Detection Of Hypermethylation Research Articles

Epigenetic Profiling of Cutaneous T-Cell Lymphoma: Promoter Hypermethylation of Multiple Tumor Suppressor Genes Including BCL7a, PTPRG, and p73

To analyze the occurrence of promoter hypermethylation in primary cutaneous T-cell lymphoma (CTCL) on a genome-wide scale, focusing on epigenetic alterations with pathogenetic significance. DNA isolated from biopsy specimens of 28 patients with CTCL, including aggressive CTCL entities (transformed mycosis fungoides and CD30-negative large T-cell lymphoma) and an indolent entity (CD30-positive large T-cell lymphoma), were investigated. For genome-wide DNA methylation screening, differential methylation hybridization using CpG island microarrays was applied, which allows simultaneous detection of the methylation status of 8640 CpG islands. Bisulfite sequence analysis was applied for confirmation and detection of hypermethylation of eight selected tumor suppressor genes. The DNA methylation patterns of CTCLs emerging from differential methylation hybridization analysis included 35 CpG islands hypermethylated in at least four of the 28 studied CTCL samples when compared with benign T-cell samples. Hypermethylation of the putative tumor suppressor genes BCL7a (in 48% of CTCL samples), PTPRG (27%), and thrombospondin 4 (52%) was confirmed and demonstrated to be associated with transcriptional downregulation. BCL7a was hypermethylated at a higher frequency in aggressive (64%) than in indolent (14%) CTCL entities. In addition, the promoters of the selected tumor suppressor genes p73 (48%), p16 (33%), CHFR (19%), p15 (10%), and TMS1 (10%) were hypermethylated in CTCL. Malignant T cells of patients with CTCL display widespread promoter hypermethylation associated with inactivation of several tumor suppressor genes involved in DNA repair, cell cycle, and apoptosis signaling pathways. In view of this, CTCL may be amenable to treatment with demethylating agents.

Molecular Biomarker in Prostate Cancer: The Role of CpG Island Hypermethylation

CpG island hypermethylation may be one of the earliest somatic genome alterations to occur during the development of multiple cancers. Recently, aberrant methylation patterns for different tumors have been reported. We present a comprehensive review of the literature describing the role of CpG island hypermethylation of DNA from prostatic tissue and bodily fluids from men with prostate cancer. We reviewed the literature to evaluate CpG island hypermethylation in tissue and bodily fluids of men with primary and metastatic prostate cancer. Additionally, we reviewed the literature with respect to CpG island hypermethylation patterns in other urological malignancies. Using modern analytic methods, CpG island hypermethylation detection can be achieved. In men with prostate cancer, correlations between specific gene regulatory region hypermethylation analyses and Gleason score, pathologic stage and tumor recurrence have been demonstrated. CpG island hypermethylation may serve as a useful molecular biomarker for the detection and diagnosis of patients with prostate cancer.

P16 expression in Barrett's esophagus and esophageal adenocarcinoma: association with genetic and epigenetic alterations

Alteration of the p16 tumor suppressor gene has been implicated as a critical lesion in the molecular pathogenesis of esophageal adenocarcinoma. The aim of this study was to characterize the spectrum of p16 alterations in surgically resected esophageal tissues, comprising histologically normal esophageal squamous and gastric epithelia, premalignant Barrett's epithelia, and associated esophageal adenocarcinomas, and to explore associations between p16 mRNA expression and p16 mutations, deletions, promoter hypermethylation, p16 protein expression, and clinico-pathologic features for the same tissues. We have shown that while p16 mutations are uncommon (2%; 1/54), hypermethylation of the p16 promoter is detected in 43% (9/21) of histologically normal epithelia, in 77% (14/18) of associated Barrett's epithelia, and in 85% (18/21) of esophageal adenocarcinomas. However, p16 mRNA levels (relative to matched normal epithelia) were variable in Barrett's epithelia and adenocarcinomas, having no clear correlation with methylation status or other molecular and clinico-pathological parameters. These findings are consistent with a role for the p16 tumor suppressor gene early in the molecular progression of Barrett's epithelium to invasive esophageal adenocarcinoma, but do not support the notion that the detection of hypermethylation is systematically associated with low levels of expression.

Detection of bladder cancer in urine by a tumor suppressor gene hypermethylation panel.

Bladder cancer is potentially curable in the majority of cases; however, the prognosis for patients with advanced disease at presentation remains poor. Current noninvasive tests such as cytology lack sufficient sensitivity to detect low-grade, low-stage tumors. Silencing of tumor suppressor genes, such as p16(INK4a), VHL, and the mismatch repair gene hMLH1, has established promoter hypermethylation as a common mechanism for tumor suppressor inactivation in human cancers. It is also a promising new target for molecular detection in bodily fluids including urine, a readily accessible fluid known to contain bladder cancer cells. Methylation-specific PCR (MSP) can determine the presence or absence of methylation of a gene locus at a sensitivity level of up to 1 methylated allele in 1000 unmethylated alleles, appropriate for identifying cancer cell DNA in a bodily fluid. We first determined the frequency of hypermethylation of the Rb tumor suppressor gene by bisulfite sequencing and of the p16(INK4a), p14(ARF), APC, and RASSF1A tumor suppressor genes by MSP in 45 bladder cancers. We then designed a panel optimal for diagnostic coverage composed of the APC, RASSF1A, and p14(ARF) tumor suppressor genes. This panel was tested for detection of hypermethylation in matched sediment DNA from urine specimens obtained before surgery from the same 45 bladder cancer patients (2 Tis, 16 Ta, 10 T1, and 17 T2-4) as well as normal and benign control DNAs. Hypermethylation of at least one of three suppressor genes (APC, RASSF1A, and p14(ARF)) was found in all 45 tumor DNAs (100% diagnostic coverage). We detected gene hypermethylation in the matched urine DNA from 39 of 45 patients (87% sensitivity), including 16 cases that had negative cytology. No hypermethylation of APC, RASSF1A, or p14(ARF) was observed in normal transitional cell DNAs or in urine DNAs from normal healthy individuals and patients with inflammatory urinary disease (cystitis). Furthermore, an unmethylated gene in the tumor DNA was always found to be unmethylated in the matched urine DNA (100% specificity). Promoter hypermethylation of tumor suppressor genes is common in bladder cancer and was found in all grades and stages of tumors examined. Hypermethylation was detected in the urine DNA from 39 of 45 (87%) patients, including cases of early-stage disease amenable to cure. MSP may enhance early detection of bladder cancer using a noninvasive urine test.

Improvement of the methylation specific PCR technical conditions for the detection of p16 promoter hypermethylation in small amounts of tumor DNA.

Methylation specific PCR (MSP) is a technique that enables the detection of hypermethylation at a CpG island. The objective of this study is the introduction of small modifications to the MSP technique to make it more suitable for the study of promoter hypermethylation at tumor suppressor genes whenever there is a shortage of material available for study. This commonly happens in the case of using archival material from the Pathology departments. Tumor DNA was extracted from a collection of 40 fresh-frozen soft tissue sarcomas and 19 paraffin-embedded PNETs (primitive neuroectodermal tumors). The MSP technique was performed to detect hypermethylation at the p16 promoter. Also, blood genomic DNA was mixed with herry sperm genomic DNA as a carrier, in nine different combinations, in order to test for the best conditions that could produce MSP bands even when low amounts of genomic tumor DNA is available for study. We demonstrate the benefit of using herry sperm carrier DNA up to 10 microg together with small quantities of tumor DNA. This result will facilitate the incorporation of paraffin-embedded samples for study of promoter hypermethylation at tumor suppressor genes. Other technical conditions for the MSP technique are also studied.

Detection of hypermethylation of thep16INK4A gene promoter in chronic hepatitis and cirrhosis associated with hepatitis B or C virus

BACKGROUND/AIM Inactivation of the p16INK4A(p16) tumour suppressor gene by promoter region hypermethylation has been demonstrated not only in many types of tumours, including hepatocellular carcinoma (HCC), but also in early...

DNA methylation in urological malignancies (review).

Three different kinds of alterations in DNA methylation have been observed in urological malignancies. DNA hypermethylation of CpG-rich promoter regions is an important mechanism involved in the inactivation of tumor suppressor and other genes in prostate, renal cell, and bladder carcinoma. Genome-wide hypomethylation is most pronounced in urothelial carcinoma, but also occurs in prostatic cancer. Loss of imprinting may be a primary event in the aetiogenesis of Wilms' tumor and probably contributes to testicular cancer. With respect to alterations in DNA methylation three tumor categories are distinguished: in the development of embryonic tumors, e.g. Wilms' tumor, loss of imprinting is important probably by upsetting the balance between genes promoting or inhibiting proliferation. In tumors with faulty DNA methylation, e.g. renal cell carcinoma, occasional errors in DNA methylation are selected for during tumor development. In tumors with deranged methylation, e. g. in most bladder and prostate carcinomas, the mechanisms establishing methylation patterns are fundamentally disturbed and multiple alterations in DNA methylation are observed. At least one of the enzymes establishing methylation patterns, viz. DNA methyltransferases and demethylases, may be deregulated. Moreover, changes in methyl group metabolism need to be considered. DNA hypermethylation and loss of imprinting act by altering the expression of selected genes, whereas hypomethylation may facilitate transcription and recombination throughout the genome by its effect on the chromatin structure. The combination of all three types of alterations may create genomic instability in tumors with deranged DNA methylation. Regarding a potential clinical use, detection of hypermethylation appears most promising in cancer diagnosis, while parameters reflecting genome-wide hypomethylation may prove useful in the prediction of prognosis. Inhibitors of DNA methylation are being improved and will presumably first be employed against tumors with hypermethylated key tumor suppressor genes.

Detection of hypermethylation of the c-abl genomic locus in the spleen of juvenile LE rats

Tumor induction by treatment with polycylic hydrocarbons depends on age and the strains of rats used. Juvenile LE rats are very sensitive to the induction of leukemia and chromosomal breaks by intravenous DMBA injection. We have previously demonstrated a chromosomal translocation in chromosome 3 and 12 in a DMBA induced LE rat leukemia cell K3D. In our present communication we have examined the c-abl expression in the leukemic cell line as well as in the LE rats at different ages. We found that in the leukemic cell K3D the c-abl expression is elevated both at the level of mRNA and protein. In the preleukemic stage, highly elevated expression of c-abl mRNA was detected exclusively in the spleen of the junvenlie LE rats. Furthermore this high expression of the c-abl gene correlates well with hypermethylation of possible cytosine residue in the c-abl genomic locus.