Application of a NanoString Technology-based TP53 Functional Assay for Prediction of Chemotherapy Response of Muscle-invasive Bladder Cancer

Platinum-based treatment (e.g., cisplatin, carboplatin) is generally the first-line chemotherapy for muscle-invasive bladder cancer (MIBC) and mediates regression in approximately 50% of the patients. The goal of this study was to improve the response rate to platinum-based treatment by developing a genomics-based method capable of classifying MIBC patients as being either likely to respond to therapy versus not likely to respond. Aberrations in TP53 and its downstream effectors play a key role in promoting bladder cancer progression, however, TP53 mutation status is neither an independent indicator of treatment response nor survival in MIBC. The hypothesis of this study was that evaluating overall TP53 function would be informative in predicting responsiveness to therapy. Towards this goal, we developed a custom-designed “TP53 functional miRGE assay”, which assesses TP53 function by simultaneously measuring the expression of 105 TP53-responsive mRNAs and miRNAs using NanoString nCounter technology. Expression profiling was performed on a panel of 34 therapy-naïve MIBC specimens with known outcomes of either being responsive or resistant. Clustering analysis identified a 7-gene subset of TP53 network genes (BAK1, BAX, BBC3, CDKN1A, MDM2, PMAIP1, TP53), which identified patients who did not respond to chemotherapy with 92.3% accuracy. Furthermore, higher TP53 function was associated more with resistant MIBCs in that these exhibited higher relative expression of TP53 target genes. Our results demonstrate a 7-gene TP53 functional assay using NanoString technology has the potential to predict patient response to platinum-based chemotherapy, and that validation of this method using a larger patient sample set is warranted. Muscle-invasive bladder cancer (MIBC) samples were obtained from patients prior to neoadjuvant chemotherapy (NAC) in accordance with a UC Davis Institutional Review Board (IRB)-approved protocol. The specimens were then processed for standard pathologic analysis, and total RNA was isolated from the formalin-fixed paraffin-embedded (FFPE) specimens. Total RNA samples (150 ng) were analyzed with a custom-designed miRGE assay CodeSet for expression profiling of TP53 pathway mRNAs (n=96) and miRNAs (n=10) using an nCounter Analysis System (NanoString Technologies, Seattle, WA), which performs all processing steps, including target and reporter probe hybridization, probe-target tripartite complex purification and immobilization, and counting. The raw data for each sample were output as Reporter Code Count (RCC) files that contain the counts for each gene in the sample. Data analysis was performed with nSolver Software (NanoString Technologies) and the following processing steps: quality control (i.e., for imaging, binding density, positive control linearity, and positive control limit of detection), background subtraction (negative control subtraction), normalization (geometric mean), fold change estimation, and agglomeration clustering. Samples that required a normalization factor of >10 were flagged and removed from further analysis.