Frequency Of Promoter Methylation Research Articles

Dysregulation of DNA epigenetic modulators during prostate carcinogenesis in an eastern Indian patient population: Prognostic implications

The role of epigenetic alteration in prostate cancer pathogenesis was reported. We aimed to analyze dysregulation of DNA methylase (DNA methyl transferase/DNMT) and demethylase (ten eleven translocase/TET) and the associated interplay between them during prostate tumorigenesis. Promoter methylation and RNA/protein expression of selected DNMT and TETs were analysed in normal prostate, benign prostatic hyperplasia (BPH), and prostate cancer (PCa). Genomic 5-hydroxymethylcytosine (5hmC) level was detected and correlated with DNMT and TET proteins. Clinicopathological association of molecular data was done. Our data revealed a very low frequency of promoter methylation for DNMT1 (5-3% and high frequency for TET1 (22–38%), TET2 (68–90 %), and TET3 (43-32 %) in BPH and PCa. The promoter methylation of DNMT1 (p = 0.019) showed a significantly decreasing trend, while that of TET1 (p = 0.0005) and TET2 (p < 0.0001) showed an increasing trend from normal prostate to BPH to PCa, indicating their epigenetic dysregulation during prostate tumorigenesis. RNA/protein overexpression of DNMT1 and reduced expression of TET1 and TET2 in PCa compared to BPH were associated with the promoter methylation status of genes. The 5hmC level was significantly lower in PCa than in BPH and correlated negatively with DNMT1 but positively with TET1 and TET2 proteins, suggesting dysregulation of DNA methylase and de-methylase activities during prostate tumorigenesis. Lastly, tumors having methylated TET1 and TET2 promoters showed advanced clinicopathological features (a higher PSA level/Gleason score) and increased risk of bone metastasis. In conclusion, DNMT1 upregulation and epigenetic silencing of TET1 and TET2 was seen during PCa development. TET1 and TET2 promoter methylation has prognostic importance.

Role of MLH1 and MSH2 deficiency in the development of tumorigenesis and chemo-tolerance of cervical Carcinoma: Clinical implications

Cervical cancer (CACX) is one of the top causes of cancer death in women globally. The involvement of several cellular pathways in carcinogenesis is still poorly understood. Here, we focused to evaluate the contributory role of Mismatch Repair (MMR) pathway genes-MLH1 and MSH2 in CACX and their association with chemo-tolerance of the disease. For this purpose, molecular profiles (expression/promoter methylation/deletion) of the genes were analysed in both normal cervical epithelium and tumour tissue, also validated in in-silico dataset as well. Later on, prognostic importance of the genes was identified through analysis of their methylation/expression status in plasma DNA of circulating tumour cells (CTCs) and cisplatin-tolerant CACX cell lines respectively. It was found that the expression profile of MLH1 and MSH2 genes was considerably reduced from undifferentiated basal-parabasal layers of normal cervical epithelium towards progression of the disease. Further analysis showed that frequent deletion [34–48%] and promoter methylation events [28–46%] of the genes were the plausible reasons for their reduced expression during tumorigenesis. Incidentally, the prevalence of MLH1 [32%] and MSH2 [27%] promoter methylation found in CTCs of plasma of the clinically advanced CACX patients implicated their prognostic importance of the disease. In addition, the patients having high alterations of those genes resulted in poor patient outcomes even after the therapy. In in-depth analysis of this result in cisplatin-tolerant CACX cell lines, we discovered that increased promoter methylation frequency of those genes at higher concentrations of cisplatin and gradual accumulation of the cells in the G2/M phase of the cell cycle were the rational causes for their reduced expression and MMR deficiency in the system. Hence, it is possible to conclude that the gradual down-regulation of MLH1 and MSH2 proteins may be a key event for MMR pathway inactivation in CACX. This might also be associated with chemo-tolerance and overall poor survival among the patients.

IDH-mutant diffuse gliomas: tips and tricks in the era of genomic tumor classification.

According to the fifth edition of the World Health Organization (WHO) Classification, diffuse gliomas typically occurring in adults are classified as oligodendroglioma IDH-mutant and 1p/19q codeleted, astrocytoma IDH-mutant, and glioblastoma IDH-wildtype. Among these, the former has the most favorable clinical course, whereas the latter has the worst prognosis. In IDH-mutant gliomas, the IDH1 p. R132H is the most frequent IDH mutation. Other mutations in IDH1 are rare and predominantly found in astrocytomas, whereas IDH2 mutations are mostly observed in oligodendrogliomas. Astrocytomas IDH-mutant display frequent immunohistochemical loss of ATRX, which is mutually exclusive with 1p/19q codeletion. They are graded based on histopathological features and the presence of CDKN2A/B homozygous deletion, whereas the criteria for grading oligodendrogliomas are less defined. DNA methylation profiling has recently shown three additional distinct tumor types among diffuse IDH-mutant gliomas: infratentorial astrocytoma IDH mutant; primary mismatch repair deficient IDH-mutant astrocytoma (PMRDIA); and oligosarcoma. Infratentorial astrocytoma IDH-mutant is enriched in IDH1 or IDH2 mutations that differ from the IDH1 p.R132H mutation and are detectable only by gene sequencing, displays less frequent ATRX loss and MGMT promoter methylation than supratentorial IDH-mutant astrocytomas, and may additionally harbor the H3 K27M mutation, which is typically found in H3 K27-altered diffuse midline glioma. PMRDIA occurs in the context of primary mismatch repair deficiency, is characterized by frequent MSH6 mutations, hypermutation, low frequency of MGMT promoter methylation, and poor clinical outcomes. Finally, oligosarcoma is a tumor featuring oligodendroglial and sarcomatous areas, and is characterized by worse outcome and frequent 1p/19q copy number loss of heterozygosity.

High frequency of MLH1 promoter methylation mediated by gender and age in colorectal tumors from Mexican patients.

Several genes determine the development of colorectal cancer (CRC), such as MLH1, which encodes a protein that participates in DNA repair. MLH1 hypermethylation has been associated with gene silencing. To analyze the methylation of five regions of MLH1 CpG island in colorectal tumors from Mexican patients. One hundred and one tumor tissue samples were obtained from Mexican patients with CRC who provided informed consent. DNA was subjected to bisulfite conversion. Methylation of all five regions of the CpG island was evaluated using methylation-specific PCR. The frequency of methylation in Mexican patients with CRC was 25%. Regions A and B methylation was the main observed pattern (60%). Female patients showed a higher frequency of methylation (71%; OR 3.085; CI: 1.85-8.03; p = 0.02), and out of total methylated samples, 80% corresponded to individuals older than 45 years (p < 0.05). We calculated a methylation frequency for the MLH1 gene of 25% in Mexican patients with CRC, with this being the first report for this population. Female patients and patients older than 45 years showed a higher frequency of methylation.

High nuclear expression of DNMT1 in correlation with inactivation of TET1 portray worst prognosis among the cervical carcinoma patients: clinical implications.

In this study, we aimed to understand the interplay of the epigenetic modifier genes DNMT1 and TET1 along with HPV infection in the cervical epithelium and how it changes during tumorigenesis. For this purpose, initially the bioinformatical analysis (methylation and expression profile) of DNMT1 and TET1 was analyzed in the TCGA dataset. Next genetic (deletion) and epigenetic profiling (promoter methylation) of DNMT1 and TET1 were done in our sample pool and also validated in CACX cell lines as well. The results were further correlated with different clinicopathological parameters. Our data revealed that HPV infection in basal/parabasal layers of cervical epithelium actually disrupts the epigenetic homeostasis of DNMT1 and TET1 proteins which ultimately leads to the high expression of DNMT1 along with further reduction in TET1 protein during the development of carcinoma. Further, in-depth look into the results revealed that comparatively low methylation frequency of DNMT1 coupled with high promoter methylation and deletion frequency [22-46%] of TET1 were the plausible reasons of their antagonistic expression profile during the progression of the disease. Interestingly, the prevalence of DNMT1 [9.1%] and TET1 promoter methylation [22.7%] found in both the plasma DNA of the respective CACX patients implicated its diagnostic importance in this study. Lastly, molecular alteration of TET1 alone or in combination with DNMT1 showed the worst overall survival among the patients. Hence, it may be concluded that an inverse molecular profile of DNMT1 and TET1 genes seen in the proliferative basal-parabasal layers of the cervical epithelium was aggravated during the development of CACX along with genetic and epigenetic changes due to HPV infection.

Novel Application of 7T MRI in CSF1R-Related Leukoencephalopathy.

<h3>Aims</h3> To evaluate how the genetic and epigenetic profile of human papillomavirus 16 (HPV16) changes from asymptomatic cervical infections to cervical cancer (CaCx) development. <h3>Methods</h3> HPV16 physical status, methylation of its early–late promoters and its upstream enhancer sequences were analysed in samples from asymptomatic cervical infections (n=89), pre-neoplastic lesions (low and high grade squamous intraepithelial lesions LSIL/HSIL, n=28) and primary CaCx (n=98). <h3>Results</h3> In asymptomatic infection (65%, 58/89) and LSIL/HSIL (57%, 16/28) samples, the episomal form of HPV16 was predominant whereas integration of HPV16 was significantly (p=0.01) higher in CaCx (59%, 57/98). The integrated viral form was also present in asymptomatic (27%, 24/89) and LSIL/HSIL (25%, 7/28) samples. The methylation of the enhancer region was comparable (29–34%) among asymptomatic, LSIL/HSIL and CaCx samples. The episomal form exhibited relatively higher methylation of the early promoter (52%) than that of the late promoter (40%) in asymptomatic infection but the integrated form in asymptomatic carriers showed the opposite methylation pattern (early promoter (42%) vs late-promoter (54%)). A similar pattern was observed in LSIL/HSIL samples, with comparable frequencies (44%) of early and late promoter methylation of the episomal form. However, irrespective of HPV16 physical status, higher methylation of late promoter than that of early promoter was observed in CaCx samples. An inverse correlation was observed between HPV16 integration and overall methylation of the early promoter–enhancer region in CaCx (p=0.05), LSIL/HSIL (p=0.09) and asymptomatic samples (p=0.09). <h3>Conclusions</h3> Our study indicates that integration of HPV16 along with changes in methylation pattern of early and late promoters is essential for neoplastic transformation of asymptomatic cervical infections.

Hypermethylation of thymosin β4 predicts a poor prognosis for patients with acute-on-chronic hepatitis B liver failure

BackgroundIt has been demonstrated that thymosin β4 (Tβ4) could inflect the severity of acute-on-chronic hepatitis B liver failure (ACHBLF), but the relationship between its methylation status and the prognosis of liver failure is not clear. This study aimed to determine Tβ4 promoter methylation status in patients with ACHBLF and to evaluate its prognostic value. MethodsThe study recruited 115 patients with ACHBLF, 80 with acute-on-chronic hepatitis B pre-liver failure (pre-ACHBLF), and 86 with chronic hepatitis B (CHB). In addition, there were 36 healthy controls (HCs) from the Department of Hepatology, Qilu Hospital of Shandong University. The 115 patients with ACHBLF were divided into three subgroups: 33 with early stage ACHBLF (E-ACHBLF), 42 with mid-stage ACHBLF (M-ACHBLF), and 40 with advanced stage ACHBLF (A-ACHBLF). Tβ4 promoter methylation status in peripheral blood mononuclear cells (PBMCs) was measured by methylation-specific polymerase chain reaction, and mRNA was detected by quantitative real-time polymerase chain reaction. ResultsMethylation frequency of Tβ4 was significantly higher in patients with ACHBLF than in those with pre-ACHBLF, CHB or HCs. However, expression of Tβ4 mRNA showed the opposite trend. In patients with ACHBLF, Tβ4 promoter methylation status correlated negatively with mRNA levels. The 3-month mortality of ACHBLF in the methylated group was significantly higher than that in the unmethylated group. Also, Tβ4 promoter methylation frequency was lower in survivors than in non-survivors. When used to predict the 1-, 2-, and 3-month incidence of ACHBLF, Tβ4 methylation status was better than the model for end-stage liver disease (MELD) score. The predictive value of Tβ4 methylation was higher than that of MELD score for the mortality of patients with E-ACHBLF and M-ACHBLF, but not for A-ACHBLF. ConclusionsTβ4 methylation might be an important early marker for predicting disease incidence and prognosis in patients with ACHBLF.

Promoter methylation analysis of DKK2 may be a potential biomarker for early detection of cervical cancer.

Dickkopf 2 (DKK2) plays an important role in multiple cancers. Its potential value in the clinical diagnosis of cervical cancer has remained unclear. To investigate the expression and promoter methylation levels of DKK2 in cervical cancer and their clinicopathological associations. We used the Gene Expression Omnibus, Oncomine, Cancer Genome Atlas, and University of ALabama at Birmingham CANcer data analysis databases, reverse transcription-PCR, and methylation-specific PCR analysis to predict and examine the expression of DKK2 mRNA and DKK2 methylation levels in cell lines and cervical cancer tissues from 79 patients with cervical cancer and 63 with cervical precancerous lesions including 25 with low-grade squamous intraepithelial lesions (LSIL) and 38 patients with high-grade squamous intraepithelial lesions (HSIL). DKK2 mRNA expression was downregulated in all cancer cell lines and cervical cancer tissues, whereas hypermethylation of DKK2 was higher in cervical cancer tissue samples. DKK2 methylation in cervical cancer was significantly higher than that in HSIL (χ2 = 8.346, P = 0.004), whereas DKK2 methylation in HSIL was significantly higher than that in normal cervical samples (χ2 = 7.934, P = 0.005) and in LSIL samples (χ2 = 4.375, P = 0.037). DKK2 silencing caused by its promoter hypermethylation was confirmed by treatment with the methyltransferase inhibitor 5-Aza-dC in cell lines. Patients with lymph node metastasis exhibited increased promoter methylation frequency (χ2 = 5.239, P = 0.022) and low DKK2 mRNA expression (χ2 = 3.958, P = 0.047) compared with patients with no lymph node metastasis. Patients with high-risk human papillomavirus infection exhibited increased promoter methylation frequency (χ2 = 6.279, P = 0.015). DKK2 epigenetic changes of DKK2 may play a key role in the development of cervical cancer, suggesting that DKK2 hypermethylation could be used as a triage test for screening, early diagnosis, or risk prediction of cervical cancer.

EPEN-27. Epigenetic dissection of spinal ependymomas (SP-EPN) separates tumors with and withoutNF2 mutation

Ependymomas encompass multiple, clinically relevant tumor types based on localization, genetic alterations, and epigenetic and transcriptomic profiles. Tumors belonging to the methylation class of spinal ependymoma (SP-EPN) represent the most common intramedullary neoplasms in children and adults. However, molecular data of SP-EPN are scarce, and clear treatment recommendations are lacking. The only known recurrent genetic events in SP-EPN are loss of chromosome 22q and NF2 mutations. Yet, it remains unclear whether SP-EPN with germline or sporadic NF2 mutations or with NF2 wild type status differ clinically or molecularly. To provide a comprehensive molecular profile of SP-EPN, we integrated epigenetic, genomic, transcriptomic, and histological analyses of up to 237 cases. Clustering of methylation data revealed two distinct molecular SP-EPN subtypes. The distribution of NF2 mutated cases differed significantly across these subtypes (p <0.0001): The vast majority of tumors harboring either a previously known NF2 germline mutation or a sporadic mutation were assigned to subtypes A, whereas subtype B tumors mainly contained NF2 wild type sequences. In addition, subtype A tumors showed a lower frequency of MGMT promoter methylation (p= 0.018) and contained almost all pediatric patients of the cohort. Whole-exome sequencing (30 cases) identified numerous mutations in NF2 wild type and mutated tumors. Mutated genes in NF2 wild type tumors were enriched for genes associated with cell cycle and cytoskeleton. RNA sequencing revealed two distinct transcriptional groups with upregulation of proliferative genes in one group and upregulation of cilial genes in the other group. The molecular subtypes displayed subtle, but significant differences in the appearance of histopathological characteristics, such as surfaces, inflammation, and hyalinized vessels. Investigation of clinical parameters is ongoing and will complete the picture of SP-EPN heterogeneity as an important basis for future clinical decision-making.

Are anaplastic lymphoma kinase (ALK) and O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation driver biomarkers of pulmonary neuroendocrine tumors (NETs) and carcinomas (NECs)?

Background: Novel targets in neuroendocrine tumors (NETs) and neuroendocrine carcinomas (NECs) are needed to improve outcome. The presence of O6-Methylguanine-DNA methyltransferase (MGMT) promoter methylation in NETs and NECs may act as a predictive marker for response on treatment with temozolomide. As anaplastic lymphoma kinase (ALK) plays an important role in the nervous system we hypothesized that ALK rearrangement can act as a biomarker in patients with NETs and NECs.Materials and Methods: We performed a retrospective analysis to establish the frequency of MGMT promoter methylation and ALK expression in tissue samples of patients with NETs and NECs.Results: 21% (14/67) of patients tested positive for MGMT promoter methylation. MGMT promoter methylation was present in 33% (3/9) patients with typical carcinoid, in 22% (2/9) patients with atypical carcinoid, in 22% (8/37) patients with small cell lung cancer and in 8% (1/12) patient with large cell neuroendocrine carcinoma. ALK- expression was present in 14% (10 of 70 patients). In all of these patients, no ALK-rearrangement nor ALK-mutation was revealed.Conclusions: Routine testing of NET and NEC samples for an ALK rearrangement is not recommended as ALK-expression is not associated with an ALK-rearrangement. Routine testing of NET and NEC samples for MGMT will detect a promoter hypermethylation in a sizable minority of patients who are eligible for a targeted treatment with temozolomide.

Significance of promoter methylation of multiple tumor suppressor genes in hepatocellular carcinoma

BackgroundMethylation of the promoter at CpG islands is a mechanism of silencing tumor suppressor genes and therefore enhances cancer progression. The study aimed to examine promoter methylation frequencies of five tumor suppressor genes in hepatocellular carcinoma and their implication on the first-year outcome of surgical resection of the tumor. Fifty specimens of hepatocellular carcinoma and the adjacent non-tumorous liver tissue were collected from the surgically resected hepatic tumor. The status of promoter methylation of tumor suppressor genes RASSF1A, CHFR, MGMT, GSTP1, and hMLH1 was investigated using methylation-specific polymerase chain reaction.ResultsThe frequency of promoter methylation of these tumor suppressors genes (TSG) genes in hepatocellular carcinoma was significantly higher than non-tumorous tissue all, P < 0.05, with a methylation rate of 80% in RASSF1A, 70% in CHFR, 46% in GSTP1, 56% in MGMT, and 10% in hMLH1. Methylation of RASSF1A, CHFR, and MGMT promoter genes was significantly associated with decreased first-year postoperative survival and increased recurrence of hepatocellular carcinoma, P < 0.05.ConclusionMethylated RASSF1A, CHRF, and MGMT promoters indicated poor prognosis among patients with hepatocellular carcinoma and may serve as potential prognostic indicators in patients with hepatocellular carcinoma.

Hypomethylation of thymosin β4 promoter is associated with glucocorticoid therapy in patients with acute-on-chronic hepatitis B-induced liver failure.

We aimed to determine whether the methylation status of thymosin β4 (Tβ4) promoter reflects the severity of acute-on-chronic hepatitis B liver failure (ACHBLF) and whether glucocorticoids affect this status. Fifty-six patients with ACHBLF, 45 with chronic hepatitis B (CHB) and 32 healthy controls (HCs), were retrospectively enrolled. Methylation-specific PCR and real-time PCR were used to detect Tβ4 methylation frequency and mRNA level. The expression of Tβ4 was measured before and after glucocorticoid treatment in patients with ACHBLF. Clinical and laboratory parameters were obtained. Tβ4 mRNA expression of patients with ACHBLF was lower than in patients with CHB or HCs, but the methylation frequency was higher. Tβ4 promoter methylation frequency was correlated with serum total bilirubin, prothrombin activity and model for end-stage liver disease score. Moreover, Tβ4 promoter methylation frequency decreased and demethylation occurred during glucocorticoid therapy. After glucocorticoid therapy, Tβ4 mRNA expression and liver function were better in patients with low levels of methylation than in those with higher levels. After 90 d, the survival of patients with low levels of methylation was significantly higher than those with high levels. Patients with ACHBLF who have low levels of Tβ4 methylation may show a more favorable response to glucocorticoid treatment.

Promoter methylation status of key genes and its implications in the pathogenesis of endometriosis, endometrioid carcinoma of ovary and endometrioid endometrial cancer.

Epigenetic processes play an important role in various physiological processes as well as in the pathogenesis of many diseases. The role of altered DNA methylation in the pathogenesis of endometriosis and associated ovarian and endometrial cancers has not been explored in detail. Therefore, this study aimed to determine the promoter methylation status of genes involved in key biological processes in the pathogenesis of these three gynecological diseases. Tissue samples of endometriosis, endometrioid carcinoma of the ovary, endometrioid endometrial cancer, and control endometrium (n = 10 each) were obtained. DNA was extracted and subjected to bisulfite conversion using commercially available kits. The methylation status of COX2, VEGF, HIF1A, TNF, MYC, and TP53 genes was checked by methylation-specific PCR. The mRNA levels of MYC and TP53 were determined using qRT-PCR in all tissue samples. The promoter methylation status of COX2, VEGF, HIF1A, and TNF genes was significantly reduced in all three diseased study subjects (P < 0.05), whereas no significant difference was observed in the promoter methylation frequency of MYC and TP53 genes. Transcriptional expression of the MYC gene was significantly increased in all diseased groups (P < 0.001) whereas, transcriptional expression of the TP53 gene was significantly reduced in endometriosis and endometrioid carcinoma of the ovary and significantly increased in endometrioid endometrial cancer subjects compared to control subjects (P < 0.001). The findings suggest that the promoter regions of pro-inflammatory and pro-angiogenic genes involved in the common molecular pathophysiology of these three disorders were significantly hypomethylated and could be the reason for their over-expression associated with them. This indicates the role of epigenetics in the pathogenesis of these three diseases.

PATH-34. MOLECULAR AND CLINICAL HETEROGENEITY WITHIN SPINAL EPENDYMOMAS

Abstract Ependymomas encompass multiple clinically relevant tumor types based on localization, genetic alterations, as well as epigenetic and transcriptomic profiles. Distinct global DNA methylation signatures serve as the most powerful diagnostic tool to distinguish these types. The methylation class of spinal ependymomas (SP-EPN) comprises mostly WHO°II tumors with slow progression and incomplete surgical resection rate. Molecular data of SP-EPN are scarce and clear treatment recommendations are lacking although these neoplasms represent the most common intramedullary tumors in children and adults. The only known recurrent genetic events in SP-EPN are the loss of chromosome 22q and mutations of the NF2 gene. However, data on the frequency of NF2 mutations range from 16 % to 71 % and originate from small series that lack epigenetic or transcriptomic characterization. Furthermore, it remains unclear whether SP-EPN with germline or sporadic NF2 mutation or with NF2 wild type status display clinical and other molecular differences. Finally, the underlying genomic and transcriptomic changes of SP-EPN without NF2 mutations are fully unclear. To provide a comprehensive molecular profile of SP-EPN, we integrated genomic and epigenetic analyses and clinical data of 170 cases. Unsupervised hierarchical clustering and t-SNE analyses of methylation data revealed three distinct molecular SP-EPN subtypes. Of the three subtypes, only subtype 1 and subtype 2 contained tumors with NF2 mutations, either as previously known germline mutations or as sporadic mutations without evidence for a syndromic disease (p&amp;lt; 0.0001). Besides the lack of NF2 mutations, subtype 3 tumors showed a higher frequency of MGMT promoter methylation (p= 0.0015) and occurred in significantly older patients compared to tumors of subtypes 1 and 2 (p= 0.0038). Further investigations such as whole-exome sequencing, copy number variation profiling, gene expression analysis, and histological evaluation are ongoing and will add to the picture of molecular and clinical heterogeneity within SP-EPN.

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

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

Mucosal melanomas of different anatomic sites share a common global DNA methylation profile with cutaneous melanoma but show location-dependent patterns of genetic and epigenetic alterations.

Cutaneous, ocular, and mucosal melanomas are histologically indistinguishable tumors that are driven by a different spectrum of genetic alterations. With current methods, identification of the site of origin of a melanoma metastasis is challenging. DNA methylation profiling has shown promise for the identification of the site of tumor origin in various settings. Here we explore the DNA methylation landscape of melanomas from different sites and analyze if different melanoma origins can be distinguished by their epigenetic profile. We performed DNA methylation analysis, next generation DNA panel sequencing, and copy number analysis of 82 non-cutaneous and 25 cutaneous melanoma samples. We further analyzed eight normal melanocyte cell culture preparations. DNA methylation analysis separated uveal melanomas from melanomas of other primary sites. Mucosal, conjunctival, and cutaneous melanomas shared a common global DNA methylation profile. Still, we observed location-dependent DNA methylation differences in cancer-related genes, such as low frequencies of RARB (7/63) and CDKN2A promoter methylation (6/63) in mucosal melanomas, or a high frequency of APC promoter methylation in conjunctival melanomas (6/9). Furthermore, all investigated melanomas of the paranasal sinus showed loss of PTEN expression (9/9), mainly caused by promoter methylation. This was less frequently seen in melanomas of other sites (24/98). Copy number analysis revealed recurrent amplifications in mucosal melanomas, including chromosomes 4q, 5p, 11q and 12q. Most melanomas of the oral cavity showed gains of chromosome 5p with TERT amplification (8/10), while 11q amplifications were enriched in melanomas of the nasal cavity (7/16). In summary, mucosal, conjunctival, and cutaneous melanomas show a surprisingly similar global DNA methylation profile and identification of the site of origin by DNA methylation testing is likely not feasible. Still, our study demonstrates tumor location-dependent differences of promoter methylation frequencies in specific cancer-related genes together with tumor site-specific enrichment for specific chromosomal changes and genetic mutations. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Epigenetic Alterations of DLL4 and Hes5 in Acute Lymphoblastic Leukemia (ALL)

Acute lymphoblastic leukemia (ALL) is a hematologic condition with more than a quarter of pediatric cancers. Aberrant promoter methylation of Notch pathway genes causes the deactivation of TSGs. The pathway is also considered a crucial factor in the pathogenesis of ALL due to its active involvement in B and T cell development. Hypermethylation of Notch pathway genes has been reported previously. In this study, the promoter methylation frequency of genes DLL4 and Hes5 of the Notch pathway were studied using methylation-specific PCR in 30 pediatric ALL blood samples against 10 healthy controls. The objective of the study was to find the subtype-specific diagnostic biomarker for ALL. Hypermethylation frequency of DLL4 in pre-B ALL and T-ALL samples was found to be 84.21% and 100%, respectively. Whereas, Hes5 showed 100% mixed methylation in both diseased and control samples. The results predicted the possible epigenetic changes of Notch pathway and the possible role of DLL4 as a diagnostic biomarker of ALL.

SCGB3A1 gene DNA methylation status is associated with breast cancer in Egyptian female patients

BackgroundIn recent years, hypermethylation of gene promoters has emerged as one of the fundamental mechanisms for the inactivation of tumor suppressor genes and has a potential role in the early detection of breast cancer. The present study is a case-control study aimed to quantify the methylation levels in the promoters of secretoglobin 3A1 (SCGB3A1), and ataxia-telangiectasia mutated (ATM) genes and evaluate their relation to clinicopathological features of the tumor in a cohort of Egyptian female patients with breast cancer.MethodsGenomic deoxyribonucleic acid (DNA) was extracted from 100 tissue samples, 50 breast cancer tissues and 50 adjacent non-cancerous breast tissues, then, it was subjected to bisulfite conversion. The converted DNA was amplified by real-time PCR; then, pyrosequencing was performed to quantify DNA methylation levels in four CpG sites in ATM and SCGB3A1 gene promoters. The methylation data were presented as the percentage of average methylation of all the observed CpG sites and were calculated for each sample and each gene.ResultsThe percentage of DNA methylation of the SCGB3A1 promoter was significantly higher in the tumor group than in the normal group (P= 0.001). However, a non-statistical significance difference was found in the DNA methylation percentage of the ATM promoter in the tumor group compared to the normal group (P = 0.315). The SCGB3A1 promoter methylation frequency was significantly associated with estrogen receptors (ER) and progesterone receptors (PR) positive tumors, lymph node metastasis, and lymphovascular invasion. However, no association was found between ATM methylation status and the different clinicopathological features of the tumor.ConclusionsThe findings of this work showed that the SCGB3A1 promoter methylation was significantly higher in the tumor group and was significantly associated with different clinicopathologic features in breast cancer. It may be considered as a suitable biomarker for diagnosis and prognosis. However, the promoter methylation levels of the ATM gene in breast cancer cases were unable to distinguish between breast cancer tissues and adjacent normal tissues, and there is no evidence that epigenetic silencing by ATM methylation has a role in breast cancer pathogenesis.

Salivary DNA Methylation as an Epigenetic Biomarker for Head and Neck Cancer. Part II: A Cancer Risk Meta-Analysis.

Aberrant methylation of tumor suppressor genes has been reported as an important epigenetic silencer in head and neck cancer (HNC) pathogenesis. Here, we performed a comprehensive meta-analysis to evaluate the overall and specific impact of salivary gene promoter methylation on HNC risk. The methodological quality was assessed using the Newcastle–Ottawa scale (NOS). Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to evaluate the strength of the association and Egger’s and Begg’s tests were applied to detect publication bias. The frequency of salivary DNA promoter methylation was significantly higher in HNC patients than in healthy controls (OR: 8.34 (95% CI = 6.10–11.39; p < 0.01). The pooled ORs showed a significant association between specific tumor-related genes and HNC risk: p16 (3.75; 95% CI = 2.51–5.60), MGMT (5.72; 95% CI = 3.00–10.91), DAPK (5.34; 95% CI = 2.18–13.10), TIMP3 (3.42; 95% CI = 1.99–5.88), and RASSF1A (7.69; 95% CI = 3.88–15.23). Overall, our meta-analysis provides precise evidence on the association between salivary DNA promoter hypermethylation and HNC risk. Thus, detection of promoter DNA methylation in saliva is a potential biomarker for predicting HNC risk.

Aberrant methylation of UBE2Q1 promoter is associated with poor prognosis of acute-on-chronic hepatitis B pre-liver failure.

Acute-on-chronic hepatitis B liver failure (ACHBLF) is one severe liver disease with rapid progression and high mortality. Identification of specific markers for the prediction of ACHBLF has important clinical significance. We explored the feasibility of UBE2Q1 gene promoter methylation as an early prediction and prognosis biomarker of ACHBLF.UBE2Q1 promoter methylation frequency was detected in 60 patients with acute-on-chronic hepatitis B pre-liver failure (Pre-ACHBLF), 40 patients with chronic hepatitis B and 20 cases of healthy control (HC). The UBE2Q1 mRNA was detected by quantitative real-time polymerase chain reaction.The methylation frequency of the UBE2Q1 promoter in pre-ACHBLF patients was 38.33%, which was significantly lower than that in chronic hepatitis B patients (60.00%) and HCs (65.00%). The UBE2Q1 mRNA expression in pre-ACHBLF patients with UBE1Q1 non-methylation was significantly higher than that in patients with UBE1Q1 promoter methylation. Further analysis showed that hypomethylation of the UBE2Q1 promoter was positively correlated with total bilirubin and international normalized ratio levels in patients with pre-ACHBLF, but negatively correlated with PTA level. COX multivariate analysis showed that the model for end-stage liver disease score and UBE2Q1 promoter hypomethylation status were potential early warning factors that can predict the progression of pre-ACHBLF to ACHBLF. The sensitivity and specificity of UBE2Q1 promoter methylation status combined with the model for end-stage liver disease score for early diagnosis of ACHBLF were 92.9% and 75.0%, respectively. The area under the receiver-operating characteristic curve was 0.895.The hypomethylation of UBE2Q1 promoter is associated with severity of Pre-ACHBLF, which could serve as a potential prognostic biomarker for pre-ACHBLF.