Correlation Of Methylation Status Research Articles

Abstract A016: Identification of methylated regions in ductal carcinoma in situ and association with disease progression

Abstract Background: The advent of breast screening has led to a 4-fold increase in the diagnosis of ductal carcinoma in situ (DCIS). Studies following the clinical outcomes of patients show variable progression free survival rates, with only up to 35% of patients progressing to invasive disease without treatment. This highlights the need to find a biomarker to accurately predict which DCIS lesions will recur as invasive tumors. Epigenetics changes are events which occur early in tumorigenesis, this makes DNA methylation a potential biomarker of DCIS progression. However, a lack of DCIS methylation profiling with long term follow-up data exists. This study investigates genome-wide methylation profiles in women with primary DCIS and associates the data with their overall recurrence free survival. Methods: DCIS was macrodissected from 89 formalin-fixed paraffin embedded (FFPE) to extract tumor enriched DNA from patients with DCIS and long term follow up. 39 women had developed an ipsilateral invasive recurrence (classified as cases) and 50 had no evidence of recurrent disease (classified as controls). Genome wide methylation was assessed using the human methylation EPIC BeadChip which, interrogates over 850,000 methylation sites. Data was assessed for quality both at the sample and probe level by the wateRmelon. Further analysis was performed in ChAMP to identify differentially methylated regions (DMR) and by DMRcate packages in R to identify variably methylated regions (VMR). Genes annotated in the most significant VMRs were analysed through Metascape, a web-based tool to perform functional gene set enrichment analysis (GSEA). A cox proportional hazards model was used to calculate the association between the methylation of the VMRs and recurrence free survival. This model was adjusted for DCIS receptor status and grade. Results: 59 samples passed data quality assessment (35 controls and 24 cases). 10 differentially methylated regions were identified. The most significant of which, was a hypomethylated region on chromosome 4, containing CDKL2 (p = 0.001), known to promote the epithelial-mesenchymal transition in breast cancer progression. 5813 VMRs were identified across the genome, (P-value range between 0 and 10-321). 82% of the VMRs aligned to the body of the gene or the surrounding regulatory features. GSEA revealed that VMRs were predominantly involved in pathways involved in cell adhesion (GO:0007156) Assessment of the significant VMRs by COX proportional hazards model showed that a VMR on chromosome 6p was associated with the development of invasive disease after adjusting for oestrogen receptor, human epidermal growth factor 2 status and grade (p=0.001). Conclusions: This preliminary study shows altered sites of methylation could be observed across the genome, in DCIS. The function of the VMRs is currently being investigated to understand how methylation in this region predisposes to invasive recurrence of DCIS. Correlation of methylation status and RNA expression data will be used to understand the biological relevance. Citation Format: Vandna Shah, Maria Roman-Escorza, Karen Clements Clements, Lennart Mulder, Esther H. Lips, Jelle Wesseling, Sarah Pinder, Alastair M. Thompson, Elinor J. Sawyer. Identification of methylated regions in ductal carcinoma in situ and association with disease progression [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr A016.

Correlation of methylation status in MTHFR promoter region with recurrent pregnancy loss

BackgroundDNA methylation is an epigenetic process for modifying transcription factors in various genes. Methylenetetrahydrofolate reductase (MTHFR) stimulates synthesis of methyl radical in the homocysteine cycle and delivers methyl groups needed in DNA methylation. Furthermore, numerous studies have linked gene polymorphisms of this enzyme with a larger risk of recurrent pregnancy loss (RPL), yet scarce information is available concerning the association between epigenetic deviations in this gene and RPL. Hypermethylation at precise DNA sequences can function as biomarkers for a diversity of diseases. We aimed by this study to evaluate the methylation status of the promoter region of MTHFR gene in women with RPL compared to healthy fertile women. It is a case–control study. Hundred RPL patients and hundred healthy fertile women with no history of RPL as controls were recruited. MTHFR C677T was assessed by polymerase chain reaction-restriction fragment length polymorphism (RFLP). Quantitative evaluation of DNA methylation was performed by high-resolution melt analysis by real-time PCR. ResultsThe median of percentage of MTHFR promoter methylation in RPL cases was 6.45 [0.74–100] vs. controls was 4.50 [0.60–91.7], P value < 0.001. In the case group, 57 hypermethylated and 43 normo-methylated among RPL patients vs. 40 hypermethylated and 60 normo-methylated among controls, P< 0.005. Frequency of T allele in C677T MTHFR gene among RPL patients was 29% vs. 23% among the control group; C allele vs. T allele: odds ratio (OR) = 1.367 (95% confidence interval (CI) 0.725–2.581). ConclusionFindings suggested a significant association between hypermethylation of the MTHFR promoter region in RPL patients compared to healthy fertile women.

The clinicopathological significance of HES1 promoter hypomethylation in patients with colorectal cancer.

Hairy/enhancer of split 1 (HES1) is a basic helix–loop–helix transcriptional repressor. Aberrant demethylation has been considered a common mechanism of tumor promoter gene activation. In the current study, we aimed to investigate the methylation status of the HES1 promoter and correlations with clinicopathological parameters and prognosis in colorectal cancer (CRC). The expression of HES1 in 50 paired CRC specimens and adjacent normal tissues was determined by using quantitative real-time polymerase chain reaction and immunohistochemical analysis. Moreover, DNA methylation status was evaluated through methylation-specific polymerase chain reaction and bisulfite sequencing. The correlation of methylation status with HES1 expression level and clinicopathological parameters was statistically analyzed in CRC patients. Our data showed that the methylation level of HES1 was significantly decreased and negatively correlated with HES1 expression in CRC tissues. Moreover, HES1 hypomethylation was associated with a poor histological grade, Dukes’ classification, lymph node metastasis, and clinical stages (P<0.05). Furthermore, survival analyses revealed that a decreased methylation status of HES1 was linked to poor prognosis of CRC patients. In conclusion, promoter hypomethylation upregulates HES1 expression and plays a critical role in the progression and prognosis of CRC patients.

Abstract 641: Correlation of methylation status of deep surgical margins with the local reccurence of head and neck squamous cell carcinoma.

Abstract Background: Locoregional recurrence occurs in as many as 40% of patients undergoing surgery for head and neck squamous cell carcinoma (HNSCC). We have been focused some molecular alterations exsiting even in histologically negative surgical margins. However, in head and neck cancer resection, surgical margins are hard to evaluate because the most of them usually consists of complicated muscular margins. In order to evaluate these deep surgical margins, before collecting some margin tissues, we often conduct tissue imprinting by using one layer nitrocellulose sheet which can collect proteins without hurting surgical samples. Method: Both 161 deep margin tissues and 166 deep margin imprints were collected from 74 histologically margin negative resections of primary HNSCC from May, 2009 to September, 2012. Bisulfited DNA from samples were amplified using quantitive methylation-specific polymerase chain reaction (QMSP) for 6 genes (DCC, EDNRB, HOXA9, KIF1A, NID2 and NR2B). The cut off value was calculated using QMSP data of tumors and normal muscles, and was applied to deep margin samples. The correlation between the QMSP status of deep margin samples and clinical outcome was evaluated. Results: The average QMSP of tumor tissues, that of normal muscles and the cut off value are 21.0, 0.6, 2.1 in DCC, 23.3, 0.6, 2.2 in EDNRB, 32.0, 0.7, 2.3 in HOXA9, 20.3, 0.4, 1.3 in KIF1A, 36.0, 3.0, 9.4 in NID2 and 10.7, 0.1, 0.4 in NR2B. Those who has the higher QMSP values of deep margin tissues than the cut off value tends to have a poor recurrence free survival (P=0.073 in DCC, P=0.018 in HOXA9, P=0.005 in KIF1A, P=0.011 in NID2) and a poor overall survival (P=0.076 in HOXA9, P=0.059 in NID2). Also, those who has the higher QMSP values of deep margin imprints than the cut off value tends to have a poor recurrence free survival (P=0.051 in HOXA9) and a poor overall survival (P=0.075 in HOXA9). Conclusion: The methylation status of HNSCC deep surgical margin samples is correlated with the local recurrence. Especially, hyper-methylated HOXA9 gene of both deep margin tissues and imprints is a significant biomarker of poor recurrence free survival and overall survival. Citation Format: Masamichi Hayashi, Gaosong Wu, Xiaofei Chang, Xiufeng Li, Julie Ahn, Zubair Khan, Belinda Akpeng, Jong–Lyel Roh, Wayne Koch. Correlation of methylation status of deep surgical margins with the local reccurence of head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 641. doi:10.1158/1538-7445.AM2013-641

Correlation between abnormal methylaiton of exon 9 in insulin-like growth factor 2 and its expression in renal clear cell carcinoma

Objective To investigate the correlation of methylation status at exon 9 CpG island of insulin-like growth factor-2 (IGF-2) with its expression in renal clear cell carcinoma ( RCC ).Methods All the 43 RCC samples and corresponding normal tissues were analyzed for the exon 9 methylated status of IGF-2 and its expression.Results Methylation rate of exon 9 was 15.00％ in RCC group and 96.77 ％ in normal tissue respectively (P ＜0.01 ) and the methylation status of exon 9 was associated with size,stage and grade of RCC (P ＜ 0.05).The biallelic expression rate of IGF-2 in unmethylated RCC tissues was significantly higher than that in methylated RCC tissues (P ＜0.01 ).Conclusion The exon 9 unmethylated status may play an essential role in renal carcinogenesis and serve as a marker for reflecting its biological behavior. Key words: Clear-cell renal carcinoma; Insulin-like growth factor 2; Exon 9; Methylation

Distinct Epigenetic Domains Separated by a CTCF Bound Insulator between the Tandem Genes, BLU and RASSF1A

BackgroundTumor suppressor gene (TSG) RASSF1A and candidate TSG BLU are two tandem head-to-tail genes located at 3p21.3. We hypothesized that there may be a concordance on their gene expression and promoter methylation status. If not, then there may be an insulator located between RASSF1A and BLU genes that provides a barrier activity.Methodology/Principal FindingsWe first identified potential transcriptionally important CpG sites using the methylation-specific oligonucleotide array in relation to mRNA expression of RASSF1A and BLU genes in primary lung tumors. We demonstrated that E2F1 bound to the potential transcriptionally important CpG sites in RASSF1A gene of a normal lung cell line expressing RASSF1A transcripts, whereas loss of E2F1 binding to RASSF1A in A549 cancer cell line was the result of DNA methylation. Both RASSF1A and BLU genes had their own potential transcriptionally important CpG regions. However, there was no correlation of methylation status between RASSF1A and BLU. Using gel shift assay and chromatin immunoprecipitation-PCR (ChIP-PCR), we found that CCCTC-binding factor (CTCF) bound to insulator sequences located between these two genes. Bisulfite sequencing and ChIP-PCR revealed distinct methylation and chromatin boundaries separated by the CTCF binding domains in normal cells, whereas such distinct epigenetic domains were not observed in cancer cells. Note that demethylation reagent and histone deacetylase inhibitor treatments led to CTCF binding and recovery of barrier effect for RASSF1A and BLU genes in cancer cells.Conclusions/SignificanceOur study dissects the potential transcriptionally important CpG sites for RASSF1A and BLU genes at the sequence level and demonstrates that CTCF binding to the insulator of BLU gene provides a barrier activity within separate epigenetic domains of the juxtaposed BLU and RASSF1A loci in the 3p21.3 gene cluster region.

Abstract 4950: Correlation of methylation status with expression levels of the genes in prostate cancer

Abstract DNA methylation abnormalities, either gain or loss of methylation in promoters or other regulatory regions, contribute to tumorigenesis by altering the activity of tumor suppressor or oncogenes. To identify the molecular alterations in prostate cancer, we have profiled gene methylation using Infinium Human Methylation27 bead chips in benign prostate tissue, primary prostate tumor tissue from patients who developed metastasis within 5 years of prostatectomy, and serum from patients who developed metastasis. Tissue DNA isolation and bisulfite conversion was done using ZR Genomic DNA kit and EZ methylation kit. Serum DNA isolation and bisulfite conversion was done using ZR serum DNA kit and EZ methylation kit. Methylation data was processed using the Beadstudio average normalization algorithm and differentially methylated genes identified using a two-group T-test p-value &amp;lt; 0.05. To evaluate the impact of methylation on mRNA expression levels, we isolated RNA from LCM-derived epithelial cells of 124 patients using Picopure RNA isolation kit and measured expression using HG-U133+2 microarrays. The gene expression data was preprocessed using GCRMA background correction, FASTLO normalization, affinities-only PM correction, and median polish summarization, and differential gene expression was defined by a two-group T-test p-value &amp;lt; 0.05, a log2 fold change &amp;gt; 1, and a maximum average expression value &amp;gt; log2 7. Comparison of the methylation and gene expression data identified 321 differentially expressed Affymetrix probesets which mapped to genes differentially methylated genes; 272 with differential methylation in the prostate cancer tissue and 97 with differential methylation in the prostate cancer serum. In general, we expected a complementary effect between gene methylation and expression, with hyper-methylation driving decreased expression, and hypo-methylation driving increased expression. This was seen in the prostate cancer tissue methylation data, where 228/272 (84%) probesets possessed this methylation-expression complementarity; 212/238 hyper-methylated genes and 16/34 hypo-methylated genes. The effect was not as strong in the prostate cancer serum methylation data, where 30/97 (31%) probesets displayed the methylation-expression complementarity; 19/79 hyper-methylated genes and 11/18 hypo-methylated genes. Overall, our results revealed a significant correlation of the DNA methylation with the down regulation in the mRNA expression levels of the genes, which could play potential roles in prostate cancer progression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4950.