Related Experiment Video
Updated: May 10, 2026
Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines
Published on: October 8, 2015
A multiplex technology platform for the rapid analysis of clinically actionable genetic alterations and validation
David L Smith1, Aude Lamy, Sylvie Beaudenon-Huibregtse
1From Asuragen Inc, Austin, Texas, (Drs Smith, Beaudenon-Huibregtse, Laosinchai-Wolf, and Labourier); the Department of Pathology, Rouen University Hospital, Rouen, France (Drs Lamy and Sabourin); and INSERM U1079, Faculty of Medicine, Rouen University, Rouen, France (Dr Sesboüé).
Context:
Current clinicopathologic assessment of malignant neoplastic diseases entails the analysis of specific genetic alterations that provide diagnostic, prognostic, or therapy-determining information.
Objective:
To develop and validate a robust molecular method to detect clinically relevant mutations in various tissue types and anatomic pathology specimens.
Design:
Genes of interest were amplified by multiplex polymerase chain reaction and sequence variants identified by liquid bead array cytometry. The BRAF assay was fully characterized by using plasmids and genomic DNA extracted from cell lines, metastatic colorectal cancer formalin-fixed, paraffin-embedded (FFPE) tissues, and thyroid nodule fine-needle aspirates.
Results:
Qualitative multiplex assays for 22 different mutations in the BRAF, HRAS, KRAS, NRAS, or EGFR genes were established. The high signal-to-noise ratio of the technology enabled reproducible detection of BRAF c.1799T>A (p.V600E) at 0.5% mutant allele in 20 ng of genomic DNA. Precision studies with multiple operators and instruments showed very high repeatability and reproducibility with 100% (98.7%-100%) qualitative agreement among 292 individual measures in 38 runs. Evaluation of 1549 representative pathologic specimens in 2 laboratories relative to independent reference methods resulted in 99.0% (97.6%-99.6%) agreement for colorectal FFPE tissues (n = 416) and 98.9% (98.2%-99.4%) for thyroid fine-needle aspiration specimens (n = 1133) with an overall diagnostic odds ratio of 10 856 (2451-48 078).
Conclusions:
The multiplex assay system is a sensitive and reliable method to detect BRAF c.1799T>A mutation in colorectal and thyroid lesions. This optimized technology platform is suitable for the rapid analysis of clinically actionable genetic alterations in cytologic and histologic specimens.
