Large amplicon droplet digital PCR for DNA-based monitoring of pediatric chronic myeloid leukemia (CML)

Background:

In the era of tyrosine-kinase inhibitor treatment stop trials in patients with CML, quantification of BCR-ABL1 fusion is an essential tool to guide treatment decisions. For detection of transcript-negative CML cells, DNA-based assays are an alternative approach. However, repeat-rich DNA sequences at the breakpoint limit the design of conventional PCR assays for minimal residual disease (MRD) monitoring.

Method:

Droplet digital PCR (ddPCR) was optimized for quantification of large amplicons (< 1330 bp) to allow primer/probe set design outside repeat regions. DNA and RNA BCR-ABL1 copy numbers of 687 specimens from 55 pediatric CML patients were compared.

Results:

Molecular characterization of genomic BCR-ABL1 fusion sites from pediatric CML patients revealed repeat elements at the breakpoint in 64% of cases. Using ddPCR, primers can be positioned outside repeat regions and large amplicons can be quantified with high sensitivity.

Conclusions:

The combination of ddPCR, double quenched probes and extended amplicons represents a valuable tool for MRD monitoring in CML and may be adapted to other translocation positive tumors.