Cell-free tumor DNA and TERT promoter mutations in bladder cancer

353 Background: Currently, there are no FDA-approved blood biomarkers for the prognosis or prediction of outcomes in urothelial carcinoma (UC). The telomerase reverse transcriptase ( TERT) promoter is recurrently mutated at high frequency in UC (50%). These mutations have been correlated with tumor recurrence and survival. Tumor cell-free DNA (cfDNA) with somatic genomic alterations can be found in the plasma of cancer patients and has the potential for use as a non-invasive cancer biomarker. Detection of TERT promoter mutations in cfDNA might be used as a prognostic tool to monitor disease outcome in UC patients. We set out to detect tumor cfDNA and TERT promoter mutations in cfDNA from patients with UC at different stages. Methods: UC patients receiving chemotherapy in the neoadjuvant, first or second-line metastatic setting had blood collected either before or during therapy. cfDNA was isolated from ~1ml plasma samples using the QIAmp (Qiagen) kit. Samples underwent ultra-low pass whole genome sequencing (ULP-WGS) to determine whether tumor cfDNA could be detected in these samples. TERT promoter mutations were detected using a sensitive qPCR assay. Results: 40 plasma samples from a total of 32 patients with urothelial carcinoma were analyzed. Sufficient amounts of plasma cfDNA were obtained for library construction and ULP-WGS in 11 patients. 6 of these 11 patients were determined to be positive for detectable tumor cfDNA and of these, all were metastatic and 50% (3/6) were positive for a TERT promoter mutation. In total, 8 out of 40 samples (20%) were positive for a TERT promoter mutation, including samples from two patients where total cfDNA yield was insufficient for library construction. A total of ~20% of patients with metastatic disease were positive for TERT promoter mutations in cfDNA. The low percentage of samples having sufficient cfDNA most likely reflects the low volume of plasma used. Conclusions: TERT promoter mutations were identified in cfDNA of UC patients. ULP-WGS showed tumor cfDNA in patients with a high tumor burden and metastatic disease. TERTpromoter mutations in cfDNA could potentially be used as a non-invasive method for detection of disease. These results have implications for the use of cfDNA in the evaluation of advanced UC.