Mutation Profiling of Bladder Cancer Using Ion Torrent Sequencing
Eva M Wojcik, Amy B Rosenfeld, Michael I Zillox, Marcus L Quek, Guliz A Barkan, Xiaowu Gai. Loyola University, Maywood, IL
Background: Urothelial carcinoma of the bladder (UC) is the second most common genitourinary malignancy in the United States. Current diagnostic tests are based on a specific biomarker or genetic alteration, and have modest sensitivity and specificity. We hypothesize that mutation profiles of known oncogenes and tumor suppressor genes collectively can distinguish different subgroups, therefore be used for accurate diagnosis, prognostic monitoring, and predicting treatment response.
Design: Histopathological examination of formalin-fixed, paraffin-embedded (FFPE) samples differentiated normal bladder tissue from tumor tissue (High grade UC, T2b). In a pilot study, DNA was isolated from pairs of UC and normal tissues of 5 patients using the AMBION Recovery all protocol. Genomic regions encompassing 739 known COSMIC mutations in 46 oncogene and tumor suppressor genes were amplified using Ion Torrent Ampliseq cancer panel protocol, which were then sequenced using the Ion Torrent Personal Genome Machine. An average base coverage depth of 570X across all target regions was achieved. Variant calls were made using the Ion Torrent Suite of software tools.
Results: We detected 7-40 genetic variants in each sample, with the majority being previously unknown somatic mutations. The overlap between the normal and cancerous tissue was less than 30% in each patient, suggesting the histologically normal tissues were possibly pre-cancerous. We found variants in exons, introns and the 3' UTR of genes that resulted in 52 silent, 83 missense and 5 nonsense coding changes. In addition, we detected 9 deletions resulting in frameshifts. Further analysis using this limited sample size suggested a positive correlation of the number of mutations with patient age. Between patients, we found the highest number of mutations in the APC, FGFR3, KDR and PDGFRA genes with few overlaps.
Conclusions: Our results revealed a large number of novel cancer mutations and confirmed 1) the highly heterogeneous nature of urothelial carcinoma, both within and between samples, 2) the presence of a large number of somatic mutations in the histologically normal surrounding tissues; 3) the lack of a common bladder cancer mutation in these oncogenes and tumor suppressor genes, and hence 4) the potential need of personalized genetic test for each patient. In addition, our preliminary results are in agreement with the hypothesis that accumulation of somatic mutations in the bladder cells as people age leads to pre-cancerous cells and eventually cancer.
Category: Special Category - Pan-genomic/Pan-proteomic approaches to Cancer
Tuesday, March 5, 2013 9:30 AM
Poster Session III # 231, Tuesday Morning