Establishment and study of different real-time polymerase chain reaction assays for the quantification of cells with deletions of chromosome 7

Abstract

The evaluation of residual disease, which has prognostic value in the treatment of hematological malignancies, is currently assessed by scoring a limited number of cells by karyotyping and molecular cytogenetics. Real-time polymerase chain reaction (PCR) is an easier and more sensitive technique, enables analysis of a larger number of cells, and decreases sampling error. However, real-time PCR has been applied only to target transcripts of fusion genes. Here, we considered two real-time PCR strategies to quantify a number of cells carrying a partial deletion of chromosome 7 mixed with normal disomic cells. The first strategy was based on the amplification of two sequences, one on chromosome 7 and the other on chromosome 14. In the second strategy residual disease was assessed by the ratio between the two alleles of a bi-allelic marker, mapped on chromosome 7, measured with allele-specific assays. Precision and accuracy of the two approaches were tested by reference samples with nominal values of residual disease ranging from 2 to 95%. As expected the second strategy resulted in more precise and accurate monitoring within the range from 5 to 95%. Furthermore, this method may be applied to assess the number of dysplastic or neoplastic clones carrying any unbalanced chromosome changes.

Figure 1

Illustration of the forward primers and the common reverse primer and probe. In square brackets, the MID1064 polymorphism on the partial sequence of 7q35.

Figure 2

Plots of the MRD functions (top) and their derivatives (bottom). ΔΔCt values range from 0 to 1 for the Chr versus Chr function and from 0 to +∞ for the two alleles function.

Figure 3

Plots of Ct values of 100, 20, 2, and 0.2 ng of genomic DNA versus the logarithm of the corresponding DNA amount. For each plot the coefficient of determination (R²), slope, and PCR efficiency are reported. Threshold was set at 0.1.

Figure 4

Calculated MRD values. A: The relative difference between nominal and calculated MRD values, SD of measured ΔΔCt (σ_ΔΔCt), and range of error at the 95% confidence interval are reported for each reference sample. Threshold was set at 0.1. Plots of calculated versus nominal values of MRD are reported in B and C for the Ch versus Ch strategy and the two alleles strategy, respectively. Stippled lines enclose the range of error. Slope and coefficient of determination (R²) of the plots are reported.