Formalin-fixed paraffin-embedded (FFPE) tissue blocks were obtained from 40 of the 43 patients with locally advanced, unresectable or metastatic solid tumors who participated in three clinical trials at the University of Wisconsin: [a phase I combination of 3-AP and doxorubicin (14
), a phase I combination of 3-AP and irinotecan (15
) and a phase II single agent study of 3-AP in pancreatic cancer (16
)]. There were thirteen tissue blocks from patients with pancreatic cancer (four from primary sites and nine from metastatic sites). Other tissue types included,:one primary bladder cancer; one primary cervical cancer; four cholangiocarcinoma (one primary, three metastatic); two primary colon cancers; three primary esophageal cancers, one primary gastric cancer, one metastatic lymphoma, three primary melanomas, two primary mesotheliomas, one primary non-small cell lung cancer (NSCLC), two primary prostate cancers, one primary sarcoma and one primary small cell lung cancer (SCLC). All samples were available for automated quantitative immunohistochemistry (AQUA) analysis. Seven individuals with pancreas cancer, two with breast cancer and one with lymphoma did not have sufficient material for RNA analysis. The Health Sciences Institutional Review Board of the University of Wisconsin approved these trials prior to their implementation, and all patients gave informed written consent.
Laser Capture Microdissection
Sections were prepared from each FFPE tissue blocks, and hematoxylin and eosin (H&E) staining was performed. H&E stained slides were reviewed by a pathologist to determine the location of tumor tissue on each slide. Laser capture microdissection (LCM) with the SL μCut Laser Microdissection System (Molecular Machines & Industries, Glattbrug, Switzerland) was utilized to ensure the isolation of only tumor cells. Briefly, the sections were melted at 60°C for 30 min and deparaffinized in xylene. Sections were then rehydrated in graded ethanols, rinsed with DEPC-treated water, stained with toluidine blue, rinsed in DEPC-treated water, dehydrated in graded ethanols followed by isopropanol and placed in a desiccator until dry.
RRM2 Gene Expression Analysis by Quantitative Reverse Transcriptase Polymerase Chain Reaction (Q-RTPCR)
After LCM, RNA extraction was performed using the Paradise Whole Transcript RT Reagent System (Arcturus Bioscience, Sunnyvale, CA) which processes FFPE tissue scrapes. Caps were placed in a microcentrifuge tube-containing proteinase K and incubated at 37°C for 16-20 hours. After centrifugation, the caps were removed and the RNA was isolated and treated with DNase following the manufacture's instructions. The total RNA was resuspended and then treated with DNase. Total RNA was stored at -80°C until analyzed. The RNA was quantified via NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE), and the total RNA extracted from FFPE tumor tissue was reverse transcribed using random primers by standard methods.
TaqMan assays were designed for the genes listed in using Integrated DNA Technologies (Coralville, IA), keeping the amplicon length < 100bp. Quantitative real-time PCR was performed using the Bio-Rad iCycler IQ system (Hercules, CA). Due to the limited sample supply, the target gene (RRM2) and the endogenous reference gene (YWHAZ) were amplified in a single well. Each well contained 5 pmol/μL of the probes, 5 pmol/μL of the primers, and 12.5 μL of iQ Multiplex Powermix (Bio-Rad) in a 25 μL final reaction mixture. The Multiplex Powermix was heat-activated for 3 min at 95°C. Each of the 50 PCR cycles consisted of 15 sec of denaturation at 95°C and hybridization of primers and probes for 45 sec at 60°C.
Sequences of the primers and probes used in quantitative PCR studies
Expression levels in the patient samples were determined by the standard curve method using standard cDNA solutions which were serially diluted 5-fold from HepG2 carcinoma cell line. The standard curve and samples were run in triplicate, and nontemplate controls were included in each run. The data was analyzed with the standard curve line equations generated by iQ5 software (Bio-Rad). The starting mass value for the gene of interest and reference gene were calculated by substituting the threshold cycle (Ct) values generated by the iQ5 software into the standard curve formula. The Ct (threshold cycle) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold. The data was expressed as target gene (RRM2)/endogenous gene (YWHAZ). The correlation coefficient (r) for each standard curve exceeded 0.99, and the coefficient of variation for the Ct values was less than 3.5% for all replicates.
Linearity was determined with five standard curves, and the mean coefficient of determination (r2) for RRM2 was 0.990 (range 0.982-0.998) and for YWHAZ was 0.996 (range 0.994-0.998). Triplicate determinations of each standard were run on a single plate on five separate occasions over a two-week period. The intra-day variability in the standard Ct readings for RRM2 averaged 0.65% (range 0.09-2.84%), while for YWHAZ the standard Ct readings averaged 0.61% (range 0.06-1.96%). The mean CV in the Ct values for RRM2 over this time ranged from 0.32% (range 0.09-0.68%) for the 400 ng standard to 1.47% (range 0.66-2.84%) for the 0.128 ng standard. For YWHAZ, the mean CV in the Ct values over the standard curve ranged from 0.54% (range 0.31-1.03%) for the 400 ng standard to 1.11% (0.33-1.96%) for the 0.128 ng standard. Triplicate determinations of Ct readings in five patient samples on a single plate had a mean CV for RRM2 of 1.18% (range 0.25-2.21%) over the mass range of 0.19-114.43 ng (all samples were diluted). In the same five patient samples, the mean CV for YWHAZ was 0.70% (range 0.03-1.14%) over the mass range of 0.13-116.12 ng (all samples were diluted). The variability did not change with concentration.
M2 Protein Analysis by Automated Quantitative Analysis (AQUA)
RRM2 expression was determined using the AQUA system (HistoRx, New Haven, CT) as previously described (17
). Initially, target compartments were localized using a fluorescently tagged rabbit anti-cytokeratin antibody (or the anti-S100 antibody for melanoma cells). 4,6-Diamidino-2-phenylindole (DAPI) was added to visualize nuclei. RRM2 was visualized with an Alexa Fluor 488 labeled tyramide which, like diaminobenzidine, is activated by horseradish peroxidase and results in the deposition of numerous covalently associated Alexa Fluor 488 dyes immediately adjacent to the horseradish peroxidase-conjugated secondary antibody (). Using this approach, classical compartments are defined on the basis of molecular co-localization. The cytokeratin compartment is equivalent to all epithelial cells in the tissue section. The S-100 compartment is equivalent to melanoma cells in tissue section. DAPI is the area defined as the cell nucleus. A matched set of H&E sections were used for locating tumor. RRM2 was tagged and measured within the subcellular compartments by the PLACE algorithm as previously described. Data is represented as a Z-score, which was calculated by subtracting the mean AQUA score from the individual AQUA score and dividing by the standard deviation (18
). Therefore a negative Z-score indicates the protein expression was less than the average and a positive Z-score means it is greater than the average.
Protein expression of ribonucleotide reductase M2 (RRM2) was determined using an automated in situ quantitative measurement of protein analysis, automated quantitative immunohistochemistry on the basis of immunofluorescence.
RRM2 gene expression and protein expression were summarized in terms of number of observations, means and standard deviations. The data were presented in graphical format using boxplots. The comparisons between groups were performed using a non-parametric Wilcoxon Rank Sum test or the Kruskal-Wallis test. Exact p-values were computed for all comparisons. All statistical tests were two-sided, and P < .05 was used to indicate statistical significance. Due to the exploratory nature of this study, no adjustments for multiple comparisons were made. Non-parametric Spearman's rank correlation analysis was used to examine the association between protein and gene expression levels. Statistical analysis was performed using SAS® (SAS Institute Inc., Cary, North Carolina) version 9.1 software.