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Optimized targeted sequencing of cell-free plasma DNA from bladder cancer patients.

ABSTRACT: Analysis of plasma cell-free DNA (cfDNA) may provide important information in cancer research, though the often small fraction of DNA originating from tumor cells makes the analysis technically challenging. Digital droplet PCR (ddPCR) has been utilized extensively as sufficient technical performance is easily achieved, but analysis is restricted to few mutations. Next generation sequencing (NGS) approaches have been optimized to provide comparable technical performance, especially with the introduction of unique identifiers (UIDs). However, the parameters influencing data quality when utilizing UIDs are not fully understood. In this study, we applied a targeted NGS approach to 65 plasma samples from bladder cancer patients. Laboratory and bioinformatics parameters were found to influence data quality when using UIDs. We successfully sequenced 249 unique DNA fragments on average per genomic position of interest using a 225?kb gene panel. Validation identified 24 of 38 mutations originally identified using ddPCR across several plasma samples. In addition, four mutations detected in associated tumor samples were detected using NGS, but not using ddPCR. CfDNA analysis of consecutively collected plasma samples from a bladder cancer patient indicated earlier detection of recurrence compared to radiographic imaging. The insights presented here may further the technical advancement of NGS mediated cfDNA analysis.

SUBMITTER: Christensen E

PROVIDER: S-EPMC5789978 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Optimized targeted sequencing of cell-free plasma DNA from bladder cancer patients.

Christensen Emil E Nordentoft Iver I Vang Søren S Birkenkamp-Demtröder Karin K Jensen Jørgen Bjerggaard JB Agerbæk Mads M Pedersen Jakob Skou JS Dyrskjøt Lars L

Scientific reports 20180130 1

Analysis of plasma cell-free DNA (cfDNA) may provide important information in cancer research, though the often small fraction of DNA originating from tumor cells makes the analysis technically challenging. Digital droplet PCR (ddPCR) has been utilized extensively as sufficient technical performance is easily achieved, but analysis is restricted to few mutations. Next generation sequencing (NGS) approaches have been optimized to provide comparable technical performance, especially with the intro ...[more]

PMID: 29382943

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Project description:ImportanceNovel sensitive methods for detection and monitoring of residual disease can improve postoperative risk stratification with implications for patient selection for adjuvant chemotherapy (ACT), ACT duration, intensity of radiologic surveillance, and, ultimately, outcome for patients with colorectal cancer (CRC).ObjectiveTo investigate the association of circulating tumor DNA (ctDNA) with recurrence using longitudinal data from ultradeep sequencing of plasma cell-free DNA in patients with CRC before and after surgery, during and after ACT, and during surveillance.Design, setting, and participantsIn this prospective, multicenter cohort study, ctDNA was quantified in the preoperative and postoperative settings of stages I to III CRC by personalized multiplex, polymerase chain reaction-based, next-generation sequencing. The study enrolled 130 patients at the surgical departments of Aarhus University Hospital, Randers Hospital, and Herning Hospital in Denmark from May 1, 2014, to January 31, 2017. Plasma samples (n = 829) were collected before surgery, postoperatively at day 30, and every third month for up to 3 years.Main outcomes and measuresOutcomes were ctDNA measurement, clinical recurrence, and recurrence-free survival.ResultsA total of 130 patients with stages I to III CRC (mean [SD] age, 67.9 [10.1] years; 74 [56.9%] male) were enrolled in the study; 5 patients discontinued participation, leaving 125 patients for analysis. Preoperatively, ctDNA was detectable in 108 of 122 patients (88.5%). After definitive treatment, longitudinal ctDNA analysis identified 14 of 16 relapses (87.5%). At postoperative day 30, ctDNA-positive patients were 7 times more likely to relapse than ctDNA-negative patients (hazard ratio [HR], 7.2; 95% CI, 2.7-19.0; P < .001). Similarly, shortly after ACT ctDNA-positive patients were 17 times (HR, 17.5; 95% CI, 5.4-56.5; P < .001) more likely to relapse. All 7 patients who were ctDNA positive after ACT experienced relapse. Monitoring during and after ACT indicated that 3 of the 10 ctDNA-positive patients (30.0%) were cleared by ACT. During surveillance after definitive therapy, ctDNA-positive patients were more than 40 times more likely to experience disease recurrence than ctDNA-negative patients (HR, 43.5; 95% CI, 9.8-193.5 P < .001). In all multivariate analyses, ctDNA status was independently associated with relapse after adjusting for known clinicopathologic risk factors. Serial ctDNA analyses revealed disease recurrence up to 16.5 months ahead of standard-of-care radiologic imaging (mean, 8.7 months; range, 0.8-16.5 months). Actionable mutations were identified in 81.8% of the ctDNA-positive relapse samples.Conclusions and relevanceCirculating tumor DNA analysis can potentially change the postoperative management of CRC by enabling risk stratification, ACT monitoring, and early relapse detection.

Dataset Information

Optimized targeted sequencing of cell-free plasma DNA from bladder cancer patients.

Publications

Optimized targeted sequencing of cell-free plasma DNA from bladder cancer patients.

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