Quantitation of gene expression in formalin-fixed paraffin-embedded (FFPE) cancer tissues is important for studying many aspects of cancer, including the initiation of tumors, their classification, progression and responsiveness to treatment, and for validating specific genes as diagnostic and/or prognostic biomarkers. Due to their impressive sensitivity and sequence specificity, RT-PCR methods are frequently used for mRNA expression profiling and for validating data obtained by microarrays. However, the random fragmentation of RNA that occurs during preparation and storage of FFPE samples degrades the sensitivity and reproducibility of RT-qPCR. To overcome this problem, we propose a novel method for assaying mRNA fragments in FFPE samples called mR-FQ (mRNA Fragment Quantification) that provides superior sensitivity and accuracy in spite of fragmentation in a cost-effective manner. Our new approach introduces three key innovations. First, we purify RNA fragments of a certain size range. Second, we modify the (fragmented) mRNA in a way that allows for more efficient reverse transcription and simultaneous pre-amplification of target mRNA sequences. Third, we use a novel PCR primer design that provides superior sensitivity and specificity while using single-dye readout (e.g., with SYBR Green), as opposed to specialized probes such as TaqMan probes. In Phase I, we will establish proof-of-concept for this new assay, apply it for detection of HER-2 mRNA in breast cancer FFPE tissue samples, and demonstrate its superior sensitivity in comparison to conventional RTR-qPCR assays. In Phase II, we will develop additional assays for all established mRNA biomarkers for breast cancer. Then, we will develop an assay in virtual PCR-array format for simultaneous expression profiling of both mRNA and miRNA biomarkers in FFPE samples. (Assaying both classes of RNA would provide a more reliable biomarker than assaying only one type of RNA). We will also co-develop breast cancer companion diagnostic assays based on this assay through collaboration with leading Pharma and diagnostic companies. Taking this assay into the clinic could be beneficial for patients and potentially improve clinical decision-making.
Breast cancer is the most common cancer among women. About 200,000 women were diagnosed and over 40,000 died from the disease last year in the US alone. The goal of this grant application is to develop a method for quantitation of fragmented mRNAs from standard histology specimens (FFPE blocks) that will provide superior sensitivity and accuracy at a reasonable cost to facilitate research in and diagnosis of breast cancer.
