title: Comparison of genetic and epigenetic alterations of primary tumors and matched plasma samples in patients with colorectal cancer.
abstract: BACKGROUND
  Although recent advances in circulating DNA analysis allow the prediction of tumor genomes by noninvasive means, some challenges remain, which limit the widespread introduction of cfDNA in cancer diagnostics. We analyzed the status of the two best characterized colorectal cancer (CRC) genetic and epigenetic alterations in a cohort of CRC patients, and then compared the degree to which the two patterns move from tissue to plasma in order to improve our understanding of biology modulating the concordance between tissues and plasma methylation and mutation profiles.
  
  
  METHODS
  Plasma and tumor tissues were collected from 85 patients (69±14 years, 56 males). KRAS and SEPT9 status was assessed by allele refractory mutation system quantitative PCR and quantitative methylation-specific PCR, respectively. Six of the most common point mutations at codon 12 and 13 were investigated for KRAS analysis.
  
  
  RESULTS
  KRAS mutations and SEPT9 promoter methylation were present in 34% (29/85) and in 82% (70/85) of primary tumor tissue samples. Both genetic and epigenetic analyses of cfDNA revealed a high overall concordance and specificity compared with tumor-tissue analyses. Patients presenting with both genetic and epigenetic alterations in tissue specimens (31.8%, 27/85) were considered for further analyses. The median methylation rates in tumour tissues and plasma samples were 64.5% (12.2-99.8%) and 14.5% (0-45.5%), respectively. The median KRAS mutation load (for matched mutations) was 33.6% (1.8-86.3%) in tissues and 2.9% (0-17.3) in plasma samples. The plasma/tissue (p/t) ratio of SEPT9 methylation rate was significantly higher than the p/t ratio of KRAS mutation load, especially in early stage cancers (p=0.0108).
  
  
  CONCLUSIONS
  The results of this study show a discrepant rate of epigenetic vs. genetic alterations moving from tissue to plasma. Many factors could affect mutation cfDNA analysis, including both presence of tumor clonal heterogeneity and strict compartmentalization of KRAS mutation profile. The present study highlights the importance of considering the nature of the alteration when analyzing tumor-derived cfDNA.
authors.affiliation: Laboratory of Clinical Biochemistry, Department of Life and Reproduction Sciences, University Hospital of Verona, Verona, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Danese
authors.affiliation: National Centre for Bowel Research and Surgical Innovation (NCBRSI), Academic Surgical Unit, Barts and The London NHS Trust, Queen Mary University of London, London, United Kingdom.
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Minicozzi
authors.affiliation: Laboratory of Clinical Biochemistry, Department of Life and Reproduction Sciences, University Hospital of Verona, Verona, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Benati
authors.affiliation: Laboratory of Clinical Biochemistry, Department of Life and Reproduction Sciences, University Hospital of Verona, Verona, Italy.
authors.forename: Martina
authors.initials: M
authors.lastname: Montagnana
authors.affiliation: Laboratory of Clinical Biochemistry, Department of Life and Reproduction Sciences, University Hospital of Verona, Verona, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Paviati
authors.affiliation: Laboratory of Clinical Biochemistry, Department of Life and Reproduction Sciences, University Hospital of Verona, Verona, Italy.
authors.forename: Gian Luca
authors.initials: GL
authors.lastname: Salvagno
authors.affiliation: Laboratory of Clinical Biochemistry, Department of Life and Reproduction Sciences, University Hospital of Verona, Verona, Italy.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Lima-Oliveira
authors.affiliation: Oncology Department, Laboratory of Pharmacology and Molecular Biology, Rovigo General Hospital, Trecenta, Rovigo, Italy.
authors.forename: Milena
authors.initials: M
authors.lastname: Gusella
authors.affiliation: Department of Medical Oncology, Rovigo Hospital, Rovigo, Italy.
authors.forename: Felice
authors.initials: F
authors.lastname: Pasini
authors.affiliation: Laboratory of Clinical Chemistry and Hematology, Academic Hospital of Parma, Parma, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Lippi
authors.affiliation: Laboratory of Clinical Biochemistry, Department of Life and Reproduction Sciences, University Hospital of Verona, Verona, Italy.
authors.forename: Gian Cesare
authors.initials: GC
authors.lastname: Guidi
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics; D000368:Aged; D015179:Colorectal Neoplasms / Q000235:genetics*; D019175:DNA Methylation / Q000235:genetics*; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000235:genetics; D044127:Epigenesis, Genetic / Q000235:genetics*; D005260:Female; D006801:Humans; D008297:Male; D017354:Point Mutation / Q000235:genetics; D011401:Promoter Regions, Genetic; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D058112:Septins / Q000235:genetics*
chemical_list: D004273:DNA, Neoplasm; C117307:KRAS protein, human; C474315:SEPTIN9 protein, human; D058112:Septins; D016283:Proto-Oncogene Proteins p21(ras)