Bladder cancer (BC) is the 5th most common cancer in Western societies with a rising global incidence. The major challenge to improving patient outcomes is to better identify patients at risk for recurrence, progression, and metastasis, and to monitor treatment response. A non-invasive monitoring assay is needed in order to interrogate the molecular features and clinical tumor heterogeneity of bladder cancers, which will potentially lead to better stratified patient management.

We developed a targeted amplicon-based re-sequencing workflow for detecting and tracking genomic mutations in cfDNA using dual molecular indexed primers for specific amplification of a panel of 20 of known (TERT, FGFR3, PIK3CA, HRAS) and novel BC mutations. Only 3hr time is needed for the total workflow for the library generation and followed by next generation sequencing (NGS) performed on Illumina MiSeq sequencing platform. Data analysis and variants calling was performed.

After a thorough test, optimization, and validation of the targeted panel and the workflow on selected samples of matched bladder tumor-blood pairs and controls, 45 samples were included for testing the workflow, which includes samples of 16 matched bladder tumor-blood pairs, technical duplicates of cfDNA DNA Reference Standards (Horizon Discovery) with mutations at known allelic frequencies. Technical duplicates of cfDNA extracted from blood from patients with metastatic BC (pre and post chemotherapy) were also included in this study. Somatic variants from matched tumor-blood pairs were detected. We showed that detection sensitivity at 1.0% allelic frequency was achieved. Our preliminary results demonstrated that we were able to detect mutation burden from cfDNA samples and track mutation burden change in response to chemotherapy for patients with metastatic BC.

Here we developed a sensitive, non-invasive targeted NGS workflow to detect somatic mutations for bladder cancers from both tumor DNA and cfDNA samples. Our early results demonstrated that we can detect and track mutational changes from patients with metastatic BC.

Citation Format: Liqin Dong, Pramit Khetrapal, Simon Rodney, Wei Shen Tan, Sheida Rezaee, Patricia de Winter, John Kelly, Andrew Feber. A targeted re-sequencing assay for molecular profiling of somatic mutations from plasma cell-free dna (cfdna) for bladder cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5702. doi:10.1158/1538-7445.AM2017-5702