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Clin Chem. 2012 Sep;58(9):1332-41. doi: 10.1373/clinchem.2012.186577. Epub 2012 Jun 27.

Nanofluidic digital PCR for KRAS mutation detection and quantification in gastrointestinal cancer.

Author information

1
Translational Research Laboratory, Catalan Institute of Oncology-ICO-IDIBELL, Hospitalet de Llobregat, Barcelona, Spain.

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.

PMID:
22745110
DOI:
10.1373/clinchem.2012.186577
[Indexed for MEDLINE]
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