RASSF1A Promoter Research Articles

DNA methylation analysis with methylation-sensitive high-resolution melting (MS-HRM) reveals gene panel for glioma characteristics.

IntroductionLocal DNA hypermethylation is a potential source of cancer biomarkers. While the evaluation of single gene methylation has limited value, their selected panel may provide better information.AimsThis study aimed to analyze the promoter methylation level in a 7‐gene panel in brain tumors and verifies the usefulness of methylation‐sensitive high‐resolution melting (MS‐HRM) for this purpose.MethodsForty‐six glioma samples and one non‐neoplastic brain sample were analyzed by MS‐HRM in terms of SFRP1, SFRP2, RUNX3, CBLN4, INA, MGMT, and RASSF1A promoter methylation. The results were correlated with patients’ clinicopathological features.ResultsDNA methylation level of all analyzed genes was significantly higher in brain tumor samples as compared to non‐neoplastic brain and commercial, unmethylated DNA control. RASSF1A was the most frequently methylated gene, with statistically significant differences depending on the tumor WHO grade. Higher MGMT methylation levels were observed in females, whereas the levels of SFRP1 and INA promoter methylation significantly increased with patients’ age. A positive correlation of promoter methylation levels was observed between pairs of genes, for example, CBLN4 and INA or MGMT and RASSF1A.ConclusionsOur 7‐gene panel of promoter methylation can be helpful in brain tumor diagnosis or characterization, and MS‐HRM is a suitable method for its analysis.

ROLE OF HYPERMETHYLATION (RASSF1A) PROMOTER GENE AMONG PATIENTS WITH HEPATOCELLULAR CARCINOMA

Background: Hepatocellular carcinoma (HCC) is one of the most aggressive malignancies worldwide and one of the main causes of cancer-related mortality globally. Its incidence is increasing at alarming rates. The presence of cirrhosis is the major risk factor and this is largely due to chronic HCV and HBV infection. Serum alpha-fetoprotein (AFP) has insufficient sensitivity and specificity for detection of hepatocellular carcinoma (HCC). Aberrant hypermethylation of tumor suppressor genes e.g(RASSF1A ) is one of the most frequent and early mechanisms involved in HCC development, so that it could help to select high-risk populations and thus to modulate the indications of screening procedures. Objectives: To evaluate the frequency of tumor suppressor gene RASSF1A hypermethylation in whole blood from HCC patients Methods: The study included eighty subjects : 30 patients with HCC and elevated AFP. ; 30 with liver cirrhosis In addition, 20 healthy subjects were included as a control group. Clinical and radiological features (abdominal ultrasonography and/or abdominal triphasic computed tomography) were recorded. Liver function tests, complete blood cell count, and serum AFP were measured.. Detection of promotor methylation status of RASSF1A using methylation specific PCR . Results: The obtained results showed a significant RASSF1A promoter hypermethylation in HCC subjects that was 83.3% in comparison to healthy control subjects as well as in comparison to subjects with non HCC chronic liver disease. Conclusion:.Detection of methylated RASSF1A promoter is useful marker for HCC screening in high risk vulnerable patients and early HCC diagnosis.

RASSF1A Tumour Suppressor: Target the Network for Effective Cancer Therapy.

The RASSF1A tumour suppressor is a scaffold protein that is involved in cell signalling. Increasing evidence shows that this protein sits at the crossroad of a complex signalling network, which includes key regulators of cellular homeostasis, such as Ras, MST2/Hippo, p53, and death receptor pathways. The loss of expression of RASSF1A is one of the most common events in solid tumours and is usually caused by gene silencing through DNA methylation. Thus, re-expression of RASSF1A or therapeutic targeting of effector modules of its complex signalling network, is a promising avenue for treating several tumour types. Here, we review the main modules of the RASSF1A signalling network and the evidence for the effects of network deregulation in different cancer types. In particular, we summarise the epigenetic mechanism that mediates RASSF1A promoter methylation and the Hippo and RAF1 signalling modules. Finally, we discuss different strategies that are described for re-establishing RASSF1A function and how a multitargeting pathway approach selecting druggable nodes in this network could lead to new cancer treatments.

Analysis of salivary detection of P16INK4A and RASSF1A promoter gene methylation and its association with oral squamous cell carcinoma in a Colombian population.

BackgroundEpigenetic factors play a fundamental role in the etiopathogenesis of oral squamous cell carcinoma (OSCC). This study evaluated if salivary detection of P16INK4A/RASSF1A gene promoter methylation might be linked to the clinical/histological features of OSCC in a Colombian population. Material and MethodsMethylation-specific polymerase chain reaction (MSP-PCR) was used to detect the methylation frequency of P16INK4A/RASSF1A genes in DNA obtained from whole saliva collected of 40 healthy controls (HC) and 43 OSCC patients. Determination of the clinical performance of MSP-PCR assay was based on standard algorithms derived from two-way contingency table analysis. The association of methylation status of targeted genes with OSCC was analyzed in a multivariate binary logistic regression model. ResultsThere were significantly higher proportions of promoter methylation of these target genes in OSCC patients when compared with HC. The analysis of single methylated genes showed high specificity, good positive and negative predictive values, but was accompanied by a low sensitivity. OSCC cases with clinical stage III/IV, poorly differentiated, and severe cellular atypia showed a significantly greater proportion of methylated than that of unmethylated targeted genes in saliva samples. Logistic regression analysis indicated an independent association of P16INK4A and RASSF1A promoter methylation with OSCC diagnosis. A significant interaction effect between ageing and P16INK4A promoter methylation was also detected. ConclusionsSalivary detection of P16INK4A and RASSF1A promoter methylation appears to be independently associated with OSCC and may be linked to the tumor activity in the present population. Consequently, the targeting of these genes in saliva samples might constitute an important tool for diagnosis and prognosis purposes. Key words:Gene methylation, oral squamous cell carcinoma, P16INK4A, RASSF1A, saliva.

The Relationship Between Promoter Methylation of RASSF1A Gene and Prognosis of Patients Affected with Breast Cancer in Khuzestan Province

Background DNA methylation is one of the most frequent molecular changes that are associated with human cancers. Tumor suppressor genes are the important targets of hypermethylation in breast cancer and thus may result in cancer by deregulation of cell growth and division.Objectives The purpose of this research was to compare the methylation pattern of the RASSF1A gene in cancerous and normal tissues.Methods Twenty breast cancer patients with known clinicopathologic characteristics and 20 healthy women as control were studied for analysis of the methylation status of the RASSF1A gene promoter by methylation-specific polymerase chain reaction (MS-PCR) technique.Results The findings showed that the frequency of RASSF1A promoter methylation was in 15% of tumor tissues and 5% of normal tissues. These results indicate that there is no statistically significant relationship between the RASSF1A promoter methylation status of tumor tissues and normal tissues (P > 0.05).Conclusions Until now, the evidence for powerful methylation biomarkers is still incomplete and the recognized biomarkers require further validation.

RASSF1A-Hippo pathway link in patients with urothelial carcinoma of bladder: plausible therapeutic target.

RASSF1A is a tumor suppressor gene, and its hypermethylation has been observed in cancers. RASSF1A acts as an upstream regulator of Hippo pathway and modulates its function. The aim of this study was to analyze expression of RASSF1A, Hippo pathway molecules (YAP, MST) and downstream targets (CTGF, Cyr61 and AREG) in bladder cancer patients. Later, the link between RASSF1A and Hippo pathway and a potential therapeutic scope of this link in UBC were also studied. MSPCR was performed to study methylation of RASSF1A promoter. Expression of molecules was studied using qPCR, Western blot and IHC. The link between RASSF1A and Hippo pathway was studied using Spearman's correlation in patients and validated by overexpressing RASSF1A in HT1376 cells and its effect on Hippo pathway was observed using qPCR and Western blot. Further therapeutic potential of this link was studied using MTT and PI assays. The expression of RASSF1A was lower, whereas the expression of YAP, CTGF and CYR61 was higher. The expression of RASSF1A protein gradually decreased, while the expression of YAP, CTGF and CYR61 increased with severity of disease. Based on Spearman's correlation, RASSF1A showed a negative correlation with YAP, CTGF and CYR61. YAP showed a positive correlation with CTGF and CYR61. To validate this link, RASSF1A was overexpressed in HT1376 cells. Overexpressed RASSF1A activated Hippo pathway, followed by a decrease in CTGF and CYR61 at mRNA, and enhanced cytotoxicity to chemotherapeutic drugs. This study finds a previously unrecognized role of RASSF1A in the regulation of CTGF and CYR61 through mediation of Hippo pathway in UBC and supports the significance of this link as a potential therapeutic target for UBC.

MUC1-C represses the RASSF1A tumor suppressor in human carcinoma cells

RASSF1A encodes a tumor suppressor that inhibits the RAS→RAF→MEK→ERK pathway and is one of the most frequently inactivated genes in human cancers. MUC1-C is an oncogenic effector of the cancer cell epigenome that is overexpressed in diverse carcinomas. We show here that MUC1-C represses RASSF1A expression in KRAS wild-type and mutant cancer cells. Mechanistically, MUC1-C occupies the RASSF1A promoter in a complex with the ZEB1 transcriptional repressor. In turn, MUC1-C/ZEB1 complexes recruit DNA methyltransferase 3b (DNMT3b) to the CpG island in the RASSF1A promoter. Targeting MUC1-C, ZEB1, and DNMT3b thereby decreases methylation of the CpG island and derepresses RASSF1A transcription. We also show that targeting MUC1-C regulates KRAS signaling, as evidenced by RNA-seq analysis, and decreases MEK/ERK activation, which is of importance for RAS-mediated tumorigenicity. These findings define a previously unrecognized role for MUC1-C in suppression of RASSF1A and support targeting MUC1-C as an approach for inhibiting MEK→ERK signaling.

MeCP2 triggers diabetic cardiomyopathy and cardiac fibroblast proliferation by inhibiting RASSF1A

Diabetes causes cardiomyopathy and increases the risk of heart failure independent of hypertension and cardiac fibrosis disease. However, the molecular mechanism of cardiomyopathy caused by diabetic (DCM) is currently unknown. Here we explore the role of the Methyl CpG binding protein 2 (MeCP2) in DCM patients and a type 1 DM (T1DM) rat model. In this study, we employed streptozotocin (STZ)-induced rats DCM and DCM patient and found that MeCP2 triggers cardiac fibroblast proliferation in DCM by inhibiting of RASSF1A expression. Moreover, the in vitro study demonstrated that high glucose inhibited RASSF1A expression, accompanied by the increases of MeCP2 expression and DNA hypermethylation in RASSF1A promoter region. MeCP2 inhibition or knockdown reversed the decrease of RASSF1A transcription induced by high glucose in cardiac fibroblasts. MeCP2 triggers cardiac fibroblasts proliferation through the activation of RASSF1A/ERK1/2 signaling pathways. Our results demonstrated that MeCP2 plays a key role in RASSF1A mediated ERK1/2 activation in DCM. Taken together, these indicate that MeCP2 acts as a key regulator of DCM and cardiac fibroblasts proliferation.

Correlation Between RASSF1A Gene Promoter Hypermethylation in Serum or Sputum and Non-Small Cell Lung Cancer (NSCLC): A Meta-Analysis.

BackgroundThe aim of this study was to evaluate the efficacy of RASSF1A promoter hypermethylation of serum or sputum in diagnosis of non-small cell lung cancer (NSCLC) by pooling open published data.Material/MethodsOpen-published studies relevant to RASSF1A promoter hypermethylation and NSCLC diagnosis were screened through Medline, EMBASE, the Cochrane Library, Web of Science, Google Scholar, and CBM. Number of cases of true positive (tp), false positive (fp), false negative (fn), and true negative (tn) by RASSF1A gene promoter hypermethylation was extracted from each of the include original studies. The combined diagnostic sensitivity, specificity, and symmetric receiver operating characteristic curve (SROC) were calculated, as was the effect size.ResultsTwelve studies with 826 NSCLC and 598 controls were included in the present work. The combined sensitivity and specificity were 0.45 (95%CI: 0.41–0.48) (random effects) and 0.99(95%CI: 0.98–1.00) (fixed effects) respectively. The pooled positive likelihood ratio (+LR) and negative likelihood ratio (−LR) were 20.27 (9.64–42.61) and 0.53 (0.42–0.66), respectively, through the random effects model. The combined DOR was 46.63 (95%CI: 17.30–125.65) through the fixed effects model. The AUC of the SROC was 0.9989, calculated through Moses’s model for RASSF1A promoter hypermethylation as a biomarker in diagnosis of NSCLC.ConclusionsThe low diagnostic sensitivity for RASSF1A gene promoter hypermethylation indicated that it is not suitable for NSCLC screening. However, the high specificity made it effective for NSCLC confirmation diagnosis, which could be used instead of pathological diagnosis.

Methylation Dynamics of RASSF1A and Its Impact on Cancer.

5-methyl cytosine (5mC) is a key epigenetic mark entwined with gene expression and the specification of cellular phenotypes. Its distribution around gene promoters sets a barrier for transcriptional enhancers or inhibitor proteins binding to their target sequences. As a result, an additional level of regulation is added to the signals that organize the access to the chromatin and its structural components. The tumor suppressor gene RASSF1A is a microtubule-associated and multitasking scaffold protein communicating with the RAS pathway, estrogen receptor signaling, and Hippo pathway. RASSF1A action stimulates mitotic arrest, DNA repair and apoptosis, and controls the cell cycle and cell migration. De novo methylation of the RASSF1A promoter has received much attention due to its increased frequency in most cancer types. RASSF1A methylation is preceded by histones modifications and could represent an early molecular event in cell transformation. Accordingly, RASSF1A methylation is proposed as an epigenetic candidate marker in many cancer types, even though an inverse correlation of methylation and expression remains to be fully ascertained. Some findings indicate that the epigenetic abrogation of RASSF1A can promote the alternative expression of the putative oncogenic isoform RASSF1C. Understanding the complexity and significance of RASSF1A methylation is instrumental for a more accurate determination of its biological and clinical role. The review covers the molecular events implicated in RASSF1A methylation and gene silencing and provides a deeper view into the significance of the RASSF1A methylation patterns in a number of gastrointestinal cancer types.

Abstract 417: Urine as an alternative to blood for germline genomics and cancer genetics

Abstract Currently, blood is the body fluid of choice to provide comprehensive germline genomics via Peripheral Blood Mononuclear Cells (PBMC) and cancer genetics via the detection of circulating tumor DNA (ctDNA, liquid biopsy) for precision medicine. In this study, we examined if urine can be an alternative to blood for comprehensive germline genomics and for liquid biopsy. We have previously shown that human urine contains DNA derived both from sloughed-off cell debris of the urinary tract (greater than 1 KB/High Molecular weight or HMW) and trans-renal DNA from the circulation (less than 1 KB/Low molecular weight or LMW). We performed whole genome sequencing (WGS) to compare the quality and comprehensiveness of the genomic data between 3 sets of matched PBMC and HMW urine DNA. There was no statistically significant difference between the NGS data obtained from PBMC and urine DNA upon comparing the number of reads, average coverage, percent aligned reads, percent reads passing Phred score Q30 (p value less than 0.05, paired t-test) and coverage of human genome. Next, we compared the detectability of ctDNA in the matching LMW urine and plasma DNA obtained from 44 HCC patients by employing a panel of HCC biomarkers including genetic mutations at TP53 codon 249 [G:C to T:A transversion (TP53 249T)], CTNNB1 exon 3 regions 32-37 (CTNNB1 32-37), and hTERT promoter region position 124 (hTERT 124) and aberrant methylation of RASSF1A (mRASSF1A) promoter. In this study, a systematic analysis of this HCC biomarker panel demonstrated that urine can complement blood for liver cancer screening. A combination of the plasma and urine biomarkers with serum AFP the current most widely used biomarker for HCC can provide higher sensitivity (up to 30% more) for liver cancer detection. In order to characterize and determine if LMW urine DNA is derived from plasma DNA, we performed WGS to compare the size profiles and coverage of these two sources of cell-free DNA in 3 sets of matching HCC urine and plasma samples. Overall shorter read lengths were obtained from urine DNA as compared to the corresponding plasma DNA. A series of peaks occurring at 10 bp periodicity was displayed in both sources of cell-free DNA Further analysis regarding the genome coverage, overlapping fraction and variant detection is ongoing. In conclusion, our data suggests that (i) HMW urine DNA can replace PBMC DNA for providing comprehensive genomic sequencing data and (ii) urine can complement blood for liver cancer liquid biopsy, precision medicine, and potential applications to other cancers. Citation Format: Surbhi Jain, Tai-Jung Lee, Adam Zhang, Jonathan Cheng, Jamin D. Steffen, Chi-Tan Hu, James P. Hamilton, Harry Luu, Hie-Won Hann, Fwu-Shan Shieh, Selena Y. Lin, Wei Song, Ying-Hsiu Su. Urine as an alternative to blood for germline genomics and cancer genetics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 417.

Abstract 831: Hypermethylation in promoter regions of CpG islands of multiple genes is associated with disease progression and molecular response in chronic myelogenous leukemia

Abstract Chronic myelogenous leukemia (CML) has a typical progressive course with transition from a less aggressive chronic phase to a terminal more aggressive blast crisis. The molecular mechanisms leading to CML disease progression remain to be elucidated. However, it has been proposed in many studies that alterations in the methylation status of various genes may contribute to the progression of different malignancies. Hence, in this study we tried to understand the role of aberrant methylation of six tumor suppressor genes in the disease progression and molecular response in CML. We investigated 100 CML patients and 100 age and sex matched healthy controls for the methylation status of six tumor suppressor genes. The methylation status of these genes was evaluated by MS-PCR. In this study, 100 CML samples and 100 controls were analyzed for the DAPK1, RASSF1A, RIZ-1, p16, p14ARF and SOCS-1 promoter methylation status. These genes, except p14ARF and SOCS-1 showed significant differences in methylation status among CML patients and healthy controls. Also DAPK1, RASSF1A, RIZ-1, p16 and p14ARF genes were observed to be hypermethylated during progression of CML disease to advanced phases. The methylation frequencies of these five genes were seen to increase progressively during CML disease progression. This increase in hypermethylation of these five genes from was statistically significant with a p-value of <0.0001, 0.001, 0.009,0.002 and 0.01 respectively. Although, there was a progressive increase in the promoter methylation of SOCS1 gene during the CML disease progression (CP= 4%, AP=8% and BC=16%) but this did not reached a statistical significance (p-value 0.1). We also assessed the molecular response of these 100 CML patients after imatinib therapy and the patients were categorized into two groups; major molecular response group and loss of molecular response group. Out of 100 CML patients on imatinib therapy 52 achieved major molecular response while 48 patients showed loss of molecular response. Out of the above studied genes RASSF1 and RIZ1 promoter hypermethylation was significantly associated with loss of molecular response in CML patients receiving imatininb therapy. Out of 48 CML patients who were in loss of molecular response, 23.0% and 16.77% showed promoter hypermethylation of RASSF1 and RIZ1 genes respectively while out of 52 patients that were in major molecular response only 9.6% and 1.92% of patients showed hypermethylation in RASSF1 and RIZ1 genes respectively. These results suggest that hypermethylation of tumor suppressor genes plays a significant role in the progression and response in CML. Note: This abstract was not presented at the meeting. Citation Format: Mamta P. Sumi, Sameer A. Guru, Imtiyaz A Najar, Mariyam Zuberi, N. Gupta, Alpana Saxena. Hypermethylation in promoter regions of CpG islands of multiple genes is associated with disease progression and molecular response in chronic myelogenous leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 831.

RASSF1A controls tissue stiffness and cancer stem‐like cells in lung adenocarcinoma

Lung cancer remains the leading cause of cancer‐related death due to poor treatment responses and resistance arising from tumour heterogeneity. Here, we show that adverse prognosis associated with epigenetic silencing of the tumour suppressor RASSF1A is due to increased deposition of extracellular matrix (ECM), tumour stiffness and metastatic dissemination in vitro and in vivo. We find that lung cancer cells with RASSF1A promoter methylation display constitutive nuclear YAP1 accumulation and expression of prolyl 4‐hydroxylase alpha‐2 (P4HA2) which increases collagen deposition. Furthermore, we identify that elevated collagen creates a stiff ECM which in turn triggers cancer stem‐like programming and metastatic dissemination in vivo. Re‐expression of RASSF1A or inhibition of P4HA2 activity reverses these effects and increases markers of lung differentiation (TTF‐1 and Mucin 5B). Our study identifies RASSF1A as a clinical biomarker associated with mechanical properties of ECM which increases the levels of cancer stemness and risk of metastatic progression in lung adenocarcinoma. Moreover, we highlight P4HA2 as a potential target for uncoupling ECM signals that support cancer stemness.

Association between methylation of DNA damage response-related genes and DNA damage in hepatocytes of rats following subchronic exposure to vinyl chloride

In the present study, we investigated the association between methylation of DNA damage response-related genes such as cyclin-dependent kinase inhibitor (CDKN)2A, Ras association (RalGDS/AF-6) domain family member (RASSF)1A, O6-methylguanine DNA methyltransferase (MGMT), Kirsten rat sarcoma viral oncogene homolog (KRAS), and spleen-associated tyrosine kinase (SYK) and DNA damage in hepatocytes of rats following subchronic exposure to vinyl chloride (VC). Sixty-four healthy rats were randomly divided into three VC exposure groups (5, 25, and 125 mg/kg) and an untreated negative control group (n = 16 each). VC was administered by intraperitoneal injection every other day for a total of three times a week. Eight randomly selected rats from each group were sacrificed at the end of 6 and 12 weeks, and liver tissue was harvested for the comet assay and for assessment of DNA methylation level and mRNA expression of related genes by PCR. Overall methylation levels in the genome of hepatocytes in VC-exposed rats were higher than those in the control group at 6 and 12 weeks (P < 0.05), although no differences were observed with regarding to dose (P > 0.05). After 12 weeks of exposure, differences in the methylation of RASSF1A and MGMT promoter regions were observed between the high-dose group and other groups (P < 0.05), whereas no differences were observed for the KRAS, SYK, and CDKN2A promoters (P > 0.05). These results suggest that DNA damage and increased genome-wide methylation are biomarkers for VC exposure and that RASSF1A and MGMT promoter methylation is related to the carcinogenic mechanism of VC.

RASSF1A promoter methylation correlates development, progression, and poor cancer-specific survival of renal cell carcinoma: trial sequential analysis.

BackgroundThis meta-analysis evaluated the clinicopathologic and prognostic significance of RASSF1A promoter methylation in renal cell carcinoma (RCC).Materials and methodsThe ORs or HRs and their 95% CIs were calculated. Trial sequential analysis was conducted.ResultsTwenty-two articles that included 1,421 patients with RCC and 724 controls were identified. RASSF1A promoter methylation correlated with RCC in tissue, blood, and urine samples. On multivariate analysis, RASSF1A promoter methylation was associated with tumor grade (grade 3–4 vs 1–2: OR=3.59), clinical stage (stage 3–4 vs 1–2: OR=2.15), T classification (pT2–4 vs pT1: OR=2.66), histologic subtypes (papillary vs clear cell: OR=2.91), and cancer-specific survival (HR=1.78), but it was not linked to age, gender, lymph node status, distant metastasis, or overall survival. The Cancer Genome Atlas data also showed that RASSF1A methylation was significantly more likely to be seen in papillary vs clear-cell RCC (OR=23.19).ConclusionRASSF1A promoter methylation may be associated with the development and progression of RCC, as well as poor cancer-specific survival. Methylation was more frequent in papillary vs clear-cell RCC. More studies are needed to confirm these findings in blood or urine samples.

DNA methylation profiling as a tool for testicular germ cell tumors subtyping.

Assess differential patterns of selected five genes' promoter methylation among testicular germ cell tumors (TGCT) subtypes. CRIPTO, HOXA9, MGMT, RASSF1A and SCGB3A1 promoter methylation levels were evaluated by quantitative methylation-specific PCR in 161 TGCT and 16 controls. Associations between clinicopathological parameters and promoter methylation levels were assessed, and receiver operating characteristics curve analysis was performed. Promoter methylation of CRIPTO/HOXA9/SCGB3A1 panel and RASSF1A best discriminated between controls and nonseminomatous tumors or seminomas, respectively, whereas HOXA9/RASSF1A panel displayed the best discriminative performance between nonseminomatous tumor and seminomas. Significant differences in CRIPTO, MGMT and RASSF1A methylation levels were depicted between pure forms and matched mixed components of seminomas and embryonal carcinoma. HOXA9, RASSF1A and SCGB3A1 promoter methylation significantly associated with tumor stage. Different combinations of five genes' promoter methylation levels discriminate among TGCT subtypes. Methylation patterns may also assist in identification of more clinically aggressive tumors.

Promoter methylation as biomarkers for diagnosis of melanoma: A systematic review and meta-analysis.

Melanoma is one of the most common skin cancer that is characterized by rapid growth, early metastasis, high malignant, and mortality. Accumulating evidence demonstrated that promoter methylation of tumor-suppressor genes is implicated in the pathogenesis of melanoma. In the current study, we performed a meta-analysis to identify promising methylation biomarkers in the diagnosis of melanoma. We carried out a systematic literature search using Pubmed, Embase, and ISI web knowledge database and found that gene promoter methylation of 50 genes was reported to be associated with the risk of melanoma. Meta-analysis revealed that hypermethylation of claudin 11 (CLDN11; odds ratio [OR], 16.82; 95% confidence interval [CI], 1.97-143.29; p = 0.010), O-6-methylguanine-DNA methyltransferase (MGMT; OR, 5.59; 95% CI, 2.51-12.47; p < 0.0001), cyclin-dependent kinase inhibitor 2A (p16; OR, 6.57; 95% CI, 2.19-19.75; p = 0.0008), retinoic acid receptorβ (RAR-β2; OR, 24.31; 95% CI, 4.58-129.01; p = 0.0002), and Ras association domain family member (RASSF1A; OR, 9.35; 95% CI, 4.73-18.45; p < 0.00001) was significantly higher in melanoma patients compared with controls. CLDN11 (OR, 14.52; 95% CI, 1.84-114.55; p = 0.01), MGMT (OR, 8.08; 95% CI, 1.84-35.46; p = 0.006), p16 (OR, 9.44; 95% CI, 2.68-33.29; p = 0.0005), and RASSF1A (OR, 7.72; 95% CI, 1.05-56.50; p = 0.04) hypermethylation was significantly increased in primary melanoma compared with controls. Methylation frequency of CLDN11 (OR, 25.56; 95% CI, 2.32-281.66; p = 0.008), MGMT (OR, 4.64; 95% CI, 1.98-10.90; p = 0.0004), p16 (OR, 4.31; 95% CI, 1.33-13.96; p = 0.01), and RASSF1A (OR, 10.10; 95% CI, 2.87-35.54; p = 0.0003) was significantly higher in metastasis melanoma compared with controls. These findings indicated that CLDN11, MGMT, p16, RAR-β2, and RASSF1A hypermethylation is a risk factor and a potential biomarker for melanoma. CLDN11, MGMT, p16, and RASSF1A promoter methylation may take part in the development of melanoma and become useful biomarkers in the early diagnosis of the disease.

Cell-Free DNA Methylation of Selected Genes Allows for Early Detection of the Major Cancers in Women.

Background: Breast (BrC), colorectal (CRC) and lung (LC) cancers are the three most common and deadly cancers in women. Cancer screening entails an increase in early stage disease detection but is hampered by high false-positive rates and overdiagnosis/overtreatment. Aberrant DNA methylation occurs early in cancer and may be detected in circulating cell-free DNA (ccfDNA), constituting a valuable biomarker and enabling non-invasive testing for cancer detection. We aimed to develop a ccfDNA methylation-based test for simultaneous detection of BrC, CRC and LC. Methods: CcfDNA from BrC, CRC and LC patients and asymptomatic controls were extracted from plasma, sodium-bisulfite modified and whole-genome amplified. APC, FOXA1, MGMT, RARβ2, RASSF1A, SCGB3A1, SEPT9, SHOX2 and SOX17 promoter methylation levels were determined by multiplex quantitative methylation-specific PCR. Associations between methylation and standard clinicopathological parameters were assessed. Biomarkers’ diagnostic performance was also evaluated. Results: A “PanCancer” panel (APC, FOXA1, RASSF1A) detected the three major cancers with 72% sensitivity and 74% specificity, whereas a “CancerType” panel (SCGB3A1, SEPT9 and SOX17) indicated the most likely cancer topography, with over 80% specificity, although with limited sensitivity. Conclusions: CcfDNA’s methylation assessment allows for simultaneous screening of BrC, CRC and LC, complementing current modalities, perfecting cancer suspects’ triage, increasing compliance and cost-effectiveness.

Association of Peripheral Blood RASSF1A and CDKN2A Methylation Status with Smoking Behaviour in Nasopharyngeal Carcinoma

BACKGROUND: Hypermethylation of RASSF1A and CDKN2A is one of epigenetic factor underlies nasopharyngeal carcinoma (NPC) development. Smoking behavior as an NPC’s risk factor causes aberrant DNA methylation. RASSF1A and CDKN2A promoter hypermethylation from peripheral blood cells correlates with smoking behavior. The use of body fluids including peripheral blood as a specimen for DNA methylation analyzes are widely developed, as less invasive method compared to the use of tissue biopsy. This study aims to observe the association between RASSF1A and CDKN2A methylation in peripheral blood and smoking behavioramong NPC patients.METHODS: Newly diagnosed NPC subjects were recruited from ear-nose-throat (ENT) outpatient clinic of Dr. Sardjito Hospital, Yogyakarta. DNA from buffycoat of 19 smokers and 20 non-smokers NPC’s patients were isolated. Bisulphite modification was applied to 500 ng of the isolated DNA. The methylation status was detected by MSP (methylation-specific polymerase chain reaction (PCR)). The association between smoking status and promoter hypermethylation was analysis using Chi-Square test.RESULTS: MSP analysis of RASSF1A showed that 68.42% smoker and 75% non-smoker NPC’s patients were methylated. MSP analysis of CDKN2A showed that 21.05% smoker and 25% non-smoker NPC’s patients were methylated. There was no association between smoking behavior with RASSF1A and CDKN2A methylation (p&gt;0.05).CONCLUSION: Statistical analysis showed that smoking behavior is not associated with methylation of RASSF1A and CDKN2A among NPC’s patients.KEYWORDS: DNA methylation, CDKN2A, RASSF1A, Nasopharyngeal carcinoma, Smoking

Study of the promoter methylation status of RASSF1A gene in oral squamous cell carcinoma

Oral cancer is one of the major global health problems. Methylation is one of the epigenetic modifications that play an important role in the transcriptional inactivation of tumor suppressor genes in human cancer. Aim: The present study was directed to assess the Ras-association domain family 1A (RASSF1 A) gene promoter methylation status in oral squamous cell carcinoma (OSCC) and its correlation to patient’s clinicopathological data. Material and methods: Formalin-fixed, paraffin-embedded tissue blocks of fifty patients with histopathologically confirmed OSCC in addition to 10 normal oral mucosa blocks were included in this study. Hematoxylin and Eosin (H&E) stain was used to confirm the diagnosis of cases as 16 well differentiated, 19 moderately differentiated and 15 poorly differentiated OSCC. Sections of each block were deparaffinized and prepared by DNA extraction for detection of RASSF1A gene methylation status by methylation-specific polymerase chain reaction (MSP) technique. Results: RASSF1A promoter methylation was not detected in normal cases, but it was found in 30% (15/50) of OSCC cases. The increase of methylation in OSCC was significant (p-value=0.045). The increase of methylation in poorly differentiated OSCC (6/15, 40%) was more than that.0 in moderately differentiated OSCC (7/19 36.8%) but both were significante in relation to normal mucosa (p-values= 0.021, 0.027 respectively). Well-differentiated OSCC was the lowest in methylation (2/16, 12.5%), while its increase than normal was not significant (p-value= 0.244). There was direct correlation between methylation and the lymph nodes metastasis. Conclusion: RASSF1A promoter methylation significantly associated with OSCC, the decrease of cell differentiation in histopathological stages and lymph node metastasis. Hence, RASSF1A promoter methylation could be used as a tumor marker in addition to histopathological stage and lymph nodes metastasis predictor in OSCC.