DNA methylation aberrancies delineate clinically distinct subsets of colorectal cancer and provide novel targets for epigenetic therapies.

Colorectal cancer (CRC) is a worldwide health concern with respect to both incidence and mortality, and as a result, CRC tumorigenesis, progression and metastasis have been heavily studied, especially with respect to identifying genetic, epigenetic, transcriptomic and proteomic profiles of disease. DNA methylation alterations are hallmarks of CRC, and epigenetic driver genes have been identified that are thought to be involved in early stages of tumorigenesis. Moreover, distinct CRC patient subgroups are organized based on DNA methylation profiles. CRC tumors displaying CpG island methylator phenotypes (CIMPs), defined as DNA hypermethylation at specific CpG islands in subsets of tumors, show high concordance with specific genetic alterations, disease risk factors and patient outcome. This review details the DNA methylation alterations in CRC, the significance of CIMP status, the development of treatments based on specific molecular profiles and the application of epigenetic therapies for CRC patient treatment.

DNA methylation profiles in diffuse large B-cell lymphoma and their relationship to gene expression status.

In an initial epigenetic characterization of diffuse large B-cell lymphoma (DLBCL), we evaluated the DNA methylation levels of over 500 CpG islands. Twelve CpG islands (AR, CDKN1C, DLC1, DRD2, GATA4, GDNF, GRIN2B, MTHFR, MYOD1, NEUROD1, ONECUT2 and TFAP2A) showed significant methylation in over 85% of tumors. Interestingly, the methylation levels of a CpG island proximal to FLJ21062 differed between the activated B-cell-like (ABC-DLBCL) and germinal center B-cell-like (GCB-DLBCL) subtypes. In addition, we compared the methylation and expression status of 67 genes proximal (within 500 bp) to the methylation assays. We frequently observed that hypermethylated CpG islands are proximal to genes that are expressed at low or undetectable levels in tumors. However, many of these same genes were also poorly expressed in DLBCL tumors where their cognate CpG islands were hypomethylated. Nevertheless, the proportional reductions in BNIP3, MGMT, RBP1, GATA4, IGSF4, CRABP1 and FLJ21062 expression with increasing methylation suggest that epigenetic processes strongly influence these genes. Lastly, the moderate expression of several genes proximal to hypermethylated CpG tracts suggests that DNA methylation assays are not always accurate predictors of gene silencing. Overall, further investigation of the highlighted CpG islands as potential clinical biomarkers is warranted.

DNA methylation profiles of female steroid hormone-driven human malignancies.

Tumor DNA contains valuable clues about the origin and pathogenesis of human cancers. Alterations in DNA methylation can lead to silencing of genes associated with distinct tumorigenic pathways. These pathway-specific DNA methylation changes help define tumor-specific DNA methylation profiles that can be used to further our understanding of tumor development, as well as provide tools for molecular diagnosis and early detection of cancer. Female sex hormones have been implicated in the etiology of several of the women's cancers including breast, endometrial, ovarian, and proximal colon cancers. We have reviewed the DNA methylation profiles of these cancers to determine whether the hormonal regulation of these cancers results in specific DNA methylation alterations. Although subsets of tumors in each of these four types of cancers were found to share some DNA methylation alterations, we did not find evidence for global hormone-specific DNA methylation alterations, suggesting that female sex hormones may participate in different tumorigenic pathways that are associated with distinct DNA methylation-based molecular signatures. One such pathway may include MLH1 methylation in the context of the CpG island methylator phenotype.

Quantitative analysis of associations between DNA hypermethylation, hypomethylation, and DNMT RNA levels in ovarian tumors.

How hypermethylation and hypomethylation of different parts of the genome in cancer are related to each other and to DNA methyltransferase (DNMT) gene expression is ill defined. We used ovarian epithelial tumors of different malignant potential to look for associations between 5'-gene region or promoter hypermethylation, satellite, or global DNA hypomethylation, and RNA levels for ten DNMT isoforms. In the quantitative MethyLight assay, six of the 55 examined gene loci (LTB4R, MTHFR, CDH13, PGR, CDH1, and IGSF4) were significantly hypermethylated relative to the degree of malignancy (after adjustment for multiple comparisons; P < 0.001). Importantly, hypermethylation of these genes was associated with degree of malignancy independently of the association of satellite or global DNA hypomethylation with degree of malignancy. Cancer-related increases in methylation of only two studied genes, LTB4R and MTHFR, which were appreciably methylated even in control tissues, were associated with DNMT1 RNA levels. Cancer-linked satellite DNA hypomethylation was independent of RNA levels for all DNMT3B isoforms, despite the ICF syndrome-linked DNMT3B deficiency causing juxtacentromeric satellite DNA hypomethylation. Our results suggest that there is not a simple association of gene hypermethylation in cancer with altered DNMT RNA levels, and that this hypermethylation is neither the result nor the cause of satellite and global DNA hypomethylation.

CpG island methylator phenotype (CIMP) of colorectal cancer is best characterised by quantitative DNA methylation analysis and prospective cohort studies.

BACKGROUND: The concept of CpG island methylator phenotype (CIMP) is not universally accepted. Even if specific clinicopathological features have been associated with CIMP, investigators often failed to demonstrate a bimodal distribution of the number of methylated markers, which would suggest CIMP as a distinct subtype of colorectal cancer. Previous studies primarily used methylation specific polymerase chain reaction which might detect biologically insignificant low levels of methylation. AIM: To demonstrate a distinct genetic profile of CIMP colorectal cancer using quantitative DNA methylation analysis that can distinguish high from low levels of DNA methylation. MATERIALS AND METHODS: We developed quantitative real time polymerase chain reaction (MethyLight) assays and measured DNA methylation (percentage of methylated reference) of five carefully selected loci (promoters of CACNA1G, CDKN2A (p16), CRABP1, MLH1, and NEUROG1) in 460 colorectal cancers from large prospective cohorts. RESULTS: There was a clear bimodal distribution of 80 microsatellite instability-high (MSI-H) tumours according to the number of methylated promoters, with no tumours showing 3/5 methylated loci. Thus we defined CIMP as having >or=4/5 methylated loci, and 17% (78) of the 460 tumours were classified as CIMP. CIMP was significantly associated with female sex, MSI, BRAF mutations, and wild-type KRAS. Both CIMP MSI-H tumours and CIMP microsatellite stable (MSS) tumours showed much higher frequencies of BRAF mutations (63% and 54%) than non-CIMP counterparts (non-CIMP MSI-H (0%, p<10(-5)) and non-CIMP MSS tumours (6.6%, p<10(-4)), respectively). CONCLUSION: CIMP is best characterised by quantitative DNA methylation analysis. CIMP is a distinct epigenotype of colorectal cancer and may be less frequent than previously reported.

Comparison of multitarget fluorescence in situ hybridization in urine with other noninvasive tests for detecting bladder cancer.

OBJECTIVES: To present a single-centre study investigating aneuploidy at chromosomes 3, 7, 17 and 9p21 (e.g. loss at 9p21) using a multitarget fluorescence in situ hybridization (FISH) system, as identifying genetic alterations in urine specimens is a promising approach for the noninvasive detection of bladder cancer. PATIENTS AND METHODS: Urine samples from 103 patients were evaluated, including those from 46 with histologically confirmed urothelial carcinoma, two with other urological malignancies, and 55 who acted as controls. The urine samples were taken before any manipulation. The validity of FISH (Urovision, Vysis, Downers Grove, Ill, USA) was compared with other noninvasive urine tests, including the BTA-Stat test, the nuclear matrix protein (NMP)-22 test, and immunocytology against 486p3/12 and LewisX. Those evaluating the tests were unaware of the clinical and histopathological data. FISH was considered positive if five or more urinary cells had gains of two or more chromosomes. The threshold for the urine tests were 10 U/mL (NMP-22), 30% positive cells (486p3/12), or 5% positive cells, respectively (LewisX). RESULTS: The sensitivity was 69% (FISH), 67% (BTA-Stat), 69% (486p3/12), 96% (LewisX) and 71% (NMP22), respectively; the respective specificity was 89%, 78%, 76%, 33% and 66%. CONCLUSION: Multitarget FISH had a better specificity than the other urine markers but because of its inadequate sensitivity it does not seem to be powerful enough to replace endoscopy. Optimizing the marker panel could provide a higher sensitivity.

Cytosine methylation in a CpG sequence leads to enhanced reactivity with Benzo[a]pyrene diol epoxide that correlates with a conformational change.

Benzo[a]pyrene (B[a]P) is a widespread environmental carcinogen that must be activated by cellular metabolism to a diol epoxide form (BPDE) before it reacts with DNA. It has recently been shown that BPDE preferentially modifies the guanine in methylated 5'-CpG-3' sequences in the human p53 gene, providing one explanation for why these sites are mutational hot spots. Using purified duplex oligonucleotides containing identical methylated and unmethylated CpG sequences, we show here that BPDE preferentially modified the guanine in hemimethylated or fully methylated CpG sequences, producing between 3- and 8-fold more modification at this site. Analysis of this reaction using shorter duplex oligonucleotides indicated that it was the level of the (+)-trans isomer that was specifically increased. To determine if there were conformational differences between the methylated and unmethylated B[a]P-modified DNA sequences that may be responsible for this enhanced reactivity, a native polyacrylamide gel electrophoresis analysis was carried out using DNA containing isomerically pure B[a]P-DNA adducts. These experiments showed that each adduct resulted in an altered gel mobility in duplex DNA but that only the presence of a (+)-trans isomer and a methylated C 5' to the adduct resulted in a significant gel mobility shift compared with the unmethylated case.