CD44 Promoter Methylation and Prostate Cancer Progression – Analysis of Microdissected Samples
JC Hanson, O Akhtar, LG Adams, FC Eberle, WM Linehan, PA Pinto, DC Edelman, MR Emmert-Buck, J Rodriguez-Canales. National Cancer Institute, Bethesda
Background: CD44 gene promoter methylation is thought to play a role in prostate cancer progression. Previous studies identified CD44 methylation using a PCR-based technique; however, no quantitative measurements were performed. The goal of the present study was to quantify CD44 promoter methylation using pyrosequencing, and to correlate methylation status with prostate cancer progression using laser capture microdissection (LCM) of tumor cells exhibiting specific Gleason patterns.
Design: 9 radical prostatectomies with cancer were analyzed. LCM epithelial samples included normal, prostatic intraepithelial neoplasia (HGPIN), and acinar prostate carcinoma with Gleason patterns ranging from 2 to 5. In one case, tumor epithelium was sampled at the apex and the base of the prostate from a single tumor focus. 2 prostate cancer cell lines (LNCaP and PC3) and 3 benign prostate cell lines (RWPE1, RWPE2 and PZ) were included as controls. DNA was extracted from the LCM samples and pyrosequencing analysis of 4 CpG in the CD44 promoter region was performed on all DNA samples. Immunostaining (IHC) for CD44 was done on the same cases used for LCM.
Results: In the cancer cell lines, LNCaP showed 95% methylation while PC3 showed 7% methylation. The 3 benign cell lines showed <8% methylation. In the tissue specimens, all normal and HGPIN samples showed <10% methylation. 5 of 9 tumor samples showed methylation >10% (15 to 53% average) and 4 of 9 tumor samples showed <10% methylation. No correlation was found between Gleason pattern and amount of methylation. In one case, LCM samples from a single tumor focus were analyzed at the apical region (9% methylation) and the base (50% methylation) of the same tumor focus. CD44 IHC expression was found only in cells of the basal cell layer, while luminal cells from normal, HGPIN and tumor were negative.
Conclusions: CD44 methylation >10% were found only in tumor epithelium (5/9 cases and 1/2 cancer cell lines), but did not correlate with Gleason pattern. CD44 methylation differences in the one tumor studied at both the apex and base of the gland suggests a regional variation of CD44 methylation that should be investigated further in a larger set of cases. CD44 IHC expression showed no difference between tumor and normal luminal cells and was not correlated with methylation percentages. These results suggest that CD44 promoter methylation may have a role in prostate carcinogenesis but it is not correlated with cancer progression per se.
Category: Genitourinary (including renal tumors)
Tuesday, March 23, 2010 1:00 PM
Poster Session IV # 115, Tuesday Afternoon