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Identification of novel high-frequency DNA methylation changes in breast cancer.

Ordway JM, Budiman MA, Korshunova Y, Maloney RK, Bedell JA, Citek RW, Bacher B, Peterson S, Rohlfing T, Hall J, Brown R, Lakey N, Doerge RW, Martienssen RA, Leon J, McPherson JD, Jeddeloh JA - PLoS ONE (2007)

Bottom Line: Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis.The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively.Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date.

View Article: PubMed Central - PubMed

Affiliation: Orion Genomics, St. Louis, Missouri, United States of America. jordway@oriongenomics.com

ABSTRACT
Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis. The inherent thermodynamic stability of cytosine methylation and the apparent high specificity of the alterations for disease may accelerate the development of powerful molecular diagnostics for cancer. We report a genome-wide analysis of DNA methylation alterations in breast cancer. The approach efficiently identified a large collection of novel differentially DNA methylated loci (approximately 200), a subset of which was independently validated across a panel of over 230 clinical samples. The differential cytosine methylation events were independent of patient age, tumor stage, estrogen receptor status or family history of breast cancer. The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively. Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date. The discovery of over 50 novel DNA methylation-based biomarkers of breast cancer may provide new routes for development of DNA methylation-based diagnostics and prognostics, as well as reveal epigenetically regulated mechanism involved in breast tumorigenesis.

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Correlation between differential DNA methylation measured by microarray and independent qPCR analyses.Log2 (tumor-adjacent normal) microarray measurements of differential DNA methylation (y-axis) are plotted against qPCR (ddCt tumor-adjacent normal) measurements (x-axis). Primer pairs designed to amplify 116 of the 220 regions predicted to be significantly differentially methylated. Delta-delta Ct values (delta Ct tumor–delta Ct adjacent normal) and differential log2 microarray values (Log2 tumor-Log2 adjacent normal) were compared for breast tumor/adjacent normal tissue pairs. Data for one representative tumor/adjacent normal pair are shown. Data points in the upper right and lower left quadrants represent hypermethylation and hypomethylation measurements that are concordant between the two independent methods, respectively. qPCR measurements within the 0.5 cycle range of variance of the qPCR platform (hatched lines) were considered discordant.
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pone-0001314-g002: Correlation between differential DNA methylation measured by microarray and independent qPCR analyses.Log2 (tumor-adjacent normal) microarray measurements of differential DNA methylation (y-axis) are plotted against qPCR (ddCt tumor-adjacent normal) measurements (x-axis). Primer pairs designed to amplify 116 of the 220 regions predicted to be significantly differentially methylated. Delta-delta Ct values (delta Ct tumor–delta Ct adjacent normal) and differential log2 microarray values (Log2 tumor-Log2 adjacent normal) were compared for breast tumor/adjacent normal tissue pairs. Data for one representative tumor/adjacent normal pair are shown. Data points in the upper right and lower left quadrants represent hypermethylation and hypomethylation measurements that are concordant between the two independent methods, respectively. qPCR measurements within the 0.5 cycle range of variance of the qPCR platform (hatched lines) were considered discordant.

Mentions: Accuracy of the microarray-based DNA methylation measurements was assessed by a quantitative PCR (qPCR) [27] as described in the Materials and Methods. The presence of purine-5mC sites within an amplified region results in digestion by McrBC and a higher cycle number at which the McrBC-treated sample crosses threshold. Therefore, higher delta Ct measurements correlate with a larger proportion of the molecules containing DNA methylation between the priming sites (Fig. 2). In total, 96 of 116 (83%) measurements were concordant between the two methods (12 hypomethylation and 84 hypermethylation events). Nine qPCR measurements fell within the 0.5 cycle region of variability of the real-time PCR platform itself, and these measurements were considered discordant. Seventeen measurements reported an increase in DNA methylation by the qPCR method that was not detected by the microarray method (approximately 15% false negative rate in the microarray experiment). Three hypermethylation microarray predictions fell within the 0.5 cycle qPCR range that was considered discordant (approximately 2% false positive rate). An overall accuracy of 83% and a higher false negative than false positive rate are consistent with results obtained in numerous independent microarray analyses ([27] and data not shown). A precisely linear relationship between the microarray and qPCR measurements is not necessarily expected because the microarray features are capable of measuring methylation of a larger local region (1 to 4 Kb) than the qPCR amplicons were designed to interrogate (400–600 bp).


Identification of novel high-frequency DNA methylation changes in breast cancer.

Ordway JM, Budiman MA, Korshunova Y, Maloney RK, Bedell JA, Citek RW, Bacher B, Peterson S, Rohlfing T, Hall J, Brown R, Lakey N, Doerge RW, Martienssen RA, Leon J, McPherson JD, Jeddeloh JA - PLoS ONE (2007)

Correlation between differential DNA methylation measured by microarray and independent qPCR analyses.Log2 (tumor-adjacent normal) microarray measurements of differential DNA methylation (y-axis) are plotted against qPCR (ddCt tumor-adjacent normal) measurements (x-axis). Primer pairs designed to amplify 116 of the 220 regions predicted to be significantly differentially methylated. Delta-delta Ct values (delta Ct tumor–delta Ct adjacent normal) and differential log2 microarray values (Log2 tumor-Log2 adjacent normal) were compared for breast tumor/adjacent normal tissue pairs. Data for one representative tumor/adjacent normal pair are shown. Data points in the upper right and lower left quadrants represent hypermethylation and hypomethylation measurements that are concordant between the two independent methods, respectively. qPCR measurements within the 0.5 cycle range of variance of the qPCR platform (hatched lines) were considered discordant.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2117343&req=5

pone-0001314-g002: Correlation between differential DNA methylation measured by microarray and independent qPCR analyses.Log2 (tumor-adjacent normal) microarray measurements of differential DNA methylation (y-axis) are plotted against qPCR (ddCt tumor-adjacent normal) measurements (x-axis). Primer pairs designed to amplify 116 of the 220 regions predicted to be significantly differentially methylated. Delta-delta Ct values (delta Ct tumor–delta Ct adjacent normal) and differential log2 microarray values (Log2 tumor-Log2 adjacent normal) were compared for breast tumor/adjacent normal tissue pairs. Data for one representative tumor/adjacent normal pair are shown. Data points in the upper right and lower left quadrants represent hypermethylation and hypomethylation measurements that are concordant between the two independent methods, respectively. qPCR measurements within the 0.5 cycle range of variance of the qPCR platform (hatched lines) were considered discordant.
Mentions: Accuracy of the microarray-based DNA methylation measurements was assessed by a quantitative PCR (qPCR) [27] as described in the Materials and Methods. The presence of purine-5mC sites within an amplified region results in digestion by McrBC and a higher cycle number at which the McrBC-treated sample crosses threshold. Therefore, higher delta Ct measurements correlate with a larger proportion of the molecules containing DNA methylation between the priming sites (Fig. 2). In total, 96 of 116 (83%) measurements were concordant between the two methods (12 hypomethylation and 84 hypermethylation events). Nine qPCR measurements fell within the 0.5 cycle region of variability of the real-time PCR platform itself, and these measurements were considered discordant. Seventeen measurements reported an increase in DNA methylation by the qPCR method that was not detected by the microarray method (approximately 15% false negative rate in the microarray experiment). Three hypermethylation microarray predictions fell within the 0.5 cycle qPCR range that was considered discordant (approximately 2% false positive rate). An overall accuracy of 83% and a higher false negative than false positive rate are consistent with results obtained in numerous independent microarray analyses ([27] and data not shown). A precisely linear relationship between the microarray and qPCR measurements is not necessarily expected because the microarray features are capable of measuring methylation of a larger local region (1 to 4 Kb) than the qPCR amplicons were designed to interrogate (400–600 bp).

Bottom Line: Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis.The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively.Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date.

View Article: PubMed Central - PubMed

Affiliation: Orion Genomics, St. Louis, Missouri, United States of America. jordway@oriongenomics.com

ABSTRACT
Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis. The inherent thermodynamic stability of cytosine methylation and the apparent high specificity of the alterations for disease may accelerate the development of powerful molecular diagnostics for cancer. We report a genome-wide analysis of DNA methylation alterations in breast cancer. The approach efficiently identified a large collection of novel differentially DNA methylated loci (approximately 200), a subset of which was independently validated across a panel of over 230 clinical samples. The differential cytosine methylation events were independent of patient age, tumor stage, estrogen receptor status or family history of breast cancer. The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively. Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date. The discovery of over 50 novel DNA methylation-based biomarkers of breast cancer may provide new routes for development of DNA methylation-based diagnostics and prognostics, as well as reveal epigenetically regulated mechanism involved in breast tumorigenesis.

Show MeSH
Related in: MedlinePlus