Extreme assay sensitivity in molecular diagnostics further unveils intratumour heterogeneity in metastatic colorectal cancer as well as artifactual low-frequency mutations in the KRAS gene

Br J Cancer. 2017 Jul 25;117(3):358-366. doi: 10.1038/bjc.2017.170. Epub 2017 Jun 15.


Background: Gene mutations in the RAS family rule out metastatic colorectal carcinomas (mCRCs) from anti-EGFR therapies.

Methods: We report a retrospective analysis by Sequenom Massarray and fast COLD-PCR followed by Sanger sequencing on 240 mCRCs.

Results: By Sequenom, KRAS and NRAS exons 2-3-4 were mutated in 52.9% (127/240) of tumours, while BRAF codon 600 mutations reached 5% (12/240). Fast COLD-PCR found extra mutations at KRAS exon 2 in 15/166 (9%) of samples, previously diagnosed by Sequenom as wild-type or mutated at RAS (exons 3-4) or BRAF genes. After UDG digestion results were reproduced in 2/12 analysable subclonally mutated samples leading to a frequency of true subclonal KRAS mutations of 1.2% (2.1% of the previous Sequenom wild-type subgroup). In 10 out of 12 samples, the subclonal KRAS mutations disappeared (9 out of 12) or turned to a different sequence variant (1 out of 12).

Conclusions: mCRC can harbour coexisting multiple gene mutations. High sensitivity assays allow the detection of a small subset of patients harbouring true subclonal KRAS mutations. However, DNA changes with mutant allele frequencies <3% detected in formalin-fixed paraffin-embedded samples may be artifactual in a non-negligible fraction of cases. UDG pre-treatment of DNA is mandatory to identify true DNA changes in archival samples and avoid misinterpretation due to artifacts.

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • Artifacts
  • Carcinoma / genetics*
  • Carcinoma / secondary
  • Class I Phosphatidylinositol 3-Kinases
  • Colorectal Neoplasms / genetics*
  • Colorectal Neoplasms / pathology
  • DNA, Neoplasm / analysis*
  • Female
  • GTP Phosphohydrolases / genetics*
  • Genes, ras*
  • Humans
  • Male
  • Membrane Proteins / genetics*
  • Middle Aged
  • Molecular Diagnostic Techniques*
  • Mutation
  • Neoplasm Metastasis
  • Oligonucleotide Array Sequence Analysis / methods
  • Phosphatidylinositol 3-Kinases / genetics
  • Polymerase Chain Reaction / methods
  • Proto-Oncogene Proteins B-raf / genetics*
  • Retrospective Studies
  • Sequence Analysis, DNA / methods
  • Young Adult


  • DNA, Neoplasm
  • Membrane Proteins
  • Phosphatidylinositol 3-Kinases
  • Class I Phosphatidylinositol 3-Kinases
  • PIK3CA protein, human
  • BRAF protein, human
  • Proto-Oncogene Proteins B-raf
  • GTP Phosphohydrolases
  • NRAS protein, human