333 Background: The current study was undertaken to determine the feasibility of detection and expression profiling of cfRNA in mUC patients treated with platinum-based chemotherapy. Methods: Plasma samples were identified from consented mUC subjects treated with platinum-based therapy at the IU Simon Cancer Center (IUSCC) from 12/2008 – 8/5/2010. The primary feasibility measure was the detection of cfRNA at any quantitative level. An IUSCC urologic cancer 48-gene array was designed. After cfRNA extraction and cDNA preparation, cDNA were amplified in a specific target amplification step using gene-specific primers for the 48 genes in the array. Gene expression analysis was performed on amplified cDNA using 96.96 format DELTAgene microfluidic Dynamic Array platform (Fluidigm). CT values were calculated for each gene after normalization against expression of internal control gene, GAPDH. To identify associations between individual gene expression and OS, a Cox proportional hazards regression analysis was performed. Results: Plasma samples and OS data were available from 15 pts. Patient demographics included: median age – 70.8, M/F – 14/1, Cisplatin/Carboplatin regimen – 9/6, lymph node/visceral mets – 13/7. Median PFS/OS were 8.7 and 23.0 months respectively. cfRNA was detectable in all 15 patients (100%). Median quantitative cfRNA concentration was 2.68 ng/ml (range 1.9 – 6.3). 96.96 Dynamic Array was performed in all patients. All 48 gene probes passed QC testing and produced CT values suitable for analysis. On univariate analysis, overall survival was significantly associated with presence of visceral metastases and plasma cfRNA expression of AKT1, AKT2, BRCA1, CCND1, HPRT1, HSD3B1, PARP1, and RPA3 (all p < 0.05). Conclusions: Successful extraction of cfRNA suitable for microarray analysis from archived mUC patient plasma samples is feasible. Intriguing hypothesis generating associations between OS and cfRNA expression of genes mediating DNA repair, apoptosis, and cell-signaling pathways were observed. Demonstration of these findings in larger independent patient cohorts is warranted.
High Throughput Gene Expression Measurement with Real Time PCR in a Microfluidic Dynamic Array
