Related Experiment Video
Updated: Jul 11, 2025
Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions
Published on: September 25, 2018
Detection of NPM1 Mutations in Acute Myeloid Leukemia by using Drop-Off Droplet Digital PCR and its Clinical
Background:
Nucleophosmin 1 (NPM1) mutations, which occur in 25 - 30% of acute myeloid leukemia (AML) and 50 - 60% of AML with normal karyotype, have been identified as an important marker for stratification of prog-nosis in AML. This study aimed to establish a new quantitative polymerase chain reaction (PCR) technique, the drop-off droplet digital PCR (ddPCR), for rapid and sensitive detection of NPM1 mutations in AML.
Methods:
We established the drop-off ddPCR system and verified its performance. NPM1 mutations were screened in 130 AML patients by drop-off ddPCR and were validated by Sanger sequencing and next-generation sequencing (NGS). Then, the NPM1 mutation burden was dynamically monitored in five patients.
Results:
The limit of blank (LOB) of drop-off ddPCR established for NPM1 mutation was 3.36 copies/μL, and the limit of detection (LOD) was 5.00 - 5.37 copies/μL in 50 ng DNA, and the sensitivity was about 0.05%, which had good linearity. Drop-off ddPCR identified 33/130 (25.4%) NPM1 mutated cases, consistent with Sanger sequencing. In 18 NPM1 positive cases selected randomly, NGS identified fourteen with type A mutation, two with type D mutation, and two with rare type mutations. The mutation burden of NPM1 mutation analyzed by NGS was consistent with the drop-off ddPCR. The sequential samples were detected for measurable residual disease (MRD) monitoring in 5 patients showed that the NPM1 mutation burden was consistent with clinical remission and recurrence. Compared with traditional ddPCR, drop-off ddPCR was also suitable for MRD monitoring.
Conclusions:
In this study, we established a drop-off ddPCR method for detecting three common mutations in AML with good sensitivity and repeatability, which can be used to screen mutations in newly diagnosed AML patients and for MRD monitoring after remission to guide treatment.
Insights
A new drop-off droplet digital PCR (ddPCR) method accurately detects Nucleophosmin 1 (NPM1) mutations in acute myeloid leukemia (AML). This sensitive technique aids in diagnosing AML and monitoring minimal residual disease (MRD) for effective treatment guidance.
Area of Science:
- Hematology
- Molecular Biology
- Genetics
Background:
- Nucleophosmin 1 (NPM1) mutations are key prognostic markers in acute myeloid leukemia (AML), found in 25-30% of cases.
- Accurate detection of NPM1 mutations is crucial for AML patient stratification and treatment planning.
Purpose of the Study:
- To establish and validate a novel drop-off droplet digital PCR (ddPCR) technique for rapid and sensitive NPM1 mutation detection in AML.
- To assess the utility of this method for screening newly diagnosed AML and monitoring minimal residual disease (MRD).
Main Methods:
- A drop-off ddPCR system was developed and performance was verified.
- NPM1 mutations were screened in 130 AML patients using drop-off ddPCR.
- Results were validated against Sanger sequencing and next-generation sequencing (NGS), with mutation burden monitored dynamically in five patients.
Main Results:
- The drop-off ddPCR demonstrated high sensitivity (0.05%), good linearity, and accurately identified NPM1 mutations in 25.4% of AML patients.
- NGS confirmed common and rare NPM1 mutation types, with mutation burden correlating between methods.
- The technique proved effective for monitoring minimal residual disease (MRD) in AML patients, aligning with clinical remission and recurrence.
Conclusions:
- A sensitive and repeatable drop-off ddPCR method for detecting common NPM1 mutations in AML has been established.
- This method is suitable for screening mutations in newly diagnosed AML and for MRD monitoring post-treatment to guide clinical decisions.
