Analysis of DNA Methylation in FFPE Tissues Using the MethyLight Technology

AbstractAmong women, ovarian cancer (OC) is one of the most severe forms of malignancy, accounting for a low 5-year survival rate, of approximately 52%. Early symptoms are unspecific and hence hard to detect. The origin of OC and its subtypes are still unclear, underlying the need for efficient diagnostic biomarkers. In that regard, epigenetics studies are emerging in cancer diagnostics, with encouraging outcomes. Among them, DNA methylation profiling has shown that the origins of the cancer epigenome are associated with molecular factors that are crucial to carcinogenesis, such as regulation of oncogenes and tumor suppressors. Furthermore, those events have been detected in abnormal cell morphology before neoplastic formation, indicating its potential crucial use in the OC diagnostics in the future. Nonetheless, studies are limited, and whether methylation analysis can be performed optimally in formalin-fixed paraffin-embedded (FFPE) preparations of OC cases is still elusive. In the present report, we investigated the performance of DNA methylation analysis in FFPE samples, compared to their matched fresh frozen tissue in a small cohort of OC samples. We found that the overall DNA methylation profile in FFPE tissue showed high concordance to that found in fresh frozen tissue, and accounting for the small cohort size, the differentially methylated sites found primarily in frozen tissue, compared to benign samples, were also reproducible in FFPE. Overall, by using samples from our current clinical setting of tissue preservation, these preliminary observations might provide insights into the clinical use of FFPE tissues in methylation studies without critically compromising the outcome.

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Characterization of Methylation Patterns in Diffuse Large B Cell Lymphoma By Genome-Wide Methylation Analysis

Blood ◽

10.1182/blood-2019-131656 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1243-1243

Author(s):

Fengyi Zhao ◽

Lei Zhang ◽

Yan Qin ◽

Ming-Zhe Han ◽

Xiaohong Han ◽

...

Keyword(s):

Dna Methylation ◽

Signaling Pathway ◽

Methylation Level ◽

Enrichment Analysis ◽

B Cell Lymphoma ◽

Mapk Signaling ◽

Mapk Signaling Pathway ◽

Pathway Enrichment Analysis ◽

Ffpe Tissues ◽

Methylation Patterns

Background: Diffuse large B cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma worldwide. Although the reference standard for identifying of the cell types is considered of gene expression profiling (GEP). But immunohistochemistry (IHC) is the most common method commercially available. The purpose of this study was to characterize the circulating cell-free DNA (cfDNA) methylation profile in DLBCL and to compare this profile with methylation observed in formalin fixed paraffin-embedded (FFPE) tissues. Additional efforts were made to correlate the observed methylation patterns with prognostic analysis and selected clinical features. Methods: The cfDNA and DNA of FFPE were extracted from 72 patients and 39 patients respectively. We assessed DNA methylation from plasma samples obtained from 29 individuals with GCB DLBCL at the time before treatment along with 43 samples of non-GCB DLBCL as controls. DNA from FFPE tissues were extracted from 11 individuals of GCB DLBCL and 28 individuals with non-GCB DLBCL. DNA methylation was analyzed with the Infinium MethylationEPIC BeadChip that quantitatively measures the methylation levels of more than 850,000 CpG sites across the genome. M values were used for visualization and intuitive interpretation of the results. Moreover, pathway enrichment analysis was performed with the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Database. Results: We found a total of 207 significant differentional differentially methylated positions (DMPs) of cfDNA between the GCB and non-GCB groups, identified with a p value of 0.001 (Fig. 1A). Of these, 65 presented at least 10% (|Δbeta| > 0.1) difference in the methylation level between GCB and non-GCB. 29 (44.6%) were found hypermethylated in GCB DLBCL, while 36 (55.4%) appeared hypomethylated (Fig. 1B). The distribution of the DMPs identified according to their location relative to CpG islands (CGI) were represented in Fig. 1C. Unsupervised clustering performed on DNA methylation values for the 207 DMPs identified is presented in Fig. 1D. These results highlight the differences between GCB and non-GCB samples. There are 1549 significant DMPs of DNA from FFPE between the GCB and non-GCB groups, identified with a p value of 0.001 (Fig. 1E). Of these, 1512 presented at least 10% (|Δbeta| > 0.1) difference in the methylation level between GCB and non-GCB . 1370 (90.6%) were found hypermethylated in GCB DLBCL, while 142 (9.4%) appeared hypomethylated (Fig. 1F). The distribution of the DMPs identified according to their location relative to CpG islands (CGI) were represented in Fig. 1G. Unsupervised clustering performed on DNA methylation values for the 1549 DMPs identified is presented in Fig. 1H. These results highlight the differences between GCB and non-GCB in FFPE samples which according with that in serum. The KEGG pathway enrichment analysis of DNA from FFPE tissue methylation revealed that the process "PI3K/Akt, Ras, MAPK signaling pathway" and "Human papillomavirus infection" are likely major contributors to Hans pathological type. In addition, the enrichment analysis of cfDNA methylation revealed that the process "MAPK signaling pathway" is likely the most important factor. Furthermore, we also have analyzed the methylation level between refractory or relapsed (R/R) DLBCL patients and individuals with a good prognosis. The differential methylation patterns were also found both in serums and FFPE tissues. Conclusions: The DNA methylation differs in GCB and non-GCB DLBCL patients. MAPK signaling pathway plays an important role in it. The mechanism needs to be further explored. Figure 1 Disclosures No relevant conflicts of interest to declare.

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DNA methylation in satellite repeats disorders

Essays in Biochemistry ◽

10.1042/ebc20190028 ◽

2019 ◽

Vol 63 (6) ◽

pp. 757-771 ◽

Cited By ~ 4

Author(s):

Claire Francastel ◽

Frédérique Magdinier

Keyword(s):

Dna Methylation ◽

Human Genome ◽

Repetitive Dna ◽

Dna Sequences ◽

Satellite Repeats ◽

Tremendous Progress ◽

Genes Encoding ◽

Dna Elements ◽

Near Future

Abstract Despite the tremendous progress made in recent years in assembling the human genome, tandemly repeated DNA elements remain poorly characterized. These sequences account for the vast majority of methylated sites in the human genome and their methylated state is necessary for this repetitive DNA to function properly and to maintain genome integrity. Furthermore, recent advances highlight the emerging role of these sequences in regulating the functions of the human genome and its variability during evolution, among individuals, or in disease susceptibility. In addition, a number of inherited rare diseases are directly linked to the alteration of some of these repetitive DNA sequences, either through changes in the organization or size of the tandem repeat arrays or through mutations in genes encoding chromatin modifiers involved in the epigenetic regulation of these elements. Although largely overlooked so far in the functional annotation of the human genome, satellite elements play key roles in its architectural and topological organization. This includes functions as boundary elements delimitating functional domains or assembly of repressive nuclear compartments, with local or distal impact on gene expression. Thus, the consideration of satellite repeats organization and their associated epigenetic landmarks, including DNA methylation (DNAme), will become unavoidable in the near future to fully decipher human phenotypes and associated diseases.

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