K. Skowron1, J. Namm5, S. Pitroda3, M. Beckett3, N. Khodarev3, M. Posner1,4, G. Steinberg2, R. Weichselbaum3 1University Of Chicago,Department Of Surgery / Section Of General Surgery,Chicago, IL, USA 2University Of Chicago,Department Of Surgery / Section Of Urology,Chicago, IL, USA 3University Of Chicago,Department Of Radiation And Cellular Oncology,Chicago, IL, USA 5Loma Linda University Health,Department Of Surgery,Loma Linda, CA, USA 4University Of Chicago,Department Of Surgery / Section Of Surgical Oncology,Chicago, IL, USA
Introduction:
Bladder cancer is the 9th leading cause of cancer death in the U.S. Treatment consists of radical cystectomy with adjuvant chemotherapy or bladder-sparing chemo-radiotherapy in selected cases. Volkmer et al. identified bladder tumor initiating cells (BTICs) in progressive stages of differentiation (1). We established patient derived xenografts (PDX) in order to study BTIC biology in vivo. We hypothesized that patient tumors with a greater population of basal or poorly differentiated BTICs would have a poor clinical outcome compared to patients with well-differentiated tumors. Gene expression analysis was performed with the goal of identifying prognostic markers.
Methods:
Patients undergoing cystectomy are recruited under an IRB-approved protocol. Tumor samples are received from pathology; fragments are injected subcutaneously into the flank of NOD/SCID mice and monitored for engraftment. Remaining tissue is analyzed with flow cytometry to quantify the BTIC populations. Any tumors which engraft are excised, reimplanted into further generations of mice, and sorted for RNA extraction of the BTIC populations. Subsequent xenograft tumors are treated with either radiation or cisplatin, and analyzed for tumor growth and changes in BTIC populations. Clinical data are gathered using the REDCap web-based data server. RNA expression is assessed using Illumina Human HT-12 array.
Results:
Of 70 patient samples, 41 tumors have engrafted in mice. There was no correlation between engraftment and survival. However, a correlation between greater basal BTIC populations and overall survival approached significance (p=0.09). If patients did not receive systemic chemotherapy (n=28), the basal BTICs conferred a greater risk to overall survival (LR 5.5, p=0.06) than among patients (n=21) who received any systemic chemotherapy (LR 0.62, p=0.74). The basal BTICs comprise an average of 1% of the tumor cells (range 0-17%). Early results indicate a differential response to cisplatin and radiation among PDX tumors for both agents. Analysis of the RNA array identified a signature of 54 genes suppressed in basal BTICs relative to the more differentiated tumor cells. This gene signature correlates with overall survival (p=0.0047) and disease-specific survival (p=0.0085) in a large cohort of patients.
Conclusion:
Our results suggest that the presence of a small percentage of basal BTICs portends a poor clinical outcome, which may be improved by the addition of chemotherapy. RNA evaluation of these BTICs has revealed potential biological markers and targets for future therapy, as well as a gene signature which may provide a tool for improved patient selection for therapy.
(1) Volkmer JP, Sahoo D, Chin RK, et al. Three differentiation states risk-stratify bladder cancer into distinct subtypes. PNAS 2012;109:2078-2083.