Preoperative Cell-Free DNA (cfDNA) in Muscle-Invasive Bladder Cancer Treatment Outcome

Abstract

Background Tumor heterogeneity and lack of personalized prognosis leads to bladder cancer (BlCa) patients’ lifelong surveillance with invasive interventions, highlighting the need for modern minimally invasive tools for disease management. Herein, we have evaluated the clinical utility of preoperative serum cell-free DNA (cfDNA) in ameliorating patients’ risk-stratification and prognosis. Methods cfDNA was purified from 190 preoperative BlCa patients and 26 healthy individuals’ serum samples and quantified by 2 assays: an in-house quantitative real-time PCR (qPCR) assay using LEP as reference control and a direct fluorometric assay using Qubit HS dsDNA. Capillary electrophoresis was performed in 31 samples for cfDNA fragment profiling. Tumor relapse/progression and metastasis/death were used as clinical endpoints for non-muscle-invasive bladder cancer and muscle-invasive bladder cancer (MIBC), respectively. Results cfDNA profiling by capillary electrophoresis highlighted that total and fragment-related cfDNA levels were significantly increased in BlCa and associated with advance disease stages. Evaluation of cfDNA levels by both Qubit/qPCR displayed highly consistent results (rs = 0.960; P < 0.001). Higher cfDNA was correlated with MIBC and stronger risk for early metastasis (Qubit:hazard ratio [HR] = 3.016, P = 0.009; qPCR:HR = 2.918, P = 0.004) and poor survival (Qubit:HR = 1.898, P = 0.042; qPCR:HR = 1.888, P = 0.026) of MIBC patients. Multivariate cfDNA-fitted models led to superior risk stratification and net benefit for MIBC prognosis compared to disease established markers. Conclusions Elevated preoperative cfDNA levels are strongly associated with higher risk for short-term metastasis and poor outcome of MIBC, supporting modern noninvasive disease prognosis and management.

MIR145 Core Promoter Methylation in Pretreatment Cell-Free DNA: A Liquid Biopsy Tool for Muscle-Invasive Bladder Cancer Treatment Outcome

“…More precisely, our group has highlighted the epigenetic regulation of miR-143/145 cluster by MIR145 core promoter methylation in bladder tumors and the lower imprinting in MIBC. 19,40 In parallel, tumor evolution into more advanced stages has been well documented to release of cfDNA into biologic fluids, because of augmented cell death, 42-44 leading to the enrichment of tumor-specific DNA fraction in circulation, while Papadimitriou et al 45 have recently reported the significantly elevated cfDNA levels in patients with MIBC compared with patients with NMIBC and HDs. Moreover, to demonstrate the BlCa-derived alterations of MIR145 promoter methylation in patients' cfDNA, we have analyzed ZNF154 methylation in cfDNA of our screening cohort, which is specifically hypermethylated in bladder tumor cells compared with normal urothelium, normal tissues, and blood/endothelial cells, acting thus as a BlCa-specific methylation marker into circulation.…”

“…More precisely, our group has highlighted the epigenetic regulation of miR-143/145 cluster by MIR145 core promoter methylation in bladder tumors and the lower imprinting in MIBC. 19,40 In parallel, tumor evolution into more advanced stages has been well documented to release of cfDNA into biologic fluids, because of augmented cell death, [42][43][44] leading to the enrichment of tumor-specific DNA fraction in circulation, while Papadimitriou et al 45…”

CDKN2A copy number alteration in bladder cancer: Integrative analysis in patient-derived xenografts and cancer patients

“…CDKN2A CNAs were calculated according to the 2 −ΔΔCT relative quantification method, using LEP as the diploid reference gene. 44 Standard curves for the identification of the assays’ limit of quantification and the determination of optimal cutoff values for CDKN2A homozygous/hemizygous deletions were constructed using diploid human gDNA as well as SK-OV-3 and RT112 cell line gDNA with known HD of CDKN2A (NCI-60/UBC-40 cell line panels 19 , 20 , 45 ). All standard curve assays were performed in triplicate.…”