We further evaluated the functional
We further evaluated the functional relation between promoter regulation of the tumor suppressor genes and in fresh frozen, microdissected tumor tissue from 55 patient with clear cell renal carcinoma. In addition, normal-appearing tissue from the same patient taken distal of the tumor and later histopathologically confirmed to be nontumorous was also evaluated. Methylation and mRNA SAR405 levels were evaluated with special regard to tumor characteristics. Followup data were available on all the patients in this study.
Materials and Methods
Tissue samples were immediately shock frozen after surgical resection and stored in liquid nitrogen at −80C. Before further processing samples were serially sectioned and stained with hematoxylin and eosin to ensure that slices used for subsequent DNA and RNA extraction predominantly contained malignant tissue of the respective tumors. Areas with the highest neoplastic cell content (greater than 80%) were selected and microdissected under microscopic guidance using a sterile needle. Histopathological staging and grading were done according to the 1997 UICC TNM classification. All patients signed a consent form approved by the Committee on Human Rights in Research at our institution.
Results Using quantitative MSP the methylation prevalence of the APAF-1 gene in tumor samples was 100% (55 of 55), while it was 62% (34 of 55) for the DAPK-1 gene. The methylation prevalence in normal tissue samples was 33% (18 of 55) for the APAF-1 and 41% (23 of 55) for the DAPK-1 gene. The expression of mRNA transcripts of the APAF-1 and DAPK-1 genes was detected in 76% of tumor tissue samples (42 of 55). In corresponding normal tissue samples the detection rate was 53% (29 of 55) for APAF-1 and 58% (32 of 55) for DAPK-1 gene mRNA expression. Overall the levels of mRNA expression of APAF-1 and DAPK-1 showed a significant correlation in tumor vs corresponding normal tissue (median 64 vs 21 RGE, p = 0.003 and 180 vs 71, p = 0.0001). Neither APAF-1 and DAPK-1 methylation levels nor mRNA expression levels correlated with individual tumor stage (table 1). When mRNA expression levels in tumor tissue were related according to differentiation grades, median mRNA expression of APAF-1 was 117 RGE (range to 438) in G1/2 tumors vs 37 RGE (range to 438) in G3 tumors (p = 0.018). For DAPK-1 median mRNA expression was 364 RGE (range to 4,815) in G1/2 tumors vs 56 RGE (range to 4,146) in G3 tumors (p = 0.05, table 1). Tumors were then classified into locally nonadvanced lesions (pT1), locally advanced lesions (pT2 or greater) and lesions smaller or larger than 4 cm. A correlation between lower mRNA expression of APAF-1 and DAPK-1 in these subgroups was not detected. When methylation and mRNA expression levels of APAF-1 and DAPK-1 were evaluated together in matched pair analysis, we observed a marked increase in the level of methylation in parallel with a decrease in mRNA expression of the APAF-1 gene. For example, median APAF-1 methylation was 83% and median APAF-1 mRNA expression was 84 RGE in the group with node negative tumors, whereas in the group with node positive tumors median APAF-1 methylation increased to 149% and mRNA expression decreased to 28 RGE (Spearman rho test p = 0.02). A significant correlation between higher methylation levels of DAPK-1 and lower mRNA expression was not observed. Although low levels of methylation of APAF-1 and DAPK-1 were detected in normal tissue, NIM levels did not correlate significantly with corresponding mRNA expression levels on matched pair analysis (Table 1, Table 2). In tumors with positive lymph node staging mRNA expression of DAPK-1 showed a significant correlation compared with expression in tumors with negative nodal staging (median 42 vs 364 RGE, p = 0.009). This was also observed for APAF-1 mRNA expression (median 28 vs 84 RGE, p = 0.04). APAF-1 and DAPK-1 mRNA expression progressively decreased from the highest in tumor tissue from patients without lymph node involvement and corresponding normal tissue to the lowest in tumor tissue from samples with lymph node involvement and corresponding normal tissue, eg median DAPK-1 RGE 364 to 126 (tumor to normal tissue, node negative) and 42 to RGE (tumor to normal tissue, node positive) (table 2).