Hypermethylation of apoptotic genes as independent prognostic factor in neuroblastoma disease

Neuroblastoma (NB) is an embryonal tumour of neuroectodermal cells, and its prognosis is based on patient age at diagnosis, tumour stage and MYCN amplification, but it can also be classified according to their degree of methylation. Considering that epigenetic aberrations could influence patient survival, we studied the methylation status of a series of 17 genes functionally involved in different cellular pathways in patients with NB and their impact on survival. We studied 82 primary NB tumours and we used methylation-specific-PCR to perform the epigenetic analysis. We evaluated the putative association among the evidence of hypermethylation with the most important NB prognostic factors, as well as to determine the relationship among methylation, clinical classification and survival. CASP8 hypermethylation showed association with relapse susceptibility and, TMS1 and APAF1 hypermethylation are associated with bad prognosis and showed high influence on NB overall survival. Hypermethylation of apoptotic genes has been identified as a good candidate of prognostic factor. We propose the simultaneous analysis of hypermethylation of APAF1, TMS1 and CASP8 apoptotic genes on primary NB tumour as a good prognostic factor of disease progression.     

Aberrant DNA methylation in pediatric patients with acute lymphocytic leukemia

BACKGROUND: Aberrant methylation of promoter-associated cystosine-guanine (CpG) islands is an epigenetic modification of DNA frequently observed in adult patients with acute lymphocytic leukemia (ALL). This epigenetic modification has been associated with gene silencing, malignant transformation, and aging. It is not known whether there are epigenetic differences between pediatric patients and adult patients with ALL. METHODS: To investigate the methylation characteristics of pediatric patients with ALL and to determine whether DNA methylation can explain prognostic or biologic differences between pediatric and adult patients, the authors analyzed the methylation status of 7 promoter-associated CpG islands in 16 pediatric patients with ALL and compared them with the methylation characteristics of a cohort of adult patients with ALL. The genes analyzed included the estrogen receptor gene (ER), multidrug resistance gene 1 (MDR1), p15, C-ABL, CD10, p16, and p73. RESULTS: The mean methylation densities of ER, MDR1, CD10, p15, and C-ABL were 25.4%, 16.4%, 5.23%, 4.24%, and 4%, respectively. P16 was methylated in 11.7% of patients, and p73 was methylated in 17.6% of patients. One patient (6.2%) had methylation of 0 genes, 15 patients (93.7%) had methylation of >/= 1 gene, and 4 patients (25%) had methylation of 3-4 genes. Methylation of all these genes was < 2% (or methylation specific polymerase chain reaction negative) in nonneoplastic tissues. A significant inverse correlation was observed between methylation of CD10 and CD10 expression. No differences were observed between the methylation characteristics of pediatric patients and adult patients. CONCLUSIONS: The results indicate that DNA methylation is common in pediatric patients with ALL and that methylation of the genes studied does not account for prognostic differences between pediatric patients and adult patients with ALL.     

抑癌基因甲基化在肾细胞癌研究中的新进展

抑癌基因启动子高度甲基化被认为是除突变和缺失以外的抑癌基因功能失活的关键机制,在肿瘤的发生和发展中起重要作用,已成为目前肿瘤病因学基础研究的热点.肾细胞癌是泌尿系统最常见的恶性肿瘤之一,近年来也有许多文献报道了肾细胞癌中抑癌基因的异常甲基化情况.肾细胞癌临床发病隐匿,出现症状时多为晚期,肾细胞癌相关基因异常甲基化检测有望为肾癌的早期无创诊断提供新的途径.针对肾癌对放、化疗效果均不敏感的特点,改变DNA甲基转移酶活性和抑癌基因甲基化状况可作为肾细胞癌辅助治疗的一种新思路.本文对抑癌基因甲基化与肾细胞癌关系的研究进展作一综述,介绍DNA甲基化在肿瘤发生过程中的作用及机理;总结近7年来肾细胞癌中抑癌基因甲基化的研究情况和肾细胞癌独特的甲基化谱,并着重介绍了新近报道的HOXB13,HAI2/SPINT2,CDH1,CTNNG/JUP四个基因启动子异常甲基化与肾细胞癌的关系;阐述DNA甲基化研究对肾细胞癌的临床意义.     

The prognostic role of epigenetic dysregulation in bladder cancer: A systematic review

BackgroundDespite adequate treatment and follow-up, around one fifth of patients with localized bladder cancer will present with disease progression. Adequate prognostic biomarkers are lacking to define patients who are at risk. Mutations in chromatin remodeling genes are more frequently found in bladder cancer than in any other solid tumor. However, the prognostic relevance of epigenetic dysregulation has not been established and may offer an opportunity for biomarker discovery.MethodsLooking for prognostic epigenetic factors, we performed a comprehensive PubMed search using keywords such as “bladder cancer”, “chromatin remodeling”, “gene methylation” and “epigenetics”. We only included studies reporting on the association of epigenetic markers with prognostic outcomes such as recurrence, progression or survival.ResultsOf 1113 results, 87 studies met the inclusion criteria, which represented a total of 85 epigenetic markers with potential prognostic relevance. No prospective studies were identified. Seventy-three percent (64/87) of the studies involved mixed cohorts of muscle invasive and non-muscle invasive bladder cancer. Promoter methylation of genes with putative prognostic value affected cellular processes such as cell cycle, apoptosis, cell-adhesion or migration, as well as critical pathways such as MAP-kinase or Wnt. Alteration of chromatin regulatory elements suggest a prognostic relevance alterations leading to a predominantly silenced chromatin state.ConclusionsThe prognostic impact of epigenetic alterations in bladder cancer is still unclear. Prospective evaluation of methylation marks and chromatin remodeling gene alterations using consistent methods and criteria is warranted.     

Prognostic significance of aberrant gene methylation in gastric cancer

Promoter methylation acts as an important alternative to genetic alterations for gene inactivation in gastric carcinogenesis. Although a number of gastric cancer-associated genes have been found to be methylated in gastric cancer, valuable methylation markers for early diagnosis and prognostic evaluation of this cancer remain largely unknown. In the present study, we used methylation-specific PCR (MSP) to analyze promoter methylation of 9 gastric cancer-associated genes, including MLF1, MGMT, p16, RASSF2, hMLH1, HAND1, HRASLS, TM, and FLNc, and their association with clinicopathological characteristics and clinical outcome in a large cohort of gastric cancers. Our data showed that all of these genes were aberrantly methylated in gastric cancer, ranging from 8% to 51%. Moreover, gene methylation was strongly associated with certain clinicopathological characteristics, such as tumor differentiation, lymph node metastasis, and cancer-related death. Of interest, methylation of MGMT, p16, RASSF2, hMLH1, HAND1, and FLNc was closely associated with poor survival in gastric cancer, particularly MGMT, p16, RASSF2 and FLNc. Thus, our findings suggested these epigenetic events may contribute to the initiation and progression of gastric cancer. Importantly, methylation of some genes were closely relevant to poor prognosis in gastric cancer, providing the strong evidences that these hypermethylated genes may be served as valuable biomarkers for prognostic evaluation in this cancer.     

Promoter methylation of RASSF1A, RARbeta and DAPK predict poor prognosis of patients with malignant mesothelioma

Hypermethylation occurs frequently in neoplastic cells and affects tumorigenesis. Malignant mesothelioma is an aggressive cancer developing in the thoracic cavity and patients have a rather bad prognosis. Our goal was to determine epigenetic alterations of a series of genes and to analyse the potential correlation of such changes with overall survival. We have analysed the methylation status of the promoter region of nine genes in serum DNA of mesothelioma patients by a nested methylation-specific PCR. Modest methylation frequencies were detected for APC1A (14.3%), RASSF1A (19.5%) and DAPK (20.0%) while hypermethylation of E-cadherin (71.4%) and FHIT (78.0%) occurred at a high incidence. Intermediate values were seen for p16(INK4a) (28.2%), APC1B (32.5%), p14(ARF) (44.2%) and RARbeta (55.8%). The methylation status of none of the single genes significantly influenced prognosis. In contrast, combining RARbeta with either DAPK or RASSF1A showed a significantly shorter overall survival of those patients who had both genes methylated compared to those with only one or no epigenetic alteration (P=0.025 and 0.040, respectively). Similarly, the combination of all three genes revealed a worse prognosis for patients with double or triple methylations compared to the group which had only one or no gene methylated (P=0.028). Our results support the idea that the prognostic value of a combination of epigenetic alterations is superior to the impact of an individual gene alone on overall survival.     

Transcriptional silencing of the Dickkopfs-3 (Dkk-3) gene by CpG hypermethylation in acute lymphoblastic leukaemia

DKK-3: is a newly characterised mortalisation-related gene and an antagonist of the Wnt oncogenic signalling pathway whose expression is decreased in a variety of cancer cell lines, suggesting that the Dkk-3 gene, located at chromosome 11p15.1, functions as a tumour suppressor gene. Although 11p15 is a 'hot spot' for methylation in acute lymphoblastic leukaemia (ALL), the role of Dkk-3 abnormalities has never been evaluated in this disease. We analysed CpG island methylation of the Dkk-3 promoter in six ALL cell lines and 183 ALL patients. We observed Dkk-3 hypermethylation in all cell lines and in cells from 33% (60/183) of ALL patients. Moreover, Dkk-3 methylation was associated with decreased Dkk-3 mRNA expression and this expression was restored after exposure to the demethylating agent 5-AzaC. Clinical features did not differ between hypermethylated and unmethylated patients. Estimated disease-free survival (DFS) and overall survival at 10 and 11 years, respectively, were 49.8 and 45.6% for normal patients and 10.5 and 15.1% for hypermethylated patients (P=0.001 and 0.09). Multivariate analysis demonstrated that Dkk-3 methylation was an independent prognostic factor predicting DFS (P=0.0009). Our data suggest that Dkk-3 methylation occurs at an early stage in ALL pathogenesis and probably influences the clinical behaviour of the disease.     

Hypermethylation of CpG island in the promoter region of CALCA in acute lymphoblastic leukemia with central nervous system (CNS) infiltration correlates with poorer prognosis

Promoter hypermethylation occurs early in leukemogenesis and seems to be associated with poor prognosis in acute lymphoblastic leukemia (ALL). The methylation status of the promoter region of six genes was analyzed in 71 children with ALL using methylation specific PCR (MSP). Calcitonin (CALCA) and E-cadherin (CDH1) were the most frequently methylated genes in this group of patients. Considering the patients with central nervous system (CNS) infiltration, the estimated 2-year overall survival (OS) was 20% for those with methylation in CALCA promoter and 85% for those without (p=0.001). Our results suggest that the hypermethylation of CALCA promoter is a promising prognostic marker and may predict a higher risk for ALL patients with CNS infiltration.     

Methylation in oral cancer and pre-cancerous lesions (Review)

There is considerable interest in the analysis of epigenetic alterations in cancer, including oral cancer and pre-cancerous lesions. These processes affect or inactivate the functions of genes without altering their structure or sequence. One example is the methylation of the promoter region of some genes involved in cell cycle control. Knowledge of methylation patterns is very important for understanding the expression of genes in normal and pathological situations. This review provides an update on research into this issue in oral cancer and pre-cancerous lesions. A greater understanding of this epigenetic alteration could not only assist the diagnosis and prognosis of oral cancer but could also open up novel therapeutic approaches. The presence of methylation in specific tumour suppressor genes could modify their function and alter cell cycle control, so the patients could have an increased risk of developing cancer and also a higher degree of malignancy. The most frequently and extensively studied methylated genes in oral premalignant lesions are p16, MGMT, RARβ2, E-cadherin and DAP-kinase.     

Combined DNA methylation and gene expression profiling in gastrointestinal stromal tumors reveals hypomethylation of SPP1 as an independent prognostic factor

Gastrointestinal stromal tumors (GISTs) have distinct gene expression patterns according to localization, genotype and aggressiveness. DNA methylation at CpG dinucleotides is an important mechanism for regulation of gene expression. We performed targeted DNA methylation analysis of 1.505 CpG loci in 807 cancer‐related genes in a cohort of 76 GISTs, combined with genome‐wide mRNA expression analysis in 22 GISTs, to identify signatures associated with clinicopathological parameters and prognosis. Principal component analysis revealed distinct DNA methylation patterns associated with anatomical localization, genotype, mitotic counts and clinical follow‐up. Methylation of a single CpG dinucleotide in the non‐CpG island promoter of SPP1 was significantly correlated with shorter disease‐free survival. Hypomethylation of this CpG was an independent prognostic parameter in a multivariate analysis compared to anatomical localization, genotype, tumor size and mitotic counts in a cohort of 141 GISTs with clinical follow‐up. The epigenetic regulation of SPP1 was confirmed in vitro, and the functional impact of SPP1 protein on tumorigenesis‐related signaling pathways was demonstrated. In summary, SPP1 promoter methylation is a novel and independent prognostic parameter in GISTs, and might be helpful in estimating the aggressiveness of GISTs from the intermediate‐risk category.     

Prognostic Relevance of SFRP1 Gene Promoter Methylation in Colorectal Carcinoma

Background: The development of colorectal carcinoma (CRC) involves many genetic and epigenetic alterations andmethylation being an important epigenetic event has been described as a diagnostic and prognostic biomarker. SecretedFrizzled- Related Protein 1 (SFRP1) gene regulates diverse physiological processes via the Wnt signaling. Promoterhypermethylation of SFRP1 gene is an epigenetic regulation mechanism that downregulates SFRP1 protein level in thetumor, and happens to be one of the significant events in colorectal carcinogenesis. We studied the clinicopathologicalrelationship of CRC including survival outcomes with SFRP1 gene promoter methylation. Methods: We evaluatedpromoter methylation status of SFRP1 gene by methylation-specific PCR (MS-PCR) in the tumor tissue in 54 casesof stage II-III CRC patients in north India. The MS-PCR result was further validated by bisulfite sequencing. Results:SFRP1 gene was methylated in 72.2% cases and un-methylated in 27.8%. We found, that SFRP1 gene methylation intumor was associated with lymph node invasion (p=0.05). The mean overall survival was 22.318 months and 45.173months respectively for patients with methylated and unmethylated SFRP1 gene (p= 0.010, log rank test), (HR = 17.313,95% CI: 2.021-148.290 P=0.009). Conclusion: Study indicates that promoter methylation of SFRP1 gene is associatedwith lymph-node metastasis and poor mean overall survival and it can be a prognostic marker in CRC.     

Expression of late cell cycle genes and an increased proliferative capacity characterize very early relapse of childhood acute lymphoblastic leukemia.

PURPOSE: In childhood acute lymphoblastic leukemia (ALL), approximately 25% of patients suffer from relapse. In recurrent disease, despite intensified therapy, overall cure rates of 40% remain unsatisfactory and survival rates are particularly poor in certain subgroups. The probability of long-term survival after relapse is predicted from well-established prognostic factors (i.e., time and site of relapse, immunophenotype, and minimal residual disease). However, the underlying biological determinants of these prognostic factors remain poorly understood. EXPERIMENTAL DESIGN: Aiming at identifying molecular pathways associated with these clinically well-defined prognostic factors, we did gene expression profiling on 60 prospectively collected samples of first relapse patients enrolled on the relapse trial ALL-REZ BFM 2002 of the Berlin-Frankfurt-Münster study group. RESULTS: We show here that patients with very early relapse of ALL are characterized by a distinctive gene expression pattern. We identified a set of 83 genes differentially expressed in very early relapsed ALL compared with late relapsed disease. The vast majority of genes were up-regulated and many were late cell cycle genes with a function in mitosis. In addition, samples from patients with very early relapse showed a significant increase in the percentage of S and G(2)-M phase cells and this correlated well with the expression level of cell cycle genes. CONCLUSIONS: Very early relapse of ALL is characterized by an increased proliferative capacity of leukemic blasts and up-regulated mitotic genes. The latter suggests that novel drugs, targeting late cell cycle proteins, might be beneficial for these patients that typically face a dismal prognosis.     

Promoter methylation of AREG, HOXA11, hMLH1, NDRG2, NPTX2 and Tes genes in glioblastoma

Epigenetic alterations alone or in combination with genetic mechanisms play a key role in brain tumorigenesis. Glioblastoma is one of the most common, lethal and poor clinical outcome primary brain tumors with extraordinarily miscellaneous epigenetic alterations profile. The aim of this study was to investigate new potential prognostic epigenetic markers such as AREG, HOXA11, hMLH1, NDRG2, NTPX2 and Tes genes promoter methylation, frequency and value for patients outcome. We examined the promoter methylation status using methylation-specific polymerase chain reaction in 100 glioblastoma tissue samples. The value for clinical outcome was calculated using Kaplan–Meier estimation with log-rank test. DNA promoter methylation was frequent event appearing more than 45 % for gene. AREG and HOXA11 methylation status was significantly associated with patient age. HOXA11 showed the tendency to be associated with patient outcome in glioblastomas. AREG gene promoter methylation showed significant correlation with poor patient outcome. AREG methylation remained significantly associated with patient survival in a Cox multivariate model including MGMT promoter methylation status. This study of new epigenetic targets has shown considerably high level of analyzed genes promoter methylation variability in glioblastoma tissue. AREG gene might be valuable marker for glioblastoma patient survival prognosis, however further analysis is needed to clarify the independence and appropriateness of the marker.     

DLEC1 and MLH1 promoter methylation are associated with poor prognosis in non-small cell lung carcinoma

The significance of chromosome 3p gene alterations in lung cancer is poorly understood. This study set out to investigate promoter methylation in the deleted in lung and oesophageal cancer 1 (DLEC1), MLH1 and other 3p genes in 239 non-small cell lung carcinomas (NSCLC). DLEC1 was methylated in 38.7%, MLH1 in 35.7%, RARbeta in 51.7%, RASSF1A in 32.4% and BLU in 35.3% of tumours. Any two of the gene alterations were associated with each other except RARbeta. DLEC1 methylation was an independent marker of poor survival in the whole cohort (P=0.025) and in squamous cell carcinoma (P=0.041). MLH1 methylation was also prognostic, particularly in large cell cancer (P=0.006). Concordant methylation of DLEC1/MLH1 was the strongest independent indicator of poor prognosis in the whole cohort (P=0.009). However, microsatellite instability and loss of MLH1 expression was rare, suggesting that MLH1 promoter methylation does not usually lead to gene silencing in lung cancer. This is the first study describing the prognostic value of DLEC1 and MLH1 methylation in NSCLC. The concordant methylation is possibly a consequence of a long-range epigenetic effect in this region of chromosome 3p, which has recently been described in other cancers.     

Methylation of CASP8, DCR2, and HIN-1 in neuroblastoma is associated with poor outcome.

PURPOSE: Epigenetic aberrations have been shown to play an important role in the pathogenesis of most cancers. To investigate the clinical significance of epigenetic changes in neuroblastoma, we evaluated the relationship between clinicopathologic variables and the pattern of gene methylation in neuroblastoma cell lines and tumors. EXPERIMENTAL DESIGN: Methylation-specific PCR was used to evaluate the gene methylation status of 19 genes in 14 neuroblastoma cell lines and 8 genes in 70 primary neuroblastoma tumors. Associations between gene methylation, established prognostic factors, and outcome were evaluated. Log-rank tests were used to identify the number of methylated genes that was most predictive of overall survival. RESULTS: Epigenetic changes were detected in the neuroblastoma cell lines and primary tumors, although the pattern of methylation varied. Eight of the 19 genes analyzed were methylated in >70% of the cell lines. Epigenetic changes of four genes were detected in only small numbers of cell lines. None of the cell lines had methylation of the other seven genes analyzed. In primary neuroblastoma tumors, high-risk disease and poor outcome were associated with methylation of DCR2, CASP8, and HIN-1 individually. Although methylation of the other five individual genes was not predictive of poor outcome, a trend toward decreased survival was seen in patients with a methylation phenotype, defined as > or =4 methylated genes (P = 0.055). CONCLUSION: Our study indicates that clinically aggressive neuroblastoma tumors have aberrant methylation of multiple genes and provides a rationale for exploring treatment strategies that include demethylating agents.     

<Emphasis Type="Italic">WT1, MSH6</Emphasis>, <Emphasis Type="Italic">GATA5</Emphasis> and <Emphasis Type="Italic">PAX5</Emphasis> as epigenetic oral squamous cell carcinoma biomarkers - a short report

Purpose

Oral squamous cell carcinoma (OSCC) is a frequently occurring aggressive malignancy with a heterogeneous clinical behavior. Based on the paucity of specific early diagnostic and prognostic biomarkers, which hampers the appropriate treatment and, ultimately the development of novel targeted therapies, we aimed at identifying such biomarkers through a genetic and epigenetic analysis of these tumors.

Methods

93 primary OSCCs were subjected to DNA copy number alteration (CNA) and methylation status analyses using methylation-specific multiplex ligation-dependent probe amplification (MS-MPLA). The genetic and epigenetic OSCC profiles obtained were associated with the patients’ clinic-pathological features.

Results

We found that WT1 gene promoter methylation is a predictor of a better prognosis and that MSH6 and GATA5 gene promoter methylation serve as predictors of a worse prognosis. GATA5 gene promoter methylation was found to be significantly associated with a shorter survival rate. In addition, we found that PAX5 gene promoter methylation was significantly associated with tongue tumors. To the best of our knowledge, this is the first study that highlights this specific set of genes as epigenetic diagnostic and prognostic biomarkers in OSCC.

Conclusions

Our data highlight the importance of epigenetically assessing OSCCs to identify key genes that may serve as diagnostic and prognostic biomarkers and, potentially, as candidate therapeutic targets.