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Glioblastomas (GBM) are aggressive brain tumors with extensive intratumoral heterogeneity that contributes to treatment resistance. Spatial characterization of GBMs could provide insights into the role of the brain tumor microenvironment in regulating intratumoral heterogeneity. Here, we performed spatial transcriptomic and single-cell analyses of the mouse and human GBM microenvironment to dissect the impact of distinct anatomical regions of brains on GBM. In a syngeneic GBM mouse model, spatial transcriptomics revealed that numerous extracellular matrix (ECM) molecules, including biglycan, were elevated in areas infiltrated with brain tumor–initiating cells (BTIC). Single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin using sequencing showed that ECM molecules were differentially expressed by GBM cells based on their differentiation and cellular programming phenotypes. Exogeneous biglycan or overexpression of biglycan resulted in a higher proliferation rate of BTICs, which was associated mechanistically with low-density lipoprotein receptor-related protein 6 (LRP6) binding and activation of the Wnt/β-catenin pathway. Biglycan-overexpressing BTICs developed into larger tumors and displayed mesenchymal phenotypes when implanted intracranially in mice. This study points to the spatial heterogeneity of ECM molecules in GBM and suggests that the biglycan–LRP6 axis could be a therapeutic target to curb tumor growth. Significance: Characterization of the spatial heterogeneity of glioblastoma identifies regulators of brain tumor–initiating cells and tumor growth that could serve as candidates for therapeutic interventions to improve the prognosis of patients.

Oncolytic viruses (OVs) are an emerging cancer therapeutic that are intended to act by selectively targeting and lysing cancerous cells and by stimulating anti-tumour immune responses, while leaving normal cells mainly unaffected. Reovirus is a well-studied OV that is undergoing advanced clinical trials and has received FDA approval in selected circumstances. However, the mechanisms governing reoviral selectivity are not well characterised despite many years of effort, including those in our accompanying paper where we characterize pathways that do not consistently modulate reoviral cytolysis. We have earlier shown that reovirus is capable of infecting and lysing both certain types of cancer cells and also cancer stem cells, and here we demonstrate its ability to also infect and kill healthy pluripotent stem cells (PSCs). This led us to hypothesize that pathways responsible for stemness may constitute a novel route for the modulation of reoviral tropism. We find that reovirus is capable of killing both murine and human embryonic and induced pluripotent stem cells. Differentiation of PSCs alters the cells’ reoviral-permissive state to a resistant one. In a breast cancer cell line that was resistant to reoviral oncolysis, induction of pluripotency programming rendered the cells permissive to cytolysis. Bioinformatic analysis indicates that expression of the Yamanaka pluripotency factors may be associated with regulating reoviral selectivity. Mechanistic insights from these studies will be useful for the advancement of reoviral oncolytic therapy.

Additional analysis LRP6 and the Wnt/β-catenin pathway activation in BTICs.

Graphical abstract. A multi-omics analysis of the glioblastoma tumor microenvironment revealed that biglycan, predominantly expressed by inflammatory BTICs, drives tumor growth. The graphics were generated with Biorender.com.

Characterization of patients whose resected tissues were used for confocal microscopy.

Legends of Supplementary figures and tables.

Glioblastomas (GBM) are aggressive brain tumors with extensive intratumoral heterogeneity that contributes to treatment resistance. Spatial characterization of GBMs could provide insights into the role of the brain tumor microenvironment in regulating intratumoral heterogeneity. Here, we performed spatial transcriptomic and single-cell analyses of the mouse and human GBM microenvironment to dissect the impact of distinct anatomical regions of brains on GBM. In a syngeneic GBM mouse model, spatial transcriptomics revealed that numerous extracellular matrix (ECM) molecules, including biglycan, were elevated in areas infiltrated with brain tumor–initiating cells (BTIC). Single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin using sequencing showed that ECM molecules were differentially expressed by GBM cells based on their differentiation and cellular programming phenotypes. Exogeneous biglycan or overexpression of biglycan resulted in a higher proliferation rate of BTICs, which was associated mechanistically with low-density lipoprotein receptor-related protein 6 (LRP6) binding and activation of the Wnt/β-catenin pathway. Biglycan-overexpressing BTICs developed into larger tumors and displayed mesenchymal phenotypes when implanted intracranially in mice. This study points to the spatial heterogeneity of ECM molecules in GBM and suggests that the biglycan–LRP6 axis could be a therapeutic target to curb tumor growth.Significance:Characterization of the spatial heterogeneity of glioblastoma identifies regulators of brain tumor–initiating cells and tumor growth that could serve as candidates for therapeutic interventions to improve the prognosis of patients.

Supplementary Figures S1-S5. Supplementary Tables S1-S5. Figure S1. Comparison of SNV mutation signatures across histological and molecular subtypes. Figure S2. Treatment timelines and radiographic outcomes. Figure S3. Mutation signature stability metrics and comparisons to known signatures. Figure S4. Structural variant mutation sigantures. Figure S5. Comparison of mutation burden between aging-driven and non-aging-driven breast tumours. Table S1. Kruskal-Wallis tests. Table S2. HRDetect prediction test metrics. Table S3. Area under the curve of HRD signatures in platinum response prediction. Table S4. Logistic regression model odds ratios of clinical improvement on platinum. Table S5. Accession IDs of genome datasets submitted to the European Genome-Phenome Archive.

Table S5: Data from this study have been submitted to the European Genome-Phenome Archive (EGA) (www.ebi.ac.uk/ega/home) under the study accession number EGAS00001001159. This table provides the accession numbers for each dataset included in this study.

Purpose: Recent studies have identified mutation signatures of homologous recombination deficiency (HRD) in over 20% of breast cancers, as well as pancreatic, ovarian, and gastric cancers. There is an urgent need to understand the clinical implications of HRD signatures. Whereas BRCA1/2 mutations confer sensitivity to platinum-based chemotherapies, it is not yet clear whether mutation signatures can independently predict platinum response.Experimental Design: In this observational study, we sequenced tumor whole genomes (100× depth) and matched normals (60×) of 93 advanced-stage breast cancers (33 platinum-treated). We computed a published metric called HRDetect, independently trained to predict BRCA1/2 status, and assessed its capacity to predict outcomes on platinum-based chemotherapies. Clinical endpoints were overall survival (OS), total duration on platinum-based therapy (TDT), and radiographic evidence of clinical improvement (CI).Results: HRDetect predicted BRCA1/2 status with an area under the curve (AUC) of 0.94 and optimal threshold of 0.7. Elevated HRDetect was also significantly associated with CI on platinum-based therapy (AUC = 0.89; P = 0.006) with the same optimal threshold, even after adjusting for BRCA1/2 mutation status and treatment timing. HRDetect scores over 0.7 were associated with a 3-month extended median TDT (P = 0.0003) and 1.3-year extended median OS (P = 0.04).Conclusions: Our findings not only independently validate HRDetect, but also provide the first evidence of its association with platinum response in advanced breast cancer. We demonstrate that HRD mutation signatures may offer clinically relevant information independently of BRCA1/2 mutation status and hope this work will guide the development of clinical trials. Clin Cancer Res; 23(24); 7521–30. ©2017 AACR.

We performed the first integrative single-cell and spatial transcriptomic analysis on HPV-negative oral squamous cell carcinoma (OSCC) to comprehensively characterize tumor core (TC) and leading edge (LE) transcriptional architectures. We show that the TC and LE are characterized by unique transcriptional profiles, cellular compositions, and ligand-receptor interactions. We demonstrate that LE regions are conserved across multiple cancers while TC states are more tissue specific. Additionally, we found our LE gene signature is associated with worse clinical outcomes while the TC gene signature is associated with improved prognosis across multiple cancer types. Finally, using an in silico modeling approach, we describe spatially-regulated patterns of cell development in OSCC that are predictably associated with drug response. Our work provides pan-cancer insights into TC and LE biologies, a platform for data exploration (http://www.pboselab.ca/spatial_OSCC/) and is foundational for developing novel targeted therapies.

Glioblastomas (GBMs) are aggressive brain tumors with extensive intratumoral heterogeneity. Here, we used spatial transcriptomics and single-cell ATAC-seq to dissect the transcriptome of distinct anatomical regions of the tumor microenvironment. We identified numerous extracellular matrix (ECM) molecules including biglycan elevated in areas infiltrated with brain tumor-initiating cells (BTICs). Single-cell RNA sequencing showed that the ECM molecules were differentially expressed by cells including injury response versus developmental BTICs. Exogeneous biglycan or overexpression of biglycan resulted in a higher proliferation rate of BTICs, and this was associated mechanistically with LDL receptor-related protein 6 (LRP6) binding and activation of the Wnt/β-catenin pathway. Biglycan-overexpressing BTICs grew to a larger tumor mass when implanted intracranially in mice. This study points to the spatial heterogeneity of ECM molecules in the GBM microenvironment and suggests biglycan-LRP6 axis as a therapeutic target to curb GBM growth.

Glioblastomas (GBMs) are aggressive brain tumors with extensive intratumoral heterogeneity. Here, we used spatial transcriptomics and single-cell ATAC-seq to dissect the transcriptome of distinct anatomical regions of the tumor microenvironment. We identified numerous extracellular matrix (ECM) molecules including biglycan elevated in areas infiltrated with brain tumor-initiating cells (BTICs). Single-cell RNA sequencing showed that the ECM molecules were differentially expressed by cells including injury response versus developmental BTICs. Exogeneous biglycan or overexpression of biglycan resulted in a higher proliferation rate of BTICs, and this was associated mechanistically with LDL receptor-related protein 6 (LRP6) binding and activation of the Wnt/β-catenin pathway. Biglycan-overexpressing BTICs grew to a larger tumor mass and higher mesenchymal phenotypes when implanted intracranially in mice. This study points to the spatial heterogeneity of ECM molecules in the GBM microenvironment and suggests biglycan-LRP6 axis as a therapeutic target to curb GBM growth. Citation Format: Reza Mirzaei, Charlotte D’Mello, Marina Liu, Ana Nikolic, Mehul Kumar, Frank Visser, Pinaki Bose, Marco Gallo, Wee Yong. Single cell spatial analysis identifies regulators of brain tumor initiating cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2435.

The focus of this paper is on labour arrangements characterized by indenture, bondage, or slavery, and the design of credit market policies to free these labourers. We examine bonded or indentured labourers that pay a fee to their patrons or employers for freedom from bondage in a multi-period game between the patron, labourers, and other lenders, where a labourer borrows the fee from a third party lender. Labourers are heterogeneous: they differ in their cost of committing strategic default in repaying the loan. The patron’s determination of the fee, and the possibility of strategic default by the labourer, are two key features that drive our results. Productivity gains are greatest when labourers with both high and low default costs pay the fee. However, there also exists a less socially optimal outcome where only the low default cost labourer participates. Importantly, we also develop a theory of the existence and persistence of bondage.

PurposeHuman papillomavirus-associated (HPV+) head and neck squamous cell carcinoma (HNSCC) is the fastest rising cancer in North America. There is significant interest in treatment de-escalation for these patients given the generally favourable prognosis. However, 15-30% of patients recur after primary treatment, reflecting a need for improved risk-stratification tools. We sought to develop a molecular test to predict the survival of patients with newly diagnosed HPV+ HNSCC.MethodsWe created a prognostic score (UWO3) that was successfully validated in six independent cohorts comprising 906 patients, including blinded retrospective and prospective external validations. Transcriptomic data from two aggressive radiation de-escalation cohorts were used to assess the ability of UWO3 to identify patients who recur. Multivariate Cox models were used to assess the associations between the UWO3 immune class and outcomes.ResultsA three-gene immune score classified patients into three immune classes (immune rich, mixed, or immune desert) and was strongly associated with disease-free survival in six datasets, including large retrospective and prospective datasets. Pooled analysis demonstrated that the immune rich group had superior disease-free survival at 5 years to the immune desert (HR= 9.0, 95% CI 3.2–25.5, P=3.6×10−5) and mixed (HR=6.4, 95%CI 2.2–18.7, P=0.006) groups after adjusting for age, sex, smoking status, and AJCC8 clinical stage. Finally, UWO3 was able to identify patients from two treatment de-escalation cohorts who remain disease-free after aggressive de-escalation to 30 Gy radiation.ConclusionsThe UWO3 immune score could enable biomarker-driven clinical decision-making for patients with HPV+ HNSCC based on robust outcome prediction across six independent cohorts. The superior survival of immune rich patients supports de-intensification strategies, while the inferior outcomes of the immune desert patients suggest the potential for intensification and/or immunotherapy. Prospective de-escalation and intensification clinical trials are currently being planned.

209 Background: NEPC includes both pure small cell carcinoma and mixed tumors with varying degrees of adenocarcinoma and neuroendocrine histology. It arises de novo or is treatment associated (TA) post androgen deprivation therapy. Clinical outcome data and prognostic biomarkers are limited and were thus explored. Methods: Patients with high grade prostate cancer and morphologic and/or immunohistochemical (IHC) NEPC features were included in this retrospective multicentre study. Clinical stage, Gleason score, and serum biomarkers were recorded. Kaplan-Meier method and log-rank test calculated and compared overall survival (OS) from time of NEPC diagnosis.Cox proportional hazards regression assessed prognostic impact of serum biomarkers at diagnosis and de novo vs TA status, adjusting for clinical stage and castration resistance. Results: 135 NEPC cases were identified. 124 (92%) were mixed tumors. 56 (41%) arose de novo. 79 (59%) were TA. 77% of those with a Gleason score (N=85/110) were grade group 5. Median PSA pre-NEPC biopsy was 11.6 ng/mL. At NEPC diagnosis, 19 (14%) had localized disease (median OS 123.0 mo); 33 (24%) non-metastatic castrate-sensitive disease (median OS 42.3 mo); 6 (4%) non-metastatic castrate-resistant disease (median OS 14.3 mo); 35 (26%) metastatic castrate-sensitive disease (median OS 17.6 mo); and 42 (31%) metastatic castrate-resistant disease (median OS 9.6 mo). Median OS for those with visceral metastases was 8.6 mo (95% CI 6.0 – 14.6), compared to patients with non-visceral metastases (11.1 mo; 95% CI 13.7 – 21.5) and no metastases (42.3 mo; 95% CI 47 – 89). Anemia (adjusted HR 1.66; 95% CI 1.05 - 2.16, p = 0.031) and NLR >3 (adjusted HR 1.51; 95% CI 1.01 - 2.52, p = 0.045) were associated with increased risk of death. De novo disease, elevated LDH, serum PSA, and Gleason score were not prognostic. Conclusions: This study identifies NEPC clinical outcomes by stage, with survival poorer than expected in pure prostate adenocarcinoma. Anemia and elevated NLR >3 are prognostic biomarkers that may help risk stratify and guide treatment intensification, including platinum-based chemotherapy. Further biomarker characterization of NEPC through IHC-staining pattern and genomic analysis is currently underway by this group.

Description, PD-1 on glioblastoma cells promotes brain tumor growth, and therapeutic antibodies cannot suppress its proliferative effects., Brain tumor–initiating cells (BTICs) drive glioblastoma growth through not fully understood mechanisms. Here, we found that about 8% of cells within the human glioblastoma microenvironment coexpress programmed cell death 1 (PD-1) and BTIC marker. Gain- or loss-of-function studies revealed that tumor-intrinsic PD-1 promoted proliferation and self-renewal of BTICs. Phosphorylation of tyrosines within the cytoplasmic tail of PD-1 recruited Src homology 2–containing phosphatase 2 and activated the nuclear factor kB in BTICs. Notably, the tumor-intrinsic promoting effects of PD-1 did not require programmed cell death ligand 1(PD-L1) ligation; thus, the therapeutic antibodies inhibiting PD-1/PD-L1 interaction could not overcome the growth advantage of PD-1 in BTICs. Last, BTIC-intrinsic PD-1 accelerated intracranial tumor growth, and this occurred in mice lacking T and B cells. These findings point to a critical role for PD-1 in BTICs and uncover a nonimmune resistance mechanism of patients with glioblastoma to PD-1– or PD-L1–blocking therapies.

Background: Cancer is the leading cause of disease-related mortality in children and malignancies are more frequently observed in individuals with primary immunodeficiencies (PIDs). This study aimed to identify and highlight the molecular mechanisms, such as oncogenesis and immune evasion, by which PID-related genes may lead to the development of pediatric cancers. Method: We implemented a novel bioinformatics framework using patient data from the TARGET database and performed a comparative transcriptome analysis of PID-related genes in pediatric cancers between normal and cancer tissues, gene ontology enrichment, and protein–protein interaction analyses, and determined the prognostic impacts of commonly mutated and differentially expressed PID-related genes. Results: From the Fulgent Genetics Comprehensive Primary Immunodeficiency panel of 472 PID-related genes, 89 genes were significantly differentially expressed between normal and cancer tissues, and 20 genes were mutated in two or more patients. Enrichment analysis highlighted many immune system processes as well as additional pathways in the mutated PID-related genes related to oncogenesis. Survival outcomes for patients with altered PID-related genes were significantly different for 75 of the 89 DEGs, often resulting in a poorer prognosis. Conclusions: Overall, multiple PID-related genes demonstrated the connection between PIDs and cancer development and should be studied further, with hopes of identifying new therapeutic targets.

Background Up to 40% of lung adenocarcinoma have been reported to lack ataxia-telangiectasia mutated (ATM) protein expression. We asked whether ATM-deficient lung cancer cell lines are sensitive to poly-ADP ribose polymerase (PARP) inhibitors and determined the mechanism of action of olaparib in ATM-deficient A549 cells. Methods We analysed drug sensitivity data for olaparib and talazoparib in lung adenocarcinoma cell lines from the Genomics of Drug Sensitivity in Cancer (GDSC) project. We deleted ATM from A549 lung adenocarcinoma cells using CRISPR/Cas9 and determined the effects of olaparib and the ATM/Rad3-related (ATR) inhibitor VE-821 on cell viability. Results IC50 values for both olaparib and talazoparib positively correlated with ATM mRNA levels and gene amplification status in lung adenocarcinoma cell lines. ATM mutation was associated with a significant decrease in the IC50 for olaparib while a similar trend was observed for talazoparib. A549 cells with deletion of ATM were sensitive to ionising radiation and olaparib. Olaparib induced phosphorylation of DNA damage markers and reversible G2 arrest in ATM-deficient cells, while the combination of olaparib and VE-821 induced cell death. Conclusions Patients with tumours characterised by ATM-deficiency may benefit from treatment with a PARP inhibitor in combination with an ATR inhibitor.

e18043 Background: Head and neck cancer is the 6th most common cancer worldwide. Oral squamous cell carcinoma (OSCC) is the most prevalent head and neck cancer that is characterized by aggressive local invasion and metastasis. Despite the leading edge (invasive front) of the tumor being a driver of OSCC pathophysiology, its biology and clinical relevance have not been fully characterized. We used spatial transcriptomics to explore signaling patterns within the leading edge and tumor core. Methods: Fresh-frozen, surgically resected OSCC samples from three HPV-negative OSCC patients were profiled using the 10x Genomics Visium Spatial Gene Expression platform. Leading edge and tumor core regions were defined by pathologist annotations and expression of previously identified edge and core gene signatures from the literature. Spatial differential gene expression (DGE) analysis and pathway analysis was performed using the Seurat package and Ingenuity Pathway Analysis (IPA), respectively. Cell-cell interaction networks were reconstructed using the CellChat package. Results: The leading edge and tumor core displayed unique transcriptional and signaling profiles that were conserved across all three OSCC patient samples. DEG analysis revealed 31 genes enriched in the leading edge and 62 genes enriched in the tumor core with a log2FC > 0.58 and adjusted p-value < 0.01. The top genes upregulated in the leading edge were FN1, COL1A1, COL1A2, IFITM3, and SPARC. Top tumor-core genes included CRCT1, LCE3D, DEFB4A, SPRR2A, and CNFN. IPA analysis of upregulated DEGs in the leading edge and tumor core predicted the activation of wound healing and GP6 signaling pathways, and activation of intrinsic prothrombin activation and MSP-RON signaling pathways, respectively. Cell communication analysis revealed that the leading edge had higher intercellular signaling than the tumor core. Upregulated leading edge cell signaling modules included collagen, CD99, CSPG4, and non-canonical WNT pathways, which have been linked to tumor invasion, metastasis, and adhesion. COL1A1 and COL1A2 ligands and CD44 and SDC1 receptors were upregulated in leading edge signaling. The tumor core was enriched for ANGPTL and PERIOSTIN cell signaling modules. Conclusions: This is the first study to characterize the tumor core and leading edge of OSCC tumors using spatial transcriptomics. Further investigation of the therapeutic potential of identified signaling pathways may improve OSCC outcomes.

Sarcomas are rare, difficult to treat, mesenchymal lineage tumours that affect children and adults. Immunologically-based therapies have improved outcomes for numerous adult cancers, however, these therapeutic strategies have been minimally effective in sarcoma so far. Clinically relevant, immunologically-competent, and transplantable pre-clinical sarcoma models are essential to advance sarcoma immunology research. Herein we show that Cre-mediated activation ofKrasG12D, and deletion ofTrp53, in the hindlimb muscles of C57Bl/6 mice results in the highly penetrant, rapid onset undifferentiated pleomorphic sarcomas (UPS), one of the most common human sarcoma subtypes. Cell lines derived from spontaneous UPS tumours can be reproducibly transplanted into the hindlimbs or lungs of naïve, immune competent syngeneic mice. Immunological characterization of both spontaneous and transplanted UPS tumours demonstrates an immunologically-‘quiescent’ microenvironment, characterized by a paucity of lymphocytes, limited spontaneous adaptive immune pathways, and dense macrophage infiltrates. Macrophages are the dominant immune population in both spontaneous and transplanted UPS tumours, although compared to spontaneous tumours, transplanted tumours demonstrate increased spontaneous lymphocytic infiltrates. The growth of transplanted UPS tumours is unaffected by host lymphocyte deficiency, and despite strong expression of PD-1 on tumour infiltrating lymphocytes, tumours are resistant to immunological checkpoint blockade. This spontaneous and transplantable immune competent UPS model will be an important experimental tool in the pre-clinical development and evaluation of novel immunotherapeutic approaches for immunologically cold soft tissue sarcomas.

Gliomas with wild type (WT) isocitrate dehydrogenase (IDH) are considerably more aggressive than those with mutant IDH. To identify putative drivers of the distinct progression trajectories of IDH WT and mutant disease, we analyzed transcriptomes of lower grade astrocytomas (LGAs; grade 2–3) with retention of 1p and 19q from The Cancer Genome Atlas (TCGA; n = 347). Compared to IDH mutant LGAs, we found that PDGF signaling was significantly enriched in IDH WT LGAs and that PDGFA was the top overexpressed gene in this pathway. We identified copy number gains of chromosome 7 in WT LGAs, and methylation of the PDGFA promoter in mutant LGAs, as candidate mechanisms for the differential expression of PDGFA. High PDGFA expression and low PDGFA promoter methylation were significantly associated with poor survival in all LGAs. We also found that PDGFA expression was positively associated with aneuploidy and extracellular matrix-related immunosuppressive features in WT LGAs, underscoring the role of PDGFA as a secreted mitogen. Finally, we show that the proportion of p53 pathway mutations increase significantly with grade in IDH WT gliomas. Taken together, our findings suggest that IDH WT LGAs evolve to higher grades, and ultimately to GBM, by progressive inactivation of the p53 pathway - functioning in concert with a background of increased PDGFA expression. These data emphasize the scope of genomic reprogramming that occurs in gliomas in relation to IDH mutations and further highlight the role of PDGFA in glioma formation, progression, and prognosis. Going forward, this work provides critical biological insight that may inspire new therapeutic strategies to suppress the transformation of IDH WT LGAs to higher-grade cancers.

Low grade astrocytomas (LGAs) are classified based on the mutational status of the isocitrate dehydrogenase (IDH) gene. While IDH wild-type (WT) LGAs evolve rapidly to glioblastoma, mutant tumors generally have a more indolent course. To identify potential drivers of the differential progression of LGAs, we analyzed transcriptomes from The Cancer Genome Atlas. Compared to mutant LGAs, platelet-derived growth factor (PDGF) signaling is enriched in WT cases, andPDGFAis the top overexpressed gene in the pathway. Putative mechanisms for differentialPDGFAexpression included copy number gains of chromosome 7 in WT cases and methylation of thePDGFApromoter in mutant LGAs. Additionally, we found that highPDGFAexpression is associated with aneuploidy, immunosuppressive features, and worse prognosis, and that WT LGAs use multiple means to inactivate the p53 pathway to progress to GBM. Our work highlights the contribution of PDGF gene family towards the unique behaviour of LGAs.STATEMENT OF SIGNIFICANCEThis study of gene expression in LGAs suggests that differential regulation of the PDGF pathway may underlie the different natural histories ofIDHWT andIDHmutant LGAs including divergent evolutionary trajectories to GBM. This insight may inspire new therapeutic strategies to suppress the transformation of LGAs to higher-grade cancers.

BackgroundThe ataxia telangiectasia mutated (ATM) protein kinase is mutated in several human cancers, presenting potential opportunities for targeted cancer therapy. We previously reported that the poly-ADP ribose polymerase (PARP) inhibitor olaparib induced transient G2 arrest but not cell death in ATM-deficient A549 lung cancer cells, while the combination of olaparib with the ATM-, Rad3-related (ATR) inhibitor VE-821 induced cell death. Here, we show that the clinically relevant ATR inhibitor, AZD6738, sensitizes ATM-deficient A549 lung, prostate and pancreatic cancer cells to olaparib.MethodsATM was depleted from A549 lung cancer cells, PC-3 prostate cancer cells and Panc 10.05 pancreatic cancer cells, and the effects of olaparib alone and in combination with AZD6738 were determined.ResultsThe combination of olaparib plus AZD6738 induced cell death in ATM-deficient lung, prostate and pancreatic cancer cells with little effect on their ATM-proficient counterparts.ConclusionsLung, prostate and pancreatic patients whose tumours exhibit loss or inactivation of ATM may benefit from combination of a PARP inhibitor plus an ATR inhibitor.

Poly-ADP ribose polymerase (PARP) inhibitors are currently used in the treatment of several cancers carrying mutations in the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2, with many more potential applications under study and in clinical trials. Here, we discuss the potential for extending PARP inhibitor therapies to tumours with deficiencies in the DNA damage-activated protein kinase, Ataxia-Telangiectasia Mutated (ATM). We highlight our recent findings that PARP inhibition alone is cytostatic but not cytotoxic in ATM-deficient cancer cells and that the combination of a PARP inhibitor with an ATR (ATM, Rad3-related) inhibitor is required to induce cell death.

Murshidabad district is one of the most highly Arsenic (As) prone areas of West Bengal, India. The predominantly rural population of this district greatly depends on traditionally used medicinal plants for treatment of various ailments and subjected to risk of arsenic contamination. The present study revealed that some naturally grown medicinal plants in this district were found to have the alarming level of concentration of arsenic and other metals (Fe, Cu) contamination. So, there should be raised more consciousness on the toxic metal contamination of medicinal plants specifically, collected from contaminated sites.

Background The incidence of human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (SCC) is increasing and has better survival than non-HPV related oropharyngeal SCC. This study compared surgical to nonsurgical treatments and demographic, clinical, and survival differences in patients with oropharyngeal SCC, stratified by p16 status. Methods We assembled a cohort of adult patients with oropharyngeal SCC diagnosed between 2000 and 2008 in Alberta. The tumor p16 biomarker was measured using fluorescent immunohistochemistry. Results In this cohort, p16 data were available for 115 of 357 patients; and 66% (n = 76) were p16-positive. Patients with p16 data had comparable outcomes to those without. Surgically treated p16-negative patients had improved 5-year disease-specific survival (DSS) and overall survival (OS) compared with nonsurgical patients. There were no differences in survival outcomes between surgical and nonsurgical treatment for patients with p16-positive disease. Conclusion Patients with p16-positive oropharyngeal SCC had similar outcomes regardless of treatment. Patients with p16-negative tumors may benefit from primary surgery with postoperative adjuvant therapy.

Myeloid cells that infiltrate into brain tumors are deactivated or exploited by the tumor cells. We previously demonstrated that compromised microglia, monocytes, and macrophages in malignant gliomas could be reactivated by amphotericin-B to contain the growth of brain tumorinitiating cells (BTICs). We identified meclocycline as another activator of microglia, so we sought to test whether its better-tolerated derivative, demeclocycline, also stimulates monocytes to restrict BTIC growth. Monocytes were selected for study as they would be exposed to demeclocycline in the circulation prior to entry into brain tumors to become macrophages. We found that demeclocycline increased the activity of monocytes in culture, as determined by tumor necrosis factor-α production and chemotactic capacity. The conditioned medium of demeclocycline-stimulated monocytes attenuated the growth of BTICs generated from human glioblastoma resections, as evaluated using neurosphere and alamarBlue assays, and cell counts. Demeclocycline also had direct effects in reducing BTIC growth. A global gene expression screen identified several genes, such as DNA damage inducible transcript 4, frizzled class receptor 5 and reactive oxygen species modulator 1, as potential regulators of demeclocycline-mediated BTIC growth reduction. Amongst several tetracycline derivatives, only demeclocycline directly reduced BTIC growth. In summary, we have identified demeclocycline as a novel inhibitor of the growth of BTICs, through direct effect and through indirect stimulation of monocytes. Demeclocycline is a candidate to reactivate compromised immune cells to improve the prognosis of patients with gliomas.

Glioblastoma multiforme (GBM) is the most deadly brain tumor, and currently lacks effective treatment options. Brain tumor-initiating cells (BTICs) and orthotopic xenografts are widely used in investigating GBM biology and new therapies for this aggressive disease. However, the genomic characteristics and molecular resemblance of these models to GBM tumors remain undetermined. We used massively parallel sequencing technology to decode the genomes and transcriptomes of BTICs and xenografts and their matched tumors in order to delineate the potential impacts of the distinct growth environments. Using data generated from whole-genome sequencing of 201 samples and RNA sequencing of 118 samples, we show that BTICs and xenografts resemble their parental tumor at the genomic level but differ at the mRNA expression and epigenomic levels, likely due to the different growth environment for each sample type. These findings suggest that a comprehensive genomic understanding of in vitro and in vivo GBM model systems is crucial for interpreting data from drug screens, and can help control for biases introduced by cell-culture conditions and the microenvironment in mouse models. We also found that lack of MGMT expression in pretreated GBM is linked to hypermutation, which in turn contributes to increased genomic heterogeneity and requires new strategies for GBM treatment.

Glioblastomas are generally incurable partly because monocytes, macrophages, and microglia in afflicted patients do not function in an antitumor capacity. Medications that reactivate these macrophages/microglia, as well as circulating monocytes that become macrophages, could thus be useful to treat glioblastoma. We have discovered that niacin (vitamin B3) is a potential stimulator of these inefficient myeloid cells. Niacin-exposed monocytes attenuated the growth of brain tumor-initiating cells (BTICs) derived from glioblastoma patients by producing anti-proliferative interferon-α14. Niacin treatment of mice bearing intracranial BTICs increased macrophage/microglia representation within the tumor, reduced tumor size, and prolonged survival. These therapeutic outcomes were negated in mice depleted of circulating monocytes or harboring interferon-α receptor-deleted BTICs. Combination treatment with temozolomide enhanced niacin-promoted survival. Monocytes from glioblastoma patients had increased interferon-α14 upon niacin exposure and were reactivated to reduce BTIC growth in culture. We highlight niacin, a common vitamin that can be quickly translated into clinical application, as an immune stimulator against glioblastomas.

Background: Atypical teratoid rhabdoid tumor of the central nervous system (CNS ATRT) is a malignancy that commonly affects young children. The biological mechanisms contributing to tumor aggressiveness and resistance to conventional therapies in ATRT are unknown. Previous studies have shown the activity of insulin like growth factor-I receptor (IGF-1R) in ATRT tumor specimens and cell lines. IGF-1R has been shown to cross-talk with other receptor tyrosine kinases (RTKs) in a number of cancer types, leading to enhanced cell proliferation. Objective: This study aims to evaluate the role of IGF-1 receptor cross-talk in ATRT biology and the potential for therapeutic targeting. Methods: Cell lines derived from CNS ATRT specimens were analyzed for IGF-1 mediated cell proliferation. A comprehensive receptor tyrosine kinase (RTK) screen was conducted following IGF-1 stimulation. Bioinformatic analysis of publicly available cancer growth inhibition data to identify correlation between IC50 of a VEGFR inhibitor and IGF-1R expression. Results: Comprehensive RTK screen identified VEGFR-2 cross-activation following IGF-1 stimulation. Bioinformatics analysis demonstrated a positive correlation between IC50 values of VEGFR inhibitor Axitinib and IGF-1R expression, supporting the critical influence of IGF-1R in modulating response to anti-angiogenic therapies. Conclusion: Overall, our data present a novel experimental framework to evaluate and utilize receptor cross-talk mechanisms to select effective drugs and combinations for future therapeutic trials in ATRT.

10035 Background: Neuroblastoma (NB) is the most common extracranial solid tumor in children. Despite the development of risk stratification tools to improve prognostication, prediction of patient survival outcomes in NB remains poor. In this study we used an unbiased machine-learning algorithm to develop and validate a transcriptomic signature capable of predicting 5-year overall (OS) and event-free survival (EFS) in these patients. Methods: The TARGET-Neuroblastoma dataset (n = 243) was used as the training set. Two independent NB cohorts, E-MTAB-179 (n = 478) and GSE85047 (n = 266) were used as validation sets. Elastic net regression was employed to identify transcripts associated with EFS. Association of the developed signature with EFS and OS was evaluated using univariate Cox proportional hazards (CoxPH), Kaplan-Meier, and 5-year receiver-operator characteristic curves in validation cohorts. Further, the independent prognostic value of the signature was assessed using multivariate CoxPH models with relevant clinicopathologic variables including age, INSS stage, and N-Myc amplification status in both validation sets. Finally, a nomogram was developed to integrate the signature with prognostic clinicopathologic variables to evaluate their combined efficacy for prediction of 5-year EFS and OS. Results: We identified a 21-gene signature that demonstrates significant association with EFS and OS in both E-MTAB-178 and GSE49710 validation cohorts. Moreover, the signature is independent of clinicopathological variables and can be effectively incorporated into a risk model, improving the prognostic performance. Several genes within the signature have been previously implicated in NB, including ECEL1, HOXC9 and ARAF1. Conclusions: To the best of our knowledge, we are the first to use an unbiased machine learning approach to generate a transcriptomic gene signature for neuroblastoma prognosis externally validated in multiple cohorts across platforms. This 21-gene transcriptomic signature significantly associated with EFS and OS in this disease. Combining this signature with current prognostic clinicopathologic variables will improve risk stratification of affected patients and may inform effective clinical decision-making.[Table: see text]

The paper models a vertically related formal and informal sector, and endogenizes the size and regulatory compliance status of the informal sector. When the formal sector can circumvent costly regulations by outsourcing polluting intermediate goods from the informal sector, stricter regulations increase (when the “composition effect” of regulation dominates its “scale effect”) or decrease total pollution, and may even have a non-monotonic impact. We identify conditions under which a partially compliant informal sector acts as a source of leakage, and examine implications for optimal enforcement policy. Further, we show that price discrimination by the formal sector, when it purchases the intermediate goods from the informal sector, can worsen regulatory compliance by the informal sector and lead to more pollution.

Among head and neck squamous carcinoma (HNSC), human papilloma virus (HPV)-driven tumors are known to enjoy more favorable prognostic outcomes compared to non-HPV-driven disease. This is especially true of HPV-positive oropharyngeal squamous cell carcinoma (OPSCC), which is associated with significantly improved outcomes compared to HPV-positive non-OPSCC and HPV-negative HNSC. Previous reports have suggested that increased immune infiltration and expression of the estrogen receptor α (ERα) may play a role in conferring improved prognosis to HPV-positive OPSCC. However, the underlying signaling pathways specifically driven by HPV in the oropharynx have remained elusive. Here we analyzed HPV-positive OPSCC and non-OPSCC tumors from The Cancer Genome Atlas (TCGA) and found that HPV-positive OPSCC is associated with significantly improved overall and disease-specific survival and progression-free interval compared to HPV-positive non-OPSCC. Among HPV-positive HNSC, we observed a distinct separation of OPSCC from non-OPSCC tumors based on unsupervised clustering of gene expression, microRNA, and methylation profiling data. Separation based on anatomic site was not observed among HPV-negative HNSC, suggesting that common biology drives the carcinogenesis of HPV-negative HNSC across subsites. In line with a recent report that ERα mRNA expression is elevated HPV-positive OPSCC, we found significantly higher ERα mRNA levels in HPV-positive OPSCC compared to HPV-positive non-OPSCC, where ERα mRNA expression mirrored that in HPV-negative HNSC. We also found that HPV induces downregulation of TGF-β signaling and extracellular matrix (ECM)-associated genes while upregulating a T-cell and dendritic cell-associated immune response signature in an OPSCC-specific manner. Our analyses suggest that HPV induces downregulation of TGF-β signaling and ECM components in OPSCC to improve immune access to these tumors, facilitating tumor clearance and improved patient survival. These findings are in line with our recent report establishing TGF-β-mediated immunosuppression through ECM deregulation and its negative effect on patient prognosis. Citation Format: Anthony Bolyos, Pinaki Bose. Improved prognosis of HPV-positive oropharyngeal cancers is associated with a suppression of TGF-β-mediated immune evasion [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr A07.

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