Next-generation sequencing (NGS) studies have demonstrated that co-occurring sporadic endometrioid endometrial carcinoma (EEC) and endometrioid ovarian carcinoma (EOC) are clonally related, ...suggesting that they originate from a single primary tumor. Despite clonality, synchronous EEC and EOC when diagnosed at early stage behave indolently, similar to isolated primary EEC or isolated primary EOC. In the present study, we compared the DNA methylation signatures of co-occurring EEC and EOC with those of isolated primary EEC and isolated primary EOC. We also performed targeted NGS to assess the clonal relatedness of 7 co-occurring EEC and EOC (4 synchronous EEC and EOC and 3 metastatic EEC based on pathologic criteria). NGS confirmed a clonal relationship in all co-occurring EEC and EOC. DNA methylation profiling showed distinct epigenetic signatures of isolated primary EEC and isolated primary EOC. Endometrial tumors from co-occurring EEC and EOC clustered with isolated primary EEC while their ovarian counterparts clustered with isolated primary EOC. Three co-occurring EEC and EOC cases with peritoneal lesions showed a closer epigenetic signature and copy number variation profile between the peritoneal lesion and EOC than EEC. In conclusion, synchronous sporadic EEC and EOC are clonally related but demonstrate a shift in DNA methylation signatures between ovarian and endometrial tumors as well as epigenetic overlap between ovarian and peritoneal tumors. Our results suggest that tumor microenvironment in the ovary may play a role in epigenetic modulation of metastatic EEC.
Abstract
BCOR is an epigenetic regulator and is genetically altered by mutation, deletion, or gene fusion in a range of cancers. “Central nervous system high-grade neuroepithelial tumor with BCOR ...alteration” is a recently described entity with characteristic internal tandem duplications within exon 15 of the BCOR gene (hereafter: CNS HGNET-BCOR ex15 ITD). In this case series of 3 patients, we report the clinicopathologic, molecular, and methylome features of gliomas with novel EP300-BCOR in-frame gene fusions, thus expanding the spectrum of BCOR alterations seen in CNS tumors. The gliomas in this series arise in children (ages 10–18), involve the supratentorial compartment, and have an infiltrative pattern of growth and a myxoid/microcystic background with frequent psammomatous calcifications and prominent chicken-wire vessels. All 3 cases had areas with low-grade morphology and 2 of them demonstrated histologic high-grade transformation. In contrast to CNS HGNET-BCOR ex15 ITD, they lack perivascular pseudorosettes. On a t-Distributed Stochastic Neighbor Embedding plot they cluster perfectly together, away from CNS HGNET-BCOR ex15ITD, consistent with a different entity. Gliomas with EP300-BCOR fusions and high-grade histology can demonstrate relatively rapid regrowth after debulking or subtotal resection.
Background and Aims
The recruitment and activation of inflammatory cells in the liver delineates the transition from hepatic steatosis to steatohepatitis (SH).
Approach and Results
We found that in ...SH, γδT cells are recruited to the liver by C‐C chemokine receptor (CCR) 2, CCR5, and nucleotide‐binding oligomerization domain‐containing protein 2 signaling and are skewed toward an interleukin (IL)‐17A+ phenotype in an inducible costimulator (ICOS)/ICOS ligand–dependent manner. γδT cells exhibit a distinct Vγ4+, PD1+, Ly6C+CD44+ phenotype in SH. Moreover, γδT cells up‐regulate both CD1d, which is necessary for lipid‐based antigens presentation, and the free fatty acid receptor, CD36. γδT cells are stimulated to express IL‐17A by palmitic acid and CD1d ligation. Deletion, depletion, and targeted interruption of γδT cell recruitment protects against diet‐induced SH and accelerates disease resolution.
Conclusions
We demonstrate that hepatic γδT cells exacerbate SH, independent of IL‐17 expression, by mitigating conventional CD4+ T‐cell expansion and modulating their inflammatory program by CD1d‐dependent vascular endothelial growth factor expression.
We validate the use of a lateral flow immunoassay (LFI) intended for rapid screening and qualitative detection of anti-SARS-CoV-2 IgM and IgG in serum, plasma, and whole blood, and compare results ...with ELISA. We also seek to establish the value of LFI testing on blood obtained from a capillary blood sample.
Samples collected by venous blood draw and finger stick were obtained from patients with SARS-CoV-2 detected by RT-qPCR and control patients. Samples were tested with Biolidics 2019-nCoV IgG/IgM Detection Kit lateral flow immunoassay, and antibody calls were compared with ELISA.
Biolidics LFI showed clinical sensitivity of 92% with venous blood at 7 days after PCR diagnosis of SARS-CoV-2. Test specificity was 92% for IgM and 100% for IgG. There was no significant difference in detecting IgM and IgG with Biolidics LFI and ELISA at D0 and D7 (p = 1.00), except for detection of IgM at D7 (p = 0.04). Capillary blood of SARS-CoV-2 patients showed 93% sensitivity for antibody detection.
Clinical performance of Biolidics 2019-nCoV IgG/IgM Detection Kit is comparable to ELISA and was consistent across sample types. This provides an opportunity for decentralized rapid testing and may allow point-of-care and longitudinal self-testing for the presence of anti-SARS-CoV-2 antibodies.
Lung adenocarcinoma (LUAD) is a clinically heterogeneous disease, which is highlighted by the unpredictable recurrence in low-stage tumors and highly variable responses observed in patients treated ...with immunotherapies, which cannot be explained by mutational profiles. DNA methylation-based classification and understanding of microenviromental heterogeneity may allow stratification into clinically relevant molecular subtypes of LUADs.
We characterize the genome-wide DNA methylation landscape of 88 resected LUAD tumors. Exome sequencing focusing on a panel of cancer-related genes was used to genotype these adenocarcinoma samples. Bioinformatic and statistical tools, the immune cell composition, DNA methylation age (DNAm age), and DNA methylation clustering were used to identify clinically relevant subgroups.
Deconvolution of DNA methylation data identified immunologically hot and cold subsets of LUADs. In addition, concurrent factors were analyzed that could affect the immune microenvironment, such as smoking history, ethnicity, or presence of KRAS or TP53 mutations. When the DNAm age was calculated, a lower DNAm age was correlated with the presence of a set of oncogenic drivers, poor overall survival, and specific immune cell populations. Unsupervised DNA methylation clustering identified six molecular subgroups of LUAD tumors with distinct clinical and microenvironmental characteristics.
Our results demonstrate that DNA methylation signatures can stratify LUAD into clinically relevant subtypes, and thus such classification of LUAD at the time of resection may lead to better methods in predicting tumor recurrence and therapy responses.
Abstract
INTRODUCTION
Pleomorphic xanthoastrocytoma (PXA) is a rare type of brain tumor that commonly affects children and young adults. PXAs are typically characterized by tumor lymphocytic ...infiltration, but the significance of the tumor immune microenvironment has not yet been well-defined. In this study, we correlated DNA methylation profiling of PXAs with clinical outcome and explored the tumor microenvironment by analyzing inflammatory cell populations.
METHODS
We retrospectively analyzed 30 tumor samples, of which 21 tumor samples from 18 subjects had a diagnosis of PXA both by DNA methylation and by histology. MethylCIBERSORT was used to deconvolute PXA inflammatory cell populations and compare them with inflammatory cell populations in previously published cohorts of IDH wildtype glioblastoma and ganglioglioma samples.
RESULTS
Median age at diagnosis was 16 years (range 7–32). 3-year and 5-year overall survival (OS) was 73% and 71% respectively. CDKN2A/B deletion was noted in 15 out of 18 subjects (83%). 10 out of the 12 subjects (83%) that had testing for BRAFV600E showed the mutation. CDKN2A/B deletion and Trisomy 7 did not show any significant association with overall survival (p = 0.39 and p = 0.69). Decreased survival was observed in subjects with tumors lacking the BRAFV600E mutation (p = 0.03). PXAs were observed to have significantly increased CD8 T-cell epigenetic signatures compared to gangliogliomas (p = 0.0019) and significantly increased CD8 T-cell and CD19 B-cell signatures compared to IDH wildtype glioblastomas (p = 0.0011 and p = 0.0011).
CONCLUSION
This research suggests that PXAs have a distinct methylation profile that correlates with clinical outcome. PXAs show significant upregulation of CD8 T-cell epigenetic signatures compared to gangliogliomas and significant upregulation of CD8 T-cell and CD19 B-cell epigenetic signatures compared to IDH wildtype glioblastomas. This distinct characterization of immune cell-types in PXAs could have an impact on future development of immunotherapy.
There is currently no targeted therapy to treat NF1-mutant melanomas. In this study, we compared the genomic and transcriptomic signatures of NF1-mutant and NF1 wild-type melanoma to reveal potential ...treatment targets for this subset of patients. Genomic alterations were verified using qPCR, and differentially expressed genes were independently validated using The Cancer Genome Atlas data and immunohistochemistry. Digital spatial profiling with multiplex immunohistochemistry and immunofluorescence were used to validate the signatures. The efficacy of combinational regimens driven by these signatures was tested through in vitro assays using low-passage cell lines. Pathogenic NF1 mutations were identified in 27% of cases. NF1-mutant melanoma expressed higher proliferative markers MK167 and CDC20 than NF1 wild-type (P = 0.008), which was independently validated both in The Cancer Genome Atlas dataset (P = 0.01, P = 0.03) and with immunohistochemistry (P = 0.013, P = 0.036), respectively. Digital spatial profiling analysis showed upregulation of LY6E within the tumor cells (false discovery rate < 0.01, log2 fold change > 1), confirmed with multiplex immunofluorescence showing colocalization of LY6E in melanoma cells. The combination of MAPK/extracellular signal‒regulated kinase kinase and CDC20 coinhibition induced both cytotoxic and cytostatic effects, decreasing CDC20 expression in multiple NF1-mutant cell lines. In conclusion, NF1-mutant melanoma is associated with a distinct genomic and transcriptomic profile. Our data support investigating CDC20 inhibition with MAPK pathway inhibitors as a targeted regimen in this melanoma subtype.
Adult granulosa cell tumor (AGCT) is characterized by the somatic FOXL2 p.C134W mutation, and recurrences have been associated with TERT promoter and KMT2D-truncating mutations. Conversely, the ...molecular underpinnings of the rare juvenile granulosa cell tumor (JGCT) have not been well elucidated. To this end, we applied a tumor-only integrated approach to investigate the genomic, transcriptomic, and epigenomic landscape of 31 JGCTs to identify putative oncogenic drivers.
Multipronged analyses of 31 JGCTs were performed utilizing a clinically validated next-generation sequencing (NGS) panel targeting 580 cancer-related genes for genomic interrogation, in addition to targeted RNA NGS for transcriptomic exploration. Genome-wide DNA methylation profiling was conducted using an Infinium Methylation EPIC array targeting 866,562 CpG methylation sites.
We identified frequent KMT2C-truncating mutations along with other mutated genes implicated in the switch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex, in addition to previously reported hotspot AKT1 and DICER1 mutations. Targeted transcriptome sequencing revealed recurrent TERT rearrangements (13%) involving partners CLPTM1L or DROSHA, and differential gene expression analysis showed FGFR1 upregulation in the TERT non-rearranged JGCTs under direct promoter control. Genome-wide DNA methylation rendered a clear delineation between AGCTs and JGCTs at the epigenomic level, further supporting its diagnostic utility in distinguishing among these tumors.
This is the largest comprehensive molecular study of JGCTs, where we further expand our current understanding of JGCT pathogenesis and demonstrate putative oncogenic drivers and TERT rearrangements in a subset of tumors. Our findings further offer insights into possible targeted therapies in a rare entity.
Background: Children with acute lymphoblastic leukemia (ALL) who relapse are more resistant to chemotherapy. We have previously identified a unique gene expression signature associated with relapsed ...leukemic blasts and showed that relapse samples have higher level of CpG methylation compared to diagnosis. We also demonstrated that treatment with DNMT inhibitor, decitabine, alone or in combination with the HDAC inhibitor, vorinostat restored blast chemosensitivity in vitro . These observations led to the hypothesis that global methylation status is a key regulator of drug resistance.
Method: To discover the pathways affected by changes in global methylation and chromosomal architecture, we performed ChIPseq for the CCCTC-binding factor (CTCF) and the histone marks H3K9ac, H3K27ac and H3K27me3 as well as RNAseq, and DNA methylation in the B-ALL cell line Reh treated with 1µM decitabine versus DMSO for 3 days in triplicates. ChIPseq data was analyzed using MACS2 broad setting for peak calling, GENCODE for gene annotations and diffbind for differences between the two groups. Promoter regions were inferred to be +/-3KB from TSS and the ROSE algorithm was used on the H3K27ac mark to define enhancers and superenhancers. Analysis of differential gene expression was performed using DESeq2 Bioconductor package and the 850k methylation data was analyzed using ‘Minfi’ Bioconductor package. For all data sets, fold changes of 1.5 or more and p<0.05 (FDR of 5% for the ChIPseq data) were considered significant.
Results: As expected, decitabine treatment resulted in a global loss of CpG methylation and there were 1286 genes upregulated and 1003 genes downregulated by 1.5 fold or more in treated versus untreated cells. Interestingly, there was a subset of CpG sites which had a significant gain of methylation, and gene ontology biological process analysis of those sites using DAVID Bioinformatics identified, among others, genes involved in mitotic cell cycle and MAPK activation. ChIPseq data analysis show that more than 90% of the peaks remain unchanged for the activation mark H3K27ac and the repressive mark H3K27me3 between decitabine-versus DMSO-treated Reh cells. However decitabine treatment resulted in a slight variation in the genomic distribution of the overall peaks. H3K27ac mark occupied 28% of the promoter region after decitabine treatment as compared to 23% in untreated state and likewise H3K27me3 occupancy changed from 11% to 8% at the promoter region with treatment with decitabine. The most impacted histone mark was H3K9ac where 40% of the global peaks called in the untreated cells were lost upon DNMTi treatment. However most of the losses of the H3K9ac mark were located outside of promoters resulting in 56% of these activation marks in promoter regions in treated cells compared to 35% in the untreated cells. Unlike the expected gain of CTCF from lower DNA methylation, there was a 30% reduction of CTCF binding sites with decitabine treatment. CTCF was disproportionately lost at promoter regions compared to gene body and intergenic regions. Integrative analysis of changes in gene expression, CpG methylation, histone marks reveal that up to 60% of their gene expression changes can be directly attributed to the observed epigenetic changes with the greatest impact from changes in DNA methylation and H3K9ac. The gene expression data was also compared to our previously published relapsed gene expression signature on a large cohort of diagnosis-relapse primary samples. We found that decitabine reversed the expression of 74 genes which are typically downregulated in blasts at relapse and 57 genes which are typically upregulated in relapsed blasts. Decitabine treatment led to two predominant epigenetic modifications: loss of CpG methylation with corresponding de-repression of genes typically downregulated in relapse samples (primarily proapototic and antiproliferative genes) and loss of H3K9ac at promoters of antiapoptotic genes.
Conclusions: We show that DNMTi not only impacts CpG methylation but also histone status, likely chromosomal architecture, and gene expression in B-ALL cells and can reverse expression of a subset of the relapse gene expression signature. Further study and validation will lead to a better description of epigenetic events in relapsed ALL that may guide future therapy.
No relevant conflicts of interest to declare.
Abstract
BACKGROUND
RNA expression and DNA methylation studies have identified different subclasses of isocitrate dehydrogenase (IDH)-wildtype (wt) glioblastoma (GBM). However, the prognostic ...significance of molecular subclasses is unclear. Although hyperglycemia has been previously associated with worse survival, attempts to lower glucose have yielded mixed responses. The role of hyperglycemia may be confounded by molecular heterogeneity and have different impact in molecularly distinct GBM subclasses.
METHODS
Clinical, laboratory, and molecular data on 89 IDH-wt GBMs profiled by clinical next-generation sequencing and treated with Stupp protocol were reviewed. IDH-wt GBMs were subclassified into RTKI (Proneural), RTKII (Classical) and Mesenchymal subtypes using DNA methylation. Average glucose was calculated by time-weighting plasma glucose measurements between diagnosis and last follow-up.
RESULTS
Patients were stratified into three groups using average glucose: tertile one (< 100mg/dL), tertile two (100-115mg/dL), and tertile three ( > 115mg/dL). Comparison across glucose tertiles revealed no significant differences in Karfnosky Performance Status (KPS), dexamethasone dose, MGMT methylation, or methylation subclass. Overall survival (OS) was not affected by methylation subclass (log-rank p=0.9) but decreased with higher glucose (log-rank p=0.015). Higher glucose tertiles were associated with poorer OS among RTK I (log-rank p=0.08) and mesenchymal tumors (log-rank p=0.05), but not RTK II (log-rank p=0.99). After controlling for age, KPS, dexamethasone dose, and MGMT status, glucose remained significantly associated with survival (adjusted hazard ratio=5.2, p=0.02). DNA methylation clustering did not identify a unique signature associated with high or low glucose levels. Metabolomic analysis of 23 tumors showed minimal variation across metabolites within the cohort with no differences across molecular subclasses.
CONCLUSION
Higher average glucose values were associated with poorer OS in RTKI and Mesenchymal IDH-wt GBM, but not RTKII. There were no discernible epigenetic or metabolomic differences between tumors in different glucose environments, suggesting a potential survival benefit with systemic glucose lowering in selected molecular subtype.
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