MethyLight Assay Research Articles

Immune microenvironmental heterogeneity according to tumor DNA methylation phenotypes in microsatellite instability-high colorectal cancers

The detailed association between tumor DNA methylation, including CpG island methylation, and tumor immunity is poorly understood. CpG island methylator phenotype (CIMP) is observed typically in sporadic colorectal cancers (CRCs) with microsatellite instability-high (MSI-H). Here, we investigated the differential features of the tumor immune microenvironment according to CIMP status in MSI-H CRCs. CIMP-high (CIMP-H) or CIMP-low/negative (CIMP-L/0) status was determined using MethyLight assay in 133 MSI-H CRCs. All MSI-H CRCs were subjected to digital pathology-based quantification of CD3 + /CD8 + /CD4 + /FoxP3 + /CD68 + /CD204 + /CD177 + tumor-infiltrating immune cells using whole-slide immunohistochemistry. Programmed death-ligand 1 (PD-L1) immunohistochemistry was evaluated using the tumor proportion score (TPS) and combined positive score (CPS). Representative cases were analyzed using whole-exome and RNA-sequencing. In 133 MSI-H CRCs, significantly higher densities of CD8 + tumor-infiltrating lymphocytes (TILs) were observed in CIMP-H tumors compared with CIMP-L/0 tumors. PD-L1 TPS and CPS in CIMP-H tumors were higher than in CIMP-L/0 tumors. Next-generation sequencing revealed that, compared with CIMP-L/0 tumors, CIMP-H tumors had higher fractions of CD8 + T cells/cytotoxic lymphocytes, higher cytolytic activity scores, and activated immune-mediated cell killing pathways. In contrast to CIMP-L/0 tumors, most CIMP-H tumors were identified as consensus molecular subtype 1, an immunogenic transcriptomic subtype of CRC. However, there were no differences in tumor mutational burden (TMB) between CIMP-H and CIMP-L/0 tumors in MSI-H CRCs. In conclusion, CIMP-H is associated with abundant cytotoxic CD8 + TILs and PD-L1 overexpression independent of TMB in MSI-H CRCs, suggesting that CIMP-H tumors represent a typical immune-hot subtype and are optimal candidates for immunotherapy in MSI-H tumors.

Targeted demethylation of the CDO1 promoter based on CRISPR system inhibits the malignant potential of breast cancer cells.

Cysteine dioxygenase 1 (CDO1) is frequently methylated, and its expression is decreased in many human cancers including breast cancer (BC). However, the functional and mechanistic aspects of CDO1 inactivation in BC are poorly understood, and the diagnostic significance of serum CDO1 methylation remains unclear. We performed bioinformatics analysis of publicly available databases and employed MassARRAY EpiTYPER methylation sequencing technology to identify differentially methylated sites in the CDO1 promoter of BC tissues compared to normal adjacent tissues (NATs). Subsequently, we developed a MethyLight assay using specific primers and probes for these CpG sites to detect the percentage of methylated reference (PMR) of the CDO1 promoter. Furthermore, both LentiCRISPR/dCas9-Tet1CD-based CDO1-targeted demethylation system and CDO1 overexpression strategy were utilized to detect the function and underlying mechanism of CDO1 in BC. Finally, the early diagnostic value of CDO1 as a methylation biomarker in BC serum was evaluated. CDO1 promoter was hypermethylated in BC tissues, which was related to poor prognosis (p<.05). The CRISPR/dCas9-based targeted demethylation system significantly reduced the PMR of CDO1 promotor and increased CDO1 expression in BC cells. Consequently, this leads to suppression of cell proliferation, migration and invasion. Additionally, we found that CDO1 exerted a tumour suppressor effect by inhibiting the cell cycle, promoting cell apoptosis and ferroptosis. Furthermore, we employed the MethyLight to detect CDO1 PMR in BC serum, and we discovered that serum CDO1 methylation was an effective non-invasive biomarker for early diagnosis of BC. CDO1 is hypermethylated and acts as a tumour suppressor gene in BC. Epigenetic editing of abnormal CDO1 methylation could have a crucial role in the clinical treatment and prognosis of BC. Additionally, serum CDO1 methylation holds promise as a valuable biomarker for the early diagnosis and management of BC.

Helicobacter pylori Eradication Can Reverse Rho GTPase Expression in Gastric Carcinogenesis.

Altered DNA methylation is a key mechanism of epigenetic modification in gastric cancer (GC). This study aimed to evaluate the changes in epigenetic and genetic expression of multiple Rho GTPases in Helicobacter pylori-related gastric carcinogenesis by comparing H. pylori-positive GCs and negative controls. The messenger RNA expression and methylation of Rho GTPases (RhoA, Rac1, DOCK180, ELMO1, and CDC42) were evaluated in H. pylori-negative (control) human gastric tissues and H. pylori-positive GCs by using real-time reverse transcription-polymerase chain reaction and the quantitative MethyLight assay, respectively. Changes in expression and methylation levels of the genes were also compared between H. pylori-eradicated and -persistent GCs at 1-year follow-up. In GCs, the methylation and expression levels of DOCK180 and ELMO1 were higher than in controls, while RhoA and Rac1 had lower levels than controls. CDC42 had the same expression pattern as DOCK180 and ELMO1 without DNA methylation. Although methylation levels of DOCK180 and ELMO1 had no difference between H. pylori-eradicated and -persistent GCs at the index endoscopic resection, those of H. pylori-persistent GCs increased and H. pylori-eradicated GCs decreased for 1 year. The expression levels of DOCK180, ELMO1, and CDC42 in H. pylori-persistent GCs were higher than those in H. pylori-eradicated GCs over 1 year, unlike those of RhoA and Rac1. The methylation levels at index and the degrees of change over time of RhoA and Rac1 had no difference between H. pylori-persistent and -eradicated GCs. Epigenetic alterations of DOCK180 and ELMO1 are involved in H. pylori-related gastric carcinogenesis. This epigenetic field could be improved by H. pylori eradication.

DNA methylation and copy number variation of the complement C4A gene in schizophrenia

The complement gene C4A has been implicated in schizophrenia through genome-wide association studies (GWAS), in vitro and animal studies. However, few studies have examined epigenetic modification of the C4A gene in schizophrenia. We hypothesized that DNA methylation levels of the C4A gene are altered in schizophrenia, and the interaction between DNA methylation and the copy number (CN) of the C4A gene leads to the higher C4 levels seen in schizophrenia. In this study, C4A DNA methylation levels, C4A CN and C4 levels were measured in the peripheral blood of 183 schizophrenia cases (SZ) and 212 healthy controls (HC). The Positive and Negative Syndrome Scale (PANSS) was used to determine the severity of psychopathology in the cases. MethyLight assay was used to measure DNA methylation levels and droplet digital polymerase chain reaction (ddPCR) was used for the calculated copy number (cCN) determination. The cCNs were also grouped into copy number variation (CNV) types: deletion, normal, and duplication. Methylation levels were measured as percentage of methylated reference (PMR) values. Both C4A PMR (rs = 0.469, p < 0.001) and C4A cCN (rs = 0.210, p < 0.001) were positively correlated with C4 levels, which in turn were higher in SZ than HC. However, we found no difference in C4A PMR values or C4A CNV types between SZ and HC. Our results suggest the potential involvement of an epigenetic modification and CNV of C4A in the upregulation of complement C4 in schizophrenia.

Association of Sat-a and Alu methylation status with HCV-induced chronic liver disease and hepatocellular carcinoma

BackgroundThe combination of epigenetic and genetic abnormalities contributes together to the development of liver cancer. The methylation status of the repetitive elements (REs) in DNA has been investigated in a variety of human illnesses. However, the methylation patterns of Sat-α and Alu REs in chronic liver disease (CLD) and hepatocellular carcinoma (HCC) caused by hepatitis C virus (HCV) have never been studied before. MethodologyIn this study, 3 groups of participants including 50 patients having HCV-induced CLD, 50 patients having HCV-induced HCC, and 46 healthy subjects were subjected to measurement of Sat-α and Alu methylation using the quantitative MethyLight assay. ResultsSat-α and Alu methylation percentages decreased significantly in both CLD and HCC, compared to control. Also, a significant Sat-α hypomethylation was detected in HCC, compared to CLD. In addition, Sat-α and Alu methylation showed a significant decline as lesion size grew. However, only Sat-α hypomethylation was significantly increased in association with portal vein thrombosis and the MELD score. Sat-α methylation percentage had the highest sensitivity and specificity for diagnosing HCC (100% and 84.4%) followed by α-fetoprotein (80% and 84.4%) and Alu methylation (66% and 61.5%). Furthermore, there was a strong positive correlation between Sat-α and Alu methylation. ConclusionsMeasuring Sat-α and Alu methylation provides us with a new tool for early detecting HCV-induced CLD and hepatocarcinogenesis. Sat-α has the potential to be utilized as an independent predictive parameter for HCC development and progression because of its ability to distinguish between CLD and HCC with their different MELD scores.

348P Prediction of sensitivity to anti-EGFR inhibitors in salvage-line treatment for metastatic colorectal cancer using a modified MethyLight assay

The genome-wide DNA methylation status in metastatic colorectal cancer (mCRC) has been reported to be associated with therapeutic sensitivity to anti-epidermal growth factor receptor (EGFR) inhibitors. We recently developed a DNA methylation assay focused on 16 CpG sites that reflect genome-wide DNA methylation status for mCRC based on the modified MethyLight and reported its ability to predict the clinical outcomes of anti-EGFR inhibitors (Ouchi et al. Cancer Sci 2022). Here, we report the predictive performance of a modified MethyLight assay on sensitivity to anti-EGFR inhibitors focused on salvage-line treatment.

Abstract 5272: Aberrant DNA 5mC and 6mA methylations increase ACE2 expression in intestinal cancer cells susceptible to SARS-CoV-2 infection

Abstract Angiotensin converting enzyme II (ACE2) is the cellular receptor of SARS-CoV-2. At present, ACE2 receptor is considered to be the key component in the SARS-CoV-2 infection and transmitting in the host. Among the cancer patients with COVID-19, the gastrointestinal cancer is the second most prevalent. The MethyLight and QASM assays were used to evaluated the genomic DNA 5mC methylation, while the CviAII enzyme-based 6mA-RE-qPCR was applied to determine motif-specific DNA 6mA methylation. The 6mA and 5mC methylation analyses of the long interspersed nuclear elements 1 (LINE1) were used to evaluate the global level of genomic 6mA and 5mC methylations, respectively. To investigate the role of ACE2 DNA methylation in regulating ACE2 expression, we performed a genome-wide methylation analysis in colorectal cancer samples collected at the Sixth Affiliated Hospital of Sun Yat-sen University. The DNA 5mC methylation of ACE2 promoter in tumor tissues were significantly lower than that in normal tissues, while the DNA 6mA methylation of ACE2 promoter in tumor tissues was significantly higher than that in normal tissues. In addition, the mRNA and protein expression of ACE2 in tumor tissues were lower than that in normal tissues. To explore the epigenetic regulation on ACE2 expression, we treated colon cancer cell lines with 5-Azacytidine and found ACE2 expression was upregulated after lowering the DNA 5mC methylation. The correlation analysis in patient cohort samples showed that ACE2 mRNA expression was positively correlated with DNA 5mC and negatively associated with DNA 6mA methylation. Next, a novel CRISPR-based tool was developed for sequence-specific 6mA editing on ACE2 promoter region, and it was applied in HCT116 cell to further confirm the regulatory role of DNA 6mA methylation in ACE2 mRNA expression. This tool was proved to be reliable with our findings that the CRISPR/dCas9-METTL3 tool could dramatically upregulate DNA 6mA methylation in ACE2 promoter, while the global level of genomic 6mA methylation remained unchanged. Both the mRNA and protein expression of ACE2 were significantly increased following a sequence-specific DNA 6mA editing in ACE2 promoter. In conclusion, we revealed the aberrant DNA 5mC and 6mA methylations in colorectal cancer, which upregulate ACE2 expression in colorectal cancer cells that may confer the susceptibility to SARS-CoV-2 infection. We developed a novel CRISPR-based tool that could realize site-directed 6mA methylation editing. Notably, the epigenetic regulation of DNA 6mA methylation on ACE2 expression provides an insight into the intersection of the biology of cancer, SARS-CoV-2 infection and organ-specific complication in COVID-19. Aberrant ACE2 methylation may serve as a biomarker and treatment target in these patients. Citation Format: Heng Wang, Yumo Xie, Gaopo Xu, Xiaolin Wang, Meijin Huang, Yanxin Luo, Huichuan Yu. Aberrant DNA 5mC and 6mA methylations increase ACE2 expression in intestinal cancer cells susceptible to SARS-CoV-2 infection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5272.

Feasibility of using P16 methylation as a cytologic marker for esophageal squamous cell carcinoma screening: A pilot study.

Early diagnosis and treatment of esophageal squamous cell dysplasia (ESCdys) and esophageal squamous cell carcinoma (ESCC) could significantly reduce the incidence and mortality of ESCC. This pilot study aimed to investigate whether P16/CDKN2A methylation could serve as a cytologic biomarker for early detection of ESCdys and ESCC. Paired esophageal biopsy and cytology specimens (exfoliated cells) were obtained from subjects at different stages of ESCC development. The methylation status of P16 gene in these two specimen types was determined using a 115-bp MethyLight assay. Categorical data were compared by the Chi-square test. Logistic regression was performed to assess adjusted odds ratios of P16 methylation associated with ESCC and ESCdys. Prediction models for identifying individuals at risk of ESCC and high-grade ESCdys (high-grade intraepithelial neoplasia, HGIN) were developed by multivariable logistic regression. Diagnostic performance was evaluated using receiver operating characteristic (ROC) analysis. Internal validation of the prediction models was performed using the 1000-bootstrap resample. A total of 105 subjects with diagnoses ranging from normal mucosa through ESCC were included in this study. An increase in P16 methylation frequency was observed with increasing severity of esophageal lesions (p for trend <0.001). In the adjusted logistic regression models, P16 methylation in cytology specimens was positively associated with ESCC and ESCdys risk, whereas P16 methylation in biopsy specimens was only associated with a higher risk of developing ESCC. The predictive capacity of base model I (AUC, 0.816) for ESCC and HGIN was significantly increased by adding P16 methylation in cytology specimens (model III; AUC, 0.882; p=0.043), but not P16 methylation in biopsy specimens (model II; AUC, 0.850; p=0.225). Bootstrap validation showed optimism-corrected AUC of 0.789 for model I, 0.822 for model II, and 0.854 for model III. P16 methylation as a cytologic marker was associated with the ESCC development and has the potential for application in minimally invasive ESCC screening.

A modified MethyLight assay predicts the clinical outcomes of anti-epidermal growth factor receptor treatment in metastatic colorectal cancer.

DNA methylation status correlates with clinical outcomes of anti‐epidermal growth factor receptor (EGFR) treatment. There is a strong need to develop a simple assay for measuring DNA methylation status for the clinical application of drug selection based on it. In this study, we collected data from 186 patients with metastatic colorectal cancer (mCRC) who had previously received anti‐EGFR treatment. We modified MethyLite to develop a novel assay to classify patients as having highly methylated colorectal cancer (HMCC) or low‐methylated colorectal cancer (LMCC) based on the methylation status of 16 CpG sites of tumor‐derived genomic DNA in the development cohort (n = 30). Clinical outcomes were then compared between the HMCC and LMCC groups in the validation cohort (n = 156). The results showed that HMCC had a significantly worse response rate (4.2% vs 33.3%; P = .004), progression‐free survival (median: 2.5 vs 6.6 mo, P < .001, hazard ratio [HR] = 0.22), and overall survival (median: 5.6 vs 15.5 mo, P < .001, HR = 0.23) than did LMCC in patients with RAS wild‐type mCRC who were refractory or intolerable to oxaliplatin‐ and irinotecan‐based chemotherapy (n = 101). The DNA methylation status was an independent predictive factor and a more accurate biomarker than was the primary site of anti‐EGFR treatment. In conclusion, our novel DNA methylation measurement assay based on MethyLight was simple and useful, suggesting its implementation as a complementary diagnostic tool in a clinical setting.

Aberrant DNA Methylation Maker for Predicting Metachronous Recurrence After Endoscopic Resection of Gastric Neoplasms.

PurposeThis study aimed to investigate whether MOS methylation can be useful for the prediction of metachronous recurrence after endoscopic resection of gastric neoplasms.Materials and MethodsFrom 2012 to 2017, 294 patients were prospectively enrolled after endoscopic resection of gastric dysplasia (n=171) or early gastric cancer (n=123). When Helicobacter pylori was positive, eradication therapy was performed. Among them, 124 patients completed the study protocol (follow-up duration > 3 years or development of metachronous recurrence during the follow-up). Methylation levels of MOS were measured at baseline using quantitative MethyLight assay from the antrum.ResultsMedian follow-up duration was 49.9 months. MOS methylation levels at baseline were not different by age, sex, and current H. pylori infection, but they showed a weak correlation with operative link on gastritis assessment (OLGA) or operative link on gastric intestinal metaplasia assessment (OLGIM) stages (Spearman’s ρ=0.240 and 0.174, respectively; p < 0.05). During the follow-up, a total of 20 metachronous gastric neoplasms (13 adenomas and 7 adenocarcinomas) were developed. Either OLGA or OLGIM stage was not useful in predicting the risk for metachronous recurrence. In contrast, MOS methylation high group (≥ 34.82%) had a significantly increased risk for metachronous recurrence compared to MOS methylation low group (adjusted hazard ratio, 4.76; 95% confidence interval, 1.54 to 14.79; p=0.007).ConclusionMOS methylation can be a promising marker for predicting metachronous recurrence after endoscopic resection of gastric neoplasms. To confirm the usefulness of MOS methylation, validation studies are warranted in the future (ClinicalTrials No. NCT04830618).

Leptin Protein Expression and Promoter Methylation in Ovarian Cancer: A Strong Prognostic Value with Theranostic Promises.

Ovarian cancer (OC) is the deadliest among all gynecological cancers. Epidemiological studies showed that obesity might influence many cancers including OC. One of the key factors that may link obesity and OC is leptin (LEP), known as an adipokine with pleiotropic effects on body homeostasis. This study aims to investigate the expression pattern of LEP, assess the methylation profiles of LEP and their associations with clinicopathological features including survival outcomes of OC patients. The protein expression of LEP was evaluated in 208 samples using both tissue microarray and immunohistochemistry techniques. The methylation profiles of LEP were measured in 63 formalin-fixed, paraffin-embedded tumor tissues by quantitative polymerase chain reaction using a MethyLight assay. Our results showed a significant association of LEP protein overexpression with several clinicopathological variables, mainly tumor subtype, LVI, age of menarche, tumor size and stage (p < 0.04). Kaplan–Meier analysis (using low expression versus high expression as a discriminator) indicated that LEP protein overexpression is a powerful positive prognosticator of both OC recurrence (DFS) and disease-specific survival (DSS) in our OC cohort (log-rank p = 0.01 and p = 0.002, respectively). This implies that patients with high LEP expression profiles live longer with less recurrence rates. Methylation analysis results demonstrated a clear association between no/low LEP protein expression pattern (38%) and LEP promoter CpG island hypermethylation (43%). Results of this study suggest that LEP is a powerful prognosticator of OC recurrence and DSS. LEP expression in OC seems to be regulated by its promoter hypermethylation through gene partial/total silencing. Further multi-institutional studies using larger cohorts are required to demystify the intricate molecular functions of this leptin-driven effects in OC pathophysiology and to accurately assess its theranostic potential and validate its prognostic/predictive power in OC onset, progression towards more effective and personalized management of OC patients.

Evaluation of the Diagnostic Potential of Candidate Hypermethylated Genes in Epithelial Ovarian Cancer in North Indian Population.

Most ovarian cancers, despite improvement in management of cancer, are still diagnosed at an advanced stage. Early detection plays an essential role in reducing ovarian cancer mortality and, therefore, is critically needed. Liquid biopsies-based approaches hold significant promise for cancer detection. The present study investigates a panel of epigenetic biomarkers for the detection of epithelial ovarian cancer. A qPCR assay has been developed based on the assessment of DNA methylation markers in circulating cell-free DNA as a minimally invasive tool. Herein, the promoter methylation of seven ovarian cancer-specific genes (RASSF1A, DAPK1, SOX1, HOXA9, HIC1, SPARC, and SFRP1) was analyzed quantitatively in 120 tissue samples by MethyLight assay. The best-performing genes were further evaluated for their methylation status in 70 matched serum cell-free DNA of cancerous and non-cancerous samples. Additionally, DNA methylation patterns of these best-performing genes were validated by clonal bisulfite sequencing. The ROC (Receiver-operator characteristic) curves were constructed to evaluate the diagnostic performances of both individual and combined gene panels. The seven candidate genes displayed a methylation frequency of 61.0–88.0% in tissue samples. The promoter methylation frequencies for all the seven candidate genes were significantly higher in cancer samples than in normal matched controls. In tissue samples, the multiplex MethyLight assay for HOXA9, HIC1, and SOX1 were the best performing gene panels in terms of sensitivity and specificity. The three best-performing genes exhibited individual frequencies of 53.0–71.0% in serum CFDNA, and the multiplex assay for these genes were identified to discriminate serum from cancer patients and healthy individuals (area under the curve: HOXA9+HIC1 = 0.95, HIC1+SOX1 = 0.93 and HOXA9+SOX1 = 0.85). The results of MethyLight showed high concordance with clonal bisulfite sequencing results. Individual genes and combined panel exhibited better discriminatory efficiencies to identify ovarian cancer at various stages of disease when analyzed in tissue and serum cell-free DNA. We report a qPCR-based non-invasive epigenetic biomarker assay with high sensitivity and specificity for OC screening. Our findings also reveal the potential utility of methylation-based detection of circulating cell-free tumor DNA in the clinical management of ovarian cancer.

Evaluation of the Impact of Intratumoral Heterogeneity of Esophageal Cancer on Pathological Diagnosis and P16 Methylation and the Representativity of Endoscopic Biopsy.

BackgroundP16 methylation is expected to be potential diagnostic and therapeutic targets for esophageal cancer (EC). The intratumoral heterogeneity (ITH) of EC has been mentioned but has not been quantitatively measured yet. We aimed to clarify the impact of ITH on pathological diagnosis and P16 methylation, and the concordance between endoscopic biopsy and the corresponding surgically resected tissue.MethodsWe designed a systematic sampling method (SSM) compared with a general sampling method (GSM) to obtain EC tumor tissue, tumor biopsy, and normal squamous epithelium biopsy. MethyLight assay was utilized to test P16 methylation. All specimens obtained by the SSM were pathologically diagnosed.ResultsA total of 81 cases were collected by the GSM, and 91.4% and 8.6% of them were esophageal squamous cell carcinomas (ESCCs) and esophageal adenocarcinomas (EADs), respectively. Nine SSM cases were 100.0% ESCCs. The positive rates of P16 methylation of the GSM tumor and normal tissues were 63.0% (51/81) and 32.1% (26/81), respectively. For SSM samples, tumor tissues were 100.0% (40/40) EC and 85.0% (34/40) P16 methylated; tumor biopsy was 64.4% (29/45) diagnosed of EC and 68.9% P16 methylated; the corresponding normal biopsies were 15.7% (8/51) dysplasia and 54.9% (28/51) P16 methylated. The concordance of pathological diagnosis and P16 methylation between tumor biopsy and the corresponding tumor tissue was 75.0% and 62.5%, respectively.ConclusionThe SSM we designed was efficient in measuring the ITH of EC. We found inadequate concordance between tumor biopsy and tissue in pathological diagnosis and P16 methylation.

Klotho promoter methylation status and its prognostic value in ovarian cancer.

Among all gynecological cancers, ovarian cancer (OC) is one of the deadliest types of cancer worldwide. Epigenetic silencing of some genes has been reported to be associated with OC. In this context, Klotho (KL) gene methylation is a promising biomarker for OC. The present study aimed to investigate the methylation profiles of KL and assess its prognostic value. A total of 63 formalin-fixed paraffin-embedded tissue samples from patients with primary OC were collected and analyzed in the present study. The methylation profiles of KL were assessed by performing DNA bisulfate treatment followed by DNA promoter methylation analysis using the MethyLight assay. The results revealed KL promoter hypermethylation in 62% of the OC cohort. Additionally, significant associations were observed between KL methylation profiles and tumor subtype (P<0.0001) and tumor site (P=0.039). Furthermore, Kaplan-Meier analysis revealed that a worse disease-specific survival was significantly associated with hypermethylated KL (P=0.03, log-rank; hazard ration, 0.58; 95% confidence interval (CI), 0.26-0.90). Cox regression multivariate analysis indicated that KL promoter methylation was an independent OC prognostic indicator (P=0.029). The current study suggested that KL may be a novel biomarker to predict prognosis in patients with OC, since patients with higher KL promoter methylation were more likely to have a poor prognosis and would therefore require frequent follow-up and integrative personalized therapeutic approaches.

Abstract 480: Aberrant promoter hypermethylation of HIC1 and SOX1 in circulating cell free DNA as a potential biomarker for noninvasive early detection of ovarian cancer

Abstract Background: Aberrant CpG island hypermethylation is an early event during carcinogenesis and a significant mechanism of tumor suppressor gene silencing. Assessment of tumor specific methylation changes in tissue or body fluids might provide insights into disease mechanisms and discovery of biomarkers. Ovarian cancer remains the most lethal gynaecological malignancy. Non-specific symptoms at early stage, presentation of disease at advanced stage and lack of sufficiently sensitive screening modalities have rendered significant challenge to its prompt diagnosis. To date, no single biomarker is able to accurately detect early OC in either tissue or body fluid. Aberrant promoter hypermethylation in cell free tumor-specific DNA can be indicative of highly specific cancer signals. Methods: Herein, we concurrently evaluated the promoter methylation of HIC1 and SOX1 gene in 120 ovarian tissue samples and validated in 70 matched serum cell free DNA of cancerous and non cancerous samples by multiplex MethyLight assay (a high throughput quantitative, TaqMan based real time PCR technique). Additionally, promoter methylation pattern of these genes were validated by clonal bisulfite sequencing. The ROC (Receiver-operator characteristic) curves were constructed to evaluate the diagnostic performances of both individual and combined gene panel. Results: In singleplex assay, high methylation sensitivity and specificity was evident for SOX1 (78.8% and 80.0%, respectively; AUC= 0.88) and HIC1 (80.0% and 77.14%, respectively; AUC=0.85) in tissue samples. In the multiplex assay, when either or both gene promoter showed methylation, the highly sensitivity and specific discrimination of EOC patients from normal controls was observed for SOX1 + HIC1 (83.53% and 88.57%, respectively; AUC=0.89). We identified DNA methylation of HIC1 and SOX1 as best discriminator between cancer and non-neoplastic tissue. Methylated SOX1 and HIC1 occurred in 53.3% and 71.1% of OC serum samples when their promoter methylation was analyzed in singleplex assay (AUC=0.81 and 0.88, respectively). In cell free DNA, this novel panel achieved a sensitivity of 80.0% and a specificity of 96.0% with an area under the ROC curve of 0.93 for discriminating OC samples from normal control. The results of MethyLight were highly concordant with those of clonal bisulfite sequencing. Methylated Individual gene as well as in combined panel exhibited better discriminatory efficiencies to identify ovarian cancer at various stages of disease when analyzed in tissue and serum cell free DNA. Conclusion: We report a QPCR based non-invasive epigenetic biomarker assay with high sensitivity and specificity for OC screening. Our findings also reveal the potential utility of methylation based detection of circulating cell free tumor DNA in clinical management of ovarian cancer. Citation Format: Alka Singh, Manisha Sachan. Aberrant promoter hypermethylation of HIC1 and SOX1 in circulating cell free DNA as a potential biomarker for noninvasive early detection of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 480.

The Correlation between Gene Expression and DNA Methylation Levels of RAS p21 Protein Activator 3 (RASA3) Gene in Saudi Autistic Children

The potential role of DNA methylation pattern in autism has been provided by revealing the differences in methylation level of multiple genes which are significantly associated with their expression and implicated in ASD pathogenesis. RASA3 is a member of GTPase-activating proteins, RASA3 is highly expressed in brain tissues and can be deregulated by different epigenetic mechanisms. Many studies reported that differentially expressed RASA3 is correlated with its aberrant methylation. Accordingly, this has been suggested that deferentially expression of RASA3 may be correlated with its methylation levels which could play a role in ASD which brought our attention to identify differentially-expressed genes that could be associated with their methylation level of ASD in Saudi population, by performing comparative gene expression of RASA3 then investigate its relation to methylation level.&#x0D; This study was conducted on 18 Saudi autistic children as well as their healthy-control siblings. Relative expression of a candidate gene (RASA3) was measured using RT-qPCR. Furthermore, MethyLight assay was performed to estimate methylation level and evaluate its impact on RASA3 expression. Interestingly, RASA3 expression has found to be dysregulated in ASD cases. In contrast, MethyLight assay result showed no differences in the methylation patterns among ASD cases in the candidate region. However, it remains an open question whether these dysregulations of RASA3 expression could be a biomarker for early screening/detection of some cases which may also suggest a role for RasGAPs in autistic brain function.

LOSS OF ADD1 METHYLATION ADDS ON BLOOD PRESSURE IN YOUNG ADULTS

Objective: To investigate the association between alpha-Adducin (ADD1) methylation and blood pressure in young adults with essential hypertension. Design and method: A total of 160 subjects (80 normotensive and 80 incident hypertensive) aged between 18 to 45 years from Kuantan, Pahang were included in a cross-sectional study by purposive sampling. They were assessed for ADD1 methylation in peripheral blood using MethyLight assay. Anthropometric measurements and biochemical parameters were also examined. Results: ADD1 methylation was inversely correlated with systolic (p = 0.006, r = –0.240), diastolic (p = 0.001, r = –0.281) and mean arterial pressures (p = 0.002, r = –0.270). Hypertensive subjects had significantly higher ADD1 methylation than normotensive control (p = 0.005). After adjusting for other relevant covariates (age, body mass index, HbA1c, high-sensitivity C-reactive protein and low density lipoprotein cholesterol), ADD1 methylation remained a significant predictor for hypertension in young adults (p = 0.020) Conclusions: ADD1 methylation is a significant predictor of hypertension in young adults. ADD1 methylation could serve as a future preventive and therapeutic target for hypertension and related cardiovascular disease.

Blood-Based Detection of Colorectal Cancer Using Cancer-Specific DNA Methylation Markers.

Cancer tissues have characteristic DNA methylation profiles compared with their corresponding normal tissues that can be utilized for cancer diagnosis with liquid biopsy. Using a genome-scale DNA methylation approach, we sought to identify a panel of DNA methylation markers specific for cell-free DNA (cfDNA) from patients with colorectal cancer (CRC). By comparing DNA methylomes between CRC and normal mucosal tissues or blood leukocytes, we identified eight cancer-specific methylated loci (ADGRB1, ANKRD13, FAM123A, GLI3, PCDHG, PPP1R16B, SLIT3, and TMEM90B) and developed a five-marker panel (FAM123A, GLI3, PPP1R16B, SLIT3, and TMEM90B) that detected CRC in liquid biopsies with a high sensitivity and specificity with a droplet digital MethyLight assay. In a set of cfDNA samples from CRC patients (n = 117) and healthy volunteers (n = 60), a panel of five markers on the platform of the droplet digital MethyLight assay detected stages I–III and stage IV CRCs with sensitivities of 45.9% and 95.7%, respectively, and a specificity of 95.0%. The number of detected markers was correlated with the cancer stage, perineural invasion, lymphatic emboli, and venous invasion. Our five-marker panel with the droplet digital MethyLight assay showed a high sensitivity and specificity for the detection of CRC with cfDNA samples from patients with metastatic CRC.

A DNA methylation panel for high performance detection of colorectal cancer

One of the most promising ways to diagnose cancer especially colorectal cancer (CRC) is to trace its epigenetic events.In this article, a discovery step for detection of methylated DNA markers (MDMs) was performed using SureSelectXT Methyl-Seq in CRC case and control groups in addition to several methylation profiling datasets (GSE48684, GSE53051, GSE77718, GSE101764, and GSE42752). In silico validation of MDMs in colorectal and other cancers was conducted by Lnc2met. MethyLight assay was run on 40 and 47 case and control formalin-fixed paraffin-embedded tissues, respectively and the performance of selected genes were classified by support vector machine (SVM).As a result, 180 regions were identified among all common genes. In addition to SEPT9 and SFRP2, the best three MDM regions were selected from SLC30A10, AKR1B1 and GALNT14. Based on all assays, the best performance was accomplished by SEPT9/AKR1B1 with 98% sensitivity, 99% specificity, 125 positive likelihood ratio, 0.02 negative likelihood ratio and 5074 diagnostic odds ratio.Our results indicate that the AKR1B1/SEPT9 methylation panel detects CRC with a higher performance than SEPT9 methylation, which is a commercial diagnostic test for CRC. However, the creation of a clinically valuable test derived from this study requires performance evaluation in liquid biopsies.

The Reprimo-Like Gene Is an Epigenetic-Mediated Tumor Suppressor and a Candidate Biomarker for the Non-Invasive Detection of Gastric Cancer.

Reprimo-like (RPRML) is an uncharacterized member of the Reprimo gene family. Here, we evaluated the role of RPRML and whether its regulation by DNA methylation is a potential non-invasive biomarker of gastric cancer. RPRML expression was evaluated by immunohistochemistry in 90 patients with gastric cancer and associated with clinicopathologic characteristics and outcomes. The role of RPRML in cancer biology was investigated in vitro, through RPRML ectopic overexpression. Functional experiments included colony formation, soft agar, MTS, and Ki67 immunofluorescence assays. DNA methylation-mediated silencing was evaluated by the 5-azacytidine assay and direct bisulfite sequencing. Non-invasive detection of circulating methylated RPRML DNA was assessed in 25 gastric cancer cases and 25 age- and sex-balanced cancer-free controls by the MethyLight assay. Downregulation of RPRML protein expression was associated with poor overall survival in advanced gastric cancer. RPRML overexpression significantly inhibited clonogenic capacity, anchorage-independent growth, and proliferation in vitro. Circulating methylated RPRML DNA distinguished patients with gastric cancer from controls with an area under the curve of 0.726. The in vitro overexpression results and the poor patient survival associated with lower RPRML levels suggest that RPRML plays a tumor-suppressive role in the stomach. Circulating methylated RPRML DNA may serve as a biomarker for the non-invasive detection of gastric cancer.