Nanofluidic Digital PCR and Extended Genotyping of RAS and BRAF for Improved Selection of Metastatic Colorectal Cancer Patients for Anti-EGFR Therapies

Mol Cancer Ther. 2016 May;15(5):1106-12. doi: 10.1158/1535-7163.MCT-15-0820. Epub 2016 Apr 1.


The clinical significance of low-frequent RAS pathway-mutated alleles and the optimal sensitivity cutoff value in the prediction of response to anti-EGFR therapy in metastatic colorectal cancer (mCRC) patients remains controversial. We aimed to evaluate the added value of genotyping an extended RAS panel using a robust nanofluidic digital PCR (dPCR) approach. A panel of 34 hotspots, including RAS (KRAS and NRAS exons 2/3/4) and BRAF (V600E), was analyzed in tumor FFPE samples from 102 mCRC patients treated with anti-EGFR therapy. dPCR was compared with conventional quantitative PCR (qPCR). Response rates, progression-free survival (PFS), and overall survival (OS) were correlated to the mutational status and the mutated allele fraction. Tumor response evaluations were not available in 9 patients and were excluded for response rate analysis. Twenty-two percent of patients were positive for one mutation with qPCR (mutated alleles ranged from 2.1% to 66.6%). Analysis by dPCR increased the number of positive patients to 47%. Mutated alleles for patients only detected by dPCR ranged from 0.04% to 10.8%. An inverse correlation between the fraction of mutated alleles and radiologic response was observed. ROC analysis showed that a fraction of 1% or higher of any mutated alleles offered the best predictive value for all combinations of RAS and BRAF analysis. In addition, this threshold also optimized prediction both PFS and OS. We conclude that mutation testing using an extended gene panel, including RAS and BRAF with a threshold of 1% improved prediction of response to anti-EGFR therapy. Mol Cancer Ther; 15(5); 1106-12. ©2016 AACR.

MeSH terms

  • Adult
  • Aged
  • Angiogenesis Inhibitors / pharmacology
  • Angiogenesis Inhibitors / therapeutic use
  • Antineoplastic Agents / pharmacology
  • Antineoplastic Agents / therapeutic use
  • Cell Line, Tumor
  • Colorectal Neoplasms / diagnosis*
  • Colorectal Neoplasms / drug therapy
  • Colorectal Neoplasms / genetics*
  • Colorectal Neoplasms / mortality
  • DNA Mutational Analysis
  • ErbB Receptors / antagonists & inhibitors
  • Exons
  • Female
  • Gene Frequency
  • Genes, ras*
  • Humans
  • Male
  • Middle Aged
  • Molecular Targeted Therapy
  • Molecular Typing
  • Mutation
  • Nanotechnology*
  • Neoplasm Metastasis
  • Neoplasm Staging
  • Polymerase Chain Reaction* / methods
  • Protein Kinase Inhibitors / pharmacology
  • Protein Kinase Inhibitors / therapeutic use
  • Proto-Oncogene Proteins B-raf / genetics*
  • Retreatment
  • Retrospective Studies
  • Sensitivity and Specificity
  • Survival Analysis
  • Treatment Outcome


  • Angiogenesis Inhibitors
  • Antineoplastic Agents
  • Protein Kinase Inhibitors
  • ErbB Receptors
  • Proto-Oncogene Proteins B-raf