Aberrant Methylation Of CpG Islands Research Articles

Age-Related DNA Methylation Changes in Normal Human Prostate Tissues

Prostate cancer is a leading cause of cancer death among the aging male population but the mechanism underlying this association is unclear. Aberrant methylation of promoter CpG islands is associated with silencing of genes and age-dependent methylation of several genes has been proposed as a risk factor for sporadic cancer. We examined the extent of gene methylation in pathologically normal human prostate as a function of age. We used pyrosequencing to quantitatively analyze the methylation status of nine CpG islands in normal prostate tissue DNA from 45 organ donors and 45 patients who had undergone cystoprostatectomy for bladder cancer. We also analyzed 12 pairs of matched benign and prostate cancer tissue DNA from patients with prostate cancer. Linear regression analysis revealed a significant increase in promoter methylation levels correlating with age for CpG islands at RARbeta2 (r = 0.4; P < 0.0001), RASSF1A (r = 0.27; P = 0.01), GSTP1 (r = 0.59; P < 0.0001), NKX2-5 (r = 0.27; P = 0.008), and ESR1 (r = 0.244; P = 0.023) in the normal prostate tissue samples studied. A calculated average methylation (z score) at all nine CpG loci analyzed in the normal prostate tissues showed a strong correlation with age (r = 0.6; P < 0.001). Comparison of the methylation level for the matched benign and prostate cancer tissues from individual patients with prostate cancer showed significantly higher methylation in the prostate cancer tissue samples for RARbeta2 (P < 0.001), RASSF1A (P = 0.005), GSTP1 (P < 0.001), NKX2-5 (P = 0.003), ESR1 (P = 0.016), and CLSTN1 (P = 0.01). Our findings show aberrant hypermethylation as a function of age in the normal prostate tissues. Such age-related methylation may precede and predispose to full-blown malignancy.

Silencing of tissue factor pathway inhibitor‐2 gene in malignant melanomas

To identify tumor-suppressor genes inactivated by aberrant methylation of promoter CpG islands (CGIs) in human malignant melanomas, genes upregulated by treatment of cells with a demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC), were searched for using oligonucleotide microarrays in melanoma cell lines, HMV-I, MeWo and WM-115. Seventy-nine known genes with CGIs were identified as being upregulated (>or=16-fold), and 18 of them had methylation of their putative promoter CGIs in 1 or more of 8 melanoma cell lines. Among the 18 genes, TFPI-2, which is involved in repression of the invasive potential of malignant melanomas, was further analyzed. Its expression was repressed in a melanoma cell line with its complete methylation, and was restored by 5-aza-dC treatment. It was unmethylated in cultured neonatal normal epidermal melanocyte, and was induced by ultraviolet B. In surgical melanoma specimens, TFPI-2 methylation was detected in 5 of 17 metastatic site specimens (29%), while it was not detected in 20 primary site specimens (0%) (p=0.009). By immunohistochemistry, the 5 specimens with promoter methylation lacked immunoreactivity for TFPI-2. The results showed that TFPI-2 is silenced in human malignant melanomas by methylation of its promoter CGI and suggested that its silencing is involved in melanoma metastasis.

Gene silencing by DNA methylation in haematological malignancies

The past decade has seen an explosion of interest in the epigenetics of cancer, with an increasing understanding that this form of genomic modification plays a critical role in pathogenesis. The malignant phenotype results from a step-wise increase of both genetic abnormalities and epigenetic modifications, leading to dysregulation of critical genes controlling cell growth, differentiation and apoptosis. The methylation of CpG islands within gene promoters is a major epigenetic transcriptional control mechanism that is frequently dysregulated in human cancer. This phenomenon (methylation of CpG islands) plays a critical role in the transcriptional silencing of tumour suppressor genes in cancer and has prompted the development and testing of several demethylating agents aimed at reversing this process. Clinical trials using epigenetically targeted therapies have yielded particularly promising results in the myelodysplastic syndromes (MDS), in which tumour suppressor gene silencing by promoter methylation is a frequent event. Several genes and gene pathways disrupted by aberrant CpG island methylation have now been identified in haematological malignancies, the most frequently studied being the cell cycle inhibitors p16 (now termed CDKN2A; mostly methylated in lymphoid malignancy) and p15 (now termed CDKN2B; commonly methylated in lymphoid and myeloid malignancies). This review will discuss the role that aberrant gene silencing by promoter hypermethylation plays in the molecular pathogenesis of haematological malignancies and assess the clinical potential of demethylating agents for the management of patients.

Epigenetic defects of hepatocellular carcinoma are already found in non-neoplastic liver cells from patients with hereditary haemochromatosis

Gene silencing through aberrant CpG island methylation is a frequent epigenetic defect in hepatocellular carcinoma (HCC). However, nothing is known as yet whether aberrant hypermethylation occurs already in non-neoplastic liver cells from patients with hereditary haemochromatosis who have a clearly elevated risk for developing HCC. Therefore, quantitative real-time PCR-based methylation analysis of six genes frequently hypermethylated in HCC (RASSF1A, cyclinD2, p16(INK4a), GSTpi1, SOCS-1, APC) was performed for liver biopsies from patients with hereditary haemochromatosis. For genotyping of the HFE gene restriction enzyme analysis and Pyrosequencing were used. Transcriptional repression of hypermethylated genes was assessed using real-time RT-PCR. Eighty-four percent of all samples with severe hepatic iron overload and a mutated HFE gene (but without HCC) had at least one gene hypermethylated. All six genes tested were affected by aberrant hypermethylation, albeit to a different extent: RASSF1A 55%, cyclinD2 45%, p16(INK4a) 32%, GSTpi1 10%, SOCS-1 6%, APC 8%. Concomitant transcriptional down-regulation was shown for RASSF1A, cyclinD2, GSTpi1 and SOCS-1. Biopsies from haemochromatosis patients showed significantly more aberrant hypermethylation than normal liver tissue or benign liver tumours (P < 0.001) and also to a higher degree. This effect is independent of patient age, cirrhosis or hepatitis infection. This is the first report demonstrating that longstanding severe iron overload is frequently associated with epigenetic defects characteristic of HCC, which reflects the increased risk of these lesions to progress to HCC. Thus, changes in DNA methylation patterns are an early event preceding morphological alterations of malignant transformation and represent promising targets for early detection.

Relational Analysis of CpG Islands Methylation and Gene Expression in Human Lymphomas Using Possibilistic C-Means Clustering and Modified Cluster Fuzzy Density

Heterogeneous genetic and epigenetic alterations are commonly found in human non-Hodgkin's lymphomas (NHL). One such epigenetic alteration is aberrant methylation of gene promoter-related CpG islands, where hypermethylation frequently results in transcriptional inactivation of target genes, while a decrease or loss of promoter methylation (hypomethylation) is frequently associated with transcriptional activation. Discovering genes with these relationships in NHL or other types of cancers could lead to a better understanding of the pathobiology of these diseases. The simultaneous analysis of promoter methylation using Differential Methylation Hybridization (DMH) and its associated gene expression using Expressed CpG Island Sequence Tag (ECIST) microarrays generates a large volume of methylation-expression relational data. To analyze this data, we propose a set of algorithms based on fuzzy sets theory, in particular Possibilistic c-Means (PCM) and cluster fuzzy density. For each gene, these algorithms calculate measures of confidence of various methylation-expression relationships in each NHL subclass. Thus, these tools can be used as a means of high volume data exploration to better guide biological confirmation using independent molecular biology methods.

Promoter methylation status of the Cyclin D2 gene is associated with poor prognosis in human epithelial ovarian cancer

Gene silencing associated with aberrant DNA methylation of promoter CpG islands is one mechanism through which several genes may be inactivated in human cancers. Cyclin D2, a member of the D-type cyclins, implicated in cell cycle regulation, differentiation and malignant transformation, is inactivated due to aberrant DNA methylation in several human cancers. In the present study, we examined the promoter methylation status and expression of Cyclin D2 in human epithelial ovarian cancer, and then determined the relationship between methylation status and various clinicopathological variables. Twelve ovarian cancer cell lines and 71 surgical specimens were examined by methylation-specific polymerase chain reaction and quantitative reverse transcription-polymerase chain reaction to evaluate the methylation status and expression of the Cyclin D2 gene. The relationship between methylation status and various clinicopathological variables was evaluated using statistical analysis. Aberrant methylation of Cyclin D2 was present in five of 12 ovarian cancer cell lines and 16 of 71 primary ovarian cancer tissues. In five cell lines with methylation, expression of the Cyclin D2 gene tended to be lower than in cell lines without methylation. In ovarian cancer tissues, methylation bands were detected in 16 of 71 cases. The methylation status of Cyclin D2 was associated with advanced stage and a residual tumor size (>2 cm) (P = 0.027 and P = 0.031, respectively). Based on univariate analysis, patients with aberrant methylation of the Cyclin D2 promoter had a significantly worse chance of disease-free survival than those without methylation (P = 0.021). Our results suggest that aberrant promoter methylation of the Cyclin D2 gene is significantly associated with patient prognosis in epithelial ovarian cancer.

Epigenetic Markers and Response to Chemotherapy in Cancer

The last ten years has seen an explosion in interest in epigenetic mechanisms of control of gene expression. This is particularly true in the field of cancer research where epigenetic alterations are now regarded as equally important as genetic alterations in the development and progression of cancer. Of particular interest is altered DNA methylation, which is a key feature of essentially all tumour types. Aberrant methylation of CpG islands represents an ideal candidate for both diagnostic and prognostic markers in cancer. It is highly prevalent, very largely tumour specific and potentially far more readily detectible than most genetic alterations. This review will discuss the genes already identified as potential epigenetic markers of drug response, as well as the rapidly improving technology for detection of methylation which has greatly expanded the potential sources of tumour specific DNA that can be used for epigenetic marker analysis.

MGMT Methylation Status: The Advent of Stratified Therapy in Glioblastoma?

Glioblastomas are the most malignant gliomas with median survival times of only 15 months despite modern therapies. All standard treatments are palliative. Pathogenetic factors are diverse, hence, stratified treatment plans are warranted considering the molecular heterogeneity among these tumors. However, most patients are treated with "one fits all" standard therapies, many of them with minor response and major toxicities. The integration of clinical and molecular information, now becoming available using new tools such as gene arrays, proteomics, and molecular imaging, will take us to an era where more targeted and effective treatments may be implemented.A first step towards the design of such therapies is the identification of relevant molecular mechanisms driving the aggressive biological behavior of glioblastoma. The accumulation of diverse aberrations in regulatory processes enables tumor cells to bypass the effects of most classical therapies available. Molecular alterations underlying such mechanisms comprise aberrations on the genetic level, such as point mutations of distinct genes, or amplifications and deletions, while others result from epigenetic modifications such as aberrant methylation of CpG islands in the regulatory sequence of genes. Epigenetic silencing of the MGMT gene encoding a DNA repair enzyme was recently found to be of predictive value in a randomized clinical trial for newly diagnosed glioblastoma testing the addition of the alkylating agent temozolomide to standard radiotherapy. Determination of the methylation status of the MGMT promoter may become the first molecular diagnostic tool to identify patients most likely to respond that will allow individually tailored therapy in glioblastoma.To date, the test for the MGMT-methylation status is the only tool available that may direct the choice for alkylating agents in glioblastoma patients, but many others may hopefully become part of an arsenal to stratify patients to respective targeted therapies within the next years.

Quantitative High-Resolution CpG Island Mapping with Pyrosequencing™ Reveals Disease-Specific Methylation Patterns of the CDKN2B Gene in Myelodysplastic Syndrome and Myeloid Leukemia

Gene silencing through aberrant CpG island methylation is the most extensively analyzed epigenetic event in human tumorigenesis and has huge diagnostic and prognostic potential. Methylation patterns are often very heterogeneous, however, presenting a serious challenge for the development of methylation assays for diagnostic purposes. We used Pyrosequencing technology to determine the methylation status of 68 CpG sites in the CpG island of the CDKN2B gene [cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)], frequently hypermethylated in myeloid malignancies, in a series of bone marrow samples from patients with myelodysplasia and myeloid leukemia (n = 82) and from 32 controls. A total of 7762 individual methylation sites were quantitatively evaluated. Precision and reproducibility of the quantification was evaluated with several overlapping primers. The use of optimized sequencing primers and the new Pyro Q-CpG software enabled precise and reproducible quantification with a single sequencing primer of up to 15 CpG sites distributed over approximately 100 bp. Extensive statistical analyses of the whole CpG island revealed for the first time disease-specific methylation patterns of the CDKN2B gene in myeloid malignancies and small regions of differential methylation with high discriminatory power that enabled differentiation of even low-grade myelodysplastic syndrome samples from the controls, a result that was confirmed in an independent group of 9 control and 36 patient samples. The precise quantitative methylation mapping of whole CpG islands is now possible with Pyrosequencing software in combination with optimized sequencing primers. This method reveals disease-specific methylation patterns and enables the development of specific diagnostic assays.

Identification of methylation-silenced genes in colorectal cancer cell lines: Genomic screening using oligonucleotide arrays

Objective. Aberrant methylation of promoter CpG islands is associated with the loss of expression of tumor suppressor genes in human cancers. The purpose of this study was to examine methylation-silenced genes in colorectal cancer (CRC) cell lines. Material and methods. Using an oligonucleotide array, we undertook a genome-wide search for genes up-regulated following treatment with a demethylating agent (5-aza-2′-deoxycytidine) in two CRC cell lines, DLD-1 and HT-29. Promoter methylation status was determined in 12 CRC cell lines and 11 CRC tissues by methylation-specific polymerase chain reaction (MSP). Results. After treatment, 350 genes were up-regulated 1.5-fold or more. Six genes (PAGE-5, VCX, MAEL, GAGED2, UCHL1, and GAGE7), which contained putative 5′ CpG islands in their promoter regions, were confirmed to be silenced in CRC cell lines. UCHL1 (also known as PGP9.5) is involved in regulation of cellular ubiquitin levels, and its promoter methylation was detected in 10 out of 12 CRC cell lines. The level of methylation of UCHL1 was significantly higher in tumors than in corresponding normal mucosae (p=0.005). Conclusions. Chemical genomic screening led to the identification of a specific promoter subject to hypermethylation in CRC. These results suggest that aberrant promoter methylation is the primary mechanism of transcriptional silencing of the UCHL1 gene and that methylation of the UCHL1 gene promoter increases during the development and progression of CRC.

Aberrant CpG Island Methylation in AML Is Associated with Disease Progression and Relapse.

The role of DNA methylation in relapse and progression of acute myelogenous leukemia (AML) is incompletely understood. We studied DNA methylation of 9 promoter-associated CpG islands of genes frequently hypermethylated in leukemic cell lines. These were NOR1, NPM2, HIN1, SLC26A4, CDH13, PGRA, PGRB, OLIG2 and the tumor suppressor gene p15INK4b. We examined bone marrow and/or peripheral blood cells collected at the time of diagnosis and at the first relapse from 32 patients (13 females, 19 males) with AML. The median age was 58 years (20–68), the median survival was 18 months (8–80), the median blast count was 64% (20–98), and 10 patients had additional solid tumors and/or lymphatic/hematologic malignancies. Bisulfite treatment of DNA, followed by PCR and pyrosequencing were used to quantitatively measure levels of cytosine methylation in promoter-associated CpG islands. We analyzed methylation data for individual genes and for a methylation index derived after Z-score (z = [value –mean]/standard deviation) transformation to equalize absolute differences between individual genes and we used paired t-tests for statistical analysis. Abnormal hypermethylation (≥10%) in bone marrow and peripheral blood cells at diagnosis was detected in all 9 investigated genes, with a range of 7/29 (24%) for HIN1 to 24/32 (75%) for CDH13. On average, an increase in methylation between diagnosis and relapse was detected in all genes, and was significant for CDH13 (mean 10%, p=0.0006), SLC26A4 (mean 7%, p=0.0012), HIN1 (mean 8%, p=0.0037), NPM2 (mean 7%, p=0.0073), p15INK4b (mean 13%, p=0.0081), NOR1 (mean 4%, p=0.0124), PGRB (mean 6%, p=0.0144), PGRA (mean 9%, p=0.0275), and OLIG2 (mean 3%, p=0.0732). When analyzed by change in methylation status: negative (methylation below 10%) turning positive (methylation ≥ 10%) and vice versa, of 238 analyses, 39 (16%) showed a negative to positive switch, 15 (6%) showed a positive to negative switch, and the remaining 184 (77%) were either positive or negative unchanged. Finally, when analyzed in individual patients, an increase in methylation was noted in 29 of 32 patients (91%). The median increase in methylation index between diagnosis and relapse calculated as a delta-Z-score was 30% (range from −10% to 147%), and was highly significant (p&lt;0.0001). In summary, abnormal hypermethylation in bone marrow and/or peripheral blood cells from AML patients was detected in all investigated genes at diagnosis. Methylation levels further increased at relapse of the disease in 29 of 32 patients in 1 to 8 of 9 investigated genes. Based on quantitative analyses, we propose that methylation of CDH13, PGRB, PGRA and OLIG2 CpG islands are early markers for AML, while hypermethylation of HIN1, NPM2 and p15INK4b CpG islands is associated with disease progression and predominantly appears at relapse. Thus, aberrant hypermethylation is clearly associated with disease progression and relapse in AML, and likely mediates drug resistance in this setting. Increase of Methylation Index in Relapse Increase of Methylation Index in Relapse

A Phase II Study of Azacitidine (Vidaza™) for Patients with Myelofibrosis (MF).

Aberrant DNA methylation of promoter-associated CpG islands is a mechanism of epigenetic silencing frequently encountered in human neoplasia. Hypermethylation of several genes such as p15INK4B, p16INK4A, progesterone receptor, and the retinoic acid receptor β have been detected in patients (pts) with advanced-stage MF. Azacitidine (Vidaza™) is a hypomethylating agent that induces reactivation of methylated genes and has been approved for the treatment of pts with myelodysplastic syndromes. The objective of this phase II trial was to study the efficacy of azacitidine in pts with relapsed or refractory MF (primary, or secondary to essential thrombocythemia or polycythemia vera) or newly-diagnosed with a Lille risk score 1 or 2 (risk factor are white blood cell [WBC] count >30x109/L or <4x109/L, and hemoglobin [Hb] <10 g/dL). Azacitidine was given at 75 mg/m_ daily (dose level [DL] 0) for 7 days every 4 weeks for 6 cycles but this could be reduced to 50 (DL -1), 25 (DL -2), or 12.5 (DL -3) or increased to 100 (DL +1) mg/m_ according to toxicity or lack of response, respectively. Thirty-four pts have been treated, median age 66 years (range 39–82), time from MF diagnosis to azacitidine therapy 21 months (range 1–361), median Hb 10.3 g/dL (range 8.2–14.3), WBC 10.7x109/L (range 1.7–57.7), and platelets 205x109/L (range 11–1216). Pts had received a median of 1 prior therapy (range 0–6), including hydroxyurea (HU; n=16), anagrelide (AG; n=8), lenalidomide (n=6), thalidomide (n=6), etanercept (n=2), and pegylated interferon (n=2). Six pts had not received any prior therapy. The JAK2 V617F mutation was detected in 19 (70%) of 27 tested pts and 12 of 34 (35%) had abnormal cytogenetics. A total of 142 cycles have been administered. All 34 patients are evaluable for response and toxicity. Responses have been observed in 10 (29%) pts so far, including complete response (CR) in 1 (normal blood counts and <5% bone marrow blasts; off HU, AG, growth factors, and no transfusions), partial response (PR) in 7 (defined as at least 2 of the following: Hb increase by ≥2 g/dL, 50% increase in platelets, decrease of bone marrow blasts or organomegaly by ≥50%, normalization of WBC count without blasts, or reduction in bone marrow fibrosis), and hematologic improvement (HI) in 2 (defined as at least 2 of the following: decrease by ≥25% of pretreatment leukocytosis, splenomegaly, or marrow blasts, or increase of Hb by ≥1g/dl or platelets by ≥25%). The median time to best response was 7.5 weeks (range, 3 to 26). Fifteen (44%) pts discontinued azacitidine due to: lack of response in 7, transformation to acute myeloid leukemia in 2, death (unrelated to azacitidine therapy) in 1, pt's decision in 1, grade 3–4 toxicity in 2, and other medical reasons in 2. Azacitidine has been generally well tolerated; 10 pts (29%) had grade 3 or 4 toxicity; neutropenia was the only grade 4 toxicity (in 4 pts). Eleven pts (32%) have required dose reduction to DL -1, of which 3 reduced to DL -2, and 9 pts (26%) escalated the dose to DL +1. In summary, azacitidine is well tolerated and has activity in a subset of pts with MF, with current overall response rate of 29%. Updated clinical results will be presented.

Detection of Residual p73 DNA Methylation Predicts for Shorter Disease Free and Overall Survival in Patients (pts) with Philadelphia (Ph) Chromosome Negative Acute Lymphocytic Leukemia (ALL) in Remission.

Aberrant DNA methylation of multiple promoter CpG islands is a common feature of ALL. Specific DNA methylation patterns involving a triad of hierarchical clustered cell cycle controlling genes including p73, p15 and p57KIP2 predict for poor prognosis in Ph negative ALL (Garcia-Manero, Blood, 2003). Of importance, this aberrant DNA methylation patterns are stable at the time of relapse in a majority of patients. This indicates that aberrant DNA methylation is a fundamental molecular feature of the ALL blast cell (Garcia-Manero, Clinical Cancer Research, 2002). Based on this, we hypothesized that the detection of residual DNA methylation in patients with standard risk (Ph negative) ALL could predict for shorter disease free survival, and worse overall survival (OS) in this patient population. To test this hypothesis, we have analyzed the frequency of p73, p15 and p57KIP2 DNA methylation at the time of initial documentation of complete remission (CR) (days 14 to 21) in 199 patients in CR with Ph negative ALL. All patients had been treated with hyperCVAD-based chemotherapy at our institution. To perform this analysis, we have developed a sensitive and specific novel real-time methylation specific PCR assay to detect DNA methylation. This method requires bisulfite DNA conversion. It uses the interferon-gamma gene as a DNA loading control and can be performed in less than 24 hours. The characteristics of the 199 patients evaluated are: median age 38 years (range 15–83), cytogenetics: 32% diploid, 5% hyper or hypodiploid, 20% insufficient metaphases and 3% MLL+, the rest others. 16% were of T cell phenotype. Using this assay, p73 methylation was detected in 18 (10%) patients, p15 in 33 (16%) and p57KIP2 in 7 (3.5%). Methylation of at least 1 gene of this triad was observed in 48 (24%) patients. The median OS of the whole group was 209 weeks (95% CI 158–514) and the disease free survival (DFS) 146 weeks (95% CI 110–335). These were representative of the overall experience with hyperCVAD at MDACC. By multivariate analysis, the presence of residual p73 methylation on days 14 to 21 in patients with Ph negative ALL was associated with a shorter DFS (hazard ratio 2.68, p=0.003) and a worse OS (hazard ratio 2.69, p=0.002). This was independent of any other patient characteristic. Other characteristic included in these models included age and platelet count for OS, and age, b2-microglobulin, LDH and platelets for DFS. The presence of p15, p57KIP2 methylation or the combination of multiple genes was not associated with shorter OS or DFS. In summary, the detection of residual p73 methylation allows the identification of a subset of patients with ALL in remission and worse prognosis. This data may allow the development of specific therapeutic interventions, such as the use of hypomethylating agents, or early allogeneic stem cell transplantation for this at-risk patient population, and suggest that inactivation of p73 is a key event in ALL.

Mutational analyses of WNT7A and HDAC11 as candidate tumour suppressor genes in sporadic malignant pancreatic endocrine tumours

We and others have reported loss of heterozygosity (LOH) on chromosome 3p25 in sporadic malignant pancreatic endocrine tumours (PETs). A common region of deletion on chromosome 3p25 contains numerous genes, including VHL and PPARgamma, that have been excluded previously as candidate tumour suppressor genes by DNA sequencing analysis. We have analysed whether WNT7A or HDAC11 was biallelically inactivated in a group of well-characterized PETs. Ten PETs from eight patients were selected from a previous study, where LOH on chromosome 3p25 was found in 11 out of 22 sporadic PETs. These tumours were examined for inactivating mutations of WNT7A and HDAC11 by direct sequencing of all exons and intron-exon boundaries. Inactivation of WNT7A expression by aberrant CpG island methylation and WNT7A protein expression were evaluated by methylation-specific polymerase chain reaction (PCR) and immunohistochemistry, respectively. HDAC11 protein expression was also examined. No point mutations, deletion or insertions were detected in either WNT7A or HDAC11 in any of the PETs. Two polymorphisms were identified in the third exon of the WNT7A gene. CpG methylation of the WNT7A gene was not detected and the WNT7A and HDAC11 proteins were normally expressed. The absence of tumour-specific somatic events in WNT7A and HDAC11 suggests that these genes are unlikely to have a classical tumour suppressor gene role in sporadic malignant PETs. The putative 3p25 tumour suppressor remains to be identified.

Preproenkephalin hypermethylation in the pure pancreatic juice compared with p53 mutation in the diagnosis of pancreatic carcinoma

Aberrant methylation of CpG islands is a common mechanism for the dysregulation of tumor suppressor genes in a variety of human malignancies. Preproenkephalin ppENK) hypermethylation is recognized in 90% of pancreatic carcinoma (PCa) tissues, but not in normal pancreas. We analyzed ppENK hypermethylation in pure pancreatic juice (PPJ) in patients with PCa, intraductal papillary mucinous neoplasms (IPMN), and chronic pancreatitis (CP), and elucidated its usefulness as a marker in the diagnosis of PCa compared with p53 mutation. PPJ was collected endoscopically from 28 patients with PCa, 15 patients with IPMN, and 20 patients with CP. DNA was extracted from the supernatant and the sediment of PPJ. Methylation-specific polymerase chain reaction was performed for hypermethylation analysis of ppENK. In addition, single-strand conformation polymorphism and direct sequencing were performed simultaneously to identify p53 mutations. The incidence of ppENK hypermethylation in the supernatant and/or the sediment of PPJ was 50% (14 of 28) in patients with PCa. In contrast, the incidence of ppENK hypermethylation was 26.7% (4 of 15) in patients with IPMN, and 5% (1 of 20) in patients with CP (P < 0.002). The incidence of p53 mutations in the PPJ was 42.9% (12 of 28) in patients with PCa and 0% (0 of 20) in patients with CP. Furthermore, the incidence of ppENK hypermethylation and/or p53 mutations in the PPJ was enhanced to 67.9% (19 of 28) in patients with PCa in the combination assay. These results suggest that ppENK hypermethylation in PPJ is specific for cancer, and the combination assay with p53 enhances the genetic diagnosis of PCa.

Epigenetic abnormalities in cutaneous squamous cell carcinomas: frequent inactivation of the RB1/p16 and p53 pathways

Aberrant methylation of CpG islands in the promoter regions of cancer-related genes has been demonstrated in many human tumours. However, the methylation profile of these regions in cutaneous squamous cell carcinomas (SCCs) has not been well studied. To examine epigenetic abnormalities of a wide range of cancer-related genes in SCCs. We investigated the methylation status of 11 candidate cancer-related genes (CDH1, p16(INK4a), p14(ARF), DAPK1, MGMT, RB1, RASSF1, p15(INK4b), PTEN, PRDM2 and p53) in 20 cases of SCC by methylation-specific polymerase chain reaction, and comparatively examined the protein production of E-cadherin (CDH1), p16, RB1, p14, BMI1 and cyclin A by immunohistochemical analysis. The frequency of cancer-related gene methylation in SCCs was: CDH1 (95%), p16 (20%), p14 (15%), DAPK1 (15%), MGMT (15%), RB1 (5%), RASSF1 (5%), p15 (0%), PTEN (0%), PRDM2 (0%) and p53 (0%). Almost all cases with hypermethylation of CDH1, p16, RB1 and p14 showed no obvious production of each protein, suggesting that promoter hypermethylation of these genes contributes to the loss of protein production. The results of methylation analysis, in combination with the results of our previous mutation analysis of CDKN2A locus and p53, revealed that 70% of SCCs have alterations in the RB1/p16 or p53 pathway. Our findings indicate that the promoter hypermethylation of cancer-related genes, especially CDH1, is frequently shown in SCCs, and dysregulation of the RB1/p16 and/or p53 pathway through either genetic or epigenetic mechanisms, except for epigenetic abnormalities of p53 itself, should contribute to the carcinogenesis of SCCs.

Promoter methylation status of the MGMT, hMLH1, and CDKN2A/p16 genes in non-neoplastic mucosa of patients with and without colorectal adenomas

The aberrant methylation of CpG islands is a common epigenetic alteration found in cancers. The process contributes to cancer formation through the transcriptional silencing of tumor suppressor genes. CpG island methylation has been observed in aberrant crypt foci (ACF) and adenomas in the colon, implicating it in the earliest aspects of colon cancer formation. In addition, some investigators have identified an age-related increase in DNA methylation of the ESR1 locus in the colon mucosa, suggesting that DNA methylation may be a pre-neoplastic change that increases the risk of colon adenomas and colon cancer. We investigated the methylation status in the promoter regions of the CDKN2A/p16, hMLH1, and MGMT genes in human non-neoplastic rectal mucosa and evaluated whether these methylation markers may predict the presence of adenomatous polyps in the colon. The promoter methylation patterns of these genes were examined in rectal biopsies (mucosa samples) of 97 colorectal adenoma cases and 94 healthy controls using methylation-specific PCR (MSP) assays. Methylation of the MGMT and hMLH1 genes was present in both cases and controls, with a frequency of 12.4% and 18.1% for the MGMT gene and 12.4% and 11.7% for the hMLH1 gene. The frequency of CDKN2A/p16 promoter methylation was very rare in normal colorectal tissue with a frequency of approximately 2%. Overall, no apparent case-control difference was identified in the methylation status of these genes, either alone or in combination. hMLH1 methylation was more frequently observed among overweight or obese subjects (BMI>/=25) with an adjusted OR of 3.7 (95% CI=1.0-13.7). Methylated alleles of the hMLH1 and MGMT genes were frequently detected in normal rectal mucosa, while the frequency of CDKN2A/p16 methylation detected was very low. The methylation status of these genes in rectal mucosa biopsies detected by MSP assays may not distinguish between patients with and without adenomas in the colon.

Epigenetic Regulation of Human γ-Glutamyl Hydrolase Activity in Acute Lymphoblastic Leukemia Cells

Gamma-glutamyl hydrolase (GGH) catalyzes degradation of the active polyglutamates of natural folates and the antifolate methotrexate (MTX). We found that GGH activity is directly related to GGH messenger RNA expression in acute lymphoblastic leukemia (ALL) cells of patients with a wild-type germline GGH genotype. We identified two CpG islands (CpG1 and CpG2) in the region extending from the GGH promoter through the first exon and into intron 1 and showed that methylation of both CpG islands in the GGH promoter (seen in leukemia cells from approximately 15% of patients with nonhyperdiploid B-lineage ALL) is associated with significantly reduced GGH mRNA expression and catalytic activity and with significantly higher accumulation of MTX polyglutamates (MTXPG(4-7)) in ALL cells. Furthermore, methylation of CpG1 was leukemia-cell specific and had a pronounced effect on GGH expression, whereas methylation of CpG2 was common in leukemia cells and normal leukocytes but did not significantly alter GGH expression. These findings indicate that GGH activity in human leukemia cells is regulated by epigenetic changes, in addition to previously recognized genetic polymorphisms and karyotypic abnormalities, which collectively determine interindividual differences in GGH activity and influence MTXPG accumulation in leukemia cells.

Predicting methylation status of CpG islands in the human brain

Over 50% of human genes contain CpG islands in their 5'-regions. Methylation patterns of CpG islands are involved in tissue-specific gene expression and regulation. Mis-epigenetic silencing associated with aberrant CpG island methylation is one mechanism leading to the loss of tumor suppressor functions in cancer cells. Large-scale experimental detection of DNA methylation is still both labor-intensive and time-consuming. Therefore, it is necessary to develop in silico approaches for predicting methylation status of CpG islands. Based on a recent genome-scale dataset of DNA methylation in human brain tissues, we developed a classifier called MethCGI for predicting methylation status of CpG islands using a support vector machine (SVM). Nucleotide sequence contents as well as transcription factor binding sites (TFBSs) are used as features for the classification. The method achieves specificity of 84.65% and sensitivity of 84.32% on the brain data, and can also correctly predict about two-third of the data from other tissues reported in the MethDB database. An online predictor based on MethCGI is available at http://166.111.201.7/MethCGI.html mzhang@cshl.edu Supplementary data available at Bioinformatics online and http://166.111.201.7/help.html.

Hypermethylation of the GATA gene family in esophageal cancer

The GATA family of transcription factors promotes the normal development of the gastrointestinal tract during embryogenesis and determines tissue differentiation in adult gut epithelium. Loss of GATA-4 and GATA-5 has been reported in human gastric and colon cancer. We examined GATA-4,-5 and -6 gene expression in established esophageal squamous cancer cell lines and the relationship to DNA methylation in the promoter region of these genes. GATA-4 and GATA-5 expression was absent in most esophageal cancer cell lines, but was restored upon treatment with the demethylating agent 5-aza-2'-deoxycytidine. For each of the cell lines without detectable GATA gene expression, aberrant methylation of the promoter region CpG-island was detected by methylation specific PCR. We confirmed these results with genomic bisulfite sequencing. GATA-6 expression was found in each of the cell lines. GATA-4/-5 promoter methylation was not detected in normal esophageal mucosa, but GATA-4 methylation was present in 27 of 44 (61%) squamous carcinomas and 31 of 44 (71%) adenocarcinoma of the esophagus, while GATA-5 methylation was present in 14 of 44 (32%) squamous carcinomas and 24 of 44 (55%) adenocarcinoma of the esophagus. Our studies demonstrate frequent silencing of GATA-4 and GATA-5, but not GATA-6, in human esophageal neoplasia associated with gene promoter hypermethylation.