Advancements in minimal residual disease detection: a practical approach using single-cell droplet PCR for comprehensive monitoring in hematological malignancy

Abstract

The identification of chromosomal abnormalities accompanied by copy number alterations is important for understanding tumor characteristics. Testing methodologies for copy number abnormality have limited sensitivity, resulting in their use only for the sample provided at the time of diagnosis or recurrence of malignancy, but not for the monitoring of minimal residual disease (MRD) during and after therapy. We developped the "DimShift" technology which enable to measure the copy number of target gene/chromosome in each cell, which is given by the single cell droplet PCR. Qualitative result of DimShift given by peripheral blood was perfectly concordant with that of bone marrow. These findings and performances are promising to be the new methodology for MRD detection in malignant diseases utilizing bone marrow as well as peripheral blood.

Keywords: chromosomal abnormalities; droplet digital PCR; minimal residual disease; myelodysplastic syndrome.

Figure 1.

(a) GM22948 cells, which have a normal karyotype, were counted and the number of cells was adjusted to approximately 5000. The cells were then mixed with PCR enzymes, buffer (ddPCR Multiplex Supermix; Bio-Rad), and specific probes/primers (IDT), and subsequently loaded on a cartridge with oil to create droplets using a specialized instrument (QX200 Droplet Generator; Bio-Rad). In the 2D readout, (1) indicates the dots obtained from these droplets, which contained one cell with enzymes and probe/primers, and (2) indicates there were more than two cells in each droplet. The droplets were transferred to a PCR tube to conduct the PCR [85°C 60 min, 95°C 10 min, (94°C 30 s, and 62°C 2 min) × 23, 98°C 10 min, 10°C hold, ramp rate = 1°C/s] using a thermal cycler (C1000 Touch; Bio-Rad). The fluorescence was analyzed by the droplet reader, and the manufacturer’s software displayed the 2D readout. (b) The same experiment was conducted using GM13721 cells with the deletion of chromosome 13. (c). The results from both cell line experiments were merged. FAM signals obtained from the GM13721 cells were reduced on the 2D readout (arrows). PCR, polymerase chain reaction.

Figure 2.

Peripheral blood was collected from a patient with MDS. The blood cells were hemolyzed and then used in the new assay to determine the copy number of chromosome 20. In the 2D readout, (1) indicates the signals from each droplet with one normal karyotype cell, (2) indicates more than two cells, and (3) indicates groups of deleted cells. The numbers of (3) dots were divided by the total values of (1), (2), and (3) to calculate the population of abnormal cells as 0.3%. The manufacturer’s software compensated for the number of droplets with more than two cells using a Poisson distribution. (a), (b), (c): each result was shown with each sample from different patients. MDS, myelodysplastic syndrome.