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.
Highlights
A tremendous progress in the understanding of the molecular processes central to cancer development has occurred during the recent years - a progress fuelled by the development and extensive use of generation sequencing (NGS)
Available mutational data of 476 bladder cancer patients from cBioPortal was used, and data was analyzed to assess the probability of detecting mutations while limiting the genomic size of the gene panel (Fig. 1a,b)
CtDNA analysis has emerged as a promising tool in cancer research
Summary
A tremendous progress in the understanding of the molecular processes central to cancer development has occurred during the recent years - a progress fuelled by the development and extensive use of generation sequencing (NGS). Substantial differences have been observed in the mutational landscapes between primary tumors and distant metastases[5]. The majority of cfDNA, originates from dying non-cancerous cells, resulting in very low frequencies of tumor-specific genomic alterations[8,9]. Circulating tumor DNA (ctDNA) has been detected in cancer patients both prior to and after intended radical surgery and found indicative of later disease recurrence with a positive lead time compared to radiographic imaging[10,11,12,13,14]. DdPCR assays are generally designed based on a priori knowledge of tumor-specific mutations and are not suited for detecting new mutations and to study tumor evolution. 25% of bladder cancer patients present with muscle-invasive disease of which up to 50% develop metastases[26]. Patients are monitored by radiographic imaging to assess treatment efficacy and disease relapse
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