Large amplicon droplet digital PCR for DNA-based monitoring of pediatric chronic myeloid leukaemia

Abstract

Quantification of tumour-specific molecular markers at the RNA and DNA level for treatment response monitoring is crucial for risk-adapted stratification and guidance of individualized therapy in leukaemia and other malignancies. Most pediatric leukaemias and solid tumours of mesenchymal origin are characterized by a relatively low mutation burden at the single nucleotide level and the presence of recurrent chromosomal translocations. The genomic fusion sites resulting from translocations are stable molecular tumour markers; however, repeat-rich DNA sequences flanking intronic breakpoints limit the design of high sensitivity PCR assays for minimal residual disease (MRD) monitoring. Here, we quantitatively evaluated the impact of repeat elements on assay selection and the feasibility of using extended amplicons (≤1330 bp) amplified by droplet digital PCR to monitor pediatric chronic myeloid leukaemia (CML). Molecular characterization of 178 genomic BCR-ABL1 fusion sites showed that 64% were located within sequence repeat elements, impeding optimal primer/probe design. Comparative quantification of DNA and RNA BCR-ABL1 copy numbers in 687 specimens from 55 pediatric patients revealed that their levels were highly correlated. The combination of droplet digital PCR, double quenched probes and extended amplicons represents a valuable tool for sensitive MRD assessment in CML and may be adapted to other translocation-positive tumours.

Keywords: BCR-ABL1 fusion; biomarkers; chronic myeloid leukaemia CML; digital PCR; disease monitoring; genomic fusion sequences; pediatric oncology; translocation.

Figure 1

BCR and ABL1 breakpoint distribution in 178 patients with pediatric CML. A, Proportion of genomic breakpoints within repeat regions in the BCR and ABL1 breakpoint cluster regions, as observed in the study cohort, and expected based on the repeat content in the respective breakpoint cluster regions. The expected proportions of breakpoints within repeat elements were calculated for expanded regions, taking into account that approximately 150 bp flanking the fusion site is necessary for primer and probe design. B, Localization of genomic breakpoints within the breakpoint cluster regions in the BCR and ABL1 genes in relation to repeat elements. Lines represent individual genomic breakpoints. Repeat elements are colour coded as indicated. C, Multiple alignment of BCR‐ABL1 fusion sequences in comparison with corresponding BCR and ABL1 wild‐type sequences. The black line indicates the fusion site between BCR (left) and ABL1 (right)

Figure 2

Performance of primer and probe sets with different amplicon lengths in ddPCR. A‐C, ddPCR sets with different amplicon lengths were compared for quantification of the single copy gene, ALB. All ddPCR sets used the same forward primer and probe, but different reverse primers, at increasing distances from the breakpoint. A, Signal amplitudes of amplicon‐positive and ‐negative droplets. B, Differences between amplitudes of amplicon‐positive and ‐negative droplets. C, Calculated concentrations (ALB copies/µL) of different ddPCR sets using the same amount of initial template DNA (50 ng). D and E, Comparison of amplitudes from ddPCR sets with varying amplicon lengths (D) and assay sensitivities (BCR‐ABL1‐positive cells/all cells) (E) for BCR‐ABL1 fusion gene quantification in samples from patients with CML. F, Amplitudes of amplicon‐positive and ‐negative droplets using identical ddPCR sets with differently quenched probes. NTC, no template control; CML, CML patient sample

Figure 3

Comparison of ddPCR results from BCR‐ABL1 fusion gene (DNA) and BCR‐ABL1 transcript (RNA) quantification. A, BCR‐ABL1 copy numbers in 687 blood or bone marrow samples from 55 patients with pediatric CML determined using RNA‐ and DNA‐based quantification. nc, non‐quantifiable; nd, non‐detectable. B, Example of disease monitoring by analysis of BCR‐ABL1 fusion transcript (RNA) and BCR‐ABL1 fusion gene (DNA) copy numbers during imatinib treatment and temporary treatment discontinuation. NC, nucleated cells