High-resolution melting analysis as a sensitive prescreening diagnostic tool to detect KRAS , BRAF , PIK3CA , and AKT1 mutations in formalin-fixed, paraffin-embedded tissues

Arch Pathol Lab Med. 2012 Sep;136(9):983-92. doi: 10.5858/arpa.2011-0176-OA.


Context: As the availability of targeted therapies for several tumor types increases, the need for rapid and sensitive mutation screening is growing. KRAS mutations constitutively activate the RAS/RAF/mitogen-activated protein kinase (MAPK) pathway and therefore play an important role in anti-epidermal growth factor receptor therapy for patients with colorectal cancers. Mutationally activated PIK3CA and AKT1 genes are promising therapeutic targets in breast cancer. In 60% to 70% of malignant melanomas, a mutation in BRAF can be found. Thus, the blocking of the oncogenic signaling induced by this mutation is now used as treatment approach.

Objective: To establish high-resolution melting assays for routinely used predictive analyses of KRAS , AKT1 , PIK3CA , and BRAF mutations.

Design: High-resolution melting assays were developed by using specifically designed primers and genomic DNA isolated either from cell lines or formalin-fixed paraffin-embedded tissues, oligonucleotides, or plasmids. Melting curve analyses were performed on the LightCyler platform and mutation analyses were additionally confirmed by Sanger sequencing.

Results: We developed high-resolution melting assays by using genomic DNA containing the desired mutation, which enabled us to detect percentages of mutated DNA (3.1% to 12.5%) mixed in a wild-type background. Assays were evaluated by hybridization probes and/or Sanger sequencing to exclude pseudogene amplification. The high-resolution melting assays were validated with genomic DNA from different tumor entities. The concordance between Sanger sequencing and high-resolution melting was 99% for KRAS exon 2 and PIK3CA exon 20 and 100% for the remaining assays.

Conclusions: High-resolution melting provides a valid and powerful tool for detecting genomic mutations efficiently.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Caco-2 Cells
  • Cell Line, Tumor
  • Class I Phosphatidylinositol 3-Kinases
  • DNA Mutational Analysis / methods
  • DNA, Neoplasm / chemistry
  • DNA, Neoplasm / genetics
  • Exons / genetics
  • Formaldehyde
  • HCT116 Cells
  • Humans
  • Mutation*
  • Neoplasms / diagnosis
  • Neoplasms / genetics*
  • Neoplasms / pathology
  • Paraffin Embedding / methods
  • Phosphatidylinositol 3-Kinases / genetics*
  • Polymerase Chain Reaction
  • Proto-Oncogene Proteins B-raf / genetics*
  • Proto-Oncogene Proteins c-akt / genetics*
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Tissue Fixation / methods
  • ras Proteins / genetics*


  • DNA, Neoplasm
  • Formaldehyde
  • Phosphatidylinositol 3-Kinases
  • Class I Phosphatidylinositol 3-Kinases
  • PIK3CA protein, human
  • AKT1 protein, human
  • BRAF protein, human
  • Proto-Oncogene Proteins B-raf
  • Proto-Oncogene Proteins c-akt
  • ras Proteins