An application case study: mutation analysis 

Development of dPCR assay to detect the presence of an NPM1 gene mutation – an application for a subset of acute myeloid leukemia

Denise Rawcliffe, PhD, Umeå University, UMU Department of Molecular Biology, Sweden

In our department of Clinical Genetics we hope to develop a digital PCR assay using QIAcuity to detect the presence of a NPM1 gene mutation that can be observed in AML.

The mutation is a 4-bp insertion where the 4 bases that are inserted can vary. The most common insertion appears in approximately 75 % of the NPM1-AML cases and is called Type A. There is also Type B, Type D, Type DD1, and several other unnamed but observed and less common insertions or assess which type of mutation is present, bone marrow samples are processed with NGS. To avoid the heavy workload of an NGS analysis the current aim is to develop a digital PCR assay where the NPM1 insertion mutations can be detected with high sensitivity. The challenge in the digital PCR assay design is to be able to detect a variety of different 4-bp insertions in the NPM1 gene and to be able to relate the output to the ABL1 gene according to established research recommendations.

Application of digital PCR in molecular pathology: multiplex mutational analysis and copy number changes

Franziska Fahlteich, University Hospital Carl Gustav Carus, Institute of Pathology, Molecular Pathology, Germany

The field of molecular pathology is regularly experiencing innovations and adaptations to clinical issues. Molecular analyses are becoming more complex and take more time in the laboratory and data evaluation. To optimize workflows, constant method enhancement is required. In melanoma diagnostics, critical timeframes need quick methods to detect therapy-relevant BRAF V600 mutations. Therefore, our goal is to develop a multiplex mutational analysis for BRAF using dPCR to detect the most frequent V600 mutations in less than 3 hours. Therefore, we developed a low error-prone, fast and straightforward workflow to overcome different obstacles. In addition, this method enables the detection of copy number alterations. In comparison to Fluorescence in situ hybridization (FISH), dPCR allows a significant reduction in workload. Here we focus on implementing copy number alterations of molecular markers such as CDKN2A. This target enables physicians to classify tumors according to their aggressiveness. In contrast to Next-Generation-Sequencing (NGS), dPCR can differentiate between hemizygous and homozygous deletion providing precise and faster results.