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A Novel Multiplex Droplet Digital PCR Assay to Identify and Quantify KRAS Mutations in Clinical Specimens

Miguel Alcaide1, Matthew Cheung1, Kevin Bushell1

  • 1Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.

The Journal of Molecular Diagnostics : JMD
|November 26, 2018
PubMed

Insights

This study introduces a novel droplet digital PCR method for simultaneous KRAS mutation and copy number alteration detection. This assay aids in assessing tumor purity and noninvasive monitoring for KRAS-driven cancers.

Area of Science:

  • Oncology
  • Molecular Biology
  • Genetics

Background:

  • KRAS mutations are key drivers in pancreatic cancer and linked to anti-EGFR therapy resistance in colorectal cancer.
  • Current KRAS genotyping has limited clinical utility in pancreatic cancer but is crucial for colorectal cancer treatment decisions.
  • Standardization of KRAS genetic testing methods is needed, especially for complex clinical specimens.

Purpose of the Study:

  • To develop and validate a droplet digital PCR (ddPCR) assay for simultaneous detection and quantification of KRAS point mutations and copy number alterations.
  • To assess the utility of the ddPCR assay in various sample types, including fresh frozen biopsies, FFPE tissues, and liquid biopsies.
  • To establish a rapid, cost-effective method for inferring tumor purity and enabling noninvasive monitoring in KRAS-mutated cancers.

Main Methods:

  • Development of a unique droplet digital PCR (ddPCR) assay.
  • Simultaneous detection and quantification of KRAS point mutations across exons 2, 3, and 4.
  • Validation of the assay for 13 specific KRAS mutations and focal amplifications using 100 DNA samples from diverse sources.

Main Results:

  • The ddPCR assay successfully detected and quantified 13 KRAS mutations and focal amplifications.
  • The assay demonstrated utility across fresh frozen tumor biopsies, FFPE tissues, and liquid biopsy specimens.
  • The method has a lower limit of detection of approximately 1%, suitable for inferring tumor purity.

Conclusions:

  • The developed ddPCR assay provides a rapid and cost-effective tool for KRAS mutation and copy number alteration analysis.
  • This assay can inform on tumor purity in challenging clinical specimens, supporting precision medicine approaches.
  • The method is valuable for noninvasive serial monitoring of circulating tumor DNA to track treatment response and detect relapse.

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