Hypermethylation Of Multiple Genes Research Articles

Abstract 5574: Retinoic acid as a therapeutic adjuvant in low-grade gliomas

Abstract Introduction: Malignant gliomas are incurable brain tumors and the most common primary brain tumors in adults. Mutations in isocitrate dehydrogenase (IDH) are enriched in a subset of malignant low-grade gliomas (MLGGs) that arise as lower grade neoplasms and steadily progress to more aggressive and universally fatal glioblastomas (GBMs) usually within 5-10 years after initial diagnosis. Gliomas with IDH1/2 mutations are characterized by hypermethylation of multiple genes, including immune genes, thereby rendering them highly immune evasive. Retinol binding protein 1 (RBP1) gene, that encodes a protein involved in retinoic acid (RA) generation, is significantly hyper-methylated in IDH mutant cells. Since RA is instrumental in the induction of multiple immune genes including immune-activating ligands such as NKG2D ligands (NKG2D-L), we hypothesized that treatment of IDH mutant cells with RA will increase the expression of NKG2D-L on the tumor cells, leading to increased recognition and lysis by immune cells. In addition, we hypothesized that RBP1 methylation was crucial to development of the IDH mutant immune-evasive phenotype. Methods: Primary glioma cells (IDH WT and mutant) were treated with 1μM RA for 48 hours. The cells were assessed for changes in expression of NKG2D-L and other type-1 immune response genes. Effect of RA treatment on NK killing and immune cell chemotaxis were evaluated. We also assessed the impact of RBP1 knock-down in IDH WT cells. Finally, we evaluated the efficacy of RA as a therapeutic modality for low-grade gliomas in a murine model. Results: RA-treated IDH mutant gliomas had a higher expression of NKG2D-L and a higher sensitivity to killing by NK cells, when compared with untreated IDH mutant cells. RA treatment of IDH mutant cells also induced increased chemotaxis of NKs and CD8+ T cells in a CCL2-dependant manner. In addition, RA demonstrated superior anti-tumor efficacy in vivo, compared with controls. Finally, we demonstrated that RBP1 knock-down in IDH WT cells was sufficient to replicate the immune evasive phenotype observed in IDH mutant cells. Conclusions: RA treatment reverses the immune evasion in IDH mutant cells primarily by inducing the upregulation of NKG2D-L and by mediating increased NK and CD8+ T cell chemotaxis via CCL2 induction. Additionally, we established that RBP1 gene silencing is crucial for the development of the immune evasive IDH mutant phenotype. Note: This abstract was not presented at the meeting. Citation Format: Aparna Rao, Xiaoran Zhang, Jason Kim, Alexander Pomerantz, Youngmi Kim, Eric Holland, Nduka Amankulor. Retinoic acid as a therapeutic adjuvant in low-grade gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5574. doi:10.1158/1538-7445.AM2017-5574

Aberrant gene methylation and bronchial dysplasia in high risk lung cancer patients

IntroductionThe risk for lung cancer is incremented in high degree dysplasia (HGD) and in subjects with hypermethylation of multiple genes. We sought to establish the association between them, as well as to analyze the DNA aberrant methylation in sputum and in bronchial washings (BW). MethodsCross sectional study of high risk patients for lung cancer in whom induced sputum and autofluorescence bronchoscopy were performed. The molecular analysis was determined on DAPK1, RASSF1A and p16 genes using Methylation-specific PCR. ResultsA total of 128 patients were enrolled in the study. Dysplasia lesions were found in 79 patients (61.7%) and high grade dysplasia in 20 (15.6%). Ninety eight patients out of 128 underwent molecular analysis. Methylation was observed in bronchial secretions (sputum or BW) in 60 patients (61.2%), 51 of them (52%) for DAPK1, in 20 (20.4%) for p16 and in three (3.1%) for RASSF1A. Methylated genes only found in sputum accounted for 38.3% and only in BW in 41.7%, and in both 20.0%. In the 11.2% of the patients studied, HGD and a hypermethylated gene were present, while for the 55.1% of the sample only one of both was detected and for the rest of the subjects (33.6%), none of the risk factors were observed. ConclusionsOur data determines DNA aberrant methylation panel in bronchial secretions is present in a 61.2% and HGD is found in 15.6%. Although both parameters have previously been identified as risk factors for lung cancer, the current study does not find a significative association between them. The study also highlights the importance of BW as a complementary sample to induced sputum when analyzing gene aberrant methylation.

DNA Hypermethylation of Cell Cycle (p15 and p16) and Apoptotic (p14, p53, DAPK and TMS1) Genes in Peripheral Blood of Leukemia Patients

Aberrant DNA methylation of tumor suppressor genes has been reported in all major types of leukemia with potential involvement in the inactivation of regulatory cell cycle and apoptosis genes. However, most of the previous reports did not show the extent of concurrent methylation of multiple genes in the four leukemia types. Here, we analyzed six key genes (p14, p15, p16, p53, DAPK and TMS1) for DNA methylation using methylation specific PCR to analyze peripheral blood of 78 leukemia patients (24 CML, 25 CLL, 12 AML, and 17 ALL) and 24 healthy volunteers. In CML, methylation was detected for p15 (11%), p16 (9%), p53 (23%) and DAPK (23%), in CLL, p14 (25%), p15 (19%), p16 (12%), p53 (17%) and DAPK (36%), in AML, p14 (8%), p15 (45%), p53 (9%) and DAPK (17%) and in ALL, p15 (14%), p16 (8%), and p53 (8%). This study highlighted an essential role of DAPK methylation in chronic leukemia in contrast to p15 methylation in the acute cases, whereas TMS1 hypermethylation was absent in all cases. Furthermore, hypermethylation of multiple genes per patient was observed, with obvious selectiveness in the 9p21 chromosomal region genes (p14, p15 and p16). Interestingly, methylation of p15 increased the risk of methylation in p53, and vice versa, by five folds (p=0.03) indicating possible synergistic epigenetic disruption of different phases of the cell cycle or between the cell cycle and apoptosis. The investigation of multiple relationships between methylated genes might shed light on tumor specific inactivation of the cell cycle and apoptotic pathways.

Patterns of aberrant DNA hypermethylation in nasopharyngeal carcinoma in Tunisian patients

BackgroundAberrant methylation in the promoter of tumor-related genes is associated closely with epigenetically mediated gene silencing. The aim of the present study was to evaluate the methylation profile of Tunisian nasopharyngeal carcinoma (NPC) and to determine the clinicopathological features of tumors showing this epigenetic alteration. MethodsThirty-six archival NPC biopsies were investigated in comparison with 19 non-tumor nasopharyngeal tissue specimens. DNA methylation status of ten tumor-suppressor and related genes was analyzed by using methylation-specific PCR. The Epstein-Barr virus (EBV) presence was verified by PCR and in situ hybridization and the LMP1 oncoprotein expression was analyzed by immunohistochemistry. Findings were then correlated with clinicopathological variables (Patients' gender and age, tumor histological subtype and stage). ResultsHypermethylation frequencies of the investigated genes in NPC biopsies were 75% for RASSFIA, 58.3% for SHP1, 47.2% for DAPK, 33.3% for P16, 31% for RARβ2, 19.4% for GSTP1 and TIMP3, 11% for APC and CDH1, and 5.5% for MGMT. In non-tumor nasopharyngeal samples, hypermethylation was detected in lower frequencies in 6 genes (SHP 26.3%, P16 21%, RARβ2 21%, DAPK 15.8%, TIMP3 10.5%, and GSTP 5.3%).Hypermethylation of RARβ2 promoter was more frequent in tumors with lymph node metastasis than those without metastasis (43.5% vs 0%, p=0.03). Methylation of RASSF1A was more frequently detected in non-keratinizing NPC than in undifferentiated subtype (100% vs 66.7%; p=0.05). A trend toward positive association was found between an increased number of methylated genes and LMP1 expression (p=0.07). However, no significant association was found for the remaining variables. ConclusionsThis study indicates that hypermethylation of multiple genes is a common alteration in nasopharyngeal carcinomas in Tunisian patients and that this epigenetic change may play a role in the nasopharyngeal carcinogenesis.

Maintenance Treatment with 5-Azacitidine for Patients with High Risk Myelodysplastic Syndr–ome (MDS) or Acute Myeloid Leukemia Following MDS (MDS-AML) in Complete Remission (CR) after Induction Chemotherapy

Around 50% of patients with high-risk MDS or MDS-AML may enter CR after induction chemotherapy, but CR duration, as well as overall survival is usually short. To address this clinical problem the Nordic MDS Group designed a prospective multicenter phase II study, which assessed the clinical feasibility and utility of long-term maintenance treatment with azaciditine. Sixty patients with high-risk MDS (IPSS intermediate-2 or high) (n=23) or AML following a previous known MDS (n=37) were enrolled between 2004 and 2006. The mean age was 68 (54–83) and patients should not be eligible for stem cell transplantation. Induction treatment consisted of standard doses of daunorubicin and ara-C. Patients in CR received low dose azacitidine subcutaneously 5/28 days until relapse, unless unacceptable toxicity developed. Methylation status of the P15ink4b (P15), E-cadherine (CDH) and Hypermethylated in Cancer 1 (HIC) gene was analysed at study start, in CR and in some patients during follow up. Last follow up was on August 1 2008, 24 months after the last CR was reported. Twenty-four patients (40%) reached CR and 23 of these started maintenance treatment with azacitidine. The initial dose of azacitidine was 75 mg/m2 but as four of the first five enrolled patients developed grade 4 cytopenia, the starting dose was lowered to 60 mg/m2, and was allowed to be reduced to 45 or 30 mg/m2 to avoid severe cytopenias. The mean dose of azacitidine was 54.3 mg/m2. Azacitidine was well tolerated. In 52% of the cases no side effects at all were reported. The most commonly reported side effect was mild rashes at the injection site (35%). Twenty-two percent developed fever or some kind of infection, mostly mild. Myelosuppression (grade 1–3) was seen in 22% of the cases. As previously reported, the probability of reaching CR was negatively correlated to promoter hypermethylation of CDH (p=0.008) and none of the 6 patients hypermethylated on all 3 genes reached CR (p=0.03) and hence only four patients hypermethylated on other genes than P15 received demethylating therapy. The median CR duration for the azacididine treated group was 13.5 months (2–49+) and median survival time from time of inclusion in the study for the same group was 20 months (4–52+). Four of 23 patients (17%) had a CR exceeding 24 months (32–52+). The two patients hypermethylated on CDH pre-induction had CR durations of only 2 and 5 months respectively. By last follow up 3 patients were still in CR. Of 10 patients without any methylation pre-treatment, all but one maintained this pattern in CR. Of the nine patients with pre treatment methylation of at least one gene, only one remained hypermethylated in CR. This patient had a CR duration of only 5 months. One patient showed development of P15 hypermethylation in the bone marrow sampled at 12 months and relapsed at 15 months. These findings support previous reports on P15 hypermethylation as a marker for minimal residual disease (MRD) and threatening relapse. In the whole group, survival was significantly shorter in patients with CDH methylation (3 vs 9 months, p=0.005), while pre-treatment p15 methylation status did not affect CR duration or overall survival. In conclusion, we show for the first time that maintenance treatment with azacytidine is feasible and associated with a median CR duration of 13.5 months, and very mild side effects. However azacytidine does not seem to prevent relapse in the majority of patients, including those with hypermethylation pre-treatment and/or in CR. Hypermethylation of multiple genes is a strong negative factor for survival, probability of CR, and CR duration. We observe a subset of patients, 17%, with a CR duration of >24 months; but no persistent pattern regarding cytogenetics, methylation or morphology could be identified in this group. The strong negative impact of E-Cadherin methylation, a gene involved in adhesion, warrants further investigation.

T2086 Hypermethylation of Multiple Genes in Intraductal Papillary Mucinous Neoplasm of Pancreas

T2086 Hypermethylation of Multiple Genes in Intraductal Papillary Mucinous Neoplasm of Pancreas

Hypermethylation of multiple genes as clonal markers in multicentric hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is highly malignant and prone to multicentric occurrence. Differentiation between a true relapse of HCC and a second primary tumour appearing is of clinical importance. At this point, no convenient method is available to determine the origin of these HCCs. Tissue samples were obtained from 19 patients and analysed for the promoter hypermethylation status of multiple tumour suppressor genes (p16, DAP-Kinase, MGMT, GSTP1, APC, RIZ1, SFRP1, SFRP2, SFRP5, RUNX3, and SOCS1) using methylation-specific PCR (MSP). Methylation status was used to determine tumour clonality. In each of the 19 cases, at least one tumour was recognised as having an aberrantly methylated gene. The frequency of the methylation in tumour tissue was 57.1% in p16, 2.4% in DAP-kinase, 23.8% in GSTP1, 90.5% in APC, 45.2% in RIZ1, 64.3% in SFRP1, 59.5% in SFRP2, 28.6% in SFRP5, 47.6% in RUNX3, and 54.8% in SOCS1, while in MGMT, no aberrant methylation was detected. The methylation status of these genes was assessed using MSP as being either positive or negative, and was used to determine the tumour clonality. The clonality of every tumour could be decided even with lesions that could not be judged by clinical diagnosis or by another molecular method (mt DNA mutation). Determining the methylation status of multiple genes in multicentric HCC was useful as a clonal marker and provided useful information for characterising the tumour. From our findings, multicentric HCCs tend to occur more independently than metastatically from the original tumour. Expanded study should be pursued further for a better understanding of the molecular mechanism of hepatocarcinogenesis.

Promoter hypermethylation of multiple genes in primary gastric lymphoma

Aberrant hypermethylation of CpG islands in the promoter region of tumor suppressor and other important genes in neoplastic cells of lymphoma has been demonstrated to be one of the mechanisms for epigenetic loss of gene function. In this study, we analyzed promoter hypermethylation of the following genes in 49 cases of primary gastric lymphoma (PGL): ATM, p16INK4a(CDKN2A), hMLH1, MGMT, DAPK, and CDH1(ECAD). The PGL cases studied included 26 (53%) cases of diffuse large B-cell lymphoma (DLBCL), 12 (25%) cases of extranodal marginal zone lymphoma (MZL), 7 (14%) cases of MZL with large cell transformation (MZL/DLBCL), 1 (2%) case of follicular lymphoma (FL), one (2%) case of Burkitt-like lymphoma (BL), one case (2%) of lymphoplasmacytic lymphoma (LPL) and one case (2%) of peripheral T-cell lymphoma. Available pathologic data regarding to extragastric involvement at the time of resection of the PGLs were reviewed and correlated. Promoter hypermethylation was detected in 6 of 49 (12.2%) cases for ATM; 13 of 49 (26.5%) for p16INK4a, 19 of 49 (38.8%) for hMLH1; 22 of 49 (44.9%) for MGMT; 27 of 49 (55.1%) for DAPK and 16 of 49 (32.7%) for CDH1. A total of 85% of the PGLs had promoter hypermethylation in at least one of these genes. With different histologic subtypes, promoter hypermethylation of DAPK, hMLH1, and CDH1 genes occurred in 70%, 42%, and 42% respectively for DLBCL, which appeared to be higher than combined MZL and MZL/DLBCL subgroup. Approximately 81% PGLs demonstrated H. pylori infection by immunohistochemistry. H. pylori status did not appear to be statistically correlated with promoter hypermethylation of the genes. Of 37 PGL cases, 19 cases had extragastric involvement at the time of resection, indicating relatively higher stage disease. The frequencies of promoter methylation in those cases were 58% for DAPK, 42% for hMLH1, 37% for CDH1, 26% for p16INK4a and 11% for ATM respectively. The promoter methylation at MGMT gene was significantly higher in the PGLs without extragastric involvement (61%) as compared to those with extragastric involvement (26%).

Quantitative Detection of Promoter Hypermethylation of Multiple Genes in the Tumor, Urine, and Serum DNA of Patients With Renal Cancer

Quantitative Detection of Promoter Hypermethylation of Multiple Genes in the Tumor, Urine, and Serum DNA of Patients With Renal Cancer

Quantitative Detection of Promoter Hypermethylation of Multiple Genes in the Tumor, Urine, and Serum DNA of Patients With Renal Cancer

Quantitative Detection of Promoter Hypermethylation of Multiple Genes in the Tumor, Urine, and Serum DNA of Patients With Renal Cancer

Quantitative Detection of Promoter Hypermethylation of Multiple Genes in the Tumor, Urine, and Serum DNA of Patients With Renal Cancer

Quantitative Detection of Promoter Hypermethylation of Multiple Genes in the Tumor, Urine, and Serum DNA of Patients With Renal Cancer

Hypermethylation of multiple genes in pancreatic cancer

Hypermethylation of multiple genes in pancreatic cancer

P-311 Aberrant hypermethylation of multiple genes in pulmonary squamous cell carcinoma

P-311 Aberrant hypermethylation of multiple genes in pulmonary squamous cell carcinoma

P-312 Aberrant promoter hypermethylation of multiple genes in pulmonary adenocarcinomas

P-312 Aberrant promoter hypermethylation of multiple genes in pulmonary adenocarcinomas

New markers for cancer detection.

Circulating tumor nucleic acids in blood have been demonstrated to reflect the biologic characteristics of tumors. During tumor progression, aberrant DNA methylation can lead to transcriptional silencing of tumor suppressor genes, DNA repair genes, and metastasis-inhibitor genes. Hypermethylation of multiple genes, detectable in the blood of cancer patients, has demonstrated increasing promise as a specific and sensitive molecular marker for detecting and monitoring cancer. In addition to these epigenetic markers, a number of mRNA markers may also enable cancer detection in the blood of patients with different cancer types. Quantification of circulating tumor cell mRNAs in cancer patients appears to be useful for monitoring cancer progression and response to treatment. DNA methylation markers and mRNA markers in the blood may open up diagnostic and prognostic possibilities.

Methylation profiling of human cancers in blood: Molecular monitoring and prognostication (Review)

Cancer is a polygenetic and polyepigenetic disease. Circulating tumor cells in peripheral blood have been demonstrated to reflect the biological characteristics of tumors including the potential of metastasis development and tumor recurrence. Aberrant promoter methylation has emergingly become a fundamental molecular abnormality leading to transcriptional silencing of tumor suppressor genes, DNA repair genes and metastasis inhibitor genes, and is linked to the predisposition of genetic alterations of other cancer-associated genes. This epigenetic inheritance has significant biological implications for cancer progression and metastasis formation. Of significance, DNA hypermethylation of multiple genes successfully detected in circulating tumor cells from cancer patients may prove valuable for disease monitoring. A number of epigenetic markers may feasibly enable the detection of circulating tumor cells from patients with different cancer types. The prognostic and therapeutic implications of aberrant DNA methylation could eventually bring forth improved outcome of cancer patients. A growing body of evidence and future advances in understanding cancer epigenetics may fuel us to monitor and treat cancers in alternative ways.