Detection Of Aberrant Methylation Research Articles

Evaluation of Immunohistochemical Expression of Enhancer of Zeste Homolog 2 (EZH2) and Its Association With Clinicopathological Variables in Carcinoma Cervix.

Carcinoma cervix is the fourth most common cancer worldwide and is one of the leading causes of cancer death in women. Recently, immunohistochemical expression of biomarkers has been utilized as indicators of disease progression, aggressiveness for predicting the prognosis in various cancers. DNA methylation of genes plays an important role in pathogenesis of carcinoma cervix and detection of aberrant methylation can be utilized for detection of carcinoma cervix and monitoring of its progression. Enhancer of Zeste Homolog 2 (EZH2) is a histone methyltransferase and catalyzes methylation of histone H3 and plays an important role in tumor cell proliferation, invasion, and metastasis. The aim of this study was to analyze the pattern, distribution, and grade of immunohistochemical expression of EZH2 in carcinoma cervix and study its association with clinico-pathological variables such as age, site and size of tumor, type of growth, tumor grade, histological subtype, lymph node metastasis, and stage of the tumor according to the Federation of Gynaecology and Obstetrics (FIGO). This observational study was carried out in the Department of Pathology & Lab Medicine, at our institute. A total of 60 consecutive histopathologically confirmed cases of carcinoma cervix from January 2018 to June 2022 were subjected to immunohistochemistry (IHC) for EZH2. The immunohistochemical score for each case was obtained by multiplying the intensity and percentage of positive cells for EZH2. An immunohistochemical score of four or greater than four was considered as high immunoexpression. The immunohistochemical results were correlated with clinico-pathological variables. The data were analyzed using relevant statistical methods using SPSS version 23 (IBM Corp., Armonk, NY). To find the significant difference (p value) and association, chi-square test along with Pearson chi-square were used, wherever necessary. A p value of <0.05 was considered as significant. High immunoexpreesion of EZH2 exhibited a significant association (p < 0.05) with the tumor grade, histologic subtype, lymphnode metastasis, and FIGO stage. The results of our study affirm that a significant association exists between immunohistochemical expression of EZH2 with tumor grade, histological subtype, lymphnode metastasis, and FIGO stage which can be utilized in future studies with larger sample size to further strengthen the association of EZH2 immunoexpression in cancer cervix patients that may aid in the development of the targeted therapy in near future.

Diagnosis of Prader-Willi syndrome and Angelman syndrome by targeted nanopore long-read sequencing

The CpG island flanking the promoter region of SNRPN on chromosome 15q11.2 contains CpG sites that are completely methylated in the maternally derived allele and unmethylated in the paternally derived allele. Both unmethylated and methylated alleles are observed in normal individuals. Only the methylated allele is observed in patients with Prader-Willi syndrome, whereas only the unmethylated allele is observed in those with Angelman syndrome. Hence, detection of aberrant methylation at the differentially methylated region is fundamental to the molecular diagnosis of Prader-Willi syndrome and Angelman syndromes. Traditionally, bisulfite treatment and methylation-sensitive restriction enzyme treatment or methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) have been used. We here developed a long-read sequencing assay that can distinguish methylated and unmethylated CpG sites at 15q11.2 by the difference in current intensity generated from nanopore reads. We successfully diagnosed 4 Prader-Willi syndrome patients and 3 Angelman syndrome patients by targeting differentially methylated regions. Concurrent copy number analysis, homozygosity analysis, and structural variant analysis also allowed us to precisely delineate the underlying pathogenic mechanisms, including gross deletion, uniparental heterodisomy, uniparental isodisomy, or imprinting defect. Furthermore, we showed allele-specific methylation in imprinting-related differentially methylated regions on chromosomes 6, 7, 11, 14, and 20 in a normal individual together with 4 Prader-Willi patients and 3 Angelman syndrome patients. Hence, presently reported method is likely to be applicable to the diagnosis of imprinting disorders other than Prader-Willi syndrome and Angelman syndrome as well.

Abstract 1154: Detection of aberrant methylation of the RASSF1A gene as a biomarker for hepatocellular carcinoma.

Abstract Hepatocellular carcinoma (HCC) is an aggressive malignancy that is responsible for between 250,000 and 800,000 deaths annually worldwide. HCC has a poor prognosis with a 5-year survival rate of less than 15% mainly due to a lack of early detection. The current biomarker for HCC is the alpha-fetoprotein (AFP), which has a low sensitivity of 40-60%. There is a necessity for non-invasive, convenient, and sensitive methods to detect the cancer in its curative stages to improve prognosis. Previous studies from our laboratory showed that the location of the CpG site methylation studied could impact the marker performance in distinguishing HCC from other liver diseases. The aberrant methylation of the tumor-suppressor RASSF1A gene has been extensively studied as a marker for a variety of cancers, including liver cancer. However, some studies found that the gene was methylated even in non-cancerous liver tissues. Thus, in order to explore the possibility of detecting aberrant methylation of the tumor-suppressor RASSF1A gene as a biomarker for HCC screening, we analyzed the methylation profiles of 57 CpG sites in three regions of the promoter - P1, P2, and E1 - in normal, hepatitic, cirrhotic, adjacent non-HCC, and HCC tissue samples through bisulfite DNA sequencing. We discovered that the P1 region was most specifically methylated to HCC. We then confirmed the findings by developing a methylation specific PCR and analyzing a larger sample size. The data were analyzed and the P1 test was found to have an area under the curve (AUROC) of 0.90, whereas the E1 and P2 tests had AUROC of 0.84 and 0.72, respectively. At a cutoff of 10 copies for our analysis, the P1 MSP gave a sensitivity of 80.8% and specificity of 85.7%. Furthermore, we found that out of 62 AFP-negative samples, mRASSF1A detected 54 (87.1%). By combining AFP and mRASSF1A, 107 of 115 (93.0%) of HCC samples were detected as cancerous. Subsequently, we developed a PCR assay to detect the methylated RASSF1A gene in the urine of patients. This assay was then used to test the urine DNA isolated from patients with hepatitic, cirrhotic, or HCC after which the data were statistically evaluated. The potential of a urine DNA test for HCC screening is discussed. Citation Format: Surbhi Jain, Lijia Xie, Batbold Boldbaatar, Ying-Hsiu Su, Wei Song. Detection of aberrant methylation of the RASSF1A gene as a biomarker for hepatocellular 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 1154. doi:10.1158/1538-7445.AM2013-1154

Abstract 4538: Semiquantitative detection of aberrant methylation as a tumor biomarker in the serum and plasma of patients with gastric cancer

Abstract Background: Epigenetic silencing of tumor-related genes, due to CpG island methylation, is considered to be an important mechanism for the development of many tumors, including gastric cancinoma (GC). DNA methylation of multiple CpG sites in promoter regions of several tumor related genes, such as hMLH1, p14, COX2, and APC has been analyzed in GC so for. Circulating tumor DNA in serum/plasma has been demonstrated to reflect the biological characteristics of tumors. Recently, the presence of gene promoter methylation in serum/plasma DNA has also been demonstrated in patients with cancers of lung, head and neck, liver, and breast. So, in the present study, we attempted to determine the feasibility and the clinical correlations of detecting tumor-associated aberrant methylation in the serum/plasma of patients with gastric cancer. Materials and Methods: We examined promoter methylation of 6 genes using methylation-specific PCR (MSP) and methylation-sensitive high-resolution melting (MS-HRM) in paired serum, plasma and tumor samples of 50 gastric cancer patients. The tumor and the paired serum/plasma were investigated for aberrant methylation in BLU, DAPK1, GADD45G, MGMT, p15 and p16. The sensitivity of the HRM analysis was tested by using dilutions of fully methylated DNA into unmethylated DNA. Results: MSP was used to assess gene methylation in tumor samples. Promoter methylation in BLU, DAPK1, GADD45G, MGMT, p15, and p16 were detected in 47%, 37%, 59%, 32%, 14%, and 43% of tumor tissues by MSP. MS-HRM was used to assess gene methylation in serum/plasma samples concurrently. In the serum/plasma of gastric cancer patients, BLU, DAPK1, GADD45G, MGMT, p15, and p16 were methylated at frequencies of 26%, 21%, 23%, 11%, 9%, and 17%, respectively. Nineteen (38%) gastric tumor samples displayed concurrent methylation in three or more tumor genes. Aberrant methylation in one or more genes was found in 74% (37/50) serum samples and 70% (35/50) plasma samples. However, there was not a significant relationship of gene methylation between gastric tumor tissues and serum/plasma samples.Conclusion: These results suggest that aberrant promoter methylation in serum/plasma can be detected in a substantial proportion of gastric cancer patients. Detection of DNA methylation in serum/plasma may be a biomarker for early detection of gastric cancer. MS-HRM is powerful technique for the analysis of promoter methylation. Application of HRM analysis to large amount of clinical samples proves to be a fast and high-throughput way to investigate the epigenetic status of genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4538. doi:1538-7445.AM2012-4538

Multiple genes are hypermethylated in intraductal papillary mucinous neoplasms of the pancreas

Ductal adenocarcinoma of the pancreas is the fourth leading cause of cancer death and is usually diagnosed late. Intraductal papillary mucinous neoplasms are an increasingly recognized precursor to invasive ductal adenocarcinoma of the pancreas. Identifying the alterations in DNA methylation that arise during intraductal papillary mucinous neoplasm development may facilitate the development of markers that could be used to differentiate intraductal papillary mucinous neoplasms from non-neoplastic pancreatic cystic lesions. Surgically resected intraductal papillary mucinous neoplasms and adjacent ductal adenocarcinomas were microdissected from 50 patients. Normal pancreas was also obtained from 27 patients with intraductal papillary mucinous neoplasms or pancreatic adenocarcinomas and 10 patients with well-differentiated pancreatic endocrine neoplasms. Methylation-specific PCR was performed on isolated DNA for seven genes (SPARC, SARP2, TSLC1, RELN, TFPI2, CLDN5, UCHL1) known to be commonly aberrantly methylated in pancreatic ductal adenocarcinomas. The mean percentage of genes methylated in invasive ductal adenocarcinomas arising in association with an intraductal papillary mucinous neoplasm (mean±s.d., 81±17%) was significantly higher than that in noninvasive-intraductal papillary mucinous neoplasms (57±26%, P=0.007) or peritumoral normal epithelial cells (22±17%, P<0.0001). Carcinomas (intraductal papillary mucinous neoplasms with carcinoma in situ or their associated infiltrating adenocarcinoma) had significantly more methylated genes (71±19%) than low-grade (low and moderate dysplasia) intraductal papillary mucinous neoplasms (44±26%, P<0.0001). The mean percentage of genes methylated in histologically normal pancreatic ductal cells from patients with ductal neoplasia (22±17%) was significantly higher than in normal ductal cells from patients with well-differentiated pancreatic endocrine neoplasms (4±7%, P=0.002). Thus, aberrant DNA methylation increases with histologic grades of intraductal papillary mucinous neoplasm. Low-level aberrant methylation in the normal ductal cells is more prevalent in patients with ductal neoplasia than in controls without ductal neoplasms and may contribute to carcinogenesis. The detection of aberrant methylation in pancreatic cystic lesions could facilitate the diagnosis of intraductal papillary mucinous neoplasms.

Detection of aberrant methylation in fecal DNA as a molecular screening tool for colorectal cancer and precancerous lesions

To investigate the feasibility of detecting methylated fecal DNA as a screening tool for colorectal carcinoma (CRC) and precancerous lesions. Methylated secreted frizzled-related protein gene 2 (SFRP2), hyperplastic polyposis protein gene (HPP1) and O6-methylguanine-DNA methyltransferase gene (MGMT) in stools from 52 patients with CRC, 35 patients with benign colorectal diseases and 24 normal individuals were analyzed using methylation-specific PCR. Methylated SFRP2, HPP1 and MGMT were detected in 94.2%, 71.2%, 48.1% of CRC patients and 52.4%, 57.1%, 28.6% of adenoma patients, respectively. The overall prevalence of fecal DNA with at least one methylated gene was 96.2% and 81.8% in patients with CRC and precancerous lesions, respectively. In contrast, only one of the 24 normal individuals revealed methylated DNA. These results indicated a 93.7% sensitivity and a 77.1% specificity of the assay for detecting CRC and precancerous lesions. Methylation testing of fecal DNA using a panel of epigenetic markers may be a simple and promising non-invasive screening method for CRC and precancerous lesions.

22 A gene promoter hypermethylation panel to detect transitional cell carcinoma of the bladder

Introduction: Promoter methylation has been established as a mechanism for silencing of tumour suppressor genes (TSGs) in TCC. Detection of aberrant methylation has been proposed as a novel diagnostic test, but the desired sensitivity and specificity has not been proven. The gene specific nature of methylation necessitates investigators to identify a panel of TSG's, that when analysed for aberrant methylation in tumour DNA, demonstrate a high sensitivity, specificity and diagnostic accuracy for clinical use. Here, we analyse a panel of 16 genes in the DNA of 96 TCCs and 30 samples of normal urothelium.Methods: DNA was obtained from 96 TCCs and 30 normal bladders. The DNA was bisulphite modified and quantitative methylation specific PCR (QMSP) was performed for 16 gene promoters.Results: Methylation was detected for all 16 genes in the 96 TCCs. This ranged from 3% to 75%. The genes with the highest frequency of methylation were RASSF1a (54%), E‐cadherin (41%), BCL2 (45%), TNFRS25 (75%), EDNRB (66%), IGFBP3 (43%) and WIF1 (56%). High levels of methylation were detected in the normal controls for RASSF1a (50%) and E‐cadherin (20%), with 11 other genes having low levels of methylation. Using a panel of 5 heavily methylated genes, a sensitivity of 57%, specificity of 89% and a diagnostic accuracy of 80% resulted.Conclusion: The frequency of methylation varied widely for different genes, confirming the gene specific nature of aberrant methylation in TCC. Using our panel of gene loci, the sensitivity and specificity of methylation DNA analysis was not adequate. However, our study does demonstrate the feasibility of TCC DNA methylation analysis and with the identification of further cancer specific gene loci, it may prove a useful early detection strategy.

Detection of aberrant methylation in apoptosis-related SARP2 gene using pancreatic juice in the diagnosis of pancreatic cancer

Detection of aberrant methylation in apoptosis-related SARP2 gene using pancreatic juice in the diagnosis of pancreatic cancer

Multigene methylation analysis of gastrointestinal tumors: TPEF emerges as a frequent tumor-specific aberrantly methylated marker that can be detected in peripheral blood.

Gene promoter methylation is a mechanism for tumor suppressor gene silencing and inactivation. The development of highly sensitive methods for revealing aberrant cancer-associated DNA methylation allows the identification of tumor markers not only in tumor samples, but also in body fluid, an approach that can be useful in the early detection of neoplasms. We analyzed the methylation status at 16 loci in tumor samples of the gastrointestinal tract and in early or pre-neoplastic lesions of the colon. Tumor samples revealed that methylation at the transmembrane protein containing epidermal growth factor and follistatin domains (TPEF) locus had the best ratio of discrimination between tumor samples versus normal tissues (83 versus 0%). Its combination with hypermethylated in cancer 1 (HIC1), death-associated protein kinase (DAPK) and O-6-methylguanine DNA methyltransferase (MGMT), allowed the detection of aberrant methylation in 98% of colorectal carcinomas and 100% of gastric carcinomas. The same alterations were also detected in colon adenomas and tissues surrounding the adenomas, indicating that hypermethylation at these loci occurred early in tumor progression. Analysis of DNA from peripheral blood revealed that TPEF methylation was detectable in colorectal tumor patients and patients with early or pre-neoplastic lesions, but not in healthy volunteers. Our results identify TPEF as a tumor marker that could be useful in the follow-up of gastrointestinal cancer patients or the screening of individuals at risk of developing gastrointestinal neoplasms.