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Radiotherapy is a major component of standard-of-care treatment for gliomas, the most prevalent type of brain tumor. However, resistance to radiotherapy remains a major concern. Identification of mechanisms governing radioresistance in gliomas could reveal improved therapeutic strategies for treating patients. Here, we report that mitochondrial metabolic pathways are suppressed in radioresistant gliomas through integrated analyses of transcriptomic data from glioma specimens and cell lines. Decreased expression of peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC1α), the key regulator of mitochondrial biogenesis and metabolism, correlated with glioma recurrence and predicted poor prognosis and response to radiotherapy of patients with glioma. The subpopulation of glioma cells with low-mitochondrial-mass exhibited reduced expression of PGC1α and enhanced resistance to radiotherapy treatment. Mechanistically, PGC1α was phosphorylated at serine (S) 636 by DNA-dependent protein kinase in response to irradiation. Phosphorylation at S636 promoted the degradation of PGC1α by facilitating its binding to the E3 ligase RNF34. Restoring PGC1α activity with expression of PGC1α S636A, a phosphorylation-resistant mutant, or a small-molecule PGC1α activator ZLN005 increased radiosensitivity of resistant glioma cells by reactivating mitochondria-related reactive oxygen species production and inducing apoptotic effects both in vitro and in vivo. In summary, this study identified a self-protective mechanism in glioma cells in which radiotherapy-induced degradation of PGC1α and suppression of mitochondrial biogenesis play a central role. Targeted activation of PGC1α could help improve response to radiotherapy in patients with glioma. Significance: Glioma cells reduce mitochondrial biogenesis by promoting PGC1α degradation to promote resistance to radiotherapy, indicating potential therapeutic strategies to enhance radiosensitivity.

Glioblastoma (GBM) is a complex ecosystem that includes a heterogeneous tumor population and the tumor-immune microenvironment (TIME), prominently containing tumor-associated macrophages (TAM) and microglia. Here, we demonstrated that β2-microglobulin (B2M), a subunit of the class I major histocompatibility complex (MHC-I), promotes the maintenance of stem-like neoplastic populations and reprograms the TIME to an anti-inflammatory, tumor-promoting state. B2M activated PI3K/AKT/mTOR signaling by interacting with PIP5K1A in GBM stem cells (GSC) and promoting MYC-induced secretion of transforming growth factor-β1 (TGFβ1). Inhibition of B2M attenuated GSC survival, self-renewal, and tumor growth. B2M-induced TGFβ1 secretion activated paracrine SMAD and PI3K/AKT signaling in TAMs and promoted an M2-like macrophage phenotype. These findings reveal tumor-promoting functions of B2M and suggest that targeting B2M or its downstream axis may provide an effective approach for treating GBM. Significance: β2-microglobulin signaling in glioblastoma cells activates a PI3K/AKT/MYC/TGFβ1 axis that maintains stem cells and induces M2-like macrophage polarization, highlighting potential therapeutic strategies for targeting tumor cells and the immunosuppressive microenvironment in glioblastoma.

Background Glioblastomas (GBMs) display striking dysregulation of metabolism to promote tumor growth. Glioblastoma stem cells (GSCs) adapt to regions of heterogeneous nutrient availability, yet display dependency on de novo cholesterol biosynthesis. The transcription factor Sterol Regulatory Element-Binding Protein 2 (SREBP2) regulates cholesterol biosynthesis enzymes and uptake receptors. Here, we investigate adaptive behavior of GSCs under different cholesterol supplies. Methods In silico analysis of patient tumors demonstrated enrichment of cholesterol synthesis associated with decreased angiogenesis. Comparative gene expression of cholesterol biosynthesis enzymes in paired GBM specimens and GSCs were performed. In vitro and in vivo loss-of-function genetic and pharmacologic assays were conducted to evaluate the effect of SREBP2 on GBM cholesterol biosynthesis, proliferation, and self-renewal. Chromatin immunoprecipitation quantitative real-time PCR was leveraged to map the regulation of SREBP2 to cholesterol biosynthesis enzymes and uptake receptors in GSCs. Results Cholesterol biosynthetic enzymes were expressed at higher levels in GBM tumor cores than in invasive margins. SREBP2 promoted cholesterol biosynthesis in GSCs, especially under starvation, as well as proliferation, self-renewal, and tumor growth. SREBP2 governed the balance between cholesterol biosynthesis and uptake in different nutrient conditions. Conclusions SREBP2 displays context-specific regulation of cholesterol biology based on its availability in the microenvironment with induction of cholesterol biosynthesis in the tumor core and uptake in the margin, informing a novel treatment strategy for GBM.

Tumor-associated macrophages (TAM) play a crucial role in immunosuppression. However, how TAMs are transformed into immunosuppressive phenotypes and influence the tumor microenvironment (TME) is not fully understood. Here, we utilized single-cell RNA sequencing and whole-exome sequencing data of glioblastoma (GBM) tissues and identified a subset of TAMs dually expressing macrophage and tumor signatures, which were termed double-positive TAMs. Double-positive TAMs tended to be bone marrow–derived macrophages (BMDM) and were characterized by immunosuppressive phenotypes. Phagocytosis of glioma cells by BMDMs in vitro generated double-positive TAMs with similar immunosuppressive phenotypes to double-positive TAMs in the GBM TME of patients. The double-positive TAMs were transformed into M2-like macrophages and drove immunosuppression by expressing immune-checkpoint proteins CD276, PD-L1, and PD-L2 and suppressing the proliferation of activated T cells. Together, glioma cell phagocytosis by BMDMs in the TME leads to the formation of double-positive TAMs with enhanced immunosuppressive phenotypes, shedding light on the processes driving TAM-mediated immunosuppression in GBM.Significance:Bone marrow–derived macrophages phagocytose glioblastoma cells to form double-positive cells, dually expressing macrophage and tumor signatures that are transformed into M2-like macrophages and drive immunosuppression.

The mechanism(s) of immune checkpoint inhibitor (ICI)-induced myasthenia gravis (MG), an immune-related adverse event (irAE) that is fatal and limits subsequent ICI use, remain unexplored. Here, through comparative genomic analysis, we identified a pathogenic p.S467C germline variant inSLC22A5in a thymoma case with ICI-induced MG, which was found to be associated with fatty acid oxidation through its regulation on L-carnitine levels. Remarkably, ICI rechallenge with L-carnitine pretreatment led to durable response without MG-related symptoms. Thus, we provide the first clinical evidence of genetic test-directed irAE management, which integrates individualized ICI treatment into the evolving paradigm of cancer management.

Treatment-resistant glioma stem cells are thought to propagate and drive growth of malignant gliomas, but their markers and our ability to target them specifically are not well understood. We demonstrate that podoplanin (PDPN) expression is an independent prognostic marker in gliomas across multiple independent patient cohorts comprising both high- and low-grade gliomas. Knockdown of PDPN radiosensitized glioma cell lines and glioma-stem-like cells (GSCs). Clonogenic assays and xenograft experiments revealed that PDPN expression was associated with radiotherapy resistance and tumor aggressiveness. We further demonstrate that knockdown of PDPN in GSCs in vivo is sufficient to improve overall survival in an intracranial xenograft mouse model. PDPN therefore identifies a subset of aggressive, treatment-resistant glioma cells responsible for radiation resistance and may serve as a novel therapeutic target.

Background Circulating tumor DNA (ctDNA) has been proven as a marker for detecting minimal residual diseases following systemic therapies in mid-to-late-stage non-small-cell lung cancers (NSCLCs) by multiple studies. However, fewer studies cast light on ctDNA-based MRD monitoring in early-to-mid-stage NSCLCs that received surgical resection as the standard of care. Methods We prospectively recruited 128 patients with stage I–III NSCLCs who received curative surgical resections in our Lung Cancer Tempo-spatial Heterogeneity prospective cohort. Plasma samples were collected before the surgery, 7 days after the surgery, and every 3 months thereafter. Targeted sequencing was performed on a total of 628 plasma samples and 645 matched tumor samples using a panel covering 425 cancer-associated genes. Tissue clonal phylogeny of each patient was reconstructed and used to guide ctDNA detection. Results The results demonstrated that ctDNA was more frequently detected in patients with higher stage diseases pre- and postsurgery. Positive ctDNA detection at as early as 7 days postsurgery identified high-risk patients with recurrence (HR = 3.90, P P Conclusions Longitudinal ctDNA surveillance integrating clonality information may stratify high-risk patients with disease recurrence and infer the evolutionary origin of ctDNA mutations.

Cutaneous T cell lymphoma (CTCL) is characterized by the accumulation of malignant T cells in the skin. However, advanced CTCL pathophysiology remains elusive and therapeutic options are limited due to the high intratumoral heterogeneity and complicated tumor microenvironment (TME). By comparing the single-cell RNA-seq (scRNA-seq) data from advanced CTCL patients and healthy controls (HCs), we showed that CTCL had a higher enrichment of T/NK and myeloid cells. Subpopulations of T cells (CXCR3

Objectives: Male breast cancer (MBC) is a rare aggressive malignant tumor that accounts for only 1% of all breast cancers and has a worse prognosis compared with female breast cancer (FBC). However, little is unveiled regarding the mechanisms of MBC occurrence and development, and there is a lack of relevant basic research and clinical studies at a global scale. The rapid development of single-cell transcriptomic technologies has helped uncover the heterogeneity within cell populations and tumor microenvironment, as well as the clonal evolution of breast cancer cells. This study aims to explore: (1) the tumor microenvironment of MBC; (2) the heterogeneity of male/female breast cancer cells and cell subgroup differences; (3) the potential specific therapeutic targets and biological characteristics differences between MBC and FBC. Methods: Single-cell transcriptome sequencing (scRNA-seq) and single cell sequencing of T cell receptors (scTCR-seq) combined with whole genome sequencing (WGS) and immunohistochemical staining were performed to deeply analysis the heterogeneity and clonal evolution of MBC, reveal the tumor microenvironment, especially the composition and function of tumor infiltrating immune cells. We performed scRNA-seq using fresh samples from three luminal type MBC patients and two post-menopausal luminal type FBC patients. Firstly, t-SNE dimensionality reduction analysis was conducted according to different genders, different patients and subpopulation of cells, and gene expression profiles were processed and visualized using heatmap. Moreover, through comprehensive analysis of scRNA-seq and scTCR-seq results of MBC and FBC, we further explored the characteristics, interrelation and dynamic changes of various types of cells in the tumor interior, and then mined novel specific therapeutic targets. Results: Based on the single cell sequencing results, we observed a striking difference in cell subsets distribution and gene expression profiles between MBC and FBC. The cell subsets were then sorted by the proportion of MBC tumor cells, and 7 subsets were selected as potential MBC specific cellular subpopulation. Further comparative analysis with Luminal type of post-menopausal FBC samples suggested that fatty acid synthase (FASN) expression was remarkably elevated in MBC, and the overall survival was significantly decreased in MBC patients with high FASN expression. Meanwhile, MBC tumors were found to have lower rates of immune cell infiltration and higher proportion of exhausted T cells, which were also validated by immunohistochemical staining of 160 cases of pathological sections. Surprisingly, we also observed the presence of some “intermediate cells”only in MBC specimens, which exhibited simultaneous expression of cancer epithelial cell marker KRT8 and T cell marker CD3E. These “intermediate cells”showed immunosuppressive status. Conclusion: In this study, we found that the high expression of FASN and the lipid metabolism pathway may play important roles in the MBC development, providing theoretical foundation and experimental basis for further clinical trials of FASN inhibitors, as well as precision therapy and prognostic evaluation for MBC. Meanwhile, further study of the “intermediate cells” may facilitate the discovery of novel tumor immune escape strategies. Our findings also played a role in promoting the comprehensive description of tumor microenvironment of MBC and the mechanism study of its occurrence and development. Key words: male breast cancer, single-cell sequencing, tumor microenvironment, intermediate cells, fatty acid synthase Citation Format: Handong Sun, Zhonglin Wang, Yanhui Zhu, Chuang Yang, Qianghu Wang, Jifu Wei, Qiang Ding. Single-cell transcriptomic analysis reveals tumor microenvironment in male breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-50.

EGFR is frequently amplified, mutated, and overexpressed in malignant gliomas. Yet the EGFR-targeted therapies have thus far produced only marginal clinical responses, and the underlying mechanism remains poorly understood. Using an inducible oncogenic EGFR-driven glioma mouse model system, our current study reveals that a small population of glioma cells can evade therapy-initiated apoptosis and potentiate relapse development by adopting a mesenchymal-like phenotypic state that no longer depends on oncogenic EGFR signaling. Transcriptome analyses of proximal and distal treatment responses identified TGFβ/YAP/Slug signaling cascade activation as a major regulatory mechanism that promotes therapy-induced glioma mesenchymal lineage transdifferentiation. Following anti-EGFR treatment, TGFβ secreted from stressed glioma cells acted to promote YAP nuclear translocation that stimulated upregulation of the pro-mesenchymal transcriptional factor SLUG and subsequent glioma lineage transdifferentiation toward a stable therapy-refractory state. Blockade of this adaptive response through suppression of TGFβ-mediated YAP activation significantly delayed anti-EGFR relapse and prolonged animal survival. Together, our findings shed new insight into EGFR-targeted therapy resistance and suggest that combinatorial therapies of targeting both EGFR and mechanisms underlying glioma lineage transdifferentiation could ultimately lead to deeper and more durable responses. Significance: This study demonstrates that molecular reprogramming and lineage transdifferentiation underlie anti-EGFR therapy resistance and are clinically relevant to the development of new combinatorial targeting strategies against malignant gliomas with aberrant EGFR signaling.

Chemoresistance and relapse are the leading cause of AML-related deaths. Utilizing single-cell RNA sequencing (scRNA-seq), we dissected the cellular states of bone marrow samples from primary refractory or short-term relapsed AML patients and defined the transcriptional intratumoral heterogeneity. We found that compared to proliferating stem/progenitor-like cells (PSPs), a subpopulation of quiescent stem-like cells (QSCs) were involved in the chemoresistance and poor outcomes of AML. By performing longitudinal scRNA-seq analyses, we demonstrated that PSPs were reprogrammed to obtain a QSC-like expression pattern during chemotherapy in refractory AML patients, characterized by the upregulation of CD52 and LGALS1 expression. Flow cytometric analysis further confirmed that the preexisting CD99+CD49d+CD52+Galectin-1+ (QSCs) cells at diagnosis were associated with chemoresistance, and these cells were further enriched in the residual AML cells of refractory patients. Interaction of CD52-SIGLEC10 between QSCs and monocytes may contribute to immune evading and poor outcomes. Furthermore, we identified that LGALS1 was a promising target for chemoresistant AML, and LGALS1 inhibitor could help eliminate QSCs and enhance the chemotherapy in patient-derived primary AML cells, cell lines, and AML xenograft models. Our results will facilitate a better understanding of the AML chemoresistance mechanism and the development of novel therapeutic strategies for relapsed/refractory AML patients.

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: Chronic lymphocytic leukemia (CLL) is a highly heterogeneous malignant lymphoproliferative B-cell disorder that can be treated using ibrutinib, a Bruton’s tyrosine kinase inhibitor. However, the ibrutinib resistance of CLL patients has caused widespread concerns, necessitating the development of novel treatment strategies. Methods: Here, we identified lectin galactoside-binding soluble 1 (LGALS1) and lymphocyte-activating gene 3 (LAG3) as potential markers for ibrutinib-resistant CLL using single-cell RNA sequencing (scRNA-seq), and the results were validated in an ibrutinib-resistant CLL cell line (MEC1-IR) and primary cells from CLL patients. Marker-gene expression was detected while functional analyses were conducted with or without OTX008, a selective Galectin-1 inhibitor. ScRNA-seq revealed that the biological features, gene expression profiles, and clonal signatures of peripheral blood mononuclear cells (PBMCs) from patients with ibrutinib-resistant CLL were distinct from those displayed by PBMCs from ibrutinib-sensitive patients. Findings: A close correlation between LGALS1 and LAG3 expression was observed and these factors were found to be highly expressed in ibrutinib-resistant CLL, with diagnostic and prognostic stratification, indicating that they may serve as drivers of ibrutinib-resistant CLL. Concordantly, LGALS1 and LAG3 expression was higher in ibrutinib-resistant CLL cells and primary cells, and OTX008 suppressed proliferation and induced apoptosis in both cells. Interpretation: LGALS1 and LAG3 gene panel is promising indicator of ibrutinib-sensitivity and prognosis marker of CLL. LGALS1 inhibitor OTX008 is effective in CLL patients, both those naive to and those resistant to ibrutinib. This work highlights the pathogenic mechanism and represents a novel therapeutic target for CLL. Funding Statement: This work was supported by grants from National Natural Science Foundation of China (Grant #81720108002, 81970146, 81770166, 81972358 and 91959113), Key Research & Development Program of Jiangsu Province (Grant #BE2017733), Basic Research Program of Jiangsu Province (Grant #BK20180036), Jiangsu Province’s Medical Elite Programme (Grant #ZDRCA2016022), Project of National Key Clinical Specialty, Jiangsu Provincial Special Program of Medical Science (Grant #BE2017751), Six Top Talent Project of Jiangsu Province (Grant #WSN-001). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: Human sample study was approved by the Medical Ethics Committee of First Affiliated Hospital of Nanjing Medical University. Written informed consent was obtained from all participants.

BackgroundCoronavirus disease 2019 (COVID-19) has rapidly spread worldwide. Systematic analysis of lung cancer survivors at molecular and clinical levels is warranted to understand the disease course and clinical characteristics.MethodsA single-center, retrospective cohort study was conducted in 65 patients with COVID-19 from Wuhan Huoshenshan Hospital, of which 13 patients were diagnosed with lung cancer. The study was conducted from February 4 to April 11, 2020.ResultsDuring the course of treatment, lung cancer survivors infected with severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) had shorter median time from symptom onset to hospitalization (P = 0.016) and longer clinical symptom remission time (P = 0.020) than non-cancer individuals. No differences were observed among indicators such as time from symptom onset to hospitalization and symptom remission time between medium-term and short-term survivors. The expression of ACE2 (P = 0.013) and TMPRSS2 (P <0.001) was elevated in lung cancer survivors as compared with that in non-cancer individuals.ConclusionsACE2 and TMPRSS2 levels were higher at resection margins of lung cancer survivors than those in normal tissues of non-cancerous individuals and may serve as factors responsible for the high susceptibility to COVID-19 among lung cancer survivors. Lung cancer patients diagnosed with COVID-19, including medium-term survivors, have worse outcomes than the general population.

Novel coronavirus disease 2019 (COVID-19) is an emerging, rapidly evolving crisis, and the ability to predict prognosis for individual COVID-19 patient is important for guiding treatment. Laboratory examinations were repeatedly measured during hospitalization for COVID-19 patients, which provide the possibility for the individualized early prediction of prognosis. However, previous studies mainly focused on risk prediction based on laboratory measurements at one time point, ignoring disease progression and changes of biomarkers over time. By using historical regression trees (HTREEs), a novel machine learning method, and joint modeling technique, we modeled the longitudinal trajectories of laboratory biomarkers and made dynamically predictions on individual prognosis for 1997 COVID-19 patients. In the discovery phase, based on 358 COVID-19 patients admitted between 10 January and 18 February 2020 from Tongji Hospital, HTREE model identified a set of important variables including 14 prognostic biomarkers. With the trajectories of those biomarkers through 5-day, 10-day and 15-day, the joint model had a good performance in discriminating the survived and deceased COVID-19 patients (mean AUCs of 88.81, 84.81 and 85.62% for the discovery set). The predictive model was successfully validated in two independent datasets (mean AUCs of 87.61, 87.55 and 87.03% for validation the first dataset including 112 patients, 94.97, 95.78 and 94.63% for the second validation dataset including 1527 patients, respectively). In conclusion, our study identified important biomarkers associated with the prognosis of COVID-19 patients, characterized the time-to-event process and obtained dynamic predictions at the individual level.

Background Coronavirus disease 2019 (COVID-19) has rapidly spread worldwide. Systematic analysis of lung cancer survivors at molecular and clinical levels is warranted to understand the disease course and clinical characteristics. Methods A single-center, retrospective cohort study was conducted in 65 patients with COVID-19 from Wuhan Huoshenshan Hospital, of which 13 patients were diagnosed with lung cancer. The study was conducted from February 4 to April 11, 2020. Results During the course of treatment, lung cancer survivors infected with severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) had shorter median time from symptom onset to hospitalization (P = 0.016) and longer clinical symptom remission time (P = 0.020) than non-cancer individuals. No differences were observed among indicators such as time from symptom onset to hospitalization and symptom remission time between medium-term and short-term survivors. The expression of ACE2 (P = 0.013) and TMPRSS2 (P

Purpose: Glioblastoma is the most frequent and lethal primary brain tumor. Development of novel therapies relies on the availability of relevant preclinical models. We have established a panel of 96 glioblastoma patient-derived xenografts (PDX) and undertaken its genomic and phenotypic characterization. Experimental Design: PDXs were established from glioblastoma, IDH-wildtype (n = 93), glioblastoma, IDH-mutant (n = 2), diffuse midline glioma, H3 K27M-mutant (n = 1), and both primary (n = 60) and recurrent (n = 34) tumors. Tumor growth rates, histopathology, and treatment response were characterized. Integrated molecular profiling was performed by whole-exome sequencing (WES, n = 83), RNA-sequencing (n = 68), and genome-wide methylation profiling (n = 76). WES data from 24 patient tumors was compared with derivative models. Results: PDXs recapitulate many key phenotypic and molecular features of patient tumors. Orthotopic PDXs show characteristic tumor morphology and invasion patterns, but largely lack microvascular proliferation and necrosis. PDXs capture common and rare molecular drivers, including alterations of TERT, EGFR, PTEN, TP53, BRAF, and IDH1, most at frequencies comparable with human glioblastoma. However, PDGFRA amplification was absent. RNA-sequencing and genome-wide methylation profiling demonstrated broad representation of glioblastoma molecular subtypes. MGMT promoter methylation correlated with increased survival in response to temozolomide. WES of 24 matched patient tumors showed preservation of most genetic driver alterations, including EGFR amplification. However, in four patient–PDX pairs, driver alterations were gained or lost on engraftment, consistent with clonal selection. Conclusions: Our PDX panel captures the molecular heterogeneity of glioblastoma and recapitulates many salient genetic and phenotypic features. All models and genomic data are openly available to investigators.

BACKGROUND: Virtually all low-grade gliomas (LGGs) will progress to high-grade gliomas (HGGs), including glioblastoma, the most common malignant primary brain tumor in adults. A key regulator of immunosuppression, fibrinogen-like protein 2 (FGL2), may play an important role in the malignant transformation of LGG to HGG. We sought to determine the mechanism of FGL2 on tumor progression and to show that inhibiting FGL2 expression had a therapeutic effect. METHODS: We analyzed human gliomas that had progressed from low- to high-grade for FGL2 expression. We modeled FGL2 overexpression in an immunocompetent genetically engineered mouse model to determine its effect on tumor progression. Tumors and their associated microenvironments were analyzed for their immune cell infiltration. Mice were treated with an FGL2 antibody to determine a therapeutic effect. Statistical tests were two-sided. RESULTS: We identified increased expression of FGL2 in surgically resected tumors that progressed from low to high grade (n = 10). The Cancer Genome Atlas data showed that LGG cases with overexpression of FGL2 (n = 195) had statistically significantly shorter survival (median = 62.9 months) compared with cases with low expression (n = 325, median = 94.4 months, P < .001). In a murine glioma model, HGGs induced with FGL2 exhibited a mesenchymal phenotype and increased CD4(+) forkhead box P3 (FoxP3)(+) Treg cells, implicating immunosuppression as a mechanism for tumor progression. Macrophages in these tumors were skewed toward the immunosuppressive M2 phenotype. Depletion of Treg cells with anti-FGL2 statistically significantly prolonged survival in mice compared with controls (n = 11 per group, median survival = 90 days vs 62 days, P = .004), shifted the phenotype from mesenchymal HGG to proneural LGG, and decreased M2 macrophage skewing. CONCLUSIONS: FGL2 facilitates glioma progression from low to high grade. Suppressing FGL2 expression holds therapeutic promise for halting malignant transformation in glioma.

Previous studies have demonstrated that Chinese lung adenocarcinoma (LUAD) patients have unique genetic characteristics, however, the specific genomic features relating to the development and treatment of LUAD in the Chinese population are not fully understood. Here, we applied the ultra-deep targeted sequencing to 66 Chinese LUAD samples, accompanied by comparative analysis with 162 Caucasian LUAD in The Cancer Genome Atlas. We focused on the 68 recurrently mutated genes and results revealed that the panel-based tumor mutational burden (pTMB) is significantly higher in the Chinese LUAD ( P=0.0017). Additionally, the percentage of smoking-associated C>A transversion is significantly lower in Chinese LUAD (15.5% vs. 39.7%, P=5.69×10 -27), while C>T transition is more frequent in Chinese LUAD (35.8% vs. 25.7%, P=2.67×10 -5), which indicated the ethnic difference in mutation types. Notably, novel driver genes ( GNAS and JAK1) that are peculiar to Chinese LUAD were identified, and a more convergent distribution of mutations was observed in the Chinese cohort ( P=0.012) compared with scattered mutations in Caucasian LUAD. Our results present a distinct genomic profile of Chinese LUAD compared to Caucasians LUAD and elucidate the ethnic difference in mutation distribution besides the type and rate.

We describe a comprehensive genomic characterization of adrenocortical carcinoma (ACC). Using this dataset, we expand the catalogue of known ACC driver genes to include PRKAR1A, RPL22, TERF2, CCNE1, and NF1. Genome wide DNA copy-number analysis revealed frequent occurrence of massive DNA loss followed by whole-genome doubling (WGD), which was associated with aggressive clinical course, suggesting WGD is a hallmark of disease progression. Corroborating this hypothesis were increased TERT expression, decreased telomere length, and activation of cell-cycle programs. Integrated subtype analysis identified three ACC subtypes with distinct clinical outcome and molecular alterations which could be captured by a 68-CpG probe DNA-methylation signature, proposing a strategy for clinical stratification of patients based on molecular markers.

Epidermal growth factor receptor (EGFR) is frequently amplified, mutated and overexpressed in malignant gliomas. Our investigation of the proximal and distal responses to EGFR inhibition identified the molecular mechanisms for the therapeutic resistance of EGFR inhibition in gliomas. Oncogenic EGFRviii-dependent gliomas initially showed cytotoxic responses upon removal of oncogenic cue. However, during the initial regression, a subset of tumor cells with activated mesenchymal subtype gene expression program emerged by lineage reprogramming which promoted tumor relapse in the absence of EGFRviii signaling. This lineage switch is dependent on YAP1 activation as a response to therapy, and is a key to EGFRviii independent tumor growth. YAP signature stratifies overall survival of recurrent glioma patients. Inhibition of YAP1 activation suppressed mesenchymal gene expression and significantly delayed recurrence of gliomas. Our findings provide mechanisms underlying inefficacies of EGFR targeted therapy in glioblastoma and suggest a new combinatorial targeting of EGFR and YAP for deeper and more durable responses.

The telomere reverse transcriptase promoter (TERTp) is frequently mutated in malignant gliomas, particularly glioblastomas (GBM, 85%), and is involved in maintaining telomere length. TERTp mutations are prognostic for patient survival. Given the evolving use of Infinium DNA methylation arrays in assaying biomarkers in glioma, we aimed to develop an accurate predictor of TERTp mutations in gliomas using this platform. We analyzed TCGA lower grade glioma (LGG) and GBM available with both TERTp sequencing and Infinium 450k data. Samples were split into training and testing set to determine predictor accuracy. Probes with potential quality issues were excluded and probe selection performed using an elastic net algorithm. Probes were selected across the genome, not limited to the TERTp locus. A logistic regression model with binomial distribution was built using the final optimized model, constructed based on 1000 CpG probes, all distinct from TERTp. Prediction accuracy was 100% (179/179, AUC=1) in the training set and 97.5% (116/119) in the test set. Comparison of the normalized frequency of selected probes by chromosome revealed enrichment for chromosomes 20 (0.4987%) and 18 (0.4859%). The majority of probes enriched in CpG islands (53.6%), followed by open sea (22.9%), shore (17.4%), and shelf (6.1%). Relative to known genes, probe enrichment occurred predominantly near transcriptional start sites (within 200–1500 bp, 31.0%), followed by gene bodies (26.3%) and intergenic regions (18.3%). Quite unexpectedly, probes within the predictor do not map near TERT or other telomere maintenance factors nor fell within known methylation signatures, such as G-CIMP, suggesting that TERTp mutation is associated with novel epigenetic changes. Moreover, this biomarker permits rapid and cost effective detection of TERTp mutation using the widely used Infinium platform.

Glioblastoma (GBM) is a primary brain cancer that contains populations of stem-like cancer cells (GSCs) that home to specialized perivascular niches comprised of vascular endothelial cells, pericytes and extracellular matrix (ECM)-rich basement membranes. Interactions with their niche influence GSC self-renewal, differentiation and drug resistance, although the pathways underlying these events remain largely unknown. Here, we report that the adhesion receptor alphavbeta8 integrin and its ECM-bound latent transforming growth factor beta1 (TGFbeta1) protein ligand play central roles in promoting niche co-option and GBM initiation. alphavbeta8 integrin is highly expressed in GSCs and is essential for self-renewal and lineage commitment in vitro. Inhibition of alphavbeta8 integrin adhesion and signaling using function-blocking antibodies inhibit sphere formation in vitro. Fractionation of beta8high cells from freshly resected human GBM samples also reveals a requirement for this integrin in sphere formation in vitro and tumor growth and invasion in vivo. In contrast, fractionated beta8low GBM cells fail to form spheroids in vitro and do not generate tumors after intracranial implantation. Whole transcriptome sequencing of primary GBM cells reveals that alphavbeta8 integrin promotes tumor initiation in the brain, in large part, by driving TGFbeta1-induced DNA replication and mitotic checkpoint progression. We show that beta8high cells activate latent TGFbeta1 in the microenvironment, resulting in enhanced TGFbeta receptor signaling in beta8low GBM cells and tumor endothelial cells via Smad transcription factors. Collectively, these data identify the alphavbeta8 integrin-TGFbeta1 signaling axis as crucial for exploitation of the perivascular niche and identify potential therapeutic targets for inhibiting tumor growth and progression in patients with GBM.

Glioblastoma expression subtypes have been previously been associated with genomic abnormalities, treatment response, and differences in tumor microenvironment. We leveraged IDH wild-type glioblastomas, derivative neurospheres, and single cell gene expression profiles to define three tumor-intrinsic transcriptional subtypes designated as proneural, mesenchymal, and classical, a revision of the previously reported TCGA subtypes. Transcriptomic subtype multiplicity correlated with increased intratumoral heterogeneity and the presence of tumor microenvironment. In silico cell sorting identified macrophages/microglia, CD4+ T lymphocytes, and neutrophils in the glioma microenvironment. NF1 deficiency resulted in increased tumor-associated macrophages/microglia infiltration. Comparison of matching primary and recurrent gliomas elucidated treatment-induced phenotypic tumor evolution, including expression subtype switching, in 45% of our cohort as well as associations between microenvironmental components and treatment response. Gene signature-based tumor microenvironment inference revealed a decrease in invading monocytes and a subtype-dependent increase in macrophages/microglia cells upon disease recurrence. Hypermutation at diagnosis or at recurrence was associated with CD8+ T cell enrichment. Frequency of M2 macrophage detection was associated with short-term relapse after radiation therapy. Our study provides a comprehensive transcriptional and cellular landscape of IDH wild-type glioblastoma during treatment modulated tumor evolution. Characterization of the evolving glioblastoma transcriptome and tumor microenvironment aids in designing more effective immunotherapy trials.

Patients with glioblastoma (GBM) have a universally poor prognosis and are in urgent need of effective treatment strategies. Recent advances in sequencing techniques unraveled the complete genomic landscape of GBMs and revealed profound heterogeneity of individual tumors even at the single cell level. Genomic profiling has detected epidermal growth factor receptor (EGFR) gene alterations in more than half of GBMs. Major genetic events include amplification and mutation of EGFR. Yet, treatment strategies targeting EGFR have thus far failed in clinical trials. In this review, we discuss the clonal and functional heterogeneity of EGFRs in GBM development and critically reassess the potential of EGFRs as therapeutic targets.

We leveraged IDH wild-type glioblastomas, derivative neurospheres, and single-cell gene expression profiles to define three tumor-intrinsic transcriptional subtypes designated as proneural, mesenchymal, and classical. Transcriptomic subtype multiplicity correlated with increased intratumoral heterogeneity and presence of tumor microenvironment. In silico cell sorting identified macrophages/microglia, CD4+ T lymphocytes, and neutrophils in the glioma microenvironment. NF1 deficiency resulted in increased tumor-associated macrophages/microglia infiltration. Longitudinal transcriptome analysis showed that expression subtype is retained in 55% of cases. Gene signature-based tumor microenvironment inference revealed a decrease in invading monocytes and a subtype-dependent increase in macrophages/microglia cells upon disease recurrence. Hypermutation at diagnosis or at recurrence associated with CD8+ Tcell enrichment. Frequency of M2 macrophages detection associated with short-term relapse after radiation therapy.

// Keeratikarn Boonyawan 1,3 , Kenneth R. Hess 2 , Jie Yang 3 , Lihong Long 3 , Qianghu Wang 3 , Ravesanker Ezhilarasan 3 , Alessandra Auia 4 , Kristin D. Alfaro-Munoz 5 , John F. de Groot 5 , Krishna P. Bhat 4 and Erik P. Sulman 3 1 Department of Radiation Oncology, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand 2 Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 3 Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 4 Department of Translational Molecular Pathology and Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 5 Department of Neuro-Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA Correspondence to: Erik P. Sulman, email: // Keywords : glioblastoma, platelets, thrombocytosis, prognosis, survival Received : June 05, 2017 Accepted : August 07, 2017 Published : October 12, 2017 Abstract Background: Thrombocytosis is triggered by and promotes tumor growth. The relationship between the change in circulating platelets after chemoradiation therapy (CRT) or adjuvant temozolomide (TMZ) and survival in glioblastoma remains unclear. We hypothesized that an increase in platelets after these treatments would be predictive of a shorter survival. Methods: We retrospectively reviewed data on 122 patients with newly diagnosed, pathologically proven glioblastoma who had been treated with surgery, followed by CRT and adjuvant TMZ, from 2007 to 2016. The association between the changes in blood count levels and survival was analyzed by the log-rank test. To adjust for confounding, we performed a multivariate analysis using known prognostic co-variates. Results: Patients were dichotomized on the basis of the relative change in platelets after CRT from the baseline: ≤30% increase, low ( n = 101) vs >30% increase, high ( n = 12). The median survival for high vs . low platelets were 11 vs 28 months ( p = 0.0062). No significant survival differences were observed on the basis of platelet changes during adjuvant TMZ. Similarly, changes in lymphocyte counts were not significantly prognostic. On multivariate analysis, MGMT, performance status, and an increase in platelets after CRT were significantly associated with survival (HR for platelets, 4.5; 95% confidence interval, 1.6-12.6). Conclusions: Increased platelet counts after CRT are predictive of poor survival in glioblastoma. The effect is platelet specific and does not reflect bone marrow changes, as lymphocyte changes were not significantly prognostic. These results suggest an interaction between platelets and tumor aggressiveness. Thus, platelets serve as a novel, minimally invasive liquid biopsy for predicting outcome.

Glioblastoma multiforme (GBM), the most common and deadliest brain tumor with a median survival of 12–15 months, has been characterized by robust angiogenesis, high invasiveness, and universal recurrence. Genomics and transcriptomics studies defined three GBM subtypes (proneuronal, classical, mesenchymal), which are associated with genomic abnormalities, treatment response, and diversity in tumor microenvironment. However, molecular mechanisms of subtype-specific genotype in contribution to tumor phenotype are less understood. We have previously generated a de novo human GBM model by inactivation of p53 and activation of AKT signaling pathways in human neural stem/progenitor cells (hNSCs). Further characterization of this model indicates that the tumors recapitulate GBM’s classical histopathogical features and exhibit a molecular profile resembling the mesenchymal subtype. Comparative transcriptomic analysis revealed that YKL-40/CHI3L1, a secreted glycoprotein, is significantly upregulated during AKT activation induced malignant transformation of hNSCs in vitro and in vivo. Pharmacological inhibition of PI3K/AKT/mTOR signaling pathway results in decreasing YKL-40 mRNA expression and protein secretion level. Mechanistically, motif enrichment analysis and functional validation revealed that YKL-40 is bidirectionally regulated by a gene regulatory network with two transcription factors, BACH2 and CEBPB. Furthermore, YKL-40 significantly activates AKT-S6 and MAPK-ERK kinase signaling pathways, resulting in enhancing tumor cells proliferation, neurosphere and soft-agar colony formation, and tumor progression. In vivo silencing YKL-40 attenuates tumor angiogenesis and prolongs animal survival in the orthotopic xenograft mouse models. Interestingly, knocking down YKL-40 expression decreases microglia/macrophages infiltration in tumor mass. Both in vitro and in vivo validation indicate that PI3K/AKT/YKL-40 mediates tumor-associated microglia/macrophages recruitment and activation during tumor progression. Taken together, these results suggest that auto- and paracrine signaling of PI3K/AKT/YKL-40 affect tumor cells and tumor microenvironment contributing to glioblastoma progression, indicating YKL-40 as a predictive biomarker for evaluation of anti- PI3K/AKT/mTOR therapy, and a potential drug target for the treatment of this devastating disease.

Although significant progresses in molecular oncology are being made using conventional cell lines, most therapies still fail in phase III clinical trials. Patient-derived models are being used more frequently as they are more faithfully representing the genomic features of primary tumors. However, one by one test of each model from large biobanks is extremely economy and time consuming. In this study, each of a panel of patient-derived glioblastoma stem cell (GSC) models was uniquely tagged by a lentiviral Cas9D10A and paired-gRNA targetable unique reporter (CAPTURE) barcoding system. Barcoded GSCs were then pooled evenly and following by radiation treatment (RT) in vitro. Amplicon sequencing was employed to count the barcodes distribution, which represent the relative cell number. The results showed that this approach faithfully identified the RT resistant GSCs from a mixing pool when comparing to the results from canonical clonogenic assay. In addition, a fluorescence marker will be switched by delivery of corresponding barcodes targeting CRISPR so that we can re-isolate interested cell models from the treated pool for investigating the treatment sensitivity and resistance mechanism. This study will provide a robust approach for therapeutic discovery take advantage of patient-derived models from large biobanks. Citation Format: Ze-yan Zhang, Yingwen Ding, Ravesanker Ezhilarasan, Jie Yang, Lihong Long, Lawrence Bronk, Qianghu Wang, Erik P. Sulman. High-throughput evaluation of treatment response in patient-derived glioma stem cell models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1917.

Cancers are composed of heterogeneous cell populations and the clonal evolution of these cells is one of the key reasons for treatment resistance and tumor recurrence. A fundamental challenge in studying clonal evolution in these tumors is the difficulty in capturing the phenotype-associated (e.g. treatment resistant) sub-populations from the heterogeneous population. We developed an approach to individually barcode and isolate specific cell subpopulations by constructing a Cas9D10A and paired-gRNA targetable unique reporter (CAPTURE) barcoding library with up to 36 million unique barcodes. This approach enabled us to uniquely barcode > 1 million cells, track barcode distribution following treatment and then isolate the resistant subpopulation using the subpopulation-specific barcode. Proof of principle studies showed that specific barcoding cells, even at as low as 0.1%, could be efficiently isolated using barcode targeting and cell sorting. Applied our barcoding approach, we found that A375 cell acquired BRAF inhibitor resistance both from pre-exiting and late-emerging mechanisms. Colony formation experiments showed that the isolated subpopulations were indeed resistant clones. Whole exome, transcriptome and methylome analysis were applied to study the captured subpopulations. Our CAPTURE barcoding approach will enable the identification of the both pre-exiting and late-emerging genetic or epigenetic changes driving treatment resistance. Citation Format: Ze-yan Zhang, Ravesanker Ezhilarasan, Yingwen Ding, Qianghu Wang, Jie Yang, Lihong Long, Roel G. Verhaak, Erik P. Sulman. Development of a CRISPR-Cas9D10A targetable, high-complexity, single-cell barcoding approach for capture of treatment resistant subclones from heterogeneous cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4698.

PURPOSE: Genomic alterations classify cancers into subtypes with distinct clinical management and prognoses. Molecular classification of gliomas, the most common and lethal primary brain tumor, define tumors into distinct biologic and clinical entities. Mutation of isocitrate dehydrogenese (IDH) is associated with hypermethylation of CpG sites in gene promotors. Other alterations, including telomerase (TERTp) mutation, ATRX mutation, chromosome 1p/19q co-deletion (1p19q codel), and gene expression subtype (Classical/CL, Mesenchymal/MES, Proneural/PN), have yet to be associated with an epigenetic signature. We hypothesized that DNA methylation signatures would classify gliomas based on genetic alterations and give insight into the development of each subtype. The resulting platform functions as a unified diagnostic (UniD) with high processivity applicable to clinical diagnosis and generalizable across molecular oncology. METHODS: Machine learning algorithms were applied to whole methylome data to build classifiers for IDH, TERTp, and ATRX mutations; 1p19q codel and gene expression subtype. Models were validated with data from a phase III trial of anaplastic gliomas. RESULTS: Individual models were generated and prediction accuracies for IDH, TERTp, and ATRX mutations, and 1p19q codel were 100%, 98.3%, 90.48%, and 99.21% in test set and 89.9%, 82.8%, 92.47%, and 89.99% in the validation clinical trial data. The prediction model for gene expression subtype, a previously reported classifier enriched for characteristic somatic alterations, achieved 72% accuracy. Analysis of the misclassified cases revealed that the characteristic alterations associated with the expression subtypes were more correctly classified by methylation than by gene expression. Methylation-determined CL subtype showed high EGFR (p-value = 0.04) and amplification (p-value = 0.00019) compared to transcriptional MES samples, and low expression (p-value = 0.08) and amplification (p-value = 0.056) in methylation-determined MES but transcriptional CL samples. CONCLUSION: Distinct DNA methylation signatures were associated with key somatic genomic alterations in gliomas. It improved the existing classifiers based on gene expression and provided a unique clinical diagnostic platform for rapid determination of glioma subtype at the time of patient diagnosis. The extensive and significant relationship between cancer epigenetic signatures indicates that this approach would have broad applicability to other tumor types and lead to similar unified diagnostic platforms. A R package (UniD) is provided for implantation of this diagnostic platform. Citation Format: Jie Yang, Qianghu Wang, Ravesanker Ezhilarasan, Lihong Long, Benedikt Wiestler, Wolfgang Wick, Yinsen Miao, Erik Sulman. DNA-methylation based epigenetic signatures predict and deconvolute somatic genomic alterations in gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4238.

Astrocytomas are the most common primary human brain tumors. Receptor tyrosine kinases (RTKs), including tyrosine receptor kinase B (TrkB, also known as tropomyosin-related kinase B; encoded by neurotrophic tyrosine kinase receptor type 2 [NTRK2]), are frequently mutated by rearrangement/fusion in high-grade and low-grade astrocytomas. We found that activated TrkB can contribute to the development of astrocytoma and might serve as a therapeutic target in this tumor type.To identify RTKs capable of inducing astrocytoma formation, a library of human tyrosine kinases was screened for the ability to transform murine Ink4aActivated TrkB cooperated with Ink4a/Arf loss to induce the formation of astrocytomas through a mechanism mediated by activation of signal transducer and activator of transcription 3 (STAT3). TrkB activation positively correlated with Ccl2 expression. TrkB-induced astrocytomas remained dependent on TrkB signaling for survival, highlighting a role of NTRK2 as an addictive oncogene. Furthermore, the QKI-NTRK2 fusion associated with human astrocytoma transformed Ink4aOur findings provide evidence that constitutively activated NTRK2 alleles, notably the human tumor-associated QKI-NTRK2 fusion, can cooperate with Ink4a/Arf loss to drive astrocytoma formation. Therefore, we propose NTRK2 as a potential therapeutic target in the subset of astrocytoma patients defined by QKI-NTRK2 fusion.

Previous studies have shown that insertion/deletion polymorphisms in the angiotensin-converting enzyme (ACE) gene and the endothelial nitric oxide synthase (eNOS) gene are associated with Henoch-Schönlein purpura nephritis (HSPN). However, further studies are required to prove the relationship between HSPN and ACE and eNOS single nucleotide polymorphisms (SNPs). We studied six ACE SNPs and two eNOS SNPs by genotyping HSPN patients. Statistical analyses indicate that four ACE SNPs and two eNOS SNPs are associated with HSPN susceptibility. A cumulative effect analysis suggested that an increased number of unfavourable genotypes may lead to an increased risk of HSPN. By comparing alleles, genotypes and haplotypes that are associated with lupus nephritis (LN) and HSPN, we found genetic heterogeneity between HSPN and LN.

The World Health Organization (WHO) classification and grading system attempts to predict the clinical course of meningiomas based on morphological parameters. However, because of high interobserver variation of some criteria, more reliable prognostic markers are required. Here, we assessed the TERT promoter for mutations in the hotspot regions C228T and C250T in meningioma samples from 252 patients. Mutations were detected in 16 samples (6.4% across the cohort, 1.7%, 5.7%, and 20.0% of WHO grade I, II, and III cases, respectively). Data were analyzed by t test, Fisher's exact test, log-rank test, and Cox proportional hazard model. All statistical tests were two-sided. Within a mean follow-up time in surviving patients of 68.1 months, TERT promoter mutations were statistically significantly associated with shorter time to progression (P < .001). Median time to progression among mutant cases was 10.1 months compared with 179.0 months among wild-type cases. Our results indicate that the inclusion of molecular data (ie, analysis of TERT promoter status) into a histologically and genetically integrated classification and grading system for meningiomas increases prognostic power. Consequently, we propose to incorporate the assessment of TERT promoter status in upcoming grading schemes for meningioma.

Adult diffuse gliomas are a diverse class of brain tumors that ultimately result in fatal malignant progression. The highly variable disease progression intervals and poor treatment response observed in glioma have been explained, in part, by the extent of heterogeneity within and across gliomas. Somatic structural variants such as deletions, duplications, insertions, inversions, translocations, and extrachromosomal DNA contribute to glioma heterogeneity by generating genomic instability, a hallmark of cancer genomes. While comprehensive approaches such as whole genome sequencing have begun to catalog the glioma structural variant landscape, the molecular mechanisms by which they contribute to tumor progression remains poorly understood. Here, we identified genomic rearrangements in the whole genomes of 147 gliomas and glioma cell lines. Among the whole-genome sequencing samples were 62 glioblastomas (TCGA), 52 low-grade gliomas (TCGA), and our own set of 33 patient-derived glioma sphere-forming cells. On average, glioblastomas demonstrated a greater frequency of high-confidence structural variants (162 per tumor) than low-grade gliomas (130 per tumor, P=0.02) with two glioblastomas displaying evidence of chromothripsis. Across the World Heath Organization's molecular classifications, the IDH-mutant 1p/19q co-deleted tumors demonstrated the lowest structural variant burden when compared with both IDH-wildtype and IDH-mutant non-co-deleted tumors. Structural variant frequencies were found to be similar between patient tumors and an independent set of patient-derived cell lines. Across all samples, a majority of the structural variant breakpoints were found in intronic (42%) and intergenic regions (55%) suggesting that rearrangements predominantly impact distal regulatory regions. To investigate the functional consequences of structural variants in these regions we integrated these samples with available RNA-sequencing and epigenetic data to characterize deregulated transcriptional circuits. Analyses examining the association between genomic rearrangements and chromatin states to determine potential mechanisms of oncogene activation in select tumor and cell line samples are now underway. Together, our study demonstrates the importance of integrating structural variant calls from whole genome sequencing with expression and epigenetic data to define functional genomic rearrangements. Citation Format: Kevin C. Johnson, Floris P. Barthel, Ming Tang, Samirkumar Amin, Qianghu Wang, Erik P. Sulman, Kunal Rai, Roel G.W Verhaak. Identification and functional characterization of glioma structural variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4355.

Glioma sphere-forming cells (GSCs) are important models for understanding glioma biology and development of novel therapies. We performed clonogenic assays to explore the relationship between methylation status and radiation response in twelve IDH wild type GSCs irradiated with 2-, 4-, and 6-Gy ionizing γ-radiation. The survival fraction at 4Gy and 6Gy (SF4 and SF6, respectively) was used to dichotomize GSCs as either radiation-sensitive or resistant. Seven (out of 12, 58%) GSCs were sensitive to radiation and five (out of 12, 42%) were resistant with SF4 > 30% and SF6 > 10%. DNA CpG methylomes of the GSCs were profiled using Illumina Infinium 450K methylation bead arrays. We observed that 304,458 out of 465,844 methylation probes (65.4%) showed increased methylation in radiation-resistant GSCs relative to radiation-sensitive GSCs (Fisher Test p-value < 1e-15). This translated to significant increases in DNA methylation of the promoter regions of 17,735 out of 21,214 (83.6%) genes in radiation-resistant compared to radiation-sensitive GSCs. RNA sequencing of the GSCs showed that 57% of genes were expressed at lower levels in radiation-resistant relative to radiation-sensitive GSCs. Through GSEA, we observed that fifteen of sixteen oxidative stress genes were methylated in radiation-resistant GSCs (p-value = 0.019), suggesting an association between radiation-resistance and reactive oxygen species metabolism (ROS). To validate our finding, we derived a methylation signature from the two GSC radiation response groups and used this to classify TCGA cases that received radiotherapy into a responders and non-responders group. We found that survival was significantly different between the two groups (median survival 84 vs. 61 weeks; HR 1.64 adjusting for patient age, p-value < 0.008), suggesting that a methylation signature could be used to predict clinical response to radiation treatment. Our study shows that GSCs can be used to determine clinical and functional properties of GBM.

Glioblastoma (GBM) is the most frequently occurring malignant primary tumor of the central nervous system, and despite aggressive clinical intervention, GBM is uniformly fatal. Data from multiple expression profiling experiments suggest that the type-I integral membrane glycoprotein podoplanin (PDPN) is an important prognostic marker in astrocytic gliomas independent of a number of relevant clinical factors. We recently discovered that radiotherapy (RT) resistance of GBM is associated with global DNA hypermethylation and hypothesized that PDPN may be involved in this phenomenon. Indeed, we found that PDPN knockdown sensitized glioma sphere-forming cells (GSCs) to RT in vitro. Furthermore, upon profiling PDPN-sorted GSC subpopulations, enrichment analysis revealed that PDPN upregulates polycomb repressive complex (PRC) activity to promote EZH2-driven global DNA hypermethylation. EZH2 is known to directly control DNA methylation, and we have previously shown that EZH2 protects GSCs from radiation-induced cell death. This is the first report implicating PDPN in EZH2-mediated RT resistance, and we believe the membrane protein may be a meaningful therapeutic target in RT resistant GBM. In summary, PDPN is a novel prognostic marker for individuals with astrocytic gliomas and promotes EZH2-mediated RT resistance in GSCs.

Glioma sphere-forming cells (GSCs) are important in glioblastoma (GBM) initiation, maintenance, and treatment resistance. We performed whole exome and transcriptome sequencing, DNA methylation profiling, DNA copy number determination, and functional characterization of 43 GSCs and matching tumors. Comparative analyses revealed that GSCs recapitulate the molecular landscape of GBM and provide a unique means for discovering the inter- and intra-tumor heterogeneity of GBM. We performed clonogenic assays to explore the relationship between methylation status and radiation response in twelve IDH wild type GSCs irradiated with 2-, 4-, and 6-Gy ionizing radiation. The survival fraction at 4Gy and 6Gy (SF4 and SF6, respectively) were used to dichotomize GSCs as either radiation-sensitive or resistant. DNA CpG methylomes of the GSCs were profiled using Infinium 450K methylation beadchip arrays. We observed that 304,458 out of 465,844 methylation probes (65.4%) showed increased methylation in radiation-resistant relative to radiation-sensitive GSCs (Fisher’s Exact Test, p < 1e-15). Using GSEA, we observed that fifteen of sixteen oxidative stress genes were methylated in radiation-resistant GSCs (p-value=0.019), suggesting an association between radiation-resistance and reactive oxygen species metabolism (ROS). To validate our finding, we derived a methylation signature differentiating the two GSC radiation response groups and used this to classify TCGA cases that received radiotherapy into a responder and non-responder group. We found that survival was significantly different between the two groups (median survival 84 vs. 61 weeks; HR 1.64 adjusting for patient age, p-value

Malignant gliomas are composed of heterogeneous cell populations and the clonal evolution of these cells is one of the key reasons for treatment resistance and tumor recurrence. A fundamental challenge in studying clonal evolution in these tumors is the difficulty in isolating the phenotype-associated (e.g. treatment resistant) sub-populations from the heterogeneous population. We developed an approach to individually barcode and isolate specific cell subpopulations using a CRISPR (clustered, regularly interspaced, short palindromic repeat)-Cas9D10A targetable, single-cell barcoding library with a complexity of 36 million unique barcodes. This approach enabled us to uniquely barcode > 1 million cells, identify enriching barcoded sub-populations following treatment and then isolate the resistant subpopulation using the subpopulation-specific barcode. Each barcode sequence carrying two guide RNAs so that CRISPR-Cas9D10A targeting can lead to “switching” (i.e. frameshift) of an EGFP (Enhanced Green Fluorescent Protein) reporter. Thus, targeted cells lose their EGFP signal and can be specifically isolated by cell sorting. Proof of principle studies showed that specific barcoding cells could be efficiently targeted to turn off EGFP and subpopulations isolated. Subsequent large-scale implementation of this approach has been performed to isolate resistant subpopulations following radiation and temozolomide. Our approach will lead to identification of the both pre-existing and acquired specific somatic genetic or epigenetic changes driving treatment resistance in patients with malignant gliomas.

Gliomas are the most common type of brain tumor in both children and adults. Several low-grade gliomas (LGG) have the ability to progress into more aggressive tumors -high-grade gliomas (HGG) including glioblastoma (GB). Although patients harboring a LGG may survive for years, after the tumor transforms to HGG, life expectancy rapidly declines to 12 to 15 months in adults and 40 months in children. Thus, inhibiting this process of malignant transformation (MT) is an attractive therapeutic strategy because of the more indolent course associated with LGGs. Immune response plays a critical role in surveillance against malignant transformation. Our previous study shows that fibrinogen-like protein 2 (FGL2) is a key hub of tumor-mediated immune suppression. Hence, we investigated the role of FGL2 in promoting tumor progression from LGG to HGG in glioma. Analysis of TCGA expression data showed that increased FGL2 expression is associated with poorer survival in LGG and GB patients. And there is a positive correlation of expression level between FGL2 and mesenchymal glioma marker CD44, and a negative correlation between FGL2 and proneural glioma marker OLIG2. Engineered expression of FGL2 in a PDGFB-dependent mouse model of oligodendroglioma, a common glioma subtype, yielded a significantly higher rate of HGGs (72% vs 29%, p=0.034) and poorer-symptom free survival (63 vs 90 days, p=0.003) than PDGFB expression alone. And HGGs from FGL2 + PDGFB expressing mice exhibited a distinct mesenchymal phenotype validating TCGA data. Further, FGL2 induced high numbers of CD4+FoxP3+ cells from an early time point of tumor formation underscoring its role in tumor progression. And FGL2 overexpression educated M2 skew in the tumors characterized by high expression of Iba1 and Arginase1 in macrophages. Finally, treatment with anti-FGL2 antibody significantly improves survival in mice, shifts the phenotype from mesenchymal HGG to proneural LGG, and rescues M2 macrophage skewing. Our results show that FGL2 is critical for malignant progression of glioma by inducing immunosuppression in tumor microenvironment, and raise the potential of FGL2 to be a promising target to suppress/reverse glioma progression and provide survival benefit in clinical. Citation Format: Khatri Latha, Jun Yan, Yuhui Yang, Loyola V. Gressot, Lingyuan Kong, Ganiraju Manyam, Ravesanker Ezhilarasan, Qianghu Wang, Erik P. Sulman, Jingda Xu, Richard E. Davis, Suyun Huang, Gregory N. Fuller, Arvind Rao, Amy B. Heimberger, Shulin Li, Ganesh Rao. Fibrinogen-like protein 2 drives malignant tumor progression in glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5606. doi:10.1158/1538-7445.AM2017-5606

Prognostic and predictive molecular diagnostics for patients with gliomas typically rely on multiple assays, requiring large amounts of tissue and high cost. DNA methylation has been utilized to identify prognostic subsets and high-throughput platforms exist that are suitable for archival tissue. Therefore, we developed a unified and integrated diagnostic pipeline that can assess multiple prognostic and predictive biomarkers using only the Illumina Infinium Methylation array. This pipeline includes two parts: data processing and diagnostic biomarkers. Data processing starts from the raw data and followed with quantitative sample, probe, and batch quality control. The diagnostic biomarkers include a glioma methylation assay that predicts radiation response (GaMA); tumor classification enriched for TCGA expression subclasses; copy number alterations including phosphatase and tensin homolog (PTEN) loss, epidermal growth factor receptor (EGFR) amplification, and chromosomes 1p/19q co-deletion; CpG island methylation phenotype (G-CIMP); isocitrate dehydrogenase (IDH) mutation, and O6-methylguanine-DNA methyltransferase (MGMT) promoter hypermethylation. WHO grade II-IV gliomas were analyzed in both publically available and institutional datasets. A signature was identified to effectively distinguished radiation resistant from radiation sensitive glioma stem-like cells (GSCs). Signature has been applied to 272 TCGA GBM samples from patients who received standard radiotherapy (RT). The survival analysis showed that the subgroup with RT-sensitive and RT-resistant have significant difference in survival time (log-rank test p-value = 0.0016). Gene expression subclasses prediction biomarker was build by using the revised TCGA gene expression subclasses as gold standard. The prediction accuracy in test data set was 83.5% in the homogeneous subgroup, 71.0% in the semi-heterogeneous subgroup, and 62.1% in the heterogeneous subgroup. The prediction accuracy decreased as tumor heterogeneity increased. Certain copy number alteration events were predicted by developing specific signatures. Revised methylation signatures were developed for IDH mutation and G-CIMP status respectively, which can identified 99% of those samples. 230 GBM samples with 450k data available were tested with MGMT methylation-specific real-time PCR for MGMT methylation status. The methylation-based MGMT prediction accuracy reached about 90%. In summary, we have developed a single, FFPE-based pipeline for unified and integrated determination of multiple biomarkers of malignant glioma. Citation Format: Jie Yang, Qianghu Wang, Lihong Long, Ravesanker Ezhilarasan, Erik Sulman. UniD: unified and integrated diagnostic pipeline for malignant gliomas based on DNA methylation data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3348. doi:10.1158/1538-7445.AM2017-3348

e13527 Background: Thrombocytosis is associated with poor survival in several malignancies and is both triggered by and promoting of tumor growth. We hypothesized that an increase in circulating platelets following standard temozolamide (TMZ) CRT and/or adjuvant TMZ would predict for decreased survival in patients with glioblastoma (GBM). Methods: We reviewed data of 122 patients with newly diagnosed, pathological-proven GBM from 2007 to 2016 with documented complete blood counts before the start of CRT, after CRT, before adjuvant TMZ, and after a minimum 2 cycles of adjuvant TMZ. The association between changes in blood count levels and patient survival was analyzed by log-rank test. Multivariate analysis was performed using known prognostic co-variates, including MGMT methylation, patient age, and performance status (PS). Results: The mean change in platelets before and after CRT was -46 k/µl (range -387 to 179 k/µl) and before and after adjuvant TMZ was -25 k/µl (range -408 to 143 k/µl). As a reference, the mean change in lymphocytes was -5.7 k/µl (range -0.7 to 1.3 k/µl) and -0.0 k/µl (range -1.6 to 0.9 k/µl) for CRT and adjuvant TMZ, respectively. Patients were dichotomized based on the relative change in platelets and lymphocytes from baseline (≤30% increase, “low”, n = 101 vs > 30% increase, “high”, n = 12). The median survival of low patients vs. high patients was 30 vs 13 months (p = 0.006). Potential confounders, such as infection or thrombosis, were not associated with the high group. However, no significant survival difference was observed between groups based on platelet changes during adjuvant TMZ. Similarly, changes in lymphocyte counts were not significantly prognostic. In multivariate analysis, extent of resection, MGMT status, PS and the increase in platelets after CRT were significantly associated with survival (HR for platelets 4.0, 95% CI 1.6-10.1). Conclusions: Increased platelet counts after CRT predict for poor survival in patients with GBM. The effect is platelet specific and does not reflect general bone marrow changes, as lymphocyte changes were not significantly prognostic. These results suggest a potential interaction between platelets and tumor aggressiveness.

Glioma sphere-forming cells (GSCs) play important roles in tumor maintenance and chemotherapy and radiation resistance, and thus serve as important models for the development of novel therapies and for understanding tumor biology. We hypothesized that genomic analyses of GSCs in the absence of normal stroma would identify novel somatic alterations not previously reported from whole tumor specimen analyses. We analyzed 42 GSCs, derived at the time of surgical resection, and their matched tumors of origin. Unsupervised consensus clustering by non-negative matrix factorization (CNMF) using whole transcriptome sequencing data identified 5 transcriptional classes. Comparison to published TCGA GBM subtypes using single-sample gene set enrichment analysis (ssGSEA) showed that 9 GSCs perfectly matched with TCGA mesenchymal subtype. The other 4 subgroups of GSCs mapped to proneural and/or classical subtypes, while none of the GSC classes matched with the neural TCGA subtype. Overall, 73% of GSCs matched the TCGA subtype of paried tumor, suggesting transcriptional consistency between GSCs and their parental tumor. A total of 88 candidate gene fusions were also identified in the GSCs, including the previously reported FGFR3-TACC3 and EGFR-SEP14 fusions. DNA copy number analysis indicated recurrent regions of frequent loss or gain, such as EGFR, MYC, and MYCN amplification, and deletion of PTEN and CDKN2A. Whole exome sequencing based analysis identified 3,918 somatic mutations in 22 GSCs. PTEN, TP53, EGFR and 17 additional genes were identified as significantly altered. Our results indicate genomic consistency between GSCs and their tumors of origin, suggesting the suitability of GSCs as a model for preclinical testing. Additional gene fusions and somatic mutations were detected in GSCs suggesting the important role of these cell models in identifying infrequent events only detectable in pure tumor cell populations.

The mesenchymal (Mes) subtype of glioblastoma (MesGBM) correlates with poor patient prognosis. Surprisingly, agents that target mesenchymal tumors, such as the antiangiogenesis drug bevacizumab, failed to show a benefit in MesGBM. The expression of MesGBM might result from both cells from the surrounding microenvironment or from the tumor cells themselves. We hypothesized that MesGBM driven by Mes-expressing tumor cells would show increased aggressiveness compared to those driven by Mes-expressing stroma. We examined 42 glioma sphere-forming cell (GSCs) lines, derived from fresh surgical specimens, by performing whole transcriptome sequencing, copy number analysis, and whole exome sequencing and compared the GSCs to the tumors from which they were derived. GSCs aggressiveness was determined by in vivo survival of orthotopic xenografted mice and in vitro radiation survival. The TCGA expression subtype of the GSCs and their matched tumor was determined by single sample gene set enrichment analysis (ssGSEA). Of the 15 tumors belonging to the mesenchymal subtype, 4 of the matched GSCs were also mesenchymal (concordant pairs, CP) and the remaining 11 GSCs were either proneural or classical subtypes (discordant pairs, DP). The normalized mesenchymal scores of the 4 CP tumors (mean 0.90, range 0.80-0.98) was higher than those the 11 DP tumors (mean 0.75, range 0.69-0.90). We determined tumor purity based on sequencing data and found it significantly higher in DP tumors than CP, suggesting a stromal contribution to the Mes signature in the DP tumors. In addition, EGFR amplification occurred in 36% of DP tumors, and 0% of TP tumors. CP tumors showed increased radiation resistance and decreased in vivo survival compared to DP tumors. In summary, stromal-driven mesenchymal tumors showed decrease aggressiveness compared to tumor cell-driven MesGBM, suggesting that therapeutics targeting the microenvironment, such as anti-angiogenesis drugs, may have a greater role in tumors where the stromal contribution dominates.

Radiation therapy (RT) remains one of the most effective treatments for patients with GBM and has been repeatedly demonstrated to improve survival; yet response to RT is variable. We explored the relationship between methylation status and radiation response to develop a predictor of RT response using the epigenetic data of glioma sphere-forming cells (GSCs). The DNA methylomes of 42 GSCs were profiled using Illumina Infinium 450K methylation bead arrays. 15 GSCs were irradiated with 2-, 4-, and 6-Gy RT and response determined using clonogenic assays. We discovered 168 CpG probes capable of distinguishing sensitive from resistant GSCs. To validate, we analyzed 362 TCGA GBM samples, 272 that received standard 60Gy RT and 90 treated with low or no RT. Using the glioblastoma methylation assay (GaMA) signature, we classified the samples as either RT sensitive or resistant. Survival was significantly different between the predicted sensitive vs resistant patients for those treated with standard RT (median 21.0m vs 14.7m, p Citation Format: Qianghu Wang, Ravesanker Ezhilarasan, Eskil Eskilsson, Joy Gumin, Jie Yang, Mona Jaffari, Ming Tang, Kenneth D. Aldape, Frederick F. Lang, Roel G.W. Verhaak, Erik P. Sulman. A glioblastoma methylation assay (GaMA) developedfrom genomic analysis of glioma spheroid cultures predicts response toradiation therapy in patients with glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1646.

Glioblastoma (GBM) is the most common primary malignant brain tumor and is invariably lethal. Past studies have shown that classification based on genomic data can potentially help in understanding this devastating tumor better. In 2013, TCGA proposed a six-subgroup classification (M1-M4, G-CIMP, and M6) based on methylation profiles. However, with the exception of G-CIMP, the subgroups did not show significant molecular differences. We aimed to develop a more informative and robust methylation classification system to better integrate molecular profiles of GBM. By using the publicly available Illumina Infinium 450K Bead Array methylation data on 414 primary GBM samples from the TCGA, a new classification was generated. The classification was validated with Glioma stem-like cells (GSCs) (n = 42). The beta-mixture quantile normalization (BMIQ) method was applied and the most variant probes were selected based on the median absolute deviation (MAD) value. Samples and probes were clustered with the consensus non-negative matrix factorization (CNMF) method and 78 representative probes were selected for each of the four clusters obtained. CNMF clustering was reapplied to all samples with these 312 probes and the final classification model was developed (named as cluster S1 to S4). Each methylation cluster exhibited unique copy number variants and somatic mutation patterns, better reflecting the biology of GBM compared to the reported TCGA methylation classification. Additionally, each methylation cluster showed enrichment of specific gene expression subtypes: S1-mesenchymal (82.1%), S2-classical (64.4%), S3-CIMP positive proneural (90.6%), and S4-CIMP negative proneural (74.3%). The GSC validation cohort reflected identical gene expression subtype, copy number variation, and somatic mutation enrichment. In conclusion, we have developed a biologically relevant methylation classification based on Illumina 450K Bead Arrays which successfully correlates to expression subtype. This classification can be used as a diagnostic tool to assign appropriate therapies based on the genomic targets identified in the methylated classes.