MPC-1 DNA methylome analysis suggested the presence of “true” IDH-wildtype lower-grade gliomas

Background: There will be significant changes in the diagnosis of IDH-wildtype adult-type gliomas in the upcoming 5th edition of the WHO Classification of Central Nervous System Tumours. IDH-wildtype lower grade gliomas (IDHwt LGGs) that harbor molecular features of glioblastoma (EGFR amplification, the combination of whole chromosome 7 gain and whole chromosome 10 loss (7+/10-), or TERT promoter mutations) will be diagnosed as glioblastomas (GBMs), while IDH-wildtype astrocytomas will not be included as a separate tumor type. However, IDHwt LGGs are a very heterogeneous group of tumors, and further investigation is warranted particularly in those without molecular features of glioblastoma. To elucidate the biology of IDHwt LGGs, we analyzed DNA methylation profile and survival time. Materials and Methods: Of the 724 adult-type diffuse glioma samples from a multi-institutional study, 64 IDHwt LGG, including 54 without any of molecular features of GBM and 10 with PDGFRA amplification or TERT promoter mutation, were examined using Infinium MethylationEPIC BeadChip. The raw data files (IDAT files) were analyzed by the web-based DNA methylation classifier provided by DKFZ (MolecularNeuropathology.org) or by R (Version 4.0.4) using the minfi (1.34.0) and Rtsne (0.15) packages. [Result] Twenty-three out of 54 IDHwt LGGs matched known methylation classes using the DKFZ methylation classifier. In t-Distributed Stochastic Neighbor Embedding clustering analysis, 20 cases formed a cluster within the methylation class family glioblastoma, IDH-wildtype, mainly subclass RTK I (“GBM” cluster). Another 29 IDHwt LGGs formed an independent cluster (“LGG” cluster) separate from any of the existing reference groups near but not overlapping with several subtypes of pediatric-type lower grade gliomas. The “LGG” cluster cases had significantly longer overall survival than the “GBM” cluster cases. Discussion: Methylation profiling showed that IDHwt LGGs without molecular features of GBM were heterogeneous group of tumors. Our data suggested the presence of “true” IDHwt LGGs with intermediate prognosis.

Accumulation of Genetic and Epigenetic Alterations in the Background Liver and Emergence of Hepatocellular Carcinoma in Patients with Non-Alcoholic Fatty Liver Disease

The incidence of hepatocellular carcinoma (HCC) related to non-alcoholic fatty liver disease (NAFLD) is increasing worldwide. We analyzed 16 surgically resected HCC cases in which the background liver was pathologically diagnosed as NAFLD. Specimens with Brunt classification grade 3 or higher were assigned as the fibrotic progression group (n = 8), and those with grade 1 or lower were classified as the non-fibrosis progression group (n = 8). Comprehensive mutational and methylome analysis was performed in cancerous and noncancerous tissues. The target gene mutation analysis with deep sequencing revealed that CTNNB1 and TP53 mutation was observed in 37.5% and TERT promoter mutation was detected in 50% of cancerous samples. Furthermore, somatic mutations in non-cancerous samples were less frequent, but were observed regardless of the progression of fibrosis. Similarly, on cluster analysis of methylome data, status for methylation events involving non-cancerous liver was similar regardless of the progression of fibrosis. It was found that, even in cases of non-progressive fibrosis, accumulation of gene mutations and abnormal methylation within non-cancerous areas were observed. Patients with NAFLD require a rigorous liver cancer surveillance due to the high risk of HCC emergence based on the accumulation of genetic and epigenetic abnormalities, even when fibrosis is not advanced.

NCMP-01. NOVEL BIOMARKERS FOR RADIATION-INDUCED NEUROTOXICITY

BACKGROUND Brain radiotherapy is the main therapeutic modality for brain metastases (BM), but carries short and long term toxicities, termed radiation-induced neurotoxicity (RIN), and classified to acute, early-delayed and late-delayed RIN according to its time onset. Although diagnosis of RIN is crucial for patient management, there is an unmet need for sensitive biomarkers for RIN. Here we report on a novel non-invasive biomarker for detection and monitoring RIN. As radiotherapy is known to induce brain cells apoptosis as well as BBB disruption, we hypothesized that circulating cell free DNA fragments derived from dying brain cells will be elevated in the blood of patients with RIN. Using comparative methylome analysis we identified 13 genomic loci showing brain-specific DNA methylation patterns, including markers for neurons, oligodendrocytes, and astrocytes. We searched for these brain-derived cfDNA (bncfDNA) in plasma samples of patients following brain irradiation. METHODS We followed 24 patients treated by brain radiotherapy for BM by clinical and radiological examinations before, during and after treatment. In addition, we serially collected blood samples for DNA analysis, and correlated bncfDNA levels with clinical and radiological assessment. RESULTS Patient`s median age was 60 years. Most common primary tumor sites were breast (25%), lung (20.8%) and melanoma (12.5%). RIN was detected in 10 patients (62%). BncfDNA levels increased up to 292.4 fold in acute RIN, up to 138533.1 in early-delayed RIN, and up to 58.4 fold in late-delayed RIN. Resolution of RIN correlated with decrease in bncfDNA. Changes in bncfDNA levels were independent of tumor response and suggested to reflect both symptomatic and asymptomatic RIN. CONCLUSION Increase in bncfDNA levels characterizes RIN independent of tumor response. Thus, BncfDNA may serve as a novel biomarker for brain cells death incurred by radiotherapy. Further studies are required to explore the clinical utility of bncfDNA as a RIN biomarker.

SPOP mutation induces DNA methylation via stabilizing GLP/G9a

Mutations in SPOP E3 ligase gene are reportedly associated with genome-wide DNA hypermethylation in prostate cancer (PCa) although the underlying mechanisms remain elusive. Here, we demonstrate that SPOP binds and promotes polyubiquitination and degradation of histone methyltransferase and DNMT interactor GLP. SPOP mutation induces stabilization of GLP and its partner protein G9a and aberrant upregulation of global DNA hypermethylation in cultured PCa cells and primary PCa specimens. Genome-wide DNA methylome analysis shows that a subset of tumor suppressor genes (TSGs) including FOXO3, GATA5, and NDRG1, are hypermethylated and downregulated in SPOP-mutated PCa cells. DNA methylation inhibitor 5-azacytidine effectively reverses expression of the TSGs examined, inhibits SPOP-mutated PCa cell growth in vitro and in mice, and enhances docetaxel anti-cancer efficacy. Our findings reveal the GLP/G9a-DNMT module as a mediator of DNA hypermethylation in SPOP-mutated PCa. They suggest that SPOP mutation could be a biomarker for effective treatment of PCa with DNA methylation inhibitor alone or in combination with taxane chemotherapeutics.

Integrative genomics analysis of nasal intestinal-type adenocarcinomas demonstrates the major role of CACNA1C and paves the way for a simple diagnostic tool in male woodworkers

Background Nasal intestinal-type adenocarcinomas (ITAC) are strongly related to chronic wood dust exposure: The intestinal phenotype relies on CDX2 overexpression but underlying molecular mechanisms remain unknown. Our objectives were to investigate transcriptomic and methylation differences between healthy non-exposed and tumor olfactory cleft mucosae and to compare transcriptomic profiles between non-exposed, wood dust-exposed and ITAC mucosa cells. Methods We conducted a prospective monocentric study (NCT0281823) including 16 woodworkers with ITAC, 16 healthy exposed woodworkers and 13 healthy, non-exposed, controls. We compared tumor samples with healthy non-exposed samples, both in transcriptome and in methylome analyses. We also investigated wood dust-induced transcriptome modifications of exposed (without tumor) male woodworkers’ samples and of contralateral sides of woodworkers with tumors. We conducted in parallel transcriptome and methylome analysis, and then, the transcriptome analysis was focused on the genes highlighted in methylome analysis. We replicated our results on dataset GSE17433. Results Several clusters of genes enabled the distinction between healthy and ITAC samples. Transcriptomic and IHC analysis confirmed a constant overexpression of CDX2 in ITAC samples, without any specific DNA methylation profile regarding the CDX2 locus. ITAC woodworkers also exhibited a specific transcriptomic profile in their contralateral (non-tumor) olfactory cleft, different from that of other exposed woodworkers, suggesting that they had a different exposure or a different susceptibility. Two top-loci (CACNA1C/CACNA1C-AS1 and SLC26A10) were identified with a hemimethylated profile, but only CACNA1C appeared to be overexpressed both in transcriptomic analysis and in immunohistochemistry. Conclusions Several clusters of genes enable the distinction between healthy mucosa and ITAC samples even in contralateral nasal fossa thus paving the way for a simple diagnostic tool for ITAC in male woodworkers. CACNA1C might be considered as a master gene of ITAC and should be further investigated. Trial registration: NIH ClinicalTrials, NCT0281823, registered May 23d 2016, https://www.clinicaltrials.gov/NCT0281823.

Transcriptome and Methylome Analysis Reveal Complex Cross-Talks between Thyroid Hormone and Glucocorticoid Signaling at Xenopus Metamorphosis

Background: Most work in endocrinology focus on the action of a single hormone, and very little on the cross-talks between two hormones. Here we characterize the nature of interactions between thyroid hormone and glucocorticoid signaling during Xenopus tropicalis metamorphosis. Methods: We used functional genomics to derive genome wide profiles of methylated DNA and measured changes of gene expression after hormonal treatments of a highly responsive tissue, tailfin. Clustering classified the data into four types of biological responses, and biological networks were modeled by system biology. Results: We found that gene expression is mostly regulated by either T3 or CORT, or their additive effect when they both regulate the same genes. A small but non-negligible fraction of genes (12%) displayed non-trivial regulations indicative of complex interactions between the signaling pathways. Strikingly, DNA methylation changes display the opposite and are dominated by cross-talks. Conclusion: Cross-talks between thyroid hormones and glucocorticoids are more complex than initially envisioned and are not limited to the simple addition of their individual effects, a statement that can be summarized with the pseudo-equation: TH ∙ GC > TH + GC. DNA methylation changes are highly dynamic and buffered from genome expression.

ASXL1 mutations are associated with distinct epigenomic alterations that lead to sensitivity to venetoclax and azacytidine

The BCL2-inhibitor, Venetoclax (VEN), has shown significant anti-leukemic efficacy in combination with the DNMT-inhibitor, Azacytidine (AZA). To explore the mechanisms underlying the selective sensitivity of mutant leukemia cells to VEN and AZA, we used cell-based isogenic models containing a common leukemia-associated mutation in the epigenetic regulator ASXL1. KBM5 cells with CRISPR/Cas9-mediated correction of the ASXL1G710X mutation showed reduced leukemic growth, increased myeloid differentiation, and decreased HOXA and BCL2 gene expression in vitro compared to uncorrected KBM5 cells. Increased expression of the anti-apoptotic gene, BCL2, was also observed in bone marrow CD34+ cells from ASXL1 mutant MDS patients compared to CD34+ cells from wild-type MDS cases. ATAC-sequencing demonstrated open chromatin at the BCL2 promoter in the ASXL1 mutant KBM5 cells. BH3 profiling demonstrated increased dependence of mutant cells on BCL2. Upon treatment with VEN, mutant cells demonstrated increased growth inhibition. In addition, genome-wide methylome analysis of primary MDS samples and isogenic cell lines demonstrated increased gene-body methylation in ASXL1 mutant cells, with consequently increased sensitivity to AZA. These data mechanistically link the common leukemia-associated mutation ASXL1 to enhanced sensitivity to VEN and AZA via epigenetic upregulation of BCL2 expression and widespread alterations in DNA methylation.