title: Cell-free DNA as biomarker and source for mutation detection in primary colorectal cancer.
abstract: OBJECTIVE
  To analyze if cell-free (cf)DNA levels and the presence of KRAS and BRAF mutations in serum could be used as diagnostic biomarkers in patients with primary colorectal cancer (CRC).
  
  
  METHODS
  This study included 92 individuals who were operated due to primary CRC (N=52;study group) and to hemorrhoids (N=40;control group). Serum cfDNA levels were measured with real-time PCR (RT-PCR) using PicoGreen dsDNA quantitation reagent. Colorectal tissue and related blood and serum samples taken at the time of surgery were subjected to DNA extraction and analysis of KRAS and BRAF mutations based on multiplex SNaPshot assay and DNA sequencing.
  
  
  RESULTS
  The average cfDNA concentration was lower in patients of the study group (20±7 ng/μL) in comparison to controls (34±9 ng/μL) and this difference was statistically significant (p<0.001). The SNaPshot analysis detected KRAS c35 mutations in colorectal tumor tissue in 14 cases, but the presence of the mutation was not confirmed in cfDNA extracted from blood samples of these patients.
  
  
  CONCLUSIONS
  The level of serum cfDNA in CRC is decreased in comparison to patients with hemorrhoids, which questions the usefulness of cfDNA as cancer biomarker. Also, cfDNA does not appear to be suitable as a source for mutation detection in this disease.
authors.affiliation: Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Nikolic
authors.forename: Marina
authors.initials: M
authors.lastname: Vlajnic
authors.forename: Momcilo
authors.initials: M
authors.lastname: Ristanovic
authors.forename: Jelena
authors.initials: J
authors.lastname: Petrovic
authors.forename: Ivan
authors.initials: I
authors.lastname: Dimitrijevic
authors.forename: Zoran
authors.initials: Z
authors.lastname: Krivokapic
authors.forename: Dragica
authors.initials: D
authors.lastname: Radojkovic
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000097:blood*; D015179:Colorectal Neoplasms / Q000235:genetics*; D004273:DNA, Neoplasm / Q000097:blood*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics
chemical_list: D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; C117307:KRAS protein, human; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras)