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Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions
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Multiplex Enrichment and Detection of Rare KRAS Mutations in Liquid Biopsy Samples using Digital Droplet

Erica D Pratt1, Robert W Cowan1, Sara L Manning1

  • 1Ahmed Center for Pancreatic Cancer Research, Department of Gastroenterology, Hepatology and Nutrition , University of Texas MD Anderson Cancer Center , Houston , Texas , United States.

Analytical Chemistry
|May 11, 2019
PubMed

Insights

A new method, Multiplex Enrichment using Droplet Pre-Amplification (MED-Amp), enhances circulating tumor DNA (ctDNA) detection for early cancer surveillance. This technique improves sensitivity for rare mutations in liquid biopsies.

Area of Science:

  • Oncology
  • Molecular Biology
  • Genetics

Background:

  • Circulating tumor DNA (ctDNA) is a promising biomarker for cancer surveillance and early detection.
  • Current ctDNA assays face limitations in sensitivity, especially for cancers with low tumor burden, due to high background DNA concentrations.
  • Accurate detection of rare mutant ctDNA is critical for effective noninvasive cancer monitoring.

Purpose of the Study:

  • To develop and validate a novel assay for highly sensitive detection of rare ctDNA variants.
  • To overcome the sensitivity limitations of existing ctDNA detection methods in challenging clinical samples.
  • To improve the utility of liquid biopsies for cancer surveillance and early diagnosis.

Main Methods:

  • Introduction of the Multiplex Enrichment using Droplet Pre-Amplification (MED-Amp) method.
  • MED-Amp combines single-molecule emulsification, short-round polymerase chain reaction (PCR) preamplification, and digital droplet PCR (ddPCR).
  • Assay performance evaluated for mutant signal amplification and allelic frequency preservation.

Main Results:

  • MED-Amp achieved over a 50-fold increase in mutant DNA signal with minimal distortion of allelic frequency.
  • The assay successfully detected as few as three mutant DNA copies within 5-50 ng of wild-type DNA.
  • KRAS mutant ctDNA was detected in 86% of plasma samples from patients with metastatic pancreatic ductal adenocarcinoma.

Conclusions:

  • The MED-Amp assay provides a significant advancement in high-sensitivity rare variant detection.
  • This method is well-suited for analyzing liquid biopsy samples and other specimens with limited DNA.
  • MED-Amp has the potential to enhance noninvasive cancer monitoring, surveillance, and early detection strategies.

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