Methods for the purification and detection of single nucleotide KRAS mutations on extrachromosomal circular DNA in human plasma

BACKGROUNDS Despite recent advances, many cancers are still detected too late for curative treatment. There is, therefore, a need for the development of new diagnostic methods and biomarkers. One approach may arise from the detection of extrachromosomal circular DNA (eccDNA), which is part of cell-free DNA in human plasma. AIMS First, we assessed and compared two methods for the purification of eccDNA from plasma. Second, we tested for an easy diagnostic application of eccDNA liquid biopsy-based assays. MATERIALS & METHODS For the comparison we tested a solid-phase silica purification method and a phenol/chloroform method with salt precipitation. For the diagnostic application of eccDNA we developed and tested a qPCR primer-based SNP detection system, for the detection of two well-established cancer-causing KRAS mutations (G12V and G12R) on circular DNA. This investigation was supported by purifying, sequencing, and analysing clinical plasma samples for eccDNAs containing KRAS mutant alleles in 0.5 mL plasma from 16 pancreatic ductal adenocarcinoma patients and 19 healthy controls. RESULTS In our method comparison we observed, that following exonuclease treatment a lower eccDNA yield was found for the phenol/chloroform method (15.7%-26.7%) compared with the solid-phase purification approach (47.8%-65.9%). For the diagnostic application of eccDNA tests, the sensitivity of the tested qPCR assay only reached ~10-3 in a background of 105 wild type (wt) KRAS circular entities, which was not improved by general amplification or primer-based inhibition of wt KRAS amplification. Furthermore, we did not detect eccDNA containing KRAS in any of the clinical samples. DISCUSSION A potential explanation for our inability to detect any KRAS mutations in the clinical samples may be related to the general low abundance of eccDNA in plasma. CONCLUSION Taken together our results provide a benchmark for eccDNA purification methods while raising the question of what is required for the optimal fast and sensitive detection of SNP mutations on eccDNA with greater sensitivity than primer-based qPCR detection.

[1]  A. Jemal,et al.  Cancer statistics, 2022 , 2022, CA: a cancer journal for clinicians.

[2]  N. Rosenfeld,et al.  Characteristics, origin, and potential for cancer diagnostics of ultrashort plasma cell-free DNA , 2021, Genome research.

[3]  Kun Wang,et al.  Extrachromosomal Circular DNAs: Origin, formation and emerging function in Cancer , 2021, International journal of biological sciences.

[4]  Selena Lin,et al.  A New Method for Improving Extraction Efficiency and Purity of Urine and Plasma Cell-Free DNA , 2021, bioRxiv.

[5]  T. Kuzel,et al.  Current Status of Circulating Tumor DNA Liquid Biopsy in Pancreatic Cancer , 2020, International journal of molecular sciences.

[6]  Anindya Dutta,et al.  Identification and characterization of extrachromosomal circular DNA in maternal plasma , 2020, Proceedings of the National Academy of Sciences.

[7]  S. Gulati,et al.  Analysis of circulating cell-free DNA identifies KRAS copy number gain and mutation as a novel prognostic marker in Pancreatic cancer , 2019, Scientific Reports.

[8]  H. Nielsen,et al.  Genome-wide cell-free DNA fragmentation in patients with cancer , 2019, Nature.

[9]  V. Babak,et al.  The Quality of DNA Isolated from Processed Food and Feed via Different Extraction Procedures , 2019, Molecules.

[10]  M. Javle,et al.  Fragment size and level of cell-free DNA provide prognostic information in patients with advanced pancreatic cancer , 2018, Journal of Translational Medicine.

[11]  M. Kerachian,et al.  Simple and cost-effective laboratory methods to evaluate and validate cell-free DNA isolation , 2018, BMC Research Notes.

[12]  A. E. del Río Hernández,et al.  Liquid biopsies for management of pancreatic cancer , 2018, Translational research : the journal of laboratory and clinical medicine.

[13]  Xianjun Yu,et al.  Critical role of KRAS mutation in pancreatic ductal adenocarcinoma , 2018, Translational Cancer Research.

[14]  B. Larijani,et al.  The presence of tumor extrachomosomal circular DNA (ecDNA) as a component of liquid biopsy in blood. , 2018, Medical hypotheses.

[15]  Michael P Snyder,et al.  Circular DNA elements of chromosomal origin are common in healthy human somatic tissue , 2018, Nature Communications.

[16]  J. Doye,et al.  Rolling circle amplification shows a sinusoidal template length-dependent amplification bias , 2017, Nucleic acids research.

[17]  Chunlei Liu,et al.  ClinVar: improving access to variant interpretations and supporting evidence , 2017, Nucleic Acids Res..

[18]  A. Bardelli,et al.  Integrating liquid biopsies into the management of cancer , 2017, Nature Reviews Clinical Oncology.

[19]  M. Abedalthagafi,et al.  Cancer diagnostics: The journey from histomorphology to molecular profiling , 2016, Oncotarget.

[20]  Stefanie S Jeffrey,et al.  Circulating tumor cell technologies , 2016, Molecular oncology.

[21]  L. Buscail,et al.  Targeting KRAS for diagnosis, prognosis, and treatment of pancreatic cancer: Hopes and realities. , 2016, European journal of cancer.

[22]  Katarzyna Sikora,et al.  Evaluation of cell-free DNA as a biomarker for pancreatic malignancies , 2015, The International journal of biological markers.

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

[24]  M A Firpo,et al.  The clinical utility of CA 19-9 in pancreatic adenocarcinoma: diagnostic and prognostic updates. , 2013, Current molecular medicine.

[25]  Lincoln D. Stein,et al.  Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes , 2012, Nature.

[26]  Matthias Meyer,et al.  Illumina sequencing library preparation for highly multiplexed target capture and sequencing. , 2010, Cold Spring Harbor protocols.

[27]  E. Birney,et al.  Mapping identifiers for the integration of genomic datasets with the R/Bioconductor package biomaRt , 2009, Nature Protocols.

[28]  G. Parmigiani,et al.  Core Signaling Pathways in Human Pancreatic Cancers Revealed by Global Genomic Analyses , 2008, Science.

[29]  S. Sommer,et al.  Microarray-based DNA resequencing using 3' blocked primers. , 2008, Analytical biochemistry.

[30]  B. Moor,et al.  BioMart and Bioconductor: a powerful link between biological databases and microarray data analysis , 2005 .

[31]  Margarita Salas,et al.  Insights into strand displacement and processivity from the crystal structure of the protein-primed DNA polymerase of bacteriophage phi29. , 2004, Molecular cell.

[32]  F. Dean,et al.  Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification. , 2001, Genome research.

[33]  A. Regev,et al.  Small polydispersed circular DNA (spcDNA) in human cells: association with genomic instability , 1997, Oncogene.

[34]  D. Schindler,et al.  Increased amount and contour length distribution of small polydisperse circular DNA (spcDNA) in Fanconi anemia. , 1993, Mutation research.

[35]  H G Claycamp,et al.  Phenol sensitization of DNA to subsequent oxidative damage in 8-hydroxyguanine assays. , 1992, Carcinogenesis.

[36]  G. Fleuren,et al.  KRAS codon 12 mutations occur very frequently in pancreatic adenocarcinomas. , 1988, Nucleic acids research.

[37]  D. Shibata,et al.  Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes , 1988, Cell.

[38]  J. Kleeff,et al.  Pancreatic cancer , 1988, Nature Reviews Disease Primers.

[39]  S. Bojesen,et al.  Identification of New Biomarkers in Patients with Pancreatic Cancer (BIOPAC): A Study Protocol of an Open Cohort Study , 2019 .