Histopathology archives of well-annotated formalin-fixed, paraffin-embedded (FFPE) tissue specimens are valuable resources for retrospective studies of human diseases. Since recovery of quality intact mRNA compatible with molecular techniques is often difficult due to degradation, we evaluated microRNA (miRNA), a novel class of small RNA molecules with growing therapeutic and diagnostic potential, as an alternative analyte for gene expression studies of FFPE samples. Analyzing total RNA yield, miRNA recovery, and robustness of real-time polymerase chain reaction for miRNA, mRNA, and rRNA species, we compared the performance of commercially available RNA isolation kits and identified a preferred methodology. We further implemented modifications to increase tissue throughput and incorporate a quantitative Armored RNA process control to monitor RNA recovery efficiency. The optimized process was tested for reproducibility as well as interoperator and interday variability, and was validated with a set of 30 clinical samples. In addition, we demonstrated that, independent of FFPE block age and RNA quality, miRNAs generate quantitative reverse transcription-polymerase chain reaction signals that are more robust and better correlate with expression levels from frozen reference samples compared with longer mRNAs. Our broad study, including a total of 272 independent RNA isolations from 17 tissue types and 65 FFPE blocks up to 12 years old, indicates that miRNAs are not only suitable but are also likely superior analytes for the molecular characterization of compromised archived clinical specimens.
Reliable prediction of recurrence in stage II colorectal cancer (CRC) patients is a major unresolved clinical need as recurrence is often the ultimate cause of death in these individuals. Several mRNA and DNA prognostic markers have been evaluated and, to date, none have been recommended by oncology groups due to poor specificity and sensitivity. MicroRNA (miRNA) have been shown to regulate fundamental cellular processes and have been implicated in several cancer types, including CRC. We used a microarray platform to examine the expression of more than 600 miRNAs in tumors from stage II patients with or without recurrence. Using an exploratory sample set of 12 tumors (6 nonrecurrent and 6 recurrent), we identified several miRNAs that were significantly deregulated in tumors from recurrent patients compared to non-recurrent patients. We used RT-qPCR to interrogate the differentially expressed miRNAs on the original 12 samples plus an additional set of 39 stage II CRC tumors that were not used in the initial miRNA discovery process. Using cross-validation, we derived a miRNA-based classifier that was strongly associated with recurrence (hazard ratio (HR), 3.83; p=0.005). We further evaluated associations of MSI status and mutations in KRAS, BRAF and PIK3CA genes with recurrence (n=39). Multivariate Cox analysis revealed that only the miRNA classifier was significantly associated with recurrence (HR 3.08; p=0.03). Interestingly, the combination of the miRNA classifier and mutant KRAS status was highly predictive of recurrence (p<0.0001). These data provide strong support for miRNAs as prognostic biomarkers for stage II CRC and warrant further validation. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A31.
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