Aberrant Gene Methylation Research Articles

Aberrant DNA methylation of apoptotic signaling genes in patients responsive and nonresponsive to therapy for cervical carcinoma

We sought to investigate CpG-island methylation profiling of apoptotic genes apoptotic protease activating factor 1, caspase 8, death-associated protein kinase (DAPK), tumor necrosis factor receptor superfamily member 6 (FAS), Survivin, and tumor necrosis factor-related apoptosis-inducing ligand receptor-1 and its role in resistance to therapy in cervical cancer (CXCA). Methylation status was performed in 85 CXCA patients comprising therapeutic nonresponses and responses using methylation-specific polymerase chain reaction. Methylation frequency of DAPK and FAS showed a statistically significant difference between therapeutic nonresponses and responses. Concurrent methylation of multiple apoptotic genes was a preferential event in CXCA. Moreover, concerted methylation of pair genes was observed in DAPK, FAS, and tumor necrosis factor-related apoptosis-inducing ligand receptor-1 and found only in nonresponses. Aberrant methylation of apoptotic signaling genes results in acquired resistance to therapy. Detection of methylation in apoptotic signaling genes is potentially useful as a molecular predictive marker for strategic planning of treatment efficacy and evaluation of therapeutic outcome in CXCA, leading to an improvement of patients' survival.

Smad Signaling Is Required to Maintain Epigenetic Silencing during Breast Cancer Progression

Breast cancer progression is associated with aberrant DNA methylation and expression of genes that control the epithelial-mesenchymal transition (EMT), a critical step in malignant conversion. Although the genes affected have been studied, there is little understanding of how aberrant activation of the DNA methylation machinery itself occurs. Using a breast cancer cell-based model system, we found that cells that underwent EMT exhibited overactive transforming growth factor beta (TGFbeta) signaling and loss of expression of the CDH1, CGN, CLDN4, and KLK10 genes as a result of hypermethylation of their corresponding promoter regions. Based on these observations, we hypothesized that activated TGFbeta-Smad signaling provides an "epigenetic memory" to maintain silencing of critical genes. In support of this hypothesis, disrupting Smad signaling in mesenchymal breast cancer cells resulted in DNA demethylation and reexpression of the genes identified. This epigenetic reversal was accompanied by an acquisition of epithelial morphology and a suppression of invasive properties. Notably, disrupting TGFbeta signaling decreased the DNA binding activity of DNA methyltransferase DNMT1, suggesting that failure to maintain methylation of newly synthesized DNA was the likely cause of DNA demethylation. Together, our findings reveal a hyperactive TGFbeta-TGFbetaR-Smad2 signaling axis needed to maintain epigenetic silencing of critical EMT genes and breast cancer progression.

Impact of the DNA methyltransferases expression on the methylation status of apoptosis-associated genes in glioblastoma multiforme

Disruption of apoptosis is considered as an important factor aiding tumorigenesis, and aberrant DNA methylation of apoptosis-associated genes could be an important and significant mechanism through which tumor cells avoid apoptosis. However, little is known about (1) the impact of methylation status of apoptosis-associated genes on the presence of apoptosis evasion phenotype in glioma; and (2) the molecular mechanism governing the aberrant methylation of apoptosis-associated genes in glioma. By analyzing human glioma biopsies, we first show that low level of apoptosis in tumor is correlated with aberrant methylation of the bcl-2, bax and XAF-1 genes, but not with the aberrant methylation of the bcl-w, survivin, TMS1, caspase-8 and HRK genes. Our work also indicates that the expression levels of DNA methyltransferase 1 (Dnmt1), Dnmt3b and Dnmt1/Dnmt3a coregulate the methylation status of survivin, TMS1 and caspase-8, whereas no correlation was observed between the expression level of Dnmts and the methylation status of the bcl-w, bcl-2, bax, XAF-1 and HRK genes. Thus, these results indicate that the epigenetic regulation of some apoptosis-regulated genes could dictate whether glioma harbors the apoptosis evasion phenotype, and provide some bases to the identification of the methylation machineries of apoptosis-associated genes for which the Dnmt expression acts as a limiting factor.

Aberrant gene methylation in the peritoneal fluid is a risk factor predicting peritoneal recurrence in gastric cancer

To investigate whether gene methylation in the peritoneal fluid (PF) predicts peritoneal recurrence in gastric cancer patients. The gene methylation of CHFR (checkpoint with forkhead and ring finger domains), p16, RUNX3 (runt-related transcription factor 3), E-cadherin, hMLH1 (mutL homolog 1), ABCG2 (ATP-binding cassette, sub-family G, member 2) and BNIP3 (BCL2/adenovirus E1B 19 kDa interacting protein 3) were analyzed in 80 specimens of PF by quantitative methylation-specific polymerase chain reaction (PCR). Eighty patients were divided into 3 groups; Group A (n = 35): the depth of cancer invasion was less than the muscularis propria; Group B (n = 31): the depth of cancer invasion was beyond the muscularis propria. Both group A and B were diagnosed as no cancer cells in peritoneal cytology and histology; Group C (n = 14): disseminated nodule was histologically diagnosed or cancer cells were cytologically defined in the peritoneal cavity. The positive rates of methylation in CHFR, E-cadherin and BNIP3 were significantly different among the 3 groups and increased in order of group A, B and C (0%, 0% and 21% in CHFR, P < 0.05; 20%, 45% and 50% in E-cadherin, P < 0.05; 26%, 35% and 71% in BNIP3, P < 0.05). In addition, the multigene methylation rate among CHFR, E-cadherin and BNIP3 was correlated with group A, B and C (9%, 19% and 57%, P < 0.001). Moreover, the prognosis was analyzed in group B, excluding 3 patients who underwent a non-curative resection. Two of the 5 patients with multigene methylation showed peritoneal recurrence after surgery, while those without or with a single gene methylation did not experience recurrence (P < 0.05). This study suggested that gene methylation in the PF could detect occult neoplastic cells in the peritoneum and might be a risk factor for peritoneal metastasis.

Consistent DNA Hypermethylation Patterns in Laryngeal Papillomas

INTRODUCTION: This study examined the contribution of promoter hypermethylation to the pathogenesis of respiratory papillomatosis (RP), including recurrences (RRP) and progression to squamous cell carcinoma (SSC). MATERIALS AND METHODS: A retrospective cohort of 25 laryngeal papilloma cases included 21 RRP, two of which progressed to SCC. Aberrant methylation status was determined using the multi-gene (22 tumor suppressor genes) methylation-specific multiplex ligation-dependent probe amplification assay and confirmed using methylation specific PCR. RESULTS: Twenty genes had altered DNA methylation in 22 of 25 cases. Aberrant methylation of CDKN2B and TIMP3 was most frequent. Promoter hypermethylation of BRCA2, APC, CDKN2A and CDKN2B was detected in 2 RRP cases with subsequent progression to SCC. Of the 25 cases, 22 were positive for HPV-6, 2 for HPV-11 and 1 for HPV-16 and 33. CONCLUSIONS: Consistent aberrant methylation of multiple tumor suppressor genes contributes to the pathogenesis of laryngeal papillomas. Persistent aberrant DNA methylation events in 2 RRP cases that progressed to cancer indicate an epigenetic monoclonal progression continuum to SCC.

Response to Hydralazine-Valproate in a Patient with Mycosis Fungoides

Histone deacetylase (HDAC) inhibitors have shown significant activity in the treatment of cutaneous T-cell lymphomas (CTCL). The epigenetic alterations of CTCL not only are limited to altered histone acetylation but also include aberrant DNA gene methylation hence, the combination of an HDAC inhibitor with a DNA demethylating agent is a promising therapy to be tested. Here we report a mycosis fungoides patient having a dramatic response to hydralazine and valproate, two repositioned drugs as HDAC and DNA methylation inhibitors, respectively.

Aberrant methylation of DNA mismatch repair genes in elderly patients with sporadic gastric carcinoma: A comparison with younger patients

Hypermethylation of promoters that regulate the expression of DNA repair genes is associated with gastric carcinoma (GC). Little is known regarding the association between age of disease onset and differences in molecular profiles. The two study groups consisted of 100 elderly patients and 100 younger patients. The aberrant DNA methylation patterns of four mismatch repair genes, hMLH1, hMSH2, hMSH3, and MGMT, were compared by bisulfite modification and methylation-specific PCR (MSP). The methylation frequencies for hMLH1 and hMSH3 were significantly higher for the elderly than for the younger GC patients (P < 0.001 and P = 0.031, respectively). A significant correlation was observed between aberrant hMLH1, hMSH3, and MGMT methylation and the loss of hMLH1, hMSH3, and MGMT protein expression (P < 0.001, P = 0.002, and P = 0.001, respectively). The prevalence of aberrant hMLH1 and hMSH3 methylation increased significantly with age. These results suggest that the methylation of hMLH1 and hMSH3 is age related and thus may play an important role in gastric carcinogenesis in the elderly. Screening for hMLH1 and hMSH3 methylation may have clinical significance for the evaluation of elderly patients with GC.

Aberrant Gene Methylation in the Lymph Nodes Provides a Possible Marker for Diagnosing Micrometastasis in Gastric Cancer

This study was designed to determine whether gene methylation is a novel diagnostic marker for micrometastasis to the lymph nodes (LNs) in gastric cancer. The gene methylation of CHFR, p16, RUNX3, E-cadherin, MGMT, hMLH1, and ABCG2 genes were analyzed in 49 primary gastric cancer tissues, corresponding to noncancerous tissues and matched LNs by quantitative methylation-specific PCR (q-MSP). CHFR, RUNX3, MGMT, and hMLH1 were more frequently methylated in primary cancer compared with the noncancerous mucosa. Further analyses investigated whether the methylation of the four cancer-specific genes was preserved in LN tissues using the 29 control cases, in which LN metastasis had been histologically confirmed. The methylation of both lesions (M/M pattern) in at least one gene, which was judged to be positive for cancer cells in LNs, was observed in 25 of 29 cases (86%). Quantitative RT-PCR (qRT-PCR) of CEA, CK19, and CK20 mRNA was conducted using the same samples. The mRNA expression of at least one of the three genes was observed in 100% of the specimens. The results of the control analysis were used to attempt to predict micrometastasis by q-MSP and qRT-PCR in the 20 test cases without histological LN metastasis. Six cases (30%) showed the M/M pattern in at least one of the four genes. Three of 20 cases (15%) exhibited both the M/M pattern and positive mRNA. The methylation analysis revealed the clinical feasibility of detecting occult neoplastic cells in the regional LNs.

Current methylation screening methods

DNA methylation is the covalent addition of a methyl group to the cytosines within a 5 -CpG-3 dinucleotide. More than 25 years ago, the methylation of the cytosines of the promoter sequences was shown to play an important role in the regulation of transcriptional activity of the genes [1]. Subsequently, it was suggested that methylation might contribute to tumorigenesis [2]. Today, methylation-dependent geneexpression aberrations have been demonstrated to contribute to all of the typical hallmarks of cancer [3]. At the same time, increasing evidence shows that aberrant methylation of cancerrelated genes could be used as a new target for effective early diagnosis and treatment of not only cancer but also many other diseases. Aberrant methylation of the cancer genes occurs in the early stages of carcinogenesis, and tumor DNA has long been shown to circulate in blood flow and other body fluids. That opens the possibility of early detection of the neoplastic process in simple, for example, blood-based, tests, once powerful and tumor-specific biomarkers have been discovered [4]. Furthermore, methylation changes, due to their nature, are potentially reversible, and therefore new compounds targeting the methylation changes can be used in the treatment of neoplastic disease [5]. The fundamental discoveries within both the fields of DNA methylation and genetics have occurred at the same time. However, for years, the field of methylation studies has lagged behind the developments within genetics due to the technological limitations and difficulties of methods for studies of methylation changes. The first technologies allowing for the investigation of methylation phenomenon relied on the endonucleases digesting the DNA, depending on the methylation status of the restriction site. The enzymatic digest was usually followed by Southern blotting. However, the fact that these methodologies require large amounts of DNA has been limiting their use, especially in cancer studies. Along with enzymatic approaches, at the beginning, field studies of total content of methylated cytosines in genomes were based on high-performance liquid chromatography and high-performance capillary chromatography techniques [6,7]. Those studies revealed the phenomena of the overall significant hypomethylation of cancer genomes. Currently, the protocols based on endonucleases and Southern blots are rarely used. However, methylationsensitive endonucleases digestion has been combined with real time (RT)-PCR technologies and integrated into genome-wide methylation study protocols (discussed below). The quantitative analysis of DNA methylation using RT-PCR (qAMP) technique is an example of the combination of methylation-sensitive endonuclease digestion and RT-PCR, where RT-PCR reveals a presence or absence of a digestion within the locus of interest [8]. During in vitro amplification of genomic DNA, all methylation information is lost. Therefore, in order to study methylation marks, the methylation pattern has to be preserved prior to the use of PCR. Revolutionary for the field was the use of sodium bisulfite, which allows to preserve methylation marks during PCR amplification. In principle, sodium bisulfite deaminates cytosines to uracil, while methylated cytosines remain unchanged. During the subsequent PCR amplification, retained 5-methylcytosines are amplified as cytosines, whereas thymine is incorporated at unmethylated cytosines sites. Current PCR-based methods for investigation of the methylation status of CpG sites within a locus if interest can be divided into two groups. One group of the technologies utilizes PCR for a

Methylation profiling of tumor suppressor genes and oncogenes in hepatitis virus-related hepatocellular carcinoma in northern India

Hepatocellular carcinoma (HCC) is the fifth most common cancer in India, and hepatitis B virus and hepatitis C virus infections are major risk factors. DNA methylation alterations have been linked to various carcinomas in different populations. Aberrant CpG island methylation of genes has been recognized in HCC, information is limited for hepatitis virus–related hepatocarcinogenesis. HCC risk has not previously been associated with gene-specific DNA methylation in India. Promoter region methylation of a panel of six tumor suppressor genes ( CDKN2A, CDKN2B, CDH1, GSTP1, SOCS1, and APC) and three oncogenes ( MYC, HRAS, and KRAS) was determined by methylation-specific PCR among 23 HCC samples and 20 control hepatitis samples. CDKN2B methylation frequency in HCC was double that for hepatitis, and methylation allele density of APC, GSTP1, and CDKN2B increased 2.2-, 2.3-, and 7.6-fold, respectively. Epigenetic silencing of tumor suppressor genes starts during viral infection and progresses toward HCC with the chronicity of the disease. Findings of altered methylation status support involvement of these tumor suppressor genes in HCC. MYC showed decreased methylation in HCC, relative to hepatitis. These observations on DNA methylation suggest the involvement of CDKN2B, SOCS1, CDH1, GSTP1, and MYC in pathogenesis of HCC in India and implicate altered DNA methylation in the molecular pathogenesis.

DNA methylation of cancer genome

DNA methylation plays an important role in regulating normal development and carcinogenesis. Current understanding of the biological roles of DNA methylation is limited to its role in the regulation of gene transcription, genomic imprinting, genomic stability, and X chromosome inactivation. In the past 2 decades, a large number of changes have been identified in cancer epigenomes when compared with normals. These alterations fall into two main categories, namely, hypermethylation of tumor suppressor genes and hypomethylation of oncogenes or heterochromatin, respectively. Aberrant methylation of genes controlling the cell cycle, proliferation, apoptosis, metastasis, drug resistance, and intracellular signaling has been identified in multiple cancer types. Recent advancements in whole-genome analysis of methylome have yielded numerous differentially methylated regions, the functions of which are largely unknown. With the development of high resolution tiling microarrays and high throughput DNA sequencing, more cancer methylomes will be profiled, facilitating the identification of new candidate genes or ncRNAs that are related to oncogenesis, new prognostic markers, and the discovery of new target genes for cancer therapy.

CpG island methylation of BNIP3 predicts resistance against S-1/CPT-11 combined therapy in colorectal cancer patients

Aberrant gene methylation is frequently observed in various cancers and plays an important role in carcinogenesis, cancer progression and drug responsiveness. The aim of this study is to identify colorectal cancer specific gene methylation determining chemosensitivity to S-1/CPT-11 therapy. The gene methylation of CHFR, p16, RUNX3, E-cadherin, MGMT, hMLH1, ABCG2, UGT1A1 and BNIP3 genes were analyzed in 27 colorectal cancer tissues by quantitative methylation-specific PCR (q-MSP). All 27 patients were postoperatively treated by S-1/CPT-11 therapy targeting the metastatic lesion and the recurrent tumor. Thereafter, the patients were divided into a responder group (RG) or a non-responder group (NRG) according to the effect of the chemotherapy. There were 13 cases of RG (48.1%) and 14 cases of NRG (51.9%). The methylation level in CHFR, RUNX3 and BNIP3 was significantly higher in cancer lesions in comparison to the non-cancerous lesion. Only methylation of the BNIP3 gene was significantly higher in primary cancer tissue of the NRG than the RG. The correlation between the BNIP3 methylation status and time to progression (TTP) suggested that the low methylation group (n=16) resulted in a significantly longer TTP, in comparison to the high methylation group (n=11; P=0.004). The methylation level of BNIP3 showed a significant inverse correlation with the mRNA expression suggesting the DNA methylation suppressed BNIP3 expression (r=-0.466, P=0.021). In conclusion, BNIP3 gene methylation is a possible marker predicting a poor response to the S-1/CPT-11 combined therapy in colorectal cancer.

A Methylation Profile of Tumor Suppressor Genes Predicts a Poorer Survival in Patients with Refractory Anemia with Ringed Sideroblasts.

Abstract 2780Poster Board II-756Patients with refractory anemia with ring sideroblasts (RARS) are considered to have good prognosis and anemia is usually managed with best supportive care and erythroid stimulating agents. Nowadays, no specific molecular marker related to outcome and disease progression has been identified. Several genes involved in cell cycle and apoptosis that may become inactivated by aberrant hypermethylation have been identified in patients with myelodysplastic syndromes (MDS) but the significance of a methylation profile (studying several genes at the same time) in RARS is unknown, mainly because the number of patients with RARS in published reports is rather low. To assess the implication of aberrant methylation in RARS, we studied the methylation status of 25 sequences of a set of tumor suppressor genes in 40 patients (median age 70 yr, 25 male gender) with RARS according to FAB criteria [WHO classification, RARS in 22 patients (55%); refractory citopenia with multilineage dysplasia and ring sideroblasts, 18 (45%)] by means of methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay. The MS-MLPA procedure (SALSA MLPA kit ME001, MRC-Holland, Amsterdam, The Netherlands) has been developed for detecting in a semi-quantitative manner changes in the methylation status of 25 tumor suppressor genes in a single experiment. Briefly, approximately 50 ng of DNA were denatured and hybridized with MLPA probes. Subsequently, the probes were ligated in half of every sample, whereas for the rest of the sample, ligation was combined with an endonuclease HhaI digestion resulting in ligation of the methylated sequences only. PCR was performed as described by the manufacturer, and then separated by capillary gel electrophoresis and quantified using the Genemapper software (ABI 310, Applied Biosystems, Foster City, CA). Quantification of the methylation status was done by dividing the peak area with the combined areas of the control probes lacking the target sequence of the HhaI. Finally, the relative peak area of each target probe from the digested sample was compared with those obtained from the undigested sample. Aberrant methylation was scored when the calculated methylation percentage was >10%. To validate the MS-MLPA method the methylation status of CDKN2B was also analyzed by methylation-specific PCR (MSP). Actuarial curves of OS were built by Kaplan-Meier method and differences between curves compared with log-rank tests. Small numbers precluded the use of multivariate methodology. The MS-MLPA analysis detected methylation of at least one gene in 17 patients (42.5%). The genes aberrantly methylated were CDKN2B (n = 8, 20%), RASSF1 (n = 7, 17%), RARB (n = 3, 7.5%), CDH13 (n = 3, 7.5%) and FHIT (n = 2; 5%). Of the 17 patients, five (12%) presented methylation in two genes (RASSF1-FHIT in 2, RASSF1-RARB in 1, RASSF1-CDH13 in 1, and CDKN2B-CDH13 in 1, who was the only patient who progressed to AML). The presence of aberrant gene methylation was not significantly associated with any clinical or biological characteristic or WHO morphological subtype. Patients with aberrant gene methylation had a significantly shorter overall survival (OS) than patients without methylated genes (median OS, 72 mo vs 114 mo, respectively; P = 0.03). Patients with hemoglobin level below 10 g/dL and platelet count below 150 ×109/L also had a significantly poorer OS (P= 0.01 and P= 0.02, respectively). As the majority of probes used in the MS-MPLA method detect methylation in only one CpG pair, the results of CDKN2B methylation were validated by MSP, which yielded the same methylation results than with MS-MPLA methodology. The 8 patients with methylated CDKN2B show a trend for a shorter survival than the remaining 32 with unmethylated CDKN2B (median 72 mo vs 114 mo, P = 0.08). The results of this study indicate that aberrant methylation of several tumor suppressor genes is observed in a substantial proportion of patients with RARS. As occurs in patients with high-risk MDS, our results suggest that aberrant gene methylation confers a poor prognosis in RARS, but these data and their potential independent value require confirmation in larger series that employ multivariate methods. Finally, these findings provide a strong rationale for the use of hypomethylating agents (e.g., azacitidine or decitabine) in patients with RARS.This work has been partially supported by ISCIII grants RD06/0020/0031 and CA08/00141. Disclosures:No relevant conflicts of interest to declare.

Aberrant DNA Methylation in Childhood Acute Lymphoblastic Leukemia as a Potential Biomarker Reflecting Disease Status.

Abstract 2637Poster Board II-613 Background:Aberrant DNA methylation of promoter-associated cystosine-guanine (CpG) islands is known to play an important role in leukemogenesis and has been reported to have association with poor prognosis in acute lymphoblastic leukemia (ALL). In adult, residual methylation at clinical remission status in patients with acute myeloid leukemia and Philadelphia chromosome-positive ALL showed significant association with poor prognosis in recent studies. We analyzed 47 patients with childhood ALL to evaluate the change of methylation status from the time of diagnosis to the time at morphologic remission. Methods:We analyzed the methylation status of CDH1, p16 and DAPK genes at the time of diagnosis and at the time of morphologic remission in 47 patients using methylation specific polymerase chain reaction method with genomic DNA extracted from bone marrow (BM) aspirates. All patients were treated with standard chemotherapy and after 4 weeks, every patient achieved morphologic complete remission. Methylation status of above three genes were also analyzed with the same method in 10 BM aspirate samples from healthy BM donor for comparison. Results:CDH1 was methylated in 30 (64%) patients, p16 in 2 (4%) patients and DAPK in 6 (13%) patients at the time of diagnosis. Thirty three (70%) patients had methylation at least one gene, and 5 (11%) had methylation of 2 genes. None of the healthy BM donors showed methylation of above genes. At the time of morphologic remission, all patients who had aberrant DNA methylation of any gene at the time of diagnosis had no detectable residual methylation showing complete resolution of aberrant DNA methylation of examined genes. Clinical prognostic factors including initial white blood cell count, immunophenotype and presence of specific translocations (TEL-AML1, BCR-ABL, E2A-PBX1) did not show any association with initial methylation status. Age was the only factor which showed correlation with methylation status; patients under 2 year old showed significantly low frequency of methylation of above three genes (p <0.001). Conclusion:Since aberrant DNA methylation of CDH1, p16 and DAPK genes were found in 70% of pretreatment ALL patients and none in healthy BM donor showing complete resolution of methylation at morphologic remission status, we cautiously suggest aberrant DNA methylation as a potential biomarker reflecting disease status in childhood ALL. Disclosures:No relevant conflicts of interest to declare.

Accumulation of aberrant CpG hypermethylation by Helicobacter pylori infection promotes development and progression of gastric MALT lymphoma

Aberrant DNA hypermethylation is an important mechanism for the inactivation of tumor-related genes in human tumors. Gastric mucosa-associated lymphoid tissue (MALT) lymphomas arise from Helicobacter pylori-associated chronic gastritis; most patients are H. pylori-positive and eradication therapy is highly effective. In the present study, we used methylation-specific PCR to analyze the DNA methylation status of 11 tumor-related genes (Kip2, p16, hMLH-1, p15, p73, MGMT, DAPK, MINT1, MINT2, MINT31 and HCAD) in 21 specimens of MALT lymphoma, 5 specimens of MALT lymphoma with large cell component (high-grade MALT lymphoma), 15 specimens of diffuse large B-cell lymphoma (DLBCL), 8 specimens of complete remission of MALT lymphoma after eradication therapy, 5 specimens with no evidence of malignancy and PBMCs from 10 healthy donors. The average number of methylated genes was significantly greater in gastric lymphomas as compared to normal controls (P<0.001). The CpG island methylator phenotype (CIMP) was observed in 93.3% (14/15) of DLBCLs, 100% (5/5) of high-grade MALT lymphomas and 61.9% (13/21) of MALT lymphomas; in contrast, CIMP was not found in the control group (0%). The average number of methylated genes and the CIMP incidence significantly increased with H. pylori infection. Furthermore, aberrant CpG methylation of specific genes, such as p16, MGMT and MINT31, was consistently associated with H. pylori infection. These findings strongly suggest that H. pylori infection causes the aberrant DNA hypermethylation of specific genes and induces CIMP, which is an important epigenetic mechanism for the development and progression of gastric MALT lymphoma; additionally, our findings provide new epigenetic markers.

TSC-22 contributes to hematopoietic precursor cell proliferation and repopulation and is epigenetically silenced in large granular lymphocyte leukemia

Aberrant methylation of tumor suppressor genes can lead to their silencing in many cancers. TSC-22 is a gene silenced in several solid tumors, but its function and the mechanism(s) responsible for its silencing are largely unknown. Here we demonstrate that the TSC-22 promoter is methylated in primary mouse T or natural killer (NK) large granular lymphocyte (LGL) leukemia and this is associated with down-regulation or silencing of TSC-22 expression. The TSC-22 deregulation was reversed in vivo by a 5-aza-2'-deoxycytidine therapy of T or NK LGL leukemia, which significantly increased survival of the mice bearing this disease. Ectopic expression of TSC-22 in mouse leukemia or lymphoma cell lines resulted in delayed in vivo tumor formation. Targeted disruption of TSC-22 in wild-type mice enhanced proliferation and in vivo repopulation efficiency of hematopoietic precursor cells (HPCs). Collectively, our data suggest that TSC-22 normally contributes to the regulation of HPC function and is a putative tumor suppressor gene that is hypermethylated and silenced in T or NK LGL leukemia.

Unraveling Breast Cancer Heterogeneity Through Transcriptomic and Epigenomic Analysis

Breast cancer diversity is histologically evident as various proliferative benign lesions, in situ carcinomas, and invasive carcinomas that may develop into distant metastases. Breast tumor molecular subtypes have been defined by genome-wide expression microarray technology and reveal associations between genetic alterations and the malignant phenotype. Early work has been conducted to use subtype-specific biomarkers to elucidate targeted treatment options early in the course of breast cancer progression. Additionally, DNA methylation is an epigenetic modification that contributes to breast cancer progression by transcriptionally silencing certain tumor suppressor genes. Among the genes characterized as targets for silencing are well-established tumor suppressors such as RASSF1A, RARB, SFN, and TGM2. Measuring elevated gene copy number and aberrant gene promoter methylation can further facilitate characterization of breast tumor molecular subtype; however, profiling of breast tumors based on epigenetic criteria has yet to be established. Epigenomic analysis has been investigated for clinical applicability, and it has great promise when used in combination with minimally invasive techniques for both diagnostic and prognostic purposes.

Aberrant Methylation of the Netrin‐1 Receptor Genes UNC5C and DCC Detected in Advanced Colorectal Cancer

UNC5C and DCC, the netrin-1 receptors, belong to the functional dependence receptors family, which shares the ability to induce apoptosis in the absence of their ligands. Recently, two reports indicated that UNC5C and DCC methylation was closely associated with loss of gene expression in colorectal cancer. These results prompted us to examine the methylation status of the UNC5C and DCC genes in the colorectal carcinomas we surgically removed. The methylation status of the UNC5C and DCC genes were examined in primary carcinomas and the corresponding normal tissues derived from 50 patients with colorectal cancer using quantitative methylation-specific polymerase chain reaction (qMSP). The correlation between the methylation status and the clinicopathologic findings was then evaluated. Aberrant methylation of the netrin-1 receptor genes were detected in 41 of the 50 (82%) primary colon cancers, suggesting that the aberrant methylation of netrin-1 receptors was frequently observed in colorectal cancer. The clinicopathologic data were then correlated with this result. A significant difference was observed in the Dukes stage (p = 0.0438). Netrin-1 receptors might act as a tumor suppressor in colorectal cancers, and thus methylation might present a malignant potential in colorectal cancer.

Prognostic Relevance of Promoter Hypermethylation of Multiple Genes in Breast Cancer Patients

Background: Methylation-mediated suppression of detoxification, DNA repair and tumor suppressor genes has been implicated in cancer development. This study was designed to investigate the impact of concurrent methylation of multiple genes in breast tumors on disease prognosis.Methods: Methylation specific PCR was carried out to analyze the methylation status of seven genes in archived breast tissues and determine the effect of aberrant methylation of multiple genes on disease prognosis and patients’ survival.Results: Promoter hypermethylation was observed in PRB 67%, ERα 64%, RASSF1A 63%, p16INK4A 51%, PRBβ2 22%, GSTP1 25% and BRCA1 27% of the breast cancers, respectively. Concurrent methylation of BRCA1, ERα, GSTP1 and RARβ2, was observed in a large proportion of breast cancers analyzed, suggesting that these genes do not appear to be methylated alone. Patients with high methylation indices had poor prognosis (p &lt; 0.001, Hazards ratio = 14.58). Cox regression analysis showed RARβ2 promoter methylation to be an independent important determinant of breast cancer prognosis.Conclusions: Our results suggest that methylation of multiple genes plays an important role in prognosis of breast cancer. Our study not only describes the association of methylation mediated silencing of multiple genes with the severity of disease, but also drives to speculate the molecular crosstalk between genes or genetic pathways regulated by them individually.

Methylation Profiles in Breast Cancer

In this study, we sought to assess the aberrant methylation of multiple tumor-suppressor genes in a single reaction by using methylation-specific multiplex ligation-dependent probe amplification. Breast tumors and corresponding normal tissues of 77 patients were analyzed. In this study, 17 of 24 genes displayed promoter methylation in one or more of the tumor samples. The most frequently methylated genes were RASSF1 and GSTP1, followed by DAPK1 and CDKN2B. Our data indicate that the methylation of specific genes is a frequent event in breast cancer and show that MS-MLPA is a powerful tool to analyze epigenetic alterations for diagnostic, as well as therapeutic, purposes.