Methylation-sensitive High-resolution Melting Analysis Research Articles

L-carnitine fails to rescue chemotherapy injured ovaries by epigenetic changes of transcription factors

Cyclophosphamide (CP), as an anti-cancer drug, is frequently used to treat various types of cancer. A decreased number of ovarian follicles impaired normal ovarian function, and subsequent premature ovarian failure (POF) presented as a side effect of cyclophosphamide usage. These events may eventually affect the fertility rate of individuals. The present study showed the effect of cyclophosphamide on ovarian reserves and the protective effect of L-carnitine (LC) as an antioxidant to prevent POF.To design the study, six to eight-week-old NMRI female mice were divided into three groups: control, cyclophosphamide (CP), and cyclophosphamide + L-carnitine (CP + LC). Mice received drugs intraperitoneally (IP) for 21 days. In the following 24 h after the last injection, both ovaries were used to evaluate the expression of Sohlh1 and Lhx8 genes by Real-time PCR. Furthermore, the alteration of Lhx8 promoter methylation was examined by Methylation-sensitive high-resolution melting analysis (MS-HRM).The present data showed the negative effect of CP on regulator genes of oogenesis including Sohlh1 and Lhx8. In addition, an examination of the epigenetic status of the Lhx8 gene showed a change in promoter methylation of this gene following cyclophosphamide injection. Although, L-carnitine is an effective antioxidant in relieving oxidative stress caused by cyclophosphamide and its damage, in the present study, however, the use of L-carnitine failed to protect the ovaries from changes caused by CP injection. So, using cyclophosphamide can alter the expression of folliculogenesis genes through its effects on epigenetic changes and may cause POF. The results of the present study showed that L-carnitine consumption can’t protect the ovaries against the adverse effects of CP.

Association between the methylation of CpG islands in JAK-STAT pathway-related genes and colorectal cancer

BackgroundAberrant promoter methylation of CpG islands plays an important role in carcinogenesis. However, the association between the DNA methylation of JAK-STAT pathway-related genes in peripheral blood leukocytes and colorectal cancer (CRC) susceptibility remains unclear. MethodsWe conducted a case-control study of 403 patients with CRC and 419 cancer free controls, and the DNA methylation levels of JAK2, STAT1, STAT3, and SOCS3 in peripheral blood samples from all subjects were assessed using a methylation-sensitive high-resolution melting (MS-HRM) analysis. ResultsCompared with controls, the methylation of the JAK2, STAT1 and SOCS3 genes increased the CRC risk (ORadjusted=1.96, 95% CI, 1.12–3.41, P=0.01; ORadjusted=5.37, 95% CI, 3.74–7.71, P<0.01; ORadjusted=3.30, 95% CI, 1.58–6.87, P<0.01). In the multiple CpG site methylation (MCSM) analysis, a high MCSM value denoted an increased CRC risk (ORadjusted=4.97, 95% CI, 3.34–7.37, P<0.01). ConclusionIn peripheral blood, the methylation of JAK2, STAT1, and high levels of MCSM are promising biomarkers for CRC risk.

Methylation-sensitive high-resolution melting analysis of the USP44 promoter can detect early-stage hepatocellular carcinoma in blood samples

Hepatocellular carcinoma (HCC) is dangerous cancer that often evades early detection because it is asymptomatic and an effective detection method is lacking. For people with chronic liver inflammation who are at high risk of developing HCC, a sensitive detection method for HCC is needed. In a meta-analysis of The Cancer Genome Atlas pan-cancer methylation database, we identified a CpG island in the USP44 promoter that is methylated specifically in HCC. We developed methylation-sensitive high-resolution melting (MS-HRM) analysis to measure the methylation levels of the USP promoter in cell-free DNA isolated from patients. Our MS-HRM assay correctly identified 40% of patients with early-stage HCC, whereas the α-fetoprotein test, which is currently used to detect HCC, correctly identified only 25% of early-stage HCC patients. These results demonstrate that USP44 MS-HRM analysis is suitable for HCC surveillance. [BMB Reports 2022; 55(11): 553-558].

Methylation analysis of APC, AXIN2, DACT1, RASSF1A and MGMT gene promoters in non-small cell lung cancer

Silencing of tumour-suppressor genes through promoter methylation is frequently observed in carcinogenesis. In this study, we determined the methylation status of RASSF1A, MGMT, APC, AXIN2 and DACT1 genes in 73 cases of non-small cell lung cancer. Methylation-sensitive high-resolution melting analysis (MS-HRM) was used to analyse the promoter methylation, which was further validated with Bisulfite pyrosequencing or Sanger sequencing. Promoter methylation of RASSF1A and APC was frequently found (56% and 49% of cases, respectively), while methylation of MGMT, AXIN2, DACT1 was observed in 30%, 19% and 16%, respectively. Concurrent gene methylation of at least two genes was observed in 55% of the examined cases, with a total of 89% of samples displaying methylation in one or more of the investigated genes. Further analysis of concurrent methylation revealed a positive correlation between AXIN2-DACT1 and an inverse correlation of APC-MGMT. Associations of methylated genes and clinicopathological features were emerged. In more detail, APC promoter methylation was correlated with smoking status (p= 0.020) and non-metastatic cases (p= 0.003). Moreover, MGMT methylation was preferentially found in TTF1-negative cases (p= 0.049). Interestingly, correlation occurred between AXIN2/DACT1 methylation and smoking status (p= 0.009) as well as tumour grade (p= 0.013), as none of these genes was methylated in the majority of smokers and one of the genes was methylated in high-grade tumours. We conclude that aberrant promoter methylation was observed in our cohort while concurrent methylation patterns were also determined. APC, MGMT and AXIN2/DACT1 methylation are potentially of clinical importance regarding prognosis and histological subtyping of NSCLC.

Detection of MSH2 Gene Methylation in Extramammary Paget's Disease by Methylation-Sensitive High-Resolution Melting Analysis.

Background Extramammary Paget's disease (EMPD) is a rare skin tumor. Hypermethylation in the MSH2 promoter resulting in the downregulation of its protein expression shows a high detection rate in EMPD tumor tissue, which indicates that the methylation of MSH2 may play an important role in the pathogenesis of EMPD. Objective This study aims to establish a rapid analysis strategy based on the methylation-sensitive high-resolution melting curve (MS-HRM) to detect the methylation level of the MSH2 promoter. Methods With the use of universal methylated human DNA products, we established the MS-HRM standard curve to quantitatively detect the methylation level of the MSH2 promoter. Then, all 57 EMPD tumor DNA samples were analyzed. Pyrosequencing assay was also carried out to test the accuracy and efficacy of MS-HRM. Besides, a total of 54 human normal and other cancerous tissues were included in this study to test the reliability and versatility of the MS-HRM standard curve. Results In this study, by using the established MS-HRM, we found that 96.5% (55/57) EMPD tumor samples had varying methylation levels in the MSH2 promoter ranging from 0% to 30%. Then, the methylation data were compared to the results obtained from pyrosequencing, which showed a high correlation between these two techniques by Pearson's correlation (r = 0.9425) and Bland–Altman plots (mean difference = −0.1069) indicating that the methylation levels analyzed by MS-HRM were consistent with DNA pyrosequencing. Furthermore, in 23 normal and 31 other cancerous tissue samples, there were two colorectal cancer (CRC) tissues that tested MSH2 methylation positive (1% and 5%) which confirmed that our established MS-HRM can be widely applied to various types of samples. Conclusion MS-HRM standard curve can be used for the detection of the methylation level of MSH2 in EMPD tumor samples and other cancerous tissues potentially, which presents a promising candidate as a quantitative assay to analyze MSH2 promoter methylation in routine pathological procedure.

Decreased promoter methylation of E-cadherin gene due to overexpression of TET enzyme by vitamin C in MCF-7 cell line

BackgroundE-cadherin (CDH1) is a well-known tumor suppressor gene. The loss of CDH1 gene function caused by genetic or epigenetic alterations may consequently result in tumorigenesis. Vitamin C is an essential factor for epigenetic regulation and its great furnished potential in epigenetic therapy in cancer patients. ObjectiveThe present study aims to evaluate the effect of vitamin C on promoter methylation of the CDH1 gene and its possible correlation with the expression of CDH1 in the MCF-7 cell line. MethodsCDH1 expression was studied by reverse transcription-polymerase chain reaction (RT–PCR) analysis in MCF-7 cell line that was treated with different concentrations of vitamin C. TET enzyme activity was measured by ELISA. Promoter methylation of CDH1 was studied by Methylation-Sensitive High-Resolution Melting (MS-HRM) analysis of bisulfite-modified DNA. ResultsOur results showed that vitamin C increase not only TET enzyme activity but also TET gene overexpressed. In the following, cause to CDH1 gene promoter demethylation and upregulation of CDH1 gene in MCF-7 cell line. ConclusionCancer cells may respond to vitamin C induces CDH1 expression by promoter demethylation due to TET enzyme overexpression.

Blood DNA Methylation Levels in the WNT5A Gene Promoter Region: A Potential Biomarker for Agitation in Subjects with Dementia.

Background:Behavioral and psychological symptoms of dementia (BPSD) cause a heavy burden for both patient and caregivers. These symptoms are diverse, and their mechanism is still unclear. Agitation is the most common and difficult to treat among BPSD. In recent years, while changes in DNA methylation levels have been receiving attention as a biomarker of aging and dementia, associations with BPSD have not been examined.Objective:Focusing on agitation, the objective of the present study was to identify a region where changes in DNA methylation levels are associated with agitation.Methods:Using genome-wide DNA methylation analysis data for 7 dementia subjects with agitation, 5 dementia subjects without agitation, and 4 normal elderly controls, we determined a signaling pathway in the WNT5A gene promoter region to be associated with agitation. Based on this result, we measured DNA methylation levels in this region for 26 dementia subjects with agitation and 82 dementia subjects without agitation by means of methylation-sensitive high-resolution melting (MS-HRM) analysis.Results:The WNT5A DNA methylation level in dementia subjects with agitation was significantly lower than in those without agitation (p = 0.001). Changes in WNT5A DNA methylation levels were not influenced by age, sex, body mass index, APOE ɛ4, medication, or inflammatory cytokines.Conclusion:Our results suggested an association of agitation with Wnt signaling, in particular with changes in WNT5A DNA methylation levels, which could be a potentially useful biomarker for predicting the appearance of agitation. It may contribute to the elucidation of the mechanism of BPSD.

Rapid semen identification from mixed body fluids using methylation-sensitive high-resolution melting analysis of the DACT1 gene.

In forensic genetics, a suspect is assigned to a component of a DNA mixture profile, and a probabilistic interpretation is then usually performed. However, it is difficult to determine what types of body fluid the component is from. Previous studies have reported that the fourth exon of the Dishevelled binding antagonist of beta catenin 1 (DACT1) gene is hypomethylated in a semen DNA-specific manner. In the present study, we evaluated whether the DACT1 gene could be effectively used to identify semen in body fluid mixtures and were able to semi-quantify the semen DNA content in mixed fluids. Our results showed that the DACT1 gene was useful in discriminating semen from venous blood and saliva. However, the amount of sperm in semen can affect semen identification. In addition, SI (the semen DNA content index), which we developed, was useful to determine whether the semen compromised majority, almost half, or was in the minority of the components in a mixed fluid. This technique is based on the methylation-sensitive high-resolution melting (MS-HRM) technology, which is time-, cost-, and labour-effective, and could be adopted in routine criminal investigations.

Abstract 1973: A novel method for highly sensitive, facile and inexpensive quantitative assessment of aberrant DNA methylation in liquid biopsies

Abstract Introduction: There remains a considerable need for the development of new, simple methods for assessing methylation of cell-free DNA obtained from liquid biopsies. In particular, methods that can achieve highly-sensitive quantitative analysis are needed to enable estimation of tumor burden, invasiveness and recurrence. While standard methylation-sensitive high resolution melt (MS-HRM) analysis methods allow for semi-quantitative assessment of DNA methylation, achieving sufficient sensitivity and accuracy to enable reliable detection of rare circulating tumor DNA (ctDNA) in a large background of predominantly healthy cfDNA remains challenging. We developed a novel method that drastically improves the analytical sensitivity of HRM analysis in order to quantify epiallelic fractions as low as 0.001%. This method was named HiQASP (Highly Quantitative Allele Specific PCR). We demonstrate use of HiQASP for ultra-high-sensitive and quantitative methylation assessment of the promoter region of SEPT9, which is currently used in colorectal cancer screening kits. Here, we utilize this method to assess SEPT9 methylation in cfDNA derived from with liquid biopsies of colorectal cancer patients. Methods: All human blood samples were obtained with patient's consent and relevant IRB approval. The plasma specimens were separated with twice centrifugation and cfDNA purified with Qiagen's QIAamp Circulating Nucleic Acid Kit. Bisulfite conversion was performed using Zymo EZ DNA Methylation-Lightning kit according to the manufacturer's instructions. Plasma DNA concentrations were calculated using DX-11Fx (DeNovix) with QuantiFluor (Promega). Analytic specificities and sample epiallelic quantification were determined using synthetic oligonucleotide standards for the bisulfite-converted sequence of methylated and unmethylated SEPT9 were obtained from IDT and GenScript, respectively. Validation of the SEPT9 HiQASP assay was performed using serial dilutions of synthetic oligonucleotides. EpiTaq HS (TaKaRa) and EvaGreen (BioTium) were used to amplify and detect the PCR products, respectively. The equipment QuantStudio 3 (ThermoFisher) was used to amplify and execute HRM analysis. Results: Custom HiQASP primers were designed to maximize assay analytical sensitivity and specificity for methylation analysis at the SEPT9 locus. Overall, the SEPT9 HiQASP assay achieved an analytical sensitivity of 0.0005% for both bisulfite-converted unmethylated and methylated synthetic oligonucleotide targets. The entire assay could be performed in only 45 minutes. The potential clinical utility of the assay was also demonstrated using cfDNA derived from of a plasma sample obtained from a colorectal cancer patient. HiQASP analysis indicated the percentage of methylated SEPT9 in this specimen to be 3.8% yielding a total copy concentration of 35 copies of methylated SEPT9 per ml of plasma. Conclusion: HiQASP is a highly-sensitive locus-specific assay for facile, rapid and low-cost quantitative assessment of methylated DNA in challenging specimens such as liquid biopsies. Citation Format: Sachio Nomura, Toshiyuki Kobayashi, Kiichi Sugimoto, Masami Arai, Thomas R. Pisanic, Hirotaka Momose, Kazuhiro Sakamoto, Okio Hino. A novel method for highly sensitive, facile and inexpensive quantitative assessment of aberrant DNA methylation in liquid biopsies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1973.

Methylation of the PKD1 Promoter Inversely Correlates with its Expression in Autosomal Dominant Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD), a multisystem disorder, is the most prevalent type of hereditary kidney disease. Here, we aimed to evaluate methylation of the PKD1 gene (PKD1) promoter and its correlation with PKD1 expression in peripheral blood. In this case-control study methylation of the PKD1 promoter was evaluated using methylation-sensitive high-resolution melt (MS-HRM) analysis. PKD1 expression was assessed by quantitative real-time PCR. The correlation was evaluated using the Pearson correlation test. Twenty subjects from both the patient and control groups (n= 40 for each) were methylated at the PKD1 promoter to various levels (18.9% in patients and 62.5% in controls). This difference was statistically significant (p< 0.0001). PKD1 expression in blood samples was significantly greater in ADPKD patients than in controls (p= 0.0081). Significant correlation was seen between PKD1 expression and its promoter methylation status in peripheral blood (r case= -0.5300, p= 0.0162, and r control = -0.6265, p= 0.0031). Methylation of the PKD1 promoter in ADPKD patients was inversely correlated with PKD1 expression.

Epigenetic changes in FOXO3 and CHEK2 genes and their correlation with clinicopathological findings in myelodysplastic syndromes

Objectives/backgroundMyelodysplastic syndromes (MDSs) are a heterogeneous disease in terms of clinical course and response to therapy. Epigenetic changes are the primary mechanism of MDS pathogenesis. FOXO3 and CHEK2 genes play significant roles in normal cellular mechanisms and are also known as tumor suppressor genes. We aimed to clarify the correlation of epigenetic changes in these genes with clinicopathologic findings in MDS. MethodsA total of 54 newly diagnosed MDS patients referred to Shariati and Firouzgar Hospitals (Tehran, Iran) were included in the study from 2013 to 2015, comprising the following cases: 26 with refractory cytopenia with unilineage dysplasia, 10 with refractory cytopenia with multilineage dysplasia, four refractory anemia with excess blasts-1 (RAEB-1), 11 refractory anemia with excess blasts-2 (RAEB-2), and three MDS associated with isolated deletion (5q-). Risk groups were determined according to the Revised International Prognostic Scoring System (IPSS-R). The methylation status of CHEK2 and FOXO3 promoters were determined by methylation-sensitive high-resolution melting analysis of sodium bisulfite-converted DNA. Expressions of CHEK2, FOXO3, and GAPDH were measured by quantitative real-time polymerase chain reaction and fold changes were calculated using the ΔΔCT method. ResultsStatistical analysis revealed no promoter methylation of CHEK2 and FOXO3 in healthy control specimens. FOXO3 promoter methylation was associated with high-risk World Health Organization subgroups (p = .017), high-risk IPSS-R (p = .007), high-risk cytogenetics (p = .045), and more than 5% blasts in bone marrow (p = .001). CHEK2 promoter methylation was correlated with more than 5% blasts in bone marrow (p = .009). ConclusionsPromoter methylation of CHEK2 and especially FOXO3 is associated with adverse clinicopathological findings and disease progression in MDS.

Identification of a four-gene methylation biomarker panel in high-grade serous ovarian carcinoma.

Background The lack of effective biomarkers for the screening and early detection of ovarian cancer (OC) is one of the most pressing problems in oncogynecology. Because epigenetic alterations occur early in the cancer development, they provide great potential to serve as such biomarkers. In our study, we investigated a potential of a four-gene methylation panel (including CDH13, HNF1B, PCDH17 and GATA4 genes) for the early detection of high-grade serous ovarian carcinoma (HGSOC). Methods For methylation detection we used methylation sensitive high-resolution melting analysis and real-time methylation specific analysis. We also investigated the relation between gene hypermethylation and gene relative expression using the 2-ΔΔCt method. Results The sensitivity of the examined panel reached 88.5%. We were able to detect methylation in 85.7% (12/14) of early stage tumors and in 89.4% (42/47) of late stage tumors. The total efficiency of the panel was 94.4% and negative predictive value reached 90.0%. The specificity and positive predictive value achieved 100% rates. Our results showed lower gene expression in the tumor samples in comparison to control samples. The more pronounced downregulation was measured in the group of samples with detected methylation. Conclusions In our study we designed the four-gene panel for HGSOC detection in ovarian tissue with 100% specificity and sensitivity of 88.5%. The next challenge is translation of the findings to the less invasive source for biomarker examination, such as plasma. Our results indicate that combination of examined genes deserve consideration for further testing in clinical molecular diagnosis of HGSOC.

DNA methylation of SFRP1, SFRP2, and WIF1 and prognosis of postoperative colorectal cancer patients

BackgroundAs biomarkers, DNA methylation is used to detect colorectal cancer (CRC) and make assessment of CRC prognosis. The published findings showed the association between the methylation of SFRP1, SFRP2, and WIF1, located in the Wnt signaling pathway, and the prognosis of CRC were not consistent. Our study aimed to explore the potential possibility of SFRP1, SFRP2, and WIF1 concomitant promoter methylation as prognostic biomarkers of postoperative CRC patients.MethodsAs a total of 307 sporadic postoperative CRC patients were followed up, we detected SFRP1, SFRP2, and WIF1 methylation obtained from tumor tissues and adjacent non-tumor tissues respectively on the basis of methylation-sensitive high resolution melting analysis. Univariate and multivariate Cox regressions were carried out so as to assess the potential possibility of SFRP1, SFRP2, and WIF1 promoter methylation as predictors of prognosis. Confounders in our study were controlled by Propensity Score (PS) analysis.ResultsThe SFRP1, SFRP2, and WIF1 methylation levels in tumor tissues were significantly higher than that in adjacent non-tumor tissues (P < 0.001). SFRP2 hypermethylation was significantly associated with a favorable clinical outcome at the hazard ratio (HR) of 0.343 [95% confidence intervals (CI): 0.164–0.718, P = 0.005] and 0.410 (95% CI: 0.200–0.842, P = 0.015) in multivariate Cox regression and PS analysis, respectively. Co-hypermethylation of SFRP1 and SFRP2 was significantly associated with a favorable clinical outcome at the HR of 0.333 (95% CI: 0.159–0.694, P = 0.003) and 0.398 (95% CI: 0.192–0.821, P = 0.013) in multivariate Cox regression and PS analysis, respectively. Co-hypermethylation of SFRP1, SFRP2 and WIF1 was significantly associated with a favorable clinical outcome at the HR of 0.326 (95% CI: 0.117–0.908, P = 0.032) and 0.401 (95% CI: 0.146–1.106, P = 0.077) in multivariate Cox regression and PS analysis, respectively.ConclusionsSFRP1, SFRP2, and WIF1 were frequently hypermethylated in CRC tumor tissues. It was apparent that the promoter hypermethylation of SFRP2 and co-hypermethylation of SFRP1 and SFRP2 might be considered as independent prognostic predictors for survival advantage of postoperative CRC patients.

DNA hypermethylation of MAL gene may act as an independent predictor of favorable prognosis in patients with colorectal cancer

Background: Aberrant DNA methylation could be used as biomarkers for colorectal cancer (CRC) detection and assessment of prognosis. The aim of our study was to investigate the potential possibility of MAL methylation as a prognostic biomarker for postoperative CRC patients. Methods: We followed up 298 sporadic postoperative CRC patients and detected MAL methylation in tumor tissues and adjacent non-tumor tissues by methylation-sensitive high resolution melting (MS-HRM) analysis. Univariate, multivariate Cox regressions were performed to evaluate the potential possibility of MAL methylation as a predictor of prognosis. Propensity score (PS) analysis was used to control confounders. Results: The MAL methylation level in adjacent non-tumor tissues was significantly lower than that in tumor tissues (P<0.001). The MAL methylation had no significant correlation with clinicopathologic characteristics. MAL hypermethylation was detected in 63.4% (189/298) tumor tissues. The overall 5-year survival rates in hypermethylation and hypomethylation group were 70.78% and 55.69% (P=0.007). MAL hypermethylation was significantly associated with a favorable clinical outcome, the hazard ratio (HR) were 0.650 [95% confidence interval (CI): 0.454–0.929, P=0.018], 0.613 (95% CI: 0.422–0.889, P=0.010) and 0.692 (95% CI: 0.481–0.996, P=0.047) in univariate, multivariate Cox and PS method, respectively. The subgroup analysis showed that MAL hypermethylation in CRC patients with lower diagnosis age (<60) and colon cancer had a lower risk of death than MAL hypomethylation patients. Conclusions: MAL was frequently hypermethylated in CRC tumor tissues. MAL hypermethylation might act as an independent prognostic predictor of survival advantage in postoperative patients with CRC.

P13.01 TERTpromoter methylation is significantly associated withTERTupregulation and tumor progression in pituitary adenomas

Abstract BACKGROUND Activation of telomerase plays a critical role in tumor development. Somatic alterations in the promoter of the telomerase reverse transcriptase (TERT) gene are a major mechanism of upregulating telomerase. Several mechanisms have been associated with TERT abnormalities, including TERT promoter mutations or methylation. Mutations in the TERT promoter have been observed in a subset of brain tumors, especially in adult gliomas. In pituitary adenomas (PAs), however, TERT abnormalities are not fully understood. The present study aimed to investigate not only mutational but also methylation status changes in the TERT promoter in PAs and to analyze their correlations with clinical variables. MATERIAL AND METHODS We retrospectively studied 70 PAs consisting of 53 primary and 17 recurrent samples. Clinical data, including age at surgery, sex, tumor size, tumor subtype, resection rate, presence or absence of postoperative irradiation, and progression-free survival (PFS), were obtained from medical records. First, we investigated TERT promoter hotspot mutations via Sanger sequencing. Next, we quantified the methylation status of the TERT promoter using methylation-sensitive high-resolution melting analysis (MS-HRM). Finally, we investigated TERT mRNA expression levels using real-time quantitative PCR. Fisher’s exact test was applied to evaluate the statistical significance between TERT promoter methylation status and tumor recurrence. PFS was calculated using Kaplan-Meier estimates and compared between methylated with ummethylated PAs with the log-rank test. The correlation between TERT promoter methylation status and mRNA levels was analyzed with the Mann-Whitney U-test. PFS was analyzed using multivariate analysis with the Cox proportional hazards model and included the following variables: age, sex, tumor size, tumor subtype, resection rate, radiation therapy, and methylation status. RESULTS TERT promoter hotspot mutations were not observed in any PA sample. Nineteen percent of PAs exhibitedTERT promoter methylation, which was significantly predominant in recurrent PA samples. PFS was significantly shorter in the methylated cases than in the unmethylated cases. Higher TERT expression levels were correlated with methylation status. CONCLUSION We found that TERT promoter methylation upregulated TERT expression and was associated with shorter PFS in PAs. Our results suggest thatTERT promoter methylation may be a potential biomarker for predicting tumor recurrence in PAs.

DNA hypermethylation of MAL gene may act as an independent predictor of favorable prognosis in patients with colorectal cancer

BackgroundAberrant DNA methylation could be used as biomarkers for colorectal cancer (CRC) detection and assessment of prognosis. The aim of our study was to investigate the potential possibility of MAL methylation as a prognostic biomarker for postoperative CRC patients.MethodsWe followed up 298 sporadic postoperative CRC patients and detected MAL methylation in tumor tissues and adjacent non-tumor tissues by methylation-sensitive high resolution melting (MS-HRM) analysis. Univariate, multivariate Cox regressions were performed to evaluate the potential possibility of MAL methylation as a predictor of prognosis. Propensity score (PS) analysis was used to control confounders.ResultsThe MAL methylation level in adjacent non-tumor tissues was significantly lower than that in tumor tissues (P<0.001). The MAL methylation had no significant correlation with clinicopathologic characteristics. MAL hypermethylation was detected in 63.4% (189/298) tumor tissues. The overall 5-year survival rates in hypermethylation and hypomethylation group were 70.78% and 55.69% (P=0.007). MAL hypermethylation was significantly associated with a favorable clinical outcome, the hazard ratio (HR) were 0.650 [95% confidence interval (CI): 0.454–0.929, P=0.018], 0.613 (95% CI: 0.422–0.889, P=0.010) and 0.692 (95% CI: 0.481–0.996, P=0.047) in univariate, multivariate Cox and PS method, respectively. The subgroup analysis showed that MAL hypermethylation in CRC patients with lower diagnosis age (<60) and colon cancer had a lower risk of death than MAL hypomethylation patients.ConclusionsMAL was frequently hypermethylated in CRC tumor tissues. MAL hypermethylation might act as an independent prognostic predictor of survival advantage in postoperative patients with CRC.

MGMT promoter methylation in patients with glioblastoma: is methylation-sensitive high-resolution melting superior to methylation-sensitive polymerase chain reaction assay?

The methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) gene promoter is a prognostic factor in adults with glioblastoma (GBM); it also yields information that is useful for clinical decision-making in elderly GBM patients. While pyrosequencing is the gold standard for the evaluation of the methylation status of MGMT, methylation-sensitive polymerase chain reaction (MS-PCR) assay continues to be used widely. Although MS-PCR results exhibited a good correlation with the prognosis of patients with GBM treated under the Stupp protocol, interpretation of the bands is based on subjective judgment, and the assay cannot be used to analyze heterogeneously methylated samples. We assessed whether methylation-sensitive high-resolution melting (MS-HRM) is an alternative to MS-PCR. The authors prepared 3 primer sets that covered CpG 72–89 for MS-HRM analysis to determine the methylation levels of 6 human glioma cell lines. The results were validated by bisulfite sequencing of cloned alleles. The authors also subjected surgical samples from 75 GBM patients treated with temozolomide (TMZ) to MS-HRM to assess the MGMT methylation status and compared the findings with MS-PCR results using receiver operating characteristic (ROC), univariate, and multivariate analyses. There was a strong correlation between the methylation levels of the 6 glioma cell lines evaluated by MSHRM and by bisulfite sequencing; with primers 1 and 2, the correlation was significant (r = 0.959 and r = 0.960, respectively, p < 0.01). Based on log-rank analysis, MS-HRM was significantly better than MS-PCR for predicting progressionfree survival (PFS) and overall survival (OS) based on the methylation status of the MGMT promoter (PFS predicted by MS-HRM and MS-PCR = 0.00023 and 0.0035, respectively; OS = 0.00019 and 0.00028, respectively). ROC analysis showed that the area under the curve was larger with MS-HRM than with MS-PCR (PFS: 0.723 vs 0.635; OS: 0.716 vs 0.695). Based on multivariate Cox regression analysis, MS-HRM was significantly better than MS-PCR for predicting the treatment outcome (MS-HRM vs MS-PCR: PFS, p = 0.001 vs 0.207; OS, p = 0.013 vs 0.135). The authors’ findings show that MS-HRM is superior to MS-PCR for the detection of MGMT promoter methylation. They suggest MS-HRM as an alternative to MS-PCR for assessing the prognosis of patients with GBM.

SA47GENETIC PREDISPOSITION TO VITAMIN B12 AND FOLATE DEFICIENCY LEADS TO THE DEVELOPMENT OF DEPRESSION IN POSTPARTUM PERIOD BY HYPO-METHYLATION OF SEROTONIN TRANSPORTER AND MONOAMINE OXIDASE-A GENE LOCI

Plasminogen activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator (uPA) play a crucial role in cancer progression. In the present study we examined the regulation of PAI-1 and uPA expressions in normal prostate epithelial cells (PrEC) and the prostate cancer cell lines LNCaP, DU-145, and PC-3. The antigen and mRNA levels of PAI-1 were down-regulated in cancer cells, especially in LNCaP and DU-145. In the presence of proinflammatory cytokines, an increase of PAI-1 mRNA levels was observed in PrEC, LNCaP and PC-3, but not in DU-145 cells. Treatment with demethylating agent, 5-aza-2′-deoxycytidine increased the level of PAI-1 transcript in DU-145 cells and restored the inducing effect of cytokines on PAI-1 expression. An aberrant methylation of PAI-1 promoter in DU-145 and LNCaP cells was shown by methylation-sensitive high resolution melting (MS-HRM) analysis. PAI-1 methylation was also significantly increased in tumor samples (23.2 ± 1.7%) in comparison to adjacent non-tumor tissue (6.0 ± 0.8%). Furthermore, the expression of uPA was increased in high invasive cell lines DU-145 and PC-3 in comparison to PrEC and low invasive LNCaP cells. MS-HRM analysis revealed aberrant methylation of uPA promoter in LNCaP cells, but not in PrEC, DU-145 and PC-3 cells, as well as in normal and prostate cancer tissue samples. In conclusion, the study shows that PAI-1 and uPA expressions were changed in opposite directions in high invasive prostate cancer cell lines resulting in a strong decrease of PAI-1/uPA ratio, which may indicate a shift towards proteolytic activities. Methylation of the PAI-1 gene is suggested as one of the molecular mechanisms involved in the cancer-associated down-regulation of the PAI-1 expression.

Investigation of DNAmethylation of TWIST Gene in Breast Cancer and Its Relationship to Histopathological Features

BACKGROUND: Promoter hypermethylation and global hypomethylation in the human genome are hallmarks of most cancers. OBJECTIVE: The aim of this study is to assess the methylation profile patterns of TWIST gene and to investigate the relationship of methylation with pathological features. METHODS: Romoter CpGisland methylation of TWIST which can be related in breast cancer was performed by methylation-sensitive high resolution melting (MS-HRM) analysis. Formalin-fixed, paraffin-embedded (FFPE) tissue samples of 80 patients with a diagnosis of primary breast cancer from Eskisehir Osmangazi University medical faculty of Oncology Clinic were included. RESULTS: In our study, the promoter hypermethylation frequency of TWIST gene was 25.0%. With these results, when the prognostic factors of the patients were analyzed, tumor stage and age were found to be meaningless with the hypermethylation of TWIST gene, but found to be significant with lymph node positivity, ER positivity, PR negativity, and HER2/NEU negativity. CONCLUSION: Our study is important as being the first study that analyzes association between histopathologic type and TWIST gene promotor methylation status in Turkish population.

TERT promoter methylation is significantly associated with TERT upregulation and disease progression in pituitary adenomas.

Alterations in the promoter of the telomerase reverse transcriptase (TERT) gene are a major mechanism of upregulating telomerase, which plays a crucial role in tumor development. Mutations in the TERT promoter have been observed in a subset of brain tumors, including adult gliomas and high-grade meningiomas. In pituitary adenomas (PAs), however, abnormalities in TERT are not fully understood. The present study aimed to investigate not only mutational but also methylation changes in the TERT promoter in PAs and to analyze their correlations with clinical variables. We retrospectively studied 70 PAs consisting of 53 primary and 17 recurrent samples. Clinical data, including age at surgery, sex, largest tumor dimension, tumor subtype, resection rate, and progression-free survival (PFS), were obtained from medical records. We investigated TERT promoter hotspot mutations via Sanger sequencing and quantified the methylation status of the TERT promoter using methylation-sensitive high-resolution melting analysis (MS-HRM). Additionally, we investigated TERT mRNA expression using real-time quantitative PCR. TERT promoter hotspot mutations were not observed in any PA sample, while 16% of PAs exhibited TERT promoter methylation. PAs with methylated TERT promoters were significantly more likely to show disease progression, shorter PFS, and higher TERT expression levels compared to those with unmethylated promoters. This is the first study showing that TERT promoter methylation is associated with disease progression and shorter PFS as well as upregulated TERT expression in PAs. Our results suggest that TERT promoter methylation may be a potential biomarker for predicting tumor recurrence in PAs.