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Differentially methylated loci distinguish ovarian carcinoma histological types: evaluation of a DNA methylation assay in FFPE tissue.

Kelemen LE, Köbel M, Chan A, Taghaddos S, Dinu I - Biomed Res Int (2013)

Bottom Line: Methylation status at each 1,505 CpG site was expressed as β-values.Methylation did not always correlate with gene expression (r(2) = -0.70 to 0.15).We found that lower bsDNA overestimates methylation, and, using higher bsDNA amounts, we confirmed a previous report of higher methylation of THBS2 in clear cell OC, which could provide new insight into biological pathways that distinguish OC histological types.

View Article: PubMed Central - PubMed

Affiliation: Department of Population Health Research, Alberta Health Services-Cancer Care, Calgary, AB, Canada T2S 3C3 ; Departments of Medical Genetics and Oncology, University of Calgary, Calgary, AB, Canada T2N 4N2.

ABSTRACT
Epigenomic markers can identify tumor subtypes, but few platforms can accommodate formalin-fixed paraffin-embedded (FFPE) tumor tissue. We tested different amounts of bisulfite-converted (bs) DNA from six FFPE ovarian carcinomas (OC) of serous, endometrioid, and clear cell histologies and two HapMap constitutional genomes to evaluate the performance of the GoldenGate methylation assay. Methylation status at each 1,505 CpG site was expressed as β-values. Comparing 400 ng versus 250 ng bsDNA, reproducibility of the assay ranged from Spearman r(2) = 0.41 to 0.90, indicating that β-values obtained with a lower DNA amount did not always correlate well with the higher amount. Average methylation for the six samples was higher using 250 ng (β-value = 0.45, SD = 0.29) than with 400 ng (β-value = 0.36, SD = 0.32). Reproducibility between duplicate HapMap samples (r(2) = 0.76 to 0.92) was also variable. Using 400 ng input bsDNA, THBS2 and ERG were differentially methylated across all histologic types and between endometrioid and clear cell types at <0.1% false discovery rate. Methylation did not always correlate with gene expression (r(2) = -0.70 to 0.15). We found that lower bsDNA overestimates methylation, and, using higher bsDNA amounts, we confirmed a previous report of higher methylation of THBS2 in clear cell OC, which could provide new insight into biological pathways that distinguish OC histological types.

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Scatter plots of β-values and Spearman correlation coefficients (r2). (a) to (f), patient data comparing 250 ng versus 400 ng bsDNA for high-grade serous ((a) and (b)), endometrioid ((c) and (d)), and clear cell ((e) and (f)) carcinomas; (g), averaged group data comparing 250 ng versus 400 ng bsDNA; (h) to (i), reproducibility plots of 250 ng bsDNA for a CEPH male (h) and a CEPH female (i).
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fig1: Scatter plots of β-values and Spearman correlation coefficients (r2). (a) to (f), patient data comparing 250 ng versus 400 ng bsDNA for high-grade serous ((a) and (b)), endometrioid ((c) and (d)), and clear cell ((e) and (f)) carcinomas; (g), averaged group data comparing 250 ng versus 400 ng bsDNA; (h) to (i), reproducibility plots of 250 ng bsDNA for a CEPH male (h) and a CEPH female (i).

Mentions: Patients with high-grade serous tumors had FIGO (International Federation of Gynecology and Obstetrics) stage classification IIIC, whereas the two patients with endometrioid tumors had FIGO stages IC and IIB. Two patients with clear cell tumors also had FIGO stages IC and IIB. Spearman correlation coefficients (r2) of Illumina background-normalized data comparing 400 ng versus 250 ng bsDNA showed a range of r2 of 0.41–0.90 for patient data (Figures 1(a)–1(f)), indicating that the findings with a lower amount of DNA did not always correlate well with the higher amount of DNA. Group data showed improved correlation of r2 = 0.90 (Figure 1(g)). Indeed, the average methylation across 1,505 CpG loci among the six samples was higher using 250 ng bsDNA (average β-value = 0.45, standard deviation = 0.29) than 400 ng bsDNA (average β-value = 0.36, standard deviation = 0.32), suggesting insufficient bsDNA leads to overestimation of methylation. Furthermore, because the reproducibility between duplicate HapMap samples using 250 ng bsDNA was r2 = 0.76 for the CEPH male (Figure 1(h)) and r2 = 0.92 for the CEPH female (Figure 1(i)), we infer that the lower amount of 250 ng can bias methylation results even with non-FFPE sources of DNA. In support of this deduction, we observed that methylation of X chromosome loci was close to zero for one replicate sample of the CEPH male, as expected, but higher for the other CEPH male replicate (Table 1). β-values were closer to one than to hemimethylation for several CpG loci for the female replicate samples.


Differentially methylated loci distinguish ovarian carcinoma histological types: evaluation of a DNA methylation assay in FFPE tissue.

Kelemen LE, Köbel M, Chan A, Taghaddos S, Dinu I - Biomed Res Int (2013)

Scatter plots of β-values and Spearman correlation coefficients (r2). (a) to (f), patient data comparing 250 ng versus 400 ng bsDNA for high-grade serous ((a) and (b)), endometrioid ((c) and (d)), and clear cell ((e) and (f)) carcinomas; (g), averaged group data comparing 250 ng versus 400 ng bsDNA; (h) to (i), reproducibility plots of 250 ng bsDNA for a CEPH male (h) and a CEPH female (i).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3794544&req=5

fig1: Scatter plots of β-values and Spearman correlation coefficients (r2). (a) to (f), patient data comparing 250 ng versus 400 ng bsDNA for high-grade serous ((a) and (b)), endometrioid ((c) and (d)), and clear cell ((e) and (f)) carcinomas; (g), averaged group data comparing 250 ng versus 400 ng bsDNA; (h) to (i), reproducibility plots of 250 ng bsDNA for a CEPH male (h) and a CEPH female (i).
Mentions: Patients with high-grade serous tumors had FIGO (International Federation of Gynecology and Obstetrics) stage classification IIIC, whereas the two patients with endometrioid tumors had FIGO stages IC and IIB. Two patients with clear cell tumors also had FIGO stages IC and IIB. Spearman correlation coefficients (r2) of Illumina background-normalized data comparing 400 ng versus 250 ng bsDNA showed a range of r2 of 0.41–0.90 for patient data (Figures 1(a)–1(f)), indicating that the findings with a lower amount of DNA did not always correlate well with the higher amount of DNA. Group data showed improved correlation of r2 = 0.90 (Figure 1(g)). Indeed, the average methylation across 1,505 CpG loci among the six samples was higher using 250 ng bsDNA (average β-value = 0.45, standard deviation = 0.29) than 400 ng bsDNA (average β-value = 0.36, standard deviation = 0.32), suggesting insufficient bsDNA leads to overestimation of methylation. Furthermore, because the reproducibility between duplicate HapMap samples using 250 ng bsDNA was r2 = 0.76 for the CEPH male (Figure 1(h)) and r2 = 0.92 for the CEPH female (Figure 1(i)), we infer that the lower amount of 250 ng can bias methylation results even with non-FFPE sources of DNA. In support of this deduction, we observed that methylation of X chromosome loci was close to zero for one replicate sample of the CEPH male, as expected, but higher for the other CEPH male replicate (Table 1). β-values were closer to one than to hemimethylation for several CpG loci for the female replicate samples.

Bottom Line: Methylation status at each 1,505 CpG site was expressed as β-values.Methylation did not always correlate with gene expression (r(2) = -0.70 to 0.15).We found that lower bsDNA overestimates methylation, and, using higher bsDNA amounts, we confirmed a previous report of higher methylation of THBS2 in clear cell OC, which could provide new insight into biological pathways that distinguish OC histological types.

View Article: PubMed Central - PubMed

Affiliation: Department of Population Health Research, Alberta Health Services-Cancer Care, Calgary, AB, Canada T2S 3C3 ; Departments of Medical Genetics and Oncology, University of Calgary, Calgary, AB, Canada T2N 4N2.

ABSTRACT
Epigenomic markers can identify tumor subtypes, but few platforms can accommodate formalin-fixed paraffin-embedded (FFPE) tumor tissue. We tested different amounts of bisulfite-converted (bs) DNA from six FFPE ovarian carcinomas (OC) of serous, endometrioid, and clear cell histologies and two HapMap constitutional genomes to evaluate the performance of the GoldenGate methylation assay. Methylation status at each 1,505 CpG site was expressed as β-values. Comparing 400 ng versus 250 ng bsDNA, reproducibility of the assay ranged from Spearman r(2) = 0.41 to 0.90, indicating that β-values obtained with a lower DNA amount did not always correlate well with the higher amount. Average methylation for the six samples was higher using 250 ng (β-value = 0.45, SD = 0.29) than with 400 ng (β-value = 0.36, SD = 0.32). Reproducibility between duplicate HapMap samples (r(2) = 0.76 to 0.92) was also variable. Using 400 ng input bsDNA, THBS2 and ERG were differentially methylated across all histologic types and between endometrioid and clear cell types at <0.1% false discovery rate. Methylation did not always correlate with gene expression (r(2) = -0.70 to 0.15). We found that lower bsDNA overestimates methylation, and, using higher bsDNA amounts, we confirmed a previous report of higher methylation of THBS2 in clear cell OC, which could provide new insight into biological pathways that distinguish OC histological types.

Show MeSH
Related in: MedlinePlus