title: Nanofluidic digital PCR for KRAS mutation detection and quantification in gastrointestinal cancer.
abstract: BACKGROUND
  Concomitant quantification of multiple mutant KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) alleles may provide information in addition to that provided by standard mutation-detection procedures. We assessed the feasibility of a nanofluidic digital PCR array platform to detect and quantify KRAS mutations simultaneously in clinically relevant samples.
  
  
  METHODS
  We assessed 2 groups of patients (colorectal and pancreatic disease): Group 1 consisted of 27 patients with colorectal carcinomas, 14 patients with adenomas, and 5 control individuals; group 2 consisted of 42 patients with pancreatic carcinoma, 4 with adenocarcinomas of the ampulla, and 6 with chronic pancreatitis). Digital PCR was performed with the Digital Array Chip (Fluidigm).
  
  
  RESULTS
  Nanofluidic digital PCR detected mutant alleles at 0.05% to 0.1%, depending on the variant analyzed. For the colorectal disease group, conventional PCR detected 9 (64%) of 14 adenomas that were positive for KRAS mutants, whereas digital PCR increased this number to 11 (79%) of 14. Sixteen (59%) of 27 carcinomas showed KRAS mutation with conventional PCR. Two additional cases were detected with digital PCR. In 5 cases (3 adenomas, 2 carcinomas), the total number of mutant alleles changed. For the pancreatic disease group, digital PCR increased the number of positive cases from 26 to 34 (81%) and identified ≥ 2 mutant alleles in 25 cases, compared with conventional PCR, which identified multiple KRAS mutant alleles in only 12 cases. A good correlation was observed between results obtained with tumor biopsies and those obtained with pancreatic juice.
  
  
  CONCLUSIONS
  Digital PCR provides a robust, quantitative measure of the proportion of KRAS mutant alleles in routinely obtained samples. It also allows a better classification of tumors, with potential clinical relevance.
authors: Translational Research Laboratory, Catalan Institute of Oncology-ICO-IDIBELL, Hospitalet de Llobregat, Barcelona, Spain.
authors: Daniel
authors: D
authors: Azuara
authors: Mireia M
authors: MM
authors: Ginesta
authors: Mireia
authors: M
authors: Gausachs
authors: Francisco
authors: F
authors: Rodriguez-Moranta
authors: Joan
authors: J
authors: Fabregat
authors: Juli
authors: J
authors: Busquets
authors: Nuria
authors: N
authors: Pelaez
authors: Jaume
authors: J
authors: Boadas
authors: Sara
authors: S
authors: Galter
authors: Victor
authors: V
authors: Moreno
authors: Jose
authors: J
authors: Costa
authors: Javier
authors: J
authors: de Oca
authors: Gabriel
authors: G
authors: Capellá
mesh_terms: D000328:Adult; D000368:Aged; D005240:Feasibility Studies; D005260:Female; D005770:Gastrointestinal Neoplasms / Q000235:genetics*; D011905:Genes, ras; D006801:Humans; D008297:Male; D044085:Microfluidics; D008875:Middle Aged; D009154:Mutation; D036103:Nanotechnology; D016133:Polymerase Chain Reaction