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

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.

[1]  K. Kinzler,et al.  Detection and quantification of rare mutations with massively parallel sequencing , 2011, Proceedings of the National Academy of Sciences.

[2]  Ashwini Naik,et al.  Phylogenetic ctDNA analysis depicts early stage lung cancer evolution , 2017, Nature.

[3]  Cassandra B. Jabara,et al.  Accurate sampling and deep sequencing of the HIV-1 protease gene using a Primer ID , 2011, Proceedings of the National Academy of Sciences.

[4]  Carlos Caldas,et al.  Multifocal clonal evolution characterized using circulating tumour DNA in a case of metastatic breast cancer , 2015, Nature Communications.

[5]  S. Linnarsson,et al.  Counting absolute numbers of molecules using unique molecular identifiers , 2011, Nature Methods.

[6]  A. Heger,et al.  UMI-tools: modeling sequencing errors in Unique Molecular Identifiers to improve quantification accuracy , 2016, bioRxiv.

[7]  Bert Vogelstein,et al.  DETECTION OF CIRCULATING TUMOR DNA IN EARLY AND LATE STAGE HUMAN MALIGNANCIES , 2014 .

[8]  Pablo Cingolani,et al.  © 2012 Landes Bioscience. Do not distribute. , 2022 .

[9]  Ash A. Alizadeh,et al.  Distinct biological subtypes and patterns of genome evolution in lymphoma revealed by circulating tumor DNA , 2016, Science Translational Medicine.

[10]  Mårten Fernö,et al.  Serial monitoring of circulating tumor DNA in patients with primary breast cancer for detection of occult metastatic disease , 2015, EMBO molecular medicine.

[11]  V. Velculescu,et al.  Insights into therapeutic resistance from whole-genome analyses of circulating tumor DNA , 2013, Oncotarget.

[12]  Ash A. Alizadeh,et al.  Integrated digital error suppression for improved detection of circulating tumor DNA , 2016, Nature Biotechnology.

[13]  G. Parmigiani,et al.  Detection of Chromosomal Alterations in the Circulation of Cancer Patients with Whole-Genome Sequencing , 2012, Science Translational Medicine.

[14]  A. McCullough,et al.  Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing , 2013 .

[15]  R. Strausberg,et al.  Circulating tumor DNA as an early marker of therapeutic response in patients with metastatic colorectal cancer. , 2015, Annals of oncology : official journal of the European Society for Medical Oncology.

[16]  M. Nykter,et al.  Circulating Tumor DNA Reveals Clinically Actionable Somatic Genome of Metastatic Bladder Cancer , 2017, Clinical Cancer Research.

[17]  Richard Durbin,et al.  A large genome center's improvements to the Illumina sequencing system , 2008, Nature Methods.

[18]  S. Horswell,et al.  Detection of ubiquitous and heterogeneous mutations in cell-free DNA from patients with early-stage non-small-cell lung cancer. , 2016, Annals of oncology : official journal of the European Society for Medical Oncology.

[19]  Peiyong Jiang,et al.  Cancer genome scanning in plasma: detection of tumor-associated copy number aberrations, single-nucleotide variants, and tumoral heterogeneity by massively parallel sequencing. , 2013, Clinical chemistry.

[20]  Yu Cao,et al.  Intratumor heterogeneity in localized lung adenocarcinomas delineated by multiregion sequencing , 2014, Science.

[21]  T. Ørntoft,et al.  Spatial and temporal clonal evolution during development of metastatic urothelial carcinoma , 2016, Molecular oncology.

[22]  M. Choti,et al.  Detection of Circulating Tumor DNA in Early- and Late-Stage Human Malignancies , 2014, Science Translational Medicine.

[23]  H. Ozen.  Bladder cancer. , 1998, Current opinion in oncology.

[24]  K. Birkenkamp-Demtröder,et al.  Re: Monitoring Treatment Response and Metastatic Relapse in Advanced Bladder Cancer by Liquid Biopsy Analysis. , 2019, The Journal of urology.

[25]  Ash A. Alizadeh,et al.  An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage , 2013, Nature Medicine.

[26]  T. Ørntoft,et al.  Genomic Alterations in Liquid Biopsies from Patients with Bladder Cancer. , 2016, European urology.

[27]  Jorge S. Reis-Filho,et al.  Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer , 2015, Science Translational Medicine.

[28]  R. Bourgon,et al.  Atezolizumab as first-line treatment in cisplatin-ineligible patients with locally advanced and metastatic urothelial carcinoma: a single-arm, multicentre, phase 2 trial , 2017, The Lancet.

[29]  Katherine Van Loon,et al.  Cell-Free DNA Next-Generation Sequencing in Pancreatobiliary Carcinomas. , 2015, Cancer discovery.

[30]  Sam Angiuoli,et al.  Direct detection of early-stage cancers using circulating tumor DNA , 2017, Science Translational Medicine.

[31]  R. Bourgon,et al.  Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy: a single-arm, multicentre, phase 2 trial , 2016, The Lancet.

[32]  C. Mathers,et al.  Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012 , 2015, International journal of cancer.

[33]  A. von Deimling,et al.  Highly prevalent TERT promoter mutations in bladder cancer and glioblastoma , 2013, Cell cycle.

[34]  S. Nelson,et al.  Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation , 2010, Nature.

[35]  A. Vincent-Salomon,et al.  Patient-Specific Circulating Tumor DNA Detection during Neoadjuvant Chemotherapy in Triple-Negative Breast Cancer. , 2016, Clinical chemistry.

[36]  N. Rosenfeld,et al.  Noninvasive Identification and Monitoring of Cancer Mutations by Targeted Deep Sequencing of Plasma DNA , 2012, Science Translational Medicine.

[37]  A. Iafrate,et al.  Anchored multiplex PCR for targeted next-generation sequencing , 2014, Nature Medicine.

[38]  P. A. Futreal,et al.  Genomic architecture and evolution of clear cell renal cell carcinomas defined by multiregion sequencing , 2014, Nature Genetics.

[39]  D. Dressman,et al.  Heteroplasmic mitochondrial DNA mutations in normal and tumor cells , 2010, Nature.

[40]  Helga Thorvaldsdóttir,et al.  Integrative Genomics Viewer , 2011, Nature Biotechnology.

[41]  T. Ørntoft,et al.  Liquid Biopsy Analysis of FGFR3 and PIK3CA Hotspot Mutations for Disease Surveillance in Bladder Cancer. , 2017, European urology.

[42]  Benjamin E. Gross,et al.  The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. , 2012, Cancer discovery.

[43]  H. Nielsen,et al.  Clinical Implications of Monitoring Circulating Tumor DNA in Patients with Colorectal Cancer , 2017, Clinical Cancer Research.

[44]  Beatriz Bellosillo,et al.  Clonal evolution and resistance to EGFR blockade in the blood of colorectal cancer patients , 2015, Nature Medicine.

[45]  R. Strausberg,et al.  Circulating tumor DNA analysis detects minimal residual disease and predicts recurrence in patients with stage II colon cancer , 2016, Science Translational Medicine.

[46]  Eric Samorodnitsky,et al.  Evaluation of Hybridization Capture Versus Amplicon‐Based Methods for Whole‐Exome Sequencing , 2015, Human mutation.

[47]  Gary L. Gallia,et al.  TERT promoter mutations occur frequently in gliomas and a subset of tumors derived from cells with low rates of self-renewal , 2013, Proceedings of the National Academy of Sciences.

[48]  T. Ørntoft,et al.  Mutational context and diverse clonal development in early and late bladder cancer. , 2014, Cell reports.

[49]  Brendan F. Kohrn,et al.  Detecting ultralow-frequency mutations by Duplex Sequencing , 2014, Nature Protocols.