Cancer classification in the clinic is primarily based on histological analysis in the proper clinical context, often supplemented by immunohistochemical and molecular studies. Recent genomic studies ...have shown the potential of integrated multiomics platforms for molecular classification. We performed unsupervised analyses of molecular platforms in The Cancer Genome Atlas data (n = 6,216 samples) in comparison with tumor type. Our data showed that mRNA signatures and DNA methylation signatures mapped to histological diagnosis with high accuracy (95% and 88%, respectively) as individual platforms. The accuracy of mRNA signatures alone for classification and subtype identification was comparable to accuracies reported in the previously published Pan-Cancer 12 analysis. When combined, mRNA and methylation revealed a set of outliers for which the mRNA- and methylation-based molecular signatures concordantly differed from the original histological diagnosis. A subset remained consistent as outliers after using alternative classification and clustering algorithms and analysis of an independent molecular platform (miRNA). Overall, our results indicate that unsupervised approaches with individual genomic platforms, especially gene expression and DNA methylation, provide substantial classification information and identify occasional outlier cases in which the molecular signature is distinct from signatures expected for a given histological diagnosis. Identification of cases in which the molecular signature correlates with a specific histology that differs from initial impressions may prompt reconsideration of tumor classification in specific cases.
Glioblastoma multiforme (GBM) contains a subpopulation of cells, GBM stem cells (GSCs), that maintain the bulk tumor and represent a key therapeutic target. Norrin is a Wnt ligand that binds Frizzled ...class receptor 4 (FZD4) to activate canonical Wnt signaling. Although Norrin, encoded by NDP, has a well-described role in vascular development, its function in human tumorigenesis is largely unexplored. Here, we show that NDP expression is enriched in neurological cancers, including GBM, and its levels positively correlated with survival in a GBM subtype defined by low expression of ASCL1, a proneural factor. We investigated the function of Norrin and FZD4 in GSCs and found that it mediated opposing tumor-suppressive and -promoting effects on ASCL1lo and ASCL1hi GSCs. Consistent with a potential tumor-suppressive effect of Norrin suggested by the tumor outcome data, we found that Norrin signaling through FZD4 inhibited growth in ASCL1lo GSCs. In contrast, in ASCL1hi GSCs Norrin promoted Notch signaling, independently of WNT, to promote tumor progression. Forced ASCL1 expression reversed the tumor-suppressive effects of Norrin in ASCL1lo GSCs. Our results identify Norrin as a modulator of human brain cancer progression and reveal an unanticipated Notch-mediated function of Norrin in regulating cancer stem cell biology. This study identifies an unanticipated role of Norrin in human brain cancer progression. In addition, we provide preclinical evidence suggesting Norrin and canonical Wnt signaling as potential therapeutic targets for GBM subtype-restricted cancer stem cells.
Abstract
Background
Chordomas are rare malignant bone cancers of the skull-base and spine. Patient survival is variable and not reliably predicted using clinical factors or molecular features. This ...study identifies prognostic epigenetic chordoma subtypes that are detected noninvasively using plasma methylomes.
Methods
Methylation profiles of 68 chordoma surgical samples were obtained between 1996 and 2018 across three international centers along with matched plasma methylomes where available.
Results
Consensus clustering identified two stable tissue clusters with a disease-specific survival difference that was independent of clinical factors in a multivariate Cox analysis (HR = 14.2, 95%CI: 2.1–94.8, P = 0.0063). Immune-related pathways with genes hypomethylated at promoters and increased immune cell abundance were observed in the poor-performing “Immune-infiltrated” subtype. Cell-to-cell interaction plus extracellular matrix pathway hypomethylation and higher tumor purity were observed in the better-performing “Cellular” subtype. The findings were validated in additional DNA methylation and RNA sequencing datasets as well as with immunohistochemical staining. Plasma methylomes distinguished chordomas from other clinical differential diagnoses by applying fifty chordoma-versus-other binomial generalized linear models in random 20% testing sets (mean AUROC = 0.84, 95%CI: 0.52–1.00). Tissue-based and plasma-based methylation signals were highly correlated in both prognostic clusters. Additionally, leave-one-out models accurately classified all tumors into their correct cluster based on plasma methylation data.
Conclusions
Here, we show the first identification of prognostic epigenetic chordoma subtypes and first use of plasma methylome-based biomarkers to noninvasively diagnose and subtype chordomas. These results may transform patient management by allowing treatment aggressiveness to be balanced with patient risk according to prognosis.
Glioblastoma (GBM) is one of the most aggressive adult brain tumors, with the histopathologic hallmark of increased abnormal vasculature. While anti-angiogenesis therapy had shown value in ...pre-clinical and early clinical studies, in particular targeting vascular endothelial growth factor (VEGF), the durability of response to anti-angiogenic therapy varies and hence efficacy remains a limitation. There is data to suggest that certain subtypes of GBM however have a more effective response to anti-angiogenic therapy. Therefore, there is a need to identify prognostic markers that can determine the subpopulation of GBM patients that response to AA therapy. We established five genetic expression profiles (group A~E) by stimulating endothelial cells (ECs) with different combinations of ionizing radiation (IR) and mesenchymal stem cells (MSCs) before performing angiogenesis array analysis. Bioinformatics analysis of Group A~E identified the combination of
HGF
and
CXCL10
alterations as the differentiators that separated the AVAglio patients into 3 groups (group 1~3). We found that GBM patients with high
HGF
and low
CXCL10
levels had the worst clinical response to AA therapy. Further qPCR analysis of gene expression levels among different cell types revealed that GBM cells have the highest expression of
HGF
and the lowest expression of
CXCL10
relative to ECs. Similar trend were detected in MSCs relative to ECs but to a lesser degree. Thus we propose that such genetic parameter in GBM could potentially be a prognostic marker for AA therapy.
Abstract
Background
Diffuse gliomas represent over 80% of malignant brain tumors ranging from low-grade to aggressive high-grade lesions. Within isocitrate dehydrogenase (IDH)-mutant gliomas, there ...is a high variability in survival and a need to more accurately predict outcome.
Methods
To identify and characterize a predictive signature of outcome in gliomas, we utilized an integrative molecular analysis (using methylation, mRNA, copy number variation (CNV), and mutation data), analyzing a total of 729 IDH-mutant samples including a test set of 99 from University Health Network (UHN) and 2 validation cohorts including the German Cancer Research Center (DKFZ) and The Cancer Genome Atlas (TCGA).
Results
Cox regression analysis of methylation data from the UHN cohort identified CpG-based signatures that split the glioma cohort into 2 prognostic groups strongly predicting survival that were validated using 2 independent cohorts from TCGA and DKFZ (all P-values < .0001). The methylation signatures that predicted poor outcomes also exhibited high CNV instability and hypermethylation of HOX gene probes. Integrated multi-platform analyses using mRNA and methylation (iRM) showed that parallel HOX gene overexpression and simultaneous hypermethylation were significantly associated with increased mutational load, high aneuploidy, and worse survival (P-value < .0001). A 7-HOX gene signature was developed and validated using the most significantly associated HOX genes with patient outcome in both 1p/19q codeleted and non-codeleted IDHmut gliomas.
Conclusions
HOX gene methylation and expression provide important prognostic information in IDH-mutant gliomas that are not captured by current molecular diagnostics. A 7-HOX gene signature of outcome shows significant survival differences in both 1p/19q codeleted and non-codeleted IDH-mutant gliomas.
Abstract
Hypoxia is a prominent characteristic of aggressive tumors, including glioblastoma (GB), and is linked to therapy resistance. Understanding the molecular mechanisms underlying ...hypoxia-induced changes in neoplastic and non-neoplastic cells is crucial for developing targeted therapies. In this study, we took a comprehensive approach to investigate the genomic, epigenomic, and spatial transcriptomic profiles of intra-tumoral hypoxia in GB at bulk and single-cell levels. Our objectives were to identify a GB-specific hypoxia gene signature and uncover novel mechanisms of GB's response to hypoxia. Pimonidazole (PIMO), a hypoxia marker, was administered to 77 GB patients. We utilized immune-guided laser microdissection to extract DNA and perform methylome profiling of PIMO+ (hypoxic) and PIMO- regions. Visium 10X spatial transcriptomics was employed to identify differentially expressed genes in PIMO+ tumor regions. Through single-nuclear multi-omics profiling, we examined gene expression and chromatin accessibility of the hypoxic neoplastic and non-neoplastic cells at the single-cell level. The hypoxia transcriptome in GB exhibited general commonalities, in addition to unique features, compared to other reported hypoxia signatures. Some of these alterations were accompanied by changes at the DNA methylome level. Importantly, we found a heterogenous molecular landscape across PIMO+ regions within each sample. While a general transition to a MES-like state was observed, we found a mixture of cell states across the different PIMO+ regions. We also found distinct sub-hypoxic microenvironments harboring unique immune cell populations that overlapped with specific cell type states, cell cycle states, and deregulation of specific cellular pathways. These sub-regions were associated with specific anatomic features and displayed prognostic utility in GBM patient cohorts. Our single-cell multi-omics analysis further revealed intra-tumoral copy number aberrations and changes in chromatin accessibility associated with hypoxic cells. Our findings demonstrate that the hypoxic microenvironment in GB is heterogenous with unique targetable features that could lead to novel treatment modalities.
Abstract
Norrin is an atypical WNT ligand that binds the Frizzled-4 (FZD4) and Low-density lipoprotein receptor-related protein (LRP5/6) receptor complex to activate canonical WNT/ β-catenin ...signaling. Norrin/FZD4 signaling is involved in the regulation of vasculature in several tissues including the retina and for blood-brain barrier function. The role of Norrin in cancer is not very well characterized. Here, we show that NDP, the gene encoding for Norrin, is expressed in a wide range of cancer types, with a particular enrichment in glioblastoma (GBM) and lower grade glioma (LGG). Kaplan-Meier survival analysis of publicly available datasets revealed a significant correlation between NDP expression and survival in GBM, LGG and neuroblastoma. To investigate the function of NDP in GBM, we performed a set of NDP and FZD4 gain and loss of function experiments in patient-derived GBM stem cell (GNS) lines. Recently Achaete-scute homolog 1 (ASCL1) expression was shown to stratify GNS lines into two cohorts with different tumorigenic, proliferation and differentiation dynamics. Surprisingly, we found that NDP manipulation resulted in opposite effects in ASCL1hi versus ASCL1lo lines. NDP inhibited proliferation and sphere formation in ASCL1lo lines through the WNT/ β-catenin pathway, while it stimulated proliferation and sphere formation in ASCL1hi lines independently of the WNT/β-catenin pathway. The NDP/FZD4 effects on growth in both GNS subtypes were recapitulated in xenografts, confirming effects of this pathway on tumor progression in vivo. To gain insight into the cellular and molecular effects of NDP/FZD4 on GNS growth we performed immunocytochemistry (ICC) and RNA-Seq analyses in GNS cells with NDP knockdown. In parallel with our in vitro and in vivo observations, we found that NDP/FZD4 signaling affects GNS proliferation index and cell cycle kinetics and the expression of cell cycle regulatory genes in both GNS subtypes, but in opposite ways. RNA-Seq analysis identified cell cycle regulatory genes and gene sets that are commonly affected by NDP in both GNS types. Interestingly, the analysis also identified several genes and genesets unique to each GNS type, consistent with the divergence of NDP functions between GNS subtypes. Collectively, our results uncover novel functions of NDP in regulating GBM stem cell progression, and that NDP functions in GBM stem cells stratify with ASCL1 expression. Additionally, our results highlight the significance of precision medicine in targeting tumor subtypes based on the molecular signature.
Citation Format: Ahmed A. Elsehemy, Hayden Selvadurai, Arturo Ortin-Martinez, Nenad Pokrajac, Yasin Mamatjan, Katherine Rowland, Kenneth Aldape, Peter Dirks, Valerie Wallace. Novel biological roles of the atypical WNT ligand, Norrin, on glioblastoma stem cells segregate with ASCL1 expression 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 4671.
Mutations in the pivotal metabolic isocitrate dehydrogenase (IDH) enzymes are recognized to drive the molecular footprint of diffuse gliomas, and patients with IDH mutant gliomas have overall ...favorable outcomes compared to patients with IDH wild-type tumors. However, survival still varies widely among patients with IDH mutated tumors. Here, we aimed to characterize molecular signatures that explain the range of IDH mutant gliomas. By integrating matched epigenome-wide methylome, transcriptome, and global metabolome data in 154 patients with gliomas, we identified a group of IDH mutant gliomas with globally altered metabolism that resembled IDH wild-type tumors. IDH-mutant gliomas with altered metabolism have significantly shorter overall survival from their IDH mutant counterparts that is not fully accounted for by recognized molecular prognostic markers of
CDKN2A/B
loss and glioma CpG Island Methylator Phenotype (GCIMP) status. IDH-mutant tumors with dysregulated metabolism harbored distinct epigenetic alterations that converged to drive proliferative and stem-like transcriptional profiles, providing a window to target novel dependencies in gliomas.
Graphical Abstract
Abstract
EGFR-mutant lung adenocarcinomas (EGFRm-LUAD) have a higher risk of developing brain metastases (BM) compared to non-EGFR-mutant tumors. BM development has significant prognostic impact and ...leads to poorer patient survival. MGMT promoter methylation is known to determine response to therapy in other cancer types including intracranial gliomas but has not been investigated in EGFRm-LUAD BM. This work aims to assess whether MGMT promoter methylation predicts patient survival or BM development in EGFRm-LUAD patients. A large cohort of 90 primary EGFRm-LUAD tumors, of which 33 (37%) developed BM, were profiled using the Illumina Infinium MethylationEPIC Bead chip. Using the previously reported MGMT-STP27 approach that uses two CpG sites to predict MGMT methylation status, Cox modeling was performed to assess whether MGMT methylation status correlates with overall survival independent of other clinical factors. MGMT methylation significantly predicted poorer survival in EGFRm-LUAD patients that developed BM (p=0.0003) and did not develop BM (p=0.003). A multivariate cox analysis, adjusting for cancer stage and smoking status as potential confounders, showed that MGMT methylation (HR=6.2, 95%CI:2.2–17.4, p=0.0005) and BM development (HR=2.6, 95%CI:1.3–5.3, p=0.007) were both independently predictive of worse overall survival in EGFRm-LUAD patients. This finding of poorer survival in MGMT methylated EGFRm-LUAD is validated in an independent LUAD patient cohort. Total mutation burden, calculated by the number of mutations per megabase of DNA, was substantially higher in MGMT methylated tumours with an interquartile range (IQR) of 58 (30–71) compared to MGMT unmethylated tumours with the IQR of 5.5 (4.3–6.1) resulting p-value of 0.01 for this comparison. Overall, this work shows that MGMT promoter methylation status is an important prognostic biomarker in LUAD patients. MGMT promoter methylation status in EGFRm–LUAD patients with BM may be used to guide patient treatment with potentially a greater extent of treatment for high-risk patients.
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