Abstract 2274: Whole-genome bisulfite sequencing of cell-free DNA (cfDNA) in muscle-invasive bladder cancer patients to predict the risk of recurrence
Authors: LU YI-TSUNG , Gooden Gerald , Tew Ben Y. , Buckley David , Siegmund Kimberly , Daneshmand Siamak , Goldkorn Amir , Salhia Bodour 
Published: August 2019

Abstract
Background: Muscle invasive bladder cancer (MIBC) is a lethal disease with high recurrence rate despite standard radical cystectomy with neoadjuvant chemotherapy. Informative new biomarker strategies are needed to accurately assess the risk of recurrence and guide treatment. Epigenetic marks play a key regulatory role in cancer initiation and progression, and methylation patterns in the tissue have been correlated with prognosis in multiple cancer types. We hypothesized that whole-genome bisulfite sequencing (WGBS) can be used to generate a cell-free DNA (cfDNA) based methylation signature prognostic of bladder cancer recurrence after curative cystectomy.
Methods: We identified a retrospective MIBC cohort with annotated clinical outcome more than 10 years after curative cystectomy. In the cohort, we selected a total of 46 patients who did not recur after 3 years of follow-up, and 40 patients who relapsed within 3 years of surgery. cfDNA was extracted from the archival plasma, and samples were pooled for WGBS based on their pathologic stages (T2a, T2b, T3a, T3b, and T4/N+) and recurrence status (recurrence vs no recurrence). A paired-end 150bp sequencing was performed on an Illumina NovaSeq platform. Sequences were aligned using the bismark tool and DNA methylation differences between groups were identified using metline.
Results: CfDNA was extracted with a median cfDNA concentration 10.6 ng/ml (range 0.97-121.5 ng/ml). After WGBS, differential methylation regions (DMRs) for each stage were called by comparing the recurrence (R) and non-recurrence (NR) groups. We identified 6412 to 9532 DMRs across stages, and greater overall hypomethylation in cfDNA before systectomy in R groups was observed in all stages. While most of the DMRs are in the gene bodies, 10.3 to 11.7% of the DMRs were in the promoter regions. DMRs were also compared with publically available datasets using Basespace correlation engine. The cfDNA methylation in R groups across all stages was positively correlated with tumor tissue methylation in a bladder cancer dataset. Similarly, the cfDNA methylation in R groups was inversely correlated with bladder cancer tissue expression in TCGA dataset. We also called the DMRs by comparing all R and NR groups, and 729 DMRs were found to be shared in all stages. Similar patterns of global hypomethylation and positive correlation to the bladder cancer tissue methylation were observed in R group.
Conclusions: cfDNA methylation in MIBC patients was concordant with multiple reported datasets. The overall hypomethylation in the cfDNA prior to the cystectomy was observed in patients who later recurred. Additional data filtering and annotation is ongoing, and these DMRs will be validated as potential biomarkers for predicting the risk of recurrence in MIBC patients.
Citation Format: YI-TSUNG LU, Gerald Gooden, Ben Y. Tew, David Buckley, Kimberly Siegmund, Siamak Daneshmand, Amir Goldkorn, Bodour Salhia. Whole-genome bisulfite sequencing of cell-free DNA (cfDNA) in muscle-invasive bladder cancer patients to predict the risk of recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2274.