Quantitative PCR Detection and Measurement of NPM1 Mutant Burden in AML

The TRUPCR® NPM1 Mutation Quantitative Kit is a CE-IVD certified, real-time RT-PCR assay designed for the qualitative detection and quantification of NPM1 mutations—specifically Types A (c.860_863dupTCTG), B (c.863_864insCATG), and D (c.863_864insCCTG)—in patients with acute myeloid leukemia (AML). The assay uses RNA extracted from clinical samples and includes a separate amplification of the ABL1 gene as a reference control for relative quantification and RNA quality. Now also available with the TRUPCR Interpretation Software, the kit offers streamlined analysis, automated reporting, and machine learning-powered accuracy.

Molecular Background

NPM1 mutations are among the most prevalent genetic alterations in AML, found in about 35% of all AML cases and up to 60% of cytogenetically normal AML (CN-AML). These mutations typically occur in exon 12 of the NPM1 gene and result in a frameshift, leading to aberrant cytoplasmic localization of the NPM1 protein (NPM1c+). This mislocalization is a defining feature of the disease and serves as a key diagnostic and prognostic biomarker.

Quantification of mutant allele burden is particularly valuable in the monitoring of minimal residual disease (MRD) and treatment response, offering greater clinical insight than qualitative testing alone. Tracking NPM1 transcript levels post-treatment can inform relapse risk, disease kinetics, and support precision-guided therapy decisions.

The TRUPCR® NPM1 Quantitative Kit specifically measures:

• Mutation A (c.860_863dupTCTG)
• Mutation B (c.863_864insCATG)
• Mutation D (c.863_864insCCTG)

These variants account for the vast majority of clinically significant NPM1 mutations.

Assay Performance & Utility

The kit delivers accurate and sensitive quantification of NPM1 mutations with a detection limit as low as 50 copies of mutant transcripts. Its integrated workflow supports:

• Quantitative monitoring of MRD and treatment response
• Differentiation of A, B, and D NPM1 mutation types
• Use of ABL1 as an internal control for normalization and RNA integrity check
• Automated data interpretation and reporting with built-in software support

The TRUPCR® kit is suitable for routine molecular testing in AML, supporting both initial diagnosis and long-term disease surveillance.

Technical Specifications

The TRUPCR® NPM1 Mutation Quantitative Kit is a real-time reverse transcription PCR (RT-PCR) assay, optimized for relative quantification of NPM1 mutant transcript levels using fluorescent probes.

• Sample Type: RNA extracted from peripheral blood or bone marrow
• Detection Method: Real-time RT-PCR
• Target Mutations: NPM1 A, B, and D (exon 12)
• Reference Gene: ABL1 (for normalization and quality control)
• Sensitivity: Detects as few as 50 copies of mutant transcripts
• Format: All-inclusive kit with reagents for cDNA synthesis and real-time PCR
• Run Time: ~90 minutes
• Compatible Instruments: ABI 7500, Bio-Rad CFX96™, Roche LightCycler®, and others

The NPM1 Quantitative  interpretation software

The RUO TRUPCR interpretation software simplifies and accelerate results analysis:

• Experiment Overview and Issue Highlighting: The software provides a comprehensive overview of experiments in a tabular format, automatically identifying and highlighting any issues encountered during the experiment.
• Automated Report Generation: Final reports are generated for all samples in the experiment, including amplification curves for visual verification, eliminating the need for manual checking.
• Improper Amplification Detection: The software uses machine learning algorithms to flag improper amplifications, which may result from incorrect threshold settings, ensuring accurate and automated data interpretation.
• Initial Copy Number Validation: It checks for the correct entry of initial copy numbers, helping to reduce human errors.