Abstract A38: Thrombospondin 2 CpG methylation is a marker of clear cell ovarian carcinomas: Evaluation of a DNA methylation assay in archival tissue
Authors: Kelemen Linda E. , Kobel Martin , Chan Angela , Taghaddos Soreh , Dinu Irina 
Published: June 2012

Abstract
Introduction: Epigenomic markers can be used to identify subsets of patients or tumors in order to determine differences in risk of disease, to account for unexplained molecular heterogeneity, to improve our ability to subtype cancers and to refine our classification of cancers at a given organ site. Carcinomas classified as ovarian are the fourth most common among female cancers, and one of the most heterogeneous of all human malignancies. Few epigenomic platforms exist that accommodate archival formalin-fixed paraffin-embedded (FFPE) tumor tissue which, when incorporated into epidemiologic studies, offers the ability to investigate somatic alterations with both etiology and prognosis.
Methods: We tested different volumes (250ng and 400ng) of bisulfite-converted (bs) DNA from each of six FFPE primary ovarian carcinomas of serous, endometrioid and clear cell histologies and two constitutional genomes from Caucasians (CEPH) from the HapMap Project using the GoldenGate™ Cancer Panel I methylation assay protocol. The methylation status at each 1,505 CpG site was determined by calculating β, which is the ratio of the fluorescent signal from the methylated allele to the sum of the fluorescent signals of both methylated and unmethylated alleles (0=completely unmethylated, 1=completely methylated). To evaluate reproducibility of the assay, Spearman correlation coefficients were used to compare β-values at 250ng vs 400ng input bsDNA for (i) each sample separately and (ii) group data. We also compared reproducibility between replicate samples of the CEPH controls at 250ng. To evaluate validity, we examined gender-specific CpG sites among CEPH male and female controls at 250ng input bsDNA in housekeeping genes from the X chromosome represented on the assay. Methylation of these genes is expected to show gene dosage between males and females owing to gene silencing on one of the two X chromosomes in female somatic cells that compensates for the single X chromosome among males. Using the 400ng input bsDNA, we used Significance Analysis of Microarrays (SAM) to find overall differences in methylation across the three carcinoma types. Results: Spearman r2 comparing 400ng vs 250ng bsDNA ranged from 0.41 − 0.90 for subject data and r2=0.90 for group data, indicating that the findings with a lower volume of DNA did not always correlate well with the higher volume of DNA. Average methylation across 1,505 CpG loci among the six samples was higher at 250ng (average β-value=0.45, SD=0.29) than at 400ng (average β-value=0.36, SD=0.32), suggesting insufficient bsDNA leads to over-estimation of methylation. Reproducibility between duplicate HapMap samples at 250ng ranged from r2=0.76 for the CEPH male to r2=0.92 for the CEPH female. Further, methylation at 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. β-values were closer to one than to hemimethylation for the female replicate samples. SAM identified two genes, ERG and THBS2, that were differentially methylated across all three carcinoma types at FDR&lt;0.1%: both genes showed higher methylation at CpG sites among clear cell carcinomas. Conclusions: The manufacturer-recommended lower volume of 250ng bsDNA can bias methylation results even with non-FFPE sources of DNA. Using 400ng bsDNA, we identified histologic-specific methylation markers, including the confirmation from a previous report of higher methylation of thrombospondin 2 in ovarian clear cell carcinomas.
Citation Information: Cancer Prev Res 2011;4(10 Suppl):A38.