of tumour prognostic markers.

Abstract 2168: Longitudinal analysis of diffuse glioma reveals cell state dynamics at recurrence associated with changes in genetics and the microenvironment

Abstract Diffuse glioma is characterized by a poor prognosis and a universal resistance to therapy, though the evolutionary processes behind this resistance remain unclear. The Glioma Longitudinal Analysis (GLASS) Consortium has previously demonstrated that therapy-induced selective pressures shape the genetic evolution of glioma in a stochastic manner. However, single-cell studies have revealed that malignant glioma cells are highly plastic and transition their cell state in response to diverse challenges, including changes in the microenvironment and the administration of standard-of-care therapy. To interrogate the factors driving therapy resistance in diffuse glioma, we collected and analyzed RNA- and/or DNA-sequencing data from temporally separated tumor pairs of over 300 adult patients with IDH-wild-type or IDH-mutant glioma. In a subset of these tumor pairs, we additionally performed multiplex immunofluorescence to capture the spatial relationship between tumor cells and their microenvironment. Recurrent tumors exhibited diverse changes that were attributable to changes in histological features, somatic alterations, and microenvironment interactions. IDH-wild-type tumors overall were more invasive at recurrence and exhibited increased expression of neuronal signaling programs that reflected a possible role for neuronal interactions in promoting glioma progression. In contrast, recurrent IDH-mutant tumors exhibited a significant increase in proliferative expression programs that correlated with discrete genetic changes. Hypermutation and acquired CDKN2A homozygous deletions associated with an increase in proliferating stem-like malignant cells at recurrence in both glioma subtypes, reflecting active tumor expansion. A transition to the mesenchymal phenotype was associated with the presence of a specific myeloid cell state defined by unique ligand-receptor interactions with malignant cells, providing opportunities to target this transition through therapy. Collectively, our results uncover recurrence-associated changes in genetics and the microenvironment that can be targeted to shape disease progression following initial diagnosis. Citation Format: Frederick S. Varn, Kevin C. Johnson, Jan Martinek, Jason T. Huse, MacLean P. Nasrallah, Pieter Wesseling, Lee A. Cooper, Tathiane M. Malta, Taylor E. Wade, Thais S. Sabedot, Daniel J. Brat, Peter V. Gould, Adelheid Wöehrer, Kenneth Aldape, Azzam Ismail, Floris P. Barthel, Hoon Kim, Emre Kocakavuk, Nazia Ahmed, Kieron White, Santhosh Sivajothi, Indrani Datta, Jill S. Barnholtz-Sloan, Spyridon Bakas, Fulvio D'Angelo, Hui K. Gan, Luciano Garofano, Mustafa Khasraw, Simona Migliozzi, D. Ryan Ormond, Sun Ha Paek, Erwin G. Van Meir, Annemiek M. Walenkamp, Colin Watts, Michael Weller, Tobias Weiss, Karolina Palucka, Lucy F. Stead, Laila M. Poisson, Houtan Noushmehr, Antonio Iavarone, Roel G. Verhaak, The GLASS Consortium. Longitudinal analysis of diffuse glioma reveals cell state dynamics at recurrence associated with changes in genetics and the microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2168.

Upregulation of Atypical Cadherin FAT1 Promotes an Immunosuppressive Tumor Microenvironment via TGF-β.

FAT atypical cadherin 1 (FAT1) promotes glioblastoma (GBM) by promoting protumorigenic inflammatory cytokine expression in tumor cells. However, tumors also have an immunosuppressive microenvironment maintained by mediators such as transforming growth factor (TGF)-β cytokines. Here, we have studied the role of FAT1 in tumor immune suppression. Our preliminary TIMER2.0 analysis of The Cancer Genome Atlas (TCGA) database revealed an inverse correlation of FAT1 expression with infiltration of tumor-inhibiting immune cells (such as monocytes and T cells) and a positive correlation with tumor-promoting immune cells [such as myeloid-derived suppressor cells (MDSCs)] in various cancers. We have analyzed the role of FAT1 in modulating the expression of TGF-β1/2 in resected human gliomas, primary glioma cultures, and other cancer cell lines (U87MG, HepG2, Panc-1, and HeLa). Positive correlations of gene expression of FAT1 and TGF-β1/2 were observed in various cancers in TCGA, Glioma Longitudinal Analysis Consortium (GLASS), and Chinese Glioma Genome Atlas (CGGA) databases. Positive expression correlations of FAT1 were also found with TGF-β1/2 and Serpine1 (downstream target) in fresh-frozen GBM samples using q-PCR. siRNA-mediated FAT1 knockdown in cancer cell lines and in primary cultures led to decreased TGF-β1/2 expression/secretion as assessed by q-PCR, Western blotting, and ELISA. There was increased chemotaxis (transmigration) of THP-1 monocytes toward siFAT1-transfected tumor cell supernatant as a consequence of decreased TGF-β1/2 secretion. Reduced TGF-β1 expression was also observed in THP-1 cultured in conditioned media from FAT1-depleted glioma cells, thus contributing to immune suppression. In U87MG cells, decreased TGF-β1 upon FAT1 knockdown was mediated by miR-663a, a known modulator. FAT1 expression was also observed to correlate positively with the expression of surrogate markers of MDSCs [programmed death ligand-1 (PD-L1), PD-L2, and interleukin (IL)-10] in glioma tumors, suggesting a potential role of FAT1 in MDSC-mediated immunosuppression. Hence, our findings elaborate contributions of FAT1 to immune evasion, where FAT1 enables an immunosuppressive microenvironment in GBM and other cancers via TGF-β1/2.