Amplichip p53 research test

DNA was isolated from the renal cortex and tumor tissues by standard phenol-chloroform extraction techniques. The level of AL-DNA adducts in the renal cortex DNA (10–20 µg) was determined using ³²P-postlabeling polyacrylamide gel electrophoresis, as previously described (13 (link)). The TP53-specific mutations were identified using the AmpliChip p53 Research Test (Roche Molecular Diagnostics, Pleasanton, CA), sensitively detecting all single base-pair substitutions and single-base deletions (13 (link)).

Evaluation of genetic alterations and protein expression has been described previously (11 (link), 14 (link), 17 (link), 30 (link)–32 (link)). Formalin-fixed paraffin-embedded (FFPE) biopsies were organized prior to R-CHOP treatment and tissue microarray was constructed for study. IHC analysis was performed for the following markers with corresponding antibodies: MYC (clone Y69, Epitomics), BCL-2 (clone 124, DAKO), p53 (clone DO-7, DAKO), and Ki-67 (clone MIB-1, DAKO). The cut-off values were 40% for MYC, 50% for BCL-2, 30% for p53 (average of the two cutoffs in previous studies; refs. 14 (link), 17 (link)), and 70% for Ki-67 as previously determined.
FISH analysis was performed on FFPE sections to detect MYC rearrangement using a Vysis LSI MYC dual-color break-apart rearrangement probe (Abbott Molecular) following the manufacturer’s instructions. Images were captured and reviewed using Cytovision software (Applied Imaging). At least 200 tumor cell nuclei were scored and a cutoff of more than 5% of positive cells was used to determine the presence of MYC rearrangement.
TP53 mutations were detected using the AmpliChip p53 Research Test (Roche Molecular Systems), which is a microarray-based assay that detects mutations in exons 2–11 (14 (link)).