Susan Cottrell

Sensitive detection of DNA methylation.

Abstract: In recent years, many molecular biomarkers have been discovered that are capable of distinguishing tumors from normal tissue. Among the different types of markers, DNA methylation markers stand out for their potential to provide a unique combination of specificity, sensitivity, high information content, and applicability to a wide variety of clinical specimens. Methylation markers are particularly suited for situations where sensitive detection is necessary, such as when tumor DNA is either scarce or diluted by excess normal DNA. One of the most widely used methods for measuring methylation levels, methylation‐specific PCR (MSP), has been proved to be a very effective tool in situations requiring sensitive detection. The addition of fluorogenic probes makes these assays more informative, quantitative, and suitable for a clinical format. The field of sensitive detection is not limited to MSP; hence, an alternative methylation‐sensitive amplification is discussed. PCR‐based methylation assays have been applied to the detection of tumor DNA in a variety of body fluids, including serum, plasma, urine, sputum, and lavage fluids. In many cases, the sensitivity and specificity of these detection assays has been impressive, but important technological issues remain in areas such as sample preparation, assay design, and marker selection. Once these technical concerns have been addressed, the sensitive detection of methylation will provide a powerful diagnostic and prognostic tool, especially for the early detection of preneoplastic and neoplastic lesions.

DNA Methylation of the PITX2 Gene Promoter Region is a Strong Independent Prognostic Marker of Biochemical Recurrence in Patients With Prostate Cancer After Radical Prostatectomy

PURPOSE Approximately 35% of patients with prostate cancer who undergo radical prostatectomy experience prostate specific antigen recurrence within 10 years of surgery. Current prognostic indicators cannot sufficiently detect who is at risk for biochemical recurrence. We evaluated DNA methylation markers for prostate cancer prognosis. MATERIALS AND METHODS We assessed the DNA methylation of 6 marker candidates that were identified in previous studies. Formalin fixed, paraffin embedded tissue sections from a cohort of 605 patients who underwent radical prostatectomy were analyzed using real-time polymerase chain reaction assays. Using a Cox proportional hazard model we determined which markers were significant predictors of biochemical recurrence. RESULTS ABHD9, Chr3-EST, GPR7, HIST2H2BF and PITX2 were significantly associated with biochemical recurrence. PITX2 methylation was the strongest predictor of biochemical recurrence, providing additional prognostic information to established clinical factors in patients treated with radical prostatectomy and especially in patients at intermediate risk (Gleason 7). Patients with greater than median PITX2 methylation in the tumors were 4 times more likely to experience biochemical recurrence within 8 years after surgery than patients with less than average methylation. CONCLUSIONS The prognostic information provided by PITX2 methylation adds significantly to currently used clinical variables such as Gleason grade and stage. Therefore, it could contribute to better counseling in patients with prostate cancer.

DNA Methylation Analysis: Providing New Insight into Human Disease

Publisher Summary The human genome contains four bases—guanine, adenine, thymine, and cytosine. The cytosines can be either methylated or unmethylated at the fifth carbon position in the pyrimidine ring. In general, they can only be methylated when they are in the context of a CpG dinucleotide that involves a cytosine immediately followed by a guanine. The methylation status of a CpG island is correlated with the chromatin structure and expression levels of nearby genes. CpG islands associated with actively transcribed genes are typically unmethylated. When a CpG island is methylated, methyl-CpG-binding domain proteins recognize the methylated CpG and recruit the necessary factors for chromatin condensation and gene inactivation. This DNA methylation state is maintained during cell division by a family of enzymes called DNA methyltransferases. Cancer was viewed as an accumulation of chromosomal aberrations and, therefore, called a “genetic disease.” However, it has become clear over time that epigenetic changes play a crucial role in carcinogenesis. While attention is focused on methylation in carcinogenesis, a similar groundswell of research is emerging on methylation in other diseases, especially autoimmune and cardiovascular conditions.

DNA Methylation Analysis

Publisher Summary The human genome contains four bases—guanine, adenine, thymine, and cytosine. The cytosines can be either methylated or unmethylated at the fifth carbon position in the pyrimidine ring. In general, they can only be methylated when they are in the context of a CpG dinucleotide that involves a cytosine immediately followed by a guanine. The methylation status of a CpG island is correlated with the chromatin structure and expression levels of nearby genes. CpG islands associated with actively transcribed genes are typically unmethylated. When a CpG island is methylated, methyl-CpG-binding domain proteins recognize the methylated CpG and recruit the necessary factors for chromatin condensation and gene inactivation. This DNA methylation state is maintained during cell division by a family of enzymes called DNA methyltransferases. Cancer was viewed as an accumulation of chromosomal aberrations and, therefore, called a “genetic disease.” However, it has become clear over time that epigenetic changes play a crucial role in carcinogenesis. While attention is focused on methylation in carcinogenesis, a similar groundswell of research is emerging on methylation in other diseases, especially autoimmune and cardiovascular conditions.

Chapter 6 – DNA Methylation Analysis: Providing New Insight into Human Disease

Publisher Summary The human genome contains four bases—guanine, adenine, thymine, and cytosine. The cytosines can be either methylated or unmethylated at the fifth carbon position in the pyrimidine ring. In general, they can only be methylated when they are in the context of a CpG dinucleotide that involves a cytosine immediately followed by a guanine. The methylation status of a CpG island is correlated with the chromatin structure and expression levels of nearby genes. CpG islands associated with actively transcribed genes are typically unmethylated. When a CpG island is methylated, methyl-CpG-binding domain proteins recognize the methylated CpG and recruit the necessary factors for chromatin condensation and gene inactivation. This DNA methylation state is maintained during cell division by a family of enzymes called DNA methyltransferases. Cancer was viewed as an accumulation of chromosomal aberrations and, therefore, called a “genetic disease.” However, it has become clear over time that epigenetic changes play a crucial role in carcinogenesis. While attention is focused on methylation in carcinogenesis, a similar groundswell of research is emerging on methylation in other diseases, especially autoimmune and cardiovascular conditions.