Analysis of CDO1, PITX2, and CDH13 Gene Methylation in Early Endometrial Cancer for Prediction of Medical Treatment Outcomes.

An observational cohort study of patients diagnosed with endometrial cancer (EC) stage IA G1, or atypical endometrial hyperplasia (AEH), undergoing organ-preserving treatment, was conducted. OBJECTIVE OF THE STUDY: To determine CDO1, PITX2, and CDH13 gene methylation levels in early endometrial cancer and atypical hyperplasia specimens obtained before organ-preserving treatment in the patients with adequate response and with insufficient response to hormonal treatment. MATERIALS AND METHODS: A total of 41 endometrial specimens obtained during diagnostic uterine curettage in women with EC (n = 28) and AEH (n = 13), willing to preserve reproductive function, were studied; 18 specimens of uterine cancer IA stage G1 from peri- and early postmenopausal women (comparison group) were included in the study. The control group included 18 endometrial specimens from healthy women obtained by diagnostic curettage for missed abortion and/or intrauterine adhesions. Methylation levels were analyzed using the modified MS-HRM method. RESULTS: All 13 women with AEH had a complete response (CR) to medical treatment. In the group undergoing organ-preserving treatment for uterine cancer IA stage G1 (n = 28), 14 patients had a complete response (EC CR group) and 14 did not (EC non-CR group). It was found that all groups had statistically significant differences in CDO1 gene methylation levels compared to the control group (p < 0.001) except for the EC CR group (p = 0.21). The p-value for the difference between EC CR and EC non-CR groups was <0.001. The differences in PITX2 gene methylation levels between the control and study groups were also significantly different (p < 0.001), except for the AEH group (p = 0.21). For the difference between EC CR and EC non-CR groups, the p-value was 0.43. For CDH13 gene methylation levels, statistically significant differences were found between the control and EC non-CR groups (p < 0.001), and the control and EC comparison groups (p = 0.005). When comparing the EC CR group with EC non-CR group, the p-value for this gene was <0.001. The simultaneous assessment of CDO1 and CDH13 genes methylation allowed for an accurate distinction between EC CR and EC non-CR groups (AUC = 0.96). CONCLUSION: The assessment of CDO1 and CDH13 gene methylation in endometrial specimens from patients with endometrial cancer (IA stage G1), scheduled for medical treatment, can predict the treatment outcome.

Lower Levels of TET2 Gene Expression, with a Higher Level of TET2 Promoter Methylation in Patients with AML; Evidence for the Role of Aberrant Methylation in AML Pathogenesis.

DNA methylation is a key epigenetic mechanism that is dysregulated in leukemia and plays a significant role in leukemogenesis. Ten-eleven translocation 2 (TET2) is one of the most frequently mutated genes among the DNA methylation regulators in hematologic malignancies, indicating its tumor-suppressor function. In this study, we investigated the expression and methylation status of TET2 in patients with AML. Quantitative RT-PCR was used to evaluate TET2 expression in peripheral blood mononuclear cells (PBMCs) from 51 newly diagnosed AML patients and 50 healthy controls. The methylation-sensitive high-resolution melting (MS-HRM) method was used in 45 patients with AML and 15 healthy controls to evaluate the promoter methylation of TET2. TET2 expression was significantly downregulated (P < 0.0001) in patients with AML compared to that in healthy controls. Furthermore, the methylation level of the TET2 promoter was significantly different between patients and controls. Aberrant methylation of the TET2 promoter was observed in 53.3% of the patients. Interestingly, a negative (- 0.3138) and significant (P = 0.0358) correlation between TET2 methylation and expression was found. The survival of patients with downregulated TET2 was poorer than that of other patients. TET2 gene expression was significantly downregulated while the promoter methylation was higher in patients, indicating that TET2 may be a tumor suppressor gene and a prognostic factor in AML and that transcriptional silencing of the TET2 gene may play a role in AML pathogenesis. Since epigenetic mechanisms are reversible, abnormal TET2 methylation could become a therapeutic target in the future.

Methylation Profile of Small Breast Cancer Tumors Evaluated by Modified MS-HRM.

The DNA methylation profile of breast cancer differs from that in healthy tissues and can be used as a diagnostic and prognostic biomarker. Aim of this study: To compare the levels of gene methylation in small malignant breast cancer tumors (<2 cm), in healthy tissue, and in fibroadenoma, and to evaluate the effectiveness of the modified Methylation Sensitive-High Resolution Melting (MS-HRM) method for this analysis. Analysis was performed using the modified MS-HRM method. For validation, the methylation levels of five genes were confirmed by pyrosequencing. The main study group included 96 breast cancer samples and the control group included 24 fibroadenoma samples and 24 healthy tissue samples obtained from patients with fibroadenoma. Breast cancer samples were divided into two subgroups (test set and validation set). The methylation of the following 15 genes was studied: MAST1, PRDM14, ZNF177, DNM2, SSH1, AP2M1, CACNA1E, CPEB4, DLGAP2, CCDC181, GCM2, ITPRIPL1, POM121L2, KCNQ1, and TIMP3. Significant differences in the validation set of samples were found for seven genes; the combination of the four genes GCM2, ITPRIPL1, CACNA1E, DLGAP2 (AUC = 0.99) showed the highest diagnostic value based on logistic regression for all breast cancer samples. Our modified MS-HRM method demonstrated that small breast cancer tumors have a specific DNA methylation profile that distinguishes them from healthy tissues and benign proliferative lesions.

Methylomic biomarkers of lithium response in bipolar disorder: a clinical utility study.

BACKGROUND: Response to lithium (Li) is highly variable in bipolar disorders (BD). Despite decades of research, no clinical predictor(s) of response to Li prophylaxis have been consistently identified. Recently, we developed epigenetic Methylation Specific High-Resolution Melting (MS-HRM) assays able to discriminate good responders (GR) from non-responders (NR) to Li in individuals with BD type 1 (BD-I). This study examined whether a combination of clinical and epigenetic markers can distinguish NR from other types of Li responders. METHODS: We recorded clinical variables that are potentially associated with Li response in 64 individuals with BD-I. MS-HRM assays were performed on DNA isolated from peripheral blood. We used backward stepwise logistic regression analyses, followed by receiver operating characteristic (ROC) curve analysis to estimate the performance of the clinical variables, alone then in combination with the epigenetic biomarkers, to identify GR and partial responders (PaR) vs NR. RESULTS: Polarity at onset, psychotic symptoms at onset and family history of BD classified correctly 70% of individuals according to their Li response (PaR + GR = 86%; NR = 35%). When combined with the epigenetic biomarkers, these three clinical variables plus alcohol misuse (and one DMR: Differentially Methylated Region) correctly classified 86% of individuals, improving the prediction of PaR + GR (93%) and of NR (70%). The ROC analysis demonstrated an improvement in the area under the curve from 0.75 (clinical variables alone) to 0.87 (combination of clinical and epigenetic markers). CONCLUSIONS: Combining clinical predictors and DNA methylation markers of Li response may have greater utility in clinical practice than relying on clinical characteristics alone.

Methylomic Biomarkers of Lithium Response in Bipolar Disorder: A Proof of Transferability Study.

Response to lithium (Li) is highly variable in bipolar disorders (BD) and no clinical or biological predictors of long-term response have been validated to date. Using a genome-wide methylomic approach (SeqCapEpi), we previously identified seven differentially methylated regions (DMRs) that discriminated good from non-responders (prophylactic response phenotype defined using the "Alda" scale). This study is a proof of transferability from bench to bedside of this epigenetic signature. For this purpose, we used Methylation Specific High-Resolution Melting (MS-HRM), a PCR based method that can be implemented in any medical laboratory at low cost and with minimal equipment. In 23 individuals with BD, MS-HRM measures of three out of seven DMRs were technically feasible and consistencies between SeqCapEpi and MS-HRM-measures were moderate to high. In an extended sample of individuals with BD (n = 70), the three MS-HRM-measured DMRs mainly predicted nonresponse, with AUC between 0.70-0.80 according to different definitions of the phenotype (Alda- or machine-learning-based definitions). Classification tree analyses further suggested that the MS-HRM-measured DMRs correctly classified up to 84% of individuals as good or non-responders. This study suggested that epigenetic biomarkers, identified in a retrospective sample, accurately discriminate non-responders from responders to Li and may be transferrable to routine practice.

Global DNA methylation profile at LINE-1 repeats and promoter methylation of genes involved in DNA damage response and repair pathways in human peripheral blood mononuclear cells in response to γ-radiation.

DNA methylation is an epigenetic mechanism, which plays an important role in gene regulation. The present study evaluated DNA methylation profile of LINE1 repeats and promoter methylation of DNA damage response (DDR) and DNA repair (DR) genes (PARP1, ATM, BRCA1, MLH1, XPC, RAD23B, APC, TNFα, DNMT3A, MRE11A, MGMT, CDKN2A, MTHFR) in human peripheral blood mononuclear cells (PBMCs) of healthy donors in response to γ-radiation. Methylation level was correlated with gene expression profile of selected DDR and DR genes (APC, MLH1, PARP1, MRE11A, TNFα, MGMT) to understand their role in gene regulation. Blood samples were collected from 15 random healthy donors, PBMCs were isolated, exposed to 0.1 Gy (low) and 2.0 Gy (high) doses of γ-radiation and proliferated for 48 h and 72 h. Genomic DNA and total RNA were isolated from irradiated PBMCs along with un-irradiated control. Methylation profile was determined from bisulphite converted DNA and amplified by methylation sensitive high resolution melting (MS-HRM) method. Total RNA was converted to cDNA and relative expression was analysed using real time quantitative-PCR. Our results revealed that at 0.1 Gy, MRE11A and TNFα showed significant (P < 0.05) increase in methylation at 72 h. At 2.0 Gy, significant increase (P < 0.05) in methylation profile was observed at LINE1, MRE11A, PARP1, BRCA1, DNMT3A and RAD23B at 48 h and 72 h. PARP1 showed significant positive correlation of methylation status with gene expression. In conclusion, low and high doses of γ-radiation have significant influence on DNA methylation status of LINE1, DDR and DR genes suggesting their potential role as epigenetic signatures in human PBMCs, which can be further explored in human populations.

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.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSION: Methylation of the PKD1 promoter in ADPKD patients was inversely correlated with PKD1 expression.

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

INTRODUCTION: Local 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. AIMS: This 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. METHODS: Forty-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. RESULTS: DNA 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. CONCLUSIONS: Our 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.

Epigenetic regulation of folate receptor-α (FOLR1) in human placenta of preterm newborns.

INTRODUCTION: Folates are essential nutrients for fetal development and pregnancy outcomes; they are transported to the fetus during gestation through specific folate transporters located in the placenta. In preterm newborns, we previously showed a lower placental mRNA expression of FOLR1 along with higher folate and lower vitamin B12 cord blood levels. Thereby we aimed to explore FOLR1 methylation in placentas of preterm newborns and hypothesized an increased FOLR1 methylation associated with cord blood folates and vitamin B12 concentrations. METHODS: FOLR1 methylation and mRNA were determined by methylation sensitive - high resolution melting (MS-HRM) and by real-time PCR respectively, in two placental sides of placental tissues: maternal (basal, BP) and fetal plates (chorionic, CP) of moderate preterm infants (32-36 gestational age) and term birth (37-41 gestational weeks). Folates and vitamin B12 were determined by electrochemiluminescence in umbilical cord blood samples from term and preterm newborns. RESULTS: We found that in preterm newborns, FOLR1 mRNA was lower in both plates of placenta compared with term newborns (p < 0,05) and was negatively associated with methylation of FOLR1 in CP. Preterm newborns presented higher folate and lower vitB12 concentrations in cord blood which correlated with increased placental FOLR1 methylation. DISCUSSION: In preterm newborns, placental FOLR1 expression is regulated by epigenetic mechanisms and presumably by maternal concentrations of folate and vitamin B12.

Assessment of PDGFRß promoter methylation in canine osteosarcoma using methylation-sensitive high-resolution melting analysis.

Platelet-derived growth factor signalling pathways play a fundamental role in inducing and sustaining the proliferative and prosurvival stimuli in canine osteosarcomas (cOSAs). The increased expression of platelet-derived growth factor receptors (PDGFRs) α and ß, and their cognate ligands, were almost invariably observed in cOSAs and OSA-derived cell lines. In particular, overexpression of PDGFRß-mediated signalling pathways was found in both the tumour microenvironment, where it drives stromal cell recruitment, and in neoangiogenesis, such as in tumour cells where it triggers aberrant proliferation, migration and local invasion. The majority of the pathological consequences of PDGFRß signalling are because of aberrant expression. In fact, epigenetic dysregulation of oncogenes throughout demethylation of their promoter has emerged as a pivotal mechanism driving oncogenesis. The aim of this study was to assess the methylation status of the PDGFRß promoter and to clarify its role in modulating the expression of the tyrosine kinase receptor in canine osteosarcoma. The CpG island of the PDGFRß promoter was identified using a mixed in silico and experimental approach, and a method based upon the methylation-sensitive high-resolution melting assay for quantitatively and precisely assessing the methylation status of the promoter was then set up. The method herein described was then exploited to assess the methylation status of the promoter in a case series of cOSAa. COSAs consistently but variably expressed PDGFRß. However, the promoter was almost completely demethylated, and its methylation status did not correlate with the expression levels. This finding supported the hypothesis that post-transcriptional regulatory mechanisms may act in cOSAs.

Do We Use Methylation of NFATC1 and FOS Genes As a Biomarker for Postmenopausal Osteoporosis?

Genetic and epigenetic factors have an important role during the development of osteoporosis. Receptor activator of nuclear factor-κ B (NF-κB) (RANK)/receptor activator of NF-κB ligand (RANKL) pathway is important for the bone remodeling, and NFATC1 and FOS are the downtargets of this pathway. Here, we report methylation status of NFATC1 and FOS genes in post- and premenopausal women. In this study, 30 premenopausal and 35 postmenopausal women were included. Methylation sensitive-high resolution melting (MS-HRM) analysis was used for identification of NFATC1 and FOS genes methylation. The NFATC1 gene was methylated in 11 of the 35 postmenopausal women, and the FOS gene was methylated in six of the postmenopausal women (p >0.005). Here, we found statistically significant association between unmethylation of the NFATC1 gene and postmenopausal status. This result explains the epigenetic regulation of osteoclasts during the menopausal transition, and for the first time, our results can be used for epigenetic explanation of postmenopausal osteoporosis in the literature. However, the limited number of studies in this field makes our results crucial. Our results showed great value of epigenetic profiles of postmenopausal women.

DNA Methylation Validation Methods: a Coherent Review with Practical Comparison.

Here, we present a practical overview of four commonly used validation methods for DNA methylation assessment: methylation specific restriction endonucleases (MSRE) analysis, pyrosequencing, methylation specific high-resolution DNA melting (MS-HRM) and quantitative methylation specific polymerase chain reaction (qMSP). Using these methods, we measured DNA methylation levels of three loci in human genome among which one was highly methylated, one intermediately methylated and one unmethylated. We compared the methods in terms of primer design demands, methods' feasibility, accuracy, time and money consumption, and usability for clinical diagnostics. Pyrosequencing and MS-HRM proved to be the most convenient methods. Using pyrosequencing, it is possible to analyze every CpG in a chosen region. The price of the instrument may represent the main limitation of this methodology. MS-HRM is a simple PCR-based method. The measurement was quick, cheap and very accurate. MSRE analysis is based on a methylation specific digestion of DNA. It does not require a bisulfite conversion of DNA as the other methods. MSRE analysis was very easy to perform, however, it was not suitable for intermediately methylated regions and it was also quite expensive. qMSP is a qPCR-based method that uses primers designed specifically for methylated and unmethylated alleles of a chosen region. This was the least accurate method and also the primer design and optimization of PCR conditions were highly demanding.

Different methylation levels in the KLF4, ATF3 and DLEC1 genes in the myometrium and in corpus uteri mesenchymal tumours as assessed by MS-HRM.

Mesenchymal tumours of the corpus uteri comprise common benign lesions - leiomyomas and very rare malignant variants - sarcomas. It can be difficult to distinguish between the particular types of mesenchymal tumours pre-surgically. Primarily, leiomyomas and the very aggressive leiomyosarcomas can be easily misdiagnosed when using only imaging devices. Therefore, a reliable non-invasive marker for these tumour types would provide greater certitude for patients that the lesion remains benign. Our collection comprises 76 native leiomyomas, an equal number of healthy myometrium samples and 49 FFPE samples of various types of sarcomas. The methylation level was assessed by MS-HRM method and we observed differences in the methylation level between healthy, benign and (semi)malignant tissues in the KLF4 and DLEC1 genes. The mean methylation levels of leiomyomas compared to myometrium and leiomyosarcomas were 70.7% vs. 6.5% vs. 39.6 % (KLF4) and 66.1% vs. 14.08% vs. 37.5% (DLEC1). The ATF3 gene was differentially methylated in leiomyomatous and myometrial tissues with 98.1% compared to 76.6%. The AUC values of the predictive logistic regression model for discrimination between leiomyomas and leiomyosarcomas based on methylation levels were 0.7829 (KLF4) and 0.7719 (DLEC1). Finally, our results suggest that there should be distinct models for the methylation events in benign leiomyomas and sarcomas, and that the KLF4 and DLEC1 genes can be considered potential methylation biomarkers for uterine leiomyomas.

Circulating ctDNA methylation quantification of two DNA methyl transferases in papillary thyroid carcinoma.

Papillary thyroid cancer (PTC) is the most common type of cancer among thyroid malignancies. Tumor-related methylation of circulating tumor DNA (ctDNA) in plasma could represent tumor specific alterations can be considered as good biomarkers in circulating tumor cells. In this study, we studied the methylation status of seven promoter regions of two DNA methyl Transferases (MGMT and DNMT1) genes as the methylated ctDNA in plasma and tissue samples of patients with PTC and goiter patients as noncancerous controls. METHODS: Both ctDNA and tissue genomic DNA of 57 PTC and 45 Goiter samples were isolated. After bisulfite modification, the methylation status was studied by Methylation-Sensitive High Resolution Melting (MS-HRM) assay technique. Four promoter regions of O6-methylguanine-DNA methyltransferase (MGMT) and three promoter regions of DNA methyltransferase 1 (DNMT1) were assessed. RESULTS: From seven candidate promoter regions of two methyltrasferase coding genes, the methylation status of ctDNA within MGMT (a), MGMT (c), MGMT (d), and DNMT1 (b) were meaningfully different between PTC cases and controls. However, the most significant differences were seen in circulating ctDNA MGMT (c) which was hypermethylated in 25 (43.9 %) of patients with PTC vs 2 (4. 4 %) of goiter samples. Between two selected DNA methyl transferase, the methylation of MGMT as the maintenance methyltransferase was significantly higher in PTC cases than goiter controls (P-value < .001). The resulting areas under the receiver operating characteristic (ROC) curve were 0.78 for MGMT (d) for PTC versus goiter samples that can represent the overall ability of MGMT (d) methylation status to discriminate between PTC and goiter patients. CONCLUSION: Among seven candidate regions of ctDNA the MGMT (c) and MGMT (d) showed higher sensitivity and specificity for PTC as a suitable candidates as biomarkers of PTC.

A rapid and accurate methylation-sensitive high-resolution melting analysis assay for the diagnosis of Prader Willi and Angelman patients.

BACKGROUND: Prader Willi (PWS) and Angelman (AS) syndromes are rare genetic disorders characterized by deletions, uniparental disomy, and imprinting defects at chromosome 15. The loss of function of specific genes caused by genetic alterations in paternal allele causes PWS while the absence in maternal allele results AS. The laboratory diagnosis of PWS and AS is complex and demands molecular biology and cytogenetics techniques to identify the genetic mechanism related to the development of the disease. The DNA methylation analysis in chromosome 15 at the SNURF-SNRPN locus through MS-PCR confirms the diagnosis and distinguishes between PWS and AS. Our study aimed to establish the MS-PCR technique associated with High-Resolution Melting (MS-HRM) in PWS and AS diagnostic with a single pair of primers. METHODS: We collected blood samples from 43 suspected patients to a cytogenetic and methylation analysis. The extracted DNA was treated with bisulfite to perform comparative methylation analysis. RESULTS: MS-HRM and MS-PCR agreed in 100% of cases, identifying 19(44%) PWS, 3(7%) AS, and 21(49%) Normal. FISH analysis detected four cases of PWS caused by deletions in chromosome 15. CONCLUSION: The MS-HRM showed good performance with a unique pair of primers, dispensing electrophoresis gel analysis, offering a quick and reproducible diagnostic.

Serum cell-free DNA methylation of OPCML and HOXD9 as a biomarker that may aid in differential diagnosis between cholangiocarcinoma and other biliary diseases.

BACKGROUND: Cholangiocarcinoma (CCA) is a fatal cancer of the bile duct epithelial cell lining. The misdiagnosis of CCA and other biliary diseases may occur due to the similarity of clinical manifestations and blood tests resulting in inappropriate or delayed treatment. Thus, an accurate and less-invasive method for differentiating CCA from other biliary diseases is inevitable. METHODS: We quantified methylation of OPCML, HOXA9, and HOXD9 in serum cell-free DNA (cfDNA) of CCA patients and other biliary diseases using methylation-sensitive high-resolution melting (MS-HRM). Their potency as differential biomarkers between CCA and other biliary diseases was also evaluated by using receiver operating characteristic (ROC) curves. RESULTS: The significant difference of methylation levels of OPCML and HOXD9 was observed in serum cfDNA of CCA compared to other biliary diseases. Assessment of serum cfDNA methylation of OPCML and HOXD9 as differential biomarkers of CCA and other biliary diseases showed the area under curve (AUC) of 0.850 (0.759-0.941) for OPCML which sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were 80.00%, 90.00%, 88.88%, 81.81%, and 85.00%, respectively. The AUC of HOXD9 was 0.789 (0.686-0.892) with sensitivity, specificity, PPV, NPV, and accuracy of 67.50%, 90.00%, 87.09%, 73.46%, and 78.75%, respectively. The combined marker between OPCML and HOXD9 showed sensitivity, specificity, PPV, and NPV of 62.50%, 100%, 100%, and 72.72%, respectively, which may be helpful to prevent a misdiagnosis between CCA and other biliary diseases. CONCLUSIONS: Our findings suggest the application of serum cfDNA methylation of OPCML and HOXD9 for differential diagnosis of CCA and other biliary diseases due to its less invasiveness and clinically practical method which may benefit the patients by preventing the misdiagnosis of CCA and avoiding unnecessary surgical intervention.

Improved locus-specific unmethylated controls for MS-HRM analysis derived from 5-aza-2-deoxycytidine-treated DNA.

We report two restriction enzyme-based approaches for generating clean locus-specific unmethylated controls for methylation-sensitive high-resolution melting (MS-HRM) analyses. These unmethylated standards are derived from DNA treated with the demethylating agent 5-aza-2-deoxycytidine (5-Aza-dc). By using them, we overcome a limitation of 5-Aza-dc treatment - incomplete demethylation at various genomic regions. When 5-Aza-dc-treated DNA is used directly as unmethylated MS-HRM standard, partially demethylated DNA can give false methylation results. MS-HRM assay differentiates between methylated and unmethylated bisulfite-treated DNA based on the different melting profiles of PCR products amplified from them. To estimate test sample methylation levels, test sample melting profiles are compared to those of methylation standards. With our pure unmethylated controls, adequate standards of known methylation levels can be prepared for single-locus MS-HRM.

Chronic lymphocytic leukemia patients with heterogeneously or fully methylated LPL promotor display longer time to treatment.

AIM: We investigated whether DNA methylation regulates expression of LPL and PI3K complex genes in chronic lymphocytic leukemia (CLL) and evaluated the prognostic significance of LPL promoter methylation in CLL patients. Patients & methods: Methylation of LPL promoter was assessed in 112 patients using methylation-sensitive high-resolution melting (MS-HRM). RESULTS: Patients with a fully or heterogeneously methylated LPL promoter had significantly longer median time to treatment (p < 0.001) and 75% lower (hazard ratio: 0.25; 95% CI: 0.15-0.42; p < 0.001) risk of requirement for treatment as opposed to patients with nonmethylated promoter. Multivariate modeling confirmed independent prognostic value of these findings. CONCLUSION: Chronic lymphocytic leukemia patients with a fully or heterogeneously methylated LPL gene promoter display indolent disease course and acquisition of heterogeneous methylation of LPL promoter is insufficient to induce gene expression.

DNA Methylation Profiling of Breast Cancer Cell Lines along the Epithelial Mesenchymal Spectrum-Implications for the Choice of Circulating Tumour DNA Methylation Markers.

(1) Background: Epithelial⁻mesenchymal plasticity (EMP) is a dynamic process whereby epithelial carcinoma cells reversibly acquire morphological and invasive characteristics typical of mesenchymal cells. Identifying the methylation differences between epithelial and mesenchymal states may assist in the identification of optimal DNA methylation biomarkers for the blood-based monitoring of cancer. (2) Methods: Methylation-sensitive high-resolution melting (MS-HRM) was used to examine the promoter methylation status of a panel of established and novel markers in a range of breast cancer cell lines spanning the epithelial⁻mesenchymal spectrum. Pyrosequencing was used to validate the MS-HRM results. (3) Results: VIM, DKK3, and CRABP1 were methylated in the majority of epithelial breast cancer cell lines, while methylation of GRHL2, MIR200C, and CDH1 was restricted to mesenchymal cell lines. Some markers that have been used to assess minimal residual disease such as AKR1B1 and APC methylation proved to be specific for epithelial breast cell lines. However, RASSF1A, RARß, TWIST1, and SFRP2 methylation was seen in both epithelial and mesenchymal cell lines, supporting their suitability for a multimarker panel. (4) Conclusions: Profiling DNA methylation shows a distinction between epithelial and mesenchymal phenotypes. Understanding how DNA methylation varies between epithelial and mesenchymal phenotypes may lead to more rational selection of methylation-based biomarkers for circulating tumour DNA analysis.