Abstract 52: Next-Gen deep amplicon sequencing of TP53 complements FGFR3 for the detection of bladder cancer-related mutations.
Authors: Millholland John M. , Campo Maria C. , Anderson Lydia D. , Fernandez Cecilia A. , Shuber Anthony P. 
Published: August 2014

Abstract
Background: We have recently described an ultra-deep amplicon sequencing method for detection of FGFR3 mutations in urine that closely matches the frequency of mutations found in bladder cancer tissue. While FGFR3 mutations are present in a large fraction of non-invasive tumors, these mutations are rarely detected in invasive bladder tumors. To complement our existing FGFR3 deep sequencing assay, we have developed a sequencing assay for TP53. Mutations in TP53 are commonly found in advanced bladder cancer, and show little overlap with FGFR3 mutations. Here, we demonstrate the detection of FGFR3 and TP53 mutations in bladder cancer tissue using a Next-Gen sequencing platform.
Methods: The FGFR3 sequencing assay was performed as described previously, permitting the detection of 9 mutations associated with bladder cancer. Similarly, amplicons were designed against TP53 exons 5-8, permitting the detection of 133 TP53 mutations previously reported in bladder cancer. Primary amplification was performed on DNA isolated from FFPE or fresh frozen tissue. The resulting PCR products were used as template for emulsion PCR and sequenced using the Ion Torrent PGM. Samples were analyzed for total DNA reads and number of mutant sequencing reads to determine percent mutation.
Results: A set of 57 bladder cancer tissue samples, comprised of 38 non-muscle invasive (NMIBC) and 19 muscle invasive (MIBC) tumors, was analyzed for mutations in both FGFR3 and TP53. Of these samples, 18 out of 38 (47.4%) NMIBC tumors were positive for FGFR3 mutations, and 2 of 38 (5.3%) were positive for TP53 mutations. 9 out of 19 (47.4%) MIBC tumors were positive for TP53 mutations, with an additional 2/19 (10.9%) positive for FGFR3 mutations. Across all tumor stages tested, 35.1% (20/57) of samples were positive for FGFR3 mutations, and 19.3% (11/57) were positive for TP53 mutations. No overlap was seen between samples positive for FGFR3 or TP53 mutations. In addition, we have now shown that these assays can detect both FGFR3 and TP53 mutations in the urine of bladder cancer patients. These studies will be expanded to a large set of urine samples to more accurately assess clinical performance of this FGFR3/TP53 deep sequencing assay in bodily fluids.
Conclusions: We have developed FGFR3 and TP53 sequencing assays that can detect mutations in a broad range of bladder cancer stages. The complementarity of mutations found in these two genes may allow for the non-invasive detection of bladder cancer in a larger fraction of undiagnosed patients.
Citation Format: John M. Millholland, Maria C. Campo, Lydia D. Anderson, Cecilia A. Fernandez, Anthony P. Shuber. Next-Gen deep amplicon sequencing of TP53 complements FGFR3 for the detection of bladder cancer-related mutations. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 52. doi:10.1158/1538-7445.AM2013-52