Prognostic relevance of methylation markers in patients with non-muscle invasive bladder carcinoma

There is increasing evidence for the role of epigenetic gene silencing in superficial bladder cancer. The aim of the current study was to investigate the prognostic value of epigenetic alterations in patients with non-muscle invasive bladder carcinoma. We checked the methylation status of 20 cancer associated genes (p14ARF, p16 CDKN2A, STAT-1, SOCS-1, DR-3, DR-6, PIG-7, BCL-2, H-TERT, BAX, EDNRB, DAPK, RASSF-1A, FADD, TMS-1, E-Cadherin, ICAM-1, TIMP-3, MLH-1, COX-2) for DNA methylation. We analysed microdissected tumour samples from 105 consecutive patients with primary non-muscle invasive bladder carcinoma. Quantitative methylation analysis of CpG sites in the promoter region of the genes was performed with methylation sensitive quantitative real time PCR ('Methylight'). Univariate analysis for association with tumour recurrence was carried out with the Kaplan-Meier analysis and the log-rank test. Follow-up data were available in 95/105 patients (91.4%). A tumour recurrence was observed in 26 patients (27.3%). We could identify six genes (SOCS-1, STAT-1, BCL-2, DAPK, TIMP-3, E-Cadherin), where methylation was associated with tumour recurrence. In Kaplan-Meier analysis, TIMP-3 showed a significant association with recurrence free survival. Methylation of TIMP-3 predicted prolonged disease free interval. In this study, we report a comprehensive analysis on prognostic relevance of gene methylation in non-muscle invasive bladder cancer. We identified one gene (TIMP-3) where methylation was associated with a more favourable outcome. Our data strongly support the usefulness of gene methylation as a prognostic marker in patients with non-muscle invasive bladder cancer.     

Epigenetic plasticity potentiates a rapid cyclical shift to and from an aggressive cancer phenotype

Highly tumorigenic, drug-resistant cancer stem-like cells drive cancer progression. These aggressive cells can arise repeatedly from bulk tumor cells independently of mutational events, suggesting an epigenetic mechanism. To test this possibility, we studied bladder cancer cells as they cyclically shifted to and from a cancer stem-like phenotype, and we discovered that these two states exhibit distinct DNA methylation and chromatin accessibility. Most differential chromatin accessibility was independent of methylation and affected the expression of driver genes such as E2F3, a cell cycle regulator associated with aggressive bladder cancer. Cancer stem-like cells exhibited increased E2F3 promoter accessibility and increased E2F3 expression that drove cell migration, invasiveness and drug resistance. Epigenetic interference using a DNA methylation inhibitor blocked the transition to a cancer stem-like state and reduced E2F3 expression. Our findings indicate that epigenetic plasticity plays a key role in the transition to and from an aggressive, drug-resistant phenotype.     

Histone H2A.Z deregulation in prostate cancer. Cause or effect?

Genetic and epigenetic changes are at the root of all cancers. The epigenetic component involves alterations of the post-synthetic modifications of DNA (methylation) and histones (histone posttranslational modifications, PTMs) as well as of those of their molecular “writers,” “readers,” and “erasers.” Noncoding RNAs (ncRNA) can also play a role. Here, we focus on the involvement of histone alterations in cancer, in particular that of the histone variant H2A.Z in the etiology of prostate cancer. The structural mechanisms putatively responsible for the contribution of H2A.Z to oncogenic gene expression programs are first described, followed by what is currently known about the involvement of this histone variant in the regulation of androgen receptor regulated gene expression. The implications of this and their relevance to oncogene deregulation in different stages of prostate cancer, including the progression toward androgen independence, are discussed. This review underscores the increasing awareness of the epigenetic contribution of histone variants to oncogenic progression.     

Ras association domain family 1A: a promising prognostic marker in recurrent nonmuscle invasive bladder cancer

IntroductionAberrant methylation of promoter CpG islands is an important inactivation mechanism of tumor suppressors and tumor-related genes. Ras association domain family 1A (RASSF1A) promoter hypermethylation was shown to be associated with bladder cancer (BC), but its prognostic value remains unclear. The aim of the present study was to investigate the value of RASSF1A methylation as a prognostic marker in BC.Materials and MethodsPrimary BC tissues were obtained from 301 patients and included 186 specimens of nonmuscle invasive bladder cancer (NMIBC) and 115 specimens of muscle invasive bladder cancers (MIBC). RASSF1A hypermethylation was assessed using methylation-specific polymerase chain reaction (MS-PCR). The association between RASSF1A hypermethylation and clinicopathologic features, and the prognostic significance of RASSF1A hypermethylation were evaluated by Kaplan-Meier and multivariate Cox regression analyses.ResultsRASSF1A promoter hypermethylation was detected in 33.6% of BCs and occurred more frequently in MIBC (46.1%) than in NMIBC (25.8%) (P < .001). In NMIBC, RASSF1A methylation was associated with advanced tumor stage (P = .026) and high grade (P < .001). Among patients with recurrent NMIBC, RASSF1A methylation was associated with shorter time to progression by Kaplan-Meier analysis (log-rank test; P = .004) and identified as an independent predictor of cancer progression by multivariate Cox regression analysis (hazard ratio [HR], 8.559; P = .014).ConclusionsOur results suggest that methylated RASSF1A may be a potential prognostic marker in patients with recurrent NMIBC.     

Epigenetic biomarkers in prostate cancer: Current and future uses

Epigenome alterations are characteristic of nearly all human malignancies and include changes in DNA methylation, histone modifications and microRNAs (miRNAs). However, what induces these epigenetic alterations in cancer is largely unknown and their mechanistic role in prostate tumorigenesis is just beginning to be evaluated. Identification of the epigenetic modifications involved in the development and progression of prostate cancer will not only identify novel therapeutic targets but also prognostic and diagnostic markers. This review will focus on the use of epigenetic modifications as biomarkers for prostate cancer.     

Epigenetische Inaktivierung von Genexpression

In addition to the multiple genetic aberrations described in hematologic neoplasias and solid tumors, epigenetic lesions in these malignancies also offer novel therapeutic targets since they are potentially reversible. Up to now, gene silencing of tumor suppressors, proteins involved in cell adhesion, metastasis and proapoptotic activity have been identified in virtually every type of human malignancy. Two major alterations have been described which can be antagonized by drugs already approved by the FDA (two azanucleosides for the treatment of myelodysplastic syndrome, the inhibitor of histone deacetylases Zolinza®/Vorinostat®/SAHA for cutaneous T-cell lymphoma). Translational studies demonstrating the in vivo inhibition of DNA methylation, and subsequent re-expression of tumor suppressor proteins such as p15/INK4b have provided proof-of-principle that demethylating agents reactivate these genes in vivo. Similarly, histone deacetylation in vivo by treatment with phenylbutyrate, valproic acid, depsipeptide and other HDAC inhibitors demonstrates the efficacy of these drugs in remodeling chromatin structure. However, ongoing studies are addressing which of the (probably multiple) targets for epigenetic therapy are reactivated in vivo in different malignancies (e.g. by methylation profiling). Combination studies to attack the “silencer” phenotype of malignant cells on different levels of epigenetic control are ongoing. Most of these clinical developments were performed with low doses of azanucleosides in hematologic malignancies, and studies to extend these prolonged, low-dose schedules to different solid tumor entities will be of great interest.     

HOXA9, ISL1 and ALDH1A3 methylation patterns as prognostic markers for nonmuscle invasive bladder cancer: Array‐based DNA methylation and expression profiling

DNA methylation patterns are associated with the development and prognosis of cancer. The aim of this study was to identify novel methylation markers for the prediction of patient outcomes using microarray analysis of DNA methylation and RNA expression patterns in samples from long‐term follow‐up patients with nonmuscle invasive bladder cancer (NMIBC). A total of 187 human bladder specimens were used for microarray array or pyrosequencing (PSQ) analyses: 6 normal controls (NC) and 181 NMIBC. Tumor‐specific hypermethylated genes were selected from a data set comprising 24 matched microarray‐based DNA methylation and gene expression profiles (6 controls and 18 NMIBC), and their clinical relevance was verified by quantitative PSQ analysis. The methylation status of Homeobox A9 (HOXA9), ISL LIM homeobox 1 (ISL1) and Aldehyde dehydrogenase 1 family, member A3 (ALDH1A3) was significantly associated with decreased gene expression levels and aggressive clinicopathological characteristics. Multivariate regression analyses showed that hypermethylation of these genes was an independent predictor of disease recurrence (HOXA9, ISL1 and ALDH1A3, either alone or in combination) and progression (ISL1 and ALDH1A3, either alone or in combination) (each p < 0.05). The results of this study suggest that these novel methylation markers are independent prognostic indicators in NMIBC patients, which may facilitate the assessment of disease recurrence and progression in NMIBC patients and inform clinical decision making regarding treatment.     

Altered epigenetic signals in human disease

The genetic information of almost all eukaryotic cells is stored in chromatin. In cancer cells, alterations in chromatin organization or in its epigenetic marks occur frequently. Among these are changes in the patterns of DNA and histone methylation. Using Acute Promyelocytic Leukemia as model system we could demonstrate a direct correlation of epigenetic events induced by the driving oncogene product PML-RARalpha and cancer progression. Several of the enzymes ultimately responsible for these events can be inhibited by small compound inhibitors and thus can serve as targets in cancer therapy. In this article, we review the role of DNA methylation, histone methylation and chromatin alterations in human diseases. A picture is emerging in which these epigenetic signals "cross-talk" and are implicated in the physiological and pathological spreading of gene silencing.     

RUNX3 inactivation by point mutations and aberrant DNA methylation in bladder tumors

RUNX3 is inactivated at high frequency in many tumors. However, in most cases, inactivation is caused by silencing of the gene due to promoter hypermethylation. Because epigenetic silencing is known to affect many major tumor suppressor genes in cancer cells, it is not clear whether RUNX3 is primarily responsible for the induction of carcinogenesis in these cases, except for the gastric cancer cases that we reported previously. We investigated genetic and epigenetic alterations of RUNX3 in 124 bladder tumor cases and seven bladder tumor-derived cell lines. Here we show that RUNX3 is inactivated by aberrant DNA methylation in 73% (90 of 124) of primary bladder tumor specimens and 86% (six of seven) of bladder tumor cell lines. In contrast, the promoter regions of 20 normal bladder mucosae were unmethylated. Importantly, one patient bore missense mutations, each of which resulted in amino acid substitutions in the highly conserved Runt domain. The mutations abolished the DNA-binding ability of RUNX3. A second patient had a single nucleotide deletion within the Runt domain coding region that resulted in truncation of the protein. RUNX3 methylation was a significant risk factor for bladder tumor development, superficial bladder tumor recurrence, and subsequent tumor progression. These results strongly suggest that inactivation of RUNX3 may contribute to bladder tumor development and that promoter methylation and silencing of RUNX3 could be useful prognostic markers for both bladder tumor recurrence and progression.     

Molecular biomarkers in urothelial bladder cancer

Bladder cancers are a mixture of heterogeneous cell populations, and numerous factors are likely to be involved in dictating their recurrence, progression, and the patient's survival. For any candidate prognostic marker to have considerable clinical relevance, it must add some predictive capacity beyond that offered by the conventional clinical and pathological parameters. None of the biomarkers reported to date have shown sufficient sensitivity and specificity for detecting the whole spectrum of bladder cancer diseases in routine clinical practice. The limitations of established prognostic markers requires us to identify better molecular parameters that could be of interest in predicting the prognosis of bladder cancer patients, in particular, the high-risk patient groups that are at risk of progression and recurrence. Recent progress in epigenetic modification and gene silencing opened a new avenue for the identification of epigenetic markers, which appears to be more useful for cancer diagnosis and prognosis. Although epigenetic markers also have limitations, the combined epigenetic marker approach may increase sensitivity and reliability. The epigenetic silencing of tumor-suppressor genes is interesting from a clinical standpoint because of the possibility of reversing epigenetic changes and restoring gene function in a cell. In addition, microarray technology provides us with additional tools for the analysis of global gene-expression analysis of tumor samples. Future microarray analyses are likely to reveal particular gene-expression signatures that predict the likelihood of bladder cancer progression and recurrence, as well as a patient's survival and responsiveness to different anticancer therapies, with great specificity and sensitivity. ( Cancer Sci 2008; 99: 646–652)     

Colorectal cancer early methylation alterations affect the crosstalk between cell and surrounding environment,tracing a biomarker signature specific for this tumor

Colorectal cancer (CRC) develops through the accumulation of both genetic and epigenetic alterations. However, while the former are already used as prognostic and predictive biomarkers, the latter are less well characterized. Here, performing global methylation analysis on both CRCs and adenomas by Illumina Infinium HumanMethylation450 Bead Chips, we identified a panel of 74 altered CpG islands, demonstrating that the earliest methylation alterations affect genes coding for proteins involved in the crosstalk between cell and surrounding environment. The panel discriminates CRCs and adenomas from peritumoral and normal mucosa with very high specificity (100%) and sensitivity (99.9%). Interestingly, over 70% of the hypermethylated islands resulted in downregulation of gene expression. To establish the possible usefulness of these non‐invasive markers for detection of colon cancer, we selected three biomarkers and identified the presence of altered methylation in stool DNA and plasma cell‐free circulating DNA from CRC patients.     

Circulating Tumor BRAF Mutation and Personalized Thyroid Cancer Treatment

Circulating tumor DNA (ctDNA) is now being extensively studied as it is a noninvasive “real-time” biomarker that can provide diagnostic and prognostic information before, during treatment and at progression. These include DNA mutations, epigenetic alterations and other forms of tumor-specific abnormalities such as microsatellite instability (MSI) and loss of heterozygosity (LOH). ctDNA is of great value in the process of cancer treatment. However, up to date, there is no strict standard considering the exact biomarker because the development and progression of cancer is extremely complicated. Also, results of the studies evaluating ctDNA are not consistent due to the different detection methods and processing. The major challenge is still the exact position of ctDNA in cancer management and the exact place of it versus tissue biopsy. actually it is still under the debate that circulating tumor markers will take the place of standard tissue biopsy or will support it to guide us to the more effective interventions?     

Consistent genomic alterations in carcinoma in situ of the urinary bladder confirm the presence of two major pathways in bladder cancer development

Bladder cancer develops through different pathways, provisionally entitled “papillary” and “invasive.” Carcinoma in situ (CIS) is thought to be the precursor of invasive bladder cancer. However, little is known about chromosomal alterations of these clinically important lesions, and the relationship between chromosomal alterations and the different pathways. We laser‐microdissected 12 CIS and 4 dysplasia samples concomitant to invasive bladder cancer. We determined genome‐wide chromosome copy number changes and loss of heterozygosity (LOH) using Mapping 10K SNP microarrays. We further examined 48 high‐risk non‐muscle‐invasive bladder cancers using SNP microarrays to reveal characteristic changes correlated with the CIS‐phenotype. DNA copy‐number changes were further validated using QPCR in 77 independent tumor samples. CIS was found to be chromosomal unstable in 8 of 12 cases. Characteristic chromosomal changes were copy number gains of chromosomes 5p, 6p22.3, 10p15.1 and losses/LOH of chromosome 5q and 13q13‐q14. Tumor samples with these alterations were significantly associated with CIS. Using FGFR3 mutations as markers of the opposing papillary phenotype, we found 5p gains and FGFR3 mutations mutually exclusive. No FGFR3 mutations were found in 23 CIS and dysplasia samples. Based on this, we classified high‐risk non‐muscle‐invasive bladder tumors according to FGFR3 mutations and chromosomal changes into papillary and CIS‐type tumors with high correlation to CIS status (p = 0.001). Furthermore, we found significant correlation to the results of molecular classifiers based on gene‐expression. We concluded that chromosomal changes may be used to characterize different pathways in bladder cancer development. © 2009 UICC     

Epigenetic alterations in the breast: Implications for breast cancer detection,prognosis and treatment

Epigenetic alterations of the genome such as DNA promoter methylation and chromatin remodeling play an important role in tumorigenesis. Recent findings indicate epigenetic modifications as key factors in breast carcinogenesis. These modifications are quite appealing as targets for preventative care and therapeutics because of their potential for reversal. Future medical care for breast cancer patients will likely depend upon a better understanding of the roles epigenetic modifications play in carcinogenesis. Here, we discuss the importance of epigenetics in breast cancer detection, prognosis, and therapy with an emphasis on mechanisms and epigenetic contributions to field cancerization effects.     

Epigenetic inactivation of SFRP genes and TP53 alteration act jointly as markers of invasive bladder cancer

In the United States each year, almost 13,000 deaths are attributable to bladder cancer, with the majority of these deaths related to higher stage, muscle-invasive solid tumors. Epigenetic silencing of the secreted frizzled receptor proteins (SFRP), antagonists of the WNT pathway, leads to constitutive WNT signaling, altering cell morphology and motility. Identifying alterations in this pathway in bladder cancer may prove useful for defining the invasive phenotype and provide targets for guiding therapy. Using a population-based study of bladder cancer (n = 355), we examined epigenetic alterations, specifically gene promoter hypermethylation, of four SFRP genes in addition to immunohistochemical staining of TP53, which has been previously shown to be a predictor of invasive disease. We observed a significant linear trend (P < 0.0004) in the magnitude of the risk of invasive disease with the number of SFRP genes methylated. Both TP53 alteration and SFRP gene methylation showed significant independent associations with invasive bladder cancer. Strikingly, in examining the joint effect of these alterations, we observed a >30-fold risk of invasive disease for patients with both altered SFRP gene methylation and intense TP53 staining (odds ratio, 32.1; P < 10(-13)). Overall patient survival was significantly poorer in patients with any SFRP genes methylated (P < 0.0003) and in proportional hazards modeling, patients with methylation of any SFRP gene had significantly poorer overall survival (hazard ratio, 1.78; P < 0.02) controlled for TP53 staining intensity and other survival-associated factors. Classifying tumors based on SFRP methylation status and TP53 protein staining intensity may be a clinically powerful predictor of invasive, deadly disease.     

Methylation of tumor suppressor genes in a novel panel predicts clinical outcome in paraffin-embedded bladder tumors

DNA methylation of tumor suppressor genes (TSGs) represents a frequent and early epigenetic event with potential applications for cancer detection and disease evolution. Our aim was to examine the stratification and prognostic biomarker role of the methylation of a novel panel of TSGs in bladder cancer. The methylation status of 18 TSGs was evaluated in bladder cancer cells (n?=?14) and paraffin-embedded primary bladder tumors (n?=?61), using a methylation-specific multiplex ligation-dependent probe amplification assay (MS-MLPA). Recurrence, progression, and disease-specific survival were analyzed using univariate and multivariate Cox models. PRDM2, HLTF, ID4, DLC1, BNIP3, H2AFX, CACNA1G, TGIF, and CACNA1A were discovered methylated in bladder cancer. The methylation of RUNX3 (p?=?0.026), TWIST1 (p?=?0.009), SFRP4 (p?=?0.002), and CCND2 (p?=?0.027) correlated to tumor stage. Univariate analyses indicated prognostic associations for recurrence (DLC1, SFRP5, H2AFX, CACNA1G), progression (DLC1, SFRP5, CACNA1G), disease-specific (PRDM2, DLC1, SFRP5, CACNA1G, and TIMP3), and overall survival (SFRP5 and TIMP3). In multivariate analyses, several TSGs remained as independent prognosticators for recurrence (SFRP5, H2AFX), progression (CACNA1G), and disease-specific survival (SFRP5). Thus, a novel set of TSGs was identified, frequently methylated in bladder cancer cells and tumors. TSG methylation allowed histopathologic and outcome stratification using paraffin-embedded tumors. This is clinically relevant by offering a strategy for the management of patients affected with uroepithelial neoplasias in pathology routine laboratories.