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Updated: Jun 29, 2026
Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions
Published on: September 25, 2018
A Multiplex Assay for Fast PIK3CA Hotspot Mutation Characterization in a Single Specimen by 3-Color Digital PCR
Jean Helmijr1, Gianmarco Motta1,2, Lisa Jongbloed1
1Department of Medical Oncology, Erasmus MC Cancer Institute, University Medical Centre Rotterdam, Rotterdam, the Netherlands.
Background:
Activating mutations in the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene have been detected often in solid tumors. Targeted therapy for mutant PIK3CA is now available in the clinic, making molecular diagnostics pivotal. Our aim was to design a multiplex digital PCR (dPCR) assay to evaluate the 4 most common PIK3CA hotspot mutations simultaneously to characterize and quantify these in liquid biopsies.
Methods:
A multiplex assay was developed to detect exon 9 p.E542K and p.E545K mutations, and exon 20 p.H1047L and p.H1047R mutations using the Stilla 3-color dPCR Naica system. The assay was evaluated on stock and pre-amplified DNA from cell lines with the above mutations as single and pooled samples, and on cell-free DNA (cfDNA) from healthy blood donors (HBDs) and breast cancer patients, to determine detection thresholds and diagnostic accuracy.
Results:
The assay distinguished all 4 PIK3CA mutations in (cf)DNA, and also when dual mutations were present. Detection thresholds of stock and pre-amplified cfDNA samples were 0.11 and 0.40 copies/uL (cp/uL) for mutant copies concentration, and 0.003% and 0.68% for variant allele frequencies (VAFs), respectively. The assay confirmed the PIK3CA (mutation) status as defined by targeted next-generation sequencing (NGS) in 82 out of 96 patients that were mutant for PIK3CA, and in 11 out of 12 patients with wild-type PIK3CA.
Conclusions:
Our designed multiplex dPCR assay detected PIK3CA mutations with high accuracy in stock and pre-amplified cfDNA. Furthermore, it is affordable and demands less cfDNA input when compared to available uniplex dPCR assays and NGS analyses.
Insights
A new multiplex digital PCR (dPCR) assay accurately detects common phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutations in liquid biopsies. This affordable method requires less cell-free DNA (cfDNA) compared to existing techniques.
Area of Science:
- Molecular diagnostics
- Cancer genomics
- Digital PCR applications
Background:
- Activating mutations in PIK3CA are frequent in solid tumors.
- Targeted therapies for PIK3CA mutations necessitate precise molecular diagnostics.
- Liquid biopsies offer a non-invasive approach for cancer mutation detection.
Purpose of the Study:
- To develop a multiplex digital PCR (dPCR) assay for simultaneous detection of four common PIK3CA hotspot mutations.
- To characterize and quantify these mutations in liquid biopsies.
- To assess the assay's performance in terms of detection thresholds and diagnostic accuracy.
Main Methods:
- A multiplex assay was designed to detect PIK3CA mutations (p.E542K, p.E545K, p.H1047L, p.H1047R) using a 3-color dPCR system.
- The assay was validated using cell line DNA (stock and pre-amplified) and cell-free DNA (cfDNA) from healthy donors and breast cancer patients.
- Detection limits and diagnostic accuracy were determined by comparing results with targeted next-generation sequencing (NGS).
Main Results:
- The multiplex dPCR assay successfully identified all four PIK3CA mutations, including dual mutations, in cfDNA.
- Low detection thresholds were achieved: 0.11 cp/uL for mutant copies and 0.003% VAF in stock cfDNA.
- The assay demonstrated high concordance with NGS, confirming PIK3CA mutation status in 93% of patients (82/96 mutant, 11/12 wild-type).
Conclusions:
- The developed multiplex dPCR assay provides accurate detection of PIK3CA mutations in cfDNA.
- The assay is cost-effective and requires minimal cfDNA input compared to uniplex dPCR and NGS.
- This assay represents a valuable tool for molecular diagnostics in oncology, particularly for guiding targeted therapy decisions.
